применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 1 – 2016 contents: 1 prospects of applying vacuum technology in the manufacture of culinary poultry meat products anna soletska, andrew krasota 3 9 2 application of d-optimal mixture design to optimize the wheat-pumpkin composite flour for bread production georgiana gabriela codină,silvia mironeasa 10 20 3 comparative study on the lipase activity from plant sources, under various conditions of ph, temperature and substrate marcel avramiuc 21 28 4 chemical analysis of fresh water from a karst area in ukraine volodymyr diychuk, anastasia sachko, igor winkler 29 35 5 ground water quality of domestic wells in cajvana town, dinu iulian oprea-gancevici, cătălin savu 36 45 6 correlations between quality of flour t-500 characteristics and bread volume viktorija stamatovska, tatjana kalevska, marija menkinoska, gjorenakov, zora uzunoska, lenche mitkova 46 54 7 tradicional production and chemical composition of bieno cheese in the republic of macedonia gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska 55 60 8 production of protein-containing preparations of natural origin nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya 61 66 9 development of complex acidifiers and analysis of impact on biochemical processes in dough tetyana silchuk, vira zuyko 67 70 10 mathematical modeling of preparing "oil / water" emulsions oksana lugovska, vasilij sidor 71 75 11 the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment оksana kochubei-lytvynenko, vyacheslav mykhailyk, anatoliy ukrainets 76 83 food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 1 – 2016 12 compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest florin-dumitru bora , alina donici, oana-mihaela ripanu 84 – 94 13 total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals valentina mancas, sonia amariei 95 100 author instructions i v subscription information vi stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xii, issue 3 30 september 2013 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava , volume xi, issue 1 – 2012 44 research on the in vivo variations of the ph values, oxidized lipids and soluble protein content of food rations based on turkey meat *ancuţa elena coşuleanu1 1 ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: cosuleanu_ancuta@yahoo.com *corresponding author received 19 january 2012, accepted 20 february 2012 abstract: the purpose of this study is to analyze the changes of ph values, soluble protein and oxidized lipid content in diets based on turkey meat after their ingestion. the biological material used was represented by mini pigs female, bred göttingen, which were alimented with diets that had in their composition turkey meat and after ingestion the stomach contents was collected in kinetics. turkey meat used was represented by the pectoralis major muscle. analyzing the data on the ph variation between 15 and 330 minutes after ingestion, there was observed a downward trend of the values, the maximum value being achieved in the first 15 minutes (6.10) and the minimum was of 1.28 at 240 minutes. in terms of protein content it has been observed a reverse trend compared to the one of ph from 0.12 mg / ml and reaching a maximum of 0.62mg / ml. the obtained results analysis indicates that as the ph decreases the amount of soluble protein increases, the animal factor having a great influence. the content of oxidezed lipids decreases with the digestion time progress. keywords: gastric ph, protein content, oxidized lipids, diet, turkey meat 1. introduction lipids play an important role in the diet because the linoleic and linolenic acids are present as polyunsaturated [1]. proteins are basic and functional components, influencing the textural, sensory and nutritional properties [2]. protein solubility is one of the most important functional propertie with applications in food industry. these properties are influenced by the environmental ph and processing temperature. the chemical composition of meat has a considerable impact on its quality because it determines the properties thar are valued by consumers. in the current context, lipid and protein daily consumption is two times higher at the meat loyal consumer compared with the ones of diversified foods [3]. 2. material and methods materials the biological matherial used was represented by 5 mini pigs females, of gottingen breed (identified as a, b, c, d, e, f), equipped with equipped with a permanent cannula at the out of the stomach. stomach content was harvested in kinetics at 15, 45, 90, 150, 330 minutes after ingestion. the surgery of cannulas placement was conducted with three weeks before the experience. with 24 hours before performing the experiment food mailto:cosuleanu_ancuta:@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava , volume xi, issue 1 – 2012 45 administration was interrupted and the access to water was halted in the morning of the experience. rations were administered after performing a stomach emptying. the turkey meat used was the muscle pectoralis major fresh, conserved 2 days in vacuum at +4° c. in order to be use the meat was subjected to grinding, weighing, vacuum operations and boiling in a baie marie at a temperature of 72 ± 0,3° c to achieve in the interior a temperature of 70 ± 1° c for 30 minutes. preparation of food rations was done by mixing (15 seconds at minimum speed, 45 seconds at medium speed) various ingredients: boiled turkey meat (120g), sunflower oil (40 ml), cellulose (7 g), starch (70 g), egg yolk (3 g), pectin (1 g), water (30 ml). ph measurements the ph was determined using a hanna digital ph-meter. soluble protein content measurement the soluble protein content was determined through the bradford method. the bradford colorimetric method is based on the absorbance reading of dye coomassie brilliant blue g-250 which forms a blue color in acid environment by binding with the proteins. 1 g of stomach content was stirred with 10 ml phosphate buffer 0,025 m (k2hpo4 + kh2po4) and kept at +4° c for 12 hours. the prepared samples were centrifuge, 100 µl of the obtained supernatant was sampled and put in contact with 5 ml bradford solution prepared in advance (1ml bradford reagent + 4 ml deionized water). the reading was performed at 595 nm using a spectrophotometer against a blank consisting in phosphate buffer. previously it will be prepared a standard range consisting in a bovine serum albumin solution of varying concentrations (0; 0.05; 0.1; 0.2; 0.5; 0.75; 1 mg/ml) to determine the extinction coefficient that will be used to determine the samples concentrations. oxidezed lipid content measurements the content in oxidezed lipids was determined by the tba test, using a spectrophotometer. this method is based on the carbonyl dosage from the decomposition of the lipid hydroxiperoxides. in acid, aldehydes such as malondialdehyde (mda) can react with acids to form a tiobarbituric pink complex that has a maximum absorbance at 535 nm. all the samples were made in duble. 3. results and discussions the analysis of gastric ph values changes showed, in general, a downward trend (table 1). the ph values determined after 15 minutes from the moment of food rations administration ranged around 6, the only exception was observed in the case of the samples colected from the mini pig e. the maximum value was determined at the mini pig a at the sampling carried out at 45 minutes (6.67), while the minimum was 1.22 (240 minutes) at the animal b. table 1 evolution of gastric ph time (min) ph values mini pig a mini pig b mini pig c mini pig d mini pig e 15 6.6 5.82 6.1 5.64 4.19 45 6.67 4.97 6.05 4.0 4.64 90 6.41 3.47 5.84 3.77 3.49 150 6.33 1.93 5.41 2.63 3.16 240 5.3 1.22 5.06 1.82 3.14 330 4.6 2.02 4.94 4.11 3.03 as the duration of digestion increases up to 330 minutes, the ph decreases, except the values determined at 330 minutes at the mini pig b (2.02), which was higher than that obtained at 240 minutes after ingestion (1.22) and the one from 330 minutes from the mini pig d food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava , volume xi, issue 1 – 2012 46 (4.11) compared to the one from 240 minues (1.82). protein solubility is crucial in the processing of meat. the amount of soluble protein increases with the digestion period progress until a certain moment (90 minutes) after ingestion. after this moment it decreases, being recorded exceptions depending on the animal. the maximum of this parameter was obtained at the mini pig c at 90 minutes after ingestion (0.62 mg/ml) and the minimum value was recorded at 330 minutes at the animal b (0.12 mg/ml) (table2). table 2 the soluble protein content evolution time (min) soluble protein content (mg/ml) mini pig a mini pig b mini pig c mini pig d mini pig e 15 0.24 0.27 0.27 0.23 0.21 45 0.26 0.24 0.17 0.28 0.21 90 0.26 0.34 0.62 0.33 0.29 150 0.22 0.31 0.28 0.3 0.26 240 0.31 0.19 0.21 0.2 0.22 330 0.22 0.12 0.21 0.2 0.2 it is generally observed that the soluble proteins are found in higher levels after 15 minutes of ingestion compared to the values determined at 330 minutes. the quantity of oxidized lipids shows great variations dependind on the collection time and animal (table 3). in general it can be observed the fact that the content of oxidezed lipids decreases with the digestion time progress. the animal factor plays a crucial role in the process. table 3 the oxidized lipid content evolution time (min) soluble protein content (eq mda/g tissue) mini pig a mini pig b mini pig c mini pig d mini pig e 15 2.22 1.39 1.79 1.60 0.66 45 1.58 2.14 0.99 1.60 1.84 90 2.11 1.20 0.85 1.23 1.04 150 0.58 1.23 1.15 0.92 0.71 240 0.73 0.71 0.82 0.73 0.59 330 0.88 0.57 1.04 0.92 0.73 the minimum values was obtained at the mini pig b (0.57 eq mda/g tissue) at 330 minutes after ingestion and the maximum was determined at the mini pig a at 15 minutes (2.22 eq mda/g tissue). 4. conclusion with the progress of digestion time, the ph value of the stomach content drops to a certain point after which it increases moderately. soluble protein contents of the digesta, obtained by the administration of diets based on turkey meat, grows with the digestion time duration to a certain point and then decreases. the content of oxidezed lipids decreases with the digestion time progress. the animal factor has a great influence, being observed a variation of the indicators determined by it. this study was performed using this biological material in order to observe the changes that occur in the stomach, being known that digestion in pigs is similar to that of humans. 5. references [1] lorrain b., dangles o., genot c., duford c., chemical modeling of heme-indused lipid oxidation in gastric conditions and inhibition by dietary polyphenols. journal of agricultural and food chemistry, 58, 676-683 (2010) [2] kinsella j.e.: protein structure and functional properties: emulsification and flavor binding effects, in food protein deterioration, mechanism and functionality, ed. j.p. cherry, washington, (1982) [3] zayos j.f.: functionality of proteins in food, ed. springer, kansas, sua, (1997) issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xv, issue 3 30 september 2016 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt (amariei), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xi, issue 1 15 march 2012 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates,bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), nationaltechnological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius,lithuania norbert kreuzinger vienna university of technology, institute for water quality,resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic ofmoldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal:@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts:@fia.usv.ro mailto:gabriela.constantinescu:@fia.usv.ro mailto:cristina.hretcanu:@fia.usv.ro microsoft word 0a. first pages.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xii, issue 1 30 march 2013 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xv, issue 4 31 december 2016 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt (amariei), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 224 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 224 – 227 influence ratio of water and oil phase of emulsions on viscosity, particle size and turbidity of products *oksana lugovska1, vasilij sidor1 1national university of food technologies, volodimirska 68, kiev, ukraine, oksana.lugovska@rambler.ru *corresponding author received august 24th 2014, accepted september 24th 2014 abstract: the paper systematically describes the basic theoretical information about the improvements in the production of emulsions. we consider the theory of a stable emulsion system, namely the particular use of raw materials and their properties analyzed the conditions necessary for the process of homogenization. and based on this, a large number of existing theories determined the most effective, which is used for the production of emulsions. much attention is paid to the use of different stabilizers receipt of test data required for the calculation formulas finished products, and technological design process of emulsions. to assign these methods using two stabilizers: resin acacia and modified starch, which when used for stability during storage of emulsions yield different results. of great importance for the stability of these products is the size of the particles. the diameter of the emulsion depends on a process of manufacturing technology. more detail the process of homogenization of emulsions. for the features of this process are a few examples that will visually see the results emulsion stability during storage. based on the processed foreign sources give modern technology and types of equipment for the homogenization process, with reference to the drawings, which will assimilate the information produced. we consider the design features of these devices, their advantages and disadvantages. keywords: emulsion, particle size, phase, stability, stabilizer 1. introduction some food, that are made using emulsions containing 1–1000 nm particle size and treated as objects of classical colloid chemistry. of great importance for the stability of these products is the size of the particles. in the case you known information about the particle size emulsion, you can control their stability and quality. it is known that the diameter of the emulsion depends on a process of manufacturing technology, the recipe emulsion. to prepare emulsions, is dispersion of one liquid in another, in practice, using mechanical means, which allow disperse phase. there is a theory about the mechanism of emulsification [1]. the first stage of this process lies in the tension drops of liquid dispersion in a field environment. pulling drops in thread accompanied by an increase of the surface and flow of work to overcome the molecular forces of surface tension. this extended liquid drop becomes so unstable that spontaneously breaks into small spherical droplets. this is the second stage of the formation of emulsions, which is accompanied by a decrease in surface and spontaneous process. then comes the next, third stage, when formed droplets on the one hand, coagulated in collisions, and the other again stretching into smaller parts to equilibrium. the basis of increasing dispersion emulsion is spontaneous decay drops learned to unstable size [2–4]. found that a stable, emulsions are closely associated with the mechanism of dispersion and depends on many factors, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 oksana lugovska, vasilij sidor, influence ratio of water and oil phases of emulsions on viscosity, particle size and muddy turbidity of products, food and environment safety, volume xiii, issue 3 – 2014, pag. 224 – 227 225 such as oil content, type and concentration of emulsifier, the route of administration phases, time and intensity and degree of dispersion and temperature. study of factors that ensure stability of emulsion, led to the conclusion that the critical degree of dispersion [5–9]. experiments found that for each type of emulsifier has its own optimum concentration that provides the highest resistance obtained emulsions [7]. for an introduction to emulsify oils (for each concentration of emulsifier) is also optimum in which the most stable emulsion is obtained, that are determining the optimal ratio between the aqueous and oil phases. excess oil in the emulsion is separation. thus for each emulsifier is its optimum concentration, the corresponding amount of oil in the emulsion [8]. the optimum concentrations of emulsifiers for certain ratios of the phases in obtaining stable emulsions are not fixed and depend on the degree of dispersion. using of highspeed mixing, and especially increasing pressure homogenizer leads to increased dispersion, viscosity and the formation of more stable emulsions [9]. 2. materials and methods the aim of the study particle size effects on the stability of emulsions during storage and use in the manufacture of beverages and their stability during 180 days. as materials for research prepared samples of emulsions with various stabilizers (gum arabic, modified starch). stability of emulsions depends of viscosity, particle size, muddy turbidity depends on the ratio of water and oil phases. for studies prepared 5 sample emulsions of varying oil phase and a constant number of gum arabic (table 1) and 5 samples emulsions of varying oil phase аnd constant number of starch as stabilizer (table 2). table 1 formulations of emulsion of varying oil phase and a constant number of gum arabic the ingredients of the emulsion content ingredient, g/kg number of emulsion 1 2 3 4 5 citrus oil 60 60 60 60 70 rezynogum (е 445) 20 40 50 60 70 gum arabic (е 414) 50 50 50 50 50 citric acid (е 330) 5 5 5 5 5 sodium benzoate (е211) 2.5 2.5 2.5 2.5 2.5 water 862.5 842.5 832.5 822.5 802.5 total 1000 1000 1000 1000 1000 table 2 formulations of emulsion of varying oil phase аnd constant number of starch the ingredients of the emulsion content ingredient, g/kg number of emulsion 6 7 8 9 10 citrus oil 40 50 55 60 70 rezynogum (е 445) 40 50 55 60 70 starch (е 1450) 120 120 120 120 120 citric acid (е 330) 5 5 5 5 5 sodium benzoate (е 211) 2.5 2.5 2.5 2.5 2.5 water 792.5 772.5 762.5 752.5 732.5 total 1000 1000 1000 1000 1000 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 oksana lugovska, vasilij sidor, influence ratio of water and oil phases of emulsions on viscosity, particle size and muddy turbidity of products, food and environment safety, volume xiii, issue 3 – 2014, pag. 224 – 227 226 preparation of emulsions 1. preparation of oil phase. weigh the required amount of flavor; add esther scales in stirrer at room temperature until esther scales completely dissolved. 2. preparation of the aqueous phase. a. weigh the required amount of water into a glass and heated to 20-50 ° c. b. attach the required amount of sodium benzoate and completely dissolve. add citric acid and dissolve completely. c. attach the required amount of stabilizer and dye solution in warm water (20-50°c). d. stabilizer mix and dissolve at a moderate speed mixer until it is completely dissolved. subject the immediate hydration; leave for a few minutes for aeration. 3. preparation of the preemulsion for the рreparation of the pre-emulsion use a high-speed mixer. slowly adding the oil phase to the aqueous phase, and then stirred at maximum speed. 4. preparation of the emulsion by homogenization. homogenization undergoing the parameters a. pressure is the first step / second step 200 /50 bar, two numbers of moves for emulsion with starch. b. pressure is the first step / second step 280 /40 bar, two numbers of moves for emulsion with gum arabic. 5. measure turbidity, viscosity and average particle size of the emulsion. investigation of the stability of emulsions was carried out by determining the size of the diameter of the particles by laser granulometry and placement on the stability of soft drink, which was used emulsion for 180 days. in the production of emulsions initially prepared aqueous and lipid phases, mixed them turbomixer and received preemulsion with particle size of about 3.0 microns. in the next step, by homogenizing the emulsion obtained with particle size from 0.1 to 1.0 microns. during the preparation of the aqueous phase in water soluble all items that are part of this phase: stabilizers (gum arabic, modified starch), acid dyes, water soluble, preservatives, antioxidants (ascorbic acid). in practice, the most important stabilizer in the manufacture of emulsions for soft drinks are: gum arabic and modified starch. to protect the product from microbial spoilage used preservative sodium benzoate. acidification lemon emulsion or malic acid to ph 4.0 bolsters preservative as well as a positive effect on the effective viscosity of the emulsion. an important factor in the production of emulsions is a significant difference in density between oil and water. essential oils have an average density of about 0.845 g/l, while the density of water is 1.0 g/l. it is therefore necessary to align low-density essential oil by adding substances that increase density. substances that increase density are rezynogum. 3. results and discussion the results of measurement of each emulsion: brookfield viscometer viscosity microscope eastcolight 92012-es (100x, 250x, 550x, 750h ) – particle size , muddy turbidity meter 2100p, lab aerometer density, lab рнmeter – ph displayed in table 3.. analyzing the figures emulsions with different stabilizers characterized by an increase in the number of oil as a part of the product shows that increasing viscosity, density, particle size of emulsion and turbidity. by continuing other components of the emulsion, the smaller the particle size, the lower the turbidity of the emulsion (but higher storage stability). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 oksana lugovska, vasilij sidor, influence ratio of water and oil phases of emulsions on viscosity, particle size and muddy turbidity of products, food and environment safety, volume xiii, issue 3 – 2014, pag. 224 – 227 227 table 3 the results of measurement of the finished product number of emulsion viscosity brookfield, cp turbidity dilution 0.025 %, ntu the average diameter of the particles of oil d, μm density, g/ cm3 рн 1 14 168 0.505 1.03 2.7 2 15 180 0.659 1.06 3.2 3 16 192 0.705 1.07 3.3 4 17 216 0.903 1.09 3.7 5 18 240 1.101 1.1 3.9 6 20 143 0.67 1.03 2.6 7 22 156 0.73 1.05 3 8 23 170 0.75 1.07 3.3 9 24 182 0.84 1.09 3.65 10 26 196 0.97 1.1 4 if the particle size less than 1 micron, the emulsion is highly robust stability and gives some turbidity but less than 1 micron particle size, the less turbidity, if the particle size is not greater than 0.3 micron, the turbidity almost any clear solution. the principle of leverage ratio of water and oil phase of emulsions with different stabilizers same. in the obtained parameters also affects the nature of emulsions stabilizer. 4. conclusion the best result of research in emulsions – is to obtain the maximum number of particles of about 1 micron. technology of preparation of emulsions with gum arabic is different from the technology of emulsifying starch. dissolve gum arabic is faster and easier than with the dissolution of starch as emulsion obtained using gum arabic, stable in quality and more expensive in value compared with emulsions prepared by using starch. different pressures for homogenization of emulsions with gum arabic and starch as the process of emulsification affects the nature of stabilizing. the results of measurement of each emulsion: viscosity, particle size, muddy turbidity depends on the ratio of water and oil phases. 5. references [1]. borisenko o.v., alekseev j.a., klimov s., methods of creating highly concentrated flavoring emulsions for soft drinks, food ingredients. raw materials and additives, 2, 18-19, (2002). [2]. phillips g.о., williams p.a., (eds.) handbook of hydrocolloids. cambridge: wood head publishing, 156 с, (2000). [3]. mckenna в.m. (ed.), texture in food — vol. 1: semi-solid foods. — cambridge: woodhead publishing, 480 с, (2003). [4]. bogach a., aromatic emulsion for manufacture of soft drinks, food & drinks. food & beverage, 4 s. 10-11, (2003). [5]. stephen p., u.s. patent for invention № 6576285, cholesterol lowering beverage, bader, fowler, 10.06.2003. [6]. imeson a. (ed.), thickening and gelling agents for food. 2nd ed., london: blackie academic and professional, 408 с, (1999). [7]. whistler r.l.b., miller j.n., paschall e.f., (eds.), starch chemistry and technology. 2nd ed. orlando, fl: academic press, 1984, 3508. galliard t. (ed.) starch; properties and potential /society of chemical industry. chichester, uk: john wiley and sons, 210 с (1987). [8]. atwell w.a., thomas d.j. starches. — st. paul, mn: american association of cereal chemists, 150 с. (1997). microsoft word 17 stefanov 2.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 103 investigation of the process of storing packaged rabbit meat in modified atmosphere *stefan stefanov1, hristo hristov1, donka stoeva1, zapryana denkova1, radosveta nikolova 1 1university of food technologies, plovdiv, stvstefanov@yahoo.com *corresponding author received 5 february 2012, accepted 10 march 2012 abstract: the article presents experimental studies with packaged in modified atmosphere and stored refrigerated rabbit meat. we have used gas environment with a composition 80% o2 and 20% co2 and vacuum. as a result of the experiment graphs of ambient temperature in cold storage were constructed. the shelf life of the packaged rabbit meat has been determined on the basis of microbiological analysis. keywords: modified atmosphere, cold storage, shelf life, rabbit meat 1. introduction meat and meat products are fast tainting foods therefore they must be stored refrigerated immediately after they are obtained. according to the european legislation of the modern meat industry, the fresh meat should be refrigerated to below 7°с. rabbit meat refers to the so-called white meat. it is obtained in specialized abattoirs. due to its low cholesterin content it is recommended to people, suffering from cardiovascular or digestive system disease. compared to other meats, it has the lowest fats content 5,1% and highest proteins content 22,77%. the human body absorbs it up to 90 %, while the beef is being absorbed up to 64%. the rabbit meat is stored in refrigerated condition up to 7 days at temperature between 0 to 4ос. the refrigeration slows down the growth of microorganisms, the metabolite processes and reactions, which can lead to worse quality of the meat. the influence of the refrigeration on the microflora depends on the temperature and the storage time. the storing effect of the refrigeration can be increased to a great extend by combining with storing in modified atmosphere. there are different approaches for applying modified atmosphere for increasing the expiry date of packaged and refrigerated stored meats. the definition of the expiry date of these foods is a complex investigation of the store temperature and their microflora. the aim of the present work is to investigate the expiry date of refrigerated stored rabbit meat, packaged in vacuum and gas environment with a composition of 20% со2 и 80%о2 . 2. materials and methods for packaging of the rabbit meat are used semi-automatic machines tray sealer “multivac 250t” and double chamber vacuum “henkelman h500”. the meat had been obtained the same day and it has been preliminary refrigerated to 4ос. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 104 when packaged with tray sealer “multivac 250t” the rabbit meat is put in a polypropylene (pp) tray with dimensions 227х178х36 mm, which is sealed with foil “bialon pp65 af” made by “wipak” company, having been preliminary filled with gas environment of 80%о2 and 20%со2. when packaged in vacuum by “henkelman h500”, the rabbit meat is put in a bag 180/220/70 iptv and is sealed in the package at the pressure of 10 mbar. each packaging machine makes five packages, as each of their sealing seams are visually checked. one of each is give for microbiological analysis as a control sample and the rest are put in refrigerator “liebherr” ct2841 and are stored in refrigerated conditions. the reading and registration of the store temperature is done by remote integrated control system (r.i.c.s.) evco italy, which has five separate temperature probes – pt 100 (fig. 1). figure 1. r.i.c.s. (remote integrated control system) during the refrigerated store (table 1) of the packaged rabbit meat, we define the microbiological indexes by the following methods: бдс en iso 4833:2004 microbiology of food and animal feeding stuffs horizontal method for the enumeration of microorganisms colony-count technique at 30 degrees c ; бдс en iso 6888:2005 microbiology of food and animal feeding stuffs horizontal method for the enumeration of coagulase-positive staphylococci (staphylococcus aureus and other species); бдс en iso 6579:2002 microbiology of food and animal feeding stuffs horizontal method for the detection of salmonella spp (iso 6579:2002); iso16649-2:2001 microbiology of food and animal feeding stuffs -horizontal method for the enumeration of beta glucuronidase-positive escherichia coli - part 2: colony-count technique at 44 degrees c using 5-bromo-4-chloro-3indolyl beta-d-glucuronide ; the reading and registration of the temperature is done by the temperature probes of r.i.c.s, which are located in the following zones: (fig. 2). figure 2. location of the temperature probes 1 lower part; 2,4,5 central part; 3 in the room. 3. results and discussion the temperature variation in the refrigerator ct 2841 liebherr has been followed for the whole store period and we have presented data for only two days (48 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 105 hours), because during the next days the temperature has no variations (fig.3 and4). figure 3. store temperature during the first day figure 4. store temperature during the second day the packaged rabbit meat is stored in the central zone of the refrigerator (probe 2, 4 and 5). during storing the outer temperature varies from 25 to 28ос (probe 3). we can see on the graph that in the beginning the packages with rabbit meat are cooled and reach store temperature around the fifth hour. the average store temperature for the whole period is 5,8ос, but the expiry date of storing is defined by the maximal temperature 6,3 ос. during store of whole packaged rabbit meats there is a change of the total microbial counts (table1 and fig.. 5а) due to the upgrowth of pshychrophilic and psychtrophic microorganisms, which appeared there during the process. the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 106 modified gas environment (80%о2 and 20%со2) has a greater effect on their growth. the data show that the whole rabbit meats, packaged in modified gas environment can be stored in refrigerated condition up to 9 days (table 1). there is no alteration in the pathogen flora. table 1 microflora of rabbit meat packaged in vacuum store time 3 days 6 days 9 days 12 days coliforms, cfu/g <102 <102 2.103 staphylococcus aureus, cfu/g <10 2 <102 <102 salmonella no no no no total microbial counts, cfu/g 3,2.10 4 1.105 >1.106 >1.108 packaged in gas environment 80%о2 and 20%со2. coliforms, cfu/g <102 <102 6.102 staphylococcus aureus, cfu/g <10 2 <102 <102 salmonella no no no no total microbial counts, cfu/g 3,2.10 3 9,4.103 8.104 >1.108 a) b) figure 4. total microbial counts of packaged rabbit meat in vacuum (a) and gas environment 80%о2 and 20%со2 (b). in the vacuum packaged rabbit meats the total microbial counts significantly increases till day 9. because of that the meat in this packaging can be stored in refrigerated conditions up to 6 days (table 1 and fig. 5b). the number of coliforms increases with 1 logarithmic unit (log n). the rest pathogenic microflora has no variations. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 107 4. conclusion the experimental studies show the possibilities of gas environment application for packaging of whole refrigerated meats and increase the expiry date of their storage time. 5. references [1]. dalle zotte a. 2002. perception of rabbit meat quality and major factors influencing the rabbit carcass and meat quality. livestock production science 75: 11-32. [2]. corino c., pastorelli g., pantaleo l., oriani g., salvatori g. 1999. improvement of color and lipid stability of rabbit meat dietary supplementation with vitamin e. meat sci. 52: 285-289 [3]. н. тинаев. разведение кроликов. м.: компания дельта,м, 2004. 48 с: ил. (серия: практические советы) isbn 5-94107-227-9 [4]. m isabel berruga, herminia vergara, m belén linares,” control of microbial growth and rancidity in rabbit carcasses by modified atmosphere packaging” journal of the science of food and agriculture volume 85, issue 12, pages 1987–1991, september 2005 study fermentation process modified sunflower husks 175 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 175 179 study on modified sunflower husk fermentation process for vermicultivation *nataliia borisivna mitina1, inna mihalivna zubareva2, alyona dmitrievna odina1 1dnepropetrovsk chemical-technological state university, dnepropetrovsk, ukraine 2national university of dnepropetrovsk, ukraine *natalimitina_68@mail.ru, zubareva95@bk.ru, *corresponding author received 5th june 2016, accepted 27th june 2016 abstract. the quantitative and qualitative composition of microorganisms, making up the modified sunflower husk microflora while fermentation, as well as a modified solid-nutrient substrate for accumulation of eisenia foetida biomass during vermicultivation, was studied. species’ composition of microorganisms changes during the fermentation process: mesophilic organisms are sequentially replaced by mycelial ones, and then by thermophilic spore-forming bacteria and actinomycetes. complex multispecies preparation em probiotic has been used to intensify fermentation. keywords: biomass, vermiculture, solidphase substrate, cultivation, microflora. 1. introduction the main objective of vermicultivation is to solve the problem of biological object's ability to adapt to a specific substrate. there is an adaptation assessment based on morphological criteria: the worm’s behavior in the substrate, the size of the worm’s body, the number of specimens being at different stages of ontogenetic development, the response to exogenous factors, body color etc. as raw materials, almost all processes for the preparation of eisenia foetida biomass use manure of livestock, sewage sludge, microbiological and food processing waste [1 7]. biochemical characteristics of vegetable raw sunflower husk (sh) were studied earlier and, taking into account accumulation of large amounts of sh and sufficient level of its nutrients, they create preconditions for effective use of sh in the development of biotechnology of protein feed additives production using vermicultivation [8]. vermicultivation process consists of two stages: fermentation of sh and development of new vermiculture population on fermented substrate under the influence of abiotic factors. the stage of substrate fermentation is very important because it includes the transfer of vegetable raw nutrients to the state, available for eisenia foetida use. such transformation of the substrate components pass through its natural microflora. from the literature it is known that the gramnegative bacteria are more important in the diet of worms than gram-positive ones, and fungi are more available nutrition source than bacteria. such types of bacteria as flavobacterium lutescens, pseudomonas fluorescens, pseudomonas putida have toxic effect on the worms [9]. http://www.fia.usv.ro/fiajournal mailto:natalimitina_68@mail.ru mailto:zubareva95@bk.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 nataliia borisivna mitina, inna mihalivna zubareva, alyona dmitrievna odina, study of modified sunflower husk fermentation process for vermicultivation, volume xv, issue 2 – 2016, pag. 175 – 179 176 optimization of fermentative transformations is necessary to intensify vermicultivation process as a whole, and to enhance the microbiological processes that take place in the substrate. it is recommended to use modified sh [8]. modification consists in grinding the starting material, thus increasing its surface area, and further improves the reactivity between glucoside bonds of sunflower husk polysaccharides and worm enzyme system. the aim of this work is to conduct research in identification of the microorganisms that make up the grinded sh microflora, laid on fermentation, as well as modified solidnutrient substrate for eisenia foetida biomass accumulation. 2. materials and methods vermiculture eisenia foetida has been used as an object of study in the experiments carried out at the institute of biotechnology of ukrainian state university of chemical technology (sri "biotechnology" shei "usuct", dnipro, tu 3336406.002-95). in the first stage of the research, sh substrate, pre-modified to the size of 200500 microns, was laid on the fermentation in containers of 50-60 cm height. the fermentation was carried out in two ways: the first without the probiotic addition to the grinded sh, and the second with the presence of biologically active substance em probiotic (produced by the commercial production center “amrita”, dnipro). moisture capacity of the material significantly affects the rate of fermentation, so grinded sh was moistened with water to achieve 70-80 % of substrate moisture capacity at liquor ratio of 1:2. during fermentation, stirring of the substrate in its entire volume was performed once a week to improve aeration, activation of sh microflora and moisture equalization. eisenia foetida biomass accumulation on nutrient substrate was being carried out for 56 days. adapted to fermented sh eisenia foetida population was laid in the amount of 100 specimens (average weight of a worm 0.1 g) quantitative and qualitative composition of microflora was assessed by plating sh washing-off on meat peptone agar (mpa) medium; and to determine micromycetes on wort agar (wa) medium, followed by cultivating at 37 (± 1) °c and 28 (± 1) °c respectively. the total number of bacteria was determined after 48 hours, and micromycetes after 7 days. three weeks later, it was defined the number of mesophilic aerobic and facultative anaerobic microorganisms (mafanm). spore forms of bacteria were determined in pasteurized washing-off of sh (fermented and non-fermented), which were plated on complex nutrient mpa+wa medium in the proportions (1:1) [10]. the results obtained were processed by methods of mathematical statistics [11]. 3. results and discussion before laying sh on fermentation, it was determined the number of mesophilic aerobic and facultative anaerobic microorganisms, as well as the presence of actinomycetes and micromycetes [10]. the results indicated that there were mainly mesophilic microorganisms and a small amount of epiphytic microflora, so-called field fungi of p. cladosporium, p. thamnidium, p. alternaria. from the spore-forming microorganisms it was identified specimens of p. bacillus, aerobic microorganisms p. cellfalcicula, p. cellvibrio, p. cytophaga and a small amount of yeast. the total number of microorganisms was 106…107 per 1 g of sh. microbiological monitoring was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 nataliia borisivna mitina, inna mihalivna zubareva, alyona dmitrievna odina, study of modified sunflower husk fermentation process for vermicultivation, volume xv, issue 2 – 2016, pag. 175 – 179 177 performed before laying the nutrient substrate and during the substrate fermentation process. the fermentation process of the modified sh was conditionally divided into four stages: mesophilic, thermophilic, cooling and afterripening. at mesophilic stage sh was at ambient temperature of 20…25 с, ph of the substrate was sub acidic 6.2 ... 6.5. on the second day, the temperature in the middle of the container was 35 с, epiphytic microorganisms dying and quickly replacing by fungi p. aspergillus, p. penicillium. the development of filamentous microorganisms contributed to raising the temperature to 40 ° c, which meant gradual transition of the fermentation process to thermophilic stage. in this case, the substrate was acidified to ph 6.0 due to the formation of organic acids. on the third day, the temperature rose to 45 °c. the gradual increase of the substrate temperature was due to the rapid development and accumulation of bacteria. at the same time, the heat release rate was equal to the heat loss rate, so that on the fifth day, the temperature reached a maximum 50 с. the composition of microflora predominantly accumulated thermophilic spore-forming microorganisms p. bacillus and actinomycetes, which participated and continued fermentation processes of substrate conversion. the substrate ph became alkaline due to the emission of ammonia formed by the breakdown of proteins. though the amount of bacteria was very large in vermisubstrate, 108…109 cells per g wet wt, they formed less than half of the total microbial biomass due to the small size (1.8 microns). actinomycetes grew much slower and became visually noticeable in the later stages of the process, when their number considerably increased, forming white plaque on the surface of the sh fermented mass. this feature is considered to be typical for actinomycetes on various substrates. their number was lower than the number of bacteria and equaled 105…108 cells per g wet wt. in the cooling stage, which followed the maximum temperature, the ph was reduced, but remained slightly alkaline at 7.3 ... 7.5. thermophilic fungi of the colder zones again filled the entire volume of the substrate and, together with actinomycetes, consumed polysaccharides, hemicellulose and cellulose, destroying these polymeric compounds to monosaccharides. at afterripening stage, when microorganisms used readily absorbed compounds, the temperature was decreasing. heat release rate became very low as the microorganisms were not breeding any more, and the substrate temperature dropped to ambient temperature in a week. therefore, sh fermentation process was carried out by microflora of diverse species composition. the quantitative ratio of microorganisms groups is represented in the figure 1. mb mesophilic bacteria, y yeast, f fungi, tb thermophilic bacteria, a – actinomycetes figure 1– species composition of microflora of modified sh fermentation process the substrate fermentation was performed in the presence of complex preparation em probiotic, which contains a set of bifidobacteria in the amount of 105-109 0,1 1 10 100 мb y f тb f а microorganisms spesies m ic ro or ga ni sm s q ua nt ity , % food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 nataliia borisivna mitina, inna mihalivna zubareva, alyona dmitrievna odina, study of modified sunflower husk fermentation process for vermicultivation, volume xv, issue 2 – 2016, pag. 175 – 179 178 microbial cells / cm3, anaerobic gramnegative bacilli (bacteroides) 103-105 microbial cells / cm3, coryneform bacteria 108 microbial cells / cm3, specimens of acetobacter 1012 microbial cells / cm3, yeast 102-104 microbial cells / cm3. em probiotic being added into the grinded sh, the time of plant substrate fermentation was reduced due to the multispecies microbial composition of the preparation. thus, without adding active microorganisms, sh substrate fermentation time equals 10-14 days in the cold season and 10-12 days in the warm period. when adding biologically active substance em probiotic, sh substrate fermentation time was reduced to 6-7 days in the cold period, and up to 5-6 days in the warm season. the research has shown that the optimum amount of em probiotic on the stage of substrate fermentation equals 100 g of active substance per 10 liters of water. increasing of the substance concentration does not affect the fermentation duration and the growth rate of biomass eisenia foetida. therefore, introduction of microorganisms complex em probiotic provided conditions of symbiotic interaction between microorganisms of sh substrate and, respectively, contributed to the rapid passage of the fermentation process. on the second stage of vermicultivation, the fermented substrate prepared by various methods was used to develop of a new population of eisenia foetida under the influence of abiotic factors. each container was laid with various types of sh fermented substrate in the amount of 250 g; substrate humidity of 70 %, ph 7.5. the substrate in each container was laid with worms specimens to study the suitability of grinded fermented sh as nutrient substrate for vermiculture breeding. visual observations indicate that the worms begin to move gradually into the substrate depth and process it. moldy microorganisms, representatives of p. aspergillus, p. penicillium, were found to appear during vermicultivation on the substrate surface in the first days after the worms’ settlement, but disappear in two weeks. the results show that harmful microorganisms are being destroyed during the worms’ processing of the investigated substrates that contributes to the substrate decontamination. further observations of the worms’ activity and survival showed that modified sh can be used as a nutrient substrate for eisenia foetida and is suitable for the culture habitat. the first worm cocoons were found in a month, the larvae hatched three weeks later. dynamics of mature eisenia foetida biomass growth, depending on the substrate fermentation process based on the modified sh is shown in figure 2. 1 – without the addition of em probiotic; 2 with the addition of em probiotic figure 2 dynamics of eisenia foetida biomass population growth depending on the kind of fermented substrate the experiental data have shown that eisenia foetida biomass on modified sh substrate without addition of probiotic cultures of em probiotic has grown by 5 6 7 8 9 10 11 12 13 14 15 7 14 28 42 56 b io m as s, g ro w th , g time, day 1 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 nataliia borisivna mitina, inna mihalivna zubareva, alyona dmitrievna odina, study of modified sunflower husk fermentation process for vermicultivation, volume xv, issue 2 – 2016, pag. 175 – 179 179 1.98 times, and with its addition – by 2.1 times comparing to the initial biomass. thus, positive dynamics of eisenia foetida biomass growth indicates the suitability of the substrate based on the modified sh. 4. conclusions therefore, sh fermentation is an exothermic biological oxidation process, wherein the mixed population of aerobic microorganisms at elevated temperature 50 с and humidity 80 % subjected the organic substrate to biodegradation. being biodegraded, the sh substrate had physical and chemical transformations followed by various decomposition products: ammonia, carbon dioxide, low molecular weight peptides. there was also accumulation of dead microorganisms biomass, some amount of living mesophilic microorganisms and products of chemical interaction between these components. the composition of the fermented sh provides vermiculture with a sufficient level of nutrition, and is a suitable habitat for eisenia foetida. the composition of microflora substrate also changes during the fermentation. at the beginning of fermentation, mesophilic aerobic bacteria dominated in sh, their number gradually decreasing. however, the number of thermophilic microorganisms, actinomycetes and fungi, was increasing. all this determined the essence of fermentation process and quality of the resulting fermented substrate. the process of substrate fermentation carried out in the presence of complex preparation em probiotic occurred more intensively and decreased almost twice compared to untreated sh. 5. references [1]. sartaj ahmad bhat. potential utilization of bagasse as feed material for earthworm eisenia fetida and production of vermicompost. sartaj ahmad bhat, jaswinder singh and adarsh palvig, 12-13, (2005) [2]. maximova s.l vermicultivation and vermicomposting as the basis of organic farming in the xxi century: achievements, problems and prospects. a collection minsk, 250,( 2013) [3]. sendetsky v.n., kolisnyk n.m., melnyk i.p., bunchak o.m., gnydiuk v.s., berdnikov o.m. technological aspects of organic waste processing with vermicultivation method in agribusiness. foliant, ivano-frankivsk, 53, (2010) [4]. vyguzova m.a, research in recycling technology of agricultural production. sc.jour. kubgau №85 (1), izhevsk, (2013) [5]. puzik v.k, rozhkov r.v, doliova t.a neutralization and utilization of waste in agricultural production, manual, kharkiv: khai, 220, (2014) [6]. vyguzova m.a, kasatkin v.v, litvinyuk n.y and etc; patent 2493139 rf. receiving of vermicompost using cattle manure as a substrate. 26, appl. 09.20.2013; 3, (2013) [7]. mitina n.b., kalashnikov s.g., frolovа l.a. research technologies of protein additives creation. 2. raw materials for vermitechnology issues of chemistry and chemical technology. 3, 32-34, (2009) [8]. mitina n.b., kulik a.p., kalashnikov s.g. research technologies of protein supplements. 3. biological purity of raw materials for vermitechnology. issues of chemistry and chemical technology. 5, 24-27, (2009) [9]. gorodnii n.m., kovalev v.b., miller i.a. vermiculture and its effectiveness. sel. host ˗ in; review. inform. ser. agriculture, agricultural chemistry with. x. reclamation. ukrniinti. 20, (1990) [10]. klymnyuk s.i., sytnyk i.a., tvorko m.s., shyrobokov v.p. applied microbiology. manual ternopil: ukrmedknyha. 440, (2004) [11]. frolovа l.a., melnikov b.i., halivets j.d., mitina n.b. mathematical modeling and optimization technology objects inorganic substances. dnepropetrovsk: jour. foundation, 208, (2010) 316 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 316 321 microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012) *florin bârlă1, lenuţa ciornei (dudici)1,2, maria poroch – seriţan1 1stefan cel mare university of suceava, faculty of food engineering, suceava – romania, florin.barla@fia.usv.ro, mariap@fia.usv.ro 2 suceava county department of public health, romania, dud_ela@yahoo.com * corresponding author received october 17th 2013, accepted december 4th 2013 abstract: the microbiological quality of drinking water has attracted great attention worldwide because of implied public health impacts. monitoring and control technologies are indispensable for the production of safe drinking water. they allow for the surveillance of source water quality and the detection of biological and chemical threats, thus defining the boundary conditions for the subsequent treatment and providing early warning in case of unexpected contamination. the aim of this study was to monitor the water distribution stations in suceava county and to better understand if the modernization projects were successfully implemented. the results showed that microbiological contamination incidence during three years monitoring was low and in some cases there were no relation between the sample-detected out of line for chlorine analysis and microbiological contamination incidence. this suggests, as a particular case, that underground water in berchisesti area is microbiologically pure. however, a relatively low and constant incidence of microbiological contamination between 1-3% was recorded during 2010-2012, among the investigated samples. the samples founded out of line for chlorine treatment, meaning insufficient treatment, recorded an average of about 25% during the same period. key words: drinking water, microbiological quality, drinking water treatment 1. introduction there is no doubt that water quality plays a crucial role on human health. in developing countries, many common and widespread health risks have been found to be associated with drinking water, a large percentage of which are of biological origin [1]. the most important aspect of drinking water in relation to waterborne diseases is its microbiological quality. detection of bacterial indicators in drinking water implies the presence of pathogenic organisms. these are considered the source of waterborne diseases that could be fatal [2]. the enteropathogenic bacteria are responsible for a variety of diseases such as cholera, typhoid, dysentery and bacillary dysentery in humans [3]. they belong to the enterobacteriaceae family of organisms that comprise members of the genera food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan, microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012), volume xii, issue 4 – 2013, pag. 316 321 317 escherichia, enterobacter, salmonella and klebsiella, among others [4]. commonly, their occurrence in water is due to faecal contamination. the faecal indicator bacterium (escherichia coli), found in the faeces of all warmblooded animals and some reptiles has been considered as a bioindicator of faecal contamination of drinking water [5]. the major pathogenic bacteria responsible for waterborne diseases are spread by the faecal–oral route, in which water may play an intermediate role. bacterial contaminants such as e. coli and fecal coliform in drinking water represent an acute health risk. there are many reports, which indicate waterborne disease outbreaks in water meeting the coliform regulations [6], [7], [8], [9]. the european union council directive states, according to world health organization guidelines, that drinking water should not contain pathogenic microorganism in such concentration able to depreciate human health. to evaluate the efficiency of drinking water treatment against microbial contamination, it is needed to estimate the adequacy of disinfection conditions, such as chlorination however, the chlorination conditions used in water distribution systems are based on the inactivation of several viruses and bacteria but not mycobacterial species or other pathogens. chlorine disinfection is often combined with storage in a safe container, in order to prevent recontamination, and educational and motivational campains [10]. as to 2011 over 86.200 people live and work in suceava county. water is one of the main sources of human exposure to microbiological hazards. pathogens are introduced into surface waters through different pathways such as industrial effluent, raw and treated sewage, storm water and animal manure runoff, in addition to industrial and household solid waste disposal [11]. although legislation establishes regulatory standards in terms of fecal indicator bacteria to assess the microbiological quality of water, these do not necessarily predict the presence of pathogens such as parasites and viruses. better surveillance and management strategies are needed to assess the risk of pathogens' waterborne transmission. underground waters usually have a high microbiological quality and can be used directly without any pretreatment prior to human consumption. on the other hand, the surface waters needs to be processed to become potable. this is a complex process and should be carefully monitored to avoid any undesirable incident. since 2008 suceava city is supplied with dinking water from two distinct sources mihoveni station based on surface water and berchisesti based on underground water. berchisesti station is situated 26 km away from city and has a capacity of 785 l/s and provides 60% of necessary consuming water of suceava city. mihoveni station is situated only 5 km away from the city and has a capacity of 320 l/s. the difference between these two water stations consists in the fact that technological fluxes are different because of the water source is different. at berchisesti where the water is from underground, there is necessary only a disinfection in a preventive way while at mihoveni station the surface water need to be well processed including sedimentationfiltration-disinfection the process being much more complex. environmental health protection requires safe dinking water means free of pathogenic bacteria, and among the pathogens disseminated in water sources, enteric pathogens are the ones most frequently mentioned. therefore, sources of fecal pollution in waters devoted to human activity must be severely controlled. e. coli o157:h7 is one of entero-pathogens commonly present at very low concentrations in environmental food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan, microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012), volume xii, issue 4 – 2013, pag. 316 321 318 waters within a diversified microflora. however, drinking water is not sterile and bacteria can be found in the distribution system and at the tap. most of these organisms are harmless, but some opportunist pathogens such as pseudomonas aeruginosa and aeromonas spp. may multiply during distribution given suitable conditions [12]. 2. experimental 2.1. detection and enumeration of intestinal enterococci we use membrane filtration method as described on iso 7899:2000. intestinal enterococci bacteria are able to reduce 2,3,5-triphenyltetrazolium chloride to formazan and hydrolyze aesculin at 44c ± 0.5c for 2h, on media slanetz and bartley medium and bile-aesculin-azide agar. the enumeration of intestinal enterococci is based on filtration of a specified volume of water sample through a membrane filter with a pore size (0.45 um) sufficient to retain bacteria. the filter is placed on a solid selective medium containing sodium azide (to suppress the growth of gramnegative bacteria) and 2,3,5triphenyltetrazolium chloride, a chlourless dye that is reduced to red formazan by intestinal enterococci. typical colonies are raised, with a red, maroon or pink color, either in the center of the colony or throughout. 2.2. detection and enumeration of escherichia coli and the coliform bacteria e. coli has been the foremost indicator of faecal contamination in water quality monitoring for many decades. e. coli has also been shown to be a significant reservoir of genes coding for antimicrobial drug resistance and therefore is a useful indicator for resistance in bacterial communities [13]. a membrane filter procedure for enumerating e. coli was developed and evaluated. the method quantifies e. coli within 24h without requiring subculture and identification of isolates. it incorporates a primary selective-differential medium for gramnegative, lactose-fermenting bacteria; resuscitation of weakened organisms by incubation for 2h at 35ºc before incubation at 44.5ºc for 18 to 22h; and an in situ urease test to differentiate e. coli from other thermotolerant, lactose-positive organisms. coliforms bacteria are lactasepositive and can produce indole from tryptophan at 44 ± 0.5c for 21-24h incubation, on medium containing caseine and agar. indole in the presence of kovac's reagent leads to the formation of red nitrozoindol. red ring appearance certifies that the presence of e. coli in the tube. 2.3. determination of chlorine and total chlorine iso 7393-2 determination with n,ndiethylphenylene-1,4-phenylenediamine, for rutine control purposes where as a reaction product a red compound result at a ph 6.2-6.5 measured at 510 nm. 3. results and discussions the results observed during three years of monitoring are shown in the figures 1, 2 and 3, as it is shown the contamination incidence was below 3%, and the total number of the investigated samples was over 300/quarter (data not shown). a slightly decrease of contamination incidence with e. coli, enterococcus and both can be observed between 2010 and 2012. however, there could not be established a correlation between the number of samples founded out of line for chloride and a the incidence of microbial food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan, microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012), volume xii, issue 4 – 2013, pag. 316 321 319 contamination, despite the fact that chlorine treatment especially for the surface water sources is essential. we also must mention that suceava area recorded large amounts of rainfall during summer figure. 1. the chlorine samples out of line and the contamination rate with e. coli, enterococcus and both, on 2010 by quarters figure. 2. the chlorine samples out of line and the contamination rate with e. coli, enterococcus and both, on 2011 by quarters food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan, microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012), volume xii, issue 4 – 2013, pag. 316 321 320 figure. 3. the chlorine samples out of line and the contamination rate with e. coli, enterococcus and both, on 2012 by quarters on 2010 and spring of 2011 that might have a significant influence on the contamination rate observed in the analyzed samples. as it is shown in the figures a relatively low rate of contamination incidence has been recorded during this time interval. on the 2011 (first, second and third quarter) the contamination incidence with enterococcus was almost none, among the analyzed samples. on the other hand on 2012 (second, third and fourth quarter) the contamination incidence with e. coli was very low. these results suggests that the modern technology and an accurate monitoring at the main water stations berchisesti and mihoveni from suceava county, were essentially to assure the high water quality for the consumers. 4. conclusion the quality of drinking water and possible associated health risks vary throughout the world. microbial contamination of drinking water remains a significant threat and constant vigilance is essential, even in the most developed countries. delivering safe and acceptable water, therefore, is a key target in improving public health in many developing countries. overall, however, it is obvious that the supply and maintenance of safe drinking water remain key requirements for public health. continuous and rigorous monitoring is required to avoid any large-scale contamination incidence. 5. acknowledgments we would like to address our thanks to the suceava county department of public health, for their help in all aspects of this investigation. 6. references: [1]. suthar s, chhimpa v, singh s., bacterial contamination in drinking water: a case study in rural areas of northern rajasthan, india, environ monit assess., 159, 43-50, (2008) [2]. yassin m.m., abu-amr s.s., al-najar h.m., assessment of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan, microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012), volume xii, issue 4 – 2013, pag. 316 321 321 microbiological water quality and its relation to human health in gaza governorate, gaza strip. public health, 120, 1177–87, (2006) [3]. ashbolt nj., microbial contamination of drinking water and disease outcome in developing regions, toxicology 198, 229–38, (2004) [4]. crichton pb., enterobacteriaceae: escherichia, klensiella, proteus and other genera. in: colle jg, fraser ag, marmion bp, simmons a, editors. mackie & mccartney practical medical microbiology. 14th ed. new york: churchill livingstone, (1996) [5]. enriquez c, nwachuku n, gerba cp., direct exposure to animal enteric pathogens, rev. environ. health, 16, 117–31, (2001) [6]. payment p., richardson l., siemiatycki j., dewar r., edwardes m., franco e., a randomized trial to evaluate the risk of gastrointestinal disease due to consumption of drinking water meeting current microbiological standards, am. j. public health, 81, 703–708, (1991) [7]. moore ac, herwaldt bl, craun gf, calderon rl, highsmith ak, juranek dd, surveillance for waterborne disease outbreaks--united states, 1991-1992, mmwr cdc surveill summ., 19, 42(5):122, (1993) [8]. reynolds k.a., mena k.d., gerba c.p., risk of waterborne illness via drinking water in the united states, rev. environ. contam. toxicol. 192,11758, (2008) [9]. craun g.f., brunkard j.m., yoder j.s., roberts v.a., carpenter j., wade t., calderon r.l., roberts j.m., beach m.j., roy s.l., causes of outbreaks associated with drinking water in the united states from 1971 to 2006, clin. microbiol. rev. 23, 507-28, (2010) [10]. arnold b., colford j., treating water with chlorine at point-of – use to improve water quality and reduce child diarrhea in developing countries: a systematic review and meta-analysis, am. j. trop. med. hyg., 76, 354-364, (2007) [11]. van den berg h., lodder w., van der poel w., vennema h., de roda husman a.m., genetic diversity of noroviruses in raw and treated sewage water, res. microbiol., 156, 532– 40, (2005) [12]. hunter p., waterborne disease. epidemiology and ecology, chichester: wiley, (1997) [13]. bucknell, d.g., gasser, r.b., irving, a., whithear, k., antimicrobial resistance in salmonella and escherichia coli isolated from horses, austrian veterinary journal, 75, 355-356, (1997) 323 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 42016, pag. 323 328 3-d modeling of water flow and cooling down within the temperature range close to inversion point roman gryshchenko1 , *yaroslav zasyadko1, andriy forsiuk1, oleksiy pylypenko1 1faculty of power engineering and energy management, national university of food technologies, 68, volodymyrska str., 01601, kyiv, ukraine, rgryshchenko@gmail.com, iaroslav@nuft.edu.ua, alex.1@i.ua , forsyuk-andrii@ukr.net *corresponding author received 9th november 2016, accepted 27th december 2016 abstract: the research and simulation of heat transfer during water refrigeration in the experimental section close to the vertical pipe which is cooled down considering abnormal character of water density change from the temperature in close to inversion point (+4°c) section were investigated. the graphs of water temperature velocity distribution throughout the height of experimental section were constructed and analyzed. the results obtained allow estimating the impact of water temperature that is closely situated to inversion point on the dynamics of water ice melting and generation as well as on the form factors of cold accumulators. such software and analytical research will allow increasing effectiveness and efficiency at the heat and bulk transfer equipment engineering. keywords: computer modeling, boundary conditions, heat transfer, cold accumulators, water ice, ice melting, ansys. 1. introduction the modern computational experiment is important at the stage of scientific research for solving various linear and nonlinear, stationary and non-stationary treedimensional problems. information, received by numerical calculations, allows understanding the observed physical effects but, in certain circumstances the only possible solution is to replace the real experiment by the computerized one. due to the further progress in computing technique development, it is expectative that in the nearest future the role of the computer modeling will increase especially at the stage of new industrial samples creation as well as in the research of complex effects and processes. currently, the computational fluid dynamics packages, heat transfer, durability and electrodynamics for engineering calculations are widespread. the problem that is being dealt with belongs to the extremely complex problems of mutual interconnected and mutually influencing problems of heat transfer and hydrodynamics. when dealing with the water moving slowly near a cooled down surface one should expect that significantly changing density of water will strongly affect the velocity distribution in the layers adjacent to the surface. the problem becomes more aggravated in case of water, since the density of water changes abnormally within the temperature range 0...+10°c, having its extremum value at the temperature of 4°c. therefore there will be reverse buoyancy in respect of layers. it is obvious that an analytical calculation of hydrodynamics of such flows is impossible http://www.fia.usv.ro/fiajournal mailto:rgryshchenko@gmail.com mailto:iaroslav@nuft.edu.ua mailto:alex.1@i.ua mailto:forsyuk-andrii@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko, 3-d modeling of water flow and cooling down within the temperature range close to inversion point , food and environment safety, volume xv, issue 4 – 2016, pag. 323 – 329 324 and yet the prognostic calculations of velocity distribution in the said region are extremely helpful, since the local velocity of water determines the onset of water crystallization (ice generation). the last is extremely important at a stage of ice accumulators designing. application of 3d computerized simulation of the process mentioned above look extremely promising on the problem on hand. 2. formulation of the problem the complex and internally interrelated heat and mass transfer problems that arise at the study of ice generation on the vertical cooled surface that is streamlined by water are significantly nonlinear physical problems. in this case, the liquid has a noticeable extremum of density at 4°c, fig. 1. it significantly affects the water flow, close to the surface with the temperature of 0°c or lower, since the adjacent layers have reverse buoyancy. it is obvious, that such difficult phenomenon is a problem for direct experiment or computer 3-d simulation. fig. 1. correlation of water density from the temperature the initial stage of studying into the processes of ice generation and melting at the vertical cylindrical surface, the modeling of water cooling down on the 3d model in the research section was carried out. it is clear that within the temperature range +10…0°c a crucial density change of water takes place. 3. experimental rig the ice melting and generation research was held at the experimental stand, which is shown in the fig. 2. ref electric heater water circ. pump water tank water to/from sect 2-3. refrig, to/ from sect 2-3 test sections 1-3 manom. condenser compr. exp. valve glass exp, valve exp. valveexp. valve te st t u be fig. 2. experimental rig the rig consists of three similar blocks (test sections 1-3). the main part of a section is a test copper tube 290 mm height, 10 mm outer diameter, 1 mm wall thickness. each tube is mounted inside of water jacket of 180 mm diameter. a water circulation contour consists of a pump, water piping, water tank, measurement and control systems including regulation and stop valves. circulating water was fed in parallel and supplied from the bottom of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko, 3-d modeling of water flow and cooling down within the temperature range close to inversion point , food and environment safety, volume xv, issue 4 – 2016, pag. 323 – 329 325 the jackets and removed from the upper part, so that an upward flow of water inside all jackets took place. the flow rate of water was controlled by a rotameter, adjusted and kept constant during every experimental run by precision needle valves, and precisely measured by the volumetric method. the water flow rate could be maintained individually for each test unit. a given temperature at the entry to the jackets has been maintained by switching on/off of a cooling coil or electric heater in the water tank. the test tubes were hooked up to the refrigeration contour in parallel on the refrigerant. the refrigeration unit has been equipped with all necessary systems allowing control, measurements and regulation of the evaporation pressure (evaporation temperature) inside each test tube and condensation pressure in the condenser. time rate of ice layer thickness formed during the experiments was measured by means visual technique. photographs of the test sections of pipes covered with ice were taken from the front of the transparent water jackets. immediately before the experiments, an adjustment session had been carried out including testing of different lighting techniques and light sources, and calibration procedures which aimed at the determination of the best measurement arrangements [1-3]. for detailed study of the processes that take place close to the cooling surface, 3-d model of the research section was built. 3. 3-d model creation geometrical model of the research area is built in the ansys software package. the core of the geometrical model is the area of coaxial cylinder, within which the water flows. four inlet and outlet pips for water are located at the bottom and in the top of cylinder respectively. cooled cylindrical surface is modeled along the central axis, fig. 3. fig. 3. geometrical model of experimental (research section) to provide the most accurate calculation at a reasonable number of integrations, the research section was generated as a 90° sector, since the whole section is a symmetrical one. a special attention was paid to the accuracy mesh creation. this enabled to generate 500000 nodes mesh in the sector which is equal to 2 million nodes for whole cylinder. for forming the boundary layer in the mesh and more accurate calculations, a special inflation limit was used, fig. 4. [4-7] to avoid possible ice-coating at the wall surface which is cooling, the temperature of inner cylinder has been programmed as a surface with 0°c. the water temperature varied within +10 to +40°c. the sst (shearstress-transport) turbulence model was chosen, as it effectively combines the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko, 3-d modeling of water flow and cooling down within the temperature range close to inversion point , food and environment safety, volume xv, issue 4 – 2016, pag. 323 – 329 326 stability and accuracy of the standard k-w model in the boundary regions and k-e model in the flow core [8-10]. fig. 4. the inflation limit in the boundary layer (cooling wall is a coolant) in order to reach the convergence of calculation results, the following physical properties of cooled water were introduced into the program model: the water density and heat capacity variation in the vicinity of the inversion point with the temperature increment of 1°c within a whole interval of experimental temperature change. the results of such program are given in fig. 5. calculations were carried out in the cfx-solver manager. the accuracy of obtained results is demonstrated by the convergence calculated equations and systems and exceeds 1520 iterations. obtained results have been imported to the cfd-post. vectorial images of liquid flow inside the research section, temperature and velocity profiles are shown in the fig. 6-7. this information allows estimating the influence of regime parameters on the process of water cooling. fig. 5. water density and heat capacity in the vicinity of inversion point fig. 6. the profile of temperature distribution in research section thus, from the data given in fig. 7, show that the intense cooling of water takes place at the bottom point of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko, 3-d modeling of water flow and cooling down within the temperature range close to inversion point , food and environment safety, volume xv, issue 4 – 2016, pag. 323 – 329 327 experimental section, fig. 6. this can be explained by the existence of vortex in the adjacent to the wall layers which is cooled and by the effect of water density abnormality, which causes the downflow of layers with the temperature of 4°c. the noticeable impact of vortex tributaries on the cooling down cooper surface is presented in the fig. 7. fig. 7. the vector of velocities in the research section volume for the more precise analyses of obtained results, the several lines in the middle of research section were positioned. the given approach allowed to built a graph of velocity distribution in the layer of 1...2 mm close to the cooled wall. the data are given in the fig. 8-9. water velocity distribution on the ascending and descending flows, i.e. a swirls formation is present. fig. 8. water velocity distribution throughout the high of experimental section in the boundary layer: 1, 2, 3, 4, 5 – velocity throughout the height of experimental section 0.1 m; 0.15 m; 0.06 m; 0.058 m; 0.059 m consequently fig. 9. velocity vector in high 0.06 m of research area it can be explained by abnormal water density redistribution. the layers of liquid that are closely situated to the cuprum surface are cooling lower than 4 °c. in this conditions water density decreases. as the result of these layers mixing with the layers whose temperature is 4°c, the local food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko, 3-d modeling of water flow and cooling down within the temperature range close to inversion point , food and environment safety, volume xv, issue 4 – 2016, pag. 323 – 329 328 water layers mixing exactly in the boundary layer is happening ( cuprum wall is a coolant). the fall of water velocity to its minimal value in the research section on the high of 6 mm is also shown in the graph. exactly at this high the velocity of the water layer approaches 0°c, so the conditions of ice generation is this very place looks more favorable, since the local shear stress on the wall is 0°c. the obtained results are proven by full-scale experiment. experimental photographs show clearly the conical shape of the ice, fig. 10. fig.10. ice generation with clear conical shape the forming of obliquity at the intensity subcooling of boundary layer which is close to cuprum surface, on the 6 mm high occurs. 4. conclusions 1.the results obtained of 3-d modeling of water flowing over and cooling down in the vicinity of cooled pipe show that the extremal character of water density change close to the inversion point, significantly affects the velocity distribution in the boundary layer. 2. the effect of reverse buoyancy causes appearing of zero velocity region on the cooled down surface which corresponds to the flow cross section at which the inverse water buoyancy compensates the momentum. 3. the inception of crystallization starts at the zero velocity cross section which is proven by the results of photo fixing of ice generation of the cooled. 5. references [1]. zasiadko y., pylypenko o., gryshchenko r., forsiuk a. experimental and theoretical study of ice formation on vertical cooled pipes, ukranian food journal, 3: 499-507, (2015). [2]. gryshchenko r., forsiuk a., zasiadko y., pylypenko o. the advisability of cold accumulators utilization in industry, refrigeration engineering and technology, 51: 12 16, (2015). [3]. zasiadko y., pylypenko o., forsiuk a., gryshchenko r. a study into ice build-up and melting on vertical cooled pipes, refrigeration engineering and technology, 52: 914, (2016). [4]. alawadhi m. finite element simulations using ansys, second edition, crc press: 426, (2015). [5]. stolarski t. nakasone y, yoshimoto s. engineering analysis with ansys software, butterworth-heinemann: 480, (2011). [6]. kent l. ansys tutorial: release 14, sdc publications: 178, (2012). [7]. kent l. ansys workbench tutorial release 14, sdc publications: 291, (2012). [8]. kleinstreuer c. modern fluid dynamics: basic theory and selected applications in macro and micro-fluidics, springer science & business media, 620, (2010) [9]. cfd-online/high-quality web services to the cfd community.31.06.16 access mode: http://www.cfd-online.com/ [10]. cadfem/ computer aided engineering. 31.06.16. access mode: http://www.cadfemus.com/ http://www.cfd-online.com/ http://www.cadfem-us.com/ http://www.cadfem-us.com/ 1. introduction 89 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 89 – 92 nanodispersed composite antimicrobial materials based on calcium hydroxylapatite *igor kobasa1, galyna bilokopyta2, larysa arsenieva3 1yu. fedkovych national university of chernivtsy, ukraine, 2bukovynian state medical university, chernivtsi, ukraine 3national university of food technologies, kyiv, ukraine i.kobasa@chnu.edu.ua * corresponding author received 5th march 2015, accepted 28th march 2015 abstract: some composite materials based on calcium hydroxylapatite and exhibiting antimicrobial activity have been investigated in the context of influence of the synthesis conditions on the phase composition of the materials and their antimicrobial activity against staphylococcus aureus and escherichia coli. it has been found that the method of synthesis involving sedimentation from the solutions of ca(no3)3 and (nh4)2hpo4 by ammonium hydroxide results in formation of the hydroxylapatite structures (са10(рo4)6(он)2) and ca2,993н0,014(po4)2 exhibiting high antimicrobial activity against the above mentioned pathogenic microbes. keywords: calcium hydroxylapatite, antimicrobial activity, staphylococcus aureus, escherichia coli 1. introduction antimicrobial protection is very topical issue related to many branches of today’s life, trade, production and other activities. even though dozens of new antimicrobial agents are being constantly developed and introduced, contagious diseases remain very serious problem affecting millions of people each year [1]. however, many of such diseases can be prevented by using various solutions that depress reproduction of pathogenic microbes. therefore, any technologies involved in such solutions seem very topical. there are numerous chemical compounds exhibiting bactericide properties: ozone, permanganates, hydrogen peroxide (all these compounds exhibit so-called oxidative bactericide mechanism involving oxidative destruction and/or deactivation of microbial proteins and ferments) and some metal ions with ‘oligodynamic’ bactericide activity. these ions can be ranged in the following row according to decrease in their activity: ag+ > hg2+ > cd2+ > cu2+ > au3+ > ni2+ > zn2+ [2]. as seen from the recent publications, the nanotechnological solutions are quite attractive and popular because physicochemical and biological properties of the nanosized materials are quite different from their macrosized analogs [3-5]. nanoparticles of calcium hydroxylapatites have been recently proposed for development of new composite antimicrobial materials. such materials can be applied to development and production of antibacterial ceramics, pigments for the dye coverings, packaging materials and so http://www.fia.usv.ro/fiajournal mailto:i.kobasa@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, g a ly na b il ok opy t a, l ar ys a a r se n i e va, n a nod i s p er s ed c om p o s i t e a n t i m i c r o bi al m at e r i a l s ba s e d o n c a l ci um h yd r o x yl pa t i t e , f oo d a nd e nvi ro n me nt sa f e t y, v olu me x i v , is s ue 1 – 2 01 5 , p ag . 89 92 90 on [6-8]. it is well known that many properties of calcium hydroxylapatite including its antibacterial activity can vary within quite a wide range depending on the synthesis conditions, presence and concentration of some arbitrary admixtures, specific surface area value, lattice structure and defectiveness class and concentration [4, 9, 10]. this paper deals with investigation of antimicrobial activity of some nanosized examples of calcium hydroxylapatite synthesized by sedimentation of calcium nitrate and ammonium hydrophosphate by ammonium hydrate (“wet” method) and by calcination of the dry mixture of calcium carbonate and ammonium hydrophosphate (“dry” method). 2. experimental the “wet” method of synthesis assumes obtaining of the calcium hydroxylapatite by its precipitation from the source solution and formation of the amorphous product according to the reaction: 10ca(no3)2 + 6(nh4)2hpo4 + 8nh4oh = ca10(po4)6(oh)2↓ + 20nh4no3+ 6h2o it should run under the temperature 45 ± 5 ºс and рн=11. the “dry” method of synthesis assumes mixing and grinding of the dry cаco3 and (nh4)2hpo4 followed by calcination at 1000 ºс during 2 hr. the following reaction occurs during the thermoprocessing: 10caco3 + 6(nh4)2hpo4 = ca10(po4)6(oh)2 + co2↑ + nh3↑ + 6h2o the final product of calcination should be ground to the uniformal state. antimicrobial activity has been determined by the diffusion (“holes”) method according to the standard iso 27447:2009(e) as described in details below. the bacterial cultures were grown using the stable nutritional medium and standard bacterial strains of staphylococcus aureus and escherichia coli from american type culture collection. the inoculation material was prepared as follows: fresh bacterial colonies were taken after the 18 hours of growing in the sterile physiological medium (0,85 % nacl) in such a way to ensure formation of the suspension with 0.5 mcfarland’s density degrees, which is approximately equal to 1-2·108 cfu/ml. then the material was ten times diluted forming the 0.05 degrees system and then ten times again to form the 0.005 degrees system. density of the systems has been determined nephelometrically. the latter (0.005 degrees) system was used for the next investigations. a cotton tampon with the inoculation material was immersed in the sterile solution, extracted and squeezed to remove the excessive solution. then the inoculation material was seeded on the substrate by triple application of the tampon with rotation 600 after each application. the substrate plates were dried and then 50 μl of various hydroxylapatite suspensions were added to investigate their antimicrobial activity. each probe contained of the 0.1; 1.0; 5.0 and 10.0 % suspensions of two hydroxylapatites: (1) – the “wet” calcium hydroxylapatite; (2) – the “dry” calcium hydroxylapatite. all the suspensions were prepared on the basis of physiological solution. the probes were placed into the incubator and stayed in the oxygen containing medium under 370 for 16-18 hours. then the diameter of the microbes decelerated growth area was measured as the source parameter to evaluate the antimicrobial activity. the standard disk impregnated by novobiocin was used as the reference sample of the antimicrobial activity. 3. results and discusion it was found that the phase composition of calcium hydroxylapatites depends on the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, g a ly na b il ok opy t a, l ar ys a ar se n i e va , n a n od i s p e rs ed c om po s i t e a n t i m i c r o bi al m at e r i a l s ba s e d o n c a l ci um h yd r o x yl p at i t e , f o od and e nv ir o n me nt sa f e t y, v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 89 92 91 synthesis conditions. the “wet” material has the composition that combines ca10(po4)6(oh)2 and ca2,993н0,014(po4)2 while the “dry” material combines ca10(po4)6(oh)2 and ca3(po4)2. this difference may be caused by different synthesis and next thermal processing conditions. it will be shown below that the structural composition of calcium hydroxylapatite affects antimicrobial activity of the composite materials comprising this compound. analysis of antimicrobial activity of hydroxylapatite samples proves that the diameter of the decelerated growth area of staphylococcus aureus depends on the synthesis method and concentration of calcium hydroxylapatite (see fig. 1). the “wet” hydroxylapatite shows the higher antimicrobial activity than the “dry” compound and activity of all the samples grows with increase in the content of calcium hydroxylapatitein in the suspension. it was found that antimicrobial efficiency of all hydroxylapatites against the agar colonies of escherichia coli prevails that against staphylococcus aureus. the total extinction of the escherichia coli colonies growing on meat infusion agar has been registered (see fig. 2). as seen from fig 1 and fig. 2, the “wet” hydroxylapatite with concentrations 0.1, 1.0, 5.0 and 10.0 % is active against the standard bacterial colonies of staphylococcus aureus and escherichia coli (bacterial suspensions with density 0,005 mcfarland degrees). antibacterial efficiency is growing with increase in the suspensions concentration. the “dry” suspensions also exhibit some antibacterial activity with concentrations 0.1, 1.0, 5.0 and 10.0 % but its level is lower than that of the corresponding “wet” suspensions. fig. 1. sensitivity of staphylococcus aureus to antimicrobial action of the “wet” (1) and “dry” (2) calcium hydroxylapatite with the following concentrations: а – 0.1, в – 1.0, с – 5.0 and d – 10 %. for instance, the 0,1 % “dry” suspension does not exhibit any antibacterial activity against staphylococcus aureus while the effect of similar “wet” suspension is quite tangible (see fig. 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, g a ly na b il ok opy t a, l ar ys a ar se n i e va , n a n od i s p e rs ed c om po s i t e a n t i m i c r o bi al m at e r i a l s ba s e d o n c a l ci um h yd r o x yl p at i t e , f o od and e nv ir o n me nt sa f e t y, v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 89 92 92 fig. 2. sensitivity of escherichia coli to the “wet” (1) and “dry” calcium hydroxylapatite with concentrations: а – 0.1, в – 1.0, с – 5.0 and d – 10 % 4. conclusion calcium hydroxylapatite materials synthesized through sedimentation from aqueous solution of calcium nitrate and ammonium hydrophosphate by ammonium hydroxide or through calcination of the dry mixtures of calcium carbonate and ammonium hydrophosphate exhibit the antibacterial activity against strains of staphylococcus aureus and escherichia coli. the activity of the “wet” material is higher than that of the “dry” samples, which can be used for development of the bactericide composite materials. 5. references [1] k.e. jones, n.g. patel, m.a. levy [et. al.] global trendsin emerging infectious diseases. nature. 451, 990-993 (2008). [2] e. vob, c. störch. evaluation of bacterial growth on various materials. the 20th international enameller congress, istanbul-turkey, 15-19 may. 194–210 (2005). [3] r.y. pelgrift, a.j. fridman. nanotechnology as a therapeutic tod to combat microbial resistance. adv. drug deliv. rev. 65, 1803-1815 (2013). [4] i.m. kobasa, w. strus, m.a. kovbasa. highly photosensitive titanium dioxide and process for forming the same. us 2008/0146441. [5] i.m. kobasa, w. strus, m.a. kovbasa. method of surface modifying titania using metal and compositions therefrom. wo 2009/052510. [6] o.v. savvova, l.l. bragina, e.v. babich. nanostructural calciumsiliconphosphate glasscrystalline materials of medical purpose. nanosystems, nanomaterials, nanotechnologies. 8, 903-910 (2010) [7] o.v. savvova, l.l. bragina, n.p. sobol. inorganic powder based on calcium phosphate for obtaining of antibacterial glassenamel covering. patents and inventions, №91878, 2010. [8] m. a. rauschmann, t.a. wichelhaus, v. stinal [et. al.]. nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. biomaterials. 26, 2677-2684 (2005). [9] v. stanic, s. dimitrijevic, j.anticstankovic [et. al.]. synthesis, characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders. applied surface science. 256, 6083-6089 (2010). [10] m.p. ferraz, a.y. mateus, j.c. sousa, f.j. monteiro. nanohydroxyapatite microspheres as delivery system for antibiotics: release kinetics, antimicrobial activity, and interaction with osteoblasts. biomedical materials research part a. 81a, 994-1004 (2007). 247 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 247 – 252 physical properties of mustard seeds and how they are influenced by packaging mihaela jarcău1 1faculty of food engineering, stefan cel mare university of suceava, suceava, românia, mjarcau @ yahoo.com * corresponding author received august 1st 2013, accepted september 5th 2013 abstract: i paid attention to how mustard seeds interact with storage container for a period of 6 months. i examined some of the physical properties of these seeds such as: length l (mm), width w (mm), thickness t (mm), geometric mean diameter dg (mm), sphericity φ (%), porosity ε (%), volume v (mm3), mass m (g), bulk density ρb (g/cm3), true density ρtr (g/cm3) before and after storage for six months at normal temperature and pressure. the samples were: sample 1 mustard seeds in a plastic bag and sample 2 mustard seeds in a plastic box with a top. the physical dimensions of the samples measured in three mutually perpendicular directions before storage had values between: for t minimum – 1.90 mm, maximum 2.61 mm, for w – minimum 1.87 mm, maximum 2.44 mm, for l – minimum 1.82 mm, maximum 2.58 mm. after the six months of storage at normal temperature and pressure in various packages, the physical dimensions of the mustard seeds varied the least for the seeds in sample 1. keywords: mustard, seeds, physical properties. 1. introduction plants, including herbs and spices, have many phytochemicals which are a potential source of natural antioxidant, e.g., phenolic diterpenes, flavonoids, alkaloids, tannins and phenolic acids [1]-[4]. many culinary herbs and spices were included in the category of medicinal plants such as cumin oil and seeds, cardamom seeds, cinnamon, cloves, dill seeds, fennel seeds and oil, garlic, ginger root, liquorice root, peppermint oil, onion, paprika, parsley root, oil and mint leaves, rosemary, sage, thyme, turmeric root, coriander seeds, mustard seeds [5]. recently, the natural spices and herbs such as rosemary, oregano, and caraway have been used for the processing of meat products [1]. most spices have been used for therapy of gastrointestinal, aphrodisiacs tonic and non-specific, some of them have antibacterial or antifungal effect, and some irritants were used for anti-inflammatory effect. because they seem to have strong antioxidant effects, some claim to be able to prevent cancer. most of the health benefits of more potent have not proven yet [6]. mustard seeds are the small round seeds of various mustard plants. mustard seeds may be colored from yellowish white to black. the seeds may come from three different plants: black mustard (brassica nigra), brown indian mustard (b. juncea), and white mustard (b. hirta/sinapis alba) [6]. mustard seeds have been used since 4000 years ago both in the kitchen for flavor and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 mihaela jarcău, physical properties of mustard seeds and how they are influenced by packaging, food and environment safety, volume xii, issue 3 – 2013, pag. 247 – 252 248 also in medicine for its beneficial effects [6]. i have accorded the same attention to the bilberry and seabucktorn fruits, analyzing the behavior during the storage period, and in different packaging (plastic box with lid, jar package, double paper wrapper, plastic bag) [7]-[8]. the objective of this study was to investigate in which way the package influences the physical proprieties of mustard seeds l (mm), w (mm), t (mm), dg(mm), φ( %), ε,(%), v(mm3), m(g), ρb (kg/m3), ρtr (kg/m3). all the information is important for designing the equipment, storage and packing to increase the work efficiency and decrease product loss. 2. materials and methods 2.1 sample preparation the analyzed seeds have been bought from suceava area, românia. i have randomly selected 10 seeds, using an electronic caliper with a precision of 0.01mm, i have measured the three major perpendicular dimensions of the fruits namely length l, width w and thickness t, after which i have packed them in: samples: 1. mustard seeds in plastic bag; 2. mustard seeds in plastic box with a top. the samples were stored for 6 months under normal conditions of pressure and temperature and then remeasured. 2.2 geometric mean diameter, sphericity, volume and surface area the geometric mean diameter dg and sphericity of seeds was calculated using the following relationship 9: dg = (lwt)1/3 (1); φ = [(lwt)1/3/l] x 100 (2); for volume and seed surface was calculated by using the following relationship [10]: = ( ) (3) = (4) where b is: = ( ) . (5) 2.3. one hundred fruits weight and the unit mass nomenclature dg-geometric mean diameter (mm) l-length (mm) m-unit mass of the seed (g) m100-100 seed mass (g) sseed surface (mm2 ) t-thickness (mm) v-single seed volume (mm3) w-width (mm) ρb-bulk density, (g/cm3) ρtr –true density, (g/cm3) ε-porosity (%) φ-sfericity, (%) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 mihaela jarcău, physical properties of mustard seeds and how they are influenced by packaging, food and environment safety, volume xii, issue 3 – 2013, pag. 247 – 252 249 to obtain the unit mass of the seeds, the mass of 100 seeds were measured with an electronic balance with an accuracy of 0.0001g. 2.4. bulk and true density the bulk density is the ratio of mass sample of the seed to its total volume. it was determined by filling a 1000 ml container with seeds from a height of about 15 cm, the excess seeds were removed using a stick and then weighing the contents [11]. the true density was determined using liquid displacement method. the seeds were used to displace toluene (c7h8) in a measuring cylinder after their masses had been measured. toluene was used instead of water because it is absorbed by seeds to a lesser extent. the true density was found as an average of the ratio of their masses to the volume of toluene displaced by seeds [11]. 2.5. porosity the porosity is the fraction of space in the bulk seeds that is not occupied by the seeds [12]. the porosity ε of bulk mustard seed was calculated using the following relationship 9: = 100 (6) where ρtr is true density in g/cm 3 and ρb is bulk density in g/cm3. 3. results and discussion 3.1. seed size the dimensions of the mustard seeds measured in three perpendicular directions, before storage had the values between two intervals: l – minimum 1.82 mm, maximum 2.58 mm, w minimum 1.87, maximum 2.44 mm, t – minimum 1.90 mm, maximum 2.61 mm. for the data analysis i have used anova single factor. in table 1 are the medium values and the dimension variance of the seeds l(mm), w(mm), t(mm) before storage, for every sample, in table 2 are the medium values and dimension variance of the same dimensions after 6 months of storage under normal conditions of pressure and temperature for both sample. table 1 the medium values and the dimension variance of the seeds l(mm), w(mm), t(mm) before storage. sa m pl e l (mm) w (mm) t (mm) a v e r a g e v a r ia n c e a v er a g e v a r ia n c e a v er a g e v a r ia n c e 1 2.31 0.046 2.19 0.04 2.23 0.06 2 2.30 0.046 2.19 0.04 2.23 0.06 tabel 2 the medium values and dimension variance of the same dimensions after 6 months of storage sa m pl e l (mm) w (mm) t (mm) a v er a g e v a r ia n c e a v e r a g e v a r ia n c e a v e r a g e v a r ia n c e 1 2.27 0.015 2.04 0.02 2.15 0.03 2 2.28 0.002 2.16 0.01 2.19 0.01 because f (observed value) > fcv (critical value) for t and w, we reject the null hypothesis. to highlight more clearly how these dimensions of mustard seeds varies over time for each sample, we have represented the relative variation of l in figure 1, the relative variation of w in figure 2 and the relative variation of t in figure 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 mihaela jarcău, physical properties of mustard seeds and how they are influenced by packaging, food and environment safety, volume xii, issue 3 – 2013, pag. 247 – 252 250 figure 1 the relative variation of l(%) figure 2 the relative variation of w(%) figure 3 the relative variation of t(%) as we can see, the variation of the all dimensions (l, w, or t) is greatest for the sample 1. 3.2 geometric mean diameter, sphericity, volume and surface area. to determinate the geometric diameter of the mustard seeds we have used eq (1). the relative variations for each sample, after 6 month of storage are presented in figure 4. figure 4 the relative variation of dg (%) to calculate sphericity, volume and surface of mustard seeds we used eq (2), (3) and (4). the relative variations of sphericity, volume and surface of seeds, after 6 month storage, we represented in figure 5. figure 5 the relative variation of s, v and φ as we can see, the variation of the sphericity, volume and surface of seeds, after 6 month storage, is greatest for sample 1. 3.3. one hundred mustard seeds weight. the mass of 100 mustard seeds before storage was: , and after storage: for sample 1 was 0.6148g, for sample 2 was 0.6125 g. figure 6 the relative variation of m100 (%) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 mihaela jarcău, physical properties of mustard seeds and how they are influenced by packaging, food and environment safety, volume xii, issue 3 – 2013, pag. 247 – 252 251 as shown in figure 6, no significant differences of the relative variations of mustard seeds weight for each samples. 3.4. bulk and true density the relative variation of bulk density, for each sample, is presented in figure 7 and the relative variation of true density for each sample, is presented in figure 8. figure 7 the relative variation of ρb(%) figure 8 the relative variation of ρtr(%) the relative variation of true density of the seeds was highest for the sample 1 10.1%, and the relative variation for bulk density was highest for sample 2 10.35%. 3.5. porosity the value of porosity were calculated with eq (6) by using the data on bulk and true densities of seeds and the results obtained are presented in figure 9. figure 9 the relative variation of ε (%) no significant variation of the porosity in none of the samples analyzed has been seen. 4. conclusion after the measurements made on physical properties of mustard seeds bought from suceava area, romania, before and after their storage for a period of six months at normal temperature and pressure in various packages, we drew the following conclusions: 1. the dimensions of the seeds measured by three perpendicular directions before storage had values between: for l – minimum 1.82 mm, maximum 2.58 mm, w minimum 1.87, maximum 2.44 mm, t – minimum 1.90 mm, maximum 2.61 mm; 2. after the six months of storage at normal temperature and pressure in various packages, the dimensions of the mustard seeds suffer different modifications that depend on the nature of the package used for each sample. 3. as we can see, the variation of the physical dimensions was greatest for the sample 1, except the relative variation of true density that was higher for the sample 2 and for mass of 100 seeds which ranged roughly the same for both samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 mihaela jarcău, physical properties of mustard seeds and how they are influenced by packaging, food and environment safety, volume xii, issue 3 – 2013, pag. 247 – 252 252 5. references [1] il-suk kim, mi-ra yang, ok-hwan lee, suk-nam kang. antioxidant activities of hot water extracts from various spices. international journal of molecular sciences, 2011; 12(6): 4120–4131. [2] amro b, aburjai t, al-khalil s. antioxidative and radical scavenging effects of olive cake extract. fitoterapia. 2002; 73:456–461. [3] cai yz, luo q, sun m, corke h. antioxidant activity and phenolic compounds of 112 chinese medicinal plants associated with anticancer. life sci. 2004; 74:2157– 2184. [4] moure a, cruz jm, franco d, dominguez jm, sineiro j, dominguez h, nunez mj, parajo jc. natural antioxidants from residual sources. food chem. 2001; 72:145–171. [5] blumenthal m. the complete german commission e monographs: therapeutic guide to herbal medicines. austin, tx: american botanical council; 1998. [6] history & special collections, ucla louise m. darling biomedical library http://unitproj.library.ucla.edu/biomed/spice/in dex.cfm?displayid=18 – 12 february 2013 [7] jarcău, m. some physical proprieties of bilberries and how the packing conditions influence them. food and environment safety, 2012, 11(2), 103-107. [8] jarcău, m. some physical proprieties of seabucktorn and how the packing conditions influence them. food and environment safety, 2012, 11(4), 36 – 42. [9] mohsenin, n. n. physical properties of plant and animal materials. new york: gordon and breach science publishers; 1970. [10] jain, r. k. physical properties of pearl millet. journal of agricultural engineering research, 54, (1997). [11] deshpande, s. b. physical properties of soybean seeds. journal of agricultural engineering research, 1993, 56, 89 – 92. [12] thompson, r. a. porosity determination of grains and seeds with air comparison pycnometer. transaction of the asae, 1967, 10, 693–696. удк 664 299 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv , issue 4 2016, pag. 299 305 ştefan cel mare university of suceava, romania volume , issue 12016, pag. ... ... research on technological properties of potato cellulose for bread production tatjana silchuk1, mariana nazar1, *tatjana golikova1 1 department of technology of nutrition and restaurant business, national university of food technologies, kyiv, ukraine, tatjana.golikova12@gmail.com * corresponding author received 12th august 2016, accepted 19th december 2016 abstract: the problem of deficiency of food fibers in food rations leads to the necessity of searching new sources of ballast compounds. one of the perspective ways of solving this problem is the use of potato cellulose to fortify by food fibers daily used products such as bread and bakery products. potato cellulose is a high dispersive and contains a high amount of food fibers. water absorption of potato cellulose has been investigated as well as its effect on the quality of dough and bread. potato cellulose has water absorption between 1.5 … 2 times higher than in wheat bran thus giving the possibility of making ready products of proper quality. the effect of food cellulose addition to the gluten content in dough has been presented. the expediency of potato cellulose used to enrich ready bread products has been proven. key words: wheat flour, potato cellulose, food fibers, bread. 1. introduction the main achievement and index of level of state development are health, efficiency and longevity of the population. according to the data of world health organization, state of human health is determined by the type of nutrition (on 67 – 74 %), factors of environment and conditions of life and only on 16 … 18 % by the genetics and 10 … 15 % by the health services. so, production and consumption of food products – is most important of acquisitions of mankind that may cause health or diseases. taking into account permanent worst of the ecology, the increasing of people diseases and shortening of human life term the search of natural additives and ingredients based on it that effect to functional properties and creating of technology of its applying in products of mass consumption is urgent task for food technologists [1, 2, 3, 4, 5]. bread is one of the main food products. it supplies the organism with full complex of necessary nutrients: herbal proteins, digestible carbohydrates, food fibers, vitamins and macroand microelements. but it is well-known that bread, especially from wheat flour of higher and first grade, is a non-balanced product by content of amino-acid, food fibers, vitamins and mineral matters. from other side, this product is consumed by all segment of population non depend on age, lifestyle, health and is suitable product for enriching with different components. daily consumption of it helps to enrich food diet, decrease effect of damage ecological factors to human organism [2, 3, 4]. http://www.fia.usv.ro/fiajournal mailto:tatjana.golikova12@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 300 food fibers take important role besides enriching ingredients. useful action of it effects in strengthening of intestine peristalsis and fastening of food moving, preventing of gallstone diseases and cancer diseases, excretion of toxic products, radionuclides, hard metals oxides, optimal functioning of useful microflora of bowels [6]. scientists have installed that using of second products of herbal raw materials and concentrates from it as source of food fibers has most physiologically effect [3, 5, 6]. from this point of view new herbal origin – potato cellulose – that consist of food fibers (70 %), mostly cellulose, hemicellulose and pectin deserve attention. increased content of food fibers in potato cellulose comparing to wheat bran that are traditionally used for enriching of products with food fibers helps to decrease it dosage in recipe of bread with ensuring of daily consumption of ballast matters in healthy/functional food [7, 8, 9, 10, 11]. due to the high dispersity of potato cellulose such products may be recommended for people with acute diseases of gastrointestinal tract because possibility of irritation of mucous tissue is excluded. bread with potato cellulose may be used for preventing of diseases linked with endocrine system, digesting and blood circulation system. important property of potato cellulose is high water retention and fat bonding ability. it allows to add it in flour culinary and bakery products [8]. so, creating of new bakery products of special and functional purpose with using of potato fibers is one of the actual direction of scientific research. 2. materials and methods. the potato cellulose (“paselli-fp”, netherland), has been used. addition of the cellulose was calculated for ensuring 30 – 50 % of daily maintenance in food fibers in case of consumption of accepted daily mass of bread enriched with food fibers. preparation of bread the dough was prepared according to recipe, which was 100 % wheat flour (containing 0.73 % ash in dry matter, 27 – 29 % wet gluten), salt 1,8 %, yeast 3 %, sunflower oil 2,0 %, potato cellulose 3, 5 and 7 % to the weight of wheat flour and water to get moisture of dough 43 %. as a control, no cellulose was added to the recipe. the ingredients were mixed during 6 minutes in mixing bowl. after 180 min fermentation, the dough was divided into 250 g loaves, formed on dough former, proofed 45 min and baked in an electric oven during 30 – 35 oc at 200 220°c with moisturing of baking camera. baking trials were performed in triplicate. content of food fibers general content of food fibers in researched products was determined by enzymatic gravimetric method [12]. this method is based on enzymatic hydrolease of starch and non-starch compounds by means of α-amylase, protease and amylohlucosidase to mono-, diand oligosaccharides and peptides. food fibers are precipitated by ethyl spirit and are dried. general content of food fibers has been calculated in percent by formula: 100 100 1 2 21 1                 m m ww m x food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 301 where x – general content of food fibers, % from low fat sediment weight; m1 – mass of dry low fat fraction of product, g; m2 – mass of sediment, g; w1 – amount of protein in sediment, %, that was determined by formula: 100 2 3 1  m m w w2 – mass of ash in sediment, %, that was determined by formula: ,100 2 4 2  m m w where m3 – mass of protein, g; m4 – mass of ash, g. water absorption water absorption of the potato cellulose was determined by means of centrifugation. the suspense of products with water (hydromodule 1:20) were made in weighted centrifugal testtube during 1 min. then suspense was mixed in laboratory mixer (frequency of rotation 50 c-1) and was leaved in thermostat during 20 min at temperature 30 oc for implementing of colloidal processes. then suspense was centrifuged in 4000 min -1 during 5 min. liquid phase was sedimentated and weighted. content of dry substances was determined by means of refractometer and calculated by formula:     100 100100     wg mfw wa , where w – amount of water in centrifugal test-tube, g; g – mass of product added in centrifugal test-tube, g; m – amount of dry maters in liquid phase, % was determined by formula 100 fdm fm  dmf – amount of dry matters (measured by refractometer), %; w – moisture mass fraction in product, %. titrated acidity, moisture of dough, porosity and volume of bread were determined by standard methods [13]. 3. results and discussions content of food fibers the content of food fibers in potato cellulose has been installed 77,1 % (table 1). it is more than in triply exceeds such index for wheat bran. it let to use less amount of potato cellulose in recipe of bread for ensuring of physiologically justified concentration of food fibers in bread products. recommended physiological daily norm of consumption of ballast substances is 25 30 g. table 1 content of food fibers and its components in raw materials, % dry matters fraction bran potato cellulose general content of food fibers, % dry matters, including 26.9 77.1 cellulose 10.3 18.9 hemicellulose 12.7 3.0 pectin 2.1 54.6 lignin 1.8 0.6 cellulose is main component of herbal cell walls. it is practically not digested in organism. main function of it is to bond significant amount of water. as experimental data of table 1 shows content in cellulose in potato fiber is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 302 almost in 2 times higher than in wheat bran. this fact causes increased water absorption of potato cellulose. hemicellulose is main component of polysaccharide complex of food fibers that has a high hydrophilicity. xylans are belong to hemicellulose that have the same process of water bonding as starch but without retrogradation. in case of increasing of content of hemicellulose in bread the fragility of crumb decreases, process of staling slows down. but hemicellulose does not effect to the volume of bread and output of bread [9]. table 1 indicates content of hemicellulose in potato cellulose in 4 times less than in wheat bran. pectin substances have complex forming properties that effect to metabolism and excretion of hard metals and radionuclides from human organism. potato cellulose contains 54.6 % of pectin substances that exceeds it content in wheat bran in 26 times. it indicates preference of potato cellulose from medical and biological aspects of evaluation of using of different sources of dietary fiber. so, the potato cellulose has high grade of dispersity and high content of food fibers comparing to wheat bran. water absorption figure 1 indicates that water absorption of potato cellulose is higher than in wheat flour in 4.4 – 4.8 times and in wheat bran – in 2.6 – 2.8 times. these meanings are characterized for temperature 30 and 60 oc. during increasing of temperature till 90 oc water absorption of cellulose is higher than in wheat flour and bran in 2.4 times. adding of small quantity of potato cellulose causes significant increasing of water absorption of dough that leads to increasing of bread volume. the water absorption of cellulose in different temperatures has been investigated with the aim of forecast of effect of potato cellulose to the technological parameters and quality of bread. temperature 30 oc refers to starting temperature of dough during kneading, temperature 60 oc refers to the starting of gelatinization of starch and proteins denaturation during baking, temperature 90 oc refers to the temperature in the piece of dough at the end of baking. as water sorption of potato cellulose is significantly higher than it in the flour it is need to made a preliminary hydration of cellulose before dough kneading. figure 1. water absorption of potato cellulose 0 100 200 300 400 500 600 700 800 900 1000 30 60 90 w at er a bs or pt io n, % temperature, ºс wheat flour wheat bran potato cellulose food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 303 studies of gluten quantity and quality of gluten have been determined for investigating of effect of potato cellulose to the physical properties of dough. gluten quality indexes depends not only on technological parameters but also on content and recipe components of dough. necessity of investigating of effect of potato cellulose to the quantity and quality of gluten is leaded to features of its chemical composition and high grade of dispersity. high grinding of product causes changes in quantity and quality of some components, enzymatic activity, digestive degree, mechanism of effect to biopolymers of dough. as results of gluten investigations strengthening of gluten with potato cellulose has been installed (table 2). gluten is more elastic and less extensible as dosage of cellulose increases. strengthening effect of potato cellulose deals with high hydrating ability of components of its polysaccharide complex that has significant de-hydrating effect to biopolymers of dough. so, hydrating ability of gluten of dough with cellulose decreases on 0,6…5,8%abs comparing to gluten of control sample dough. table 2 effect of potato cellulose to the quality indexes of gluten index control sample substitute of flour with potato cellulose, % 3 5 7 content of raw gluten,% to the weight of flour 25.10 25.10 24.14 23.64 content of dry gluten, % 9.29 9.22 9.19 8.99 humidity of gluten, % 63.7 63.4 62.8 61.0 hydrating ability of gluten,% the weight of dry gluten 175 171 163 162 extensibility, cm 13.0 13.0 12.5 11.0 elasticity of gluten, unit of device ddk 69.3 63.4 59.0 55.6 as a result of decreasing of content of raw gluten the gas retention of dough also decreases that effects to lowering of volume and saving of form of bread. research of quality of wheat bread made by direct process with different dosage of potato cellulose shows increasing of acidity of dough on 0.2 – 0.4 grade and, as a result, acidity of bread – increases on 0.3 – 0.6 grade (table 3). increasing of initial acidity of dough encourages intensification of spirit fermentation that is accompanied with increasing of carbon dioxide, exhausting during fermentation. saving of form of bread betters in case of using of potato cellulose «paselli fp» in amount of 3 … 7 %, but volume of bread decreases on 11 … 12 %, porosity of crumb worsts. so, adding of potato cellulose in amount more 7 % is not expediently. results of sensorial investigations shows pleasant taste and flavor of bread with potato cellulose, but crumb gets grey colour. thus, in case of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 304 dosage of potato cellulose 5 % to the weight of flour bread has acceptable quality and daily requirement in food fibers is ensured by 25 %. table 3 the effect of potato cellulose on the technological parameters and quality of wheat bread index control sample dosage of potato cellulose in bread, % to the weight of flour 3.0 5.0 7.0 dough duration of kneading, min 10 10 10 10 duration of fermentation, min 180 180 180 180 duration of proofing, min 40 40 45 45 duration of baking, min 40 40 40 40 titrated acidity, grade initial 1.4 1.5 1.6 1.7 final 2.0 2.2 2.3 2.4 bread titrated acidity, degree 1.4 1.4 1.5 1.6 porosity, % 75 75 74 73 volume, см3/г 3.7 3.1 3.3 3.4 moisture content, % 42 43 44 45 crust colour light brown surface smooth texture middle, uniform compacted elasticity middle, uniform middle, compacted taste and flavor proper to bread 4. conclusions the expedient of using of potato cellulose «paselli fp» (netherland) as new raw materials for baking has been proven. potato cellulose is a perspective source of food fibers in bread production. this additive has advantages in high content of food fibers and high dispersity comparing to food fibers of wheat bran, fruits and vegetables. taking to account of dispersity of potato cellulose bread with it may be recommended for people with diseases of gastrointestinal tract. potato cellulose is advisable to add to the recipe of bread made from wheat flour in amount 5 % to the weight of flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 tatjana silchuk, mariana nazar, tatjana golikova, research of technological properties of potato cellulose for bread production, food and environment safety, volume xv, issue 4 – 2016, pag. 299 – 305 305 5. references [1] shchelkunov l.v., dudkin m.s., korzun v.n. food and ecology. – оdessa: “optimum», 2000. – 517 p. (in ukrainian). [2] lusyik g.m., oliynyk s.g., samohvalova o.v., kucheruk z.i. technological aspects of creating of bread and pastries of special purpose // food science and technology. – 2009 № 1(6) – p. 25-30. (in ukrainian). [3] iliina o. dietary fiber – most important component of bread and pastry products // bread products. № 9. -2002. p. 34-36. (in ukrainian). [4] steigman a. all dietary fiber is fundamentally functional // cereal foods world. – 2003. – vol. 48, № 3. – р. 128 – 132. [5] ignatova l.v., kochetkova a.a., shubina o.g., dukhu t.a. physiological and technological aspects of using of dietary fiber // food ingredients and additives. – 2004. – № 1. – с. 14 – 17. (in russian.) [6] arsenieva l.y., borysenko o.v., dotsenko v.f. theoretical and practical aspects of using of concentrates of dietary fiber of high dispersity in technology of rye-wheat bread // scientific works of nuft. 2008. no 25. p. 115-119. (in ukrainian). [7] drobot v.i., grishchenko a.n. using of potato cellulose in technology of bread products / хранителна наука, техника и технологии 2013: scientific works. – plovdiv, 2013. – т. lx. – p. 105-108 (in ukrainian). [8] drobot v.i., bilyk o.a., savchuk n.i., bakhtiriov s. potato flakes in bread products / baking and confectionery industry of ukraine.-к., 2009. № 7-8. p. 43-44. (in ukrainian). [9] drobot v.i., dotsenko v.f., ustinov y.v., arsenieva l.y., sukhodol v.f., savchuk n.i. using of potato flour in bread production // baking and confectionery industry of ukraine. 1997. № 6. с. 34-38. (in ukrainian). [10] potato cellulose / [electronic resource]. http://www.avefood/pasellifp [11] the new potential of potato fiber, international food ingredients, 2005, vol.6, p.48. [12] state standard р 54014 – 2010 functional food. determination of soluble and unsoluble dietary fiber by enzymatic gravimetric method. [13] drobot v.i., arsenieva l.y., bilyk o.a. laboratory practicum of bread and macaroni products technology. к .: centre of scientific literature, 2006. 341 p. (in ukrainian). 342 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 342 346 variation of nitrite content in meat products during the shelf life *sonia amariei1, sorina ropciuc1, domitian mugurel scripca1 1faculty of food engineering, stefan cel mare university of suceava, romania gutts@usv.ro * corresponding author received november 17th 2013, accepted december 7th 2013 abstract: in the meat industry nitrite is used, together with the salting mixture, to maintain pink red color of meat, for bacteriostatic effects, antioxidant effect and for amplification of products flavor. current standards imposed the use a concentration of nitrite that would be within the limit of 7 mg/100g product without negative effects on the human body. two meat products were analyzed, smoked chest and gypsy steak, to monitor the development of nitrite concentration. during monitoring nitrate concentrations increased with 14% when compared to the first analysis of smoked chest, and with 7.7% in the case of gypsy steak. during the ten days of analysis nitrite concentration did not exceed the maximum allowable dose. key words: nitrite, griess method, porkloin, smoked chest 1. introduction nitrates and nitrites are commonly used in meat products because they have positive effects such as: maintaining of pink-red meat color, antioxidant effects, bacteriostatic and flavor enhancement of products. in order to a better distribution in the products nitrates and nitrites are usually added in solid form or in brine at a ratio of 1 kg nitrate or 0.5 kg nitrite per 100 kg of solid salt. nitrites have an effect on the meat flavor [1] and they inhibit the oxidation of lipids [2]. thus, some substances resulting from the metabolism of lipids, valerian aldehyde and hexanol (fat rancidity specific product) are formed in small quantity and flavor of meat is more enjoyable [3]. this explains the weak antioxidant effect of nitrite, thus resulting in inhibition of formation of substances with taste and unpleasant odor. nitrates and nitrites are used in meat preservation not only as a source of nitric oxide, also for their bacteriostatic properties, increasing the salt preservative action. they are stronger preservatives than salt destroying some groups of microorganisms against which salt has no effect. after highlighting the cumulative effect of nitrites and the possibility of formation of carcinogenic nitrosamines [4] toxicological implications of these chemicals have become more complex [5]. nitrites formed with amines exogenous and endogenous nitrosamines [3] highly carcinogenic compounds [6] that turns in stomach in nitrosoguanidine (this substance ranks second in terms of carcinogenic action). nitrites reduce reserves of vitamin a in the liver and disrupt thyroid function, but they have good effects on blood pressure [7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sonia amariei, sorina ropciuc, domitian mugurel scripca, variation of nitrite content in meat products during the shelf life, volume xii, issue 4 – 2013, pag. 342 – 346 343 deficiencies in vitamin e and b are also due to the nitrites action. nitrites, in a normal diet, are coming from drinking water in a proportion of 21%, from fruits and vegetables [8] more than 70%, but can also come from meat products, in which nitrite is used as a preservative and improving color agent, at a rate of 6%. nitrite has both bacteriostatic and bactericidal effect that can be explained by the so-called perigo, (combination of nitrite and amine groups of microorganisms citoplasma protein structure) is dependent on ph / rh, former of hno2 and that no is greater at ph <5.8. the antibacterial effect is manifested in an initial concentration of 150 mg nano2 /100 g. in the absence of nitrite, sausages after a heat treatment have a gray color, unattractive from the organoleptic point of view. specific red color of fresh meat is determined as the ratio of meat pigments in reduced and oxidized state, myoglobin concentration in meat, and the level of residual hemoglobin influenced by the degree of bleeding [9]. 2. materials and methods determination of nitrite in meat products was performed by griess method. the method is based on the fact that nitrites at acid ph may be combined with a primary aromatic amine to form a diazonium salt. on switching to another primary amine forms a colored complex with maximum absorption at 540 nm. 2.1 reagents the solution to precipitate proteins: i potassium ferrocyanide solution, 10.6%: in a 1000 ml flask is placed 106 g crystallized potassium ferrocyanide [k4fe (cn) 6 • 3h2o] weighed to the nearest 0.01 g dissolved in water; ii zinc acetate solution 22%: in a 1000 ml flask is placed 220 g crystallized zinc acetate [zn (ch3coo) • 2h2o] weighed to the nearest 0.01 g and 30 ml glacial acetic acid, dissolved in 300-400 ml of water and dilute to the mark with water. iii saturated sodium borate solution: 50 g sodium tetraborate crystallized placed in a 1000 ml volumetric flask, dissolve in warm water (40 50 ° c), cooled at room temperature and dilute to the mark with water. griess reagent: mixture of equal volumes of solution i and solution ii. the mixture was prepared immediately before use. solution i is dissolved by heating on a water bath, 6 g sulfanilic acid, accurately weighed of 0,001 g, in 200 ml glacial acetic acid and 400 ml water. after cooling is added 200 ml of 10% sodium chloride and diluted to 1000 ml with water. solution ii is dissolved by heating in a water bath, 0.3 g of alpha-naphthylamine in 100 ml of water is filtered if necessary is cooled and added to 200 ml of glacial acetic acid and diluted with water in a volumetric flask of 1000 ml. solutions i and ii shall be kept in brown bottles, sealed one week. note: solution ii is handled carefully avoiding contact with skin. solution of sodium nitrite standard prepared at the time of use. measurement equipment and materials: uv-vis spectrophotometer, analytical balance, tubes, 100 ml cylindrical glass. 2.2 procedure. preparing filtrate for analysis. the sample for testing weighs about 10 g, with an accuracy of 0,001 g, is passed quantitatively into a 200 ml flask with about 100 ml of warm water (70 80oc), and 5 ml of saturated sodium borate solution is added and the flask, is heated for 15 minutes in boiling water bath, stirring from time to time. the sample is cooled at to room temperature and subsequent is added 2 ml of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sonia amariei, sorina ropciuc, domitian mugurel scripca, variation of nitrite content in meat products during the shelf life, volume xii, issue 4 – 2013, pag. 342 – 346 344 potassium ferrocyanide and 2 ml of zinc acetate, stirring after each addition. the sample stands 20 to 30 minutes and then is made up to volume. contents of the flask are stirred and filtered off a filter paper with a high porosity. the filtrate should be clear. the extinction of the standard sample is measured by the spectrophotometer at a wavelength of 520 nm and the calibration curve is plotted. 2.3. samples analyzed were smoked chest and gypsy steak. the products were monitored for 10 days, and analyzed every two days. 3. results and discussion in the analyzes conducted during the ten days has been found that the product has undergone some changes monitored in terms of the amount of nitrite, figure 1. figure 1. variation of nitrate in smoked chest if in the first day it was an amount of 2.98 mg nano2/100g product on the last day the nitrite concentration reached to 3.4 mg/100 product. this increase is due to the decrease in the water content of the product during the ten days of storage. a greater increase in nitrite can be seen following the results of the fifth days, registering an increase of 10.7% during the first days of processing, product humidity drops significantly, and the concentration of other ingredients contained in the product increases. five days after the product has stability in the variation of nitrite and the increase was 3%. following quantitative analysis that were made in the ten days it was recorded a maximum of 3.4 mg/100g nitrite, dosage compliance with current standards of maximum dose of 7mg/100g product. the other product analyzed was gypsy steak., which was determined also the variation of nitrite. the results are presented in figure 2. figure 2. variation of nitrate in gypsy steak following the analysis made, the variation of nitrite in gypsy steak during the ten days is 7.7%. in the first three days was the largest increase of 3.3% due to lower of humidity in the product followed by the increasing of other substances from chemical composition. in the last 5 days, variation in nitrite content was 2%. during the 10 days the concentration of nitrite was within the limit of 7 mg/100g product. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sonia amariei, sorina ropciuc, domitian mugurel scripca, variation of nitrite content in meat products during the shelf life, volume xii, issue 4 – 2013, pag. 342 – 346 345 figure 3. comparison of nitrite content in the two products smoked chest and gypsy steak present different concentrations of nitrite but the variation in the ten days is similar. in the smoked chest the maximum concentration determined was 3.4mg nano2 /100g product with a 14% increase. gypsy steak contains 5.12 mg/100g and recorded an increase of 7.7%. the two products have different contents of nitrites due to the different production process. the raw material for the two products is different, for getting smoked chest is using pork neck and spinal muscles, products resulting from the cutting of carcases and for gypsy steak is used boneless pork bellies. nitrites concentration in the product depends on nitrites and nitrates added to the salt mixture used in the meat industry and on injection mode. the two products have a different injection efficiency given that it used the same mixed of injection. pork neck has a higher capacity of brine retention due chemical composition than pork breast. another factor is the size of parts that are the subject to the injection process. 4. conclusions in this study was analyzed quantitatively the presence of nitrite by griess method in two meat products smoked chest and gypsy steak and the variation of their concentration in a period of ten days after processing. the nitrite concentration in gypsy steak on the first day was 4.75 mg/100g product, and increased up to 5.12 mg/100g product. this increase is largely due to loss of moisture from the product and transformation reactions of nitrates. the analyzis of smoked pork chest showed a concentration of 2.98 mg / 100g of product on the first day, and an increase up to 3.4 mg/100g product. nitrite concentrations increased in smoked chest with 14% for the entire period of study. nitrite concentration in the two products was within the permissible limits for this additive. from a quantitative point of view, nitrite concentrations of the two samples are quite large, gypsy steak recorded a maximum of 5.12 mg/100g product because it has a higher yield of injection than the smoked chest which was 3.4 mg/100g product. it is very important for producers to comply with limits on the use of food additives, as they can present serious risks for consumers. it is feasible to use essential oils to reduce initial nitrite levels in meat products(t.l. coutinho de oliveira, 2012 et al.). nitrate daily intake is 5 mg / kg body weight and 0.2 mg / kg body weight for nitrite. lethal dose for children is 0.2 0.5 g [10]. in conclusion, it is recommended that meat products obtained with nitrates and nitrites are not used for feeding infants and for children and adults is preferable not to exceed the maximum of two classes of compounds (through moderate consumption of such foods). this goal can be achieved only by reasonable eating, balanced and varied. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sonia amariei, sorina ropciuc, domitian mugurel scripca, variation of nitrite content in meat products during the shelf life, volume xii, issue 4 – 2013, pag. 342 – 346 346 5. references [1]. thomas c., mercier f., tournayre p., j.-l. martin, j.-l. berdagué, effect of nitrite on the odourant volatile fraction of cooked ham, food chemistry 139, (2013), pag. 432-438 [2]. leandro coutinho de oliveira t., malfitano de carvalho s., de araújo soares r., aparecida andrade m., das graças cardoso m., mendes ramosc e., hilsdorf piccolia r., antioxidant effects of satureja montana l. essential oil on tbars and color of mortadella-type sausages formulated with different levels of sodium nitrite, lwt food science and technology 45 (2012), pag. 204-212 [3]. parthasarathy d. k., bryan n. s., sodium nitrite:the “cure” for nitric oxide insufficiency, meat science, 92, (2012), pag. 274279. [4]. drabik-markiewicz g., dejaegher, e. de mey, s. impens, t. kowalska, h.paelinck, y. vander heyden, evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on n-nitrosamine formation when heating cured meat, analytica chimica acta, volume 657, issue 2, pag.123-130 [5]. honikel k.o., the use and control of nitrate and nitrite for the processing of meat products, meat science 78, (2008), pag. 68–76 [6]. van den maagdenberg g., de mey e., deprez s., kowalska t., paelinck, role of proline and hydroxyproline in nnitrosamine formation during heating in cured meat, meat science, volume 81, issue 3, (2009), pag. 479-486 [7]. liu a. h., bondonno c. p., croft k. d., puddey i.b. , woodman r. j., rich l., natalie c. ward,joseph a. vita, jonathan m.hodgson, effects of a nitraterich meal on arterial stiffness and blood pressure in healthy volunteers, nitric oxide, volume 35, (2013) pag. 123–130 [8]. du s.-t, zhang y.-s, lin x.-y, accumulation of nitrate in vegetables and its possible implications to human health, agricultural sciences in china, 6(10), (2007), pag. 1246-1255 [9]. barbieri g., m. bergamaschi, ge. barbieri, m. franceschini, kinetics of nitrite evaluated in a meat product, meat science, 93, (2013), pag. 282-286. [10]. tofană m., food additives food interactions (in romanian: aditivi alimentari – interacțiunea cu alimentul), editura academic press, cluj – napoca, (2006) 117 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 117 122 comparative study of proximate composition of nile tilapia (oreochromis niloticus) (linnaeus, 1758) from fresh water and brackish water in niger delta region, nigeria *olaniyi alaba olopade¹, iyabode olusola taiwo², oba sunday siyeofori1 and nathanael bamidele2 1faculty of agriculture, department of fisheries, university of port harcourt, rivers state, nigeria.olaniyi.olopade@uniport.edu.ng ²institute of food security, environmental resources and agricultural research, federal university of agriculture, abeokuta, nigeria *corresponding author received 5th april 2016, accepted 25th may 2016 abstract:the aim of this study was to determine and compare proximate composition and mineral content of oreochromisniloticus from fresh water and brackish water. fish samples were collected from bayalsa state (fresh water samples) an rivers state (brackish samples) and analyzed for their nutrient compositions. the results of proximate composition of the o. niloticus from fresh water and brackish water showed that the moisture content was of 73.92% in brackish water species and 72.07% in freshwater,respectively, crude protein was of 17.90 % in brackish samples and 22.23% in the fresh water ones. ash content had a percentage composition of 3.82 % in the brackish water and2.50% in the fresh water species.the level of lipid content of o. niloticus in fresh water (1.17±0.37) was found significantly (p<0.05) higher than that in brackish water (0.39±0.04) samples. further results showed that the levels of zinc in both fish samples were found to be very close to each other (p<0.05) while the level of potassium content of o. niloticus in fresh water was found significantly (p <0.05) higher than that of samples from brackish water with the value of 2.38±0.30mg/100g and 0.33±0.07mg/100g respectively. the results show that o.niloticus from the two sources of water has a good supply of fish nutrients and minerals. keywords:orechromis niloticus, fresh water, brackish water, proximate composition 1. introduction fish distribution and abundance in different habitats is associated with availability and abundance of food and substrate types in a particular habitat [1]. habitat selection by fishes is not extremely rigid as most species of fish exhibit some degree of plasticity and therefore spread to various habitat types [2]. niger delta region of nigeria is blessed with fresh and brackish water bodies that are rich in fin fish and shellfish. faced with an array of aquatic habitat, a fish species can choose which habitats to use. this is because a single habitat seldom provides all necessary resources for long termsurvival and reproduction and couple with the rate of oil pollution in the niger delta. the cichlid family, with a total of 1330 species, is the second largest family in the perciformes order [3]. nile tilapia (oreochromis niloticus) is one cichlids widely distributed in the inland water bodies in nigeria. although, they are mainly fresh water fish o. niloticus has been described as euryhaline and can disperse along brackish coastalines between rivers [3].the species is esteemed as food; affordable and supporting both small scale subsistence and commercial fisheries in nigeria. it grows to a http://www.fia.usv.ro/fiajournal mailto:olaniyi.olopade@uniport.edu.ng food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 olaniyialaba olopade, iyabodeolusola taiwo, oba sunday siyeofori,nathanael bamidele, comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish waterin nigerdeltaregion, nigeria, volume xv, issue 2 – 2016, pag. 117 – 122 118 maximum length of 62 cm, weighing 3.65 kg (at an estimated 9 years of age). the average size (total length) of o. niloticus is 20 cm [5]. demand for fish and fishery products are on the increase because of the excellent nutritional quality and the beneficial effects on health.nutritional quality of a fish is the detailed analysis of nutrients contained in it. fish meat consists of several components that all contribute to its overall chemical composition and is used as an indicator of nutritional value. fish muscle is rich in protein, unsaturated fatty acids and mineral elements [6, 7]. consumers have often wanted to know if there are nutritional differences in various fish species from different sources [8] particularly same fish species from difference habitats. it is against this background this study was designed to determine and compare the proximate composition and mineral contents of fresh oreochromis niloticus from the fresh water and brackish water. 2. materials and methods fish samples the brackish water fish samples (o.niloticus) used for this study were purchased from landing site of oba river, rivers state (4° 45′n 6° 50′ e)while the fresh water fish samples (o.niloticus) were purchased from landing site of swali market, bayelsa state (4° 45′ n, 6° 5′ e). the average weight and length of sampled fish ranged between 190.7 198.9g 185.34±9.66 and 18.68 -19.57±2.13cm, respectively were collected in polyethylene bags (cold stored) and transported early in the morning to the national agency for food and drug administration control (nafdac) in port harcourt, rivers state .the fish were then gutted and thoroughly washed with clean tape water and ready for chemical analysis. fish samples were homogenized separated by taking only meat portion (fillet) used in human consumption to determine crudeprotein, total lipid, moisture, carbohydrate and ash content. crude protein was determined by kjeldahl method (%nx6.25), which involves digestion of sample and then distillation for n determination while crude fibre moisture and lipid content were determined according to the method described in aoac[9] . ash was estimated by burning in a muffle furnace at 500ºc for 24 hours, and the incinerated sample was cooled in desiccator and weighed for ash determination. the mineral content of the fishes were determined from the solution obtained after dissolving the ash in distilled water containing a few drops of concentrated hcl. carbohydrate content was obtained by difference from the combined percent of moisture, protein, ash and fat from 100. the assessment of proximate composition and mineral contents of each sample was performed in triplicate. 3. data analysis data collected for the proximate analysis in triplicate are expressed in means (±sd) and compared by student's ttest (p <0.005) of sas[10]. 4. results and discussion proximate composition table 1 presents the proximate composition of the o. niloticus from fresh water and brackish water . moisture content was 73.92% in the brackish water samples and 72.07% in the fresh water, crude protein was 17.90 % in the brackish o. niloticus and 22.23% in the fresh water o. niloticus. the percentage composition ofash content was 3.82 % in the brackish water samples and 2.50% in the fresh food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 olaniyialaba olopade, iyabodeolusola taiwo, oba sunday siyeofori,nathanael bamidele, comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish waterin nigerdeltaregion, nigeria, volume xv, issue 2 – 2016, pag. 117 – 122 119 water species. the proximate composition of carbohydrate in the brackish (1.59%) was slightly lower than in the fresh water species (1.84%) and also the lipids content in brackish water species (0.39%) was lower than in the o. niloticus in fresh water (1.17%). the fibre content in the fresh water was 0.11% and in the brackish water specieswas 0.04%. table 1 proximate composition of o. niloticus from brackish water and fresh water proximate composition (%) brackish water samples fresh water samples minimum maximum mean minimum maximum mean ash 0.25 7.14 3.82±2.14 0.55 4.94 2.5±1.33 moisture 49.43 74.21 73.92±9.26 48.48 74.50 72.07±6.34 protein 11.15 20.25 17.90±2.83 19.54 24.87 22.23±2.19 carbohydrate 0.24 2.10 1.59±8.88 1.47 2.90 1.84±6..20 lipid 0.21 0.64 0.39±0.13 0.12 3.11 1.17±1.10 fibre 0.02 0.05 0.04±0.01 0.02 0.30 0.11±0.10 the mineral composition of o.niloticus from the two sources are presented in tables 2 and 3. large amount of potassium was found in the fresh water (2.38 mg/100g) and the brackish species recorded low value of potassium 0.33 mg/100g. obtained results demonstrate that the value of zinc in the fresh water species was 0.14 mg/100g and in the brackish species was 0.33 mg/100g. the value of copper in brackish species (0.36mg/100g) was lower than in the fresh water species (0.77 mg/100g). table 2 mineral composition of o. niloticusfromfresh and brackish water mineral composition (mg/100g) brackishwater fresh water minimum maximum mean minimum maximum mean copper 0.04 1.01 0.36±0.32 0.24 1.82 0.77±0.05 zinc 0.11 0.64 0.30±0.21 0.04 0.28 0.14±0.08 potassium 0.08 0.60 0.33±021 1.15 4.05 2.38±0.09 table 3 presents the comparative of proximate composition of o. niloticus from brackish and fresh water .in the table significant differences were observed only for fibre and lipid contents. the level of lipid content of o. niloticus in fresh water (1.17±0.37) was found significantly (p<0.05) higher than of brackish water (0.39±0.04) samples. the level of fibre in o.niloticus in fresh water was found to be statistically higher than that of brackish water samples with mean value of 0.11±0.03% and 0.30±0.07%, respectively (p <0.05) in the food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 olaniyialaba olopade, iyabodeolusola taiwo, oba sunday siyeofori,nathanael bamidele, comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish waterin nigerdeltaregion, nigeria, volume xv, issue 2 – 2016, pag. 117 – 122 120 presen tstudy. the levels of zinc in both fish samples were found to be very close to each other (p<0.05) while the level of potassium content of o. niloticus in freshwater was found significantly (p <0.05) higher than that of samples from brackish water with the value of 2.38±0.30mg/100g and 0.33±0.07mg/100 g, respectively. table 3 comparative result of the proximate composition and mineral contents ofo. niloticus from brackish and fresh water proximate composition brackish water mean±sd fresh water mean±sd ash 3.82±0.71 2.50±0.44 moisture 73.92±3.09 72.07±2.11 protein 17.90±0.94 22.23±8.05 carbohydrate 1.59±2.96 1.84±2.07 lipid 0.39±0.04 b 1.17±0.37 a fibre 0.04±0.00 b 0.11±0.03 a copper 0.36±0.11 0.77±0.17 zinc 0.30±0.07 a 0.14±0.03b potassium 0.33±0.07b 2.38±0.30 a means ± sd within the same row that have different letter is significantly different (p<0.05). the values obtained for proximate composition on the o.niloticus from both sources during the study agreed with the findings of suzuki [11] who reported that the main constituents of fresh fish are water (65-80%), protein (15-24%), fat (0.1-22%), carbohydrate (1-3%) and inorganic substances (0.8-2%) [12] also observed that the chemical composition of fish varied from 16-21% proteins, 0.1-25% lipids, 0.4-1.5% ash and moisture 60-81% with extremes of 96%. [13] described fish meat, in general, as having a proximate composition of 77.2% water, 19% meat protein, 2.5% lipid and 1.3% ash. the ranges of values of the proximate composition of the o.niloticus from the brackish water were slightly different from freshwater samples. variation of biochemical composition of fish flesh may also occur within same species depending upon the fishing ground, fishing season, age and sex of the individual and reproductive status.the habitat and food intake of these species are equally diverse. these widely different environmental conditions of temperature, salinity, pressure, availability of food etc. have profound influence on the biochemical composition. factors that play a role in the meat composition can be both endogenous (genetic) and exogenous (related to diet and the environment)[14]. fish meat is very nutritious food source, because of its high content of protein and essential amino acids [15, 16].the protein content of the fish samples ranged from17.90 % in the brackish species to 22.23% in the fresh water species. this indicates that fresh water fishes are better sources of protein in the body when consumed. these values were in agreement with those reported earlier [17].the relatively high to moderate percentage crude protein may be attributed to the fact that fishes are good source of pure protein, but the differences observed in values obtained could also be as a result of fish consumption or absorption capability and conversion potentials of essential nutrients from their diets or their local environment into such biochemical attributes needed by the organisms body[18]. the results in table1 followed the general rule of proximate composition of fish which states that there is an inverse relationship between the water, protein and fat contents of fish flesh. when the fat content is low, the moisture level and protein content are higher.o.niloticus in the two water bodies is generally low in food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 olaniyialaba olopade, iyabodeolusola taiwo, oba sunday siyeofori,nathanael bamidele, comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish waterin nigerdeltaregion, nigeria, volume xv, issue 2 – 2016, pag. 117 – 122 121 lipid. brackish water samples had a significantly lower (p< 0.05) lipid content (0.39 ±0.04%) than fresh water (1.17± 0.37%).[19] attributed low lipid content in fishes to migration and span due to low feeding ability in fishes at such periods. ash content had a percentage composition of 3.82 % in the brackish water and 2.50 % in the fresh water species. the observed range of ash content from the two water bodies indicates that the species is a good source of minerals. the variations in ash content could be attributed to the water body as fresh water fish are lower in sodium than salt water fish. the carbohydrate content in the brackish water species (1.59%) was slightly lower than in the fresh water species (1.84%). fish flesh contains negligible quantities of carbohydrates because glycogen presents in living fishisrapidlyconverted to lactic acid after death. the relatively low values of carbohydrate could be due to higher values of moisture and a relatively high value of protein content. the proportion of water in the fish varies widely, though in a majority of cases the variation is always between 70-80%. in the present study, moisture content was 73.92% in the brackish water species and 72.07% in the fresh water. the fishes had moisture ranging from 65.0 to 75.8 % indicating that the percentage moisture in fish muscles was within the acceptable level. the variations in the proximate composition in o.niloticus from fresh water and brackish water could be attributed to widely different environmental conditions of temperature, salinity, pressure, availability of food etc. have profound influence on the biochemical composition. many studies have shown that the concentration in minerals is greatly affected by different environmental factors (food availability, salinity, temperature) and intrinsic such as species, age, sex and physiological factors [20].the level of potassium in the o.niloticus from fresh water (2.38±0.30) was found to be higher than that of brackish water samples (0.33±0.07) which is far below fao range of 19-502mg/100g. the amount of cu and zn in flesh of o.niloticus in this study differed from each other. the value of zinc in the fresh water species was 0.14 mg/100g and in the brackish species was 0.33 mg/100g. these results are in agreement with the fao ranges of 0.23 2.1 mg/100 g recommandation. according to [21], the recommended daily requirement of copper in human nutrition ranges between 1.5 2.5 mg. the value of copper in this study ranged from 0.36 mg/100g in brackish species to 0.77 mg/100g in the fresh water species. the amounts of copper in fish flesh range from 0.04 to 0.6mg 100g with an average 0.25mg [22]. this is much lower than the recommended daily intake, assuming a single serving of 100g fish per day. in conclusion, the results clearly indicate nutritional potential of o.niloticus from the two study sites for human consumption. however, in terms of protein, lipid and fiber contents fresh water species has higher nutritional value than brackish water species. similarly the amounts copper and potassium in fresh water o.niloticus are higher than the brackish water species. 5. references [1].welcomme, r.l., ecology of flood plain river fisheries. fao fisheries technical paper, 262pp, (1985). [2]. ribbink, a. j., distribution and ecology of cichlids of the african great lakes,in; cichlid fishes behaviour, ecology and evolution. chapman and hall, london & new york, pp 890897, (1991). [3]. nelson ,j.s. fishes of the world. john wiley and sons, new york, 601 pp, (2006). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 olaniyialaba olopade, iyabodeolusola taiwo, oba sunday siyeofori,nathanael bamidele, comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish waterin nigerdeltaregion, nigeria, volume xv, issue 2 – 2016, pag. 117 – 122 122 [4].fao cultured aquatic species information programme. oreochromis niloticus.cultured aquatic species information programme.text by rakocy, j. e. in: fao fisheries and aquaculture department [online]. rome. updated 18 december 2015.[cited 28 december, 2015, (2005-2012). [5].bwanika, g.n., makanga, b., kizito, y., chapman, l.j. and balirwa, j. observations on the biology of nile tilapia, oreochromi sniloticus, l., in two ugandan crater lakes.africanjournal of ecology 42: 93–101, (2004). [6].zhao, f., zhuang, p., song, c., shi, z.h. and zhang, l.z.,aminoacid and fattyacid compositions and nutritionalquality of muscle in the pomfret, pampuspunctatissimus. food chem. 118: 224-227, (2010). [7]. ana, f., isabel, f.s., juan, a.s. and josé, m.b.,comparison of wild and cultured seabass (dicentrarchuslabrax) quality. food chem. 119: 1514-1518, (2010). [8].okonji, v.a. and afegbua, b.h., production and marketing of pond raised fish in southern part of delta state, nigeria. journal of agriculture, fisheries and forestry, 5(2): 3841(2004). [9].aoac., (association of official analytical chemists) (2000). official methods of analysis. 17th edition,washington d.c. 1: 234 p, (1990). [10]. sas sasuser‟s guide statisyics. sas inc. cary, north carolina 1998 edition, (1998). [11]. suzuki, t., fish and krill protein: processing technology. applied science, london, isbn: 0853349541,(1981). [12]. muraleedharan, v., antony, k.p. perigreen, p.a. and gopakumar, k., utilization of unconventional fish resources for surimi preparation. in: pillai vk, abidi sah, ravindran v, balachandran kk, agadi vv, editors. proc of the second workshop on scientificresults of forv sagar sampada. new delhi: department of ocean development. pp. 539–543, (1996). [13]. jacquot, r., organic constituents of fish and otheraquatic animal foods. fish as food. academic press,vol-1, (1961). [14].shearer, k. d., factors affecting the proximate composition of cultured fishes with emphasis on salmonids. aquaculture, amsterdam v. 119, p. 63– 88,(1994). [15].geiger, e. and borgstrom, g., fish protein-nutritive aspects. in fish as food (ed. g. borgstrom), new york: academic press. pp. 29– 114, (1962). [16].yanez , e., ballester, d. and monckeberg, f., enzymatic fish protein hydrolyzate: chemical composition, nutritive value and use as a supplement to cereal protein. j. food sci. 41: 1289-1292, (1976). [17]. fao nutritional elements of food and agricultural organization fish. rome,(2010). [18].adewoye, s.o. and omotosho, j.s., nutrient composition of some freshwater fishes in nigeria. biosci. res. commun. 11 (4) 333-336, (1997). [19]murray, j. and burt, j.r., the composition of fish.torry advisory note no. 38. ministry of agriculture, fisheries and food, aberdeen, 14 pp, (1977). [20].roy, p.k. and lall, s.p., mineral nutrition of haddock melanogram musaeglefinus(l.).acomparision of wild and cultured stock. j. fish biol. 68:1460-1472, (2006). [21].wildma,r. e. c., and medeiros d. m., advanced human nutrition. boca raton: crc press. chicago (author-date, 15th ed.),(2000). [22]. love r.m., the biochemical composition of fish. in the physiology of fish, margaret e. brown, editor. academicpress inc., new york, (1957). microsoft word 8 art 2 daniela borda.doc 53 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 53 58 thermal inactivation kinetics of lactoperoxidase in model system, milk and whey daniela borda 1*, mihaela ghermăneanu1, iulia bleoancă1, corina neagu1 1faculty of food science and engineering, ”dunarea de jos” university of galaţi, corina.neagu@ugal.ro 111 domneasca street, 800201, romania, daniela.borda@ugal.ro, iulia.bleoanca@ugal.ro * corresponding author received 6 january 2013, accepted 10 february 2013 abstract: inactivation of lactoperoxidase (lpo) in model system, milk and whey at atmospheric pressure was studied in a temperature range of 60-70 °c. the first order kinetics model allowed the estimation of the inactivation rate constants (k) and the thermal death times (d). dand k-values decreased and increased, respectively with increasing temperature, indicating a more rapid lpo inactivation at higher temperatures. at 70°c the inactivation of lpo was achieved in milk after 6 minutes and in whey after 4 minutes of thermal treatment. at 67.5°c lactoperoxidase was completely inactivated after 14 minutes in phosphate buffer. in all systems studied the temperature dependence of lactoperoxidase inactivation in milk, whey and model system versus the reaction rates could be accurately described by the arrhenius equation. the estimated activation energies were of 155.67 kj/mol for phosphate buffer, 217.79 kj/mol for milk and 235.57 kj/mol for whey. the correspondent zt values were estimated with the thermal death model and the values obtained were very close for all the three systems studied. for all the loglinear regression equations calculated sas system for windows 9 software was used. lactoperoxidase is an important antimicrobial system and knowing its thermostability in milk, byproducts and model systems allows a better control of the enzyme activity. keywords: lactoperoxidase, inactivation, enzymatic activity, kinetics 1. introduction lactoperoxidase (ec 1.11.1.7) is a member of the peroxidase family that has antimicrobial properties. the enzyme, naturally occurring in milk is part of the lactoperoxidase (lpo) system made of lactoperoxidase–thiocyanate–hydrogen peroxide. when activated, lpo catalyses, in the presence of h2o2, the oxidation of thiocyanate to compounds, such as hypothiocynate (oscn−) or higher oxyacids [1]. not only the reaction products but also the intermediate ones are known to have antimicrobial effects against bacteria, fungi and viruses [2]. lpo activity in milk is variable due to the difference in thiocyanate concentration directly related with cyanglucozide presence. a high cyanglucozide concentration is associated mostly with feeding from green pastures. lpo activity is enhanced by the presence of xanthineoxidase and sulhydryl oxidase. the activation of the lpo has a bacteriostatic effect on the raw milk and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu, thermal inactivation kinetics of lactoperoxidase in model system, milk and whey, food and environment safety, volume xii, issue 1 – 2013, pag. 53 58 54 effectively extends the shelf life of raw milk for 7–8 hours under ambient temperatures of around 30°c. this ensures adequate time for the milk to be transported from the collection point to a processing centre without refrigeration. in developing countries lpo system is still used for milk preservation. lpo is effective in refrigerated raw milk. researches have demonstrated that the activated lpo is successful also in prolonging the quality of raw milk for up to 5–6 days in refrigerated (+4°c) conditions [3]. in various experimental studies, the bacteriostatic or bactericidal effect of the lpo has been demonstrated against several human pathogenic microorganisms, such as streptococcus mutans, aeromonas hydrophila, candida albicans and helicobacter pillory [3]. recently new products as dental creams and chewing gums were launched on the market, intended to improve the dental hygiene by increasing the enzyme activity in saliva [4] lactoperoxidase is considered one of the enzymes that could be used and an indicator for the high pasteurization treatment and it is inactivated at 80°c in 2.5 s. the aim of this research study was to quantify the thermal inactivation kinetics of the lpo in milk, whey and buffer, in terms of rate constants and their associated temperature sensitivity. 2. experimental assay of enzyme activity the lpo activity was measured spectrophotometrically (uv-vis cintra) at 412 nm, using 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid or (abts) as a substrate. a volume of (2,5 ml) abts 0,001 mol/l, ph=6, was mixed with 100 µl of sample (enzyme, milk or whey) and the reaction was initiated by the addition 100 µl of freshly prepared hydrogen peroxide (0.3%). the enzymatic activity was determined for 10 minutes as the slope of the absorbance versus reaction time. one unit of activity (u) is defined as the amount of enzyme that catalyses the oxidation of l mol of abts per min at 20°c. all tests were performed in duplicate. thermal treatment the first experiment considered a thermostability screening study, whereby the samples are treated at different temperatures (25-70°c) for a fixed time interval (5min). in order to determine the thermostability of lpo, glass blaubrand capillary tubes were filled with 200 µl, to obtain “quasi” isothermal conditions. the tubes without air were sealed at both ends and incubated in a thermostated water bath at temperatures from 60°c to 70°c for fixed time intervals. the maximum period for the treatment was 15 minutes. immediately after the treatment samples were could in an icebath to prevent the inactivation produced by the remanent thermal effect. kinetic analysis primary models thermal and pressure inactivation of enzymes frequently follows first-order kinetics. in this case, the decrease of enzyme activity as a function of the time, at constant processing conditions, can be described by eq.(1):  ktaa  exp0 (1) which can be linearized by a logarithmic transformation in eq. (2) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu, thermal inactivation kinetics of lactoperoxidase in model system, milk and whey, food and environment safety, volume xii, issue 1 – 2013, pag. 53 58 55 kt a a       0 ln (2) where a is the enzymatic activity at time t; a0 is the initial activity; t is the treatment time and k is the first-order inactivation rate constant. next to the inactivation rate constant, the decimal reduction time (d) can be used to characterize the inactivation process. the relation between the decimal reduction time and the inactivation rate constant is given by eq. (3): kd /303.2 (3) secondary models the temperature dependency (at constant pressure) of the rate constant and the decimal reduction time can respectively be characterized by the activation energy (based on the arrhenius equation, (4)) and the zt-value (thermal death time equation, (5)).                  ref a refobs ttr e kk 11 lnln (4) (5) where kref is the rate constant at reference temperature tref, ea is the activation energy, r is the universal gas constant (r= 8.314 j/mol k), dref is the decimal reduction time at reference temperature and zt is the z-value. the activation energy and z-value were estimated using a linear regression analysis. the thermal inactivation data were analyzed both according to the arrhenius equation and the thermal death time model (in order to compare our data with existing literature data), while the high pressure thermal inactivation data were analyzed according to the arrhenius approach. to model the results linear regression equations were applied for the data using sas system for windows 9 software. 3. results and discussion the thermostablity screening studies indicated that lpo in phosphate buffer started to be inactivated at 50°c after 10 min of treatment; at 60°c a half of the initial activity was registered, while at 65°c the inactivation was almost ended after 10 min (fig 1, fig. 2). a similar behavior could be noticed for the milk samples. however in this case the inactivation is very close to the one in phosphate buffer. at temperatures lower than 62.5°c lpo displays in milk a resistance to the inactivation similar to the one of the enzyme in buffer system. this behavior could be explained by the presence in milk of protective enzymes such as xanthine-oxidase and sulhydryl oxidase, up to the temperatures at which these enzymes starts to be inactivated. following the screening experiment, a detailed kinetic study (inactivation as function of temperature and time) was carried out on the lpo system in phospahate buffer, in milk, and in whey to obtain the inactivation rate constants (figs. 3, 4 and 5). detailed thermal inactivation kinetics of the lpo system was studied at temperatures between 60°c and 70°c. the applicability of the first-order kinetic model, which is frequently reported in the literature for enzyme inactivation [1] was confirmed for the lpo system under study in all media. inactivation rate constants and decimal reduction time values were estimated by linear regression analysis and are presented in table 1. as expected, decimal reduction time decreases with temperature increase for each of the systems studied. )/)(()(log)(log 1010 trefref zttdd  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu, thermal inactivation kinetics of lactoperoxidase in model system, milk and whey, food and environment safety, volume xii, issue 1 – 2013, pag. 53 58 56 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 20 30 40 50 60 70 temperature (°c) lp o a ct iv ity 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 20 30 40 50 60 70 temperature (°c) lp o a ct iv ity figure 1. lpo activity at different temperatures after five minutes of isothermal treatment in phosphate buffer ph 6. figure 2. lpo activity at different temperatures after five minutes of isothermal treatment in milk the inactivation of lpo goes very quickly in whey at 70°c, in 4 min., while it might take more than 6 min for the same enzyme to be inactivated in milk. the difference could be explained by the protective effect of milk fat on the lpo system in milk. the enzyme activity was experimentally measured up to 67.5°c in phosphate buffered and up to 70°c in milk and whey. at 62.5°c the inactivation rate constant is 0.0579 min-1 and is 1.03 fold higher in milk at the same temperature and 1.38 fold higher in whey. the inactivation rate constant at 67.5°c is 0.1599 min-1 in phosphate buffer, 1.06 fold higher in milk and 1.76 fold higher in whey. tabel 1. d-values for lpo inactivation in phosphate buffer, milk and whey a) standard deviation in general the purified enzyme in phosphate buffer displays a higher thermostablity in comparison with milk and whey. the decimal reduction time is decreasing with the temperature increase. in phosphate buffer the deacrese from 60°c to 70°c was of 5.14, while in milk for the temperature range 62.5-70°c the deacrese in d-value was 6.38 fold. in whey the inactivation at 70°c goes 7.05 fold faster than at 62.5°c. to express the temperature dependence of the inactivation rate constant, the arrhenius equation (eq. 4) was applied and the activation energies were estimated. good correlation coefficients were obtained for the regression equations in all three systems studied (r2 was 0.96 for phosphate buffer, 0.94 for milk 0.96 for whey). the highest activation energy estimated was registered for lpo in whey (235.5 kj/mol) and the lowest for lpo in phosphate buffer (156.67 kj/mol). tabel 2. the activation energies and zt values for lpo inactivation variable phosphate buffer milk whey ea (kj/mol) 155.67±10.3a 217.79±12.5 235.57±19.5 zt 10.10±0.5 10.12±0.7 9.354±0.6 a) standard deviation d (min) tempe rature (°c) phosphate buffer milk whey 60.0 74.05±2.8a 62.5 39.78±1.06 38.43±1.8 28.72±0.01 65.0 18.06±1.6 15.75±0.5 10.92±0.009 67.5 14.40±0.14 13.56±0.4 8.15±0.007 70.0 6.06±0.05 4.07±0.001 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu, thermal inactivation kinetics of lactoperoxidase in model system, milk and whey, food and environment safety, volume xii, issue 1 – 2013, pag. 53 58 57 y = -0.0311x + 0.0968 y = -0.1275x + 0.1584 y = -0.057x + 0.117 y = -0.1599x + 0.0513 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0 2 4 6 8 10 12 14 16 ln ( a /a 0 ) time (min ) 60°c 65°c 62.°c5 67.5°c y = -0.0599x + 0.111 y = -0.1462x + 0.1414 y = -0.1698x + 0.0452 y = -0.379x + 0.042 r² = 0.912 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0 2 4 6 8 10 12 14 l n ( a /a 0 ) time (min ) 62.5°c 65°c 67.5°c 70°c figure 3. thermal inactivation kinetics of the lpo in phosphate figure 4. thermal inactivation kinetics of the lpo in milk figure 5. thermal inactivation kinetics of the lpo in whey the zt values expressing the increase in temperature necessary for a 10 fold reduction in d-values obtained with eq. 5 were very close for all three systems studied (table 2). the zt values for lpo in milk and phosphate buffer are similar. for whey there is less than 1 unit difference between the zt value in whey and the other two estimated values. the standard deviation values for each of the estimated variables shows good estimation as indicate table 1 and table 2. 4. conclusion the present paper studied the thermal inactivation of lpo in model system, in milk and in whey. the kinetic study was done in the temperature range of 69°c to 70 °c. based on primary kinetic equations the inactivation rate constant and the decimal time were estimated with linear regression. the fastest lpo inactivation was obtained for the whey system at 70°c of 4 minutes. the lowest inactivation time registered was for lpo in phosphate buffer at 60°c (74 minutes). thermostability studies showed that the enzyme was more stabile in phosphate buffer, followed by milk and whey. secondary kinetic equations allowed to accurately estimate the activation energies and the d-values using arrhenius equation and thermal death model. y = -0.0802x + 0.0849 y = -0.2825x + 0.1816 y = -0.2109x + 0.2084 y = -0.5655x + 0.1642 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0 2 4 6 8 10 12 14 time (min) ln ( a /a 0) 62.5°c 65°c 67.5°c 70°c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu, thermal inactivation kinetics of lactoperoxidase in model system, milk and whey, food and environment safety, volume xii, issue 1 – 2013, pag. 53 58 58 the lowest activation energy was of 155.67 kj/mol for the enzyme in phosphate buffer and the highest was in whey 235.57 kj/mol. the zt values were very similar for all of three systems studies: milk-10.13, whey9.35 and buffer10.10. the results obtained are in line with other studies of lpo inactivation kinetics in goat milk [1]. 5. acknowledgments the authors wish to thank re-spia project, smis code 11377, for the infrastructure provided in this study. 6. references [1]. trujillo a.j., pozo p.i., guamis b., effect of heat treatment on lactoperoxidase activity in caprine milk, small ruminant research, 67. 243–246 (2007) [2]. seifu e., buys e.m., donkin, e. f., significance of the lactoperoxidase system in the dairy industry and its potential applications: a review, trends in food science & technology, 16. 137–154 (2005) [3]. **** benefits and potential risks of the lactoperoxidase system of raw milk preservation, report of an fao/who technical meeting, rome, italy, 28 november 2 december (2005) [4]. marín e., sánchez l., pérez m.d., puyol p., calvo m., effect of heat treatment on bovine lactoperoxidase activity in skim milk: kinetic and thermodynamic analysis, journal of food chemistry and toxicology, 68. 89-93 (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 contents: 1. wild berry plants contamination with technogenic radionuclides in northern bukovina yevgenia volyns’ka, natalia omel’chenko, мyron rogozynskyi, oksana mykytyuk 214 217 2. a cas e study o f the j uice -makin g fact or y w astew ater treatment and p ossible w ays of i ts optim izati on alla cho bs an, ig o r winkler 218 224 3. highly active freeze-dried probiotic concentrates with long shelf life rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva 225 232 4. atom-absorption method of determination of selenium content in some raw materials and food igor kobasa 233 239 5. determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts micşunica rusu 240 246 6. physical properties of mustard seeds and how they are influenced by packaging mihaela jarcău 247 252 7. eryt hr os ine b in t he en vir o nm ent. rem oval p r oces ses laura carmen apostol, maria gavrilescu 253 264 8. the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters silviu-gabriel stroe, gheorghe gutt, silvia mironeasa 265 270 9. the quality of wastewater in the dairy industry sonia amariei, aspazia rusu 271 – 277 10. calcium lactate influence on some non-pathogenic microorganisms octavian baston, octavian barna 278 283 11. author instructions i v 12. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 48 study on the influence of sex and breed on bovine meat quality *ancuţa elena coşuleanu1 1 ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: cosuleanu_ancuta@yahoo.com *corresponding author received 25 march 2012, accepted 22 may 2012 abstract: the purpose of this study is to analyze the variations in chemical composition of two muscle groups collected from three bovine breeds. the biological material was represented by the gluteus and semimembranosus muscles collected from bovine of fleckvieh, pinzgauer and black spotted romanian breed. the average protein content from gluteus muscle ranged between 21.46% at the muscle collected from the pinzgauer females and 21.83% at the males from the same breed. the average lipid content in gluteus muscle has been above 2.0% in all breeds. the semimembranosus muscle presented a minimum average protein content of 21.75% at the muscle collected from black spotted romanian breed and a maximum of 21.84% at pinzgauer breed. the lipid content from semimembranosus muscle varied between 1.85% at the black spotted romanian males and 2.50% at fleckvieh breed. for both muscles there have not been obtained significant differences for the two parameters studied according to the sex of the animal. the breed has influenced the lipid content from the gluteus muscle collected from females. analysis of chemical composition of meat revealed the influence of sex, race, anatomical positioning of the muscle. meat obtained has a poor quality, which revealed the need of refurbishment of these animals before slaughter. keywords: muscle, protein content, lipid content, breed 1. introduction meat is an important component in human nutrition, providing through its composition the essential nutrients that the organism needs for a balanced diet. animal sex has important implications on all meat production, both during growth and development and also on the further processing [1]. commercial processing of cattle meat is under dual dependence of quantitative (weight) and qualitative criteria regarding the carcass and muscle composition. the quality of muscle groups depends on their lipid and protein composition [2]. proteins are basic and functional components, influencing the textural, sensory and nutritional properties [3]. lipids play an important role in the diet because the linoleic and linolenic acids are present as polyunsaturated [4]. the breed influences both the quantitative production of meat and the carcass composition, respectively the main muscle groups [5]. the porpoise of this study was to analyze the basic chemical composition of two muscles collected from three bovine breeds slaughtered for the current human consumption, although the breeds aren’t specialized for meat production. 2. experimental the research material used was represented by bovines, belonging to races fleckvieh, pinzgauer and black spotted mailto:cosuleanu_ancuta:@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 49 romanian, which were slaughtered for sale as cut portions. the cattle were classified according to their gender; any other factor was not taken into account since we refer to cattle sold on the current markets. from these animals muscle samples were taken. the muscular tissue was represented by gluteus and semimembranosus muscles. the muscle samples were collected after the operations included in the slaughter and refrigeration flow were performed. the measurements were performed at 24 hours after cutting the half-carcass in different anatomical regions and about 5 to 6 days after slaughter. meat samples of about 100 g were properly packaged, labeled and transported in cold conditions to the laboratory for analysis. the automatic meat analyzer is an infrared spectrophotometer that is used for composition analysis, using infrared absorbance characteristics of the sample spectra (fig. 1). figure 1. automatic meat analyzer data collected from the conducted research were processed using tabular msexcel computing applications. usual statistical estimators were calculated. anova analysis was used to determine the influence of the sex factor on the variation of protein and lipid muscle content. to determine the differences between the races of the same sex fisher test was used. for the data were we have found distinct differences at the fisher test we have applied the student test to determine the pairs that caused these differences. 3. results and discussion the proportion of protein content participation in the chemical composition of gluteus muscle presented values that ranged between 21.46% at the pinzgauer females and 21.83% at the males from the same breed (table 1). the homogeneity of the protein content was very good, the values found for the variation coefficient being below 10%. table 1 estimators of chemical composition (%) of gluteus muscle, depending on bovines sex and breed specification bovine sex females males ba1 p2 bnr3 ba1 p2 bnr3 n 5 5 9 5 4 5 p ro te in s (% ) x xs± 21.67±0.09 21.46±0.09 21.68±0.06 21.60±0.04 21.83±0.17 21.60±0.06 s 0.38 0.21 0.19 0.10 0.33 0.14 v% 1.75 0.97 0.86 0.46 1.51 0.65 min÷max 21.50÷21.90 21.20÷21.70 21.40÷22.00 21.30÷22.00 21.40÷22.20 21.50÷21.70 l ip id s (% ) x xs± 2.00±0.27 2.48±0.31 2.62±0.21 2.80±0.18 2.43±0.43 2.80±0.13 s 0.61 0.69 0.63 0.40 0.85 0.28 v% 30.41 27.70 24.10 14.29 35.21 10.10 min÷max 1.60÷2.70 2.10÷3.70 1.70÷3.50 2.40÷3.20 1.90÷3.70 2.60÷3.00 note: 1 bovines fleckvieh breed, 2 bovines pinzgauer breed, 3 bovines black spotted romanian breed. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 50 the minimum value of the protein content was obtained at the pinzgauer females (21.20%) and the maximum at the males from the same breed (22.20%). after data analyses of the average protein content it has been observed that at females it has been, in general, lower than the one obtained at males, with the exception of fleckvieh breed. the lipid content presented a high variability between the individuals of the same breed, but also between breeds (table 1). the variation limits of the lipid content were 1.60% at the fleckvieh females and 5.50% at the females from the black spotted romanian breed. females presented, in general higher values than males. statistical interpretation of the obtained results for the protein and lipid muscle content has not revealed statistical differences between the two sexes (table 2). table 2 the significance of differences in chemical composition (%) of gluteus muscle recorded in bovines between the sexes based on race chemical parameter (%) significance proteins ba1: f̂ = 0.25; f5% (1;8) = 5.32; f̂ < f5% => ns p2: f̂ = 4.15; f5% (1;7)= 5.59; f̂ < f5% => ns bnr3: f̂ = 0.30; f5% (1;12)= 4.75; f̂ < f5% => ns lipids ba1: f̂ = 3.62; f5% (1;8) = 5.32; f̂ < f5% => ns p2: f̂ = 0.01; f5% (1;7)= 5.59; f̂ < f5% => ns bnr3: f̂ = 0.14; f5% (1;12)= 4.75; f̂ < f5% => ns note: 1 bovines fleckvieh breed, 2 bovines pinzgauer breed, 3 bovines black spotted romanian breed, ns not significant (p<0.05), * p>0.05, ** p>0.01, *** p>0.001 the results of fisher test showed that between the females of different breeds regarding the proteins content there are no significant differences and significant for the lipid content. at males the obtained differences were not significant for all the studied parameters (table 3). table 3 analysis of the significance of values differences recorded by the chemical composition (%) of gluteus muscle in bovines, based on race by sexes chemical parameter (%) significance proteins f1: f̂ = 2.12; f5%(3;20) = 3.10; f̂ < f5% => ns m2: f̂ = 0.92; f5%(2;11) = 3.98; f̂ < f5% => ns lipids f1: f̂ = 3.71; f5%(3;20) = 3.10; f1%(3;20) = 4.94; f5%< f̂ < f1% => * m2: f̂ = 0.36; f5%(2;11) = 3.98; f̂ < f5% => ns note: f females, m males, ns not significant, * significant table 4 student test results on lipid content (%) in gluteus muscle in females rasa p2 bnr3 ba1 average 2.48 2.62 3.72 ba1 3.72 tù = 1.98; t5%= 1.90; t1%= 3.00; t5% < tù < t1% => * tù = 2.69; t5%= 1.78; t1%= 2.68; t0.1%= 3.93; t1%< t ù < t0.1% => ** bnr3 2.62 tù = 0.25; t5%= 1.80; tù < t5% => ns p2 2.48 note: 1 bovines fleckvieh breed, 2 bovines pinzgauer breed, 3 bovines black spotted romanian breed, ns not significant, * significant, ** distinct significant. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 51 in order to determine the female breeds between these differences appear the student test was applied. there were found not significant differences between the pairs pinzgauer – black spotted romanian, significant between pinzgauer – fleckvieh and between black spotted romanian – fleckvieh distinct significant. the data on the chemical characteristics of the semimembranosus muscle revealed a protein content with relatively closed values between the studied breeds. the protein content showed a high homogeneity of the studied populations (v%< 10%) (table 5). the minimum of the protein content was found at pinzgauer males (21.20%) and the maximum at the fleckvieh females and black spotted romanian males (22.20%). the variation of the average lipid content was between 1.85% at the black spotted romanian males and 2.50% at the fleckvieh females (table 5). the lipid percentage, like the one of proteins, at the semimembranosus muscle was higher at females than males, with the exception of pinzgauer breed that presented an average value with 0.10% higher at males than females (table 5). table 5 estimators of chemical composition (%) of semimembranosus muscle, depending on bovines sex and breed specification bovine sex females males ba1 p2 bnr3 ba1 p2 bnr3 n 5 5 9 5 4 5 p ro te in s (% ) x xs± 21.83±0.14 21.84±0.07 21.77±0.07 21.77±0.14 21.80±0.20 21.75±0.28 s 0.32 0.15 0.20 0.31 0.41 0.64 v% 1.47 0.69 0.92 1.40 1.87 2.93 min÷max 21.60÷22.20 21.70÷22.10 21.50÷22.10 21.50÷22.10 21.20÷22.10 21.30÷22.20 l ip id s (% ) x xs± 2.50±0.43 2.30±0.14 2.30±0.21 2.43±0.46 2.40±0.86 1.85±0.47 s 0.96 0.32 0.64 1.03 1.71 1.06 v% 38.57 13.75 28.01 42.18 71.28 57.33 min÷max 1.40÷3.20 2.00÷2.80 1.40÷3.10 1.30÷3.30 1.20÷4.90 1.10÷2.60 min÷max 74.90÷75.50 75.30÷76.10 75.00÷76.50 74.90÷76.50 73.50÷76.60 75.10÷76.40 note: 1 bovines fleckvieh breed, 2 bovines pinzgauer breed, 3 bovines black spotted romanian breed. the statistical comparison of the studied bovine populations belonging to the two sexes showed not significant differences for the protein and lipid content (table 6). table 6 the significance of differences in chemical composition (%) of semiembranosus muscle recorded in bovines between the sexes based on race chemical parameter (%) significance proteins ba1: f̂ = 0.07; f5% (1;8) = 5.32; f̂ < f5% => ns p2: f̂ = 0.04; f5% (1;7)= 5.59; f̂ < f5% => ns bnr3: f̂ = 0.005; f5% (1;12)= 4.75; f̂ < f5% => ns lipids ba1: f̂ = 0.006; f5% (1;8) = 5.32; f̂ < f5% => ns p2: f̂ = 0.02; f5% (1;7)= 5.59; f̂ < f5% => ns bnr3: f̂ = 0.67; f5% (1;12)= 4.75; f̂ < f5% => ns note: 1 bovines fleckvieh breed, 2 bovines pinzgauer breed, 3 bovines black spotted romanian breed, ns not significant (p<0.05), * p>0.05, ** p>0.01, *** p>0.001 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 52 table 7 analysis of the significance of values differences recorded by the chemical composition (%) of semimembranosus muscle in bovines, based on race by sexes chemical parameter (%) significance proteins f1: f̂ = 2.87; f5%(3;20) = 3.10; f̂ < f5% => ns m2: f̂ = 0.01; f5%(2;11) = 3.98; f̂ < f5% => ns lipids f 1: f̂ = 3.06; f5%(3;20) = 3.10; f̂ < f5% => ns m2: f̂ = 0.12; f5%(2;11) = 3.98; f̂ < f5% => ns note: f females, m males, ns not significant. the results of fisher test showed that between the females and males of different breeds for the chemical parameters studied the differences were not significant (table 7). 4. conclusion analysis of chemical composition of meat revealed the influence of sex, race, anatomical positioning of the muscle. the lipid content varied around 2.50% depending on race, sex and muscle. the chemical parameters that define the chemical composition of semimembranosus muscle had not varied significant. meat obtained has a poor quality, which revealed the need of refurbishment of these animals before slaughter. 5. references [1] lawrie r. a., lawrie’s meat science, technomic publishing company, lancaster, (1998) [2] micol d., robelin j., geay y., composition corporelle et caractéristiques biologiques des muscles chez les bovins en croissance et a l’engrais, inra production animale, 6(1):61-69, (1993) [3] jiang s.t., contribution of muscle proteinases to meat tenderization, proceedings of national science council, roc, 22(3), 97-107, (1998) [4] lorrain b., dangles o., genot c., duford c., chemical modeling of heme-indused lipid oxidation in gastric conditions and inhibition by dietary polyphenols, journal of agricultural and food chemistry, 58, 676-683, (2010) [5] picard b., jurie c., bauchart d., dransfield e., ouali a., martin j. f., jailler r., lepetit j., culioli j., caractéristiques des muscles et de la viande des principales races bovines allaitantes du massif central. journée science du muscle et technologies des viandes, 11:183-190, (2006) influence of κ-carrageenan, agar-agar and starch on the rheological properties of blueberries yogurt food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 29 hon ey cla ssi fi cation us ing co lour me asu reme n t *mircea oroian1, ana leahu 2, cristina damian 3, amelia buculei4 1-4food engineering faculty, stefan cel mare university of suceava, romania 1m.oroian@fia.usv.ro, 2analeahu@fia.usv.ro, 3cristina.damian@fia.usv.ro, 4ameliab@fia.usv.ro *corresponding author received 15 march 2012, accepted 2 may 2012 abstract: the purpose of this work is to classify honey to their floral source based on their colour. for this study were chosen 15 samples of honey from different floral sources (5 samples of acacia, 5 samples of tilia and 5 samples of sun flower honey). the colour of honey is influenced by the floral source, due to the minerals and other minor components presented; exposure to heat and storage time may affect honey’s colour. one of the easiest way to determine the honey colour is using the reflectance measurement in cielab coordinates. the coordinates in cielab dimension are l*, a* and b* values, which explain a 3-dimensional colour space. the l* value is the vertical axis and defines the lightness, and a* and b* values are perpendicular horizontal axes and define red-to-green and blue-to-yellow, respectively. in addition, hue angle (h) and colour intensity (c*) can be calculated from a* and b*. the honeys with the greatest purity of colour were the sun flower and tilia samples; the acacia samples exhibited the least purity of colour. the colour intensity of the samples is different from a floral source to another one. the sun flower sample had the highest colour intensity, followed by tilia and acacia honeys. all the colour parameters were significantly different. keywords: luminosity, colour intensity, hue angle 1. introduction the colour of honey is one of its most variable features. honeys show very different colours, varying from white or pale yellow to dark red or even black [1]. the colour of honey is characteristic of its floral source due to minerals and other minor components. exposure to heat and storage time may affect honey’s colour. honey appears lighter in colour after it has granulated. the colour of a specific sample of honey, after it granulates depends on the crystal size. the final crystals give the lightest appearance. for this reason, most cremed honeys are opaque and light in colour. honey can become darker as a result of storage, although at widely differing rates. this depends upon the composition of the honey (acidity, nitrogen, and fructose contents) and its initial colour. generally, the darkening of honey is temperature sensitive and occurs more rapidly when honey is stored at high temperatures [2]. many studies have dealt with the relation of honey colour to the floral origin, industrial processing methods, and the temperature and/or time of storage [3, 4, 5, 6]. in other study, the influence of the pollen grains, their morphology and colour, on the honeycolour has been considered [7]. however, in spite of the great importance of the colour of honey as an indicator of its origin and quality, there is no official method for its determination. thus, as for other foods, the method proposed by food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 30 the organisation internationale de la vigne et du vin [8] for the colourimetric analysis of grape-derived products, which is a simplification of the cieyxy [9] with the cie 1931 standard observer (2° visual field) based on the consideration of the whole visible spectrum, is used. the l*, a*, and b* values explain a 3dimensional colour space. the l* value is the vertical axis and defines the lightness, and a* and b* values are perpendicular horizontal axes and define red-to-green and blue-to-yellow, respectively. in addition, hue angle (h) and chroma (c*) can be calculated from a* and b*. h is distributed in the 4 quadrants of the a* and b* plane, and c* is higher the further it is from the origin of the coordinate [10]. the purpose of this work is to classify the honey (from different unifloral sources) based on the colour profile in cielab coordinates. 2. materials and methods 2.1. materials 15 samples of honeys (5 samples of acacia, 5 samples of tilia and 5 samples of sun flower) were purchased from the local market of suceava county. before being used they were warmed up to 55 °c to dissolve any crystals, and kept in flasks at 30 °c to remove air bubbles that could interfere the colour studies. 2.3. colour measurement the colour measurement was achieved with using spectrometer ocean optics (usa). the samples were fitted in plastic flask with 3.8 cm height and 6 cm diameter. to measure the reflected light is opened a diagram of 8 mm. the reflection specters are registered and are calculated the cielab parameters are calculated at a 100 angle, using the d65 light: l*(luminosity, between 0[black], at 100 [white]), a*(+a*[red], -a* [green]), b*(+b*[yellow], -b*[blue]) and h* [tone]. in figure 1 is presented the l*, h and c*. the colour intensity c* (eq.1) is calculated as: (1) the hue angle (eq. 2) is computed as: (2) in figure 1 is presented the l*, h and c*. fig.1. l*, c*, h* colour space [11] 2.7. statistical analysis an analysis of variance (anova) (α=0.05) with least significant difference (lsd) test using statgraphics plus 5.1 were performed on the data colour parameters. the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. 3. results and discussion the colour of honey is different from a floral source to another one. to characterize the honey samples in terms of colour, they were plotted in their corresponding positions food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 31 on the a*-b* and a*-l* colour spaces (fig. 2 and 3). on the a*-b* colour space, the nearer a honey is to the origin, the less purity of colour it has, and the further away it from the origin it is, the greater its purity is. the honeys with the greatest purity of colour were the sun flower and tilia samples; the acacia samples exhibited the least purity of colour. sun flower and tilia samples had the greatest yellow component (highest b* values). sun flower and tilia samples had a high red component (a*), while the tilia sample had weak green component (-0.43, tab. 1).the diference between honeys in function of the components a* and b* are statistical semnificative (p<0.001). figure 2 shows tilia and acacia honeys were clearer (higher l* value) than the other varieties. sunflower honey is not so clear then the other ones (l* values are much smaller than in the case of acacia and tilia). the luminosity of honey samples was significantly different (p<0.001). tab.1. colour parameter of honey colour parameter honey type f-value acacia sun flower tilia l* 56.61a 44.88b 50.01b 7.13*** a* -0.43c 6.13a 3.33b 18.66*** b* 17.76b 23.07ab 24.43a 3.39* c* 17.89b 24.10a 24.88a 4.53* h -1.15b -2.57b 3.36a 7.34** the colour intensity of the samples is different from a floral source to another one. the sun flower sample had the highest colour intensity, followed by tilia and acacia honeys. the colour intensity could be influenced by the presence of the pigments from the floral source into the sun flower honeys. the colour intensity was significantly different (p<0.05). the hue angle is significantly different (p<0.05) from a floral source to another one. the highest hue angle is achieved in the case of tilia honeys, while the acacia and sun flower had a negative hue angle. fig.2. a*-b* colour components of honeys – blue circle acacia, red rhombus – tilia, green triangle sunflower the colour values obtained were within the expected ranges for each of the honeys studied. the most colour values reported in the literature generally correspond to measurements made by the pfund scale (mm; [12, 13]). although only a few studies had used cielab (l*, a*, b*) to measure colour in nectar and honeydew honey [14, 15] their results were similar to this study. fig.3. a*-b* colour components of honeys– blue circle acacia, red rhombus – tilia, green triangle – sunflower food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 32 4. conclusion the colour of honey could be chosen as reliable indicator of its floral origin. the honeys with the greatest purity of colour were the sun flower and tilia samples; the acacia samples exhibited the least purity of colour. sun flower and tilia samples had the greatest yellow component (highest b* values). the sun flower sample had the highest colour intensity, followed by tilia and acacia honeys. the colour intensity could be influenced by the presence of the pigments from the floral source into the sun flower honeys. the highest hue angle is achieved in the case of tilia honeys, while the acacia and sun flower had a negative hue angle. all the colour parameter were significantly different. 5. references [1] gonzalez-miert, m.l., ayala, f., terrab, a., echavarri, j. f., negueruela, a.i. heredia, f. j., 2007, simplified method for calculating colour of honey by application of the characteristics vector method, food research international, 40, 1080-1086 [2] usa national honey board (303) 776-2337 [3] crane, e. (1984). bees, honey and pollen as indicators of metals in the environment. bee world, 65, 47–49. [4] fe´ller-demalsy, m. j., vincent, b., & beaulieu, f. (1989). mineral content and geographical origin of canadian honeys. apidologie, 20, 77–91. [5] free, j. b., williams, i. h., pinsent, r. j. f. h., townshend, a., basi, m. s., & graham, c. l. (1983). using foraging honeybees to sample an area for tracemetals. environmental entomology, 9, 9–12 [6] pereyra, a. g., burin, l., & buera, m. p. (1999). colour changes during storage of honeys in relation to their composition and initial colour. food research international, 32, 185–191. [7] terrab, a., gonza´lez-miret, m. l., & heredia, f. j. (2004). colour characterisation of thyme and avocado honeys by diffuse reflectance spectrophotometry and spectroradiometry. european food research and technology, 218, 488–492. [8] oiv (1979). recopilacion de los metodos internacionales de analisis de vinos. madrid: publicaciones del ministerio de agricultura. [9] cie 15:2004, technical report colourimetry, 3rd ed. commission internationale de l’eclairage central bureau. [10] bakker j, bridle p, timerlake f. 1986. tristimulus measurements (cielab 76) of port wine colour. vitis 25:67–78. [11]www.konicaminolta.com/about/research/core_tec hnology/optical/instrument_001.html [12] corbella, e., & cozzolino, d. (2006). classification of the floral origin of uruguayan honeys by chemical and physical characteristics combined with chemometics. lwtfood science and technology, 39, 534–539. [13] persano-oddo, l., gioia-piazza, m., & zellini, g. (1995). caratteristiche cromatiche dei mieli uniflorali. apicoltura, 10, 109–120. [14] terrab, a., gonzalez, g. a., diez, m. j., & heredia, f. j. (2003a). mineral content and electrical conductivity of honeysproduced in northewet morocco and their contribution to the characterisation of unifloral honeys. journal of the science of food and agriculture, 83, 637–643. [15] lazaridou, a., biliaderis, c. g., bacandritsos, n., & sabatini, a. g. (2004). composition, thermal and rheological behaviour of selected greek honeys. journal of food engineering, 64(1), 9–21. 15 samples of honeys (5 samples of acacia, 5 samples of tilia and 5 samples of sun flower) were purchased from the local market of suceava county. before being used they were warmed up to 55 °c to dissolve any crystals, and kept in flasks at 30 °c to remove air bubbles that could interfere the colour studies. 15 samples of honeys (5 samples of acacia, 5 samples of tilia and 5 samples of sun flower) were purchased from the local market of suceava county. before being used they were warmed up to 55 °c to dissolve any crystals, and kept in flasks at 30 °c to remove air bubbles that could interfere the colour studies. 15 samples of honeys (5 samples of acacia, 5 samples of tilia and 5 samples of sun flower) were purchased from the local market of suceava county. before being used they were warmed up to 55 °c to dissolve any crystals, and kept in flasks at 30 °c to remove air bubbles that could interfere the colour studies. microsoft word 10 torosian 2 n_kazlauskiene.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 66 assessment of heavy metal (ni, cu) interactions in binary mixture to rainbow trout (oncorhynchus mykiss) at early life stages *nijolė kazlauskienė1, gintaras svecevičius1, edvinas taujanskis1, raimondas leopoldas idzelis2, aistė liekytė2 1institute of ecology of nature research centre, akademijos 2, vilnius-21, lt-08412, lithuania kazlauskiene.nijole@gmail.com 2 vilnius gediminas technical university, sauletekio ave 11, vilnius, lt-10223, lithuania e-mail: aiste.liekyte@st.vgtu.lt *corresponding author received 10 june 2011, accepted 25 november 2011 abstract. lethal toxicity test and sublethal short-term and long-term toxicity tests on embryos and larvae of rainbow trout oncorhynchus mykiss (beginning from “eye-egg” embryos and from 1-day old larvae) were performed. lethal and sublethal toxic effects of the 1.0, 0.5, 0.25, 0.125 and 0.06 portion of the lc50 of cu, ni singly and their binary mixtures (cu+ni) were analyzed. the mixtures according to 96-hour lc50 for embryos and 96-hour lc50 for larvae were prepared. during exposure such biological parameters of the embryos and larvae as: mortality; gill ventilation frequency, behavioral responses of larvae were evaluated. lethal toxicity test showed that the 96hour lc50 values for embryos and larvae were different, and depended on the mixture preparation mode, heavy metal concentration in the mixture, and development stage of organism. sublethal exposure of mixture disordered gill ventilation function in larvae, affected behavioral reactions, and depended on the mixture of preparation mode, heavy metal concentration in the mixture, duration of exposure and affected development stage of organism. obtained data showed that toxicity assessment in fish at early stages of their development is important for evaluation of water pollution with heavy metals and prediction of their risk to aquatic animal populations. keywords toxicity, nickel, copper, binary mixture, fish 1. introduction copper is toxic to aquatic animals at levels marginally in excess of those found in many unpolluted aquatic environments [1]. nickel as a water-soluble metal is easily absorbed and accumulated by the aquatic organisms resulting in metal-induced disturbances in the structure and function of various tissues and organs [2, 3]. the background concentrations of copper and nickel in lithuanian water bodies are comparatively low. levels of these metals, especially copper, can frequently increase from industrial pollution or from anthropogenic sources [4]. the toxic effects of copper alone have been widely studied [5, 6]. the data on nickel toxicity in fish is very scarce, however impact of this metal was partially investigated for other freshwater organisms [7]. nevertheless, environmental contaminants are frequently encountered as mixtures, and the behavior of chemicals may not correspond to data predicted for pure compounds [8]. data for heavy metal binary mixture toxicity depend largely on different organisms/methods applied, sensitivity of parameters studied etc. for the majority of the binary combinations, food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 67 the interactions were of synergistic nature [9, 10, 11, 12 and 13]. the aim of this study was to undertake investigations into the lethal and sublethal effects of copper, nickel singly and to ascertain the toxic effect of cu+ni mixtures depending on the mixture preparation mode, on the heavy metal concentration in the mixture, on the duration of exposure and on affected development stage of organism; to assessment of copper and nickel interactions in their binary mixture to rainbow trout (oncorhynchus mykiss) at early life stages of development. 1. materials and methods toxicity tests were conducted at the laboratory of ecology and physiology of hydrobionts institute of ecology of nature research centre. rainbow trout embryos and larvae were obtained from the žeimena hatchery. dilution water was from high quality artesian water. the average hardness of dilution water was approximately 284 mg/l (caco3), alkalinity 244 mg/l (hco3) , the mean ph was 8.0, temperature was 10° ± 0.5°c and the oxygen concentration was between 8 to 10 mg/l. the lethal and sublethal toxic effects of the 1.0, 0.5, 0.25, 0.125 and 0.06 portion of the lc50 of cu, ni singly and their binary mixtures (cu+ni) were analyzed (table 1). the mixtures according 96-hour lc50 for embryos and 96-hour lc50 for larvae were prepared (table 2) [14, 15]. the mixtures were equal to 1%. solutions were made from chemically pure copper and nickel salts (cuso4·5h2o; niso4 7h2o). concentrations of ni and cu 0.2; 0.5 mg/l, respectively are accepted as the maximum-permissible-discharge into inland water bodies according to the european parliament and council directive 2000/60/ec [16]. control water and solutions were renewed on alternate days. table 1 concentrations of heavy metals studied concentration, heavy metal concentration (mg/l) portion of lc50 embryos larvae cu ni cu ni 1 1.1 3.4 0.4 1.2 0.5 0.5 1.7 0.2 0.6 0.25 0.25 0.8 0.1 0.3 0.125 0.125 0.4 0.05 0.15 0.06 0.06 0.2 0.025 0.08 lethal toxicity test (96-hour) and sublethal short-term (96 hour) and long-term (38, 24 days) toxicity tests on embryos and larvae (beginning from “eye-egg” embryos – for the first test and beginning from 1-day old larvae – for the second test) were performed. all tests were conducted under semi-static conditions. one hundred embryos were exposed to each concentration of hm, and their mixtures. studies with embryos and larvae were performed in three replications. biological [mortality; gill ventilation frequency (gvf, counts/min); behavioral responses of larvae (the number of individuals making nests in % and the number of individuals responding to external stimuli in %); parameters of the larvae were evaluated using routine methods [14]. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 68 table 2 lethal toxicity of heavy metals to rainbow trout embryos and larvae [14] heavy metal 96-hour lc50 (mg/l) 95% confidence interval (mg/l) embryos cu 1.13 1.0  1.27 ni 3.39 2.98  3.85 larvae cu 0.44 0.39  0.49 ni 1.25 1.07  1.47 median acutely lethal concentration (lc50) values and their 95% confidence intervals were estimated by use spearmankarber methods [17]. the toxic effect of the mixtures was calculated according to the mixture toxicity index (mti), and it was estimated according to the presented classification [18]. the significance of all the data obtained was determined by use of student‘s t-test with p ≤ 0.05. table 3 lethal toxicity of cu+ni mixtures, mixtures toxicity indexes (mti) and effects of the mixtures to rainbow trout embryos and larvae hm concentration in mixture (portion of lc50) 96-hour lc50 (%/mg/l) 95% confidence interval (%) mti effect of mixture embryos (mixture prepared according to 96-hour lc50 for embryos, the acute test beginning from “eye-egg” embryos till hatching period) 1:1 0.32/0.36+1.08 0.29  0.44 1.65 more-than additive 0.5:0.1 0.43/0.48+1.46 0.48  1.46 1.23 more-than additive larvae (mixture prepared according to 96-hour lc50 for larvae, the acute test beginning from “eye-egg” embryos including hatching period) 1:1 0.28/0.11+0.35 0.22  0.36 1.92 more-than additive 1:0.1 0.42/0.18+0.5 0.36  0.58 1.27 more-than additive larvae (mixture prepared according 96-hour lc50 for larvae, the acute test beginning from 1-day larvae) 1:1 0.38/0.17+0.45 0.30  0.42 1.42 more-than additive 1:0.1 0.48/0.21+0.60 0.39  0.50 1.06 more-than additive 3. results and discussion 2.1. lethal toxicity the data obtained showed that the 96-hour lc50 values for embryos and larvae were different, and depended on the mixture preparation mode, on the heavy metal concentration in the mixture, and on affected development stage of the organism (table 3). hatching larvae were more sensitive to cu+ni mixture as compared to embryos and 1-day old larvae. 2.2. sublethal toxicity short-term effect of 0.125 portion of the 96-hour lc50 for larvae concentration of cu and ni did not induce alterations in the gvf of the larvae. meanwhile, the same concentration of cu+ni mixture induced significantly increase in the gvf of the larvae at the end of the first test (table 4). short-term effect of 0.125 portion of the 96-hour lc50 for larvae concentration of. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 69 table 4 sublethal toxic effects of cu, ni and their binary mixture on gvf and behavioral responses of rainbow trout larvae (mean±sem) gvf (counts/min) the number of individuals making nests (%) the number of individuals responding to external stimuli (%) chemicals after 4 days after 38 days after 4 days after 38 days after 4 days after 38 days 0.125 portion of the 96-hour lc50 for larvae beginning from “eye-egg” stage embryos cu 90.0 ± 4.1 112.8 ± 3.2 86 ± 4.6 86 ± 6.2 88 ± 3.8 88 ± 3.8 ni 92.6 ± 5.7 104.2 ± 2.6* 80 ± 4.4 56 ± 4.8* 86 ± 4.2 48 ± 4.2* cu+ni 98.4 ± 4.8* 106.0 ± 2.2* 72 ± 4.0* 42 ± 4.4* 78 ± 4.4* 46 ± 4.2* control 87.4 ± 4.4 115.0 ± 3.4 91.6 ± 8.4 90.4 ± 9.4 92.4 ± 7.6 94.4 ± 5.6 after 4 days after 20 days after 4 days after 20 days after 4 days after 20 days 0.125 portion of the 96-hour lc50 for larvae beginning from 1 day old larvae cu 90.2 ± 2.8 112.4 ± 2.4 86 ± 4.6 86 ± 6.2 88 ± 3.8 88 ± 3.8 ni 90.4 ± 2.6 114.8 ± 3.0 80 ± 4.4 88 ± 4.8 86 ± 4.2 84 ± 4.2 cu+ni 92.4 ± 2.4 127.6 ± 2.2* 82 ± 4.0 72 ± 4.4* 90 ± 4.4 66 ± 4.2* control 87.4 ± 4.4 115.0 ± 3.4 91.6 ± 8.4 90.4 ± 9.4 92.4 ± 7.6 94.4 ± 5.6 *values significantly different from controls (p<0.05; p<0.001) cu, ni and of cu+ni mixture did not induce alterations in the gvf of the larvae at the end of the second test long-term effect of 0.125 portion of the 96-hour lc50 for larvae concentration of cu did not induce disorders in the gvf of the larvae. meanwhile, the same concentration of ni and mixture induced significantly a decrease in the gvf of the larvae at the end of the first test. longterm effect of 0.125 portion of the 96-hour lc50 for larvae concentration of cu and ni did not induce alterations in the gvf of the larvae. however, the same concentration of cu+ni mixture induced significantly increase in the gvf of the larvae at the end of the second test (table 4). other obtained short-term behavioral data (table 4) could be defined as contradictive. for example, larvae significantly demonstrated only weak response to external stimuli and intensively formed the nests, do not scattering in the test mixtures. long-term exposures also confirmed vulnerable behavioral responses of larvae. this proves that respiratory responses of larvae, apparently, are much more sensitive indicators than other behaviors of exposure to sublethal or even lethal heavy metal mixtures. the lethal toxicity test showed that the 96hour lc50 values for embryos and larvae were different, and depended on the mixture preparation mode, the heavy metal concentration in the mixture, and affected development stage of organism. our previous results indicated that lethal toxic effects of hm singly and their mixture on fish early life stages of development depended on the type of metal and development stages. the impact of heavy metals (cu, zn, ni, cr, and fe) on testorganisms of different phylogenetic and ontogenetic level showed that in the most cases cu is the more toxic metal to rainbow trout (onchorchyncus mykiss) at all stages of development [14]. the sensitivity of different life stages of rainbow trout (based on 96-h lc50) might be indicated in the following sequences: larvae>adult fish>eggs [15]. hatching larvae were more sensitive to cu+ni mixture as compared to embryos and 1-day old larvae (table 3). fish at early food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 70 stages of development are more sensitive to the impact of hm and their binary and multinomial mixtures than adults [19, 12, 20 and 21]. mixture toxicity indices (mti) and effects of cu+ni mixtures to rainbow trout embryos and larvae (the lethality tests beginning from “eye-egg” embryos and from 1-day larvae) were similar and did not depend on the mixture preparation mode, on the heavy metal concentration in mixture, and on affected development stage of organism (table 3). these results are consistent with our previous data. the effects of cu, cr, zn, ni, and fe mixtures prepared according to the 96-hour lc50 for embryos and larvae were the same – more than additive and did not depend on the mixture preparation, the ratio of the metals in mixture, heavy metal concentration in the mixture, and affected development stage of organism [22, 19, 12]. according xua [13] the majority of the binary combinations, the interactions were of synergistic nature, but in ternary or quaternary mixtures, the joint action was mainly concentration additive, while antagonism was only observed for two mixtures (cu+pb and zn+cd) among all the 11 combinations. the sublethal short-term and long-term toxicity data showed that hm (ni, cu) singly induced effects on larvae gill ventilation frequency and behavior responses (the number of individuals making nests and the number of individuals responding to external stimuli in %) were less as compared to the effect of mixture (cu+ni) and depended on the mixture preparation mode, on the heavy metal concentration in the mixture, on the duration of exposure and on affected development stage of organism. our previous sublethal long-term toxicity test results indicated that the hm (ni, cu) singly induced effects on rainbow trout larvae mortality were less as compared to the effect of mixture. long-term exposure to hm and their mixture not only reduced survival of larvae of rainbow trout, but disordered the function of the most important vital systems (cardio-respiratory), disturbed all early ontogenesis processes (hatching of larvae), their growth, caused various disorders, and affected behavior reactions of larvae. the results indicated that toxic effects of hm (ni, cu) singly and their mixture on fish early life stages of development depended on the type of metal, its concentration, and development stages [21]. obtained data showed that toxicity assessment in fish at early stages of their development is important for evaluation of water pollution with heavy metals and prediction of their risk to aquatic animal populations. 4. acknowledgements this work was funded by research council of lithuania, project no. mip-58/20102011. 5. references [1]. tailor e.w., beamont m.w., butler p.j., mair j., mujallid m.i. lethal and sub-lethal effects of copper upon fish: a role for ammonia toxicity? in: tailor e.w. (1996) ed.; toxicology of aquatic pollution: physiological, cellular and molecular approaches. cambridge university press, cambridge, england, (1996). [2]. deleebeeck m.e., schampheleare a.c., janssen c.r. a bioavailability model predicting the toxicity of nickel to rainbow trout (oncorhynchus mykiss) and fathead minnow (pimephales promelas) in synthetic and natural waters, ecotoxicology ant environmental safety. 67. 113, (2006). [3]. deleebeeck m.e., schampheleare a.c., heijerick d.g., bossuyt t.a., janssen c.r. the acute toxicity of nickel to daphnia magna: predictive capacity of bioavailability models in artificial and natural waters, ecotoxicology and environmental safety. 70. 6778, (2008). [4]. anonymous. annual report of water quality of rivers of lithuania. ministry of environment of the lithuanian republic. vilnius (in lithuanian), (2004). [5]. handly r.d. chronic effects of copper exposure versus endocrine toxicity: two sides food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 71 of the some toxicological process? comp. biochem. physiol,. a 135(1). 25, (2003). [6]. clearwater s.j., faraga a.m., meyer j.s. bioavailability and toxicity of diet borne copper and zinc to fish, comp. biochem. physiol. c 132. 269, (2002). [7]. sigel h., sigel a. metal ions in biological systems. nickel and its role in biology, university of toronto, canada, 23. 13-18, (2006). [8]. alterburger r., nendza a.m., schuurmann n.g. mixture toxicity and its modeling by quantitative structure – activity relationships, environ. toxicol.chem, 22(8). 1900, (2003). [9]. ince n.h., dirilgen n., apikyan i.g., tezcanli g. and üstün b. assessment of toxic interactions of heavy metals in binary mixtures: a statistical approach, archives of environmental contamination and toxicology, 36(4). 365372, (1999). [10]. adebayo a.o. evaluation of the jointaction toxicity of binary mixtures of heavy metals against the mangrove periwinkle tympanotonus fuscatus var radula (l.), ecotoxicology and environmental safety, 53(3). 404-415, (2002). [11]. otitoloju a.a., don-pedro k.n. determination of types of interactions exhibited by binary mixtures of heavy metals tested against the hermit crab, clibanarius africanus, toxicological & environmental chemistry, 88(2). 331–343, (2006). [12]. kazlauskienė n., vosylienė m.z. characteristic features of the effect of cu and zn mixture on rainbow trout oncorhynchus mykiss in ontogenesis, polish journal of environmental studies, 17(2). 291– 293, (2008). [13]. xua x., lib y., wangc y. and wanga y. assessment of toxic interactions of heavy metals in multi-component mixtures using sea urchin embryo-larval bioassay, toxicology in vitro, 25(1). 294-300, (2011). [14]. kazlauskienė n., burba a., svecevičius g. acute toxicity of five galvanic heavy metals to hydrobionts, ekologija, 1. 33, (1994). [15]. marčiulionienė d., montvydienė d., kazlauskienė n., svecevičius g. comparative analysis of the sensitivity of test-organisms of different phylogenetic level and life stages to heavy metals, environ. & chem. physics, 24 (2). 73, (2002). [16]. european parliament and council directive 2000/60/ec. [17]. hamilton m.a., russo r.c., and thurston r.w. trimmed spearmankarber method for estimating median lethal concentrations in toxicity bioassays. environ. sci. and technol,. 12. 417-719, (1978). [18]. kanemann h. fish toxicity tests with mixtures of more than two chemicals. a proposal for a quantitative approach and experimental results, toxicology, 19. 229-238, (1981). [19]. vosylienė m.z., kazlauskienė n., svecevičius g. complex study into the effect of heavy metal model mixture on biological parameters of rainbow trout oncorhynchus mykiss, environ. sci. & pollut. res, 10(2). 103, (2003). [20]. jezierska b., ługowska k., witeska m. the effects of heavy metals on embryonic development of fish (a review), fish physiol biochem, 35. 625–640, (2009). [21]. idzelis r.l., kazlauskienė n., liekytė a., taujanskis e. peculiarities of sublethal effects of heavy metals (ni, cu) and their binary mixture to rainbow trout in early ontogenesis, journal of environmental engineering and landscape management. in press, (2011). [22]. kazlauskienė n., burba a. investigations of the effect of heavy metal mixture on hydrobionts, ekologija, 2. 7-11, (1997). microsoft word 4 ciobanu articol 2.doc ` 24 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 24 29 changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold rodica ciobanu1* 1„alexandru ioan cuza” university, faculty of biology, iaşi, romania e-mail: ciobanu.rodica1981@yahoo.com * corresponding author received 14 november 2012, accepted 5 february 2013 abstract: the objective of this study was to estabilish the changes produced in the total water and dry matter content in peach leaves from cultivars springcrest and springold infected under natural conditions by taphrina deformans (berk.) tul. the infection with the pathogenic fungus produced an increasing in total water content in leaves of this two peach cultivars and a decreasing of dry matter content when compared with the values of this two physiological parameters recorded in healthy leaves. both in healthy and in curled leaves, the dry matter content is increasing with the leaves aging. total water content decreases in healthy and diseased leaves with the leaves age, but the values remained higher in attacked leaves. a linear negative correlation was found between the total water and dry matter content, in both healthy and infected leaves at this two peach cultivars. keywords: leaf curl, peach, fungi, phytopathogen agent 1. introduction taphrina deformans (berk.) tul. is an obligate parasite, the causal agent of peach leaf curl; this biotrophic fungus develops an intercellular, septate mycelium which induces an abnormal growth of the cells and produces the hypertrophy and hyperplasia of peach leaves tissues. peach leaf curl is a disease devastating to both crop and tree longevity. a severe attack of taphrina deformans generates early defoliation of peach trees followed by a massive fruit drop and if untreated, in a few years this disease can do to the drying of peaches. this study is important because in romania peaches are appreciated in alimentation for their high nutritive value and flavour and also used in therapy and such disease like peach leaf curl influence the fruits growth and their quality. studies concerning the biology of fungus taphrina deformans and peach leaf curl were made by many authors such as mary syrop [1], roselli et al. [2], melnicenco [3], testone et al. [4], in romania studies were carried out by ene ileana et al. [5], stoian elisabeta et al. [6], georgescu mihaela et al. [7, 8, 9] ivascu antonia et al. [10, 11], teodorecu georgeta [12] etc. cultivar (cv.) sprincrest is very susceptible to taphrina deformans [13] and it was obtained in california in 1969 [14]; cv. springold in considered susceptible at the attack of fungus taphrina deformans, it food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 rodica ciobanu, changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold, food and environment safety, volume xii, issue 1 – 2013, pag. 24 29 25 was obtained at fort valley (sua) and distributed in production in 1966 [15, 16]. 2. materials and methods the material used in this study was represented by fresh peach leaves. leaves showing curl simptoms were collected from cultivars sprincrest and springold cultivated in the didactic orchard „vasile adamachi” iaşi. for control, healthy leaves, were also collected. the estimation of total water and dry matter content was made by using the gravimetric method described by boldor et al. the fresh leaves were fixed for a few minutes at 105 ºc then dried out at 60ºc until the constant weight was achieve [17]. data obtained in this experiment were statistically processed using the linear correlation model [18] of microsoft excel program. 3. results and discussion the values of total water content from healthy leaves and from leaves attacked by taphrina deformans at cv. springcrest, are presented in fig. 1, from which it can be observed that the highest value of the total water content – 69.785 g% was registered at 4.05.2009, followed in decreasing order by the values recordered at 18.05.2009 (68.5551 g%), 25.05.2009 (66.0404 g%) at 1.06.2009 (65.3545 g%). in the leaves infected by taphrina deformans, the total water content had the smallest value at 1.06.2009 (76.8214 g%), followed in increasing order by the next values: 77.6348 g% (25.05.2009), 80.2711 g% (18.05.2009), 83.0569 g% (4.05.2009). both, in healthy leaves and in diseased ones, the total water content is decreasing with the leaves ageing. in diseased leaves total water content is decreasing at one time with the disease evolution. dry matter content from healthy leaves recorded the smallest value – 30.2149 g% at 4.05.2009 and it was followed in increasing order by the values observed at 18.05.2009 (31.4448 g%), 25.05.2009 (33.9595 g%), 1.06.2009 (34.6454 g%); in the leaves infected with în taphrina deformans, dry matter content is increasing at one time with the evolution of the fungus attack and it recorded the following values: 16.943 g% (4.05.2009), 19.7289 g% (18.05.2009), 22.3651 g% (25.05.2009) şi 23.1785 g% (1.06.2009). the dry matter content at cv. springcrest had smaller values in diseased leaves compared with the healthy ones, as evidence are the values of diseased/healthy ratio: d/h=0.5607 (4.05.2009), d/h=0.6274 (18.05.2009), d/h=0.6585 (25.05.2009), d/h=0.6690 (1.06.2009); total water content recorded higher values in diseased leaves when compared with the healty leaves and they registered specifically variation from one period to another, the d/h had the following values: d/h=1,1901 (4.05.2009), d/h=1.1708 (18.05.2009), d/h=1.1755 (25.05.2009), d/h=1.1754 (1.06.2009). 0 10 20 30 40 50 60 70 80 90 4.05.2009 18.05.2009 25.05.2009 1.06.2009 g % total water healthy leaves total water infected leaves dry matter healthy leaves dry matter infected leaves figure 1. total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at cv. springcrest the percenage differences of dry matter content in attacked peach leaves over healthy leaves at cv. springcrest were of: 43.9251% (4.05.2009), -37.2586% (18.05.2009), -34.1418% (25.04.2009) and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 rodica ciobanu, changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold, food and environment safety, volume xii, issue 1 – 2013, pag. 24 29 26 -33.0979% (1.06.2009); results revealed that the % differences in diseased leaves over healthy ones for total water content were: +15.9793% (4.05.2009), +14.5955% (18.05.2009), +14.9345 % (25.05.2009) and +14.9267% at 1.06.2009. analysing the regression equations (fig. 2, 3) it was found that between total water content (the dependent variable) and the dry matter content (the independent variable) of the leaves at cv. springcrest, a high negative linear correlation was observed both in healthy leaves (r=0.9783*) and in leaves attacked by taphrina deformans (r=-0.9764*); at one time with the increasing of dry matter content of the leaves it take place an decreasing of total water content. y = -1.5806x + 71.385 r2 = 0.9572 62 63 64 65 66 67 68 69 70 71 30.2149 31.4448 33.9595 34.6454 g% dry matter g% t ot al w at er c on te nt healty leaves linear (healty leaves) figure 2. correlation analysis between dry matter and total water content in healthy leaves at cv. springcrest (* the correlation is significant at p < 0.05) y = -2.1343x + 84.782 r2 = 0.9534 72 74 76 78 80 82 84 16.9430 19.7289 22.3651 23.1785 g% dry matter g% t ot al w at er infected leaves linear (infected leaves) figure 3. correlation analysis between dry matter and total water content in leaves infected by taphrina deformans at cv. springcrest (* the correlation is significant at p < 0.05) the total water content from diseased leaves is modifying at same time with the increasing of dry matter content, this modification is more intense in compare with changes which take place in healthy leaves, the regression coeficient had the following value b=-2.1343 in the case of diseased leaves and b=-1.5806 for the healthy leaves. the results concerning the total water content and dry matter from healthy and diseased leaves at cv. springold are presented in fig. 4. the highest value of total water content in healthy leaves was recorded at 4.05.2009 (68.6174 g%), followed in decreasing order by the values recorded at: 18.05.2009 (67.7404 g%), 25.05.2009 (65.5511 g%) and at 1.06.2009 (65.1461 g%). total water content from the leaves attacked by taphrina deformans is decreasing at the same time with the disease evolution, so were recorded the next values: 81.8561 g% (4.05.2009), 80.8951 g% (18.05.2009), 77.432 g% (25.05.2009) and 77.1027 g% (1.06.2009). dry matter content, at the same peach cultivar, had the smallest value -31.3825 g% at 4.05.2009 and was followed in increasing order by the values recorded at: 18.05.2009 (32.2592 g%), 25.05.2009 (34.4488 g%), the highest value for this parameter -34.8538 g% was registered at 1.06.2009. in the leaves attacked by taphrina deformans the dry matter content is increasing at one time with the development of the disease, this parameter recorded the smallest value -18.1438 g% at 4.05.2009, followed in increasing order by the values: 19.1048 g% (18.05.2009), 22.5679 g% (25.05.2009) şi 22.8972 g% (1.06.2009). the d/h ratio for total water content at cv. springold registered values higher then control at all dates when the determinations were made: d/h=1.1929 (4.05.2009), d/h=1.1941 (18.05.2009), d/h=1.1812 (25.05.2009), d/h=1.1835 (1.06.2009); dry matter content, registered food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 rodica ciobanu, changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold, food and environment safety, volume xii, issue 1 – 2013, pag. 24 29 27 an decreasing in leaves attacked by taphrina deformans when compared with the values from the healthy leaves, the ratio d/h had the following values: d/h=0.5781 (4.05.2009), d/h=0.5922 (18.05.2009), d/h=0,6551 (25.05.2009), d/h=0.6569 (1.06.2009). 0 10 20 30 40 50 60 70 80 90 4.05.2009 18.05.2009 25.05.2009 1.06.2009 g % total water healthy leaves total water infected leaves dry matter healthy leaves dry matter infected leaves figure 5. total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at cv. springold at cv. springold, the percentage differences in attacked leaves over healthy leaves for dry matter content had the next values: -42.185% (4.05.2009), -40.7772% (18.05.2009), -34.4886% (25.05.2009), 34.305% (1.06.2009); total water content was higher in infected leaves than in healthy ones, the difference percent was of: +16.1731% (4.05.2009), +16.2614% (18.05.2009), +15.3436% (25.05.2009) and of +15.5073% at 1.06.2009. from the regression equation (fig. 6, 7) between dry matter and total water content it was observed that a negative linear correlation estabilished between this two variables, both in healthy leaves (r = -0,9679*) and in those infected by taphrina deformans (r = -0.9502*). thus, with the leaves maturation it take place an increasing of dry matter content which is accompanied by an decreasing of total water content. the modification of total water content is more intense in attacked leaves that in the healthy leaves, this is proved by the values or regression coefficients for diseased leaves b = -1.7724 and for the healthy ones b = -1.2603. the results of this study, carried out at cvs. springcrest and springold revealed higher values in total water content in diseased leaves when compared with the values from healthy leaves at both cultivars. y = -1.2603x + 69.915 r2 = 0.9369 62 63 64 65 66 67 68 69 31.3825 32.2592 34.4488 34.8538 g% dry matter g% t ot al w at er healty leaves linear (healty leaves) figure 6. correlation analysis between dry matter and total water content in healthy leaves at cv. springcrest (* the correlation is significant at p < 0.05) y = -1.7724x + 83.752 r2 = 0.903 74 76 78 80 82 84 18.1438 19.1048 22.5679 22.8972 g% dry matter g% t ot al w at er infected leaves linear (infected leaves) figure 7. correlation analysis between dry matter and total water content in leaves infected by taphrina deformans at cv. springcrest (* the correlation is significant at p < 0.05) the higher content of total water in attacked leaves can be attributed to the degradation of cellulose, hemicellulose and pectic compounds of the cell wall by taphrina deformans. it is well known from literature that taphrina deformans hyphae which are growing in the intercellular spaces cause a partial dissolution of the leaves cell walls by secretion of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 rodica ciobanu, changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold, food and environment safety, volume xii, issue 1 – 2013, pag. 24 29 28 polysaccharide degrading enzymes, such as cellulase [19]. the increasing of dry matter content in diseased leaves at cvs. springcrest and springold is probably due to the presence and the development of mycelium of taphrina deformans in intercellular spaces and due to ascus with ascospores formation. with the leaves aging, the total water content is decreasing at both cultivars studied; this could be due to the increased transpiration of affected leaves [20] which after this stage, gradually turn brown because of tissues necrosis which occures as the disease develops. in infected plants leaves lose their turgor and finally die and become dessicated. in both healthy and disease leaves, with aging of the tissues it take place an general increasing of dry matter content and a decreasing of total water content. the results obtained in this study are simillar with those carried out at persica vulgaris by raggi and booth, who observed that the leaves infected with taphrina deformans, even dried leaves have a higher water content then the healthy ones [21, 22]; simillar results were mentionated and by shehu and aliero in the case of the attack of alternaria porri on onion leaves [23]; the result are in contradiction with those mentionated by nicolae mariana and mitrea rodi at cv. superbă de toamnă infected by taphrina deformans [20] and with those observed by tang et al. in the case of foliar attack of cercospora moricola and phyllactinia corylea at morus alba [24, 25]; the reusults obtained are in discrepancy with those presented by oerke et al. at cucumis sativus infected with pseudoperonospora cubensis [26] and with those observed by aldesuquay and baka in the case of senecio glaucus infected by puccinia lagenophorae and albugo candida infecting cakile maritima [27]. 4. conclusions total water content is higher in attacked leaves at both peach cultivars then the values of this physiological parameter recorded in the healthy leaves. dry matter content had higher values, at both cultivars, in healthy leaves when compared with diseased ones. between this two variables (total water and dry matter content) it establishing a negative liniar correlation in both healthy and diseased leaves from cvs. springcrest and springold. total water content is decreasing with leaves age at both cultivars and in the same time dry matter content is increasing with leaves aging. 5. references [1].syrop mary, leaf curl disease of almond caused by taphrina deformans (berk.) tul. an electron microscope study of the host/parasite relationship, protoplasma, 85 (1): 57-69, (1975) [2].roselli g., cantini c., mariotti p., susceptibility to peach leaf curl (taphrina deformans (berk.) tul.) in a peach germplasm collection, journal of horticultural science an biotechnologi, 72 (6): 863-872, (1997) [3].melnicenco l., rezistenţa soiurilor şi hibrizilor de nectarin la taphrina deformans, genetics and breeding of plants, animals and microorganisms, reports and abstracts of the viii genetics breeder’s congress of moldova, academia de ştiinţe a moldovei, chişinău, (2005) [4].testone g., bruno l., condello e., chippetta adriana et al., peach [prunus persica (l.) batsch] knope 1, a class 1 knox orthologue to arabidopsis brevipedicellus knat 1 is misexpressed during hyperplasia of leaf curl disease, jornal of experimental botany, 59 (2): 389-402, (2008) [5].ene ileana, matei iulia, bobîrnac b., consideraţii etologice privind unele soiuri de piersic la atacul bolilor şi insectelor dăunătoare în zona nisipoasă a olteniei, an. univ. craiova, ix ( xix), 253 256, craiova, (1978) [6].stoian elisabeta, burloi niculina, ivaşcu antonia, combaterea deformării frunzelor de piersic produsă de ciuperca food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 rodica ciobanu, changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold, food and environment safety, volume xii, issue 1 – 2013, pag. 24 29 29 taphrina deformans (berk.) tul., horticultura, nr. 1, 14-16, (1991) [7].georgescu mihaela., iorgu mădălina, delian elena, pătrunderea şi dezvoltarea miceliului ciupercii taphrina deformans (berk.) tul. în frunza de piersic, ahb, bucureşti, 203206, (1998) [8].georgescu mihaela, chira elena., delian elena, iorgu mădălina, modificări produse de ciuperca taphrina deformans (berk.) tul. în structura şi fiziologia frunzelor de piersic, lucr. şt. usamv, bucureşti, seria b, vol. xli-xlii, 201-210, (1998) [9].georgescu mihaela ioana, delian elena, modificări morfologice şi fiziologice în frunzele de piersic atacate de taphrina deformans, analele icpp, vol. xxx, bucureşti, p. 57-62, (1999-2000) [10]. ivaşcu a., balan v., toma s., oprea m., severin v., mircea i., sonica d., isac m., strategy of peach beeding for resistance to diseases in romania, acta hort. (ishs) 525:489-498, (2000) [11]. ivaşcu antonia, buciumanu a., lazar v, tamaş d., recent trends in peach breeding in romania, acta hort (ishs) 760: 467-472, (2007) [12]. teodorescu georgeta, observaţii morfologice şi histo-anatomice privind influenţa ciupercii parazite taphrina deformans (berk.) tul. asupra limbului foliar, bul. grăd. bot., iaşi, 5: 41-45, (1995) [13]. kaymak s., boyraz n., baştaş k. k., susceptibility of some peach and nectarine varieties to leaf curl disease (taphrina deformans (berk.) tul.) in field conditions, j. turk. phytopath., 37 (1-3): 27-37, (2008) [14]. cociu v., soiuri noi de piersic şi nectarine, producţia vegetalăhorticultura, 11: 23-25, (1988) [15]. ivascu antonia, hoza d., catalogul soiurilor de piersic, medro, (2003) [16]. minoiu n., lefter gh., bolile şi dăunătorii speciilor sâmburoase, ed. ceres, bucureşti, (1987) [17]. boldor o., raianu o., trifu m., fiziologia plantelor (lucrări practice), edit. did. şi pedag., bucureşti, 217-220, (1983) [18]. varvara m., zamfirescu ş., neacşu p., lucrări practice de ecologie, edit. univ.”al. i. cuza”, iaşi, (2001) [19]. bassi maria, conti g.g., barbieri nicoletta, cell wall degradation by taphrina deformans in host leaf cells, mycopathologia, 88, 2-3, 115-125, (1984) [20]. nicolae mariana, mitrea rodi, physiological modifications in prunus persica as a result of the attack produced by taphrina deformans, seria: biologie, horticultură, tehnologia prelucrării produselor agricole, ingineria mediului vol. xiv (xlx ): 517-522, universitatea din craiova, (2009) [21]. raggi v., water relations in peach leaves infected by taphrina deformans (peach leaf curl)—diffusive resistance, total transpiration and water potential, physiological and molecular plant pathology, 30 (1): 109120, (1987) [22]. booth c., taphrina deformans [description of fungi and bacteria], 72, 711 p, (1981) [23]. shehu k., aliero a. a., effects of purple blotch infection on the proximate and mineral contents of onion leaf, international journal of pharma sciences and research, 1(2): 131-133, 2010. [24]. tang a.k., salam k.a., samad m.a., absar n., nutritional changes of four varieties of mulberry leaves infected with fungus (cercospora moricola), pakistan journal of biological science 8 (1): 127-131, (2005) [25]. tang a.k., samad m.a., aynul haque akand a.s.m., azahar babyl sabina, absar n., nutritional changes of four varieties of mulberry leaves infected with fungus (pyllactinia corylea), pakistan journal of biological science 9 (3): 355-359, (2006) [26]. oerke e-c., steiner u., dehne hw., lindenthal m., thermal imaging of cucumber leaves affected by downy mildew and environmental conditions, journal of experimental botany, 57 (9). 2121-2132 (2006) [27]. aldesuquy h. s., baka z. a. m., physiological and biochemical changes in host leaf tissues associated with the growth of two biotrophic fungi growing in egypt, phyton. (horn, austria) 32 (1): 129-142, (1992) 321 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 321 326 the influence of frozen storage on some commercial mushrooms *octavian baston1, eugenia mihaela pricop1, cornelia lungu1 1dunarea de jos university of galati, 111, domnească street, 800201, galati, romania, phone: 0336 130177, fax: 0336 130 281, octavian.baston@ugal.ro, mihaela.pricop@ugal.ro, cornelia.lungu@yahoo.com *corresponding author received october 14th 2014, accepted november 25th 2014 abstract: the aim of our study was to determine some specific parameters of two mushroom species that were frozen and stored under freezing conditions for six months. the sensorial variation of thawed mushrooms was also analyzed. with reference to macronutrients, reducing sugars and total protein were determined. we found that agaricus mushrooms had a larger amount of reducing sugars than pleurotus and after six months of frozen storage, agaricus showed a decrease in reducing sugars amount by 2.38% and pleurotus by 7.69%. the total protein content was higher for agaricus than for pleurotus, and after six months of frozen storage agaricus showed a diminution of 5.17% and pleurotus a diminution of 8.74%. a biochemical analysis was made in order to study peroxidase enzyme. the initial value of peroxidase was 0.78 abs/g for agaricus and 0.48 abs/g for pleurotus. the variation of two micronutrients: niacin and ascorbic acid were also determined. niacin was found in high amount in both mushroom species and after frozen storage it decreased slightly. ascorbic acid decreased dramatically at frozen storage. after the first month of storage the ascorbic acid amount in agaricus decreased by 64.43% and in pleurotus by 34.56%. after six months of storage, it decreased by 76.73% and by 76.91% respectively. the sensorial analysis showed that the firmness, appearance and taste of agaricus and pleurotus were lower after six months of storage. keywords: agaricus bisporus, pleurotus ostreatus, niacin, peroxidase. 1. introduction in romania pleurotus ostreatus and agaricus bisporus are the most commercialized species of mushrooms in markets. mushrooms are largely consumed due to their great nutritional value and content in proteins and vitamins. agaricus bisporus, lentinula, pleurotus and flammulina are the most cultivated mushrooms worldwide [1, 15] because those mushrooms require shorter growth time compared to other edible mushrooms, also they demand few environmental controls and can be cultivated in a simple and cheap way [5]. raw mushrooms are a very perishable food and its shelf life varies between 3 and 5 days when stored at 0 to 2°c [11, 13]. but when stored at ambient conditions, raw, fresh mushrooms have a shelf-life of 24 hours [8]. after harvesting, mushrooms loose quality because of: enzymatic browning, microbial decay, moisture loss, tissue softening, development of some off-flavors,etc. therefore, mushrooms need to be preserved properly to extend use time. dehydrated and drying can be used as preservation methods [10]. these two methods of water elimination from mushrooms are the most commonly used for preservation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 octavian baston, eugenia mihaela pricop, cornelia lungu, the influence of frozen storage on some commercial mushrooms, volume xiii, issue 4 – 2014, pag. 321 – 326 322 giri [8] remarks that dehydrated mushrooms are valuable ingredients in a variety of sauces and soups. arora [2] and tuley [19] mention that dehydrated mushrooms are used as an important ingredient in several food formulations including instant soups, pasta, salads, meat, snack seasonings, stuffing, casseroles and rice dishes. they can also be preserved by tinning (canning) [9, 10, and 17]. guillamon [9] sustains that freeze-dried mushrooms have a long shelf-life, present better structural integrity and flavor retention. bernas [4] and czapski [6] state that freezing is one of the best postharvest preservation methods. sloan [17] affirms that the dominant trend in modern food processing technology is towards convenience foods of the “readyto-cook” type, which can be consumed after thermal treatment, as well as “readyto-heat” and “ready-to-eat” type foods which can be preserved by frozen or canning. usually, before applying the mentioned preservation methods, mushrooms are blanched. blanching is an important operation during mushroom processing because it inactivates the polyphenol oxidase enzyme, which is responsible for browning of mushroom. the objective of this work is to determine some physicochemical parameters and sensorial characteristics of frozen stored mushrooms cultivated in romania. 2. materials and methods materials the studied mushrooms are: agaricus bisporus and pleurotus ostreatus, first quality, purchased in refrigerated and packaged state from a local market. after a minimum processing: washing in cold water, blanching in hot water at 95 to 99°c for 10 minutes, water cooling at 15 to 20°c, cutting in slices of two millimeter thick, cooling in an ice-water mixture at final temperature of 2 to 4°c, water draining with a strainer, the mushrooms were frozen at -20°c using platilab 500 freezer (angelantoni industrie, italy) and stored in frozen state for six months. physicochemical evaluation fresh and thawed samples of pleurotus ostreatus and agaricus bisporus were subjected to several analyses: dry matter by aoac 984.13 method [3], reducing sugar by schrool method [16], total protein by kjeldahl method [16] using udk 130 d distilling unit (velp scientifica, italy), ascorbic acid [16], niacin by iodometric method [14], and peroxidase by colorimetric method [16], expressed as absorbance/g mushroom (abs./g). the dry matter and the chemical analyses of mushrooms were made on 3 samples, in triplicates. sensorial evaluation we applied the romanian standard referring to the general guidance on establishing a sensorial profile (sr en iso 13299/2010). five trained panelists gave scores for each sample with respect to their perception attributes as follows: 1 (worst) to 5 (best), by using the attributes mentioned in table 1. in order to familiarize the panel with different intensities for the different sensory properties of mushrooms, and to assimilate the scoring scale, two sessions of 1 hour each were carried out. the sensory profile used was obtained from two previous open sessions. evaluations were conducted in a sensory testing room. statistical data treatment the statistic analysis was made using microsoft office excel to determine the mean and standard deviations of the chemical evaluation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 octavian baston, eugenia mihaela pricop, cornelia lungu, the influence of frozen storage on some commercial mushrooms, volume xiii, issue 4 – 2014, pag. 321 – 326 323 table 1. sensorial attributes and quality of mushrooms 3. results and discussion the values of chemical parameters, after mushroom blanching and before frozen state, are shown in table 2. mushrooms have a high amount of protein and a good amount of reducing sugars. among the two species of mushrooms, agaricus bisporus has higher amounts of protein, reducing sugars and ascorbic acid than pleurotus ostreatus. after blanching, the enzyme peroxidase remained in higher quantity in agaricus. table 2. the initial parameters of mushrooms (only mean values) mushroom reducing sugars (g/100g dry weight) total protein (g/100g dry weight) peroxidase (abs./g) ascorbic acid (mg/100g dry weight) niacin (mg/100g dry weight) agaricus 22.62 44.03 0.78 28.37 49.18 pleurotus 10.24 27.66 0.48 17.33 45.65 the data of table 2 show that these the following figures have zero value on the frozen storage axis, meaning that the values are determined after blanching and before freezing operation of mushrooms. the values of 1 to 6 months are the frozen storage times for mushrooms and the determinations were made after thawing the products. all values were calculated by reporting to dry weight values (d.w.). fig. 1.reducing sugars amount of frozen mushrooms. attributes mushroom characteristics 1 2 3 4 5 flavor moldy, advanced altered product specific to the beginning of alteration pleasant well typical taste specific to advanced alteration specific to the beginning of alteration well typical appearance mould stains over 50% of product surface little moldy spots pleasant well typical firmness specific to altered product very low low well typical food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 octavian baston, eugenia mihaela pricop, cornelia lungu, the influence of frozen storage on some commercial mushrooms, volume xiii, issue 4 – 2014, pag. 321 – 326 324 fig. 2.total protein amount of frozen mushrooms. figure 1 shows the variation of reducing sugars for both species of mushrooms. the agaricus mushroom has a larger amount of reducing sugars than pleurotus. the values of 1 to 6 months are the frozen storage times for mushrooms and the determinations were made after thawing the products. all values were calculated by reporting to dry weight values (d.w.). figure 1 shows the variation of reducing sugars for both species of mushrooms. the agaricus mushroom has a larger amount of reducing sugars than pleurotus. the mean values of reducing sugars have slightly decreased after thawing, due to drip losses, for both mushrooms species. after frozen storage for six months agaricus presented a decrease in reducing sugars amount by 2.38% reported to the initial mean value (month 0). also, pleurotus presented a decrease by 7.69%. figure 2 shows that the total protein amount is higher in agaricus than in pleurotus, and all along the frozen storage time the mean values decrease is probably due to drip loss after the thawing of the mushrooms. the total protein losses for agaricus after frozen storage for six months reported to the initial mean value is of 5.17%. pleurotus presented a loss in total protein of 8.74%. fig. 3.peroxidase activity of frozen mushrooms. according to figure 3, the peroxidase activity has decreased for both mushroom species. the enzymatic activity of mushrooms was reduced by blanching operation that had happened before freezing. frozen state of mushrooms also inhibited peroxidase activity that could affect the color of mushrooms. the decreasing value of the enzymatic activity can be due to the drip loss at thawing. agaricus and pleurotus mushrooms presented a decrease in ascorbic acid values especially after frozen storage. after the first month of storage the ascorbic acid amount in agaricus decreased by 64.43%, in pleurotus by 34.56% and after six months of storage, it decreased by 76.73% and by 76.91% respectively. this is due both to drip losses after thawing and to freezing of mushrooms. according to fennema [7], ascorbic acid losses are temperature dependent. according to the scientific literature [12] ascorbic acid in pleurotus can vary from none to 144 mg/100g d.w. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 octavian baston, eugenia mihaela pricop, cornelia lungu, the influence of frozen storage on some commercial mushrooms, volume xiii, issue 4 – 2014, pag. 321 – 326 325 fig. 4. ascorbic acid amount of frozen mushrooms. we believe that the amount of vitamins present in mushrooms is due to the chemical composition of the substrate as nutritive feed and the postharvest attack of phenol oxidase. fig. 5. niacin amount of frozen mushrooms. both mushroom species are rich in niacin. niacin amount in the case of frozen storage varied slightly. for agaricus it varied (as mean values) around 48 to 49 mg/100g d.w. and for pleurotus it decreased after six months of frozen storage by 8.12%. the scientific literature [12] shows the niacin content for agaricus of 36 to 57 mg/100g d.w. and for pleurotus of 33 to 108 mg/100g d.w. fig. 6. agaricus bisporus sensorial analysis. fig. 7. pleurotus ostreatus sensorial analysis. the sensory analysis of mushrooms showed a very good quality for agaricus after the first month of frozen storage; only firmness was slightly lower as compared with the fresh product. for pleurotus, firmness and flavors were not at their best values after the first month of storage. the reduction of firmness is due to freezingthawing cycle that affected the mushrooms due to the transformation of ice crystals into water at thawing. after six month of storage, the panelists determined that firmness, appearance and taste were lower for agaricus and pleurotus. only appearance was pleasant for both mushroom species, possibly due to some oxidations made by air and enzymes that were not inactivated by blanching. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 octavian baston, eugenia mihaela pricop, cornelia lungu, the influence of frozen storage on some commercial mushrooms, volume xiii, issue 4 – 2014, pag. 321 – 326 326 4. conclusion our study proves that in the case of frozen mushrooms stored for six months (agaricus bisporus and pleurotus ostreatus) no major losses take place with reference to the macronutrient amounts. as for the micronutrients studied, the ascorbic acid makes an exception, its values being dramatically diminished on frozen storage. after six months of storage, the frozen mushrooms were not rejected by the trained assessors, only that their sensorial quality was slightly diminished. frozen state and frozen storage are methods that can be successfully used for mushrooms preservation. 5. references [1]. aida, f.m.n.a., shuhaimi, m., yazid, m., maaruf, a.g., mushroom as a potential source of prebiotics: a review, trend. food sci. technol,. 20. 567–575, (2009). [2]. arora, s., shivhare, u. s., ahmed, j., raghavan, g. s. v., drying kinetics of agaricus bisporus and pleurotus florida mushrooms. transactions-american society of agricultural engineers, 46(3). 721-724, (2003). [3]. association of official analytical chemists (aoac), official methods of analysis (16th ed.), washington dc, (1995). [4]. bernaś, e., jaworska, g., effect of preservation method on amino acid content in selected species of edible mushroom, lwt-food science and technology, 48(2). 242-247, (2012). [5]. bonatti, m., karnopp, p., soares, h.m., furlan, s.a., evaluation of pleurotus ostreatus and pleurotu ssajor-caju nutritional characteristics when cultivated in different lignocellulosic wastes, food chem., 88. 425–428, (2004). [6]. czapski, j., szudyga, k., frozen mushrooms quality as affected by strain, flush, treatment before freezing, and time of storage, journal of food science, 65(4). 722-725, (2000). [7]. fennema, o. r., effects of freezepreservation on nutrients. in: r. s. harris and e. karmas (eds) nutritional evaluation of food processing, avi, westport, connecticut, 244–288, (1975). [8]. giri, s. k., prasad, s., drying kinetics and rehydration characteristics of microwave-vacuum and convective hot-air dried mushrooms, journal of food engineering, 78(2). 512-521, (2007). [9]. guillamón, e., garcía-lafuente, a., lozano, m., rostagno, m. a., villares, a., martínez, j. a., edible mushrooms: role in the prevention of cardiovascular diseases, fitoterapia, 81(7). 715-723, (2010). [10]. jaworska, g., bernaś, e., & mickowska, b., effect of production process on the amino acid content of frozen and canned pleurotus ostreatus mushrooms. food chemistry, 125(3). 936-943, (2011). [11]. martine, b., gaelle, l. p., ronan, g., post-harvest treatment with citric acid or hydrogen peroxide to extend the shelf life of fresh sliced mushrooms. lebensmittel-wissenschaft undtechnologie, 33(4). 285–289, (2000). [12]. mattila, p., könkö, k., eurola, m., pihlava, j. m., astola, j., vahteristo, l., & piironen, v., contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms, journal of agricultural and food chemistry, 49(5). 2343-2348, (2001). [13]. mohapatra, d., frias, j. m., oliveira, f. a. r., bira, z. m., kerry, j., development and validation of a model to predict enzymatic activity during storage of cultivated mushrooms (agaricus bisporus spp.), journal of food engineering, 86(1). 39-48, (2008). [14]. popa, g., musca, l., biochimie medicalăanalize de laborator, editura fundaţiei universitare „dunărea de jos”, galaţi, 114-115, (2003). [15]. reis, f. s., barros, l., martins, a., & ferreira, i. c., chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study, food and chemical toxicology, 50(2). 191197, (2012). [16]. segal, r., vâţă, c., muscă, l., îndrumar de lucrări practice pentru biochimia produselor alimentare, uniersitatea „dunărea de jos”, galaţi, 19-22, 65-69, 104-105, 112-114, (2000). [17]. sloan, e., with better-for-you menu options, fresh fast fixin’s, and heart friendly foods, the global food industry retools for health. top 10 global food trends, food technology, 59(4). 2032, (2005). [18]. sr en iso 13299:2010, sensory analysis methodology. general guidance for establishing a sensory profile. [19]. tuley, l., swell time for dehydrated vegetables, intern. food ingredients, 4(1). 23–27, (1996). microsoft word 11 art_2 adrian ioana.doc 73 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 73 77 analysis of the environmental performance indicators for the emas regulation *adrian ioana1, augustin semenescu1, cezar florin preda1 1university politehnica of bucharest, spl. independentei 313, bucharest, s6, 060042, adyioana@gmail.com * corresponding author received 7 january 2013, accepted 15 february 2013 abstract. this paper presents the main environmental performance indicators for the ecomanagement and audit scheme (emas), which are used by the specific international regulations. they should therefore be cost-effective and appropriate to the size and type of organization and its needs and priorities. the paper also deals with the indicators’ functions, quality and basic categories of environmental performance indicators. the originality of the paper lies in establishing the correlation: environment – environment performance indicators (epi) – organization. keywords: environmental performance indicators; eco-management audit scheme 1. introduction environment protection consists of: environmental programs, objectives and targets, training, incentive schemes, audit frequency, site inspections, administration and community relations. the environmental management and performance of organizations is helped by the selection and use of environmental performance indicators (epi). the guidance no. 2001/680/ec, included in the emas environment project, also contains regulations concerning the selection and use of environmental performance indicators. organizations should make optimum use of the environmental information they collect [1]. to this end the indicators should fulfill the dual purpose of assisting the management of the organization and providing information to stakeholders. getting environmental information can be expensive and time consuming. environmental performance indicators (epi) should therefore be cost-effective and appropriate to the size and type of organization and its needs and priorities. they should address primarily those environmental impacts that are most significant and which the company can influence by its operations, management, activities, products and services. they should also be sensitive enough to reflect significant changes in environmental impacts [2]. depending on an organization’s capabilities and resources, the use of environmental performance indicators may initially be limited to those aspects considered most relevant, with the initial scope being gradually widened over time. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, analysis of the environmental performance indicators for the emas regulation, food and environment safety, volume xii, issue 1 – 2013, pag. 73 – 77 74 organizations should make the optimum use of the environmental information they collect. to this end the indicators should fulfill the dual purpose of assisting the management of the organization and providing information to stakeholders [3, 4]. 2. materials and methods categories of environmental performance indicators the environmental indicator systems are:  comparability: indicators should enable a comparison and show changes in the environmental performance.  balance between problematic (bad) and prospective (good) areas.  continuity: indicators should be based on the same criteria and should be taken over comparable time sections or units.  timeliness: indicators should be updated frequently enough to allow action to be taken.  clarity: indicators should be clear and understandable [5]. usually, three categories of environmental indicators are defined for evaluating and reporting the environmental performance of an organization. a. operational performance indicators (opis). this focus on the aspects associated with an organization’s operations including activities, products or services and can cover such topics as emissions, product and raw material recycling, fuel consumption of vehicle fleet, or energy usage. operational performance indicators can be subdivided into:  input indicators.  physical facilities and equipment indicators.  output indicators. they focus on planning, controlling and monitoring the environmental impacts of the organization’s operations. operational performance indicators are also a tool for communicating environmental data through environmental reports or environmental statements, in accordance with the eco-management and audit scheme (emas) regulation. by integrating cost aspects into them, they furthermore represent a basis for environmental cost management. b. management performance indicators (mpis). these focus on the efforts of management to provide the infrastructure for environmental management to succeed and can, among others, cover environmental programs, objectives and targets, training, incentive schemes, audit frequency, site inspections, administration and community relations. these indicators serve primarily as internal control and information measurements, but do not by themselves provide sufficient information to give an accurate picture of the organization’s environmental performance. c. environmental condition indicators (ecis). these give information on the quality of the environment surrounding the organization or the local, regional or global state of the environment. examples include the water quality of a nearby lake, the regional air quality, concentrations of greenhouse gases or the concentration of certain pollutants in the soil. while they may be quite wide-ranging they can be used to focus the attention of the organization on the management of the environmental aspects associated with significant environmental impacts. the condition of environmental media (air, water, land) and the environmental problems that arise from it often depend on a variety of influences. examples are emissions from different organizations, private households or transport. data about the condition of environmental media are usually measured and recorded by governmental institutions. these data are used to derive specific environmental food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, analysis of the environmental performance indicators for the emas regulation, food and environment safety, volume xii, issue 1 – 2013, pag. 73 – 77 75 indicator systems for the main environmental problems [6-9]. figure 1 presents the basic principles of environmental indicator systems, also the categories of environmental indicators and the correlation levels. figure 1. the basic principles of environmental indicator systems, the categories of environmental indicators and correlation levels opis – operational performance indicators; mpis – management performance indicators; ecis – environmental condition indicators; i) – correlations between the basic principles; ii) – correlations between the categories indicators; iii) – general correlation between the basic principles and categories indicators; iv) punctual correlation between the basic principles and categories indicators. in connection with environmental policy goals, public environmental indicators can be used by organizations as an orientation for setting priorities in determining their own indicators and objectives. this is especially the case when the organization is one of the main sources of an environmental problem, for example the impact of an airport on its surroundings with respect to noise or the impact of a direct effluent discharge on local water quality. for these cases in particular, ecis enable the measurement of environmental impacts of the organization. these three categories of environmental indicators have become widely accepted and organizations should consider a combination of these indicators in order to be able to demonstrate, that:  they understand the environmental impacts associated with their activities, products and services (ecis).  they are taking appropriate measures to ensure the management of environmental aspects associated with the environmental impacts (mpis) and  the result of the management of environmental aspects is improved environmental performance of their operations (opis). the main functions of the environmental performance indicators are:  converting raw data into information that can be easily understood by the intended audience ;  summarizing extensive environmental data to a limited number of significant key information sets;  quantifying and reporting environmental performance;  assisting organisations in the management of their environmental aspects and impacts. the quality of environmental performance indicators consists of:  being cost – effective;  being appropriate to the size and type of organization and its needs and priorities;  being sensitive enough to reflect significant changes in environmental impacts. environmental performance indicators should fulfil a dual purpose:  assisting the management of the organization;  providing information to stakeholders. it is recognized however that for organizations with less significant basic principles categories indicators comparability balance timeliness continuity clarity op m ec i i i i food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, analysis of the environmental performance indicators for the emas regulation, food and environment safety, volume xii, issue 1 – 2013, pag. 73 – 77 76 environmental impacts and a less complex environmental management system the most important indicators will be those relating to operational performance. 3. results and discussion the correlation environment – environmental performance indicators (epi) – organisation the main correlations between the organization and environment are ensured, in order, by: offers data (od); epi and organization management (om). the om must be correlated with the following three parameters: type; measure and priorities of the organization. in figure 2 are presented the main correlations concerning these indicators. figure 2. the correlation environment – environmental performance indicators (epi) – organization epi – environment performance indicators; om – organization management od – offers data 4. conclusions in selecting environmental performance indicators for a particular environmental aspect an organization should ask itself the following questions:  what are the organization’s main environmental aspects and impacts?  where can most improvements be achieved?  where can environmental improvements also lead to cost reduction? the selected environmental indicators should comply with environmental policy priorities:  how does the organization affect the local or regional environmental situation in relation to important local or regional environmental policy issues?  what environmental problems dominate the current political discussions?  what external requirements, for example from interested parties, affect the organization? organizations should select indicators which enhance their management. indicators which do not contribute to the management of the organization will ultimately not be incorporated in day-today management and hence will have little effect in improving performance. in short, only those indicators which enable the employees and management to perform their tasks better are the ones which are most appropriate to the organization. the three categories of environmental indicators have become widely accepted and organizations should consider a combination of these indicators in order to be able to demonstrate, that:  they understand the environmental impacts associated with their activities, products and services (eci);  they are taking appropriate measures to ensure the management of environmental aspects associated with the environmental impacts (mpi);  the results of the management of environmental aspects are improved environmental performance of their operations (opi). organisation  type  measure  priorities e ee e o o o d o food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, analysis of the environmental performance indicators for the emas regulation, food and environment safety, volume xii, issue 1 – 2013, pag. 73 – 77 77 it is recognized however that for organizations with less significant environmental impacts and a less complex environmental management system the most important indicators will be those relating to operational performance. 5. references [1]. ioana, a., nicolae, a., 2001. possibilities of reducing the electric arc furnace’s impact over the environment, cnm’ 2001, bucharest. [2]. barbbit, b., 1999. ecosystem management for sustainability, university of tennessee. [3]. ioana, a., 2007. production management in metallic materials industry. theory and applications, ed. printech, bucharest. [4]. ioana, a., 2007. management elements for optimization of steel elaboration in eaf, fascicle of management and technological engineering, annals of the oradea university, vol. vi (xvi)/2007, 315-319. [5]. ioana, a., nicolae, a. et al, 2002. optimal managing of electric arc furnaces, ed. fairs partners, bucharest. [6]. ioana, a., 1998. the electric arc furnaces (eaf) functional and technological performances with the preheating of the load and powder blowing optimisation for the high quality steel processing, doctoral degree paper, university „politehnica” of bucharest, bucharest. [7]. ***, 2003. official journal of the european union, brussels. [8]. ***, http://www.ec.europa.eu.com microsoft word 13 murariu_gontariu lucrare.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 82 cold to ler an ce in eu ro pean o a t gen etic res ou rc es danela murariu1, marius murariu2, *ioan gontariu3 1suceava genebank , b-dul 1 decembrie 1918, nr. 17, suceava, romania, e-mail dmurariu@suceava.astral.ro 2agricultural research and development station of suceava, b-dul 1 decembrie 1918 nr. 17 suceava, romania, e-mail marius_murariu2005@yahoo.com 3faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania, e-mail ioang@fia.usv.ro *corresponding author received 19 january 2012, accepted 20 february 2012 abstract: oat is a crop with an important european history and tradition. the high value of oat in human nutrition, which is unique among cereals, is widely recognized and confirmed by health claims issued in various countries. it is based on a high content and quality of proteins, considerable content of fat with high proportion of polyunsaturated fatty acids, high contents of dietary fiber, especially the soluble, highly viscous mixed linked (1->3)(1->4)-ß-d-glucans, which hypo-cholesterolemic effects, minerals and antioxidants, especially tocopherols, tocotrienols and avenanthramides. oat is considered moderately susceptible to fusarium spp. infection and mycotoxin contamination. nevertheless t2/ht-2 toxins raise considerable concerns in the european health administration (european commission, 2006). among winter cereals, oat is the most frost sensitive and its insufficient level of winterhardiness is the most important factor limiting the sowing of winter oat in northern and central europe (cattivelli et al., 1998). resistance to low temperatures, which limited yield potential and sustainable oat growing in many major oat growing regions in europe, was the main aspect which have been evaluated in this paper. the results of the evaluation of frost tolerance at first leaf stage, accomplished in the laboratory of experimental institute for cereal research, fiorenzuola d’arda, italy were compared with the data of winter hardiness obtained in the field experiments carried out by suceava genebank in environments more frequently subjected to severe frost events, as compared to the italian site keywords: cold tolerance, winter hardiness, genotypes 1. introduction in order to improve the utilisation of the oat germplasm collection in breeding programs, apart from biochemical and molecular characterisation, an extensive evaluation of important agronomic traits is also required. resistance to abiotic stress is a crucial aspect for cultivar adaptation to agriculture environments. among winter cereals, oat is the most frost sensitive and its insufficient level of winterhardiness is the most important factor limiting the sowing of winter oat in northern and central europe [1]. winter hardiness can be defined as the ability to survive throughout the winter. by the virtue of the wide range of stressful conditions that a plant may experience during the cold season, winter hardiness is a complex trait. freezing temperature is the most relevant stress factor, although other stress situations, such as anoxia due to excess water or to ice encasement and photoinhibition due to the combination of light and low temperature, may also occur. in temperate areas, winter cultivars are preferred, whenever possible, as winter food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 83 varieties are higher-yielding than spring ones, and the identification of new gene sources for frost tolerance is still an important task for oat improvement [2]. despite the fact that genetic diversity has also been observed for freezing tolerance of non-acclimated barley plants, the ability of overwintering plants to withstand cold is mainly based on an adaptive response, known as cold acclimation or hardening, activated during growth at low nonfreezing temperatures. frost resistance can be assessed through field evaluation methods, a strategy depending of the occurrence of natural conditions which satisfactorily differentiated genotypes, or through artificial freezing tests such as percentage of post-stress survival, lt50 (temperature at which 50% of the population is killed), integrity of cell membranes after freezing [3], evaluation of the fv/fm ratio the maximum quantum yield of the psii photochemistry after freezing [4]. a number of physiological traits such as proline accumulation, aba and crown fructan content have been associated with the development of frost tolerance. generally, the amount of these metabolites increases during acclimation, being higher in frost-tolerant genotypes versus frostsusceptible ones [5]. a comparison of a number of frost resistance evaluation methods in different crop species was carried out by [6]. the value of the laboratory tests appears to vary with the plant species, although for cereals frost resistance evaluation methods based on freezing assays provide the highest correlation with field survival. 2. materials and methods winter hardiness was assessed in a field experiment at suceava for 104 oat accessions (including 9 standards mures, ivory, jaak, argentina, mina, belinda, saul, genziana, auteuil). the seeds were sown manually into one row of 1m length (50 seeds per row) in two replications with a distance between accessions of 25 cm. time of sowing was october 1st 2009. the following data were recorded:  number of emerged seeds.  number of survived plants after winter  the field injury have been visually estimated on a 0-9 scale according to rizza et al. (1994), where: 0: no damage; 1: slightly yellowed leaf tips; 2: half yellowed basal leaves; 3: fully yellowed basal leaves; 4: whole plants slightly yellowed; 5: whole plants yellowed and some plants withered; 6: whole plants yellowed and 10% plant mortality; 7: whole plants yellowed and 20% plant mortality; 8: whole plants yellowed and 50% plant mortality; 9: all plants killed.  the environmental conditions were registered in the target period (october 2009 – march 2010). all obtained results in the field were compared with obtained results in the laboratory of the experimental institute for cereal research, fiorenzuola d’arda, italy the used method for frost tolerance was the chlorophyll fluorescence evaluation (fv/fm) on a set of 104 oat cultivars, at the temperature on -10°c and -12°c. 3. results and discussion a minimum air temperature of -24.7°c (-31°c soil temperature) was measured in january and severe freezing conditions persisted from december 2009 to march 2010 (table1). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 84 table 1 the decadal temperatures registered at suceava meterological centre, romania, during period october 2009march 2010 high damage occurred for most accessions. for 91 out of 104 accessions all plants were killed showing a visual score of 9 (table 2): for 8 accessions a score of 8 (whole plants yellowed and 50% plant mortality) was recorded. only five accessions were identified as superior for winter hardiness capacity showing a value october, 2009 aer temperatures soil temepratures p d ec ad es average max min 7 h. a.m avera ge max min rh (%) sun light (h) mm decade 1 12.4 19.3 7.3 8.7 12.2 23.4 5.9 79 39.0 12.4 decade 2 6.0 10.3 2.5 4.4 5.9 12.3 2.1 83 27.7 35.2 decade 3 7.8 11.8 5.0 5.1 7.6 14.6 4.0 86 40.4 15.0 monthly average 8.7 13.7 4.9 6.0 8.5 16.7 4.0 83 107.1 62.6 november 2009 decade 1 3.3 5.5 1.4 2.7 3.6 6.4 1.8 93 4.4 7.9 decade 2 6.8 10.5 3.7 4.2 6.0 10.8 3.0 87 22.3 4.4 decade 3 6.5 12.8 2.2 1.0 4.3 12.3 0.1 80 50.2 6.3 monthly average 5.5 9.6 2.4 2.6 4.7 9.8 1.6 87 76.9 18.6 december 2009 decade 1 2.4 4.7 0.6 1.4 2.5 5.2 0.9 97 9.2 0.2 decade 2 -8.0 -6.1 -9.8 -1.2 -0.9 -0.2 -1.3 93 6.0 20.9 decade 3 -1.3 2.4 -5.3 -4.1 -2.8 1.4 -5.9 87 22.7 16.0 monthly average -2.3 0.4 -4.8 -1.4 -0.5 2.1 -2.2 92 37.9 37.1 january 2010 decade 1 -2.4 0.0 -4.6 -3.8 -2.8 -0.6 -5.2 96 3.8 8.7 decade 2 -5.1 -3.7 -5.8 -5.0 -4.4 -2.3 -6.0 97 0.0 19.0 decade 3 -13.1 -9.2 -16.9 -17.5 -13.5 -6.0 -20.3 86 30.3 3.7 monthly average -7.0 -4.4 -9.4 -9.1 -7.1 -3.1 -10.8 93 34.1 31.4 february 2010 decade 1 -7.9 -4.3 -11.4 -12.3 -8.9 -2.6 -14.6 87 36.5 1.4 decade 2 -2.1 0.8 -5.3 -4.6 -2.6 1.6 -6.6 98 15.4 30.0 decade 3 0.3 4.4 -2.0 -2.3 -0.6 3.4 -3.4 93 18.6 3.8 monthly average -3.5 0.0 -6.5 -6.7 -4.3 0.6 -8.5 92 70.5 35.2 march 2010 decade 1 -2.9 1.3 -5.8 -5.9 -3.2 2.7 -8.4 86 27.4 decade 2 0.6 5.8 -4.2 -3.9 -0.9 6.0 -7.0 73 49.6 2.2 decade 3 9.2 15.5 4.3 5.6 8.7 18.1 2.6 69 81.9 16.8 monthly average 2.6 7.8 -1.7 -1.2 1.8 9.2 -4.0 76 158.9 28.6 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 85 of 6 (plants yellowed and up to 10% plant mortality) for 83/200-cr (coming from bulgaria) and local 7 (coming from russia) and value of 7 (whole plants yellowed and between 10 to 20% plant mortality) for millennium (coming from great britain), ava and donata (coming from italy). table 2 the winter hardiness capacity of oat analysed genotypes st at us o f s am pl e g en ot yp e n o. o f e m er ge d se ed s n o. o f s ur vi va l pl an ts a ft er w in te r sc or e ac co rd in g r iz za , 1 99 4, im m ed ia te ly a ft er w in te r st at us o f s am pl e g en ot yp e n o. o f e m er ge d se ed s n o. o f s ur vi va l pl an ts a ft er w in te r sc or e ac co rd in g r iz za , 1 99 4, im m ed ia te ly a ft er w in te r modern cavallo 14.5 0 9 modern raven 19.5 0 9 modern monarch 14.5 0 9 modern flaming 30 0 9 modern efesos 18.5 0 9 modern flamingsg 21 0 9 modern effektiv 21.5 0 9 modern nelson 27 0 9 modern ehostar 21 0 9 standard ivory 34 0.5 9 standard mina 19.5 0 9 modern tyfhon 20.5 0 9 cultivated 83/200-c 11.5 8.5 6 modern pergamon 18 0 9 cultivated bgr 250 17.5 0 9 cultivated konradin 14.5 0 9 cultivated sofia 121 14 0 9 cultivated nuernberg 25.5 0 9 cultivated bgr 7982 22 3.5 8 cultivated mayer a 22.5 0 9 modern veli 11 0 9 cultivated lueneburg 31.5 0 9 cultivated brnensky 20.5 1 8 modern sandokan 32.5 0 9 modern abel 21 0 9 modern kaplan 25.5 0 9 modern auron 22 0 9 modern leo 22 0 9 modern neklan 16.5 0 9 cultivated alo 29.5 0 9 modern istra 14.5 0.5 9 modern nelson 27 0 9 modern izak 21.5 0 9 cultivated platek 18.5 0 9 cultivated dalimil 19.5 0 9 modern breton 15 0 9 standard saul 17 0 9 modern cwal 9.5 0 9 cultivated konradin 14.5 0 9 modern hetman 3 0 9 cultivated nuernberg 25.5 0 9 modern sam 11.5 0 9 cultivated mayer a 22.5 0 9 cultivated acmariu 3 29.5 0 9 cultivated lueneburg 31.5 0 9 standard mures 30.5 0 9 modern sandokan 32.5 0 9 cultivated sacel 25 0 9 modern kaplan 25.5 0 9 cultivated lunca il 9.5 0 9 modern leo 22 0 9 cultivated baisoara 1 24.5 0 9 cultivated alo 29.5 0 9 cultivated local 1 21 0 9 standard jaak 37 0 9 cultivated winterhaf 17 0 9 cultivated jogeva 33 0 9 cultivated local 2 16.5 0 9 modern miku 26 0 9 cultivated local 3 10.5 0 9 modern villu 24.5 0 9 cultivated local 4 19.5 0.5 9 cultivated noire 31 0.5 9 cultivated mulyat 22.5 0 9 cultivated noire ri. 15 0 9 cultivated jari oves 28.5 0 9 cultivated neu gro 14 2 8 cultivated beltckii 1 23.5 0 9 cultivated grise 31.5 0 9 cultivated kinelskjj 29 1.5 8 cultivated jaune 16 0 9 cultivated local 5 20 0.5 9 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 86 the results of the evaluation of frost tolerance at first leaf stage, accomplished in the laboratory of experimental institute for cereal research, fiorenzuola d’arda, italy were compared with the data of winter hardiness obtained in the field experiments carried out by suceava genebank in environments more frequently subjected to severe frost events, as compared to the italian site. correlation coefficients (r) are reported in table 3 between the frost tolerance data obtained in two independent experiments under controlled conditions at stress temperature of -10 and -12°c and the winter hardiness evaluated during 20092010 in suceava, romania, (by visual score and number of plants that survived) the correlations between the different evaluations of winter hardiness are also reported to compare the results obtained in the different fields and with different methods. table 3 correlations of frost tolerance results on the oat accessions analyzed in the laboratory conditions (experimental institute for cereal research, fiorenzuola d’arda, italy) and in field conditions (suceava genebank) (2009 – 2010) winterhardiness suceava genebank field 2009-10 visual score (0-9 rizza et al.) winterhardiness suceava genebank field 200910) number survived plant frost tolerance fv/fm means two experiments (-10 and -12°c) -0.447*** 0.432*** winterhardiness suceava genebank field 2009-10 visual score (0-9 rizza et al.) -0.951*** *,**,*** significant at the 0.05, 0.01 and 0.001 probability level, respectively; ns non significant ( 0.05 level) cultivated joanette 26.5 0 9 cultivated local 6 33.5 0 9 modern chantilly 23.5 0 9 cultivated local 7 19 12.5 6 standard auteuil 32 0 9 cultivated signal 26.5 0 9 modern aintree 0 0 9 cultivated anchar 10 0 9 modern lennon 11 0 9 cultivated vendelin 22.5 0 9 modern millenium 10.5 5.5 7 cultivated gagybator 21 3 8 modern ava 10.5 4 7 modern detvan 8.5 0 9 modern donata 12.5 4.5 7 cultivated zvolen 14.5 0 9 modern primula 9.5 0 9 cultivated svkpol 16.5 0.5 9 modern bionda 9.5 2 8 cultivated ukrkar 25.5 0 9 modern teo bd40 16.5 0 9 cultivated sisko 26 0 9 standard gentiana 13 2.5 8 cultivated fyris 16.5 0 9 modern novella 6.5 0 9 cultivated seger li 13 0 9 standard arg 14.5 2 8 cultivated purhavre 12 0 9 cultivated 1404-11 15 0 9 cultivated nidar li 24.5 0 9 modern jaugila 17.5 0 9 modern cilla 27 0 9 cultivated stendes 21.5 0 9 standard belinda 26 0 9 cultivated pulawski 28 0 9 modern sw betw 12 0 9 cultivated gorski 25.5 0 9 modern kentucky 22 0 9 cultivated persidskij 13.5 0 9 modern sw iborg 20.5 0 9 cultivated platek 18.5 0 9 cultivated omskij 27.5 0 9 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 87 4. conclusion 1. the high severity of the conditions recorded in the site of suceava genebank did not allow to record high variability among the accessions (visual score 9 for 87.5% of the accessions) but it was very useful in showing few genotypes with plant survival (6, 7) that were identified as superior for frost tolerance also in the laboratory tests recorded in the experimental institute for cereal research, fiorenzuola d’arda, italy. 2. results obtained from laboratory tests on frost tolerance of 104 accessions were highly correlated with data for winter hardiness obtained in romania for the same group of accessions. 5. references [1]. cattivelli l., crosatti, c., rizza f., 1998, oats. in: italian contribution to plant genetics and breeding. (g.t. scarascia-mugnozza and m.a. pagnotta, eds.). tipografia quatrini a. & f. snc viterbo, italy, publisher. pp. 257-308. [2]. stanca a m, romagosa i, takeda k, lundborg t, terzi v, cattivelli l, 2003, diversity in abiotic stresses. in: von bothmer r, knüpffer h, van hintum t, sato k (eds.) diversity in barley (hordeum vulgare l.). elsevier, pp. 179-199. [3]. rizza f., crosatti c., stanca a. m., cattivelli l., 1994 studies for assessing the influence of hardening on cold tolerance of barley genotypes. euphytica, 75: 131-138. [4]. rizza f., pagani d., stanca a. m., cattivelli l., 2001 use of chlorophyll fluorescence to evaluate the cold acclimation and freezing tolerance of winter and spring oats. plant breeding, 120: 389396. [5]. murelli c., rizza f., marinone f., albini a., dulio v., terzi v., and cattivelli l, 1995, metabolic changes associated with coldacclimation in contrasting cultivars of barley. physiol. plant. 94: 87-93. [6]. pulli s., hjortsholm k., larsen a., gudleifsson b., larsson s., kristiansson b., hömmö l., tronsmo a.m., ruuth p., and kristensson c., 1996, development and evaluation of laboratory testing methods for winterhardiness breeding. nordic gene bank. 156 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 156 160 comparative study of oxidative stability for different types of vegetable oils *sonia amariei (gutt)1, elena sănduleac (todosi sănduleac)1, simona ciornei (ștefăroi)1 1faculty of food engineering, ştefan cel mare university of suceava, 13 universităţii str., 720229, suceava, romania gutts@fia.usv.ro, sanduleacelena@yahoo.com, simona_stefaroi@yahoo.com * corresponding author received april 18th 2013, accepted may 29th 2013 abstract: use of oils in the diet requires their protection against oxidation under conditions of high temperature and light. the expiration date is different depending on the type of oil. the presence of light and heat affects its quality: acid value, peroxide value and antioxidant capacity due to the presence of natural oils, antioxidants such as tocopherols, polyphenols, vitamin c, and so on, or artificial, added during the technological processes of obtaining them. changes in acid value and peroxide may be linked to the antioxidant content assessed by measuring antioxidant activity by dpph method. oxidative stability of sunflower oil, corn oil, extra virgin olive oil, grape seed oil and nut oil was studied before and after keeping them in oven for 5 days at 50 º c. under these conditions of accelerated oxidation, oxidative stability was evaluated by measuring peroxide value, antioxidant activity of free acidity. from analyzes it came out that coconut oil has the highest antioxidant capacity and the lowest variation of peroxide value and free acidity in mentioned conditions. keywords: vegetable oils, antioxidant activity, peroxide value, free acidity, 1. introduction free radicals are atoms or molecules that have an unpaired electron with a high chemical instability and aggression, able to react with other molecular structures. their presence was associated with a lot of diseases such as cancer, autoimmune cardiovascular, pulmonary, rheumatic diseases, and nervous system disorders, [1].therefore, eating non-oxidized food and antioxidants consumption play an important role in protecting against these degenerative processes. extra-virgin olive oil produced by the application of mechanical pressure on the fruit olea europea l., the high proportion of monounsaturated fatty acids, for example oleic acid, and the modest presence of polyunsaturated fatty acids contain natural antioxidants such as tocopherols, carotenoids, sterols, and phenolic compounds [1]. the effects of polyphenols and tocopherols on oxidative stability during oil storage and the processes of heating have been extensively evaluated in different studies [1]. oil walnut (juglans regia l.), by the content of essential fatty acids is a good source of monounsaturated fatty acids (oleic acid mainly) and polyunsaturated fatty acids (linoleic and alpha-linolenic acid) [3]. oil walnut (juglans regia l.) by the content of essential fatty acids is a good source of monounsaturated fatty acids food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), elena sănduleac (todosi sănduleac), simona ciornei (ștefăroi), comparative study of oxidative stability for different types of vegetable oils, food and environment safety, volume xii, issue 2 – 2013, pag. 156-160 157 (oleic acid mainly) and polyunsaturated fatty acids (linoleic and alpha-linolenic acid) [3]. according to the study of simopoulos (2002) walnut oil has a perfect balance of n-6: n-3 polyunsaturated acids, a 4:1 ratio, which has been shown to reduce the incidence of cardiovascular risk [3]. sunflower oil has about 70% linoleic acid [2] and is very sensitive to lipid oxidation [2]. polyunsaturated acids, are exposed to environmental factors such as air, light and temperature oxidation reactions produce undesirable flavors, odors and rancid, discoloration and other forms of deterioration. primary autoxidation products are hydroperoxides, having no taste or flavor, but their degradation products (aldehydes, ketones) have very strong taste and aroma are changed [2]. besides their chemical composition, the susceptibility to oxidation of oils depends on the processing, packaging and storage conditions. in a recent paper [2] it was established that, in general, oils are highly susceptible to degradation in the presence of light. oils stored in transparent bottles, exposed to fluorescent light (1100 lux) and ambient temperature, without addition of any antioxidant, could maintain acceptable quality for up to two months of storage, a very short shelf life. although synthetic antioxidants are widely used as food additives, their safety has been questioned [4] the boost search of natural resources, of compounds with antioxidant properties has taken a large amplitude. free radicals concentration and the amount of antioxidants in lipids are important factors for predicting oil stability. dpph method for estimating the stability of edible oils can be applied to determine the antioxidant activity of fat-soluble compounds. the objective of this study is to compare the thermal oxidative stability of sunflower oil, corn oil, grape seed oil, extra virgin olive oil and coconut oil. 2. experimental the oils used in this study were purchased commercially (kaufland store suceava) and herbal pharmacies inside this store: sunflower oil from "unisol" valid until 11.07.2013, corn oil for frying, valid until 07.25.2013, extra virgin olive oil obtained by cold pressing from "solaris" valid until 10.16.2014, grape seed oil, packed in sleeves, available until march 2014, walnut oil packed in manic, validity until march 2014. the reagents used for chemical determinations were analytically pure reagents and laboratory equipment used belongs to the faculty of food engineering suceava: oven spectrophotometer ocean optics fiber optic, laboratory utensils. the oils were stored in an oven for 5 days at a temperature of 50 ° c, in the presence of light and atmospheric oxygen. the methods used to determine their stability was: titrimetric method (peroxide index, acidity) and spectrophotometric methods (antioxidant capacity, dpph method). 2.1. determination of antioxidant activity of oils by spectrophotometric method dpph (2,2-diphenyl-1-picrilhidrazil) dpph is one of the most stable organic radicals and commercially available and has a maximum absorption of 515 nm in the vis. when the solution is discolored and reduced forward, the reaction is monitored spectrophotometrically (spectrophotometer ocean optics). in this way we can evaluate the antioxidant capacity of a system. inhibition % = 100 [(at5/ at0) * 100 in which: at0 dpph is the absorbance at t = 0 min. at5 is the absorbance of dpph + oil sample after t = 5 min. titration methods for the determination of peroxide value and acidity were compliant with the standards in force. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), elena sănduleac (todosi sănduleac), simona ciornei (ștefăroi), comparative study of oxidative stability for different types of vegetable oils, food and environment safety, volume xii, issue 2 – 2013, pag. 156-160 158 2.2. determination of peroxide-standard sr en iso 3960/2009 reagents and tools required: -bottles of 250 ml dry glass stopper, chloroform, glacial aceticmacid,ki, saturated ,na2s2o3 solution, 0.1 n starch 1% solution mode: more than ~ 5 g of an oil, weighing analytical balance, was added 12 ml of chloroform, 18 ml of glacial acetic acid and 1 ml of ki, saturated. close the bottle, shake 1 minute, allow to stand 15 minutes, then add 30 ml distilled water. in parallel is prepared the blank. titrate the liberated i2 with sodium thiosulfate solution, 0.1 n, in the presence of starch as an indicator. calculations and interpretation of results: i.p= 1000 (meq/kg) m-mass of the sample (g) v1volume na2s2o3 (sample) v2volume na2s2o3 (blank) 2.3. determination of acidity oil according to nf iso 660-1999 mode: over ~ 5g sample plus solvent is titrated with koh solution in the presence of phenolphthalein as indicator. principle of the method: free fatty acids contained in a known volume of oil are titrated with alcoholic koh solution of known concentration, using phenolphthalein as an indicator. 56.11 the number of mg of koh contained in one milliliter of solution of koh,0,1n. n-normality of the koh v, the amount of koh 0.1 n used in the titration the amount of oil to be taken of the sample, g in which:nnormality of na2s2o3 solution method of calculation: acidity index= 56,11*n/m [mgkoh / g sample] titratable acidity: 0.1% -0.4% admitted values expressed as oleic acid. acid value is twice acidity. table 1. results from initial samples type of oil acidity (%) rds (%) peroxid value ( meq/kg) rds (%) antioxidant activity (%) rds (%) sunflower oil 0.165 1.365 3.51 1.986 20 1.876 corn oil 1.94 1.895 2.38 2.132 42 1.753 extra virgin olive oil 0.61 1.257 11.34 1.587 24 2.233 grape seed oil 0.28 2.008 10.24 1.180 10 1.540 coconut oil 18.5 1.789 19.1 1.711 49 1.999 table 2. results before the storage oven 5 days, 50 º c type of oil acidity (%) rds (%) peroxid value ( meq/kg) rds (%) antioxidant activity (%) rds (%) sunflower oil 0.24 1.664 8.15 1.956 10 1.567 corn oil 2.05 1.887 3.062 1.833 12 1.775 extra virgin olive oil 0.64 1.341 12.06 2.090 6 1.980 grape seed oil 0.5 2.213 11.9 1.893 4 1.342 coconut oil 19.2 1.891 20.3 1.333 15 2.099 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), elena sănduleac (todosi sănduleac), simona ciornei (ștefăroi), comparative study of oxidative stability for different types of vegetable oils, food and environment safety, volume xii, issue 2 – 2013, pag. 156-160 159 fig. 1. variation in acidity fig. 2. variation peroxide value fig. 3. variation in antioxidant activity 3. results and discussion the presence of natural antioxidant (polyphenols, tocopherols, vitamin c) in vegetable oils like sunflower oil, corn oil, olive oil, grape seed oil and walnut, influenced changes in the peroxide and thus free acidity. table 1 and table 2 contain the results of peroxide value, free acidity and antioxidant activity of the oils, before and after accelerated storage for 5 days at 50 º c in an oven. for the sunflower oil the peroxide value increased after accelerated storage by 132% from 3.51 eq / kg 8.15 eq / kg, titratable acidity rose with 45.45% from 0.165% to 0.24 %, and the antioxidant activity decreased due to the influence of light and heat by 50%, from 20% to 10%. corn oil and peroxide changed to 41.26% from 2.38 eq / kg 3062 eq / kg, a free acidity increased by 5.67% from 1.94% to 2.05%, and the antioxidant activity decreased by 71.42%, from 42% to 12%. extra virgin olive oil has modified peroxide value with 6.34% from 11.34 meq / kg 12.06 meq / kg, free acidity increased with 4.91% from 0.61% to 0, 64%, and the antioxidant activity decreased by 75%, from 24% to 6%. grape seed oil has modified peroxide value with 16.21% from 10.24 meq / kg to 11.9 meq / kg, free acidity increased with 78.57% from 0.28% to 0,5%, and the antioxidant activity decreased by 60% from 10% to 4%. coconut oil has modified peroxide value with 6.28% from 19.1 meq / kg to 20.3 meq / kg, free acidity increased from 3.78% to 18.5% at 19 2% and the antioxidant activity decreased by 69.38% from 49% to 15%. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), elena sănduleac (todosi sănduleac), simona ciornei (ștefăroi), comparative study of oxidative stability for different types of vegetable oils, food and environment safety, volume xii, issue 2 – 2013, pag. 156-160 160 4. conclusion deterioration resulting from oxidative processes that take place in the seed, in the course of manufacturing steps, as well as during storage, reduces the nutritional value of the oil and results in additional losses relating to the need for their reconditioning. edible oils with higher content of unsaturated fatty acids, especially polyunsaturated fatty acids are more susceptible to oxidation. lipid oxidation of oils can produce not only rancid odors, flavors and color changes, but it can also reduce the quality of nutritional values and safety due to the degradation of the products, with harmful effects on human health. as a result of the oxidative degradation appears the taste of "rancid" due to the presence of secondary compounds found in very small amounts, usually less than 0.1% and sometimes up to 1%. of the oils analyzed, the walnut oil has the highest antioxidant capacity and the best chemical stability. it follow olive oil, grape seed oil, corn oil, sunflower oil, order that reflects their chemical composition and they recommended to be used. 5. references [1]. silva l. , pinto j. , carrola j. , paiva-martins f. , oxidative stability of olive oil after food processing and comparison with other vegetable oils, food chemistry 121 (2010) 1177–1187. [2]. smith s. a., king r. e., min d.b., oxidative and thermal stabilities of genetically modified high oleic sunflower oil, food chemistry 102 (2007) 1208–1213. [3]. martínez m. l. , penci ma. c, ixtaina v. , ribotta p. d. , maestri d. , effect of natural and synthetic antioxidants on the oxidative stability of walnut oil under different storage conditions. [4]. lee j. , chung h. , chang p. s. , jaehwan s. , development of a method predicting the oxidative stability of edible oils using 2,2-diphenyl-1-picrylhydrazyl (dpph) [5]. silva l., garcia b., martins f. p. , oxidative stability of olive oil and its polyphenolic compounds after boiling vegetable process. [6]. albi t., lanzón a., guinda a. , león m. , pérez-camino m. c. (1997). microwaveand conventional heating effects on thermoxidative degradation of edible fats. journal of agriculture and food chemistry, 45, 37953798. [7]. baldioli m., servili m., perreti g., montedoro g. f. (1996). antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. journal of the american oil chemistry society, 73, 1589-1593. [8]. beltrán-maza g., jiménezmárquez a., garcía-mesa j. a., fríasruiz l. (1998). evolution of extra virgin olive oil natural antioxidants during continuous frying.grasas y aceites, 49, 372-373. [9]. brand-williams w., cuvelier, m. e., berset c. (1995). use of a free radical method to evaluate antioxidant activity. lebensmittel-wissenschaft und-technologie, 28, 25-30. [10]. nf iso 660-1999, determination of oils acidity [11]. sr en iso 3960/2009, determination of peroxide value 130 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 130 134 researches regarding development body and cutting reports at oncorhynchus mykiss species *gabriel vasile hoha1, ionut bogdan pagu1, catalin emilian nistor1, emanuel măgdici1, benone păsărin1 1university of agricultural sciences and veterinary medicine, animal science faculty, 8 mihail sadoveanu alley, 700489, iaşi, romania, gabihoha@yahoo.com * corresponding author received january 8th 2013, accepted april 15th 2013 abstract: from fish are important for human consumption only certain body regions. from trout we are interested by the muscular tissue which has the greatest proportion. the purpose of this research is to analyze the development of body and cutting reports oncorhynchus mykiss species of different ages (one year, two year and three year). the best efficiency at slaughter yield, respectively 82.15% was recorded at the third batch of rainbow trout, while at the first batch we obtain the lower 78.96%. setting component parts indicate an increase of the trunk beside total body weight, at a rate of 66.63% for trout aged 1 year to 72.69% at 3 years old trout. visceral mass accounted for 18.45% of trout with a yearling trout beside 14.25% at 3 years old. visceral mass accounted for 18.25% at aged 1 year trout beside 14.45% at 3 years old trout. keywords: trout, body, cutting, oncorhynchus mykiss 1. introduction salmoniculture is the second important branch of pisciculture, and assure the diversification of food products and proteins with a high biological value through trout destined for humans’ consumption, very easy digestible products. because worked hard to improve the production of salmonids species today are produced trouts throughout the year, in a relatively short time (compared with cyprinids) and increasing density [1]. from the angle of rainbow trout meat chemical composition, must mention mainly the protein content, which at this breed is higher in comparison with meat gathered from other fish breeds [2]. researches say that fish and fish products, even in low quantities, could have a positive impact in diet improving with proteins, by supplementation with essential amino acids (lysine and methionine, which are in present small quantities of the diets based on vegetable products. trout meat have a high nutritive value, offered by the high content in proteins, lipids with a high unsaturated degree, together with lipo and hidrosolubles, content in mineral salts (which include a, d vitamins, b group vitamins, phosphorous, magnesium, selenium, cobalt and iodine) [3, 4]. fish meat quality, generally, and trout meat, especially, justifies the increased demand of those type of meat on market, face to these breed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 gabriel vasile hoha, ionut bogdan pagu, catalin emilian nistor, emanuel măgdici, benone păsărin researches regarding development body and cutting reports at oncorhynchus mykiss species, food and environment safety, volume xii, issue 2 – 2013, pag. 130 134 131 rainbow trout is one of the most important salmonids species of interest in commercial culture worldwide and trout feed costs account for over 40% of production costs [5]. 2. materials and methods the current research were realized on three batches of oncorhynchus mykiss (30 individuals/batch), of different ages (one year, two year and three year) growth in the specific environmental conditions from ne area of romania. during research trout from the three batches were grown in identical environment conditions, being slaughtered in april 2012, when were determined: body length; the maximum circumference of the body; live weight; weight of carcass; slaughter yield (%); participation of the slaughtered parts in carcass (head, body, fins, skin and bones) and the weight of visceral mass and internal organs (liver, kidneys and heart) for all three age categories (one year, two year and three year). biometric data used to determine the increase in length of the fish and to determine general physiological condition are obtained by measurements made using special tools (ihtiometers) or other measuring tools (ruler, caliper) [1, 2]: total body length is measured from the tip of the snout to the line joining the tips of the caudal fin lobes; maximum body circumference measured at the maximum thickness and maximum height, before ridge. live weight and weight of carcass were determinate on live individuals, measured by weighting with the help of kern precision balance. determination of rainbow trout live weight was realized at the fishery unit after maximum one hour from gathering, and for determination of carcasses weight after slaughter and refrigeration after 24 hours at 40c. the obtained data were used for concretion of slaughter yield, reported as a rate between the weight of carcass and live weight of studied fishes. to determinate slaughter yield were used the following formulas: r (%) = (carcass weight : live weight) × 100 in the case of rainbow trout breed (oncorhynchus mykiss) carcass is represented by fish body which was eviscerated and removed the gills. evisceration is the process from which are removed the uneatable parts from inside the trout and have the role to prolonged the preservation period of fish and to offer presentation possibilities of the final product in very good conditions. this operation must be effectuated quickly because in fish body starts the proteolysis processes. evisceration process implies opening of fish along median ventral line, from gills level up to anal orifice. were effectuated gravimetric measurements regarding participation of sliced portions in carcass’ structure (head, body, fins, skin). fish is considered to be more valuable as much as the gravimetric composition is in the favor of eatable parts. research involved also the establishment of weight of internal organs and visceral mass. the gathered organs were: liver, kidneys and heart. kidneys which at oncorhynchus mykiss breed are placed at the level of backbone were removed with the help of a spatula. the rate of the organs for fish corporal mass have a significant influence on slaughter yield, this one being different food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 gabriel vasile hoha, ionut bogdan pagu, catalin emilian nistor, emanuel măgdici, benone păsărin researches regarding development body and cutting reports at oncorhynchus mykiss species, food and environment safety, volume xii, issue 2 – 2013, pag. 130 134 132 function of corporal dimensions, and also by the development of trout skeleton. the obtained data after gravimetric measurements were statistically processed by calculating the values of some statistical estimators, appreciation of variance, its limits, differences and their signification. 3. results and discussion the research began with biometric measurements: total body length, maximum body circumference and live weight. after measurements was calculated an average of 30 trouts in each experimental batch, the results are presented in table 1. table 1. size and body weight average in oncorhynchus mykiss specification batch l1 batch l2 batch l3 x v% limits x v% limits x v% limits body length (cm) 18.5 12.35 16.421.3 27.30 13.10 25.132.4 38.73 11.54 28.3 46.4 body circumference (cm) 9.4 8.70 8.2-11.3 15.30 9.20 13.3 – 17.4 23.17 11.21 18.5 28.2 live weight (g) 171.2 11.22 142184 540.70 14.70 460590 853.90 17.48 7251124 from measurements made to rainbow trout specimens analyzed were obtained values close to those presented in the literature consulted [1, 2, 3], which demonstrates that experimental batches had appropriate developments. coefficients of variation, lower to batches l1 and l2 and higher to batch l3, revealed every time a good homogeneity of the three groups of trout analyzed. after making measurements and weighing, trouts from each experimental group were slaughtered and eviscerated being weighed before and after evisceration. based on live weight and carcass weight was calculated slaughter yield, the results are presented in table 2. table 2. slaughter yield calculated for oncorhynchus mykiss species specification batch l1 batch l2 batch l3 x v% x v% x v% live weight (g) 171.20 11.22 540.70 14.70 853.90 17.48 weight of carcass (g) 135.18 12.47 435.25 13.40 701.50 13.71 slaughter yield (%) 78.96 9.40 80.49 10.20 82.15 11.42 the data presented in table 2 shows great values of yield slaughter which increases according to the age of the experimental trout. the best values was recorded to the batch l3 (82.15%) followed by batch l2 (80.49%) and batch l1 (78.96%). the coefficient of variation of yield at slaughter had values of 10-11% indicating a good homogeneity for experimental batches. the calculated values for slaughter yield at all three batches are in the limits cited in the literature, 66.56% – 86.48 % [1, 2, 4, 6]. the main components of body fishes are the head, trunk, visceral organs and internals organs. results obtained in the experimental batches are presented in table 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 gabriel vasile hoha, ionut bogdan pagu, catalin emilian nistor, emanuel măgdici, benone păsărin researches regarding development body and cutting reports at oncorhynchus mykiss species, food and environment safety, volume xii, issue 2 – 2013, pag. 130 134 133 table 3. the weight of internal organs and visceral mass at oncorhynchus mykiss breed specification batch l1 batch l2 batch l3 x v% x v% x v% live weight (g) 171.20 11.22 540.70 14.70 853.90 17.48 head (g) 21.14 4.87 50.13 5.13 80.78 7.29 % 12.34 5.03 9.27 5.19 9.46 7.09 trunk (g) total, of which: 114.04 10.43 385.12 11.24 620.72 12.53 % 66.63 11.54 71.23 11.35 72,69 12.62 fins 3.12 3.26 6.12 5.82 8.29 7.21 tegument 10.47 5.42 27.15 5.61 42.17 8.23 bones 8.12 4.67 21.32 5.58 33.14 7.25 visceral mass (g) 31.25 9.25 88.30 11.42 123.47 12.35 % 18.25 9.28 16.33 11.51 14.45 12.42 internal organs (g) total, of which: 4.77 5.17 17.15 5.45 28.93 6.49 % 2.78 5.21 3.17 5.52 3.38 6.56 liver 2.54 5.21 10.10 5.52 17.31 6.82 kidneys 2.02 5.14 6.17 5.64 10.24 6.25 heart 0.21 4.26 0.88 5.11 1.38 5.84 the heads which at the studied individuals from oncorhynchus mykiss species presents a percent of 12.34% for the trout of first batch, 9.27% for trout of second batch and 9.46 % for the individuals of third batch. trunk have the greatest rate with an ascendant evolution at the same time with the age, so the lowest percent of 66.63 % was recorded at rainbow trout individuals of first batch, and the highest rate of 72.69 %was recorded at third batch. the fins’ mass related with the total weight of body represents in the case of rainbow trout, values between 3.12 g and 8.29 g, and tegument represents between 10.47g and 42.17g from the total mass of total body mass. analysing the visceral mass of those three batches we notice that its weight increases with age trouts, being 31.25 g for batch l1, 88.30 g for batch l3 and 123.47 g for batch l3. the percentage of internal organs it was between 2.78% and 3.38%% from the total corporal mass of rainbow trout. in descending order of internal organs was liver weighing between 2.54g and 17.31g, followed by kidney weighing between 2.02g and 10.24g, while the heart was much lower values between 0.21g and 1.38g. the values at all three experimental batches are in the limits mentioned in the literature for oncorhynchus mykiss species [4-7]. 4. conclusions an overall analysis of the weight of the specimens of rainbow trout in the 3 experimental groups indicate that the results obtained are slight differences according to age trout. measurements on specimens of rainbow trout studied to determine body size trout showed that the groups analyzed had a good development fits within the limits described in the literature for this species. the rainbow trout specimens studied, the most significant percentage change occurred with increasing age and weight were recorded at the mass of the trunk, where the share increased from 66.63% to trout aged one to 72, 69% to trout by the age of three years. the data obtained from research conducted showed that rainbow trout as age and weight increases as the percentage of meat from the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 gabriel vasile hoha, ionut bogdan pagu, catalin emilian nistor, emanuel măgdici, benone păsărin researches regarding development body and cutting reports at oncorhynchus mykiss species, food and environment safety, volume xii, issue 2 – 2013, pag. 130 134 134 carcass has an upward trend, reflected and calculating the yield on the three different slaughter ages. 5. references [1] păsărin b.: salmonicultură. editura “ion ionescu de la brad”, iaşi, 2007. [2] bud i., vlădău v.v., ştefan reka, pop s.n., ladoşi, daniela: contributions concerning the species and age influence on fish meat qualitative index, bulletin usamv cluj, animal science and biotechnologies, 65(1-2), pages 288-292, 2008. [3] pulcini d., wheeler p.a., cataudella s., russo t., thorgaard g.h.: domestication shapes morphology in rainbow trout oncorhynchus mykiss, journal of fish biology, volume: 82, issue: 2, pages: 390-407, 2013. [4] cocan d.i., mireşan vioara, răducu camelia, feştilă iulia, coroian aurelia, constantinescu r., negrea o., ranga, r.: the share of internal organs and viscera in rainbow trout (oncorhynchus mykiss) reared in different growth systems, bulletin usamvb timişoara, scientific papers: animal science and biotechnologies, 45 (2), 2012. [5] ostaszewska t., dabrowski k., kwasek k., verri t., kamaszewski m., j. sliwinski, l. napora-rutkowski: effects of various diet formulations (experimental and commercial) on the morphology of the liver and intestine of rainbow trout (oncorhynchus mykiss) juveniles, aquaculture research, volume:42, issue: 12, pages:1796-1806, 2011. [6] merkin g., bjorn r., gjerstad c., dahl-paulsen e., nortvedt ragnar: effect of pre-slaughter procedures on stress responses and some quality parameters in sea-farmed rainbow trout (oncorhynchus mykiss), aquaculture, volume: 309, issue: 1-4, pages: 231-235, 2010. [7] roth b., torrissen o.j., slinde e.: the effect of slaughtering procedures on blood spotting in rainbow trout (oncorhynchus mykiss) and atlantic salmon (salmo salar), aquaculture, volume: 250, issue: 3-4, pages: 796-803, 2005. 108 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 12015, pag. 108 116 con tr i bu t ions to a me thod a nd a new t ech niqu e for dete rm ina tion o f som e i m por tan t m echan ic al propert ies of m as s iv e w ood t extu re *ştefan beşliu¹, gheorghe gutt¹, sonia amariei¹ ¹faculty of food engineering, stefan cel mare university of suceava, romania besliustefan@yahoo.com *corresponding author received 17th march 2015, accepted 31st march 2015 abstract: this article proposes and describes a new method and tehnique for testing and determining of some important mechanical characteristics of massive wood. the method is based on the use of a loading-unloading continuous cycle of steel indenter until its penetration to a predetermined depth in the wooden material. during the cycle the force-displacement values are recorded in real time, values that are shown in a graph by an adequate software that can determine the hardness (by measuring the force in correlation to the displacement it causes) and also the following mechanical characteristics: hardness under load, modulus of elasticity, total deformation energy, elastic deformation energy, plastic deformation energy, relaxation and creep of samples.the equipment used to implement the method are a mix of field and laboratory equipment which have a constant speed of continuos loading-unloading system of indenter, an electronic sensor system made of a dynamometer cell used for measuring the force applied to the indenter and an incremental electronic displacement sensor for measuring the depth of penetration for the sperical indenter of 5.5 mm in the wooden material, as well as a pc equiped with a specialized software to interpret the experimental data. keywords: wood, universal hardness, texture, anisotropy 1. introduction wood penetrations are made to determine it’s hardness. wood hardness is a mechanical property that expresses the resistance to penetration from a metalic element of a certain geometry called indenter. from a technological point of view wood hardness reflects it’s resistance to machine cutting and resistance through friction. to determine wood hardness the usual methods that can be used are: the classic brinell hardness test [1],[2],[3], the modified brinell hardness test [3],[4] and the janka test [5],[6]. the classic brinell test uses optical measurements of the spherical imprint left on the test material after removing the indenter; it has the disadvantage that it doesn’t show the elastic deformation component of the test material. the modified brinell hardness test takes into consideration the depth of displacement of the indenter under a number of set forces. because measurments are made under stress the elastic component for the test material is highlited. it’s disadvantage is that it requires two sensors: one for the applied force and one for measuring the displacement. the janka method is based on determining wood hardness by measuring the force needed to penetrate wood with a 11.28 mm steel indenter up to a depth of half the diameter (5.64 mm). this method of hardness testing determines both the elastic and the plastic deformation components. it has the advantage, compared to the modified brinell method, of using one sensor, the one used to measure force. the common disadvantage of the three presented methods is that all three http://www.fia.usv.ro/fiajournal mailto:besliustefan@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 109 determine only one mechanical parameter, respectively wood hardness. this article removes this disadvantage by, besides measuring the force and displacement parameters for a steel indenter in a constant speed loading-unloading cycle used to determine wood hardness, it also determines important material properties such as: modulus of elasticity, total deformation energy, elastic deformation energy, plastic deformation energy, and for multiple penetration the anisotropy coefficient of wood. 2. materials and methods to have a unitary hardness scale that follows the same test method so called ”universal hardness” was promoted [7], [8],[9],[10] in which hardness is considered the ratio between the force applied to the indenter and the measured displacement for the test material. in 2003, in honor of the person who in 1898 foresaw the need for a hardness test under load for metals, this hardness was called martens hardness. it takes into consideration both the elastic and plastic deformation. the evolution of precision and sensitivity for electronic force and displacement sensors opened a wide field of so called ”instruments for measuring hardness” [9],[11]. the result of such a test is a loading-unloading graph for the indenter as a result of continuous reading the pairs of force-displacement. this being a mechanical test that goes through both the elastic and plastic domain most mechanical material parameters can be deduced [11]. because of the pronounced wood anisotropy, but also because the longest time, the brinell and janka [12] methods were considered sufficient, neither the universal hardness test nor the martens hardness test so far haven’t been used for the advanced mechanical characterization of this material. the objective of this experimental and theoretical research is to characterize and promote a new method that is based on measuring the hardness under a load that, alongside the hardness itself allows the determining of values for other important mechanical characteristics of wood. the method proposed by the collective is also an experimental method [13],[14] [15],[16] that consists of using spherical shaped steel indenters and a system for the continuous progressive load of the indenter until a set displacement is reached, followed by a progressive unloading of the force until it reaches zero. the acquisition and data processing system consists of electronic sensor for measuring force, electronic sensor for measuring displacement as well as a specialized software for the automated memorizing of the force-displacement pairs. the same software allows the realization of the loading-unloading graph as well the calculus for the mechanical characteristics: hardness, modulus of elasticity, total deformation energy, elastic deformation energy, plastic deformation energy. the recommended equipment for laboratory use is not in fact a hardness tester but an universal machine for material testing using small loads and a single column on which the device with the sperical indenter is mounted fig.1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 110 fig.1. universal machine for material testing under small loads 1. sample 2. indenter 3. dynamometer 4.displacement sensor 5. electric motor 6.speed reduction unit 7. electronic unit and the measuring system for force and displacement. 8. computer. a. loading-unloading graph. b. relaxation graph. c. creep graph 3. results and discussion 3.1 experimental data to establish the viability of the proposed test method a universal machine for material testing under small loads was used fig.1. which corresponds to all the requirements for mechanical penetration testing. the method used was the incremental increase of the load for a spherical steel indenter with a diameter of 5.5 mm until reaching a displacement of 2.82 mm. the load speed for the indenter was 0.02 mm/s. the wood specimens had identical dimensions: 200 mm length, 40 mm width, 12 mm thickness. they were all dried and thermostated to a final humidity of 10.5%. ten diffrent species of wood were used: fir , beech, ash , larch , spruce, walnut , maple , pine, acacia , oak . following the experiments using a prescribed depth the loading-unloading graphs were obtained fig.2.-11.: fig.2. loading-unloading graph for fir fig.3. loading-unloading graph for beech fig.4. loading-unloading graph for ash food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 111 fig.5. loading-unloading graph for larch fig.6. loading-unloading graph for spruce fig.7. loading-unloading graph for walnut fig.8. loading-unloading graph for maple fig.9. loading-unloading graph for pine fig.10. loading-unloading graph for acacia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 112 fig.11. loading-unloading graph for oak 3.2 data interpretation taking into consideration the phenomenology for penetrationwithdrawal of indenter fig.12. the obtained graph for this process fig.14. as well as the fact that the described hardness test is actually a mechanical experiment with a progressively increasing/decreasing load [din en iso 14577] for which the wood material under the indenter passes from the elastic deformation domain to the plastic deformation domain, from this graph the same material properties usually deducted from a tensile testing are calculated. the definitions and calculus equations for these properties are described next. fig.12. phenomenology for penetrationwithdrawal for the indenter fig.13. the spherical steel indenter with the dome detail fig.14. example for a force-displacement diagram for wood hh hardness, [n/mm], is determined from the ratio between the maximum load force fmax and the surface of the dome made by the indenter in the wood specimens ac for the maximum displacement of 2.82 mm. the spherical indenter has a radius r= 2.75 mm.   h 1,59 h31,4 50 ha f hr2π f a f h maxmax c max h       22 (1) ha hardness, [n/mm2], is determined by the ratio between the maximum load force for the spherical indenter with a radius of r=2.75 mm and ap the surface for the projection of the contact area surface between the test material and the spherical indenter, a surface situated at a distance of h from the indenter extremity. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 113 22 p hh31,4 50 hhr2π f a f h a     (2) hm hardness is determined from the m slope of the load curve. according to [din en iso 14577] the m slope can be determined from the graph or for the regression equations of the load curve. applying that method for the load curves in fig.3.-fig.12. the data in tab.1. resulted. fmh  (3) 22 / 1 ham h s m  (4) the fact that the linear regression equation is of the y =mx+c type, where c has a certain value different from zero (the linear regression equation doesn’t go through the origin) is because the first reading of the dynamometer for the displacement h is made the first time the force f resulted from the contact between the indenter and the specimen appears (f>0). the advantage for determining the hm hardness from the gradient of the load curve is that it is independent from the imprecision given by determining the point of zero penetration (h0) as well as the geometrical deviations and the specimen roughness. the penetration modulus of elasticity ep is determined mathmaticly from the foloowing equations from [din en iso 14577] , [gutt],[weiler] : i 2 r 2 e )i(ν1 e 1 )s(ν1e p     (5) pa2c π er  (6) where: sν poisson’s number for the tested wood with values between 0.0035 and 0.59, iν poisson’s number for the steel die with values between 0.27 and 0.30, er the reduced penetration elasticity modulus, ei – the elasticity modulus for the steel indenter, has a value of 210.000 n/mm2, c – the value for the reciprocity contact rigidity, is calculated by deriving dh/df for the indenter unload graph on the point for fmax , ap – the surface of the contact area projection between the test material and the spherical indenter ap, a surface situated at a distance of h from the indenter extremity. because of the fibers orientation wood is an orthotropic material with a pronounced transversal anisotropy. using eq.5. to determine the penetration modulus of elasticity ep resulted variations between 2% and 75% which represent differences higher than an order of magnitude, an unacceptable situation from a measuring point of view. the calculation for the penetration modulus of elasticity from the load graphs in fig.3.-fig.12. can also be made with a sufficient low error through other methods. one of them consists of drawing a tangent to the graph’s load curve in the following coordinates force (f)surface (ac). from there the penetration modulus of elasticity can be calculated from the tangent of the α angle created by this line and the abscissa:    2c h31,4δ δf hr2πδ δf δa δf tgαep     (7) where: ac – the surface of the dome created by the indenter in the tested material, r – the indenter’s radius (2.75 mm), h – the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 114 displacement created by the indenter in the wooden tested material. another method which can be used to determine the penetration modulus of elasticity ep, graphic method that does not involve the exercise described, consists of taking into consideration the liniar regression equation for the graph’s load curve in coordinates force (f)-surface (ac). where the slope m of the linear regression is in fact the penetration modulus of elasticity the total deformation energy wt it is the sum of the total elastic deformation energy wel and the total plastic deformation energy wpl. the plastic and elastic components for the machine work are best visible in fig.3.fig.12., but each can be determined through integration:   maxh 0 dhfwt (11)   pl pl h dhfw 0 (12)   max pl el h h dhfw (13) or by difference from the total deformation energy wt : plelt www  (14) elplt www  (15) table.1. the linearreression equations for each wood speciment, highlighting the correlation coefficient r², the m gradient and calculating the hm hardness test linear regression equation r² m [n/mm] fmax [n] hm x 107 [n/mm] hm x 107 [n/mm] fir test 1 y=95.703x-6.1062 0.9976 95.70 253 178.20 fir test 2 y=193.41x-13.571 0.9969 193.41 544 15.47 79.83 fir test 3 y=112.38x+5.448 0.9979 112.38 310 45.80 beech test 1 y=480.97x-64.149 0.9982 480.97 1261 2.50 beech test 2 y=502.81x-70.34 0.9977 502.81 1327 2.28 2.46 beech test 3 y=472.3x-1.4658 0.9991 472.30 1288 2.59 ash test 1 y=532.29x-126.36 0.9894 532.29 1437 2.04 ash test 2 y=590.36x-90.864 0.9975 590.36 1536 1.66 1.89 ash test 3 y=541.46x-107.29 0.9941 541.46 1413 1.97 larch test 1 y=314.65x-88.389 0.9763 314.65 727 5.84 larch test 2 y=342.76x+20.415 0.9903 342.76 903 4.92 5.71 larch test 3 y=301.31x-12.839 0.9917 301.31 792 6.37 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 115 table.2. the different types of calculated hardnesses, the penetration modulus of elasticty and the transversal anizotropy coefficient species hh [n/mm] ha [n/mm2] hm x 107 [n/mm] ep [n/mm2] wt [n∙m] 1. fir (abies alba) 131.79 18.87 79.83 21.59 492.07 2. beech (fagus sylvatica) 461.29 54.38 2.46 40.95 1737.00 3. ash (fraxinus excelsior) 522.14 61.53 1.89 38.41 1778.00 4. larch (larix decidua) 288.33 33.98 5.71 22.68 1170.40 5. spruce (picea albis) 145.71 17.17 28.63 19.01 546.17 6. walnut (juglans regia) 402.38 47.42 3.1 44.13 1474.67 7. maple (acer pseudoplatanus) 392.02 46.20 3.32 36.54 1339.67 8. pine (pinus sylvestris) 290.12 34.19 6.31 20.75 1063.33 9.acacia (robinia pseudoacacia) 482.14 53.92 2.19 32.96 1444.33 10. oak (quercus robur) 384.40 45.30 3.63 33.09 1329.67 4. conclusion the instrumental penetration test using a continuous loading-unloading cycle is a new performant method for the advanced characterization of wood concerning the main physical dimensions for materials like hardness under load, modulus of elasticity, total deformation energy, elastic deformation energy, plastic deformation energy, surface anisotropy modulus of elasticity, work of deformation and surface anisotropy.taking into consideration the other known methods for hardness testing, the brinell and janka hardness tests, the testing under load, spruce test 1 y=126.76x-1.9482 0.9979 126.76 347 36.02 spruce test 2 y=177.67x-33.335 0.9918 177.67 496 18.33 28.63 spruce test 3 y=135.47x+8.0828 0.9986 135.47 381 31.54 walnut test 1 y=403.03x-60.591 0.9968 403.03 1031 3.56 walnut test 2 y=475.38x-83.572 0.9978 475.38 1241 2.56 3.15 walnut test 3 y=415.59x-64.282 0.9981 415.59 1108 3.35 maple test 1 y=411.02x-85.756 0.9916 411.02 1065 3.42 maple test 2 y=420.16x-50.989 0.9977 420.16 1142 3.27 3.32 maple test 3 y=420.05x-92.75 0.9946 420.05 1086 3.28 pine test 1 y=276.4x-8.754 0.9978 276.40 738 7.57 pine test 2 y=341.1x-72.302 0.9930 341.10 868 4.97 6.31 pine test 3 y=301.08x-46.913 0.9935 301.08 831 6.38 acacia test 1 y = 479.69x-22.524 0.9978 479.69 1280 2.51 acacia test 2 y = 552.15x-106.59 0.9945 552.15 1435 1.89 2.19 acacia test 3 y = 515.58x-102.62 0.9954 515.58 1334 2.17 oak test 1 y = 367.08x-53.144 0.9979 367.08 994 4.29 oak test 2 y = 429.43x-59.593 0.9965 429.43 1144 3.13 3.63 oak test 3 y = 408.07x-52.474 0.9973 408.07 1091 3.47 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 ştefan beşliu, gheorghe gutt, sonia amariei, contributions to a method and a new technique for the determination of some important mechanical properties of massive wood texture, food and environment safety, volume xiv, issue 1 – 2015, pag. 108 – 116 116 defined by the ration between the force applied to the indenter and displacement caused to the wooden material is superior because it can be used for any type of shape for the indenter and for any values for the maximum force and displacement. from the different ways of expressing hardness under load the most notable is the hardness determined from the slope of the load curve of the graph. it has the advantage that it’s value is independent from the imprecisions resulted from determining the point of zero displacement as well as geometrical errors and the surface roughness of the tested wooden materials. considering the high spread of the values for the material characteristics mentioned beforehand, to get a good reproducibility for the experimental data the mediation of three of the five values for certain dimensions, the two most extreme values being overlooked. 5. references [1]. weiler w., hardness testing of metals and plastics, expert verlag, ehningen bei böblingen, 1990, p.109-131, isbn 3-8169-0552-8 [2].gutt g., palade d.d., gutt s., klein f., schmitt-thomas kh, g., experiments and characterisation of metals, editura tehnica bucuresti, 2000, p.245-261, isbn 973-31-1574-6, [3]. gutt g.,. gutt s., schmitt.th. kh, universal hardness tester part. i, constructia de masini, nr.1112. s.8-15, bucuresti, 1997, [4]. gutt g., gutt s., hader b., portable hardness based on force measurement, metrologia.nr.3, p.76-85,bucuresti, 1997. [5].gutt g., modern methods for materials testing and characterization, vol. i., editura universitatii suceava , 1997, isbn 973-98210-7-3., isbn 9739408-43-5 [6]. gutt g., modern methods for materials testing and characterization, vol.ii., p.100-145, editura universitatii suceava suceava, 1997, isbn 973-98210-7-3, isbn 973-9408-43-5 . [7].wulfmeyer h., gutt g., a process for the automatic loading in static and dynamic hardness, tehnomus, suceava, b. vi,1991, p.159167. [8]. gutt g., apparatus for hardness testing, patent ro-91680/86. [9]. xxx, din en iso 14577, instrumental hardness testing, 2004 [10]. ullner cr., standars din en iso 14577,. first accepted rules in the world for instrumental hardness testing, berlin, 2005, [11]. weiler w., behnke h., requirements for hardness indenter used in universal hardness testing, materialprüfung, 32(1990)10, p. 301-303 , [12] wayne c. lewis. hardness modulus as an alternate measure of hardness to the standard janka ball for wood and wood-base materials. usda, forest service, forest products laboratory research note fpl-0189 [13]. dorner m., nix w.,u.a., a method for interpreting the data from depth-sensing indentation instruments .j mater res. 1(1986)4, p.601-609, [14]. gutt g., gutt s., a new process for hardness testing based on force measuring, analele universităţii suceava, ii, 1993, nr 1-6, p.80-86 [15]. oliver w.,pharrr g. an improved technique for determing hardness and elastic modulus using load and displacement sensing indentation experiments j. maters res. 7 (1992)6, p.1564-1583, 55 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 55 60 traditional production and chemical composition of ’’bieno cheese” in the republic of macedonia *gordana dimitrovska1, sonja srbinovska2, stefce presilski 1, violeta manevska3, ljupce kochoski 1 , elena josheska1 1faculty of biotechnical sciences, university st.’’kliment ohridski’’-bitola, partizanska b.b, bitola, r.macedonia, gordanadimit@yahoo.com 2faculty of agricultural science and food, university ss.’’ cyril and methodius’’-skopje, blvd. aleksandar makedonski bb, skopje, r.macedonia 3faculty of information and communication technologies, university st.’’kliment ohridski’’-bitola, partizanska b.b, bitola, r.macedonia *corresponding author received february 17th 2015, accepted march 18th 2016 abstract: bieno cheese is an autochtonous dairy product in our country with indigenous production technology which dates back from the ottoman empire. the research includes and presents results of the chemical composition and safety of raw milk for traditional production of bieno cheese, technology and physico-chemical quality of bieno cheese. milk samples quality was determined within the permissible maximum according to data legislation. after 45 days of ripening the bieno cheese registered average results for the following parameters: moisture (38.96%), dry matter (61.13%), milk fat (26.30%), milk fat in dry matter (43.02%), protein (26.21%), ash (9.37%), salt (5.09%) and average of yield (9.14%). in this experiment the microbiological quality of bieno cheese cheese maturing in 45 days is studied. the technology, physico-chemical and microbiological quality could be used in the protection of the origin and geographical label based on its unique technology. keywords: traditional production, raw milk, chemical composition, quality. 1. introduction in the republic of macedonia today three kinds of cheese are mainly being produced: cheese, white brined cheese and hard (bieno cheese). these types of cheese are autochthonous products typical to a particular region and specific to the geographic region. bieno cheese is a typical indigenous dairy product in macedonia from the central area of mariovo region whose production dates back from the ottoman empire. bieno cheese is manufactured in industrial conditions, but in some places it is still homemade, formerly produced from sheep milk, but today mostly from cow milk. in rural and environmentally unpolluted regions, such as mariovo, the production of traditional cheese is preserved today. the future of small farmers is to preserve the traditional technologies, production of authentic and traditional products, protection and marketing of domestic and foreign markets. given its solid consistency and extremely salty taste it is often consumed by the poor and therefore called 'poor cheese'. bieno cheese is hard, low fat cheese with spongy appearance, which matures in brine, with a great diversity in production and nonstandard quality. from this perspective, the study finds a particular practical application and economic viability in the http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska, tradicional production and chemical composition of bieno cheese in republic of macedonia, food and environment safety, volume xv, issue 1 – 2016, pag. 55 – 60 56 new conditions, plans and policies for the development of agriculture and rural development of certain regions in the country. 2. matherials and methods 2.1 analysis and sampling this paper presents the results of the milk quality, technology and physico-chemical quality of bieno cheese. sampling of milk and cheese, as well as production technology, were done in the mariovo region in republic of macedonia. the technology of bieno cheese includes the following steps: raw milk squeezing through cloth lurch heating to 35-360c and coagulation with liquid rennet processing of curd and heating of curd (by adding boiling water t = 75-80 0 c) kneading and collecting of curd in ball self pressing with hanging in the cloth 1618 h ripening at 25-300 c, 5-7 days, sunbathing of balls cutting strips (4-5cm) dry salting (1-2 days) salting in brine (20%) ripening and storage (45 days) 18-200 c 2.2 physico-chemical analysis milk samples were analysed for the content of dry matter, fat, protein, lactose and solids non fat by infrared analyser lactoscopе. active acidity was measured with ph meter (testo 206) and titratable acidity according to the soxhlet henkel method (carić et al., 2000). total count of somatic cell was analysed with a somascope, and total count of bacteria with bactoscan. for the whey there were conducted the following analysis: active acidity of the milk with a digital ph meter (testo 206) ; titrable acidity (0sh) by the method of soxhlet henkel (carić et al., 2000); chemical analisys of whey (milk fat, protein, lactose and dry matter) with an infrared analyzer lactoscopе. the cheese composition was analysed by standard methods: dry matter (aoac,1995), fat soxhlet henkel method (carić et al., 2000), protein (aoac, 1995), salt and ash (inihov method, 1971). the contents of moisture and fat in the dry matter (fdm) were calculated (codex, 1978), and also the yield of bieno cheese (balthadzieva,1993). 2.3 microbiological analysis the cheese was subject to the following microbiological analysis:  iso 6579, microbiology of food and feed-horizontal method for the enumeration of salmonella spp (iso6579: 2002 / cor: 2004);  iso 16649, microbiology of food and feed-horizontal method for the enumeration of β glucuronidase positive escherichia colli iso 16649 1, 1: material of counting colonies of 44 0c using membranes and 5 bromo-4-chloro3-indolyl β d glucoronide (iso 21528-1: 2001);  iso 21528-2, microbiology of food and animal feed horizontal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska, tradicional production and chemical composition of bieno cheese in republic of macedonia, food and environment safety, volume xv, issue 1 – 2016, pag. 55 – 60 57 method for the detection and enumeration of enterobacteriaceae, part 2: method of counting colonies (iso 21528 1: 2004);  iso 11290-1, microbiology of food and animal feed horizontal method for the enumeration of listeria monocitogenes, part 1: detection method (iso 11290-1: 1996/ amd.1: 2004);  iso 6888-1, microbiology of food and feed-horizontal method for the detection of staphylococcal koagulasa positive (staphyloccocus aureus and other species), part 1: technique using baird-parker agar medium (iso 6888-1: amd 1: 2003) 3. results and discussion the quality parameters of raw milk used for the production of bieno cheese are shown in table 1. table 1 physico-chemical parameters and hygienic quality of milk for bieno cheese raw cow milk parameters % �̅� min max sd cv milk fat 3.46±0.04 3.38 3.55 0.06 1.81 proteins 3.19±0.05 3.11 3.24 0.06 1.89 lactose 4.49±0.02 4.46 4.53 0.03 0.57 dry matter 12.60±0.10 12.42 12.77 0.13 1.03 рн 6.57±0.08 6.46 6.70 0.10 1.53 oѕн 6.73±0.06 6.65 6.85 0.08 1.16 *tcsc/ml 364600 323000 389000 28500.88 7.82 *tcb/ml 237800 221000 273000 20510.97 8.63 *tcsc/ml (total count of somatic cells) *tcb/ml (total count of bacteria) the milk used for the production of indigenous bieno cheese has good chemical composition; hygiene is right with allowable number of somatic cells and the total number of bacteria in accordance with the special requirements for raw milk. our results regarding the chemical composition of cow’s milk were in accordance with the results from other authors [1, 2, 3]. in the indigenous technology of production of bieno cheese, must pay particular attention to all the factors that affect the microbiological quality of the milk and the hygienic correctness, namely: hygienic milking, milk cans, cooling devices (tankers) were the milk is held at a temperature of 5°c, transport and storage. it is necessary to respect the hygienic-sanitary norms related to cultivation, preservation and care of the milking herds, and with the right technology of milking, [4,5]. all these factors play a major role for the hygienic correctness and the microbiological quality of the milk. according to the author levkov et.al [6] in milk used for cheese production the total number of bacteria is 2.7 x 106 to 1,0 x 108 / ml. the total number of bacteria depends on the season and the standard of hygiene on the farm. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska, tradicional production and chemical composition of bieno cheese in republic of macedonia, food and environment safety, volume xv, issue 1 – 2016, pag. 55 – 60 58 the total number of bacteria in the milk used for the manufacture of ''bieno'' cheese varies in the range of 2.0 to 3.5 x 107 / ml by the author [7] and that depends on milking hygiene, storage and transportation of milk. whey is a product obtained in the manufacture of cheese. it is more or less clear, yellowish liquid with a distinctive sweet flavor. the chemical composition and properties of whey depend primarily on the quality of milk and the technological process of producing cheese. the results of chemical composition and acidity of the whey can be seen in table 2. whey is an important by product in the manufacture of ’’bieno’’ cheese which can be used to obtain fresh albumin cheese. table 2 chemical composition of whey whey parameters % �̅� min max sd cv milk fat 0.50±0.03 0.45 0.55 0.04 8.16 proteins 1.05±0.05 0.94 1.10 0.06 6.04 lactose 4.63±0.03 4.57 4.64 0.03 0.69 dry matter 6.91±0.07 6.76 7.01 0.10 1.39 рн 6.67±0.07 6.52 6.72 0.09 0.01 oѕн 4.95±0.11 4.80 5.20 0.10 0.01 from the table 2 can be seen that the high content of milk fat (0.50±0.03) in whey likely due to the higher heat treatment or during mating in the curd, resulting in major losses occurring fat. lost of fat in milk whey are essential effect on the production and quality of the cheese produced. protein is an important component in the chemical composition of whey and average mean values ranging from 1.05 ± 0.05%. most % of the dry matter constitutes lactose whey, followed by protein and minerals and finally, with the lowest number was fat. the active acidity of the whey has an average mean (6.67±0.07) and the titrable acidity has an average mean (4.95±0.11). similar results are found in whey obtained in the production of cheese and soft white cheese in research of [2,8]. from table 3, the bieno cheese contains 38.96±0.24 of moisture, 61.13±0.35 of dry matter, 26.30±1.04 fat, 43.02 ±1.56 fat in dry matter, proteins 26.21±0.72, ash 9.37±0.15 and salt 5.09±0.06 after 45 days of ripening. those results coincide with results of scientific papers whose topic was hard cheese and that show that moisture content ranges from 38 to 40%, fat 21 26% and fat in dry matter 40 43% [9,10]. yield is a complex variable because it depends upon a number of factors, the major effect on the quality of milk, the protein content and milk fat, [11]. the greater degree of the distribution of the components of milk into cheese, primarily protein and milk fat affect the majority of the cheese dressing percentage. this statement can be seen in our results obtained in our research and average cheese yield of bieno cheese was (9.14 %). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska, tradicional production and chemical composition of bieno cheese in republic of macedonia, food and environment safety, volume xv, issue 1 – 2016, pag. 55 – 60 59 table 3 chemical composition of bieno cheese after 45 days of ripening bieno cheese parameters % �̅� min max sd cv moisture 38.96±0.24 38.60 39.56 0.37 0.94 dry matter 61.13±0.35 60.35 61.60 0.48 0.79 milk fat 26.30±1.04 24.63 27.43 1.23 4.69 fat in dry matter 43.02±1.56 40.11 44.78 1.96 4.55 proteins 26.21±0.72 25.26 27.30 0.90 3.45 ash 9.37±0.15 9.21 9.60 0.17 1.83 salt 5.09±0.06 5.01 5.23 0.08 1.64 the research specified the microbiological quality of “bieno” cheese after 45 days of ripening in accordance with the special requirements of food safety regarding the microbiological criteria. table 4. microbiological analisys of bieno cheese after 45 days of rippening entereobacteriaceae cfu/ml e. coli cfu/ml coagulase positive staphylococcus cfu/ml listeria monocytogenes cfu/ml salmonella spp. cfu/ml i 1200 252 30 / / ii 2000 840 112 / / iii 1200 720 6 / / iv 3000 960 70 / / v 2000 1280 50 / / from the results of table 4 can be concluded that it was not determined the presence of listeria monocytogenes and salmonella spp. the presence of enterobacteriaceae set possibly due to the production of bieno cheese from raw milk. for cheeses manufactured from raw milk, according to the regulation on microbiological quality, allowed the presence of coagulase positive staphylococcus. in our study, these values are much lower in all variants in all iterations of the experiment; it can be connected with the fact that at the time of mating the cheese dough temperature of 75-80 0c comes to the destruction of coagulase positive staphylococci as confirmed in tests of [12] and [13]. e. coli whose limits are allowed (10 to 100 cfu / ml) was determined in all samples in limits given refer to each tested unit sample. [6], following the dynamics of coliform bacteria during the whole technological process for hard cheese, 45 th day of ripening in brine, found the presence of coliform bacteria in height 104 -106/ml.in tests of [7], 45 th day of ripening of hard cheese in brine, the number of coliforms ranged from 1,0×106 to 2,8×106/ ml. the presence of enterococci under [14] in their food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 gordana dimitrovska, sonja srbinovska, stefce presilski , violeta manevska, ljupce kochoski , elena josheska, tradicional production and chemical composition of bieno cheese in republic of macedonia, food and environment safety, volume xv, issue 1 – 2016, pag. 55 – 60 60 ripening, due to their tolerance to temperature of 10 to 45 0c, ph value of 4.0 to 9.0 as a result of tolerance to certain concentrations of salt ( 6.5%). 4. conclusion bieno cheese from the region mariovo was indigenous product with specific and recognizable properties. the research results show that cow’s milk, as the raw material for cheese production, is of satisfactory quality. bieno cheese has a good chemical composition: dry matter was (61.13 %), milk fat (26.30 %), the content of fat in dry matter (43.02 %), protein (26.21 %), ash (9.37 %), salt (5.09 %). according to dry matter content cheese belongs with hard cheeses, and according to their storing belongs to the sour brined cheese. average yield of bieno cheese was (9.14 %). the data obtained may serve as the basis for creating standardized production procedures, leading to the uniform quality of these products. therefore, traditional dairy should not be seen as a return to the past, but as an effort to preserve the indigenous technology, to gain their organized form, the ethnographic richness of a given region so distinctive, a time stamp to the development of a nation. 5. references [1] srbinovska s., hygiene and quality of milk in the republic of macedonia in accordance with the legislation, modern agriculture, 56, (5): 61 68, (2007). [2] mateva n., z., naletoski., b. palasevski., technology and chemical composition of beaten cheese in r. macedonia. biotechnology in animal husbandy, 24(1)-2:139147, (2008). [3] talevski g., tradition production of beaten cheese, third international scientific symposium "agrosym jahorina", 524-528, (2012). [4] dozet n., macej, o., indigenous white cheeses in brine, monograph: faculty of agriculture, beograd – zemun, (2006). [5] samaržija d., antunac n., pecina m., havranek j., quality of artisanal hard cheeses produced in the mediterranean area of croatia, milchwissenschaft, 58,1/2, 43-46, (2003). [6] kakurinov v., composition and dynamics of microflora of beaten cheese, phd thesis, agricultural faculty, university ss. ‘’cyril and methodius’’, skopje, (2002). [7] levkov v., kakurinov v., microbial characteriristics of beaten cheese made in tetovo region. proceedings of iii symposiym of livestock production with international participation, 379388, (2007). [8] sulejmani e., hayaloglu aa, rafajlovska v., study of the chemical composition, proteolysis, volatile compounds, and textural properties of industrial and traditional beaten (bieno cheese) ewe milk cheese, j. dairy sci. 97(3):1210-24, (2014). [9] kapac-parkačeva n., čižbanovski t., lazarevska d., chemical composition, characteristics and rheological properties of bieno cheese were produced from the area of the federal republic of macedonia, mljekarstvo 24, (4), (1974). [10] kamber u., úelik h.t., some microbiological and chemical characteristics of gorcola cheese. yyü vet. fak. derg., 18, (1), 8792, (2007). [11] guinee, t.p.,salting and the role of salt in cheese. int.j.dairy tech.,vol 57, no 2/3, 99-109, (2004). [12] lima carla d., las casas., monica m.o.p.cerquera., elaine g.ferreira. cesar l.l.faria jr., david lee nelson., luiz s.carmo., carlos a.rosa., microbiological, physical-shemical and sensory evaluation of a tradicional brazilian cheese during ripening process. world j.microbiol.biotechnol. 24: 2389-2395, (2008). [13] gomezlucia e., goyache j., orden a.j. domenech a., javier hernandez, f., riz – santa quiteria j.a., lopez b., blanko j.l., suarez g., growth od staphiloccocus aureus and synthesis of enterotoxin during ripening of experimental manchego – type cheese. journal of dairy science, 75,19-26, (1990). [14] giraffa g., styding the dynamics of microbial populations during food fermentation. fems microbiology reviews 28, 251-260, (2004). 1. introduction [8] sulejmani e., hayaloglu aa, rafajlovska v., study of the chemical composition, proteolysis, volatile compounds, and textural properties of industrial and traditional beaten (bieno cheese) ewe milk cheese, j. dairy sci. 97(3):1210-24, (2014). 365 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 365 371 medical and biological estimation of grain small loaves on the basis of whole wheat grain with the inclusion of vegetable additives * marina mardar¹, rafaela znachek¹ ¹ faculty of marketing, enterprise and commerce odessa national academy of food technologies, ukraine e-mail address: marina_mardar@mail.ru, rafaehlab@yandex.ru *corresponding author received november 28th 2014, accepted december 29th 2014 abstract: the results of medical and biological estimation of grain small loaves with the inclusion of vegetable additives (silybum mariánum and green tea extract) are presented in the article. from the analysis of medical and biological data investigations on new grain small loaves, it is obvious that they possess liver-protecting action, therefore they can be recommended both in mass and in prophylactic nutrition of people suffering from diseases of endocrine system, disorder of metabolism, obesity. keywords: nutrition, medical and biological estimation, grain small loaves, vegetable additives, whole wheat grain 1. introduction grain products are the most important group of products in the world population’s nutrition and are included as the obligatory component of its daily diet. at the expense of application of grain products in nutrition, demand in proteins is supplied up to 40 %, in carbohydrates – up to 60 %, in mineral substances – up to 30 %, in vitamins of group b – up to 40 % and in a man’s energy – up to 50 %. in the diet the share of grain products makes up 40–45 % on average from the general diet. it testifies to an especially significant role of grain products in a man’s nutrition [1]. in spite of the significant application of grain products in nutrition, their food and biological value is unbalanced by the main food substances [2]. it’s stipulated by the fact that food value of grain products depends, to a large extent, on the degree of macroand micronutrients of the initial grain being preserved in them. in the traditional approach to the grain processing, aimed at maximum removing of seed-coats and obtaining the flour of the top quality, the content of healthy components inevitably decreases. furthermore, this process continues under the influence of the technological parameters at all stages of production and storage of products. in the process of the technological processing of the raw materials into food products, the significant amount of micronutrients is lost. during grinding of grain into flour, the most part of them is removed with the seed-coats. the flour of the top quality is much poorer in vitamins and minerals, than the flour of lower grades. at the stage of production of finished products, further decrease of content of physiologically valuable substances, in particular vitamins в1, в6, рр, magnesium, potassium, phosphorus, iron, occurs. increase in food value and concentration of physiologically functional substances of grain products, can be provided by two ways: 1. preservation of seed-coats and aleurone food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 366 layer, containing the largest amount of micronutrients and biologically active substances of grain; 2. enrichment of the products with functional ingredients in the process of their production. the first method provides application of food products on the basis of the whole grain – whole grain bread, grain small loaves, flakes, cereals from unground grain, as well as products from coarse-ground flour. the second method is connected with the inclusion of vitamin and mineral complexes, vegetable additives, sources of food fibres, prebiotics, phospholipides, proteins and other enriching ingredients into flour, cereals or finished products. one of the topical directions is application of products on the basis of whole grain in a man’s nutrition. the whole-grain food is rich in vitamins, minerals and cellulose, that’s why it can be a good choice for people, suffering from cardiovascular diseases, diabetes, obesity and other diseases, caused by the unhealthy diet. medical and biological research, carried out in the clinic of harward school of public catering, testify that for the people, suffering from the ii-nd type diabetes and using a great amount of the whole-grain products, the risk of death from cardiovascular diseases has decreased by 35% [3]. the scientists of london imperial college and university of leeds, as a result of the carried out research, have stated, that daily consumption of 10 grams of cellulose reduces chance of intestine tumour appearance by 10%, and the additional 90 grams of whole-grain products a day reduced the risk of the disease up to 20% [4]. consumption of whole-grain products can help the patients to reduce the increased blood pressure. as a result, the risk of occurrence of other diseases, such as a heart infarction, a cerebral thrombosis or a stroke, a heart asthenia, can be reduced. according to the researchers, actually, the matter is in vegetable ingredients, which are included in the composition of uncleaned whole-grain products. worthless substances, magnesium, potassium, calcium, and especially antioxidant substances in the husks, have a beneficial influence on blood vessels properties [5]. the researchers of harward university in the usa have confirmed the research of the english scientists, who discovered, that the consumption of the products on the basis of whole grain, contributes to reduce of the high blood pressure [6]. the investigations, carried on with the animals, have shown that application of products from cleaned cereals in contrast to whole-grain products, can cause increase in adipose tissue formation, even if the total energy consumption doesn’t change and the weight of the body remains constant [7]. on the basis of the carried out medical and biological research it has been stated that wholegrain products, which possess low glycaemic index, cause a feeling of satiation and shorten the consumption of energy, this they contribute to a weight decrease [8]. the investigations of the american scientists, made from 1984 to 1996, (74091 women were under observation) with the aim of establishment the intercommunication between consumption of whole-grain products and weight changing, development of obesity among women of middle age, have shown that weight gaining is in inverse dependence on consumption of whole grain products and in direct dependence on consumption of cleaned cereals and so, it proves distinction between whole-grain products and cleaned cereals for solving the problem of checking the weight [9]. besides medical and biological investigations in the field of influence of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 367 whole-grain products on the condition of a man’s health, the marketing research is carried out with the aim of revealing consumers’ preferences concerning the given products. the research, carried out in three european countries of great britain, italy and finland testify that nowadays consumers care about their health more and prefer the products on the basis of ground whole grain [10, 11]. in ukraine the marketing investigations of the customers’ preferences during choice of grain products, which also testify that a modern customer orients himself, first of all, in the products of sanitary purpose, namely, the products, obtained on the basis of the whole grain, grain products, enriched with different kinds of natural enriching additives, have been conducted [12]. 2. material and methods nowadays, in odessa national academy of food technologies, the research in the field of working out the recipes and complex merchandising estimation of the products quality on the basis of a whole grain, namely grain small loaves with the inclusion of vegetable additives (silybum mariánum and green tea extract), the inclusion of which stipulates improvement of organoleptic indices of small loaves quality, giving additional prophylactic properties and extension of storage terms of the finished product. the given products are received on the basis of a whole grain according to the special technology, in spite of all this they do not contain residual products of fermentation and are fully ready for consumption. small loaves refer to lowcalorie products, though their calorie content doesn’t differ very much from that of ordinary bread. the calorie content of small loaves makes up approximately 300 kcal by 100 grams of the product. however, they contain so called «slow» carbohydrates, which are slowly assimilated by a man’s body and together with this they preserve the feeling of satiation for a long time. physiological value of grain small loaves is determined by the ability to influence digestive, nervous, cardiovascular systems of a man. for example, grain small loaves contain vitamins of b-group, which normalize work of the nervous system. due to the cellulose small loaves are able to improve the condition of our intestine, remove slags from our organism, regulate work of a gall-bladder and contribute to its cleaning. at the expense of the additives which are included in the small loaves recipe, the additional. volume of minerals and vitamins, necessary for our organism, appears. the vitamins, which this product contains, increase metabolism of carbohydrates and fats, and macroand microelements maintain healthy condition of the cardiovascular system, provide optimum acid-and-alkaline balance of the organism. inclusion of the vegetable additives into a composition of small grain loaves from the wheat whole grain, gives an opportunity of their usage in mass and prophylactic nutrition, which it’s nessary to estimate from the point of view of medical and biological research. 3. results and discussion the aim of the work was to conduct medical and biological research of small loaves from wheat whole grain with inclusion of vegetable additives, as well as substantiation of the possibility of using them in the prophylactic nutrition of the population. for this, their medical and biological estimation in the conditions of «in vivo» on the laboratory animals (white rats of vistar line) has been conducted. the research was conducted on the basis of the laboratory of odessa state institute food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 368 of stomatology of the academy of medical sciences of ukraine. for every investigation, the experimental animals were fed with a special mixed fodder, in the composition of which a control or experimental sample of grain small loaves 10 % in number from the total mass of the diet, was included. biological investigations of the small loaves were conducted on 35 white rats (males, 4 months, average living mass 250 g), which were distributed into 5 groups: i group – intact group of rats (norm); ii group – rats with the triple pathology (high-fat diet + aids+ dysbacteriosis), subsequently «pathology»; iii group – pathology + grain small loaves without inclusion of biologically active substances; iv group – pathology + grain small loaves, containing fine-crumbled up silybum mariánum (lat.); v group – pathology + grain small loaves, containing green tea extract. high-fat diet was received by adding some 15 % oil to the mixed fodder. aids was created by introducing the cytostatic of cyclophosphane in a dose of 25 mg/kg in a day during 20 days. dysbacteriosis was caused by introducing the antibiotic of lincomicine in a dose of 60 mg/kg with drinking water every day during 5 days. euthanasia of the animals was carried out on the 21-st day during thiopenthal anaesthesia (20 mg/kg) by the total bloodletting from the heart. blood serum was obtained and a part of liver was slashed to pieces. in the blood serum the following «liver» markers were determined: the content of bilirubin, activity of alaninetransaminase and activity of alkaline phosphatase. in the liver homogenate (50 mg/ml 0,05м tris-hcl-buffer ph 7,5), the level of inflammation markers was determined: activity of elastase, content of malon dealdehyde, activity of urease (index of microbe sowing), activity of lysozyme (marker of non-specific immunity), activity of antioxidant enzyme of catalase. according to the correlation of catalase activity and content of malon dealdehyde, the antioxidant – prooxidant index was calculated, and by the correlation of relative activities of urease, the degree of dysbacteriosis, according to levitskiy, was calculated [13]. in figure 1 the results of determining the level of «liver» markers in the blood serum are presented. fig. 1 influence of food additives on the level of «liver» markers in the rats’ blood serum (1 – norm, 2 – pathology (p), 3 – p + small loaves, 4 – p + small loaves with silybum mariánum (lat.), 5 – p + small loaves with green tea), * – p<0.05 in comparison to graph № 1; ** – p<0.05 in comparison to graph № 2 bi l i rubin,µmol /l 0 1 2 3 4 5 6 1 2 3 4 5 pat n * ** * ** alt,µkat/l 0 0,1 0,2 0,3 0,4 0,5 0,6 1 2 3 4 5 pat n * ** * ** * ap, µka t/l 0 0,5 1 1,5 2 2,5 1 2 3 4 5 pat n * ** * * food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 369 from the obtained data, it can be seen that during modelling of the triple pathology (high-fat diet, aids and dysbacteriosis) all three «liver» markers really increase their level. feeding rats with small loaves without dietetic additives decreases a little the level of the markers (however, in all cases p>0.05). at the same time, small loaves, containing finely crumbled up silybum mariánum, really reduce the level of liver markers practically to the norm, and it testifies to high liver protecting efficiency of finely crumbled up silybum mariánum. it’s known, that the leaves of silybum mariánum contain flavolignans [14], which are widely used in medicine in a number of liver protectors. as for the group of animals, which received small loaves with the additive of green tea extract, the level of bilirubin and activity of alaninetransaminase really reduce. in fig. 2 the results of determining activity of elastase in the liver tissue have been given. from it we can see that increased activity of inflammation marker significantly reduces during small loaves action, even if they don’t contain dietetic additives. 0 80 160 240 320 400 480 560 1 2 3 4 5 e la st as e, µ ka t/ kg * ** ** **pathology norm * fig. 2 influence of food additives on the activity of elastase in the rat’s liver with the combined pathology (1 – norm, 2 – pathology (p), 3 – p+small loaves, 4 – p+ small loaves with silybum mariánum (lat.), 5 – p+small loaves with green tea ), * – p<0.05 in comparison to graph № 1; ** – p<0.05 in comparison to graph № 2 the level of the second marker of inflammation, malon dealdehyde, reduces in rats’ liver, which received small loaves with the additive of finely crumbled up silybum mariánum (lat.) (fig. 3). 0 10 20 30 40 50 60 1 2 3 4 5 m d a , m m ol /k g * * ** *pathology norm fig. 3 influence of food additives on content of malon dealdehyde in rats’ liver with combined pathology (1 – norm, 2 – pathology (p), 3 – p+small loaves, 4 – p+ small loaves with silybum mariánum (lat.), 5 – p+small loaves with green tea ), * – p<0.05 in comparison to graph № 1; ** – p<0.05 in comparison to graph № 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 370 in table 1 the results of determining activity of urease and lysozyme in rats’ liver are given. table 1 influence of food additives on the activity of urease and lysozyme in rats’liver with combined pathology (all groups consist of 7 rats each) № groups urease, µkat/kg lysozyme, unit/kg 1 norm 0.20±0.05 167±15 2 pathology (p) 0.97±0.11 p<0.001 62±20 p<0.001 3 p + small loaves 83±10 p<0.01 p1>0.3 102±10 p<0.01 p1<0.05 4 p + small loaves with silybum mariánum 73±9 p<0.01 p1>0.05 p2>0.1 103±10 p<0.01 p1<0.05 p2>0.8 5 p + small loaves with green tea extract 75±11 p<0.01 p1>0.05 p2>0.5 83±10 p<0.01 p1>0.3 p2>0.1 note: p – in comparison to group №1; p1 – in comparison to group №2; p2 – in comparison to group №3 from the obtained data we can see that during pathology in liver, activity of urease significantly increases (almost by 5 times!) and it testifies to increase of microbe sowing of this organ. using small loaves decreases activity of urease, to a greater degree with the additive of finely crumbled-up silybum mariánum (lat.). the results of determining the degree of dysbacteriosis in liver (fig. 4) show, the degree of dysbacteriosis increases by 12 times and really decreases during feeding with small loaves with vegetable additives. 0 2 4 6 8 10 12 14 1 2 3 4 5 th e de gr ee o f d ys ba ct er io si s * ** ** ** * * fig. 4 influence of food additives on the degree of dysbacteriosis in rats’ liver with the combined pathology (1 – norm, 2 – pathology (p), 3 – p+small loaves, 4 – p+ small loaves with silybum mariánum (lat.), 5 – p+small loaves with green tea ), * – p<0.05 in comparison to graph № 1; ** – p<0.05 in comparison to graph № 2 in table 2 the results of determining the activity of catalase and antioxidantprooxidant index are given. table 2 influence of food additives on activity of catalase and antioxidant-prooxidant index (api) in rats’liver with combined pathology (all groups consist of 7 rats each) № groups catalase, µkat/kg api 1 norm 5.93±0.20 1.57±0.08 2 pathology (p) 5.78±0.09 p>0.03 1.14±0.09 p<0.005 3 p + small loaves 5.82±0.14 p>0.3 p1>0.6 1.21±0.10 p<0.05 p1>0.3 4 p + small loaves with silybum mariánum 5.92±0.25 p>0.09 p1>0.3 p2>0.3 1.44±0.11 p<0.4 p1<0.05 p2>0.05 5 p + small loaves with green tea extract 5.80±0.05 p>0.3 p1>0.8 p2>0.8 1.27±0.08 p<0.05 p1>0.3 p2>0.4 note: p – in comparison to group №1; p1 – in comparison to group №2; p2 – in comparison to group №3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 marina mardar, rafaela znachek, medical and biological estimation of grain small loaves on the basis of the wheat whole grain with the inclusion of vegetable additives, food and environment safety, volume xiii, issue 4 – 2014, pag. 365 – 371 371 4. conclusion enriched small loaves of the cereals are the products of the new generation. that’s why growth of production and development of scientifically grounded new types of grain products in ukraine becomes necessary for our society. the development of the new compositions at the expense of introducing the additives of vegetable origin into the composition, will allow to increase their food and biological value as well as to balance the correlation of the main food substances at most. analysing the data of medical and biological investigations of the new grain small loaves from the wheat whole grain with inclusion of vegetable additives, it was determined that they possess liver protecting activity, that’s why they can be recommended both in mass and in prophylactic nutrition of the population. 5. references [1]. zadorozhnyi i.m., gavrilishin v.v. merchandise knowledge of supplies. grain flour products : textbook.-lviv: «compact lv», p. 304 (2004). [2]. functional food products. introduction into the technologies. edited by а.а. kochetkova.m.: deli print, p. 288 (2009). [3]. wholegrain products protect diabetics from premature death. russian scientific centre of surgery named after b.v. petrovskiy rams [electronic resource]. — access: http://www.proctosite.ru/news/show-96.htm, (2011). [4]. wholegrain products protect from intestine cancer [electronic resource]. — access: http://news.vdv-s.ru/beauty/?news=221018, (2011). [5]. cardiovascular prophylactic. national recommendations were worked out by the committee of experts of all-russian scientific society of cardiologists, appendix 2 to the journal: «cardiovascular therapy and prophylactic», №10 (6), (2011). [6]. liflyandskiy v.g. nutrients and food products in prophylactic and treatment of cardiovascular diseases, svop №2: 14-23, (2011). [7]. denyer g, pawlak d, jiggins j, et al. dietary carbohydrate and insulin resistance: lessons from humans and animals. proc. nutr. soc. austr., 22:158–67, (1998). [8]. roberts s.b., heyman m.b. dietary composition and obesity: do we need to look beyond dietary fat? j. nutr.; 130: p. 267 (2000) (editorial). [9]. simin liu, walter c willett, joann e manson, frank b hu, bernard rosner, graham colditz relation between changes in intakes of dietary fiber and grain products and changes in weight and development of obesity among middle-aged women, american society for clinical nutrition, 78: 920–927, (2003). [10]. arvola a., lahteenmaki l., dean m., vassalo m., winkelmann m., claupein e., saba a., shepherd r. consumers beliefs about whole and refined grain products in the uk, italy and finland, j. cereal sci., 46(3): 197-206, (2007). [11]. dean m., shepherd r., arvola a., raats m.m., saba a., j. cereal sci., 46(3): 188-196, (2007). [12]. mardar m.r. marketing research of market demand for grain products of a healthy purpose. storage and processing of grain, 7(184): 45 -49, (2014). [13]. levitskiy a.p., makarenko o.a., selivanskaya i.a. [and others], gfts, mzy, p. 26 (2010). [14]. kurkin v.a. silybum mariánum – a source of medicines (review). chemical and pharmaceutical journal, v. 37.(4): 27-41, (2003). title …………………… 29 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 29 35 chemical analysis of fresh water from a karst area in ukraine * volodymyr diychuk 1 , anastasiya sachko 1, igor winkler 1,2 1department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine v.diychuk@chnu.edu.ua 2department of medicinal chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine i.winkler@chnu.edu.ua *corresponding author received january 26th 2016, accepted march 24th 2016 abstract: the chemical composition analysis was performed for fresh water samples taken from a karst area located in the region of chernivtsi, ukraine. water samples were taken from private wells, springs and open ponds near verenchanka. total water hardness exceeded the sanitary limits for all the well samples and ranged from 11 to 29 mmol-eq/l (respective sanitary limit is 7 mmol-eq/l). this can be caused by the close-to-surface karst formations located under the village which release massive amounts of water hardness ions in the well water as a result of karst ablation. however, the heavy metals content (fe, zn, cu, mn) was within the sanitary limits excepting two samples which slightly exceeded zinc content. total hardness of the surface spring and pond waters ranged from 6.2 to 11 mmol-eq/l and the heavy metals ions contents were comparatively low. keywords: fresh water, total hardness, heavy metals content, karst area, trilonometry, atomabsorption spectrometry. 1. introduction availability of fresh water is a key issue for any inhabitant of municipal or rural areas and it is an indicative parameter of the life quality and safety, keeping the public health and improvement of the life conditions. this problem has gained the primary importance for many countries and regions all over the world. it is known that quality of approximately 10 % of the drinking water in ukraine remains below sanitary standards and requirements set for its chemical composition and bacterial parameters [1]. moreover, a lot of inhabitants of rural areas of ukraine traditionally consume the untreated water, which is often contaminated by chemical and/or germ agents, which results in worsening of the total organism resistance and immune status [2]. it seems interesting to investigate possible effects of the karst formation and development processes on the drinking water quality in the nearby areas. karst is an integral watercycling rock geosystem enriched with numerous pores and channels formed as a result of dissolution/sedimentation of the primary rock materials by circulating natural waters. the near-surface subterranean karst formation causes numerous specific phenomena in the local relief and hydrology related to significant voidage and channels penetrability of the rock. all these factors result in formation of some specific karst landhttp://www.fia.usv.ro/fiajournal mailto:v.diychuk@chnu.edu.ua mailto:i.winkler@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 30 scapes. changes in the hydrology regime of the area can lead to dynamic changes in the karst character, which causes changes in the water cycling followed by possible changes in the local natural waters composition [3]. fig. 1. approximate location of vil. verenchanka in chernivtsi region (encircled) and the same in bigger scale (arrowed) in the map of the west of ukraine (embedded). the karst rocks can perform a strong adsorption action since they are formed by quite porous materials capable to extract various heavy metals while water is infiltrating through karst. on the other hand, the karst rocks is represented mainly by a comparatively easy-soluble limestone and gypsum, which results in releasing of significant amounts of calcium and magnesium (hydro)carbonates, sulfates and other salts into water. as a result, the overall influence of the karst rocks on the drinking water quality and composition depends on numerous local specifics and should be investigated thoroughly for each locality of interest. the village of verenchanka is located in zastavna district of chernivtsi region, ukraine (see fig. 1) at the well-known subsurface gypsum karst formation. numerous occurrences of karst phenomena have been continuously reported in the nearby area and within the village. besides, there is a gypsum opencast mine near verenchanka. the 20-30 meters thick sub-surface karst formations are mostly responsible for the active karst manifestations in the middle part of pruth-dnister interfluve. local karst manifestations in the area of verenchaka are related to the block tectonic structure of the local rock and different karst opening depth by the draining rivers incuts [4]. therefore, it is expected that karst phenomena can provide quite a tangible influence on the well-water quality in the village. basing on this assumption we food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 31 planned and carried out a systematic investigation of some physico-chemical parameters of the water samples taken from various sources inside and around the village. water quality parameters have been determined using the following methods: organoleptic evaluation (taste, odor and color), trilonometric determination of the total water hardness and atom-absorption spectrometry (aas) measurement of some heavy metals contents. 2. materials and methods water sampling has been performed according to [5, 6] using the 0.5 liter polyethylene containers washed preliminary with nitric acid and then distilled water. 5 ml of the highly purified nitric acid have been added to each water sample for the purpose of conservation. total series involved 32 samples taken from the wells, springs and ponds inside and nearby the vil. verenchanka. total water hardness has been determined using the well-known method of trilonometry based on formation of stable complexes of ethylenediamine tetraacetic acid (edta) with ca2+ and mg2+ ions. ph of the solution was kept at 9–10 by the ammonia buffer solution (nh4cl + nh4oh) and eriochrome black-t was used as an indicator. then the heavy metals content has been measured by aas. it should be noted that this method has proved its superior sensitivity, high accuracy and requires comparatively short time. on the other hand, it requires preliminary chemical decomposition of the samples. the ‘matrix’ composition should also be taken into consideration. qualitative determination of fe, zn, mn and cu in all the samples has been done according to the method [7, 8] using the air-acetylene flame and standard solutions approach. details of the determination regimes for different elements are available from table 1. table 1. determination regimes for fe, zn, mn and cu element wavelength, nm determination limit, mg/l fe 248.3 0.08 zn 213.9 0.012 mn 279.5 0.025 cu 324.8 0.04 3. results and discussion as seen from a map (see fig. 2), the wells are scattered practically all around the village area. they are located both in valleys and uplands. the valley wells are relatively shallow (7, 8, 20, 21 and 28) and their depth is limited by 9 meters while the upland wells (1, 13÷19, 22÷27, 29÷32) are much deeper and their depth can reach 25 m (see table 2). an average well depth in the area is 12.9 m. as seen from table 2, the water quality parameters are ranged within quite wide limits. however, no reliable dependence between well locations and the water quality parameters has been found. in our opinion, this can be caused by different geological conditions and water feeding regimes for each of the wells. our results are in good agreement with a concept of geological structure of the region. therefore, high total hardness can be caused by an intense circulation of the well-feeding waters inside the miocene gypsum rocks forming karst structures in the area. the total water hardness was found very high for all the wells and it exceeds substantially the sanitary value. more detailed results of the water quality investigation and explanation of the sampling points location are given in table 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 32 table 2. well parameters in the area of vil. verenchanka № parameter averaged value minimal value maximum value permissible sanitary values 1 total hardness, mmol-eq/l 18.52 11 29 7 2 fe content, mg/l < 0.08 < 0.08 0.09 0.3 3 zn content, mg/l 0.38 0.082 1.887 1.0 4 cu content, mg/l 0.031 0 0.265 1.0 5 mn content, mg/l < 0.02 < 0.02 < 0.02 0.1 6 well depth, m 12.875 7 25 – fig. 2. locations and total hardness of the water sampling points it is known that calcium and magnesium sulfates, chlorides, carbonates, bicarbonates and other soluble forms are responsible for the natural water hardness. soft water is conditionally limited by the total hardness 3.5 mmol-eq/l; moderately hard – by 7; hard – by 10, and very hard water is over 10 mmol-eq/l. it should be understood, that this classification is based mostly on the organoleptic characteristics since water tastes bitter if it is excessively hard. therefore, it is desirable to have drinking water of the soft or moderately hard category. it was found that the 10 well (7, 15, 16, 20, 21, 25, 29÷32) water samples had water hardness higher than the averaged value (18.52 mmol-eq/l). they are scattered all over the village but part of them are located as a group (16, 25, 30, 31, 32). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 33 table 3. complete results of analysis of the water samples sample content, mg/l total hardness, mmoleq/l organoleptic parameters well depth type of the sampling point fe mn zn cu smell, points taste, points and character colour 1 – – 0.11 0.065 18 1 1, bitter-sour no 15 well 2 – – 0.016 – 6.2 2 – yellowish – pond 3 – – 0 – 11.4 1 0 no – spring 4 – – 0.021 0.025 15.6 1 1, bitter-sour no 25 well 5 – – 0.02 – 6.2 2 1 yellowish – pond 6 – – 0.031 0.035 9 1 1 no – spring 7 – – 0.082 – 26 1 2 bitter-sour no 7 well 8 – – 0.143 0.015 17 1 0 no 8 well 9 – – 0.094 0.02 16.4 1 1, bitter-sour no 20 well 10 – – 0.032 0.025 10 1 – no – spring 11 0.16 – 0.064 0.015 10 1 – yellowish – pond 12 0.08 – 0.055 0.015 7 1 – no – spring 13 0.08 – 0.106 – 11 1 0 no 12 well 14 – – 0.134 – 11 1 0 no 15 well 15 – – 0.196 – 22 1 1, bitter-sour no 15 well 16 – – 0.137 – 22 1 1, bitter-sour no 20 well 17 – – 0.746 0.015 18 1 1, bitter-sour no 15 well 18 – – 0.097 0.01 14 1 0 no 12 well 19 – – 0.111 0.04 15 1 1, bitter-sour no 12 well 20 0.09 – 0.941 0.265 26 1 2 bitter-sour no 9 well 21 0.08 – 0.143 0.02 24 1 2 bitter-sour no 8 well 22 – – 0.144 0.045 14 1 0 no 12 well 23 0 – 0.805 – 14 1 1 no 12 well 24 0.08 – 0.281 0.035 14 1 1 no 15 well 25 – – 0.159 0.02 21 1 2 bitter-sour no 15 well 26 – – 0.179 0.035 16 1 1, bitter-sour no 12 well 27 – – 1.887 – 13 1 1 no 15 well 28 – – 1.379 0.01 12 1 1 no 7 well 29 – – 0.3 0.045 29 1 2 bitter-sour no 12 well 30 – – 0.214 0.025 20 1 1, bitter-sour no 12 well 31 – – 0.263 0.025 23 1 1, bitter-sour no 14 well 32 0.08 – 0.48 0.065 28 1 2 bitter-sour no 15 well concentration of zn is another particular local feature, which even exceeded the sanitary value in some wells. the highest zn contents have been found in the samples number 17 (0.745 mg/l), 20 (0.941 mg/l), 23 (0.805 mg/l), 27 (1.887 mg/l – exceeding) and 28 (1.379 mg/l – exceeding). in the contrary, the concentration of cu is within the sanitary values for all the samples. the concentration of fe was also far food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 34 below its sanitary limits. no correlation between the total hardness and concentrations of the heavy metals ions has been established for all the wells except the well 20. this sample has showed both high total hardness (26 mmol-eq/l) and quite high contents of fe, zn and cu (0.09, 0.941 and 0.265 mg/l respectively). this well is located quite close to the area of the former spirits production factory that has been closed because of the karst activation in the nearby area years ago. in our opinion, former and continuous spills and discharges of the factory’s technological liquids and wastewaters could be accumulated in the local soil and caused high contents of the salts in this sample. spring and pond waters analysis results there are some springs and ponds within the village and the organoleptic and physico-chemical parameters of their waters have also been analyzed in the same manner as for the wells in order to enhance our survey of natural water quality in the area. as seen from comparison of the analysis results, the spring and pond water composition is quite different from that of the well water (see fig. 3). for instance, the total hardness of the spring and pond water sample is ranged within 6.2-11 mmol-eq/l while this parameter for the well water samples is significantly higher – its minimal value for the well waters is 11 mmoleq/l. this is an evidence of difference in water feeding sources of local wells and springs/ponds. the wells are fed mostly from the underground water sources, which seem to be enriched with ca and mg salts. it is quite obvious since the region is located on the thick and comparatively high (6-22 m from the surface) bed of gypsum [9-11]. this is the most probable source of the water hardness ions released to the well waters. alternatively, the surface springs and ponds are fed mostly by the rain and snowmelt waters, which are much softer. 0 2 4 6 8 10 12 1211106532 sample total water hardness, mmol/l fig. 3. total hardness of the spring and pond water samples 0.00 0.03 0.06 0.09 0.12 0.15 mg/l fe zn cu 1211106532 sample fig. 4. contents of the heavy metals ions (fe, zn, cu) in the spring and well waters. more detailed results of investigation of the heavy metals ions in the spring and pond waters are represented in fig. 4. as seen from this figure, the samples 11 and 12 have the highest fe (0.16 and 0.08 mg/l respectively) and zn (0.064 and 0.055 mg/l) contents. however, even these values are far below their sanitary levels. 4. conclusion the well water of village verenchanka has shown comparatively high total hardness (11-29 mmol-eq/l), which exceeds significantly the sanitary limits. this problem can food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 volodymyr diychuk, anastasia sachko, igor winkler, chemical analysis of the freshwater from a karst area in ukraine, food and environment safety, volume xv, issue 1 – 2016, pag. 29 – 35 35 be originated from the thick and close-tosurface karst formations in the village area. according to the aas results, the subthreshold or exceeding contents of zn have been registered in the samples 17, 23, 27 and 28 (0.746, 0.941, 0.805 and 1.887 mg/l respectively). the highest zn content in the sample 28 that exceeds the sanitary limits can be resulted by the influence of the old spirit production factory spills and wastewater discharges that took place in the nearby area. concentrations of fe, mn and cu in all the samples were far below the sanitary limits. on the other hand, the total hardness of the surface water objects (springs and ponds) in the area of investigation was found much lower than that of the well water. this hardness was ranged within 6.2-11 mmol-eq/l and, in our opinion, this is an evidence of different water feeding sources for the surface water objects and the wells. the former are fed preliminary by the softer rain and snowmelt water while the latter are fed by the karstoriginated hard ground water. concentrations of fe, zn, mn and cu in all the surface samples were also much lower than the sanitary limits. taking into account the character of the well water feeding in the area of investigation, no solution can be proposed to bring the total water hardness back to its sanitary limits except intense utilization of water softener agents whenever this parameter seems critical (cooking, washing, using water in the home heating systems network). karst area is much wider than the area of the village and finding a spot to establish a soft water source near the village does not seem realistic. on the other hand, the surface water springs can provide water with appropriate hardness but they cannot cover required water output while soft water from the local ponds can hardly meet other sanitary requirements (mostly bacteriological). 5. references [1]. a law of ukraine “about drinking water and water supply”. supreme council of ukraine: official edition. 36 p. kyiv, (2005). (in ukrainian). [2]. voloshkina o. s., yakovlev e. o., udod v. m., to environmental safety of surface water bodies. council of national security and defense of ukraine, institute for national security problems: official edition. 139 p. kyiv, (2007). (in ukrainian). [3]. klimchouk a. b., subsidence hazards in different types of karst: evolutionary and speleogenetic approach. envir. geol., 48(3): 287-296, (2005). [4]. ridush b., technogenic karst activization in zastavna karst area. sci. bull. of chnu (geography), 395: 189-196, (2006). (in ukrainian). [5]. sharmal b., tyagi s., simplification of metal ion analysis in fresh water samples by atomic absorption spectroscopy for laboratory students. j. lab. chem. edu., 1(3): 54-58, (2013). [6]. bader n. r., sample preparation for flame atomic absorption spectroscopy: an overview. rasayan j. chem., 4(1): 49-55, (2011). [7]. welz b., sperling m., atomic absorption spectrometry, wiley-vch, weinheim, germany, 941 p., (1999) [8]. ramadan n., bader, n. r., zimmermann b., sample preparation for atomic spectroscopic analysis: an overview. adv. in app. sci. res., 3(3): 1733-1737, (2012). [9]. klimchuk l. m., information bulletin on activization of dangerous exogenic geological processes in ukraine according to egp monitoring data. geoinformation of ukraine. 4: 22p, (2007). [10]. klimchouk a. b., hypogenic speleogenesis, its hydrogeological importance and role in the karst evolution, diaipi, simferopol, 411 p., (2013) (in russian). [11]. klimchouk a. b., tokarev s. v., problems of drinking water sources protection at the karst exposure. ukr. j. geogr. 1: 43-52 (2014) (in russian). 1. introduction 4. conclusion the use of stabilized frying fat in the production of potato chips 249 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 3 2016, pag. 249 254 the use of stabilized frying fat in the production of potato chips *olena kovalenko1, vladimir kovbasa1, irina radzievska1 1 department of bakery and confectionery goods technology, national university of food technology volodymyrska str. 68, kyiv, ukraine, 01601, alenkala@ukr.net *corresponding author received july 3th 2016, accepted september 28th 2016 abstract: the influence of natural and synthetic antioxidants of phenolic nature on the oxidation process of blended fats in deep-frying conditions was studied. it was established that heat treatment leads to accumulation of products of thermal destruction in all investigated samples. to slow the oxidation process down during frying potato chips, antioxidants were added to the investigated samples of frying fat. all the antioxidants used slow down effectively the process of oxidation, but their effectiveness varies. from the analysis of the dynamics of oxidation process it is possible to conclude that the use of the complex additive bht + bha-pd 50070-5e as antioxidant in frying fat is a potential advantage. keywords: antioxidant, chips, frying fat, grease, snacks. 1. introduction over the past 10 years, persistent adverse changes in the structure of nutrition of the population of ukraine were observed, including a sharp decline in consumption of biologically valuable products meat, milk, eggs, fish, vegetables, fruits, vegetable oils, while relatively stable high level of consumption of bread, potatoes and animal fats. modern trends in nutrition and high pace of life contribute to the growth of consumption of fast food. deepfried products, like chips, cakes, donuts, sweet twigs, etc. have great success among consumers. a common product in many sectors of the population, especially the young generation became potato chips, which is a kind of snack. consumption of potato chips in ukraine is estimated at 0.5 kg capita per year. in europe the figure ranges from 1 to 5 kg, in the united states it reaches 10 kg per year [1]. despite the fact that sunflower oil is commonly used for deep-frying, in its physical, chemical and consuming properties, it is not ideal for this purpose. prolonged heating, at high temperatures, causes profound changes in its quality. therefore, in the process of frying it is better to use special heat-resistant oils, which allows preparing five times more products than with the same volume of sunflower oil. the largest suppliers of frying fat on the market of ukraine are «bears company» and «trilini international». the raw material for the production of frying fat is vegetable hydrogenated fat, sunflower oil, palm oil and transesterificated grease. most types of frying fat for frying in an intensive mode are produced from selectively hydrogenized sunflower or other oil, which is determined by the prices of raw materials and its availability in the market. ukrainian scientific research institute of oils and fats developed recipes to cooking fat "frytyurniy", the raw material for the production of which is hydrogenated fat, http://www.fia.usv.ro/fiajournal mailto:alenkala@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 olena kovalenko, vladimir kovbasa, irina radzievska, the use of stabilized frying fat in the production of potato chips food and environment safety, volume xv, issue 3 – 2016, pag. 249 – 254 250 palm oil, palm olein and transesterificated grease [2], [3]. prolonged deep-frying at the temperature of 140 180°c is changing the quality of fats: they darken, acquire a sharp odor and a bitter taste [4]. these effects are caused by the formation of primary and secondary oxidation products. the primary ones are peroxides, hydroxides; their content is determined by the peroxide number (pn). they irritate the walls of the digestive tract and liver, resulting in inflammation of these organs in severe forms. secondary products of oxidation of fats are aldehydes, ketones, epoxides, polymeric compounds; their content is correlated with the value of the anisidine number (an). dangerous are the products of radical oxidation, the concentration of which exceeds maximum content standards. recent studies have found that the high content of products of radical lipid oxidation in the tissues of people provoke diseases such as atherosclerosis, cancer, premature aging, alzheimer's disease and parkinson's disease. reducing the rate of accumulation of oxidation products is possible by adding antioxidants into fat. butylated hydroxyanisole, propyl gallate, and polymethyloxane added in amount of 0.52.0 mg / kg slow down significantly oxidation and polymerization processes that occur during frying. inhibition of oxidative processes with antioxidants helps to preserve food and biological value of both the oil and the fried product. quality control of frying fat is a crucial element of reducing risks to the health of consumers of these products. research of deep-frying fat is conducted to confirm its quality and the quality of deep-fried products. 2. materials and methods this research is devoted to finding an active antioxidant for frying fat for the production of potato chips. the research was conducted on samples of liquid frying fat of the following composition: №1 palm olein 40% + corn oil 30% + rapeseed oil 30% and №2 -corn oil 50% + rapeseed oil 50%. as a control sample the sunflower oil, which is widely used in ukraine for frying was chosen. experimental and control samples were used for frying potato petals at a temperature of 160°c for 210 ± 30 sec. to prevent the oxidation of fats under thermal impact, antioxidants: tocoblendl 70 ip by company "bipraym" a mixture of tocopherols (α-tocopherol 920%, β-tocopherol 1-4%, γ-tocopherol 5065%, δ-tocopherols 20-35 %) in sunflower oil were used; bht (butylated hydroxytoluene 100%) of the company "bipraym"; complex antioxidant bht + bha-pd 50070-5e (see composition) of the company «marisa». the control of oxidation was determined by monitoring the value of the pn and an by standard methods [5], [6]. roasting was stopped when pn reached over 10 mmole ½o/kg, and/or an over 6 conventional units. 3. results and discussion through the influence of a number of factors that initiate thermal oxidation of fats today we do not have a universal method of preventing oxidative deterioration of fat-containing products [7]. for this reason, it is necessary to assess the main factors of oxidation of fats and choose the appropriate measures of their inactivation in each case. currently, the main way to increase the oxidative stability is the addition of antioxidants and their synergists. however, the approach to the antioxidant protection of each food system should be individual. practically, potato chips can contain up to 42% fat [iso 4608: 2006], which is in contact with air and exposed to high temperatures during frying. therefore, in this case an effective way to protect food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 olena kovalenko, vladimir kovbasa, irina radzievska, the use of stabilized frying fat in the production of potato chips food and environment safety, volume xv, issue 3 – 2016, pag. 249 – 254 251 products from oxidation is to add antioxidants into frying fat before the beginning of the process and in the process of frying. scientific results obtained in previous studies allowed choosing samples of blended frying fats: №1 palm olein 40% + corn oil 30% + rapeseed oil 30%, and №2 corn oil 50% + rapeseed oil 50%, which are characterized by high biological value due to the balanced fatty acid composition. to slow the oxidation during frying of potato chips antioxidants were added in grease. natural and synthetic antioxidants of phenolic nature authorized by the ministry of health of ukraine for use in food in ukraine and europe were used. when deep-frying potato slices weighing 70 ± 15 g in oil with the addition of antioxidants and without them for 5 h, period of possible application of oil in these conditions, at a temperature of 160°c, pn and an were monitored (table 1) every 60 minutes. to study the effectiveness of antioxidants of blended oils they were added according to the recommendations of the manufacturer in the amount of 0.02% of weight before frying. table 1. dynamics of thermal oxidation of samples of frying fats sample the duration of frying, min. pn, [mole½о/kg] an, [conventional units] 0 60 180 300 0 60 180 300 control sample – sunflower oil 2.00 4.00 9.04 15.30 4.17 5.60 8.17 11.15 №1 – palm olein 40% + corn oil 30% + rapeseed oil 30% without additives 3.80 6.29 9.36 11.90 0.67 1.96 2.90 6.63 tocoblendl 70 ip 2.72 5.70 9.43 11.11 0.50 2.07 3.29 5.50 bht+bha-pd 50070-5e 2.22 4.05 6.75 7.40 0.48 2.13 3.44 4.08 bht 2.22 4.60 7.53 10.62 0.50 2.09 3.62 5.25 №2 – corn oil 50% + rapeseed oil 50% without additives 4.82 7.24 9.70 14.36 0.74 4.18 6.17 9.25 tocoblendl 70 ip 4.50 6.12 8.60 13.10 0.58 4.18 5.93 6.57 bht+bha-pd 50070-5e 4.50 5.21 7.95 10.36 0.46 2.39 3.37 4.37 bht 4.53 6.39 8.09 12.90 0.46 3.15 4.28 5.69 n=3, δ±0.5 by analyzing the results of the above table, we can see that all the additives slow down the oxidation of blended fats, but their effectiveness varies. in our studies the most effective antioxidant appeared to be the bht + bha-pd 50070-5e in both studied samples of fat. this comprehensive antioxidant shows the inhibitory effect in the processes of both primary and secondary oxidation, which is set by the values of pn and an. obviously, this is explained by inactivation of free radicals, which rate is higher than the processes that occur when adding other antioxidants. other additives also show high stabilizing properties. specifically, it was found that bht and tocoblendl 70 ip, added in the same dose, actively stabilize the thermal oxidation of blended fats, when the first is relatively a more active antioxidant. it is possible to explain the discovered impact of studied antioxidants in terms of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 olena kovalenko, vladimir kovbasa, irina radzievska, the use of stabilized frying fat in the production of potato chips food and environment safety, volume xv, issue 3 – 2016, pag. 249 – 254 252 the chemistry of oxidation. the process of oxidation of vegetable oil is a free radical chain process that can be described by the following stages [8], [9]: initiation: rh + o2 → r· + oh development: r· +o2 → roo· roo· + rh → rooh + r· breakage: r· + r· → rr r· + roo· → roor roo· + roo· → roor + o2 in the first initiation phase a free radical is formed from the lipid substrate under the influence of the initiator. branching chain is a result of the radical collapse of the hydroperoxides that are the only primary oxidation products. with the presence of an inhibitor in oils an additional channel of consumption of peroxide radicals appears: roo· + inh → rooh +in· roo· + in· → molecular products it is known that antioxidants destroy free radicals in fats, giving them hydrogen and turning into relatively stable radicals with lower recovery potential [10]. higher stability of the antioxidant radical compared to the radicals of fat is the result of resonance delocalization in the structure of the phenolic ring of the antioxidant, which is lost in the first place [11], [12]. during the oxidation of saturated fatty acids saturated hydroperoxides are formed, while during the oxidation of unsaturated fatty acids unsaturated hydroperoxides. in this case the oxidation is not due to the addition of oxygen to the double bond of the acid, but as a result of the separation of hydrogen from the methylene group located adjacent to the double bond. during the oxidation of linoleic acid hydroperoxides with the two conjugated double bonds can be formed. the formation of cyclic peroxides happens as a result of addition of oxygen at the place of the double bond of the acid. in the thermal oxidation unstable primary products hydroperoxides are formed, which break down to form secondary oxidation products epoxides, aldehydes, ketones and other products. simultaneous control of the content of oxidation products is possible by a combined indicator of full fat oxidation [13]. totox, the integral indicator of the oxidation that takes into account the total number of primary and secondary oxidation products, was calculated as тотох= 2pn+an. figure (1) shows the progress of the oxidation of the studied blended frying fats with added antioxidants and without them. а) b) figure 1. oxidation of blended frying fats with addition of antioxidants (a), and without them (b). 4 8 12 16 20 24 28 32 36 40 0 min 60 min 180 min 300 min т о т о х time control sample sunflow er oil №1 palm olein 40% + corn oil 30% + rapeseed oil 30% 8 12 16 20 24 28 32 36 40 0 min 60 min 180 min 300 min т о т о х time contr ol sampl e sunflo wer oil №2 corn oil 50% + rapese ed oil 50% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 olena kovalenko, vladimir kovbasa, irina radzievska, the use of stabilized frying fat in the production of potato chips food and environment safety, volume xv, issue 3 – 2016, pag. 249 – 254 253 figure (1) shows that heat treatment leads to accumulation of thermal degradation in all investigated samples. all applied antioxidants effectively slow down the process of oxidation, but their effectiveness varies. analyzing the dynamics of the process of oxidation it is possible to conclude about the potential advantage of the use of the complex additive bht + bha-pd 50070-5e as an antioxidant for frying fat. in particular, after 300 minutes of heat treatment the rate of accumulation of oxidation products decreased from 41.75 conventional units in the control sample to 18.88 conventional units in the №1 blend and to 25.09 conventional units in the №2 blend. thus, the effectiveness of the antioxidant impact of this additive when added to a blend №1 is 2.2 times and when added to a blend №2 – 1.7 times. the discovered effects of prolongation of slowing the degradation of blended fats can be explained in terms of the mechanism of processes of oxidation and stabilization of lipids. in our view, a comprehensive antioxidant bht + bhapd 50070-5e inactivates free radical oxidation products in the fat turning at the same time in relatively stable radicals with low recovery potential. probably, this higher activity of this additive compared to other used antioxidants is the result of mutual influence of atoms in the structure of the rings of phenolic molecules of butylated hydroxytoluene, butylated hydroxyanisole, butylated hydroquinone in its composition [11], [12]. in addition, the high efficiency of inhibiting free radicals can be explained by relatively lower dissociation energy of the o-h phenol connection in the structure of these molecules. since the antioxidant with low dissociation energy is a more efficient hydrogen donor in the reactions of oxidation chain breakage, therefore he is a more effective antiradical agent [14], [15]. so, higher (better) antiradical activity of the complex antioxidant bht + bha-pd 50070-5e is explained by the synergistic effect between the molecules of its active components. this leads to a rapid transfer of electrons from these molecules to peroxyl radicals of fat and as a result to a rapid breakage of the reaction of formation of oxidation products. the observed synergy phenomenon is explained by the ability of molecules of butylated hydroxytoluene, butylated hydroxyanisole and butylated hydroquinone in a complex antioxidant to transfer hydrogen of a phenolic group simultaneously to peroxyl, alkoxyl and alkyl radicals, thus slowing down the formation of both primary and secondary oxidation products. 4. conclusion it is shown that one of the ways to increase the quality of potato chips is the use of antioxidant supplements during frying. mechanisms of oxidation and stabilization of blended frying fats in terms of thermal oxidation were analyzed. it is obvious high efficiency of the complex antioxidant bht + bha-pd 50070-5e, consisting of butylated hydroxytoluene, butylated hydroxyanisole, tertiary butylated hydroquinone and ether of citric acid of monoand diglycerides of fatty acids (е471). 5. references [1]. bondarchuk a.a., potatoes, bila tserkva, т.з. – 536 p., (2007) [2]. o'brien r. fats and oils. production, composition and properties, usage / r. o'brien; translated from english. 2-nd edition.v.d. shirokova, d.a. babeikina, n.s. selivanova, n.v. magda. – st. petersburg: “professiya”– 752 р., (2007) [3]. collection of recipes. margarine, confectionery, baking, cooking and dairy industry fats, hydrogenated fats 25660944.010-2003.: – ukrainian research institute of oils and fats – 50 p., (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 olena kovalenko, vladimir kovbasa, irina radzievska, the use of stabilized frying fat in the production of potato chips food and environment safety, volume xv, issue 3 – 2016, pag. 249 – 254 254 [4]. man, y. c., tan, c. p.,effects of natural and synthetic antioxidants on changes in refined, bleached, and deodorized palm olein during deepfat frying of potato chips, journal of the american oil chemists' society, 76.3: 331-339, (1999) [5]. iso 3960-2001 animal and vegetablefats and oils. determination of the peroxide number (iso 3960:1998, idt). [6]. iso 6885 –2002 animal and vegetablefats and oils. determination of theanisidine number. [7]. sarkisian, v.a., smirnova, e. a., kochetkova, a. a., bessonov, v. v., synergistic interaction of antioxidants in fatty foods, food processing industry, 3: 14-17, (2013) [8]. fomin, v. m., the radical chain oxidation of organic compounds and itsdeceleration by phenolic inhibitors, nizhegorodskii university, nizhnii novgorod, 37, (2010) [9]. denisov, e. t., cyclic mechanisms of chain termination reactions in the oxidation of organic compounds, russian chemical, 65.6: 547-563, (1996) [10]. choe, e., min, d. b., chemistry and reactions of reactive oxygen species in foods, journal of food science, 70.9: 142-159, (2005) [11]. choe, e., min, d. b., mechanisms and factors for edible oil oxidation, comprehensive reviews in food science and food safety, 5.4: 169186, (2006) [12]. amorati, r., cavalli, a., fumo, m.g., masetti, m., kinetic and thermochemical study of the antioxidant activity of sulfur-containing analogues of vitamin e, chemistry a european journal, 13.29: 8223-8230, (2007) [13]. lisitcin, a. n., alymova, t. b., prokhorova, l. t., some factors determining the stability of vegetable oils to oxidation, oilseed industry, 5: 14–15, (2005) [14]. shahidi, f., janitha, p. k, wanasundara, p. d., phenolic antioxidants, critical reviews in food science & nutrition,32.1(1):67-103, (1992) [15]. noor, n., augustin, m. a., effectiveness of antioxidants on the stability of banana chips, journal of the science of food and agriculture, 35.7: 805-812, (1984) 67 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 67 – 73 the influence of storage temperature and time on total phenolic content in a soft wheat cultivar *marcel avramiuc1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received 5th march 2015, accepted 29th march 2015 abstract: the influence of storage temperature and time on total phenolic content (tpc) in a soft wheat cultivar, was the purpose of this paper. the biological material was represented by soft wheat grains from which there were formed control and working samples (grains, flour and bran). the control samples were stored at 10°c, and the working ones at four thermal thresholds (40ºc, 60ºc, 120ºc and 180ºc) for 15 days. aftet 5, 10 and 15 days, total phenolic content (tpc) has been estimated by a colorimetric assay, by measuring the ability of phenols to reduce folin-ciocalteu reagent. in all analyzed samples, compared to controls (samples stored at 10ºc), the largest decreases of total phenolic content were registered after 15 days of storage, with significant percentage reductions at 180°c and 120°c, in flour type 480 (wf), and in whole-wheat flour (wwf). after 10 days of storage, significant percentage reductions of tpc were registered in flour type 480 (wf), and in whole-wheat flour (wwf), and after 5 days in wf (in all cases in samples stored at 180°c and 120°c). the percentage decrease of tpc values was emphasized more when the storage temperature increased from 120°c to 180°c, compared with the thresholds 40-60°c or 60-120°c. the keeping of soft wheat samples under high temperature conditions led to a greater reduction in the total content of phenolic compounds,as compared to grains or bran. keywords: storage, temperature, wheat, grains, flour, bran, total phenolic content 1. introduction phenolic compounds are organic substances widespread in plants. present in more than 4000 different types of plants [1], these compounds highlight various physiological properties such as: antiallergenic, antioxidant, anti-artherogenic, anti-inflammatory, anti-microbial, antithrombotic, cardio-protective and vasodilatory effects [2-7]. wheat is an important component of the human diet, and an important source of phenolic antioxidants [8], wheat cultivars having a wide variation of phenolic content [9]. in cereal grains, the phenolic acids are found in three forms: free, soluble conjugate and insoluble bound [9]. bound phenolics are considered to have have more health benefits because they escape from the upper gastrointestinal digestion conditions with cell wall materials, and after they are absorbed into blood plasma previous hydrolysis from esterases present in the intestinal microflora [10 ct. by 8]. thus, wheat bran consumption increases total phenols and antioxidant capacity in plasma to a comparable extent to some other phenol-rich foods [11]. it is known that heat processing influences http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am iu c , t he i n f l u e nc e of st or a g e t e m p er a t ur e a nd t i m e o n t ot a l p h en o l i c c on t e n t i n a s o f t wh ea t c u l t i v a r , f oo d a nd e n vi ro nme nt s a f et y, v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 67 73 68 the concentration and quality of food nutrients (carbohydrates, lipids, proteins), but there are less information, in this regard, on natural antioxidants and their activity. it seems that thermal treatment significantly reduces concentration of natural antioxidants, but the overall antioxidant properties of food products were maintained or even enhanced by the development of maillard products [12]. because of vulnerability to rancidity or for reducing number of microorganisms, sometimes is necessary a preheat treatment of wheat bran, germ or flour. the temperature used in the preheat treatment is relatively low not exceeding 100°c, and heat exposure time is short [13]. in this paper it has searched the influence of storage temperature and time on total phenolic content (tpc) in four samples, belonging to a soft wheat cultivar, stored 15 days at four different thermal thresholds. 2. experimental the biological material, supplied by suceava agricultural research and development station, was represented by grains (wg), belonging to a local cultivar of soft wheat (triticum aestivum l. spp. aestivum), from which, by grinding, there were formed the other samples of this research, such as: integral flour made from ground beans (whole-wheat flour, wwf), bran (wb) and flour type 480 (wf). type 480 signifies the maximum content of ashes in flour (0,480) multiplied by 1000. before the beginning of experiment, the moisture content of wheat samples was between 8 and 10%. control samples were stored at 10ºc, and work samples at four different thermal thresholds (40ºc, 60ºc, 120ºc and 180ºc) for 15 days, using thermostats and ovens set at temperatures above mentioned. to determine total phenolic contents (tpc), first it was obtained an extract for each sample, weighting each 1 g of grain (flour or bran) which were finely ground and subjected to extraction with a mixture methanol and water 80:20 (v/v), by stirring, centrifuging and recovering the supernatant [14]. the estimation of total phenolic content in seed extracts was carried out through a colorimetric assay, by measuring the capacity of phenols to reduce folinciocalteu reagent. for this pupose, it has generated a standard curve (fig.1) representing the absorbance values of gallic acid standard solutions in relation to their concentrations [15]. tpc was expressed as mg gallic acid equivalent per gram dry matter (gae/g d.m.). the data obtained from four replications were analyzed using statistical package for social science software, version 16.0. the correlation analyses were performed at the probability levels of 95% and 99%. the differences between mean values of tpc were tested using analysis of variance anova one-way. in order to highlight the degree of influence of storage temperature and duration and the interaction between them on tpc in each wheat samples, there was applied factorial analysis of variance [16]. further, in tab. 1 there are shown mean values of total phenolic content in the 4 samples of soft wheat, kept at 10ºc (control samples = blank). fig. 1. standard curve for tpc, using gallic acid y = 0,0011x + 0,0031 r2 = 0,9964 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 100 200 300 400 500 600 gallic acid conce ntration (μg/ml) a bs or ba nc e (7 65 n m ) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am iu c , t he i n f l u e nc e of st or a g e t e m p er a t ur e a nd t i m e o n t ot a l p h en o l i c c on t e n t i n a s o f t wh ea t c u l t i v a r , f oo d a nd e n vi ro nme nt s a f et y, v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 67 73 69 table 1 total phenolic content mean values (±sd) of the control wheat samples test tpc (mg gae/g d.m.) wheat samples wg wwf wb wf 1.95±0.035 2.01±0.03 2.76±0.08 0.79±0.03 sd=standard deviation; tpc=total phenolic content; gae=gallic acid equivalent; wg=wheat grains; wwf=whole-wheat flour; wb=wheat bran; wf=white flour (type 480) 3. results and discusion from table 1 it can observe that the highest mean values of tpc were in bran samples, wb (2.76 ± 0.08 mg gae/g), and the lowest ones in flour type 480 samples, wf (0.79 ± 0.03 mg gae/g). fig. 2 reproduces the distribution of tpc mean values in the four samples of soft wheat, compared to average values of the control (blank) samples, after 5 days of storage at thermal thresholds analyzed. compared with the control (1.95 ± 0.035 mg gae/g), tpc of wheat grain (wg) has changed very little, dropping only 2% at 180°c (1.91 ± 0.03 mg gae/g) . tpc mean values in whole-wheat flour samples (wwf) has decreased by 2%, compared to the control, at 40°c (1.97 ± 0.024 mg gae/g), by 5.5 % at 120°c (1.90 ± 0.08 mg gae/g), and by 8.4% at 180°c (1.84 ± 0.05 mg gae/g). during the five days, tpc correlated negatively with storage temperature (r = –0.494, p>0.05). after 5 days of storage, tpc mean values from bran (wb) fell by 2.2% at 120°c (2.70 ± 0.06 mg gae/g), and by 5.4% at 180°c (2.61 ± 0.045 mg gae/g) (r = – 0.363, p>0.05). tpc mean values in flour (wf) were reduced, compared to the blank, by 8.8% at 120°c (0.72 ± 0.04 mg gae/g) and by 21.5% at 180°c (0.62 ± 0.02 mg gae/g). during the five days, in wf samples, the correlation analysis between tpc and temperature has shown negative correlations (r = –0.892, p<0.05). fig. 3 plays the mean values of tpc in the four samples of soft wheat, compared to average values of the control (blank samples), after 10 days of storage. fig. 2. mean values of phenols total content after 5 days keeping of soft wheat samples wg=wheat grains; wwf=whole-wheat flour; wb=wheat bran; wf=white flour (type 480) 0 0,5 1 1,5 2 2,5 3 3,5 blank 40°c 60°c 120°c 180°c storage temperatures tp c m ea n va lu es (m g g a e /g ) wg wwf wb wf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am iu c , t he i n f l u e nc e of st or a g e t e m p er a t ur e a nd t i m e o n t ot a l p h en o l i c c on t e n t i n a s o f t wh ea t c u l t i v a r , f oo d a nd e n vi ro nme nt s a f et y, v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 67 73 70 fig. 3. mean values of phenols total content after 10 days keeping of soft wheat samples wg=wheat grains; wwf=whole-wheat flour; wb=wheat bran; wf=white flour (type 480) compared with the control, after 10 days tpc of wheat grains (wg) fell by 2.5% at 60°c (1.90 ± 0.04 mg gae/g) and 120°c (1.90 ± 0.03 mg gae/g) and by 5.1% at 180°c (1.85 ± 0.05 mg gae/g) (r = – 0.347, p>0.05). tpc mean values in whole-wheat flour samples (wwf) have decreased by 4.5% at 40°c (1.92 ± 0.04 mg gae/g), by 6.5% at 60°c (1.88 ± 0.06 mg gae/g), by 8.9% at 120°c (1.83 ± 0.05 mg gae/g), and by 13.4% at 180°c (± 1.74 0.03 mg gae/g). in wwf samples, the correlation analysis between tpc and temperature has shown negative correlations (r=–0.865, p<0.05). after 10 days of storage, tpc mean values in bran (wb) fell by 1.8% at 60°c (2.71 ± 0.075 mg gae/g), by 4.3% at 120°c (2.64 ± 0.06 mg gae/g), and by 8.7% at 180°c (2.52 ± 0.09 mg gae/g). during the ten days of storage, tpc correlated negatively with storage temperature (r = –0.548, p>0.05). compared to the control, tpc mean values in flour (wf) were reduced by 6.3% at 40°c (0.74 ± 0.045 mg gae/g) and 60°c (0.74 ± 0.03 mg gae/g), by 17.7% at 120° c (0.65 ± 0.025 mg gae/g), and by 39.2% at 180°c (0.48 ± 0.04 mg gae/g) (r = –0.947, p<0.01). in fig. 4 are reproduced mean values of tpc in wheat samples after 15 days of storage. fig. 4. mean values of phenols total content after 15 days keeping of soft wheat samples wg=wheat grains; wwf=whole-wheat flour; wb=wheat bran; wf=white flour (type 480) 0 0,5 1 1,5 2 2,5 3 blank 40°c 60°c 120°c 180°c storage temperatures tp c m ea n v al ue s (m g g a e /g ) wg wwf wb wf 0 0,5 1 1,5 2 2,5 3 3,5 blank 40°c 60°c 120°c 180°c storage temperature tp c m ea n va lu es (m g g a e /g ) wg wwf wb wf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am i u c , th e i n f l u e nc e of s t or a g e t e m p er at ur e a nd t i m e o n t o t a l p he n o l i c co n t e nt i n a s o f t wh ea t c u l t i v a r , f oo d and e n vi ro nme nt s af et y, v ol ume x iv , is s ue 1 – 20 1 5, p ag . 6 7 7 3 71 after 15 days, compared with the control, tpc in wheat grains (wg) fell by 4.6% at 60°c (1.86 ± 0.03 mg gae/g), by 5.6% at 120°c (1.84 ± 0.04 mg gae/g) and by 12.8% at 180°c (1.75 ± 0.025 mg gae/g). in wg samples, tpc correlated negatively with storage temperature (r = –0.624, p>0.05). compared with the control, tpc mean values in whole-wheat flour (wwf) has decreased by 5.9% at 40°c (1.89 ± 0.025 mg gae/g) by 9.4% at 60°c (1.82 ± 0.04 mg gae/g), by 14.4% at 120°c (1.72 ± 0.06 mg gae/g) and by 20.4% at 180°c (± 1.60 0.03 mg gae/g). after 15 days of storage, in wwf samples, the correlation analysis between tpc and storage temperature has shown significant negative correlations (r = –0,925, p<0,01). after 15 days of storage, tpc in bran samples (wb) decreased by 4% at 60°c (2.65 ± 0.06 mg gae/g), by 8.7% at 120°c (2.52 ± 0.08 mg gae/g) and by 13% at 180°c (2.40 ± 0.07 mg gae/g) (r = –0.597, p>0.05). after 15 days, tpc mean values in flour samples (wf) were reduced compared to the control, by 11.4% at 40°c (0.70 ± 0.035 mg gae/g), by 24% at 60°c (0.60 ± 0.04 mg gae/g), by 35.4% at 120°c (0.51 ± 0.025 mg gae/g) and by 59.5% at 180°c (0.32 ± 0.05 mg gae/g). in wf samples, the correlation analysis between tpc and storage temperature has shown significant negative correlations (r = – 0,995, p<0,01). the value of f test has indicated a significant cumulative effect (p=0.000) of the factors temperature and storage time on tpc in wf and wwf. tpc mean values in blank differ significantly (p<0.01) of those ones from wf samples (stored 5, 10 and 15 days), anf from wwf (stored 10 and 15 days), the storage temperature significantly influencing these values. in the table 2 are centralized the decreasing percentages of tpc mean values in wheat samples. table 2 percent reduction of tpc mean values in wheat samples kept at different temperatures test tpc reduction percent duration 5 days 10 days 15 days st* wg wwf wb wf wg wwf wb wf wg wwf wb wf 40°c 2% 4.5% 6.3% 5.9% 11.4% 60°c 2.5% 6.5% 1.8% 6.3% 4.6% 9.4% 4% 24% 120°c 5.5% 2.2% 8.8% 2.5% 8.9% 4.3% 17.7% 5.6% 14.4% 8.7% 35.4% 180°c 2% 8.4% 5.4% 21.5% 5.1% 13.4% 8.7% 39.2% 12.8% 20.4% 13% 59.5% st*= storage temperature; wg=wheat grains; wwf=whole-wheat flour; wb=wheat bran; wf=white flour (type 480) as it can see in all samples analyzed, the largest decreases of tpc were after 15 days of storage, with the largest percentage reductions at 180°c and 120°c, in wf, followed by wwf. although tpc reductions were also in wb and wg (with higher values at 180°c and 120°c), they did not differ significantly from controls, and therefore could not be taken into account. compared to controls, after 10 days of storage, significant percentage reductions of tpc were in wf and wwf, but after 5 days only in wf (in the samples stored at 180°c and 120°c). from table 3 one can see that the percent decrease of tpc values was emphasized more when the storage temperature increased from 120°c to 180°c, compared with the thresholds 40-60°c or 60-120°c. except wg sample, the other samples (wwf, wb and wf) were stored at time intervals and working temperatures as powder with particles of different sizes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am i u c , th e i n f l u e nc e of s t or a g e t e m p er at ur e a nd t i m e o n t o t a l p he n o l i c co n t e nt i n a s o f t wh ea t c u l t i v a r , f oo d and e n vi ro nme nt s af et y, v ol ume x iv , is s ue 1 – 20 1 5, p ag . 6 7 7 3 72 exposure of wheat bran to high temperature (100°c) resulted in partial loss of total phenolic compounds and significant decline in antioxidant activity [17]. according to cheng et al., 2006 [17], the effect of temperature under storage on total phenolic compounds and antioxidant activity varied significantly depending on the material stored, particle size, wheat variety, or assay methods used for antioxidant activity reduction of wheat bran particle size facilitates phytochemical release, thus enhancing the available and maybe bioavailable antioxidant activity [18]. if are not protected, phytochemicals are exposed (through grinding) to oxidation, resulting in shorter shelf life and loss of antioxidant activity [17]. in this paper, of the wheat samples exposed for 15 days at the four thermal thresholds, wf (wheat flour type 480) had the smallest particle size, which made that the phenolic acids from its composition to be exposed to oxidation, accelerated once with increasing of temperature. the phenolic compounds from whole wheat grain were the most protected, being less exposed to the direct oxidation process, but in the other two samples (milled) the phenolic compounds have not undergone major changes, during storage at thermal thresholds and intervals analyzed (wwf – after 5 days, and wb – after 5, 10 and 15 days). cheng et al., 2006 [17] have stored wheat grains under different temperatures (25, 60, and 100°c) for 9 days, and found that tpc and antioxidant activity have not changed during the whole tested period, regardless of the assay methods used. 4. conclusion the storage at four different thermal thresholds (40°c, 60°c, 120°c and 180°c) of some hard wheat samples belonging to a romanian cultivar for 5, 10 and 15 days, has revealed changes in the total content of phenolic compounds, depending on storage temperature, duration, and type of sample. in all analyzed samples, compared to controls (samples stored at 10ºc), the largest decreases of total phenolic content were after 15 days of storage, with significant percentage reductions at 180°c and 120°c, in flour type 480 (wf), and in whole-wheat flour (wwf). after 10 days of storage, significant percentage reductions of tpc were in flour type 480 (wf), and in whole-wheat flour (wwf), and after 5 days in wf (in all cases in samples stored at 180°c and 120°c). the percentage decrease of tpc values was emphasized more when the storage temperature increased from 120°c to 180°c, compared with the thresholds 4060°c or 60-120°c. keeping of soft wheat samples under high temperature as flour caused a greater reduction in the total content of phenolic compounds, compared to grains or bran. 4. acknowledgments many thanks to dr. eng. dumitru bodea, director of suceava agricultural research and development station, for the biological material supplied. 5. references [1] hollman p.c., hertog m.g.l., katan m.b., analysis and health effects of flavonoids. food chemistry, 57, 43-47 (1996) [2] benavente-garcia o., castillo j.,marin f.r., ortuno a., del rio j.a. – uses and properties of citrus flavonoids. journal of agricultural and food chemistry, 45, 4505-4515 (1997) [3] samman s., lyons wall p.m., cook n.c. flavonoids and coronary heart disease: dietary perspectives. in c.a. riceevans & l. packer (eds.), flavonoids in health and disease (pp. 469-482), new york: marcel dekker (1998) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 m a rc e l av r am i u c , th e i n f l u e nc e of s t or a g e t e m p er at ur e a nd t i m e o n t o t a l p he n o l i c co n t e nt i n a s o f t wh ea t c u l t i v a r , f oo d and e n vi ro nme nt s af et y, v ol ume x iv , is s ue 1 – 20 1 5, p ag . 6 7 7 3 73 [4] middleton e., kandaswami c., theoharides t.c. – the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. pharmacological reviews, 52, 673-751 (2000) [5] puupponen-pimiä r., nohynek l., meier c., kähkönen m., heinonen m., hopia a., et al. – antimicrobial properties of phenolic compounds fom berries. journal of applied microbiology, 90, 494-507 (2001) [6] manach c., williamson g., morand c., scalbert a., rémésy c. – bioavailability and bioefficacy of polyphenols in humans. i. review of 97 bioavailability studies. american journal of clinical nutrition, 81 (suppl), 230s – 242s (2005) [7] balasundram n., sundram k., samman s. – phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. food chemistry 99, 191-199 (2006) [8] hernández lia, afonso desirée, rodríguez elena m., díaz carlos phenolic compounds in wheat grain cultivars. plant foods for human nutrition © springer science+business media, lc 2011 10.1007/s 11130-011-0261-1 (2011) [9] li l, shewry p., ward j. phenolic acids in wheat varieties in the health grain diversity screen. j agric food chem 56, 9732–9739 (2008) [10] andreasen m, kroon p, williamson g, garcía-conesa m.t. intestinal release and uptake of phenolic antioxidant diferulic acids. free radical biol med 31, 304-314 (2001) [11] price r.k., welch r.w., lee-manion a.m., bradbury l, strain j.j. total phenolics and antioxidant potential in plasma and urine of humas after consumption of wheat bran. cereal chem 85, 152–157 (2008) [12] slavin j.l., jacobs d., marquart l. crit. rev. food sci. nutr., 40 (4), 309-327 (2000) [13] lin c.j., guo g., mennel d.l. effects of postharvest treatments, food formulation, and processing conditions on wheat antioxidant properties in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 78-79 (2008) [14] adom, k. k., liu r. h. antioxidant activity of grains. j. agric. food chem. 50, 6182-6187, (2002) [15] moore j., yu liangli (lucy) methods for antioxidant capacity estimation of wheat and wheat-based food products in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 147-150 (2008) [16] tabachnick b.g., fidell l.s. – using multivariate statistic (5th ed.) pearson, london, new york (2007) [17] cheng z., su l., moore j., zhou k., luther m., yin j.j., yu l. j. agric. food chem. 53, 2433– 2440. (2006) [18] martinez-tome m., murcia a., frega n., ruggieri s., jimenez a.m., roses f., parras p. j. agric. food chem. 52, 4690–4699 (2004) 278 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 278 283 calcium lactate influence on some non-pathogenic microorganisms octavian baston1, *octavian barna1 1dunarea de jos university of galati, 111, domneasca street, 800201, galati, romania, phone: 0336 130 177, fax: 0336 130 281, octavian.baston@ugal.ro, octavian.barna@yahoo.com corresponding autor octavian barna, octavian.barna@yahoo.com * corresponding author received august 29th 2013, accepted september 14th 2013 abstract: after calcium lactate was produced by chemical reaction between lactic acid and calcium carbonate from eggshells, it was tested on some non-pathogenic microorganisms. the influence of calcium lactate powder on non-pathogenic microorganisms varied as group and species. all the nonpathogenic bacteria growth was inhibited by calcium lactate, the inhibition diameter ranged between 1.92 ± 0.23 to 2.35 ± 0.12 cm. among yeasts only candida mycoderma and pichia sp. growth was inhibited. calcium lactate showed no influence on the studied moulds: aspergillus niger, penicillium sp. and rhizopus sp. calcium lactate is a substance that has an inhibition effect on some nonpathogenic micoorganisms. it can be used alone, without being combined with other substances, having an acidifying effect on the microorganism nutritive media. keywords: radial diffusion, cup plate method, bacteria, mould, yeast. 1. introduction lactic acid can be produced biochemically by fermentation or chemically by synthesis of petrochemical compounds [1]. according to hofvendahl [2], the biotechnological production of lactic acid has the following advantages over the chemical synthesis: environment protection, the natural resources used for biotechnological production are renewable, the lactic acid isomer produced can be chosen because it is well known that only l(+)-lactic acid isomer is used in food industry and pharmaceutical compounds. calcium lactate is now used to preserve various foodstuffs and prolong shelf life of food. its main applications include preservation of vegetables, fruits, meat and meat products, also being added in beverages, jelly, chewing gum, candy products [3-5]. moreover, calcium lactate can be used in food industry as calcium source, dietary supplement, for preserving fresh food [6-7], as antioxidant and stabilizer [4, 8], as antimicrobial [5, 9-11]. it can be produced by chemical reaction between lactic acid and calcium carbonate. the calcium carbonate can be used as a pure, commercial substance, or can be obtained from egg shells or crustacean shells. it is recommended to use calcium from eggshells because eggshells are considered to be a waste product, and according to omi [12], the calcium from eggshell powder is more easily absorbed by the rat small intestine than the commercial calcium carbonate. calcium lactate has antimicrobial activity against aerobic and anaerobic microorganisms that can be found in meat. although calcium lactate, sodium lactate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 octavian baston, octavian barna, calcium lactate influence on some non-pathogenic microorganisms, food and environment safety, volume xii, issue 3 – 2013, pag. 278 – 283 279 and potassium lactate have some bactericidal activity, their ability to control bacteria and the pathogens from fresh and processed meat is due to their use together with other antimicrobial agents such as : malic acid, glutathione, acetyl-cysteine [13, 14]. the mixture of the previously mentioned substances acts upon e. coli o157: h7, mesophilic bacteria, psychrophilic bacteria, yeasts and moulds [14]. calcium lactate activity on fruits and vegetables resulted in reduced water activity (for melon) when it was immersed in 2.5 % calcium lactate solution at 4 °c [7]. when applied at high temperature (50 ... 60 °c), there was diffusion of calcium in tissues [15] and the formation of calciumbridges between pectin molecules. the calcium lactate alone has some effect on the microorganisms and it can be used in combination with other substances such as sodium diacetate and sodium chloride. the objective of this study was to produce calcium lactate by chemical reaction between lactic acid and calcium carbonate from eggshells powder. the calcium lactate thus produced was tested on various non-pathogenic microorganisms. 2.experimental 2.1.lactic acid solution the lactic acid was produced by fermentation from jerusalem artichoke flour, according to the method presented by baston [16]. after having introduced the nutrients and optimized the fermentation parameters, we could increase the lactic acid yield from 4.5 g/l to 30 g/l. the lactic acid solution was then filtered by centrifugation with a centrifugal force of 6,026g for 10 minutes, using the centrifuge universal 320r (hettich gmbh, germany). the solution was treated with active charcoal and then centrifuged again at 3,075g for 15 minutes. 2.2. calcium lactate production for calcium lactate production we used white eggshells that were initially cleaned by washing with water at 40-50 °c, then sanitized at 130 °c for 15 minutes using ae-110 dry autoclave (raypa, spain) and dried at 50 °c for 60 minutes, using the stericell 111 (mmm medcenter einrichtungen gmbh, germany) drying stove. the dried eggshells were then converted into powder and immersed in the lactic acid solution prepared as described above. two grams of eggshell powder were introduced into a 500 ml beaker together with 100 ml of lactic acid solution. after homogenization, the mixture was left for 12 hours at 25 °c for chemical reaction. to ease the gravimetric filtration and for pasteurization purpose, the mixture (calcium carbonate solution) was heated at 70 °c for 10 minutes and filtered on whatman paper no. 1. the mixture was then freeze-dried using alpha 1-4 ld plus (martin christ gmbh, germany) lyophilization unit. the produced substance is calcium lactate powder, as can be seen in figure 1. figure 1.the freeze drying vials with calcium lactate powder food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 octavian baston, octavian barna, calcium lactate influence on some non-pathogenic microorganisms, food and environment safety, volume xii, issue 3 – 2013, pag. 278 – 283 280 the purity of the calcium lactate was determined according to food chemical codex [17]. the calcium lactate purity was of 88 ± 2.35 %. the ph of calcium lactate was determined for the 10 % solution of calcium lactate using s20 phmeter (mettler toledo, spain). 2.3.testing the antimicrobial activity of calcium lactate for the bacteria growth we used the plate count agar (pca) media from liofilchem, italy, and for all the moulds and yeasts, except saccharomyces cerevisiae, we used the malt extract agar (mea) media from scharlau chemie, spain. for saccharomyces cerevisiae we used sabouraud agar (scharlau, spain). we made a slant culture in the tubes. using a sterile loop we have drawn the following microorganism colonies: -bacteria: bacillus (b. subtilis), sarcina sp., lactobacillus sp. -yeasts: saccharomyces (s. cerevisiae), candida (candida mycoderma), pichia sp., rhodotorula sp., torulopsis sp. -moulds: aspergillus (a. niger), penicilium sp., rhizopus sp. the studied microorganisms were provided by the collection of microorganisms of bioaliment platform. using a bf 4000 oven (binder, germany) at 37 °c for bacteria and clw 32 (poleko aparatura, poland) oven at 25 °c for moulds and yeasts, we incubated the pure culture colonies on slant culture media. lactobacillus was anaerobically incubated. with a sterile loop, 9 ml of sterile suspension were taken from every slant media and every 1 ml of this suspension was inoculated in petri dishes with nutritive media characteristic to the type of microorganism. the petri dishes were incubated according to tofan [18]. the purity of pure microorganism was verified according to tofan [18]. as regards the antimicrobial activity of calcium lactate we used agar well diffusion method according to toba [19]. the well diameter was of 10 mm. the calcium lactate powder was sterilized using an ultraviolet light lamp. we used the calcium lactate powder to fill every well. the petri dishes were incubated as mentioned above, for every type of microorganism. we worked in duplicate with one more repetition of the experiment. we used microsoft excel software from microsoft office 2003 to determine the mean value and standard deviation. 3. results and discussion the results of the incubation period for every type of microorganisms are shown in table 1. table 1 the calcium lactate effect on tested microorganisms microorganism calcium lactate action bacteria sarcina sp. bacillus subtilis lactobacillus sp. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 octavian baston, octavian barna, calcium lactate influence on some non-pathogenic microorganisms, food and environment safety, volume xii, issue 3 – 2013, pag. 278 – 283 281 yeasts pichia sp. rhodotorula sp. torulopsis sp. candida mycoderma saccharomyces cerevisiae moulds aspergillus sp. rhizopus sp. penicilium sp. as can be seen in table 1, some of the non-pathogenic microorganisms studied were affected by calcium lactate, whereas others were not. all the studied bacteria showed a zone of inhibition, the extent of this zone being different, as can be seen in figure 2. figure 2.the inhibition extent of bacteria due to calcium lactate influence of all the non-pathogenic bacteria studied, we can state that bacillus subtilis registered the longest diameter, followed by lactobacillus. the inhibition of bacteria was due to the ph of the calcium lactate that diffused into the culture media and changed the pca ph value. the pca ph media was of 7.0 ± 0.2. an interesting fact occurred in the case of moulds, namely calcium lactate did not develop any inhibition zone in all the studied moulds. it seems that these ones food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 octavian baston, octavian barna, calcium lactate influence on some non-pathogenic microorganisms, food and environment safety, volume xii, issue 3 – 2013, pag. 278 – 283 282 have the capacity to adapt to ph change of the culture media and develop colonies. as regards yeasts’ inhibition, two of the studied species were affected only, as shown in figure 3. figure 3.the influence of calcium lactate on some yeast species as seen in figure 3, only candida mycoderma and pichia were affected by calcium lactate. they were more susceptible than the other studied yeasts to even calcium or carbonate ions added in the nutritive media, or to ph change of the culture media (mea). the ph of the mea culture media was of 5.4 ± 0.2 and for sabouraud agar was 5.6 ± 0.2. taking into consideration the fact that the calcium lactate produced had a ph value of 4.3 ± 0.13, we can state that the calcium lactate influence is due to the acidifying activity on nutritive media. 4. conclusion calcium lactate is a substance that has an inhibition effect on some non-pathogenic micoorganisms. eleven microorganisms (bacteria, yeasts and moulds), were subjected to calcium lactate activity. all the bacteria were inhibited by calcium lactate. only candida mycoderma and pichia sp. yeasts were inhibited. calcium lactate showed no influence on the studied moulds: aspergillus niger, penicillium sp. and rhizopus sp. calcium lactate as antimicrobial substance can be used alone, without being combined with other substances, having an acidifying effect on the microorganism nutritive media. further studies are needed to observe the effect of calcium, and lactate ions on various microorganisms. 5. acknowledgments this work has benefited from financial support through the 2010 posdru/89/1.5/s/52432 project „organizing the national interest postdoctoral school of „applied biotechnologies” with impact on romanian bioeconomy”, project co-financed by the european social fund through the sectoral operational programme human resources development 2007-2013. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 octavian baston, octavian barna, calcium lactate influence on some non-pathogenic microorganisms, food and environment safety, volume xii, issue 3 – 2013, pag. 278 – 283 283 6. references [1]. wee y-j., kim j-n., ryu h-w., biotechnological production of lactic acid, food technol. biotech., 44. 163–172, (2006). [2]. hofvendahl k., hahn-hägerdal b., factors affecting the fermentative lactic acid production from renewable resources, enzyme microb. tech., 26. 87–107, (2000). [3]. rodrigues d.a.c., miguel m.g., cavaco a.m., dandlen s., nunes s., antunes m.d.c., influence of citric acid, ascorbic acid and calcium lactate applications on the shelf life of minimally processed horticultural products, ishs acta horticulturae, 858. 369-372, (2008). [4]. kim h., keeton j.t., smith s.b., maxim j.e., yang h.s., savel j.w., evaluation of antioxidant capacity and colour stability of calcium lactate enhancement on fresh beef under highly oxidising conditions, food chem., 115. 272-278, (2009). [5]. mohammed s. h., williams s.k., sodium diacetate and sodium lactate affect microbiology and sensory and objective characteristics of a restructured turkey breast product formulated with a fibrin cold-set binding system, poultry sci., 89. 594-602, (2010). [6]. yavas e., bilgin b., effect of calcium lactate, sodium diacetate and sodium chloride mixture on the microbiological, chemical and sensory properties of chicken nuggets stored in refrigeration and under modified atmospheres, international journal of poultry science, 9. 6671, (2010). [7]. luna-guzmán i., barrett d.m., comparison of calcium chloride and calcium lactate effectiveness in maintaining shelf stability and quality of fresh-cut cantaloupes, postharvest biol. tec., 19. 61-72, (2000). [8]. tian m., hu w-z., zhu b-w., jiang a-l., pang k., physiobiochemical changes of fresh-cut pear treated with different concentrations of calcium lactate, food science and technology, 6. 226-230, (2008). [9]. yeh j-y., hoogetoorn e., chen j., influence of calcium lactate on the fate of spoilage and pathogenic microorganisms in orange juice, j. food protect., 67. 1429-1432, (2004). [10]. yuk h-g., jo s-c., seo h-k., par sm., lee s-c., effect of storage in juice with or without pulp and/or calcium lactate on the subsequent survival of escherichia coli o157:h7 in simulated gastric fluid, int. j. food microbiol., 123. 198-203, (2008). [11]. aran n., the effect of calcium and sodium lactates on growth from spores of bacillus cereus and clostridium perfringens in a 'sous-vide' beef goulash under temperature abuse, int. j. food microbiol., 63.117-23, (2001). [12]. omi n., ezawa i., effect of eggshell ca on preventing of bone loss after ovariectomy. journal of home economics of japan, 49. 277– 282, (1998). [13]. shelef l.a., antimicrobial effects of lactates: a review, j. food protect., 57. 445-450, (1994). [14]. raybaudi-massilia r.m., mosqueda-melgar j., solivafortuny r., martin-belloso o., control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials, compr. rev. food sci. f., 8. 157-180, (2009). [15]. martin-diana a.b., rico d., frias j., henehan g.t.m., barat j.m., mulcahy j., effect of calcium lactate and heat-shockon texture in fresh-cut lettuce during storage, j. food eng., 77. 1069–1077, (2006). [16]. baston o., constantin o.e., selection of lactic acid bacteria able to ferment inulin hydrolysates, the annals of the university dunarea de jos of galati, fascicle vi–food technology 36. 31-40, (2012). [17]. ***, food chemical codex, national academy press, washington dc., 62 – 63. (1996). [18]. tofan c., bahrim g., nicolau a., zara m., microbiologia produselor alimentare. tehnici si analize de laborator (food microbiology. techniques and laboratory determinations), agir, bucuresti, 42-43, 70-72. (2002). [19]. toba t., yoshioka e., itoh t., acidophilucin a, a new heat-labile bacteriocin produced by lactobacillus acidophilus lapt 1060. lett. appl. microbiol., 12. 106-108, (1991). 342 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 342 348 an i nv es t igat ion o n t he ex tracti on c once nt ra t ion of mi croe le m en ts from aque ous s olu t ion for atom abso rbt io n an alys is *igor kobasa1, vasyl bilogolovka1, mariya vorobets1, oxana panimarchuk2 1 yury fedkovych national university of chernivtsi, ukraine, i.kobasa@chnu.edu.ua 2 bukovynian state medical university, ukraine, imk-11@hotmail.com *corresponding author received november 27th 2014, accepted december 29th 2014 abstract: some sodium diethyldithiocarbamate and 8-oxyquinoline based mixtures were tested as potential extractant to concentrate solutions containing ions of cu2+, co2+, pb2+, ni2+, zn2+, fe3+, cd2+ while preparing the samples for atom-absorption analysis. the mixture of butylacetate/sodium diethyldithiocarbamate has shown the best performance. the method of the metal ions determination that combines the sample concentration using this extractant followed by the flame atom-absorption spectroscopy provides high sensitivity and selectivity outperforming characteristics as compared to the classical method of evaporation the concentration of samples. keywords: extraction, heavy metal ions, atom-absorption analysis, natural waters 1. introduction the atom-absorption spectroscopy method (aas) is a powerful analytical tool used widely in investigations of various environmental objects: soils, natural waters and wastewaters, air, biomaterials, foodstuff and raw materials. a number of macroand microelements can be successfully identified using this method along with proper chemical preprocessing of the samples. since regular contents of some microelements in the natural water samples and some other probes can be under the aas sensitivity threshold, the preliminary concentrating of the probes is required prior to aas measurements. in case of heavy metals, such concentrating can be performed through extraction by dithiocarbamates, 8-oxyquinoline, dithizone and some other [1, 2] while chloroform, carbon tetrachloride, a mixture of the polar oxygen-containing (spirits, ketones) or non-polar oxygen-free (benzene) compounds can be used as the organic solvents [3]. however, these organic compounds are hardly combustible and can produce side colorization of the flame hindering accurate aas determination of the ingredients. besides, some of them release toxic products of incomplete combustion [4]. this paper reports the results of investigation of performance of new extraction mixtures based on the butyl and amyl esters of acetic acid as extractants used for the aas preconcentrating of the heavy metals compounds from the probes taken from natural waters. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 343 2. materials and methods the following mixtures were used as extractants for the heavy metals ions: butylacetate/sodium diethyldithiocarbamate; amylacetate/sodium diethyldithiocarbamate; butylacetate/8-oxyquinoline; amylacetate/8-oxyquinoline. the 10 % solution of sodium diethyldithiocarbamate prepared on deionized water and 0.01 m solution of 8-oxyquinoline prepared on the heavy metals-free butylacetate or amilacetate [5] were used throughout all experiments as components for the working mixtures. according to [6], all extractants were used in excessive amounts (this does not effect the extraction degree), then the volatile organic compounds were distilled off and the heavy metals contents were analyzed in the distillation residue by aas method. aqueous solutions of some heavy metals (cu2+, co2+, pb2+, ni2+, zn2+, fe3+, cd2+) with concentrations close to the usual values occurring in natural water objects were used as model samples. all solutions were prepared using deionized water with specific resistance 15 mohm or above. ph levels were adjusted by adding some amounts of metals-free hcl or nh4oh. all ph measurements were performed by ph-meter ph-150 equipped with the glass electrode ecl-4707 and silver-chlorine reference electrode evl-1m. all extraction processes were carried out according to the method [7] and the flame atom absorption spectrometer kas-120-m1 was involved in the direct determination of metal ions concentrations. 3. results and discussion the atom absorption spectra of the pure extractants combustion products were investigated preliminary using the deionized water as the reference. the results of this investigation are shown in fig. 1. fig. 1. light absorbance for combustion products of amylacetate (1) and butylacetate (2). as seen from fig. 1, light absorbance of the butylacetate and amylacetate combustion products within the range 200-240 nm is lower than that of water. then both light absorbance values remain close within 240-320 nm. the former light absorbance becomes positive only after 320 nm. this unfavorable situation impedes accurate determination of aas using water as the reference. however, pure extractants still can be used as reference solutions in case of the heavy metals ions contents analysis. there were two experimental series performed in the framework of this investigation. free metals ions were analyzed within one series while their chelate complexes were analyzed within the other. it was found that aas of both series were quite close and lie within the experimental errors. besides, an influence of ph on the extraction degree of the ions cu2+, co2+, pb2+, ni2+, zn2+, fe3+, cd2+, mn2+, cr3+ has also been investigated (figs 2-8). as seen from fig. 2, the highest (almost 100 %) extraction degree can be reached for the ions pb2+ within quite a wide range of ph (2-9) for the system sodium diethyldithiocarbamate/butylacetate (curve 1) while only about 50 % of pb2+ ions can be extracted out by the mixture sodium diethyldithiocarbamate/amylacetate within the ph range 2-5 (curve 2). the extraction activity of the mixtures amylacetate/8-oxyquinoline and butylacetate/8-oxyquinoline (curves 4 and 3) within the range of ph 6-8 is lower. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 344 fig.2. an influence of ph on the pb2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline (4). fig.3. an influence of ph on the cu2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline. details of extraction of cu2+ ions are shown in fig. 3. it is seen that the pattern of both curves for the sodium diethyldithiocarbamate containing system is similar to the previous case while the degree of extraction for the 8-oxyquinoline containing systems is significantly higher and reaches 80 % for the system butyl acetate/8oxyquinoline (see curve 3). the ph range of effective extraction is quite wide (2-6) for all the cu2+ containing systems. details of extraction of ni2+ ions are shown in fig. 4. the range of effective extraction (about 90 %) by the mixture sodium diethyldithiocarbamate/butylacetate is also quite wide (see curve 1). the sodium diethyldithiocarbamate/amylacetate mixture provides only 55 % efficiency of extraction within the same range of ph. low solubility of the ni2+/8-oxyquinoline chelates causes very low efficiency of the corresponding systems (see curves 3 and 4). fig.4. an influence of ph on the ni2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline. fig.5. an influence of ph on the cd2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline. the highest extraction degrees for the cd2+-systems can be reached for the system sodium diethyldithiocarbamate/butylacetate (see fig. 5, curve 1). this extractant remains active within very wide range of ph (1.8-9.0). similarly to the above case, the amylacetate/sodium diethyldithiocarbamate mixture ensures extraction degree about 50 % within the same ph range (curve 2) while the 8oxyquinoline based systems are inactive (curves 3 and 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 345 fig.6. an influence of ph on the zn2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline. fig. 7. an influence of ph on the co2+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8oxyquinoline (3); amylacetate/8-oxyquinoline. both sodium diethyldithiocarbamate mixtures ensure almost complete extraction of zn2+ under ph 3.5-8.5 (with butylacetate) or 4.5-7.5 (with amylacetate) (see fig. 7, curves 1 and 2). both 8-oxyquinoline based systems in this case are also inactive (curves 3 and 4). the extraction degree of ions co2+ by the mixture sodium diethyldithiocarbamate/butylacetate within the ph range 1.58.0 is about 80 % (see fig. 8, curve 1) and by the mixture sodium diethyldithiocarbamate/amyacetate – about 40 % (same ph range, see curve 2). the extraction degrees by the 8-oxyquinoline systems are also quite fair: 60 % for the system butylacetate/8-oxyquinoline (cobalt oxynate is formed within ph values 6.5-9.5, curve 3) and 30 % for the system amylacetate/8oxyquinoline, where the oxynate copound is formed within ph values 7.8-9.0 (curve 4). fig.8. an influence of ph on the fe3+ extraction degree for the mixture of butylacetate/sodium diethyldithiocarbamate (1); amylacetate/sodium diethyldithiocarbamate (2); butylacetate/8-oxyquinoline (3); amylacetate/8-oxyquinoline. the data related to extraction of fe3+ ions are shown in fig. 8. it is seen that about 40 % of the ions can be extracted by sodium diethyldithiocarbamate/butylacetate mixture for the range of ph 1.5-5.0 while the mixture sodium diethyldithiocarbamate/amylacetate ensures extraction of about 40 % of fe3+ only (ph = 3.0-7.0, see curves 1 and 2). the 8-oxyquinoline based systems exhibit higher extraction degrees: 70 % for the system butylacetate/8oxyquinoline (ph = 5.0-8.5, see curve 3) and 55 % for the system amylacetate/8oxyquinoline (weakly acid media, see curve 4). no tangible extraction activity has been determined for all the four mixtures under investigation and ions cr3+ and mn2+. therefore, the highest extraction degrees were found for the sodium diethyldithiocarbamate based systems. the heavy metals ions chelates are formed in such systems and then dissolve in butylacetate or amylacetate. it should also be noted that the chelates solubility in butylacetate is better than in amylacetate. besides, butylacetate also facilitates further atomization and improves light absorbance (1.33.1 times depending on the metal nature). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 346 the best extraction degrees can be achieved for the weakly acid (ph=4.0-6.0) solutions (see figs 2-8). the above results were applied at the next stage when a series of experiments was carried out in order to build the calibrating lines for all the metal ions. the butylacetate/sodium diethyldithiocarbamate was used as the extracting while the same system without any heavy metals was used as the reference solutions. all experimental results were compared with the similar data obtained using the classical method of concentrating by evaporation of the samples. the concentrating coefficients were similar for the extraction and evaporation methods. the “extraction” calibrating lines are represented in figs 9 and 10. they all are close to linear meaning that the experimental method was correct and adequate. the higher is the line slope, the better is the sensitivity of determination related to the corresponding metal. in this context we can state the this method exhibits the highest sensitivity towards ions zn2+ and cd2+ while the sensitivity of determination of pb2+ is the worst. the sensitivities of determination of co2+, cu2+, ni2+ and fe3+ are very close. fig. 9. calibrating curves for the ions fe2+ (1), ni2+ (2), co2+ (3), cu2+ (4) and pb2+ (5) after their extraction by the mixture butylactate/sodium diethyldithiocarbamate. fig. 10. calibrating curves for the ions zn2+ (1) and cd2+ (2) after their extraction by the mixture butylactate/sodium diethyldithiocarbamate. then the calibrating lines were applied to determine the ions concentrations in the model solutions and then the results of the “extraction” analyses were compared to the results of evaporation” analyses. all related data and results of their statistical analysis are shown in table 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 347 table 1 results of analysis of the model solutions metal tested concentration mg/l found concentration, mg/l statistical dispersion, s2 confidence interval limits evaporation extraction evaporation extraction evaporation extraction co2+ 0.065 0.065 0.064 4.917·10-6 2.425·10-5 0.0625 ± 7.823·10-6 0.066 ± 3.858·10-7 0.063 0.069 0.060 0.063 0.061 0.057 0.125 0.112 0.120 5.00·10-5 1.076·10-4 0.096 ± 7.955·10-5 0.112 ± 1.712·10-4 0.115 0.11 0.054 0.10 0.104 0.12 fe3+ 0.065 0.048 0.056 1.7·10-4 1.500·10-5 0.061 ± 2.705·10-4 0.060 ± 2.38·10-5 0.053 0.062 0.066 0.065 0.077 0.059 0.125 0.122 0.11 3.933·10-4 1.87·10-4 0.130 ± 6.257·10-4 0.107 ± 2.975·10-4 0.09 0.162 0.12 0.123 0.11 pb2+ 0.125 0.124 0.118 4.090·10-5 1.00·10-5 0.124 ± 6.507·10-5 0.121 ± 1.591·10-5 0.120 0.119 0.134 1.122 0.121 0.125 0.250 0.245 1.806·10-4 1.250·10-5 0.0256 ± 2.865·10-4 0.249 ± 1.989·10-5 0.250 0.249 0.268 0.249 cu2+ 0.065 0.060 0.059 3.3·10-4 4.56·10-5 0.57 ± 5.25·10-4 0.56 ± 1.453·10-4 0.031 0.055 0.073 0.048 0.064 0.064 0.125 0.146 0.110 1.795·10-4 4.360·10-5 0.127 ± 2.856·10-4 0.117 ± 6.33·10-5 0.142 0.121 0.115 0.118 0.125 ni2+ 0.065 0.061 0.044 8.667·10-6 9.963·10-9 0.060 ± 1.379·10-5 0.057 ± 1.585·10-4 0.057 0.064 0.060 0.057 0.068 0.125 0.116 0.125 4.667·10-6 9.667·10-6 0.119 ± 7.425·10-6 0.120 ± 1.538·10-5 0.119 0.123 0.120 0.118 0.121 0.120 zn2+ 0.013 0.024 0.0125 4.823·10-5 7.507·10-7 0.016 ± 7.673·10-5 0.012 ± 1.194·10-6 0.013 0.011 0.021 0.013 0.02 0.0125 0.025 0.039 0.026 1.805·10-4 2.0·10-6 0.029 ± 2.87·10-4 0.024 ± 3.182·10-6 0.023 0.025 0.020 0.0265 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk, an investigation of the extraction concentrating of microelements from aqueous solution for atom-absorbtion analysis ,volume xiii, issue 4 – 2014, pag. 342 – 348 348 as seen from table 1, the extraction method ensured lower deviation and better sensitivity for the majority of samples of cu, fe, pb, zn. therefore, we can conclude that the extraction method of concentrating the traces of co, cu, fe, ni, cd, zn, pb using butylacetate/sodium diethyldithiocarbamate extractant and aas determination can ensure better concentrating degree, shorter processing time, higher selectivity and sensitivity (up to 1.3-3.1 depending on the metal nature). 4. conclusion the mixture of butylacetate and sodium diethyldithiocarbamate ensures the highest concentrating degree for the heavy metals ions extraction from aqueous solutions. almost complete extraction of the metal ions can be reached within the ph range 46. this result is based on rise in the aas analytical signal intensity (1.3-3.1 times) caused by atomization of the chelate complexes formed by the heavy metals ions and butylacetate or amylacetate molecules. the extraction method also ensures less laborious and more sensitive and selective analysis than the classical evaporation method. besides, lesser number of the treatment stages in the method of extraction lowers chances of unwilled and uncontrollable probes pollution while processing. 5. references [1]. korenman i. extraction in organic compounds analysis. khimiya. moscow. 392 p. (1977). [2]. goncharova n. et al. atom-absorption determination of metals-microelements in natural and industrial waters. chem. and tech. of water. 6. 152-157. (1984). [3]. granzhan a.. kunuk g.. charykov a. extraction atom-absorption determination of the heavy metals ions. j. analyt. chem. 4. 711-717. (1991). [4]. novikov yu.. lastochkina k.. boldina z. methods of the natural waters quality investigations. medicine. moscow. 181 p. (1990). [5]. staryi m. extraction of chelates. mir. moscow. 392 p. (1966) [6]. novikov m. application of atomabsorption method of heavy metals analysis in natural objects. khimiya. moscow. 189 p. (1989). [7]. kobasa i. atom-absorption method of determination of selenium content in some raw materials and food. j. food and environment safety of the suceava university. 12. 233-239. (2013). 200 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 200 206 analysis methods of tasting as an instrument of marketing during quality forming of new grain products *marina mardar1 1odessa national academy of food technologies, ukraine e-mail address: marina_mardar@mail.ru *corresponding author received august 29th 2014, accepted september 25th 2014 abstract: the questions on application of the analysis method of tasting as an instrument of marketing during quality forming of the new, food products with the improved consumers’ qualities, have been considered in the article. when combining marketing methods of the consumers’ estimation with a descriptive and profiling method of the tasting analysis, one can obtain an expected “ideal gustatory and aromatic image” of the new product and application of visualization of these images gives an opportunity of obvious and quick comparison of the main indices of the product. the bread on the basis of the whole grain of wheat was taken as an example and application of the analysis methods of tasting during creating of “an ideal image” of the products, has been considered. it was used during the working out of recipe composition of the new product later on. keywords: “an ideal image” of the products, bread on the basis of a whole grain, descriptors, marketing investigations, tasting analysis 1. introduction one of the most important tasks concerning the improvement of the nutrition structure of the population is increase of consumer goods products with the increased food and biological value. the modern nutrition must satisfy not only physiological demands of a man’s organism in energy, nutritious and biologically active substances, but also carry out prophylactic functions and must certainly be absolutely safe. we can say that one of the main principles of the modern development of food industry is working out of the new products, taking into account the consumers’ interests with the functional directions which will contribute to improving and preserving of the population’s health. during creation of a new product, a researcher is quite often supported by the subjective estimation that sometimes leads to putting goods on the market which do not meet the consumers’ requirements. that’s why, for successful development of a new product, it is necessary to determine as soon as possible what a new product should give to a consumer, what advantages it has got, what desired properties it should have, application, its expiry date, its importance. the conception of a new product should be worked out together with a consumer, imagining the product in such a way, in which a consumer would like to see it [1]. it’s necessary that this product will be in demand among the consumers, that it will have profitable competitive advantages (using the terms of the market researcher – food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 201 “a unique trade offer”). so, it’s necessary to involve in the development of the new products not only engineers – technologists but also specialists of marketing. only active joint work gives an opportunity to create a strong competitive brand [2, 3]. an indispensable part of the work of marketing experts is market analysis and seeing to the range of the main competitors, determining of the market segment and positioning of a new product, determining of the prime cost, an optimal retail price and product profitability, methods of introduction into the market and methods of merchandising. marketing specialists must also give primary recommendations about the taste of the product, its size, package design and take an active part in composing of the verbal “image’ of the product [3]. 2. material and methods we have developed a model of forming of the consumers’ characteristics of a new food product with the improved consumers’ characteristics [4], which foresees a complex approach to the development of a new competitive product. on the one hand, this model foresees a complex of measures concerning forming of culture of consuming products with the aim of developing of a consumer himself in the questions of nutrition and healthy way of life. on the other hand, this model foresees a complex of steps, which should be done by a manufacturer himself concerning development of a new product with the improved consumers’ characteristics which will meet the requirements and wishes of the potential consumers. we will consider in detail the process of forming the quality of a new product with the improved consumers’ characteristics. according to the worked – out model it should be started with studying of prevalence and depth of deficit of the food and biologically active substances (fig. 1). at this stage it’s necessary to carry out the analysis of actual nourishment of the population, reveal its disadvantages and causes of origin of some alimentary – dependent diseases, carry out the research with the aim of determining of the indispensable nutritive substances (vitamins, macro-and microelements and so on), the deficit of which is the deepest in the particular region and embraces the most part of the population. it’s necessary to take into account influence of the environmental factors which are characteristic for this particular region. the obtained results are bases for choice of the nutritive and biologically active substances, which are additionally introduced in the composition of the product, and their amount as well. simultaneously, a marketing specialist carries out the analysis of the market of the existing analogues of goods, raw materials, materials and determines the possibilities of the enterprise concerning the production of the developed products with the aim of working out the demands to the products meeting of which will contribute to its realization. creation of a high – quality product is impossible without carrying out the marketing research of the consumers, both existing and potential, which includes investigation of the economic, social, geographic, demographic and other characteristics of the consumers and revealing of their motivations and demands in the process of buying a product. therefore, the second stage of the product development includes carrying out marketing research of the consumers’ motivations and preferences. in the process of marketing research, the consumers’ preferences are determined; first of all they are gustatory preferences of the potential consumers, possible amounts of a new product consumption, the main and additional sources of the indispensable food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 202 nutritive substances introduction, which influence choice of the amount of the enriching additive introduction. on the basis of the revealed consumers’ preferences the technical task for the industry is formed, that is the consumers’ conception of the product is formed. simultaneously, tasting of the products – analogues is carried out; their weak and strong points are revealed and these points concern social, esthetic, ergonomic, logistical and other peculiarities of the given products. carrying out of the given measures is aimed at the development of such a product, as a result, which will be competitive on the market, that is due to its consumers’ characteristics it will satisfy definite contingent of the consumers, not being inferior and even being superior to the products of the analogous purpose, which are produced by the competitive enterprises. if a researcher is guided by the market demands, then the significant contribution into the product development is made by the consumers as well, who become its researchers in the reality as well. we can say, that the most rational way of forming of the consumers’ market of the food products with the improved consumers’ characteristics, according to the developed by us model [4], is the technology, due to which, the samples demanded by particular social groups of population or even by every separate consumer, are introduced into the market. such system suggests revealing of the consumers’ preferences of the population by carrying out the marketing research, forming of the technical task for industry on the basis of the revealed preferences, manufacturing of the production and carrying out its address distribution. registration of the individual demands of the potential consumers can be carried out on the basis of tasting analysis as well. it’s well known that tasting analysis allows to solve different tasks during the whole life cycle of the goods: from forming its quality to introducing the product to the consumers and tracing the quality of the product at each stage of logistics. we will study in detail the application of the tasting method of the analysis during forming the quality of a new grain product, in particular bread on the basis of wheat whole grain. according to a lot of investigations the gustatory qualities of the product occupy the second place after its price in forming a decision whether to buy it or not [2]. modern investigations in the theory and practice of the organoleptic analysis allow to apply qualitatively new methods in developing of the new food products. these particular methods include carrying out comparative tastings of the competitive products, forming of the “ideal image” or the image of the new product, creating the panel of descriptors of the product being developed on the grounds of consumers’ expectations, working out the variants of the recipes, carrying out expert tastings (fig. 1). created during the working out, the individual signs of a food product (descriptor) allow to change gustatory and aromatic characteristics of the product depending on their qualitative value. so, the qualitative individual indices, relating to gustatory, olfactory or tactile stimuli, can be expressed quantitatively. in the opinion of some authors [3, 5], this methodology in addition to added practical skills, is necessary in developing of the new food products, including functional ones, socially significant. 3. results and discussion the great popularity in developing of the new food products has lately acquired an organoleptic method of forming “an ideal image” of the developed product [3]. in connection with it, in developing of the new bread on the basis of wheat whole grain with introduction of the additives of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 203 plant origin (root – crops of celery, parsley and parsnip), the tasting methods were used, in particular the method, based on creating of the product “ideal image”. the aim of creating “an ideal image” is getting a visual complex characteristic of the developed product, which allows to express advantages of these or those characteristics quantitatively [3]. combining the marketing methods of the consumers’ value with a descriptive – profile method of the tasting analysis, we can get the expected “ideal gustatoryaromatic image” of a new product, and application of visualization of the given images gives an opportunity of visual and quick comparison of the main indices of the product. a complex “ideal image’ of a product can be formed from some “components” – a gustatory profile, an aromatic profile, an emotional (hedonistic) profile [3]. for forming an organoleptic “ideal image” of the new bread on the basis of the wheat whole grain, its gustatory – aromatic profile, it is necessary, first of all, to work out panel of descriptors, that is the main signs of the product, forming its perception. for this purpose, opinion-polls were carried out in focus – groups. during the opinion-polls, the respondents were offered to descried verbally those characteristics of the bread, which they consider to be optimal for the developed product and which they would like to see in the new product. as not all respondents can correctly describe their gustatory sensations they were offered already formed panels of descriptors, from which a respondent had to choose 10 ones, at most, meeting his requirements to an “ideal product”. then, on the basis of the filled out tasting questionnaires, those signs were choser, which repeated as often as possible with their division into the following groups: gustatory, aromatic, emotional and so on. so, the most significant signs for this, having a special purpose, group, characterizing its understanding of the “ideal product”, were chosen. m ar ke ti ng r es ea rc h in f or m in g of th e ne w p ro du ct s qu al it y carrying out of the marketing research of the consumers’ motivations and preferences (estimation of gustatory preferences of having a special purpose audience; tasting of products-analogues; revealing of their strong and weak points the analysis of the market of the existing analogues of goods, raw material, materials and determining of free market niche studying of the reasons of origin, prevalence and depth of deficit in nutritious and biologically active substances in particular groups of population figure 1. the complex of measures on forming the quality of the new products with the improved consumers’ characteristics o rg an ol ep ti c m et ho ds o f t he a na ly si s in fo rm in g th e qu al it y of th e ne w p ro du ct s optimization of the recipes taking into account the degree of its advisability for a consumer comparison of the obtained “images” of the worked-out products with a product “ideal image” modeling of the variants of the product recipes and creating of the complex “image” of every variant with the help of tasting analysis forming of the product “ideal image” forming of the panel of descriptors on the basis of the consumers’ preferences food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 204 on the second stage, the coefficients of descriptors importance of the bread new types, were determined. for this purpose the respondents had to rank, by the degree of importance, the product descriptive characteristics and without assistance to determine a bearing interest share of importance for them, the sum of which must be equal to 100%. on the basis of these data the coefficients of importance were calculated (table 1). the coefficients of importance were calculated, taking into account that their sum should be equal to 20, so, that ratable five-mark scales can easily be transformed into 100-markscales and total marks can be expressed in percentage. table 1. coefficients of descriptors significance (csi) taste csi aroma cs i emotional perception csi harmonious 5 baked bread 5 usefulness 5 saturated 4 saturated 3 rate of satisfying one’s hunger 4 sweet 2 intensive 4 quality of satisfying one’s hunger 3 bread-like 4 grain 3 restoring effect 3 grain 2 caramel 2 prophylactic effect 5 spicy 3 spicy 3 sum total 20 20 20 then, using the calculation coefficients of importance of every descriptor, their grading was carried out and a complex panel of descriptors was composed (this is the most important condition of forming the product ideal image). on the basis of the coefficients of importance it is easy to create “an ideal image” of the product, in which every descriptor is significant. this is, so – called, ideal or expected by the consumer image of a product, reflecting its competitive advantages, an obtained profile diagram is given in fig. 2. as we can see from the diagram, first of all a consumer would like to see usefulness and its prophylactic orientation in the new product, then the bread should possess pleasant organoleptic indices, namely a pronounced aroma of the baked bread and harmonious, saturated taste. after the panel of descriptors has been formed and an “ideal image” of a new product is clear, the work of engineers – technologists begins concerning development of the recipe composition of a new product. it is done by the variation choice of the initial components with application of the expert methods of the tasting analysis. figure 2. «ideal image» of the bread on the basis of the wheat whole grain with adding root-crops: 1 – harmonious taste, 2 – saturated taste, 3 – bread-like taste, 4 –spicy taste, 5-aroma of baked bread, 6 –intensive aroma, 7 usefulness, 8 –rate of satisfying one’s hunger, 9 – prophylactic effect the final stage of the work is creating the image of the properly developed product, namely the new bread on the basis of the whole grain with introduction of root-crops and with the following comparison with an “ideal image” of the product. the organoleptic analysis was carried out by tasting. during tasting, every taster was offered tasting questionnaires, a panel of descriptors and made in the laboratory conditions 3 samples of bread on the basis of the wheat whole grain with adding food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 205 different percentage of root-crops: sample 1 – bread on the basis of the wheat whole grain with adding 7.5% root-crops; sample 2 – bread on the basis of the wheat whole grain with adding 10,0% root-crops; sample 3 – bread on the basis of the wheat whole grain with adding 12.5% root-crops. during tasting the descriptors were estimated, which were important for consumers and which were included in the complex “ideal image” of the product. by the results of the carried out tasting with the help of descriptive – profile method, the “images” or gustatory – aromatic profiles of the introduced variants of the production were composed and it was revealed that sample 2 corresponds to the “ideal image“ most of all. in fig. 3 the profile diagram of comparison of the bread “ideal image” from the point of view of the consumers (dotted line) and “the image” of the sample of the new type of bread on the basis of the wheat whole grain with adding 10% root-crops (continuous line). as we can see, the profiles practically coincide, except the index of bread-like and spicy taste of bread, that can be easily corrected, slightly changing the recipe of the bread. figure 3. comparison of the profiles of bread “ideal image” on the basis of the whole grain witch adding of root-crops and an experimental sample: 1 – harmonious taste, 2 – saturated taste, 3 – bread-like taste, 4 –spicy taste, 5-aroma of baked bread, 6 –intensive aroma, 7 usefulness, 8 –rate of satisfying one’s hunger, 9 – prophylactic effect 4. conclusion on the basis of the given results in the laboratory conditions, the experimental samples of bread on the basis of the wheat whole grain witch adding of root crops, were worked out and their complex merchandising estimation by studying the organoleptic indices of quality, food and biological value, structure peculiarities, indices of safety and medical and biological characteristics, was carried out [6]. as a result it was established, that the developed bread is characterized by high consumers’, namely improved organoleptic indices, increased content of cellulose, vitamins and mineral substances, which allows to create the balance of nutrients in the products, which is adequate to the demands of the prophylactic nutrition. and the most important thing is that the obtained product fully meets the requirements, which were raised by potential consumers during marketing research, and it, in its turn, rectifies about competitiveness of the new product. we can make a conclusion that such method is very visual and simple and it allows to see disadvantages of the products in the complex and reveal ways of achievement of the necessary organoleptic properties of the product. developing new products, manufacturers often underestimate methods of the tasting analysis, counting on the aggressive promotion. however, it is the professional application of sensory methods of estimation of gustatory advantages of the product that allows to significantly reduce the budget of marketing and technical research and to avoid mistakes. 5. references [1]. earl m., earl r., andersen a. development of food products, saint petersburg: profession, p. 384 (2004). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 marina mardar, the analysis methods of tasting as an instrument of marketing during quality forming of the new grain products, food and environment safety, volume xiii, issue 3 – 2014, pag. 200 – 206 206 [2]. kantare v.m., matison v.a., promenko m.a. consumers’ estimation of products is the most important component of marketing research, meat industry, (2002). [3]. chugunova o.v., zavoroina n.v. application of methods of the tasting analysis during modeling of the recipes of food products with the set consumers’ characteristics, yekaterinburg, the publishing house of ural state economical university, p. 148 (2010). [4]. yegorov b.v., mardar m.r. development of production principles for functional foods, novi sad (serbia): institute of food technology, 319323, (2012). [5]. stone h., sidel j. l. sensory evaluation practices, san diego: academic press, p. 338, (1993). [6]. mardar m.r., grain products on the basis of a whole grain – are the products of health improving nutrition, food processing industry magazine, 31-34, (2013). 127 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag.127 137 modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun *verdier n. abouo1,2, clément d. akmel1, ernest k. kakou1, emmanuel n. assidjo1, georges n. amani2, benjamin k. yao1 1laboratoire des procédés industriels de synthèses de l'environnement et des energies nouvelles (lapisen), inphb bp 1093 yamoussoukro 2laboratoire de biochimie alimentaire et de transformation des produits tropicaux (lbatpt), una 02 bp 801 abidjan 02 v.abouo@gmail.com *corresponding author received april 1st 2015, accepted june 5th 2015 abstract: the purpose of this work was to make a drying experiment of fermented cocoa beans concomitantly in the sunlight (using the experimental tray as a recommended device) and in a microwave oven (mo) to simulate the process by empirical models. the gradient method was used to estimate the coefficient of empirical models used in the study. the experimental data were adjusted from eleven equations published in the literature. the best suitable model was evaluated using the coefficient of determination (r²), reduced chi square (χ2), the root mean square error (rmse), the sum square error (sse) and the mean relative deviation (mrd) in percentage (%) between the experimental and predicted values. the diffusion approach model was selected for the solar drying because this model presents the best statistics characteristics (0.991; 2.49 e-04; 0.036; 0.037 and 3.401%). for the microwave oven drying, high r2 values (higher than 0.95), low values of χ2, rmse, sse and the mrd values below 10% are given by the page and newton models proposed: 0.981; 0.002; 0.080; 0.080; 3.718% for the device mo 2400w; 0.974; 0.003; 0.096; 0.067; 7.708% for the mo 2800w and 0.982; 0.002; 0.069; 0.107; 8.496% for mo 3200w. keywords: drying, microwave, sun, empirical models, gradient method. 1. introduction the cocoa bean is the raw base material of the chocolate industry. it is used to obtain chocolate products [1]. the beans must undergo different processing steps including drying [2]. it is one of the oldest methods of preserving agricultural products [3]. it consists in water evaporation, thus reducing the potential growth of microorganisms and some undesirable chemical reactions such as the enzymatic browning in order to increase the product life [3]. it provides a dry and homogeneous product [4]. at harvest which occurs just after the maturity of the pods, the beans are fermented and dried before any other form of industrial transformations. the fermentation lasts for approximately six days and can be done with banana leaves or wooden crates [5, 6]. after fermentation two drying methods of beans are used. natural drying in the sunlight and artificial drying by forced convection. the beans are dried until they reach the accepted limit of moisture (8%) and the establsihed limit for free fatty acid (≤1.75%) [2, 7]. natural drying has a high http://www.fia.usv.ro/fiajournal mailto:v.abouo@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 128 dependence on weather conditions, which could influence the quality of cocoa beans [8]. artificial drying meanwhile is carried out with hot air that is forced convection. several authors report the influence of different crops post-treatment on the quality of agricultural products [8, 9]. many research studies were conducted in order to provide solutions [7]. drying is a crucial step. sunlight drying of cocoa beans is done on tray at free air with a relatively long time (7-21 days) [10]. the recovery of the moisture beans causes the change of the constituents of fat depending on seasons’ variability [11]. in industry, the drying of beans is carried out by the forced convection mode in 24 to 48 hours which is a relatively short time in comparison with solar drying [12, 13]. however, this heat transfer mode has the disadvantage of retaining volatile acids in the cotyledons [12-14]. this fact can change the physical and chemical properties, leading to an acid and unstructured cocoa butter [11]. among other reported disadvantages of hot air drying, one can notice the increase of bitterness, appearance and smell caused by the presence of acrolein and an undesirable flavor of burnt beans [15]. several studies of artificial drying in electromagnetic environment have shown excellent results on various products and in various fields. derya and mehmet showed that the onion drying kinetics by electromagnetic waves has better characteristics than those obtained by sun-drying modes (drive) and the hot air oven (forced convection). this study showed that the mineral constituents (k+, ca2+) are higher and have better rectangular coordinates l * a * b *. concentrations of phenolic compounds are also more important for the electromagnetic wave drying as compared to the others mentioned above [16]. chekroune showed that the dates are likely to be dried with the aid of electromagnetic waves while maintaining their physicochemical characteristics [17]. furthermore, the work done by kone has shown that the tomato could be dried in electromagnetic environment intermittently with a specific power steering [18]. the results of this work showed improved retention rate of the lycopene. lucchesi demonstrated the efficiency, speed (reduction factor extraction time 9) and selectivity without inertia in the heating mints by using microwave, revolutionizing the vegetable oil extraction process [19]. their work has been the subject of a study patent in europe and the united states. microwave drying generally has enough benefits including speed, uniformity of the dried material and more effectiveness in terms of energy transfer compared to convection (forced) and infrared. it accelerates moisture loss from the inside to the outside of the material [20]. microwave technology is quickly growing. indeed, it is variously used for drying, either directly to dry agricultural products and or as an adjunct to other drying methods (convection hot air) in industry [18, 21, 22, 23]. a good practice and monitoring of the drying process is necessary to obtain good quality of cocoa beans by the formalization of the phenomenon [2]. in this regard, several empirical models (mathematical equations) were exploited and described in the literature [2, 22]. these models establish relations between reduced water content and drying time in order to obtain the characteristic curves and describing the phenomenon [24]. the aims of this work are to describe drying characteristics and to model the cocoa beans drying as an experimental tray device and microwave oven by the empirical models of thin layer. 2. matherials and methods 2.1 sample preparation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 129 thirty kilograms of fresh cocoa beans were purchased in the month of october 2013 from growers (3) yamoussoukro (a town in central cote d'ivoire, west africa) and put into fermentation for six days in banana leaves. 2.2 drying procedure the obtained fermented beans were removed from waste before starting the drying process. during this period, a thermo-hygrometer (auriol ian 71010, france) was used to raise the ambient temperature. relative humidity and air velocity were recorded using an anemometer (pce-81 am, france). solar drying operation took place five hours on experimental tray of one square meter raised of one meter in relation to the ground during seven days. for artificial drying device, a microwave oven (guangzhou qualiway ltd 510630, china) at three power levels (2400w, 2800w and 3200w) was used. thirty (30) samples of the same mass (200 g) were spreaded in thin layer of 3 to 4 cm thick in experimental tray [7, 24]. the weight loss was recorded regularly every hour using scales (sartoruis, a200s, france) until stability thereof ie the difference between three successive weighing not more than 0.001g. for drying oven at mo, three sets of 30 samples of 200 g were dried in the three powers mentioned above. the differential mass loss was regularly recorded every minute (application time) under the same conditions as above [25]. the initial and final water content was performed according to aoac methods [26]. 2.3 mathematical modelling the reduced water content and the drying rate were determined using the following equations (1 and 2): ∗ = (1) = (2) where xt is the water content at t; xe is the water content at equilibrium and x0 is the initial water content [27, 28]. the table 2 below presents eleven models used to model the drying kinetics of cocoa beans in thin layer. they are semiempirical models used in the literature [22]. these equations contain coefficients that need to be adjusted from experimental data [29]. to do the calculation, a program implemented in matlab r2014a was developed. it is based on the gradient method to estimate model coefficients and linear regressio to calculate the following statistical criteria: the coefficient of determination (r²), the reduced chi-square (chi-square), the root mean square error (rmse), the sum square error (sse) and mean relative deviation (mrd) [30]. they have been used for the selection of models that fit best the experimental values and values predicted by the model [23, 29]. table 1 nomenclature classifications a, b, c, drying coefficients rmse root mean square error mrd mean relative deviation sse sum square error k, g, n, h constants drying n number of observations r² coefficient of determination x* dm reduced water content dry matter greek symbols χ² reduce chi square subsrcipts i at time i e equilibrium o initial exp experimental pre predicted food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 130 table 2 description of the empirical models in thin layer drying tested in the study equation n°. model name expression of the model references i newton ∗ = ( ) [31] ii iii page page i modified ∗ = ( ) ∗ = (( ) ) [32] [24] iv v henderson and pabis logarithmic ∗ = ( ) ∗ = ( )+c [33] [34] vi vii two term two term exponential ∗ = ( )+ ( ) ∗ = ( )+ (1 − ) ( ) [29] [24] viii ix wang and singh diffusion approach ∗ = 1 + + ∗ = ( )+ (1 − ) ( ) [35] [3] x verma et al., ∗ = ( )+ (1 − ) ( ) [36] xi midilli and kucuk ∗ = ( ) + [37] with a, b, c (coefficients) and g, h, k, k0, k1, n (parameters) to be determined. statistical criteria for choosing models: coefficient of determination ² = ∑ ( ∗ , ∗ , ) ∑ ∗ , ∗ , ∗[∑ ( ∗ , ∗ , ) ] (3) reduced chi square: ² = ∑ ( ∗ , ∗ , ) (4) root mean square error: = [ ∑ ( ∗ , − ∗ , ) ] (5) sum square error: = ∑ ( ∗ , − ∗ , ) (6) mean relative deviation: = ∑ ∗ , ∗ , ∗ , (7) 3. results and discussion 3.1 drying characteristics the initial water content of the cocoa beans is 53.52 ± 2.93% and is reduced to a value less than 8% at end of the drying [1, 2, 7]. the mean levels of the end of drying beans vary from 5.49 to 5.77%. they are respectively: mo 2400w (5.77 ± 0.17%); mo 2800w (5.63 ± 0.20%); mo 3200w (5.60 ± 0.14%) and 5.49 ± 0.11% at the tray device. the figure 1 shows the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 131 evolution of aerodynamic parameters during 35hours of solar drying. these are: the relative humidity which fluctuated between 52-74%, the temperature flucted to 27.4-36.2°c and the velocity air of the drying varied from 0.2-2ms-1. fig. 1: evolution of aerodynamic parameters (solar drying) similarly, the evolution of the temperatures of dried cocoa beans with different powers in the microwave oven is shown in figure 2. fig. 2: evolution of cocoa beans drying temperature in mo the curves reflecting changes in the temperatures during microwave drying of cocoa beans generally have the same allure. the trend of evolution can be seen in six (6) minutes followed by falling to the tenth (10th) minutes. then some stabilization thereof is noticed until the end for mo 2400w device. at the drying mo 2800w, the temperature increases during first four (4) minutes, and then decreases food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 132 until the end of drying with a phase slight increase in the eleventh to twelfth minute. an increasing speed to phase ((3) first three minutes) followed by a lasting stable phase one (1) minute and a decrease thereof to 3200w. following the determination of the water content of the beans after drying, successive readings of the masses permitted to establish the curves of the differential mass loss of samples of fermented cocoa beans. they are obtained by expressing the reduced water content as a function of drying time [x* = f (t)] and are plotted for triplicates (figure 3 and 4). fig. 3: curve of cocoa beans drying in tray fig. 4: curve of cocoa beans drying in the microwave oven the end of the experiment is reached after thirty-five hours (35) on drying experimental tray, 16, 12 and 8 minutes in the microwave oven as shown in figures 3 and 4. all drying curves have the same shape. three (3) drying periods are observed: the heating period, the constant rate period and the falling rate; except that obtained at the 3200w power that has almost no heating temperature. the heating period observed during the first 2 hours of drying on tray, 4th and 3rd minute respectively for the mo 2400w and mo 2800w devices. that operates at a constant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 133 rate of 2-25 hours of sunshine cumulated on tray, between 4-13th minute for the device mo 2400w; 3-10th minute at mo 2800w and for mo 3200w device during 1-7th minute. the third period is observed during 25-35 hours in the sun, 13-16th minute at 2400w; the 10-12th minute at 2800w and of 7-8th minute at 3200w. thus, the maximum rate reached during drying of cocoa beans in the microwave oven is 7.59 kg of h2o/kg dm/minute to 2400w; 9.19 kg of h2o/kg dm/minute to 2800w; 13.12 kg of h2o/ kg dm/minute to 3200w and 6.24 kg of h2o/kg dm/hour at sun (tray). the drying characteristics curves fitting allow to obtain the equations of drying rate in the form of polynomial of degree 3 with the determination coefficients higher than 0.95 (0.982 for sun drying; 0.993 at 2400w; 0.984 at 2800w and 0.982 at 3200w) [24,38]. mathematical expressions of drying rates of cocoa beans of two devices are: 1.078x*3 – 1.753x*² + 1.631x* ; (claie) 0.665x*3 – 0.516x*² + 0.825x* ; (2400w) 2.454x*3 – 2.995x*² + 1.508x* ; (2800w) 2.367x*3 – 3.003x*² + 1.563x* ; (3200w) the drying time in the microwave oven has an incomparable application time to the sun. the drying of cocoa beans by microwave oven shows a fast decrease of the mass with the increased level of power. dried beans in the microwave oven have higher drying rates than those dried in the sun in the descending order of power level. dried beans in the microwave oven have higher drying rates than those dried in the sun in descending order of power level. this fast loss of mass of the different samples (beans) can be explained by the large amount of dipolar molecule such as water (53.59 ± 2.93%, initial water content). their polarization then allows them to orient themselves in the alternating electromagnetic field creating a rotational movement and resulting in the conversion of kinetic energy into heat energy dissipation [39, 40, 41]. to this end, a fast gradual rise of the temperature is observed. they evolve from: 30 to 80.2°c; 30 to 84.4°c and 30-98,5c° respectively for the mo 2400w, mo 2800w and mo 3200w (fig. 2). the beans become source of energy for the simple reason that the heating system is in volume. in fact, the difference vapor pressure between the inside and outside of the beans is high [21]. thus, the matter and energy spread from the inside to the outside of the product subject to radiation [18, 27, 39]. thus, from the standpoint of speed, the microwave drying mode can be a good alternative to solar drying in experimental tray mode which moreover is strongly influenced by the weather [11]. 3.2 mathematical modelling the table 3 below presents the estimated coefficients of the eleven (11) thin layer drying models of the experimental data used, and the accuracy of the models by comparing the settings: the r2, the reduced χ2, rmse, the sse and mrd (%).the analysis of the above table (2) shows that the values of the parameter r2 fluctuate from 0.855 to 0.991; from 0.903 to 0.981; from 0.883 to 0.974; from 0.895 to 0.982, respectively, for the tray, mo 2400w, mo 2800w and mo 3200w. those (values) of the reduced χ2 fluctuate from 2.49e-04 to 0.017 for the tray; from 0.002 to 0.065 at mo 2400w; from 0.002 to 2.717 at mo 2800w and from 0.002 to 0.034 at mo 3200w. while it is recorded rmse of 0.036 to 0.370 for drying on tray; 0.080 to 0.497 at 2400w; 0.096 to 2.760 at 2800w and 0.069 to 0.932 for the mo 3200w drying. as for the sse, they fluctuate from 0.037 to 13.529 for solar drying; 0.004 to 7.807 at 2400w; 0.001 to 5.672 at food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 134 2800w and 0.011 to 8.247 at 3200w. finally, the mrd fluctuate from 3.401 to 15.200 for the tray device; from 3.718 to 18.309; from 0.249 to 13.628 and from 3.070 to 13.974 in ascending order of powers in microwave oven. based on what the model that best describes the drying kinetics thin layer is the one with the highest value of the determination coefficient (r2), low values of reduced χ2, rmse, sse and a lower mrd 10% [23, 29]. the model of the approach of diffusion is retained for solar drying (experimental tray). those page and newton are retained respectively for levels of 2400w, 3200w and 2800w in microwave oven. other models which have been selected are reported in the literature during solar drying and would be the result of the influence of experimental conditions [8, 9, 42, 43]. indeed, akmel et al, retained the logarithmic model while hii et al, a new model that is similar to page model at two terms. this difference in selected models is due to the influence of the following parameters such as temperature, relative humidity, wind speed and initial water content of the cocoa beans [2, 24]. table 3 coefficients of models and statistical parameters of models choice model number devices coefficients & models parameters r² χ² rm se sse mrd (%) i claie k=0.070 0.991 2.91e-04 0.046 0.022 8.834 2400w k=0.078 0.975 0.003 0.112 0.274 8.544 2800w k=0.115 0.974 0.003 0.096 0.067 7.708 3200w k=0.072 0.966 0.014 0.161 1.135 11.346 ii claie k=0.078 n=0.091 0.982 0.014 0.315 13.523 13.782 2400w k=0.171 n=0.901 0.981 0.002 0.080 0.080 3.718 2800w k=0.219 n=0.598 0.970 0.006 0.137 0.206 7.670 3200w k=0.148 n=1.029 0.982 0.002 0.069 0.107 8.496 iii claie k=0.093 n=0.148 0.978 0.002 0.119 0.174 5.702 2400w k=0.013 n=0.521 0.970 0.013 0.211 2.071 10.248 2800w k=0.112 n=0.052 0.955 0.011 0.169 0.001 0.249 3200w k=0.104 n=0.080 0.948 0.010 0.141 0.021 3.070 iv claie a=1.040 k=0.009 0.881 0.012 0.304 12.903 14.128 2400w a=0.966 k=0.009 0.915 0.022 0.267 4.330 11.495 2800w a=0.906 k=0.010 0.894 0.025 0.263 3.094 11.208 3200w a=0.958 k=0.009 0.936 0.041 0.281 3.471 12.609 v claie a=-0.654 c=0.338 k=0.135 0.977 0.014 0.232 7.533 4.906 2400w a=-0.324 c=0.286 k=0.157 0.980 0.031 0.263 4.980 6.157 2800w a=1.421 c=0.069 k=0.060 0.943 0.027 0.296 5.239 13.628 3200w a=-1.391 c=0.420 k=0.064 0.963 0.370 0.909 6.358 13.974 vi claie a=0.928 b=0.452 ko=0.009 k1=0.043 0.931 0.017 0.370 9.174 15.200 2400w a=1.444 b=0.150 ko=0.005 k1=0.443 0.954 0.065 0.497 7.807 14.556 2800w a=0.839 b=0.003 ko=0.010 k1=0.514 0.899 0.021 0.246 2.332 10.526 3200w a=1.363 b=1.585 ko=0.006 k1=0.105 0.974 0.348 0.932 8.247 7.237 vii claie a=0.942 k=0.009 0.881 0.011 0.289 11.578 13.907 2400w a=0.870 k=0.009 0.915 0.024 0.279 4.947 11.799 2800w a=0.809 k=0.010 0.894 0.301 0.291 4.431 12.044 3200w a=0.862 k=0.009 0.936 0.046 0.301 3.996 12.926 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 135 viii claie a=-7.49e-6 b=2.39e-11 0.855 0.018 0.359 7.961 14.327 2400w a=-1.27e-3 b=7.49e-6 0.903 0.031 0.319 6.619 12.128 2800w a=-1.52e-2 b=2.45e-5 0.883 0.039 0.325 5.673 12.319 3200w a=-6.96e-2 b=4.56e-3 0.922 0.055 0.33 4.799 13.138 ix claie a=0.271 b=0.27 k=0.305 0.991 2.49e-04 0.036 0.037 3.401 2400w a=0.613 b=0.162 k=0.261 0.962 0.003 0.098 0.004 4.441 2800w a=0.401 b=0.208 k=0.174 0.971 0.007 0.148 0.602 9.477 3200w a=0.747 b=0.132 k=0.253 0.971 0.003 0.077 0.081 7.726 x claie a=0.668 g=0.288 k=0.023 0.981 0.002 0.114 1.417 10.688 2400w a=0.591 g=0.289 k=0.025 0.966 0.005 0.139 0.376 7.981 2800w a=0.472 g=0.105 k=0.066 0.962 0.005 0.127 0.377 9.199 3200w a=0.565 g=0.377 k=-0.003 0.895 0.017 0.171 1.104 10.552 xi claie a=1.425 b=-0.301 k=0.119 n=0.992 0.976 0.011 0.267 9.245 13.264 2400w a=1.308 b=-0.047 k=0.145 n=1.067 0.976 0.014 0.23 3.814 18.309 2800w a=0.815 b=-0.698 k=0.162 n=0.671 0.887 2.717 2.760 5.016 13.001 3200w a=1.540 b=-0.109 k=0.159 n=1.401 0.981 0.054 0.326 3.954 4.944 4. conclusion the water content of the cocoa beans is less than 8% after 35 hours in sunshine. for the mo, we have respectively 16 min at 2400w, 12 min at 2800w and 8 min at 3200w. the estimated coefficients models and the statistical parameters determined describe well the cocoa beans drying kinetics showed that the model of the diffusion approach is retained for the solar drying. on the other hand, the models page and newton have been retained to describe the kinetics of drying thin layer of cocoa beans in electromagnetic environment with high r2 values (greater than 0.95); low values of χ2 ; rmse; sse and less than 10% for mrd. 5. references [1] afoakwa e., cocoa and chocolate consumptionare there aphrodisiac and other benefits for human health? african journal clinic nutrition. 21(3): 107-113, (2008) [2] hii c.l, law c.l. and cloke m., modelling of thin layer drying kinetics of coca beans during artificial and natural drying. journal of engineering sciences and technolgy, 3(1): 1-10, (2008) [3] gowen a, abu-ghannam n. and oliviera j., modelling dehydratation and rehydratation of cooked soybeans subjected to combined microwave-hot air drying. innovative food science and emerging technologies. 9: 129137, (2008) [4] bonazzi c, dumoulin e. and bimbenet j., le séchage des produits alimentaire. industrie alimentaire agricole, 125 (03-04): 12-22, (2008) [5] afoakwa e, kongor j, takrama j. and budu a., changes and nib acidification and biochemical composition during fermentation of pulp pre-conditioned cocoa (theobroma cocoa) beans. international food research journal, 20(4): 1843-1853, (2013) [6] lagunes g, loiseau g, paredes j, barel m. and guiraud j., study on the microflora and biochemistry of cocoa fermentation in the dominican republic. international journal of food microbiology, 114: 124-130, (2007) [7] akmel d., assidjo n. and yao b., effet des dispositifs de séchage à l’air libre sur la qualité des fèves de cacao bord champ, revue ivoire sciences technologies, 11 : 45-58, (2008) [8] hii c., rahman r., jinap s. and man y., quality of cocoa beans dried using direct solar dryer at different loadings. journal of the science of food and agriculture, 86: 1237-1243, (2006) [9] diamante l., ihns r, savage g. and vanhanen l., a new mathematical model for thin layer drying of fruits. international journal of food science and technology. 45(9):1956-1962, (2010) [10] asiedu j., la transformation des produits agricoles en zone tropicale, approche technologique. editions karthala (paris) et cta (wageningen), 39-60, (1991) [11] pointillon j., cacao et chocolat : production, utilisation et caractéristiques. tech and doc/ lavoisier. collection sciences et techniques agroalimentaires. paris. 96-115, (1997) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 136 [12] augier f., nganhou j., barel m., benet j.c. and berthomieu g., réduction de l'acidité du cacao lors du séchage. plantations recherche développement, 5(2): 127-133, (1998) [13] jacquet m., vincent j-c., hahn j. and lotode r., le séchage artificiel des fèves de cacao, café cacao thé, 24(1): 43-56, (1980) [14] guehi t., zaouli i., ban-koffi l., fae m. and nemlin j., performance of different drying methods and their effects on the chemical quality attributes of raw cocoa material. international journal of sciences and technology, 45: 1564-1571, (2010) [15] afoakwa e., paterson a., fowler m. and ryan a., flavor formation and character in cocoa and chocolate. critical review in food sciences and nutrition. 48: 840-857, (2008) [16] derya a. and mehmet m., study the effect of sun, oven and microwave drying on quality of oinion slices, food sciences and technology, 43: 1121-1127, (2010) [17] chekroune m., 2009. etude comparative de deux types de séchage (convection et microonde) par application des plans d’expériences : cas des fruits de datte, master, université m’hamed bougara boumerdes, algérie, 127 p. (2009) [18] kone k., amélioration de la qualité de la tomate séchée par microondes assisté par air chaud avec pilotage de la puissance spécifique. thèse de doctorat, paris tech, 168p, (2011) [19] luchessi m., extraction sans solvant assistée par micro-onde. conception et application à l’extraction des huiles essentielles. thèse de doctorat, université de la reunion, france. 146 p, (2005) [20] wang j., wang j. and yu y., microwave drying characteristics and dried quality of pumpkin. international journal of food science and technology, 42(2): 148-156, (2007) [21] krysiak w., effects of convective and microwave roasting on the physicochemical properties of cocoa beans and cocoa butter extracted from this material. grasas y aceites. 62(4): 467-478, (2011) [22] harish a., rashmi m., krishna m., blessy b. and ananda s., mathematical modeling of thin layer microwave drying kinetics of elephant foot yam (amorphophallus paeoniifolius). international food research journal, 21(3): 1081-1087, (2014) [23] sorour h. and el-mesery h., 2014. effect of microwave and infrared radiation on drying of onion slices. international journal of research in applied, 2(5): 119-130, (2014) [24] akmel d., assidjo n., yao b. and kouame p., mathematical modelling of sun drying kinetics of thin layer cocoa (theobroma cacao) beans, journal of applied sciences research, 5(9): 1110-1116, (2009) [25] belahmidi m., belghit a., mrani a., mir a. and kaoua m., approche expérimentale de la cinétique de séchage des produits agro-alimentaires. revue générale de thermique, 380-381: 444-453, (1993) [26] aoac, official methods of analysis (18th ed.), association of official analytical chemists. washington, dc, moisture content in plants, 1: 949, (2005) [27] wang j. and sheng k., far infrared and microwave drying of peach. lebensmittelwissenschaftund-technologie, 39: 247-255, (2006) [28] ozbek b. and dadali g., thin-layer drying characteristics and modeling of mint leaves undergoing microwave treatment. journal of food engineering, 83(4): 541-549, (2007) [29] roberts j., kidd d. and padillazakour o., drying kinetics of grape seeds, journal of food and engeneering, 89: 460-465, (2008) [30] griva i., nash g. and sofer a., linear and nonlinear optimization, edition siam, 581p, (2009) [31] yaldiz o., ertekin c. and uzun h.b., mathematical modelling of thin layer solar drying of sultana grapes. energy, 26: 457-465, (2001) [32] jurendic t., determination of controlling resistance to moisture transfer during drying. journal of food sciences technologies. 4(1): 34-45, (2012) [33] akpinar e.k., bicer y. and yildiz c., thin layer drying of red pepper. journal of food engineering, 59(1): 99-104, (2003) [34] togrul i. and pehlivan d., modeling of thin layer drying of some fruits under open-air sun drying process. journal of food engineering, 65: 413-425, (2004) [35] wang c. and singh r., use of variable equilibrium moisture content in modelling rice drying. asae meeting paper no 78-6505, st. joseph, (1978) [36] verma l., bucklin r., endan j. and wratten f., effects of drying air paramet ers on rice drying models. transactions of the asae, 28: 296-301, (1985) [37] midilli a. and kucuk h., mathematical modelling of thin layer drying of pistachio by using solar energy. energy conversion and management, 44(7): 11111122, (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao, modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun, food and environment safety, volume xiv, issue 2 – 2015, pag. 127 – 137 137 [38] touati b., etude théorique et expérimentale du séchage solaire des feuilles de la menthe verte (mentha viridis). thèse de l’insttitut national des sciences appliquées de lyon, france, p13, (2008) [39] lucchessi m., chemat f. and smadja j., solvent-free microwave extraction of essential oil from aromatic herbs: comparison with conventional hydro-distillation. journal of chromatography a, 1043: 323-327, (2004) [40] sarimeseli a., microwave drying characteristics of coriander (coriandrumsativum l.) leaves. energy conversion and management, 52: 1449-1453, (2011) [41] arikan m., ayhan z., soysal y. and esturk o., 2012. drying characteristics and quality parameters of microwave-dried grated carrots. food bioprocess technology, 5: 32173229, (2012) [42] doymaz i., sun drying of figs: an experimental study. journal of food engineering.71, 403-407, (2005) [43] jayas d.s., cenkowski s., pabis s. and muir w.e., review of thin layer drying and wetting equations. drying technology, 9(3): 551588, (1991) 76 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1 2016, pag. 76 83 the use of glass transition temperature in forecasting whey powder storage stability obtained by electro-spark treatment *оksana kochubei-lytvynenko1, vyacheslav mykhailyk2, anatoliy ukrainets1 1nationaluniversity of food technologies, 68 volodymyrska str., kyiv, ukraine, okolit@email.ua 2institute of engineering thermal physics, national academy of sciences of ukraine, 2 acad. bulakhovskii str., kyiv, ukraine, mhlk45@gmail.com *corresponding author received februay 18th 2016, accepted march 28th 2016 abstract: this study investigated the behavior during storage of dry whey obtained using different approaches that prevent caking (by adding silicon dioxide, electro-spark treatment of whey). forecasting the stability of quality whey powder was carried out based on a glass transition temperature.the glass transition temperature of whey powder was measured by differential scanning calorimetry.we showed that the highest glass transition temperature (+18.5 °с) and,subsequently, the smallest difference between product temperatures and glass transition temperature were discovered in dry whey, obtained with the use of electro-spark treatment, which proved to be stable during storage.we have found that the other samples of dry whey under normative storing conditions (from 0 till 20 °с) are predominantly in rubbery state, which can influence negatively the stability of the product during storage. based on the glass transition temperature we determined that electro-spark treatment used in the technology of whey powder allowed reaching anti-caking effect due to physico-chemical processes resulting from electro-spark charges. this was proved by calculating the stickiness and caking sensibility index and the degree of caking (to 2 %). it was experimentally proved that the dry milk whey produced with electro-spark treatment differed positively from other samples by absence of non-fermentative darkening.whey powder produced from whey treated with electro-spark charges was characterized by the highest degree of whiteness, which did not significantly change in 8 month storage. keywords: wheypowder, electro-spark treatment, glass transition temperature, caking, stickiness and caking sensibility index. 1. introduction dry milk whey belongs to amorphous metastable products, which is very sensitive to temperature and humidity changes. spoiling of low humidity products during storage is determined by chemical, biochemical or/and physical changes, including migration and loss of humidity which can cause caking, fermentative and non-fermentative reactions displayed by product darkening. conditions of outer environment (temperature, moisture content) have big influence upon these processes, as well as product structure, active acidity, water activity index etc. in order to increase quality, avoid spoiling of food products and to increase their shelf life, various technological measures are used, as well as nontraditional treatment measures, processing methods and storage techniques. at the same time for long shelf life products, including dry whey concentrate, important issue is the choice of qualities and characteristics used for objective and reliable forecasting of quality index changes during storage. in a range of works [1-5] correlation was proved between some qualities of dry http://www.fia.usv.ro/fiajournal mailto:okolit@email.ua mailto:mhlk45@gmail.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 77 products (such as stickiness, caking) and glass transition – phase transition of separate nature, particular to amorphous material. the process of glass transition is a process of material transition from glass-like to rubber-like state and vice versa which is accompanied by changes in its thermodynamic and mechanical qualities, molecular mobility and dielectric penetrability[6]. the range of temperatures at which this transition is observed is called the glass transition temperature (tg). the concept of glass transition together with the concept of water activity is lately widely used to forecast stability of food products under drying and storage[7]. it is considered that stability of organoleptic, chemical and physicochemical qualities of dried food products depends on storage temperatures. at temperatures higher than glass transition temperature may occur oxidation, crystallization and recrystallization processes, and increase in adhesive qualities, because is increasing in molecular mobility and lowering viscosity of the material. this in turn leads to changes in chemical and physical state, sticking of particles on the solid surface and caking of the product. the speed of such changes is determined by the difference in product temperatures (т) and tg [2, 7, 8]. itisknownthatthelowerthedifferencebetwee nproductstoragetemperatureandglass transition temperature, thebetterisitsstorage [2, 3, 6]. humidity content of the product has a decisiveinfluenceupontheglass transition temperature [8, 9, 14, 18, 22]. the change in chemical content, phase state of sugars and biopolymers (casein, whey proteins) has its influence upon dependence of tgfrom product humidity content [13-22]. scientific sources predominantly pay attention to glass transition temperature of vegetative origin products, separate components (including various carbohydrates) and model samples of dry products. at the same time, not much data is available on multicomponent products, produced in industrial conditions with the use of modern technological approaches, including demineralization of whey, use of electrophysical treatment procedures and anti-caking agents. however, this data is necessary to determine or prove storage regimes of dry products and to forecast stability of their quality indexes. theaimoftheworkwastodetermineglass transition temperature ofdrymilkwhey, obtained with the use of various approaches that prevent caking (adding silicon dioxide, electro-spark treatment of whey); tostudythebehaviorofexperimentalsamples duringpackaging, transportationandstoragebyparametersdiap asonchange [t – tg]. 2. materials and methods 2.1. objects of research. demineralizedwheypowder(wpd) wasderivedbydemineralizationofcheesewh eyatnanofilterutility(gea, denmark), thickening(end up with mass fraction of milk solidsto 50-52 %), coolingofthethickenedmilkwheyandcrystall izationoflactose (10-12 hours, under 15 °с) and further drying at spraying drier (vzduchotorg, slovak republic). whey powder enriched with particles of magnesium and manganese (wpepmgmn)wasobtainedbytheabovementi onedscheme,butdemineralized whey wastreatedatexperimentalelectrosparktechnologicalinstallationbeforethicke ning.thelatter consisted of charge impulse generator, control panel, running charge chamber, measuring and supplementary tools[10]. electro-spark treatment was conducted under such conditions of charge contour: recharge condenser voltage – (75 ± 5) v; condenser volume – 100 uf; food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 78 resistance of granules layer in charge chamber – 0.15–1.5 om; interval between metal granules – to 0,1 mm; impulse frequency – 0.2–2.0 khz; exposition – 1 min for magnesium electrode system; 0.5 min – for manganese electrode system. in result of volumetric electro-spark dispersion of metal granules (mg and mn), whey was enriched by these components (table 1). content of mg and mn in whey samples was determined at atomic-absorption spectrometer ааs1n (carl-zeiss jena, germany), equipped with burner for acetylene-air flame and lamps with hollow cathode for magnesium and manganese. table 1 content of magnesium and manganese inwheypowders whey powders contentmg, g/kg contentmn, mg/kg wpd 0.94±0.03 1.1±0.04 wpepmgmn 2.9±0.15 12.9±0.5 demineralized whey powder with anticaking agent (wpdaca) 1.0% of anticaking agent was added to demineralized whey powder and thoroughly mixed. silicon oxide sio2 (e-551 silicon dioxide amorphous) was used as an anti-caking agent. 2.2. chemical and physical analysis glass transition temperatures and heat capacity changes (δcp) were determined using differential scanning microcalorimeter (dsc-2m) made at special design office of biological instrument making in pushchino (russia) and equipped with a thermcap data acquisition and processing program written in the delphi programming language. micro calorimeter temperature scales were graded by to two benchmark points: -95.0 °с (melting temperature of chemically pure toluene) and 0 °с (melting temperature of water after double distillation). cooling of calorimeter block was performed with the use of liquid nitrogen. accuracy of temperatures measurement was not worse than ±0.1 °с. the samples were hermetically canned in aluminum containers. weighting was performed at microanalytical scales vlm1 with accuracy ±0.01 mg. overall content of water in samples was determined after measurements by dehydration till constant mass in the drying box at 104-105 °с. in order to avoid artifacts connected to humidity condensation in calorimeter chambers, measurement block was filled with dried gas-like helium, which flow was controlled during measurements. samples were cooled at –50 °c at scanning rate 16 к/min. temperature intervals (tg), beginning temperatures s gt and end temperatures of glass transition f gt were determined by dsc-curves, obtained under heating of samples at scanning rate 16 к/min from –50 till +35 °c. typical dsc heating curve is presented at fig.1. tg was determined as tg/2. the change in heat capacity under glass transition cp was obtained by the difference in heat capacity at f gt and s gt . each sample had undergone at least three measurements. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 79 fig. 1 typical dsc heating curveforwhey powder showing the glass transition the degree of caking was measured using the method described by pisecky [11]. in the beginning amount of whey power (5 g) was kept in desiccators under artificially created conditions of high humidity (above 80 %) until the mass increase stopped. afterwards samples were dried in drying box during 1 hour at 104±2 °с. after cooling in a desiccators, the sample was weighed, quickly transferred to a stainless steel sieve (250 µm), and shaken for 5 minutes in a shaking apparatus. the powder that passed through the sieve was weighed, and the caking index was determined by the equation 1. 100 1 21    m mm сз (1) whereм1is the total mass of powder (g) and м2is the mass of fines that passed through the sieve (g). ability of dry product particles to stickiness and/or caking during treatment and storage was forecasted under a stickiness and caking sensitivity index (scsi). scsi (in diapason from 0 to 10) was determined by equation 2 according to parameters changes diapason [t – tg] (t – temperature of dry product) and δcp [3]. scsi = number of points [t – tg] + number of points [δcp], (2) wheretg is the glass transition temperature, с; t – dry product temperature (at the exit from drier, in the dry powder batch mixer and under storage), с; δcp – change in heat capacity, j/(g∙к). this index simultaneously integrates the values of [t – tg] (ranging between 0 and 5) and δcp (ranging between 0 and 5) (tab. 2) [3]. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 80 table 2 calculation of stickiness and caking sensitivity index [3] t – tg, с δcp, j/(g∙к) number of points [t – tg]≤5 δcp<0.1 0 5<[t – tg]≤10 0.1≤ δcp<0.2 1 10<[t – tg]≤15 0.2≤ δcp<0.3 2 15<[t – tg] ≤20 0.3≤ δcp<0.4 3 20<[t – tg]≤30 0.4≤ δcp<0.5 4 30<[t – tg] 0.5≤ δcp 5 non-fermentative darkening of dry milk whey was determined by the change of product whiteness during storage. whiteness of the product was estimated in relative points at blik-p3 (russia) instrument for measurement of directed zonal reflection index and determining whiteness. 3. results and discussion for research objects dsccurves of heating were determined (fig.2), according to which characteristic temperatures of glass transition were found out (table 3). fig.2 dsc heating curvesforwheypowder: 1 – wpd, 2 – wpepmgmn, 3 – wpdаса. results obtained showed the difference in studied samples by glass transition temperatures. the control sample of demineralized whey powder had the lowest tg level. addition of silicon dioxide as an anti-caking agent to wpd leads to increaseglass transition temperature on 10 с. it was observed highest тg value for whey powder enriched with magnesium and manganese particles. significant тg grow in whey powder, produced with the use of electro-spark food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 81 treatment, and most likely is explained by physicochemical processes taking place in whey components, as a result, of electrospark charge. among them: accumulation of magnesium and manganese particles in colloidal form in case of their transition from electrodes to solution; their interaction with water and whey components (including proteins and thus increasing its molecular mass); transition of lactose and creation of its derivatives (lactulose, lactobionic acid) [12]. table 3 wheypowdersampleshumiditycapacityandtemperaturesofglasstransition sample moisture content, g/gdrywhey s gt , c f gt , c glass transition temperature, tg, c wpd (control) 0.044 −18.0±0.2 +12.0±0.2 −3.0±0.2 wpepmgmn 0.031 +10.0±0.2 +27.0±0.2 +18.5±0.2 wpdаса 0.034 −6.0±0.2 +19.0±0.2 +6.8±0.2 the conduct of researched whey powder samples during packaging, transportation, mixing with other components (sticking of particles to hard surfaces) and storage (caking) was forecasted by the change in index diapasons [t – tg] and δcp, established by dsc-curves. the scsi value calculated from equation 2. it is known that scsi allows to forecast the dry product behavior during drying, transportation and storage, from the most favorable case (scsi ≤ 4: no stickiness and/or no caking) to the most unfavorable (scsi ≥ 6: high to very high stickiness or caking hazard) [3]. for measurements with the aim of forecasting product behavior during packaging, transportation (stickiness) and storage (caking), such temperatures were taken for the dry product (т): on the exit from dryer – +30 с; storage – +20 с. calculated results are presented in table 4. table 4 wheypowderbehaviorduringpackaging, transportationandstorage as a functionof [t – tg] andδcp values the analysis of received values showed that the highest ability to caking and high stickiness was particular for control sample of whey powder. adding silicon dioxide decreased product caking risk, which was proved by scsi and level of caking, measured experimentally. based on the glass transition temperature measurement and change of specific heat capacity, it was determined that including electro-spark treatment in technology of whey powder allowed reaching anti-caking effect due to physicochemical processes resulting from electro-spark charges. this was proved by stickiness and caking sensibility calculation index (scsi =2) as well as experimental data of the degree of caking (to 2 %). sample δcp, j/(g∙к) stickiness caking degree of caking, % (experimental data) [t – tg], c scsi (calculated data) [t – tg], c scsi (calculateddata) wpd (control) 0.38 33.0 8 23.0 7 16.4±0.7 wpepmgmn 0.23 11.5 4 1.5 1 2.0±0.04 wpdаса 0.32 23.2 7 13.2 5 13.4±0.4 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 82 research results and scsi levels, calculated on their basis fully correspond to observations over research samples in the process of storage under 18±2 с temperatures and relative humidity not more than 80 %. it is worth mentioning that besides stability to caking of whey powder particles produced with the use of electro-spark treatment, these samples positively differed from other samples by the absence of nonfermentative darkening. researchsamplesproducedfromthewheytre atedwithelectro-spark charges was characterized by highest degree of whiteness, which did not significantly lower in 8 months storage (table 5). other samples suffered from lowering whiteness index by 8.8-10.5 conventional units depending on the type of product.lossofwhitenesstestifiestheflowof maillardreaction, whichasknowncannotretardevenunderlowh umidityunlikeotherbiochemicalprocesses.n evertheless,its speed decreases by reducing the difference between the storage temperature andtg[2, 3]. it was observed in whey samples prepared using electric spark treatment. table 5 change of whiteness in whey powder research samples during storage sample whey powder whiteness, conventional units, in storage 1 month 6 months 8 months wpd 90.6±2.0 83.6±2.3 80.1±1.7 wpepmgmn 97.4±1.0 95.7±1.1 95.1±0.8 wpdаса 90.9±1.6 85.4±1.0 82.1±1.2 4. conclusions this study investigated the behavior during storage of dry whey obtained using different approaches that prevent caking (adding silicon dioxide, electro-spark treatment of whey). forecasting the stability of quality whey powder was carried out based on a glass transition temperature. we showed that the highest glasstransitiontemperature (+18.5 °с) and,subsequently,smallestdifferencebetwee n product temperatures and glass transition temperaturewere discovered in dry whey, obtained withtheuseofelectrosparktreatment, which proves its stability while in storage. we found that the other samples of dry whey under normative storing terms (from 0 till 20 °с) are predominantly in rubbery state which can negatively influence on stability of the product while in storage. based onthe glass transition temperaturewe determinedthatincludingelectrosparktreatmentintechnologyofwheypowder allowed reaching anti-caking effect due to physicochemical processes resulting from electro-spark charges. this was proved by calculation stickiness and caking sensibility index and thedegreeofcaking(to 2 %). itwasexperimentallyprovedthat the drymilkwheyproducedwithelectrosparktreatmentpositivelydifferedfrom other samples byabsenceofnonfermentativedarkening. whey powderproducedfromthewheytreatedwithel ectro-spark charges was characterized by highest degree of whiteness, which did not significantly lower in 8 months storage. other samples suffered from lowering food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 1 – 2016 оksana kochubei-lytvynenko, vyacheslavmykhailyk, anatoliy ukrainets, the use of glass transition temperature in forecasting whey powder storage stability, obtained by electro-spark treatment, food and environment safety, volume xv, issue 1 – 2016, pag. 76 – 83 83 whiteness index by 8.8-10.5 conventional units depending on the type of product. 5. references [1] silalai n., roos y.h. roles of water and solids composition in the control of glass transition and stickiness of milk powders, journal food science, 75 (5): 285-296, (2010) [2] roos y.h. importance of glass transition and water activity to spray drying and stability of dairy powders, lait, 82: 478–484, (2002). [3] schuck p., blanchard e., dolivet a., méjean s., onillon e., jeantet r. water activity and glass transition in dairy ingredients, lait, 85: 295-304, (2005). [4] kylie d. foster the prediction of sticking in dairy powders, b.tech (hons) : 170, (2002) [5] kalichevsky m.t., blanshard j.m.v., tokarczuk p.f. effect of water content and sugars on the glass transition of casein and sodium caseinate, international journal food science technology, 28: 139–151, (1993). [6] karel m., lund d.b. physical principles of food preservation. 2th edition, revised and expanded, ny: marcel dekker: 400, (2003). [7] schwartzberg h.g., hartel r.w. physical chemistry of foods, ny: marcel dekker: 747, (1992). [8] kilcast d., subramaniam p. the stability and shelf-life of food. chapter 2, cambridge: woodhead publishing: 344, (2000). [9] mauer l.j., smith d.e., labuza t.p. effect of water content, temperature and storage on the glass transition, moisture sorption characteristics and stickiness of -casein, international journal food properties,. 3: 233– 248, (2000). [10] kochubey-litvinenko o.v., bilyik e.a., olishevskiy v.v., marinin a.i., lopatko k.g. method of enrichment ofwhey by colloidal particles ofmagnesium and manganese, prospects of its use, food industry: science and technology,3 (29): 36-42, (2015) [11] pisecky j. handbook of milk powder manufacture, copenhagen: niroa/s: 261, (1997). [12] kochubey-litvinenko o.v., ishchenko v.m., lopatko k.h., fomenko v.v. transformation of whey components in the electric-treatment, proceedings of hduht "progressive technique and technology of food production and trade restaurant business, 2:316-326, (2015). [13] roos y.h., effect of moisture on the thermal behavior of strawberries studied using differential scanning calorimetry, journal of food science, vol. 52(1): 146–149, (1987). [14] khalloufi s., el-maslouhi y., ratti c., mathematical model for prediction of glass transition temperature of fruit powders, journal of food science, vol. 65(5): 842–848, (2000). [15] wang h., zhang s., chen g., glass transition and state diagram for fresh and freezedried chinese gooseberry, journal of food engineering, vol. 84: 307–312, (2008). [16] moraga g., martinez-navarrete n., chiralt a., water sorption isotherms and phase transitions in kiwifruit, journal of food engineering, vol. 72: 147–156, (2006). [17] fabra m.j., talens p., moraga g., marti.neznavarrete n., sorption isotherm and state diagram of grapefruit as a tool to improve product processing and stability, journal of food engineering, vol. 90: 52–58, (2009). [18] mrad n.d., bonazzi c., boudhrioua n., kechaou n., courtois f., moisture sorption isotherms, thermodynamic properties, and glass transition of pears and apples, drying technology, vol. 30: 1397–1406, (2012). [19] mikhailik v.a., dmitrenko n.v., snezhkin yu.f., change in the specific heat capacity of parenchymal tissues of apples due to dehydration, journal of engineering physics and thermophysics, vol. 87, № 1: 48–53, (2014). [20] goula a.m., karapantsios t.d., achilias d.s., adamopoulos k.g. water sorption isotherms and glass transition temperature of spray dried tomato pulp, journal of food engineering, vol. 85(1): 73–83, (2008). [21] mikhailikv.a., snezhkinyu.f., korinchevska t.v. , the impact of treatment on crystallinity convective drying powders with apple and sugar beet, industrial heat engineering,vol. 37 (5): 23-37, (2015). [22] mikhailikv.a., snezhkinyu.f., korinchevska t.v., research of glass transition tissues dried apples and sugar beet, industrial heat engineering, vol.37 (7): 148-149, (2015). 290 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 4 – 2014, pag. 290 298 contributions to achieve a composite material for advanced electromagnetic shielding of living and workspaces first partshielding material cristian florin alexuc1, *gheorghe gutt1, sonia amariei1 stefan cel mare university of suceava, romania *corresponding author received december 5th 2014, accepted december 18th 2014 abstract: the paper refers to a new material for electromagnetic shielding of living and workspaces in order to reduce the electro smog. the material and manufacturing technology described comes in support of the activities carried on by electromagnetic compatibility of high frequency radiation emitted by mobile antennas, radar, cell phones, microwaves, computers, wireless networking, on the environment. the shielding material described by the authors is of composite type and it is in the form of nets with fine meshes made of thin steel wires on which a high-purity copper shirt, coated in its turn by a polymer shirt, is deposited, the latter one’s aim is to protect against oxidation of copper coating. nets, supplied in rolls, can be integrated under the plaster of working and living spaces performing an advanced electromagnetic shielding throughout the duration of building those spaces belong to. key words: electromagnetic compatibility with the environment, electromagnetic shielding nets. 1. introduction electromagnetic radiations are mixed waves containing electric and magnetic alternative fields. electric field is generated by potential differences and magnetic field is generated by current flow. those radiations are present everywhere in the environment, they forms there the electricity is produced, consumed and distributed. wavelengths  covers the field of 10-11 m (gamma radiation) up to 103 m (radio waves) and their frequency cover a total of 14 orders of magnitude from 1 hz to 10 000 thz. electromagnetic radiation need not matter to propagate. in vacuum the speed of propagation is those of the light and a propagation speed in given environment is limited due to its refractive index. the interference extent of electromagnetic radiation with matter, including the living matter, depends on frequency and wavelength thereof [1],[2]. electromagnetic radiation is divided into two main areas after wavelength: ionizing and non-ionizing radiation. the wavelength of ionizing radiation is between 0.01 nm to 100 nm and is represented by gamma radiation, x-ray radiation (x-rays) and ultraviolet radiation. the wavelengths of non-ionizing radiation food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 291 is between 1 mm and 1000m and are represented by infrared radiation, microwaves, radio waves and lowfrequency waves of power supply networks. between the two types of radiation it is the visible range (neutral white light) of electromagnetic radiation, domain considered to be a buffer zone between ionizing radiation and nonionizing radiation. when the visible spectrum is split by an optical system in different wavelength, it is perceived colorful and named visible color spectrum, figure 1, [3]. fig.1.wavelength range covered by electromagnetic radiation. recovering files [3] due to the very small wavelength and extremely high frequency effect of ionizing radiation on matter is pronounced causing its ionization. the effect of ionizing radiation on living organisms is destructive leading to rapid death in the case of their relatively short exposures to gamma radiation and tumor diseases more likely in the case of a longer exposure to x-ray and uv radiation. [4],[5],[6]. higher wavelength and lower frequency of nonionizing radiation makes their effect on living organisms to be not so pronounced as that of ionizing radiation, but far from being neglected. the fact that the low intensity of this radiation effect on living organisms is not as strong as those ionizing radiation and that the harmful effects of this radiation occurs after long periods of the order of years or even decades, has led and lead today many people to a tolerant viewpoint, often even the negation of the effect of this radiation. the latest scientific research clearly shows that non-ionizing food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 292 radiation acting on living organisms long time, leading to cell damage and advanced affecting of nervous system [7],[8]. as a protection against non-ionizing electromagnetic radiation in the electromagnetic compatibility dealing with the extent to which electrical devices influence each other by electromagnetic fields unwanted was defined a subdomain called electromagnetic compatibility with environment [9],[10],[11] that defines compatibility emission of electromagnetic fields with the environment, especially with the man and sets limit values to ensure his safety and to prevent his disease due to these radiations. measuring instruments of electromagnetic radiation development, characterization, testing and production of special materials [12],[13] for shielding the high and low frequency fields and measuring their intensity are also under the environment protection measures and [14],[15],[16]. in the electromagnetic compatibility of environmental the general term of electrosmog is used to express the pollution which refers both at forms of appearance and manifestation of electric fields, magnetic and electromagnetic fields and their undesirable effect on living matter. viewed through the concept of electrosmog non-ionizing electromagnetic radiation can be divided into lowfrequency radiation (0 300 hz) and high frequency radiation (300 hz 300 ghz). low frequency radiation are produced by power lines, electric trains, electric generators, transformers, electrical household appliances and high-frequency radiation from radio broadcasting antennas, telephone, television and mobile, radar and microwaves, tvs, computers, etc. 2. shielding material used to shield living and workingspaces against electromagnetic radiation of low and high frequency corresponding frequency domain of nonionizing electromagnetic radiation shielding means are divided into means of shielding for low frequency and high frequency. this paper deals with a shielding material both for low frequency of electromagnetic radiation and those of high frequency. the low-frequency electromagnetic radiation shielding is performed by means of faraday cage, a mesh of metal wires binding connected to the ground. [1], [2]. the materials used for the faraday cage consumes the electric component of the electromagnetic field by two mechanisms. a mechanism consists of earth leakage part of the electricity and the other mechanism is to use a part of the electric current through the resistive effect. the later effect makes for achieving faraday cage to be used and metallic material having a lower electrical conductivity than copper, such as for example, the steel. high-frequency electromagnetic radiation shielding is achieved by eddy currents which final effect is local heat generation. the most important condition to be fulfilled in this case by the shielding materials is their high electrical conductivity. for shielding high frequency electromagnetic radiation are used two solutions. first solution consists food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 293 in the shielding with copper sheet of all areas of a space [16]. on the one hand this type of shielding is provided a shielding factor of more than 100 db and, on the other hand is a very expensive solution, gives real problems of integration the copper plate under the plaster of living or working space. besides these disadvantages the oxidation phenomena of copper sheet surface determine a sensitive lowering of electrical conductivity of surface layer and its negative manifestation of the effect skin [17], [18] in order to reduce the shielding capacity. currently, this solution is mainly used for shielding of high generating frequency devices and less for shielding workspaces (apart from some military applications) or those for living. a second solution refers to the development of some schielding nets with small meshes, with a matrix made of natural or synthetic wires. in this matrix are integrated by various methods electrically conductive materials such as carbon nanofibers, copper and silver[19], [20], [21], 22]. when using shielding nets besides high conductivity of the material must be respected and electrical resonance condition. the size of the mesh side should be less than half of the wavelength (λ/2) of the electromagnetic wave that is desired to be schielded. compared to massive copper shielding, iron nets presents the advantage of lower cost price, high mechanical flexibility, as well as that of perfect compatibility with various coatings can be easily integrated together in finishing of walls structures or in fibrous wood boards used for making furniture, paneling etc. the disadvantage of this type of shielding mesh is that of a lower electrical conductivity than that of copper sheet. this technology is mainly due to the use of metallic and non-metallic powders (carbon nanofibers), usually in the form of inserts in a polymer matrix from which is then pulling the wires in order of nets achieving. even if copper or silver carbon nanoparticles touch each other, in the wires constituting the mesh shielding, the contact is only in one point that leading to a local electrical resistance increased and finally to a weaker closing of eddy currents in the mesh shielding. this is why the shielding efficiency of such mesh shielding is at the maximum values of about 30 db. in order to obtain the best values of shielding efficiency the percentage of silver and copper particulates in mesh wires is greatly increased that leads to values of 30-70 db of shielding, at the same time, however, the price of each m2 of schield mesh is over an order of magnitude, and values over 70 db of shielding lead to an increased price by more than two orders of magnitude. 2.1. shielding mechanism in order to achieve the new type of shielding material proposed [23] and studied by the authors it is use primary metallic nets of thin steel wire mesh, small squares with the mesh side of millimeter order. in the first phase, the net wires are coated uniformly, by electrochemical or chemical way, with a thin layer of high conductivity of pure copper, and in the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 294 second phase, which follows immediately after the first stage, with a layer of monomer that is converted by ultraviolet irradiation in a thin polymer coat. the final product is a composite material in a net form which is based on steel wire coated with a thin layer of electrolytic copper which in turn is coated with a polymeric structure that protects it against oxidation. the novelty character of this advanced shielding material results from the fact that the structure of steel wire core wrapped in a pure copper shirt so it shields both electromagnetic radiation of low frequency and high frequency. shielding at low frequency occurs by earth leakage of the electric field captured by the copper coating and by the resistive power consumption achieved by the steel core of low-frequency current. as already mentioned, high-frequency electromagnetic radiation shielding is achieved by eddy currents whose final effect is local heat generation. given the fact that at high frequency currents of contact, including eddy currents inductive generated, moving to the area and not in the depth of conductors, metallic material thickness that provides the shielding may decrease more, in terms of ensuring an advanced shielding with increasing of frequency (skin effect). based on this consideration, electrolytic copper coatings thicknesses of several μm, made around some thin steel wires, are an excellent electrical conductor, and these threads woven in small meshes constitute an advanced electromagnetic shielding means. at these advantages is added a price of low cost compared to other shielding materials and a durability provided by the polymer coating of metallic wires that delivers an advanced protection against oxidation of copper from the shielding nets throughout the duration of building structure which includes the space shielded. 3. technology and flow realization of composite material for shielding electromagnetic radiation of low and high frequency in order to achieve the new electromagnetic shielding material authors have developed three ways of achieving nets of composite material. the first method is based on electrochemical deposition, galvanically, a pure copper coating on some nets of thin wire steel, figure 1. a second way of achieving shielding meshes is chemically reducing a thin layer of pure copper on meshes made of thin steel wire, figure 2., and the third way consists in electrochemical deposition without an external power of thin pure copper layer on steel wire of nets, the same figure 2. 3.1. flow and continuous line production of metallic nets for electromagnetic shielding obtained by galvanic deposition of high-purity copper on wire steel meshes the manufacturing flow consists in, figure 1, a power supply unit which comprises a drum 1 rolling a primary net 2 with meshes of thin steel wire, a pickling bath 3 for steel wire primary net 2, a bath 4 for food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 295 rinsing the net 2, a bath 5a galvanic thermostated for electrochemical deposition of copper containing an galvanic electrolyte 6 based on copper cyanide for the electrolytic deposition of a thin coating of pure copper on steel wire of primary mesh 2, a power source 7 for supplying electric current of soluble electrolytic copper anode 8 and metallic drum 9 that powering primary steel mesh 2 that form the cathode of galvanic bath 5, figure 2, another rinsing bath 11 for intermediate metal mesh 12 of steel wire wrapped in a shirt of pure copper, a tunnel 13 for drying intermediate metallic net 12, a impregnating bath 14 for wire of intermediate metallic net 12 with a monomer in order to provide advanced protection from oxidation of copper deposited electrolytically, a line 15 in uv for polymerization of monomer deposited on intermediate metal wire mesh 12, a start drum 16, and a drum winder 17 of the final shielding net 18 having a composite structure of the type: thin steel wire electrolytic copper shirt polymeric shirt. production line includes some drums tg with guidingstretch role and some drums tt with tractionreturn role. operator mode to obtain electromagnetic shielding nets by galvanic deposition of copper is as follows: the drum 1 is installed for rolling the primary net 2 of thin steel wire meshes, the end of the primary net 2 is attached by means of a cross-clamps belonging to a net of polymeric material. the latter net has the same width with the net of steel wire, its role only for traction it up when metallic net passes through all stages of process. the net of polymeric material is driven by the opening drum 16 that is under winding drum 17. after connecting the two nets it commands the starting of continuous manufacturing operation of final shielding nets 18 respecting the forward speed prescribed for their high quality. fig.1. flow and continuous line of manufacturing metallic electromagnetic shielding nets obtained by galvanic deposition of high-purity copper on nets of wire steel. 1-rolling drum, 2-primary net with steel wire, 3pickling bath, 4rinsing bath, 5agalvanic bath thermostated, 6galvanic electrolyte, 7continuous current power supply, 8electrolytic copper soluble anode, 9metallic drum for powered on, 10thermostat, 11rinsing bath, 12intermediate net with steel wire wrapped in a shirt of pure copper, 13drying tunnel, 14 impregnating bath with monomer, 15line of monomer polymerization with ultraviolet radiation, 16 opening drum of rolling, 17winder drum, 18final net shielding with a composite structure type: thin steel wire electrolytic copper shirt polymeric shirt, tgdrums of guiding stretch role, ttdrums of tractionreturn role. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 296 fig.2. flow and continuous production line of metallic nets for electromagnetic shielding obtained by electrochemical deposition of high-purity copper on nets of wire steel, without using external power sources or deposition of copper by chemical reduction.1rolling drum, 2-primary net of steel wire, 3pickling bath, 4 rinsing bath, 5b-galvanic bath thermostated, 11rinsing bath, 12intermediate net with steel wire wrapped in a shirt of pure copper, 13drying tunnel, 14impregnating bath with monomer, 15line of monomer polymerization with ultraviolet radiation, 16opening drum of rolling, 17winder drum, 18final net shielding with a composite structure type: thin steel wire electrolytic copper shirt – polymeric shir when the end of final net shielding 18 reaches the winding drum 17 clip cross coupling of two nets automatically unfolds and the end of the final net shielding 18 is routed to the winding drum 17, operation taking place until the primary net 2 is entirely consumed from the rolling drum 1. in figure 2 is shown a flow of continuous production of the metallic net shielding based on the achievement of high-purity copper deposit through electrochemical process without external power and chemical reduction of copper in a copper sulfate bath, on thin steel wire, forming the primary meshes that is presented in turn, as well as that of the first embodiment, as a winding drum. the manufacturing flow based on chemical reduction of copper consists in a power supply unit which comprises a rolling drum 1 of a primary net 2 with meshes from thin steel wire, a pickling bath 3 for steel wire of primary mesh 2, a rinsing bath 4 of primary net 2, a thermostatic bath 5b for depositing copper by chemical reduction of copper from a sulfate electrolyte 6c on the basis of copper sulftate, another rinsing bath 11 of the metallic intermediate net with the coated steel wire in a shirt of pure copper, a tunnel 13 for drying the intermediate metallic net, a bath 14 for impregnation of metallic intermediate net wire with a monomer in order to provide superior protection to oxidation of copper electrolytically deposited, a line 15 for polymerization in uv radiation of the monomer deposited on the metal wire of intermediate net, drum 16 to open roling and a winding drum 17 of final shielding net 18, the latter having, like in the first embodiment, a composite structure of the same type: thin steel wire electrolytic copper shirtpolymeric shirt. the operating mode of this example is identical to that of the first embodiment. 4. conclusion the composite material for shielding of living and workspaces to electromagnetic radiation of low and high frequency presented in the paper is an achievement of authors team concerned to ensure an acceptable electromagnetic compatibility with the environment using for this purpose modern and efficient solutions.the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 297 solution described combine into a single material in the form of nets, a high shielding capacity both against lowfrequency radiation and high frequency radiation. by shielding performance and low cost price, shielding nets described are recommended for advanced shielding of all six sides of living or workspace. material forming shielding meshes of nets consists in thin steel wires coated with high-purity copper and outer polymeric coating for protection against copper oxidation. the nets can be invisible placed under the plaster of a certain space making its advanced electromagnetic shielding throughout the duration of the building to which it belongs. full description of the manufacturing process flow of shielding nets enables immediate addressing of their production. 5. references [1]. wolfsperger h., a., www. schirmungstechnik.de, abschirmung elektromagnetischer felder. grundlagen und beispiele [2]. wolfsperger h.,a.,elektromagnetische schirmung, springer verlag, berlin, 2008, p.18-26 [3].xxx.http://de.wikipedia.org/wiki/nichtionisier ende_strahlung#mediaviewer/datei:spectre.svg [4]. crasson m., 50-60hz electric and magnetic field effects on cognitiv function in humanas, a rewiew. radiat protect dosim.106, 2003,p.333-340 [5]. reilly j.p. neuroelectric mechanism applied to low frequency electric and magnetic field exposures guidlines -part1-sinusoidal waveforms. health phys.83, 2002, p.341-355 [6]. saunders r.d., jefferys j.g. weak electric field interactions in the central nervous system, healt phys.83, 2002, p.366-375 [7]. cook c.m. saucier d.,m., s.a. exposure to elf magnetic and elf modulated radiofrequency fields: the time-cours of physiological and cognitive effects observed in recent studies (2001-2005). biolectromagnetics, 27, 2006, 613-628 [8]. xxx. international agency for reseach on cancer. static and extremly low frequency electric and magnetic fields. luon, france. iarc monographs on the evaluation of carcenigenic risc to humanas volume 80, 2002 [9]. xxx.guidelines for limiting exposures to time varying electric and magnetic fields (1 hz 100 khz), icnirp (international commission on nonionizing radiation protection), health physics, 99(6),2010, p. 818-836 [10]. xxx. international commission on nonionizing radiation protection. guidelines on limiting exposures to static magnetic fields. health phys. 96, 2009, p.504-514 [11]. xxx international electrotechnical commission. exposures to electric or magnetic fields in the low and intermediate frequency rangemethods for calculating the current density and internal electric field in the human body. geneva, iec, 62226, 2004 [12]. stuchly m.,a., dawson t.w., human body exposure to power lines. relation of induced quantities to external magnetic field. health phys. 83,2002, p.33-340 [13]. kaden h., wirbelströme und schirmung in der nachrichtentechnik , springer verlag berlin, 2006, p.36 [14]. xxx. mil-std-285/1997 (military standard), attenuation measurements for enclosures, electromagnetic shielding, for electronic test purposes [15]. gutt g., popa. v, alexuc c., f., warning system for low frequency electromagnetic radiations, proposal of invention, osim file a00601/2014 [16]. xxx.http://www.aaronia.de/grundlagen/gru ndlagen-wissen/grundlagen-abschirmleistung/ [17]. gutt s., gutt g., zerstörungsfreie werkstoffprüfung, editura didactica şi pedagogica r.a, bucuresti, 2010, p.159-162 [18]. xxx.http://de.wikipedia.org/wiki/skineffek t#mediaviewer/datei:skin_depth_by_zureks.png food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 4 – 2014 cristian florin alexuc, gheorghe gutt, sonia amariei, contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material, volume xiii, issue 4 – 2014, pg. 290 – 298 298 [19]. dadfar a.,n., s.a.seyyed ebrahimi, et al., microwave absorption properties of 50%srfe12o19–50% tio2 nanocomposites with porosity, m.r., journal of magnetism and magnetic materials, 324, (2012), 42044208 [20]. mohammed h. al-saleh a., genaro a, et al, carbon nanofiber/polyethylene nanocomposite: processing behavior, microstructure and electrical properties, materials and design, 52, (2013), p.128–133 [21]. jung yoon seo, hyun woo kang, dae soo jung, hye moon lee, seung bin park, one-step synthesis of copper nanoparticles embedded in carbon composites, materials research bulletin 48 (2013) 1484–1489 [22]. fusheng wen a.,n., fang zhang a., jianyong xiang a., microwave absorption properties of multiwalled carbon nanotube/feni nanopowders as light-weight microwave [23]. alexuc c.,f., gutt g., amariei s., metallic material for shielding of electromagnetic radiations and its realization technology, proposal for invention, osim file a00 684/214 применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 3– 2016 contents: 1 heat transfer modeling in down flowing laminar films with the developed wavy structure with co-current steam flow valentyn petrenko, yaroslavzasyadko 203 215 2 the influence of lupin flour addition on bread quality georgiana gabriela codină, andreea roxana marineac, elena todosisănduleac 216 226 3 the impact of different diet and environmental conditions on chemical composition of rainbow trout (oncorhynchus mykiss walbaum, 1792) from macedonian aquaculture facilities biljana sivakova, dijana blazhekovikj dimovska 227 233 4 physi cochemic al and t e xt ural pr oper ties of hone ys f rom n or th east part of romani a mircea oroian, elena todosi sănduleac, sergiu pădureț 234 239 5 traditional leafy vegetable combinations enhance growth performance and improve health status of new zealand white x californian rabbits franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke 240 248 6 the use of stabilized frying fat in the production of potato chips olena kovalenko, vladimir kovbasa, irina radzievska 249 254 7 souring dynamics of the milk samples with different fat content igor winkler, marinela opaeț 255 258 8 analysis of physical parameters of natural juices mihaela jarcău, maria poroch-seriţan 259 265 9 validation of 3-dimentional models of natural gas combustion and temperature measurements pavlo zasiadko, mykola pryadko 266 275 author instructions i v subscription information vi 359 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 359 368 statistical studies on the lexicon used in sensory evaluation of soy-added beef hamburger *cristina-elena hrețcanu1 , alice-iuliana roșu1, ioana zelezneac1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania *cristina.hretcanu@fia.usv.ro, alicer@fia.usv.ro, *corresponding author received november 5th 2013, accepted november 28th 2013 abstract: the aim of this paper was to study consumers’ perception of three samples of soy added beef hamburger in different percentages, using some helpful methods to capture consumers’ perception sensory characteristics, such as free listing, check–all-that-apply (cata) question and intensity scaling in order to determine the intensity of some sensorial characteristics (taste, odor, flavor and texture). for this purpose we prepared three samples of soy-added beef hamburger in different percentages (25%, 50% and 75% soy bean of total mass) and we study the effect of soy addition in beef hamburger on the sensory properties was studied. keywords: check-all-that-apply (cata), free listing, lexicon for sensory characterization, sensory evaluation, hamburger 1. introduction sensory characteristics such as odor, taste, appearance, flavour and texture are important quality factors of food products industry. tools that describe data regarding consumer liking are sensory lexicons and descriptive sensory profiling methods, used to capture consumers’ perception of food products. moreover, methods such as free listing and check–all that – apply (cata) are increasingly used in sensory analysis, because they can be helpful for information that could be used to develop new food products [1]. with these tools, we could start new studies to determine which sensory attributes drive consumers’ satisfaction and determine the perception of potential consumers. a free listing method is used to develop a lexicon describing the product space. cata is a method used in the food sensory evaluation to gather information about sensory and non sensory perception, based on the selection of appropriate words from a predefined list of descriptors. also, cata method can be used to determine the perception of product sensory attributes and it may lead to a better product differentiation among food products of the same category [2]. cata has been applied in analyzing many food products, such as: salty snacks [3], strawberry cultivars [4], vanilla ice cream [5], chocolate milk dessert [6-7], orange-flavored powdered drinks [6-7] and texture perception of milk desserts [8]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 360 we applied the above mentioned methods to capture consumers’ perception of sensory characteristics of soy protein added beef hamburger. hamburgers most likely first appeared in the 19th or early 20th centuries, they were firstly made in hamburg, germany [9]. they are usually regarded a feature of fast food restaurants, where they are grilled on a flat-top or on a gas flame grilling process [10]. still the addition of soy in ground beef has some advantages and disadvantages for hamburgers’ production. first of all, several reasons that justify the advantages of the addition of soy protein in ground beef hamburger can be the following:  the consumers’ demand for healthier meat products that are generally low in fats and calories and contain in addition health-promoting bioactive components [11];  soy is considered healthful, contains well balanced amino acids, being a source of protein [12];  detectable amounts of soybean lecithin were found in soy-based edible products such as hamburgers [13];  textured soy protein content was found to be the most important factor to minimize fat and moisture loss of hamburger patties after cooking [14];  soybean proteins can act as emulsifiers in preventing coagulation of fats during heating of the meat when the content of lean meat is low [15];  soy paste is a non-meat protein source which has a great potential use in the manufacture of emulsion type meat products with good acceptability and cost benefit [16];  soybeans have a low beany flavor, better color and consistency with a composition of 77.43% moisture, 13.09% protein and 5.95% fat [17]. on the other hand, the use of soy as a food has been limited because of its odor generated during processing, known as "bean odor" or "smell of green beans" [18]. 2. material and methods the descriptive analysis was used to define and quantify the sensory characteristics ascertained by panelists who provided intensity ratings for a set of selected attributes. it involves three main steps [19]. in the first step, a lexicon that describes the product space was developed, where synonyms were regrouped in a single term, using a free listing as a simple qualitative technique [20]. introduced to food consumer science by hough and ferraris, in 2009, [20-21], the free listing method is a qualitative technique, used for the first time specially in anthropology by rusell bernard, in 2005, [22], which consists in asking participants to ‘‘list all the x they know about the studied product”. in the present study 50 participants recruited from students of the faculty of food engineering, stefan cel mare university of suceava, romania, ranging in age between 18 and 26 years old, 76% female and 24% male, were asked to list some sensory characteristics of hamburger. in the second step, the panelists were familiarized with the sensory concepts of the panel (using iso 5492: 2008 and [23-24]). the terms obtained using free listing method, identified as most relevant for consumers, have been used during consumer studies from the third step, in which the intensity scale or cata questions methods were considered to evaluate the sensory characteristics of soyadded beef hamburger in different percentages (25%, 50% and 75%, respectively). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 361 the third step consists in tasting the three samples by each taster. the sensory analysis phases took the following steps: 1. sampling samples of the same type was made to obtain identical samples in terms of quantity, shape, consistency, color, appearance and temperature; the sensory samples analyzed should be similar to each other. 2. presentation of samples: sample temperature ranged between 60 and 65 0c. samples were coded (with three digit numbers) so that the tasters could not get any information regarding the identity of the sample. evaluators were given three versions of the product to be tasted (hamburger with 25% soy, 50% soy and, 75% soy respectively). 3. appreciation of descriptors was made using a 6 point value scale for taste, odor and flavor of soy in hamburger, from 0 to 5, ordered by intensity (where 0 is equivalent to the absence of perception and the 5 represents the maximum intensity). also, the influence of soy on the texture of hamburger, using a 5 point value scale from 1 to 5, ordered by intensity (where 1 corresponds to soft texture and 5 corresponds to 5 texture) was study. 4. while the testing sessions ran on, evaluators marked on a scale of intensity, based on the terms of the check-all-thatapply (cata) question list. 5. statistical analysis of results was made according to iso 11035:2007 and iso 4121:2008. samples were made as follows: the first hamburger sample consisted of 75% beef and 25% soy, the second hamburger sample consisted of 50% beef and 50% soy and the third one consisted of 25% beef and 75% soy respectively. 1500 g of ground beef, 1500 g dry soy granules (hydrated with water), salt and pepper were used to prepare the samples. a mixture was prepared from these ingredients, but with different amounts of soybean and beef, as follows: sample 1 contains 750g ground beef and 250g soy granules, sample 2 contains 500g ground beef and 500g soy granules and sample 3 contains 250g ground beef and 750g soy granules. these raw materials and ingredients were well mixed, allowed to stand for 10 minutes, afterwards meat patties were formed. after having been prepared, the hamburgers were cooked on the grill. figure 1 shows sections of hamburgers cooked on the grill. a). b). c). figure 1. section of hamburgers prepared with addition of: a). 25% soy (sample 1), b). 50% soy (sample 2), c. 75% soy (sample 3) three samples of beef hamburger were evaluated by 10 assessors (50% female and 50% male), recruited from students of the faculty of food engineering, stefan cel mare university of suceava, romania, ranging in age between 22 and 26 years old. samples were presented nomadically and water was available for rinsing between samples. to obtain more accurate data sensory examination was performed in a bright, clean room, free from foreign odors and at room temperature of about 200 c. sensory examination was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 362 performed in daylight and did not alter the colors of the products (iso 6658:2007). the plates with samples have been placed side by side on a white surface. the sensory characteristics appreciated in the examination session of soy-added hamburger were the following: smell, taste, color and texture. samples were coded with different 3-digit codes and were served at room temperature in a randomized order. principal component analysis (pca) of data was computed for sensory analysis of samples. all analyses were performed by using the software program xlstat (trial version 2013.10.08, ©addinsoft). 3. results and discussion the results obtained using free listing method consists of all the words elicited by participants. a search for recurrent terms was performed, grouping different word classes for the same term (i.e. adjectives and nouns) [22]. only the most frequently mentioned 5 terms were considered in our analysis. thus, we obtain a list of word which can describe taste (oily, metallic, raw, prick, frying), odor (fried, soy milk, rancid, caramel, bean), flavor (cereals, dried, boiled, cooked on the grill, no flavour), texture (soft, hard, elastic, wet, dry) and appearance respectively (fresh, appearance densely, homogeneous composition, crunchy, crumbly) of soy-added beef hamburgers. the data analysis resulted from the testing sessions contains two parts. in the first part, the panelists scored the products sensory characteristics (taste, odor, flavor and texture) on the basis of each descriptive attribute on a 6 point intensity scale for taste, odor, flavor, and 5 point intensity scale for texture respectively. table 1 shows the results (mean ± standard deviation) regarding the influence of soy addition on the taste, odor, flavor and texture of hamburger, in terms of intensity of soy taste, odor, flavor (0 = imperceptible, 1 = very low, 2 = low, 3 = medium, 4 = relatively strong, 5 = strong or very intense) and texture (1 = very soft, 2 = soft, 3 = medium, 4 = hard, 5 = very strong). table 1. the score obtained in terms of intensity soy presence in sensory characteristics of soy-added hamburger in different percentages (25% for sample 1, 50% for sample 2 and 75% for sample 3) samples taste odour flavour texture mean sd mean sd mean sd mean sd sample 1 0.200 0.400 0.300 0.900 0.600 0.917 4.000 0.447 sample 2 1.800 1.166 1.800 1.249 2.000 1.095 3.300 0.458 sample 3 3.000 1.000 3.100 1.221 3.000 1.414 1.900 0.700 sd = standard deviation using bartlett's sphericity test, correlations between these three samples of soy-added hamburger in different percentages (25% for sample 1, 50% for sample 2 and 75% for sample 3), in terms of the intensity of soy taste, odour or flavour, are significant, at the level of significance alpha=0.050. significant values of correlation at the level of significance alpha=0.050 (twotailed test) was obtained for the sensory parameters studied, as follows: a stronger correlation between taste (r=0.857) and odor (r=0.866) of the samples 2 and 3 respectively, between flavor of the samples 2 and 3 (r=0.904) and that of the samples 1 and 2 (r=0.896). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 363 also, texture is well correlated to the samples 2 and 3 (r=0.717) and the samples 1 and 3 (r=0.639). in the second part of the testing sessions, a check-all-that-apply cata question [25] consists of a list of 25 attributes which justify the influence of adding soy protein in ground beef of hamburger. assessors selected all attributes they consider appropriate to describe the three samples of soy-added hamburger. for each category consumers were asked to check the number 1, if the sensory characteristic attribute was present and the number 0 if this attribute was not present. figure 2 presents the taste characteristics checked (oily, metallic, raw, prick, frying), the ones that were preferred over by the assessors after the sensory examination session. figure 2. the number of taste characteristics chosen in cata method we noticed that the adjective “frying” is the most used in all three taste samples, in a percentage of 100% for the sample 1, 80% for the sample 2 and of 90% for the sample 3 respectively. figure 3 is presents the odor characteristics checked (fried, soy milk, rancid, caramel, bean), the ones that were preferred by the assessors after the sensory examination session. figure 3. the number of odor characteristics chosen in cata method we noticed that the adjective “fried” is the most used in the sample 1, “soy milk” odor is the most used in the sample 2 and, in the sample 3 the most used adjectives are “soy milk” odor and “bean” odor respectively (equally distributed on a percentage of 40%). figure 4 presents flavor characteristics checked (cereals, dried, boiled, cooked on the grill, offflavour), the ones that were preferred over by the assessors after the sensory examination session. figure 4. the number of flavor characteristics chosen in cata method we noticed that the expression “cooked on the grill” flavor is the most used in the sample 1 (in a percentage of 90%) and also in sample 2 (in a percentage of 60%), in the sample 3 the most used adjectives are “cereals” flavor (in a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 364 percentage of 40%) and “boiled” respectively (in a percentage of 30%). figure 5 presents the texture characteristics checked (soft, hard, elastic, wet, and dry) the ones that were preferred over by the assessors after the sensory examination session. figure 5. the number of texture characteristics chosen in cata method we noticed that the adjective “strong” texture is the most used in sample 1 (in a percentage of 80%), in sample 2 the most used adjectives are “soft” texture (in a percentage of 50%) and “elastic” texture respectively (in a percentage of 40%), and in sample 3 the adjective “soft” texture is the most used one (in a percentage of 90%). figure 6 presents the appearance characteristics checked (fresh, appearance densely, homogeneous composition, crunchy, crumbly), the ones that were preferred over by the assessors after the sensory examination session. figure 6. the number of appearance characteristics chosen in cata method we noticed that in sample 1, the most used expressions for appearance are “appearance densely” (in a percentage of 50%) and the adjective “fresh” respectively (in a percentage of 40%), the expression “homogeneous composition” is the most used in sample 2 (in a percentage of 70%), while in sample 3, the adjective “fresh” is the most used one (in a percentage of 60%). the biplot graphics [26] based on principal component analysis (pca) for the most important factors (f1 and f2), resulted from the above studied sensory characteristics are given in figures 7 11. figure 7 shows the biplot graphic for the taste characteristics chosen in cata method, making obvious the fact that all the three samples tested are very similar regarding their taste characteristics. figure 7. biplot graphic for the taste characteristics chosen in cata method the variation of the parameters studied implies the existence of 3 factors. f1 (2.984) is the principal component which registers an eigenvalue higher than 1. the percentage of variability represented by the first two factors is high (99.82 %). the first factor (f1) explains 99.45 % of the total variance with a significant parameter “frying” with an important contribution of 79.53%, for the observations for the factor f1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 365 figure 8 shows the biplot graphic for the odor characteristics chosen in cata method, making obvious the fact that samples 2 and 3 are very similar in the odor characteristics, but they are different in sample 1. f1 (2.042) is the principal component that registers an eigenvalue higher than 1. the percentage of variability represented by the first two factors is high (98.09 %). figure 8. biplot graphic for the odor characteristics chosen in cata method the first factor (f1) explains 68.07 % of the total variance with significant parameters “soy milk” and “bean” odor, having important contributions of 38.89% and of 20.57% respectively for the observations made for the factor f1. the second factor (f2) explains 30.02% of the total variance with a significant parameter “fried” having a contribution of 46.06% for the observations made for the factor f2. figure 9 presents the biplot graphic for the flavor characteristics chosen in cata method, making obvious the fact samples 1 and 2 are very similar in the flavor characteristics, but they are different in sample 3. f1 (2.268) is the principal component that registers an eigenvalue higher than 1. the percentage of variability represented by the first two factors is high (98.26 %). figure 9. biplot graphic for the flavor characteristics chosen in cata method the first factor (f1) explains 75.60 % of the total variance with the significant parameter “cooked on the grill” flavor, having important contributions of 69.91% for the observations for the factor f1. the second factor (f2) explains 22.66% of the total variance with significant parameters “cereal” and “boiled” flavour, having contributions of 25.47% and 8.1% respectively for the observations for the factor f2. figure 10 shows the biplot graphic for the texture characteristics chosen in cata method, making obvious the fact that samples 2 and 3 are very similar in the texture characteristics, but they are different in sample 1. f1 (1.792) is the principal component that registers an eigenvalue higher than 1. the percentage of variability represented by the first two factors is high (89.24 %). the first factor (f1) explains 59.72 % of the total variance with the significant parameter “soft” texture, having important contributions of 62.97% for the observations for the factor f1. the second factor (f2) explains 29.52% of the total variance with a significant parameter food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 366 “strong”, having the contributions of 56.71% for the factor f2. figure 10. biplot graphic for the texture characteristics chosen in cata method figure 11 shows the biplot graphic for the appearance characteristics chosen in cata method, making obvious the fact that all samples are different in the appearance characteristics. figure 11. biplot graphic for the appearance characteristics chosen in cata method f1 (1.526) and f2 (1.206) are the principal components that register higher eigenvalues than 1. the percentage of variability represented by the first two factors is high (91.06 %). the first factor (f1) explains 50.87% of the total variance with the significant parameter “homogeneous composition” appearance, having important contributions of 54.78% for the observations for the factor f1. the second factor (f2) explains 40.19% of the total variance with a significant parameter “fresh”, having contributions of 56.37% for the factor f2. 4. conclusions hamburgers are intensively consumed food products, especially by young people. the results obtained using free listing method consist of a list of terms describing taste characteristics (oily, metallic, raw, prick, frying), odor characteristics (fried, soy milk, rancid, caramel, bean), flavor characteristics (cereals, dried, boiled, cooked on the grill, offflavour), texture characteristics (soft, hard, elastic, wet, dry) and appearance characteristics respectively (fresh, appearance densely, homogeneous composition, crunchy, crumbly) of soyadded beef hamburger. our study was made on three samples of soy-added beef hamburger in different percentages (25%, 50% and 75% soybean of total mass) and its aim was to analyze the effect of soy addition in beef hamburger on sensory properties. after the testing session, the following results were obtained: in sample 1 (hamburger with 25% soy added) the taste, smell and flavor of soy are indistinguishable and the hamburger texture was graded as crusty (hard); in the case of sample 2 (hamburger with 50% soy added) the taste, odor and flavor of soybean in hamburger has been described as low or very low, and the texture was chosen as medium (neither hard nor soft); for sample 3 (hamburger with 75% soy added), the taste, smell and flavor of soy were defined as being low and texture was chosen as soft. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 367 after using the check-all-that-apply (cata) question method, the influence of adding soy protein in ground beef hamburger is as follows: the adjective “frying” is the most used in all three samples taste; the adjective “fried” is the most used for the odor in sample 1, “soy milk” odor is the most used in sample 2 and, whereas in sample 3, the most used adjectives are “soy milk” odor and “bean” odor respectively; the expression “cooked on the grill” flavor is the most used in samples 1 and 2, while in sample 3, the most used adjectives are “cereals” flavor and “boiled” flavour respectively; the adjective “hard” texture is the most used in sample 1, in sample 2 the most used adjectives are “soft” and “elastic” texture respectively and in sample 3 the adjective “soft” texture is the most used one; in sample 1 the most used expressions for appearance are “appearance densely” and “fresh” respectively, the expression “homogeneous composition” is the most used in sample 2, and in sample 3 “fresh” is the most used adjective. in conclusion, consumers expect that these meat products added with soy protein to taste, look and smell in the same way as the traditional hamburgers, prepared only with ground beef. 5. references [1]. vázquez-araújo l., chambers d., carbonell-barrachina a. a., development of a sensory lexicon and application by an industry trade panel for turrón, a european protected product, journal of sensory studies, 27 (2012), pag. 26–36. [2]. reinbach h. c., giacalone d., machado ribeiro l., wender l.p. b., frøst m.b., comparison of three sensory profiling methods based on consumer perception: cata, cata with intensity and napping, food quality and preference, 32 (2014), pag. 160–166. [3]. adams j., williams, lancaster a. b., foley m., advantages and uses of check-allthat-apply response compared to traditional scaling of attributes for salty snacks, in: 7th pangborn sensory science symposium, (2007), pag. 12–418. [4]. lado j., vicente e., manzsioni a., ares g., application of a check-all-that-apply question for the evaluation of strawberry cultivars from a breeding program, journal of the science of food and agriculture, 90, (2010), pag. 2268–2275. [5]. dooley l., lee y.s., meullenet, j.f., the application of check-all-that-apply (cata) consumer profiling to preference mapping of vanilla ice cream and its comparison to classical external preference mapping, food quality and preference, 21, (2010), pag. 394–401. [6]. ares g., gimenez a., barreiro c., gambaro a., use of an open-ended question to identify drivers of liking of milk desserts: comparison with preference mapping techniques, food quality and preference, 21, (2010), pag. 286– 294. [7]. ares g., deliza r., barreiro c., gimenez a., gambaro a., comparison of two sensory profiling techniques based on consumer perception, food quality and preference, 21, (2010), pag. 417–426. [8]. bruzzone, f., ares, g. & gimenez, a. consumers’ texture perception of milk desserts. comparison with trained assessors’ data. journal of texture studies, volume 43, issue 3, (2012), pag. 214–226. [9]. mcdonald ronald l., the complete hamburger: the history of america's favorite sandwich (1st ed.). london: citadel, (1997), isbn 1-55972-407-2. [10]. william o. giles, method for preparing hamburger patties, u.s. patent 5,484,625 issued january 16, (1996). [11]. weiss j. , gibis m., schuh v., salminen h., advances in ingredient and processing systems for meat and meat products, meat science, volume 86, issue 1, (2010), pages 196–213 [12]. t. schyver, c. smith, reported attitudes and beliefs toward soy food consumption of soy consumers versus nonconsumers, natural foods or mainstream grocery stores journal of nutrition education and behavior, volume 37, issue 6, (2005), pag. 292– 299 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 cristina-elena hrețcanu, alice-iuliana roșu, ioana zelezneac, statistical studies on the lexicon used in sensory evaluation of soy added beef hamburger, volume xii, issue 4 – 2013, pag. 359 – 368 368 [13]. rizzi c., galeoto l., zoccatelli g., vincenzi s., r. chignola r., peruffo a.d.b, active soybean lectin in foods: quantitative determination by elisa using immobilised asialofetuin, food research international, volume 36, issue 8, (2003), pag. 815–821 [14]. h. m. velioğlu, s. d. velioğlu, i̇. h. boyaci, i̇. yilmaz,, ş. kurultay, investigating the effects of ingredient levels on physical quality properties of cooked hamburger patties using response surface methodology and image processing technology, meat science, volume 84, issue 3, (2010), pag. 477–483 [15]. cederroth c. r., nef s., soy, phytoestrogens and metabolism: a review molecular and cellular endocrinology volume 304, issues 1–2, 25, (2009), pag. 30–42 [16]. das a. k., anjaneyulu a.s.r., gadekar y.p., singh r.p., pragati h., effect of full-fat soy paste and textured soy granules on quality and shelf-life of goat meat nuggets in frozen storage, meat science, 80, (2008), pag. 607– 614 [17]. das, a. k., anjaneyulu, a. s. r., verma, a. k., & kondaiah, n. , effect of fullfat soy paste and soy granules on quality of goat meat patties, international journal of food science and technology, 43, (2008), pag. 383–392. [18]. moon s.y., li-chan e.c.y., changes in aroma characteristics of simulated beef flavour by soy protein isolate assessed by descriptive sensory analysis and gas chromatography, food research international, volume 40, issue 10, december 2007, pag. 1239–1248 [19]. valentin d., chollet s., lelievre m.l., abdi h., quick and dirty but still pretty good: a review of new descriptive methods in food science, international journal of food science and technology, volume 47, issue 8, (2012), pag. 1563–1578. [20]. hough, g. e.; ferraris, d. free listing: a method to gain initial insight of a food category. food quality and preference, barking, v. 21, (2010), pag. 295-301., [21]. hough g., ferraris d. free listing: a method to gain initial insight of a food category. food quality and preference, 21, (2009), pag. 295– 301. [22]. rusell b. h., free listing. h. rusell bernard (ed.), research methods, anthropology: qualitative and quantitative approaches (4th ed). lanham: altamira press., (2005) , pag. 301–311. [23]. antmann g., ares g., varela p., salvador a., coste b., fiszman s. m. , consumers’ texture vocabulary: results from a free listing study in three spanish-speaking countries, food quality and preference, 22, (2011), pag. 165– 72 [24]. carr, b. t., craig-petsinger, d., & hadlich, s., a case study in relating sensory descriptive data to product concept fit and consumer vocabulary, food quality and preference, 12 (2001), pag. 407–412. [25]. ares g., giménez a., bruzzone f., identifying consumers’ texture vocabulary of milk desserts. application of a check-all-that-apply question and free listing, braz. j. food technol., 6, (2010), pag. 98-105 [26]. gabriel k.r, 1971, the biplot graphic display of matrices with application to principal component analysis, biometrika , 58 (3), pag. 453467 [27]. iso 5492:2008 – sensory analysis vocabulary [28]. iso 6658:2007 sensory analysis. methodology. general principles [29]. iso 4121:2008 sensory analysis. principles for the use of quantitative response scales [30]. iso 11035:2007 – sensory analysis. identification and selection of descriptors for establishing a sensory profile by a multidimensional approach 225 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 225 232 highly active freeze-dried probiotic concentrates with long shelf life *rositsa denkova1, zapryana denkova2, velichka yanakieva2, lyubka georgieva 1 department of “biotechnology”, sofia university “st. kliment ohridski”, sofia, bulgaria, rositsa_denkova@mail.bg 2department of “microbiology”, university of food technologies, plovdiv, bulgaria, zdenkova@abv.bg * corresponding author received august 21st 2013, accepted september 10th 2013 abstract: the application of probiotics in the composition of products intended to have long shelf life is limited due to their mandatory refrigeration storage. increase in the resistance of probiotics to the production conditions and improvement of their viability throughout storage is achieved by microencapsulation. the preservation of probiotic strains for a long period of storage and low cost transportation and thus, resulting in ready-to-use cultures for dairy and other food-related industries including in the field of probiotics is facilitated by freeze-drying using appropriate cryoprotectant media. the used cryoprotectants are hydrocolloids which are natural, vegetable raw materials, with numerous beneficial physiological effects on the body. the aim of the present study is obtaining highly active probiotic freeze-dried bioproducts of selected probiotic strains by applying a combined modern biotechnology with the following main stages: selection, immobilization of the selected strains in the hydrocolloid matrix, cryoprotection, freezing and freeze-drying. the selected probiotic strains lactobacillus plantarum x2 and lactobacillus paracasei rn5 are immobilized and freeze-dried using combined hydrocolloid matrix sodium alginate and high-ester pectin. the survival of the two lyophilized microorganisms after lyophilization and in the course of storage for nine months is monitored – both strains retain high concentrations of active cells. the used hydrocolloids are appropriate for the mechanical immobilization of cellular cultures and as cryoprotectants. the new combined biotechnology allows the preparation of probiotic bioproducts with long shelf life and intact probiotic potential for application as supplements for foods with dietary purpose and in the prophylaxis and treatment of gastrointestinal disorders. keywords: probiotic, polyand oligosaccharides, immobilization, cryoprotection, freezing, freezedrying (lyophilization), lactobacillus 1. introduction when incorporating a probiotic strain into a food matrix there are two main problems to be addressed, namely the resistance of the strain to the technological conditions of food production [1] and the maintenance of cellular viability up to the expiring date of the food, since the manufacturer has to ensure an optimal administration of the probiotics throughout the whole shelf life of the product. that could lead to limitations of the use of probiotics in longlife products especially if they are not stored in a refrigerator. freeze-drying is one of the most common methods used to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 226 preserve probiotics. however, this method is not considered optimal, as it just protects probiotics from the humidity but does not confer protection from certain technological and environmental conditions such as varying temperatures, oxygen toxicity or passage through the intestinal tract. decreased viability of the probiotics during or after freeze-drying is also a problem. in this sense, microencapsulation, which is defined as a process in which the cells are retained within an encapsulating matrix or a membrane, may provide an approach for protecting probiotics. this technique would allow the isolation of probiotics from the environment thus increasing their resistance to the conditions of production and would also improve their viability throughout the storage. the protective effect of microencapsulation is due to the limited diffusion of inhibitory substances such as metabolic products from starter cultures, h2o2, lactic acid, and bacteriocins into the capsules [2, 3]. freeze-drying of lactic acid bacteria (lab) facilitates their preservation for a long period of storage, their low cost transportation resulting in ready-to-use cultures for dairy and other food-related industries, particularly in the emerging and continuously growing field of probiotics. in order to apply low rates of freezing, which are considered advantageous in industrial production of lactic acid starters because of their lower cost than high freezing rates (e.g. liquid nitrogen), suitable cryoprotective agents should be employed [4]. various materials have been used for microencapsulation of probiotics, such as alginate [5], κ-carrageenan [6], cellulose acetate phthalate [7], gelatin [8] and pectin [2, 9]. the hydrocolloids used in the present study are sodium alginate and high-ester pectin. they act as matrices for the immobilization of the cell material and as cryoprotectants. they are natural, vegetable raw materials, with well known content of biologically active substances and beneficial physiological effects on the body [10, 11, 12]. alginate is a linear heteropolysaccharide of d-mannuronic and l-guluronic acid extracted of various species of algae [13] which has interesting characteristics like its relative inexpensiveness, structural simplicity, and biocompatibility [3]. the use of alginate is limited due to its low physical stability in the presence of ca+2chelating agents, monovalent ions and harsh environmental conditions [13]. entrapment of probiotic bacteria in alginate matrices is among the most widely used immobisation techniques [14, 15]. this approach has been shown to enhance bacterial cell tolerance to alcohols, phenols, antibiotics or quaternary ammonium sanitizers [16] and resistance to adverse processing techniques such as freezing [17] and freeze-drying [18] and hostile environments such as simulated gastric environment [19]. blending with other polymers such as starch [20] provides to some extent alginate gels with additional features and it has been reported as an effective method for the encapsulation of probiotics [3]. pectins are high-molecular acid mucopolysaccharides composed of polygalacturonic acid in which some of the carboxyl groups are esterified with methyl alcohol residues. several forms of pectin are produced – low-, mediumand highester pectin, depending on the methylation of the cooh-groups. when esterification affects less than 30% of the total number of carboxyl groups the pectin is called lowfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 227 ester pectin. when methylation concerns 30% to 60% of the -cooh-groups, the esterification is medium, and at esterification of 60% to 95.0% of the carboxyl groups the pectin is called highester pectin. fruit pectins (citrus, apple) possess gelling ability, related to their high degree of esterification. the result of this activity is their sorption capacity, to which their hypolipidemic, detoxifying and radioprotective action are attributed. a number of dietetic foods like milk enriched with pectin, functional foods and herbal medicines, creams and others are produced in bulgaria on the basis of pectin. the main objective of the present study is to obtain highly active probiotic lyophilisates of selected strains of lactic acid bacteria by applying combined modern biotechnology. 2. materials and methods microorganisms: probiotic microorganisms: lactobacillus plantarum x2 and lactobacillus paracasei rn5, isolated from naturally fermented sourdough. media: mrs-broth. composition (g/dm3): peptone from casein 10, yeast extract 4; meat extract 8, glucose 20; k2hpo4 2, sodium acetate 5; diammonium citrate 2; mgso4 0.2; mnso4 0.04; tween 80 1 cm3/dm3; ph is adjusted to 6.5. sterilization 15 minutes at 118ºc. mrs-agar. composition (g/dm3): mrsbroth + 2% agar. sterilization 15 minutes at 118ºc. saline solution. composition (g/dm3): nacl 5. sterilization 20 minutes at 121ºc. hydrocolloid matrices: pectin + sodium alginate / 1:1 / 1.2% solution of sodium alginate, and 4% solution of pectin. the concentrations of the hydrocolloid solutions are determined on the basis of the physicochemical parameters of the hydrocolloids used, namely (table 1): table 1 physicochemical parameters of the hydrocolloids used total moisture (%) from 2.0 to 13.00% molecular weight – according to the certificate from 5000 to 800 000 ph from 3.0 to 8.0 viscosity (mpa) from 50.00 to 58.00 dissolution temperature (ос) nа – alginate 18–25; pectin /hep/ – 20-22 the application of hydrocolloids of plant origin is in compliance with the requirements for physiological activity, safety and microbial stability [21]. lyophilization the new bioproducts are processed using a combined method of technological treatment that includes the following stages: primary processing of the cellular suspension, freezing and freeze-drying. in the primary processing the cellular suspensions of the selected lactobacillus strains are diluted, equilibrated, dosed and immobilized by inclusion in the polymer matrix that acts as a cryoprotectant. these processes are performed before freezing. a mechanical method for the immobilization of the microbial cells is applied. the immobilization includes the following processing steps: 1) obtaining cellular suspensions by cultivation of the strains in mrs-broth for 24 hours at 37°c. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 228 2) mixing the obtained cellular suspensions with the prepared polymer solution at 40-45°c and homogenization for 1.5 hours in a bioreactor at 500 rpm. samples are pre-frozen in chambers with forced convection of the air environment at a temperature of -30°c to -35°c for 12-15 hours. the freeze-drying is performed in a vacuum sublimation installation “hochvakuum-tg 16.50” with contact heating of the plates in icft institute of cryobiology and food technologies, sofia, bulgaria. the temperature regime for freeze-drying is programmed so as to obtain optimum drying speed that would ensure the good quality of the final products. the final drying off temperature ranges from +30°c to +35°c. after lyophilization, the lyophilizates are digested in the granulator "erveka". the digested lyophilized bioproducts are packed in three layer aluminum foil, sealed under vacuum. a summary description of the results of the technological tests related to the whole cycle of freeze-drying of the specimens is given in table 2. table 2 parameters of freeze-drying parameters unit value indicators і. object of study cellular suspensions lactobacilli and bifidobacteria in a cryoprotectant medium – highester apple pectin and sodium alginate іі. layer thickness mm 11 ііі. freezing 1.temperature of freezing °с -30°с to – 35°с 2. rate of freezing °с/sec slow method 0.05 0.06 3. eutectic temperature °с – 37.00 4. hardening temperature °с – 40.00 іv. freeze-drying 1. load bearing surface kg/m 2 10.60 2. temperature of final drying off °с – 30.0 3. temperature of the desublimer °с from 55.0 to 60.0 – before drying from 65.0 to 70.0 – in final drying off 4. partial pressure ра from 30.0 to 34.0 5. chamber pressure ра from 20.0 to 27.0 6. temperature of final drying off °с + 30 7. maximum temperature of the product °с 30 8. residual moisture content of the product % from 1.37 to 3.09 /according to bs – no more than 6.0%/ 9. duration of the process h from 12 to 14 10. storage conditions in three layer bags of aluminum foil, sealed under vacuum, stored at a relative humidity less than 35% at 20°c – 22°c microbiology 1. the microbiological status of the native and the freeze-dried samples acc. bs 1670-82 and ordinance № 5 of the mh sg 39/84, bs en iso 4833. indicators: • lactic acid bacteria cfu/g; • total number of mesophilic aerobic and facultative anaerobic microorganisms cfu/g; • coliforms in 0.1 g of product; • pathogens including salmonella sp. in 25.0 g of product; • pathogenic staphylococci in 1.0 g of product; food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 229 • sulfite reducing clostridia in 0.1 g of product; • spores of microscopic molds, cfu/g; • yeasts, cfu/g; 2. the number of viable cells is determined through appropriate serial dilutions of the samples and spread plating on coloured laptg10 – agar medium (to determine the number of viable lactobacilli cells) or on elective media (for the enumeration of the specific microorganisms). 3. the petri dishes are incubated for 72 hours at 37±1°с until the appearance of countable single colonies. the count of the colonies is then used to estimate the number of bacteria in the original sample. bacterial counts are transformed to log values. 4. the survival of lactobacilli in % is determined according to the following formula: survival, % = 100 [(xn / x0) * 100], wherein: xn – the concentration of viable cells in the moment of reporting of the survival x0 – the concentration of viable cells before lyophilization. 3. results and discussion the strains lactobacillus plantarum x2 and lactobacillus paracasei rn5, isolated from naturally fermented sourdough with proven probiotic properties [22, 23, 24, 25] are cultured in mrs-broth in a bioreactor with a volume of 2 dm3 at constant stirring at 100 rpm at 37±1°c. immobilization in a hydrocolloid matrix consisting of highester apple pectin and sodium alginate, followed by freeze-drying of the two strains is carried out. the resulting lyophilized bioproducts are stored at 20°c 22°c and the changes in the concentration of viable cells of lactobacilli for nine months of storage is monitored (fig. 1 and fig. 2). the examinations of the lyophilized bioproducts according to the standard methods show absence of insemination with pathogenic microflora (table 3): table 3 microbiological status of the probiotic lyophilisates type of pathogens norm according to bs number of tested microorganisms, cfu/g 1. general number of mesophilic aerobic microorganisms, cfu/g no more than 800 310 2. coliform bacteria in 0.l g of the product not to be found not found з. sulfite reducing clostridia in 0.l g of the product not to be found not found 4. salmonella sp. in 25.0 g of the product not to be found not found 5. staphylococcus aureus in 1.0 g of the product not to be found not found 6. spores of microscopic molds, 100 cfu/g no more than 100 35 – 40 7. yeasts, cfu/g no more than 100 32 – 48 no pathogenic microorganisms are found in the probiotic lyophilisates. the obtained lyophilizates meet the standard requirements for microbial purity of food. the absence of pathogenic insemination evidences that the overall process is carried out in compliance with the sanitary standards and requirements. the freezedrying process has a bactericidal effect and does not lead to the development of conditions for insemination of the freezedried products. moreover, the higher the degree of dehydration is, as in our bioproducts (residual moisture content food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 230 below 5.0%), the lower the survival of the pathogenic microorganisms is. table 4 survival of probiotic lactobacilli upon lyophilization (in %) strain before lyophilization after lyophilization survival rate, % lactobacillus plantarum x2 9.00x10 13 1.90x1013 95.26 lactobacillus paracasei rn5 6.00x1013 1.60x1013 96.25 the results of the microbiological tests prove the high survival rates of the two strains after lyophilization 95.26% for lactobacillus plantarum x2 and 96.25% for lactobacillus paracasei rn5 (table 4). the results in table 4 show a slight decrease of the number of viable cells in the process of freeze-drying, which is due to the optimally conducted process, including the use of a combined cryoprotectant hydrocolloid matrix. the observed decrease is by about 0.68 logn for lactobacillus plantarum x2 and by 0.57 logn for lactobacillus paracasei rn5. the hydrocolloids used for the mechanical immobilisation of the probiotic strains of microorganisms, as well as as a cryoprotectant agent, sodium alginate and high-ester apple pectin, prove to be highly efficient, as evidenced by the high survival rate of the lyophilized microorganisms. the probiotic lyophilized concentrates of viable cells of lactobacillus plantarum x2 and lactobacillus paracasei rn5, isolated from naturally fermented sourdough, are stored at 20°c 22°c for 9 months, taking samples every three months and determining the number of viable cells. the results of this examination are shown on fig. 1 for lactobacillus plantarum x2 and fig. 2 for lactobacillus paracasei rn5. experimental data indicate that in the course of storage for nine months the concentrations of viable cells of the two probiotic concentrates are above 109cfu/g (fig. 1 and fig. 2). figure 1. survival of the cells of lactobacillus plantarum x2 after lyophilization and during storage of the freeze-dried bioproduct for 9 months at 20°c 22°c. figure 2. survival of the cells of lactobacillus paracasei rn5 after lyophilization and during storage of the freeze-dried bioproduct for 9 months at 20°c 22°c. according to wolfson, 1999 [26] 109cfu/g is the required minimum concentration of cells of probiotic bacteria food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 231 in order for a concentrate to be defined as highly effective probiotic preparation. the obtained results demonstrate that the main objective of our research is achieved probiotic lyophilisates with high survival rate of the beneficial microflora after lyophilization are obtained. the number of viable cells before lyophilization is over 1013cfu/cm3 and after freeze-drying it remains over 1013cfu/cm3 i. e. the survival rate is high 95.26% for lactobacillus plantarum x2 and 96.25% for lactobacillus paracasei rn5. this effect is due to the effectiveness of the hydrocolloids used as a cryoprotectant medium a combined hydrocolloid matrix, consisting of sodium alginate and highester apple pectin. a number of effects are attributed to the use of alginate and pectin as a hydrocolloid matrix for the immobilization of probiotic cultures: significant stabilization of the enzyme activities of the immobilized cells; stabilization and increase of the overall activity of the immobilized probiotic system, which is beneficial for their survival after lyophilization and during storage. the high survival rate of the tested lactobacilli in the lyophilized biological products are due to the optimal conduction of the whole process freezing conditions, applying suitable cryoprotectant medium and process parameters, proper determination of the duration of the cycle, which provides low residual moisture content in the final lyophilizates, resistance to the thermal processes and extension of their storage. 4. conclusions the survival and the viability of the probiotic lactobacilli bioconcentrates obtained through lyophilization are high – 1.60x1013 for lactobacillus paracasei rn5 (survival percentage – 96.25%) and 1.90x1013 cfu/g for lactobacillus plantarum x2 (survival percentage 95.26%) as a result of the applied technological approach. the combined hydrocolloid matrix of sodium alginate and high-ester apple pectin prove to be a highly effective cryoprotectant of endocellular type that increases the survival of lactobacilli during lyophilization. the absence of pathogenic microflora and the low residual moisture content of the new freeze-dried bioproducts determine the prolonged storage. during the monitored 9-month period no adverse changes in the microbiological safety and activity of the lyophilized probiotic strains are established. 5. references [1] saarela m., mogensen g., fondén r., mätö j., mattila-sandholm t., probiotic bacteria: safety, functional and technological properties, journal of biotechnology, 84. 197 – 215, (2000). [2] heidebach t., först p., kulozik u., microencapsulation of probiotic cells for food applications, critical reviews in food science and nutrition, 52. 291 – 311, (2012). [3] martin m. j., lara-villoslada f., ruiz m.a., morales m.e., effect of unmodified starch on viability of alginateencapsulated lactobacillus fermentum cect5716. lwt food science and technology, 53. 480 – 486, (2013). [4] chavarri f. j., depaz m., nunez m., cryoprotective agents for frozen concentrated starters from non-bitter streptococcus lactis strains, biotechnology letters, 10(1). 11–16, (1988). [5] mandal s., puniya a. k., singh k., effect of alginate concentrations on survival of microencapsulated lactobacillus casei ncdc-298, international dairy journal, 16. 1190–1195, (2006). [6] adhikari k., mustapha a., grün i. u., survival and metabolic activity of microencapsulated bifidobacterium longum in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, lyubka georgieva, highly active freezedried probiotic concentrates with long shelf life, food and environment safety, volume xii, issue 3 – 2013, pag. 225 232 232 stirred yoghurt, journal of food science, 68. 275– 280, (2003). [7] fávaro-trindade c. s., grosso c. r. f., microencapsulation of l. acidophilus (la-05) and b. lactis (bb-12) and evaluation of their survival at the ph values of the stomach and in bile, journal of microencapsulation, 19. 485–494, (2002). [8] annan n. t., borza a. d., hansen l. t., encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic bifidobacterium adolescentis 15703 t during exposure to simulated gastro-intestinal conditions, food research international, 41. 184–193, (2008). [9] gerez c. l., font de valdez g., gigante m. l., grosso c. r. f., whey protein coating bead improves the survival of the probiotic lactobacillus rhammnous crl 1505 to low ph, letters in applied microbiology, 54. 552–556, (2012). [10] murgov i., denkova z., probiotics on the threshold of the new millennium, journal “milk”, 27-29.35, (2001). [11] idris a., wahidin s., effect of sodium alginate concentration, bead diameter, initial ph and temperature on lactic acid production from pineapple waste using immobilized lactobacillus delbrueckii, process biochemistry, 41(5): 11171123, (2006). [12] georgieva l., development of new cryobiotechnologies as a scientific basis for obtaining effective lyophilized foods for health, dsc thesis, institute of cryobiology and food technologies, sofia, bulgaria, (2009). [13] smidsrød o., skajak-braek g., alginate as immobilization matrix for cells, trends in biotechnology, 8. 71 – 78, (1990). [14] champagne c.p., gaudy c., poncelet d., neufeld r.j., lactococcus lactis release from calcium alginate beads, appl. environ. microbiol., 58. 1429–1434, (1992). [15] klinkenberg g., lystad k.q., levine d.w., dyrset n., cell release from alginate-immobilized lactococcus lactis ssp. lactis in chitosan and alginate coated beads, j. dairy sci. 84. 1118–1127, (2001). [16] lacroix c., grattepanche f., doleyres y., bergmaier d., immobilised cell technologies for the dairy industry. in: nedovic v., willaert r. (eds.), applications of cell immobilisation biotechnology, springer, the netherlands, pp. 295–321, (2005). [17] sheu t.y., marshall r.t., heymann h., improving survival of culture bacteria in frozen desserts by microentrapment, j. dairy sci. 76. 1902–1907, (1993). [18] kearney l., upton m., loughlin a.m.c., enhancing the viability of l.plantarum inoculum by immobilizing the cells in calciumalginate beads incorporating cryoprotectants, appl. environ. microbiol., 56. 3112–3116, (1990). [19] cui j.h., goh j.s., kim p.h., choi s.h., lee b.j., survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles, int. j. pharm., 210. 51–59, (2000). [20] sultana k., godward g., reynolds n., arumugaswamy r., peiris p., kailasapathy, k., encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt, international journal of food microbiology, 62. 47 – 55, (2000). [21] willemer h., quality control during freeze-drying of biological products, 16th international congress of refrigeration, paris, intrnational institute of refrigeration, 78-83, (1983). [22] denkova r., georgieva l., denkova z., urshev z., yanakieva v., biochemical and technological properties of lactobacillus plantarum x2 from naturally fermented sourdough, journal of food and packaging science technique and technologies, year i, №1, 2012, 59 – 64, (2012a). [23] denkova r., yanakieva v., denkova z., ilieva s., petkova a., antimicrobial activity of l.plantarum strains isolated from different sources, scientific conference with international participation “food science, technique and technologies 2012”, 77 – 82, (2012b). [24] denkova r., ilieva s., nikolova d., evstatieva y., denkova z., yordanova m., yanakieva v., antimicrobial activity of l.plantarum x2 against pathogenic microorganisms, bulg. j. agric. sci., supplement 2, 19. 108–111, (2013a). [25] denkova r., yanakieva v., denkova z., galenova e., identification and survival in the model conditions of the gastrointestinal tract of the probiotic strain l.casei rn5, 23rd international scientific conference “challenges for the scientists in relevance to the new program for science and innovations of eu “horizon 2020” (in press), (2013b). [26] wolfson, n. p., a probiotics primer, nutrition science news, 4(6). 276-280, (1999). 146 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 22016, pag.146 154 the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages *maria poroch serițan1, nicoleta – gabriela popescu2 1faculty of food engineering, ”ștefancel mare” university of suceava, universitatii str., no. 13, 720229, suceava, romania, mariap@fia.usv.ro, 2popescu.nicoletagabriela@yahoo.com *corresponding author received 25th april 2016, accepted 27th may 2016 abstract: the purpose of this paper is to assess the meat product quality (especially summer sausage type) during storage based on relationship between nitrite concentration and colour parameters. nitrites are used for conservation of meat products, being responsible for colour characteristics, formation of distinct flavours which allows the differentiation from nitrite-free products. it also prevents and controls the oxidation of lipids and serves as antimicrobial ingredient composition by it self or through synergy with other ingredients. nitrite reacts with myoglobin through nitrogen monoxide resulting pink-red nitroso-myoglobin compound associated by customers with a tasty and nutritious food. concentrations of nitrites were quantified by molecular absorption spectrometry method using griess reagents. the colour reflectance of meat products was focused on determination of l*, a*and b*. also, ph was measured by applying the potentiometric method and moisture content of the meat product was determined by drying in the oven, as they can influence both the colour and nitrite concentration. our results show that during storage nitrate concentrations did not differ greatly, and the colour does not have a major influence because it remains pink – red. measurements of colour parameters showed that during storage components l * and b * registered slight decreases and chromatic component a * a significant difference respectively. keywords: griess method, colour reflectance, quality of meat products. 1. introduction nitrites and nitrates are added to meat products for technological and antibacterial reasons: (1) act as fixers of color and prevents meat browning [1], (2) help to development flavor [2, 3], (3) have antioxidant properties, which hampers oxidation of lipids responsible for the rancidity [4 – 7] and (4) protection against the spreading of clostridium botulinum [8, 9]. salting mix which include nitrites is added in meat products for maturing red obtained after a number of reactions, until no myoglobin (fe2+) is formed [10]. the red system of cycle no porphyrin exists in meat products heated to 120°c, although by heating no myoglobin of moiety the protein is denatured. an advantage for the customer is that the thermally stable red color will be changed to alter bacteria, therefore, the consumer will recognize alteration by changing the color [11]. as an unintended consequence of maturation nitrite formation is discussed much in literature n nitrosamines (na), which could be carcinogens [12 21] and therefore the controlling and checking of the added dosage is compulsory. in order to secure the meat products quality and safety for customers the addition of nitrates http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 147 and especially that of nitrites is strictly monitored. the maximum admitted quantity of these additives is determined by the law [22, 23]. the purpose of this paper is to assess the quality of the meat product summer sausage type during storage by analyzing the relationship between nitrite concentration and colour parameters. 2. matherials and methods they were analyzed samples of meat products (sausages summer cooked product smoked) by three meat factories in suceava. the analyzes of the meat products have been made on the 1st day, 7 days and 15 days using the product provided by the local manufacturers. determinations of nitrites content were performed by molecular absorption spectrometry method using jenway spectrophotometer, model 6400. nitrites were determined based on their diazotization reactions, in acid medium, with sulphanilic acid and subsequent coupling with alpha naphthylamine, which results in a complex with red color, which measure the absorbance at λ =520 nm. nitrite content was calculated from the standard curve prepared with standard sodium nitrite solution (1 ml working standard solution = 0.001 mg no2-), as the standard method (griess). the curve equation obtained is shown in equation 1: y = 0.0668x – 0.0521, r2 = 0.9985. (1) proteins were eliminated from samples by heating at temperatures of 60ºc 70ºc for 20 minutes and using solutions of potassium ferrous cyanide 10%, zinc acetate 22%, considered to be the most efficient. in order to determine the moisture content of summer sausage samples we used the drying method oven until a constant weight [24], and the loss of weight is calculated with equation 2: (2) where: g – vial mass (g), g1 – vial sample weight before drying (g) și g2 – vial sample mass after drying (g). the color is measured using reflection spectrum of a sample and comparison with a standard illuminant. the amount of light energy from the sample reflects is manipulated and reduced to tristimulus values x, y and z. these values correspond to the physiological response of the three types of color receptors in the human eye. x, y and z values are combined into the uniform color space values: l*, a* and b* [25]. the chroma, considered a quantitative attribute of coloring, is used to determine the difference degree of the hue angle (h*) in comparison with a grey color of the same luminosity. the description of a color using the hue angle attribute refers to how humans perceive an object’s color red, orange, green, blue, etc. the 0º or 360º angle represents the red hue, while angles of 90º, 180º and 270º represent yellow, green and blue hues [26, 27]. within the study, the hr 4000 cg-uv-nir spectrometer (ocean optics inc., dunedin, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 148 fl) which is based on the reflection property of samples was used. in this respect, a scanning with 0.025 nm resolution has been carried out for the entire wavelength spectrum ranging from 200 up to 1100 nm. a tungsten halogen light source (uv-vis-nir light source dh-2000, mikropack) was used. the experimental data was displayed and stored using the operating software ooi base32 including ooicolor from ocean optics. the light from light source dh2000 was focused to the sample through an array of optical fibres (qr400-7-uv/bx, ocean optics inc., dunedin, fl). reflectivity was measured as against a reference standard – wsdiffuse reflectance standard [25]. all the equipment used in this work belongs to the food safety research centre of the faculty of food engineering, ștefan cel mare university of suceava, romania. 3. results and discussion nitrates and nitrites are used in meat curing process in order to maintain its red. myoglobin meat pigment and hemoglobin the blood pigment that reacts with nitrogen oxide which is the degradation product of nitrite and nitrate, forming nitrozomioglobina (no-mb) and nitrozohemoglobina (no-hb), two pigments salting. the pigments have a high stability over time and under the action of heat treatment are formed nitroso-chromogens (nitrosomiochromogen and nitrosohemochromogen) by denaturing the protein part of the pigments, ie globin. the formation mechanism of nitrosopigment is [28]: nano3+ 2h → nano2 + h2o reduction bacterial reduction due to their muscle enzymes ph acid nano2 → hno2 + nar 2hno2 → no+ no2+h2o reductants meat or added acidic ph, reducing bacteria no+ mb (hb) → nomb sau nohb from the reactions results that nitrate has no direct effect coloring meat, but it serves as a source of nitrite. denitrifying bacteria secrete nitrate reductase and thereby the conversion of nitrate to nitrite at ph values greater than 5.8. it can be considered and action reductases own muscle tissue of the meat on the conversion of nitrate into nitrite [29]. following the test results obtained on the first day it was found that the three producers have complied with the provisions of the legislation which stipulates a maximum of 7 mg/kg sodium nitrite and 45 50% humidity. during storage we have changed organoleptic characteristics, the product dries significantly and so was recorded and a change in the content of sodium nitrite. as can be observed from figure 1 it is evident that the sodium nitrite quantity increased in all three samples analyzed during storage, with the decrease in moisture content and modification of the organoleptic characteristics. the results obtained in this study are consistent with those in the field, similar results were obtained by [30]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 149 fig. 1. variation content of nitrites and humidity in three samples during storage immediately after salting nitrate it was observed that at the surface, the meat has bright red color, while in depth the meat color is red purple. an occurrence of nitrite and salting pigment that forms in the air oxidizes and becomes a gray color. in a more advanced stage meat is red and has an acid reaction. nitrite disappears immediately and has no effective action on the formation of color [29]. color evaluation is more than a numerical expression. color is an important quality attribute in food industry and it influences the consumers’ choice and preferences. the food color measurement can be used as indirect quality indicator of other attributes such as: flavor and pigment content [27, 31]. the aspect is one of the most important sensory attributes of fresh and processed food. the color of food is the first quality parameter appraised by consumers and it is momentous for product acceptance. food aspect, greatly determined by color, is the first sensation that the consumer perceives and uses as a tool to accept or reject a certain type of food. food visual aspect has a strong influence on consumer’s opinion on food quality. color can be correlated with other quality attributes such as: sensory, nutritional and visual or non-visual defects and helps control the products’ quality. color is a perceptual phenomenon which depends on the observer and on the color observing conditions. l*, a*, b*, hue angle (h*) and chroma parameters for the samples subjected to the assay analysis were measured. lightness, figure 2, all three samples showed values similar to the analysis of the first day, while after seven days of storage under refrigeration have made some changes in color parameters. thus was observed a slight decrease in lightness, color becomes darker sausages summer samples, and 15 days after the change was very visible parameters degradation products due to changes. l* parameter recorded the lowest values observed since the closure sharp color products. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 150 fig. 2. variance based on the retention period of lightness in the case of a* parameter in the first day the values obtained from the three samples were negative, the lowest value obtained for sample 1 and sample 2 at the most, as shown in figure 3. darkening, which was observed after 7 days of storage, it was also reflected in the change of the parameter values a*. for all the evidence is on the rise and so when determining all values were positive, the most visible change is observed in sample 3. after 15 days of storage in a refrigerator, sample 1 and sample 2 had similar values to a* and the increase being higher in sample 1, while for sample 2, the value in the seventh day of storage the difference was small. thus, during storage organoleptic characteristics of the products have changed, the most visible change being the darkening pink initial samples of the product, sausages summer, and after expiry acquiring a dark red. fig. 3. variance based on the retention period of a* parameter food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 147 another parameter which describes the b* color of a product whose variation in time is shown in figure 4. the values of b* were positive for the first two samples analyzes from day one, but with significant differences, because for the first sample value is much higher, while the value for the third sample is slightly negative with a value of 0.4. the b* value has dropped for all samples after seven days of storage; the most significant decrease observed for the samples 1 and 3, resulting in a negative value. following the intensification of degradation processes for all 3 samples b* parameter has a significant decline, all values are negative. fig. 4. variance based on the retention period of b* parameter the variation of quality of the color attribute, hue angle, is represented in figure 5. a larger angle indicates a less yellow in the sample. sample 1 on the first day of storage had the largest angle; indeed not visible yellowish hue. at the opposite pole sample 3 yellowish hue is observable with the naked eye. after seven days the angle register with a fall in the first two tests, the most visible being the sample 1. for sample 3 is a slight increase angle. after 15 days of storage yellowish nuances come into sharper focus, registering approximately equal to three samples. while decreasing the angle may connote a rancid fat [32] which causes the yellow color. fig. 5. variance based on the retention period of hue angle parameter food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 152 the parameter variation of chroma obtained are shown in figure 6. regarding chroma parameter after the first day of storage, it seems that the color intensity of sample 1 is most perceived by the human eye, with the highest value. at the opposite pole is the color intensity of the sample 3. for sample 1 chroma parameter decreased after 7 days of storage, while the sample 2 and sample 3 was a slight increase. chroma parameter has registered the highest values after a 15-day shelf life, color intensity is easily perceived by the human eye due to the formation brown color. fig. 6. chroma parameter variation depending on storage period fig. 7. the difference in color during storage period δe* – represents the color difference magnitude between the value of the initial color and the value of the final color and gives the distance between the points representing the color, being calculated according to the equation [27]: δε*= [(δl*)2 + (δa*)2 + (δb*)2]1/2 (3) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 153 perceptible color differences can be classified analytically as very distinct (δe* >3), distinct (1.5 < δe* < 3) and less distinct (1.5 < δe*) [27]. in some cases the total color difference and chromaticity are considered the most sensitive parameters for measuring color degradation. as can be seen from figure 7, in the fifteenth day color difference is very noticeable, and the highest value is recorded sample 1, although all samples inregistrated color difference great on the fifteenth day storage. 4. conclusion the residual amount of nitrite increased after 15 days of storage in all the three samples of the sausages summer products. for the second sample an amount slightly higher than the permitted quantity was obtained. the increase in residual nitrite after storage is closely correlated with dehydration products, which is visible and determines the concentration of sodium nitrite. during the 15 days of products storage the organoleptic characteristics have significantly changed, the color is easily observed that goes from pale pink to deep red and after the 15th day of storage becomes dark red-brown. therefore, the lightness decreased during storage due to darkening. also, by decreasing the content of water and increasing the amount of residual nitrite the a* value increased in time, while the parameter b* decreased due to degradative processes. chroma parameter, responsible for perceiving color intensity to the human eye, has grown and presented elevated values after 15 days of storage of the product. products with more pronounced tinged pink, with shades of yellow were observed hue angle parameter values are greater, while the sample with a higher fat content were obtained lower values of this parameter. also, after 15 days of storage of the product in all samples was registred a decrease in the parameter hue angle and thus the appearance of yellow shades. so it is obvious that there is a close correlation between the amount of sodium nitrite in samples sausages summer humidity and color parameters changes during storage. 5. acknowledgments we would like to address our thanks to the societatea comercială s.c. killer s.r.l. horodnic de jos, suceava, for their help in all aspects of this investigation in this project ”studiu privind influența calității materiei prime asupra calității nutriționale a produselor finite din carne”, no. 276 / 9.01.2013. 6. references [1]. wirth f., restricting and dispensing with curing agents in meat products, fleischwirtschaft, 71, 1051−1054, (1991) [2]. jira w., chemical reactions of curing and smoking — part 1: curing, fleischwirtschaft, 84, 235−239, (2004) [3]. fischer a., bristle a., gehring u., herrmann k., gibis m., reddening of emulsion type sausage without nitrite curing salt — part 1: colour, colour stabilisation, nitrite and nitrate concentrations, sensory properties, fleischwirtschaft, 85, 110−115, (2005) [4]. freybler l. a., gray j. i., asghar a., booren a. m., pearson a. m., buckley d. j., nitrite stabilization of lipids in cured pork, meat science, 33, 85−96, (1993) [5]. arneth w. chemistry of curing meat flavour. fleischwirtschaft, 81, 85−87, (2001) [6]. arneth w., münch, s., contents of cholesteroloxides in heated meat produtcs — 2. possibility of technological influence, fleischwirtschaft, 82, 87−91 (2002) [7]. shahidi f., prevention of lipid oxidation in muscle foods by nitrite and nitrite free compositions, acs symposium series, 500, 161−182, (1992) [8]. lücke f. k., use of nitrite and nitrate in the processing of meat from organic production — benefits and risks, fleischwirtschaft, 83, 138−142, (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 maria poroch serițan, nicoleta – gabriela popescu, the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, volume xv, issue 2 – 2016, pag.146 – 154 154 [9]. kabisch j., scheuer r., roedel w., gareis m., influence on the microbial effect of sodium nitrite in raw fermented sausage, mitteilungsblatt der fleischforschung kulmbach, 47, 99−105, (2008) [10]. honikel k. o., the use and control of nitrate and nitrite for the processing of meat products, meat science, 78, 68−76, (2008) [11]. andrée s., jira w., schwind k.-h., wagner h., schwägele f., chemical safety of meat and meat products, review, meat science, 86, 38–48, (2010) [12]. fiddler w., pensabene j. w., piotrowski e. g., phillips j. g., keating j., mergens w. j., newmark h. l., inhibition of formation of volatile nitrosamines in fried bacon by use of cure-solubilized alpha-tocopherol, journal of agricultural and food chemistry, 26, 653−656, (1978) [13]. mirvish s. s., formation of n-nitroso compounds: chemistry, kinetics and in vivo occurrence, toxicology and applied pharmacology, 31, 325−351, (1975) [14]. ward f. w., coates m. e., dietary fats and n-nitrosation in the rat, british journal of nutrition, 58, 221−231, (1987) [15]. tricker a. r., n-nitroso compounds and man: sources of exposure, endogenous formation and occurrence in body fluids, european journal of cancer prevention, 6, 226−268, (1997) [16]. iarc iarc monographs on the evaluation of carcinogenic risks to humans overall evaluations of carcinogenity an updating of iarc monographs, vol. 1-42, supplement 7, (1987) [17]. drabik markiewicz g., van den maagdenberg k., de mey e., deprez s., kowalska t., paelinck h., role of proline and hydroxyproline in n-nitrosamine formation during heating in cured meat. meat science, 81, 479−486, (2009) [18]. bulushi i. a., poole s., deeth h., dykes g., biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation — a review. critical reviews in food science and nutrition, 49, 369−377, (2009) [19]. warthesen j., scanlan r., bills d., libbey l., formation of heterocyclic nnitrosamines from the reaction of nitrite and selected primary diamines and amino acids, journal of agricultural and food chemistry, 23, 898−902, (1975) [20]. fiddler w., pensabene j. w., gates r. a., adam r., nitrosamine formation in processed hams as related to reformulated elastic rubber netting. journal of food science, 63, 276−278, (1998) [21]. helmick j. s., fiddler w., thermal decomposition of the rubber vulcanization agent zinc dibenzyldithiocarbamate, and its potential role in nitrosamine formation in hams processed in elastic nettings, journal of agricultural and food chemistry, 42, 2541−2544, (1994) [22]. ec (2006). directive 2006/52/ec of the european parliament and of the council of 5 july 2006 amending directive 95/2/ec on food additives other than colours and sweeteners and directive 95/35/ec on sweeteners for use in foodstuffs. official journal of the european union, l 204 of 26.7.2006. [23]. regarding the use of nitrites sodium or potassium (e249-e250), regulation (eu) no. 1129/2011 of the commission of 11.11.2011 amending annex ii to regulation (ec) no. 1333/2008 of the european parliament and the council by establishing a union list of food additives make the following clarifications [24]. sahleanu c., îndrumar pentru analiza cărnii şi produselor din carne: lucrări de laborator, editura universităţii din suceava, suceava, (1998) [25]. ocean optics. ocean optics inc., (2007), product catalog, available from: http://oceanoptics.com/knowledge-support/productcatalog/. [26]. x-rite. a guide to understanding color communication, © x-rite, incorporated (2007), accessed may 2014, available from: http://www.x_rite.com. [27]. pathare p.b., opara u.l., al-said f.al-j., colour measurement and analysis in fresh and processed food, food bioprocess technol, 6, 36 – 60, (2013) [28]. banu c., alexe p., vizireanu c., procesarea industrial a cărnii, editura tehnică, bucurești, (1997) [29]. banu c., butu n., lungu c., alexe p., rasmerita d., vizireanu c., aditivi și ingrediente pentru industria alimentară, editura tehnică, bucurești, (2000) [30]. amariei s., ropciuc s., scripca d. m., variation of nitrite content in meat products during the shelf life, food and environment safety, xii:4, 342-346, (2013) [31]. mureșan a., lecture 14. evaluation of color, (in romanian), (2013), available from: http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/ prof.%20dr.%20ing.%20augustin%20muresan/, accessed 01-aug-2013 [32]. zanardi e.; dorigoni v.; badiani a.; chizzolini r., lipid and colour stability of milanotype sausages: effect of packing conditions, meat science, 61: 7-14, (2002) http://oceanoptics.com/knowledge-support/product-catalog/ http://oceanoptics.com/knowledge-support/product-catalog/ http://www.x_rite.com/ http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/prof.%20dr.%20ing.%20augustin%20muresan/ http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/prof.%20dr.%20ing.%20augustin%20muresan/ 1. introduction 4. conclusion food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 5 probiotic properties of lactobacillus casei ssp. rhamnosus lbrc11 *rositsa denkova1, lyubka georgieva, zapryana denkova 2, svetla ilieva 1, velichka yanakieva2 1 sofia university „st. kliment ohridski”, faculty of biology, department of biotechnology, rositsa_denkova@mail.bg 2 university of food technologies, department of organic chemistry and microbiology, zdenkova@abv.bg *corresponding author received 12 april 2012, accepted 25 may 2012 abstract: restoring the balance of the intestinal microflora is achieved by consummation of foods and concentrates, containing beneficial bacteria lactobacilli and bifidobacteria known as functional foods and probiotics. not all lactobacilli can be included in the composition of probiotics and probiotic foods, but only those who possess certain properties. identification of the strain lactobacillus lbrc11 is achieved through the application of physiological and biochemical (api 50 chl) and molecular genetic (ardra) methods. the antimicrobial activity against saprophytic microorganisms (bacillus subtilis, bacillus mesentericus, aspergillus niger, penicillium sp., rhizopus sp., saccharomyces cerevisiae) is determined using the agar diffusion method and the antimicrobial activity against pathogens (e.coli atcc 25922, e.coli 8739, salmonella abony, salmonella sp., staphylococcus aureus atcc 25093) is investigated by carrying out joint cultivation. the profile of antibiotic resistance is determined by the method of diffusion in agar. the enzymatic profile of the strain is studied with the test kit api zym. by applying api 50 chl and ardra the strain lactobacillus lbrc11 is identified as lactobacillus casei ssp.rhamnosus. the strain possesses antimicrobial activity against the saprophytes bacillus mesentericus, aspergillus niger, penicillium sp., rhizopus sp. and against all of the pathogens included in the study. lactobacillus casei ssp.rhamnosus lbrc11 is resistant to 8 of the 22 antibiotics used in the study and is sensitive to 10 of them. the results of the tests on some probiotic properties of lactobacillus casei ssp.rhamnosus lbrc11 make it suitable for incorporation in probiotics and probiotic foods. keywords: probiotics, ardra, cultivation, pathogen, saprophytic microorganism 1. introduction maintaining the balance of the flora in the stomach and the intestines is a necessary condition for good health. restoring the balance of the intestinal microflora is achieved by consummation of foods and concentrates, containing beneficial bacteria lactobacilli and bifidobacteria known as functional foods and probiotics. fao defines probiotics as live microorganisms which when administered in adequate amounts confer a health benefit on the host [1, 2]. the main components of probiotics are lactic acid bacteria (lactobacillus, enterococcus, pediococcus, lactococcus, streptococcus, leuconostoc) and bifidobacteria, which are applied in the manufacture of probiotic foods [3, 4, 5, 6, 7], with lactobacilli being the largest part. not all lactobacilli can be included in the composition of probiotics and probiotic foods, but only those that possess certain mailto:rositsa_denkova:@mail.bg mailto:zdenkova:@abv.bg food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 6 properties [3,4,5]: to be part of the natural microflora in humans and animals; to have the ability to adhere to epithelial cells or cell lines; to survive under the conditions of the stomach and the intestines, i.e. to survive at acidic ph values in the stomach and to withstand the action of bile [8, 9]; to be able to reproduce in the gastrointestinal tract, predominantly utilizing the substrate to suppress and expel from the biological niche the pathogenic and toxigenic microorganisms; to allow industrial cultivation; to have antimicrobial activity against pathogens and carcinogens; to produce antimicrobial substances; to modulate the immune response and to be safe for clinical and food applications. the studies of saxelin et al. [10, 11], donohue & salminen [12], salminen et al. [3, 4, 5] indicate that the safety of lactic acid bacteria and bifidobacteria has been shown and strains belonging to the genera lactobacillus, lactococcus and bifidobacterium are most often with gras status (generally recognized as safe). the purpose of the present article is to investigate the presence of some probiotic properties of the strain lactobacillus lbrc11 antimicrobial activity against pathogenic and saprophytic microorganisms, antibiotic resistance, the presence of surface layer proteins and determination of the enzyme profile. 2. experimental 1. media: 1.1. saline. composition (g/dm3): nacl 5 g; distilled water 1litre. sterilization 20 minutes at 121ºc. 1.2. liquid laptg10 medium. composition (g/dm3): peptone 15, yeast extract 10; tryptone 10, glucose 10. the ph is adjusted to 6.6 6.8 and tween 80 1cm3/l is added. the medium is sterilized for 20 minutes at 121ºc. 1.3. laptg10-agar. composition (g/dm3): medium l-broth + 2% agar. sterilization 20 minutes at 121ºc. 2.physiological and biochemical methods 2.1. determination of the biochemical profile. the determination of the biochemical profile is performed, using the system api 50 chl (biomerieux sa, france) for identification of species belonging to the genus lactobacillus on the basis of their ability to utilize 49 carbon sources. fresh 24-hour culture of the studied strain is centrifuged for 15 minutes at 5,000 x g. the resulting biomass sludge is washed twice with pbsbuffer and resuspended in l resuspension medium, which is an integral part of the used kit. the api strips are placed in incubation boxes and the microtubules are inoculated with the prepared cell suspension and sealed with sterile liquid paraffin. the results are reported on the 24th and on the 48th hour of incubation at optimum temperature for the development of the studied strain. reporting is done according to colour change in comparison to the control (microtubule 0). positive results are reported in the case of a colour change from green to bright yellow. the results are processed with apiwebr identification software. 2.2. determination of the profile of antibiotic sensitivity. the profile of antibiotic resistance is determined by the disk diffusion method of bauer, kirby et al. [13]. fresh 24-hour culture of the tested strain is used to inoculate the plates with laptg10-agar. standard discs impregnated with antibiotics are placed in the plates. the plates are incubated for 48 hours at optimal temperature. the diameters (in mm) of the sterile zones formed around each of the antibiotic discs are recorded. then they are subjected to the following designations: r resistant (zone < 8 mm), sr intermediately sensitive (zone 8-16 mm), s sensitive (zone > 16 mm). 2.3. determination of the antimicrobial activity against saprophytic food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 7 microorganisms. to determine the antimicrobial activity of the tested strain against saprophytic microorganisms liquid culture (lc), acellular supernatant without ph adjustment (asn) and neutralized acellular supernatant (nasn) (ph 6.5), obtained from a 48 hour culture of the tested strain are used. the antimicrobial activity is tested against the following test microorganisms: bacteria bacillus subtilis, bacillus mesentericus; yeasts saccharomyces cerevisiae, molds aspergillus niger, penicillium sp., rhizopus sp. each of the test microorganisms (106-107 cfu/cm3) is inoculated in a petri dish with agar medium and after the hardening of the agar wells (6 mm) are prepared. 0,06 cm3 of kt, asn or nasn are inoculated in the wells of the plates and the plates with the test microorganisms are incubated at 300c or 37ºc for 24 to 48 hours, and then the inhibition zones in mm are reported. 2.3. determination of the antimicrobial activity against pathogenic microorganisms. to determine the antimicrobial activity of the studied strain against pathogens a 48 hour culture of the lactobacillus strain is used. in the mixtures 0,5 ml of the suspension of the lactobacillus strain, 0,5 ml of the suspension of the pathogen and 9 ml of culture medium (laptg10) are mixed, while in the control of the lactobacillus strain and in the control of the pathogen 9,5 ml of the liquid laptg10 medium are mixed with 0,5 ml of the suspension of lactobacillus casei ssp.rhamnosus lbrc11 and of the suspension of the pathogen respectively. the following pathogens are used: e. coli atcc 25922, e.coli 8739, salmonella abony, salmonella sp., staphylococcus aureus atcc 25093. joint cultivation of the strain lactobacillus casei ssp.rhamnosus lbrc11 and pathogenic microorganism under static conditions in a thermostat at 370c for 60 to 72 hours, taking samples at 0, 12, 24, 36, 48, 60 and 72 h and monitoring the change of the titratable acidity and the concentration of viable cells of both the pathogen and the lactobacillus strain is performed. 2.4. determination of the profile of enzyme activity. the determination of the profile of enzyme activity is performed, using the test kit api zym (biomericux, france) for semi-quantitative determination of the enzyme profile of the studied strain. fresh 24-hour culture of the tested strain is centrifuged for 15 minutes at 5000 x g, the obtained biomass precipitate is washed twice and resuspended in api suspension medium. the api zym strips are placed in the incubation boxes and the microtubules are inoculated with the prepared cell suspension. the sample is incubated for 4 to 4,5 hours at 370c. after the incubation one drop of reagent a and one drop of reagent b are pipetted into each microtubule. after 5 min staining is reported according to the color scheme described in the manufacturer's instructions. the enzyme activity is determined according to the color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 3. genetic methods 3.1. isolation of total dna. the isolation of dna is performed by the method of delley et al. [14]. 3.2. pcr reactions and visualization. all pcr reactions are performed using the pcr kit ready to gotm pcr beads (amersham biosciences), in a volume of 25 l in a progene cycler (techne, uk). the resulting products are visualized on a 2% agarose gel stained with ethidium bromide solution (0.5 g/ml), using an uvp documentation system (uk). 3.3. 16s rdna amplification and 16s rdna ardra (amplified ribosomal dna restriction analysis). the method ardra involves enzymatic multiplication of gene encoding the 16s rrna, using food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 8 primers complementary to the conservative regions at both ends of the 16s rrna gene and the product of the multiplication is then restricted with restriction enzymes. the resulting profile is highly specific for the particular studied species. dna of the studied strain is amplified using universal primers for the 16s rdna gene fd1 and rd1 [15]. the amplification program includes: denaturation 95°c for 3 minutes, 40 cycles 93°c for 30 s, 48°c for 60 s, 72°c for 60 s, final elongation 72°c for 5 min. the resulting pcr product from the 16s rdna amplification of the tested strain is treated with the endonucleases ecori, haeiii and hapii (boehringer mannhem gmbh, germany). reactions are carried out according to the following quantities: pcr products 10 l, enzyme solution 10 l (1 l of the respective enzyme, 2 l buffer, 7 l dh2o). incubation for 1 night at 370c is performed. the resulting restriction products are visualized on a 2% agarose gel. 4. characterization of the surface layer proteins by sds-page. untreated bacterial cells or cells treated with 5m licl, are resuspended in 1% sds and are incubated for 30 min at 37ºc to isolate the surface layer proteins. after centrifugation for 5 min at 9000 × g, the supernatant is analyzed using sds-polyacrylamide electrophoresis [16], using 10% polyacrylamide gel. protein bands are visualized by staining with coomassie blue r-250. 3. results and discussion 1. physiological and biochemical characteristics of the strain lactobacillus lbrc11 the biochemical profile of the strain lactobacillus lbrc11 is determined using the system for rapid identification api 50 chl (biomerieux, france). according to its ability to utilize the 49 carbon sources, included in the test kit (table 1) the strain belongs to the species lactobacillus casei ssp.rhamnosus with a rate of reliability 99.9%. the strain lactobacillus lbrc11 is subjected to ardra-analysis to confirm the results of the species identification obtained by the conventional methods for identification. the results of these genetic experiments are shown in fig. 1 and fig. 2. using the restriction enzymes hae iii and hap ii the strain’s belonging to the species lactobacillus casei ssp.rhamnosus is confirmed. table 1: api 50 chl of the strain lactobacillus lbrc11 # carbohydrate lbrc11 1 glycerol +/(25-30%) 2 erythriol 3 d-arabinose 4 l-arabinose 5 ribose + (90-100%) 6 d-xylose 7 l-xylose 8 adonitol 9 -metil-dxyloside 10 galactose + (90-100%) 11 d-glucose + (90-100%) 12 d-fructose + (90-100%) 13 d-mannose + (90-100%) 14 l-sorbose + (90-100%) 15 rhamnose + (90-100%) 16 dulcitol 17 inositol + (50%) 18 manitol + (90-100%) 19 sorbitol + (90-100%) 20 -methyl-dmannoside 21 -methyl-dglucoside + (90-100%) 22 n-acetylglucosamine + (90-100%) 23 amigdalin + (90-100%) 24 arbutin + (90-100%) 25 esculin + (90-100%) # carbohydrate lbrc11 25 esculin + (90-100%) 26 salicin + (90-100%) 27 cellobiose + (90-100%) 28 maltose + (90-100%) 29 lactose + (90-100%) 30 melibiose 31 saccharose + (50%) 32 trehalose + (90-100%) 33 inulin 34 melezitose + (90-100%) 35 d-raffinose 36 amidon 37 glycogen 38 xylitol 39 -gentiobiose + (90-100%) 40 d-turanose + (90-100%) 41 d-lyxose 42 d-tagarose + (90-100%) 43 d-fuccose 44 l-fuccose 45 d-arabitol 46 l-arabitol 47 gluconate + (70-75 %) 48 2-ketogluconate 49 5-keto-gluconate food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 9 figure 1: restriction profile with hae iii figure 2: restriction profile with hap ii 1. lactobacillus casei ssp. rhamnosus lmg 6400 2. lactobacillus lbrc11 3. 100 bp antimicrobial activity of lactobacillus casei ssp. rhamnosus lbrc11 against saprophytic microorganisms the antimicrobial activity of lactobacillus casei ssp. rhamnosus lbrc11 against saprophytic microorganisms is studied by the method of diffusion in agar. for this purpose, cultural liquid is used to determine the inhibitory effect of the cells of the strain on the test microorganisms; acellular supernatant obtained from the cultural liquid after centrifugation without ph adjustment (with acidic ph) is used to determine the influence of the acidic ph on the saprophytes. in a parallel experiment the activity of the neutralized acellular supernatant after neutralization to ph 6.5 to eliminate the inhibitory effect of the lactic acid produced by the lactobacillus strain. test microorganisms belonging to different groups of microorganisms are used: bacteria bacillus subtilis, bacillus mesentericus; yeasts saccharomyces cerevisiae, molds aspergillus niger, penicillium sp., rhizopus sp. incubation is conducted for 24 to 48 h at 30°c and/or 37°c. the results of the repeated twice experiments are summarized in table. 2. table 2: antimicrobial activity of the strain lactobacillus casei ssp. rhamnosus lbrc11 against saprophytic microorganisms. the values are in mm. diameter of the well 6 mm. cl cultural liquid; asn acellular supernatant without ph adjustment and nasn – neutralized acellular supernatant (ph = 6.5). saprophytic microorganism lbrc11 cl asn nasn aspergillus niger 1,2x107cfu/cm3 300 10 9 9 370 16 10 rhizopus sp. 1,8x107cfu/cm3 30 0 14.5 11 8 penicillium sp. 5,2x107cfu/cm3 30 0 10 8 8 bacillus subtilis 1,9x107cfu/cm3 300 370 bacillus mesentericus 4x106cfu/cm3 300 10 10 9 370 12 10 10 saccharomyces cerevisiae 9,2x106cfu/cm3 300 370 15 lactobacillus casei ssp.rhamnosus lbrc11 possesses antimicrobial activity against rhizopus sp. and penicillium sp., and the cultural liquid has greater antimicrobial activity than the acellular supernatant. the strain exhibits antimicrobial activity against aspergillus niger and bacillus mesentericus, both at 300c and 370c; the antimicrobial activity of the cultural liquid is higher than that of the supernatant. the cultural liquid of the strain exhibits antimicrobial activity against saccharomyces cerevisiae only at 370c, which means that the suppression of the test organism is due to competition for nutrients and/or places of attachment. the strain does not possess antimicrobial 1 2 3 1000 bp 500 bp 1 2 3 -1000 bp -500 bp food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 10 activity against bacillus subtilis at 300c or at 370c. determination of the presence of surface layer proteins the strain lactobacillus casei ssp.rhamnosus lbrc11 does not possess surface layer proteins. antibiotic resistance knowledge on the antibiotic resistance of the lactobacilli with probiotic potential is essential. it can be seen as an important criterion for the selection of probiotic cultures due to the possibility of conducting a combined therapy with antibiotics and probiotics in order to restore the normal microflora of the gastrointestinal tract and/or the urogenital tract [17]. for this purpose, 22 antibiotics with different mechanisms of action most frequently used in medical practice are selected and the sensitivity of lactobacillus casei ssp.rhamnosus lbrc11 is determined. the results of the diffusion method of bauer, kirby et al. [13] for 24-48 hours are summarized in table 3. lactobacillus casei ssp.rhamnosus lbrc11 is resistant to 8 out of the 22 antibiotics used in the study penicillin, ampicillin, oxacillin, amoxicillin, vancomycin, cefemandole, chloramphenicol and nalidixic acid. intermediate sensitivity is observed to four antibiotics gentamicin, streptomycin, kanamycin and tobramycin. the strain shows sensitivity to 10 out of 22 antibiotics azlocillin, piperacillin, tetracycline, doxycycline, clindamycin, amikacin, rifampin, lincomycin, erythromycin and ciprofloxacin. it is noteworthy that lactobacillus casei ssp.rhamnosus lbrc11 is resistant to 6 out of 8 antibiotics inhibiting the synthesis of the cell wall, but it is sensitive to 7 out of 12 antibiotics that inhibit protein synthesis, and to other 4 of this group it shows intermediate sensitivity. table 3: antibiotic sensitivity of the strain lactobacillus casei ssp.rhamnosus lbrc11. legend: rresistant, sr – intermediately sensitive (zone 716 mm), s sensitive (zone > 16 mm) mechanism of action antibiotic concentration lbr c11 in hi bi tio n of th e sy nt he si s of th e ce ll w al ls penicillin p 10 e/disc r azlocillin az 75 µg/disc s piperacillin p 100 µg/disc s ampicillin a 10 µg/disc r oxacillin o 1 µg/disc r amoxicillin ax 25 µg/disc r vancomycin v 30 µg/disc r cefamandole cm 30 µg/disc r in hi bi tio n of th e pr ot ei n sy nt he si s tetracycline t 30 µg/disc s doxycycline d 30 µg/disc s gentamicin g 10 µg/disc sr streptomycin s 30 mg/disc sr clindamycin cl 2 µg/disc s kanamycin k 30 µg/disc sr tobramycin tb 10 µg/disc sr amikacin am 30 µg/disc s rifampin r 5 µg/disc s lincomycin l 15 µg/disc s chloramphenicol c 30 µg/disc r erythromycin e 15 µg/disc s in hi bi tio n of th e sy nt he si s of d n a an d/ or t he c el l di vi si on nalidixic acid nx 30 µg/disc r ciprofloxacin cp 5 µg/disc s the enzyme profile of lactobacillus casei ssp.rhamnosus lbrc11 is studied by determining the presence of a set of 19 enzymes involved in the system for semiquantitative determination of key enzyme activities api zym (biomerieux, france). the results of these studies are shown in table 4. lactobacillus casei ssp.rhamnosus lbrc11 possesses the following enzymes: alkaline phosphatase, esterase, esteraselipase, lipase, leucine-aminopeptidase, valine-aminopeptidase, cysteineaminopeptidase, chymotrypsin, acid phosphatase, phosphohydrolase, alphagalactosidase, beta-galactosidase, betaglucuronidase, alpha-glucosidase, betaglucosidase, alpha-glucoseaminidase and alpha-fucosidase (table 4). food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 11 table 4: enzyme profile of lactobacillus casei ssp.rhamnosus lbrc11 enzyme activity* lbrc11 1 control 2 alkaline phosphatase 3 3 esterase 3 4 esterase-lipase 2 5 lipase 0.5 6 leucine-aminopeptidase 5 7 valine-aminopeptidase 5 8 cysteine-aminopeptidase 3 9 trypsin 10 chymotrypsin 2 11 acid phosphatase 4 12 phosphohydrolase 2 13 -galactosidase 3 14 -galactosidase 5 15 -glucuronidase 1 16 -glucosidase 3 17 -glucosidase 5 18 -glucoseaminidase 3 19 -manosidase 20 -fucosidase 4 *the enzyme activity is determined according to the colour scale from 0 (lack of enzyme activity) to 5 (maximum enzyme activity) the antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against pathogenic microorganisms is examined. in co-cultivation of the lactobacillus strain with e.coli atcc 25922 in an incubator under static conditions an increase of the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 and of the pathogen e.coli atcc 25922 in the first 24 hours is observed; the rates of the increase are commensurable. then the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 remains unchanged while the one of the pathogen reduces rapidly, reaching 0 on the 60th hour (fig. 3). in determining the inhibitory action of lactobacillus casei ssp.rhamnosus lbrc11 against e.coli 8739 an increase in the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 and of the pathogen e.coli atcc 25922 in the first 24 hours is established, but the rate of the increase in the lactobacillus strain is higher than that of the pathogen, in contrast to the results obtained with e.coli atcc 25922. then the concentration of viable cells of the lactobacillus strain remains the same while the concentration of viable cells of the pathogen decreases rapidly, reaching 0 on the 60th hour, as observed in e.coli atcc 25922 (fig. 4). figure 3: antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against e.coli atcc 25922 figure 4: antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against e.coli 8739 in tracing the changes in the titratable acidity it is noticeable that both the two controls of the pathogens are with significantly lower titratable acidity in comparison to the control of lactobacillus casei ssp. rhamnosus lbrc11 and to the titrable acidity of the mixtures (lactobacillus casei ssp.rhamnosus lbrc11 and e.coli atcc 25922; lactobacillus casei ssp. rhamnosus lbrc11 and e.coli 8739), and the values of the titratable acidity of the mixtures are food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 12 lower for each hour than the values of the control of the lactobacillus strain. it is noted that during the first 24 hours the titratable acidity of the mixture lactobacillus casei ssp.rhamnosus lbrc11 and e.coli 8739 has higher values than the mixture lactobacillus casei ssp.rhamnosus lbrc11 and e.coli atcc 25922, while on the 48th hour the acidity of the mixture lactobacillus casei ssp.rhamnosus lbrc11 and e.coli 8739 is lower than that of the mixture lactobacillus casei ssp.rhamnosus lbrc11 and e.coli atcc 25922, and this trend is maintained until the 72th hour (fig. 5). figure 5: change in titrable acidity in testing the antimicrobial activity of the cells of lactobacillus casei ssp.rhamnosus lbrc11 against the cells of e.coli 8739 and e.coli 25922 in studying the inhibitory activity of lactobacillus casei ssp.rhamnosus lbrc11 against salmonella abony an increase in the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 and of the pathogen salmonella abony during the first 12 hours is observed, and the rates of the increase are commensurable. then the concentration of viable cells of the lactobacillus strain remains the same while the one of the pathogen decreases more slowly up to 24th hour, after which the rate of reduction in the number of viable cells increases significantly and the concentration of viable cells reaches 0 by the 60th hour (fig. 6). in co-cultivation of lactobacillus casei ssp.rhamnosus lbrc11 and salmonella sp. an increase in the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 and of the pathogen salmonella sp. in the first 12 hours is established, but the rate of the increase of the lactobacillus strain is lower than that of the pathogen. then the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 continues to increase while that of the pathogen decreases, reaching 104 cfu/cm3 on the 60th hour (fig. 7). figure 6: antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against salmonella abony figure 7: antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against salmonella sp. the change in titratable acidity indicates that the values for the titrable acidity of the controls of both the two pathogens are significantly lower than the values of the control of the lactobacillus strain and food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 13 those of the mixtures (lactobacillus casei ssp.rhamnosus lbrc11 and salmonella abony; lactobacillus casei ssp.rhamnosus lbrc11 and salmonella sp.). it is noted that during the first 24 hours the change in the titratable acidity of both the two mixtures is commensurable, while on the 48th hour the acidity of the mixture lactobacillus casei ssp.rhamnosus lbrc11 and salmonella abony is lower than that of the mixture lactobacillus casei ssp.rhamnosus lbrc11 and salmonella sp., and this trend is maintained up until the 60th hour (fig. 8). figure 8: change in the titrable acidity in testing the antimicrobial activity of the cells of lactobacillus casei ssp.rhamnosus lbrc11 against the cells of salmonella abony and salmonella sp. in studying the antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against staphylococcus aureus atcc 25093 an increase in the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 and of the pathogen during the first 24 hours is observed, the growth rate of the lactobacillus strain is higher than that of staphylococcus aureus atcc 25093. then the concentration of viable cells of lactobacillus casei ssp.rhamnosus lbrc11 continues to grow at a slower rate, while that of the pathogen decreases slowly, reaching 105 cfu/cm3 on the 72nd hour (fig. 9). figure 9: antimicrobial activity of lactobacillus casei ssp.rhamnosus lbrc11 against staphylococcus aureus atcc 25093 the data, reflecting the changes in the titratable acidity shows that the titratable acidity of the control of the pathogen is significantly lower than the titrable acidity of both the control of the lactobacillus strain and of the mixture (lactobacillus casei ssp.rhamnosus lbrc11 and staphylococcus aureus atcc 25093) (fig. 10). figure 10: change in titrable acidity in testing the antimicrobial activity of the cells of lactobacillus casei ssp.rhamnosus lbrc11 against the cells of staphylococcus aureus atcc 25093 the strain lactobacillus casei ssp.rhamnosus lbrc11 exhibits antimicrobial activity against all of the pathogens included in the study. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 14 4. conclusion the strain lactobacillus lbrc11, identified by contemporary physiological and biochemical and molecular genetic methods as a representative of the species lactobacillus casei ssp.rhamnosus, has good antimicrobial activity against saprophytic and pathogenic microorganisms. the results of the testings on the possession of some probiotic properties make the strain lactobacillus casei ssp.rhamnosus lbrc11 suitable for inclusion in probiotic preparations for prevention and treatment of various conditions. 5. references [1] food and agriculture organization of the united nations (fao). health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, (2001). [2] food and agriculture organization of the united nations (fao). guidelines for the evaluation of probiotics in food, (2002). [3] salminen s., ouwehand a. c., isolauri e., clinical applications of probiotic bacteria. int. dairy j., 8, 563-572, (1998). [4] salminen s., von wright.a, current probiotics – safety assured, scandinavian university press, issn 0891-060x, (1998). [5] salminen, s.; bouley, m.c.; boutronrualt, m.c.; cummings, j.; franck, a.; gibson, g.; isolauri, e.; moreau, m.-c.; roberfroid, m.; rowland, i. functional food science and gastrointestinal physiology and function. br. j. nutr., suppl., 1, 147–171, (1998). [6] wolfson, n. p., a probiotics primer. nutrition science news, 4(6), 276-280, (1999). [7] gibson g.r., (2004). from probiotics to prebiotics and a healthy digestive system. j.of food science, 69, (5), m 141 m 143, (2004). [8] kashtan h.et al (1990), manipulation of faecal ph by dietary means. prev. med., 19 (6), 607 – 613, (1990). [9] segal i. et al., faecal short chain fatty acids in south african urban africans and whites. dis. colon rectum, 38 (7), 732 – 734, (1995). [10] saxelin m., h. rautelin, b. chassy, s. l. gorbach, s. salminen and h.makela, lactobacilli and septic infections in southern finland. clinical infectious diseases 22, 564 – 566, (1996a). [11] saxelin m., s. salminen, the safety of commercial products with viable lactobacillus strains. infectious diseases clinical practice 5, 331 – 335, (1996b). [12] donohue d. c., s. salminen, safety assessment of probiotic bacteria. asia pac. j. clin. nutr. 5, 25 – 28, (1996). [13] bauer a.w., kirby w.m., sherris j.c., turck m, antibiotic susceptibility testing by a standardized single disk method. american journal of clinical pathology 36, 49-52, (1966). [14] delley, m., b. mollet, and h. hottinger, dna probe for lactobacillus delbrueckii. appl. environ. microbiol. 56:1967– 1970, (1990). [15] weisburg w.g., barns s.m., pelletier d.a., lane d.j., 16s ribosomal dna amplification for phylogenetic study. j bacteriol;173:697-703, (1991). [16] laemmli, u.k., clevage of structural proteins during the assembly of the head of bacteriophage t4. nature (london) 227, 680–685, (1970). [17] charteris w.p., p.m. kelly, l. morelli and j.k. collins, selective detection, enumeration and identification of potential probiotic lactobacillus and bifidobacterium species in mixed bacterial populations, international journal of food microbiology 35, pp. 1–27, (1997). s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r am e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 176 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 169 175 s ta t is ti cal s tudy of th e de pende nc e b etw ee n conce nt rat ion of me ta ll ic e l em ents m igra ted fro m s ta inl ess s te e l gr ad e ais i321 and worki ng param ete rs *silviu-gabriel stroe1, gheorghe gutt1 1faculty of food engineering, stefan cel mare university of suceava, universitatii str. 13, suceava, romania, silvius@fia.us.ro; g.gutt@fia.usv.ro * corresponding author received april 27th 2013, accepted may 25th 2013 abstract: one of the main problems concerning food safety is the possible migration of ions in metallic materials intended to come into contact with food. the aim of this paper is to find and apply the mathematical modeling of experimental data which should describe as accurately as possible the dependence between the variables used in the experimental plan. the research made and presented in this study also aims to create a real possibility for rapid intervention in the process control when one of the parameters cannot be maintained at a predetermined value. in this paper we used some experimental data obtained by testing the migration of metal ions from austenitic stainless steel grade aisi 321 samples in solutions with concentrations of 3%, 6% and 9% acetic acid. to find an accurate mathematical model describing the phenomena of diffusion, anova method, known as the variance analysis, was used. in order to obtain the mathematical model we used a polynomial model with independent variables: corrosive environment temperature (x1), exposure time (x2), stirring environment (x3) and the dependent variables y concentrations of elements ti, cr, mn, 56fe and ni found in solutions. values of the regression coefficients very close to the value 1 (the dependent variables coefficients are valid) were obtained, which demonstrates the validity of the applied method. keywords: austenitic stainless steel, mathematical modeling, anova method, dependent and independent variables, coded values, validity of the model. 1. introduction stainless steels are commonly used as metallic materials intended to come into contact with food environments. this widespread use of stainless steels led to increase the importance of studying the diffusion processes of metal ions in food environments, research on the interaction between corrosive environments and stainless steels being the subject of scientific research for a long time [1], [2], [3], [4]. in this context, one of the most used grade of stainless steel is 300 series such as aisi 321, both containing 11wt% ni [5]. diffusion of metal ions in foodstuffs is a slow process that occurs in different types of environments, often in acidic environments [6], [7], [8], [9], [10]. in the last decade, based on several scientific researches, the modeling of migration metallic constituents of materials in contact with foodstuffs has played an important part in the quality assurance system in the food industry. by classical food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o rg he g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nv ir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 177 methods or by using specialized software, mathematical modeling has become a relatively inexpensive tool for scientists aiming to minimize the number of experiments and to determine the influence of various parameters influencing the safety and quality processes [11-13]. since the beginning we must analyze if the problem is correctly defined and consistent, if its solution to provide useful information for the study and influence factors correspond to the characteristics that must be satisfied. to find an accurate mathematical model describing the phenomena of diffusion, the anova method, known as the analysis of variance, was used. anova is widely used because it is recommended when studying a larger number of levels of the independent variables, making possible to observe with greater accuracy the effect of the independent variables on the dependent ones and their combined effect. the aim of this work was to apply the anova method for modeling the experimental data obtained from the migration test, which describes exactly the dependence between the variables used in the experimental plan. 2. experimental 2.1. materials and samples preparation in this study metal samples of aisi 321 stainless steel grade were used. the chemical composition of the steel is given in table 1 (en 10088-2:2005). table 1 chemical composition of austenitic stainless stell aisi 321 (wt %) fe c mn p s si cr ni ti 68 0.08 2 0.045 0.03 1 18 11 0.4 metal samples were cut from unused sheet, free from deformation or scratches. sample sizes were established according to the d.m. 03/21/1973, which stipulates that the ratio of the exposed surface of the sample and the volume of solution should be between 0.5 ... 2. sample sizes were 40 x 40 x 2.5 mm. acetic acid is recommended to test metal alloys in contact with foodstuffs. acetic acid solutions 3%, 6% and 9%, were used as corrosive environments. to avoid contamination of corrosive solutions with foreign compounds, the surface of metal samples was washed with a detergent solution at 40°c and rinsed in double distilled water. ultrasonic cleaning was performed at 45°c for 15 minutes in the ultrasonic. the samples were dried in oven at 50°c. profile roughness evaluation, complying with en iso 4287, was determined with an optical profilometer (nano focus µscan) for contactless 2d and 3d measurement of microscopic surface structures. the surface roughness was ra 0.7816±0.019µm. 2.2. migration test in the experimental studies the following working parameters were used:  testing temperature t [ °c]  stirring environment n [rot/min]  exposure time t [min] variation levels of working parameters are given in table 2. table 2 variation levels of working parameters working parameters minimum value central value maximum value testing temperature 22 28 34 stirring environment 0 125 250 exposure time 30 60 90 in the case of the experiments made in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o rg he g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nv ir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 178 stationary environment and at different temperatures of the environment, the samples were maintained in the oven while for those requiring temperatures of 28°c and 34°c and stirring environment, a magnetic heating stirrer heidolph mr heitec was used. 2.3. chemical analysis of corrosive environments the concentrations of metal ions ti, cr, mn, 56fe and ni from acid solutions were analyzed using mass spectrometry and inductively coupled plasma icp-ms agilent 7500 model. 3. results and discussion the concentrations of metallic elements ti, cr, mn, 56fe and ni obtained by mass spectrometry and inductively coupled plasma were used in mathematical modeling of diffusion phenomena. since the literature has not provided a sufficiently accurate mathematical apparatus to describe the interdependencies mentioned we chose to develop a mathematical model with statistical means by anova method. to achieve this mathematical model design expert® software and a polynomial model with multiple variables were used:  independent variables: testing temperature (x1), exposure time (x2), stirring environment (x3);  dependent variables (response functions) y1, y2, y3, y4 and y5: concentrations expressed in ppb of elements ti, cr, mn, 56fe and ni. 3.1. mathematical modeling of concentration of metallic elements migrated in 3% acetic acid solution. after having made the analysis of variance anova, a polynomial model with the lowest value of the critical probability p for all dependent variables: ti, cr, mn, 56fe and ni, was chosen. it is known that the null hypothesis of the dependent variables is rejected if the value of the variables of p is less than the chosen significance threshold (α=0.05). the statistical summary of mathematical models found to describe dependent variables ti, cr, mn, 56fe and ni is given in table 3. the mathematical models are presented in equations (1), (2), (3), (4) and (5) and comparative graphical representation of measured values and the values obtained by modeling anova are shown in figures 1-5. table 3. model summary statistics dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.033 cubic 0.24 0.9905 0.9440 y2 0.016 quartic 1.62 0.9925 0.9512 y3 0.015 quadratic 1.20 0.9878 0.8872 y4 0.046 cubic 91.33 0.9898 0.8876 y5 0.025 quartic 4.13 0.9903 0.9812 the mathematical model of the response function y1 (ti concentration) is given in the equation (1): y = 1.71 + 0.97 ∙ + 0.94 ∙ x − 0.50 ∙ x − 0.22 ∙ − 0.094 ∙ x − 0.19 ∙ x − 0.17 ∙ ∙ x − 0.19 ∙ ∙ x − 0.40 ∙ x ∙ x − 0.33 ∙ ∙ x − 0.17 ∙ x ∙ x − 0.23 ∙ ∙ x − 0.36 ∙ x ∙ x − 0.05 ∙ x ∙ x − 0.13 ∙ x ∙ x + 0.05 ∙ ∙ x ∙ x (1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 179 to check the validity of the mathematical model obtained, the experimental values vs model values were plotted (figure 1). figure 1. validity check of the mathematical model obtained the mathematical model of the response function y2 (cr concentration) is given in equation (2): y = 7.26 + 3.72 ∙ x + 1.44 ∙ x + 2.67 ∙ x + 3.61 ∙ x + 8.11 ∙ x + 2.11 ∙ x + 0.25 ∙ x ∙ x + 2.50 ∙ x ∙ x − 2.00 ∙ x ∙ x + 1.58 ∙ x ∙ x + 0.50 ∙ x ∙ x − 0.58 ∙ x ∙ x + 1.17 ∙ x ∙ x + 2.50 ∙ x ∙ x + 1.83 ∙ x ∙ x + 0.75 ∙ x ∙ x ∙ x − 6.92 ∙ x ∙ x + 6.50 ∙ x ∙ x ∙ x − 0.17 ∙ x ∙ x − 1.00 ∙ x ∙ x ∙ x + 1.00 ∙ x ∙ x ∙ x − 0.67 ∙ x ∙ x (2) the validity of the mathematical model obtained for cr has been plotted by experimental values vs model values (figure 2). figure 2. validity check of the mathematical model obtained for chromium the mathematical model of the response function y3 (mn concentration) is given in equation (3): y = 8.57 + 1.90 ∙ x + 2.40 ∙ x − 0.92 ∙ x − 0.20 ∙ x + 0.33 ∙ x − 1.78 ∙ x + 1.68 ∙ x ∙ x − 0.60 ∙ x ∙ x − 0.24 ∙ x ∙ x (3) the validity of the mathematical model obtained for mn has been plotted by experimental values vs model values (figure 3). figure 3. validity check of the mathematical model obtained for manganese the mathematical model of the response function y4 (56fe concentration) is given in equation (4): y = 861.85 + 293.33 ∙ x + 145.56 ∙ x − 50,00 ∙ x − 108.89 ∙ x − 3.89 ∙ x − 310.56 ∙ x + 67.50 ∙ x ∙ x − 9.17 ∙ x ∙ x + 28.33 ∙ x ∙ x + 54.17 ∙ x ∙ x + 107.50 ∙ x ∙ x − 27.50 ∙ x ∙ x − 157.50 ∙ x ∙ x + 5.00 ∙ x ∙ x − 43.33 ∙ x ∙ x + 37.50 ∙ x ∙ x ∙ x (4) the validity of the mathematical model obtained for 56fe has been plotted by experimental values vs model values (figure 4). r² = 0.9785 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0.00 1.00 2.00 3.00 4.00 m od el v al ue , t i[ pp b] experimental value, ti [ppb] r² = 0.9925 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 0.00 10.00 20.00 30.00 40.00 m od el v al ue , c r [p pb ] experimental value, cr [ppb] r² = 0.908 0.00 5.00 10.00 15.00 20.00 0.00 5.00 10.00 15.00 20.00m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 180 figure 4. validity check of the mathematical model obtained for 56fe the mathematical model of the response function y5 (ni concentration) is given in equation (5): y = 35.56 + 9.00 ∙ x + 6.33 ∙ x + 3.44 ∙ x − 3.89 ∙ x + 0,11 ∙ x − 7.89 ∙ x + 7.50 ∙ x ∙ x − 2.50 ∙ x ∙ x + 0.002 ∙ x ∙ x + 4.00 ∙ x ∙ x − 1.17 ∙ x ∙ x + 0.003 ∙ x ∙ x − 3.00 ∙ x ∙ x − 1.17 ∙ x ∙ x + 2.00 ∙ x ∙ x + 1.25 ∙ x ∙ x ∙ x + 0.83 ∙ x ∙ x + 0.50 ∙ x ∙ x ∙ x + 7.83 ∙ x ∙ x + 2.00 ∙ x ∙ x ∙ x − 6.50 ∙ x ∙ x ∙ x + 0.83 ∙ x ∙ x (5) the validity of the mathematical model obtained for ni has been plotted by experimental values vs model values (figure 5). figure 5. validity check of the mathematical model obtained for nickel 3.2. mathematical modeling of concentration of metallic elements migrated in 6% acetic acid solution. statistical summary of mathematical models found to describe dependent variables ti, cr, mn, 56fe and ni in 6% acetic acid solutions is given in table 4. the mathematical models are presented in equations (6), (7), (8), (9) and (10) and comparative graphical representation of measured values and the values obtained by modeling anova are shown in figures 6-10. table 4. model summary statistics dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.0001 cubic 0.23 0,9931 0,9820 y2 0.0061 cubic 1.29 0,9803 0,9488 y3 0.0670 quartic 0.61 0,9949 0,9669 y4 0.0145 quartic 13.79 0,9988 0,9921 y5 0.0516 quartic 28.65 0,9952 0,9688 the mathematical model of the response function y1 (ti concentration) is given in equation (6): y = 1.44 + 1.03 ∙ x + 0.58 ∙ x + 0.31 ∙ x + 1.25 ∙ x + 0.017 ∙ x − 0.05 ∙ x + 0,42 ∙ x ∙ x + 0.75 ∙ x ∙ x + 0.45 ∙ x ∙ x + 0.85 ∙ x ∙ x + 0.47 ∙ x ∙ x − 0.06 ∙ x ∙ x − 0,12 ∙ x ∙ x − 0,03 ∙ x ∙ x + 0.81 ∙ x ∙ x ∙ x (6) the validity of the mathematical model obtained for ti has been plotted by experimental values vs model values (figure 6). r² = 0.9568 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 1400.00 1600.00 0.00 500.00 1000.00 1500.00 m od el v al ue ,5 6 f e [p pb ] experimental value, 56fe [ppb] r² = 0.9803 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 10.00 20.00 30.00 40.00 50.00 60.00 m o de l v al ue , n i [ pp b] experimental value, ni [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 181 figure 6. validity check of the mathematical model obtained for titanium the mathematical model of the response function y2 (cr concentration) is given in equation (7): y = 10.33 + 3.83 ∙ x + 8.06 ∙ x + 2.83 ∙ x − 0.17 ∙ x + 0.17 ∙ x − 0.83 ∙ x − 0,75 ∙ x ∙ x + 0.083 ∙ x ∙ x + 1.08 ∙ x ∙ x − 3.08 ∙ x ∙ x − 1.75 ∙ x ∙ x − 0.75 ∙ x ∙ x − 0,25 ∙ x ∙ x + 1.25 ∙ x ∙ x − 1.58 ∙ x ∙ x − 0.25 ∙ x ∙ x ∙ x (7) the validity of the mathematical model obtained for chromium has been plotted by experimental values vs model values (figure 7). figure 7. validity check of the mathematical model obtained for chromium the mathematical model of the response function y3 (mn concentration) is given in equation (8): y = 6.46 + 1.36 ∙ x + 1.57 ∙ x − 0.17 ∙ x − 0.39 ∙ x − 0.99 ∙ x − 0.29 ∙ x + 1.37 ∙ x ∙ x − 1.07 ∙ x ∙ x − 0.075 ∙ x ∙ x + 0.68 ∙ x ∙ x + 1.37 ∙ x ∙ x + 0.84 ∙ x ∙ x − 0.058 ∙ x ∙ x + 0.23 ∙ x ∙ x + 0.77 ∙ x ∙ x + 1.13 ∙ x ∙ x ∙ x + 1.06 ∙ x ∙ x + 1.72 ∙ x ∙ x ∙ x + 0.21 ∙ x ∙ x + 0.40 ∙ x ∙ x ∙ x + 0.075 ∙ x ∙ x ∙ x + 1.16 ∙ x ∙ x (8) the validity of the mathematical model obtained for manganese has been plotted by experimental values vs model values (figure 8). figure 8. validity checkof the mathematical model obtained for manganese the mathematical model of the response function y4 ( 56fe concentration) is given in equation (9): y = 298.89 + 120.56 ∙ x + 108.89 ∙ x + 93.33 ∙ x + 11.67 ∙ x − 3.33 ∙ x + 66.67 ∙ x + 47.50 ∙ x ∙ x + 2.50 ∙ x ∙ x + 5.00 ∙ x ∙ x + 19.17 ∙ x ∙ x − 32.50 ∙ x ∙ x + 14.17 ∙ x ∙ x − 30.83 ∙ x ∙ x + 5.00 ∙ x ∙ x − 8.33 ∙ x ∙ x + 37.50 ∙ x ∙ x ∙ x + 42.50 ∙ x ∙ x + 27.50 ∙ x ∙ x ∙ x − 67.50 ∙ x ∙ x + 27.50 ∙ x ∙ x ∙ x − 25.00 ∙ x ∙ x ∙ x + 0.001 ∙ x ∙ x (9) the validity of the mathematical model obtained for 56fe has been plotted in (figure 9). r² = 0.9931 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 0.00 2.00 4.00 6.00 8.00 10.00 m od el v al ue , t i[ pp b] experimental value, ti [ppb] r² = 0.9803 0.00 5.00 10.00 15.00 20.00 25.00 0.00 5.00 10.00 15.00 20.00 25.00 m od el v al ue , c r [p pb ] experimental value, cr [ppb] r² = 0.9949 0.00 5.00 10.00 15.00 20.00 25.00 0.00 5.00 10.00 15.00 20.00 25.00 m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 182 figure 9. validity check of the mathematical model obtained for 56fe the mathematical model of the response function y5 (ni concentration) is given in equation (10): y = 603.04 + 151.11 ∙ x + 83.33 ∙ x + 117.22 ∙ x − 95.56 ∙ x + 54.44 ∙ x − 100.56 ∙ x + 22.50 ∙ x ∙ x + 30.00 ∙ x ∙ x + 7.50 ∙ x ∙ x − 30.00 ∙ x ∙ x − 85.83 ∙ x ∙ x − 1.67 ∙ x ∙ x + 0.83 ∙ x ∙ x + 16.67 ∙ x ∙ x + 10.00 ∙ x ∙ x − 18.75 ∙ x ∙ x ∙ x − 46.67 ∙ x ∙ x − 21.25 ∙ x ∙ x ∙ x + 135.83 ∙ x ∙ x − 6.25 ∙ x ∙ x ∙ x − 1.25 ∙ x ∙ x ∙ x − 26.67 ∙ x ∙ x (10) the validity of the mathematical model obtained for nickel has been plotted in (figure 10). figure 10. validity check of the mathematical model obtained for nickel 3.3. mathematical modeling of concentration of metallic elements migrated in 9% acetic acid solution. statistical summary of mathematical models found to describe dependent variables ti, cr, mn, 56fe and ni in 9% acetic acid solutions is given in table 5, the mathematical models are presented in equations (11), (12), (13), (14) and (15), and comparative graphical representation of measured values and the values obtained by modeling anova are shown in figures 11-15. table 5. model summary statistics dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.042 quartic 0.032 0.9920 0.9948 y2 0.017 cubic 3.78 0.9850 0.9746 y3 0.009 quartic 0.11 0.9996 0.9975 y4 0.052 cubic 23.76 0.9921 0.9794 y5 0.048 quadratic 4.56 0.9885 0.9365 the mathematical model of the response function y1 (ti concentration) obtained using anova methodis is given in equation (11): y = 1.39 + 0.36 ∙ x + 0.43 ∙ x + 0.24 ∙ x − 0.072 ∙ x + 0.028 ∙ x + 0.028 ∙ x + 0,10 ∙ x ∙ x − 0.075 ∙ x ∙ x + 0.075 ∙ x ∙ x − 0.12 ∙ x ∙ x − 0.058 ∙ x ∙ x − 0.017 ∙ x ∙ x − 0,042 ∙ x ∙ x + 0.042 ∙ x ∙ x + 0.001 ∙ x ∙ x ∙ x − 0.017 ∙ x ∙ x − 0.025 ∙ x ∙ x ∙ x + 0.008 ∙ x ∙ x + 0.10 ∙ x ∙ x ∙ x + 0.025 ∙ x ∙ x ∙ x − 0.092 ∙ x ∙ x (11) r² = 0.9988 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 0.00 200.00 400.00 600.00 800.00 m od el v al ue , 5 6 f e [p pb ] experimental value, 56fe [ppb] r² = 0.9952 250.00 350.00 450.00 550.00 650.00 750.00 850.00 200.00 400.00 600.00 800.00 m od el v al ue , n i[ pp b] experimental value, ni [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 183 the validity of the mathematical model obtained for titanium has been plotted in (figure 11). figure 11. validity check of the mathematical model obtained for titanium the mathematical model of the response function y2 (cr concentration) obtained using anova methodis is given in equation (12): y = 13.93 + 2.39 ∙ x + 13.17 ∙ x + 0.50 ∙ x − 4.44 ∙ x + 4.06 ∙ x − 0.11 ∙ x + 4.75 ∙ x ∙ x + 4.67 ∙ x ∙ x + 3.42 ∙ x ∙ x − 4.75 ∙ x ∙ x + 7.00 ∙ x ∙ x + 3.42 ∙ x ∙ x − 0,33 ∙ x ∙ x + 2.75 ∙ x ∙ x − 2.25 ∙ x ∙ x + 2.75 ∙ x ∙ x ∙ x (12) to check the validity of the mathematical model obtained, the experimental values vs model values were plotted (figure 12). figure 12. validity check of the mathematical model obtained for chromium the mathematical model of the response function y3 (mn concentration) obtained using anova methodis is given in equation (13): y = 5.64 − 0.78 ∙ x + 1.83 ∙ x + 0.46 ∙ x − 1.73 ∙ x + 1.12 ∙ x + 0.37 ∙ x + 0,57 ∙ x ∙ x − 0.53 ∙ x ∙ x + 0.10 ∙ x ∙ x + 0.30 ∙ x ∙ x + 0.48 ∙ x ∙ x + 0.75 ∙ x ∙ x + 0.40 ∙ x ∙ x + 0.058 ∙ x ∙ x − 0.17 ∙ x ∙ x − 0.41 ∙ x ∙ x ∙ x + 0.42 ∙ x ∙ x + 0.31 ∙ x ∙ x ∙ x − 0.23 ∙ x ∙ x − 0.24 ∙ x ∙ x ∙ x + 0.24 ∙ x ∙ x ∙ x − 0.46 ∙ x ∙ x (13) to check the validity of the mathematical model obtained for the y3 variable, the experimental values vs model values were plotted (figure 13). figure 13. validity check of the mathematical model obtained for manganese the mathematical model of the response function y4 ( 56fe concentration) obtained using anova methodis is given in equation (14): y = 522.96 + 123.33 ∙ x + 58.33 ∙ x + 96.67 ∙ x − 103.89 ∙ x − 13.89 ∙ x − 12.78 ∙ x + 16.67 ∙ x ∙ x − 5.83 ∙ x ∙ x + 6.67 ∙ x ∙ x + 5.00 ∙ x ∙ x + 12.50 ∙ x ∙ x + 0.001 ∙ x ∙ x + 42.50 ∙ x ∙ x − 15.00 ∙ x ∙ x − 15.00 ∙ x ∙ x − 17.50 ∙ x ∙ x ∙ x (14) r² = 0.9992 0.50 0.70 0.90 1.10 1.30 1.50 1.70 1.90 2.10 2.30 2.50 0.50 1.00 1.50 2.00 2.50 m od el v al ue ,t i[ pp b] experimental value, ti [ppb] r² = 0.9645 -10.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 20.00 40.00 60.00 m od el v al ue ,c r [p pb ] experimental value, cr [ppb] r² = 0.9996 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 2.00 4.00 6.00 8.00 10.00 m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o r ghe g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nvir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 184 the validity of the mathematical model obtained has been plotted by experimental values vs model values (figure 14). figure 14.validity check of the mathematical model obtained for 56fe the mathematical model of the response function y5 (ni concentration) obtained using anova method is given in equation (15): y = 21.29 + 12.72 ∙ x + 10.72 ∙ x + 10.78 ∙ x + 3.72 ∙ x + 2.72 ∙ x + 3.22 ∙ x + 3.25 ∙ x ∙ x + 5.50 ∙ x ∙ x + 5.83 ∙ x ∙ x (15) to check the validity of the mathematical model obtained for y5 variable, the experimental values vs model values were plotted (figure 15). figure 15. validity check of the mathematical model obtained for nickel 4. conclusions the aim of this study was to find and apply a method for modeling the experimental data obtained by migration test, which describes how exactly the dependence between variables used in the experimental design is. in order to establish the dependences between the working parameters of migration test and the metal concentrations of ti, cr, mn, 56fe and ni, found in corrosive solutions using mass spectrometry icp-ms inductively coupled plasma, the anova method was used; by utilizing this statistical method to obtain mathematical models that gives dependence of the working parameters and ti, cr, mn, 56fe and ni concentrations, these models can be used to estimate the objective function values. so, the simulation process may be obtained by using mathematical models, but they are produced with certain approximations and assumptions, allowing however, a description of processes for a wide range of values; for the mathematical models we obtained values of the regression coefficients very close to the value 1 (the dependent variables coefficients are valid), which demonstrates the validity of the applied statistical method; the comparative graphical representation of the experimental values vs values obtained by modeling shows that the deviation is less than 5%. these small differences give a real opportunity to use mathematical models in real intervention in production processes. 5. references [1]. asami k., hashimoto k., surface analytical study of atmospheric corrosion of stainless steel, proc. 13th international corrosion congress, paper 038, melbourne (1996); [2]. beddoes j., bucci k., the influence of surface condition on the localized corrosion of 316l stainless steel orthopaedic implants, r² = 0.9921 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 100.00 300.00 500.00 700.00 900.00 m od el v al ue , 5 6 f e [p pb ] experimental value, 56fe [ppb] r² = 0.9585 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 0.00 20.00 40.00 60.00 80.00 m od el v al ue , n i[ pp b] experimental value, ni [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug ab r ie l s t r oe , g he o rg he g u tt , st a t i s t i ca l s t u d y of t he d e pe nd en c e be t we en c o nc e nt r at i on o f m e t a l li c e l e m e n ts m i g r at ed f r om s t a i n le ss s t e e l g r ad e ai s i 3 21 a nd wo r k i n g p a r a m e t e r s , fo od a nd e nv ir o nm e nt sa f e t y, v o l ume x i i, is s ue 2 – 2 0 13 , p a g. 1 76 -1 85 185 journal of materials science: materials in medicine, 10, 389 (1999); [3]. hudson r. m., freeman p. f., allan g. k., corrosion of stainless steel above and below ground level, proc. 4th european stainless steel science and market congress, paris, france (2002); [4].wallinder d., odnevall wallinder i., leygraf c., influence of surface treatment of type 304l stainless steel on atmospheric corrosion resistance in urban and marine environments, corrosion, 59, 220 (2003); [5]. astm a321-90, standard specification for steel bars, carbon, quenched and tempered (2001); [6]. herting gunilla, wallinder i. o., leygraf c., factors that influence the release of metals from stainless steels exposed to physiological media, corrosion science, 48 2120-2132 (2006); [7]. barnes k. a., sinclair c. r., watson d.h., chemical migration and food contact materials, crc press, woodhead publishing limited, cambridge, england, 180-202 (2007); [8]. herting g., bioaccessibility of stainless steels-importance of bulk and surface features, doctoral thesis, division of corrosion science school of chemical science and engineering royal institute of technology stockholm, sweden (2008); [9] herting g., wallinder i. o., leygraf c., corrosion-induced release of chromium and iron from ferritic stainless steel grade aisi 430 in simulated food contact, journal of food engineering 87 291-300 (2008); [10] okazaki y., gotoh e., metal release from stainless steel, co-cr-mo-ni-fe and ni-ti alloys in vascular implants, elsevier, corrosion science 50, doi: 10.1016/j.corsci.2008.09.002 3429-3438 (2008); [11] sanches silva a., cruz j. m., sendor garcia r., franz r., paseiro losada, p., kinetic migration studies from packaging films into meat products, sciencedirect, meat science 77 238-245 (2007); [12].tehrany e. a., desobry s., partition coefficient of migrants in food stimulants/polzmers systems, sciencedirect, food chemistry 101 1714-1718 (2007); [13]. soojin j., irudayaraj j.m., food processing operations modeling design and analysis, second edition, crc presstaylor&francis group, 1-2 (2009). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 contents: 1. genetic, biochemical and physiological characteristics of the recombinant strain lactobacillus rl15, obtained by intergeneric hybridization between lactobacillus acidophilus 2 and bifidobacterium bifidum l1 rositsa denkova, i. dobrev, zapriana denkova, velichka yanakieva, zoltan urshev, maria yordanova, svetla ilieva 5 2. determination of pcb in water using microextraction and capillary gas chromatography ján ilavský, danka barloková 15 3. ecological problems at realization of active influence on hail processes suren hovsepyan, yeva torosyan, gagik torosyan 23 4. analysis trichloroand tetrachloro-ethylene in water ján ilavský, danka barloková 29 5. im pa ct o f s to rag e t em p era ture s a nd sto rag e o n red uc ing sug ar acum ulat io ns in s ug ar b ee t octa vi a n ba rna, o cta vi a n ba st on, aur a da ra bă 39 6. research on the in vivo variations of the ph values, oxidized lipids and soluble protein content of food rations based on turkey meat ancuţa elena coşuleanu 44 7. rai n fall in th e suh ard m o unt ai ns fr o m b uco vin a ioan gontariu, iontănasă, amelia buculei 47 8. inv est ig atio n o f th e p ro ce s s o f s t o r i ng p ack ag e d pum pki n in m o dif ie d a t m o sph e r e stefan stefanov, hristo hristov, donka stoeva, zapryana denkova, radosveta nikolova 54 9. identification and examination of some probiotic properties of the strain lactobacillus lbrz12 rositsa denkova, svetla ilieva, dilyana nikolova , zapriana denkova, velichka yanakieva 60 10. assessment of heavy metal (ni, cu) interactions in binary mixture to rainbow trout (oncorhynchus mykiss) at early life stages nijolė kazlauskienė, gintaras svecevičius, edvinas taujanskis, raimondas leopoldas idzelis, aistė liekytė 66 11. assessment of the action of deposit mycoflora on vicia faba l beans from suceava genebank’s collection diana batîr rusu, danela murariu 72 12. effects of dry lager brewing saccharomyces cerevisiae strain on the fermentation process and beer quality georgiana gabriela codină, silvia mironeasa 76 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 13. co ld to lera nce in eu ro pe an o at g en etic r e so u rce s danela murariu, marius murariu, ioan gontariu 82 14. assessment of the action of deposit mycoflora on phaseolus vulgaris l . beans from suceava genebank’s collection diana batîr rusu, danela murariu 88 15. studies concerning influence of the inoculum upon amyloglucosidase’s biosynthesis at aspergillus niger adriana dabija, cristina-gabriela constantinescu (pop), amelia buculei 93 16. ef fect o f ad dit io n o f s tar ch an d ag ar-ag ar o n rh eo lo g ic al b eh avio ur o f y o g urt cristina damian, mircea-adrian oroian, ana leahu, iuliana cioarbă 97 17. investigation of the process of storing packaged rabbit meat in modified atmosphere st e fa n st e fa nov, hr i st o hri st o v, d on ka st oe va, za pr ya n a de nko va, ra dos vet a nik o lov a 103 18. study reg a rd ing th e infl uence of metals m ig r atio n u pon t h e q ualit y o f b ee r at sto rag e amelia buculei, sonia amariei, stefan stefanov, gheorhe gutt, adriana dabija 108 19. author instructions 114 20. subscription information 119 microsoft word 0b. contents 1_2013 corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 contents: 1. certai n tex tural pro perties o f c hicken me at, invest igated through histology a nd ph otonic mi crosco py techniques răzvan mihail radu-rusu, ancuţa elena prisacaru (coşuleanu) tahsin sady hussen, gabriel vasile hoha 5 11 2. phase transfer catalysis for green chemistry yeva torosyan, andranik avetisyan, gagik torosyan 12 17 3. the effect of intense light pulsed treatment on aspergillus flavus (mi 148) spores corina neagu, liliana gîtin, daniela borda 18 23 4. changes of total water and dry matter content in healthy leaves and in leaves infected by taphrina deformans at peach cultivars springcrest and springold rodica ciobanu 24 29 5. temporal changes of corrosion losses and corrosion rates in water pipes vanda dubová, marek sokáč 30 37 6. eaf environmental impact analisys through mathematical modeling and fuzzy logic adrian ioana, augustin semenescu, cezar florin preda 38 46 7. is the laying hens rea ring system relevant fo r table eggs chemical a nd nut rit ional fe atures? cristina gabriela radu-rusu (şestaliuc), christelle plocus, céline dandois, răzvan mihail radu-rusu 47 52 8. thermal inactivation kinetics of lactoperoxidase in model system, milk and whey daniela borda, mihaela ghermăneanu, iulia bleoancă, corina neagu 53 58 9. the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour georgiana gabriela codină, silvia mironeasa 59 63 10. studies regar ding th e dil ute aci d pretre atment a nd e n zy mati c hyd rolysi s o f wheat str aws fo r bioethanol prod uction eufrozina albu, gheorghe gutt, mircea -adrian oroian 64 72 11. analysis of the environmental performance indicators for the emas regulation adrian ioana, augustin semenescu, cezar florin preda 73 77 12. modi fied prussia n blue screen pri nted electro des fo r h 2 o 2 detection florentina hutanu, gheorghe gutt 78 83 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 13. aspects concerning aflatoxins incidence in milk and milk products adriana dabija, iuliana sion, mihaela-adriana tita, ovidiu tita 84 88 14. incidence of food contamination with staphylococcus aureus in suceava county, romania florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton) 89 94 15. in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco biljana gveroska 95 104 16. biochemical aspects of water decontamination with ozone iryna belykh, sergiy samoylenko, myron rogozynskyi, igor winkler 105 108 17. author instructions i v microsoft word wd0000019.doc 95 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 95 104 in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco *biljana gveroska scientific tobacco institute-prilep, republic of macedonia bgveros@yahoo.com *corresponding author received 17 january 2013, accepted 20 february 2013 abstract biological control is an environmentally friendly approach to the plant protection from diseases. it reduces use of a chemical pesticides and it is a contemporary and reliable model which can be introduced into integrated pest management system. the biocontrol activity of trichoderma harzianum was evaluated against some fungal pathogens on tobacco. in vitro assays confirmed the main mechanisms involved in biocontol-mycoparasitism, antibiosis and competition for food and space. t. harzianum has a biocontrol activity against all tested fungi. it reduced the pathogen growth and completely overgrowth its colony. their relative growth in the presence of the biocontrol agent was 13,75 to 62,66%. the percentage inhibition of radial growth ranged from 37,34 to 93,83%. inhibition of the radial growth of r. solani is 61,10%. inhihition of the leaf pathogen a. alternata is very high -86,64%. t. harzianum showed the highest antagonistic potential (96,95%) against p. parasitica var. nicotianae, and the smallest (68,67%) – in p. debarianum. this result suggests that t. harzianum can be used in biological control of economically most important fungal diseases on tobacco. keywords: t. harzianum, pathogen, interaction, inhibition 1. introduction tobacco is a crop of a great interest and benefit for many countries. it has a big agricultural, economical and social impact. total world tobacco production is still increasing over recent years, mostly in developing countries and toward economic objectives [29]. but tobacco yield, quality and profitability are often affected by disease. fungal plant pathogens are among the most important factors that cause serious losses to agricultural products every year [14]. the pathogenic fungi attack different parts of the tobacco plant and cause a various damages. the most common fungal diseases affecting the tobacco in our country, from seedling production to mature leaves are: the damping-off on seedling caused by rhizoctonia solani and pythium debarianum, the black shank caused by phytophthora parasitica var.nicotianae and the brown spot, by alternaria alternata. the damping-off disease on tobacco seedlings is a very destructive and causesconsiderable damages in tobacco production. the pathogenic fungus attacks the plants on the ground part of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 96 seedling's stem. the tissue at this point necrotized, died and the following development is not enabled. infected plants lie down on the soil as they are “cuted”,. the disease is spreading to the neighboring plants and the big infected areas appears in the seed beds. damages and losses are obvious because of seedling's importance for quality production. the disease is caused by pathogenic fungi r.solani and p. debarianum. these are worldwide pathogens causing the damages of many horticultural and agricultural crops. symptomsnoticeable from the each fungi is almost the same. only microscopic observation and then, isolation from infected material may help in the true determination of the causing agent. although chemicals are used, control of these soil borne pathogensis almost impossible. black shank affects tobacco plants at all growth stages.the causing agent is a soilbornefungusp. parasitica var. nicotianae.the root system and lower stalk of infected plantsare usually black, from several inches above the soil. the disease development is followedbywilting, yellowing of leaves, development of stem lesions, and plant death. according to [29], the objective for an ideal cultural system and post-harvest management is not only for high yield, but also for leaf usability and desirable smoke quality. therefore, the brown spot disease (caused by the pathogenic fungus a. alternata) plays a significant role among some economically important diseases on tobacco because of severe economic losses in tobacco production. the recognizable symptom is the presence of brown spots, mainly on tobacco leaves, but on the other parts of plant, too. their presence causes biochemical changes that strongly reduce the quality of raw tobaccoaccompaniedby non-pleasant aroma. the available technique of disease control is fungicidal treatment, though often are ineffective. at the same time, they cause harmful consequences to human health and environmental security [19]. therefore, alternative control measures are focused on the use of biocontrol agents which reduce the disease and are less harmful than conventional fungicides. they have a high level of safety and minimal environmental impacts [21]. fungi of the genus trichoderma are broadly effective against various plant pathogens and a range of plant species. they successfully suppresssoil pathogens, as well as leaf pathogens[11,13]. there must be hundreds of separate genes and gene products involved in the biocontrol processes. the main mechanisms involved in biocontol are: antibiosis, mycoparasitism and food competition[10,12,19,14]. the use of trichoderma with all mechanisms of biological control will help suppress these pathogens. there are many data aboutthe biocontrol activity of the trichoderma fungi against above mentioned pathogens. biological control effect of trichoderma sp. on r. solani is confirmed by küçük and m. 16, 10,11,12,24,21]. these fungi are used in the control of many soil pathogens, including r. solani and p. debarianum[2]. [1] researched in vitro antagonism of trichoderma species against six fungal pathogens, including r. solani, p. aphanidermatumand p.parasitica var. nicotianae. t. viride and t. harzianum have antagonistic properties against phytophthora nicotianae[29]. according to [8], these two trichoderma strains controlled phytophthora spp. in some crops, including tobacco. t. harzianum is a biocontrol agent used in control of a.alternata [22,19,23]. it is a strong bca and the base of many commercial products [10]. it can produce lytic enzymes and antifungal antibiotic, it can be competitors of fungal pathogens and, in promoting plant growth [16]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 97 the first and quickest screening technique for antibiotic production and/or mycoparasitism is a petri dish assays [12]. therefore, the aim of this in vitro investigation is to take a certain knowledge about the biocontrol activity of t. harzianum against tobacco fungal pathogens. even that there are different pathogenic mechanism, spreading of the pathogen and disease, and finally, a various environmental conditions for the biological control, understanding the mechanisms which are involved and making possible to suppress them enable the use of biological control as an effective and eco-friendly measurement of tobacco disease control. 2. materials and methods the pure cultures of pathogenic fungi on tobacco were obtained from the culture collection of scientific tobacco instituteprilep. t. harzianum was isolated from the root zone of tobacco rhizosphere, by a method of dilution, using czapek agar.then, it was transmitted and maintained inpotato-dextrose agar medium (pda), as well as pathogens. the dual culturing technique was used for in vitro investigations of t. harzianum biocontrol activity against the pathogens.a 5 mm diameter mycelial disc from the margin of the trichoderma 10 days-old culture and the pathogen were placed on the opposite sites, in the center of each half of the plate with a pda. only in p. parasitica var.nicotianae, because of its very slow development(very fast overgrowing by the biocontrol agent), the other variant was used, also.t. harzianum was placed after 5 days in the plates with this pathogen. trials for each pathogen were set up in three replications, with five petri dishes for the controls and dual culture plates. they were incubated at 25oc and the diameter of the colonies was daily measured at 10 days, or the fulfillment of control plates for pathogens with faster development. interactions between pathogens and biocontrol agent were evaluated according changes in growth of mycelium in pure and mixed cultures, with a modified scale of the index of sensitivity (0-10) [15]: 0 good growth and stop before contact with other fungi 6 faster growth before and stop at contact 1 slower growth before and stop after contact 7 faster growth before and growth over other fungi mycelium after contact 2 slower growth before and growth over other mycelium after contact 8 slower growth before and stop before contact 3 good growth before and stop at contact 9 faster growth and stop before contact 4 good growth before and overgrowth, above other mycelium, after contact 10 faster growth before and growth over other fungi before contact 5 good growth before and grow beneath the colony/mycelium after contact relative development of a pathogen in the presence of biocontrol agent (% rd) was estimated by the method [17]: (1) where gp = growth of the pathogen and bca = biocontrol agent percentage inhibition of radial growth of the pathogen (% pirg) was calculated using the following formula (2) [18]: (2) where c= radial growth of pathogen in the absence of biocontrol agent (control) and t= radial growth of pathogen in the presence of biocontrol agent for p. parasitica, (the second case) we used the modified formula[26]: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 98 x100 (3) where rpc= radius pathogen colony control, rfp= radius of final pathogen colony in dual culture with trichoderma, rip – radius of initial pathogen colony still without the antagonist. degree of antagonism was evaluated according a scale [1]: class 1= trichoderma had completely overgrown the pathogen and covered the entire medium surface, class 2= trichoderma over grew at least two-thirds of the medium surface, class 3= trichoderma and the pathogen each colonized approximately one-half of the medium surface (more than one-third and less than two-third) and neither organism appeared to dominate than another, class 4 =the pathogen colonized at least two-thirds of the medium surface and appeared to withstand encroachment by trichoderma, and class 5 = the pathogen completely overgrew the trichoderma and occupied the entire medium surface. the antagonistic potential of the biocontrol agent (% ap) was evaluated by the formula[26]: where % mp: mycoparasitism potential of trichoderma – determined by its growing capacity on the pathogen colony in pairs culture. this capacity is 100% against four tested pathogens. % atp: antibiosis potential expressed in percentage of inhibition on the pathogen. 3. results and disscusion dual culturing test in the dual culture with biocontrol agent, r.solani started developmentalmost the same with a check culture, but slowly than t.harzianum. but, at the period of incubation, it is falling behind them (table.1). their contact is already realized at the second day (fig 1). from this point, a pathogen was continuing its development (like an any attemptingtowards the opposite direction) until the fourth day and its colony take a lengthened, deformed look (fig 2). at the contrary, the check colony fulfilght the plate on the fourth day, as well as t. harzianum. table 1. effect of t. harzianum on development of r. solani variant day 1 2 3 4 r. solaniø 18,70 52,53 82,47 110,00 r. solani 20,40 32,99 40,93 42,80 t. harzianum 30,47 68,58 105,80 110,00 t. harzianumø 29,20 82,89 94,52 110,00 t. harzianum started its development more rapidly than a pathogen. after the contact with them, it was continuing to develop without obstacles besides the presence of the pathogen (fig. 2,3). fulfillment of the petri dishes is achieved at control plots (table 1). it was developed over the pathogen's colony, covering them (fig. 4). figure 1 – 4. (upper left to down right) development of r. solani and t. harzianum food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 99 in dual culture the inhibitory effect of t. harzianumagainst the growth of pathogenic fungi was through the nonvolatile and volatile metabolites and r. solani was one of the most sensitive fungi to all tested t. harzianum isolates [16]. [21] also confirmed that t. harzianum is the best antagonistic fungi against r. solani. isolates of t. harzianum can produce antifungal antibiotics and lytic enzymes with it suppreses the pathogens [10,3].[24] described phases, or more correctly, the mechanisms employed in the mycoparasitic activity. p. debarianum started to grow as well ast.harzianumbut it has grown very quickly and passed its half of the plate. (table 2, fig. 5). but, after the contact with a biocontrol agent, its development became very poor. it can be seen a change of the pathogen's colony color but in a dense, too. it's become yellow to green and look like “melted” (fig 6,7).[5] founded that culture filtrates of t.harzianumcause changes of the mycelial growth andinhibition of zoospore germination.the mycoparasitism was evident in this case. it was concluded there was the lytic activity fromβ-(l, 3) -glucanase against cell walls of pythium spp.[4]. table 2. effect of t. harzianum on development of p. debarianum variant day 1 2 3 4 5 6 p. debarianumø 24 ,8 7 78 ,2 0 11 0, 0 0 p. debarianum 29 ,7 3 65 ,7 3 68 ,9 3 t. harzianum 25 ,6 0 55 ,4 7 74 ,4 7 81 ,0 0 90 ,1 3 11 0, 00 t. harzianumø 25 ,3 1 65 ,6 7 94 ,7 6 11 0, 00 besides a slower growth, t. harzianum grow well.it continued to grow after the contact, over the pathogen's colony and very soon fully covered them (table 2, fig 8). p. parasitica var. nicotianae has a very slow development in the control plates. the mycelial growth rate is very small and it doesn’t fill the plates in the period of incubation. figure 5 – 8. (upper left to down right) development of p. debarianumand t. harzianum in dual culture therefore, it was “captured” and overgrown by a biocontrol agent before it can really develop. even it was allowed to grow before the biocontrol agent was put onto plates with them, it stopped the development very soon, i.e. after the contact with them (table 3). it is realised at the second day, as well as in the other pathogens. t. harzianum developed as well as in the check and filled the plates, besides the pathogen presence (fig 9-12). phytophthora changes its colony color before it was covered by the biocontrol agent (fig 10,11). at the end of incubation it is very hard to recognize them (fig 12). these results are in contrary to [10] who stated that t.harzianum isolate t 22 has not ability to control phytophthora spp.because it has no mechanism to intercept or attack zoospores.but, the presented biocontrol activity of t. harzianum against p. parasitica var. nicotianae is in agreement with [20] who food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 100 founded that competition, mycoparasitism and antagonism are mechanisms of trichoderma spp. against p.nicotianae. table 3. effect of t. harzianum ondevelopment of p. parasitica var. nicotianae variant day 1 2 3 4 5 6 7 8 9 1 0 p. parasitica var. nicotianaeø + 13 ,0 2 22 ,2 4 28 ,0 4 36 ,6 9 43 ,4 2 56 ,6 7 65 ,0 4 73 ,5 5 77 ,0 9 p. parasitica (at the same time with t.harzianum ) + 10 ,6 0 p. parasitica (with t.harzianum after 5 days) 3 9, 86 41 ,4 3 43 ,5 7 44 ,5 7 t.harzianum 19 ,2 9 55 ,7 1 83 ,1 4 11 0, 00 t.harzianumø 21 ,2 1 67 ,0 4 94 ,3 6 11 0, 0 0 figure 9–12. (upper left to down right) development of p.p. var. nicotianae and t. harzianum in dual culture it was previously stated that trichoderma spp. are effective against leaf pathogens, too. accordingly, t. harzianumdeveloped more vigorously than a.alternatain the control plates, as well as in the dual culture(table 4).because of that, trichoderma passed the own half of medium and approached to the pathogen's colony as early as in the second day. (fig 14). futher growth of a.alternata is seen only in some cases when it makes an effort to develop towards the opposite site (until the third or fourth day).the pathogen's colony had got a white zone and a very light colorwith reduced number of conidia.this is in accordance with the statement of [22] that diffusible as well as volatile substances induced morphological abnormalities in fungal structure.a. alternata was “captured”, surrounded and covered by t. harzianum (fig. 15, 16). table 4. effect of t. harzianum on development of a. alternata variant day 1 2 3 4 5 6 7 8 9 10 a. alternata ø 12 ,5 3 26 ,9 3 39 ,6 7 49 ,4 7 59 ,5 3 71 ,4 7 84 ,1 1 99 ,6 0 10 5, 13 10 7, 73 a. alternata 12 ,1 3 21 ,6 0 27 ,7 3 28 ,8 0 t. harzianum 19 ,1 3 59 ,5 3 10 1, 40 11 0, 00 t. harzianum ø 18 ,2 7 59 ,2 0 10 5, 60 11 0, 00 figure 13–16. (upper left to down right) development of a. alternata and t. harzianum in dual culture the incidence of mycoparasitism against a. alternata is of a big importance in biological control. the pathogenicity of a. alternata is decreased when the solution of chitinaseis applied in tobacco seedlingsand there is a significantly better effect of crude than purified chitinase [6]. interactions between pathogens and biocontrol agent food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 101 according to the presented datain table 1 4, and a scale of the index of sensitivity (010), t. harzianum showed index 7 in the cases of three pathogensr. solani, p. parasitica var. nicotianae and a. alternata, whose is was 1 (table 5).after plating t. harzianum started to grow faster than these pathogens (especially in the case of phytophthora).after contact (which is being very soon-at second day) trichoderma continued to grow over other fungi mycelium. mycoparasitism was evidenced in the all investigated pathogens. table 5. index of sensitivity of some fungal pathogens on tobacco and t. harzianum pathogen t. harzianum index of sensitivity (is) r. solani 1 7 p.debarianum 6 4 p. parasitica var. nicotianae 1 7 a. alternata 1 7 in the case of p. debarianum, t.harzianum showed is 4 because of a very fast growing of the pathogen, who has an index 6.at the beginning, it developed faster than a biocontrol agent, but stop at contact. besides the presence and very quick growth of the pathogen, t. harzianum has good growth and overgrowth the pathogen. trichoderma sp.in vitro inhibits growth and sporulation of several soil-borne plant pathogenic fungi.trichodermal antagonism may involve mycoparasitism, antibiosis antimicrobial metabolites and volatile compounds and competition for food and space[20]. because of competition, trichoderma overgrowth the parasite [24].but, trichoderma can sense the presence of target fungi and appeared to grow tropically towards them. when paired with a target fungus, the endochitinase and exochitinase is activated. the diffusion of this enzyme catalyzes the release of cell wall fragments of target fungi. these fragments are highly potent inducers of enzymes and induce a cascade of physiological changes within the fungus, including an enhancement in trichoderma growth [12]. relative development of a pathogen in the presence of biocontrol agent each pathogen in double culture was developed weaker than in check (table 14).but, the relative growth in the presence of biocontrol agent differs between them. it ranges from 13,75 to 62,66%. the growth of r. solani in the presence of antagonist is ranged from 37,6 to 51,1% [17]. in this assay, rhizoctonia's growth is 38,91%, which is in accordance with this.the weakest growth was estimated in p. parasitica var.nicotianae and, the highest, in p. debarianum (figure 1). 0 10 20 30 40 50 60 70 alternaria altern ata rhizoctonia solani pythium debarian um p. parasitica var. nicotianae 26,73 38,91 62,66 13,75 p e rc e n ta g e graph 1. relative growth of the pathogen % figure 1. relative growth of the pathogen (%) inhibition of radial growth of the pathogen accordingly to the last values of pathogen's colony diameter in double culture, radial growth of pathogens are inhibited in the presence of t. harzianum.the percentage of inhibition varies between pathogens (figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 102 0 10 20 30 40 50 60 70 80 90 100 altern ar ia alternata rhizoctonia solan i pythium debarian um p. parasitica var. nicotian ae 86,64 61,1 37,34 93,89 p e rc e n ta g e graph 2. inhibition of radial growth of the pathogen % figure 2. inhibition of radial growth of the pathogen (%) the inhibition of radial growth of r. solani is 61,10%.this result is in agreement with results of [20] who estimated that t. harzianum cause 61-63% reduction of mycelial growth of r. solani.simillar of this, siameto et al [25] estimated that different isolates oft.harzianum have different radial growth inhibition of r.solani.it ranged from 4,31 to 71,37%. the control plates were filled with r. solani in 4 days and the pathogen was overgrown by antagonist in this period.therefore, these results are in accordance with the data obtained by [21] who found that t. harzianum after 4 days showed antifungal activity of 63% against r. solani. the biocontrol agent achieved the smallest percentage inhibition of radial growth in p. debarianum.[18] reported that inhibition of radial growth of p. aphanidermatum by two isolates of t.harzianumwas 60.3%to 69.8%, but in the other (isolate t.harzianum-4)was 38.5%, which is similar to the shown inhibition of p. debarianum growth -37,34%. p. aphanidermatumis one the causing agent of damping-off in tobacco seed beds [27]. percentage of reduction in the leaf pathogen a. alternata was very high86,64%. similar of this, [7]founded that t. harzianum showed 65,88 % inhibition of a.alternata due the production of diffusible metabolites. degree of antagonism t. harzianum has shown an antagonistic abilities against each pathogen.it has completely overgrown the pathogen and covered the entire medium surface. this antagonism of t. harzianum against the pathogens is characterized by class 1.it is the highest degree of in vitro antagonism by the used scale.belonging to class 1 of our isolate of t. harzianum is of a big importance for successful biocontrol of pathogens, especially in a case of phytophthora as a destructive parasite.inoculation with the two pure cultures of p. parasitica from anatgonism class 1did not induce disease during the 12 days [1]. antagonistic potential antagonistic potential (resulted from mycoparasitism and antibiosis potential) has a very high value (above 80%) in the three investigated pathogens (figure 3). 0 10 20 30 40 50 60 70 80 90 100 alternaria alternata rhizoctonia solani pythium debarianum p. parasitica var. nicotianae 86,64 80,55 6 8,67 96,95 percentage graph 3. antagonistic potential of t. harzianum against pathogenic fungi % figure 3. antagonistic potential t. harzianum has the highest potential against p. parasitica var. nicotianae, but the smallest antagonistic potential was against p. debarianum.[18] stated that metabolites released in culture filtrates of t. harzianum strongly inhibited pathogen growth, which suggest that antibiotic compounds play an important role in biocontrol activity.but, in some cases purified antibiotics mimic the effect of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 103 whole agent [3]. the combination of hydrolytic enzymes and antibiotics results in a higher level of antagonism that obtained by other mechanisms alone. it is also noted that direct interaction between the pathogen and bca must be established before the synthesis of hydrolytic enzymes, toxic compounds and/or antibiotics.establishment the interaction and the cell-wall degradation is needful because of lower synergizam when the enzymes were added after the antibiotics.individual strains may produce antibiotics in addition or in conjunction with mycoparasitism[9]. therefore, the conclusion that all pathogens developed until the contact with biocontrol agent, are in accordance with these findings. according to [11], different mechanisms might be responsible for biocontrol in different plants, and with different pathogens. although there are different pathogens, obviously the most important mechanisms are involved in the shown biocontrol activity competition, antibiosis and mycoparasitism and they are efficient through the mutual action.themechanisms are integrated and what has been defined as biocontrol is the final result of different mechanisms acting synergistically to achieve disease control [14]. 4. conclusions in conclusion, this study confirmed that t. harzianum has a biocontrol effect against the four investigated fungal pathogens. it reduced development of the certain pathogens through the main mechanisms mycoparasitism, antibiosis and competition for food and space. the shown biocontrol activity of t. harzianum ensures a possibility to use it as a biological control agent against the most important fungal diseases in tobacco. this offers incorporation into cultural practice and environmentally friendly approach to the control management of tobaccofungal diseases. 5. references [1]. bell d.k., wells h.d, markham c.r., in vitro antagonism of trichoderma species against six fungal plant pathogens, phytopathology, 72, 379-382, (1982) [2]. baličević r., paradziković n., ćosić j., rozman v., šamota d., suzbijanje zemljišnih parazita (pythium debarianum, rhizoctonia solani) na salati biološkim pripravkom, proceeding 43rd croatian and 3rd international symposium on agriculture, opatija, croatia, 465-468, (2008) [3]. benitez t., rincón m.a., limón c.m, codó c.a, biocontrol mechanisms of trichodermastrains, international microbiology, 7, 249-260. (2004) [4]. devaki n.s, shankara b.s., bhat s.g., manjunatha k.r., antagonistic activities of trichoderma harzianumagainst pythium aphanidermatum and pythium myriotylum on tobacco, journal of phytopathology, 136, 82–87, (1992) [5]. fajola a.o., alasoadura s.o., antagonistic effects of trichoderma harzianum on pythium aphanidermatum causing the damping-off disease of tobacco in nigeria, mycopathologia, 57, 47-52, (1975). [6]. gang t., xing l., wang g.e., shidong l., role of chitinase by trichoderma sp. in biocontol of tobacco brown spot caused by alternaria alternata, http://www.cababstractsplus.org/abstracts, (2008) [7]. gildiyal a., pandey a., isolation of cold tolerant antifungal strains of trichoderma sp. from glacial sites of indian nimalayan region, research journal of microbiology, 3 (8), 559-564,(2008) [8]. ha t.n, using trichoderma species for biological control of plant pathogens in vietnam, j. issaas, 16, 17-21, (2010) [9]. harman g.e., trichoderma for biocontrol of plant pathogens: from basic research to commercialized products. conference on biological control, cornell community, april 11-13, (1996) [10]. harman g.e., myths and dogmas of biocontrol. changes in perceptions derived from research on trichoderma harzianum t 22. plant disease84: 377-393, (2000). [11]. harman g.e., trichoderma speciesopportunistic avirulent plant symbionts, nature review microbiology, 2, 43-56, (2004) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 biljana gveroska, in vitro biocontrol activity of trichoderma harzianum against some pathogenic fungi on tobacco, food and environment safety, volume xii, issue 1 – 2013, pag. 95 – 104 104 [12]. harman g.e., overview of mechanisms and uses of trichoderma spp, phytopathology, 96, 190-194, (2006) [13]. heydari a., pessarakli m., a review on biological control of fungal plant pathogens using microbial antagonists, journal of biological sciences, 10, 273-290, (2010) [14]. howell c.r, mechanisms employed by trichoderma species in the biological control of plant diseases: the history and evolution of current concepts, plant disease, 87, 4-10, (1987) [15]. keča n.,in vitro interactions between armillaria species and potential biocontrol fungi. glasnis šumarskog fakulteta, beograd, 100,129-142, (2009) [16]. küçük ç., kivan ç m., isolation of trichoderma spp. and determination of their antifungal, biochemical and physiological fetures, turk j biol, 27, 247-253, (2003) [17]. melo i.s., faul j.l., parasitism of rhizoctonia solani by strains of trichoderma spp., scientia agricola, 57, 55-59, (2000) [18]. mishra v.k., in vitro antagonism of trichoderma species against pythium aphanidermatum, journal of phytology, 2, 2835, (2010) [19]. monte e.,understandingtrichoderma: between biotechnology and microbial ecology, int. microbiol, 4, 1-4, (2001) [20]. okhovvat m., in vitro antagonistic effects of trichoderma spp. on several soil-borne plant pathogenic fungi, j.sci.i. r. iran, 8, 8695, (1997) [21]. osman m.e.h., el-sheekh m.m., metwally m.a., ismail a.e., ismail m.m., antagonistic activuty of some fungi and cyanobacteria species against rhizoctonia solani, international journal of plant pathology, 2, 101-114, (2011) [22]. roco a., pérezl.m., in vitro biocontrol activity of trichoderma harzianum on a.alternata in the presence of growth regulators, plant biotechnology, 4 (2), (2001) [23]. sempere f., santamarina m. p.,. in vitro biocontrol analysis of alternaria alternata (fr.) keissler under different environmental conditions, mycopathologia, 163, 183-190, (2007) [24]. shalini k.p., narayan l., kotasthane a.s., genetic relatedness among trichoderma isolates inhibiting a pathogenic fungi rhizoctonia solani, african journal of biotechnology, 5, 580-584, (2006) [25]. siameto e.n., okoth s, amugune n.o., chege n.c., antagonism of trichoderma harzianumisolates on soilborne plant pathogenic fungi from embu district, kenya, journal of yeast and fungal research, 1, 47-54, (2010) [26]. simoes m.l.g., tornisielo s.m.t., niella g.r., tapua d.m.t., evaluation of trichoderma spp. for the control of moniliopthora perniciosa subgroup 1441, journal of biology and life science, 3, 18-36, (2012) [27]. subhashini d.v., biological control of damping off (pythium aphanidermatum) using streptomyces lavendulae isolate 21 on tobacco seed beds, journal of biological control, 24, 338-342, (2010) [28]. tso t.c., production, physiology and biochemistry of tobacco plant. ideals, inc. beltsvile, maryland, (1990) [29]. yi x., ding w., li y., antagonistic mechanisms of trichoderma spp. against phytophthora nicotianae, zhongguo zhong yao za zhi, 35, 1386-90, (2010) 3 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12015, pag. 3 9 prospects of applying vacuum technology in the manufacture of culinary poultry meat products * anna soletska1 , andrew krasota1 1department of meat, fish and seafood technologies, odessa national academy of food technologies,ukraine anna.soletska@gmail.com *corresponding author received january 8th 2016, accepted march 19th 2016 abstract: this work contains a comparative analysis of organoleptic, physico-chemical and microbiological parameters of food chicken meat products which were made using the vacuum technology and the traditional technology of the ukrainian producers. the products are made accordingly to a technological scheme, which is developed on the basis of similar traditional technology being normalized by the standards. the evacuation of the product was carried out in industrial conditions, the way of heat treatment of vacuum technology was chosen in accordance with the thickness of the product. the results showed that culinary products made from chicken meat, cooked using the vacuum technology, have better appearance, taste, smell and texture, the output being of 93%, reducing losses by a factor of 4, increasing the shelf life to 5 days. the microbiological research has proven the high reliability of vacuum chicken products and the promise for this technology to be widely applied into industry. the application of vacuum technology opens new prospects of diversifying the range of natural culinary products made from poultry meat, being far superior as regards their nutritional value, organoleptic properties and shelf life, much more convenient in terms of packaging than traditional products. keywords: technology sous vide, the chicken, heat treatment, culinary readiness. 1. introduction in the manufacture of culinary products from meat one of the methods most often used is the surface heating at moderate temperatures (up to 100 oc, preferably 8090 oc). the most characteristic and important changes, caused by damp heat in the moderat temperature are heat denaturation of soluble proteins, joining and hydrothermal collagen breakdown, the changing of extractives and vitamins, the death of vegetative forms of microorganisms. [1] the term “sous vide” literally from french means “under vacuum” and involves the placement of products in food-grade plastic bag, vacuum them, and then together with the product processing in a water bath at strictly controlled temperatures. the method provides that food is cooked in a sealed environment without oxygen at temperatures below 70 oc for a long time to achieve culinary readiness. [2, 3] nowadays catering is in a state of intensive development, using technological innovations. heat treatment of raw materials is accompanied by significant changes in the organoleptic, nutritional and biological value, as well as technological losses of mass. in this regard, a priority for the development of food production is to minimize these drawbacks by improving technology. a promising direction of improvement is the handling of raw materials at moderate temperatures prior to http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 4 vacuum packing in plastic shrink-wrap, known as sous vide technology, which allows you to get food while maintaining weight, nutritional value increase shelf life. the aim of the project was the application of sous vide technology for culinary products from poultry meat and its comparison with the traditional technology of similar product. the composition of meat is approximately 75% water, 20% protein and 5% fat and other substances. the main purpose of heat treatment is denaturation of proteins. the depth of this process depends on its temperature and, to a lesser extent time. the meat proteins are divided into three main groups: myofibrillar (50-55%), sarcoplasmic (30-34%) and the connective tissue proteins (10-15%). the myofibrillar proteins (mostly myosin and actin) and the connective tissue proteins (mostly collagen) contract when heated, while the sarcoplasmic proteins expand when heated. during heating, the muscle fibers shrink transversely and longitudinally, the sarcoplasmic proteins aggregate and gel, and connective tissues shrink and solubilize. muscle fibers begin to shrink at a temperature of 35-40 oc and the shrinkage increases almost linearly with increasing temperature up to 80 oc. aggregation and gelation of proteins sarcoplasmic begins approximately at a temperature of 40 oc and ends at 60 oc. the connective tissue proteins begin to shrink at a temperature of about 60 oc but a more intensive decrease takes place at 65 oc. about 80% of water in muscle tissue is contained in between thick myosin and thin actin fibers. in the temperature range from 40 oc to 60 oc muscle fibers shrink in the transverse direction, reinforcing fibers. at temperatures above 60-65 oc muscle fibers shrink longitudinally and cause substantial water loss increase with increasing temperature. [4] nowadays, there is a trend of using the meat and poultry of younger and lean animals. that's why the meat is dry and products are tasteless. to keep natural product made of poultry meat succulent and tender, the temperature of heat treatment shouldn’t be exceeded over 6065 oc. traditional methods of cooking poultry are using the temperatures 70-80 oc depending on the type of a bird and parts of a carcass. the tougher is the meat, the higher are the temperature and the duration of treatment. [5] until now culinary readiness of meat products was associated with the temperature of 72 oc in the center of the product, and the temperatures of an environment higher than 85-90 oc. the main purpose of this heat treatment is the denaturation of all proteins and destruction of vegetative microorganisms. however, it is now clear that the temperature range of 70-80 oc greatly affects the number of technological indicators, in particular organoleptic properties, mass loss, and nutritional value. [4, 6] the advantages of using the sous vide technology for the production of meat products following: 1) the product is produced in a sealed environment without oxygen at temperatures below 70 oc, 2) the absence of oxygen and water increase the amount of nutrients and consequently increase the nutritional value of the product, 3) through a plastic barrier significantly reduces oxidation, retains the properties of the essential polyunsaturated fatty acid lot, reduced the loss of moisture and extractives, whereby the flavors are enhanced, so less spices and salt, 4) the technology of sous vide allows us to get useful natural product with completely new flavor-aromatic properties and a new structure in a convenient package for long term storage. [7, 8] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 5 2. matherials and methods the experimental part of the work was conducted in the scientific laboratories of the department of technology of meat, fish and seafood and department of biochemistry, microbiology and physiology of nutrition onaft. as the object of research was selected chicken, culinary product according to the traditional technology – cooked chicken, evacuation of the product was performed in an industrial environment. in table 1 provides a list of methods for determination used in the experimental studies. [9] table 1 methods of research in the experimental part of scientific work the name of the indicator control methods mass fraction of moisture, % according to govermment standart 9793 mass fraction of sodium chloride, % according to govermment standart iso 1841-1:2004 yield, % according to [9] loss, % according to [9] the amount of mesophile and optional-anaerobic microorganisms, colony-generating iteames in 1 g. of the product according to govermment standart 10444.15 3. results and discussion to achieve the goal of scientific work it was necessary to solve several problems: 1) to justify the production of culinary products from poultry meat using the sous vide technology; 2) to make samples of culinary products from poultry meat using the sous vide technology and traditional techniques; 3) conduct comparative analysis of organoleptic, physicochemical and microbiological properties of the samples manufactured by two technologies. as the object of research was selected chicken, culinary product according to the traditional technology – cooked chicken. [9] in fig. 1 shows a flow diagram for the manufacture of chicken fillet with both technologies. in case of manufacturing the product using sous vide technology, after meat was added into salt and species, it was carried out for the evacuation in food heatresistant plastic bag and after storage for ripering treatment it was being handled in a water bath at a temperature of 63-65 oc for 60 minutes. it should be noted that the modes of thermal processing was determined in accordance with the thickness of the product [6]. the main purpose was to bring the product to the culinary readiness at extremely low temperature for a time that would ensure full denaturation of the protein with the least damage to the nutritional value microbiological stability. meanwhile, when traditional technology was used, the product was being boiled in water at a temperature 95-98 oc during 30 minutes until it became culinary ready. in fig. 2 shows the context of the product manufactured by both technologies that indicate significant differences of organoleptic characteristics. diffenerces between sous vide and traditional technology are clearly seen in fig. 3. radar chart shows organoleptic evaluation of samples, manufactured according to the traditional technology and the technology of sous vide. the sample, which was made using the sous vide technology, had more impressive taste and smell, firm and juicy texture, colour and appearance of cut resembled ham product. physico-chemical parameters determined the content of moisture, salt and yield and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 6 loss after heat treatment. the results of the comparative characteristics of the indicators are given in table 2, indicate that plastic bag, and low temperature heat treatment prevent loss of water and salt product, which confirms the results of the organoleptic studies, and explains the increase in the yield by 19% and the reduction of losses in 4 times sample, made using the sous vide technology. 1. acceptance of chilled raw material t=1 ос ↓ 2. the formation of fillets of chicken ↓ 3. add: salt – 2%, spices – 0.5% ↓ ↓ 4. bringing to the condition τ=12 hours, t=2…4 ос 4. determination of thickness of raw materials and vacuum ↓ ↓ 5. cooking in water at t=95…98 ос, τ=30 min 5. bringing to the condition τ=12 hours, t=2…4 ос ↓ ↓ 6. cooling at t=4 ос to t=8 ос 6. water bath at t=63…65 ос, τ=60 min ↓ ↓ 7. storage not above 8 oc for not more τ=24 hours 7. cooling at t=4 ос to t=8 ос ↓ 8. storage of not more than 8 ос τ=6 days fig. 1 – the technological scheme of production of chicken in the following technologies: ─ ─ ─ ─ traditional; ─ ─ ─ ─ sous vide а b fig. 2 – view the cut chicken fillets are made: a – according to the traditional technology, b – using the sous vide technology food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 7 an important step in the research was to prove that the selected modes of heat treatment of the pattern manufactured by using the sous vide technology, provide adequate microbiological safety and duration of storage of finished products. in table 3 shows the results of microbiological research of samples manufactured by two technologies, which were stored at a temperature of 2-4 oc during 6 days. samples for analyses were taken immediately after manufacture, through the day and on the sixth day of storage. for comparison, similar analyses were conducted for raw meat. qualitative reaction for anaerobic microorganisms on the environment kitt-tarocci on the first day of research gave negative results, as in raw meat and in the samples that were made according to the traditional technology and the sous vide technology. research the amount of mesophile and optional-anaerobic microorganisms, colony-generating iteames in 1 g. of the product indicates that the microbiological safety of sous vide technology satisfies the standard requirements for finished products manufactured in ukraine and extends the shelf life to five days compared to products produced by traditional technology. fig. 3 – the radar chart of organoleptic evaluation on a 5-point scale specimens, manufactured according to the traditional technology and the technology of sous vide the content of moisture and salt, the yield and the product loss after temperature treatment were determined by phisycochemical parametres. the results of the comparative characteristics of the indicators are given in table 2, indicate that plastic bag and low temperature heat treatment prevent loss of water and salt product, which confirms the results of the organoleptic studies, and explains the increase in the yield by 19% and the reduction of losses in 4 times sample, made using the sous vide technology. 0 1 2 3 4 5 appearance the color and appearance of meat on the cut the smelltaste consistency traditional technology technology sous vide food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 8 table 2 comparative characteristics of physico-chemical properties of the samples made according to the traditional technology and the technology of sous vide the name of the index the sample made according to the technology the value of the index according to govermment standart 4531:2006 traditional sous vide mass fraction of moisture, % 66.3 74.9 not normalized mass fraction of sodium chloride, % 0.6 0.8 1.2 yield % 74.2 93.4 not normalized loss, % 25.7 6.5 not normalized table 3 microbiological properties of the samples, which were stored at t=2-4 oc the amount of mesophile and optional-anaerobic microorganisms, colony-generating iteames in 1 g. of the product raw meat the sample made according to the technology standart traditional sous vide storage period, days 0 1 6 0 1 6 0 1 6 210*103 240*103 ─ not allowed 0.66* 103 1.2*103 not allowed 0.61* 103 0.37* 103 < 1*103 4. conclusion sensory characteristics of chicken which was made by the technology sous vide greatly surpassed them in comparison with the sample which was made by traditional technology. namely, the meat was juicier, tender, and solid on the cut, taste was expressed more brightly. the output of chicken fillet which was made by sous vide technology increased by 19% in comparison with the sample which was manufactured by the conventional technology, also the product loss decreased in 4 times. microbiological studies have proved microbiological trustworthiness chicken, made by a technologist sous vide on the 6th day of storage compared to the sample made according to traditional technology, the shelf life which is according to govermment standart 4531:2006 ‒ not more than 1 day. so, the technology of sous vide allows you to get natural (no additives or preservatives) culinary products from poultry meat of high nutritional value, with improved organoleptic properties safe for the consumer during storage for 6 days. this technology is promising for domestic manufacturers of meat products, because gives you the opportunity to expand the range, to provide high yield and extend the shelf life of the finished product. 5. references [1]. shalimova o. a., gorkova i. v., innovative technologies in the production of quality and safe food products from meat, orelgau, 549, (2007). [2]. kowalyk c., sous vide cooking method. patent us №0034361 a1 (2012). [3]. baldvin d. e., sous vide for the home cook, paradox press, 271, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 anna soletska, andrew krasota, prospects of application of sous vide technology in the manufacture of productsfrom poultry meat, food and environment safety, volume xv, issue 1 – 2016, pag. 3 – 9 9 [4]. tornberg e. effect of heat on meat proteins − implications on structure and quality of meat products. meat science, №70, 493–508 (2005). [5]. soletska a. d., optimization of heart treatment modes for boiled sausages, fod science and technology, №3(28), 73–76, (2014). [6]. ghazala s. sous vide and cook-chill processing for the food industry. an aspen publication, 358, (1998). [7]. church i. j., parsons a. l. the sensory quality of chicken and potato products prepared using cook-chill and sous vide methods. international journal of food science and technology, №35, 155–162, (2000). [8]. garcia-linares m. c., gonzalezfandos e., garc´ıa-fern´andez, m. c., garc´ıaarias, m. t. microbiological and nutritional quality of sous vide or traditionally processed fish: influence of fat content. journal of food quality, №27, 371–387, (2004). [9]. antipova l. v. research methods of meat and meat products. moscow: kolos, 376, (2001). 1. introduction 4. conclusion 276 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 276 282 microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation natia j. kouadio1, *bernadette g. goualie1, honoré g. ouattara1, sevérin k. a. kra1, sébastien l. niamke1 1laboratoire de biotechnologies, filière biochimie-microbiologie, unité de formation et de recherche en biosciences, université félix houphouet-boigny, cocody, abidjan-côte d’ivoire. 22 bp 582 abidjan 22, bettygoualie@yahoo.fr *corresponding author received september 19th 2014, accepted september 29th 2014 abstract: the objective of the present study was to investigate microorganisms associated with traditional plantain-based food dockounou during spontaneous fermentation. five samples including rice and maize flour, over ripe banana pulp, and over ripe banana pulp/rice or maize flour mixture were fermented separately during 4 hours. microbial load was estimated by plate count using decimal dilution on selective medium depending of each microbe. the microorganisms isolated from all samples included yeast, lactic acid bacteria and bacillus sp. the yeast was detected at levels of values ranged 2.54 log10 to 3.50 log10 at biginning of spontaneous fermentation and increased with value ranged from 4.03 log10 to 4.56 log10 at the end of the process. the bacterial load during this spontaneous fermentation decreased to 4.50 log10 to 2.72 log10 for some samples. acetic acid bacteria were not detected in all samples during the spontaneous fermentation. the metabolic activities of yeast, bacillus sp and lactic acid bacteria during fermentation could improve the sensory, nutritive and safety quality of dockounou. key words: plantain, dockounou, fermentation, yeast, lactic acid bacteria, bacillus sp 1. introduction bananas (musa aaa, aa) is one of the major food resources in the world. with an average production of 106 million tones per year [1, 2], bananas occupy the fourth in the world after rice, wheat and milk [3, 4]. sub-saharan africa accounts for over a third of global production of plantains, with nearly 7 million tones per year. in côte d’ivoire, plantain is the third food product after yam and cassava [5]. interest in plantain is mainly related to its nutritional quality. in fact, plantain constitutes an agriculture product with carbohydrates accounting for 22% of fruit weight and rich in vitamins a, b6, c, minerals and dietary fibre [6, 7]. this vegetable resource contributes significantly to food security and provides more than 25 and 10% of the daily intake of carbohydrates and calories, respectively, for more than 70 million people in sub saharan africa [8, 9, 10]. however, plantain is a highly perishable fruit and difficult to keep. what is causing huge post-harvest losses estimated at nearly 60% of the annual harvest. the difficulties of plantain preservation result from its easy ripening at ambient temperature, leading to a qualitative and quantitative degradation along the distribution chain [11]. according to palmer [12], the ripening of banana fruit is associated with a massive breakdown of starch, pectin, cellulose and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 277 hemicellulosic substances concomitant with a rise of simple sugars. in côte d’ivoire, to reduce these post-havest losses, plantain fingers are rapidly consumed boiled, roasted and fried [7, 13, 14, 15, 16]. the over ripe fruits of plantain are transformed by farming women into dish called dockounou in côte d’ivoire [14] and “ofam” in ghana [16] to reduce these losses. the dockounou is a dish consists of over ripe banana supplemented with other starchy source mainly rice or maize flour [17]. production process of this dish followed seven steps including fermentation which is still optional [18]. to date, importance of fermentation and microorganisms involved in this process during the dockounou production are still unknown. however, akoa et al. [19], in optimization of dockounou manufacturing process, showed that the best sensory quality of this dish was obtained after 4 hours of fermentation of over ripe banana and rice or maize mixture. therefore the aim of this study is a first time to investigate the microorganisms present in the dough of dockounou during this fermentation. 2. experimental 2.1. samples collection senescent plantains fingers, maize and rice grains used in this study were purchased from adjame market (abidjan). 2.2. samples preparation samples preparation was performed according to dockounou optimization method described by akoa et al. [19]. the rice and maize grains were soaked in water respectively for 2 and 10 hours. after drying, the grains were grinded in a home wooden mortar and the flour was sieved (500 μm). after that, senescent plantain fingers were washed with potable water to eliminate all impurity and peeled manually using ordinary knife. pulp crushing was made in traditional hygienic wood mortar and 450 g of this mushy pulp was mixed with 50 g of rice or maize flour. in all, five (5) samples were made including 500 g of rice flour, 500 g of maize flour, 500 g of over ripe banana dough, 500 g of banana pulp and rice flour mixture and 500 g of banana pulp and maize flour mixture. 2.3. sample fermentation process each sample was puted in a sterile container which hermetically closed with a top. all containers were placed at room temperature that was set at 25 o c28 °c and allowed to ferment for 4 hours [19]. each hour (t0, t1, t2, t3 and t4) 10 g of each sample was tacked and analyzed for monitoring, isolation, and enumeration of fermenting flora according to afnor v 08-015 guidelines. briefly, 10 g of each sample was added to 90 ml of peptone water buffer contained in a sterile stomacher bag that was shaken for 2 min to obtain a uniform homogenate. samples (1.0 ml) of the homogenate were serially diluted 10-fold in trypton salt buffer, from which aliquots (0.1 ml) were plated on different selective agar media that were incubated at 30 °c for 1 to 3 days in a standard incubator (jouan, st. herblain, france). specific medium was used for each microorganism: mygp (malt yeast glucose, peptone) supplemented with chloramphenicol (1 %) for yeast, mrs (man rogossa and sharpe) for lactic acid bacteria, duthathai et pathom-aree [20] agar (0.5 % d-glucose, 1 % yeasts extract, 1 % peptone, 2 % glycerol, 1.5 % potato and 4 % ethanol (v/v) and 0.0016 % of bromocresol green) for acetic acid bacteria and nutrient agar (biorad, france) for bacillus sp isolation. morphologically different colonies of acetic acid bacteria, lactic acid bacteria and bacillus were picked up from each sample on specific medium and purified through subculturing and confirmed by using of both phenotypic (colony and cell morphology, mobility, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 278 gram staining) and biochemical analyzes (catalase activity and oxidase activity). while yeast were identified by morphological characteristics (colony and cell morphology) after pick on mygp agar. 2.4. colonies counting after the incubation periods, colonies were counted by using a colony counter (jp selecta, spain) and results were expressed as colony forming unit per gram (cfu/g) of sample. 3. results and discussion the evolution of microbial cell counts in all samples during fermentation step can be seen in figure 1 and 2. figure 1: microbial growth dynamic during fermentation of banana (a), rice flour (b) and maize flour (c) 2 2.5 3 3.5 4 4.5 0 1 2 3 4 n um be r o f m ic ro or ga ni sm s in m ai ze fl ou r ( lo g 1 0 cf u /g ) time (hours) bacillus sp. lactic acid bacteria yeast 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 nu m be r o f m ic ro or ga ni sm s i n ba na na ( lo g cf u /g ) time (hours) bacillus sp. lactic acid bacteria yeast (a) 2 2.5 3 3.5 4 4.5 5 5.5 6 0 1 2 3 4 n um be r o f m ic ro or ga ni sm s i n ri ce flo ur (l og 10 cf u /g ) time (hours) bacillus sp. lactic acid bacteria yeast (c) (b) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 279 yeast, lactic acid bacteria and bacillus sp were detected in all samples throughout the fermentation period. additionnally, result show that yeast population increase during this process while the bacterial load decrease. indeed, microbial load of yeast increased to a value of 2.54 to 4.56 log10 (cfu/g) in banana pulp (figure 1 a), 3.43 to 4.03 log10 (cfu/g) in rice flour (figure 1 b) and 3.03 to 4.22 log10 (cfu/g) in maize flour (figure 1 c). in mixture samples corresponding to dockounou dough, the yeast population increased also during fermentation step with microbial ranged from 4.06 to 4.56 log10 (cfu/g) for over ripe banana pulp and maize flour mixture and 3.50 to 4.46 log10 (cfu/g) for over ripe banana and rice flour mixture (figure 2). figure 2: microbial growth dynamic during banana/rice flour mixture (a) and banana/maize flour mixture fermentation 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4n um be r o f m ic ro or ga ni sm s i nm ix tu re of b an an a an d m ai ze fl ou r ( lo g 1 0 cf u /g ) time (hours) bacillus sp. lactic acid bacteria yeast (b) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 n um be r o f m ic ro or ga ni sm s in m ix tu re o f b an an a an d ri ce fl ou r (lo g 1 0c fu /g ) time (hours) bacillus sp. lactic acid bacteria yeast (a) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 280 soro-yao et al. [21] was also reported that yeast, lactic acid bacteria and bacillus sp are the major microorganisms implicated in production of many ivoirian fermented food. in additionally kim et al. [22] indicated an increase of yeast throughout the fermentation process of korean traditional rice wine supplemented with banana during fermentation. the increase of yeast population in this study could be related to the fact that banana, maize and rice contain favorable substrates (such as glucose) for the growth of yeast. indeed during ripening of banana, starch is converted in glucose through αamylase activity [12] while rice and maize contain starch essentially [23] which will be converted in glucose eventually [24]. according to amoa-awua and jakobsen [25], an environment containing high sugar constituted a favorable medium for yeast. concerning the bacillus sp population, evolution of microbial load in banana, maize and rice presented globally a own phase. indeed, the bacterial population decreased slightly during fermentation with microbial load ranged 4.50 -3.66 log10 (cfu/g) for the banana pulp (figure 1 a), 3.65 -3.45 log10 (cfu/g) for the rice flour (figure 1 b) and 4.08-3.58 log10 (cfu/g) for the maize flour (figure 1 c). lactic acid bacteria population presented similar variation in the three samples. in mixture samples, the bacterial load increase within 3-4 hours for banana pulp and rice flour mixture (figure 2 a) and remain stable during fermentation period in banana pulp and maize flour mixture (figure 2 b). so our results indicated that lactic acid bacteria and bacillus sp are predominant at beginning of this spontaneous fermentation. this phenomen was also reported by other researchers during fermentation of variable food [21, 26, 27, 28, 29]. the slightly decrease of bacterial load during all samples fermentation is probably due to yeast activity during fermentation mainly alcohol production [30]. on the over end, variable acid (malic, succinic, lactic and acetic acids…) which rate increase during fermentation of banana [22] contributed also to this decrease of bacterial population. however, the increase of bacterial load observed within 3-4 hours during mixture samples fermentation imply that these microorganisms could be competitive and capable to survive under certain stress conditions and eventually to express their fermentative potential during the fermentation process. lactic acid bacteria, bacillus sp and yeast are involved in many fermentation to a varying extent [31, 32, 33]. kim et al. [22] and rhee et al. [30] for example demonstrated the contribution of these microorganisms in improvement of asian fermented product quality. according to soro-yao et al. [21], these microorganisms allowed improvement of food quality during fermentation step by affecting sensory properties (aroma, taste, colour, texture), enhancing food safety by inhibition of pathogens bacteria and removal undesired compounds such as mycotoxins, improving the nutritive value, and reducing energy required for cooking. yeast are characterized by their pectinase activity and capacity to ferment glucose or other sugar into alcohol and co2. these technological properties contributed probably in the first time to improve the texture of dockounou by hydrolysis of pectin contain in banana and in second time to inhibit the growth pathogen bacteria in this dish during fermentation. moreover, bouatenin et al. [24], in their studies on cassava dough fermentation, reported that yeast, lactic acid bacteria and bacillus are able to produce some enzymes mainly amylases. consequently, these microorganisms contributed probably of improvement of dockounou quality by hydrolysis of starch contained in over ripe banana and in cereals by production of α-amylases. that food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 281 allowed improving the digestibility of dockounou and availability of fermentable sugar for ulterior acidification. amoa-awua et al. [34] showed also the ability of some bacillus sp to produce organics acid mainly lactic acid from starch and fermentable sugar. in additionally, it well-know that lactic acid bacteria produced from fermentable sugar the amount varies acid depends of the product type during fermentation [35]. this food acidification during fermentation could have many advantages notably renders food resistant to microbial spoilage and the development of pathogenic microorganisms in food, produce desirable flavors and often improves nutritional value [30, 36]. therefore, this activity of lactic acid bacteria and bacillus during dokounou dough fermentation contribute certainly to improving the taste, aroma and nutritional and health qualities of the product at the end of cooking process. what could explain that dockounou baked from fermented dough were the best sensory qualities according testers in akoa et al. [19] studies. in this study, no colony corresponding to acetic acid bacteria was identified on the plate at this time during fermentation. absence of characteristic colony of acetic acid bacteria at the beginning (0-24 hours) of fermentation as also reported by some researchers [37, 38]. it well knows that acetic acid bacteria growth in aerobic atmosphere so the low level of oxygen in our fermenting medium is probably responsible of this growth inhibition. 4. conclusion this study allowed to known that yeast, lactic acid bacteria and bacillus sp implicated in dockounou fermentation. however, further studies are needed to characterize these microorganisms and to investigate the technological properties of each microbial group to understand their role during dockounou dough fermentation. 5. acknowledgments this work was supported by the biotechnology laboratory of university of félix houphouët-boigny. 6. references [1]. lescot t., banane: production, commerce et variété. fruitrop, 63, 13-16, (1999) [2]. fao, faostat statistics database agriculture. roma: fao, (2004) [3]. inibap,. banana diversity. international network for the improvement of banana and plantain. http://www.inibap.org/publications/factsheet-eng.htm. (2002) [4]. fao, faostat statistics database agriculture. roma: fao, (2002) [5]. lescot t., locillet d., banane et environnement: vers une production plus propre à l‟horizon 10 ans. fruitrop, 153: 3-4, (2008) [6]. chandler s., the nutritional value of bananas. in: gowen s., ed. bananas and plantains. chapman & hall. london. pp. 468-480, (1995) [7]. honfo f.g., tenkouano a., coulibaly o, banana plantain-based fo odsconsumption by children and mothers in cameroon and southern nigeria: a comparative study. afr. j. food sci., 5 (5): 287-291, (2011) [8]. john p., marchal j, ripening and biochemistry of the fruit. in s. r. gowen (ed.) bananas and plantains (pp. 122-156). london, (1995) [9]. iita, improving plantain and banana based. international institute of tropical agriculture. project 2, annual report, ibadan, p. 67, (2000) [10]. bakry f., didier c., ganry j., lebellec f., lescot t., pinon a., vannicre m, fruits species. in: cirad (ed), guide of agronomist (pp. 960-974). paris, (2002) [11]. chia c.l., huggins c.a. bananas. in ctahr (ed.), community fact sheet fruit (pp. 23-29). hawaii, (2003) [12]. palmer j.k, the banana. in ac hulme, ed, the biochemistry of fruits and their products. academic press, london, pp 65-105, (1971) [13]. n’guessan a., yao n., kehe m, la culture du bananier plantain en côte d’ivoire. spécial bananes ii: systèmes de production du bananier plantain. fruits 48(2):133-143, (1993) [14]. koffi k.s, rôle des ressources génétiques dans l’essor du secteur bananier plantain en côte d’ivoire. in: plant genetic resources and food security in west and central africa. regional conference, ibadan, nigeria, 26-30 april, 2004 2007. vodouche r, attakrah k, achigan-daka ge, eyog-matig (eds), (2007) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke, microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation, food and environment safety, volume xiii, issue 3 – 2014, pag. 276 – 282 282 [15]. lassois l., jean-pierre b., haïssam j, la banane : de son origine à sa commercialisation. univ. liège gembloux agro-bio tech. plant pathology unit. passage des déportés, 2. b-5030 gembloux (belgium). biotech. agron. soc. environ. 13(4), 575-586, (2009) [16]. dzomeku b. m., dankyi a.a., darkey s.k. socioeconomic importance of plantain cultivation in ghana. j. anim. plant sci., 21 (2): 269273, (2011) [17]. akoa e.e.f., megnanou r.m., kra k.a.s., ahonzo-niamke l.s, technical variation in the processing of dockounou, a traditional plantain derivate dish of côte d’ivoire. american journal of research communication. vol 1(5) 81-97, (2013) [18]. kra k.a.s., akoa e., megnanou r-m., yeboue k., akpa e. e., niamke l. s, physicochemical and nutritional characteristics assessement of two different traditional foods prepared with senescent plantain. afr. j. food sci. 7(3) : 51-55, (2013) [19]. akoa e.f.e., kra k.a.s., mégnanou r.m., kouadio n.j., niamké l.s, (2014) optimization of dockounou manufacturing process parameters sustainable agriculture research; vol. 3, no. 1. [20]. duthathai, f., pathom-aree w, application of chemical dyes as colour indicator for selective isolation of acetic acid bacteria. research journal of microbiology, 2: 885-888, (2007) [21]. soro-yao a.a., brou k., koffi-nevry r., djè k.m, microbiology of ivorian fermented products: a review. asian journal of agriculture and food science. 1(2):37-47, (2013) [22]. kim e., chang y.h., ko j.y., jeong y, physicochemical and microbial properties of the korean traditional rice wine, makgeolli, supplemented with banana during fermentation. prev. nutr. food sci;18 (3):203-209, (2013) [23]. cruz j.f., troude f., griffon d., hebert j.p, conservation des grains en régions chaudes techniques rurales en afrique. ministère de la coopération & du développement (2nd eds), f. paillart abbeville, paris (france), 1-545, (1988) [24]. bouatenin k.m.j-p., djeni n.t., ouassa t., zinieu e., menan h., dje k. m, characterization and enzyme activies of microrganismes from a traditional cassava starter used for the production of adjoukrou attieke (côte d’ivoire). journal of food technology 11 (1): 4-13, (2013) [25]. amoa-awua w.k., jakobsen m, the third biennal seminar on african fermented food. the role of microorganisms in the fermentati on of agbélima cassava, dough. pp. 1-7, (1996) [26]. padonou s.w., nielsen d.s., hounhouigan d.j., thorsen l., nago c.m., jakobsen m, the microbiota of lafun, an african traditional cassava food product. int j food microbiol 133, 22–30, (2009) [27]. bouatenin m.k.j-p.,. djéni n.t., aka s., brou k., djè k.m, the contribution of microorganisms to the fermentation of cassava dough during attiéké processing in côte d’ivoire, food, vol. 6, no. 1, pp. 58-64, (2012) [28]. afoakwa e.o.,. kongor j.e, takrama j.f., budu a.s, changes in acidification, sugars and mineral composition of cocoa pulp during fermentation of pulp pre-conditioned cocoa (theobroma cacao) beans. international food research journal 20 (3): 1215-1222, (2013) [29]. ouattara d.h., ouattara h.g., goualie b.g., kouame l.m., niamke s.l, biochemical and functional properties of lactic acid bacteria isolated from ivorian cocoa fermenting beans. j. appl. biosci. 77:6489– 6499 (2014) [30]. rhee s.j., lee j-e., lee c-h.. importance of lactic acid bacteria in asian fermented foods . microbial cell factories. 10(suppl 1):s5. doi:10.1186/1475-2859-10-s1-s5, (2011) [31]. hounhouigan d.j., nout m. j.r., nago c.m., houben j.h., rombouts f.m, composition and microbiological and physical attributes of mawe, a fermented maize dough from benin. int. j. food sci. technol. 28, 513-517, (1993) [32]. oyewole o.b, characteristics and significance of yeasts involved in cassava fermentation for ‘fufu’ production. int j food microbiol 65, 213–218, (2001) [33]. crafack m., mikkelsen m., saerens s., kundsen m., blenbow a., lowor s., takrama j., swiegers j., petersen g.b., heimdal h., nielsen d. s, influencing cocoa flavor using pichia klutveri and kluyveromyces marxianus in a defined starter culture for cocoa fermentation. int. j. food microbiol. 167: 103-116, (2013) [34]. amoa-awua w.k., frisvad j.c., sefadedeh s., jakobsen. (1997). the contribution of moulds and yeasts to the fermentation of ‘agbelima’ cassava dough. j appl microbiol 83, 288–296. [35]. lee c. h, food biotechnology. in food science and technology campbell-platt, g., wiley-blackwell 2009, 85-113 (2009) [36]. steinkraus k.h, acid fermented cereal gruels, p. 189–198. in k. h. steinkraus (ed.), handbook of indigenous fermented foods. microbiology series, vol. 9. marcel dekker, inc., new york (1983) [37]. moreira i.m., da veiga da cruz m.g., miguel p., w.f. duarte, dias d.r., schwan r.f, microbial succession and the dynamics of metabolites and sugars during the fermentation of three different cocoa (theobroma cacao l.) hybrids. food research international 54: 9–17 (2013) [38]. yao w., ouattara h.g., goualie b., soumahoro s., niamke s,. analysis of some functional properties of acetic acid bacteria involved in ivorian cocoa fermentation. j. appl. biosci. 75:6282– 6290 (2014) 227 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 3 2016, pag. 227 233 the impact of different diet and environmental conditions on chemical composition of rainbow trout (oncorhynchus mykiss walbaum, 1792) from macedonian aquaculture facilities biljana sivakova1, *dijana blazhekovikj dimovska2 1food and veterinary agency of republic of macedonia bitola, macedonia, bnajdovska0509@ yahoo.com, 2faculty of biotechnical sciences, university „st. kliment ohridski” bitola, macedonia, dijanablazekovic@ yahoo.com *corresponding author received september 2nd 2016, accepted september 22nd 2016 abstract: the main purpose of the research was to make an analysis of the influence of ambient conditions and nutrition on the chemical composition of rainbow trout (oncorhynchus mykiss walbaum, 1792) from various aquaculture facilities in macedonia, in different ambient conditions in the aquatic environment, and with the use of pelleted food from different manufacturers. this research produced additional analyzes that determine the physico-chemical properties and also a microbiological analysis of the water in which the rainbow trout grows, the chemical composition of the food used for its feeding, as well as, the energy value of fish meat from these two aquaculture facilities (fishpond a and fishpond b). the results obtained during the examination of the chemical composition of the rainbow trout meat from the fishpond a determined the mean value of 71.700% water, 20.366% proteins, 5.633% fats and 1.21% ash, while the ones from the fishpond b determined the mean value of 74.533% water, 20.600% proteins, 3.366% fats and 1.38% ash. the energy value of the rainbow trout meat from the fishpond a was 568.941kj/100 g, while the one from the fishpond b was 484.635 kj/100 g. overall, by a comparative analysis of the chemical composition of the rainbow trout from both aquaculture facilities, as well as energy value of fish meat, the ambient conditions prevailing in the aquatic environment, and the forage mixtures used for feeding fish, it can be concluded that both aquaculture facilities are producing high nutritional quality fish. keywords: rainbow trout (oncorhynchus mykiss walbaum, 1792), chemical composition 1. introduction the rainbow trout (oncorhynchus mykiss walbaum, 1792) is one of the widely distributed species of fresh water in europe [1]. according to [2] [3] it is one of the most appreciable fish in human nutrition thanks to its low fat content, proteins of high biological value and relatively low cholesterol content as well as valuable quantities of essential fatty acids. researchers [4][5] considered that the most important parameter which influences fish growth, feed conversion and chemical composition of fish meat is proper nutrition by offering suitable quantities of high-quality feed, especially in intensive fish farming. [6] noticed that in spite of this constatation, salmonid fish species, where the rainbow trout belongs, are the most demanding ones, because nutrition may affect, not only the ratio of proteins, fat and water, but the nutritional value of meat as well, and the fatty acid composition. however, besides that, [7] [8] concluded that the fish nutritional quality might be influenced by genetic factors, ph, oxygen content, water quality and its temperature, type of feed and feeding technology. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 228 the main purpose of the research was to make an analysis of the influence of ambient conditions and nutrition on the chemical composition of the rainbow trout (oncorhynchus mykiss walbaum, 1792) from various aquaculture facilities in macedonia at different ambient conditions in the aquatic environment, and with the use of pelleted food from different manufacturers. the main purpose of the research produced additional analyzes that determine the physical chemical properties and also a microbiological analysis of the water in which the rainbow trout resides, the chemical composition of food used for feeding of the rainbow trout, as well as, the energy value of fish meat from these two aquaculture facilities (fishpond a and fishpond b). 2. matherials and methods examinations were performed on the rainbow trout (oncorhynchus mykiss walbaum, 1792) with consumption size of 250-300g from two aquaculture facilities coldwater fishponds (fishpond a and fishpond b). the fishpond a is located in the demir hisar region (macedonia) and it is supplied with water from the river crna, the spring zheleznec. in this fishpond pelleted food aller silver (6 mm) from manufacturer aller aqua (poland) is used, with following content (per specification): fish meal, wheat, fish oil, soy, hemoglobin powder, hydrolyzed protein, protein concentrate from sunflower and oil rape. besides the main components, the food contains the following components (per specification): phosphate (1%), calcium (0.8%), sodium (0.2%), vitamin a (10,000 ie/kg), vitamin d3 (1000 ie/kg), antioxidant e 324 ethoxyquin (100 mg/kg), trace elements e2 iodine (3 mg/kg), e4 copper (5 mg/kg), e5 manganese (12 mg/kg), e6 zinc (70 mg/kg), and pigments astaxanthin e161j (50 mg/kg) and canthaxanthin e161g (25 mg/kg). the average mass of rainbow trout samples from fishpond a was 285 g, while the average length, 29 cm. the fishpond b is located near the city of bitola (macedonia) and it is supplied with water from the accumulation strezhevo. in this fishpond pelleted food troco prime 18 (4.5 mm) from manufacturer coppens international (netherlands) is used, with following content (per specification): fish meal, wheat, fish oil, soy, wheat gluten, hemoglobin powder and oil rape. besides the main components, the food contains the following components (per specification): phosphorus (0.96%), calcium (1.6%), sodium (0.3%), vitamin a (10.000 ie/kg), vitamin c (150 mg/kg), vitamin e (200 mg/kg), vitamin d3 (799 ie/kg), antioxidants e 324 ethoxyquin (100 mg/kg) and e321 butilied hidroksitulen (40 mg/kg), as well as trace elements of e1 iron (75 mg/kg), e2 iodine (5 mg/kg), e4 copper (5 mg/kg), e5 manganese (20 mg/kg) and e6 zinc (80 mg/kg). the average mass of the rainbow trout samples from fishpond b was 267 g, while the average length, 27.2 cm. during examinations, the following methods for determination of the chemical composition of fish meat were used: determination of moisture content iso 712:2009; spectrophotometric determination of total nitrogen according kjeldahl hach dr 400 procedure method 2410; determination of the total fat by gravimetric method (soxhlet extraction) аоаc method 2003.6; determination of ash in an oven at 700°c iso 3593:1981; the methods for physical – chemical properties of the water which are used in the fishponds are: ph determination iso 1052:1994; chloride determination iso 9297:1989; food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 229 spectrophotometric determination of nitrates hach dr 400 procedure method 8039; spectrophotometric determination of iron hach dr 400 procedure method 8365; spectrophotometric determination of nitrites hach dr 400 procedure method 8507; turbidity determination of translucency iso 7027:1999; spectrophotometric determination of ammonia hach dr 400 procedure method 8038; determination of chemical oxygen demand merck method spectroquant 1.18752.0001; total nitrogen determination merck method spectroquant 1.14537.0001. methods for microbiological analyses of water that are used: detection and enumeration of coliform bacteria and escherichia coli iso 9308 1:2000; detection and enumeration of intestinal enterococci and streptococcus faecalis iso 7899 2:2000. 3. results and discussion considering the results of the physical – chemical and microbiological analysis of the water in fishpond a and fishpond b, we`ve obtained the following results (tab. 1 and tab. 2): table 1. physical – chemical properties of water in fishpond a and fishpond b parameters fishpond a fishpond b represent of oxygen saturation 76 % 78 % 5-day biochemical consumption of o2 at 20 °c 1.25 mg/l 1.30 mg/l chemical oxygen demand 4.95 mg/l 3.00 mg/l dry residue of filtered water 36.0 mg/l 39.0 mg/l ph 8.3 7,15 visible waste no no visible color no no noticeable odor no no fe 0.00 mg/l 0.030 mg/l nitrites 0.0015 mg/l 0.0960 mg/l nitrates 0.00 mg/l 0.00 mg/l ammonia 0.00 mg/l 0.160 mg/l turbidity 1.0 ntu 1.0 ntu chlorides 6.40 mg/l 6.80 mg/l total phosphorous 0.0048 mg/l 0.0070 mg/l total nitrogen 0.320 mg/l 0.300 mg/l the water quality is a combination of chemical, physical and biological parameters affecting the growth and development of farmed fish. the success of the activities of the commercial aquaculture is dependent on the optimal environmental conditions for the accelerated fish growth and the development at the lowest cost of resources used. the water quality affects the general fish condition and determines the conditions for the growth and health of the farmed fish. as a result, water quality food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 230 is a major factor to be taken into account when planning aquaculture production. based on the physical chemical and microbiological properties of the water from these two aquaculture facilities,the water is classified into class ii (according to the regulation on water classification official journal of rm 18/99), which is allowed for fish production. table 2. microbiological properties of water in fishpond a and fishpond b parameters fishpond a fishpond b the probable number of thermo-tolerant coliform bacteria in 100 ml 10 30 streptococcus of faecal origin in 100 ml 0 0 considering the results of the chemical analysis of pelleted food used in fishpond a (aller aqua, poland) and fishpond b (coppens international, netherlands), we`ve obtained the following results (tab. 3): table 3. chemical composition of pelleted food used in fishpond a and fishpond b parameters percent (%) fishpond a aller aqua (poland) aller silver (6 mm) fishpond b coppens international (netherlands) troco prime 18 (4.5 mm) proteins 41 42 fats 24 18 carbohydrates 18 16 ash 7 6.2 by comparing the content of the basic chemical components in the food that is used for rainbow trout feeding, it can be established that the food is characterized by similar composition in both fishponds, with deviation only in the amount of fat which in the food from fishpond a (24%) is greater than in the one from fishpond b (18%). considering the results of the chemical composition and energy value of the rainbow trout meat from fishpond a and fishpond b, we`ve obtained the following results (tab. 4): table 4. comparative indicators of the chemical composition (%) and energy value (кј/100 g) of the rainbow trout meat from fishpond a and fishpond b parameters water proteins fats ash energy value fishpond a 71.700а ± 1.003 20.366 ± 1.268 5.633b ± 0.555 1.210c ± 0.157 568.941d fishpond b 74.533а ± 0.573 20.600 ± 0.571 3.366b ± 0.880 1.380c ± 0.120 484.635d *the differences in the values with the same superscripts are statistically significant on level р<0,001 , the result are x ± sd (mean value ± standard deviation) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 231 the results obtained during the examination of the chemical composition of the rainbow trout meat from the fishpond a show the mean value of 71.700 % water, 20.366 % proteins, 5.633 % fats and 1.21% ash. on the other hand, the results of the rainbow trout meat from fishpond b are as following: 74.533 % water, 20.600 % proteins, 3.366 % fats and 1.380 % ash. in our tests, the differences in the values of certain chemical parameters and the energy value of rainbow trout meat from fishpond a and fishpond b are statistically significant on level (p<0.001) (tab. 4). the rainbow trout meat from the fishpond a contains significantly less water (71.700%) compared with the one from fishpond b (74.533%). the differences in proteins content in the rainbow trout from fishpond a (20.366%) and fishpond b (20.600%) is quite minimal, and were not statistically significant. fat content in the rainbow trout meat from the fishpond a (5.633%) is greater compared with the one from fishpond b (3.366%). the average amount of ash is slightly higher in rainbow trout from fishpond b (1.380%) compared with the one in fishpond a (1.210%). higher energy value is established in the rainbow trout meat from fishpond a (568.941 kj/100 gr) compared to the meat from fishpond b (484.635 kj/100 gr). our results obtained during the examination of the chemical composition of rainbow trout meat correlated with the findings of [9] (water 71.65%, proteins 19.6%, fats 4.43%, ash 1.36%), [10] (water 74.18%, proteins 20.33%, fats 4.1%, ash 1.22%), [11] (water 75%, proteins 20%, fats 3.8%, ash 1.2%), [12] (water 74.77%, proteins 20.78%, fats 3.18%, ash 1.27%) and [13] (water 73.03%, proteins 20.39%, fats 4.73%, 1.37% ash). proteins content determined in our results is higher compared with the results cited in [14] (17.13%), [15] (16.9%) and [16] (17.34 17.81%), and lower than the ones in [17] (22.96%). in terms of fat content, our results show higher values than the ones presented by [18] (2.17%) and [16] (1.28 2.28%), and lower, compared with the results in [19] (7.02-8.27%), [14] (9.07%), [15] (10.1%) and [20] (11.6%). the variations in the chemical composition of the fish meat depend on many factors, but primarily on the diet, the ambient temperature, the geographical location, the gender, the fish age, the physical chemical parameters of the water, the rearing conditions, the physical activity, etc. it is established that the fat content in fish meat is directly related to the nature and fat content in the food, while the water content of fish meat is inversely proportional to the fat content [21]. this statement correlates with our findings that the higher fat content (24%) in the food consumed by fish from the fishpond a leads to higher fat content in fish meat (5.633%), compared to fat content (18%) in the food used in fishpond b which results in lower fat content in fish meat (3.366%). the results obtained for the energy value of fish meat indicate that the energy of the rainbow trout meat from fishpond a (568.941 kj/100 g) is higher compared to the one from fishpond b (484.635 kj/100 g). these results are directly related with the fat content in fish (5.633% fat in the fish meat from fishpond a and 3.366% fat in the fish meat from fishpond b). higher production results obtained in fishpond a compared with the ones from fishpond b arise as a result of the higher fat content and higher energy value of the relevant diet used in fishpond a. therefore, the higher fat content in fish from the fishpond a compared with the one from the fishpond b stems does not depend only on the difference in the type of nutrients and their participation in the diet, but of the season, gender and water temperature. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 232 due to the presence of essential fatty acids in fish fat, the higher fat content can be assessed as an important parameter for the fish quality and should not be considered as a negative impact on nutrition. overall, by a comparative analysis of the chemical composition of the rainbow trout from the both aquaculture facilities, as well as energy value of fish meat, the ambient conditions prevailing in the aquatic environment, and the used forage mixtures for feeding fish, it can be concluded that the both aquaculture facilities are producing fish with high nutritional quality. 4. conclusion the results obtained during the examination of the chemical composition of the rainbow trout meat from the fishpond a determined the mean value of 71.700% water, 20.366% protein, 5.633% fat and 1.21% ash, while the ones from the fishpond b determined the mean value of 74.533% water, 20.600% protein, 3.366% fat and 1.38% ash in fish meat. the energy value of the rainbow trout meat from the fishpond a was of 568.941kj/100 g, while the one from the fishpond b was of 484.635 kj/100 g. the differences in the values of certain chemical parameters and the energy value of rainbow trout meat from fishpond a and fishpond b are statistically significant on level (p<0.001). overall, by a comparative analysis of the chemical composition of the rainbow trout from the both aquaculture facilities, as well as energy value of fish meat, the ambient conditions prevailing in the aquatic environment, and the forage mixtures used for feeding fish, it can be concluded that both aquaculture facilities are producing high nutritional quality fish. fish breeding in quality water with well balanced food and proper implementation of health care, will give better results compared with the ones in conditions of insufficient and poor quality food, and too dense plantation. 5. references [1] fao, the state of world fisheries and aquaculture opportunities and challenges. isbn 978-92-5-108275-1, (2014). [2] conor w.e., importance of n-3 fatty acids in health and disease. american journal of clinical nutrition, 71, 171-175, (2000). [3] sidhu k.s., health benefits and potential risks related to consumption of fish or fish oil. regulatory toxicology and pharmacology, 38, 336–344, (2003). [4] valente l.m.p., bandarra n. m., figueiredo-silva a.c., rema p., vazpires p., martins s., prates j.a.m., nunes m.l., conjugated linoleic acid in diets for large-size rainbow trout (oncorhynchus mykiss): effects on growth, chemical composition and sensory attributes. british journal of nutrition, 97, 289–297, (2007). [5] almeida i., martins h.m., santos s., freitas s.g., bernardo f., mycobiota in feed for farmed sea bass (dicentrarchus labrax). biotechnology in animal husbandry, 27, 93-100, (2011). [6] caballero m.j., obach a., rosenlund g., montero d., gisvold m., izquierdo m.s., impact of different dietary lipid sources on growth, lipid digestibility, tissue fatty acid composition and histology of rainbow trout, oncorhynchus mykiss. aquaculture, 214, 253-271, (2002). [7] skalli a., robin j.h., le bayon n., le delliou h., person-le ruyet j., impact of essential fatty acid deficiency and temperature on tissues’ fatty acid composition of european sea bass (dicentrarchus labrax). aquaculture, 255, 223– 232, (2006). [8] dube p.n., hosetti b.b., behaviour surveillance and oxygen consumption in the freshwater fish labeo rohita (hamilton) exposed to sodium cyanide. biotechnology in animal husbandry, 26, 91-103, (2010). [9] çelik m., diler a., küçükgülmez a., a comparison of the proximate compositions and fatty acid profiles of zander (sander lucioperca) from two different regions and climatic conditions. food chem., 92: 637–641, (2005). [10] korkmaz a.ş., kirkagac m., fillet yield, body composition and energy content of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 biljana sivakova, dijana blazhekovikj dimovska, the impact of different diet and environmental conditions on chemical composition of rainbowtrout (oncorhynchus mykiss walbaum,1792) from macedonian aquaculture facilities, food and environment safety, volume xv, issue 3 – 2016, pag. 227 – 233 233 rainbow trout (oncorhynchus mykiss) reared in sea net-cages and fresh water concrete ponds, ankara üniversitesi ziraat fakültesi tarım bilimleri dergisi, 14 (4) 409-413, (2008). [11] grujić r., nauka o ishrani čoveka, izdavač tehnološki fakultet, atlantik, banja luka, (2000). [12] coroian c.o., coroian a., răducu c.m., atodiresei a.c., cocan d.i., mireşan v., influence of various fat levels on meat quality in rainbow trout (oncorhynchus mykiss) and brook trout (salelinus fontinalis). aacl bioflux, volume 8, issue 6, (2015). [13] stevanovski v., uticaj hraniva sa razlićitim proteinskim sadržajem na krmnu sliku kalifornijske pastrve na ribnjaku, "vrutok". magisterski rad. poljoprivredni fakultet, sveućilište u zagrebu, (1981). [14] savić n., mikavica d., grujić r., bojanić v., vučić g., mandić s., đurica r., hemijski sastav mesa dužičaste pastrmke (oncorhynchus mykiss wal.) iz ribnjaka gornji ribnik, tehnologija mesa, 45, 1–2, 45–49, (2004). [15] rasmussen r. s., ostenfeld t. h., effect of growth rate on quality traits and feed utilisation of rainbow trout (oncorhynchus mykiss) and brook trout (salvelinus fontinalis). aquaculture, 184 (3), 327-337, (2000). [16] vranić d., trbović d., đinović j., mažić z., spirić d., milićević d., spirić a., nutritivna vrednost kalifornijske pastrmke (oncorhynchus mykiss) i šarana (cyprinus carpio) iz akvakulture. tehnologija mesa 51 2, 159–168, (2010). [17] tokur b., çakli ş., polat a., the quality changes of trout (oncorhynchus mykiss w., 1792) with a vegetable topping during frozen storage (-18°c). e.u. journal of fisheries and aquatic sciences, 23:345–350, (2006). [18] hadzinikolova l., hubenova t., chemical composition and energy value of meat of rainbow trout (oncorhynchus mykiss w.) from dospat dam lake free aquatory. bulg. j. agric. sci., supplement 1, 21: 186–189, (2015). [19] plavša n., baltić m., sinovec z., jovanović b., kulišić b., petrović j., uticaj ishrane obrocima različitog sastava na kvalitet mesa kalifornijske pastrmke (oncorhynchus mykiss walbaum). savremeno ribarstvo jugoslavije – monografija, radovi saopšteni na iv jugoslovenskom simpozijumu „ribarstvo jugoslavije“ – vršac, beograd, (2000). [20] timberg l., kuldjärv r., koppel k., paalme t., rainbow trout composition and fatty acid content in estonia. agronomy research 9 (special issue ii), 495–500, (2011). [21] kaushik s. j., nutrient requirements, supply and utilization in the contest of carp culture. aquaculture, 129, 225–241, (1995). 252 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 252 262 study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire patricia d. oulai1, *lessoy t. zoue1, athanase k. otchoumou1, sébastien l. niamke1 1laboratoire de biotechnologies, ufr biosciences, université félix houphouët-boigny, 22 bp 582, abidjan 22, côte d’ivoire. y.lessoy@yahoo.fr *corresponding author received september 14th 2014, accepted september 29th 2014 abstract: african leafy vegetables (alvs), also known as african spinach, contribute significantly to household food security and add variety to cereal-based staple diets. five leafy vegetable species (amaranthus hybridus, andasonia digitata, ceiba patendra, hibiscus sabdariffa and vigna unguiculata) that are used for sauce preparation in northern côte d’ivoire were subjected to roasting in order to evaluate the impact of this non conventional processing method on their nutritive value and antioxidant properties. this study showed that longer time (higher than 2 min) of roasting at 180200°c caused negative impact with nutrients losses but positive impact by reducing anti-nutrients such as oxalates and phytates. the registered losses at 2 min were as follow: ash (0.09 – 6.58 %), proteins (1.22 – 29.31 %), vitamin c (77.56 – 89.01 %), carotenoids (11.24 – 45.16%) oxalates (2.30 – 20.51 %) and phytates (5.20 – 63.82 %). roasting processing of the studied leafy vegetables highlighted a significant increase (4.77 – 32.71 %) of polyphenols contents coupled with increasing of antioxidant activity. moreover, after 2 min of roasting time, the residual contents of minerals were: calcium (294.78 879.74 mg/100g), magnesium (175.87 480.54 mg/100g), potassium (159.72 371.33 mg/100g), iron (15.54 84.97 mg/100g) and zinc (13.27 38.85 mg/100g). all these results suggest that roasting of leafy vegetables (less than 2 min) may be used as alternative method of cooking in order to minimize nutrients losses and to contribute efficiently to the food security of ivorian population. keywords: antioxidant properties roasting processing leafy vegetables nutritive value 1. introduction leafy vegetables are plants which leafy parts (young stems, flowers and young fruits) are used as vegetables. these plants have a unique place among vegetables because they are rich sources of many nutrients and antioxidant compounds [1,2]. socio-economic surveys conducted in various parts of africa indicate that alvs are important commodities in household food and nutrition security [3]. indeed, african leafy vegetables (alvs) are the cheapest and most readily available sources of micronutrients and they provide also important sources of employment in peri urban areas because of their short labour-intensive production system [4,5]. leafy vegetables are obtained by harvesting or cultivation and their high moisture render them perishable and seasonal availability limits their utilization all round the year. hence, there is a need to preserve this nature's store house of nutrients through proper processing techniques for safe storage with efficient nutrient retention [6]. to extend the period during which they are available, different ways of preserving food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 253 these vegetables have been developed. the two main methods are the sun-drying of fresh leaves and the sun-drying of blanched or cooked leaves. both of these methods transform the leafy vegetables into dry products that have long shelf lives [7]. electrification of the rural areas has introduced new preservation technology, including the freezing of leafy vegetables [8]. among the twenty hundred and seven (207) leafy vegetables widely consumed in tropical africa, about twenty (20) species of leafy vegetables belong to 6 botanical families, are widely consumed and cultivated by ivorian population [9,10]. furthermore, the consumption of these leafy vegetables is linked to the region and ethno-botanical studies have stated that most people in northern côte d’ivoire consume indigenous green leafy vegetables such as amaranthus hybridus “boronbrou”, andasonia digitata “baobab”, ceiba patendra “fromager”, hibiscus sabdariffa “dah” and vigna unguiculata “haricot” [10,11]. earlier reports have highlighted the nutritive potential of these fresh leafy vegetables [12]. for these species, the tender leaves are prepared as potherbs or as relishes, primarily to accompany starchy paste foods as cassava, maize and sorghum. these leafy vegetables may be prepared from a single species or from a combination of them. for cooking, the mature and freshly leaves are boiled in water for about 30 min in order to reduce bitter taste and then used, after discarding boiled water, for sauce preparation. in a lesser extent, blanching is also used to inactivate oxidative enzymes, destroy vegetative microbial cells, reduce or eliminate the bitterness and to remove any residual pesticides [13,14]. even if some adverse effects such as nutrient losses have been reported [15,16] by using boiling or blanching processing, there is any scientific data with regards to the effect of roasting (oven-cooking) processing on the physicochemical and nutritive characteristics of leafy vegetables consumed in northern côte d’ivoire. therefore, the aim of this study is to evaluate the effect of roasting on the nutritive value of these selected leafy vegetables in order to provide necessary information for their wider utilization and contribution to food security of ivorian population. 2. material and methods 2.1. samples collection leafy vegetables (amaranthus hybridus, andasonia digitata, ceiba patendra, hibiscus sabdariffa and vigna unguiculata) were collected fresh and at maturity from cultivated farmlands located at dabou (latitude: 5°19′14″ north; longitude: 4°22′59″west) (abidjan district). the samples were harvested at the early stage (between one and two weeks of the appearance of the leaves). these plants were previously authenticated by the national floristic center (university felix houphouët-boigny, abidjan-côte d’ivoire). 2.2. samples processing the fresh leafy vegetables were rinsed with deionized water and the edible portions were separated from the inedible portion. the edible portions were chopped into small pieces (500 g) and allowed to drain at ambient temperature. each sample was divided into two lots. the first lot (raw, 250 g) was dried in an oven (memmert, germany) [17], ground with a laboratory crusher (culatti, france) equipped with a 10 μm mesh sieve. the powdered samples were stored in a clean dry air-tight sample bottle in a refrigerator (4°c) until further analyses. the second lot (250 g) was roasted (oven-cooking) for 2, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 254 4 and 6 min at 180-200°c. the roasted samples were cooled at ambient temperature and subjected to the same treatment using for raw samples. 2.3. chemicals all solvents (n-hexane, petroleum ether, acetone, ethanol and methanol) were purchased from merck. standards used (gallic acid, β-carotene) and reagents (metaphosphoric acid, folin-ciocalteu, dpph) were purchased from sigmaaldrich. all chemicals used in the study were of analytical grade. 2.4. nutritive properties 2.4.1. proximate analysis proximate analysis was performed using official methods [18]. the moisture content was determined by the difference of weight before and after drying the sample (10 g) in an oven (memmert, germany) at 105°c until constant weight. ash fraction was determined by the incineration of dried sample (5 g) in a muffle furnace (pyrolabo, france) at 550°c for 12 h. the percentage residue weight was expressed as ash content. for crude fibres, 2 g of sample were weighed into separate 500 ml round bottom flasks and 100 ml of 0.25 m sulphuric acid solution was added. the mixture obtained was boiled under reflux for 30 min. thereafter, 100 ml of 0.3 m sodium hydroxide solution was added and the mixture were boiled again under reflux for 30 min and filtered through whatman paper. the insoluble residue was then incinerated, and weighed for the determination of crude fibres content. proteins were determined through the kjeldhal method and the lipid content was determined by soxhlet extraction using hexane as solvent. carbohydrates and calorific value were calculated using the following formulas [19]: carbohydrates: 100 – (% moisture + % proteins + % lipids + % ash + % fibres). calorific value: (% proteins x 2.44) + (% carbohydrates x 3.57) + (% lipids x 8.37). the results of ash, fibres, proteins, lipids and carbohydrates contents were expressed on dry matter basis. 2.4.2. mineral analysis minerals contents were determined by the icp-ms (inductively coupled argon plasma mass spectrometer) method [20]. the dried powdered samples (5 g) were burned to ashes in a muffle furnace (pyrolabo, france). the ashes obtained were dissolved in 10 ml of hcl/hno3 and transferred into 100 ml flasks and the volume was made up using deionized water. the mineral composition of each sample was determined using an agilent 7500c argon plasma mass spectrometer. calibrations were performed using external standards prepared from a 1000 ppm single stock solution made up with 2% nitric acid. 2.4.3. anti-nutritional factors oxalates content was performed using a titration method [21]. one (1) g of dried powdered sample was weighed into 100 ml conical flask. a quantity of 75 ml of sulphuric acid (3 m) was added and stirred for 1 h with a magnetic stirrer. the mixture was filtered and 25 ml of the filtrate was titrated while hot against kmno4 solution (0.05 m) to the end point. phytates contents were determined using the wade’s reagent colorimetric method [22]. a quantity (1 g) of dried powdered sample was mixed with 20 ml of hydrochloric acid (0.65 n) and stirred for 12 h with a magnetic. the mixture was centrifuged at 12000 rpm for 40 min. an aliquot (0.5 ml) of supernatant was added with 3 ml of wade’s reagent. the reaction mixture was incubated for 15 min and absorbance was measured at 490 nm by using a food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 255 spectrophotometer (pg instruments, england). phytates content was estimated using a calibration curve of sodium phytate (10 mg/ml) as standard/ 2.5. antioxidant properties 2.5.1 vitamin c and carotenoids determination vitamin c contained in analyzed samples was determined by titration [23]. about 10 g of ground fresh leaves were soaked for 10 min in 40 ml metaphosphoric acidacetic acid (2%, w/v). the mixture was centrifuged at 3000 rpm for 20 min and the supernatant obtained was diluted and adjusted with 50 ml of bi-distilled water. ten (10) ml of this mixture was titrated to the end point with dichlorophenolindophenol (dcpip) 0.5 g/l. carotenoids were extracted and quantified by using a spectrophotometric method [24]. two (2) g of ground fresh leaves were mixed three times with 50 ml of acetone until loss of pigmentation. the mixture obtained was filtered and total carotenoids were extracted with 100 ml of petroleum ether. absorbance of extracted fraction was then read at 450 nm by using a spectrophotometer (pg instruments, england). total carotenoids content was subsequently estimated using a calibration curve of β-carotene (1 mg/ml) as standard. 2.5.2. polyphenols determination polyphenols were extracted and determined using folin–ciocalteu’s reagent [25]. a quantity (1 g) of dried powdered sample was soaked in 10 ml of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min. an aliquot (1 ml) of supernatant was oxidized with 1 ml of folin–ciocalteu’s reagent and neutralized by 1 ml of 20% (w/v) sodium carbonate. the reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm by using a spectrophotometer (pg instruments, england). the polyphenols content was obtained using a calibration curve of gallic acid (1 mg/ml) as standard. antioxidant activity antioxidant activity assay was carried out using the 2,2-diphenyl-1-pycrilhydrazyl (dpph) spectrophotometric method [26]. about 1 ml of 0.3 mm dpph solution in ethanol was added to 2.5 ml of sample solution (1 g of dried powdered sample mixed in 10 ml of methanol and filtered through whatman no. 4 filter paper) and was allowed to react for 30 min at room temperature. absorbance values were measured with a spectrophotometer (pg instruments, england) set at 415 nm. the average absorbance values were converted to percentage antioxidant activity using the following formula: antioxidant activity (%) = 100 – [(abs of sample – abs of blank) x 100/abs positive control] statistical analysis all the analyses were performed in triplicate and data were analyzed using excell and statistica 7.1 (statsoft). differences between means were evaluated by duncan’s test. statistical significant difference was stated at p < 0.05 3. results and discussion the proximate composition of the roasted leafy vegetables is presented in table 1. the physicochemical parameters generally differ significantly (p<0.05) from a roasting time of a leafy vegetable to another. the ash content after 2 min of roasting ranged from 8.19 ± 0.00% (a. hybridus) to 23.90 ± 0.24% (c. patendra). these values were closed to 7.66 ± 0.18% food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 256 and 21.69 ± 1.56% after 6 min of roasting. the observed decrease rate at 2 min of roasting ranged from 0.09 to 6.58 % in the following order: v. unguiculata (0.09%) > a. digitata (1.55%) > a. hybridus (4.65%) > h. sabdariffa (5.43%) > c. patendra (6.58%). these losses are lower than that (9.78 – 28%) reported for boiled nigerian leafy vegetables [27]. therefore, roasting processing could be advantageous for mineral quality preservation of leafy vegetables. as concern protein contents, roasting processing caused 1.22 to 29.31% reduction after 2 min. these proteins losses increased in the order: v. unguiculata (1.22%) > a. hybridus (3.16%) > h. sabdariffa (10.29%) > c. patendra (12.56%) > a. digitata (29.31%). this reduced protein contents could be attributed to the severity of thermal process (180-200°c) which leads to protein degradation [28]. however, this thermal processing could enhance the digestibility of proteins by degradation of anti-nutritional factors such as tannins [29]. with regards to their protein contents (12.78 – 21.69%) at 2 min, roasted leaves of the selected species could be considered as non negligible sources of proteins for human nutrition. roasting of the studied leafy vegetables resulted in a slight increase (p > 0.05) in their crude fibres content (0.9 – 4.27%) after 2 min of heat application. table 1. proximate composition of raw and roasted leafy vegetables consumed in northern côte d’ivoire ash (%) fibres (%) proteins (%) lipids (%) carbohydrates (%) calorific value (kcal /100g) h. sabdariffa raw 10.30 ± 0.10a 14.27 ± 1.70b 14.47 ± 0.10a 4.75 ± 0.15a 56.21 ± 1.78b 275.71 ± 0.55a 2 min 9.74 ± 0.09b 14.40 ± 1.99b 12.98 ± 0.01b 4.87 ± 0.01a 59.73 ± 6.88a 271.28 ± 4.68a 4 min 9.60 ± 0.23b 14.67 ± 1.56b 12.36 ± 0.00b 4.92 ± 0.03a 59.11 ± 4.30a 276.85 ± 5.60a 6 min 9.26 ± 0.64b 16.00 ± 1.24a 12.15 ± 0.00b 4.95 ± 0.01a 54.64 ± 2.41b 266.13 ± 8.49b a. hybridus raw 8.59 ± 1.34a 17.80 ± 0.30a 13.25 ± 0.13a 2.15 ± 0.01b 58.21 ± 1.78a 305.19 ± 7.73a 2 min 8.19 ± 0.00a 17.19 ± 2.21a 12.83 ± 0.04b 2.48 ± 0.03b 60.21 ± 2.26a 259.46 ± 7.85c 4 min 8.03 ± 1.43a 17.28 ± 1.24a 12.24 ± 0.00b 2.54 ± 0.06b 59.91 ± 3.87a 265.02 ± 3.34c 6 min 7.66 ± 0.18b 17.54 ± 0.84a 11.99 ± 0.06c 3.79 ± 0.03a 59.02 ± 2.41a 271.69 ± 2.41b a. digitata raw 10.97 ± 0.40a 12.56 ± 0.45a 18.08 ± 0.10a 2.18 ± 0.03c 56.23 ± 1.25b 267.03 ± 4.00d 2 min 10.80 ± 0.12a 12.80 ± 3.49a 12.78 ± 0.40b 2.56 ± 0.01c 61.06 ± 3.77a 270.60 ± 2.37c 4 min 9.82 ± 1.39a 13.33 ± 1.15a 12.60 ± 0.00b 3.91 ± 0.03b 60.35 ± 0.28a 278.90 ± 0.75b 6 min 7.82 ± 0.22b 14.79 ± 1.51a 8.20 ± 0.28c 4.73 ± 0.04a 64.46 ± 5.96a 289.71 ± 2.34a v. unguiculata raw 11.17 ± 0.25a 18.00 ± 0.92a 21.96 ± 0.30a 4.23 ± 0.25c 44.64 ± 1.72b 248.35 ± 1.33c 2 min 11.16 ± 0.14a 18.77 ± 0.94a 21.69 ± 0.08a 5.81 ± 0.04b 42.57 ± 0.93b 253.52 ± 2.76b 4 min 11.11 ± 0.15a 19.55 ± 2.47a 20.86 ± 0.12b 6.11 ± 0.01a 42.38 ± 2.76b 253.31 ± 9.43a 6 min 10.79 ± 0.02a 19.68 ± 1.15a 17.09 ± 0.00c 6.43 ± 0.03a 46.01 ± 1.11a 259.77 ± 4.18a c. patendra raw 25.67 ± 1.12a 31.50 ± 1.50a 15.20 ± 0.05a 1.39 ± 0.22c 26.30 ± 0.11b 142.61 ± 7.74c 2 min 23.90 ± 0.24b 32.44 ± 2.28a 13.29 ± 0.41b 6.62 ± 0.03b 23.67 ± 2.09c 172.34 ± 8.69b 4 min 22.56 ± 0.00b 33.21 ± 1.70a 12.98 ± 0.14c 7.15 ± 0.03a 24.10 ± 2.88c 177.54 ± 7.94b 6 min 21.69 ± 1.56b 33.56 ± 1.09a 7.42 ± 0.00d 7.46 ± 0.05a 29.87 ± 2.70a 187.14 ± 7.48a data are represented as means ± sd (n = 3). means in the column with no common letter differ significantly (p<0.05) for each leafy vegetable. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 257 indeed, the increased temperature leads to breakage of weak bonds between polysaccharides and the cleavage of glycosidic linkages, which may result in solubilization of the dietary fibres [30]. with regard to their fibres content (12.80 32.44%) after 2 min, adequate intake of roasted leafy vegetables could lower the risk of constipation, diabetes, colon and breast cancers [31]. the relatively low values of lipids contents at 2 min of roasting (2.48 – 6.62%) in the studied leafy vegetables corroborate the findings of many authors which showed that leafy vegetables are poor sources of fat [32]. in addition, the estimated calorific values (172.34 – 271.28 kcal/100g) agree with general observation that leafy vegetables have low energy values due to their low crude fat and relatively high level of moisture [33]. mineral composition of roasted leafy vegetables used in this study is shown in table 2. the residual contents of minerals after 2 min of roasting were significantly different (p < 0.05): calcium (294.78 879.74 mg/100g), magnesium (175.87 480.54 mg/100g), potassium (159.72 371.33 mg/100g), iron (15.54 84.97 mg/100g) and zinc (13.27 38.85 mg/100g). compared to the values of raw leafy vegetables, these observed reductions may be due to the losses of ashes as observed previously. with regards to these values, the consumption of roasted leafy vegetables (2 min) could cover at least 50% of the standard mineral requirements for human. table 2. mineral composition of raw and roasted leafy vegetables consumed in northern côte d’ivoire ca mg p k fe na zn h. sabdariffa raw 402.21±0.55a 295.93±0.41a 407.59±0.00a 816.19±1.12a 102.08±0.14a 23.46±0.03a 26.06±0.04a 2 min 371.84±2.43b 241.61±1.80b 371.33±5.16b 711.42±3.00b 56.18±3.41b 22.82±0.91a 17.32±9.38b 4 min 368.74±8.90c 111.98±1.53c 232.49±7.70c 629.75±7.23c 35.62±0.35c 21.30±3.81a 12.25±0.31c 6 min 257.32±3.51d 111.85±3.70c 208.79±2.85d 583.76±9.40d 32.43±0.74c 20.05±1.79a 10.12±0.10d a. hybridus raw 932.6±0.55a 497.75±0.49a 368.69±0.00a 1989.32±2.12a 77.88±0.05a 94.39±0.04a 31.73±0.04a 2 min 879.74±6.44b 480.54±4.74b 366.74±4.36a 1943.57±8.65b 72.57±0.00b 94.38±0.00a 30.11±0.38b 4 min 843.65±0.00c 476.88±0.00c 356.28±0.00b 1826.41±0.00c 68.52±0.58c 94.37±1.33a 26.41±0.00c 6 min 755.01±0.00d 337.24±8.26d 274.86±4.92c 1690.04±1.71d 49.95±3.49d 90.33±5.64b 20.04±0.92d a. digitata raw 496.26±2.20a 264.36±1.17a 761.63±0.00a 1856.90±8.23a 106.27±0.47a 37.13±0.12a 22.61±0.10a 2 min 464.74±8.39b 175.87±3.74b 275.88±5.54b 1571.84±4.90b 15.54±0.22b 37.00±2.46a 20.68±0.44b 4 min 439.84±4.80c 157.7±2.23c 269.01±3.80c 1496.44±2.14c 15.15±0.32b 35.97±1.36b 12.06±0.14c 6 min 407.66±5.88d 147.35±2.06d 249.03±5.29d 1370.45±6.98d 15.05±2.87b 32.17±1.11c 11.97±0.83c v. unguiculata raw 439.54±0.56a 341.34±0.18a 309.04±0.00a 718.11±0.91a 91.45±0.12a 33.32±0.02a 40.83±0.04a 2 min 426.94±0.54b 315.39±0.25b 300.02±4.45b 678.50±2.03b 84.97±3.74b 31.75±0.17b 38.85±0.43b 4 min 424.92±5.57c 298.81±2.49c 297.34±0.46c 622.33±2.70c 82.38±0.33b 26.99±1.09c 31.52±0.39c 6 min 417.65±5.36d 293.19±2.46d 293.94±3.69d 549.18±1.44d 78.29±3.56c 25.59±1.07c 23.04±0.02d c. patendra raw 997.02±0.55a 773.55±0.43a 570.85±2.11a 1585.58±0.87a 219.84±0.12a 42.69±0.02a 35.68±0.02a 2 min 294.78±5.63b 224.82±4.48b 159.72±2.34b 983.00±9.71b 20.74±4.19b 27.96±0.99b 13.27±2.68b 4 min 266.36±9.42c 191.69±0.00c 105.90±0.00c 854.30±0.00c 20.70±0.60b 22.06±4.46c 9.75± 0.00c 6 min 245.09±0.00d 162.08±6.20d 83.27±3.15d 729.74±2.60d 18.11±0.00c 21.08±0.00c 7.78± 0.26d data are represented as means ± sd (n = 3). means in the column with no common letter differ significantly (p<0.05) for each leafy vegetable. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 258 indeed, these standard requirements are: calcium (1000 mg/day); magnesium (400 mg/day), iron (8 mg/day) and zinc (6 mg/day) [34]. calcium and phosphorus play important role in growth and maintenance of bones, teeth and muscles [35]. as concern magnesium, this mineral is known to prevent cardiomyopathy, muscle degeneration, growth retardation, congenital malformations and bleeding disorders [36]. iron plays important role in prevention of anemia while zinc is important for vitamin a and vitamin e metabolism [37,34]. to predict the bioavailability of calcium and iron, antinutrients to nutrients ratios of blanched leafy vegetables were calculated (table 3). the calculated [phytates]/[ca] ratio in all the roasted species were below the critical level of 2.5 which is known to impair calcium bioavailability [38]. the effect of roasting on anti-nutritional factors (oxalates and phytates) contents is depicted in figure 1. levels losses (p < 0.05) at 2 min were 2.30 – 20.51% and 5.20 – 63.82% for oxalates and phytates, respectively. oxalates losses increased in the order: a. hybridus (2.30%) > a. digitata (12.30%) > v. unguiculata (13.49%) > h. sabdariffa (18.54%) > c. patendra (20.51%) while phytates losses increased in the order: a. digitata (5.20%) > v. unguiculata (20.92%) > c. patendra (24.54%) > a. hybridus (61.48%) > h. sabdariffa (63.82%).the reductions in oxalates and phytates contents during roasting could be advantageous for improving the health status of consumers. indeed, oxalates and phytates are antinutrients which chelate divalent cations such as calcium, magnesium, zinc and iron, thereby reducing their bioavailability [39]. table 3. anti-nutritional factors/mineral ratios of raw and roasted leafy vegetables consumed in northern côte d’ivoire phytates/ca phytates/fe oxalates/ca h. sabdariffa raw 0.21 0.85 3.26 2 min 0.08 0.56 2.86 4 min 0.07 0.78 2.71 6 min 0.10 0.80 3.69 a. hybridus raw 0.03 0.41 0.07 2 min 0.01 0.17 0.07 4 min 0.01 0.16 0.06 6 min 0.01 0.18 0.04 a. digitata raw 0.04 0.19 1.57 2 min 0.04 1.21 1.47 4 min 0.03 1.09 1.25 6 min 0.03 0.90 1.11 v. unguiculata raw 0.04 0.19 1.66 2 min 0.03 0.16 1.48 4 min 0.03 0.15 1.35 6 min 0.02 0.11 1.35 c. patendra raw 0.04 0.17 0.78 2 min 0.10 1.39 2.10 4 min 0.09 1.22 1.46 6 min 0.09 1.30 1.34 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 259 roasting also resulted in a decrease of carotenoids and vitamin c contents in the studied leafy vegetables (figure 2). for carotenoids, losses at 2 min were estimated to 11.24 to 45.16%. carotenoids losses increased in the order: a. digitata (11.24%) > a. hybridus (15.28%) > h. sabdariffa (17.87%) > c. patendra (22.02%) > v. unguiculata (45.16%). the decrease in the concentration of total carotenoids could be attributed to the oxidation and isomerization of β-carotene [40]. for vitamin c content, a significant reduction (77.56 89.01%) was highlighted after 2 min during roasting processing (figure 2). figure 1: oxalates (a) and phytates (b) contents of raw and roasted leafy vegetables consumed in northern côte d’ivoire this decrease in vitamin c could be attributed to the fact that this important micronutrient is not stable at high temperature [41]. with regard to the vitamin c decrease, consumption of roasted leafy vegetables may be supplemented with other sources of vitamin c such as tropical fruits to cover the daily need for humans (40 mg/day) as recommended by food agriculture organization [34]. figure 2: vitamin c (a) and carotenoids (b) contents of raw and roasted leafy vegetables consumed in northern côte d’ivoire food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 260 the effect of roasting on polyphenols content and antioxidant activity of the selected leafy vegetables is depicted in figure 3. it was observed a high increase of polyphenols contents varying from 4.77 to 32.71%. the percent gain in the total phenol content during blanching may be due to the breakdown of tough cell walls and release of phenolic compounds trapped in the fibres of green leafy vegetables [42]. polyphenols compounds of the roasted leafy vegetables could be advantageous for lower cellular oxidative stress which has been implicated in the pathogenesis of various neurodegenerative diseases, including alzheimer’s disease, parkinson’s disease, and amyotrophic lateral sclerosis [43,44]. figure 3: polyphenols contents (a) and antioxidant activity (b) of raw and roasted leafy vegetables consumed in northern côte d’ivoire 4. conclusions leafy vegetables consumed in northern côte d’ivoire contain significant levels of nutrients that are essential for human health. the present study showed that roasting at 2, 4 and 6 minutes may increase their antioxidant activity and decrease the contents of proteins, mineral, vitamin c, carotenoids, and anti-nutritional factors (oxalates and phytates). the reduction of anti-nutritional factors might have a beneficial effect on bioavailability of minerals like calcium, iron and zinc. thus, the study suggests that the recommended time of roasting leafy vegetables must be less than 2 min in order to contribute efficiently to the nutritional requirement and to the food security of ivoirian population. 5. references [1]. gupta s., lakshmi j.a., manjunath m.n. prakash j. analysis of nutrient and antinutrient content of underutilized green leafy vegetables. lwt food sci. technol. 38. p. 339– 345. (2005). [2]. gupta s., prakash j. studies on green leafy vegetables for their antioxidant activity. plant food hum. nutr. 64. p. 39-45. (2009). [3]. mnzava n.a. vegetable crop diversification and the place of traditional species in the tropics. in proceedings of the ipgri international workshop on genetic resources of traditional vegetables in africa. guarino l. (ed.), icraf-hq, nairobi, kenya. p. 1-15. (1997). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 261 [4]. martin f.w., meitner l.s. edible leaves of the tropic, educational concerns for hunger organization. (1998). [5]. schippers r.r . african indigenous vegetables. an overview of the cultivated species. technical centre for agriculture and rural coperation. chatham . (2000). [6]. gupta s., jyothi l.a., prakash j. effect of different blanching treatments on ascorbic acid retention in green leafy vegetables. nat. prod. radiance. 7. p. 111-116. (2008). [7].vorster h.j, jansen-van-rensburg w.s., venter s.l., van-zijl j.l. recreating awareness of traditional leafy vegetables in communities. regional workshop on african leafy vegetables for improved nutrition, ipgri, nairobi, kenya. (2005). [8]. jansen van rensburg w.s., van averbeke w., slabbert r., faber m., van jaarsveld p., van heerden i., oelofse a. african leafy vegetables in south africa. 2007. water sa. 33. p. 317-326. (2007). [9]. prota. ressources végétales de l’afrique tropicale. volume 2 : légumes. grubben g.j.h. et denton o.a. (eds). fondation prota / backhuys publishers / cta, wageningen. (2004). [10]. fondio l., kouamé c., n’zi j.c., mahyao a., agbo e., djidji a.h. survey of indigenous leafy vegetable in the urban and periurban areas of côte d’ivoire. acta hort. 752 p. 287289. (2007). [11]. soro l.c., atchibri l.o., kouadio k.k., kouame c. evaluation de la composition nutritionnelle des légumes-feuilles. j. appl. biosci. 51. p. 3567– 3573. (2012). [12]. oulai p., zoué l., mégnanou r.m., doué r., niamké s. proximate composition and nutritive value of leafy vegetables consumed in northern côte d’ivoire. eur. sci. j. 10. p. 212-227. (2014). [13]. canet w. quality and stability of frozen vegetables. in s. thorne (ed.). developments in food preservation (vol. 5). new york: elsevier science publishing inc. (1989). [14]. prestamo g., fuster c., risueno m.c. effects of blanching and freezing on the structure of carrots cells and their implications for food processing. j. sci. food agric. 77 p. 223–229. (1998). [15]. murcia m.a., lopez-ayerra b., garcia-carmona f. effect of processing methods and different blanching times on broccoli: proximate composition and fatty acids. lebensmittel-wissenschaft technol. 32. p. 238–243. (1999). [16]. oboh g. effect of blanching on the antioxidant properties of some tropical green leafy vegetables. lebensmittel-wissenschaft technol. 38. p. 513–517. (2005). [17]. chinma c.e., igyor m.a. micronutriments and anti-nutritional content of select tropical vegetables grown in south-east, nigeria. nig. food j., 25. p. 111-115. (2007). [18]. aoac. official methods of analysis. association of official analytical chemists ed., washington dc. 684 p. (1990). [19]. fao. food and agriculture organization. food energy-methods of analysis and conversion factors. fao ed, rome. (2002). [20]. ceaeq. détermination des métaux. méthode par spectrométrie de masse à source ionisante au plasma d’argon. ma 200 – met 1.2, rev 4. quebec. (2013). [21]. day r.a., underwood a.l. quantitative analysis. 5th ed. prentice hall. 701 p. (1986). [22]. latta m., eskin m. a simple method for phytate determination. j. agric. food chem. 28. p. 1313-1315. (1980). [23]. pongracz g., weiser h., matzinger d. tocopherolsantioxydant. fat sci. technol. 97. p. 90-104. (1971). [24]. rodriguez-amaya d.b. a guide to carotenoids analysis in foods. ilsi press, washington dc. 64 p. (2001). [25]. singleton v.l., orthofer r., lamuela-raventos r.m. analysis of total phenols and other oxydant substrates and antioxydants by means of folin-ciocalteu reagent. methods enzymol. 299. p. 152-178. (1999). [26]. choi c.w., kim s.c., hwang s.s., choi b.k., ahn h.j., lee m.z., park s.h., kim s.k. antioxydant activity and free radical scavenging capacity between korean medicinal plant and flavonoids by assay guided comparison. plant sci. 163. p. 1161-1168. (2002). [27]. lola a. the effect of boiling on the nutrients and anti-nutrients in two non conventional vegetables. pak. j. nutr. 8. p. 14301433. (2009). [28]. lund d.b. effects of heat processing on nutrients. in: nutritional evaluation of food processing, (r. harries and e. karmas, eds). the avi publishing co. inc westport. p. 205 – 203. (1997). [29]. gibson, r.s., perlas l., hotz c. improving the bioavailability of nutrients in plant foods at the household level. proceedings of the nutrition society. 65. p. 160–168. (2006). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke, study of roasting effect on nutritive and antioxidant properties of leafy vegetables consumed in northern côte d’ivoire, food and environment safety, volume xiii, issue 3 – 2014, pag. 252 – 262 262 [30]. svanberg s.m., nyman e.m., andersson l., nilsson r. effects of boiling and storage on dietary fiber and digestible carbohydrates in various cultivars of carrots. j. sci. food agric. 73. 245–254. (1997). [31]. ishida h., suzuno h., sugiyama n., innami s., todokoro t., maekawa a. nutritional evaluation of chemical component of leaves stalks and stems of sweet potatoes (ipomea batatas). food chem. 68. p. 359-367. (2000). [32]. ejoh a.r., tchouanguep m.f., fokou e. nutrient composition of the leaves and flowers of colocasia esculenta and the fruits of solanum melongena. plant food hum. nutr. 49. p. 107-112. (1996). [33]. sobowale s.s., olatidoye o. p., olorode o.o., akinlotan j.v. nutritional potentials and chemical value of some tropical leafy vegetables consumed in south west nigeria j. sci. multidisciplinary res. 3. p. 55-65. (2011). [34]. fao. human vitamin and mineral requirements. fao ed. 361p. (2004). [35]. turan m., kordali s., zengin h., dursun a., sezen y. macro and micro-mineral content of some wild edible leaves consumed in eastern anatolia. plant soil sci. 53. p. 129-137. (2003). [36]. chaturvedi v.c., shrivastava r., upreti r.k. viral infections and trace elements: a complex trace element. curr. sci. 87. p. 15361554. (2004). [37]. trowbridge f. and martorell m. forging effective strategies to combat iron deficiency. j. nutri. 85. p. 875-880. (2002). [38]. hassan l.g., umar k.j. and umar z., antinutritive factors in tribulus terrestris (linn) leaves and predicted calcium and zinc bioavailability. j. trop. biosci. 7. p. 33-36. (2007). [39]. sandberg a.s. bioavailability of minerals in legumes. brit. j. nutr. 88. p. 281-285. (2002). [40]. speek a.j., temalilwa g.r., schrijver j. determination of β-carotene content and vitamin a activity of vegetables by hplc. food chem. 19. p. 65-74. (1986). [41]. nagy s., smooth j.m. temperature and storage effects on percent retention and percent u.s. recommended dietary allowances of vitamin c in canned single-strength orange juice. j. agric. food chem. 25. p. 135-138. (1977). [42]. oboh g., rocha j.b. polyphenols in red pepper and their protective effect on some prooxidants induced lipid peroxidation in brain and liver. eur. food res. technol. 225. p. 239-247. (2007). [43]. rice-evans c., miller n.j. antioxidants: the case for fruit and vegetables in the diet. brit. food j. 97. p. 35-40. (1995). [44]. amic d., davidovic-amic d., beslo d., trinajstic n., structure-radical scavenging activity relationship of flavonoids. croatia chem. acta. 76. p. 55-61. (2003). food and environment safety journal of faculty of food engineering, ştefancel mareuniversity suceava volume xiv, issue 2– 2015 contents: 1. modelling of thin layer drying kinetics of cocoa beans in a microwave oven and sun verdier n. abouo, clément d. akmel, ernest k. kakou, emmanuel n. assidjo, georges n. amani, benjamin k. yao 127-137 2. assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu 138-143 3. seasonal variations’ influence on chemical composition of cow milk and vidraru semi hard cheese ancuţa elena prisacaru, laura apostol 144-147 4. prebiotic effects of inulin and gum acacia (review) gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova 148-156 5. the influence of processing by crushing and boiling on ascorbic acid content in some plant species marcel avramiuc, silvia mironeasa 157-161 6. influence of temperature on the physical properties of vegetable oils mircea oroian, gheorge gutt 162-170 7. assessment of environmental impacts from municipal solid waste management iasi, romania cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu 171-180 8. study on the influence of samples’ dimensions on the analysis of smoked cheese texture properties elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache) 181-189 9. the use of texture destructive methods to assess the state of pork freshness sergiu paduret, gheorghe gutt 190-195 10. biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké 196-205 11. the use of plant materials in culinary products out of yeasted dough oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk 206-211 12. development of operational quality control method for meat products olha lyubchyk, mykola mykyjchuk, mariуa vorobets 212-217 food and environment safety journal of faculty of food engineering, ştefancel mareuniversity suceava volume xiv, issue 2– 2015 13. study on flour quality assessment designed to obtain biscuits adriana dabija, anca magda păiuş 218-222 14. strategic alliances between firms – a model of competitive strategy in food industry ciprian ionel hretcanu, cristina elena hretcanu 223 232 15. mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments silviu-gabriel stroe 233 240 16. author instructions i v 17. subscription information vi применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 4– 2016 contents: 1 activating the glutathione system of eiseniafetidaduring exposure to contaminated silt sludge nataliya mitina, nina khromykh, larisa shupranova, inna zubareva 276 283 2 heat transfer in down flowing turbulent evaporating liquid films with developed wavy structure and co-current steam flow valentynpetrenko, yaroslavzasyadko 284 – 298 3 research on technological properties of potato cellulosefor bread production tatjana silchu, mariana nazar, tatjana golikova 299 – 305 4 nanosized titanium dioxide as an antibacterial admixture for the food packaging materials igorkobasa, mariiavorobets, larysaarsenieva 306 311 5 use of emulsions in production restaurants and food industry oksana lugovska, vasilij sidor 312 322 6 3-d modeling of water flow and cooling down within the temperature range close to inversion point roman gryshchenko, yaroslav zasiadko, andriy forsiuk, oleksiy pylypenko 323 328 7 mathematical modeling of thermal processing of meat products volodymyr pavelko, serhiy hrybkov, arkadiy zaslsvsky, daria dmytrenko 329 333 8 the influence of berry puree on microbiological indicators of cheese product during storage olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko 334 340 9 the influence of proteins on the technological process of bread making with fructose vera drobot, anastasiia shevchenko, olha marchenko 341 346 author instructions i v subscription information vi применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 2– 2016 contents: 1 performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient waste water – simulation with asal 1 model aleksandar slavov 101 107 2 physico-chemical characteristics, antioxidant activity and mineral content of hawthorn fruits from suceava county silvia mironeasa, elena sănduleac todosi, mădălina iuga 108 116 3 comparative study of proximate composition of nile tilapia (oreochromisniloticus) (linnaeus, 1758) from fresh water and brackish water in niger delta region, nigeria olaniyi alaba olopade, iyabode olusola taiwo, oba sunday siyeofori, nathanael bamidele 117 122 4 evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc 123 130 5 the influence of culinary processing on the content of some secondary metabolites in flint corn seeds marcel avramiuc 131 140 6 relevance of nutritional intervention of canned milk soldiers’ daily diet natalija valeriivna ryabokon, oksana valerjanivna kochubeylytvynenko, dmitroviktorovich rindyuk 141 145 7 the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages maria poroch serițan, nicoleta – gabriela popescu 146 154 8 contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia katerina bojkovska, trajan dojchinovski, nikolche jankulovski,elena joshevska, blagojche najdovski 155 164 9 studies on the influence of quinoa flour addition on bread quality georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac 165 –174 10 study on modified sunflower husk fermentation process for vermicultivation natalija borisivna mitina, inna mihalivna zubareva,alyonadmitrievnaodina 175 179 11 total phenolic content and antioxidant activity of infusions from two byproducts: walnut (juglans regia l.) shell and onion (allium cepa l.) sonia amariei, valentina mancas 180 186 food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xv, issue 2– 2016 12 study on effects of artichoke flour addition in bread sorina ropciuc, ana leahu, cristina damian, laura carmen apostol 187 195 13 description of the influence of freezing on the quality of egg-white creams for pastries julya kambulova, iryna sokolovska 196 – 202 14 author instructions i v subscription information vi stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xii, issue 4 20 december 2013 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved microsoft word 7 gontariu precipitatii suhard_en[1].doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 47 ra in fa ll in the s uha rd m ou nta ins fr om bucov in a *ioan gontariu1, ion tănasă2, amelia buculei3 1faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania, e-mail ioang@fia.usv.ro 2meteorological station suceava, b-dul 1 decembrie 1918 nr.15, suceava, 720262, romania, e-mail ion_tanasa.meteo@yahoo.com 3faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania, e-mail ameliab@fia.usv.ro *corresponding author received 9 december 2011, accepted 5 february 2012 abstract: in the study of the rainfall from suhard mountain, we used data from meteorological stations and rainfall stations situated in the immediate vicinity, which ensured a relatively satisfactory interpretation of this parameter. the altitude difference registered in suhard, 1262 m (between the 670m, at the confluence of maria river with someşul mare and 1932 m of omu peak), generates a remarkable spatial variation of rainfall. as a true reflection of the local geographical conditions, the rainfall quantities graphs don’t describe anymore a proportional linear increase with the altitude, but mildly resembling a parable. the type of precipitations which characterizes especially the cold season increases in days proportionally with the altitude. the consequence, besides a large amount of solid water, is also noticeable in the termic regime. keywords: rainfall, variation, climatic hazards, humidity excess, rainfall, deviation, frequency. 1. introduction suhard’s role of climatic barrier complicates rainfall regime and distribution [1]. the orientation of the main peak, almost perpendicular to the direction of movement of air masses, the increase in altitude in north-west determines the reactivation of cloud systems and larger quantities of rainfall in this direction, which is totally opposite of bistriţa’s lane, situated in the “underwind” area, or dornelor’s depression, where the phenomenon of foehn takes place, which destroys the descending cloud systems from the eastern peaks of bârgău’s mountain [2]. a study on rainfall differences from between the eastern and western parts of eastern carpathians, realized by n. barbu and m. apăvăloaiei (1975), highlights the part of orographic obstacle played by this carpathian branch, in the western region of it being recorded larger quantities, with a difference of 200-300 mm from the eastern part. 2. materials and methods because the weather and rainfall stations are missing from the inside of the mountainous area we study, monthly and daily data about rainfall from the higher or lower areas around this one were used [3]. based on the use of gis and the corrections to real data recorded around suhard mountain, annual and semiannual maps were developed, de martonne dryness index and mountains exhibition maps, both with important roles in the local differentiations of rainfall [4]. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 48 3. results and discussion the reality on the field is proved by data registered in the neighboring rainfall and weather stations: ciocăneşti, (840 m) with 606.3 mm, poiana stampei (915 m) with 651 mm, vatra dornei (800 m) with 657 mm, cîrlibaba (930 m) with 699 mm, iezer (1735 m) with 1247 mm, rarău (1632m) with 907 mm. we mention that we didn’t take into account the data from reţitis weather station (2020 m) which, given the location and relatively short string of observations, registers only an annual average rainfall quantity of 524.6 mm (1991-2009) [7]. figure 1. map of annual average rainfall of suhard mountain from bucovina average annual rainfall amounts from areas in close proximity are located between 651 mm in poiana stampei and 1267 mm in iezer [5]. the inferior limit of the mountain, at contact with the depression, registers a smaller quantity than areas from lower altitudes, but positioned in the west, as a proof being the 650 mm from poiana stampei in comparison with the 684 mm from bistriţa’s station. nevertheless, the 700 mm value can be found in almost the entire area of suhard mountain (figure 1). in altitude, the increase is slower until 1600 m, where values can reach up to 1000 mm or even more above this height. an increase of values in north-west is added up, reaching 1250-1300 mm in omu peak [6]. even though some sources present higher values, with approximately 100 mm (rpr monography, 1960), the situation can be explained by the fact that the analysis involves another interval of time, possibly more humid. it is highlighted the patchy nature of rainfall, with quite large variations from year to year. for example, in iezer, they were from 951 mm, which is 75% from the annual average, and 1935 mm, 152% from annual average. the semiannually situation indicates that in the entire area, 60% of the total quantities of rainfall take place in the warm semester (between april and september). for lower areas, we can notice higher weights to 70%, with a slight decrease towards south-north. in altitude, the degree of continental is perfectly illustrated: at iezer just 60% (weight approaching the one of bistriţa), while in rarău the weight of 72% exceeds even the one of vatra dornei, 70%. in the cold semester, the average quantities vary from 197mm in vatra dornei, to 497 mm in iezer (table1, fig. 2). compared to the annual situation, the graph of rainfall distribution, in the cold semester, according to the altitude, describes a more pronounced parabola, showing the important part of local conditions in registering rainfall quantities. the entire area we study is delimited by the values of 200-250 mm, and at higher altitudes, of 1600 m, the increases are more noticeable, from 400 mm to over 600 mm. the continental degree was analyzed according to the martonne dryness index (table 1 fig 31), which expresses the deficit/excess of humidity, calculated by the formula ia=p/(t+10) (p is the average annual rainfall, t is the average annual temperature) and whose higher values emphasizes an humidity excess. for the studied area it is obvious the growth tendency with the altitude of the martonne index. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 49 the annual average quantities increase with the altitude, being at minimum in winter and maximum during summer. this way, from 24 mm in poiana stampei, in february and 67mm at iezer in january, in june it can be reached 104 mm in poiana stampei and 160 mm in iezer. analyzing the variation of annual average quantities of rainfall, it is noticeable the maximum from june, no matter the altitude, and the minimum from january-february. quantitatively speaking, the increase is constant starting with winter months (with small differences among them and among stations) and continues until the beginning of summer. we can talk about the summer maximum (june-july), noticeable at all stations disregarding the altitude. a b figure 2. the half-year repartition of average rainfall (cold season-a, warm season-b) at the end of summer, the quantities constantly decrease until the winter interval. besides the increase in quantities according to the altitude, the major difference between station at higher altitude and the lower ones consists in more constant values, proportionally with the altitude. so, at iezer, besides the winter minimum, the rest of the months register more than 800mm and monthly average of over 100mm 6 moths a year. in rarău, station located with only 200 m lower, but towards east, the quantities not only represent 70% from those in iezer but are there less than 50 mm and average values over 100 mm appear only in 4 months. (table 2, figure 4). at lower stations, the values are of 70mm, at poiana stampei half of the year has a minimum of 50mm and the maximum is of over 100mm. 24 hours maximum quantities are related to the continental influence and local particularities of cloud-burst rainfall and were registered valleys and depressions. here are a series of values representing national records (table 3). it’s the case of the 280.4 mm registered in cîrlibaba, on july, 16 1938, and reported in the newsletter edited by inmh, the fourth most large in the country, the third of the month of june and second in the mountain area. table 1. annual average sums of rainfall, cold semester (sr), warm semester (sc) and de martonne index station an sr sc de martonne bistriţa 684 251 433 37.17391 campulung 709 172 537 42.71084 vatra dornei 657 197 460 54.76821 poiana stampei 651 198 453 45.13889 rarău 907 247 660 73.14516 iezer 1267 497 769 109.386 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 50 figure 3. the distribution of de martonne index of aridity table 2 mounthly average quantities of rainfall, registered at the weather stations around suhard staţia alt.(m) i f m a m i i a s o n d interval bistriţa 365 42 33 33 57 75 93 78 68 52 46 46 52 1950-2000 vatra dornei 825 27 28 34 48 86 99 95 82 50 46 35 28 1988-1995 poiana stampei 915 29 24 30 50 78 104 98 72 51 41 35 38 1970-2009 cîrlibaba 960 32 24 42 63 97 93 87 75 55 52 40 40 1969-1998 rarău 1536 39 39 47 76 122 144 138 110 71 46 38 40 1961-2000 iezer 1735 67 69 81 101 127 160 155 127 101 99 96 85 1958-2006 recorded values appear in a series of works on the surrounding landscape units, which we haven’t found published in professional newsletters. this way, in vatra dornei, on the 5th of september 1912, the quantity of 260 mm was recorded, considered to be the largest quantity produced in other period than summer, at country level. there are also values from the rainfall station of gura ţibăului, 140,6 mm at july, 18 1951 and iacobeni, 63,1 mm, on june, 14 1949. figure 4 monthly average rainfall table 3. monthly maximum rainfall registered in 24 hours, absolute maximum and the date of occurrence station i f m a m i i a s o n d max. data poiana stampei 23.4 19.6 26.3 23.5 59.4 48.8 68.1 44.6 43.0 37.1 32.3 25.6 68.1 9.07.1999 iezerul rodnei 34.4 37.1 38.4 45.3 66.1 61.0 80.8 60.5 62.1 70.6 50.5 48.1 80.8 29.07.1966 vatra dornei 20.0 27.9 23.2 44.5 62.1 51.0 65.2 49.7 26.0 34.2 34.3 19.2 65.2 5.09.1912 ciocăneşti 19.0 24.7 17.8 20.2 42.7 37.8 54.5 35.8 43.5 24.0 23.2 24.0 54.5 22.07.1974 cîrlibaba 20.0 20.1 21.5 32.5 50.0 280.0 36.6 70.0 61.5 28.2 29.0 31.7 280.0 16.06.1938 rarău 30.9 34.9 33.4 88.7 56.0 110.6 91.0 71.5 60.2 38.5 27.6 29.7 110.6 29.06.1978 the average of the days with rainfall quantities 0,1 were analyzed based on the data from poiana stampei and iezer stations, which are considered representative for the high area of suhard mountain (table 4). for the number of days with rainfall bigger or equal with 0,1 mm, we food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 51 can observe the increase in it according to the altitude, from 160 days at the boundary with depression, which is over 40% of the year) to 194 days at 1700m, representing more than half a year. therefore, it is an increase with approximately 10% at a difference of 800 m in altitude, which leads us to the conclusion that at the omu peak, the number of rainfall days doesn’t exceed with much the value of 200. liquid rainfall, speaking in number of days, predominates in poiana stampei between april and october and in iezer between may and october. rain represents the most important type of liquid rainfall, which can appear in each month of the year, in the entire mountainous area (table 5, fig. 5). table 4. number of days with rainfall bigger or equal with 0,1mm station i f m a m i i a s o n d total poiana stampei 12.5 11.6 12.2 14.0 16.0 17.5 16.1 13.3 11.1 9.6 12.0 14.2 160.1 iezer 15.9 16.0 17.7 17.9 18.4 18.8 17.8 14.2 13.1 12.3 15.4 17.0 194.5 table 5. number of days with liquid and solid rainfall station liquid i f m a m i i a s o n d total poiana stampei 2.4 2.5 5.6 11.2 15.7 16.6 15.8 12.7 10.6 8.8 6.5 4.1 112.5 iezer 0.3 0.8 1.7 6.2 14.6 17.7 17.9 13.6 11.6 8.7 4.6 1.9 97.1 solid poiana stampei 11.7 10.5 9.5 5.6 0.6 0.0 0.0 0.0 0.2 2.3 7.5 12.1 60.0 iezer 15.6 15.0 16.3 15.7 6.0 3.5 2.3 1.5 5.2 7.2 12.4 16.1 110.6 the number of days with solid rainfall at the contact with the depression reaches the maximum in december, while in altitude happen between march and april (figure 5). a b figure 5. number of days with liquid (a) and solid (b) rainfall number of days with snow the type of precipitations which characterizes especially the cold season increases in days proportionally with the altitude. the consequence, besides a large amount of solid water, is also noticeable in the termic regime. from analyzing the data, for the suhard mountain, a weight of this phenomenon is found between 33% from marginal areas and 45% at over 1700%. these values may be further discussed both because of the particular situations of the stations which recorded them and the field configuration (table 6, fig. 6). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 52 figure 6. number of days per month with layer of snow starting from the map of the mountains’ orientation (figure 7), in our analysis, we can see that more than one third of the peaks in suhard go towards north, north – east, north-west, which helps maintain the snow for a long period of time. adding also the vast surfaces of forests, we can infer that the data from iezer are valid for a much larger area than the altitude level they are registered at. from our observations, yearly speaking, on the north-eastern peak of omu, the layer of snow exists even after the 15 of june and under ouşoru peak it can maintain itself until the beginning of the same month. table 6. number of days with layer of snow station i f m a m i i a s o n d total poiana stampei 29.6 27.1 23.4 5.4 0.2 1.4 11.5 25.8 124.4 iezer 30.7 28.2 30.6 22.1 8.0 0.6 0.1 0.1 1.0 5.0 16.8 25.6 167.2 figure 7. exposure of peaks from suhard mountain these are the areas where the shattered snow in quantities big enough to produce avalanches. meanwhile, on the sunny peaks, there are chances for the snow to be missing even in the middle of winter, fact depicted by the intensity of gelifractie process. lower, at the forests level, the same orientation (south east, south and south west) determines shrinkage in the snowy interval of time during spring, but also a possible later installation of snow. 4. conclusions in the area of the suhard’s mountain from bucovina, the quantities of annual, semiannual, monthly and daily rainfall have a great variability, both in space and time. thus, annual average sums are between 700 mm at lower altitudes and over 1250 mm around omu peak (1932 m). variation of precipitations is between 0mm, in clear, without rainfall days and 280 mm, the maximum quantity registered in cîrlibaba, on june, 16 1938. most of the rainfall occurs in the warm semester, approximately 60%, when the maximum number of liquid rainfall days is also registered. the maximum number of days with solid rainfall occurs in the cold season. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 53 5. references [1]. straton i., mircia i., 2010, contributions to the improvement of permanent grasslands in the s-e of the suhard massif, which belongs to the second stage nemoral (summary of thesis), iaşi. [2]. apăvăloaie m., apostol, l., 1984, characteristics of the thermal inversions in depresiunea dornelor, paper. semin. geogr. d. cantemir,nr. 4, 1983 iaşi, univ. al. i. cuza. [3]. wilks d.s., 1995, statistical methodes in the atmospheric sciences, academic press, inc., san diego, california, usa, 465 p. [4]. apostol l., 1987, considerations on the relationship between the quantities of romaniamonthly rainfall, paper. semin. geogr. d. cantemir,nr. 7, 1986, univ. al. i. cuza, iaşi. [5]. alth a., 1958, ein ausflug in die marmaroscher karpathen. mitt. geogr. gess., wien. [6]. rusu c., 2002, masiff rarău-study of physical geography , editura romanian academy, bucharest. [7]. *** 2008, romanian climate, editted by a collective of nma, editure of romanian academy, bucharest. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 108 stu dy r eg ar di ng the influ enc e of me ta ls m ig ra tion upon the quality of beer at storage *amelia buculei1, sonia amariei1, stefan stefanov2, gheorhe gutt1, adriana dabija1 1stefan cel mare university, faculty of food engineering, 13th university street, 720229,suceava – romania, e-mail : ameliab@fia.usv.ro, gutts@fia.usv.ro, g.gutt@fia.usv.ro, dadianadabija@yahoo.com 2 university of food technologies, plovdiv, bulgaria, stvstefanov@yahoo.com *corresponding author received 12 february 2012, accepted 7 march 2012 abstract: for some substances maximum rates of concentration in food and beverages are set. essential for consumer safety is to study the mechanisms of metal migration in packaging products. very often, sources of migration are elements of the packaging and this pays scant attention. the aim of this work was to determine the content of heavy metals form different kinds of beer from market and to observe: ph of the beer at different periods of time and different temperatures of storage, beer sensorial analysis at different periods of time and different temperatures of storage, physical-chemical properties of beer at different periods of time and different temperatures of storage. we studied four types of beer for the content of certain metals as fe, cu, cd, pb, al and mn. the test was made at 4°c (for cold beer) and at 20°c for the beer in the shop. for purposes of this study used modern methods and equipment were used. they allow to determine with accuracy the content of various substances in food and beverages. the results of experimental work showed increased content of various metals. this is the result of migration, which is different for different beers tested. during the preservation period of products in metallic packages, lead and cadmium migration from the packaging material into product takes place with a rate directly proportional with increasing of storage time. the data obtained allow determining the conditions under which drinks are safe for consumers. key words: heavy metals, migration, beer, aluminium can storage 1. introduction the stability of the system food product/ package when food products are preserved in metallic packages is determined by more factors among which the migration of compounds from package into the product preserved holds an important role. metallic containers (cans) are often used for food, and in particular for beverage packaging [1]. most of the beer cans and refreshments are made of aluminium. in comparison with the steel the aluminium and the recipients made of it are lighter more resistant to corrosion easier to mould less resistant in general and more expensive. all the aluminium recipients have lacquer systems that prevent very well the contact between the food and the metal. in this way all the aluminium levels are generally very low but occasionally they can affect the sensitivity of the products such as beer causing a change of their colour. the problem of aluminium in beer has been elaborated in several papers over the last decade. however, the effect of aluminium on organoleptic properties of beer has been observed in few papers where it has been stated that aluminium gives beer a »metallic« and bitter flavour mailto:ameliab:@fia.usv.ro mailto:gutts:@fia.usv.ro mailto:g.gutt:@fia.usv.ro mailto:dadianadabija:@yahoo.com food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 109 without any observations on particular aroma compound changes. also, the number of reports on precise changes of aroma components throughout different storage conditions is surprisingly scarce. [2] furthermore, after filling maturated beer into bottles, kegs or cans this process goes on, especially in aluminium cans and kegs. the process of aluminium corrosion and migration from can to beer has been elaborated over the last decade in several papers. the factors of the main influence are: the type and quality of cans, the type and thickness of protective can coating, ph of the beer, the length of contact between the can and the beer, thermal treatment, storage temperature, and presence of any corrosive substances[3]. although it is well known that dissolved oxygen concentration has decisive influence on beer stability, primary packaging material can facilitate various processes with negative influence on colloidal and/or flavour stability of beer. beer filled in glass bottles is sensitive to light-struck flavour formation [4]. canned beer is protected from the influence of light, but aluminium migration from can to beer could aggravate colloidal and flavour stability [5]. steel beverage can fabrication consists of dwi process, organic application and seaming operation. the seaming operation consists of the junction of body can with the end (can closing). they are widely used as containers of various kinds of beverage. the body of a two-piece can is formed by a draw and wall ironing (dwi) process [1–3]. overlaying tin on the base steel (tinplate) consists the basic material more used to make food can to conditioner food [4,5]. during end seaming operation, organic discontinuity can be formed at the body hook and at the end hook (internal seam area) caused by small frictions. in the packaging internally lacquered, the interaction product/packing occurs mainly through these discontinuities. the main consequence of this discontinuity is the iron, which can be dissolved in the food. the reactions involved can modify the flavour characteristic of the product conferring to it metallic flavour. the combination of sensory, nutritional and hygienic characteristics will provide the quality of a food product [6]. for the case of the beverage cans made from tinplate, the control of the migration of iron acquires an even greater importance, because extremely small levels of iron migrated to the drink (0.5 ppm) can already compromise the flavour of the drink [7]. the lacquer coating is the most widely used method for reducing tinplate can corrosion [8-10]. a need for rapid development of new internal coatings on tinplate cans arise in the food and beverage industry, mainly as a response to proposed volatile organic compound regulations [11]. the pressure control is important in achieving the optimal level of carbon gasification of the product and avoiding the excessive creation of foam that leads to the loss of liquid and high air levels in the recipient (affecting the internal corrosion and degrading the quality of the product). if these technological factors are respected there shouldn’t appear in beer any organoleptic or physical-chemical modification when being stored at different temperatures and periods of time [12]. 2. materials and methods for experiments it was used blonde beer samples on a lot taken from the market. analyses were conducted periodically throughout six months of storage on for different brands of beer (a, b, c and d brand). brand a was standard lager beer and brands b, c and d were premium lager beers. samples were stored in a food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 110 refrigerator (4 °c) or in a thermostatic chamber (22°c). for estimate beer’s quality there was made the following methods: total acidity determinationthe titration method in the presence of phenolphthalein as indicator; sensorial analysisthe marking scale method; color of beer – observing of view comparison of the analyzed sample’s color with the iodine solution’s color having a known concentration; foam stability – using the times measuring for the persistent foam, starting from the moment when the beer touches the tasting glass till to its total vanishes; co2 determination – standard method; heavy metals content. prior to heavy metals concentration determination, 40 ml of beer were withdrawn from cans. containers and laboratory materials were washed with warm, diluted nitric acid and subsequently rinsed with double distilled water (conductivity=0.055 ms/cm). beer sample was placed in ultrasonic water bath to eliminate carbonation(60 min. at 45hz, t=20 c, p=100%) and then 5 ml of degassed beer were diluted to 50 ml with 1 % nitric acid (in double distilled water). after wards, the sample was placed in the auto sampler cup and shortly of icp-ms agilent seria 7500ce. 3. results and discussion 3.1. the sensorial analysisthe marking scale method the results of the appreciations led by the tasting committee members were written in the tasting papers for each sample of beer can presented and then the average percentage was calculated. on the basis of the average total mark the evaluation of the quality level of beer can samples was led taking on account the organoleptic point of view respectively the place in the quality levels on the 0-20 scale as shown in table 1. table 1. the evaluation of the quality level of the beer can samples analyzed samples’ name total average mark mark product characterization sample a, b, c, d initially verygood the beer can has positive organoleptic traits specific and well determined. sample a 1 month 16,5 good the beer can has positive organoleptic traits specific and well determined but it presents insignificant taste of heavy metals.3months 16,3 6 months 16 sample b satisfac tory the beer can has organoleptic traits slightly determined and it presents …due to which the product is situated under the standard admitted level for the product. 1 month 15,5 3 months 14,5 6 months 12,5 sample c very good the bear can has positive organoleptic traits specific and well determined. 1 month 17 3 months 16,5 6 months 16 good the beer can has positive organoleptic traits specific and well determined but it presents insignificant taste of heavy metals. sample d good the beer can has positive organoleptic traits specific and well determined but it presents insignificant taste of heavy metals. 1 month 15,5 3 months 15 6 months 14,5 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 111 3.2 physical-chemical analysis foam checking. one of the quality characteristics of the beer appreciated by many consumers is the height of the foam when pouring the liquid into the glass and its persistence. the results of the determinations led are presented in table 3. table 3 . checking the foam for the superior beer grades analyzed sample storage time foam checking height of the foam layer [mm] foam stability [minutes] sample a 1 month 72 15 3 months 60 12 6 months 45 5 sample b 1 month 65 15 3 months 62 10 6 months 55 5 sample c 1 month 70 15 3 months 64 13 6 months 48 8 sample d 1 month 72 15 3 months 68 11 6 months 52 7 table 4. the physical-chemical properties of the beer at different periods of time and different temperatures of storage sample storage time (months) acidity [ml na oh/100ml beer] co2 [g/cm3] colour [ebc units] at 4°c 22°c at 4°c 22°c at 4°c 22°c sample a 1 month 1.8 1.9 0.42 0.46 9.6 9.6 3 months 2.1 2.2 0.48 0.50 9.8 9.8 6 months 2.9 3.2 0.52 0.56 9.9 10.2 sample b 1 month 2.4 2.8 0.68 0.70 10.7 11 3 months 2.5 3.1 0.72 0.74 10.8 11.7 6 months 2.9 3.3 0.76 0.78 11.3 12.7 sample c 1 month 1.9 2.1 0.48 0.51 8.8 9.6 3 months 2.3 2.4 0.50 0.56 9.8 10.1 6 months 2.6 2.8 0.52 0.60 10.2 10.6 sample d 1 month 2.6 2.8 0.69 0.74 10.9 11.3 3 months 2.8 3.3 0.71 0.78 11.5 11.9 6 months 2.5 3.6 0.74 0.80 12.3 12.7 3. heavy metal content figure1. amendment of lead concentration figure.2. amendment of cadmium concentration in the product at 4 and 22 degreesfor beer a in the product at 4 and 22 degreesfor beer a food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 112 figure 3. amendment of lead concentration in the product at 4 and 22 degreesfor beer b figure 4.amendment of the cadmium concentration in the product at 4 and 22 degreesfor beer b figure 5. al content in the product during storage of 4 types of beer figure 6. mn content in the product during storage of 4 types of beer figure 7 cu content in the product during storage of 4 types of beer figure 8 fe content in the product during storage of 4 types of beer figure 9 comparison of lead in different beers figure 10 comparison of cadmium in different beers food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 1 – 2012 113 4. conclusion the results just confirm that heavy metals concentration in finished beer differs among samples of different brands and also among samples of the same brand because heavy metals in beer is derived from various raw materials, equipment and brewing processes. it was assumed that during upside down storage more prominent migration could occur due to the fact that beer is in contact with the edges of the can body and its lid on which more corrosion points could arise. generally, temperature of storage influenced heavy metals migration from can to beer. it is obvious that heavy metals vessels in contact with beer have to be well coated to protect the quality of beer. analyzed samples showed certain aroma stability, which was more expressed for samples stored at 22°c. experimental results show that the contents of various metals in beer for the duration of storage. some beers presence of metals fast rise, which means that any element of the package contains high quantity. particularly disturbing are the results for the presence of cadmium in beer b. rapid increase of its content in the product means the wrong choice of one of the elements. with this type of beer is likely at the end of the storage period to exceed the maximum rate of heavy metals from 60 mg/l. 5. references [1]. robertson gl (2006) food packaging: principles and practice. crc press/taylor and francis, london [2]. bernardo pem, dos santos jlc, costa ng (2005) prog org coat 54:34 [3]. montanari a, pezzani a, cassara` a, quaranta a, lupi l (1996) prog org coat 29:159 [4]. catala` r, cabanes jm, bastidas jm (1998) corros sci 40:1455 [5]. bonora pl, deflorian f, fedrizzi l (1996) electrochim acta 41:1073 [6]. dewit jhw (1995) in: marcus p, oudar g (eds) corrosion mechanisms in theory and practice. marcel dekker, new york [7]. marcus p, mansfeld f (eds) (2006) analytical methods in corrosion science and engineering. taylor and francis, london [8]. f. deflorian, s. rossi, m.d.c. vadillo, m. fedel, (2009) electrochemical characterisation of protective organic coatings for food packaging, j appl electrochem 39:2151– 2157 doi 10.1007/s10800-009-9818-1 [9]. f. ivu[i] et al. (2006) aluminium and aroma compound concentration in beer during storage at different temperatures, food technol. biotechnol. 44 (4) 499– 505 [10]. p. viñas, n. aguinaga, i. lópez-garcía, m. hernández-córdoba, determination of cadmium, aluminium, and copper in beer and products used in its manufacture by electrothermal atomic absorption spectrometry, j. aoac int. 85 (2002) 736–743. [11]. m. eruga, j. grgi, z. grgi, b. eruga, aluminium content of beers, z. lebensm. unters. forsch. 204 (1997) 221–226. [12]. m.m. vela, r.b. toma, w. reiboldt, a. pierri, detection of aluminium residue in fresh and stored canned beer, food chem. 63 (1998) 235– 239. 214 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 214 217 wild be rr y pl ants co nt am i nati on w ith tec hnog eni c radio nu cl i des in nor th ern b uk ovi na yevgenia volyns’ka1, natalia omel’chenko1, *мyron rogozynskyi1, oksana mykytyuk2 1national technical university “khpi”, chernivtsi faculty, chernivtsi, ukraine, rmyron@ukr.net 2 bukovinian state medical university, chernivtsi, ukraine, oksana_mp@mail.ru * corresponding author received may 21st 2013, accepted august 15th 2013 abstract: chernivtsi region, part of northern bukovina, is an attractive one for development of both national and international tourism. investigation of radionuclide migration in “soil wild berry plants” chain was carried out in our research project in order to check safety of forest berry products in chernivtsi region. we established that content of radionuclide — 137cs and 40k stayed within acceptable levels in blueberry and cowberry from putyla district. hence, all berries are safe and can be eaten both raw and processed. keywords: radionuclides, 137cs, 40k, forest berry, blueberry, cowberry, post-chernobyl’ period 1. introduction radioactive contamination of wild berry plants in post-chernobyl’ period attracts greater interest of investigators. investigation of mentioned topic is especially valuable in chernivtsi region that has large square woodlands and great quantity of natural mineral water sources (incl. medical ones). that makes region an attractive one for development of both national and international tourism. tourists, primarily foreigners, are interested logically whether berried and their processed products from chernivtsi region are edible. great number of publications was dedicated to this problem in recent years therefore [1, 2, 3]. significant differences in radionuclide content are demonstrated in different country regions as well as in miscellaneous species of wild berry plants in them. such investigations contributed into important practical conclusion about existence of tight connection between radionuclide content in certain raw type and this radionuclide contamination of the soil in each exact edatop. 2. materials and methods investigation of radionuclide migration in soil-wild berry plants chain was carried out in our research project. representatives of cowberry species (vaccinium myrtillus, vaccinium vitis-idaea) and soils where they germinate were objects of our investigations. samples collection and preparation for -spectrophotometric analysis were done according to standard food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 yevgenia volyns’ka, natalia omel’chenko, мyron rogozynskyi, oksana mykytyuk, wild berry plants contamination with technogenic radionuclides in northern bukovina, food and environment safety, volume xii, issue 3 – 2013, pag. 214 217 215 methods [4, 5, 6]. γ-spectrophotometric complex including scintillation detector sdu-2-23 and amplitude-to-digital spectrophotometric trans-former adc-8к2м was used for detection of cesium-137 and potassium-40 nuclides. γ-spectrums processing was conducted employing original акw in software. 3. results and discussion results of investigation of radionuclide content in wild berry plants collected in putyla district are demonstrated in table 1. table 1 content of radionuclides in wild berry plants investigation object specific activity, bq/kg accumulation quotient 137cs 40k 137cs 40k blueberries: berries 443 1123 3.04 2.94 leaves 764 997 5.23 2.61 stems 329 416 2.25 1.09 soil 146 382 – – cowberries: berries 315 974 3.21 2.79 leaves 523 935 5.34 2.68 stems 227 408 2.32 1.17 soil 98 349 – – leaves of plants are contaminated with radioactive cesium to the biggest extent and stems are the least. coincidence was observed between corresponding accumulation quotients for both berry plants that confirms the findings of lviv investigators [6] about similarity of processes of radionuclides accumulation by single species plants from soils of a same type and same pollution origin. re-distribution of radioactive cesium-137 during period of blueberry fruiting was studied by ukrainian investigators [7]. radioactive cesium activity was distributed as follows: 31% of total radionuclide activity was detected in leaves, 26% in stems, 25% in berries, and 18% — rhizomes and roots. our investigations revealed maximal content of cesium-137 in blueberry and cowberry leaves (764 and 523 bq/kg correspondingly), characteristic for fruiting period (pic. 1). but, radioactive cesium content in stems was a little bit lower comparing berries in our case that did not match data of previously done investigations. inconsistency may be caused by complex of reasons: carrying out food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 yevgenia volyns’ka, natalia omel’chenko, мyron rogozynskyi, oksana mykytyuk, wild berry plants contamination with technogenic radionuclides in northern bukovina, food and environment safety, volume xii, issue 3 – 2013, pag. 214 217 216 investigations in different seasons after accident at chernobyl atomic station, measurements in different geographical district or under another weather conditions, employing different measuring methods etc. figure 1. cesium-137 content (bq/kg) in organs of investigated berry plants. especial specialists’ attention is attracted to intensity of radioactive cesium accumulation by berries of wild plants. a range of berry species distributed according to cesium-137 income level was provided in literature sources [8, 9]: cranberry > cowberry > blueberry > raspberry. our results demonstrated that cowberry has slightly higher accumulation quotient (3.21) comparing blueberry (3.04). cooking processing of berries from vacciniaceae family can significantly decrease specific activity of 137cs. in partial, washing fresh berries decreases their content of radionuclide by 5-15% at the expense of its partial leaching. according to results of joint research of belarus and radiologists-ecologists from european union (kenigsbers, buglova, 1994; kenigsberg et al., 1996) quotients of 137cs activity changes in berries depending on type of culinary processing were determined: 0,9 for washing; 0,5 — for jam cooking; 0,65 — grinding with sugar; 9,0 — drying. soaking the cowberries decreases 137cs activity in them by 71%, cooking jam — by 52% [8]. в activity of 137cs in fruit compote decreases by 50% comparing fresh berries. drying berries causes increase of specific activity by 7-10 times — proportionally to decrease of their weight. obtained results may be applied practically both in food industry and in private subsistence farming for decrease of internal exposure dose got by population from forest food products. 4. conclusion as follows, our investigations demonstrate that radionuclide content in wild berry plants collected in putyla district, chernivtsi region does not exceed standards for fresh plants according to order of ministry of health protection of ukraine “acceptable levels of 137cs and sr-90 radionuclide content in food products and water» [10], agreed with codex alimentarius statements [11]. content of radionuclide — 137cs (normal level — below 500 bq/kg) and 40k (normal level — below 1480 bq/kg) stayed within acceptable levels in blueberry and cowberry from putyla district. all berries are safe and can be eaten both raw and processed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 yevgenia volyns’ka, natalia omel’chenko, мyron rogozynskyi, oksana mykytyuk, wild berry plants contamination with technogenic radionuclides in northern bukovina, food and environment safety, volume xii, issue 3 – 2013, pag. 214 217 217 5. references [1] orlov а.а., kaletnik n.n, korotkova е.z. intensity of accumulation of cs-137 by berries species from vacciniaceae family in different types of habitat conditions of polessie, ukraine // science. chernobyl-96. – res.pract.conf. 11-12.02.1997г. – abstacts. – kiev, 1997. – p.403. [2] orlov а.а., krasnov v.p. possibility of phytoindication application for quantitative estimation of 137cs accumulation from soils by medicinal plants from ukrainian polessie woods. // plant resources. – 1999. – v. 35, iss. 4. – p. 79-86. [3] orlov а.а., iraklienko s.p, krasnov v.p. et al. patterns of accumulation of 137cs by wild mushrooms and berries of polessie, ukraine // hygiene of settlements. – iss. 36, part i. – kiev, 2000. – p. 431-445. [4] metodic recommendations on sanitary control of radioactive substances content in environmental object / ed. by а.n. marey and a.s. zykova. – м.: меdicine, 1980. – 121 p. [5] му 5779–91 cesium-137. determination in food products, approved by ministry of health protection (ussr) 04.01.1991. [6] му 5778–91 potassium-40. determination in food products, approved by ministry of health protection (ussr) 04.01.1991. [7] grabovskyi v.а., dzendzeliuk о.s., оhrimenko s.v. contamination flora representatives with cesium-137 in western regions of ukraine // ecotechnology and resource conservation – 2000. – №2. – p.60-64. [8] korotkova е.z., orlov а.а. accumulation of 137cs with phytomass of berry plants in different density of radioactive contamination of soils // forestry and agroforestry. — iss. 95. woods health. — kharkiv: rvp “оriginal”, 1999. – p. 16-23. [9] krasnov v.p. radioecology of the woods of polessie, ukraine. – zhytomir: volyn, 1998. – 112 p. [10] bulavik i.m. accumulation of cs-137 in food products of forests/ problems of ecology of forests and forest management in polessie, ukraine / res. works of polessie alnis. – iss. 4. – zhytomir, 1996. – p. 31-35. [11] гн-6.6.1.1-130-2006 acceptable levels of radionuclides cs-137 and sr-90 content in food products and water. — 1997, mhp, ukraine, kyiv, 1997. [12] joint fao/who food standart programme, codex alimentarius, vol.1 (1991), section 6.1, "levels for radionuclides", 1991. microsoft word 11 bg diana1 corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 72 assessment of the action of deposit mycoflora on vicia faba l beans from suceava genebank’s collection *diana batîr rusu1, danela murariu1 genebank suceava, romania dia_sv@yahoo.com, d_murariu@yahoo.com *corresponding author received 20 november 2011, accepted 10 january 2012 abstract: this study consisted in a phytopathological evaluation of epiphyte and endophyte mycological flora which appeared on vicia faba seeds placed on two types of substrates (cga medium and blotting paper). the 30 populations of faba bean resulted from the active collection of suceava genebank and conserved for different time intervals (8, 18 and 23 years), in controlled atmosphere storages (t=+40c; relative air humidity = 30 40%). micromycets were evaluated by counting the infected seeds and the attack frequency was expressed as a percentage, by visual estimation of seeds surface. the target objectives of the study were to establish the influence of the conservation period on the activity of micromycets placed on stored seeds and to settle the influence of the substrate type cga medium (potato dextrose agar) and blotting paper on the development of fungal pathogens. keywords: micromycets, landraces, cga medium 1. introduction size variation of pathogen colonies kept in constant environmental conditions reflects the differences in quantity, viability and location of inoculums on seed. the development of colony around each seed on growth medium, and the intensity of symptoms on germs in case of blotting paper tests are closely dependent on the amount of inoculum on seed number of spores or mycelium abundance [1]. generally, the correlation between inoculum (spores load/seed) and colony size (the amount of mycelium) developed on cga medium (potato dextrose agar) is very significant [2]. micromycets existing on stored legume seeds can cause during storage a wide range of changes, with negative consequences from a technological, nutritional, hygienic and commercial point of view [3]. beratlief and collaborators in a study concerning the deposit ecosystem characteristics, revealed the mycological flora evolution and sequence on legume seeds stored with high moisture content [4]. the purposes of this study are:  to establish the influence of the conservation period on the activity of micromycets placed on stored seeds  to settle the influence of the substrate type cga medium (potato dextrose agar) and blotting paper on the development of fungal pathogens. 2. experimental we have performed the phytopathological characterization of local germplasm represented by 30 populations of vicia faba, conserved for 8, 18 and 23 years at t = +4°c, which come from collecting expeditions realized by the collecting department from suceava genebank during a term of 15 years (19862001). lab experiments were carried on suceava genebank by using the genetic food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 73 seminal material from the active collection of the institution, which was placed on the cga medium and blotting paper. to make possible the assessment of the micromycets present on vicia faba seeds, we implemented the following research methods: macroscopic analyses of the seeds; ulster method [5] on cga medium (potato dextrose agar). 3. results and discussion the seeds of vicia faba, placed on cga medium and blotting paper, presented after the incubation period the following characteristics concerning the presence of fungal microorganisms: a) cga medium (potato dextrose agar) on cga medium, the presence of deposit mycoflora on the 30 samples of vicia faba seeds conserved at +40c temperature, for 8, 18 and 23 years was different, as follows: on the samples stored for 8 years at +40c temperature, we identified 11 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride, botrytis fabae, stachybotrys atra, stemphylium botryosum) which showed a different attack degree on each sample of the 7 analyzed, registering an infection rate of 78% (table 1). on 17 samples stored at +40c temperature. for a period of 18 years we identified 11 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, mucor sp., trichothecium roseum, trichoderma viride, botrytis fabae, stemphylium botryosum). the 510 seeds submitted to macroscopic and microscopic analysis presented an infection rate of 66 %. table 1 proportion of micromycets isolated on vicia faba beans placed on cga medium other 6 seed samples. conserved for a period of 23 years, have been infected by a smaller number of fungal microorganisms (penicillium sp., aspergillus sp., rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride) and the infection percentage on the 180 seeds analyzed was much lower (40.5 %). in table 2 are presented the micromycets placed in three experimental experimental conditions seeds stored at t+ 40c, for 8 years seeds stored at t+ 40c, for 18 years seeds stored at t+40c, for 23 years isolated micromycets attack frequency (%) penicillium sp. 13.3 16 11.6 aspergillus sp. 6.2 10.6 6.1 rhizopus sp. 25.7 18.4 7.2 epicoccum sp. 8.1 0.4 2.7 cladosporium herbarum 3.8 1.9 3.3 alternaria alternata 11.4 11.4 5.5 mucor sp. 0 0.4 0 trichothecium roseum 0.5 2.7 2.7 trichoderma viride 0.9 0.8 1.1 botrytis fabae 2.8 2.9 0 stachybotrys atra 0.9 0 0 stemphylium botryosum 4.3 0.2 0 total 77.9 65.7 40.2 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 74 conditions (8. 18 and 23 years) at +40c temperature: table 2 action mode of micromycets identified on vicia faba beans in 3 experimental conditions on cga medium b) blotting paper analyzing the 30 seeds samples of vicia faba stored at +40c temperature for 8, 18 and 23 years, we have identified the following infection percentages caused by fungal pathogens: on the samples stored for 8 years at + 40c temperature we have identified 6 fungal pathogens (penicillium sp., aspergillus sp., alternaria alternata, rhizopus sp., cladosporium herbarum, botrytis fabae). which had a different attack degree on each sample of the 7 analyzed, registering an infection rate of 68.6% (table 3). table 3 proportion of micromycets isolated on vicia faba beans placed on blotting paper on 17 samples conserved at +40c temperature for a period of 18 years, we have identified 7 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., cladosporium herbarum., alternaria alternata., trichothecium roseum, botrytis fabae). the 255 seeds submitted to macroscopic and microscopic analysis presented an infection rate of 30.9 %. the 6 seed samples with a storage period of 23 years have been infected by a smaller number of micromycets (penicillium sp., aspergillus sp., rhizopus sp. and alternaria alternata). the infection percentage on the 90 seeds analyzed being more low (24.4 %). in table 4 are presented the micromycets placed in 3 experimental conditions (8, 18 and 23 years) at +40c temperature. analyzing the number of experimental conditions seeds stored at t +40c, for 8 years seeds stored at t +40c, for 18 years seeds stored at t +40c, for 23 years isolated micromycets total number of infected seeds penicillium sp. 28 82 21 aspergillus sp. 13 54 11 rhizopus sp. 54 94 13 epicoccum sp. 17 2 5 cladosporium herbarum 8 10 6 alternaria alternata 24 58 10 mucor sp. 0 2 0 trichothecium roseum 1 14 5 trichoderma viride 2 4 2 botrytis fabae 6 15 0 stachybotrys atra 2 0 0 experimental conditions seeds stored at t +40c, for 8 years seeds stored at t + 40c, for 18 years seeds stored at t +40c, for 23 years isolated micromycets attack frequency (%) penicillium sp. 19 10.9 10 aspergillus sp. 2.8 2.7 6.6 rhizopus sp. 13.3 5.0 5.5 cladosporium herbarum 7.6 1.6 0 alternaria alternata 20.9 3.9 2.2 trichothecium roseum 0 4.7 0 botrytis fabae 4.7 1.9 0 total 68.3 30.7 24.3 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 75 infected seeds, we can observe that all micromycets genus of legume seeds were isolated on a more smaller number of seeds when samples were incubated on blotting paper, in comparison to the number of seeds placed on cga medium. table 4 action mode of micromycets identified on vicia faba in 3 experimental conditions on blotting paper 4. conclusions deposit mycoflora developed on faba bean seeds taken in this study was analyzed according to genotype period of seed conservation and type of substrate used. from our study resulted the following conclusions: 1. the seed samples of vicia faba stored in 3 experimental conditions placed on cga medium, were infected in different proportions by fungal pathogens. the species stachybotrys atra was identified only on the samples conserved for 8 years at +40c temperature and the species botrytis fabae, stemphylium botryosum were identified only on the seeds with a storage period of 8 and 18 years. other types of micromycets were detected in all storage conditions, but on a different number of seeds. the species mucor sp. was isolated only on the seed samples with a storage period of 18 years. 2. by placing the same seed samples of vicia faba in 3 experimental conditions on blotting paper, we observed that samples were infected in a smaller proportion compared to cga medium. the fungal pathogens mucor sp., epicoccum sp., trichoderma viride, stachybotrys atra, stemphylium botryosum identified on cga medium, were not isolated on blotting paper. 5. references [1]. raicu c., baciu d., 1978. seed pathology. ceres publishing. bucharest. 1 50. 109 111. 170 – 1 [2]. hulea a., negru al., severin v., 1973. main seed crop diseases. ceres publishing. bucharest. 35 – 70 [3]. nagy e., trif v., 1998. research regarding [4]. prevention and control of seed borne diseases at cereals in terms of transylvania. plant protection issues.bucharest. 26: 48 53 [5]. beratlief c., oprea m., 1994. ecosystem characteristics of agricultural products storages and health implications. plant protection issues. bucharest. 22: 13. 44 [6]. malone j.p., muskett a.e., 1941. the ulster method for the examination of flax seed for the presence of seed borne parasites. annals appl. biol.. 8 13. 28 experimental conditions seeds stored at t+40c, for 8 years seeds stored at t+40c, for 18 years seeds stored at t +40c, for 23 years isolated micromycets total number of infected seeds penicillium sp. 20 28 9 aspergillus sp. 3 7 6 rhizopus sp. 14 13 5 cladosporium herbarum 8 4 0 alternaria alternata 22 10 2 trichothecium roseum 0 12 0 botrytis fabae 5 5 0 329 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 42016, pag. 329 333 mathematical modeling of thermal processing of meat products *volodymyr pavelko1, serhiy hrybkov2, arkadiy zaslsvsky1, daria dmytrenko1 1national university of food technologies, thermal fluids and industrial refrigeration department, 68, volodymyrska str., 01601kyiv,ukraine 2national university of food technologies, information systemsdepartment, 68, volodymyrska str., 01601kyiv,ukraine volodymyrpavelko@gmail.com *corresponding author received 11th november 2016, accepted 27th december 2016 abstract: the article presents the mathematical modeling for sausage products heat treatment during thermal processing in steamer cooking chambers in meat processing plants.the sausage and meat cooking processing period has been divided into several stages, each stage duration being predefined by heat transfer analytical solving for any particularly sized sausage product. sequential thermal process stages include drying stage, smoking stage and the cooking sausage product stage. the duration of each sausage thermal processing stage can be calculated by proposed empirical equations. calculating the duration of the sausage product thermal cooking stages, the initial temperature condition of the product is considered to be characterized as the temperature averages. specially elaborated thermal calculation algorithm for sausage product heat treatment allows estimating the thermal energy consumption for any given cooking processing, considering the changes in technological parameters.thus, the mathematical model is proposed to be applied to thermal (structural) design and engineering calculations for the above named steamer cooking chambers. keywords: heat treatment, meat (sausage) products, mathematical modeling, thermal (structural) design calculation of the cooking chambers. 1. introduction the process of heat treatment of meat (sausage) products is carried out in a steamer cooking chamber and contains three (3) steps: warming through(drying), (smoking)roasting and cooking. the process is completed when the core temperature reaches 72 ° c [1]. selecting heat treatment modes(for different stages) not only the recommendations of equipment manufacturers have to be taken into account, but also theoretical knowledge for each stage of treating meat products is essential. the aim of this work is to create a mathematical model of the meat (sausage) product thermal processing, and implement it to optimize of thermal and structural design procedure of steamercookingchambers. 2. matherials and methods any sausage product can be considered to have oval-cylindrical shape,and be http://www.fia.usv.ro/fiajournal mailto:volodymyrpavelko@gmail.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 4 – 2016 volodymyr pavelko, serhiy hrybkov, arkadiy zaslsvsky, daria dmytrenko, mathematical modeling of thermal processing of meat products, food and environment safety, volume xv, issue 4 – 2016, pag. 329 – 333 330 introduced as a figure (fig.1), where rh radius. fig. 1. sausage product shape where d is sausage product diameter, i.e.d=2∙rh, and acceptinglength l being 1meter. in case the product has an oval, square or rectangular form in cross-section, we take the diameter of the smallest radius(fig.2) for a diameter. a) b) fig. 2. cross-section of the sausage product: а) rectangular shape; b) oval shape considering 0rrh  , where 0r – is the diameter of the product at the initial stage of heat treatment. the total duration of the heat treatment process of cured/boiled and/or smoked raw-cooked sausage products is specified using the following formula: 𝜏𝑡𝑜𝑡𝑎𝑙 = 𝜔1 ∙ 𝜏𝑑𝑟𝑦𝑖𝑛𝑔 + 𝜔2 ∙ 𝜏𝑠𝑚𝑜𝑘𝑖𝑛𝑔 + +𝜔3 × 𝜏𝑐𝑜𝑜𝑘𝑖𝑛𝑔 , (1) where drying −the warming-though (drying) time; smoking –the smoking and roasting time; cooking –the time for sausages cooking, i –availability ratio of i –stage of heat treatment (i=1 – warming-through (drying), i=2 – smoking and roasting; i=3 – cooking) in the product treatment processing. the numerical value of the coefficient is  1.0i where i 0 – in the nonheat treatment processing stage, and i 1 – in the heat treatment processing stages. at each stage of the sausage product heat treatment process its diameter (radius rh)changes its value according to the istage of processing and is calculated by the empirical formula: 𝑅𝑠𝑖 = 𝐾𝑖 ∙ 𝑅0, (2) where ik – the coefficient characterizing the increase (growth) of the sausage product radius during its heat treatment. therefore accepted values are 11 k , (і=1), 023.12 k (і=2), 045.13 k (і=3), [1]. 3. results and discussion let us consider the duration of each stage of the heat treatment. 1) the drying sausage processing stage. the duration of the stage of sausage product warming-through (drying) is the amount of time spent on the curingwarming-through and drying of the product (τdrying) and the time required to evaporate condensation from sausage casing developinginchamber (τcondensation): 𝜏𝑑𝑟𝑦𝑖𝑛𝑔 = = 𝜏𝑤𝑎𝑟𝑚−𝑡ℎ𝑟(𝑑𝑟𝑦𝑖𝑛𝑔) + 60 ∙ 𝜏𝑐𝑜𝑛𝑑𝑒𝑛𝑠𝑎𝑡𝑖𝑜𝑛 , (3) time calculating τwarm-thr(drying)is carried out using the following formula: rh d rh d rh food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 4 – 2016 volodymyr pavelko, serhiy hrybkov, arkadiy zaslsvsky, daria dmytrenko, mathematical modeling of thermal processing of meat products, food and environment safety, volume xv, issue 4 – 2016, pag. 329 – 333 331 𝜏(warm−thr(drying) = 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 ∙ 𝑅𝑠 2 𝑎 = = 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔∙𝑅0 2 𝑎 = 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 ∙𝑅𝑠2 2 𝑎 , (4) where f0,drying–the duration of drying in dimensionless form; a − temperature conductivity coefficient of the sausage product; for cured/boiled and/or smoked raw-cooked sausages а=5.10-4 m/min, [1]. fourier criterion value f0, drying, corresponding to the time required to reach the set-pointcore temperature of the product (in the center of sausage loaf) is calculated using the following formula: 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 = ( 𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 + 4 8 ∙ 𝐵𝑖 ) × × (𝑙𝑛 ( 2 (𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔+2)∙(1−𝑇𝑛,𝑑𝑟𝑦𝑖𝑛𝑔) ) + 𝐹0 ´ ), (5) where dryingbi – biot criteria for drying stage; dryingnт , – dimensionless value of the surface product temperature during drying; 0f − the time of the “temperature front” passage, defined in nomogram [1,5]. the value of the biot criteria is specified using the following formula: 𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 = 𝛼𝑑𝑟𝑦𝑖𝑛𝑔 𝜆 ∙ 𝑅0 = 25.47 ∙ 𝑅0,(6) where αdrying − the heat-transfer coefficient during heat treatment; − thermal conductivity coefficient ( =0.465 w/m*k is accepted for cured/boiled and/or smoked rawcooked sausages), [1,6]. the heattransfer coefficient can be specified by the following expression: 𝛼𝑑𝑟𝑦𝑖𝑛𝑔 = 𝛼𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 ∙ (1 + 1.9 ∙ 𝑑) = = (6.16 + 4.49 ∙ 𝑊) ∙ (1 + 1.9 ∙ 𝑑) = = 11.84w/(m2∙k), (7) where αenvironment − the heat-transfer coefficient of the vapor environment in a heat chamber, defined by the empirical yurhes formula [2,3]: 𝛼𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 = 6.16 + +4.49 ∙ 𝑊w/(m2∙k), where d – the moisture content of the product (sausage product) calculated in kg of moisture(water) per kg of the environment with chamber environment temperature of tenvironment=100°c and relative air humidity φ=10 % the moisture content d=0,076 kg/kg. 0f −the time of the “temperature front” passage can be calculated by the above nomogram [1,6], but having defined the specification of the bio criteriabidrying according to the formula (6), can also be specified using the following expression: 𝐹0 ´ ≈ 0.7 ∙ ( 1 12 + 1 3∙𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 − 2 3∙(𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔) 2 × × 𝑙𝑛(1 + 0.5 ∙ 𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 )) = 0.7 × × ( 1 12 + 1 3∙25.47∙𝑅0 − 2 3∙(25.47∙𝑅0) 2 × × ln(1 + 0.5 ∙ 25.47 ∙ 𝑅0)) = 0.7 ∙ ( 1 12 + 1 76.41∙𝑅0 − 2 1946.16∙𝑅0 2 × 𝑙𝑛 (1 + 12.47 ∙ 𝑅0)), (8) the dimensionless temperature value of the product surface while drying is specified using the following formula: 𝑇𝑛,𝑑𝑟𝑦𝑖𝑛𝑔 = 𝑡𝑒𝑛𝑑,𝑑𝑡𝑦𝑖𝑛𝑔−𝑡0,𝑑𝑟𝑦𝑖𝑛𝑔 𝑡𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡,𝑑𝑡𝑦𝑖𝑛𝑔−𝑡0,𝑑𝑡𝑦𝑖𝑛𝑔 , (9) where dryingendt , − the temperature of the product surface (sausage loaf) at the end of the drying process, °с; dryingt ,0 −the initial temperature of the sausage product surface (it is assumed that dryingt ,0 =15°с),°с. the environment temperature dryingtenvironment , is specified by the technological requirements foreachof appropriate stage of sausage product heat treatment. substituting the equations (6) and (9) in the formula (5), we obtain the fourier criterion value corresponding to the time required to reach the set-point temperature in the center of sausage product: 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 = ( 25.47 ∙ 𝑅0 + 4 8 ∙ 25.47 ∙ 𝑅0 ) × (𝑙𝑛 ( 2 (25.47∙𝑅0+2)∙(1− 𝑡𝑒𝑛𝑑,𝑑𝑡𝑦𝑖𝑛𝑔−𝑡0,𝑑𝑟𝑦𝑖𝑛𝑔 𝑡𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡−𝑡0,𝑑𝑡𝑦𝑖𝑛𝑔 ) ) + 𝐹0 ´ ), (10) otherwise, 0f value can be determined using nomogram and taking into   food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 4 – 2016 volodymyr pavelko, serhiy hrybkov, arkadiy zaslsvsky, daria dmytrenko, mathematical modeling of thermal processing of meat products, food and environment safety, volume xv, issue 4 – 2016, pag. 329 – 333 332 accountcriteria dryingbi and the parameter    dryingndrying dryingdryingndrying тbi biтbi , , 1 2    , that allow you to determine 0f. the time required for evaporation of condensate from the outer surface of the sausage product can be specified using the following formula: 𝜏𝑐𝑜𝑛𝑑𝑒𝑛𝑠𝑎𝑡𝑒 = (2 − 0.46 ∙ 𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 ) × × (21 ∙ 𝑇𝑛,𝑑𝑟𝑦𝑖𝑛𝑔 − 8) = = (2 − 0.46 ∙ 25.47 ∙ 𝑅0) × × (21 ∙ 𝑇𝑛,𝑑𝑟𝑦𝑖𝑛𝑔 − 8), (11) taking into accountthe equations (4) and (11), the drying stageduration can be specified using the following formula: 𝜏𝑑𝑟𝑦𝑖𝑛𝑔 = 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 ∙𝑅0 2 𝑎 + 60 ∙ 𝜏𝑐𝑜𝑛𝑑𝑒𝑛𝑠𝑎𝑡𝑒 = ( 𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 +4 8∙𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 )∙(𝑙𝑛( 2 (𝐵𝑖𝑑𝑟𝑦𝑖𝑛𝑔 +2)∙(1−𝑇𝑛,𝑑𝑟𝑦𝑖𝑛𝑔 ) )+𝐹0 ´ )∙𝑅0 2 𝑎 + 60 ∙ 𝜏𝑐𝑜𝑛𝑑𝑒𝑛𝑠𝑎𝑡𝑒 , (12) 2). the smoking (roasting) stage of the sausage product the duration of the smoking(roasting) stage τfryingcan be specified using the following formula: 𝜏𝑓𝑙𝑦𝑖𝑛𝑔 = 𝐹0,𝑠𝑚𝑜𝑘𝑖𝑛𝑔∙𝑅𝑠2 2 𝛼𝑠𝑚𝑜𝑘𝑖𝑛𝑔 , (13) wherers2=r0∙k2−the average diameter (radius) of the product at the end of thesmoking(roasting) stage. the value of the biot criteria for the smoking(roasting) stage is specified using the following formula 𝐵𝑖𝑠𝑚𝑜𝑘𝑖𝑛𝑔 = 𝛼𝑠𝑚𝑜𝑘𝑖𝑛𝑔 𝜆 ∙ 𝑅𝑠2 = 𝛼𝑠𝑚𝑜𝑘𝑖𝑛𝑔 𝜆 ∙ 𝑅0 ∙ 𝐾2, (14) where 𝛼𝑠𝑚𝑜𝑘𝑖𝑛𝑔 – is the heat-transfer coefficient of the vapor environment to the sausage product at the smoking (roasting) stage, defined by the above empirical yurhesformula (7): 𝛼𝑠𝑚𝑜𝑘𝑖𝑛𝑔 = 𝛼𝑑𝑟𝑦𝑖𝑛𝑔 = 𝛼𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 × × (1 + 1.9 ∙ 𝑑) = 11.84w/(m2∙k); − is the thermal conductivity coefficientofcured/boiled and / or smokedsausages, which is also accepted for the warming-through and dryingstage = 0.465 w/m∙k) [1]. the smoking/ roasting stage duration of sausage products can be finalized by the following expression: 𝐹0,𝑠𝑚𝑜𝑘𝑖𝑛𝑔 = ( 𝐵𝑖𝑠𝑚𝑜𝑘𝑖𝑛𝑔 + 4 8 ∙ 𝐵𝑖𝑠𝑚𝑜𝑘𝑖𝑛𝑔 ) × × (𝑙𝑛 ( 2 (𝐵𝑖𝑠𝑚𝑜𝑘𝑖𝑛𝑔 +2)∙(1−𝑇𝑛,𝑠𝑚𝑜𝑘𝑖𝑛𝑔 ) ) + 𝐹0 ´ − −𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 ), (15) where 𝑇𝑛,𝑠𝑚𝑜𝑘𝑖𝑛𝑔 = 𝑡𝑒𝑛𝑑,𝑠𝑚𝑜𝑘𝑖𝑛𝑔−𝑡0,𝑠𝑚𝑜𝑘𝑖𝑛𝑔 𝑡𝑠𝑚𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 −𝑡0,𝑠𝑚𝑜𝑘𝑖𝑛𝑔 , (16) 3). the cooking stage of the sausage product. the dimensional cooking stage duration τcooking of the sausage products is specified using the formula (4), and the biot criteria using the formula (6). the diameter (radius) of the sausage product (loaft) at the cooking stage is specified using the empirical relation: 𝑅𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = 𝑅0 ∙ 𝐾3 = 1.045 ∙ 𝑅0 the heat-transfer coefficient value of the vapor environment to the outer sausage product surface can be specified using the following equation: 𝛼𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = 𝛼𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 ∙ (1 + 1.9 ∙ 𝑑) = = 35.68w/(m2∙k), where d – the moisture content of the product (sausage product) with vapor environment temperature in the chamber; tenvironment=85 °c and relative air humidity φ=90 %. according to the technological requirements for the heat treatment process of sausage products, the above parameters of the environment tenvironment and φ must be steadily maintained during the whole cooking stage [3]and have to meet following values: the moisture content of d=0.7653 kg/kg, tenvironment=85 °c and φ=90%.   food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 4 – 2016 volodymyr pavelko, serhiy hrybkov, arkadiy zaslsvsky, daria dmytrenko, mathematical modeling of thermal processing of meat products, food and environment safety, volume xv, issue 4 – 2016, pag. 329 – 333 333 the value of the biot criteria for the cooking stage is calculated usingthe following formula: 𝐵𝑖𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = 79.42 ∙ 𝑅0, (17) the fourier criterion value for the cooking stage is given by formula (8), substituting though biot value (the equation (17): 𝐹0,𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = 0.7 ∙ ( 1 12 + 1 238.26∙𝑅0 − − 2∙𝑙𝑛(1+39.71∙𝑅0) 18922.61∙𝑅0 2 ), (18) the sausage product cooking stage duration in the dimensionless value is specified using the following formula: 𝐹0,𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = ( 𝐵𝑖𝑐𝑜𝑜𝑘𝑖𝑛𝑔 + 4 8 ∙ 𝐵𝑖𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ) × × [𝑙𝑛 ( 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 − 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔0 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 − 𝑡𝑒𝑛𝑑𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ) +𝐹0 ´ ] = ( 79.42 ∙ 𝑅0 + 4 8 ∙ 79.42 ∙ 𝑅0 ) × × [𝑙𝑛 ( 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 −𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔0 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 −𝑡𝑒𝑛𝑑𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ) + +𝐹0 ´ ], (19) where endcookingt the core product temperature( in the center of sausage loaf) at the end of the cooking stage. calculating the cooking stage duration of the sausage product, the initial condition of the product is considered to be characterized 0,cookingt by the temperature averages: 𝑡𝑐𝑜𝑜𝑘𝑖𝑛𝑔𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 ,0 = 𝑡𝑓𝑙𝑦𝑖𝑛𝑔 +𝑡𝑛,𝑓𝑙𝑦𝑖𝑛𝑔 2 ,(20) the dimensional cooking stage duration τcooking (of the sausage loaf) is specified using the following formula: 𝜏𝑐𝑜𝑜𝑘𝑖𝑛𝑔 = 2142.45 ∙ 𝐹0,𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ∙ 𝑅0 2, (21) 4. conclusion thus we obtain the mathematical heat treatment model for the entire thermal processing of cured/ boiled and/or smoked raw-cooked sausages as follows: 𝜏 = 2000 ∙ 𝐹0,𝑑𝑟𝑦𝑖𝑛𝑔 ∙ 𝑅0 2 + (2 − 11.72 × × 𝑅0 2) ∙ (21 ∙ 𝑇𝑛 − 8) + 2048.29 × × 𝐹0,𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ∙ 𝑅0 2 + 2142.45 × × 𝐹0,𝑐𝑜𝑜𝑘𝑖𝑛𝑔 ∙ 𝑅0 2, (22) 5. references [1]. brazhnikov a., karpychev v., peleev a., analytical methods for the study of thermal processing of meat products, learning guidem.: food industry: 1–365, (1974) [2]. ivashkin yu., modelling of production processes of meat and dairy industry, learning guidem.: agropromizdat: 1− 236, (1987) [3]. pavlov k., romankov p., noskov a., examples and problems on the course of processes and devices of chemical technology, learning guide, l.: chemistry: 1–576, (2007) [4]. pavelko v., heating supply of the meat and dairy processing industries, learning guide vinnytsia: nivaknyha: 156–166, (2007) [5]. pavelko v., sokolenko o., zaslsvsky a., the influence of sometechnologicalfactors on theduration of theprocess of heattreatment of sausages, k.: researchpapers nuft: 31-36, (2012) [6]. brazhnikov a., the theory of thermal processing of meat products, m.: agropromisdat: 1-270, (1987) 1. introduction 372 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 372 375 application of titrimetric technique to determine phosphates in cheeses *mariуa vorobets1, igor kobasa1, aureliуa cheban1 1yu. fedkovych national university of chernivtsi, ukraine, m.vorobets@chnu.edu.ua *corresponding author received november 19th 2014, accepted december 29th 2014 abstract: a method of the titrimetric determination of phosphate (food additives e338 phosphate acid, e339 sodium phosphate, e340 potassium phosphate, e341 calcium phosphate, e450 sodium and calcium pyrophosphate,) in cheeses has been developed. the research experimental results regarding the phosphate ion content in hard and spread cheeses (in terms of phosphate acid, %) obtained by means of the developed methodology are described. the correctness and accuracy of the results obtained by developed technique are confirmed by the definition of phosphates in the studied samples of cheeses by photometric method (by the method according to dstu iso 3360: 2008). a comparative analysis of the two methods has been performed. keywords: food additives, phosphates, hard and spread cheeses, titrimetric method, photometric method 1. introduction today, the use of biological and food additives in the food industry has become an integral component technology [1]. to improvement of the quality of many products and for perfection of the process in the milk industry the phosphates are used. phosphorus-containing food additives are also used in the production of soft drinks, bakery, confectionery and meat products as stabilizers, acidity regulators, baking powder and a latches color. but most of them are used in the production of hard and spread cheeses. food additives e338 (phosphate acid h3po4), e339 (sodium phosphate nah2po4; na2hpo4; na3po4), e340 (potassium phosphate kh2po4; k2hpo4; k3po4), e341 (calcium phosphate ca(h2po4)2; cahpo4; ca3(po4)2) e450 (pyrophosphates na2h2p2o7; na3hp2o7; na4p2o7; ca2p2o7; cah2p2o7) have a variety of technological properties: soluble alkali phosphate have a pronounced buffer potential to regulate acidity; polyphosphates inhibit growth of grampositive bacteria, which can significantly increase the shelf life of products; tricalcium phosphate used in the production of bakery products, special drinks, powdered and condensed milk, ice cream, fish and minced meat, alcoholic beverages, convenience foods, breakfast cereals, instant food, confectionery, baking powder, spread cheeses [2]. phosphates are used as acidity regulator, emulsifier and stabilizer. these salts perform essential function in the production of cheese, because without salt-fin its production is impossible [2, 3]. however, phosphates are found in many foods and can cause adverse reactions (allergy), especially in children. the result of allergy is an altered mental reaction, impulsivity, motor restlessness, impaired concentration, increased aggressiveness food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 mariуa vorobets, igor kobasa, aureliуa cheban, application of titrimetric technique for determination of phosphates in cheeses, volume xiii, issue 4 – 2014, pag. 372 – 375 373 sometimes. frequent use of food additives e338, e339, e340, e341 in food leads to disturbances in the gastrointestinal tract, vomiting, diarrhea, nausea and aversion to food, weight loss [3, 4]. if a healthy person with food gets a lot of phosphates in her body disturbed calcium metabolism, begins osteoporosis (bones become brittle and relatively easily broken). the products with the addition of phosphate are very dangerous for pregnant women because it is likely to have a child with a variety of disorders in the brain and respiratory system [5, 6]. according to the committee on the rights of consumers over 80 % of food products, which are sold in the markets of ukraine, falsified. therefore, the development and improvement of methods of analysis, including analysis of the food additives content in the food products is an actual problem nowadays. for researches phosphates (food additives e338, e339, e340, e450, e452, etc.) traditionally are using the photometric method [7]. however, this method requires expensive reagents and instruments as well as highly qualified specialists. not all laboratories can to have such complex providing. volumetric analysis is a simple and affordable method, which is often used for studies of liquids. the aim of our investigation was to develop (or to adapt) known the titrimetric method of determination of phosphates in milk for research phosphates in hard and spread cheeses. 2. materials and methods the determination of phosphates in hard and spread cheeses by photometric method includes the next points: complete destruction of organic compounds under the action of sulfuric acid and hydrogen peroxide; addition of an ammonium molybdate solution in ascorbic acid; measurement of the optical density of blue phosphorus molybdenum complex [8]. the sample was prepared immediately before the definition. sample of cheese 50±1 g was crushed and stirring until a pasty mass. the sample we put in a kjeldahl flask, added concentrated sulfuric acid and were carried out a mineralization: were heated to form the minimum foam by avoiding local overheating and the release of foam from flask; cooled to room temperature; added 3÷4 drops of solution of hydrogen peroxide and heated again. this procedure repeated for as long as the content of the flask will become uncolored and transparent. to remove traces of hydrogen peroxide continued to heat the flask for 30 min, were cooled. the contents of the flask were transferred to a measuring flask of 100 cm3 and volume was adjusted to the mark with distilled water. using a pipette selected 1 cm3 of obtained mineralize substance and put into measuring flask of 50 cm3 volume; added a mixed solution of sodium molybdate and ascorbic acid. the volume of solution was adjusted to the mark with distilled water. the flask was put in a boiling water bath for 15 min. after cooling, the optical density was measured at the wavelength 820 nm in cuvettes with optical path length of 10 mm. the titrimetric method is simple, cheap and affordable. for develop a methodology of determining phosphates in cheeses by titrimetric method chosen method-prototype for milk. to find the mass of samples cheeses were taken into account: 1) for determination of phosphates in milk for analysis selected 20 cm3 of milk (known [9] that in 100 cm3 of milk is 90÷100 mg of phosphorus, i.e. in 20 cm3 of milk can be determined ≈20 mg of phosphorus); 2) based on acceptable standards: 100 g of spread cheese includes 2074 mg of phosphorus additives. the mass of cheese sample was found by calculation method: ).(1 074,2 02,0100 gx    food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 mariуa vorobets, igor kobasa, aureliуa cheban, application of titrimetric technique for determination of phosphates in cheeses, volume xiii, issue 4 – 2014, pag. 372 – 375 374 the sample preparation was carried out similarly as in the photometric definition. from the prepared samples were taken 1 g of cheese, added 20 cm3 of 0.1 m solution of sodium carbonate. ashing in a muffle furnace is an integral part of the preparation of samples. the methodology for milk about ashing of samples says nothing. based on experimental studies, we made adjustments. we propose before ashing samples in a muffle furnace to make the melting of the samples during slow heating (sand bath or electric stove), preventing the release of foam to full drying of cheese mass. then, make the ashing in a muffle furnace at a temperature 600°c for 1÷2 min. further analysis was performed according to the method for milk: an ash quantitatively transferred to a conical flask, was added 30 cm3 of 0.1 m solution of hydrochloric acid. the solution were evaporated on a sand bath to a volume of ≈15÷20 cm3 (all ash dissolves). according to the method for milk to solution add 2 drops of 0.1 % solution of methyl orange indicator. the contents of the flask was cooled and titrated by 0.1 m solution of sodium hydroxide to change color to yellow and then by drops add of 0.1 m solution of hydrochloric acid to a neutral reaction (orange color). our correction: instead of indicator methyl orange to use indicator phenolphthalein. it is necessary to pay special attention to bring the reaction to neutral. in liquid was added 20 cm3 neutral with respect to phenolphthalein and methyl orange 40 % solution of calcium chloride. the liquid was boiled for 2 min, adding 1÷2 drops of phenolphthalein and titrated by 0.1 m solution of sodium hydroxide to change color from yellow to pink. again were boiled for 2 min, titrated by 0.1 m solution of sodium hydroxide. this process repeats for as long as after heating, the solution remains pink. then added 2 drops of phenolphthalein and titrated by 0.1 m solution of sodium hydroxide to change the color of the contents of the flask with pink to red. if color for 10 min weakens to add a few drops of titrant. the content of phosphate acid (w, %) were calculated by the formula: %,100 1000 43 43     m env w pohnaohnaohpoh where naohv the amount solution of alkali (mean value), which went on titration, cm3; naohn concentration of alkali, mol/dm3; 43poh e molar equivalent weight of phosphoric acid (32.67 g/mol); m mass of sample product, g. 3. results and discussion we carried the experimental determination of phosphate in the samples of hard and spread cheeses of known domestic manufacturers by the developed methodology. were studied three samples of hard cheeses and three samples of spread cheeses. to confirm the correctness of the methodology and the reliability of the results, we conducted determination the content of phosphates in the same samples by photometric method according to [7]. the results are shown in table 1. table 1 phosphates in cheeses number of the sample phosphates (in terms of phosphoric acid). % titrimetric method photometric method hard cheeses 1. 1.16±0.05 1.19±0.04 2. 1.58±0.02 1.60±0.05 3. 1.09±0.01 1.10±0.015 spread cheeses 4. 1.80±0.03 1.85±0.02 5. 1.40±0.025 1.37±0.03 6. 1.60±0.03 1.59±0.035 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 mariуa vorobets, igor kobasa, aureliуa cheban, application of titrimetric technique for determination of phosphates in cheeses, volume xiii, issue 4 – 2014, pag. 372 – 375 375 comparative analysis of the results obtained by the developed method (titrimetric) and by the method according to [7] (photometric) show that the value of phosphates correlate with each other and differ only in the limits of errors. this clearly comparison chart demonstrates in fig. 1. fig. 1. the comparison chart of content of phosphates in the samples of cheeses 4. conclusions based on the methodology of the titrimetric determination of phosphates in milk developed a method for the titrimetric determination of phosphates in cheeses. by calculation method found mass of the samples (1 g) made adjustments for sample preparation and analysis. the correctness of the methodology and reliability of the results confirmed by determining the content of phosphate in the studied samples photometric method in according to dstu iso 3360: 2008 the method of titrimetric determination of phosphates can be used to determine their аs well as in hard and in spread cheeses. 5. references [1]. buldakov a. food additives. moscow: «ut», 240 p. (1996). [2]. e-resource http://dobavkam.net/products [3]. rosival l., engst r. foreign materials and food additives in foods. moscow: light and food industry, 264 p. (1982). [4]. smolar v. toxic effects of food additives. problems of nutrition, 5-15. (2005). [5]. sanpin 2.3.2.1293-2003. hygiene requirements for the use of food additives. (2003). [6]. donchenko l., nadykta v. safety of food raw materials and food. moscow: food industry, 352 p. (1999). [7]. dstu iso 3360:2008. phosphoric acid and sodium phosphates specifications (including for the food industry) (2008). [8]. inihov s., brio n. methods of analysis of milk and milk products. moscow: food industry, 156 p. (1971). [9]. milk and milk products: normat. documents: argument. / ed. v. ivanova. l.: nip leonorm, in 3 vols, 402 p. (2000). microsoft word 14 bg diana2 corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 88 assessment of the action of deposit mycoflora on phaseolus vulgaris l . beans from suceava genebank’s collection *diana batîr rusu1, danela murariu1 genebank suceava, romania dia_sv@yahoo.com, d_murariu@yahoo.com *corresponding author received 10 november 2011, accepted 25 february 2012 abstract: this study consisted in a phytopathological evaluation of epiphyte and endophyte mycological which appeared on phaseolus vulgaris seeds, placed on two types of substrates (cga medium and blotting paper) and emphasing corellations between the evolution of isolated micromycets on phaseolus vulgaris samples and different periods of storage. the 30 populations of bean resulted from the active collection of suceava genebank, were conserved for different time intervals (6 and 15 years), in controlled atmosphere storages (t=+40c; relative air humidity = 30 40%). micromycets were evaluated by counting the infected seeds, seeds and the attack frequency was expressed as a percentage, by visual estimation of seeds surface. the target objectives of the study were:  to establish the influence of the conservation period on the activity of micromycets placed on stored seeds;  to settle the influence of the substrate type cga medium (potato dextrose agar) and blotting paper on the development of fungal pathogens;  to establish a complementar action of identified micromycets on phaseolus vulgaris seeds in two storage periods, by determining the correlation coefficients between the action of fungal pathogens identified on the samples taken in study. keywords: micromycetse, landraces, cga medium, correlation coefficient 1. introduction the transmission of fungal pathogens by seeds has always been the most rapidly spreading of diseases from one region to another and therefore it’s need to know the symptoms, the biology and mode of transmission of pathogens, for once reported to be ensured effectively preventing and fighting them [1]. generally, the correlation between inoculum (spores load/seed) and colony size (the amount of mycelium) developed on cga medium (potato dextrose agar) is very significant [2]. viability of inoculum seed borne is in some cases directly related to interspace, harvesting storage analysis. some fungal pathogens are present and can be found in high percentages in freshly harvested seeds, but their infection it’s reducing significantly after one year of storage [3]. micromycets existing on stored legume seeds can cause during storage a wide range of changes, with negative consequences from a technological, nutritional, hygienic and commercial point of view [4]. beratlief and collaborators, in a study concerning the deposit ecosystem characteristics, revealed the mycological food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 89 flora evolution and sequence on legume seeds stored with high moisture content [5]. the purposes of this study are: to establish the influence of the conservation period on the activity of micromycets placed on stored seeds; to settle the influence of the substrate type cga medium (potato dextrose agar) and blotting paper on the development of fungal pathogens; to establish the complementary action of identified micromycets on phaseolus vulgaris seeds in two storage periods, by determining the correlation coefficients between the action of fungal pathogens identified on the samples taken in study. 2. experimental we have performed the phytopathological characterization of local germplasm represented by 30 populations of phaseolus vulgaris, conserved for 6 and 15 years at t = +4°c, which come from collecting expeditions realized by the collecting department from suceava genebank during a term of 10 years (19932003). lab experiments were carried on suceava genebank by using the genetic seminal material from the active collection of the institution, which was placed on the cga medium and blotting paper. to make possible the assessment of the micromycets present on phaseolus vulgaris seeds, we implemented the following research methods : macroscopic analyses of the seeds; ulster method [6] on cga medium (potato dextrose agar). interpretation of results concerning identified micromycets evolutions on bean seeds taken in study was achieved by analyzing correlations and regressions accordingly with experimental factors [7]. 3. results and discussion the seeds of phaseolus vulgaris, placed on cga medium and blotting paper, presented after the incubation period the following characteristics concerning the presence of fungal microorganisms: a) cga medium (potato dextrose agar) on cga medium, the presence of deposit mycoflora on the 30 samples of phaseolus vulgaris seeds conserved at +4 0c temperature, for 6 and 15 years was different, as follows: on the samples stored for 6 years at +4 0c temperature, we identified 10 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride, stachybotrys atra, stemphylium botryosum) which showed a different attack degree on each sample of the 21 analyzed, registering an infection rate of 70,5 % (444 infected seeds of 630 analyzed) (table 1). on 9 samples stored at +40c temperature, for a period of 15 years we identified 9 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride, stemphylium botryosum). the 270 seeds submitted to macroscopic and microscopic analysis presented an infection rate of 31,4 %, being infected 85 seeds. in these storage conditions, the genus stachybotrys not expressed at all. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 90 table 1 proportion of micromycets isolated on phaseolus vulgaris beans placed on cga medium b) blotting paper for emphasing the role of substrate used for analysis of micromycets occuring on phaseolus vulgaris seeds after different periods of storage, we used also blotting paper substrate. analyzing the 30 seed samples of phaseolus vulgaris stored at +40c temperature for 6 and 15 years we have identified the following infection percentages caused by fungal pathogens: on the samples stored for 6 years at +40c temperature we have identified 6 fungal pathogens (penicillium sp,. rhizopus sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride) which had a different attack degree on each sample of the 21 analyzed registering an infection rate of 57.7 % (table 2). on 9 samples conserved at +40c temperature for a period of 15 years we have identified 7 fungal pathogens (penicillium sp., aspergillus sp., rhizopus sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride). the 135 seeds submitted to macroscopic and microscopic analysis presented an infection rate of 30.3 %. table 2 proportion of micromycets isolated on phaseolus vulgaris beans placed on blotting paper revealing correlations between isolated micromycets evolution on phaseolus vulgaris samples taken in study and different storage periods (6 and 15 years) for establishment complementary action of micromycets identified on phaseolus vulgaris seeds in the two storage periods, we have determined correlation coefficients between fungal pathogens action identified on samples tahen in study (table 3). in the analyzed samples of phaseolus vulgaris, the results from the table related a few statistical correlations in both storage periods. after 6 years of storage of phaseolus vulgaris seeds at +4°c temperature, it’s noticed that there is one significant positive correlation between micromycets action: stemphylium botryosum x alternaria alternata. experimental conditions seeds stored at t + 40c, for 6 years seeds stored at t + 40c, for 15 years isolated micromycets attack frequency (%) penicillium sp. 11.1 11.8 aspergillus sp. 0.8 1.5 rhizopus sp. 20.3 6.3 epicoccum sp. 5.5 0.7 cladosporium herbarium 2.7 1.1 alternaria alternata 17.1 2.6 trichothecium roseum 5.8 1.5 trichoderma viride 2.5 3.7 stachybotrys atra 0.5 0 stemphylium botryosum 3.9 2.2 total 70.2 31.4 experimental conditions seeds stored at t + 40c, for 6 years seeds stored at t + 40c, for 15 years isolated micromycets attack frequency (%) penicillium sp. 7.6 8.8 aspergillus sp. 0 2.9 rhizopus sp. 18.1 6.6 cladosporium herbarium 3.2 3.7 alternaria alternata 14.3 5.2 trichothecium roseum 8.2 2.2 trichoderma viride 6.3 0.7 total 57.7 30.1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 91 tabel 3 correlation coefficients between micromycets action identified on phaseolus vulgaris samples stored at +4°c, for 6 years after 15 years of storage of phaseolus vulgaris seeds at +4°c temperature, there is only one very significant positive correlation between fungal pathogens action rhizopus sp. x aspergillus sp. (table 4). therefore, after 15 years of conservation is observed a constant presence of two fungal pathogens rhizopus sp. and aspergillus sp. for setting of the two micromycets action (aspergillus sp. x rhizopus sp.) present on seeds after 15 years of storage we traced the suitable regression straight (figure 1). this regression straight line out complementary action of the two saprophytic micromycets, meaning that while maintaining seeds at +40c temperature alongside genus aspergillus appears also rhizopus sp. table 4 correlation coefficients between micromycets action identified on phaseolus vulgaris samples stored at +4°c temperature. for 15 years r el at ed ch ar ac te rs p en ic ill iu m sp . a sp er gi llu s sp . r hi zo pu s sp . e pi co cc um sp . c la do sp or iu m h er ba ru m a lte rn ar ia al te rn at a t ri ch ot he ci um ro se um t ri ch od er m a vi ri de st ac hy bo tr ys at ra st em ph yl iu m bo tr yo su m penicillium sp. 1 aspergillus sp. 0.21 1 rhizopus sp. 0.10 0.18 1 epicoccum sp. -0.03 -0.18 -0.21 1 cladosporium herbarum -0.09 -0.00 -0.21 -0.19 1 alternaria alternata 0.01 0.11 -0.43 -0.04 -0.01 1 trichothecium roseum -0.26 -0.24 -0.08 0.27 -0.09 -0.47 1 trichoderma viride -0.40 -0.24 -0.42 -0.10 0.23 -0.05 0.27 1 stachybotrys atra 0.12 -0.12 -0.01 -0.06 -0.04 0.26 -0.13 -0.32 1 stemphylium botryosum -0.02 0.57 -0.36 -0.06 -0.12 0.56* -0.06 0.03 -0.16 1 r el at ed ch ar ac te rs p en ic ill iu m sp . a sp er gi llu s sp . r hi zo pu s sp . e pi co cc um sp . c la do sp or iu m h er ba ru m a lte rn ar ia al te rn at a t ri ch ot he ci um r os eu m t ri ch od er m a vi ri de penicillium sp. 1 aspergillus sp. 0.48 1 rhizopus sp. 0.43 0.923 *** 1 epicoccum sp. -0.16 -0.12 0.01 1 cladosporiu m herbarum -0.63 -0.25 -0.38 -0.25 1 alternaria alternata 0.30 -0.30 -0.46 -0.30 0.17 1 trichotheciu m roseum 0.06 -0.16 -0.25 -0.16 -0.33 -0.14 1 trichoderma viride -0.27 -0.06 -0.33 -0.06 0.69 0.47 -0.09 1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 92 y = 2,5625x + 0,75 r = 0,925 *** 0 2 4 6 8 10 12 0 1 2 3 4 5 număr seminţe infectate de aspergillus sp . n um ăr s em in ţe in fe ct at e de r hi zo pu s sp . fig. 1 the regression straight for correlation between number of infected seeds of aspergillus sp. and number of infected seeds of rhizopus sp. on phaseolus vulgaris samples stored in controlled environmental conditions (+ 40c) for 15 years 4. conclusions deposit mycoflora developed on bean seeds taken in this study was analyzed according to genotype period of seed conservation and type of substrate used. from our study resulted the following conclusions : 1. the seeds samples of phaseolus vulgaris stored in 2 experimental conditions placed on cga medium, were infected in different proportions by fungal pathogens. the species stachybotrys atra was identified only on the samples conserved for 6 years at + 40c temperature and the other types of micromycets (penicillium sp., aspergillus sp.,. rhizopus sp., epicoccum sp., cladosporium herbarum, alternaria alternata, trichothecium roseum, trichoderma viride, stemphylium botryosum) were detected in all storage conditions, but on a different number of seeds. 2. by placing the same seed samples of phaseolus vulgaris in 2 experimental conditions on blotting paper, we observed that samples were infected in a smaller proportion compared to cga medium. the fungal pathogens epicoccum sp., stemphylium botryosum, stachybotrys atra identified on cga medium were not isolated on blotting paper. 3. phaseolus vulgaris seeds stored for 6 years at +4°c temperature presented additional infections with the following micromycets: stemphylium botryosum x alternaria alternata, existing significant correlations between the action of these two fungal pathogens. 4. after 15 years of storage of phaseolus vulgaris seeds at +4°c temperature, there is a strong attack of rhizopus sp. x aspergillus sp., being a very significant correlation between the action of these two fungal pathogens. 5. references [1]. baker k.f., 1966. dynamics of seed transmission of plant pathogens. ann. rev. phytopathol.. 4. 311 [2]. hulea a., negru al., severin v. 1973. main seed crop diseases. ceres publishing. bucharest. 35 – 70 [3]. van der spek j., 1965. botrytis cinerea als parasiet van vals. wageningen. 146 [4]. nagy e., trif v., 1998. research regarding prevention and control of seed borne diseases at cereals in terms of transylvania. plant protection issues.bucharest. 26: 48 53 [5]. beratlief c., oprea m. 1994. ecosystem characteristics of agricultural products storages and health implications. plant protection issues. bucharest. 22: 13. 44 [6]. malone j.p., muskett a.e., 1941. the ulster method for the examination of flax seed for the presence of seed borne parasites. annals appl. biol.. 8 13. 28 [7]. ceapoiu n., 1968. statistical methods applied in agricultural and biological experiments. agro-forestry publishing. bucharesti title …………………… 255 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 3 2016, pag. 255 258 souring dynamics of the milk samples with different fat content * igor winkler1,2, marinela opaeț1 1department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine i.winkler@chnu.edu.ua 2department of medicinal chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine *corresponding author received july 17th 2016, accepted september 27th 2016 abstract: an influence of butterfat content on the souring dynamics has been evaluated for some commercial samples of drinking cow milk using the standard turner’s degree as an index of milk souring. no additional measurement of milk fat content was made, and density and other parameters were determined, being taken as indicated in milk packaging or described in the standards. it has been found that some buffering capacity of milk can be related to the butterfat content and the souring dynamics of high-fat milk is slower than that of low-fat milk. this effect can be caused by absorption of lactic acid and/or other acidic products formed by the souring processes on the butterfat globules, which is facilitated by close structural affinity between the butterfat and lactic acid molecules. even though the difference in the milk samples souring rate is not very significant, it can be influential within last days of the lowand high-fat milk validity term. no effect of the butterfat content on the final milk acidity value has been revealed – the samples with various acidity values exhibit various souring rates only. keywords: milk storage; milk souring; butterfat content; turner’s grade 1. introduction milk and various milk products is very important component of the nutrition since they contain a number of vital compounds required for normal functioning of the human organism [1-3]. on the other hand, raw milk and many untreated milk products are perishable and can spoil completely just within several days or weeks. an expiration date can be sufficiently postponed by various preservatives, which, however, can depreciate the consumer’s value of the products. that is why it is important to understand a dynamics of the changes occurring in the milk products during their storage period, especially near its end. milk acidity is an important index showing freshness and general value of the product. bad taste and odor are not the only problems of the soured milk. excessive acidity is usually associated with excessive amount of microbes and overstepping in some other milk product quality parameters. therefore, acidity of milk is a key indicator showing its conditions [4]. in this context, the dynamics of the milk souring has been investigated within the framework of the present work. milk acidity can be measured using various methods and units such as: turner’s http://www.fia.usv.ro/fiajournal mailto:i.winkler@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 igor winkler, marinela opaeț, souring dynamics of the milk samples with different fat content, food and environment safety, volume xv, issue 3 – 2016, pag. 255 – 258 256 degree (0t), soxhlet-henkel degree, dornic acidity degree and others. according to the legislation of ukraine [5], the turner’s degree should be used as a measure of the milk acidity and this requirement has been observed in our work. all technical details of 0t determination are described in [5]. souring of milk is a complex biochemical and chemical process depending on numerous conditions: temperature, other storage conditions, lactic acid bacteria nature and population, butterfat content and so on. various lactic sugars (mainly lactose) are being gradually transformed into lactic acid ch3-ch(oh)-cooh in course of the milk souring process. this is a weak hydroxy-acid (pка = 3.86 at 25 0с) [6]) causing a foul taste and odor, coagulation and layering of the spoiled milk. this acid is being produced as a result of the lactic bacteria activity and, according to the standards [5], its content in the drinking milk should be under 20 0t. on the other hand, there are several buffering systems of milk capable to restrain (to some extent) growing of the lactic acid content [7-9]. the following milk compounds can exhibit the buffering activity: phosphates with various hydrogen substitution degrees (monoand di-hydrogen phosphates), citric acid salts with various substitution degrees and hydrogen generating/accepting proteins [4, 7]. all these compounds can partially mitigate accumulation of lactic acid and retard a process of milk souring. it should also be mentioned that chemical composition of lactic acid and the butterfat compounds is similar. indeed, the butterfat compounds are mainly triglycerides with a ‘carboxyl’ head and three long aliphatic chains [10]. similar structure is known for lactic acid even though its aliphatic part is very short (fig. 1). figure 1. comparison of structural formulas of lactic acid (left) and butterfat (right). both compounds contain the carboxyl and the aliphatic parts. it is known that structural likeness between adsorbate and adsorbent facilitates better adsorption and, therefore, one can expect that lactic acid will be adsorbed on butterfat, which should counteract and retard gradual acidification of milk with higher fat content. therefore, the basic idea of this work was to check if there is any tangible effect of the milk fat content on the dynamic of its souring and, if so, how influential is this effect. these data can be useful in the storage management and consumption regime of different milk brands near the end of their validity terms. 2. experimental three ukrainian-made commercial drinking milk brands were taken as samples for this investigation. we used molochar” drinking milk (fat content 1.5 and 2.6 %), “molokiya” (1.6 and 2.5 %), “prostokvashyno” (2.5 %) and “bila liniya” (1.5 %). no additional determinations of the milk fat content, density and other parameters were made as we relayed on the milk manufacturers’ information provided on the packaging. such parameters should be strictly controlled during the milk production processes and it was beyond our food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 igor winkler, marinela opaeț, souring dynamics of the milk samples with different fat content, food and environment safety, volume xv, issue 3 – 2016, pag. 255 – 258 257 intention to substitute these standard milk quality control procedures. all the milk packs were purchased before the expiration date. then the milk samples were taken according to the following scheme: the “-1” sample was taken from a pack that was opened one day before its expiration date. the “0” sample was taken from a pack that was opened on its expiration date. the “+1” sample was taken from a pack that was opened one day after its expiration date. the “+2” sample was taken from a pack that was opened two days after its expiration date. all the packs were stored unopened under the temperature recommended for each type of milk. acidity of milk was determined in turner’s degree following the standard experimental procedure [5]. all acidity determinations were repeated at least 5 times for the reason of better reproducibility and then the relative error values were calculated for all the samples. none of them has exceeded 10 % throughout the entire series. 3. results and discussion all experimental results are summarized in table 1. table 1. acidification dynamics of various samples of drinking milk (*) brand sample (**) relative increase of acidity, % -1 0 +1 +2 molochar, 1.5 % 17.3 18.3 19 19.9 15.0 molochar, 2.6 % 17.1 17.4 18.1 18.9 10.1 prostokvashyno, 2.5 % 18.8 19.4 20.1 21.1 12.3 molokiya, 1,6 % 19.8 20.4 21.8 22.3 15.9 molokiya, 2.5 % 19.1 21.8 23.1 23.9 24.9 bila liniya, 1.5 % 20 20.6 21.3 22.2 10.8 (*) all low-fat milk data are given in italic for the reason of clarity. (**) calculated as a difference between the “+2” and “-1” values divided by the former. example: the molochar, 1.5 % value is obtained as: (19.9-17.3)/17.3 =0.15 (15 %). let us analyze the data of table 1. first of all, it can be noted that quality of the “molokiya” brand is very poor both for the low-fat and high-fat samples. these samples show the sub-threshold acidity even one day before the expiration date. one day later it exceeds the limit value and keeps growing rapidly for the both samples. this can be caused by inappropriate quality of the raw materials or incompliance between the standards and real production scheme for this milk. acidity level of the “bila liniya” brand was close the limit on the “-1” day and then it kept growing but in this case the acidity growing rate was quite slow and two days after the expiration date this value became only 10 % higher than the limit. this can be caused by a deeper pasteurization or by preservatives added to this milk brand. now we can compare dynamics of the milk acidity growing for the lowand high-fat samples. the mean value of the low-fat samples acidity growing is 13.9 % and it is 11.2 % for the high-fat samples. in our opinion, this difference can be caused by additional buffering effect of the butterfat globules as described above. that is why it can be concluded that some retardation effect of the butterfat globules actually takes place during its souring. milk with the higher food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 igor winkler, marinela opaeț, souring dynamics of the milk samples with different fat content, food and environment safety, volume xv, issue 3 – 2016, pag. 255 – 258 258 butterfat content resists acidification better than the low-fat milk. this fact should be taken into consideration to ensure proper storage management of different drinking milk batches. it should also be noted that there is no effect of the milk fat content on its final acidity level – only the rate of souring is influenced by the milk acidity value. 4. conclusion determination of the turner’s acidity degree can be used to evaluate milk souring dynamics close to its shelf life. this dynamics is higher for low-fat milk as compared to that of high-fat samples. this means that acidification of low-fat milk takes place faster than the process of the high-fat milk if other storage and pretreatment conditions are similar. in our opinion, this difference is caused by adsorption of lactic acid on the butterfat globules. 5. references [1]. nikerson t. a., chemical composition of milk. j. dairy sci., 43(5): 598-606, (1960). [2]. bodkowski l., et al, lipid complex effect on fatty acid profile and chemical composition of cow milk and cheese. j. dairy. sci., 99(1): 57-67, (2016). [3]. claeys w. l., et al, consumption of raw or heated milk from different species: an evaluation of the nutritional and potential health benefits. food control, 42: 188-201, (2013). [4]. walstra p., et al, dairy technology: principles of milk properties and processes. marcel deker, inc. 794 p. new york, basel, (1999). [5]. dstu 2661:2010, drinking caw milk. general technical conditions. state standards of ukraine. 6 p. kyiv, (2011) (in ukrainian). [6]. chemist’s reference book. vol. 2. ed.: nikolsky b. p., khimiya. p. 82. moscow, (1965) (in russian). [7]. salaun f., mietton b., gaucheron f., buffering capacity of dairy products. int. dairy j., 15(2): 95-109, (2005). [8]. hejlasz z., nicpon j., buffer systems, protein and electrolyte indices in calves fed with beestings and milk substitutes. polish arch. weter., 24(2): 229-237, (1984). [9]. park y. w., relative buffering capacity of goat milk, cow milk, soy-based infant formulas, and commercial non-prescription antacid drugs. j. dairy sci., 74(10): 3326-3333, (1991). [10]. kuksis a., mccarthy m. j., beveridge m. r., quantitative gas liquid chromatographic analysis of butterfat triglycerides. j. amer. oil chem. soc., 40(10): 530-535, (1963). 1. introduction 4. conclusion 322 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 322 327 effect of citrus fibers addition on wheat flour dough rheological properties *silvia mironeasa1, georgiana gabriela codină1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania *silviam@fia.us.ro; codina@fia.usv.ro *corresponding author received october 15th 2013, accepted december 5th 2013 abstract: nowadays, demand for food products with high fiber content is increasing because fiber consumption improves health problems such as diabetes, cardiovascular diseases, colon cancer etc. the citrus such as orange, lemon, and grapefruit can been used as source of fiber for bread-making products. the quality of bakery products is influenced by the wheat flour dough rheological properties. the addition of different ingredients like citrus fiber affects dough rheology and dough processing as function of the fiber level addition. the effects of citrus fiber addition to wheat flour at the levels of 0, 2, 4 and 6 % on the rheological behaviour of the dough obtained were investigated using as rheological devices farinograph and amylograph. dough rheological parameters measured were water absorption (wa), development time (dt), stability (st), degree of softening (sdg), gelatinization temperature (g_t) and peak viscosity (p_v). the fiber addition has the effect of increasing farinograph water absorption simultaneously with the increase of the addition level. an increase in development time and stability were recorded upon addition of citrus fiber  4 %. also, the amylograph parameter peak viscosity increased with increase of the citrus fiber level. keywords: citrus fibers, rheological dough parameters, wheat flour, farinograph, amylograph 1. introduction in the recent decades, dietary fiber has received great attention from researchers in food science and nutrition. the term dietary fiber comprises to major classes: soluble fiber in water such as pectin, gum and mucilage, which cannot be digested by the human digestive enzymes and water insoluble fiber, i.e. cellulose, lignin and some of the hemicelluloses to absorbable components in the upper alimentary tract [1]. many studies dealing with the nutritional and physiological aspects of dietary fiber has led to their incorporation in food products, such as bakery, breakfast cereals, meat products, pasta and yogurts [2]. the high functional value of dietary fiber has been well studied over the years [3-5]. a large number of studies have been reported on the physiological actions of fiber addition in foods such as, the maintenance of gastrointestinal health, reduction of intestine transit time, protection against colon cancer, lowering of total and low-density lipoprotein cholesterol in the blood serum, reduction of postprandial blood glucose levels, increase of calcium bioavailability and reinforcement of the immunological system [1], [6]. also, the increased of food consumption with dietary fiber are associated with the prevention, reduction and treatment of some diseases, such as diverticular and coronary heart diseases [7], improves blood glucose control in diabetes, promotes regularity, aids in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silvia mironeasa, georgiana gabriela codină, effect of citrus fibers addition on wheat flour dough rheological properties, volume xii, issue 4 – 2013, pag. 322 327 323 weight loss, and appears to improve immune function [8]. nowadays, demand for foods products with ingredients which contain bioactive compounds that provide additional health benefits are increasing. bread is the most popular food consumed daily by the majority of the population and to meet this requirement, the development of enriched bread with bioactive compound such as, dietary fiber should be the best way to increase the fiber intake. from the point view of the daily intake of total fiber for adults, is recommended 38 g for men and 25 g for women [9]. dietary fiber plays an essential role in human health not only for their nutritional properties, but also for their functional and technological properties. the functional properties of dietary fiber derived from different sources should be studied in order to obtain the individual characteristics of each one [10]. adding different fibers in wheat flour dough, in order to improve the nutritional quality of bread will affect the rheological properties of dough. the evaluation of rheological properties of wheat flour dough is decisive for bread making technology because its provide information’s regarding dough behaviour during the bread making process and the quality of the finished bakery products [11] bread can be enriched with many form of dietary fiber such as wheat bran, oat bran, rice bran, corn bran, carob fiber, peanut hull, sunflower hull, flaxseed [12-15] e.g. when added to bread dough, the fiber can change its consistency, texture, rheological behavior and the properties of the finished products [16-18]. many studies have been reported on rheological properties of dough supplemented with fibers from different sources [19-25]. in recent years, dietary fiber from fruits has received increasing attention from researchers due to their considerable beneficial effects on the human body. the citrus fiber from oranges, lemons and grapefruits constitute rich sources of dietary fiber with well-balanced proportion of soluble and insoluble fiber fractions [26]. comparatively with the dietary fibers from other sources, such as cereals, the main advantage of citrus fiber is its higher proportion of soluble dietary fiber, about 33%, while only 7% is present in wheat bran [27], [28]. also, citrus fibers have an additional advantage due the presence of associated bioactive compounds such as flavonoids, polyphenols, vitamina c and others [29] with antioxidant properties, which may exert further health promoting effects in addition to those of the dietary fiber itself [30]. therefore, citrus fiber can named as antioxidant dietary fiber. in the preparation of bread, some amount of wheat flour can be replaced with citrus fiber, but the main problem of this substitution in bread dough is the detrimental effects on dough handling due to changes in dough rheological properties. incorporation of fiber into wheat flour interacts directly with structural elements of the three dimensional gluten network and disrupts the starch-gluten matrix, affecting the rheological behaviour of blended dough during mixing, sometime causing negative effect on the finished bread quality [31]. the effect of citrus fiber on dough and bread quality depends greatly on citrus fiber properties. the aim of this experimental work was to assess the rheological properties of wheat flour-citrus fiber blends (0, 2, 4, and 6%, w/w of flour addition with citrus fiber) by using the rheological devices farinograph and amylograph. in addition, the amylase activity in the samples has been tested. 2. materials and methods 2.1. materials the wheat flour used in the research are 550 type wheat flour (harvest 2012) milled at s.c. dizing s.r.l. brusturi, neamţ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silvia mironeasa, georgiana gabriela codină, effect of citrus fibers addition on wheat flour dough rheological properties, volume xii, issue 4 – 2013, pag. 322 327 324 county, romania. as a fiber source was used commercial citrus fiber purchased from s.c. enzymes@derivates romania (costisa, neamt, românia). 2.2. preparation of wheat flour citrus fiber blends citrus fiber (cf) was added in wheat flour in different doses: 0, 2, 4 and 6 (g/100g). the wheat flour sample without fiber was considered as the control one. 2.3. wheat flour quality evaluation the control flour sample was analyzed from proximate composition by near infrared reflectance spectroscopy (nir) technique using inframatic flour analyzers, model 9140, perten instruments. the determined values of it analytical characteristics are the following: moisture 14.20%, protein content 12.50%, water absorption 56.40%, zeleny sedimentation index 40.10% and wet gluten content 29.10%. also, the hagberg falling number method (icc method 107/1) to measure flour -amylase activity was used. 2.4. dough rheological properties measurement by farinograph and amylograph the rheological properties of the wheat flour and various flour blends with cf were conducted using a farinograph with a 300 g capacity (brabender gmbh and co. duisburg, germany) according to sr iso 5530-4:2005. water absorption (wa) is the amount of water required for dough to reach an arbitrarily chosen consistency (500 brabender unit (bu) consistency), dough development time (dt), dough stability (st) and the dough degree of softening (sdg) were analyzed. wheat flour dough viscometric characteristics were carried out using an amylograph (brabender ogh, duisburg, germany) device according to sr iso 7973:2000. peak viscosity and gelatinization temperature were the parameters analyzed. 2.5. statistical analysis all analyses were performed in triplicate and the data obtained were analyzed by the statistical software spss 16.0 (spss inc., chicago, il, usa). the figures plotted with spss shows the variation of the rheological parameters as a function of citrus fiber addition. 3. results and discussion 3.1. effect of citrus fiber addition on farinograph properties the effect of citrus fiber addition on farinograph parameters of wheat flour with different fiber addition is showed in figure 1. figure 1. farinograph characteristics of wheat flour-citrus fiber blends: st, dough stability (min); dt, dough development time (min); sdg, softening degree of dough (bu); wa, water absorbtion (%). the results show that the addition of cf significantly influenced all the wheat flour dough rheological properties as measured by the farinograph. as the amount of citrus fiber increased, the water absorption food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silvia mironeasa, georgiana gabriela codină, effect of citrus fibers addition on wheat flour dough rheological properties, volume xii, issue 4 – 2013, pag. 322 327 325 increase for all the samples with citrus fiber added. the water absorption (wa) of the blended dough increased as function of citrus fiber concentration and followed a linear relationship (1): wa = 3.34  cf + 56.13; r2 = 0.99 (1) the wa of control sample was 56.6 % which gradually increased to 76.8 % for the sample with 6 g/100g cf incorporated. the increased is due to probably of a great number of hydroxyl groups existing in the fiber structure, which allow more water interactions through hydrogen bonding [32], [33]. this behaviour is attributed to citrus fiber ability to retain water within its matrix. these results are in agreement with the results obtained by many studies made on various fiber added in wheat flour doughs [34-36]. highest values in the farinograph parameters development time (dt) and dough stability (st) were observed in wheat flour dough in were was incorporated 4% cf. addition of citrus fiber above a dose of 4% decreased dough development time and stability. softening degree of dough was also found to increase with the addition of the citrus fiber. these results may be explained by the fact that citrus fiber addition reduces the wheat flour gluten network formation rate. similar results on the effect of addition of dietary fibres on the farinographic properties of wheat flour were reported by almeida, chang & steel (2010). 3.2. effect of citrus fiber addition on amylograph properties incorporation of citrus fiber at 0%, 2%, 4% and 6% levels showed differences on the dough pasting properties as measured by amylograph. the results are indicated in figure 2. the presence of the citrus fiber significantly affected the pasting properties of the flour blends. figure 2. amylograph characteristics of wheat flour-citrus fiber blends: g_t, gelatinization temperature (oc); p_v, peak viscosity (bu). the results show that the increasing addition level of cf reduced the gelatinization temperature (g_t), the lowest gelatinization temperature values being recorded to a 6% dose of citrus fiber addition. also, the peak viscosity (p_v) increased simultaneously with the increase dose of citrus fiber addition in wheat flour dough. these fact may be attributed to a more quickly starch gelatinization due to a higher water level in the dough samples with a higher fiber content incorporated. 3.3. effect of citrus fiber addition on falling number index value effect of citrus fiber on the amylase activity and consequently on dough viscosity are illustrated in figure 3. figure 3. falling number index (fn) as a function of citrus fiber (cf) addition. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silvia mironeasa, georgiana gabriela codină, effect of citrus fibers addition on wheat flour dough rheological properties, volume xii, issue 4 – 2013, pag. 322 327 326 with the increase dose of citrus fiber content to 6%, the fn index increased from 351 to 398 s. therefore, an increase in fn index indicates that an increase in citrus fiber content in the wheat flour dough has decreased it’s -amylase activity. this may be due to the ability of the starch granule and of the citrus fiber fraction to retain water and swell freely, transforming into a mass with increasing viscosity. 4. conclusions citrus fiber additions had significant effects on dough rheological behavior, increasing the farinograph parameter water absorption capacity. the farinograph parameters of fiber-enriched dough provided useful information on the suitability of citrus fiber incorporation levels, from its parameters development time, dough stability and degree of softening. it clearly indicates that citrus fiber incorporation above 4g/100g has negative impact on dough development time and dough stability. incorporation of citrus fiber did also influence the amylograph parameters gelatinization temperature and peak viscosity, showing a decrease in -amylase activity. the results obtained showed the fact that citrus fiber can be added to wheat flour at a level of 4% without negative influence on rheological properties of the wheat flour dough. 5. references [1]. thebaudin j. y., lefebvre a. c., harrington m., bourgeois c. m., dietary fibres: nutritional and technological interest, trends in food science and technology, 8. 41–49, (1997) [2]. gelroth j., ranhotra g. s., food uses of fibre. in: s. sungsoo cho, m. s. dreher (eds.), handbook of dietary fibre, new york: taylor and francis (2001) [3]. eshak e.s., iso h., date c., kikuchi s., watanabe y., wada y., wakai k., tamakoshi a., dietary fiber intake is associated with reduced risk of mortality from cardiovascular disease among japanese men and women, journal of nutrition, 140. 1445-1453, (2010) [4]. king d.e., dietary fiber, inflammation, and cardiovascular disease, molecular nutrition & food research, 49. 594-600, (2005) [5]. o’neil c.e., zanovec m., cho s.s., nicklas t.a., whole grain and fiber consumption are associated with lower body weight measures in us adults: national health and nutrition examination survey 1999-2004, nutrition research, 30. 815-822, (2010) [6]. tungland b.c., meyer d., nondigestible oligo and polysaccharides (dietary fibre): their physiology and role in human health and food, comprehensive reviews in food science and food safety, 1. 73–92, (2002) [7]. figuerola f., hurtado m.l., estevéz a.m., chiffelle i., asenjo f., fibre concentrates from apple pomace and citrus peel as potential fibre sources for food enrichment, food chemistry, 91. 395401, (2005) [8]. anderson j.w., baird p., davis r.h., ferreri s., knudtson m., koraym a., waters v., williams c.l., health benefits of dietary fiber, nutrition reviews, 67. 188-205, (2009) [9]. trumbo p., schlicker s., yates a., poos m., dietary reference intakes for energy, carbohydrate, fibre, fat, fatty acids, cholesterol, protein, and amino acids, journal of the american dietetic association, 102. 1621–1630, (2002) [10]. lópez g., ros g., rincón f., periago m.j., martínez m.c., ortuňo j., relationship between physical and hydration properties of soluble and insoluble fiber of artichoke, journal of agricultural and food chemistry, 44. 2773–2778, (1996) [11]. bloksma a.h., bushuk w., rheology and chemistry of doughs, p. 131–218 in: wheat: chemistry and technology, y. pomeranz, ed. 3rd ed. aacc, st. paul, mn, usa (1988). [12]. anil m., usage of flaxseed as a source of dietary fiber in breadmaking, phd thesis, ondokuz mayis university, samsun, turkey, (2002) [13]. őzkaya b., effects of sugar beet dietary fiber of different particle size on rheological properties of dough and quality of bread, gıda teknolojisi, 2. 57–64, (1997) [14]. glitsøt l. v., bach knudsen k.e., milling of whole grain rye to obtain fractions with different dietary fibre characteristics, journal of cereal science, 29. 89–97, (1999) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silvia mironeasa, georgiana gabriela codină, effect of citrus fibers addition on wheat flour dough rheological properties, volume xii, issue 4 – 2013, pag. 322 327 327 [15]. uzunkaya d.f., ercan r., the possibilities of using high fiber additives in hamburger buns, gıda, 24. 369–377, (1999) [16]. wang j., rosell c.m., benedito c., effect of the addition of different fibres on wheat dough performance and bread quality, food chemistry, 79. 221-226, (2002) [17]. sangnark a., noomhorm a., effect of dietary fiber from sugarcane bagasse and sucrose ester on dough and bread properties, lwt food science and technology, 37. 697-704, (2004) [18]. sangnark a., noomhorm a., chemical, physical and baking properties of dietary fiber prepared from rice straw, food research international, 37. 66-74, (2004) [19]. angioloni a., collar c., physicochemical and nutritional properties of reduced-caloric density high-fibre breads, lwt food science and technology, 44. 747-758, (2011) [20]. anil m., using of hazelnut testa as a source of dietary fiber in breadmaking, journal of food engineering, 80. 61-67, (2007) [21]. sudha m.l., baskaran v., leelavathi k., apple pomace as a source of dietary fiber and polyphenols and its effect on the rheological characteristics and cake making, food chemistry, 104. 686–692, (2007) [22]. barros f., alviola j. n., rooney l.w., comparison of quality of refined and whole wheat tortillas, journal of cereal science, 51. 5056, (2010) [23]. gomez m., moraleja a., oliete b., ruiz e., caballero p.a., effect of fibre size on the quality of fibre-enriched layer cakes, lwtfood science and technology, 43. 33-38, (2010) [24]. salmenkallio-marttila m., katina k., autio k., effects of bran fermentation on quality and microstructure of highfiber wheat bread, cereal chemistry, 78. 429-435, (2001) [25]. sullivan p., o’flaherty j., brunton n., arendt e., gallagher e., fundamental rheological and textural properties of doughs and breads produced from milled pearled barley flour, european food research and technology, 231. 441-453, (2010) [26]. kohajdová z., karovičová j., jurasová m., influence of grapefruit dietary fibre rich powder on the rheological characteristics of wheat flour dough and on biscuit quality, acta alimentaria, 42. 91-101, (2013) [27]. gorinstein s., martín-belloso o., park y.s., haruenkit r., lojek a., ciz m., caspi a., libman i., trakhtenberg s., comparison of some biochemical characteristics of different citrus fruits, food chemistry, 74. 309–315, (2001) [28]. griguelmo-miguel n., martiｴ nbelloso o., comparison of dietary fibre from byproducts of processing fruits and greens and from cereals, lebensmittelwissenschaft und technologie, 32. 503–508, (1999) [29]. schieber a., stintzing f. c., carle r., by-products of plant food processing as a source of functional compounds-recent developments, trends in food science & technology, 12. 401–413, (2001) [30]. marín f.r., frutos m.j., pérezalvarez j.a., martínez-sánchez f., del río j.a., flavonoids as nutraceuticals: structural related antioxidant properties and their role on ascorbic acid preservation. in e. atta-ur-rahman (ed.), studies in natural products chemistry, p. 741, amsterdam, elsevier, (2002) [31]. ahmed j., almusallam a.s., alsalman f., abdulrahman m.h., alsalem e., rheological properties of water insoluble date fiber incorporated wheat flour dough, lwt food science and technology, 409–416, (2013) [32]. wang j., rosell c.m., benedito de barber c., effect of the addition of different fibres on wheat dough performance and bread quality, food chemistry, 79. 221–226, (2002) [33]. rosell c.m., rojas c., benedito de barber c., influence of hydrocolloids on dough rheology and bread quality, food hydrocolloids, 15. 75-81, (2001) [34]. sudha m.l., baskaran v., leelavathi k., apple pomace as a source of dietary fiber and polyphenols and its effect on the rheological characteristics and cake making, food chemistry, 104. 686-692, (2007) [35]. ajila c.m., leelavathi k., rao u., improvement of dietary fiber content and antioxidant properties in soft dough biscuits with the incorporation of mango peel powder, journal of cereal science, 48. 319–326, (2008) [36]. larrauri j. a., rupe´rez p., bravo l., saura-calixto f., high dietary fibre powders from orange and lime peels: associated polyphenols and antioxidant capacity, food research international, 29. 757–762, (1996) [37]. almeida e.l., chang y.k., steel c.j., effect of adding different dietary fiber sources on farinographic parameters of wheat flour, cereal chemistry, 87. 566-573, (2010) microsoft word 12 hutanu florentina.doc 78 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 78 83 m odified prus sian blu e scr een pr inted electrod es fo r h2 o2 detec tion florentina hutanu1, gheorghe gutt1* 1stefan cel mare university of suceava, faculty of food engineering, universității street, 720229, suceava, romania, g.gutt@usv.ro *corespending author received 10 january 2013, accepted 21 february 2013 abstract in this work, presents recent developments in the electrochemical application of disposable screen-printed sensors, to the type of materials used to modify the working electrode.the sensor was based on the electrocatalytic reduction of h2o2 on prussian blue modified screen-printed electrode. a comparative study regarding different procedures for modifying the carbon screen-printed electrodes (spe) with prussian blue (pb) was carried out in this work. two procedures for pb deposition on the spe electrodes were tested: electrochemical deposition (galvanostatic, cyclic voltammetry) and chemical deposition by the reaction of k3[fe(cn)6] with fecl3.also, the influence of the pretreatment of spe (+1.7v for 3 min in pbs, ph 7.4) and of the stabilization of pb deposited on spe (by heating at 100°c) were evaluated. the developed sensors were optimized with respect to the lowest limit of detection achieved for amperometric detection of h2o2. analytical parameters, such as detection limit, linearity range and sensitivity have been evaluated, together with operational and storage stability. the improved electro-deposition methods, ph stability and permeability of the optimized pb film provide a further boost in its sensitivity for h2o2 detection, which is a critical parameter in biosensor design and application. keywords: screen printed electrodes, h2o2 determination, prussian blue. 1. introduction in 1978, neff [1] reported a thin layer of prussian blue deposition on platinum foil by chemical method. prussian blue, is a prototype of mixed-valence transition metal hexacyanoferrates, has been widely used as an electron transfer mediator in amperometric biosensor due to its for electrocatalysis.[2-5] only 4 years later, itaya et al. [6] showed that the reduced from of prussian blue (also called prussian white, pw) had a catalytic effect on the reduction of both o2 and h2o2, many publications have appeared exploring its electrocatalytic, electromagnetic and electrochemic properties [7]. itaya et al. [8] demonstrated the most important feature of prussian blue (in terms of analytical application). it was in fact shown that the reduced from of prussian blue (also called prussian white) had a catalytic effect for the reduction of o2 and hydrogen peroxide. many efforts have been made to improve the selectivity of carbon based electrochemical sensors, mainly through their modification with redox mediators [9]. however, direct hydrogen peroxide food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florentina hutanu, gheorghe gutt, modified prussian blue screen printed electrodes for h2o2 detection, food and environment safety, volume xii, issue 1 – 2013, pag. 78 83 79 amperometric detection at conventional electrodes is only possible 0.6 v vs. ag/agcl. at this potential, the presence of easlily oxidisable compounds such as ascorbate, bilirubin, urate etc. can easily interfere in the measurements, being oxidised at the electrode together with hydrogen peroxide [10]. prussian blue modified electrodes represent very attractive detectors for hydrogen peroxide because they work at an applied potential around 0 v vs. ag/agcl with no other electrochemical interferences [11]. objectives the aim of this work was to compare three procedures for pb film formation on the working electrode of screen printed carbon electrodes in order to prepare sensitive and robust pb sensors for h2o2 determination. the tested procedure were based on: chemical, galvanostatic and cyclic voltammetry based deposition. recently it was reported that the surface modification of electrodes with pb in presence of added surfactant offers enhanced film growth, improved stability and excellent electrochemical reversibility [13]. this approach also increases the pb sensor stability in neutral and basic media. although, studies to date have focused mainly on the use of cationic surfactants like cetyltrimethyl ammonium bromide (ctab), in this work we have studied the influence of the anionic surfactant dioctyl sulfo-succinate sodium salt (aot) on the electrochemical properties of the pb film deposed on screen printed carbon electrode. 2. materials and methods 2.1. apparatus electrochemical measurements were carried out using a µ autolab type iii potentiostat/galvanostat computer controlled by the gpes software, as well as a portable palmsens potentiostat/ galvanostat controlled via the palmsenspc software. 2.2. reagents all chemicals from commercial sources were of analytical grade. iron chloride (fecl3), potassium ferricyanide k3[fe(cn)6], hcl 37%, sodium chloride, hydrogen peroxide (30%), were purchased from sigma-aldrich. aot (dioctyl sulfosuccinate sodium salt) was from carlo erba. double-distilled water was used throughout. 2.3. electrodes screen-printed carbon electrodes (spces) model drp-110 purchased from dropsens (spain) were used for electrochemical measurements. in this case the electrochemical cell is composed by a graphite working electrode (d = 4mm), a graphite auxiliary electrode and a silver pseudoreference electrode, with silver electric contacts deposed on a ceramic substrate. in this case the electrochemical cell is composed by a graphite working electrode (d = 4 mm), a graphite auxiliary electrode and a silver pseudoreference electrode, with silver electric contacts and ceramic substrate. these electrodes are for working with microvolumes, for decentralized assays or to develop specific sensors.the electrodes were produced in 75 units packs. each sensor consists of three printed electrodes, a carbon working and two silver electrodes, acting as counter and pseudoreference, respectively. screen-printed electrodes [12] are frequently used in analytical applications because of their unique properties such as small size, low detection limit, fast response, high reproducibility, etc. [13]. advantages of screen-printed electrodes is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florentina hutanu, gheorghe gutt, modified prussian blue screen printed electrodes for h2o2 detection, food and environment safety, volume xii, issue 1 – 2013, pag. 78 83 80 that they are inexpensive, simple to prepare, versatile and suitable for the mass-production of disposable electrodes [14] 2.4 chemical deposition of prussian blue prior to prussian blue modification, screen-printed electrodes were pretreated in presence of 50 mm phosphate buffer in 0.1 m kcl, ph 7.4, by applying the potential of + 1.7 v versus ag/ag cl for 3 minute [15]. for the chemical deposition of pb films two solutions were prepared. solution 1: 100 mm k3[fe(cn)] in solution 10 mm hcl. solution 2: 100 mm fecl3 in solution 10 mm hcl prussian blue modification of screen printed was then accomplished by placing 10 µl onto the working electrode area. the drop was carefully placed exclusively on the working electrode area, in order to avoid the formation of pb on the reference and counter electrodes which may increase the internal resistance of the system. the solution was left onto the electrode for 10 min and then rinsed with a few militers of 10 mm hcl. the electrodes were then left 90 min in the oven at 100° c to obtained more stable and active layer of prussian blue. [14] 2.5. electrochemical deposition of prussian blue prior to prussian blue modification, screen-printed electrodes were pretreated as in previous section. pb modification of spe eas a accomplished by placing a drop (40 ml of total volume) of precursor solution onto the working electrode area. the deposition was made in a mixture kcl 100 mm of 0.1 m k3[fe(cn)6,], 0.1m fecl3 prepared in 100 mm kcl and 100 mm hcl solution by applying the potential of 0.4v for 40 sec [15]. after a gentle rinsing with water, the sensor was placed in a solution of 100 mm kcl in 100 mm hcl and a number of 20 cycles, between 0.2 and 0.4 v, at a scan rate of 50 mv/s. after some deposition cycles, the pb modified electrode was dried at 100° for 1 h to obtain a more stable and active pb layer [16] 3. results and discussion 3.1. prussian blue deposition the influence of the pre-treatment and thermal stabilization steps in the chemical and electrochemical procedures for pb deposition on the spe electrodes were tested. -0.1 0.0 0.1 0.2 0.3 -1.5x10-4 -1.0x10-4 -5.0x10-5 0.0 5.0x10-5 1.0x10-4 i( a ) e(v) vc in kcl 0.1m in hydrochloric acid 0.1m spe-ii-pretreated spe-ii a (procedure i) -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -2.0x10-4 -1.5x10-4 -1.0x10-4 -5.0x10-5 0.0 5.0x10-5 1.0x10-4 1.5x10-4 i( a ) e(v) p ii with pretreatment p ii without pretreatment b (procedure ii) fig. 1. cyclic voltammograms obtained for the spce modified pb using the chemical andelectrochemical procedure 0.1 m kcl in 0.1m hcl; 50mv/s. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florentina hutanu, gheorghe gutt, modified prussian blue screen printed electrodes for h2o2 detection, food and environment safety, volume xii, issue 1 – 2013, pag. 78 83 81 in fig. 2 are presented the cyclic voltammograms obtained for the spce modified pb using the chemical procedure in presence and absence of the anionic surfactant aot at concentartion h2o2 3mm. the pb sensor prepared in presence of aot showed for the first redox couple higher iox and ired. -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 -2,0x10-4 -1,5x10-4 -1,0x10-4 -5,0x10-5 0,0 5,0x10-5 1,0x10-4 1,5x10-4 2,0x10-4 i( a ) e(v) spe/pb deposition chemical without aot vc h 2 o 2 3mm spe/pb deposition chemical with aot vc h 2 o 2 3mm figure 2. cyclic voltamograms, in kcl 0.1 m in hcl 0.1 m; the scane rate 50 mv/s, modified pb/aot using the chemical procedure. -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 -2,5x10-4 -2,0x10-4 -1,5x10-4 -1,0x10-4 -5,0x10-5 0,0 5,0x10-5 1,0x10-4 1,5x10-4 2,0x10-4 2,5x10-4 i( a ) e(v) p-i vc in h2o2 1mm in phosphate buffer ph6,5 p-ii vc in h2o2 1mm in phosphate buffer ph6,5 p-iii vc in h2o2 1mm in phosphate buffer ph6,5 p-iv vc in h2o2 1mm in phosphate buffer ph6,5 figure 3. cyclic voltammograms of pb sensor 0.1 m kcl in hcl; e= -50mv/s, phophate buffer ph 6,5, h2o2 1mm. the influence of the thermal stabilization by keeping the electrodes at 100° for 90 min was also studied [17] no evident differences between the treated and nontreated pb electrodes were observed regarding the response of the electrodes in kcl, phosphate buffer or for h2o2, but the operational stability was greater improved for the electrodes stabilized via the thermal treatment. 0 5 10 15 20 25 -600 -400 -200 0 200 400 i( a ) v1/2 p ii without pretreatment i ox =-41.1321+18.2348x r=0.9991 ired=32.3735-22.9104x r=0.9998 figure 4. variation of oxidation and reduction vs. square root of scan rate in electrolyte solution 0.1m kcl, 0.1m hcl plotting the oxidation and reduction vs. square root of scan rate showed a linear relationship (fig. 4), the result indicating that diffusion of electrolyte across the pb layer controls the electrode process. this behaviour was observed for all the two tested procedures used for pb deposition. 3.2. electrocatalytic reduction of h2o2 the ph influence on the electrochemical determination of h2o2 using the pb modified spce was studied at ph ranging from 6.5 (fig. 3). for all the tested electrodes the highest reduction peaks were obtained for the ph 6.5. in order to select the working potential to be applied when measuring h2o2 it was used the amperometry technique. therefore, we selected the -50 mv as working potential, considering both the response intensity and operational stability. the applied potential used -50 mv was selected in our previous paper [18] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florentina hutanu, gheorghe gutt, modified prussian blue screen printed electrodes for h2o2 detection, food and environment safety, volume xii, issue 1 – 2013, pag. 78 83 82 0 20 40 60 80 100 0 200 400 600 800 1000 i( na ) h 2 o 2 (m) amperometry pii-a (pretreated) e=-50mv figure 5. amperometry response procedure ii, from these sensors was evaluated by amperometric measurement in turbulent solution. 0 20 40 60 80 100 0 200 400 600 800 1000 1200 1400 1600 i( na ) h 2 o 2 (m) deposition electrochemical e=-50mv; 120s 1-100 m h 2 o 2 y=6.8071+15.1112x r=0.9986 figure 6. calibration curves obtained by successive additions of solution h2o2 phosphate buffer ph 6.5, e= -50 mv/s. 0 20 40 60 80 100 0 100 200 300 400 500 600 i( na ) h 2 o 2 (m) depositon galvanostatic cronoamperometrie e=-50mv 1 100 m h 2 o 2 y=-1.7741+5.5562x r=0.9996 figure 7. calibration curves obtained for h2o2detection performed at electrodes with pb layers in the 1-100 µm concentration range. applied potential -50mv/s . equation obtained for the linearity is y= 1.77+5.55, with a correlation coeficient r2 = 0.9996 deposition galvanostatic for the deposition electrochemical y=-6.8+15 r2=0.998 the time required to achieve 90% of corresponding steady state current was 10 s. 3.3. performances of the pb sensor the developed pb sensors was used for h2o2 determination using as electrochemical techniques: amperometry and chronoamperometry. according to the measurement results, the linear range of the pb sensors was from 1-100 µm, h2o2 with the liniar corelation of 0.9996 for the deposition galvanostatic and chemical deposition r2=0.998. for both type of sensors the detection limit was 0.5 µm h2o2. the relative standard deviation (rsd) was 3.6 %, for pb sensors prepared according to the procedures mentioned. 4. conclusions two methods for preparation of pb modified spces were compared in order to design a sensitive and robust sensor for hydrogen peroxide as a platform for oxidase based biosensors, resulting in the selection of the galvanostatic procedure (spe ii-a) as the most efficient. 5. acknowledgments the authors acknowledge to the project posdru/cpp107/dmi1.5/s/78534 for financial support. 6. references [1].v. d. neff, electrochem. soc. 125 (1978) 886. [2].a.a. karayakin, o.v. gitelmacher, e.e. karayakina, anal. chem. 67 (1995) 2419. [3].g. zhao, j.j. feng, q.l. zhang, s.p. li, h.y. chen, chem. mater. 17 (2005) 3154 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florentina hutanu, gheorghe gutt, modified prussian blue screen printed electrodes for h2o2 detection, food and environment safety, volume xii, issue 1 – 2013, pag. 78 83 83 [4].f. ricci, a. amine, d. moscone, g. palleschi, biosens. bioelectron. 22 (2007) 854 [5].l.v. lukachova, e.a.kotel’nikova, d.d’ ottavi, e.a. shkerin, e.e. karyakina, d. moscone, g.palleschi, a. curulli, a.a. karayakin, iee sens. j. 3 (2003) 3. [6].k. itaya, k.shibayama, h. akahoshi, s. toshima, j. appl. phys. 53 (1982) 804 [7].f. ricci, , g.palleschi, biosens. bioelectron. 21 (2005) 389-407. [8].itaya, k., shoji, n., uchida, i., 1984a. catalysis of the reduction of molecular oxygen to water at prussian blue modified electrodes. j. am. chem. soc. 106, 3423-3429 [9].cass, a.e.g., davis, g., francis, g.d., hill, h.a.o., aston, w.j., higgins, i.j., plotkin, e.v., scott, l.d., turner, a.p.f., 1984. ferrocene mediated enzyme electrode for amperometric determination of glucose. anal. chem. 56, 667-671. [10]. scheller, f.w., pfeiffer, d., schubert, f., reneberg, r., kirstein, d., 1987. application of enzyme amperometric biosensors to analysis of real object. in turner, a.p.f., karube, i., wilson, g.s. (eds.), biosensors: fundamental and applications. oxford university press, oxford, uk(chapter 18). [11]. f. ricci, , g.palleschi, biosens. bioelectron. 21 (2005) 389-407. [12]. q. dominguez renedo, m.a. alonso-lomillo, m.j. arcos martinez recent developments in the field of screen-printed electrodes and their related applications talanta 73 (2007) 202-219 [13]. ivanildo luiz de mattos, lo gorton, tautgirdas ruzgas biosensors and bioelectronics 18 (2003) 193-200 [14]. ricci, a. amine, g. palleschi, d. moscone biosens. & bioelectronics 18 2003 165-174 [15]. f. ricci, a. amine, d. moscone, g.palleschi, prussian blue based screen printed biosensors with improved characterics of long-term liftemie and ph stability . in: biosensors bioelectron. (2003) vol. 18, p. 165174. [16]. f. ricci, , g.palleschi, biosens. bioelectron. 21 (2005) 389-407. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 33 inf luence o f ma tura tion o n te chno log ica l par ameters of whit e win e chard on nay an d pino t gris *ana leahu1, sonia amariei(gutt)1, marcel avr miuc1, cristina-elena hre canu1, cristina damian1, mircea oroian1 1 tefan cel mare” university, faculty of food engineering, 13th university street, suceava, romania; e-mail: analeahu@fia.usv.ro *corresponding author received 15 march 2012, accepted 2 may 2012 abstract: the aim of the study was to determine, for two white wine varieties chardonnay and pinot gris, the influence that wine maturation has on its chemical properties. for the experimental study we used two varieties of sweet wine from the same vineyard and the same year, with different periods of maturation. harvesting took place when the grapes reached technological maturity, at a sugar content of 210-225g/l. we intended to highlight the improvement of wine quality during the aging period on the basis of the physico-chemical analysis made at the beginning and, after 24 months, at the end of the aging period. we analyzed the influence of aging on the chemical composition of wine, measuring its alcohol concentration, total acidity, volatile acidity, fixed acidity, ph (acidity real/ion), free and total so2 content, total dry extract and non-reducing extract. the maturation had a significant effect on volatile acidity, fix acidity, ph, so2 free, so2 total, sugars, conductivity, total dry extract and non-reducing extract, while alcoholic concentration, total acidity and density were not significantly affected. keywords: wine maturation, physico-chemical parameters, correlation matrix. 1. introduction wine maturation is the process that develops quality attributes of wine: clarity, colour, stability. during this phase, the process takes place under the action of oxygen, which combines fast enough with oxidizable constituents such as polyphenols, sulfur compounds, sulfur amhidrida. [1]. the transformations taking place are running at a relatively high redox potential, with limited participation of oxygen and consist in: the dissolution of some constituents of wood barrel, oxidation, condensation and partial precipitation of phenolic compounds and colloids, modifying the content of alcohols, aldehydes, acetals and esters, partial evaporation of volatile components, partial hydrolysis of (poliozide) and (heterozide) and finally, the process of insolubilisation accompanied by sedimentation of unstable components. wines produced by fermentation and maturation in oak barrels have different flavour characteristics to those which have undergone barrel maturation only after fermentation in stainless steel. one reason for this phenomenon is that actively growing yeasts are capable of transforming volatile flavour components, extracted from oak wood, into other volatile metabolites. [2] oxidation reaction rate increases with temperature under the action of oxidase enzymes, so aging is influenced by storage temperature. [3] mailto:analeahu:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 34 j. maric and m. first-baca, 2003 studied the evolution of chemical and sensory characteristics of bottled chardonnay white wine matured at 12ºc and 75% humidity. wine reaches its best quality at 12 months after bottling, longer periods, 24 respectively 36 months, can lead to a significant loss of quality due to reductive processes. [4] chardonnay is the most popular variety of white grapes used for wine production today, well known around the world; unlike other white wine varieties of vitis vinifera l. chardonnay’s prominent aroma comes mainly from its maturation in oak barrels. 2. materials and methods the grapes were harvested in panciu vineyard, vrancea county. the analysed wines (samples 1 ÷ 2, according to data from table 1) were obtained using white wines specific technology. after fermentation, clarification and fining, the wine was kept in wooden pots. the vineyard from which the wine varieties come has plantations located as follows: 70% on the slope, 20% on set, and 10% on the plain. the great heliothermic resources have a positive influence on the quality of the wine. each wine received an identification code, namely: c 01 chardonnay white wine at the beginning of maturation; c 02 chardonnay white wine matured after 24 months; pg 01 pinot gris wine at the beginning of maturation; pg 02 pinot gris wine matured after 24 months; the physical-chemical analyses (alcohol, total acidity, total dry extract, nonreducing extract, ph, so2 free, so2 total, density and conductivity) were made according to the methods indicated by in force state and international standards (***1988; *** 2005). statistical analysis a multifactor analysis of variance (anova) (using statgraphics plus version 5.1.) was carried out to study the influence of the type of wine and the maturation time. two factors were taken into consideration: the type of wine and maturation time. the double interactions between these factors were also considered. the method used for multiple comparisons was the lsd test (least significant difference) with a significance level = 0.05. the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. to achieve the correlation and the significance between the physico-chemical parameters the pearson coefficient has been computed. 3. results and discussion during wine aging, alcoholic concentration can decrease but also increase due to the partial hydrolysis of esters. further analyses noted that the alcohol concentration of chardonnay wine increased during maturation from 12.0 to 12.22º alc because of the fermentation of sugar residues, while the total acidity decreased from 6.3 to 6.135 g/l tartaric acid giving the wine physico-chemical stability, colour brilliance and a less astringent and softer taste. in table 1. shows physicochemical parameters analysed in sample before and after maturation of wines (chardonnay and pinot gris); anova f-ratio for each of two factors (maturation time and wine type) and their respective interaction food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 35 table 1. physicochemical parameters analysed in sample before and after maturation of wines (chardonnay and pinot gris); anova f-ratio for each of two factors (maturation time and wine type) and their respective interaction physicochemical parameters wine type f-value maturation time [months] f-value w xm interaction chardonnay pinot gris 0 24 alcoholic concentration, [% v/v] 12.08a 11.91b 31.81** 11.92b 12.07a 24.77** 5.39ns total acidity [g/l sulphuric acid] 6.20b 6.58a 566.4*** 6.51a 6.28b 209.3*** 17.64* volatile acidity [g/l sulphuric acid] 0.60b 0.87a 19758*** 0.62b 0.84a 13005.9*** 175.85*** fixed acidity [g/l sulphuric acid] 5.60b 5.72a 68.10*** 5.88a 5.44b 987.8*** 8.91* ph 3.26a 3.23b 9.40* 3.18b 3.31a 274.15*** 0.38ns so2 free , mg/l 27.90a 25.74b 1027*** 28.44a 25.19b 2335.8*** 47.38** so2 total, mg/l 140.74a 130.72b 860.7*** 145.87a 125.86b 3532.7*** 6.03ns sugars g/l 12.97a 11.97b 961.9*** 15.46a 9.47b 34629*** 0.0ns density g/cm3 0.9914a 0.9949a 1.91ns 0.9926a 0.9937a 0.21ns 0.07ns conductivity w/mxgr 46.53a 46.58a 0.18ns 46.18b 46.93a 41.10** 8.95* total dry extract,g/l 32.57a 31.87b 73.82** 35.86a 28.58b 8028.3*** 0.0ns non-reducing extract,g/l 19.60b 19.90a 36.51** 20.40a 19.10b 685.5*** 0.0ns the density of wine depends on the content of extractive substances and alcohol, but also on temperature. fixed acidity decreases due to the precipitation of salts of tartaric, malic and citric acid metabolism during malolactic fermentation, whence the combination of fatty alcohols. compared with the total acidity, volatile acidity of wines increased during storage, as well as the ph of wine, which fits in values up to 3.5 prevents attack microorganisms, tartaric precipitation and disposal oxidase. such ph, and preservation of free and total so2 maximum limits prevents unpleasant taste and smell of sulfur in wine. the higher the f-ratio (quotient between variability due to the considered effect and the residual variance), the greater the effect that a factor has on a variable. according to this, alchol, total acidity and density were most affected by the type of wine. the factor “maturation” has an influence on volatile acidity, fix acidity, ph, so2 free, so2 total, sugars, conductivity, total dry extract and nonreducing extract. table 2 shows the correlation matrix obtained for each pair of variables. the food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 36 number in brackets is the p-value which tests the statistical significance of the estimated correlations at the 95.0% confidence level. the highest passively correlation was observed between sugars and so2 total, so2 total and so2 free, total dry extract and sugars, total dry extract and so2 total, total dry extract and fix acidity, non-reducing extract and total dry extract. the highest negative correlation was observed between the ph and the fix acidity, conductivity and sugars, nonreducing extract and ph, total dry extract and conductivity, so2 total and volatile acidity . table 2. correlation matrix (pearson correlation coeffiecints) between physicochemical parameters paramet er alchol total acidity volatile acidity fix acidity ph so2 free so2 total sugars density conducti vity total dry extract nonreduc ing extraalchol 1 total acidity -0.886* (0.114) ** 1 volatile acidity -0.136 (0.864) 0.343 (0.657) 1 fix acidity 0.741 (0.259) 0.722 (0.278) -0.403 (0.597) 1 ph -0.165 (0.835) -0.664 (0.336) 0.474 (0.526) -0.996 (0.004) 1 so2 free -0.260 (0.740) -0.056 (0.944) -0.954 (0.046) 0.648 (0.352) -0.709 (0.291) 1 so2 total -0.506 (0.494) 0.081 (0.919) -0.908 (0.092) 0.748 (0.252) -0.799 (0.201) 0.989 (0.011) 1 sugars -0.506 (0.494) 0.368 (0.632) -0.747 (0.253) 0.908 (0.092) -0.939 (0.061) 0.907 (0.093) 0.957 (0.043) 1 density -0.524 (0.476) 0.603 (0.397) 0.906 (0.094) -0.080 (0.920) 0.142 (0.858) -0.749 (0.251) -0.686 (0.314) -0.461 (0.539) 1 conduct ivity 0.653 (0.357) -0.351 (0.649) 0.646 (0.354) -0.817 (0.183) 0.862 (0.138) -0.831 (0.169) -0.853 (0.147) -0.902 (0.098) 0.264 (0.736) 1 total dry extract -0.561 (0.439) 0.431 (0.569) -0.699 (0.301) 0.935 (0.065) -0.961 (0.039) 0.876 (0.124) 0.934 (0.066) 0.998 (0.002) -0.399 (0.601) -0.906 (0.094) 1 nonreducin g extract -0.777 (0.223) 0.690 (0.310) -0.438 (0.562) 0.995 (0.005) -0.998 (0.002) 0.683 (0.317) 0.774 (0.226) 0.924 (0.076) -0.093 (0.907) -0.868 (0.132) 0.948 (0.052) 1 *pearson correlation, **p-value at 95.0% food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 37 4. conclusions wine maturation took place from the first decanting and lasted until the wine has acquired characteristics (bottling). it was influenced by: oak barrels, climate change, oxidation, storage temperature, so2 content, etc. white wines made from pinot gris and chardonnay varieties showed a slight increase in concentration during the maturation alcoholic fermentation due to the sugar residue, offering high alcoholic wine and a slightly sweet taste. although there was a slight increase during maturation, because the ph value of the wine did not exceed 3.5, wines from pinot gris and chardonnay varieties are prone to attack microorganisms, precipitation of tartaric or quashing oxidase. the unpleasant taste and smell of sulfur and microorganisms attack for both types of wines are prevented by framing free and total so2 values in the maximum limits for the entire period of storage of the wine. non reducing extract values have decreased slightly during the ripening period due to condensation and deposit of phenolic compounds, and partial coagulation of protein substances. the highest passively correlation was observed between sugars and so2 total, so2 total and so2 free, total dry extract and sugars, total dry extract and so2 total, total dry extract and fix acidity, nonreducing extract and total dry extract. 5. acknowledgement this research was supported by the faculty of food engineering at “ tefan cel mare” university of suceava, romania . the authors would like to thank them for the financial support of this work. 6. references [1]. a. popa – the secret of the good wine (in romanian „secretul vinului bun – contribu ii i restituiri”) , editura alma, craiova, (2008). [2]. s. herjavec, a. jeromel, a. da silva, s. orlic, s. redzepovic, the quality of white wines fermented in croatian oak barrels. food chemistry 100 124–128 (2007). [3]. petrie, pr, and sadras, v.o., advancement of grapevine maturity in australia between 1993 and 2006: putative causes, magnitude of trends and viticultural consequences. australian journal of grape and wine research, (2008). [4]. j. mari , m. firšt-ba a, 2003. sensory evaluation and some acetate esters of bottle aged chardonnay wines, plant soil environ., 49, (7): 332–336(2003). http://www.agriculturejournals.cz/web/pse. *** catalog oficial al soiurilor de plante de cultur din românia, edi ia 2006, bucure ti, ministerul agriculturii, p durilor i dezvolt rii rurale, institutul de stat pentru testarea i îngrijirea soiurilor. *** 1988 colec ie de standarde pentru industria vinului si b uturilor alcoolice. ministerul industriei alimentare, bucuresti. http://www.agriculturejournals.cz/web/pse. 347 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 347 353 variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum) *ana leahu1, cristina damian1, mircea oroian1, veronica miclescu1, sorina ropciuc1 1faculty of food engineering, stefan cel mare university of suceava, universitatii str. 13, suceava, romania, analeahu@usv.ro * corresponding author received november 25th 2013, accepted december 8th 2013 abstract: the aim of this study was to determine the content of polyphenolics, antioxidant activity and vitamin c in the extracts of three medicinal and aromatic plants: basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum). the content of total polyphenols determined for the three aromatic plants can be estimated as high. the 1,1´-diphenyl-2-picrylhydrazyl (dpph) test were applied to determine antioxidant activity, which was also high in comparison with other herbs species. with regard to ic50 values (%), the order in dpph radical-scavenging were dried parsley (14.45) > fresh basil (9.03) > and fresh parsley (8.62). the ascorbic acid content varied between 347.60 mg/100g in the fresh parsley and 16.53 from dried dill. these results suggest the ocimum basilicum extract with highest polyphenolic content has more antioxidant activity against protein oxidation. keywords: basil, parsle, dill, total polyphenols content, scavening activity dpph, ascorbic acid. 1. introduction the importance of oxidation processes in the body and the food was widely recognized in many studies. defense mechanisms against excessive oxidation effect is provided by the action of antioxidants. the oxidative stress plays a role in the development of many diseases with high mortality. according to the bahramikia s. et al., 2009 oxidative stress refers to an imbalance between the production of free radicals and the antioxidant defense system, which results in functional tissue damage. it has long been recognized that naturally occurring substances in higher plants have antioxidant activities [2]. antioxidants such as vitamin c and phenolic compounds, vitamin e and carotenoids present in vegetables contribute to the reduce oxidative stress. as a result, they protect the cell from oxidative damage, and thus can prevent chronic diseases, such as cancer, cardiovascular diseases, diabetes [3]. in recent years, there is an increasing interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free radical reactions, protect the human body from diseases[4]. the most effective components seem to be flavonoids and phenolic compounds of many plant raw materials, particularly in herbs, seeds, and fruits [4]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 348 many studies have been shown that the presence of natural antioxidants from various aromatic and medicinal plants is closely related to the reduction of chronic diseases such as dna damage, mutagenesis, and carcinogenesis [2]. polyphenols are considered protective factors against degenerative diseases and cardiovascular disease. herbs may contain phenolic compounds that contribute to the intake of natural antioxidants that protect important cellular components such as adn, proteins and lipid membranes against the action of reactive oxygen species. levels of phenolic compounds from plants, fruits, vegetables and herbs are influenced by genotype, agronomic practices, maturity at harvest and postharvest storage, climate, region and processing conditions [5]. parsley (petroselinum crispum l.), dill (anethum graveolens) and basil (ocimum basilicum), are the most important romanian seasoning herbs. the aromatic herbs most consumed romania are parsley, dill and lovage and romania is one of the leading european spice consumer, according to eurostat and faostat [6]. it is widely used, both fresh and dry, to enhance the flavor of many different foods [7]. spices leaves deterioration occurs during processing and storage and is related to oxidative processes. amounts of nutrients are removed or destroyed components during storage of raw materials or technological processing (drying, grinding). spices and dry herbs are used as ingredients in a variety of products prepared in different ways, this fact suggests the need to provide a control system to improve the quality of herbs and spices [8]. essential oils are known to possess multifunctional properties other than their classical roles as natural food additives and/or fragrances [2]. the commercial essential oil of parsley is largely derived from the seed or the herb harvested at seed formation, prior to ripening [2]. wahba nm et all., 2010 evaluated the addition of some edible plants including cayenne, green pepper, parsley, and dill to kareish cheese and to evaluate the antimicrobial activity of these plant materials against natural microflora, coliforms, molds, and staphylococcus aureus. therefore, this study revealed that pepper, parsley, and dill exhibited antibacterial activity against natural microflora in kareish cheese, and the addition of these plants is acceptable to the consumer and may contribute to the development of new and safe varieties of kareish cheese [9]. lisiewska z. et al., 2003, evaluate the effect of temperature and storage period on the preservation of vitamin c, thiamine and riboflavin in leaves and whole plants (leaves with petioles and stems) of dill, harvested with a plant height of 25 cm. the treatment of blanching affected a decrease in the level of vitamin c by 35-48% [10]. the purpose of this paper is to evaluate and compare the total polyphenol content, antioxidant capacity and the content of vitamin c parsley, dill and basil fresh and dry. these plants were chosen because they are represented as native herbs, and spices that presents a common variable consumption also shows a strong pharmacological activity, and have high antioxidant content. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 349 2. materials and methods 2.1. plant material a fresh spice (table 1) was purchased at a commercial establishment in suceava in the spring 2013. the leaves were removed from the stems and were refrigerated at 5 °c for analysis fresh. dried vegetables were obtained from a local market and are from different companies. table 1 botanical and common names of spices used for the analysis botanical name common name ocimum basilicum basil anethum graveolens dill petroselinum sativum parsley 2.2. chemicals all chemicals used for experiments were of analytical grade and procured from sigma merck, aldrich and fluka. deionizer water was used. absorption determination for total polyphenols content was made using uv–vis spectrophotometer. 2.3. chemical analyses the determination of moisture in samples was effectuated according to the european standard en iso 665/2000 by the drying process in a drying chamber at the temperature of 103 °c. total polyphenol were assayed according to the folin-ciocalteu method. fresh herbs was cleaned, carefully washed with tap water was extracted with 80% ethanol (0.1 g sample/10 ml of 80% ethanol), and total phenol contents (tpc) of the extracts were determined. the results were expressed as mg of gallic acid equivalents (mg gae).the correlation coefficient (r2) for the calibration curve was 0.9954. 2,2-di (4-tert-octylphenyl) -1 pycrilhydra zyl (dpph) scavenging capacity assay the method used for determining the antioxidant activity of spices herbs extracts is based on scavenging 2,2-di (4-tertoctylphenyl)-1-picrylhydrazyl (dpph) radicals. the spices samples aliquot (0.5 ml) was added to freshly prepared dpph reagent. after incubating for 5 min, the absorbance of the resulting solutions was measured at 517 nm using a spectrophotometer. the control was conducted in the same manner, except that distilled water was used instead of sample. the ic50 is the concentration of an antioxidant that is required to quench 50% of the initial dpph radicals under the experimental conditions given. the dpph scavenging capacity assay value is calculated according to the formula: ic50 % = [1 – (asamples/ acontrol)] × 100. ascorbic acid determination was done on acid extracts of samples. extraction of ascorbic acid from samples the extracts were obtained following rodriguez-saona and wrolstad (2001) modified protocol: 4 gram of parsley was extracted with 12 ml of acidified solutions (perchloric acid and ophosphoric acid 1%) using a ceramic mortar and a pestle. the residue was re-extracted until the extraction solvents remained colorless (the total solvent volume was 50 ml).the extract was filtered through a whatman no. 1 filter paper. the extracts were kept at -20°c until further analysis. ascorbic acid separation, identification and dosage the ascorbic acid in the samples was separated, identified and dosed in a hplc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 350 shmadzu system coupled with uv–vis detector (dad). zorbax c18 column (5μm, 250x4,6) was used. the column was eluted in isocratic system with a mobile phase consisted of phosphate buffer ph = 3.5 (tfa): solution 0.02 m/l of monopotassium phosphate and orthophosphoric acid 10%, adjusted to ph = 3.5. at a flow rate of 0, 6 ml/min. the chromatograms were registered at 245 nm. for ascorbic acid identification standard lascorbic acid (sigma 99% standard l ascorbic acid) was used. for dosage of ascorbic acid in the samples, a calibration curve was constructed using dilutions of standard l-ascorbic acid in bidistilled water. 3. results and discussions phenolic compounds widely exist in plants and they are highly effective antioxidants [1,11]. the contents (mg/g of dried extract) of phenols in ethanolic extracts of dill flower, leaf and seed was higher in the order of flower > leaf > seed extracts [11]. 12 samples were formed, and table 2 shows the values obtained for moisture, total phenols and dpph values of aromatics herbs. table 2. moisture, phenolic, and free radical scavenging activities (dpph) values in selected herbs samples moisture (g/100 g) tpa (mg gae/100g) ic50 basil (ocimum basilicum) s1 fresh 79.82 245.2 9.03 s2 dried 0.97 109.6 29.07 s3 0.98 92.8 55.63 s4 0.98 63.2 67.05 dill (anethum graveolens) s5 fresh 82.25 228.1 26.51 s6 dried 0.99 92.8 84.24 s7 0.98 111.8 92.77 s8 0.99 57.7 86.56 parsley (petroselinum sativum) s9 fresh 83.24 211.9 8.62 s10 dried 1.00 181.9 14.45 s11 0.99 178 23.84 s12 1.00 160.3 16.76 avalues are referred to mg/100 g fresh weight vegetable. tp = content of total phenols (mg gae/100 g). ic50 = free radical scavenging activities moisture from dried samples presented the lower values. important to maintain the quality of dried spices herbs is low moisture content, which decreases the likelihood of microbial growth and late biochemical reactions associated with these processes. moisture content from fresh samples was higher in s9 parsley (83.24 %) and lower in s1 basil (79.82 %). the use of freshly harvested vegetables in human nutrition is of fundamental importance today as modern storage or transforming systems allow the conservation of fresh vegetables for long periods of time in refrigerated cells under a controlled atmosphere, although the longstorage or mildly processed vegetables possess a significantly lower phenolic and ascorbic acid content than fresh vegetables and their antioxidant capacity decreases proportionally [12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 351 total phenols. the comparison of selected aromatics herbs reveals impressive differences. from table 2, total phenolic contents of three dried basil extracts were relatively lower and their total antioxidant capacities were weaker. the three dried dill spice extracts contained lower poliphenols, (mean = 87.43 mg/100 g of dw) than the three dried parsley spice extracts contained higher poliphenols, (mean=173.4 mg/100g of dw). basil had the highest content of poliphenols 245.2 mg gae/100 g. the correlation value for the parameters moisture and total phenols measured in selected aromatics herbs is 0.740, reveled a stronger correlation between these two parameters (table 3). table 3 correlation matrix for the variablesmeasured in aromatics herbs: moisture(g/100 g), tp (mg gae/100g) and ic50 moisture (g/100 g) tp (mg gae/100g) ic50 moisture (g/100 g) 1 tp (mg gae/100g) 0.740 1 ic50 -0.333 -0.753 1 values in bold are different from 0 with a significance level alpha=0,05 the highest negatively correlation have been observed in the case of total poliphenols with antioxidant activity against the dpph* free radical ic50 (r = -0.753). figure 1. the relationship between the content of total phenols of analyzed samples the analyses show a significant relationship between total phenols content values on samples of study (see figure 1). dpph scavenging capacity assay methanolic extracts of fresh basil, dill and parsley had exceptionally high scavenging activity. a stronger radical-quenching agent resulted in a lower ic50 value (scavenging of 50% dpph radical). table 2 shows that the dpph radical scavenging ability of samples can be ranked as dried parsley > dried basil> dried dill. the scavenging abilities on dpph radicals at 0.1 mg of dried extract/ml were 14.45%, 16.46% and 23.84% for the dried parsley, and 29.07%, 55.63% and 67.05% for the dries basil. the ascorbic acid extracts of samples are presented in figure 2. the ascorbic acid content in aromatics herbs has recorded different discounts, depending the samples analyzed fresh or dried. thus, the greatest values of acid ascorbic concentration were registered in the fresh parsley 347.60±6.2 mg/100g. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 352 greatest reduction in the content of ascorbic acid was in the dried dill samples (89.33 %), followed by parsley (75.41 %). as seen in the figure 2, the ascorbic acid content in samples analyzed was higher in fresh samples. figure 2. ascorbic acid concentrations mg /100g of analyzed samples cătunescu g.m. et al., 2012 evaluate the ascorbic acid in parsley, dill and lovage samples packed in poliethylene stored in bags at 4°c for up to 12 days. the results showed the content of ascorbic acid diminished by 18% for parsley samples, 8% for dill and 3% for lovage after 12 day of storage. our results illustrated that ascorbic acid should be responsible for the effective antioxidant properties of the parsley fresh extract. principal component analysis (pca) was used to test for the correlation between the variables: moisture (g/100 g), tp (mg gae/100g) and ic. the variation of the parameters studied implies the existents of 3 factors. the principal component that has the eigenvalue more than 1 is f1 (2.235). the eigenvalue for f2 and f3 are less than 1 that implies that these factors are not important. the percentage of variability represented by the first two factors is high (96.75 %). figure 3. biplot graphic for the parameters studied in analyzed samples the first factor (f1) explains 74.52 % of the total variance with a significant parameter tp, having a factor loading 0.871. an important contributions of the observations (%) for the factor f1 have basil fresh (23.9 %), parsley fresh (19.3 %) and dill dried (18.7 %). the second factor (f2) explains 22.23 % , of the total variance with a significant parameter moisture(g/100 g). an important contributions of the observations (%) for the factor f2 has dill fresh (36.2 %). 4. conclusions antioxidant capacities of basil, dill and parsley fresh and dried were analyzed. during storage important quality losses occur. spices herbs are important sources of valuable and essential nutrients such as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc, variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), volume xii, issue 4 – 2013, pag. 347 – 353 353 vitamins and minerals. these are the richest potential sources containing different antioxidants. various research studies have shown that a higher consumption of plants containing antioxidant sources is associated with a lower incidence of cardiovascular disease, cancer and other chronic diseases. following the analysis of samples, total phenols content was 245.2±2.3mg/100 g in the fresh basil but the value decreased to 63.2±3.6mg/100 g. in the dried basil. both remarkably high phenolic content and radical scavenging activities were found in the fresh dill. in conclusion, this study suggests that the processing and storage temperature significantly affect the antioxidant capacities of spices herbs analyzed. 5. references [1]. bahramikia s., ardestani a., yazdanparast r., protective effects of four iranian medicinal plants against free radicalmediated protein oxidation, food chemistry vol. 115, p. 37–42 (2009). [2]. zhang h., feng c., xi w., hui-yuan y., evaluation of antioxidant activity of parsley (petroselinum crispum) essential oil and identification of its antioxidant constituents, food research international vol. 39, p. 833–839 (2006). [3]. podsedek, a., natural antioxidants and antioxidant capacity of brassica vegetables: a review. institute of technical biochemistry, faculty of biotechnology and food sciences, technical university of ło´dz ,́ poland (2005). [4]. ebrahimzadeh m.a., nabavi s.m., nabavi s.f., bahramian f. and bekhradnia a.r., antioxidant and free radical scavenging activity of h. officinalis l. var. angustifolius, v. odorata, b. hyrcana and c. speciosum, pak. j. pharm. sci., vol.23, no.1, pp.29-34, (2010). [5]. luthria, d., mukhopadhyay s., kwansa a.l., a systematic approach for extraction of phenolic compounds using parsley (petroselinum crispum) flakes as a model substrate, journal of the science of food and agriculture, 86:1350–1358 (2006). [6]. cătunescu g.m., tofană m., mureşan c., ranga f., david a., muntean m., the effect of cold storage on some quality characteristics of minimally processed parsley (petroselinum crispum), dill (anethum graveolens) and lovage (levisticum officinale), bulletin uasvm agriculture, 69(2)/(2012). [7]. díaz-maroto m.c., pérez-coello m.s., cabezudo m.d., effect of different drying methods on the volatile components of parsley (petroselinum crispum l.), eur food res technol 215:227–230 (2002). [8]. sospedra i., jose m. soriano & mañes j., assessment of the microbiological safety of dried spices and herbs commercialized in spain, plant foods hum nutr, 65:364–368 (2010). [9]. wahba nm, ahmed, as, ebraheim, zz, antimicrobial effects of pepper, parsley, and dill and their roles in the microbiological quality enhancement of traditional egyptian kareish cheese, foodborne pathogens and disease, vol.7 pages 411-418(2010). [10]. liseiwskay, z., jacek słupski, emilia zuchowicz, influence of temperature and storage period on the preservation of vitamina c, thiamine and riboflavin in frozen dill (anethum graveolens), food science and technology, vol. 6, 2003; [11]. yung-shin shyu, jau-tien lin, yuan-tsung chang, chia-jung chiang, deng-jye yang, evaluation of antioxidant ability of ethanolic extract from dill (anethum graveolens l.) flower, food chemistry 115, 515– 521(2009). [12]. ninfali p., gloria mea, giorgini s., marco rocchi and bacchiocca m., antioxidant capacity of vegetables, spices and dressings relevant to nutritio � �n, br. j. nutr., 93, 257−266 (2005). звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 53 effect of addition of carrageenan on rheological behaviour of yogurt *cristina damian1, mircea-adrian oroian2, ana leahu3, nicolae carpiuc4 1-4ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1cristinadamian@fia.usv.ro *corresponding author received 25 march 2012, accepted 12 may 2012 abstract: the purpose of this work is to study the effect of addition of k-kappa carrageenan and iiota carrageenan on the rheological behaviour of different types of yogurts. the role of concentration as well as the type of carrageenan used (k-kappa carrageenan and i-iota carrageenan) has been analysed.the most common technique used for rheological liquids is viscometers, measuring shear strength, described by the coefficient of viscosity. the most common technique used for rheological liquids is viscometers, measuring shear strength, described by the coefficient of viscosity. for this study, the yogurt samples were obtained in the laboratory using the cultures starter diprox yba 986. the effect of addition of carrageenan in yoghurt was investigated by a rotational viscometer, brookfield viscometer (brookfield engineering inc, model rv – dv i prime) with rv spindles. the brookfield viscometer dv i prime with disk spindles represents an easy and cheap method for the rheological characterization of non-newtonian fluids, in this case of yoghurt. the cluster analysis was performed using unsrambler x version 10.1 (camo process as, oslo, norway, 2005), all the rheological and physicochemical parameters were weighed and normalized for performing the cluster analysis. in the case of yogurt samples obtained in the laboratory, the addition of carrageenan increases the viscosity. i-iota carrageenan has been evaluated as more effective for increasing the viscosity of yogurt in comparison with k-kappa carrageenan (with the same concentration). keywords: yogurt, viscosity, viscometer 1. introduction rheology is the deformation and flow of matter [1]. polysaccharides are widely used in food systems as, e. g. thickeners [2]. polysaccharides (pectin, modified starch, xanthan gum, locust bean gum, guar gum, alginate, etc.) are often added to dairy products to stabilise their structure, enhance viscosity and alter textural characteristics [3]. carrageenans are anionic linear polysaccarides extracted from the red seaweed (rhodophyceae), consisting of alternating α-1,4 and β-1,3 linked anhydrogalactose residues. there are three major fractions (k-kappa, i-iota and λlambda) with varying number and position of sulphate groups on the galactose dimer. k-carrageenan and i-carrageenan undergo a temperature-dependent coil (disordered state) to helix (ordered state) transition in aqueous solution. both of these carrageenans are able to form gels (the process of gelation is closely associated with helix formation). k-carrageenan usually forms firm, britlle gels and icarrageenan usually generates soft, elastic gels. the strength of the gels formed by both polysaccarides is strongly influenced by the presence of cations. k-carrageenan is especially sensitive to potassium and icarrageenan to calcium ions. on the other hand, λ-lambda carrageenan is not able to build up stable gels [4]. yogurts are dairy products obtained by fermentation with lactic acid bacteria food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 54 during which a weak protein gel develops due to a decrease in the ph of the milk. following the fermentation of lactose lactic acid is formed causing the coagulation of milk proteins (casein) to form a gel-like structure, in which fat globules and the aqueous phase are embedded [5]. in the liquid milk, casein micelles are presented as individual units. as the ph reaches ph 5.0, the casein micelles are partially broken down and become linked to each other under the form of aggregated and chains forming part of a three-dimensional protein matrix in which the liquid phase of the milk is immobilized. this gel structure contributes substantially due to the overall texture and organoleptic properties of yoghurt and gives rise to shear and time dependent viscosity [6]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions [7]. the most frequent defects related to yogurt texture that may lead to consumer rejection are apparent viscosity variations and the occurrence of syneresis [8]. yogurt rheological characterization is required for product and process acceptability [9]. this characterization can be made using either instrumental or sensory measurements. the firmness of yogurt and the viscosity of just-stirred gel are greatly influenced by the amount of heat treatment the yogurt mix receives. heating unfolds the globular whey proteins and exposes sulphydryl groups, which react with other sulphydryl groups and disulfides and induce linkages and protein-casein aggregates [10, 11]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions. the binding of δ-lacto globulin to the casein micelle seems to be responsible for the increase of gel strength [12, 13, 14]. 2. materials and methods 1.1. materials uht milk, lactobacillus bulgaricus and streptococcus thermophilus pure starter culture diprox yba 986 provided by enzymes & derivates, piatra neamț, românia; k-kappa carrageenan and i-iota carrageenan provided by enzymes & derivates, piatra neamț, românia; orbital shaker; thermostat; brookfield viscometer model rv dv ii pro, with disk spindle, rv3, rv4, rv5, rv6 type. 1.2. sample preparation the yogurt samples were made using uht milk, having the physical and chemical parameters in tabel 1. table 1. milk properties fat, g/100g 3 protein, g/100 g 3 sugar, g/100g 4.5 ash, % 0.72 acidity, 0t 18 table 2. ph of yogurt samples sample ph s 1 4.0 s 2 4.5 s 3 4.2 s 4 4.0 s 5 4.5 300 ml milk was inoculated using 0.015 g starter culture. after inoculation with starter culture, the samples were homogenised with an orbital shaker for 15 min. at 100 rpm. after shaking the samples were thermostated at 420c for 6 hours. carrageenans were added in the next concentration to the yogurt sample: 0.9 g k-kappa carrageenan (s1), 0.9 g i-iota carrageenan (s2), 0.6 g k-kappa carrageenan and 0.3 g i-iota carrageenan mailto:fiajournal:@fia.usv.ro mailto:gutts:@fia.usv.ro mailto:cristina.hretcanu:@fia.usv.ro mailto:gabriela.constantinescu:@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 55 (s3), 0.45 g k-kappa carrageenan and 0.45 g i-iota carrageenan (s4) and 0.3 g kkappa carrageenan and 0.6 g i-iota carrageenan (s5). the ph of the yogurt samples is presented in table 2. 2.3. determination of rheological properties viscosity measurements were carried out on the yogurt samples at ambient temperature (250c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 0.5, 1, 2, 2.5, 4, 5 and 10 rpm with rv spindle (rv3, rv4, rv5, rv6 type). the spindle no was used in accordance with the sample nature to get all readings within the scale [15]. the samples in 300 ml of beaker with a 8.56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain desirable temperature of 250c. first measurements were taken 2 min. after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression σ = k·γn (1) where: σ – shear stress (n/m2), γ is the shear rate (s-1), n is the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. the shear stress is calculated using the next equation: τi = kτ·αi·c (2) where: τi – shear stress, (dyne/cm2) kτ = 0.119, this constant is for the spindle nos 2 αi – torque dial, (%) c – 7.187 dyne/cm for rv viscometer the shear rate is calculated using the next equation: γi = kγ(n)·ni (3) where: γi – shear rate, (s-1) kγ(n) – constant, depends by the value of n ni – rotational speed, (rpm). statistical analysis the cluster analysis was performed using unsrambler x version 10.1 (camo process as, oslo, norway, 2005), all the rheological and physico-che mical parameters were weighed and normalized for performing the cluster analysis. 3. results and discussion the yogurt samples exhibited a nonnewtonian behaviour. the power law model is a suitable one for predicting the rheological parameters. table 3. viscosity for yogurt samples sample viscosity* [cp] s1 60947 s2 107000 s3 213000 s4 106000 s5 144000 *viscosity was measured at 0.5 1/s in general, addition of carrageenan increases the viscosity of yogurt, as shown in table 3. it achieved a stabilization of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 56 aqueous dispersions whose continuous phase is the water and the dispersed one is solid or liquid and tends to separate. through the addition of carrageenan, the viscosity is increased and the tendency of destabilization by separation of components is reduced. upon cooling and in the presence of appropriate cations, kappa and iota carrageenan polymers align themselves to form individual helices. these helices can further associate with divalent cations that are present, e.g. calcium, to form a gel matrix. figure 1 is a schematic representation of the gelling mechanism for carrageenan. figure 1. gelation mechanism [16] the gel texture of the basic carrageenans can be modified by blending carrageenans to meet specified gel texture parameters. figure 2 illustrates the penetration or elasticity of carrageenan gels before the gels are fractured. the gels can be made more elastic or less elastic by combining kappa and iota carrageenans until the desired texture is achieved. figure 2. gel texture [17] gels prepared with carrageenan are thermally-reversible. these gels will become fluid when heated above the melting point of gels and will reset into a gel when cooled, with minimal loss of the original gel strength. in food systems, one of the most important properties that truly differentiate carrageenan from other hydrocolloids is its ability to complex or interacts with proteins. in milk protein systems, at peripheral locations on the casein micelle there is a concentration of positive charges. this positive electrostatic charge attracts the negatively-charged sulfate groups of the carrageenan molecule to form linkages among the dispersed casein micelles as illustrated in figure 3. this reaction, in combination with the normal water gelling capabilities of carrageenan, can increase the gel strength about 10-fold. the ability of carrageenan to complex with milk proteins, combined with its water gelling properties enhances the functionality of carrageenan, e.g. increased gel potential. figure 3. interaction of milk protein with carrageenan figure 4 shows the evolution of viscosity with the two hidrocolloid with its concentration and shear rate. it can be observed that the viscosity of the yogurt sample with i-iota carrageenan (s2) is higher than that of the yogurt that contains the same amount of k-kappa carrageenan added (s1). carrageenan mixtures have a http://www.fia.usv.ro/fiajournal/food_and_environment_safety_copyright.doc http://www.fia.usv.ro/fiajournal/files/referent_report_for_fens_fia.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 57 different rheological behavior on the samples of yogurt. thus, the sample with a mixture of both types of echimolecular carrageenan has the lowest viscosity, followed by the sample with a smaller amount of k-kappa carrageenan. sample s5 has the highest viscosity and also a higher content of i-iota carrageenan. figure 4. viscosity profile of yogurt sample with 0.9 g k-kappa carrageenan (s1), 0.9 g i-iota carrageenan (s2), 0.6 g k-kappa carrageenan and 0.3 g i-iota carrageenan (s3), 0.45 g k-kappa carrageenan and 0.45 g i-iota carrageenan (s4) and 0.3 g k-kappa carrageenan and 0.6 g i-iota carrageenan (s5) the results of cluster analysis shows that the samples with k-kappa carrageenan and i-iota carrageenan added are in the same group, while the samples with the mixture of the two types of carrageenans make a different group – see figure 5. figure 5. diagram of yogurt samples clustered according to rheological and physicochemical properties 4. conclusion due to large molecular sizes, to the specific configuration, the charge and possible formation of intraand intermolecular links, gums are able to reduce water mobility. this reduction in water mobility increases the viscosity of gum solutions in water. in the case of yogurt samples obtained in the laboratory, the addition of carrageenan increases the viscosity. by means of carrageenan addition, yogurt viscosity is increased and reduces the tendency to destabilize the separation of components. i-iota carrageenan has been evaluated as more effective for increasing the viscosity of yogurt in comparison with kkappa carrageenan (with the same concentration). the results of cluster analysis shows that the samples with k-kappa carrageenan and i-iota carrageenan added are in the same group, while the samples with the mixture of the two types of carrageenans make a different group. 5. references 1. oroian m. a., gutt g., influence of total soluble content, starter culture and time period on rheological behaviour of cultured buttermilk, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, year ix, no 2, 73 p, (2010) 2. klein, c., venema, p., sagis, l., van der linden, e., rheological discrimination and characterization of carrageenans and starches by fourier transform-rheology in the non-linear viscous regime, j. non-newtonian fluid mech., 151, 145-150, (2008) 3. černiková, m., buňka, f., pavlínek, v., březina, p., hrabě, j., valášek, p., effect of carrageenan type on viscoelastic properties of processed cheese, food hydrocolloids, 22, 1054-1061, (2008) 4. imeson, a. p., carrageenan, in g. o. philips, & p. a. williams (eds.), handbook of hydrocolloids (p. 87-102), boca raton, fl: woodhead publishing limited, crc press. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 58 5. banu, c. (coordinator), aditivs and ingredients in food industry (in romanian: aditivi şi ingrediente pentru industria alimentară), editura tehnică, bucureşti, (2000) 6. oroian m. a., escriche i., gutt g., rheological, textural color and physicochemical properties of some yoghurt products from the spanish market, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, year x, no 2, 24 p, (2011) 7. damian, c., oroian, m.a., șmadici, a., effect of addition of corn flakes on rheological behaviour of some yogurt, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, volume x, issue 4, 84 p, (2011) 8. kroger, m. quality of yogurt. journal of dairy science 59(2): 344-350. (1975) 9. benezech, t., maingonnat, j.f. characterization of the rheological properties of yogurt-a review. journal of food engineering. 21, 447-472. (1994) 10. sawyer wh. complex between –lacto globulin and γ-casein. a review. journal of dairy science 52:1347–55. (1969) 11. kinsella h. milk proteins: physiochemical and functional properties. crc crit rev food sci nutr 21(3):197. (1994) 12. bonomi f, iametti s, palgliarini e, peri c. a spectrofluorometric approach to estimation of the surface hydrophobicity modifications in milk proteins upon thermal treatment. milchwissenschaft 43:281–5, (1998) 13. mottar j, bassier a, joniau m, baert j. effect of heat-induced association of whey proteins and casein micelles on yogurt texture. journal of dairy science 72(9):2247– 56, (1989) 14. bonomi f, iametti s. real-time monitoring of the surface hydrophobicity changes associated with isothermal treatment of milk and milk protein fractions. milchwissenschaft 46:71–4, (1991) 15.http://www.brookfieldengineering.com/products /accessories/spindles/rv_ha_hb_spindles.asp (7.09.2011) 16.http://www.fmcbiopolymer.com/food/ingredien ts/carrageenan/functionalityandrheology.aspx 17.http://www.fmcbiopolymer.com/food/ingredien ts/carrageenan/geltexture.aspx 18.http://www.fmcbiopolymer.com/food/ingredien ts/carrageenan/milkproteininteraction.aspx mailto:gutts:@fia.usv.ro mailto:cristina.hretcanu:@fia.usv.ro mailto:gabriela.constantinescu:@fia.usv.ro the following points are important in order to warrant paper publishing in the journal food and environment safety. the following points are important in order to warrant paper publishing in the journal food and environment safety. the following points are important in order to warrant paper publishing in the journal food and environment safety. the following points are important in order to warrant paper publishing in the journal food and environment safety. the following points are important in order to warrant paper publishing in the journal food and environment safety. the following points are important in order to warrant paper publishing in the journal food and environment safety. poor average good excellent the contribution is original the paper communicates are relevant for the journal food and environment safety 180 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2015, pag. 180 186 total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.) *sonia amariei1, valentina mancas1 1faculty of food engineering, stefan cel mare university of suceava, romania gutts@fia.usv.ro *corresponding author received 25th april 2016, accepted 27th june 2016 abstract: total phenolic content and antioxidant activity of two by-products of onion (allium cepa l.) and walnut (juglans regia l.) processing were studied in order to establish the possibility of capitalization as antioxidant sources. the by-products analyzed were walnut shell and onion peel. from these by-products, infusions were prepared using water as solvent. total phenolic content was determined using folin-ciocalteu method, while the radical scavenging ability of the infusions was monitored using the stable free radical dpph (1,1-diphenyl-2-picrylhydrazyl). walnut shell infusion had a relatively low content in phenolic compounds (32.08 mg gae/g), below the level reported for other walnut by-products. given the low phenolic content, walnut shell infusion exhibited a reduced radical scavenging capacity (7.82%). on the contrary, the onion peel infusion had both high phenolic content (280.32 mg gae/g) and antioxidant activity (69.38%). based on these two results it was confirmed that onion peels resulted from bulb processing are suitable for reuse in antioxidants’ extraction in both food and pharmaceutical industries. keywords: walnut, onion, antioxidants, dpph, folin-ciocalteu 1. introduction official surveys indicate that the european food-processing industry produces vast volumes of wastes every year, and despite the efforts to reduce solid waste volume the impact of food industry on the environment remains unchanged. apart from the environmental challenges posed, such streams represent considerable amounts of potentially reusable materials and energy [1]. fruit and vegetable processing has the highest wastage rate of any food, rising up to 16.4% for fruits and to 25.8% in the case of vegetables [2]. according to food and agriculture organization (fao), nearly 50% of all fruits and vegetables in the eu go to waste, 5.5% only being lost during postharvest handling and processing [3]. byproducts discharged from manufacturing of fruit or vegetable-based products depend on the fresh produce used and include peels, skin, hulls, pips, and cores. fruit and vegetable waste can be capitalized in added-value products such as animal feed, single-cell protein (scp) and other fermented products, baker’s yeast, organic acids, amino acids, enzymes (e.g. lipases, amylases, and cellulases), flavors and pigments, gums and polysaccharides [4,5]. walnut (juglans regia l.) is a tree native to a region stretching from the balkans eastward to the western himalayan chain [6,7]. the walnut is traditionally cultivated throughout europe for its valuable wood and seeds (walnuts). seeds are of high http://www.fia.usv.ro/fiajournal mailto:gutts@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 181 economic interest to the food industry and are valued globally for the nutritional, health and sensory attributes [8]. romania is among the first ten world producers of walnut. in order to maximize the productivity and reduce the waste from processing, several by-products derived from the walnut tree, such as green walnuts, shells, kernels, bark, green walnut husks, and leaves have been used in both cosmetic and pharmaceutical industries [9,10]. walnut shells and green walnut husk have various possible uses in the industry. recent research has reported on the great potential of green walnut husk to remove lanaset red g from aqueous solution [11]. it was also investigated the ability of a powdered activated carbon (pac) derived from walnut shell to adsorb mercury from aqueous solutions [12,13], and the reuse of walnut shell as a support material (biosorbent) for effective cesium adsorption from aqueous solution [14]. in addition to non-food applications, the central point of recent research is the phenolic composition and antioxidant activity of extracts from byproducts. while several studies reported on the bioactive compounds of extracts from green walnut husk [10, 15-17], no data is available on the composition of aqueous extracts from walnut shells. onion (allium cepa l.) is one of the major vegetable crops grown worldwide, after potato and tomato, with a production that was estimated to 84.75 mt in 2013 [18] and increases constantly. onions possess strong, characteristic aromas and flavours [19] that made the vegetable (bulb or scallion) an important ingredient of food. on account of the whopping production and use of onion, bulbs and scallions processing raises some concern over the large quantity of industrial waste produced and its disposal. furthermore, the perishable nature make onion waste not suitable for fodder or landfill disposal due to the rapid growth of phytopathogens, e.g. sclerotium cepivorum (white rot) [20]. in these conditions, the only suitable decision in onion waste management is the capitalization of by-products. the main onion by-products include onion skins generated during industrial peeling, and undersized, malformed or damaged bulbs [21]. onion peel importance in bioactive compounds extraction is given by its high levels of two chemical groups with proven health benefits for humans, namely flavonoids and alk(en)yl cysteine sulfoxides (acso) [22]. the main flavonoid present in onion peel is quercetin aglycon [23], a compound known for its antioxidant and free radical scavenging activity, cardioprotective effect, anticancer, antiinflamatory and antiviral activity, as well as inhibitory activity against bacterial ureases [24-26]. naturally occurring ascos in onion peel are trans-(+)-s-(1propenyl)-l-cysteine sulphoxide, (+)-smethyl-l-cysteine sulphoxide, and (+)-spropyl-l-cysteine of sulphoxide [27]. although romania has a high production of walnuts and onion, the capitalization of waste resulted from processing has been less studied. the aim of this work was to analyze the composition in antioxidants of infusions from walnut shells, and onion peel in order to establish the possibility of reuse in romanian industry according to the by-product composition. the solvent used in the extraction process was water. no alcoholic solvent was added to enhance the extraction. the parameters analyzed were total phenolic content and scavenging effect on dpph (2,2-diphenyl-1-picrylhydrazyl). 2. matherials and methods 2.1. plant material onions were purchased from an organic farmer located in suceava county, in northeastern romania. the bulbs were from the 2015 harvest. walnuts were collected in september 2015 from a single tree that had not received any insecticide treatment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 182 the walnut shell was cracked and the kernel was removed, while the outer skin of the onion was sectioned, and then peeled. both by-products were rinsed prior to being used. to enhance the extraction of bioactive compounds onion peels were cut into chunks and walnut shells were crushed. 2.2. preparation of individual infusions the infusion (fig. 1) was prepared as follows: 80 g of freshly separated onion peels or walnut shells was placed in a stainless steel pot and 100 ml of water brought to the boiling point (100 °c) was added. the pot was quickly covered leaving the mix to infuse for about 15 minutes. the cover was then removed and the content was allowed to rest for about 4 hours. finally, peels and shells were retained in a filter and the infusion was stored in a closed container until its composition was analyzed. 2.3. total phenolic content determination total phenolic content of the obtained infusions was determined colorimetrically using the method described by singleton and rossi (1965) with some modifications [28]. briefly, 50 µl of infusion was mixed with 250 µl of folin-ciocalteu reagent. after 3 minutes 500 µl of saturated sodium carbonate solution (20%, w/w) was added and mixed again. the mixture was allowed to stand for 30 minutes in the dark prior to measuring the absorbance at 765 nm in a single beam uv-vis-nir spectrophotometer (shimadzu corporation, japan). total phenolic content was expressed in mg gallic acid equivalents (gae)/g. for gallic acid, absorbance is described by the following equation: y = 0.0012x – 0.0345 (r2 = 0.9997) 2.4. antioxidant activity the radical scavenging ability of the infusions was monitored using the stable free radical dpph (2,2-diphenyl-1picrylhydrazyl) following a procedure similar to those proposed by mraicha et al. (2010) and bouaziz et al. (2008) in previous studies [29,30]. aqueous solutions of samples were prepared by mixing various concentration of infusion with ethanol solution, as follows: 60 µl onion peel infusion and 2940 µl solvent, and 30 µl walnut shell infusion in 2970 µl ethanol. the aliquots were added to 5 ml of a 0.004% (w/w) freshly prepared methanol solution of dpph. the mixtures were left to stand at room temperature for 60 minutes in the dark (until stable absorbance values were obtained). the absorbance was measured against a blank at 517 nm in a single beam uv-vis-nir spectrophotometer (shimadzu corporation, japan). free radicals inhibition (i%) was calculated using the equation: i% = [(ablank – asample)/ablank] ×100, where: ablank – absorbance of the control reaction, containing all the reagents except the analyzed infusion asample – absorbance of the infusion fig. 1 walnut shell infusion (left) and onion peel infusion (right) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 183 fig. 2 absorbance (left) and total phenolic content (right) of infusions 3. results and discussion 3.1. total phenolic content of infusions the folin-ciocalteu assay, used for the determination of the total phenolic content of infusions from walnut shells and onion peel is a simple and widespread method that has been employed for a long time as a measure to evaluate total phenols of natural products. extraction of phenolic compounds from plant material is influenced by the chemical nature of the compound, the extraction method employed, sample particle size, the solvent used, extraction conditions (time, temperature), as well as the presence of interfering substances [31]. unlike the extraction with alcoholic solvents (methanol, ethanol), the use of water in the solubilization of bioactive compounds does not rise concern regarding the toxicity and recovery of the solvent from the extract. recent studies on the extraction of anthocyanins, carried out on synthetic flavylium salts showed that the molecules require neutralizing their own electrostatic repulsions with water molecules so that the dimerisation can be carried out [32]. therefore, water plays a fundamental role in phenolic compounds extraction. total phenolic content of walnut shell infusion and onion peel infusion, alongside the absorbance measured is presented in fig. 2. as it was expected, total phenolic content of walnut shell infusion (32.08 mg gae/g) was relatively low but exceeded the mean of 18.04 mg gae/g reported for the phenolic content of methanolic extract from powdered walnut hull [33]. higher phenolic content was determined for extracts from green walnut husk (50.18 mg gae/g), leaves (94.39 mg gae/g), and kernel (116.22 mg gae/g) [34]. for onion peel infusion, total phenolic content measured was 280.32 mg gae/g, a level that was close to the content obtained by boo et al. (2012) for water extracts from powdered onion peel [35]. regarding the influence of plant section and growth on the phenolic content of onion, it was noted that the greenish part of the onion had a higher phenolic content in comparison with the white part of the bulb [36]. 3.2. antioxidant activity antioxidants are essential to preserve the biological system from free radicals damage to biological molecules [37]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 184 fig. 3 absorbance (left) and scavenging effect (right) of infusions the capacity of antioxidants to protect a biological system against the harmful effects of oxidative processes is defined as antioxidant activity. in this study, the ability of the investigated walnut shell infusion and onion peel infusion to act as donors of hydrogen atoms or electrons in dpph radical transformation into its reduced form of dpph•-h was investigated in dpph• assay. as fig. 3 shows, both samples exhibited a radical scavenging activity below 70%. for walnut shell infusion the scavenging activity measured was 7.82%, a value that was greater than an inhibition of 7.19%, reported by other authors for the same by-product [33]. compared to other by-products of walnut processing, the scavenging activity on dpph radicals of infusions from shells was lower than the one obtained for water:ethanol extracts from green walnut husk [38]. onion peel infusion, which had the highest content of phenols, exhibited a high dpph free radical scavenging capacity, namely 69.38%. this value is close to the antioxidant activity of 69.7%, measured for methanolic extracts from red onion peel [39]. as in the case of polyphenols, the antioxidant activity is influenced by the distribution of the analyzed segment in vegetable material. it was reported that anthocyanins, a group of compounds with high antioxidant potential are heavily concentrated in onion skin [40]. moreover, it was observed that peel shredding led to an increase in the content of quercetin 4'-oβ-glucopyranoside [41], a flavonoid with documented radical scavenging activity. these two last observations confirm the increase in antioxidant activity from the inside to the outer part (peel) of the bulb. 4. conclusion the present study reports on total phenolic content and antioxidant activity of two infusions prepared with by-products of onion and walnut processing. the byproducts used were walnut shells and onion peels, and the solvent of extraction was water. walnut shell infusion had a low content of phenolic compounds, and therefore exhibited free radicals scavenging activity close to the levels reported in other similar studies. on the other hand, onion peel infusion presented a balanced phenolic content, and increased radical scavenging activity. between the two by-products investigated, onion peel has a great potential for capitalization. given the high food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 185 content of antioxidants, the reuse could imply both food and non-food applications. 5. references [1]. kosseva, m. r., processing of food wastes, advances in food and nutrition research, 58:57-136, (2009) [2]. wadhwa, m., bakshi, m. p. s., utilization of fruit and vegetable wastes as livestock feed and as substrates for generation of other valueadded products, rap publication, 4, (2013) [3]. prokopov, t. v., utilization of byproducts from fruit and vegetable processing: a review, food and packaging, 49, (2014) [4]. joshi, v. k., food processing industries’ waste: opportunities, technologies, challenges and future strategies, biotechnology in agriculture and environment, 129-148, (2002) [5]. marwaha, s. s., arora, j. k. (eds.), food processing: biotechnological applications, asiatech publishers, (2000) [6]. taha, n. a., al-wadaan, m. a., utility and importance of walnut, juglans regia linn: a review, african journal of microbiology research, 5(32):5796-5805, (2011) [7]. fernandez-lopez, j., aleta, n., alias, r., forest genetic resources conservation of juglans regia l., ipgri, rome, italy, 20-25, (2000) [8]. martínez, m. l., labuckas, d. o., lamarque, a. l., maestri, d. m., walnut (juglans regia l.): genetic resources, chemistry, by‐products, journal of the science of food and agriculture, 90(12):1959-1967, (2010) [9]. oliveira, i., sousa, a., ferreira, i. c., bento, a., estevinho, l., pereira, j. a., total phenols, antioxidant potential and antimicrobial activity of walnut (juglans regia l.) green husks, food and chemical toxicology, 46(7):2326-2331, (2008) [10]. stampar, f., solar, a., hudina, m., veberic, r., colaric, m., traditional walnut liqueur-cocktail of phenolics, food chemistry, 95(4):627-631, (2006) [11]. çelekli, a., birecikligil, s. s., geyik, f., bozkurt, h., prediction of removal efficiency of lanaset red g on walnut husk using artificial neural network model, bioresource technology, 103(1):64-70, (2012) [12]. zabihi, m., ahmadpour, a., asl, a. h., removal of mercury from water by carbonaceous sorbents derived from walnut shell, journal of hazardous materials, 167(1):230-236, (2009) [13]. zabihi, m., asl, a. h., ahmadpour, a., studies on adsorption of mercury from aqueous solution on activated carbons prepared from walnut shell, journal of hazardous materials, 174(1):251256, (2010) [14]. ding, d., zhao, y., yang, s., shi, w., zhang, z., lei, z., yang, y., adsorption of cesium from aqueous solution using agricultural residue walnut shell: equilibrium, kinetic and thermodynamic modeling studies, water research, 47(7):2563-2571, (2013) [15]. jakopic, j., veberic, r., extraction of phenolic compounds from green walnut fruits in different solvents, acta agriculturae slovenica, 93(1):11-15, (2009) [16]. ghasemi, k., ghasemi, y., ehteshamnia, a., nabavi, s. m., nabavi, s. f., ebrahimzadeh, m. a., pourmorad, f., influence of environmental factors on antioxidant activity, phenol and flavonoids contents of walnut (juglans regia l.) green husks, journal of medicinal plants research, 5(7):1128-1133, (2011) [17]. popovici, c., gîtin, l., alexe, p., characterization of walnut (juglans regia l.) green husk extract obtained by supercritical carbon dioxide fluid extraction, journal of food and packaging science technique and technologies, 1(1):5-9, (2012) [18]. faostat, production/crops for onions, http://faostat3.fao.org/browse/q/qc/e (2016) [19]. ye, c. l., dai, d. h., hu, w. l., antimicrobial and antioxidant activities of the essential oil from onion (allium cepa l.), food control, 30(1):48-53, (2013) [20]. roldán, e., sánchez-moreno, c., de ancos, b., cano, m. p., characterisation of onion (allium cepa l.) by-products as food ingredients with antioxidant and antibrowning properties, food chemistry, 108(3):907-916, (2008) [21]. benítez, v., mollá, e., martíncabrejas, m. a., aguilera, y., lópezandréu, f. j., cools, k., downes, k., terry, l. a., esteban, r. m., characterization of industrial onion wastes (allium cepa l.): dietary fibre and bioactive compounds, plant foods for human nutrition, 66(1):48-57, (2011) [22]. park, j., kim, j., kim, m. k., onion flesh and onion peel enhance antioxidant status in aged rats, journal of nutritional science and vitaminology, 53(1):21-29, (2007) [23]. downes, k., chope, g. a., terry, l. a., effect of curing at different temperatures on biochemical composition of onion (allium cepa l.) skin from three freshly cured and cold stored ukgrown onion cultivars, postharvest biology and technology, 54(2):80-86, (2009) [24]. bonaccorsi, p., caristi, c., gargiulli, c., leuzzi, u., flavonol glucosides in allium species: a comparative study by means of http://faostat3.fao.org/browse/q/qc/e food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sonia amariei, valentina mancas, total phenolic content and antioxidant activity of infusions from two by-products: walnut (juglans regia l.) shell and onion (allium cepa l.), food and environment safety, volume xv, issue 2 – 2016, pag. 180 186 186 hplc–dad–esi-ms–ms, food chemistry, 107(4):1668-1673, (2008) [25]. caridi, d., trenerry, v. c., rochfort, s., duong, s., laugher, d., jones, r., profiling and quantifying quercetin glucosides in onion (allium cepa l.) varieties using capillary zone electrophoresis and high performance liquid chromatography, food chemistry, 105(2):691-699, (2007) [26]. shabana, s., kawai, a., kai, k., akiyama, k., hayashi, h., inhibitory activity against urease of quercetin glycosides isolated from allium cepa and psidium guajava, bioscience, biotechnology, and biochemistry, 74(4):878-880, (2010) [27]. mallor, c., thomas, b., resource allocation and the origin of flavour precursors in onion bulbs, the journal of horticultural science and biotechnology, 83(2):191-198, (2008) [28]. singleton, v. l., rossi, j. a., colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, american journal of enology and viticulture, 16(3):144-158, (1965) [29]. mraicha, f., ksantini, m., zouch, o., ayadi, m., sayadi, s., bouaziz, m., effect of olive fruit fly infestation on the quality of olive oil from chemlali cultivar during ripening, food and chemical toxicology, 48(11): 3235-3241, (2010) [30]. bouaziz, m., lassoued, s., bouallagui, z., smaoui, s., gargoubi, a., dhouib, a., sayadi, s., synthesis and recovery of high bioactive phenolics from table-olive brine process wastewater, bioorganic and medicinal chemistry, 16(20): 9238-9246, (2008) [31]. naczk, m., shahidi, f., extraction and analysis of phenolics in food, journal of chromatography a, 1054(1):95-111, (2004) [32]. castañeda-ovando, a., de lourdes pacheco-hernández, m., páezhernández, m. e., rodríguez, j. a., galán-vidal, c. a., chemical studies of anthocyanins: a review, food chemistry, 113(4):859-871, (2009) [33]. akbari, v., jamei, r., heidari, r., esfahlan, a. j., antiradical activity of different parts of walnut (juglans regia l.) fruit as a function of genotype, food chemistry, 135(4):2404-2410, (2012) [34]. carvalho, m., ferreira, p. j., mendes, v. s., silva, r., pereira, j. a., jerónimo, c., silva, b. m., human cancer cell antiproliferative and antioxidant activities of juglans regia l., food and chemical toxicology, 48(1):441447, (2010) [35]. boo, h. o., hwang, s. j., bae, c. s., park, s. h., heo, b. g., gorinstein, s., extraction and characterization of some natural plant pigments, industrial crops and products, 40:129135, (2012) [36]. marinova, d., ribarova, f., atanassova, m., total phenolics and total flavonoids in bulgarian fruits and vegetables, journal of the university of chemical technology and metallurgy, 40(3):255-260, (2005) [37]. mishra, n., dubey, a., mishra, r., barik, n., study on antioxidant activity of common dry fruits, food and chemical toxicology, 48(12):3316-3320, (2010) [38]. fernández-agulló, a., pereira, e., freire, m. s., valentao, p., andrade, p. b., gonzalez-alvarez, j., pereira, j. a., influence of solvent on the antioxidant and antimicrobial properties of walnut (juglans regia l.) green husk extracts, industrial crops and products, 42:126-132, (2013) [39]. singh, b. n., singh, b. r., singh, r. l., prakash, d., singh, d. p., sarma, b. k., upadhyay, g., singh, h. b., polyphenolics from various extracts/fractions of red onion (allium cepa) peel with potent antioxidant and antimutagenic activities, food and chemical toxicology, 47(6):1161-1167, (2009) [40]. gennaro, l., leonardi, c., esposito, f., salucci, m., maiani, g., quaglia, g., fogliano, v., flavonoid and carbohydrate contents in tropea red onions: effects of homelike peeling and storage, journal of agricultural and food chemistry, 50(7):1904-1910, (2002) [41]. abouzid, s. f., elsherbeiny, g. m., increase in flavonoids content in red onion peel by mechanical shredding, journal of medicinal plants research, 2(9):258-260, (2013) 1. introduction получение белоксодержащих препаратов природного происхождения 61 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1 2016, pag. 61 66 production of protein-containing preparations of natural origin *nataliia borisivna mitina1, inna mihalivna zubareva2, olga ivanivna tkalya1 1“dnepropetrovsk chemical-technological state university”, dnepropetrovsk, ukraine 2national university of dnepropetrovsk, ukraine *natalimitina_68@mail.ru , zubareva95@bk.ru *corresponding author received february 26th 2015, accepted march 28th 2016 abstract. the possibility of increasing the yield of mycelial biomass and protein substances of higher edible mushroom pleurotus ostreatus in the conditions of submerged cultivation, as well as the productivity increase of the culture eisenia foetida as producer of protein biomass on the nutrient substrate mixture of sunflower and rice husk modified to 200–500 microns have been studied. the greater amount of deep pleurotus ostreatus biomass was found to accumulate on media containing hydrolysates of buckwheat, corn or wheat flour combined with gluten. it is on these media that the highest protein content of biomass is observed. the presence of soy milk as part of nutrient medium provided pleasant mushroom smell specific to pleurotus ostreatus of natural growth. fermented substrate of sunflower and rice husk mixture was established to increase vermiculture yield at densities ranging between 10–15 thousand per m2 and to optimize biochemical composition of the eisenia foetida biomass tissue. keywords: higher edible mushroom, biomass, vermiculture, fermented substrate, culture media. 1. introduction protein-containing products are widely used in medical, veterinary, food, pharmaceutical industry, animal husbandry and in the research area. today's society shows a special interest to the protein products of natural origin [1]. industrial production of protein-containing natural products is possible by extraction from plant, animal or microbial raw materials [2, 3]. however, this method does not allow to produce sufficiently large quantities of protein-containing preparations, because the method is limited by the deficiency of vegetable and animal raw materials, crop areas, weather and climatic conditions. in addition, to extract proteins from a corresponding raw material it is necessary to use chemical reagents (acids, bases) having negative influence on the native properties of proteins in finished product. thus, the industrial production of protein biopreparations by this method appears to be not profitable. the production of protein-containing preparations with appropriate producer organisms turns out to be the most expedient both from the economic and technological point of view. various commercial protein products produced industrially in various countries are known [2]. among the producers of such preparations yeast are the most common (bacteria less common). thus, preparations of food yeast, therapeutic beer yeast are produced by saccharomyces cerevisia. the basis for such preparations as paprin (yeast feed protein) and torutin is candida maltosa and candida utilis, respectively. protein preparation gaprin http://www.fia.usv.ro/fiajournal mailto:natalimitina_68@mail.ru mailto:zubareva95@bk.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya, production of protein-containing preparations of natural origin, food and environment safety, volume xv, issue 1 – 2016, pag. 61 – 66 62 (bacterial feed protein) is produced on the basis of methylophilus methylotropus bacteria. there are a few examples of using organisms from other taxonomic groups as producers of protein-containing products. the technologies for producing proteincontaining preparations of mycelial origin are also developed. for example, digitatin and food mycoprotein are prepared using fungi penicillium digitatum and fusarium graminearum, respectively. saprophytic fungi paecilomyces varioti are used to prepare the mushroom feed mass. mycelial organisms as industrial producers have some advantages. in particular, it is easier to separate mycelium from the culture liquid by filtration; the amount of protein in the cells of many fungi is higher than that of yeast or bacterial organisms [1]. organisms from other taxonomic groups as producers of protein products are not studied enough. so, with the help of representatives of higher edible mushrooms (pleurotus ostreatus, champignon, etc.) it is possible to produce not only fodder products, but also quality foodstuffs with high protein content possessing racy flavour and aroma of natural fungi [4–6]. vermiculture eisenia foetida can be used as a basis for protein products of medical, pharmacological and fodder purpose [3]. however, the use of these organisms as industrial producers of protein-containing products requires comprehensive research including selection and optimization of culture media and substrates for growing them in vitro. thus, this problem is urgent and requires further investigations. the aim of this work is selection and optimization of liquid media for submerged cultivation of pleurotus ostreatus as well as modification of solid-state nutrient substrate for increasing biomass of eisenia foetida culture. 2. materials and methods in these experiments, carried out at the institute of biotechnology of the ukrainian state university of chemical technology and in the research laboratory at the department of microbiology, virology and biotechnology of dnipropetrovsk national university n.a. o. honchar, we used as objects of study: higher edible mushroom pleurotus ostreatus strain nc-35 (department of mycology, institute of botany, kiev) as a producer of protein substances and vermiculture eisenia foetida (research institute "biotechnology" of ihe "usctu" dnipropetrovsk tech. specs. тu 3336406.002-95) as a producer of protein biomass, sunflower and rice husk (tech. specs. tu-18 ussr6280). for storage of museum culture pleurotus ostreatus large microbiological test-tubes with wort-agar media (6 °c) were used. subsequent fermentation was performed in 300 ml capacity shake flasks containing 50 ml of nutrient medium. the composition of the fermentation media was different. test media contained (%): corn steep liquor – 6, hydrol (high-green syrup) – 6, potassium dihydrogen phosphate – 0.05, thiamine chloride – 0.0002, sunflower oil – 4. experimental nutritive media included enzyme hydrolysates of different types of flour or husking bran and gluten or soya milk at different ph concentrations (6.8– 6.9). the main raw material of the experimental culture media (different types of flour) was subjected to enzymatic prehydrolysis using amylolitic enzyme preparations of domestic production. hydrolysis conditions were as follows: temperature 57–60 °c, 10–15 minutes duration, concentration of alphaamylase preparation 1:1900 and that of glucoamylase – 1:2000. the prepared nutrient media were autoclaved for 45 minutes at 120 °c [7]. fungus cultivation was carried out in submerged conditions for 120 hours at the temperature of 26–28 °c with stirring at 240 r/min. in the shaker of fhc-12-250 type. the resulting culture medium (cm) was analyzed for protein and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya, production of protein-containing preparations of natural origin, food and environment safety, volume xv, issue 1 – 2016, pag. 61 – 66 63 biomass content [7, 8] after its separation in the centrifuge k-23 at 3000 rpm for 10 minutes. experiments were carried out in 7 replications. the results were processed by mathematical statistics methods [9]. modification of sunflower and rice husk was carried out in a mill with rotary beater having cylindrical elements to fractions 200–500 microns. cultivation was carried out on eisenia foetida fermented substrate of sunflower and rice husks in the ratio 1:0.5 at a temperature of 20–25 °c; ph 6.5–7.5; stocking density – 10–15 thousand worms per 1 m2 (to speed up the growth of biomass), 15-30 thousand worms per 1 m2 (to increase the number of eisenia foetida population); frequency of making fresh substrate – once every 10 days. biological value of eisenia foetida biomass was determined by amino-acid analyzer n1200e, by bernstein, lowry spektrometric method. potassium was defined by the weight of potassium tetraborate which was formed in acidic medium; phosphorus – by colorimetric briggs’ method; calcium – by conductormetric method [7, 8, 10–12]. gross energy and digestible energy of eisenia foetida biomass was determined by v.i. ivanov’s formula [13]. 3. results and discussion increasing of fungus productivity at the basic parameters was possible by optimizing the qualitative and quantitative composition of the culture media. for the growth of the producer, media of different composition were proposed. the extract molasses medium containing starch production waste was corn extract (a source of nitrogen and growth factors) and hydrol (carbon and energy source). for comparison, culture media including other sources of producer development (starchy flour production waste as a source of carbon and energy as well as gluten or soya milk as a nitrogen supply source of the fungus) were studied. starchy components were subjected to enzymatic prehydrolysis. the concentration of the investigated components in the medium was calculated by the content of sugar (0.15 %) and nitrogen (0.22–0.24 %), respectively. the results on the accumulation of biomass and protein by edible higher mushroom of pleurotus ostreatus (strain tc-35) which were identified in the conditions of this experiment are presented graphically in fig. 1 and 2. fig. 1. pleurotus ostreatus biomass yield depending on the quality of carbon and nitrogen sources in culture media: 2, 4, 5, 6, 7, 8 – hydrolysates of corn, buckwheat, wheat flour, wheat husking bran, oats and rye flour, respectively fig. 2. protein yield of pleurotus ostreatus depending on culture media quality: 1, 3, 4, 5, 6, 7 – hydrolysates of corn, buckwheat, wheat flour, wheat husking bran, oat and rye flour, respectively these charts indicate that more of depth pleurotus ostreatus biomass was accumulated on the media containing hydrolysates of buckwheat, corn or wheat 1 1.5 2 2.5 3 1 2 3 4 5 6 7 8 сarbon sources soybean milk gluten 0 20 40 60 80 1 2 3 4 5 6 7 p ro te in c on te nt , % сarbon source gluten soybean milk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya, production of protein-containing preparations of natural origin, food and environment safety, volume xv, issue 1 – 2016, pag. 61 – 66 64 flour combined with gluten. the highest protein content in the biomass was observed on these particular media. the combination of these hydrolysates and soya milk provided obtaining of smaller amount of biomass by a factor of 1.5; 1.3 and 1.4 times respectively than that produced on gluten. similar pattern was observed in the analysis of protein content in the deep fungal biomass. thus, obtaining pleurotus ostreatus biomass with high protein content (up to 60%) is possible. to do this, enzymatic hydrolysates of various starch-containing materials could be used. but the use of buckwheat, corn or wheat flour hydrolysates seemed to be optimal. the nitrogen source was found to be more important as a component of the culture medium for the accumulation of pleurotus ostreatus biomass and protein, in the conditions of the experiment involved. thus, gluten provided a greater accumulation of both biomass and protein therein as compared with soya milk. it was also noted that the quantitative values for biomass and protein on all hydrolysis media in combination with soya milk have no significant differences between themselves and as compared with the control (the extract-molasses medium). but in the presence of soya milk as a part of the culture medium, an important feature of mushroom mycelium, namely the emergence of a pleasant mushroom smell characteristic of natural growth pleurotus ostreatus, was revealed. the presence of the appropriate taste and smell were indispensable features of deep fungal mycelium as biotechnological product of food quality. it was also found that the cultivation of these mushrooms on the extract-molasses medium did not provide the highest yield of the fungus protein-containing biomass. the concentration of the mycelium in liquid culture reached 1.82±0.091 g/100 ml and the protein content was 45.99±2.01 %, which is 1.5 and 1.3 times lower than that on the enzymatic hydrolysate of buckwheat flour in combination with gluten. the analysis of the obtained data indicated a need for further research on the optimizing of culture media composition, which would ensure the yield increase of proteincontaining biomass having characteristic aroma of natural pleurotus ostreatus mushroom. to do this one must probably combine nitrogen sources under investigation in one nutrient medium, but it is necessary to optimize quantitatively media for nitrogen-containing components. during the investigations it was established that the mixture of natural polymers of sunflower and rice husks crushed on a rotary mill to 200–500 microns appeared to be optimal medium for breeding eisenia foetida, met the international requirements for the content of the necessary materials and components to ensure the nutritional value of the substrate: crude protein – 5– 6 %; crude fat – 4–4.5 %; crude fiber – 40– 45 %; calcium – 4.5 g/kg; phosphorus – 0.8 g/kg. to determine the optimal conditions for keeping and breeding eisenia foetida investigations concerning effect of humidity, temperature, worms stocking density on the fruitfulness and growth of the eisenia foetida population biomass were carried out. optimum conditions for keeping and breeding eisenia foetida worm on the substrate of modified sunflower and rice husk were as follows: 70 % to 80 % humidity, the ecological valence of humidity 40–98 %, the normal life activity area 65– 90 %. the optimum temperature for growth and functioning of eisenia foetida biomass was 20–25 °c; normal activity area was 15– 30 °c, the environmental temperature valence was 5–35 °c. the acidity of the substrate should be within 6.2–7.8. observations on the activity and survival of eisenia foetida showed that the modified substrate consisting of natural polymers mixture was suitable as a habitat and a growth medium. the first cocoons were found a month later, larvae hatched out two weeks later. microorganisms (mould) not food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya, production of protein-containing preparations of natural origin, food and environment safety, volume xv, issue 1 – 2016, pag. 61 – 66 65 typical for a living environment appeared on the substrate surface during composting process in the first days after the introduction of worms, but they disappeared in two weeks. these results showed that during the processing of eisenia foetida substrates mouldy microflora was destroyed, thereby disinfecting vermicompost. we conducted the experiments to determine the effect of worm introduction density on the increase in eisenia foetida population biomass. introduction density during three experiments was 10000/m2, 15000/m2 and 30000/m2, with an average weight of worms 210 mg. the experiment has been conducted for three months. data on the effect of introduction density on the growth of eisenia foetida population biomass is given in the table. table 1. the impact of introduction density on the growth of eisenia foetida population biomass indicator research period the number of worms per 1 m 2 10000 15000 30000 the average weight of a single worm, mg initiation of study 210±1.47 210±1.47 210±1.47 after 1 month 427±5.12 425±5.52 369±6.27 after 2 months 756±13.60 661±13.88 581±12.78 after 3 months 1048±24.10 979±22.52 876±20.58 the gain since study initiation, mg after 1 month 217±2.60 215±2.79 159±2.22 after 2 months 546±9.83 451±9.47 371±8.16 after 3 months 838±19.27 769±17.69 666±15.65 thus, we can conclude that with greater introduction density the number of eisenia foetida vermiculture and substrate processing speed increased. to intensify the eisenia foetida biomass yield, introduction density may be in the range of 10–15 thousand / m2. the intensity of the substrate processing by worms is 3.2–3.6 kg per day per 1 m2; the yield of the protein additive is 50–60 %. as a result of processing of organic substances by vermiculture, the mixture of plant substrate extracted from the worms’ digestive tract – coprolites – appeared to be nutritious: the total amount of humic acid constituted 60,36–60,95 %, that of nitrogen – 1.5–2.7 %, of phosphorus – 1.25–1.71 %, potassium 2,18–2,41 %; there were 17 amino acids, including essential ones – lysine, methionine, tryptophan; macroand trace elements – magnesium, iron, copper, manganese and zinc, vitamins b1, b2, pp. it was determined that dry matter in the eisenia foetida biomass tissue, adapted on the modified sunflower and rice husks, constituted 17–23 %, crude protein up to 60 %, lipids 6–9 %, nitrogen extractives from 7 to 16 %. the criterion of gross and digestible energy served to assess quality of eisenia foetida biomass for animal feed . 1 kg of eisenia foetida biomass produced by culturing on sunflower and rice husk contained 1.03 of feed units and 14.4 mj of digestible energy. 4. conclusions thus, the selection and optimization of liquid nutrient media for submerged cultivation of pleurotus ostreatus provided increased yield of both biomass and protein in it; modification of solid-phase nutrient substrates based on sunflower and rice husks for growing eisenia foetida allowed to obtain a high quality protein containing mass with quality biochemical composition comprising all essential amino acids, microelements, vitamins, enzymes. consequently, the above said organisms can be used as effective producers of proteinfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 nataliia borisivna mitina, inna mihalivna zubareva, olga ivanivna tkalya, production of protein-containing preparations of natural origin, food and environment safety, volume xv, issue 1 – 2016, pag. 61 – 66 66 containing products of natural origin. 5. references [1]. becker m.e., liepinsh g.k., raypulis e.p. biotechnology. moscow. agropromizdat, 334, (1990) [2]. yelinov n. fundamentals of biotechnology. st. petersburg. nauka, 600, (1995) [3]. titov i.n., usoev v.m. vermiculture as a renewable source of animal protein from organic waste. bulletin of the tomsk state university. biology. 2 (18), 74-80, (2012) [4]. simakhina h. prospects for the use of edible fungi as the complete proteins. products & ingredients. 6, 106-109, (2008) [5]. solomko i. nutritional value and healing properties of cultivated mushrooms.vegetables. 3, 70-73, (2009) [6]. velichko t.a., mitina n.b., zubareva i.m tkalya o.i., shatalin db. optimization of culture media for the cultivation of pleurotus ostreatus proceedings onaft 2, 165-168, (2011) [7]. egorov n.s .guide to practical training in microbiology. moscow state university, 256, (1983) [8]. guidelines for zootechnical feed analysis. dnepropetrovsk: dgau, 47, (2007) [9]. frolov l.a., melnikov b.i., halivets j.d., mitina n.b. mathematical modeling and optimization technology objects inorganic substances. dnepropetrovsk: jour. foundation, 208, (2010) [10]. gorodnii n.m., kovalev v.b., miller i.a. vermiculture and its effectiveness. sel. host ˗ in; review. inform. ser. agriculture, agricultural chemistry with. x. reclamation. ukrniinti. 20, (1990) [11]. kucherenko m.e. modern methods of biochemical research. kiev. phyto ˗ center, 424, (2001) [12]. mitina n.b., kulik a.p., kalashnikov s.g. research technologies of protein supplements. 3. biological purity raw materials for vermitehnolohiy. questions of chemistry and hymycheskoy technology. 5, 24-27, (2009) [13]. nozdrin m.t., karpus m.m., karavashenko v.f. detailed norms for farm animals feeding. handbook, 130, (1991) 161 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 161 168 some chemical aspects during white cabbage pickling process *marcel avramiuc1 *faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro * corresponding author received: april 18th 2013, accepted may 25th 2013 abstract. in this work was searched the evolution of some chemical parameters (salt content, ph and ascorbic acid content) during cabbage pickling. the biological material was white cabbage purchased from commercial network, iodized and non-iodized salt and water. in various glass jars were introduced different samples of cabbage (whole and minced), over which was poured brine (iodized and non-iodized salt), for each type of cabbage. the samples were left to ferment, and at 3-4 days were made determinations of salt content, ascorbic acid and ph. during pickling, the evolution of both types of salt (iodized and iodized), accumulated both in the whole and in the minced cabbage, showed a progressive increase in the first 5-8 days, after which the values decreased, reaching minimum, in all cases, at 26 days of pickling. throughout the trial, in both types of cabbage (whole and chopped) non-iodized salt was accumulated in larger quantities than iodized one. for the both types of cabbage, the ph of sauce, obtained with iodized salt, recorded greater reductions than the ph of sauce with non-iodized salt. the higher ph values were recorded in the sauce derived from whole cabbage. during pickling process, the biggest losses of ascorbic acid were recorded in minced cabbage, whose brine was prepared with iodized salt. the evolution of the ascorbic acid concentration in the two types of cabbage sauce highlights, in all cases, greater increases in chopped cabbage brine, processed with iodized salt. keywords: cabbage, salt, sauce, pickling, ascorbic acid, ph 1. introduction for the human diet, vegetables and fruits are fundamental sources of water-soluble vitamins (vitamin c and group b vitamins), provitamin a, phytosterols, dietary fibres, minerals and phytochemicals [1 cited by 8]. according to some authors [2, 3], cooking, pasteurization and the addition of chemical preservatives are the main technology options guaranting vegetables and fruits safe, but bringing a number of (not always) desirable changes in their physical characteristics and chemical composition. in order to preserve vegetables and fruits for a long time without modifying their properties, there were developed some conservation methods based on growth of lactic acid bacteria from microbiota of these plant species over other existing organisms. this preservation methods are known as: acidification or pickling methods. lactic acid fermentation is considered as the simple and valuable biotechnology to keep and/or enhance the safety, nutritional, sensory and shelf life properties of vegetables and fruits [4, 5, 6, 7, 8 ]. lactic acid fermentation undoubtedly represents the easiest and the most suitable way for increasing the daily consumption of freshlike vegetables and fruits [8]. some fermented pickles, such as: seasonal leafy vegetables, radish, cucumbers and young tender bamboo shoots, obtained through indigenous techniques of biofood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 162 preservation, are very popular in asian and african countries, representing fundamental components of their daily diet [9]. some fermented pickles, such as: seasonal leafy vegetables, radish, cucumbers and young tender bamboo shoots, obtained through indigenous techniques of biopreservation, are very popular in asian and african countries, representing fundamental components of their daily diet [9]. there are many scientific works describing the lactic acid fermentation of cabbage to get sauerkrauts and of olives to make table olives [10]. in romania, pickles (cucumbers, cabbage, cauliflower, carrots, green tomatoes, fruits) are very popular and appreciated, being consumed especially in winter. of these, pickled white cabbage is, for many families, a component of the daily diet (spice and / or food), being almost customary in the diet. in this work was studied the evolution of some chemical parameters (salt content, ph and ascorbic acid content) during pickling of whole and minced white cabbage, using two types of brine: iodized and non-iodized salt. 2. experimental 2.1. research materials in this paper, the biological material was white cabbage purchased from commercial network, selected to meet the quality parameters necessary to conservation. the cabbage was fresh, well made, no damage and no attack by insects or parasites, clean, odorless and taste, with a head weight of between 1-1.5 kg. fresh raw material (prior to curing by pickling) was analyzed, determining the ph, whose value was 6.94, and the content of ascorbic acid (36.21 mg %). for experiments it used iodized and noniodized salt recrystallized and water from the distribution network of the suceava city. the salt used was provided by the national salt company, salina cacica branch. 2.2. the preparation of pickles whole cabbages were clean and were placed in two jars of colored glass, of 15 liters each, well stuffed (pressure) do not run goals. using the two types of salt (iodized and noniodized) there were prepared in 2 pots, two types of brine, introducing of 45 g salt per liter of water. among the rows of cabbage there were placed pieces of horseradish root and flourished over dill and dried, because after banu et al. (2000) the additions improved the flavor of the product and the respective broth with preservative role. before closing the jars, there were placed two small wooden boards (cross) to prevent cabbage to rise above, then the jars were left to ferment at 15-18ºc. for anaerobic environment may favor butyric fermentation, the cabbage brine was subjected to periodic aeration [12], through introduction of the air into the brine by means of a hose or by pouring from one container to another in prolonged contact with air. after completion of pickling tank, pickled cabbage was stored at 8-10°c. minced cabbage was obtained from fresh cabbage, healthy, clean, without outer leaves, which was cut with a knife steel in long thin strips. the cabbage fragments were transferred within two metal flasks and were salted, separately, with iodized and non-iodized salt (25-30 g of salt per 1 kg of cabbage), and then were rubbed for leaving enough juice. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 163 after approx. 30-60 minutes, the shredded and salt cabbage from containers was introduced in two jars of colored glass (5 liters each), one for each type of salt, squeezed by hand as well to cover the cabbage with soup made (2-3 cm liquid). above they have put a few celery leaves for flavor and two small wooden boards cross. the initial fermentation temperature was 1820°c for 3 days, after which the jars were transferred to a room at a temperature of 810°c. during fermentation it was added brine, made up of 15 g of salt per liter of water. the determinations of chemical and biochemical parameters were carried out to each 3-4 days, using the whole or chopped pieces of cabbage or brine, in both types. 2.3. research methods the evaluations of the three parameters (salt concentration, ph and concentration of ascorbic acid) were made at specific intervals (2, 3 or 4 days), the first measurement being every three days from the beginning of the pickling process. determination of salt (nacl) concentration was made from pickled products, by titrimetric method, based on sodium chloride titration with silver nitrate in the presence of potassium chromate as indicator. the result was expressed in grams of sodium chloride in 100 g (100 ml) of product [13]. determination of ph was made with a digital ph meter supplied by hanna instruments. determination of ascorbic acid was made from products and its sauce according to official aoac methods [14], based on the reduction by ascorbic acid of 2.6dichlorphenol-indophenol (2.6-dcfif) to the corresponding leucoderivate. the result was expressed as mg ascorbic acid per 100 g (100 ml) of product. 3. results and discussion in fig. 1 is shown the evolution of the salt concentrations (iodized and non-iodized) of the cabbage samples. evolution of salt accumulated in whole cabbage during pickling showed a progressive increase of this parameter, which reached a maximum after eight days for both types of salt: 2.48 g nacl/100g of product for non-iodized salt and 2.19 g nacl/100g of product for iodized salt. 1.3 1.6 1.75 2.32 2.19 2.48 1.46 2.33 1.41 2.18 1.25 1.9 1.16 1.67 1.02 1.26 0 0,5 1 1,5 2 2,5 g n ac l/1 00 g 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d num ber of days (d) is cb s cb fig. 1. the evolution of the salt values in whole cabbage during pickling process is cb = iodized salt in cabbage; s cb = non-iodized salt in cabbage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 164 further, since the 12th day, both types of salt concentration values were decreased with time, reaching a minimum after 26 days (1.26 g nacl/100g of product for noniodized salt and 1.02 g nacl/100g of product for iodized salt). at all periods examined, the highest values were recorded for iodized salt. in the fig. 2 are reproduced the salt concentration values acquired in minced cabbage during pickling. as seen, in minced cabbage the concentration of the both salt types reached maximum values after five days of pickling (2.74 g nacl/100g of product for non-iodized salt and 2.58 g nacl/100g of product for iodized salt). the concentration of both types of salt decreased, to finally reach the same amount (1.35 g nacl/100g of product). 1.85 2.04 2.58 2.74 2.37 2.74 2.4 2.55 1.82 2.19 1.58 1.83 1.35 1.6 1.35 1.35 0 0,5 1 1,5 2 2,5 3 g n ac ll/ 10 0g 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d num ber of days (d) is mcb s mcb fig. 2. the evolution of the salt values in minced cabbage during pickling process is mcb = iodized salt in minced cabbage; s mcb = non-iodized salt in minced cabbage throughout the process of pickling, the iodized salt was accumulated in the largest quantities in minced cabbage. in the whole cabbage, the iodized salt concentration has an evolution with similar values of chopped cabbage, especially in the second part of the pickling period (15-26 days). the non-iodized salt has accumulated in higher quantities in chopped cabbage samples, only in the range 3-12 days of pickling. next 15-26 days, the concentration values of non-iodized salt were very close in the both types of pickled cabbage. the greater amount of salt (iodized and noniodized) acquired into minced cabbage, compared to the whole cabbage, can be explained by the grinding process, which, on the one hand, increases the area of contact between the cells and brine, and on the other side, destroys many cells facilitating the easier ingress of salt in the tissues fragments of cabbage. according to banu et al. (2003), the lactic fermented foods (pickled cabbage, pickled cucumbers) partial loss nutrients and biominerals of raw materials, passing the liquid (fermentation broth). this may explain the progressive decrease of the amount of salt in cabbage during pickling process in this experiment. the ph of the two samples (whole and minced cabbage) has changed during pickling, registering, in all cases, cut-off, larger or smaller, depending on the type of salt used and the analysed interval (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 165 table 1 the ph values of cabbage sauce during pickling process number of days (d) 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d samples ph cabbage sauce with iodized salt 6.75 6.67 6.07 4.51 4.10 4.02 3.97 3.87 cabbage sauce with non-iodized salt 6.68 6.45 5.40 4.49 4.15 3.95 3.98 4.15 minced cabbage sauce with iodized salt 6.13 6.07 4.66 3.90 3.77 3.80 3.81 3.95 minced cabbage sauce with non-iodized salt 6.00 5.47 4.71 4.10 3.95 3.92 3.96 3.99 from the table 1 it can see that the ph of cabbage sauce, with iodized salt, showed (after 26 days) the largest decreases: 3.07 units for whole cabbage and 2.99 units for minced one, versus 2.53 units for whole cabbage, and 2.01 units for minced cabbage, in non-iodized salt sauce. for both types of salt, the broth (sauce) acidification was more pronounced in the first 15 days of the pickling process. the comparative evolution of ph values during pickling process of whole and chopped cabbage samples (with iodized and non-iodized salt) showed that the highest ph values were recorded in the sauce derived from whole cabbage. the stronger acidification of the broth from minced cabbage, compared to broth from whole cabbage, can be explained by the greater amount of nutrients released from damaged cells through grinding, resulting in increased fermentative activity of the microflora present in the environment, which produces a larger amount of lactic acid. fig. 3-4 show the ascorbic acid values in the cabbage samples during pickling. in whole cabbage, after 26 days from the beginning of the pickling process, the content of ascorbic acid was reduced by 49.6% in cabbage with iodized salt, and by 34.8% in cabbage with non-iodized salt (fig. 3). in whole cabbage, the highest percentage reduction of ascorbic acid were recorded in the first 19 days of pickling (by 37.8% in cabbage with iodized salt, and by 28.39% in cabbage with noniodized salt). 33,19 34,71 29,55 32,85 28,97 30,56 28,85 27,71 26,07 27,65 22,52 25,93 20,9 25,96 18,25 23,61 0 5 10 15 20 25 30 35 a sc or bi c ac id ( m g/ 1 00 g) 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d number of days (d) cb is cb s fig. 3. the ascorbic acid evolution in cabbage during pickling process cb is = cabbage in iodized salt; cb s = cabbage in non-iodized salt food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 166 in minced cabbage, after 26 days of pickling, the ascorbic acid content decreased by 55.6% in cabbage with iodized salt, and by 43.85% in cabbage with non-iodized salt (fig. 4). at minced cabbage, the highest percentage reduction of ascorbic acid were recorded in the first 15 days of pickling (by 42.4% in chopped cabbage with iodized salt, and by 31.9% in chopped cabbage with non-iodized salt) 29.68 30.78 24.33 27.78 22.46 26.08 21.18 25.52 20.85 24.65 19.29 24.41 19.9220.62 16.08 20.34 0 5 10 15 20 25 30 35 a sc or bi c ac id (m g/ 10 0g ) 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d number of days (d) mcb is mcb s fig. 4. the ascorbic acid evolution in minced cabbage during pickling process mcb is = minced cabbage in iodized salt; mcb s = minced cabbage in non-iodized salt through analysis of ascorbic acid evolution during pickling of the two types of cabbage, it results that the highest values of this parameter were recorded in the samples with non-iodized salt. for the both types of salt, throughout the study (26 days) the highest values of ascorbic acid were recorded in the whole cabbage. one explanation may be that, during shredding, the cabbage loses, by oxidation, some of ascorbic acid (vit. c), process continued, perhaps, with a lower intensity throughout the pickling. according to banu et al. (2003), simply cut of cabbage, carrots leads to losses of up to 75% for vitamin c. also, iodine in the salt composition probably contributed to the degradation of this vitamin, because in all samples analyzed and at all intervals the ascorbic acid concentration was lower when using iodized salt, as compared with noniodized salt. the sauce of the pickled products evidenced different levels of ascorbic acid, depending on the type of salt and the characteristics of the biological material used (table 2). table 2 the ascorbic acid values in cabbage sauce during pickling process number of days (d) 3 d 5 d 8 d 12 d 15 d 19 d 22 d 26 d samples ascorbic acid (mg/100 g) cabbage sauce with iodized salt 7.92 8.81 11.48 13.06 13.86 13.88 15.04 16.63 cabbage sauce with non-iodized salt 7.31 8.33 10.29 11.06 11.84 12.37 12.74 13.02 minced cabbage sauce with iodized salt 8.71 11.27 12.65 14.44 14.61 15.44 15.94 16.76 minced cabbage sauce with non-iodized salt 7.92 10.84 11.85 12.30 12.78 13.30 13.90 14.04 as seen in the table 2, in the whole cabbage sauce, pickled in iodized salt, the ascorbicacid content has increased by 105% (from the first to the last determination), while in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 167 sauce pickled in non-iodized salt the ascorbic acid content has increased by 89%. also, in the table 2 can be seen that ascorbic acid in the sauce of minced cabbage, processed with iodized salt, recorded, after 23 days, an increase of ascorbic acid values by 96.2%, and in the sauce of minced cabbage, processed with non-iodized salt, an increase by 88.64%. from the data of table 2 results that ascorbic acid concentration recorded, in all cases, higher increases in pickles sauce processed with iodized salt, which shows that higher vitamin losses from these samples are partially found in the sauce of these pickles. in the both types of salts, the highest increases of ascorbic acid were in the sauce derived from minced cabbage. there were calculated the simple correlations between ph and ascorbic acid values in the cabbage samples and its sauce, during pickling process with non-iodized salt, because the lowest losses of ascorbic acid were found in pickled samples with this type of salt. if in the samples of cabbage (whole or minced) between ph values and ascorbic acid content not significant correlations could be established, in sauce samples were negative correlations more significant in the samples from the whole cabbage (r2=0.9522). 4. conclusions the analysis of salt content, the ph values and the ascorbic acid content of in the samples of white cabbage (whole and cubed) during pickling with iodized and non-iodized salt, some differences were found between samples, depending on the type of cabbage and type of salt used. during pickling, the evolution of both types of salt (iodized and iodized), accumulated both in the whole and in the minced cabbage, showed a progressive increase of this parameter up to the maximum values at 8 and 5 days, after which the values decreased, reaching minimum, in all cases, at 26 days of pickling. throughout the trial, in both types of cabbage (whole and chopped) non-iodized salt was accumulated in larger quantities than iodized one. for the both types of cabbage, the ph of sauce, obtained with iodized salt, recorded (during pickling) greater reductions than the ph of sauce with non-iodized salt. the higher ph values were recorded in the sauce derived from whole cabbage. during pickling, the biggest losses of ascorbic acid were recorded in minced cabbage, whose brine was prepared with iodized salt. the evolution of the ascorbic acid concentration in the two types of cabbage sauce highlights, in all cases, greater increases in chopped cabbage brine, processed with iodized salt. 5. references [1] gebbers j.o. atherosclerosis, cholesterol, nutrition, and statins – a critical review, german medical science, 5 (2007), pp. 1–11 [2] zia-ur-rehman z., islam m., shah w.h. effect of microwave and conventional cooking on insoluble dietary fibre components of vegetables, food chemistry, 80 (2003), pp. 237–240 [3] zhang d.l., hamauzu y. phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking, food chemistry, 88 (2004), pp. 502–509 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 marcel avramiuc, some chemical aspects during white cabbage pickling process, food and environment safety, volume xii, issue 2 – 2013, pag. 161 168 168 [4] steinkraus k.h. handbook of indigenous fermented foods revised and enlarged (second ed.) marcel dekker, new york, ny (1996), pp. 776-777 [5] buckenhüskes h.j., p.d. doyle, l.r. beuchat, t.j. montville (eds.) fermented vegetables, food microbiology: fundamentals and frontiers (second ed.), asm press, washington, dc (1997), pp. 595–609 [6] karovičová j., kohajdová z. lactic acid fermented vegetable juices, horticultural science, 30 (2003), pp. 152–158 [7] demir n., bachçeci k.s., acar j. the effects of different initial lactobacillus plantarum concentrations on some properties of fermented carrot juice, journal of food processing and preservation, 30 (2006), pp. 352–364 [8] di cagno raffaella, coda rossana, de angelis maria, gobbetti marco, 2013 exploitation of vegetables and fruits through lactic acid fermentation, food microbiology, volume 33, issue 1, february 2013, pp. 2–4, 15-20 [9] tamang j.p. plant-based fermented foods and beverages of asia, in: z. zalán, a. halász, á. baráth (eds.)y.h. hui (ed.), fermented red beet juice, handbook of plant-based fermented food and beverage technology (second ed.), crc press, boca raton, usa (2012), pp. 48–91 [10] hurtado a., reguant c., bordons a., rozes n. lactic acid bacteria from fermented table olives, food microbiology, 31 (2012), pp. 1–9 [11] banu c. (coord.), butu n., rasmerita d., sahleanu v. biotechnologies in food industry, ed. tehnica, bucuresti, 2000, pp. 110-126 [12] guţulescu i.the processing technology of vegetables and fruits, editura didactică şi pedagogică, bucureşti, 1973, pp. 45-86 [13] sr 9065-5-73 [14] indyk h. and konings e., eds. official methods of analysis of aoac international, 17th ed., aoac international, gaithersburg, md, 2000, pp. 45-60 [15] banu c., iordan m., nour v, musteaţă g., the processing of raw materials and the loss of biologically active substances. ed. „tehnica” utm. chişinău, 2003, pp. 58-59, 91-92 microsoft word 5 dubova sokac corrosion.doc 30 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 30 37 temporal changes of corrosion losses and corrosion rates in water pipes vanda dubová1, *marek sokáč 2 1department of sanitary and environmental engineering, faculty of civil engineering, slovak university of technology in bratislava, radlinského 11, 813 68 bratislava, slovak republic, vanda.dubova@stuba.sk 2 department of sanitary and environmental engineering, faculty of civil engineering, slovak university of technology in bratislava, radlinského 11, 813 68 bratislava, slovak republic, marek.sokac@stuba.sk * corresponding author received 7 january 2013, accepted 15 february 2013 abstract: water distribution systems are often constructed of metal tubes, which in addition to its good properties, especially mechanical, has also tendency to corrosion. increased activity of corrosion processes significantly affect not only the timelife of the pipe, its failure rate and reliability, but also changes hydraulic conditions of water supply system and may result in poor water quality. price increase of the water caused a reduction of water consumption and the extension of the travel time of the water in distribution system. this also influences the interaction of the transported water and pipe material. for the reasons, stated above, we observed the corrosive effect of the water from water source pernek on the pipe with the shortand longterm corrosion tests. tests are based on measurements of the weight loss difference of the test sample after 30 and 60 day exposure to flowing water. since short time (30-60 day) test does not always give an accurate picture of the interaction between water and pipe, we extend the test up to two years. during these long term test we have observed not only the corrosion losses and the corrosion speed, but also the formation of the corrosion layer and its thickness. corrosion losses between 30 and 60 day were almost in linear course, but during longer tests corrosion losses speed decreases and the average corrosion rate for 2year test reached only about 39% of the corrosion rate determined from short term tests. keywords: corrosion of water pipes, corrosion rate, corrosion losses, corrosion layer 1. introduction during the transport of drinking water to the consumer, an interaction between transported water and pipe material occurs, which often results in increased activity of the corrosion process. increased activity of corrosion processes significantly affect not only the lifetime of the pipeline, its failure rate and reliability, but also changes the hydraulic conditions of water supply system and may result in a poor water quality. in general it can be stated that the problems caused by corrosion cause problems for all operators of water supply systems, whether in the public drinking water supply or the industrial water supply and therefore the interaction of water and transported material pipe should be in spotlight of all water systems operators [15]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 31 2. matherials and methods determination of water aggressiveness – used materials and methods one of the most important chemical equilibrium in water chemistry and water technology and water is calciumcarbonate balance, which is important in assessing the aggressive and incrusting effects of water [6, 7, 8, and 9]. water aggressiveness can be determined from chemical analyses of water, direct test for solid caco3 (heyer’s test), by a variety of formulas and tables, or corrosion tests [6]. since the calculations of water aggressiveness, according to various authors [8,9] take into account only the water aggressiveness caused by co2, but do not consider the amount of dissolved oxygen in the water, which may be the dominant factor in the corrosion rate or the flow velocity of the water. because these factors substantially affect the corrosion processes, results of direct corrosion tests are the most objectives, but very troublesome and also highly timeconsuming [10]. the test methodology is presented in the slovak technical standard (stn) 75 7151 "quality requirements for water transported in pipes" [12] and is based on weight loss measurement of the test sample after 30 and 60 days after exposure of the sample to the flowing water [11]. the measured corrosion losses (g / sample) can be converted to the unit g/m2 using following equation: (1) where m0 is the weight of the sample before exposure in grams, m is the weight of the sample after exposure in grams, s is the surface area of the sample in square meters. following, using the steel density (7.86 g.cm-3), the corrosion losses can be converted from unit g.m-2 to the unit µm/(exposure time) (10-6 m / exposure time). this means that the conversion of corrosion losses from unit g/sample to µm/exposure time (10-6 m/exposure time) leads to idealization in which a corrosion loss is equal over the sample surface (uniform corrosion). actually, this is not true in generally, because in most cases non-uniform corrosion occurs and local losses are greater. according to slovak technical standard stn 75 7151 for each experiment / measurement shall be 5 test samples used, each with dimensions 42 x 42 x 1 mm, and the averaged value is used to calculate the corrosion rate. based on the measured corrosion losses the corrosion rate is calculated, which basically means the thickness reduction of the pipe wall, related to the original thickness. based on the corrosion rate (expressed in in micron for 1 year between the 30th and 60 day), the level of water aggression is determined. stn 75 7151 recognises the three levels of water aggressiveness and their assessment (table 1): table 1 water categorisation according the aggressiveness level aggressiveness level corrosion rate (µm. year-1) assessment anticorrosive measures: i. less than 50 moderate aggressiveness not required ii. from 50 up to 150 medium aggressiveness based on results of technologic and economic analysis iii. more than 150 heavy aggressiveness necessary food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 32 if we want to check also the corrosion type (in addition to the corrosion loses), ie whether the pipeline corrosion is uniform (areal flat) or non-uniform (local, indepth), respectively, what type of corrosion layer will be formed, the test period shall be extended up to one year. advantage of the test is that the water complex effect on the test specimen as the pipe. 3. results and discussion field experiments were carried out in cooperation with the bratislava water company, inc. (bvs, a.s.). corrosion tests were performed on the water source pernek, whose water shows relatively high aggressive effects. the water source pernek supplied water in the past to the záhorský water system. after interconnection of the záhorský water system with the bratislava water system, the záhorský water system is supplied with water of very high quality from the water resources in the bratislava region. from the pernek water source is currently only one well used (hl-1) to supply municipalities jablonové and pernek. [11]. corrosion tests on the wells were started in april 2010 and conducted two years long. during the experiments, groundwater has shown following ranges of parameters (parameters related to the calcium carbonate equilibrium of the water): saturation index: -0.52 till -0.74 free co2: 6.60 till 10.12 mg.l-1 balanced co2: 2.9 till 4.52 mg.l-1 aggressive co2: (aggressive to iron, fe):4.41 till 5.60 mg.l-1 aggressive co2 (aggressive to marble, caco3 ) 2.58 till 3.24 mg.l-1 groundwater ph value was in range from 7.30 till 7.42, groundwater temperature was 9.8 till 10.5 oc, acid neutralization capacity (anc4.5) 2.10 till 2.43 mmol.l-1, base neutralization capacity (bnc8.3) 0.15 till 0.23 mmol.l-1, fe concentration was below 0.03 mg.l-1, mn below 0.01 mg.l-1, ca2+ concentration was 69 till 74 mg.l-1, nh4+ below 0.02 mg.l-1, no318 till 22 mg.l-1, so4274 till 78 mg.l-1, a cl10 till 14 mg.l-1. in the well hl-1 we installed a test device with a 1.5 m long glass tube, into which we put up to 6 racks of test samples. 5 samples were retracted into each rack, and each sample was separated with a nonconductive cylinder. the samples were made of class 11 steel, 1 mm thick, with the size of 42 x 42 mm. water in the test device was pumped out from the well and after flowing through the racks (experiment device) water flow out through an outlet to a surface infiltration ditch. all corrosion tests were evaluated in accordance with stn 75 7151 for corrosion losses, corrosion rate, corrosion layer as well as the velocity of water flowing in the test facility. under the corrosion layer we understand a poorly adhered layer of corrosion products, which includes also substances from water in addition to corroded pipe material. the method of determining the amount of corrosion products permanently attached on test samples was performed according to stn 75 7151. after finishing the exposure of the samples, the rack with the five samples was taken out of the glass pipe. the test samples were carefully removed from the rack in order to avoid loss due to fractions peel-off or fall-off from the corrosion layer. the samples with corrosion layer are dried in an oven at the temperature of 105° c until the weight was constant. after weighting the samples (with corrosion layer) the corrosion layer has been mechanically removed from the samples and the test samples were cleaned same way as during the procedure for the corrosion losses determination (5-minute submerging in 20% hydrochloric acid solution with a concentration of hexamethylenetetramine 5 g.l-1, rinsing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 33 with distilled water and ethanol, subsequent drying at 105 ° c and weighing). the amount of corrosion products permanently attached on a test samples were calculated from the difference of the test sample weight (with corrosion layer and weight of the test sample after cleaning). following measurements have been completed and evaluated so far: two twoyear, three one-year, three six-month, one four-month, one three-month, eight 60-day and sixteen 30-day measurements, respectively. each 60-day test consists of two 30-day and one 60-day measurement. the 60-day tests two highest placed racks of samples were used, which were after removal being replaced by racks with new samples. for the sixmonth tests the third rack and for the annual test the fourth racks were used, with samples that have also been replaced after removal by new samples. the fifth and sixth racks were used for the twoyear tests. with the aim to check, if there isn’t a gross fault in the corrosion measurements (whether the minimum or maximum) and thus avoid a possible distortion of the results of the average corrosion loss of the tests, the measured values of corrosion losses (g per sample, respectively. g.m-2), were checked by the extreme deviations tests. we used the dixon and grubbs test. of the total of 170 tested values 8 values did not comply with the tests criteria and were excluded from further evaluation (whereas all values were close to the maximum). measurements results are shown in tab. 2 and on the fig. 1 and fig. 2. table 2 corrosion rates achieved during the test period corrosion speed vt (µm.year-1 = 10-6 m. year-1) days interval average % 30 60 90.94–219.14 134.86 100.0 % 30 180 48.31 – 87.92 73.00 54.1 % 30 365 59.85 – 67.27 63.10 46.8 % 30 730 50.29 – 53.84 52.23 38.7 % 365 – 730 41.52 – 43.54 42.25 31.3 % in table 2 the ranges of corrosion speed as well as the average corrosion speed obtained during the two-year corrosion tests are presented. the biggest and significant variance of corrosion rates was found during the short 30 to 60 day corrosion tests (fig. 1); whereas with increasing time of the test period variance of determined corrosion speed decreased significantly. this is due to the forming of the corrosion layer, which creates a barrier between the transported water and pipeline and thus reduce the corrosion rate. as can be seen on figure 1, water aggressiveness changes during the test period. a significant increase in the corrosion rate occurred mainly during the second 60-day test, which was probably due to the significant increase of the groundwater level due to long-term heavy rainfalls. in contrast, the corrosion rates measured in the same month, but with annual shift (experiment nr. 1 and 7) are very similar. the completed and evaluated 60-day measurements have shown that the tested water is classified in ii. and iii. aggressiveness level. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 34 0 25 50 75 100 125 150 175 200 225 1 2 3 4 5 6 7 8 c or ro si on sp ee d (µ m / ye ar ) test nr. figure 1. achieved corrosion rates during i. -vii. 60-day cycle increases of the corrosion losses and corrosion products are not linear, but are decreasing with increase of the exposure time. their trends are shown in fig. 2. however, the chart shows that the measured amounts of corrosion products and corrosion losses were during the 30 or 60day measurements not the same, but changed significantly during the year, and these changes were not influenced by the flow rate. e.g. for eight 60-day tests were standard corrosion losses determined in the range from 0.397 to 0.804 g / sample and corrosion products in the range from 0.479 to 1.095 g / sample. significantly smaller variance occurs during annual tests, the corrosion losses ranged from 1.755 to 1.974 g / sample and corrosion products from 2.815 to 2.978 g / sample. r² = 0.9296 r² = 0.9393 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 c or ro si on lo ss es a nd c or ro si on pr od uc ts (g / sa m pl e) corrosion losses (g /sample) corrosion products (g /sample) days figure 2. trend of corrosion losses and corrosion products the corrosion rates for long term annual test are significantly lower than corrosion rates of the short tests (30 60 days test), which are required by the standard stn 75 7151. short test results of the corrosion speeds are more than half lower then longterm tests and water can be classified just above the criteria for the 2nd aggressiveness level, respectively in the bottom range of the 2nd aggressiveness level. in the two-year test the results even decreased to the criteria between the 1st and 2nd aggressiveness level and represents only 39% of the results from short tests (30-60 day tests). there is approximately double increase of the values of corrosion food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 35 losses and corrosion products between 30 and 60-day tests. the decrease of the corrosion losses shows that during the long-term tests the corrosion layer takes some protective effect, although usually there is only a poorly adherence and the corrosion layer fall off very easily even with a careful handling with the samples. in terms of the corrosion rate development and corrosion losses the formation of corrosion layer and its protective effects is important. this layer changes the hydraulic roughness and cross-section of the pipe; therefore we focused our research also on the mapping of their development. the thickness of the corrosion layer was determined with use of the digital photo microscope (180 up to 300 times magnification). the corrosion layer is not uniformly distributed across the test samples surface, but mostly concentrated along the samples edges, on places, where the samples are attached to the frames. with increasing exposure duration during the corrosion tests increases the area of corrosion layer with bigger thickness. averaged surface "with" and "without" of samples with thick corrosion layer are shown in table 3. table 3 average area, covered with thick corrosion layer exposure time sample area without thick corrosion layer (%) sample area with thick corrosion layer (%) 30 days 68 32 60 days 51 49 6 months 35 65 1 year 22 78 2 years 17 83 assuming development according (fig. 3), the average corrosion loss, which represents uniform corrosion, reached after 10 years, the value of about 9.9 g/sample, respectively 357 µm/10 years. in fact, the local corrosion losses are several times larger. r² = 0.9296 r² = 0.9393 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 0 1 2 3 4 5 6 7 8 9 10 c or ro si on lo ss es a nd c or ro si on pr od uc ts (g / sa m pl e) corrosion losses (g /sample) corrosion products (g /sample) years figure 3. trend of corrosion losses and corrosion products and their extrapolation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 36 figure 4. development of corrosion layer over time (30, 60, 180, 365 days). plastic racks with samples are ordered by exposure time, whereas the lowest is the shortest time exposure (30 days). figure 5. detail of the corrosion layer after two years exposition. figure 6. photomicroscope’s detail of the corrosion layer after two years exposition. figure 7. comparison of the amount of peeledoff fragments from corrosion layer after 30, 60, 180 and 365 days of exposition of the test samples in water. 4. conclusion from the measured corrosion rate, it is clear that the aggressiveness of the tested water varied during the test period. the results of the two-year monitoring of the water aggressiveness of the water source pernek show a decrease in the corrosion rate during the long-term tests (30 to 730 day tests) compared to a standard tests (30 60 days). while in normative tests, corrosion rates classified the water to the 2nd up to the 3rd level of aggressiveness and therefore it is seriously suggested to consider and adopt some anti -corrosive measures, for longer-term corrosion tests the speed decreased significantly and also the water aggressiveness decreased to the 1st up to the 2nd level of water aggressiveness. the average corrosion rate measured during the annual test reached only 48.4% of the corrosion rate of 30-60 days test (and only 38.75% for the two years test) even on the basis of these tests, we can conclude, that the 30-60 day corrosion tests may overestimate the aggressiveness of the water. if the water shows in a standard test significant effects of aggressiveness, we recommend to perform long-term measurements to examine these aggressive effects to confirm it or to modify the original test results. with increasing exposure time during the corrosion tests increases also the sample surface, which is covered with thick corrosion layer. after oneyear tests the thick corrosion layer covered in average 78% of the samples surfaces and after two years tests up to 83% of the sample surfaces were covered with thick corrosion layer. these values are more than 2.5 times bigger than the 30-day test results and more than 1.5 times comparing with the 60-day tests. so far, the corrosion layer on samples after 60 days, 4 and 6 months exposition were documented with photo food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 vanda dubová, marek sokáč, temporal changes of corrosion losses and corrosion rates in water pipes, food and environment safety, volume xii, issue 1 – 2013, pag. 30 – 37 37 microscopic snapshots. in april will be the tests completed, that should demonstrate, how thickness of corrosion layer and its shape changes on samples from annual and biennial tests. although corrosion losses between the 30th and 60th day shows nearly a linear dependency and corrosion layer after two years exposure was only poorly adhered on the samples surface, during the longer test the growth of corrosion losses and hence the corrosion rate was slowing down. 5. acknowledgment experimental measurements were carried out with the financial support of the scientific grant agency within the project apvv-0379-07 in cooperation with the bratislava water company, inc., division of water production west in the town malacky, as well as with the support of the scientific grant agency of the ministry of education within the project vega 1/1243/12. 6. references [1]. bartoníček, r. et al.: koroze a protikorozní ochrana kovů. (corrosion and anticorrosion protection of metals, in czech language) academia praha, 1966. [2]. pierre r. roberge: handbook of corrosionengineering, copyright © 2000 by the mcgraw-hill companies, isbn 0-07076516-2. [3]. lin j, ellaway m, adrien r study of corrosion material accumulated on the inner wall of steel water pipe. in corrosion science, volume 43, issue 11, november 2001, pages 2065-2081. [4]. volk, ch. et al. (2000) : practical evaluation of iron corrosion control in a drinking water distribution system. in: water research, volume 34, issue 6, 1 april 2000, issn: 00431354,p. 1967-1974. [5]. ehlers laura j. et al. (2005): public water supply distribution systems: assessing and reducing risks, national academy of sciences. isbn: 0-309-54967-1, 58 pages. [6]. ilavský, j., barloková, d. galvanická úprava vod. (galvanic water treatment, in slovak language). in: sborník z xii. medzinárodnej vodohospodárskej konferencie voda zlín 2008, zlín, marec 2008, s. 131-136, isbn 978-80-254-1348-7. [7]. dubová, v.: životnosť potrubia. (pipe lifetime, in slovak) in zborník z odbornej konferencie s medzinárodnou účasťou nové trendy v zásobovaní vodou, odkanalizovaní a nakladaní s domovým odpadom, žiar, september 2006, s. 21-28. isbn 80-227-2469-6. [8]. pitter p. hydrochemie, fourth ed., institute of chemical technology press, praha, 2009 (in czech). [9]. ilavský j., barloková d., biskupič f. chémia vody a hydrobiológia. (water chemistry and hydrobiology, in slovak), bratislava 2008, isbn 978-80-227-2930-7 [10]. dubová, v., ilavský, j., barloková, d. spolupôsobenie prepravovanej vody a materiálu potrubia – vyhodnotenie koróznych skúšok. (interaction between transported water and pipe material evaluation of corrosion tests, in slovak). in: zborník z konferencie 10. zdravotno-technické stavbymalé vodné diela – krajina a voda. tatranská javorina, sr, 23.-25.11. 2011, elsewa, s.r.o., 201, s. 18-25, isbn 978-8089385-17-1. [11]. dubová, v., ilavský, j., barloková, d.: evaluation of annual corrosion test for aggressive water. in slovak journal of civil engineering, vol. xix, 2011, no 4, p. 24-27, issn 1210-3896. [12]. stn 75 7151: požiadavky na kvalitu vody dopravovanej potrubím. (quality requirements for water transported in pipes). 228 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 32014, pag.228 237 statistical studies on consumer’s perception of fruit *cristina-elena hrețcanu1 , alice – iuliana roșu1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania cristina.hretcanu @ fia.usv.ro, alicer @ fia.usv.ro *corresponding author received july 15th 2014, accepted september 12th 2014 abstract: the purpose of this paper is to demonstrate that an important criterion in determining consumers’ fruit choice is not only the sensory properties, but consumer’s previous information acquired on fruit which can be turned into beliefs and preferences. the starting point consists of the following aspects: to establish the frequency of consumption of fruit, to determine fruit varieties associated with the colour preferred by consumers; to identify the considerations according to which a person would choose a certain type of fruit and to analyse consumer’s feedback on product sensorial properties, to study consumer’s attitudes in the choice of a fruit and to understand the influence of colour on consumer’s choice of fruit. keywords: crossmodal correspondence, focus group, repertory grid method, sensory properties 1. introduction consumer’s response to a given fruit depends on the positive or negative response to its sensory properties, the knowledge and opinions about it and a behavioral component, involving intentions to consumption of some fruit. [1] cross-overs between the vision and the taste pathways occur to create color and taste associations [2]. thus, we can state that the choice of a fruit depends on crossmodal correspondences between several factors. “crossmodal correspondences have been defined as a tendency for a sensory feature, or attribute, in one modality, either physically present or merely imagined, to be matched (or associated) with a sensory feature in another sensory modality” [3] crossmodal associations among taste, flavor or odour and other sensory dimensions have been reported in many papers regarding food product acceptance ([4-11]), where this term is used to refer to compatibility effect between some attributes, such as colour and different other qualities of food product. as non-verbal elements, food colours can influence consumer’s sensory and hedonic expectations. also, if these expectations are high, consumers may be interested in consuming these food products [12-15]. we have tried to answer the question “why are colors so important for consumers?”. first of all, fruit colour is important because it shows the presence of beneficial substances for the human body. thus, fruit contains bioactive substances (such as phytochemicals) that have beneficial properties [16] and also produce bright colors in fruit [17]. for example, fruit of green colour (such as green kiwi fruit, green aples, avocados, limes) contain high antioxidant activity [18]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 229 the phytochemicals responsible for orange colour in fruit are carotenoids which are involved in immune responses of organisms [19]. flavonoids together with β-cryptoxanthin are responsible for the light orange to yellow colour of fruits (such as peach, papaya or orange) and they possess antiviral, anti-inflammatory, antihistamine and antioxidant properties [20]. red-purple colour of fruit (such as grapes, prunes, berries, raspberries and cranberries) is caused by anthocyanins which are very important in preventing carcinogenesis and mutagenesis [21]. also, red fruit (such as raspberries, cherries, strawberries) are rich in antioxidants such as lycopene and anthocyanins, which reduce the risk of several types of cancer [17]. secondly, colours have important physiological properties. thus, colours which are in the red area of the colour spectrum, such as orange and yellow, tend to make consumer think of warmth and energy. colours which are in the blue side of the colour spectrum such as blue, purple and green are often described as calm, cool but sometimes they induce feelings of sadness or indifference. [22] last, but not at least, colour interferes with judgments of flavour identification and influences the pleasantness and acceptability of foods [23] 2. matherials and methods the main objective of this survey is to identify the messages that fruit colour convey to consumers. the basic tool in this survey is the use of a questionnaire which reflects the objectives set out above. the questionnaire consists of simple questions, arranged in a logical order, which attempted to discover the main characteristics of local consumers, focusing on certain statements regarding respondents’ perception of fruit. participants were 81 (19 male, 62 female), in the age range 18–40 years (mean 22.37 years). participants were recruited from the students of the faculty of food engineering, stefan cel mare university of suceava, romania, in april 2014. focus group session (fgs) [24] and repertory grid method (rgm) [25-27] are used in this study in order to establish the features of fruit which are relevant to fruit consumers and to determine what fruits colour may affect fruits consumers’ perception. the fruit brought into discussion by the questionnaire are chosen among fruit frecvently purchased from the romanian supermarkets or fruit markets, as follows: apples, pears, bananas, oranges, grapes, cherries, strawberries, berries, plums and kiwi fruit. a focus group interview is a qualitative research tool designed to get the perceptions of consumers [28]. conducted by a researcher who introduces the topic, the focus group interview consists in discussions which start with general questions and continues with the more specific ones. the repertory grid method is used to study consumers’ beliefs concerning colour influence on the perception of different types of fruit. statistical analysis is conducted to process the interpretation of results obtained by questionnaire. the analysis is based on principal component analysis (pca) and hierarchical cluster analysis (hca) of a set of opinion questions. the data regarding the acceptance of the fruit under investigation are evaluated using variance analysis (anova), followed by the test of averages (tukey, p -values < 0.05). statistical analysis is performed using the program xlstat™ (trial version 2014.3.04, addinsoft©, u.s.a.) and data analisys in ms excel 2010. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 230 3. results and discussion in the survey, participants are firstly asked to report information on demographics and purchasing fruit habits. therefore, respondents are asked about their height (m) and weight (kg) to determine the body mass index (bmi). the results are given in figure 1. fig. 1. body mass index (bmi) for respondents using bmi categories, we have found out that 16.05% of respondents are underweight (i.e. bmi index is less than 18.5), 65.43% of respondents have normal weight (bmi is between 18.5 and 24.9), 16.05% of respondents are overweight (bmi is between 25 and 29.9) and 2.47% of respondents are in the first grade of obesity (bmi is between 30 and 34.99). the focus group interview aims at object questions regarding fruit consumption habits. the results of the questions: “are you a frequent fruit consumer? how often do you eat fruit?” are given in the fig. 2. one can easily observe that 60.5% of respondents consume fruit several times a week. fig. 2 the frequncy of fruit consumption the results of the question “where do you generally purchase fruit from?” are given in the fig. 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 231 fig. 3 locations where respondents generally purchase fruit from one can remark that about 49.38% prefer to purchase fruit from markets and supermarkets. the results of the question “what is the main criterion you choose a certain type of fruit?” are given in the fig. 4. from fig. 4(a) one can remark that the main reason for which consumers choose a certain type of fruit is its content rich in vitamins. in terms of why consumers would choose fresh fruit, one can notice that among the top preferences on the variety of fruit are apples, followed by grapes and berries. the values are very close in terms of the fruit variety preference, not only for the first three classes (apples, grapes and berries), but also for other fruit set in the questionnaire. from fig. 4(b) one can remark the biplot graphic based on principal component analysis (pca) for the most important factors (f1 and f2) show inter-unit distances among units as well as display variances and correlations of the variables aimed at [30]. (a) (b) fig. 4 (a). the main consumers’ reasons to choose a fruit (b). pca for these reasons the variation of the parameters studied implies the existence of two factors and the percentage of variability represented by the first two factors is high (77.86 %). the first factor (f1) explains 49.06 % of the total variance with significant parameters “rich in vitamins” and “rich in nutrients”. the second factor (f2) explains 28.8 % of the total variance with significant parameters “rich in minerals” and “rich in antioxidants”. the results of the question “how do you like to eat the following fruit?” are given in the fig. 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 232 (a) (b) fig. 5 (a). the form consumers prefer to eat fruit (b). pca for these consumers’ preferences from fig. 5(a) we remark that 53.5% of consumers surveyed prefer to eat fresh fruit. from fig. 5(b) one can remark the biplot graphic for the most important factors. the variation of the parameters studied implies the existence of 2 factors and the percentage of variability represented by the first two factors is high (76.58 %). the first factor (f1) explains 42.8 % of the total variance with significant parameter “fresh fruit”. the second factor (f2) explains 33.79 % of the total variance with significant parameters “passed fruits” and “compotes”. the results of the question “what colours do you associate with the fruit presented?” are given in the fig. 6. (a) (b) fig. 6 (a). the colour associated with fruit (b). pca for associatins of colours to fruits food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 233 from fig.6 (a) we remark that yellow is the most popular colour of fruit (21.30%), followed by red (18.3%) and green (18.08%). from fig. 6 (b) one can remark the biplot graphic based on principal component analysis (pca) for the most important factors (f1 and f2). the variation of the parameters studied implies the existence of 2 factors and the percentage of variability represented by the first two factors is medium (55.33 %). the first factor (f1) explains 34.51 % of the total variance with significant parameters “purple” and “blue”. the second factor (f2) explains 20.82 % of the total variance with significant parameters “orange” and “red”.the question “how much do you like this fruit?” was rated on a likert scale, marked with 5 descriptors, where the quantifications of descriptors are the following: “dislike much” was quantified with 1 point, “dislike moderately” with 2 points, “neither like nor dislike” with 3 points, “like moderately” with 4 points, and “like very much” with 5 points. the average of scores given by consumers regarding the palatability of every fruit is presented in the fig. 7. data sets are evaluated using standard descriptive statistics (average, standard deviation) and one-way analysis of variance (anova). we have observed that strawberries, cherries, grapes, oranges and berries are consumers’ favourite fruit. significant differences between individual samples are determined using anova (p-value < 0.05). fig. 7 mean values of consumers’ responses to quantification of fruit palatability means with common superscripts did not differ significantly (p-value< 0.05). as regards palability of fruit, we remark that three groups of fruit can be formed: the first one contains strawberry, cherries, grapes, oranges and berries which obtained scores between 4.48 poins (nearby “like very much” ) and 4.17 (nearby “like moderately”), the second group contains cherries, grapes, oranges,berries, bananas, apples, kiwi and pears which obtained scores between 4.23 (nearby “like moderately”) and 3.72 points, and the last one which contains apples, kiwi, pears and plums which obtained scores between 3.98 (nearby “like moderately”) and 3.49 points. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 234 one can remark from fig. 8 that the variation of the parameters studied implies the existence of 2 factors and the percentage of variability represented by the first two factors is very high (92.61 %). the first factor (f1) explains 88.87 % of the total variance and the second factor (f2) explains 3.74%. all the fruit under investigation can be seen as significant parameters of the first factor f1, while for the second factor f2, the significat factors are “apples” and “strawberries”. fig. 8 pca for consumers’ responses to fruit palatability in the following statement respondents are asked to complete a word association task as a useful qualitative methodology commonly used in psychology and sociology [30]. requirement results of the task “assign words refreshing, exotic, seasonal, grown in our area, fresh, healthy with the fruit: apples, pears, bananas, oranges, grapes, cherries, strawberries, berries, plums and kiwi fruit” are given in the fig. 9. most respondents replied that they consume fruit because they are healthy, fresh and grown in our area. from fig. 9(a) we remark the number of word associations with the fruit discussed above. all fruit types are considered as being healthy and fresh. apples, pears, grapes, cherries, strawberries, berries and plums are regarded as seasonal and grown in our area fruit, whereas bananas, oranges and kiwi are considered exotic fruit. in order to group the fruit sharing similar features of proposed words, a hierarchical cluster analysis (hca) is performed and the resulting dendrograms are shown in the fig. 9(b). three groups have been identified: the first group (exotic fruit), the second group (refreshing, fresh and healthy fruit) and the third group (seasonal and grown in our area fruit). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 235 (a) (b) fig. 9 (a) results of word association task (b). hierarchical cluster analysis (hca) for word association words: refreshing, exotic, seasonal, grown in our area, fresh, healthy fruit: apples, pears, bananas, oranges, grapes, cherries, strawberries, berries, plums and kiwi fruit the results of the question “what aspects do you like most of the following fruit?” are given in the fig. 10 where the numbers of responses regarding the aspects of fruit sensorial analysis (taste, odour, flavour, texture and colour) are represented. (a) (b) fig.10 (a) profile of consumers’ perception of sensory analysis (b). pca of consumers’ perception from fig.10 (a) we remark that taste is the most appreciated sensory characteristic of fruit for all the fruit taken into consideration, except for oranges which are appreciated first of all for their odour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 236 from fig. 10 (b) one can remark the variation of the parameters studied which implies the existence of 2 factors and the percentage of variability represented by the first two factors is medium (67.88%). the first factor (f1) explains 42.69 % of the total variance with significant parameters “texture”, „colour”, „freshness flavour”and “taste”. the second factor (f2) explains 25.19 % of the total variance with significant parameters “odour” and “taste”. 4. conclusion in conclusion we can remark the following aspects of fruit consumption: most respondents consume fruit several times a week; about 50% of them prefer to purchase fruit from markets and supermarkets; the main reason for which consumers choose a certain type of fruit is its content rich in vitamins; more than 50% of the consumers surveyed prefer to eat fresh fruit; yellow, red and green are the most popular fruit colours; strawberries, cherries, grapes, oranges and berries are the most appreciated fruit for their taste; respondents consume especially healthy, fresh and grown in our area fruit and taste is the most appreciated fruit sensory characteristic for all the fruit brought into discussion, except for oranges which are appreciated mainly for their odour. 5. references [1]. costell e., tárrega a., bayarri s., food acceptance: the role of consumer perception and attitudes, chem. percept., 3: 42–50, (2010) [2]. spence c., levitan carmel a., shankar m.u., zampini m., does food color influence taste and flavor perception in humans?, chemosensory perceptions, 3: 69-84, (2010) [3]. spence c., managing sensory expectations concerning products and brands: capitalizing on the potential of sound and shape symbolism. journal of consumer psychology, 22: 37–54, (2012) [4]. crisinel a.s., spence c., implicit association between basic tastes and pitch, neuroscience letters, 464: 39–42, (2009). [5]. crisinel, a.s., spence c., a sweet sound? food names reveal implicit associations between taste and pitch. perception, 39: 417–425, (2010) [6]. crisinel a.s., spence c., as bitter as a trombone: synaesthetic correspondences in nonsynaesthetes between tastes and flavours and musical instruments and notes, attention, perception, & psychophysics, 72: 1994–2002, (2010) [7]. demattè m. l., sanabria d., spence c., cross-modal associations between odors and colors. chemical senses, 31: 531–538, (2006) [8]. parise c. v., spence c., audiovisual crossmodal correspondences and sound symbolism: a study using the implicit association test, experimental brain research, 220 (3-4): 319-333, (2012) [9]. spence c., crossmodal correspondences: a tutorial review, attention, perception, & psychophysics, 73: 971–995, (2011) [10]. spence c., gallace, a., tasting shapes and words, food quality and preference, 22: 290– 295, (2011). [11]. oram n., laing d.g., hutchinson i., et al., the influence of flavor and color on drink identification by children and adults, developmental psychobiology, 28.4: 239-246, (1995) [12]. dubose c., cardello a., maller o., effects of colorants and flavorants on identification, perceived flavor intensity, and hedonic quality of fruit-flavored beverages and cake, j food sci 45:1393–1399, (1980) [13]. garber l., hyatt e., starr r., placing food color experimentation into a valid consumer context. j food prod mark, 7:3–24, (2001) [14]. yeomans m., chambers l., blumenthal h., blake a., the role of expectancy in sensory and hedonic evaluation: the case of smoked salmon ice-cream, food quality and preference, 19:565–573, (2008) [15]. zampini m., sanabria d., phillips n., spence c., the multisensory perception of flavor: assessing the influence of color cues on flavor discrimination responses, food quality and preference, 18:975–984, (2007) [16]. the european food information council (eufic) http://www.eufic.org/ article/en/show/spotlight/rid/colours-of-fruits-andvegetables-and-health/ , accesed in april 2014 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 cristina-elena hrețcanu, alice – iuliana roșu, statistical studies on consumer’s perception of fruit, food and environment safety, volume xiii, issue 3 – 2014, pag. 228 – 237 237 [17]. dolson l., eat your colors! (all about phytonutrients), (2004), http://members.cruzio.com/~dolson/healthtips/color s.html, accesed in april 2014 [18]. halimoon n., abdul hasan m., determination and evaluation of antioxidative activity in red dragon fruit (hylocereus undatus) and green kiwi fruit (actinidia deliciosa), american journal of applied science, 7 (11): 1432-1438, (2010) [19]. hounsome n., hounsome b., tomos d., edwards-jones g., plant metabolites and nutritional quality of vegetables, journal food of science, 73(4): 48-62, (2008) [20]. heber d., bowerman s., applying science to changing dietary patterns. american institute for cancer research 11th annual research conference on diet, nutrition and cancer, (2001) [21]. lazzè m. c., savio m., pizzala r., cazzalini o., perucca p., scovassi a.i., stivala l. a., bianchi l., anthocyanins induce cell cycle perturbations and apoptosis in different human cell lines, carcinogenesis, 25 (8): 1427—1433, (2004) [22]. kendra c., how colors impact moods, feelings, and behaviors, color psychology, http://psychology.about.com/od/sensationandpercep tion/a/colorpsych.htm , accesed in april 2014 [23]. clydesdale f.m., color as a factor in food choice, critical reviews in food science and nutrition, 33: 83–101, (1993) [24]. brug j., debie s., assema p., weijts w., psychological determinants of fruit and vegetable consumption among adults: results of focus group interviews, food quality and preference, 6:99-107, (1995) [25]. deliza r, macfie h., hedderley d., an investigation using the repertory grid and focus group methods of the package features affecting consumer perception of fruit juice, braz. j. food technol., 2(1,2):63-71, (1999) [26]. thomson d.m., mcewan j.a., an application of the repertory grid method to investigate consumer perceptions of foods, appetite, 10(3):181-93, (1988) [27]. thomson d.h.m., mcewan j.a., an application of repertory grid method to investigate consumer perceptions of foods, appetite, 10(3): 181-193, (1988) [28]. casey m.a., krueger, r.a., focus group interviewing, macfie, h.j.h., thomson, d.m.h (ed). measurement of food preferences. essex, england: elsevier science publishers ltd, p.77-96, (1994). [29]. gabriel k.r, the biplot graphic display of matrices with application to principal component analysis, biometrika , 58 (3): 453-467, (1971) [30]. ares g., gimenez a., deliza r., influence of three non-sensory factors of consumer choice of functional yogurts over regular ones, food quality and preference, 21: 361-367, (2009) [31]. ares g., gimenez a., gámbaro a., does information about the source of functional ingredients influence consumer perception of functional milk desserts?, journal of the science of food and agriculture, 88(12): 2061–2068, (2008). 135 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 135 142 changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans *rodica ciobanu1 1alexandru ioan cuza university,faculty of biology, iaşi, romania ciobanu.rodica1981@yahoo.com *corresponding author received june 9th 2013, accepted june28th 2013 abstract: the influence of taphrina deformans (berk.) tul., associated with peach leaf curl disease,on glucose dehydrogenase (ec 1.1.99.10), isocitrate dehydrogenase (ec 1.1.1.42), αketoglutarate dehydrogenase (ec 1.2.4.2) and malate dehydrogenase (ec 1.1.1.37) activitities in the leaves harvested from peachcultivar springcrest, was investigated. samples of both healthy and diseased leaves were analyzed. the resultus of this study suggests that the leaves infection with the biotrophic fungus taphrina deformans lead to the decreasing of glucose dehydrogenase and malate dehydrogenase activities and to a significantlly increasing of isocitrate dehydrogenase and αketoglutarate dehydrogenase activities as an attempt of host plant tissues to limit the damages caused by the fungus attack. data obtained in this study revealed significant differences in these enzymes activities depending on the type of theenzyme, the age of the leaves and the presence or absence of fungus attack. keywords:peach leaf curl, glucose dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase 1. introduction persica vulgaris mill. is one of the major fruit crop in romania, in present it is occuping the third place after apples and plums [1]. peach leaf curl disease caused by taphrina deformans (berk.) tul.is predominant in all the peach growing areas of the world [2] and is one of the most dangerous disease for peach because it can cause the defoliation and major crop loss at nearly all cultivars of peach trees.the infection is favoured by low temperature and high humidity from the time of bud swellen; the infection occurs mainly during a short period after the buds open when the new tissues are susceptible and as all organs grow older they become resistant to infection [3]. dehydrogenases are oxidizing enzymes which catalyze the electron transfer from the donor to an acceptor other than molecular oxigen. glucose dehydrogenase (ghd, d-glucose: acceptor 1-oxidoreductase, ec 1.1.99.10) is a fad-dependent enzyme. glucose dehydrogenase is anoxidoreductase that catalyze the first hydroxyl group of glucose and other sugar molecules, utilizing fad as primaryelectronacceptor. fad gdhs utilize a variety of external electron acceptors, but not oxygen; glucose dehydrogenase has been found as extracellular enzyme in fungi, such as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 136 aspergillus sp., and it has ahigh specificityfor glucose [4]. intracellular fad-dependent gdh is involved inmetabolic pathways, such as the glycan metabolism and the biosynthesis of secondary metabolites; they are suggested to play a role in the pentose phosphate pathway involving glucose turnover for the production of nadh as reducing equivalents and pentoses as integral parts of nucleotides. the biological function of extracellular fad-dependent glucose dehydrogenase is still unclear, but a role during fungal attack on the host-plant is proposed. by reducing quinones and phenoxy radicals glucose dehydrogenase is able to neutralize the action of plant laccases, phenoloxidases or peroxidases, which are used by infected plant tissues to limit the fungal attack [5]. isocitrate dehydrogenase (idh, ec 1.1.1.42) is a nadp-dependent enzyme, that controls the carbon flux between the krebs cycle and the glyoxylate bypass via its activation and inactivation by the bifunctional idh kinase/phosphatase. thus, the activation of isocitrate dehydrogenase forces the flow through the krebs cycle, causing a decrease in the intracellular isocitrate level and an increase in the α-ketoglutarate level [6]. α-ketoglutarate dehydrogenase (α-kgdh, ec 1.2.4.2), a key regulatory point of tricarboxylic acid cycle, plays vital roles in the multiple pathways of energy metabolism and biosynthesis [7]. αketoglutarate dehydrogenase is an enzyme which catalyses the non-equilibrium reaction converting α-ketoglutarate, coenzyme a and nad+ to succinyl-coa, nadh and co2, requiring thiamine pyrophosphate as a cofactor [8]. it transfers four-carbon aldehyde group from α-ketoglutarate to thiamine phyrophosphate to form hydroxyethylthiamine phyrophosphate. malate dehydrogenase (mdh, l-malate: nad+ oxidoreductase, ec 1.1.1.37) catalyzes the conversion of oxaloacetate and malate utilizing the nad or nadp coenzyme system. malate dehydrogenase is found in cytosol where it participates in malate/aspartate shuttle and in the mitochondrial matrix where it has a key role in the citric acid cycle and are naddependent enzymes; the malate dehydrogenase found in plant chloroplasts has nadp as coenzyme [9]. 2. materials and methods vegetal material used in this study was represented by fresh healthy leaves and leaves naturally infected with the fungus taphrina deformans, harvested, starting to middle of april until late june in year 2008, from peach cv. springcrest, from the experimental orchard “vasile adamachi” iaşi. the determinations of the dehydrogenases activity were made at: 19 april (i), 7 (ii), 19 (iii) and 27 (iv) may, 3 (v), 10 (vi) and 22 (vii) june. springcrest cv. is considered to be very susceptilbe to this pathogen attack [10,11]. the leaves were harvested early in the morning and enzymes activity was estimated in the same day. the dehydrogenases activity was determinated by sîsoev and krasna spectrophotometric method, modified by artenie. this method has at basis the ability of dehydrogenases to transfer hydrogen from various substrate (glucose, isocitric acid, αketoglutaric acid and malic acid) to 2,3,5triphenil-tetrazolium-chloride (ttc) which is reduced to triphenyl formazan colored in red. samples collected were first washed with distilled water, then the enzymes were extracted using 3 ml of phosphate buffer ph-7,4. the assay mixture of dehydrogenases contained: 0,25 ml of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 137 crude enzyme extract, 0,2 ml of specific substrate 0,2 m, ph-7,4, 0,75 ml distillated water and 0,2 ml of standard solution of 2,3,5-triphenil-tetrazolium-chloride 1%. in the control tests the specific substrate was replaced with the same quantity of phosphate buffer. the tests were incubated 18 hours in a thermostat at 28˚c, then separated by centrifugation at 4000 rotations per minute; supernatants were discarded and in the tests was added 5 ml of dissolvent for the extraction of triphenyl formazan; samples were centrifugated again andthe absorbance was readed spectrophotometrically at 540 nm; the color intensity is proportional with dehydrogenases activity. dehydrogenase activities were expressed as μg formazan per gram fresh vegetal material[12]. 3. results and discussion the dynamics of glucose dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities have been studied in healthy and curled leaves of peach cv. springcrest and are presented in figs. 1-4. the activity of glucose dehydrogenase at cv. springcrest is presented in fig. 1, from which it can be seen that in healthy leaves this enzyme had the highest value – 0,0640 μg formazan/g mat. in the last stage (vii) and it was followed in decreasing order by the values: 0,0356 μg formazan/g mat. (ii), 0,0251 μg formazan/g mat. (iv), 0,0232 μg formazan/g mat. (vi), 0,0212 μg formazan/g mat. (iii), 0,0182 μg formazan/g mat. (v), 0,0156 μg formazan/g mat. (i). in the leaves infected by taphrina deformans, glucose dehydrogenase activity recorded the smallest value in the third stage of infection 0,0146 μg formazan/g mat., followed in increasing order by the values: 0,0156 μg formazan/g mat. (iv), 0,0166 μg formazan/g mat. (v), 0,0168 μg formazan/g mat. (i), 0,0193 μg formazan/g mat. (ii), 0,0217 μg formazan/g mat. (vi), 0,0830 μg formazan/g mat. (vii). the activity of glucose dehydrogenase in attacked leaves, recorded values higher than the control (enzyme activity in healthy leaves), in the first stage (d/h=1,0769) and in the last stage (d/h=1,2968); in stage ii (d/h=0,5421), stage iii (d/h=0,6886), stage iv (d/h=0,6215), stage v (d/h=0,9120) and stage vi (d/h=0,9353) glucose dehydrogenase activity in diseased leaves was smaller than the enzyme activity recorded in healthy ones. glucose dehydrogenase has an important role during fungal attack on the host plant, it can reduce quinones and phenoxy radicals and is able to neutralize the action of host plant peroxidases and polyphenoloxidase, which are used by plants to block the fungal attack [13]. glucose dehydrogenase activity recordet at cv. springcrestwas higher in diseased leaves at the begining and at the end of fungal attack; at the other dates of the determinations glucose dehydrogenase activity was higher in healthy leaves when compared with the activity from attacked peach leaves; the results obtained in this study indicated that taphrina deformans was not able to produce high amounts of glucose dehydrogenase and to stop the action of the enzymes responsible for plant protection against the oxidative stress caused by fungus attack; these results are in opposition with those mentionated in literature, which say that in injured plants the enzymes from pentose phosphate pathway are increasing their activity [14]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 138 figure 1 the influence of taphrina deformans berk. (tul.) attack on the dynamics of glucose dehydrogenase activity in fig. 2 are presented the results concerning the isocitrate dehydrogenase activity in healthy and in infected leaves by taphrina deformans in cv.springcrest. in healthy leaves, isocitrate dehydrogenase activity recorded the next values, presented in decreasing order: 0,0649 μg formazan/g mat. (i), 0,0514 μg formazan/g mat. (vii), 0,0368 μg formazan/g mat. (v), 0,0317 μg formazan/g mat. (vi), 0,0268 μg formazan/g mat. (iv), 0,0238 μg formazan/g mat. (iii) and 0,0157 μg formazan/g mat. (ii). isocitrate dehydrogenase activity in healthy peach leaves, had the highest value at the begining of the fungus attack and the smallest value of it’s activity was recorded in stage ii. in diseased leaves the activity of isocitrate dehydrogenase had the highest value– 0,0546 μg formazan/g mat. in the last stage (vii) and was followed in decreasing order by the values: 0,0512 μg formazan/g mat. (iv), 0,0455 μg formazan/g mat. (v), 0,0447 μg formazan/g mat. (vi), 0,0239 μg formazan/g mat. (i), 0,0159 μg formazan/g mat. (ii), 0,0138 μg formazan/g mat. (iii). the activity of isocitrate dehydrogenase in the leaves infected by the fungus taphrina deformans had smaller values in compare with those recorded in healthy ones at stages i (d/h=0,3682) and iii (d/h=0,5798); at stages ii (d/h=1,0127), iv (d/h=1,9104), v (d/h=1,2364), vi (d/h=1,4100) and vii (d/h=1,0622) the isocitrate dehydrogenase activity from diseased leaves was higher than the activity recorded in healthy leaves. isocitrate dehydrogenase activity is increasing in the leaves naturaly infected by taphrina deformans as the disease simptoms develops, this enzymes is provideing the substratum necessary for αketoglutatate dehydrogenase activity, which recorded the same dymanic in it’s activity. isocitrate dehydrogenase is the enzyme that reflects the increased respiratory rate from diseased peach leaves. the enhanced isocitrate dehydrogenase activity in the leaves infected by taphrina deformans correlatd with a low photsynthesis activity [15] could be due to the synthesis of oxaloacetic acid by phosphoenolpyruvate carboxylase in the cytosol with subsequent transport into mitochondria where it serves as substratum for isocitrate dehydrogenaseactivity [16]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 139 figure 2 the influence of taphrina deformansberk. (tul.) attack on the dynamics of isocitrate dehydrogenase activity in fig. 3 are presented the results concerning the activity of α-ketoglutatate dehydrogenase from which it can bee seen that the highest value of this enzyme activity, in non-infected leaves, was registered in the last stage (vii) 0,0813 μg formazan/g mat. and it was followed in decreasing order by next values: 0,0376 μg formazan/g mat. (iv), 0,0359 μg formazan/g mat. (vi), 0,0188 μg formazan/g mat. (iii), 0,0183 μg formazan/g mat. (ii), 0,0162 μg formazan/g mat. (v), 0,0148 μg formazan/g mat. (i). in diseased leaves were recorded the following values of α-ketoglutatate dehydrogenase activity: 0,1055 μg formazan/g mat. (vii), 0,0698 μg formazan/g mat. (iv), 0,0587 μg formazan/g mat. (vi), 0,0434 μg formazan/g mat. (i), 0,0377 μg formazan/g mat. (v), 0,0185 μg formazan/g mat. (ii), 0,0153 μg formazan/g mat. (iii). the activity of α-ketoglutatate dehydrogenase had higher values in diseased leaves when compared with the enzyme activity from the healthy ones in the stages: i (d/h=2,9324), ii (d/h=1,0109), iv (d/h=1,8563), v (d/h=2,3271), vi (d/h=1,6350), vii (d/h=1,2976); in stage iii (d/h=0,8138) this dehydrogenase activity recorded a decreasing in its activity, which was higher in healthy leaves. this dehydrogenase activity is increasing in the same time with the disease development. α-ketoglutatate dehydrogenase it is found in it’s soluble form in the mithocondrial matrix and it is considered one of the main center able to generate reactive oxygen species [17] in response to biotic stress caused by the pathogen attack. the increased α-ketoglutatate dehydrogenase activity from infected leaves suggest an increase in respiratory rate, dependent of the age of the leaves and fungus, and an intense activity of the enzymes from the antioxidant defense line, knowing that a high metabolic rate is followed by the increase of oxidative stress markers that are responsible for the aging of mitochondria, which are the main source of reactive oxygen species [18] due to multiple reactions that transfer electrons. the decreased α-ketoglutatate dehydrogenase activity recorded at iii in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 140 diseased leaves could be due to the influence of reactive oxygen species, this enzyme in known to be one of the major target enzyme of this radicals, when inhibition of this enzyme take place and this limits the nadh availability and, as a result, the respiratory function of mitochondria [8, 19, 20, 21]. 3 the influence of taphrina deformans berk. (tul.) attack on the dynamics of α-ketoglutatate dehydrogenase activity the last stage in the krebs cycle, in which l malate is oxidize to oxaloacetate is catalysed by the malate dehydrogenase. the activity of malate dehydrogenase (fig. 4), in healthy leaves had the smallest value – 0,0214 μg formazan/g mat. in the first stage of the determinations and it was followeed in increasing order by the next values: 0,0246 μg formazan/g mat. (iii), 0,0293 μg formazan/g mat. (vi), 0,0333 μg formazan/g mat. (v), 0,0352 μg formazan/g mat. (iv), 0,0428 μg formazan/g mat. (ii) and 0,1069 μg formazan/g mat. (vii). in the leaves attacked by the fungus taphrina deformans, malate dehydrogenase activity had the highest value – 0,0967 μg formazan/g mat. in the final stage of the attack (vii) and it was followeed in decreasing order by the values: 0,0512 μg formazan/g mat. (iv), 0,0492 μg formazan/g mat. (vi), 0,0221 μg formazan/g mat. (ii), 0,0216 μg formazan/g mat. (v), 0,0127 μg formazan/g mat. (i), 0,0045 μg formazan/g mat. (iii). malate dehydrogenase activityat peach cv. springcrest recorded smaller values in diseased leaves in compare with the control in stages: i (d/h=0,5934), ii (d/h=0,5163), iii (d/h=0,1829), v (d/h=0,6486), vii (d/h=0,9045); in stages iv (d/h=1,4545) and vi (d/h=1,6791), this enzyme activity recorded higher values in leaves infected by the pathogenic fungus than the activity recorded in healthy leaves at the same dates. malate dehydrogenase activity recorded, in general, smaller values in curled leaves comparative with the values recorded in control, these results are similar with those presented in literature at nicotiana tabacum plants infected by viruses, where it was observed a decrease of malate dehydrogenase activity in diseased plants food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 141 [22]. the decreased malate dehydrogenase activity from diseased leaves, can be correlated with the big amount of oxalate which is known to inhibit this enzyme activity [23]. the results obtained in this study show that the infection of peach leaves with taphrina deformans, is followed by an enhancement of the activity of the enzymes of mitochondrial resiration except malate dehydrogenase activity. figure 4 the influence of taphrina deformans berk. (tul.) attack on the dynamics of malate dehydrogenase activity 4. conclusions glucose dehidrogenase activity was smaller in diseased leaves, these results suggest that the host plant tissues were able to mobilize theirs defensive mechanisms against taphrina deformans and to limit its attack. isocitrate dehydrogenase and αketoglutatate dehydrogenase activities recorded the same dynamics and were,in general, higher in the leaves infected by taphrina deformans, than the activity recorded in healthy leaves. malate dehydrogenase activity recorded specific variations from one date to another, but the enzyme activity was in general, smaller in diseased leaves. the results obtained in this study suggest that these dehydrogenases play important roles in defence machanisms against peach leaf curl infection; these reflects the ability of host plant to moblize its defensive enzymes and to limit the fungus attack and the damages produced by infection. 5. references [1]. chira l., chereji v., roman m., apricot and peach,m.a.s.t., bucureşti,(2008) [2]. naqvi s.a.m.h., diseases of fruits and vegetables (diagnosis and management), volume ii, p. 488-490, kluwer academic publishersdordrecht, (2004) [3]. agrios g.n., plant pathology, elsevier academic press, p. 445-447, (2005) [4]. ferri s., kojima k., sode k., review of glucose oxidases and glucose dehydrogenases: a bird’s eye view of glucose sensing enzymes, journal of diabetes science and technology, vol. 5 (5): 10681076, (2011) [5]. sygmund c., staudig petra, klausberg miriam, pinotsis n., djinovic-carugo kristina, gorton lo et al., heterologous overexpression of glomerella cingulata fad-dependent glucosefood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rodica ciobanu, changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans, food and environment safety, volume xii, issue 2 – 2013, pag. 135 142 142 dehydrogenase in escherichia coli and pichia pastoris, microbial cell factories, 10:106, (2011) [6]. roy o. sylvie, packard t.t., nadpisocitrate dehydrogenase from pseudomonas nautica: kinetic constant determination and carbon limitation effects on the pool of intracellular substrates, applied and environmental microbiology, 64(12): 4958–4964, (1998) [7]. qi feng, pradhan r.k., dash r.k., beard d.a.,modeling the kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase , the faseb journal, 25:732.9, (2011) [8]. tretter l., adam-vizi vera,alphaketoglutarate dehydrogenase: a target and generator of oxidative stress, philos trans r soc lond b biol sci.360(1464): 2335–2345, (2005) [9]. minárik p., tomáková n., kollárová m., antalík m., malate dehydrogenases structure and function (minireview), gen. physiol. biophys., 21: 257-265, (2002) [10]. morarita sinziana venera, frasin neagu loredana beatrice, behaviour of some varieties of peaches and nectarines when attacked by main pathogens, the annals of “valahia” university of targoviste,agriculture, 7 (vii): 48-52, (2012) [11]. kaymak s., boyraz n., baştaş k. k., susceptibility of some peach and nectarine varieties to leaf curl disease (taphrinadeformans(berk.) tul.) in field conditions, j.turk. phytopath., 37 (1-3): 27-37, (2008) [12]. cojocaru d. c., practical enzymology, tehnopress, iaşi, (2005) [13]. sygmund c., klausberger m., felice a., ludwig r.,reduction of quinones and phenoxy radicals by extracellular glucose dehydrogenase from glomerella cingulatasuggests a role in plant pathogenicity,microbiology, 157(11): 3203-3212, (2011) [14]. danson j., wasano k., nose a., infection of rice plants with the sheath blight fungus causes an activation of pentose phosphate and glycolytic pathways, european journal of plant pathology, 106 (6): 555-56, (2000) [15]. nicolae mariana, mitrea rodi, physiological modifications in prunus persica as a result of the attack produced by taphrina deformans, seria: biologie, horticultură, tehnologia prelucrării produselor agricole, ingineria mediului vol. xiv (xlx): 517-522, universitatea din craiova, (2009) [16]. mackenzie sally, mcintos i., higher plant mitochondria, the plant cell, 11: 571–585, (1999) [17]. starkov a.a., fiskum g., chinopoulos c., lorenzo b.j., browne s.e., patel m.s., beal m.f.,mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species,j. neurosci., 24: 7779–7788, (2004) [18]. tahara e. b., barros m. h., oliveira a. graciele, netto l. e. s., kowaltowski j. alicia, dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in saccharomyces cerevisiae aging, the faseb journal, 21(1):274-283, (2007) [19]. cabiscol e., piulats e., echave p., herrero e.r.j,,oxidative stress promotes specific protein damage in saccharomyces cerevisiae, j biol chem., 275(35): 27393-27398, (2000) [20]. tretter l., adam-vizi vera, generation of reactive oxygen species in the reaction catalyzed by α-ketoglutarate dehydrogenase, j. neurosci., 24(36): 7771-7778, (2004) [21]. chinopoulos ch., gerencser a.a., mandi m., mathe katalin, torocsik beata, et al., forward operation of adenine nucleotide translocaseduring f0f1-atpase reversal: critical role of matrix substrate-level phosphorylation, the faseb journal,24(7): 24052416, (2010) [22]. nayudu m.v., plant viruses, tata mc grawl –hillpublising company, (2008) [23]. fahien l.a., kmiotek e.h., macdonald m.j., fibich b., milka m., regulation of malate dehydrogenase activity by glutamate, citrate,α-ketoglutarate, and multienzyme interaction, j. biol. chem., 263: 10687-10697, (1988) 45 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 45– 52 contributions to achievement of a composite material for advanced electromagnetic shielding of living and work spaces second part quantitative aspects cristian florin alexuc1, *gheorghe gutt1, sonia amariei1 1faculty of food engineering, stefan cel mare university of suceava, romania g.gutt@usv.ro *corresponding author received 7th march 2015, accepted 27th march 2015 abstract: quantitative aspects about the composition of copper baths of steel wire nets and technological parameters used are presented in the second part of the paper. the mechanism of copper plating process is described and also the theoretical material balance for the three copper deposition technologies respectively galvanic deposition, electrochemical deposition without external power and deposition by chemical reduction, all described in the first part of the paper. the issue of automation copper plating bath for the latter technology is presented. keywords: copper plating recipe, technological parameters, reaction mechanism, material balance, productivity 1. introduction in the first part of the paper [1] the authors presented the need for electromagnetic shielding of living and workspaces and the issue of this important environmental need. in the purpose of advanced shielding to electromagnetic radiations of low and high frequency the authors propose a new composite material that takes the form of some fine nets made of thin steel wires. over these nets are submitting a high purity copper shirt wrapped in turn by a polymer shirt made in order to protect the copper coating against the oxidation. the nets, supplied in rolls, can be integrated under the plaster of working and living spaces performing their advanced electromagnetic shielding throughout the duration of the building which the space belongs [3], [4]. the second part of the paper is dedicated to the description of copper baths composition, technological work parameters, mechanism of galvanic copper deposition and material balances and carry out a benchmark considering quantitative and qualitative aspects of the three technologies proposed for copper plating. 2. quantitative aspects of steel wire nets coppering for shielding of living and workspaces against electromagnetic radiations of low and high frequency. 2.1. steel wire nets coppering by galvanic deposition of copper http://www.fia.usv.ro/fiajournal mailto:g.gutt@usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 46 the authors recommend two types of galvanic copper plating baths, one with alkaline electrolyte based on copper cyanide( recipe 1 with the eletrolyte 6a, fig.1), and the other with acid electrolyte based on copper sulphate(recipe 2 electrolyte 6b, fig.1) [1], [2]. the two galvanic baths composition and operating mode are as follows: 1. recipe 1alkaline electrolyte 6a based on copper cyanide with the following composition for 1 liter of electrolyte:150 g cucn , 100 g nacn, 20 g naoh. current density 1 a/dm2 (the surface refer to the steel wire from which it is made the net and it does not refere to the steel net) working temperature 60oc feed of advancement of steel wire net of 6m / minute immersion length of the net in galvanic bath of 2 linear meters electrolyte recirculation flow rate 2 liters / second galvanic bath capacity 500 liters thickness of copper deposited about 10 µm reactions that occur during galvanic deposition of copper from cyanide bath on steel wire net are: bathgalvanicondissociatiohhoh bathgalvanicondissociatiohnanaoh bathgalvanicondissociaticnnanacn bathgalvanicondissociaticncucucn 2 aq aq aq         emissionanode2h2h2e2h depositioncathodecu2e2cu ndissolutioanode2cu2ecu bathgalvanicondissociati2cu(oh)oh 2cu     to perform the theoretical material balance, expressing the theoretical consumption coefficients, the faraday's law governing galvanic processes is used: ηtikm  (1) where: k electrochemical constant k =a/zf a atomic mass z valence f faraday number, (96.500 c) m mass of metal deposited on the cathode or dissolved from the anode [g] i galvanic current intensity [a] t time passing of electrical current through the cell [s] η current efficiency taking into consideration theoretical material balance as 100% in the case of coppering from cyanide baths of thin steel wire nets, the quantity of copper theoretical deposited is: (2)0,013320i 96.500 64 20i 96.500 a m 1 i 11 cu cu1  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 47 where: mcu1 mass of copper deposited on the cathode, respectively dissolved at the anode in conditions of 100% current yield η of anode and cathode 64 atomic mass of copper 96.500 faraday number 20 time, in seconds, as electric current passes through a segment of thin steel wire net from its entry to the exit of galvanic bath (feed rate of the mesh in the galvanic bath of 6m / minute, immersion length in galvanic bath 2 meters), [s], i1 calculated current intensity of electrolysis at a current density of 1a / dm2 considered on the surface of the steel wire, diameter of 0,3 mm, which form meshes of metal net, with an immersion length of 2 meters, at a given moment in the galvanic bath. fig. 1. constructive details of electroplating bath from the technological process of copering of wire nets on galvanic way, work flow represented in the papers [1], [2]. 2-primary steel wire net, 5athermostated galvanic bath used for copper galvanic deposition[1], electrolyte 6a, 7-dc power supply, 8-soluble anode from electrolytic copper, 9-metal drum to power-up, 10-thermostat, 12intermediate metallic net having steel wire encased in a copper shirt, tg-drums of guidingstretching role, tt-drums of tensilereturn role 2. recipe 2with acid electrolyte 6b, based on copper sulphate (electrolyte composition for 1 liter of galvanic solution): 150 g cuso4 · 5h2o 20 ml h2so4 96% 100 ml ethanol 96% current density 0.5 a / dm2 (area refers to steel wire that mesh is made and not on the surface of the steel net) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 48 working temperature 45oc feed speed of steel wire net 4m / minute immersion length of the net in galvanic bath2 linear meters electrolyte recirculation flow 2 liters / second galvanic bath capacity 500 liters deposited copper thickness about 4 μm reactions that occur during galvanic deposition of copper from sulfate bath on steel wire net are: release anodeh2h2e2h deposition cathodecu2ecu dissolving anodecu2ecu bath galvanic in theon dissociaticu(oh)ohcu bath galvanic in theon dissociatiohhoh bath galvanic in theon dissociatisocuaq4cuso 2 2 2 2 2 2 2 4 2             for the case of electrolytic coppering of thin steel wire net from sulfate baths, theoretical mass balance expressed by weight deposited on the cathode mcu2 or dissolved from the anode, under a current efficiency η of the anode and cathode of 100%, has the same general expression that of equation (2): (3)i0,019930i 96.500 64 m 22cu2  the difference from coppering with copper cyanide is in working mode, respectively the time that electric current passes through a net segment from its entry to the exit from galvanic bath, in this case in 240 seconds (speed advance of the net 0.3m/ minute). the intensity of the electrolysis current value i2 is calculated at a current density of 0.5 a/dm2. whereas the yield of anodic dissolution of copper is not equal to that of cathodic deposition at an interval of approximately 48 hours a chemical analysis of galvanic bath on copper ion content is required. depending on the result of the analysis copper salts is added or electrolyte is extracted from galvanic bath, and the remaining electrolyte is diluted to the optimal concentration. 2.2. electrochemical coppering of steel wire nets without external power the embodiment [1-fig.2] is a manufacturing flux of continuous production of the metal nets for shielding based on electrochemical deposition of copper of high purity, without external power, on the thin steel wire that forms meshes of primary net that in its turn is in a form of winding drum. copper deposition on iron without exterior power is based on electrochemical potential difference of the two metals in the electrochemical potential series of metals. in this serie the normal electrochemical potential for fe/fe2+ is 0.440 v and the normal potential for cu/cu2 + is + 0.337 v. in these conditions the ions from the more electronegative metal surface (iron) go into solution, and on iron are reduced the ions of more electropositive metal (copper), the deposition are taking place only to obtain an uniform layer of copper on steel wire metal net. manufacturing flux food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 49 consists of a power unit comprised a rolling drum 1 of a primary net 2 of thin steel wire, a pickling bath 3 for steel wire of primary net 2, a rinse bath 4 of primary net, a thermostated bath 5b, fig.2 from [1] and fig.2 from the present paper for electrochemical deposition of copper without external power, containing an electrolyte 6c based on copper sulphate used for electrolytic deposition of a thin coating of pure copper on the steel wire of primary net 2 on the basis of electrochemical potential difference between copper and iron, another rinsing bath 11 of the intermediate metal mesh with steel wire wrapped in a shirt of pure copper, a drying tunnel 13 of intermediate metalic net, an impregnating bath 14 for wire of intermediate metalic net with a monomer in order to provide advanced protection from oxidation of electrodeposited copper, a line 15 for polymerization with ultraviolet radiations of the monomer deposited on the metalic wire of intermediate net, an initiating drum 16 and winding drum 17 of final shielding net 18, the latter having a composite structure type thin wire steel electrolitic copper shirt –polymeric shirt. the operative mode to obtain electromagnetic shielding nets by electrochemical deposition without external current is the same as the galvanic deposition of copper with the difference that the bath 5a, fig.1 [1] and 5a from fig.1 in the present paper, equipped with dc power source and soluble copper anodes is replaced with a thermostated bath 5b, fig.2 comprising a thermostat 10, an automatic system for analyzing of copper ion concentration from the thermostatic bath 5b. composition of 6c electrolyte, based on 1 liter of the solution used for the electrochemical deposition without external current, is as follows: 200g cuso4. 5 h2o, 10ml h2so4 98%, 1g naoh, 5 ml hcl working temperature 50oc feed rate of steel wire net 7m / min length immersion of net in galvanic bath 2 linear meters electrolyte recirculation debit 2 liters / second thickness of the deposited copper layer of the order of nm the reactions that occur during electrochemical deposition without external current of copper on steel wire net are: tionreac globalaqsofecufeaqsocu reductioncu2eaqcu oxidation2efefe bath copperingin on dissociati socucuso 2 4 22 4 2 2 2 aq 2 4 2 aq4         in this case the theoretical balance of materials corresponds to the real balance of materials. performing material balance aims to determine the time at which the coppering bath must be filled with copper sulfate for it to remain in work setpoints. in this regard will be taken into account as may be allowed a decrease in the concentration of copper ions by 20% which still allows uniform deposition of a layer of 300 nm thickness as (about two copper atomic diameter) pure copper on the thin steel wire that forms meshes of metalic nets. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 50 2.3. coppering steel wire nets by chemical reduction of copper cell for electrochemical copper deposition without external current on steel wire mesh shown in figure 2 of the present paper, can be used to copper deposition by its chemical reduction on steel wire mesh. for this purpose it is used an electrolyte 6d, based on copper sulphate and copper chloride and a reducing agent, in the specific case of formaldehyde. the coppering bath composition 6d by chemical reduction and operating mode are: 10g cuso4. 5 h2o 5g cucl2. 2h2o 10 ml formaldehyde 1g naoh 15g edta working temperature 60oc feed rate of steel wire net 4m / min length immersion of net in galvanic bath 2 linear meters electrolyte recirculation debit 2 liters / second thickness of the deposited copper layer about 10 µm as in the case of copper depositing electrochemically without external power also in chemical reduction of copper on steel wire net theoretical balance of materials corresponds to the real balance of materials. in this type of deposit the balance of materials aims to determine the time at which the coppering bath must be filled with copper sulfate or copper chloride as the copper concentration variation does not exceed 5% which is the limit which provides another deposition copper in prescribed parameters. for a concrete example the net of steel wire with a diameter of 0.3 mm, with net width of 1m and side meshes of 2 mm, the feed rate of 4m /minute, in an hour copper is deposited on a net with 240 m length of linear steel wire-containing of 19,200 m length. taking into account that on this wire is deposited a pure copper jacket of about 10 micrometres thick (thickness required technology) also considering the copper density of 8.92 g/cm3, in one hour it is consumed about 28 g of copper from galvanic bath which requires the completion of electrolyte for coppering with copper sulfate and copper chloride. given that in this type of coppering, copper chloride has the role of ensuring a fine crystalline structure and increasing the adhesion of copper to the steel substrate and that copper ions are not consumed in equal ratio from copper sulphate and chloride, a system of automatic control of copper concentration in bath is needed correlated with an automatic dosing of the two substances providing copper ions. this system, fig.2, consists of spectrometer 19, a flow cell 20 continuously fed by a peristaltic pump 21 with solution of electrolyte 6c from bath 5b of deposition. the spectrometer is coupled by means of microprocessor 22 to an automatic dosing system 23 of concentrated solution of copper sulphate and a system 24 for automatically dosing the copper chloride solution. in the system of control and automatic dosing the coppering bath is filled continuously with two copper salts maintaining the prescribed concentration, according to the recipe of the coppering by means of microprocessor. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 51 fig.2 constructive details of electrolytic bath from coppering process of thin steel wire nets [1], [2], using chemical reduction process and automation related the coppering bath to maintain the nominal operating parameters. 2-primary steel wire net, 5b-reduction thermostatic bath for depositing copper used either without external current (electrolyte 6c) or for depositing copper using chemical reduction process (electrolyte 6d), 10-thermostat, 12intermediate metalic net of coated steel wire having a copper jacket, 19the spectrometer, 20-flow cell, 21-peristaltic pump, 22microprocessor, 23 automatic system of dosing automatically concentrated solution of copper sulfate, 24automatic dosing system of copper concentrated chloride solution. tg-drums of guiding –stretching role, tt-drums of tensilereturn role. 3. conclusions steel wire net coated with copper according to the technology, methods and recipes for coppering described is an advanced composite material that through the steel net provides advanced electromagnetic shielding of lowfrequency currents, through the copper layer deposited on the steel wire mesh provides shielding of high-frequency currents, and by the thin film of polymer reduced on the copper layer provides protection thereof against oxidation allowing fixing nets shielding under plaster of workspaces or housing thereby assuring a lifetime equal to that of the building. these nets from composite material can be used in the furniture industry to be secured non-detachably and invisible in stratified wood panels, coated with melamine, being mounted during manufacture of plates by hot pressing between core laminated wood food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 c r i st i a n f l or i n al e x u c, g he r o ghe g utt , so nia am ar i e i, c on t r i b u t i o ns t o ac h i e vem n t a c o m p os i t e m a t er i a l f or ad v a nc e d e l e ct r om ag n et i c s h ie l d i n g of l i vi ng an d wor k s pa c es , f o o d a n d e nv i r o nme n t s af e t y , v ol ume x iv , is s ue 1 – 2 01 5 , p a g. 45 52 52 and melamine plate.detailed description of recipes for coppering and theoretical material balances as well as advanced description of process flow of manufacturing enable immediate approach of production of this shielding material. the comparative study of three coppering methods that may be used reveals that the most efficient in terms of quality, productivity and layer thickness is the method of galvanic deposition of copper from cyanide solutions instead the lowest price presents electrolytic deposition method without external power. 4. references [1]. alexuc c., gutt g., amariei s., contributions to achievement a composite material for advanced electromagnetic shielding of living and workspaces, first part shielding material, food and environment safety 12 (4), 283-289, (2014) [2]. alexuc c.,f., gutt g., amariei s., metallic material for electromagnetic shielding of radiations and its achieving technology, proposal for invention, osim file a00684/09.09.2014 [3]. gutt g., gutt s., stroe s., alexuc c.f., a process for electromagnetic shielding, patent ro 126.682/2011 [4]. gutt g., gutt s., alexuc c.f., building materials for housing electromagnetic shielding, patent, ro 126.683/2011 291 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 291 299 chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts natalya cherno1, sophya osolina2, *aleksandra nikitina3 1 odessa national academy of food technologies, odessa, 65039, ukraine, cherno_n_k@mail.ru 2 odessa national academy of food technologies, odessa, 65039, ukraine, ossofol@mail.ru 3 odessa national academy of food technologies, odessa, 65039, ukraine, alex.nikitina@gmail.com *correspoding author received november 4th 2013, accepted december 11th 2013 abstract: the chemical composition of two edible mushroom species was investigated. agaricus bisporus is a saprotrophic spice. pleurotus ostreatus belongs to the wood-rotting mushrooms. they are the most popular among mushrooms that are cultivated in europe. it is found that mushrooms contain carbohydrates, protein, lipids, phenolic compounds and mineral elements. p. ostreatus has more carbohydrates than a. bisporus. trehalose dominates among the low molecular weight carbohydrates. the main monosaccharide in the hydrolysates of easily hydrolysable polysaccharides is glucose. only glucose and glucosamine are detected in the hydrolysates of hardly hydrolysable polysaccharides. glucan and chitin are present in the composition of this polysaccharide fraction. a. bisporus contains 2.0 times more chitin than p. ostreatus. all essential amino acids are found in the mushroom protein hydrolysates. nonprotein nitrogen compounds are present in the mushrooms. lipid level is 1.4 times higher in a. bisporus than in p. ostreatus. unsaturated fatty acids dominate in the lipid composition. phenolic compounds of mushrooms include low molecular weight compounds and melanin. the low molecular weight phenolic compounds levels in the mushrooms are the same. a. bisporus contains significantly more melanin than p. ostreatus. mushrooms differ in the mineral element content. mushroom morphological parts have the different chemical composition. carbohydrates are mainly concentrated in the stipes, and protein can be found in the caps. a bisporus caps contain more lipids than the stipes. morphological parts of p. ostreatus do not significantly differ in this index. the phenolic compounds are mainly concentrated in a. bisporus caps. keywords: white button mushroom, oyster mushroom, cap, stipe, carbohydrate, protein, lipids, phenolic compounds, mineral elements 1. introduction increase in the number of people suffering from the so-called "diseases of civilization" has changed the attitudes to the foodstuff. now they can be only an energy source but also a promising means of non-drug correction of certain physiological processes in a human body. it has caused the fact that a new type of food products – functional food was proposed. it often includes the foodstuff containing some functional ingredients. these components are able to correct some physiological processes in a human body [1]. mushrooms are a promising source of such ingredients. some ecotoxicants in the wild food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 292 mushrooms, the possibility of biological mutations due to the environmental pollution, seasonality and difficulties of prediction the stable amounts of provision interfere using these types of mushrooms. cultivated mushrooms do not have these defects [2]. agaricus bisporus (white button mushroom) and pleurotus ostreatus (oyster mushroom) are mainly cultivated in europe [2]. the content of protein, lipids and mineral elements in mushrooms was evaluated [3-7]. however, determination of carbohydrate content as the difference between the total dry weight and weight of these substances does not give enough information about carbohydrate composition. it was not investigated what monoand oligosaccharides, polysaccharides are present in the mushrooms. however, some researches pay attention to the mushroom polysaccharides because of their high physiological activity [8]. mushroom fruiting bodies are composed of two morphological parts: caps and stipes. they probably have different chemical composition. however, few investigations have focused on the content of some compounds in the mushroom morphological parts. thus, information about the chemical composition of these mushrooms is not sufficient for an objective evaluation of prospects for using their as a source of biologically active compounds, extraction and making dietary supplements on their basis. the purpose of this research was to determine the chemical composition of agaricus bisporus and pleurotus ostreatus fruting bodies and their morphological parts. 2. experimental two spices of edible mushrooms: agaricus bisporus (white button mushroom) and pleurotus ostreatus (oyster mushroom) were purchased at the farm that is cultivated mushrooms and used in this research. the fruit bodies were cleaned, separated into caps and stipes, dried and milled. analysis of total dry matter content was carried out at 65 °c using a vacuum oven [3]. for determination easily hydrolysable polysaccharides (ehp) content mushroom samples were being hydrolyzed with 2 % hcl solution at the boiling temperature for 4.0 hours. then the residues after hydrolysis were washed with water up to neutral value ph, dried up to constant weight. for determination hardly hydrolysable polysaccharides (hhp) content these residues were being treated with 72 % h2so4 solution at the ambient temperature for 2.5 hours. the water was added. then they were being hydrolyzed for another 5 hours at the boiling temperature [9]. in the all hydrolyzates the total neutral carbohydrate was determined by the anthrone method with glucose as standard [10], the glucoasamine content was evaluated with 3-methyl-2benzothiazolone-hydrazone-hydrochloride according to [11]. for identification of monosaccharides the sugars were converted into their corresponding alditol acetates [12] and identified by gas-liquid chromatography. gas-liquid chromatography was performed on hewlett-packard 5890 a chromatograph with a flame ionization detector and integrator 3393 a with a ultra-1 capillary column (25 m x 0.2 mm) from 175 °c to 270 °c at 10 °c/min in a stream of nitrogen. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 293 extraction of the mannitol was carried out according to [13]. sample was suspended in the water and being thoroughly homogenized for 30 min. the suspension was being boiled for 15 min. after freezedrying, the material was redissolved in the pyridine and being heated for 30 min. the mannitol solution in pyridine was thoroughly mixed with arabitol solution. this solution was mixed with a derivatizing agent (the mixture of n,obistrimethylsilyl trifluoroacetamide and trimethylchlorosilane). the mixrure was vortexed and allowed to stand at room temperature for 15 min. then it was injected into the gas-liquid chromatograph. analysis of total nitrogen content was done according to the kjeldahl method [3]. for determination non protein nitrogen 0.3 g of sample was transferred to the glass. then 25 cm3 of distilled water was added while stirring. it was heated up to the boiling temperature and 5 cm3 of 50 % trichloroacetic acid solution was added stirring thoroughly. this mixture was left for 0.5 – 1 hour, filtered through ashless filter. the precipitate was washed with small portions of 2 % trichloroacetic acid solution. the filtrate was mineralized with sulphuric acid and nonprotein nitrogen content was determined by the kjeldahl method [3]. chitin nitrogen level in mushroom was obtained by multiplying the chitin content by the coefficient 0.069 (in the natural chitin there is 6.9 % n) [14]. total protein nitrogen content was calculated as the difference between total nitrogen level and nonprotein nitrogen, chitin nitrogen values. total protein content was calculated by multiplying the protein nitrogen content by 6.25 [3]. the level of amino acids was determined with using the t 339 chromatographic amino acid analyser. liquid-phase hydrolysis of sample was being performed in 6m hcl solution at 110 °c for 24 h in an argon atmosphere [15]. the hydrolysate was lyophilised, dissolved in an appropriate volume of dilution buffer and filtered. the determination of the sulphurcontaining amino acids (methionine and cysteine) was carried out by means of oxidative hydrolysis, using a mixture of formic acid and hydrogen peroxide (9:1) at 4 °c for 16 h, followed by standard hydrolysis procedure with hcl. alkaline hydrolysis for tryptophan determination was done according to [15]. the calculations were carried out with reference to external standards. the composition of amino acids was expressed as grams per 100 g of protein. on the basis of the amino acid composition, the chemical score index was calculated [16]. the lipid content was determined by soxhlet extraction [17]. fatty acids, transmethylated with 5 % sulphuric acid in methanol, were analyzed by gas liquid chromatography (hp 6890 plus) equipped with a flame ionization detector [18]. the level of the total phenolic compounds was estimated in the ethanol extracts using folin–chiocalteu method [19]. for preparation these extracts sample (2 g) was being extracted by stirring with 20 cm3 of 70 % ethanol solution at 65 ºc for 30 min and filtered. this operation was repeated one time again. then 1 cm3 of the combined extract was added to 10 cm3 of distilled water and 2 cm3 of folinciocalteu phenol reagent. the mixture was allowed to stand at room temperature for 5 min and then 2 cm3 of 20 % sodium carbonate solution was added to the mixture. the resulting blue complex was measured at 730 nm. chlorogenic acid from sigma was used as a standard for the calibration curve. melanin from mushrooms was being extracted by 2 % naoh solution for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 294 2 hours at the boiling temperature. the extract was cooled, acidified to ph 2.0. concentrated pigment was separated by centrifugation at 8000 g for 15 min [20]. the precipitate was dissolved in the 2 % naoh solution. melanin content was calculated from the calibration curve on the basis of the photometry of the solution at the wavelength of 490 nm. melanin from sigma was used for the calibration curve. ash content was evaluated by weighing before and after treatment of the dried mushrooms in a chinaware crucible at 550 °c to constant weight. total ash content was expressed in percentage of dry weight [3]. analytical determinations were conducted in three simultaneous replications. 3. results and discussion the water content in mushrooms and in their morphological parts was 91.5-92.1 %. chemical composition of the mushroom morphological parts is given in table. 1. carbohydrates are one of the important mushroom components. they include monoand oligosaccharides, easily and hardly hydrolysable polysaccharides (ehp and hhp respectively). notably, their content in p. ostreatus is 2.1 times higher than in a. bisporus. table 1 the chemical composition of mushroom morphological parts (% of the dry weight) components agaricus bisporus pleurotus ostreatus cap stipe cap stipe mono-and oligosaccharides 1.2 1.4 16.0 11.0 mannitol 30.4 28.9 7.5 5.2 polysaccharides including easily hydrolysable 13.7 21.9 25.0 27.0 hardly hydrolysable 7.2 9.2 8.3 24.0 including chitin 4.0 7.9 2.6 2.9 total nitrogen 5.3 4.7 4.8 2.9 including nonprotein nitrogen 2.0 1.7 1.9 1.1 protein 18.9 15.3 17.0 10.0 lipids 4.0 1.8 2.5 2.0 phenolic compounds 8.3 4.0 4.9 3.2 including melanins 6.4 2.1 3.1 1.6 mineral elements 9.0 7.6 7.9 6.7 ehp dominate among the carbohydrates. their content in a. bisporus is 64.7 % of the total carbohydrate level, in p. ostreatus is 47.2 % respectively. glucose is a main monosaccharide in their hydrolysates (table 2). the levels of mannose, fucose and xylose that are present in the small amounts are almost the same in the studied mushrooms. however, the galactose content is higher in a. bisporus than in p. ostreatus. it is possible that ehp contain glucan and heteropolysaccharides. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 295 table 2 monosaccharide composition of the easily hydrolysable polysaccharides hydrolysates of mushroom morphological parts (% of the monosaccharide residues) monosaccharide agaricus bisporus pleurotus ostreatus cap stipe cap stipe galactose 4.6 3.2 1.0 0.5 glucose 79.5 84.0 82.5 88.0 mannose 3.9 4.8 6.5 4.5 xylose 5.2 3.3 5.0 5.0 fucose 6.8 4.7 5.0 2.0 p. ostreatus has 1.9 times more hhp than a. bisporus. only glucose and glucosamine are identified in their hydrolysates, so hhp contain glucan and chitin. however, the ratio of these polysaccharides depends on the mushroom type. chitin dominates in a. bisporus (69.5 % of hhp), glucan prevails in p. ostreatus (82.0 % of hhp). this difference is associated with the mushroom nutrition type: saprotrophic spice (a. bisporus) synthesizes more chitin than wood-rotting one (p. ostreatus) [7, 21]. the trehalose dominates among the low molecular weight carbohydrate. p. ostreatus has 10.8 times more trehalose than a. bisporus. probably this disaccharide makes reserve function. the reserve substance in a. bisporus is polyol – mannitol [22]. its content is 4.6 times higher than in p. ostreatus. it is shown that morphological parts of mushrooms contain different quantity of carbohydrates. the total level of polysaccharides is 1.3-1,5 times higher in stipes in comparison with caps. however, the caps have more low molecular weight carbohydrates and mannitol than stipes. it can be explained by the different biological roles of mushroom morphological parts. stipes makes the transport and support function. that is why they contain welldeveloped transport and mechanical tissue structures. polysaccharides are their main components. caps accumulate a large amount of storage nutrition compounds that are necessary for forming mushroom reproductive organs – basidiospores [7, 21]. the various nutrition types of studied mushrooms cause the difference in the quantity of protein in their composition. a. bisporus has 1.3 times more protein than p. ostreatus. 18 amino acids are found in the mushroom protein hydrolysates (table 3). table 3 amino acid composition of protein of mushroom morphological parts (% of total protein content) amino acid agaricus bisporus pleurotus ostreatus cap stipe cap stipe lysine 5.7 5.7 5.6 6.0 histidine 2.8 2.8 2.1 2.1 arginine 4.9 4.8 5.6 5.4 aspartic acid 11.8 10.9 11.4 10.4 threonine 4.9 4.6 4.5 3.7 serine 5.8 5.9 5.4 5.2 glutamic acid 20.0 18.1 19.2 17.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 296 proline 5.3 5.9 4.1 5.7 glycine 5.0 5.7 4.3 4.0 alanine 5.7 5.7 6.8 4.3 cysteine 3.7 4.4 3.9 3.8 valine 3.9 4.4 5.4 5.9 methionine 1.1 1.8 2.3 1.9 isoleucine 3.6 3.6 3.7 3.8 leucine 7.2 7.3 6.7 9.2 tyrosine 3.4 2.7 3.5 4.9 phenylalanine 4.2 4.2 4.3 5.0 tryptophan 1.2 1.4 1.3 1.4 the most abundant amino acid is glutamic acid, which plays an important role in the organism metabolism. the part of essential amino acids gets 39.4 – 43.2 % of the total amino acids level. leucine is the main essential amino acid. the levels of methionine and cysteine, phenylalanine and tyrosine, tryptophan, threonine, leucine, lysine of both spices of mushrooms are higher than in the ideal protein (table 4). lysine is a deficient amino acid for the most food rawmaterials. blood circulation is disrupted and the number of red blood cells reduces while it lacks in a human body [23]. p. ostreatus contains more valine and isoleucine than a. bisporus. p. ostreatus has less nonprotein nitrogen compounds than a. bisporus (1.2 times). these intermediate products of protein metabolism include free amino acids, urea, organic and purine bases, ammonia nitrogen that is free ammonia and ammonium-magnesium salts of phosphoric acid [7, 21]. table 4 amino acid chemical score of protein of mushroom morphological parts (%) amino acid agaricus bisporus pleurotus ostreatus cap stipe cap stipe lysine 103.6 104.0 100.9 109.5 threonine 121.5 114.3 111.5 93.3 methionine + cysteine 137.4 177.1 176.9 162.3 valine 78.4 80.7 108.4 118.8 isoleucine 89.8 90.3 93.0 96.0 leucine 102.9 104.3 96.3 131.1 phenylalanine + tyrosine 126.3 115.3 130.7 164.5 tryptophan 120.0 140.0 130.0 140.0 mushroom morphological parts differ in the total amount of protein and the intermediate products of protein metabolism. they get the highest level in caps. it can be explained by their biological role. a significantly difference in the quantity of protein and nonprotein compounds is found in p. ostreatus morphological parts. stipes contain more essential amino acids in the protein hydrolysates than caps. this is typical for p. ostreatus and a. bisporus. the amount of lipids in a. bisporus is 1.4 times higher than the one in p. ostreatus. a. bisporus caps have more these compounds than stipes. however, the morphological parts of p. ostreatus contain almost the same quantity of lipids. it is shown that unsaturated fatty acids dominate in the lipid composition. their food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 297 level in a. bisporus is 82.8 % of the total fatty acids, in p. ostreatus is 71.5 %. a. bisporus and olive oil have almost the same level of unsaturated fatty acids [24]. their content in p. ostreatus is approximately equal to the level of these acids in soybean and cotton oil [24]. mushrooms are also different in abundant unsaturated fatty acids. the oleic acid is in a. bisporus, linoleic one is in p. ostreatus. the latter together with the linolenic acid are the essential acids. they are involved in the synthesis of cell membranes, prostaglandins, in the regulation of cells metabolism [24]. table 5 fatty acid composition of lipids of mushroom morphological parts (% of the total fatty acids) fatty acids agaricus bisporus pleurotus ostreatus cap stipe cap stipe tridecanoic traces traces 2.8 10.5 myristic 0.4 traces traces 19.1 pentadecanoic 1.1 2.0 1.9 traces palmitic 8.7 7.6 11.2 7.1 stearic 5.9 9.1 1.5 3.2 oleic 54.8 38.7 18.4 17.6 linoleic 24.1 41.9 61.9 36.6 linolenic 5.0 0.7 0.7 5.2 the amount of certain types of fatty acids and their ratio in the products are important for the normal functioning of a human body. a. bisporus and p. ostreatus contain 34.4 % and 53.5 % polyunsaturated, 48.4 % and 18.0 % monounsaturated, 17.2 % and 27.3 % saturated acids respectively. the ratio of linoleic and linolenic acids is 9.6 for a. bisporus and 18.9 for p. ostreatus. that is approximately equal to the recommended norms [24]. this fact allows us to offer mushrooms as food ingredients to the elderly and patients with cardiovascular disease. however, mushroom morphological parts have the different levels of these acids. it is found the level of monounsaturated acids is 1.4 times higher in a. bisporus caps than in stipes. but they contain less polyunsaturated acids. the level of saturated acids is almost the same in caps and stipes of this mushroom. morphological parts of p. ostreatus have approximately the same content of monounsaturated acids. the amount of polyunsaturated in caps is 1.5 times more than in stipes. the level of saturated acid is 2.3 times less. phenolic compounds in mushrooms include high molecular weight compounds – melanin and low molecular weight substances. the latter content is approximately the same for mushrooms and their morphological parts. they show high antioxidant and antimicrobial properties [25]. identification of melanin was carried out by qualitative reactions and the pattern of absorption in the uv and visible regions of the spectrum. they are oxidized by h2o2, kmno4 solutions and formed a precipitate reacting with fecl3 solution. it is characteristic for all pigments of this nature, regardless of their origin. the absorption spectrum of mushroom melanins is typical [20]. the highest absorption intensity is observed in the uv food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 298 region of the spectrum and then with increasing wavelength it gradually decreases. the amount of melanin in p. ostreatus is far less than in a. bisporus. caps have higher levels of these polymers. it is known [7, 21] that the level of mineral elements in mushrooms depends on the type of mushrooms. the studied mushrooms differ in their content. mushroom morphological parts contain various amounts of mineral elements. caps have them more than stipes. 4. conclusion the various nutrition types of studied mushrooms cause the differences in their chemical composition. it is shown, that carbohydrates are dominating macrocomponent in the mushrooms. p. ostreatus contains 2.1 times more these compounds than a. bisporus. trehalose level in p. ostreatus is 10.8 times higher than that in a. bisporus. the latter contains more mannitol. the easily hydrolysable polysaccharides mainly compose of glucose units. hardly hydrolysable polysaccharides contain glucan and chitin. p. ostreatus is richer in this fraction than a. bisporus. the level of the essential amino acids is 39.4 – 43.2 % of the total amino acids. the content of total protein and nonprotein nitrogen compounds is higher in a. bisporus in comparison with p. ostreatus. a. bisporus contains 1.4 times more lipid than p. ostreatus. the part of unsaturated fatty acids is 71.5-82.8 %. the oleic acid dominates in a. bisporus, the linoleic one is the main fatty acid in p. ostreatus. the total amount of phenolic compounds is higher in a. bisporus than in p. ostreatus. melanins dominate among these substances. the studied mushrooms contain the different amount of mineral elements. it is shown that among mushroom morphological parts stipes have higher level of carbohydrates. caps contain more mannitol, protein, nonprotein nitrogen compounds, lipids, phenolic compounds. attention should be paid to difference in chemical composition of mushrooms and their morphological parts while the technologies for obtaining a number of functional components from them are developed. 5. acknowledgments we thank larisa karpynskya for help in translation. 6. references [1] grajek w., olejnik a., sip a., probiotics, prebiotics and antioxidants as functional foods, acta biochim. pol., 52(3). 665-71, (2005) [2] marshall e., nair n., make money by growing mushrooms, infrastructure and agroindustries division, food and agriculture organization of the united nations, italy, 62 p, (2009). [3] manzi p., gambelli l., marconi s., nutrients in edible mushrooms: an interspecies comparative study, food chem., 65. 477-482, (1999). [4] manzi p., aguzzi a., pizzoferrato l., nutritional value of mushrooms widely consumed in italy, food chem., 73. 321-325, (2001) [5] mattila p., salo-väänänen p., känkö k., aro h., jalava t., basic composition and amino acid contents of mushrooms cultivated in finland, j. agric. food chem., 50. 6419-6422, (2002) [6] dikeman c., cauer l., flickinger e., fahey g., effects of stage of maturity and cooking on the chemical composition of selected mushroom varieties, j. agric. food chem., 53. 1130-1138, (2005) [7] andres s., baumann n., mushrooms: types, properties and nutrition, nova science publishers, new york, 381 p, (2012) [8] wasser s., medicinal mushrooms as a source of antitumor and immunomodulating food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 natalya cherno, sophya osolina, aleksandra nikitina, chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts, volume xii, issue 4 – 2013, pag. 291 – 299 299 polysaccharides, appl. microbiol. biotechnol., 60. 258-274, (2002) [9] whister l., wolfrom m., methods in carbohydrate chemistry, academic press, new york and london, 512 p, (1962-1965) [10] chung c., nickerson w., polysaccharide synthesis in growing yeast, j. biol. chem., 208. 395-407, (1954) [11] smith r., gilkerson e., quantification of glycosaminoglycan hexosamine using 3-methyl-2benzothiazolone hydrazone hydrochloride, anal. bioch., 98. 478-480, (1979) [12] laine r., esselman w., sweely c., gas-liquid chromatography of carbohydrates, methods enzymol., 18. 156-167, (1972) [13] tan y., moore d., high concentrations of mannitol in the shiitake mushroom lentinula edodes, microbios, 79. 31-35, (1994) [14] ofenbeher-miletic i., stanimirovic d., stanimirovic s., on a determination chitin content in mushrooms, plant foods for human nutrition, 34. 197-201, (1984) [15] darbre a., practical protein chemistry, chichester [sussex], new york, wiley, 620 p, (1986). [16] osborne d., voogt p., the analysis of nutrients in food, academic press, london, u.k., 240 p, (1978) [17] sanmee r., dell b., lumyong p., izumori k., lumyong s., nutritive value of popular wild edible mushrooms from northern thailand, food chem., 84. 527-532 (2003) [18] johnson a., davenport j., biochemistry and methodology of lipids, john wiley and sons inc., new york, ny, 590 p, (1971) [19] ragazzi e., veronese g., quantitative analysis of phenolics compounds after thin layer chromatographic separation, j. chromotogr., 77. 369-375, (1973) [20] selvakumar p., rajasekar s., periasamy k., raaman n., isolation and characterization of melanin pigment from pleurotus cystidiosus (telomorph of antromycopsis macrocarpa), world j. microbiol. biotechnol., 24. 2125-2131, (2008) [21] chang, s.-t., miles p., mushrooms. cultivation, nutritional value, medicinal effect, and environmental impact, 2nd ed., crc press, boca raton, london, new york, washington, 451 p, (2004) [22] stoop j., williamson j., pharr d., mannitol metabolism in plants: a method of coping with stress, trends plant science, 5. 139-144, (1996) [23] wu g., amino acids: biochemistry and nutrition, crc press, boca raton, london, new york, washington, 503 p, (2013) [24] fao/who, fats and fatty acids in human nutrition. report of an expert consultation, fao food and nutrition paper, 91. fao, rome, (2010) [25] jong, s., birmingham j., medicinal and therapeutic value of the shiikate mushroom, adv. in applied microbiology, 39. 153-184, (1993) 101 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 101 107 blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity georgiana gabriela codină1, maria poroch-seriţan1, silvia mironeasa1,* 1faculty of food engineering, stefan cel mare university, suceava, romania, codina@fia.usv.ro; mariap@fia.usv.ro; silviam@fia.usv.ro *corresponding author received 14th march 2015, accepted 31st march 2015 abstract: sunflower oil (sfo) was blended with grape seed oil (gso) in order to improve its quality. if sunflower oil is a traditional one on romania market the grape seed oil is a non-conventional one which has a high content in unsaturated fatty acids like oleic and linoleic ones and a large amount of tannins like the oligomeric proanthocyanosides. also, grape seed oil is rich in bioactive compounds like phenolic components, tocopherols, e.g. thus making it a valuable source in order to improve the nutritional quality of the blended oils. the main goal of the present work was to compare and correlate the physical-chemical and radical scavenging activity of the sfo blended with gso in different proportions: 10:0, 9:1, 8:2, 7:3 and 0:10, w/w. the physical-chemical properties analyzed in this study were refractive index, iodine value, peroxide value, saponification value and acid value. the radical scavenging activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (dpph) method. the results showed that by increasing the proportion of gso in sfo, the physico-chemical parameters values decreased, while the radical scavenging activity increased. oxidative stability of oil blends was improved with the level increase of gso. this study showed that the quality of sunflower oil may be improved with the addition of grape seed oil. key words: oil blends, sunflower oil, grape seed oil 1. introduction vegetable oils from seed sources are of important interest in different food and application industries. one of these vegetable oils, well known worldwide due to its functional properties, is the grape seed oil. this oil is obtained from the seed of grapes (vitis vinifera l.), a by-product of the winemaking industry, which makes up to 15% of the solid waste produced in wine industries [1]. various extraction methods like solvent extraction, hydraulic pressing, supercritical carbon dioxide [2, 3, 4] e.g., are used to recover the grape seed oil. the oil content of the grape seeds range between 12-20% of its dry weight [5, 6] depending on the pressing condition [7] used. grape variety, degree of ripening, soil quality, climate and the technological procedure of grape processing are some of the factors that influence the grape seed oil yield [8]. grape seed oil is a valuable source of dietary fat due to its high content in unsaturated fatty acids (85–90%) [1], which has many benefits such as the prevention of thrombosis, dilation of blood vessels, inhabitation of cardiovascular diseases, reduction of cholesterol in serum and the oxidation of low-density lipoproteins e.g. http://www.fia.usv.ro/fiajournal mailto:codina@fia.usv.ro; mailto:mariap@fia.usv.ro; mailto:silviam@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 102 [9]. due to its compounds that have beneficial health effects, grape-seed oil has a high potential to be used in pharmaceutical and food applications [10, 11]. its content is high in unsaturated fatty acids, oleic and linoleic acids [12] that are very important for the stability of oils due to the chemical reactions in the double bonds. the rates of those oxidation reactions depend on the number of double bonds in the carbon chain [13]. grape seed oil contains a large amount of tannins, like the oligomeric proanthocyanosides (opcs), in a higher level than in the other seed oils [14]. also, grape seed oil is rich in bioactive compounds like phenolic components that are known for their antioxidant properties [15]. also, it contains tocopherols, especially α-tocopherols [13] that have the highest vitamin e activity [16], thus making it important for human health and biological activity [17]. grape seed oil has a high antioxidant potential, fernandes et al. (2013) reporting that dpph radical scavenging activities varied between 38.68 and 69.89% depending on grape genotype. due to its content, grape seed oil can be used in blends with other vegetable oils, especially in the cases when a high level of antioxidant protection is needed. therefore, considering the above mentioned, this study was focused on grape seed oil and also due to the fact that this oil has a high content of unsaturated fatty acids such as linoleic acid (72–76%, w/w), which exceeds those from the sunflower oil (60–62%) accordingly to c. ghisalberti, pct int. appl. wo 2001018161 a2 (15 march, 2001). in addition, grape seed oil has, unlike the sunflower one, low levels of linolenic acid [20] which is desired in edible oils because it improves the oxidative stability [21]. having three double bonds on its hydrocarbon chain, the linolenic acid is readily oxidized and therefore, the stability of an oil rich in linolenic acid would be too short [22]. sunflower oil is a vegetable oil with poor oxidative stability which is prone to flavor degradation due to its high proportion of polyunsaturated fatty acids [23]. this oxidation process of unsaturated fatty acids represents one of the major causes which determine the development of off-flavor compounds and decrease the nutritional value of the food products [24]. lipid oxidation changes the physicochemical parameters of the oil, thus influencing directly the quality of the vegetable oil and has harmful effects on food quality and human health [25]. sunflower oil is not quite suitable at high temperature (e.g. for frying) due to the higher magnitude of oxidation [26]. one way to improve the oxidative stability of this oil is by blending it with oils that have high antioxidant levels, like grape seed oil. the blending of these oils changes the functional properties without any chemical or biological process [27]. various studies have been reported on the blending of vegetable oils with different physicochemical properties in order to improve some quality parameters such as, peroxide value, oxidative stability, e.g. [25, 28]. although the quality of the above mentioned vegetable oils, such as sunflower oil and grape seed oil has been evaluated by many researchers, to our knowledge, their quality parameters, in different blends have not been extensively studied. the objective of the present study was to investigate the effects of grape seed oil with sunflower oil in different blends proportion on the some physical-chemical values like refractive index (ri), iodine value (iv), acid value (av), peroxide value (pv) and radical scavenging activity by means of the dpph method. this study may be useful in the development of healthy blended oils with an improved quality. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 103 2. materials and methods 2.1. materials sunflower oil and grape seed oil was purchased from the local market (suceava, romania). sunflower oil (sfo) was blended with grape seed oil (gso) at the following sfo:gso mixing ratios: 10:0; 9:1, 8:2, 7:3 and 0:10 (w/w). all reagents and chemicals that were used in this work were of analytical grade. 2.2. methods of analysis the analysis of the physical and chemical parameters was carried out according to the methods described in the romanian or international standard methods. all tests were performed in duplicate. the refractive index (ri) of the oil samples at room temperature was determined according to the sr en iso 6320:2002/ac:2006 by using an a abbé refractometer (leica plus). the ri is a factor by which the wavelength and the velocity of the radiation are reduced with respect to their vacuum values [29]. the iodine value (iv), expressed as grams of i2 absorbed by 100 g oil (sr en iso 3961:2013), was determined using the hanus method. saponification value (sv) is expressed as mg koh required for the saponification of the free fatty acids and esterified from 1 g of oil sample and it was determined according to the sr en iso 3657:2013. acid value (av) was determined according to the sr en iso 660:2009 and is expressed in mg koh required to neutralize the free fatty acids present in 1 g vegetable oil. the av is a measure of the amount of carboxylic acid groups in a chemical compound, such as a fatty acid, or in a mixture of compounds [29]. peroxide value (pv) measures the peroxides and hydroperoxides that are in the initial stages of lipid oxidation. milliequivalents of active oxygen per kg of oil sample are measured by titration with sodium thiosulfate solution (0.01n) according to the sr en iso 3960:2010. extraction of the phenolic fraction the extraction was carried out with the method reported by mraicha et al. (2010). in short, 4 g of the oil sample was added to 2 ml of n-hexane and 4 ml of a methanol/water (60:40, v/v) solution in a 20 ml centrifuge tube. after vigorous mixing, the sample was centrifuged for 10 min at 1500 rpm. the hydroalcoholic phase was collected, and the hexanic phase was reextracted twice with 4 ml of methanol/water (60:40, v/v) solution each time. finally, the hydroalcoholic fractions were combined, washed with 4 ml of n-hexane to remove the residual oil, and then concentrated. free radical scavenging activity the free radical scavenging activity of the extracts was measured by the 1,1-diphenyl2-picrylhydrazyl (dpph) method proposed by bouaziz et al., 2008. in succinct terms, aliquots (50 µl) of various concentrations from the tested compound were added to 5 ml of a 0.004% methanol solution of dpph. when dpph reacts with an antioxidant compound, which can donate hydrogen, it is reduced. the changes in colour (from deep violet to light yellow) were read at 518 nm after 30 min of reaction at room temperature using a uv vis nir spectrometer (shimadzu, 3600, japan). methanol was used to zero the spectrophotometer. the inhibition of free radicals dpph in percent (i%) was calculated in the following way: i% = [(ablank asample)/ablank] × 100, where ablank is the absorbance of the control reaction (containing all reagents except the test compound) and asample is the absorbance of the test compound. 2.3. statistical analysis the experimental results were expressed as means ± standard deviation (sd) of the food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 104 duplicate measurements. differences among the means were evaluated by the analysis of variance (anova) using spss v.16.0 software. mean values with a statistical difference of p  0.05 were considered as significant. 3. results and discussion physicochemical characteristics refractive index fig. 1 shows the obtained results of the refractive index (ri). significant differences at p  0.05 were obtained between gso and sfo, and their blends with 1%, 2 % gso. it can be seen that sfo presents the highest ri while gso has the lowest ri. fig. 1. variation of refractive index (ri) in oil samples formulation: sfo – sunflower oil; gso – grape seed oil. line show means. the low ri value for gso can be due to its higher content in linoleic acid, comparatively with the sfo. it can also be related to the nature of the fatty acids present, since ri decreases with the molecular weight of the fatty acids and with unsaturation [31]. the decrease of ri with the increase level of gso from blends may be due to the increase in the number of double bonds, the ri of oils being related with the degree of unsaturation in a linear way [32]. iodine value the results for iodine value (iv) of the oil samples are shown in fig. 2. grape seed oil has positive effect on the iv of the oil blends and led to a significant decrease (p  0.05) of the iv with the increase of the gso level from 10 to 30%. fig. 2. variation of iodine value (iv) in oil samples formulation: sfo – sunflower oil; gso – grape seed oil. bars show mean. the low iv for gso may have contributed to its higher oxidative stability. therefore, the oxidative stability of the oil blend increased with the increase of gso level in the oil blend because the gso is the most resistant oil to oxidation due to its highest natural antioxidants, namely tocotrienols, which are 40-60 times in a higher level than tocopherols [33]. saponification values the saponification value (sv) of the grape seed oil is lower comparatively with the sfo (fig. 3), probably due to the fatty acids present in the gso that have a larger number of carbon atoms. therefore, this lower value of sv may be due to the higher molecular weight of the fatty acids from gso, knowing that sv is inversely related to the average molecular weight of them. the sv of the binary blends were found to food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 105 be in decrease with the increase of gso mixing proportion. fig. 3. variation of saponification value (sv) in oil samples formulation: sfo – sunflower oil; gso – grape seed oil. bars show mean. peroxide value the peroxide value (pv) calculated for sunflower oil, grape seed oil and binary oil blends are shown in fig. 4. the pv of gso shows a relative good quality compared to the sfo. the high pv of sfo indicates an early lipid phase peroxidation. fig. 4. variation of peroxide value (pv) in oil samples formulation: sfo – sunflower oil; gso – grape seed oil. bars show mean. the addition of gso to the sfo significantly decreases (p  0.05) the pv value, this results in an enhancement of the oxidative stability of the formulated oil blends. therefore, the pv value indicates an improvement of the product quality. acid value acid value (av) is a parameter used for the assessment of the oil quality, representing free fatty acid content due to its enzymatic activity. an increase in acid value can be due to the hydrolysis of glycerides, due to humidity and temperature. higher av for sfo gives an idea about increased susceptibility to rancidity due to the free fatty acid present in the oil. on the other hand, gso contains high levels of phenolic compounds that have antioxidant and antihydrolitic effects. therefore, a significant decrease (p  0.05) in the av of the sfo when it is blended with gso (fig. 5) was obtained. gso blended with sfo improved the quality of the oil blend as compared with pure sfo, this fact suggesting low levels of hydrolytic and lipolytic activities in the oil blends. fig. 5. variation of acid value (av) in oil samples formulation: sfo – sunflower oil; gso – grape seed oil. bars show mean. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 106 radical scavenging activity to evaluate the antioxidant activity of the phenolic fraction, the well known dpph antiradical test was performed. the inhibition of free radicals dpph in percent (i%) is higher for the grape seed oil (32.39%) in comparison with the sunflower oil (24.6%) (fig. 6). these results indicate that the grape seed oil showed the highest antioxidant activity in comparison with the sunflower sample. also, from this study it was observed that the inhibition of free radicals dpph in percent (i%) increases with the increasing amount of added grape seed oil in sunflower oil (28.24%; 28.59%; 29.78%). therefore, the antioxidant activity increased in the sunflower oil, with the increasing level of grape seed oil in the oil blends. fig. 6. spectrum in the vis range of the dpph radical in presence of oils: 1 – blank; 2 sfo:gso (10:0); 3 sfo:gso (0:10); 4 sfo:gso (9:1); 5 sfo:gso (8:2); 6 sfo:gso (7:3). 4. conclusion various physical and chemical parameters have been studied and may be used for the quality control of edible oil samples. the physical and chemical parameters decrease in oil blends with the increase proportion of grape seed oil mixed in sunflower oil, while radical scavenging activity increased. quality parameters of oil blends were better than for the sunflower oil, probably due to the changes in the fatty acids profile of oil blends, and to the minor bioactive lipids that the grape seed oil presents. the grape seed oil was found to be edible oil with high qualitative properties that can be used in order to improve the quality of sunflower oil. 5. references [1]. luque-rodríguez j.m., castro m.d.l., perez-juan p., extraction of fatty acids from grape seed by superheated hexane, talanta 68, 126– 130, (2005) [2]. beveridge t.h.j., girard b., kopp t., drover j.c.g., yield and composition of grape seed oils extracted by supercritical carbon dioxide and petroleum ether: varietal effects, journal of agricultural and food chemistry 53(5), 1799-1804, (2005) [3]. gómez a.m., lópez c.p., ossa e.m., recovery of grape seed oil by liquid and supercritical carbon dioxide extraction: a comparison with conventional solvent extraction. chemical engineering journal 61, 227–231, (1996) [4]. rosenthal a., pyle d.l., niranjan k., gilmour s., trinca l., combined effect of operational variables and enzyme activity on aqueous enzymatic extraction of oil and protein from soybean, enzyme and microbial technology 28(6), 499–509, (2001) [5]. kim s.y., jeong s.m., park w.p., nam k.c., ahn d.u., lee s.c., effect of heating conditions of grape seeds on the antioxidant activity of grape seed extracts, food chemistry 97, 472–479, (2006) [6]. bravi m., spinoglio f., verdone n., adami a., aliboni a., d’andrea a., de santis a., ferri d., improving the extraction of α-tocopherol-enriched oil from grape seed by supercritical co2. optimization of the extraction conditions, journal of food engineering 78, 488493, (2007) [7]. schieber a., muller d., rohrig g., carie r., influence of grape cultivar and processing method on the quality of cold-pressed grape seed oils, mitteilungen klosterneuburg 52(1-2), 29-33, (2002) food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa, blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity, food and environment safety, volume xiv, issue 1 2015, pag. 101 107 107 [8]. mironeasa s., seminţe de struguri: studii şi cercetări, pim, iaşi, romania (2013) [9]. cao x., ito y., supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography, journal of chromatography, 1021, 117–124, (2003) [10]. bail s., stuebiger g., krist s., unterweger h., buchbauer g., characterisation of various grape seed oils by volatile compounds, triacylglycerol composition, total phenols, food chemistry 108, 1122–1132, (2008) [11]. furiga a., lonvaud-funel a., badet c., in vitro study of antioxidant capacity and antibacterial activity on oral anaerobes of a grape seed extract, food chemistry 113, 1037–1040, (2009) [12]. crews c., hough p., godward j., brereton p., lees m., guiet s., winkelmann w., quantitation of the main constituents of some authentic grape-seed oils of different origin, journal of agricultural and food chemistry 54(17), 6261–6265 (2006) [13]. baydar n.g., özkan g., çetin e.s., characterization of grape seed and pomace oil extracts, grasas aceites 58, 29–33 (2007) [14]. rao p.u., nutrient composition of some less familiar oil seeds, food chemistry 50, 379-382, (1994) [15]. morin o., corn and grapeseed oil. in: a. karleskind (ed.), oil and fats manual, hampshire, andover, 143-146, (1996) [16]. baydar n.g., akkurt m., oil content and oil quality properties of some grape seeds, turkish journal of agriculture and forestry 25, 163–168, (2001) [17]. guthrie n., kurowska e.m., anticancer and cholesterol lowering activities of tocotrienols. in: wildman rec, ed. handbook of nutraceuticals and functional foods, boca raton, crc press, florida, 269-280, (2000) [18]. fernandes l., casal s., cruz r., pereira j.a., ramalhosa e., seed oils of ten traditional portuguese grape varieties with interesting chemical and antioxidant properties, food research international 50,161-166, (2013) [19]. ghisalberti c., pct international application wo 2001018161 a2, (2001) [20]. popovici c., capcanari t., deseatnicova o., sturza r., study of quality indices of functional vegetal oil mixture, the annals of the university dunarea de jos of galati, fascicle vi – food technology 34(1), 17-24, (2009) [21]. sineiro j., dominguez h.,. nunez m.j., pepitas de uva como fuente de aceite y proteína, alimentación, equipos y tecnología 14, 49–56, (1995) [22]. warner k., orr p., glynn m., effect of fatty acid composition of oils on flavor and stability of fried foods, journal of the american oil chemists' society 74(4), 347-356, (1997) [23]. gordon m.h., oils & fats: taint or flavour? chemistry in britain, 1020-1022, (1991) [24]. hemalatha g.s., sesame lignans enhance the thermal stability of edible vegetable oils, food chemistry 105, 1076-1085, (2007) [25]. li y., ma w.j., qi b.k., rokayya s., li d., wang j., feng h.x., sui x.n., jiang l.z., blending of soybean oil with selected vegetable oils: impact on oxidative stability and radical scavenging activity, asian pacific journal of cancer prevention 15, 2583-2589, (2014) [26]. anwar f., hussain a.i., iqbal s., bhanger m.i., enhancement of the oxidative stability of some vegetable oils by blending with moringa oleifera oil, food chemistry 103, 1181– 1191, (2007) [27]. liu h.r., white p.i., high temperature stability of soybean oils with altered fatty acid compositions, journal of the american oil chemists' society 4, 161-167, (1992) [28]. monfreda m., gobbi l., grippa a., blends of olive oil and sunflower oil: characterisation and olive oil quantification using fatty acid composition and chemometric tools, food chemistry 134, 2283–2290, (2012) [29]. atabani a.e., mahlia t.m.i., lee k.t., investigation of physical and chemical properties of potential edible and non-edible feedstocks for biodiesel production, a comparative analysis, renewable and sustainable energy reviews 21, 749– 755, (2013) [30]. bouaziz m., lassoued s., bouallagui z., smaoui s., gargoubi a., dhouib h., sayadi s., synthesis and recovery of high bioactive phenolics from table-olive brine process wastewater, bioorganic and medical chemistry 16, 9238–9246, (2008) [31]. ngassapa f.n., othman o.c., physicochemical characteristics of some locally manufactured edible vegetable oils marketed in dares salaam, tanzania journal of science 27, 4958, (2001) [32]. rudan-tasic d., klofutar c., characteristics of vegetable oils of some slovene manufacturers, acta chimica slovenica 46, 511–521, (1999) [33]. barisheva o.s., techniceskie masla v pishevoi promishlennosti – prestuplenie, pishevaia promishlennosti 11, 51-53, (2011) 58 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 58 – 66 comparison of aluminium sorption using inorganic and organic natural sorbents danka barloková1, ján ilavský1, eleonóra franková1, tomáš molnár 1 department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovakia danka.barlokova@stuba.sk, jan.ilavsky@stuba.sk, *corresponding author received 5th march 2015, accepted 29th march 2015 abstract: aluminium is the third most abundant element on the earth. most often it is tightly bound in rocks. on acidification of the environment with acid rain or civilization interventions, there is a release of aluminium in rocks bound to the soluble form of aluminum salts. the effects of acidification are in the final phase of ecosystems visibly unsafe (damage to crops, mortality of fish in lakes and rivers, etc.). the results of laboratory tests with a strain of penicillium glabrum and natural zeolit – cliniptilolit to estimate the sorption efficiency for removal of aluminum from water are presented. keywords: water treatment, removal of aluminium, penicilium glabrum, natural zeolites, drinking water 1. introduction in the earth´s crust, aluminum is the most abundant (8.3% by weight) metallic element and the third most abundant of all elements (after oxygen and silicon). because of its strong affinity to oxygen, it is almost never found in the elemental state; instead it is found in oxides or silicates. there are more than 270 different minerals containing aluminum. after acidification of the environment by acid rain or civilization interventions leads to the release of aluminum bound in rocks to form soluble aluminum salts. aluminum concentration in the waters are significantly affected by ph, at neutral ph values are generally low, in the range 1-50 μg/l and more acidic waters are rising up to 500-1000 μg/l, the concentration greater than 700 μg/l al it is toxic to fish [1]. the concentration 0.52 mg/l aluminum greatly reduces the growth of fish. in seawater, the concentration of aluminum depends on the salinity of the water, normally is around 1-2 μg/l [2]. in the open oceans are aluminum concentration of about 0.5 μg/l [3]. aluminum may be present in different forms, at a higher ph is dominated by ion al(oh)4-, whereas the lower ph prevalent toxic al3+ [4]. higher concentrations of aluminum in surface water are in a period of high water levels, storm after rain or spring snowmelt in [5]. http://www.fia.usv.ro/fiajournal mailto:danka.barlokova@stuba.sk, mailto:jan.ilavsky@stuba.sk, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 59 aluminum levels in drinking-water vary according to the levels found in the source water and whether aluminum coagulants are used during water treatment [6]. in germany, levels of aluminum in public water supplies averaged 0.01 mg/l in the western region, whereas levels in 2.7% of public supplies in the eastern region exceeded 0.2 mg/l. in a 1993–1994 survey of public water supplies in ontario, canada, 75% of all average levels were less than 0.1 mg/l, with a range of 0.04 0.85 mg/l. in a large monitoring program in 1991 in the united kingdom, concentrations in 553 samples (0.7%) exceeded 0.2 mg/l. in a survey of 186 community water supplies in the usa, median aluminum concentrations for all finished drinking-water samples ranged from 0.03 to 0.1 mg/l; for facilities using aluminum sulfate coagulation, the median level was 0.1 mg/l, with a maximum of 2.7 mg/l. in another us survey, the average aluminum concentration in treated water at facilities using al2(so4)3 coagulation ranged from 0.01 to 1.3 mg/l, with an overall average of 0.16 mg/l. despite its natural abundance, aluminum has no known function in biology. it is remarkably nontoxic, aluminum sulfate having an ld50 of 6207 mg/kg (oral, mouse), which corresponds to 500 grams for a 80 kg person [7]. despite the extremely low acute toxicity, the health effects of aluminum are of interest in view of the widespread occurrence of the element in the environment and in commerce. some toxicity can be traced to deposition in bone and the central nervous system, which is particularly increased in patients with reduced renal function. because aluminum competes with calcium for absorption, increased amounts of dietary aluminum may contribute to the reduced skeletal mineralization (osteopenia) observed in preterm infants and infants with growth retardation. in very high doses, aluminum can cause neurotoxicity, and is associated with altered function of the blood–brain barrier [8,9]. a small percentage of people are allergic to aluminum and experience contact dermatitis, digestive disorders, vomiting or other symptoms upon contact or ingestion of products containing aluminum, such as deodorants or antacids. in those without allergies, aluminum is not as toxic as heavy metals, but there is evidence of some toxicity if it is consumed in excessive amounts [10]. aluminium in food may be absorbed more than aluminium from water [11]. aluminium is presented at low concentration in foodstuff. it is being used in conservation of the food in a powder (instant coffee, milk-powder, firming agent for whipped cream, etc.). there is increased content of aluminium in the conditioner dough, tea leaves and in the drugs used against ill-digestion. higher aluminium concentrations, that may toxically affect the organisms, enter the foodstuff by abrasion or by leaching of aluminium cookware. aluminium tocixocy is an serious factor that is limitating the growth of the plants and woody plants in the acid soils. moreover, the toxicity of the aluminium is being expressed also in microorganisms and animals [12]. in a kidney function disorder diseases, the neurotoxical effects of aluminium on organism was proved. the utterances of aluminium intoxication represent the seizures, logagnosia and dementia utterances that is reffered to as alzheimer's disease. even if the opinions of the neuroendokrinologists are not completely united, they are affraid of water and food aluminium contamination may cause the emergence of alzheimer's disease, which is the degenerative kind of disease related to reduction of the brain cells and by that also to decrease of brain food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 60 functional capabilities, including the intelect. there was recorded the aluminosis occurence in germany (a severe lungs disease that is similar to silicosis). aluminosis is caused by inhalant aluminium exposure or by alumina exposure. moreover, in the presence of air the organometallic compounds of aluminium are self-igniting. the effect of aluminium organometallic compounds on the skin represents the epidermis irritation and after inhalation of these compounds the bleeding into lungs may occur. encephalopathy (brain fever), osteodistrofia and anemia (increase in white blood cells) are among intoxication, which may be caused by aluminum. for alluminium salts toxicity determination, analogous to gallium and indium, the crutial point represents anion.among the most toxic anions include cl-, no3and so4 2[13]. the regulation of the government of the slovak republic no. 269/2010 sets the limit concentration for aluminum in surface water 0.2 mg/l, limit concentration for al in streams intended for drinking water abstraction 0.2 mg/l in the category a1 (water requiring simple physical treatment and disinfection or rapid filtration and disinfection), 0.2 mg/l in the category a2 (water requiring physicalchemical treatment and disinfection, e.g. coagulation, flocculation, filtration and chlorine disinfection) and 1.0 mg/l in the category a3 (water requiring intensive physical-chemical treatment and disinfecttion, e.g. coagulation, flocculation, filtration, adsorption using active carbon, chlorine and ozone disinfection). the regulation of the government of the slovak republic no. 496/2010 establishing the requirements for water intended for human consumption and water quality monitoring sets the permissible limit concentration 0.2 mg/l for aluminium in drinking waters. this limit complies with the recommendations of the who and the council directive 98/83/eu. at the end of 80-ies were found in our deposit of natural zeolite with a high content of mineral zeolite clinoptilolite in nižný hrabovec, in eastern neovol-canites. besides this deposit, the zeolite accumulations were discovered in the complex of the central slovakian neovolcanites, in the south – western border of kremnické vrchy and in zemplínske pohorie on the territory of the slovak republic. natural zeolites were created by prolonged exposure of mineral alkaline solutions at different petrographic types of rocks of different ages at elevated temperatures. not surprisingly, therefore, that these aluminosilicates bearings are close to hot dilute mineral or volcanic craters. environment in which they were created, reflecting their structure and chemical composition. zeolites are naturally not in a pure state, but usually in conjunction with other minerals and rocks that are part of them. every deposit contains a rock of specific mineralogical composition. mineralogical and chemical analysis of zeolitic material – clinoptilolite is stated in table 1 and table 2. table 1 mineralogical analysis of zeolite from deposit nižný hrabovec [14] mineral content [%] clinoptilolite 84 cristobalite 8 feldspar 3-4 illite 4 quartz traces minerals of carbonates traces (<0.5 %) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 61 table 2 chemical analysis of clinoptilolite from deposits nižný hrabovec [14] compound content [%] sio2 66.4 al2o3 12.2 k2o 3.33 cao 3.04 fe2o3 1.45 mgo 0.56 na2o 0.29 mno 0.02 tio2 0.15 p2o5 0.02 loss by ignition 12.2 the primary structural unit of the zeolite is an octahedron, formed of silicon and aluminum atoms with oxygen atoms coordinated tetratedricky. representation of silicon and aluminum to oxygen is usually 1:2. this creates the crystal structure of relatively large cavities interconnected channels, which effectively averages range from 0.2 to 0.7 nm, and the total volume is empty space as 20 to 50% of the mineral. these cavities are stored mobile single or divalent cations of alkali metals and alkaline earth metals (ca, k, na) are surrounded by water molecules, which can be exchanged for other from the surrounding solution [15,16]. wide range of uses of natural zeolites in various sectors of the economy is determined by their structure and physicochemical properties. one of the most important properties of zeolite minerals is their catalytic properties, the ability to exchange ions and adsorb organic or inorganic molecules of certain sizes [17,18]. in the last decades the zeolitic mineral – clinoptilolite has started to be also used in drinking water treatment. zeolites are not to be found in the grained condition in the natural deposits, and before their use, zeolites require crushing and sorting at the required fractions in the waterworks engineering. sufficient mechanical strength, chemical stability and abrasion values, by which, though, they are classified among the soft filtering materials, and which permit to use the natural zeolites as a filtering material [19]. table 3 filtration materials and some of their parameters material clinoptilolite filtration sand activated carbon grain size [mm] 0.3 – 2.5 0.3 – 1.0 0.3 – 1.5 specific weight [g.cm-3] 2.39 2.66 2.19 apparent density [g.cm-3] 0.84 1.55 0.40 porosity [%] 64.8 41.7 81.7 abrasion [%] 8.2 0.57 39.08 the specific weight of clinoptilolite is lower as compared with the siliceous sand, and porosity and sludge capacity exceeds by 1.5-times the values of filtering sand. clinoptilolite in combination with siliceous sand is more effective in elimination of fe, al, nh4, etc. during slow sand filtration it enabled increasing the filtration speed up to quadruply. clinoptilolite as compared with the classical materials used in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 62 dressing and reparation engineering, has got the properties, which predestine it for utilization in the water treatment processes, and it meets the demands made upon the granular filtering materials. 2. experimental 2.1aluminium sorption using inorganic natural sorbents the model laboratory experiments we observed sorption of aluminum on two natural sorbents of inorganic and organic origin. in our experiments we tested the natural zeolite clinoptilolite, and compared the sorption capacity of the sorption capacity penicillium glabrum. adsorption was studied for different initial concentrations of the aluminum in water. a milled clinoptilotite of 1,2-1,6 mm granularity from nižný hrabovec deposite was used for alluminium sorption monitoring. the process of adsorption was discouous. the water containing alluminium (400 ml) was in a contact with adsorbent for a certain period of time (table 4, fig.1). solution of the monitored substance, co=50 mg.l-1 initial concentration, was mixed up with 100 g of clinoptilolite in a jar equipped with mixer. samples were taken away in a certain latency and the alluminium content of the samples was subsequently analysed. 2.2 aluminium sorption using organic natural sorbents three fungus tripes were used in monitoring of the alluminium sorption from water environment on a organic sorbent. penicillium glabrum is one of the worldwide species [20]. it was isolated from various substrates, from a soil, plants, cereals, flavourings, wood mass, paper, composts, texture substances, fuelling and also from natural water, drinking and waste water [21-23]. its occurence in air microflora is seasonal and this occurence was noticed also in a radioactive environment of the uran mines. the species, that were we working with it, was isolated from a moisty environment of drinking water treatment plants. during testing the growing of some micromycetes, that were isolated from treatment plants, on a cultivation media containing zinc and alluminium, we have noticed the same growing rate of p. glabrum species in both types of media non-alluminium and alluminium containing media. we inoculated this species by spore inoculation to the roux flasks with a sabouraud media (sab) in which we added the solution of alluminium sulphate that way as the final alluminium concentration in particular flasks was : 65, 125, 250, 500 and 1 000 mg.l-1. flasks were incubated at the day-light and laboratory temperature for 3 months. the growth of the penicillium glabrum in the experimental flasks with alluminium sulphate lasted initially for 7-9 days and in consideration with the control sample, this growth was slowed-down, than the growth rate and the character of the growth of the experimental samples were in equivalence with the control samples in such way, no difference in mycelium mass increase and in sporulation was noticeable. after the end of the experiment the biomass was filtered out and dried at 105°c. dried biomasses as well the cultivation media filtrates were mineralized with the same measures, in hno3 environment, at the temperature of 300°c and the pressure of 130 bar in a high-pressure decomposition system hpa-s company of anton paar (austria). 3. results and discusion the results of the alluminium sorption from water environment on the inorganic sorbent (clinoptilolite) for the initial alluminium concentration of 50 mg.l-1 are shown in the table 4 and fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 63 fig. 1 relation of alluminium concentration on the time, after the zeolite addition table 4 the removal of aluminium from water by using clinoptilolite t [min] 10 20 30 40 60 120 cm [mg/l] 32.5 13.0 8.0 6.0 4.5 3.5 η[%] 35.0 74.0 84.0 88.0 91.0 93.0 at [mg/g] 60 148 168 176 182 186 instanteneous adsorption capacity at (1) and adsorption efficiency (2) we calculated by the following formula:  0 m t c c v a = m [mg/g] (1)  0 m 0 c c 100 η = c [%] (2) where co (mg/l) represents the alluminium concentration before adsorption, cm (mg/l) represents the alluminium concentration after adsorption at the time t, v (l) represents a volume of the water solution of the polutant, m (g) represents the weight of the adsorbent, η (%) represents the adsorption efficiency, at (mg.g -1) represents the instanteneous adsorption capacity; the amount of the polutant which is adsorbed on the mass unit of the adsorbent at a time. aes –icp was used for analysis of the alluminium content in the biomass samples and in media after the mineralisation. results are shown in table 5. according to the results presented by table 5 it is obvious that considering the biomass growth (1,8-2,0 g) of the experimental samples and the growth of control samples, the growth of experimental samples was not inhibited (2,1 g) in fact. initial ph value 4.5 of the growing solution has been changed in all the samples, during the cultivation. whereas the control medium stayed at the acidic reaction ph 5.2 during and also after the experiment, the ph value of the experiment samples has increased up to the neutral positions of ph spectrum (6.9-7.8). this fact may mean an active defence of penicillium glabrum against a toxic effect of the alluminium, as it is known that in general the toxicity is being significantly decreased in neutral environment. the differencies between the concentrations of the alluminium that is being added to experimental samples from adulterated stock solution and the measured alluminium concentrations can be explained by higher sensitivity and accuracy of the measuring device and aes-icp method (in consideration with the other used and trace concentrations of the alluminium in used chemicals – were not included in added dose). we noticed an non-inhibited growth of penicillium glabrum in our laboratory experiments. this growth was noticed in a total testing range of high al2(so4)3 concentrations, see (tab.5) and in fact its complete (100%) sorption on a mycelia biomass. it is an interesting finding, in regard to the general knowledge, that alluminium is toxical when it is presented at the higher concentrations. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 64 table 5 alluminium accumulation in a biomass of penicillium glabrum samples control 1 2 3 4 5 g ro w in g m ed iu m before the inoculation ph 4.5 ± 0.1 after the cultivation ph 5.2 6.9 7.2 7.3 7.8 7.5 a l 2( s o 4) 3 added [mg.l -1] al 0 1000 500 250 125 65 measured after kultivation v [mg.l-1 ] al 0 ± 3.6 b io m as a total dry mass [g] pri 105 ºc 2.10 1.90 1.89 1.80 2.00 1.95 dose of dry mass for analysis 0.232 0.244 0.253 0.232 0.189 0.195 al [mg.l-1] measured 2.867 141.4 78.83 38.78 13.75 8.341 al [mg.l-1] adsorbed from the solution and adjusted for total biomass 4.5 1007 506 259 128 67 fig. 2 alluminium content in the biomass, method of aes analysis with inductance-coupled plasma as the driving source, after the mineralisation of the samples (1-5) in hydrogen nitrate in high-pressure decomposition system hpa-s company a. paar (austria). fig. 3. alluminium content in growing media no.1 and no.2, after the grown biomass separation, determined by aes-icp method and the consideration with the standard and blank food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 65 although many of the authors in slovakia and abroad deal with the sorbtion characteristics of filamentous micromycets, especially in terms of the case of heavy metals and cations of the toxic and radioactive elements [24,25]. 4. conclusion at this experiment work was tested two natural sorption materials of inorganic and organic origin in removal of aluminium from water. sorption capacity of natural zeolite – clinoptilolite was compared with sorption capacity of organic material penicilliumglabrum, one of the worldwide species. adsorption tests were studied for different initial concentrations of the aluminum in water. clinoptiloliteachieved 93 % efficiency after two hours of contact with water containing aluminium, penicilliumglabrum needed longer time – from seven7 to 9 days to achieve 100% efficiency. results obtained from laboratory experiments shown, that both tested materialsclinoptilolite and penicilliumglabrumeffectively removed aluminium from water. 5. references [1] who: environmental health criteria 194, aluminium. geneva, 1997 [2] becaria a., campbell a., bondy s.c. aluminium as a toxicant. toxicology and industrial health 18, 309 (2002). [3] barabasz w., albińska d., jaśkowska m., lipiec j. ecotoxicology of aluminium. j. of environ. studies 11 (3), 199 (2002). [4] totten, g. e.; mackenzie, d. s. handbook of aluminum. marcel dekker. p. 724. (2003). [5] caballero b., allen l., prenkice a. aluminium. encyklopedia of human nutrition, 2006. [6] who: aluminium in drinking-water. background document for development of who guidelines for drinking-water quality, who/sde/ wsh/03.04/53 (2003). [7] helmboldt o., hudson l.k.: aluminum compounds, inorganic. ullmann´s encyclopedia of industrial chemistry. wiley-vch, p. 527 (2007). [8] banks w.a., kastin a.j. aluminuminduced neurotoxicity: alterations in membrane function at the blood–brain barrier. neurosci biobehav rev. 13(1), 47– 53 (1989). [9] ferreira p.c, piai k.a., takayanagui a.m., seguramuñoz s.i. aluminum as a risk factor for alzheimer's disease. rev. lat. am. enfermagem 16, 151 (2008). [10] gitelman h. j. physiology of aluminum in man. in: aluminum and health, crc press, p. 90, (1988). [11] yokel r.a., hicks c.l., florence r.l. aluminum bioavailability from basic sodium aluminum phosphate, an approved food additive emulsifying agent, incorporated in cheese. food and chemical toxicology 46 (6), 2261 (2008). [12] flaten t.p., alfrey a.c., birchall j.d., savory j., yokel r.a.: status and future concerns of clinical and environmental aluminium toxicology j. toxicol. environ. health 48, 527 (1996). [13] vulterin j., štablová r. toxikológia, bezpečnosť a hygiena práce v chemii. toxicology, safety and hygiene work in chemistry. karolinum, praha (1991). [14] horváthová e. properties and elimination of ammonium ions from water with natural zeolites. (in slovak). vodní hospodářství 7, p. 173-178 (1989). [15] barloková, d., ilavský, j.: natural zeolites in water treatment (in slovak). vodní hospodářství, 57/6, 213-215 (2007). [16] šamajová e., kraus i.: prediction of areas of slovak zeolites and the possibilities of utilizing them (in slovak). in: proceeding of the conference slovzeo”84, vysoké tatry, 1984, pp. 6-10. [17] reháková m., čuvanová s., gavaľová z., rimár j. utilization of the clinoptilolite type of natural zeolite in agrochemistry and agriculture (in slovak). chem. listy 97, 260-264 (2003). [18] barloková d. natural zeolites in the water treatment proces. slovak journal of civil engineering vol. xvi, issue 2, 2008, pp. 8-12, slovak university of technology in bratislava 2008, issn 1210-3896. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 d a n ka b ar l o ko vá , j án i l avs k ý , e l eo nór a f r an k o v á, t o máš m ol ná r , c om pa r i s o n o f a l u m i n u m s o r p ti o n u s i n g i n or g a ni c a nd o rg a ni c n a t ur a l s or b e n t s , f o od and e n v ir o nme nt s af e t y, v o lu me x iv , is s ue 1 – 20 1 5, p ag . 5 8 6 6 66 [19] merkle p.b., knocke w.r, gallagher d., et al: characte-rizing filter media mineral coatings, journal of awwa, 12/1996, s.62-73. [20] samson, r.a., hoekstra, e.s., frisvald j.c., filtenborg o.: introduction to foodborne fungi. centralbureau voor schimmel-cultures baarn, ponsen and looyrn (1996). [21] tóthová, l. occurrence of microscopic fungi in the slovak section of danube river. biologia, bratislava 54/4, 379 (1999). [22] franková, e.: soil micromycetes in the water environment. život. prostr., vol. 35, no. 3, 156 (2001). [23] franková e., simonovičová a. geochemically microscopic fungi in polluted environment (in slovak). mineralia slovaca, 29, 10 (1997). [24] frank, v., támová, g., farkas, v., characterization of white mutant of trichoderma viride obtained by cadmium treatment. microbiological research 149, 61–64 (1994). [25] gadd, g. m. interaction of fungi with toxic metals. the new phytologist 124, 2560 (1993). 123 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 123 130 evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species *ana leahu 1 , cristina elena hretcanu 1 , mircea oroian 1 , sorina ropciuc 1 1 food engineering faculty, stefan cel mare university of suceava, romania analeahu@fia.usv.ro *corresponding author received 29 th march2016, accepted 17 th may 2016 abstract: this paper analyzes the total phenolic compounds and ascorbic acid in water, methanol extracts from four selected wild fruits originally from the bukovina area, namely, sea-buckthorn (hippophae rhamnoides l), cranberry bush (viburnum opulus l.), hawthorn (crataegus monogyna l.), and rosehip (rosa canina l.), used as tea beverages or as decoction for medicinal purposes. ascorbic acid was separated, identified and dosed in fruit extracts by means of high-performance liquid chromatography (hplc); the total phenolics were determined as gallic acid equivalents gae/100 g fw, by the folin-ciocalteu method. the levels of berries mean ascorbic content in the four different families ranged from 112.87 (viburnum opulus l) to 224.21 mg/kg of fresh weight (crataegus monogyna l.). the total phenolic content ranges from 4.38 mg gae/kg fw (hippophae rhamnoides l) to 11.09 mg gae/kg fw (crataegus monogyna l.). these results suggest that the crataegus monogyna l. extract is a rich source of natural antioxidants. keywords: drying, freezing, sea-buckthorn, cranberry bush, hawthorn, rosehip, ascorbic acid. 1. introduction the consumption of forest fruits has beneficial therapeutic effects such as hypotensive, anxiolytic and sedative and it reduces the risk of contaminating cardiovascular diseases, some cancers, lung diseases. furthermore it has antioxidant and immune enhancing effects. these properties are attributed to a variety of constituents, including micro and macronutrients such as flavonoids, tannins, sugars and organic acid (citric and malic acid, ascorbic acid and phenolic acids), vitamins and minerals. sea buckthorn grows in the form of a shrub. buckthorn plants are very resistant to frost and drought. the fruits are orange and have an astringent, sour, but pleasant taste. in romania sea buckthorn berry is widespread. it is harvested from spontaneous flora and has been used for medicinal and nutritional purposes. sea buckthorn's natural distribution area includes china, mongolia, india, nepal, pakistan, russia, latvia, romania, great britain, france, denmark, netherlands, germany, poland, finland, sweden, norway and canada. hippophae rhamnoideshas been further divided into eight subspecies [1]. the fruits with a rich oil content in seeds, contain fat soluble vitamins (a, k, and e), fatty acids, lipids, organic acids, amino acids, carbohydrates, vitamins c, b1, b2, folic acid, tocopherols and flavonoids, phenols, terpenes and tannins [2]. sea buckthorn berry is an excellent source of vitamin c, approximately 400 mg/100 g [3], fat soluble vitamins, antioxidants [4] and bioactive phenolic compounds that both individually and synergistically may help protect against cardiovascular diseases, mailto:analeahu@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123– 130 124 cancer, inflammation, obesity, diabetes, skin diseases, and other chronic diseases. fructose and glucose were the major sugars in sea buckthorn berry, and the dominating acids were malic and quinin acids [5]. the total anthocyanin content of sea buckthorn was negligible (0.84 mg/100 g) [6]. the orange color of fruits of sea buckthorn is also due to the large content of carotenes that are powerful antioxidants. sea buckthorn are used to prepare juices, oil, jellies, marmalade, fruit pulp, ice-cream, vitamin c tablets, liqueur, wine, alcoholic beverages, tincture or decoction. cosmetic applications for sea buckthorn include moisturizing, dandruff control and hair loss prevention lotions, anti-ageing skin creams and lotions, and sun care cosmetics [7].the fruits used for medicinal purpose are collected after cooking and can be used either fresh or dried. their drying is done naturally or artificially (50 – 60°c). fruits may be kept frozen, a process carried out in two stages (at 1-2 °c, then at 3536°c). the cranberry bush (viburnum opulus l.) grows as a bushy shrub, and in romania is found in moist woods and thickets, from lowland and hill, but also in the alpine and sub alpine areas. in autumn, the plant makes red fruit and the leaves change their color in red. the flowers are pale green with a diameter of 5-7 cm bud blooms in may-june. the fruits are red, fleshy and spherical with a diameter of 8 mm and contain one flat seed, grow in september and have a sour-bitter like flavour. the flowers, fruits and the crust of the cranberry bush contain valeric acid and tannin and is used in the pharmaceutical industry and the preparation of antispasmodic drugs and other drugs. the fruits contain: ascorbic acid, citric acid, b, c, k, p, vitamins, esters, oxalic acid, free and esterified sterols that are important to human nutrition [8]. the fruits are sometimes used as syrups, tinctures, decoct jellies and marmalade. rosehip (rosa canina l.) is a 1-3 m shrub that has spikes, a wide base and curved tip down sickle. the flowers are pink, sometimes white 2-3, located at the tip of the branches. the fruit – the globular or ovoid receptacle is red-orange or even deep red depending on the stage of ripeness [9]. rose hip fruits are rich in carotenoids which vary depending on harvesting time [10], and are considered a particularly rich source of ascorbic acid [11, 12]. the fruits contain biologically active components such as vitamin a, b complex, e, also minerals like mg, k, s, ca, fe, se, mn and si [13], polyphenols [14], amino acids, citric acid, malic acid, flavonoids, pectin, lecithin, sugars, tannins, and volatile fatty oil. carotenoids provide immunological and antimutagenic protection and prevent tumors [15]. vitamin c supplementation is known to have a protective effect against several disease conditions, most notably the common cold. it can strengthen the immune system against some infections, cardiovascular diseases and some cancers [13, 16]. hawthorn (crataegus monogyna jacq) has special qualities in treating heart disease and is known from ancient times for its therapeutic effects. it grows like bushes, especially at the border of mountain forests, low land and pastures, sometimes grown for ornamental purpose [17]. hawthorn fruit can be eaten fresh (which are available in this form) but the decoction of dried fruits can be made jam, marmalade and liquors, mixed with other herbs or added to some chicken or fish dishes. the hawthorn fruits contain vitamin c, flavonoids, tannins, tartaric acid, ursolic, citric, oxalic, nicotinic acid, chlorogenic acid, choline acetyl choline, pectin, glucose, fructose, sucrose, maltose and minerals [18, 19]. hawthorn fruits are used food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123– 130 125 to produce jams, jellies, juices, alcoholic beverages and other drinks [21]. the objective of the present study was to investigate the antioxidant activity and study the possible interdependence of a series of wild berries fruits commonly used in the folk medicine in bucovina (romania). the fruits are preserved fresh, dried and frozen and are used as teas or decoctions, for the treatment of several diseases (table 1). 2. materials and methods 2.1. solvents and reagents reagents and solvents were procured from sigma chemical. deionizer water was used throughout. 2.2. plants source and extracts preparation the berry fruits were collected at the fully ripe mature stage (late september and october) 2014 from hillsides and hedgerows in cacica forest, located in western part of the bucovina region in romania. table 1 the studied botanical species and their traditional uses plant name common name traditional uses hippophae rhamnoides l seabuckthorn cardio tonic, antiinflammatory viburnum opulus l cranberry bush asthma, antitussive, bronchial diseases and coughs crataegus monogyna l. hawthorn sedative, hypotensive, antiarhytmic, heart palpitation rosa canina l. rosehip cardiotonic,vitam inic, antioxidant, antiinflammatory the fruits were sorted visually for color (maturity), size and physical damage in the laboratory. the test samples were divided into three equal parts as follows: the first one was packed into plastic bags and was stored in a refrigerator at 4 0 c overnight; the second part of fruits was dried in the natural form and the third one was kept in a freezer at −20°c. 2.3. chemical analyses the initial moisture content of fruits was determined according to the european standard en iso 665/2000 by drying at the temperature of 103 °c. total ash composition was obtained by calcinations of 5g of sample at 600 °c for 240 min. protein content was analysed by using the kjeldahl method. total phenolics were measured in duplicate samples of each extract, using the folin-ciocalteu method: briefly 1.0 ml of the extracted sample, 10 ml of folin−ciocalteu reagent, and 9 ml 7.5% na2 co3 solution was added. the solutions were mixed in the cuvette by using a pipet and allowed to equilibrate at room temperature for 2 hours. the absorbance was recorded at 750 nm using a spectrophotometer t70 uv-vis pg instruments ltd. the results are expressed as milligrams of gallic acid equivalents (gae) per gram of fresh weight (fw) of fruit material. ascorbic acid determination was done on acid extracts of samples. extraction of ascorbic acid from samples the extracts were obtained following the protocol: 4 gram of fruit was extracted with 12 ml of acidified solutions (perchloric acid and ophosphoric acid 1%) using a ceramic mortar and a pestle. the residue was re-extracted until the extraction solvents remained colorless (the total solvent volume was 50 ml). the extract was filtered through a whatman no. 5 filter paper. the extracts were kept at -20°c until further analysis. ascorbic acid separation, identification and dosage food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123– 130 126 the ascorbic acid in the samples was separated, identified and dosed in a hplc shmadzu system coupled with uv–vis detector (dad). zorbax -c18 column (5μm, 250x4.6) was used. the column was eluted in isocratic system with a mobile phase that consisted of phosphate buffer ph = 3.5 (tfa): solution 0.02 m/l of monopotassium phosphate and orthophosphoric acid 10%, adjusted to ph = 3.5. the pump flow rate was set at 0.6 ml/min. the chromatograms were registered at 245 nm. for ascorbic acid identification standard lascorbic acid (sigma 99% standard l ascorbic acid) was used. for dosage of ascorbic acid in the samples, a calibration curve was constructed using dilutions of standard l-ascorbic acid in bidistilled water [20]. 2.4. statistical analysis statistical analysis was done using the analysis toolpak of m.s. excel 2010. two factor anova without replication was used for analysing the significant differences of ascorbic acid concentrations and total phenolics mg gae/100 g of fresh fruits, respectively air dried and frozen wild berries fruit samples. all samples were drawn from normally distributed populations having a common variance. for this statistical analysis the samples were drawn independently from each other. 3. results and discussions table 1 shows the values obtained for crude protein, ash, moisture, ascorbic acid and total phenolics content of fruit samples. macronutrients the data presented as a preliminary study on protein content of sea-buckthorn, cranberry bush rose hip and hawthorn from the romanian ethnoflora, shows these fruits were not a very rich source of proteins showing maximum values of 7.68 g/100g fw (rosa canina l.) and 6.36 g/100 g fw (crataegus monogyna jacq) (table 2). the proteins contained in fruits contribute to the regeneration of tissues. the viburnum opulus l. and hippophae rhamnoides l. fruits reach the highest values of moisture (75.66 respectively 72.34 %) compared to those of rosa canina l. and crataegus monogyna jacq. alternatively, the hippophae rhamnoides l. fruits attain the lowest values of ash 1.099%. the significance of the drying condition is mentioned in table 2. table 2 moisture (g/100 g of fresh weight), protein content (g/100 g of dry weight) and ash content of fruits used for analysis samples moisture% protein content % s.u. ash% sea-buckthorn hippophae rhamnoides l. 72.34 4.3 1.099 cranberry bush viburnum opulus l. 75.66 4.87 1.3997 rosehip rosa canina l. 46.76 7.68 1.8426 hawthorn crataegus monogyna jacq 55.49 5.36 2.5737 unal, h. guran, and kamilsacilik [18] found that in the case of hawthorn fruits dried at 50, 60 and 70 0 c air temperatures, the air temperature and blanching in hot food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123– 130 127 water had a significant effect on the moisture content of samples. in contrast to animal protein that favours the atherosclerosis and the accumulation of cholesterol in the blood plasma; vegetable protein contribute to maintaining low cholesterol levels. the impact of frozen storage and air drying on ascorbic acid content was evaluated and compared to the ascorbic acid content in fresh samples that were frozen and stored at -20 0 c for six months. ascorbic acid c level in fresh berry varies between 112.87 (cranberry bush) and 224.21 mg/100 kg fw of fruits (hawthorn), whereas for rosehip the concentration is 216.62 mg/kg of fruit and for sea-buckthorn, 222.25 mg/kg of fruit (figure 1). the total phenolics content determined by spectrophotometric methods for the mentioned fruits is depicted in the figure 2. figure1. the ascorbic acid concentrations in fresh, air dried and frozen wild berries fruit samples figure2.total phenolics variation content in fresh, air dried and frozen wild berries fruit samples frozen samples presented a slightly higher reduction in ascorbic acid content than fresh fruits. however, the difference was not statistically significant. vitamin c is a dietary nonenzymatic antioxidant required as a co-factor for many enzymes and can be obtained exogenously, as a part of a diet rich in wild berries. other reports show rosehip fruits pulp contains between 53 and 563 mg/100g fresh substances [21]. comparatively, leahu a et al., [20] found the rosehip jam is rich in vitamin c; the products analyzed ranging from 37 mg/100 g and 32.25 mg/100 g [20]. these results are in agreement with other literature data, such as: roman et al., [22] who obtained 360.22 mg/100 g frozen pulp (var. transitoria f. ramosissima, altitude 1250 m), ropciuc et al., [23] who obtained values ranging from 374.12 mg/100g and 663 mg/100g rosehip powder, and barros, lillian, et al., [24], who reported 220.24 mg/100 g dry weight in crataegus mono gyna ripened fruits. sea-buckthorn and cranberries have a great importance in 0 100 200 300 fresh dried frozen fresh dried frozen fresh dried frozen fresh dried frozen h . rh a m n o id e s l . v . o p u lu s l . r . c a n in a l . c . m o n o g y n a ja c q ascorbic acid mg/kg -200 0 200 400 600 fresh dried frozen fresh dried frozen fresh dried frozen fresh dried frozen h . rh a m n o id e s l . v . o p u lu s l . r . c a n in a l . c . m o n o g y n a ja c q total phenolics mg gae/100 g food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 – 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. – 128 strengthening human immunity; this effect have been associated with the presence of vitamin c. the poliphenols’ content varies, only the cranberry bush has higher content (figure 3). the content of polyphenols in buckthorn, hawthorn and rosehip is correlated with the content with vitamin c. all these fruits have a prophylactic and therapeutic activity against a wide range of diseases, including the inflammatory, flu, colds and infection. therefore, the overall drying process, had a negative effect on the phenolic contents of sea buckthorn, reducing with 88.51%, similar result were obtain for cranberry bush, total phenolics content is decreasing with 87.75%. on the other hand, drying process had a greater effect on total phenolics loss in rosehip (82.04%) and hawthorn (79.09%). total phenolic contents of frozen samples were found stable over 1 month of storage (figure 3). the fruits, which were stored frozen for 1 month, showed no significant difference in total phenolic content from the fresh berries. lillian barros et al., [24], also reported the phenolic were the major antioxidant components (247.03-701.65 mg gae/g 393 of extract); unripe fruits revealed the highest content in phenolics. rosu c. m. et al. [11] founded that the ascorbic acid content in rose hips decreased after four month freezing, compared with the level quantified in the fresh fruits. using two factor anova without replication we analyzed if there exist significant differences for ascorbic acid concentrations and respectively, total phenolics (mg gae/100 g) for fruits in fresh, air dried and frozen wild berries fruit samples. there are two null hypotheses: one for the rows which contain the factor a (fruit samples fresh, air dried and frozen wild berries) and the other for the columns which contain the factor b (type of fruits used for analysis: sea-buckthorn, cranberry bush, rosehip and hawthorn, respectively. we note by h0 the null hypotheses which consist in the following assumption: there is no significant difference in yield between the (population) means of the blends. the results regarding significant differences for ascorbic acid concentrations and respectively, total phenolics (mg gae/100 g) are given in table 3. table 3 analysis of variance for ascorbic acid and phenolic content in wild berries (factor b), used fresh, air dried and frozen (factor a) total ascorbic acid total phenolics mg gae/100 g source d.f. mms f-value mms f-value factor a 2 9021.098 80.17*** 52667.23 12.90** factor b 3 7048.312 62.64*** 21497.9 5.26* error 6 112.5194 4081.219 d.f. = degree of freedom; mss = mean sum square; significant at 5% level (p*), 1% (p**) and 0.1% (p***) . since the p-value (factor a samples fresh, air dried and frozen wild berries) = 4.6910 -5 < 0.001 = α, we can reject the factor a null hypothesis and we conclude (with 99.9% confidence) that there are significant differences between ascorbic acid concentrations in fresh, air dried and frozen wild berries fruit samples (table 3). also, f = 80.17 > 5.14 = f-critic we reject the null hypothesis, and conclude that ascorbic acid concentrations for fruit samples fresh, air dried and frozen wild food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 – 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123 – 130 129 berries are statistically different. since the p-value (factor b -type of fruits used for analysis) = 6.4 10 -5 < 0.001 =α, we can reject the factor b null hypothesis and we conclude (with 99.9% confidence) that there are significant differences between ascorbic acid concentrations for fruits used for analysis (table 3). also, f= 62.64 > 4.75 =f-critic we reject the null hypothesis and conclude that ascorbic acid concentrations for types of fruits used for analysis are statistically different. since the p-value (factor a samples fresh, air dried and frozen wild berries) = 0.0067 < 0.05 = α, we can reject the factor a null hypothesis and we conclude (with 95% confidence) that there are significant differences between total phenolics mg gae/100 g for fruits used for analysis in fresh, air dried and frozen wild berries fruit samples (table 4). also, f = 12.90 > 5.14 = f-critic we reject the null hypothesis, and conclude that total phenolics mg gae/100 g for fruits samples fresh, air dried and frozen wild berries are statistically different. since the p-value (factor b -type of fruits used for analysis) = 0.04< 0.05 = α, we can reject the factor b null hypothesis and we conclude (with 95% confidence) that there are significant differences between total phenolics mg gae/100 g for fruits used for analysis (table ). also, f= 5.26> 4.75 =f-critic we reject the null hypothesis and conclude that total phenolic mg gae/100 for fruits used for analysis are statistically different. 4. conclusions all analyzed fruit samples in this study represent a significant source of phenolic compounds and ascorbic components that together bring a huge benefit to the human body, fight free radicals, chronic diseases and are revitalizing strong. the results of this study indicate that the contents of ascorbic acid in hawthorn fresh fruit were higher than in cranberry bush. the ascorbic acid concentrations and the total phenolics mg gae/100 g, respectively for fruit samples (fresh, air dried and frozen wild berries) and, respectively for types of fruits (seabuckthorn, cranberry bush, rosehip, hawthorn) are statistically different and we remark that slightly higher reduction in ascorbic acid content was observed in frozen samples than in fresh fruits, but the overall drying process had a negative effect on the phenolic contents of these fruits. the cranberry bush presents higher content of polyphenols, whereas for the other studied fruits it varies. for buckthorn, hawthorn, rosehip, the content of polyphenols is proportional with the content of vitamin c. the present study revealed the high content of total phenolic content in cranberry bush (v. opulus l.) from bucovina region in romania and recommend them as excellent source of antioxidants. 5. references [1] suryakumar, g., & gupta, a., medicinal and therapeutic potential of sea buckthorn (hippo phae rhamnoides l.) .journal of ethnopharmacology, 138(2), 268-278, (2011). [2] bal, l. m., meda, v., naik, s. n., & satya, s., sea buckthorn berries: a potential source of valuable nutrients for nutraceuticals and cosmoceuticals. food research international, 44(7), 1718-1727, (2011). [3] gutzeit, d., baleanu, g., winterhalter, p., & jerz, g., vitamin c content in sea buckthorn berries (hippophae rhamnoides l. ssp. rhamnoides) and related products: a kinetic study on storage stability and the determination of processing effects. journal of food science,73(9), c615-c620, (2008). [4] papuc, c., diaconescu, c., & nicorescu, v., antioxidant activity of sea buckthorn (hippophae rhamnoides) extracts compared with common food additives. rom biotechnol lett, 13(6), 4049-4053, (2008). [5] yang, b., sugars, acids, ethyl β-dglucopyranose and a methyl inositol in sea food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 – 2016 ana leahu, cristina e. hretcanu, mircea oroian, sorina ropciuc, evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, volume xv, issue 2 – 2016, pag. 123 – 130 130 buckthorn (hippophae rhamnoides) berries. food chemistry,112(1), 89-97, (2009). [6] hosseinian, farah s., and trust beta, saskatoon and wild blueberries have higher anthocyanin contents than other manitoba berries. journal of agricultural and food chemistry 55, no. 26 (2007): 10832-10838, (2007). [7] kalia, rajwant k., rohtas singh, manoj k. rai, gyan p. mishra, sharbati r. singh, and a. k. dhawan. biotechnological interventions in sea buckthorn (hippophae l.): current status and future prospects. trees 25, no. 4: 559-575, (2011). [8] bubulica m. v., i. anghel, a. m. grumezescu, c. saviuc, g. a. anghel, m. c. chifiriuc, i. gheorghe, v. lazar, and a. popescu. in vitro evaluation of bactericidal and antibiofilm activity of lonicerata tarica and viburnum opulus plant extracts on staphylococcus strains. farmacia 60, no. 1: 80-91, (2012). [9] sorina, r., iuliana, c., ana, l., & giancarla, v., biometric and chemical characteristics of the species rosa canina l. used as a natural and functional food. in 48. hrvatskii 8.međunarodni simpozij agronoma, dubrovnik, hrvatska, 17.-22. veljač 2013.zbornik radova. poljoprivredni fakultet sveučilište josipa jurja strossmayera u osijeku (pp. 425-429), (2013). [10] andersson, staffan c., k. rumpunen, e. johansson, and m. e. olsson. carotenoid content and composition in rose hips (rosa spp.) during ripening, determination of suitable maturity marker and implications for health promoting food products. food chemistry 128, no. 3: 689-696, (2011). [11] rosu, c. m., olteanu, z., truta, e., ciornea, e., manzu, c., &zamfirache, m. m., nutritional value of rosa spp. l. and cornusmas l. fruits, as affected by storage conditions. analelestiintifice ale universitatii" alexandru ioan cuza" din iasi sec. ii a. genetica si biologie moleculara, 12(4), 147-155 (2011). [12] iuliana, c., sorina, r., & leahu, a., evaluation of rosehip fruit productivity and total acidity in response to climatic factors. romanian biotechnological letters, 18(4), 8403-8412, (2013). [13] patel seema, rose hips as complementary and alternative medicine: overview of the present status and prospects." mediterranean journal of nutrition and metabolism 6, no. 2 (2013): 89-97, (2013). [14] kilicgun h. and dehen a., correlation between antioxidant effect mechanisms and polyphenol content of rosa canina. pharmacognosy magazine 6, no. 23 (2010): 238, (2010). [15] machmudah s., yukari k., mitsuru s., and motonobu g., process optimization and extraction rate analysis of carotenoids extraction from rosehip fruit using supercritical co2.the journal of supercritical fluids 44, no. 3 (2008): 308-314, (2008). [16] cunja v., m. mikulic-petkovsek, a. zupan, f. stampar, and v. schmitzer, frost decreases content of sugars, ascorbic acid and some quercetin glycosides but stimulates selected carotenes in rosa canina hips. journal of plant physiology 178 (2015): 55-63, (2015). [17] braşovan, a., campean, r., mandroc, v., codrea, v., & olah, n. (2012). determination of calcium, magnesium and polyphenols in hawthorn fruits from vulcan coal dump. studia universitatis babes-bolyai, chemia, 57(2) 2012, p. 71 81, (2012). [18] unal h. guran, and kamil sacilik. drying characteristics of hawthorn fruits in a convective hot‐air dryer. journal of food processing and preservation 35, no. 2 (2011): 272279, (2011). [19] nechifor, ghe., m. pascu, d. e. pascu, g. a. trăistaru, and v. i. panait. comparative determinations for certain phytoterapeutical substances from indigenous herbs, u.p.b. sci. bull., series b, vol. 76, iss. 1, (2014). [20] a. leahu, c. damian, m. oroian, s. ropciuc, r. rotaru. influence of processing on vitamin c content of rosehip fruits. scientific papers animal science and biotechnologies 47, no. 1 (2014): 116-120, (2014). [21] soare, r., bonea, d., iancu, p., &niculescu, m., biochemical and technological properties of rosa canina l. fruits from spontaneous flora of oltenia, romania. bulletin of university of agricultural sciences and veterinary medicine cluj-napoca. horticulture, 72(1), (2015). [22] roman i., a. stănilă, and s. stănilă. bioactive compounds and antioxidant activity. chemistry central journal, 7:73, (2013). [23] ropciuc, s., tănase, i., crețescu, i., & velicevici, g. the variation of the acid ascorbic content in cynosbati fructus influenced by stationary factors, food and environment safety, volume x, issue no.4 (2011). [24] barros, l., a. m. carvalho, and i. cfr ferreira. comparing the composition and bioactivity of crataegus monogyna flowers and fruits used in folk medicine. phytochemical analysis 22, no. 2 (2011): 181-188, (2011). 84 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 84-88 influence of hydrocolloids in oil-in-water emulsions during storage of food *oksana lugovska1, vasilij sidor1 1national university of food technologies, volodimirska 68, kiev, ukraine, oksana.lugovska320@gmail.com *corresponding author received 7th march 2015, accepted 31st march 2015 abstract: this paper describes the physico-chemical principles underlying the functional role of hydrocolloids in oil-in-water emulsions. basic terms such as emulsifier and stabilizer are defined, and the origins of electrostatic and steric interaction potentials are explained. the paper discusses the main factors controlling flocculation, creaming, coalescence and ostwald ripening, distinguishing between the differing effects of adsorbing and non adsorbing hydrocolloids. the attention is specifically paid to new understanding concerning the rheological and microstructural control of emulsion stability by non-adsorbing hydrocolloids, and the great potential of electrostatic protein-polysaccharide interactions at the oil-water interface to enhance emulsion properties. keywords: emulsion stability, depletion flocculation, rheology and microstructure, hydrocolloid emulsifiers 1. introduction an emulsion is a dispersion of one liquid (the dispersed phase) as small spherical droplets in another immiscible liquid (the continuous phase). the two main types of emulsions are oil-in-water (o/w) and water-in-oil (w/o). food emulsions of the oil-in-water type include, for example, milk, cream, salad dressings, sauces and beverages; examples of the water-in-oil type are butter and margarine. the oil phase is either a flavor oil (in soft drinks) or triglyceride oil (in dairy emulsions). in the latter case the oil phase is semi-solid at ambient temperatures due to the presence of dispersed fat crystals. some emulsion products also contain other kinds of dispersed phases gas bubbles (in whipped cream), starch granules (in cake batter) and ice crystals (in ice-cream). despite the wide diversity of structures, compositions and textures of food emulsions, there are some basic underlying principles that can be reliably used to understand and predict their general behavior. one generally important factor influencing their stability properties is the presence of hydrocolloids [1]. emulsions are thermodynamically unstable. with time they tend to break down into their constituent oil and aqueous phases. the term ‘emulsion stability' therefore refers to the ability of an emulsion to resist this breakdown, as indicated by growth in average size of droplets or change in their spatial distribution within the sample. the more slowly that these properties change, the more stable is the emulsion. in practice, stability is a relative term which depends on the context. for some food emulsions, http://www.fia.usv.ro/fiajournal mailto:oksana.lugovska320@gmail.com food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 oksana lugovska, vasilij sidor, influence of hydrocolloids in oil-in-water emulsions during storage of food, food and environment safety, volume xiv, issue 1, pag. 84-88 85 such as cake batters or cooked sauces, the required time-scale for stability is only a few minutes or hours. but for other products, such as soft drinks and cream liqueurs, emulsion stability must be maintained over a period of several months or years [2]. a hydrocolloid ingredient may act as an emulsifying agent, as a stabilizing agent, or in both of these roles. an emulsifying agent (emulsifier) is a surfaceactive ingredient which adsorbs at the newly formed oil-water interface during emulsion preparation, and it protects the newly formed droplets against immediate coalescence. given that polysaccharides are predominantly hydrophilic in molecular character, and most hydrocolloids are not surface-active, they cannot act as primary emulsifying agents. there is really only one hydrocolloid namely, gum arabic which is commonly employed as an emulsifying agent. the main emulsifying agents used in food processing arc the proteins, especially those derived from milk or eggs. a stabilizing agent (stabilizer) is an ingredient that confers long-term stability on an emulsion, possibly by a mechanism involving adsorption, but not necessarily so. in o/w emulsions, the stabilizing action of hydrocolloids, such as xanthan, carboxymethycellose, carrageenan etc., is traditionally attributed to the structuring, thickening and gelation of the aqueous continuous phase [3]. the expression ‘emulsifying agent' is to be preferred over the more concise ‘emulsifier’. this is because the latter term normally implies membership of the class of small-molecule surfactants, comprising lipid-based ingredients such as monoglycerides, phospho-lipids (lecithin) and polysorbates (tweens). the functional role of these smallmolecule emulsifiers in food technology is typically not for emulsion making, but for other reasons: controlling fat morphology and crystallization; promoting shelf-life through interaction with starch; and destabilizing emulsions by competitive protein displacement from the oil-water interface [4, 6]. a stable emulsion is one where the droplets remain sufficiently small and well separated that brownian motion alone keeps them evenly dispersed throughout the continuous phase. the physic-chemical principles of o/w emulsion stability arc based on the classical colloid theories of electrostatic and steric stabilization. electrostatic stabilization arises from the presence of electrical charge on the surface of the droplets, or more usually on the adsorbed stabilizer layer at the surface of the droplets. the greater the charge density at the surface, and the lower the ionic strength (electrolyte concentration) of the continuous phase, the more stable is the emulsion. steric stabilization arises from the presence of a polymeric (steric) barrier at the droplet surface. to confer long-term stabilization, this polymer must be present at sufficient concentration to cover the oilwater interface completely, and it must remain permanently attached to the surface, with at least part of the molecule projecting away from the surface into the aqueous medium. steric stabilization is increasingly supplemented by electrostatic stabilization in emulsions containing adsorbed proteins at ph values well away from the protein’s isoelectric point (p)[7]. whether emulsion droplets will remain dispersed or will tend to stick together depends on the nature of the antiparticle pair potential between the droplet surfaces. generally speaking, colloidal stability requires that the antiparticle repulsion should be of sufficient range and strength to overcome the combined effects of gravity, convection. brownian motion and the ubiquitous short-range attractive forces together drive the system towards its final food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 oksana lugovska, vasilij sidor, influence of hydrocolloids in oil-in-water emulsions during storage of food, food and environment safety, volume xiv, issue 1, pag. 84-88 86 and inevitable phase-separated equilibrium condition. figure 1 shows two possible forms of the interaction potential (measured in units of the thermal energy, kt) as a function of the surface-to-surface separation (measured in arbitrary units of the order of nanometers). the shape of the potential u(d) at any separation d tells us about the nature and strength of the interaction force between the surfaces. that is. the force f is equal to the (negative) derivative of the potential, i.e., f = —6u(d)/<ld. potential a in fig. 2.2 corresponds to a stable system where the interaction force is repulsive or zero (f> 0) at all droplet separations. potential b corresponds to a more complicated situation in which the interaction force is strongly attractive at close separations (f < 0, d < 5), repulsive at medium separations (f> 0), and weakly attractive or zero at larger separations (f< 0, d> 12). in this latter case, the system is stable with respect to coagulation (i.e., coalescence of emulsion droplets) but unstable with respect to reversible flocculation. in any particular emulsion, what determines whether the potential u(d) is of type a or type b, or if it has some other functional form, is the delicate balance between the various types of molecular forces contributing to the overall interaction. fig. 1 theoretical interaction potential between a pair of spherical emulsion droplets. the energy u(d) (in units of kt) is plotted as a function of surface-to-surface separation d (arbitrary units). curve a corresponds to a pure repulsive interaction. curve b corresponds to a dlvo-type potential having a potential energy barrier more than sufficient to confer colloidal stability (pmin = primary minimum. pmiax = primary maximum. s min = secondary minimum). the aim is to study particle size effects on the stability of emulsions during storage and use in the manufacture of beverages and their stability during 180 days [5, 8]. 2. material and methods as materials for research are prepared samples of drinks with emulsions (o/w) orange, lemon, peach, grapefruit in which use stabilizers (gum arabic, modified starch). when consistency and stability of the emulsion in the beverage were verified by preparing drinks and placing them in direct sunlight, for at least 72 hours, an oily ring is formed. then this emulsion is homogenized again for particles to 1 micron. drink this emulsion based not put on stability during storage as oily ring will increase. if within 72 hours oily ring is formed in drinks, such drinks are investigated for stability following manner. forstudiesprepared samples of drinks with emulsions (o/w): non-alcoholic beverages orange (1), lemon (2), peach (3), grapefruit (4)(table 1) alcoholic beveragesorange (5), lemon (6), peach (7), grapefruit(8) (table 2). preparation of drinks preparation of the sugar syrup. a. weigh the required amount of water into a glass and heated to 50 -80° c. b. attach the required amount of sugar and completely dissolve. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 oksana lugovska, vasilij sidor, influence of hydrocolloids in oil-in-water emulsions during storage of food, food and environment safety, volume xiv, issue 1, pag. 84-88 87 dissolving citric acid and sodium benzoate in water (t=20 ºc). attach the required amount of sodium benzoate and completely dissolve. add citric acid and dissolve completely. attach the required amount of emulsion and completely dissolve. weigh the required amount of ingredients of the drinks add in stirrer, mix at room temperature until ingredients completely dissolved in water with co2. table 1 formulations of non-alcoholic beverages orange (1), lemon (2), peach(3), grapefruit (4) the ingredients of the drinks content. g/100g number of drinks 1 2 3 4 emulsion 1 1 1 1 sugar 100 100 100 100 juice bx 70% vodka 40% 3.5 3.5 3.5 3.5 citric acid (e330) 0.15 0.15 0.15 0.15 water 895.4 895.4 895.4 895.4 total 1000 1000 1000 1000 table 2 formulations of alcoholic beverages orange (5), lemon (6), peach (7), grape fruit(8) the ingredients of the drinks content. g/100g number of drinks 5 6 7 8 emulsion 2 2 2 2 ugar 180 180 180 180 juice bx 70% 23 23 23 23 vodka 40% 450 450 450 450 citric acid (e330) 2.5 2.5 2.5 2.5 water 342.5 342.5 342.5 342.5 total 1000 1000 1000 1000 for alcoholic beverages add required amount of vodka 40%.measure density, рн of the drinks. the results of measurement of each emulsion: density lab density meter, рнlab рн meter. 3. results and discussion figure 2 illustrates the four main kinds of instability processes exhibited by o/w emulsions: creaming, flocculation, coalescence and ostwald ripening. (for w/o emulsions, the processes are the same, except that sedimentation replaces creaming). flocculation is probably the most subtle and complicated phenomenon to control, because it can be triggered by so many different factors, and the resulting emulsion properties can be quite different depending upon whether the flocculation is weak or strong. in dairy-type o/w emulsions, at or below ambient temperature, instead of the full coalescence of liquid droplets, we have so-called ‘partial coalescence’ (‘clumping’) of semicrystalline globules. in practice, two or more of the phenomena shown in fig. 1 may happen at the same time, and the presence of one mechanism (flocculation) may trigger or enhance another (creaming or partial coalescence). emulsion phase inversion, as in the shear-induced transformation of cream (o/w) into butter (w/o), is a multi-mechanism process. if the particle size is less than 1 micron, the emulsion is highly robust stability and gives some turbidity but less than 1 micron particle size, the less turbidity, if the particle size is not greater than 0.3 micron. the principle of leverage ratio of water and oil phase of emulsions with different stabilizers is the same. in the obtained parameters also affects the nature of emulsions stabilizer. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceava volume xiv, issue 1 2015 oksana lugovska, vasilij sidor, influence of hydrocolloids in oil-in-water emulsions during storage of food, food and environment safety, volume xiv, issue 1, pag. 84-88 88 fig. 2 schematic representation of the key mechanisms of o/w emulsion instability: (a) stable dispersion of droplets; (b) creaming; (c) flocculation (weak); (d) flocculation (strong); (e) coalescence; (f) ostwald ripening. investigation of the stability of emulsions was carried out by determining the size of the diameter of the particles by laser granulometry and placement on the stability of soft drink, which was used emulsion for 180 days. during storage of beverages prepared from emulsions studied, there was no formation of oil ring or “creaming” bottled, indicating the stability of emulsion systems. 4. conclusion the use of aromatic emulsions in the manufacture of soft drinks has several benefits, including: reduced duration blending, as there is no need to pick up the flavor, color and taste. the process of storing drinks not observed the emergence of oil rings on the surface of the drink, there was no change in color, taste and aroma of the drink. complete the emulsion must have the following parameters: the size of the oil particles (up to a micron); organoleptic appearance, color, smell (aroma) and taste according to recipes; density (1,0301,100), g/cm3; ph (3, 3 ± 0, 7); stability in the drinks. the best result of research in emulsions is to obtain the maximum number of particles of about 1 micron. creating a stable emulsion system is a pressing issue in the food industry, so these studies are useful and important for the development of new food products. 5. references [1]. borisenko o.v., alekseev j.a., klimov s., methods of creating highly concentrated flavoring emulsions for soft drinks, food ingredients. raw materials and additives, 2, 18-19, (2002). [2]. phillips g.о., williams p.a., (eds.) handbook ofhydrocolloids. cambridge: wood head publishing, 156 с, (2000). [3]. mckenna в.m. (ed.), texture in food — vol. 1: semi-solid foods. — cambridge: woodhead publishing, 480 с, (2003). [4]. bogach a., aromatic emulsion for manufacture of soft drinks,food & drinks. food & beverage, 4 s. 10-11, (2003). [5]. stephen p., u.s. patent for invention № 6576285, cholesterol lowering beverage, bader, fowler, 10.06.2003. [6]. imesona. (ed.), thickening and gelling agents for food. 2nded., london: blackie academic and professional, 408 с, (1999). [7]. whistlerr.l.b., millerj. n., paschall e.f., (eds.), starch chemistry and technology. 2nded. orlando, fl: academic press, 1984, 3508. galliard t. (ed.) starch; properties and potential /society of chemical industry. chichester, uk: john wiley and sons, 210 с (1987). [8]. atwellw.a. thomas d.j. starches. — st. paul, mn: american associationof cereal chemists,150с.(1997). 369 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 369 375 portable equipment for advanced characterization of food texture first part equipment georg gutt1*, sonia amariei1, mircea adrian oroian1, cristina elena hretcanu1 1faculty of food engineering, stefan cel mare university of suceava, romania g.gutt@fia.usv.ro * corresponding author, received november 8th 2013, accepted december 15th 2013 abstract: the main objective of the paper is the harmonization of instrumental testings of food texture with methods of human sensory analysis as they are defined and described in reference standard iso 11036. lack of dual standardized system of advanced food texture characterization in admission of instrumental methods and means besides human sensory methods of characterization and the impossibility of numerical quantifying, with high-resolution of human perceptions, subjective assessment of texture characteristics and low reproducibility of testing, makes human sensory tests remain far behind those of texture establishing. the paper presents a concept of multifunctional electronic equipment, with force and displacement sensors and complex devices for an advanced characterization of food texture. key words: texturometer, modular electronic device, force sensor, displacement sensor 1. introduction iso 11036 standard [1] and iso 5492, [2] define texture of solid and semisolid food as all the mechanical, geometrical and surface properties of product, perceptible by touch or mechanic receptors, visual and auditory, where it is appropriate. according to this reference standard properties that can be received tactile or by mechanical receptors are reactions of tested material to a certain type of mechanical load being divided into primary characteristics such as: hardness, cohesion, viscosity, elasticity and adhesiveness and secondary characteristics such as fracturability, chewiness, gumminess. currently, in establishing the texture of raw material or finished food product are contributing more the objective interpretation, automated of their response to various mechanical loads exerted on them by instrumental way than subjective interpretation, human sensorial of their response to mechanical tests, [3],[4],[5],[6]. using advanced instrumental analysis of food texture conclusions can be drawn on elastic, plastic, visco-plastic behavior, tensile strength, state crunchy, brittle state, pasty state, state creamy gelatinous state, state of grain, condition of fiber, toughness, extrusion capacity, thereof, [7],[8],[9],[10],[13]. also by the texture instrumentally measured, it can be appreciated fruit ripeness, deadline for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 370 storage of fruits and vegetables, level of cheeses [12] and meat products maturation, level of dryness, doneness of bakery and pastry. 2. materials and methods the current situation regarding the establishment of texture and textural state characterization of solid and semisolid foods 2.1 an example of establishing texture on the way of human sensory currently, for food texture characterization based on primary and secondary parameters are used by assessors, in most cases, reference scales described in detail by iso 11036, [1]. the disadvantage of this system of characterization consists of its subjectivity and in extreme low resolution quantification of intermediate state between two graduations of the assessment panel. these two drawbacks are added to the impossibility of acquisition and automatic data processing. for example: annex a1 of reference standard iso 11036/2007 provides for the assessment of food hardness a scale of increasing hardness with numbers from 1 to 9 for as many specific foods. on this scale, the hardness value of 1 is assigned to cream cheese and 9 value to candies. between these extremes are foods such as egg white with value 2, cheese with value 4, nuts value 6, etc. the situation is somewhat similar to the beginning of the last century when it was used to estimate the hardness of materials a hardness scale of minerals after mohs with 10 units, hardness value 1 is assigned to talc and value 10 to diamond. currently, there are a variety of means for advanced hardness testing of metallic and nonmetallic materials and accurate numerical expression, high resolution, of this property. probably a close route will be covered and by the instrumental equipment of measurement and quantification the structural parameter of hardness characterizing the food texture. example above only refers to one of the five primary texture parameters, namely hardness. for all other primary parameters situation of appreciation on human sensory way of texture is similar in terms of subjectivity and reproducibility of the assessment of food hardness. 2.2 establishment of texture on instrumental sensory way characterization of texture by mechanical instrumental testings and their automated interpretation of electronic sensory response is achieved at present by two types of equipment. the firsts are mechanical testing machines, loading motorized with small task, equipped with force sensors and displacement sensors, texture of food being defined by different characteristic sizes that are determined or calculated from characteristic curves of type force displacement obtained by electronic processing of values supplied by the force sensor and the displacement sensor. in the market, this type of equipments are actually classic machines for materials testing with low loads, from current production machinery of manufacturing companies for testing materials, equipped with various specific devices such as: platters, pliers, knives, penetraters, hooks for covering a diverse and specific issue of food texture determination. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 371 with these testing machines and adaptation of specific devices it provides a wide range of mechanical tests such as: compression, tensile, crushing, bending, shearing, twisting and penetration. automatic interpretation of the response of the two sensors allows a quite advanced texture characterization of a large raw materials and food products variety under different conditions. the second type of equipment is represented by automatic systems that determine visco-elastic or visco-plastic properties by measurement of parameters such as: resistive shear modulus, rotary element speed variation of a specific geometry immersed in the food investigated, changing of electric current power supply for a motor that drives a rotatable element of specific geometry submerged in the tested food. 3. results and discussion portable universal equipment for mechanical testing of food and electronic sensorial measuring of its texture response the equipment described above are laboratory equipment with complicated structure and kinematics, they also means bringing food to device and not device to food. these facts are also the main disadvantage of these equipments for testing and characterization food texture. another disadvantage is the cost of the laboratory equipment for food texture analysis, price being situated in the tens of thousands euro / unit. the problem solved by the equipment described in the paper is to provide a portable and universal electronic structure which allows a wide range of mechanical tests such as: compression, bending, shearing, penetration, carried out on raw materials in the field of vegetables and fruits, meat and sausages, hard cheese, bakery and pastries products, all these tests using to the definition of food texture and to obtain numerical values expressing resistance of food tested to different types of mechanical tests. also, using the equipment described by authors solve the problem of interpretation, harmonization and correlation of resistance characteristics of foods tested with international standard reference on human sensorial analysis of food texture. on the portable test equipment conceived, designed and built by team of authors represented in figure 1, figure 2 and figure 3, can be fitted by simple operations, in a few seconds, six different devices used for determining food texture according to iso 11.036/2007 [1] but also for advanced characterization of food behavior to other types of mechanical testing than those stipulated in the standard. these devices will be presented in the second part of the paper entitled: specific devices from a functional viewpoint the portable device has a kinematic of loading and unloading carried out with a manual drive screw nut and two parallel jaws that at nut rotation towards the right mechanically load the test food and at nut rotation to the left download it mechanically, lower mobile jaw of the device being equipped with an electronic displacement sensor, and fixed upper jaw with a power electronic sensor. food texture assessment is done by interpreting the values that compose forcedisplacement characteristic curves or force-penetration depth curves obtained by mechanical testings of food with equipment designed by the authors and equipped with specific devices. the results are automatically displayed in a calculation unit which is connected with electronic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 372 unit measuring of force applied and measurement of food deformation or appropriate of depth penetration of a penetrator in it. figure 1. cross-section on side view of the portable device for determining the texture of food and advanced characterization of their behavior to the mechanical testings. 1-screw, 2-nut, 3-liner rubber, 4lower jaw, 5,6,7,8-screws, 9-displacement sensor, 10-upper jaw, 11-electronic dynamometric cell, 14-nut, 15-spherical rubber body, 16,17-knurled screws, c-guiding channel, o1. o2-holes, [11] constructively portable device consists of a screw 1 provided with a guide channel c, a nut 2, a rubber jacket 3, a lower jaw 4 provided with four screws 5,6,7 and 8, two holes o1 and o2 and an incremental position sensor 9, upper jaw 10, an electronic dynamometric cell 11, two guide screws 12 and 13, a nut 14 on which is molded a spherical body rubber 15, two knurled screws 16 and 17 for mounting specific test devices, two knurled screws 18 and 19 for mounting on a metal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 373 stand 20 provided with four rubber supports 21, two electrical connectors 22 and 23, an electronic unit 24, and a computing unit 25. figure 2. side view of apparatus for determining food texture and advanced characterization of their behavior to mechanical tests and laboratory version with supporting apparatus stand: 1-screw, 2-nut, 3liner rubber, 4-lower jaw, 8.12-guide screws 9-displacement sensor, 10-upper jaw, 11-electronic dynamometric cell, 14-nut, 15-spherical body rubber, 16,17,18,19knurled screws, 20-metallic stand, 21rubber supports, 22.23-electrical connectors, [11] on the two jaws of equipment can be fitted by a simple operation of clamping with a knurled nut different testing specific devices for determining a wide range of texture characteristics such as: elasticity, adhesion, fracturability, mastication and a number of mechanical characteristics such as compressive strength, resistance to crushing, bending behavior and resistance to bending, shear resistance, viscoelastic behavior, visco-plastic behavior and resistance to extrusion, characteristics whose values are important in the processing, transportation, storage and sale of food. to work rapidly in series the device according to the invention can be food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 374 easily vertically mounted with two screws on a solid metallic table stand. figure 3. schematic diagram and kinematic of device for determining food texture and advanced characterization of their mechanical behavior. 1-screw, 2-nut, 4-lower jaw, 9-displacement sensor, 10upper jaw, 11-electronic dynamometric cell, 24-electronic unit, 25-computing unit, [11] on the two jaws of the device can be mounted by a simple operation of clamping with a knurled nut various specific devices, for determining a wide range of texture characteristics. 4. conclusions texture tests on instrumental way and automatic interpretation of solid and semisolid food response to specific mechanical stress is now a performant means of objective interpreting with high resolution and reproducibility. using a portable electronic device of the type described in the paper allows in situ texture tests for a wide range of raw materials and foods allowing the determination and interpretation of primary and secondary parameters of texture. 5. references [1]. sr iso 11036 2007 analiza senzorială. metodologie. profil de textură [2]. iso 5492 2008 senzory analysis vocabulary [3]. iso 6658 -1985 senzory analysis methodology general guidance [4]. kealy t., application of liquid and solid rheological technologies to the textural characterisation of semi-solid foods, food research international 39 (2006), p. 265 276 [5]. jamesa b., younga a., smitha b., , kimb e., wilson a., morgenstern m.,p., texture changes in bolus to the “point of swallow” fracture toughness and back extrusion to test start and end points, procedia food science 1 (2011), p. 632 – 639 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 georg gutt, sonia amariei, mircea adrian oroian, cristina elena hretcanu, portable equipment for advanced characterization of food texture. first part – equipment, volume xii, issue 4 – 2013, pag. 369 – 375 375 [6]. herrero a.,m., ordonez j.a., avila r., et. al., breaking strength of dry fermented sausages and their correlation with texture profile analysis (tpa) and physico-chemical characteristics, meat science 77 (2007), p. 331 338 [7]. sasaki k., motoyama m., yasuda j., et. al., beef texture characterization using internationally established texture vocabularies in iso5492:1992: differences among four different end-point temperatures in three muscles of holstein steers, meat science 86 (2010), p.422 429 [8]. funami t., next target for food hydrocolloid studies: texture design of foods using hydrocolloid technology, food hydrocolloids 25 (2011), p. 1904 1914 [9]. sayaka i., satomi n., et. al., compression test of food gels on artificial tongue and its comparison with human test, journal of texture studies , 44, (2013), p. 104 -114 [10]. xianzong x., daquan l., et. al., jaw movement patterns during incisor penetration on different textural foods, journal of texture studies , 44, (2013), p. 124 -131 [11]. amariei s., gutt g., hretcanu c.e., oroian m.a., apparatus and devices for determination of food texture and advanced characterization of its behavior upon mechanical strain. romanian patent ro129025/2013 [12]. amariei s., gutt g., apparatus and process for determining the texture and the ripening degree of hard cheese, romanian patent ro129115/2013 [13]. gutt g., method and apparatus for determining cutting behavior of raw materials and food, patent proposal nr. a01018/2012 349 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 349 353 antioxidant activity and total phenolic content in allium ursinum and ranunculus ficaria gheorghe-florin bârlă1,2, *maria poroch seriţan2, elena sănduleac (tudosi)2, simona elena ciornei (ştefăroi)2 1old dominion university, department of civil engineering, norfolk, virginia, usa 23529-0241 2“ştefan cel mare” university of suceava, faculty of food engineering, romania, 13th university street, 720229, suceava – romania, fbarla@odu.edu, mariap@fia.usv.ro *corresponding author received november 17th 2014, accepted december 9th 2014 abstract: allium ursinum and ranunculus ficaria, some perennial herbs that are consumed in domestic regions in romania especially during springtime, are considered efficient spring tonic, giving a boost to our immune system. ethanol extracts from their leaves were tested in vitro in order to evaluate their antioxidant potential by 2, 2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging assay and the total phenolic content was also determined. a. ursinum leaves extract exhibit antioxidant activity over 77% with an ec50 value of 322 g/ml while r. ficaria leaves extract exhibit antioxidant activity over 80% with an ec50 value of 88.6 g/ml respectively, however higher than that of epigallocatechingallate (ec50 value of 1.2 g/ml) and gallic acid (ec50 value of 0.8 g/ml) which were used as positive control in this experiment. the amount of total phenolic content was 1.420.09 g gae/100g extract in the case of a. ursinum leaves extract and 1.790.11 g gae/100g extract in the case of r. ficaria leaves extract. these results indicate that a. ursinum and r. ficaria possess an important potential to be used as functional food ingredient or nutraceutical. keywords: antioxidant activity, total polyphenol, allium ursinum, ranunculus ficaria 1. introduction many antioxidants compounds are vital substances, which possess antiinflammatory, antiathero-screlotic, antiploriferative, antitumor, antimutagenic, anticarcinogenic, antibacterial or antiviral activities to a grater or lesser extent [12]. there is an increasing interest in the efficacy and use of naturally derived antioxidants, therefore functional foods and nutraceuticals has received much attention in the recent years. polyphenols present in medicinal and dietary plants possess an ideal chemical structure for free radical scavenging activity some have been shown to be more effective than tocopherols and ascorbate when tested in vivo. polyphenols possess a high reactivity as hydrogen or electron donors, and from the ability of the polyphenol derived radical to stabilize and delocalize the unpaired electron and from their ability chelate transition metal ions [34]. dietary antioxidants protect against free radicals such as reactive oxygen species, in the human body, which are continuously produced in vivo results in cell death and tissues damage [5]. also, those reactive oxygen species can damage dna, which causes mutations and chromosomal damage, the production of excessive free radicals stimulate the oxidative damage and such situation led to more than one hundred disorders in humans including atherosclerosis, coronary heart disease, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 g h e o r g h e f l o r i n b â r l ă , m a r i a p o r o c h s e r i ţ a n , e l e n a s ă n d u l e a c ( t u d o s i ) , s i m o n a e l e n a c i o r n e i ( ş t e f ăr o i ) , an t i o x i d a n t a c t i v i t y a n d t o ta l p h en o l i c c o n t en t i n al l i u m ur s i n u m a n d ra n u n c u l u s fi c a r i a , volume xiii, issue 4 – 2014, pag. 349 – 353 350 neurodegenerative disorders, cancer and they also play a major role in aging process [6]. due to detection of many bioactive compounds in food with possible antioxidant activity, there has been increased interest in the relationship between antioxidants and disease risks [7]. therefore, the present study was undertaken to evaluate the antioxidant capacity of leaves extracts of a. ursinum and r. ficaria, some perennial herbs as compared with some compounds with high antioxidant capacity, and the total phenolic content in these extracts was measured. allium ursinum l., known also as wild garlic or bear’s garlic is a wild growing allium species found in the forests of europeas well as in romania. the species belong to allium family have been reported, to be used as a remedy for the prevention and treatment of certain diseases [8]. a. ursinum has not yet been cultivated and it did not gained any particular importance. the fresh leaves or dried herb is used in local cuisines especially in eastern europe. the leaves are edible and can be used as a salad or as spice, or they can be boiled as vegetable. the bear’s garlic is also a common “wild” vegetable in ukraine and russia. it is sold on local markets as fresh, pickled, or salted and is becoming increasingly popular in the czech republic and germany [9]. a. ursinum is widely used as spice and traditional folk medicine recommends the use of bear’s garlic asan antiscorbutic, fever-fighting, also recommended in problems with intestines. in medieval medicine, the leaves of a. ursinum were used as a therapy for cardiovascular diseases [10]. a “in vitro” cardioprotective action of a. ursinum was first described back in 1993 [11]. it has been reported that wild garlic has a greater effect on lowering the blood pressure of rats than regular garlic [12]. several biological activities of a. ursinum plants, such as antioxidative, [13] cytostatic, and antimicrobial, [14] and antifungal properties [15], were also reported. the potential health benefits of bear’s garlic have been attributed mainly to the sulfur-containing compounds. high amounts of volatile compounds, such as sulfides and disulfides, which had been identified in bear’s garlic, have a direct impact on the quality of a. ursinum as a medicinal plant and as a spice [16]. recently, xu et al., confirmed that a. ursinum water extract can inhibit proliferation and induce apoptosis in ags (gastric cancer cell line) cells, indicating that in most of the investigated cases, diallyl sulfites are responsible for this effect [17]. because of the content of allin, allicin, and other sulfuric compounds, the plant possesses parasite-killing, fungicidal, and antibacterial properties. other components, such as lectins and flavonoids, have been found in a. ursinum [18 19]. flavonoids were found to be responsible for the inhibition of platelet aggregation in humans and to have antioxidant activity [20]. a. ursinum is found to be more beneficial than a. sativum in in vivo and in vitro studies. thus, a. ursinum showed a higher effect in increasing high-density lipoprotein (hdl) and decreasing total cholesterol, as well as lowering the systemic blood pressure [21]. while all portions of a. ursinum were foundto exhibit antioxidant property, the leaves were found to have the highest activity [20]. this activity could be caused by the high content of flavonoids. however, the chemical profile of flavonoids in the leaves, stalks, and seeds of a. ursinum has not been fully studied. there are only five flavonoids isolated from a. ursinum by carotenuto 1996 [18]. traditionally, early leaves of r. ficaria are edible fresh or preapared as ingredient of different cusine. however, is not recommended to use more than 50-100 g of fresh leaves. the plant is used to treat food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 g h e o r g h e f l o r i n b â r l ă , m a r i a p o r o c h s e r i ţ a n , e l e n a s ă n d u l e a c ( t u d o s i ) , s i m o n a e l e n a c i o r n e i ( ş t e f ăr o i ) , an t i o x i d a n t a c t i v i t y a n d t o ta l p h en o l i c c o n t en t i n al l i u m ur s i n u m a n d ra n u n c u l u s fi c a r i a , volume xiii, issue 4 – 2014, pag. 349 – 353 351 haemorrhoids, early leaves are high in vitamin c can prevent scurvy. the plant contains protoanemonina mild toxin, however, the process of heating or drying turns the toxin to anemonin, which is nontoxic and has antispasmotic and analgesic properties [22]. there are few scientific reports to date about r. ficaria leaves extract. 2. materials and methods chemicals 1,1-diphenil-2-picryl-hydrazyl (dpph) was purchased from sigma-aldrich. gallic acid and epigallocatechinegallate were from sigma-aldrich. preparation of vegetable samples fresh a. ursinum and r. ficaria were purchased from the local market in suceava city. the vegetables were washed with tap water measured and suspended into a 35% v/v ethanolic solution then extracted at 80°c for 2 hours. after evaporating to dryness and freeze drying the crude extract was obtained. the dry powder extract was used to assay the antioxidant activity and the total polyphenol content. dpph radical scavenging activity assay radical scavenging activity against the 1,1-diphenil-2-picryl-hydrazyl (dpph) was measured as previously described by barla 2009 [23]. briefly the sample was dissolved in ethanol and mixed with a solution of 0.4 mg/ml dpph, 0.5 ml. after incubation at room temperature for 30 min. the absorbance was measured at 520 nm. the ec50 value, denoting the effective concentration of sample required to scavenge 50% of dpph free radicals, was calculated by graphical regression analysis, and expressed as g/ml. total phenolic content the total phenolic content of the aqueous ethanolic extract of each sample was obtained using the folin–ciocalteau method as previously described by velioglu 1998 [24], with some modifications. one hundred microliters of sample extracts were mixed with 100 µl of the folin–ciocalteu reagent, vortexed for 5 min. following this, 200 µl of sodium carbonate solution (9%) and 1600µl of deionized water was added and the reactants were mixed and left at room temperature for 30 min. absorbance was determined at 760 nm against the blank, and a gallic acid calibration curve (0–500 mg/l) was constructed and used to determine the total phenolic content of the samples, expressed as gallic acid equivalents (gae). 3. results and discussions the antioxidant property of ethanol leaves extract of a. ursinum and r. ficaria were evaluated. dpph radical is a commonly used substrate for easy evaluation of antioxidant activity based on its stability in the radical form and simplicity of the assay. the principle of this assay consists in the color change of dpph solution from purple to yellow as the antioxidant compound quenches the radical found in the extract. the color changes can be measured quantitatively by spectrophotometer absorbance at 520 nm. the a. ursinum and r. ficaria ethanolic extracts showed good antioxidant capacity. for the same extract amount r. ficaria showed a slight stronger antioxidant activity (figure 1), r. ficaria extract showed an ec50 lower than that of a. ursinum extract this was in accord with the fact that the total phenolic content was higher in r. ficaria extract, as shown in the table 1. however, the antioxidant power of r. ficaria extract was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 g h e o r g h e f l o r i n b â r l ă , m a r i a p o r o c h s e r i ţ a n , e l e n a s ă n d u l e a c ( t u d o s i ) , s i m o n a e l e n a c i o r n e i ( ş t e f ăr o i ) , an t i o x i d a n t a c t i v i t y a n d t o ta l p h en o l i c c o n t en t i n al l i u m ur s i n u m a n d ra n u n c u l u s fi c a r i a , volume xiii, issue 4 – 2014, pag. 349 – 353 352 over 3 times stronger than that of a. ursinum extract. 80.9 77.9 0.0 20.0 40.0 60.0 80.0 100.0 r. ficaria (1 mg/ml) a. ursinum(1 mg/ml) a nt io xi da nt a ct iv it y (% ) fig. 1. the antioxidant activity of a. ursinum and r. ficaria crude extracts (concentration of 1 mg/ml). this result indicates that this effect can be correlated with the polyphenols content in r. ficaria. however, the ec50 of a. ursinum and r. ficaria extracts were higher than that of gallic acid and epigallocatechinegallate, which were used as positive controls in this experiment. results suggest, in the case of a. ursinum extract, that the observed antioxidant activity might be given by the various kinds of flavonols present in the extracts and possible mainly kaempferol derivatives as reported by oszmiaski on 2013 [25]. table 1. antioxidant activity and total phenolic content of a. ursinum and r. ficaria antioxidant activity total phenolics ec50 (µg/ml) (g gae/100g extract) allium ursinum 322 1.42 ± 0.09 ranunculus ficaria 88.6 1.97 ± 0.11 egcg* 1.2 − galic acid 0.8 − *egcg-epigallocatechinegallate plant phenolic constituents are one of the major groups of compounds acting as antioxidants. phenolics are able to scavenge reactive oxygen species due to their electron donating properties. the total phenolic content was determined using folin-ciocalteu method, reported as gallic acid equivalents by reference to standard curve. total phenolic content in a. ursinum extract was 1.42 0.09 g gae/100g of extract and 1.97 0.11g gae/100g of extract in the case of r. ficaria extract. the total phenolic content did not differ significantly in these pants extract. 4. conclusion in conclusion a. ursinum and r. ficaria exhibit antioxidant capacity and its extracts contain substantial amounts of polyphenolic compounds. generally, high levels of phenolic compounds are responsible for strong antioxidant capacity. consumption of these vegetables especially during springtime can provide a good source of antioxidants. therefore, these plants extract seams to possess an important potential to be used as functional food ingredient or nutraceutical. finally, this report showed that despite the fact that r. ficariais is even less common than a. ursinum, exhibited interesting properties and further investigation are to be done concerning some particular biological activities of r. ficaria extracts. 5. references [1]. liu m., li x.q., weber c., lee c.y., brown j., liu r.h., antioxidant and antiproliferative activities of raspberries, j agric food chem., 50, 2926–2930, (2002) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 g h e o r g h e f l o r i n b â r l ă , m a r i a p o r o c h s e r i ţ a n , e l e n a s ă n d u l e a c ( t u d o s i ) , s i m o n a e l e n a c i o r n e i ( ş t e f ăr o i ) , an t i o x i d a n t a c t i v i t y a n d t o ta l p h en o l i c c o n t en t i n al l i u m ur s i n u m a n d ra n u n c u l u s fi c a r i a , volume xiii, issue 4 – 2014, pag. 349 – 353 353 [2]. ratnam v.d., ankola d.d., bhardwaj v., sahana d.k., kumar r.m.n.v, role of antioxidants in prophylaxis and therapy: a pharmaceutical perspective, j control release., 113, 189–207, (2006) [3]. rice-evans c.a., miller n.j., paganga g., antioxidant properties of phenolic compounds, trends plant sci., 2, 152–159, (1997) [4]. javanraedi j., stushnoff c., locke e., vivanco j.m., antioxidant activity and total phenolic content of iranian ocimum accessions., food chem., 83, 547–550, (2003) [5]. nihal t., ferda s., sedat y.v., the effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables, food chemistry, 93. 713-718, (2005) [6]. subramanion l.j., zakaria z., streenivasan s., phytochemicals screening, dpph free radical scavenging and xanthine oxidase inhibitory activities of cassia fistula seeds extract, j. medicinal plants research, 5, 1941-1947, (2001) [7]. nilsson j., stegmark r., akesoon b., total antioxidant capacity in different pea (pisum sativum) varieties after blanching and freezing, food chemistry, 86, 501-507, (2004) [8]. bagheri f., gol a., dabiri s., javadi a., preventive effect of garlic juice on renal reperfusion injury, j. kidney dis., 5, 194–200. (2011) [9]. blazewicz-wozniak m., michowska a., the growth, flowering and chemical composition of leaves of three ecotypes of allium ursinum l., acta agro bot., 64, 171−180, (2011) [10]. richter t., baerlauch in medizin und mythologie, pharm. ztg., 144, 2197−2198, (1999) [11]. rietz b., isensee h., strohbach h., makdessi s., jacob r., cardioprotective actions of wild garlic (allium ursinum) in ischemia and reperfusion., mol. cell. biochem., 119, 143−150, (1993) [12]. preuss h.g., clouatre d., mohamadi a., jarrell s.t., wild garlic has a greater effect than regular garlic on blood pressure and blood chemistries of rats, int. urol. nephrol., 32, 525−530, (2001) [13]. wu h., dushenkov s., ho c.t., sang s., novel acetylated flavonoid glycosides from the leaves of allium ursinum, food chem., 115, 592−595, (2009) [14]. ivanova a., mikhova b., najdenski h., tsvetkova i., kostova i., chemical composition and antimicrobial activity of wild garlic allium ursinum of bulgarian origin, nat. prod. commun., 4, 1059−1062, (2009) [15]. parvu m., parvu a.e., vlase l., rosca-casian o., parvu o., antifungal properties of allium ursinum l. ethanol extract., j. med. plants res., 5, 2041−2046, (2011) [16]. sendl a., elbl g., steinke b., redl k., breu w., wagner h., comparative greater effect than regular garlic on blood pressure and blood chemistries of rats, int. urol. nephrol., 32, 525−530, (1992) [17]. xu x., song g., yu y., ma h., jin y., apoptosis and g2/m arrest induced by allium ursinum (ramson) watery extracts in an ags gastric cell line, onco targets and therapy, 6, 779-783, (2013) [18]. carotenuto a., feo v.d., fattorusso e., lanzotti v., magano s., circala c., the flavonoids of allium ursinum, phytochemistry, 41, 531−536, (1996) [19]. smeets k., van damme e.j., van leuven f., peumans w.j., isolation, characterization and molecular cloning of a leafspecific lectin from ramsons (allium ursinum l.)., plant mol. biol., 35, 531−535, (1997) [20]. stajner d., popovic b.m., canadanovic brunet j., stajner m., antioxidant and scavenger activities of allium ursinum, fitoterapia, 79, 303−305, (2008) [21]. preuss h.g., clouatre d., mohamadi a., jarrell s.t., wild garlic has a pharmacological investigations of allium ursinum and allium sativum, planta med., 58, 1−7, (2001) [22]. parvu c., universul plantelor, editura asab, bucuresti, 2006 (in romanian) [23]. barla f., higashijima h., funai s., sugimoto k., harada n., yamaji r., fujita t., nakano y., inui h., inhibitive effects of alkyl gallates on hyaluronidase and collagenase, biosci. biotechnol. biochem., 73, 2335-2337, (2009) [24]. velioglu y.s., mazza g., gao l., oomah b.d., antioxidant activity and total phenolics in selected fruits, vegetables and grain products, jurnal of agricutural and food chemistry, 46, 4113-4117, (1998) [25]. oszmianski j., kolniak-ostek j., wojdyło a., characterization and content of flavonol derivatives of allium ursinum l., plant j. agric. food chem., 61. 176−184, (2013) 109 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 109 114 sucrose cooling crystallization modelling valeriy myronchuk1, oxana yeshchenko2, *maryna samilyk3 1national university of food technologies, kyiv, ukraine, mironcuk@nuft.edu.ua 2national university of food technologies, kyiv, ukraine, oxayes@mail.ru 3national university of food technologies, kyiv, ukraine, m.samilyk@ukr.net * corresponding author received april 8th 2013, accepted may 15th 2013 abstract: based on the material balance equations and understanding of the final-grade massecuite cooling crystallization process as the technology object, a simulation model of the process has been built by which the computational experiments have been conducted. by results of these experiments, analytical exponential dependences of the massecuite characteristics change during cooling crystallization have been obtained, namely, grain content, weight, purity and dry solids weight ratio of massecuite syrup. the constructed model has been used to study the industrial cooling crystallization process. it is proved that the results of the experiment of the developed simulation model fully reflect the nature of the industrial process of final-grade massecuite cooling crystallization of sucrose. typical scheme industrial cooling crystallization with massecuite water or impure sugar solution dilution and authors’ scheme with an intermediate heat massecuite have been considered. it’s experimentally shown that the use of final-grade massecuite intermediate heating after cooling to 5052 ºc by 7-10 ºc increases the effect of crystallization to 8.4% by reducing the viscosity of massecuite syrup, the surface tension and alignment of the massecuite cooling rate at sucrose crystalization rate. consequently, the exclusion of dilution of massecuite by water or impure sugar solution and its replacement by intermediate heating provides a more complete depletion of molasses and increases the amount sugar grains in the massecuite. keywords: massecuite, syrup, grains, dilution, mixer-crystallizer, the intermediate heat. 1. introduction additional sucrose crystallization process by cooling has complex dynamics and depends on many factors [1] – [7]. that is why there are many difficulties in regulating the cooling of the massecuite [8]. in addition, the long duration of the process generates complex physical and chemical transformations of the system. despite numerous studies in this area, today there are gaps in the optimal mode of heat and mass transfer in the crystallization of the massecuite in the mixercrystallizers. the study of this process using physical modelling is difficult in large part due to its duration (more than 30 hours). this can be eliminated by the use of simulation [9] – [11]. the objective of our research was to create a mathematical model of the process of crystallization by cooling, the ability to regulate it, to bring the best mode in which the crystallization rate corresponds to the cooling rate. 2. experimental we presented the crystallization process as a technology object (fig. 1) with the input, control and output parameters. the basis of the mathematical description of the sucrose cooling crystallization food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 valeriy myronchuk, oxana yeshchenko, maryna samilyk, sucrose cooling crystallization modelling, food and environment safety, volume xii, issue 2 – 2013, pag. 109 114 110 process accepts the material balance of the process. figure 1. the sucrose cooling crystallization process in mixers-crystallizers as a technology object: input parameters: mm – massecuite mass, dsm – massecuite dry solids weight ratio, g1 – grain content at the beginning of crystallization, pm – massecuite purity; control parameters: t1 – the initial temperature of the process, t2 – the final temperature of the process, hss – supersaturation coefficient, hsi – solubility index; output parameters: g2 – grain content at the end of crystallization, ms – syrup mass, dss – syrup solids weight ratio, ps – syrup purity. general material balance equation: 2211 sgsgm mmmmm  , (1) balance equation by sucrose: 222 111 gss gssmm mscm mscmscm   , (2) balance equation by nonsugar: 2211 ssssmm nsmnsmnsm  , (3) balance equation by dry solids: 222 111 gss gssmm mdsm mdsmdsm   , (4) balance equation by water: 2211 ssssmm wmwmwm  , (5) where mm , 1gm , 2gm , 1sm , 2sm – massecuite mass, the mass of crystals in the massecuite and syrup at the beginning and at the end of the cooling crystallization process, kg; msc , 1ssc , 2ssc , mns , 1sns , 2s ns , mds , 1sds , 2sds , mw , 1sw , 2sw – sucrose, non-sugars, dry solids and water weight fractions in the massecuite and molasses at the beginning and at the end of process, %/ the mathematical description of the sucrose cooling crystallization is necessary to determine the sucrose amount in the syrup. it is determined from equation   sssiwss hpthm scm m , %100  , (6) then   %100 , s sssiw s m hpthm sc m , (7) where sm – the syrup mass, kg, ssc – sucrose weight ratio in syrup,%, mw m – water mass in the massecuite, kg,  pth si , – solubility index of sucrose as a function of temperature and purity, ssh – supersaturation coefficient of the massecuite. variation of sucrose solubility index with temperature and the solution is determined by the regression equation of the third order, we have received the least square method:   25 26 36 36 3 23 25 10821,2 10317,8 10244,5 10429,6 101505,4 10926,6 10084,6 01827,0 1903,00502,1, tp pt p t tp p t p tpth si                 , (8) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 valeriy myronchuk, oxana yeshchenko, maryna samilyk, sucrose cooling crystallization modelling, food and environment safety, volume xii, issue 2 – 2013, pag. 109 114 111 where t – the product temperature, ºc, p – sugar solution purity, %. we believe that in the cooling crystallization process water and nonsugars content in syrup remains unchanged. then at the beginning of cooling crystallization, we have syrup mass:   %100%100 , 111 mmmm sssiws wmnsm hpthmm m   , (9) grain mass: 11 smg mmm  , (10) grain content in the massecuite: %10011 m g m m g  . (11) similarly, we find the syrup and crystals masses at the end of the cooling crystallization process:   %100%100 , 222 mmmm sssiws wmnsm hpthmm m   , (12) 22 smg mmm  , (13) %10022 m g m m g  . (14) in general, for any temperature in the cooling process massecuite 21 ttti  we have   %100%100 , mmmm ssiisiws wmnsm hpthmm mi   , (15) ii smg mmm  , (16) %100 m g i m m g i . (17) the sucrose, non-sugars, dry solids and water mass fractions in the syrup, and its purity were determined by the equations:   %100 , i m i s ssiisiw s m hpthm sc  , (18) %100 i i s mm s m nsm ns  , (19)   %100 , i m i s mmssiisiw s m nsmhpthm ds   , (20) %100 i m i s wm s m mm w  , (21) %100 i i i s s s ds sc p  , (22) equations (15)–(22) is a mathematical description of the model of sucrose cooling crystallization process. on the basis of computational experiments on the model (15)–(22) we have obtained analytical dependence of the massecuite characteristics (grain content, mass, purity, and the dry solids weight ratio of syrup) on time and massecuite purity:          mg pcmgmgm epbpapg , , (23)          mмm м м pc mm mm ms epb pa pm    1 , , (24)          msp s s pc mp mp ms epb pa pp    1 , , (25)          msds s s pc mds mds ms epb pa pds    1 , , (26) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 valeriy myronchuk, oxana yeshchenko, maryna samilyk, sucrose cooling crystallization modelling, food and environment safety, volume xii, issue 2 – 2013, pag. 109 114 112 where c i     – the relative time, i current time, c the total cycle time, mp – massecuite purity. 3. results and discussion the above model we used to simulate the industrial sucrose cooling crystallization process in the mixer-crystallizers, which requires water or impure sugar solution dilution of massecuite. although this method to some extent, can improve the crystallization conditions, the water addition in the massecuite violates isohydric conditions of crystallization. this reduces the final crystallization effect, increases the molasses amount, and hence the sucrose content in it, as well as energy costs in sugar house. for this reason, it is advisable to carry out massecuite heated to a definite temperature instead of water dilution. the constructed sucrose cooling crystallization model is used to study the process of industrial sucrose crystallization in two modes, flow graphs of which are presented in fig. 2. the amount of water or impure sugar solution for dilution of massecuite is calculated as:   dmd mdmm d dsds dsdsm m    , (27) where md – mass of water or impure sugar solution for dilution of massecuite, mm – masscuite mass, dsd, dsm, dsdm – dry solids weight ratio of sugar solution for the dilution, of massecuite before and after dilution, respectively; if massecuite is diluted with water, then 0dds . a b figure 2. flow graph of the massecuite cooling crystallization process: a – with water or impure sugar solution dilution, b – with an intermediate heat, rm – reception mixer, mc – mixer-crystallizer, h – heater, c – centrifuge. temperature range of the cooling crystallization process is described by dependencies: when massecuite is diluted with water or impure sugar solution:     1 1 1 1 ceb a t   (28) when intermediate heating is used               heatingafter , 1 heatingbefor , 1 2 1 2 2 1 1    c c eb a eb a t . (29) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 valeriy myronchuk, oxana yeshchenko, maryna samilyk, sucrose cooling crystallization modelling, food and environment safety, volume xii, issue 2 – 2013, pag. 109 114 113 35 40 45 50 55 60 65 70 75 80 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 ti/tc t, o c figure 3. temperature range of final massecuite cooling crystallization: – with an intermediate heat, – to the standard mode. it stands to reason that the water dilution not only reduces the grain content in the massecuite at the end of crystallization, but also increases syrup purity (fig. 4). for bringing of massecuite to a given dry solids weight ratio impure sugar solution for dilution of its must be much more than water. this may explain the smallest content of the grains in the massecuite after dilution and at the end of crystallization for the scheme with the impure sugar solution dilution. besides that decrease the purity syrup in this case is not achieved at the expense of its desugarization, but due to high content of non-sugars which were added during dilution (fig. 4). 24 26 28 30 32 34 36 38 40 42 44 4045505560657075 t, oc g , % a. 84,0 84,5 85,0 85,5 86,0 86,5 87,0 87,5 88,0 88,5 4045505560657075 t, oc d s s, % b. 52 54 56 58 60 62 64 4045505560657075 t, oc s c s , % c. 63 64 65 66 67 68 69 70 71 72 73 4045505560657075 t, oc p s , % d. figure 4. changing massecuite technological characteristics during the cooling crystallization process: – with water dilution; – with impure sugar solution dilution; – with an intermediate heat; а – grain content in the massecuite; b – dry solid weight ratio in the syrup; c –sucrose weight ratio in the syrup; d – syrup purity to analyze the effectiveness of intermediate heating final massecuite in the mixer-crystallizers we carried out a series of research in which besides the addition of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 valeriy myronchuk, oxana yeshchenko, maryna samilyk, sucrose cooling crystallization modelling, food and environment safety, volume xii, issue 2 – 2013, pag. 109 114 114 dilution water massecuite or molasses, massecuite intermediate heating by 5, 7, 10 and 12 °c has been used. the results suggest that the best effect is got when the temperature of the intermediate heating of massecuite in the mixer-crystallizers after cooling to 50-52 ºc is 7-10 °c (fig. 4). in this case, the viscosity of the syrup is reduced almost by half, the surface tension is also reduced. decreases the viscosities of syrup increases the sucrose molecules diffusing from solution to the crystal surface, and reduce the surface tension increases the rate of crystallochemical reaction at the phase interface during the transition of sucrose dissolved in the crystal. crystallization effect in this case is 8.4%. in addition, we found that if the purity of the initial massecuite decreases, the temperature of its intermediate heating must be increase. also a significant improvement in the sugar grains size moves up in fractions of 0.63-1.0 mm and greater than 1.0 mm 4. conclusion alternative water dilutions of final massecuite in the cooling crystallization process in mixers-crystallizers is to use an massecuite intermediate heating by 8 ºc-10 ºc after reducing its temperature by 50-52 ºc. the use of massecuite intermediate heating reduces the syrup viscosity and the surface tension at phase interface "solution" – "solid", which increases the intensification of sucrose crystallization. in this case greater molasses desugarization and better grain size of sugar crystals in the final massecuite are achieved. 5. acknowledgments we thank elena vygran for assistance in translation. 6. references [1] s. ouiazzane, b. messnaoui, s. abderafi, j. wouters, t. bounahmidi. estimation of sucrose crystallization kinetics from batch crystallizer data // journal of crystal growth, volume 310, issue 4, 15 february 2008, pages 798-803 [2] lie-ding shiau. the distribution of dislocation activities among crystals in sucrose crystallization // chemical engineering science, volume 58, issues 23–24, december 2003, pages 5299-5304 [3] a. ferreira, n. faria, f. rocha. roughness effect on the overall growth rate of sucrose crystals // journal of crystal growth, volume 310, issue 2, 15 january 2008, pages 442-451 [4] håkan gros, teuvo kilpiö, juha nurmi. continuous cooling crystallization from solution // powder technology, volume 121, issue 1, 5 november 2001, pages 106-115 [5] issam a. khaddour, luís s.m. bento, antónio m.a. ferreira, fernando a.n. rocha kinetics and thermodynamics of sucrose crystallization from pure solution at different initial supersaturations // surface science, volume 604, issues 13–14, 15 july 2010, pages 1208-1214 [6] giuseppe vaccari, giulio sgualdino, elena tamburini, giorgio pezzi, piercarlo citterio, roberto verardi, krzysztof urbaniec. new eco-friendly proposal for the crystallization of beet raw juice // journal of cleaner production, volume 13, issue 15, december 2005, pages 1447-1460 [7] m. quintas, t.r.s. brandão, c.l.m. silva, r.l. cunha. rheology of supersaturated sucrose solutions // journal of food engineering, volume 77, issue 4, december 2006, pages 844-852 [8] p.m. martins, f. rocha. new developments on size-dependent growth applied to the crystallization of sucrose // surface science, volume 601, issue 23, 1 december 2007, pages 5466-5472 [9] n. faria, s. feyo de azevedo, f.a. rocha, m.n. pons. modelling agglomeration degree in sucrose crystallisation // chemical engineering and processing: process intensification, volume 47, issues 9–10, september 2008, pages 1666-1677 [10] c. campañá cué, a.r. ruiz salvador, s. aguilera morales, f.l. falcon rodriguez, p. pérez gonzález. raffinose–sucrose crystal interaction modelling // journal of crystal growth, volume 231, issues 1–2, september 2001, pages 280-289 [11] n. a. f. a. samad, ravendra singh, gürkan sin, krist v. gernaey, rafiqul gani. a generic multi-dimensional model-based system for batch cooling crystallization processes // computers & chemical engineering, volume 35, issue 5, 11 may 2011, pages 828-843. microsoft word 3 torosyan in fia romania _1_.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 23 ecological problems at realization of active influence on hail processes suren hovsepyan1, yeva torosyan2, gagik torosyan3* 1 state commercial organization “ the service of active influence on atmospheric processes ”, yerevan, ра, hovsepyansuren@mail.ru 2european regional educational academy, yerevan, armenia, yevatorosyan@yahoo.com 3state engineering university of armenia, department of chemical technology & environmental engineering, yerevan, armenia, gagiktorosyan@seua.am *corresponding author received 10 november 2011, accepted 15 february 2012 abstract: the problems of environmental pollution are discussed at realization of activities of active influencet on hail. the results on application effective and rather ecological secure method against hail of protection in terrain of republic armenia are leaded. it`s necessary to realize activities on hail the continuous radar control of changes of aircraft attitude of clouds evolution. technology of protection from hailstones beaten, as any technologies of environmental impact, should be ecological secure. therefore one of prominent aspects for an estimation of technologies of protection from hail is the estimation of levels of an ill effect on an environment, collateral effects and ecological safety of used technologies. an effect is recognized successful, if the radar parameters of a cloud descend or terminate to grow, in this case cloud passes in stage of dissipation. one of the symptoms of a dissipation of polycell cloud is the termination of originating of filial cells or transition in one cell with cloud tendentious destructions of last cell. the agriculture of armenia is responsive to instability of a climate and consequently requires to protection. the agriculture is in econiomical trouble and has low adaptation. the purpose of the present paper is: detection of contamination of an ambient environment in region against hails activities. keywords: environmental pollution, hail, hailstones, cannon, radar, cloud dissipation, noise, 1. introduction hail can cause serious damage for society and most commonly for agriculture, for farmer`s crops. hail occurs most frequently within continental interiors at mid-latitudes and is less common in the tropics, despite a much higher frequency of thunderstorms than in the midlatitudes [1 ]. when hail stones exceed 13 mm in diameter the automobiles, aircraft, skylights, glasses and other planes can be seriously damaged within seconds [2]. rarely, massive hailstones can to cause concussions or fatal head trauma [ 3 ], for instance, hail is one of india`s, china`s and canada's most expensive hazards [ 3,4 ]. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 24 hail is formed in space and time an incidentally, but it`s a possible to insure against this damage. one of method for hail insuring hail formation and against hail fall processes are used the hail cannons. the cannon is a large impressive apparatus, it makes a lot of noise, and it comes with confidence-building detailed instruction. more ever, any times that are used the acetylene explosion for sound and infrared waves burning. that make also ecological problem for surrounding. in the present paper the problems of early warning of ecological danger in region of realization of fissile effects are discussed. the agriculture of armenia is responsive to instability of a climate and consequently requires to protection. the agriculture is in econiomical trouble and has low adaptation. the extreme weather phenomena invoke sometimes loss of crop. so, in 2009 around 9 dangerous phenomena of hail took place, when diameter of hailstones made 20 mms and more, from behind that agricultural branch of country the huge damage was is marked. because of hail activities, with 2005 for 2010 from behind hail the people three have perished technology of protection from hailstones beaten, as any technologies of environmental impact, should be ecological secure, therefore one of prominent aspects for an estimation of technologies of protection from hail is the estimation of levels of an ill effect on an environment, collateral effects and ecological safety of used technologies. discussion in the present paper the problems of early warning of ecological danger in region of realization of active influence on hail are discussed. it is known, the interplay implements on hail processes by operating depositing ice formed reactants directly in on cloudy medium, in area laying ahead hail formation [5]. the hail cannons are used for that process. the cannon are large impressive apparatus, it makes a lot of noise, and it comes with confidencebuilding detailed instruction. more ever, any times that are used the acetylene explosion for rocket moving. that make also ecological problem for surrounding. the rockets used against hails basically consist of a nose cone, engine, deflocculant with pens and system of safety control. the nose cone of rockets is supplied with the pyrotechnic generator of crystallizing aerosols of silver iodide. the engines are equipped with ballistic firm fuel. the security system contains a charge of explosives with hollow-charge extractions ensuring splitting of cotton-bakelite bodies of rockets on small-sized debris. for example, the rocket „alazav” against hails has a duraluminium body, engine and parachute system of safety control. the generator of crystallizing fragments is the sustainer equipped with mixed fuel with %2 -s' contents of silver iodide (agl), creating indispensable thrust force on all flight path. and, the rocket „wr-98z” automatically starts to reduce landing speed of a rocket. so, the pollution of the environment in case of activities against hailstones with application of rocket technology of protection is conditioned by the following factors: 1. contamination of free air, soil and water of the open pools by combustion products of pyrotechnic structures, reactant of an inoculation (agl), charge of engines and explosive of a system of selfliquidation of bodies (debris and oddments of bodies) against hailstones of rockets; 2. the derivated fumes of combustion products particulate is outwashed by precipitations and fall in soil, in the open pools in atmosphere, are diffused by airflows and are born for limits of defended terrains. the formation of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 25 iodate acid and its potassium salt is connected to interplay the iodine with strong oxidants, much more likely with ozone. it is necessary to mark, that much more likely, the oxidation is promoted by strong ozone formation during a thunderstorm. it was earlier rotined, that the level of contamination of environment at realization anti tail activities by iodides, reaction products is lower than the sanitary regulations, though the writers approve there can be strong local contaminations [6]. agi -------agjo3 + hjo3+ i2 + hj the derivated materials are toxic in a miscellaneous degree toxic activity. for example, even such widespread material as the iodine has toxic effect on the people at absorption more than 1 milligram. the maximum tolerable dose of the iodine for the miscellaneous age qualifications of the people is determined in the field of doses 0,150 and 1,000 mg per day. the elementary iodine is toxic, and its fume stimulates of an eyes and pulmones. all iodides also are toxic in reduced limits, as the iodine, on the chemical nature is easily freed at stand from an iodide anion containing compounds [7]. moreover, known isotope iodine 131 one of radioactive materials, live in atmospheric test of automatic weapons. reduced causes to transition to new technologies of processing of hail clouds. the rocket-artillery method on active influence hail processes is the most expensive method. besides there are also other of this method: -the inoculation of clouds is made antihail by shells or rockets issued with cannon kc-19, rocket installation tkb-40, or other / anti-hail installations /this military weapon/. the rockets and the shells are unguided, after launch a rocket or shell can knock dawn from a pathway and detonate in the other place, that can result in tragical consequences. the statistics of country of cis on the average annually spend 187500 rockets, rockets firings with tragical outcome 94 the usages of this methods, before launch anti-hail items are necessary need the sanction from bodies(organs) of aircraft, it detains spent activities on anti-hail to protection in a consequence of realization of perennial activities on anti hail protection there is, a contamination of free air, soil, water of the open pools etc., are diffused by airflows and are born for limits defended територий. there is account documentation: the stuffs of overseeings by a condition of atmosphere in a site of negotiations of activities on active influence on hail processes on paper and electronic carriers, the concludings of organizations and experts on stuffs of observations, minutes of commissions extending the concludings of organizations and the specialists. for a super-power cloud of hail one of the first symptom for discontinuance of hail formation in a cloud is the steady lowering of a zone heightened radar reflection on a wavelength λ = 10 cm. the reduction of the geometrical sizes of a shed, and also absence of a zone of a gentle radio echo are symptoms of the discontinuance of new development of a cloud and formation in a cloud of new blastemals of hailstones. the basic symptom of transition of hail cell in stage of dissipation are: the steady reduction of the size of falling out hailstones; the steady reduction of probability of loss of hails or complex yardstick of hail risk; food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 26 the steady reduction maximum radar reflection of a cloud; the downturn of a radio echo maximum; the reduction of horizontal gradients radar reflection in a zone at a level of 1-3 kms overland: the downturn of high bound of a zone heightened radar reflection λ = 10 cm is lower than a level of an isotherm -10oс; the activity in a mode of exposure implements so long as above defended or accumbent terrains are watched hail risk of a cloud. the outcomes of experiment are estimated by commission, in a structure which one included the specialists in the field of atmospheric physics and meteorology. recently large application is found audiosignal generators with a directional sound wave technological “know-how” in this area. in these generators as fuel will be used either gas of acetylene or propanebutane gas. though acetylene has a high scale of endothermicity and at incineration the plenty of heat is excreted, usage of this gas for some reasons should be eliminated. on our view the reasonable causes for transition to system propane butane, which one ecological cleaner in many aspects. usage of acetylene in fuel grade as contrasted to this gas creates following troubles: 1. acetylene in a mixture with oxygen blasts out in a very broad band of concentrations. the explosion-hazard decreases at a dilution of acetylene with inert gases, for example n2. 2. acetylene blasts out at the temperature of about 500oc or pressure above 0,2 mp at long-lived stand in copper medium will be formed copper acetylenide, that blasts out at impact or temperature rise. therefore at storage of acetylene the stuffs keeping cuprum (for example, valves of bottles) will not be used. 3. difficulty of storage, / store and transport it in filled inert porous weight (for example, char coal) steel bottles of white colour (with a red placard "«a") by the way of solution in an acetone under pressure 1,5-2,5 mp./ 4. toxicity / 0.3 mg/m3 /. though it is considered, that of acetylene has gentle toxic operating, in matching about a propane butane fraction, that despite of a smaller endothermicity, is less toxic on 1000 times / 300 mg/m3 /, is less explosion-hazard, storage is less problematic. it is necessary to mark, that at combustion of using organic compounds the same materials are excreted water and carbon dioxide. however, for acetylenes is formed more carbon dioxide / about 15 %/: 2c2h2 + 5o2 ------ 4co2 + 2h2o 2c3h8 + 10 o2 ------ 6co2 + 8h2o 2c4h10 + 13 o2 ------ 8co2 + 10 h2o now the problem to made everything for reduction quantity of carbon dioxide / kioto`s protocol / . so that, and in this aspect, application of a mixture the propane butane is more justified. the propane butane fraction is liquefied easily (below 0oc and on normal pressure or at heightened pressure and customary temperature – volatile liquid) and transported, nor creates of any difficulties with stuffs of storing systems. the application of acetylene is connected also to high operating cost. so, 40 liter bottles acetylene contain 7,4 kgs of acetylene, with the cost about 110 000amd /300 us dollars /, in that врема as 40 liters жжиженного the propane butane of a fraction from 20 kgs of gas costs 20 000 amd /about 55 us dollars /. acetylene it is possible to store no more than 6 months, then the bottle can be blasted. in case of explosion the trotil equivalent of an acethylene bottle is peer 2,75кг. the explosion of an acetylene food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 27 bottle results to let of metallical debris up to 300м. therefore, from many aspects, and with ecological, there is a necessity of replacement of acetylene on propane butane a fraction. by the way, it 5-6 times more cheaply acetylene, and is easy to application. the reduced data convincingly testify to expediency of usage of propane butane in fuel grade for audio-signal generators. the efficiency of a method makes approximately 70 %. the noise problem: the noise is new danger from environment. the noise hinders strain-relief crystallization, dream, dialogue, also to activity, therefore can lower a quality of life and damage to health. it is necessary to mark, that the who / world health organization/ now prolongs researches on influencing a noise on a human body. it is necessary to mark, million of the people live in constant acoustic discomfort, because of the proximity to the places of residence of transportation facilities automobile, rail-way, aircraft. strong acoustic influencing have also means of military engineering. the measurements on definition of a noise are conducted at explosions on devices rft-000/17 and rft000/28. the measurements are conducted as in a building, where is located acoustic antihail of a cannon, and outside of a building. the greatest noise effect on regime lin and c in a building in case of an acethylene bottle made 130 db, and case a propane butane of a fraction is 164 db. in escaping the acoustic end on 0,5 m in impulse mode the sound strength makes 174дб, and in a mode lin and c 168. in case of acetylenes are 149 db and 139 db accordingly. estimation of efficiency anti-hail activities: essential variability of repeatability and intensity of hails phenomena in space and time during a season, and also their annual variability, make an estimation of efficiency of simulated interference to extremely problem. it can be executed only on the basis of the analysis and confrontation of the perennial data. in this case at an estimation of those or diverse methods of simulated effect on hail processes are planned an experiment with a randomization of simulated interference. it allows to receive two independent series of observations, in which one the clouds subjected and which are not subjected to effect, selected are assorted on the basis of definite and identical to both series of yardsticks. the value of efficiency is calculated under the formula most often used in global practice of active effects for such estimations: e = (1 si/s0) 100 %, where are: si – the area of damage by hail of agricultural cultures in the season of realization of anti hail activities, reduced to a unit area. s0 will use or perennial mean area of damage by hail in terrains, defended from hail prior to the beginning activities on hail safety, or area of damage by hailstones in monitoring terrains for the compared season, also reduced to a unit area. the selection of monitoring terrain implements pursuant to the requirements: identical with terrains, defended from hail physical geographical and climatic features of terrains comparable hail dangers, including on the areas hail beating. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 28 4. conclusion on fulfilment of the present investigation it is detected following for usage influence against hail means on environment: the most effective way of strife against hail is the method, though a noise level little bit high as contrasted to acethylene analog. considerable effect on health of the person because of contamination by movable sources though in the article the given problem is not discussed, however it is necessary to take into account: a/ low efficiency of verification of quality monitoring systems of air b/ the old equipment low technological level of are resulting in to considerable emissions. 5. acknowledgments this paper was supported in framework of project “climate change, contamination and risk” in the field of scientific researches on environment in ministry of extremal situation ra and seua 6. references [1]. w.h. hand, cappelluti g. (2011). a global hail climatology using the uk met office convection diagnosis procedure (cdp) and model analyses". meteorological applications (meteorological applications. wiley)., 18 (4), 446. [2]. damir p., željko v., janez st. (2009) hail characteristics of different regions in continental part of croatia based on influence of orography". atmospheric research, 93 (1– 3),516. [3]. john e. o. (2005).encyclopedia of world climatology, springer.401. [4]. dongxia l., guili f., shujun wu. (2009) the characteristics of cloud-to-ground lightning activity in hailstorms over northern china". atmospheric research, 91 (2–4): 459– 465. [5]. abshaev m.t.( 1081) opretaive control of hail influeneffectivity vgi. n. 72, 81-99. [6]. abesashvili l.sh., sunatishvili g.d., iodine and containing in kakhetia soil, igan georgia, ( 1982) n.19, 6-93,. [7]. pennington j.a.t., ( 1990): a review of iodine toxicity reports..journal of the american dietetic association, v90.n11, 1571(11). the method of determination of the optimal parameters of dispersed materials granulation through consolidation food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 15 the method of determination of the optimal parameters of dispersed materials granulation through consolidation dmitro viktorovich rindyuk1, svyatoslav yuriyovich lementar1 1national university of food technologies, kiev, ukraine rel_dv@ukr.net *corresponding author received 10 may 2012, accepted 15 june 2012 abstract: the method of determination of the optimal constructive and technological parameters of dispersed materials granulation through consolidation is represented within the task of nonlinear mathematical programming. the method is based on mathematical modeling of dispersion materials extrusion through the granulator matrix holes. the method allows taking into account constructive features of technological equipment and rheological properties of the raw material. key words: extrusion, disperse materials, mathematical modeling. 1.introduction recently in food and processing industry appeared a problem of the rational usage of the offals, such as sunflower husk, straw, bagasse, beer pellet, wood shavings and other. according to the ukrainian law about the stimulation of biofuel production and considering the world energy crisis, such offals should be used as biofuel, compound feed and other. but because of the low bulk density, the transportation of such materials is not profitable. it stipulates increasing of bulk density of these materials with briquetting consolidation, baling, granulation and other. according to the analysis of different consolidation technologies we conclude that granulation is the most rational way since it is a continuous process that allows getting the product with the maximum density and provides versatility of further usage of granules (biofuel, compound feed of different factions.) the analysis of the latest researches and publications [1] shows that effective realization of pressing technologies through extrusion depends a lot on constructive parameters of the equipment, which provide the required performance. herewith the application of modern information technologies of design allows determining of quantitative and qualitative patterns of consolidation of disperse materials of different structural and mechanical characteristics. considering the wide range of raw materials while determining the constructive parameters of the equipment there appears a new problem. it is the finding of interconnection between the basic constructive and technological parameters of a specific pressing node. since the problem includes lots of parameters, it is important to find their optimal distribution. 2. materials and methods the goal of this paper is to create the method of determination of basic elements of technological equipment for disperse materials granulation through consolidation [2]. the method is schematically represented in figure 1. consolidation technology is implemented by raw material extrusion through matrix holes. figure 1 shows, that to set the optimization task within the general mathematical programming theory, mailto:rel_dv:@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 16 it is necessary to find the interconnection between all the parameters which describe the special granulation process. so there is a necessity to make a research to define quantitative dependences, which describe thermodynamic process and mass transfer in the material during its granulation through consolidation. the effectiveness of granulating press is evaluated according to the granules quality (density), productivity and power consumption. so appear several optimization criteria: 1) maximize granules density 2) minimize power consumption 3) maximize productivity ( ) ( ) ( ) max; min; max; ® ® ® a a ar q w (1) where, ( )ar – granules density, ( )aw – power of granulator drive, ( )aq – granulator productivity. having analyzed the construction of granulator pressing node and granulation technology [4], we found out the following optimization restrictions: 40 mm ≤ l≤ 90 mm, 8 mm ≤ d ≤ 28 mm, (2) 50 mpa ≤ р ≤ 300 mpa, where l – length of matrix dies, d – dies diameter, p – shrinkage pressure. lets rewrite (1) considering (2): ( ) ( ) ( ) 30050 288 9040 max;,, min;,, max;,, ££ ££ ££ ® ® ® p d l pdlq pdlw pdlr (3) so, the problem with three target functions is formulated. and we face the multicriterion problem – making the solution in the presence of number of target functions f = {fi (a)} (i = 1, 2, …, m), where a is some alternative implying continuous vector variable, which belongs to the convex closed range which is usually set by system of inequalities [3]. fig 1. the scheme of determination of optimal constructional and technological parameters of disperse materials granulation through consolidation food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 17 it is clear that the optimum after each target function criteria can not always be reached with the same control parameter a value. that’s why it is necessary to stipulate how to make a decision. there appears a problem of choice of one of the effective alternatives – search of compromise (compromise decision). 0a alternative can be considered as effective if there are no other alternatives better on at least one criteria and not worse on the others [3]. in our case f set criterion possess different physical content. some are maximized and some are minimized. it should be noted that if 0a is the effective alternative of criterion set f = {fi (a)} (i = 1, 2, …, m), than 0a is an effective alternative of function set ψ = {ωi(fi(a))} (i = 1, 2, …, m), where ωi(fi(a)) is a monotonic function fi(a). to find the effective points it is necessary to transform the functions ωi(fi(a)) on to dimensionless ones or such, when ωi(fi(a))→ min. that’s why the following monotonic transformations for maximizing criterion should be introduced: .m,...,1i, ff )a(ff ))a(f( (min)i 0 i i 0 i ii ==w (4) for minimizing criterion .m,...,1mi, ff f)a(f ))a(f( 0 i(max)i 0 ii ii +==w (5) 0 if – optimal value of the i-criteria (min)if the smallest value of maximizing criteria (max)if the biggest value of minimizing criteria so the parametrical problem should be solved: } ff f)a(f ff )a(ff {min)(man m 1i m 1mi 0 i(max)i 0 ii (min)i 0 i i 0 i v u å å= += îa îa g+ g=ay (6) for each { }å =+ =î mi iii 1 1,0 gggg f . 3. results and discussion lets use such method when the compromise solution is considered as the one, which allows the minimal relative deviation from the optimum parameter values depending on weighting coefficient δі, such that { }å =+ =>î mi iii 1 1,0 dddd . let’s solve the problem using this method. according to (2) and (3) let’s deceive the obtained dependences to dimensionless and minimizing ones: , minrr rr r =* opt opt where 222 0065.009.053.0049.0 25.472.158.904.58 pldpd pld ---× -+++=r ρopt – optimal value of ρ function, ρmin – minimal possible value of ρ function considering restrictions (2) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 18 , qq qq q minopt opt =* where 22 p018.0l298.0pl0443.0pd132.0p96.10l2.18d78.355.1745q --×-×++++= qopt – optimal value of q function, qmin minimal possible value of q function considering restrictions (2) , ww ww w optmax opt =* where wopt optimal value of w function, wmax– minimal possible value of w function considering restrictions (2). so for (3) case having considered (4), (5) and (6) we get: [ ] [ ] [ ] ïþ ï ý ü ïî ï í ì g+ g+ r-r r-r g=y î î î optmax opt 3 minopt opt 2 minopt opt 1 300,50p 90,40l 30,8d ww ww qq qq min)p,l,d(min where { }å = => 3 1 1,0 i ii gg . basing on levenberg–marquardt algorithm, in mathcad 14 pack was created a program to calculate compromise values of control parameters according to the proposed method. 4. conclusion the represented method allows formulating and solving of the problem of multicriterion optimization for the set of control parameters of pressing node within the nonlinear mathematic programming. solving of the problem allows getting the appropriate optimal parameters of the granulator pressing node in order to formulate the further project solutions. 5. references: [1] zayets y. development of information practice of food technologies equipment design. chisa-96, 119, (1996). [2] mikrailov o., serdyuk g., yepifanseva t., shtefan e. numerical simulation of powder materials extrusion. euro pm-2005, 427, (2005). [3] himmelblau, d. applied nonlinear programming, mcgraw-hill, new york. 11.,534, (1972). 207 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 207 – 213 physicochemical monitoring and performance evaluation of sbr municipal wastewater treatment *tsvetko prokopov1, dasha mihaylova2, nikolay mihalkov3 1department of environmental engineering, university of food technologies, 26 maritsa blvd., 4000 plovdiv, bulgaria, e-mail: tsvetko_prokopov@abv.bg 2university of food technologies, department of biotechnology, e-mail: dashamihaylova@yahoo.com 3wwtp-hisarya town, bulgaria, e-mail: nmihalkov@abv.bg *corresponding author received august 29th 2014, accepted september 28th 2014 abstract: hisarya town is one of the famous bulgarian resort with mineral springs and spa-centers attracting thousands of tourists where is installed one of the newly wastewater treatment plants (wwtp) designed on the base of sequencing batch reactor (sbr) technology. in recent years, sbr has been applied as an efficient technology for wastewater treatment. monitoring and control of sbr is a very attractive field of research nowadays. present study are dealt with monitoring of physicochemical characteristics viz. bod5, cod, suspended solids (ss), total nitrogen (tn) and total phosphorus (tp) and performance evaluation of sbr municipal wastewater treatment at wwtp-hisarya. significant reduction (p<0.05) in the physicochemical characteristics of the sbr treated wastewater was established. the average removal of bod5 (95.1±0.6 %), ss (95.1±0.6 %), cod (93.2±0.7 %), tn (80.3±8.0 %) and tp (53.4±7.2 %) were recorded. the values obtained of physicochemical parameters in treated wastewater were considerably below permitted emission limits which indicate that wwtp-hisarya is working perfectly. thus it is helpful in the control of the pollution of “blue river” as a discharging water body. keywords: characteristics, effluent, influent, pollutants, purification 1. introduction water environment pollution has become a major environmental problem recently. continuous development of urbanization, tourism and industry leads to increasing of worldwide water consumption. on the other hand the volume of wastewater effluents into water intakes containing a variety of pollutants is continuously growing. wastewater treatment before its discharging into water bodies is an important assignment of any civilized society, central and local government. it is an urgent task for mankind to improve the wastewater treatment efficiency. domestic and industrial wastewater incoming into the urban treatment plants are characterized by irregularity in the amount and type of the pollutants. therefore, the facilities for the treatment of this type of water are combined and typically include a mechanical, biological and in some cases, chemical steps [1]. the sequencing batch reactor (sbr) represents a modern approach to wastewater treatment. unlike the traditional continuous flow activated sludge process, where different reactions are carried out in separated tanks, sbr allows using a single tank for the whole process. sbr is a modified activated sludge process used to treat a variety of wastewaters. in recent years, sbr has been employed as an efficient technology for wastewater treatment, especially for domestic wastewaters, because of its simple configuration and high efficiency in food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 208 bod5 and suspended solids removal, nitrification, denitrification and phosphorus removal [2]. there are several literatures supporting the applicability of this promising reactor in different wastewater treatment [3-15]. monitoring and control of sbr is a very active field of research nowadays. the quality and quantity of influent wastewater are floating with season, temperature and weather. meanwhile wastewater treatment systems have characteristics of high dimensional data and strong nonlinearity. therefore precise monitoring and control for sbr is still complex for research and practice [16]. the efficiency of wastewater treatment plant (wwtp) can be illustrated by a study on the evaluation of pollution levels of the influent and the effluent at the treatment plant discharging into the environment [17]. on the other hand, process monitoring of wastewater treatment systems can be used successfully for the wwtp optimization [18]. in the town of hisarya, which is one of the famous bulgarian resorts with its mineral springs and spa-centers attracting thousands of tourists especially in summer, is installed one of the newly wastewater treatment plants designed on the base of sbr-technology. bulgarian experience in wastewater treatment for complete removal of bod5, nitrogen and phosphorus is relatively new and limited. recent projects for new wwtp are developed by mathematical models and programs. these plants are not susceptible to mathematical verification. for examination of plant’s design and efficiency of operation can only be used the results from the wastewater analysis at the inlet and the outlet of the already constructed plant during its exploitation. monitoring and performance evaluation will also help for the better understanding of design and operating difficulties in wwtp and also to assess reuse potential of treated water. this work aimed the monitoring of physicochemical characteristics of municipal wastewater before and after treatment at sbr plant installed in hisarya town, bulgaria and the performance evaluation of sbr treatment. 2. materials and methods wastewater treatment plant (wwtp) general description the object of this study was wwtp designed on the base of sbr-technology installed in hisarya town, bulgaria (fig. 1). the plant was put into operation in 2011. design values of the performance of the plant are: load 10000-25000 pe, wastewater dry weather flow 7250 m3/d, wastewater wet weather flow up to 2000 m3/h, daily treatment volume in wet weather 1080 m3/h, organic load as bod5 up to 1500 kg/d, total nitrogen load 275 kg/d and total phosphorus load 45 kg/d. wwtp includes three aeration basins (sbrs), aerobic stabilization of sludge, dewatering machine (centrifuge) and lime conditioning, with installed capacity of about 430 kw, daily consumption of electricity at full load about 2000 kwh/d, specific consumption of electric energy per unit volume of wastewater 0.27 kwh/m3 and specific electricity consumption equivalent per capita per year 29 kwh/pe. analytical methods wastewater sampling was conducted according to bds iso 5667-10 standard. samples were collected automatically into the plastic bags and were brought to the laboratory for analysis. physicochemical monitoring of the wastewater treatment was carried out by the values of the following standard parameters: five-day biological oxygen demand (bod5), chemical oxygen demand food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 209 (cod), total nitrogen (tn), total phosphorus (tp) and suspended solids (ss). values of cod, tn and tp were determined, spectrophotometrically by using of standard cuvette tests (hach lange), according iso 6060, en iso 11905-1 and iso 6878-1, respectively and dr3900 spectrophotometer acomplished with termostat lt200. values of bod5 were determined by using of bodtraktm ii respirometric bod apparatus (hach lange). quantity of suspended solids was measured by filtration through glass filter, according to bds en 872 standard. fig. 1. schematic flow diagram of the wwtphisarya: 1 input shaft, 2 mechanical pretreatment units, 3 sbrs, 4 uv-disinfection, 5 excess sludge stabilization tanks, 6 excess sludge dewatering machine, 7 conditioning of dewatered excess sludge, 8 pumping station performance evaluation performance evaluation of the sbr wastewater treatment was carried out by assessment of percentage degree of purification (dp, %) calculated by the following equation: 100. с сс dp inlet outletinlet= (1) where: inletс – concentration of the respective pollutant at the inlet of the sbr, mg/l; outletс – concentration of the respective pollutant at the outlet of the sbr, mg/l. individual emission limits for controlled pollutants which are specified in the permit for the use of water body "blue river" – hisarya for discharge of the wastewater were used for the wwtp performance evaluation. statistical analysis all experiments were performed in triplicate. the data were analyzed and presented as mean values with standard deviation. statistical analysis was conducted by using of statgraphics centurion xvi version 16.2.04 software. values are considered at a significance level of 95 %. 3. results and discussion the processes in the wwtp and the sequence are presented in the schematic flow diagram shown in fig. 1. results for average wastewater influent in the wwtp for 2013 year are presented in fig. 2. the established average wastewater daily influent for 2013 year was 2995.3±614.2 m3/d. for monitoring of the sbr operation and to calculate pollutants degree of purification, physicochemical characteristics of the wastewater influent and effluent were determined. table 1 presents physicochemical characteristic of the sbr wastewater influent. average bod5 of the inlet was 100.1±11.7 mg/l with maximum 121 mg/l and minimum 90 mg/l. average values of the inlet for cod, tn, tp and ss were 272±26 mg/l, 25.3±2.0 mg/l, 3.65±0.27 mg/l and 74.1±8.2 mg/l respectively. the results in fig. 2 and table 1 show that the actual load of the wwtp is less than the design, which allows for the treatment of additional volumes of wastewater if any expansions of the business in the town and to increase the number of tourists. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 210 fig. 2. average wastewater influent in the wwtp-hisarya for 2013 year table 1 physicochemical characteristic of the sbr wastewater influent month/ year 2013 bod5 (mg/l) cod (mg/l) tn (mg/l) tp (mg/l) ss (mg/l) january 119±23 302±34 24.5±2.4 3.3±0.5 82.6±13.3 february 121±14 310±35 23.5±2.9 3.4±0.4 76.0±17.0 march 98±7 299±40 25.0±1.4 3.4±0.3 77.8±12.6 april 117±29 282±27 23.8±1.8 3.4±0.2 75.0±13.0 may 90±3 258±26 24.4±2.7 3.7±0.2 71.0±18.0 june 92±6 261±24 23.7±2.0 3.4±0.3 64.0±12.0 july 92±9 236±28 22.0±1.9 3.7±0.3 63.0±12.0 august 93±9 285±51 28.7±3.0 3.8±0.1 80.0±30.0 september 98±9 291±23 27.2±1.2 3.7±0.1 89.0±38.0 october 95±6 257±43 26.2±2.8 3.9±0.2 75.1±13.8 november 93±6 244±37 27.0±1.9 4.0±0.2 73.8±14.8 december 93±6 239±35 27.5±1.3 4.1±0.1 61.8±16.3 values of standard parameters of the sbr treated wastewater for the investigated period of time are presented in tables 2 and 3. statistical analysis indicated that there are not significant differences (p>0.05) between the average values of physicochemical parameters determined for the effluents from sbr 1 and sbr 2, respectively. contrary, values of bod5, cod, tn, tp and ss were decreased to be significantly (p<0.05) in finally treated (outlet) wastewater (tables 2 and 3) in comparison to inlet (table 1). for example, the average bod5 decreasing was from 100.1±11.7 mg/l to 4.7±0.7 mg/l. for cod, tn, tp and ss it was from 272±26 mg/l to 18.2±1.4 mg/l, from 25.3±2.0 mg/l to 4.4±1.6 mg/l, from 3.65±0.27 mg/l to 1.67±0.15 mg/l and from 74.1±8.2 mg/l to 3.5±0.2 mg/l, respectively. the obtained results are in accordance to [4, 7, 9, 14, 17] which described such significantly decreasing of pollutants after sbr treatment of different wastewaters. table 4 presents percentage degree of purification of sbr municipal wastewater treatment. reached degree of purification 0 500 1000 1500 2000 2500 3000 3500 4000 ja nu ary fe br ua ry m arc h ap ril m ay ju ne ju ly au gu st se pte mb er oc tob er no ve mb er de se mb er a ve ra ge w as te at er fl ow , m 3/ d food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 211 for bod5 was in the range from 93.2 % to 95.9 %, average 95.1±0.6 %. for cod this degree was in the range from 92.1 % to 95.0 % (93.2±0.7 %). for tn it was from 60.0 % to 94.5 % (80.3±8.0 %). such intervals for tp and ss were from 44.5 % to 66.8 % (53.4±7.2 %) and from 93.7 % to 96.2 % (95.1±0.6 %), respectively. similar degree of purification of municipal wastewater treatment plant as follow: bod5 (91.31 %), cod (91.84 %), tn (71.40 %) and ss (88.37 %) was reported by [7]. recently, degree of purification of sbr municipal wastewater treatment of bod5 (89-98 %), cod (80 %), total ss (85 97 %), tn (>75 %) and bio-p (57-69 %) was reported by [8] also. fig. 3 represents the results for performance evaluation of sbr municipal wastewater treatment based on the average pollutant’s concentration in the wastewater and emission limit. the average values of standard parameters bod5, cod, tn and ss of treated wastewater were considerably below emission limits (25 mg/l, 125 mg/l, 15 mg/l and 35 mg/l, respectively) specified in the discharge limit. average values of tp of the effluents from sbr 1 (1.69±0.17 mg/l) and sbr 2 (1.67±0.15 mg/l) were closed to the permitted emission limit (2 mg/l). table 2 physicochemical characteristic of wastewater effluent from sbr 1 month/year 2013 bod5 (mg/l) cod (mg/l) tn (mg/l) tp (mg/l) ss (mg/l) january 5.3±0.7 17.9±8.5 7.43±0.97 1.83±0.05 4.2±1.1 february 5.4±0.8 17.5±1.5 9.40±2.20 1.85±0.07 3.6±0.5 march 4.9±0.1 18.1±1.3 5.80±2.70 1.78±0.09 3.5±0.5 april 5.5±1.3 20.1±3.2 7.95±4.98 1.87±0.13 3.7±0.3 may 4.1±0.4 19.6±1.2 3.75±1.37 1.70±0.42 3.7±0.6 june 3.8±0.3 16.9±0.1 4.72±0.48 1.81±0.12 3.4±0.4 july 4.8±0.6 18.7±1.6 4.37±1.11 1.75±0.15 3.3±0.4 august 4.1±0.7 20.8±3.8 4.11±0.76 1.72±0.14 3.6±0.4 september 4.0±0.3 18.6±3.1 3.17±0.96 1.76±0.11 3.6±0.6 october 5.1±0.5 18.2±1.5 5.07±1.79 1.51±0.03 3.6±0.5 november 4.7±0.1 17.7±1.3 3.72±0.97 1.40±0.16 3.8±0.6 december 4.7±0.1 18.8±1.2 4.85±0.48 1.36±0.16 3.9±0.6 table 3 physicochemical characteristic of wastewater effluent from sbr 2 month/year 2013 bod5 (mg/l) cod (mg/l) tn (mg/l) tp (mg/l) ss (mg/l) january 5.9±0.6 15.2±3.0 5.8±2.0 1.82±0.13 3.8±0.6 february 5.4±2.1 18.2±1.9 7.2±3.3 1.86±0.04 3.3±0.5 march 5.5±0.4 18.3±1.2 3.8±2.4 1.78±0.12 3.5±0.6 april 5.4±0.9 19.2±2.2 6.9±4.0 1.59±0.08 3.6±0.4 may 6.1±0.1 19.5±1.9 3.2±0.3 1.61±0.43 3.7±0.6 june 4.1±0.4 19.3±2.0 4.4±1.3 1.79±0.11 3.2±0.4 july 4.6±0.4 17.1±2.9 3.8±1.3 1.72±0.18 3.3±0.5 august 4.2±0.3 20.0±2.6 4.0±0.7 1.79±0.14 3.4±0.4 september 4.2±0.1 18.6±1.0 1.5±0.1 1.72±0.07 3.4±0.4 october 4.6±0.4 18.2±1.8 4.2±0.9 1.49±0.05 3.5±0.5 november 5.2±0.1 16.4±1.8 3.9±1.2 1.44±0.19 3.8±0.4 december 5.2±0.1 17.8±0.9 4.6±0.4 1.46±0.09 3.7±0.1 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 212 table 4 degree of purification of sbr municipal wastewater treatment month/year 2013 dpbod5 (%) dpcod (%) dptn (%)) dptp (%) dpss (%) sbr 1 sbr 2 sbr 1 sbr 2 sbr 1 sbr 2 sbr 1 sbr 2 sbr 1 sbr 2 january 95.5 95.0 94.1 95.0 69.7 76.3 44.5 44.8 94.9 95.4 february 95.5 95.5 94.4 94.1 60.0 69.4 45.6 45.3 95.3 95.7 march 95.0 94.4 93.9 93.9 76.8 84.8 47.6 47.6 95.5 95.5 april 95.3 95.4 92.9 93.2 66.6 71.0 45.0 53.2 95.1 95.2 may 95.4 93.2 92.4 92.4 84.6 86.9 54.1 56.5 94.8 94.8 june 95.9 95.5 93.5 92.6 80.1 81.4 46.8 47.4 94.7 95.0 july 94.8 95.0 92.1 92.8 80.1 82.7 52.7 53.5 94.8 94.8 august 95.6 95.5 92.7 93.0 85.7 86.1 54.7 52.9 95.5 95.8 september 95.9 95.7 93.6 93.6 88.3 94.5 52.4 53.5 96.0 96.2 october 94.6 95.2 92.9 92.9 80.6 84.0 61.3 61.8 95.2 95.3 november 94.9 94.4 92.7 93.3 86.2 85.6 65.0 64.0 94.9 94.9 december 94.9 94.4 92.1 92.6 82.4 83.3 66.8 64.4 93.7 94.0 fig. 3. performance evaluation of sbr municipal wastewater treatment recorded maximum values of the standard parameters for discharged wastewater (tables 2 and 3) during the investigated period of time were below emission limits also. these results indicates a significantly better performance of sbr wastewater treatment compared the requirements for the discharge into the water body and also potential of treated water for non-potable reuses, like gardening, cooling water and etc. 4. conclusion the results obtained in present research and performance evaluation of the sbr municipal wastewater treatment indicated that wwtp installed in hisarya town, bulgaria is working perfectly. the significant reduction (p<0.05) was observed in the physicochemical characteristics viz. bod5, cod, tn, tp and ss. the average removal of bod5 (95.1±0.6 %), ss (95.1±0.6 %), cod (93.2±0.7 %), tn (80.3±8.0 %) and tp (53.4±7.2 %) of municipal wastewater were recorded after sbr treatment. the values obtained of standard parameters for treated wastewater were considerably below emission limits which indicate a significantly better performance of sbr wastewater treatment compared the requirements for the discharge into the water body and also reuse potential of 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 bod5 cod tn tp ss c on ce nt ra ti on o f po llu ta nt , m g/ l influent effluent from sbr 1 effluent from sbr 2 emission limit food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 t s ve t ko p r o kop o v, d as ha m ih ay l o va, n ikol a y m ih alk o v , ph y s i c o ch e m i c a l m o n i t o ri ng a nd p e r f o r m an c e e va l u a ti o n of sb r m un i c i p al wa s t e wa t e r t r e a t m e nt , food and environment safety, volume xiii, issue 3 – 2014, pag. 207 213 213 treated water. thus it is helphul in the control of the pollution of “blu river” – hisarya. 5. acknowledgments the authors express their most sincere gratitude to the municipality and mayor of hisarya town, bulgaria for their kind assistance and permission to access the wwtp. 6. references [1]. davis m.l., water and wastewater engineering: design principles and practice. mcgraw-hill inc., usa, (2010). [2]. mahvi a.h., sequencing batch reactor: a promising technology in wastewater treatment. iran. j. environ. health sci. eng. vil. 5, no 2. 7990, (2008). [3]. de nardi i.r., del nery v., amorim a.k.b., dos santos n.g., chimenes f., performances of sbr, chemical-daf and uv disinfection for poultry slaughterhouse wastewater reclamation. desalination 269. 184-189, (2011). [4]. elmolla e.s., ramdass n., chandhur m., optimization of sequencing batch reactor operating conditions for treatment of high-strength pharmaceutical wastewater. journal of environmental science and technology vol. 5, issue 6. 452-459, (2012). [5]. janczukowicz w., szewczyk m., krzemieniewski m., pesta j., settling properties of activated sludge from a sequencing batch reactor (sbr). polish journal of environmental studies vol. 10, no. 1. 15-20, (2001). [6]. jia y., gao ch., zhang l., jiang g., effects of pre-fermentation and influent temperature on the degree of purification of cod, nh4+-n and po43—p in slaughterhouse wastewater by using sbr. energy procedia 16. 1964-1971, (2012). [7]. kumar v., chopa a.k., monitoring of physico-chemical and microbiological characteristics of municipal wastewater treatment plant, haridwar city (uttarakhand) india. journal of environmental science and technology 5(2). 109118, (2012). [8]. kumbhar s., main j., desai m., reuse potential of treated sewage from sequencing batch reactor. international journal of applied engineering research 8(7). 2011-2016, (2013). [9]. kundu p., debsarkar a., mukherjee s., treatment of slaughter house wastewater in a sequencing batch reactor: performance evaluation and biodegradation kinetics. biomed research international volume 2013, article id 134872. 11 p., (2013). http://dx.doi.org/10.1155/2013/134872. [10]. mace s. mata-alvarez j., utilization of sbr technology for wastewater treatment: an overview. ind. eng. chem. res. 41. 5539-5553, (2002). [11]. mohan s.v., rao n.ch., prasad k.k., madhavi b.t.v., sharma p.n., treatment of complex chemical wastewater in a sequencing batch reactor (sbr) with an aerobic suspended growth configuration. process biochemistry 40. 1501-1508, (2005). [12]. mojiri a., aziz h.a., aziz s.q., zaman n.q., review on municipal landfill leachate and sequencing batch reactor (sbr) technique. archives des sciences 65(7). 22-31, (2012). [13]. obaja d., mace s., mata-alvarez j., biological nutrient removal by a sequencing batch reactor (sbr) using an internal organic carbon source in digested piggery wastewater. bioresource technology 96. 7-14, (2005). [14]. sathian s., rajasimman m., radha g., shanmugapriya v., karthikeyan c., performance of sbr for the treatment of textile dye wastewater: optimization and kinetic studies. alexandria engineering journal 53. 417-426, (2014). [15]. spangi a., marsili-libelli s., nitrogen removal via nitrite in a sequencing batch reactor treating sanitary landfill leachate. bioresource technology 100. 609-614, (2009). [16]. fan l., xie y., optimization control of sbr wastewater treatment process based on pattern recognition. procedia environmental sciences 10. 20-25, (2011). [17]. wakode p.n., sayyad s.u., performance evaluation of 25 mld sewage treatment plant (stp) of kalyan. american journal of engineering research vol. 03, issue 03. 310316, (2014). [18]. yoo ch.k., villez k., van hulle s.w.h., vanrolleghem p.a., enhanced process monitoring for wastewater treatment systems. environmetrics 19. 602-617, (2008). microsoft word 16 winkler.doc 105 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 105 108 bioch em ica l as pec ts o f water deco ntam in a tion w ith o zon e iryna belykh1, sergiy samoylenko1, myron rogozynskyi2, *igor winkler3 1national technical university “khpi”, kharkiv, ukraine, ari74@meta.ua 2national technical university “khpi”, chernivtsi faculty, chernivtsi, ukraine, rmyron@ukr.net 3yu. fedkovych national university of chernivtsi, chernivtsi, ukraine, igorw@ukrpost.ua * corresponding author received 27 january 2013, accepted 5 march 2013 abstract: an article deals with the mechanism of ozone effect on microorganisms. it is known that ozone can destruct various bacteria. kinetics of bacteria inactivation under the influence of ozone depends on ozone content in the ozone-air mixture, dissolved ozone concentration, amount of ozone absorbed by bacteria, time of the microorganisms’ exposure and their type. it is suggested that in the biochemical sense, ozone acts through the catalytic oxidation of bacterial proteins and their destruction to amino acids. an influence of the oxidized products of bacteria decay on the kinetics of the residuary bacteria dying in the process of water ozonization is evaluated. it is proved that the water ozonization promotes better population health conditions. keywords: ozone, fluorescence, bovine serum albumin, conformations changes, microorganisms 1. introduction new effective technologies of the municipal wastewater decontamination have been developed in the beginning of 20th century. these technologies involve infiltration fields, jet filters with sand and broken stone filling and forced ventilation air-tanks. the latter equipment is used as the key stage at the municipal wastewater treatment plants. simple wastewater decontamination was considered as the main target of the treatment technologies before. however, better understanding of complexity of the clean water problem has been developed later and pushed more efforts towards higher quality of the water treatment and protection of all kinds of the natural water and water bodies. modern approaches to the high quality wastewater treatment assume almost complete decomposition of the organic pollution agents. according to the actual legislation, the content of the residual organic compounds in water should not exceed 10 mg/l [1]. bacterial contamination of water with municipal, meat-and-dairy, other food processing, tannery, sugar, wood-pulp, pharmaceutical, microbiological and other industrial wastewater is considered as one of the most dangerous types of the biological pollution. dangerous bacterial contamination is also generated at the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 iryna belykh, sergiy samoylenko, myron rogozynskyi, igor winkler, biochemical aspects of water decontamination, food and environment safety, volume xii, issue 1 – 2013, pag. 105 108 106 landfill areas. pollution of the drinking and service water sources with wastewater prevents normal usage of these sources and brings serious sanitary and epidemic threats as water is considered as one of the main ways of the bacterial and virus infections proliferation [1]. new type of contamination became important in ukraine after the chornobyl nuclear catastrophe, which left strong radionuclide anthropogenic pollution in many areas. this caused qualitative and quantitative changes in some natural microbiocenoses and resulted higher aggressivity of some enteropathogenic strains [2]. all these factors contribute into higher risk of contagion with various enteric infections. moreover, a chance contamination with genetically modified organisms becomes more probable and this can cause new, uninvestigated diseases [2]. besides, some bacteria can gain chlorine tolerance as a result of the genetic changes and survive after the chlorine water disinfection, which is still widely used in ukraine as a stage of the drinking water production. water chlorination has some serious shortcomings [3]: chlorine does not provide needful effectiveness against some types of microbes and ensure only selective contamination of some vital centres of bacteria. this contamination is quite slow because of the low diffusion rate of chlorine into cytoplasm. chlorine is also almost completely passive against viruses. many countries have stopped a practice of chlorination and use water ozonization instead. ozone is very effective disinfection agent and it reveals strong bactericide and virucide activity together with quite high effectiveness in decomposition of many organic pollutants to rather nontoxic products [3-5]. therefore, this work was aimed onto investigation of the destruction kinetics of some microbial taxonomic groups under effect of ozone and influence of ozone on some microbial proteins. 2. materials and methods ozone was generated by the barrier and non-barrier discharge tube generators designed in institute of cryobiology and cryomedicine problems of nas of ukraine and institute of the plasma electronics and new methods of acceleration of kharkiv physicotechnical institute. ozone concentration was measured by the spectrophotometry method through the hartley light absorption band (255 nm) value. this method can be applied to the ozone concentration measurement in the gaseous and liquid phases [6]. water samples were contaminated with the following microbial cultures escherichia coli, staphylococcus aureus, candida albicans, bacillus subtilis and then gaseous mixtures with the ozone concentration 6,87,0 mg/l were barbotaged through the samples. the barbotage time was different for various microbes [7]. it was found that the complete deactivation of the bacteria escherichia coli, staphylococcus aureus and yeast-like fungi candida albicans required 60 min of barbotage while the spore forms of bacillus subtilis required 120 min [7]. the area of ozonization is widening but details of its antimicrobic activity are still disputable. it is supposed that biochemical bactericide effect of ozone can be caused by the catalytic oxidation of the bacterial proteins. ozone exerts an impact on the entire redox system of bacteria and their protoplasm while chlorine influences ferments of the microbial cell only. this concept can be proved by the high antiviral activity of ozone. bactericide effect of low doses of ozone is rather insignificant for the bacteria cultures in the organic-free water but it jumps to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 iryna belykh, sergiy samoylenko, myron rogozynskyi, igor winkler, biochemical aspects of water decontamination, food and environment safety, volume xii, issue 1 – 2013, pag. 105 108 107 much higher level as soon as concentration of ozone reaches some critical value. then further deactivation of bacteria goes on with significantly lower intensity. for example, two separate phases can be identified in a process of escherichia coli deactivation in the bidistilled water: initial fast deactivation followed by continuous and much slower decontamination. this pattern depends on several parameters: amount of ozone consumed by bacteria, concentration of the dissolved ozone in water and content of ozone in the ozone-air mixture. it was found [6] that kinetics of the initial fast deactivation of escherichia coli is governed by the amount of ozone consumed by the bacteria. on the other hand, this parameter was found noninfluential for the second phase of deactivation. long and less active “tailoring deactivation” can be related to protection of the survived bacteria with the bacterial cells lysis products. initial consumption of ozone is very active and the majority of bacteria die out during this stage. besides, kinetics of deactivation also depends on the microbes’ nature and presence of some organic agents. they can react with ozone and form secondary products with higher bactericide activity. for instance, ethylenes can react with ozone and form hydroperoxides which reveal higher bactericide activity than ozone. in contrary, humic acids react with ozone and do not form any secondary bactericides so, amount of ozone required for effective decontamination should be increased in case any humic compounds are present in the system. the higher is concentration of the humic acids, the longer latent stage without any tangible bactericide activity will be registered after ozonization [6]. 3. results and discussion the model investigation of influence of ozone on the conformational changes in the microbial proteins (serum albumin) has been carried out to clarify details of the ozone effect on bacteria [8]. figure 1. the intrinsic fluorescence spectra of bsa after bubbling of the ozone-oxygen mixture (ozone content 7,0 mg/l) during various periods of time. 1 – control experiment (no ozone mixture bubbled); 2 – 0,5; 3 – 1; 4 – 2,5; 5 – 4; 6 – 6 min. exciting radiation wavelength – 280 nm. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 iryna belykh, sergiy samoylenko, myron rogozynskyi, igor winkler, biochemical aspects of water decontamination, food and environment safety, volume xii, issue 1 – 2013, pag. 105 108 108 the bull serum albumin (bsa) has been exposed to the high doses of ozone that should result in deactivation of microbes. bsa samples were dissolved in the bufferfree physiological solution and then the ozone-oxygen mixture with 7,0 mg/l of ozone was bubbled during various time periods. activity of bsa was measured after the bubbling using the fluorescence spectra [8]. these spectra are shown in fig. 1 for various times of the ozone exposition. the wavelength of the exciting radiation was 280 nm and the wavelength of the fluorescence maximum was 338 nm. as seen in fig. 1, the fluorescence intensity decreases after the ozone exposition and no fluorescence can be registered after 6 min long exposition [8]. 4. conclusion analysis of the fluorescence spectra shows that destruction of the protein globule results in its complete decomposition down to amino-acids. it can be supposed that ozone acts on the other bacterial proteins similarly. the results of this investigation can be helpful for better understanding of the biochemical effect of ozone on microbes, which promotes developing of more effective water decontamination ozone technologies. it is well known that environmental safety is one of the main competitive advantages of ozone. 5. references [1]. prokopov v. a., tolstopyatov g. v. and maktaz e. d. possible solutions for the kiev municipal landfills filtrate treatment, chem. and tech. of water. 17, 43-45 (1995). [2]. grigorieva l. v., korchak g. n., bey t. v and antonov m. yu. hygienic aspects of changes in the micribiocenoses under radioactive contamination, chem. and tech. of water. 17, 88-91 (1995). [3]. goncharuk v. v., potapchenko n. g. and vakulenko n. f. ozonization as a method of the drinking water treatment: possible side effects and the toxicology assessment. chem. and tech. of water. 17, 3-33 (1995). [4]. kozhinov v. f. and kozhinov i. v. ozonization of water, stroyizdat, moscow. 160 p. (1973). [5]. orlov v. a. ozonization of water, stroyizdat, moscow. 88 p. (1984). [6]. belykh i. a. and vysekantsev i. p. toxic effect of ozone on bacteria e.coli., modern. problems of toxicology 1, 49-53 (2009). [7]. belykh i. a., vysekantsev i. p. and grek a. m. toxic effect of ozone on microbes staphylococcus aureus, yield-like fungi candida albicans and spore forms bacillus subtilis, modern. problems of toxicology 2, 45-49 (2010). [8]. belykh i. a., zinchenko v. d., zinchenko o. v. and sakharov g. v. an interaction of the human serum albumin with ozone, ukr. biochem. j. 74, 21 (2002). 299 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 299 308 effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with de-oiled rice bran *tanuja srivastava1, d.c. saxena2, renu sharma3 1* bhai gurdas institute of engineering & technology, sangrur, punjab, india, tanusriva @ yahoo.co.in 2deptt. of food technology, sliet, longowal, punjab, india, dcsaxena@yahoo.com 3deptt. of applied sciences, bgiet, sangrur, punjab, india, renu.sharma6286@gmail.com *corresponding author received november 28th 2014, accepted december 29th 2014 abstract: mechanical properties of ready-to-eat extrudates are perceived by the consumers as quality criteria. texture quality of any product has a strong influence on the sensory evaluation as well as on the acceptability of the product. the main texture characteristics influencing the product acceptability are crispness, elasticity, hardness and softness. in the present work, we investigate one of the most important texture characteristics of extrudates i.e. hardness. a five-level, four-factor central composite rotatable design was employed to investigate the effect of temperature, screw speed, feed moisture content and feed composition mainly rice bran content and their interactions, on the mechanical hardness of extrudates. among these, feed moisture was found to be a prominent factor affecting the product hardness. it was found that with the increase of feed moisture content, the rice bran proportion leads to increase in hardness of extrudates whereas the increase of temperature leads to decrease of hardness of product. a good agreement between the predicted (26.4899 n) and actual value (28.73n) of the response confirms the validation of response surface methodology (rsm)-model. keywords: texture, crispness, barrel temperature, screw speed, feed moisture, response surface methodology 1. introduction deoiled rice bran, a co-product of rice milling and rice bran oil extraction, is rich source of proteins, carbohydrates, dietary fibers like beta-glucan, pectin and gum and micronutrients like oryzanols,tocopherols, tocotrienols, phytosterols [1-4]. after extraction of oil, the remained defatted rice bran possesses unique functional and nutritional properties and has great potential to add value to food products. it keeps humans healthy due to low fat content. rice bran obtained from different coloured varieties of rice are rich in antioxidants compounds namely polyphenols, carotenoids, vit.e and tocotrienol help in preventing the body tissue damage and oxidative damage of dna [5]. due to presence of proteins, dietary fibers and bioactive compounds [13], rice bran helps in reducing the risk of coronary heart disease, lowering blood cholesterol [7,8], decrease of artherosclerosis disease [9] and it posses laxative effect [6]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 300 although the nutritional profile and health benefits of deoiled rice bran have been recognized, but it is still used as animal food. very few studies concerning the incorporation of rice bran into the food products have been available. keeping in view, the nutritional composition of rice bran, it must be provided opportunity for incorporation into extruded products. the substitution of rice bran in food products will increase the nutritional value as well as provide health benefits to consumers. so, value added, edible food products can be obtained by utilizing defatted rice bran through extrusion cooking. the technology of extrusion is a latest, continuous, high temp., short-time processing, used in various food industries due to several advantages like fast process, significant reduction in energy consumption, more production and final products at lower prices. this process involves the use of corn meal, rice, wheat or potato flour to produce variety of snack foods in many shapes and variety of textures. published literature shows the use of extrusion process in cereals, vegetables, fruits and legumes to improve their nutritional profile. the extrusion processing of food is growing day by day due to its economical production with attractive texture, size and shape [10]. twin screw extruder is the most commonly used for extrusion process. the present work was designed to implement deoiled rice bran in the preparation of ready-to-eat extrudates. rsm was used to optimize the process conditions for the production of easily consumable, highly nutritive, physically attractive extrudates with good taste and flavor and also used to study the effect of operating conditions like moisture content, barrel temperature, screw speed etc. upon the mechanical properties of the product. 2. material and methods experimental design response surface methodology (rsm) was adopted in the experimental design [11]. the main advantage of rsm is reduced number of experimental runs needed to provide sufficient information for statistically acceptable result. a five-level, four-factor central composite rotatable design was employed. table 1 shows independent variables selected for the experiments. the variables and their levels were chosen by taking trials of samples. hardness was considered as response variable. the five levels of the process variables were coded as -2, -1, 0, 1 and +2 [11]. and design in coded (x) form and at the actual levels (x) is given in table 2 & table 3. preparation of sample ingredients for the production of highly nutritious ready-to-eat snack food consisted of deoiled rice bran, corn flour, rice flour. deoiled rice bran (drb) used for present study was procured from m/s. ap solvex ltd., dhuri. corn flour (cf) and rice flour (rf) were purchased from local market sangrur, punjab, india. ingredient formulations for extrusion products are given in table 3. in the blend preparation rice bran (rb) at levels of 100, 200, 300, 400g were used while corn flour at level of 10% was kept constant in all samples. the moisture was adjusted by sprinkling the stilled water in all the dry ingredients. all the ingredients were weighed and then mixed in the food processor with mixer attachments for 20 min. this mixture was then passed through a 2 mm sieve to reduce the lumps formed due to addition of moisture. after mixing samples were stored in polyethylene bags at room temperature for 24h [12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 301 the moisture content of all the samples was estimated before extrusion by using the hot air oven method [13]. preparation of extrudates extrusion trials were performed using a corotating twin-screw extruder (g.l. extrusion systems pvt. ltd., delhi). the main drive is provided with 7.5 hp motor (400 v, 3ph, 50 cycles). the extruder is provided with standard design of screw configuration, automatic cutting knife fixed on rotating shaft and a temperature sensor. it was kept running for suitable period of time to stabilize the set temperatures and sample were then poured in to feed hopper and the feed rate was adjusted to 4kg/h for easy and non-choking operation. the die diameter of 4 mm was selected as recommended by the manufacturer for such product. the product was collected at the die end and packed in for proper storage. table 1 values of independent variables at five levels of the ccrd design independent variables unco ded levels in coded form -2 -1 0 +1 +2 feed composition (%) x1 75:15:10 70:20:10 65:25:10 60:30:10 55:35:10 feed moisture (%) x2 13 14 15 16 17 screw speed (rpm) x3 275 300 325 350 375 die temperature (°c) x4 100 110 120 130 140 feed composition (rf:rb:cf) table 2 experimental design in coded form for response surface analysis code ‘0’ is for centre point of the parameter range investigated ‘±1’ for factorial points, and ‘±2’ for star points; x1 feed composition (%), x2 feed moisture(%), x3 screw speed (rpm) and x4 die temperature (°c) coded variables combin ations replicati ons no. of expt. x1 x2 x3 x4 ±1 ±1 ±1 ±1 16 1 8 ±2 0 0 0 2 1 2 0 ±2 0 0 2 1 2 0 0 ±2 0 2 1 2 0 0 0 ±2 2 1 2 0 0 0 0 1 6 6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 302 table 3 experimental combination in coded and uncoded levels for extruded snacks sr. no. coded variables uncoded variables x1 x2 x3 x4 x1 feed proportion (rf:rb:cf) x2 moisture content(%) x3 screw speed (rpm) x4 temperature (°c) 1 -1 -1 -1 -1 70:20:10 14 300 120 2 1 -1 -1 -1 60:30:10 14 300 120 3 -1 1 -1 -1 70:20:10 16 300 120 4 1 1 -1 -1 60:30:10 16 300 120 5 -1 -1 1 -1 70:20:10 14 350 120 6 1 -1 1 -1 60:30:10 14 350 120 7 -1 1 1 -1 70:20:10 16 350 120 8 1 1 1 -1 60:30:10 16 350 120 9 -1 -1 -1 1 70:20:10 14 300 130 10 1 -1 -1 1 60:30:10 14 300 130 11 -1 1 -1 1 70:20:10 16 300 130 12 1 1 -1 1 60:30:10 16 300 130 13 -1 -1 1 1 70:20:10 14 350 130 14 1 -1 1 1 60:30:10 14 350 130 15 -1 1 1 1 70:20:10 16 350 130 16 1 1 1 1 60:30:10 16 350 130 17 -2 0 0 0 75:15:10 15 325 125 18 2 0 0 0 55:35:10 15 325 125 19 0 -2 0 0 65:25:10 13 325 125 20 0 2 0 0 65:25:10 17 325 125 21 0 0 -2 0 65:25:10 15 325 125 22 0 0 2 0 65:25:10 15 325 125 23 0 0 0 -2 65:25:10 15 325 125 24 0 0 0 -2 65:25:10 15 325 125 25 0 0 0 0 65:25:10 15 325 125 26 0 0 0 0 65:25:10 15 325 125 27 0 0 0 0 65:25:10 15 325 125 28 0 0 0 0 65:25:10 15 325 125 29 0 0 0 0 65:25:10 15 325 125 30 0 0 0 0 65:25:10 15 325 125 evaluation of textural property of extrudates mechanical properties of the extrudates were determined by a crushing method using a ta – xt2 texture analyzer (stable micro systems ltd., godalming, uk) equipped with a 500 kg load cell. an extrudate 40 mm long was compressed with a probe sms – p/75mm diameter at a crosshead speed 5 mm/sec to 3 mm of 90% of diameter of the extrudate. the compression generates a curve with the force over distance. the highest first peak value was recorded as this value indicated the first rapture of snack at one point and this value of force was taken as a measurement for hardness [12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 303 statistical analysis of responses the responses such as hardness for different experimental combinations were related to the coded variables (xi, i=1,2,3 and 4) by a second degree polynomial (equation 1) as given below: y = β0 + β1x1 + β2x2 + β3x3 + β4x4 + β11x12 + β22x22 + β33x3 2 + β44x4 2 + β12x1.x2 + β13x1.x3 + β14x1.x4 + β23x2.x3 + β24x2.x4 + β34x3.x4 + ε (1) where x1, x2, x3 and x4 are the coded values of rice flour, corn flour and rice bran mixture, moisture content of feed (%), screw speed (rpm) and temperature of die (0c). the coefficients of the polynomial were represented by β0 (constant), β1, β2 , β3, β4 (linear effects); β12 , β13 , β14 , β23 , β24 , β34 (interaction effects) ; β11 , β22, β33, β44 (quadratic effects) ; ε (random error). multiple regression analysis was used for data modeling and statical significance of the terms was examined by analysis of variance. design expert 6.0 (version 6.0, by stat-ease inc., usa) was used for statistical analysis of the data to check the adequacy of the regression model, r2, adjusted r2, adequate precision and ficher,s f-test were used [11]. regression coefficients were used for statistical calculations and for the generation of three dimensional surface plots. 3. results and discussion variation of responses such as hardness of extrudates with independent variables (corn flour, rice flour and rice bran proportion, moisture content, die temperature and screw speed) were analysed. a complete second order model (equation 1) employed for its adequacy to decide the variation of response with independent variables. for visualization of variation in hardness with respect to processing variables, three dimensional response surfaces were drawn using design expert 6.0 (version 6.0, by stat-ease inc., usa) was used . diagnostic checking of the fitted models regression analyses indicated that the fitted quadratic model accounted for more than 85.7% of the variations in the experimental data, which was found to be highly significant. all main effects, linear, quadratic and interaction were calculated. the regression coefficients are shown in table 4. the correlation coefficients for the response was 0.9353 was quite high for response surfaces. table 4 estimated coefficients of the fitted quadratic equation for hardness based on t-statistic coefficients hardness coefficients hardness x0 34.58*** x42 0.062 ns x1 2.43*** x1 x2 -0.37 ns x2 1.99*** x1 x3 -0.59 ns x3 0.26 ns x1 x4 -1.68** x4 -1.04** x2 x3 0.92** x12 -0.68** x2 x4 -0.27 ns x22 0.78** x3 x4 -0.54 ns x32 -1.38** r2 = 0.9353 *p < 0.1 , **p < 0.05 , ***p<0.0001, ns : non significant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 304 analysis of variance analysis of variance was calculated to evaluate the goodness of the model. the fvalue for model was 15.47 (table 5) and significant at 99% level. on the other hand, the lack of fit (2.0) was found to be non-significant. on this basis, it can be concluded that the selected models adequately represent the data for the response. table 5 analysis of variance for hardness source df f value hardness x0 14 15.47*** x1 1 71.66*** x2 1 48.29*** x3 1 0.83 ns x4 1 13.13** x1 2 1 6.41** x2 2 1 8.51** x3 2 1 26.62** x4 2 1 0.053 ns x1 x2 1 1.12 ns x1 x3 1 2.84 ns x1 x4 1 22.81** x2 x3 1 6.86** x2 x4 1 0.58 ns x3 x4 1 2.35 ns residual 15 2.0 lack of fit 10 pure error 5 total 29 *p < 0.1 , **p < 0.05 , ***p<0.0001 ns : non significant effect of process variables on texture of extrudates (hardness) the textural property of extrudates was determined by measuring the force (newton) required to break the extrudates. hardness of the extrudates varied between 26.49 and 41.53 n. table 6 and 7 shows the coefficients of the model and other statistical attributes of hardness. regression model fitted to experimental results of hardness (table 7) shows that model f-value of 15.47 was significant (p<0,0001) whereas lack-of-fit f-value of 2.0 was not significant (p > 0.2314). the fit of model expressed by the coefficient of determination, r2 (0.9352), indicating that 93.52 % of the variability of the response could be explained by the model, whereas adjusted r2 (0.8748) and adequate precision (14.786) suggests that model may be used to navigate the design space. the quadratic model for hardness (h) in terms of coded levels of variables was developed as follows: h = 34.58 + 2.43xl + 1.99x2 +0.26x3 1.04x4 0.68x1 2 + 0.78x2 2 1.38x3 2 + 0.062x4 2 0.37xix2,0.59xix3 -1.68xix4 + 0.92x2x3 0.27x2x40.54x3x4 (2) the analysis of variance of above equation predicting that hardness of the extrudates had highly significant (p < 0.0001) positive linear affect of feed composition (x1) and feed moisture (x2) whereas, barrel temperature (x4) had significant (p<0.005) negative linear effect on hardness of product. with the increase in deoiled rice bran and moisture content, hardness of product also increases. this happens due to rupture of gas cells by dietary fiber content of rice bran [14]. while increase of temperature leads to decrease of hardness. the results are similar to findings of [15]. the quadratic terms of feed composition (x12) and screw speed (x32) had significant (p<0.05) negative quadratic effect indicating convex shaped variation on product hardness. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 305 table 6 analysis of variance for hardness (h). source coefficient of model terms sum of squares mean square df f value prob>f model 34.58 427.89 30.56 14 15.47 <0.0001*** x1 2.43 141.57 141.57 1 71.66 <0.0001*** x2 1.99 95.40 95.40 1 48.29 <0.0001*** x3 0.26 1.65 1.65 1 0.83 0.3754 x4 -1.04 25.94 25.94 1 13.13 0.0025** x12 -0.68 12.67 12.67 1 6.41 0.0230** x22 0.78 16.81 16.81 1 8.51 0.0106** x32 -1.38 52.59 52.59 1 26.62 <0.0001*** x42 0.062 0.10 0.10 1 0.053 0.8217 x1 x2 -0.37 2.21 2.21 1 1.12 0.3067 x1 x3 -0.59 5.61 5.61 1 2.84 0.1128 x1 x4 -1.68 45.06 45.06 1 22.81 <0.0002*** x2 x3 0.92 13.56 13.56 1 6.86 0.0193** x2 x4 -0.27 1.15 1.15 1 0.58 0.4573 x3 x4 -0.54 4.63 4.63 1 2.35 0.1465 *significant at p<0.1, ** significant at p<0.05,*** significant at p<0.001, df: degrees of freedom table 7 analysis of variance results of equation 2 response source sum of squares df mean squares f-value p-value hardness regression 427.89 14 30.56 15.47 <0.0001* lack of fit 23.70 10 2.37 2.0 0.2306 pure error 5.93 5 1.19 residual 29.63 15 1.98 total 457.52 29 r2-value 0.9352 adjusted r2 0.8748 adeq. precision 14.786 *significant at p<0.05, df: degrees of freedom f-value for interaction term of feed composition and barrel temperature (x1x4) was 22.81 and p value 0.0002 predicting the term is significant. since coefficient of term is negative, it will show convex shaped variation with the change in value of variables. response surface plot (fig.1), showing that hardness increased with increasing feed composition (x1) and decreased with increasing barrel temperature (x4). therefore, a crispy texture was obtained with increasing temperature due to decrease in hardness. this result is in agreement with [16, 17]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 306 fig. 1. response suface plot for the hardness value of extrudates as function of feed composition (x1) and barrel temperature (x4) the interaction term of feed moisture and screw speed (x2x3) was significant at 95% confidence level (f-value 6.88 and p-value 0.0192). coefficient of x2x3 was positive so causes concave shaped variation in product hardness. response surface plot (fig.2) showing variation in hardness with increase in feed moisture and screw speed. with increasing feed moisture content (x2), the hardness of the extrudates increased. the results are same as observed by [18]. fig.2. response surface plot for the hardness value of extrudates as a function of feed moisture content (x2) nad screw speed (x1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 307 the purpose of verification was done to test the adequacy of the response surface model for predicting response values. the predicted value of hardness obtained is 26.4899 n while actual value is 28.73n. a good agreement between the predicted and actual value of the response confirms the validation of rsm model in the preparation of ready-to-eat extrudates enriched with de-oiled rice bran. 4. conclusion. the use of extrusion process for the production of extrudates not only utilize rice milling by-product but also add value to commercialized product with health benefits. the application of response surface methodology serves as the useful tool in the present research. analysis of regression model equations helps to interpret the relationship between the effects of extrusion operating variables on the mechanical properties of the product. temperature of the barrel section, screw speed, moisture content of raw feed and feed composition i.e. rice bran proportion were found to have significant effect on the hardness of extrudates. among these factors, feed moisture content is found to have prominent effect upon the product hardness. increasing feed moisture content and rice bran proportion leads to increase in hardness of extrudates while increasing temperature leads to decrease of hardness of product. 5. references [1]. hernandez n., rodriguez-alegria m.e., gonzalez f., lopez-munguia a., enzymatic treatment of rice bran to improve processing, journal of the american oil chemists’ society, 77. 177-180, (2000). [2]. jiang y., wang t., phytosterols in cereal by-products, journal of the american oil chemists’ society, 82. 439-444, (2005). [3]. piironen v., lindsay d.g., miettinen t.a, toivo j. and lampi a.m., plant sterols: biosynthesis, biological function and their importance to human nutrition, j sci food agric., 80. 939-966, (2000) . [4]. hu w., wells j.h., tai-sun s. and godber j.s., comparison of isopropanol and hexane for extraction of vitamin e and oryzanols from stabilized rice bran, journal of the american oil chemists’ society, 73.1653-1656, (1996). [5]. frei m. and becker k.a., novel correlation between lipid components and all-transβ-carotene in differently colored rice landraces, submitted to journal of the science of food and agriculture. [6]. saunders r.m., the properties of rice bran as a food stuff, cereal foods world, 35. 632-636, (1990). [7]. chotimarkorn c., silalai, oxidative stability of fried dough from rice flour containing rice bran powder during storage, lwt, 41. 561-568, (2007). [8]. kahlon t.s., chow f.i.,sayre r.n., cholesterol lowering properties of rice bran, cereal foods world, 39. 99-103, (1994). [9]. saunders r.m., rice b.. composition and potential food uses, food rev int., 1. 465-495, (1985). [10]. smith a.c. , singh n., new applications of extrusion technology, indian food ind., 15. 1423, (1996). [11]. montgomery d.c., design and analysis of experiments, new york wiley, pp. 416-419, (2001). [12]. stojceska v., ainsworth p., plunkett a., ibanoglu e., ibanoglu s., cauliflower by-products as a new source of dietary fiber, antioxidants and proteins in cereal based ready-to-eat expanded snacks, journal of food engineering, 87. 554-563, (2008). [13]. ranganna s., handbook of analysis and quality control for fruit and vegetable products, india: tata mc graw hill publishing company limited, new delhi, (1995). [14]. charunuch c., limsangouan n., prasert w. , wongkrajang k., optimization of extrusion conditions for ready-toeat breakfast cereal enhanced with defatted rice food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 tanuja srivastava, d.c. saxena, renu sharma, effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with deoiled rice bran, food and environment safety, volume xiii, issue 4 – 2014, pag. 299 – 308 308 bran, international food research journal, 21. 713722, (2014). [15]. bhattacharya s., choudhary g.s., twin screw extrusion of rice flour: effect of extruder length-to-diameter and barley temperature on the extrusion parameters and product characteristics, j. food pro. preserve., 18. 389-94, (1994). [16]. ryu g.h. , walker c.e., the effects of extrusion conditions on the physical properties of wheat flour extrudates, starch – starke, 47. 33-36, (1995). [17]. duizer l.m., winger r.j., instrumental measures of bite force associated with crisp products, journal of texture studies, 37. 1-15, (2006). [18]. ding q.b., ainsworth p., plunkett a., tucker g., marson h., (2006). the effect of extrusion conditions on the functional and physical properties of wheat based expanded snacks, j. food eng., 73. 142-148, (2006). 253 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 253 – 264 er yth ros ine b in the envi ron m ent. re mov al proc ess es *laura carmen apostol1, maria gavrilescu2,3 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania; laura.apostol@fia.usv.ro 2gheorghe asachi technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof.dr.docent d. mangeron street, 700050 iasi, romania 3academy of romanian scientists, 54 splaiul independentei, ro-050094 bucharest, romania mgav@tuiasi.ro * corresponding author received march 11st 2013, accepted september 5th 2013 abstract: this paper presents a comparison of the resultes obteined for different methods used for the decolorization of the food dye erythrosine b. erythrosine b is a red odorless powder used in food industry as a colloring substance. erythrosine b, also known as e 127, consists essentially of disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl) benzoate monohydrate and subsidiary coloring matters together with water, sodium chloride and sodium sulphate as the principal uncolored components. the methods considered in this paper were sorption, biodegradation and photodegradation. sorption demonstrated good removal efficiency in the presence of low-cost activated carbon from agro-waste but the treatment increases the operation cost. because of erythrosine b toxicity aerobic biodegradation processes showed to be inefficient in the most studies. considering this, erythrosine b degradation could be performed by photodegradation process using an adequate catalyst in order to reduce the operation cost. keywords: food dyes, performance comparison, removal processes 1. introduction food coloring is a substance that is added to food or drink to change its color. erythrosine b (fd & c red no. 3) is the only xanthene dye listed for use in food and ingested drugs. it is exclusively authorised for use in cocktail and candied cherries, and bigarreaux cherries (94/36/ec). the paper presents an overview including: erythrosine b presentation in terms of its use as a xanthene food coloring substance; the most used methods used for the possible decolorization of aqueous solutions containing the food dye erythrosine b. 1.1. erythrosine b (e 127), a coloring substance in eu synthetic food colors, or coal-tar/ petroleum colors, represent a special class of dyes with application in food industries. the main dye categories used in food industry are: azo dyes, such as: tartrazine (fd&cyellow no. 5)., ponceau (fd&c red no. 4), sunset yellow (fd&c yellow no. 6); food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r ia gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 254 non azo dyes, like brilliant blue (fd&c blue no. 1), erythrosine (fd&c red no. 3), indigotin (fd&c blue no. 2). unlike other food additives, these compounds need to be tested and certified by the official chemical examination bodies. dyes approved by the food dye and coloring act (fd&c) are coal tar derivatives, contain aromatic rings. erythrosine b (e 127) is a red odorless powder or granules with a calculated log p (octanol-water) of 4.95 at 25°c, which is soluble in water (≤ 9% w/w) and ethanol [2]. the molecular weight of e 127 is 879.84 g mol-1. it’s full chemical name is disodium 2-(2,4,5,7-tetraiodo-6-oxido-3oxoxanthen-9-yl)benzoate. erythrosine b structural formula is presented in fig. 1. at least 78 synonyms of the compound are in use. as european food safety authority (2011) related the most commonly synonyms used in literature for erythrosine are: ci food red 14, fd & c red no. 3, c.i. 45430, ins no. 127 and erythrosine sodium [1]. erythrosine b is a red odorless powder or granules with a calculated log p (octanolwater) of 4.95 at 25°c, which is soluble in water (≤ 9% w/w) and ethanol [2]. specifications have been defined in the directive 2008/128/ec7 and by jecfa (2006) (table 1) [3]. figure 1. chemical structure of erythrosine b erythrosine b (e 127) consists essentially of disodium 2-(2,4,5,7-tetraiodo-6-oxido3-oxoxanthen-9-yl) benzoate monohydrate and subsidiary coloring matters together with water, sodium chloride and sodium sulphate as the principal uncolored components. the phosphorescence of erythrosine b is due to the xanthene ring with four iodine atoms. erythrosine b has phosphorescence emission time scale of 10-5s to 10-3s corresponding to motion in glassy environment and is sensitive to oxygen [4]. table 1 specification for erythrosine according to commission directive 2008/128/ec and jecfa (2006) [3] purity commission directive 2008/128/ec jecfa (2006) inorganic iodides calculated as sodium iodide ≤ 0.1% ≤0.1% fluorescein ≤20 mg kg-1 <20 mg kg-1 subsidiary colouring matters ≤ 4.0% ≤4.0% water insoluble matter ≤0.2% ≤0.2% ether extractable matter ≤0.2%a ≤0.2%b arsenic <3 mg kg-1 lead ≤10 mg kg -1 ≤2 mg kg-1 zinc ≤50 mg kg -1 mercury ≤ 1 mg kg -1 cadmium ≤ 1 mg kg -1 heavy metals as pb ≤ 40 mg kg -1 loss on drying at 135°c together with chloride and sulphate calculated as sodium salts ≤13% tri-iodoresorcinol ≤0.2% ≤0.2% 2-(2,4-dihydroxy-3,5-diodobenzoyl) benzoic acid ≤0.2% ≤0.2% a from a solution of ph from 7 through 8 b from a solution of ph not less than 7 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 255 erythrosine b, like other xanthene compounds, exhibits unusual spectroscopic and photochemical properties like huge absorption coefficient or molar extinction coefficients (λmax: ε524nm = 67,282 m−1 cm−1 for erythrosine b; ε510nm = 60,826 m −1 cm−1 for eosin y) in the visible region and a high tendency for intersystem crossing to produce a photochemically active triplet excited state [5]. 1.2. short overview on the synthesis process and analysis erythrosine b, the tetraiodoanalogue of fluorescein, is produced by iodination (the reaction of iodine or potassium iodate in an ethanolic solution converted to the sodium salt) of fluorescein followed by the condensation of resorcinol with phthalic anhydride (eq. 1) [6]. erythrosine b may be converted to the corresponding aluminium lake by reacting aluminium oxide with coloring matter [1]. undried aluminium oxide is usually freshly prepared by reacting aluminium sulphate or aluminium chloride with sodium carbonate or sodium bicarbonate or aqueous ammonia. following lake formation, the product is filtered, washed with water and dried [3]. in the literature few methods for the determination of erythrosine b in foods are described. these methods include high performance liquid chromatography (hplc) and capillary electrophoresis [7]. 1.3. stability, reaction and fate in the environment a low number of data on the fate and reaction of erythrosine b in food is available. in general, the dyes used as additives are changeable in combination with oxidizing/reducing compounds in food. dyes depend on the existence of a conjugated unsaturated system. any compound which modifies the system will affect the dye (e.g. oxidising or reducing agents, sugars, acids, and salts) [8]. for exemple when cherries colored with erythrosine b are stored in uncoated steel cans, fluorescein is readily formed (the production of fluorescein from erythrosine b occurs in the presence of iron and/or tin and free organic acid as a result of electrochemical reduction in the can [9]). 1.4. use and toxicology authorized use levels have been defined in the directive 94/36/ec8 on colors for use in foodstuffs (table 2). as a synthetic food coloring substance erythrosine b is permitted in the eu for certain limited uses only. table 2 give the main foodstuffs that are permitted to contain erythrosine b up to specified maximum permitted levels (mpls) set by directive 94/36/ec. erythrosine b has been used as a food dye since its approval by the u.s. department of agriculture in 1907. it is used in maraschino cherries, sausage casings, oral drugs, baked goods, and candies. erythrosine b has been evaluated several times by jecfa and by the scientific committee for food. (1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 256 table 2 food dye certification [10] food dye pounds of total dye certified percentage of total blue 1 711 659 4.7 blue 2 550 883 3.7 citrus red 2 1 764 0 green 3 15 817 0.1 orange b 0 0 erythrosine b 216 235 1.4 red 40 6 203 374 41.3 yellow 3 3 765 331 25 yellow 6 3 338 351 23.7 total 15 016 634 100 table 3 maximum permitted usage levels of erythrosine b according to european parliament and council directive 94/36/ec [11] foodstuffs maximum permitted level (mg/kg) cocktail cherries and candied cherries 200 bigarreaux cherries in syrup and in cocktail 150 the us code of federal regulation, states that fd&c red no.3 (erythrosine b) may be safely used as a coloring in general foods in amounts consistent with good manufacturing practice (gmp) [12]. erythrosine b is permitted in the usa for general use in sweets and foods marketed to children such as candies, popsicles, cake frosting and cake-decorating gels. the report provided by united states food and drug administration (us fda) indicate that under experimental conditions erythrosine b at high dose levels (4% in the diet) can affect the level of circulating thyroid hormones in rats, thus leading to an increase in the incidence of thyroid tumors. the response of the us fda in 1990 was withdrawal of permission to use erythrosine b lakes (salts), but not erythrosine b, in all foods, drugs and cosmetics, and to withdraw the use of erythrosine in cosmetics and externally applied drugs [1]. in australia, the code restricts the use of erythrosine b in foods. it is used just to preserved cherries (maraschino cherries), cocktail cherries or glace cherries. the dye is used prior to processing. food standards australia new zealand (fsanz) have proposed extending the permitted uses to products such as icing and frostings used in other foods that are more widely consumed (e.g. cakes, biscuits, fancy breads) [13]. in eu, erythrosine b is used in manufacturing photographic plates, for microscopic stains, pharmaceuticals and cosmetics. in june 2010, the scientific committee on consumer safety published an opinion on erythrosine use in toothpaste products. glaxosmithkline (gsk) considered erythrosine b safe for consumers when used as a colorant in toothpaste products with a maximum concentration of 0.0025% (25 ppm) and estimated exposure from this use to be 0.0002 mg/kg bw/day (table 4) [1]. table 4 toxicological parameter of xanthene dye erythrosine b [13] accepted daily intake = 0 0.1 mg kg-1 bw-1 day-1 noel = 60 mg person-1 day-1 (mg kg-1 bw-1 day-1) toxicological parameter value [mg kg-1] ld50 (rat), intraperitoneal ldlo (rat), intravenous ldlo (rabbit), intravenous ldlo (mouse), oral ld50 (mouse), intravenous 300 200 200 2500 370 2. removal of erythrosine b from wastewater food once was colored only with natural dyes, coming mainly from various plants. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 257 by the 19th century, colors began to derive from other chemicals so that they came into use with sometimes serious health consequences. erythrosine b is still permanently listed for use in ingested drugs and food, such as baked goods, cherries, dairy products, desserts, dietary supplements, food seasonings, jellies, jams, and vegetable products. acid dye like erythrosine b can be lost in effluents in percentage varying from 5 to 20%, since low adsorption occurs for acid and reactive dyes, while high adsorption occurs with basic and direct dyes and high to medium for disperse dyes [14; 15]. ryvolova et al. [16] and ramakrishnan et al. [17] found that the concentration of colorants in foods and drugs, respectively were between 0.3 and 0.03 mg ml−1. in this situation, it was estimated that a high amount of dye results as waste and has to be treated (table 5) [16; 17]. erythrosine b contained in aqueous solutions was subjected to different decolorization methods, including physical, chemical, and biological processes. some of them are discusses below. table 5 amount of erythrosine b contained in different products product erythrosine b (µg ml-1) cherry 235 cream biscuits 316.65 gems 177.8 candies 36.74 ibuprofen 5.6 (µg/tablet) 2.1. sorption sorption is widely used for dye removal from wastewaters. activated carbon, the universal adsorbent used for pollutant removal, although reasonably effective at removing dyes from aqueous streams, needs either regeneration or disposal, once it is fully loaded. other limitations are the high cost and 10-15% loss of adsorbent during reactivation [14]. in recent years, many investigations have been undertaken to evaluate inexpensive alternative materials of biological origin (biosorbents) as potential adsorbents for dyes, which include feathers [18], de-oiled mustard [19], montmorillonite [20], bottom ash and de-oiled soya [21], fungi [22]. due to its low cost and widespread availability, biomass has been extensively investigated as sorbent for removing color, with promising results. in this case, biomass refers to dead plant and animal matter resulting from agriculture, forest, fermentation and shellfish by-products or wastes. other unconventional biosorbents used for sorptive removal of different dye classes are pinus bark powder [23], hazelnut shells [24], nut shells [25], or bagasse pith [26]. the main mechanisms found responsible for the decolorization of aqueous solutions containing dyes are adsorption and ion exchange. agricultural wastes can be good sorbents for the removal of pollutants, since they involve reduced costs for operation and waste disposal, providing a cheap alternative to existing commercial activated carbons. vegetal hull/husk was applied as a raw material for producing sorbents with the following advantages: (1) appropriate chemical composition; (2) low cost; (3) high dispersity; (4) scaly structure and developed porous surface ensuring a high surface-to-volume ratio [27]. several studies reported chemical modifications of celluloses and lignocelluloses extracted from cotton waste, sawdust and corn stalks in order to increase the number of active centers for dye immobilization on sorbent surface [28]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 258 table 6 sorbents tested for xanthene dye removal dye initial dye conc. (x 10-5 mol l-1) sorbent q (x 10-5 mol g-1) ref. rhodamine b 2 200 fungi 8.15 [22] 5 50 activated carbon 4.57 [33] 4 400 algae 16.7 [34] 10 50 baryte 34.22 [35] eosin y sun-dried jute fiber 4.55 [36] 1 70 chitosan hydrobeads 11.57 [37] erythrosine b 1 – 6 bottom ash de-oiled soya 2.37 1.20 [21] 1 – 6 hen feathers 2.31 [18] 1 9 de-oiled mustard activated carbon 13.15 19.9 [19] 5 10 montmorillonite 1.5 [20] 5 40 activated carbon 47.28 [38] adsorption of reactive dyes by sawdust chars and activated carbon [29]; methylene blue by waste rosa canina sp. seeds [30]; anionic dyes by modified coir pith [31]; and methylene red by acid-hydrolysed beech sawdust [32] has also been reported. table 6 presents a summary of the biosorbents used for xanthene dye removal. 2.2. biodegradation biological processes involve the aerobic (presence of oxygen) or anaerobic (absence of oxygen) degradation of organic substances by microorganisms. anaerobic biological reduction of dyes has been investigated from different perspectives, i.e. degradation and color removal. anaerobic reducing conditions found in the environment include sediments at the bottom of streams of certain sections of landfills where there is no oxygen. anaerobic bioremediation of soluble dyes to undergo decolorization by breaking them into less toxic compounds has been widely investigated [14]. the decolorization in the case of azo dyes occurs due to azo reduction [39]. additional carbon is required for decolorization to proceed the process at a viable rate: this is converted into methane, hydrogen sulphide and carbon dioxide [40]. this additional carbon source may be a limiting factor from a commercial perspective. in many situations, decolorization of reactive dyes under anaerobic conditions is due to the action of a ‘reductase’ enzyme. if complete mineralization occurs, conversion of organic contaminants into methane and oxygen leads to production of bioga,s which is a major attraction because of heat, power and reduced energy costs [14]. in the case of toxic compounds interactions with living microorganisms two pathways can be considered: i) when biodegradation starts with chemical interactions, the sorption rate limits the biodegradation; ii) when biodegradation starts after some lag period, the chemical has time to sorb before degradation begins, and slow sorption causes biodegradation to be desorption-rate limited [41]. however, the toxicity of certain dyes inhibits the complete mineralization. xanthene dyes have been reported to be toxic to various species tested in laboratory conditions. several reports on enzymatic oxidation of xanthene dyes are available [42; 43; 44; 45; 46; 47]. enzymatic reduction of xanthene compounds was not reported. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 259 borgerding and hites (1994) reported the presence of xanthene dye erythrosine b in wastewater from food and cosmetic industry and affirmed that the dye was adsorbed by the sludge [48]. itoh and yatome (2004) studied the decolorization of six xanthene dyes by a white rot fungus, coriolus versicolor but only three of them were degraded [45]. table 7 presents a summary of xanthene dyes removal by enzymatic mechanism. 2.3. photodegradation because processes like adsorption or biodegradation can generate secondary pollutants, aop like photocatalytic degradation represented an alternative for the removal of hazardous xanthene dye from effluents [49]. aops are effective for detoxification and mineralization of dyes from wastewaters and research studies have shown promising results as these processes appear to have the ability to completely decolorize and partially mineralize the pollutants from dye-industry in short reaction time [50; 5]. table 7 xanthene dyes removal by biodegradation dye initial dye conc. (x 10-5 mol l-1) biomass r (%) r (μm min−1 mg−1)* ref. fluorescein 10 fungus coriolus versicolor 85.0 10.2 [45] 4 amino fluorescein 10 fungus coriolus versicolor 95.0 6.7 5 amino fluorescein 10 fungus coriolus versicolor 91.9 7.2 rhodamine b 10 fungus coriolus versicolor 0 5 laccase mediator system (lms) 80% [46] 40 lip of fungus phanerochaete chrysosporium 46.0 [44] rhodamine 123 10 fungus coriolus versicolor 0 [45] rose bengal fungus strain aspergillus wentii 89.3 [43] erythrosine b 10 mold neurospora crassa [47] aerobic sludge 0 [48] 30 anaerobic granular sludge 20 – 70 1.5 h-1* [38] *first order degradation rate table 8 xanthene dye removal by chemical degradation process dye initial dye conc. (x 10-5 mol l-1) catalyst (uv) r (%) ref. eosin y 10 tio2 p25 78 [51] rhodamine b 5 0.65 g l -1 tio2 80 [52] 1 sio2@tio2 90 [53] rhodamine 6g 5 1 g l-1 zno 80 [54] fluorescein zno 44.4 [55] phloxine b 10 0.04 g l-1 tio2 82 [19] erythrosine b 10 2 g l-1 tio2 p25 35 [56] 10 g l-1 zno 60 10 0.5 g l-1 tio2 aeroxide 99 [57] 20 electrochemical degradation 95 [58] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 260 photodegradation is based on the degradation of a molecule mediated by the absorption of photons. photocatalytic degradation of several xanthene contaminants using large bandgap semiconductor particles (such as tio2, zno, wo3) has been extensively studied. table 8 shows data on xanthene dye photodegradation in the presence of different catalysts. 3. comparison of physico-chemical and bological processes applied for the decontamination of aqueous sollutions polluted with erythrosine b physico-chemical and biological methods that are effective in the elimination of dye have been studied. each process has its own constraints in terms of cost, feasibility, practicability, reliability, stability, environmental impact, sludge production, operational difficulty, pretreatment requirements, the extent of the organic removal and potential toxic byproducts. even if a process is reported to be successful in decolorizing a particular effluent, the same may not be able to other type of colored aqueous solution. the use of a single process may not be efficient in the complete decolorization of the polluted effluent. the comparison of different processes used for dyes removal from aqueous solution is of interest to establish the conditions for removal performance (the most efficient experimental conditions for the elimination of compound from solution) and to provide useful information for the essential aspects of the combination of different processes [59]. sorption, biodegradation and photodegradation processes where tested to evaluate erythrosine b removal from aqueous solution. table 9 summarizes the conditions and the performances of the processes obteined for 300 mg l-1 erythrosine b (the higer dye concentration tested) removal from aqueous solution. sorption process resulted in 65% – 90% removal efficiency at 50°c and natural ph of the solution (ph=5.6) for a contact time of around 21h [60]. photodegradation using fixed or suspended tio2 yielded very high color removal (95%), within the same contact time like sorption study also at natural ph and resulting nontoxic products. anaerobic biodegradation treatment has demonstrated to be affected by erythrosine b toxicity at high concentration (0.4 mm) and low color removal was achieved for the same biomass amount as with sorption study (20 g l-1) [38]. sorption process demonstrated to have good removal efficiency especially at higher temperature (50°c) where the dye amount adsorbed per sorbent unit was between 14.1 and 16.4 mg g-1 for bh and psh respectively. sorption of erythrosine b demonstrated a ~5-fold increase of dye adsorption in the presence of low-cost activated carbon in the study conducted by jain and sikarwar (2009) but agro-waste treatment increases the operation cost [19]. table 9 erythrosine b removal using physico-chemical and biological methods under different condition process applied experimental condition amount of dye uptake (mg g-1) removal efficiency (%) observation sorption using agrowaste ci=300 mg l -1 contact time = 21h 14.1 16.4 mg g-1 (bh and psh) 65 – 80 (bh and psh) csorbent= 20 g l -1 t=50°c anaerobic biodegrdation 3 mg g -1 30 cbiomass= 20 g l -1 t=37°c photocatalytic degradation 95 ctio2= 0.5 g l -1 ti=25°c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 261 the results obtained for biodegradation study confirmed the biomass capacity to adsorb erythrosine b based on the biomass color at the end of the study. similar result was obtained by chamam et al. (2007) for a sulphuric textile dye, cassulfon cmr, using activated sludge for the batch sorption test [61]. for the range of erythrosine b concentrations studied (30 and 300 mg l-1) dye photodegradation in the presence of tio2 represent the most efficient method than sorption and biodegradation in the removal of ery b in aqueous solution. ong et al. (2009) compared the sorption and photodegradation of bb3 and ro16 and observed also a higher efficiency for photodegradation using tio2 that in the sorption study using modified rice hull [62]. the decolorization rate was observed to be different for each system, for the first 60 min of reaction the relative order evaluated was: sorption > photodegradation >> biodegradation. the combination of different methods for the removal of dyes from aqueous solution sounds to be an ideal solution for the present need of time. a combination of aop and biological treatment showed better results for the color removal from dyeing effluents containing hazardous compounds [63]. the scientific knowledge represented an important element for managing the direction and improvement of techniques applied for dye removal from effluents. in view of the need for a technically and economically satisfying treatment technology, an abundance of technologies were proposed and tested [40]. 4. conclusions erythrosine b or fd&c red no.3 is a cherry-pink/red synthetic coal tar dye. it is most popularly used as food coloring and a host of other applications, such as printing inks, biological stain, and for extractionphotometric determination of k, cd, pb, mn, zn, ag. erythrosine b was the subject of different decolorization methods applied to aqueous solutions containing dye. sorption demonstrated good removal efficiency in the presence of low-cost activated carbon from agro-waste but the treatment increases the operation cost. because of erythrosine b toxicity aerobic biodegradation processes showed to be inefficient in the most studies. considering this, erythrosine b degradation could be performed by photodegradation process using an adequate catalyst in order to reduce the operation cost (with irradiation time). 5. acknowledgments this work was supported by the romanian national authority for scientific research, cncs-uefiscdi, project number pn-iiid-pce2011-3-0559, contract 265/2011 and brain project (id 6681, european social found and romanian government). 6. references [1] european food safety authority, efsa panel on food additives and nutrient sources added to food (ans), scientific opinion on the reevaluation of erythrosine (e 127) as a food additive, efsa journal, 9. 1854, on line at: www.efsa.europa.eu/efsajournal (2011) [2] molinspiration, cheminformatics on the web, on line at: www.molinspiration.com, (2007) [3] jecfa (joint fao/who expert committee on food additives), combined compendium of food additive specifications all specifications monographs from the 1st to the 65th meeting (1956-2005), fao jecfa monographs series. 1-3, (2006) [4] you y., modulation of molecular mobility in sucrose-based amorphous solids detected by phosphorescence of erythrosin b, the state university of new jersey phd thesis, (2007) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 262 [5] fithol m.z.m., application of experimental design for photodegradation of rose bengal (acid red 94), universiti malaysia pahang, on line at: http://umpir.ump.edu.my/737/1/muhamad_zulh elmi_mohamad_fithol.pdf, (2009) [6] mai h.t., brodie d.l., meyers m.b., baldo a.l., krantz z., weisz a., determination of 2,4,6-triiodoresorcinol and other side reaction products and intermediates in the color additive fd & c red no. 3 (erythrosine) using high-performance liquid chromatography, food additives and contaminants, 23. 547–551, (2006) [7] yoshioka n., ichihashi k., determination of 40 synthetic food colors in drinks and candies by high-performance liquid chromatography using a short column with photodiode array detection, talanta, 70. 14081413, (2007) [8] scotter m.j., castle l., chemical interactions between additives in foodstuffs: a review. food additives and contaminants: part a, 21. 93–124, (2004) [9] dickinson d., raven t.w., stability of erythrosine in artificially coloured canned cherries, journal of the sciences of food and agriculture, 13. 650-652, (1962) [10] food and drug administration (fda), on line at: http://www.fda.gov/downloads /food/foodsafety/retailfoodprotection/foodc ode/foodcode2009/ucm188533.pdf, (2009) [11] european parliament and council directive 94/36/ec of 30 june 1994 on colours for use in foodstuffs. 10.09.1994. 13, (1994) [12] us code of federal regulation, code of federal regulations, part 74 listing of color additives subject to certification, 1. sec. 74.303 fd&c red no. 3, (2006) [13] food standards, initial assessment report, application a603, erythrosine b erythrosine in food colouring preparations, on line at: http://www.foodstandards.gov.au/_srcfiles/a60 3%20erythrosine%20iar%20final.pdf#sear ch=%22a603%20final%22, (2008) [14] joshi m., purwar r., developments in new processes for colour removal from effluent, rev. prog. color, 34. 58-71, (2004) [15] hlihor r.m., apostol l.c., smaranda c., pavel v.l., căliman f.c., robu b.m., gavrilescu m., bioavailability processes for contaminants in soils and their use in risk assessment, environmental engineering and management journal, 8. 1199-1206, (2009) [16] ryvolova m., taborsky p., vrabel p, krasensky p., preisler j., sensitive determination of erythrosine and other red food colorants using capillary electrophoresis with laser-induced fluorescence detection, journal of chromatography a, 1141. 206–211, (2007) [17] ramakrishnan s.p., lakshmi j.b., surya p.r., estimation of synthetic dye erythrosine in food stuff and formulation and effect of dye on the protein binding of drug in bsa, der pharmacia lettre, 3. 361-373, (2011) [18] gupta v.k., mittal a., kurup l., mittal j., adsorption of a hazardous dye, erythrosine, over hen feathers, journal of colloid and interface science, 304. 52–57, (2006) [19] jain r., sikarwar s., adsorptive removal of erythrosine dye onto activated low-cost deoiled mustard, journal of hazardous materials, 164. 627–633, (2009) [20] rytwo g., huterer-harari r., dultz s., gonen y., adsorption of fast green and erythrosin-b to montmorillonite modified with crystal violet, journal of thermal analysis and calorimetry, 84. 225–231, (2006) [21] mittal a., mittal j., kurup l., singh a.k., process development for the removal and recovery of hazardous dye erythrosine from wastewater by waste materials—bottom ash and de-oiled soya as adsorbents, journal of hazardous materials, 138. 95 – 105, (2006) [22] das s.k., bhowal j., das a.r., guha a.k., adsorption behavior of rhodamine b on rhizopus oryzae biomass, langmuir, 22. 72657272, (2006) [23] ahmad r., studies on adsorption of crystal violet dye from aqueous solution, journal of hazardous materials, 171. 767–773, (2009) [24] ferrero f., dye removal by low cost adsorbents: hazelnut shells in comparison with wood sawdust, journal of hazardous materials, 142. 144-152, (2007) [25] de oliveira brito s.m., carvalho andrade h.m., soares l.f., de azevedo r.p., brazil nut shells as a new biosorbent to remove methylene blue and indigo carmine from aqueous solutions, journal of hazardous materials, 174. 84 – 92, (2010) [26] gad h.m., el-sayed a.a., activated carbon from agricultural by-products for the removal of rhodamine-b from aqueous solution, journal of hazardous materials, 168. 1070-1081, (2009) [27] abdullin i.s., kudinov v.v., highly effective sorbents obtained by treating agrowaste products in cold plasma, journal of guangdong non-ferrous metals, 15. 6-13, (2005) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 263 [28] mtui g.y.s., recent advances in pretreatment of lignocellulosic wastes and production of value added products, african journal of biotechnology, 8, 1398-1415, (2009) [29] gan q., allen s.j., matthews r., activation of waste mdf sawdust charcoal and its reactive dye adsorption characteristics, waste management, 24. 841-848, (2004) [30] gürses a., doar ç., karaca s., açikyildiz m., bayrak r., production of granular activated carbon from waste rosa canina sp. seeds and its adsorption characteristics for dye, journal of hazardous materials, 131. 254-259, (2006) [31] namasivayam c., sureshkumar m.v., anionic dye adsorption characteristics of surfactant-modified coir pith, a waste lignocellulosic polymer, j. appl. poly. sci., 100. 1538-1546, (2006) [32] batzias f.a., sidiras d.h., dye adsorption by prehydrolysed beech sawdust in batch and fixed-bed systems, bioresour. technol., 98. 1208-1217, (2007) [33] vasu a.e., studies on the removal of rhodamine b and malachite green from aqueous solutions by activated carbon, ejournal of chemistry, 5. 844-852, (2008) [34] hii l.s., yong s.-y., wong c.-l., removal of rhodamine b from aqueous solution by sorption on turbinaria conoides (phaeophyta), j appl phycol, 21. 625–631, (2009) [35] vijayakumar g., yoo c.k., elango g.k.p., dharmendirakumar m., adsorption characteristics of rhodamine b from aqueous solution onto baryte, clean, 38. 202 – 209, (2010) [36] porkodia k., kumar k.v., equilibrium, kinetics and mechanism modeling and simulation of basic and acid dyes sorption onto jute fiber carbon: eosin yellow, malachite green and crystal violet single component systems, journal of hazardous materials, 143. 311-327, (2006) [37] chatterjee s., chatterjee s., chatterjee b.p., das a.r., guha a.k., (2005), adsorption of a model anionic dye, eosin y, from aqueous solution by chitosan hydrobeads, journal of colloid and interface science, 288, 30–35. [38] apostol l.c., pereira l., pereira r., gavrilescu m., alves m.m., biological decolorization of xanthene dyes by anaerobic granular biomass, biodegradation, 23. 725–737, (2012) [39] dubrow s.f., boardman g.d., michelsen d.j., chemical pretreatment and aerobic-anaerobic degradation of textile dye wastewater, in: environmental chemistry of dyes and pigments, reife a, freeman h.s. (eds.), wiley-interscience n.y., (1996) [40] anjaneyulu y., sreedhara chary n., samuel suman raj d., decolourization of industrial effluents – available methods and emerging technologies – a review, reviews in environmental science and bio/technology, 4. 245–273, (2005) [41] karapanagioti h.k., gossard c.m., strevett k.a., kolar r.l., sabatini d.a., model coupling intraparticle diffusion sorption, non-linear sorption and biodegradation processes, journal of contaminant hydrology, 48. 1-21, (2001) [42] krylov s.n., chebotareva a.b., peroxidase-catalyzed co-oxidation of indole-3acetic acid and xanthine dyes in the absence of hydrogen peroxide, febs lett, 324. 6–8, (1993) [43] chan li, jinghua bai, youqi liu, identification and properties of xanthene dye decolorization of aspergillus wentii wehmer hd_1, mycosystema, 1-11, (1999) [44] lan j., huang x., hu m., li y., qu y., gao p., wu d., high efficient degradation of dyes with lignin peroxidise coupled with glucose oxidase, j biotechnol, 123. 483–490, (2006) [45] itoh k., yatome c., decolorization and degradation of xanthene dyes by a white rot fungus, coriolus versicolor, journal of environmental science and health part a toxic/hazardous substances & environmental engineering, a39. 2383–2389, (2004) [46] khammuang s., sarnthima r., mediator –assisted rhodamine b decolourization by tramates veriscolor laccase, pakistan j biological sci, 12. 616 – 623, (2009) [47] jesus g.j., corso c.r., campos a., franchetti s.m.m., biodegradation of erythrosin b dye by paramorphic neurospora crassa 74a, brazilian arch biol technol, 53. 473-480, (2010) [48] borgerding a.j., hites r.a., identification and measurement of food and cosmetic dyes in a municipal wastewater treatment plant, environ. sci. technol., 28. 1278–1284, (1994) [49] vinu r., madras g., environmental remediation by photocatalysis, journal of the indian institute of science, 90. 189-230, (2010) [50] rauf m.a., ashraf s.s., fundamental principles and application of heterogeneous photocatalytic degradation of dyes in solution, chemical engineering journal, 151. 10–18, (2009) [51] saquib m., muneer m., photocatalytic degradation of two selected textile dye food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 l a ur a c a r me n ap o s to l , ma r i a gav r ile sc u. er yt h r os i ne b i n t h e e nvi r o nm e nt . c o mpa r is o n o f r e mo va l p r oc e s s e s, fo o d a nd e nv i r o nm e nt s a f e t y, v o l u me x ii, i ss ue 3 – 2 01 3 , p a g. 25 3 2 6 4 264 derivates, eosin yellowish and prosaniline, in aqueous suspension of titanium dioxide, journal of environmental science and health, a38. 2581-2598, (2007) [52] soares e.t., lansarin m.a., moro c.c., a study of process variabiles from the photocatalytic degradation of rhodamine b, brazilian journal of chemical engineering, 24. 29-36, (2007) [53] wilhelm p., stephan d., photodegradation of rhodamine b in aqueous solution via sio2@tio2, journal of photochemistry and photobiology a: chemistry, 185. 19-25, (2007) [54] kansal s.k., singh m., sud d., studies on photodegradation of two commercial dyes in aqueous phase using different photocatalysts, journal of hazardous materials, 141. 581-590, (2007) [55] bardhan m., mandal g., ganguly t., interaction and photodegradation characteristics of fluorescein dye in presence of zno nanoparticles, journal of nanoscience and nanotechnology, 11. 3418-3426, (2011) [56] uddin m.m., hasnat m.a., samed a.j.f., majumdar r.k., influence of tio2 and zno photocatalysts on adsorption and degradation behaviour of erythrosine, dyes and pigments, 75. 207 – 212, (2007) [57] pereira l., pereira r., oliveira c.s., apostol l., gavrilescu m., pons m.n., zahraa o., alves m.m., uv/tio2 photocatalytic degradation of xanthene dyes, photochemistry and photobiology, 89. 33–39, (2013) [58] jain r., bhargave m., sharma n., electrochemical degradation of erythrosine in pharmaceuticals and food product industrial effluent, journal of scientific and industrial research, 64. 191-197, (2005) [59] hsing h.-j., chiang p.-c., chang e.-e., chena m.-y., the decolorization and mineralization of acid orange 6 azo dye in aqueous solution by advanced oxidation processes: a comparative study, journal of hazardous materials, 141. 8–16, (2007) [60] apostol l.c., pereira l., alves m., gavrilescu m., agro waste used as natural sorbents for acid red 51 uptakes, proceedings of the 3rd international conference on advanced materials and systems, incdtpincpi, september 16-18, 2010, bucharest – romania, p. 351-356. (2010) [61] chamam b., heran m., amar r.b., grasmick a., comparison of textile dye treatment by sorption and membrane bioreactor, environmental technology, 28. 1325-1331, (2007) [62] ong s.t., lee c.k., zainal z., a comparison of sorption and photodegradation study in the removal of basic and reactive dyes, australian journal of basic and applied sciences, 3. 3408-3416, (2009) [63] harrelkas f., paulo a., alves m.m., el khadir l., zahra o., pons m.n., van der zee f.p., photocatalytic and combined anaerobic–photocatalytic treatment of textile dyes, chemosphere, 72. 1816–1822, (2008) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 contents: 1. utilization of experimental design for specific surface area optimization of a pillared bentonite ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot 284 290 2. chemical composition of agaricus bisporus and pleurotus ostreatus fruiting bodies and their morphological parts natalya cherno, sophya osolina, aleksandra nikitina 291 299 3. study of the mechanism of the retarding effect on bread staling by using natural gums svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko 300 310 4. research on chemical composition of cow raw milk sorina ropciuc 311 315 5. microbial contamination of the drinking water distribution system in suceava county, romania three years of monitoring (2010-2012) florin bârlă, lenuţa ciornei (dudici), maria poroch – seriţan 316 – 321 6. effect of citrus fibers addition on wheat flour dough rheological properties silvia mironeasa, georgiana gabriela codină 322 327 7. polyphenol content and antioxidant activity of commercial beers from the romanian market mircea oroian, cristina damian, ana leahu, oana gherasim 328 331 8. effects of work-related factors on workers' health in bulgaria dobromir kralchev, donka taneva, tsvetko prokopov 332 341 9. variation of nitrite content in meat products during the shelf life sonia amariei, sorina ropciuc, domitian mugurel scripca 342 346 10. variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum) ana leahu, cristina damian, mircea oroian,veronica miclescu, sorina ropciuc 347 – 353 11. study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods silviu-gabriel stroe 354 358 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 12. statistical studies on the lexicon used in sensory evaluation of soy-added beef hamburger cristina-elena hrețcanu , alice-iuliana roșu, ioana zelezneac 359 368 13. portable equipment for advanced characterization of food texture. first part equipment georg gutt, sonia amariei, mircea adrian oroian, cristina elena hrețcanu 369 375 14. on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra marine arsen hayriyants 376 381 15. author instructions i v 16. subscription information vi 186 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 186 191 lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers octavian baston1, *octavian barna1 1dunarea de jos university of galati, 111, domneasca street, 800201, galati, romania, phone: 0336 130 177, fax: 0336 130 281, octavian.baston@ugal.ro , octavian.barna@yahoo.com * corresponding author received: may 18th 2013, accepted june 15th 2013 abstract: we isolated lactic acid bacteria from the rhizospheric soil of dahlia tubers. lactic acid was produced by batch fermentation of dahlia hydrolysate. the selection of the best homolactic acid productive strain was made by biotechnological characteristics: ph tolerance, lactic acid yield and lactic acid productivity. the selected strains produced lactic acid at maximum yield after 48 hours of fermentation. the productivity was better after 48 hours of fermentation. for all investigated strains, the lbd2 strain showed the best lactic acid production yield of 6.30 ± 0.02 after 96 hours of fermentation and was the most acid-tolerant bacteria, due to the ph value which was 3.3±0.06. the lbd2 lactic acid productivity after 48 hours of fermentation was 0.10 g· l-1· h-1, being the highest value obtained of all the strains studied. keywords: inulin, polyfructans, fructose, acid hydrolysis. 1. introduction polyfructans (fructosans or fructans) are found as reserve carbohydrates in plants, being in high amounts in jerusalem artichoke, dahlia and chicory [1]. inulin, levan and graminan are polyfructans, the difference between those chemicals being due to the type of bonds between the fructose [2]. dahlia sp. is a decorative plant that contains polyfructans in the tubers. polyfructans, and especially inulin, can be chemically or enzymatically hydrolyzed to fructose and sucrose. these are fermentable sugars that can be transformed into lactic acid or other substances by fermentation. lactic acid is a substance with high importance in human life. it has applications in food, pharmaceutical, textile, leather, and other chemical industries. it is recognized as safe for human consumption and in food industry is used as acidulant, flavoring, buffering agent or inhibitor of bacterial spoilage in a wide variety of processed foods. the advantages of the biotechnological and chemical production are the optical purity of isomers, low cost of substrates, low production temperature, and low energy consumption [3, 4]. lactobacillus acidophilus, l. amylovorus, l. casei, l. fermentum, l. johnsonii, l. gasseri, l. paracasei, l. plantarum, l. rhamnosus, l. delbrueckii can use fermentable sugars (especially fructose) as carbon source [5, 6]. these bacteria are called lactic acid bacteria (or lab). the technique of lactic acid fermentation is important because it can reduce the time of fermentation or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 octavian baston, octavian barna, lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers, food and environment safety, volume xii, issue 2 – 2013, pag. 186 191 187 increase the yield of lactic acid. so, it can be used in the separate hydrolysis and fermentation, the simultaneous saccharification and fermentation or combined hydrolysis and fermentation. our goals are to isolate the homolactic acid bacteria from the rhizospheric soil of dahlia tubers and to test the isolated strains for lactic acid production. 2.materials and methods materials the polyfructans rich feedstock used in this study was dahlia flour. dahlia singer tubers were purchased from s.c. agrosel s.r.l., romania. dahlia flour was produced in the laboratory by cleaning and cutting the roots followed by freezing at -70°c in platinum 500 freezer (as biomedical, germany), drying using alpha 1-4 ld plus lyophilizer (martin christ, germany) and finally grinding with vc2011 grinder (victronic, prc). hydrolysis 3% (w/v) suspension was prepared by mixing the dahlia flour with distilled water. for chemical hydrolysis the ph of dahlia flour was subsequently adjusted to 2 using 98%, 1n and 0,1n sulphuric acid solutions, then heated at 100 ± 2°c for 30 minutes. the ph was measured using a ph meter s20 (mettler toledo, usa). to reduce sugars, the hydrolysate was cooled at 23 ± 2°c and the ph was increased until 6.0 ± 0.2 using 33%, 1n and 0.1n sodium hydroxide solutions. the hydrolysate was then inoculated with bacteria. lactic acid bacteria isolation the lactic acid bacteria strains were isolated from rhizospheric soil of dahlia tubers. soil samples were collected with sterile spoons, and saved into clean bags. two grams of soil sample were inoculated in 100 ml mrs (deman, rogosa and sharpe) broth and then incubated at 37°c, for 48 hours in a bf 4000 incubator (binder gmbh, germany). samples were serially diluted and plated onto mrs agar supplemented with 1% calcium carbonate using the double-layer method, as described in barbu [7]. the mrs agar and mrs broth and calcium carbonate were purchased from sigma-aldrich, germany. only the colonies showing a clear halo on mrs agar were selected. the isolates were tested for characteristics of cell and colony morphology to certify the existence of lactic acid bacteria, according to tofan [8]. their isolation was carried out in pure cultures in mrs broth, according to tofan [8]. preservation of isolates all strains were stored in the laboratory freezer platinum 500 (as biomedical, germany), at -70°c in mrs broth supplemented with 10% (v/v) glycerol. biotechnological characterization of isolated lactic acid bacteria the type of fermentation has been determined using the liquid mac cleskey medium according to barbu [7]. the lactic acid bacteria that showed homolactic fermentation were isolated from the rhizospheric soil of dahlia roots and were identified as lbd1 to lbd3. as fermentation medium, we used the dahlia flour chemical hydrolysate prepared as described above. the hydrolysate was then pasteurized at 80°c for 30 minutes using stericell 111 oven (mmm, germany) and cooled at 21 ± 2°c. the hydrolysate was inoculated with 1% of each isolated lactic acid bacteria and immediately incubated at 37°c using a bf 4000 incubator. the fermentations were conducted in 100 ml flat bottom flasks. the lactic fermented samples were taken every 24 hours and analyzed for ph, reducing sugars and acidity. the conversion yield of substrate to product and the productivity were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 octavian baston, octavian barna, lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers, food and environment safety, volume xii, issue 2 – 2013, pag. 186 191 188 determined according to pirt [9]. the determinations were made in triplicate. physical and chemical analysis the dry matter was determined by a standard drying method in an oven at 105°c to constant mass, according to aoac official methodology of analysis [10]. the concentration of fructose was estimated by 3,5-dinitrosalicylic acid method using 6505 uv-vis spectrophotometer (jenway, uk), according to miller [11]. fructose was used to establish a standard curve. the polyfructans amount was determined using fructan assay procedure for the measurement of fructo-oligosaccharides (fos) and fructan polysaccharide (megazyme international, ireland), according to mccleary and blakeney [12]. the romanian standard sr 90:2007 [13] was used for ph determination. the lactic acid was determined at λ = 470 nm with hydroquinone using the spectrophotometer 6505 uv-vis, according to the method described in banu [14]. all the chemicals used were of analytical grade. all the determinations were made in triplicate. 3.results and discussion three lactic acid bacteria strains were isolated from rhizospheric soil of dahlia tubers. the colonies distinguished clearly because they had around them a clear halo, while the rest of the medium in the plate was opalescent. the halo, that is a transparent surface around the colonies, is due to the production of lactic acid by the isolated bacteria. table 1 presents different characteristics of the isolates. table 1. characteristics of the isolated bacteria bacteria colony characteristics cell morphology cells image lbd1 3 mm cream circular colony, convex, opaque, smooth long curved rods, rounded ends, single, pairs, short chains lbd2 2-3 mm cream circular colony, convex, opaque, smooth long rods, rounded ends, single, pairs, in palisades lbd3 2 mm whitecream colony, convex, glistening, opaque short rods, rounded ends, single, pairs, in palisades the separate hydrolysis and fermentation technique was used for the lactic acid production, which consisted in 30 minutes of acid hydrolysis, followed by 72 hours of lactic acid fermentation. fructose, glucose food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 octavian baston, octavian barna, lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers, food and environment safety, volume xii, issue 2 – 2013, pag. 186 191 189 and sucrose formed after hydrolysis were fermented into lactic acid by bacteria isolated from the rhizospheric soil of dahlia tubers. the composition of the dahlia flour and the yield of hydrolysis are presented in table 2. table 2. chemical composition of the dahlia flour before and after hydrolysis (values presented as mean ± standard deviation) product dry matter (%) flour composition flour hydrolysate polyfructans hydrolysis yield (%) fructose (% dry weight) polyfructa ns (% dry weight) fructose (% dry weight) dahlia flour 94.36 ± 2.17 1.20 ± 0.24 74.27 ± 3.78 37.90 ± 1.92 51.03 the initial dahlia flour polyfructans (inulin and oligosaccharides) amount is 74.27 ± 3.78 g/100 g flour. the amount of fructose after chemical hydrolysis increased from 1.2 to 37.9 g/100 g flour. also, the hydrolysis yield was of 51.03 %. usually, the yield of the acid hydrolysis of polyfructans from tuber plants to fructose is around 50%. razmovski [15] stated that 30 minutes and a ph = 2.0 were needed for 52 % hydrolysis of jerusalem artichoke inulin. our findings comply with the ones from the scientific literature due to the identical hydrolysis parameters used. figure 1.ph variation of lactic acid fermented dahlia hydrolysate after 96 hours of lactic acid fermentation with bacteria isolated from rhizospheric soil of dahlia tubers we observed a ph variation as presented in figure 1. all the studied microorganisms are resistant to low ph value, but lbd2 are produced after 24 and 48 hours of fermentation in a more acidic media compared with the other microorganisms. also, it is tolerant to lowest ph value of the fermentation medium (ph = 3.3±0.06). figure 2.lactic acid yield variation for dahlia hydrolysate the reduced value of ph is proportionally with the increasing value of the lactic acid in the fermentation media because of the lactic acid accumulation. in figure 2 we food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 octavian baston, octavian barna, lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers, food and environment safety, volume xii, issue 2 – 2013, pag. 186 191 190 can observe that the maximum lactic acid yield is produced by lbd2, increasing from 2.16 ± 0.6 g/l after 24 hours of fermentation to 6.30 ± 0.2 g/l after 96 hours. lbd2 presented the best lactic acid yield of the homolactic acid bacteria studied. lbd3 had the lowest lactic acid yield of all the homolactic acid bacteria studied. for the lactic acid production we used batch fermentation and separate hydrolysis and fermentation techniques. it is known the fact that in batch cultures lactic acid concentrations can be higher than in continuous cultures [16]. comparing the lactic acid yield produced by lbd2 with ones produced by batch fermentation [16] we can say that our values are very low because the researchers used fermentation media enriched with nutrients or they used lactic acid bacteria selected for lactic acid production or another substrate for fermentation than dahlia hydrolysate. figure 3. lactic acid productivity from dahlia hydrolysate the productivity of dahlia hydrolysate, as shown in figure 3, increased for lbd1 and lbd2 after the first 48 hours of fermentation and decreased until the end of lactic acid fermentation. this is due to the increasing in the period of time between 48 and 96 hours of fermentation simultaneously with the slow accumulation of lactic acid in the fermentation media. lbd2 showed the best lactic acid productivity after 48 hours of fermentation with a value of 0.10 ± 0.01 g·l-1·h-1. comparing the productivity of the homolactic bacteria isolated, the lowest values were registered by lbd3. 4.conclusions we isolated three strains of homolactic acid bacteria from rhizospheric soil of dahlia tubers. lbd2 showed the best biotechnological production of lactic acid using batch fermentation and separate hydrolysis and fermentation technique, with no other nutrients added to the fermentation medium. also, it has high toleration to acid environment. it can be used to produce lactic acid from chicory hydrolysate. further studies needs to be made to improve the lactic acid production by enrichment of the fermentation medium with nutrients. 5.acknowledgements this work has benefited from financial support through the 2010 posdru/89/1.5/s/52432 project „organizing the national interest postdoctoral school of „applied biotechnologies” with impact on romanian bioeconomy”, project cofinanced by the european social fund through the sectoral operational programme human resources development 2007-2013. 6. references [1] chi z.m., zhang t., cao t.s., liu x.y., cui w., zhao c.h., biotechnological potential of inulin for bioprocesses, bioresource technol., 102. 4295-4303, (2011) [2] anan´ina n.a., andreeva o.a., mycots l.p., oganesyan e.t., standardization of inulin extracted from dahlia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 octavian baston, octavian barna, lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers, food and environment safety, volume xii, issue 2 – 2013, pag. 186 191 191 single tubers and some physicochemical properties of inulin, pharmaceutical chemistry journal, 43. 157-159, (2009) [3] john r.p., nampoothiri k.m., pandey a., fermentative production of lactic acid from biomass: an overview on process developments and future perspectives, appl. microbiol. biotechnol., 74. 524–534, (2007) [4] hofvendahl k., hahnhägerdal b., l-lactic acid production from whole-wheat starch hydrolysate using strains of lactobacilli and lactococci, enzyme microb. tech., 20. 301–307, (1997) [5] makras l., van acker g., de vuyst, l., lactobacillus paracasei subsp. paracasei 8700:2 degrades inulin-type fructans exhibiting different degrees of polymerization, appl. env. micro., 71. 6531-6537, (2005) [6] patil s.s., kadam s.r., bastawde k.b., khire j.m., gokhale d.v., production of lactic acid and fructose from media with cane sugar using mutant of lactobacillus delbrueckii ncim 2365, lett. appl. microbiol., 43. 53–57, (2006) [7] barbu v., phenotypical characterization of several lactic acid bacteria strains isolated from wheat’s epiphyte microbiota, rom. biotechnol. lett., 13. 4074-4085, (2008) [8] tofan c., bahrim g., nicolau a., zara m., food microbiology. techniques and laboratory analysis, agir, bucharest, 34-61 p, 192-195 p, (2002) [9] pirt s.j., principles of microbe and cell cultivation, blackwell scientific publications, oxford, 4-14 p, (1985) [10] aoac, official methods of analysis of aoac international, gaithersburg, md, usa, (2000) [11] miller g.l., use of dinitrosalicylic acid reagent for determination of reducing sugar, anal. chem., 31. 426-428, (1959) [12] mccleary b.v., blakeney, a.b., measurement of inulin and oligofructan, cereal foods world, 44. 398–406, (1999) [13] sr 90:2007, wheat. methods of analysis, romanian standard, (2007) [14] banu c., ionescu a., berza, m., control methods and techniques for fish and fish products, dunărea de jos university of galati, galati, 131 p, (1991) [15] razmovski r.n., šćiban m.b., vučurović v.m., bioethanol production from jerusalem artichoke by acid hydrolysis, rom. biotechnol. lett., 16. 6497-6503, (2011) [16] wee y.j., kim j.n., ryu h.w. biotechnological production of lactic acid and its recent applications, food technol. biotech., 44. 163–172, (2006) stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiii, issue 4 31 december 2014 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xi, issue 2 22 june 2012 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), nationaltechnological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius,lithuania norbert kreuzinger vienna university of technology, institute for water quality,resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic ofmoldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal:@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts:@fia.usv.ro mailto:gabriela.constantinescu:@fia.usv.ro mailto:cristina.hretcanu:@fia.usv.ro звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 54 in ves t ig at io n o f th e pro c ess o f s tor in g p ack ag ed pum pk in in mod ified a tm osphe r e *stefan stefanov1, hristo hristov1, donka stoeva1, zapryana denkova1, radosveta nikolova1 1university of food technologies, plovdiv, bulgaria, stvstefanov@yahoo.com *corresponding author received 10 december 2011, accepted 5 february 2012 abstract: the article presents experimental studies with packaged in modified atmosphere and stored refrigerated grated pumpkin and chopped pumpkin. we have used three types of gas environment: 30% со2 / 70% n; 100%со2 and vacuum. we have drawn up time temperature curves during refrigerator storage. we have defined the variation of со2 in the packages during storage. on the base of microanalysis we have defined the shelf life. keywords: modified atmosphere, cold storage, shelf life, pumpkin 1. introduction fruits and vegetables are still alive after being harvested. their shelf-life is defined by several factors, such as harvest ripeness, store temperature, content of the gas environment, type and material of the packaging, availability of microorganisms and others [1, 2, 3, 4]. their storing in refrigeration units increase the possibility of longer quality lasting periods of the products. the packaging in modified atmosphere is one of the ways for additional increase the shelf-life (1) or allows their storing at higher temperature. the defining of shelf-life of a product is a complex investigation of the store temperature, the gas environment content and microflora. the aim of the present work is to define the most suitable out of the three gas environment types for storing of grated pumping and chopped pumpkin at same refrigeration temperature. 2. materials and methods for packaging of grated pumpkin and intact pieces of pumpkin is used semi automatic packaging machine tray sealer fibosa with two trays – fig. 1. figure 1. chamber of the packaging machine with packaging trays figure 2. control desk of the packaging machine mailto:stvstefanov:@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 55 polypropylene (pp) trays with dimensions 130 x 215 mm (fig. 3) are used for packaging. the upper foil, which seals the tray has high stoppage effect on gases and vapours. it is multi ply lintop pp dan. this is a foil, which has very high stoppage effect on oxygen, carbon dioxide and water vapours. the values of oxygen permeability are under 15 cm3/m2.24h.bar at 23oc and rh 0% according to astm d 3965, the values of carbon dioxide permeability are under 75 cm3/m2.24h.bar at 23oc and rh 0% according to astm d 3965 and the values of water vapours permeability is under 3 g/m2.24h.bar at 23oc and rh 85% according to iso 2528. the foil thickness is 50 μm and the basis weight is 46 g/m2. the strength characteristics are defined according to astm d882. the sealing temperature of the upper oil to the base, which can be pp as well as pe, is 170 °с. figure 3. packaging tray figure 4. upper foil lintop pp dan the grated pumpkin and intact pieces pumpkin is packed in three different packages in condition of each gas environment, as the sealing seams are visually inspected. one of them is put aside for microbiological analysis as a control sample and the rest are put in a refrigerator „liebherr” ct2841and are stored in refrigeration conditions. the reading and registration of the temperature is done by r.i.c.s. (remote integrated control system) evco italy, which has 5 separate temperature probes – pt, placed as it is shown on fig. 5. during storage the gas environment content is measured by portable gas analyzer pbi dansenzor „checkpoint”. after expiration of the 5 days set storage period of the pumpkin, we separate the microbiological availability by the following methods: bds en iso 4833:2004 microbiology of food and animal feeding stuffs horizontal method for the enumeration of microorganisms colony-count technique at 30 degrees c ; bds en iso 7954:2002 microbiology -general guidance for enumeration of yeasts and moulds -colony count technique at 25 degrees c yeasts and molds; the reading and registration of the temperature is done by the temperature probes of r.i.c.s. which are located in the following zones (fig. 5) figure 5. location of the temperature probes 1 lower part; 2,4,5 central part; 3 in the room. 3. results and discussion the temperature variation in the refrigerator ct 2841 liebherr has been followed for the whole store period and we food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 56 have presented data for only two days (48 hours), because during the next days the temperature has no variations(fig.3 and 4) figure 6.store temperatures during the first day figure 7.store temperatures during thesecond day the packaged pumpkin is stored in the central zone of the refrigerator (sensor 2, 4 and 5). we can see on fig. 6 that the upper part of the refrigerator (sensor 3) has the highest temperature variation. this is observed only during the first 10 hours, until the input packages are cooled down. the average temperature of the stored pumpkin for the whole period is 5.5 ос, but the shelf life is defined by the maximal temperature of 6.5 ос. the breathing of fruits and vegetables is a biochemical process, related to evolving heat and carbon dioxide. the higher store temperature is related with more intensive breathing of the packaged fruits. this means reduction of o2 quantity and increasing the со2 quantity in packages, made of high barrier property materials. using the gas analyzer we reported the со2 content of each sample during the separate days of the refrigerator storage (5,5 ± 1ос). you can see the results for the co2 content in table 1. fig. 8 and fig.9 graphic of co2 content at storing in vacuum packaging of intact and grated pumpkin. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 57 table 1 pumpkin, packaged with upper foil lintop packaging environment content condition of the pumpkin № of sample co2, content % third day fourth day air grated 1 2,4 2,9 vacuum intact 2 2,6 3,3 intact 3 2,5 3,1 grated 4 3 3,2 intact 5 3,1 4,4 intact 6 2,9 3,7 intact 7 2,5 2,8 30% co2 :70% n2 intact 8 3,7 4,6 intact 9 11,1 10,6 grated 10 14,8 13,5 intact 11 4,5 5,8 100% co2 intact 12 7,6 7,2 grated 13 43,6 39,3 intact 14 39,7 31,5 intact 15 7,8 4,0 intact 16 10,7 9,6 intact 17 9,3 8,9 figure 8. co2 content in packages with intact pieces of pumpkin (upper foil lintop ) packaged in vacuum figure 9. co2 content in packages with grated pumpkin (upper foil lintop ) packaged in vacuum during storage in vacuum in grated condition as well as intact pieces condition of the pumpkin we observer similar со2 content variation. the breathing is an aerobic process which is sopped in case of oxygen absence it is stopped. in our case the process is due to the incoming through the foil oxygen and even the bigger surface of the grated pumpkin has no effect. on fig. 10 and 11 we can see a graphic of the со2 content of packages with gas environment of grated and intact pieces of pumpkin. figure 10. co2 content in packages of pumpkin (upper foil lintop ) packaged in gas environment 30% co2 :70% n2 figure 11. co2 content in packages of pumpkin (upper foil lintop )packaged in gas environment 100% co2 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 58 on the graphics of fig 10 and 11 we can see the decreasing со2 in the packages, as in the case with the grated pumpkin the breathing process is more intensive and the со2 content is higher in the end of the storage period. when storing fruits and vegetables, for better quality of the end product it is necessary to observe the microbiological pollution as well as the maintaining of low temperature. we can see on table 2 experimental results from microflora of pumpkin (grated and intact) packaged in different gas environment: 100% со2, 70:30% n2: со2 and vacuumed after 5 days storing in refrigerator (5,5 ± 1ос). table 2.packaging of pumpkin grated pumpkin intact pumpkin total microbial counts, cfu/g yeasts and molds, cfu/g total microbial counts, cfu/g yeasts and molds, cfu/g 100% со2 7,7. 103 over 10 1,87. 103 over 10 30% со2 / 70% n2 3,1. 104 10 7,5. 104 over 10 vacuum 1,6. 104 over 20 over 104 over 10 figure 12.total microbial counts in grated pumpkin after refrigerator storing(5,5±1ос) 5 days figure 13. yeasts and molds content in grated pumpkin after refrigerator storing(5,5±1ос) 5 days on table 2 and fig. 12 and 14 we can see that gas environment containing 100% со2, is more suitable for packaging grated pumpkin or intact pumpkin pieces, because the со2 itself has antimicrobial effect. when it is mixed with n2 its effect is being reduced. the less suitable for such products with high aqua activity aw is packaging in vacuum – fig. 13 figure 14. total microbial counts in intact pumpkin pieces after refrigerator storing (5,5 ± 1ос) 5 days food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 59 4. references [1]. stefanov st. at all. explore options to extend the berries by store packaging in modified atmosphere. international conference “biotechnologies, present and food engineering, “stefan cel mare” university of suceava, romania. fourth edition, november, 19-20, 2010. [2]. stefanov st., n. arabadjieva, а. hristova, z. denkova, y. stefanova, ch. sazdov – prilojenie na perforirani folia za opakovane na sveji plodove v modifizirana atmosfera. nauchni trudove na rusenskia universitet 2010, tom 49, seria 9.2. str. 50 (in bulgarian) [3]. stefanov st., h. hrisov, g. kostov, y. stefanova, ch. sazdov, n. arabadjieva, d. stoeva. sahranenie na opakovani hranitelni producti v modifizirana atmosfera. nauchni trudove na rusenskia universitet 2009, tom 48, seria 9, str. 125-129. (in bulgarian) [4]. stefanov st., n. arabadjieva, а. hristova, y. stefanova, z. denkova, ch. sazdov opakovaneto v modifizirana atmosfera sp. “propak”, issn 1313-0986, br. 6, хіі, 2010 г. str. 20-26. (in bulgarian) genetic, biochemical and physiological characteristics of the recombinant strain lactobacillus rl15, obtained by intergeneric food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 5 genetic, biochemical and physiological characteristics of the recombinant strain lactobacillus rl15, obtained by intergeneric hybridization between lactobacillus acidophilus 2 and bifidobacterium bifidum l1 *rositsa denkova1, i. dobrev1, zapriana denkova2, velichka yanakieva2, zoltan urshev1, maria yordanova1, svetla ilieva1 1 department „biotechnology”, faculty of biology, sofia university „st. kliment ohridski”, sofia, bulgaria, rositsa_denkova@mail.bg 2department „organic chemistry and microbiology”, university of food technologies, plovdiv, bulgaria, zdenkova@abv.bg *corresponding author received 10 december 2011, accepted 5 february 2012 abstract: a successful intergeneric recombination by protoplast fusion between lactobacillus acidophilus 2 and bifidobacterium bifidum l1 is performed. the strain lactobacillus rl15 with high reproductive capacity and moderate acidity, that doesn’t cause sliming of milk is selected . using modern molecular genetic methods it has been found that the hybrid inherits the genome of one of the parental strains. it is shown that in the united genome occur changes, which are expressed in the hybrid’s phenotypic properties, enzyme profile and technological parameters. key words: intergeneric recombination, protoplast fusion, lactobacillus, bifidobacterium, ardra, aflp 1.introduction hybridization by protoplast fusion of cells belonging to the same or different types of microorganisms allows to unite in one genome characteristics of both the parental strains [1, 2, 3, 4, 5, 6]. thus conditions for obtaining a heterogeneous population are created, and recombinants with new useful properties, such as increased proliferation, bacteriocin producing ability, higher lipase, amylase, b-galactosidase activity, with the ability to develop in a wide temperature range or other properties, might be selected. according to yeehn et al., 1996 [6] genetic recombination through protoplast fusion as a result of the union of the genomes of the parental cells allows not only to improve the features of the producers, but also to acquire new ones. the aim of the present study is to reveal the similarities and the differences in the genome, biochemical and technological properties between the recombinant strain lactobacillus rl15, obtained by intergeneric genetic recombination by protoplast fusion between lactobacillus acidophilus 2 and bifidobacterium bifidum l1, and each of the parental cultures. 2. materials and methods determination of the biochemical profile the system api 50 chl (biomerieux sa, france) is used for the identification of the species of the genus lactobacillus based on their ability to utilize 49 carbon sources. fresh 24-hour culture of the studied strain is centrifuged for 15 min at 5000xg. the obtained sludge, containing biomass, is washed twice with pbs-buffer and resuspended in medium api 50 chl, an integral part of the used kit. the api strips are placed in the incubation boxes, the microtubules are inoculated with the prepared cell suspension and sealed with mailto:rositsa_denkova:@mail.bg mailto:zdenkova:@abv.bg food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 6 sterile liquid paraffin. the results are reported on the 24th and the 48th hour of incubation at 37°c ± 1°c. reporting is done, based on the colour change of each microtubule, compared to the colour of the control microtubule (microtubule 0). positive results were recorded in the cases of color change to green or bright yellow. the obtained results are processed with apiweb ® identification software. profile of enzyme activity the system api zym (biomericux, france) is used for semi-quantitative determination of the enzyme profile of the studied strains. fresh 24-hour culture of the studied strain is centrifuged for 15 min at 5000xg, the resulting sludge, containing biomass, is washed twice and resuspended in api suspension medium. the api zym strips are placed in the incubation boxes and the wells are inoculated with the prepared cell suspension. the samples are incubated for 4 hours at 37°c. then one drop of reagent a and reagent b are added to each well. after 5 min staining is recorded as described in the color scheme in the manufacturer's instructions. enzyme activity is determined by the color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). genetic methods isolation of total dna is performed by the method of delley et al., 1990. ardra (amplified ribosomal dna restriction analysis) the method ardra involves pcramplification of the gene encoding 16s rrna, using primers complementary to the conservative regions at both ends of the 16s rrna gene, followed by restriction with restriction enzymes. the resulting profile is strictly specific for the particular tested species. dna of the studied strain is amplified using universal primers for the 16s rdna gene fd1 and rd1 (weisburg wg, 1991). the amplification program includes: denaturation 95°c for 3 min, 40 cycles 93°c for 30 s, 48°c for 60 s, 72°c for 60 s, final elongation 72°c for 5 min. the resulting pcr product from the 16s rdna amplification of the tested strain is treated with endonucleases: ecori, alui, hapii and taqi (boehringer mannhem gmbh, germany). the resulting restriction products are visualized on a 2% agarose gel. aflp (amplified fragment length polymorphism) aflp is developed and performed for the two pairs of endonucleases: bamhi/psti and hindiii/mboi. aflp is a genomic typing technique based on selective amplification of a set of fragments after restriction of genomic dna. the method consists of 3 steps: 1. dna restriction and ligation of adapters (linkers) to the corresponding restriction sites. 2. selective amplification of a set of fragments. 3. amplified fragment analysis using polyacrylamide electrophoresis. pcr amplification is achieved by using the common sequence of the restriction site and the adapter, ligated to it, as a target site for the primers. selective amplification is achieved using primers that have one or more selective nucleotides in their 3' end. for detection of the pcr product one of the primers needs to be fluorescently (or radioactively) labeled. after amplification fragments of about 50-100 are obtained, for which there is no preliminary information about their sequence. the fragments are then separated using polyacrylamide gels. periodic cultivation bioreactor and cultivation conditions food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 7 the laboratory bioreactor is a cylinder with a geometric volume of 2 dm3 and working volume 1,5 dm3. fig. 1. scheme of the laboratory bioreactor 1 vessel with geometric volume of 2 dm3; 2-four repulse devises ; 3–thermo-strength pt100 ; 4–heater ; 5-heat exchanger for cold water ; 6–turbine stirrer ; 7–ph electrode ; 8–exit for co2; 9–filter ; 10–peristaltic pump for ph correction ; 11– reagent for ph correction – 20% koh; 12–motor ; 13-control links ; 14–control device "applikon" periodic cultivation processes are carried out using two media: skimmed cow's milk or pasteurized goat's milk without ph adjustment. skimmed milk medium is sterilized at 121°c for 20 min. after cooling to 37°c the prepared media in the bioreactor is inoculated with 5% (v/v) inoculum from a fresh 24-hour culture of the studied strain, cultivated in skimmed cow's milk or pasteurized goat's milk. the process of cultivation is carried out at 37ºc, stirring speed of 100 rpm, without air supply. the duration of the cultivation is different (up to the 7th hour, up to the 24th hour), and samples of the cultural medium are periodically taken in order to determine the number of viable cells of the strain (cfu/cm3) and the titratable acidity. the bioreactor is equipped with sensors for ph and eh (oxidation reduction potential), so at each sampling these parameters are recorded as well. along with the cultivation conducted in dynamic conditions (in a bioreactor), cultivation at static conditions (in a thermostat) was conducted as well. 3. results and discussion a successful intergeneric hybridization was performed between the cells of lactobacillus acidophilus 2 and bifidobacterium bifidum l1. from the regeneration medium recombinants resistant to the antibiotics streptomycin and neomycin sulfate were isolated. recombinants, which are characterized by moderate titratable acidity, relatively short coagulation time of milk, without slime formation, and no change in morphology, were selected among them. the proliferation ability of the selected recombinants and the parental cultures in microaerophilic and anaerobic static cooling water food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 8 conditions was determined. the results show that during the time from the 6th to the 12th hour all the recombinants achieve high concentration of viable cells 2 log units higher in comparison with the concentration of viable cells in anaerobic conditions. in that capacity, they resemble the parental strain l.acidophilus 2. the best growth in obligate (anaerobic) conditions show two recombinants: lactobacillus rd6 (2,0.1011 cfu/cm3) and lactobacillus rl15 (1,0.1010 cfu/cm3), which resemble the other parental strain b.bifidum l1. biochemical profile of the recombinant lactobacillus rl15, obtained by hybridization between l. acidophilus 2 and b.bifidum l1. genetic recombination through protoplast fusion results in uniting in a hybrid cell the genetic structures of the two parental cells. the changes that occur in the integration are reflected in the phenotype of the newly obtained recombinants. along with the physiological peculiarities, changes in their biochemical properties are observed as well. using the api 50 chl (biomerieux sa, france) the biochemical profile of the recombinant strain and the parental strains is defined. the degree of similarity between the hybrid and the parental strains, according to the ability of each strain to utilize 49 carbohydrate sources, is determined. table 1 biochemical profile of the hybrid lactobacillus rl15 and the parental cultures l.acidophilus 2 and b.bifidum l1 # carbohydrates l.acidophilus 2 b. bifidum l1 lactobacillus rl15 1 glycerol 2 erythriol 3 d-arabinose 4 l-arabinose 5 ribose + (80-85%) + ( 80-100 %) 6 d-xylose 7 l-xylose 8 adonitol 9 b-metil-d-xyloside 10 galactose + (90-100%) 11 d-glucose + (90-100%) + ( 80-100 %) + (50-60%) 12 d-fructose + (90-100%) 13 d-mannose + (90-100%) + ( 80-100 %) 14 l-sorbose + (90-100%) 15 rhamnose + (50-60%) + ( 80-100 %) 16 dulcitol + (90-100%) 17 inositol 18 manitol + (90-100%) + ( 80-100 %) 19 sorbitol + (80-85%) + ( 80-100 %) 20 a-methyl-d-mannoside 21 a-methyl-d-glucoside + (80-85%) 22 n-acetyl-glucosamine + (90-100%) 23 amigdalin + (80-85%) 24 arbutin + (80-85%) 25 esculin 26 salicin + (90-100%) + ( 80-100 %) 27 cellobiose + ( 80-100 %) 28 maltose 29 lactose + (90-100%) + ( 80-100 %) + (90-100%) 30 melibiose 31 saccharose + ( 80-100% ) 32 trehalose + (90-100%) 33 inulin 34 melezitose 35 d-raffinose 36 amidon 37 glycogen 38 xylitol 39 b-gentiobiose 40 d-turanose 41 d-lyxose food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 9 42 d-tagarose + (50-60%) 43 d-fuccose 44 l-fuccose 45 d-arabitol 46 l-arabitol 47 gluconate + (50-60%) + (60-70%) 48 2-keto-gluconate 49 5-keto-gluconate the recombinant strain lactobacillus rl15 has suffered profound changes in the genome that affect some regulatory mechanisms. unlike the parental cultures he has the ability to assimilate only glucose and lactose (table 1). examination of the enzyme profile the enzyme profile is important in determining the technological characteristics of lactobacilli and their applicability as starter cultures for production of various dairy, meat and bakery products. therefore, the enzyme activity of the strains l. acidophilus 2, b. bifidum l1 and lactobacillus rl15 is determined using api zym (biomerieux, france). the results of these studies are shown in table 2. the hybrid lactobacillus rl15 has a weak alkaline phosphatase, c8 lipase, trypsin and chymotrypsin, and α-glucoseaminidase activity. the similarity between its enzyme activities and the enzyme activities of the parental strain b.bifidum l1 is logical.the hybrid’s β-galactosidase and leucineaminopeptidase enzyme activity, moderate acid phosphatase, naphthol-as-blphosphohydrolase, cysteineaminopeptidase activity resemble the profile of the parental strain l.acidophilus 2. table 2 enzymatic profile of the strains b.bifidum l1, lactobacillus rl15 and l.acidophilus 2 enzyme activity* b.bifidum l1 activity* lactobacillus rl15 activity* l.acidophilus 2 1 control 2 alkaline phosphatase 0.5 3 lipase с4 1 0.5 0.5 4 lipase с8 1 0.5 5 lipase с14 0.5 1 0.5 6 leucine-aminopeptidase 5 4 4 7 valine-aminopeptidase 5 3.5 3 8 cysteine-aminopeptidase 1 3 3.5 9 trypsin 0.5 10 chymotrypsin 0.5 0.5 11 acid phosphatase 3 1.5 1 12 naphthol-as-bl-phosphohydrolase 3 1 1 13 α – galactosidase 3.5 14 β-galactosidase 5 5 5 15 β-glucoronidase 16 α – glucosidase 3.5 17 β-glucosidase 4 18 α-glucoseaminidase 2 0.5 19 α-manosidase 20 α-fucosidase 0.5 the ability of the strains to synthesize enzymes is essential for the uptake of substrates and the formed metabolites are involved in the formation of the tastearomatic complex of dairy and meat foods. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 10 ardra the hybrid lactobacillus rl15, obtained by protoplast fusion between l. acidophilus 2 and b.bifidum l1, has the same restriction profile of the gene encoding 16s rrna with the restriction enzyme taqi as the acidophilic parent (fig.1). most likely the hybrid lactobacillus rl15 has inherited the gene for 16s rrna from the parental culture l. acidophilus 2. the parental strains l. acidophilus 2 and b.bifidum l1, as well as their recombinant have a restriction site for the enzyme ecori, but the restriction profile of the hybrid shows greater similarity with the restriction profile of the parental culture l.acidophilus 2 (fig. 1b). a) taqi b) ecori c) hapii d) alui 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 fig. 1. restriction profile: 1 b. bifidum l1; 2 l. acidophilus 2; 3 lactobacillus rl15; 4 kbase plus marker (usb) the dependencies between the restriction profiles of the recombinant strain and the parental cultures, obtained with the restriction enzymes hapii and alui, are similar to the dependencies between the profiles, obtained with ecori and taqi (fig. 1c and fig. 1d). the applied molecular genetic method shows that the hybrid has inherited the 16s rrna gene from one of its parents l.acidophilus 2. these results show the predominant presence of one of the parental strains in the genetic recombination that occurred in the process of merging of the protoplastic cultures.therefore, the next step in the molecular-genetic typing is the conduction of aflp analysis of genomic dna. aflp aflp – analysis of the hybrid lactobacillus rl15 and the two parential strains l.acidophilus 2 and b.bifidum l1 with two pairs of endonucleases bamhi/psti and hindiii/mboi was conducted. the results of these studies are shown on fig. 1 and fig. 2. the obtained after computing dendrograms confirmed the results of the ardra analysis. this recombinant resembles to a greater degree the parental strain l.acidophilus 2 (fig. 2b and fig. 3b). changes that occurred in the genome of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 11 the hybrid, although low (15%), find expression in its phenotypic features – its biochemical and physiological properties. la rl15 а) l1 b) fig. 2. aflp strip (а) and dendrogram (b) of the aflp analysis with the endonuclease pair bamhi/psti of lactobacillus rl15; l. acidophilus 2; b. bifidum l1; rl15 la а) l1 b) fig. 3. aflp strip (а) and dendrogram (b) of the aflp analysis with the endonuclease pair hindiii/mboi of lactobacillus rl15; l. acidophilus 2; b. bifidum l1 dynamics of proliferation of the parental strains and the recombinant under static conditions and in a bioreactor with continuous stirring the main parameters of the fermentation process – coagulation time, titratable acidity of the medium, proliferation ability, oxidation-reduction potential (eh) and ph during the cultivation of the parental strains and the hybrid in a bioreactor with continuous stirring and at static conditions were tracked. in the cultivation of l.acidophilus 2 at static conditions at 37±1ºc for 12 hours and for 24 hours the concentration of viable cells reached 8.1010cfu/cm3 and 5.1011cfu/cm3, respectively. the titratable acidity starts to grow with the entry of the culture in the exponential growth phase and continues up to the 24th hour from the beginning of the process, reaching 180°t (fig. 4a). in the bioreactor better conditions for the development of the strains are created. at the 6th hour the microbial content is food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 12 8.1010cfu/cm3. lactobacilli need a limited amount of oxygen because they are microaerophiles. in the studied strain long lag-phase (about 4h) is observed, then the exponential growth phase starts. 0 6 12 18 24 7 8 9 10 11 0 6 12 18 24 25 50 75 100 125 150 175 log n lo g n , c fu /c m 3 tk, ot tk ,0 t 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 25 30 35 40 45 50 55 60 65 70 75 80 0 1 2 3 4 5 6 265 270 275 280 285 290 295 300 305 0 1 2 3 4 5 6 5.8 5.9 6.0 6.1 6.2 6.3 log n lo g n , c fu /c m 3 tk, ot t k ,o t eh, mv e h, m v ph p h ti me, h ti me, h а) b) fig. 4. dynamics of proliferation and accumulation of organic acids in cultivation of l.acidophilus 2 under static conditions (a) and in a bioreactor with continuous stirring (b). the curve, reflecting the titratable acidity is similar to the growth curve. it continues to increase after the culture enters the stationary phase of growth and reaches a value of 85°t. the accumulated acid is almost two and a half times less than that under static conditions (fig. 4b). up to 2nd hour after the start of the fermentation process oxidation increases, reaching + 300 mv, then changes its direction towards reduction with the culture entering the exponential growth phase. at the end of logarithmic growth phase the value of the redox potential is about +265 mv. the cells of bifidobacteria significantly differ from the acidophilic strains in their development. under static conditions b.bifidum l1 has a short lag-phase and exponential growth up to 12th hour from the start of the fermentation process. at the end of the process high concentration of viable cells (1015cfu/cm3) and titratable acidity (65°t (fig. 5a)) are achieved. the same concentration of active cells accumulates in the medium for a shorter time (7h) in the cultivation of b.bifidum l1 in a bioreactor with continuous stirring (fig. 5b). 0 6 12 18 24 10 11 12 13 14 15 16 0 6 12 18 24 25 50 75 100 125 150 175 200 log n lo g n , c fu /c m 3 tk, ot t k ,0 t 0 1 2 3 4 5 6 7 10 11 12 13 14 0 1 2 3 4 5 6 7 30 35 40 45 50 55 60 65 0 1 2 3 4 5 6 7 320 325 330 335 340 345 350 355 360 365 0 1 2 3 4 5 6 7 5.0 5.5 6.0 log n lo g n , c fu /c m 3 tk, ot t k ,o t eh, mv e h, m v ph p h time,h time,h а) б) fig. 5. dynamics of proliferation and accumulation of organic acids in b.bifidum l1 under static conditions (a) and at periodic cultivation in a bioreactor with continuous stirring (b). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 13 for the same time the titratable acidity of the medium increased to 65ºt. the results confirm the studies of schiraldi et al., 2003, that under microaerophilic conditions biomass is accumulated, and under anaerobic conditions primarily lactic acid is accumulated. during the phase of adaptation to the conditions of the medium the oxidative potential increases up to +370 mv, but with the entering of the culture in the exponential phase of growth it starts to decline and keeps this tendency until the end of the process when it reaches +320 mv, probably due to the reducing capacity of the cells. distinct phases of growth are detected in lactobacillus rl15 under static growth conditions. a concentration of 2,0.1011 cfu/cm3 viable cells is reached for 12 hours under static conditions at 37°c (fig. 9a). during this time the titratable acidity reaches 160°t. with the further development of the culture up to the 24th hour the concentration of viable cells in the medium reaches 1,0.1012cfu/cm3, without any significant change in the values of the titratable acidity (fig. 6a). a concentrate of 1,0.1010cfu/cm3 with titratable acidity 90°t is obtained by periodic cultivation of lactobacillus rl15 in a bioreactor with continuous stirring for 5h (fig. 9b). in this strain during the lag-phase the redox potential decreases and in the phase of active multiplication during the exponential phase continues to decrease until the passage of the microbial cells in the stationary phase of growth, then it gradually increases. this is a result of the changes in the ratio of oxidized and reduced forms in the medium (fig. 6b). 0 6 12 18 24 7 8 9 10 11 12 0 6 12 18 24 25 50 75 100 125 150 175 log n lo g n , c fu /c m 3 tk, ot tk ,0 t 0 1 2 3 4 7 8 9 10 0 1 2 3 4 25 50 75 100 0 1 2 3 4 325 350 375 400 425 0 1 2 3 4 4.8 5.2 5.6 6.0 log n lo g n , c fu /c m 3 tk, ot t k ,o t eh, mv e h, m v ph p h time, h time, h a) б) fig. 6. dynamics of proliferation and accumulation of organic acids in cultivation of lactobacillus rl15 under static conditions (a) and in a laboratory bioreactor with continuous stirring (b). such concentrates with beneficial microorganisms to human health are applied as liquid probiotic drinks. 4. conclusion 1. through molecular genetic methods (ardra and aflp) the genomic similarities and differences between between the hybrid and the parental cultures are revealed: 1.1. the recombinant strain lactobacillus rl15 derived from the hybridization of l.acidophilus 2 and b.bifidum l1 inherits the gene for the 16s rrna from the parent culture l.acidophilus 2. 1.2. it is shown that the recombinant strain retained the genome of one of the parents to a great extent, and in its genome changes occur as a result of the conducted protoplast fusion, giving individual characteristics to the hybrid. 1.3. it is revealed that in the united genome occur changes in the regulatory food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 14 mechanisms, which are expressed by the loss of the ability of the recombinant strain to utilize carbon sources. 2. high concentration of viable cells (1012 1015 cfu/cm3) in the static and the dynamic process of cultivation is obtained. 3. the patterns of the change of the oxidation-reduction potential of the media in the cultivation of the recombinant strain and the parental cultures are revealed. in lactobacilli the redox potential increases during the lag-phase, decreases during the exponential phase until the cultures enter the stationary phase of growth, with subsequent retention. in b.bifidum l1 it increases during the lag-phase, continues slowly to grow during the exponential phase and gradually decreases during the stationary phase. in the recombinant derived from the intergeneric hybridization between l.acidophilus 2 and b.bifidum l1, the oxidation-reduction potential decreases during the lag-phase and the logarithmic growth phase, slightly increases during the transition of the culture in the stationary phase of growth. 5. references: [1] gupta r. k. and v. k. batish, (1992a) "genetic evidence for plasmid-encoded lactococcin production in l. lactis subsp. lactis 484". curr. microbiol., 24: 231-238. [2] gupta r. k. and v. k. batish, (1992b), "protoplast-induced curing of bacteriocin plasmid in l. lactis subsp. lactis 848". y. appl. bacteriol., 73: 337-341. [3] gupta r. k. and v. k. batish, (1992c), "lytic response of l lactis subsp. lactis 484 of muralytic enzymes". enzyme microbiol. technol., 14: 156-160. [4] hayes f., e. caplice, a. mcsweny and daly., (1990), "pamb1-associated immobilization of proteinase plasmids from l. lactis subsp. lactis uc317 and l. lactis subsp. cremoris uc205". environ. microbiol., 56 (1): 195201. [5] kim. h. and m. chassy, (1994), study in the plasmid gene transformation of lactobacillus casei – 36 (21) 212-216. [6] yeehn y., young bae io and oh chang, (1996), protoplast fusion between l.casei and l.acidophilus "rwou, biotechnology letters, v. 8 (7) p 805-808. звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 93 studies concerning influence of the inoculum upon amyloglucosidase’s biosynthesis of aspergillus niger *adriana dabija1, cristina-gabriela constantinescu (pop) 1, amelia buculei1 1faculty of food engineering, stefan cel mare university, adriana.dabija@fia.usv.ro *corresponding author received 12 december 2011, accepted 15 february 2012 abstract: amyloglucosidase is used in food industry in the saccharification processes of starch raw materials. in the alcohol and beer industry amyloglucosidase is used to obtain glucose syrup and in the baking industry, to obtain crystallized sugar etc. this enzyme can be prepared by biosynthesis from culture molds of genus aspergillus, rhizopus or yeasts of genus endomycopsis and catalyzes splitting of bonds α-1, 4, α-1,6, α-1,3 glucosidic from starch, dextrin, glycogen, maltose, with the successive release of glucose molecules at a rate dependent on the degree of polymerization and structure of substrate. this paper researched the influence of vegetative and sporiferous inoculum on the accumulation dynamics of amyloglucosidase, under the conditions of cultivating selected mould of genus aspergillus. following the experiments performed to obtain amyloglucosidase under the conditions of cultivating the genus aspergillus mold (ac-6), it is recommended the use for insemination basic nutrient medium of the vegetative inoculum at a rate of 10% for which it is achieved amyloglucosidase activity 10 times higher than using at insemination spore suspensions. keywords: mould cultures, spore suspensions, vegetative inoculum 1. introduction the amyloglucosidase (α1,4 glucohydrolase, glucoamylase, glucogenase, γ-amylase) is biosynthesis produced by the mould cultures like genus aspergillus, rhizopus, or yeasts endomycopsis fibuliger, endomycopsis bispora [1, 2]. the structures of multiple forms of aspergillus niger glucoamylase have been shown to be closely related. it is known that aspergillus niger is industrially useful in view of their use in the production citric acid and enzymes such as glucosidase, glucoamylase etc. production processes have been thoroughly optimized by improvements in process technology and by strain breeding. classical improved strains have been obtained using random mutagenesis, recombination and genetic modification based on transformation of protoplasts [3, 4, 5, 6]. amyloglucosidase (amg) (1,4-α-d glucan glucanohydrolase, ec 3.2.1.3.) is an industrially important biocatalyst. amg is a multi-domain, exo-acting enzyme that catalyzes the hydrolysis of starch and related substrates from the non-reducing ends mainly by cleaving α-1,4glycosidic linkages and a few α-1,6 linkages, but at a very slow pace [7]. also, the amyloglucosidase catalyses the scission of glucosidal links α-1,4, α-1,6, α1,3 in starch, dextrin, glycogen, maltose producing glucose molecules successively disengaging at a rate dependent of mailto:adriana.dabija:@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 94 polymerization’s degree and substratum’s structure on. the bond α-1,4 is faster hydrolyzed than the α-1.6, while maltotriose and maltose are faster hydrolyzed than the oligosaccharides. the enzyme is widely used in baking and the production of ethanol, molasses, and crystalline glucose. the enzyme has been immobilized using different methods including alginate entrapment. the micromycetes can produce multiple shapes of enzyme, which is differentiating among them by weight molar, kinetics constant, substratum specification, extracellular enzymes which concentration depend on medium’s composition, growing conditions, microbic agent’s nature. the amylo glucosidase, beside αand β-amylase, has a lot of industrial applications in saccharifying processes of starchy raw, in alcohol industry and breweries, in obtaining glucose syrups and crystallized glucose, in bakery industry. for instance, amyloglucosidase is used when the brewers want to obtain beer having low carbohydrates content (diet beer). in bakery field, for the higher glucose contents of dough the amyloglucosidase is used too, because that provides for a better yeast multiplication and encourages the obtaining of bread with good-looking crust and soft core. the optimum conditions for the enzyme activity are: temperature 55÷60°c and ph 4.5÷5.5. the enzyme is inactived by heating to 80°c on 5 minutes or to 75°c on 40 minutes long. the enzyme activity is canceled using the procedure of exchanger ions’ crossing or the treating with active carbon [1]. in this present work we have observed the influence of vegetative sporiferous inoculum upon the accumulation dynamics of amyloglucosidase under growing’s conditions for a selected mould belonging to the aspergillus genus. 2. experimental for the obtaining of sporiferous inoculum, the spores from a growing on inclined malt must with agar (mma) have been carried away by sterile physiological serum, and the determination of spores number was performed using the chamber thoma method. for the obtaining of vegetative inoculum, spores suspensions have been inseminated on the medium having the following composition [g/%]: ▪ maize flour 5 ▪ yeast extract 0.25 ▪ ph medium (hcl addition) 5.5 the inseminated medium has been maintained under stirring condition at 25°c for 48 hours long having as effect the spores’ germination and growth of vegetative hyfa. for the experiments, it has been used different quantities of vegetative or sporiferous inoculum, which was inseminated on basic medium having the composition [g/%] as following: ▪ maize flour 15 ▪ malt flour 0.4 ▪ ph medium (hcl addition) 5.5 the growing was made under stirring conditions at 25÷28°c for 96 hours long. for the fermented medium, which contains extra-cellular amyloglucosidase, it was determined values for the ph, refractometric degree and amyloglucosidase activity. an amyloglucosidase activity unit means the glucose quantity [mg] obtained for 1 cm3 enzyme filtrate at 60°c in one hour long from starch soluble 4% solution having ph=4.2. 3. results and discussion the insertion of 5·107÷20·107 spores/cm3 suspension on basic medium and growing medium under same conditions and the analysis of enzyme filtrate after that, gave food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 95 the opportunity to show the synthesized results in table 1. table 1. the influence of sporiferous inoculum upon amyloglucosidase’s activity v ar ia nt sporesno./cm3 mediu m enzyme filtrate’s characteristics ph refractometric degree enzyme activity [agu/cm3 enzyme filtrate] relative activity % optimum variant 1 5·107 3.8 12.8 54.4 100.0 2 10·107 4.0 12.5 41.6 76.4 3 15·107 3.5 12.2 28.2 52.9 4 20·107 4.5 12.0 24.0 44.1 among the four used alternatives for the experiments, only the first one (5·107 spores/cm3) shows a maximum enzyme activity, than, gradually, the activity cuts down so that the doubling of spores number for inoculum leads a reduction by 23% of enzyme activity, while for the four times growth of spores number leads to the decrease of enzyme activity with 66% (fig.1). through the determination of extract using refractometric way it can see when the inoculated spores number start growing in medium the soluble substances’ consumption, of medium become more intensive. this fact it not become favorably for amyloglucosidase’s elaboration. 0 20 40 60 80 100 a g u /c m 3 5 10 15 20 spores no/cm3 medium [x107] enzyme activity relative activity fig.1. the influence of sporiferous inoculum upon amyloglucosidase’s activity and relative activity. for the study of vegetative inoculum’s influence it has been used 4 variants having the variation of inoculum quantity between 5÷30% given basic medium. the results for analysis of enzyme filtrate are shown in table 2. table 2. the influence of vegetative inoculum upon the amyloglucosidase’s activity variant inoculum quantity [%] enzyme filtrate’s characteristics ph enzyme activity [agu/cm3 enzyme filtrate] relative activity [% optimum variant] 1 5 3.0 363 67 2 10 2.8 541 100 3 20 2.5 521 96 4 30 2.5 482 89 using the vegetative inoculum in quantity of 10%, it was obtain maximum amyloglucosidase’s activity by ten times more than the sporiferous inoculum’s using. the doubling of inoculum quantity (variant 3) it was not lead to the enzyme activity’s growth. through the exaggerated growth of inoculum quantity (variant 4), the enzyme activity decreases with 11% (fig.2). 0 100 200 300 400 500 600 a g u /c m 3 5 10 20 30 inoculum quantity [%] relative activity enzyme activity fig.2. the influence of vegetative inoculum upon the amyloglucosidase' activity and relative activity the lower values of ph obtained for the vegetative inoculum is owing to the metabolically activity of it, which is more intense than spores activity because the last food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 96 one needs additionally adaptation and germination periods when its own enzyme activity could have a common evolution. 4. conclusion for the amyloglucosidase’s obtaining under conditions of selected mould growing which belongs to aspergillus genus, it is recommended, for the insemination of basic nutritive medium, to use vegetative inoculum in 10% proportion having as effect an amilogluosidase’s activity of 10 times more than the utilization for insemination of spores’ suspensions. 5. references [1]. banu c., et al., biotechnology of food industry, tehnica, bucharest, (2000) [2]. brigs d.e., et al., malting and brewing science, chapman et hall, london, (1995) [3]. giesen r., fungal starter cultures for fermented foods: molecular aspects, trends in food science et technology, 8, 251-254, (1993) [4]. dan v., food microbiology, alma, galati, (2001) [5]. rocktotpal, k., et al., magnetically recyclable, antimicrobial and catalytically enhanced polymer-assisted green monosystem immbolized aspergillus niger amyloglucosidase, applied microbiology and biotechnology, 6, 1983-1992, (2010) [6]. sarma usha, fascinating potential of aspergilli. indian journal of chemical biochemistry, vol.25, 4, 331-334, (2010) [7]. svensson, b., et al., characterization of two form of glucoamilase from aspergillus niger, carlsberg research communications, 1, 55-69, (1992) compositional aspect of quality wines produced in silvaniei vine growing center of șimleul silvaniei, harvest of 2013-15 84 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 84 94 compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest * florin-dumitru bora 1 , alina donici 1, oana-mihaela ripanu 1 1research station for viticulture and enology, g-ral eremia grigorescu, târgu bujor, galați, românia, boraflorindumitru@gmail.com, *corresponding author received february 28th 2016, accepted march 29th 2016 abstract: wine is a food product produced exclusively by partial/total alcoholic fermentation of fresh grapes. from a chemical point of view, wine is a complex mixture consisting of water, sugar, ethanol, amino acids, polyphenolic compounds, anthocyanins, organic/inorganic materials. viticulture depends on meterorological conditions. the wine industry from romania is particularly involved in the controversial effects generated by climate change. although the overall effects of climate change on romanian viticulture are uncertain, it is known that grapevine yields diminish with the occurrence of abiotic stress, such as freezing temperatures, increasing soil salinity and drought because of the varying effects on grape quality. the purpose of this work is to present data relating to the composition characteristics of some quality wines from the șimleul silvaniei in the new climate conditions over the last few years. the biological material consisted of the varieties: fetească regală (f.r.), fetească albă (f.r.), italian riesling (r.i.) and furmint (f.m.). the weight values obtained for 100 grains (182.06±5.98 g f.a. 2014), sugar (202.28±2.98 g/l f.a 2013), titratable acidity (9.39±0.03 g/l c4h4o6 f.m 2014), acidity (4.70±0.09 g/l h2so4 f.m. 2014), ph (3.92±0.24 r.i. 2014) in grapes and reducing sugars (2.63±0.17 g/l f.a. 2015), total dry extract (27.73±1.29 g/l r.i. 2013), nonreducing extract (26.62±0.46 g/l r.i. 2013), total acidity (7.65±0.11 g/l c4h4o6 f.r. 2013), volatile acidity (0.57±0.04 g/l ch3cooh) of wine, are specific to the four varieties analyzed. the correlation analysis revealed a number of strong correlations between the qualitative characteristics of wine and composition of grapes. keywords: grapes, wine, vitis vinifera. 1. introduction wine is food product produced exclusively by partial/total alcoholic fermentation of fresh grapes, whether or not pressed or by must fermentation (o.i.v). from a chemical point of view, wine is a complex mixture consisting of sugar, water, ethanol, amino acids, polyphenolic compounds, anthocyanins, organic/inorganic materials [1, 2, 3, 4]. today, the vines are grown throughout the world; europe has the highest percentage (51%) of the global planted with vines, followed by america and asia [5]. in romania the vineyard area has decreased since the 1990s and it currently ranks fifth in europe after italy, spain, france, portugal, and in 2013 romania has an area of 229 000 hectares of vineyards [6, 7]. europe encompasses the largest vineyard area in the world (oiv, 2012). large scale analyses have demonstrated that climate change effects in europa are spatially variable: water deficits and severe dry conditions are expected to decrease wine quality and increase annual fluctuations in yields in the romanian zone. conversely, in northern europe and central, warming conditions are forecast for the future, and http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 85 this should improve wine quality [8]. wine industry from romania is particularly involved in the controversial effects generated by climate change. although the overall effects of climate change on romanian viticulture are uncertain, it is known that grapevine yields diminish with the occurrence of abiotic stress, such as freezing temperatures, increasing soil salinity and drought becase of the varying effects on grape quality. aridity would likely affect viticulture from romania, especially during the crop-growing season [9]. the spatial variability of climate change effects in the wine industry is recognizable also to the local scale. this implies the adoption of a finer scale of the resolution in simulations of future climate conditions. the micro-climatic and meso-climatic characteristics of a given winemaking zone are considered key factors of the wine production performance [10]. moreover, soil structure and chemistry, as well as vineyard management practice, are factors varies also at a local scale and there is evidence that they influence very significantly wine performance [11]. soil is one of the most important factors for vine [12] it supports the root system, which absorbs water, accumulates carbohydrates and other nutrients, being crucial for grapevine growth, physiology and yield attributes [13]. soil water retention properties are also important, as they can affect grapevine quality [14]. past, current and future changes in global climatic conditions are condensed in the fourth assessment report of the intergovernmental panel on climate change, ipcc [15]. according to the ipcc report, the global mean temperature has increased by 0.740c±0.180c from 1906 to 2005 in non-linear way: the warming rate over the last 100 years is 0.070c±0.020c per decade, over the last 50 years 0.130c±0.030c and over the last 25 years near surface temperature increased by 0.180c±0.050c per decade. from the 1996 years, all years from 1995 to 2006 rank among the 11 warmest years on record since 1850. the number of cold nights (lowest 10%, based on 1961-1960) has decreased in the period of 1951 to 2003, whereas the number of warm night (highest 10%) has increased [16]. these changes affect agriculture in general and viticulture in particular. amongst others the vegetative period lengthens when temperatures, especially spring temperatures are increasing in last several years. clear changes in the dates of phonological vine stages are observable in many coutries form europe [17]. in alsace region, budburst and flowering events occurred about two weeks earlier in 2003 compared with 1965. the period between flowering and change of colour of berries shrunk by 8 days and change colour of the berries occurred almost 23 days early [18]. viticulture depends on meteorological conditions. evidence of this phenomen on is provided by extensive and worldwide empirical literature. adopting different scale of analysis and new methods, researchers have demonstrated that climate affects vineyard yields [19, 20] wine quality [21, 22, 23, 24]. some studies have considered effects on winegrower’s profitability in terms of net revenue of profit [25, 26, 27]. other studies have shown that ecoclimate conditions change impacts on grapevines are highly heterogeneous across varieties [28]. in romania the effect of ecoclimatic conditions on the vine culture was studied by [29, 30, 31, 32, 33, 34]. the purpose of this work is: i) present data relating to the composition characteristics of some quality white wines that may be obtained from the vine varieties grown frequently in the șimleul silvaniei center for the silvania vineyard in the new climate conditions from the last few years as a result of global warming, that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 86 increasing the amount of useful temperatures both during the growing season and the maturation of the grapes and ii) to also present the climatic conditions (temperature, insolation, raifall) and theier interaction, expressed by some viticultural indices and coefficients form șimleul silvaniei. 2. matherials and methods four vine varieties for high quality white wines were used in the research: fetească regală, fetească albă, italian riesling and furmint grafted on the rootstock berlandieri x riparia kobber 5 bb and cultivated in the area of șimleul silvaniei (47°05' north, 47°35' east), satu mare county, nw romania. grape samples was collected in 2013 at full maturity and 10 kg of grapes/variety were collected from 10 vines/repetition. three repetitions/variety were used, placed in randomized blocks. the grapes were harvested from the middle, top and lower, of each vine, grapes exposed to the sun, but also from shaded, thus obtaining a homogeneous sample [35]. after sampling, the samples were placed in sealable plastic bags, they were numbered and shipped as soon as possible to the laboratory. the grape samples was pressed with the laboratory press (manually) and the must was obtained, followed by the process of microvinification which resulted in the samples of wine. in order to characterize the areas of șimleul silvaniei center for the silvania vineyard meteorological data from the national meteorological agency has been used. based on their specific formulas, ecoclimatic indicators were determined, important for the growth and the fruition of vines, such as global thermal balance (σtog); active thermal balance (σtoa); useful thermal balance (σtou); thermal coefficient (ct); annual and monthly rainfall amount; amount of hours of sunshine (σir) and real sunburn coefficient (ci). to get a wider picture on how climatic factors influence the growth and fruition of vines, the heliothermic index (hi), hydrothermal coefficient (ch) and bioclimatic index (ibcv) were calculated [36]. statistical analyses was performed using the statistical software package spss (version 23.0; spss inc., chicago, il., usa). the data were expressed as mean ± standard deviation (sd) of three replications foe each sample analyzed. in order, for determination the significance differences among values, analysis of variance (anova) and duncan multiple range test (mrt) was performed. pearson ̍s corelation was done using version 23.0 of spss (spss inc. chicago, il., usa). 3. results and discussion analysis of the main climate data. global warming has caused a disruption in the natural evolution of climatic conditions in the vineyards ecosystem, therefore summers have become extremely dry and autumns have become cold, wet, or warmer. as an indicatior of the vocation of a vineyard region, but also for establish the direction of production, the thermal balance and the amount of temperature degrees and have an absolute importance. the length of the vegetation period is within the normal cultivation limits of vines, over 170 days. in all three years studied can be seen that the length of the vedetation period has exceeded this limit: 196 (2013); 193 (2014) and 194 (2015). the thermal balance with the highest food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 87 values was recorded in 2013, the global thermal balance (∑t0g) 3683, the active thermal balance (∑t0a) 3394 and the active useful thermal balance (∑t0u) 1549, the opposite with the thermal balance which has the lowest values was recorded in 2014, the global thermal balance (∑t0g) 3652, the active thermal balance (∑t0a) 3287 and the active useful thermal balance (∑t0u) 1538. we can see that from this point of view the years 2013 and 2014 are extremes of this research, while 2015 shows average values compared whit 2013 and 2014 (table 1). to appreciate the thermal resource and for interpreting the interaction of climatic factors from a vineyard area, is calculated the thermal coefficient (ct). given that 2013 was the warmest year of all, the highest thermal coefficient (18.1 (ct) (table 1) was registered. when assessing the favorability of sun light which is used in viticulture, the following parameters are taken into consideration: insolation (sun shining) potential and actual (real (∑ir)) and coefficient of insolation (ci). the highest values of insolation were recorded in 2013 (1594 (∑ir)) and (7.81 (ci)), followed by the values obtained in 2015 (1563 (∑ir)) and (7.69 (ci)), whereas the lowest ones were recorded in 2014 (1498 (∑ir)) and (7.53 (ci)) (table 1). precipitation is expressed in mm height of the layer of water, respectively l/m2. the amount of rainfall is the average of daily values of calendar year (∑pp). coefficient of precipitation (cp) is the ration between the amount of precipitation and the number of days. to establish correlations between grape production and precipitation, it is recommended to consider wine growing in on year. the highest values of precipitations was recorded in 2014 (536.9 (∑pp); 1.83 (cp)), followed by 2015 (521.9 (∑pp); 1.79 (cp)), the lowest values of precipitation was recorded in 2013 (489.6 (∑pp); 1.72 (cp)) (table 1). in most centers, vineyards, not exceeding 550 mm precipitation annually (∑pp) and in four of them do not reach even 500 mm. this suggests that vine growth and fructification, does not need much water and that irrigation would be unnecessary. this view may be supported by the negative influence of abundant precipitations in areas with moderate temperatures on product quality wine. scientific research shows that vines have need more water than the quantity of precipitation infiltrates in the soil (table 1). in nature, ecoclimatic factors (temperature, insolation and precipitation) do not act independently, but in a complex connection. the values of real heliothermic index (ihr) it is between values 1 and 5. it is considered optimal for vineyard when values of real heliothermic index (ihr) exceed 2.6. in romania real heliothermic index (ihr) has values between 1.35 and 2.70 [37]. from this point of view values of real heliothermic index (ihr) are between 1.75 (2013), 1.56 (2014) and 1.69 (2014) (table 1). hydrothermal coefficient (ch) it shows how vines are satisfied in terms of water in a certain temperature regime. express the degree of suitability for a particular year. may have values between 0.3-3.4 but normal value for our country is between 0.5-2.5 [38]. hydrothermal coefficient (ch) values are between 1.35 (2013), 1.48 (2014) and 1.41 (2015). it can be seen on hydrothermal coefficient (ch), that year with most precipitations is 2014 (1.48 (ch)) (table 1). bioclimatic index (ibcv) and express the interaction between temperature, insolation and humidity. it indicated the possibility of an area for one or the other directions production of vine. bioclimatic index (ibcv) has values between 8.3 (2013), 7.6 (2014) and 8.1 (2015) (table 1). the ecoclimatic conditions from șimleul silvaniei vine growing revealed the exceptional character food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 88 of this area and nature of wine from din vineyard present in large variety of wine produced in the area studied. table 1 climate data from șimleul silvaniei area in 2012-2014 area year studies elements specific optimal condions for cultivation of vines average vales extreme limits min. max. șimleul silvaniei 2013 the vegetation period days 196 191 199 150-170 thermal balance global (∑t0g) 3683 3456 3731 2700-3600 active (∑t0a) 3394 3305 3697 2600-3500 useful (∑t0u) 1549 1541 1561 1000-1700 thermal coefficient (ct) 18.1 17.3 19.2 16-19 insolation (hours) real (∑ir) 1594 1456 1661 1200-1600 coefficient of insolation (ci) 7.81 6.84 8.31 7-9 hours precipitations (mm) total annual (∑pp) 489.6 459.4 549.3 500-700 coefficient of precipitation (cp) 1.72 1.36 2.01 0.9-2.7 the interaction of climatic factors real heliothermic index (ihr) 1.75 1.05 2.31 1.35-2.70 hydrothermal coefficient (ch) 1.35 1.25 1.70 0.6-1.8 bioclimatic index (ibcv) 8.3 7.9 8.9 4-15 2014 the vegetation period days 193 189 196 150-170 thermal balance global (∑t0g) 3652 3369 3697 2700-3600 active (∑t0a) 3287 3219 3654 2600-3500 useful (∑t0u) 1538 1531 1594 1000-1700 thermal coefficient (ct) 17.6 17.0 18.2 16-19 insolation (hours) real (∑ir) 1498 1420 1643 1200-1600 coefficient of insolation (ci) 7.53 7.59 8.01 7-9 hours precipitations (mm) total annual (∑pp) 536.9 521.9 578.6 500-700 coefficient of precipitation (cp) 1.83 1.52 1.98 0.9-2.7 the interaction of climatic factors real heliothermic index (ihr) 1.56 1.09 2.23 1.35-2.70 hydrothermal coefficient (ch) 1.48 1.12 1.53 0.6-1.8 bioclimatic index (ibcv) 7.6 6.9 8.1 4-15 2015 the vegetation period days 194 191 195 150-170 thermal balance global (∑t0g) 3676 3484 3869 2700-3600 active (∑t0a) 3321 3532 3846 2600-3500 useful (∑t0u) 1551 1493 1676 1000-1700 thermal coefficient (ct) 16.9 15.3 19.0 16-19 insolation (hours) real (∑ir) 1563 1395 1569 1200-1600 coefficient of insolation (ci) 7.69 7.53 7.96 7-9 hours precipitations (mm) total annual (∑pp) 521.9 512.1 568.3 500-700 coefficient of precipitation (cp) 1.80 1.70 1.99 0.9-2.7 the interaction of climatic factors real heliothermic index (ihr) 1.69 1.51 2.06 1.35-2.70 hydrothermal coefficient (ch) 1.41 1.36 1.82 0.6-1.8 bioclimatic index (ibcv) 8.1 8.0 8.4 4-15 analysis of the main qualitative indicators of grapes. the content in sugar of the grapes it was between values of 202.28±2.98 (2013) (g/l) and 201.32±1.59 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 89 (2015) (g/l) obtained by variety f.a. the opposite is variety f.r. 179.72±2.46 (2014) (g/l) and f.m. 185.72±5.59 (2014) (g/l). we can see that the accumulation of sugar in this case was influenced by factor years (f=11.711, p ≤ 0.000) and variety (f=20.640, p ≤ 0.000). titratable acidity expressed in g/l (c4h4o6) has the following values: the highest values was registered to f.m variety (9.34±0.08 (g/l) 2013) and (9.39±0.03 (g/l) 2014), the lowest values was registered to f.a. (8.70±0.04 (g/l) 2013) and (8.68±0.04 (g/l) 2015). acidity expressed in g/l (h2so4) in this case it was influenced by factor years (f=64.777, p ≤ 0.000) and variety (f=11.803, p ≤ 0.000) and the higest values was obtained to f.m. (4.70±0.09 g/l h2so4 (2014)). values of ph it was included in the normal limits for the variety analyzed. mass of 100 gains had values between 134.39±1.47 (g) (2015) and 182.06±5.98 (g) (2014) (table 2). table 2 the main features of the composition of grapes area va riet y years weight of 100 grains (g) sugar (g/l) titratable acidity (g/l c4h4o6) acidity h2so4 (g/l) ph ș.s. 2013 174.75±4.26 c α 191.72±2.46 cde α 9.14±0.04 b α 4.59±0.15 ab α 3.22±0.05 bcd β f.r. 2014 182.06±5.98 a α 179.70±2.45 f β 9.22±0.05 b α 4.58±0.11 ab α 3.92±0.24 a α 2015 181.60±1.75 ab α 187.68±3.45 de α 9.18±0.02 b α 4.43±0.05 c α 3.84±0.10 a α 2013 129.32±2.19 f α 202.28±2.98 a α 8.70±0.04 e β 4.28±0.04 c β 3.12±0.03 d β f.a. 2014 130.16±1.96 f α 196.27±5.78 abc α 9.05±0.06 c α 4.56±0.07 b α 3.21±0.04 bcd α 2015 129.91±1.39 f α 201.32±1.59 a α 8.68±0.04 e β 4.21±0.02 c β 3.19±0.02 cd α 2013 134.63±5.02 ef α 193.21±4.77 bcde β 9.17±0.01 b α 4.55±0.03 b α 3.42±0.02 b β r.i. 2014 140.84±4.97 e α 190.43±0.84 cde β 9.17±0.02 b α 4.54±0.06 b α 3.92±0.24 a α 2015 134.39±1.47 ef α 199.87±1.39 ab α 8.79±0.05 d β 4.25±0.04 c β 3.84±0.10 a α 2013 167.97±1.47 d α 187.92±7.46 de α 9.34±0.08 a α 4.67±0.05 ab α 3.39±0.03 bc α f.m . 2014 169.67±4.23 cd α 185.72±5.59 f α 9.39±0.03 a α 4.70±0.09 a α 3.27±0.03 bcd β 2015 175.39±5.14 bc α 193.41±3.03 bcd α 9.19±0.02 b β 4.27±0.05 c β 3.25±0.04 bcd β f (fisher factor) 109.531 8.615 87.231 17.268 24.372 sig. p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 year f1 4.270 11.711 96.520 64.777 23.882 sig. * *** *** *** *** variety f1 395.055 20.640 211.922 11.803 53.009 sig. *** *** *** *** *** years x variety f1 1.856 1.571 21.790 4.164 10.217 sig. ns ns *** ** *** f.r. = fetească regală; f.a. = fetească albă; r.i. = italian riesling; f.m. = furmint; ș.s. = șimleul silvaniei f1 = fisher factor food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 90 analysis of the main qualitative indicators of white wine. the reducing sugar (g/l) was very significantly influenced by variety factor (f=12.952, p ≤ 0.000), the interaction the year x variety had a distinctly significant influence (f=4.595, p = 0.003), but the year factor had not influenced this character. the highest values were registered to f.a. variety (2.63±0.17 (g/l) 2015) and the lowest values were registered to f.m. (1.70±0.17 (g/l) 2015). the highest content of total dry extract (g/l) was recorded in wines from r.i. variety (27.73±1.29 (g/l) 2013); (25.66±1.92 (g/l) 2014) and f.a. variety (25.84±1.48 (g/l) 2013) this variants are equal in statiscal terms. the lowest content of total dry extract was recorded to wine obtained from f.m. (19.32±1.88 (g/l) 2013; 19.42±1.00 (g/l) 2014 and 19.80±0.75 (g/l) 2015). the differences between variants were statistically assured (f=12.442, p ≤ 0.000). the biggest influence on the non-reducing extract content was given by variety factor (f=50.244, p ≤ 0.000), followed by year factor (f=6.92, p = 0.004) and interaction of factors years x variety (f=2.677, p = 0.039), this had a distinctly significant influence on this character. the highest content of non-reducing extract was recorded in wine obtained from r.i. variety (26.62±0.43 (g/l) 2013 (table 3). table 3 the main features of the composition of white wine are a vari ety years reducing sugars (g/l) total dry extract (g/l) non-reducing extract (g/l) total acidity (g/l c4h4o6) volatile acidity (g/l ch3cooh) ș.s. 2013 1.94±0.08 de α 22.75±0.84 bc α 19.70±0.78 d α 7.65±0.11 a α 0.45±0.02 cde α f.r. 2014 2.15±0.19 bcd α 21.47±0.93 bcd αβ 20.58±0.58 cd α 7.53±0.10 ab αβ 0.50±0.03 abcd α 2015 2.08±0.08 d α 20.48±0.91 cd β 19.91±1.18 d α 7.38±0.06 b β 0.52±0.09 ab α 2013 2.48±0.43 ab α 25.84±1.48 a α 24.83±0.31 b α 7.30±0.15 bc αβ 0.41±0.02 ef α f.a. 2014 2.12±0.11 cd α 21.35±1.09 bcd β 23.78±0.32 b α 7.11±0.05 cd β 0.46±0.03 bcde α 2015 2.63±0.17 a α 23.30±1.72 b αβ 24.31±1.83 b α 7.35±0.06 b α 0.44±0.03 de α 2013 2.46±0.13 abc α 27.73±1.29 a α 26.62±0.46 a α 6.92±0.12 de α 0.53±0.03 a α r.i. 2014 2.14±0.05 bcd α 25.66±1.92 a α 25.48±0.88 ab α 6.89±0.05 de α 0.57±0.04 a α 2015 2.50±0.30 ab α 21.86±1.63 bcd β 24.42±1.91 b α 7.01±0.03 de α 0.51±0.04 abc α 2013 1.99±0.13 de α 19.32±1.88 d α 24.57±0.92 b α 6.61±0.12 f α 0.39±0.02 ef α f.m. 2014 2.16±0.03 bcd α 19.42±1.00 d α 21.86±0.68 c β 6.82±0.15 ef α 0.37±0.04 f α 2015 1.70±0.17 e β 19.80±0.75 d α 21.10±0.48 cd β 6.61±0.31 f α 0.36±0.03 f α f (fisher factor) 6.168 12.442 16.421 21.321 10.140 sig. p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 p ≤ 0.000 yea rs f1 0.709 11.712 6.920 0.237 1.631 sig. ns *** ** ns ns vari ety f1 12.952 28.771 50.244 71.752 32.981 sig. *** *** *** *** *** yea r x vari ety f1 4.595 4.521 2.677 3.138 1.556 sig. ** ** * * ns f.r. = fetească regală; f.a. = fetească albă; r.i. = italian riesling; f.m. = furmint; ș.s. = șimleul silvaniei f1 = fisher factor food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 91 the total acidity (g/l) c4h4o6 and volatile acidity (g/l) ch3cooh recorde very significant differences between variants total acidity (g/l) c4h4o6 (f=21.321, p ≤ 0.000) and volatile acidity (g/l) ch3cooh (f=10.140, p ≤ 0.000). in both cases the factor who influencing most was the variety (f=71.752, p ≤ 0.000 total acidity (g/l) c4h4o6) and (f=32.981, p ≤ 0.000 volatile acidity (g/l) ch3cooh). the highest content was recorded in f.r. (7.65±0.11 (g/l) 2013 total acidity c4h4o6) and r.i. (0.53±0.03 (g/l) 2013; (0.57±0.04 (g/l) 2014 volatile acidity ch3cooh) (table 3). pearson correlation coeficients of composition of grapes and qualitative characteristics of white wine. to reveal if the composition of the wine are influenced by the composition of grape, in this sense we have performed person correlation between composition of grapes (weight of 100 grains, sugar, titratable acidity, acidity and ph) and qualitative characteristics of wine (reducing sugars, total dry extract, non-reducing extract, total acidity and volatile acidity). values greater than 0.5 represent a strong correlation between variables, a positive correlation means that, when a variable increases, correlated variables increase also, while a negative correlation means an increase in the primary variable causes a decrease in the correlated variables. fig. 1. pearson correlation coeficients of composition of grapes and qualitative characteristics of white wine 200190180 9,3 9,0 8,7 200190180 4,8 4,5 4,2 9,39,08,7 4,8 4,5 4,2 180160140 2,8 2,4 2,0 9,39,08,7 2,8 2,4 2,0 180160140 30 25 20 200190180 30 25 20 180160140 25,0 22,5 20,0 200190180 25,0 22,5 20,0 9,39,08,7 25,0 22,5 20,0 200190180 25,0 22,5 20,0 4,03,53,0 0,5 0,4 0,3 titratabile acidity *sugar acidity *sugar acidity *titratabile acidity reducing sugars*weight of 100 grains reducing suga rs*titrata bile acidity total dry ex tract*weight of 100 gra ins total dry ex tract*sugar non-reducing ex tract*weight of 100 gra ins non-reducing ex tract*sugar non-reducing ex tra ct*titratabile acidity non-reducing ex tract*sugar volatile acidity *ph food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 92 they have obtained a large number a string relationships between the qualitative characteristics of wine and composition of grapes: titratabile acidity & sugar (0.835**); acidity & sugar (-0.821**); acidity & titratabile acidity (0.715*); reducing sugars & weight of 100 grains (0.526*); reducing sugars & titratabile acidity (-0511*); total dry extract & weight of 100 grains (-0.666*); total dry extract & sugar (0.529*); non-reducing extract & weight of 100 grains (-0.874**); nonreducing extract & sugar (0.559*); nonreducing extract & titratabile acidity (0.641); non-reducing extract & sugar (0.698*) and volatile acidity & ph (0.673*). 4. conclusion the ecoclimatic conditions from silvaniei vine growing revealed the exceptional character of this area and nature of authenticity of wine from vineyard present in large variety of wine produced in the area studied. regarding qualitative assessment of varieties taken in testing, based on the results it can be observed that the varieties have a good suitability in the area studied and quality determinations show particular characteristics but also influence of the ecoclimatic and ecopedologic on wine quality. the quality of wine obtained for f.r., f.a., r.i. and f.m. in years 2013, 2014 and 2015 in șimleul silvaniei center, was particulary influenced by the balance between alcoholic strength, acidity and residual sugar. in the conditions of silvaniei vineyard, at the șimleul silvaniei center can be obtained wines fresness and flavour, giving it a speial personality that is appreciated by connoisseurs and casual consumers. person ̉s correlation analysis revealed a number of strong correlation between the qualitative characteristics of wine and composition of grape. 5. acknowledgments this paper was published under frame of romanian ministry of agriculture and rural development project ader no.14.2.2. „quantitative studies on assessment and monitoring contaminants, on the chain of viticulture and winemaking to minimize the amount of pesticides and heavy metals as principal pollutants”. 6. references [1]. voica c., dehelean a., pamula a., method validation for determination of heavy metals in wine and slightly beverages by icp-ms, journal of physics, 182(1) : 1–5, (2009) [2]. dalipi r., borgese l., zacco a., tsuji k., sangiorgi e., piro r., bontempi e., depero l.e., determination of trace elements in italian wines by means of total reflectation x-ray fluorescence spectroscopy. international journal environmental anal chemistry, 95(1): 1–11, (2015) [3]. karataș d., aydin f., aydin i., karataș h., elemental composition of red wines in southeast turkey. czech journal food sci 33: 228–236, (2015) [4]. marini f., bucci r., magri a., magri a., authentication of italian cdo wines by class-modeling techniques. chemometrics and intelligent laboratory systems 84: 164–171, (2006) [5]. gonçalves da silva a.m., pavan m.a., muniz a.s., tonin t.a., pelizer t., nutrient availability in the soil and its absorption, transport, and redistribution in vines. communications in soil science and plant analysis 39: 1507–1516, (2008) [6]. lădaru g.r., beciu s., vlad i.m., analysis on the evolution of surfaces under vine in romania (2003-2013). scientific papers series food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 93 management, economic engineering in agriculture and rural development 14(4): 153–156, (2014) [7]. tudorache a., pircalabu l., porumb r., tomoiaga l., iliescu m., enache v., simion c., dorina d., petrescu a., ghica m., description des climats dans les centres viticoles principaux de roumanie. oiv 86:45–58, (2013) [8]. fraga h., santos j.a., malheiro a.c., oliveira a.a., moutinho-pereira j., jones g.v., climatic suitability of portuguese grapevine varieties and climate change adaptation. international journal of climatology. doi: 10.1002/joc.4325.2015. [9]. paltineanu c., mihailescu i. f., seceleanu i., dragota c., vasenciuc f., using aridity indices to describe some climate and soil features in eastern europe: a romanian case study. theoretical and applied climatology 90: 263–274, (2007) [10]. carbonneau a., ecofisiologie de la vigne et terroir. terroir, zonizzazione, viticoltura. trattato internazionale phytoline 1: 61–102, (2003) [11]. mackenzie d.e., christy a.g., the role of soil chemistry in wine grape quality and sustainable soil management in vineyards. water sci. technol. 51: 27–37 (2005) [12]. magalhaẽs n., tratado de viticultura: a videira, a vinha e o terroir. lisboa, portugal: chaves ferreira. (2008) [13]. morlat r., jacquet a., grapevine root system and soil characteristics in a vineyard maintained long-term with or without interrow sward. american journal of enology and viticulture 54: 1–7 (2003) [14]. field s.k., smith j.p., holzapfel b.p., hardie w.j., emery r.j.n., grapevine response to soil temperature: xylem cytokinins and carbohydrate reserve mobilization from budbreak to anthesis. american journal of enology and viticulture 60: 164–172 (2009) [15]. solomon s., qin d., manning m., chen z., marquis m., averyt k.b., tignor m., miller h.l., climate change 2007: the physical science basis. in: contribution to working group i to the fourth assessment report of the intergovernmental panal on climate change. cambridge university press, chap 3 and 11 (2007). [16]. urhausen s., brienen s., kapala a., simmer c., climatic conditions and their impact on viticulture in the upper moselle region. climatic change 109:349-373. doi 10.1007/s10584-011-0059-z (2011) [17]. menzel a., a 500 year phenoclimatological view on the 2003 heat wave in europe assessed by grape harvest dates. meteorologische zeitschrift 14(1):75–77 (2005). [18]. duchêne e., schneider c., grapevine and climatic changes: a glance at the situation in alsace. agronomy for sustainable development 25:93–99 (2005) [19]. adams r.m., wu j., houston l.l., the effects of climate change on yields and water use of major california crops. appendix ix to climate change and california. california energy commission, public interest energy research (pier), sacramento, ca (2003) [20]. lobell d.b., field c.b., cahill k.n., bonfils c., impacts of future climate change on california perennial crop yields: model projections with climate and crop uncertainties. agric. for. meteorol. 141: (2–4), 208–218 (2006) [21]. jones g.v., duchêne e., tomasi d., yuste j., braslavaska h., schultz h., martinez c., boso s., langellier f., perruchot c., guimberteau g., changes in european winegrape phenology and relationships with climate. in: xiv international gescoviticulture-congress, geisenheim 1: 55–61 (2005) [22]. storchmann k., english weather and rhine wine quality: an ordered probit model. j. wine res. 16(2): 105–119 (2005) [23]. alston j.m., fuller k.b., lapsley j.t., soleas g., too much of a good thing? causes and consequences of increases in sugar content of california wine grapes. j. wineecon. 6(2): 135–159 (2011) [24]. de orduna r.m., climate change associated effects on grape and wine quality and production. food res. int. 43(7): 1844–1855 (2010) [25]. haeger j., storchmann k., prices of american pinot noir: climate, critics, craftsmanship. agric. econ. 35: 67–78 (2006) [26]. ashenfelter o., storchmann k., measuring the economic effect of global warming on viticulture using auction, retail and wholesale prices. rev. ind. organ. 37: 51–64 (2010) [27]. marinoni o., navarro garcia j., marvanek s., prestwidge d., clifford d., laredo l.a., development of a system to produce maps of agricultural profit on a continental scale: an example for australia. agric. syst. 105(1) : 33–45 (2012) [28]. webb l.b., whetton p.h., barlow e.w.r., climate change and winegrape quality in australia. clim. res. 36(2): 99 (2008) [29]. bunea c.i., mucaciu m.l., pop n. the influence of green works on seyve-villard 18402 grape quality, vine with biological resistance. bulletin uasvm horticulture 70(1): 60–67 (2013) [30]. suciu l., puia c., florian v., miclea r., bunea c.i., the behaviour of vine varieties to downy mildew attack (plasmopara viticola berk&curt) in ampelegraphic collection in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 florin-dumitru bora, alina donici, oana-mihaela ripanu, compositional quality assessment of wines produced in silvaniei vine growing center of șimleul silvaniei, 20132015 harvest, food and environment safety, volume xv, issue 1 – 2016, pag. 84 94 94 the conditions of usamv cluj-napoca. proenvironment 5: 221–226 (2012) [31]. iliescu m., popescu d., comșa m., the impact of climatic factors on the rootstock quality, in the blaj vineyard center. bulletin uasvm horticulture 72(1): 219–220 (2015) [32]. bora f.d., pop t.i., bunea c., popescu d., iliescu m., pop n., the assessment of the main climatic conditions in northwest of romania for viticulture (1991-2013). bulletin uasvm horticulture 72(2): 445–447 (2015) [33]. bora f.d., pop t.i., bunea c.i., urcan d.e., babeș a., mihaly-cozmuța l., mihaly-cozmuța a., pop n., influence of ecoclimatic and ecopedological conditions on quality of white grape varieties from north-west of romania. bulletin uasvm horticulture 71(2): 218–225 (2014) [34]. bora f.d., bunea c.i., rusu t., pop n., vertical distribution and analysis of micro-, macroelements and heavy metals in the system soilgrape-wine in vineyard from north-west romania. chemistry central journal doi 10.1186/s13065015-0095-2 9–19 (2015) [35]. bunea c.i., pop n., bora f., popescu d., bunea d., the behaviours of wine grape varieties with biological resistant, in blaj vineyard conditions. bulletin uasvm horticulture 71(2): 343–344 (2014) [36]. țârdea c., dejeu l., viticulture. editura didactică și pedagogică. bucurești (1995) [37]. oșlobeanu m., general and special viticulture. edp bucurești (1980) [38]. pop n., ecological viticulture. ed. academicpress. cluj-napoca. romania (2005) 1. introduction 4. conclusion 263 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 263 266 vi rgin o l iv e oil adu lte rat ion w ith other edib le o ils: in flu ence of s ub st i tu t ion on phys ico ch emic al prope rti es *mircea oroian1, ana leahu1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received september 7th 2014, accepted september 16th 2014 abstract:adulteration is a common practice in the field of virgin olive oil industry because of its greater demand at european and international level. the olive oil represents around of 4% of the edible oil at international level.the aim of this paper is to study the influence of olive oil adulteration by substitution with other types of edible oils (sunflower, corn and groundnut) in different percentages (10%, 20%, 30%, 40% and 50%, respectively). the physicochemical properties studied were: density, refraction index and saponification index. the suitable parameter for the adulteration identification is the density, at percentages of substitution greater than 5% it can be observed that the parameter values is getting out of the domain from the eu regulations. the refractive index is not a proper tool for the adulteration identification, because its values are in the normal range in all the substitutions levels. the saponification index can be used for the identification of virgin olive oil adulteration with sunflower and corn oils in substitutions greater than 30%. keywords:olive oil, adulteration, substitution, physicochemical properties 1. introduction extra virgin olive oil is obtained from the olive fruit named olea europaea. the extra virgin oil (evoo) is extracted by only mechanical procedure without application of refining process [1]. the evoo is practically the only vegetable oil that can be consumed directly in its raw state as well as it contains important nutritional elements (fatty acids, vitamin, sterols, etc.) [2]. the quality of olive oil ranged from the high quality of evoo to the low quality olive pomace oil [3]. it is one of the primary ingredients of the mediterranean diet [4]. adulteration of food products involves the replacement of high-cost ingredients with lower grade and cheaper substitutes [5]. due to its sensory quality andnutritional benefits extra-virgin olive oil is often adulterated withless expensive oils. adulteration of olive oil is a serious problemfor regulatory agencies, oil suppliers and could also threat healthof consumers. the substitution of adulteration of evoo with cheaper ingredients is not only economic fraud, but may also on occasion have severe health implications for consumers [6]. because of the high value of evoo, it can be adulterated with other oils of lower commercial value. the most common adulterants found in evoo are refined olive oil, seed oils and nut oils.actually, blend edible oils can be prepared only forsuitable products, but if the resulting blend deviates from the mixture proportions given on the label, or if the blend is traded as genuine, it means the oil is adulterated [7,8]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 mircea oroian, ana leahu, virgin olive oil adulteration with other edible oils: influence of substitution on physicochemical properties, food and environment safety, volume xiii, issue 3 – 2014, pag. 263 – 266 264 the aim of this study is to investigate the influence of virgin olive oil substitution with other edible oils (sunflower, corn and groundnut oils) on the physicochemical parameters. 2. materials and methods materials olive oil, sunflower oil, corn oil and groundnut oil were purchased from the local market of suceava. methods density measurements density (ρ) of the oils samples was measured using pycnometer with an accuracy 10−4 gm/cm3. the calibration of pycnometer was made with ultrapure water. temperature was kept constant within ±0.1 °c using pid controller and circulating water using thermo static-fluid bath. refraction index the refraction index of the analysed samples was measured using an abbe refractomer leica at 20 °c. saponification index saponification value is expressed by potassium hydroxide, in mg, required to saponify one gram of fat [9]. it depends on the kind of fatty acid contained in the fat. the sample is first saponified by adding 0.5 mol/l potassium hydroxide ethanol, and then the excessive potassium hydroxide is titrated with 0.5 mol/l hydrochloric acid until the end point is reached. the reactions which occur are: c3h5(oocr)3 + 3koh 3rcook + c3h5(oh)3 3. results and discussions the influence of different percentage of olive oil substitution of its physicochemical properties has been studied. firstly, the olive oil purchased from the local market of suceava was studied to see if the density, refraction index and saponification index, respectively, are according to the range established by the eu regulations [10]. the density of virgin olive oil must range between 0.914 and 0.919 g/cm³, the refraction index must range between 1.467 and 1.470 and the saponification index must range between 184 and 196. in the table 1 are presented the density, refraction index and saponification index of virgin olive oil purchased from the local market. table 1. physicochemical parameters of virgin olive oil parameter value density 0.915 g/cm3 refraction index 1.467 saponification index 187.51 according to the values presented to the table 1, the virgin olive oil meets the threshold set by the eu regulations [10]. substitution influence on the virgin olive oil density in table 2 are presented the evolution of virgin olive oil density with different percentages substitution. table 2. oil samples density: avirgin olive oil substituted with sunflower oil, bvirgin olive oil substituted with corn oil and c – virgin olive oil substituted with groundnut oil substitution percentage a (g/cm3) b (g/cm3) c(g/cm3 ) 0 0.9150 0.9150 0.9150 5% 0.9204 0.9250 0.9350 10% 0.9225 0.9252 0.9350 20% 0.9226 0.9261 0.9354 30% 0.9227 0.9271 0.9356 40% 0.9230 0.9280 0.9359 50% 0.9235 0.9282 0.9365 according to the eu regulations [10], the virgin olive oil density must range between food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 mircea oroian, ana leahu, virgin olive oil adulteration with other edible oils: influence of substitution on physicochemical properties, food and environment safety, volume xiii, issue 3 – 2014, pag. 263 – 266 265 0.914 and 0.919 g/cm³. it can be observed that the substitution of the virgin olive oil with other types of edible oil with 5%, the density is increasing greater than the maximum allowable value for the density (0.919 g/cm3). the highest density value was observed in the case of the sample c (virgin olive oil substituted with 50% groundnut oil). it can be observed a positively correlation between the substitution of virgin olive oil and density. substitution influence on the virgin olive oil refractive index in figure 1 is presented the evolution of the virgin olive oil refractive index – pure and substituted. fig. 1. refractive index evolution: avirgin olive oil substituted with sunflower oil, bvirgin olive oil substituted with corn oil and c – virgin olive oil substituted with groundnut oil according to the eu regulations [10], the refractive index must range between 1.467 and 1.470. the evoo substation with groundnut oil (in all the substitution percentages) decreases the value of the refractive index. the substitution of evoo with 10%, 20%, 30%, 40% and 50% corn oil and sunflower oil maintain the refractive index at normal levels. the substitution of evoo with sunflower oil had the highest influence on the refractive index. substitution influence on the virgin olive oil saponification index in table 3 is presented the evolution of the saponification index of the virgin olive oil. table 3 oil samples saponification index: avirgin olive oil substituted with sunflower oil, bvirgin olive oil substituted with corn oil c – virgin olive oil substituted with groundnut oil substitution percentage (%) a b c 0 187.5 187.5 187.5 5 206.1 200.2 185.2 10 216.2 203.1 183.2 20 223.2 205.8 180.9 30 228.6 217.8 178.2 40 230.2 219.2 175.4 50 232.5 223.6 176.1 the values of saponification index for the virgin olive oil, according to the eu regulations [10], range between 184 and 196. the most visible adulteration is that with sunflower oil, which at a percentage of substitution of 50%, the saponification index is reaching the value of 240. in smaller percentages, the virgin olive oil adulterated with sunflower oil is maintained the saponification index value closer to the normal ones. for observing the adulteration of virgin olive oil with sunflower oil is needed at least a substitution percentage of 30%. in the case of evoo adulteration with corn oil, the saponification index values are smaller than in the case of sunflower oil, but the adulteration can be observed if the substitution percentage is greater than 30%. in the case of evoo adulteration with groundnut oil, the saponification index cannot be used as an indicator, because the values of the adulterated samples are in the range of evoo. 1.4655 1.466 1.4665 1.467 1.4675 1.468 1.4685 1.469 1.4695 0 20 40 60 r ef ra ct iv e in de x substitution percentage (%) a b c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 mircea oroian, ana leahu, virgin olive oil adulteration with other edible oils: influence of substitution on physicochemical properties, food and environment safety, volume xiii, issue 3 – 2014, pag. 263 – 266 266 4. conclusions food adulteration is a common practice for the olive oil industry due to its great demand at european and international level because of the great positively influence of it on the human body. the adulteration of virgin olive oil with different edible oils can be observed using the values for density, refractive index and saponification index. the substitution of virgin olive oil with sunflower can be observed using: the density determination for substitutions greater than 5%, the saponification index greater than 5%, while the refractive index determination cannot be used. in the case of the substitution of evoo with corn oil, the density and saponification index can be used for the identification of the adulteration greater than 5%. the saponification index cannot be used for the identification of the substitution of evoo with groundnut oil. 5. references [1] apetrei, i. m., apetrei, c., detection of virgin oil adulteration using a volatammetric e-tongue, computers and electronics in agriculture, 108, 148-154, (2014) [2] jabeur, h., zribi, a., abdelhedi, r., bouaziz, m., effect of olive storage conditions on chemical olive oil quality and the effective role of fatty acids alkyl esters in checking olive oils authenticity, food chemistry, 169, 289-296, (2015) [3] tsimidou, m. z., olive oil quality. in: boskou, d., (ed.), olive oil: chemistry and technology, second ed. champaign, illinois: aocs publishing, 93-112, (2006) [4] psaltopoulou, t., naska, a., orfanos, p., trichopoulos, d., mountokalakis, t., trichopoulou, a., olive oil, the mediterranean diet, and arterial blood pressure: the greek european prospective investigation into cancer and nutrition (epic) study. american journal of clinical nutrition, 80, 1012–1018, (2004) [5] tay, a., singh, r. k., krishnan, s. s., gore, j. p., authentication of olive oil adulterated with vegetable oils using fourier transform infrared spectroscopy.lwt-food science and technology, 35, 99–103, (2002) [6] toreccilla, j. s., rojo, e., dominguez, j. c., rodriguez, f., linear and non linear chemometric models to quantify the adulteration of extra virgin olive oil, talanta, 83, 404-409, 2010 [7] ulberth, f., buchgraber, m., authenticity of fats and oils. european journal of lipid science technology, 102, 687–694, (2000) [8] gurdeniz, g., ozen, b., detection of extravirgin olive oil by chemometric analysis of mid-infrared spectral data, food chemistry, 116, 51-525, (2009) [9] iso 3657 – saponification value of fat and oil [10] commission implementing regulation (eu) no 1348/2013 of 16 december 2013 amending regulation (eec) no 2568/91 on the characteristics of olive oil and oliveresidue oil and on the relevant methods of analysis food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 38 the study of heavy metals (ni, zn, cu, pb) in the vegetative organs, harvest and growing soil of potatoes, wheat and wild blackberry *rafael h. yedoyan1, t. v. yedoyan2 1vanadzor state pedagogical institute,* info@vspi.am 2yerevan state university of architecture and construction, armenia *corresponding author received 6 may 2012, accepted 26 june 2012 abstract: here was discussed the researches of heavy metals transition to the root, steam and leaf from sprout growing soil of potatoes, wheat and wild blackberry. the growing soil data comparison shows that soils differ with heavy metals content. heavy metals content differs a little in potato leaf and tube. this gives an opportunity to suggest a new method that is to define heavy metals content in potato tube before harvesting, which will have great organizational and economical importance. according to our study in different organs of potato, wheat and blackberry the quantity of zn, cu, ni, pb is proportional to their content in soil; zn. cu. ni. pb contents in vegetative organs of potato, wheat and blackberry and harvest are different; zn, cu, ni, pb contents in potato tube, wheat grain and blackberry fruit is the least as compared with vegetative organs; the contents of heavy metals in potato tube can be defined by means of new method, before harvesting, by analysis data of green leaf. growing soils have different contents of heavy metals. keywords: heavy metals, root, green leaf, potato, sprout, juicy fruit, crop. 1. introduction biosphere and human populatio n health need protection and conservation. the population health depends on production of ecologically pure products. ecologically pure product is a priority problem of modern agricultural product evaluation, where is significant not only the production of many and qualified agricultural products and raw materials, but also the ecological evaluation of pollution. the kinds of crop harvest pollution are of different nature from this viewpoint. the wide usage of chemicals lead as agricultural products fertilizers, herbicides, insecticides, fungicides, different growth stimulators, as well as cultivation of trans-genetic sorts. the environment safety, improvement of sanitary conditions of anthropogenic factors, ecological wastes treatment and reduction, toxic elements in ecosystems and ecologically pure product receiving is one of the main problems, separate issues of which are solved by many scientists of the world. the high rate of populatio n growth on the earth, advanced stage of motor transport, power engineering, food, heavy and light industr y development essentially increases humane influence on biosphere. there mailto:info:@vspi.am food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 39 were used as many raw materials as for the whole human history on the earth during the last 30 years [1]. the science has not created such ecological terms and methods that would give an opportunity to abolish the emerging negat ive ecological problems. indeed, some technological processes are being improved in industry, but ecological damage is irreversible from the ecological viewpoint [1,2]. 2. experimental plant pollution is directly connected with water, atmosphere, especially soil pollution. water, atmosphere and soil pollution factors are divided into 3 groups: 1. physical (temperature, noise, electromagnetic field, radioactive materials), 2. chemical (chloral-organic, aromatic combinations, carbonic acid, heavy metals, radio nucleotides, mineral fert ilizers, herbicides, various chemicals), 3. biological (microbiological, biogenous, gene engineering). the sources of water, atmosphere and soil pollution are different, where nowadays heavy metals rank high term with their harmfulness [3,6,7,8]. according to the level of harmfulness these metals are divided into 3 groups: 1. as, cl, mg, zn, f, hg; 2. b, co, mo, cu, cr; 3. ba, v, w, mn. the main source of heavy metal distribution for agriculture is the soil, the ways of pollution of which are different and we won't touch upon it. but we wish to state by the scientific data of that sphere that they influence on the soil solution reaction, physicochemical properties, mechanical structure, absorption complex, changing soil fertility and crop yield [4,5]. heavy metals get into the human organism both from crop yield, and wild plants used by people and reflect on their health. plant pollution with heavy metals is conditioned by their quantity and dissemination in the growth soil. in this concern the main thing is the initial rock content, airing of which resulted soil was formed. along with this the main sources of soil pollution with heavy metals is mining, heavy metallurgy, engineering industry, galvanic industry, production of cement, leather, light industry and food production waste and water used for irrigation in agricultural regions. the sources of water pollution are very different. heavy metals get into plants from soil and into our organism by food chain and infect it, causing various illnesses. their danger also consists in that influencing on genetic system they cause genetic illnesses, which are inherited to generations. the danger of heavy metals increases. the rates of harmfulness of separate heavy metals are not fully defined by separate metals calculation in plants and especially wild plants. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 40 3. results and discussion according to data of definite illnesses monitoring carried out by medical centers and corresponding organizations in lori region they are more spread in lori region and heavy metals have their role here. according to international literature data cancer often occurs in developed countries [6,7,8]. if so, lori region is not the most developed in ra. in the result of anthropogenic pollution in lori region the presence of color metallurgy is specified (coppermo lybdenum factory of alaverdi, ajrum branch and small companies), as well as teghut. it is possible that there can be other sources of illnesses which are not revealed yet. from this point of view it must be stated that both citizens of vanadzor and region use wild spicy, food and herbal plants, the number of which is more than twenty. it is not excepted, that part of them can contain more heavy metals which can influence upon our health. the aim of this paper is to determine heavy metals content in food plants used by people and suggest a method to define hm content before harvesting. the research was carried out in 2009-2011. from cultured plants we took potatoes and wheat, and blackberry bush from wild ones. we followed the principles that both mentioned plants occupy the largest sowing area. wild blackberry is widely used especially by lori region population from which preserves and syrups are made and they are very rich of vitamins, sugars and mineral elements. selecting these plants we also followed the botanical variety of crop formation, that is – underground potato crop tuber modification to sprout, wheat grain is a single-seed real fruit, and blackberry is multi-seed juicy fruit. the contents of heavy metals in different organs of plants were defined before harvesting. average samples were taken from 20 plants. during the ripening of potato tops the leaves fall, which were dried in laboratory condit ions, as well as other samples. the content of heavy metals was defined by un-diction cuploidal plasmomass-spectrum ether analysis method. the researches were carried out upon potato «morphona» and wheat «bezostaya 1» sorts, and the wild plants of blackberry of pambak region forest. the samples were taken from our agricultural industrial and experimental sowing area in darpas. the cultivation of plants was carried out by the technology approved in the region for the same plants cultivation (land plowing and sowing, plant care and harvesting). content of ni, zn, cu, pb (mg/kg) was studied in all plants. the study results contents of heavy metals in different organs and growing soil of wheat and potatoes are shown in tables [1,2]. in table 1 is shown heavy metals contents in green leaf, sprout, root, tube and growing soil of potato. it was food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 41 revealed that in all cases from heavy metals the contents of zn is the most, pb is less, cu and ni have average content. it is remarkable, that this regularity is condit ioned by growing soil. table 1. quantitative data of ni, zn, cu, pb in potato tube and vegetative organs (mg/kg) n sample heavy metals ni zn pb cu 1. leaf 1.5943 8.7320 1.8435 6.8684 2. sprout 3.6053 91.2308 2.2054 19.1473 3. root 14.1301 55.5524 3.5985 22.2364 4. tube 0.1206 0.33385 0.00915 0.044887 5. soil 38.815 72.3573 2.6621 50.3459 in addition. the less contents of heavy metals is in tube. then in leaf. in other organs this regulation is not observed. those heavy metals which are many in soil proportionally get into potato plant. in table 2 is shown the same data for wheat grain, leaf, sprout root and growing soil. table 2. quantitative data of ni. zn. cu. pb in wheat grain. leaf. tube and vegetative organs (mg/kg) n sample heavy metals ni zn pb cu 1. grain 0.0554 32.1754 0.0005 3.4084 2. leaf 6.1682 17.0842 2.2689 4.7428 3. sprout 2.5916 15.2739 0.2936 2.6427 4. root 6.4893 21.4806 1.8036 11.8458 5. soil 51.7345 90.2153 13.3052 75.0361 in this case also heavy metals content in different organs of plant is proportional to their content in soil. but heavy metals contents do not differ in quantity. though in grain heavy metals are the less. except zn and cu. where the difference is insignificant. heavy metals content in decreasing sequence is as follows: zn, cu, ni, pb. in table 3 is shown data for wild blackberry. content of heavy metals in blackberry is also conditioned by their content in soil and what is important the heavy metals content is less as compared with cult ivate plants and their content in decreasing sequence is as follows: zn, cu, ni, pb. the heavy metals content in case of blackberry o f in leaf. sprout. root are less in comparison with crop. it is interesting that zn. cu. ni. pb are more in leaf. in root and sprouts there is no regulatio n regarding their quantitative content. table 3. the heavy metals contents in blackberry fruits leaf. sprout. root and growing soil (mg/kg) no. sample heavy metals ni zn pb cu 1. berry 2.8728 41.8593 1.6366 19.5595 2. leaf 9.6428 66.3545 6.5276 36.3678 3. sprout 4.7645 52.2056 1.7386 14.0042 4. root 2.6425 48.1776 1.7584 53.6446 5. soil 30.4895 1111.6418 76.2079 417.4815 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 2 – 2012 42 thus, heavy metals zn. cu. ni. pb migration in various organs of plant and harvest is different and mainly depends on heavy metals quantity in soil and the most important – in potato tube, wheat grain and multi-seed fruit of blackberry these metals are the least. 4. conclusion the growing soil data comparison shows that soils differ wit h heavy metals content. heavy metals content differs a little in potato leaf and tube. this possibility gives an opportunity to suggest a new method that is to define heavy metals content in potato tube before harvesting. which will have great organizational and economical importance. according to our study the following is concluded: 1. in different organs of potato. wheat and blackberry the quantity of zn, cu, ni, pb is proportional to their content in soil. 2. zn. cu. ni. pb contents in vegetative organs of potato. wheat and blackberry and harvest are different. 3. zn. cu. ni. pb contents in potato tube. wheat grain and blackberry fruit is the least as compared wit h vegetative organs. 4. the contents of heavy metals in potato tube can be defined by means of new method. before harvesting. by analysis data of green leaf. 5. growing soils have different contents of heavy metals. 5. references [1]. v. e. stepanyan, m. s. galstyan, m. v. azaryan, m. v. khanbabyan, a. a. avakyan. environmental-geological and socio economic frameworks for the assessment nature-technical negative changes in the environment of urbanized territory of armenia. yerevan, 2001. 419p. [2]. r. a. yedoyan. a study of some heavy metals (ni. zn. pb. cu) in the vegetative organs and potato tubers. proceedings of xx international scientific symposium. simferopol. 2001. 311-316 p. [3]. r. a. yedoyan, v. a. avagyan, h. g. kochinyan. agriculture and ecology. yerevan 1999. 95 p. [4]. t. v. yedoyan, g. sh. hovsepyan. wastewater treatment from heavy metals and the utilization of the wastes. international congress "etewk-2009”. collection of reports. ukraine. yalta, 2009. 247-248 p. [5]. a. k. saghatelyan. features of the distribution of heavy metals in the territory of armenia. monograph. yerevan. 2004. 241 p. [6]. k. j. netter. heavy metals in the environment. toxicology. volume 72. issue 2. 1979. 235-236 p. [7]. e. a. merian. effect of heavy metal pollution on plants. chemosphere. volume 11. issue 4. 1982. 30-31 p. [8]. m. g. sharon. heavy metals. handbook of toxicologic pathology (second edition). volume 1. 2002. 701-733 p. 155 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 22016, pag.155 164 contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of republic of macedonia *katerina bojkovska1, trajan dojchinovski1, nikolche jankulovski1, elena joshevska1, blagojche najdovski1 1faculty of biotechnical sciences bitola.university „st. kliment ohridski” bitola partizanska bb, bitola, republic of macedonia, katerina.bojkovska@uklo.edu.mk *corresponding author received 15th april 2016, accepted 25th june 2016 abstract: the management and protection of the environment is a problem and companion to the development of human civilization, but in the recent decades is becoming more popular. the european union supports the development of ecologically adapted and competitive economy, which aims to achieve the objectives of the concept of sustainable development. the ecological awareness today is a planetary phenomenon because it comes as an expression of knowledge about the danger for humanity from pollution of soil, water and air due to the carelessness of the consequences of certain activities of the environment. consumers are increasingly more aware of the consequences of global pollution and the constant disorder of the environment. people around the world show concern for the environment and change their behavior. the ecological awareness and environmentally responsible behavior of the consumers is a necessity and obligation of the modern world and is part of the social responsibility. the green consumers as one of the most important features of the modern ecological marketing are environmentally responsible consumers who require producers to express their ecological responsibility. keywords: consumer, segmentation, ecological responsibility, green marketing. 1. introduction during the last three decades, the issues for environment and sustainable development of marginal topics have become a central theme for thinking and research. the increased media attention, high level of awareness about ecological issues influenced by reports of various ecological disasters, increasing activities of interest groups focused on issues of the environment and more legal regulations on a national and international level, are factors that are the driving force of those changes. determining the economic and ecological interests has become part of every segment of modern life and the consumption. in the market this resulted with the appearance of a new segment called green consumers. ecologically responsible manufacturers now are able to achieve key competitive advantage in the market because the ecological awareness is gradually integrated into all aspects of social, business, political and ethical thinking and acting. has become undoubted the fact that there is an ecologically responsible segment of consumers in the market. http://www.fia.usv.ro/fiajournal mailto:katerina.bojkovska@uklo.edu.mk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 156 however, with the increase of bidders exactly in the green market is no longer enough to be aware of that, but it also requires a complex strategy of segmentation, targeting and positioning. as in the case with any other market segment, there is a need to identify those target consumers to be able for more effective access and to meet their needs based on specifically designed marketing programs. based on the above, this research is based on 4 key assumptions: 1. the social attraction to encourage and build ecological responsibility of individuals, companies and society in general, is undeniable; 2. the ecological responsibility of the individual in the role of consumer (making decisions for buying and consumption in general) is essential for this social transformation; 3. educational programs and communication strategies for changing attitudes and beliefs of consumers towards ecological responsibility must be based on knowledge of the factors that affect and the strength of their influence on the level of ecological responsibility that consumers express, and the knowledge of demographic, socioeconomic and psychographic characteristics of green consumers; 4. current approaches for segmentation of customers according to the level of ecological responsibility helps in the decision making (outside) of the economic entity and designing programs for the market in the process of creating ideas, product modeling (design), determining prices and the way of distribution and communication adjusted to the individual segment. therefore it is necessary to create a specific, comprehensive and dynamic knowledge base for macedonian green consumers. the goal of this research is to contribute to find answers to the following research questions: who are the macedonian green consumers? how many are they? how educated are they? how do they live? do they react to promotional messages? are they ready to pay more for green products? from the answers of these questions arise some indirect answers that present implications for commercial and nonprofit entities: how to reach green consumers? how should we communicate with them? it is necessary to emphasize the fact that according to that criteria, in republic of macedonia till now have not been conducted researches related to ecological responsibility of consumers and their segmentation. the attention of most authors was directed to the producers and the level of their understanding and application on green marketing. defining of green consumers and previous researches green consumer can identify as the one whom in satisfying their needs and desires search product and has a minimal negative impact on the human environment. these are educated consumers and as target market are very interesting, although much larger market are those others passive towards the preservation of the environment [1]. business dictionary defines the green consumers as consumers that intensively reflect on the questions related to the environment, that support ecological objectives and actions and are ready to switch to the other manufacturer or supplier of ecological reasons but even if it includes a higher costs [2]. elkington [3] defines green consumers through features that are undesirable for them. researchers food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 157 are still trying to understand behavior of green consumers from three different perspectives: first examines consumers through demographic and socio-economic variables, the second takes into account the amount of information and knowledge that people have about environmental problems and issues, and the third includes the values, lifestyles, personality traits and attitudes [4]. in addition to trying to determine their number or relative share of green consumer market, a large number of research aimed determining demographic and other characteristics of green consumers [5]. in contemporary literature can be found studies that cover a wide range of topics: from the attempts of assessment and awareness of environmental issues, analyzing the perceived importance of the environment, the importance of environmental awareness, thinking about the actual impact of an individual on the environment, willingness to pay higher prices for environmentally friendly products, etc. [6]. some studies show only limited influence of cognitive factors, such as knowledge of the environment, and demonstrate a significant impact of affective factors in the adoption of environmentally conditioned purchase decisions [7]. in addition to attitudes and knowledge, significant attention has been paid to research scientists such as fraj and martinez in 1987, esty and winston in 2002 and pafa and associates from 2008 dedicated to the relationship between lifestyles and value system of consumers and their attitude towards environmental issues [8]. in certain cases it can be realized with it and direct personal benefits such as health benefits brought about organically grown food, monetary savings resulting from energy saving, etc. [9]. segmentation of consumers according to numerous conducted researches have been made various attempts for segmentation of consumers regarding to different criteria. the most known and acceptable is that one that was offered by the company roper starch worldwide. according to roper starch worldwide, consumers toward their interest in the question of the environment and activities that are undertaken about that can be divided into five segments [10]: 1. real green (true-blue greens) 2. green (greenback greens) 3. young (sprouts) 4. grousers 5. basic browns real greens and greens belong to the group of active agents of the environment, and the difference between them is that real greens are ready to devote their time and energy in solving ecological problems, while the greens are ready to contribute in financial terms, for a price premium of green products or financial contribution to ecological organizations. young people show their concern for the environment if this one does not require much effort of them or costs. grousers believe that governments and large commercial entities have to take care for the environment and to negotiate to do things that contribute to the environmental protection (e.g. separating waste for recycling). but they consider that they are too busy or do not have money for that and so there is nothing significant to change. basic browns opposed to grousers do not try to find an excuse for their inaction in terms of environmental protection. they simply do not care because they think ecological problems are not serious and that there are more important things to take care [11]. the new approach for green consumer segmentation has been used by the consulting firm the hartman group in washington. their previous studies have food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 158 been focused on consumer segmentation in unique ecological groups that follow the appropriate demographic characteristics. however, recent studies have found that consumers with the purchase of green products primarily take care of their own health and wellbeing, and do not take care about the environment, because it is much more tangible and therefore they developed a model of segmentation based on lifestyle [12]. along with concepts linking to quality, other dimensions of consumption drive the values that inform consumer behavior in the world of wellness and include price, convenience, brand, experience, expert opinion, fun/enjoyment, knowledge, authenticity, and sustainability. 2. matherial and methods it is estimated that the method used to collect the required data for this research is the examination, considering its advantages over other methods such as varied ability to apply the methods of testing, relative speed of data collection and relatively low costs. regarding to the model of examination it is chosen a personal examination. as a tool of the research is applied structured questionnaire. shaping tools of research is a complex process that is implemented in several steps:  after defining the information that we want to get, based on relevant literature from the subject area is designed a questionnaire.  in creating questionnaire were used closed questions with the modalities of the features, and most of them are measured by likert scale with five levels, where 1 means ‘completely disagree with the statement’ and 5 means ‘completely agree with the statement’. samples are the households from republic of macedonia. this model is suitable for obtaining indicative results and making general conclusions about the level of ecological awareness of consumers, as well as some important implications for economic entities in republic of macedonia. the description of the researched sample is shown in table 1. table 1 description of researched sample n % gender 552 100 male 275 50.2 female 273 49.8 age 552 100 15-24 92 16.7 25-34 93 16.9 35-44 92 16.7 45-54 89 16.2 55+ 185 33.6 education 552 100 elementary education 72 13.1 secondary education 297 54.1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 159 university education and more 180 32.8 marital status 552 100 not married 167 30.4 married 315 57.4 divorced or widow 67 12.2 number of household members 548 100 1 55 10.0 2-3 236 43.1 4-5 231 42.2 6 and more 26 4.7 the environment in which they grew up 552 100 city 269 48.9 suburban 91 16.5 country 190 34.5 household income 550 100 to 10.000 den 43 7.8 10.00120.000 den 82 14.9 20.001 – 30.000 den 127 23.1 30.001 – 40.000 den 97 17.6 40.001 – 50.000 den 72 13.1 more than 50.000 den 129 23.5 in the analysis of the data are applied methods of descriptive and inferential statistics. category analysis is used where under categorization is meant the classification of the variables. then it is made a cross-tabulation i.e. it is calculated chi-square, lambda, and contingency coefficient and pearson correlation coefficient. 3. results and discussion based on the study of relevant sources of literature, especially the previously conducted researches related to the subject matter as well as prior researches, it is formed tool for research or questionnaire consisting of 30 questions. the aim is to measure the overall level of ecological responsibility of consumers, and consists of three parts (each includes ten questions), to be appropriately measured: attitude, knowledge and taken actions. the famous scale for measuring the ecological responsibility of consumers ecoscale [13] is used as a source of ideas for setting the formulation of questions, because it covers a variety of factors affecting the ecological responsibility of consumers, but it is not used as a measuring tool, because food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 160 conceptually does not match the objectives of this research [14]. in order and in accordance with the objectives of the research, to contribute towards creating a clearer picture of the macedonian green consumer, was necessary first to identify green consumers by differentiating them from consumers who show a relatively lower level of ecological responsibility, which are called neutral and the consumer group with the lowest level of environmental responsibility that are called brown consumers. the scale according which consumers are classified into one of these three segments, based on the average score of all thirty questions is given in table 2. in the process of coding for each answer was given a grade from 1 to 5 in order to calculate the average. chosen is the main (most severe) division of the three segments of consumers (green, neutral and basic browns) in order to define the basic differences by the demographic characteristics making possible a higher level of significance. in setting the scale is paid attention for that the average ratings of distribution in particular segment to be relatively high in order thereby to neutralize the impact of the partiality of the interviewee due to the perceived social desirable responses that maintain a high level of ecological responsibility. considering the fact that it is a likert scale with five grades, the score of 3 is taken as an indication of a neutral position on the offered statement and therefore the average grade 3 is defined as the minimum grade to be considered the consumer as neutral. however, due to the mentioned impact of social attraction on the answers this limit has increased by 10% or 0.3 and so the minimum average score for classification of respondents in the segment neutral consumers amounted to 3.3. in the further modeling of the scale (green segment), was taken into account for that the classes of average ratings to be equally high. the average risk categories are equally high. table 2. the scale of consumer segmentation with the segmentation of consumers in accordance with the conditions given in table 2 was obtained structure of consumers as shown in figure 1. as it was expected, the smallest group consists of green consumers (17.2%), while the neutrals are the biggest segment. extremely low ecological responsibility was expressed by 21% of consumers, and they make up the basic browns consumer segment. figure 1. participation of green, basic brown and neutral consumers in the total sample in order to develop a profile or create a clearer picture of each segment, especially for green consumers has been made a cross-tabulation (cross tabulation) with demographic characteristics of respondents. by using the chi-square test was made an attempt to demonstrate existence of statistically significant differences when comparing the given segment structure and the demographic characteristics of respondents. if the level of significance is above 0.05 then there name of segment average rate green 4.20 – 5.00 neutral 3.30 – 4.19 basic browns less than 3.29 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 161 cannot exist statistically significant differences. comparing the structure of segments according to the variable ‘gender’ is not noticeable statistically significant difference (signif. > 0.05). however, according to the participation in all three segments, it is evident that female respondents are slightly disposed for ecologically responsible behavior, because their participation in the segment of greens is insignificantly high, and the participation in basic brown segment was significantly lower. at the variables that represent the age group there is statistically significant difference (signif.<0.05), and therefore cannot be said that green consumers generally have 55 or more years. it indicates to the fact that members of this age group are mostly in the segment of green consumers while their participation was significantly lower in the segment of neutral and basic brown consumers. it is surprising the very small participation of the youngest age group in the segment of green consumers. the members of the age group from 15-24 years are the most included in the segment of basic brown customers. for that purpose the future researches should be directed to the identification of the reasons for this phenomenon. due to the structure of the sample (in the sample is a group of subjects with more than 55 years) it is designed complementary comparison that shows the structure of the age groups of users according to the defined segments in order to remove any doubts for the structure of green consumers and to confirm the statement that consumers over 55 years are really the ‘greenest’ segment. it shows that the participation of green consumers is significantly higher in the group of consumers over 55 years compared to all other age groups, and the fact that the participation of green consumers in the youngest age group is the smallest. in the variable ‘level of education’ the difference was also statistically significant (signif. <0.05), and can be concluded that the level of ecological responsibility grows with the level of education, because the participation of respondents with higher education decreases from green to basic brown while the participation of respondents with primary education increases. the ‘marital status’ is also statistically significant variable and at the significance level of 0.001 can be concluded that green consumers mostly live in a marriage. persons who are not married often belong to the basic brown segment. this may be associated with the variable ‘age group’ and can be concluded that they are members of the younger age groups. for other demographic variables, such as ‘household size’ (signif. = 0.172), ‘environment in which consumers raised’ (signif. = 0.513) and ‘household income’ (signif. = 0.379) was not found statistically significant difference, and it is the reason why we do not specifically note the structure of the segments of consumers according to these variables. however, significant is the fact that these variables are not significant, and indicates to the future researches that are supposed to answer the question why the number of household members, the environment in which raised respondents and household income have a significant impact on the level of ecological responsibility of consumers. particularly interesting variable is the ‘household income’ because intuitively can be assumed that individuals and families with solved subsistence needs should be more inclined to focus their time and resources on ‘higher purpose’. however the income of households obviously does not have such predictable and direct impact food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 162 on the ecological responsibility of consumers. according to leinberg servey, ‘household income’ obviously does not have such a predictable and immediate impact on the environmental responsibility of consumers. therefore consumers with the highest revenue do not belong to the "greenest" segment [15]. besides demographic variables for the development of green consumer profile are used two important questions that are often taken as the main measure of the level of ecological responsibility of consumers 1. have you ever bought a product motivated by the promotional message which says that the product is safe for the environment? 2. are you ready to pay more for product (e.g, detergent) that is ecologically more acceptable than competing products? these questions are cross tabulated with the identified segments to get two more determinants of green consumers. these criteria must be followed in dynamics, as this may indicate a change in the level of ecological responsibility of consumers and measuring the impact of certain social events or activities. from the performed statistical processing of collected data can be seen that green consumers mostly periodically buy products stimulated by promotional message that states that the product is safe for the environment, but the percentage of respondents that do that often is quite high (40 %) (figure 3). this result indicates that green consumers, although compose a relatively small segment quite are active and ready to act in accordance with their ecological attitudes of the place of purchase, where need to be informed about the ecological characteristics of products. this fact justifies the investment in promotional messages and green marketing campaign that lately are increasingly found in republic of macedonia. 1.7% 10.9% 40% 25% 43.1% 47.4%45.7% 38.7% 12.6% basic browns neutrals greens figure 3. purchaseing of goods fueled by promotional activities in which states that the product is safe for the environment (χ2 = 134.137, signif. = 0.000) another important indicator for the marketing decision-making and marketing theorists that can be measured in a number of studies of green consumers is a willingness to pay a price premium for green products. therefore, can be concluded that the most of the green consumers are ready to pay 20% higher price for green products. at the same time, it is not insignificant the percentage (15%) of those who are ready to pay 40% more for a product that is ecologically more acceptable than competing products (figure 4). 1.7% 10.9% 40% 25% 43.1% 47.4% 45.7% 38.7% 12.6% basic browns neutrals greens figure 4 the willingness to pay higher prices for the environmentally friendly product readiness (χ2 = 87.593, signif. = 0.000) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 163 4. conclusion regardless of the type or specific area to which it applies, the customer is always the target of the marketing. on the market was identified a growing and potential profitable segment of the green consumers, which on their decision making moments, they are considering all available information and all their knowledge how they would choose the right alternative, that for them is optimal combination for satisfying their own needs and welfare of the environment, as well as the hole community. based on the survey results, the conclusion is that the average green consumer in republic of macedonia is a person older than 55 years and has higher education, is married and reacts to promotional messages in which is highlighted that the product is safe for the environment and under it influence temporary or often makes buying decision and is ready to pay 20% more for environmental acceptable product. these results, for the economic entities, should make easier the decisions making and creating the program for market appearance in the whole process for creating the idea, product design, price determination and the ways of distributions of the product. in addition, the research results may have implications to the other groups of participants to the green marketing. this applies especially to the environmental associations and organizations, as well as government agencies that have a role in encouraging changes, providing the necessary information through education and directing changes in domestic and international laws and regulations. the state authorities especially should react with warning for the low level of ecological responsibility for the youngest age group that undoubtedly lead to be made necessary changes in the educational system and should make certain efforts about it. it is necessary to separate the data that shows that the youngest age group between 15 to 24 years is minimum ecologically conscious. this information is alarming, especially if we take into consideration the fact that in the most of the researches in other countries, this age groups is the most environmentally conscious. the future researches should tend to identify all the factors that influenced in such a way, as well as developing a strategy for positive changing in that sense. based on the survey results, can be concluded that the green consumers in republic of macedonia, although they are relatively small seqment, they are very active and ready to act with accordance of their ecological position on the place where they buy, but need to be previously informed for the ecological characteristics of the products. this information justifies the investment in more promotional messages in the campaign of the green marketing that lately are using in republic of macedonia. also the expressed willingness to pay a price premium for ecological acceptable product is an important indicator for making marketing decisions. for full implementation of the green marketing in republic of macedonia, it is necessary to create a comprehensive knowledge base for the macedonian green consumers, their socio-demographic characteristics, attitudes, needs, desires and expectations. this knowledge must be in dinamic category, but the research should be systematic and continuous. only on the basis of knowledge about green consumers may develop a strategy for the development of green marketing at macro and micro level in republic of macedonia. worldwide and in europe, there are very few ecological oases, whereby with strategic determination on the green marketing on national level in republic of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 katerina bojkovska, trajan dojhinovski, nikolche jankulovski, elena joshevska, blagojche najdovski, contemporary approaches for consumer segmentation according to the level of ecological responsibility: the case of the republic of macedonia, food and environment safety, volume xv, issue 2 – 2016, pag. 155 164 164 macedonia, only in this field can be achieved competitive advantage in the international market. it is necessary systematic thinking and to use synergistic effects of natural capital that is available in republic of macedo nia, as well as complementarity with the growing phenomenon ecotourism, with a strong growth of the market of ecological produced food and market green products in general. with the appropriate system investments and economic cooperation, as well as with help of knowledge and government entities, republic of macedonia can create strong and credible global green brand. 5. references [1]. grbac, b., identity marketing: basic principles the new reality, ekonomic faculty rieka, 2005, pp. 476. [2].http://www.businessdictionary.com/definition/g reen-consumer.html (assessed 27.06.2016). [3]. elkington, j.: toward the sustainable corporation: win-win-win business strategies for sustainable development, california management review, vol. 36, no. 2: 92, (1994). [4] fraj, e., martinez, e., ecological consumer behaviour: an empirical analysis, international journal of consumer studies, vol. 31: 26-33 (2007). [5] paff ogle, j., hyllegard, k. h., dunbar, b. h.: predicting patronage behaviors in a sustainable retail environment: adding retail characteristics and consumer lifestyle orientation to the belief-attitude-behavior intention model, environment and behavior, vol. 36: 717, (2004). [6] jain, s.k., kaur, g.: green marketing: an attitudinal and behavioural analysis of indian consumers, global business review, vol. 5: 187205, (2004). [7]. hartmann, p., apaolaza ibanez, v., forcada sainz, f.j.: green branding effects on attitude: functional versus emotional positioning strategies, marketing intelligence & planning, vol. 23, no. 1: 10, (2005). [8]. fraj, e., martinez, e.: environmental values and lifestyles as determining factors of ecological consumer behaviour: an empirical analysis, journal of consumer marketing, vol. 23, no. 3: 133-144, (2006). [9]. hartmann, p., apaolaza ibanez, v., green value added, marketing intelligence & planning, vol. 24, no. 7: 673-680, (2006). [10]. jain, s. k., kaur, g., green marketing: an attitudinal and behavioural analysis of indian consumers, global business review, vol. 5: 187 (2004). [11]. kalafatis, s. p., pollard, m., east, r., tsogas, m. h.: green marketing and ajzen’s theory of planned behaviour: a cross-market examination, journal of consumer marketing, vol. 16, no. 5: 441-460, (1999) [12]. mccarty, j.a., shrum, l.j., a structural equation analysis of the relationships of personal values, attitudes and beliefs about recycling, and the recycling of solid waste products, advances in consumer research, vol. 20: 641-655, (1993) [13]. stone, g., barnes, j.h., montgomery, c. ecoscale: a scale for the measurement of environmentally responsible consumers, psychology & marketing, vol. 12, no. 7: 595-612 (1995) [14]. ham, m., forjan, j., frajmanjaksic, a., measuring consumer environmental responsibility, 4 internationalconference “an enterprise odyssey: tourism governance and entrepreneurship”, june 11-14, 2008, dubrovnik croatia). paper published in proceedings, an enterprise odyssey: tourism governance and entrepreneurship, pp. 1448-1459. [15]. leinberger, p., roper green gauge 2000, presentation at sixth national green power marketing conference, portland oregon, august 1st, 2001 prema: holt, e.a., holt, m.s.: green pricing resource guide, 2 ed., prepared for american wind energy association, ed holt & associates, inc., london, 2004. http://www.businessdictionary.com/definition/green-consumer.html http://www.businessdictionary.com/definition/green-consumer.html 1. introduction звіт з ндр 29-81 за 2007 – 2009 р 234 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 3 – 2016, pag. 234 239 ph ysicochem ic al and tex tu ra l pro per ties o f ho n e ys fr om nor th eas t par t o f roma nia *mircea oroian1, elena todosi sănduleac1, sergiu pădureț1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received july 22nd, accepted september 23rd abstract:the aim of this study is to evaluate the physico-chemical (ph, aw, free acidity, brix concentration, moisture content, ash content, colour parameters (l*, a*, b*, chroma, hue angle and yellow index, glucose, fructose and sucrose content) and textural (hardness, viscosity, adhesion, cohesiveness, springiness, gumminess and chewiness)) properties of 5 samples of honey of different floral origins (acacia, tilia, sunflower, honeydew and polyfloral). the physico-chemical parameters of honey were in agreement with those reported by other scientists. the moisture content of all the five samples do not exceeded the maximum allowable level of 20% established by the european commission. the acacia and tilia honeys presented green components, while all the honey presented yellow components. the highest yellow index has observed in the case of tilia honey. the data were submitted to principal component analysis (pca) and it was found that the two principal components (pcs) explained 100% of the variations in the data set. the pc1 explains 99% of the variability and the pc2 explains 1%. the pc1 separates the honey into two groups: one group below the pc1 represented by acacia, sunflower and polyfloral honeys and the second group below the pc1 represented by tilia and honeydew honeys. keywords: honey, physico-chemical parameters, texture parameters 1. introduction according to the european council directive 2001/110 ec [1], honey is “the natural sweet substances produced by apismellifera bees from the nectar of plants or from secretions of living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature”. one of the parameter used by the specialist in the food industry for the assessment of food quality and acceptability is food texture. from all the food texture parameters, hardness or firmness is the most used and important one for fruit and vegetables [2]. texture profile analysis (tpa) sets up a ‘bridge’ from objective measurement to subjective sensation and makes food texture characteristics more predictable [3]. honey contains approximately 80% carbohydrates (35% glucose, 40% fructose, and 5% sucrose) and 20% water, serving as an excellent source of energy [4]. in addition, it contains more than 200 components, including amino acids, vitamins, minerals, enzymes, organic acids and phenolic compounds [4, 5]. the characterization of honey from the physico-chemical point of view has been http://www.fia.usv.ro/fiajournal mailto:m.oroian@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 m i r c e a o ro i an, el e na to do si sănd ule ac, se r g i u p ădure ț , ph y s i c o c h e mi c a l a n d t ex t u r a l p r o p er t i es o f h o n ey s f r o m n o r t h ea s t p a r t of r o ma n i a , fo od a n d en vi r on me n t sa fe t y, v ol u me xv, is s u e 3 – 2 0 1 6 , p a g. 2 3 4 2 3 9 235 conducted on honey from algeria [6], india [7], slovenia [8], malaysia [9] and bangladesh [10]. the aim of this study is to investigate the physico-chemical and textural properties of some honeys from the north east part of romania. 2. materials and methods materials 5 honey samples of five different botanical origins (acacia, tilia, sunflower, polyfloral and honeydew) were purchased from local beekeepers from the suceava county. physico-chemical properties the physico-chemical properties (ph, aw, free acidity, refraction index, moisture content, ash content, colour parameters (l*, a*, b*, chroma, hue angle and yellow index, glucose, fructose and sucrose content) were determined accordingly to the harmonised methods of the international honey commission [11]. texture profile analysis (tpa) the tpa was carried out at 20 °c with mark 10 texture analyzer (mark 10 corporation, usa) equipped with a 50 mm disc probe, the flask diameter was of 70 mm. the tpa was operated at a constant speed of 150 mm/min, until a depth of 12.5 mm (the honey column had 25 mm). the tpa can offer a great number of texture parameters, such as: hardness (h), viscosity (v), adhesion (a), cohesiveness (co), springiness (s), gumminess (g) and chewiness (ch) [12]. 3. results and discussion physico-chemical properties physico-chemical properties (ph, aw, free acidity, refraction index, moisture content, ash content, colour parameters (l*, a*, b*, chroma, hue angle and yellow index, glucose, fructose and sucrose content) of the honey analysed are presented in table 1. table 1. physico-chemical properties of honey parameter acacia tilia polyfloral honeydew sunflower ph 4.81 5.36 4.00 5.16 4.17 aw 0.50 0.52 0.54 0.54 0.51 freeacidity (meq acid/k) 4.10 3.40 32.00 17.00 14.40 °brix 81.30 79.80 81.90 81.90 82.40 moisture content, % 17.28 18.84 16.83 16.60 16.16 electrical conductivityµs/cm 136.10 219.60 504.20 937.80 353.20 ash content, % 0.07 0.11 0.25 0.46 0.17 l* 45.10 32.37 38.89 21.52 36.75 a* -0.67 -0.17 4.96 5.65 2.78 b* 15.11 14.57 13.72 6.90 12.03 chroma 15.12 14.57 14.59 8.92 12.35 hue angle 3.74 3.96 -2.54 0.36 0.41 yellow index 47.87 64.32 50.40 45.82 46.78 fructose (g/100g) 48.31 43.14 35.14 40.21 39.40 glucose (g/100g) 22.06 30.67 34.12 36.40 36.15 sucrose (g/100g) 1.90 1.09 1.97 0.00 0.80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 m i r c e a o ro i an, el e na to do si sănd ule ac, se r g i u p ădure ț , ph y s i c o c h e mi c a l a n d t ex t u r a l p r o p er t i es o f h o n ey s f r o m n o r t h ea s t p a r t of r o ma n i a , fo od a n d en vi r on me n t sa fe t y, v ol u me xv, is s u e 3 – 2 0 1 6 , p a g. 2 3 4 2 3 9 236 the ph values of honeys ranged between 4.00 – 5.36, all the honey are acidic as the literature present [13]. the acidic nature of honey is the result of the acids present naturally in it. the water activity ranged between 0.50 – 0.54; a low water activity product (aw< 0.70) means that the product is microbiologically safe [14] because this product does not support the growth of foodborne pathogens [15]. the honey free acidity indicates if the honey started to ferment. the maximum allowable level for the free acidity is established to 40 meq acid/kg honey in the case of mono and polyfloral honeys and 50 meq acid/kg in the case of honeydew honeys. all the samples respect this limit. the moisture content ranged between 16.60 – 18.84 %; all the samples respect the maximum allowable level of 20% established by the european commission [1] .a moisture content higher than 20% will accelerate the fermentation processes and decrease the stability of honey. the electrical conductivity ranged between 136.10 – 937.80 µs/cm, as it was normal, the honeydew honeys have the highest electrical conductivity and it was in agreement with the literature (honeydew honeys must have electrical conductivity higher than 800 µs/cm) [16]. ash content represents the mineral content presented into honeys. the values of the mineral content ranged between 0.07 – 0.46%, in agreement with the literature [17]. an important parameter for the food is the colour. the colour of honey represents the first attribute and for this reason is an important parameter for its commercialization and authentication. table 1 present the values of honey colour in ciel*a*b* coordinates: l* (luminosity), a* (negative values indicate green while positive values indicate magenta), b* (negative values indicate blue and positive values indicate yellow), hue angle, chroma and yellow index. the highest luminosity had the acacia honey, followed by polyfloral, while honeydew honey had the lowest luminosity level, being the darkest one. the acacia and tilia honeys presented green components, while all the honey presented yellow components. the highest yellow index has observed in the case of tilia honey. the major contributors of the honey sugars are fructose and glucose (inverted sugar). the sum of the fructose and glucose content into the honey should not be less than 60 g/100 g honey (european commission). all five honeys s have inverted sugar content higher than 60 g/100g honey so they comply with the ue regulation. of all the three sugars determined in honey, fructose was in a higher concentration than glucose, followed by sucrose, which was in lower concentrations. in all the cases the ratio fructose/glucose was higher than 1. the honeydew honey does not contain sucrose. texture profile analysis in figure 1 the texture parameters magnitude are presented for each honey. it can be observed that honeydew honey has the highest chewiness, springiness, adhesion and viscosity. the polyfloral honey had the highest gumminess and hardness. accordingly to the pearson correlation, the texture parameters are influenced positively as: hardness with gumminess (r = 0,991**), hardness with chewiness (r = 0.932**), viscosity with adhesion (r = 0.949*), viscosity with springiness (r = 0.817*), adhesion with refraction index (r = 0.996**), gumminess with chewiness (r = 0.969*), and negatively: hardness with fructose content (r = -0.741*), viscosity with sucrose (r = food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 m i r c e a o ro i an, el e na to do si sănd ule ac, se r g i u p ădure ț , ph y s i c o c h e mi c a l a n d t ex t u r a l p r o p er t i es o f h o n ey s f r o m n o r t h ea s t p a r t of r o ma n i a , fo od a n d en vi r on me n t sa fe t y, v ol u me xv, is s u e 3 – 2 0 1 6 , p a g. 2 3 4 2 3 9 237 0.854*), springiness with water activity (r = -0.782*). figure 1.texture parameters of honey: ssunflower, h – honeydew, p – polyfloral, t – tilia, aacacia the principal component analysis was conducted to evaluate the global effect of the textural and physico-chemical parameters, from a descriptive point of view. figures 1 and 2 present the scores and compound loadings of pca analysis performed. it was found that the two principal components (pcs) explained 100% of the variations in the data set. the pc1 explains 99% of the variability and the pc2 explains 1%. it can be observed in the figure 2 that the samples are grouped into two groups separated by the principal component 1: one group below the pc1 represented by the acacia, sunflower and polyfloral honeys and the second group below the pc1 represented by the tilia and honeydew honeys. the two groups can be separated by the electrical conductivity parameter, which is an important parameter for the classification of honeys; the value of this parameter are greater in the case of honeydew and tilia honeys. pca scores are presented in the figure 3. the refraction index and adhesion have the lowest influence on the projection. 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 hardness [n] viscosity [n] adhesion springiness [mm] cohesiveness [n] gumminess chewiness [n] s h p t a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 m i r c e a o ro i an, el e na to do si sănd ule ac, se r g i u p ădure ț , ph y s i c o c h e mi c a l a n d t ex t u r a l p r o p er t i es o f h o n ey s f r o m n o r t h ea s t p a r t of r o ma n i a , fo od a n d en vi r on me n t sa fe t y, v ol u me xv, is s u e 3 – 2 0 1 6 , p a g. 2 3 4 2 3 9 238 fig. 2.principal component analysis – loadings fig. 3. principal component analysis scores 4. conclusions the honey analysed were of five different botanical origins (acacia, tilia, sunflower, polyfloral and honeydew). the honey samples complied with the regulations established by the council directive 2001/110/ec. the principal component analysis (pca) permitted the separation of honey samples into two different groups based on conductivity level (one group represented by acacia, sunflower and polyfloral honeys, and the other one represented by tilia and honeydew honeys). acknowledgement this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 m i r c e a o ro i an, el e na to do si sănd ule ac, se r g i u p ădure ț , ph y s i c o c h e mi c a l a n d t ex t u r a l p r o p er t i es o f h o n ey s f r o m n o r t h ea s t p a r t of r o ma n i a , fo od a n d en vi r on me n t sa fe t y, v ol u me xv, is s u e 3 – 2 0 1 6 , p a g. 2 3 4 2 3 9 239 – uefiscdi, project number pn-ii-rute-2014-4-0110. 5. references [1].council directive 2001/110/ec council directive of 20 decemberrelatingtohoney 2001/110/ec. official journal of the european communities. [2].konopacka, d., plocharski, w.j., effect of storage conditions on the relationship between apple firmness and texture acceptability. postharvest biology and technology 32 (2), 205– 211, (2004). [3].chen, l., opara, u. l., approaches to analysis and modelling texture in fresh and processed foods – a review, journal of food engineering, 119, 497-507, (2013). [4].roshan, a. r. a., gad, h. a., elahmady, s. h., abou-shoer, m. i., khanbash, m. s., & al-azizi, m. m., characterizationanddiscrimination of the floral origin of sidrhoneybyphysicochemical data combinedwithmultivariateanalysis. foodanalyticalmethods, 1-10, (2016) [5].de rodríguez, g. o., de ferrer, b. s., ferrer, a., &rodríguez, b., characterization of honeyproduced in venezuela. foodchemistry, 84(4), 499-502, (2004). [6].ouchemoukh, s., louaileche, h., & schweitzer, p., physicochemical characteristics and pollen spectrum of some algerian honeys. food control, 18(1), 52-58, (2007). [7].saxena, s., gautam, s., & sharma, a., physical, biochemical and antioxidant properties of some indian honeys. food chemistry, 118(2), 391397, (2010). [8].bertoncelj, j., doberšek, u., jamnik, m., & golob, t.,evaluation of the phenolic content, antioxidant activity and colour of slovenian honey. food chemistry, 105(2), 822828, (2007). [9].aljadi, a. m., & kamaruddin, m. y.,evaluation of the phenolic contents and antioxidant capacities of two malaysian floral honeys. food chemistry, 85(4), 513-518, (2004). [10]. islam, a., khalil, i., islam, n., moniruzzaman, m., mottalib, a., sulaiman, s. a., & gan, s. h., physicochemical and antioxidant properties of bangladeshi honeys stored for more than one year. bmc complementary and alternative medicine, 12(1), 1, (2012). [11]. bogdanov, s., harmonised methods of the international honey commission (swiss bee research centre, fam, liebefeld, ch-3003 bern, switzerland), (2002). [12]. chen, l., opara, u. l., approaches to analysis and modelling texture in fresh and processed foods – a review, journal of food engineering, 119, 497-507, (2013). [13]. oroian, m., physicochemical and rheological properties of romanian honeys. food biophysics, 7(4), 296-307, (2012). [14]. gurtler, j. b., doyle, m. p., & kornacki, j. l.,the microbiological safety of spices and low-water activity foods: correcting historic misassumptions. in the microbiological safety of low water activity foods and spices (pp. 3-13). springer new york, (2014). [15]. cordier, j. l.,methodological and sampling challenges to testing spices and lowwater activity food for the presence of foodborne pathogens. in the microbiological safety of low water activity foods and spices (pp. 367-386). springer new york., (2014). [16]. bogdanov, s., ruoff, k., & persano oddo, l., physico-chemicalmethods for thecharacterisation of unifloralhoneys: a review. apidologie, 35(suppl 1), 4-17, (2004). [17]. chakir, a., romane, a., marcazzan, g. l., & ferrazzi, p., physicochemicalproperties of somehoneysproducedfromdifferentplants in morocco. arabian journal of chemistry., (2011). 328 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 328 331 poly phen ol con ten t an d ant io xidant a cti v it y o f com merc ia l beer s from the roman ian m a rke t *mircea oroian1, cristina damian1, ana leahu1, oana gherasim1 1 faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro * corresponding author received november 7th 2013, accepted december 5th 2013 abstract: the aim of this study is to evaluate the polyphenol content and antioxidant activity of 10 different samples from the romanian market. the samples were purchased from the local market of suceava town, suceava county. polyphenolic compounds are ubiquitous in all plant organs and are, therefore, an integral part of the human diet. they are synthesised by the phenylpropanoid biosynthetic pathway, which produces the large variety of plant phenols. the polyphenol content of the beer samples was measured using the folin ciocalteau method. the antioxidant activity of beer samples were measured using the dpph assay. the two method were applied using a uv-vis spectrometer. the 10 beer samples analyzed showed considerable differences between them among total polyphenol content, ranging between 61.40 mg gae/l for landbier beer to 361.40 mg gae/l for skol beer. all the beer samples showed strong activities of the dpph radical at the concentration test. the values of pi% are rangind between 10.24 and 33.68%. tuborg beer showed the highest antioxidant activity, while landbier showed the smallest one. keywords:polyphenol, antioxidant activity, beer 1. introduction beer is a natural beverage, with no fat and low caloric content, with organic acids and vitamins, proteins, hop and water. beer has a higher nutritional value than other alcoholic beveragesm because of its mineral value and essential nutrients such as potassium, magnesium, calcium and sodium. the use of cereals and malt to produce beer may also contribute for the ingestion of naturally occuraing antioxidant compounds, such as polyphenols [1]. polyphenolic compounds are ubiquitous in all plant organs and are, therefore, an integral part of the human diet. they are synthesised by the phenylpropanoid biosynthetic pathway, which produces the large variety of plant phenols. in the last decades, the interest of polyphenolic compounds was related to the ability of them to bind and precipitate marcomolecules (dietary protein, carbohydrate, digestive enzymes), but in the last part of xx century, the interest in food phenolics has increased due to the antioxidant and free radical-scavenging food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 mircea oroian, cristina damian, ana leahu, polyphenol content and antioxidant activity of commercial beers from the romanian market, volume xii, issue 4 – 2013, pag. 328 331 329 abilities [2]. polyphenols may exert an indirect antioxidant effect, by protecting endogenous antioxidant enzymes in the human body [3, 4]. stevenson & hurst [3] suggest in their review that it appears that polyphenols can provide significant protection from oxidative stress in vitro at concentrations much lower than would be required for chemical antioxidant protection [5]. the antioxidant properties of phenolics is mainly because of their redox properties, which allow them to act as reducing agents, hydro-gen donators and singlet oxygen quenchers [6]. a polyphenol substance can be defined as an antioxidant only if it fulfills two conditions, firstly, when present in low concentration relative to the substrate to be oxidized it can delay, retard or prevent the oxidation or free radical mediated oxidation of a substrate and secondly that the resulting radicals formed after scavenging must be stable [7]. the aim of this study is to evaluate the polyphenol content and antioxidant activity of 10 different samples from the romanian market. 2. materials and methods 2.1. materials for this study were purchased 10 different commercial beers from the romanian market. 2.2. polyphenols content folinciocalteau reagent preparation reagents: 7.5% na2co3 solution, foli ciocalteau solution 1:10, methanol. 100 g of na2wo42h2o and 25 g of na2moo42h2o are dissoved into 700 ml of warmed bidistilled water. 50 ml of 85% o-phoshoric acid and 100 ml of 37% clorhidric acid are mixed with the previous substances. the mix is boilled for 10 h. 150 of li2so4h2o and some goats of br are added, and the boilling is continued for 15 min. the mix is puted into 1 l flask and filled up with bidistilled water. the reagent is kept in dark. beer polyphenol determination 0.2 ml of beer are added into a tube, 2 ml of folin ciocalteau 1:10 and 1.8 ml of 7.5 % na2co3 are added. the mixture is agitated and the absorbance in measured at 750 nm using the ocean optics hr4000 spetrometer. polyphenol calibration curve is made using a known polyphenol (gallic acid) of 1, 2, 3, 4 and 5 mg/l concentration, respectively. the calibration curve is presented into figure 1. fig.1. polyphenol calibration curve 2.3. antioxidant activity the antioxidant activiy was determined using the dpph assay. the antiradical power determination is made by y = 0,114x r² = 0,984 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 2 4 a bs or ba nc e acid galic mg/l food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 mircea oroian, cristina damian, ana leahu, polyphenol content and antioxidant activity of commercial beers from the romanian market, volume xii, issue 4 – 2013, pag. 328 331 330 comparing with a stable radical, 1,1difenil-2–picrilhidrazil, dpph. fig.2 dpph chemical structure 5 μl of beer sample are added into a spectrohotometer cuvette and after it 3 ml of dpph solution (25 mg/l methanol) are added. the sample is left for 5 min and the absorbance is measured at 517 nm using a ocean optics hr4000 uv-vis spectrometer. the inhibition power of free radical (pi %) is measured by comparing the dpph absorbance with the sample absorbance using the equation: pi % = [1 – (asample/ ablank)] × 100. the absorbance for the samples asample were measured at 517 nm, and for ablank for the dpph solution. 2.4.statistical analysis the statistical analysis was made using the next software packs: excel 2007. the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. 3. results and discussions the polyphenol content of the 10 beer samples were examined using the folin ciocalteau method, and the results are presented in table 1. the 10 beer samples analyzed showed considerable differences between them among total polyphenol content, ranging between 61.40 mg gae/l for landbier beer to 361.40 mg gae/l for skol beer. guinness, gauloise and suceava beers showed a high content of polyphenol (>100 mg gae/l). the polyphenols play a critical role into the flavor stability and for the colloidal stability too. table 1. polyphenol content of beer samples no. commercial name acid galic mg/l 1 skol 361,40 2 suceava 107,01 3 ursus 96,49 4 timişoareana 94,74 5 tuborg 68,42 6 silva brună 78,95 7 ursus brună 70,18 8 gauloise 115,79 9 guinness 131,58 10 landbier 61,40 the antioxidant activity of the 10 beer samples are presented into the table 2. all the beer samples showed strong activities of the dpph radical at the concentration test. the values of pi% are rangind between 10.24 and 33.68%. tuborg beer showed the highest antioxidant activity, while landbier showed the smallest one. all the results showed that the raw materials and the brewering process have significant influence on the dpph radical activity of beer samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 mircea oroian, cristina damian, ana leahu, polyphenol content and antioxidant activity of commercial beers from the romanian market, volume xii, issue 4 – 2013, pag. 328 331 331 table 2. antioxidant activity no. commercial name pi % 1 skol 11.29 2 suceava 13.12 3 ursus 12.06 4 timişoareana 17.79 5 tuborg 33.68 6 silva brună 13.12 7 ursus brună 12.61 8 gauloise 11.16 9 guinness 21.57 10 landbier 10.24 4. conclusions phenolic compounds play a critical role into arome stability and in the coloidal stability of beer. the phenolic compounds are considered, in general, the most important source of antioxidants in beer. even the individuals phenolics responsible for the antioxidant activity of the beer were not determined the general contribution of the phenolic compounds at the antioxidant activity of the beer ranged between 10.24 and 33.68%. the results obtained showed that the raw materials and the brewing process can influence significantly the antioxidant activity. the polyphenol content ranged between 61.40 mg gae/l for landbier beer to 361.40 mg gae/l for skol beer. 5. references [1] tafulo. p. a. r., quieros. r. b.. delerue-matos. c. m.., sales. m. g. f., control and comparison of the antioxidant capacity of beers. food research international. 43. 1702-1709. (2010) [2] bravo. l.. polyphenols: chemistry. dietary sources. metabolism. and nutritional significance. nutrition reviews. 50 (11). 317333.. (1998). [3] stevenson. d.e.. hurst. r.d.. polyphenolic phytochemicals – just antioxidants or much more? cellular & molecular life sciences. 64: 2900–2916. (2007). [4] aron. p.m.. kennedy. j.a. flavan-3-ols: nature. occurrence and biological activity. molecular nutrition & food research 52: 79– 104. (2008). [5] weichselbaum. e.. buttriss. j. l.. polyphenols in the diet. nutrition bulletin. 33. 157-164. (2010). [6] rice-evans. c. a.. miller. n. j.. & paganga. g. structure-antioxidant activity relationships of flavonoids and phenolic acids. free radical biology and medicine. 20(7). 933–956. (1996). [7] kaur. c.. kapoor. h.c.. antioxidants in fruit and vegetables the millenium’s health. international journal of food science and technology. 36. 703-725. (2001) 354 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, stefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 354 358 study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods *silviu-gabriel stroe1 1 faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania *silvius@fia.usv.ro *corresponding author received november 17th 2013, accepted december 10th 2013 abstract: the aim of this work was the study of corrosion behavior of the aisi304 stainless steel samples in 3%, 6% and 9% acetic acid solutions, using accelarated electrochemical methods. in order to achieve accelerated corrosion tests was been used a system consisting of a potentiostat-galvanostat wenking hp 96-20, an electrochemical cell, two multimeters hameg hm 8112-3 for monitoring the voltage values and a pc system for programming, command, acquisition and interpretation of the values recorded on the multimeter. in order to developing the mass balance of the migration process of metal ions were calculated the theoretical masses of 56fe, mn, cr and ni ions migrated in the acidic simulant solutions. chemical analysis of corrosive solutions, after carrying out the electrochemical tests, was carried out using icp-ms method. were calculated the dissolution rates η for each metal ion studied. regarding the corrosion behavior of metal ions in corrosive solutions was observed a decrease of dissolution rate with the increasing of acetic solution concentration. keywords: electrochemical cell, stainless steel, acetic acid, mass balance, dissolution rate 1. introduction in this paper was used an advanced method to study the transfer of heavy metal ions from stainless steel samples intended to come into contact with the food simulated environments, using in addition to a corrosive environment concentration gradient and the current gradient resulting from the application of continuous current to the electrodes, both formed of the same stainless steel studied. given that one of the most aggressive environments of food processing industry is the acid environments [1], [2], [3], [4], [5], and taking into account the ph values encountered in current industrial practice (wine industry, vinegar or canning industry), in the tests presented in this paper, as food simulant corrosive environments, acetic acid in double distilled water solutions (simulant b) was used [6], [7], [8], [9]. the aim was to increase the dissolution rate with direct effect on the limit of detection of heavy metal ions in the food environment, the accuracy of measurement of the concentration of metal ions and analytical research on productivity growth by reducing the time required for corrosion and diffusion process. to accomplish the intended purpose, has been developed the material balance of metal elements that dissolve in acidic food simulant solutions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silviu-gabriel stroe, study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods, volume xii, issue 4 – 2013, pag. 354 – 358 355 by determining the theoretical and real masses of iron, manganese, chromium and nickel ions. 2. materials and methods 2.1. metallic samples, corrosive environments and equipments to study the corrosion behavior of the aisi304 stainless steel were used discs with a diameter of ø40 and 1 mm of thickness. chemical composition of metal samples are shown in the table 1 (according to en 10088-2:2005) [10]. to achieve accelerated corrosion tests were used a system consisting in a potentiostat-galvanostat model wenking hp 96-20 (bank elektronik, germany), a thermostated electrochemical cell with an working and a counter electrode, both of the stainless steel subjected to the corrosion tests, two multimeters hm81123 (hameg, germany) for monitoring the voltage values and a pc system for programming, command, acquisition and interpretation of the values recorded on the multimeter. (figure 1). chemical analysis of corrosive solutions, after carrying out the electrochemical tests, were been carried out using icp-ms method (icp-ms agilent 7500). table 1. chemical composition of aisi304 stainless steel [wt %] fe c mn p s si cr ni 67 0.07 1-2 0.045 0.03 1 18 8 figure 1. potentiostat-galvanostat system for studying the corrosion behavior of aisi304 stainless steel samples: (1) potentiostat-galvanostat wenking hp 96-20 (bank elektronik, germany); (2) – electrochemical cell; (2a) – working electrod disk ø40 (aisi304 stainless stell); (2b) – counter electrod disk ø40 (aisi304 stainless stell); (3) hameg hm8112-3 multimeter for working voltage monitoring; (4) hameg hm8112-3 multimeter to record the current intensity values in the electrochemical cell; (5) pc system for programming, command, acquisition and interpretation of the values recorded on the multimeter (4); (6) thermostatic bath (therm phoenix ii) 2.2. experimental conditions and methods the experimental conditions used for the electrochemical tests, in each of the three food simulant corrosive environments, are shown in the table 2. the volume of the electrolyte solutions was 300 ml. the exposure time of the metal samples in corrosive environments was 90 min. tabel 2. experimental conditions used to perform the electrochemical tests concentration of ch3cooh solutions, [%] 3 6 9 tensions in electrochemical cells, [v] 6 7 6.5 temperature of electrolyte solutions, [ºc] 22 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silviu-gabriel stroe, study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods, volume xii, issue 4 – 2013, pag. 354 – 358 356 2.3. developing material balance in order to developing the mass balance of the metal ions migrated from the aisi304 stainless steel samples, the theoretical and real masses of 56fe, mn, cr and ni ions in acidic simulant solutions were determined. to calculate the theoretical mass of metal ions dissolved, the equation (1) was used, using weighted atomic mass (ap) (3), based on the ion concentration in the studied stainless steel. = ∙ ∙ (1) where, m amount of metal dissolved from the anode, [g]; k – gram equivalent (electrochemical equivalent) of the species; i the measured intensity of the electric current in the electrochemical cell [a]; t time of electrolysis, [min.]. gram equivalent of the metal was calculated using the relation (2): = ∙ (2) where, ap weighted atomic mass of the species studied, [g]; z valence species (the number of electrons involved in the reaction); f faraday's constant (f=96500 c/g. echiv.). = ∙ % + ∙ % + ∙ % + ∙ % 4 (3) calculated masses of the dissolved metal ions in the solution were used to develop the theoretical mass balance. for developing of the real mass balance, the simulant solutions were analyzed by icp-ms. for the calculation of the dissolution rate (η) was used the relation (4): = (4) where, mp practical masses calculated by the equation (5), obtained by the icp-ms analysis (concentration of the electrolytic bath after 90 min.), [g]; mt – theoretical masses calculated by relation (1), [g]. = → = ∙ (5) where, c concentration of metal species, [ppb]; v volume of test solution, [ml]; 3. results and discussion anodic current intensity values measured with the hameg hm8112-3 multimeter reflect the anodic dissolution kinetic of the 56fe, mn, cr and ni metal ions from the aisi304 stainless steel samples at 90 min. exposure time (table 3). table 3. the electric current values of the aisi304 stainless steel samples in 3%, 6% and 9% ch3cooh solutions current values, [a] exposure time, [min.] 3% 6% 9% 90 0.1038 0.1522 0.1765 the masses theoretically dissolved, calculated using the relation (1), provided that the dissolution rate would be 100%, for the metal ions of the aisi304 stainless steel samples studied are shown in table 4. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silviu-gabriel stroe, study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods, volume xii, issue 4 – 2013, pag. 354 – 358 357 table 4. theoretical masses possible to be dissolved of metal ions in acidic simulant solutions from aisi304 stainless steel samples (after 90 min.), [g] elem. conc. 56fe mn cr ni 3% 0.0381 0.0381 0.0254 0.0381 6% 0.0558 0.0558 0.0372 0.0558 9% 0.0647 0.0647 0.0431 0.0647 the values of real mass dissolved [g] of the of metal ions studied, found in food simulant acid solution, obtained by analyzing the solutions through icp-ms method are shown in table 5. table 5. real masses dissolved of the metal ions in acidic simulant solutions from aisi304 stainless steel samples (after 90 min.), [g] elem. conc. 56fe mn cr ni 3% 0.0001 0.0016 0.0146 0.0025 6% 0.00018 0.0017 0.0198 0.0027 9% 0.0001 0.0017 0.0199 0.0028 by determining the theoretical and real masses values of 56fe, mn, cr and ni ions, were calculated the dissolution rates  [%] (relation 4). the values of the dissolution rates are shown in table 6. tabel 6. the dissolution rates of the metal ions in the solution with the concentrations: 3%, 6% and 9% ch3cooh [%] η56fe ηmn ηcr ηni 3% 0.0026 0.0420 0.5748 0.0656 6% 0.0032 0.0305 0.5323 0.0484 9% 0.0015 0.0263 0.4617 0.0433 after obtaining the numerical values of the dissolution rate, can be achieve a comparative study that can provide informations on the influence of the solution concentrations on the dissolution rate of the metal ions in acidic food simulant solutions. regarding the corrosion behavior of metal ions in corrosive solutions is observed a decrease of dissolution with the increasing of solution concentration. because the migration of nickel ions in solution shows a great interest, the study of corrosion behavior by electrochemical methods has been the main purpose of this research. from studying the dissolution rate of nickel ions in the three solutions, we can observe that the maximum value was obtained at 3% ch3cooh solution (0.0656), and the minimum value was obtained at 9% ch3cooh (0.0433). 4. conclusions it may be noted that the electrochemical method used to study the behavior of aisi304 stainless steel in acidic environment, studied and presented in this paper, is less expensive and a fast tool that provides very accurate information about the nature of migration processes of metal species in corrosive solution. the obtained data make possible the development of the theoretical material balance, results that have been used with those obtained by icp-ms analysis method for the calculation of the dissolution rate of metallic ions in the acid simulant solutions. the experiments performed on aisi304 stainless steel samples, in accelerated corrosion conditions using an electric gradient overlapped on the concentration gradient, have validated the validity of the method and also recommended for use in similar research. the higher dissolution rate increases the spectrometric analytical method sensitivity in very low concentration fields of heavy metals, with direct effect on the limit of detection and precision of determination. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 silviu-gabriel stroe, study of corrosion behavior of aisi304 stainless steel samples using accelerated electrochemical methods, volume xii, issue 4 – 2013, pag. 354 – 358 358 5. references [1]. kumar r., srivastava p.k., srivastava s.p., leaching of heavy metals (cr, fe and ni) from stainless steel utensils in food simulants and food materials. bull. environmental contamination toxicology, 53, 259–266, (1994); [2]. katan, l. l., migration from food contact materials. aspen publishers, inc., frederick, maryland, (1996); [3]. jellesen m. s., rasmussen a. a., hilbert l. r., a review of metal release in the food industry, materials and corrosion, 57, no. 5, doi: 10.1002/maco.200503953, 387393, (2006); [4]. barnes k. a., sinclair c. r., watson d.h., chemical migration and food contact materials, woodhead publishing limited, isbn-13: 978-1-84569-029-8, cambridge, england, (2007); [5]. herting g., odnevall wallinder i., leygraf c., corrosion-induced release of chromium and iron from ferritic stainless steel grade aisi 430 in simulated food contact, journal of food engineering 87, 291–300, (2008); [6]. efsa, european food safety authority food contact materials, note for guidance, updated on 30/07/2008, (2008); [7]. grob k., the future of simulants in compliance testing regarding the migration from food contact materials into food, food control, 19, 263–268, (2008); [8]. ningshen s., mudali u.k., amarendra g., raj b., corrosion assessment of nitric acid grade austenitic stainless steels, corrosion science 51, 322–329, (2009); [9]. stroe s.g., gutt g., poroch-seriţan m., contributions to the development of the material balance of migration process of metal ions from the aisi304 stainless steel in acetic acid solutions, food and environment safety journal of faculty of food engineering, stefan cel mare university of suceava, suceava, romania, volume xii, issue 2 – 2013, 207-213, (2013); [10]. en 10088 2 : 2005, stainless steels. technical delivery conditions for sheet/plate and strip of corrosion resisting steels for general purposes, (2005). звіт з ндр 29-81 за 2007 – 2009 р 306 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 4 – 2016, pag. 306 311 nan osized titan ium d io xide as an a ntiba cter ial admixtur e for foo d pack ag in g mater ia ls *igor kobasa1, mariia vorobets1, larysa arsenieva2 1yu. fedkovych national university of chernivtsy, ukraine, * imk-11@hotmail.com 2national university of food technologies, kyiv, ukraine * corresponding author received 15th november 2016, accepted 29th december 2016 abstract: the article deals with research on antibacterial activity of the composite materials based on titanium dioxide. the influence of synthesis condition on phase composition of titania as well as its photocatalytic activity and antibacterial effect against the pathogenic microbes such as staphylococcus aureus and escherichia coli have been investigated. it was proved that the structure of nanosized titania samples synthesized by the high temperature hydrolysis of titanium tetrachloride in the air-hydrogen atmosphere at 700-1100 0c is non-stoichiometric. these samples consist of both anatase and rutile crystalline modifications and exhibit comparatively high antibacterial activity. keywords: tio2, photocatalytic activity, antimicrobial activity, staphylococcus aureus, escherichia coli. 1. introduction contagious diseases set acute challenge for the health care field all over the world despite rapid progress achieved in pharmacy and new drugs research and development [1]. that is why special attention is paid to the search of new technological solutions related to antibacterial protection. in this context, it seems topical to design and develop new non-toxic packaging materials providing protection of food from pathogenic microbes and other adverse effects during the production and storage stages. main efforts are being spent to development of the antibacterial composite materials to be used in the bactericide coverings for medical, pharmaceutical, sanitarytechnical and household needs. it has been reported that some antibacterial glass/enamel coverings were synthesized on the base of copper and silver compounds [2], titanium dioxide [3] and calcium hydroxylapatite [4, 5]. other authors reported new antibacterial composite materials containing the ions of some noble or heavy metals ag+, au3+, hg2+, cd2+, cu2+ [6] or nanosized particles of silver and gold [7, 8]. tio2 nanoparticles have been recently proposed as a source material for synthesis of the composites with high antibacterial activity [9]. such composites can be used as components of antimicrobial ceramics, lacquer/dye coverings or microbe-proof packaging. it is known [10-12] that antibacterial and photocatalytic activity (pa) of tio2 can vary depending on the conditions of its synthesis, temperature of preliminary treatments, nature and concentrations of the admixtures added to the source synthehttp://www.fia.usv.ro/fiajournal mailto:imk-11@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 igor kobasa, mariia vorobets, larysa arsenieva, nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, food and environment safety, volume xv, issue 4 – 2016, pag. 306 – 311 307 sis composition, specific surface area, presence or absence of the crystalline inclusions, tio2 structure defects, their nature and concentration. therefore, it is possible to govern photocatalytic and antibacterial activity of tio2 through changes in these parameters and obtain either highly or moderately active products. it is known that anatase is the most photocatalytically active modification of titania (rutile and brookite are two other modifications) [13]. different crystal lattices, various degree of the lattice imperfectness, presence of some admixtures can be mentioned as the factors causing different photocatalytic activity of various titania modifications. tio2 is one of the semiconducting photocatalysts used most widely because of stability of its aqueous mixtures, cheapness, non-toxicity and high activity of the material that is based on strong oxidation efficiency of the photogenerated holes. besides, tio2 is registered as a food additive e171. this interest towards tio2 has initiated numerous investigations aimed onto development of the tio2-based materials with predefined photocatalytic and antibacterial characteristics, phase composition and texture. our efforts also followed this direction and were targeted onto synthesis of the tio2 material with some required properties. this paper reports results of investigation of the activity of the anatase and rutile modifications of titania and the nonstoichiometric product containing a mixture of anatase and rutile that has been carried out in order to develop the antibacterial packaging materials. 2. experimental three kinds of the tio2 samples were used in this work: (1) nanosized titania synthesized by the high temperature hydrolysis of titanium tetrachloride vapors in the airhydrogen flame at 700-1100 0c according to [13], specific surface area (sbet) = 50 m2/g; the anatase (2) and rutile (3) modifications of tio2 with sbet of 20 and 10 m2/g respectively, which have been synthesized by the low temperature hydrolysis of titanium chloride and sulfate [10]. the phase composition and particles size of all the samples were checked prior other investigations. the powder surface was analyzed with xray photoelectron spectroscopy (xps) also referred to as electron spectroscopy for chemical analysis (esca) using escala™ xi+ x-ray photoelectron spectrometer microprobe. xps is an elemental analysis technique which is capable of detecting all elements except for h and he and it has a nominal detection limit of ~0.1 atom%. spectral interferences may prohibit the detection of some elements in relatively low concentrations. samples were measured at a 90° take-off-angle yielding a sampling depth of ~10 nm. the analysis area was ~500 µm in diameter. analyses were performed with a monochromatic al kα x-ray source. the powder particle size analysis was performed with a field emission scanning electron microscopy (fesem) using hitachi su70 electron microscope. fesem images depict topographic features of the sample surface. fesem imaging was performed at 2 kev. one hundred particles of each powder were measured to provide an average particle size. both powders were coated with ~100 å of gold to facilitate analysis. a reaction of methylene blue reduction has been used to measure photocatalytic activity of the samples [14]. antibacterial activity of the samples has been investigated using the diffusion method (also known as the disks method) according to the standard iso 27447:2009(e) and methods [4]. the bacterial colonies were cultivated from the atcc standard strains staphylococcus aureus and escherichia coli while a paper disk impregnated with antibiotic novobiocin was used as a control experiment. antimicrobial activity has been determined food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 igor kobasa, mariia vorobets, larysa arsenieva, nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, food and environment safety, volume xv, issue 4 – 2016, pag. 306 – 311 308 by the diffusion (“holes”) method according to the standard iso 27447:2009(e) as described in details below. the inoculation material was prepared as follows: fresh bacterial colonies were taken after the 18 hours of growing in the sterile physiological medium (0.85 % nacl) in such a way to ensure formation of the suspension with 0.5 mcfarland’s density degrees, which is approximately equal to 1-2·108 cfu/ml. then the material was ten times diluted forming the 0.05 degrees system and then ten times again to form the 0.005 degrees system. density of the systems has been determined nephelometrically. the latter (0.005 degrees) system was used for the next investigations. a cotton tampon with the inoculation material was immersed in the sterile solution, extracted and squeezed to remove the excessive solution. then the inoculation material was seeded on the substrate by triple application of the tampon with rotation 600 after each application. the substrate plates were dried and then 50 μl of various nanodispersed tio2 suspensions were added to investigate their antimicrobial activity. each sample consisted of 0.1; 1.0; 5.0 and 10.0 % suspensions of the nanosized titanium dioxide. all the suspensions were prepared on the basis of physiological solution. the probes were placed into the incubator and stayed in the oxygen containing medium under 370 for 16-18 hours. then the diameter of the microbes decelerated growth area was measured as the source parameter to evaluate the antimicrobial activity. the standard disk impregnated by novobiocin (5 μg) was used as the reference sample of the antimicrobial activity. 3. results and discussion it was found that phase composition and photocatalytic activity of the tio2 samples depend on the method used to synthesize each sample (see table 1). the pyrogenic tio2 obtained by the high temperature synthesis from ticl4 vapors is a highly defective material that contains both rutile and anatase crystalline modifications. there are many paramagnetic centers in such material, which serve as active photocatalytic centers [15]. the size of these particles ranged between 45 to 174 nm (see fig. 1). a mechanism of photocatalytic and antibacterial activity of nanosized tio2 depends on the process or origination of the photoexcitated electrons initiated by the light quanta. these electrons can be transferred from the allocated valence band of a semiconductor into its unallocated molecular orbital in the conductivity band. a pair electron-hole (е-–h+) is formed as a result of these processes and then further transformations can run in different directions [16]: photoexcitation: tio2 + hν → e+ h+; absorption of electron by oxygen:   22 oeo ; ionization of water: h2o → oh+ h+; peroxide formation by protonation of the ion-radical:   hooho2 . the hydroperoxide radical formed in the latter process can take part in the following processes: hoo• + e→ ho2or hoo+ h+ → h2o2. therefore, the processes of oxidation and reduction can run simultaneously on the surface of photoexcitated semiconducting photocatalyst. an electron-hole recombination can take place if oxygen is capable to accept electrons and form peroxide ( 2o ), which participates in the follow-up protonation and form hydro peroxide radical ( hoo ) and then hydrogen peroxide (н2о2). particles sizes for the anatase (2) and rutile (3) samples of tio2 synthesized by the liquid phase hydrolysis are 0.2 and 0.5 m simultaneously, these samples are lowdefective and exhibit low pa. as it will be explained below, phase composition and structure of tio2 also provides a signififood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 igor kobasa, mariia vorobets, larysa arsenieva, nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, food and environment safety, volume xv, issue 4 – 2016, pag. 306 – 311 309 cant influence on antibacterial activity of the titania-based composites. for instance, no antibacterial activity has been found for the tio2 samples synthesized by the liquid phase hydrolysis of titanium chloride and sulfate. table 1 photocatalytic activity and phase composition of the tio2 samples synthesized by various methods synthesis method phase composition pa*, mg/(ml×min×m2) liquid phase hydrolysis of ticl4 anatase 1.2×10-2 ± 0.05 liquid phase hydrolysis of ti2(so4)3 rutile 1.7×10-3± 0.05 high temperature hydrolysis of ticl4 vapour anatase 50 % + rutile 50 % 1.2 ± 0.09 * all the data are obtained in the 5-repetitions series and the mean values with standard errors are shown in the column as seen from the antibacterial activity investigation results (see fig. 2), the colony extension inhibition area depends on tio2 concentration in suspension. figure 1. sem image of the tio2 sample. antibacterial activity of tio2 suspensions containing 1.0, 5.0 and 10.0 wt % of tio2 as against the gram-negative microbes escherichia coli is higher than that of novobiocin (see fig. 2, c and d); the novobiocin deceleration area diameter was of 13 mm, while the nanosized tio2 suspensions areas were 7, 15, 16 and 20 mm for tio2 concentrations 0.1; 1.0; 5.0 and 10 % respectively. therefore, three later compositions exhibit higher antibacterial activity than antibiotic novobiocin. antibacterial activity of tio2 suspensions containing 5.0 and 10.0 wt % of tio2 against the gram-positive microbes staphylococcus aureus is higher than that of novobiocin (see fig. 2, c and d): the novobiocin deceleration area diameter was 12 mm while the nanosized tio2 suspensions areas were 1, 7, 13 and 15 mm for tio2 concentrations 0.1; 1.0; 5.0 and 10 % correspondingly. therefore, two later compositions exhibit higher antibacterial activity than antibiotic novobiocin. а b c d e figure 2. antibacterial activity of various compositions against escherichia coli: а – 0.1, в – 1.0, с – 5.0 and d – 10 % of tio2; e – novobiocin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 igor kobasa, mariia vorobets, larysa arsenieva, nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, food and environment safety, volume xv, issue 4 – 2016, pag. 306 – 311 310 the activity rises with increase in the concentration so that antibacterial activity of the 1.0 % tio2 suspension is approximately similar to that of the standard sample test performed with antibiotic novobiocin (5 μg) (fig. 2 e). moreover, antibacterial activity of the 5.0 and 10.0 % tio2 suspensions in relation to escherichia coli (fig. 2 c, d) is higher than that of novobiocin. antibacterial activity of the nanosized titania-based compositions against grampositive staphylococcus aureus cultivated in the beef-extract agar is less noticeable than the activity against escherichia coli. only after the concentration of tio2 in the suspensions becomes equal to 5.0 %, the activity reaches same value as for the reference novobiocin sample (fig. 3). a b c d e figure 3. sensitivity of staphylococcus aureus to the nanodispersed suspensions of tio2 with concentrations: а – 0.1, в – 1.0, с – 5.0 і d – 10 %; e – novobiocin. as seen from analysis of experimental results of the tio2 samples antibacterial activity investigation, only nanosized titania reveals some activity against pathogenic organisms. that is why such material can be used in further antibacterial research as a component of special bactericide coverings. in this context, the antibacterial activity of tio2 samples was tested by adding 15 wt % of this source material to acryl-based lacquer/dye compositions. an aqueous suspension of the composition was applied by spraying over the metal surface in such a way to ensure formation of a thin homogeneous layer that covers the entire surface. then the covering was dried in the open air at the room temperature during 24 hours and used to carry out the antibacterial activity test according to iso/wd 17094: “fine ceramics (advanced ceramics , advanced technical ceramics) – test method for antibacterial activity of semiconducting photocatalytic materials under indoor lighting environment” [17]. all samples were preliminary disinfected by 70 % ethanol and then kept under the following incubation conditions: 4 or 8 hours at 25 0c, humidity 65 %. illumination intensity was 1000 lx. all the coverings received by this method ensured comparatively high antibacterial activity against pathogenic microbes escherichia coli and staphylococcus aureus. it was found that 99.4 and 93.7 % of the above colonies respectively were extinguished after 4 hours long contact with the surfaces containing the nanodispersed tio2 while 8 hours long contact caused total extinguishing of the colonies of both bacterial species. no antibacterial activity has been revealed in the control experiments with the same coverings containing no tio2. that is why it can be assumed that the nontoxic bactericide packaging materials can be obtained by adding some tio2 as a source component. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 igor kobasa, mariia vorobets, larysa arsenieva, nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, food and environment safety, volume xv, issue 4 – 2016, pag. 306 – 311 311 4. conclusion the tio2 samples synthesized by high temperature hydrolysis of ticl4 vapors in the air/hydrogen mixture have shown high antibacterial activity against the standard strains of staphylococcus aureus and escherichia coli (total extinguishing of both microbial colonies has been achieved after the 8 hours long application of the tio2 samples). a comparatively high antibacterial activity has also been registered by the acryl-based lacquer-dye compositions containing some nanosized tio2. these materials can potentially find wide applicability in the area of food packaging materials production. 5. references [1]. k.e. jones, n.g. patel, m.a. levy [et. al.] global trends in emerging infectious diseases. nature. 451, 990-993 (2008). [2]. j. prochazka. manufacturing of photocatalytic, antibacterial, selfcleaning and optically non-interfering surfaces on tiles and glazed ceramic products. wo 2004080918 a1 (2004). [3]. i. kobasa, i. kondratyeva. antibacterial activity of highly porous tio2. e-mrs spring meeting symposium v: bioinspired and biointegrated materials as frontiers nanomaterials, lille, france, 11-15 may, v-14 (2015). [4]. i. kobasa, g. bilokopyta, l. arsenieva. nanodispersed composite antimicrobial materials based on calcium hydroxylapatite. j. food and environment safety of the suceava university. food engineering. 14, 89-92 (2015). [5]. m. a. rauschmann, t.a. wichelhaus, v. stinal [et. al.]. nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. biomaterials. 26, 2677-2684 (2005). [6]. e. vob, c. störch. evaluation of bacterial growth on various materials. the 20th international enameller congress, istanbulturkey, 15-19 may. 194-210 (2005). [7]. s. srinivasan, p.t. kumar, s.v. nair [et. al.]. antibacterial and bioactive αand βchitin hydrogel/nanobioactive glass ceramic/nano silver composite scaffolds for periodontal regeneration. journal of biomedical nanotechnology. 9, 1803-1816 (2013). [8]. m.s. holden, j. black, a. lewis [et. al.]. antibacterial activity of partially oxidized ag/au nanoparticles against the oral pathogen porphyromonas gingivalis w83. journal of nanomaterials. 2016 (2016). article id 9605906, 11 pages. [9]. i. kobasa. antibacterial activity of the highly dispersed tio2. in: «resources and energy saving technologies of food production and packaging – main competitiveness trends» proceedings of third international conference. kyiv, 10 september, 2015 (in ukrainian). [10]. i. kobasa. semi-conductive materials based on the titanium dioxide doped with zinc: catalytic activity for copper deposition and effect of uv-irradiation. polish journal of chemistry 78 (4), 553-560 (2004). [11]. ya. s. mazurkevich, i.m. kobasa. tio2-bi2o3 materials. inorganic materials. 38 (5), 522-526 (2002). [12]. ya. s. mazurkevich, i.m. kobasa. zro2-tio2 materials. inorganic materials. 37 (12), 1285-1288 (2001). [13]. i.m. kobasa, w. strus, m.a. kovbasa. highly photosensitive titanium dioxide and process for forming the same. us 2008/0146441. [14]. i.m. kobasa, i.v. kondratyeva, yu.i. gnatyuk. photocatalytic reduction of methylene blue by formaldehyde in the presence of titanium dioxide and cadmium sulfide sensitized by (1-phenyl-5,6 benzoquinoline-2)-2,4-dihydroxystyryl iodide. theoretical and experimental chemistry. 44 (1), 42-47 (2008). [15]. i.m. kobasa. materials based on tio2menom (menom = zno, bi2o3, zro2, v2o5, wo3). ukrainian chemistry journal. 69 (6), 88-92 (2003). [16]. k. vingopal. photochemistry on surfaces: photodegradation of 1,3diphenylisobenzofuran over metal oxide particles. j. phys. chem. 96, (12), 5053-5059 (1992). [17]. iso 17094:2014: fine ceramics (advanced ceramics , advanced technical ceramics) – test method for antibacterial activity of semiconducting photocatalytic materials under indoor lighting environment http://www.iso.org/iso/iso_catalogue/catalogu e_tc/catalogue_detail.htm?csnumber=58094 http://link.springer.com/journal/10789 http://link.springer.com/journal/10789 http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=58094 http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=58094 microsoft word 12 codina_mironeasa corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 76 effects of dry lager brewing saccharomyces cerevisiae strain on the fermentation process and beer quality georgiana gabriela codină1, *silvia mironeasa1 1ştefan cel mare university, faculty of food engineering, 13th university street, 720229, suceava romania, e-mail: codina@fia.usv.ro; silviam@fia.usv.ro *corresponding author received 8 february 2012, accepted 5 march 2012 abstract: this paper describes the use of the dry lager brewing saccharomyces cerevisiae (saflager s-189) yeast strain in a beer factory from the moment of inoculation until the secondary fermentation stage. the research follows the physico-chemical and microbiological parameters of the inoculum, primary fermentation, secondary fermentation and unfiltered beer. special attention was paid to the way the yeast was prepared for inoculation. in the laboratory measurements made on wort and beer during the fermentation process, different devices existing in the laboratory factory from s.c. bermas s.a. suceava were used, such as: saccharimeters, thermometers, oxygen content, microscop, thoma camera, carbon dioxide, refractometer for beer analysis, viscosimeter, spectrophotometer e.g. the yeast presents good flocculation ability, a quick adaptation to the medium conditions (wort composition) and the beer obtained with this strain yeast presents good physical-chemical parameters and organoleptic ones. keywords: yeast, fermentative process, beer quality 1. introduction to make beer four ingredients are necessary: water, malt, yeast and hop. therefore, brewer’s yeast is a principal element in beer production [1]. especially, ale beers are made using saccharomyces cerevisiae strains and lager beers are made using saccharomyces pastorianus (formerly called saccharomyces carlsbergensis then as saccharomyces uvarum) yeast strains [2]. beer quality is strongly influenced by the biochemical performance of the yeast during fermentation [3]. the selection of a certain yeast strain for beer production is made taking into account the main specific characters of yeast: final fermentation grade and fermentation rate, the ability of yeast to metabolize all of the available substances, the flocculation and sedimentation capacity, the range and the amounts of secondary products resulted from secondary fermentation with implications in beer aroma and flavor, its resistance to degeneration and contamination e.g. [4]. before yeast is added, the breweries must provide a suitable growth medium for it. yeast fermentation performance and beer quality is influence especially by the wort composition, oxygen prior to pitching, fermentation temperature, size and shape of the fermentor vessels e.g [5]. the fermentation environment must be highly aerobic (that’s the way the wort is first aerated) [6] and the fermentation tanks must be cooled and maintained at 8°c to 15°c for lagers and 15°c to 22°c for ales. during the fermentation process different factors that affects beer yeast activity like oxygen concentration, osmotic potential, ph, ethanol concentration, nutrient availability and temperature are in continues fluctuations [7]. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 77 because yeast growth conditions are never constant, the brewers must always control the fermentation process (i.e. temperature, ph, co2, extract decomposition, ethanol content e.g.) to avoid a stressed yeast which may lose its ability to replicate, become unable to ferment or die. yeast quality is measured by its viability (the percentage of live cells within a population) and/or vitality (metabolically active yeast). at the end of the fermentation process when yeast fermented all the fermentable sugars from the medium, its flocculations occurs [8] and the beer is considered to be fully attenuated. once the yeasts have flocculated, yeasts are collected and removed from the beer [9]. beer flavor, clarity, and overall quality depend on the moment when yeast flocculate. even if the yeast cells are removed after their sedimentation, the beer will still contain some yeast cells that will led to undesirable flavor compound. therefore, it is essential to have a secondary fermentation to eliminate the remaining yeasts and haze-forming proteins, to produce beer with a mature or finished flavor and some amount of co2 gas formation. the aim of this paper is to evaluate the quality of a lager brewing saccharomyces cerevisiae strain quantitatively and qualitatively with the highlight of the technological parameters that changes during the fermentation process with implications for the evolution of the fermentative process and the quality of the beer obtained. 2. materials and methods 2.1. yeast and wort. all experiments were carried out with an industrial dry lager brewing strain of saccharomyces cerevisiae (saflager s-189) (fermentis, belgium) (humidity 3.5 to 6; dry matter 94 to 96.5%) on eighteen malt hopped wort at s.c. bermas s.a. suceava. the malt hopped wort quality parameters (mean and standard deviation) used in this study are shown in table 1. table 1 physico-chemical and microbiological properties of cooled hopped wort characteristics avarage value standard deviation original extract, °p 10.2 0.15 ph 5.2 0.1 acidity, ml naoh 1n 1.7 0.16 turbidity, ebc 69.5 1.5 color, ebc 8.2 0.2 final fermentation grade, % 88.4 1.6 live cell/g none 2.2. propagation and fermentation conditions. for yeast propagation, 40 kg of saccharomyces cerevisiae (saflager s-189) strain were rehydrated in a fermentor vessel with a capacity of 200 hl with approximately 150 l of sterile malt hopped wort at 23°c temperature. obtained cream was spilled over 40 hl wort to a temperature of 23°c in a cooling tank with internal cooling coils. the yeast mixture obtained was left to grow for 3 h in the wort medium for its regeneration and for the initiation of the fermentation process. finally the process continues by pouring a quantity of 100 hl of cooled hopped wort at 8.0÷8.5°c temperature above the yeast inoculum. thus, the amount of yeast inoculum that was obtained is of 140 hl with the following parameters: apparent extract 10°p, temperature 12°c and the number of viable cells/ml of 32.5. 2.3. fermentation analysis. the number of yeast cells was counted using a cytometry camera thoma. the yeast cell viability was assessed using the methylene blue staining method [10]. the physico-chemical parameters of yeast inoculum, wort and beer quality were determined according to the romanian standard methods or by using the technological instructions from s.c. bermas food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 78 s.a. suceava: wort apparent extract using a saccharimeter device (technological instructions), wort temperature using a termometre device (technological instructions), oxygen content (ebc method), original (sr 13355-5:2005), density (sr 13355-5:2005), alcohol concentration (sr 13355-3:2005), ph (sr 6182-14:2009), color (sr 13355-7:2005), acidity (sr 13355-6:2000) bitterness value (sr 13355-9:2003), viscosity (ebc method), carbon dioxide (sr 133558:2003) and fermentation grade for sale using a saccharimeter device (technological instructions). 2.4. data analysis. all analytical determinations were performed at least in triplicate. the values of different parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95%. all data was processed with microsoft excel 2003. also, statistica 6.0 software was used for graphical representation. 3. results and discussion physical and chemical analysis. the yeast inoculum parameters (temperature, apparent extract, yeast cell number, oxygen content, ph) were determined during 28 h time period. the results obtained are shown in table 2. table 2 the yeast inoculum parameters parameters (average value ± standard deviation) time (h) temperature (°c) apparent extract (°p) yeast cells number (x 106 cel/ml) oxygen content (mg/l) ph 0 13.00 ± 0.01 10.00 ± 0.01 35.00 ± 0.20 9.60 ± 0.10 5.20 ± 0.20 4 13.50 ± 0.05 9.40 ± 0.02 17 14.00 ± 0.02 8.10 ± 0.03 57.50 ± 0.50 0.60 ± 0.10 5.00 ± 0.10 24 12.50 ± 0.05 7.20 ± 0.02 85.00 ± 0.50 28 12.00 ± 0.02 6.60 ± 0.02 85.40 ± 0.40 from the figure 1 we can see an increase of the yeast cell number correlated with a decrease of the apparent extract. figure 1. time (t) variation of the yeast cells number (y_cell no.) and of the apparent extract (e_ap.). the yeast population in this phase increased almost 2.5 times from the starting inoculum quantity. this fact is explainable because in this phase yeast uses the amino acids as a source of nitrogen and glucose and the fermentable sugars as a source of energy and carbon [18] leading to a decrease of the apparent extract with almost 34%. according to the recommendations made by the yeast producers the yeast inoculum temperature did not exceed 14°c. in the last hours of yeast inoculum fermentation the temperature was decreased until 12°c to avoid a decrease to high of the apparent extract (producer's recommendation in this phase for apparent extract values was that it should varied only between 6-7°p). the ph of the hopped wort is favorable to yeast food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 79 growth (5.2 to 5.0) and its decrease from 5.2 to 5.0 it can be explained by the amino acids yeast metabolization and the formation in the wort medium of the organic and volatile fixed acids [19]. oxygen consumption rate is very high (about 16 times in the first 17 hours) due to its extensive utilization by the yeast cells [6]. the amount of yeast inoculum obtained (140 hl) with the parameters mentioned above was transfuse on a 130 hl of cooled hopped wort. over this another quantity of cooled hopped wort of 130 hl was added. both quantities of cooled hopped wort had the same technological parameters (temperature 8°c and original extract 10.2°p). the parameters evolution during the fermentation process is shown in table 3. table 3 the tank fermentation parameters parameters (average value ± standard deviation) time (h) temperature (°c) apparent extract (°p) yeast cells number (x 106 cel/ml) oxygen content (mg/l) 0 10.00 ± 0.01 8.00 ± 0.01 25.00 ± 0.10 0.53 ± 0.01 7 10.80 ± 0.02 7.70 ± 0.03 35.00 ± 0.15 11 11.50 ± 0.05 7.50 ± 0.05 47.00 ± 0.20 24 11.50 ± 0.05 6.80 ± 0.04 55.40 ± 0.20 31 12.00 ± 0.02 6.20 ± 0.02 84.50 ± 0.50 35 12.00 ± 0.02 5.80 ± 0.03 48 12.00 ± 0.02 5.00 ± 0.04 93.70 ± 0.30 0.45 ± 0.01 59 12.00 ± 0.02 4.10 ± 0.01 72 10.00 ± 0.01 3.50 ± 0.05 41.25 ± 0.25 83 9.50 ± 0.05 3.10 ± 0.03 96 9.50 ± 0.05 2.80 ± 0.02 27.50 ± 0.02 107 9.50 ± 0.05 2.60 ± 0.04 23.50 ± 0.03 120 9.50 ± 0.05 2.40 ± 0.02 18.75 ± 0.25 133 7.00 ± 0.04 2.40 ± 0.02 144 5.50 ± 0.05 2.30 ± 0.01 12.50 ± 0.02 148 5.00 ± 0.02 2.30 ± 0.01 7.50 ± 0.01 0.43 ± 0.01 figure 2. time (t) variation of the yeast cells number (y_cells no.) and of the temperature (t). figure 3. time (t) variation of the apparent extract (e_ap) and of the yeast cells number (y_cells no.). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 80 this phase is characterized by an intense fermentative metabolism of yeast with the production of ethanol, co2 and other fermentation products. the yeast population from the beginning of fermentation process increases from 25 x 106 cel/ml to 93.70 x 106 cel/ml at 48 h of fermentation while the apparent extract decreases from 8°p to 4,1°p as is shown in figure 2. when the wort temperature increases until 12°c the fermentation tank is cooled gradually until it reached 5°c at the end of the fermentation process. as figure 3 shows we can observe a slower decrease of the apparent extract and of the yeast population leading to the end of the fermentation process to 2.3°p and 7.5 x 106 cel/ml the moment when primary fermentation is considered complete. the fermentation process continues during the secondary fermentation in tanks held at lower temperatures for a longer time.the secondary fermentation beer parameters evolution is given in table 4. table 4 the secondary fermentation beer parameters parameters value apparent extract (°p) at the beginning of secondary fermentation 2.30 ± 0.12* yeast cell number (mil cel/ml) at the beginning of secondary fermentation 7.00 ± 1.00* tank pressure (barr) 0 to 0,9 temperature (°c) 5.8 to 2 apparent extract (°p) after 14 days of fermentation 1.80 ± 0.10 * * average value ± standard deviation the secondary fermentation process was held for 30 days after which the laboratory tests were performed on unfiltered beer quality parameters as it shown in table 5. from it we can noticed that the secondary fermentation process has normal regarding the decrease of extract from the beginning of the process until the end and thus we can explained why the fermentation grade for sale determined in the beer factory is very close to the fermentation grade for sale determined in the laboratory. this shows that in the secondary fermentation tank, an optimal amount of yeast cells passed, allowing the fermentation process to continue to take place and therefore to the decrease of beer apparent extract during the secondary fermentation from 2.3° p (at the beginning of the process) to 1.8°p after 14 days of fermentation and 1.7°p when the fermentation ends. the diagram of the secondary fermentation respectively the correlation temperature / pressure on the tank was respected fact that contributed to the good beer quality parameters like carbon dioxide content of 0.56 g/100 ml beer and to the very good organoleptic qualities like taste and smell nice, clean with a bitterness tint, persistent foam e.g. table 5 the unfiltered beer parameters parameters average value standard deviation original extract (°p) 10.20 0.08 apparent extract (°p) 1.70 0.09 density (g/ml) 1.0008 alcohol concentration % (v/v) 4.60 0.30 acidity (ml naoh 1n/100 ml beer) 1.80 0.07 ph 4.20 0.18 color (ebc) 8.00 0.80 carbon dioxide (g/100 ml beer) 0.56 0.04 bitterness value (be) 20.00 0.60 viscosity (cp) 1.35 0.03 fermentation grade for sale (%) 83.30 1.76 yeast cell number (x 103 cel/ml) at the end of the secondary fermentation 500 132.34 4. conclusions the dry lager brewing saccharomyces cerevisiae (saflager s-189) yeast strain used food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 81 in our research has been quickly adapted to the environmental conditions (wort composition) its growth is continuous during all the fermentation phases form the starting inoculum until the secondary fermentation stage. the yeast presents good flocculation ability because at the end of the primary fermentation phase the yeast cell population was 7.5 x 106 cel/ml enough for the sugars metabolisation during the secondary fermentation phase. the diagram of primary and secondary fermentation was well conducted, the fermentation grade for sale determined in the beer factory being very close to the fermentation grade for sale determined in the laboratory target that must be achieved by the brewers. regarding the beer physical-chemical parameters and also the organolpetic ones the yeast strain used improved them. 5. acknowledgements the authors thank to s.c. bermas s.a. for the technical support. 6. references [1]. sohrabvandi, s., razavi, s.h., mousavi, s.m., mortazavian, a.m., characteristics of different brewer’s yeast strains used for non-alcoholic beverage fermentation in media containing different fermentable sugars, iranian journal of biotechnology 8. 178-185, (2010) [2]. davydenko, s.g., yarovoy, b. f., stepanova, v. p., afonin, d. v., batashov, b. e., dedegkaev, a. t., a new yeast strain for brewery: properties and advantages, russian journal of genetics 46. 1295–1305, (2010) [3]. powell, c.d., quain, d.e., smart, k.a., the impact of brewing yeast cell age on fermentation performance, attenuation and flocculation, fems yeast research 3. 149-157 (2003) [4]. banu, c. (ed.), the science and technology of malt and beer, bucharest, agir press, (2000) [5]. speer, r.a., stokes, s., effects of vessel geometry, fermenting volume and yeast repitching on fermenting beer, j. inst. brew., 115.148–150 (2009) [6]. depraetere, s.a., delvaux, f., de schutter, d., williams, i.s., winderickx, j., delvaux, f.r., the influence of wort aeration and yeast preoxygenation on beer staling processes, food chemistry 107. 242–249, (2008) [7]. briggs, d.e., boulton c.a., brookes, p.a. & stevens, r., brewing: science and practice, woodhead, cambridge, uk., (2004) [8]. bauer, f.f., govender, p., bester, m.c., yeast flocculation and its biotechnological relevance, appl microbiol biotechnol 88. 31–39, (2010) [9]. van hamersveld, e.h., van der lans, r. g. j. m., caulet, p. j. c., luyben, k. ch. a. m., modeling brewers’ yeast flocculation, biotechnology and bioengineering 57. 330-341, (1998) [10]. anonymous, ebc-analytica microbiologica, j. inst. brew. 83. 109-118, (1977) [11]. sr 13355-5:2005, beer. analysis methods. extract determination, standardization association of romania, bucharest, romania. [12]. sr 13355-3:2005, beer. analysis methods. determination of alcoholic concentration, standardization association of romania, bucharest, romania. [13]. sr 6182-14:2009, wine. part 14: determination of ph-value, standardization association of romania, bucharest, romania. [14]. sr 13355-7:2005, beer. analysis methods. color determination, standardization association of romania, bucharest, romania. [15]. sr 13355-6:2000, beer. analysis methods. determination of total acidity, standardization association of romania, bucharest, romania. [16]. sr 13355-9:2003, beer. analysis methods. determination of bitterness of beer, standardization association of romania, bucharest, romania. [17]. sr 13355-8:2003, beer. analysis methods. determination of carbon dioxide, standardization association of romania, bucharest, romania. [18]. gibson, b.r., lawrence, s.j., leclaire, j.p.r., powell, c.d., smart, k.a., yeast responses to stresses associated with industrial brewery handling, fems microbiol rev. 31. 535– 569, (2007) [19]. lei, h., zhao, h., yu, z., zhao, m., effects of wort gravity and nitrogen level on fermentation performance of brewer’s yeast and the formation of flavor volatiles, appl biochem biotechnol. 166. 1562–1574 (2012) microsoft word 13 dabija_nr.1_2013.doc 84 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 84 88 aspects concerning aflatoxins incidence in milk and milk products * adriana dabija1, iuliana sion 2, mihaela-adriana tita3, ovidiu tita 3 1faculty of food engineering, stefan cel mare university of suceava, romania 2d.s.v.s.a. bacau, romania, 3faculty of agricultural sciences ,food industry and environment protection , “lucian blaga” university of sibiu , romania adriana.dabija@fia.usv.ro *corresponding author received 12 january 2013, accepted 13 february 2013 abstract: mycotoxins production was found most frequently in pre-harvest grains that are harvested under high temperatures, prolonged drought and high insect activity. aflatoxine incidence in milk and milk products has become a typical issue for many countries, as there were detected amounts over legal limits in consume milk. origins of aflatoxins in milk and dairy products are mainly resulted from feeds supplied to animals. this paper proposes a study on aflatoxin incidence in compound feed and milk raw material in the dairy industry. for determinations was used elisa enzymeimmunity-test. records should be maintained for all feeds, feeding practices, milk contamination and animal health and performance for all cases of aflatoxin contamination of milk. keywords: safety, carcinogenicity, maximum residue limit, monitoring 1. introduction production and marketing of quality and health-safety foods are primary objectives for food industry. recently, the aspect of aflatoxins contamination for milk producers was coming back, due to the serbia milk contamination event. panic has been induced among consumers, and that was the moment when authorities had to recognize the overtaking level for aflatoxins, much higher than legal limits, in drinking milk. in 2012, in united states, was found high level of aflatoxins, half of the corn harvest being compromised. in our country, national sanitary veterinary and food safety authority has specified that in 2012, 81 samples of milk were analyzed for aflatoxins determination, all in conformity with regulations and also, that the country hadn’t received contaminated milk from serbia [1,2]. last years were the most favorable for mycotoxins development, in whole europe, including romania. the mycotoxins european contamination level is about 20%, even in economic developed countries, the percent being much higher through less competitive agriculture conditions. in these countries has been estimated a harvest loss of 5-10%, because of molds’attack, that’s why have been required special measures [3, 4, 5]. the mycotoxins analysis of feedstuffs or foodstuffs represents initial stadium of mycotoxicologic research. an ideal model should be analysis for each sample, in initial medium conditions. unfortunately, the phenomenon of apparition, growth, developing and regression of mould species are not known completely. that is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adriana dabija, iuliana sion, mihaela-adriana tita, ovidiu tita, aspects concerning aflatoxins incidence in milk and milk products, food and environment safety, volume xii, issue 1 – 2013, pag. 84 88 85 why mycotoxins investigation has limited area and must be completed with mycotoxicological research for monitoring vegetables safety. hereinafter are presented the main conditions for molds developing and mycotoxins production: high values for temperature and moisture, favorable parameters for the substrate, water activity. the food contamination with aflatoxins producing molds could compromise the food safety and could affect the consumers’ health, by producing intoxication, cancer, even death [6]. european and international researchers have found over 3500 molecules inside mycotoxins group. trying to synthesize and classify these substances has encountered a problem with the fact that the same mould could synthesize more mycotoxins and, vice versa, the same mycotoxin could be obtained from different molds. all groups of mycotoxins are produced from 5 (five) genus of alteration molds: aspergillus, penicillium, fusarium, alternaria and claviceps. milk and milk products could contain aflatoxins from biosynthesis by aspergillus flavus, aspergillus parasiticus and aspergillus nomius. these molds could get consumer illness by their development on different foods. in the case of cheese assortments, we could meet many types of mycotoxins, so derived from environment molds, as well as mold culture used in cheese fabrication [7,8]. high values were found in the case of cheese assortments with long time of preservation and advanced proteolysis degree. aflatoxins could be present even in melted cheese, with lower incidence. aflatoxins are among those most risky and carcinogenic substances, because their methabolisation through liver, toward intermediate substances, m1 or m2. the highest concentration of aflatoxins is formed during cereal cultivation and storage, but also during their processing. growth of aflatoxins in diet and increase in the rate of these toxins from the critical range (20 in diet and 0.5µg/kg in milk) cause decrease in fertility and reproductive ability in animals [9]. in 1987, world health organization classified group 1 of aflatoxins as carcinogen, with a great impact on liver health and dna destroyer agents, m1 aflatoxin being considered the most aggressive. the incidence of m1 aflatoxin in milk could be explained by molded feed (b1) consumed by animals. short-time ingestion of large quantities of aflatoxins can lead to acute poisoning, which can manifest through bleeding, acute liver failure and even death. lethal dose depends on several factors, the amount of aflatoxin ingested, age, health and nutritional status of the individual. eating a small amount, but for a long period of time may cause chronic poisoning. effects and symptoms are often difficult to identify, both because of low intensity but mostly because of their nonspecific character [10]. consumption of food contaminated with aflatoxins can have serious consequences such as cirrhosis and hepatocarcinoma, a particular form of liver cancer. epidemiological studies developed in india and some african countries have shown an association between the consumption of food contaminated with aflatoxin and increased incidence of liver cancer. in humans, aflatoxins have caused a number of sub-acute and chronically effects. these effects mainly include liver cancer, chronic hepatitis, jaundice, hepatomegaly and cirrhosis caused by repeated ingestion of small quantities of aflatoxins. this paper proposes a study of aflatoxin contamination in milk and dairy products compared with their presence in feed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adriana dabija, iuliana sion, mihaela-adriana tita, ovidiu tita, aspects concerning aflatoxins incidence in milk and milk products, food and environment safety, volume xii, issue 1 – 2013, pag. 84 88 86 2. materials and methods in present, the immununologic tests consists time one of the rapid methods for aflatoxin and other micotoxin detection in foodstuffs. immunological tests were succesful used since 1970 (pestka et al., 1995), for mycotoxins detection. for qualitative or semi-quantitative analyses of majority food matrix it can be used mycrotitration boards or elisa membranes with polyclonal or monoclonal antibodies. however, interference matrix or the presence of mycotoxins could interfere with conjugated antibodies that could leads to errors for quantitative results of mycotoxins. elisa kit is used for routine analyses for majority of food matrix. the principle of method the kit board consists of 12 stripes with 8 places, coated with rabbit antibody and anti igg mouse. in the first step, are added specific antibody (mouse anti aflatoxine), aflatoxine marqued with enzyme conjugated and afm1 standards and also the samples to be analysed. specific antibody were binded by rabbit antibody strike-bound, and, in the same time, free aflatoxin(unbinded), from standards or samples and marqued aflatoxin are in competition for binding situs of specific antibody, that is immunoenzyme competitive test. after one hour, the reagents (marqued with enzyme) unbinded, were washed. the enzyme quantity that is binded was revealed by adding chromogen substrate (tetramethylbenzidine, tmb). this will turn chromogen in a coloured one. the reaction was stopped by adding 100 µl h2so4 1m solution. the colour intensity was measured by spectrophotometry, at 450 nm, with elisa reader system, taking account that the aflatoxine m1 concentration is in inverse ratio with sample colour intensity. in the same time was established a calibrating curve with standard solutions of m1 aflatoxine: 5(s1); 10(s2); 25(s3); 50(s4); 100(s5) şi 200(s6) ng/l. the sample preparation the samples were prepared in compliance with technical informations of r-biopharm ag, ridascreen kit for afm1. milk samples were centrifugated for fat carrying-off for 10 min / 3500 g and after centrifugation, the kit contain the following: microtitration board (96 places), 12 stripes with 8 places, coated with mouse antibody anti-igg; 6 aflatoxine m1 flasks (1,3 ml) with concentration 0 ng/l (zero standard), 5 ng/l , 10 ng/l , 25 ng/l , 50 ng/l , 100 ng/l, 200 ng/l; 1 conjugate solution flask (aflatoxine marqued with peroxidase) manifold drying antibody antim1 (manifold drying 1,3 ml); chromogen substrate solution flask (tetramethylbenzidine) , (10 ml); buffer solution flask ph 7, (20 ml); stopping solution flask, h2so4 1n ( 14 ml); washing buffer solution flask( 30 ml). reagents and solutions with kits indicated for elisa method could be effectuated 96 determinations, including standards. reagents that are used, out of kit: methanol n-heptane diclormethan buffer solution, ph 7.2: (0.55 g nah2po4 x h2o + 2.85 g na2hpo4 x 2 h2o + 9 g nacl în 1000 ml distilled water) equipments and materials: elisa microstrip reader 303 centrifuge eba-20 micropipete de 20-200 µl şi respectiv 200-1000 µl food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adriana dabija, iuliana sion, mihaela-adriana tita, ovidiu tita, aspects concerning aflatoxins incidence in milk and milk products, food and environment safety, volume xii, issue 1 – 2013, pag. 84 88 87 pasteur pipette graduate pipette gloves method proceeding 100 µl buffer solution from standard dilution consists as blank (places a1 and a2). than, was putting 50 µl buffer dilution, s0 standard (places b1 and b2) and 50 µl standard dilution in downward disposal for concentration, duplicate form (places s1 and s6), and for sample analyses we had put 50 µl in other places (p1, p2, etc.) 3. results and discussion regulation nr. 1881/2006 establishes maximum levels for different types of contaminants in food products. in table 1 are presented the maximum limits accepted for aflatoxin in foods. they were processed 10 samples of rawmilk, from many collecting centers in bacau county. the results are presented in table 2. regulation nr. 574/2011 establishes maximum levels for different types of additives and/or contaminants in feed products and also suggested the prohibition of those feeds that contains forbidden substances in overtaken quantities. in table 3 are presented the maximum limits accepted for aflatoxin in feeds. table 1 maximum admitted level for micotoxins in food products table 2 determination of aflatoxin m1 in milk sample provenience aflatoxin m1 µg/kg[ppb] conclusion 1 serbesti 0.002 comply with regulation 2 gheorghe doja less than lod comply with regulation 3 negri 0.003 comply with regulation 4 sascut 0.003 comply with regulation 5 onesti less than lod comply with regulation 6 targu ocna 0.004 comply with regulation 7 filipesti less than lod comply with regulation 8 racaciuni 0.002 comply with regulation 9 valea seaca 0.001 comply with regulation 10 nicolae balcescu less than lod comply with regulation table 3 maximum level for micotoxines in feeds type of feed measure unit maximum level for feeds with moisture content 12% b1 aflatoxine raw feeds mg/kg[ppm] 0.02 supplementary and complete feeds mg/kg[ppm] 0.01 combined feeds for milk cattle and calfs, milk ovine and lambs, milk goats and billy goats, piglets and young poultries mg/kg[ppm] 0.005 combined feeds for bovine(exception milk bovine), ovine (except milk ovine), poultries (except young poultries), swine(except piglets) mg/kg[ppm] 0.02 type of micotoxin measure unit limit value aflatoxine b1 µg/kg[ppb] 0.10 aflatoxine m1 µg/kg[ppb] 0.025 ochratoxine a µg/kg[ppb] 0.50 patuline µg/kg[ppb] 10 deoxinivalenol µg/kg[ppb] 200 zearalenone µg/kg[ppb] 20 fumonisine µg/kg[ppb] 200 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adriana dabija, iuliana sion, mihaela-adriana tita, ovidiu tita, aspects concerning aflatoxins incidence in milk and milk products, food and environment safety, volume xii, issue 1 – 2013, pag. 84 88 88 table 4 determination of b1 aflatoxin in feeds sample type of feed aflatoxin b1 [mg/kg] conclusion 1 lucerne 0.005 comply with regulation 2 hay1 less than lod comply with regulation 3 complete feed for goats 0.004 comply with regulation 4 complete feed for adult bovine less than lod comply with regulation 5 complete feed for calfs less than lod comply with regulation 6 hay2 0.003 comply with regulation 7 complete feed for milk ovine less than lod comply with regulation 8 alfalfa 0.005 comply with regulation 9 complete feed for milk goats less than lod comply with regulation 10 lucerne 0.004 comply with regulation for aflatoxin b1 determinations in feeds were analyzed various types of animal feeds, also from bacau county farmers, as well as, complete feeds and raw feeds (table 4). 4. conclusions our country has implemented the most important regulations in many fields of activity, since 2005, especially in those domains with great impact in our trade collaboration with ue members or non ue members. monitoring of mycotoxins content in foods is a constant concern of the authorities, and mainly for food processors in this industry. milk and dairy products are foods susceptible to contamination with aflatoxins, reasons why preventive measures should be taken, such as: ensuring optimal feed storage, monitoring of mycotoxin levels in feeds, risk assessment in accordance with the requirements of applicable law and achieve traceability to prevent aflatoxins contamination of milk and dairy products. it is also necessary to develop procedures and guidelines of good management practices to prevent, reduce and / or eliminate contamination with aflatoxin b1 in feed, respectively aflatoxin m1 in milk and dairy products. 5. references [1]. dragacci, s., grosso, f., gilbert, j., immunoaffinity column clean-up with liquid chromatography for determination of aflatoxin m1 în liquid milk: collaborative study, j. assoc. off. anal. chem. int., no.84, 437, (2001) [2]. elgerbi, a. m., aidoo, k. e., kandlish, a. a., tester, r. f, occurrence of aflatoxin m1 în randomly selected north african milk and cheese samples. food additives and contaminants, no.21(16), 592–597, (2004) [3]. malone, b.r., humphrey, c.w., romer, t.r., richard, j.l., determination of aflatoxins în grains and raw peanuts by rapid procedure with fluorometric analysis, j. assoc. off. anal. chem. int., no. 83, 95, (2000) [4]. nachtmann, c., gallina, s., rastelli, m., ferro, g. l., decastelli, l., regional monitoring plan regarding the presence of aflatoxin m1 în pasteurized and uht milk în italy, food control, no.18(6), 623–629, (2007) [5]. mokoena m. p., chelule p. k., gqaleni n., the toxicity and decreased concentration of aflatoxin b în natural lactic acid fermented maize meal. j appl microbiol, no.100, (2006) [6]. peltonen k., elnezami h., haskard c., ahokas j., salminen s., aflatoxin b1 binding by dairy strains of lactic acid bacteria and bifidobacteria. j dairy sci, no. 84, (2001) [7]. govaris, a., roussi, v. , koidis, p.a., and botsoglou, n.a., distribution and stability of aflatoxin m1 during processing, ripening and storage of telemes cheese, food additives and contaminants, no.18, 437, (2001) [8]. movassagh, m.h., khodabandehloo, e., movassagh, a., detection of aflatoxin m1 in cow’ raw milk in miandoab city, west aazerbaijan province, iran, global veterinaria, no 6(3), 313-315, (2011) [9]. pirestani, a., toghyani, m., the effect of aflatoxin levels on milk production, reproduction and lameness in high production holstein cows, african journal of biotechnology, vol.9 (46), 79057908, (2010) [10]. battacone, g., nuda, a., cannas, a., cappio, b. a., bomboi, g., pulina, g., excretion of aflatoxin m1 in milk of dairy ewes treated with different doses of aflatoxin b1, journal dairy science, no. 86: 2667-2675, (2003) 218 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 218 224 a cas e s tud y of t he j uice-m aki ng fac tory wast ew ate r tr eatm e nt a nd pos si ble w ays o f its o pt im i zat ion alla choban1, *igor winkler1 1yu. fedkovych national university of chernivts, 2 kotsyubynsky st., chernivtsi, 58012, ukraine, igorw@ukrpost.ua * corresponding author received august 31st 2013, accepted september 15th 2013 abstract: a general analysis of the typical juice making factory wastewater composition and its treatment technology is carried out. since the technology used at the moment does not ensure effective decontamination and complete renovation of the wastewater treatment equipment is not feasible, some simple actions are proposed to improve the treatment quality. possible efficiency of these actions is also analyzed and discussed. keywords: juice making; wastewater treatment; active sludge; biogenic components 1. introduction availability and quality of water resources are known to be the key factors of sustainable development of any country and normal health conditions of its population (world health organisation, 2010). many water bodies in ukraine have been degrading because the natural self-cleaning processes cannot cope with the amount of pollution they receive. as a result, the quality of water in many reservoirs that were previously used for drinking water supply has now dropped to the third class now (dmitrieva et al, 2003). this process is caused mainly by massive discharge of untreated or poorly treated wastewater and every effort should be made to minimize this wrong practice. various food processing facilities are quite widely distributed in ukraine and their wastewater treatment equipment is often very old and worn, which results in malfunctioning of the water cleaning technologies. besides, food factories are often equipped with the wastewater treatment lines that were projected for the public (municipal) wastewater that does not always ensure the required cleaning of the industrial effluents. juice making factories can be referred to as an example of such improper realization of the food processing wastewater treatment. the main problem is caused by the strong acid reaction of the wastewater and rather low content of the biogenic elements (mainly n and p). under these conditions, “normal” biotreatment of the wastewater is too slow and inefficient. as reported (el-kamah et al, 2010), the highest efficiency of the orange juice manufacturing wastewater decontamination can be achieved only in a combined technology with initial treatment in a two-stage up-flow anaerobic sponge reactor followed by the activated sludge reactor with approximately similar treatment periods in the both reactors. traditional wastewater treatment technology includes only the latter of the above mentioned operations. therefore, it is obvious that even thorough planning and realization of such technology cannot ensure the required level of the juice manufacturing wastewater decontamfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 219 ination. on the other hand, there are references (shepherd et al, 2001, mosse et al, 2011) to quite successful functioning of the simpler wastewater decontamination systems. shepherd et al (2001) reported a 98 % decrease in the chemical oxygen demand and 97 % in the total suspended solids content for the high-strength acidic winery wastewater treatment at a simple wetland landfill processing system combined with the sand prefilter. extended reviews related to various winery and distillery wastewater treatment problems and technologies (mainly, decolourization) using various biological methods have been published by pant, adholeya (2007) and mosse et al (2011). juice factories production has good market and constantly growing demand, causing rise in the production capacities and the amount of wastewater formed and discharged by this branch. therefore, insufficient wastewater treatment seems quite a topical problem for any region of ukraine with active fruit and vegetable processing. it should also be emphasized that more popular orange and some other tropical fruit juices technologies are analyzed in the majority of investigations related to this issue while many juice making facilities in ukraine work with apple juice and their wastewaters are much more acidic and poorer with organic components. therefore, it seems topical to investigate possible advances in treatment of this type of wastewater. some related and close problems of the wastewater treatment and its environmental effects have been discussed by choban and winkler (2008, 2011, 2012), choban et al (2012) in this paper we analyze general problems of the juice making wastewater treatment. some solutions for the wastewater quality improvement are also proposed and discussed. 2. materials and methods general description of the actual wastewater collection and treatment technologies this investigation deals with an analysis of the wastewater treatment technology, problems, environmental effects and possible ways of optimization on example of “bmb” juice making factory located in vil. kobolchin, region of chernivtsi, ukraine. this is a typical juice manufacturer and many similar factories are still working all over ukraine. the factory is equipped with separated sewage systems for industrial and public wastewaters collection and transportation. a composition of the public effluent is typical for the moderately polluted wastewater (bod5 = 180 – 200 mg/l and ph = 6,5 – 8,5) while the industrial effluent is more concentrated and acidic (ph = 4,4 – 5,2; bod5 = 3500 – 4000 mg/l). the total projected discharge is 168,57 m3/day with only 31,11 m3/day of the public wastewater. therefore, industrial wastewater is a key factor that determines the quality and the quantity of the factory’s discharge. untreated industrial wastewater quality parameters are shown in table 1 (column 3). the general flowchart of the wastewater treatment technology is shown in fig. 1. according to this technology, all collected wastewater is self-flowing to the treatment station. then it is pumped to the receiver tank and then to the multisectioned settlerflotator. apple peels and seeds are filtered out in the first section at the arch sieve and then removed periodically. then the wastewater flows to the next section for the acidity neutralization and adding some phosphorus and nitrogen compounds. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 220 figure 1. a technological flowchart of the current wastewaters treatment technology food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 221 the maximum calculated daily load of the nitrogen compounds is 32 kg and phosphorus compounds is 6,4 kg. ammonia water (25 % of nh3 or 20,6 % of n) is mainly used as a source of nitrogen and the partial alkalization agent. the calculated amount of this reagent is 156,3 kg/day. superphosphate (20 % of p2o5 or 8,7 % of p) is used as a source of phosphorus and the calculated daily consumption of this reagent is 73,6 kg. all amounts of the required reagents are determined in fact by the periodical lab analysis of the wastewater compositions. any overdose is unwanted since it causes raise in the content of p and n in the treated wastewater. caustic soda or lime water are used for the wastewater neutralization. lab analysis of the wastewater is also involved in determining the amount of these reagents required to bring ph to the range 6,5 – 8,5. the combined settler-flotator (diameter 4,0 m) ensures removal of the coarse dispersed pollution by settling and then the remained suspended particles are removed by the pressure floatation with working liquid recycling. the required technological regime of the pressure floatation is maintained by saturation of the working fluid with air under pressure 0,4 mpa and its constant pumping to the floatation chamber. the working fluid is formed initially from the primary treated wastewater after the settler-flotator and then from the clarified wastewater taken from the upper part of the secondary settler at the second stage of bioaeration. saturation of the working fluid with air is realized in the pressure tank. biotreatment of the wastewater is realized by the two-stage technology. most of the biopollution decomposes in the primary bioaeration tank while fine biocleaning and separation of the sludge mixture take place in the secondary bioaeration unit. a vertical baffle separates the latter unit into the central settling area and the peripheral area of aeration. the activated sludge does not receive high working load in this unit that ensures stabilization of the excessive sludge. both primary and secondary bioaeration tanks are equipped with highly effective stream aerators, and the atmosphere air is being actively captured by the working liquid stream, which results in its good saturation. the near-bottom activated sludge mixture is pumped out from the bioaeration unit and used as the working liquid for biocleaning. an additional foamed polystyrene psv filter equipped with the grains ranged from 8 to 20 mm is used for the wastewater fine cleaning. finally, the solution of sodium hypochlorite is used for the bacterial decontamination of the wastewater. this reagent is added directly to the decontamination tank and its daily consumption is about 3,5 l/day (pure chlorine content is 170 g/l). the stabilized excessive activated sludge is collected from the secondary bioaeration tank and then returned to the receiving chamber of wastewater treatment station. this solution is effective for biocoagulation of the mechanical pollution and its better settling. then excessive sludge is filtered out in the settler-flotator together with the rest of the coarse dispersed particles. this equipment produces about 4,2 m3/day of the sediments and 0,2 m3/day of the floatation sludge. these components are being removed from the settler-flotator and sent for dehydration to the sludge storage area. the treated wastewater is discharged to a small pool connected to the nameless stream. the projected parameters of the treated wastewater quality are as follows: sediments – under 6,0 mg/l; bod5 – 6,0 mg/l; ammonia form of nitrogen – 0,5 mg/l; nitrites – 0,08 mg/l; nitrates – 40 mg/l; solid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 222 residue – 1000 mg/l; sulfates – 100 mg/l; phosphates – 0,17 mg/l and chlorides – 300 mg/l (see table 1, column 4). however, our results of the wastewater quality control prove that the actual quality parameters are much worse than the planned values (see table 1, column 5). data in table 1 show that the wastewater treatment equipment is far from its full efficiency and this technology needs in serious improvement and modification. table 1 some parameters of wastewater quality № pollution agent concentration, mg/l before treatment after treatment projected values actual values of the treated wastewater expected values after implementation of our suggestions 1 рн 4.4 – 5.2 6.5 – 8.5 5.8 6.5 – 8.5 2 sediments 853.0 6.0 54.3 15.0 3 bod5 3562 6.0 98,6 15.0 4 cod 8480.0 30.0 306.0 80.0 5 nitrogen (ammonia form) 3.2 0.5 1.8 0.5 6 nitrites 0.007 0.08 0 0.08 7 nitrates 5.48 40.0 3.48 40.0 8 solid residue 3226.0 1000.0 846.0 <1000 9 sulphates 180.0 100.0 168.3 <500 10 phosphates 0.625 0.17 5.8 0.5 – 1.0 11 chlorides 629.0 300.0 172.0 <350 3. results and discussion in our opinion, the realization of the current wastewater treatment technology at this juice making factory is poor because it was initially planned and designed for meat processing factories wastewater containing much more easily oxidizable components, biogenic elements (aminoacids, proteins, etc.) and with substantially milder ph (choban, winkler, 2008, zapolsky et al, 2000). another wastewater treatment technology providing discharge of the effluents with some amount of the added chlorella to the landfill areas seems more effective because this alga is capable to ensure biodegradation of the hardly oxidizable organics even in the acid solutions. the efficiency of this technology for the high-strength acid wastewater from wine producing factories has been reported by shepherd et al (2001). on the other hand, complete renovation of the existing wastewater treatment equipment and technology would require very serious investments and does not seem feasible. since similar situation is typical for many other juice making factories in ukraine and some other countries, it is important to pay attention to possible ways of improvement of the wastewater treatment using the existing equipment and technology. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 223 therefore, the following actions can be suggested to ensure the wastewater treatment improvement without major equipment renovation: 1. an equalizing tank should be added and used in the wastewater treatment; 2. neutralization agents dosage should be automatically monitored and controlled; 3. the active sludge concentration should also be controlled automatically. it is proposed to rise the activated sludge concentration at the biocleaning stage up to 4-6 g/l instead of the actual value 2,4 g/l. this suggestion is supported by the fact of better decontamination from the organic pollution performed by more concentrated sludge. bod and cod values would decrease as a result of this operation as well as the ammonia nitrogen concentration (in case of active propagation of nitromonads). however, some additional amounts of the biogenic elements should be provided by adding superphosphate and the ammonia water to ensure additional activated sludge propagation. on the other hand, excessive alkalization of the effluents disturbs normal development of the sludge (zapolsky et al, 2000) and should be avoided. acid reaction of the wastewater causes swelling of the sludge, which slips out from the secondary settlers and results in additional secondary mechanical pollution of the treated wastewater. so, the wastewater ph should be kept within the range 6,5-8,5 where the sludge is still active and can be effectively deposited under such values. badly controlled mechanical dosage of the reagents often brings ph outside of this range with either passivation or swelling of the sludge. an accurate automatic reagents dosage and/or installation of the equalizing tank can mitigate this problem and normalize conditions of the active sludge propagation. 4. conclusion the general assessment of the results of the above mentioned steps realization is shown in table 1 (column 6). it can be seen that the most dangerous parameters would drop after implementation of our suggestions while the increase of some other parameters (solid residues, sulphates content) would not bring them above of the maximum permissible levels. it should also be emphasized that the ammonia nitrogen content decreases too and this causes better protection of the natural water bodies from eutheriphication. mathematical simulation of changes in the receiving water body proved that it would remain conforming to the fish-farming class even receiving discharged wastewater treated in the proposed way. 5. references [1] mcdougall f. r., white p. r, franke m. and hindle p. integrated solid waste management: a life cycle inventory, blackwell publishing company, oxford, uk. doi: 10.1002/9780470999677, p. 33-85, (2007). [2] troschinetz a. m., mihelcic j. r. sustainable recycling of municipal solid waste in developing countries, waste management. 29, 915-923, (2009). [3] williams p. t. waste treatment and disposal, john wiley & sons, ltd, chichester, uk. doi: 10.1002/0470012668, 375 p, (2005). [4] reinhart d. r. and al-yousufi a. b. the impact of leachate recirculation on municipal solid waste landfill operating characteristics, waste manag. res. 14, 337-346, doi: 10.1177/0734242x9601400402, (1996). [5] korte f. (ed). environmental chemistry, mir, moscow. 396 p, (1996). [6] stolberg f. (ed). municipal ecology, kharkiv, 560 p, (2000). [7] choban a. and winkler i. chemical and ecological problems of small reservoirs at designing of wastewater treatment installafood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 alla chobsan, igor winkler, a case study of the juice-making factory wastewater treatment and possible ways of its optimization, food and environment safety, volume xii, issue 3 – 2013, pag. 218 224 224 tions. in: dangerous pollupants (xenobitics) in urban water cycle, hlavinek p., bonacci o., marsalek j., mahrikova i. (eds), springer, the netherlands, 257-265, (2008). [8] choban a. and winkler i. featherprocessing wastewater: composition, influence on the natural water objects and decontamination technologies. croatian journal of food technology, biotechnology and nutrition. 6(12), 58-62, (2011). [9] choban a, winkler i and rogozynskyi m. environmental safety analysis of the projected municipal landfill area in a small city in ukraine. journal of food and environment safety of the suceava university. 11(3), 47-51, (2012). [10] choban a. and winkler i. juice making wastewater treatment problems. noosfera. 6-7, 211-212, (2012). [11] dmitrieva o. o., kalashnikova v. o. and koldoba i. v. wastewater treatment in cities of ukraine and possible ways of its enhancing. environment protection and life safety, 3 (3), 63 – 68, (2003). [12] el-kamah h., tawfik a., mahmoud m and abdel-halim h. treatment of high strength wastewater from fruit juice industry using integrated anaerobic/aerobic system. desalination, 253, 158-163 (doi: http://dx.doi.org/10.1016/j.desal.2009.11.013), (2010). [13] mosse, k. p. m., patti, a. f., christen, e. w. and cavagnaro, t. r. review: winery wastewater quality and treatment options in australia. australian journal of grape and wine research, 17, 111–122. (doi: 10.1111/j.1755-0238.2011.00132.x), (2011). [14] pant d. and adholeya a. biological approaches for treatment of distillery wastewater: a review. bioresource technology, 98, 2321-2334, (2007). [15] progress and challenges on water and health: the role of the protocol on water and health (2009) world health organization. 2010, copenhagen, denmark. [16] shepherd h. l., grismer m. e. and tchobanoglous g. treatment of highstrength winery wastewater using a subsurfaceflow constructed wetland. water environment research, 73, 394-403, (2001). [17] zapolsky n. a., mishkovaklimenko n. a., astrelin i. m., brik m. t., gvozdyak p. i. and knyazkova t. v. physico-chemical principles of the wastewater treatment technologies. libra, kiev, (2000). звіт з ндр 29-81 за 2007 – 2009 р 101 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2-2016, pag. 101 107 performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient waste water – simulation with asal1 model *aleksandar slavov1 department environmental engineering university of food technologies, plovdiv, bulgaria aleksander_slavov@yahoo.com *corresponding author received april 13th 2016, accepted 24th may 2016 abstract: based on modified ludzack-ettinger process, the performance of biological complete-mix and plug-flow units are compared during simulative treatment of low loaded nitrogen deficient wastewater. bod-based model asal1 is applied. different variants of nitrogen deficiency, anoxicaerobic volume distributions, as well as diverse ssvi3.5 are manipulated. it is shown that plug-flow reactors give better effluent quality over complete-mix systems in relation to indicators: total nitrogen, total bod and total suspended solids. nitrogen removal is primarily diminished with the increase of anoxic against aerobic zone, while ssvi3.5 up to 200 ml/g has a less pronounced effect on this parameter. however,the optimal nutrient composition gives the lowest nitrogen in the outcome stream – 95.53 % for plug-flow and 94.75 % for complete-mix series. keywords: biological nitrogen removal, complete-mix, plug-flow, stoat, asal1 1.introduction elevated amounts of nitrogen compounds in wastewaters have harmful influence over the life in aquatic ecosystems. to meet water quality standards different methods for nitrogen removal are applied [1,2]. common technologies include activated sludge processes with various nitrification and denitrification steps realized as complete-mix or plug-flow reactors [3,4,5]. one of the most widespread systems is modified ludzack-ettinger process where nitrogen removal is carried out in pre-anoxic and aerobic zones without the necessity of easily biodegradable carbon supplementation [6]. nevertheless beneficial effects of biological nitrogen elimination, microorganisms involved in activated sludge have special requirements on the basis of wastewater composition. proper biomass development and effective contaminant elimination requires the bod:n:p ratio in the secondary influent to be 100:5:1 [7]. however, many sewage and industrial waste streams are below such ratio, including food production ones with high nitrogen deficiency – brewery, fruit processing, beverage [8,9,10], which can lead to operational problems and low quality effluents [11]. laboratory investigations of many different conditions on the behavior of wastewater treatment plant during purification of high bod:n containing wastewater is an expensive and time-consuming work. it can be realized considerably faster with the application of mathematical modeling of biological nitrogen removal [12], where diverse initial conditions can be applied http://www.fia.usv.ro/fiajournal mailto:aleksander_slavov@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 102 within models for various design options and analyses. the aim of this work is to simulate and compare the performance of complete-mix and plug-flow systems in the treatment of low loaded wastewater with diverse amounts of nitrogen deficiency. nomenclature bod – biological oxygen demand mlss – mixed liquor suspended solids ssvi3.5 – stirred sludge volume index at mlss 3500 mg/dm3 wrc – independent centre of excellence for innovation and growth 2.methods 2.1. biological unit description the biological wastewater treatment unit is designed graphically in the modeling and simulation environment stoat® 5.0 [13] to purify wastewater with a constant flowrate of 2400 m3/d (fig. 1). figure 1. schematic representation of biological wastewater treatment unit in stoat®. it consists of a biological activated sludge reactor with a total working volume of 1000 m3 and a secondary sedimentation tank with a surface area of 500 m2. initial conditions for activated sludge reactor and secondary sedimentation tank are represented in table 1. activated sludge reactor is divided into anoxic and aerobic zones (a/o) on the basis of modified ludzack-ettinger process. three a/o percent volume ratios are investigated: 25/75, 50/50 and 75/25. sludge wastage and internal cycle flowrates are 5 m3/h and 500 m3/h, respectively. these parameters are maintained constant in all simulation variants. table 1. initial conditions in: a) activated sludge reactor; b) secondary sedimentation tank. a) parameter anoxic stage aerobic stage soluble bod, mg/dm3 o2 5 5 nh3, mg/dm3 1 1 dissolved oxygen, mg/dm3 0 2 mlss, mg/dm3 3000 3000 viable autotrophs, mg/dm3 100 100 viable heterotrophs, mg/dm3 1000 1000 b) parameter stages 1-3 4-7 8 soluble bod, mg/dm3 o2 5 5 5 nh3, mg/dm3 1 1 1 dissolved o2, mg/dm3 2 2 2 mlss, mg/dm3 0 300 6000 viable autotrophs, mg/dm3 0 100 2000 viable heterotrophs, mg/dm3 0 10 200 2.2. wastewater treatment plant modeling wrc’s activated sludge model: asal1 is chosen as a model for the description of bacterial growth and decay processes, both of autotrophs and heterotrophs. it is bodbased and is recommended for normal activated sludge processes with hydraulic retention time higher than 4 h and nh3 concentration lower than 40 mg/dm3. the model is applied for complete-mix and plug-flow reactors according to author’s instructions [13]. asal1 works with the respective settling tank model: ssed1. model asal1 doesn’t include the growth and decay of filamentous microorganisms, solely, although these heterotrophs directly affect development of activated sludge with good settling properties in high bod/n ratios. activated sludge bulking as a result of nitrogen deficite in wastewater is achieved with the variation in ssvi3.5, being higher than 150 ml/g. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 103 2.3. influent characterization the wastewater characteristics include bod-based profile for constant influent pattern, included in stoat®. influent indicator values are pointed at table 2. table 2. influent characteristics parameter value flow, m3/h 100 temperature, °c 15 soluble bod, mg/dm3 o2 150 particulate bod, mg/ dm3 o2 90 volatile solids, mg/dm3 180 non-volatile solids, mg/dm3 60 nh3, mg/dm3 vary, according to bod/n ratio assuming that free nh3 in the influent at ph 7 and temperature 15° c is negligible small (less than 0.0027 mole/dm3), nitrogen concentration is calculated on total bod basis for bod/n ratio: 100/5, 100/3 and 100/1, respectively, in the form of nh4+-n. all other parameters, included in the model, are kept at default values. 3. results and discussion. in a set of 48 h simulations the ability of complete-mix and plug-flow activated sludge units to remove nitrogen from low nutrient wastewater is investigated. in addition, 25/75, 50/50 and 75/25 a/o volume distributions and different ssvi3.5 values are analysed. summarized results are presented in fig. 2a, fig. 2b and fig. 2c. model data show high purification levels over 70 %. close results are obtained for complete-mix and plug-flow reactors in all bod/n ratios. however, plug-flow units show slightly better performance over complete-mix series which is probably due to the reactor differences in equation for steady state effluent concentration, described by [14]. exceptions are at bod/n ratio: 100/3 and at bod/n ratio: 100/5, both with anoxic/aerobic volume relation: 75/25 in the range of 2.5 and 5.9 %, respectively higher than other variants (fig. 2b, fig. 2c). low effluent nitrogen is a result of consecutive nh3 oxidation and no3¯ reduction from wastewater, which can be achieved with proper anoxic/aerobic volume calculations. results obtained show that lower or equal dimensions of anoxic basin towards aerobic give better performance of biological nitrogen removal unit, notwithstanding the reactor type. this counteracts with the conclusions, received by [15] and [16], where optimal a/o ratios are 0.75 and 0.64, respectively. however, the mentioned authors analyse processes with sufficient amount of influent nitrogen. at analysed bod/n ratios exceptions are also observed, but the differences are less of the percent (fig. 2a, fig. 2b, fig. 2c). switching to bigger anoxic vs. aerobic zone reflects over nh3 transformation into no3¯, which rises total n in the effluent. this process is apparent at the highest bod/n ratio with differences over 16 % for complete-mix and more than 22 % for plug-flow reactors against other two a/o zones (fig. 2c). to simulate the effect of sludge bulking ssvi3.5 values of 150, 200 and 220 ml/g at which effluents fit standards for total nitrogen, total bod and total suspended solids are explored (fig. 2a, fig. 2b). obtained results show that development of filamentous organisms in large numbers doesn’t reflect nitrogen removal capacity of the system for the investigated ssvi3.5 interval and period of simulations. the highest nitrogen removal is achieved at optimal bod/n ratio 100/5 and equal a/o volumes – 95.53 % for plug-flow reactor and 94.75 % for complete-mix unit (fig. 2c). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 104 a) b) c) figure 2. total nitrogen removal in biological complete-mix and plug-flow unit from wastewater with bod/n ratio: a) 100/1; b) 100/3; c) 100/5. 8 9 .7 5 8 9 .8 1 8 7 .8 2 9 0 .5 5 9 0 .7 3 8 8 .9 8 8 9 .5 6 8 9 .9 9 8 7 .8 2 9 0 .5 5 9 0 .7 3 8 8 .9 8 8 9 .1 5 8 9 .3 9 8 7 .4 1 9 0 .4 5 9 0 .7 3 8 8 .9 8 0.00 20.00 40.00 60.00 80.00 100.00 a/o = 25/75 completemix a/o = 50/50 completemix a/o = 75/25 completemix a/o = 25/75 plug-flow a/o = 50/50 plug-flow a/o = 75/25 plug-flow t o ta l n r e m o v a l, % anoxic/aerobic zone volume distribution, % nitrogen removal at bod/n ratio: 100/1 ssvi3.5 = 150 ml/g ssvi3.5 = 200 ml/g ssvi3.5 = 220 ml/g 9 3 .8 3 9 3 .9 3 8 7 .9 4 9 4 .5 6 9 4 .7 5 8 4 .6 1 9 3 .7 6 9 3 .8 9 8 7 .9 1 9 4 .5 0 9 4 .7 5 8 5 .1 2 9 3 .5 3 9 3 .6 5 8 7 .9 9 9 4 .4 0 9 4 .7 2 8 5 .7 0 0.00 20.00 40.00 60.00 80.00 100.00 a/o = 25/75 completemix a/o = 50/50 completemix a/o = 75/25 completemix a/o = 25/75 plug-flow a/o = 50/50 plug-flow a/o = 75/25 plug-flow t o ta l n r e m o v a l, % anoxic/aerobic zone volume distribution, % nitrogen removal at bod/n ratio: 100/3 ssvi3.5 = 150 ml/g ssvi3.5 = 200 ml/g ssvi3.5 = 220 ml/g 9 4 .6 4 9 5 .3 4 9 4 .7 5 9 5 .5 3 7 8 .0 2 7 3 .1 7 0.00 20.00 40.00 60.00 80.00 100.00 complete-mix plug-flow t o ta l n r e m o v a l, % anoxic/aerobic zone volume distribution, % nitrogen removal at bod/n ratio: 100/5 a/o = 25/75 a/o = 50/50 a/o = 75/25 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 105 at bod/n ratio 100/3 the best nitrogen reduction is the same for plugflow reactor with a/o ratio 50/50 at ssvi3.5 from 150 to 200 ml/g – 94.75 %. complete-mix system gives better performance at equivalent a/o volume and ssvi3.5 200 ml/g – 93.89 % (fig. 2b). at bod/n ratio 100/1 the lowest nitrogen values are obtained with identical a/o ratios at all three ssvi3.5 – 90.73 %. complete-mix type achieves 89.99 % at same a/o distribution and ssvi3.5 200 ml/g (fig. 2a). figure 3. total nitrogen in the effluent at different reactor system and bod/n ratio. figure 4. total bod in the effluent at different reactor system and bod/n ratio. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 1 6 11 16 21 26 31 36 41 46 t o ta l n it ro g e n , m g /d m 3 time, h total nitrogen at different bod/n ratio complete-mix and bod/n: 100/5 plug-flow and bod/n: 100/5 complete-mix and bod/n: 100/3 0.00 5.00 10.00 15.00 20.00 25.00 1 6 11 16 21 26 31 36 41 46 t o ta l b o d , m g /d m 3 o 2 time, h total bod at different bod/n ratio complete-mix and bod/n: 100/5 plug-flow and bod/n: 100/5 complete-mix and bod/n: 100/3 plug-flow and bod/n: 100/3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 106 figure 5. total suspended solids in the effluent at different reactor system and bod/n ratio. a comparison between 48 h effluent profiles is done for the best obtained values from complete-mix and plug-flow reactors for all bod/n ratios. the obtained results are represented in fig. 3, fig. 4 and fig. 5. analysed indicators: total nitrogen, total bod and total suspended solids from all variants cover european standards for effluent quality from wastewater treatment plants – 2 mg/l, 25 mg/l o2 and 35 mg/l, respectively [17]. furthemore, total nitrogen concentration in the purified wastewater is less than 0.7 mg/l (fig. 3). nevertheless, it should be taken into account that very low effluent nitrogen under 0.5 mg/l can provoke additional sludge bulking even in well performed wastewater treatment plant [18]. better performance of plug-flow reactors over complete-mix ones have been achieved in all simulation series, which confirms the comparative statements of the two systems, summarized by [11]. stationary conditions in total nitrogen removal are attained after 21st h. only complete-mix system for bod/n: 100/5 reaches earlier equilibrium – after 11th h (fig. 3). obtained result may be explained with optimal nutrient composition of the wastewater. plug-flow systems don’t differ too much in their effluent bods. faster substrate biodegradation by heterotrophs corresponds with earlier bod balance in the outgoing stream, gained between 16th and 21st h (fig. 4). complete-mix units don’t reach dynamic equilibrium for 48 h. only the effluent at bod/n: 100/1 has two times higher bod loading at 7th h in comparison with the other ideal-mixed reactors. the result is connected with the bulking sludge which increases particular bod, giving higher total bod in the effluent (fig. 2a, fig. 4). complete-mix and plug-flow series for bod/n ratios: 100/5 and 100/3 are equal in their effluent profiles for total suspended solids. average balance is reached around 21th h. at bod/n: 100/1 the plug-flow unit follows the same behavior. in contrast, bigger ssvi3.5 overloads the complete-mix system – about 4 h total suspended solids are over permissible for the effluent quality – 35 mg/dm3 (fig. 5). 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 1 6 11 16 21 26 31 36 41 46 t o ta l s u sp e n d e d s o li d s, m g /d m 3 time, h total suspended solids at different bod/n ratio complete-mix and bod/n: 100/5 plug-flow and bod/n: 100/5 complete-mix and bod/n: 100/3 plug-flow and bod/n: 100/3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 aleksandar slavov, performance of complete-mix and plug-flow systems during treatment of low loaded nitrogen deficient wastewater – simulation with asal1 model, food and environment safety, volume xv, issue 2 – 2016, pag. 101 – 107 107 4. conclusion as a result of investigated analysеs on the biological nitrogen removal in completemix and plug-flow reactors the following more important conclusions can be made: 1. plug-flow reactors give better performance over complete-mix units with lower effluent concentrations of total nitrogen, total bod and total suspended solids; 2. better nitrogen removal can be achieved if anoxic basin is smaller or equal to aerobic; 3. rising ssvi3.5 up to 200 ml/g doesn’t affect nitrogen elimination from wastewater; 4. the best nitrogen removal is reached in a plug-flow reactor with equal anoxic and aerobic zones, at optimal nutrient composition bod:n = 100:5 – 95.53 %. 5. references [1]. luo x., yan q., wang c., luo c., zhou n., jian c., treatment of ammonia nitrogen wastewater in low concentration by twostage ozonation, international journal of environmental research and public health, 12: 1197511987, (2015) [2]. breisha g. z., bio-removal of nitrogen from wastewaters – a review, nature and science, 8 (12): 210-228, (2010) [3]. zhu g., peng y., wang s., wu s., ma b., effect of influent flow rate distribution on the performance of step-feed biological nitrogen removal process, chemical engineering journal, 131: 319-328, (2007) [4]. ma b., wang s., cao s., miao y., jia f., du r., peng y., biological nitrogen removal from sewage via anammox: recent advances, bioresource technology, 200: 981-990, (2016) [5]. liotta f., chatellier p., esposito g., fabbricino m., van hullebusch e. d., lens p. n. l., hydrodynamic mathematical modelling of aerobic plug flow and nonideal flow reactors: a critical and historical review, critical reviews in environmental science and technology, 44: 2642-2673, (2014) [6]. jeyanayagam s., true confessions of the biological nutrient removal process, florida water resources journal, january: 37-46, (2005) [7]. marshall r., best management practices guide for nutrient management in effluent treatment, marshall environmental training and consulting: 8, (2008) [8]. patterson r. a., nitrogen in wastewater and its role in constraining on-site planning, in future directions for on-site systems: best management practice. proceedings of on-site ’03 conference by patterson, r.a. and jones, m. j. (eds). held at university of new england, armidale 30th september to 2nd october 2003. published by lanfax laboratories armidale. isbn 0-9579438-1-4: 313-320, (2003) [9]. fatone f., bolzonella d., battistoni p., cecchi f., removal of nutrients and micropollutants treating low loaded wastewaters in a membrane bioreactor operating the automatic alternate-cycles process, desalination, 183: 395-405, (2005) [10]. davies p. s., the biological basis of wastewater treatment, strathkelvin instruments ltd: 3, (2005) [11]. tchobanoglous g., burton f. l., stensel h. d., wastewater engineering: treatment and reuse (4th ed.), metcalf & eddy inc, boston: mcgraw-hill, eisbn 0-07-112250-8, (2003) [12]. vandekerckhove a., moerman w., van hulle s. w. h., full-scale modeling of a food industry wastewater treatment plant in view of process upgrade. chemical engineering journal, 135: 185-194, (2008) [13]. stoat® 5.0. computer software, retrieved from http://www.wrcplc.co.uk/ps-stoat, (2014) [14]. hartley k., tuning biological nutrient removal plants, iwa publishing, eisbn 978-178040-4837, (2013) [15]. chen x., yuan l., lv j., nie k., influence of anoxic to aerobic volume ratio on sludge settleability and bacterial community structure in a denitrifying – nitrifying activated sludge system, desalination and water treatment, 56 (7): 114, (2014) [16]. luo y., zhang x., zhang h., guo z., influence of volume ratio of anoxic zone to aerobic zone on denitrifying phosphorus removal in carrousel 2000 oxidation ditch process, china water and wastewater issn 1000-4602, 7: 22-24, (2015) [17]. european economic community, council directive 91/271/eec: urban wastewater treatment, official journal of the european union, 135: 40-52, (1991) [18]. lever m., operator’s guide to activated sludge bulking, 35th annual qld water industry workshop community sports centre, cq university – rockhampton, australia: 9, (2010) http://www.wrcplc.co.uk/ps-stoat 259 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 32016, pag. 259 265 analysis of physical parameters of natural juices mihaela jarcău1, *maria poroch-seriţan1 1faculty of food engineering, stefan cel mare university of suceava, adressuniversitatii 13, 720229 suceava, romania, mihaela.jarcau @ fia.usv.ro, mariap@ fia.usv.ro *corresponding author received august 7th 2016, accepted september 27th 2016 abstract: the purpose of this paper was to investigate the change in density, electrical conductivity, ph, refractive index, dry soluble solids, as well as the parameters specific to the color of natural juices stored at + 4°c and normal atmospheric pressure in two types of containers. the physical parameters were determined immediately after the obtaining of juice, at 2 days and 5 days of storage respectively. from the analysis of experimental results, there is an increase in density, in all samples, while the ph changes only slightly for the orange juice. storage at temperatures of + 4°c reduces the risk of contamination and decreases speed of biochemical changes, but it is not sufficient to guarantee the nutritive and organoleptic qualities of the juice and its acceptability by the consumer. keywords: electrical conductivity, ph, refractive index, color, juice. 1. introduction physical and chemical analyzes of fruit and vegetables juices (density, electrical conductivity, ph, refractive index, dry soluble solids), and the parameters specific for the color, represent investigating methods of the beverage quality examined to storage at the temperature of 0 +4°c and normal atmospheric pressure in various kinds of container on a period of up to five days. knowing the physical parameters is very important in determining the optimum storage conditions of fruit and vegetables juices, the sizing of storage and the conditioning installations. specific parameter values of fruit and vegetables juices and concentrates are useful in preparing thermal balance. current requirements of consumers are turning to juices of superior quality that are cheap, with nutritional value, flavor, texture and a color corresponding to fresh fruits without chemical preservatives and yet wholesome and safe. keeping juices at low temperatures is carried out in order to preserve them (prevents fermentations). refrigeration is used to temperatures between -5ºc and +5ºc without changing the state of aggregation and the freezing temperatures are used between -10ºc and -30ºc [1-2]. using artificial cold for the processing, storage and marketing of perishable foods is a method widely used in almost all branches of food industry. due to its preservative and inhibitory action on agents that cause degradation, cold artificial processes used in refrigeration, freezing or freeze-drying [1]. refrigeration operation comprises two phases, the actual refrigeration (cooling from the initial temperature to the storage temperature) and keeping the refrigeration. it has been found that by lowering the temperature of juice from 25 30ºc to 0ºc, the preservation period is increased to approximately 15 times [2]. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 260 fruits are particularly valuable for the content of carbohydrates, vitamins, minerals, pigments and other biologically active substances. fruits have an alkalizing effect in the body (like vegetables) and they represent the food that influence the health of the organism and reduce the risk of chronic diseases. fruits rich in carbohydrates are contraindicated for diabetics and overweight people, but also healthy people with normal weight should not consume excess. they are suitable for children and athletes who require immediate energy. vegetables are an important source of vitamins, minerals, carbohydrates (~ 5% for leafy vegetables and 10 12% for root vegetables, melons, solano fruiting), fiber (1 2%), and other biologically active substances [3]. vegetable juices with pulp can be used to feed infants from the age of 4 months as excellent sources of minerals and vitamins. the most commonly used juices are: carrots, tomatoes and beetroot, alone or in combination with each other and with other vegetable juices: parsley, spinach, celery [4]. the researches presented in this paper followed the variation of density, electrical conductivity, ph, refractive index, and the parameters specific for the color of orange (citrus sinensis), lemon (citrus limon), kiwi (actinidia chinensis), carrot (daucus carota) and beet (beta vulgaris) juices stored at +4ºc and normal atmospheric pressure in two types of container (a brown bottle and polyethylene terephthalate, respectively) at 2 days and 5 days of storage respectively. the main objective of this survey is to investigate the change in density, electrical conductivity, ph, refractive index, dry soluble solids, as well as the parameters specific for the color of natural juices stored at +4°c and normal atmospheric pressure in two types of container. 2. matherials and methods 2.1. samples and equipment juices were prepared from fruits and vegetables purchased from the local market in suceava, romania, using a vital juicer and they were divided into 25 samples, from which 5 were analyzed immediately and the remaining 20 samples were stored individual in brown glass containers respectively polyethylene terephthalate (pet) opaque in the cooler at +4ºc and normal atmospheric pressure. samples of fruit and vegetables are coded according to table 1. equipment used was from the food safety research center of the faculty of food engineering, stefan cel mare university of suceava, romania. table 1. coding the juice samples studied type of juice coding fresh juice juice stored 2 days juice stored 5 days brown glass bottle pet container brown glass bottle pet container orange (citrus sinensis) 1 2 3 4 5 lemon (citrus limon) 1 2 3 4 5 kiwi (actinidia chinensis) 1 2 3 4 5 carrot (daucus carota) 1 2 3 4 5 beet (beta vulgaris) 1 2 3 4 5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 261 2.2. determination of density density of juices was calculated by using density determination kit (xa, radwag) of analytical balance (partner xa 160). 2.3. determination of conductivity and ph conductivity of juices were determined by using a conductivity meter (accumet xl30, fisher scientific) and the ph of samples was measured with a calibrated digital ph meter (hq11d ph by hach), by the glass electrode being immersed in samples. 2.4. refractive index and % solids measurement refractive index (nd) and % solids in juices were measurement by using abbe refractometer (leica mark ii plus). the dry soluble solids content (% solids) of a sample is estimated from its refractive index, with reference to the refractive index of a pure sugar solution. the refractive index is proportional to the solution concentration (following the theory of lorentz and lorenz). in fruit juices the refractive index is therefore dependent upon sugar concentration and also upon the concentration of other soluble materials (organic acids, minerals, amino acids etc.). 2.5. color measurement the color measurement involves the determining of the reflection spectrum of a sample and applying it to a standard illuminant. the amount of light energy the sample reflects is manipulated and reduced to tristimulus values x, y and z. these values correspond to the physiological response of the three types of color receptors in the human eye. x, y and z values are combined into the uniform color space values such as l*, a* and b* [5-6]. the chroma, considered a quantitative attribute of coloring, is used to determine the difference degree of a hue in comparison with a grey color of the same luminosity. the description of a color using the hue (h*) attribute refers to how humans perceive an object’s color red, orange, green, blue, etc. a 0º or 360 º angles represent the red hue, while angles of 90º, 180º and 270º represent yellow, green and blue hues [7-8]. δe* – represents the color difference magnitude between the value of the initial color and the value of the final color and gives the distance between the points representing the color, being calculated according to the equation: 2*2*2** lbae  (1) within the study, the hr 4000 cguv-nir spectrometer (ocean optics inc., dunedin, fl) which is based on the reflection property of samples was used. in this respect, a scanning with 0.025nm resolution has been carried out for the entire wavelength spectrum ranging from 200 up to 1100 nm. a tungsten halogen light source (uv-vis-nir light source dh-2000, mikropack) was used in this study. the experimental data was displayed and stored using the operating software ooi base32 including ooicolor from ocean optics. the light from light source dh-2000 was focused to the sample through an array of optical fibres (qr400-7-uv/bx, ocean optics inc., dunedin, fl). reflectivity was measured as against a reference standard – wsdiffuse reflectance standard [5-6]. 2.6. statistical analysis all the assays were conducted at least in duplicate, and the results were expressed as the mean ± standard deviation. statistical significance was accepted at p < 0.05. 3. results and discussion according to figure 1 a), from the analysis of experimental results, there is an increase in density over time, for all samples. the highest increase was observed for beet, while the lowest variation was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 262 observed in carrot. also, analyzing the behavior depending upon the nature of juice container, it can be seen an increase in density for kiwi and lemon juice stored in a brown glass bottle compared to the pet container. for the beet juice there is a greater variation in sample density packaged in pet. a) b) c) d) e) figure 1. variation a) density, b) ph, c) conductivity, d) refractive index, e) dry soluble solids for the analyzed juice samples from the analysis of experimental results, according to figure 1 b), there is an increase in ph only for orange juice, while neither for the other juices is nor observed its variation neither is observed the ph dependence upon the nature of the container. there is a decrease in conductivity at the samples analyzed in time, from the analysis of experimental results, according to figure 1c). we can observe the biggest 0 0.2 0.4 0.6 0.8 1 1 2 3 4 5 d e n s it y [ g /m l] sample orange lemon kiwi carrot beet 0 1 2 3 4 5 6 7 8 1 2 3 4 5 p h sample orange lemon kiwi carrot beet 0 2 4 6 8 10 12 14 16 1 2 3 4 5 c o n d u c ti v it y [ u s /c m 2 ] sample orange lemon kiwi carrot beet 1.33 1.335 1.34 1.345 1.35 1.355 1.36 1 2 3 4 5 r e fr a c ti v e i n d e x sample orange lemon kiwi carrot beet 0 2 4 6 8 10 12 14 16 18 1 2 3 4 5 % s o li d s sample orange lemon kiwi carrot beet food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 263 variation in the carrot juice and beet juice and the least for kiwi. there are no changes in conductivity for samples 2 (juice stored 2 days in brown glass bottle) and 3 (juice stored 2 days in pet container) or samples 4 (juice stored 5 days in brown glass bottle) and 5 (juice stored 5 days in pet container) where we conclude that conductivity is not influenced by the container material. for orange and kiwi is irrelevant. many people consume fruit juices on a daily basis. fruit juices are a convenient way for people to receive the benefits of various fruits. however, they may also have high sugar content. although the sugar is natural, it may not be healthy in high quantities. it was suggested that too much sugar could pose harmful health effects, as people could develop diabetes, obesity, heart disease, and other complications from excess consumption [9]. a significant variation is found from the analysis of experimental results for beet, carrot and lemon juices and irrelevant for orange and kiwi juices, according to figure 1 d) and e), for the refractive index and dry soluble solids. in the food industry, colour is a quality attribute that affects consumers in terms of choice and food preferences. from the physical point of view, the colour is a numerical expression which is evaluated as the difference in colour against a known standard. instead, from the consumer's point of view, the colour is the first quality parameter and is even more important as it influences the acceptability or the unacceptability of the product. the appearance of the food, which is one of the most important sensory attribute of the food products, both fresh and processed is in close correlation with the visual appearance and hence the colour of the food. the consumer uses colour as a measure of food quality and associates it with other sensory attributes such as flavor and pigment content [7], [10]. food visual aspect has a strong influence on consumer’s opinion on food quality. color can be correlated with other quality attributes such as: sensory, nutritional and visual or non-visual defects and helps control the products’ quality. color is a perceptual phenomenon which depends on the observer and on the color observing conditions. l*, a*, b*, hue angle (h*) and chroma parameters for the samples subjected to the assay analysis were measured [6]. as it can be seen from figure 2 a), there is an increase for l* for the stored juices comparing to the fresh ones. a significant increase is found after 2-day storage of samples (2 and 3). considering that the a* parameter has positive values for red colors and negative values for green colors and the b* parameter has positive values for yellowish colors and negative values for bluish colors [8-9], it can be observed from figure 2 b) and figure 2 c) that all samples tend to modify their color (to become brownish). significant variations are observed in all the studied juices. analyzing the chroma parameters, figure 2 d) it can be observed a growth for juices stored for 2 days in both the glass bottle (samples 2), and the pet container (samples 3), and then a decrease after a storage for 5 days (samples 4 and 5). hue (h*) attribute, figure 2 e) presents negative values, which is consistent with the parameters a* and b* which indicate that the changes are perceptible by the human eye, and thus accepts the consumer to consume the product or not. in figure 2 f) it can be seen δe* for all the juice samples that represent the color difference magnitude between the value of the initial color and the value of the final color. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 264 a) b) c) d) e) f) figure 2. the variation in color parameters for the analyzed samples: a) l*, b) a*, c) b*, d) chroma, e) hue angle, f) δe* due to storage without preservatives biochemical changes occur that causes changes in the color and in the other physical parameters, regardless of the storage containers. 4. conclusion this paper presents an experimental study on the change of density, electrical conductivity, ph, refractive index, and on the parameters specific to the color of natural juices stored at a temperature of +40c and normal atmospheric pressure in two types of containers. from the analysis of experimental results, it results that there is an increase in density, in all samples, while the ph changes only slightly in orange juice. storage temperatures of +4ºc reduce the risk of contamination and 40 50 60 70 80 90 100 1 2 3 4 5 l * sample orange lemon kiwi carrot beet -50 -40 -30 -20 -10 0 10 20 1 2 3 4 5 a * sample orange lemon kiwi carrot beet -35 -30 -25 -20 -15 -10 -5 0 5 1 2 3 4 5 b * sample orange lemon kiwi carrot beet 0 10 20 30 40 50 60 1 2 3 4 5 c h ro m a sample orange lemon kiwi carrot beet -200 -150 -100 -50 0 50 1 2 3 4 5 h * sample orange lemon kiwi carrot beet 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 δ e * sample orange lemon kiwi carrot beet food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 mihaela jarcău, maria poroch-seriţan, analysis of physical parameters of natural juices, food and environment safety, volume xv, issue 3 – 2016, pag. 259 265 265 decrease the speed of biochemical changes, but this fcat is not sufficient to guarantee the nutritive and organoleptic qualities of the juice and its acceptability by the consumer. 5. references [1]. tofan i., tofan c., utilizarea frigului artificial la procesarea, depozitarea și comercializarea produselor alimentare perisabile, editura agir: bucurești, românia, (2002). [2]. glevitzky m., bogdan i., brusturean g. a., perju d., silaghiperju d., studiul procesului de inhibare a degradării sucurilor naturale supuse la tratament termic prin pasteurizare, revista de chimie, bucurești, 59, 5, 595 – 60, (2008). [3]. banu c., nour v., stoica a., bărascu e., alimentație pentru sănătate, editura asab, bucurești, românia, (2009). [4]. costin g.m., alimente pentru nutriție specială, editura academica, galați, românia, (2001). [5]. *** 2007, ocean optics, inc 2007 product catalog [6]. poroch – serițan m., popescu n. g., the relationship between nitrite concentration and colour parameters during storage of meat product summer sausages, food and environment safety, xv, 146 – 154, (2016). [7]. pathare p.b., opara u.l., al-said f. al-j., colour measurement and analysis in fresh and processed food, food bioprocess technol., 6, 36 – 60, (2013). [8]. *** 2007, a guide to understanding color communication, accesed in may 2014: [http://www.x_rite.com] © x-rite, incorporated [9]. serpen j. y., comparison of sugar content in bottled 100% fruit juice versus extracted juice of fresh fruit, food and nutrition sciences, 3, 1509 – 1513, (2012). [10]. mureșan a., curs 14, evaluarea culorii, information on: http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/ prof.%20dr.%20ing.%20augustin%20muresan/, (2013) 01-aug-2013. http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/prof.%20dr.%20ing.%20augustin%20muresan/ http://www.tex.tuiasi.ro/biblioteca/carti/cursuri/prof.%20dr.%20ing.%20augustin%20muresan/ activating the glutathione system of eisenia fetida during exposure to silt contaminated by different toxic compaunds 276 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv , issue 4 2016, pag. 276 283 ştefan cel mare university of suceava, romania volume , issue 12016, pag. ... ... activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge *nataliya mitina1, nina khromykh2, larisa shupranova2, inna zubareva1 1dnipropetrovsk chemical-technological state university, 8 gagarin avenue, dnipro, ukraine, natalimitina_68@mail.ru, 2oles gonchar dnipropetrovsk national university, 72 gagarin avenue, dnipro, ukraine, *corresponding author received 24th october 2016, accepted 23rd december 2016 abstract. in order to identify effective and inexpensive method of purifying wastewater sludge, we tested the hypothesis about the ability of earthworm eisenia fetida to detoxify the silt complex of different chemical components through the activation of glutathione system. in the laboratory conditions, earthworms were exposed to silt from the sedimentation tanks during 112 days in the boxes, while the control earthworms were on a sunflower husk substrate. glutathione s-transferase (gst) activity and reduced glutathione (gsh) content in coelomic fluid of earthworms were measured spectrophotometrically. the results revealed biomass of the experimental e. fetida worms was declined by 69% compared to control after the exposure. heavy metals content in the silt decreased in the range from 13% (for zinc and lead) to almost 100% in the case of cadmium compared to beginning of the experiment. the gst activity increased significantly (87% above the control level), as well as gsh content (36% above the control). induction of 4 new proteins biosynthesis was found by gel-electrophoresis exploration of coelomic fluid proteins of the worms exposed to contaminated silt. study results indicate a high level of toxicant-induced activation of the earthworm glutathione-dependent system, and confirmed the efficiency of e. fetida using for detoxification of silt from the sedimentation tanks in the case of complex contamination. key words: earthworm, wastewater sludge, heavy metals, detoxification, glutathione stransferase, glutathione. 1. introduction in ukraine, the annual accumulation of compacted sewage sludge (css) in the sedimentation tanks reaches 40 million tons, implying a need to occupy 120 hectares of natural land for storage silt on the drying beds and burning [1]. such actions ultimately lead to pollution of atmosphere, surface and ground water, soil, and vegetation, and thereby deteriorate the quality of human life. silt wastewater sludge utilization is also actual problem in many countries [2], [3], where the css application includes the addition to agricultural and other soils. silt wastewater sludge contains a high amount of various organic compounds [4], heavy metals and trace elements [5], so the pretreatment is required for the successful application of css [3]. one of the affordable and effective methods of silt sludge cleaning might be ensured by vermiculture, in particular eisenia fetida culture. the final product of processing is the vermicompost (or biohumus), which can be used as fertilizer, and for the soils restoration and cleaning from radionuclides, heavy metals and pesticide residues as well. it has been found [6] that earthworms can biohttp://www.fia.usv.ro/fiajournal mailto:natalimitina_68@mail.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 277 accumulate, biodegrade or biotransform the heavy metals and a wider range of other xenobiotics, such as herbicides [7] and veterinary drugs [8]. it was established [9], [10], that resistance of e. fetida to the toxins action has always been associated with activation of the metabolic defense including the glutathione-dependent system. in particular, the increase in glutathione-s-transferase (gst) activity was found towards the pesticides [7], [11] and heavy metals [12], [13]. on the other hand, the high inhibition of e. fetida gst activity was observed due to the action of carbamate pesticide carbaryl [14]. the goals of our work were (1) to evaluate the detoxifying ability of e. fetida in the case of integrated substrate contamination and (2) to identify the role of earthworm glutathione-dependent system in the substrate cleaning processes. 2. materials and methods the object of the study (vermiculture of earthworm eisenia fetida) was provided by the research institute “biotechnology” state university, dnipropetrovsk; specification 3336406.00 2–95. substrates for vermiculture were prepared on the basis of modified sunflower husk (sh, control); mixed husk of sunflower, rice and buckwheat (mh), and compacted sewage sludge (css) from the sedimentation tanks. each substrate was ground, and the fraction 200 – 500 microns was subjected to fermentation in the clamps 50 – 60 cm high, while maintaining 70 – 80% humidity. process of substrates fermentation provided by microflora activity, lasted 14 days at a daily stirring. the fermented substrates were used for vermiculture, and the e. fetida worms were placed in substrate with layer thickness 30 cm at a density of 5 – 10 thousand of the earthworms per square meter. the following conditions been complied during the experiment: ph level of substrate was in the range 6.5 – 7.5, humidity reached 70 – 80%, and the temperature fluctuation was within 20 – 25° c. for each substrate, three replicates were prepared. the worms was selected at 112 day and placed on wet filter paper to remove the gut content. after 3 days with daily replacement of filter paper coelomic fluid was extracted, frozen, and stored at – 20° c. the average biomass weight of e. fetida from each substrate was determined by weighing a group on analytical scales. earthworm’s weight measurement was carried out during the whole experiment, selecting samples every 14 days. parameters of growth inhibition for the various exposure periods were computed according to [15]. the content of heavy metals was determined by atomic absorption method in the substrates before placing of earthworms and after their removal at the end of 112 days. activity of glutathione-s-transferase (gst, ec 2.5.1.18) in the coelomic fluid was measured according to the method described by habig et al. [16] and saint-denis et al. [9] with 1-chloro2,4-dinitro-benzene (cdnb) as a substrate. the assay mixture (100 μl of 0.1 m/l tris buffer, ph 8.0, 100 μl gsh, and 200 μl sample) was incubated during 10 min at 30° c, and the reaction was initiated by addition of 100 μl cdnb. optical density change was detected at 340 nm during four minutes, and gst activity was expressed in nmol cdnb sec-1ml-1 (nkatal ml-1). the reduced glutathione (gsh) content determination in the coelomic fluid was conducted by the method of anderson [17], based on spectrophotometric food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 278 registration of the glutathione reaction with the ellman’s reagent (dithiobis-2nitrodenzoic acid). optical density of assay mixture (110 μl of 0.1 m/l kphosphate buffer contained edta, and 300 μl of sample) was detected at 412 nm after the addition of ellman’s reagent. gsh content was calculated by using calibration graph, and expressed in nmol gsh ml-1. protein spectrum study of e. fetida coelomic fluid was carried out by a modified sds-page method of laemmly [18] in 7.5 – 17.5% gradient acrylamide gel. protein samples (50 g ml-1) were boiled for 5 min at 95° c and applied to the respective wells in the gel. analysis was run at 20 ma in the electrophoresis unit ve-20 (russia). protein standards were run in parallel with the samples. bands were stained with 0.25% coomassie brilliant r-250. all measurements were performed in triplicate at least. data present mean values and standard deviation (m±sd). significance of the differences between control and treated samples was estimated with student’s t-test (p < 0.05). fisher's test was used for analysis of variances, fcrit.(0.05; 4; 4) = 6.39. 3. results and discussion the biomass growth of e. fetida exposed to the sunflower husk, as well as to the mixed husk, was a positive trend during the experiment, whereas decrease in weight observed in the case of worms exposed to the silt wastewater sludge (figure 1). fig 1. wet weight of e. fetida earthworms exposed to different substrates: sunflower husk (sh, control), mixed husk (mh), and compacted sewage sludge (css) the increase in e. fetida biomass at both substrates sh and mh occurred 98 days until the experiment and reached, respectively 198% and 220% compared to initial weight of the worms. at the same time, the wet weight of the earthworms exposed to substrate css diminished up to 112 days; weight loss was 16% of control already at 14 days of the experiment, and the final weight loss was 69% of baseline. the results obtained indicated, that growth of e. fetida worms exposed to compacted sewage sludge was inhibited drastically. decrease in the wet weight was observed during whole experiment, however, the most significant decrease was observed up to 42 days, then becoming a less pronounced. a similar non-linear decrease in growth rate of e. 0 5 10 15 20 25 0 14 28 42 56 70 84 98 112 w et w ei gh t, g day of the experiment sh mh css food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 279 fetida was occurred under the action of herbicide acetochlor [19]. likewise, the growth of earthworms aporrectodea caliginosa was decreased noticeably after 7 and 14 days of exposure to insect growth regulators flufenoxuron and pyriproxyfen [20]. in general, growth inhibition is a good indicator of chemical stress, which may link effects to energy dynamics and ultimately inhibit growth of the organisms [15]. in our study, the content of heavy metals discovered in the substrate css before the earthworm’s introduction, was strongly reduced at the end of the experiment (table 1). table 1 effect of e. fetida vermiculture on heavy metal content in compacted sewage sludge (css) metal content of metal in css, mg/kg before the experiment after the experiment cu 76.21±2.54 5.40±0.20 zn 218.04±8.07 17.32±0.61 mn 794.10±30.17 54.74±1.86 ni 29.20±1.05 0.70±0.03 cr 38.12±1.49 2.54±0.12 pb 39.03±1.40 3.02±0.10 cd 5.12±0.19 the smallest drop was found for the zinc and lead levels (respectively, 12.6 and 13 times compared to baseline). decrease in the levels of copper, manganese and chromium was greater (14.1, 14.5 and 15.2 times, respectively). nickel content was reduced to 41.4 times; however, the most striking was the almost 100% reduction in level of cadmium, which was not identified in the substrate after the experiment. that is, the worm’s biomass reduction was accompanied by a sharp decrease in heavy metal content in contaminated substrate. it is obvious that inhibition of e. fetida growth could be due to enormous amplification of detoxification processes of heavy metals. our results are consistent with data [6] about earthworm’s ability to accumulate high concentrations of mercury, lead, copper, manganese, zinc, and the extreme cadmium content (up to 100 mg per kg dry weight). it was established [21], that accumulation of a single heavy metal does not affect the worms’ physiology; while in our study, the sharp decline in biomass could be caused by presence of several heavy metals and possibly other toxicants in the substrate. increase in activity of glutathione-stransferase was revealed in coelomic fluids of e. fetida exposed to both substrate mh (insignificant difference with control) and css (187% compared to control, p < 0.05), as it shown in figure 2. f-test indicated the difference of data dispersions for two different substrates mh and css. effective metabolic degradation of different xenobiotics by the earthworms based on the functioning of glutathiones-transferase, which can catalyze the conjugation of toxic compounds with reduced glutathione, and is involved in antioxidant defense [22]. in our study, almost 2-fold activation of gst was found in coelomic fluid of e. fetida after 112 days of exposition to complex contaminated substrate. this result confirms the involvement of e. fetida glutathione-s-transferase in detoxification of various components as well as the capacity for long-term food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 280 functioning under the influence of substrate with an integrated pollution. fig. 2. gst activity (nkatal/ml) in coelomic fluid of e. fetida exposed to sh (sunflower husk), mh (mixed husk), and css (compacted sewage sludge) outcomes are comparable with the data [13] about significant increasing gst activity of e. fetida in soils with copper after a period of contaminants accumulation in body tissues, as well, as about gst activation with longer duration and increasing concentrations of herbicide acetochlor [23]. in our study, the control level of reduced glutathione content in the e. fetida coelomic fluid was exceeded in the worms exposed to substrate mh (10 % above control, p < 0.05), and to substrate css (36% above control, p < 0.05) as well (figure 3). f-test revealed the difference of data dispersions for substrates mh and css. fig. 3. effect of substrates on gsh content (nmol/ml) in coelomic fluid of e. fetida exposed to sh (sunflower husk), mh (mixed husk), and css (compacted sewage sludge) thus, amount of reduced glutathione in the e. fetida coelomic fluid increased by 1.4 times, indicating enhancement the biosynthesis of glutathione and recovery of the oxidized glutathione during exposition to the contaminated substrate. reduced glutathione carries out many functions besides conjugation with xenobiotics; in particular, in the worms’ cells, gsh was involved in the biosynthesis of metallothioneins due to the action of copper [13] and cadmium 0 20 40 60 80 100 120 140 160 sh mh css g s t a ct iv ity , nk at /m l substrate 0 100 200 300 400 500 600 sh mh css g s h c on te nt , nm ol /m l substrate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 281 [24]. in our experiment, gsh pool enhancement was less than gst activation level; this could be attributed to glutathione spending for the synthesis of metallothioneins. anyway, the glutathione content was sufficient to ensure the growth of gst activity, which is consistent with the assertion [25] about the absence of gst activation in all earthworm species with low gsh levels. in our study, sds-page analysis of e. fetida coelomic fluids revealed increase in intensity of certain protein bands in the spectra of the worms exposed to both substrates mh and css, compared with the control spectrum (figure 4). mh css sh st fig. 4. polypeptide content of coelomic fluid of the e. fetida worms exposed to different substrates: mh – mixed husk; css – compacted sewage sludge; sh – sunflower husk (control). st – standard proteins (cytochrome c, mr =12 kd; egg albumin, mr = 45 kd; albumin bovine serum, mr = 67 kd). e. fetida coelomic fluid proteins have been shown to have molecular masses ranging from 14.3 kda to 97.8 kda. noteworthy, the components with mr=18.2; mr=78.5, and mr=81.3 kda were absent in the spectrum of the worms exposed to contaminated silt. at the same time, strengthening the bands corresponding to low-molecular polypeptides with mr=14.3; mr=16.6, mr=21.4 kda, and to high-molecular zone with mr=97.8 kda was observed in the spectrum of the worms exposed to silt compared to control spectrum. beside them, proteins with mr=16.6; mr=41.3; mr=84.1, and mr=87.1 kda were presented only in the samples exposed to scc. therefore, the appearance of these proteins in the worm’s coelomic fluid reflects the changes in the protein metabolism including induction and / or enhancement of biosynthesis of enzymes responsible for detoxification of contaminated substrate. so, it can be concluded the metabolic systems of e. fetida generally actively reacted to substrate content, providing a high adaptive capacity of worm organism to polluted environment by means of the xenobiotics detoxification. 4. conclusion summing up, the studies confirmed the ability of e. fetida to simultaneously reduce the content of copper, zinc, manganese, nickel, chromium, lead and cadmium in contaminated substrate. processes of the substrate cleaning against nickel and cadmium were the most effective. induction of four new proteins biosynthesis along with activation of glutathione-s-transferase mr 67 kd 45 kd 12 kd food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 282 was found in the coelomic fluid of earthworms exposed to polluted silt. moreover, increasing pool of reduced glutathione was also observed in the coelomic fluid at the end of earthworms’ exposition to contaminated substrate. hence, vermiculture of e. fetida can be an effective way of sewage sludge purification in the case of integrated pollution by enhancing the activity of glutathione-dependent system. 5. acknowledgements the present research was conducted under the grants of ministry of education and science of ukraine (0113u003034 and 0116u001723). 6. references [1] markin v., utilization of domestic sewage sludge, donbas national academy of civil engineering and architecture, 3(101): 138-140, (in russian), (2013) [2] sigua g.c, adjei m.b., rechcigl j.e., cumulative and residual effects of repeated sewage sludge applications: forage productivity and soil quality implications in south florida, usa, environ sci. pollut. res. int., 12 (2), 8088, (2005) [3] smith s.r., a critical review of the bioavailability and impacts of heavy metals in municipal solid waste composts compared to sewage sludge, environment international, 35(1), 142–156, (2009) [4] zhang h., zhang q., yang b., wang j., compacted sewage sludge as a barrier for tailings: the heavy metal speciation and total organic carbon content in the compacted sludge specimen, plos one., 30: 9(6), (2014) [5] babel s, del mundo dacera d., heavy metal removal from contaminated sludge for land application: a review, waste manag., 26 (9), 988-1004, (2006) [6] sinha r.k., chandran v., soni b.k., patel u., ghosh a., earthworms: nature’s chemical managers and detoxifying agents in the environment: an innovative study on treatment of toxic wastewaters from the petroleum industry by vermifiltration technology, environmentalist, 32, 445-452, (2012) [7] aly m.a, schroder p., effect of herbicides on glutathione s-transferases in the earthworm, eisenia fetida, environonmental science and pollution research international, 15(2), 143-149, (2008) [8] torkhani a.l., kozuh erzen n., kolar l., virant c.t., lestan d., does ivermectin attract earthworms, journal of soils and sediments, 11(1), 124-128, (2011) [9] saint-denis m., labrot f., narbonne j.f., ribera d., glutathione, glutathione-related enzymes, and catalase activities in the earthworm eisenia-fetidaandrei, arch. environ. contam. and toxicol., 4 (35), 602-614, (1998) [10] wang j.h., zhu l.s., liu w., wang j., xie h., biochemical responses of earthworm (eisenia foetida) to the pesticides chlorpyrifos and fenvalerate, toxicol. mech. methods, 22(3), 236-241, (2012) [11] xiao n.-w., song y., ge f., liu x.h., ou-yang z.-y., biomarkers responses of the earthworm eisenia fetida to acetochlor exposure in oecd soil, chemosphere, 65 (6), 907–912, (2006) [12] lukkari t., earthworm responses to metal contamination. tools for soil quality assessment, jyvaskyla university printing house, jyvaskyla. 64 p. (2004) [13] cataldo j.r., hidalgo m.e., neaman a., gaete h.o., use of molecular biomarkers in eisenia foetida to assess copper toxicity in agricultural soils affected by mining activities, journal of soil science and plant nutrition, 11(3), 57-70, (2011) [14] ribera d., narbonne j.f, arnaud c, saint-denis m., biochemical responses of the earthworm eisenia fetida andrei exposed to contaminated artificial soil, effects of carbaryl, soil biology and biochemistry, 33 (7– 8), 1123–1130, (2001) [15] badawy m.e., kenawy a., elaswad a.f., toxicity assessment of buprofezin, lufenuron, and triflumuron to the earthworm aporrectodea caliginosa, international journal of zoology, article id 174523, 9 p., (2013) http://www.ncbi.nlm.nih.gov/pubmed/?term=adjei%20mb%5bauthor%5d&cauthor=true&cauthor_uid=15859114 http://www.ncbi.nlm.nih.gov/pubmed/?term=rechcigl%20je%5bauthor%5d&cauthor=true&cauthor_uid=15859114 http://www.ncbi.nlm.nih.gov/pubmed/15859114 http://www.sciencedirect.com/science/article/pii/s0160412008001153 http://www.sciencedirect.com/science/journal/01604120 http://www.sciencedirect.com/science/journal/01604120/35/1 http://www.sciencedirect.com/science/journal/01604120/35/1 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhang%20h%5bauthor%5d&cauthor=true&cauthor_uid=24979755 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhang%20q%5bauthor%5d&cauthor=true&cauthor_uid=24979755 http://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20b%5bauthor%5d&cauthor=true&cauthor_uid=24979755 http://www.ncbi.nlm.nih.gov/pubmed/?term=wang%20j%5bauthor%5d&cauthor=true&cauthor_uid=24979755 http://www.ncbi.nlm.nih.gov/pubmed/24979755 http://www.ncbi.nlm.nih.gov/pubmed/?term=del%20mundo%20dacera%20d%5bauthor%5d&cauthor=true&cauthor_uid=16298121 http://www.ncbi.nlm.nih.gov/pubmed/?term=aly%20ma%5bauthor%5d&cauthor=true&cauthor_uid=18380233 http://www.ncbi.nlm.nih.gov/pubmed/?term=schr%c3%b6der%20p%5bauthor%5d&cauthor=true&cauthor_uid=18380233 http://www.ncbi.nlm.nih.gov/pubmed/18380233 http://www.ncbi.nlm.nih.gov/pubmed/18380233 http://academic.research.microsoft.com/author/45006554/alja-livio-torkhani http://academic.research.microsoft.com/author/3592676/nevenka-kozuh-erzen http://academic.research.microsoft.com/author/19838801/lucija-kolar http://academic.research.microsoft.com/author/45006556/tina-virant-celesetina http://academic.research.microsoft.com/author/23659701/domen-lestan http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/article/pii/s0045653506004073 http://www.sciencedirect.com/science/journal/00456535 http://www.sciencedirect.com/science/journal/00456535/65/6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 4 – 2016 nataliya mitina, nina khromykh, larisa shupranova, inna zubareva, activating the glutathione system of eisenia fetida during exposure to contaminated silt sludge, food and environment safety, volume xv, issue 4 – 2016, pag. 276 – 283 283 [16] habig w.h., pabst m.j., jakoby w.b., glutathione s-transferase. the first step in mercapturic acid formation, j. biol. chem., 249(22), 7130 – 7139, (1974) [17] anderson m.e., determination of glutathione and glutathione disulfide in biological samples, methods enzymol., 548 – 555, (1985) [18] laemmli u.k., cleavage of structural of bacteriophage t-4, nature, 227, 680-685, (1970) [19] xiao n., jing b., ge f, liu x., the fate of herbicide acetochlor and its toxicity to eisenia fetida under laboratory conditions, chemosphere, 62(8), 1366 – 1373, (2006) [20] nasr h.m., badawy m.e., biomarker response and biomass toxicity of earthworms aporrectodea caliginosa exposed to igrs pesticides, j environ. anal. toxicol., 5, 332, (2015) [21] hartenstein r., neuhauser e.f., collier j., accumulation of heavy metals in the earthworm eisenia foetida, j. environ. qual., 9, 23-26, (1980) [22] martin-diaz m., blasco j., sales d., delvalls t., field validation of a battery of biomarkers to assess sediment quality in spanish ports, environ. pollut., 151, 631-640, (2008) [23] xiao n.w., liu x.h., li w., ge f., effect of herbicide acetochlor on cytochrome p450 monooxygenases and gst of earthworms eisenia fetida, j environ sci. (china)., 18(1), 135-140, (2006) [24] gillis p., reynoldson t., dixon g., natural variation in a metallothioneinlike protein in tubifex tubifex in the absence of metal exposure, ecotox. environ. safe, 58, 2228, (2004) [25] hermanm j., stenersen v, qien n.i., glutathione s-transferases in earthworms (lumbricidae). substrate specificity, tissue and species distribution and molecular weight, comparative biochemistry and physiology. part c comparative pharmacology, 69(2), 243-52, (1981) http://www.sciencedirect.com/science/article/pii/s0045653505009495 http://www.sciencedirect.com/science/article/pii/s0045653505009495 http://www.sciencedirect.com/science/article/pii/s0045653505009495 http://www.sciencedirect.com/science/article/pii/s0045653505009495 http://www.ncbi.nlm.nih.gov/pubmed/?term=xiao%20nw%5bauthor%5d&cauthor=true&cauthor_uid=20050562 http://www.ncbi.nlm.nih.gov/pubmed/?term=liu%20xh%5bauthor%5d&cauthor=true&cauthor_uid=20050562 http://www.ncbi.nlm.nih.gov/pubmed/?term=li%20w%5bauthor%5d&cauthor=true&cauthor_uid=20050562 http://www.ncbi.nlm.nih.gov/pubmed/?term=ge%20f%5bauthor%5d&cauthor=true&cauthor_uid=20050562 http://www.ncbi.nlm.nih.gov/pubmed/20050562 https://www.researchgate.net/profile/jorgen_stenersen2 https://www.researchgate.net/profile/nils_oien https://www.researchgate.net/journal/0306-4492_comparative_biochemistry_and_physiology_part_c_comparative_pharmacology https://www.researchgate.net/journal/0306-4492_comparative_biochemistry_and_physiology_part_c_comparative_pharmacology [2] sigua g.c, adjei m.b., rechcigl j.e., cumulative and residual effects of repeated sewage sludge applications: forage productivity and soil quality implications in south florida, usa, environ sci. pollut. res. int., 12 (2), 80-88, (2005) [4] zhang h., zhang q., yang b., wang j., compacted sewage sludge as a barrier for tailings: the heavy metal speciation and total organic carbon content in the compacted sludge specimen, plos one., 30: 9(6), (2014) [7] aly m.a, schroder p., effect of herbicides on glutathione s-transferases in the earthworm, eisenia fetida, environonmental science and pollution research international, 15(2), 143-149, (2008) [15] badawy m.e., kenawy a., el-aswad a.f., toxicity assessment of buprofezin, lufenuron, and triflumuron to the earthworm aporrectodea caliginosa, international journal of zoology, article id 174523, 9 p., (2013) [23] xiao n.w., liu x.h., li w., ge f., effect of herbicide acetochlor on cytochrome p450 monooxygenases and gst of earthworms eisenia fetida, j environ sci. (china)., 18(1), 135-140, (2006) microsoft word 1 razvan radu rusu.doc 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 5 11 certain textura l pr operties o f ch ick en m ea t, in ves tig ated th roug h histolo gy an d pho ton ic m icro sco py tech n iqu es *răzvan mihail radu-rusu1, ancuţa elena prisacaru (coşuleanu)2 tahsin sady hussen3, gabriel vasile hoha1 1university of agricultural sciences and veterinary medicine, animal science faculty, 8 mihail sadoveanu alley, 700489, iaşi, romania, rprobios@yahoo.com 2”ştefan cel mare” university, food engineering faculty, 13 universităţii str., 720229, suceava, romania 3university of salahadin, college of agriculture, animal production department, erbil, iraq * corresponding author received 10 january 2013, accepted 15 february 2013 abstract: this study is included within a wide range of researches regarding poultry meat quality, consisting in some comparative researches referring to the sensorial features of the skeletal musculature as well as to the nutritional value of the meat produced by several modern broiler brands: cobb 500, ross 308, shaver starbro, hybro-pg. the results in the paper reveal some of the poultry muscles histological parameters: myocytes (muscle fibers) thickness and cross-section area, muscular fibers density, and, finally, the proportion of main tissues (pure muscular and connective) in whole muscle structure. 100 broilers (50♂ + 50♀), selected from a shelter accommodating 9500 cobb 500 chickens of 42 days old, served as biological material in order to elect the samples from five representative muscles: pectoralis profundis et superficialis, biceps brachii, semimembranosus, gastrocnemius medialis. the tissue samples were used to obtain histological smears on cross-section, which were then analyzed using photonic microscopy. white muscles (breast fillet) were found to have the highest values for the myocytes’ thickness (41.11µ), while the contractile cells of the red muscles were thinner (the thinnest within the brachial biceps – 27.9µ) and more dense per surface unit (≈1062 myocytes/mm2 of muscle). the highest proportion of pure muscular tissue has been found in males’ pectoralis superficialis muscles (65.22%). keywords: chicken broiler, myocytes, density, texture, meat quality 1. introduction scientific literature, mainly those publications dealing with human nutrition and customer safety, emphasizes on some aspects which pass over the quantitative side of meat production in poultry. while most of the data spread by the companies producing high value commercial broilers refers to some technological and economical features of their product (microclimate, nutritional requirements, weight gains, feed conversion ratio, slaughtering efficiency), this paper brings some partial results from a study onto the textural quality of the meat produced by high performance hybrids. one of the curiosities among scientists and practitioners in poultry industry is how carcass cuts develop in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 6 superintensive muscle growth during the normal 42 day rearing period – basing on muscle cell hypertrophy or on myocytes “hyperplasia” (increase of muscle cells amount per cross section tissual surface unit) [1]?. from poultry meat qualit y traits, texture is a useful parameter for food scientisits, because is straightly reşated to tenderness or toughness of meat. usually, instrumental methods are used to assess tenderness on cooked meat (warner-bratzler, tensile tests, or allo-kramer shear press) [2, 3]. other relevant parameter for meat tenderness in pre-rigor and post-rigor stages is the sarcomere length [4], also assessed through histological methods. such techniques could also be applied in differentiating pale soft exudative (pse) meat from normal meat and to determine the best ante mortem treatments for broilers in order to avoid pse occurrence [5] why not using histological techniques to assess the development tendency in meat structure, related to chickens age, genotype and rearing technique, in order to predict the tissual and textural features of the meat prior to commercial broiler choosing for intensive farming or prior to slaughterhouse options in relation with products needed to be processed starting from raw meat? the paper aims to reveal those histological techniques that might be used to predict textural and other technological properties of the meat. 2. experimental a group of 100 „cobb 500” broilers (50 males + 50 females), issued from a shelter accommodating 9500 chickens, have been used as biological material to sample five studied muscles: pectoralis superficialis et profundis, biceps brachii, semimembranosus and gastrocnemius medialis. the feed consisted in classical corn-soymeal diet (3012kcal me and 24% cp starter; 3175kcal me and 22.5% cp grower; 3226kcal me and 20% cp finisher). muscular samples have been processed after techniques specified in literature [6, 7] through formalin 10% fixation, paraffin impregnation at +56° c, acid fuchsine and evans blue coloration, resulting histological smears. these have been studied at a motic dmwb1-223 photonic microscope, calibrated for three obxoc associations: 10x10; 20x10 and 40x10. an ocular micrometer served to run the assessments, while an 8mp digital camera has been used to take microphotography shots, which were subsequentially processed in the motic images plus ml software. the studies comprised cytometric and histometric measurements of myocytes and muscular fascicles, whose results have been introduced in several mathematical relations, in order to achieve some histological indexes: fibers mean thickness, cross section areas, myocytes density per muscular tissue unit, proportions of pure muscular/connective tissues. the formulas are listed below: *mean thickness:   2/)(xd dd  , meaning: d=large diameter, d=small diameter; *cross section area: 4/)s( 2   dd , meaning: π = 3.1416; *myocytes density: /mfi area..n m.f. 0000001 muscle) ofmm(myocites/ dens. m. 2   , meaning: n m.f. = amount of muscular fibers per 1st order muscular fascicle (mfi), mfi area = area of the measured 1st order muscular fascicle (mm2); * tissual categories proportion: mfi aream.f. arean m.f. /100(%) mt  )100(%) ct -mt (% , meaning: mt (%) proportion of pure muscular tissue; ct (%) proportion of connective tissue, m.f. area= myocytes area (µ2). the achieved average values of the studied parameters have been statistically processed running the anova single factor algorithm. 3. results and discussion figure 1 presents screenshots from microscopic field, while table 1 and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 7 figures 2-4 reveal data concerning dimensional features of the muscular fibers, their density within muscles an also the proportion of pure muscular and connective tissues. mean myocytes diameter in the pectoralis superficialis muscles has been found of 35.22±0.46µ at cockerels and of 35.29±0.29µ at pullets. muscular fibers were thicker into the profound pectoral muscles, both in males and females (41.11±0.56µ and 36.51±0.45µ) and distinguished statistical significance occurred. the homogeneity was average (v=10.91-13.63%). as compared to breast fillet (white muscles), the texture of red studied muscles proved to be thinner. the lowest values for muscular fibers diameter were measured within the biceps brachialis samples (26.51±0.34µ at pullets and 27.90±0.38µ at cockerels) (v% varied between 12.7213.72%). table 1 myocytes thickness, density and proportion of main tissual categories within some of the breast, wings, thighs and drumsticks muscles studied muscles broilers gender statistics myocytes mean thickness (µ) myocytes density (myocytes /mm2 of muscle) pure muscular tissue (mt%) connective tissue (ct%) x 35.22 683.90 d ± xs 0.46 18.02 ♂ v% 12.93 13.18 65.22 d 34.78 a x 35.29 583.73 a ± xs 0.39 20.12 p ec to ra l i s su pe rf ic ia lis ♀ v% 10.91 17.24 56.12 a 43.88 d x 41.11 d 407.51 a ± xs 0.56 18.18 ♂ v% 13.63 22.31 53.49 a 46.51 b x 36.51 a 604.46 d ± xs 0.45 36.60 p ec to ra l i s pr of un di s ♀ v% 12.28 30.28 62.56 b 37.44 a x 27.90 c 838.25 a ± xs 0.38 27.42 ♂ v% 13.72 16.36 50.43 a 49.57 b x 26.51 a 1061.94 d ± xs 0.34 47.72 b ic ep s br ac hi i ♀ v% 12.72 22.47 58.19 b 41.81 a x 30.89 a 801.63 d ± xs 0.32 28.06 ♂ v% 10.41 17.50 59.16 40.84 x 32.44 b 722.01 a ± xs 0.28 15.70 se m im em br an os us ♀ v% 8.64 10.88 58.81 41.19 x 29.77 a 810.15 d ± xs 0.41 32.31 ♂ v% 13.88 19.94 56.20 43.80 x 37.38 d 518.34 a ± xs 0.38 19.59 g as tr oc ne m iu s m ed ia lis ♀ v% 10.15 18.90 56.49 43.51 anova test – applied to each studied variable and muscle, as compared between genders, per column: ab significant differences ( f̂ >f α 0.05); ac distinguished significant differences ( f̂ >f α 0.01); ad high significant differences ( f̂ >f α 0.001) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 8 pectoralis superficialis (breast) pectoralis profundis (breast) biceps brachialis (wing) semimembranosus (thigh) gastrocnemius medialis (shank – drumstick) figure 1. muscle fibers (myocites) and 1st order muscular fascicles in skeletal muscles of chicken broilers (100 x magnification, oc 10xob 10, sampling: left-males, right-females) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 9 the anova test applied to red muscles cytometric values revealed various levels of statistically significance between genders: significant differences for thighs muscles (semimembranosus, thicker fibers in females); distinguished significance for wings muscles (biceps brachialis, thicker fibers in males) and high significant differences between drumsticks muscles (gastrocnemius medialis, thicker fibers in females). 35.22 35.29 41.11 36.51 27.9 26.51 30.89 32.44 29.77 37.38 0 5 10 15 20 25 30 35 40 45 psm psf ppm ppf bbm bbf smm smf gcmm gcmf m ea n th ic kn es s of th e m us cu la r fib er s (µ ) 683.9 583.73 407.51 604.46 838.25 1061.94 801.63 722.01 810.15 518.34 0 200 400 600 800 1000 psm psf ppm ppf bbm bbf smm smf gcmm gcmf m yo ci te s de ns ity (m .f. /m m 2) figure 2. mean thickness of the myocytes (µ) figure 3. myocytes density (m.f./mm2 of muscle) (ps = pectoralis superficialis; pp = pectoralis profundis; bb = biceps brachii; sm = semimembranosus; gc = gastrocnemius medialis; m=muscles from males, f=muscles from females) mal e s 34.78 65.22 pure muscular tis sue connective tissue fe m al e s 56.12 43.88 pure mus cular tiss ue connective tis sue mal e s 46.51 53.49 pure muscular tissue connective tissue fe m al e s 62.56 37.44 pure mus cular tiss ue connective tissue pectoralis superficialis muscles pectoralis profundis muscles male s 50.4349.57 pure muscular tis s ue connective tis sue fem al e s 58.19 41.81 pure mus cular tis s ue connective tis s ue mal e s 59.16 40.84 pure muscular tis sue connective tis sue fe mal e s 58.81 41.19 pure muscular tis sue connective tissue biceps brachii muscles semimembranosus muscles mal e s 56.20 43.80 pure mus cular tis sue connective tissue fe m al e s 56.49 43.51 pure mus cular tis sue connective tissue gastrocnemius medialis muscles figure 4. proportions of pure muscular and connective tissues within breast, wing, thigh and drumstick muscles (%) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 10 myocytes density values varied conversely than those obtained for the average diameter. thus, the lowest density could be observed into the males pectoralis profundis muscles (407.51±18.18 m.f./mm2), while highest amount of muscular fibers has been found into the pullets biceps brachialis muscles (1061.94 m.f./mm2). very significant differences occurred for all studied muscles, when both genders were compared. widest variation amplitude of the differences between cockerels and pullets was observed in gastrocnemius medialis muscles (513.84 m.f./mm2 in females and 810.15 m.f./mm2 in males). the calculated variability showed high heterogenic characteristics (v=10.88-30.28%). the acquired data are consistent with those previously reported by other scientists [8], which showed higher myofibres density in males, in most analysed skeletal muscles of commercial broiler strains. proportions of main tissual categories in muscles structure revealed that highest amount of pure muscular tissue was found into the superficial pectoral muscles at males (65.22%) and into the profound pectoral muscles at females (62.56%). very high significant differences occurred between genders when pectoralis superficialis muscles have been compared, while other significant differences have been observed for cockerels vs. pullets pectoralis profundis and biceps brachialis comparisons. thinnest texture has been observed in biceps brachialis muscles, while the thickest fibers have been measured within the pectoralis profundis muscles, fact also observed during mastication. these results confirm that those myocytes having mainly glycolitical metabolism, usually found in white muscles (pectorals) are thicker than those with oxidative preponderant metabolism (red muscles – limbs) which have higher density [9, 10]. as compared to chicken broilers, in waterfowl domestic species (duck and goose), all skeletal muscles are mainly made of red fibers, which are thicker in wings [11, 12, 13]. although previous researches recommend pectoral muscles as high qualitative, mainly concerning the physical, chemical and nutritional features (ph value, high protein content, low energy level) [14, 15, 16, 17], these researches showed that, for cobb-500 broilers, textural features are better in red muscles (especially in wings and thighs ones), which have, consequently, improved tenderness. 4. conclusion the quality sensorial features of meat (texture, tenderness, flavour) are influenced by the dimensional myocytes features, as well as by the proportion between main tissual categories and by lipids content, hence the possibility and usefulness in applying histological and microscopy methods in assessing them. further researches have to be carried on, in order to elect those components of the connective tissue which affect tenderness (eg. % of collagen, % of adipous tissue etc.), as another goal to reach in commercial broilers meat quality knowledge. 5. acknowledgments the authors wish to thank prof. vasile teuşan, phd, from animal science faculty, u.a.s.v.m. iaşi, for sharing his expertise in histology processing and microscopy analysis. 6. references [1]. velleman s.g., nestor k.e., coy c.s., harford i., anthony n.b., effect of posthatch feed restriction on broiler breast muscle development and muscle transcriptional regulatory factor gene and heparan sulfate proteoglycan expression. international journal of poultry science, 9(5).417-425, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 r ă z v a n m i h a i l r a d u r u s u , a n c u ţ a e l e n a p r i s a c a r a ( c o ş u l e a n u ) , t a h s i n s a d y hu s s e n , g a b r i e l v as i l e h o h a , c e r t a i n t e x t u r a l p r o p e r t i e s of c h i c k e n m e at , i n v e s t i g a t e d t h r o u g h hi s t o l o g y a n d p h ot o n i c m i c r o s c o p y t e c h n i q u e s , f o o d a n d e n v i r o n m e n t s a f e t y , v o l u m e x i i , i s s u e 1 – 2 0 1 3 , p a g . 5 1 1 11 [2]. lyon b.g., lyon c.e. meat quality: sensory and instrumental evaluation, in: sams a.r. (ed.) poultry meat processing, pp. 97-120, crc press, new york, ny, (2001). [3]. cavitt l.c., meullenet j.f., gandhapuneni r.k., youm g.w., owens c.m., rigor development and meat quality of large and small broilers and the use of allokramer shear, needle puncture, and razor blade shear to measure texture. poultry science, 84(1).113-118, (2005). [4]. petracci m., baéza e., harmonization of methodologies for the assessment of poultry meat quality features. world's poultry science journal, 67.137-153, (2011). [5]. guarnieri p.d., soares a.l., olivo r., schneider j.p., macedo r.m., ida e.i., shimokomaki m., preslaughter handling with water shower spray inhibits pse (pale, soft, exudative) broiler breast meat in a commercial plant. biochemical and ultrastructural observations. journal of food biochemistry, 28.269-277, (2004). [6]. diaconiţă g., eskenasy a., hagi-paraschiv a., tehnica histopatologică, editura de stat pentru literatură ştiinţifică, bucureşti (1953). [7]. bancroft j.d., gamble m., theory and practice of histological techniques, 6th edition, elsevier ltd., (2008) [8]. scheuermann g.n., bilgili s.f., hess j.b., mulvaney d.r., breast muscle development in commercial broiler chickens. poultry science, 82.1648–1658 (2003). [9]. piccard b., jure c., cassar-malek i., hocquette j.f., lefaucheur l., berry c., duclos m. j., alami-durante h., rescan p.y., typologie et ontogenèse des fibres musculaires chez différentes espèces d’intérêt agronomique. inra productions animales, 16(2).115-120, (2003). [10]. berri c., duclos m.j., typologie et ontogenèse des fibres musculaires chez les oiseaux. inra productions animales, 16 (2).137-143, (2003). [11]. radu-rusu r.m., teuşan v., voicu p., researches concerning the thickness and the cross-section area of the miocytes in the domestic waterfowl’s pectoral muscles, lucr. ştiinţ. u.s.a.m.v. iaşi, seria zootehnie, 49.156-164, (2006). [12]. radu-rusu r.m., teuşan v., teuşan a., comparative researches concerning some histometric features of the miocytes in somatic musculature of the domestic chicken and waterfowl (i). pectoral muscles. lucr. stiint. u.s.a.m.v. iasi, seria zootehnie, 50.115-120, (2007). [13]. radu-rusu r.m., teuşan v., vacaru-opriş i., comparative researches concerning some histometric features of the miocytes in somatic musculature of the domestic chicken and waterfowl (ii). wing and thigh muscles. lucr. stiint. u.s.a.m.v. iasi, seria zootehnie, 50.107-114, (2007). [14]. banu c. et al., quality control of food products, agir publishing house, bucharest, romania, (2002). [15]. radu-rusu r.m., boişteanu p.c., acţiunea unor factori asupra metabolismului muscular in-vivo şi influenţa acestora asupra calităţii finale a cărnii de pasăre, revista de zootehnie, 1.75-82, (2007). [16]. ristic m., meat quality of organically produced broilers, world poultry, 20(8).30-31, (2004). [17]. suchy p., jelinek p., strakova e., hucl j., chemical composition of muscles of hybrid broiler chickens during prolonged feeding. czech journal of animal science, 47(12).511-518, (2002). microsoft word 10 avramiuc.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 59 the influence of hypothermic shocks and additions during processing, on some yoghurt features *marcel avramiuc1 1ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1avramiucm@fia.usv.ro *corresponding author received 25 march 2012, accepted 12 may 2012 abstract: in this work there was searched the influence of some hypothermic shocks and compound admixtures, during yoghurt processing, on some yogurt features (ph, titratable acidity and curd aspect). the biological material used in this work was represented by cow milk, inoculated with a starter culture of thermophile lactic bacteria, including two different species: streptococcus termophilus and lactobacillus bulgaricus. the experiments during yoghurt processing have consisted in subjecting of samples to hypothermic shocks, on the one hand, and introducing of some admixtures (starch or gelatin), in certain proportions, on the other hand, to see the influence of these experiments on the evolution of ph, titratable acidity and quality characteristics of yoghur curd particles. the application of hypothermic shocks in the early period of thermostating has determined the decrease of fermentation activity of lactic bacteria (expressed by reduction of titratable acidity values) and has made the coagulation process of milk to be blocked. the later applied hypothermic shock, the lower effect on titratable acidity and coagulation process was. the addition of gelatine or starch during yoghurt processing has influenced, indirectly, the fermentation activity of lactic bacteria. the gelatin addition has led to the increase of titratable acidity values, comparing to blank. the addition of gelatine or starch has not rushed the milk coagulation during yoghurt processing. compared to the control, in samples with admixtures the curd formation was slower, especially in those ones with starch addition. keywords: yoghurt, hypothermic shocks, titratable acidity, ph, starch, gelatin. 1. introduction in order to avert food unbalances caused by various deficiency states, last time some nourishing substances are introduced within foods, being an efficient way to ensure an optimum healthy state of people (1, 2, 3). the admixtures of active biological compounds within products poor in nutritional substances is the method to get fortifiated foods, having as result the ensurance of a body maximum protection (4, 5). in need, the dairy products can be supplemented with liposoluble provitamins / vitamins (a and/or d), with hydrosoluble vitamine (niacine, tiamine, riboflavin, ascorbic acid) or with biominerals, such as: iron, iodine, fluor etc. (6, 7). some works tried to evidence the effect of certain admixtures on biochemical processes, having, indirectly, in view the changes occured within transformations of carbohydrates and proteins made by lactic bacteria (8). in this work there was searched the influence of some hypothermic shocks and admixtures of starch or gelatin, during yoghurt processing, on some yogurt food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 60 features (ph, titratable acidity and curd aspect). 2. experimental the biological material used in this work, was represented by cow milk, whose biochemical features are rendered below: ph: 6.90 acidity (törner degrees): 20t fats: 1.99% proteins: 3.50% lactose: 4.40% dry matter: 9.85% minerals: 0.08% the milk was inoculated with a starter culture of thermophile lactic bacteria, used for direct inoculation within the tub for yoghurt. the starter culture (a fine liofilisated powder) has included two lactic bacteria different species: streptococcus termophilus and lactobacillus bulgaricus. the thermostating temperature was 43c during 5 hours, and the inocul dose was 5ua/100 l. the milk was conditioned with powder milk (20 g/l) and pasteurised 20-30 minutes at 90○c. for some work variants there were used only milk or milk with different admixtures. the experiments during yoghurt processing have consisted in subjecting of samples to hypothermic shocks, on the one hand, and introducing of some admixtures (starch or gelatin), in certain proportions, on the other hand, to see the influence of these experiments on the evolution of ph, titratable acidity and quality characteristics of curd particles of yoghurt. thermal shocks have been carried out by subtracting fast of temperature milk sowed and thermostated after certain time intervals, in order to achieve in the end t=20○c. samples were kept in the freezer for 10 minutes, then in the refrigerator for 5 minutes. thermal shocks have been performed after: 30, 90, 150 and 210 of thermostating minutes at temperature 43○c, and determinations of above mentioned indices, have been carried out hour by hour during yoghurt processing. the percentage of admixtures used are rendered below: table 1 the percentage of admixtures admixtures starch % 0.5 1.5 2.5 gelatin % 0.5 1.5 2.5 the samples with admixtures for trials are played in the table no. 2: table 2 the samples with admixtures for trials the determination of the titratable acidity, expressed in thőrner degree (○t), was made according to aoac standard (9), and the ph values were estimated according to stas 8201/82 (10). as seen in the table 1, in blank (p1) ph values fell from 6.6 (after first time keeping in the oven), to ph 4.6 (after 5 hours) the difference being 2 ph units. the acidity of milk (sowed with starter culture) after the first thermostating hour was 25○t, and in the second hour this index has grown very little (with only 4○t). visible changes occurred between 2 hour (29○t) and 3 hours of termostating process (56○t) – an extra of 27○t, respectively between 4 hour (61○t) and the 5th termostating hour (82○t) that an increase by 21○t. in blank, between the initial and the final titratable acidity value reveals a difference of 57○t. admixture sample starch (%) gelatin (%) p1 p2 0.5 p3 1.5 p4 2.5 p5 0.5 p6 1.5 p7 2.5 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 61 table 3 values of ph and titratable acidity of yogurt samples, subjected to thermal shocks in sample p2, ph values fell from ph 6.56, after the first thermostating hour, to ph 6.15 (after 5 hours) – a more pronounced decrease being registered from 3 to 5 hours of processing (0.27 ph units). as to titratable acidity, the growth of this index was higher between the 3rd and the 5th thermostating time (5○t) compared with the interval 1-3 hours (3.3○t). compared with the blank, in sample p2, which suffered thermic shock after 30 minutes of thermostating, it can see that the hypothermic impact (suffered in that time), was much felt by the starter bacteria, whose enzymatic activity has been drastically reduced, manifested by growth, in the end, of the titratable acidity with only 8.3○t. sample p3, which underwent hypothermic shock after 90 minutes of thermostating, recorded values very close to those of the sample p2, in terms of ph and titratable acidity. thus, the ph values fell from ph 6.47, after the first thermostating hour, to ph 6.2 (after 5 hours) – a more pronounced decrease being registered from 3 to 5 hours of processing (0.25 ph units). in sample p3, the growth of the titratable acidity was higher between the 3rd and the 5th thermostating time (5○t) compared with the interval 1-3 hours (2.5○t). when the hypothermic shock has been applied after 150 minutes of termostating (sample p4), the ph value has decreased, in the end, with 1.60 ph units; a more pronounced reduction being in the range of 1 to 3 hours (0.9 ph units beside 0,7 ph for range of 3 to 5 hours). in p4 sample, the titratable acidity has increased from 25.2○t, after the first time, at 72○t, after 5 hours, that is with 46.8○t. in this case, the biggest increase of this index was in the range of 1 to 3 hours of thermostating (with 28.2○t). sample p5 underwent hypothermic shock after 210 minutes of thermostating. in this sample, ph values has decreased, in the end, with 1.7 ph units, a more pronounced reduction being in the range of 1 to 3 hours (0.9 ph units beside 0.8 ph for range of 3 to 5 hours). the sample number and range of application of thermal shock from the start of thermostating process analyses made thermostating duration 1 h 2 h 3 h 4 h 5 h p1 (blank) p2 (hypothermic shock after 30 thermostating minutes) p3 (hypothermic shock after 90 thermostating minutes) p4 (hypothermic shock after 150 thermostating minutes) p5 (hypothermic shock after 210 thermostating minutes) ph acidity (t) ph acidity (t) ph acidity (t) ph acidity (t) ph acidity (t) 6.60 6.48 5.65 4.80 4.60 25 29 56 61 82 6.56 6.51 6.42 6.39 6.15 22.0 23.5 25.3 26.5 30.3 6.47 6.40 6.35 6.40 6.20 22.5 23.5 25.0 29.5 30.0 6.50 6.48 5.60 5.20 4.90 25.2 35.5 53.4 58.2 72 6.50 6.45 5.60 5.10 4.80 25.5 34 56 65 78 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 62 table 4 noting of curd is played as follows: in the same sample, the titratable acidity has increased from 25.5○t, after the first time, at 78○t, after 5 hours, that is with 52.5○t. in this last case, the biggest increase in this index was in the range of 1 to 3 hours of termostating (with 30.5○t). in the table 5 is reproduced the evolution of milk coagulation under hypothermic shocks. table 5 the comparative evolution of milk coagulation during yoghurt processing, in terms of thermal shocks application the coagulation evolution of milk sowed with starters cultures, during thermostating period the sample number and range of application of thermal shock from the start of thermostating process 1 h 2 h 3 h 4 h 5 h p1 (blank) 0 + + + + + + + + + + + p2 (hypothermic shock after 30 thermostating minutes) 0 0 0 0 0 p3 (hypothermic shock after 90 thermostating minutes) 0 +  +  + + p4 (hypothermic shock after 150 thermostating minutes) 0 + + + + + + + + p5 (hypothermic shock after 210 thermostating minutes) 0 + + + + + + + + + + as seen in table 2, the milk coagulation during yoghurt processing had a different evolution in samples subjected to hypothermic shocks compared to blank. in blank (p1), the curd was: formed (after 2 and 3 hours), well-formed (after 4 hours) and very well-formed (after 5 hours). if in the sample p2 the curd has not appeared at all during yoghurt processing, in the other samples the curd had a different evolution, depending on the application of hypothermic shock. thus, in p3 after 2 and 3 hours the curd has been very weak, and after 4 and 5 hours its aspect was of curd which begins to form. in the sample p4, the curd was formed after 2 hours, and it was maintained in this form up to the end of process (without being well-formed). in the sample p5, the curd was formed after 2 hours, then it bacame very wellformed after 4 hours, and it was maintained in this form up to the end of process. application of hypothermic shock in the early period of thermostating (the sample p2) has made the coagulation process of milk to be blocked. the later applied hypothermic shock, the lower effect on coagulation process was (samples 4 and 5). the table 3 reproduces ph and acidity values of yoghurt samples, with addition of starch or gelatin. according to the table 3, in blank (p1) ph values fell from 6.6 (after first hour) to 4.6 (after 5 hours of thermostating). the tiratable acidity of blank registered a difference of 57○t between the initial value (25○t) and the final value (82○t) of this index. 0 negative +  very weak curd + curd begins to form + + curd formed + + + well-formed curd + + + + very well-formed curd food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 63 table 3 biochemical index values of yoghurt samples, with addition of starch and gelatin, in certain proportions in the sample p2, (with 0.5% starch) ph values fell from ph 6.7, after the first thermostating hour, to ph 5.06 (after 5 hours). as to titratable acidity, the growth of this index was 45.5○t, and its final value (in the end of process) was 70○t. in the sample p3 (with 1.5% starch) the ph values fell from ph 6.60, after the first thermostating hour, to ph 4.7 (after 5 hours). the titratable acidity has grown from 25 to 78○t (in the end of process), that is a difference of 53○t. the sample p4 (with 2.5% starch) has registered a decrease of ph values from ph 6.72, after the first thermostating hour, to ph 4.69 (after 5 hours). the titratable acidity has grown from 24.6 to 79○t (in the end of process), that is a difference of 54.4○t. in the sample p5 (with 0.5% gelatin) ph values fell from ph 6.68, after the first thermostating hour, to ph 4.45 (after 5 hours). as to titratable acidity, the growth of this index was 65○t the final value of this index (after 5 hour of thermostating) being 90○t. the sample p6 (with 1.5% gelatin) has registered a decrease of ph values from ph 6.50, after the first thermostating hour, to ph 4.52 (after 5 hours). the titratable acidity has grown from 30.4 to 88○t (in the end of process), that is a difference of 58.6○t. in the sample p6 (with 2.5% gelatin) ph values fell from ph 6.48, after the first thermostating hour, to ph 4.40 (after 5 hours). as to titratable acidity, the growth of this index was 60.4○t the final value of this index (after 5 hour of thermostating) being 93○t. comparing the values of the indexes in tab. 3, it can see that in the case of samples with gelatin admixture ph values were lower, and the titratable acidity higher than blank. along with the increasing of gelatin amount added, the titratable acidity has grown too. in all samples with starch added, the titratable acidity value was less then blank. it is known that ph and titratable acidity are biochemical indices expressing, indirectly, the fermentation activity of lactic bacteria. since no starch or gelatin can be used directly as a source of carbon and energy by lactic bacteria cultures, this increasing of the fermentation activity (in thermostating duration the sample number and the compound added analyses made 1 h 2 h 3 h 4 h 5 h ph 6.60 6.48 5.65 4.80 4.60 p1 (blank) acidity (○t) 25 29 56 61 82 ph 6.70 6.48 6.01 5.15 5.06 p2 (0.5% starch) acidity (○t) 24.5 30 404 57 70 ph 6.60 6.50 5.50 4.95 4.70 p3 (1.5% starch) acidity (○t) 25 31.2 57 59.4 78 ph 6.72 6.50 5.54 4.93 4.69 p4 (2.5% starch) acidity (○t) 24.6 31 54 60.4 79 ph 6.68 6.42 5.48 4.89 4.45 p5 (0.5% gelatin) acidity (○t) 25 34.8 56.2 60 90 ph 6.50 6.34 5.89 5.10 4.52 p6 (1.5% gelatin) acidity (○t) 30.4 39.8 49.6 58.4 88 ph 6.48 6.35 5.52 5.06 4.40 p7 (2.5% gelatin) acidity (○t) 32.6 38.8 54.2 58 93 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 64 the case of gelatin addition) could be explained by the role of oxygen barrier that plays the gelatin inside emulsions (7). limiting or blocking access of oxygen within emulsion (milk) mass, favors the fermentation activity of lactic cultures. in the table 4 is rendered the evolution of milk coagulation during yoghurt processing. as seen in table 4, the milk coagulation during yoghurt processing had a different evolution. in blank (p1), the curd was formed (after 2 and 3 hours), well-formed (after 4 hours) and very well-formed (after 5 hours). in the sample p2 (0.5% starch) the curd was very weak after 2 and 3 hours, it began to form after 4 hours and is formed in the end of process. at sample p3 (1.5% starch), the curd was very weak after 2 and 3 hours, it began to form after 4 hours, an was formed after 5 hours (in the end). the curd of sample p4 (2.5% starch) was very weak after 2 and 3 hours, it began to form after 4 hours, and was wel-formed in the end of process. table 4 the comparative evolution of milk coagulation during yoghurt processing, in terms of some compound additions the coagulation evolution of milk sowed with starters cultures, during thermostating period the sample number and the compound added 1 h 2 h 3 h 4 h 5 h p1 (blank) 0 + + + + + + + + + + + p2 (0.5% starch) 0 +  +  + + + p3 (1.5% starch) 0 +  +  + + + p4 (2.5% starch) 0 +  +  + + + + p5 (0.5% gelatin) 0 +  + + + + + + p6 (1.5% gelatin) 0 +  + + + + + + + p7 (2.5% gelatin) 0 + + + + + + + + table 5 noting of curd is played as follows: 0 negative +  very weak curd + curd begins to form + + curd formed + + + well-formed curd + + + + very well-formed curd at sample p5 (0.5% gelatin), the curd was very weak after 2 hours, it began to form after 3 hours, it bacame formed after 4 hours, and well-formed after 5 hours (in the end). in the sample p6 (1.5% gelatin) the curd was very weak after 2 hours, it began to form after 3 hours, was formed after 4 hours, and very well-formed in the end of process. the curd of sample p7 (2.5% gelatin) began to form after 2 and 3 hours, it became formed after 4 hours, and very well-formed in the end of process. analysing the data in the table 4, it seems the addition of gelatine or starch has not food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 65 rushed the milk coagulation process during yoghurt processing. compared to the control, in samples with admixtures the curd formation was slower, especially on samples with starch addition. 4. conclusions during yoghurt processing, the application of some hypothermic shocks and the introduction of some compounds has influenced the evolution of acidity and the milk coagulatin process. application of hypothermic shocks in the early period of thermostating has determined the decrease of fermentation activity of lactic bacteria (expressed by reduction of titratable acidity values) and has made the coagulation process of milk to be blocked. the later applied hypothermic shock, the lower effect on titratable acidity and coagulation process was. the addition of gelatine or starch during yoghurt processing has influenced, indirectly, the fermentation activity of lactic bacteria. the gelatin addition has led to the increase of titratable acidity values, comparing to blank. the addition of gelatine or starch has not rushed the milk coagulation during yoghurt processing. compared to the control, in samples with admixtures the curd formation was slower, especially in those ones with starch addition. 5. references [1].juillet m.t., bornet f., 1998 – assurer une alimentation équilibrée popur maintenir la santé. ind. alim. agric., 115, 12, 19 [2] mincu i. et.al., 1989 – orientări actuale în nutriţie. ed. medicală, bucureşti [3] segal b. et al., 1987 – metode moderne privind îmbogăţirea valorii nutritive a produselor alimentare. ed. ceres, bucureşti [4] nicol m., 1995 – vitamines antioxydantés et bêta-caroténe: activités prevéentives en pathologie humaine. réunion commentée, mèd. et nutr., 31, 1, 41-46 [5] nagy k.,1999 – the role of food fortification in combating micronutrient deficiences. f hoffmann-la roche basleswitzerland [6] banu c. et al.., 2003 – procesarea materiilor prime alimentare şi pierderile de substanţe biologic active. ed. „tehnica” utm, chişinău, 138 [7] segal rodica, 1999 – alimente fortifiate, in alimente funcţionale de g.m. costin şi rodica segal (edit.) et.al.,1999, ed. academica, galaţi, 308 [8] avramiuc m., leahu a., 2009 – the influence of some metabolits addition on fermentation process, during butter milk manufacturing. journal of agroalimentary processes and technologies, timişoara, vol. xv, nr. 1, 88-93 [9] cunniff p. (ed), 1995 – official methods of analysis of aoac international (16th ed., vol 1,2), association of officinal analytical chemists international, arlington, va. [10] costin g.m. si segal r. (edit.) et..al., 2003 – ştiinţa şi ingineria fabricării brânzeturilor. ed. academica, galaţi, 502, 503, 504, 510. microsoft word 9 codina georgiana.doc 59 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag.59 63 the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour *georgiana gabriela codină1, silvia mironeasa1 1faculty of food engineering, stefan cel mare university, suceava, romania, codina@fia.usv.ro; silviam@fia.usv.ro *corresponding author received 10 january 2013, accepted 26 february 2013 abstract: lecithin is a natural surfactant, which could be used like bread improver in order to improve dough rheological behavior, bread quality characteristics and it sensorial properties. the aim of this study was to investigate the effect of lecithin which was added in different doses (0.1-0.5%) into flour with a very good quality for bread making on dough rheological properties and bread quality. dough rheological properties measured were dough resistance (p), dough extensibility (l), index of swelling (g), baking strength (w) and configuration ratio (p/l) using a chopin alveograph. the presence of lecithin influences the alveogram characteristics by increasing dough resistance, baking strength, the configuration ratio p/l and by decreasing dough extensibility and index of swelling. the bread quality characteristics like loaf volume, porosity and elasticity were improved by lecithin addition. also, bread sensorial characteristics like external appearance, crust aspect, firmness and taste were better evaluated in the sample with lecithin addition. therefore, by using lecithin on bread making, the effects of baked products are visible in terms of loaf volume, porosity and elasticity increasing, the freshness preserving, as well as the crumb structure improvement. all the results obtained in this study suggested that lecithin can be used in small concentrations as an excellent improver in bread making. keywords: lecithin, rheological properties, bread characteristics, wheat flour 1. introduction the lipids content in wheat flour is minor and varied between 1.5% and 2.0% most of them being located in the endosperm, germ and aleurone tissue of the wheat kernels [1]. it has been demonstrated that the lipid fractions may be involved in the glutenic complex either through hydrophilic bonds or through hydrophobic interactions. as far as starch-lipid interacttions are concerned, the lipid fractions appear as inclusions in the matrix of amylase polyglucans or are chemically tied to carbohydrates [2]. the three main sources of lipids in a typical bread formula are wheat flour, shortening and surfactants [3]. lecithin, a mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol [4] is a naturally surfactant frequently used in bread making. this type of polar lipid is one of the most widespread phospholipids in nature and is contained in practically all living cells [5]. it can be isolated on an industrial scale from plant sources like soybean, rapeseed, sunflower, e.g. and can be used as improvers in breadmaking, in concentrafood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 georgiana gabriela codină, silvia mironeasa, the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour, food and environment safety, volume xii, issue 1 – 2013, pag. 59 63 60 tions up to 6 g/kg of flour [6]. because of its phospholipids content it can act as emulsifiers, viscosity regulators and dispersing agents [7] in bread making and it improve dough fermentation behaviour, the wheat flour dough rheological properties [8], bread quality characteristics like loaf volume, crumb structure and bread preservation [6] and desirable textural properties such as tenderness, richness, and improved mouth feel [9]. the aim of the present study was to investigate the effect of lecithin on wheat flour with a very good potential for breadmaking like raw material. the objectives were to (1) investigate the effect of lecithin on alveograph rheological characteristics of wheat flour dough chosen like raw material; (2) to determine the bread characteristics (loaf volume, porosity, and elasticity) made with different addition levels of lecithin, and (3) to asses the bread sensorial characteristics made with and without exogenous dose of lecithin addition. 2. materials and methods the experiments were carried out on a very good flour quality for bread making. like flour improver was used crude lecithin from s.c. enzymes@derivates romania (costişa, neamţ, românia). the physical-chemical composition of the control flour was analysed according to romanian standards methods: sr 90:2007, sr en iso 3093:2007, sr en iso 2171:2002, sr iso 711:1999, sr 130133:1994. the determined values of its physical-chemical properties are the following: acidity 2.2, wet gluten content 27.2 %, gluten deformation index 8 mm, falling number 296 s, ash content 0.64%, humidity 14.3% and protein content 12.42%. the rheological properties of the control sample and of the flours with different addition levels of lecithin were made on chopin alveograph according to sr iso 5530-4:2005. the parameters obtained from the alveograph curve are: p, the maximum pressure needed to blow the dough bubble, expresses dough resistance; l, length of the curve, expresses dough extensibility; p/l, configuration ratio of the alveograph curve; g, index of swelling; w, baking strength (surface area of the curve) [10]. starting from the flour chosen like row material in experiments, different doses of lecithin were used as follows:  m control sample (sample, without exogenous lecithin);  p1 sample, with 0.1% addition level of lecithin;  p2 sample, with 0.2% addition level of lecithin;  p3 sample, with 0.3% addition level of lecithin;  p4 sample, with 0.4% addition level of lecithin;  p5 sample, with 0.5% addition level of lecithin. bread quality characteristics were determined according to sr 91:2007 (ref.). sensory characteristics for the bread with lecithin addition, that had the best score for the bread making characteristics, compared with the control sample, was selected. the representative sensorial attributes of bread qualities in conformity with the method described by [11] the romanian diagram of evaluation for determining bread quality up to 20 points were: external appearance, crust aspect, firmness, flavor and taste. statistical analysis. all analyses were carried out in duplicates and the data obtained were analyzed using excel software. values of the parameters are expressed as the mean ± standard deviation to a confidence interval of 95%. 3. results and discussion dough rheological characteristics measurement by alveograph food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 georgiana gabriela codină, silvia mironeasa, the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour, food and environment safety, volume xii, issue 1 – 2013, pag. 59 63 61 table 1 shows the flour parameters samples with and without lecithin addition recorded by the alveograph device. the curves performed by dough rheological behaviour through alveograph method are shown in fig. 1. table 1 alveograph characteristics of flour samples with and without lecithin addition samples parameters symbol m p1 p2 p3 p4 p5 maximum pressure (mm) p 76 ± 0.1 84 ± 0.1 94 ± 0.1 113 ± 0.2 117 ± 0.2 121 ± 0.2 extensibility (mm) l 85 ± 0.2 81 ± 0.2 76 ± 0.1 66 ± 0.2 64 ± 0.1 64 ± 0.1 swelling index (mm) g 20.5 ± 0.1 20.0 ± 0.1 19.4 ± 0.2 18.1 ± 0.1 17.8 ± 0.2 17.8 ± 0.2 deformation energy (10−4 j) w 211 ± 1 225 ± 2 252 ± 1 264 ± 1 275 ± 1 286 ± 1 alveograph ratio p/l 0.89 1.04 1.24 1.71 1.83 1.89 as it can be seen from the table 1 data the amphiphilic nature of lecithin addition in wheat flour dough conducted to an increase of dough strength in the way of a higher tenacity compared to a lower extensibility which resulted in an increase of the alveograph ratio p/l from 0.89 to 1.89. lecithin by it lipophilic and hydrophilic part may form transversal bindings between granular starch and gluten and between gliadin and glutenin forming lipoprotein complexes between starch, gluten and other hydrophobic components. depending on the binding’s complexity it can be formed cross-links which contribute to dough compaction and stabilization and to the formation of the interface resistant films to gas expansion. the dough strengthening effect of lecithin it may be caused by displacement of the flour lipid binding by lecithin in the dough system. binding of their lipophilic part to proteins during mixing increases protein aggregation and dough manageability [3]. this fact may predict good dough handling properties and a large fermentation tolerance, especially during final proof [8]. figure 1. alveograms of the analyzed samples (m, p1, p2, p3, p4, p5) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 georgiana gabriela codină, silvia mironeasa, the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour, food and environment safety, volume xii, issue 1 – 2013, pag. 59 63 62 bread making characteristics bread loaf volume, porosity and elasticity of the samples with and without lecithin addition are shown in figure 2. with the increase dose of lecithin addition, loaf volume, porosity and elasticity increase up to 0.3% lecithin addition, for all the samples the values obtained being higher compared to the control one. this positive effect of lecithin on bread characteristics is due especially to its phospholipids content [6]. the phospholipids increase the dough ability of gas-retaining which depends on the foaming properties and stability of the aqueous phase in dough influenced by them [12]. also phospholipids play an important role in maintaining a stable gas cell structure via its interaction with protein at the gas/liquid interface of liquid lamellae surrounding gas cells in the bread dough [1]. 0 50 100 150 200 250 300 350 400 m p1 p2 p3 p4 p5 volume (cm3/100g product) elasticity (%) porosity (%) figure 2. loaf volume, elasticity and porosity of analyzed samples bread sensorial characteristics the sensorial characteristics of the control sample (m) and of the sample with 0.3% addition level of lecithin (p3) are shown in figure 3. the bread supplemented with lecithin presented a better evaluation regarding external appearance, crust aspect, firmness and taste. the crumb structure became more uniform, less gumminess with a brighter color. this fact is explainable because water absorption and swelling of the granules is postponed through interaction of lecithin with starch during baking, which creates a softer crumb structure [3], and therefore improve the bread firmness and its external appearance. 0 2 4 6 external appearance crust aspect firmnessflavor taste m p3 figure 3. sensory evaluation of wheat bread samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 georgiana gabriela codină, silvia mironeasa, the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour, food and environment safety, volume xii, issue 1 – 2013, pag. 59 63 63 4. conclusions flour replacement at different levels (0.1– 0.5%) by lecithin significantly changes the measured dough properties by an alveograph device. lecithin reduced the dough extensibility and increase dough resistance in the alveograph, which indicates its strengthened effect on the dough. regarding the effects that lecithin has on the baking quality characteristics, we concluded that for a 0.3% dose of lecithin addition, we obtained the best quality in terms of bread characteristics. 5. reference [1]. sun h., yan s., jiang w., li g., macritchie f., contribution of lipid to physicochemical properties and mantoumaking quality of wheat flour, food chemistry, 121. 332-337 (2010) [2]. leahu a., codină g.g., mironeasa s., roşu a.i., effects of a2 phospholipase on dough rheological properties and bread characteristics, food and environment safety, x. 66-71 (2011) [3]. pareyt b., finnie s.m., putseys j.a., delcour j.a., lipids in bread making: sources, interactions, and impact on bread quality, journal of cereal science, 54. 266-279 (2011) [4]. ai, y., hasjim j., jane j.l., effects of lipids on enzymatic hydrolysis and physical properties of starch, carbohydrate polymers, 92. 120-127 (2013) [5]. campanella l, pacifici f., sammartino m.p., tomassetti m., a new organic phase bienzymatic electrode for lecithin analysis in food products, bioelectrochemistry and bioenergetics, 47. 2538 (1998) [6]. helmerich g., koehler p., functional properties of individual classes of phospholipids in breadmaking, journal of cereal science, 42. 233-241 (2005) [7]. comas d.i., wagner j.r., tomás m.c., creaming stability of oil in water (o/w) emulsions: influence of ph on soybean protein–lecithin interaction, food hydrocolloids, 20. 990–996 (2006) [8]. stampfli l., nersted b., molteberg e.l., effects of emulsifiers on farinograph and extensograph measurements, food chemistry, 57. 523-530 (1996) [9]. huschka b., bonomi f., marengo m., miriani m., seetharaman k., comparison of lipid effects on structural features of hard and soft wheat flour proteins assessed by front-face fluorescence, food chemistry, 133. 1011-1016 (2012) [10]. codină g.g., mironeasa s., mironeasa c., popa c.n., tambaberehoiu r., wheat flour dough alveograph characteristics predicted by mixolab regression models, journal of the science of food and agriculture, 92. 638-644 (2012) [11]. bordei d., bahrim g., pâslaru v., gasparotti c., elisei a., banu i., georgescu l., codină g., quality control in bakery industry methods of analysis, academica, galati, 783 p, (2007) [12]. ukai t., urade r., cooperation of phosphatidylcholine with endogenous lipids of wheat flour for an increase in dough volume, food chemistry, 102. 225-231 (2007) [13]. sr 90:2007. wheat flour. analysis method, standardization association of romania (asro), bucharest, romania [14]. sr en iso 3093:2007. wheat, rye and respective flours, durum wheat and durum wheat semolina determination of the falling number according to hagberg-perten, standardization association of romania (asro), bucharest, romania [15]. sr en iso 2171:2002. cereals, pulses and by-products determination of ash yield by incineration, standardization association of romania (asro), bucharest, romania [16]. sr iso 711:1999. cereals and cereal product. determination of moisture content (basic reference method), standardization association of romania (asro), bucharest, romania [17]. sr 13013-3:1994. cereal, milled cereal and panification products, biscuits and flour pastes. raw protein determination, standardization association of romania (asro), bucharest, romania [18]. sr iso 5530-4:2005. wheat flour (triticum aestivum l.). physical chraracteristics of doughs. part 4: determination of rheological properties using an alveograph, standardization association of romania (asro), bucharest, romania [19]. sr 91:2007. bread and fresh pastry products. methods of analysis, standardization association of romania (asro), bucharest, romania 376 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 376 384 new fri end ly env iron me nta l etchi ng t echno log ies for som e st e els an d co pper a lloys vira vodyanka1, sergiy boruk2, *igor winkler2 1kyiv national university of commerce and economy, chernivtsi college of commerce, vera-79@ukr.net 2yu. fedkovych national university of chernivtsi, ukraine, i.winkler@chnu.edu.ua *corresponding author received november 18th 2014, accepted december 29th 2014 abstract: new compositions containing some organic inhibitors were tested in the carbon steels and copper alloys etching processes. the analysis of the etching efficiency and harmful products emission proves that significant reduction in the specific discharge of the waste waters and emission of nitrogen oxides can be achieved at the appropriate etching quality. this reduction is caused mainly by adsorption of the organic inhibitors on the metal surface by decreasing its excessive dissolution. on the other hand, the effective aggregation and dispersion of the corrosion products promotes faster transportation of the rust and dross particles away from the treated metal surface reducing the total processing time. keywords: environmental safety; corrosion; etching; carbon steels, copper alloys 1. introduction the galvanic industry is the second highest pollution source after the surface transport branch and it causes continuous and longlasting contamination of the entire environment (air, water and soil). therefore, the extensive efforts should be directed to prevent this dangerous contamination bringing many aggressive components of the galvanic (including metals etching) composition into the enevironment [1-7]. among the others, the metal etching technologies produce significant amounts of the air and water pollution agents and extended activity is directed onto investigation of possible mitigation steps, which should provide same etching quality and efficiency at lower emission of pollutants. sulfuric, hydrochloric and nitric acids are used as the main components for the lowalloy carbon steel etching compositions [2 3]. significant amounts of iron can dissolve in sulfuric acid during this process while dissolution of iron in the hydrochloric acid compositions is less influential. on the other hand, a rate of the metal rust and scale dissolution in the latter compositions is higher than in the former, therefore, the hydrochloric compositions are more suitable for the low-allow carbon steel etching [2, 3, 8]. an optimal content of sulfuric acid in the regular etching compositions is 20-22 %; hydrochloric acid – 18-20 %. additional organic substances are usually added to the both types of the compositions to counteract excessive metal dissolution, decrease emission of the gaseous pollutions agents and minimize amount of the wastewater formed. thiocarbamidehydrosulfite-, sulfite-, thiosulfateand thiocyanate compounds are used regularly in the nitric acid based etching compositions in order to protect the metal surface from its dissolution. however, these compounds are quite toxic and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 377 can provide effective surface protection within quite a narrow ph range only [2, 3, 8-11]. it is obvious, that such inhibitors should be substituted with different, less toxic substances with the wider working ph range. the phosphoric acid etching compositions are also used in industry. trace amounts of the unwashed acid can provide additional corrosion protection since iron phosphates are insoluble and form quite dense surface protection film. on the other hand, this acid is weaker and effective etching requires the solution to be heated up to 85 0c, causing rise in the processing cost. due to this shortcoming, the phosphoric compositions have quite limited usage and are recommended mostly for the fine etching of the surfaces to be dyed. a number of the mixed acids compositions are also used in the modern industry for etching [2, 3]. two types of compositions are used for the chemical polishing of copper and its alloys. the first type consists of various mixtures of nitric (5-20 vol. %), phosphoric (30-80) and acetic (10-50) acids and the second type is based on hydrogen peroxide. the higher is concentration of nitric acid, the worse is the surface glitter while efficiency of the polishing decreases with rise of the phosphoric acid content. excessive concentration of the acids may result in the unwanted etching process running along with the polishing. most polishing compositions are effective only for the temperatures under 323-373 k and for the copper contents below 30-36 g/l [2, 3 1214]. this process is unsafe environmentally and causes emission of harmful nitrogen oxides [2, 3]. various nitrogenand sulfur-containing substances can be used in the polishing technology as inhibitors. for instance, urotropine is widely used in various composite inhibitors together with catapine, tribenzenamine or thiocarbamide. the former substance exhibits own inhibition activity and promotes higher activity of the other components. however, such compositions are used at the temperatures above 298 k, which increases the production cost. as reported in [15-19], some nitrogen-containing heterocycles (derivatives of quinoline, pyridine, acrydine, indole and others) can provide an effective inhibition for the acidic polishing mixtures. however, development of specific polishing compositions is still required in various industrial branches. this paper deals with investigation of some new effective, nontoxic, environment-friendly, inexpensive compositions for the acidic etching of the carbon steels, copper and its alloys, which produce reduced amounts of the harmful products. as stated above, the carbon steel etching compositions are usually based on the mixture of hydrochloric and nitric acids. as the latter agent is the strong oxidizer, the proposed inhibition compositions should remain active and stable in such aggressive solution. two or more different inhibition agents are usually mixed in various compositions [2, 3. 14-19] in order to cover all technological range of temperatures, ph and other parameters of the solutions. the protective activity of the compositions and the range of their usability can be changed by selection of various concentrations of the components. on the other hand, changes in these concentrations would also influence the technological characteristics of the etching process and emission of the harmful compounds. therefore, such an investigation should be aimed onto finding of the environmental friendly compositions ensuring high technological efficiency food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 378 2. experimental two groups of parameters have been measured in order to evaluate influence of the admixtures on the process of the metal surface etching. a: environmental parameters: specific emission of nitrogen oxides; concentration of metal ions in the wastewater; specific amount of the wastewaters formed; b: technological parameters: etching quality; mass loss and rate of the metal dissolution. all experiments were carried out at 293±2 k during 5 minutes (for the steel samples) or 30 seconds (copper and copper alloys samples) using the etching compositions with two different inhibitors. the first inhibitor was taken from the group i: benzenesulfonic acid; sulfosalicylic acid; sulfanilic acid while the second agent was taken from the group ii: thiosemicarbazide; acetylcarbamide; pyrimidone; carbamide; n-n-etoxyphenylcarbamide. the most optimal concentrations of the inhibitor i and inhibitor ii for the carbon steels st10 and st3 were sought for the following acidic etching composition (1) (g/l): hydrochloric acid (ρ = 1.19 g/ml) 200; nitric acid (ρ = 1.34 g/ml) 120; inhibitor i up to 10 inhibitor ii up to 2.0. as seen from the electrochemical data (see table 1, 2), the majority of the group ii inhibitors show good efficiency for etching the st10 steel samples in the composition (1) with sulfosalicylic acid (10 g/l). the group i inhibitors exhibit the mixed type of activity and can decelerate both cathodic (hydrogen release) and anodic (metal dissolution) processes (see table 1). a technologically sufficient metal protection can be ensured at 293 k by adding of 2 g/l of acetylcarbamide or 0.2 g/l of thiosemicarbazide together with 10 g/l of sulfosalicylic acid to the composition (1) (table 2). for example, the corrosion retardation coefficient γ = 4,8 and 15.0 and the metal surface protection degree was 79.2 % and 93.3 % for acetylcarbamide and thiosemicarbazide simultaneously. since effective corrosion protection requires γ ≥ 8 or the metal protection degree over 87 %, thisosemicarbazide can be classified as very effective inhibitor while efficiency of acetylcarbamide is lower but still sufficient. both substances contain atoms of nitrogen and oxygen (or sulfur) with the opposite effective charges, which facilitates possible adsorption on both anodic and cathodic areas on the metal surface and formation of the dense adsorption layer. sulfosalicylic acid provides additional retardation of the metal dissolution because it contains the functional groups so3hі oh-, which exhibit some inhibition effect on the anodic areas. all these properties ensure good inhibition effect and prove the assumption of higher efficiency of the two-component inhibitors protecting the metal surface during its etching [2, 3]. table 1 some electrochemical parameters of the st10 steel etching processes with or without inhibitors of the group i inhibitor і,a/dm2 φs, v z, % γ сa, % wc, g/mm2∙year source composition (no inhibitor used) 0.024 0.391 40 1.68 benzenesulfonic acid 0.017 0.370 29.17 1.4 33 1.16 sulfosalicylic acid 0.016 0.382 33.3 1.5 37.5 1.12 sulfanilic acid 0.014 0.382 41.7 1.7 44 0.96 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 379 3. results and discussion the inhibition efficiency has been estimated (see tables 2, 3) using the following parameters: i – current density (a/dm2); φs – potentials of the metal sample surface (v); wc – index of the metal corrosion dissolution (g/mm2 hour); z – inhibition activity coefficient (%); γ – corrosion retardation coefficient; ca – anodic control degree (%). current density (i) and potentials of the metal sample surface (φs) were determined through direct instrumental measurements. index of the metal corrosion dissolution (wс) was calculated by the formula: wс = ∆m /(s×t), where ∆m – change in the steel or copper sample mass, g; t – duration of the process, h; s – surface area of the metal sample, m2 (we used samples with s = 0.1 m2); wс – rate of the metal surface dissolution, g/m2·h. the value (z) – metal surface protection degree characterizes depth of the corrosion retardation and can be calculated by the formula: %100 0 0      z , where  and 0 – corrosion rates with and without inhibitor simultaneously. the dimensionless corrosion retardation coefficient γ shows the ratio of the corrosion rate retardation by the given inhibitor and can be calculated as: ,0    where  and 0 are similar to the formula above. anodic control degree (ca) was calculated by the formula: ac = %100 0e ea   , where ae – is a shift of the working anodic potential from its initial value under the given electric current; 0e – the initial potentials difference between the cathodic and anodic areas. table 2 some electrochemical parameters of the st10 steel etching process in the solution consisting sulfosalicylic acid and some inhibitors of the group ii inhibitor і,a/dm2 φs, v z, % γ сa, % wc, g/mm2∙year 1 g/l acetylcarbamide 0.014 0.362 41.7 1.7 50 0.96 2 g/l acetylcarbamide 0.005 0.380 79.2 4.8 50 0.36 1 g/l carbamide 0.01 0.396 58.3 2.4 50 0.68 2 g/l carbamide 0.006 0.397 75.0 4.0 50 0.4 1 g/l n-netoxyphenylcarbamide 0.018 0.376 25.0 1.3 50 1.24 2 g/l n-netoxyphenylcarbamide 0.02 0.375 0.17 1.2 50 1.4 1 g/l pyrimidone 0.01 0.382 58.3 2.4 67 0.68 2 g/l pyrimidone 0.009 0.391 62.5 2.7 60 0.64 0.1 g/l thiosemicarbazide 0.002 0.397 91.7 12.0 50 0.12 0.1 g/l thiosemicarbazide 0.0016 0.398 93.3 15.0 60 0.12 table 3 represents some parameters of st10 and st 3 etching in the acidic composition (1) with some inhibitors of the group i. as seen from table 3, efficiencies of benzenesulfonic acid and sulfosalicylic acid are similar and higher than efficiency of sulfanilic acid. therefore, the two former agents can ensure more efficient etching with better environmental parameters. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 380 since sulfosalicylic acid is cheaper and used widely in the industry, it was selected for our further investigations as the most effective inhibitor of the group i. table 3 some parameters of st10 and st 3 etching in the composition (1) with some inhibitors of the group i (*) inhibitor c. g/l (δm.mg)/(d. μm) wc. g/m 2·h vnox. mg/l vs. l/m 2 composition (1). no inhibitors 98/1.4 117.6 112.3 38 benzenesulfonic acid 10.0 28/0.40 33.6 52.9 14 sulfosalicylic acid 10.0 29/0.41 34.8 41.7 15 sulfanilic acid 10.0 51/0.73 61.2 71.3 24 (*) c – inhibitor concentration in the etching composition; δm – changes in the sample mass after etching; d – thickness of the dissolved metal layer; wc – rate of the metal dissolution; vnox – specific emission of nox; vs – specific volume of the wastewater formed. an influence of the group ii agents together with sulfosalicylic acid has been investigated at the next stage (see results in table 4). table 4 some parameters of st10 and st3 steels etching in the composition (1) with sulfosalicylic acid and the group ii agents (*) inhibitor c. g/l (δm.mg)/(d. μm) wc. g/m 2·h vnox. mg/l vs. l/m2 carbamide 2.0 20/0.29 24.0 24.3 10.0 acetylcarbamide 2.0 7/0.10 8.4 4.5 3.5 pyrimidone 2.0 10/0.14 12.0 5.6 5.0 n-n-etoxyphenylcarbamide 1.5 42/0.60 50.4 34.4 21.0 thiosemicarbazide 0.2 3/0.04 3.6 3.75 3.0 (*) all notations are similar to those in table 3. the compositions with acetylcarbamide (2.0 g/l) and thiosemicarbazide (0.2 g/l) together with sulfosalicylic acid prove the best inhibition efficiency since their inhibition and dispersing properties ensure better environmental and technological parameters of surface treatment of the carbon steels, copper and its alloys (see table 4 and 5). specific emission of the nitrogen oxides reduces tenfold (from 0.0082 to 0.00048 mg/m3 for the carbon steel etching and down to 0.088 mg/m3 for the copper alloys). amount of the wastewaters formed reduces 3-4 times, the rate of the metal dissolution decreases by one order of magnitude and quality of the surface after the treatment becomes significantly higher comparing to the results achieved by the traditional technologies. therefore, the following two compositions can be recommended for the carbon steels etching: table 5 the compositions recommended for the carbon steels etching composition №1 (g/l): composition №2 (g/l): composition №3 (g/l): hydrochloric acid (ρ=1.19 g/ml) – 200; nitric acid (ρ=1.34 g/ml) – 120; sulfosalicylic acid – 10; acetylcarbamide – 2.0; distilled water – up to 1 l hydrochloric acid (ρ =1.19 g/l) – 200; nitric acid (ρ =1.34 г/см3) – 120; sulfosalicylic acid – 10; thiosemicarbazide – 0.2; distilled water – up to 1 l phosphoric acid (ρ = 1.834 g/ml) – 275; acetic acid (ρ = 1.049 g/ml) – 125; nitric acid (ρ = 1.340 g/ml – 100; thiosemicarbazide – 0.15; distilled water up to 1 l. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 381 a series of the thioamides derivatives was checked for their inhibition efficiency in the copper and copper alloys etching and polishing (see table 6) and phenylthiosemicarbazide and thiosemicarbazide (0.15 g/ml) have shown the best inhibition and brightening performance. these substances also facilitate the significant reduction in nitrogen oxides emission and in amount of the wastewaters formed. table 6 some parameters of the copper alloys polishing with and without inhibition agents (*) inhibition agent с. g/l δm. mg / d. μm wc. g/sm2·hour vnox. mg/l vs l/m 2 ∆. % polishing mixture. no inhibitor 430/6.14 860.0 181.0 18.2 +2 thiocarbamide 0.15 250/3.57 500.0 76.1 4.0 +15 thioacetamide 0.15 190/2.71 380.0 65.0 3.0 +4 rubeanic acid 0.15 170/2.43 204.0 59.0 12.5 -7 thiosemicarbazide 0.15 130/1.86 260.0 7.6 0.65 +17 phenylthiosemicarbazide 0.15 115/1.64 230.0 7.5 0.6 +12 (*) all notations are similar to those in table 3. ∆ means changes in the surface glitter. %. therefore, the following composition can be recommended for polishing of copper and its alloys (column 3 in table 5) . a character and nature of the inhibition activity of various agents can be understood from analysis of changes in the associative interaction between the group i and group ii compounds. this interaction causes some changes in the spectral parameters of the solution described in details below. at the next stage, an influence of the corrosion inhibitors (sulfosalicylic acid. thiosemicarbazide, carbamide, acetylcarbamide and pyrimidone) on environmental safety and performance of the metal etching compositions has been investigated. special attention has also been given to the predicted associative interaction between the group i agent (sulfosalicylic acid) and the group ii agents (thiosemicarbazide, carbamide, acetylcarbamide and pyrimidone). results of this investigation are represented in the tables 7 and 8. it can be seen that pyrimidone and thiosemicarbazide do not form any associates with sulfosalicylic acid since the characteristic light absorbance maximum of sulfosalicylic acid does not shift after mixing with both agents (see tables 6 and 7). therefore, these inhibitors adsorb on the metal surface independently. in contrary, formation of the associates between sulfosalicylic acid and carbamide has been determined and position of the light absorbance peak shifts for about 80 nm – from 250 to 330 nm (see fig. 1). sulfosalicylic acid and acetylcarbamide also form associates causing the acetylcarbamide maximum absorbance peak in the mixture to shift for 55 nm in comparison to the pure acetylcarbamide solution (see fig. 2). table 7 position of the light absorbance peak and its intensity for sulfosalicylic acid, pyrimidone and their mixture compound λmax lgε sulfosalicylic acid 337.5 4.83 pyrimidone and sulfosalicylic acid 337.5 4.90 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 382 table 8 position of the light absorbance peak and its intensity for sulfosalicylic acid. thiosemicarbazide and their mixture compound λmax lgε sulfosalicylic acid 337.5 4.83 sulfosalicylic acid and thiosemicarbazide 338.0 4.79 the above mentioned associates are comparatively instable since the shifted absorbance peaks were found returned to the previous positions in 2-3 hours. for instance. the pure acetylcarbamide spectral data showed the peak in the position 1. admixture of sulfosalicylic acid caused its shifting to the position 2 and then the peak was recorded in the position 1 again in 2-3 hours (see fig. 1). since c/a dependence (c – adsorbate concentration; a – adsorption value) on adsorbate concentration is linear (see fig. 3). the process of adsorption can be represented by the monomolecular langmuir mechanism. fig.1. dependence of the optical density (d) on wavelength (λ) for aqueous solutions of carbamide. and its mixture with salicylic acid; 1 – carbamide; 2 – mixture of sulfosalicylic acid and caramide (concentration of both compounds is 1•10-5 m/l). fig. 2. dependence of the optical density (d) on wavelength (λ) for aqueous solutions of acetylcarbamide. and its mixture with sulfosalicylic acid; 1 – acetylcarbamide; 2 – mixture of sulfosalicylic acid and acetylcaramide (concentration of both compounds is 1•10-5 m/l). as seen from fig. 4. sulfosalicylic acid adsorption on the iron oxides powder is higher than on iron and steel. similar comparison for acetylcarbamide proves its better adsorption on iron, then on steel and iron oxides (see fig. 5). as seen from the fig. 4 and 5, according to the descending adsorption activity in relation to sulfosalicilic acid (a) and acetylcarbamide (b), the adsorbents can be arranged in the following sequences: (a): feo > fe2o3 > fe > st10 (b): fe > st10 > feo > fe2o3. it was found that the descending adsorption activity sequence related to carbamide. pyrimidone and thiosemicarbamide is similar to the row (a). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 383 fig. 3. near-to-linear dependencies of c/a on the adsorbate (sulfosalicilic acid) concentration proves the langmiur mechanism of its adsorption on the following samples: steel st10 (1); iron powder (2); fe2o3 powder (3) and feo powder (4). fig. 4. adosrption isotherms of sulfosalcylic acid on: 1 – feo. 2 – fe2o3. 3 – fe. 4 – steel st10. effective aggregation and dispersion of the corrosion products are essential to ensure higher environmental safety of the etching composition. fast aggregation and dispersion of the rust and dross particles promote faster transportation of these products away from the metal surface, increase in the etching surface area and in general, higher rate of the process. therefore, total etching time decreases causing reduced emission of the hazardous etching gases. in this context, an influence of sulfosalicylic acid, thiosemicarbazide and their mixtures on the aggregation stability of the aqueous suspensions containing iron oxides and some corrosion products has been investigated. fig. 5. adosrption isotherms of acetylcarbamide on: 1 – fe. 2 – steel st10. 3 – feo. 4 – fe2o3. fig. 6. dependence of the sedimentation stability coefficient (k) on the concentration of sulfosalicylic acid for: 1 – st10 steel corrosion products; 2 – particles of fe2o3; 3 – particles of feo. the data of fig. 6 prove that increase in the inhibitor concentration results in higher sedimentation stability. an optimal etching mode can be achieved at some ratio between two inhibitors (sulfosalicylic acid and acetylcarbamide or sulfosalicylic acid and thiosemicarbazide) when the inhibitors provide sufficient metal surface protection and high dispersion of the corrosion products. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 vira vodyanka, sergiy boruk, igor winkler, new environment friendly etching technologies for some steels and copper alloys, food and environment safety, volume xiii, issue 4 – 2014, pag. 376 – 384 384 4. conclusion an effective and environment friendly composition is proposed for the etching solutions to be used for steel and copper alloys. these compositions ensure significant reduction in the harmful gases emission and decrease amount of the technological waste water formation. specific emission of nitrogen dioxide can be nine times less and formation of the spent solution – four time less comparing to the standard etching compositions. besides. no special surface cleaning reagents are required in the proposed compositions because of the enhanced surface activity of the components used. a mechanism of the components adsorption on the metal surface is the main factor determining the overall performance of the proposed compositions. 5. references [1]. “industry of bukobyna in 2005-2010”. a statistical reference. department of statistics of chernivtsi region. 184 p. (2011). [2]. reshetnikov s. m., inhibitors of the acid corrosion of metals. khimiya. leningrad. 144 p. (1986). [3]. antropov l. i., makushkin e. m, panasenko v. f., metals corrosion inhibitors. technics. kyiv. p. 17-48. (1981). [4]. zapolsky a. k., mishkovaklimenko n. a., astrelin i. m., physicochemical grounds of the wastewater processing technologies. libra. kyiv. p. 337-365. (2000). [5]. novikov yu. v., ecology, environment and human, khimiya. moscow. 398 p. (1998). [6]. ganuschak m. i., karp’yak v. v. , functional derivatives of carboxylic and carbon acids. publishing centre of lviv university. lviv. 292 p. (1965). [7]. chemist’s reference, khimiya, moscow. leningrad. 920 p. (1965). [8]. filatov v. a. (ed.). hazardous chemicals. inorganic compounds of the groups v-viii. reference. khimiya. leningrad. 592 p. (1988). [9]. filatov v. a. (ed.). hazardous chemicals. inorganic compounds of the groups i-iv. reference. khimiya. leningrad. 512 p. (1988). [10]. mamashvili t. n. et al., synthesis of thyosemicarbazone the copper complex of methyglyoxale and investigation of its dnainhibition and antitumor activity. chem. pharm. j. 11. p. 9-11. (1999). [11]. popov v. v., postornko a. i.. bludova a. a., utilization of the alkybromides production wastes in manufacturing of corrosion inhibitors, science without borders, proceedings of ii int. conf. p. 45-47. (2005). [12]. el warraky a. a., the effect of sulphide ions on the corrosion inhibition of copper in acidic chloride solutions. anti-corrosion methods and materials. 50(1). p. 40-46. (2003). [13]. cicileo g. p. et al., inhibitory action of 8-hydroxyquinoline on the copper corrosion process. corr. sci. 40(11). p. 1915-1926. (1998). [14]. quartarone g., using indole to inhibit copper corrosion in aerated 0.5 m sulfuric acid. corrosion. 54(8). p. 606-618. (1998). [15]. ramesh s., corrosion inhibition of copper by new triazole phosphonate derivatives. app. surf. chem. 224(1-4). p. 214-225. (2004)/ [16]. du t., luo. y., desai. v., the combinatorial effect of complexing agent and inhibitor on chemical-mechanical planarization of copper. microelectr. eng. 1(1). p. 90-97. (2004). [17]. little b., ray. r., a perspective on corrosion inhibition by biofilms. corrosion. 58(5). p. 424-428. (2002). [18]. ekilik v. v., berezhnaya a. g., svyataya m. n., acridine derivatives as inhibitors of copper dissolutions. russ. j. app. chem. 74(9). p. 1500-1505. [19]. cicileo g. p. et al. comparative study of organic inhibitors of copper corrosion. corrosion. 41(7). p. 1359-1375. (1999). 334 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 42016, pag. 334 340 the influence of berry puree on microbiological indicators of cheese product during storage olena grek1, tetiana pshenychna1, *olena krasulya1, yuliia pakhomova1, kateryna ivashchenko2 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str.68, kyiv, ukraine, olena_krasulya@ukr.net 2odessa institute of postgraduate education nuft 65110 balkovskaya 54 str., odessa, ukraine *corresponding author received 14th october 2016, accepted 27th december 2016 abstract: a contemporary trend is providing people with wholesome foods enriched with vitamins and microelements by improving food products’ composition by addition of functional ingredients. this trend may be successfully achieved by innovative solutions related to the technologies of cheese products. one of the most promisive ways to increase significantly nutrition value and consumptional properties of cheese product is based on the enrichment of cheese by dairy-protein and berry raw materials. the modernization of the technical process consists in the fact that the process of milkprotein clot coagulation is carried out by processed berries. the process needs to be carried out under the strict observation of microbiological parameters. similarly the product stability should be rigorously kept during its storage. since the products’ organoleptic, physico-chemical properties and their shelf-life depend on initial contamination, composition and state of bacterial flora, these indicators should also be controlled. taking into account the above mentioned aspects, the effect of black currant as coagulant in the form of frozen milled berries in homogenized and sterilized puree on development of bacterial flora (lactic acid microorganisms, yeasts and moulds) in dairy-protein products during storage was examined. safety and compliance with the regulatory requirements of received samples are proven. the positive impact on dairy-protein foundation’ stability and quality of berry coagulant is proven.it was confirmed that the use of black currant in homogenized puree which has stable indicators is more appropriate and safe. keywords: cottage cheese, black currant, heat treating, thermo acid coagulation. 1. introduction the task to provide consumers with balanced nutrition requires relevant approaches and solutions in terms of milk processing. modern food products have to supply human organism with vitamins, macroand microelements and other essentials for normal functioning of substances. the development of modern food product technologies capable of the above mentioned qualities is an essential problem. such technologies might be based on the addition of various ingredients in the process of cheese production in some stages. such technologies involve the addition of not only traditional ingredients but raw berry materials which contain biologically active substances. the analysis of the information sources shows that not only the enrichment of dairy ready products by raw berry materials is possible, but also the addition http://www.fia.usv.ro/fiajournal mailto:olena_krasulya@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 335 of berry materials on a stage of protein component coagulation as well [1]. berries contain from 80 to 90 % moisture. carbohydrates are represented by fructose and sucrose; among minerals we mention ferrum, potassium, sodium, magnesium, phosphorus, calcium, zink, cuprum, etc. the biochemical composition of berry crops is shown in table 1 [2-11]. table 1. biochemical composition of berry crops according to the table data, black currant berries have the biggest amount of minerals (464.15…478.48 mg/100 g) and vitamin c (464.15…478.48 mg/100 g). high content in vitamin c, in comparison with other berry crops, determines its high antioxidant properties. nevertheless, black currant berries fall short of the carbohydrates amount to bilberry (8.6…15.49 g/100 g) and red currant (7.7…15.8 g/100 g). black currant is one of the most widespread berry crops that grow in ukraine. medicinaland-prophylactic properties are determined by fact that the berries contain vitamins, macro and microelements, polysaccharides (pectin), polyphenols and others which are necessary for humans. berries contain a big amount of iron, phosphorus and calcium salts in the form of organic compounds, which are easily digestible by the human body. the physicochemical indicators of black currant berries are given in table 2 [1, 12]. table 2. physico-chemical indicators of black currant berries moisture mass fraction, % pectin substances mass fraction, % tanning and coloring substances mass fraction, % organic acids mass fraction, % flavonoid s mass fraction, mg % р – active substanses, mg% titratable acidity,% sugaracid index 85.60… 86.99 7.400… 11.100 0.70…0.90 1.90…3.66 245… 1047 319…560 2.0…4.3 2.0…4.8 black currant berries may be provided in the native or frozen state and in the form of paste. they are widely used in food production: jam, jelly, juice, syrup, wine etc. in cheese product manufacturing we recommend to use frozen and sterile berries, homogenized liqberry puree. black currant puree is recommended for use as an additional source of biologically active, pectin substances, vitamins, micro and macroelements, fatty saturated and unsaturated berry carbohydrates content, g/100 g saccarides content, % vitamin с content, mg/100 g microand macroelements content, mg/100 g black currant 7.30…15.38 4.50…11.02 157.70…181.00 464.15…478.48 red currant 7.70…15.80 7.02…10.75 41.00…74.80 367.52…374.35 rasberry 8.30…11.97 5.00…11.00 21.80…35.00 229.87…239.14 blackberry 4.40…9.61 6.20…6.90 21.50…28.40 235.96…242.87 bilberry 8.60…15.49 6.00…7.18 9.70…21.30 101.83…108.24 cranberry 3.70…6.80 4.40…6.80 13.30…23.80 113.77…118.50 guelder 7.90…12.3 7.31…9.56 15.00…27.60 20.20…28.60 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 336 acids. nutrition and energy value of the homogenized puree per 100 g of the product, should not be less than: proteins – 1.0 g; carbohydrates – 8.0 g, fibers – 3.0 g; 36.0 kcal/150.6 k. the physico-chemical indicators of the homogenized black currant puree are given in table 3. table 3. physico-chemical indicators of the homogenized black currant puree dry soluble substanses, % not less than рн value, no higher than polyphenols, mg/100 g organic acids, g/100 g vitamin c, mg/100 g pectins, g/100 g 10.0 3.5 350…400 2.0…2.5 20…40 0.9…1.1 there have been data reported about the use of berries not only as cheese products filler, but as a coagulant in the process of coagulum obtaining [13]. in our opinion, the use of berry coagulant is a promising area of combination in the dairy-protein products manufacturing and requires further research. according to the classical technology of cottage cheese production, coagulation of the casein occurs at рн 4.6 at a temperature (30±2) °c. the duration of fermentation lasts for (8…10) hours. in the process of berry-protein foundation processing, it is expedient to approach the ph of fermented milk mixture to the corresponding value by correction of the process by adding berry raw materials. according to the regulatory documents, shelf life period of the cottage cheese without additional treatment is of 72 hours at a temperature (0…6) °c. 2. matherials and methods during the development of technology for cheese product based on dairy-protein, the characteristics of all components providing nutritional and biological value, safety requirements including microbiological parameters of the product were considered. the aim of the research is to study the effect of different types of black currant on the shelf life and microbiological parameters of berry-protein foundation. object of research – cheese product, which is received by using black currant as coagulant. the following conventional methods are used in the work: identification of viable lactic microorganisms and their most probable number; determination of coliform bacteria; identification of yeasts and fungi; determination of the amount of mesophilic aerobic and facultative anaerobic microorganisms. dairy-protein clot was produced by the classical technology from whole milk with the following indicators: solids mass fraction – 12.3 %, fat mass fraction – 2.6 %, protein mass fraction – 2.8 %, active acidity – 6.9 units. ph, density – 1029 kg/m3 [14]. besides, frozen berries or homogenized black currant puree produced by liqberry (ukraine technical conditions 15.3-24110704-003:2011) were added to the milk. the amount of berry coagulant was determined by leading up normalized milk with fermented compound to the classical ph value. on the average, the mass of pasty currant was (10±1) % from the mass of normalized mixture. for cottage cheese production vivo ferment was used, which contains the following strains of microorganisms: lactococcus lactis subsp. lactis, lactococcus lactis subsp. cremoris, lactococcus lactis subsp. lactis biovar. diacetylactis. fermentation control (monitoring) was carried out at a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 337 temperature 32 °с for 4.5 hours to reach the value of рн 4.5…4.6. before introducing in normalized mixture, frozen currant berries needed an additional processing. they were defrosted and milled in a blender for 2.5...3.0 min to the puree consistence, with a particle size of 200…250 microns. in parallel, the samples with homogenized blackcurrant puree were prepared. it was produced in industrial conditions by advanced technology using hydrodynamic (cavitation) processing of raw materials on a tek-sm device. the berries were treated in the above mentioned setup according to designed regime to a necessary level of homogenization and industrial sterility [15-16]. the samples of cheese product obtained were kept at the temperature (4±2) °с. microbiological parameters in as-prepared product, after the storage for 72 and 144 hours were determined. the microbiological research to assess the safety of cheese products with increased biological value was carried out by standard methods on the base of subsidiaries and dependent companies "odessa institute of postgraduate education nuft" laboratory [17]. microbiological analysis of berry-protein foundation includes the determination of: coliform bacteria – by signs of growth (turbidity, gas formation, change of colour) in the kessler broth and growth of colonies on endo fueler; moulds and yeasts – by the growth of colonies on nutrient saburo agar fueler; mesophilic aerobic and facultative anaerobic microorganisms (qmafanm) by counting the colonies which grew up on meat-and-peptone agar. 3. results and discussion the research results of lactic acid bacteria amount in cheese products during storage are shown in the table 4. table 4. the research results of lactic acid bacteria amount in cheese product samples during storage, cfu in 1 g of product the norm, according to the state standard, not less than (for cottage cheese) cottage cheese (control) sample 1 cheese product with frozen milled currant berries sample 2 cheese product with homogenized black currant puree sample 3 asprepared after storage asprepared after storage asprepared after storage 72 hours 144 hours 72 hours 144 hours 72 hours 144 hours 1·106 1.1·1010 0.7·109 1.1·109 1.1·1010 0.7·108 1.1·107 1.1·1010 0.9·107 1.1·107 according to the data shown in table 4, the amount of lactic acid bacteria in cheese product has decreased to (1.0·103) cfu with the addition of berry puree. after 72 hours of storage, the amount of lactic acid bacteria in all samples has decreased as compared to asprepared product. this confirms that currant puree is not a nutritious substrate for lactic acid microorganisms, as it inhibits their growth during product storage. to determine the final characteristics and morphological properties of dairy products’ microflora, the research of preparations with their dilution under the microscope after gram staining were conducted. when investigating smear sample 1 (dairy-protein foundation), a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 338 big amount of rod bacteria and few cocci were determined, namely: microorganisms painted in blue (gr +), oval, which have a capsule and are grouped in pairs. (fig. 1a); microorganisms painted in blue (gr +) in the form of long rods placed in pairs. (fig. 1b). а b fig. 1. photomicrographs of preparations with the breeding of dairyprotein foundation (1000х magnification): а – photomicrograph of oval microorganisms; b – photomicrograph of microorganisms in the form of long rods during microscopic examination of sample 2 (cheese product with frozen milled currant berries) a big amount of long rods (gr+) with rounded ends, which are located singly or in pairs were found. in sight of sample, single oval cells, which have a capsule, are visible. microscopy of the sample 3 smear (cheese product with homogenised currant puree) showed the presence of long sticks in large numbers placed in chains or pairs. the next task of research was to identify e. coli bacteria, which mainly belong to the escherichia genus, ferment lactose in culture medium with acid and gas creation. according to the regulatory document requirements, the presence of coliforms in the cottage cheese and cheese products are not allowed in 0.01 g of product with shelf life of more than 72 hours. during the research no coliform bacteria in the berryprotein foundation samples obtained by different methods were found. to confirm the samples accordance to the state standard of ukraine, the presence of yeast and mold fungi were tested. when present in food products moulds produce mycotoxins that cause the development of various toxicosis in humans. the presence of these microorganisms in berry-protein foundation leads to its rapid deterioration and has negative impact on the level of safety for human consumption. the results of microbiological research of cheese products samples safety are presentedin the table 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 339 table 5. the content of moulds and yeasts in model samples of cheese products during the whole period of storage in sample 1 (cottage cheese without currant berries) and sample 3 (with homogenized puree), no colonies of yeast and moulds were found; as-prepared sample 2 (berry protein foundation with black currant berries milled to a paste state) contained 18·102 cfu of mould fungi, which significantly exceeded the norm. the obtained results prove that the product contamination by mould fungi happened as a result of using defrosting of crushed currant berries without additional heat treatment. this has been confirmed in the process of determining the microbiological quality indicators of raw material (black currant). the number of mesophilic aerobic and facultative anaerobic microorganisms, yeasts and mould fungi in black currant were verified. research showed the following: a significant contamination of defrosted berries qmafanm was 45·102, mould fungi 27·103 cfu, yeast – were not found. the research of sample 2 after 72 and 144 hours of storage has shown the absence of these microorganisms. such results may be explained by the formation of nisin antibiotic in the process of streptococcus lactic vital activity, in the presence of which the number of outside microorganisms, except of lactic acid, decreases. 4. conclusion according to the research results of microbiological indicators in the process of berry-protein foundation storage, no negative dynamics was found. cheese product with homogenized blackcurrant puree complies with the cheese product regulatory requirements at the beginning and end of storage. the sample, which is produced by addition of milled defrosted black currant berries, turned out to be contaminated with mould fungi, making thus the product unsafe for consumers and requiring mandatory additional heat treatment. therefore, it is more appropriate and safe for the given technology to use sterile, homogenized blackcurrant puree. in the case of fresh berries or defrosted raw berry material use, preparatory operations should be carried out carefully. storage time, hours mould amount, cfu in 1 g yeasts amount, cfu in 1 g the norm according to dstu 4554: 2006, no more than control cheese produc t with black currant berries berryprotein foundation with blackcurrant puree the norm accordin g to dstu 4554: 2006, no more than control cheese produc t with black currant berries berryprotein foundati on with blackcur rant puree immediately after production 5,0·101 not found 18·102 not found 1·102 not found not found not found 72 not found 120 not found food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 olena grek, tetiana pshenychna, olena krasulya, yuliia pakhomova, kateryna ivashchenko, the influence of berry puree on microbiological indicators of cheese product during the storage., food and environment safety, volume xv, issue 4 – 2016, pag. 334 – 340 340 5. references [1] makarkina m.a., yanchuk t.v., the characteristics of the black currant grades by sugars and organic acids content, modern gardening, 2: 9– 12, (2010). [2] makarov v.n., vlazneva v.n., bioactive substanses in berry crops and products of their processing, storage and processing of the agricultural raw materials,12: 75–82, (2008). [3] yushkov a. n., savelyev n.i., akimov m.y., antioxidant activity and biochemical content of berry crops, science and apk technique achievements,8: 5–6,(2010). [4] isaeva e.l., cranberries and blueberries. the overcoming bacteria berries, healthy living and longevity, 64p., (2010). [5] popova e.i., khromov n.v., vynnytskaya v.f., biochemical evaluation of viburnum accessions and prospects of it use in the manufacture of functional foods, scientific sheets: ser. natural sciences, 21: 127–131, (2012). [6] simakhina g.a., basic bio-components of raspberry and their behavior during freezing, food & ingredients, 4: 44–46, (2009). [7] petrova s.n., composition of black currant’s fruits and leaves chemistry of plant raw materials, 4: 43–50, (2014). [8] streltsina s.a., tikhonova o.a., nutrient and bioactive substances of black currant berries and leaves in the conditions of the northwest russia, agricultural russia, 1: 1–16, (2010). [9] kamzalova a.m., dmitrieva v.t., gumenyuk s.l., lipskaya o.i., the biochemical composition of currant berries in the conditions of belarus, fruit growing, 215p., (2005). [10] artemovа e.n., physico chemical properties of red currant jelly, food industry, 7: 58–59 (2006). [11] huseynova b.m., daudova t.i., environmental aspects of the biocomplex formation in fruits as wild blackberries and blackthorn, biological resources: flora, 675–678 (2010). [12] shevchuk l.m., the quality of black currant in the carpathian region, ukrainian academy of agricultural sciences, 10: 24–26, (2008). [13] shchetinin m.p., koltyugina o.v., kosynkina a.a., cheese product with berry ingredients, dairy industry 10: 58 (2011). [14] dstu 4837:2007 quick-frozen fruits and berries, state committee for technical regulation and consumer policy, 28 p. (2009). [15] ivashchenko k.y., improving of homogenized fruit products technology using hydrodynamic processing: synopsis of a thesis..phd,speciality 05.18.13. «technology of canned and chilled products» nuft: 20 p., (2015). [16] bessarab o.s., osypenko s.b., stoyanova l.o., pakhomova k.y. innovative technology for homogenized fruit products with increased biological value based on hydrodynamic processing of raw materials, equipment and technology of food production, 32: 74–19, (2014). [17] dstu 7357:2013 milk and dairy products, state committee for technical eegulation and consumer policy, 39 p. (2014). 238 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 238 243 th e opt im um de ns it y for wi nt er rape und er suce av a pla te au c on di t ion s *ioan – cătălin enea1, ioan gontariu 2 1agricultural research and development station of suceava, b-dul 1 decembrie 1918 no.15, suceava, 1faculty offood engineering, 13 universitatii st., 720229, suceava, romania, catalin_i75@yahoo.com, ioang@fia.usv.ro * correspondingauthor received june15th, accepted september 25th2014 abstract:important autumn oilseed rape has been bred considerably in the last period as a result of the fact that it provides raw materials for the manufacture of oil, which is an alternative to unconventional fossil sources of energy, as well as for themaking ofsome oils of superior quality used in human nutrition and not only. the experimental results from suceavaarea during the period 2011-2012 regardingtwo winter rape genotypes bred at different densities are presented. the results show that at lower densities, the collum development is better and an increased resistance to wintering of the rape plant is noticed. at lower densities, higher plants, with a great number of branches per plant and a greater number of seeds and capsule per plant havebeen obtained. the largest grain production was achieved in both genotypes at the density of 50 plants/ m2. keywords:rape genotypes, grain production, annual precipitation, plants’densities 1. introduction three field trials and one greenhouse trial were conducted from 2008 to 2010 at two locations in south west and northern germany to assess the effect of cultivar, location and year on dormancy characteristics of open-pollinated winter oilseed rape cultivars. a simple selection was performed in the field and in the greenhouse for low-dormant individual plants, and the offspring of these plants (potential volunteers) were tested for the stability of the trait under field conditions [1]. due to the high demand for rape, the profitability of its culture on surface unit,as well as any special requirementsfor these cultivars to be grown and developed under the conditions from the big island of braila, makes this culture adapted in this area, which has been temporarily excluded from the agricultural circuit for many years to come. by continuous improvement of this technology crops with a view to obtaining maximum yields and performance from all points of view, it must be clear, accurate and flexible [2]. winter rape cropwas introduced recently in the suceava plateau area and the technological elements were accordingly to those recommended in areas where this crop was introduced some time ago, or even from the recommendations of seed production companies [3]. this crop tends to expand in this area because the new used hybrids have a high resistance to frost, the losses during the winter being very small and in good food and environment safety journal of faculty of food engineering, ştefancelmareuniversity–suceava volume xiii, issue 3 – 2014 i oan – c ăt ăl i n e nea, ioa n g ont ar iu , t h e o pt i mum d en si t y f o r wi n t e r rap e un d e r s uc ea va pl at eau con di t i on s, food and environment safety,volume xiii, issue 3 – 2014, pag. 238 – 243 239 technical conditions high yields per hectare are obtained. the importance of these crops for the big output derives from the fact that there are in the first place certainties of capitalization at competitive prices, production settlement shall be carried out immediately before wheat, while, at the same time profitability much higher than other agricultural crops [4]. for old varieties, the following recommendation is made:to use the density of 150-200 plants /ha, whereas for new hybrids, the density recommended being much lower, namely 35-60 plants/m 2. the behavior regarding density is different from hybrid to hybrid depending on the plant height and capacity of the branch, which leads to research on different varieties, as the recommendation of the crop density on different genotypes could be made [5]. 2. materials and methods the experiment was set on faeoziomchernozem soil with a clay content of 31.6% in the 0-20 cm layer, weakly acidic with a ph in water of 5.6 to 5.8, a humus content of 3.0% and medium stocked in phosphorus (32 ppm) and potassium (150 ppm). the conditions from the experimental period (2011-2012) are shown in table 1. the average annual temperature was 8.40oc in 2010/2011 and 9.30o c in 2011/2012, being with 0.60oc and respectively 1.5 higher than the annual average. the average monthly temperature in both years was higher than the annual average during the entire period, with values between 0.5 and 4.40o c, except for the months of october and february when it was lower than 1.60o c and 3.30o crespectively [6]. annual precipitation totalized 678.8 mm in the first year and 469.4mm in the second one, their average being lower than the annual average with 11.7 mm [7]. in autumn, (september and october) and in spring (march, april) the monthlyprecipitation average was higher than the annual one, while in the other months the rainfall was lower than the annual average. the experiment was bi-factorial, with the following graduations: factor a – advanced cultivar or hybrid a1 – scelni cs mid-sized and medium branching a2 –triptics high sized and very good branching factor b-plant density with 5 levels b1= 30 plants /m 2 b2= 40 plants/ m2 b3=50 plants/ m2 b4=60 plants/ m2 b5= 70 plants/ m2 the aim of this work is to ensure optimum density of oilseed rape fall under the conditions of north of the country, having regard to climatic conditions. the seeding was carried out at a distance of 25cm between rows, with a larger quantity of seeds, of 5kg/ha. after emergence, the plants have been depleted by 13, 10, 8, 7, 6 cm distances to provide the proposed densities aimed at by experiments. in autumn and spring the counting was done in order to determine the losses over winter (fig. 1). during the growing season, observations were made concerning the diameter of the collum, plant height, the height of insertion of the first branch, the number of branches, the number of capsules/plant, the average number of seeds/capsule and seed production/ha. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity–suceava volume xiii, issue 3 – 2014 i oan – c ăt ăl i n e nea, ioa n g ont ar iu , t h e o pt i mum d en si t y f o r wi n t e r rap e un d e r s uc ea va pl at eau con di t i on s, food and environment safety,volume xiii, issue 3 – 2014, pag. 238 – 243 240 table 1. climatic conditions in the period 2011-2012 at ards suceava specify viii 2011 ix 2011 x 2011 xi 2011 xii 2011 i 2012 ii 2012 iii 2012 iv 2012 v 2012 vi 2012 vii 2012 average or total precipitation (mm) decade i 23.3 23.6 7.2 0.6 0.3 0.9 9.9 4.3 14.8 1.6 26.9 1.9 decade ii 0.1 0.4 7.3 0.3 10.4 5.6 4.6 5.4 45.9 36.8 13.1 34.2 decade iii 2.8 2.8 1.4 17.7 5.7 3.4 29.8 42.7 6.3 12.9 monthly total 23.4 26.8 17.3 0.9 15.4 24.2 20.2 13.1 90.5 81.1 46.3 49.0 457.2 annual average 62.8 40.8 29.5 30.6 26.5 24.2 25.6 36.2 48.2 80.2 93.6 88.6 586.8 standard deviation ± -39.4 -14.0 -12.2 -29.7 -11.1 0.0 -5.4 -23.1 +42.3 +0.9 -47.3 -39.6 -129.6 temperature (0c) decade i 18.6 14.8 13.3 4.5 4.4 1.5 -17.0 -2.8 6.7 15.7 18.4 24.6 decade ii 19.2 18.1 5.5 0.4 1.5 -2.6 -9.7 4.9 9.0 13.0 20.6 20.8 decade iii 19.4 14.5 3.9 0.8 -1.2 -8.4 -1.2 7.9 15.1 15.7 20.1 22.5 monthly total 19.1 15.8 7.6 1.9 1.5 3.2 -9.3 3.3 10.3 14.8 19.7 27.2 9.2 annual average 18.3 14.2 8.4 2.4 -1.9 -4.1 -2.9 1.2 0.8 13.7 16.9 18.4 7.1 standard deviation ± +0.8 +1.6 -0.8 -0.8 +3.4 +7.3 -6.4 +2.1 +2.3 +1.1 +2.8 +4.3 +2.1 ards suceava fig. 1 location of experiment in the experimental field at ards suceava food and environment safety journal of faculty of food engineering, ştefancelmareuniversity–suceava volume xiii, issue 3 – 2014 i oan – c ăt ăl i n e nea, ioa n g ont ar iu , t h e o pt i mum d en si t y f o r wi n t e r rap e un d e r s uc ea va pl at eau con di t i on s, food and environment safety,volume xiii, issue 3 – 2014, pag. 238 – 243 241 3. results and discussion a) winter resistance before enteringthe dormant period, the plants’ number in one row and plants’ number/plot were counted. in spring, after the beginning of vegetation, the same plants were counted; the percentage of dead plants due to winter conditions was determined. the data in table 2 show relatively low losses in the two experiment years at this crop. the winter conditions were difficult, the temperatures were very low, and namely bellow -31oc in 2012, but the plants have survived very well because they were covered with a thick layer of snow. the two testedgenotypeshad small losses, the variety scelnisc up to 7.2% and 12.3% the variety tripti cs. also we noticed that the smallest losses were registered by the plants oflow densities. the losses were higher when the plants’densities have been increased, reaching more than double at the plants’ highest densities. the reason is due to the development of autumn plants,plants’ low densities caused the plants to develop their foliage system better and the collum was developed better containing many more reserve substances, thusproviding passage over winter in good conditions. table 2 frost resistance of winter rape by plant densities genotype density (pl number/m2) losses over winter (%) 2011 2012 average 2011-2012 average on genotype scelni cs 30 4.0 2.0 7.2 40 5.0 5.5 5.2 50 7.2 8.4 7.8 60 8.5 10.0 9.3 70 10.2 12.5 11.5 trepti cs 30 5.5 7.2 6.4 12.3 40 8.2 10.4 9.3 50 10.6 13.5 12.1 60 17.0 15.5 16.3 70 17.5 17.0 17.2 b) plant growing during the growing season we made a series of observations concerning plant development in two varieties of rape: collum diameter, plant height, and number of branches, number of capsules/ plant and the average number of seeds in a capsule (table 3). collum diameter was primarily influenced by plant density and less by genotype. so at the variety scelni, the collum diameter was 10.6 mm to the density, 70 plants/m2, and 14.4mm at 30 plants /m2 and at the variety trepti, the collum diameter was 10 mm at density 70 plants/m2 and 13.9 mm at a density of 30 plants/ m2. the collum diameter is important in order to create a support base for future plant and resistance to harsh conditions during winter time. -plant height of winter rape was between 127 cm and 142 cm in variety scelni and between 165 cm to 137cm in tripti variety and at higher densities the plant height was lower. on the other hand the height of the insertion of the first branches of the two genotypes was higher in higher densities. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity–suceava volume xiii, issue 3 – 2014 i oan – c ăt ăl i n e nea, ioa n g ont ar iu , t h e o pt i mum d en si t y f o r wi n t e r rap e un d e r s uc ea va pl at eau con di t i on s, food and environment safety,volume xiii, issue 3 – 2014, pag. 238 – 243 242 the number of branches was higher at fewer densities, ranging from 5 to 10.8 for scelni variety and between 8.3 and 12.5 for the other variety. number of capsules/ plant was 139 capsules/plant at density of 30 plants/m2 at the variety scelni, and decreased to 64 capsule at a density of 70 plants/m2, at the variety tripti, number of capsule per plant was 156 at the density of 30 plants/m2and continuously decreased to 100 capsules/plant at density of 70 plants/m2. the average number of seeds in a capsule in both varieties was higher at low densities (24 to 24.6) and lower at higher densities (19-20). table 3 biometric measurements at winter rape hybrids according to density (average of the years 2011-2012) geno type density plants/m2 collum diameter (mm) plant height (cm) the height of insertion of the first branch (cm) number of branches number of capsules/ plant medium number of seeds in a capsule scelni cs 30 14.4 142 58.8 10.8 139 24.6 40 13.2 139 59.0 10.4 148 25.4 50 13.0 128 58.9 10.4 126 24.6 60 12.8 127 63.4 6.4 90 21.6 70 10.6 127 73.4 5.0 64 20.0 tripti cs 30 13.9 165 60.0 12.5 156 24.0 40 12.8 158 65.0 11.4 152 23.0 50 12.8 142 65.5 10.9 146 20.0 60 11.6 140 72.0 8.5 120 20.0 70 10.0 137 80.0 8.3 100 19.0 c) the yield of seeds in the two varieties of winter rape according to plant densities is shown in table 4. high potential for production of the varietytripti (3573 kg / ha) in comparison with the varietyscelmi (3187 kg/ha) can be noticed. table 4 the yield of seeds (kg/ha) genotype density (pl/m2) the yield of seeds (kg/ha) 2011 2012 ave rage diferences kg/ha % signification scelni cs 30 2880 2700 2790 -397 87 00 40 3560 3180 3370 183 106 50 3840 3280 3560 373 112 xx 60 3260 3025 3143 -44 99 70 3340 2804 3072 -115 96 the average of the variety 3376 2998 3187 100 trepti cs 30 3220 2017 3019 -554 84 000 40 4150 3285 3718 145 104 50 4060 3876 3968 395 111 xx 60 3850 3514 3682 109 103 70 3615 3340 3478 -95 97 the average of the variety 3779 3366 3573 100 dl 5 % 203 dl 1 % 304 dl 0.1 % 407 food and environment safety journal of faculty of food engineering, ştefancelmareuniversity–suceava volume xiii, issue 3 – 2014 i oan – c ăt ăl i n e nea, ioa n g ont ar iu , t h e o pt i mum d en si t y f o r wi n t e r rap e un d e r s uc ea va pl at eau con di t i on s, food and environment safety,volume xiii, issue 3 – 2014, pag. 238 – 243 243 the two varieties grown at different densities, have produced the highest yield of seeds at the density of 50 plants/ m2, being of 3560 kg / ha for scelni and 3968 kg/ha for tripti. the yield increased in both varieties, being statistically assured. by increasing the rape density, an yielddecrease was registered at the cultivars scelniandtripti, more exactly the greatest decrease being noticed at the density of 30 plants/m2, the absolute value of this reduction was the following:397 kg/ha and 554 kg/ha. 4. conclusion in the two experiment years, the winter rape genotypes, scelni and trepti, behaved differently to the wintering conditions, depending on density. the biggest losses in the variety tripti were recorded at lower densities; the plants were well developed, registering lower losses over winter period. the winter rape plants’ density influenced their development, in the sense that at the lower densities the collum diameter, plant height and the number of branches were bigger, and the height of the main branches was lower. production information: the number of capsules/plant and the number of seeds per plant were larger at low densities. the highest losses were registeredat the density of 50 plants/m2.when the density varied between 40 and 60 plants /m2, the losses were statistically assured. the biggest losses were recorded at the density of 30 plants /m2,taking into consideration that the optimum density of autumn rape culture is to be determined according to the area to be bred in. 5. references [1]. heinrich thöle, antje dietzpfeilstetter, 2012, molecular markerbased identification of oilseed rape volunteers with different secondary dormancy levels in oilseed rape fields, european journal of agronomy, volume 43, november 2012, pag. 194-200. [2]. buzdugan, l.g., 2011, research on optimization of rape culture technology of the autumn in enclosure with dig insula mare a brăilei, work for phd degree,usamv bucharest. [3]. buzdugan, l. g., 2011 – nutrients management for increasing he soil fertility and the crop productivity în the great island of brăila, meeting the fertilizer demand on a changing globe: biofuels, climate change and contaminants, 15th world fertilizer congress of the international scientific centre for fertilizers 29.08.– 02.09.2010, romanian academy publishing house, bucharest. [4]. bălteanu gh., salontai al.,vasilică c., bârnaure v., borcean i., 1991 plant cultivation technology, vol. ii, ceres publishing house, bucharest; [5]. râşnoveanu l., buzdugan l., 2011 somme aspects infuence of sowing time of winter oliseed rape production,in the condition north-east baragan, scietific papers, vol. 54, agronomy series,usamv bucharest. [6]. suceava meteorological station, the register monthly weather observation, 2011-2012. [7]. suceava meteorological station, synthetic tables rainfalls, 2011-2012. heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow 203 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv , issue 3 2016, pag. 203 215 ştefan cel mare university of suceava, romania volume , issue 12016, pag. ... ... heat transfer modeling in down flowing laminar films with the developed wavy structure with co-current steam flow valentyn petrenko1, *yaroslav zasyadko1 1national university of food technologies, thermal fluids and industrial refrigeration dept. 68, volodymyrska st.; 0160 kyiv; ukraine petrenkovp@ukr.net, yaroslav@nuft.edu.ua *corresponding author received 12th july 2016, accepted 23rd september 2016 abstract: the result of heat transfer modeling in down flowing viscous laminar films with the developed wavy structure at the regime of evaporation from film interface at free falling and with co-current steam flow, a heat transfer model, which takes into account a cyclic process of temperature field relaxation with the periodic mixing of film by big waves, has been presented. a mathematical heat transfer model in laminar saturate liquid film with the developed wavy structure on its surface has been developed. the model takes into consideration cyclic relaxation of transient temperature field which happens right after the passage of a powerful big wave. the developed mathematical model describes the time history of the two dimensional temperature fields as a function of the peclet number and the core characteristic of the wavy motion (the length of big waves). based upon the proposed model a set of correlations has been obtained. these ones are proposed as a means for the generalization of heat transfer experimental data, obtained within the experimental studies of liquid films, heated to the saturation temperatures and evaporation from the interface. a generalized equation has been derived, which can be used for the calculations of heat transfer coefficients (htc) to the saturated sugar solutions liquid films. this equation contains wavy characteristics of down flowing films and are valid within the range of parameters characteristic of sugar industry evaporators, namely: concentrations – 0…70 % dry matter; liquid mass flow rate density–0.01×103…0.6×10-3 m2/sec, the peclet number range – 400…25000. key words: films, waves, heat transfer, temperature, evaporation. 1. introduction. a structure of liquid films down flowing over the vertical tubular surface of a film evaporator changes along the length of a channel. this happens as a result of a continuous development of wave structure and due to the growth of the co-current steam velocity. therefore, the experimental results of studies on the heat and hydrodynamic processes at models with different channel geometry may differ significantly. the predominant amount of theoretical papers related to the wavy movement of films [1, 2, 3] is dedicated to the research into the development of regular waves structure. the part of http://www.fia.usv.ro/fiajournal mailto:petrenkovp@ukr.net mailto:yaroslav@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 204 such waves on the full length of heat transfer tubes of industrial evaporators is negligibly small if compared to the regions of big waves. in the available papers dedicated to the research of heat transfer in down flowing films the effect of wavy structure on heat transfer has been taken into account indirectly through the parameters of turbulence and film interface shear stress. predominant number of experimental works dedicated to the research of heat transfer to the films, heated to the saturation temperature, has been obtained in the experimental units modeling industrial evaporators [4, 5, 6]. thus, the experimental data obtained reflect the effect of number of factors, which influence simultaneously heat transfer, without separation of components. therefore, the experimental correlations thus obtained, which are recommended for the calculations of the heat transfer to the saturated films in industrial evaporators are limited in terms of the applicable ranges of regime parameters similar to the model ones, as well as in terms of the geometry of the experimental unit. keeping in mind the statements mentioned above the development of adequate physical model of film flows relevant to a concrete structural form of films looks of current interest. according to [7], in case of free falling films over the vertical surface the saturation of wavy movement with fully developed big wave structure occurs at a distance approximately 2…2.5 m from the film forming devise. these big waves move on the film interface with the phase velocity, which 1.5…2 times meant film velocity [8]. an important factor, which affects seriously hydrodynamic film structure, is the fact that big waves contain a central swirl. due to this the liquid in the bulk of film is periodically mixed, which in turn causes the velocity, temperature and concentration profiles deformation. it should be mentioned also that in tubular channels of 7…12 m long industrial film evaporators there is a continuous growth of co-current steam core due to the gradual film evaporation. this, in turn, causes a continuous growth of interface shear stress, which distorts wavy and hydrodynamic structure of film flow. taking into account all mentioned above, the heat transfer in liquid films may be treated as a cyclic process of temperature field relaxation, which happens after each consequent passage of a big wave. this big wave due to the existence of a powerful central swirl, which transports slightly, superheated liquid from the boundary region to the external side of film. according to this mechanism the peak of a big wave will be constantly fed with the slightly superheated liquid and transfer it to the wave surface, from which evaporation takes place, thus transferring heat to the steam core. it is clear that the more powerful the wave, the bigger the deformation of temperature profile in the vicinity of heated surface right after the wave is. within the periods between the big waves passage a relation of temperature field takes place. the process of relaxation is stretched along the film movement until a consecutive mixture of liquid happens due to the passage of the consecutive wave. 2. materials and methods a direct experiment of heat transfer in down flowing liquid films heated to the saturation temperatures with sugar solutions as model liquids has been carried out at the experimental unit with the independent formation of phases’ mass flow rates and heat flux. the main core of the experimental unit was represented by a stainless still pipe food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 205 with the inside diameter of 20 mm and 1.8 m long. the experimental tube was separated into the initial 1.5 m stabilization section and 0.3 m measurement section. the down flowing of water (sugar solutions) film has been formed by means of overflowing over the tube’s upper rim. in the event of steam-liquid flow modeling, dry saturated steam has been supplied in co-current regime. the liquid falling film has been heated by dry saturated steam which was supplied into outside heating sections attached to the experimental tube. the heating chambers were designed in such a way as to provide an individual heating of the stabilization section and the experimental one. the mentioned sections were hooked up to the individual vacuum-condensation sections which allowed for the keeping of different pressures in each chamber. such arrangement allowed also the maintaining of vacuum down to 0.8 bars and thus, to vary the temperature head between the heating steam temperature and evaporation temperature. special probes for taking samples of liquid to determine its concentration and measurements of temperatures were positioned directly after the measurement section. a detailed description of the experimental unit is given in [11] and its schematic is given in fig.1. figure 1. scheme of the experimental stand 1,6,15,41,47,53 – manometer; 2,42,48,54 – vacuum manometer; 3,39,45 – sensor for vacuum control; 4,40,46,52 – sensor for pressure control; 5,9 – collector flow control; 7,16 – heat exchanger; 8,26,32 – tank; 10,11,21 – diaphragm; 12,13,22,43 – differential pressure gage; 14,30 – sampler; 17 – gage probe; 18,23,24,25,28,29 – hotwell; 19,20 – condenser; 27 – pump; 31,33 – vacuum pump; 34 – tank for sampling; 35 – tank of solution’s temperature measurement on the exit of pipe; 36 – module analog input 7018р; 37 – module 7520; 38 – computer; 44,50 – heating chamber; 49 – equaling tank; 51 – separator of heating steam; 55 – experimental pipe; 56 – top chamber. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 206 3. results and discussion according to the proposed heat transfer model the rolling of a big wave along the film surface causes local mixing of liquid and deformation of velocity and temperature fields and equalizing the field of concentrations, respectively. it could be suggested that right prior to the passage of a big wave a velocity profile is close to the parabolic one, although to a certain extent deformed due to the interface friction of the steam core. the temperature profile close to the linear is shown in fig. 2a. right after a big wave passage, velocity becomes constant across the film thickness, whereas the temperature profile will be curved, fig. 2b. figure 2. schematic of big wave passage on the film surface a) velocity and temperature profiles prior to big wave passage; b) the same – afterwards since the amplitude of big waves is insignificant, the distortion of temperature curve right behind the wave could be not deep and 0 1      . in this case the evaporation from the interface right behind the wave reaches its minimum value and, further on, its growth. in the event of big wave amplitudes and powerful swirls inside the bulk of the film, respectively, there could be a noticeable transfer of unheated liquid in the direction towards the wall. under this condition there would be cessation of evaporation until the moment temperature gradient appears on the surface. thus, a maximum heat flux on the wall will occur in the moment of a big wave passage, when temperature of the boundary layer will reach its maximum value. further on in the process of film heating up the wall flux will decrease and that one on the interface will grow, asymptotically approaching the value, at which temperature profile becomes linear. in light of the above, a process of temperature field relaxation could be split into two periods: the first one lasts until the temperature gradient on the interface appears, the second one – after that. y u t t u c u u c a б 2 2 x b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 207 it is clear that the first period starts right after the passage of a big wave, fig. 2b. the liquid in film is mixed at a constant temperature; velocity is constant across the thickness of the film, the presence of interface friction the velocity in the film could be determined as the mean integral across the film                         0 2 2 322 1 g dyy g y g u ii , (1) where 2 2 y g y g u i              – velocity distribution across laminar field with interface friction due to the steam core movement. within this period the heat transfer equation is as follows 2 22 ),(),( 32                      a gi , (2) where     satw sat tt t,t ,    – dimensionless temperature;   y ,   x – dimensionless transverse and longitudinal coordinates;  – film thickness; vг – volumetric liquid flux; a – temperature conductivity;  – cinematic viscosity coefficient; i – shear stress on the film interface; satt – saturation temperature; wt – wall temperature. since the film thickness and mean fluid velocity are interrelated as a cubic equation       32 32 g uг iv , (3) then (2) we read 2 2 4      ),(),(pe , (4) where a u a г pe v   44 – the peclet number. the solution of (4) with the boundary conditions   00,0  ,   1,0  ,   00,1  , 0 ),(      , (5) would be              pe erfc, 4 . (6) the profile (6) will be developed until the temperature gradient at a distance ξm appears on the interface. further on, the temperature on the film surface will remain constant as a result of evaporation. the limiting temperature curve, which signifies a change of temperature field ( 0 1     ) can be determined from the heat transfer equation with the parabolic velocity profile. it should be kept in mind that within the process of film heating up its velocity will change 2 2 32 ),(),( 2                              a ggi . (7) having substituted the left hand side of (7) by the mean integral velocity, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 208                                             avii a g d a gg 1 32 ),( 2 321 0 3 2 and combining (3), one obtains (7) as     2 2 , 4          avpe . (8) double integration of (8) with the boundary conditions 0 , 1 ; 1 , 0   , (9) yields     1 24 , 2                  av pe . (10) a derivative       av may be found from the equation for mean film temperature                            1 0 2 1 0 32 2 ,,                 d g gg d u u i i av . (11) integrating (11) one obtains        g gpe i iav av 23 1625 80 1      .(12) denoting   g gpe d i i 23 1625 80    , and substituting in (12) yields first order differential equation ddav av 11       , (13) which can be solved with the following boundary conditions: ξ = 0 , 0av ,        dav   exp1 . (14) substituting the derivative of (14) into (10) one obtains a temperature distribution in the film at m :   1 2 exp 1 4 , 2                         dd pe m m . (15) the coordinate ξm may be denominated from (15) at a suggestion that at m the dimensionless temperature equals 0,   01  m,        d pe lndm 8 . (16) beginning with m , the second period of the temperature field development starts. during the whole second period the film surface temperature remains constant to the fact that evaporation from the surface goes on. within this period, the boundary conditions (9) change into 0 , 1 ; 1 , 0 ; m , (17)   1 2 exp 1 4 , 2                         dd pe m m integration of (8) with the boundary condition (17) yields:      1 8 , 2         av pe . (18) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 209 the derivative     av will be found by the determination of the film mean temperature at a section m , which gives                                 1 0 2 2 32 2 1 8                  d g gg pe i i av av . (19) integration of (19) yields             g gpe g g i icp i i av 23 107 16023 4 3         . (20) having introduced the following     16023 107 pe g g r i i      , peg g r i i o 40 107 34      ,   g g rrr i i o 23 4 3 1    , a differential equation is obtained oav av r r    1   , (21) which has its solution        r cr oav   exp1 . (22) the constant of integration c0 is determined through the films mean temperature at a moment when a temperature field starts development at m with boundary condition (17)                                           1 0 2 2 . 32 2 1 2 exp 1 4                 d g gg dd pe i i m mav . (23) further integration of (23) yields               dgd gpe m i i mav     exp 23 2516 80 1. now substituting m.av in (22), the integration constant c0 is                       rdg g d pe rc mm i i o    expexp 23 2516 80 1 1 , and respectively, film’s mean temperature in the region m will be                         r exp d exp g g d pe rr mm i i av 23 2516 80 1 11 . (24) differentiating of (24) by ξ yields food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 210                           r exp d exp g g rd pe rr r mm i iav 23 2516 80 11 . (25) after substitution of the derivative (25) into the initial equation (18) we obtain the temperature distribution in the film in the region m         1 23 2516 80 1 8 21                           r exp d exp g g rd pe r rpe , mm i i . (26) graphic interpretation of (26) within m , and (6) at m , and (15) at m is given in fig. 3. the bigger the interface shear stress i , the thinner the film will be. 1 2 3 4 5 0.2 0.2 0.6 0.6 1.0 1.0   ,  figure 3. temperature distribution in water film calculated by (6, 15, 26) at t = 100 0c; i = 0; γv =0.5∙10-3 s m 2 , ; 1 – 167 , equation(6); 2 – , equation(15); 3 – 367 ; 4 – 467 ; 5 – ; 3, 4, 5 – equation (26). a relative distance ξ will be bigger at the same mass flow rate, and distance between the peaks of waves. therefore, linearization of temperature profile will happen much quicker in the presence of interface shear stress, then in the case of film free fall, fig. 4. 0.2 0.2 0.4 0.6 0.6 0.8 1.0 1 2 3 1.0    , figure 4. temperature distribution in water film calculated by (6, 15, 26) at t = 100 0c; гv = 0.5∙10-3 s m 2 ; bwx = 0.1 m; 1 – 270,0  i ; 2 – 4003 2  , m n i ; 3 – 61010  ,i . a derivative on the wall ( 0 ) of (26) gives       1 23 2516 80 1 8 1 0                              r exp d exp g g rd pe r rpe d ,d mm i i . (27) 267m m 767 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 211 the wall heat flux ( 0 ) during the second period of heating within the section m is                                       1expexp 23 2516 80 1 8 1 0 02 rdg g dr pe r rpett d dtt q mm i isatw satw            , (28) and during the first period m from (6) will come to             pett d dtt q satwsatw 20 01 . (29) if the process of liquid film hating up starts right after the passage of a big wave at o and finishes right before the incoming successive big wave at v , the mean heat fluxes at sections ( om  ) and ( mv  ) will be      pepettdqq om om satw om av m o           011 1 , (30)                 mv satw mv av tt dqq v m 022 1                                                     mv i ivmmvm g g r exp d exp d pe r expr pe 23 1625 1 640 11 8 2 1 . (31) now the mean heat flux at the both sections may be given as mean weighted     ov mvavomav av qq q    21 , (32) then combining (30, 31) we get                                                                      mv i ivm mvm om ov satw av g g r exp d exp d pe r expr pe pepe tt q 23 1625 1 640 11 8 1 2 1 . (33) if heat transfer coefficient is defined as iw av tt q   , then                                                                      mv i ivm mvm om ov g g r exp d exp d pe r expr pe pepe 23 1625 1 640 11 8 1 2 1 , (34) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 212 where it – liquid film saturation temperature on the interface. for water films sati tt  . when evaporating from the free surfaces of solution films fcsati tt  , where fc physicalchemical depression. it should be pointed out that ov  corresponds to the dimensionless  bwx , which is to be passed by the last layer of the film when moving with the velocity llu between the peaks of big waves                  1 ll bw llbw ll ll bwll llbw u c f c ucf сu uc u ux , (35) where c , bw , bwf – face velocity, length and big waves frequency, respectively, ll bw uc    – time, in which the peaks of big waves cross the film cross section, which, in turn, is moving with the llu . the values c , bw , bwf are the function of wetting density, interface shear stress and channels geometry. therefore, the analysis of heat transfer in liquid films covered by big waves can be done with the adequate accuracy with the application of data characterizing wave processes only in channels with the relevant geometry and the experimental conditions that correspond to those, taken for the analysis. the length of big waves bw on the water film surface in a tube with 25 mm diameter is according to [8]. the reynolds number r=40…400 remains approximately constant within bw =100…120 mm, with the increase of the reynolds number to 4000 the length of big waves also increased gradually to 140 mm at distance 2.38 m from the distributing device. at these conditions the phase velocity c was 0.5…2.5 times the mean film velocity llu . from (35) follows that given lluc 2 the distance, which the film passes within the time of a big wave passage will be equal to the length of a big wave, that is bwbwx  . therefore, it is possible to assume approximately that    bwov  . as it may be concluded from (33), the decrease in o entails growth of the heat flux, insofar there is a noticeable growth of the temperature gradient on the wall, and vice versa. therefore, for a given bw the value o remains the only sought function in (34), which correlates the relationship between the modeled heat transfer characteristics with experimental data. the function o has been derived based upon the comparison of the calculated by the correlation (34) heat transfer coefficients with the experimental data, obtained when studying heat transfer to the saturated liquid films at the regime of evaporation from free surface. in [12] the expression for o applicable to the free falling water and sugar solution films in a tube of 20 mm diameter and 1.8 m at bw =120 mm is given 2.043  yo при 115.0y , 1331150 98.0  yo , при 115.0y , (36) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 213 where 01.0 9.0 3 3                           wat v g г y ; wat – water cinematic coefficient of viscosity;     g – capillary constant;  – surface tension. at opepe  , 01.09.0 3 / 4 3                     wato o g ape y , 2.043  oo y ,at 115.0y 1331150 98.0  oo y , at 115.0y in case of the co-current steam flow a shear stress appears on the interface, which is determined as 8 2 2 2 u i  , (37) where 2u – steam core velocity; 2 – steam density. the value of interface friction coefficient can be determined from [11]. the correlation generalizes the experiments on the determination of friction pressure loss in steamand gasliquid flows of water and sugar solutions in tubes with diameter 13…55mm 1 26.1 2 1 1,1 2 5.1226.1 21 1025.1exp627                                    fr d d hfrkfr d d o o  , (38) where    kc 23 1 104103 – hydraulic friction coefficient for the first region – region of weak interaction; 25,0 2re 316.0 c – hydraulic friction on dry wall;     g g г k v6 2 3 ; 2 2 2    dg h ; dg u fr 2 2 2  ; 2 22 2    du re ; мdo 013.0 ; 2 – steam dynamic viscosity; d – tube diameter. transfer between the first and second regions happens at a condition 0112  o , d d hfr . a comparison of experimental data on heat transfer coefficients to water and sugar solutions with the concentration up to 70% dm ( sm /1077.3 26 ) has been made. the data obtained from the regime of falling dawn films over the vertical surface with the interface interaction from [11] with the results calculated by (3, 34, 36, 37, 38) are given in fig. 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 214 a w кm2, 1000 100 20 30 2000 3000 4000 5000 6000 7000 1 1 22 3 3 4 4 5 5 6 6 u2 m/s, figure 5. effect of steam core velocity upon heat transfer coefficient. water and sugar solutions at t = 100 0c. 1, 2 – water; 3, 4 – sugar solution, dm=40%; 5, 6 – sugar solution, dm = 70%. λbw =120 mm. lines – calculated by (3, 34, 36, 37, 38) the derived equations (34, 36) reflect the heat transfer mechanism in films, in case there is no co-current steam core (u2=0, i =0). fig. 6 illustrates the comparison of calculations by (34, 36) with the experimental heat transfer coefficients obtained from free falling water and sugar solutions (up to 70% dm) on the vertical surface [11]. 4000 3000 2000 1000 1 2 3 4 5 6 10 4 1 2 3 4 5 6 a w кm2, m2,гv 1 2 3 4 5 6 s figure 6. comparison of experimental heat transfer coefficients (htc) to water and sugar solutions free falling films with the calculated results by (34, 36) at λbw=120 mm. 1 – water, t = 100 0c; 2 – sugar solution, dm = 30 %; 3 – 40 %; 4 – 50 %; 5 – 60 %; 6 – 70 %. as it can be seen from the given plots, the correlations (34, 36) are also valid within the turbulent range (r=6500 for water) despite the fact that the turbulence parameters were not used explicitly in the derived equations. α α α α food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer modeling in downflowing laminar films with the developed wavy structure with co-current steam flow, food and environment safety, volume xv, issue 3 – 2016, pag. 203 – 215 215 4. conclusions 1. a model of heat transfer to the liquid films with the developed wavy structure down flowing on the vertical surfaces has been developed. the proposed model is based on the suggestions that the periodic relaxation of temperature field, disturbed as a result of a mixing effect of big waves. the comparison of experimental results with the calculated within the proposed model ones seem to be acceptable. this coincidence proves the applicability of the proposed model and describes adequately the mechanism of heat transfer to the films heated to the saturation temperature with the developed wavy structure. 2. the equations (3, 34, 36, 37, 38) are recommended to be used for the calculation of htc in water and sugar solutions (up to 70% dm) at a regime of free falling and the presence of cocurrent steam flow, as well. the equations may be applied to the films at a regime of evaporation from the interface of laminar and turbulent films within the marked above range of flow rates. 5. references [1]. holpanov l.p., shkadov v. ya., hydrodynamics and heat-mass exchenge with interface, nauka, moscow, (1990),271 p. [2]. alekseenko s.v., nakoryakov v. e., pokusayev v. e., wave flow of liquid films, nauka, novosibirsk, (1992), 256 p. [3]. nakoryakov v. e., pokusayev v. e., shreyber i.r. wave dynamics gazo-and vapor-liquid environments, energoatomizdat, moscow, (1990), 248 p. [4]. ardashev v.a., research of heat exchange at evaporation gravitationally the flowing-down liquid films in vertical pipes, dissertation, kyiv, (1982), 188 p. [5]. vasylenko s.m., heat transferin vapor-liquid flows of food productions heat exchangers, dissertation, kyiv, (2003), 379 p. [6]. globa o.v., heat hydrodynamic processes in pellicle evaporating apparatus of apple juice, dissertation, kyiv, (2011), 218 р. [7]. ganchev b. g., cooling of nuclear reactor of elements by film flow, energoatomizdat, moscow, (1987), рр. 192. [8]. kulov n. n., hydrodynamics and a mass exchange in the film and disperse streams descending, dissertation, moscow, 1984,409 p. [9]. pryadko м.о., globa о.v., forsyuk а.v., globa v.z., film flows in tubes of apple juice evaporators, scientific works of national university of food technologies, vol. 21, №2, (2015),pp. 88 – 95. [10]. demekhin e.a., kalaidin e.h., rastaturin a.a. effect of wavy regimes upon the mass transfer in down flowing liquid film. termophysics and aerodynamics . v. 12, no 2, p. (2005), 259-269. [11]. riabchuk о. м., thermal and hydrodynamic processes evaporation in downstream film flows of sugar solutions, dissertation, kyiv, (2013) , 167 р. [12]. petrenko v, prydko m, zasyadko y, miroshnyk m., modeling of heat transfer in free down flowing laminar liquid films with development wavy structure at the regime of evaporation from the interface, ukrainian food journal, volume 5. issue 1. (2016), pp. 162 – 173. http://enuftir.nuft.edu.ua/jspui/browse?type=author&value=forsyuk%2c+andriy [9]. pryadko м.о., globa о.v., forsyuk а.v., globa v.z., film flows in tubes of apple juice evaporators, scientific works of national university of food technologies, vol. 21, №2, (2015),pp. 88 – 95. 67 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1 2016, pag. 67 70 development of complex acidifiers and analysis of impact on biochemical processes in dough tetyana silchuk1, vira zuyko2* 1faculty of hotel, restaurant and tourist business, national university of food technologies volodymyrska street, 68, kyiv, ukraine, tsilchuk@mail.ru 2*faculty of hotel, restaurant and tourist business, national university of food technologies volodymyrska street, 68, kyiv, ukraine, viviensmile@yandex.ua received february 25th 2016, accepted march 29th abstract. nowadays there are many different changes in all branches of human practice. this is engaged with runaway of science and technique and is dictated by consumer`s demand. similarity of products, low capacity to adaptation and changes may lead to a decrease of products’ popularity with consumers. nutrition technology, especially production of rye and rye-wheat bread, is also under great pressure from the point of view of growing rivalry. but this product apart from its popularity with consumers is not only an integral part of rational nutrition, but also a part of clinical nutrition. in modern conditions it is necessary to find ways to reduce the length of production of rye-wheat bread and to expand the range of products in restaurant enterprises and mini-productions. a formula of acidifiers, based on organic acids, dry milk whey, rye fermented malt and enzymes was developed in view of applying it in the technology of rye and rye-wheat bread. it contains organic acids and enzymes and provides significant shortening of dough fermentation and proper quality bread. the addition of supplements can reduce the duration of the process to 2,5-3 times and provide improvement of the structural and mechanical, physical and chemical properties of dough and bread. the influence of elaborated complex additives on biochemical processes in the dough and on bread quality was analyzed. a comparative study was made on the products obtained by traditional technology, using thick sourdough and those obtained by accelerated technology using of complex acidifiers.the changes in the balance of reducing sugars in the dough with complex acidifiers were analyzed as well. the addition of recently developed complex additives provides shortening of production process without quality loss of the finished products. these results allow the using of complex acidifiers in restaurant enterprises and mini-productions. keywords: rye-wheat bread, accelerated technology, biochemical properties, complex acidifiers. 1. introduction. in recent years the development of restaurants and mini technology influenced significantly the production of bakery products. previously bakery was produced mainly on industrial bakeries, which are equipped with specialized equipment. now a part of the bakery products are producing at mini bakeries or in restaurant industry. this makes it possible to produce a wide range of bakery products and sell them in their outlets being hot during the day. nowadays bread from rye flour (rye bread) is in demand among the people and has high nutritional value, thanks to the flour in essential amino acids, vitamins, micro and macro elements. a special taste and smell of rye bread increases its physiological value. these properties allow including the bread from rye flour to category of healthy nutrition products. http://www.fia.usv.ro/fiajournal mailto:tsilchuk@mail.ru mailto:viviensmile@yandex.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 tetyana silchuk, vira zuyko, development of complex acidifiers and analysis of impact on biochemical processes in dough, food and environment safety, volume xv, issue 1 – 2016, pag. 67 – 70 68 taking into account the characteristics of carbohydrate and amylase complex, protein and proteinase complex of rye flour, technology of making bread is very sophisticated and labor-intensive (demanding intensive labor). on ukrainian market acidifiers supplying additives are imported, but there are problems of economical and resource availability of these additives, the lack of scientific evidence of their use. these reasons make necessary to develop new acidifying additives and technological principles of their application. therefore, the creation of complex acidifying additives with a substantiate structure for the rapid production of rye-wheat bread and a study their impact on basic dough processes is actual and up-to-date [14] , [6]. to create the proper conditions of biochemical processes in the dough and shortening manufacturing rye-wheat bread it is necessary to provide proper acidity of dough and fermentation activity of yeast. for this purpose it is necessary to develop the complex acidifiers and explore their effects on biochemical processes in dough and bread quality. 2. materials and methods citric acid, dry milk whey, rye fermented malt, enzymes with different principles of action were used to create compositions of acidifiers. all components are selected taking into account the features of ryewheat dough and products of it. dough was prepared from a mixture of wheat flour first grade and peeling rye flour in proportion 50:50(1:1). the humidity of dough was 47%. there were prepared dough on traditional technology on thick rye sourdough as a control sample. the quality of semi-finished product and bread was evaluated by organoleptic and physico-chemical parameters with standard methods [5]. the intensity of fermentation of dough was determined by its ability gasification, sugar content research was carried out by shorly’s iodometric method. 3. results and discussion using methods of mathematical-statistical modeling and trial baking, three options of compositions by complex acidifiers were developed: "effect plus", "optimal-1" and "optimal-2". there were defined their influence on technological process and the quality of bread [7, 8]. there were investigated 4 samples of dough: 1 – control on thick rye sourdough, 2, 3, 4 – developed acidifiers "effect plus", "optimal-1", "optimal-2" respectively. there were established (table. 1), that production of rye-wheat bread for accelerated technology with using developed acidifiers initial acidity of dough was greater than the control sample on 0.4 – 0.8 degrees. this is explained acidifiers’ great acidity, which composed of organic acids. during fermentation acidity of dough is increasing due to the intensification of the fermentation of dough. thus, in samples of dough with the established acidifiers process of acids accumulation occurred more intensively. for less time of fermentation there were accumulated more acids respondent substances as in control sample. this fact can be explained by more optimal ph and the presence of pressed yeast in dough’s samples with acidifiers’ addition. the intensive of fermentation promotes to cut down the length of maturation products that are relevant in the accelerated technologies of rye-wheat bread. bread, which was made by accelerating technology, satisfied the requirements of regulatory documents, had good organoleptic properties, pleasant taste and flavor. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 tetyana silchuk, vira zuyko, development of complex acidifiers and analysis of impact on biochemical processes in dough, food and environment safety, volume xv, issue 1 – 2016, pag. 67 – 70 69 table 1 effect of acidifiers on figures of technologic process and rye-wheat bread’s quality figures control with acidifiers effect plus optimal-1 optimal-2 dough initial acidity . grad. 6.6 7.4 6.8 7.0 duration of fermentation. min. 90 30 30 30 duration of maturation. min. 50 40 40 35 final acidity. grad. 7.4 8.6 7.7 8.0 bread specific volume . сm3/g 2.4 2.2 2.2 2.3 porosity. % 66 64 65 66 acidity. grad. 6.0 6.6 6.4 6.8 look right shape, smooth crumb quality non-rigid porosity equable taste and flavor natural for rye-wheat bread one of the features, which indicators the intensity of fermented dough, is its ability of gasification property. this figures images activity of yeast cells and the availability of sugars and nitrogencontaining substances. since compositions of acidifiers contain significant amount of these substances, there was conducted determination of number of selected carbon dioxide during 300 minutes of fermentation. there was found that gas production in the dough with acidifiers is 11% 32% stronger than in the control sample. compositions developed acidifiers provide more intensive gas production. there is associated with more accumulated sugars under the influence of enzymes in dough’s fermentation. the dynamic of gas production in the rye-wheat dough shows that acidifiers’ addition in dough intensifies the accumulation of carbon dioxide during the period of fermentation. maximum of carbon dioxide’s emission in samples with acidifiers is observed much earlier than in the control sample. it is known that the biochemical processes in dough can be characterized by changes in the balance of sugars in semi-finished product. the depth of their changes effects on gas production’s ability of dough during fermentation. there were prepared four samples (1-control thick rye sourdough, 2, 3, 4 – with developed acidifiers "effect plus", "optimal-1", "optimal-2" respectively) to determining the balance of sugars of rye-wheat dough. to avoid the influence of yeast’s enzymes and fermentation on content of sugars, yeasts are not made in dough. similarly, there were prepared four samples of dough but with addition of 3% of yeast. accumulation of sugars was determined immediately after mixing and after 1.5 hours of dough’s fermentation at temperature of 32° c. in the dough after mixing and after 1.5 hours of fermentation sugars’ content were determined by shorly’s iodometric method. the accumulation of sugars was determined by the difference between their content in the dough after mixing and after 1.5 hours of fermentation. the comparison between sugars formed in non-yeast dough, and the residual of sugars in dough after 1.5 hours of fermentation, helps to determine amount of sugars that were fermented (tab. 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 tetyana silchuk, vira zuyko, development of complex acidifiers and analysis of impact on biochemical processes in dough, food and environment safety, volume xv, issue 1 – 2016, pag. 67 – 70 70 table 2 content of reducing sugars in rye-wheat dough subject reducing sugars samples without yeast samples with yeast 1 2 3 4 1 2 3 4 dough after mixing 3.62 4.46 5.1 4.67 3.59 4.58 5.2 4.8 dough after 1.5 hours of fermentation 6.0 7.06 7.8 8.0 4.28 5.4 5.9 6.0 accumulated sugars 2.38 2.6 2.7 3.33 0.69 0.82 0.7 1.2 fermented sugars 1.69 1.78 2.0 2.13 the table 2 shows that in dough with acidifiers accumulation of sugars and their fermentation exceed the level of control in all dough samples. after 1.5 hours of fermentation there were formed in samples of dough with the addition of 10-40% more reducing sugars than in control sample. this can be explained by increased activity of enzymes by reduction the ph of dough acidifiers, leading to a deeper hydrolysis of starch and dextrins. the number of fermented sugars in dough with acidifiers was by 26% higher than in the control due to the intensification of the process of fermentation. this creates conditions to reduce the period of fermentation of semi-finished products. thus, intense accumulation and fermentation of sugars in dough samples with complex acidifiers is important foundation for intensifying the process of fermenting dough. 4. conclusions the development of integrated manufacturing new acidifiers for rye-wheat bread’s manufacture is appropriate and relevant. production of rye-wheat bread by accelerated technology with the addition of complex acidifiers promotes more intensive acids accumulation in dough. it increases the gas production process and accelerates ripening dough reduces the duration of proofing dough pieces and has positive effect on the taste qualities of bread. developed acidifiers, based on organic acids and enzymes provide significant shortening of dough fermentation and proper quality bread. the use of elaborated complex acidifiers is a perspective in the production of ryewheat bread by accelerated technology in discrete modes of production in restaurant enterprises and mini-productions. 5. references [1]. puchkova l.i.. malofeeva y.n. matveeva i.v. yudina t.a.. the use of enzyme preparations in the production of bread from a mixture of rye and wheat flour. food ingredients. raw materials and additives. 2: 68 – 71. (2001). [2]. matveeva i. the concept and application of technological solutions of baking improvers. food and beverage. 5: 20 – 23. (2006) [3]. sylchuk t.a.. kulinich v.i.. sidorenko a.a.. application acidifiers the production of rye-wheat bread. bakery and confectionery industry ukraine. 05 (126): 3 – 5. (2015) [4]. gorban n.. enzyme preparations and their influence on the flour. bakery and confectionery industry ukraine. 10 (35): 30 – 31. (2007). [5]. drobot v.i.. bakery production technology. kyiv. logos. 368. (2002). [6]. goesaert h.. brijs k.. veraverbeke w.s.. courtin c.m.. gebruers k.. delcour j.a. wheat flour constituents: how they impact bread quality. and how to impact their functionality. trends in food science & technology. 16:12 – 30. (2005) [7]. sylchuk t.a.. drobot v.i.. kulinich v.i.. tsyrulnikova v.v.. patent 100480 ua. ipc a21d 8/02 (2006.01) ingredients acidifiers complex "optimal-1"; applicant and patentee national university of food technologies. u201501349. (2015) [8]. sylchuk t.a.. drobot v.i.. kulinich v.i.. tsyrulnikova v.v.. patent 100478 ua. ipc a21d 8/02 (2006.01) ingredients acidifiers complex "optimal-2": applicant and patentee national university of food technologies. u201501346. (2015) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 contents: 1. effect of treatment parameters on the carotenoid extraction from tomato peels of bulgarian industrial varieties milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova 283 289 2. contributions to achieve a composite material for advanced electromagnetic shielding of living and workspaces. first partshielding material cristian florin alexuc, gheorghe gutt, sonia amariei 290 298 3. effects of twin screw parameters upon the mechanical hardness of ready-to-eat extrudates enriched with de-oiled rice bran tanuja srivastava, d.c. saxena, renu sharma 299 308 4. improvement of wheat flour dough rheology by αlpha -amylase and protease combination georgiana gabriela codină, silvia mironeasa 309 316 5. chlorophyll content and antioxidant activity of sunflower oil with aromatic raw materials svitlana usatiuk, liubov pelekhova 317 320 6. the influence of frozen storage on some commercial mushrooms octavian baston, eugenia mihaela pricop, cornelia lungu 321 -326 7. the evolution of some biochemical indices during chilling of four freshwater fish species camelia-zaraza avrămiuc, marcel avrămiuc 327 – 334 8. influence of citrus fibre addition on textural and rheological properties of yogurt mircea oroian, sergiu paduret, gheorghe gutt 335 341 9. an i n vesti gat i on on t he e xtr acti o n con cent r ati on of mi cr oel eme nts fr o m aq ueou s so l uti o n for at o mabs orb ti o n an al ysis igor kobasa, vasyl bilogolovka, mariya vorobets, oxana panimarchuk 342 348 10. anti oxi da nt a cti vit y a nd t o tal p h enoli c co nt e nt i n alliu m ursin u m a nd r a nu nc ulu s fi ca ri a gheo r ghe -florin bârlă, maria p oroc h seriţan, elen a săndu leac (t ud os i), si mo na elen a ciornei (şt efăro i) 349 353 11. t echn ol o gi cal f eat u r es of co mb i ni ng co n de ns ed can n ed mil k wit h s u gar a n d frui t-b er r y s yr u ps du ri ng p r oc essi ng natal ija val eriivn a ry abokon, dmit ro vik to rovi ch ri ndy uk, maks i m serg i yo vu ch sh pak, kseniy a ro manivna k omisar 354 359 12. molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage remzi cholakov, rositsa denkova, desislava teneva, velichka yanakieva, iliyan dobrev, zapryana denkova, zoltan urshev 360 364 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 13. medical and biological estimation of grain small loaves on the basis of whole wheat whole grain with the inclusion of vegetable additives marina mardar, rafaela znachek 365 371 14. application of titrimetric technique to determine phosphates in cheeses mariуa vorobets, igor kobasa, aureliуa cheban 372 – 375 15. new friendly environmental etching technologies for some steels and copper alloys vira vodyanka, sergiy boruk, igor winkler 376 384 16. influence of starch and oil phase ratio on the quality of emulsions oksana lugovska, vasilij sidor 385 391 17. author instructions i v 18. subscription information vi 143 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 143 147 antioxidant activity of some essential oils * sonia amariei (gutt) 1, simona ciornei (ștefăroi)1, elena sănduleac (todosi)1 1 faculty of food engineering, ştefan cel mare university of suceava, 13 universităţii str., 720229, suceava, romania guts@fia.usv.ro, sanduleacelena@yahoo.com, simona_stefaroi@yahoo.com * corresponding author received april 28th 2013, accepted june 5th 2013 abstract: the paper presents the antioxidant activity of some essential oils: clove, basil, coriander, sage, dill, oregano, juniper, rosemary and thyme, as determined by the dpph (2,2-diphenyl-1picrilhidrazil). clove oil has not only the greatest antioxidant activity but also a strong antimicrobial, antiseptic, astringent, anti-inflammatory analgesic properties, given by its main component namely eugenol. following the results, the other essential oils, present an antioxidant activity with lower values. keywords: essential oils, antioxidant activity, dpph , 1. introduction essential oils are volatile, natural, complex compounds, that are characterized by a strong odor and are formed in aromatic plants as secondary metabolites. antioxidants are molecules with electron stable up or with the ability to receive additional electrons. they are the body's natural defense system against the harmful effects of free radicals, electron capture seekers [26]. antioxidants protect cell structures and dna against free radicals. antioxidants fail or receive an extra electron to neutralize free radicals and stop the cascade effect of oxidation. because free radicals are created not only in the local geography, but also in our body, for health and longevity, it is essential to have a continuous supply of antioxidants, so that our body can benefit from a healthy dietary plan and a healthy lifestyle. essential oils which are concentrated in powerful antioxidants are called plant essences. they come from raw plant material such as flowers, leaves, wood, bark, roots, seeds and shells. essential oils have been used for thousands of years for their healing and purifying effects on the body. today, consumers are demanding even more food quality and the demand for natural and organic food is also increasing. the food industry is required in these circumstances to replace synthetic food additives with natural additives, namely natural antioxidants, to retard oxidative degradation of lipids in foods and improve their nutritional quality and value. aromatic and medicinal plants and their extracts have a growing interest for the food industry and scientific research due to the default content with antioxidant properties and antimicrobial substances. cloves are a spice made from the buds of a flowering exotic tree (syzygium aromaticum or eugenia caryophyllata) mirtaceelor family, which includes among food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), simona ciornei (ștefăroi), elena sănduleac (todosi), antioxidant activity of some essential oils, food and environment safety, volume xii, issue 2 – 2013, pag. 143 147 144 others myrtle and eucalyptus. clove oil is obtained by distillation of the leaves, flowers and stems [1]. clove oil is a strong antimicrobial, antiseptic, astringent and anti-inflammatory analgesic that is used in dental diseases. the antioxidant activity of clove oil is given by its main component namely eugenol (76.8%) [2]. rosemary (rosmarinus officinalis l.) is a plant that has been cultivated since ancient times due to strong antioxidant activity and antimicrobial activity. rosemary has other important qualities such as antiviral, antiinflammatory and anticarcinogenic [3], [4]. the composition of rosemary essential oil was a special research topic in recent years. it contains mainly monoterpenes and monoterpene derivatives (95-98%) [5], [6]. the main volatile compounds in rosemary are camphor and 1,8-cineole (díaz-maroto et al, 2007). genus salvia (sage) is one of the largest and most important type of medicinal and aromatic plant in the lamiaceae family, which includes about 900 species, widespread throughout the world [7]. previous studies on antimicrobial activity, antioxidant and antifungal showed that plants of this kind are particularly used as a natural preservative and antioxidant potential for the food industry and cosmetics [8]. the antioxidant activity of sage oil has been extensively studied with other plants of this kind [8]. dill (anethum graveolens l.) is an annual or biennial plant of the parsley family (apiaceae or umbelliferae), is native to southwest asia and southeast europe and is cultivated since ancient times [9]. the specialty literature speaks of the fact that the use of dill leaves in alimentation may reduce the risk of cancer and cholesterol levels [10]. moreover, the essential oil of dill is a good antioxidant [11], [12]. thyme (satureja hortensis) is a perennial plant of the genus satureja, labiates family (lamiaceae). it is a shorter plant, that doesn’t reach more than 20 to 30 cm in height. the main component of thyme oil is thymol, a powerful antiseptic that is considered to be quite toxic if used inappropriately. aromatherapy is known in the ranks as one of the most powerful antiseptic. thymol has been researched extensively for its antibacterial, antiviral and anti-fungal properties [13]. coriander (coriandrum sativum) is a species of annual herbaceous plant from the umbeliferelor family, exclusively of culture, 30-100 cm high. the addition of coriander in food increases its antioxidant content and may inhibit unwanted oxidation processes [14]. studies on the chemical composition of coriander oil showed as major components linalool (75.30%) geranyle acetate (8.12%) and αpinene (4.09%) [15]. oregano (origanum vulgare l.), also known as wild marjoram, basil forest, is a medicinal plant of the lamiaceae family, genus origanum. the composition of oregano essential oil determined by gas chromatography shows a high content of carvacrol and thymol, which showed a high antioxidant activity when tested [16] . other research results show that the essential oil of oregano may be used as a source of antioxidants to the food industry, so it is interesting to consider application of this natural antioxidant as an additive in some products [17]. common basil (ocimum basilicum l., o. basilicum), belongs to the family lamiaceae and is an annual plant that grows in many regions around the world. contains a broad range of essential oils rich in phenolic compounds including polyphenols such as flavonoids and anthocyanin [18], [19], [20]. basil essential oil is widely used for flavoring foods such as sauces, pickles, beverages, spices and pastries, but it is also used in perfumery, medicine, pharmaceutical, etc. [21]. juniper (juniperus communis l.) is a conifer belonging to the genus juniperus of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), simona ciornei (ștefăroi), elena sănduleac (todosi), antioxidant activity of some essential oils, food and environment safety, volume xii, issue 2 – 2013, pag. 143 147 145 the family cupressaceae. dried fruits of juniper (berries large, black, shriveled) are used in cooking as a spice, especially in marinades for cabbage, and as an ingredient in the manufacture of gin. in many studies, there are results which indicate that juniper has antioxidant properties that can be exploited to inhibit lipid oxidation [22]. the main chemical components of the oil of juniper are apinene, camphene, b-pinene, sabinene, aphellandrene, a-terpinene, y-terpinene, 1,4cineole, and traces of limonene, camphor, linalool, linalyl acetate , borneol and nerol, and to all of these substances juniper oil is an important antioxidant [23]. 2. experimental the essential oils used in this study were purchased from a herbal pharmacy in suceava, marketed and manufactured by solaris in 10 ml glass bottles with shelf life until 16/03/2014. the dpph reagent that was used at determination was pure from an analytical point of view, and the equipment used belongs to the faculty of food engineering suceava: ocean optics fiber optic spectrophotometer, other laboratory utensils. determination of antioxidant activity of essential oils by spectrophotometric method dpph (2,2-diphenyl-1picrilhidrazil). the dpph test (2,20diphenylpicrylhydrazyl) is based on the reaction of reduction of dpph of violet color to pale yellow difenlpicrilhidrazină, the moment they react with the antioxidants in the sample [1]. dpph is one of the most stable organic radicals and commercially available and has a maximum absorption at 515 nm in the vis. when the solution starts to reduce its volume it begins a discoloration process and before that reaction, the solution is being monitored spectrophotometrically (spectrophotometer ocean optics). this is an alternative method for the determination of antioxidant capacity of a system. inhibition % = 100 [(at5/ at0) *100 in which: at0 dpph is the absorbance at t = 0 min. at5 is the absorbance of dpph + oil sample after t = 5 min. table 1. results for antioxidant activity type of essential oil inhibition (%) thyme oil 23.41 rosmary oil 3.3 basil oil 2.2 sage oil 4.35 dill oil 2.3 juniper oil 2 coriander oil 4.8 oregano oil 29.35 clove oil 93.69 antioxidant activity of essential oils 23,41 3,3 2,2 4,35 2,3 2 4,8 29,35 93,69 0 10 20 30 40 50 60 70 80 90 100 1 types of oils a nt io xi da nt a ct iv ity (% ) thyme oil rosemary oil basil oil sage oil dill oil juniper oil coriander oil oregano oil clove oil fig. 1. antioxidant activity 3. results and discussion there are many methods used to evaluate the antioxidant capacity of foods and dietary supplements, herbal extracts and pure compounds. however, few of them have been widely used because of the difficulty of measurement associated food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), simona ciornei (ștefăroi), elena sănduleac (todosi), antioxidant activity of some essential oils, food and environment safety, volume xii, issue 2 – 2013, pag. 143 147 146 methodologies and sources of free radical [24], [25], [1]. after calculations it was found that clove oil has the highest antioxidant activity, or percent inhibition, reaching 93.69%. this could be observed even when added to dpph solution because it has faded. high speed response seen in this oil shows a high content of antioxidants, able to act against free radicals. in the literature, this content is represented mostly by eugenol, which is a fenilpropen a substitute for guaiacol. it is an oily liquid, yellowish and clear which is extracted from certain essential oils is slightly soluble in water and soluble in organic solvents. discoloration of other types of essential oils was perceived by the naked eye, but there was oregano oil with antioxidant activity of 29.35% and 23.41% of the thyme. all oils showed antioxidant activity in different proportions. 4. conclusion essential oils have a high density of antioxidants and also participate in the destruction of pathogens, microbes, viruses, having bactericidal action. phenolic compounds, including polyphenols, tocopherols and fat-soluble vitamins with antioxidant activity are the main components of essential oils, antioxidant components which ensure potential. in terms of chemical content, eugenol has the highest proportion in oil of cloves, the thyme oil thymol prevails in coriander oil is linalool, carvacrol and thymol found in oregano oil, thujone and camphor in the essential oil sage, cineol and eugenol in basil oil, rosemary oil is found in camphor and 1,8-cineole, borneol, and that these substances are essential to provide an outstanding use in many fields such as medicine, food, perfumery and body care, pharmaceuticals etc.. 5. references [1]. gülҫin a. i., mahfuz elmastas b, hassan y. a-e. , antioxidant activity of clove oil – a powerful antioxidant source, arabian journal of chemistry (2012) 5, 489–499 [2]. jirovetz l. , buchbauer g. , stoilova i. , stoyanova a. , krastanov a. , schmidt e. , chemical composition and antioxidant properties of clove leaf essential oil, 2006 [3]. mengoni e. s., vichera g., rigano l. a., rodriguez-puebla m. l., galliano s. r., cafferata e. e., et al. (2011). suppression of cox-2, il-1beta and tnfalpha expression and leukocyte infiltration in inflamed skin by bioactivecompounds from rosmarinus officinalis l. fitoterapia, 82, 414e421. [4]. barni m. v., carlini m. j., cafferata e. g., puricelli l., & moreno s. (2012). carnosic acid inhibits the proliferation and migration capacity of human. oncology reports, 27(4), 1041e1048. [5]. angioni a., barra a., cereti e., barile d., coïsson j.d., arlorio m., dessi s., coroneo v., cabras p., chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of rosmarinus officinalis l. [6]. diaz-maroto m. c. , perezcoello e. sanchez-palomo m. a. gonzales v. , impact of drying and storage time on sensory characteristics of rosemary (rosmarinus officinalis l.), 2007 [7]. walker j.b., sytsma k.j., treutlein j., wink m., 2004. salvia (lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of salvia and tribe mentheae. am. j. bot. 91, 1115–1125. [8]. tenore g. c. , ciampaglia r. , apostolides arnold n. , piozzi f. napolitano f. , rigao d , senatore f. , antimicrobial and antioxidant properties of the essential oil of salvia lanigera from cyprus, 2010 [9]. bailer j., aichinger t., hackl g., de hueber k., dachler m. (2001). essential oil content and composition in commercially available dill cultivars in comparison to caraway. industrial crops and products—an international journal, 14, 229–239. [10]. yang y., huang c. y., peng s. s., & li j. (1996). carotenoid analysis of several darkgreen leafy vegetables associated with a lower risk of cancers. biomedical and environmental sciences, 9, 386–392. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 sonia amariei (gutt), simona ciornei (ștefăroi), elena sănduleac (todosi), antioxidant activity of some essential oils, food and environment safety, volume xii, issue 2 – 2013, pag. 143 147 147 [11]. delaquis p. j., stanich k., girard b., & mazza, g. (2002). antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. international journal of food microbiology, 74, 101–109. [12]. yung-shin shyu a, jau-tien lin b, yuan-tsung chang c, chia-jung chiang d, deng-jye yang, evaluation of antioxidant ability of ethanolic extract from dill (anethum graveolens l.) flower, 2008 [13]. zehra karagöz emiroğlu, gökçe polat yemiş , betül kodal coşkun, kezban candoğan, antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties, 2010 [14]. wangensteen h., anne berit samuelsen a. b., malterud k. e., antioxidant activity in extracts from coriander, 2004 [15]. gurdip s., sumitra maurya, de lampasona m. p., cesar a., catalan n. , studies on essential oils, part 41. chemical composition, antifungal, antioxidant and sprout suppressant activities of coriander (coriandrum sativum) essential oil and its oleoresin, 2006 [16]. lagouri v. et al. composition and antioxidant activity of essential oils from oregano plants grown wild in greece, 1993 [17]. kulisic t., radonic a., katalinic v., milos m., use of different methods for testing antioxidative activity of oregano essential oil, 2004 [18]. simon j. e., quinn j., and murray r.g. basil: a source of essential oils. in: j. janick and j.e. simon (eds.), advances in new crops. timber press, portland, p: 484–489, 1990. [19]. phippen w. b. simon j. e. 1998. anthocyanins in basil (ocimum basilicum l.). j. agr. food chem. 46:1734–1738. [20]. phippen w. b., simon j. e.. 2000. anthocyanin inheritance and instability in purple basil (ocimum basilicum l.). j. hered. 91:289–296 [21]. muhammad asif hanif, masoud yahya al-maskari, awatif almaskari, ahmed al-shukaili, ahmed yahya al-maskari, jamal nasser alsabahi, essential oil composition, antimicrobial and antioxidant activities of unexplored omani basil, 2011 [22]. elmastaş m, gülçin i̇. , beydemir ş., küfrevioğlu i. ö, enein a., hassan y, a study on the in vitro antioxidant activity of juniper (juniperus communis l.) fruit extracts, 2006 [23]. theodore t., borek j. , hochrein m., irwin a. n., composition of the essential oils from rocky mountain juniper (juniperus scopulorum), big sagebrush (artemisia tridentata), and white sage (salvia apiana), 2003 [24]. prior r.l., wu x., schaich k., 2005. standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. j. agric. food chem. 53, 4290–4302. [25]. schauss a.g., wu x., prior r.l., ou b., huang d., owens j., agarwal a., jensen g.s., hart a.n., shanbrom e., 2006. antioxidant capacity and other bioactivities of the freeze-dried amazonian palm berry, euterpe oleraceae mart. (acai). j. agric. food chem. 54, 8604–8610. [26]. bakkali f. a,. averbeck a,. idaomar m., biological effects of essential oils – a review. применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 contents: 1. probiotic properties of lactobacillus casei ssp. rhamnosus lbrc11 rositsa denkova, lyubka georgieva, zapryana denkova, svetla ilieva, velichka yanakieva 5 2. the method of determination of the optimal parameters of dispersed materials granulation through consolidation dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar 15 3. char acte r is ti cs o f t hre e ye ast st rains fo r wastewater treatment aleksander slavov, zapryana denkova, rositsa denkova 19 4. ho ney cl ass i ficat io n usi ng co lo u r meas u reme nt mircea oroian, ana leahu, cristina damian, amelia buculei 29 5. in flue nce o f m atu rat io n o n te ch nol og ical par ameters of whit e wi ne ch a rdo nna y an d pi not gri s ana leahu, sonia amariei(gutt), marcel avrămiuc, cristina-elena hreţcanu, cristina damian, mircea oroian 33 6. the study of heavy metals (ni, zn, cu, pb) in the vegetative organs, harvest and growing soil of potatoes, wheat and wild blackberry r. h. yedoyan, t. v. yedoyan 38 7. li mitat io n fact ors o f th e agri cu lt ural produ ction i n caci ca vi ll age , s ucea va elena-viorica rotaru, ioan gontariu 43 8. study on the influence of sex and breed on bovine meat quality ancuţa elena coşuleanu 48 9. effect of addition of carrageenan on rheological behaviour of yogurt cristina damian, mircea-adrian oroian, ana leahu, nicolae carpiuc 53 10. author instructions 59 11. subscription information 64 169 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 169 175 estimation of biochemical properties of walnuts from the region of suceava romania *ana leahu1, cristina damian1, mircea oroian1, cristina elena hreţcanu1 1faculty of food engineering, stefan cel mare university of suceava, universitatii str. 13, suceava, romania, analeahu@usv.ro * corresponding author received april 5th 2013, accepted 8th may 2013 abstract: the purpose of this paper was to evaluate the biochemical characteristics of walnuts in nine areas of suceava county. the walnuts have been stored in a cabinet at ambient temperature and we analyzed the physical and chemical parameters of the walnuts: fruit weight, moisture content, ash content, total oil content, protein and total carbohydrates. the predominate component in the kernel, varying between 55.1 and 66.1% was oil, followed by carbohydrate which varied between 12.1 and 24.38 %, and protein with values between 13.81 and 17.22 %. the values obtained from kernel weight varied between 3.42 and 4.84 g, water activity from 0.564 and 0.555, moisture from 3.77 and 4.58 %, ash content from 1.7 and 2.05 %, crude protein. extracts were prepared /methanol/ water and total polyphenol content (tpc) was in the range of 23.52-17.28 mg gae/g dry weight walnuts kernel. keywords: kernel weight, chemical composition, total polyphenol content, antioxidants, total oil; 1. introduction walnut (nux juglandes) is harvested from walnut tree (juglans regia), walnuts are rich sources of nutrients, and excellent sources of phosphorous, magnesium, iron, selenium, zinc and calcium, and walnuts oil is a consistent oil, rich in unsaturated fatty acids. sina cosmulescu, 2009 et al., in analyzing mineral composition in nine walnut cultivars with origin in romania, has found rich mineral composition, especially potassium, magnesium, calcium. walnut kernel contains about 60% lipids, but there may be differences between different varieties of walnut between 50-70 % [2-3], it is a good source of macronutrients and micronutrients and other bioactive. walnuts also contain 15.8% protein, 13.7% carbohydrate, 4.1% water, and 1.8 % ash [4]. walnuts contain relevant micronutrients, such as folic acid, niacin and vitamins e and b6, and minerals such as magnesium, copper, zinc, selenium, phosphorus and potassium. [5,6]. jun yang, 2009 reported brazil nuts are unique in that they are the highest known food source of selenium (se). the consumption of nuts and nut oil is recommended for those who suffer from hypercholesterolemia and are prone to atherosclerosis [6,7,3]. according to some authors [9] walnuts contain several phytosterols, which have been used as “nutraceuticals” as it appears that they can inhibit intestinal absorption of cholesterol. the high linoleic acid content of walnut oil food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 170 makes it undesirable for use in cooking as it is more prone to charring, but walnuts are a perfect ingredient in a variety of breads, muffins, cakes, and biscuits [10]. a. serrano et al. (2007) evaluated the influence of various cooking methods on the composition and physicochemical characteristics of restructured beef steaks formulated as 20% added walnut. thus, according to the his research the texture of restructured beef steaks with added walnut was softer and the addition of walnut improved the yield of cooked restructured beef steaks as compared to those formulated with beef fat [11,12]. according b. olmedilla-alonso et al. (2008), the consumption of the walnutenriched meat-based steaks (ca. 19.4 g walnuts/d) over five weeks provoked, on average, decreases in serum total cholesterol and ldl cholesterol concentrations of 4.5% and 5.1%, respectively. iwamoto m et al. (2002) evaluated the effect of walnut (diet had 12.5% of the energy derived from walnuts (44-58 g/day), comparative with reference and similar decreases were reported in normolipidaemic subjects (cholesterol decreased by 4.4% in men and women and ldl cholesterol by 11% in women). the restructured meat products with added walnuts supplied can be considered functional foods [13]. secondary products such as green nuts, shells and leaves are used in cosmetics, pharmaceutical and in folk medicine. the j. regia leaf extracts can be used as an easily accessible source of natural antioxidants [15]. a. fernandezagullo et al. (2013) analyse the effect of the solvent on the properties of walnut green husk extracts. according to this research, the solvent used resulted to be a significant factor on the total phenols content. the extracts with the highest total phenols content were obtained with 50% ethanol (wee), followed very closely by 50% methanol and the lowest value was obtained with water. [16] different works have characterised the phenolic composition of walnut byproducts [6 9]. after gathering nuts were dried at room temperature and were subsequently selected two samples for each region studied according to their sensory and size quality characteristics. in the present work, nine walnuts samples were characterized in respect to their chemical composition and the antioxidant potential.the samples were analyzed for proximate constituents: moisture content, ash content, oil content, protein and total carbohydrates of walnut. 2. materials and methods 2.1. samples and storage conditions in this study the fruit weight, moisture content, ash content, oil content, protein and total carbohydrates of nine walnut (juglans regia l.) samples (straja-1, radauti-2, solca-3, paltinoasa-4, campulung moldovenesc-5, suceava-6, falticeni-7, malini-8 and dolhasca-9) grown in suceava were determined. the samples of the walnuts (200g) used in this work were gathered in september 2011 from the suceava plateau, an area of romania. dried nuts were shelled manually and the parameters were analyzed for each sample. since the beginning of the analyses, in april 2012, the walnuts samples were stored in shell in paper bags, in a dark room at approximately 12°c for 5 months. 2.2. physical analysis physical analysis includes kernel properties such kernel weight and kernel ratio. 2.3. chemical analysis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 171 the determination of moisture and volatile matter content in kernel was effectuated according to the european standard en iso 665/2000 by the drying process in a drying chamber at the temperature of 103 °c. water activity plays an important role in the oxidation of walnuts in storage. the water activity of walnuts was evaluated with the device aqua lab. total ash composition was obtained by calcinations of 5g of sample at 600 °c for 240 min. protein content was analyzed by kjeldahl method. oil content was determined in a soxhlet apparatus, extracting the lipids from 10 g of kernel sample with petroleum ether, followed by extract evaporation to dryness and gravimetric determination according to the european standard en iso 659/1998. carbohydrate content was estimated by difference of the other components using the following formula: carbohydrate content = 100% (% moisture + % protein + % fat + % ash) [9]. energy was expressed as kilocalories/100g, using the factors mentioned in the romain legislation: energy (kcal) = 4.1x (g protein+g carbohydrate) + 9.3x (g lipid). the content of total polyphenolic compounds in walnuts methanol extracts diluted 1/10 was determined by folinciocalteu method. for the preparation of the calibration curve 0.5 ml aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 μm/ml aqueous gallic acid solution were mixed with 10.0 ml folin-ciocalteu reagent (diluted ten-fold) and 1.0 ml sodium carbonate (20.0%) and the volume made up to 10.0 ml with h2o. the absorption was read after 2 h at 25°c, at 760 nm. all determinations were performed in triplicate. total phenols were determined as gallic acid equivalents on a dry weight (mg gae g-1 d.w.). all chemicals used in this study were supplied by merck companies. distilled water was used throughout. 2.4. statistical analysis three replications were used to obtain average values and standard deviations for proximate biochemical properties and results are given as averages  sd. 3. results and discussion the results obtained from proximate composition of walnut samples were shown in table 2. the kernel weight (g), ranged from 3.42(falticeni) to 4.84 (dolhasca) g. the highest positively correlation have been observed in the case of kernel ratio with kernel weight (r = 0.832). significant differences in kernel ratio were found between nine selected walnuts samples kernel weight (p <0.01). walnuts also have low water content, with water activity (aw) ranged from: for s 2 radauti 0.658 to 0.665 to s1 straja. the low water content of nuts is related to their relatively easy storage over long periods of time, while the high unsaturated fat content has two major effects: it increases their chances of becoming rancid during cooking at high temperatures, and it also leads to a significant loss of flavor during storage. however, significant differences were not found in water activity of walnuts samples (p > 0.05). moisture presented the lower values. moisture content was higher in s9 dolhasca (4.58%) and lower in s5 campulung moldovenesc and s6 suceava (3.85%). important to maintain the quality of walnuts is low moisture content, which decreases the likelihood of microbial food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 172 growth, premature germination of seeds and late biochemical reactions associated with these processes. ash content was higher in s5 campulung moldovenesc (2.2%) and lower in s4 paltinoasa (1.7%). the negatively correlation have been observed in the case of ash with kernel weight (r=-0.784). if the results were compared with the ones previously obtained by pereira j. et al. (2008), some differences can be observed namely ash, total oil content (lower in the present work). its differences could be attributed to the harvesting year and environmental condition, with different temperatures, rainfall and light, which can influence the chemical composition of fruits [17]. the results indicate that walnut consumption conduce to a high input level of energetic value, ranged from 662.95 kcal in sample falticeni and 726.9 kcal in sample malini (table 1). protein content of the studied walnuts ranges from 13.81% (solca) to 17.22% (suceava) (table 1). walnuts are good sources of vegetable protein; they may be used in vegetarian diets. in this case walnuts have been associated with a low cardiovascular risk compared to that of animal protein. the sample with the highest content is sample s4 (suceava) 17.22 %. according to antoanela patraş, 2010 the proteins content of the romanian walnut types is between 12% 24%. table 1 physico-chemical parameters walnut samples kernel characteristics samples of walnuts s1 s2 s3 s4 s5 s6 s7 s8 s9 fruit weigh, (g) 11.93± 0.21 13.12± 0.35 13.42± 0.41 14.26± 0.52 12.94± 0.41 13.98± 0.52 12.03± 0.37 13.45± 0.33 14.52± 0.62 kernel weight (g) 4.03± 0.21 4.71± 0.11 4.75± 0.23 4.78± 0.22 3.75± 0.27 4.21± 0.23 3.42± 0.25 4.35± 0.30 4.84± 0.31 kernel ratio (%) 43,0924 36,3861 42,8465 52,6395 39,7822 40,1387 41,1929 42,0074 42,368 water activity (aw %) 0.555 0.565 0.558 0.564 0.561 0.562 0.565 0.556 0.560 moisture % 4.05± 0.25 3.77± 0.05 3.98± 0.07 4.02± 0.18 3.85± 0.25 3.85± 0.03 4.5± 0.25 4.22± 0.25 4.58± 0.25 ash % 2.0± 0.13 2.1± 0.13 1.8± 0.11 1.7± 0.09 2.2± 0.13 1.95± 0.012 2.05± 0.015 1.92± 0.13 2.05± 0.13 crude protein % 15.2± 0.15 15.59± 0.2 13.81± 0.2 16.12± 0.1 14.81± 0.2 17.22± 0.2 15.26± 0.1 15.06± 0.2 15.26± 0.3 total oil % 59.9± 1.33 57.85± 1.22 60.07± 1.20 59.07± 1.16 55.1± 0.47 56.68± 0122 53.81± 1.24 65.7± 0.90 66.1± 1.38 carbohydrate content (cho) % 20.85 20.69 20.34 19.09 24.04 20.3 24.38 13.1 12.1 total phenols mg gae/kg d.w 20.15± 1.02 23.22± 0.92 19.26± 1.27 19.82± 1.18 22.17± 1.25 23.52± 1.30 18.12± 1.09 17.28± 1.20 20.15± 1.28 values represent mean ± sd [dry weight]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 173 the highest negatively correlation have been observed in the case of total oil with carbohydrate content (r= 0.992). the obtained results were compared with the ones previously obtained for fruits collected in the same cultivars and in the same orchard by [17]. valuable edible nuts produced by walnut trees are well appreciated because they are enriched with unsaturated fat (linoleic, oleic acid) [18]. the percentage values of protein, carbohydrates and lipids, respectively the variation of energetic values (kcal) of walnut samples are presented in figures 1 and 2. figure 1. the values of protein (%), carbohydrates (%) and lipids (%) figure 2. the variation of energetic values of walnut samples the arithmetic mean of the energetic values (kcal) for the 9 walnut samples is 694.78 (± 22.23). total oil content fat was the highest constitute in all samples, ranging from 53.81% in falticeni (s7) to 66.1% in dolhasca (s9). these values were similar to the results reported in [9], [19]. oil contents of this study were lower than those reported by other [17]. pereira et al., 2008, reported total oil contents from 78.83 to 82.4%. variation of total oil % in the walnut samples is presented in figure no. 3. figure 3. variation of total oil (%) in the walnut samples total phenols among all the samples analyzed, walnuts of suceava contained the highest total phenols 23.52 ± 1.30 mg gae/kg d.w, followed by radauti, campulung moldovenesc, straja, dolhasca, solca, paltinoasa, falticeni and malini walnuts. based on the folin–ciocalteu method, the total phenolic content mg of gae per 100 g of nut reported by jun yang et al. (2009) was in the order walnuts > pecans peanuts food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 174 > pistachios > macadamias > cashews > hazelnuts almonds> pine nuts > brazil nuts, which shows a different pattern from our data. however, the results obtained for total phenols content were in accordance with previous studies [19]. 4. conclusions in conclusion, walnut (juglans regia) is characterized by increased biological and nutritional value.walnuts have various bioactive components and the consumption of walnuts has many health benefits such as: reducing "bad" cholesterol ldl in blood and also the triglycerides, they are anti-diabetic and a valuable protective factor against the risks of cardiovascular disease, they also have anti-oxidant and anti-inflammatory effect. these results demonstrate some differences in chemical and physical properties among the nine studied samples of walnuts from suceava plateau. these differences could be attributed to environmental and cultivation conditions. the present study revealed that walnut kernels are mainly rich in protein and total oil content. total phenols content of walnuts varied from 23.52 mg gae/g d.w. to 17.28 mg gae/g d.w. at drying, the moisture content of kernels varied from 3.85% to 4.58 % (w/w), but the differences among cultivars were not significant. these data should help in selecting cultivars that are suitable for commercial production of walnut oil in romania. 5. references [1]. cosmulescu s., adrian baciu a., gheorghe achim gh., mihai botu m., ion trandafir i., mineral composition of fruits in different walnut (juglans regia l.) cultivars. not. bot. hort. agrobot. cluj 37 (2) 2009, 156-160. [2]. patraş a., dorobanţu p., physical and chemical composition of some walnut (juglans regia l) biotypes from moldavia. lucrări ştiinţifice – vol. 53, nr. 2, seria agronomie, (2010). [3]. bakkalbas e., yilmaz ö. m., javidipour i., artik n., 2012. effects of packaging materials, storage conditions and variety on oxidative stability of shelled walnuts. lwt food science and technology 46, 203-209 (2012). [4]. shahidi f. and miraliakbari h., tree nut oils, bailey’s industrial oil and fat products, sixth edition, six volume set.175-193(2005). [5]. brufau g., boatella j. and rafecas m., nuts: source of energy and macronutrients, british journal of nutrition, 96, suppl. 2, s24–s28, (2006). [6]. yang j., brazil nuts and associated health benefits: a review. lwt food science and technology 42, 1573–1580, (2009). [7]. mexis s.f., badeka a.v., riganakos k.a., karakostas k.x., kontominas m.g., effect of packaging and storage conditions on quality of shelled walnuts. food control 20, 743–751,(2009). [8]. rabrenovic b., dimic e., maksimovic m., sobajic s. and gajickrstajic l., determination of fatty acid and tocopherol compositions and the oxidative stability of walnut (juglans regia l.) cultivars grown in serbia. czech j. food sci. vol. 29, no. 1: 74–78(2011). [9]. amaral j. s., casal s., joseä a. pereira, rosa m. seabra, and beatriz p. p. oliveira, determination of sterol and fatty acid compositions, oxidative stability, and nutritional value of six walnut (juglans regia l.) cultivars grown in portugal. 7698 j. agric. food chem., 51, 7698-7702 (2003). [10]. savagea g.p., duttab p.c., mcneila d.l., fatty acid and tocopherol contents and oxidative stability of walnut oils. journal of the american oil chemists society, vol. 76, no. 9 (1999). [11]. serrano a., j. librelotto, s. cofrades, f.j. sa´nchez-muniz, f. jime´nez-colmenero, composition and physicochemical characteristics of restructured beef steaks containing walnuts as affected by cooking method. meat science, 77, 304–313, (2007). [12]. serrano a, cofrades s, ruizcapillas c, olmedilla-alonso b, herrero-barbudo c, jime´nezcolmenero f., nutritional profile of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu, estimation of biochemical properties of walnuts from the region of suceava – romania, food and environment safety, volume xii, issue 2 – 2013, pag. 169-175 175 restructured beef steak with added walnuts. meat sci 70:647–654, (2005). [13]. olmedilla-alonso b., pharmd, phd, granado-lorencio f., herrerobarbudo c., blanco-navarro i., blazquez-garcia s., mchem, b. perezsacrista n, consumption of restructured meat products with added walnuts has a cholesterollowering effect in subjects at high cardiovascular risk: a randomised, crossover, placebocontrolled study. journal of the american college of nutrition, vol. 27, no. 2, 342–348 (2008). [14]. iwamoto m, imaizumi k, sato m, hirooka y, sakai k, takeshita a, kono m., serum lipid profiles in japanese women and men during consumption of walnuts. eur j clin nutr 56: 629–637, (2002). [15]. almeida, i.f., fernandes, e., lima, j.l.f.c., costa, p.c., bahia, m.f.,. walnut (juglans regia) leaf extracts are strong scavenger of pro-oxidant reactive species. food chemistry 106, 1014–1020, (2008). [16]. fernandez-agullo a., pereira, m.s. freire e., valentao p., andrade p.b., gonzalez-alvareza j., pereira j.a., influence of solvent on the antioxidant and antimicrobial properties of walnut (juglans regia l.) green husk extracts, industrial crops and products 42 (2013) 126– 132. [17]. pereira j. a., oliveira i., sousa a., ferreira i. c.f.r., bento a., estevinho l., bioactive properties and chemical composition of six walnut (juglans regia l.) cultivars, food and chemical toxicology 46, 2103–2111, (2008). [18]. muhammad ali, amin ullah, hidayat ullah, fawad khan, fruit properties and nutritional composition of some walnut cultivars grown in pakistan, pakistan journal of nutrition 9 (3): 240-244, (2010). [19]. jun yang, rui hai liu, linna halim, antioxidant and antiproliferative activities of common edible nut seeds, lwt food science and technology 42 1–8 (2009). 233 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 233 239 atom-absorption method of determination of selenium content in some raw materials and food igor kobasa1 1yu. fedkovych national university of chernivtsi, ukraine, imk-11@hotmail.com * corresponding author received august 31st 2013, accepted september 14th 2013 abstract: a novel atom-absorption method of determination of selenium in some raw materials, biological objects and food is proposed. this method involves preliminary extraction and concentrating of the components to be analyzed with butylacetate/sodium diethyldithiocarbamate mixture. the highest extraction degree for se(iv) and se(vi) ions is reached for ph ranged from 1 to 6. investigation of influence of high concentrations of iron and zinc on accuracy of the se determination proved only insignificant (under 5%) blurring of the se atom-absorption peaks for zn admixture contents 0÷6 g/kg and 0÷10 g/kg of fe. keywords: selenium; atom-absorption analysis; environmental objects; food 1. introduction selenium and its compounds are known as biologically active and important components with clear limits of toxicity, sufficient and insufficient supply. toxic concentration of se disturbs normal functioning of the cell structures through formation of stable and reactionless compounds of se and s. selenites are easily reducible and can form free se followed by active reactions between se and organic compounds and its incorporation in the structure of proteins. some authors report higher content of se found in malignant tumours. other results prove radioprotection activity of the low-concentrated se and some counteraction to negative effects of the heavy metals and irradiation after treatment with the se microdoses. therefore, it is essential to find the clear limits between useful microdoses and harmful overdosage of this element. this issue is related to various methods available for continuous and precise analytical determination of se content in environmental objects and other materials. a series of experimental methods are available for experimental determination of se content in water [1, 2], biomaterials [3], blood plasma and serum [4, 5] and food [5, 6]. however, all these methods are very complex and require deficient and expensive reagents and equipment. on the other hand, sensitivity and accuracy of some of these methods seem insufficient. our investigation deals with development of the quantitative determination of se content in the above objects using a method of the extraction concentrating of se followed by the atom-absorption spectroscopy determination of this element at the acetylene-air atomizer. 2. experimental and methods dithiocarbonates and dithizone are used traditionally as extraction agents [7] and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 234 chloroform, tetrachlorated carbon, various mixtures of the polar oxygen-containing and non-polar oxygen free liquids [8] are used as organic solvents for preliminary extraction of se from aqueous solution required for spectral determination methods. however, most of the above mentioned organic agents can complicate fine analytical determination of se since they are hardly combustible and form yellowish and smoking flame. besides, emission of harmful products is an unavoidable result of combustion of the chlorine-containing compounds. methylisobutylcetone (mibc) is proposed in [9] as an alternative solvent. however, this agent cannot ensure sufficient concentrating degree. in our experiments, we used the butyl ether of acetic acid as a solvent. physicochemical properties of this compound are similar to mibc while concentrating performance is significantly higher. technically, all extractions have been performed in two systems: butylacetate/sodium diethyldithiocarbamate and butylacetate/dithizone. extraction mixtures have been prepared using the 10 % aqueous solution of sodium diethylldithiocarbamate and 0.01 m solution of dithizone in butylacetate. a series of the model solutions with predetermined contents of se has been prepared. all concentrations of se were ranged within the values expected in real analytical samples. these solutions were prepared from the deionized water and analytically pure selenium acid. hydrochloric acid and analytically pure ammonium solution were used to adjust ph of the systems. a process of extraction was performed according to [10]. the flame atom-absorption spectrophotometer c-115-m1 has been used for determination of se content. the stage of atomization was realized with the acetyleneair flame at the temperature up to 2600 °c. a high frequency gas discharge tube filled with microamount of se was used as a source of the discrete spectrum. all atom absorption determinations were performed at the wave length 196.1 ± 0.4 nm. rest of the samples preparation stages were made according to [11]. 3. results and discussion first, we had to select the most effective atomization mode and a value of electric current of the gas discharge tube. the mode of atomization had to provide the highest values of the wanted signal and signal/noise ratio. results of the atomization mode selection are shown in fig. 1. as seen in fig. 1, the most optimal composition of the combustible mixture can be reached at feeding the system with acetylene at the pressure 0.4 kg/sm2 and air at 0.75 kg/sm2. this composition of the combustible mixture insures the highest and comparatively stable value of the wanted signal (light absorption value). a dependence of the wanted signal value on the tube current is shown in fig. 2. it can be seen that the highest wanted signal was obtained at the lowest current value (90 ma). however, under this current we had to feed the photocell with almost the highest possible current, which caused generation of quite intense ‘noise’. taking into account the above conditions, we set the photocell feeding current value at 100 ma. this value was still close to the most optimal wanted signal area and did not cause generation of excessive interfering noise. this mode of the gas discharge tube feeding was realized using the superhigh frequency electric generator 2pbl3mv. it was also found that the hollow cathode tube produced less relevant results. at the next stage we analyzed a character of influence of ph on the se extraction degree. a series of extractions has been performed for solutions with predetermined food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 235 content of seо3 2-. then the content of the extracted se in the organic phase has been determined using similar atom absorption method and the degree of absorption (r) was calculated using the formula below: r=(c0v0/cвvв+c0v0)·100 %, where v0 and vb – volumes of the aqueous and organic phases; c0 and cb – concentrations of se in the aqueous and organic phases respectively. 0 50 100 150 200 250 300 0,4 0,45 0,5 0,55 0,6 0,65 0,7 0,75 0,8 0,85 oxygen pressure, kg/sm2 li gh t a bs or pt io n, c .u . 1 2 3 4 5 figure 1. a series of dependencies of the light absorption (conventional units, c.u) on the oxygen pressure (kg/sm2) for the following acetylene pressures: pac = 0.40 kg/sm 2 (curve 1); pac = 0.45 kg/sm 2 (curve 2); pac = 0.50 kg/sm2 (curve 3); pac = 0.55 kg/sm 2 (curve 4); pac = 0.60 kg/sm 2 (curve 5). 0 50 100 150 200 250 300 350 400 90 95 100 105 110 115 120 125 130 i, ma l ig ht a bs o rp tio n , c .u . figure 2. dependence of the wanted signal value on the tube feeding current. it should be noted that accurate determination of se distribution ratio between the phases was quite difficult due to very low initial concentration in the aqueous phase. a dependence of the distribution ratio values on ph is shown in fig. 3. one can see that the mixture butylacetate/sodium diethyldithiocarbamate (fig. 3, line 1) shows the highest extraction performance in the range of ph values 1-6 where this ratio is between 90-95 %. extraction performance of the mixture dithizone/butylacetate is much lower and its highest value (about 10 %) is reached within ph range 0-1.5 (see fig. 1, line 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 236 figure 3. dependence of the se extraction ratio (r) on ph for the system butylacetate sodium diethyldithiocarbamate (line 1) and dithizone/butylacetate (line 2). therefore, one can conclude that the extraction mixture butylacetate/sodium diethyldithiocarbamate ensures very high extraction ratio of se and should be used for this purpose. the sediethyldithiocarbamate chelate is well soluble in butylacetate and this facilitates better atomization of the element. the narrower ph range of the best se extraction is 4-6 and our next experiments were performed within this range. a series of the model solutions was prepared and se contents were ranged within the limits that can be expected for the real analytical systems. a model solution with some predefined se content was mixed with the extraction mixture butylacetate/sodium diethyldithiocarbamate with ph 4-6. after the extraction process completed, the organic mixture with extracted se was analyzed at the atom-absorption spectrophotometer and the dependence of its optical density on initial se concentration is shown in fig. 4 (line 1). this dependence is almost exactly linear, which proves correct selection of the working concentrations range. a slope of the line is determined by the spectrophotometer’s sensitivity and in our case it seems quite sufficient for quantitative determination of the selected concentrations of se. it should be taken into account that many other inorganic elements would also be coextracted from real experimental systems after their mineralization with strong inorganic acids. these side elements may significantly raise the noise level of the analytical signal complicating reliability of se determination in the real systems. our next experiments were aimed onto investigation of relevance of the se determination in more ‘noisy’ systems containing se on the background of some side element(s). a series of biological samples were mineralized and then some known amounts of selenite were added to the probes. all natural samples contain some background amounts of se and this admixture method has to show relevance of determination of small extra amounts of se on the background of its natural content in presence of the other elements. results of this investigation are represented in fig. 4 (line 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 237 0 200 400 600 800 1000 1200 0 5 10 15 20 c, mg/dm3 li gh t a bs or pt io n, c .u . 1 2 figure 4. dependence of light absorption on se concentration in the initial model solution for extraction mixture butylacetate/sodium diethyldithiocarbamate. line 1 – a mixture prepared with the pure deionized water; line 2 – a mixture prepared from a real biological sample with some admixture of extra selenite. it is seen that the line 2 (‘noisy’ samples taken from the biological objects) is similar to the pure one (line 1) but has slightly lesser slope. this effect can be caused by influence of the ‘matrix’ of other elements present in the natural objects and some slackening of the wanted signal by this matrix. it was found that the bottom limit of this method is 0.2 mg/kg se in the organic extract. according to peculiarities of the method of the samples preparation, this concentration corresponds to the content of se in the source biological objects or food 0.04 mg per 1 kg dry weight. this sensitivity is about one order of magnitude below background and maximum permissible contents of se in these objects. therefore, this analytical method can be recommended for analytical determination of se in food and other biological objects. as reported in [12], ions of iron, nickel, magnesium and zinc can also be intensively co-extracted by the mixture butylacetate/sodium diethyldithiocarbamate in the weakly acid medium. many biological objects can release significant amounts of these ions so, it is essential to investigate possible overlapping and shadowing of the wanted signal because of presence of these ions. 950 955 960 965 970 975 980 985 990 995 0 2 4 6 8 10 c, mg/dm3 li gh t a bs or pt io n, c .u . ряд1 ряд2 figure 5. influence of iron (line 1) and zinc (line 2) on light absorption of the test solution with the constant concentration of se. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 238 influence of ions of iron and zinc on the wanted signal value at the given (unchanged) concentration of se is represented in fig. 5. we ranged contents of the both interfering elements from 0 to 6 g/kg (zn) and 10 g/kg (fe). it should be understood that such concentrations are about 100 times higher than the concentrations found usually in any real extractions taken from the biological objects. it can be seen that despite some decrease of the analytical signal it remains quite strong and easily detectable. maximum slackening of the wanted signal does not exceed 1-2 % of its value. such high selectivity is caused by absence of any characteristic lines close to 196.1 nm (se characteristic line of light absorption) in the spectra of fe and zn. the spectrophotometer is equipped with precision monochromator that allow to distinguish the lines with accuracy up to 0.4 nm. then the calibration line (fig. 4, line 2) was employed to recalculate results of determination of se content in a series of the soil samples taken from the grass cover in a park located in chernivtsi, ukraine. in order to assess relevance and accuracy of the method each sample has been analyzed in a row of 5 consequent tests. results of the se content determination are shown in table 1. as seen, statistical relevance of the atom absorption determination of se content is quite high. relative error of the method is 1.26-4.47 %, statistical dispersion of the results is ranged from 1.7∙10-4 to 3.68∙10-3 within the confidence interval 0.0334 0.0697. table 1 results of the atom absorption determination of se content in the soil samples added selenium. mg/kg determined selenium. mg/kg relative error. % statistical dispersion average* experimental content of se (mg/kg) 0.40 0.395 0.418 0.408 0.384 0.398 1.4 1.678∙10-4 0.4006 ± 0.0334 0.80 0.792 0.792 0.808 0.812 0.806 4.47 9.79∙10-4 0.802 ± 0.0359 1.20 1.26 1.14 1.10 1.14 1.18 2.33 3.68∙10-3 1.164 ± 0.0697 1.60 1.64 1.58 1.48 1.48 1.52 1.39 2.325∙10-3 1.54 ± 0.054 2.0 2.0 1.92 1.86 1.92 1.88 1.26 2.88∙10-3 1.916 ± 0.062 * _ x ± n st  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 igor kobasa, atom-absorption method of determination of selenium content in some raw materials and food, food and environment safety, volume xii, issue 3 – 2013, pag. 233 239 239 4. conclusion it can be concluded that the method of the atom absorption determination of se with preliminary extraction by the butylacetate/sodium diethyldithiocarbamate mixture proves high relevance and accuracy and can be recommended for monitoring of content of this metal in food and other biological objects. this method is characterized by high selectivity and relatively low errors even for the ‘noisy’ samples containing some side elements. 5. references [1]. nazarenko i.i. and kislova i.v. determination of various forms of se in water. lab and technological investigations and concentrating of mineral raw materials. 6. 1-8. (1977). [2]. nazarenko i.i. and kislova i.v. a highly sensitive method of determination of the migrating forms of selenium in natural water. j. anal. chem.. 32. 1857-1859. (1978). [3]. watkinson j.h.. fluorometric determination of selenium in biological material with 2. 3 diaminonaphthalene. anal. chem.. 38. 9297. (1966). [4]. bayfield r.f.. romalis l.f.. ph control in the fluorometric assay for selenium with 2.3-diaminonaphthalene. anal. biochem.. 144. 569-576. (1985). [5]. lebedev p.a and lebedev a.a.. a modification of the spectral fluorimetric method of determination of selenium content in blood. j. chem. and pharm.. 30. 54-55. (1996). [6]. golubkina n.a.. et al.. determination of selenium in wheat flour from various regions of ussr. nutrition problems.. 59. 64-66. (1990). [7]. pilipenko a. and samchuk a.. extraction-atom-absorption analysis of natural objects. j. anal. chem.. 61. 957-961. (1989). [8]. gindin l. extraction process and their usage.. nauka. moscow. 481 p. (1984). [9]. grandzhan a.. kuchuk a. and charykov a.. extraction-atom-absorption determination of heavy metals. j. anal. chem.. 63. 711-717. (1991). [10]. zolotov yu. and kuzmin n.. extraction concentrating.. khimiya. moscow. 320 p. (1971). [11]. bilogolovka v. t. and kobasa i. m.. atom-absorption analysis of heavy metals content in raw materials. food and environmental objects.. chernivtsi university. chernivtsi. 108 p. (2010). [12]. zolotov yu. extraction in inorganic analysis.. moscow university. moscow. 18-21. (1988). 187 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 22016, pag. 187 195 study on effects of artichoke flour addition in bread *sorina ropciuc1, ana leahu1, cristina damian1, laura carmen apostol1 1faculty of food engineering, stefan cel mare university, 13th university street, 720229, suceava, romania sorina.ropciuc@fia.usv.ro; sorinaropciuc@yahoo.com *corresponding author received 23th april 2016, accepted 29th june 2016 abstract: the increase of the fiber content in bread is a major challenge in the baking industry. in this study, the quality characteristics of flour and bread mixture obtained by adding artichoke flour , as a source of fiber, was determined. the physico-chemical characteristics of flour were analyzed: gluten content, acidity of artichoke flour added at 0%, 10%, 20%, 30 % and 40%. bread made with and without the addition of artichoke flour has been evaluated for specific volume, color and porosity. the addition of artichoke flour decreased water absorption capacity of the flour, reduced the amount of gluten in the dough and increased acidity of the flour mixture. the bread samples obtained by substituting the whole wheat flour by artichoke flour were analyzed in terms of acceptability and quality. for this objective five bread samples were analyzed and the color of bread and a series of physical parameters (diameter, sample mass, height / diameter core porosity) were determined. keywords: artichoke flour, inulin, quality bread, wheat flour. 1. introduction bread is considered one of the important foods from the nutritionists because is a source of carbohydrates, protein, fiber, vitamins, micronutrients and antioxidants. food fibers, in particular the soluble, have a number of beneficial effects including prebiotic activity, increased mineral absorption and prevention of diseases such as type ii diabetes, cardiovascular disease and colorectal cancer [1]. whereas the fiber content of white bread flour is only 23%, the feeding ensures full intake of fiber, 7-8% of dry matter [2]. however, these propositions cover only a small part of recommended daily intake. because of this the present study aims at increasing the fiber content of bread by total or partial replacement of wheat flour with flours from other sources [3]. artichoke flour has a low calorific value, nearly fat free and starch. the interest component of the artichoke is inulin, a dietary fiber with linear structure that is included of fructans class. the inulin content of artichoke flour exceed 50% of dry matter. the use of inulin is not considering only increase the fiber content of products, but also numerous beneficial effects on human health. thereby, inulin is recommended in the prevention and treatment of obesity and diet followed by people suffering from diabetes. inulin also has the prebiotic activity, role in improving bowel function and increase immunity [4]. the enrichment bread with functional ingredients is important for the consumers and for the baking industry. in order to increase the fiber content of bread it may be carried addition of inulin content products at high levels, such as artichoke flour. artichokes is classified in the genus helianthus l., family asteraceae; the plant is also known as the "artichoke", "jerusalem artichoke", "turnip". the name artichoke was attributed due to the similarity that exists between taste and http://www.fia.usv.ro/fiajournal mailto:sorina.ropciuc@fia.usv.ro mailto:sorinaropciuc@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 188 texture of the boiled artichoke tubers (cynara scolymus l.). artichoke tubers contain about 80% water, 15% carbohydrates and 1-2% protein. the principal carbohydrate from artichoke is inulin of which proportions varies from 730% of tuber fresh weight (about 50% dry) [5-7]. the use of inulin from artichoke is not considering only the increase of the fiber content from the products but also many health benefits evidenced by previous studies [8]. therefore, inulin is recommended in the treatment of obesity and diets followed by people suffering from diabetes. inulin has also prebiotic activity that is involved in the improvement of intestinal function and the growth of immunity and plays a role in preventing osteoporosis and maintaining bone health. in technological terms, partial substitution of wheat flour with artichoke flour in different percentages significantly change the quality and mechanical properties of the dough [6]. the addition of fiber to the flour leads to changes in the technological parameters, changes that directly determines the quality of the bread. a potential effect of the addition of fiber volume is the decrease of the volume by reducing the percentage of gluten content from the dough and consequently reducing the capacity of the dough to retain fermentation gases [7-9]. another aspect to be evidenced and studied is the fact that most of the fibers determine the increase of hydration capacity of the flour, which in economic terms, it constitutes an advantage because from the same amount of flour you can obtain more products. the study has as its objective: the effect of the addition artichoke flour and the quality of bread from whole wheat flour. 2. materials and methods 2.1. materials 2.1.1. wheat flour for the proposed determination whole wheat flour sample derived from s.c. mopan suceava s.a. were used. the flour chemical composition was determined according to romanian standard methods: moisture (sr en iso 712:2010), ash content (sr en iso 2171:2010), wet gluten content, (sr 90:2007), acidity (sr 90:2007). artichoke flour in order to obtain artichoke flour tubers were washed, peeled and cut into thin slices. drying was carried out in an oven at a temperature of 40°c. after cooling, flour obtained by milling the dried slices of artichoke got used to prepare bread. 2.2. methods preparing the mixture of wheat flour meal with artichokes flour the amount of artichoke flour that was a flour-specific substitution of whole wheat was at the rate of 0%, 10%, 20%, 30% and 40%. the dough was prepared using the technique of direct manufacturing. for dough preparation food processor was used. the dough for each bread sample was prepared separately using the amounts from the ingredients listed. the dry ingredients (flour and salt) were introduced into the food processor bowl, and the yeast was first dissolved in water and then added over the rest of the ingredients. the dough was left to rise for 30 minutes. after initial fermentation, the dough was shaped imparting to specific loaf shape and put in baking tray. the final step was the fermentation in pan for 15 minutes. the baking process of the bread was held in an electric furnace at 200°c for 30 minutes. after baking the bread was left to cool to room temperature before being analyzed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 189 physical and quality parameters of bread for assessing the influence of the characteristics of artichoke flour, bread samples were evaluated to determine elasticity, volume, porosity and other characteristics. determinations were made at 6 hours after baking for all the bread samples. bread volume, elasticity and porosity were determined according to sr 91:2007. the determination of bread sample colors was measured using portable spectrophotometer. for the assay were used two slices of each bread sample and the measurement was repeated at least twice. results are expressed in the parameters of the cartesian system's ciel * a * b * namely l * brightness (0 black 100 white), a * green (-) and red (+) and b * blue () and yellow (+). statistical analysis to calculate the mean values and standard deviations of the analysis results, microsoft office excel software was used. in order to achieve the correlative links and to establish the optimal proportion of artichoke flour xlstat statistical software was used. 3. results and discussion 3.1. physical and chemical properties of wheat flour based on the flour acidity there can be assessed the level of freshness, alteration or compliance storage. the acidity of the flour is determined by processes of decomposition of fats from the wheat germ, in wheat flour's case or tuber plant composition, or from artichoke flour. values obtained for the acidity of wheat flour (blank) and flour mixtures with artichoke flour are expressed in table 1. the variation of acidity according to the proportion of added artichoke flour is shown in fig. 1. table 1. acidity of flour used to obtain samples of bread sample acidity level wheat flour 2.6 10% artichoke flour mix 3.2 20% artichoke flour mix 3.8 30% artichoke flour mix 4.2 40% artichoke flour mix 4.4 100 0 1 2 3 4 5 0 % 1 0 % 2 0 % 3 0 % 4 0 % % wheat flour a ci di ty l ev el s % artichoke mix 0-1 1-2 2-3 3-4 4-5 fig. 1. variation of acidity according to the proportion of artichoke flour from the mixture the addition of artichoke flour mixture causes an acidity increase of the mixture wheat flour and artichoke flour up to 40%. for the latter one the determined acidity is lower than 30% artichoke flour mixture one. the capacity of hydration the wheat flour used in the production of the control sample has a water absorption capacity of 53%. in contrast, the hydration ability of artichoke flour is 31%. the variation of the hydration capacity according to the modification of the composition of the mixture of the flour is shown in fig. 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 190 fig. 2. the variation of hydration capacity of the mixture according to the proportion of artichoke flour partial substitution of wheat flour with artichoke flour changes the hydration capacity [10]. moreover, the greater the proportion of artichoke flour, the amount of water required is reduced [11]. so a substitution of 10% of wheat flour changes the hydration artichoke flour reduce capacity by 5%. for artichoke flour proportion of 20%, 30% and 40% of the mixture the water absorption decreases by 3% for each 10% increase in the amount of flour artichoke. values and similar trends were obtained by gedrovica and karklina (2011) for mixtures of wheat flour and artichoke flour in identical proportions to those examined in this study [12]. the decrease in water uptake can be attributed to the content of low molecular weight carbohydrates and oligosaccharides typically of the artichoke wheat flour [13]. another factor that causes a reduced hydration capacity of mixtures with artichoke flour is the high inulin content, which account for about 50% of dry matter [14]. inulin forms a barrier around the starch molecule limiting the possibility of fixing [15-16]. for this reason artichoke flour has a hydration capacity of 31%, almost twice lower than the wheat flour's one. wet gluten content another important parameter on which depends the quality of flour and which influence the development of bread is the gluten content. the modification of this parameter with the increase of the addition of flour artichoke is the shown in fig. 3. wheat flour 10% artichoke flour 20% artichoke flour 30% artichoke flour 40% artichoke flour fig. 3. variation of wet gluten content according to the proportion of added arthicoke flour from the mixture in the formation of gluten are involved water and two characteristic protein of wheat flour: gliadin and glutenin. first determine extensibility, and the latter dough elasticity. from the wheat flour a wet gluten content of 24.4% was determined. as artichoke flour does not contain glycine and glutenin, it has been expected a reduction in the proportion of gluten with the increase of the amount of artichoke flour used. therefore, a wet gluten content of 23.4%, as determined for the sample with 10% artichoke flour is brought to one of 11.4% in the case of wet gluten sample with 40% arthicoke flour. role in reducing wet gluten content has inulin from the artichoke flour, which prevents water fixing and thereby determining the formation of gluten network. in order to obtain a high quality bread there should be used mixtures of wheat flour with acidity, hydration capacity, and the optimal gluten content. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 191 3.2. bread flour quality sensory analysis the score obtained for each feature senzorial bread samples in fig. 4. for the blank, represented by wholemeal bread wheat was obtained the maximum score for the cores. core and shell color appearance were less popular, while porosity and taste convened lowest scores (4.6 points). sensory analysis showed that the most popular bread samples made from a mixture of wheat flour and meal of artichokes flour were the proportions of 10% and 20% meal of artichokes flour. they were given the maximum score for the color of flesh. compared with the sample of wholemeal bread, bread crumb color with the addition of 10% and 20% was considered superior by tasters. another highly appreciated sensorial feature (denoted by 5 points) if 20% of the sample was bread peel appearance. samples with 10% and 20% meal of artichokes flour obtained equal scores for the cores (4.8 points) and taste (4.6 points). less than appreciated by tasters were 30% bread with artichoke flour and the 40% meal of artichokes flour. the latter obtained the lowest score (3.2 points) for the cores, porosity and taste. scoring is justified by the fact that with the increase of the amount of wheat flour dough substituted structure is changed and thereby the degree of development of bread during the baking process. in this case the addition of artichoke flour has a significant influence on core, general appearance and on its porosity. based on these observations it can be concluded that in the case of bread made from flour mix is not recommended the proportions greater than 30% of arthicoke flour. 3.3 physical parameters of quality in order to assess the development of artichoke bread with flour after baking the mass and diameter of each sample, the ratio height / diameter and core porosity was measured. the results are expressed in table 2. it is noted that the addition of artichoke flour results in a decrease in the weight of the bread. the variation is justified by the reduced hydration capacity of the flour, as well as low-gluten content of the dough obtained in a high quantity of flour. similar values obtained for the bread weight and diameter decrease with increasing content arthicoke flour. 0 1 2 3 4 5 peel appearance the cores color of fleshporosity taste control sample 10% artichoke flour mix 20% artichoke flour mix 30% artichoke flour mix 40% artichoke flour mix fig. 4. graphical representation of the values obtained for the samples sensory characteristics of bread with added artichoke flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 192 table 2. the values of the physical parameters of bread samples sample weight, g diameter, cm height / diameter relation porosity, % vol. wheat bread 155 8 1.03 71 bread with 10% artichoke flour 146.5 7.4 1.06 68 bread with 20% artichoke flour 135.9 6.5 1.07 53 bread with 30% artichoke flour 128.7 6.1 1.10 50 bread with 40% artichoke flour 96.9 5.2 1.13 30 there is generally no significant differences between height and diameter of bread; exceptions are samples with 30% and 40% arthicoke flour at low height and larger diameter highlights their inappropriate development. last but not least, it has been observed a reduction in porosity determined by increasing the amount of wheat flour substituted with the artichoke flour. the difference between the highest and the lowest is 13%, which shows that the effect is not very pronounced. the porosity of the bread is in direct correlation with the gluten content of the flour used so that the use of large amounts of artichoke flour bread leads to a low porosity [17]. bread color in order to study the effect of the addition of artichoke flour has on the color of bread samples was used for color measurement system ciel * a * b * values obtained are noted in table 3. the analysis followed cartesian measurement of the brightness of the system, namely (l *) and hue color (a *, green-red and b * the blue-yellow) and determining the differences in these parameters between the blank (wholemeal bread wheat) and samples of bread with artichoke flour. the amounts presented in the table shows that the addition of artichoke flour leads to a significant change in the color of bread. except for the sample with 30% artichoke flour, bread characteristic brightness decreases with increasing amounts of flour from artichoke. therefore, 40% flour bread with artichoke flour presents the darker color than the rest of the evidence. a similar trend of brightness on bread samples was observed by rubel et al. (2014) after an addition of 2.5 to 5 g of flour artichoke. the value obtained by researchers for the 5g flour-artichoked bread was the lowest (61.51 ± 2.05). for all measured parameters, color sample meal on bread with artichoke 20% was significantly different from that of other samples. this sample is darker than 30% added artichokes flour and presents the most prominent shades of red and yellow. consequently, the measured values for the difference in color and shades of red and yellow with 20% of artichokes flour and blank were higher. determined color difference was 8.56, amounting to almost three times higher than that obtained for the remaining samples. the measured value for the difference between reds was 3.74, while the difference in shades of yellow was 7.11. modifying the color of bread with artichoke flour has been attributed to the high content of inulin, which undergoes decomposition during the baking process, resulting in strong maillard reactions [14]. the sample showing color close to that of the control sample is 10% artichok flour. if you prefer bread with characteristics similar to full it is recommended proportions of 10% and 30% artichokes flour. the use of smaller quantities of artichoke flour also provides a corresponding development of the bread, ensuring a diameter, height and an optimal porosity of the core. incidentally, bread with added 10% and 20% artichoke flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 193 samples were most appreciated sensory attributes. 3.4 the statistical interpretation of experimental data pearson matrix offers the opportunity to see which are the statistically significant correlations between variables taken two by two. for example, bond strong positive correlation is indicated by the correlation coefficient r 2 = 0.974 between gluten and porosity. it is estimated that the higher gluten content, porosity increases (in the study, which has no general condition, strong gluten flours don’t lead to high porosity).inverse correlation stands between hydration capacity and the height / diameter relation (r2 = -0.955). the principal component analysis (pca) were plotted correlations fig. 5, the types of bread obtained and analyzed variables. pca analysis shows that bread with added 10 and 20% artichokes flour are blends best correlate significantly with the most important features we appreciate the flour mixture and those bread: glute, hydration capacity, porosity and ground. from optimal mathematical model (fig. 6) the addition of artichoke flour mixed with wheat flour is between 18.2 to 20.6%, the content leads to bread with quality features ideal of sensory and technological standpoint. fig. 5. graphical representation of the main components table 3. influence of the addition of artichoke flour on the bread color sample l* a* b* dl da db de1 wheat bread 61.8 4.9 17.5 bread with 10% artichoke flour 61.0 4.8 20.82 -0.7 0.4 3.3 3.4 bread with 20% artichoke flour 58.8 8.1 24.6 -2.9 3.7 7.1 8.5 bread with 30% artichoke flour 59.8 4.88 19.88 -1.9 0.49 2.37 3.12 bread with 40% artichoke flour 58.3 4.37 19.14 -3.4 -0.01 1.64 3.84 1color difference food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 194 fig. 6. regression curve of the optimal artichoke flour addition 4.conclusion the use of artichoke flour as an ingredient in the preparation of bread aimed to increase the fiber content and to obtain a product with beneficial effects on consumer health. to study the effect of the addition of artichoke flour on product quality five samples of bread were used. a sample obtained from wheat flour and four samples of bread from a mixture of wheat flour and artichokes flour in different proportions varring between 10% and 40% were prepared. issues involved were characteristics of mixtures of flour and dough, acceptability and quality of finished products. a first condition for obtaining a quality bread is to use an flour with optimal composition. for the analysis of the quality of wheat flour mixtures, artichoke flour the ph, hydration ability and wet gluten was determined. it was concluded that the samples with a lower acidity presents artichoke flour and optimal hydration capacity and high gluten content. therefore, the use of mixtures with 10% and 20% artichoke flour produced a corresponding porosity volume. finished product quality assessment was performed by sensory analysis, color measurement and determination of physical parameters (diameter and mass of the sample, the height / diameter core porosity). the obtained data supporting previous observations, that the addition of 10-20% artichoke flour gives a bread with improved quality and sensory attributes. 5. references [1]. abou-arab, a. a., talaat, h. a. & abu-salem, f. m. “physico-chemical properties of inulin produced from jerusalem artichoke tubers on bench and pilot plant scale”, australian journal of basic & applied sciences, 5(5), 1297-1309, (2011) [2].abrams, s. a., griffin, i. j., hawthorne, k. m., liang, l., gunn, s. k., darlington, g., ellis, k. j., a combination of prebiotic short-and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young, 82(2), 471-476, (2005) [3]. hager, a. s., ryan, l. a., schwab, c., gänzle, m. g., o’doherty, j. v., arendt, e. k. influence of the soluble fibres inulin and oat β-glucan on quality of dough and bread”, european food research and technology, 232(3), 405-413, (2011) [4].rouillé, j., della valle, g., lefebvre, j., sliwinski, e., shear and extensional properties of bread doughs affected by their minor components, journal of cereal science, 42(1), 45-57, (2005) [5].sezik, e., yeşilada, e., honda, g., takaishi, y., takeda, y., tanaka, t., traditional medicine in turkey x. folk medicine in central anatolia, journal of ethnopharmacology, 75(2), 95-115, (2001) [6].baltacioğlu, c. & esin, a., crisp production from jerusalem artichoke (helianthus food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 sorina ropciuc, ana leahu, cristina damian, laura apostol, study on effects of artichoke flour addition in bread, food and environment safety, volume xv, issue 2 – 2016, pag. 187 – 195 195 tuberosus l.) and investigation of quality parameters, academic food journal/akademik gida, 11(2), 14-20, (2013) [7].chen, f., long, x., yu, m., liu, z., liu, l., shao, h., phenolics and antifungal activities analysis in industrial crop jerusalem artichoke (helianthus tuberosus l.) leaves, industrial crops and products, 47, 339-345, (2013) [8]. gedrovica, i., karklina, d., fras, a., jablonka, o., boros, d., the non–starch polysaccharides quantity changes in pastry products where jerusalem artichoke (helianthus tuberosus l.) added, procedia food science, 1, 1638-1644, (2011) [9].karolini-skaradzinska, z., bihuniak, p., piotrowska, e., wdowik, l.’ properties of dough and qualitative characteristics of wheat bread with addition of inulin, polish journal of food and nutrition sciences, 57(4), 267-270, (2007) [10]. ilga gedrovicaa, daina karklinaa, anna frasb, olga jablonkab, danuta boros, non–starch polysaccharides quantity changes in pastry products where jerusalem artichoke (helianthus tuberosus l.) added, procedia food science 1, 1638 – 1644, (2011) [11]. poinot, p., et al.,. influence of inulin on bread: kinetics and physico-chemical indicators of the formation of volatile compounds during baking. food chemistry, 119(4), 1474– 1484, (2010) [12]. caprita r. & caprita a. & julean c., biochemical aspects of non-starch polysaccharides. scientific papers: animal science and biotechnologies, 43 (1), 368–374, (2010) [13]. d. every, l. simmons, k.h. sutton, m. ross, studies on the mechanism of the ascorbic acid improver effect on bread using flour fractionation and reconstitution methods. journal of cereal science, volume 30, issue 2, september 1999, pages 147-158, (1999) [14]. halina gambuś, marek sikora, rafał ziobro, the effect of composition of hydrocolloids on properties of gluten-free bread, acta sci. pol., technol. aliment. 6(3) 61-74, (2007) [15]. i.a. rubel , e.e. pe´rez , g.d. manrique , d.b. genovese, fibre enrichment of wheat bread with jerusalem artichoke inulin: effect on dough rheology and bread quality. food structure, volume 3, january 2015, pages 21–29, (2015) [16]. wanga j., rosella c.m., barbera c.b. effect of the addition of different fibres on wheat dough performance and bread quality. food chemistry, 79: 221–226, (2002): [17]. sr iso, standardization association of romania (asro), bucharest, romania: 90, 91, 11036, (2007) [18]. sr iso, standardization association of romania (asro), bucharest, romania: 712, 2171, (2010) 1. introduction 376 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 376 381 on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra 1marine arsen hayriyants 1yerevan m. heratsi state medical university, armenia mhayriyants@mail.ru * corresponding author, received november 3rd 2013, accepted december 5th 2013 abstract: the prevalence of glaucoma in ra is studied in this paper, including inherently conditioned. frequency of symptoms of glaucoma and used instrumental methods for diagnostics of the given pathology is also studied. the efficiency of screening research of patients with glaucoma, with a glance of risk groups – age, iop, vascular diseases, anamnestic record is shown, too. all of obtaining results provide a basis for more detailed surveys of the prevalence of glaucoma in view of climate geographical zones and ecology. keywords: glaucoma, screening of enhanced risk, ophthalmologic researches. 1. introduction glaucoma is a term used for designation of a group of diseases, the general symptom of which is chronically progressing neuropathy of optic nerve, at the same time structural changes of dvn appear – excavation increases, defects of visual field and intraocular pressure are observed. according to literature data the average prevalence of glaucoma at the age of more than 40 is 1.6% among europeans and 4.6% among black people. during screening research single tonometry in healthy people is less informed, i.e. it can reveal invariably normal parameters even if there are characteristic peculiarities of primary glaucoma (changes of optic nerve and visual field). more exact methods are still unsearchable for screening research, and traditional methods include visiometry, perimetry, tonometry and ophthalmoscopy. but if there is heredity through glaucoma, the first line must seriously be examined after the age of 40. in the absence of some change the following examination must be done every year. the primary glaucoma progresses clinically unsuspected, unless changes of peripheral vision are observed. the injuries take place gradually. though the disease usually progresses as a double-sided process, asymmetry is often observed, that is why changes of vision field are usually revealed in one eye, and in less degree – in the other one. even patients being very attentive towards themselves cannot notice evident perimetritic changes, and earlier defects can be revealed only accidentally. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 marine arsen hayriyants, on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra, volume xii, issue 4 – 2013, pag. 376 – 381 377 the main aim of the primary glaucoma therapy is prevention of abnormality of visual function during the whole life of the patient. at present the best method of achievement of this aim is considered compensation of iop. and concrete informative parameters for the control over progressing pathologic process are visiometry, and condition of optic medium, biomicroscopy, tonometry, gonioscopy, ophthalmoscopy and perimetry – the choice of its kind depends on existing apparatus, the age of the patient and indices of visiometry. in case of change of optic medium the perimetry is done in conditions of mydriasis medicamentosus [1-5]. 2. material and methods taking into consideration the importance of the given pathology a research of the disease of glaucoma in ra during 2004-2012 is done. in the period of the research glaucoma in ra gave sharp variations.: 2004 – 1.00; 2005-0.46; 2006-0.06; 2007 10.14; 2008-6.4; 2009-4.83; 2010-34.2; 2011-5.03; 20125.4. at the same time preglaucoma is revealed in 13.8%, primary – in 34.2%, developed – in 27.6%, fargone – in 22.1%, absolute – in 2.3%. the base for diagnosing the patients examined by us were the data of generally accepted ophthalmologic researches (visiometry, biomicroscopy, gonioscopy, tonometry, tonography, perimetry, ophthalmoscopy).the complaints that had these patients were lowering of the sharpness of sight, dimming of the sight in the mornings, appearing iridescent circles when looking at light, quick progressing of presbyopia. the disturbance of hydrodynamics were in the limits: pq more than 20 mm mercury column, c-0.14 and lower, f-1,5 and less, cb – more than 100. for pathology in the visual field peripheral limits constriction was accepted more than 5o by one or several meridians, availability of microscope in central part of visual field, any increase of boundaries of blind spat in comparison with the initials. while doing ophthalmoscopy the following data were taken into consideration: dilation of physiological excavation of dvn, bleaching of dvn, shift of vascular fascicle to the nose side, atrophy and excavation (marginal) of dvn. our research also included the study of family anamnesis of patients, those who had had hospital treatment within eight years, besides, 217 patients (17.4%) out of the total number of examined patients are revealed (1583 individuals) with aggravation among total number of patients with primary glaucoma. the symptoms of existence of hereditary predisposition were small front camera, narrow corner of front camera, strengthening the pigmentation of corneoscleral trabecules, dystrophic changes of iris, including pseudoecofoliation on the edge of the pupil of the eye and on front сapsule of lens, dimming the reaction of the pupil towards light, lenticular opacity of different degree. the study of anamnestic and clinical data among patients examined by us with aggravation brought to the conclusion that glaucoma is inherited by multifunctional type, characteristic for both diseases of hereditary predisposition, it gave a chance to consider that glaucoma is genetically a determined, not a monogene hereditary disease, but rather polygene, i.e. a disease with a hereditary predisposition. in the light of modern ideas of medical genetics of our material, and also considering the opinion of some authors, who think that there exist genetic and hereditary factors predisposing the development of glaucoma, however, exact food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 marine arsen hayriyants, on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra, volume xii, issue 4 – 2013, pag. 376 – 381 378 mechanisms, bringing to its origin, aren’t ascertained yet, the role of genetic hereditary is difficult, predisposition is conditioned by the influence of many genes and in the absence of other risk factors, the disease may not be revealed. and we consider such an idea nearer to the reality. among the relatives of examined patients glaucoma is met oftener than among usual people. and that is why they must be examined systematically with the purpose of exposure of earlier stage of the disease. 3. results and discussion ther main quantity of people with glaucoma are those of old age. that’s why we confined ourselves to research the genealogy of three generations: i. parents of the examined patient, ii. the examined patient, his brothers and sisters, iii. children of the examined patient. while examining the genealogy with age distribution we have noted, that men get this disease earlier, than women. however, mothers oftener are transmitters of heredity. in research of the total number of genealogically examined patients (the patient, his parents, brothers and sisters), in 217 (17.4%) cases genealogically conditioned glaucoma is revealed, in 89 (41%) cases transmitters of disease were mothers, and in 82 (37.7%) patients – fathers. while examining the second generation of the patients, it is revealed almost the same frequence of disease (brothers – 22 cases (10.1%), sisters – 21 cases (9.6%). out of 82 cases (37.7%) the fathers’ diseases were passed to 42 men and 40 women. the latter is indicative of the fact, that the man who has glaucoma, it nearly equally passes on sons and daughters. as to 89 women (41%) with glaucoma – mothers of patients – in 39 cases (43. 8%) the disease hereditarily appeared in sons, and in 50 cases (56.1%) – in daughters, i.e. in this case we can talk with confidence about the fact, that in case of having glaucoma the greater probability is observed in its passing to female line genealogically. though, totally, the number of men patients with glaucoma prevails over the women-patients. while examining genealogically the second generation, some equilibrium is observed between the number of patients with glaucoma – those of brothers and sisters. the sisters of the patients generally inherit the disease from mothers. in revealing the disease in the second generation we can confidently speak if not about the inherited disease, but about reliable inherited predisposition to this disease, which can be revealed reaching the appropriate age. proceeding from the data of our researches, we consider it necessary, in detection of the mentioned disease to take the relatives under the control of the doctor ophthalmologist. out of 217 individuals with aggravation only three children had glaucoma. however, it must be noted that the examination of inherent youth glaucoma isn’t included in the frames of the present theme. our researches let us only state the data, do some suppositions and conclusions. that is, first, the necessity of systematic examinations of presence of glaucoma of close relatives of the examined patients, as in that case it is possible to expose the earliest stages of the disease. such a direction of prophylactic examinations promotes less danger of the disease, as the examinations aren’t limited by age. in researching the disease incidence of glaucoma of the mentioned contingent of patients there was also revealed the presence of somatic diseases and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 marine arsen hayriyants, on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra, volume xii, issue 4 – 2013, pag. 376 – 381 379 combination of glaucoma with other ophthalmopathology. in addition there were diagnosed with hypertension – 26.2% of patients, ischemic disease of heart – 9%, insular diabetes – 16.1%, and other somatic diseases were also noted. such a combination of glaucomatic process with vascular diseases gives reason to agree with the opinion of those authors (nesterov a.p., 1982), who mentioned, that up to now there haven’t been firm evidences of causal dependence between primary glaucoma and other diseases or of direct participation of the latter in pathogenesis of glaucomatous process. the existence of common reasons, that call forth diseases in people of old age, including glaucoma, isn’t excluded. finally, 42.6% of examined individuals had signs of other ophthalmopathology. those are: anomalies of development of eyes, traumas of eye ball, residual phenomena of old uveitis, cataract (congenital and age-specific), lesion of retinal, vascular membrane and visual nerve, myopia, astigmatism. glaucomatic symptoms and frequency of usage of instrumental research for diagnostics are studied in the article. a) depending on the gender (1) – as you can see in the table 1. table 1 distribution of glaucomatic symptoms and used diagnostic methods depending on the gender of patients in ra during 2004-2012 (in %) gender symptoms men women pains in the eye 5.40 6.36 lowering of sight 13.80 14.07 iridescent circles 1.64 2.10 intraocular pressure 13.24 13.10 tonography 12.77 12.89 biomicroscopy 11.14 11.48 ophthalmoscopy 7.26 6.49 vision field 13.82 14.02 gonioscopy 8.42 6.56 the data brought testify that certain symptoms of glaucoma are met both among women and men approximately equally. instrumental researches are used almost with the same frequency. b) changes of glaucomatic symptoms and used research methods during the years of the research. retrospective analysis shows, that during 2004-2012 glaucomatic symptoms was noted, but not with the same frequency, as you can see in the table 2. . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 marine arsen hayriyants, on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra, volume xii, issue 4 – 2013, pag. 376 – 381 380 table 2 frequency of glaucomatic symptoms of patients and used diagnostic methods in ra during 2004-2012 (in %) years symptoms 2004 2005 2006 2007 2008 2009 2010 2011 2012 pains in the eye 22.0 22.8 00.0 44.8 66.6 88.7 55.7 44.8 66.5 lowering of sight 113.8 112.0 55.9 112.7 113.3 115.3 114.1 113.7 116.6 iridescent circles 00.4 11.9 00.0 00.9 55.2 33.6 00.7 11.2 11.3 intraocular pressure 113.8 112.0 55.9 112.4 112.7 114.5 112.8 112.7 114.5 tonography 113.4 113.0 55.9 112.9 112.0 113.8 112.8 112.4 113.4 biomicroscopy 88.3 112.0 00.0 113.4 110.1 44.9 111.9 112.4 111.9 ophthalmoscopy 99.1 113.0 55.9 55.5 55.1 33.9 77.9 99.6 110.4 vision field 113.8 112.0 335.3 116.4 115.7 114.9 114.9 114.8 00.5 gonioscopy 111.8 88.3 117.6 88.4 77.4 66.4 66.6 66.3 111.9 daily iop 113.8 113.0 223.5 112.7 111.9 113.9 112.6 112.1 113.0 more often on the assumption of subjective sensation there was lowering of sharpness of sight. it practically didn’t give special variation during the whole period of research. among diagnostic methods tonometry, as well as tonography, perimetry, gonioscopy, ophthalmoscopy and biomicroscopy were used, as you can see in figure 1. figure 1. dynamics of revelation of certain symptoms of glaucoma and diagnostic methods used in ra during 2004-2012 (in %) 4. conclusions we can draw the following conclusions: for diagnosing “glaucoma” the complaints of the patients were considered and always instrumental methods of reaearch were used. in plan of principle particular deviations in the frequency of use of diagnostic methods aren’t noted. their main set is generally food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 marine arsen hayriyants, on screening of patients with primary glaucoma and some aspects of diagnostics of primary glaucoma in ra, volume xii, issue 4 – 2013, pag. 376 – 381 381 accepted. taking into account the aforesaid and also proceeding from the fact, that screening examination of asymptomatic patients is lately especially stressed, with the purpose of revelation of early stages, as always there exists the best prognosis at the early beginning of treatment. but at the same time it must be mentioned, that small total prevalence of the disease brings to economic non-efficiency of screening0 examination of a large group of population. groups of high risk must be exposed to screening. the main factors of glaucoma risk are the age, high iop, miopy, heartvascular diseases (arterial high pressure, angiospasm), diabetes and anamnestic record. we have also made an attempt to establish patterns and relationships between the external environment and the human body and in our opinion within specific geographic areas and ecology, these effects can be specifically. all of this provides a basis for more detailed surveys of the prevalence of glaucoma in view of climate geographical zones and ecology. study the dependence of the spread of glaucoma on the mineral composition of drinking water, soil composition, ecology and other physical geographical features we could not establish. in this regard, requires new research on a number of areas of armenia. 5. references [1]. bourne rr, sorensen ke, klauber a, foster pj et. al., glaucoma in east greenlandic inuit—a population survey in ittoqqortoormiit (scoresbysund), acta ophthalmol scand. 2001 oct; (5): 462-467. [2]. distelhorst j.s., hughes g.m., open-angle glaucoma, am fam physician. 2003 may 1;67(9): 1937-1944. [3]. joachim s.c., grus f.h., pfeiffer n. , analysis of autoantibody repertoires in sera of patients with glaucoma. eur j ophthalmol. 2003 nov.-dec; 13(9-10): 752-758 . [4]. nesterov a.p. epidemiology of glaucoma. regular medical service of patients with glaucoma.in book: primary glaucoma. m., 1982, pp.269-277. [5]. zhao j, sui r, jia l, ellwein lb. prevalence of glaucoma and normal intraocular pressure among adults aged 50 years or above in shunyi county of beijing, zhonghua yan ke za zhi. 2002 jun; 38(6): 335-339. [6]. spelton d.g., hitching r.a., hunter p.a., primary glaucoma, atlas on clinical ophthalmology. 2007, pp. 195 200. stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xii, issue 2 30 june 2013 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved sensory evaluation 10 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1-2016, pag. 10 20 application of d-optimal mixture design to optimize the wheat-pumpkin composite flour for bread production georgiana gabriela codină 1, silvia mironeasa*1 1faculty of food engineering, stefan cel mare university, suceava, romania, codina@fia.usv.ro; silviam@fia.usv.ro *corresponding author received january 4th 2016, accepted february 18th 2016 abstract: as compared to wheat flour, pumpkin flour is considered a high quality raw material in terms of nutrients and bioactive compounds essential in providing human health benefits. due to its potential, pumpkin flour can be used as a functional ingredient and in bread making can be used in composite blends with wheat flour. in this study, the effect of wheat-pumpkin composite flour bread on some physical parameters of bread was investigated. for this purpose, three raw materials pumpkin pulp flour, pumpkin seed flour, wheat flour were used and as statistical program the d-optimal mixture design (design expert 7.0) was used. as response variables, some physical parameters such as bread porosity, elasticity and h/d ratio were evaluated. the experimental data obtained were used to carry out the analyses of variance (anova) and to develop the regression models. special cubic model were used to explain all physical parameters considered, highlighting that the composite flour components interacted with each other and had effects on bread porosity, elasticity and h/d ratio. the best formulation of composite flour formula was chosen depending on the desirability function which showed a value of 0.94. the optimized formulation which showed desirable physical parameters contained 78.3% wheat flour, 15% pumpkin pulp flour and 6.70% pumpkin seed flour. key words: wheat flour, pumpkin flour, bread quality, mixture design approach, formula optimization 1. introduction bread is an important food product based on wheat flour which is consumed worldwide due to its competitiveness cost, ease of preparation, versatility, sensory and nutritional characteristics [1]. from the nutritional point of view, bread is a source of vitamins, minerals, dietary fiber and proteins [2 5]. however, by milling and processing, many of the nutrients from the wheat are destroyed, afectting from the nutritional point of view the quality of the wheat flour as raw material. also, some vitamins such as a, c, d, and b12 lacked from the wheat flour, decreasing it nutritional quality [6]. the quality of raw materials used in bread formulation play an important role, influencing not only the nutritional value of bread but also functional, sensory and physicochemical quality of the final bread product [7]. various ingredients can be added in bread products to improve wheat flour processing and/or nutritional value of bread. it is known that wheat flour proteins are of a lower nutritional quality than the milk, soy, pea and lupin proteins [8], e.g. because it is deficient in essential amino acids such as lysine, tryptophan and threonine [8 11]. therefore, nowadays, the trend is to improved bread quality by incorporation of new types of raw materials to produce composite breads as functional foods [10], made from blend of wheat flour and non-wheat or pseudohttp://www.fia.usv.ro/fiajournal mailto:codina@fia.usv.ro; mailto:silviam@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 11 cereals flours [12] since they are rich in nutrients such as essential amino acids, dietary fiber, fatty acids, minerals, vitamins, phenolic compounds, e.g., with great potential for the human health. the blends between wheat flour and non-wheat flours can be successfully used to obtain a composite bread with a better overall balance of essential amino acids [13], especially the lysine content, significant in a balances diet [14], improving its nutritional characteristics. in this way, the used in wheat flour of not just one nonwheat flour but a mixture of legumes and seeds, can have as result from bread products an improved taste, aroma and nutritional quality. some studies made in order to improve bread quality from the nutritional point of view by supplementation of wheat flour with nonwheat or pulses flours have been made by mohammed et al. (2012) [13]; harinder et al. (1999) [15]; see et al. (2007) [16]; güemes-vera et al. (2004) [17]. also, some researches [18; 19] reported that the application of vegetable flours as functional ingredients in breads have in addition, many beneficial effects in controlling and preventing various metabolic diseases [20 22]. pumpkin is an economically and agriculturally valuable vegetable cultivated around the word. pumpkin fruits are suitable after processing for consumption, making a major contribution to human nutrition due to their composition which varies from one species or cultivar to another. pumpkin pulp provides a valuable source of vitamins such as vitamin a, vitamin b2, and vitamin c [23 25], carotenoids [23; 25; 26] that have significant roles in human nutrition. also, pumpkin pulp is rich in bioactive compounds with high antioxidant activity such as phenolics and flavonoids [23; 27], minerals, pectin, dietary fiber [28] and other compounds which are beneficial to the human health [29; 30]. among the dietary fiber, pumpkin flour contains cellulose, hemicelulose and lignin [31]. therefore, by adding in composite flour, the pumpkin flour leads to a fiber enrichment of bread-making products, improving its nutritional quality [31]. different levels of pumpkin pulp flour used for wheat flour substitution was used in preparation of various bakery products such as cakes and cookies [32; 33], biscuits [34], toast breads [35], having various effects on bakery products. it was showed that higher levels of pumpkin pulp caused an unpleasant aroma and taste. noor and komatchi (2009) [36] investigated the potential of peeled and unpeeled pumpkin pulp as a raw material for the production of flour for use in composite blend with wheat flour or as a functional ingredient in food products. processed into flour which has a longer shelf-life, pumpkin can be use as functional ingredients in bakery products, supplementing cereal flours. the incorporate of pumpkin powder for preparation of cake lead to a increased of fiber content and -carotene [32]. the carotene content increased with the increasing dose of pumpkin powder addition, increasing the level of vitamina a [33]. same effects on pumpkin blend in biscuits were reported by kulkarni and joshi, 2012 [34]. in addition, they reported an increased of minerals content in biscuits as compared to the sample without pumpkin powder. due to its highlydesirable flavor, sweetness and deep yellow-orange color, pumpkin flour can be use as a natural coloring agent in composite flour [35]. pumpkin seeds are an interesting raw material for food application, highly nutritious than cereals due to its high protein content (39.25%), oil (27.83%), minerals (4.59%) and dietary fibers 16.84% [1; 37 39]. by the addition of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 12 pumpkin seed powder in bread the protein content of the product increases [1]. pumpkin seed flour is recognized as a good source of lysine [40] improving nutritional value of products by incorporated them in bakery products, especially bread [41]. the addition of pumpkin seed flour to this product, conducted to an increased of the total essential amino acids, protein, fat and mineral content, showing that the pumpkin seed flour is a good source of protein and nutrients for bread fortification [40]. the level effects of pumpkin seed flour added in wheat flour on the bread quality were also studied [16], showing that the level of 5% is a feasible level to produce bread with good nutritional value and sensory characteristics. taking into account it nutritionally value, the pumpkin pulp and seeds flours can be a good supplement to wheat flour which can be used in formulation of the composite flours in order to improve quality of bread. by using in wheat flour not just one nonwheat flour but a mixture of vegetables and seeds oils we can obtain a better result regarding the quality of bread products. although pumpkin pulp flour or pumpkin seed flour had been used in composite flour formulation, after our knowledge the addition of both pumpkin pulp and pumpkin seed flours in wheat flour in various ratio had not been studied before. the used of statistically designed experiments in the bread-making processes to develop the new products or to improve the existing one is one of the priorities of the baking industry and not only that can lead to a lower general manufacturing costs. mixture design is a statistical technique that can be used for evaluating the quality of final product based on the relative proportions of the components in the mixture. this technique uses an equation that describes how the independent variables in a mixture experiment affect the response variables [42]. d-optimal mixture design was applied to find the optimal response for any mixture of the components of composite flour, and to obtain the influence on the response of each component singly and in combination with other components. the objective of this work was to investigate the effect of varying ratios of pumpkin pulp flour, pumpkin seed flour and wheat flour and their interaction on some physical parameters of bread by using a d-optimal mixture design approach and to determine the optimal mixture of wheat-pumpkin composite flour. also, the main objective of this study was to optimize the mixture of the three ingredients used, wheat flour, pumpkin pulp flour and pumpkin seed flour in the composite flour recipe, with the purpose of achieving the optimum physical parameters of produced composite breads. d-optimal criteria were applied to determine the optimal blend for the best physical parameters of composite bread obtained. 2. materials and methods 2.1. materials 2.1.1. wheat flour the research study has been carried on 650 flour type (harvest 2015) obtained from s.c. mopan s.a. (suceava, romania). the flour used in this study presents the following characteristics: moisture content 14.5%, ash content 0.65%, protein content 12.6%, wet gluten content 34%, gluten deformation index 8 mm, falling number 380 s. the flour chemical composition was determined according to romanian standard methods: moisture (sr en iso 712:2010), wet gluten content (sr 90:2007), gluten deformation index (sr 90:2007), ash content (sr en iso food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 13 2171:2010) and falling number index (sr en iso 3093:2010). 2.1.2. pumpkin flour fresh pumpkins fruits (cucurbita moschata) and commercial wheat flour were obtained from the local supermarket. in order to obtain the pumpkin pulp and seed flours, the unpeeled pulp and seeds of fresh pumpkins was manually separately and then washed under running tap water. the pumpkin pieces cut into slices of 2-3 mm thickness and the seeds were dried in hot air oven to a moisture content of 10 12% at 50oc for 24 hours. the shelled seeds and the dried pumpkin slices was grounded using a mechanical mill, and then sieved through a 150 m mesh sieve in order to obtain fine flour. 2.2. methods 2.2.1. experimental design d-optimal mixture design with three components was used to optimize the composite pumpkin wheat flour bread formula. the wheat flour (wf or x1) as a bread composite flour base, pumpkin pulp flour (ppf or x2) used as a source of fiber, and pumpkin seed flour (psf or x3) used as a source of proteins and minerals are the mixture components which was taken as independent variables. the components proportions from composite flour formula, established from preliminary studies, are subject to multiple-components constraints [cornell, 2002], as follows: x1 + x2 + x3 = 1; 0.71 x1  0.92; 0.05 x2  0.20; 0.03 x3  0.09, yielding a polyhedral constrained region. the dependent variables selected as the responses were some physical parameters of composite pumpkin wheat flour bread such as porosity, elasticity and d/h ratio. the stat-ease design expert 7.0.0 software package (trial version) was used to achieve as well as to analyze the experimental design. the variations of wheat-pumpkin composite flour formulations and their replications including a total of 12 formulations are shown in table 1 and for each treatment combination the sum of the component proportions was equal to one. table 1. a d-optimal mixture design for the composite flour formulated with wheat flour (wf), pumpkin pulp flour (ppf) and pumpkin seed flour (psf) in various proportion formulation number formulation variables x1 (wf) x2 (ppf) x3 (psf) 1 0.920 0.050 0.030 2 0.855 0.055 0.090 3 0.831 0.108 0.061 4 0.766 0.200 0.034 5 0.842 0.128 0.030 6 0.710 0.200 0.090 7 0.766 0.144 0.090 8 0.876 0.083 0.041 9 0.799 0.138 0.063 10 0.855 0.055 0.090 11 0.766 0.200 0.034 12 0.710 0.200 0.090 according to the d-optimal approach effect of proportions of wheat flour (wf), pumpkin pulp flour (ppf) and pumpkin seed flour (psf) on some physical parameters of composite pumpkin wheat flour bread as response variables was evaluated and optimum combination was determined. the combination of independent variables in optimal proportions that lead to the best responses from bread physical parameters in terms of porosity, elasticity and height/diameter (d/h) ratio was determined. 2.2.3. preparation of composite pumpkinwheat flour bread the pumpkin-wheat composite flour including pumpkin pulp flour, pumpkin seed flour and wheat flour at different food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 14 proportions (table 1) were used for the bread production. the 1.8 g compressed baker’s yeast, 1% sodium chloride and 67% water (on 100% weight of mixed flour) were the other ingredients used for prepared the bread. all the ingredients were kneading in a mixer for 7 min at 28oc and then the modeled samples was proofed for 90 min at 30oc, 85% relative humidity and baked for approximately 30 min at 250oc. a 30 min after removal from the oven, the baked bread samples were evaluated. 2.2.4. physical parameters of bread the bread elasticity, porosity and h/d ratio are the physical parameters determined for assessed the effects of pumpkin-wheat composite flours formulation on bread quality. loaf height, diameter, elasticity and porosity were determined according to sr 91:2007 and h/d ratio was calculated. 2.2.5. statistical analysis and optimization in this study, fitting response values was made using the linear, quadratic and cubic models (eqs. (1) – (3)). analysis of variance (anova) at p-value of the model  0.05, p-value of lack of fit  0.05 and coefficient of determination, r2  0.85 are the criteria’s used for the selection of predictive model for each response. y = β1x1+ β2x2+ β3x3 +  (1) y = β1x1+ β2x2+ β3x3+ β12x1x2+ β13x1x3 + β23x2x3 +  (2) y = β1x1+ β2x2+ β3x3+ β12x1x2+ β13x1x3 + β23x2x3 + β123x1x2x3 +  (3) where y is the predictive response (bread elasticity, porosity and h/d ratio);  is the error of estimation; β1, β2, β3, β12, β13, β23 and β123 are the constant coefficients for each linear and interaction term in the predicted models; and x1, x2, x3 is proportion of pseudo-components. also, the regression coefficients of individual linear, interactive and cubic terms were determined. the significances of all terms in the regression models were assessed statistically by computing the fvalue at a level of significance of 95% or 99%. after evaluating the regression models that reveal the relationship between flours mixture components and response variables, the desirability function approach was used to simultaneously optimize bread porosity, elasticity and h/d ratio. the optimal levels of mixture components of the composite flour required to yield final optimized physical parameters of bread were obtained by numeric analysis based on criteria of maximizing porosity, elasticity and the value of 8 for h/d ratio. the stateease design expert 7.0.0 software package (trial version) was used to statistical analysis, to generate the surface response of the fitted regression equations and to determine the optimum proportions of the components from composite pumpkin-wheat flour bread. 3. results and discussion 3.1. fitting for the best model the best model for predicting physical parameters of composite flour bread is the regression model that has a low standard deviation, a low predicted sum of squares, and high r-squared [42]. therefore, the results of anova shown that special cubic model was found the best fitted for all response variables, bread porosity, elasticity and h/d ratio (table 2). the r2 value obtained from special cubic model was found to be greater than 0.85, indicating a great fitting model. the mathematical response surface models food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 15 were carried out using design expert software by applying pseudo components. table 2. regression coefficients of predicted special cubic models for physical parameters of composite flour bread formulation using data in pseudo components component physical parameters of bread sample porosity (%) elasticity (%) h/d ratio β1 (wf) 80.33 82.38 0.96 β2 (ppf) 91.02 85.06 0.96 β3 (psf) -303.45 -146.73 2.99 β12 (wf x ppf) -57.58 -21.44 -0.095 β13 (wf x psf) 535.55* 311.73* -3.15 β 23 (ppf x psf) 474.63* 297.52* -3.39 β123 (wf x ppf x psf) 280.19 162.14 -0.64 p-value 0.0270 0.0471 0.0133 lack-of-fit 0.7765ns 0.3072 ns 0.1473 ns r2 0.8897 0.8596 0.9184 β1 – wheat flour (wf) value coefficient, β2 – pumpkin pulp flour (ppf) value coefficient, β3 – pumpkin seed flour (psf) value coefficient; ns no significant effect at level  0.05; * significant at the level of p  0.05. the physical parameters of bread sample were significantly predicted by independent variables pumpkin pulp flour, pumpkin seed flour and wheat flour. the values of linear and interaction terms show that pumpkin seed flour is the component which most affects the bread porosity, following the elasticity and h/d ratio. the pumpkin seed flour was again the most important variable in predicting all physical parameters of bread. residual analysis was also carried out in order to evaluate the adequacy of the prediction models. the residual values for porosity, elasticity and h/d ratio, explained through the difference between the observed and predicted values of these parameters, are normally distributed (figure 1a), b) and c)). the plots showed that the special cubic model is adequate to describe the each response surface. the predicted models were used to generate contour plots for response variables significantly predicted by independent variables. figures 2-4 show the estimate response surface graphs for the porosity, elasticity and h/d ratio. 3.2. effect of component from composite flour on quality measurements of bread the bread samples produced with different proportion of wheat, pumpkin pulp and pumpkin seeds flours shows differences exist among the samples in porosity, elasticity and h/d ratio. bread porosity bread porosity is an important parameter of bread quality which indicate it assimilability that can be higher or lower. the influence of composite flour components on bread porosity has been shown in the response surface graph in figure 2. regarding to porosity, a high correlation (r2 = 0.8897) was obtained. the formulation components, wheat flour (wf), pumpkin pulp flour (ppf) and pumpkin seed flour (psf), independently not influenced significantly the porosity, however, the interactions between wf and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 16 psf, between ppf and psf, significantly (p  0.05) influenced bread porosity. (a) (b) (c) fig. 1. residual plots for porosity % (a), elasticity % (b) and h/d ratio (c) the value of linear parameter psf showed negative coefficient for porosity which indicated a decrease of this physical parameter. fig. 2. the response surface plot of porosity this result is in agreement with the results obtained by see et al. (2007) [16] who concluded that addition of 5% pumpkin seed flour in wheat flour resulted in bread with high loaf volume are most acceptable. after this level, the specific volume decreased. the increased level of psf addition in bread composite flour up of 5% leaded to a decrease of bread porosity. it was evident from this study that at lower psf proportion the bread porosity increased. an increase in wf level in composite flours lead to an increase of bread porosity values. the increase in porosity value with increasing level of ppf in composite flour has also been reported previously [35], the bread obtained beeing acceptable at 10% level. porosity value is proportional to the increase level of ppf and wf in composite flour. therefore, high proportions of ppf and wf in composite flour may increase the bread porosity, by improving gas-cell stability according to ptitchkina et al. (1998) [31] which affirm that the enhanced stability comes from surface activity of pumpkin pectin. pectin constitutes about 30% of the dry weight of pumpkin tissue. the increase in aeration is tentatively attributed to surface activity of the highly acetylated pectin present as a major component (about 30% of the total drymatter content) of pumpkin tissue. różyło food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 17 et al. (2014) [27] showed that bread with pumpkin pulp had smaller and more compact pores. elasticity the bread having high elasticity are considered to be having desirable features. figure 3 shows response surface plot of elasticity as a function of wheat flour (wf) proportion, pumpkin pulp flour (ppf) proportion and pumpkin seed flour (pps) proportion in composite flour. fig. 3. the response surface plot of elasticity elasticity was maximum at intermediate to higher flours proportions. addition of ppf to composite flour showed a positive coefficient indicated that ppf had a synergistic effect on porosity. the wf/psf and ppf/psf combinations showed a positive significant influence on porosity, while the three component combination is insignificant. also, in present study, interaction of wf/ppf showed a negative coefficient indicating an antagonistic effect on elasticity, while the interaction wf/psf and ppf/psf has a synergistic effect on this physical parameter. h/d ratio the predictive model for h/d ratio showed the three variables, wf, ppf and psf hadn’t significant effects, but which was positive. all the three flours showed not significant quadratic negative effects on h/d ratio. comparatively with other components of composite flour, pumpkin seed flour has a high contribution on h/d ratio. interactive effects between wheat flour and pumpkin pulp flour, pumpkin seeds flour and, triple interaction effect between wheat flour, pumpkin pulp flour and pumpkin seeds flour had a negative effect on h/d ratio of the bread. according to anova, there was no significant effect of the regression model on h/d ratio (table 2), but a high correlation (r2 = 0.9184) was detected. in h/d ratio, the contour plot can be observed that the reduction of wheat flour content and increasing of pumpkin seed flour in composite flours formulation resulted in lower h/d ratio values (figure 4). fig. 4. response surface plot showing effects of composite flour formulations on h/d ratio 3.3. optimized composite flour formulation upon comprehensive evaluation of available design expert solutions, the optimum mixture ratio of flours with wheat flour, pumpkin pulp flour and pumpkin seed flour was determined on the basis of desirability function, using the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 18 optimization process suggested by derringer and suich [43]. simultaneous optimization was performed by imposing some constraints such as h/d ratio of 8, and maximum porosity and elasticity. the best combinations between the proportion of wheat flour, pumpkin pulp and pumpkin seeds flours used in this study in order to obtain optimum values for some physical parameters of bread were extracted by state-ease design expert software that performs from the thousands of iterations and calculation the maximum desirability value and the conditions on which it was arrived. on the basis of these calculations, a total desirability value (d) of 94.1% was obtained for the optimal formulation of composite flour with wheat flour (wf), pumpkin pulp flour (ppf) and pumpkin seed flour (psf) in proportion of 78.30%, 15.00% and 6.70% (w/w). the responsesurface plot corresponding to d value is represented in figure 5, and it was used to predict final mixture proportion. fig. 5. desirability function response surface under these mixture proportions, the predicted h/d ratio of 0.81, bread porosity of 89.80% and elasticity of 88.53% were obtained. 4. conclusion this study highlights the usefulness of doptimal design in the assessment of the simultaneous influence of composite flour components and their interaction on bread physical parameters. the effects of composite flour components on response variables could be accurately predicted by special cubic type models. a high linearity was observed between predicted and actual value of response variables. by using numerical technique, the optimized pumpkin-wheat composite flour was obtained. the optimized composite flour formula chosen on the basis of desirability function revealed that the composite flour with 78.30% wheat flour, 15.00% pumpkin pulp flour and 6.70% pumpkin seed flour (w/w) is the best formulation which can be used to obtain pumpkin-wheat flour bread with the desirable physical parameters. acknowledgements this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs – uefiscdi, project number pn-ii-ru-te-2014-4-0214. 5. references [1] shemi g., nazni p., formulation and optimization of functional bread by employing response surface methodology, international journal of current research, 4: 008-014, (2012) [2] charmet g., masood-quraishi u., ravel c., romeuf i., balfourier f., perretant m.r., joseph j.l., rakszegi m., guillon f., sado p.e., bedo z., saulnier l., genetics of dietary fibre in bread wheat, euphytica, 170: 155-168, (2009) [3] steer t., thane c., stephen a., jebb s., bread in the diet: consumption and contribution to nutrient intakes of british adults, proceedings of the nutrition society, 67: e363, (2008) [4] malinowska e., szefer p., concentration of several bioelements in bread and other plant supplements (in polish), roczn. pzh, 56(2): 171178, (2005) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 19 [5] borawska m., omieljaniuk n., markiewicz r., witkowska a., total dietary fibre content in selected baker’s goods (in polish), bromat. chem. toksykol., 1: 25-28, (1999) [6] hoseney r.c., principles of cereal science and technology, 2nd ed., aacc international: st. paul, mn, (1992) [7] duodu k.g., minnaar a., legume composite flours and baked goods: nutritional, functional, sensory, and phytochemical qualities. in flour and breads and their fortification in health and disease prevention (v.r. preedy, r.r. watson and v.b. patel, eds.) pp. 193–203, academic press, london, u.k., (2011) [8] morens c., bos c., pueyo m.e., benamouzig r., gausseres n., luengo c., tome d., gaudichon c., increasing habitual protein intake accentuates differences in postprandial dietary nitrogen utilization between protein sources in humans, journal of nutrition, 133: 2733-2740, (2003) [9] wrigley c.w., bietz j.a., proteins and amino acids. in: pomeranz y. (ed.), wheat: chemistry and technology, (pp. 159–275). st. paul, mn, usa, american association of cereal chemists, (1988) [10] dewettinck k., van bockstaele f., kühne b., van de walle d., courtens t.m., gellynck x., nutritional value of bread: influence of processing, food interaction and consumer perception, journal of cereal science, 48: 243-257, (2008) [11] jideani v., onwubali f., optimisation of wheat-sprouted soybean flour bread using response surface methodology, african journal of biotechnology, 8(22): 6364-6373, (2009) [12] fang s.e., physico-chemical and organoeptic evaluations of wheat substituted with different percentage of pumpkin flour (cucurbita moschata), thesis submitted in fulfillment of the requirements for the degree of master of science, university sains malaysia, (2008) [13] mohammed i., ahmed a.r., senge b., dough rheology and bread quality of wheat– chickpea flour blends, industrial crops and products, 36:196–202, (2012) [14] kadam m.l., salve r.v., mehrajfatema z.m., more s.g., development and evaluation of compositeflour for missi roti / chapatti, food process & technology, 3(1): 134-140, (2012) [15] harinder k., kaur b., sharma s., studies on baking properties of wheat: pigeon pea flour blends, plant foods for human nutrition, 54(3): 317 – 326, (1999) [16] see e.f., wan nadiah w.a., noor aziah a.a., physico-chemical and sensory evaluation of breads supplemented with pumpkin flour, asean food journal, 14: 123–130, (2007). [17] güemes-vera n., arciniegaruizesparza o., dávila-ortiz g., structural analysis of the lupinus mutabilis seed, its flour, concentrate, and isolate as well as their behavior when mixed with wheat flour, lwt-food science and technology, 37: 283–290, (2004) [18] hefnawy t.m.h., el-shourbagy g.a., ramadan m.f., impact of adding chickpea (cicer arietinum l.) flour to wheat flour on the rheological properties of toast bread, international food research journal, 19(2): 521-525, (2012) [19] dhinda f., lakshmi j.a., prakash j., dasappa i., effect of ingredients on rheological, nutritional and quality characteristics of high protein, high fibre and low carbohydrate bread, food and bioprocess technology, 5(8): 2998-3006, (2011) [20] arab e.a.a., helmy i.m.f., barch g. f., nutritional evaluation of functional properties of chickpea (cicer arietinum l.) flour and the improvement of spaghetti produced from its, the journal of american science, 6(1): 1055-1072, (2010) [21] angioloni a., collar, c., high legumewheat matrices: an alternative to promote bread nutritional value meeting dough viscoelastic restrictions, european food research and technology, 234(2): 273-284, (2012) [22] de la hera e., ruiz-paris e., oliete b., gómez m., studies on the quality of cakes made with wheat-lentil composite flours, lwtfood science and technology, 49(1): 48-54, (2012) [23] jacobo-valenzuela n., zazuetamorales j.j., gallegos-infante j.a., aguilar-gutierrez f., camachohernández i.l., rocha-guzmán n.e, gonzález-laredo r.f., chemical and physicochemical characterization of winter squash (cucurbita moschata d.), notulae botanicae horti agrobotanici cluj-napoca, 39(1): 34-40, (2011) [24] roura s. i., del valle c.e., aguero l., davidovich l.a., changes in apparent viscosity and vitamin c retention during thermal treatment of butternut squash (cucurbita moschata duch) pulp: effect of ripening stage, journal of food quality, 30: 538–551, (2007) [25] gonzález e., montenegro m.a., nazareno m.a., carotenoid composition and vitamin a value of an argentinian squash (cucurbita moschata), archivos latinoamericanos de nutrición, 51(4): 395–399, (2001) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 georgiana gabriela codină, silvia mironeasa, application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production, volume xv, issue 1 – 2016, pag. 10 – 20 20 [26] noseworthy j., loy b., improving eating quality and carotenoid content of squash. in m. pitrat (ed.), proceedings of the ixth ucarpia meeting on genetics and breeding of cucurbitaceae, inra, avignon (france), may 21–24th: 521–528, (2008) [27] różyło r., gawlik-dziki u., dziki d., jakubczyk a., karaś m., różyło k., wheat bread with pumpkin (cucurbita maxima l.) pulp as a functional food product, food technology and biotechnology, 52(4): 430–438, (2014) [28] tamer c.e., i̇ncedayi b., parseker a.s., yonak s., çopur ö.u., evaluation of several quality criteria of low calorie pumpkin dessert, notulae botanicae horti agrobotanici cluj-napoca 38: 76-80, (2010) [29] jun h.i, lee c.h., song g.s., kim y.s., characterization of the pectic polysaccharides from pumpkin peel, lwt food science and technology, 39: 554–561, (2006) [30] djutin k.e., pumpkin: nutritional properties, potatoes and vegetables 3:25-26 (in russian), (1991) [31] ptitchkina n.m., novokreschonova l.v., piskunova g.v., morris er., large enhancement in loaf volume and organoleptic acceptability of wheat bread by small additions of pumpkin powder: possible role of acetylated pectin in stabilizing gascell structure, food hydrocolloids, 12: 333–337, (1998) [32] bhat m.a., bhat a., study on physicochemical characteristics of pumpkin blended cake, food processing & technology, 4(9): 1-4, (2013) [33] pongjanta j., naulbunrang a., kawngdang s., manon t., thepjaikat t., utilization of pumpkin powder in bakery products, songklanakarin journal of science and technology, 28: 71-79, (2006) [34] kulkarni a.s., joshi d.c., effect of replacement of wheat flour with pumpkin powder on textural and sensory qualities of biscuit, international food research journal, 20(2): 587591, (2013) [35] el-deremy m.e., evaluation of physicochemical properties of toast bread fortifi ed with pumpkin (cucurbita moschata) flour, the 6th arab and 3rd international annual scientific conference on development of higher specific education programs in egypt and the arab world in the light of knowledge era requirements, faculty of specific education, mansoura university, mansoura, egypt, 13–14, (2011) [36] noor a.a.a., komatchi c.a., physicochemical and functional properties of peeled and unpeeled pumpkin flour, journal of food science, 74(7): 328–333, (2009) [37] mansour e.h., dworschak e., peredi j., lugasi a., evaluation of pumpkin seed (cucurbita-pepo, kakai-35) as a new source of protein, acta alimentaria, 22(1): 3-13, (1993) [38] alfawaz m.a., chemical composition and oil characteristics of pumpkin (cucurbita maxima) seed kernels, res. bult., no. (129), food sci. agric. res. center, king saud univ.: 5–18, (2004) [39] simwaka j.e., huiming z., masamba k.g., amino acid profile, mineral, pasting, thermal and protein solubility characteristics of sorghum-finger millet based complementary food as affected by fermentation, journal of academia and industrial research (jair), 3(10): 504-510, (2015) [40] el-soukkary f.a.h., evaluation of pumpkin seed products for bread fortification, plant foods for human nutrition, 56: 365-384, (2001) [41] gan z., angold r.e., williams m.r., ellis p.r., vaughan j.g., galliard t., the microstructure and gas retention of bread dough, journal of cereal science 12: 15–24, (1990) [42] cornell j.a, experiment with mixtures, designs, models and the analysis of mixture data, 3rd edition, john wiley & sons , inc., usa. (2002) [43] derringer g., suich r., simultaneous optimization of several responses variables, journal of quality technology, 12(4): 214–219, (1980) 3.3. optimized composite flour formulation microsoft word 6 art_1 adrian ioana.doc 38 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 38 46 eaf environmental impact analisys through mathematical modeling and fuzzy logic *adrian ioana1, augustin semenescu1, cezar florin preda1 1university politehnica of bucharest, spl. independentei 313, bucharest, s6, 060042, adyioana@gmail.com * corresponding author received 10 january 2013, accepted 15 february 2013 abstract. the original optimisations mathematical model of the electric arc furnace’s charge preheating process mainly takes into consider 2 thermo-technological aspects: the heat transfer between fluids and particles and the heat transfer between the fizz layer and an exchange surface. according the energetically balance at the gaseous environment level, the conductive transfer model is also analysed through the finished elements method. the results of the mathematical model are presented as the analysis and quantification of the thermo gradients obtained during the charge preheating process. these thermo gradients are determined for various temporal moments and for different capacities of the electric arc furnace. the results of the mathematical model are presented as the analysis and quantification of the thermo gradients obtained during the charge preheating process. these thermo gradients are determined for various temporal moments and for different capacities of the electric arc furnace. keywords: environmental impact, electric arc furnace (eaf), modelling, fuzzy logic 1. introduction the electric arc furnaces (eafs), as powerful energy consumers, are also polluting emissions generators with an important environmental impact. the most significant polluting emissions of the eaf furnace are metallic and oxides powders driven by emergent gases [1-6]. the powders are produced during the following technological operations: raw materials loading, steel melting, refining, alloying and evacuation. generally, the driven powders contain heavy metals (cr, ni, zn, cd, pb, cu etc) and some metal oxides (iron, manganese, aluminum and silicon oxides) and they can reach values of more than 15 kg/t steel [7-8]. fuzzy logic (fl) is a powerful problemsolving methodology with wide applications in economical control and information processing [9-10]. it provides a simple way to draw definite conclusions from vague, ambiguous or imprecise information. it resembles human decision making with its ability to work from approximate data and find precise solutions [11, 12]. the neuro-fuzzy approach is to use neural networks and fuzzy set theory to model practical systems. a pattern match or recognition system is a black box constructed using multiple layers of neurons called neural networks. neurons have the ability of memory and selflearning by training. fl algorithms are implemented as an inference engine which can automatically infer from facts (data) [13-15]. mathematical modelling of the electric arc furnace’s processes (eafp) for the optimisation of the functional and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 39 technological performances of this complex unit is based on the next principles [16-19]: a. the principle of analogy – consists in observing and analysing competently the modelated reality, using both analogy with other fields of research and logical homology. according to this principle, for mathematical models making were used the following steps: • the modelated subject definition – represents the first phase of the modelation analysis. this step must satisfy both the purpose and the simultaneous system’s aims, assuring their compatibility; • the effiency criteria’s definition – is a step imposed on the correct definition of the system’s aims and allows the optimisation of the modelling solutions; • making the options – basing on accessing some realistically, original and efficient solutions; • choices evaluating – related to the established efficiency criterials; • choosing the final solution – based on the analysis between the different solutions of the modelling. b. the principle of concepts is based on the sistems’theory, including the feedback concept. c. the principle of hierarchisation consists of making hierarchical models systems, for structuring the decision and coordinating the interactive subsystems [20-22]. fuzzy logic has been found to be very suitable for embedded control applications [23-25]. several manufacturers in the automotive industry are using fuzzy technology to improve quality and reduce development time. in aerospace, fuzzy enables very complex real time problems to be tackled using a simple approach. in consumer electronics, fuzzy improves time to market and helps reduce costs. in manufacturing, fuzzy is proven to be invaluable in increasing equipment efficiency and diagnosing malfunctions. the gaseous phase (burnt gases) that comes out of the eaf mainly results from the melting and refining procedures and contains carbon monoxide, carbon dioxide together with nitrogen and sulfur oxides (nox and sox); however, in practice it also contains very toxic other components, such as fluorides or volatile organic compounds (dioxine, chloride derivatives of benzene or phenol) resulted from burning of organic oils that are introduced as contaminants together with the raw materials. 2. materials and methods environmental impact of eaf from the total polluting emissions, over 90% are generated during the technological operations of melting and refining. the chemical composition of these emissions is extremely variable and directly dependent on multiple factors, as followings [25-29]: composition of the raw materials that make up the loading; the melting managing way; type of refining process that is used (with gaseous oxygen or ore); time duration of the melting and refining steps; desired quality degree of the elaborated steel. figure 1 presents the main scheme of the environment polluting system through the eaf. table 1 presents the variation limits of chemical composition for the powders generated during the steel elaboration in electric arc furnaces (eaf) in the usa and germany, from loading that consists of scrap iron, only. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 40 figure 1. environment polluting system through the eaf table 1 variation limits of chemical composition for the eaf powders variation limits, % germany no. component sua plain basic steel alloy steel 1 fetotal 16.4 – 38.6 21.6 – 43.6 35.3 2 si 0.9 – 4.2 0.9 – 1.7 17.0 3 al 0.5 – 6.9 0.1 – 1.5 x) 4 ca 2.6 – 15.7 6.6 – 14.5 0.4 5 mg 1.2 – 9.0 1.0 – 4.5 1.2 6 mn 2.3 – 9.3 0.9 – 4.8 2.0 7 p 0 – 1.0 0.1 – 0.5 x) 8 s 0– 1.0 0.3 – 1.1 0.1 9 zn 0 – 35.3 5.8 – 26.2 1.4 10 cr 0 – 8.2 0 – 0.1 13.4 11 ni 0 – 2.4 x) 0.1 12 pb 0 – 3.7 1.3 – 5.0 0.4 environmental modelling system of eaf the modelling system’s central element of the eaf processes conceived consists of the system’s criteria function. knowing that the technological processes study for eaf is subordinated to high quality steel obtaining, the modelling system’s criteria function (cf) is the ratio between quality and price: max        price quality cf (1) the maximum of the criteria function is assured by the mathematical model of prescribing the criteria function (m.p.c.f.) the mathematical model of prescribing the criteria function concept consists of transforming the criteria function (cf) in a quality-economical matrice mqe, as in the scheme presented in figure 2. waste gases electric arc furnace (eaf) liquid steel nox co co2 powde r slag scrap iron carburizing agents lime iron alloys o2 air . . . noise food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 41 figure 2. the modelling system’s criteria function’s evaluation the levels of prescribing the criteria function could be obtained by using a composition algorithm for three vectors: • vector – technical parameters’ vector (ti); • vector – economical parameters’ vector (ej); • vector – weight vector (p1). in figure 3 there is presented the general logical scheme used for the eaf’charge preheating. fuzzy logic modelling for ecological impact of eaf patterns reflect the behavioral characteristics of how a person or a system acts in a certain environment. a spending pattern may represent the way a consumer spends money on different goods, such as travels, cars, or food. a defect pattern in a semiconductor equipment may indicate the way in which a part or assembly fails. by matching various patterns, a marketing specialist at a credit card company is able to better understand consumers spending habits, and therefore he can tailor his or her marketing strategies targeted to different consumer groups. figure 3. general logical scheme by the same token, scientists study and match various patterns of machine faults in star read: h, , cp, , q, t,  t , , x, y, n, m n = 1 n  n [t] = [f], [k]-1 n y read xjn,yin i= 1…3, j = 1…3 write [t] (n+2, n+2) stop bin = yi+1,n – yi+2,n cjn = xj+2,n – xj+1,n i= 1…3, j = 1…3 an = ½ (x2n  y3n – x3n  y2n – x1n  y3n + x3n  y1n + x1n  y2n) 213 132 )( )( nnn nnn xxi xxi   for i = 1…3 and j = ki,j = ksni, j+ki,j for j = 1,..., n + 2 do n = n fi,j = fsni, j+fi,j t t1 t2 … tn p p1 p2 … pn e e1 e2 … em mce (mu,w)   ti  ej  pk i = 1 … n; j = 1 … m; k = 1 … l food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 42 order to be able to predict and control the performance of equipment, including advanced warning. the application of the fuzzy logic (fl) is based on 3 simple steps defined below (figure 4): figure 4. the steps of the fl fls – fuzzy logic steps; f – fuzzification; a – aggregation; d defuzzification neuro-fuzzy modeling is to use neural networks and fuzzy set theory to model practical systems. neuro-fuzzy technology was developed that can automatically extract business rules from the neuro-fuzzy model. these rules can explain which and why people tend to fraud, or why a customer price increases. mathematically, the fuzzy utility function is a more accurate measure on the consumption utility. it can describe the relationships between: spending (s), price (p), consumption composition (cc), preference and subjective measure on commodity (c) or service values (sv). these relationships are described by the fufc matrix concept (figure 5). fuzzy consumption utility functions (fcuf) are based on utility theory (ut). fuzzy utility function for consumption (fufc) is described by the fufc matrix concept. this concept is based on the following vectors: s (spending vector); p (price vector); cc (consumption composition vector); c (commodity vector) and sv (service values vector). figure 5. fucf matrix concept fucf – fuzzy utility function for consumption; ofm – objective factors matrix; sfm – subjective factors matrix; s – spending vector; p – price vector; cc – consumption composition vector; c – commodity vector; sv – service values vector. 3. results and discussion the correlation between the criteria function’s (c.f.) prescribed levels and t vector’s components’ variation is presented in figure 9. the execution of the eaf’s charge preheating modelling (cpm) was made both for a 10t eaf (fig.11a, b, c) and for a 50t eaf (fig. 11d). it was considered to be a load with medium permeability ε = 0.45. the scheme of the eaf environmental impact is presented in figure 6. in figure 7 is presented the co concentration in the evacuated gas during the melting in the eaf. fls f a d [fufc [s [p [cc [c [sv [ofm [sfm] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 43 e x p e r i m e n t a l r figure 6. scheme of the eaf environmental impact t im e 10 20 30 40 50 60 70 80 90 l i l iv 0 2000 4000 6000 8000 10000 12000 14000 16000 co[%] time [min] l i l ii l iii l iv figure 7. co concentration in the evacuated gas during the melting in the eaf figure 8 presents the carbon dioxide impact of eaf. figure 8. carbon dioxide impact of eaf the correlation between the criteria function‘s (cf) prescribed levels and the t vector’s components’ variation ( t 1, t 2, vector’s components’ variation ( t 1, t 2, t 3 t 3) are presented in figure 9. figure 9. the correlation between the criteria function ‘s (cf) prescribed levels and the t the cumulated correlation between the criteria function’s (c.f.) prescribed levels and t and e vectors’ variation are presented in figure 10. figure 10. the cumulated correlation between the criteria function’s (cf) prescribed level and t and e vectors’ variation. eaf waste gases powder slag noise ea f food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 44 figure 11 presents the main cpm’s results. a) c) d) b) figure 11. the main cpm’s results food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 45 the components of two vectors t and e which are considered to have important weight in the criteria function’s evaluation are:  t1 – the steels chemical composition;  t2 – the steels purity (in gases);  t3 – the steels purity (inclusions );  e1 – the specific consumption of basic material and materials;  e2 – the specific consumption of energy;  e3 – the elaboration process’s productivity in eaf. the best level (no) for each component of the 2 vectors is: for t1 – the prescribed variation limits of the elaborated steel quality composition arithmetical mean. the cumulated correlation between the criteria function’s (c.f.) prescribed levels and t and e vectors’ variation are presented in figure 10. for e1 the minimum content specific consumption prescribed of basic materials for e2 the minimum prescribed specific energy consumption. for e3 the maximum prescribed productivity of the elaboration process. we can notice the obtaining of:  the criteria function’s maximum level fot,max = 43,76 for the t vector’s variation (t1 component the prescribed variation limits of the elaborated steel quality composition arithmetical mean).  the criteria function’s maximum level foe,max = 55,31 for the e vectors’ variation (e3 component the maximum prescribed productivity of the elaboration process). and respective the criteria function’s maximum level focum,max = 19,85 for the t and e vectors’ cumulated variation. 4. conclusions from the total polluting emissions of eaf, over 90% are generated during the technological operations of melting and refining. the gaseous phase (burnt gases) that comes out of the eaf mainly results from the melting and refining procedures and contains carbon monoxide, carbon dioxide together with nitrogen and sulfur oxides (nox and sox); however, in practice it also contains very toxic other components, such as fluorides or volatile organic compounds (dioxine, chloride derivatives of benzene or phenol) resulted from burning of organic oils that are introduced as contaminants together with the raw materials. fuzzy logic has been found to be very suitable for embedded control applications. every application can potentially realize some of the benefits of fl: performance, productivity, simplicity and lower cost. fl algorithms are implemented as an inference engine which can automatically infer from facts. the application of the fuzzy logic is based on three steps: fuzzification; aggregation and defuzzification. neuro-fuzzy modeling is to use neural networks and fuzzy set theory to model practical systems. neuro-fuzzy technology was developed that can automatically extract business rules from the neuro-fuzzy model. mathematically, the fuzzy utility function is a more accurate measure on the consumption utility. the levels of prescribing the criteria function could be obtained by using a composition algorithm for three vectors: • vector – technical parameters’ vector (ti); • vector – economical parameters’ vector (ej); • vector – weight vector (p1). the execution of the eaf’s charge preheating modelling (cpm) was made both for a 10t eaf and for a 50t eaf. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 adrian ioana, augustin semenescu, cezar florin preda, eaf environmental impact analisys through mathematical modeling and fuzzy logic, food and environment safety, volume xii, issue 1 – 2013, pag. 38 – 46 46 5. references [1]. a., ioana, the electric arc furnaces (eaf) functional and technological performances with the preheating of the load and powder blowing optimisation for the high quality steel processing, ph d thesis, politehnica university of bucharest, 1998, pp. 21-187. [2]. a., ioana, ecological aspects and solutions in eaf’s line of work, the danube river environment & education, tulcea, nr. 3, 2005, pp. 17-20. [3]. a., ioana, technical – economical analysis options for the quality of the steel elaborated in the eaf, int. conf. imt, oradea, proc., fasc. management and technol. eng., vol. v (xv), 2006, pp. 132-135. [4]. a., ioana, environment & eaf, proc. 5th iasme / wseas int. conf. on heat transfer,thermal engineering and environment hte’07, august 25-27, vouliagmeni, wseas press, athens, 2007, pp. 51-54. [5]. a., ioana, managementul producţiei în industria materialelor metalice. teorie şi aplicaţii., editura printech bucureşti, isbn 978-973-7581232, 2007, pp. 17-232. [6]. n., mardani, modeling the impact of environmental variables on type and density of landccover, carpathian journal of earth and environmental sciences, february 2012, vol. 7, no. 1, 2012, pp. 137 – 148. [7]. a., ioana, c., bălescu, management elements of the materials costs, isi book of the „3rd international conference on management, marketing and finances”, [isi-web of science/science citation index expanded], huston, usa, 2009, pp. 71-75. [8]. a., ioana, c., bălescu, optimization of steel elaboration in eaf through mathematical modelling, junior euromat conf., proc., lausanne, 48 sept., 2006, pp. 85-87. [9]. a., ioana, v., mirea, c., bălescu, economic processes study through fuzzy logic, journal of economic computation and economic cybernetics studies and research, no. 2., [issn 0424267x, isi-web of science/science citation index expanded], academy of economic studies publishing, bucharest, 2010, pp. 129-138. [10]. a., ioana, v., mirea, c., bălescu, analysis of service quality management in the materials industry using the bcg matrix method, amfiteatru economic review, vol. xi, nr. 26, [issn 1582-9146, isi-web of science/science citation index expanded], bucureşti, 2009, pp. 270-278. [11]. m., cabeza, m.b., arau, r.j., wilson, c.d., thomas, m.j.r. cowley, a., moilanen, combining probabilities of occurrence with spatial reserve design. ecology, 2004, pp. 41. [12]. m.p., austin, spatial prediction of species distribution an interface between ecological theory and statistical modeling, ecol. model., 2002, pp. 101118. [13]. a., ioana, a., nicolae, optimal working of eaf , ed. fair partners, bucharest, 2002, pp. 51-117. [14]. a., ioana, c., bălescu, sustainable development and risk management, computers and simulation in modern science, vol. v, isi book wseas, [isi-web of science/science citation index expanded], 2011, pp. 35-41. [15]. a., ioana, a., nicolae, c., bălescu, elements of electric arc furnaces’ management, metalurgia international nr. 7/2008, vol. 13, [issn 1582-2214, isi-web of science/science citation index expanded], bucureşti, 2008, pp. 12-15. [16]. gh., boldur–lãţescu, gh., ciobanu, i., bãncilã, analiza sistemelor complexe, editura ştiinţificã şi enciclopedicã, bucureşti, 1982. [17]. b.v., barnes, forest ecology, john wiley and sons, inc., 1998, pp. 610-773. [18]. d., taloi, c., bratu, e., floian, e., berceanu, optimizarea proceselor matalurgice, editura didacticã şi pedagogicã, bucureşti, 1983, pp. 15-127. [19]. d., mãrgulescu, i., vãduva, analiza economico – financiarã asistatã de calculator, editura tehnicã, bucureşti, 1985, pp. 27-83. [20]. r., ristić, environmental impacts in serbian ski resorts, carpathian journal of earth and environmental sciences, vol. 6, no. 2, 2011, pp. 125 – 134. [21]. b.b., salem, application of gis to biodiversity monitoring. journal of arid environments, 54(1), 2003, pp. 91-114. [22]. d.a., sarr, d.e., hibbs, m.n.a., huston, hierarchical perspective of plant diversity, quarterly review of biology.80, 2005, pp. 187-212. [23]. a., ioana, c., bălescu, environmental study of the formation of evacuated burnt gases from a steels making plant, revista de chimie nr.5, [issn 0034-7752, isi-web of science/science citation index expanded], bucureşti, 2009, pp. 468471. [24]. r., dirzo, e., mendoza, biodiversity. encyclopedia of ecology, 2008, pp. 368-377. [25]. a., guisan, n.e., zimmermann, predicting habitat distribution models in ecology. ecological modeling, 2000, pp. 147-186. [26]. k.h., chi, m.b., chang, s.h., chang, measurement of atmospheric pcdd/f and pcb distributions in the vicinity area of waelz plant during different operating stages, science of the total environment 391 (1), 2008, pp. 114-123. [27]. da silva, machado, j.g.m., brehm, f.a., moraes, c.a.m., dos santos, c.a., vilela, characterization study of electric arc furnace dust phases, materials research, 9 (1), 2006, pp. 41-45. [28]. c.a., pickles, thermodynamic analysis of the separation of zinc and lead from electric arc furnace dust by selective reduction with metallic iron, separation and purification technology 59 (2), 2008, pp. 115-128. [29]. f., skov, j., svenning, predicting plant species richness in a managed forest. forest ecology and management, 180(1-3), 2003, pp. 583-593. 214 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 214 – 217 study on the adhesion of lactobacillus plantarum strains with probiotic properties to mdck *rositsa denkova1, hristina strinska1, *zapriana denkova2, georgi dobrev1, daniel todorov3, kirilka mladenova4, stoyan shishkov3 1university of food technology, department of biochemistry and molecular biology, rositsa_denkova@mail.bg 2university of food technology, department of microbiology, zdenkova@abv.bg 3sofia university “st. klimentohridski”, laboratory of virology 4sofia university “st. klimentohridski”, department of biochemistry *corresponding author received august27th 2014, accepted september 15th 2014 abstract: one of the requirements for probiotic strains is to adhere to epithelial cells or cell lines. the presence of s-layer proteins in three lactobacillus plantarum strains with probiotic properties was examined as well as their ability to adhere to the epithelial monolayer model non-cancerous cell line mdck. the three strains lacked s-layer proteins, but lactobacillus plantarum x2 and lactobacillus plantarum lbrz12 adhered to the cells of mdck, while lactobacillus plantarum f3 didn’t. along with their other probiotic properties these make them suitable for inclusion in the composition of probiotics and probiotic foods. keywords: adhesion, lactobacillus, mdck, probiotic, s-layer proteins 1. introduction probiotics are live microorganisms that have beneficial effects on the health of the host, when administered in adequate amounts [1, 2]. due to their proven health beneficial effect the strains most frequently included in the composition of probiotics belong to the genera lactobacillus, bifidobacterium and propionibacterium [3]. probiotic microorganisms contribute to the restoration and maintenance of intestinal balance, play an important role in maintaining the overall health condition of the organism and improve significantly the quality of the foods they are incorporated into [4]. not all strains can be included in probiotic formulas but only those that meet certain requirements [5]. one of these requirements for probiotic strains is to be able to adhere to the intestinal mucosa and cell lines, thereby competitively excluding pathogens [3; 6]. adhesion may be either non-specific, related to specific physicochemical factors, or based on specific adhesion molecules located on the surface of the microbial cells and receptor molecules on the surface of the epithelial cells. adhesion is species specific. lactic acid bacteria possess moderate to good adhesion properties on human cell lines [7]. adhesion of probiotic strains to the surface of the gut is of milestone importance as it is related to the subsequent colonization in the human intestinal tract which in turn determines the longer retention time of probiotic bacteria in the intestinal tract and the realization of their inherent immunomodulatory action. a number of in vitro studies demonstrating adherence of different strains of lactic acid bacteria to human epithelial cells have been conducted [8]. s-layer proteins are a strain-specific trait. surface layer (s-layer) proteins with molecular masses between 40 and 60 kda [9, 10], responsible for their food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceavavolume xiii, issue 3 – 2014 rositsa denkova, hristina strinska, zapriana denkova, georgi dobrev, daniel todorov, kirilka mladenova, stoyan shishkov, study of theadhesion of lactobacillusplantarumstrainswithprobioticpropertiesto mdck,food and environment safety,volume xiii, issue 3 – 2014, pag. 214 217 215 adhesion to epithelial cells or cell lines, are described in lactobacilli [11]. the purpose of the present study was to examine the presence of s-layer proteins and the ability of three strains of lactobacillus plantarum with probiotic properties to adhere to the cells of the epithelial monolayer model non-cancer cell line mdck. 2. materials and methods microorganisms the studies in this work were performed with three strains of lactobacillus plantarum with probiotic properties designated as lactobacillus plantarum x2 and lactobacillus plantarum f3, isolated from spontaneously fermented sourdough, and lactobacillus plantarum lbrz12, isolated from spontaneously fermented vegetables. determination of the adhesion of the lactobacillus plantarum strain to mdck the used non-cancer, kidney cell line mdck (madin-darbey canine kidney) is cultured as a monolayer in a 24-well plate in dmem without antibiotics. the single-strain cultures of each lactobacillus plantarum strain were incubated overnight at 37°c and centrifuged thrice, the biomass sludge was washed with pbs-buffer. 0,5 cm3 of each cell suspension, suspended in pbs-buffer, with concentration of 109cfu/cm3 was resuspended in 1 cm3 dmem and 0,5 cm3 of the mixture were pipetted into the wells with the developed monolayer cell line mdck. after 3 hours of incubation at 37°c the mdck monolayer was washed with 250 μl pbs-buffer. 250 μl of fixating agent (96% ethanol : and acetic acid = 3: 1) were pipetted in each well and the plate was incubated for 20 minutes at room temperature. it was washed once with saline solution. a few drops of gimza stain (diluted in a ratio of 1: 7) as to cover the bottom of the wells were pipetted in each well. after 5 minutes, the dye was removed and the cells were washed twice with 300 μl of saline solution. a light microscope (dundee), at a magnification of 100x, was used for each well of the plates characterization of surface layer proteins by sds-page [9]. untreated bacterial cells were resuspended in 1% sds, incubated for 30 min at 37ºc for the isolation of surface layer proteins, centrifugedfor 5 min at 9000 x g and the supernatant was analyzed by sdspolyacrylamide gel electrophoresis using 10% polyacrylamide gel. the used marker for the electrophoresis was precision plus proteintm standards (biorad, cat. # 161-0373). protein bands were visualized by staining with coomassie blue r-250. 3. results and discussion the presence of surface layer (s-layer) proteins with molecular masses between 40 and 60 kda is described in lactobacilli. the examination of the presence of s-layer proteins showed that the strains lactobacillus plantarum x2, lactobacillus plantarum f3 and lactobacillus plantarum lbrz12 did not possess s-layer proteins (fig. 1). the ability of lactobacillus plantarum x2, lactobacillus plantarum f3 and lactobacillus plantarum lbrz12 to adhere to the cells of the monolayer model non-cancerous epithelial cell line mdck was examined. in the control the cells were arranged in a dense monolayer, their shape and intercellular contacts being clear. the strains lactobacillus plantarum x2 and lactobacillus plantarum lbrz12 adhered to mdck cells, while lactobacillus plantarum f3 didn’t (fig. 2). food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceavavolume xiii, issue 3 – 2014 rositsa denkova, hristina strinska, zapriana denkova, georgi dobrev, daniel todorov, kirilka mladenova, stoyan shishkov, study of theadhesion of lactobacillusplantarumstrainswithprobioticpropertiesto mdck,food and environment safety,volume xiii, issue 3 – 2014, pag. 214 217 216 marker 1. marker 2. l. plantarum f3 3. l. plantarum lbrz12 4. l. plantarum x2 fig. 1. presence of s-layer proteins in lactobacillus plantarum x2, lactobacillus plantarum f3 and lactobacillus plantarum lbrz12 when examining the adhesion of lactobacillus plantarum lbrz12 and lactobacillus plantarum x2 to the cells of mdck there was visible mounting of the cells of the lactobacillus plantarum strains to the cells of the monolayer. furthermore disturbances in the structure, as well as the appearance of individual cell line cells were observed. 4. conclusion the ability of lactobacillus plantarum strains of different origin with probiotic properties to adhere to the epithelial monolayer model non-cancerous cell line mdck was studied. control lactobacillus plantarum x2 lactobacillus plantarum f3 lactobacillus plantarum lbrz12 fig. 2. adhesion of l. plantarum strains to mdck cell line food and environment safety journal of faculty of food engineering, ştefancelmareuniversity – suceavavolume xiii, issue 3 – 2014 rositsa denkova, hristina strinska, zapriana denkova, georgi dobrev, daniel todorov, kirilka mladenova, stoyan shishkov, study of theadhesion of lactobacillusplantarumstrainswithprobioticpropertiesto mdck,food and environment safety,volume xiii, issue 3 – 2014, pag. 214 217 217 the three studied strains did not possess slayer proteins, but lactobacillus plantarum x2 and lactobacillus plantarum lbrz12 adhered to the monolayer model noncancerous cell line mdck, which is a prerequisite for their inclusion in the composition of probiotics and probiotic foods. 5.references [1]. charalampopoulos d, pandiella s, webb c, evaluation of the effect ofmalt, wheat and barley extracts on the viability of potentially probiotic actic acidbacteria under acidic conditions. int. journal of food microbiology 82: 133–141 (2003). [2]. stanton c, ross r, fitzgerald g, van sinderen d, fermented functional foods based on probiotics and their biogenic metabolites. current opinion inbiotechnology 16: 198–203 (2005). [3]. collado m, meriluoto j, salminen s, in vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus. food research international 40:629–636 (2007). [4]. de lacey aml, pérez-santín e, lópez-caballero e, montero p, survival and metabolic activity of probiotic bacteria in green tea. lwt food science and technology 55: 314322 (2014). [5]. salminen s, bouley m, boutronrualt m, cummings j, franck a,gibson g, isolauri e, moreau m-c, roberfroid m, rowland i, functional foodscience and gastrointestinal physiology and function. br. j. nutr., suppl. 1: 147–171 (1998). [6]. salminen s, ouwehand a, benno y, lee y,probiotics: how should they be defined? trends food sci. tecnol. 55:1901-1906 (1999). [7]. ouwehand a, tuomola e, tolko s, salminen s, assessment of adhesion properties of novel probiotic strains to humen intestinal mucus. int. j. of food microbiology 64:119 – 126 (2001). [8]. schillinger u, guidas c, holzapfel w, in vitro adherence and other properties of lactobacilli used in probiotic yoghurtlike products. int. dairy j.15(12):1289-1297 (2005). [9]. kos b, suskovic j, vukovic s, simpraga m, frece j, matosic s, adhesion and aggregation ability of probiotic strain lactobacillus acidophilus m92. journal of applied microbiology 94:981–987 (2003). [10]. van der mei h, van de beltgritter b, pouwels p, martinez b, busscher h, cell surface hydrophobicity is conveyed by s-layer proteins a study in recombinant lactobacilli. colloids surf. b biointerfaces 28:127-134 (2003). [11]. meng j, zhu x, gao s-m, zhang q-x, sun z, lu r-r. characterization of surface layer proteins and its role in probiotic properties of three lactobacillus strains. int. j. of biol. macromolecules 65:110–114 (2014) 144 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 22015, pag. 144147 seasonal variations’ influence on chemical composition of cow milk and vidraru semi hard cheese *ancuţa elena prisacaru1, laura apostol1 1faculty of food engineering, ştefan cel mareuniversity, university street 13, suceava, românia, ancuta.prisacaru@fia.usv.ro *correspondingauthor received april 4th 2015, accepted june 8th 2015 abstract: raw milk is a valuable food whose quality is important regarding both milk consumption and manufacturing of other dairy products. in this study, principles of traditional production and the characteristics of cow vidraru hard cheese are presented. the aim of this study was to investigate the effect of different seasons on the variations of row milk and vidraru cheese composition. the technology process of hard cheese production was described in details. different samples of raw milk and cheese were taken during spring, summer, autumn and winter and their physico-chemical properties were examined. the chemical composition of cow milk is influenced by seasonal feeding. vidraru semi hard cheese is a traditional cheese characterized by ripening at ambient conditions and smoking. keywords: semi hard cheese, cow milk, chemical compostion 1. introduction milk is the ancient food known by humans and it represents a complex mixture of fats, proteins, carbohydrates, minerals, vitamins and other compounds dispersed in water [1]. there are various factors that influence milk chemical composition (fat, proteins, water and mineral substances) within the same species. some of these factors relate to genetic, stage of lactation, feeding, age, animal health and season [2]. production and the quality of milk products, like cheese, are influenced by these factors [3]. there is a negative correlation between the environment temperature and the quantity of proteins and fat in milk. when the temperature is high the fat content tends to decrease. the content of fat, proteins, and casein are influenced by the seasonal variations [4]. the content of fat and protein is higher in the autumn and winter and smaller in spring and summer [5]. high environment temperature negative influence the fabrication yield and cheese properties by rising the clotting time and the rate of clot formation [5]. semi hard cheese is one of the most popular cheeses with hard paste in many european countries. semi hard cheese production is dating since eleventh century. although the historical references suggest that this dairy product has an older tradition [6]. this type of cheese is different from others because milk utilization and the scalding of the obtained curd provide specific properties regarding its structure and flavor. the aim of this study was the analysis of the chemical composition variation of milk, raw material at semi hard cheese fabrication and its influence on the final product. http://www.fia.usv.ro/fiajournal mailto:ancuta.prisacaru@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 2 – 2015 ancuţa elena prisacaru, laura apostol, seasonal variations’ influence on chemical composition of cow milk and vidraru semi hard cheese, food and environment safety, volume xiv, issue 2 – 2015, pag. 144 – 147 145 2. matherials and methods milk samples were collected from the dorna lactate dairy products factory, working point târguneamţ. this unity is supplied with raw material from the local farmers from suceava, neamţ, botoşani and iaşi counties from the 126 centers of collection. the research was conducted during an entire year, monthly being analyzed 30 milk and 10 hard cheese samples. milk samples were collected direct from the cooling tanks in sterile recipients of 200 ml, stored at +8° c and immediately transported to the analysis laboratory. total fat was determined according to the romanian standards using a milk analyzer (ekomilk) and dry matter by gravimetry. titratable acidity and specific gravity of milk samples were determined as described by kurt et al (2003) [7]. the chemical composition of hard cheesecheese was determined according to reference standards: dry matter (sr en iso 5534:2004), salt (mohr’s method, santa and srbinovska, 2014), and fat (sr en iso 1735:2005) [8, 9, 10]. milk and cheese samples were analyzed during the fourth seasons: spring, summer, autumn and winter. 3. results and discussion cheesemaking process the production process of vidraru semi hard cheese is subdivided into several steps as shown in a table 1. table 1. production process of vidraru semi hard cheese step description 1 raw cow’s milk 2 normalization (3,8-3,9 % fat) 3 clotting (32-34° c, 40 minutes) 4 coagulum machining 5 way partial draining 6 second heating (41-43° c, 14-20 minutes) 7 curd verification 8 progressive pressing of the curd (20-30 minutes) 9 curd pieces formation 10 curd ripening (26-28° c, 8-10 hours) 11 cutting the curd in slices (0,3-0,4 cm thickness) 12 curd scalding (74-75° c, 50-60 seconds) 13 semi hard cheese formation 14 semi hard cheese ripening (4-5 days, 14-18° c) 15 smoking (2-4 hours) 16 packing raw cow milk is first filtered, normalized to a fat percent of 3,8-3,9%, heated at 3234° c, renneted wit rennet and left to coagulate within 40 minutes. curd is stirred at the surface, cut, chopped and left standing for about 5 to 10 minutes. about 10% of the whey is drained. the next step is the second heating at 43-45° c for 15-20 minutes. after the heating the fresh curd is pressed for 20-30 minutes with a force of 15-25 kgf/kg. afterwards the curd is cut into pieces of 4-6 kg. the process of ripening starts and lasts for approximately 10 hours at 26-28° c. then the curd is sliced into very thick pieces (0.3-0.4 cm) that are immersed in hot brine (10-12% nacl, 74-75° c) for about 50-60 seconds. the sliced curd is gently stirred in order to achieve plastic and workable consistency. the next step is semi hard cheese formation by casting the heated curd into round forms. the ripening of the semi hard cheese takes about 4 to 5 days at 14-18° c. the next step is semi hard cheese smoking with smoke from hard essence wood for 34 days at 20-25° c. after this the smoked semi hard cheese is ripened one more time at at 16-18° c for 10-12 days. the vidraru food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 2 – 2015 ancuţa elena prisacaru, laura apostol, seasonal variations’ influence on chemical composition of cow milk and vidraru semi hard cheese, food and environment safety, volume xiv, issue 2 – 2015, pag. 144 – 147 146 semi hard cheese is usually packed in vacuum polymer foil. physico-chemical parameters of cow milk used for the production of vidraru semi hard cheese the physic-chemical parameters of raw cow milk used for vidraru semi hard cheese production are presented in table 2. table 2 physico-chemical parameters of cow milk used for the production of vidraru semi hard cheese parameter sample number season spring summer autumn winter fat (%) 30 3.59±0.02 3.45±0.08 3.64±0.04 3.78±0.03 dry matter (%) 30 8.39±0.17 8.25±0.24 8.32±0.15 8.54±0.12 acidity (° t) 30 15.7±0.36 17.0±0.27 17.2±0.48 16.8±0.36 density (g/cm3) 30 1.0279±0.004 1.0282±0.005 1.0279±0.005 1.0276±0.004 the quality and quantity of milk products such as semi hard cheese is highly dependent on the quantity and quality of fat contained in the original milk. the average fat content of cow milk was 3.59±0.02% for spring and 3.64±0.04% in the autumn. the total fat percent between winter and summer season has been shown differences. the mean fat content of milk was 3.45±0.08% during warm months and 3.78±0.03% in winter. similar results were obtained by sevi a. et al (2001). the average content of dry matter in summer was 8.25±0.24%. the percentage of milk dry matter decreased from winter to summer gradually. the titratable acidity (° t) had higher values in summer (17.0±0.27° t) and autumn (17.2±0.48° t) than winter (16.8±0.36° t) and spring (15.7±0.36° t). the average density of cow milk was about 1.027 g/cm3 in spring, autumn and winter and a bit higher in summer 1.0282 g/cm3. table 2 shows the chemical composition of cow’s milk is affected by seasonal feeding in winter, spring, summer and autumn. the changes in fat content of milk are explained by colombari et al (1999)who correlated milk fat content to the rate of fiber digestion with consequent ruminal synthesis of fat precursors [11]. hattem h. e. et al (2012) mentioned that the increased fat content in milk composition is due to the type of feeding and correlated the highest values for the pre-mentioned constituent with the starting of green feeding [12, 13]. physico-chemical parameters of vidraru semi hard cheese table 3 shows the chemical composition of vidraru semi hard cheese as influenced by season. moisture content was affected by the seasonal variations and the recorded values were higher in summer (44.026±0.31%) as compared to autumn and winter time (42.952±0.39%; 42.848±0.42%). table 3 physico-chemical parameters of vidraru semi hard cheese parameter sample number season spring summer autumn winter moisture (%) 30 43.076±0.24 44.026±0.31 42.952±0.39 42.848±0.42 fat (%) 30 28.661±0.95 27.433±0.91 28.853±0.82 28.901±0.74 % fat/dry matter 30 50.35±0.31 49.012±0.29 50.578±0.38 50.57±0.58 nacl (%) 30 1.96±0.02 2.24±0.29 1.988±0.05 2.26±0.58 concerning fat/dry matter content, the recorded average values ± standard deviation of vidraru semi hard cheese were the highest in the autumn and winter food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 2 – 2015 ancuţa elena prisacaru, laura apostol, seasonal variations’ influence on chemical composition of cow milk and vidraru semi hard cheese, food and environment safety, volume xiv, issue 2 – 2015, pag. 144 – 147 147 season (50.78±0.38%; 50.57±0.58%), thought to be due to the different feedings. the salt content was higher in summer period (2.24±0.29%) and winter (2.26±0.58%) as compared to spring (1.96±0.02%) and autumn (1.988±0.05%). regarding the influence of milk quality on the cheese quality, based on the obtained data we can mention that the highest content of fat in milk determined in winter season was also found in the final product, vidraru semi hard cheese respectively. 4. conclusion vidraru semi hard cheese is made from cow milk in a special way characterized by ripening at ambient conditions and smoking which results into a product of an exceptional taste. the results of this study showed that the seasonal changes had an impact on milk and cheese composition. as the season progressed, milk fat decreased from winter to summer. fat recoveries in the cheese are not very different over the season. seasonal variations result in varied composition of milk predominantly due to animal feeding. 5. acknowledgments the author is very grateful to the anonymous referees for their valuable comments. 6. references [1]. ozrenk e., inci o. s., the effect of seasonal variations on the chemical composition of cow milk in van province. pak. j. nutr., 7(1): 161164, (2008) [2]. haenlein g. f. w., goat milk in human nutrition. small ruminant research, 51(2): 155163, (2004) [3]. lindmark-mansson h. u., svensson m., paulsson g., alden b., johnson f., johnson g., influence of milk components, somatic cells and supplemental zinc on milk process ability.int. dairy j., 10: 423-433, (2000) [4]. ng-kwai-hang k. f., hayes j. f., moxley j. e., monardes h. g., variability of test-day milk production and composition and relation of somatic cells counts with yield and compositional changes of bovine milk.j. dairy sci., 67: 361-366, (1984) [5]. sevi a., annicchiorico g., albenzio m., taibi l., muscio a., dell’aquila s.,effects of solar radiation and feeding time on behavior, immune response and production of lacting ewes under high ambient temperature. j. dairy sci., 84: 629-640, (2001) [6]. kindstedt p., coric m., milanovic s., pasta filate cheese. in: fox p. f., mcsweeney p. l. h., cogan t. m., guinee t. p., cheese: chemistry, physics and microbiology, u.k. elsevier academic press 2, 251-277, (2004) [7]. kurt a., cakmakci s., coglar a., 2003. guide of inspection and analysis methods in milk and milk products, erzurum 284, (2003) [8]. sr en iso 5534:2004 cheese and cheese products: determination of dry matter content. (reference method) [9]. santa d., srbinovska s., traditional production and main characteristics of galichkikashkaval. mljekarstvo. 64(2): 119-126, (2014) [10]. sr en iso 1735:2005 cheese and cheese products: determination of fat content. (reference method) [11]. colombari g., borreani g., crovetto g.m., comparison of lucerne silage and ventilated hay in maize silage-based ratios for dairy cows for the production of milk destined for grana cheese. grass and forage sci, 54: 184-194, (1999) [12]. hattem h. e., taleb a. t., manal a. n., hanaa s. s., effect of pasteurization and season on milk composition and ripening of ras cheese. j. brew. distilling, 3(2): 15-22, (2012) [13]. ropciuc s., research on chemical composition of cow raw milk. j. food and environment safety, xii(4): 311-315, (2013) 327 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 327 334 the evolution of some biochemical indices during chilling of four freshwater fish species camelia-zaraza avrămiuc1, *marcel avrămiuc2 1food industry technical college of suceava, romania 2faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received october 8th 2014, accepted november 15th 2014 abstract: the purpose of this paper was to study the evolution of some biochemical parameters in freshwater fish, subjected to chilling for 72 hours in fresh and after smoking. the biological material was represented by four species of fish (catfish, trout, carp and grayling) from which there were prepared fresh and hot smoked (60 minutes at +70°c) samples, then chilled at +5°c for 72 hours. after 12, 48 and 72 hours of refrigeration the following aspects were determined: acidity index (ai) and peroxide index (pi) of the fat, and amine nitrogen (an). both after 12 and after 72 hours of chilling, the largest percentage increases of biochemical indices, as compared to controls (fresh and smoked samples before refrigeration), were recorded in unsmoked samples. during the 72 hours of refrigeration, in all analyzed cases, the an has recorded the highest growth, followed, in order, by ai and pi. the evolution of pi values during chilling of the four freshwater fish species, have shown the freshness maintenance of fat just in catfish and carp, both in fresh samples and in smoked ones. regarding an of the four analyzed fish species, the values obtained have indicated a relative freshness of meat after 72 hours (catfish and carp) or even after 48 hours of refrigeration (trout and grayling). keywords: fish, chilling, smoking, acidity index, peroxide index, amine nitrogen. 1. introduction the chilling and freezing are conservation methods using low temperatures, which seeks to prevent or limit the modification of nutritional and sensory qualities of raw materials and foodstuffs. regarding fishes, the deterioration of quality of both wild and farmed fish species is mainly due to action of intrinsic enzymes and microbes [1,2,3]. according to pigott and tucker (1990), in order to extend the keeping quality of fish it should lower their body temperature. the freezethaw accelerated protein and lipid oxidation, changed the structure of the myofibrillar protein, caused muscle discoloration, and led to the loss of myofibrillar protein function [5,6]. according to kong et al. (2013), due to their high levels of long-chain polyunsaturated fatty acids, the fish products are very susceptible to oxidation leading to the formation of lipid hydroperoxides and free radicals. by arvanitoyannis and kotsanopoulos (2012), the smoking process has been used for hundreds of years either to preserve and extend the shelf-life of foods or to give them specific organoleptic characters (taste, flavour). in recent decades, smoking was widely applied because of the inactivation effect food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 328 of smoke (and thermal processing) on harmful enzymatic compounds and microorganisms [9]. in this paper it has investigated the evolution of biochemical indices (acidity and peroxide indices of fats and amino nitrogen) in four species of freshwater fish, subjected to chilling for 72 hours in fresh and after smoking. 2. experimental the biological material was represented by four species of fish caught in streams in romania: catfish (silurus glanis l.), trout (salmo trutta l.), carp (cyprinus carpio l.) and grayling (thymallus thymallus l.) with weights ranging from 0.600 kg (trout) and 1.6 kg (catfish). fishes were brought fresh (in containers of water) at laboratory for analysis, where they were slaughtered, and after evisceration fresh samples were prepared, which were refrigerated (at + 5°c) for 72 hours. in parallel, form the same biological material were prepared samples subjected to hot smoking process (60 minutes at +70°c), and then were chilled under the same conditions as the fresh ones (not smoked). all samples were placed in individual sealed plastic bags and then refrigerated. from fish fat (extracted by pressing) were evaluated peroxide index (pi) and acidity index (ai), and from meat amino nitrogen (an). fresh and smoked samples were analyzed both before and during the chilling, after 12, 48 and 72 hours. the ph values were determined with a digital ph-meter type hanna. the peroxide index pi (g iodine/100g fat) was determined by titration with na2s2o3 0.01 n solution of iodine released from ki by peroxides formed in oxidized fat [10,11]. the assessment of the degree of impaired fat oxidation is as follows: pi<0.03 is considered fresh fat; ip=0.03 to 0.06 fat is fresh, but not recommended for storage; ip=0.06 to 0.1 fat is questionable freshness, and the ip>0.1 fat is altered. the determination of acidity index ai (mg koh/g) was made through a titration method, based on measurement of volume of koh 0,1 n solution, which neutralizes free fatty acids from one gram of fat [10]. the amine (trimethylamine) nitrogen an (mg%) was determined by the difference between the nitrogen content of volatile bases and the nitrogen content of the ammonia, and primary amines [10]. after castell and triggs (cited by beschea and toma [10]), the admissibility limits of fish depending on the content of trimethylamine are: 0-1 mg/100 g (fresh fish); 1-5 mg/100 g of product (relative freshness of fish); above 5 mg/100 g (altered fish). four replicates for each determination represented the data of experiments, which were statistically processed, the analysis of variance being used to calculate differences between results. the differences at p <0.05 were considered significant. 3. results and discussion in the table 1 are reproduced the values of ph, acidity index (ai), peroxide index (pi) and amine nitrogen (an) from fresh and smoked samples, belonging to: catfish, trout, carp and grayling. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 329 table 1 the mean values of biochemical indices of fish species before and after smoking * fresh; ai=acidity index; pi=peroxide index; an=amine nitrogen from table 1 it is observed that, in all four analyzed species, as result of smoking process, ph values, ai, pi and an have registered more or less pronounced increases compared to fresh samples. the table 2 restores the values of ai, pi and an in samples of fresh and smoked fish, which have been kept refrigerated for 72 hours. table 2 the mean values of biochemical indices during chilling of the four fish species fish species status of chilled cooling duration ai (mg koh/g) pi (g iodine/100 g an (mg%) catfish unsmoked 12 2.35±0.01 0.019±0.002 0.50±0.006 48 4.94±0.04 0.027±0.003 0.62±0.003 72 7.40±0.09 0.043±0.001 1.27±0.001 smoked 12 4.25±0.07 0.025±0.003 0.38±0.002 48 5.30±0.08 0.032±0.006 0.42±0.008 72 6.08±0.02 0.048±0.005 0.81±0,005 trout unsmoked 12 5.35±0.01 0.031±0.001 0.88±0.001 48 9.90±0.07 0.043±0.002 1.72±0.003 72 16.64±0.72 0.064±0.007 3.04±0.007 smoked 12 8.82±0.06 0.039±0.003 0.62±0.001 48 10.36±0.04 0.052±0.008 1.14±0.003 72 14.23±0.03 0.073±0.004 1.56±0.009 carp unsmoked 12 4.12±0.04 0.028±0.007 0.63±0.005 48 8.23±0.07 0.027±0.002 0.93±0.002 72 14.68±0.05 0.058±0.005 1.85±0.006 smoked 12 7.15±0.02 0.034±0.009 0.52±0.007 48 10.53±0.07 0.037±0.001 0.78±0.003 72 13.90±0.01 0.056±0.002 1.03±0.002 grayling unsmoked 12 6.02±0.04 0.033±0.008 0.75±0.009 48 8.45±0.02 0.045±0.003 1.35±0.004 72 12.38±0.03 0.061±0.006 2.43±0.005 smoked 12 7.45±0.08 0.043±0.005 0.69±0.003 48 9.67±0.02 0.049±0.001 1.02±0.002 72 11.74±0.05 0.062±0.002 1.23±0.009 ai=acidity index; pi=peroxide index; an=amine nitrogen fish species fish status ph ai (mg koh/g) pi (g iodine/100 g fat) an (mg%) catfish unsmoked* 6.45 2.03±0.04 0.019±0.004 0.22±0.002 smoked 6.62 3.74±0.01 0.022±0.006 0.24±0.005 trout unsmoked* 6.35 3.70±0,09 0.024±0.004 0.42±0.003 smoked 6.53 6.25±0.07 0.032±0.001 0.48±0.008 carp unsmoked* 6.31 3.57±0.08 0.021±0.002 0.25±0.007 smoked 6.54 6.08±0.03 0.028±0.005 0.36±0.001 grayling unsmoked* 6.27 4.35±0.05 0.025±0.001 0.31±0.001 smoked 6.50 6.39±0.01 0.037±0.003 0.38±0.003 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 330 as seen from the table 2, in unsmoked samples of catfish ai has increased, during chilling, with percentages between 15.7% (after 12 hours) and 254.5% (after 72 hours), as compared to fresh sample before chilling (tab. 1). also at chilled catfish, but in smoked samples, ai has increased by 13.6% (after 12 hours) and by 62.6% (after 72 hours), compared to smoked samples before chilling (tab. 1) (p<0.05). in unsmoked catfish samples pi has increased during chilling with percentages between 0% (after 12 hours) and 126.3% (after 72 hours), compared to fresh samples prior to chilling. in smoked catfish sample ip increased, during chilling, by 13.6% (after 12 hours) and by 118.2% (after 72 hours), as compared to smoked sample before chilling (p<0.05). pi values of catfish shows that, during chilling, the fat of this fish maintained this state of freshness, both in smoked and fresh samples. also in catfish (tab. 2), the amine nitrogen values (an) in unsmoked samples have increased, during the refrigeration, with percentages between 127.3% (after 12 hours) and 577.3% (after 72 hours) as compared to fresh samples before chilling. in smoked and chilled samples, an has increased by 58.3% (after 12 hours) and by 237.5% (after 72 hours), as compared to smoked samples before chilling (p<0.05). after 72 hours of refrigeration, an values have indicated in fresh samples (unsmoked) a fish (catfish) with relative freshness. in unsmoked trout samples (tab. 2), ai has increased, during chilling, with percentages between 44.6% (after 12 hours) and 349.7% (after 72 hours), as compared to fresh sample before chilling (tab. 1). also in chilled trout, but in smoked samples, ai has increased by 41.1% (after 12 hours) and by 127.7% (after 72 hours), compared to smoked samples before chilling (tab. 1) (p<0.05). in unsmoked trout samples pi has increased, during chilling, with percentages between 29.2% (after 12 hours) and 166.7% (after 72 hours), compared to fresh samples prior to chilling (tab. 1). in smoked trout samples pi has increased, during chilling, by 21.9% (after 12 hours) and by 128.1% (after 72 hours), as compared to smoked sample before chilling (p<0.05). pi values of trout shows that, after 72 hours of chilling, both fresh samples but especially the smoked ones have shown a dubious freshness fat. also in trout, an values in unsmoked samples have increased, during refrigeration, with percentages between 109.5% (after 12 hours) and 623.8% (after 72 hours), as compared to fresh samples before chilling. in smoked and chilled trout samples, an has increased by 29.2% (after 12 hours) and 225% (after 72 hours) compared to smoked samples before chilling (p<0.05). the extension of refrigeration state over 48 hours has led to higher an values, both in fresh and in smoked samples, indicating, in both cases, a fish with relative freshness. in unsmoked carp samples (tab. 2), ai has increased, during chilling, with percentages between 15.4% (after 12 hours) and 311.2% (after 72 hours), as compared to fresh sample before chilling (tab. 1). also in chilled carp, but in smoked samples, ai has increased by 17.6% (after 12 hours) and by 128.6% (after 72 hours), compared to smoked samples before chilling (tab. 1) (p<0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 331 in unsmoked carp samples pi has increased, during chilling, with percentages between 33.3% (after 12 hours) and 166.7% (after 72 hours), compared to fresh samples prior to chilling (tab. 1). in smoked carp samples pi has increased, during chilling, by 21.4% (after 12 hours) and by 100% (after 72 hours), as compared to smoked sample before chilling (p<0.05). analyzing pi values in carp samples, it is observed that throughout the chilling the fat of this fish has maintained its freshness status, both in fresh and smoked samples also in carp, an values in unsmoked samples have increased, during refrigeration, with percentages between 152% (after 12 hours) and 640% (after 72 hours), as compared to fresh samples before chilling (tab. 1). in smoked and chilled carp samples, an has increased by 44.4% (after 12 hours) and by 186.1% (after 72 hours), compared to smoked samples before chilling (p<0.05). after 72 hours of refrigeration, an values have indicated in fresh samples (unsmoked) a fish with relative freshness. in unsmoked grayling samples (tab. 2), ai has increased, during chilling, with percentages between 38.4% (after 12 hours) and 184.6% (after 72 hours), as compared to fresh sample before chilling (tab. 1). also in chilled grayling, but in smoked samples, ai has increased by 16.6% (after 12 hours) and by 83.7% (after 72 hours), compared to smoked samples before chilling (tab. 1) (p<0.05). in unsmoked grayling samples pi has increased, during chilling, with percentages between 32% (after 12 hours) and 144% (after 72 hours), compared to fresh samples prior to chilling (tab. 1). in smoked grayling samples pi has increased, during chilling, by 16.2% (after 12 hours) and by 67.6% (after 72 hours), as compared to smoked sample before chilling (p<0.05). after 72 hours of refrigeration, pi values have indicated, both in fresh and smoked samples, a fat with dubious freshness. also in grayling, an values in unsmoked samples have increased, during refrigeration, with percentages between 141.9% (after 12 hours) and 683.9% (after 72 hours), as compared to fresh samples before chilling (tab. 1). in smoked and chilled grayling samples, an has increased by 100% (after 12 hours) and by 223.7% (after 72 hours), compared to smoked samples before chilling (p<0.05). 48 hours of refrigeration, in fresh samples (not smoked), and 72 hours, in smoked samples, have made that an values indicate, in both cases, a fish (grayling) with relative freshness. according to horner (1997), besides surface drying and salting process, the food protection is provided by antioxidant compounds and several antimicrobial agents like phenols, nitrites etc. phenolic compounds generated by the combustion combined with the temperature and the conditions of smoking can reduce the microbiological development and the oxidation [13]. kyriazi-papadopoulou et al. (2003) evaluated the qualitative and safety characteristics of mediterranean mussel (mytilus galloprovincialis) during storage at 2–3°c, under vacuum packaging, after washing and steaming (at 80°c, for 10 min), salting (brine 4%, 15 min), draining (60–65°c for 13 min), and smoking (at 65– 80°c for 17 min). under these conditions, the product could be preserved for up to 70 days; the authors found a dropping of ph, an increase of thiobarbituric acid, and a marked increase of total volatile basic nitrogen (tvb-n) values. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 332 searching the effects of hot smoking in combination with marinating and vacuum packaging, on the qualitative characteristics of mackerel (scomber scombrus) stored at 4°c, ozogul and balikci (2011) found several fluctuations of total volatile basic nitrogen (tvb-n) level, throughout storage period, while the level of fatty acids enhanced. smoking in combination with marinating and vacuum packaging preserved the fish quality, the shelf-life of products was 9 months. fig. 1 illustrates, comparatively, the percentage increases of the three biochemical indices, after 12 hours of refrigeration. 0,00% 20,00% 40,00% 60,00% 80,00% 100,00% 120,00% 140,00% 160,00% u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed ai pi an ai pi an ai pi an ai pi an catfish trout carp grayling biochemical indices of fish species in cr ea si ng p er ce nt ag es ( % ) co m pa re d to co nt ro ls ai=acidity index (mg koh/g); pi=peroxide index (g iodine/100 g fat), an=amine nitrogen (mg%) fig. 1. percentage increases of biochemical indices in fish species after 12 hours of refrigeration, compared to controls (fresh samples) from the figure it is observed that, after 12 hours of fish chilling, the largest percentage increases of biochemical indices, compared to controls (fresh and smoked samples before chilling), were recorded in unsmoked samples. in unsmoked samples, one can see that an has recorded the largest increases in carp (152%) and the lowest in trout (109.5%), ai in trout (44.6%) and the lowest in catfish (15.7%) and carp (15.4%), and pi the biggest in carp (33.3%) and the lowest in catfish (0%). in smoked samples, an has recorded the largest increases in grayling (100%) and the lowest in trout (29.2%), ai in trout (41.1%) and the lowest in catfish (13.6%), and pi the higher in trout (21.9%) and lowest in catfish (13.6%). fig. 2 reproduces, comparatively, the percentage increases of the biochemical indices, after 72 hours of refrigeration. after 72 hours of refrigeration of the four fish species, the largest percentage increases of the analyzed biochemical indices, compared to controls (fresh and smoked samples before chilling), were recorded in unsmoked samples too. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 333 0,00% 100,00% 200,00% 300,00% 400,00% 500,00% 600,00% 700,00% 800,00% u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed u ns m ok ed s m ok ed ai pi an ai pi an ai pi an ai pi an catfish trout carp grayling biochemical indices of fish species in cr ea si ng p er ce nt ag es ( % ) co m pa re d to co nt ro ls ai=acidity index (mg koh/g); pi=peroxide index (g iodine/100 g fat), an=amine nitrogen (mg%) fig. 2. percentage increases of biochemical indices in fish species after 72 hours of refrigeration, compared to controls (fresh samples) in unsmoked samples, an has recorded the largest increases in grayling (683.9%) and the lowest in catfish (577.3%), ai in trout (349.7%) and the lowest in grayling (184.6%), and pi the biggest in carp (176.2%) and lowest in catfish (126.3%). in smoked samples, an has recorded the largest increases in catfish (237.5%) and the lowest in carp (186.1%), ai in carp (128.6%) and trout (127.7%), and the lowest in catfish (62.6%), and pi the higher in trout (128.1%) and lowest in grayling (67.6%). 4. conclusions the evolution of the peroxide index (pi), acidity index (ai) and amine nitrogen (an) during chilling (6°c) of four freshwater fish species (catfish, trout, carp and grayling) has shown differences, both between fresh (not smoked) samples and smoked of the same species, and between different species. so after 12 and after 72 hours of refrigeration, the largest percentage increases of biochemical indices, compared to controls (fresh and smoked samples before chilling), were recorded in unsmoked samples thus, after 12 hours of refrigeration of unsmoked samples, an has recorded the highest increases in carp and the lowest in trout; ai in trout and the lowest in catfish and carp, and pi the largest increases in carp and lowest in catfish. at smoked samples, an has recorded the most marked increases in grayling and the smallest in trout; ai in trout, and the smallest in catfish, and pi the largest increases in trout and lowest ones in catfish. after 72 hours of refrigeration of unsmoked samples, an has recorded the largest increases in grayling and the smallest in catfish; ai in trout, and the lowest in grayling, and pi the largest in carp and the lowest in catfish. at smoked samples, an has recorded the highest increases in catfish and the smallest in carp; ai in carp and trout, and the lowest in catfish, and pi the largest increases in trout, and the lowest in grayling. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 camelia-zaraza avrămiuc, marcel avrămiuc, the evolution of some biochemical indices during chilling of four freshwater fish species, volume xiii, issue 4 – 2014, pag. 327 – 334 334 during the 72 hours of refrigeration, in all analyzed cases, an has recorded the highest growth, followed, in order, by ai and pi. the evolution of pi values during chilling of the four freshwater fish species, has shown the freshness maintenance of fat just in catfish and carp, both in fresh samples and in smoked ones. regarding an of the four analyzed fish species, the values obtained have indicated a relative freshness of meat after 72 hours (catfish and carp) or even after 48 hours of refrigeration (trout and grayling). 5. references [1]. pigott g.m., tucker b.w. science opens new horizons for marine lipids in human nutrition. food rev int 3, pp. 105-138 (1987) [2]. hsieh r., kinsella j.e. oxidation of polyunsaturated fatty acids: mechanisms, products and inhibition with emphasis on fish. adv food nutr res 33, pp. 233-341 (1989) [3]. ehta naresh kumar, elavarasan k., reddy manjunatha a., shamasundar b.a. effect of ice storage on the functional properties of proteins from a few species of fresh water fish (indian major carps) with special emphasis on gel forming ability. journal of food science and technology© association of food scientists & technologists (india) 201110.1007/s13197-011-0558y, published online: 12 october 2011 (2011) [4]. pigott g.m., tucker b.w. sea food: effect of technology on nutrition. marcel dekker, new york and basel inc., p. 362 (1990) [5]. xia x.f., kong b.h., liu q., liu j. meat sci. 83 (2), pp. 239-245 (2009) [6]. xia x.f., kong b.h., xiong y.l., ren y.m. meat sci. 85 (3), pp. 481-487 (2010) [7]. kong b., guo y., xia x., liu q., li y., chen h. cryoprotectants reduce protein oxidation and structure deterioration induced by freeze-thaw cycles in common carp (cyprinus carpio) surimi. food biophysics© springer science+business media new york 201310.1007/s11483-012-9281-0, published online: 9 january 2013 [8]. arvanitoyannis i. s., kotsanopoulos k.v. smoking of fish and seafood: history, methods and effects on physical, nutritional and microbiological properties. food bioprocess technol (2012) 5:831-853, doi 10.1007/s11947-011-0690-8, springer science + business media, llc 2011, published online: 23 september 2011 (2012) [9]. šimko p. determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives. journal of chromatography b, 770, pp. 3-18 (2002) [10]. beschea magda, toma gabriela practical textbook of organic chemistry and special biochemistry (fascicule 1 and 2), galaţi, pp. 7075; 101-103 (1984) [11]. sahleanu v., sahleanu e. guidelines for analysis of meat and meat products. university publishing of suceava, pp. 34-37 (1998) [12]. horner w.f.a. fish processing technology (2nd ed.). london: hall gm, blackie academic & professional (1997). [13]. varlet v., prost c., sérot t. volatile aldehydes in smoked fish: analysis methods, occurrence and mechanisms of formation. food chemistry, analytical, nutritional and clinical methods, 105, pp. 1536-1556 (2007) [14]. kyriazi-papadopoulou a., vareltzis k., bloukas j.g., georgakis s. effect of smoking on quality characteristics and shelf-life of mediterranean mussel (mytilus galloprovincialis) meatunder vacuum in chilled storage. italian journal of food science, 15(3), pp. 371-381 (2003) [15]. ozogul y, balikci e. effect of various processing methods on quality of mackerel (scomber scombrus). food and bioprocess technology.doi:10.1007/s11947-011-0641-4 (2011) 265 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 265 270 the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters *silviu-gabriel stroe1, gheorghe gutt1, silvia mironeasa1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania *silvius@fia.us.ro; g.gutt@fia.usv.ro; silviam@fia.usv.ro *corresponding author received july 25th 2013, accepted august 5th 2013 abstract: the aim of this study was to apply statistical techniques to analyze the experimental data obtained from accelerated migration tests of metal ions from different stainless steels grades in simulated environments foods. for this purpose it was used principal component analysis (pca) to the metal ions analyzed in acetic acid solutions (3%, 6% and 9% in double-distilled water) at different working parameters, in order to get an overview of the data obtained and to find which variables are related, and which variables are the most important in distinguishing between samples. for aisi304 stainless steel grade was studied the migration of cr, mn, fe and ni ions and for aisi321 stainless steel grade was studied the migration of cr, mn, fe, ni and ti ions. the correlations between all determined varibles were studied using pearson's coefficients with statistical significance at p<0.05. significant correlations between metal ions released in food simulant solution of 3%, 6% and 9% concentrations for aisi304 and aisi321 were found. this study demonstrated that the principal component analysis (pca) is useful for interpreting data set, providing informations about the relationships between metal ions analyzed release in food simulant solutions from both stainless steel grades, at various working parameters. the results indicate a high cr and mn content comparatively with iron and nickel content released from aisi304 in food simulant 6% concentration. high iron content comparatively with chromium and nickel content was realeased from aisi321 in acid food simulant 3% concentration and a high fe and ni content comparatively with cr and mn content was realeased from same stainless steel grade in food simulant 9% concentration. keywords: acetic acid, metal migration, principal component analysis, correlation matrix 1. introduction although the presence of metal ions in food plays often an essential role in metabolic processes, they can easily exceed toxicity limits, if the choice of metallic material is not done correctly, taking into account the specific conditions of work. current research on food contamination led to an increased awareness of the behavior of metal ions that are released into food during their industrial processing or storage. at the same time, the consumer concerns about food safety have greatly increased. thus, at national and international level, this result has led to strictly impose of the new higher limits on the migration of chemical elements that can contaminate the food raws in the food chain and processed food [1], [2], [3]. aisi304 and aisi321 stainless steel grades are the most common metallic alloys, both in the manufacture of food processing equipment components, as well in the construction of various storage containers. to study the behavior of these materials in food corrosive environments, the tests were performed under accelerated food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 silviu-gabriel stroe, gheorghe gutt, silvia mironeasa, the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3 – 2013, pag. 265 -270 266 conditions and the release of metal ions provides quickly and relevant informations. in these migration tests a great importance has the choice of working parameters, taking into account their influence on the migration process. in order to distinguish between different compounds who migrate into food corrosive environments, various authors have used different statistical techniques [4], [5], [6]. mathematical modeling was performed to assess the concentration of metallic elements migrated in acetic acid solution of various concentration as function of working parameters [7]. the aim of this study was to apply of the principal component analysis (pca) to some metal ions that migrated in acetic solution at 3%, 6% and 9% concentration at diferent working parameters, to get an overview of the data obtained and to find which variables are related, and which variables are most important in distinguishing between samples. 2. materials and methods the present study was carried out for 27 samples of each grades of stainless steel, aisi304 and aisi321. as corrosive environments were used solutions of 3%, 6% and 9% acetic acid in double-distilled water, which it is recommended as a simulant solution in the migration testing of metal alloys in contact with acidic foods [8], [9]. in order to prevent the contamination of corrosive environment with foreign compounds from the surface of metal samples, they were washed with a solution of detergent at 40°c, rinsed in double-distilled water and then subjected to ultrasonic at a temperature of 45°c in the ultrasonic bath elma d-78224 for 15 min. the samples were then dried in oven at a temperature of 40°c. for aisi304 stainless steel was studied the migrations in the solution of cr, mn, fe and ni ions and for aisi321 stainless steel was studied the cr, mn, fe, ni and ti ions. in the immersion tests the following working parameters were used: the testing temperature ranged from 22 to 34ºc, the stiring environment varied from 0 to 250 rot/min and the exposure time ranged from 30 to 90 min. the concentrations of metal ions in solution were analyzed by inductively coupled plasma mass spectrometry (icp-ms). the concentrations of metal ions obtained by icp-ms were used in multivariate analysis. the interdependence among all determined varibles metal ions release in food simulant solutions of 3%, 6% and 9% concentration from aisi304 stainless steel grade, respectively aisi321 grade at various working parameters – were studied using pearson’s coefficient with statistical significance at p  0,05 and principal component analysis (pca). pca is a multivariate analysis technique that can be applied to original data to reduce the number of variables by preserving as much as possible the variance of the original data. this technique transforms the variables under investigation into new variables, namely principal components (pcs) minimizing the correlation among the original variables. the new variables are arranged in decreasing order of importance, are determined and expressed as linear combination of the original variables: the first pc has maximum variance, while the second principal component has a variance as high as possible but less than that of the first principal component. it will retain the pcs that have an eigenvalue > 1 because they bring a lot more information than the initial variables [10]. the variables were compared and interpreted through two dimensional bi-plots that show the position of determined variables and identify how the variables relate to both the pcs and other input. the elements of pcs can be food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 silviu-gabriel stroe, gheorghe gutt, silvia mironeasa, the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3 – 2013, pag. 265 -270 267 divided into fewer groups, underlining the differences between the variables and similarities between them. pearson’s correlation analysis and pca were carried out using spss v.16.0 software. 3. results and discussion minimum and maximum values, mean and standard deviation for each of metal ions of chromium, manganese, iron and nickel which migrated from stainless steel samples aisi304 and for each of metal ions of titanium, chromium, manganese, iron and nickel which migrated from aisi321 stainless steel samples in the acid food simulant solution of 3%, 6% and 9% concentration are showed in table 1 and 2. correlation coefficients between ions released in simulant solution of 3%, 6% and 9% concentration from aisi304 are presented in table 3. table 1. specific values of metal ions which released from the aisi304 stainless steel samples in 3%, 6% and 9% acetic acid solutions metal ions released in solutions * notations used for metal ion released in the solutions 3%, 6% and 9% acetic acid calculated value [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd chromium cr_3 4.00 238.00 33.51 58.23 cr_6 5.00 42.00 18.22 8.45 cr_9 1.00 36.00 6.33 7.01 manganese mn_3 1.80 43.00 8.77 11.15 mn_6 1.47 15.67 4.45 4.07 mn_9 0.72 9.72 2.03 1.91 iron fe_3 100.00 4450.00 865.18 1159.47 fe_6 100.00 610.00 304.07 149.31 fe_9 10.00 1140.00 178.88 254.30 nickel ni_3 6.80 237.20 38.59 50.96 ni_6 121.00 561.00 326.77 118.67 ni_9 3.30 16.80 7.53 2.85 * cr_3, mn_3, fe_3, ni_3 – metal ions of chromium, manganese, iron and nickel which release in 3% acetic acid; cr_6, mn_6, fe_6, ni_6 – metal ions of chromi um, manganese, iron and nickel which release in 6% acetic acid; cr_9, mn_9, fe_9, ni_9 – metal ions of chromium, manganese, iron and nickel which release in 9% acetic acid; table 2. specific values of metal ions which released from the aisi321 stainless steel samples in 3%, 6% and 9% acetic acid solutions metal ions released in solutions * notations used for metal ion released in the solutions 3%, 6% and 9% acetic acid calculated value [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd titanium ti_3 0.40 3.50 167 0.99 ti_6 0.58 8.28 2.24 1.70 ti_9 0.68 2.28 1.33 0.44 chromium cr_3 5.00 36.00 13.03 7.31 cr_6 1.00 22.00 9.77 5.69 cr_9 0.00 49.00 13.59 12.42 manganese mn_3 3.77 16.57 7.47 3.20 mn_6 2.93 19.73 6.42 3.37 mn_9 2.49 8.99 5.36 2.15 iron fe_3 140.00 1370.00 579.62 272.40 fe_6 110.00 760.00 337.77 155.20 fe_9 140.00 710.00 452.96 165.66 nickel ni_3 13.30 58.30 32.00 11.54 ni_6 306.00 806.00 536.37 162.25 ni_9 7.70 77.70 27.73 18.09 * ti_3, cr_3, mn_3, fe_3, ni_3 – metal ions of titanium, chromi um, manganese, iron and nickel which release in 3% acetic acid; ti_6, cr_6, mn_6, fe_6, ni_6 – metal ions of titanium, chromium, manganese, iron and nickel which release in 6% acetic acid; ti_9, cr_9, mn_9, fe_9, ni_9 – metal ions of titanium, chromium, manganese, iron and nickel which release in 9% acetic acid. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 silviu-gabriel stroe, gheorghe gutt, silvia mironeasa, the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3 – 2013, pag. 265 -270 268 table 3. pearson correlation matrix of ions released in 3%, 6% and 9% acetic acid solution from aisi304 stainless steel grade cr_3 cr_6 cr_9 mn_3 mn_6 mn_9 fe_3 fe_6 fe_9 ni_3 ni_6 ni_9 cr_3 1.000 cr_6 0.259 1.000 cr_9 0.219 0.787a 1.000 mn_3 0.984a 0.290 0.263 1.000 mn_6 0.082 0.638a 0.588a 0.128 1.000 mn_9 0.274 0.791a 0.941a 0.325 0.664a 1.000 fe_3 0.855a 0.336 0.437 b 0.911a 0.220 0.506a 1.000 fe_6 0.483b 0.648a 0.387b 0.492a 0.300 0.454b 0.467b 1.000 fe_9 0.331 0.819a 0.930a 0.391b 0.660a 0.971a 0.573a 0.469b 1.000 ni_3 0.977a 0.299 0.262 0.959a 0.092 0.298 0.799a 0.480b 0.329 1.000 ni_6 0.011 -0.285 -0.306 -0.007 -0.412b -0.377 -0.022 0.014 -0.425b 0.055 1.000 ni_9 0.126 0.864a 0.876a 0.175 0.527a 0.810a 0.308 0.401b 0.830a 0.169 -0.324 1.000 bold values represent correlation with significance a significant correlations at a 0.01 level b significant correlations at a 0.05 level among the ions released from stainless steel samples grade aisi 304 in the acidic solution of concentration 3%, table 3, the ions of manganese, iron and nickel showed significant positive correlations, at a level of significance p = 0.01, with chromium ions. significant correlations were obtained at a level p = 0.01 between the manganese, iron and chromium ions that were released in solution of 6% concentration and between manganese, iron, nickel and chromium ions that were released in the solution of 9% concentration was found. the interdependence among metalic ions migrated from stainless steel grade aisi304 in acid simulant solution of 3%, 6% and 9% concentration are represented in figure 1. the first two pcs represent 98.13 % of the total variance (pc1 = 94.76% and pc2 = 3.37%). the plot of pc1 vs. pc2 loadings shows, along the pc1 axis, a close association between ions of manganese and chromium which were released in the acidic solution of concentration 6% and ions of manganese, chromium, iron and nickel which were released in acetic solution of concentration of 9%, variables strongly associated with the specific axis. the correlations between this varaibles is positive, so as shown by their position in the same quadrant. the second principal component pc2 show a strong association between the metal ions of chromium, manganese, nickel and iron which migrated in the acidic solution of concentration 3%, variables perfectly differentiated from the rest, since they have lower values on the first component. figure 1. pca loadings plot for ions released in the 3%, 6% and 9% acetic acid solutions from aisi304. symbols and definition: о ions released in solution of concentration 3%; ions released in solution of concentration 6%; δ ions released in solution of concentration 9%. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 silviu-gabriel stroe, gheorghe gutt, silvia mironeasa, the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3 – 2013, pag. 265 -270 269 also, pc2 underlines the opposition between the nickel, chromium and manganese ions which migrated in the 3% acetic solution and same metal ions which migrated in the 9% acetic solution. chromium and manganese metal ions were more predominantly released in 6 % acetic acid comparatively with some metals like iron and nickel. from the correlation matrix of metal ions that migrate from aisi321 stainless steel grade in acid simulant solution of 3%, 6% and 9% concentrations, table 4, it can be noted that was obtained significant positive correlations at a significance level of 0.01 between the manganese, nickel and titanium ions that migrate in acid food simulant of 3% concentration. table 4. pearson correlation matrix of ions released in 3%, 6% and 9% acetic acid solution from aisi321 stainless steel grade ti_3 ti_6 ti_9 cr_3 cr_6 cr_9 mn_3 mn_6 mn_9 fe_3 fe_6 fe_9 ni_3 ni_6 ni_9 ti_3 1.000 ti_6 0.254 1.000 ti_9 0.362 0.720a 1.000 cr_3 0.030 0.718a 0.780a 1.000 cr_6 0.420b 0.663a 0.911a 0.672a 1.000 cr_9 0.066 0.703a 0.808a 0.789a 0.727a 1.000 mn_3 0.707a 0.471b 0.651a 0.299 0.601a 0.485b 1.000 mn_6 0.263 0.901a 0.788a 0.792a 0.681a 0.825a 0.568a 1.000 mn_9 0.098 0.362 0.638a 0.563a 0.618a 0.702a 0.526a 0.564a 1.000 fe_3 0.475b 0.543a 0.699a 0.464b 0.601a 0.593a 0.769a 0.582a 0.489a 1.000 fe_6 0.338 0.829a 0.939a 0.823a 0.856a 0.824a 0.604a 0.876a 0.614a 0.658a 1.000 fe_9 0.184 0.506a 0.863a 0.733a 0.705a 0.660a 0.443b 0.596a 0.476b 0.592a 0.794a 1.000 ni_3 0.571a 0.770a 0.837a 0.638a 0.786a 0.649a 0.757a 0.809a 0.604a 0.686a 0.853a 0.602a 1.000 ni_6 0.369 0.596a 0.890a 0.726a 0.797a 0.619a 0.546a 0.642a 0.391b 0.601a 0.818a 0.882a 0.744a 1.000 ni_9 0.168 0.831a 0.888a 0.860a 0.832a 0.816a 0.457b 0.829a 0.496a 0.513a 0.884a 0.795a 0.753a 0.832a 1.000 bold values represent correlation with significance a significant correlations at a 0.01 level b significant correlations at a 0.05 level significant correlations exist between the titanium, chromium, manganese, iron and nickel ions which migrate in acid solution of 6% concentration. figure 2. pca loadings plot for ions released in acidic solution of concentration 3%, 6% and 9% from aisi321. symbols and definition: о ions released in solution of concentration 3%; ions released in solution of concentration 6%; δ ions released in solution of concentration 9%. for all the ions studied that migrate from aisi321 stainless steel grade in acid food simulant solution of 9% concentration, significant correlations were obtained between chromium, manganese, iron and nickel ions. the relationship between the metal ions released from the aisi321 stainless steel grade samples in 3%, 6% and 9% acid food simulant solution are shown in figure 2. the first two principal components represent here 77.86% and 16.51% of the total variance. in regard to principal component pc1, we can notice a strong association between some metal ions which migrated from aisi321 stainless steel grade in acid food simulant solution of 6% and 9% concentration. these variables are closely associated with the first principal component. the second principal component is strongly correlated food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 silviu-gabriel stroe, gheorghe gutt, silvia mironeasa, the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3 – 2013, pag. 265 -270 270 to the iron, manganese and titanium metal ions which migrated from aisi321 stainless steel grade in acid food simulant solution of 3% concentration. strong significant correlations (r  0,7) at a level of significance of p  0,01 between titanium, iron and manganese ions was found (table 4). with respect to pc2, this principal component distinguishes between iron and chromium metal ions released in 3% acetic acid solution. iron ion was more predominantly released in 3% acetic acid comparatively with some metals like chromium and nickel. nickel ion was more predominantly released in 9% acetic acid comparatively with some metals like chromium and manganese. 4. conclusion correlation analysis showed that are significant correlations (p  0,5) between some metal ions released in 3%, 6% and 9% solution from both stainless steel grades. the pca method is useful for interpreting data set, providing informations about the relationships between metal ions released in 3%, 6% and 9% solution from aisi304 and aisi321 stainless steels, at various working parameters. a close relationship was found between manganese and chromium ions which were released from aisi304 stainless steel in 6% and 9% solution. the distribution of ions which were released from aisi304 in solution of concentration 3% indicated a high association between ions of nickel, chromium, manganese and iron. regarding the ions released from aisi321 stainless steel grade, a high correlation was obtained between chromium, iron and manganese ions in acetic acid solution of 6% concentration and between nickel and iron ions which were released in 9% solution. 5. references [1]. european parliament and council of the european union. regulation (ec) no 1935/2004 of 27 october 2004 on materials and articles intended to come into contact with food and repealing directive 80/590/eec and 89/109/eec. official journal of the european communities, l338, 4–17, (2004); [2]. grob k., stocker j., colwell r. assurance of compliance within the production chain of food contact materials by good manufacturing practice and documentation-part 1: legal background and general requirements. food control, 20, 476–482, (2009); [3]. grob k., stocker j., colwell r., assurance of compliance within the production chain of food contact materials by good manufacturing practice and documentation – part 2: implementation by the compliance box; call for guidelines, food control, 19, 483–490, (2009); [4]. ubuoh e. a., analysis of metal concentrations in selected canned beers consumed in owerri urban, imo state, nigeria, international journal of chemistry and material science vol. 1(5), pp. 090095, issn 2327-5553, academie research journals, (2013); [5] roots o., zitko v., principal component analysis of polychlorinated biphenyls and polycyclic aromatic hydrocarbons in seals, estonian environmental research centre, 10617 tallinn, estonia fisheries and oceans canada, marine environmental sciences, canada, экологическая химия 2003, 12(1): 57–66, (2002); [6]. bodroža-solarov m. i., latković d. s., berenji j. j., brlek t. i., nićin s. d., vučković j. n., application of principal component analysis in assessment of mineral content of nine cultivars of buckwheat grain, food & feed research, journal of the institute for food technology in novi sad, (2012); [7]. stroe s. g., gutt g., statistical study of the dependence between concentration of metallic elements migrated from stainless steel grade aisi321 and working parameters, food and environment safety, volume 12, issue 2, (2013); [8]. grob k., the future of simulants in compliance testing regarding the migration from food contact materials into food, food control, 19, 263–268, (2008); [9]. herting g., wallinder o., leygraf i. c., corrosion-induced release of chromium and iron from ferritic stainless steel grade aisi 430 in simulated food contact, journal of food engineering 87, 291–300, (2008); [10]. chen k., jiao j., huang j., huang r., multivariate statistical evaluation of trace elements in groundwater in a coastal area in shenzhen, china, environmental pollution 147, 771–780, (2007). 309 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 309 316 improvement of wheat flour dough rheology by αlpha -amylase and protease combination *georgiana gabriela codină1, silvia mironeasa1 1faculty of food engineering, stefan cel mare university, suceava, romania, codina@fia.usv.ro ; silviam@fia.usv.ro *corresponding author received november 5th 2014, accepted december 19th 2014 abstract: the objective of this study was to determine the optimum combination of α-amylase and protease on alveograph wheat flour dough rheological properties. response surface methodology (rsm) was used to relate the enzymes used like independent variables on alveograph rheological parameters maximum pressure (p), extensibility (l), ratio configuration (p/l), index of swelling (g) and deformation energy (w). the wheat flour used in the research as control sample was a strong one with a low α amylase content. each independent variable was tested at five levels: 0.000, 0.020, 0.040, 0.060, 0.080% for α-amylase and 0.000, 0.002, 0.004, 0.006 and 0,008 % for protease respectively. the results showed that the enzyme combinations weakened the dough (by increasing dough extensibility and reducing dough tenacity and deformation energy). among the enzymes used like variables, protease has a more effective effect on weakening the dough as compared to α-amylase. the best results obtained on alveograph rheological parameters were for 0.060% α-amylase and 0.001% protease combination. key words: wheat flour, α-amylase, protease, alveograph, optimization 1. introduction wheat flour contains enzymes like amylases, proteases, lipases, phosphatases and oxidases [1]. from these enzymes the main role corresponds to the amyllolytic and proteolytic enzymes. normally, wheat flour contains enough amounts of -amylase but little or no amylase. -amylase can produce some maltose without the presence of amylase, but the amount produced is relatively small, so -amylase must be added to optimize the amylase activity. the hydrolysis of starch carried out by amylase favor the production of low molecular weight dextrins, degraded by the endogenous -amylase in maltose. during baking, dextrins won’t form gels, will increase the amount of free water from the bread crumb, will conduct to a softer crumb, will reduce the starch ability to retrogradate and will increase the shelf life of the finished backery products [2, 3, 4]. however an activity too intense of amylase may conduct to a sticky mass form by dextrins which will conduct to a stickiness bread crumb with a wet, and unbaked aspect. therefore, in wheat flour dough -amylase activity must be an optimum one, not too moderate and not too intense. proteases may be used to reduce the mixing time, to decrease dough consistency, to reduce gluten strength and to improve the bread flavor [5]. therefore it may be adding in bread making in order to improve dough extensibility, machining properties, gas retention, mixing time and bread sensorial characteristics [6]. it food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 310 should be used only for processing strong gluten flours with high resistance and elasticity and low extensibility [7]. the practice of adding different enzymes in bread technology is a very common one in order to optimize the wheat flour dough rheological properties and therefore to improve the quality characteristics of the finished bakery products. in order to improve dough rheological properties the effect of -amylase and protease enzymes was evaluated on strong wheat flour with a low -amylase activity in a single and in different combinations by using response surface methodology in order to establish an optimized enzyme combination. 2. materials and methods 2.1. basic ingredients the chemical composition of the flour was determined according to the specific romanian standards: moisture content (sr iso 711:1999), wet gluten content (sr 90:2007), gluten deformation index (sr 90:2007), ash content (sr en iso 2171:2010), and the falling number (se en iso 3093:2010). enzymatic agents (commercial names clarase g plus and belpan bi) commercialized by s.c. enzymes & derivates romania was used. the effect of enzymatic agents was evaluated at five levels: 0.000, 0.020, 0.040, 0.060, 0.080% for α-amylase and 0.000, 0.002, 0.004, 0.006 and 0,008 % for protease respectively. clarase g plus is a fungal α-amylase derived from a selected strain of aspergillus oryzae and belpan bi is a standard protease derived from a selected strain of bacillus subtillus purchased from enzymes & derivates (neamt, romania). 2.2. rheological tests the alveograph parameters were determined according to romanian standard sr en iso 27971:2008. each alveograph curve was analyzed for the fallowing parameters: p, the maximum pressure needed to blow the dough bubble, expresses dough resistance; l, length of the curve, expresses dough extensibility; p/l, configuration ratio of the alveograph curve; g, index of swelling; w, baking strength (surface area of the curve) [8, 9]. 2.3. statistical analysis to study the effects of the enzymatic agents on wheat flour dough rheological properties, the two types of this, the most used in the bread making technology namely α-amylase (c_gp) and protease (b_bi) were used as independent variables. levels of enzymes added in wheat flour were selected at a level range from 0.000 to 0.080 % for α-amylase and a level range from 0.000 to 0.008 % for protease, respectively. response surface methodology (rsm) and central composite design (ccd) with two factors and five different levels was generated by the stat ease design expert 7.0.0 software package (trial version). the complete experimental design required 11 experimental runs that consist of 4 factorial points, 3 centre points and 4 axial. the centre point in the design was repeated three times to allow the adequate estimation of the model. the experimental results were analyzed by multiple regression method. the following equation (1) was proposed for each of the response variables: 1212 2 222 2 11122110 xbxbxbxbxbby  (1) where y is the predicted response, b0 a constant, b1, b2 coefficient of linear effects, b11, b22 coefficient of quadratic effects, b12 coefficient of the interaction effects and x1, x2 are the independent variables. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 311 the statistical significance of the coefficients in the regression equations and the quality of the models’ fitness was evaluated by analysis of variance (anova). the fit of each model to the experimental data was given by the lack of fit test, coefficient of determination, r2 and adjusted coefficient of determination, adjusted-r2 and coefficient of variation, cv. 2.4. optimization procedure after the surface-response results, optimization of the mix of enzymes of αamylase and protease in wheat flour was performed using a multiple response method called desirability [10]. the desirability functions involve transformation of each predicted response into an individual desirability function, dn, which includes the desires and researcher’s priorities to build an optimum procedure for each of the independent variables. the value of individual desirability function, dn ranges between 0, for a completely undesired response, and 1, for a fully desired response. for simultaneous optimization of the alveograph rheological parameters: maximum pressure (p), extensibility (l), ratio configuration (p/l), index of swelling (g) and deformation energy (w) were used following modified desired function (2) developed by derringer and suich [11]. dn = 0 if yn  a s n n ab ay d          if a  yn  b (2) dn = 1 if yn  b where a and b are the constraints of the response, respectively the lowest and the highest values of yn and s is a weighing factor. the individual desirability functions are then combined into a single composite response, namely total desirability function, d (0  d  1) defined as the geometric mean of the individual desirability function dn, which can be expressed as: d = (d1  d2  …  dn)(1/k) (3) where dn, n = 1, 2, … k is the individual desirability function for each response. a high value of d indicates the more desirable and the best combination of enzymes doses, which is considered as the optimal solution of this formulation mix that generated the best results for alveograph rheological characteristics. 3. results and discussion 3.1. analytical characteristics the wheat flour used in this study presents the following chemical characteristics: moisture content 14.30%, wet gluten content 29.10%, gluten deformation index 2.00 mm, ash content 0.65% and falling number 462 s. the alveograph wheat flour rheological properties of the control sample are: maximum pressure (p) 138.00 mm, extensibility (l) 67.00 mm, ratio configuration (p/l) 2.06, swelling index (g) 18.20 mm; deformation energy (w) 342.0010-4 j. according to the chemical and rheological characteristics of the control sample it represents a strong wheat flour quality for bread making with low alphaamylase content. 3.2. effect of process variables on the alveograph rheological characteristics the experimental design consisted of five levels for each of the variables, α-amylase and protease used in this study, and two replications for each experimental condition. a central composite design (ccd) was employed in this study to perform the experiments by varying simultaneously all the variables (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 312 table 1. central composite design: coded and actual values of independent variables experimental design points process variables c_gp (% ) b_bi (%) coded actual coded actual 1 -1 0.020 -1 0.002 2 1 0.060 -1 0.002 3 -1 0.020 1 0.006 4 1 0.060 1 0.006 5 -α 0.000 0 0.004 6 α 0.080 0 0.004 7 0 0.040 α 0.000 8 0 0.040 α 0.008 9* 0 0.040 0 0.004 10* 0 0.040 0 0.004 11* 0 0.040 0 0.004 *centre point repeated 3 times; c_gp α-amylase clarase g plus; b_bi – protease belpan bi the estimated regression coefficients of the models for de response variables, maximum pressure (p), extensibility (l), ratio configuration (p/l), index of swelling (g) and deformation energy (w), are shown in table 2. analysis of variance (anova) for each of the response variables shows that the two factor interaction (fi) models for p, w and p/l ratio, and quadratic models for l and g alveograph parameters adequately represented the data obtained for wheat flour dough rheological properties. table 2. significant coefficients (95% confidence interval) of the design factors of the regression fitting model for alveograph rheological parameters factor p (mm) l (mm) p/l g (mm) w (10-4j) constant 85.18 83.00 1.00 20.30 239.36 c_gp 8.66 13.92 0.24 1.61 ns b_bi 14.60 8.57 0.24 0.99 33.98 c_gp  b_bi ns ns ns ns ns c_gp2 5.06 0.54 b_bi2 ns ns r2 0.95 0.96 0.98 0.97 0.88 adjusted-r2 0.93 0.93 0.97 0.94 0.84 lack of fit ns ns ns ns ns cv % 4.77 4.39 4.96 2.06 5.57 ns no significant effect at the 5% level; p maximum pressure; l – extensibility; p/l ratio configuration; g index of swelling; w deformation energy; c_gp α-amylase, clarase g plus; b_bi – protease, belpan bi; c_gp  b_bi interaction between α-amylase, clarase g plus and protease, belpan bi; c_gp2 quadratic effect of α-amylase, clarase g plus; b_bi2 quadratic effect of protease, belpan bi; r2 coefficient of determination; adj. r2 adjusted coefficient of determination; cv % coefficient of variation. the regression models were highly significant for all alveograph parameters with coefficients of determination (r2) exceeding 85%, which indicates that a high proportion of variability is well explained by the each model. the lack-of-fit tests did not result in a significant f-value, indicating that the models obtained for alveograph parameters are sufficiently accurate for predicting the wheat flour dough rheological properties obtained by blending with these enzymes. the coefficients of variation (cv) for each alveograph rheological parameter show a small value, giving a better reproducibility. the response surfaces of the maximum pressure (p) as functions of the doses of αamylase (c_gp) and protease (b_bi) showed that the α-amylase and protease had a negative effect on p value, while the interaction between this had a nonsignificant effect on the p value. as can be seen in fig. 1a the p parameter decreased with the increased dose of α-amylase and protease, but the effect of interaction food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 313 between α-amylase and protease is nonsignificant (p  0.05) (table 2). positive linear effect of the used enzymes was observed on l (table 2). these effects might be attributed to both enzymes used in combinations due to the hydrolyze effect on the peptide bonds of the protease enzymes and to the intensification of the starch degradation process of the amylase enzymes. the statistical analysis of the coefficients of the model for l parameter revealed that the interaction coefficients were non-significant. it indicated that independent variables individually affected the response variable, l. also, the quadratic effect of protease on the l was insignificant (p  0.05). fig. 1b shows the response and contour plot for effect of αamylase and protease on l parameter. negative linear effect of α-amylase and protease, respectively was observed on p/l alveograph ratio (table 2), while the interaction between these had a nonsignificant effect on the p/l value. the effect of the α-amylase and protease added in wheat flour on p/l ratio is shown in fig. 1c. an increase of α-amylase and protease level in wheat flour conducted to a decrease in the alveograph ratio value p/l. as can be seen in table 2, the effect of quadratic term of protease on index of swelling (g) is insignificant, but the quadratic term of α-amylase has a positive significant influence on g, possibly due to the hydrolyze effect on the starch polymer which will exerts a hydrolytic release of water from the gluten substrate, which reduces the dough viscosity [12]. g was significantly affected (p < 0.05) by the linear terms of α-amylase and protease concentrations added in wheat flour. the interaction term between α-amylase and protease (c_gp  b_bi) was found to be non-significant (p  0.05). increasing concentrations of α-amylase and protease in wheat flour caused an increase in the index of swelling of dough (fig. 1d). as shown in fig. 1e, with the addition of protease (b_bi), the deformation energy of dough significantly decreased. the results of the recent study support previous reports of the caballero et al. [13] showing that protease addition in wheat flour dough decrease alveograph deformation energy. it was observed in table 2 that protease added in wheat flour had significant negative linear effect (p  0.05) on w, while the linear term of α-amylase and the interaction term between α-amylase and protease had a non significant effect (p  0.05). 3.3. optimized solution for the formulation multiple response optimizations were performed to determine the optimum levels of enzymatic agent’s α-amylase (clarase g plus) and protease (belpan bi) to achieve the desired response goals. simultaneous optimization was performed by imposing some constraints for the alveograph parameters according to banu et al. [14] like: the optimal values for maximum pressure (p) in range 80-100 mm and for deformation energy (w) in range from 250 mm to 300 mm, while for extensibility (l) the target value of 100 mm is desirable. the best combinations between these enzymes used in this study in order to obtain optimum values for alveograph rheological parameters were extracted by design expert software that performs from the thousands of iterations and calculation the maximum desirabilit y value and the conditions on which it was arrived. on the basis of these calculations, a total desirability value (d) of 0.811 was obtained for the following concentrations of enzymes used in wheat flour: 0.060% α-amylase and 0.001% protease (table 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 314 a) b) c) d) e) fig. 1. response surface plots showing the combined effects of enzymes α-amylase clarase g plus (c_gp) and protease belpan bi (b_bi) on alveograph rheological parameters food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 315 the response-surface plot corresponding to this d value is represented in fig. 3, where the best combination of enzymes used in this study is obtained at the top of the graph. the coordinates of d value represent the optimum conditions and the corresponding predicted responses for rheological parameters are shown in table 3. table 3. simultaneous optimization of proves variables by desirability approch independent process variables actual levels p (mm) l (mm) p/l g (mm) w (10 -4 j) total desirability value α-amylase (c_cp), (%) 0.060 84.910 94.523 0.905 21.607 250.000 0.811 protease (b_bi), (%) 0.001 c_cp α-amylase clarase g plus; b_bi – protease belpan bi; p maximum pressure; l – extensibility; p/l – alveographic ratio; g index of swelling; w deformation energy. fig. 2. response surface plot of the total desirability function 4. conclusions in the present study, response surface methodology was found to be effective in predicting models for alveograph rheological parameters and to establish a relationship between processing variables α-amylase clarase g plus and protease belpan bi and responses maximum pressure (p), extensibility (l), index of swelling (g), deformation energy (w) and p/l ratio of wheat flour dough rheology. the results showed that, α-amylase and protease have a significant effect on the p, l, g and p/l, while w is especially significantly influenced by protease addition. by using simultaneous optimization of the multi-response method by desirability function in order to obtain an optimum of α-amylase and protease combination we concluded that the best results on the alveograph rheological properties were obtained for a formulation of 0.060% α-amylase and 0.001% protease. 5. references [1]. lindahl l., eliasson a.c., influence of added enzymes on the rheological properties of a wheat flour dough. cereal chemistry 69 (5). 542546, (1992) [2]. defloor i., delcour j.a., impact of maltodextrins and antistaling enzymes on the differential scanning calorimetry staling endotherm of baked bread doughs. journal of agricultural and food chemistry 47. 737–741, (1999) [3]. martin m, hoseney r., a mechanism of bread firmining. role of starch hydrolysis enzymes. cereal chemistry 68. 503-507, (1991) [4]. min b.c., yoon s.h., kim j.w., lee y.w., kim y.b., park k.h., cloning of novel maltooligosaccharide-producing amylases as antistaling agents for bread. journal of agricultural and food chemistry 46. 779-782, (1998) [5]. hassan a.a., mansour e.h., el bedawey a. e. a., zaki m.s., improving dough rheology and cookie quality by protease enzyme. american journal of food science and nutrition research 1 (1). 1-7, (2014) [6]. mathewson p.r., enzymatic activity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 georgiana gabriela codină, silvia mironeasa, improvement of wheat flour dough rheology by αlpha -amylase and protease combination, volume xiii, issue 4 – 2014, pag. 309 – 316 316 during bread baking. cereal foods world 45. 98101, (2000) [7]. david i., mişcă c., the monitoring of enzyme activity of protease on the bread dough. journal of agroalimentary processes and technologies 18 (3). 236-241, (2012) [8]. codină g.g., mironeasa s., mironeasa c., popa c.n., tama berehoiu r., wheat flour dough alveograph characteristics predicted by mixolab regression models, journal of the science of food and agriculture, 92. 638-644, (2012) [9]. codină g.g., mironeasa s., the effect of lecithin on alveograph characteristics, baking and sensorial qualities of wheat flour, food and environment saftey, xii (1). 59-63, (2013) [10]. myers r.h., montgomery d.c., response surface methodology: process and product optimization using designed experiments. john wiley & sons, new york, ny, (1995) [11]. derringer g., suich r., simultaneous optimization of several response variables, journal of quality technology, 12(4), 214–219, (1980) [12]. lewicki pp, water as the determinant of food engineering properties, a review. j food eng, 61. 483–495, (2004) [13]. caballero p.a., gómez m., rosell c.m., bread quality and dough rheology of enzyme-supplemented wheat flour, european food research and technology, 224 (5). 525-534, (2007) [14]. banu i., stoenescu g., ionescu v.s., aprodu i., vasilean i., studies concerning the quality of bread wheat varieties from romania, scientific study & research, x (2). 171-178, (2009) [15]. sr 90:2007. wheat flour. analysis method, standardization association of romania (asro), bucharest, romania [16]. sr en iso 3093:2010. wheat, rye and respective flours, durum wheat and durum wheat semolina determination of the falling number according to hagberg-perten, standardization association of romania (asro), bucharest, romania [17]. sr en iso 2171:2010. cereals, pulses and by-products determination of ash yield by incineration, standardization association of romania (asro), bucharest, romania [18]. sr iso 711:1999. cereals and cereal product. determination of moisture content (basic reference method), standardization association of romania (asro), bucharest, romania [19]. sr en iso 27971:2008. cereals and cereal products common wheat (triticum aestivum l.) determination of alveograph properties of dough at constant hydration from commercial or test flours and test milling methodology, standardization association of romania (asro), bucharest, romania 36 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 36 45 ground water quality of domestic wells in cajvana town * dinu iulian oprea-gancevici1, cătălin savu2 1faculty of history and geography, ștefan cel mare university, universității 13, suceava, romania, dinuo@atlas.usv.ro, 21faculty of history and geography, ștefan cel mare university *corresponding author received february 4th 2016, accepted march 7th 2016 abstract: the purpose of this paper is to analyze the water quality in some wells of cajvana town, uniformly distributed in the built area. the water quality is analysed for the following parameters: nh4+-n, no2--n, no3--n, ppo43and s2-. a hach–lange spectrofotometer was used in order to analyze the chosen parameters of the water samples collected in august 28th, 2015 (the chosen time is random, for expeditionary measurements purposes). the measured values were converted into interpolation maps of the quality classes of each monitored parameter. consistent above average (of the built area) concentrations of ammonium and nitrite near the main graveyard of the settlement caused by the decaying organic matter were measured. nitrite pollution is stronger along cajvana valley. nitrate pollution has values above average in the western half of the town. orthophosphate pollution, caused by domestic wastewaters, has high values in the central part of cajvana. the water pollution by sulfides is almost absent. the worst quality class recorded for each parameter is: very good for ammonium, average for nitrite and mediocre for nitrate and orthophosphate. the increasing standard of living and human pressure on the environment will most probably cause increased groundwater pollution in the near future if a proper sewerage will not be implemented. keywords: nitrites, sulfide, wastewater, graveyard, interpolation. 1. introduction water quality for drinking purposes is a management issue that requires continuous or, at least, repeated measurements of water parameters. in rural areas of romania, water quality in domestic wells is often not suitable for human consumption. septic tanks continue to be a source of groundwater pollution in the developed world [1]. phosphorus and nitrogen based pollutants are a persistent pollution issue of surface waters and groundwaters in both rural and urban environments, from point and diffuse sources and from agricultural to domestic sources [1-4]. the romanian landscape is not an exception [5]. even if cajvana is officially classified in the urban category of settlements, it is, in fact, a larger village due to the lack of public infrastructure meant to improve the life quality, as its landscape proves. numerous villages in suceava plateau were previously investigated concerning the groundwater quality and they have persistent and consistent water pollution with nutrients [6-8]. the aim of this paper is to measure the concentration of some nutrients and sulfides in domestic wells of the studied area and to classify the analysed waters into quality classes. this study was undertaken because there is a lack of case studies that especially analyse the water http://www.fia.usv.ro/fiajournal mailto:dinuo@atlas.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 37 quality in the built areas of various environments. the various studies concerning the water quality in areas that incidentally include our study area do not contain data that specifically refers to the built area. the romanian legislation concerning the water quality follows the eu directives. however, the new european water framework directive insists on eliminating any anthropogenic contamination of the ground waters, no matter the national chemical quality standards. 2. study area cajvana is a small town located in suceava county (fig. 1). the administrative area covers 24.83 km2 populated with 6901 inhabitants, according to the 2011 census. according to this data, the population density was of 277.9 inhabitants/km2, but the official built area covers only 7.52 km2, implying a much greater human footprint, of 917.7 inhabitants/km2 in the area occupied by local people on a daily basis. fig. 1. geographic location of the studied town the local surface geology is represented by volhynian deposits partially covered by quaternary strata. the alternation of clays, marls, sands and sandstones represents the main lithology. there are some fluvial deposits composed of gravel, sands and clays in beldean hill (s). loessic elluvial deposits can be found in the upper area of the local hills. holocene deposits lie along the small rivers that spring in the built area. these rivers belong to 2 catchments: solca catchment in the north, containing crivăț and berbec rivers, and soloneț catchment, which covers the main part of the studied territory and contains cajvana (the main local river) and rușcanul rivers. the homoclinal geological strata were modelled by the hydrographic network into food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 38 cuestas. the town was built on the dip slope of the cuesta between solca and soloneț and a small part extended on the escarpment facing north of the same cuesta. the hillsides with high slopes are covered by landslide bodies. these bodies generate springs from small aquifers. the depth to groundwater level is higher on the hills (>5 m) and smaller in the valleys. the main road crossing the city is dj 178d, which links some communal roads. the impervious surfaces constituted by roads, roofs and other constructed areas are highly dispersed, as can be seen in any aerial/satellite imagery; therefore, no strong surface water drainage is expected, together with its implications in pollutant movement [9]. the climate is continental temperate, roughly similar to that of the nearby suceava city [10, 11]; however, an increase in the annual precipitation amount and in the number and intensity of the torrential rains is expected [12], possibly leading to increased land erosion and pollutant movement. 3. materials and methods the main objective of this survey is to identify the degree of groundwater contamination in the built area of cajvana; the measured parameters are ammonium, nitrites, nitrates, orthophosphates and sulfides. in order to fulfill the objective, 15 water samples were collected (no replicates) from cajvana on august 28th, 2015. fig. 2. location of the sampling points within the built area the sampling points are represented by wells drilled in geologic strata containing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 39 phreatic waters. their territorial distribution is homogenous and almost ideal for obtaining interpolation maps of the studied parameters (fig. 2). the water samples were collected with polyethylene bottles and analysed within 24h from sampling. a hach-lange dr2800 spectrophotometer was used with reagents that have the following detection ranges: 0.015-2 mg/l nh4+-n, 0.002-0.3 mg/l no2--n, 0.1-10 mg/l no3--n, 0.022.5 mg/l po43and 5-800 µg/l s2-. the chemical analysis methods are specific to the hach-lange dr2800 spectrofotometer [13] and are based on standard methods for the examination of water and wastewater [14]. the described methodology was also previously used by other authors for analyzing the groundwater quality in suceava plateau [68]. the maps showing the spatial distribution of the values of analysed parameters were done by using the methodology described by briciu and oprea-gancevici [8], which is mainly a simple interpolation based on the “natural neighbors” function in the arcgis software. the interpolated values are those of the quality classes corresponding to each sampling point per studied parameter, according to the official water quality classification described in order 161/2006, issued by the romanian government. 4. results and discussion the ammonium concentration in wells is very low (fig. 3) and all sampling points have a very good water quality for this parameter. fig. 3. territorial distribution of the ammonium concentration and quality classes no value is higher than the maximum admissible concentration (mac) of 0.5 mg/l for potable water. nitrite water pollution is higher in cajvana valley, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 40 where average groundwater quality can be found (fig. 4). however, the mac threshold of 0.5 mg/l is far from being reached. one can observe that the highest nitrite contamination corresponds to the highest ammonium contaminantion, meaning that at least a part of the actual nitrite pollution results from ammonium pollution via biological oxidation of ammonium into nitrite. the orthodox graveyard is within 200 m from the sampling point number 12 (recording the maximum ammonium and nitrite concentrations) and this can explain the higher ammonium concentration, possibly generated by the decaying corpses in the graves. on the other side, there is another graveyard in the town, belonging to the pentecostals, located within 100 m from the sampling point no. 13, where low ammonium and nitrite concentration were recorded. the different size of the two graveyards is the key point, the orthodox graveyard being ~5 times higher than the other one (the graveyards surface ratio is approximately similar to the percentage of the people belonging to the implied confessions: 71.48% orthodox, 26.71% pentecostals – according to the 2011 census). the bigger graveyard seems to have reached the critical mass and density to become an environmental hazard. fig. 4. territorial distribution of the nitrite concentration and quality classes the area of high nitrite concentration (from human and animal manure) in cajvana valley could be explained by the terrain slope, permeable soils and geologic strata which favour the nutrient concentration from the surrounding hills into the valley. also, this is partly an explanation for the spatial distribution of the nitrates in groundwater (fig. 5). the mac value of 50 mg/l nitrate for potable water is not reached in any point, but the groundwater quality is not in a good state when nitrate is concerned; 86.6% of the sampling points have water quality oscillating from average to mediocre quality. at least in cajvana valley, some food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 41 nitrates originate from the chemical oxidation of the locally abundant nitrites. in the entire town, the main source of nitrates could be the n-based fertilizers used by people in their own gardens. according to the 2011 census, from 3341 people engaged in economic activities in cajvana, 2549 (76.3%) were involved in agriculture, silviculture and fishing. fig. 5. territorial distribution of the nitrate concentration and quality classes the western half of the town is also more polluted when analyzing the groundwater pollution with orthophosphates (fig. 6). the water pollution with phosphorus is caused by p-based fertilizers and by wastewaters rich in detergents. the pbased water pollution has certainly aggravated in time because of the increasing standard of living that was not, unfortunately, accompanied by the construction of a centralized wastewater collection system. therefore, the local people are pumping pheatic water, use it and, then, send it back into background with all types of domestic water pollutants. the increased standard of living over time is proven by the increased number of buildings in the built area: 1569 (1992 census), 1878 (2002 census) and 2201 buildings (on december 31st, 2013 [15]). the inhabitable surface has risen from 57371 m2 (1992) to 91038 m2 (2002) and 128004 m2 (on december 31st, 2013 [15]). the higher density of constructed spaces implies a higher wastewater volume, even if the population number is in a slight decline (7069 inhabitants in 1992, 7263 inhabitants in 2002 and 6901 inhabitants in 2011). the total number of residents on july 1st, 2014, was 9279 [15]. public water distribution and wastewater collection networks are planned in the near future for cajvana town. it is likely that, due to the lack of funds, the potable water system will be realized first, leading to an increased groundwater pollution with orthofosphates, nitrites and nitrates until food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 42 the wastewater collection network will be finished. fig. 6. territorial distribution of the orthophosphate concentration and quality classes only one sampling point (no. 1) showed sulfide concentration inside the detection limits of the used method (fig. 7). no groundwater contamination with anthropogenic sulfides is to be suspected and the water quality is very good. the values of groundwater pollution with ammonium, nitrite, nitrate, orthophosphate and sulfide is similar to those often found in other rural settlements in suceava plateau, meaning that waters are not of better quality than in fratautii noi and not as bad as in veresti [8] (table 1). considering the worst quality class identified for all parameters per each sampling point, one can observe that the dominant water quality is the mediocre one and that covers ~75% of the built area (fig. 8). if we try to mediate the values of quality classes in each monitoring point (1 is the best quality, 5 is the worst), instead of choosing the worst identified quality class at each sampling point, the average water quality class is dominant. moreover, it is useful to analyze the average concentration of the analysed parameters for the entire town: 0.04 mg/l nh4+-n, 0.013 mg/l no2-n, 6.23 mg/l no3--n, 0.134 mg/l po43-p and 0.6 µg/l s2(if we take into account all values, including those that did not fit in the high precision detection range). according to these values, within the built area, there are very good waters for the ammonium criterion, good waters for the nitrite and orthophosphate criteria, average waters for the nitrate criterion; the sulfides in groundwater are almost absent. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 43 fig. 7. territorial distribution of the sulfide concentration table 1. comparative water quality analysis of some settlements in suceava plateau (according to this study and other sources [7, 8]) frătăuții noi cajvana verești parameter average quality in all sampling points ammonium-n very good very good very good nitrite-n good good deteriorated nitrate-n good deteriorated average orthophosphate-p good good good worst quality in all sampling points ammonium-n very good very good very good nitrite-n mediocre average deteriorated nitrate-n mediocre deteriorated mediocre orthophosphate-p deteriorated mediocre deteriorated concerning the real spatial distribution of the different quality classes, it should be noted that it slightly differs from the mapped one because of the heterogenous distribution of the geologic strata and because of the variable lithology. the various slopes of the topographic surface contribute to the complexity of the real spatial distribution of groundwater pollutants. predictive and empiric numeric models (such as mnp [16]) applied to groundwater quality issues are necessary in order to perfectate the maps of the territorial distribution of the quality classes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 44 fig. 8. the worst water quality classes (colour palette for: 1 – very good… 5 – deteriorated) and the arithmetic average (displayed numeric values on map) of all recorded water quality classes per each sampling point the groundwater generates the small rivers withn the built area and the groundwater pollution is transferred into them. when these rivers have high levels due to surface runoff at torrential rains (which can wash some pollutants from the topographic surface), the rivers can feed the groundwater and new amounts of pollutants will be inserted into the ground, complicating the process of identifying their source and their spatial distribution. also, the diurnal thermal regime of the rivers [17] is influencing the same groundwater regime along the rivers and the chemical reactions in the underground. 4. conclusion in conclusion, we can remark that the most important water pollution in wells is caused by nitrates. there is a distinct groundwater pollution with ammonium and nitrite in the extreme west built area caused by the main graveyard of the town. the lack of a public sewerage is causing water pollution with orthophosphate, which will most probably worsen in the near future due to the increasing standard of living. the average water quality of the domestic wells in cajvana town is different depending on the analysed parameter: very good – ammonium, good – nitrite, deteriorated – nitrate and good – orthophosphate. very small concentration of sulfides was detected. it is necessary for the future studies to map in detail the various sources of water pollution in order to identify the best local water management strategy. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 dinu iulian oprea-gancevici, cătălin savu, ground water quality of domestic wells in cajvana town, food and environment safety, volume xv, issue 1 – 2016, pag. 36 – 45 45 5. acknowledgments we thank andrei-emil briciu (department of geography, ștefan cel mare university) for the scientific guidance offered for writing this article. 6. references [1]. withers p. j. a., jarvie h. p., stoate c., quantifying the impact of septic tank systems on eutrophication risk in rural headwaters, environment international, 37:644653, (2011) [2]. judová p., janský b., water quality in rural areas of the czech republic: key study slapanka river catchment, limnologica ecology and management of inland waters, 35:160-168, (2005) [3]. jarvie h. p., neal c., withers p. j. a., sewage-effluent phosphorus: a greater risk to river eutrophication than agricultural phosphorus?, science of the total environment, 360:246-253, (2006) [4]. liang l., nagumo t., hatano r., nitrogen flow in the rural ecosystem of mikasa city in hokkaido, japan, pedosphere, 16(2):264272, (2006) [5]. briciu a.-e., mihaila d., mihaila doina, short, medium and long term stochastic analysis of the suceava river pollution evolution in the homonymous city, sgem2012 conference proceedings, 3:809-816, (2012) doi: 10.5593/sgem2012/s13.v3039 [6]. briciu a.-e., oprea-gancevici d., study concerning the waters quality in berchişeşti area, analele universității ștefan cel mare, secțiunea geografie, 19(2):43-56, (2010) [7]. briciu a.-e., oprea-gancevici d., the effect of rural settlements on water quality in northern suceava plateau, food and environment safety, 10(2):88-96 (2011) [8]. briciu a.-e., oprea-gancevici d., rural hydrology study regarding the phreatic water quality in dragomirna plateau, romania, present environment and sustainable development, 5(2): 279-293, (2011) [9]. briciu a.-e., suceava anthropic torrential basin prolegomena, analele universității ștefan cel mare, secțiunea geografie, 19(1):165-179, (2010) [10]. mihăilă d., tanasa i., particularitati climatice ale semestrului rece la suceava, analele universității ștefan cel mare, secțiunea geografie, 15:61-72, (2006) [11]. mihăilă d., tanasa i., particularitati climatice ale semestrului cald la suceava, analele universității ștefan cel mare, secțiunea geografie, 16, (2007) [12]. mihaila d., briciu, a.-e., actual climate evolution in the ne romania. manifestations and consequences, sgem2012 conference proceedings, 4:241-252 (2012) doi: 10.5593/sgem2012/s17.v4001 [13]. http://www.hach.com, last accessed 01.08.2015 [14]. https://www.standardmethods.org, last accessed 01.08.2015 [15]. i.n.s.s.e. – statistical yearbook of suceava county 2014, revista romana de statistica publishing house, bucuresti, (2015) [16]. briciu a.-e., the attempt of systematical space changes modelling and the numerical predictive models in physical geography (l’essai de la modélisation systématique de la transformation de l’espace et les modèles numériques prédictifs dans la géographie physique), analele universității ștefan cel mare, secțiunea geografie, 18:243-250, (2009) [17]. briciu a.-e., oprea-gancevici d. i., diurnal thermal profiles of selected rivers in romania, sgem2015 conference proceedings, 1:221-228, (2015) doi: 10.5593/sgem2015/b31/s12.029 http://www.hach.com/ https://www.standardmethods.org/ 1. introduction 4. conclusion 115 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 115 122 information technology in designing high-performance equipment for biomass compacting *dmitro viktorovich rindyuk1, svyatoslav yuriyovich lementar1 1national university of food technologies, kiev , ukraine, rel_dv@ukr.net * corresponding author received: may 14th 2013, accepted june 14th 2013 abstract: the basic principles and methods of constructing a mathematical model for the processes of particular materials processing are presented in this paper. an example of the practical application of the developed model in the technology of biomass extrusion, which is used for the production of fuel pellets, is considered. a mathematical model of the process for particular materials extrusion is developed and a new approach to the extrusion granulator unit design is offered with the developed methods taken as a basis. the effect of technological and design factors of extrusion granulator unit to the extrusion process regularity are also studied. keywords: design information technology, mathematical modeling, particulates, granulation, fuel pellets. 1. introduction food processing industry has recently faced a problem of rational use of waste products such as sunflower husk, straw, brewer's grain, wood chips, etc. it is advisable to use this waste as biomass fuel, or feed compound, etc. transporting these materials is considered economically unjustified because of their small bulk density. this fact becomes an agent for bulk density increase of the materials through compression (briquetting, embaling, granulation, etc.) the analysis of various compression technologies showed that the granulation technology is the most rational one as it can be used for continuous product processing. it also allows obtaining the products of the highest density and versatility (biofuel, feed compounds of different fractions). granular materials have greater storage stability and better mixture homogeneity. they also better preserve vitamins and trace elements, occupy 2-3 times less space, and are less exposed to environmental influences if compared to bulk materials. intensification of particular materials compression processes, which recently has become most noticeable in the field of solid biofuels production, results in more stringent requirements for performance indicators of main technological systems. therefore, the connection between both design (active zone sizes, shape and speed of the movable elements, etc.) and technological (machine performance, pressure, temperature, physical and mechanical characteristics of the material, etc.) factors should necessarily be considered when designing such machines and devices. the traditional approach to the design of this type of equipment is based on empirical food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 116 dependences and experimental experience [1] and does not allow quantifying the interference between technological and design factors of processing and structural and mechanical properties of materials. therefore, applying the modern methods of mathematical modeling to the process of extrusion granulation of particulate materials is considered to be a topical issue. the analysis of the recent research studies in the field of granulation technology testifies that for the effective designing of the particular technologic equipment it is necessary to take into consideration the structural and mechanical characteristics of the processed materials and in the first place their such rheological properties as elasticity, plasticity and viscosity. works [2-3] show the principles of mathematical model construction for the granulation process of dispersed materials by extrusion. to increase efficiency of practical use of the given model it is essential to further improve it with the aim to embrace the maximum number of design and technological parameters. taking into account the broad range of raw materials types, the problem of proper interdependence between main design and technological parameters of a given extrusion unit comes to the front when defining design properties of the appropriate equipment. figure 1. information technology scheme of designing equipment for extrusion compacting. input parameters: angular velocity of matrix, geometric dimensions of the matrix and rollers the geometrical dimensions of the matrix holes, thickness of the matrix analysis of mechanisms of interaction in the system: extruding unit raw materials design scheme of the process extrusions through the matrix hole mathematical model of extrusion process carrying out numerical experiments for selected types of raw materials rheological properties conditions of surface interaction between raw material and the matrix during extrusion process conditions force loading during pelleting of raw materials the analytical model algorithmic model numeric model a strained strain state of raw materials during extrusion change in pressure during extrusion dependence of the process on the geometry of holes in the matrix the load on the elements of process equipment analysis of results the conducted experiments database food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 117 2. experimental the main aim of this work is to develop the design information technology (dit) of processing equipment for dispersed materials by extrusion as well as to use the developed (dit) when designing an extrusion granulator unit when applying biomass granulation technology to produce fuel pellets. the article describes the (dit) type [2]: “mathematical model intellectual expert system automatic design system “which is represented in figure 1. (dit) represents technological extrusion process as a multi-component system of interconnected objects under study: raw material mass, technological equipment elements, mechanic loading, etc. when examining particular processing technology we use the conception of introducing dispersed mass as a two-phase mixture of the porous or solid granular deformed structure with liquid or gas. to describe the behavior of dispersed mass, the notions of tension, deformation, density and also the velocity of changing these parameters were used. as a basis of analytical model the momentum retention equation at macrocoordinates was used for: a) a solid phase         0gradgrad t  11 ffσuuu 11111   (1) b) a gas-liquid phase         ,gradgrad t 0ffpvvv 2222222   (2) where 1, 2  volume content of solid and gas-liquid phase; 1, 2  corresponding average phase density; u, v  vectors of average displacement speed of solid particulates and liquid correspondingly; p  hydrostatical pressure at a gas-liquid phase; f1, f2  vectors of volume forces in solid and liquid phases correspondingly;   a tensor of tensions at a solid phase; f(1), f(2) – forces of interphase interaction. as a result of equality nσnp  , at division points of inner surface of gasliquid and solid phases (n normal vector to the division surface) the following condition is fulfilled:     012 fff  (3) formal addition of equation (1) and (2) describes, apparently, momentum retention at all macropoints of dispersed environment :       0gradgrad t   21 ffvpσ vvuuvu 2 221 22112211     (4) however, when doing practical calculations, it is more convenient to consider balance momentum equations separately in the forms of equation relating to movement of a gas-liquid and a solid phase. if to set the force of interphase interaction in the form of     2 gradprff=f 210 (5) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 118 and to take into account that 1 = 1 – 2, then the relative movement equation of the solid phase will be represented as follows:   0grad dt dd          g r σ vu 211 2 f 211 dt , (6) where  uvr  212a effective viscous resistance force;  -coefficient of viscosity at a liquid phase; а – interphase friction coefficient. the equation of relative movement of liquid phase we can describe in the following form: g r p v 2 22 αdt d  grad , (7) where g vector of acceleration of the free falling. it should be mentioned that for the processes which take place at a low pace, there are no inertial effects: 0; 22  g v  0 dt d ), the equation (7) represents the law of filtration within isotropic porous environment: puv grad 1 2 μα a  (8) the experimental study shows that at a small pressure gradients or filtration velocity the linear darcy’s law acts. puv grad k 1 p   , (9) where pk environment permeability coefficient. to loop a system of obtained equations (1-9), it is necessary to define the mode of solid phase deformation (determinant correlation) with corresponding spatial and temporal modes of boundary condition changes. determinant correlations are taken within a framework of rheological model of elastic, viscous and plastic body. besides, degree of deformation of a liquid phase is carried out by setting kinetics of volumetric phase content change, determined by filtration mechanism. (7) algorithmic model consists of the following main parts: solution to a specified problem is based upon the principle of application of projecting and grid methods: finite elements to spatial variables and finite-differences by time argument. calculated algorithms, which realize the most typical extrusion rheological processes of dispersed systems. the developed algorithms were realized in the form of “plast-grn” programming complex (digital model) [4]. programming complex is aimed to simulate non-balanced deformation processes of two-phase dispersed structures under given technological mode. within the framework of the developed design information technology, to obtain fuel pellets the granulation process of dispersed masses by extrusion was considered [5]. the major impact on the granulation process is made by the granulator design (especially, the holes profile of granulator unit matrix [6,7] and the rheology of the matter being granulated food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 119 figure 2. granulator unit with annular matrix besides, one of the typical structural schemes of granulation by extrusion was considered, the one, which contains pressing roller mounted within annular matrix. for the given structural scheme of the granulator unit it is possible to point out main parameters, which determine the production process such as roller and matrix size, the holes size in matrix, surface roughness in holes, rotation speed of granulator matrix, extrusion pressure through holes, rheological properties of the matter, productivity and quality of the finished products. figure 3. scheme of work of the granulator unit figure 4. calculated schemes of the granulation process by extrusion. the key technologic operation takes place within granulator pressing unit, i.e., the operation of granulating the raw materials through matrix holes by extrusion. figure 4 shows the calculations of the granulation process. the first calculated scheme in figure 4 (a) presents the part of granulator matrix with holes, where raw material is confined in the upper part by a pressing roller. scheme takes into account both the granulator matrix geometry (its diameter, and distance between holes and pressing roller diameter as well as air gap between matrix and roller. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 120 the second calculated scheme in figure 4 (b) shows granulator matrix channel. the scheme takes into account geometry size of the channel, namely, the length, the diameter and the bevel size. 3. results and discussion the given study we are concerned with the processes which go on at the very time of extrusion, that is, we are especially interested in the behavior of the raw material in the holes of granulator matrix. the question arises as to what hole we should examine at a certain time and where the process of pressing flows most intensively. in order to find such a hole the necessary calculations were conducted (calculated scheme figure 4.a), which resulted in the diagram creation, that allows defining the specific size of the hole. the diagrams of movement allocation at x and y axes show that maximal movement we can notice in hole 3 and hole 4, figure 5, correspondingly. figure 5. diagram of movement allocation at y axis. the diagram of pressure gives possibility to define the values of pressure which act in the particular area. data obtained after calculation the first model were used to construct the following model (calculated scheme figure 4.b). in this case, we are interested in the behavior of raw material in the hole of matrix which will result in the opportunity to define dependence between the holes geometry and raw material behavior under granulation process. figure 6 shows the example of the obtained results after numeric modeling of the extrusion process of the raw material through matrix. figure 6. allocation of a) movements and b) porosity in the bulk volume of dispersed material. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 121 based on the obtained data, taken as an example, the graphs of pellet density dependence on main design and technological parameters of the granulator unit under different values of pressure were constructed so, the figure 7 shows the dependences for sunflower husk. figure 7.a. density dependence of ρ pellets on d hole diameter ● 50mpa, ■ 100mpa, ▲ 150mpa figure 7.b. density dependence of ρ pellets on l hole matrix length ● 50mpa, ■ 100mpa, ▲ 150mpa figure 7.c. density dependence of ρ pellets on  hole matrix angle ● 50mpa, ■ 100mpa, ▲ 150mpa figure 7.d. density dependence of ρ pellets on dr roller diameter ● 50mpa, ■ 100mpa, ▲ 150mpa figure 7.e. density dependence of ρ pellets on t air gap size between roller and matrix figure 7.f. density dependence of ρ pellets on friction coefficient between dm and matrix food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar, information technology in designing highperformance equipment for biomass compacting, food and environment safety, volume xii, issue 2 – 2013, pag. 115 122 122 4. conclusion so, the developed mathematical model gives possibility to define interdependences between main design and technological parameters of pressing granulator taking into account type of raw materials. it is advisable to use the gained results as recommendations in designing granulating equipment. 5. references [1] shtefan j., behavioral modeling of dispersed system within non-balanced processes in food production. scientific works of nuft, #8, pp. 63-66, 2000. [2] shtefan j., equipment design information technology for mechanical processing of dispersed materials. international periodic collection of scientific works. processing of dispersed materials and environments. theory, research, technology, equipment. issue #12, odessa: votum spa, pp. 72-78, 2002. [3] shtefan j., blazhenko s., constructing analytical model of deformation processes of dispersed materials. international periodic collection of scientific works. processing of dispersed materials and environments. theory, research, technology, equipment. issue #13, odessa: votum spa, pp. 26-33, 2003. [4] ryndiuk d., shtefan j., developing method of optimal structural and technological parameters of a press granulator. scientific works of odessa naft. ministry for education and science of ukraine. odessa. issue 28.volume 2. pp. 202-205, 2006. [5] patent for a useful model #30058 mpk (2006) b01j2/00. the method of determining optimal structural and technological parameters of a press-granulator. shtefan j., ryndiuk d. [6] shtefan j., zajets j., ryndiuk d., deternination of structural and technological parameters of food products processing by cold extrusion. mixed fodder industry of ukraine.#5, pp. 16-20, 2006. [7] shtefan j., ryndiuk d., utilizing mathematical models for designing pressgranulator unit. mechanics and informatics. collection of scientific works of young scientists. khmelnytskiy. khnu, pp 172-175, 2005 [8] valentas k., food engeneering: reference book with calculation samples.translation from english by valentas k.,rotshtein j,singh p., st.petersburg, profession, p. 848, 2004. [9] fengel d., vegener g., woodwork: translation from english by obolenskaya a., elnitskaya p., under supervision of doctor of technical sciences leonovichaa., moscow, forestry, p. 540, 1988. 284 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 284 290 utilization of experimental design for specific surface area optimization of a pillared bentonite *ana-maria georgescu1,2, gheorghe brabie3, *ileana denisa nistor1, françoise nardou2, claude penot2 1« vasile alecsandri » university of bacău, faculty of engineering, ica department, 157 calea mărăşeşti, 600015, bacău, romania, georgescu_am@yahoo.com , dnistor@ub.ro 2 university of limoges / cec, spcts, umr 7315 cnrs, 12 rue atlantis, 87068, limoges cedex, france, francoise.nardou@unilim.fr 3« vasile alecsandri » university of bacău, faculty of engineering, imsi department, 157 calea mărăşeşti, 600015, bacău, romania, g-brabie@ub.ro *corresponding author received november 11th 2013, accepted december 2nd 2013 abstract: statistically designed experiments were used to study the effects of relevant factors, such as calcination temperature and duration, in obtaining of al-pillared clays with a high specific surface area. experiments have been planned by the factorial 32 design methods. the optimal conditions to obtain the maximum specific surface area of al-pillared bentonite were deduced as follows: calcination temperature 326°c and calcination duration 63 min, for a specific surface area of 152.49 m2/g. the results from bet (brunauer-emmett-teller) method showed that the pillared smectite clay possesses a specific surface area up to 155.65 m2/g. besides, chemical treatment of smectite clay by pillaring process produces an adsorbent with optimal porosity and other adsorption properties suitable for many industrial processes. keywords: calcium bentonite, factorial design, mathematical optimization, pillaring, bet method 1. introduction pillared interlayer clays (pilcs) are an interesting class of 2-dimensional microporous materials. due to their high surface area and permanent porosity, they are very attractive solids for adsorption and catalysis purposes. this research was focused on the use of inorganic hydrated polyhydroxocations as pillaring agents. such pillaring agents, when calcined, dehydrate and dehydroxylate to form a fixed metal oxide pillar with a high thermal stability and high surface area. the use of the al13 polyhydroxocation was chosen as it is extensively researched and easily prepared [1-6]. the concept of pillaring is very straightforward and consists of two main steps: first, small interlamellar cations are exchanged for other, bulky ions. a second step, calcination, converts the inorganic polyoxocation precursors into rigid, stable metal oxide pillars, tightly bonded to clay layers [7]. mathematical modeling through factorial design procedure was used by now in different domains for optimizing and modeling of different processes such: water depollution using some unconventional procedures, biogas food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 285 production, etc. the advantages of this optimization method consist in the fact that not only the individual (simple) effect of each variable is taken into account, but also the interaction and/or their possible synergy effects. this method allows studying the influence of a large number of variables. the methods of mathematical planning of experiments aimed the obtaining of quantitative relationships of y = f (x1, x2, x3, ...) type, respectively of mathematical models that are associated to the processes, and the systematical, efficiently and economical investigation of significant factors of the process. the interest of factorial program of kn type, appears because of the difficulty to establish, by conventional ways, the mathematical relationship between the variables of studied system-process couple [8, 9]. in this study, the procedure for synthesis of al-pillared clays consists in five steps: clay purification, ionic exchange of clay with cu2+ ions, preparation of pillaring agent, intercalation of ionic exchanged clay with pillaring agent and calcination. the last step is very important, for the conversion in stable pillared clays. the aim of this paper is to establish an optimal domain for calcination parameters in the case of al-pillared clays, using an experimental design study. two factors were varied (calcination temperature and calcination duration) and evolution of specific surface area was achieved as the response function. 2. experimental 2.1. materials and devices the calcium bentonite from oraşu nou (romania) used as raw material in this study, was purified. all chemicals used in this work were supplied by alfa-aesar. the specific surface area (determined by bet technique), was obtained from n2 adsorption–desorption isotherm at 77 k, measured in a micromeritics asap 2010. the samples were degassed at 473 k, for 16 h. 2.2. synthesis 2.2.1. purification the aim of purification process for romanian natural calcium bentonite is the obtaining of well-defined granulometric fractions, with particles of less than 2 µm. the purification process consists in dispersion of some quantity of natural calcium bentonite into a volume of distilled water, for obtaining of 0.4 wt % suspension [10]. the suspension was stirred for 3 hours at room temperature, till a complete homogenization. montmorillonitic fractions, which were recuperated on the basis of stokes’ law, were centrifuged, and the recovered clay was dried in an oven at 105oc, for 15 h. the purified bentonite was ground and sieved below 63 μm, in order to use only the finer grains. 2.2.2. ionic exchange with 0.1 m cucl2 the bentonite samples were copperexchanged, by its treatment with 0.1 m cucl2 solution, with a solid:liquid ratio of 1:20, the suspension being stirred at room temperature, for 24 h [11]. this procedure was repeated two times for obtaining a complete copper-exchanged bentonite. between copper-exchanged operations, the samples were washed with distilled water, until modified montmorillonite was free of chloride anions (0.1 n agno3 test). after sample washing, the suspension was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 286 centrifuged. next, the sample was dried in an oven at 80oc for 16 h, ground and sieved in order to obtain particles size less than 63 μm. 2.2.3. al-pillaring solution preparation the dilute al-pillaring solution was prepared by drop-wise addition of a 0.2 m naoh solution to a 0.2 m alcl3 solution, at room temperature with vigorous stirring, till the (oh/al) molar ratio was 2, for obtaining the maximum content of keggin cation (al13) [2]. the pillaring agent was kept at room temperature, for 48 h. 2.2.4. intercalation of cu-clay with alpillaring agent cu-montmorillonite was dispersed in distilled water (2 wt % slurry) and the suspension was stirred for 1 h, at room temperature. further, this slurry was mixed with the pillaring solution at room temperature, until the molar ratio was 10 mmoles al/g clay. the clay was then washed with distilled water for the removal of chloride ions, and centrifuged. the pillared clays were then dried at 80oc for 15 h, and then hand-ground into a mortar. 2.2.5. calcination the calcination of intercalated samples was realized using a device with a tubular furnace under nitrogen. the nitrogen flow was maintained at 100 ml n2/min, at a heating rate of 2oc/min. the calcination temperatures were varied from 300oc, 400oc to 500oc for 1, 2 and 3 h. 2.3. factorial design the most often used experimental programs are kn factorial design type, where k is the variation number of parameters level, and n is the number of parameters [9, 12]. in this study, it was investigated the influence of two parameters for the preparation of al-pillared bentonites, which are expressed in terms of calcination temperature (x1) and calcination duration (x2) upon one response function, the specific surface area, in the variation ranges summarized in table 1. table 1. parameters that influence pillaring process and their variation domain parameters (xi) reduced variable minimal level (ximin) median level (xi med) maximal level (xi max) δxi calcination duration [min] x1 60 120 180 60 calcination temperature [oc] x2 300 400 500 100 3. results and discussion the response function was the specific surface area of al-pillared bentonites that is presented in table 2. in parenthesis are noted the reduced values of the variables. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 287 table 2. factorial 32 experiment design and response function for specific surface area of al-pillared bentonites run calcination duration [min] calcination temperature [oc] specific surface area (sbet) [m2/g] x1 x2 y 1 60 (-1) 300 (-1) 155.65 2 400 (0) 141.67 3 500 (+1) 122.51 4 120 (0) 300 (-1) 143.21 5 400 (0) 140.98 6 500 (+1) 125.58 7 180 (+1) 300 (-1) 133.12 8 400 (0) 128.51 9 500 (+1) 96.36 to calculate the significance of the program, three other tests in the central point of the domain (0, 0) were also realized; the obtained values are shown in the table 3. table 3. values in the central point of the domain yk0 y10 y20 y30 value for specific surface area [m2/g] 141.32 140.78 142.18 3.1. elaboration of the mathematical model the particular form of response function for factorial program of type 32 is presented in equation (1). in table 4 are presented the coefficients values of polynomial mathematical model. these were calculated according to the literature specifications [8, 9, 12]. 2222 2 11121122211 xaxaxxaxaxaay o  (1) table 4. values of the polynomial coefficients coefficient value coefficient value a0 141.689 a12 -0.905 a1 -14.588 a11 -7.648 a2 -10.307 a22 -6.953 according to the polynomial coefficients, the mathematical model which describes the response function of the optimizing criteria is: 2 2 2 12121 953.6648.7905.0307.10588.14689.141 xxxxxxy  (2) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 288 3.2. determination of the coefficients significance in order to determine the significance of the polynomial coefficients it was used the t student test. for this, it is necessary first to calculate the average value of the three response functions and of the average value of the measurement errors according to the algorithm presented in the literature [7, 10]. the t-student test results are used to determine the insignificant coefficients (table 5). it will be calculated the average specific surface area in the central point of the domain (0, 0): 427.141 3 3 1 0 0   i i med y y (3) the average error square is calculated, knowing that the control samples number, n, is 3, using the equation:         n i medi n yy 1 200 2 499.0 1  (4) it will be calculated the error for control samples: 706.0 (5) the calculation of coefficients significance, s, is realized with the equation (6), knowing that the number of experiments, n, is 9: 235.0 n s  (6) the significance of the coefficients will be tested using student t-test, with the equation: sat jj / (7) the t-student test values for each coefficient are presented in table 5. the t-student test results show that the term that can be eliminated is x12. table 5. t-student test results tj t0 t1 t2 t12 t11 t22 value 602.019 61.984 43.792 3.845 32.497 29.544 the mathematical model that describes the response function of optimization criterion, after the removal of insignificant term using t-student test, is: 2 2 2 121 953.6648.7307.10588.14689.141 xxxxy  (8) forward, it will be discussed the effects of parameters. the ao value (141.689) indicates that the optimal specific surface area is close to this value. because the coefficients a1 and a2 are negative, the variables x1 and x2 have an unfavorable individual action on the pillaring process. the individual effect of the x12 term that was determined with tstudent test, was insignificant, it will not be discussed. analyzing the quadratic coefficients a11 and a22, which values are negative, thus the response function is characterized by a maximum in relation with the variables x1 and x2. for response function obtained after the insignificant terms elimination, using tstudent test, the partial derivatives of first order will be calculated, in rapport with each variable: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 289 2 2 1 1 953.62307.10 648.72588.14 x x y x x y       (9) by equating the partial derivatives of first order with 0, the linear system resulted will be resolved. the optimal point searched, represented in dimensionless coordinates, is (-0.953; 0.741). as it can be seen, the optimal values for x1 and x2 are within the limits of the domain (-1, 1) that were initially supposed. knowing the range of variation of calcination temperature and calcination duration, it can be obtained the real values of the optimal point using the following equations: med med xxxx xxxx 2222 1111   (10) where: x1, x2 – real values of optimum; x1, x2 – dimensionless values of optimum; δx1, δx2 – step of each variation domain; x1med, x2med – real average value of parameters. cx x o9.325 min82.62 2 1   (11) in this study, the specific surface area dependence was established according to the two factors, temperature and duration calcination in order to obtain the alpillared clays. this dependence can be illustrated using the curve shown in the diagram presented in figure 1. figure 1. influence of calcination duration (x1) and calcination temperature (x2) on specific surface area (y) as long as both coefficients of quadratic terms have negative signs, the quadratic terms determine a maximum and the response surface (figure 1), which corresponds to the model is concave. the optimal value of specific surface area obtained by factorial planned experiments was 152.49 m2/g. 4. conclusions from 3d representation of concave shape, it can be distinguished the roundness of area and the maximum due to the quadratic coefficients effects. this representation evidences the obtained results of experimental design. the real optimal values of varied parameters are: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 ana-maria georgescu, gheorghe brabie, ileana denisa nistor, françoise nardou, claude penot, utilization of experimental design for specific surface area optimization of a pillared bentonite, volume xii, issue 4 – 2013, pag. 284 – 290 290 325.9oc for the optimal calcination temperature; 62.82 minutes as the optimal calcination duration, and give an optimal specific surface area value of 152.49 m2/g. this optimal specific surface area value could be still increased by the application of experimental design to the other steps for al-pillared clays obtaining, like: purification, ionic exchange and intercalation. 5. acknowledgements this paper was realized with the support of brain project: “doctoral scholarships as an investment in intelligence”, financed by the european social found and romanian government. 6. references [1]. mrad, i., ghorbel, a., tichit, d., lambert, j.f., optimization of the preparation of an al-pillared clay: thermal stability and surface acidity, applied clay science, 12, 349-364, 1997. [2]. carriazo, j., guélou, e., barrault, j., tatibouët, j.m., molina, r., moreno, s., synthesis of pillared clays containing al, al-fe or al-fe-ce from a bentonite: characterization and catalytic activity, catalysis today, 107–108, 126– 132, 2005. [3]. maes, n., heylen, i., cool, p., vansant, e.f., the relation between the synthesis of pillared clays and their resulting porosity, applied clay science, 12, 43-60, 1997. [4]. brotas de carvalho, m., pires, j., carvalho, a.p., characterisation of clays and aluminium pillared clays by adsorption of probe molecules, microporous materials, 6, 65 – 77, 1996. [5]. gil a., assis f.c.c., albeniz s., korili s.a., removal of dyes from wastewaters by adsorption on pillared clays, chemical engineering journal, 168, 1032–1040, 2011. [6]. timofeeva m.n., panchenko v.n., gil a., doronin v.p., golovin a.v., andreev a.s., et al, effect of the acid–base properties of zr, al-pillared clays on the catalytic performances in the reaction of propylene oxide with methanol, applied catalysis b: environmental, 104, 54–63, 2011. [7]. kloprogge, j.t., duong, l.v., frost, r.l., a review of the synthesis and characterization of pillared clays and related porous materials for cracking of vegetable oil to produce biofuels, environmental geology, published online, doi 10.1007/s00254-005-1226-1, march 3, 2005. [8]. nistor, i.d., jinescu, c., azzouz, a., elemente şi strategii în designul proceselor tehnologice, editura alma mater, bacău, isbn 978-606-527-008-4, 120-169, 2008. [9]. zivorad r. lazic, design of experiments in chemical engineering, wiley-vch, verlag gmbh & co kgaa, new york, isbn 3-52731142-4, 2004. [10]. khalaf, h., bouras, o., perrichon, v., synthesis and characterization of al-pillared and cationic surfactant modified al-pillared algerian bentonite, microporous materials, 8 (3-4), 141-150, 1997. [11].cseri, t., bekassy, s., bodas, z., agai, b., figueras, f., acetylation of b15c5 crown ether on cu modified clay catalysts, tetrahedron letters, 37 (9), 1473-1476, 1996. [12]. azzouz, a., concepte de modelare şi elemente de strategie în designul industrial, editura tehnică-info, chişinău, 2001. 300 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 300 310 study of the mechanism of the retarding effect on bread staling by using natural gums *svitlana palyvoda1, vira urchak1, tatjana golikova1, veniamin fomenko2 1 department of “bakery and confectionery goods technology”, national university of food technologies, kyiv, ukraine, sv_palivoda@bigmir.net 2department of “general and inorganic chemistry”, national university of food technologies, kyiv, ukraine, tanyayev@ukr.net * corresponding author received november 4th 2013, accepted december 7th 2013 abstract: the mechanism of natural gums' effect on the preservation of bread freshness has been studied. the stage of bread staleness was estimated from the deformation of bread’s crumb. hydrophilicity and texture of bread crumb were also considered. the mechanism of gum’s effect on the process of bread staling was studied by determining the respective change in the state of flour starch crystal structure, which was assessed by qualitative roentgen phase analysis and the change of bread matter moisture bonds with the addition of gums, which was determined by the method of differential thermal analysis. as the result of the research, it was found that addition of natural gums accommodates bread shelf-life extension by means of crumb texture change, as well as the effect on bread biopolymer moisture bonds. keywords: gums, bread, freshness, structure, starch, crystallinity, moisture bonds. 1. introduction extension of shelf freshness of bread always has been and to this day remains a problem for the baking branch of food industry. now, with the introduction of accelerated technology, slowing down the process of baked product staling is a priority for bread manufacturers. the process of bread staling is generally associated with changes to its properties, caused by changes of the state of starch, proteins and changes to moisture bonds of the bread [1, 2]. some researchers [1, 3] believe that primary causes of bread staling are changes to starch structure. the process of bread staling occurs due to the process of starch retrogradation after gelatinization during baking. this explains the changes to physical and chemical properties of the crumb and hydrophilicity in particular. reduction of crumb elasticity is explained by an increase of starch density that occurs due to transition from amorphous to crystalline state. knyahinichev [4] sees the process of bread staling as changes in moisture bond forms (bound and unbound) during preparation and storage of food. the author believes the reduction of unbound moisture in bread samples leads to slower formation of a single structural system of water molecules, starch and protein. this system is compacted by the convergence of starch chains due to intermolecular interaction of starch food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 301 and protein. thus, it is possible to keep the system less dense by reducing the amount of unbound water in bread samples. a definite relationship between the content of bound water and bread staling can be established. in a study [5], it is shown that, during storage, unbound moisture content decreases as a result of an increase in capillary specific surface area that occurs due to starch retrogradation. thus, factors that affect staling are the formulation, the technological parameters of production, ingredient quality, and its storage conditions. food additives application appears as one of the effective ways of the bread quality improvement and shelf life extension [6, 7, 8]. hydrophilic colloids (carbullose, carboximethylcellulose) are being used in baking aiming at the slowing of bread staling and improvement of crumb structure of frozen bread products [9, 10, 11, 12, 13]. natural gums are widely used as structure forming and water bonding ingredients in food production. it is easy to suggest that they will also effect the dough component moisture bonding and, probably, influence the process of bread staling. it is also known that adding guar gum has a positive impact on the quality of defrosted bread in comparison to products without such additives [14]. however, the exact mechanism of the influence of gums on the process of bread staling is yet to be researched. among gums, gum from the carob tree (ceratoniasiligua), guar gum (cyamopsis tetragonolobus) and gum from tara trees (cesalpinia spinosa), which are mostly contained in the seeds of these plants and prevent their dehydration, have industrial importance [11]. chemical structure of the aforementioned natural gums is that of neutral polysaccharides consisting of (1,4) βglycosidically bound mannose residues to which side chains of α-d-galactose residues are attached at regular intervals via 1,6links. the mentioned galactomannans have different ratios of mannose to galactose, which vary between 1,6:1 (for guar gum) and 3,5:1 (for carob gum) [11, 12, 15]. galactose content in carob gum is 17 26%, tara gum about 25%, guar gum 33 40%. galactomannan guar, tara and carob gums are all non-ionic [15, 16]. the water absorbing ability and effect to the water system properties are one of the functional peculiarities of gums. galactomannans also regulate texture, affect crystallization of starch containing products, prevent stratification or sedimentation, increase resistance to freezing and thawing processes, prevent syneresis, and retrogradation [12, 16]. due to these properties, natural gums have found application in bakery products and pastry production in quantities around 1 5 gram of gum per 1 kilogram of product. the mechanism of the influence of gums on the process of staling may be caused by the mentioned technological functional properties. however, current literature does not offer enough data to reveal the exact mechanism of the influence of natural gums on the processes of bread staling, so a task of systematization of data on their influence on the quality of baked products and extension of their term of freshness was undertaken by the authors by course of examination of the impact natural gums have on the changes in bread crumb structure during staling, and of the investigation of the process of starch retrogradation, as well as of changes of moisture states in baked products during staling. 2. materials and methods decelerating effects of natural gums (guar gum, tara gum and carob gum) on the process of bread staling were studied. gum food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 302 samples of 0.25 0.50 % of the weight of wheat flour were used. preparation of bread the gums used were: guar gum vidogum gh (е 412), tara gum vidogum sp (е 417) and carob tree gum vidogum l (е 410) unipektin (switzerland). the dough was prepared according to formulation, which was 100% wheat flour (containing 0.73% ash in dry matter, 27 – 29 % wet gluten), salt 1,8 %, yeast 3 %, sunflower oil 2,0 %, gums 0.25 0.50 % of the weight of wheat flour and water to get moisture of dough 44.5 %. as a control, no gums were added to the formulation. the ingredients were mixed during 6 minutes in mixing bowl. after 170 min fermentation, the dough was divided into 250 g loaves, formed on dough former, proofed 45 min and baked in an electric oven during 30 – 35 oc at 200 220°c. baking trials were performed in triplicate. bread crumb structure physical and mechanical properties of crumb were assessed based on its compressibility, which was measured on a model 'aп 4/1' automated penetrometer. readings were taken after 4, 24, 48 and 72 hours during storage. baked products were stored at room temperature in conditions that prevent drying. measurements were taken at 10 points on the bread slice 40 mm thick on both sides. the loading tip had a hemisphere form of 25 mm diameter. it could be loaded with the removable weights. total mass of pressing system reached 300 g. during the experiments, total, plastic and elastic deformations of the crumb were been determined. total deformation was been determined based on the reading of load penetration (h1) and was expressed in penetration units. after these, an additional weight was been removed and, as a result, the tip partially moved back under the action of the crumb elasticity. the respective reading of the penetrometer (h2) refers to a value of elastic deformation. the difference (h1-h2) refers the value of plastic deformation [2]. crumb fragility evaluation of the freshness of bread was also carried out by taking measurements of crumb fragility after 4 hours and during the 3 days of storage and the amount of absorbed water. crumb fragility was been determined in the following way: 2 rectangular pieces of bread with the mass of 5 g each were placed in a conical glass and shaken during 5 minutes on the vibration shaker. after that, the crumb particles were weighted with an accuracy of 0,01 g. crumb fragility was been determined as the relationship of crumb particles mass to the total mass of bread samples and was expressed in percent points. hydrophilicity of bread crumb the amount of moisture absorbed was been determined by the addition of water which was delivered by drops at total quantity of 17 cm 3 from the pipette during 5 minutes. water drops were delivered to the sample of 3 g of crumbs which was placed on the laboratory sieve. the sample moisture was been removed from the sieve and weighted. the amount of water absorbed by bread, in percent to dry measure, was been determined as follows:   , )100( 100100 2 21 wg gg v    (1) where g2 – sample mass after moisturisation, g; де g1 – sample mass before moisturisation, g; w – moisture mass fraction in bread, %. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 303 radiographic research effect of gums on the process of starch retrogradation in bread was investigated by radiographic procedures conducted on a 'дрон-ум' diffractometer. the pieces of bread were placed in the standard container for taking diffractograms of powder samples and were sealed in the container [17]. all laboratory samples (with gums and control sample) were roentgenographed in one regime. therefore, the results could be compared in terms of diffraction maximum (reflections) for the qualitative characteristics in the starch structure. diffractograms of the samples were first taken 16 hours after baking and after 7 days of storage of bread, because changes in diffractograms during the first 2-3 days are insignificant. they were compared with the diffractograms of the flour. water bond forms in bread study of the forms of moisture in the bread crumb and their relative change during storage were carried out by a model "derivatograph q-1000" differential-thermal analysis unit for temperatures ranging from 0 200 °c. samples weighing 1 g were heated at 1.25 °c min-1. differentialthermal analysis readings were taken 16 hours after baking and after 3 days of storage. analysis of temperature curves (ta) and mass change curves (tg), as well as their derivatives (dta, and dth, respectively) allowed to distinguish 4 temperature ranges with varying rates of moisture evaporation, which are seen as a measure of the strength of connection with the material. statistical analysis measurements of penetration values, crumb fragility and hydrophyllic qualities of crumb were carried out 5 times in 3 experimental series. experimental data were processed using the mathematical statistic methods within a 95 confidence interval %. roentgenographic and thermalgravimethrical measurements were triplicated with the averaging of data thus obtained. 3. results and discussion 3.1. bread crumb texture during storage as is widely accepted, determining bread crumb compression with a penetrometer is one of the most veracious indicators during the assessment of the freshness of bread [1, 2, 18]. as shown by our study (table 1), supplementing the dough with a mass fraction of gums in quantities of 0.25 and 0.5% marginally affects the overall, plastic and elastic deformation of crumb immediately after baking, but contributes to a significant improvement of overall and plastic strain indicators after 48 hours of storage, when compared to the control. elastic deformation of bread test samples does not differ from the control, though on the 72 hours of storage, this effect is effectively lost. this illustrates that gums can be used as additives in bread in order to constrain the process of staling during the first and second days of storage. the best staling retarding effect is observed with the use of tara and carob gums. a more pronounced effect of freshness preservation of baked goods is obtained when using 0.5% mass fraction of gums. it is known that during staling crumb pore walls lose their strength, which increases their tendency to crumble. increase of crumb pore fragility during storage lies at the base of currently used methodology for bread freshness evaluation. characteristics of hydrophilic colloids in bread are used as a basis for a crumb swelling in water method of freshness assessment [19]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 304 table 1 indicators of crumb deformation of bread with natural gum additives indicators control (no additives) with the addition of gums in the amount of 0.25% by weight of flour control (no additives) with the addition of gums in the amount of 0.5% by weight of flour guar tara carob guar tara carob total deformation of crumb, h1, instrument units after 4 hours 107±2 103±2 106±2 101±2 90±2 92±2 113±2 90±2 after 24 hours 56±1 73±1 74±1 76±1 45±1 52±1 57±1 59±1 after 48 hours 47±1 51±1 51±1 49±1 31±1 37±1 49±1 45±1 after 72 hours 35±1 34±1 37±1 33±1 30±1 33±1 38±1 35±1 plastic deformation of crumb, h2, instrument units after 4 hours 86±2 82±2 83±2 81±2 69±2 70±2 90±2 67±2 after 24 hours 45±1 60±1 61±1 62±1 35±1 41±1 46±1 48±1 after 48 hours 38±1 40±1 42±1 40±1 24±1 29±1 42±1 38±1 after 72 hours 28±1 27±1 32±1 27±1 23±1 26±1 30±1 28±1 elastic deformation of crumb, (h1-h2), instrument units after 4 hours 21±1 21±1 24±1 21±1 20±1 22±1 24±1 22±1 after 24 hours 11±1 13±1 13±1 14±1 10±1 11±1 11±1 11±1 after 48 hours 9±1 11±1 9±1 10±1 7±1 8±1 8±1 7±1 after 72 hours 6±1 7±1 5±1 6±1 7±1 6±1 7±1 7±1 it is also proven that bread staling is accompanied by a decrease of hydrophilic properties of its crumb [1]. it was determined that crumb fragility of bread samples with gum additives is lower than in controls, both after 24 hours of storage and after 72 hours (fig. 1). hydrophilicity of bread samples with gum additives is higher when compared to control (table 2) both 4 hours after baking and after 48 hours of storage. after 72 hours, this difference levels out. a more evident effect on crumb hydrophilicity was achieved with tara and carob gums. therefore, gums contribute to a prolongation of bread freshness within two days of storage when compared with a control sample. fig.1. change of bread crumb fragility during storage:1 control (no additives), 2, 3, 4 with addition of 0.5% guar, tara, carob gum respectively the role of additives in enhancing hydration and crumb fragility reduction was apparently caused by their ability to absorb moisture, possible creation of complexes with amylose starch and the influence on starch retrogradation. table 2 changes in the hydrophilicity of crumb of bread with gum additives during storage bread samples with the addition of gums in the amount of 0.25% by weight of flour amount of absorbed water,% of bread dry matter after 4 hours after 24 hours after 48 hours after 72 hours control (no additives) 300.0±5 284.5±5 258.5±5 249.4±5 with guar additives 334.5±5 317.8±5 298.9±5 239.2±5 with tara tree gum additives 340.8±5 319.6±5 309.3±5 256.6±5 with carob gum additives 344.0±5 323.6±5 299.0±5 246.7±5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 305 3.2. radiographic research it is widely known that during baking, starch granules undergo partial gelatinization, thus binding unbound moisture and water that is released as a result of protein coagulation. at the same time, starch partially changes from crystalline to amorphous state, its grains swell, causing an increase in volume. during storage of bread, the reverse process is known to take place. gelatinized starch transforms from amorphous state to partially crystalline, as its retrogradation takes place. radiographic studies of this process [1, 18] show that native flour starch results in diffractograms typical of crystalline structure of starch granules. during baking, starch partially gelatinizes, its structure undergoes changes, so diffractograms of the bread crumb show a combination of amorphous starch elements with elements in their crystalline state. diffractograms of stale bread crumb combine the elements of the previous two. the staler the bread, the closer the diffractogram is to that of flour. the diffractogram of stale bread has shown that starch returns into its initial crystalline condition, i.e. retrogrades. analysis of diffraction patterns obtained during research (fig. 2) shows a significant difference between diffractograms of freshly baked bread and wheat flour. diffractograms of the crumb structure of freshly baked bread without gum additives show domination by an x-ray amorphous phase both after 16 hours of storage (fig. 2a), and after 7 days (fig. 2b). 2θ – diffraction angles 1 – wheat flour, premium class 2 – bread with no additives (control) a) b) 2θ – diffraction angles r el at iv e m ax im um in te ns ity re la tiv e m ax im um in te ns ity 2θ – diffraction angles fig.2. bread crumb diffractograms of: a) bread 16 hours after baking, and b) bread 7 days after baking food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 306 a) b) а) b) а) b) fig. 2. continuation. bread crumb diffractograms with gum additives: а) bread 16 hours after baking; б) bread 7 days after baking 3– bread with addition 0,5 % guar gum 4 – bread with addition 0,5 % tara gum 5 – bread with addition 0,5 % carob tree gum r el at iv e m ax im um in te ns ity 2θ – diffraction angles 2θ – diffraction angles r el at iv e m ax im um in te ns ity 2θ – diffraction angles r el at iv e m ax im um in te ns ity 2θ – diffraction angles r el at iv e m ax im um in te ns ity 2θ – diffraction angles r el at iv e m ax im um in te ns ity 2θ – diffraction angles r el at iv e m ax im um in te ns ity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 307 this indicates that the starch of the sample is almost completely gelatinized. on diffraction diagrams of bread with gum additives, crystalline structure of starch remains evident, especially after 16 hours of storage. after 7 days diffractograms of bread with gum additives indicate a dominant presence of starch of amorphous structure. because, as is widely accepted [1, 18], the nature of bread crumb diffraction diagrams and the rate of crystal structure formation depends on the moisture of the starch gel, it can be assumed that due to significant water absorbing capability of natural gums less starch undergoes gelatinization, as can be clearly seen on diffractograms of bread with gum additives. probably, as a result of the lower quantity of free moisture, partial gelatinization of starch, in the process of bread with gums baking, takes place. during storage of this bread partially gelatinized starch removes moisture from gums and swells. with this its level of amorphousness increases. thus, the crumb of the bread with gums preserves its freshness significantly longer. so, prolongation of the freshness of bread with natural gum additives is associated with preservation or even increasing of amorphousness level of starch granules. 3.3. water bond form changes in bread during storage according to modern concepts, the process of staling is a set of several simultaneously flowing processes of starch retrogradation, denaturation of proteins and complex redistribution of moisture between the polymers in flour. the latter aspect is less studied and is quite controversial in terms of the distribution of moisture bond forms. the majority of conducted studies have determined that bread storage is accompanied by a decrease of bound moisture in the crumb [1, 5]. the influence of structure forming additives on changes in bond forms of moisture in bread during staling was not yet researched. in the present work the method of differential-thermal analysis has been applied, which appears to be the most objective and approved testing method. results transcription is given in table 3. during the first stage of moisture removal from food samples (table 3) there is a significant loss of water. in the temperature range, corresponding to the first stage of drying, unbound moisture, moisture in macroscopic capillaries and immobilized water are removed. the amount of moisture removed during the first stage of drying varied in the different samples 12.2 19.2% by weight of the sample, which amounts to 27 43% by weight of moisture in the dough. in samples of bread with gum additives, after 24 hours of storage, moisture content was lower when compared to the control sample of bread without additives. during storage, the amount of unbound water decreases, moreover in samples of bread with gum additives this reduction is less noticeable. in bread with carob gum additives, moisture content that corresponds to this temperature range virtually does not change during storage. in the second temperature interval, ranging from 96 105 °c, the rate of moisture change of the samples is slightly lower, which is obviously due to removal of moisture from micro capillaries. during this interval, smaller amounts of moisture are removed, amassing to 2.8 7.0% of sample mass or 6.3 15.7% of the total mass of water in the dough. moreover, content of this form of moisture bond in samples of bread with gum additives is also lower than in the control sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 308 table 3 differential thermal analysis of bread with natural gum additives during storage thus, the total amount of moisture that is removed in the 15 105°c temperature range for the control sample on the first day of measurements is 26% of the sample total mass, or 58.5% of the total mass of the content of water in the dough. for bread samples with gum additives, losses of moisture in the 15 105°c temperature range on the first day of measurements were 19.2 20, 8% of the sample total mass or 43.1 46.8% of the total mass of water in the dough. after 72 hours of the storage of the samples, moisture content of these bond forms in the control decreased by 4.4%, while moisture content in samples of bread with gum additives remained virtually unchanged. the third and fourth temperature intervals, ranging from 105 117 °c and 110 146°c respectively are marked with an endothermic peak on the dta curve, i.e., in these bounds an endothermic process takes place and it may be associated with the removal of moisture with substantial binding energy, such as osmotically bound moisture and adsorbed moisture. as can be seen from table 3, the amount of moisture removed during the third temperature range is higher for different samples of bread with gum additives by 1.8 3.2% when compared to a control sample of bread and amounts to 13.2 14.6% of the sample weight or 29.7 32.8% of the total mass of water in the dough. after 72 hours of storage, the amount of moisture of this form of bond in bread without additives is slightly reduced while reduction in samples of bread with gum additives is much more evident. bread with tara gum additives is an exception. amount of water bound by adsorption, which corresponds to the fourth temperature range, during the first day of storage of samples totals to 5.6 6.4% of the sample weight or 12.6 bread samples bread moisture loss during dehydration in different temperatures ranges interval i interval ii interval iii interval iv temperature, с moisture loss temperature с moisture loss temperature, с moisture loss temperature, с moisture loss mg % of sample mass % of over all mois ture mass mg % of sam ple mass % of over all mois ture mass mg % of sample mass % of over all mois ture mass mg % of sample mass % of over all moi s ture mass no additives (control) after 24 h 15-99 192 19.2 43.15 99-105 68 6.8 15.28 105-116 114 11.4 25.62 116-139 56 5.6 12.58 after 72 h 16-96 160 16.0 35.96 96-104 56 5.6 12.58 104-113 108 10.8 24.27 113-138 60 6.0 13.48 with 0,5% guar gum after 24 h 17-99 176 17.6 39.55 99-101 32 3.2 7.19 101-114 138 13.8 31.01 114-139 58 5.8 13.03 after 72 h 19-98 152 15.2 34.16 98-105 54 5.4 12.13 105-116 120 12.0 26.97 116-141 56 5.6 12.58 with 0,5% tara gum after 24 h 17-96 130 13.0 29.21 96-103 62 6.2 13.93 103-117 132 13.2 29.66 117-144 62 6.2 13.93 after 72 h 16101 122 12.2 27.42 0 0.0 0.00 101-119 210 21.0 47.19 119-146 62 6.2 13.93 with 0,5% carob gum after 24 h 17101 174 17.4 36.85 101102 28 2.8 6.29 102-114 146 14.6 32.81 114-139 60 6.0 13.48 after 72 h 18-98 166 16.6 37.30 98-104 70 7.0 15.73 104-114 102 10.2 22.92 114-137 64 6.4 14.38 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 309 14.4% of the total mass of water in the dough both in the control and samples with additives. after 72 hours of storage, the amount of water bound by adsorption in all of the samples of bread is almost identical. thus, the analysis of forms of moisture bonds in samples of bread with gum additives suggests that a reduction in the speed of staling of these samples is connected with a lower content of unbound moisture, lower content of water in macroand micro capillaries after baking and an increase of the amount of osmotically bound moisture. tara tree gum shows a profound slowing effect on staling, which correlates well to the data received about a decrease in moisture content that was removed from samples during the first and second temperature intervals. 4. conclusions use of natural gum additives in the amount of 0.25 0.5% of the weight of flour improves the elasticity of crumb, slows down staling, judging from the parameters of crumb deformation, crumb fragility and hydrophilicity. best improving effect is observed in the case of tara tree gum. a more evident effect on the preservation of the freshness of bread is achieved with gum dosages of around 0.5%. roentgenographic studies and studies of moisture bonds form during bread storage allowed an insight of the mechanism of natural gums' staling slowing effect. diffraction patterns obtained of bread with gum additives showed that prolongation of the freshness of bread with natural gums takes place due to the increase in amorphousness level of starch granules. the slowdown of staling that took place in the studied samples can be explained as a result of a lower content of unbound moisture, lower content of water in macroand micro capillaries at the beginning of the storage period and an increase of the amount of osmotically bound moisture. 5. acknowledgments research was conducted by the authors in collaboration with scientists from the institute of colloid and water chemistry of national academy of sciences of ukraine. 6. references [1] horyacheva, a.f., kuzminskiy, r.v.. bread freshness extension. moscow: light and food industry . – 240 р. (in russian). (1983) [2] drobot, v.i.. technology of baking producing. kiev: logos. – 365 р. (in ukrainian). (2002) [3] kim, l.v. about bread staling. baking and pastry industry, vol. 5, рр. 19 – 22. (in russian). (1975). [4] knyahinichev, m.i., horyacheva, a.f., ignashyna, n.m. research of the changes in bread during storage. baking and pastry industry, vol. 11, рр. 8 – 9. (in russian). (1970). [5] puchkova, l.i., sugrobova, l.l., arkhangelskiy, i.v. the state of water in bread crumb and its components. bread and pastry industry, vol. 10, рр. 31 – 32 (in russian). (1980). [6] rodge, a.b, sonkamble, s.m, salve, r.v, syed imran hashmi effect of hydrocolloid (guar gum) incorporation on the quality characteristics of bread. food processing&technology volume 3, issue 2, 2012, p. 2 – 7. (2012). [7] rosell, c. m., rojas, j. a., & benedito de barber, c. influence of hydrocolloids on dough rheology and bread quality. food hydrocolloids, 15, рр. 75–81. (2001). [8] kohajdova z., koravicova j.. influence of hydrocolloids on quality of baked goods. asta sci., pol., technol., aliment., 7 (2), рр. 43 – 49. (2008) [9]. andreev, a.n., shylenko, a.v.. influence of the hydrocolloids vakuceli and guar gum on the quality of enriched bread. problems of technique and technologies of food production, рр. 148 – 153. (in russian). (2005) [10]. arsenieva, l.y. the developing of bread from poor gluten content flour by means of hydrophilling additives using (doctoral dissertation). kiev technological institute of food industry. – 20 p. (in russian). (1987). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 svitlana palyvoda, vira urchak, tatjana golikova, veniamin fomenko, study of the mechanism of the retarding effect on bread staling by using natural gums , food and environment safety, volume xii, issue 4 – 2013, pag. 300-310 310 [11]. kushnir y. substances that increase adhesive and water bounding ability. products&ingredients, vol. 6, рр. 20 – 22. (in russian). (2004). [12]. nechaev, a.p., kochetkova, a.b., zaicev, a.n.. food additives. moscow: kolos, kolos-press. – 256 р.(in russian). (2002) [13]. despinoy p., ormenese r. c. s. c., faria e.v. the use of guar or xanthan gums in the production of “light” low fat cakes. zambranofrancy. int. j. food sci. and technol., 39, vol.9, p. 959–966. (2004). [14] mandala i.g.. physical properties of fresh and frozen stoped, microwavereheated breads, containing hydrocolloids. j. food eng., vol. 3, рр.. 291 – 300. (2005) [15] phillips, g.o., williams, p.a.. hydrocolloids. s. –petersburg: гиорд. – 536 р. (in russian). (2006) [16] funami t., kataoka y., omoto t., goto y., asai i., nishinari k.. effects of non-ionic polysaccharides on the gelatinization and retrogradation behavior of wheat starch. food hydrocolloids, 19, 1–13. (2005) [17] gorelyk, s.s., rastorguev l.n., skakov, y.a.. roenthgen and electronical optical analysis. – 368 p. (in russian). (1970) [18] auerman, l.y. technology of baking production. moscow: food industry. – 515 р. (in russian). (2010) [19] martynenko, n.s. research of the improvers’ effect to prolonging of the wheat bread shelf life (doctoral dissertation). moscow (in russian). moscow: kolos. – 23 р. (in russian). (1976) articol porumb 131 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 131 140 the influence of culinary processing on the content of some secondary metabolites in flint corn seeds *marcel avramiuc1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received 25th april 2016, accepted 30th may 2016 abstract. the influence of culinary processing on the content of phenolic and phytic acid in flint corn seeds, belonging to ten romanian local populations, was the purpose of this paper. the biological material, used in experiments, was represented by flint corn seeds (zea mays l. var. indurata) belonging to ten romanian local populations. there were conducted three culinary processing of seeds (boiling, steaming and roasting,) followed by the determination of total phenolic content and phytic acid in thermally processed grains and the water used. the culinary processing of corn seed samples has significantly influenced the seeds’ content in phenols and the phytic acid. thus, both the boiling and the steaming process led to the decrease of phenolic compounds and of the phytic acid in corn seeds, through thermal destruction (more) and through releasing into used waters (less), which could be the sources of such metabolites. the roasting process has generally led to significant increase of phenolic compounds, and to decrease of phytic acid. keywords: culinary, phenolic content, phytic acid, corn seeds 1. introduction consumption of fruits, vegetables, and unpolished grains is strongly associated with the reduced risk of developing chronic diseases such as cancer and cardiovascular disease [1,2] cited by [5]. phytochemicals in food are beneficial to health due to their high antioxidant properties, which is related to prevention of cardiovascular disease, diabetes or obesity and mutagenesis or cancer [3,4] cited by [5]. plant phenolics have potential health benefits mainly due to reactive oxygen species scavenging and inhibition, electrophile scavenging, and metal chelation [6]. the interest in antioxidant activities, bioactive properties and anti-radical scavenging activities of anthocyanins and phenolics has increased [7,4], being, according to epidemiological and in vitro research [8], inverse relationships between the incidence diseases such as cardiovascular, cancer and cataracts, that come with aging, and consumption of vegetables and fruits containing anthocyanins and phenolics. like other secondary metabolites (anthocyanins and carotenoids), phenolics are influenced by plant species and varieties [9,10,11,12,13], and are primarily synthesized through the pentose phosphate pathway (ppp), shikimate and phenylpropanoid pathways [14]. phenolic compounds exhibit pharmacological properties such as: antitumor, antiviral, antimicrobial, antiinflammatory, hypotensive and http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 132 antioxidant activity [15,16], being a relationship between the consumption of phenolic-rich food products and a low incidence of coronary heart disease, atherosclerosis, certain forms of cancer and stroke [17,18,19,20]. the phytic acid (inositol hexaphosphate or phytate) is a major component of all plant seeds [21,22], and in cereal grains, most of the minerals form salts with this acid, that act as a storage compound of phosphorous [23]. phytate inhibits lipid peroxidation by forming a complex in which all six coordination sites of fe (iii) are occupied by phytate [24]. according to shamsuddin and ullah (1989), the phytic acid can have anticancer effects at both the initiation and promotion stages of colon cancer; it significantly reduced colon tumor number, size and mitotic rate, when compared to the control group. considered as antinutritional factors, because they interact with food constituents such as minerals which make them unavailable [26,27], phytate and polyphenols can be reduced by processing methods such as: soaking, sprouting, cooking, malting and fermentation [28,27,29,30,31,32]. corn (zea mays ssp. mays) is a major cereal used to produce grain and fodder that are the basis for a number of foods, feed, pharmaceutical and industrial products. due to its adaptability and productivity, it is the third most cultivated field crop after wheat and rice [14]. in our diets, corn is a source of macroand micronutrients [33,34], a rich source of many phytochemicals, including carotenoids [34,35,36,37,38,13,39], phenolic compounds [34,35,40,41,13,39], anthocyanins [35,41,42], tocopherols [34,43] and phytic acid, which have multiple functional roles, for example, as antioxidants [35,11,12,24], as antimutagens [11], and as inhibitors of colorectal carcinogenesis [11,10,25]. cooking induces changes in physiological and chemical composition, influencing the concentration and bioavailability of bioactive compounds in food [44]. turkmen et al. (2005) found that thermal treatments decreased the total phenolics in squash, peas and leek, while in sweet corn cooking led to an increase in the level of phenolic compounds [45]. according to oliveira et al., 2010 [65], cited by [46], to understand better the effects of cooking on antioxidant levels in food, it is necessary to test real cooking conditions, because the behavior of any food cannot be predicted. in this paper there was studied to what extent the culinary processing influences the content of phenolic compounds and phytic acid in corn seeds, belonging to ten romanian local populations. 2. experimental 2.1. research materials the biological material, used in this work, was represented by flint corn seeds (zea mays l. var. indurata) belonging to ten romanian local populations (lp). working and control samples from each lp grains were made up, using dried seeds (moisture content = 8-10 %) coming from crops of the last two years. 2.2. procedure and research methods three culinary processings were conducted, as follows. for boiling, there were taken 100 corn seeds of each sample, which have been placed in a stainless steel vessel with three liters capacity. the boiling was done in one liter of tap water in the pot covered, for 30 minutes (timed from the moment when the water began to boil). for steaming, in each pot, a dense mesh fixed to the walls was mounted, to twofood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 133 thirds of the vessel bottom. after pouring of one liter of tap water into the bowl, on the sieve were placed 100 seeds that were steamed, within covered pot, for 30 minutes (timed from the moment when the water began to boil). for roasting, 100 seeds from each sample were roasted for 20 minutes at 150-190°c, in a covered stainless steel vessel. in order to determine total phenolic content (tpc), first an extract for each seeds sample was obtained, weighing 1 g of grains, which were finely ground and subjected to extraction with a mixture methanol and water (80/20), by stirring, centrifuging and recovering the supernatant [47]. the estimation of total phenolic contents in seeds extract was carried out through a colorimetric assay, by measuring its reducing capacity with folinciocalteu reagent. tpc was expressed as mg gallic acid equivalent/g dry seeds ie dry weight (mg gae/g dw). for this purpose, a standard curve was generated, representing the absorbance values of gallic acid standard solutions in relation to their concentrations [48]. the phytic acid (pa) determination was done using garcia-villanova method, based on treating the sample with a ferric salt and titration of fe3+ ions (which has not precipitated with phytic acid), with edta solution, in the presence of sulfosalicylic acid as an indicator [49]. for this assay, 1 g of grains was finely ground, adding hcl-na2so4 solution for phytic acid extraction. after some operations (filtration, adding of hcl-na2so4, clorhidric solution of feric clorure, and sulfosalicylic acid solution, boiling, filtration) the mixture obtained was heated at 70°c and fe3+ ions were titrated (la cald), according to the method above mentioned. phytic acid content was expresed as mg per 100 g dry weight (mg% dw). 2.3. statistical analysis. the data of experiments, consisting in four replicates for each determination, were statistically processed using sas version 8.02 [50]. in order to analyze the significance of differences among samples, generalized linear model analysis was carried out, and for multiple comparisons was used duncan’s multiple range test (p<0.05). 3. results and discussion in the table 1 are rendered the values of the total phenolic content (tpc) in the ten flint corn seed samples, subjected to culinary processing. table 1 comparative values of the total phenolic content in flint corn seeds culinary processed corn local populations (lp) tpc (mg gae/g dw) raw material* boiled steamed roasted seeds seeds water seeds water lp1 (white)*** 7.5±0.5c** 4.3±0.6e** 0.9±0.07fg 6±0.8d 0.2±0.02g 8.6±0.9c lp2 (yellow) 8.2±0,7c 4.9±0.3de 0.8±0.05fg 6.5±0.3d 0.3±0.03g 9.1±0.6bc lp3 (yellow) 7.9±0.4c 4.7±0.8de 1.1±0.09fg 6.2±0.7d 0.2±0.01g 9.4±0.7bc lp4 (red) 8.7±0.9bc 4.9±0.5de 0.8±0.06fg 7.1±0.5cd 0.3±0.05g 8.5±0.5c lp5 (purple) 9.1±0.6bc 5.3±0.6de 0.9±0.06fg 6.9±0.3cd 0.5±0.06g 10.2±0.8b lp6 (white) 6.5±0.7d 4.1±0.2e 0.6±0.07g 5.3±0.6cd 0.3±0.01g 7.4±0.5cd lp7 (red) 10.2±0.8b 5.9±0.4d 1.2±0.06fg 8.1±0.7c 0.4±0.02g 11.8±1.2a lp8 (red) 8.8±0.6bc 5.3±0.6de 1.1±0.08fg 7±0.4cd 0.2±0.03g 9.7±0.8b lp9 (yellow) 6.8±0.8cd 3.8±0.5e 0.9±0.07fg 5.6±0.7d 0.2±0.04g 7.5±0.6c lp10 (purple) 9.7±0.9b 5.9±0.7d 1.2±0.09fg 7.7±0.9c 0.3±0.05g 9.5±0.9bc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 134 *unprocessed seeds; **means with different letters are statistically diferent (p<0.05); ( )***kernel colour as seen from tab. 1, the total phenolic content (tpc) in seeds of the ten maize local populations (lp) ranged between 6.5±0.7 and 10.2±0.8 mg gae/g dw, the highest values being found in red and purple grains. according to some authors [13,39,51], cited by [5], the pigmented corn contains (more) anthocyanins, carotenoids, phenolic compounds, and antioxidant activity than non-pigmented corn compared with control samples (raw seeds), tpc of seeds has been modified after processing by boiling, steaming or baking (roasting). thus, boiling for 30 minutes led to a significant decrease (p<0.05) of the total content of phenolic compounds from the seeds of lp, with percentages between 38% (lp6) and 43% (lp4 and lp9). analyzing the boiling water of seeds, the presence of phenolic compounds was found, whose percentages were between 10% (lp2, lp4, lp5 and lp6) and 14% (lp3 and lp9). gathering (mathematically) the content of phenolic compounds from the seeds of each boiled sample and from its boiling water and comparing it with the control (unprocessed seeds), significant differences were found (p<0.05), whose explanation is that a part of the phenolic compounds was destroyed by cooking. these losses of total content of phenolic compounds in the analyzed samples ranged between 26% (lp3) and 33% (lp4), i.e. about 2.5 times greater than phenols released in the boiling water. these first results of this work are consistent with some data reported by some researchers. thus, studying antioxidant components, antioxidant activity, and their changes during traditional cooking of fresh purple waxy corn, harakotr et al. (2014a) found a higher content of phenolics in the boiling water, than in steaming one, attributed, by authors, to the losses of phenolic in cooking water. since the sum of phenolics in cooked samples and cooking water consistently differed from their content in raw samples, differences in phenolic content could be, by harakotr et al. (2014a), due to breakdown of phenolics, which was greater than losses due to leaching into cooking water. regarding seeds steaming, the table 1 shows that this thermal processing for 30 minutes significantly reduced (p<0.05) the total phenols from analyzed seed samples, with percentages between 17% (lp9) and 24% (lp5) . the analysis of water used for seeds steaming, showed the presence of phenolic compounds, with percentages ranging from 2% (lp8) and 5% (lp5 and lp6). gathering (mathematically) the content of phenolic compounds from the seeds of each steamed sample and from its steaming water, and comparing it with the control (raw seeds), significant differences were (p<0.05) caused by the destruction of phenolic compounds through steaming. the phenolic compounds losses, through this type of thermal processing, have ranged between 13% (lp6) and 19% (lp3 and lp5), i.e. about 4-6 times greater than phenols released into steaming waters. comparing the results of the two types of thermal processing, we can see that steaming has caused losses of phenolic compounds significantly lower (p<0.05) within analyzed corn seeds. reffering to corn seeds, harakotr et al. (2014a), reported that boiling had a more detrimental effect on phenolic acids than steaming. the boiling treatment causes of disruption cellular components with the consequent release of these molecules into the cooking water ([52], cited by [46]), while steaming food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 135 treatment causes matrix softening and increases extractability of antioxidant components from the raw materials [53]. by xu and chang, (2009), the thermal treatment could cause changes in phenolic substances, in the corn, by liberating the bound phenolic compounds into free form. analyzing the total content of phenolic compounds in roasted corn seeds and comparing them with control samples (raw seeds), it appears that, except three samples (lp1, lp4, and lp10), in all other samples this parameter has registered significant increases (p<0.05), with percentages between 10.2% (lp8) and 19% (lp3). this result is consistent with observations and data reported in some scientific papers. thus, han and koh (2011), researching antioxidant activity of hard wheat flour, dough and bread prepared using addition of different phenolic acids, found that the antioxidant activity and residual free phenolic acid content of flour were reduced by mixing, but increased by fermentation and baking. by cheng et al. (2006), the thermal treatment causes phytochemical degradation, oxidation, and maillard reactions resulting in changes in antioxidant property. maillard reaction products may protect phytochemicals from oxidation [57], and those products, derived from mixtures of glucose or fructose and cysteine or glutathione, greatly inhibit activities of polyphenoloxidases and oxidoreductases [58]. heat treatment at 150°c for 40 min. liberated bound phenolics in citrus peels having as result a significant increasing of tpc after treatment [59]. in the table 2 are rendered the values of the phytic acid content in the ten flint corn seed samples, subjected to culinary processing. table 2 comparative values of the phytic acid content in flint corn seeds culinary processed corn local polulations (lp) phytic acid (mg% dw) raw material* boiled steamed roasted seeds seeds water seeds water lp1 (white)*** 0.9±0.02b** 0.6±0.05bc** 0.15±0.01c 0.8±0.09b 0.05±0.002d 0.72±0.09bc lp2 (yellow) 0.7±0.05bc 0.5±0.07bc 0.1±0.02c 0.6±0.03bc 0.04±0.005d 0.55±0.03bc lp3 (yellow) 1.8±0.07a 1.2±0.15b 0.4±0.03bc 1.6±0.12ab 0.09±0.01cd 1.31±0.15ab lp4 (red) 1.3±0.09ab 1±0.09b 0.15±0.02c 1.2±0.08b 0.06±0.004cd 0.97±0.1b lp5 (purple) 1.2±0.14b 0.9±0.06b 0.2±0.05c 1±0.1b 0.08±0.01cd 0.94±0.08b lp6 (white) 0.9±0.07b 0.7±0.09bc 0.15±0.01c 0.8±0.05b 0.04±0.006d 0.76±0.05bc lp7 (red) 1.5±0.08ab 1±0.16b 0.3±0.02c 1.4±0.16ab 0.06±0.002cd 1.14±0.09b lp8 (red) 1.7±0.06a 1.3±0.11ab 0.3±0.05c 1.5±0.12ab 0.1±0.04c 1.34±0.14ab lp9 (yellow) 0.8±0.09b 0.6±0.02bc 0.1±0.01c 0.7±0.08bc 0.08±0.005cd 0.64±0.03bc lp10 (purple) 1.9±0.08a 1.4±0.17ab 0.2±0.04c 1.8±0.2a 0.06±0.007cd 1.46±0.16ab *dried seeds; **means with different letters are statistically diferent (p<0.05); ( )***kernel colour tab. 2 shows that, compared with the control samples (raw seed), the boiling process for 30 minutes led to a significant decrease (p<0.05) of the phytic acid content in seeds of eight lp, with percentages between 18% (lp9) and 32% (lp3). analyzing the phytic acid content in the boiling water of seeds, the presence of this compound was found, whose percentages were between 12% (lp4) and 20% (lp3). adding the phytic acid content from seeds of each sample and from its boiling water, and comparing it to the control sample (raw seeds), significant differences were found (p <0.05), explained by the destruction of this compound through boiling. these losses (percentage) of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 136 phytic acid in the analyzed samples ranged between 5% (lp9) and 13% (lp5 and lp7), of about 1.5-2.4 times lower than phytic acid released into the boiling waters. gupta and sehgal (1991) have observed a decrease in phytic acid contents of cereal grains used for preparing weaning foods as a result of soaking and germination. the decrease in the level of phytic acid during soaking may be attributed to leaching the acid out into soaking water under the concentration gradient [26,32, cited by 61]. according to sathe and venkatachalam (2002), being soluble in water, phytate can be reduced if a soaking step is carried out at temperatures above 45°c but below 60°c, a significant percent of phytate hydrolysis can take place due to activation of endogenous phytases and acid phosphatases, during the early part of the cooking phase. if we take into consideration those mentioned by sathe and venkatachalam (2002), in the experiment of this work, the content of phytic acid in boiled seeds would be reduced during their immersion, at temperatures below 60°c, through the phytate hydrolysis caused by phytases and endogenous acid phosphatases activated during this part of the cooking process. it is about the phytate identified in the boiling water. but phytic acid losses should be attributed to its destruction by heat treatment (boiling). from tab. 2 one can note that, through seeds steaming for 30 minutes, the content of phytic acid was significantly reduced (p<0.05) in seeds of only four samples (from ten analyzed), with percentages between 8% (lp3) and 15% (lp9). the analysis of water used to seeds steaming showed the presence of phytic acid, with percentages between 3% (lp10) and 10% (lp9). adding the phytic acid content from the seeds of each steamed sample and from its steaming water, and comparing it with the control (raw seeds), significant differences were found (p<0.05) caused by the phytate destruction. the losses of this compound, by means of this type of thermal processing, have ranged between 3% (lp3) and 8% (lp8), i.e. about 2-2.5 times lower than phytate released into steaming waters. if we compare the content of phytic acid in roasted seeds with control samples (raw seeds), one can see that this parameter has registered significant decrease (p<0.05), with percentages between 15% (lp6) and 27% (lp3). this result is consistent with observations and data reported by some authors. according to fretzdorff and brümmer (1992), the ph and cooking temperature are important factors reducing the content of phytic acid, because in doughs with ph 4.3–4.6 phytic acid is more effectively reduced than in doughs with higher ph. in roasted sliced french toast the baking temperature has reduced phytic acid content by 86% [64]. by some authors [66,67,68,69,70,71,72] cited by [73], the possible beneficial effects of food phytates include lowering of serum cholesterol, triglycerides and protection against certain diseases such as cardiovascular diseases, renal stone formation, and certain types of cancers. because phytates can chelate minerals (copper, iron, zinc, magnesium etc.), and many of these minerals are essential cofactors for numerous oxidoreductases, phytate may act as antioxidant in vivo [74,75,76], whose activity would be responsible for free radical scavenging action in vivo, leading to anticancer activity [69]. according to studies on animals and humans, the phytates decrease mineral bioavailability through forming complexes with these minerals [77,78]. based on the result obtained in the experiments of this work, all culinary processing used on corn seeds (boiling, steaming and roasting) may contribute, by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 137 means of phytate reduction, to the increase of the minerals bioavailability. 4. conclusions the culinary processing of corn seeds, belonging to ten local populations of flint corn (zea mays l. var. indurata) has significantly influenced the phenols and the phytic acid seeds content. thus, as compared to control samples, the thermal processing of corn seeds by boiling and steaming for 30 minutes, resulted in significant losses (mainly through thermal destruction), higher at boiling, both of phenolic compounds and of phytic acid. in cooking water the presence of these compounds was found (more in boiling and less in steaming ones), thus these waters can be exploited as sources of such metabolites. in the case of roasted corn seeds (20 min. at 150-190°c), compared to the control samples, the phenolic compounds have generally registered significant increases, apparently due to maillard reaction products, which have protected phenols against oxidation, on the one hand, and due to the release of bound phenols, by the action of high temperatures and exposure time, on the other hand. regarding the phytic acid, the roasting of maize seeds, in the same experimental conditions, has led to significant reductions of this metabolite concentration in all maize samples analyzed. by careful choice of culinary processing methods of maize seeds would retain a higher level of useful metabolits. 5. acknowledgments many thanks to suceava genebank and to suceava agricultural research and development station for the biological material provided. 6. references [1]isabelle m, lee b.l., lim m.t., koh w.p., huang d., ong c.n. antioxidant activity and profiles of common fruits in singapore. food chem 123, 77–84 (2010) [2]liu r.h. potential synergy of phytochemicals in cancer prevention: mechanism of action. j. nutr 134, 3479s–3485s (2004) [3]he j., giusti mm. anthocyanins: natural colorants with health-promoting properties. annu rev. food sci. technol. 1, 63–187 (2010) [4]tsuda t., horio f., uchida k., aoki h., osawa t. dietary cyanidin 3-0-b-d glucosiderich purple corn color prevents obesity and ameliorates hyperglycemia in mice. j. nutr. 133, 2125-2130 (2003) [5]harakotr b., suriharn b., scott m.p., lertrat k. genotypic variability in anthocyanins, total phenolics, and antioxidant activity among diverse waxy corn germplasm. euphytica doi 10.1007/s10681-014-1240-z, springer science+business media dordrecht (2014b) [6]huang m.t., ho c.t., lee c.y. – phenolic compounds in food and their effects on helth ii: antioxidants and cancer prevention. american chemical society symposium series 507. washington, dc: american chemical society, pp 2-7 (1992) [7] setchell k.d.r., aedin c. j. nutr. vol. 129, p. 758-767 (1999) [8]ames b.n., shigenaga m.k. and hagen t.m. p. natl. acad. sci. usa vol. 7915-7922, p. 90 (1993) [9]abdel-aal e.-s.m., hucl p. a rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats. cereal chem., 76, 350–354 (1999) [10]zhao z.h., egashira y., sanada h. phenolic antioxidants richly contained in corn bran are slightly bioavailable in rats. j.agric.food chem., 53, 5030–5035 (2005) [11]pedreschi r., luis c.-z. antimutagenic and antioxidant properties of phenolic fractions from andean purple corn (zea mays l.). j. agric. food chem., 54, 4557–4567 (2006) [12]li w., wei c.v., white p.j., beta t. high-amylose corn exhibits better antioxidant activity than typical and waxy genotypes. j. agric. food chem., 55, 291–298 (2007) [13]lopez-martinez l.x., oliart-ros r.m., valerio-alfaro g., lee c.-h., parkin k.l., garcia h.s antioxidant activity, phenolic compounds and anthocyanins content of eighteen strains of mexican maize. lwt-food sci. technol. 42, 1187–1192 (2009) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 138 [14]randhir reena, shetty kalidas developmental stimulation of total phenolics and related antioxidant activity in lightand darkgerminated corn by natural elicitors. process biochemistry, 40, 1721-1732 (2005) [15]cowan m.m. plant products as antimicrobial agents. clin. microbiol. rev. 12, 564-582 (1999) [16]shetty k. biotechnology to harness the benefits of dietary phenolics: focus on lamiaceae. asia pac. j. clin. nutr. 21, 79-102 (1997) [17]hertog m.g.l., fesrens e.j.m., hollmann p.c.h., katan m.b., krombout d. dietary antioxidant flavonoids and risk of coronary hearth disease: the zutphen elderly study. lancet 342, 1007-1011 (1993) [18]diaz m.n., frei b., vita j.a, keaney j.f. antioxidants and atherosclerotic heart disease. new engl. j. med., 337, 408-416 (1997) [19]ito n., hirose m. antioxidantscarcinogenic and chemo-preventive properties. adv. cancer res. 53, 247-302 (1989) [20]ness a.r., powles j.w. fruit and vegetables and cardiovascular disease: a review. int. j. epidemiol. 26, 1-13 (1997) [21]dendougui f., schwedt g. in vitro analysis of binding capacities of calcium to phytic acid in different food samples, european food research and technology © springer-verlag 2004 10.1007/s00217-004-0912-7 original paper, published online: 27 july 2004 [22]kamao m., tsugawa n., nakagwa k. – j. nutr. sci. vitaminol. 46, 34–41 (2000) [23]nielsen m.m., damstrup m.l., thomsen a.d., rasmussen s.k., hansen å. phytase activity and degradation of phytic acid during rye bread making, european food research and technologyzeitschrift für lebensmitteluntersuchung und forschung a © springerverlag 2006 10.1007/s00217-006-0397-7 original paper, published online: 26 july 2006 [24]graf, e. and eaton, j.w. antioxidant functions of phytic acid. free radicals in biol. med. 8, 61–69 (1990) [25]shamsuddin, a.m. and ullah, a. inositol hexaphosphate inhibits large intestinal cancer in f344 rats 5 months after induction by azoxymethane. carcinogenesis 10, 625–626 (1989) [26]abd el rahaman s.m., el maki h.b., idris w.h., hassan a.b., babiker e.e., el tinay a.h. antinutritional factors content and hydrochloric acid extractability of minerals in pearl millet cltivars as affected by germination. int. j. food sci. nutr. 58, 6-17 (2007) [27]idris w.h., abd el rahaman s.m., el maki h.b., babiker e.e., el tinay a.h. effect of malt pretreatment on phytate and tannin level of two sorghum (sorghum bicolor) cultivars. int. j. food sci. technol 41, 1229-1233 (2006) [28]el maki h.b., babiker e.e., el tinay a.h. changes in chemical composition, grain malting, starch and tannin contents and protein digestibility during germination of sorghum cultivars. food chem. 64, 331-336 (1999) [29]lewu m.n, adebola p.o., afolayan a.j. effect of cooking on the mineral contents and anti-nutritional factors in seven accessions of colocasia esculenta (l.) schott growing in south africa. j. food comp. anal. 23, 389-393 (2010) [30]obizoba i.c, atii j.v. evaluation of the effect of processing techniques on the nutrient and antinutrients contents of pearl millet (pennisetum glaucum) seeds. plant foods hum. nutr. 45, 23-34 (1994) [31]osman m.a. effect of traditional fermentation process on the nutrient and antinutrient contents of pearl millet during preparation of lohoh. j. saudi soc. agric. sci. 10, 1-6 (2011) [32]vadivel v., stuetz w., scherbaum v., biesalski h.k. total free phenolic content and health relevant functionality of indian wild legume grains: effect of indigenous processing methods. j. food comp. anal. 24, 935-943 (2011) [33]gonzalez r., reguera e., figueroa j.m., sanchez-sinencio f. on the nature of the ca binding to the hull of nixtamalized corn grains. lebensm.-wiss. technol. 38, 119–124 (2005) [34]chander s., meng y., zhang y., yan j., li j. comparison of nutritional traits variability in selected eighty-seven inbreds from chinese maize (zea mays l.) germplasm. j. agric. food chem. 56, 6506–6511 (2008) [35]de la parra c., serna saldivar s.o., liu r.h. effect of processing on the phytochemical profiles and antioxidant activity of corn for production of masa, tortillas, and tortilla chips. j. agric. food chem. 55, 4177-4183 (2007) [36]kopselld d.a., armel g.r., mueller t.c., sams c.e., deyton d.e., mcelroy j.s., kopsell d.e. increase in nutritionally important sweet corn kernel carotenoids following mesotrione and atrazine applications. j. agric. food chem. 57, 6362–6368 (2009) [37]kean e.g., hamaker b.r., ferruzzi m.g. carotenoid bioaccessibility from whole grain and degermed maize meal products. j. agric. food chem. 56, 9918–9926 (2008) [38]li s.s., tayie f.a.k., young m.f., rocheford t., white w.s. retention of provitamin a carotenoids in high β-carotene maize (zea mays) during traditional african household food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 139 processing. j. agric. food chem. 55, 10744–10750 (2007) [39]montilla e.c., hillebrand s., antezana a., winterhalter p. soluble and bound phenolic compounds in different bolivian purple corn (zea mays l.) cultivars. journal of agricultural and food chemistrry, 59, 7068-7074 (2011) [40]santiago r., reid l.m., arnason j.t., zhu x.y., martinez n., malvar r.a. phenolics in maize genotypes differing in susceptibility to gibberella stalk rot (fusarium graminearum schwabe). j. agric. food chem. 55, 5186-5194 (2007) [41]pedreschi r., cisneros-zevallos l. phenolic profiles of andean purple corn (zea mays l.). food chem., 100, 956–963 (2007) [42]li c.-y., kim h.-w., won, s.-r., min h.k., park k.-j., park j.-y., ahn m.-s., rhee h.-i. corn husk as a potential source of anthocyanins. j. agric. food chem. 56, 11413– 11416 (2008) [43]ibrahim k.e., juvik, j.a. feasibility for improving phytonutrient content in vegetable crops using conventional breeding strategies: case study with carotenoids and tocopherols in sweet corn and broccoli. j. agric. food chem. 57, 4636–4644 (2009) [44]turkmen n., sari f., velioglu y.s. the effect of cooking methods on the total phenolics and antioxidant activity of selected green vegetables. food chem., 93, 713-718 (2005) [45]dewanto v., wu x., liu r.h. processed sweet corn has higher antioxidant activity. journal of agricultural and food chemistry, 50, 4959-4964 (2002) [46]harakotr b., suriharn b., tangwongchai r., scott m. p., lertrat k. anthocyanin, phenolics and antioxidant activity changes in purple waxy corn as affected by traditional cooking, food chemistry, 164, 510–517 (2014a) [47]adom, k.k., liu r.h. antioxidant activity of grains. j. agric. food chem. 50, 6182–6187, (2002) [48]moore jeffrey and yu liangli (lucy) methods for antioxidant capacity estimation of wheat and wheat-based food products in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 147-150 (2008) [49]bordei d. (coord.), bahrim g., pâslaru v., gasparotti c., elisei a., banu i., ionescu l., codină g quality control in the bakery industry. analytical methods. ed. academica, galaţi, pp: 236-237 (2007) [50] sas institute (2005) sas user’s guide. statistical analysis system institute, cary, nc, [51]žilić s., serpen a., akilhoğlu g, gōkmen v., vančetović j. phenolic compounds, carotenoids, anthocyanins and antioxidant capacity of colored maize (zea mays l.) kernels. j agric. food chem. 5, 1224–1231 (2012) [52]miglio c., chiavaro e., visconti a., fogliano v., & pellegrini n. effects of different cooking methods on nutritional and physicochemical characteristics of selected vegetables. journal of agricultural and food chemistry, 56, 139–147 (2008) [53]huang y.c., chang y.h., & shao y.y. effects of genotype and treatment on the antioxidant activity of sweet potato in taiwan. food chemistry, 98, 529–538 (2006) [54]xu b. and chang s.k.c. total phenolic, phenolic acid, anthocyanin, flavan-3-ol, and flavonol profiles and antioxidant properties of pinto and black beans (phaseolus vulgaris l.) as affected by thermal processing. journal of agricultural and food chemistry, 57, 4754–4764 (2009) [55]han hye-min and koh bong-kyung antioxidant activity of hard wheat flour, dough and bread prepared using various processes with the addition of different phenolic acids. j. sci. food agric. 91: 604–608 (2011) [56]cheng z., su l., moore j., zhou k., luther m., yin j.j., yu l. j. agric. food chem., 53, 2433–2440 (2006) [57]lin c.j., guo g., mennel d.l. effects of postharvest treatments, food formulation, and processing conditions on wheat antioxidant properties in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 78-79 (2008) [58]billaud c., maraschin c., peyratmaillard m.n., nicolas j. ann. n. y. acad. sci. 1043, 876–885 (2005) [59]jeong s.-m., kim s.-y., kim d.-r., jo s.c., nam k.c., ahn d.u., lee s.-c. effect of heat treatment on the antioxidant activity of extracts from citrus peels. journal of agricultural and food chemistry, 52, 3389-3393 (2004) [60]gupta c., sehgal s. development, acceptability and nutritional value of weaning mixtures. plant foods hum. nutr. 41, 107-116 (1991) [61]sokrab a.m., mohamed ahmed i.a., babiker e.e. effect of germination on antinutritional factors, total, and extractable minerals of high and low phytate corn (zea mays l.) genotipes. journal of the saudi society of agricultural sciences, 11, 123-128 (2012) [62]sathe s.k., venkatachalam m. influence of processing technologies on phytate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 marcel avramiuc, the influence of culinary processing on content of some secondary metabolits in flint corn seeds, volume xv, issue 2 – 2016, pag. 131 – 140 140 and its removal in: food phytates, ed. by rukma reddy, shridhar k. sathe, crc press llc, 2000 n.w. corporate blvd., boca raton, florida 33431, (2002) [63]fretzdorff b., brümmer j.-m. cereal chem. 69, 266–270 (1992) [64] garcía-estepa r.m., guerrahernández e., garcía-villanova b. food res. int. 32, 217–221 (1999) [65]oliveira c., amaro l.f., pinho o., ferreira i.m.p.l.v. cooked blueberries: anthocyanin and anthocyanidin degradation and their radical-scavenging activity. journal of agricultural and food chemistry, 58, 9006-9012 (2010) [66]thompson l.u. potential health benefits and problems associated with antinutrients in foods. food res. intern. 26, 131-148 (1993) [67]rickard s.e. and thompson l.u. interactions and biological effects of phytic acid. in antinutrients and phytochemicals in food, shahidi, f. (ed.), acs symposium series 662, american chemical society, washington, dc, 294-313 (1997) [68]zhou j.r. and erdman j.w. -. phytic acid in health and disease. crit. rev. food sci. nutr. 35, 495-508 (1995) [69]shamsuddin a.m., vucenik i., and cole k.e. ip6: a novel anti-cancer agent. life sci. 61, 343-354 (1997) [70]grases f., march j.g., prieto r.m., simonet b.m., costa-bauza a., garciaraja a., and conte a., urinary phytate in calcium oxalate stone formers and healthy people. dietary effects on phytate excretion. scan. j. urology nephrology 34,162-164 (2000) [71]urbano g., lopez-jurado m., aranda p., vidai-valverde c., tenorio e., and porres j. the role of phytic acid in legumes: antinutritional or beneficial function? j. physiol. biochem.56, 283-293 (2000) [72]graf e. applications of phytic acid. j. amer. oil chem. soc. 60, 1861–1867 (1983) [73]reddy n.r., sathe s.k., (eds.) food phytates, crc press llc, 2000 n.w. corporate blvd., boca raton, florida 33431 (2002) [74]graf e., eaton j.w. antioxidant functions of phytic acid. free radical biol.med.8,61-69 (1990) [75]graf e., mahoney j.r., bryant r.g., and eaton j.w. iron-catalyzed hydroxyl radical formation. stringent requirement for free iron coordination site. j. biol. chem. 259, 3620-3624 (1984) [76]rimbach g., pallauf j. phytic acid inhibits free radical formation in vitro but does not affect liver oxidant or antioxidant status in growing rats. j. nutr. 128, 1950-1955 (1998) [77]reddy n.r., pierson m.d., sathe s.k., and salunkhe d.k. phytates in cereals and legumes. crc press, boca raton, fl. (1989) [78]krebs n.f. overview of zinc absorption and excretion in the human gastrointestinal tract. j. nutr. 130, 1374s-1377s (2000) 354 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 354 359 technological features of combining condensed canned milk with sugar and fruit-berry syrups during processing natalija valeriivna ryabokon1, *dmitro viktorovich rindyuk1, maksim sergiyovuch shpak1, kseniya romanivna komisar1 1national university of food technologies, kiev, ukraine rel_dv@ukr.net *corresponding author received november 29th 2014, accepted december 29th 2014 abstract: a mathematical model of combining milk and plant products was built on the basis of dependency of mass fraction of solids condensed canned milk (ccm).with sugar and fbs of mass fraction of solids milk-sugar mixture and the temperature of intensified lactose crystallization. taking into consideration the rheological characteristics and constructive-technological parameters, the degree of homogeneity consistency and duration of formation of condensed milk with sugar and fruitberry syrup "cranberry-blueberry" were studied. keywords: condensed canned milk with sugar, milk-sugar base, fruit-berry syrup, mathematical model, homogeneity, duration. 1. introduction dairy industry in ukraine is characterized by a monotonous assortment; finished products are unbalanced with high content of calories. such development is contrary to the newest industry food concepts, does not meet the needs of the modern consumer [1, 2]. today, scientists and researchers are searching new ways and methods of the changing nature of food while preserving the fresh and natural raw materials; ensuring rational, balanced products according to the physiological needs of the human body. a.g. galstyan, l. v. golubeva, k. k. polyansky, i. a. radayeva, l. v. chekulayeva, and others [1] at different times were engaged in development and improvement of technologies and structure of ccm. after detailed literature analysis, the authors recommended to review two possible ways of dynamic development of dairy industry: creating new types of specialized canned milk; improving and developing of technologies of canned milk with wider assortment and with new combinations of new components and adding of taste aromatic substances. at the same time, the proposed development of the industry using new raw material is more efficient because it does not limit the needs of people of all categories and a wide audience of consumers. it was identified that usage of nontraditional raw materials of plant origin in ccm, such as fruit-berry syrup (fbs), will allow to create products of combined carbohydrate composition with reduced glycemic, with improved and stable quality characteristics during storage [3, 4, 5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 na t a l i j a va l e r i i vna ry ab o k o n, dmi t r o vi kt or o vi c h ri n dy uk , m a ks i m se r g i y o vuc h sh p ak , k s e ni ya r o ma ni vna k o m i sar, t ec h n o l o g i c a l f e a t u r es o f c o mb i n a t i o n d u r i n g p r o d u c t i o n o f c o n d en s ed c a n n ed mi l k wi t h s u g a r a n d f r u i t b er r y s y r up s , volume xiii, issue 4 – 2014, pag. 354 – 359 355 it also will help producers to diversify taste range of products, expand the sphere of using ccm (in the confectionery industry as fillers in catering organizations – as filler like "topping"). but the production of condensed canned milk with sugar and fruit-berry fillings has several features which are affected by different physico-chemical properties of two environments: milk-sugar base (msb) and fbs ("cranberry-blueberry", "rosehips-hawthorn", "rosehips-ehinatseyamint"). therefore, further in the work experimental study of combining operations, the parameters of which primarily depends on the mass fraction of solids milk and plant base’s; temperature crystallization of lactose; quality and duration of cooling milk and sugar environment; structural and technological conditions combination msb with fbs is presented. 2. material and methods according to classical technology milksugar mixture is concentrated to a mass fraction of solids 71...72%, due to the fact that during the cooling product thickens at 0,088% with a decrease of 1 ° c, ie on the 3-3.5% overall [1]. thickening of msb in the production of ccm with sugar and fbs cannot be made on the same principle as the mass fraction of fbs solids in accordance with experimental results are 67...70%. with this purpose influence of mass fraction of solids milk-sugar mixture and fruit-berry syrup on the mass fraction of solids in products was determined. milk-sugar mixture was thickened to the state of dry matter content of 70...76%, and after cooling (20 °c) to its composition contributed 15% of fruit-berry syrup with solids mass fraction not less than 69%. table 1 shows the results of the experiment. table 1 dependence the mass fraction of solids ccm with sugar and fbs of mass fraction of solids milk-sugar mixture no. mass fraction solids of milk-sugar mixture, % mass fraction solids of cooled milk-sugar mixture, % mass fraction solids of ccm with sugar and fbs, % 1 70 73 69 2 71 74 70 3 72 76 72 4 73 77 73 5 74 78 74 6 75 79 74 7 76 80 76 analysis of the data shows that the adding of fbs to the milk-sugar mixture that thickens to 70...72% of solids as required by classical technology of ccm, leads to appearing of defects of "liquid consistency" and production of finished product with irregular quality characteristics. thickening of milk-sugar base to the mass fraction of solids over 75% resulted in loss of fluidity of final products that is explained by the increase of protein content in the mixture. as it is known, under these conditions, particles of casein-calcium-phosphate complex are converged, due to which there appear conditions for coagulation of protein [1] and as a result, an abrupt increase in viscosity, which is unacceptable. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 na t a l i j a va l e r i i vna ry ab o k o n, dmi t r o vi kt or o vi c h ri n dy uk , m a ks i m se r g i y o vuc h sh p ak , k s e ni ya r o ma ni vna k o m i sar, t ec h n o l o g i c a l f e a t u r es o f c o mb i n a t i o n d u r i n g p r o d u c t i o n o f c o n d en s ed c a n n ed mi l k wi t h s u g a r a n d f r u i t b er r y s y r up s , volume xiii, issue 4 – 2014, pag. 354 – 359 356 consequently, to obtain ccm with sugar and fbs with standard indicators milksugar mixture it is recommended thickening for mass fraction of solids 74 75%. at the next stage of work conditions to adequately process the lactose crystallization during cooling were specified. since in ccm with sugar and fbs lactose concentration decreased to 22.1 22.6% comparing with the classical ccm with sugar need to establish intensified crystallization temperature of lactose appeared. in condensed milk with sugar lactose is crystallized according to the kinetic molecular theory in two stages: at the first stage emergence of centers of crystallization happens, at the second increase of crystals quantity appears [1, 2]. therefore, in a heterogeneous formation mechanism of germs seed is needed for basic surface crystallization. in the role of seed in industrial conditions refined crystalline lactose is used, content of which is 0.02% of the weight of the product [3]. according to the schedule of the hudson (fig.1) temperature of intensified crystallization of lactose in the investigated samples of condensed canned milk with sugar and selected fruit-berry syrups was determined. for investigated samples of milk-sugar base condensed milk with sugar and various fbs "lactose number" was calculated (formula 1). received values were compared with lactose number of classic sweetened condensed milk. l.n (100l ) / (l w )pr pr prl   (1) fig.1. temperature of lactose intensified crystallization in ccm with sugar and fbs on schedule of the hudson food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 na t a l i j a va l e r i i vna ry ab o k o n, dmi t r o vi kt or o vi c h ri n dy uk , m a ks i m se r g i y o vuc h sh p ak , k s e ni ya r o ma ni vna k o m i sar, t ec h n o l o g i c a l f e a t u r es o f c o mb i n a t i o n d u r i n g p r o d u c t i o n o f c o n d en s ed c a n n ed mi l k wi t h s u g a r a n d f r u i t b er r y s y r up s , volume xiii, issue 4 – 2014, pag. 354 – 359 357 it was established "lactose number" for model samples: condensed milk with sugar: l.nl (100 12.3) / (12.5 26) 33.4%    condensed milk with sugar and fbs "cranberry-blueberry": l.nl (100 12.3) / (8.59 22.5) 39.5%    condensed milk with sugar and fbs "rosehips-hawthorn ": l.nl (100 12.3) / (8.6 23) 38.9%    condensed milk with sugar and fbs "rosehips-echinacea-mint": l.nl (100 12.3) / (8.64 23) 38.8%    via graph it was determined that intensified temperature of lactose crystallization for condensed canned milk with sugar and fruit-berry syrups should be 37...38 °c. while in the classic technology the temperature is 30...34 °c [6]. it was established that the adding of the seed at temperature treatment of 37...38 °c will allow a complete crystallization of lactose that will prevent the appearing of large crystals with size bigger than 15 microns. 3. results and discussion in order to ensure adequate crystal formation in the ccm with sugar and fbs further it was necessary to investigate the process of combining msb cooled to a temperature of 20...22 °c with fruit-berry syrup. for this purpose based on software complex flowvision. the mathematical model of combining milk and herbal products was constructed. since used higher fbs have similar rheological and physic-chemical characteristics. further studies performed on the example of ccm with sugar and fbs "cranberry blueberry." a mathematical model based on the dependencies described below [7. 8. 9]: navier-stokes equation: 1 ( ) ( ) ( ) (1 ) t hydt v p v v t v v g                        (2) flow continuity equations 0v ; (3) where v the vector of relative velocity. m/s; t time. s; p relative pressure. pa;  density. kg/m3; µ. µt respectively dynamic and turbulent viscosity. pa·s; hyd hydrostatic density. kg/m 3; g vector force of gravity. m / s2. the model of raw materials movements is based on usage of k ε turbulence model of the first circuit. within use of which for locking system it is required to get the formula for the coefficient of eddy viscosity t. today for achieving of this goal the socalled two-parameter model is used most frequently. they are called two parameters since t of these models is defined via two parameters. which are solved via additional differential equations in partial derivatives. the equation for the turbulent viscosity has the form: 2 с       (4) model also includes equations for the turbulent energy k and dissipation rate of turbulent energy ε: ( k) ( ) k d vk k g dt                        (5) 2 1 2 ( ) ( ) d v g dt c g c k k                                 (6) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 na t a l i j a va l e r i i vna ry ab o k o n, dmi t r o vi kt or o vi c h ri n dy uk , m a ks i m se r g i y o vuc h sh p ak , k s e ni ya r o ma ni vna k o m i sar, t ec h n o l o g i c a l f e a t u r es o f c o mb i n a t i o n d u r i n g p r o d u c t i o n o f c o n d en s ed c a n n ed mi l k wi t h s u g a r a n d f r u i t b er r y s y r up s , volume xiii, issue 4 – 2014, pag. 354 – 359 358 where below mentioned expression is indicated by g: 2 ; ; 3 i ij ij ij ij j dv g d d s v dx             ji ij j i dvdv s dx dx   where the value of the parameter is: σk=1; σε=1.3; сµ=0.09; с1=1.44; с2=1.92. transferring mixing components is solved via equation of convection diffusion transfer: 1 ( ) c c dc vc c d s s                    (7) where c concentration of the substance; sc schmidt number. the equation of transfer of filling function f has the form: 0 f v f t      . (8) via usage of developed mathematical model combining of two environments "condensed milk-sugar base – fruit-berry syrup" comprehensive experimental and theoretical studies were done. as limiting (output) of data in real production conditions rheological properties were selected (density. viscosity. mass fraction of solids) and were chosen constructiontechnological parameters (volume. shape and design of the vacuum mold. the speed scraping like rotary body). the results are presented in the form of field distribution uniformity consistency of milkplant environment in a vacuum crystallizer fig. 2. a. b. c. d. fig. 2. change of homogeneity of milk-plant consistency of protection in time: a) 10 seconds; b) 150 seconds; a) 300 seconds; d) 700 seconds. on figure 2 gradient filling of process combining milk-sugar base with fruit-berry syrup "cranberry-blueberry" is shown which allows to state the following: scraper agitator speed of 16 rev / min. provides a combination of two environments for 11.7 min to form a uniform consistency virtually all volume of vacuum crystallizer. heterogeneity consistency was observed in a conical piece of equipment to which does not get scraper agitator. for leveling consistency food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 na t a l i j a va l e r i i vna ry ab o k o n, dmi t r o vi kt or o vi c h ri n dy uk , m a ks i m se r g i y o vuc h sh p ak , k s e ni ya r o ma ni vna k o m i sar, t ec h n o l o g i c a l f e a t u r es o f c o mb i n a t i o n d u r i n g p r o d u c t i o n o f c o n d en s ed c a n n ed mi l k wi t h s u g a r a n d f r u i t b er r y s y r up s , volume xiii, issue 4 – 2014, pag. 354 – 359 359 for the whole mass authors the process of creating ccm with sugar and fbs in time was studied. analysis of fig. 3 showed that the combination of milk-sugar and plant basis to form the final product in an amount of 99.17% lasts for 17 minutes. at the same time in classic technology for ccm with sugar and fillers (coffee. cocoa. chicory) a similar process lasts 20 minutes. 0.83% of the total weight accounted for inhomogeneous systems that are not available for mixing through the adhesive properties of ccm and designed features of vacuum crystallizer. fig. 3. the duration of the formation condensed milk with sugar and fruit-berry syrup "cranberryblueberry" in a vacuum crystallizer 4. conclusion summarizing the above mentioned information we can conclude that the adding of fruit-berry syrups to condensed canned milk promotes expansion of the range; increase biological value of products for vitamins and minerals; to savings of raw milk by 30%. correctly done processes of cooling and combining of msb and fbs with simultaneous reduction of process time for 17 % ensures energy conservation for dairy industry enterprises. 5. references [1] semipyatny vk, production system in milk industry, semipyatny vk. malova ti. karapetyan vv, dairy industry. 2014. № 4. pp 42 43. [2] skorchenko t., pukhlyak a.. ryabokon n.,. patent for a useful model #56598 mpk (2011) a23c9/00. method dandified canned milk with fruit fillings. [3] herrington b. l., some physic-chemical properties of lactose., the spontaneous crystallization of supersaturated solutions of lactose b. l. herrington, journal of dairy science. 2000. – vol. 17. p. 501–518. [4] u.s. patent 2007/0020368 al. low glycemic. high fiber composition of all natural compounds that provides a sweet flavor profile for use in foods. beverages or as a sugar substitutes. sugar sense inc. #, maiullo d.a.. maletto p.s.. palat h. w. 25.01.2007. [5] heilbronn l., the effect of highand lowglycemic index energy restricted diets on plasma lipid and glucose profiles in type 2 diabetic subjects with varying glycemic control: translation from english by heilbronn l.. noakes m. clifton p.. p. 120. (2002). [6] shevchuk v. b. gnezdilova a. i , temperatures seeding in the crystallization of lactose in the condensed milk with sugar, proceedings of the universities. 2005. № 1. s. 68-70. [7] gausier. s. & bonnet. m. a k-ε model for turbulent mixing in shock-tube flows induced by rayleightaylor instability. physics of fluids a. vol.2. no 9. pp. 1685-1694. 1990. [8] young. y. n. , tufo h., dubey.a., rosner r., on miscible rayleigh-taylor instability: two and three dimensions. journal of fluid mechanics. vol. 447. pp. 377408. 2001. [9] kucherenko yu.a. et al, . experimental investigation into the evolution of turbulent mixing of gases by using the osa facility. laser and particle beams. vol. 21. pp. 389-392. 2003. stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiv, issue 2 30 june 2015 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava http://www.fia.usv.ro/fiajournal editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:fiajournal@fia.usv.ro mailto:gutts@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 43 lim ita tion facto rs of the agr icu ltur al produ c tion in cac ica v ill ag e, suc eava elena-viorica rotaru1, *ioan gontariu2 1,2 faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania *corresponding author received 26 march 2012, accepted 12 may 2012 abstract: the cacica village area has according to the mayor office documents a total surface of 6755 ha, from which 3067 ha are agricultural areas. the mapped surface has 224.74 ha and represents the entire agricultural surface taking on account the terrain of the neighboring places and the non productive terrain surfaces. among the limitative factors of the agricultural production the most important are: the with 1389.77 ha affected land (61.97% of the mapped surface), the strongly acid and acid reaction (80.60% of the mapped surface), the water erosion with 831.11 ha affected land (37.06% of the mapped surface), the landslides with 589.72 ha affected land (26.29% of the mapped surface) and the depth erosion (160.83 ha – 7.17% of the mapped surface). for the eroded fields we recommend the avoiding of pasturage in the humid times, the over seeding with perennial herbs mixtures in which the vegetables predominate, the establishing of a small number of cattle admitted for pasturage/ ha. in the case of the fields affected by strong and very strong erosion there are recommended protection crops. keywords: water erosion, affected land, eroded fields, protection crops 1. introduction cacica (polish: kaczyka) is a village situated in suceava county, romania, 21.7% of its inhabitants are polish, who began settling there at the turn of the 19th century. the commune is composed of five villages: cacica, maidan, pârteştii de sus (the commune center), runcu, soloneţu nou [1]. the cacica village area due especially to the climate factors and to the pedological and relief factors has a low plowing potential of the soil instead having a high one for the pastures and hays [2]. the limitative factors that cannot be modified and that limit the agricultural production are: climate, relief, exposure and solidification rocks. thus, the characteristics of the soil, temperature and rainfall quantity constitute the main factors that determine the way of using the fields as hay or agricultural field [3]. besides the above mentioned factors we can also add the geological and geomorphologic (the slope) ones [4]. the main enhancing measures that are imposed for decreasing the limitative action of the above factors are: collecting the costal springs, drainage, capital leveling, draining, limestone arrangement, radical fertilization, and damming water course regularization, terracing, anti erosion arrangement and anti erosion systems, protection crops, ploughing in parallel with the level curve. 2. materials and methods the object of the present paper is the study of the limiting factors of the field area of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 44 cacica village, suceava county. the study refers to the field situated in terrain. the purpose of the study is establishing, identifying, setting the boundaries for and classifying the types of soil/ land degradation, establishing the land restrictions for different purposes and the agro pedo ameliorating and anti erosion proper measures. at these we can add the differentiation between the agro technical and enhancing measurements, the establishing of the agro productive potential of the soil and the total or temporary exclusion of certain fields from the agricultural circuit. the way in which the paper was elaborated and presented had as purpose the fact that the pedological study would become a working instrument for the farmers offering them the best information and measures for a good managing of the fields at the plots, household, farm and village level. the universal equation of soil loss (usle), soil erosion is a factor “k” whose value is determined quantitatively both, by direct measurements in standardized parcels, and indirectly through mathematics [5]. erosion factor that k is the usle soil loss due to raindrops and surface flow concentrated in the appropriate field of rainfall erosion index unit as measured in standard plots for flow control. this factor includes of soils to be eroded, and flow rate, measured in terms of standardized plots [6]. taking on account the utility type the mapped area is divided in: table 1 the use of land utility agricultural surface (ha) ploughing land 1138.85 pastures 376.24 hays 692.02 orchards 10.24 3. results and discussion the human involvement in decreasing the limitative factors of the agricultural production can be made in the case of the edaphic factors. taking the right measures we can intervene upon them the final result being the increasement of the evaluation marks and the opportunity for different crops. according to the nature and intensity of the limitative factors the fields can be grouped as it follows: surface erosion limitations. due to the relief conditions (high slope) but also to the improper using the water eroded fields have a high percentage (37.06% of the mapped surface) from which 254.42 ha with low erosion, 254.50 ha with moderate erosion, 259.23 ha with high erosion, 62.96 ha with very strong erosion. the slopes can be hardly affected by depth erosion and landslides. direct methods for estimating soil erosion know a great variety, which are grouped by moţoc m. and morărescu v. (2000) as follows: physical research laboratory models revealed that erosion mechanism drops and gullies and erosion resistance of soils with different properties; research in the field using mobile sprinkler systems of different sizes. they are used to calibrate erosion models to estimate and quantify its; research plots with drainage [7]. the eroded surfaces taking on account the degrees of erosion are presented in table 2. depth erosion limitations. these limitations were identified on the sides with big slopes where the conditions favourable for the erosion process met. some of the surfaces that present depth erosion are also affected by the stabilized or semi stabilized landslides. the affected surface is of 160.83 ha, meaning 7.17% of the mapped surface, the erosion formations being drips, gullies (138.06 ha), and ravines (22.77 ha), as shown in table 3. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 45 table 2 wind and water eroded fields total agricultural surface mapped agricultural surface affected surface water eroded low moderate strong verystrong excessiv e 3067 ha 2242.74 831.11 254.42 254.5 259.23 62.96 0 % 73.12 37.06 30.61 30.62 31.19 7.58 0 wind eroded 3067 ha 2242.74 0 0 0 0 0 0 % 73.12 0 0 0 0 0 0 table 3 depth erosion affected fields total agricultural surface mapped agricultural surface affected surface eroded by differnt form : drips,gull ies torrents ravines 3067 ha 2242.74 160.83 138.06 0 22.77 % 73.12 7.17 85.84 0 14.16 table 4 landslides affected fields total agricultural surface mapped agricultural surface landslide surface from which : furrows waves steps mounds flows collapse 3067 2242.74 589.72 98.48 0 0 491.24 0 0 % 73.12 26.29 16.7 0 0 83.3 0 0 table 5 fields with gleyed and pseudo gleyed soils total agricultur al surface mapped agricultur al surface pseudogleyzed affected surface from which (ha, %): low moderate strong very strong excessive pseudogleyzed soils 3067 ha 2242.74 1389.77 200.32 813.49 309.33 66.63 0 % 73.12 61.97 14.41 58.53 22.26 4.79 0 gleyzed soils 3067 ha 2242.74 40.58 0 0 0 0 40.58 % 73.12 1.81 0 0 0 0 100 landslides due to erosions the affected surface is of 589.72 ha, that represents 26.29 % of the mapped surface as it is shown in table 4. the landslides appear due to the coastal spring’s action and to the small depth phreatic water. these soils are situate don the coast with slopes between 14-30%. the main types of landslides are food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 46 with the furrows (98.48 ha) and with the mounds (491.24 ha). the nivo-pluvial excess due limitations on these surfaces the stagnogleyzation phenomena appears duet to the reduced slope of the field, to the fine or average fine texture and to some local conditions that prevent the water draining. the combination of these factors leads to a faulty drainage of the water coming from the vertical raining. the stagnogleyzation affected surface overpasses half of the mapped area being of 1389.77 ha. the affected surface on degrees of stagnogleyzation is presented in table 5. we recommend as measurements a deep loosening, surface or deep draining all according to the stagnogleyzation degree. this category of fields includes the low drainage surfaces the pluvial water stagnation affecting the agricultural production. the stagnogleyzation is from low to extremely intense. as agro pedo amelioration measurements we recommend the scarification at distances of 1.5-2 m, perpendicular on the roads and level curves if the slope is < 2%, and diagonal on the roads if the slope is between 2 – 12 %. there are also necessary draining works and surface draining. another possible measure is the systematic drain gullies at low depths (15-20 cm) that can be remade annually. on small slopes (< 1.5%) they are placed on the biggest slope line an don big slopes in the way that the average gully slope to be between 0.50.8%. on the fields with clay texture (> 40% clay) the mole drainage system can be applied at depths of 40-80 cm, with the distance between them of 8-10 cm. this is a complementary draining method as a subsidiary for the asanation-draining systems. the essential condition is the existence of fields with slopes of 1-2%. in the case of the fields with slopes lower than 1-2 %, we recommend the molding of earth strains that suppose the molding of curved strains separated by draining gullies [8]. they are executed on the biggest slope line of the field. fields with flooding limitations the flooding surface is situated in the proximity of the water courses or in the inner meadows. the total flooding surface is of 182.56 ha, being presented on flooding degrees in table 6. for these surfaces we recommend the damming and water courses regularization. table 6 surfaces affected by flooding total agricultural surface mapped agricultur al surface flooding surface from which (ha/%): rare frequent very frequent 3067 ha 2242.74 182.56 40.2 77.76 64.6 % 73.12 8.14 22.02 42.59 35.39 4. conclusions the cultivation and growth of crops is conditioned by several limited factors. some of them such as the climate (temperature and raining) relief (slope and position) and parental material cannot be intervened upon. other factors can be influenced or changed by measurements of soil erosion prevention, landslides, phreatic or pluvial humidity excess or other different agro pedo amelioration works. for preventing and controlling the landslides we recommend the collecting of the costal springs that are numerous especially in the complex soil units. we also recommend the asanation and draining food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 47 works and surface draining followed by a proper maintenance. another factor that would lead to good results is the cultivation of perennial and water resistant ameliorative crops (or over seeding in the meadow areas). it is also recommended the growth stimulation of these crops with the aid of organic or chemical fertilizers. there can also be done leveling for the regularization of the water drain on the slopes. the works of deep raising that lead to the soil aeration, the increase of apparent density and the creation of better crop developing conditions. the scarification will be done in parallel with slope direction at slopes smaller than 2 – 3% and diagonal to this direction for slopes bigger than 2 – 3%. the organic fertilizers incorporation (cobs and straws in a dose of 40-50 t/ha) or other agricultural secondary products. in the inter mountains areas due to the water accumulation tendency the deep raising is done only in the cases in which the fields aren’t drained. on the fields with depth erosion we recommend the same measures as for the surface erosion as well as protection crops for the ravine. 5. references [1]. popp n., i. iosep i., paulencu d., (1973) – „suceava county”, academy publishing house, bucharest. [2]. brânduş i., (2008) – „ soil study land territory the cacica commune, suceava county, sc 1: 10000, ospa suceava. [3]. puiu st. and. all., (1983) – „the pedology”, bucharest. [4]. ungureanu i., (1978) -„geomorphological maps”, publisher junimea, iasi [5]. wells m., (1998) – „a method of assesing water erosion risk in land capability studies”, resource management tehnical report no. 73 gov. of western australia. [6]. le bissonnais y., le souder christine, (1995) – „measure the structural stability of soils to assess the sensitivity batance and the erosion”, study and management of soils, vol. 2, no. 1. [7]. moţoc m., morărescu v., (2000) – „soil erosion problems on the surface”, soil science series iii, no. 1, vol. xxxiv. [8]. xxx., (1987) – „development methodology soil survey” – i.c.p.a. bucharest (vol. i, ii, iii). effect of addition of starch and agar-agar on rheological behaviour of yogurt food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 97 effect of add it ion o f sta rch a nd ag araga r on rhe olo gi ca l b eh av io ur o f y og ur t *cristina damian1, mircea-adrian oroian1, ana leahu1, iuliana cioarbă1 1ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1cristinadamian@fia.usv.ro *corresponding author received 12 february 2011, accepted 5 march 2012 abstract: the purpose of this work is to study the effect of addition of starch and agar-agar on the rheological behaviour of different types of yogurts. the most common technique used for rheological liquids is viscometers, measuring shear strength, described by the coefficient of viscosity. for this study, the yogurt samples were obtained in the laboratory using the cultures starter diprox yba 986. the effect of addition of starch and agar-agar in yoghurt was investigated by a rotational viscometer, brookfield viscometer (brookfield engineering inc, model rv – dv i prime) with rv spindles. the brookfield viscometer dv i prime with disk spindles represents an easy and cheap method for rheological characterization of non-newtonian fluids, in this case of yoghurt. the cluster analysis was performed using unsrambler x version 10.1 (camo process as, oslo, norway, 2005), all the rheological and physicochemical parameters were weighetd and normalized for performing the cluster analysis. in the case of yogurt samples obtained in the laboratory, the addition of starch and agar-agar increases the viscosity, proportional to the doses added. the results of cluster analysis shows that the samples with agar added are in the same group, while the sample with 12.04 g starch is much appropiate to the samples with agar-agar then with the sample with 10.61 g starch. keywords: yogurt, viscosity, viscometer, starch, agar-agar 1. introduction for processed food the composition and the addition of ingredients to obtain a certain food quality and product performance requires profound rheological understanding of individual ingredients, their relation to food processing, and their final perception [1]. yogurts are dairy products obtained by fermentation with lactic acid bacteria during which a weak protein gel develops due to a decrease in the ph of the milk. following the fermentation of lactose lactic acid is formed causing the coagulation of milk proteins (casein) to form a gel-like structure, in which fat globules and the aqueous phase are embedded [2]. in the liquid milk, casein micelles are presented as individual units. as the ph reaches ph 5.0, the casein micelles are partially broken down and become linked to each other under the form of aggregated and chains forming part of a three-dimensional protein matrix in which the liquid phase of the milk is immobilized. this gel structure contributes substantially due to the overall texture and organoleptic properties of yoghurt and gives rise to shear and time dependent viscosity [3]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions [4]. for yoghurt production a variety of gums is being used with more functional roles: mailto:1cristinadamian:@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 98 they give the product the rheological and organoleptic characteristics desired in acidic ph conditions; prevent the process of syneresis; they participate in gel formation in the case of gelificate yoghurt; -they are designed to promote formation of a structural network by interacting with compounds of structural constituents of milk and fermentation products. agar is extracted from red algae. agar is unique among gums in the sense that gelling occurs at temperatures much lower than the temperature at which agarose gels melt. agar is compatible with many polysaccharides and proteins, in the sense that when their dispersions are mixed flocculation or other degradations do not occur. starch is a complex food hydrocolloid (polymer of α-d-glucose and partially crystalline polymer). starch granules absorb water resulting in swelling up to several times their original size and lose their crystallinity. the complete process is known as gelatinization. gelatinization of starch involves changes in amylase and amylopectin [5]. it has been reported that amylase undergoes aggregation at a much more rapid rate than amylopectin does[6]. starch gelatinisation involves the following steps: reversible hydration and swelling of the granules; loss of birefringence in pasting temperature; increasing the transparency of the paste; the rapid increase in viscosity and achievement of maximum viscosity; leaching of linear molecules of amylose fully hydrated and circulated through the grain; the cooling takes place with formation of gelatinised starch retrogradation pasta, and then a gel. an elevated starch concentration enhanced the strength of the custard gels considerably [7]. the most frequent defects related to yogurt texture that may lead to consumer rejection are apparent viscosity variations and the occurrence of syneresis [8]. yogurt rheological characterization is required for product and process acceptability [9]. this characterization can be made using either instrumental or sensory measurements. the firmness of yogurt and the viscosity of just-stirred gel are greatly influenced by the amount of heat treatment the yogurt mix receives. heating unfolds the globular whey proteins and exposes sulphydryl groups, which react with other sulphydryl groups and disulfides and induce linkages and protein-casein aggregates [10, 11]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions. the binding of δ-lacto globulin to the casein micelle seems to be responsible for the increase of gel strength [12, 13, 14]. 2. materials and methods 2.1. materials uht milk, lactobacillus bulgaricus and streptococcus thermophilus pure starter culture di prox 986 provided by enzymes & derivates, piatra neamț, românia; commercial starch, agar-agar provided by enzymes & derivates, piatra neamț, românia; orbital shaker; food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 99 thermostat; brookfield viscometer model rv dv ii pro, with disk spindle, rv3, rv4, rv5, r v6 type. 2.2. sample preparation the yogurt samples were made using uht milk, having the physical and chemical parameters in tabel 1. tabel 1. milk properties dry substance 10,5% fat, g/100g 3 protein, g/100 g 3 sugar, g/100g 4.5 ash, % 0.72 acidity, 0t 18 tabel 2. ph of yogurt samples sample ph s1 4.0 s2 4.5 a1 4.2 a2 4.0 a3 4.5 300 ml milk was inoculated using 0.015 g starter culture. after inoculation with starter culture, the samples were homogenised with an orbital shaker for 15 min at 100 rpm. after shaking the samples were thermostated at 420c for 6 hours. starch and agar-agar were added in the next concentration to the yogurt sample: 10.61 g starch (s1), 12.04 g starch (s2), 0.27 g agar (a1), 0.3 g agar (a2) and 0.33 g agar (a3). the ph of the yogurt samples are in table 2. 2.3. determination of rheological properties viscosity measurements were carried out on the yogurt samples at ambient temperature (250c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 0.5, 1, 2, 2.5, 4, 5 and 10 rpm with rv spindle (rv3, rv4, rv5, rv6 type). the spindle nos was used in accordance with the sample nature to get all readings within the scale [15]. the samples in 300 ml of beaker with a 8,56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain desirable temperature of 250c. first measurements were taken 2 min after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression σ = k·γn (1) where: σ shear stress (n/m2), γ the shear rate (s-1), n the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. the shear stress is calculated using the next equation: τi = kτ·αi·c (2) where: τi – shear stress (dyne/cm2) kτ = 0.119, this constant is for the spindle nos 2 αi – torque dial, % c – 7,187 dyne/cm for rv viscometer food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 100 the shear rate is calculated using the next equation: γi = kγ(n)·ni (3) where: γi – shear rate, s-1 kγ(n) – constant, depends by the value of n ni – rotational speed, rpm. 2.4 statistical analysis the cluster analysis was performed using unsrambler x version 10.1 (camo process as, oslo, norway, 2005), all the rheological and physicochemical parameters were weighetd and normalized for performing the cluster analysis. 3. results and discussion the yogurt samples exhibited a nonnewtonian behaviour, the flow index (n) is under 1 for all the samples. the power law model is a suitable one for predicting the rheological parameters, the regression coefficient is near 1 (r2>0.95) – see table 2. table 2. viscosity, flow index, consistency index and regression coefficient for yogurt samples sample viscosity * [cp] n–flow index k-consistency index [nsn/m2] r2 s1 42231 0.63 27831 0.982 s2 106000 0.96 48317 0.96 a1 91900 0.86 46770 0.989 a2 97899 0.78 54213 0.985 a3 125000 0.81 69153 0.995 *viscosity was measured at 0.5 1/s in general, addition of starch increases the viscosity of yogurt in proportion to the dose added, as shown in figure 1. it achieved a stabilization of aqueous dispersions whose continuous phase is the water and the dispersed one is solid or liquid and tends to separate. through addition of starch, the viscosity is increased and the tendency of destabilization by separation of components is reduced. the starch improved the rheological properties of the yogurt samples, due to the water-bind capacity, high molecular weight and due to the forming of the casein-starch system, the water-bind capacity. various studies have reported the existence of phase separated networks in casein–polysaccharide mixtures [16, 17], and the amylopectincasein system in particular [18], but not in the samples with low levels of amylopectin added. depending on the weight ratio of the two biopolymers in the mixture, as well as their relative affinities for water, a casein– starch system may either have the protein or the polysaccharide-rich domain forming the continuous phase, with the second component dispersed as the discontinuous phase. as a result, only a fraction of the total water content will be available to one biopolymer phase (e.g. casein), with the remaining water being bound by the second phase (e.g.starch) [19]. it seems that the addition of hydrocolloids (starch or agar-agar) lead to an increasing of consistency index (tab. 2), while the flow index increases in the case of additio of starch but in the case of agar agar it is not observed an increasing or decreasing of this parameter. in figure 1 and figure2 is presented the evolution of viscosity with the two hidrocolloid with concentration of it and shear rate. the highest the concentration of hydrocolloid, the highest is the viscosity. figure 1. viscosity profile of yogurt sample with starch (s1 – 10.61g starch, s2 – 12.04 g starch) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 101 figure 2. viscosity profile of yogurt sample with agar-agar (a1 – 0.27 g agar, a2 – 0.3 g agar, a3 – 0.33 g agar) the results of cluster analysis shows that the samples with agar added are in the same group, while the sample with 12.04 g starch is much appropiate to the samples with agar-agar then with the sample with 10.61 g starch. figure 3. diagram of yogurt samples clustered according to rheological and physicochemical properties 4. conclusion due to large molecular sizes, to the specific configuration, the charge and possible formation of intraand intermolecular links, gums are able to reduce water mobility. this reduction in water mobility increases the viscosity of gum solutions in water. in the case of yogurt samples obtained in the laboratory, the addition of starch and agar-agar increases the viscosity, proportional to the doses added. starch carries stable aqueous dispersions whose continuous phase and the dispersed one is solid or liquid and tends to separate. by means of starch addition, yogurt viscosity is increased and reduces the tendency to destabilize the separation of components. 5. references [1]. fischer, p., windhab, e.j., rheology of food materials, current opinion in colloid & interface science, 16, 36-40, (2011) [2]. banu, c. (coordinator), aditivi și ingrediente pentru industria alimentară, editura tehnică, bucurești, (2000) [3]. oroian m. a., escriche i., gutt g., rheological, textural color and physicochemical properties of some yoghurt products from the spanish market, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, year x, no 2, 24 p, (2011) [4]. damian, c., oroian, m.a., șmadici, a., effect of addition of corn flakes on rheological behaviour of some yogurt, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, volume x, issue 4, 84 p, (2011) [5]. ahmed, j., ramaswamy, h.s., ayad, a., alli, i., thermal and dynamic rheology of insoluble starch from basmati rice, food hydrocolloids, 22, 278-287, (2008) [6]. chang, y.h., lim, s.t., yoo, b., dynamic rheology of corn starch-sugar composites, journal of food engineering, 64, 521-527, (2004) [7]. keršienė, m., adams, a., dubra, a., de kimpe, n., leskauskaitė, interactions between flavour release and rheological properties in model custard desserts: effect of starch concentration and milk fat, food chemistry, 108, 1183-1191, (2008) [8]. kroger, m. quality of yogurt. journal of dairy science 59(2): 344-350. (1975) [9]. benezech, t., maingonnat, j.f. characterization of the rheological properties of yogurt-a review. journal of food engineering. 21, 447-472. (1994) [10]. sawyer wh. complex between –lacto globulin and γ-casein. a review. journal of dairy science 52:1347–55. (1969) [11]. kinsella h. milk proteins: physiochemical and functional properties. crc crit rev food sci nutr 21(3):197. (1994) [12]. bonomi f, iametti s, palgliarini e, peri c. a spectrofluorometric approach to estimation of the surface hydrophobicity food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 102 [13]. modifications in milk proteins upon thermal treatment. milchwissenschaft 43:281–5, (1998) [14]. mottar j, bassier a, joniau m, baert j. effect of heat-induced association of whey proteins and casein micelles on yogurt texture. journal of dairy science 72(9):2247– 56, (1989) [15]. bonomi f, iametti s. real-time monitoring of the surface hydrophobicity changes associated with isothermal treatment of milk and milk protein fractions. milchwissenschaft 46:71–4, (1991) [16]. http://www.brookfieldengineering.com/prod ucts/accessories/spindles/rv_ha_hb_spindles.asp (7.09.2011) [17]. bourriot, garnier, & doublier, phase separation, rheology and microstructure of micellar casein–guar gum mixtures. food hydrocolloids, 13, 43–49, (1999) [18]. schorsch, clark, jones, &norton, behavior of milk protein/polysaccharide systems in high sucrose. colloids and surfaces b, 12, 317–329, (1999) [19]. de bont, van kempen, & vreeker, phase separation in milk protein and amylopectin mixtures. food hydrocolloids, 16, 127–138, (2002) [20]. shiu-kin chan p., chen j., ettelaie r., law z., alevisopoulos s., day e., smith s., study of the shear and extensional rheology of casein, waxy maize starch and their mixtures, food hydrocolloids 21 716–725, (2007) 95 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 95 100 total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals valentina mancas1, *sonia amariei1 1faculty of food engineering, stefan cel mare university of suceava, romania gutts@fia.usv.ro *corresponding author received february 28th 2016, accepted march 29th 2016 abstract: traditional medicine offers a wide range of plant based products with multiple claimed benefits in health preservation, as well as disease treatment and prevention. although known for their therapeutic role in various health conditions, syrups prepared with flower petals have not been thoroughly studied. in this paper total phenolic content and antioxidant activity of peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petal syrups were analyzed in order to confirm the beneficial effects on health that the consumption of syrups with high antioxidant content has. the ingredients used for syrup preparation were flower petals, water and acacia honey.the total phenolic content was determined using folin-ciocalteu method, while the antioxidant activity was evaluated by dpph (1,1-diphenyl-2-picrylhydrazyl) method. peony petal syrup had the highest phenolic content (642.03 mg gae/g) and also a greater antioxidant activity (inhibition of 87.13%). dpph radical scavenging activity measured for rose petal syrup (62.95%) reflected the influence of phenolic content (437.91 mg gae/g) of the product on antioxidant activity. these results show that syrups from medicinal flower petals might be helpful in the treatment of common diseases and the prevention of oxidative stress related illnesses. keywords: rose, peony, antioxidants, dpph, folin-ciocalteu 1. introduction for thousands of years nature has been a source of numerous plants with health benefits and important role in the fight against frequent diseases. plant composition and especially active compounds have been thoroughly investigated in order to validate their use. herbal medicine is the oldest from of healthcare known to mankind [1], also serving as base for the development of new antimicrobial drugs. the controversies and debates that surround commercially available drugs, as well as the high cost of medical treatment led patients and consumers toward alternative natural therapies involving medicinal plants. as antioxidants have been reported to prevent oxidative damage caused by free radicals [2 -3], scientific developments in the antioxidant activity of phytochemicals from medicinal plants increased significantly. given the geographical position and favorable climate, romania has a large medicinal flora comprising over 3600 species of spontaneous and cultivated plants. peony (paeonia officinalis) and rose (rosa centifolia) are two flowering plants used as traditional remedies mostly for respiratory tract illnesses. species of peonia genus are widely distributed in european, mediterranean and asia region and known for the ornamental and economic value of the entire plant as well as the medicinal properties of their http://www.fia.usv.ro/fiajournal mailto:gutts@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 valentina mancas, sonia amariei, total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals, food and environment safety, volume xv, issue 1 -2016, pag. 95 – 100 96 roots and petals [4]. the biological properties of peonies have been attributed to a group of monoterpenes with common cage-like pinane skeleton with hemiketalacetal system, namely paeoniflorin and its derivatives [5-6]. more recent studies have reported considerable amounts of polyphenols (paeonol and gallic acid derivatives), monoterpenes and monoterpene glycosides, triterpenoids, and steroids as the main phytochemicals [7]. peony seed oil is rich in 𝛼-linolenic acid [8], which has beneficial effects on human nutrition and health, and a great value on the chinese market. moreover, in peony pod were determined increased amounts of apigenin and luteolin, two flavonoids with antioxidant, anticancer, and antiinflammatory effects [9]. both compounds have manifested in vitro capacity to induced cell death in two aggressive human pancreatic cancer cell lines [10]. furthermore, a large number of scientific observations suggest that luteolin could be a chemotherapeutic agent for other types of cancer. rosa centifolia, a specie native to asia and middle east belongs to the family rosaceae [11]. this cultivar has medium sized colorful flowers that are rich in fragrance. rose petals contain acids, essential oils, tellimagrandin i, rugosin b, carotenoids, anthocyanins and proanthocyanidins. the main constituent of the oil extracted from rose is geraniol, an alcohol with a rose-like odor that gives upon oxidation aldehyde citral, a compound also present in rose petals. furthermore, the oil fraction of rose contains l-citronellol in a proportion of about 20% [12]. a small fraction of both citronellol and geraniol is combined in ester form. other compounds found in rose are tannins, mineral salts, mallic acid and tartaric acid salts, pectin, riboflavin, vitamin c, sugars, and purgative glycosides (multiflorin a and b) [13]. many chemical constituents of rose have proven antioxidant activity. although the health benefits of rose and their mechanism have not been widely explored, the plant is used traditionally in the treatment of diabetes, conjunctivitis, and aroma therapy [14]. most of the research on antioxidant activity and phenolic profile of peony and rose is conducted on extracts from different plant segments obtained through conventional or modern extraction techniques. the present study takes a more practical approach on the matter, using traditionally prepared syrups from peony and rose petals that are also commercially available. the advantages of syrups over petal extracts are the superior nutritional value, reduced toxicity, and improved digestibility. in the current case, the purpose of the study was to determine the total phenolic content and antioxidant activity of freshly prepared peony and rose petal syrups. 2. matherials and methods 2.1. plant material to assure the preparation of high quality syrup, the plants were purchased from an organic farmer and were free of fertilizers and insecticides. roses were from rosa centifolia specie, while peonies originated from the romanian cultivated specie paeonia peregrina mill. var. romanica. petals were removed from the corolla, rinsed and then dried at 40 °c into an oven (to inactivate microbial cells but also avoid phytochemicals damage). 2.2. syrup preparation the syrup was prepared as follows: 1 g of peony or rose petals was added to a beaker containing 100 ml of water. after a slow stir, the beaker was placed over a heat source where the petal and water mix was allowed to simmer for about 15 minutes. the beaker was then removed from the heat food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 valentina mancas, sonia amariei, total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals, food and environment safety, volume xv, issue 1 -2016, pag. 95 – 100 97 allowing the mix to cool. when the temperature of the mix decreased to 35 °c, 64% (w:w) acacia honey was added to petal infusion and mixed thoroughly. the beaker was covered again leaving the content to rest for about 10 hours. finally, flower petals were removed using a filter and the syrup was refrigerated until its composition was analyzed. 2.3. total phenolic content determination the total phenolic content of the syrups was measured colorimetrically using the folinciocalteu method. 50 µl of syrup was transferred into a test tube and mixed thoroughly with 250 µl of folin-ciocalteu reagent. the content of the tube was shaken and then 500 µl of sodium carbonate (20%, w/v) was added and mixed again. the mixture was allowed to stand for 30 minutes prior to measuring the absorbance at 765 nm in a single beam uv-vis-nir spectrophotometer (shimadzu corporation, japan). total phenolic content was expressed in mg gallic acid equivalents (gae)/g. for gallic acid, absorbance is described by the following equation: y = 0.0012x – 0.0345 (r2 = 0.9997) 2.4. antioxidant activity the antioxidant activity of the syrups was measured using the dpph (1,1-diphenyl-2picrylhydrazyl) method. the dpph radicalscavenging effect was evaluated through a procedure similar to those proposed by mraicha et al. (2010) and bouaziz et al. (2008) in previous studies [15,16]. the samples analyzed were prepared by mixing various concentration of syrup with ethanol solution, as follows: 1200 µl peony syrup and 1800 µl solvent, and 300 µl rose syrup in 2700 µl ethanol. the aliquots were added to 5 ml of a 0.004% (w/w) freshly prepared methanol solution of dpph. after 30 minutes of incubation at room temperature, the absorbance was measured against a blank at 517 nm in a single beam uv-visnir spectrophotometer (shimadzu corporation, japan). free radicals inhibition (i%) was calculated using the equation: i% = [(ablank – asample)/ablank] ×100, where: ablank – absorbance of the control reaction, containing all the reagents except the analyzed syrup asample – absorbance of the analyzed syrup 3. results and discussion two syrups with therapeutic benefits, respectively rose petal syrup and peony petal syrup, were analyzed to determine the total phenolic content and the antioxidant activity of the product. the results obtained for both parameters are presented in table 1. the absorbance spectra for phenolic compounds and antioxidants are shown in figures 1and 2. as the results show, peony petal syrup had both the highest phenolic content and antioxidant activity. total phenolic content of peony petal syrup was 642.03 mg gae/g, while the concentration determined for rose petal syrup was 437.91 mg gae/g. very few studies were conducted on the antioxidants level of peony and rose petals, and no research was found on the phenolics and antioxidant activity of syrups prepared from petals. the phenolic content reported for polar extract obtained from roots of paeonia rockii was 160.2 mg/g, and its soluble fraction had a total phenolic content of 256.0 mg/g [4]. for an extract from a mixture of astragalus membranaceus and paeonia lactiflora plant powder, the maximum phenolic content determined was 275.062 mg gae/g [17]. in the case of rose, total phenolic content reported for fresh petals and essential oil extracted from rose exceeded the level measured in petal syrup. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 valentina mancas, sonia amariei, total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals, food and environment safety, volume xv, issue 1 -2016, pag. 95 – 100 98 fig.1 absorbance spectra of phenolic compounds from peony petal syrup (▬) and rose petal syrup (▬) fig.2 absorbance spectra of antioxidants from peony petal syrup (▬) and rose petal syrup (▬) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 valentina mancas, sonia amariei, total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals, food and environment safety, volume xv, issue 1 -2016, pag. 95 – 100 99 therefore, rose petals had a total phenolic content of 1,171.06 mg gae/g [18], but this concentration was below the level determined in the essential oil from rose, namely 2,134.3 mg gae/g [19]. table 1 total phenolic content and antioxidant activity of petal syrups sample total phenolic content (mg gae/g) antioxidant activity (% inhibition) peony (paeonia peregrina mill. var. romanica) petal syrup 642.03 87.13 rose (rosa centifolia) petal syrup 437.91 62.95 it is well documented that phenolic compounds are the major bioactive components contributing to the antioxidant activity of many herbs. moreover, the phenolic content was positively correlated with antioxidant capacity [20]. given the high content of phenolics in peony and rose petal syrup, to further investigate its beneficial effects, the antioxidant activity of the products was analyzed. phenolic compounds contribution to the antioxidant activity of the syrup was particularly highlighted in the case of peony petals, for which was determined an inhibition of 87.13%. antioxidant activity of rose petal syrup (62.95% inhibition of reactive species) was comparable and slightly higher than dpph radical scavenging activity reported for alcoholic extracts (70% ethanol) from rose petals (about 60% inhibition) [21]. 4. conclusion this paper presents the results of a study on total phenolic content and antioxidant activity of two syrups used traditionally for therapeutic benefits. syrups were prepared with rosa centifolia and paeonia peregrina mill. var. romanica petals from a local organic culture. the data obtained showed that peony plant syrup has a higher phenolic content than rose syrup, therefore exhibiting a greater antioxidant activity through the scavenging of free radicals. taking note that peony petal syrup is less known among consumers and also rarely used, the study proves the importance of promoting its importance as a source of natural antioxidants, and pointing out the role of these compounds in health promotion and disease prevention. 5. references [1]. preethi, r., devanathan, v. v., loganathan, m., antimicrobial and antioxidant efficacy of some medicinal plants against food borne pathogens, advances in biological research, 4(2): 122-125, (2010) [2]. patel, v. r., patel, p. r., kajal, s. s., antioxidant activity of some selected medicinal plants in western region of india, advances in biological research, 4(1): 23-26, (2010) [3]. kratchanova, m., denev, p., ciz, m., lojek, a., mihailov, a., evaluation of antioxidant activity of medicinal plants containing polyphenol compounds. comparison of two extraction systems, acta biochim polonica, 57(2): 229-234, (2010) [4]. picerno, p., mencherini, t., sansone, f., del gaudio, p., granata, i., porta, a., aquino, r. p., screening of a polar extract of paeonia rockii: composition and antioxidant and antifungal activities, journal of ethnopharmacology, 138(3): 705-712, (2011) [5]. abdel-hafez, a. a., meselhy, m. r., nakamura, n., hattori, m., watanabe, h., mukarami, y., el-gendy, m. a., mahfouz, n. m., mohamed, t. a., effects of paeoniflorin derivatives on scopolamine-induced amnesia using a passive avoidance task in mice; structure-activity relationship, biological and pharmaceutical bulletin, 21(11): 1174-1179, (1998) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 valentina mancas, sonia amariei, total phenolic content and antioxidant activity of syrups from peony (paeonia peregrina mill. var. romanica) and rose (rosa centifolia) petals, food and environment safety, volume xv, issue 1 -2016, pag. 95 – 100 100 [6]. duan, w. j., yang, j. y., chen, l. x., zhang, l. j., jiang, z. h., cai, x. d., zhang, x., qiu, f., monoterpenes from paeonia albiflora and their inhibitory activity on nitric oxide production by lipopolysaccharide-activated microglia, journal of natural products, 72(9): 15791584, (2009) [7]. wu, s. h., wu, d. g., chen, y. w., chemical constituents and bioactivities of plants from the genus paeonia, chemistry and biodiversity, 7(1): 90-104, (2010) [8]. yinmao, w. zhang, p., dong, y., the technique of the extraction of oil from peony seed with supercritical co2. extraction method and the analysis of the composition, journal of the chinese cereals and oils association, 8(21), (2009) [9]. wang, h., yang, l., zu, y., zhao, x., microwave-assisted simultaneous extraction of luteolin and apigenin from tree peony pod and evaluation of its antioxidant activity, the scientific world journal, (2014) [10]. johnson, j. l., mejia, e. g., flavonoid apigenin modified gene expression associated with inflammation and cancer and induced apoptosis in human pancreatic cancer cells through inhibition of gsk‐3β/nf‐κb signaling cascade, molecular nutrition and food research, 57(12): 2112-2127, (2013) [11]. shabbir, m. k., nadeem, r., mukhtar, h., anwar, f., mumtaz, m. w., physico-chemical analysis and determination of various chemical constituents of essential oil in rosa centifolia, pakistan journal of botany, 41(2): 615620, (2009) [12]. hirulkar, n. b., antimicrobial activity of rose petals extract against some pathogenic bacteria, international journal of pharmaceutical and biological archive, 1(05): 478-484, (2010) [13]. jitendra, j., vineeta, t., ashok, k., brijesh, k., singh, p., rosa centifolia: plant review, international journal of pharmaceutical chemistry research, 2(3): 794-796, (2012) [14]. sankar, r. a., nikhila, c., lakshmiprasanna, v. c., mobeena, s. k., karunakar, k., bharathi, n., evaluation of anti-tussive activity of rosa centifolia, international journal of pharmaceutical sciences and research, 2(6): 1473-1475, (2011) [15]. mraicha, f., ksantini, m., zouch, o., ayadi, m., sayadi, s., bouaziz, m., effect of olive fruit fly infestation on the quality of olive oil from chemlali cultivar during ripening, food and chemical toxicology, 48(11): 3235-3241, (2010) [16]. bouaziz, m., lassoued, s., bouallagui, z., smaoui, s., gargoubi, a., dhouib, a., sayadi, s., synthesis and recovery of high bioactive phenolics from table-olive brine process wastewater, bioorganic and medicinal chemistry, 16(20): 9238-9246, (2008) [17]. xu, x., li, f., zhang, x., li, p., zhang, x., wu, z., li, d., in vitro synergistic antioxidant activity and identification of antioxidant components from astragalus membranaceus and paeonia lactiflora, plos one, 9(5), (2014) [18]. leja, m., mareczek, a., nanaszko, b., antyoksydacyjne właściwości owoców wybranych gatunków dziko rosnących drzew i krzewów, roczniki akademii rolniczej w poznaniu, 383: 327331, (2007) [19]. ulusoy, s., boşgelmez-tinaz, g., seçilmiş-canbay, h., tocopherol, carotene, phenolic contents and antibacterial properties of rose essential oil, hydrosol and absolute, current microbiology, 59(5): 554-558, (2009) [20]. cai, y., luo, q., sun, m., corke, h., antioxidant activity and phenolic compounds of 112 traditional chinese medicinal plants associated with anticancer, life sciences, 74(17): 2157-2184, (2004) [21]. selvan, c. t., velavan, s., milton, m. j., antioxidant activity of rosa centifolia flowers, international journal of research in plant science, 4(3): 68-71, (2014) 1. introduction odstraňovanie železa a mangánu z vody filtráciou prírodnými materiálmi food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 29 analysis trichloroand tetrachloro-ethylene in water 1ján ilavský, 1danka barloková 1department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovakia jan.ilavsky@stuba.sk, danka.barlokova@stuba.sk *corresponding author received 10 december 2011, accepted 25 february 2012 abstract: the method for determination of trichloroethylene (tce) and tetrachloroethylene (ttce), also known as perchloroethylene (pce), in water at 1 – 30 mg.l-1 concentrations was elaborated. water (1000 ml) is extracted by manual shaking (5 minutes) at 5-7 oc with n-pentane (0,5 ml) in the presence of internal standard (1-cl-n-hexane, 1-cl-n-octane) and the extract chromatographed using split-splitless injection technique. the recoveries of 28 chlorinated hydrocarbons in water at 5 mg.l-1 concentrations for each compound relative to internal standard (r=100%) are also introduced. the fid and ecd detectors (in the case of ecd detector without of internal standard) were used in final stage of determination. keywords: capillary gas chromatography, microextraction, water analysis, chlorinated hydrocarbons 1. introduction organic compounds of chlorine and other halogens present in water predominantly come from human activities or are influenced by them. it is mainly chemical industry (production of synthetic polymers, fibers, organic solvents, etc.) engineering (degreasing and surface treatment of metals), metallurgical and electrical industry (microelectronics), agriculture (pesticides), communal undertaking (chemical cleaning of clothes) and others that are responsible for abrupt increase in contamination of all kinds of water including rain water. moreover, the organohalogen compounds are formed in the course of water disinfection itself as undersirable products of chlorination of different hydrocarbons of biogenic or synthetic origine. on the basic of present knowledge, these substances are considered to by very dangerous for environment as well as for human being itself. many organohalogen compounds are very resistent to oxidative, photolytic and other physicochemical degradation processes and therefore they stay in aqueous medium for a long time. the half-times of decay of some chlorinated hydrocarbons in aqueous medium are given in table 1 [1,2]. table 1 half-times of decay of some chlorinated hydrocarbons in aqueous medium [1,2] compound half-time of decay tetrachloroethylene 6 years trichloroethylene 9 months chloroethylenes 6 – 12 weeks chloromethanes 10 – 33 weeks chloroethanes 10 – 33 weeks as organohalogens belong among substances detrimental to aqueous medium and biological life, they should not be present in water. however, the requirement of absolute zero concentration of these substances even in drinking water is hardly realizable for the present. the concentration ranges of some chlorinated hydrocarbons in different kinds of mailto:jan.ilavsky:@stuba.sk mailto:danka.barlokova:@stuba.sk food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 30 water statistically processed according to literary resources [3] are given in table 2. table 2 concentration ranges of trichloro-ethylene (tce) and tetrachloroethylene (ttce) in different kinds of water [3] water tce [µg/l] ttce [µg/l] surface water 0.01 11 0.1 1300 drinking water 0.1 15 0.1 200 ground water 0.1 150 0.1 1000 rain water 0.1 13 0.1 50 waste water up to 2000 0.1 6000 the international agency for research on cancer has classified trichloroethylene and tetrachloroethylene as a group 2a carcinogen, which means that it is probably carcinogenic to humans [4]. maximum contaminant level (usepa) for tce and ttce in drinking water is 0,005 mg/l (according to the requirements of the regulation of the government of the slovak republic no. 496/2010 on drinking water, the contaminant level is 0,01 mg/l). the granular activated carbon in combination with packed tower aeration are required for effective removing tce and ttce from water [5]. the analysis of volatile organohalogene compounds (voc) in water samples requires a several steps: a) isolation and preconcentration of the pollutants from water, b) trace organic analysis by capillary gas chromatography (gc), c) identification and quantitative determination individual compounds. sample preparation methods include static headspace, dynamic headspace (purge and trap), solvent extraction and solid-phase microextraction techniques, or direct injection of water into gas gromatograph. the advantages and disadvantages of these methods are presented and discussed [6]. the chlorinated hydrocarbons in water can by analyzed by injection water directly into a gas chromatograph [7-14]. this method is profitable because of simplicity. the rests of salts deposited in the injector after evaporation of water bringcauses the problems. however, such problems can be solved by simple exchange of glass tube of the injector after 50 – 100 analyses (according to quantity of the dosed volume of sample). the sensitivity of this method reaches 0,1 mg/l h2o. the chlorinated hydrocarbons in water can be determined by using the “headspace” method. this method is either static, i.e. carried out in closed system [15-17] or dynamic, the so-called stripping or purge and trap, with adsorption, i.e. on tenax [18-20]. the dynamic method is more sensitive and if we use a detector ecd, we can analyze these componds even when they are present in concentrations under 0,01 mg/l h2o. the static headspace method is an attractive method regarding its rapid times and simplicity, i.e., no sample workup is required outside the vial [21]. in static headspace method a sample of water is placed into a vial, sealed, and heated to a specific temperature. all of the components volatile at or below the pre-set temperature escape from the sample to form a gaseous "headspace" above the sample. after a certain period of time, the headspace gas is extracted from the vial and injected into a gas chromatograph with selective detector. the sensitivity for static headspace is typically in the sub microgram range. however, it depends on the volatility of the compounds. in dynamic headspace method the sample is purged with ultra pure nitrogen while being heated in a teflon vessel. as the nitrogen stream exits the vessel it passes through the thermal desorption tube filled with an adsorbent material. the outgassed products are collected onto the adsorbent material. following the predetermined collection time, the tubes are transferred to a thermal desorption unit which is inline http://en.wikipedia.org/wiki/international_agency_for_research_on_cancer http://en.wikipedia.org/wiki/international_agency_for_research_on_cancer http://en.wikipedia.org/wiki/carcinogen http://en.wikipedia.org/wiki/tetrachloroethylene%23cite_note-2 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 31 with the gas chromatograph and selective detector (gc/ms or gc/ecd). the thermal desorption unit heats the individual tubes while a flow of gas is applied through the tube. the collected analytes are flushed from the sorbent and collected onto a cold trap within the thermal desorption unit. the cold trap is heated rapidly after purging the entire sample from the sample tube and collected in the cold trap. the collected analytes are then swept from the cold trap into the gc/ms for analysis as a volatile sample. the static headspace method is less expensive than the purge-and-trap since no expensive purging equipment is used here. with the headspace method, multiple runs can be performed on a single sample vial, whereas the purge-and trap method is essentially destructive; the sample may only be purged and analysed once [21]. the disadvantage of the purge-and-trap method is associated with the problems related to the use of adsorbents, such as overloading, carryover, and breakdown with repeated heating and purging cycles. solid-phase microextraction (spme) is a relatively new technique, first published in 1989 [22]. a thin fused silica fiber coated with a layer of polymeric adsorbent material is introduced directly into the aqueous sample, whereupon the analytes diffuse into the fibre coating until equilibrium is established, thus being extracted in amounts determined by their distribution coefficients and concentrations. subsequently, the analytes are desorbed thermally in gc injector. the method is effective, sensitive to wide range of compounds (detection limits are typically from 20 ng/l to 200 ng/l, except for the very light vocs), less expensive and easier to use then well-established methods such as purge and trap and traditional head-space analysis techniques. solid-phase microextraction has been applied to quantitative analysis of organohalogens in drinking water [23-29]. the temperature has a very direct influence on extraction. a higher temperature reduces the time to reach equilibrium because of faster diffusion in the water. however, it lowers the total amount absorbed on the fibre as the distribution coefficients decrease. for this reason the spme method requires examination of the extraction time, fibre coating-water distribution coefficient, equilibration curves for vocs compound, adsorption heat and analytes volatility. the extraction methods are frequently used for determining the chlorinated hydrocarbons present in water. in literature issued in our country as well as abroad many methods of extraction of the halogenated organic compounds from water have been described. they differ from each other by kind and amount of the used extractant (ratio water : solvent) as well as by the recovery of extraction. n-pentane [30-38], n-hexane [31,33,39,40], methylcyklohexane [31,33,42], isooctane [31-33], petroleum ether [43], mixture of n-hexane + diisopropylether [39,41], etc are used for extraction. by comparing the efficiencies of extraction methods with each other, it has been found that no significant differences appear if different extractants are used [44]. the effectiveness of extraction mainly depends on affinity of dissolved substance to extractant (the measure of which is distribution coefficient, i.e. the ratio of concentration of organohalogen in extractant to its concentration in water) and phase ratio (the ratio of extractant volume to sample volume) as well as on number of extraction steps. the extraction can be carried out either in one step or in several steps in series (the extraction is performed either with successive addition extractant or in countercurrent of water and solvent). the efficiency of extraction is affected by extent of the interface contact, solubility of the investigated substances in both phases, ionic strenght and ph of the medium. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 32 a comparison of extraction methods with the headspace or stripping methods is presented in papers [45,46]. for instance, the stripping method is about ten times more sensitive method then microextraction for compounds with boiling points up to 200 oc. on the other hand, the stripping method necessitates special apparatus and is slow and therefore it is less convenient for routine analysis of organohalogens in water when compared with microextraction. in most cases the cappilary gas chromatography with an ecd detector or the gc/ms combination is used in final stage of determination. if a fid detector appropriate for the chlorinated hydrocarbons containing 1 or 2 chlorine atoms in a molecule is used, we must choose a volitale solvent (or a column with more polar phase) so that the solvent should not interfere with waves of the analyzed substances. in view of the above – mentioned facts, we have paid attention to the possibility of using microextraction for the analysis of the chlorinated hydrocarbons in water. the experimental part of this paper comprises the results of investigation represented by the recovery of 28 chlorinated hydrocarbons obtained by microextraction with n-pentane. 2. experimental 2.1 instrumentation gas chromatograph carlo erba (vega 6000) equipped with an ecd or fid detectors and split-splitless injector system was used. for the chromatographic separation (in the case of fid detector) a glass capillary columns with stationary phase peg 400, triton tx-305 and ucon lb550+ igepal co 880 (4:1) were used. for the chromatographic separation (in the case of ecd detector) a silica capillary column db-5 wet with silicone stationary phase se-54 was used. chromatograms were integrated with dp 700 (carlo erba) integrators. 2.2 microextraction one litre of water containing defined content of chlorinated hydrocarbons was subjected to microextraction (at + 5-7 oc) with 0,5 ml of n-pentane containing internarnal standard (in the case when a fid detector was used) by intense manual shaking 5 minutes. the glass extraction flask, equipped with a male joint (1) and conical stopper (5) was used to extraction (a). after extraction (b) the solvent thin layer separator (2) containing side arm for water (3), capillary for extract (4) was connected to the extraction flask (figure 1) [47]. in this case the n-pentane extracts are easily accessible and can be injected immediately by means of a syringe into a gas chromatograph. fig.1 solvent thin layer separator for microextraction of water 2.3 chemicals for the analysis of the chlorinated hydrocarbons certified standards from slovak metrology institute and standards from supelco (purity at least 98 %) were used. as the internal standards 1-cl-nhexane, 1-cl-n-octane or 1-cl-n-dodecane food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 33 were used (purity at least 98% supelco, bellefonte, usa). solvent (n-pentane) was highly purified and checked chromatographically (e. merck, darmstadt, germany). 3. results and discussion it results from literature review that npentane is most frequently used as extractant in extraction methods. because of volatility of lower chlorinated hydrocarbons and boiling point of npentane we may expect lower losses of the analyzed components for this solvent than for extractants with higher boiling point. for this reason, it is serviceable to add into extractant an internal standard exhibiting similar volatility as analyzed compounds. the recoveries (average of five analysis) of some chlorinated hydrocarbons with respect to 1-cl-n-hexane as internal standard (r=100%) for equal concentration of each component (5 mg/l of water) are given in table 3. table 3 the recoveries of volatile chlorinated hydrocarbons from water by microextraction with n-pentane compound recovery [%] fid detector 5 mg/l % rsd dichloromethane 1.74 5.63 trichloromethane 3.79 5.42 dichlorobromomethane 4.27 5.86 chlorodibromomethane 3.051 6.31 tribromomethane 6.921 6.58 tetrachloromethane 57.20 5.12 1,1-dichloroethane 4.32 4.39 1,2-dichloroethane 1.05 6.92 1,1,1-trichloroethane 1,1,2-trichloroethane 2.71 5.38 1,1,2,2-tetrachloroethane 7.141 4.92 1,2-dichloropropane 6.12 5.14 1,1-dichloroethylene 1,2-dichloroethylene (trans) 4.71 4.95 trichloroethylene 34.75 3.82 tetrachloroethylene 85.98 3.73 1 the recovery with respect 1-cl-n-octane (is) table 3 continued 1,3-dichloropropene (trans) 1.16 6.38 1,3-dichloropropene (cis) 3.44 4.78 2-chloroethylvinylether 4.62 5.41 chlorobenzene 1.68 5.17 1,3-dichlorobenzene 58.171 3.32 1,4-dichlorobenzene 54.861 3.60 1,2-dichlorobenzene 53.231 3.98 1,3,5-trichlorobenzene 77.622 4.09 1,2,4-trichlorobenzene 77.102 4.18 1,2,3-trichlorobenzene 76.972 4.37 1,2,3,5-tetrachlorobenzene 85.772 3.89 1,2,4,5-tetrachlorobenzene 85.862 3.54 pentachlorobenzene 94.852 3.84 hexachlorobenzene 97.712 3.32 compound recovery [%] ecd detector 5 mg/l % rsd dichloromethane 8.12 7.58 trichloromethane 9.75 7.21 dichlorobromomethane 11.62 6.89 chlorodibromomethane 12.20 6.45 tribromomethane 14.68 6.14 tetrachloromethane 76.20 6.06 1,1-dichloroethane 8.65 6.37 1,2-dichloroethane 9.84 6.18 1,1,1-trichloroethane 27.92 6.52 1,1,2-trichloroethane 14.58 6.20 1,1,2,2-tetrachloroethane 15.32 6.36 1,2-dichloropropane 11.26 6.78 1,1-dichloroethylene 9.41 7.15 trichloroethylene 41.92 5.89 tetrachloroethylene 86.56 5.22 1,3-dichloropropene (trans) 7.68 7.34 1,3-dichloropropene (cis) 7.82 7.21 chlorobenzene 1,3-dichlorobenzene 39.72 5.76 1,4-dichlorobenzene 38.98 5.92 1,2-dichlorobenzene 38.58 4.89 1,3,5-trichlorobenzene 1,2,4-trichlorobenzene 1,2,3,5-tetrachlorobenzene 1,2,4,5-tetrachlorobenzene pentachlorobenzene hexachlorobenzene 2 the recovery with respect 1-cl-n-dodecane food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 34 the values obtained for the investigated compounds show that microextraction into n-pentane is convenient for tetrachloromethane, trichloroethylene, tetrachloroethylene, dichlorobenzene and other higher chlorinated benzenes. the recovery of other chlorinated hydrocarbons are relatively low which is due to stronger hydrogen bonds between the oh groups of water and the chlorine atoms in a molecule of hydrocarbon. the efficiency of isolation is significantly affected by polarity of the extracted compounds (its solubility in water). therefore the solubilities of some chlorinated hydrocarbons in water taken from literature are given in table 4. the values of recovery given in table 3 were obtained by reverse extraction of 0,5 ml of n-pentane containing 5 mg of eachcomponent (including 1-cl-n-hexane as internal standard) with water (1 litre). the extraction was performed by intense manual shaking for 5 minutes. table 4 the solubilities of some chlorinated hydrocarbons in water [48,49,50] compound solubility [g/l at 20 oc] chloromethane 7.25 dichloromethane 19.80 trichloromethane 8.20 tetrachloromethane 0.78 chloroethane 4.1 1,1-dichloroethane 5.5 1,2-dichloroethane 8.7 1,1,1-trichloroethane 0.48-4.4 1,1,2-trichloroethane 1.1-4.6 1,1,2,2-tetrachloroethane 2.87 pentachloroethane 0.5 hexachloroethane 0.05 1,2-dichloropropane 2.8 1,3-dichloropropane 2.7 1,2,3-trichloropropane 1.9 tribromomethane 3.1 chloroethylene 0.40-2.66 1,1-dichloroethylene 0.4 table 4 continued chloroethylene 0.40-2.66 1,1-dichloroethylene 0.4 1,2-dichloroethylene (trans) 8.8 1,2-dichloroethylene (cis) 3.5 trichloroethylene 1.1 tetrachloroethylene 0.149 chlorobenzene 0.502 1,3-dichlorobenzene 0.143 1,4-dichlorobenzene 0.065 1,2-dichlorobenzene 0.137 1,3,5-trichlorobenzene 0.006 1,2,4-trichlorobenzene 0.031 1,2,3-trichlorobenzene 0.018 1,2,3,5-tetrachlorobenzene 0.0051 1,2,4,5-tetrachlorobenzene 0.00046 1,2,3,4-tetrachlorobenzene 0.00592 if we compare data in table 3 with table 4, we can see that the recovery of chlorinated hydrocarbons are consistent with their solubility in water. the highest recovery appear if the solubility falls under 1 g/l of water. the data in table 4 may serve as orientation for determination of other chlorinated hydrocarbons in water by using microextraction as well. furthermore, it results from table 3 that the recovery increases with number of the chlorine atoms in a molecule of hydrocarbon which manifests itself e.g. in the series di-, triand tetrachloromethane or mono-, and as far as hexachlorobenzene. the recovery of extraction are usually affected by concentration of the analyzed substance in water which is most conspicuous if the concentration is very low. it is very important to taken this fact into consideration, especially if the recovery are low which is usual in the case of microextraction. the errors due to the change in recovery produced by varying concentration may be reduced by drawing the analytical curve for a given concentration range. the values for analytical curve of trichloroethylene and tetrachloroethylene food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 35 in the concentration range 1 – 30 mg/l of water were obtained by the use of microextraction of model sample of water (1 litre) containing know quantity of the analyzed substances with 0,5 ml of npentane containing 5 mg of 1-cl-n-hexane. the analytical curves are represented in figure 2 which shows that it is practically possible to execute linearization of the curve in the investigation concentration region and thus to neglect the variation of recovery with concentration. fig. 2 the analytical curves of trichloroethylene (tce) and tetrachloroethylene (ttce) in the concentration range 1 – 30 mg/l of water ptce – area of tce elution peak, pttce – area of ttce elution peak, pis – area of is elution peaks we have also had the reason for paying attention to tce and ttce because these two substances belong among the most frequent contaminants of ground waters. we analyzed real samples of water in the same way as described for determining the analytical curve. this method was used to determine the areal and vertical distribution of groundwater contaminated by chlorinated hydrocarbons water source, the red willows in piestany. water source yielding 80 l / s was located 350 m from the plant site, there was used organic solvents based on chlorinated hydrocarbons in manufacturing process. inadequate provision of storage facilities and handling facilities to the leakage of chlorinated hydrocarbons caused degradation of groundwater and soil in the area of this plant site. the pollution spread to an area 4.2 square kilometers with an average depth range of 15 m. within the monitoring we analyzed 164 samples, 88 samples in tce content and content ttce 47 was equal to or higher than 0.01 ug/l. from the total number of samples the limit value of 0.01 mg/l for drinking water (gr 496/2010) exceeded for tce 27 samples and for ttce 3 samples. repeated analyzes of groundwater samples in the following years confirmed the existing pollution of groundwater and water resource red willow was declared unsuitable for public drinking water supply. the chromatogram of a model sample of water containing chlorinated hydrocarbons represented in figure 3 may serve as an example of the determination of trichloroethylene and tertachloroethylene in water frequent contaminants of ground waters. the developed method was used for analytical determination tce and ttce in ground waters of accident regions. fig. 3 the gas chromatogram of a model sample of water containing trichloroethylene and tetrachloroethylene together with aromatic and saturated hydrocarbons elution peaks (fig. 3): 1 benzene, 2 octane, 3 – trichloroethylene, 4 methylbenzene, 5 tetrachloroethylene, 6 nonane, 7 1-cl-n-hexane (is), 8-ethylbenzene, 9 1,3-dimethylbenzene, 10 1,4-dimethylbenzene, 11-1,2-dimethylbenzene, 12-decane, 13-iso-propylbenzene, 14-n-propylbenzene, 15-sek-buthylbenzene, 16iso-buthylbenzene, 17undecane, 18n-buthylbenzene food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 36 figure 4 shows the gas chromatogram of chlorinated hydrocarbons mixture with identification of individual elution peaks. identification of components is based on certified standard allowance into n-pentane solvent. for the analysis of chlorinated hydrocarbons in water a microextraction with the following gas capillary chromatography equipped with ecd can be used. ecd detector is more sensitive to amount of chlorine atoms in compound. therefore, the recoveries of trichloroethylene and tetrachloroethylene (in table 3) are higher when using of ecd detector rather than fid detector. fig. 4 gas chromatogram of the model mixture of chlorinated hydrocarbons (concentration of each component of 1 mg) in 0,5 ml of n-pentane before the microextraction elution peaks (fig. 4): 1– 1,1-dichloroethylene, 2– dichloromethane, 3– trichloromethane, 4– 1,1,1-trichlorothane, 5– 1,2-dichloroethane, 6– tetrachloromethane, 7– 1,2-dichloropropane, 8– trichloroethylene, 9–dichlorobromomethane,10–1,1,2-trichloroethane, 11–dibromochloromethane,12– tetrachloroethylene, 13–tribromomethane, 14–1,1,2,2-tetrachloroethane. 4. conclusions the above-mentioned considerations indicate that the microextraction methods of trichloroethylene and tetrachloroethylene isolation is very rapid, simple, economically profitable and with low detection limit (0,1 mg/l of water). the recovery of trichloroethylene into npentane is above 40% and for tetrachloroethylene it is higher than 85%. these values are influenced by number of chlorine atoms in molecule and their solubility in water. the results of this study give provide data important for quantitative analysis of these compounds in water and they can be used for routine quantitative analysis involving microextraction and capillary gas chromatography. 5. references [1] mc connel g., ferguson d.m., pearson c.r.j. chlorinated hydrocarbons and the environment. endeavour 34, 13-18, (1975). [2] dilling w.l., tefertiller n.b., kallos g.j. evaporation rates and reactivities of methylene chloride, chloroform,1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, and other chlorinated compounds in dilute aqueous solutions environ. sci. technol. 9, 833, (1975). [3] atri, f.r. chlorierte kohlenwasserstoffe in der umwelt.iii. landschaftsentwicklung und umweltforschung 34, 134, (1985). [4] iarc monograph. tetrachloroethylene, vol. 63, p. 159. last updated may 20, 1997. last retrieved june 22, (2007). [5] eur 21680 en european union risk assessment report. volume 57. tetrachloroethylene. part1 – environment. institute for health and consumer protection, final report, pp. 154, (2005). [6] marczak m., wolska l., chrzanowski w., namieśnik j. microanalysis of volatile organic compounds in water samples – methods and instruments. microchimica acta 155, 331-348, (2006). [7] harris l.e., budde w.l., eichelberger j.m. direct analysis of water samples for organic pollutants with gas http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://pubs.acs.org/doi/abs/10.1021/es60107a008 http://www.inchem.org/documents/iarc/vol63/tetrachloroethylene.html https://springerlink3.metapress.com/content/?author=marcin+marczak https://springerlink3.metapress.com/content/?author=lidia+wolska https://springerlink3.metapress.com/content/?author=wojciech+chrzanowski https://springerlink3.metapress.com/content/?author=jacek+namie%c5%9bnik https://springerlink3.metapress.com/content/0026-3672/ https://springerlink3.metapress.com/content/0026-3672/155/3-4/ http://pubs.acs.org/doi/abs/10.1021/ac60349a001 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 37 chromatography-mass spectrometry. anal. chem. 46(13), 1912, (1974). [8] fuji t. j. direct aqueous injection gas chromatography-mass spectrometry for analysis of organohalides in water at concentrations below the parts per billion level. j. chromatogr. 139(2), 297, (1977). [9] nicholson a.a., meresz o., lemyk b. determination of free and total potential haloforms in drinking water. anal. chem. 49(6), 814, (1977). [10] grob k., habich a. trace analysis of halocarbons in water; direct aqueous injection with electron capture detection, j. high. resol. chromatogr. 6(1), 11, (1983). [11] pfaender f.k., jonas r.b., stevens a.a., moore l., hass j.r. evaluation of direct aqueous injection method for analysis of chloroform in drinking water, environ. sci. technol. 12(4), 438, (1978). [12] zlatkis a., wang f. s., shanfield h. direct gc analysis of aqueous samples at the part-per-billion and part-per-trillion levels. anal. chem. 55(12), 1848, (1983). [13] jolley r. l., suffet i. h. concentration techniques for isolating organic constituents in environmental water samples. advances in chemistry 214, 3, (1987). [14] mehran m.m., cooper w.j., mehran m. comparison of direct headspace and aqueous injection techniques for halogenated hydrocarbons in water. j. chromatogr. sci. 24, 142, (1988). [15] diets e.a., singie k.f. determination of chlorinated hydrocarbons in water by headspace gas chromatography. anal. chem. 51(11), 1809, (1979). [16] kolb b. application of an automated headspace procedure for trace analysis by gas chromatography. j. chromatogr. 122, 553, (1976). [17] kuráň p., soják l. environmental analysis of volatile organic compounds in water and sediment by gas chromatography. j. chromatogr. 733(1-2), 119-141, (1996). [18] mcnally m.e., grob r.l. determination of the solubility limits of organic priority pollutants by gas chromatographic headspace analysis. j. chromatogr. 260, 23-32, (1983). [19] castello g., gerbino t.c., kanitz s. gas chromatographic analysis of halocarbons in drinking water by headspace extraction and mixed column separation. j. chromatogr. 247(2), 263, (1982). [20] mindrup r. trace analysis of organics in water by gas chromatography. pergamon ser. environ. sci. 3, 325, (1980). [21] roe v.d., lacy m.j., stuart j.d., robbins g.a. manual headspace method to analyze for the volatile aromatics of gasoline in groundwater and soil samples. anal. chem., 61(22), 2584, (1989). [22] belardi rg, pawliszyn j. the application of chemically modified fused silica fibers in the extraction of organics from water matrix samples and their rapid transfer to capillary columns. water qual. res. j. can. 24, 179, (1989). [23] arthur c.l., pratt k., motlagh s., pawliszyn j. environmental analysis of organic compounds in water using solid phase micro extraction. j. of high resolution chromatogr. 15(11), 741, (1992). [24] chai m., arthur c.l., pawliszyn j., belardi r.p., pratt k.f. determination of volatile chlorinated hydrocarbons in air and water with solid-phase microextraction analyst, 118, 150, (1993). [25] arthur c.l., chai m., pawliszyn j. solventless injection technique for microcolumn separations. j. microcol. sep. 5(11), 51, (1993). [26] page b.d., lacroix g. application of solid-phase microextraction to the headspace gas chromatographic analysis of halogenated volatiles in selected foods. j. chromatogr., 648(1), 199, (1993). [27] niri v.h., bragg l., pawliszyn j. fast analysis of volatile organic compounds and disinfection by-products in drinking water using solid-phase microextraction–gc/timeof-flight mass spectrometry. j. chromatogr. 1201, 222, (2008). [28] eichelberger j.w., budde w.l. method 524.2 measurement of purgeable compounds in water by capillary column gas chromatography/mass spektrometry.us epa, cincinnati, ohio, (1989). [29] nilsson t., pelusio f., larsen b., montanrella l., facchetti s., madsen j.ø. an evaluation of solid-phase microextraction for analysis of volatile organic compounds in drinking water. j. high. resol. chromatogr. 18(10), 617, (1995). [30] hagenmaier h., werner g., jäger w., quantitative determination of volatile halogen hydrocarbons in water samples by capillary gas chromatography and electron capture detector. z. wasser abwasser forsch. 15, 195, (1982). [31] mehran m.f., slifker r.a., cooper w.j. a simplified liquid-liquid extraction method for analysis of trihalomethanes in drinking water. j. chromatogr. sci. 22, 241, (1984). http://pubs.acs.org/doi/abs/10.1021/ac60349a001 http://pubs.acs.org/doi/abs/10.1021/ac60349a001 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44b861m-p7&_user=10&_coverdate=09/21/1977&_rdoc=8&_fmt=high&_orig=browse&_origin=browse&_zone=rslt_list_item&_srch=doc-info(%23toc%235248%231977%23998609997%23269982%23flp%23display%23volume)&_cdi=5248&_sort=d&_docanchor=&_ct=24&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=4337dcdad23cb9cddc039decafefe920&searchtype=a 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http://www.sciencedirect.com/science?_ob=articleurl&amp;amp;amp;amp;_udi=b6tg8-44b861m-p7&amp;amp;amp;amp;_user=10&amp;amp;amp;amp;_coverdate=09/21/1977&amp;amp;amp;amp;_rdoc=8&amp;amp;amp;amp;_fmt=high&amp;amp;amp;amp;_orig=browse&amp;amp;amp;amp;_origin=browse&amp;amp;amp;amp;_zone=rslt_list_item&amp;amp;amp;amp;_srch=doc-info(%23toc%235248%231977%23998609997%23269982%23flp%23display%23volume)&amp;amp;amp;amp;_cdi=5248&amp;amp;amp;amp;_sort=d&amp;amp;amp;amp;_docanchor=&amp;amp;amp;amp;_ct=24&amp;amp;amp;amp;_acct=c000050221&amp;amp;amp;amp;_version=1&amp;amp;amp;amp;_urlversion=0&amp;amp;amp;amp;_userid=10&amp;amp;amp;amp;md5=4337dcdad23cb9cddc039decafefe920&amp;amp;amp;amp;searchtype=a http://pubs.acs.org/doi/abs/10.1021/ac50014a036 http://pubs.acs.org/doi/abs/10.1021/ac50014a036 http://pubs.acs.org/doi/abs/10.1021/es60140a016 http://pubs.acs.org/doi/abs/10.1021/es60140a016 http://pubs.acs.org/doi/abs/10.1021/es60140a016 http://pubs.acs.org/doi/abs/10.1021/ac50047a047 http://pubs.acs.org/doi/abs/10.1021/ac50047a047 http://pubs.acs.org/doi/abs/10.1021/ac50047a047 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44y41rt-rb&_user=10&_coverdate=07/07/1976&_alid=1601279525&_rdoc=2&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_st=13&_docanchor=&view=c&_ct=2&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=da4d00011324fd219b53bfc131c91de9&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-44y41rt-rb&amp;_user=10&amp;_coverdate=07/07/1976&amp;_alid=1601279525&amp;_rdoc=2&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_st=13&amp;_docanchor=&amp;view=c&amp;_ct=2&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=da4d00011324fd219b53bfc131c91de9&amp;searchtype=a 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http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-3vjrr06-b&amp;_user=10&amp;_coverdate=05/10/1996&amp;_alid=1601273876&amp;_rdoc=5&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_sort=r&amp;_st=13&amp;_docanchor=&amp;view=c&amp;_ct=41&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=975c8fad097a1072e34ca03c154e6cf6&amp;searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;amp;_udi=b6tg8-3vjrr06-b&amp;amp;_user=10&amp;amp;_coverdate=05/10/1996&amp;amp;_alid=1601273876&amp;amp;_rdoc=5&amp;amp;_fmt=high&amp;amp;_orig=search&amp;amp;_origin=search&amp;amp;_zone=rslt_list_item&amp;amp;_cdi=5248&amp;amp;_sort=r&amp;amp;_st=13&amp;amp;_docanchor=&amp;amp;view=c&amp;amp;_ct=41&amp;amp;_acct=c000050221&amp;amp;_version=1&amp;amp;_urlversion=0&amp;amp;_userid=10&amp;amp;md5=975c8fad097a1072e34ca03c154e6cf6&amp;amp;searchtype=a 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http://www.sciencedirect.com/science?_ob=articleurl&amp;amp;_udi=b6tg8-44wcy84-3&amp;amp;_user=10&amp;amp;_coverdate=12/31/1983&amp;amp;_alid=1601273876&amp;amp;_rdoc=16&amp;amp;_fmt=high&amp;amp;_orig=search&amp;amp;_origin=search&amp;amp;_zone=rslt_list_item&amp;amp;_cdi=5248&amp;amp;_sort=r&amp;amp;_st=13&amp;amp;_docanchor=&amp;amp;view=c&amp;amp;_ct=41&amp;amp;_acct=c000050221&amp;amp;_version=1&amp;amp;_urlversion=0&amp;amp;_userid=10&amp;amp;md5=3c60028002e825677d5c635e405bfdaf&amp;amp;searchtype=a 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http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44cpwjh-ph&_user=10&_coverdate=10/01/1993&_alid=1605190996&_rdoc=1&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_sort=r&_st=13&_docanchor=&view=c&_ct=1&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=fc9a902c2cf675fae17776802039cdb8&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-44cpwjh-ph&amp;_user=10&amp;_coverdate=10/01/1993&amp;_alid=1605190996&amp;_rdoc=1&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_sort=r&amp;_st=13&amp;_docanchor=&amp;view=c&amp;_ct=1&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=fc9a902c2cf675fae17776802039cdb8&amp;searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;amp;_udi=b6tg8-44cpwjh-ph&amp;amp;_user=10&amp;amp;_coverdate=10/01/1993&amp;amp;_alid=1605190996&amp;amp;_rdoc=1&amp;amp;_fmt=high&amp;amp;_orig=search&amp;amp;_origin=search&amp;amp;_zone=rslt_list_item&amp;amp;_cdi=5248&amp;amp;_sort=r&amp;amp;_st=13&amp;amp;_docanchor=&amp;amp;view=c&amp;amp;_ct=1&amp;amp;_acct=c000050221&amp;amp;_version=1&amp;amp;_urlversion=0&amp;amp;_userid=10&amp;amp;md5=fc9a902c2cf675fae17776802039cdb8&amp;amp;searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;amp;amp;_udi=b6tg8-44cpwjh-ph&amp;amp;amp;_user=10&amp;amp;amp;_coverdate=10/01/1993&amp;amp;amp;_alid=1605190996&amp;amp;amp;_rdoc=1&amp;amp;amp;_fmt=high&amp;amp;amp;_orig=search&amp;amp;amp;_origin=search&amp;amp;amp;_zone=rslt_list_item&amp;amp;amp;_cdi=5248&amp;amp;amp;_sort=r&amp;amp;amp;_st=13&amp;amp;amp;_docanchor=&amp;amp;amp;view=c&amp;amp;amp;_ct=1&amp;amp;amp;_acct=c000050221&amp;amp;amp;_version=1&amp;amp;amp;_urlversion=0&amp;amp;amp;_userid=10&amp;amp;amp;md5=fc9a902c2cf675fae17776802039cdb8&amp;amp;amp;searchtype=a http://www.sciencedirect.com/science/journal/00219673 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235248%232008%23987989997%23694831%23fla%23&_cdi=5248&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=42496118a982b8c8c87407c2e1571bbc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 38 [32] richard j.j., junk g.a. liquid extraction for rapid determination of halomethanes in water. j. awwa 69, 22, (1977). [33] varma m.m., siddique m.r., doty k.t., machis a. analysis of trihalomethanes in aqueous solutions: a comparative study. j. awwa 71, 389, (1979). [34] biziuk m., przyjazny a. methods of isolation and determination of volatile organohalogen compounds in natural and treated waters. j. of chromatogr. 733(1-2), 417, (1996). [35] weil l., jandik j., eichelsdörfer d. organic halogenated compounds in swimmingpool water, i. determination of volatile halogenated hydrocarbons. z. wasser abwasser forsch. 13, 141, (1980). [36] trussell a.r., umphres m.d., leong l.y., trussell r.r. precise analysis of trihalomethanes. j. awwa 71, 385, (1979). [37] peruzii p, cursi v, griffini v. application of capillary gas chromatography with electron capture detection and splitsplitless injection to the evaluation of volatile halogenated hydrocarbon removal using hydrogen peroxide after break-point chlorination in drinking water treatment. j. high. resol. chromatogr. 8, 450, (1985). [38] eklund g., josefsson b., roos c. determination of volatile halogenated hydrocarbons in tap water, seawater and industrial effluents by glass capillary gas chromatography and electron capture detection. j. high. resol. chromatogr. 1, 34, (1978). [39] norin h., renberg l. determination of trihalomethanes (thm) in water using high efficiency solvent extraction. water research 14(10), 1397, (1980). [40] rensburg j.f.j., huyssteen j.j., hassett a.j. a semi-automated technique for the routine analysis of volatile organohalogens in water purification processes. water research 12(2), 127, (1978). [41] rensburg j.f.j., hassett a.j. a lowvolume liquid-liquid extraction technique. j. high. resol. chromatogr. 5(10), 574, 1982. [42] mieure j.p. a rapid and sensitive method for determining volatile organohalides in water. j. awwa 69, 60, (1977). [43] reunanen m., kroneld r. determination of volatile halocarbons in raw and drinking water, human serum, and urine by electron capture gc. j. chromatogr. sci. 20(10), 449, (1980). [44] dressman r.c., stevens a.a., fair j., smith b. comparison of methods for determination of trihalomethanes in drinking water. j. awwa 71, 392, (1979). [45] shottmeister r.c., engervald w. einige aspekte der isolierung und anreicherung flüchtiger organischer mikroverunreinigungen aus wasser. acta hydrochim. hydrobiol. 9(5), 479, (1981). [46] otson r., williams d.t., bothwell p.d. a comparison of dynamic and static head space and solvent extraction techniques for the determination of trihalomethanes in water. environ. sci. technol. 13(8), 936, (1979). [47] hrivňák j. solvent thin layer separator for microextraction of water. anal. chem. 57(11), 2159, (1985). [48] hutchinson t.c., hellebust j.a., mackay d., mascarenkas r.a., shin w.y. the correlation of the toxicity to algae of hydrocarbons and halogenated hydrocarbons in the aquatic environment, edited by b.h. afghan and d. mackay, plenum press, p. 581, (1978). [49] banerjee s. solubility of organic mixtures in water. environ. sci. technol. 18(8), 587, (1984). [50] connor m.s. comparison of the carcinogenic risks from fish versus groundwater contamination by organic compounds. environ. sci. technol. 18(8), 628, (1984). http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-3vjrr06-14&_user=10&_coverdate=05/10/1996&_alid=1606348776&_rdoc=2&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_docanchor=&view=c&_ct=2&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=bdcca2b0a2c928ea3d636edad351a2e0&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-3vjrr06-14&amp;_user=10&amp;_coverdate=05/10/1996&amp;_alid=1606348776&amp;_rdoc=2&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_docanchor=&amp;view=c&amp;_ct=2&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=bdcca2b0a2c928ea3d636edad351a2e0&amp;searchtype=a 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р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 60 identification and examination of some probiotic properties of the strain lactobacillus lbrz12 *rositsa denkova1, svetla ilieva 1, dilyana nikolova 1, zapriana denkova2, velichka yanakieva2 1 department „biotechnology”, faculty of biology, sofia university „st. kliment ohridski”, sofia, bulgaria, rositsa_denkova@mail.bg 2department „organic chemistry and microbiology”, university of food technologies, plovdiv, bulgaria, zdenkova@abv.bg *corresponding author received 19 december 2011, accepted 25 february 2012 abstract: in order to be included in the composition of probiotic preparations, a strain should have certain probiotic characteristics, so that when administered as a probiotic it would exercise its beneficial effects on the health of the host. a lactobacillus strain, lactobacillus lbrz12, was identified using contemporary biochemical (api 50 chl) and molecular genetic (ardra) methods. the antimicrobial activity of the strain was determined by the diameter of the hydrolysis zones against 4 saprophytic microorganisms – bacillus subtilis, bacillus mesentericus, aspergillus niger and saccharomyces cerevisiae. its antibiotic resistance was determined with the use of 22 of the most commonly used in medical practice antibiotics. the resistance of lactobacillus lbrz12 to the conditions in the gastrointestinal tract was tested in vitro with solutions with ph=2 + pepsin, ph+8 + pancreatin, as well as with solutions with different concentrations of bile salts – 0.15%, 0.3%, 0.6%, 1% . the lactobacillus strain, isolated from fermented cabbage, was identified as lactobacillus plantarum. some of the probiotic properties of the selected culture were examined. lactobacillus lbrz12 has high antimicrobial activity against saprophytic microorganisms, resistance to most of the antibiotics, currently applied in medical practice, resistance to the conditions in the gastrointestinal tract (ph 2 + pepsin, ph 8 + pancreatin, more than 0.3% bile salts). the probiotic properties of lactobacillus lbrz12 define the strain as a potentially probiotic culture suitable for inclusion in the composition of starter cultures for meat and bakery products. keywords: api 50 chl, api zym, antimicrobial activity, antibiotic resistance, stomach juice, intestinal juice, bile salts 1. introduction lactobacilli belong to the natural microflora of human and animal organisms. together with bifidobacteria they maintain the balance between the microbial species in the gastrointestinal tract through the production of short chain acids (lactic, acetic, etc.), bacteriocins [2] and other metabolites that inhibit enteropathogens [4, 6, 7]. in acidic medium lactobacilli stop the putrefactive processes, thus ensuring the normal functioning of the digestive system [9, 10]. the intake of lactic acid bacteria in the composition of fermented foods or probiotics helps to restore the gastrointestinal microflora and to overcome disbacteriosis [8]. not all lactobacilli can be included in the composition of probiotics and probiotic foods, but only those that have certain properties [9, 10]. some of these properties are the ability of the strains to survive in the conditions of the gastrointestinal tract, mailto:rositsa_denkova:@mail.bg mailto:zdenkova:@abv.bg food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 61 sustaining high concentration of viable cells, antimicrobial activity against saprophytes and antibiotic resistance. the aim of this study is to examine some of the probiotic properties of the strain lactobacillus lbrz12 of plant origin: sustainability in artificial gastric and bile juice, antimicrobial activity and antibiotic resistance. 2. experimental determination of the biochemical profile the system api 50 chl (biomerieux sa, france) is used for the identification of the species of the genus lactobacillus based on their ability to utilize 49 carbon sources. fresh 24-hour culture of the studied strain is centrifuged for 15 min at 5000xg. the obtained sludge, containing biomass, is washed twice with pbs-buffer and resuspended in medium api 50 chl, an integral part of the used kit. the api strips are placed in the incubation boxes, the microtubules are inoculated with the prepared cell suspension and sealed with sterile liquid paraffin. the results are reported on the 24th and the 48th hour of incubation at 37°c ± 1°c. reporting is done, based on the colour change of each microtubule, compared to the colour of the control microtubule (microtubule 0). positive results were recorded in the cases of color change from blue to green or bright yellow. the obtained results are processed with apiweb® identification software. determination of the profile of antibiotic sensitivity the profile of antibiotic susceptibility is determined by the disc diffusion method according to bauer, kirby et al., 1966 [1]. fresh 24-hour culture of the tested strain is used to inoculate the plates with mrs-agar medium. standard discs impregnated with antibiotic are placed in the plates. then the plates are incubated for 48 hours at optimal temperature. the diameter (in mm) of the sterile zones formed around each antibiotic disc is recorded. the following indications are used: r resistant (zone diameter < 8 mm), sr intermediate sensitive (zone diameter 8-16 mm), s sensitive (zone diameter > 16 mm). determination of the antimicrobial activity to determine the antimicrobial activity of the studied strain cultural liquid (cl), cultural acellular supernatant without ph adjustment (casn) and neutralized acellular supernatant (nasn) (ph 6.5), obtained from a 24 hour culture of the strain are used. the antimicrobial activity of the strain is tested against the following test microorganisms: bacillus subtilis, bacillus mesentericus, saccharomyces cerevisiae and aspergillus niger. each of the test microorganisms (108cfu/cm3) is inoculated in agar medium and then the medium is poured in the plates. after hardening of the agar, wells are prepared (6 mm). 0,1 cm3 of cl, casn and nasn are dropped in the wells and the plates are placed at 4ºc for 1h to allow diffusion in the agar. for each combination of saprophyte + lactobacillus strain two plates are prepared. then one of the plates is incubated at 30ºc and the other plate at 37ºc for 24 h and the inhibition zones in mm are reported. determination of the survival at low ph in the presence of pepsin and at weakly alkaline ph in the presence of pancreatin [3]. fresh 24-hour culture of the tested strain is centrifuged for 15 min at 5000xg. the sludge, containing biomass, is washed twice with pbs-buffer and resuspended to the initial volume in pbs-buffer. 0.2 cm3 of the cell suspension is incubated with 5 cm3 buffer solution with ph = 2, containing 0,5% nacl and pepsin (at a concentration of 3.2 g/dm3) (sigma, 2,5003,500 u/mg protein) and buffer solution with ph=8 containing 0,5% nacl and pancreatin (at a concentration 1 g/dm3) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 62 (sigma, 2,500-3,500 u/mg protein) at a suitable temperature for the studied strain for 24h. aliquots are taken for determination of the total number of viable cells (cfu/cm3) at the 0, 2, 4 and 24th hour. determination of the tolerance to bile salts (method modified by denkova z., 2005 [5]). mrs-broth medium, containing bile salts at different concentrations (0%, 0.15%, 0.3%, 0.6% and 1%), is inoculated with 4% inoculum of a 24 hour culture of the tested strain. during the incubation for 24h at the optimum temperature for the tested strain aliquots are taken to determine the total number of viable cells (cfu/cm3) at the 0, 2nd, 4th, 6th, 8th and 24th hour. profile of enzyme activity the system api zym (biomericux, france) is used for semi-quantitative examination of the enzyme profile of the studied strain. fresh 24-hour culture of the strain is centrifuged for 15 min at 5000xg, the resulting sludge, containing biomass, is washed twice and resuspended in api suspension medium. the api zym strips are placed in the incubation boxes and the wells are inoculated with the prepared cell suspension. the samples are incubated for 4 hours at 37°c. then one drop of reagent a and reagent b are added to each well. after 5 min staining is recorded as described in the color scheme in the manufacturer's instructions. enzyme activity is determined by the color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 3.results and discussion biochemical characteristics of lactobacillus lbrz12 the biochemical profile of the strain lactobacillus lbrz12 is examined using the system for rapid identification api 50 chl (biomerieux, france). table 1. ability of lactobacillus lbrz12 to utilize the 49 carbon sources included in api 50 chl # carbohydrates lactobacilluslbrz12 1 glycerol 2 erythriol 3 d-arabinose 4 l-arabinose + (90-100%) 5 ribose + (90-100%) 6 d-xylose 7 l-xylose 8 adonitol 9 b-metil-d-xyloside 10 galactose + (90-100%) 11 d-glucose + (90-100%) 12 d-fructose + (90-100%) 13 d-mannose + (90-100%) 14 l-sorbose 15 rhamnose 16 dulcitol 17 inositol 18 manitol + (90-100%) 19 sorbitol + (90-100%) 20 a-methyl-d-mannoside + (90-100%) 21 a-methyl-d-glucoside 22 n-acetyl-glucosamine + (90-100%) 23 amigdalin + (90-100%) 24 arbutin + (90-100%) 25 esculin + (90-100%) 26 salicin + (90-100%) 27 cellobiose + (90-100%) 28 maltose + (90-100%) 29 lactose + (90-100%) 30 melibiose + (90-100%) 31 saccharose + (90-100%) 32 trehalose + (90-100%) 33 inulin 34 melezitose 35 d-raffinose + (90-100%) 36 amidon 37 glycogen 38 xylitol 39 b-gentiobiose + (90-100%) 40 d-turanose + (90-100%) 41 d-lyxose +/(40-45%) 42 d-tagarose +/(40-45%) 43 d-fuccose +/(30-35%) 44 l-fuccose 45 d-arabitol + (65-70%) 46 l-arabitol 47 gluconate + (90-100%) 48 2-keto-gluconate 49 5-keto-gluconate according to the strain’s ability to utilize 49 carbon sources (table 1) is determined its belonging to the species lactobacillus plantarum 1 with the corresponding percentage of accuracy (99.9%). in order to confirm the result of the biochemical study the molecular genetic method ardra with the restriction enzymes ecori, hap ii and haeiii is applied. the restriction profile of the strain food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 63 lactobacillus lbrz12 with each of the three restriction enzymes coincides with the profile of the type strain lactobacillus plantarum dsm 20174. the results of the molecular genetic analysis confirmed the identity of lactobacillus lbrz12 as a representative of the species lactobacillus plantarum. in a series of experiments some of the strain’s probiotic properties have been established. antimicrobial activity of lactobacillus lbrz12 the antimicrobial activity of lactobacillus lbrz12 is studied using the method of diffusion in agar. the results of the double retries are summarized in table. 2. table 2. antimicrobial activity of lactobacillus lbrz12 against saprophytic microorganisms. the values are in mm. diameter of the wells 6 mm. saprophytic microorganism concentration of the suspension cultural liquid cultural acellular supernatant neutralized acellular supernatant 300c 370c 300c 370c 300c 370c bacillus subtilis 1,9x107cfu/cm3 bacillus mesentericus 4x106cfu/cm3 9 11,5 9 11 saccharomyces cerevisiae 9,2x106cfu/cm3 aspergillus niger 1,2x107cfu/cm3 12 12 10 12 antimicrobial activity against bacillus mesentericus and aspergillus niger, but not against bacillus subtilis and saccharomyces cerevisiae is established. for bacillus mesentericus the diameters of the sterile zones of the cultural liquid and the cultural acellular supernatant are comparable, which means that the inhibition of the strain lactobacillus plantarum lbrz12 is due to the production of acids, which leads to a decrease in ph. for aspergillus niger the antimicrobial activity of the cultural liquid is higher than that of the cultural acellular supernatant and the neutralized acellular supernatant, which means that the suppression of the test microorganism is a result of the competition for nutrients and/or attachment sites as well as of the lowering of ph due to the acids synthesized by lactobacillus plantarum lbrz12. antibiotic resistance of lactobacillus plantarum lbrz12 the initial concentration of viable cells of lactobacillus plantarum lbrz12 is 1.3x1013cfu/cm3. table 3. antibiotic resistance of lactobacillus lbrz12 m ec h an is m of a ct io n # antibiotic co ncentration lactobacillus lbrz12 in h ib ito rs o f t h e sy nt he si s of th e ce ll w al ls 1 penicillin p 10 e/ disc r 2 azlocill in az 75 µg/disc r 3 piperacillin pi 100 µg/ disc r 4 ampicilli n a 10 µg/disc s 5 oxacillin o 1 µg/disc r 6 amoxicillin ax 25 µg/disc r 7 vancomyci n v 30 µg/disc r 8 cefamando le cm 30 µg/disc sr in hi bi to rs o f th e pr ot ei n sy nt h es is 9 tetracycl ine t 30 µg/disc r 10 doxycycline d 30 µg/disc s 11 gent amycin g 10 µg/disc r 12 streptomycin s 30 mg/disc r 13 cli ndоmyci n cl 2 µg/disc s 14 kana mycin k 30 µg/disc r 15 to bramycin tb 10 µg/disc sr 16 amikacin am 30 µg/disc r 17 rifampin r 5 µg/disc sr 18 lincomycin l 15 µg/disc s 19 chloramphenicol c 30 µg/disc sr 20 erythro mycin e 15 µg/disc s in hi bi to rs o f th e sy nt he si s of d n a an d/ or o f th e bi na ry fis sio n 21 nalidixic ac id nx 30 µg/disc r 22 ciprofl oxacin cp 5 µg/disc r legend: r-resistant, sr intermediate (zone 7-16 mm), s sensitive (zone> 16 mm) in order to study the spectrum of antibiotic resistance of the strain 22 antibiotics with different mechanisms of action belonging to the main groups used in medical practice were selected. the results of the conducted diffusion method according to bauer, kirby et al., 1966 [1] for 24 h are summarized in table. 3. the strain lactobacillus plantarum lbrz12 is resistant to penicillin, cefamandole, nalidixic acid, ampicillin, amoxicillin, tetracycline, oxacillin, gentamycin, food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 64 kanamycin, amikacin, vancomycin, rifampin, streptomycin and chloramphenicol and ciprofloxacin but it is sensitive to clindomycin, doxycycline, lincomycin, tobramycin, azlocillin, piperacillin and erythromycin. in vitro determining the ability of lactobacillus plantarum lbrz12 to survive in the conditions simulating the departments of the gastrointestinal tract in order to be included in the composition of probiotic preparations, the probiotic strains must possess the ability to survive in the conditions of the gastrointestinal tract. the stability of the cells of lactobacillus plantarum lbrz12 in artificial conditions, simulating the conditions of the gastrointestinal tract ph = 2 + pepsin and ph = 8 + pancreatin, is examined. in a parallel experiment the tolerance of the strain to different concentrations of bile salts is tested. all three experiments survival in artificial gastric and pancreatic juice and tolerance to high concentrations of bile salts were held for 24 h. the results of the experiments are presented on fig. 1 and fig.2. figure 1. survival of the cells of lactobacillus plantarum lbrz12 at low ph (ph = 2) + pepsin and weakly alkaline ph (ph=8) + pancreatin. higher sensitivity to low ph (ph=2) + pepsin than to ph=8 + pancreatin is observed (fig. 1). for the residence time of food in the stomach 1.5 to 3 hours in the acidic environment the number of viable cells of lactobacillus plantarum lbrz12 is reduced by about 2 log units by the fourth hour and by about 3 log units by the 24th hour, while at ph=8 and pancreatin the number of viable cells decreases by about 1.5 log units by the 24th hour. another important factor that influences the survival of probiotic strains in the intestinal tract is the concentration of bile salts. it is known that about three hours after ingestion of food the concentration of bile salts in the small intestine reaches about 0.3%. this requires a study of the influence of different concentrations of bile salts on the development of the strain. this study is conducted in a liquid medium (mrs) with different concentrations of bile salts 0%, 0.15%, 0.3%, 0.6% and 1% for 24-hour incubation (fig. 2). figure 2. survival of the cells of lactobacillus plantarum lbrz12 at different concentrations of bile salts. the cells of the strain lactobacillus plantarum lbrz12 are resistant to the presence of bile salts in the medium (fig. 2). the number of active cells decreases more rapidly in the first two hours at all concentrations of bile salts. then the reduction of the number of viable cells continues up to the 24th hour, when the concentration of colony forming units is over 108cfu/cm3 at all concentrations of bile salts (fig. 2). in the gastrointestinal tract of humans, however, such extreme conditions rarely occur. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 65 examination of the enzyme profile the enzyme profile is important for determining the technological characteristics of lactobacilli and their applicability as starter cultures. therefore, characterization of the strain lactobacillus plantarum lbrz12 included determining the presence of a set of 19 enzyme activities using the kit api zym (biomerieux, france). the results of this study are shown in table.4. table 4. enzymatic profile of lactobacillus plantarum lbrz12 enzyme activity* 1 control 2 alkaline phosphatase 0.5 3 esterase 0.5 4 esterase-lipase 1 5 lipase 1.5 6 leucine-aminopeptidase 5 7 valine-aminopeptidase 5 8 cysteine-aminopeptidase 4.5 9 trypsin 10 chymotrypsin 11 acid phosphatase 3.5 12 phosphohydrolase 2.5 13 α-galactosidase 14 β-galactosidase 5 15 β-glucoronidase 16 α-glucosidase 4 17 β-glucosidase 2.5 18 α-glucoseaminidase 5 19 α-manosidase 20 α-fucosidase * enzyme activity is determined by the color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity) the strain lactobacillus plantarum lbrz12 has the following enzyme activities: alkaline phosphatase, esterase, esterase-lipase, lipase, leucineaminopeptidase, valine-aminopeptidase, cysteine-aminopeptidase, acid phosphatase, phosphohydrolase, βgalactosidase, α-glucosidase, βglucosidase, α-glucoseaminidase (table 4). the results of the conducted experiments determine the strain lactobacillus plantarum lbrz12 as potentially probiotic. 4.conclusion the strain lactobacillus lbrz12 of plant origin has been identified as lactobacillus plantarum through the application of physiological, biochemical and molecular genetic methods. the results of the strain for the possession of some probiotic properties antimicrobial activity, antibiotic resistance and survival in the conditions of artificial gastric and intestinal juice – show the potential of the strain for inclusion in the composition of probiotic preparations. 5. references [1] bauer a.w., kirby w.m., sherris j.c., turck m, antibiotic susceptibility testing by a standardized single disc method. american journal of clinical pathology 36, 49-52, (1966). [2] bhatia s. s., kochar n., abraham p., lactobacillus acidophilus inhibits growth of campylobacter pylori in vitro, s. clin. microbial., 27, 23282330, (1989). [3] charteris w.p., p.m. kelly, l. morelli, j.k. collins, development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and bifidobacterium species in the upper human gastrointestinal tract, journal of applied microbiology 84 (5), pp. 759–768, (1998). [4] davidson r.h., s. e. duncan, probiotic culture survival and implications in fermented frozen yoghurt characteristics. j. dairy sci., 83, 666-673, (2000). [5] denkova z., “production and application of probiotics” doctor of science thesis, (2005). [6] fang he et al., adhesion of bifidobacterium spp. to human intestinal mucus. microbiol. immunol., 45(3), 259 – 262, (2001). [7] kmeitel w., ulberth f., erhard f., doris j., aroma profiles and yoghurt related products. screening of commercially available starter cultures, milchwissenscaft, 47, 6, 362-365, (1992). [8] marteau p., pochart p., flourie b. et al, effect of chronic ingestion of a fermented dairy product containing l. acidophilus and bifidobacterium bifidum on metabolic activities of the colonic flora in humans, am. s. clin. nutr., 52, 685688, (1990). [9] salminen s., von wright.a, current probiotics – safety assured, scandinavian university press, issn 0891-060x, (1998). [10] salminen, s., isolauri, e., salminen, e., probiotics and stabilization of the gut mucosal barrier. pacific j. clin. nutr. 5, 53-56, (1996a). 37 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 37 44 bioch e mica l a nd m ol ec ular-ge ne t ic char ac t eriz ati on of a s tra in iso la t ed from a ther mal he ali ng s pr ing i n staro zagorski m ine ra ln i bani, sta ra zag ora re gion, bu lga ria *nedyalka vylcheva-zhekova1, zapryana denkova2, radosveta nikolova2 1 pht “aleksander paskalev”, haskovo, 2university of food technology, department of microbiology zdenkova@abv.bg *corresponding author received 5th february 2015, accepted 30th march 2015 abstract: mineral waters of springs near the village of starozagorski mineralni bani, stara zagora region, are well known for their healing effect in diseases of bones and joints, peripheral nervous system, gynecological, kidney and urological, gastro-intestinal and hepatic and biliary diseases. the physico-chemical and microbiological parameters of healing spring water in the village of starozagorski mineralni bani, stara zagora region were determined. according to its physicochemical and microbiological parameter, spring water meets the standard requirements for quality of drinking water. a strain was isolated from the examined healing spring as a pure culture and its colonial and morphological characteristics were examined. the isolated strain smb was rod-shaped, gram positive, motile, sporeforming. it formed a precipitate on the surface of the liquid culture medium, did not turbidify the liquid culture medium and formed a precipitate on the bottom of the tube. strain smb was a facultative aerobe. it was identified by biochemical (api 50 chb) and molecular genetic methods (sequencing of the gene for the 16s rrna) as a representative of the species bacillus thuringiensis. after performing further examinations of its antimicrobial activity against phytopathogenic molds strain smb would be incorporated in the composition of biological preparations against plant diseases. keywords: mineral water, physico-chemical, microbiological, identification, sequencing, 16s rrna 1. introduction mineral water from springs near starozagorski mineralni bani, stara zagora region, come to the surface with a temperature of about 40°c from about 1600 m in depth. they are known to have healing effect in diseases of bones and joints, peripheral nervous system, gynecological, kidney and urological, gastro-intestinal and hepatic and biliary diseases [1 8]. mineral water is slightly mineralized, hyperthermal, with neutral ph (ph=6.9) due to the presence of 60 mg of carbon dioxide in it. it contains hydrocarbons, sulfates, calcium, magnesium, silicon, fluorine, and other trace elements; the mineral content is 0.498 g/l to 2 g/l; the content of metasilicic acid in colloidal state is 34 mg/l, which makes it effective in a number of diseases. potable water used in enterprises of the food and microbiological industry must meet the requirements for potable water. http://www.fia.usv.ro/fiajournal mailto:zdenkova@abv.bg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 38 thus the total bacterial abundance (tba), the number of coliform bacteria and pathogenic bacteria should be determined and monitored periodically. pathogenic bacteria are controlled by hygienic epidemiology laboratories. in bulgaria there are a number of mineral springs with unexamined microflora. studies have shown that microorganisms with valuable properties can be isolated from healing and spring waters. the thermal healing spring with water temperature of 40°c in starozagorski mineralni bani, stara zagora region has not been examined so far. the purpose of the present research was the physico-chemical and microbiological analysis of the spring water from the thermal healing spring with water temperature of 40°c in starozagorski mineralni bani, stara zagora region and the physiological, biochemical and molecular-genetic identification of a strain isolated from the spring. 2. materials and methods 2.1. physico-chemical methods the colour was determined according to the rublyovska scale method bs 8451: 1977; the odor was determined according to the the method for determination of the odor at 20°c method bs 8451: 1977, technical means – mercury thermometer, conditions № 21; the turbidity was determined according to en iso 7027, technical means turbidity meter type turb 355 ir id № 200807088; the ph was determined according to bs 3424: 1981, technical means ph meter type ub10 id №ub10128148; the oxidation was determined according to bs 3413: 1981; method for determination of chlorides bs 3414: 1980; the nitrates were determined according to vlm no3 №2, technical means – photometer “nova 60 a” id № 08450505; the nitrites were determined according to vlm no2 №3, technical means – photometer “nova 60 a” id № 08450505; the ammonium ions were determined according to vlm nh4 №1, technical means – photometer “nova 60 a” id № 08450505; the total hardness was determined according to iso 6058; the sulphates were determined according to vlm so4 №4, technical means – photometer “nova 60 a” id № 08450505; the calcium was determined according to iso 6058; the magnesium was determined according to bs 7211: 1982; the phosphates were determined according to vlm po4 №5, technical means – photometer “nova 60 a” id № 08450505; the manganese was determined according to vlm mn №7, technical means photometer “nova 60 a” id № 08450505; the iron was determined according to vlm fe № 6, technical means photometer “nova 60 a” id № 08450505; the fluoride was determined according to vlm f № 8, technical means photometer “nova 60 a” id № 08450505; the electrical conductivity was determined according to bs en 27888, technical means conductivity inolab cond № 720 id 11081137. 2.2. microbiological methods the applied methods for microbiological indicators were in accordance with ordinance № 9/2001 darjaven vestnik, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 39 issue 30 and decree № 178 / 23.07.2004 on the quality of water intended for drinking purposes. the presence of escherichia coli and coliform bacteria were determined according to bs en iso 9308-1: 2004; the presence of enterococci were determined according to bs en iso 78992; the presence of spores of sulfite reducing anaerobes was determined according to bs en 26461-2: 2004; the total number of aerobic and facultative anaerobic bacteria was determined according to bs en iso 6222: 2002; the presence of pseudomonas aeruginosa was determined according to bs en iso 16266: 2008; 2.3. determination of the sulphytereducing anaerobic bacteria (clostridium perfringens) in water the samples were heated in a water bath at 80°c for 15 min and pour plated in tubes. the inoculated tubes were incubated at 37°c for 24 h until the appearance of black colonies and cavities in the medium. the amount of sulphytreducing sporeforming anaerobes (clostridium perfringens) was determined by their titer (the smallest volume of water in which they were established) using standard tables. 2.4. identification methods determination of the biochemical profile the system api 50 chb (biomerieux sa, france) was used for the identification of the species of the genus bacillus based on their ability to utilize 49 carbon sources. fresh 24-hour culture of the studied strain was suspended in api 50 chb medium, an integral part of the used kit. the api strips were placed in the incubation boxes, the microtubules were inoculated with the prepared cell suspension and sealed with sterile liquid paraffin. the results were reported on the 24th and the 48th hour of incubation at 37±1°c. reporting was done, based on the colour change of each microtubule, compared to the colour of the control microtubule (microtubule 0). positive results were recorded in the cases of color change from red to orange or bright yellow. the obtained results were processed with apiweb® identification software. molecular-genetic methods isolation of total dna [1] 16s rdna amplification all pcr reactions were performed using the pcr kit – pcr vwr, in a volume of 25 µl in a progene cycler (techne, uk) according to the manufacturer’s instructions. 50 ng total dna of the studied strain and 10 pmol primers were mixed in each reaction. dna of the studied strain was amplified using universal primers for the 16s rdna gene 27f (5’agagtttgatcmtggctcag3’) [2] and 1492r (5’accttgttacgactt3’) [2]. the amplification program included: denaturation 95°c for 3 minutes, 40 cycles 93°c for 30 s, 55°c for 60 s, 72°c for 2 min, final elongation 72°c for 7 min. the resulting pcr product from the 16s rdna amplification of the tested strain was visualized on a 2% agarose gel, stained with ethidium bromide solution (0.5 µg/ml), using an uvp documentation system (uk) [3]. purification of the product of the pcrreaction – 16s rdna – from taeagarose gel the purification of 16s rdna was conducted using dna-purification kit (gfx microspintm) according to the manufacturer’s instructions. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 40 dna-sequencing sequencing of the gene encoding the 16s rrna was conducted by „macrogen europe laboratory”, the netherlands using the sanger method for dna-sequencing. the obtained forward and reverse partial sequences of the two ends of the 16s rrna gene were assembled using the software clc sequence viewer. the total gene sequence of the 16s rrna gene was compared with the sequences available in the online genbank database through the online software blastn and the species identification was determined by the rate of correspondence between the squence of the studied strain and the reference strain from the online database [4]. 3. results and discussion determination of the physicochemical characteristics of thermal healing spring in the village of starozagorski mineralni bani, stara zagora region with water temperature of 40°c the values of the main physico-chemical parameters of the healing spring in the village of starozagorski mineralni bani, stara zagora region were compared with the requirements set by the standards (table 1). table 1 physico-chemical analysis of the healing spring water in the village of starozagorski mineralni bani, stara zagora region controlled parameter unit maximum value result colour according to the rublyovska scale colour degrees acceptable for the consumer acceptable odor at 20 °с total result acceptable for the consumer acceptable turbidity ntu acceptable for the consumer acceptable рн ph units ≥ 6,5 and ≤ 9,5 6,9 oxydation mgo2/dm 3 5,0 0,6 chlorides mg/dm3 250 26 nitrates mg/dm3 50 7 nitrites mg /dm3 0,50 0,00 ammonium ions mg/ dm3 0,50 0,00 total hardness mgekv/dm3 12 7 sulphates mg/dm3 250 14 calcium mg/dm3 150 72 magnesium mg /dm3 80 30 phosphates mg/dm3 0,5 0,0 manganese mg/dm3 50 0 iron µg/dm3 200 16 fluorides mg/dm3 1,5 0.0 conductivity µs/dm3 2000 750 experimental data showed that the thermal healing spring waters met the required values for all controlled parameters of ordinance № 9/2001 dyrjaven vestnik, issue 30 and decree № 178/23.07.2004 for the quality of potable water. determination of the microbiological safety of thermal healing spring in the village of starozagorski mineralni bani, stara zagora region with water temperature of 40°c the experimental data from the microbiological examination of the thermal healing spring water indicated that the water met the standard criteria for microbiological quality of mineral and potable water (table 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 41 microflora of the thermal healing spring water from the spring in the village of starozagorski mineralni bani, stara zagora region with water temperature of 40°c one strain was isolated from the spring water. the colonial and morphological characteristics of the isolated bacterial strain were determined (table 3). the isolated strain was rod-shaped, gram positive, motile, sporeforming. the strain formed a precipitate on the surface of the liquid culture medium, did not turbidify the liquid culture medium and formed a precipitate on the bottom of the tube. the strain was a facultative aerobe (table 4). the ability of the isolated strain to absorb the 49 carbon sources included in the kit system for rapid identification of bacilli api 50 chb/e was examined. after processing of the test results with apiweb® the species identification of strains with the corresponding percentage of reliability was obtained (table 5). the strain bacillus smb belongs to the species bacillus thuringiensis with percentage of reliability of 99%. table 2 microbiological analysis of healing spring water from the spring in the village of starozagorski mineralni bani, stara zagora region with water temperature of 40°c controlled parameter norm, cfu/cm3 result, cfu/cm3 coliforms 0/100 0/100 escherichia coli 0/100 0/100 enterococci 0/100 0/100 sulphyte reducing anaerobic bacteria (clostridium perfringens) 0/100 0/100 tba at 22 °с 100 0 tba at 37 °с 20 0 pseudomonas aeruginosa 0/250 0/250 table 3 colonial characteristics of the isolated strain strain description of the colonies visualization description of the cells visualization smb round, soft, smooth and shiny, whitish, drop-like colonies with wave-like edges, size – 2 – 3 mm gram-positive short, thick rods with round edges, motile, sporeforming, arranged singly, in pairs and in long chains table 4 growth of the isolate from the thermal healing spring water in the village of starozagorski mineralni bani, stara zagora region in liquid medium for 24-48 h, at temperatures 3°c 45°c (p=precipitate; t=turbidification; s=sludge) strain 3 °с 25 °с 30°с 37 °с 45 °с attitude to oxygen p t s p t s p t s p t s p t s smb ─ ─ ─ + ─ + + ─ + + ─ + ─ ─ ─ facultative aerobe + occurance of precipitate, turbidity, sludge; ─ absence of precipitate, turbidity, sludge. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 42 table 5 api 50 chb/e of the strain bacillus smb # carbohydrates smb 1 glycerol 2 erythriol 3 d-arabinose 4 l-arabinose 5 ribose + (90-100%) 6 d-xylose 7 l-xylose 8 adonitol 9 -metil-d-xyloside 10 galactose 11 d-glucose + (50%) 12 d-fructose + (50%) 13 d-mannose 14 l-sorbose 15 rhamnose 16 dulcitol 17 inositol 18 manitol 19 sorbitol 20 -methyl-d-mannoside 21 -methyl-d-glucoside 22 n-acetyl-glucosamine + (90-100%) 23 amigdalin 24 arbutin + (90-100%) 25 esculin + (90-100%) 26 salicin + (90-100%) 27 cellobiose 28 maltose + (90-100%) 29 lactose 30 melibiose 31 saccharose + (90-100%) 32 trehalose + (90-100%) 33 inulin 34 melezitose 35 d-raffinose 36 amidon + (50%) 37 glycogen + (90-100%) 38 xylitol 39 -gentiobiose 40 d-turanose 41 d-lyxose 42 d-tagarose 43 d-fuccose 44 l-fuccose 45 d-arabitol 46 l-arabitol 47 gluconate 48 2-keto-gluconate 49 5-keto-gluconate identification bacillus thuringiensis % of reliability 99 bacillus thuringiensis b62, 16s ribosomal rna gene, partial sequence; sequence id: gb|jx010983.1| length: 1455 number of matches: 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 43 score expect identities gaps strand 2773 bits(1442) 0.0 1444/1445(99%) 0/1445(0%) plus/plus * query 10 cggcgtgcctatacatgcagtcgagcgaatggattaagagcttgctcttatgaagttagc 69 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 7 cggcgtgcctatacatgcagtcgagcgaatggattaagagcttgctcttatgaagttagc 66 query 70 ggcggacgggtgagtaacacgtgggtaacctgcccataagactgggataactccgggaaa 129 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 67 ggcggacgggtgagtaacacgtgggtaacctgcccataagactgggataactccgggaaa 126 query 130 ccggggctaataccggataatattttgaactgcatggttcgaaattgaaaggcggcttcg 189 |||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||| sbjct 127 ccggggctaataccggataacattttgaactgcatggttcgaaattgaaaggcggcttcg 186 query 190 gctgtcacttatggatggacccgcgtcgcattagctagttggtgaggtaacggctcacca 249 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 187 gctgtcacttatggatggacccgcgtcgcattagctagttggtgaggtaacggctcacca 246 query 250 aggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggc 309 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 247 aggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggc 306 query 310 ccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacgga 369 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 307 ccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacgga 366 query 370 gcaacgccgcgtgagtgatgaaggctttcgggtcgtaaaactctgttgttagggaagaac 429 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 367 gcaacgccgcgtgagtgatgaaggctttcgggtcgtaaaactctgttgttagggaagaac 426 query 430 aagtgctagttgaataagctggcaccttgacggtacctaaccagaaagccacggctaact 489 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 427 aagtgctagttgaataagctggcaccttgacggtacctaaccagaaagccacggctaact 486 query 490 acgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcgta 549 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 487 acgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcgta 546 query 550 aagcgcgcgcaggtggtttcttaagtctgatgtgaaagcccacggctcaaccgtggaggg 609 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 547 aagcgcgcgcaggtggtttcttaagtctgatgtgaaagcccacggctcaaccgtggaggg 606 query 610 tcattggaaactgggagacttgagtgcagaagaggaaagtggaattccatgtgtagcggt 669 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 607 tcattggaaactgggagacttgagtgcagaagaggaaagtggaattccatgtgtagcggt 666 query 670 gaaatgcgtagagatatggaggaacaccagtggcgaaggcgactttctggtctgtaactg 729 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 667 gaaatgcgtagagatatggaggaacaccagtggcgaaggcgactttctggtctgtaactg 726 query 730 acactgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccg 789 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 727 acactgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccg 786 query 790 taaacgatgagtgctaagtgttagagggtttccgccctttagtgctgaagttaacgcatt 849 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 787 taaacgatgagtgctaagtgttagagggtttccgccctttagtgctgaagttaacgcatt 846 query 850 aagcactccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcc 909 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 847 aagcactccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcc 906 query 910 cgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtct 969 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 907 cgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtct 966 query 970 tgacatcctctgaaaaccctagagatagggcttctccttcgggagcagagtgacaggtgg 1029 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 967 tgacatcctctgaaaaccctagagatagggcttctccttcgggagcagagtgacaggtgg 1026 query 1030 tgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaa 1089 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1027 tgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaa 1086 query 1090 cccttgatcttagttgccatcattaagttgggcactctaaggtgactgccggtgacaaac 1149 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1087 cccttgatcttagttgccatcattaagttgggcactctaaggtgactgccggtgacaaac 1146 query 1150 cggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgt 1209 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1147 cggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgt 1206 query 1210 gctacaatggacggtacaaagagctgcaagaccgcgaggtggagctaatctcataaaacc 1269 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1207 gctacaatggacggtacaaagagctgcaagaccgcgaggtggagctaatctcataaaacc 1266 query 1270 gttctcagttcggattgtaggctgcaactcgcctacatgaagctggaatcgctagtaatc 1329 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1267 gttctcagttcggattgtaggctgcaactcgcctacatgaagctggaatcgctagtaatc 1326 query 1330 gcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacacc 1389 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1327 gcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacacc 1386 query 1390 acgagagtttgtaacacccgaagtcggtggggtaaccttttggagccagccgcctaaggt 1449 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1387 acgagagtttgtaacacccgaagtcggtggggtaaccttttggagccagccgcctaaggt 1446 query 1450 ggacc 1454 ||||| sbjct 1447 ggacc 1451 fig. 1. comparison of the nucleotide sequence of the 16s rrna gene of the strain bacillus smb and the partial sequence of the 16s rrna gene of bacillus thuringiensis b62. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 n e d yal k a vy l c he vaz hek o va , z a pr ya na d e n ko va, r ad o s ve t a n i k ol o va b i oc h e m i c al a nd m o l e c u l ar g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n s t a ro z a go r s k i m i n e r al ni ba n i , s t a r a z a go r a r e gi o n , b ul g a r i a , foo d a nd e nv ir o nm e nt sa f e t y , v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 3 7 4 4 44 the 16s rrna gene was sequenced and the resulting sequence was processed using the software blastn and it was confirmed that the studied strain belongs to the species bacillus thuringiensis (fig. 1). 4. conclusion the physicochemical and microbiological parameters of thermal healing spring water in the village of starozagorski mineralni bani, stara zagora region were examined. the spring water met the requirements laid down in the ordinance № 9/2001 dyrjaven vestnik, issue 30 and decree № 178/23.07.2004 for the quality of potable water when it comes to its physicochemical parameters. from a microbiological point of view the spring water was safe to use. the strain smb isolated from the spring was identified using physiochemical, biochemical (api 50 chb) and molecular-genetic (sequencing of the gene for the 16s rrna) methods as a representative of the species bacillus thuringiensis. after further examinations of its antimicrobial activity against phytopathogenic molds strain smb would be incorporated in the composition of biological preparations against plant diseases. 5. references [1]. bender t, bálint p, bálint g, a brief history of spa therapy. ann. rheum. dis. 61,949 (2002). [2]. bender t, karagülle z, bálint g, gutenbrunner c, bálint p, sukenik s, hydrotherapy, balneotherapy and spa treatment in pain management. rheumatol. int. 4:220–224 (2005). [3]. geytenbeek j, evidence for effective hydrotherapy. physiotherapy 9:514–529 (2002). [4]. riyaz n, arakkal f,spa therapy in dermatology. indian j. dermatol. venereol. leprol. 77:128–134 (2011). [5]. seite s, thermal waters as cosmeceuticals: la roche-posay thermal spring water example. clin. cosmet. investig. dermatol. 6:23–28, (2013). [6]. sukenik s, flusser d, abu-shakra m, the role of spa therapy in various rheumatic diseases. rheum. dis. clin. n. am. 25:883–897 (1999). [7]. tishler m, rosenberg o, levy o, elias i, amit-vazina m, the effect of balneotherapy on osteoarthritis. is an intermittent regimen effective? eur. j. intern. med. 15:93–96 (2004). [8]. tonko c, király a, mizsey p, patzay g, csefalvay e, limitation of hardness from thermal water by means of nanofiltration. water sci. technol. 67:2025–2032 (2013). [9]. delley m, mollet b, hottinger h, dna probe for lactobacillus delbrueckii. appl. environ. microbiol., 56,1967–1970 (1990). [10]. lane dj, 16s/23s rrna sequencing. in: nucleic acid techniques in bacterial systematics. stackebrandt, e., and goodfellow, m., eds., john wiley and sons, new york, ny, pp. 115-175 (1991). [11]. denkova r, georgieva l, denkova z, urshev z, yanakieva v, biochemical and technological properties of lactobacillus plantarum x2 from naturally fermented sourdough. journal of food and packaging science technique and technologies 1(1):59 64 (2012a). [12]. denkova r, yanakieva v, denkova z, urshev z, goranov b, sotirova e, identification and examination of some probiotic properties of lactobacillus plantarum f3. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava 9 (4):22 – 29 (2012b). microsoft word 14 poroch barla.doc 89 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 89 94 incidence of food contamination with staphylococcus aureus in suceava county, romania *florin bârlă1, maria poroch – seriţan1, victoria domnica săvuţ (straton)2 1“ştefan cel mare” university of suceava, faculty of food engineering, romania, 13th university street, 720229, suceava – romania, e-mail address: mariap@fia.usv.ro, florin.barla@fia.usv.ro 2 laborator de analize medicale -synevo romania suceava, e-mail: victoriasavut@yahoo.com *corresponding author received 10 january 2013, accepted 11 february 2013 abstract: staphilococcus aureus is one of the most wide spread bacterial pathogens initiating foodborne disease worldwide. an investigation was conducted, between 2002 and 2011, in order to evaluate s. aureus contamination in various types of animal origin food, commonly consumed in suceava county. a total of 781 samples were examined and 15.6% were found contaminated with s. aureus. prevalence rates varied, recording the highest rate on 2006 when over 50% among the investigated food samples and over 20% among the food handlers (nasal cavity and hands) samples were contaminated. of a particular case from 76 samples analyzed on 2002 and 2006 at suceavabucsoaia students’ camp 38.1% were confirmed positive in both food samples and food handlers samples. none of the samples analyzed on 2011 were contaminated. our findings indicate a substantial improvement of gmp in food processing units and catering divisions as well as the benefit of food safety systems implemented during this period in suceava county. keywords: staphylococcus aureus, food contamination, food-borne illness 1. introduction contaminated food consumption frequently results in the illness, which is called food-borne illness of food poisoning. s. aureus contamination is recognized as a major cause of food-borne illness worldwide, being indicated as one of the most prevalent among the bacterial pathogen agents in both communityacquired as well as nosocomial infections [1]; [2]. it has been suggested that over 30%-50% of the population represent the numbers of carriers [3]. kluytmans and werheim [4] reported that in fact only 20% of people almost never carry s. aureus. foods storage at improper temperature, inadequate handling as well as the capacity of microorganism to develop in a wide range of ph conditions and salt concentrations indicate that a wide range of food products are the main epidemiological features to provide appropriate conditions for an epidemic of s. aureus food poisoning. meat and milkbased products were found likely to be the most frequently involved matrices of food poisoning during the investigations done after an outbreak [5]; [6]. work surfaces and equipment used to prepare foods are an important source of indirect contamination. a study carried on 2003 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton), incidence of food contamination wit h staphylococcus aureus in suceava county, romania, food and environment safety, volume xii, issue 1 – 2013, pag. 89 94 90 [3], reveled that 25% of swabs taken from work surfaces were contaminated with s. aureus and 71.7% of ready-to-eat products handled after heat treatment were contaminated. some s. aureus strains are capable to produce one or more enterotoxins this consist in pathogenity of this microorganism. it is estimated that the enterotoxigenic strains account for about 25% of all isolated strains [5]. staphylococci are catalase positive, oxidase-negative, facultative anaerobes [7]. enterotoxin production is adversely affected by anaerobic condition far more than growth [8]. staphylococcal food poisoning is caused by the; ingestion of foods containing enterotoxins produced by some species of staphylococci [9]. the disease is described by sudden start of symptoms, including nausea, vomiting, abdominal cramps, and diarrhea within few hours after ingestion of toxin-contaminated foods. staphylococcal food poisoning is generally considered a mild, self-limited illness with low mortality rate lasts only a few hours with no consequence. however, the hospitalization rate has been reported to be as high as 10% in united states of america [10]. the diagnosis of this foodborne illness is based primarily on recovering enterotoxigenic staphylococci and enterotoxins from leftover food. staphylococci are common in nature can be found in the air, in dust, in water, and on humans and animals. the main human reservoirs of these organisms are the skin, nasal cavities and throat [11]. about 40 to 44% of healthy humans carry staphylococci in the nose [12]. strains present in the nose often contaminate the back of hands, fingers and face; nasal carriers can easily become skin carriers. although it is difficult to determine the origin of the strains involved in staphylococcal food poisoning outbreaks, food handlers are usually regarded as one of the primary source of these organisms [13]; [14]. it has been reported that, one of the important pathogens often transmitted via food contaminated by infected food handlers is s. aureus [15]. health risks are also linked with subsequent contaminations by the workers during handling. staphylococci are included in the bacterial group that contaminates food products in this way [16]. for many years, s. aureus was the only staphylococcal species known to produce enterotoxins [9]. an important characteristic that differentiates s. aureus from most staphylococcal species is its ability to produce coagulase, an enzyme that clots blood plasma. staphylococcal food poisoning is widespread and quite frequent. the incidence of staphyloenterotoxicosis cases is probably underestimated considerably, and there may be a lot of reasons for that: not calling medical services by many ill people due to the short duration of the disease or mild symptoms, improper both sample collection and laboratory examination [17]. the aim of the present study was to investigate microbiological quality and to detect the presence of the pathogenic of s. aureus in various kinds of animal origin food, as well as in food handlers, occurred in suceava country during 2002 2011. although, food safety is one of the most important issues for maintaining human health therefore, to prevent s. aureus contamination becomes an important apprehension. 2. materials and methods according to the method of lancette and tatini [18], 25 g of each implicated food were homogenized with in 225 ml of 0.1% buffered peptone water. a 0.1 ml aliquot of the suspension was then spread on the surface of a baird-parker agar plate. additional plates were prepared with successive decimal dilutions. the plates were incubated for 48 hours at 37ºc, and the suspected colonies were counted. ten food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton), incidence of food contamination wit h staphylococcus aureus in suceava county, romania, food and environment safety, volume xii, issue 1 – 2013, pag. 89 94 91 typical colonies (jet black to dark grey, smooth, convex, well-defined contours, off-white edge, presenting an opaque zone and/or a clear halo beyond the opaque zone) and 10 colonies classified as atypical (gray and mucoid showing one halo) were transferred to tubes containing nutrient agar (stock culture) for further testing. for detecting small numbers of s. aureus in raw food ingredients and non-processed foods expected to contain large numbers of competing organisms, incubation is in trypticase soy broth containing 10 % nacl and 1 % sodium pyruvate before transferring to baird-parker agar plates. for detecting relatively large numbers of staphylococci, the food extract is plated directly on baird-parker agar. typical colonies of s. aureus on baird-parker agar are circular, smooth, convex, moist, cca. 1.5 mm in diameter, gray-black to jetblack, off-white edges and may show an opac zone with a clear halo extending beyond it. isolation and identification of s. aureus: for the bacterial colony isolation serial dilutions of the samples were made and diluted sample (100µl) was transferred on baird-parker agar supplemented with egg yolk tellurite enrichment suspension (oxoid-england) and incubated at 37°c for 48 hours as previously described elsewhere [19]. the physical identification characteristics of the bacterial colonies such as black, smooth, convex to uniform outline with one or two halos were recorded [20]. s. aureus was confirmed by colonial morphology, gram staining, catalase activity, and coagulation of citrated rabbit plasma (sigma – aldrich group) isolation of staphylococci from food handlers each swab collected from the nasal cavity, throat and from under the fingernails was introduced in tubes containing 5 ml of triptic soy broth (tsb) with 10% of nacl. the tubes were incubated for 24 hours at 37ºc. the cultures were streaked on baird-parker plates and incubated for 48 hours at 37ºc. ten typical and 10 atypical colonies were selected for further testing as described above. 3. results and discussion a total of 781 samples of animal origin, from suceava county were collected and examined between 2002 and 2011, of which 15.6% were found contaminated with s. aureus. the highest peak was recorded on 2006 when over 50% among the investigated food samples and over 20% among the food handlers sample were contaminated with s. aureus as can be seen in the figure 1. also, a slight increase of the incidence rate can be observed during monitoring; however on 2011 there was no contaminated samples recorded. in 2004, the incidence of food handlers samples riches the highest incidence over 37% among the analyzed samples were positive. the staphylococci sp. is omnipresent microorganisms that cannot be eliminated completely from our environment. as it was mentioned by di ginatnatale [3], at least 30-50% of individuals are carriers of these types of microorganisms in their nasal cavity or throats, or on their hands. the possibility that the food to be contaminated with s. aureus, is strongly correlated with the expose to the human handling therefore, the risk of contamination with staphylococci that can produce enterotoxins can rich at least 30% 50%. the contamination incidence of food sample and food handler samples by year are summarized in the table 1. the highest incidence was recorded during 2004 2006, and the incidence decreased considerably from 2007. recently, oliveira [21], show that in korea on 2007 the incidence of raw milk samples contaminated with s. aureus was 0.34% among the investigated samples, in norway in 2005, 11 samples were found positive, in 2010 in united states, 29% of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton), incidence of food contamination wit h staphylococcus aureus in suceava county, romania, food and environment safety, volume xii, issue 1 – 2013, pag. 89 94 92 samples were positive for s. aureus in cow’s milk. 0,0 20,0 40,0 60,0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 in ci de nc e of s . a ur eu s co nt am in at io n (% ) food s amples food handlers figure1. the incidence of s. aureus contamination (foods and food handlers) occurred in suceava county between 2002 2011 table 1. summary of the contamination incidence with s. aureus in food samples and food handlers occurred in suceava county between 2002 – 2011 year samples no. of examined samples no. of sample positive for s. aureus % of isolation food samples 66 6 9.09 2002 food handlers 82 4 4.87 food samples 51 7 13.72 2003 food handlers 85 2 2.35 food samples 27 8 29.62 2004 food handlers 27 10 37.04 food samples 56 0 0.00 2005 food handlers 59 10 16.95 food samples 54 28 51.85 2006 food handlers 105 25 23.81 food samples 7 3 42.86 2007 food handlers 12 0 0.0 food samples 14 4 28.57 2008 food handlers 12 3 25.0 food samples 14 3 21.43 2009 food handlers 21 4 19.05 food samples 15 5 33.33 2010 food handlers 9 0 0.0 food samples 35 0 0.0 2011 food handlers 30 0 0.0 the same study show that in turkey in 2007, 18.18% had, in morocco in 2004, registred 40% and in india in 2008, with a 61.7% infestation respectively from raw milk samples were found contaminated with s. aureus. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton), incidence of food contamination wit h staphylococcus aureus in suceava county, romania, food and environment safety, volume xii, issue 1 – 2013, pag. 89 94 93 table 2. summary of the contamination incidence with s. aureus in food samples and food handlers occurred in suceava bucsoaia student's camp on 2002 and 2006 year samples no. of examined samples no. of sample positive for s. aureus % of isolation food samples 35 14 40.0 2002 food handlers 16 0 0.0 food samples 20 10 50.0 2006 food handlers 5 5 100.0 in romania according to ivana 2010 [22], the situation is worrying and the s. aureus contamination incidence rises to over 50%. on the other hand, of a particular case from 76 samples analyzed on 2002 and 2006 at suceava-bucsoaia students’ camp 38.1% were confirmed positive in both food samples and food handlers samples, the results are summarized in the table 2. bucsoaia students’ camp is organized usually during summer. the warmer summertime temperatures may complicate the situation resulting in increasing the contamination incidence. another factor as a particular summer problem is that if the food products are not stored under adequate refrigeration until consumption may also increase the risk. 4. conclusion in conclusion, this study provides a summary of the evaluation regarding prevalence of s. aureus contamination occurred in various food of animal origin, in suceava county during the past decade. a decrease of contamination incidence with s. aureus can be observed during 2002 2011. these results show a low incidence of food contamination in suceava county as compared with the average of contamination incidence in romania, in this case the level rises 50% according to literature. these results, in our opinion, suggest that the improvement in applied haccp and gmp in food processing units in suceava county were implemented successfully. 5. references [1]. chambers h.f., the changing epidemiology of staphylococcus aureus, emerg. infect. dis., 7, 178-182, (2001). [2]. karlowsky j.a., jones m.e., drraghi d.c., thornsberry c., sham d.f., volturo g.a., prevalence and antimicrobial susceptibilities of bacteria isolated from blood cultures of hospitalized patients in the united states in 2002, ann.clin. microbiol. antimicrob., 3, 7-14, (2004). [3]. di giannatale e., prencipe v., tonelli a., characterization of staphylococcus aureus strains isolated from food for human consuption, veterinaria italiana, 47, 165-173, (2011). [4]. kluytmans a.j.w., wertheim h.f.l., nasal carriage of staphylococcus aureus and prevention of nosocomial infection, infection, 33, 3–7, (2005) [5]. le loir y., baron f., gautier m., staphylococcus aureus and food poisoning, genet. mol.. res., 2, 63-76, (2003) [6]. zschock m., botzelr d., blocher s., sommerhauser j., hamann h.p., detection of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst) in mammary isolates of staphilococcus aureus by polymerase chain reaction, int. dairy j., 10, 569-574, (2000). [7]. frankham h., howard e., margery o., causes of food spoilage. fitzhenery and whiteside press ltd., 294, (1994). [8]. lidsay j., staphylococcus: molecular genetics. caister academic press, 395, (2008) [9]. bergdoll m.s., staphylococcus aureus. in, doyle m.p., (ed): foodborne bacterial pathogens, marcel dekker, inc., new york, 463523, (1989). [10]. holmberg s.d., blake p.a., staphylococcal food poisoning in the united states: new facts old misconceptions. jama, 251, 487489, (1984) [11]. jay j.m., staphylococcal gastroenteritis, in, jay j.m. (ed): modern food microbiology. 3rd ed., van nostrand reinhold company inc., new york, 437-458, (1986) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 florin bârlă, maria poroch – seriţan, victoria domnica săvuţ (straton), incidence of food contamination wit h staphylococcus aureus in suceava county, romania, food and environment safety, volume xii, issue 1 – 2013, pag. 89 94 94 [12]. williams r.e.o., healthy carriage of staphylococcus aureus its prevalence and importance, bacteriol rev., 27, 56-71, (1963) [13]. bryan f.l., factors that contribute to outbreaks of food borne disease, j. food prot., 41, 816-827, (1978) [14]. genigeorgis c.a., present state of knowledge on staphylococcal intoxication, int. j. food microbiol., 9, 327-360, (1989). [15]. ollinger snyder p., matthews m.e., food safety: review and implications for dietitians and dietetic technicians, j. am. dietetic assos., 96, 163-171, (1996). [16]. fadel h.m., ismail j., prevalence and significance of staphylococcus aureus and enterobacteriaceae species in selected dairy products and handlers. int. j. dairy sci., 4, 100108, (2009). [17]. paciorek m.l., kochman m., piekarska k., grochowska a. windyga b., the distribution of enterotoxin and enterotoxinlike genes in staphilococcus aureus strains isolated from nasal carriers and food samples, int. j. food microbiol., 117, 319-323, (2007). [18]. lancette g. a., tatini s. r., staphylococcus aureus. in: eds vanderzant, c. and splittstoesser, d. f. (eds.). compendium of methods for the microbiological examination of foods, washington, american public health association. 533-550, (1992). [19]. speck ml, compedium of methods for examination of food microbiological. american public health associations, washington dc, 417423, (1976). [20]. lancette g, bennett r., staphylococcus aureus and staphylococcal enterotoxins. in: downes f, ito k. (editors.), compendium of methods or the microbiological examination of foods, apha, washington, 387-403, (2001). [21]. oliviera l.p., soares e., barros l.s., silva v.c., cirqueira m.g., study of staphylococcus aureus in raw and pasteurized milk consumed in reconcavo area of the state of bahia, brazil, j. food process technol. 2, 128-133, (2011). [22]. ivana s. (coord.), microbiologia alimentelor vol. i, ed. asclepius, bucureşti (2011) odstraňovanie železa a mangánu z vody filtráciou prírodnými materiálmi food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 15 determination of pcb in water using microextraction and capillary gas chromatography *ján ilavský1, danka barloková1 1department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovakia jan.ilavsky@stuba.sk, danka.barlokova@stuba.sk *corresponding author received 19 december 2011, accepted 20 january 2012 abstract: capillary gas chromatography and fid detector were used in the study of the recoveries of pcb microextraction of the delor 103 mixture and selected congeners of pcb (8, 28, 52, 101, 118, 138, 153, 180, 203) from 1 litre of water (at 7 °c) into hexane and toluene (1 ml) by vigorous mechanical shaking for 5 minutes. the recoveries of pcb from the delor 103 microextraction (at concentrations from 0.01 to 10 mg/l h20) related to the internal standard 1,2,3,4-tetrachlornaphtalene (recovery = 100%) were above 60%. the similar results were obtained also in the case of the individual pcb congeners. the pcb recovery is influenced with number of chlorine atoms in biphenyl, concentration of the individual congeners in the mixture delor 103 and their solubility in water. the results of recoveries showed, that the method described above can be used for the purposes of the routine pcb analysis in waters. keywords: capillary gas chromatography, polychlorinated biphenyls, microextraction, water analysis 1. introduction pcbs have been identified in almost every environmental compartment or matrix. detected levels depend on the nature and location of the particular environmental sample [1]. environmental legislations approved by federal agencies like who, us epa, or by european commission directives [2,3,4], aim at the most severe reduction of the pcb levels in water and soils (< 1 ng.kg-1 in the original sample). this implies, for a pcb analytical method to be validated, the achievement of very low detection limits (< 0.5 ng.kg-1 in the extract). analysis of polychlorinated biphenyls (pcb) is being discussed very often, as for the result evaluation several ways giving not always the same results can be used. technical mixtures of pcb are composed of tens of individual components (theoretically of 209 so called congeners), contents of which depends on a degree of chlorination and conditions of the respective reaction [5-8]. it is necessary to stress, that the composition of the technical mixtures produced in chemko strážske, slovakia (delor from 103 till 106) is markedly different from the compositions of the mixtures produced by another producers (aroclor, clophen, kaneclor, etc.). this fact is a source of some problems arising in the quantitative evaluation of pcbs using capillary gas chromatography (gc). exact quantitative analysis of pcb in waters needs identification of all present congeners and on the base of a detector response also calculation of each concentration. from the practical point of view this fact means the necessity of using high resolution capillary gc and to dispose of all individual congeners or their mailto:jan.ilavsky:@stuba.sk mailto:danka.barlokova:@stuba.sk food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 16 mixtures applicable for calibration of each component, detector and the respective conditions. the result of analysis can be than calculated as the sum of the respective congener contents. the work [9] is in the closest touch with this exacting evaluation. its author used silica capillary column se-54 (50 m x 0.2 mm) equipped with the electron capture detector (ecd) and was succeed in separation of 187 congeners pcb. the work [10] has introduced a method for the quantitative analysis of pcb using the highly selective specific hall´s electrolytic detector, response of which depends on the content of chlorine in pcb molecule. application of this detector in certain conditions enables to analysis quantitatively the content of pcb isomers without a need of individual standards. calibration is related mainly to the samples of aroclor. because of the problems mentioned above some simpler methods are being used. i.e. all pcbs in the extract are chlorinated using the respective reactant and dekachlorbiphenyl is obtained. this compound has in the chromatographic spectrum the only one peak [11,12]. the procedure depends on the quality of reactant having been used for chlorination (sbcl2, so2cl2) and on the degree of chlorination. moreover, dekachlorbiphenyl determination can be disturbed with some accompanying compounds. next procedure being used for pcb determination is a summarizing of all identified peaks of the sample and standard giving the same response in detector. this method is best worked out for the aroclor standard [13,14,15]. this method does not take into account the different degree of congeners transformation [16]. thanks to the very different responses of individual congeners in ecd a standard choice must be done very carefully. summarizing of all identified peaks is recommended by a lot of official methods. another widespread method of quantitative evaluation is the method of representative peaks. it is based on the choice of a few peaks, the highs or areas of which represent the whole quantity of pcb. as the representative peaks present in each chromatogram of pcb having been isolated from water are chosen. the authors have named these peaks as characteristic for each pcb mixture. evaluation must be completed with the congeners, which are the results related to. nowadays, these the most typical peaks are described in literature -28,52,101,138,153, 180. in the same conditions as an unknown sample also a pcb standard is analysed. the most reliable method of pcb analysis is the combination of gas chromatography and mass spectrometry. a lot of works have been published, in which mass spectrometry (gs-ms) is presented as a method of individual pcb´s identification. the most usable extractant of pcb´s from water is hexane, dichloromethane, toluene or petrol ether. extraction is performed either in one batch with the biggest amount of extractant, or in multiplied batches with a smaller volumes of solvent [17-22]. a standardized method epa 608 [23] uses for extraction of pcb together with organochloric pesticides dichlormethane as the extractant. 1 l of water is extracted 3 times with 60 ml of ch2cl2. all extracts, poured together, are dried with natrium sulphate anhydride. in the kuderna-danish (kd) concentrator they are concentrated to the volume of 1 ml and after addition of 50 ml of n-hexane they are preconcentrated into hexane. pcbs are separated from chlorinated pesticides in the column packed with silicagel or fluorisil. in a triple n-hexane extraction of 2 l of water or pouring together of two parallel hexane extracts and their concentrating together with using ecd a level 0.01 mg/l is achievable [24]. ecd is suitable first of all for multichlorinated pcb. for the components with food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 17 less content of chlorine a combination of ecd and fid is advantagable [25]. pcb analysis in waters can be performed also with stripping in a close cycle. the work [26] describes, that the recovery of 2,2 ,́4,5,5´-pentachlorbiphenyl from water achieves nearly 20%, if these parameters are used: the volume of water sample is 3.8 litre, water sample temperature is 30 °c, stripping time 2 h. the pentachlorbiphenyl concentration in water was 21.4 ng.l-1. in spite of the non-optimized conditions the recovery is surprisingly high, because pentachlorbiphenyl is not very volatile and has a high boiling point. relatively high recovery is caused with the minimal solubility and thus with the high activity coefficient of pcb in water. recovery of pcb in the process of their separation from water can be improved with the sample temperature during stripping. in the same way also a temperature of a filter filled with charcoal is necessary to increase. thus a condensation of water can be avoided. i.e. at the water sample temperature 70 °c (the charcoal filter temperature 90 °c and the stripping time 2 h) the recovery between 30 and 65% (related to the degree of biphenyl chlorination) was achieved. the method must be calibrated with the same mixture of pcb in a standard water solution as in the case of a contaminated water sample. for the purposes of the charcoal extract analysis capillary columns wet with nonpolar stationary phase on the base of polysiloxane (ov-101, se-54, se-52) are usually used. detection is performed with ecd, however, the concentration factor of this method is so high, that the trace concentrations of pcb in water can be analysed either with fid (there is a danger of disturbing the analysis with the common hydrocarbons) or mass detector. with the help of gc-ms an identification of pcb and their differentiation from the possible interfering components is possible. for the purposes of the charcoal extract analysis capillary columns wet with nonfor the purposes of the charcoal extract analysis capillary columns wet with nonpolar stationary phase on the base of polysiloxane (ov-101, se-54, se-52) are usually used. detection is performed with ecd, however, the concentration factor of this method is so high, that the trace concentrations of pcb in water can be analysed either with fid (there is a danger of disturbing the analysis with the common hydrocarbons) or mass detector. with the help of gc-ms an identification of pcb and their differentiation from the possible interfering components is possible. moreover, distinguishing of fractions with the different degree of biphenyl chlorination or identification of respective chemical individuals is possible. continual destilation and extraction are also possible to use for pcb extraction from water. if the concentrations of pcb were at about 10 mg/l, the average achieved recoveries of pcb in water (aroclor 1260) were approximately 95%. the volume of sample was 50 ml, extractant (pentane) 2.5 ml and time of destilation-extraction one hour. gc analysis was done in a capillary column with ov-101 and ecd [27]. 2. experimental 2.1 instrumentation gas chromatograph carlo erba (vega 6000) equipped with a fid detector and split-splitless capillary injector system was used. for the chromatographic separation a silica capillary column db-5 (30 m x 0.25 mm i.d.) wet with silicone stationary phase se54 was used. the column temperature during injection was 40 °c and after 1 min programmed at the temperature gradient 30 °c/min till 200 °c and then linearly programmed at 3 °c/min till 260 °c. at this temperature the column was kept 10 min. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 18 the detector and injector temperature were 300 °c. fid detector was used together with nitrogen as the carrier gas and makeup. for injecting a splitless technique was used, i.e. during injection of extract (1.5ml) a sample splitter was closed within 30 sec. for the analysis of selected (representative) congeners the chromatographic conditions were the same. the only one change was in a linear programming of the column temperature from 200 °c. program of 5 °c/min was used till the temperature value 270 °c. chromatograms were integrated with dp 700 (carlo erba) integrator. 2.2 chemicals for the analysis of the mixture delor 103 certified standards from the slovak metrology institute were used. the concentration of delor 103 in n-heptane was 1 mg/ml. the mixture of selected (representative) congeners was produced in the research institute for drugs in modra and is being sold by the slovak metrology institute. concentration of the respective congeners was 15 mg/ml of n-heptane. for extract (4) was connected to the extraction flask (figure 1) [28]. in this case the n-hexane or toluene extracts are easily accessible and can be injected immediately by means of a syringe into a gas chromatograph. fig.1 solvent thin layer separator for microextraction of water 3. results and discussion solvents (n-hexane, toluene) were highly purified and checked chromatographically. as the internal standard (is) 1,2,3,4tetrachlornaphtalene was used (purity at least 98%). a stock solution with the concentration of 1 mg/ml was prepared by weighing into hexane and toluene. working solution was prepared by its gradual diluting to the concentration 0.1 m g/ml of n-hexane and toluene. 2.3 microextraction one litre of water containing defined content of organic substances was subjected to microextraction (at + 5-7 °c) with 1 ml of n-hexane or toluene containing internal standard (is) by intense manual shaking 5 min. the glass extraction flask, equipped with a male joint (1) and conical stopper (5) was used to extraction (a). after extraction (b) the solvent thin layer separator (2) containing side arm for water (3), capillary for the analysis of polychlorinated biphenyls in waters a microextraction with the following capillary chromatography equipped with fid can also be used. for the sake of the fact that in our country delor 103 was mostly used (approximately 77% from the total production), this work pays attention to this mixture of pcb. figure 2 shows the gas chromatogram of delor 103 (the pcb mixture with the content of chlorine between 42 and 43% with the prevailing content of trichlor derivatives) after microextraction of 1 litre h2o into 1 ml of n-hexane with is (0.1 mg. ml-1 1,2,3,4-tetrachlornaphtalene). identification of delor 103 components (elution peaks) is based on the data already published [29,30] and obtained standard from the slovak metrology institute. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 19 fig. 2 chromatogram of the delor 103 mixture table 1 describes the relative recoveries of individual congeners of delor 103 at the concentration of 1 mg/l of water related to the internal standard 1,2,3,4-tetrachloronaphtalene (r=100%) obtained with the extraction of 1 l of water (at + 5-7 °c) into 1 ml of n-hexane with intensive hand shaking within 5 min. the recoveries are the arithmetical average values from 5 measurements. table 1 relative recoveries of pcb congeners after microextraction into 1 ml of n-hexane. peak no. structure of congener recovery [%] % rsd 4 2,2´ 70.48 1.05 9 2,5 69.27 0.34 7 2,4 69.53 0.22 6 2,3´ 73.15 0.23 5, 8 2,3 ; 2,4´ 79.85 3.97 19 2,2´,6 75.78 0.31 18 2,2´,5 80.20 2.83 15, 17 4,4´ ; 2,2´,4 76.51 1.84 27 2,3´,6 76.32 0.42 16, 32 2,2´,3 ; 2,4´,6 80.43 2.72 26 2,3´,5 76.79 0.56 25 2,3´,4 74.88 0.45 31 2,4´,5 80.31 4.12 28 2,4´,4 79.36 7.98 33, 53 2´,3,4 ; 2,2´,5,6´ 79.55 12.78 21, 51 2,3,4´ ; 2,2´,4,6´ 80.14 2.14 45 2,2´,3,6 77.02 0.59 46 2,2´,3,6´ 77.15 0.80 tab. 1 continued 52 2,2´,5,5´ 82.79 3.57 49 2,2´,4,5´ 82.92 2.73 48, 47 2,2´,4,5 ; 2 ,2´,4,4´ 80.25 3.06 44 2,2´,3 ,5 80.78 1.89 37, 42 3,4,4´; 2,2´,3,4´ 78.63 2.10 41, 64, 71, 72 2,2´,3,4 ; 2,3´,4,6 ; 2,3´,5,5´ ; 2,3´,4,6 79.10 1.51 40 2,2´,3,3´ 76.82 0.60 67 2,3´,4,5 72.34 0.19 63 2,3,4´,5 73.58 0.17 74, 94 2,4,4´,5 ; 2,2´,3,5,6´ 77.28 1.34 70, 76 2,3´,4´,5 ; 2´,3,4,5 78.06 1.79 66 2,3´,4,4´ 77.81 2.73 56, 60 2,3,3´,4 ; 2,3,4,4´ 76.11 1.84 101 2,2´,4,5,5´ 70.48 0.62 99, 113 2,2´,4,4´,5 ; 2,3,3´,5´,6 69.14 0.36 97 2,2´,3´,4,5 68.25 0.22 87 2,2´,3,4,5´ 68.78 0.35 111 2,3,3´,5,5´ 68.47 0.32 77, 110 2,3´,4,4´ ; 2,3,3´,4´,6 69.68 0.49 118 2,3´,4,4´,5 69.51 0.52 the individual recoveries of congeners in the mixture delor 103 are influenced with the concentrations of respective congeners in the mixture and their solubility in water. from the data in table 1 the possibility of using pcb separation from water (without concentration) and following extract analysis with capillary gas chromatography is apparent. the recoveries of individual congeners in the mixture delor 103 are higher than 60%. as pcb´s represent the rich mixture of chlorinated derivatives of biphenyl, the content of each of them can be determined from the areas (highs) of elution peaks belong to the respective components without the need of identification of each component. a lot of official methods recommend summarizing of all separated congeners. that was the reason we studied the recoveries of microextraction of the mixture delor 103 at the concentrations from 0.01 m to 10 mg/l of water related to 1,2,3,4-tetrachlornaphtalene as the internal standard. water with the defined content of delor 103 was extracted into 1 ml of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 20 hexane containing 0.1 mg of 1,2,3,4-tetrachlornaphtalene. figure 3 shows the graphical dependence of the recoveries of pcb microextraction on the delor 103 concentration. fig. 3 graphical dependence of the delor 103 microextraction recoveries in dependence on the concentration. the recoveries of delor 103, calculated from the summarised area of its elution peaks, are at the concentration of delor 103 above 0.5 mg/l higher than 60%. it can be concluded, that for the content of delor 103 above 0.5 mg/l a microextraction into hexane can be used. it can also be seen, that at the delor 103 concentration below 0.5 mg the recovery rapidly falls and is not a linear function of the concentration. figure 4 shows the analytical curve of the ratio of the area sum of elution peaks for the mixture delor 103 (pdelor 103) and area of the internal standard (pis) in the dependence of concentration. fig. 4 analytical curve of the mixture delor 103 in the concentration range 0.01 10 mg/l h2o. apart from the summarizing of all peaks a method of representative peaks nowadays often used. literature describes the most typical congeners as the representative peaks. they are compared with analysed samples, whereas the chromatogram and the procedure of isolation were performed in the identical conditions. figure 5 shows the gas chromatogram of these congeners at concentration 50 ng/l of water. fig. 5 chromatogram of the selected pcb congeners in the concentration of 0.05 mg/l h2o. table 2 shows the relative recoveries of the individual congeners mentioned above (concentration of each component is 50 ng/l of h2o) related to the internal standard (0.1 mg 1,2,3,4-tetrachlornaphthalene). they were obtained with the microextraction of 1 litre of water (at 7 °c) into 1 ml of n-hexane or toluene performed by intensive hand shaking within 5 min. the recoveries are the arithmetical average values from 5 measurements. as there is a need to have a continual control over these congeners, the proposed method can be used for the quantitative determination of pcb´s in waters (the recovery is above 75%). the advantage of this method is in the fact, that in spite of its simplicity and speed, it is suitable for concentrations recommended as maximal acceptable in waters. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 21 table 2 relative recoveries of the selected congeners at concentration 50 ng/l h2o related to is (r=100%) obtained with microextraction. peak no. structure of congener recovery [%] 1 ml n-hexane 1 ml toluene 8 2,4´ 75.41 82.67 28 2,4´,4 76.38 83.58 52 2,2´,4,4´ 81.66 85.09 101 2,2´,4,5,5´ 85.92 89.14 118 2,3´,4,4´,5 84.69 91.86 138 2,2´,3,4,4´,5 89.78 92.11 153 2,2´,4,4´,5,5 88.02 91.58 180 2,2´,3,4,4´,5,5´ 94.39 96.73 203 2,2´,3,4,4´,5,5´,6 91.27 95.14 next conclusion of this table is the fact, that with increasing the number of chlorine in biphenyl the recovery of microextraction increases too. this is in accordance with the solubility of these compounds in water (see table 3). the higher is the degree of chlorination the lower is the solubility of respective congeners in water. the higher values of the recoveries were obtained using toluene as the extractant. when toluene was used for the extraction, the extract was concentrated (via dissolving the part of toluene in water). the higher recovery of toluene extraction is also caused with the similarity of the structures of extractant and biphenyl. table 3 solubilities of the searched congeners in water [31,32,33] peak no. structure of congener solubility in water [µg/l] 8 2,4´ 538 – 1880 28 2,4´,4 67 – 260 52 2,2´,4,4´ 54.1 – 68 101 2,2´,4,5,5´ 4.3 – 31 118 2,3´,4,4´,5 13.4 138 2,2´,3,4,4´,5 7.3 153 2,2´,4,4´,5,5 0.9 – 9.1 180 2,2´,3,4,4´,5,5´ 3.85 203 2,2´,3,4,4´,5,5´,6 0.1 – 3.0 4. conclusion the results of recoveries showed, that the microextraction with n-hexane or toluene and capillary gas chromatography with detector fid can be used for the pcb analysis in waters. the pcb recovery is influenced with number of chlorine atoms in biphenyl, concentration of the individual congeners in the mixture delor 103 and their solubility in water. when the analytical curve for the selected congeners is constructed, the method mentioned above can be successfully used, similarly as in the case of the mixture delor 103, for the purposes of the routine analysis of pcb in water. also a method of an internal standard is suitable for evaluation of the analysed components. references [1] lang v. polychlorinated biphenyls in the environment. j. chromatogr. a, 595(1-2), 143, (1992). [2] federal register epa. method 1668, revision a. chlorinated biphenyl congeners in water, soil and sediment by hrgc/hrms. epa821-r-00-002, (1999). [3] european commission directive 1996/59/ eec and updates; directive 2002/201/ec and updates. [4] who. polychlorinated biphenyls and terphenyls. environmental health criteria, 140. geneva, switzerland: world health organization, 48-52, 444-479, (1993). [5] alford-stevens a.l. analyzing pcbs. environ. sci. technol. 20(12), 1194, (1986). [6] erickson m.d. analytical chemistry of pcbs, 2nd ed.; lewis publishers: boca raton, 667 p., (1997). [7] polychlorinated biphenyls manufacturing, processing, distribution in commerce and use prohibition," 40 code of federal regulations, part 761, (1998). [8] mcdonald, c. j., tourangeau r. e. pcbs: question and answer guide concerning polychlorinated biphenyls. commercial chemical branch, eps, environment canada, ottawa, ontario, (1986). [9] mullins m.d., pochini c.m., crindle s.mc., romkes m., safe s.h., safe l.m. high-resolution pcb analysis: synthesis and chromatographic properties of all 209 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44cpw55-12m&_user=10&_coverdate=03/20/1992&_alid=1594949816&_rdoc=3&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_docanchor=&view=c&_ct=39&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=271523fa1faca22162c732609d81a169&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-44cpw55-12m&amp;_user=10&amp;_coverdate=03/20/1992&amp;_alid=1594949816&amp;_rdoc=3&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_docanchor=&amp;view=c&amp;_ct=39&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=271523fa1faca22162c732609d81a169&amp;searchtype=a http://pubs.acs.org/doi/abs/10.1021/es00124a014 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 22 pcb congeners. environ. sci. technol. 18(6), 466, (1984). [10] ramus t.l., hein s.l., thomas l.c. determinations of polychlorinated biphenyl isomers by response factor calibration. j. chromatogr. 404, 155, (1987). [11] duinker j.c., hillebrand m.t.l., palmork k. h., wilhelmsen s. an evaluation of existing methods for quantitation of pcbs in environmental samples and suggestions for an improved method based on measurement of individual components. bull. environ. contam. toxicol. 25(1), 956, (1980). [12] copland g.b., gohmann c.s. improved method for polychlorinated biphenyl determination in complex matrices. environ. sci. technol. 16(2), 121, (1982). [13] kimbrough d.e., chin r., wakakuwa j. industry-wide performance in a pilot performance evaluation sample program for hazardous materials laboratories. 2. precision and accuracy of pcbs. environ. sci. technol. 26, 2101, (1992). [14] frame g.m. congener-specific pcb analysis. anal. chem. 69, 468a-475a, (1997). [15] frame g.m., cochran j.w., bowadt s.s. complete pcb congener distributions for 17 aroclor mixtures determined by 3 hrgc systems optimized for comprehensive, quantitative, congener-specific analysis. j. hrc & cc 19(12), 657-668, (1996). [16] onuska f.i., mudroch a., davies s. application of chemometrics in homologspecific analysis of polychlorinated biphenyls. j. hrc & cc 8, 747, (1985). [17] moret i., gambaro a., piazza r., ferrari s., manodori l.: determination of polychlorobiphenyl congeners (pcbs) in the surface water of the venice lagoon. mar pollut. bull. 50(2), 167, (2005). [18] font g., mañes j., molto j. c., pico y. current developments in the analysis of water pollution by polychlorinated biphenyls. j. chromatogr. 733(1-2), 449, (1996). [19] ballschmiter k., zell m. analysis of polychlorinated biphenyls by glass capillary gas chromatography: composition of technical aroclor and clophen-pcb mixtures. fresenius z. anal. chem. 302, 20-31, (1980). [20] federal register epa. method 1668, revision a. chlorinated biphenyl congeners in water, soil, sediment, and tissue by hrgc/hrms. epa no. epa-821-r-00-002, (1999). [21] kannan n., petrick g., schultzbull d.e., duinker j.c. chromatographic techniques in accurate analysis of pcb´s. j. chromatogr. 642(1-2), 425-434, (1993). [22] astm d6160-98. standard test method for determination of polychlorinated biphenyls (pcbs) in waste materials by gas chromatography, (2008). [23] federal register epa. method 608. u.s. epa. washington, october 26, (1984). [24] hutzinger o., safe s., zitko v. the chemistry of pcbs. crc press, inc.: cleveland, ohio, (1974). [25] cooper s.d., moseley m.a., pellizzari e.d. surrogate standards for the determination of individual pcb using high-resolution gas chromatography with electron capture detection. anal. chem. 57(13), 2469, (1985). [26] coleman w.e., melton r.g., slater r.w., kopfler f.c., voto s.j., allen w.k., aurand t.a. determination of organic contaminants by the grob closedloop-stripping technique. j. awwa 73(2), 119, (1981). [27] godefroot m., stechele m., sandra p., verzele m. a new method for the quantitative analysis of organochlorine pesticides and polychlorinated biphenyls. j. hrc & cc 5(2), 75, (1982). [28] hrivňák j. solvent thin layer separator for microextraction of water. anal. chem. 57(11), 2159, (1985). [29] krupčík j., kočan a., petrík j., leclercq p.a., ballschmiter k. on the use of reference standards for quantitative analysis of pcbs by hrgc. analyses of technical pcb formulations by hrgc/fid. chromatographia 33(11-12), 514, (1992). [30] krupčík j., kočan a., petrík j., leclercq p.a., ballschmiter k. reference standards for quantitative trace analysis of pcb's by gc. technical pcb formulations for the calibration of ecd and msd responses, chromatographia 35(7-8), 410, 1993. [31] murphy t.j., mullin m.d., meyer j.a. equilibration of polychlorinated biphenyls and toxaphene with air and water. environ. sci. technol. 21(2), 155, (1987). [32] opperhulzen a., gobas f.a.p.c., steen j.m.d., hultzinger o. aqueous solubility of pcbs related to molecular structure. environ. sci. technol. 22(6), 638, (1988). [33] mackay d., shiu w.y., ma k.c. illustrated handbook of physical-chemical properties and environmental fate for organic chemicals. volume i+ii. lewis publishers inc.: boca raton, fl, usa, pp. 608, 1992. http://pubs.acs.org/doi/abs/10.1021/es00124a014 http://pubs.acs.org/doi/abs/10.1021/es00124a014 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44c8ydy-3b&_user=10&_coverdate=12/31/1987&_rdoc=16&_fmt=high&_orig=browse&_origin=browse&_zone=rslt_list_item&_srch=doc-info(%23toc%235248%231987%23995959999%23270547%23flp%23display%23volume)&_cdi=5248&_sort=d&_docanchor=&_ct=56&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=21b1757191030691f73e9fde4cd099d8&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-44c8ydy-3b&amp;_user=10&amp;_coverdate=12/31/1987&amp;_rdoc=16&amp;_fmt=high&amp;_orig=browse&amp;_origin=browse&amp;_zone=rslt_list_item&amp;_srch=doc-info(%23toc%235248%231987%23995959999%23270547%23flp%23display%23volume)&amp;_cdi=5248&amp;_sort=d&amp;_docanchor=&amp;_ct=56&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=21b1757191030691f73e9fde4cd099d8&amp;searchtype=a http://www.springerlink.com/content/?author=j.+c.+duinker http://www.springerlink.com/content/?author=m.+t.+j.+hillebrand http://www.springerlink.com/content/?author=k.+h.+palmork http://www.springerlink.com/content/?author=s.+wilhelmsen http://www.springerlink.com/content/x6x5w51282v3155l/ http://www.springerlink.com/content/x6x5w51282v3155l/ http://www.springerlink.com/content/x6x5w51282v3155l/ http://www.springerlink.com/content/x6x5w51282v3155l/ http://www.springerlink.com/content/x6x5w51282v3155l/ http://pubs.acs.org/doi/abs/10.1021/es00096a013 http://pubs.acs.org/doi/abs/10.1021/es00096a013 http://pubs.acs.org/doi/abs/10.1021/es00096a013 http://pubs.acs.org/doi/abs/10.1021/es00035a004 http://pubs.acs.org/doi/abs/10.1021/es00035a004 http://pubs.acs.org/doi/abs/10.1021/es00035a004 http://pubs.acs.org/doi/abs/10.1021/es00035a004 http://www.ncbi.nlm.nih.gov/pubmed?term=%22moret%20i%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gambaro%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22piazza%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ferrari%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22manodori%20l%22%5bauthor%5d http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-3vjrr06-15&_user=10&_coverdate=05/10/1996&_alid=1594949816&_rdoc=4&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_docanchor=&view=c&_ct=39&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=cfbb3b175dca65067491bf7391cc03c5&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-3vjrr06-15&amp;_user=10&amp;_coverdate=05/10/1996&amp;_alid=1594949816&amp;_rdoc=4&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_docanchor=&amp;view=c&amp;_ct=39&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=cfbb3b175dca65067491bf7391cc03c5&amp;searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6tg8-44cprv8-1b&_user=10&_coverdate=07/16/1993&_alid=1594949816&_rdoc=1&_fmt=high&_orig=search&_origin=search&_zone=rslt_list_item&_cdi=5248&_docanchor=&view=c&_ct=39&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=eb649cacb8486b661e22dcb2d65cd686&searchtype=a http://www.sciencedirect.com/science?_ob=articleurl&amp;_udi=b6tg8-44cprv8-1b&amp;_user=10&amp;_coverdate=07/16/1993&amp;_alid=1594949816&amp;_rdoc=1&amp;_fmt=high&amp;_orig=search&amp;_origin=search&amp;_zone=rslt_list_item&amp;_cdi=5248&amp;_docanchor=&amp;view=c&amp;_ct=39&amp;_acct=c000050221&amp;_version=1&amp;_urlversion=0&amp;_userid=10&amp;md5=eb649cacb8486b661e22dcb2d65cd686&amp;searchtype=a http://onlinelibrary.wiley.com/doi/10.1002/jhrc.1240050203/abstract http://onlinelibrary.wiley.com/doi/10.1002/jhrc.1240050203/abstract http://onlinelibrary.wiley.com/doi/10.1002/jhrc.1240050203/abstract 332 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 332 – 341 eff ects of work -related factors on work er s’ health in b ulgar ia *dobromir kralchev 1, donka taneva 2, tsvetko prokopov 2 1department of informatics and statistics, university of food technologies 2department of environmental engineering, university of food technologies, 26 “maritsa” blvd., 4002 plovdiv, bulgaria, dobromir_kralchev@abv.bg; tsvetko_prokopov@abv.bg * corresponding author received november 7th 2013, accepted december 13th 2013 abstract: work plays a significant role in people’s lives, in the functioning of companies and society. many workers believe that their work affects their health. occupational health problems are crucial economic and social issues. the 5-th european working conditions survey (ewcs) was carried out in 2010 in all the countries in the eu. our research presents a secondary analysis of the bulgarian data set and identifies working conditions associated with workers’ perception that their job is detrimental to their health. keywords: work-related health problems, healthy working environment, job factors, ergonomics. 1. introduction work-related health problems attract a lot of attention due to their high frequency and the significant impact they usually have on life. about 2 mln. work-related fatalities and 330 million work-related accidents still occur each year in the world [1]. in 2010 there were 109 work-related fatalities and 3086 work-related accidents in bulgaria [2]. working conditions of high quality and low health risk are both a legal requirement and a powerful stimulus for innovations and productivity of labour. to improve working conditions is a constant concern of the eu. during the past years, health-safety at work has often been considered an integral part of the individual and social prosperity as well as a determinant of quality management, competitive power, employment growth, economic progress etc. in a european survey conducted in 2009, 42% of bulgarians answered that their work had some negative effect on their health, whereas another 29% found this negative effect strong. in the same survey, 47% of bulgarians said that health-safety at work had deteriorated for the previous five years, 9% considered the change significant, and only 15% reported that working conditions had improved. many interviewees (60%) blaimed the deterioration of working conditions on the economic crisis [3]. a recent research [4] has demonstrated that working conditions may cause several health problems rarely recognized as workassociated diseases. medical specialists should pay attention to the relation between patients and their jobs. the aim of this research is to identify working conditions that have a strong impact on workers’ health. food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 333 2. materials and methods the data have originated from the fifth european working conditions survey [5] that was carried out in 2010 by the european foundation for the improvement of living and working conditions. this survey has been conducted every five years since 1991. the questionnaire covers a broad range of working conditions, work characteristics and workers’ sense of satisfaction and perception of different aspects of their jobs. in the 5-th ewcs, 1014 participants from bulgaria were interviewed. their profiles are presented in table 1. a stratified sample was used and a post-stratification weighting was carried out. throughout this paper, all percentages are weighted after the w4 variable in the data set. table 1 interviewees’ profiles age men (53 %) women (47 %) total (1014) under 30 09 % 07 % 16 % 30 – 49 29 % 26 % 55 % over 50 15 % 14 % 29 % length of service in the same company (q12) under 5 years 24 % 20 % 44 % 05 – 10 years 11 % 10 % 21 % 10 – 20 years 11 % 10 % 21 % over 20 years 07 % 07 % 14 % level of education primary 00.5 % 00.4 % 01 % lower secondary 06.3 % 04.3 % 11 % upper secondary 36.0 % 27.6 % 63 % semi-higher 01.0 % 02.2 % 03 % higher 09.5 % 12.3 % 22 % disclaimer: the european foundation for the improvement of living and working conditions and the uk data archive bear no responsibility for our further analysis and interpretation. 3. results and discussion 3.1. important correlations there were 1014 bulgarian participants in the survey; 4 of them refused to answer the q67 question, “does your work affect your health, or not?”, and 58 participants had no opinion. the remaining 952 interviewees answered as presented on fig. 1 and fig. 2. no 61 % yes, mainly positively 5 % yes, mainly negatively 34 % figure 1. answers of the bulgarian participants to the q67 question, “does your work affect your health, or not?” food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 334 men 5 % 39 %56 % women 5 % 27 % 68 % figure 2. answers by gender of the bulgarian participants to the q67 question, “does your work affect your health, or not?” the p-value returned by the 2-test is 0.0006, which means that the difference between the two distributions is unlikely to have occurred by chance. a strong correlation exists between the gender of an interviewee and his or her answer to q67. men report negative influence of work over health more often than women. there is a strong correlation between the age of interviewees and their answers to q67 (p = 0.008). young people’s health is the least frequently affected by work. the positive influence of work over health is common for interviewees aged 30 – 49 years. in the age group of 50+ years, the negative influence of work increases. a strong correlation (p = 0.003) exists between the level of education and q67. however, this correlation is controversial. while higher levels of education correspond to positive influence of work over health, negative influence can’t be excluded either. different kinds of education are best suited to different kinds of work with different risks (i.e. many other factors interfere and make difficult to deduce a simple rule). the length of service (q12) is another significant factor (p = 0.01). its impact on health increases after 10 or more years. generally, health is negatively affected by manual work occupations more often than by clerical occupations (p = 0.00002). the activity of the organisation where an interviewee works (industry or services) is not very strongly correlated (p = 0.13) with q67. nevertheless, health of industrial workers is negatively affected by their work a little more often. with respect to health, no significant difference exists (p = 0.46) between the public sector and the private sector. and yet, a negative influence of work on health is reported a little more often by those who work in joint private-public companies. work affects the health of employees (positively or negatively) less often than the health of the self-employed (p = 0.0004). but the kind of the employment contract is not significant (p = 0.14). the positive influence of work over health is almost doubled (from 4% to 8%) by new processes and technologies and by reorganization carried out at the workplace. this effect is somewhat obscured by the fact that the same absolute difference (i.e. 4%) is relatively smaller when compared with the other two percentages (about 34% for the negative influence and 60% for the absence of influence of work over health), which gives p  0.10 > 0.05 (the standard significance level). therefore, the effect of new processes and technologies and the effect of reorganization on workers’ health is insignificant as a whole (the negative influence of work on health decreases little); but it is significant with respect to the positive influence (almost doubled). the number of employees working together has little influence on their health (p = 0.13). work in big companies that have over 100 employees affects health most (positively or negatively). next to it in negative influence comes the case when an interviewee works alone. the gender of workers at the same workplace with the same job title as the interviewee is more important (p = 0.06). the negative influence of work is more frequent when the workers in the same job title are men. in this case, however, the positive influence of work is also strongest. (the positive influence depends not on the gender but on the number of workers; it is weakest when nobody else has the same job title as the interviewee.) food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 335 as expected, those who work more hours per week report a stronger (positive or negative) influence of work on their health (p = 0.0007). the influence is significant when the number of working hours per week is greater than 60; work that takes up 45 or less hours per week does not have a strong impact on health. working at night (p = 0.0002) or more than 10 hours a day (p = 0.00001) correlates with the negative influence of work on health. working in free time to meet work demands correlates with both the positive and negative influence of work (p ≈ 10 – 13 ). a second paid job affects health little (p = 0.30). an occasional second job affects health positively; a regular one affects it negatively. less than 10 hours per week are insignificant; 15 or more hours affect health negatively. both positive and negative outcomes of work on health increase (p < 10– 8 ) when a job involves working to tight deadlines most of the time. working at a very high speed less than 1/4 of the time affects health positively; otherwise the effect is negative (p < 10 – 12 ). interruptions of the current task due to unforeseen tasks increase both the positive and the negative influence of work on health (p = 10 – 6 ). disruptive interruptions are those that increase the effect of work (p = 0.0003); positive and neutral interruptions do not. precise quality standards (p = 0.10), complex tasks (p = 0.00002) and solving unforeseen problems (p = 0.03) correlate strongly with the positive influence of work, but weakly with the negative one. learning new things correlates with the positive impact of work on health (p = 10 – 3 ). monotonous tasks seem to increase the positive influence of work on health, but the difference is insignificant (p = 0.28). assessing the quality of one’s own work has no impact on health (p = 0.50). 3.2. geographical distribution there is a significant correlation (p = 0.03) between the influence of work on health and geographical regions. the negative impact is more often reported in northern bulgaria whereas the positive influence and the absence of influence are most frequently reported in southern bulgaria (table 2). there is an obvious difference between towns and villages (p = 0.0005). in villages, work does not affect health much (table 2). the positive influence of work on health is most frequent in cities with 50000+ inhabitants, and the negative one — in small towns (10000 – 50000). table 2 geographical distribution size of locality q67 does your work affect your health, or not? yes, positively yes, negatively no total up to 500 inhabitants 00 % 14 % 86 % 4 % 501– 9999 03 % 30 % 67 % 33 % 10 – 50 thousands 03 % 40 % 57 % 15 % over 50 000 07 % 36 % 57 % 48 % total 05 % 34 % 61 % 100 % 3.3. occupational safety and health naturally enough, the negative influence of work on health jumps from 13.4% to 64.2% when workers’ health and safety are at risk because of their work (p ≈ 10 – 62 ). jobs that require personal protective equipment are more frequently associated (44% vs. 28%) with the negative influence of work (p ≈ 10 – 7 ) but also with the positive one (6.8% vs. 3.8%), which is a paradox. food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 336 about 90% of workers always use their protective equipment, and 10% do not. of the second group, nobody reported a positive effect of work on his or her health; but 62.4% were affected negatively. contrariwise, those who always use their protective equipment reported either a positive (7.5%) or no impact (50.5%) of work on their health (p = 0.056). only those workers are positively affected by work who are well-informed about health and safety risks. the negative effect is most frequent (56.7%) among those who are not very well-informed. surprisingly, those who are not at all wellinformed most often say (80.6%) that work does not affect their health (p = 0.0007). perhaps their jobs are free of risk. 3.4. classification of factors factors can be divided into groups. a) factors that are correlated with the negative influence of work on health:  breathing in smoke, fumes, powder, dust or vapours such as solvents and thinners.  contact with chemical products.  repetitive hand or arm movements. (these all have p < 10–9).  standing upright (p = 0.00002).  breathing in tobacco smoke from other people (p = 0.0001).  direct contact with materials that can be infectious (p = 0.0005). some of these factors seem to be correlated also with the positive influence of work. most probably this correlation is a false one due to the small frequencies associated with the positive influence that violate one of the constraints of the 2-test. b) factors that are correlated with the positive influence of work on health:  dealing directly with people who are not employees, such as customers, patients, pupils etc. (p = 0.02).  working with computers (p = 0.0004).  using internet / email (p = 0.03). c) factors correlated with both the positive and negative influence of work:  vibrations (q23a) and noise (q23b).  high or low temperatures (q23c,d).  tiring or painful positions (q24a).  handling angry clients (q24g).  short repetitive tasks (q44). the p-values are smaller than 10 – 6, i.e. the correlation is very strong. the nature of the correlation must be carefully interpreted. as a typical example, consider the impact of vibrations on health (table 3). table 3 influence of vibrations on health exposed to vibrations q67 does your work affect your health, or not? yes, positively yes, negatively no total (almost) all of the time 10 % 57 % 33 % 12 % over 50% of the time 04 % 49 % 47 % 0 7 % about 25% of the time 02 % 51 % 47 % 0 7 % (almost) never 04 % 27 % 69 % 74 % total 05 % 34 % 61 % 100 % answers of those workers who are not exposed to vibrations have almost the same distribution as the set of all answers. interviewees exposed to vibrations report a negative influence of work more often (51%, 49%, 57%) when they work in such conditions for a longer period. suprisingly, the percentage of the positive influence of work (2%, 4%, 10%) also grows. however, factors correlated with some kind of influence may or may not be its cause. such factors may just accompany agents that affect health. for example, physical activity itself is healthful, even if involving tiring positions. not all factors of this group raise such problems. some of them (e.g. q24g) are dual in nature (their influence on a worker’s health depends on his or her reaction to the factors). food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 337 d) factors whose influence on health depends on their intensity:  carrying or moving heavy loads (p < 10– 9).  lifting or moving people (p = 0.00002). these comply with the well-known principle that moderate physical activity is healthful, but overstrain is harmful. 3.5. health problems the questionnaire of ewcs 2010 contains a question (q69) with 14 items to check for different health problems. table 4 shows how they depend on q67 (the smaller the p-value, the stronger the correlation). the thirteen health problems in table 4 are both frequently reported and work-related (their p-values are smaller than 0.05). other health problems are rarely reported (0.7%) and not workrelated (p = 0.21 is too great). all the health problems listed in table 4 correlate with the negative influence of work. typically, about 30% of workers who do not have some health problem think that work affects health negatively (the percentage is almost the same for all the health problems). however, from 50% to 70% of workers who do have some health problem say that their work affects their health negatively (the percentage now varies from one health problem to another). health problems correlate with the positive influence of work too. however, the correlation itself can be either positive or negative, i.e. those who are sick may report a positive influence of work on their health either more frequently or less frequently than those who are healthy. the second column of the table contains both percentages and the correlation type (where  stands for a positive correlation and  stands for a negative one). table 4 health problems health problems positive influence among q67 p-value frequency healthy sick hearing problems 4.5 %  10.6 % 0.0004 04.2 % skin problems 4.6 %  0 8.0 % 0.0004 05.2 % backache 5.2 %  0 4.0 % 10 – 15 33.1 % muscular pains in neck, shoulders and upper limbs 5.5 %  03.4 % 10 – 16 33.1 % muscular pains in lower limbs 5.1 %  03.9 % 10 – 9 30.0 % headache, eyestrain 4.6 %  0 5.2 % 10 – 7 37.4 % stomach-ache 4.8 %  0 4.3 % 0.0002 10.7 % respiratory difficulties 5.0 %  0 0.9 % 0.0010 05.3 % cardiovascular diseases 4.7 %  0 5.9 % 0.0002 08.9 % injury(ies) 4.8 %  0 4.3 % 10 – 10 06.7 % depression or anxiety 4.9 %  0 4.5 % 10 – 8 14.0 % overall fatigue 4.9 %  0 4.6 % 10 – 15 48.8 % insomnia 4.5 %  0 6.0 % 10 – 7 19.5 % other 4.9 %  0 0.0 % 0.21 00.7 % food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 338 a positive correlation can be interpreted differently:  work affects this particular health problem positively.  work has a positive influence on other health problems accompanying this one.  people suffering from the health problem find a suitable job more easily. not surprisingly, those who work when they are sick find their health affected by work twice as frequently as those who don’t (table 5). this is a very strong dependence (p < 10 – 11 ). table 5 working when sick w or ki ng w he n si ck q67 does your work affect your health, or not? yes, positively yes, negatively no total yes 07 % 57 % 36 % 023 % no 03 % 28 % 69 % 077 % total 04 % 35 % 61 % 100 % 3.6. correlations between health problems and factors of the working environment multiple positive correlations exist between health problems and factors of the working environment. injuries correlate with tiring / painful positions, carrying or moving heavy loads, working with machines or hand tools. tiring / painful positions and repetitive hand or arm movements are correlated with backache, overall fatigue and muscular pains in shoulders, neck, upper limbs and lower limbs. muscular pains in lower limbs are also correlated with standing upright and with carrying or moving heavy loads (but not with lifting or moving people). there is an association between headache and interruptions of the current task due to unforeseen tasks. stress at work corresponds with anxiety, depression, insomnia and overall fatigue. tasks which are in conflict with a worker’s personal values correlate with insomnia. 3.7. correlations between workers’ profiles and factors of the working environment men more frequently than women are exposed to vibrations from machinery or hand tools, high or low temperatures, breathing in dust, smoke, fumes or powder. men’s jobs more often than women’s include carrying or moving heavy loads. the higher the level of education, the less the exposition to the last factor (this also holds for standing upright). higher levels of education are also correlated positively with the following characteristics of work:  complex tasks and learning new things;  working with computers and internet;  dealing with customers, patients etc. 3.8. correlations between different health problems backache and muscular pains in shoulders, neck, upper limbs and lower limbs correlate strongly with each other and somewhat less strongly with headache, eyestrain, injuries, overall fatigue and insomnia. overall fatigue, headache, anxiety, depression and insomnia often go together. insomnia is also correlated with cardiovascular diseases. stomach-ache is associated with injuries. food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 339 3.9. correlations between different factors of the working environment loud noise, vibrations from hand tools or machinery, high and low temperatures, skin contact with chemical products, breathing in vapours (such as solvents and thinners), dust, fumes, powder, smoke (incl. tobacco smoke from other people); tiring or painful positions, carrying or moving heavy loads, standing upright, repetitive hand or arm movements — these all are correlated positively with one another and negatively with working with computers and internet. vibrations and noise are correlated positively (though not very strongly) with precise quality standards. vibrations and high temperatures are correlated negatively (again not very strongly) with dealing with customers, patients, pupils etc. direct contact with materials that can be infectious is associated with contact with chemical products and breathing in vapours such as solvents and thinners as well as with lifting or moving people (such working conditions are often present in hospitals). tiring / painful positions are connected with stress at work. repetitive hand or arm movements are associated with monotonous tasks and precise quality standards. 3.10. are most health problems work-related? the detailed analysis made above reveals the impact of each factor on workers’ health as part of the overall influence of work. the question if work as a whole has a strong influence on health is important in itself and serves as a base for all other inferences: its answer, if negative, will make the discussion useless. work is commonly believed to have a strong influence on health, but this belief still needs to produce evidence. workers’ answers to the q67 question, “does your work affect your health, or not?” provide some piece of information, but not all we need. indeed, the three percentages (5%, 34%, 61%) tell us how often work affects health, not how strongly it does. health is also strongly affected by factors other than work, such as:  age;  conditions of life;  regimen of diet;  sports;  social relations. for example, older people often suffer from cardiovascular diseases. the regimen of diet, conditions of life, sports and social relations are also known to have a strong effect (both positive and negative) on health. therefore, it is important to know which health problems are caused by work and which are not. if many health problems are work-related, then work affects health strongly. to settle it, consider the q68 question, “how is your health in general?” its correlation with the q67 question, “does your work affect your health, or not?” is presented in table 6. table 6 q67 – q68 correlation q 68 h ow is yo ur h ea lt h in g en er al ? q67 does your work affect your health, or not? yes, positively yes, negatively no total very good 02 % 16 % 82 % 24.3 % (fairly) good 06 % 39 % 55 % 73.3 % bad 02 % 53 % 45 % 02.2 % very bad 00 % 79 % 21 % 00.2 % total 05 % 34 % 61 % 100 % food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 340 the p-value returned by the  2 test is smaller than 10 – 10, i.e. it is almost certain that there is a correlation between the two distributions. most workers whose health is good or very good think that work does not affect their health (55%, resp. 82%). those workers whose health is bad or very bad generally find (53%, resp. 79%) that their work affects their health negatively. if work did not have a strong influence on health, this polarization would be smaller. the last statement is further supported by the following correlation (p = 0.03): the longer a worker is absent from work for reasons of health problems, the greater the probability that work affects his or her health negatively. only 4% of the workers who were absent from work for reasons of health problems reported that this was due to an accident at work. the last group is further divided as follows:  about 29% had a minor health problem (up to 10 days of absence). most of them thought that their work affected their health positively. nobody reported a negative influence of work.  about 71% had a major health problem (over 10 days of absence); 88% of them thought that their work affected their health negatively. nobody reported a positive influence of work. a strong polarization in workers’ answers is noticed again. it suggests that most health problems are work-related. to sum it up, there is strong evidence that most health problems are work-related. indeed, a lot of correlations were discussed in the previous sections. they can be used now as an explanatory basis for social phenomena or as a set of criteria to estimate efficiency of legal measures. 4. conclusions work affects health very strongly. actually, work (together with age) is one of the most important determinants of human health. overall fatigue, headache, eyestrain, backache, muscular pains — these are the most frequent health problems in bulgaria. all of them are work-related. factors of the working environment that have a strong negative influence on health are: moving heavy loads or people, standing upright, repetitive hand or arm movements, contact with chemical products or infectious materials, breathing in smoke (incl. tobacco smoke), fumes, powder, dust or vapours such as solvents and thinners. working at night, working more than 10 hours a day and working at a very high speed more than 1/4 of the time affect health negatively. creating legal conditions for a safe and healthy working environment is a constant concern of every government. employers are required to take preventive measures and employees are expected to strictly follow these regulations. special attention should be paid to avoid overstrain and protect workers exposed to the adverse factors listed above. it is of prime importance that workers should be kept very well-informed about all health and safety risks and use their personal protective equipment whenever required. precise quality standards, modern technologies and new processes should be widely introduced where possible because of their strong positive influence on health. 5. acknowledgements the authors would like to thank the european foundation for the improvement of living and working conditions and the uk data archive for providing the initial data. food and environment safety – journal of faculty of food engineering, ştefan cel mare university – suceava volume xii, issue 4 – 2013 dobromir kralchev, donka taneva, tsvetko prokopov, effects of work-related factors on workers’ health, volume xii, issue 4 – 2013, pag. 332 – 341 341 6. references [1]. international labour office, geneva. ilo standards on occupational safety and health. (promoting a safe and healthy working environment.) geneva, ilo publications, 2009. [2]. national social security institute of bulgaria. work-related accidents, 2010. (in bulgarian.) from: http://www.noi.bg. [3]. european agency for safety and health at work. european survey of public opinion on occupational safety and health, 2009. from: http://osha.europa.eu/en/safety-health-in-figures/ eu-poll-slides-2009/package_bulgaria.pdf. [4]. consortium “national working conditions survey”, sofia. national survey of working conditions in bulgaria, 2011. from: http://bilsp.org/ documents/547_2_final_report_nwcs-bg%20 pages_1-201.pdf. [5]. european foundation for the improvement of living and working conditions, european working conditions survey, 2010 [computer file]. colchester, essex: uk data archive [distributor], february 2012. sn: 6971. 138 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag.138 143 assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania *maria-alexandra hoaghia1,2, erika levei1, cecilia roman1, marin senila1, claudiu tanaselia1, dumitru ristoiu2 1 incdo-inoe 2000, research institute for analytical instrumentation, 67 donath, ro-400293 cluj-napoca, romania 2 babeş-bolyai university, faculty of environmental science and engineering, 30 fantanele, ro-400294 clujnapoca, romania, alexandra.hoaghia@icia.ro *corresponding author received april 24th 2015, accepted june 16th 2015 abstract: the levels of inorganic contaminants in well waters used as drinking water sources from urban and nonferrous mining areas were studied. well water samples from mining area are circumneutral and present high concentrations of metals (1.0-132 µg/l as, 20-73 µg/l cr, 4.0-272 µg/l mn), in some samples exceeding the maximum admissible concentrations set by romanian law 311/2004, regarding the drinking water quality, while well water samples collected from urban area are circumneutral to alkaline and show high nitrates (31-271 mg/l), nitrites (0.1-3.3 mg/l) and sulphates (51-245 mg/l), but low metal concentrations. the metal contamination reflects the influence of nonferrous mining activities, while household activities and agriculture imprint high values of no2-, no3and so42-. keywords: well water, inorganic contaminants, mining area, urban area, metal contamination, nitrite, nitrate. 1. introduction water is an essential resource for maintaining life. well waters sources are used as drinking water sources in areas with lack of other fresh water sources or water distribution supply. the main pollution sources of well waters are anthropogenic activities, such as mining activities (exploiting and metallurgy), industry, household (dry toilets, collecting tanks), impairment of wastewater treatment systems (sewage discharges) and agricultural practices (use of fertilizers, manure deposits) [1-3]. nitrate and nitrite contamination of well water is found in many urban areas, because of leak from sewage effluent, dry toilets, collecting tanks, cattle manure [4, 5], while metal contamination of well waters is determined mostly by mining activities. drinking water contamination by mining activities represents a significant problem worldwide [6, 7]. in african countries, such as ghana, tanzania the mining activities impact on water resources quality is serious; well waters being unsuitable to be used as drinking water due to their high toxic metal contents [8]. asian countries http://www.fia.usv.ro/fiajournal mailto:alexandra.hoaghia@icia.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu, assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania, food and environment safety, volume xiv, issue 2 – 2015, pag.138 – 143 139 (china, india) are also confronting with exposure to toxic metals by well waters near mining areas [2, 9]. in romania the mining activities determined local pollution of aquatic systems, especially in the north-western part of the country [10, 11]. the aim of the present study is to assess and compare the inorganic contaminants of well water from nonferrous mining (tarna mare, turulung and turt, satu mare county) and urban areas (medias, sibiu county). 2. matherials and methods 2.1. study area and sampling the studied mining area is localized near oas mountains, part of the north-western volcanic chain, romanian carpathian. the area is nearby (about 50 km) baia mare, the main centre for nonferrous metallurgy and mining (cu, zn, pb, au, ag) and metals pollution “hot spot” [12-14]. the nonferrous complex ores are exploited from three main ore deposits (ghezuri, penigheri-turt and socea-tarna mare) [15]. ghezuri and penigheri-turt mines are localized near turt village, 20 km away from satu-mare. socea turt mine is situated near tarna mare, and 8 km north west from turt village [16]. turulung is situated at 8 km south-west from turt and 16 km south from tarna mare. inhabitants of the three small localities use well water as drinking water source, because of lack of other fresh water sources. mining activities started in 1973, as modern exploiting; but since middle ages ores containing precious and nonferrous metals were exploited from the volcanic oas mountains [16]. surface water pollution assessment studies of the area indicate acidic ph and pollution with highly toxic elements as as, cd, pb, mn and zn [11]. the studied urban area is medias, a medium size town situated in the northern part of sibiu county, in central romania. although the town has water supply system, not all households have access to it. thus, private water wells are used as drinking water supply. from the nonferrous mining area five well water samples (m1-m5) were collected from tarna mare, one sample (m6) from turt and two (m7, m8) from turulung. nineteen well water samples (u1-u19) were collected from urban area, medias. all samples were collected in august 2014 in clean 500 ml bottles and stored at 4 °c until analysis. the ph was measured on site and samples were filtered using 0.45 µm acetate cellulose membrane filters. the anions were measured directly in filtered samples, while for the determination of metals; samples were and acidified to ph ≤ 2 with 63% hno3. 2.2. instrumentation elements (cr, as, zn, mn) were measured using inductively coupled plasma mass spectrometry (icp-ms) using an elan drc ii (perkin-elmer, canada) spectrometer. the content of no2-, no3 and so4 2were analysed by ion chromatography using a 761 compact (methrom a.g., switzerland) ion chromatograph. the ph was measured by a 350i multiparameter (wtw, germany). 3. results and discussion the ph value of well water samples was circumneutral in the mining area and circumneutral to alkaline in the urban area. mean ph values for water samples from the mining area was 6.73, while that from the urban area was 7.42. the so42 concentrations for all well water samples were below acceptable limits set by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu, assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania, food and environment safety, volume xiv, issue 2 – 2015, pag.138 – 143 140 romanian law 311 [17], with highest values for urban well waters (figure 1). fig.1 the ph values and so4 2-, no2 -, no3 concentrations in well water samples there are several regulations that set guideline values for drinking waters, such as the romanian law 311/2004, who (world health organization), and u.s. epa (environmental protection agency), guidelines [17-19]. the source of nitrites and nitrates could be the collecting tanks, dry toilets, cattle manure localized near water wells used as drinking water source. except five samples (u4, u11, u14, u15, u18) all water samples from medias exceed mac (maximum admissible concentration) for no2-, while only one water sample from mining area (m7 from turulung) exceeds the mac (figure 1). table 1 measured and guideline concentrations of inorganic contaminants in studied well water samples used as drinking water chemical parameters measured concentrations mac guideline threshold min sample max sample law 311/2004 who u.s. epa ph ph units 6.27 m6 7.81 u18 6.5-9.5 6.5-8.5 6.5-8.5 as µg/l <0.8 u2-u4, u6, u12, u17-u19, m3-m6 132 m8 10 10 10 cr 0.90 u12 73 m2 50 50 100 mn <1.0 u2, u9,u18, m5 272 m1 50 50 zn 7.1 u3 549 m6 5000 5000 no2mg/l <0.05 u4, u14 3.3 m17 0.5 3.0 1.0 no30.92 m2 271 u10 50 50 10 so4213 m1 245 u18 250 0 1 2 3 4 5 6 7 8 9 m 1 m 2 m 3 m 4 m 5 m 6 m 7 m 8 u 1 u 2 u 3 u 4 u 5 u 6 u 7 u 8 u 9 u 10 u 11 u 12 u 13 u 14 u 15 u 16 u 17 u 18 u 19 ph (ph units) sample points 0 50 100 150 200 250 300 m 1 m 2 m 3 m 4 m 5 m 6 m 7 m 8 u 1 u 2 u 3 u 4 u 5 u 6 u 7 u 8 u 9 u 10 u 11 u 12 u 13 u 14 u 15 u 16 u 17 u 18 u 19 so42(mg/l) mac sample points 0 0.5 1 1.5 2 2.5 3 3.5 m 1 m 2 m 3 m 4 m 5 m 6 m 7 m 8 u 1 u 2 u 3 u 4 u 5 u 6 u 7 u 8 u 9 u 10 u 11 u 12 u 13 u 14 u 15 u 16 u 17 u 18 u 19 no2(mg/l) mac sample points 0 50 100 150 200 250 300 m 1 m 2 m 3 m 4 m 5 m 6 m 7 m 8 u 1 u 2 u 3 u 4 u 5 u 6 u 7 u 8 u 9 u 10 u 11 u 12 u 13 u 14 u 15 u 16 u 17 u 18 u 19 no3(mg/l) mac sample points food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu, assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania, food and environment safety, volume xiv, issue 2 – 2015, pag.138 – 143 141 the no3concentrations are higher for well water samples (range 31 271 mg/l, mean 101 mg/l) from the urban area than for well water samples (range 0.92 50 mg/l, mean 22 mg/l) from the mining area (figure 1). the elements considered in the current study include as, cr, mn and zn. the elements content for well water samples from nonferrous mining area present higher concentrations that those from the urban area. as, cr and mn concentrations in waters from mining area exceed the macs set by drinking water guidelines [17 19] while those from urban area do not exceed the corresponding macs (table 1). chromium concentrations ranged between 0.90 µg/l and 73 µg/l. well water samples from medias present lower cr concentrations than the well water samples from turulung and tarna mare. one water sample (m2) from the mining area shows contamination with cr, exceeding mac, while other seven samples indicate cr concentrations between 20 µg/l and 42 µg/l (figure 2). fig. 2 cr, as, mn and zn concentrations in well water samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu, assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania, food and environment safety, volume xiv, issue 2 – 2015, pag.138 – 143 142 the as content in four well water samples (m1, m2, m7 and m8) were found to be more than ten times higher than the corresponding, while samples from medias are lower than mac. studied well water samples present mn contamination, two well water samples (u11 and u12) from medias, three well waters from tarna mare (m1, m2, m4) and one sample m7 from turulung exceeding the mac (figure 2). the zn concentrations do not exceed mac, as shown in figure 2. samples from medias show generally higher zn concentrations (mean 207 mg/l) than samples from mining area (mean 97 mg/l as). source for high zn concentrations could be a metallurgical facility (zn, pb, sn) localized at about 13 km from medias. 4. conclusion growing population and anthropogenic activities (mining, industry, agriculture and household) imply interference with the natural ecosystems and influence the well water quality used as drinking water source. in the current study one was found that no2-, no3-, so42-, and as, cr, mn concentrations exceed the limits set by national and international drinking water guidelines. well water samples from turulung and tarna mare present higher cd, as and mn concentrations than the well water samples from medias. the ph values, measured for well water samples from the nonferrous mining area, indicate circumneutral waters, while well water samples from the urban area are characterized by circumneutral to alkaline ph. however, no2 -, no3 and so4 2 concentrations are significantly higher compared with the values measured in the turt region, especially no2-, no3values which are noteworthy higher as mac. 5. acknowledgments this paper is a result of a doctoral research made possible by the financial support of the sectorial operational program for human resources development 20072013, co-financed by the european social fund, under the project posdru / 159 / 1.5/s/133391 “doctoral and postdoctoral excellence programs for training highly qualified human resources for research in the fields of life sciences, environment and earth”. 6. references [1] kostyla c., bain r., cronk r., bartram j. seasonal variation of fecal contamination in drinking water sources in developing countries: a systematic review, science of the total environment, vol. 514, 333-343 (2015). [2] prasad b., kumari p., bano s., kumari s. groundwater quality evaluation near minig area and development of heavy metal pollution index, applied water science., vol. 4, 1117 (2014). [3] briciu a.e., oprea-gancevici d., the effect of rural settlements on water quality in northern suceava plateau, food and environment safety, vol. 10, 2, 88-96, (2011). [4] widory d., kloppmann w., chery l., nonnin j., rochdi h., guinamant j.l. nitrate in groundwater: an isotopic multi-tracer approach, journal of contaminant hydrology, vol. 72, 1-4, 165-188 (2004). [5] nola b.t., ruddy b.c., hitt k.j., helsen d.r. a national look at nitrate contamination of groundwater, water conditioning and purification magazine, vol. 39, 12, 76-79 (1998). [6] armendariz-villagas e.j., covarrubias-garcia m.a., lagunes e., arreola-lizarraga a., nieto-garibay a., beltran-morales l.f., ortegarubio a. metal mining and natural protected areas in mexico: geographic overlaps and environmental implications, environmental science and policy, vol. 48, 9-19 (2015). [7] lopez-merino l., cortizas a.m., reher g.s., lopez-saez j.a., mighall t.m., bindler r. reconstructing the impact of human activities in a nw iberian roman mining food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 maria-alexandra hoaghia, erika levei, cecilia roman, marin senila, claudiu tanaselia, dumitru ristoiu, assessment of inorganic contaminants in well waters from nonferrous mining and urban areas in north-west and central romania, food and environment safety, volume xiv, issue 2 – 2015, pag.138 – 143 143 landscape from the last 2500 years, journal of archaeological science, vol. 50, 208-218 (2014). [8] tay c. and momade f. trace metal contamination in water from abandoned mining and non-mining areas of the northern parts of the ashanti gold belt, west african journal of applied ecology, vol. 10, 187–207 (2006). [9] cai l.m., xu z.c., qi j.y., feng z.z, xiang t.s. assessment of exposure to heavy metals and health risks among residents near tonglushan mine in hubei, china, chemosphere, vol. 127, 127-135 (2015). [10] lacatus r. contributions regarding heavy metals flow within soil-plant-animal system in polluted areas, acta metallomica-metal elements in environment, medicine and biology, vol. 1, 73-88 (2014). [11] nagy i., fodorpataki l., weiszburg t., bartha a. preliminary results on environmental impact of mining activity on the turt creek, satu mare county, romania, flora si fauna rezervatiei naturale “raul tur” / eds. sike t., mark-nagy j.), university of oradea publishing house, 17-26 (2008). [12] cordos e.a., frentiu t., rusu a.m., vatca g. elemental speciation of pb, zn and cu in sedimented dust and soil using a capacitively coupled atomic emission spectrometer as detector, analyst, vol. 20, 725 (1995). [13] frentiu t., ponta m., anghel s.d., simon a., cordos e.a. cadmium determination in sedimented dust by atomic emission spectrometry with a new radiofrequency capacitively coupled plasma source, analytical letters, vol. 33, 2, 323-335 (2000). [14] cordos, e., rautiu, r., roman, c., ponta, m., frentiu, t., sarkany, a., fodorpataki, l., macalik, k., mccormick, c., weiss, d. characterization of the rivers system in the mining and industrial area of baia mare, romania, european journal of mineral processing and environmental protection, vol. 3, 3 324–335 (2003). [15] kovacs m., edelstein o., gabor m. neogene magmatism and metallogeny in the oasgutain-tibles mts.; a new approach based on radiometric datings, romanian journal of mineral deposits, vol. 78, 35-45 (1997). [16] clain e., haake r. the lead-zinc site from turt, oas mountains, romania (die bleizink-lagerstatte von turt, oasgebirge, rumanien), mineralien-welt, vol. 17, 5, 52-64 (2006). [17] law 311 from 6rd june 2004 that improves and complement 458 law from 29 july 2002 regarding the quality of drinking water. official gazette 2004, part i, no. 582/30.06.2004 [in romanian]. [18] guidelines for drinking water quality, fourth edition, world health organization, 2011. accessed online at 04 march 2015 from: http://www.who.int/water_sanitation_health/publica tions/2011/dwq_guidelines/en/ [19] u.s. environmental protection agency (epa) national primary and secondary drinking water regulations (npdwrs and nsdwrs or secondary standards): guidance for nuisance chemicals, 2006. accessed online at 04 march 2015 from: http://water.epa.gov/drink/contaminants/#lis. http://www.who.int/water_sanitation_health/publica http://water.epa.gov/drink/contaminants/#lis. alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 192 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 192 206 an interdisciplinary approach on the influence of packaging design on emotional response to chocolate alice-iuliana roșu1, *cristina-elena hrețcanu1, ana leahu1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania alicer@fia.usv.ro, *cristina.hretcanu@fia.usv.ro , analeahhu@fia.usv.ro *corresponding author received april 15th 2013, accepted 18th may 2013 abstract: emotions have been generally associated with food products but there has been little research on emotions related to chocolate package. culturally, emotions may be described in different ways and every culture may have its own sensory lexicon, giving the possibility for feelings to be socially shared. by means of three analysessensory, semiotic and statistical – we designed 6 types of chocolate packages which allow chocolate consumers to describe their feelings while watching or consuming chocolate. the objective of this paper was to study the influence of packaging design of chocolate in attracting consumers’ attention and generating hedonic and sensory expectations from a semiotic, statistical and sensory point of view. to demonstrate these, six images of chocolate packages with different design and color were presented to 123 consumers, who were asked to complete a questionnaire and to score their expected liking as against the chocolate presented. the first part of practice is given in experiment 1 and is referred to the perception of its image resulting from the statistical study conducted using a questionnaire applied to a number of 123 people. in the second practice the sensory analysis was performed for six kinds of chocolate: mint chocolate, milk chocolate, dark chocolate with coffee, dark chocolate with orange, luxurious chocolate and white chocolate, using 12 tasters. key words: word association, hedonic and sensory expectations, semiotic analysis 1. introduction word association can be used to study the influence of package design (shape, color, images) on consumer expectations of desserts, which could affect their product perception and purchase decisions [1]. visual perception of a product packaging image conducts to symbolic meanings associated with sensorial characteristics of a food product [2]. one can affirm that the design packaging has a semantic symbolism which contributes to create a given expectancy about the product [3]. nowadays we can remark a growing interest on the study of the emotional response to foods, due to both the potential for emotional responses to provide important information about products and differences between a set of products that go beyond traditional sensory and liking food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 193 variables. in many academic papers, questionnaire approaches have been used to assess emotional responding to foods by feelings and emotion related terms that can be used [4]. jaeger & cardello [4] use the term emotion words to describe emotions (fear, anger, shame, jealousy, sadness, energetic, alert, stimulated, etc.), feelings (inspiration, pity, respect, disappointment, etc.), personal characteristics (selfconscious, unfulfilled, helpless, encouraged, etc. [5]), personality based terms (friendly, warm [6]) or masculine and sociable [7]), as well as terms for image-based characteristics of products (mild, free and whole [6] or comforting and luxurious [7]) or associations that people may have in mind when responds to labels, images, on packaging of a food product. managing sensory expectations concerning products and brands, charles spence, ([3], [9], [10]) discussed in his paper about multisensory perception relates to the topic of crossmodal correspondences (e.q. a tendency for a feature, or attribute, in one sensory modality to be matched (or associated) with a sensory feature, or attribute in another sensory modality). he remarks that knowing about shape symbolism effects in the food sector may allow sensory marketers to set-up the appropriate associations in the minds of consumers through the shapes used on the packages. gastón ares et al. states in the paper [11] that “sensory expectations are related to consumers’ beliefs about the sensory characteristics of the product, whereas hedonic expectations refer to how much the product will be liked or disliked. if the sensory and hedonic expectations shaped by the packaging are high, consumers may be interested in the product and would choose to buy it. “ the objective of this paper is to demonstrate the influence of packaging design of chocolate in attracting consumer attention and generating hedonic and sensory expectations. talking about chocolate packages, some researchers remark that people match dark chocolate with angular shapes and milk chocolate with rounded shapes [9]. 2. material and methods chocolate is a complex multi-stage particle (sugar, cocoa, some components of the milk) with a continuous phase (cocoa butter, milk fat and emulsifiers). its production and refining determine the particle size, consistency and viscosity, leading to specific textural and sensory characteristics. depending on cocoa content, the main types of chocolate are: white chocolate, milk chocolate and dark semi bittersweet chocolate. these types of chocolate may be produced by conventional cocoa beans (product of mass and cost) and the specialty of cocoa beans (aromatic and expensive), or a mixture of these two types. mixture composition, origin cocoa beans, and roasting treatment, and the types and quantities of additives used will significantly affect the final chocolate taste and price. the first part of practice is given in experiment 1 and is referred to the perception of its image resulting from the statistical study conducted using a questionnaire applied to a number of 123 people from suceava romania and the surrounding villages of suceava, aged between 14 and 25 years, undergraduate students or graduated students of the faculty food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 194 of food engineering, stefan cel mare university of suceava, and students from ciprian porumbescu high-school of suceava). 2.1. experiment 1: questionnaire approaches have been used to assess emotional responding to seeing the design of chocolate packages. the questionnaire was based on 6 images describing six types of chocolate. the purpose of this questionnaire was to identify consumers’ perception (thoughts, feelings, associations, words, descriptions) on the image of chocolate; frequency of chocolate consumption; what color is associated with chocolate; preference on packaging; preference on the type of chocolate (of 6 types); type of chocolate packaging association. six simulated chocolate labels were designed by the authors. the objective was to create six chocolate labels capable of conveying some distinct messages. the labels comprised different combination of non-verbal elements. the labels were used as stimuli for a consumer survey, as in the paper [11]. the six designed model labels are displayed in figure no. 1. (a) (b) (c) (d) (e) (f) figure 1. model labels of chocolate used in the study. (a) chocolate 1; (b) chocolate 2; (c) chocolate 3; (d) chocolate 4; (e) chocolate 5; (f) chocolate 6. 2.2. experiment 2: sensory profiling in the second practice the sensory analysis was performed for six kinds of chocolate: mint chocolate, milk chocolate, dark chocolate with coffee, dark chocolate with orange, luxurious chocolate and white chocolate. this study emphasizes on one hand the great popularity of this product related to its high consumption and on the other hand how consumers’ perception of six types of chocolate packages can be statistically and semiotically analyzed to assess emotional responding to foods by feelings and emotion related terms. the sensory analysis was performed using 12 tasters (undergraduate students of the ` food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 195 faculty of food engineering, stefan cel mare university of suceava – romania). experiment 2 was performed in two stages, between 10-12 am and 15-17 in the afternoon, and the meeting time last for more than 2 hours. consumers were told to take the portion of chocolate served and let it melt on the tongue some seconds. after tasting each sample, an approximately 3 minute –break was taken. chocolate mass is 100 g and its caloric content (as may be observed in table 2) varies depending on the type of chocolate. thus, a chocolate may have 400 kcal and 550 kcal per 100g based on its contents. table 2. the nutritional value of the six types of chocolate type of chocolate energeti c values kcal and kj pro tein s (g) in 100 g car boh ydr ates (g) in fats (g)1 in 100 g 00g conte nt of cocoa (%) mint chocolate 449 kcal 1887 kj 4.00 70.0 17 .0 51% milk chocolate 530 kcal 2225 kj 6.60 58.5 29.5 30% dark chocolate with coffee 547 kcal 2289 kj 6.12 46.8 37.9 > 50% dark chocolate with oranges 529 kcal 2214 kj 6.93 48.4 36.7 > 50% white chocolate 540 kcal 2255 kj 4.80 63.0 29.5 0% dark chocolate with almonds 552 kcal 2302 kj 8.10 45.0 36.3 > 47% the type of chocolate used in the study and ingredients are: sample 1 mint chocolate (40% cream mint, cocoa-butter, emulsifier: soya lecithin and e 476, oil-mint, vanilla flavor), sample 2 milk chocolate (sugar, cocoa butter, skimmed milk powder, cocoa mass, whey powder, milk fat, soy lecithin), sample 3 dark chocolate with coffee (sugar, butter and cocoa mass coffee powder 2%, emulsifier: soy lecithin, flavors, natural vanilla extract and espresso), sample 4 dark chocolate with orange (sugar, cocoa mass and butter, candied orange peel 10%, glucose syrup, emulsifier: soy lecithin, natural vanilla extract and orange), sample 5 dark chocolate with almonds (sugar, cocoa mass, almonds 20%, cocoa butter, milk fat, flavor) and sample 6 white chocolate (sugar, cocoa butter, whole milk powder, whey powder, fat soybean, soybean lecithin, flavor). the sensory appreciation of food is made with five analyzers with which man is endowed by nature: taste, smell, sight, hearing, and tactile (touch) [17-21]. the analyzer consists of three segments: peripheral segment (includes the receiver and is designed to receive specific stimuli and in turn excitations), management segment (represented by those nerves leading to excitation of the sensory cortex center) and the central segment (represented by a certain place of the cerebral cortex that transforms stimuli received into sensations). the technical equipment consists of: sample preparation room equipped with necessary appliances; the tasting room separated from the training room should be sound and heat proofed, provided with air conditioning system. tasting room is equipped with: glass or earthenware crockery. tasters were selected so as to meet certain physiological and psychological conditions, respectively, to avoid factors that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 196 may influence negatively the well functioning of sensory analyzers, as well as attention and concentration. the sensory analysis method applied in this study was the method of quality assessment by scoring the responds from 1 to 4 (as one can see in table 3). table 3 scores of chocolate sensory quality ([16]) sensory characteristics description of the products examined scores accorded general appearance smooth, contoured designs. 4 the surface is slightly matt 3 show small irregularities of form and design 2 matt surface with faint spots and drawings 1 color uniform feature 4 too intense 3 too weak 2 presents uncharacteristic shades 1 odor well-defined characteristic 4 highly expressed 3 less clear 2 foreign uncharacteristic 1 taste pleasant, typical assortment 4 very sweet 3 poor bitter 2 unpleasant, uncharacteristic 1 flavor specific to chocolate 4 highly expressed 3 poorly expressed 2 missing, uncharacteristic 1 texture creamy 4 sticky 3 oil 2 brittle 1 tasters’ choice was analyzed to assess their sensory abilities and tasting experience and their training as well. tasters did not feel hungry an hour before tasting, so they did not eat or drink while the test was being carried out, except for water, were not allowed to smoke or use perfumes, deodorants and cosmetics that might affect the sensory analysis. to remove the residual taste, drinking water has been used. samples were prepared in the same manner for all tasters and were distributed in equal amounts in the same dish. the temperature at which the examination was made was identical in all samples analyzed ranging between 20-24 celsius degrees. the team leader presented the chocolate samples analyzed, purpose, sensory characteristics to be tested, the order and manner in which the analysis was to be made. 3. results and discussion 3.1 experiment 1 the analysis of the demographic characteristics of the respondents revealed some very useful conclusions for the study under analysis: 65 respondents living in urban areas and 58 living in rural areas, the sex ratio of respondents is 86 females and 37 males, having an average education level: 43 high school students, 57 under graduate university students and 23 graduate university students (as one can see in the figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 197 (a) (b) figure 2. respondents’ demographic characteristics: (a) urban / rural; (b) men / women the answers to the question: what color can be generally associated with chocolate? are shown in the figure 3: figure 3. color association with chocolate labels shown in fig. 1 “colors in a food label are usually used to differentiate between products and make them more attractive to consumers. attractive colors are able to catch consumers’ attention even when seen from a long distance” [11] according to the results in figure 3, chocolate is associated by most people with brown (46%) and black (20%). a low percentage is held by "other colors" such as pink, purple, red, green and yellow, the last one being especially chosen by students who associate chocolate color with cream. the results of the semiotic analysis of the images of the six chocolate labels are presented bellow:  chocolate 1 the main image of the label referred to a dark chocolate with mint. the feelings and emotions conveyed by mint leaf are associated to calm, tranquillity and freshness. the dark from the fundal may suggest the idea of an elegant and luxurious product [11]  chocolate 2 the visual perception of this label is mainly influenced by some chocolate types similar to milka © brand with strawberry. the background color is lilac, generally associated with femininity, dreamlike states and relax [12]; what draws the viewer’s attention at first sight is the plastic sign of heartshaped and red-colored strawberries. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 198 they are considered a symbol of venice, the goddess of love, of purity and sensuality, of fertility and abundance. therefore, the perception mechanism and the powerful message conveyed by strawberries can be easily deciphered by the semiotic analysis of these iconic signs: if one tastes this particular type of chocolate, they will feel like a goddess, happy and loved.  chocolate 3 – the viewer’s attention is caught by a white cup of coffee, “surrounded” by coffee beans, centrally placed, based on a strong chromatic contrast between white and brown color, conveying the idea of special moments somebody can share with. coffee beans function as strong smell and taste stimuli, being associated with dynamism and energy. moreover, they have a double function: from the hedonic point of view, coffee -chocolate stands for the “perfect match”, the ultimate taste combination, and semiotically they work as indices for taste and ingredients. all these non-verbal iconic elements speak for themselves, conveying the message that it is a refreshing dark chocolate which signifies invitation, special and pleasant moments to share with, friendship, etc.  chocolate 4the image is based on a strong color contrast between black and orange, inferring the idea the product would have a positive effect on health, taking into consideration the ingredients used oranges and a high content of cocoa. orange functions semiotically from a double perspective: visuallythe color itself and olfactory the fruit. as such, orange is considered one of the healing colours. it is believed to increase the craving for food. from this point of view it works as a strong stimulus. moreover, the plastic sign of orange color and fruit corresponds to desire, pleasure, domination, optimism, stimulating enthusiasm and creativity. the use of orange color in the word chocolate written with special orange shade characters, as against a dark background, emphasizes once more the high quality sensory characteristics of this product. orange communicates optimism and energy [11]  chocolate 5 the use of a nonconventional image (rings have been used throughout history on many levels to convey a similar message to that signified by the wedding ring, generally being perceived as a marriage proposal, commitment, used on special happy occasions) conveys the message that it is an authentic and elegant product, meant to a targeted group of people. rings stand for high esteem, whether that be wealth, marriage or power, being a universally understood symbol. in this context, it carries respect, authenticity, high quality product.  chocolate 6 the focus in this image goes straight to the beautiful white cherry flower, conveying freshness, naturalness, calm, relaxation, peace and purity. the background is white, the name of the product written in pink, stylised characters, reinforce the idea of pure and natural, light or low-calorie product [11]. as regards the cultural significance of cherry blossom, it symbolizes friendship and transience of human life. in this context, it stands for a high quality product, with special nutritional values. word association – the answers to the question: what are the first words, descriptions, links, thoughts or feelings that come to your mind when you look at these chocolate images? are shown in figure 4. the results of word associations made by studying the six types of chocolate labels are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 199 expressed by: feelings, descriptions, at the morphological level by positive adjectives and nouns; most people associated the chocolate image with the sensations caused iconic signs: fruit, colors, characters, and ways of spelling, as you can see in the figure 4. figure 4. word association with chocolate labelling models the answers to the question what type of chocolate do you associate the following design package with?are shown in figure 5. thus, one can see that the package 5 (a) is associated by a large number of respondents with bittersweet chocolate filled with mint, whereas the chocolate package 5 (b) is mainly associated with milk chocolate type and filled with strawberry or white chocolate type, the package 5 (c) is associated with dark chocolate and filled with coffee, the package 5 (d) is associated predominantly with dark chocolate filled with orange cream, the package 5 (e) is associated mostly with aerated chocolate type with milk and bittersweet, as a luxury food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 200 chocolate package and the package 5(f) is associated by most people with white chocolate with milk respectively. (a) (b) (c) (d) (e) (f) figure 5. types of chocolate associated with design packages food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 201 the hedonic scores regarding the six chocolate labels (figure 1) is shown in figure 6. figure 6. classification of appreciations on chocolate packages’ design the chocolate 2 (fig. 1 (b)), which displays a similar background with the milka chocolate brand, was the most appreciated (by 36 respodents). means scores given by people participating in the study can be seen in the table 1. using the one way anova, we obtained the values of fisher ratio f= 0.22 < f critical = 3.07 (p=0,79 > 0.05) thus we can affirm that there are significant differences between the 3 groups of people (high school students, undergraduate and graduate student, respectively) in the way of appreciation the design of the six chocolate packages (presented in fig. 1). table 1 the overall assessment of chocolate packages data are presented as mean ± standard deviation. the same letters within a row indicate no significantly different among means (p < 0.05). the biplot association for the hedonic scores regarding the six chocolate labels (from figure 1) is shown in figure 7. the biplot (proposed by gabriel (1971; 1981)) [12 15] displays the observations and variables in the same plot, in a way that depicts their joint relationships. the biplot data result in a matrix, y (of order n x m ), that can be represented in 2 dimensions ( or 3 ) as the product of a two matrices, a ( n x 2 ), and b ( m x 2 ), where model labels of chocolates used in the study mean scores given by: total mean scores standard deviation of total mean scores high school students under graduate university students graduate university students (a) chocolate 1 7.23a ± 2.26 8.05a±1.96 7.13a±2,00 7.47 0.51 (b) chocolate 2 8.07b± 2.27 8.12b±1.69 7.39b±2,51 7.86 0.41 (c) chocolate 3 7.81c± 2.40 8.05c±1.96 8.39c±2,18 8.09 0.29 (d) chocolate 4 7.95d±1.96 7.48d±2.20 7.17d±2,36 7.54 0.39 (e) chocolate 5 8.09e±1.87 8.12e±1.95 8.09e±1,83 8.10 0.02 (f) chocolate 6 7.67f±2,04 7.74f±2.19 8.04f±2,45 7.82 0.20 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 202 the rows of a represent the observations (the scores – from 1 = minimum to 10 = maximum) given by people in a 2 dimensional space, and the columns of b represent the variables (the six labels of chocolates proposed in the study). figure 7. the biplot association of the hedonic responses regarding chocolates’ labels the configuration of points is essentially as scores the first two principal components. the variables are plotted as vectors from the origin. the angles between the vectors represent the correlations among the variables [12]. 3.2 experiment 2. chocolate has the following quality features [16]: appearance (exterior gloss surface without blemishes, scratches or air pockets (bubbles), interior unfilled chocolate mass to be homogeneous, matte (not glossy) without air bubbles, chocolate filled, filling or addition is evenly distributed and have homogeneous consistency), color (uniform variable depending on composition, light brown to dark brown); consistency (relatively hard and brittle at break); taste and odor (pleasant, aromatic, characteristic chocolates without foreign taste or odor) and, respectively texture (should give the feeling of oily product, does not cause teeth grinding). the representative features of the analysis are: general appearance, color, odor, taste, aroma and texture (at a temperature of 20 ± 2 ˚ c). the sensory profiles of the 6 types of chocolate studied are shown in figure 8: (a) (b) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 203 (c) (d) (e) (f) figure 8. sensory profiles of the six types of chocolate as we can see in figure 8, from the analysis of the six types of chocolate studied using six sensory descriptors: appearance (general aspect), color, odor, taste, and flavor and, texture respectively, we obtain the following remarks: • the general appearance has a variation scored between 3.08 to 3.83, which means that the six types of chocolate describe a smooth, slightly matt, embossed clear-cut (significant differences for the 6 types of chocolates in terms of assessing the overall appearance is determined using one-way anova method, fisher ratio f = 2.36> f critical = 2.35 and the probability of error is p = 0.0487 < 0.05 = α); • the variation of the color scores is between 3.00 to 3.83, which means that the six types of chocolate describes a solid color, but some of their assortment characteristics such as the color of mint chocolate is too high (no significant differences for the 6 types of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 204 chocolates in terms of color is determined using one-way anova method, fisher ratio f = 1.72 < f critical and the probability of error is p > 0.05); • the variations of the odor scores are from 2.50 to 3.50, which means that the 6 types of chocolate describe odor varied from specific odor to the strong odor characteristic assortment expressed (in the case of chocolate mint) specified in less odor as if white chocolate, (significant differences for the 6 types of chocolates in terms of odor is determined using one-way anova method, fisher ratio f = 2.56 > f critical and the probability of error is p = 0.0351 < 0.05); • taste has a variation scored between 2.67 to 3.67, which means that the six types of chocolate described taste varies from pleasant taste, characteristic types, to a very sweet taste (for example milk chocolate) and the weak bitter taste as house coffee or chocolate (significant differences for the 6 types of chocolates in terms of taste is determined using one-way anova method, fisher ratio f = 2.78 > f critical = 2.35 and the probability of error is p = 0.024 < 0.05=α); • flavor has a variation scored between 2.75 to 3.58 which means that the six types of chocolate described a specific flavor of the product (no significant differences for the 6 types of chocolates in terms of flavor is determined using one-way anova method, fisher ratio f = 2.14 < f critical and the probability of error is p > 0.05); • texture has a variation scored between 1.58 to 3.67 which means that the six types of chocolate describe the texture varies from brittle (in the case of dark chocolate coffee), an oily texture (in the case of milk chocolate) a sticky texture (white chocolate), chocolate with a creamy texture and luxury chocolate mint filling, so in terms of texture are significant differences (f=9,49 > f critic = 2.35). from the figure 9, one can remark that sample 1 and sample 5 obtained more than 20 points in assessment session by scoring sensory quality chocolate. by comparing the 6 types of chocolates in the experiment 2, the most preferred chocolate types were: dark chocolate with mint, followed by dark chocolate with almond (luxurious chocolate). figure 9. total scores resulted from assessment by scoring sensory quality chocolate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 205 4. conclusions this research based on an interdisciplinary approach – sensory, statistical and semiotic shows that people, if properly questioned, are capable of identifying a circumscribed range of terms within the sensory lexicon that describe emotional aspects elicited by chocolate. such an analysis emphasizes how important is the influence of food package design (shape, color, images) on consumers’ expectations, which could affect their product perception and purchase decisions. visual perception of a product packaging image conducts to symbolic meanings associated with sensorial characteristics of a food product. the sensory analysis reveals itself to be particularly helpful in characterizing chocolates, allowing the delineation of different sensory profiles according to the type of chocolate. it facilitates the comprehension of human behavior, how this one is influenced by taste/olfactory and visual stimuli in eliciting interior feelings and expectations. from a semiotic point of view, all the nonverbal iconic signs analyzed speak for themselves that is they convey the proper message intended to. charged with cultural significance and symbolism, iconic and plastic signs stand for positive feelings and attitudes and high quality products as well underlining that they play a huge role in human communication. the results of word associations made by studying the six types of chocolate labels are expressed by: feelings, descriptions, at the morphological level by positive adjectives and nouns; most people associated the chocolate image with the sensations caused by iconic signs: fruit, colors, characters, and ways of spelling. 5. references: [1] ares g., deliza r., studying the influence of package shape and color on consumer expectations of milk desserts using word association and conjoint analysis, food quality and preference 21, 930–937, (2010) [2] becker l., thomas j.l. van rompay, hendrik n.j. schifferstein, galetzka m., tough package, strong taste: the influence of packaging design on taste impressions and product evaluations, food quality and preference 22 , 17–23, (2011). [3] spence c. , gallace a., tasting shapes and words, food quality and preference 22 290–295, (2011) [4] jaeger s. r., cardello a.v., schutz h.g., emotion questionnaires: a consumer-centric perspective, food quality and preference 30 , 229–241, (2013) [5] laros, f. j. m., & steenkamp, b. e. m. emotions in consumer behavior: a hierarchical approach. journal of business research, 58, 1437–1445, (2005). [6] king, s., & meiselman, h. l. development of a method to measure consumer emotions associated with foods. food quality and preference, 21, 168–177, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 alice-iuliana roșu, cristina-elena hrețcanu, ana leahu, an interdisciplinary approach on the influence of packaging design on emotional response to chocolate, volume xii, issue 2 – 2013, pag. 192 206 206 [7] thomson, d. m. h., & crocker, ca datadriven classification of feelings. food quality and preference, 27, 137–152, (2013). [8] thomson, d. m. h., crocker, c., & marketo, c. g. , linking sensory characteristics to emotions: an example using dark chocolate. food quality and preference, 21, 1117–1125, (2010). [9] spence c., kim ngo m., percival b., smith b., crossmodal correspondences: assessing shape symbolism for cheese, food quality and preference 28, 206–212, (2013). [10] spence c., managing sensory expectations concerning products and brands: capitalizing on the potential of sound and shape symbolism, journal of consumer psychology 22, 37–54, (2012). [11] ares g., piqueras-fiszman b., varela p., morant marco r., martín lópez a., s. fiszman, food labels: do consumers perceive what semiotics want to convey?, food quality and preference 22, 689–698, (2011). [12] giovannetti v., m. d. el mundo del envase. manual para el diseño y producción de envases y embalajes. barcelona: editorial gustavo gili., (1995). [13] friendly m., statistical graphics for multivariate data, paper presented at the sas sugi 16 conference, york university apr, 1991, accessed online on http://www.math.yorku.ca/scs/sugi/sugi16paper.html#h1_6:biplot, accesed in april 2013 [14] gabriel k. r. the biplot graphic display of matrices with application to principal components analysis. biometrics, 58(3), 453-467, (1971). [15] gabriel, k. r. biplot display of multivariate matrices for inspection of data and diagnosis. in v. barnett (ed.), interpreting multivariate data. london: wiley. (1981). [16] pop c., pop i. m., science of food commodity, publisher edict , iași, romania, (2006) [17] iso 5492, sensory analysisvocabulary [18] iso 6658, sensory analysis methodology general guidance [19] iso 85861, sensory analysisgeneral guidance for the selection, training and monitoring of assessorspart 1: selected assessors [20] iso 85862, sensory analysisgeneral guidance for the selection, training and monitoring of assessorspart 2: experts [21] iso 8587, sensory analysismethodology ranking 165 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 165 174 studies on the influence of quinoa flour addition on bread quality *georgiana gabriela codină 1 , simona geanina franciuc 1 , elena todosi-sănduleac 1 1 faculty of food engineering, stefan cel mare university, 13 th university street, 720229, suceava, romania codina@fia.usv.ro; codinageorgiana@yahoo.com *corresponding author received 27 th may 2016, accepted 27 th june 2016 abstract: the aim of this research was to investigate the effect of quinoa flour addition to wheat flour on bread quality. a part of wheat flour type 000 was replaced by red and white quinoa flour in various proportions from 0% to 20% as follows: 0%, 5%, 10%, 15% and 20% respectively. once this prototype was established, we wanted to study the influence of the quinoa flour on physical, colour, and crumb cell, textural and sensory characteristics of the bread products. for this purpose motic stereo microscope, konica minolta cr–700 spectrophotometer and mark-10-esm301 texture analyzer were used. the experimental data showed statistically significant differences between samples. increasing amounts of quinoa flour both white and red, lead to stronger and less extensible doughs. the volume as well as the elasticity of samples decreased with a higher percentage of quinoa flour addition. the porosity decreased as well. the results also showed that blends containing either 5 or 10% white or red quinoa flour presented better physical properties than those with higher proportions of quinoa flour. the colour of samples was visibly modified with the increasing amounts of quinoa flour, the most evident being in red quinoa samples especially from the l* parameter point of view of cie lab method. the obtained results were statistically interpreted. the statistical analysis was done using two-factor anova without replication and microsoft excel 2007 software. the tasters appreciated the samples with 5 and 10% quinoa flour, the overall acceptance score being close to that of control or even higher. key words: wheat flour, quinoa flour, bread quality, colour profile, crumb structure 1. introduction bread and bakery products represent an important sector of the food industry. they are known and consumed throughout the world for thousands of years. however, we can say that their production is relatively new only thinking of how fast the technology evolves from year to year. lately, the bread can be considered an exceptional product, and so is, considering the continued growth of its market. bread is a source of vitamins and minerals, especially phosphorus and copper [1]. however, from the nutritional point of view, the bread has a low content of essential amino acids, e.g. lysine, tryptophan, threonine [2]. also, a, c, d and b12 found in a low content in bread decrease the nutritional quality of it [3]. adding other types of flour in bread helps to improve its nutritional properties. bread with composite flour maybe made from other types of flour than wheat flour or may be made from flours derived from pseudo-cereals mixed with wheat flour [4]. el-soukkary f. a. h. (2001) shows that the addition of flour from pseudo-cereals can increase the protein content of bread mailto:codina@fia.usv.ro mailto:codinageorgiana@yahoo.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 166 and therefore can improve its nutritional quality, particularly by increasing the lysine content in which bread is deficient [5]. on the other hand, the addition of pseudo-cereal flour increased the soluble fiber content of the product. there are two reasons for which the adding of fibers to bread is essential: fibers contribute to the decrease of caloric value of the product and help in increasing there commended intake of soluble fiber [6]. making bread and bakery products using composite flour have been developed in recent years, attracting the attention of many researchers. after many years of studying in this area, they found that products made with mixture of several types of flour have the same characteristics with products made entirely of wheat flour. the use of composite flour has direct impact on the finished products in terms of physical and chemical effects on the human being health [7]. in addition, breadmaking using composite flour is a new approach to the use of cereals that are lesser used or known and have higher nutritional characteristics. in order to make composite flour, many other types of flour can be used with wheat flour. these include: corn and chickpea flour [8–10], pumpkin flour [11], lupine flour [12–13], quinoa and amaranth flour [14–18] e.g. quinoa (chenopodium quinoa willd.) is a pseudo-cereal found at high altitudes in regions of south america. it belongs to the dicotyledons class, the chenopodiaceae family, genus chenopodium, species quinoa. quinoa is known for its high protein content. more exactly it contains all essential amino acids with large amounts of lysine and methionine [19]. it contains significant amounts of fiber, minerals, especially calcium and iron [20] and antioxidants [21]. moreover, it doesn’t contain gluten protein, so products made exclusively from this type of flour can be consumed by people with gluten intolerance or those allergic to wheat [22]. with these characteristics, quinoa is distinguished from other conventional grain which makes it appreciate worldwide. quinoa seeds are small (about 350 grains weight a gram), round, slightly flattened, colour ranging from pale yellow to pink or even black. quinoa, like other andean grain, is consumed in different ways, today presenting numerous innovations at the industry level. thus, it can be eaten cooked, milled or extruded. in the present study, composite flour bread was prepared using wheat flour type 000 and whole quinoa flour both white and red. the objective of this work was to investigate the effect of different ratios of quinoa flour addition to wheat flour on bread quality. 2. materials and methods 2.1.materials 2.1.1. wheat flour the research study has been carried on 000 flour type obtained from a local hypermarket. the flour used in this study presents the following characteristics: moisture content 14.2%, ash content 0.48%, wet gluten content 27%, gluten deformation index 6 mm and 3.3 acidity. the flour chemical composition was determined according to romanian standard methods: moisture (sr en iso 712:2010), ash content (sr en iso2171:2010), wet gluten content, gluten deformation index (sr 90:2007), acidity (sr 90:2007). 2.1.2. quinoa flour red quinoa seeds (bolivia origin) and white seeds (ecuador origin) were purchased from specialized shops and then were grounded separately with a laboratory grinder in order to obtain flour. prior to grinding, the grains were washed with cold water to remove the layer of saponins. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 167 the process was repeated several times until water clarification and disappearance of foam. the seeds were dried and then grounded. 2.2. methods 2.2.1. preparation of composite quinoawheat flour bread the bread samples (fig. 2) were made using direct method for preparation of the dough. the ingredients were mixed in a mixer for about 15 minutes. yeast and salt were added in doses of 3% respectively 1.5% reported to the mass of the wheatquinoa flour and water up to 56% wheat flour hydration capacity. the doughs obtained were manually moulded into pieces of 250 grams each. samples were then put into special trays and left to rise for some time until the samples have doubled their volume before being baked in an electric oven with thermo humidification and ventilation (piron caboto pf8004 d, italy) 30 minutes at 180°c. after taking out of the trays, bread samples were cooled for analysis. 2.2.2. physical and quality parameters of bread for assessing the influence of the characteristics of quinoa flour, bread samples were evaluated to determine elasticity, volume, porosity and other characteristics. measurements were performed for all samples under the same conditions and according to the same parameters. the analysis was performing in proper time so that not to exceed the shelf life of the product. bread volume, elasticity and porosity were determined according to sr 91:2007. photometric analysis of bread samples was made using konica minolta cr – 700 (japan) portable colorimeter from the food safety research laboratory of the faculty of food engineering of suceava. the measurement of colour was carried out by colour system method cie lab, where l* indicates the brightness of the colour, measured in a range from 0 (black light minimum) to 100 (maximum brightness), a* denotes the position of the colour on a scale ranging from pure green (-) to pure red (+) and b* coordinate indicates the colour position on a scale ranging from pure blue (-) to yellow pure (+). the parameters of this method, l* (hunter lightness), a* (redness), b* (yellowness), offered concrete results on the effectiveness and sensitivity of colorimetric sensor, absorption of electromagnetic radiation being achieved in the uv-vis range. the readings for each sample separately were carried out under laboratory conditions, after device calibration by targeting its light path on a specific area of white (obtaining white spectrum, δ65). to appreciate the texture of bread samples we used a texture analyzer (mark-10esm301) in order to obtain primary and secondary texture parameters resulting from a mechanical action applied to the product. the electronic texture analyzer is equipped with a sensor that measures the force, displacement and analysis time. texture parameters analyzed were: hardness, gumminess, cohesiveness, elasticity and chewiness. bread samples have passed through a double compression between the discs of texture analyzer (aluminium disk of 50 mm diameter), compression being carried out on half of the thickness of the bread slice. the samples were analysed for 2.5 to 4 minutes depending on composition and structure. interpretation of the data obtained was performed using a mesur gauge software of mark 10 texture analyzer. in order to establish their relevance, it was also emphasized the relation between primary and secondary texture parameters analysed. on this line, we used data analysis package provided by ms excel. to study the microstructure of bread samples binocular stereo microscope food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 168 motic smz-140 was use. bread sample slices of 8 mm thick were placed on the special board of microscope. for analysis, a 20x objective was used. the captured images were sent to an external hard drive and then were saved in jpg format using colour model blue at a resolution of 2048x1536 pixels. the most effective way we can appreciate the quality of bread is sensory analysis. for this method bread samples with different levels of quinoa flour addition were cut into small pieces and placed on plastic dishes to be tasted. the tasters were 20 semi-trained judges, students of the faculty of food engineering of stefan cel mare university (representative sample given multiple students’ knowledge in this area). assessing the value of the product was done with a scale score of 1 (i dislike very much) to 9 (i like very much), pursuing the following characteristics: appearance, colour, taste, smell, flavour, firmness and overall acceptance. for a better appreciation of the influence of quinoa flour on bread samples, the results of this analysis were statistically interpreted, results being expressed as means. 2.2.3. statistical analysis to calculate the mean values and standard deviations of then analysis results, microsoft office excel software was used. to determine the significance of these obtained results, the two-factors without replication anova was used, and fisher's test was used to assess significant differences (p < 0.05) among samples. the significance level for all tests was 0.05. it was calculated pearson's coefficient (r) to measure the linear relationship existing between two variables. 3. results and discussion 3.1. physical and chemical properties of wheat flour the results obtained from the flour analysis correspond to standards. white flour 000 type is of a very good quality for bread making. due to its low nutrients content we chose to mix it with quinoa flour more rich in nutrients. 3.2. bread volume quinoa flour significantly influences the volume of the bread sample in which is added. according to the results fromfigure1, we can note that the relation between the variation of quinoa flour percentage and bread volume is described by a regression line with descending slope. the correlation coefficient, r = 0.985 for white quinoa and r = 0.966 for red quinoa indicates a strong inverse connection between these parameters. small amounts of flour did not significantly affect the volume of bread, samples having similar characteristics to that of the control one. quinoa, both white and red has a slightly amylase activity which may lead to an increase in the production of gas and thus the volume of bread [23]. however, samples with the addition of quinoa flour over 15% have a smaller volume compared to the control sample. the results corresponded with those of morita, n. et al. (2001) [24]. this can be attributed to gluten reduction in the product the dough couldn’t retain so well the fermentation gases. park, h.s. et al. (2005) [15] studied under a microscope samples of wheat flour bread mixed with 30% quinoa flour and observed that structured gluten matrix encompassing aggregate grains starch which retain gases was missing. as we can see in figures 1 and 2 compared to the control, samples loaf bread volume decreases with increasing concentration of quinoa flour added. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 169 0 5 10 15 20 fig. 3. bread samples with red quinoa flour 0% 5% 10% 15% 20% analysis of variance shown no significantly differences (p < 0.5) between the two types of quinoa flour regarding the bread samples volume (f = 3.47 < fcrit = 7.7). from this results we can conclude that the type of quinoa flour, white or red, does not influence the decreasing of bread volume, but the percentage of flour added (f = 65.46 > fcrit = 6.38). 3.3. elasticity and porosity the quinoa flour influence on the elasticity and porosity of the bread is shown in figure 3. as seen, bread samples of 5% and 10% quinoa flour, both white and red, have elasticity close to that of control (0% quinoa flour added). higher concentrations of quinoa flour resulted in a less elastic crumb. correlation coefficients obtained in both cases, r = 0.973 for white quinoa flour and r = 0.948 for red quinoa flour indicate strong inverse connections between samples, both quinoa white flour samples elasticity and red quinoa flour samples elasticity decreasing (p < 0.05) with the increasing amount of flour added. this is explained by mechanical damage of gluten by fibers in quinoa flour. samples porosity significantly differs from the control sample. regression lines obtained in both cases indicate strong inverse connections between samples. following statistical analysis of variance (anova) we can note a decreasing samples porosity (p < 0.05) with increasing amounts of quinoa flour added, both white and red (f = 65.46 < fcrit = 6.38). this is explained by mechanical damage of gluten by fibers in quinoa flour. 3.4. colour samples the colour of the crumb has been also an important parameter for characterising quinoa-wheat flour bread. colour values of bread crumb samples are presented in table 1. lower l* value indicates darker crumb, a* positive value is associated with crumb redness, whereas b* positive value indicates yellow colour. fig. 1.loaf volume of bread samples with different levels of quinoa flour addition fig. 2. bread samples with white quinoa flour 0% 5% 10% 15% 20% (a) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 170 0 5 10 15 20 0 5 10 15 20 (b) control sample has the greatest lightness. in general, the brightness values l* decrease (p < 0.05) with increasing concentration of quinoa flour added to bread. this dark colour is owed to the darker quinoa flour colour, both white and red, compared to white wheat flour. the darkest colour presents the sample with highest concentration of red quinoa flour. compared with the control, coordinate a* indicates lower values of white quinoa bread samples, where as in case of red quinoa bread samples, the values turn to the positive area, that is red colour. coordinate b* of the cie lab colour system method indicates close and positive values of the samples colour. 3.5. bread texture primary (hardness, cohesivity, elasticity) and secondary (gumminess, chewiness) texture parameters of samples are presented in table 2. quinoa flour in different concentrations has significant effects on the bread samples texture profile. for example, the hardness of the bread increases to a certain level of quinoa flour added. above this concentration (15% for white quinoa and 10% for red) hardness begins to decrease. bread samples cohesivity is largely of interest, with a preference for a high cohesivity so during mastication product not disintegrate. in our case, control sample has the highest cohesivity and compared to it, the other samples cohesiveness is relatively low (p < 0.05), due to the reduction of the wheat flour amount in the product. as for chewiness, the things remain the same as in the case of hardness. up to 15% quinoa white flour and 10% red quinoa flour added, chewiness (less than in case of control) increases. over these values it begins to fall. to establish links between texture parameters, a pearson correlation was established, table 3. as seen, the correlation coefficients obtained indicate fig. 3. effect of quinoa flour addition on bread elasticity (a) and porosity (b) (a) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 171 the presence of close and statistically significant links between some parameters. 3.7. sensory analysis attributes that have the greatest influence on the reaction of tasters were colour and overall acceptance. tasters prefer samples of bread with the addition of 5 and 10% quinoa flour, both white and red, overall acceptance score exceeded that of the control. table 1. colour values of bread samples with quinoa flour addition parameters sample l* a* b* δl δa δb δe control sample 74.46 -1.87 13.35 38.64 -15.22 -0.87 41.54 5% wqf 69.39 -2.23 14.8 33.57 -15.58 0.58 37.01 10% wqf 65.84 -2.07 13.24 30.02 -15.42 -0.99 33.76 15% wqf 61.46 -1.33 15.17 25.64 -14.69 0.94 29.67 20% wqf 67.11 -1.4 16.82 31.29 -14.75 2.6 34.69 5% rqf 68.41 -0.22 12.02 32.59 -13.57 -2.2 35.37 10% rqf 62.21 0.99 13.19 26.39 -12.36 -1.03 29.16 15% rqf 59.16 1.79 12.34 23.34 -11.56 -1.88 26.11 20% rqf 55.2 3.02 12.74 19.38 -10.33 -1.48 22.01 wqf – white quinoa flour; rqf – red quinoa flour table 2. texture profile analysis for quinoa-wheat flour composite bread table 3. pearson correlation for primary and secondary texture parameters correlation is significant at the 0.05 level tpa parameters sample hardness (n) cohesivity (dimensionless) elasticity (dimensionless) gumminess (n) chewiness (n) control 16.22 0.69 0.86 11.19 9.62 5% wqf 16.78 0.54 0.80 9.06 7.25 10% wqf 18.48 0.61 0.84 11.27 9.47 15% wqf 54.62 0.57 0.84 31.13 26.15 20% wqf 31.16 0.67 0.36 20.88 7.52 5% rqf 18.80 0.63 0.82 11.84 9.71 10% rqf 32.72 0.56 0.84 18.32 15.39 15% rqf 28.42 0.64 0.83 18.19 15.10 20% rqf 15.38 0.68 0.85 10.46 8.89 hardness cohesivity elasticity gumminess chewiness hardness 1 cohesivity -0.377 1 elasticity -0.146 -0.266 1 gumminess 0.986 -0.229 -0.248 1 chewiness 0.892 -0.395 0.305 0.846 1 food and environment safety journal of georgiana gabriela codină, simona of quinoa flour addition on bread quality, 4,5 6,5 8,5 10,5 appearance colour smellflavour firmness overall acceptance control 5% 15% 20% these samples were analysed for differences between them regarding influence of the percentage of quinoa flour added on the overall acceptance product. the averages scores of the 5 samples were 7.94 (for control (for 5% and 10% white quinoa 8.41 respectively (for bread with 10% red quinoa flour). the two-factor without replication anova revealed a significant difference between these averages, f = 1.84 (higher than fcrit = 1.64), p < 0.05 for type of flour, white or red, and f = 8.35 (greater than fcrit = 3.13), p < 0.05 for the concentration of the quinoa flour. there are statistically differences between samples made with white and red quinoa flour. the two influencing factors were type of quinoa flour (independent variable) and percentage of quinoa flour added 15 and 20% (dependent variable). 0.05, f = 2.17 > fcrit = 1.52 (for the type of flour), and f = 9.99 > fcrit = 2.44 percentage of the flour added). as for both of these factors than fcrit, it is clear that both white flour or red and their percentage significant statistically influence the taste of the product. (a) journal of faculty of food engineering, ştefancel mare university volume xv, issue 2 2016 simona geanina franciuc, elena todosi-sănduleac, quality, food and environment safety, volume xv, issue 2 – 2016, 172 colour taste smell 10% 4,5 5,5 6,5 7,5 8,5 9,5 appearance flavour firmness overall acceptance control 5% 15% 20% these samples were analysed for regarding the influence of the percentage of quinoa flour nce of the final product. the averages scores of the 5 for control) 8.29, 8.76 white quinoa), 8.17 and for bread with 5% and factor without replication ant difference = 1.84 (higher 0.05 for type of flour, 35 (greater than 0.05 for the concentration statistically significant samples made with quinoa flour. the two influencing factors were type of quinoa flour (independent variable) and percentage of quinoa flour added, 0, 5, 10, 15 and 20% (dependent variable). for p < for the type of = 2.44 (for the percentage of the flour added). as for both of these factors f is greater white flour or percentage significant taste of the fig. 4. sensory profile of wheat bread (a) and (b) wheat 3.6. bread microstructure the analysis of microstructure of samples is shown in figure fig. 5.crumb structure of bread samples a control; b 5% wqf; c d 15% wqf; e 20% wqf; f g 10% rqf; h 15% rqf; i tefancel mare university suceava sănduleac, studies on the influence 2016, pag. 165– 174 appearance colour taste smell 10% profile of wheat-quinoa white wheatquinoa red bread bread microstructure the analysis of microstructure of bread figure 5. structure of bread samples 5% wqf; c 10% wqf; 20% wqf; f 5% rqf; 15% rqf; i 20% rqf; food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 173 it is notices that the bread samples with higher concentrations of quinoa flour presents a crumb structure made up of thin films and filaments, absent to the control sample. this is may be due to the destruction of gluten protein network by quinoa flour fibers. the results are in agreement to those obtained by others [25– 26]. 4. conclusions the addition of rich-in-fiber quinoa integral flour to wheat flour causes a number of changes to bread technological parameters, changes that have direct impact on the quality of the finished products. thus, the volume, elasticity and porosity of the bread samples decrease with increasing amount of quinoa seed flour addition, regression lines obtained in all cases indicating very strong intensity reverse link. the two-factor without replication anova showed no statistically significant difference (p < 0.05) in what concerns the type of quinoa flour added on texture and physical parameters analysed. the colour of bread with quinoa flour white or red, the volume, elasticity and porosity are influenced in especially by the percentages of quinoa flour mixed with wheat flour type 000. a percent of 5-10% quinoa flour mixed with wheat flour type 000 of a very good quality for bread making has beneficial influence on the bread quality, firstly by the obtained values, values close to those of the control sample, and secondly by the improving sensory characteristics of bread or increasing it nutrient content. 5. references [1]. steer t., thane c., stephen a., jebb s., bread in the diet: consumption and contribution to nutrient intakes of british adults, proceedings of the nutrition society, 67: e363, (2008) [2]. dhingra, s., jood, s., organoleptic and nutritional evaluation of wheat breads supplemented with soybean and barley flour, journal of food chemistry, 77: 479-488, (2001) [3]. hoseney r.c., principles of cereal science and technology, 2 nd ed., aacc international: st. paul, mn, (1992) [4]. aghamirzaei, m., heydari-dalfard, a., karami, f., fathi, m., pseudo-cereals as a functional ingredient: effects on bread nutritional and physiological properties-review, intl j agri crop sci., 5(14): 1574-1580, (2013) [5]. el-soukkary, f. a. h., evaluation of pumpkin seed products for bread fortification, plant foods hum nutr, 56: 365-384, (2001) [6]. stauffer, c. e., functional additives for bakery foods. van nostrand reinhold, new york; 1990 [7]. noorfarahzilah, m, lee, j. s., sharifudin, m. s., mohd fadzelly, a. b., hasmadi, m., applications of composite flour in development of food products, international food research journal, 21(6):2061-2074, (2014) [8]. hussein, a. m. s., kamil m. m., hegazy, n. a., el-nor, a. s. a. h., effect of wheat flour supplemented with barely and/or corn flour on balady bread quality, pol. j. food nutr. sci., 63(1):11-18, (2013) [9]. man, s., păucean, a., muste, s., pop, a., effect of the chickpea (cicerarietinum l.) flour addition on physicochemical properties of wheat bread, bulletin uasvm food science and technology, 72(1): 41-49, (2015) [10]. wani, i. a., sogi, d. s., sharma, p., singh gill, b., physicochemical and pasting properties of unleavened wheat flat bread (chapatti) as affected by addition of pulse flour, cogent food & agriculture, 2: 1-9, (2016) [11]. wongsagonsup, r., kittisuban, p., yaowalak, a., suphantharika, m., physical and sensory qualities of composite wheatpumpkin flour bread with addition of hydrocolloids, international food research journal, 22(2): 745752, (2015) [12]. ahmed, h. f., effect of fortification pan bread with lupine flour on the chemical, rheological and nutritional properties, j. food and dairy sci., mansoura univ., 4 (3): 65-75, (2013) [13]. correia, p. m. r., gonzaga, m., batista, l. m., beirão-costa, l., guiné, 250μm food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xv, issue 2 2016 georgiana gabriela codină, simona geanina franciuc, elena todosi-sănduleac, studies on the influence of quinoa flour addition on bread quality, food and environment safety, volume xv, issue 2 – 2016, pag. 165– 174 174 r. f. p., development and characterization of wheat bread with lupin flour, international journal of biological, biomolecular, agricultural, food and biotechnological engineering, 9(10): 923-927, (2015) [14]. enriquez, n., peltzer, m., raimundi, a., tosi, v., pollio, m. l., characterization of wheat and quinoa flour blends in relation to their breadmaking quality, the journal of the argentine chemical society, 91(4/6): 47-54, (2003) [15]. park, s. h., morita, n., dough and breadmaking properties of wheat flour substituted by 10% with germinated quinoa flour, food sci. technol. int., 11: 471-476, (2005) [16]. rosell, c. m., cortez, g., repocarrasco, r., breadmaking use of andean crops quinoa, kañiwa, kiwicha, and tarwi, cereal chem., 86(4): 386-392, (2009) [17]. berghofer, e., schonlechner, r., utilization of amaranth and quinoa in europe, ernahrung, 24: 303-308, (2000) [18]. kong, x., bao, j., corke, h., physical properties of amaranthus starch, food chem., 113: 371-376, (2009) [19]. abugoch james, l. e., quinoa (chenopodium quinoa willd.): composition, chemistry, nutritional, and functional properties, advances in food and nutrition research, 59: 131, (2009) [20]. ando, h., chen y. c., tang, h., shimizu, m., watanabe, k., mitsunaga, t., food components in fractions of quinoa seed, food science and technology research, 8(1): 8084, (2002) [21]. hirose, y., fujita, t., ishii, t., ueno, n., antioxidative properties and flavonoid composition of chenopodium quinoa seeds cultivated in japan, food chemistry, 119(4): 13001306, (2010) [22]. caperuto, l. c., amaya-farfan, j., camargo, c. r. o., performance of quinoa (chenopodium quinoa willd) flour in the manufacture of gluten-free spaghetti, journal of the science of food and agriculture, 81(1): 95-101, (2001) [23]. lorenz, k., roewe-smith, p., kulp, k., bates, l., preharvest sprouting of winter wheat. ii. amino acid composition and functionality of flour and flour fractions, cereal chem, 60: 360-366, (1983) [24]. morita, n., hirata, c., park, s. h., mitsunaga, t., quinoa flour as a new food stuff for improving dough and bread, j. appl. glycosci., 48: 263-270, (2001) [25]. bordei, d., burluc, r. m., technology and quality control in the bakery industry, dunărea de jos university foundation publishing, galaţi, (2003) [25]. farcaş, a. c., research on the identification and recovery of biologically active compounds from biomass resulting as a byproduct from brewing, cluj-napoca, (2014) [26]. sr iso, standardization association of romania (asro), bucharest, romania: 90, 91, 11036, (2007) [27]. sr iso, standardization association of romania (asro), bucharest, romania: 712, 2171, (2010) звіт з ндр 29-81 за 2007 – 2009 р 312 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 4 – 2016, pag. 312 322 use of emulsions in production restaurants and food industry *oksana lugovska1, vasilij sidor1 1national university of food technologies, volodimirska 68, kiev, ukraine, oksana.lugovska320@gmail.com *corresponding author received9th august 2016, accepted 27th december 2016 abstract: the paper focuses on the application of hydrocolloids, which are stabilizers, emulsifiers, gum arabic and starch technology aromatic emulsions. the regularities of their transfer to the active emulsion and stabilization of emulsion state laws and the formation and stabilization of emulsions to ensure their stability during storage were investigated. grounded rational prescription content, main components and technological parameters of the production of emulsions are presented with in this research.thus, the technology and range of aromatic emulsions and drinks with their use were studied hereby. the complex of consumable and technological properties, reasonably terms and conditions. regulatory and technological documentation, introduction of new technologies implemented in institutions restaurant industry, the economic effect of their introduction were developed and approved. keywords: aromatic emulsions, hydrocolloids, stabilizers, starch, gum arabic, particle size 1.introduction the key point of the efficiency of the institutions restaurant industry and food industry is the introduction of resourcesaving and competitive technologies based on the use of high quality components of food and beverages (aromatic emulsions). a special group of products consists of drinks. in restaurants and industry exclusive approaches are used for their manufacture. the use of emulsions in the manufacture of food and beverages is associated with the possibility of reducing the length of the production process while reducing power consumption compared to traditional production methods. this difference is particularly useful in restaurants and food industry because emulsions are necessary ingredients that provide food with flavor, color and aroma, thus making greatly easier their production technology. the use of aromatic emulsions and food additives as ingredients or represented as aromatic essences and extracts that have a short shelf life involves much more expensive price. having in view the major trends of the food market, this situation is a deterrent and requires substantial improvement in terms of product range. market analysis of ingredients and food additives indicates that the production of emulsions that can be used in foods and their promotion on the food market ukraine is constrained by insufficient levels of basic and applied research related mainly to the support of their colloidal stability during storage. this leads to the need for scientific and applied research aimed at using and implementing functional and technological properties of the components of the prescription components and structure-stabilizer (emulsifier) emulsions, aromatic oils, flavors, narrowing agents, dyes, preservatives, acids. the studies made by phillips g.o., williams p.a.[1-2], rebinder p.a. [3], kremnev l.a., soskin http://www.fia.usv.ro/fiajournal mailto:oksana.lugovska320@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 313 s.a., kuprika b.c.[4-6], p.stephen[7], a. aymesonand others [8-10], a.b.horalchuk [12], highlight the scientific foundations and practical aspects of the production of emulsions. but systematic studies aimed at obtaining stable emulsions based on these ingredients have not been found. not the basic patterns of their formation and technological parameters of production, there are no recommendations for their use. having in view the development of science-based technologies aromatic emulsions based on specified raw materials, which ensure the stability of food product during shelf life, significant improvement of efficiency of zrh-food industry and their implementation, one may state that the existing range of food and beverages using aromatic emulsion will expand the range of culinary and industrial products with their use. 2.material and methods construct implement quality control parameters emulsions at all stages of the process according to tu 10.8-02070938209: 2015 "emulsions aromatic food industry." raw analyzed at reception by qualitative indicators of regulatory documents and certificates of quality from producers under water gost "drinking water" 4077-2001. sampling according to iso 4856, preparation of samples for testing for laboratory tests in accordance with gost 26671, to determine the content of toxic elements in accordance with iso 7670. the quality of packaging and labeling checks visual aspects. test methods for quality organoleptic properties and volume according to iso 7099 ph gost 26188physico-chemical characteristics of the emulsion must meet the requirements by tu 10.8-02070938209: 2015. determination of toxic elements carried out according to the methods specified in table 4 tu 10.802070938-209: 2015. sampling for microbiological analysis according to gost 26668, preparation of samples according to gost 26669, methods of culturing microorganisms according to gost 26670, preparation of solutions, reagents, culture media according to iso 5093, microbiological tests carried out according to the methods specified pursuant tu 10.8-02070938-209: 2015. control of preservatives and artificial colors carried out in accordance with methods approved. to determine ph we used a laboratory ph meter (anion 4100), density laboratory density meter (hydrometer purpose aon1, 2-aon, aon-3, 4-aon, aon-5); turbidity emulsions were determined using (2100 p), and viscosity brookfield viscometer temperature electronic terometrom htc-2. the average particle size was investigated microscope eastcolight 92012-es (100x, 250x, 550x, 750 x) and measured flatware ls ™ 13 320 by laser granulometry. the data worked out methods of mathematical statistics using software mathcad, xl. 3. results and discussion by experimental research on emulsification technology homogenization two immiscible liquids with the use of arabic gum (e 414) proved the feasibility of using arabic gum as an emulsifier, which is a carrier of functional and technological components arabian galantines protein fractions, which gives emulsion stability. accordingly the developed innovative strategy should ensure stability of emulsions proteinpolysaccharide complexes. they generated a controlled transfer of dryness in the water-soluble active state to form steric food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 314 barriers on the border of two distribution phases oilwater, ensuring their functional and technological properties in the formation of direct emulsions with desired characteristics. on the basis of innovative strategies formulated a working hypothesis, which is that the use of arabic gum as an emulsifier, a source of polysaccharides and proteins, provided directional regulation of functional and technological properties solubility, the ability of protein-polysaccharide complex formation and stability during storage, surface activity, emulsifying, complexing ability of its major constituents (polysaccharides, proteins) will provide a stable emulsion. gum arabic is characterized by a highly branched structure with arabinohalaktan protein fraction in the center, thus forming a solution that provides high-quality emulsifying properties. studies have shown that acacia gum containing monomers d-galactose linked β (1,3)hlykozyd connection with numerous ramifications, which consist of αand βgalactose and other sugars or uronic acids. in instant and highly purified form, it absorbs moisture and rapidly dissolves in water. it is proved that the transfer of gum arabic is soluble active state, requires a two-stage process preparation of pre emulsion is formed by mixing oil and water phase of the emulsion particle size, more than 3 microns. (fist stage), crushing her to obtain a stable emulsion by homogenization to enhance the content and complexing ability (cp) gum arabic to give an emulsion with a particle size of less than 1 micron (second stage). it has been proved that mixing two liquids oil-water carried through the use of an emulsifier gum arabic reduces the value of the surface tension on the surface phase distribution, oil, water, providing better mixing of oil and water phases. temperature, time, speed and mixing respective phases affect the degree of dissolution and foaming ingredients. the emulsions based on citrus oil were used for the investigations. gum-arabic (produced by cni, france) and modified starch (national starch, germany) were the stabilizers. to study the emulsifying properties different types of emulsions were prepared. according to the specification [10] the emulsions contain gum-arabic of high purity (4–7 wt %) and oil phase of 8– 14 wt %. the emulsions with starch contain 8–14 wt % of e1450 and 8– 14 wt % of oil phase. the emulsions contain different amount of oil phase and constant amount of stabilizer (tables 1 and 3), as well as constant amount of oil phase with variable amount of stabilizer (tables 2 and 4). table 1 emulsions with different amount of oil phase and constant amount of gum-arabic emulsion ingredients ingredients content, wt % emulsion number 1 2 3 4 5 citrus oil 6.000 6.000 6.000 6.000 7.000 resingum (е445) 2.000 4.000 5.000 6.000 7.000 gum-arabic (е414) 5.000 5.000 5.000 5.000 5.000 citric acid (е330) 0.500 0.500 0.500 0.500 0.500 sodium benzoate (е211) 0.250 0.250 0.250 0.250 0.250 dye (е124) 0.150 0.150 0.150 0.150 0.150 dye (е110) 1.400 1.400 1.400 1.400 1.400 antioxidant (е320, е321) 0.003 0.003 0.003 0.003 0.003 water 84.698 82.698 81.698 80.698 78.698 total 100.000 100.000 100.000 100.000 100.000 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 315 table 2 emulsions with constant amount of oil phase and different amount of gum-arabic emulsion ingredients ingredients content, wt% emulsion number 6 7 8 9 10 citrus oil 6.000 6.000 6.000 6.000 6.000 resingum (е445) 4.000 4.000 4.000 4.000 4.000 gum-arabic (е414) 4.000 5.000 5.500 6.000 7.000 citric acid (е330) 0.500 0.500 0.500 0.500 0.500 sodium benzoate (е211) 0.250 0.250 0.250 0.250 0.250 dye (е124) 0.150 0.150 0.150 0.150 0.150 dye (е110) 1.400 1.400 1.400 1.400 1.400 antioxidant (е320, е321) 0.003 0.003 0.003 0.003 0.003 water 83.698 82.698 82.198 81.698 80.698 total 100.000 100.000 100.000 100.000 100.000 table 3 emulsions with different amount of oil phase and constant amount of starch emulsion ingredients ingredients content, wt% emulsion number 1 2 3 4 5 citrus oil 4.000 5.000 5.500 6.000 7.000 resingum (е445) 4.000 5.000 5.500 6.000 7.000 starch (е1450) 12.000 12.000 12.000 12.000 12.000 citric acid (е330) 0.500 0.500 0.500 0.500 0.500 sodium benzoate (е211) 0.250 0.250 0.250 0.250 0.250 dye (е124) 0.150 0.150 0.150 0.150 0.150 dye (е110) 1.400 1.400 1.400 1.400 1.400 antioxidant (е320, е321) 0.003 0.003 0.003 0.003 0.003 water 77.698 75.698 74.698 73.698 71.698 total 100.000 100.000 100.000 100.000 100.000 table 4 emulsions with constant amount of oil phase and different amount of starch emulsion ingredients ingredients content, wt % emulsion number 6 7 8 9 10 citrus oil 5.500 5.500 5.500 5.500 5.500 resingum (е445) 5.500 5.500 5.500 5.500 5.500 starch (е1450) 8.000 10.000 11.000 12.000 14.000 citric acid (е330) 0.500 0.500 0.500 0.500 0.500 sodium benzoate (е211) 0.250 0.250 0.250 0.250 0.250 dye (е124) 0.150 0.150 0.150 0.150 0.150 dye (е110) 1.400 1.400 1.400 1.400 1.400 antioxidant (е320, е321) 0.003 0.003 0.003 0.003 0.003 water 78.698 76.698 75.698 74.698 72.698 total 100.000 100.000 100.000 100.000 100.000 we determined that the effective dissolution rates are gum arabic solution temperature increase heating gum arabic from 20 ° c to 25 ... 30 ° c, providing the increase of soluble gum arabic of 3 ± 0,03% to 5,03%. established rational dissolving gum arabic zone temperature 25 ... 30 ° c, the rate of mixing an aqueous solution of gumarabic50 ... 100 rev / min., which provides a growing share of gum arabic dissolved in 1,5 times. as the temperature of the aqueous phase above food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 316 the established range 25 ... 30ºc and stirring speed 50-100rev / min., will increase the degree of foaming, resulting in the inability to use the aqueous phase further production increase significantly as the duration of its upholding of 4 ... 5 hours up to 10 hours. with decreasing temperature, stirring speed and time of the aqueous phase, compared to the defined range, decrease the degree of dissolution of the ingredients of the aqueous phase. when preparing the aqueous phase determined the best water-soluble dye dissolves when it prepared to make in the water and not with emulsifiers, as they are to some extent prevent the dissolution of dyes. given the fact that the period of introducing sodium benzoate and citric acid is very large, the following sequences were taken in making ingredients in the aqueous phase, preparing (purified) water; sodium benzoate; dyes soluble emulsifier (gum arabic); citric acid. a mixture of gum arabic is used for 4-5 hours until the foam disappears. we established sequence making oil phase components: the capacity is number of citrus oils 9-10%, which will ensure the best taste and flavor emulsions compared to the prototype. the increase in the number of citrus oil above this range will result in a need to increase the amount of gum arabic to ensure the stability of the emulsion during storage that will increase greatly its value. the temperature introduction of citrus oil in an oil phase 25 ... 30 ° c is justified by the fact that an increase in the temperature range of cooking oil phases increases as well energy costs. another added antioxidant which previously dissolved in a little oil and emulsifier is vidvazhena number (the range of esther or more). the mixing was performed by using the mixer to dissolve components (a solution should be transparent, monitored visually), 1-2 hours. the preparing of pre-emulsion requires that the temperature of the oil and water phase is in the same temperature range 25 ... 30 ° c. thus we mixed water with acacia and oil phases, given the availability of other types of mixers reduce 8000 rev / min. 2500 ... 3000 rev / min. and increase the mixing of 3 minutes and 15 ... 20 min to obtain emulsion particles larger than 3 ... 5 microns. we also conducted the experimental research technology emulsification and homogenization of the two immiscible fluids using starch (e 1450) based on the working hypothesis; experimental data which developed scientific principles and defined technological parameters of the production of emulsions. the modified starch-emulsifier (e 1450), due to oktenilyantarn acid esterification starch (e 1450) has emulsifying properties [13-14]. the prototype is a method of preparing an emulsion of gum arabic (9). this method involves preparing the aqueous phase with the addition of emulsifier, cooking oil phase, mixing, double stage homogenization pressure to obtain a concentrated emulsion, by introducing it in food products. the disadvantage of this method is the impossibility of complete dissolution of ingredients not listed as dissolution in the respective phases, parameter preparation and homogenization process and the high price of imported gum arabic as an emulsifier. the invention is based on the task design improvement method of preparation of emulsions with starch by replacing gum arabic at a cheaper price for the starch and the introduction of new process parameters to obtain a stable emulsion with the maximum number of particles as small as 1 micron. рreparation of emulsion oil/water with starch includes the preparation of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 317 aqueous phase with the addition of emulsifier, cooking oil phase preparation pre-emulsion mixing, double-stage homogenization pressure to obtain a concentrated emulsion, the introduction of a food product according to the invention, the preparation of the aqueous phase is carried out of emulsifier starch in an amount of 8-14% at a temperature of 20-25 ° c, mixing is performed at a rate of mixing 40-50rev / min., the resulting aqueous phase uphold 10-12 hours. to the disappearance of the foam, in the preparation of the oil phase using citrus oil, the process is carried out at a temperature of 25-30 ° c, pre-emulsion preparation is carried out at the speed of stirring 25003000 rev / min. for 15-20 min., homogenization is carried out under pressure of 200/50 bar. providing mixing two liquids oil-water carried through the use of an emulsifier starch in an amount of 8-14% (according to the technical documentation for this product), which reduces the value of the surface tension on the surface phase distribution, oil, water, providing better mixing and water phases. temperature, time, speed and mixing respective phases affect the degree of dissolution and foaming ingredients. as the temperature of the aqueous phase above the defined range 20-25ºc and stirring speed 40-50 rev / min., will increase the degree of foaming, resulting in the inability to use the aqueous phase further production increase significantly as the duration of its assertion of 10-12 hours up to 20 hours. with decreasing temperature, stirring speed and time of the aqueous phase, compared to the defined range, decrease the degree of dissolution of the ingredients of the aqueous phase. citrus oil is required to ensure the taste and aroma of citrus emulsion. temperature introduction of citrus oil in an oil phase of 25-30 ° c is justified by the fact that an increase in the temperature range of cooking oil phases, increasing energy costs. preparation of pre-demulsify requires that the temperature of the oil and water phase were in a temperature range of 25-27 ° c; to prevent foaming during preparation of pre-emulsion at high speed, we used stirring 2500-3000ob / min. for 15-20 min. speed mixing 2500-3000ob / min. gives a maximum number of shares emulsion of 3 microns. speed mixing at least at this range contributes to the formation of particles larger than 3 microns, subsequently; the homogenization will cause the formation of particles larger than 1 micron, and therefore will not be provided with stability during storage of the finished product. speed mixing above this range increases the foaming system which is not desirable. the method is presented as follows below. preparation of aqueous phase. the main components for the preparation of the aqueous phase are: water, sodium benzoate, starch, citric acid and other materials according to the recipe 60-70% of water used by prescription. when enabled mixer is set previously sodium benzoate and stirred until dissolved. soluble dyes are the next ingredient obtained. starch in an amount of 8-14% is added manually, dispelling it on the water surface when running at low to 40-50 / min., turning the mixer through a funnel, preventing suction of air in the system. the temperature of dissolving starch ranged between20-25 °c. the mixture is stirred until complete dissolution of starch. when enabled mixer given by citric acid. a mixture of starch is to defend for 10-12 hours prior to the disappearance of the foam. preparation of oil phase. in the mixer an amount of 4-7% citrus oil is added, then heated to a temperature of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 318 25-30 ° c, antioxidant previously diluted in a little oil is added, then an amount of 47% is weighed. the mixing is done until the components are dissolved (solution should be transparent, monitored visually) 1-2 ours. preparation of pre-emulsion. preparation of pre-emulsion is made in the tank, which is equipped with high-speed mixer. aqueous phase is weighed, which fed with constant stirring oil phase. residual water is added. pre-emulsion mixing time is 15-20 minutes at a speed of mixer speed of 2500-3000 rev/min. preparation of finely dispersed emulsion (homogenization). for fine dispersed emulsion, a mixture is subject to two-stage homogenization process processing under pressure 200/50 bar. however, due to the sharp drop in head pressure homogenizer, oil crushed particles uniformly distributed in the emulsion stabilizing starch emulsifier. the emulsion is considered stable if the size of the oil particles does not exceed 1mikrona. in the case of non-compliance of product this indicator requires repeated homogenization of a corresponding change in pressure. the characteristics of the emulsions obtained are shown in tables 5 and 6. table 5 characteristics of emulsions with gum-arabic emulsion number viscosity, cp dissolution turbidity 0.025 %, ntu particles size d, m density, g/cm3 ph 1 14.00 168.00 0.51 1.03 2.70 2 15.00 180.00 0.66 1.06 3.20 3 16.00 192.00 0.71 1.07 3.30 4 17.00 216.00 0.90 1.09 3.70 5 18.00 240.00 1.10 1.10 3.90 6 14.00 192.00 0.75 1.04 2.60 7 15.00 180.00 0.66 1.06 3.20 8 15.50 174.00 0.60 1.07 3.30 9 16.00 168.00 0.56 1.09 3.40 10 17.00 154.00 0.50 1.10 3.80 table 6 characteristics of emulsions with starch emulsion number viscosity, cp dissolution turbidity 0.025 %, ntu particles size d, m density, g/cm3 ph 1 20.00 143.00 0.67 1.03 2.60 2 22.00 156.00 0.73 1.05 3.00 3 23.00 170.00 0.75 1.07 3.30 4 24.00 182.00 0.84 1.09 3.70 5 26.00 196.00 0.97 1.10 4.00 6 19.00 210.00 0.98 1.04 2.70 7 21.00 196.00 0.91 1.06 3.10 8 22.00 184.00 0.83 1.07 3.20 9 23.00 170.00 0.75 1.07 3.30 10 25.00 157.00 0.68 1.10 3.80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 319 there have been developed and approved the following regulations tu 10.8 02070938-2009: 2015 "emulsions aromatic food industry" instructure technology of their production, their drinks recipes developed using emulsions. new technologies tested in the restaurant business establishments of "nautilus". its principle of operation and advantages to clarify the parameters of the technological process of water and oil phases, pre emulsions and emulsions, equipment selection, namely homogenizer for qualitative process of emulsification and homogenization, improved water treatment, namely the introduction of mechanical filters and germicidal lamp to obtain stable product in terms of its shelf life storage. we developed the technological scheme (fig. 1 ) and prescription composition of six emulsions "orange" (ph 6.2 ... 6.4); "pineapple", "grapefruit", "tropic", "ghost" (ph 5.0 ... 5.2) with shelf life of 180 days in a closed area, protected from getting direct co-rays at room temperature 5 to 18 ° c and relative humidity of 75%. fig. 1 technological scheme of emulsions preparation. purified water is stored in tanks with a capacity of 1 to 2 m3 prepared water. the aqueous phase is prepared in a container 2, which is equipped with a stirrer, a log scale and the probe. oil phase is prepared just before cooking pre emulsion in the tank 3, which is equipped with a stirrer, a log scale and the probe. oil phase is pumped via pump capacity 3 to 4 capacity with constant stirring. after making the oil phase mixing is necessary for it to rest for 10 minutes. preparation of pre emulsion is made in a container 4, which is equipped with high-speed stirrer "silverson" (to 3000ob. / min), measuring funnel probe. its principle of operation and advantages to clarify the parameters of the technological process of water and oil phases, pre emulsions and emulsions, equipment selection, namely homogenizer for qualitative process of emulsification and homogenization, improved water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 320 treatment, namely the introduction of mechanical filters and germicidal lamp to obtain product stable for the duration of storage. 4. conclusion 1. when increasing oil phase content of 8 to 14% of gum arabic emulsion, the average particle size increased from 0.51 to 1.10 microns, viscosity from 14 to 18 cp, turbidity from 168 to 240 ntu, density from 1.03 to 1.10 g / cm3. however, increasing the particle size should be limited the size of 1 micron, as a small number of particles larger than 1 micron, can lead to the formation of unstable emulsion during storage. so, the best emulsion should be selected: № 2 of gum arabic (oil phase 10%). increasing the amount of oil phase in the system at a constant amount of stabilizer condition occurs when the amount of the stabilizer is not enough to stabilize the emulsion system. it is therefore important to find the optimal concentration of stabilizer, the optimal amount of oil phase. to this end, five samples of emulsions were examined, a constant number of oil phases changing the amount of stabilizer. 2. the research on the effect of the number of characteristics of stabilizer emulsion obtained showed that the increase in the amount of gum arabic from 4 to 7% (the oil phase content 10%) average particle size is reduced from 0.75 to 0.50 microns, turbidity from 192 to 154 ntu. however emulsion viscosity increases from 14 to 17 cp density from 1.04 to 1.10 g / cm3. thus, the increase in the number of stabilizer at a constant amount of oil phase emulsion stability improves as particle size decreases, but has little effect on toughness. however, the increase in the concentration of the stabilizer in the emulsion system leads to increased cost emulsion. therefore, given the level of stability and moisture product, and taking into account the price of the emulsion obtained, the optimum water content and stabilizer for oil phase emulsions of gum arabic are 5% and 10% emulsion (№ 7) respectively. 3. the third stage of the study is to establish the optimal homogenization pressure for a particular formulation of emulsions with the best performance. four identical emulsions according to selected recipes with gum arabic (№ 7) were prepared and homogenized at different pressures. successive top pressure homogenization, as it affects the particle size and lowers the pressure, the stability of emulsion system remains unchanged. according to the research on four emulsions, the best rates and pressure homogenization of emulsions is defined as the optimum pressure homogenization under these conditions. optimal performance for homogenization of emulsions of gum arabic pressure first step / second step [bar] 280/40 [bar], the number of cycles of homogenization 2; finished emulsion particle size less than 1 micron, since the pressure sinister this range can lead to the formation of particles greater than 1 micron, unstable emulsion is formed, which is stratified during storage. pressure above this range can lead to the formation of particles less than 0.3 microns, which will reduce moisture emulsion. by using the proposed method it is possible to ensure complete dissolution of the ingredients by choosing the dissolution temperature conditions, speed and time of mixing the components in the respective phases, obtaining parameters for preemulsion and homogenization. this will ensure the reduction of time when preparing emulsions at lower cost of energy compared to the prototype. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 321 4. emulsions with starch increase the oil phase from 8% to 14%, leading to an increase in the average particle size of 0.67 to 0.97 microns, viscosity from 20 to 26 cp, turbidity from 143 to 196 ntu, density from 1.03 to 1.10 g / cm3. however, increase in the particle size should be limited to the size of 1 micron, as a small number of particles larger than 1 micron, can lead to the formation of unstable emulsion during storage. so, the best performance selected emulsion is № 3 with starch (oil phase 12%). the increase in the amount of oil phase in the system at a constant amount of stabilizer condition occurs when the amount of the stabilizer is not enough to stabilize the emulsion system. it is therefore important to find the optimal concentration of stabilizer, the optimal amount of oil phase. 5. the emulsion with starch, the stabilizer increases from 8 to 14% (if the content of the oil phase is of 11%) leading to a decrease in the average particle size of 0.98 to 0.68 microns, turbidity from 210 to 157 ntu, the increase in viscosity from 19 to 25 cp and density from 1.04 to 1.10 g / cm3. thus, increasing the number of stabilizer at a constant amount of oil phase emulsion stability improves as particle size decreases, but has little effect on moisture and toughness. however, increasing the concentration of the stabilizer in the emulsion system leads to increased cost emulsion. therefore, given the level of stability and moisture of the product and taking into account the price of the emulsion stabilizer obtained optimum water content and oil phase emulsion of starch is of 12% and 11%, respectively, compounding the emulsion number 9. 6. the third phase of the study is to establish the optimal homogenization pressure for a particular formulation of emulsions with the best performance. four identical emulsions according to selected recipes, with starch (№ 9) were prepared and homogenized at different pressures. successive top pressure homogenization, as it affects the particle size and the lower the pressure, supporting the stability of emulsion system remains unchanged. step change top pressure of 20 bars. according to the research of four emulsions, the emulsion is elected the best rates and pressure homogenization of emulsions is defined as the optimum pressure homogenization under these conditions. optimal performance for homogenization of emulsions with starch: pressure first step / second step [bar] 200/50 [bar], the number of cycles of homogenization 2; finished emulsion particle size less than 1 micron. pressure destroying this range can lead to the formation of particles greater than 1 micron, unstable emulsion is formed, which is stratified during storage. pressure above this range can lead to the formation of particles less than 0.3 microns, which will reduce moisture of emulsion [15-16]. by using the proposed method it is possible to reduce the cost of emulsions by using a cheaper emulsifier starch compared to arabic and ensure complete dissolution of the ingredients by choosing dissolution temperature conditions, speed and time of mixing the components in the respective phases. this will ensure reduction of time in preparing emulsions at lower energy consumption and reduce the cost of emulsions in comparison with the prototype. the practical significance of the results. the technology used is based on theoretical and experimental research, including a range of aromatic emulsions, which are regulated by tu 10.8-02070938209: 2015 "emulsions aromatic food industry" and technological instructions of their production. technological recipes for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4– 2016 oksana lugovska, vasilij sidor, use emulsions in production restaurants and food industry, food and environment safety, volume xv, issue 4 – 2016, pag. 312 – 322 322 drinks and industry zrh with aromatic emulsions were used. an organizational and technological measure was used to introduce new products into production. compliance tests of products from the organoleptic, physico-chemical point of view and safety parameters and indicators were made. the research was implemented by introducing developed technologies in the production of "nautilus" (12.01.15). thus the results of the research were implemented in the educational process nuft. 5. references [1]. phillips g.o., williams p.a., (eds.) handbook of hydrocolloids. cambridge: wood head publishing, 156 pp, (2000) [2]. mckenna v.m. (ed.), texture in food vol. 1: semi-solid foods. cambridge: woodhead publishing, 480 pp, (2003) [3]. rehbinder. p.s. the theory of emulsions. "colloidal magazine." t.vii. vyp.z, 1946, p. 157. [4]. kremnev la, soskin sa, homogenization highly concentrated emulsions. "colloidal magazine" th vyp.z, 1948, s.209. [5]. kremnev l.y, kuprik b.c. homogenization in the capillaries, "colloidal magazine." t.hr /., vyp.2, 1952, s.98. [6]. kremnev.l.ya., ravdel aa on the mechanism of emulsification. "colloidal magazine." t xvi. vol. 1.1954, p. 17 [7]. stephen p., u.s. patent for invention № 6576285, cholesterol lowering beverage, bader, fowler, 10.06.2003. [8]. imeson a. (ed.), thickening and gelling agents for food. 2nd ed., london: blackie academic and professional, with 408 (1999). [9]. aymeson, food thickeners, stabylyzators, publishing "profession", st. petersburg, 2012. century. 24-44. [10]. aymeson,thickeners, stabylyzator, art. 351-382, profession publishing house, st. petersburg 2012 [11]. g.m.lyavynets, a.v.gavrish, o.v.nyemirich, l.y.arsenyeva "technology emulsion sauces such high nutritional value" (science and innovation. 2013. t. 9. № 6. p. 15-19) [12]. horalchuk a.b. technology termostabylity emulsion sous vegetables based raw materials. manuscript. [13]. whistlerr.l.b.,millerj.n., paschall e.f., (eds.), starch chemistry and technology. 2nded. orlando, fl: academic press, 1984, 3508. galliard t. (ed.) starch; properties and potential /society of chemical industry. chichester, uk: john wiley and sons, 210 с (1987). [14]. atwellw.a. thomas d.j. starches. — st. paul, mn: american association of cereal chemists, 150 с. (1997). [15]. borisenko o.v., alekseev j.a., klimov s., methods of creating highly concentrated flavoring emulsions for soft drinks, food ingredients. raw materials and additives, 2, 18-19, (2002). [16]. bogach a., aromatic emulsion for manufacture of soft drinks,food& drinks. food & beverage, 4 s. 10-11, (2003). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 contents: 1. sucrose cooling crystallization modelling valeriy myronchuk, oxana yeshchenko, maryna samilyk 109 114 2. information technology in designing high-performance equipment for biomass compacting dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar 115 122 3. antimic r obial a ctivit y of t he pr ob i otic s train lactobacil l us delbru eckii ss p. bulgaricus gb of h uman or i gin a g ainst p ath o ge ns rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva 123 129 4. researches regarding development body and cutting reports at oncorhynchus mykiss species gabriel vasile hoha, ionut bogdan pagu, catalin emilian nistor, emanel măgdici, benone păsărin 130 134 5. changes of some dehydrogenase activities in the leaves of peach cultivar springcrest naturally infected with the fungus taphrina deformans rodica ciobanu 135 142 6. antioxidant activity of some essential oils sonia amariei (gutt), simona ciornei (ștefăroi), elena sănduleac (todosi) 143 147 7. prussian blue based screen printed biosensors with improved characteristics of long term and ph stability florentina huțanu, maria marcu, gheorghe gutt 148 155 8. comparative study of oxidative stability for different types of vegetable oils sonia amariei (gutt), elena sănduleac (todosi), simona ciornei (ștefăroi) 156 160 9. some chemical aspects during white cabbage pickling process marcel avramiuc 161 168 10. estimation of biochemical properties of walnuts from the region of suceava romania ana leahu, cristina damian, mircea oroian, cristina elena hreţcanu 169 175 11. statistical study of the dependence between concentration of metallic elements migrated from stainless steel grade aisi321 and working parameters silviu-gabriel stroe, gheorghe gutt 176 185 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 12. lactic acid production by bacteria isolated from rhizospheric soil of dahlia tubers octavian baston, octavian barna 186 191 13. an interdisciplinary approach on the influence of packaging design on emotional response to chocolate alice-iuliana roșu, cristina-elena hrețcanu, ana leahu 192 – 206 14. contributi ons to t he developm ent of t he m aterial balan ce of mi g rati on p rocess of metal i ons f r om t he ais i3 04 stainless st eel in acetic a cid s oluti ons silviu-gabriel stroe, gheorghe gutt, maria poroch-seriţan 207 213 15. author instructions i v 16. subscription information vi microsoft word 12 dabija adriana_1 articol cu citari.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 74 study on the the amount starter culture used in yog ur t m an u fac tur ing *adriana dabija1 faculty of food engineering, stefan cel mare university od suceava, romania adriana.dabija@fia.usv.ro *corresponding author received 25 march 2012, accepted 11 may 2012 abstract: among fresh dairy products, yogurt holds the largest share of value and is classified as a functional food with health benefits. researches conducted worldwide have highlighted the beneficial effects of acid dairy product consumption on health, due to the viable microorganisms present in the finished product when sold to consumers. yogurt is a fermented milk product with controlled technology benefiting from the activity of specific lactic bacteria, which have a well known taxonomy and morphology, making them easily detected and differentiated. the role of lactic bacteria in yogurt is that of milk acidification, the synthesis of flavor, texture and viscosity development. interactions between lactic bacteria in yogurt are very complex and beneficial for stimulating the growth of biotechnological properties.the technology of yogurt manufacturing uses pure bacterial cultures selected in specialized laboratories, which are delivered to milk processing companies in either liquid or dry form. this paper proposes a study on the influence of the amount of added leaven towards the quality of the finished product. it was used as raw material cow's milk with 2.8% fat, which was then subjected to processing according to classical method, fermentation in packaging. . it was found that yogurt made with the smallest amount of leaven, only 1%, was appreciated by tasters and from a physico-chemical point of view it is a product within the normal parameters of quality keywords: functional food, viable microorganisms, lactic bacteria 1. introduction milk and dairy products are foods with exceptional nutritional value, but also a source of bioactive substances with multiple physiological activities beneficial to the human body. yogurt is a fermented dairy product obtained from milk by the action of some lactic bacteria; a product particularly appreciated by consumers. worldwide various kinds of yogurt are produced, some locally using traditional methods, while others are produced by more elaborate industrial processes, with strict control for each manufacturing phase [1]. 1.9 billion gallons of milk are consumed globally, the demand for milk having increased in the last 7 years by 14%, higher, for example, than the increase in demand for oil, which is only 13% for the same period [2]. this trend stems from the fact that people became aware of the beneficial influence of milk and dairy products in general. yogurt quality depends on the characteristics of the milk raw material, as well as numerous technological factors (technology adopted, equipment used, selected lactic bacteria starter cultures, etc.) [3, 4]. for milk fermentation and obtaining a product wirh specific biotechnological properties, the milk is seeded with a culture of lactic bacteria by "direct inoculation" with thermophilic lactic food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 75 bacteria: streptococcus thermophilus and lactobacillus bulgaricus. the role of lactic bacteria in yogurt manufacturing is that of milk acidification, the synthesis of flavor, development of texture and viscosity [5]. the technology of yogurt manufacturing uses pure bacterial cultures selected in specialized laboratories, which are delivered to milk processing firms in liquid or dry form. this paper proposes a study on the influence of the amount of lactic bacteria starter culture on yogurt quality. 2. experimental to obtain the yogurt was used as raw material cow's milk with 2.8% fat, which was then subjected to processing according to classical method, fermentation in packaging. after pasteurization and refrigeration, milk was distributed in plastic packaging of 250 ml and inoculated with a specific culture for yogurt in varying amounts (1 ÷ 3%), resulting in the final 5 samples of yogurt finished product: sample 1 yogurt with 1% leaven, sample 2 yogurt with 1.5% leaven, sample 3 yogurt with 2% leaven, sample 4 yogurt with 2.5% leaven; sample 5 yogurt with 3% leaven. after seeding, samples were subjected to thermostatic incubation at 45°c / 3 hours, during which the milk clotting occurred. after the incubation, samples were cooled to 20°c, then stored in the refrigerator for 12 hours at 6°c and then were analyzed. the yogurt samples studied were subjected to the following determinations: sensory analysis method of rating scale; fat content gerber butirometric acid-method; protein substances content kjeldahl method; total solids content the oven drying method; acidity titrimetric method. 3. results and discussion the five samples of yogurt were subjected to sensory analysis, followed by the physical and chemical analysis. the basic principle of sensory analys was to assess each organoleptic characteristic by comparison with the scale score of 0-5 points and obtain the average score given by the group of tasters (table 1). table 1 rating scale used in sensory analysis (costin, g.m., et al, 2005) number of points to be granted rating product features that constitute the basis for assessing the organoleptic characteristics 5 very good specific product characteristics (texture, flavor, surface brightness, acidity), pregnant, very well defined, no defects 4 good specific product features positive, fairly shaped, of very small defects 3 satisfactory positive specific product characteristics, rather shaped and contoured, show small defects 2 nonsatisfactory product characteristics has gaps or defects, which leads some users to repel 1 inappropriate the product has gaps or obvious defects different characteristics which leads consumers to reject it sensory analysis of the 5 types of yogurt in the study was conducted with the help of seven tasters. each taster appreciated the yoghurts’ sensory qualities, for each product noting the compliance with the scoring scale. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 76 the results obtained in sensory analysis, detailed for each taster in part and for each product and for each sensory property are presented in the tables below. for sample 1, the sensory analysis results are presented in table 2. table 2 organoleptic analysis results – sample 1 no. taster degree of pleasure acidity consistency surface brightness smell total rating 1 3 2 5 4 3 17 2 3 2 3 4 3 15 3 5 3 4 4 4 20 4 4 3 4 3 4 18 5 4 2 3 2 3 14 6 2 3 4 4 2 15 7 4 3 4 4 3 18 total 25 18 27 25 22 117 the maximum score for this type of yogurt was 20 which was given by taster number 3. the best sensory quality of the product is its consistency, which received a score of 27 points, followed by the degree of pleasure and surface brightness 25 points, followed by smell with 22 points. acidity received the lowest score 18 points, this product seemed too sweet to tasters. sensory analysis results for sample 2 are summarized in table 3. table 3 organoleptic analysis results – sample 2 no. taster degree of pleasure acidity consistency surface brightness smell total rating 1 5 3 4 3 3 18 2 2 3 2 2 3 12 3 4 3 4 3 3 17 4 2 1 2 2 3 10 5 4 1 2 4 3 14 6 4 2 2 3 4 15 7 3 2 2 4 4 15 total 24 15 18 21 23 101 the maximum score obtained for sample 2 is 18 points and was given by taster no. 1. as with sample 1, sample 2 was best appreciated in terms of degree of pleasure (acceptability), this characteristic totaling 24 points. the smell is ranked second place with a score of 23 points, followed by surface brightness with 21 points and consistency with 18 points. for sample 3 sensory analysis results are presented in table 4. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 77 table 4 organoleptic analysis results – sample 3 no. taster degree of pleasure acidity consistency surface brightness smell total rating 1 4 3 4 4 3 18 2 4 2 3 5 3 17 3 1 1 2 4 2 10 4 4 2 4 4 4 18 5 3 1 4 2 2 12 6 4 2 3 3 3 15 7 4 3 4 4 3 18 total 24 14 24 26 20 108 the panel awarded the maximum score of 18 points, given by three tasters, and obtained the minimum score of 10 points which was granted by taster no. 3. surface brightness received the maximum score of 26 points, followed by consistency and degree of pleasure by 24 points. the lowest score was received for acidity, only 14 of the maximum score of 35. sensory analysis results for sample 4 are summarized in table 5. table 5 organoleptic analysis results – sample 4 no. taster degree of pleasure acidity consistency surface brightness smell total rating 1 2 3 2 3 2 12 2 2 3 2 2 5 14 3 4 3 3 4 3 17 4 3 3 2 4 3 15 5 3 3 4 4 3 17 6 3 3 1 2 3 12 7 2 4 1 5 3 15 total 19 22 15 24 22 102 surface brightness obtained the highest score of 24 points, followed by acidity and smell both with 22 points. the degree of pleasure gained 19 points, followed by consistency with the lowest score of 15 points. for the last sample, sample 5 sensory analysis results are presented in table 6. table 6 organoleptic analysis results – sample 5 no. taster degree of pleasure acidity consistency surface brightness smell total rating 1 4 3 3 3 4 17 2 2 3 2 2 4 13 3 2 3 3 4 4 16 4 4 1 2 2 2 11 5 2 3 3 2 4 14 6 5 3 3 3 3 17 7 5 2 4 4 4 19 total 24 18 20 20 25 107 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 78 analyzing the data shows that the maximum score calculated for this test is 19 points and was awarded by taster no. 7, and the minimum score is 11 points and is given by taster no. 4. the most appreciated the quality of this product is the smell with 25 points, followed by the degree of pleasure with 24 points and tied, the surface brightness and consistency with 33 points each. figure 1 shows that the sample of yogurt that has obtained the highest score for the sensory characteristics is to use the smallest amount of starter culture, 1%. 117 101 108 102 107 90 95 100 105 110 115 120 n um be r o f p oi nt s p1 p2 p3 p4 p5 sample figure 1. summary of organoleptic evaluation of yogurt samples physico-chemical analysis results are summarized in table 7. table 7 physico-chemical characteristics of the studied yogurt samples yogurt sample physico-chemical characteristic s1 s2 s3 s4 s5 fat content, % 2,80 2,85 2,80 2,74 2,80 protein, % 3,20 3,25 3,18 3,12 3,20 solids, % 11,50 11,85 11,05 11,09 11,90 acidity, °t 74,5 77 80,2 78 81 examination of the data obtained the following: in terms of fat content of yogurt, only one sample sample 4 does not fit the standardized fat content (2.74% instead of 2.8%); protein content and solid matter content that are close in value, registering small differences between samples. samples 3 and 4 do not meet the conditions imposed by romanian standards, namely min. 11.3% total solids for this type of yogurt, with 2.8% fat; acidity of the samples studied are within normal limits for such products. there is a correlation of this indicator with the amount of starter cultures used to obtain the yogurt (increasing the amount of starter culture led to higher acidification of yogurt samples analyzed). 4. conclusion yogurt is the best known fermented dairy product that is produced in a wide range of varieties that differ by texture, taste and aroma. one of the determinants of the quality of yogurt finished product is the quality and quantity of lactic bacteria starter cultures specific in yogurt manufacturing, respectively a culture consisting streptococcus thermophilus and lactobacillus bulgaricus. technology for obtaining the starter culture varies from manufacturer to manufacturer, but it is essential that both species are present. this paper presented a study on the influence of the amount of leaven of lactic bacteria used in yogurt obtainment, a study performed by assessing organoleptic characteristics and determining the main food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 79 physico-chemical characteristics of yogurt samples obtained with varying amounts of leaven. analyzing the data obtained it was found that yogurt made with the smallest amount of leaven, only 1%, was appreciated by tasters and from a physico-chemical point of view it is a product within the normal parameters of quality, as with current standards. an increasing amount of leaven over this value led to a worsening quality of yogurt, and in technological practice this may negatively affect the production cost of the finished product. fermented dairy products are considered functional foods, especially due to the beneficial physiological effects on the human body, effects resulting also from the biochemical activity of starter cultures used to obtain them. 5. references [1]. banu c., et al., the influence of technological processes on foodstuffs quality (influenţa proceselor tehnologice asupra calităţii produselor alimentare), editura tehnică, bucureşti, (1974) [2]. costin, g.m., et al., fermented dairy product (produse lactate fermentate), editura academica, galaţi, (2005) [3]. banu c., vizireanu, c., industrial processing of milk (procesarea industriala a laptelui), editura tehnică, bucureşti, (1998) [4]. banu c., et al., selected cultures and preparation quide and microbiological examination in dairy (îndrumător pentru prepararea culturilor selecţionate şi examenul microbiologic în industria laptelui, editura tehnică, bucureşti, (1986) [5]. collado m.c., et al., food research international, 39, p.530-535, (2006) microsoft word 0b. contents 3_2012.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 3 – 2012 contents: 1. analysis of chlorinated phenols in water ján ilavský, ján hrivňák, danka barloková 5 2. probiotic properties of lactobacillus acidophilus z10, isolated from naturally fermented sourdough rositsa denkova, svetla ilieva, donka dimbareva, zapryana denkova 15 3. experiments concerning physico-chemical and microbiological control of bakery yeast industrial production adriana dabija, oana elena buzatu 21 4. study of a new coordination compounds based on cr(iii) and succinimide in aqueous solution mihaela dana tutulea (anastasiu), igor cretescu, doina sibiescu, ioan rosca 27 5. removal of humic substances in water by coagulation danka barloková, ján ilavský 31 6. pseudomonas sp. and aeromonas sp. selection for treatment of goose down washing wastewater aleksander slavov, zapryana denkova, mima hadjikinova, nikolai mihalkov 37 7. separation of selected pesticides by an hplc technique; performance parameters and validation camelia popa 42 8. enviro nme ntal s a fety an alysis of the pro jecte d mu nici pal lan d fill a rea i n a small c ity in uk r ai ne alla choban, igor winkler, myron rogozynskyi 47 9. copper ions concentration using ion exchange resins petru bulai, camelia popa 52 10. researches on the stability and shelf-life of canned tomatoes amelia buculei 56 11. effe ct o f di ffere nt milk substitu tes on rheological pro perties o f pud dings cristina damian 62 12. fish pollution with heavy metals camelia popa, petru bulai 68 13. author instructions 72 14. subscription information 77 звіт з ндр 29-81 за 2007 – 2009 р 71 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1 2016, pag. 71 75 mathematical modeling of preparing "oil / water" emulsions *oksana lugovska1, vasilij sidor1 1national university of food technologies, volodimirska 68, kiev, ukraine, oksana.lugovska320@gmail.com *corresponding author received march 6th 2016, accepted march 29th 2016 abstract: past studies on the effect of the number of emulsifiers in emulsion characteristics obtained showed that the change in the fate of particle distribution depended on the particle size emulsion close to gaussian distribution. therefore, the designed factor model depends on the particle size emulsion of their number. keywords: emulsion, phase, starch, arabic gum, mathematical modeling 1. introduction nowadays, the emulsion is widely used in different sectors of the food industry. getting a stable emulsion system is an important and promising issue. as the stabilizing and emulsifying ingredient in the manufacture of scented oil emulsions using gum arabic (e 414) and starch (e 1450). starch is one of the most widely used thickeners and emulsifier. gum arabic provides better emulsion stability [1]. found that a stable, emulsions are closely associated with the mechanism of dispersion and depends on many factors, such as oil content, type and concentration of emulsifier, the route of administration phases, time and intensity and degree of dispersion and temperature. study of factors that ensure stability of emulsion, led to the conclusion that the critical degree of dispersion [2–3]. experiments found that for each type of emulsifier it has its own optimum concentration that provides the highest resistance obtained emulsions [4]. for an introduction to emulsify oils (for each concentration of emulsifier) is also optimum in which the most stable emulsion is obtained, that are determining the optimal ratio between the aqueous and oil phases. for each emulsifier has its own optimal concentration for the stability of the emulsion. [5-6]. the optimum concentrations of emulsifiers for certain ratios of the phases in obtaining stable emulsions are not fixed and depend on the degree of dispersion [7]. to determine the most effective parameters of technological processes of production emulsions to build a mathematical model of conduct and its optimization of defined parameters. model a conventional image of the object which displays its most significant characteristics required for the study. • any model performs predictive function without which it would build inappropriate for theory and practical use. • experimental models based only on the laws of probab ility theory. • in constructing these models studied processes conventionally regarded as deterministic, but the model is introduced elements of evaluating the probability of obtaining a certain result. http://www.fia.usv.ro/fiajournal mailto:oksana.lugovska320@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 oksana lugovska, vasilij sidor, mathematical modeling of preparing "oil / water" emulsions, food and environment safety, volume xv, issue 1 – 2016, pag. 71 – 75 72 determined factor analysis -it analysis of factors resulting indicator can be presented as a product, part or the algebraic sum of factors [8]. past studies on the effect of the number of emulsifiers in emulsion characteristics obtained showed that changing the fate of particle distribution depending on the particle size emulsion close to gaussian distribution probability distribution, which is set in one-dimensional case, the probability density function. 2 2 2 )( 2 1 )(       x exf where μ expectation (average), median and mode of distribution, and the parameter σ standard deviation (σ ² variance) distribution. if the variable has a normal distribution, then it there (end) points for all values such that in different parts of the domain, it can be given different analytical expressions or piecewise given function. piecewise given function is a function that is defined on the set of real numbers given on each of the intervals, the domain of the components, a separate formula. in general terms:          33 22 11 ),( ),( ),( bxaifx bxaifx bxaifxf y   where function defined on a certain interval the lower limit of the range upper limit of the range, the analysis of the experimental data shows that the value of the particle size can be determined at such intervals (0, 0.1); [0.1, 1) and (1, 10). in the interval (0, 0.1) function is a linear regression on the interval (0.1, 1) and (1, 10) function is polynomial model norder. if we denote the fate of particle distribution variable y, a particle size emulsion x, then the general model takes the form.          .101, ;11,0, ;1,00, 5 5 4 4 3 3 2 210 5 5 4 4 3 3 2 210 xifxbxbxbxbxbb xifxaxaxaxaxaa xifc y calculation of the model is the method of least squares (mls). the method is based on minimizing the sum of squared deviations of certain functions of the unknown variables. it is used to "solve" overriding systems of equations (equations when the number exceeds the number of unknowns), to find a solution in the case of conventional systems of nonlinear equations to approximate point values of some function. mls is a basic regression analysis methods for the estimation of the unknown parameters of regression models for the sample data.objective research to conduct mathematical modeling study of the influence quantity of emulsifier gum arabic or starch and the oil phase to final figures emulsions "oil / water" 2. material and methods we built a model of distribution of particles emulsions containing emulsifiers equal amount of gum arabic (5%) and different amounts of oil phase.for oil content of 14.0% phase model is:                101,0001,0002,0 017,0041,0308,0867,1 11,0,381,78106,299 517,339261,97381,27974,3 1,00,0 54 32 54 32 1 xifxx xxx xifxx xxx xif y for each oil phase built their system, describing the experimental data for predicting the future of individual particle size similarly, processed research data at constant oil phases and changes in the content and formed stabilizer system of mathematical models describing the formation of particles of different size stabilizer. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 oksana lugovska, vasilij sidor, mathematical modeling of preparing "oil / water" emulsions, food and environment safety, volume xv, issue 1 – 2016, pag. 71 – 75 73 similarly, there was a process of modeling the distribution of the fate of particle size emulsion particles at different pressure homogenization. 3. results and discussion figure 1 shows the experimental and calculated curves of particle size distribution with stable emulsions containing emulsifier gum arabic 5% oil content and a different phase (8.0%; 10.0%; 11.0%; 12.0%; 14 0%). a) b) fig. 1. curves size distribution of particles emulsions with constant content of emulsifier gum arabic 5% and oil content varying phases: 1 8.0%; 2 10.0%; 3 11.0%; 4 12.0%; 5 14.0%: a) experimental data; b) estimates construct models of the size distribution of particles emulsions with constant emulsifier starch content (12%) and different content oil phase. figure 2 shows the experimental and calculated curves of size distribution of particles emulsions with constant emulsifier starch content of 12% oil content and a different phase (8.0%; 10.0%; 11.0%; 12.0%; 14 0%. a) b) fig.2. curves size distribution of particles emulsions with constant emulsifier starch content of 12.0% and oil content varying phases: 1 8.0%; 2 10.0%; 3 11.0%; 4 12.0%; 5 14.0%: a) experimental data; b) estimates food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 oksana lugovska, vasilij sidor, mathematical modeling of preparing "oil / water" emulsions, food and environment safety, volume xv, issue 1 – 2016, pag. 71 – 75 74 construct models of the size distribution of particles emulsions with a constant number of oil phase 10.0% and different content emulsifier gum arabic. figure 3 shows the experimental and calculated curves of size distribution of particles emulsions with a constant number of oil phase 10.0% and different content emulsifier gum arabic (4.0%; 5.0%; 5.5%; 6.0%; 7.0%. a) b) fig. 3. curves size distribution of particles emulsions with a constant number of oil phase 10.0% and different content emulsifier gum arabic 6 4.0%; 7 5.0%; 8 5.5%; 96.0%; 10 7.0%: a) experimental data; b) estimates construct models of the size distribution of particles emulsions with a constant number of oil phase 11.0% and different content emulsifier starch. for example, the starch content of 8.0% emulsifier model is: a) b) fig. 4. curves size distribution of particles emulsions with a constant number of oil phase 11.0% and different content emulsifier starch 6 8.0%; 7 10.0%; 8 11.0%; 9 12.0%; 10 14.0%: a) experimental data; b) estimates food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 oksana lugovska, vasilij sidor, mathematical modeling of preparing "oil / water" emulsions, food and environment safety, volume xv, issue 1 – 2016, pag. 71 – 75 75 4. conclusion the best result of research in starch emulsion – is to obtain the maximum number of particles of about 1 micron. in order to determine the most effective process parameters of food production to build a mathematical model of optimization of certain parameters. analysis of a priori information and experience on current technology and conditions of production of aromatic emulsions allow for process modeling approaches applied regression analysis and choose the regression equation of the second order. the results of computational experiments show that almost all indicators emulsion stabilizer content is more important. the relative error of calculations does not exceed 3%. for all models fisher criterion settlement does not exceed the tabular value that indicates the adequacy of research models and can be used for decision making. unlike emulsion stabilizer acacia, modeling parameters emulsions with starch show that oil phase is more influentual. the relative error of calculations does not exceed 4.5%, and the estimated fisher criterion is less than the table and all models also adequately describe the study process for all models of criterion fisher estimated value does not exceed tabular ftabl. = 4.96/. therefore all the models adequately describe the study process and can be used for decision-making). 5. references [1]. phillips g.о., williams p.a., (eds.) handbook ofhydrocolloids. cambridge: wood head publishing, 156 с, (2000). [2]. mckenna в.m. (ed.), texture in food — vol. 1: semi-solid foods. — cambridge: woodhead publishing, 480 с, (2003). [3]. stephen p., u.s. patent for invention № 6576285, cholesterol lowering beverage, bader, fowler, 10.06.2003. [4]. imesona. (ed.), thickening and gelling agents for food. 2nded., london: blackie academic and professional, 408 с, (1999). [5]. whistlerr.l.b.,miller j.n., paschall e.f., (eds.), starch chemistry and technology. 2nded. orlando, fl: academic press, 1984, 3508. galliard t. (ed.) starch; properties and potential /society of chemical industry. chichester, uk: john wiley and sons, 210 с (1987). [6]. atwellw.a. thomas d.j. starches. — st. paul, mn: american association of cereal chemists, 150 с. (1997). [7]. a. aymeson, food thickeners, stabilizers, gelling agents, "profession" publishers, st. petersburg, 2012.art. 24-44 issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xv, issue 1 31 march 2016 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:fiajournal@fia.usv.ro mailto:gutts@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro 341 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 4 2016, pag. 341 346 the influence of proteins on the technological process of making bread with fructose vera drobot1, *anastasiia shevchenko1, olha marchenko1 1 department of bakery and confectionery goods technology, national university of food technologies volodymyrska str. 68, kyiv, ukraine, 01601, nastyusha8@ukr.net * corresponding author received 15th november 2016, accepted 28th december 2016 abstract: the article discusses the necessity of enriching products by proteins for patients suffering from diabetes. the influence of casein, egg albumin and whey protein on microbial and structural and mechanical processes in the dough and on the quality of the finished bread for diabetics was studied. the aim of our research was to determine the influence of animal proteins on the technological process of making bread with fructose. the studies included recommendations on nutrition for patients with diabetes. as indicators of the technological process there were investigated gas producing, gas holding ability of dough and its viscosity as well as the quantity and quality of gluten, quality of finished products. it has been established that it is technologically advisable to enrich bakery products for people with diabetes by fructose and by studied animal proteins due to their high biological value. keywords: bread, diabetes, casein, egg albumin, whey protein. 1. introduction nowadays the actual problem is diabetes mellitus because there are millions of people who suffer from this disease. however, published data do not reflect the true dissemination of the disease. it is believed that for each registered person there are 2 2.5 undiagnosed patients [1]. a key role in maintaining patients’health with diabetes plays nutrition. one of the main food products is bread, but there are a few types of bread for diabetics enriched by nutrients with a low glycemic index. according to the who expert committee patients with diabetes need products with the low contents of easily digestible carbohydrates, i.e. which contain ingredients with a low glycemic index, and especially sugar free or with reduced sugar content. scientists found appropriate to replace sugar by fructose when producing bread, because the monosaccharide fructose is sweeter than sucrose 1.5-1.7 (1.5 – 1.7) times, it does not require insulin for assimilation, does not affect significantly blood sugar level. the glycemic index of fructose is 20 whereas sucrose 70 [2]. traditional breads, especially from topgrade flour are characterized by low nutritional value, including restricted anabolic protein targets due to low content of lysine and threonine [3]. it is known that bread from top-grade flour is a product with a low content of complete protein; its limited amino acid is lysine. the variety of bread commercially available is quite wide, but there is limited number of bakery products for special purposes, enriched by proteins. when there is a growing prevalence of diabetes this problem is particularly acute because such patients should have complete proteins in daily diet [4]. therefore, the actual problem is the increase in the biological value of bakery products especially because of this disease [5]. http://www.fia.usv.ro/fiajournal mailto:nastyusha8@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 vera drobot, anastasiia shevchenko, olha marchenko, the influence of proteins on the technological process of making bread with fructose, food and environment safety, volume xv, issue 4 – 2016, pag. 341 – 346 342 this problem can be solved by using natural raw materials that contain a number of biologically active substances. leading scientists conducted research in this field, but data on the impact of proteins on bread manufacturing process and the quality of finished products with fructose have not been found yet. as it is known, the works of many researchers are focused on studying the possibility of using these natural raw materials in end products of processing sunflower, beans, and a variety of nontraditional supplements for enrichment bread by protein [6]. a number of studies are devoted to such sources of protein like soya, pea, and lupine [7], [8]. raw vegetables are effective in terms of protein enrichment, but in most cases their use is limited because of the negative impact on the manufacturing process and products’ quality. almost all protein sources of enrichment impair the structural and mechanical properties of dough, bread volume and porosity. its main reason is considered to be technological incompatibility of proteins from different materials [9]. it is believed that proteins of bread enrichers interact with gluten proteins and seal their structure due to the formation of new disulphide bonds (-s-s-). an important role in a balanced diet belongs to animal proteins, because they are the most valuable ones due to the balance of amino acid composition [10]. native scientists conducted research concerning the enrichment of bread by these proteins to improve the biological value of bakery products [11], [12], [13]. foreign scientists found that the best sources of enrichment by animal protein are eggs and milk proteins. their assimilation reaches 96 98% [14], [15]. therefore, the aim was to investigate the effect of animal protein such as egg albumin, casein and whey protein on the technological process of making bread with fructose for people who suffer from diabetes. 2. materials and methods the influence of egg albumin, casein and whey protein on the technological process of making bread from top-grade flour containing fructose 5% to the weight of flour has been researched. proteins were dosed in an amount that satisfies 20, 30 and 40% of the daily dose of protein when consuming 277 grams of bread. the effect of supplements on yeast microflora, structural and mechanical properties of dough and the quality of finished products have been investigated. as indicators of the technological process there were investigated gas producing, gas holding ability of dough, its viscosity, quantity and quality of gluten [16], [17]. the indicators of the quality of finished products were: specific volume, porosity, shape stability and acidity of bread. control a sample with fructose without addition of protein enrichers. 3. results and discussion as a result of the experiments it has been found that the reduction of dedicated carbon dioxide during the fermentation of dough when adding proteins is explained by decrease of gas producing ability (table 1). it has been found that in compounds with casein (20%) gas producing ability of dough deteriorated by 7.1% as compared with control, with albumin (20%) – by 6.1%, with whey protein (20%) – by 9.3%. while adding 30% of casein gassing deteriorated by 8.2% compared with control, albumin (30%) – by 7.2%, whey protein (30%) – and by 11.1% respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 vera drobot, anastasiia shevchenko, olha marchenko, the influence of proteins on the technological process of making bread with fructose, food and environment safety, volume xv, issue 4 – 2016, pag. 341 – 346 344 table 1. the influence of proteins on gas producing ability of dough holders of protein number of enrichers, % to provide the daily protein intake the total gas production in dough, [cm3, co2] control 0 1368 casein 20 1271 30 1256 40 1232 egg albumin 20 1277 30 1263 40 1237 whey protein 20 1244 30 1220 40 1204 while enriching by casein (40%) the studied indicator declined by 9.9% as compared with control, with albumin (40%) – by 9.0%, with whey protein (40%) – by 12.2% respectively. the addition of animal proteins to the dough affects adversely the fermentation activity of yeast. it can be explained by high protein buffering, thus creating unfavorable ph of dough for fermentative hydrolysis of starch and accumulation of maltose, which is a reason of reducing the intensity of fermentation. in the case of enrichment products by proteins the important factors are structural and mechanical properties of dough (smp). smvp are characterized primarily by the quantity and quality of gluten. the determination was conducted after softening the dough for 20 minutes at 30˚c for swelling of proteins. the impact of added proteins for this indicator is given in table 2. table 2. the influence of proteins on gluten properties holders of protein number of enrichers, % to provide the daily protein intake w et g lu te n, [% ] d ry g lu te n, [% ] v al ue id k , u ni ts o f in st ru m en t h yd ra ta tio n ab ili ty , [% ] e xt en si bi lit y % , [c m ] control 0 25.52 9.55 73.7 168.1 15.4 casein 20 23.5 8.70 70.3 170.5 15.1 30 21.5 8.03 63.5 168.1 14.9 40 21.1 7.72 56.7 163.6 14.8 egg albumin 20 23.2 8.70 72.0 168.5 15.5 30 23.1 8.63 67.2 167.8 15.3 40 22.8 8.61 63.1 165.2 15.0 whey protein 20 19.04 7.1 52.1 168.0 15.1 30 18.4 6.8 36.4 161.7 14.9 40 not washed thus, the trend of reducing wet gluten when adding proteins is traced, namely in samples with albumin (20%, 30%, 40%) the quantity of wet gluten decreased by 7.8 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 vera drobot, anastasiia shevchenko, olha marchenko, the influence of proteins on the technological process of making bread with fructose, food and environment safety, volume xv, issue 4 – 2016, pag. 341 – 346 344 17.2% compared with control; with casein – by 9.1 10.3%; with whey protein (20% and 30%) – by 25.4 27.9%. when adding 40% of whey protein gluten was not washed. reduction in the quantity of gluten can be explained by the fact that milk proteins are not involved in the formation of gluten and form complexes that affect dough viscosity. when adding albumin the percent of reduction is the least and whey protein – is the highest. however, gluten is highlighted by indicator idk. visco-elastic properties of dough were determined by its specific volume (figure 1). figure 1. gas holding ability of dough. it has been established that the addition of proteins in dough reduces gas holding capacity, in particular casein by 0.8 7.1% depending on the dosage, egg albumin – by 0.8 3.9%, whey protein – by 1.6 6.3 %, apparently due to the negative influence of proteins on fibrinous frame, resulting in the deterioration of co2 holding. elastic properties of dough were determined by spreading the dough balls (table 3). table 3. shape holding ability of dough holders of protein number of enrichers, % to provide the daily protein intake spreading dough balls, [mm] control 0 94 casein 20 112 30 120 40 130 egg albumin 20 106 30 118 40 132 whey protein 20 118 30 122 40 126 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 vera drobot, anastasiia shevchenko, olha marchenko, the influence of proteins on the technological process of making bread with fructose, food and environment safety, volume xv, issue 4 – 2016, pag. 341 – 346 345 when adding proteins to the dough, a trend of spreading and increasing especially with whey protein was observed. it is explained by the decrease in dough viscosity: whey protein reduces the water absorbing capacity of dough by dehydrating action of milk sugar lactose. dilution is observed mostly with increasing of dosage of protein in the recipe. there have been studied the indicators of bread when adding sources of protein. the results are shown in the table 4. table 4. the influence of proteins on the quality of finished products holders of protein number of enrichers, % to provide the daily protein intake indicators specific volume [cm3/100 g] shape stability, [h/d] porosity, [%] end acidity, hail control 0 337 0.42 76 2.0 casein 20 311 0.41 71 2.1 30 299 0.39 71 2.1 40 285 0.38 72 2.3 egg albumin 20 309 0.39 71 2.2 30 296 0.35 72 2.3 40 281 0.33 72 2.5 whey protein 20 301 0.37 70 2.2 30 288 0.36 71 2.2 40 262 0.30 71 2.6 when increasing the dosage of proteins the specific volume of bread, shape stability and porosity decrease. 4. conclusion as a result of studies on the impact of animal proteins on the technological process of making bread it has been found that enrichment of diabetic bakery products by fructose and by protein reduces fermentation activity of yeast microflora and affects adversely the structural and mechanical properties of dough, including reducing the intensity of flatulence, gas producing, gas holding ability, quantity of gluten in dough. however, it is strengthened. but due to the valuable amino acid composition of studied proteins they are considered to be advisable to be used in order to provide patients suffering from diabetes with complete proteins. to ensure the quality of diabetic bakery products enriched by animal proteins there should be applied technological measures to increase the intensity of fermentation and improve the structural and mechanical properties of dough. 5. references [1]. tronko m., efimov a., kravchenko v. epidemiology of diabetes, kyiv: 152, (2000) [2]. polumbryk m., litvyak v., lovkys z., kovbasa v. carbohydrates in food products: monograph, nat. university of pysch. techn., ukraine; sci. and practical centre of nat. acad. of sciences of belarus in food, republic of belarus. minsk: minfina: 592, (2016) [3]. arsenyeva l., dotsenko v., momot a. methodological approaches to the development of new types of baked goods with a balanced chemical composition, food industry, vol. 4: 5 – 8, (2005) [4]. davidenko o., darcel n., fromentin g., tome d. control of protein and energy intake brain mechanisms, european journal of clinical nutrition, № 67: 455-461, (2013) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 4 – 2016 vera drobot, anastasiia shevchenko, olha marchenko, the influence of proteins on the technological process of making bread with fructose, food and environment safety, volume xv, issue 4 – 2016, pag. 341 – 346 346 [5]. kaprelyants l., iorhachova k. functional foods: monograph, odessa, press: 312, (2003) [6]. lysyuk g. increase protein and reduced carbohydrates by adding to the bakery kernel of sunflower seeds, bakery and confectionery industry ukraine, № 6: 40 – 42, (2008) [7]. arsenyeva l., borisenko a., cooper n. composition and digestibility of proteins bean product processing, proceedings of the national university of food technologies, № 15: 51 – 54, (2004) [8]. kaprelyants l, shpыrko t., trufkaty l. soy products and ingredients, chemistry, technology, using: monograph, odessa: tes: 196, (2014) [9]. mahynko v., chernish l. high protein additives in bakery, refining storage and grain, № 6 (183): 57 – 60, (2014) [10]. kretovich v., tokarev r. the problem of food usefulness of bread, moskow, nauka: 147 – 149, (1978) [11]. dotsenko v., shydlovska a., tkachuk y. with food albumin bakery acquire high biological value, bakery & confectionary industry, ukraine, № 2:22 – 26, (2010) [12]. ishchenko t., shydlovska a., tkachuk y., dotsenko v. milk casein as the most effective fortificant of bread, bakery and confectionery industry of ukraine, № 10 (59): 4 – 8, (2009) [13]. tkachuk y., gavrish a., nyemirich a., ishchenko t., dotsenko v. improvement technology bread increased biological value of casein for use, equipment and technology of food production, № 30: 186 – 192, (2013) [14]. hoffman jay r., michael j. falvo protein which is best?, j sports sci med. № 3: 118 – 130, (2004) [15]. marion j. franz protein controversies in diabetes, diabetes spectrum, № 3, v. 13: 132, (2000) [16]. drobot v., yurchak v., bilyk a. chemical control of raw bakery and pasta: teach, nat. university of food. techn, kyiv, condor: 972, (2015) [17]. iso 7517 – 2014 bread from wheat flour. general specifications. 53 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 – 2015, pag. 53 57 nan os iz ed im plant s o f ag + , cu+ and cu 2 + ions into t he s urfa ce laye r of polyc rys tal l ine cds phot ocat al yst fo r th e ox id atio n r eact io n of i od ide ion s *igor kobasa1, lyubomyra odosiy2 1yu. fedkovych national university of chernivtsy, ukraine, 2petro sagaydachnyi academy of ground forces, lviv, ukraine i.kobasa@chnu.edu.ua * corresponding author received 5th march 2015, accepted march 31st 2015 abstract: the process of construction of the nanosized photoactive systems based on cadmium sulfide has been developed. additional ions ag+, cu+, cu2+ can be implanted into the surface layer of cds microcrystals leading to the formation of nanosized particles of corresponding sulfides. high redox photocatalytic efficiency was determined in the model reaction of potassium iodide oxidation for the low-doped samples of cds containing silver and copper ions and the composite nanostructured cds-based sulfides containing nanoparticles of ag2s, cu2s і cus. this effect is caused by the depression of electron-hole recombination because of reduction of the dope agent ions by free electrons, which results in the formation of reactive neutral atoms. alternatively, this effect can also be caused by the transfer of photogenerated charges between likely charged clusters in nanoand microcomponents of the composite material. keywords: cadmium sulfide, potassium iodide, photocatalytic activity, nanostructured composite material, implantation. 1. introduction photocatalytic decontamination of some environment pollution agents through the reaction of oxidative destruction is quite a topical issue discussed widely in some articles [1, 2] and reviews [3, 4]. as seen from analysis of the recent publications, the processes of photocatalytic decontamination (by oxidation or reduction) of various inorganic and organic pollution agents by highly effective semiconducting photocatalysts is interesting for many scientific groups worldwide and the problem of development of such photoactive materials seems very topical [5-8]. the process of electron-hole recombination is naturally inherent for any photoexcited semiconductor and this is the key limiting factor that put obstacles on the catalyst’s efficiency. therefore, the decrease in the photocatalytic efficiency of the composite materials containing two different semiconductors can be caused by spontaneous electrons transfer between the conductivity bands or the analogous process of the holes transfer between the valence bands [9, 10]. the above mentioned transfer processes can take place only if they are allowed thermodynamically. moreover, since the back transfer of the charges is prohibited thermodynamically, the excited semiconducting particles with divided http://www.fia.usv.ro/fiajournal mailto:i.kobasa@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, l yu bo m yr a o d o si y, n a no s i z ed i m pla nt s of t h e i on s a g+ , c u + a n d c u 2 + i nt o t he s u r f ac e l a y er o f p o l y cr y s t al l i n e cd s ph o t oc a t a l y s t f o r t h e r ea c t i o n of ox id at i o n o f i od id e i o ns , f o od a nd e nvir o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , p ag . 5 3 5 7 54 charges can exist during the period of time that is required for interaction between the reaction components causing rise in the quantum yield. various model systems were used in investigations of the photogenerated charges separation [11] but the entire photocatalytic process has been thoroughly investigated for the two model systems only – photodecomposition of water and some alcohols, which produces molecular hydrogen [12] and reduction of methylene blue to its leucoform [13, 14]. it should be emphasized that the photoefficiency of both process is quite high. that is why it seems important to understand if the similar approach can be widened on some other redox systems, for instance, photocatalytic oxidation of iodides. 2. experimental the following source materials were used to build the nanostructured composites ag2s/cds, cu2s/cds and cus/cds: cadmium sulfide with specific surface area sbet = 4.0 m2/g, agno3, cu2cl2, cuso4·5h2o. the solubility product of ag2s, cu2s and cus is much lower than that of cds (these values are 1,0·10-51, 2,5·10-50, 4,0·10-38 and 4,0·10-29 simultaneously [15]). therefore, the former ions can substitute the latter one in the materials. the reaction of substitution has been carried out by stirring of cds suspension in the solution containing the ions of the above mentioned less soluble sulfides. reaction dynamics has been controlled by periodic sampling of the solution followed by the atom-absorption determination of concentrations of the free ions ag+, cu+, cu2+ and cd2+ in the samples. the substituted suspension was centrifuged and the sediment was washed by warm water in order to separate the unreacted components and then dried at the room temperature. redox photocatalytic activity of the composite sulfides was evaluated through amount of the free iodine formed in the reaction of potassium iodide oxidation according to the method described in [16, 17]. efficiency of the free iodine formation (r, %) was calculated by the methods reported in [18]. oxidation of potassium is a suitable model reaction because it is comparatively simple and can be widely applied in the solar cells production technologies [19, 20]. it was considered that the specific surface area of the source cadmium sulfide remained unchanged throughout all chemical modifications. 3. results and discussion similarly to syntheses of bi2s3/cds composites [13], the above mentioned method ensured obtaining of the cds-based materials with exact preplanned values of the surface ions substitution ratio. it was considered that the distance between the surface ions of cadmium and sulfur was equal to the sum of their ionic radiuses while the total surface area of all nanocrystals was equal to the sum of all their ionic radiuses. the former parameter of the 1 g sample makes its specific surface area. this relation was used to calculate the total number of ions cd2+ in the surface monomolecular layer. fig. 1. dependence of the surface occupational ratio for cds and the following substitute ions: ag+ (a), cu+ (b) and cu2+ (c) on their concentration in the reacting mixture. on the other hand, results of quantitative analysis of the surface contents of silver food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, l yu bo m yr a o d o si y, n a no s i z ed i m pla nt s of t h e i on s a g+ , c u + a n d c u 2 + i nt o t he s u r f ac e l a y er o f p o l y cr y s t al l i n e cd s ph o t oc a t a l y s t f o r t h e r ea c t i o n of ox id at i o n o f i od id e i o ns , f o od a nd e nvir o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , p ag . 5 3 5 7 55 and copper ions provided the data required for evaluation of the surface occupational ratios for each substitute ion throughout a series of the cds samples [13]. a dependence of the surface occupational ratio on concentration of the substitute ions in the reaction mixture is shown in fig. 1 (rest of the synthesis conditions was kept constant). at the initial stages of embedding of the substitute ions into the surface layer of cds, only few separated defects could appear. due to the chemical bonds formed between the defects and the substrate lattice, they can be considered either as molecular ions ags-, cusor as molecules ag2s, cu2s and cus. the number of the defects is rising in course of the substrate processing, and interaction between them appears and grows resulting formation of the nanosized semiconducting islands of the substitutes on the surface of cds. thus, two separate groups can be identified among the substituted cds materials. the low concentrated substitute products fall into the first group. as the substitute components concentrations are low, they can be considered as two-component systems: molecule (or molecular ion)/semiconductor. the second group includes the highly concentrated substituted products. as content of silver and copper sulfides are high in the products, they exhibit no molecular properties and play role of classic semiconductors only. according to [11, 12], the latter materials are nanostructured semiconducting composites containing a mixture of nanoparticles of cds and the other sulfides. investigation of photocatalytic activity of the above mentioned materials was carried out using the model reaction of oxidation of iodides. it proved that the activity depends on the nature and concentration of the substitute ions in the material (see fig. 1). fig. 2. efficiency of the free iodine formation by the implanted cds products with ions of ag + (a), cu+ (b), cu2+ (c) as a function on the surface monolayer occupational ratio. as seen from fig. 2, the rise in the free iodine production is registered even for the slightly substituted examples. it can hardly be expected that any semiconducting islands of ags or cus can be formed on such low-substituted material. therefore, the first class of substituted materials appears to have some advanced photoactivity similarly to the two-semiconductor nanocomposite materials as it was described for the process of the molecular hydrogen production [12] or reduction of methylene blue on bi2s3/cds [13]. the process of phototransformation involving the second class material will run similarly to that for the first class. the only difference in the process scheme is related to the charge separation stage involving transfer of the charges between the likely charged areas. as seen from the energy transformation diagram analysis [10], the conductivity band of the nanosized ag2s is located above the conductivity band of cds while the gap between the valence bands of these components is 0.4-0.6 ev wide. that is why the electron transfer processes are thermodynamically hindered while the hole transferring is not. the latter process will result in separation of the photogenerated charges. as a result, the electron-holes recombination will be inhibited in such a particle meaning that its redox activity will rise. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, l yu bo m yr a o d o si y, n a no s i z ed i m pla nt s of t h e i on s a g+ , c u + a n d c u 2 + i nt o t he s u r f ac e l a y er o f p o l y cr y s t al l i n e cd s ph o t oc a t a l y s t f o r t h e r ea c t i o n of ox id at i o n o f i od id e i o ns , f o od a nd e nvir o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , p ag . 5 3 5 7 56 analysis of the energy transformations during light absorption by the synthesized sulfides shows that two different mechanisms of photocatalysis can actually take place. the first mechanism is exhibited by the materials with low content of ag+, cu+ and cu2+ ions sitting in the lattice and bonded chemically with sulfur. this mechanism implies transfer of the photogenerated electrons from the conductivity band to the above mentioned ions and formation of highly reactive atoms of silver and copper. the second mechanism is more intrinsic for the copper and silver-enriched materials. in involves separation of the photogenerated charges by their transfer between the likely charged areas of cadmium sulfide and nanoparticles ag2s, cu2s, cus resulting in oppressed charges recombination. 4. conclusion new cds-based catalysts were developed on the basis of the low-doped or composite nanostructured sulfides containing nanoparticles of ag2s, cu2s, cus. an extremal pattern has been found for the dependencies of the materials photoactivity on their composition and synthesis conditions. the first class of photocatalytic materials includes the polycrystalline cds-based products with ag+, cu+ and cu2+ surface implants similar to those applied to technologies of molecular hydrogen production by photodecomposition of water or photoreduction of methylene blue. our results prove that potential applicability of such photoactive materials can also be widened to some other processes. besides, new reliable methods of synthesis of various implanted cds-based materials with preplanned photocatalytic activity have been developed and tested. 5. references [1]. s. fukahori, h. ichiura, t. kitaoka, h. tanaka. photocatalytic decomposition of bisphenol a in water using composite tio2-zeolite sheets prepared by a papermaking technique. environ. sci. technol., 37, 1048-1051 (2003). [2]. s. kaniou, k. pitarakis, i. barlagianni, i. poulis. photo-catalytic oxidation of sulfamethazine. chemosphere. 60, 372-380 (2005). [3]. d.f. bahnemann. ultrasmall metal oxide particles: preparation, photophysical characterisation and photocatalytic properties. israel journ. chem. 33, 115-136 (1993). [4]. h. fujii, m. ohtaki, k. eguchi. synthesis and photocatalytic activity of lamelar titanum oxide formed by surfactant bilayer templaying. j. am. chem. soc. 120, 6832–6833 (1998). [5]. i. kobasa. semi-conductive materials based on the titanium dioxide doped with zinc: catalutic activity for copper deposition and effect of uv-irradiation. polish j. chem. 78, 553-560 (2004). [6]. i. kobasa, i. kondratyeva, l. odosiy. tio2/biscyanine and cds/biscyanine heterostructures – influence of the structural composition on the photocatalytic activity. canad. journ. of chem. 88, 659–666 (2010). [7]. i. kobasa, i. kondratyeva. sensitizing of semiconducting photocatalysts by cyanine pigment with two conjugated chromophors. polish. j. chem. 82,1639-1648 (2008). [8]. i.m. kobasa, ya.s. mazurkevich, n. i. zozulya. influence of photochemical and reductive activation of titatania on its catalytic activity in the process of metallic copper deposition. theor. and exper. chem. 40, 110-115 (2004). [9]. a.i. kryukov, s.ya. kuchmiy, v.d.pokhodenko. molecular design in photocatalysis: basic physico-chemical principles in design of highly effective photocatalytic redox systems. theor. and exper. chem. 30, 175–191 (1994). [10]. a.i. kryukov, s.ya. kuchmiy, v.d.pokhodenko. energy transformation in electronic processes in semiconducting photocatalytic systems. theor. and exper. chem. 36, 69–89 (2000). [11]. p.v. kamat. photochemistry of nonreactive and reactive (semiconductor) surfaces. chem. rev. 93, 267-300 (1993). [12]. a.i. kryukov, s.ya. kuchmiy, v.d.pokhodenko. nanostructured composite photocatalysts based on polycrystalline cadmium food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 i g o r k ob as a, l yu bo m yr a o d o si y, n a no s i z ed i m pla nt s of t h e i on s a g+ , c u + a n d c u 2 + i nt o t he s u r f ac e l a y er o f p o l y cr y s t al l i n e cd s ph o t oc a t a l y s t f o r t h e r ea c t i o n of ox id at i o n o f i od id e i o ns , f o od a nd e nvir o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , p ag . 5 3 5 7 57 sulfide. theor. and exper. chem. 33, 306-321 (1997). [13]. i.m. kobasa, g.p. tarasenko. photocatalysis in the reaction of methylene blue reduction by nanocomposites bi2s3/cds. theor. and exper. chem. 38, 249-252 (2002). [14]. i.m. kobasa, g.p. tarasenko. the products of implantation of ions ag+, cu+ and cu2+ on surface of the polycrystalline cadmium sulfide as photocatalysts in the redox reaction between methylene blue and formaldehyde. theor. and exper. chem. 39, 107-110 (2003). [15]. d. dobosh. electrochemical constants. moscow, mir, 365 p (1980). [16]. n. husiak, i. kobasa, s. kurek. nature inspired dyes for the sensitization of titanium dioxide photocatalys. chemik. 67, 11941198 (2013). [17]. husiak, i. kobasa, s. kurek. new dyes for dye-sensitized solar cells and photocatalysis: verifying thermodynamic requirements for electron transfer. funct. mater. lett. 7, 1450030-1450034 (2014). [18]. c. berberidou, i. poulios, n. p. xekoukoulotakis, d. mantzavinos. sonolytic photocatalytic and sonophotocatalytic degradation of malachite green in aqueous solutions. app. catal., b. 74, 63-72 (2007). [19]. h. he, ch. lui, k. d. dubois [et al.] charge separation in nanostructured tio2 materials for photocatalytic and photovoltaic applications. eng. chem. res. 51., 11841−11849 (2012). [20]. n. gusiak, i. kobasa, s. kurek. organic dye sensitisers for solar cells and semiconructor fotocatalyst – a metod for suitability evaluation explained. czasopismo techniczne. chemia 108, 59-73 (2011). microsoft word 11 article_camelia popa_v11_iulie 2012.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 66 studies regarding soil quality and its impact on environment *camelia popa1 1ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: cameliamai_2010@yahoo.com *corresponding author received 25 march 2012, accepted 11 may 2012 abstract: the purpose of this study is to make an analysis of the soil form the area of agricultural research and development resort suceava, regard the content of pesticide and a risk assessment of their environmental threats. after analyzing the sample harvested from this area there were identified qualitative and quantitative the acid 2,4d and the florasulam, compounds of the pesticide called mustang, post emergent pesticide, used previous years in combating weeds with broad leaf from cereal crops. studies have revealed that emphasizes the fact that samples taken from soil surface have higher concentrations both in florasulam and 2,4d than harvested from depth, because along with the leaching of pesticides and under the action of micro-organisms in the soil, their concentration decreases.2,4d has remained in the ground less than the florasulam which, under the action of micro-organisms or soil passes into hydroxylated derivative compound with similar structure to florasulam but with persistence and accumulation in soil. keywords: environment, pesticide, mustang 1. introduction soil, through his position, nature and her role, is a product of interaction between biotic and abiotic environment, representing a live organism, where an intense life takes place and a certain ecological equilibrium has established. soil, through their properties of maintaining and develop life, to regenerate, filter the pollutants, absorb and transform them. if the air and water represent vectors of pollutants transmissions, soil represents the environment of accumulating them. the principal chemical process that takes place in soil is alteration. soil physic-chemical conditions of alteration depend on chemical constituents that reach the soil. they can come from atmosphere, lithosphere, biosphere or chemical reactions of alteration nature which can take place on chemical, biochemical or biological way [1]. soil, naturally, presents the capacity of defense and auto-build which annihilate the natural pollutant factor, exceeded capacities, undetectable in our days because of pollution [2]. with industrial development, population growth and their needs, soil degradation has taken on worrying. ways through which the soil pollution are many and complex but they all lead to degradation by the penetration of elements from outside [3]. a special case in the pollution of soils is the pollution with pesticides. their time of waiting in the soil depends on the nature of the soil as well as the link with the biodegradability [4]. pesticides from the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 67 soils go into the vegetal life and then into the animal life, in the end being accumulated in the body’s fatty tissue. due to the high degree of persistence and toxicity, pesticides have taken the name of organic polluted polluters (pop). soil fertility and the ability to auto generate is provided by the activity of micro-organisms and mezofauna [5]. the behavior of pesticides in soils is dictated by physical factors, chemical and biologic processes such as absorptiondesorption, chemical degradation, volatile, leaching, absorption by plants. the geographic position of the city of suceava specific climate, are decisive factors in affecting the soils by natural ways: rain, storms, frost, snowstorm, massive snow fall. on all these, the atrophic factor is added also. suceava county occupies a distinct place in the romanian economy because of diversity and in some cases because of the abundance of its natural resources. over 52% of the county’s surface belongs to the forests found, respectively aprox. 7% of the country surface, in the underground there can be found deposits of manganese, copper ores, sulphur, barite, salt, natural gas, mineral, uranium ore, and aprox. 42% of the surface is represented by arable land, the majority being located along the valleys of siret,moldova and suceava [6]. representative industries in the county are: timber industry, developed in direct correlation with forests fond surface; pulp and paper industry, represented by one of the largest company in the country in this domain, sc “ambro” s.a suceava; the food industry, which develops in direct correlation with the county, because agriculture is based mainly on the processing of animal products (meat, milk),light industry, represented by clothing companies and knitwear, leather and footwear, machine building industry, represented by companies that produce tools and bearings; mining in the county, represented by exploiting and processing of ore (ores, cuprifere, polimetalic, uranifere, manganese salt), industry in decline over the past decade [6]. all these activities have led to the impairment of the quality of soil ground by: storage for waste wood chips, waste from domestic, mining etc.; soil pollution by waste and waste from food processing industry; accidental overflow pollution by oil products, chemicals used in technological flows; pollution with pesticides; massive deforestation which lead to the emergence or accentuation of slip phenomena-erosion, acidifications, etc. the resort of agricultural research and development is individualized by climate conditions, characterized by specific restrictions, non-uniformity of thermal-water regime of the failure to excess-and by a wide variety of soil types, from low fertility soils with up to those with good fertility. the consequence of this ecological diversity, along with the need to diversify-related, research in response to solve the main problems of agriculture in the suceava plateau [7]. the land on which the cores were taken from the soil type mold was grown in 2010 – 2011 with wheat. to combat the weed has been used a herbicide, mustang ". the pesticide mustang is part of the herbicides class and was produced in the usa by the company dow agro sciences llc in 2007. is used in weeding wide leaf crops of grain. for effective weed control (over 95%) should be applied in the early stages of vegetation. the recommended rate is 0,4-0,6 l/ha, diluted in 200-400 l water/ha; active substance: 6,25 g/l florasulam + 300 g/l 2,4-d [8]. environmental risk:florasulamis a very mobile compound, not persistent in soil, airy, is a fast biodegrade in derivative analogue 5-oh-xde-570, which in turn is much harder to biodegradable co2. florasulam is not significantly degraded by abiotic processes. easily degrade in aquatic food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 68 systems, where also the derivative hidroxilat is bio-transforming. this derivative 5-ohxde-570 is much more mobile in the ground than the original compound, florasulam. persistence and accumulation of the derivative is more variable than that of florasulam, indicating that it can persist in the lowerpedogenetic layers. data submitted indicates that florasulamul has a very low potential for bioaccumulation in aquatic organisms. with reference to the risk of contamination of groundwater by florasulam has shown that it can take place, but in a lesser extent due to biodegradation under the action of the micro-organisms, but the potential for derivative analogue, infiltration is greater due to its solubility in water but it's slower. 2,4-d acid has reduced persistence in soils. the half-life in soil is more than 7 days. micro-organisms in the soil are primarily responsible for its demise. in aquatic environments, microorganisms degrade slightly 2, 4-d acid. despite short time,half life in soil and aquatic environments, the chemical has been detected in the groundwater in canada and at least five other states. 2. experimental sampling to a depth of 5 cm, is aimed at identification of pollutants and the impact on the soil surface, and taking samples from 2530 cm depth, you can determine the degree of accumulation of pollutants in the layer being plowed, and the maximum concentration of agricultural plant roots [10]. 8 soil samples were taken randomly, choose from 4 points at depths of 5 to 25 cm. for sampling methodology was followed, in accordance with order no 184/97 a.n.p.m. [11]: 1) vegetation has been completely removed from the area sampling; 2) a sampling instrument was used for assuring a sufficient volume of the sample for analysis; 3) sampling was carried out at two different depths ranging from 5 to 25-30 cm from the soil surface. once collected, the soil samples were labeled by numbering the i1, 2 to iii1,2 immediately transported to the laboratory in order to be prepared to perform qualitative analysis. determination of ph: iso 10390:2005 (12). the ph value of aqueous suspension of determined in soil is an analytical index easily obtainable, which characterizes the reaction of the soil and the acid-basic properties of soil-water system. in an aqueous suspension or alkaline soil, at equilibrium, the h + ions are distributed between the solid and liquid phase. taking into account the variation of soil ph values, both in site and in aqueous suspensions of varying concentrations (in the solid phase) where the measurements are taken, the definition of soil ph has a conventional, depending on values and soil/water system where the measurements are taken. in the present case there has been a ground/water ratio 1:100 the mass/volume. after soil samples were dried, they were mojarated, homogenized and from each sample were weighed on a glass plate, 1 g of the ground and were dissolved in 100 ml of distilled water in erlenmayer glasses. it has shaken off the solution and it was left for 1 h to sediment and filtered on the filter paper. for measuring ph meter was used 11 d portable hach hq. determination of pesticides in soil: in order to determine the presence of pesticides in soil samples taken, i.e. their determination, they were first prepared, weighted and then order a few rounds: drying in the memmert oven at 105oc at a constant weight, shredding, sieving. the next stage was to pesticides in soil extraction food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 69 with light petroleum to soxhlet, evaporation to dryness and retrieve residue in 5 ml of acetonitrile and undergo analysis by high-performance liquid chromatography (hplc) performance. contents in acid and 2,4-d in the samples thus processed florasulam is determined by high pressure liquid chromatography, reverse phase, using water as the mobile phase and acetonitrile, acidified with acetic acid in the following terms: analysis: 280nm wavelength; the ambient temperature; injection volume: 20 µl; working pressure: 150-180 barr; the gradient according to table 1 [13]. table 1 composition of mobile phase (gradient) nr. time (min) mobile phase a (%) mobile phase b (%) 1 0 95 5 2 9 95 5 3 17 50 50 4 40 50 50 * a – sour water (1% acetic acid); b – sour acetonitrile (1% acetic acid). the analyses were performed on a liquid chromatograph type schimadzu. the concentration of the acid 2,4-d were calculated using the standard method, using external relationship 1.   100 5 pg ea 25 eacidg pa %c     (1) where: ap surface of 2,4d acid peak from sample chromatogram geacid grams of 2,4d acid standard ae  surface of 2,4d acid peak from standard chromatogram; gp the amount of soil taken in the work (g); 25  standard dilution; 5  sample dilution. 3. summaries and discussions measuring the ph of the aqueous extract 1/100 for the 6 soil samples is observed that the samples taken from the soil surface (5 cm) have more acidic ph compared to those taken from deep, respectively 25 cm. results are shown in table 2. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 70 table 2 soil acidity for 8 samples harvested from different points and depths no. analysed samples the value of ph soil acidity 1. sample no. i1 6,27 slightly acid 2. sample no. i2 6,44 slightly acid 3. sample no. ii1 5,10 moderately acid 4. sample no. ii2 6,13 slightly acid 5. sample no. iii1 5,94 moderately acid 6. sample no. iii2 6,11 slightly acid *i1surface sample (5cm); i2 – depth sample (25cm). for the extraction of pesticides were used each (approximately) 40 g of dry soil to constant weight at a temperature of 105 0c, which were subjected to soxhlet extraction with each 200 ml petroleum ether.the extract was evaporated to dryness on water bath, was taken in 5 ml acetonitrile and analysed by liquid chromatography using the method of section 2. to identify the components were utilized certified reference materials. after analysing soil samples by hplc were qualitative identified 2, 4-d acid and florasulam with appropriate retention times, as can be seen in the chromatograms presented in figs. 1 and 2. the order of elution is florasulam, 2, 4-d acid. figure1. chromatogram of soil sample iii2. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 71 figure 2. chromatogram of soil sample iii2enriched with acid 2,4d. it can be seen that the peak of florasulam is not pure, it has a shoulder. this tells us that florasulam goes in chromatogram together with a component with a very similar structure and polarity, i.e. hydroxylated derivative of florasulam. literature data indicates that hydroxylated derivative is formed in soil from florasulam, under the action of microorganisms. the results of analysis of the 6 samples are shown in table 3. from the results obtained in this study, results the following conclusions: the samples collected from the surface of the soil 5 cm, have concentrations in both the 2,4-d acid and florasulam greater than those harvested from deep (25 cm), which means that with the leaching of pesticides, soil micro-organisms under the influence they lost in their concentration; samples taken from soil surface have higher concentrations in the florasulam than in 2, 4d acid, according to the studies of literature [8] which affirm that florasulam in soil is greater than 2, 4d acid, the latter being more easily biodegradable, with a half-life of 7 days, compared to florasulam, that once in the form of derived analogue persists long periods of time. results obtained to determine the ph of come in support of above affirmations; namely, samples of the surface have ph more acidic than depth, in other words, a content to 2, 4d above to the surface. although the concentration of product in florasulam, conditioner is 50 times less than 2, 4d acid, its concentration in the soil is often 100 times greater. degradation of florasulam is lower net depravity 2, 4d acid. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 72 table 3 the concentration of acid 2, 4-d and florasulam obtained from calculations analysed samples components retention time peak area sample mass dilution 2,4-d acid concentration (µg /kg) florasulam concentration (µg /kg) florasulam 17.85 12412004.50 43.05 5 52.54 sample no. i1 2,4dacid 25.99 6849.80 43.05 5 2.97216 ∙ 10 -2 florasulam 17.85 7642256.10 43.36 5 38.82 sample no. i2 2,4dacid 25.18 7645567.90 43.36 5 32.939272 florasulam 17.85 7116538.80 44.58 5 29.78 sample no. ii1 2,4dacid 24.69 169714.90 44.53 5 0.711843 florasulam 17.86 7411629.30 45.83 5 29.26 sample no. ii2 2,4dacid 25.70 58269.30 45.83 5 0.237484 florasulam 17.81 10648238.30 44.84 5 43.856 sample no. iii1 2,4dacid 25.98 75269.30 44.84 5 0.313531 florasulam 17.84 7070500.00 44.87 5 28.376 sample no. iii2 2,4dacid 26.01 62289.90 44.87 5 0.259295 4. conclusions following the experimental measurements demonstrated the presence of components of mustang herbicide in collected soil samples. have been identified active substances in herbicide (florasulam acid and 2,4-d acid) emphasizing at the same time and concentration. florasulam is an extremely mobile compound, do not persist in the soil airy and well under the action of micro-organisms in the soil, the derivative is biodegrade in the compound with analogue structure, similar to that of florasulam. persistence and accumulation of the derivative product is significantly different from that of florasulam, indicating that it can persist long into the lower pedogenetice layers. with reference to the risk of contamination of groundwater by florasulam, it was found that this can take place, but in a lesser extent due to biodegradation under the action of the micro-organisms, but for the derivative product (analogue), potential flow is higher due to its solubility in water but also to his slower metabolism. 2,4-d acid has much less persistence in soils, also thanks to the action of microorganisms present in the ground, given the half-life of less than 7 days. concentrations of 2,4d acid are quite small ranging between 2.97216 ∙ 10-2 µg/kg and 32.939272 µg/kg compared with those of florasulam food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 73 28.372416-52.546237 mg/kg mg/kg although the concentration of product in florasulam, conditioner is 50 times less than 2,4d acid, its concentration in the soil is often 100 times greater. degradation of florasulam acid is net lower depravity 2,4d acid. in general, due to the high degree of persistence and toxicity, pesticides have been referred to as persistent organic pollutants (pops). pops are chemicals that persist in the environment, bioaccumulated in living organisms and runs the risk of causing adverse effects on the environment and even human health. several studies concerning the pesticides widely used have already demonstrated that their application leads to changes in soil nutrient levels and changes in the activity of microbial diversity in soil and/or genetic soil structure. accordingly, disorders of microbial communities that ensure more soil-friendly key processes, such as the degradation of organic matter and nutrient cycle, could influence the proper fertilization soil and sustainable agricultural productivity. excessive use of the extensive and often even inadequate synthesis of pesticides, constitute the main source of pollution, with serious risks for human health and also led to a new type of which jeopardizes the rationale for their use, namely the emergence and development of species tolerant of harmful pesticides. the immediate trend now was to increase the rates charged to offset the decline in the efficacy of pesticides. in the case of the using pesticides there are attacked not only the species to be destroyed (pests), as well as many other species that are required for soil fertilization. only 3% of the pesticides used to secure. sometimes, under the action of pesticides is the inhibition of certain groups of microorganisms and on the other hand, the development of others. thus, the microbial balance is not altered, resulting in a even stimulating micro-organisms. remaining of pesticides in soil depends on the type of link between them and the ground but in particular the biodegradability of pesticides. up to 80% of pesticides are adsorbed by humus, a fact which, due to the persistence in the soil increases much. 5. references [1]. lupaşcu g., jigău g., vârlan m., general pedology, junimea publishing, iaşi, (1998) [2]. cojocaru i, sources, processes and products for remediation, junimea publishing, iaşi, (1995) [3]. puiu ş. pedology, ceres publishing, bucureşti, (1980) [4]. arias-este´vez m. et al., the mobility and degradation of pesticides in soils and the pollution of groundwater resources, agriculture ecosystems & environment agr ecosyst environ, 123 (4), 247-260, (2008) [5]. stoian l., an alternative: biological agriculture, hortus, 4, (1996) [6]. national environmental protection agency suceava – report on the state of the suceava environment in 2010: on-line at http://www.mmediu.ro/legislatie/substante_pericul oase.html [7]. united states environmental protection agency (us epa), pesticide fact sheet: florasulam washington, d.c. 20460, office of prevention, pesticides and toxic substances: on-line at: www.epa.gov/opprd001.com [8]. united states environmental protection agency (us epa), – 2,4-d red facts: on-line at www.epa.gov/oppsrrd1/reds.com [9]. papacostea p., soil biology, scientific and encyclopedic publishing, bucharest, (1976) [10]. http://apmdj.anpm.ro/legislations [11]. iso 10390:2005, soil quality. determination of ph. standardization association of romania, bucharest, romania. [12]. popa c. et al., invention patent. methode for determinind the content of de 2, 4dichlorphenol, dicllorphenoxi acetic acids and 3,6 diclhor-2 metoxibenzoic acid (1994) microsoft word 2 torosyan 2013 corectat.doc 12 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 12 17 phase transfer catalysis for green chemistry yeva torosyan1, andranik avetisyan1, *gagik torosyan2 1 european regional educational academy, yerevan, armenia, gagiktorosyan@seua.am 2 faculty of chemical technology and environmental engineering, state engineering university of armenia, * corresponding author received 10 february 2013, accepted 5 march 2013 abstract: the problem of “green chemistry” using like example phase transfer catalysis has been discussed in the present paper. nowadays catalysis plays a very important role in the new green chemical industry. catalysis can reduce the environmental impact of processes and therefore can reduce the costs of these processes. application of new catalysts and catalytic systems aim to achieve both environmental protection and economical benefits. the ptc technology has been chosen and is used in these applications, because it provides many compelling benefits, primarily related to the cost reduction of organic manufacture chemicals and secondly because it prevent the environmental pollution. keywords: green chemistry, sustainable development, phase transfer catalysis ptc, pollutions. 1. introduction globalization and countries industrial development creates fast changes on environment fact that conduct to the opportunity of occurrence of new principles for definition of tasks of a society. moreover, the increases changes of the extreme climatic phenomena are worsening more and more the situation on a global scale. it is widely acknowledged that there is a increase need of the human society for a more environmentally acceptable processes in the chemical industry. in the present paper the idea of green chemistry develops as a philosophy of chemical research that tried to minimize the use and the production of dangerous substances. the focus is to minimize the substance chemical danger and to maximize the efficiency of any chemical choose. catalysis has played such a vital role in the success of the chemical industry in the 20th century, the application of this process ranging from pharmaceuticals to petroleum processing fields. more than 90% of all industrial processes are based on catalysis process. the widespread utilization by industry of catalytic processes has a positive influence on the economic and environmental safety [1]. one of the best way to realize of chemical processes, specially in organic reactions is the phase transfer catalysis ptc [2,3]. cost reduction and pollution prevention are the two most powerful driving forces in the chemical industry today, and they match precisely the strengths and benefits provided by phase transfer catalysis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 yeva torosyan, ashot avetisyan, gagik torosyan , phase transfer catalysis for green chemistry , food and environment safety, volume xii, issue 1 – 2013, pag. 12 17 13 phase transfer catalysis conduct to high productivity, increase environmental performance, improved safety environment, and increase plant operability in hundreds of commercial manufacturing processes for organic chemicals in dozens of reaction categories [2, 3]. 2. materials and methods green chemistry and the principles the developing of green chemistry methodologies is a challenge that can be presented through the framework of the “twelve principles of green chemistry” [4]. the principles of green chemistry [4] compared with the advantages of ptc are presented in table 1. the principle number 9 identifies the catalysis as one of the most important tools for implementing green chemistry. nowadays, organic synthesis, especially fine chemicals and pharmaceuticals use “stoichiometric” technologies. some of these reactions types were transformed in phase transfer catalytic (ptc) system. ptc is one of the most efficient methodologies in organic synthesis and therefore it. it was widely used for the synthesis of organic compounds. ptc uses as phase transfer catalyst onium salts (as quaternary or phosphonium ammonium salts /quat/, crown ethers and others/, who facilitate the transport of one reactant into the other and interaction between reagents in two immiscible phases. ptc is widely used in the organic chemicals synthesis for both two and triphase systems. the use of ptc can be combined with other rate enhancement techniques like microwaves, sonochemistry and others. ptc has made possible to use cheaper and easily available alternative raw materials, avoiding in these way the need of using severe anhydrous conditions, expensive solvents, and dangerous bases such as metal hydrides and organo-metallic reagents. ptc uses much fine organic chemistry instead of toxic alkali metal alkoxides, amides, and hydrides. table 1 a comparison of the principles of green chemistry and ptc advantages n principles of green chemistry 1. waste prevention instead of remediation 2. atom efficiency 3. less hazardous and toxic chemicals 4. safer products by design 5. innocuous solvents and auxiliaries 6. energy efficient by design 7. preferably renewable raw materials 8. shorter syntheses 9. catalytic rather than stoichiometric reagents 10. design products for degradation 11. analytical methodologies for pollution prevention 12. inherently safer processes n advantages of ptc 1. minimization of industrial wastes 2. high reactivity and selectivity 3. less dangerous, inconvenient and expensive reactants 4. high yields and purity of products 5. less inconvenient and expensive organic solvents 6. low energy consumption 7. simplicity of technology 8. possibility to mimic counter-current process an important issue in organic chemistry technology is the use of organic solvents. the principle of ptc also proposes the use of usual and environmental friendly solvent that coincides with the number 5 principle for green chemistry. for both of them it necessary to use the solvents with different characteristics like: low toxicity, easy recyclability (no disposal) and further desirable characteristics easy removal from the product and. in this case some solvents should never be used: tetrachlorocarbon, 1,2-dichloroethane, 1,1-dichloroethane and some solvents are preferred like water, co2 , heptane, tertbutyl methyl ether, ethyl acetate, tert-butyl alcohol, ethanol. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 yeva torosyan, ashot avetisyan, gagik torosyan , phase transfer catalysis for green chemistry , food and environment safety, volume xii, issue 1 – 2013, pag. 12 17 14 we are mentioned bellow the solvents are more convenient to be used for green chemistry technologies: 2.1. water as a reaction medium economically and environmentally attractive inexpensive and abundantly available non-inflammable and non-toxic odorless and colorless highly polar reaction medium facile product separation reduced product contamination. 2.2. supercritical co2 as a reaction medium tc 31.0 °c, pc 73.8 bar, dc 0.477 kg l-1 • low viscosity (more like a gas than like a liquid); hence, fast mass transfer • cheap and abundantly available • easy to remove • non-toxic, non-inflammable, inert. ptc tries to use no toxic reagents, solvents and other materials as we seen in the number 3 principle for green chemistry. one of the other advantages of ptc is it lower energy and simplified technology – as we can see from the number 6 principle. however, the main disadvantages of ptc, especially in commercial applications, are the necessity to separate the catalyst from the organic phase. further, is important to show the difference between homogenous and heterogeneous catalysis. phase transfer catalysts can be either homogeneous (soluble in one or both solvents) or heterogeneous. the place ptc in catalytic systems between homogenous and heterogeneous, because of its catalytic system allowed to transfer heterogeneous system into the homogenous one – as in ptc liquid-liquid system. in this case appeared the possibility to use the advantages of homogenous catalysis. quaternary ammonium and similar onium salts (quat), crown ethers with alkaline metal cation and other provide a source of singly charged lipophilic cations. generally, catalyst efficiency is influenced by the large number of carbon atoms and the symmetry of the carbon atom chains around the heteroatom that provide high lipophilicity [2,3]. homogenous ptc is based on the mechanism proposed [2]. table 2 advantages and disadvantages of catalytic systems catalytic system advantages disadvantages homogeneous mild reaction condition, high activity and selectivity, efficient heat transfer cumbersome separation and recycling of catalyst heterogeneous facile separation of catalyst continuous processing product contamination heat transfer problems low activity and selectivity products not readily adapted biphasic homogeneous catalysis integrates reaction and products and catalyzes separation into one single operation: other possible solutions; supported liquid phase catalysis; -thermo-regulated biphasic catalysis supported liquid phase catalysis. ptc is used also in solid-liquid system, which is coming more as a heterogeneous system and even more heterogeneous when we have tri-phase system. ptc linked to a polymer or inorganic matrix are described as heterogeneous catalysis in this system the catalysts play role as like a third system, for instance, the immobilized on alumina / and other inorganic material/ or polymers quaternary ammonium salts or crown ethers. here can include also polyethylenglicols (peg) [2, 3] and polypropargyl alcohols [2]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 yeva torosyan, ashot avetisyan, gagik torosyan , phase transfer catalysis for green chemistry , food and environment safety, volume xii, issue 1 – 2013, pag. 12 17 15 for quat and crown ethers the situation is: catalyst is bonded to a matrix forming a third immiscible solid phase between the organic and aqueous ones involving a swelling, mixing and diffusion during the reaction. due to diffusion retardation, reactions with slow intrinsic reaction rates are much slower with a tri-phase catalyst than with its homogeneous counterpart [2,3]. the use of tri-phase ptc simplifies the removal of the catalyst after the reaction which can be re-used until they lose their mechanical stability. here we discussed about c–c bond formation as michael condensation in ptc system. 3. results and discussions ptc is efficiently applicable for baseinduced reactions of organic anions as michael reaction. this reaction mentioned concern the chemists from the perspective point of view. unsaturated compounds (acceptor groups), and compounds with donor group (ch-acid) receive new connections with the most different groups [5, 6]. o h2n nuh + nu h2n o nuн – the molecule of conforming сн acid (see table 3). this reaction one of two major categories of ptc reaction, when anion generated in situ/there is an anion from acetoacetic acid ethyl ether that reacts with acrylamide. here is studied the interaction of acrylamide with some accessible classic сн acids – malonic ester or diethylmalonate (me), acetoacetic ester or ethyl acetoacetate (aae), ethyl ether of cyanaoacetic acid (eca) and acetylacetone or pentanedione-2,4 (aa). all these reagents react with acrylamide as the type of 1,4-addition for michael reaction. the conversion of acrylamide with сн acids is implemented in a water-alcaline medium, in conditions of ptc as catalyst of phase transfer carry were applied with different quats. table 3 the molecule of conforming сн acid nuh nu malonic ester (me) oet oet o o ethyl ether of cyanoacetic acid (eca) oet nc o acetoacetic ester (aae) me oet o o acetylacetone (aa) me me o o influencing of сн acids structure and catalysts on a reaction rate constant and yields of product is studied. it is visible from the results in table 4 me and eca in conditions of michael addition to acrylamide are very close on reactivity. the aae reacts with acrylamide a little bit more slowly, than eca or, in particular me. the reaction for aa is more slowly. at the same time, outgoing from values of dissociation constants of this ch acids, diverse sequence of reaction speeds was expected: me (рк~13) < cae (рк~11) < aae (рк~11) < of an aa (рк~9). probably, the main role in the control of reaction speed is played by solvation of сh-acids or their complexes with catalyst in aqueous phase, where an addition reaction flows past. in this case, the anion of more strong acid and slowing down for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 yeva torosyan, ashot avetisyan, gagik torosyan , phase transfer catalysis for green chemistry , food and environment safety, volume xii, issue 1 – 2013, pag. 12 17 16 reacting, because of solvation effects is most solvated for that is also more. the speed of michael addition in ptc conditions depends not only on a сh-acid, but also, at largely, from used catalyst (table 4). table 4 the yield of addition products of сh-acids to acrylamide the yield michael addition product (%) catalysts me cae aae aa without catalyst 4.0 3.0 3.75 1.75 tmacl 60 40 75 28 tmhacl 80 58 80 40 tmbacl 85 70 80 45 tmdhacl 90 85 90 60 outgoing from an extreme length of radical alkyl chain of l, the best catalytic activity shown trimethylhexadecylammonium or trimethylcetylammonium chloride (tmdhacl), is objective, as well as least, shown by tetramethylammonium chloride. to activity of chlorides trimethylhexyl and trimethylbenzylammonium (tmbacl) are rather close, despite of a little bit large length hexyl radical, as contrasted to benzyl. despite of a little bit smaller, than for tmdhacl catalytic activity, (tmdhacl), nevertheless, is perspective catalyst because of the availability that can have crucial importance, in case of organization for a commercial production. on our point of view, in a reduced example of michael addition, the important organic reaction, ptc demonstrates actual chance for an approaching to principles green chemistry. it is necessary to mark also, that here for the first time is described new reaction of acrylamide for synthesis of the very relevant products, in particular for synthesis physiologically and bacteriological active compounds. in this paper have not gone deep into mechanistic estimations of the conducted reaction, as it implements in called of an inverse ptc. 4. conclusion the experiments were conducted in the reactor of ideal mixture at 300с. initial concentrations of a сh-acid and acrylamide at 1,5-1,75 mol/l; it was compared yields of experiments by confrontation constants of reaction speeds in the supposition, that at enough high concentration of catalyst 0,1 mol/l, what, for utilized of quat, in 1,75 times for above critical concentration micelle formation (cmf) change of concentration for catalyst cease is appreciable to have an effect for reaction speed. current concentration of the conforming сh-acid determined (glch) through 10 minutes from a beginning of reaction. the yields of addition reaction product was determined on expiration of an estimated time of reacting indispensable for achievement of 95% transformation for reagents (receiving conditionally selectivity for reacting equal 100 %). in tab. 4 the yields of addition products for investigated сh-acids to acrylamide are described. due to the features and advantages of ptc here was considered as an efficient for green chemistry. 5. references [1]. m.s. simmons, in: p.t. anastas, t.c. williamson (eds.), green chemistry: designing chemistry for the environment, american chemical society, washington, dc, (1996), ch. 10, p. 116 [2]. a.t. babayan., g.h. torosyan., the phase transfer catalysis development stages. journal of mendeleeev society, (1986) n.12, p. 129 [3]. starks, c.; liotta, c.; halpern, m.; "phase-transfer catalysis: fundamentals, applications and industrial perspectives," food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 yeva torosyan, ashot avetisyan, gagik torosyan , phase transfer catalysis for green chemistry , food and environment safety, volume xii, issue 1 – 2013, pag. 12 17 17 chapter 16, chapman & hall, new york , (1994). [4]. p.t. anastas, j.c. warner, green chemistry: theory and practice, oxford university press, new york, (1998), p. 30. [5]. torosyan g., harutyunyan a., isakova l., beyleryan n., chobnyan j., simonyan g., reaction of acetoacetic acid ethyl ester with acryland αmethylacrylamides in conditions of inverse phase transfer catalysis. effect of sufractant's type// oxid. commun.-2007.v. 30, №3, pр. 548-552. 93 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 93 – 100 application of infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials volodumur strebezhev1, viktor strebezhev1, *george vorobets1, ruslan hurjui1, roman rogov 1yury fedkovych national university of chernivtsi, ukraine, g.vorobets@chnu.edu.ua *corresponding author received 16th march 2015, accepted 31st march 2015 abstract: the presence in food and the environment of nitrates, organic and other substances harmful to humans can be identified by their characteristic molecular spectra in the near infrared radiation. when developing optical sensors, whose work is based on the determination of the spectral absorption characteristics of organic matter, it is necessary to apply cutting optical filters for the selection of certain infrared ranges of the spectrum. the work carried out to develop multilayer thin film interference filters with replaceable cutting position limits infrared radiation depending on the concentration and type of the studied compounds. the peculiarity of these filters is the use of semiconductor crystals in4se3, in4te3 and cdsb as substrates for thin-film multilayer coating. high stability of the spectral characteristics, resistance to chemicals and atmospheric action of single crystals in4se3, in4te3 and cdsb enables the design based on these filters with =1,7-6,5 um different positions of shortwave radiation limits cutting range and a maximum transmittance tmax=91-95%. the method of sem studies depending on the structure of thin films component filters zns, sio, ge, te, srf2 on technological factors and on spectral and mechanical stability of the multilayer coating are used. the requirements to design solutions of the active cell optical biosensor developed with the use of infrared filters. the method and simulation modeling to build adaptive reconfigurable computer information-measuring system of rapid analysis of qualitative composition and quantitative parameters of molecular spectra of bioactive substances are proposed. keywords: optical sensor, multilayer thin films, interference filter, adaptive reconfigurable computer information-measuring system 1. introduction in developing biosensors use of organic complexes and materials change their electrical or optical properties under the influence of external physical factors (temperature, electromagnetic radiation, mechanical stress, etc.), or when they are surface or bulk chemical interaction with other chemicals elements and complexes in aerate or liquid phase [1-3]. feature of optical biosensors are subject to change spectral characteristics of reflection or transmission of electromagnetic radiation sensitive layer of the sensor as in the visible, i.e. the optical and ultraviolet (uv) and infrared (ir) spectral region [4, 5]. ir spectroscopy in comparison with uv and visible range into wider use in biotechnology, medicine, pharmacology studies for quantitative and qualitative composition of substances on the content http://www.fia.usv.ro/fiajournal mailto:g.vorobets@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 94 of amino acids, proteins, bacteria, inorganic compounds and other constituents analysis [6]. this is due to greater information content of ir spectra concerning the molecular structure of matter. in addition, a high-energy radiation shortwave visible and uv range when irradiated with certain organic components can stimulate photochemical processes and lead to the destruction of the sensitive layer of the biosensor. certain restrictions imposed on the permissible level of background effects of radiation nearby areas infrared wavelengths measured in information signals. to improve the resolution ir method and the exclusion of undesirable constituents spectrum in the middle infrared region advisable to use in the optical path of the measuring channel thin-film multilayer interference filters that are applied to the respective semiconductor crystals. in the development of optical biosensors, whose work is based on the determination of the spectral absorption characteristics of organic matter, there is a need to apply cutting optical filters for selecting specific ranges of infrared spectrum. it is possible to distinguish two modes of investigation of the spectra of biosensor materials: 1) measurement and analysis of spectra own sensing elements; 2) measurement and analysis of changes in the spectra sensitive elements in their interaction with other monitored substances. unlike biosensors with electrical conversion information where the sensor element must have a galvanic connection with semiconductor radiation detectors, may be by the part of the structure of the detector in optical biosensors or can confine the optical channel between the sensing element and the detector. for example in [7] presented data from a study using human urine absorption infrared spectroscopy, which allows the characteristic absorption bands to determine the qualitative characteristics of bioliquids in general. the authors used the method of sample preparation means "dried drop". with the contrasting observations in the spectrum of urea revealed characteristic bands with peaks in 3440, 3345, 3261, 1680, 1605, 1464, 1155, 1056 cm-1 and 557 cm-1, corresponding to the wavelength range from 2.5 to 18 microns. the obtained experimental results are typical for stretching and deformation vibrations of complexes (nh2)2–co and their derivatives. the observed shift of the position of the maxima on the value of 1020 cm-1 in the absorption spectra of the authors explain the different options for different pathology specimens that own properties biomaterial. but apparently similar changes may be characteristic spectra and the results of the interaction of urea with outside groups –co. for other biosensitive materials [8] may be important changes in their spectra due to their natural aging. we believe this underlines the importance of dynamic updating databases spectra used biosensors and periodic calibration of measuring paths of control devices during their operation. this approach will improve the resolution of information-measuring system for improved detection and differentiation of the components of the studied mixtures. increasing contrast investigated spectra can be achieved as the use of high contrast optical ir filter that would cut light noise (interference) in a given wave bands. therefore, this work is dedicated to designing and testing new interferenceabsorption filters for short and medium infrared region, according to the measurement of the spectra of various organic liquids and create models of hardware and software reconfigurable specialized computer system for solving control problems tract spectrometer and measuring information processing in realtime. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 95 2. experimental the feature of cutting off ir filters, which were developed in this paper are used as substrates for thin film multilayer coating of semiconductor crystals in4se3 and in4te3 [9]. the fundamental absorption is caused by an appropriate of band gap (0,65 ev for in4se3 and 0,48 ev for in4te3), and provides absorption component functioning filter. as substrates for filters used in4se3 single crystals and in4te3, grown by the czochralski method using pelt'ye effect of which was pinned on cleavage plane of the plate thickness of 0,6–0,8 mm. high quality spectral characteristics, stability, resistance to chemical agents and atmosphere monocrystals in4se3, in4te3 enable design based on these filters with different placement of short-cut boundary radiation in the range b =1,7–7 um and maximum transmittance tmax=91%–95%. applied to the surface of the crystals in4se3 thin film and the in4te3 antireflaction and multilayer filter coatings can be made efficient cutting and bandpass filters for applications in optical devices [10, 11]. calculation of thin-film interference structures of crystals for application to in4se3, in4te3 and in4(se3)(1-x)(te3)x conducted standard matrix method of equivalent layers in combination with the method of smoothing the spectral characteristics in relation to the function that simulates the appearance of a given filter. cadmium antimony have similar to indium telluride crystals in the electronic properties and also can be used for ir signal filtering [10]. in addition, the use of indium selenide and telluride as their basis for photo-sensitive hetero structure has advantages [12, 13]. this allows combine constructively semiconductor filters and detectors in one device. the developed software allows calculate the spectral properties of multilayer interference coatings and analyze spectra reflectance and transmittance coatings for different values of the number of layers and their thicknesses and refractive indices. the input data of the program are the refractive indices of the individual layers nj (j=1...m, where m number of layers in the system), their optical thickness dj=njlj, where lj – geometric layer thickness, and refractive index of the medium n0 and the substrate ns. 3. results and discusion the corresponding spectral characteristics of systems b=2,5; 3,3; 3,9 microns shown in fig. 1 (curves 1, 2, 3). it is known that contrast filters and slope cutting limits are largely determined by the difference in refractive indices of layers with high nh and low nl refractive indices [14]. as a film-forming substance was chosen wellknown combination of materials ge-sio (respectively nh=4,0 and nl=1,85; δn=nh– nl=2,15), as well as materials te-srf2 (nh=4,5 and nl=1,35; δn=3,15), which is much less used. to filter which can be made of layers of te-srf2, provided the desired large difference of refractive indices of films at the same time in the literature little information on the use of these materials for forming cutting filters, especially in in4se3 crystals and in4te3. such filters are realistic to achieve high optical characteristics – steep short-cut border kp=0,93–0,96, the maximum bandwidth in the workspace tmax≥90% average transmittance in the region lock background τ≤10-2 %. optimal matching the measured characteristics of the calculated filter with b=3,3 microns was achieved when used as a substrate crystals in4se3 (fig. 1, curve 5). such films had high adhesion and no degradation during thermal cycling to nitrogen temperatures. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 96 this technology temperature determines the structure and density of the film, which is lower compared to the massive crystal and affect properties such as thermal stability, optical thickness, refractive index, strength, mechanical stresses. fig 1. dependence of the transmission coefficient t filters based in4(se3)(1-x)(te3)x with b=2,5; 3,3; 4,0 microns of wavelength : curves 1, 2, 3 theoretically calculated curves; 4, 5, 6 – experimental according to research in the scanning electron microscope sem-100ua and diffraction eg-100m was found that at substrate temperatures ts=403 k derived amorphous and fine-grained polycrystalline films te (fig. 2, a), and at ts=523 k – oriented texture (fig. 2, b). such films provide better optical properties interference system. this mode of deposition was used to produce dense, mechanically and temperature stable te films with low density of drops, that were part of the filter on crystals in4te3. these values correspond to transmittance requirements industry-removals interference filters. in fig. 3 shows the spectral transmission characteristics of the 14-layer filter based on ge-sio films, deposited on a plate of in4se3. on the reverse side the substrate by a three-layer lighting antireflaction lining sio-ge-sio was coated. this design interference-absorption filter provides suppression of background radiation in comparison a narrow range, according to the value of the difference nh-nl for these materials. as can be seen from fig. 3 (curve 1), filters, obtained at low substrate temperatures and in the absence of ion etching, containing defects (fig. 2, a), had deep dips in spectral characteristic to 50– 60% bandwidth. at the same time filter, obtained at the above advanced technology, had spectral characteristics which were average transmittance in the workspace at > 80% (fig. 3, curve 2). a) b) fig 2. the structure of thin films at different substrate temperatures: a) ts=403 k; b) ts=473 k; c) ts=493 k; d) ts=523 k for filters with a long wavelength limit cut the substrate in4te3 was applied. this material has a band gap of 0,48 ev, corresponding to the position of the absorption edge of 2,5 microns. since the range of background suppression in nonworking area of the filter is determined food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 97 by absorption in the substrate and in layers, as well as by a range of highly reflective mirrors of interference system, which significantly increases for films with a large value of δn=nh-nl, were set filters based on the films of te-srf2. it is possible to obtain cutting filters below cut λb=4,0 microns and λb=6,5 microns more longwave region (fig. 4, curve 1) compared with filters on films ge-sio. fig 3. spectral transmittance characteristics of filters on substrates in4se3: curve 1 at ts=363k; curve 2 at ts=423 k fig 4. spectral transmittance characteristics of filters on substrates in4te3: curves 1, 3 at ts=300 k; curve 2 at ts=84 k investigation of the temperature dependence of the spectral characteristics of the filters showed that cooling to 84 k there is a shift λb by 3-4% in the longwavelength region, as well as some reduction in the average transmission in the workspace (fig. 4, curve 1 and 2). this phenomenon can be explained by higher absorption of the films it by cooling and thermal compression of all the films at low temperatures, which causes the difference of optical thickness and index nh from those values that appear in the theoretical modeling of spectral curves of filters. it should be noted that the magnitude of the displacement of spectral curves does not exceed the values that are put forward appropriate standards for interferenceabsorption filters. have also been developed for two-channel interference filters cdsb had a different design depending on the requirements for conditions of use (fig. 5). the simplest filter design, which should operate at room temperature, is a combination of absorption filter b =2,5 microns (first channel) and on top of it within a given geometric area multilayer interference filter b=3,9 um (second channel). a) b) c) fig 5. design (a, b) and experimental transmission characteristics (c) of the interference absorption 24-layer filter on cdsb single crystals: a) single-channel filter, b) two/four-channel coordination-sensitive filter pattern for environmental monitoring (curve 1 – at 293 k, curve 2 – t=83k) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 98 the block diagram of a prototyping of the digital module data processing (fig. 6) for rapid analysis of changes in transmission or reflection spectra biosensors implemented with dual channel scheme similar to [15, 16]. in this configuration, the first channel is the reference and the other information. when in multiple dimensions, or previous calibration of measuring channel for "zero" indicators both channels can be informative. the converter of inputting information signal can be implemented as a two-component spectrometric converter. one part is a twolayer structure consisting of reflectivity or missing bases which caused a translucent layer of bio-sensitive material (fig. 6, area 1 "biosensor" which gets radiation (h (1)). fig 6. implementation example reconfigurable microprocessor control module of measurements and digital processing of biosensors signals another part – a two-channel semiconductor detector based barrier metal-semiconductor or heterostructure to which cutting through infrared filters of different band (1) and (2) gets reflected from "biosensor" radiation (h (2). in fig. 6 inset photo shows the layout of dualchannel scaling amplifier. dynamically change the gain, managing the analog-todigital conversion, data exchange with peripherals, ram and special cpu based fpgas and general management of measurements and data processing is governed by the cpu based on arm microcontroller. communication with the computer server is provided through the usb interface. library files of reconfiguration is downloaded from the server and stored in eeprom. reconfiguration of special cpu can be performed manually from the keyboard when you have selected the module or spectrometric transducer, and /or offline according algorithm of the management program. library files in reconfiguration includes software modules designed for calibration information-measuring device path, numerical calculations of integrated performance ratios reflection / transmission at different wave bands for various test substances and biosensors, and applications of simulation modeling spectra for basic measurements given points in the wave range. the results of rapid processing and analysis can be controlled with a graphical display. the overall indicator value transmission / absorption of radiation in a given wave range is defined as the difference value signals s0 and s, measured according to the channels containing the studied range, in which he "cut" ir filter. thus the measured signal values are adjusted by an amount that takes into account the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 99 deviation spectra radiation source, whose rays irradiated the sample, ir filter transmission spectra and spectra photos sensitivity photodetectors from ideal rectangular shape [15]. the experimental spectra of two samples of urea in the range of 2,5 to 12,5 microns (fig. 7, a) show some deviations in the range of 2,8–5,5 and 8,0–12,5 microns. in [7] the first change in the spectra range from 2.8 to 3.1 microns explain the absorption bands of high protein component of urea, in 10–12,5 micron may manifest elements and compounds penicillin. a) b) fig 7. changes in absorption spectra due to urea protein complexes and of penicillin components (a), and (b) an example of cutting off infrared filters applying for rapid analysis of changes in the spectral characteristics of urea integrated performance measurement bandwidth in the area 2,7–4,0 um for both samples differ within no more than 3–4%, which is within the measurement error and does not allow state changes in real spectra. apply filters from outside cutoff of 2,6 and 3,3 mm (fig. 7, b) allows the shortwave infrared range characteristic of the protein component. the default value is the difference between the measured transmission performance in this case is 12–15%, significantly higher than the total measurement error path device and is sufficient for rapid analysis of the spectra of the samples. as seen in fig. 7, and is similar to the difference of these indicators for penicillin compounds in the range of 40–50 microns 10–12,5%. for its fixation can be used with the filter cut-off limit of 6,5 microns. this measurement error of the order is consistent with measurement error path, as in the range of 6,5 to 10 microns no "overcompensation" spectra. of course, to identify specific elements and their teams should conduct a comparative analysis of the fine structure spectroscopy and rapid method it can not replace. to improve the accuracy of rapid analysis should decrease the width of the strip controlled waves and analyze different ranges which manifested the characteristic vibrations of the same group of elements and compounds. 4. conclusion at this experiment work was tested two natural sorption materials of inorganic and organic origin in removal of aluminium from water. sorption capacity of natural zeolite – clinoptilolite was compared with sorption capacity of organic material penicilliumglabrum, one of the worldwide species. adsorption tests were studied for different initial concentrations of the aluminum in water. clinoptiloliteachieved 93 % efficiency after two hours of contact with water containing aluminium, penicilliumglabrum needed longer time – from seven7 to 9 days to achieve 100% efficiency. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov, application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials, food and environment safety, volume xiv, issue 1 – 2015, pag. 93 100 100 results obtained from laboratory experiments shown, that both tested materialsclinoptilolite and penicilliumglabrumeffectively removed aluminium from water. 5. references [1] who: environmental health criteria 194, aluminium. geneva, 1997 [2] becaria a., campbell a., bondy s.c. aluminium as a toxicant. toxicology and industrial health 18, 309 (2002). [3] barabasz w., albińska d., jaśkowska m., lipiec j. ecotoxicology of aluminium. j. of environ. studies 11 (3), 199 (2002). [4] totten, g. e.; mackenzie, d. s. handbook of aluminum. marcel dekker. p. 724. (2003). [5] caballero b., allen l., prenkice a. aluminium. encyklopedia of human nutrition, 2006. [6] who: aluminium in drinking-water. background document for development of who guidelines for drinking-water quality, who/sde/ wsh/03.04/53 (2003). [7] helmboldt o., hudson l.k.: aluminum compounds, inorganic. ullmann´s encyclopedia of industrial chemistry. wiley-vch, p. 527 (2007). [8] banks w.a., kastin a.j. aluminuminduced neurotoxicity: alterations in membrane function at the blood–brain barrier. neurosci biobehav rev. 13(1), 47– 53 (1989). [9] ferreira p.c, piai k.a., takayanagui a.m., seguramuñoz s.i. aluminum as a risk factor for alzheimer's disease. rev. lat. am. enfermagem 16, 151 (2008). [10] gitelman h. j. physiology of aluminum in man. in: aluminum and health, crc press, p. 90, (1988). [11] yokel r.a., hicks c.l., florence r.l. aluminum bioavailability from basic sodium aluminum phosphate, an approved food additive emulsifying agent, incorporated in cheese. food and chemical toxicology 46 (6), 2261 (2008). [12] flaten t.p., alfrey a.c., birchall j.d., savory j., yokel r.a.: status and future concerns of clinical and environmental aluminium toxicology j. toxicol. environ. health 48, 527 (1996). [13] vulterin j., štablová r. toxikológia, bezpečnosť a hygiena práce v chemii. toxicology, safety and hygiene work in chemistry. karolinum, praha (1991). [14] horváthová e. properties and elimination of ammonium ions from water with natural zeolites. (in slovak). vodní hospodářství 7, p. 173-178 (1989). [15] barloková, d., ilavský, j.: natural zeolites in water treatment (in slovak). vodní hospodářství, 57/6, 213-215 (2007). [16] šamajová e., kraus i.: prediction of areas of slovak zeolites and the possibilities of utilizing them (in slovak). in: proceeding of the conference slovzeo”84, vysoké tatry, 1984, pp. 6-10. [17] reháková m., čuvanová s., gavaľová z., rimár j. utilization of the clinoptilolite type of natural zeolite in agrochemistry and agriculture (in slovak). chem. listy 97, 260-264 (2003). [18] barloková d. natural zeolites in the water treatment proces. slovak journal of civil engineering vol. xvi, issue 2, 2008, pp. 8-12, slovak university of technology in bratislava 2008, issn 1210-3896. [19] merkle p.b., knocke w.r, gallagher d., et al: characte-rizing filter media mineral coatings, journal of awwa, 12/1996, s.62-73. [20] samson, r.a., hoekstra, e.s., frisvald j.c., filtenborg o.: introduction to foodborne fungi. centralbureau voor schimmel-cultures baarn, ponsen and looyrn (1996). [21] tóthová, l. occurrence of microscopic fungi in the slovak section of danube river. biologia, bratislava 54/4, 379 (1999). [22] franková, e.: soil micromycetes in the water environment. život. prostr., vol. 35, no. 3, 156 (2001). [23] franková e., simonovičová a. geochemically microscopic fungi in polluted environment (in slovak). mineralia slovaca, 29, 10 (1997). [24] frank, v., támová, g., farkas, v., characterization of white mutant of trichoderma viride obtained by cadmium treatment. microbiological research 149, 61–64 (1994). [25] gadd, g. m. interaction of fungi with toxic metals. the new phytologist 124, 2560 (1993). 283 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 283 289 effect of treatment parameters on the carotenoid extraction from tomato peels of bulgarian industrial varieties milena nikolova1, *tsvetko prokopov 1, daniela ganeva 2, galina pevicharova 2 1department of environmental engineering, university of food technologies, plovdiv, bulgaria 2maritsa vegetable crops research institute, plovdiv, bulgaria tsvetko_prokopov@abv.bg *corresponding author received october 15th 2014, accepted december 29th 2014 abstract: tomato by-products are an attractive source of natural carotenoids. this study was carried out to investigate the extractability of carotenoids from tomato peels of two bulgarian industrial varieties, named “stela” and “karobeta”, and to assess the effects of the extraction parameters (time, extraction steps, temperature, solid/liquid ratio and solvent type) on the yield of carotenoids. individual carotenoid identification in dried tomato peels was carried out by using a hplc system. the content of total carotenoids, lycopene and -carotene in the obtained extracts was measured spectrophotometrically. carotenoid recovery was significantly (p < 0.05) affected by the investigated extraction parameters. the maximum amounts of total carotenoids (111.58±2.11 and 76.59±2.13 mg/100g), -carotene (54.69±2.15 and 64.70±1.42 mg/100g) and lycopene (49.07±0.19 and 6.51±0.57 mg/100g) extracted from dried tomato peels of “stela” and “karobeta” varieties, respectively, were obtained by using acetone as solvent, for three successive extraction steps of 30 min, at 40oc and solid/liquid ratio of 1:30. it was experimentally established that tomato peels of “stela” variety are suitable for lycopene and -carotene recovery, while those of “karobeta”variety for the recovery of -carotene only. keywords: vegetable, processing, waste, by-products, lycopene, -carotene, extractability 1. introduction the food processing industry produced large quantity of waste by-products. onetird of the used product in fruit and vegetable processing was reported that being discarded which creates a significant environmental problem [1]. today, food wastes including fruit and vegetable by-products are considered as a cheap source of valuable components since the existent technologies allow the recovery of target compounds and their recycling inside food chain as functional additives in different products [2]. for example, tomato is a food product containing large amounts of high addedvalue compounds such as carotenoids, mainly in the form of lycopene and -carotene. at present, large quantities of tomato skin and outer pericarp tissue are discarded as a waste product from the peeling operation. about 10-40 % of the total tomato processed for tomato products are as skins and seeds. the quantity of wastes generated during tomato production and processing, combined with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 284 the beneficial characteristics of components of these wastes justifies the great interest of researchers and manufacturers in extracting carotenoids from tomato wastes [3]. by-products of tomato processing are attractive source of valuable bioactive components and colour pigments. these products can be used as functional foods, dietary supplements and can also be applied to cosmetic and pharmaceutical products. extraction with organic solvents is a well-established method in the food industry. as the main tomato carotenoids are lipid-soluble, common organic solvents such as acetone, hexane, ethanol, ethyl acetate, methanol, petroleum ether and solvent mixtures in different ratios (e.g. 50:50 hexane-ethyl acetate, hexaneacetone, 50:25:25 hexane-acetone-ethanol) have been tested for carotenoids extraction. although carotenoids extraction yields obtained from tomato by-products and comparison of efficiency among different solvents are presented in the literature [38] there are limited data about the effect of treatment parameters on the carotenoids extraction from tomato by-products including the influence of tomato varieties. there are needs for further research concerning optimization of tomato carotenoids extraction and factors affecting carotenoids recovery from tomato byproducts which could be applied on a commercial scale. this study was carried out to investigate the extractability of carotenoids from peels of two bulgarian industrial tomato varieties and to assess the effects of extraction parameters, such as time, successive extraction steps, temperature, solid/liquid ratio and type of organic solvent on the yield of total carotenoids, lycopene and -carotene. 2. materials and methods 2.1. raw materials two bulgarian industrial tomato varieties named “stela” and “karobeta” were used in present study. raw tomatoes were grown under field conditions within the maritsa vegetable crops research institute, plovdiv, bulgaria. tomatoes were harvested from the selected plants in technological maturity for each variety and were transported to the laboratory within 24 h. 2.2. preparation of tomato peels raw tomatoes were blanched at 95oc for 2 min, cooled on tap water and hand peeled. the obtained tomato peels were subsequently air dried at 25±1oc, ground in a laboratory mill (bosh mkm 6003, germany) and sieved through a 1.00 mm sieve. moisture content of ground dry tomato peels was determined by gravimetric method at 105oc and was 4.61±0.21 % and 5.30±0.32 % for “stela” and “karobeta” variety, respectively. the obtained dry ground material was kept in glass jars closed with aluminium caps and wrapped with aluminium foil at -20oc until conduction of experiments. 2.3. chemicals acetone, n-hexane, ethanol, methanol, tetraclormethane, acetonitrile and methyl tert-butyl ether (mtbe) in analytical grade were purchased from sigma (germany). used standards of lutein, lycopene and -carotene were purchased from extrasynthese (france). 2.4. carotenoids extraction the extraction of carotenoids was performed into 250 ml conical glass flask wrapped with aluminium foil. the flask was plased in a temperature-controlled (±1oc) water bath and continuously agitated with magnetic stirrer (velp food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 285 scientifica aluminium hot plate stirrerare, italy) at 400 rpm. ground dry tomato peels (1.00 g) were plased in the extraction flask and stirred with extraction solvent at different extraction conditions, such as time (5, 10, 15, 30 and 40 min), temperature (20, 30, 40 and 50oc) and tomato peels/solvent ratio (1:5, 1:10, 1:20, 1:30 and 1:50). the obtained extract was vacuum filtered through filter paper mn640de and analysed for carotenoids content. the effect of extraction steps was examined as follows: successive extractions with acetone were conducted under the same conditions as above for 30 min each at 20oc and peels/solvent ratio 1:10. at the end of the first extraction step, the mixture was vacuum filtered, the residue was collected, put again in the flask and re-extracted with another quantity of acetone. the whole procedure was repeated four times to complete the successive extraction steps. the carotenoids content of the extract in each successive extraction were determined. when the effect of type of organic solvent was studied the obtained extracts were vacuum filtered, the liquid fraction was vacuume dried (t < 40oc), redisolved in acetone and analysed for carotenoids content. 2.5. determination of carotenoids content total carotenoids, lycopene and carotene contents in the extracts were measured spectrophotometrically (uvvis helios omega spectrophotometer, thermo fisher scientific, madison, wi, usa) at max 448 and 472 nm against acetone as blank, according to manuelyan [9]. carotenoids content was expressed as mg/100g dry weight. 2.6. carotenoids analysis for the identification of individual carotenoids dryed tomato peels were analysed by hplc system (waters, milford, usa) composed of a uv-vis deterctor (waters 2487 dual ), a waters 1525 binary pump and thermostat (lco 102), according to the method proposed by [10]. the hplc system was equipped with a (supelco discovery hs) c18 column (25 cm x 4.6 mm, 5 m particle). a mobile phases of methanol:acetonitrile in ratio 8:2 (a) and mtbe (b) with following gradient elution were used: 95 % (a) and 5 % (b) initially, 95 % (a) and 5 % (b) in 3 min, 80 % (a) and 20 % (b) in 4.5 min, 65 % (a) and 35 % (b) in 10 min, 95 % (a) and 5 % (b) in 15 min. the flow rate was maintained at 1 ml.min-1, the column temperature at 30oc and detection was carried out at 270 nm and 290 nm. the analysis of the chromatographic data was carried out on a breeze 3.30 (waters, milford, usa) software. the identification of major carotenoids in tomato dry peels was carried out by comparing the retention times and absorption spectra with reference standards as described by [10]. the calibration curves were linear from 5 to 50 g/ml (r2 > 0.99). the percentage of hplc identified carotenoids in dried tomato peels was calculated as the ratio of the concentration of each carotenoid (based on the respective standard curve) to the sum of all identified carotenoids in the chromatogram, multiplied by 100 [4]. 2.7. statistical analysis all experiments were run in triplicate. the data were analysed and presented as mean values with standard deviation. statistical analysis was conducted by using of statgraphics centurion xvi version 16.2.04 software (statpoint technologies inc., usa). the analysis of variance technique, incl. lavene’s test, anova, and duncan’s multiple range test were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 286 used to determine significant differences at 95 % confidence (p < 0.05) level. 3. results and discussion 3.1. carotenoids identification hplc analysis of dried tomato peels of the two investigated varieties indicated that the main carotenoid was -carotene, while lycopene and lutein are also presented (table 1). this is contrary to the results obtained of [3], who reported lycopene as the main carotenoid of processing tomato waste composed of skins and seeds. tomato peels of “stela” variety were riched in -carotene (61.8 %) and lycopene (35.3 %), while these of “karobeta” variety were riched in -carotene (86.7 %). table 1 carotenoids concentration (%) in dried tomato peels tomato varieties carotenoids concentration (%) lycopene -carotene lutein stela 35.34 61.78 2.86 karobeta 8.09 86.72 5.17 3.2. effect of extraction time the effect of time on carotenoids extraction from dried tomato peels was investigated by conducting experiments during the extraction with acetone in solid:liquid ratio 1:10 at 20oc. 0 10 20 30 40 50 60 70 80 5 10 15 30 40 time, min c ar ot en oi ds c on te nt , m g/ 10 0g total caroten oids beta-carotene lycopene fig.1. effect of time on carotenoids extraction (peels/acetone 1:10, t = 20oc) from dried tomato peels of “stela” variety the obtained results are shown in figure 1 and figure 2 for “stela” and “karobeta” variety, respectively. as it was observed, carotenoids content of the extracts depended on extraction time, showing a high intial rate of extraction that decreased with time until an almost equilibrium was riched. the experimental results indicated the suitable extraction time of 30 min for total carotenoids, lycopene and -carotene extraction from peels of the two tomato varieties. this is in accordance with the results obtained by another research [3]. 0 10 20 30 40 50 60 70 5 10 15 30 40 time, min c ar ot en oi ds c on te nt , m g/ 10 0g total carotenoids beta-carotene lycopene fig.2. effect of time on carotenoids extraction (peels/acetone 1:10, t = 20oc) from dried tomato peels of “karobeta” variety 3.3. effect of extraction steps the effect of successive extraction steps on carotenoid extraction is illustrated in table 2. it is clearly indicated that the total carotenoids, lycopene and -carotene contents in the obtained extracts were significantly (p < 0.05) affected by the number of extractions. as it was observed, no significant differences (p < 0.05) or in some cases decreasing was obtained for the carotenoids contents between third and fourth extraction steps. triple extraction was found as adequate for all experiments. 3.4. effect of temperature the carotenoids content obtained by three successive extraction of 30 min each with acetone in solid/liquid ratio 1:10 at food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 287 temperatures ranging from 20 to 50oc is presented in table 3. limiting factor for the choise of extraction temperature was the boiling point of acetone (56oc) and the need to avoid isomerisation and/or oxidation of carotenoids. the increase in extraction temperature generally increased the extractability of carotenoids. the contents of total carotenoids, lycopene and -carotene in the obtained extracts were significantly (p < 0.05) higher at 40oc than at 20oc. however, when the extraction temperature rose to 50oc, an important extractability of carotenoids was not noticed. finally, at 50oc decreasing of the carotenoids content was oserved, excluding the content of -carotene in the extract from tomato peels of “karobeta” variety. following the obtained results, a suitable extraction temperature of 40oc was chosen for adequate extraction of carotenoids. 3.5. effect of solid/liquid ratio solid/liquid ratio is another factor which affects the extraction of carotenoids. an equilibrium between the use of high and low solid/liquid ratios, involving a balance between high costs and solvent wastes, on the one side, and avoidance of insufficient mixing and saturation effects, on the other side, has to be found to optimized value [5]. table 2 carotenoids content (mg/100g) in extracts from dried tomato peels (peels/acetone 1:10, 30 min extraction, t = 20oc) depending of successive extraction steps parameter tomato variety extraction steps 1 2 3 4 total carotenoids, mg/100g stela 31.84±0.09a 44.45±1.05b 54.17±1.00c 54.58±0.82c karobeta 12.05± 0.07a 14.78±0.43b 23.92±1.78c 20.38±1.26d lycopene, mg/100g stela 6.03±0.34a 10.59±0.30b 12.62±0.44c 12.71±0.35c karobeta 0.61±0.06a 0.66±0.03a 1.62±0.07b 1.44±0.21b β-carotene, mg/100g stela 24.07±0.81a 34.16±1.02b 41.44±0.57c 42.11±1.52c karobeta 10.80±0.23a 13.96±0.27b 22.27±1.71c 18.94±1.05d the values are mean of three replicates  sd. values bearing different lowercase letters (a, b, c, d) in the same row differ significantly (p < 0.05). table 3 carotenoids content (mg/100g) in extracts from dried tomato peels (peels/acetone 1:10, triple extraction of 30 min) depending of temperature parameter tomato variety temperature of extraction, oc 20 30 40 50 total carotenoids, mg/100g stela 10.17±1.38a 23.62±1.65b 31.16±1.21c 26.92±1.10d karobeta 19.28±0.28a 21.45±0.25b 23.35±0.55c 23.59±0.32c lycopene, mg/100g stela 1.36±0.31a 3.54±0.37 b 5.29±1.08c 3.36±0.28b karobeta 1.59±0.07a 2.79±0.36b 3.61±0.03c 1.33±0.03a β-carotene, mg/100g stela 8.08±0.97a 18.41±1.17b 27.4±1.10c 21.66±0.75b karobeta 16.34±0.19a 17.15±0.12b 18.21±0.43c 20.39±0.53d the values are mean of three replicates  sd. values bearing different lowercase letters (a, b, c, d) in the same row differ significantly (p < 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 288 table 4 presents the effect of solid/liquid ratio on carotenoids extractability. the increased organic solvent volume increased the carotenoids extractability up to solid/liquid ratio of 1:30, than at ratio 1:50 it decreased. the solid/liquid ratio of 1:30 produced significantly (p < 0.05) higher contents of total carotenoids, -carotene and lycopene in all samples in comparison with the other ratios. table 4 carotenoids content (mg/100g) in extracts from dried tomato peels (triple extraction of 30 min with acetone, t = 20oc) depending of solid/liquid ratio parameter tomato variety solid/liquid ratio 1:5 1:10 1:20 1:30 1:50 total carotenoids, mg/100g stela 42.13±0.25a 59.92±0.21b 84.41±0.19c 98.26±0.67d 58.16±5.54b karobeta 26.40±0.46a 27.02±0.18a 29.26±0.30b 64.08±0.25c 27.17±0.32a lycopene, mg/100g stela 6.92±0.18a 10.75±0.55b 13.65±0.64c 16.94±1.01d 12.50±1.19c karobeta 0.17±0.04a 1.63±0.26b 2.01±0.12c 3.9±0.12d 1.09±0.16 e β-carotene, mg/100g stela 32.30±0.79a 41.65±2.57b 64.25±0.29c 73.98±1.89d 44.87±0.6 b karobeta 24.81±0.18a 23.63±0.34b 25.21±0.22a 56.06±1.40c 23.88±0.98b the values are mean of three replicates  sd. values bearing different lowercase letters (a, b, c, d, e) in the same row differ significantly (p < 0.05). 3.6. effect of extraction solvent solvent selection is usually considered as the most important factor [5]. the contents of total carotenoids, lycopene and carotene in the extracts obtained by three successive extractions of 30 min with two different solvents and one solvents mixture at 40oc and solid/liquid ratio of 1:30 are presented in table 5. the extraction efficiency was affected by the solvent type and its polarity. as indicated in table 5, acetone presented significant (p < 0.05) higher content of carotenoids extracted from tomato peels of “karobeta” variety, compared to the hexane and hexane/acetone/ethanol mixture, possibly due to better penetration of the acetone to plant cells where carotenoids are enclosed. concerning “stela” variety, no significant differences (p > 0.05) was observed for carotenoids content extracted from tomato peels using acetone and hexane, respectively. table 5 carotenoids content (mg/100g) in extracts from dried tomato peels (solid/liquid ratio 1:30, triple extraction of 30 min, t = 40oc) depending of used organic solvent parameter tomato variety type of solvent acetone hexane hexane/acetone/ ethanol 50/25/25 total carotenoids, mg/100g stela 111.58±2.11a 108.42±1.06a 96.83±2.11b karobeta 76.59±2.13a 69.14±1.07b 65.95±0.01c lycopene, mg/100g stela 49.07±0.19a 49.05±0.21a 41.18±2.35b karobeta 6.51±0.57a 4.66±0.25b 5.45±2.46a b β-carotene, mg/100g stela 54.69±2.15a 51.78±0.77a 48.86±0.39b karobeta 64.70±1.42a 59.63±0.75b 55.39±1.98c the values are mean of three replicates  sd. values bearing different lowercase letters (a, b, c) in the same row differ significantly (p < 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 milena nikolova, tsvetko prokopov, daniela ganeva, galina pevicharova, effect of treatment parameters on the carotenoids extraction from tomato peels of bulgarian industrial varieties, food and environment safety, volume xiii, issue 4 – 2014, pag. 283 – 289 289 from the experimental results presented in table 5 can be concluded that extraction of total carotenoids, lycopene and -carotene from tomato peels of “stela” and “karobeta” varieties with acetone is adequate. 4. conclusion the extractability of carotenoids from dried peels of two bulgarian industrial tomato varieties, named “stela” and “karobeta”, was investigated. the effects of extraction time, extraction steps, temperature, solid/liquid ratio, two organic solvents and one solvents mixture on the contents of total carotenoids, lycopene and -carotene in the obtained extracts were established. the maximum amounts of total carotenoids, lycopene and -carotene extracted from tomato peels of the investigated tomato varieties were obtained in our experiments using acetone for three successive extractions of 30 min, at 40oc and solid/liquid ratio of 1:30. carotenoids contents in the extracts obtained at these optimal extraction conditions, for “stela” and “karobeta” varieties, respectively were as follow: total carotenoids of 111.58±2.11 and 76.59±2.13 mg/100g, -carotene of 54.69±2.15 and 64.70±1.42 mg/100g and lycopene of 49.07±0.19 and 6.51±0.57 mg/100g. experimentally was established that tomato peels of “stela” variety are suitable for lycopene and -carotene recovery, whiles these of “karobeta”variety for the recovery only of -carotene. 5. references [1]. prokopov ts., utilization of byproducts from fruit and vegetable processing: a review, journal of food and packaging science, technique and technologies, 3. 49-54, (2014) [2]. galanakis ch.m., recovery of high added-value components from food wastes: conventional, emerging technologies commercialized applications, trends in food science and technology, 26. 68-87, (2012) [3]. strati i.f., oreopoulou v., effect of extraction parameters in the carotenoid recovery from tomato waste, international journal of food science and technology, 46. 23-29, (2011) [4]. strati i.f., oreopoulou v., process optimisation for recovery of carotenoids from tomato waste, food chemistry, 129. 747-752, (2011) [5]. strati i.f., oreopoulou v., recovery of carotenoids from tomato processing by-products – a review, food research international, (2014), http://dx.doi.org/10.1016/jfooders.2014.09.032 [6]. calvo m.m., dado d., santamaria g., influence of extraction with ethanol or ethyl acetate on the yield of lycopene, -carotene, phytoene and phytofluene from tomato peel powder, eur. food res. technol., 224. 567-571, (2007). [7]. nikolova m.i., prokopov ts.v., characteristics and functional properties of natural origin lycopene: a review, journal of food and packaging science, technique and technologies, 2. 115-120, (2013) [8]. rizk e.m., el-kady a.t., el-biali r.el., characterization of carotenoids (lyco-red) extracted from tomato peels and its uses as natural colorants and antioxidants of ice cream, annals of agricultural science, 59(1). 53-61, (2014) [9]. manuelyan h., express methods for assessing the carotenoid composition of tomato fruits (in g.kallo. genetic improvement of tomato) spring –velag, 193-195, (1991) [10]. georgieva l., marchev a., ivanov i., ganeva d., bojinov b., pavlov a., improved hplc methods for determination of carotenoids and tocopherols in different varieties of tomatoes, scientific works of the university of food technologies-plovdiv, volume lx. 632-637, (2013) 266 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 32016, pag. 266 275 validation of 3-dimentional models of natural gas combustion and temperature measurements pavlo zasiadko, mykola pryadko thermal fluids and indastrial refrigeration dept., national university of food technologies, adress, kyiv,ukraine, info@nuft.edu.ua, corresponding author: pavlo zasiadko, iaroslav@nuft.edu.ua received september 7th 2016, accepted september 29th 2016 abstract: technological combustion processes present serious environmental problems due to the emission of harmful substances, predominantly in gaseous state. according to the generally accepted view, the nitrogen oxides mostly originate in combustion plants in the high temperature zones. therefore, determination of such zones within the combustion applications at a stage of design calculations or by means of directs measurements are of a major importance. the problems associated with the thermocouple errors at temperature measurements in the flames and combustion product flows are being dealt with. a model has been derived aimed at the evaluation of correction factors that are to be applied, when processing data of the direct thermocouple readings. the model takes into account convective heat transfer, irradiation from the combustion products on a part of the thermocouple bead. it also takes into consideration that a part of the thermocouple bead irradiates in the direction of the cooled surrounding. as a result, a transcendent fourth order equation has been obtained and solved numerically by mathcad. a set of correction coefficient was obtained which, when added to the direct readings of thermocouples, have shown a close conformity to the results obtained from the 3-d modeling of natural gas combustion in the cylindrical downflow reactor. thus developed correction methodology may be recommended for use in practice, when temperature measurements are performed by bare thermocouples, when bead sheathing or suction pyrometer can not be used. key words: thermocouple, heat flux, convection, radiation, flame, reradiation. 1. introduction the problem of flame temperature measurements with bare thermocouples has been dealt with in [1-8, 10, 11]. they mainly address the measurement of temperature in combustion processes within the flame or near it in the vicinity of cooled walls or adversely temperature measurements of relatively cold gas flows in the presence of high-temperature enclosures that are in direct exposure to the thermocouple. if a thermocouple measures the temperature of gas flow outside of the radiating flame, it most likely gives too high temperature readings, since the thermocouple bead receives not only the convective heat from the flow itself but irradiative flux from the flame as well. because the radiant flux is proportional to the 4th temperature degree, it is clear that thermocouple measurements in high temperature processes, particularly in combustion ones, the input of radiant flux will be much more significant than that from convection. similarly, when measurements take place in a close vicinity of flame with the enclosure at a lower temperature and with optically transparent medium, the thermocouple readings will be lower than the actual flame temperature. this is due to the effect that the thermocouple bead, http://www.fia.usv.ro/fiajournal mailto:info@nuft.edu.ua mailto:iaroslav@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 267 being a participant of complex heat transfer, will receive heat as a result of convection from the gas flow and radiation from the flame, will also irradiate towards the cooled enclosure. shannon and butler [1] suggested a mathematical model that takes into account the heat balance of the thermocouple bead: (𝜀𝑓𝑙 ϭ𝑇𝑓𝑙 4 -𝜀𝑏 ϭ𝑇𝑏 4)𝐹𝑓𝑙−𝑏 −(𝜀𝑏 ϭ𝑇𝑏 4 − 𝜀∞ϭ𝑇∞ 4 )(1 − 𝐹𝑏 ) = ℎ𝐴𝑏 (𝑇𝑓𝑙 − 𝑇𝑏 ) , (1) where f – a function that takes into account the relative exposure according to subscripts flame-bead or beadsurrounding; h, a heat transfer coefficient to the thermocouple bead and its surface area, respectively; ε, ϭ –respective surface emissivity (absorptivity) and stefan-boltzmann constant. in [2] an extremely simplified model was accepted, according to which the thermocouple radiates all the heat, obtained by the convection from the flow: (𝑇𝑓𝑙 − 𝑇𝑏 ) = 𝜀ϭ ℎ (𝑇𝑏 4 − 𝑇∞ 4 ), (2) where h – heat transfer coefficient from the gas flow calculated by: 𝑁𝑢 [ 1 2 (𝑇𝑓𝑙−𝑇𝑏) 𝑇𝑏 ] −0.17 = 0.24 + +0.56( 𝑤𝑑 𝜈 )0.45 , (3) combining (2) and (3) one can approximately determine the thermocouple error from the re-radiation: (𝑇𝑓𝑙 − 𝑇𝑏 )~ 𝑑0.55 𝑈0.45 (𝑇𝑏 4 − 𝑇∞ 4 ). (4) from (4) it follows that the reduction of the error can be achieved by reducing the diameter of the thermocouple bead or by decreasing u the proportion of radiant heat transfer between the gas flow and the bead. walker-stokes [3] proposed the method of measuring temperature with a thermocouple set, whose diameter is decreased progressively. this method has allowed to reduce inertia of thermocouple readings and to determine the effect of radiation basing upon the expression (4). the method becomes unrealizable due the temperature instability of flows and necessity to install the thermocouple in the same place of a flow. most often, the protective screens are used to shield thermocouple beads [1-4]. protective screens can be accompanied with the suction of flow through a cylindrical screen, which leads to an increase of flow speed around the thermocouple bead and, as a result, increasing convective heat transfer coefficient thus bringing closer bead actual temperature to that of flow. in practice, the speed of sucked flow is limited by the experimental considerations (prevention of significant disturbance in the flow).comparative results of the temperature measurement in industrial furnaces by means of unprotected thermocouples of different diameters, two types of shielded thermocouples, that differed by the place of suction, were given in [1, 2, 8]. the authors have shown that the greatest error occurs at measuring the temperature of medium with the thermocouple irradiation. from the data given in [1-8] follows that the error of "cold" flow measurement by bare thermocouple within the radiating environment reaches 250%. errors of temperature measurements by bare thermocouples in “hot” flows surrounded by cooled walls reach 25%. installing of a dual screen can reduce the error to 25% and 7%. the main objective of the work is to derive a method to correct the direct readings obtained when measuring temperatures of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 268 hot combustion products by bare thermocouples in the presence of cooled walls 2. matherials and methods experiments have been conducted in the experimental rig designed and erected at the coal energy technology institute of the nas of ukraine. the scope of experimentation covered the following: obtaining experimental data of the direct temperature and components” concentration distribution within the reactor when burning natural gas, which then will be used for the 3-d cfx and fluent models validation by the comparison of the measured and modeled data; co-combustion of coal and solid biomass aimed at the determination of the optimal process conditions; combustion of solid biomass at different regime parameters aimed at the determination of the effect of the various types of biomass burning kinetics, its conditions and properties aimed at the determination of the optimal process conditions. since the experimental rig closely models the conditions which exist in the lower radiation section of industrial boiler furnaces in the vicinity of burners, the experimental results obtained thus may be applicable for the designing optimal combustors. the layout of the stand is shown in figure 1. thermal capacity of the unit when burning coal with air as an oxidant reaches 100 kw. the main part of the unit is a down flow test channel with four diagnostic sections in which combustion takes place. each of three initial sections is 0.6 m long with the internal diameter 0.28m. the last one has the same length and 0.2 m internal diameter. the internal walls of all test sections have a tree layer protective covering consisting of consecutive layers of zircon dioxide, fire clay and asbestos. each section has a water cooled jacket with the individual supply and water flow measurement, which in turn allows thecalorimetry of each section. the burner head is equipped with two natural gas burners. the main burner has an additional (ground) solid biomass or coal together with ng and initial air. fig.1. experimental down flow unit vgp-100 b 1-experimental sections, water cooled jackets for calorimetry; 2-burner head with multiple air inlets; 3-ash collector; 4-cyclon for fly ash removal; 5-flue gases duct. the secondary burner is designed to inject natural gas and air only. there are also additional inlets allowing injection of any of the combustion components with the respective measurements of the component’s flow rate. each section also equipped with the inlets allowing inserting probes and thermocouples allowing direct food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 269 measurements of local temperature and flue gases composition. the experiments of natural gas combustion were carried out in the down flow cylindrical reactor vgp-100, figure1. the temperature of test sections’ walls was measured by chromel-alumel (type k) thermocouples that were built in the fire proof lining 10 mm deep from the fire layer. periodical measurements of fire layer of lining were performed with the pyrometer “dpr-1” through the diagnostic openings. the temperature of the gas flow on the axis of the reactor was measured by platinum-platinum-rhodium thermocouples (type r) with 0.2 mm diameter wires in corundum protective cover with 0.5 mm diameter channels positioned in probes at a midsection of each test section. hot thermocouple junctions were covered with a layer of corundum making the bead diametr-5 mm. thus obtained data related to the combustion of natural gas of a known composition were used for the validation of the developed 3-d cfx model of the process along with the direct temperature measurements which were used as benchmark data for developed model of measured temperatures correction. in order to carry out the validation, a complex 3-d meshes of the reactor has been developed. the mesh represents the experimental stand vgp in general and in minor details. the main problem of the mesh development consisted in finding an optimum compromise between the number of mesh cells (which eventually determines accuracy and calculations convergence time and, thus, the required computer capacity) on the one hand, and the necessity to represent minor stand details which affect the actual process and are to be represented in the mesh – on the other. (a) (b) (c) fig.2. reactor vgp a vgp reactor; b geometry model with and without insulation; c 3-d mesh of the reactor core and insulation it was found that the gas temperature measured in 4 points in the middle of each diagnostic section on the axis were 1201500c lower than respective cfx – fluent simulated values. the difference in the obtained data can be explained by re-radiation of thermocouple beads towards the cooled walls. thermal balance of thermocouple bead, that considers both convective and radiant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 270 flows, can determine the value of error of flow temperature measurement on the axis of the reactor. it should be noted that the model under consideration becomes significantly complex as a result of taking into account radiant heat transfer between the thermocouple and gas flow, since gases are characterized by selective spectral energy absorption and emission. the absorption and emission of energy by monatomic and diatomic gases, including nitrogen, oxygen, and hydrogen are insignificant and may be neglected. polyatomic gases, including carbon dioxide co2, water vapour h2o, sulphur dioxide so2, ammonia nh3, and others, have a large capacity for absorption and emission of radiant energy. carbon monoxide co also has a significant level of emission and absorption, but this gas occurs only as traces in the products of methane combustion at a proper air – fuel ratio. the model of radiant heat exchange of bead with the environment has been derived under the assumption that there exists a local thermodynamic equilibrium bead-environment, so we can assume the equality absorption – emission according to the kirchhoff’s law. flue gas and the walls are considered gray bodies. radiation of the wall is characterized by a continuous spectrum and the medium (combustion gases) has selective radiation in a form of separate bands. the results of direct temperature measurements with bare thermocouples during the experimental combustion of natural gas in a vertical reactor vgp-100 (100 kwth) of the coal energy technology institute were taken as benchmark values for the validation of the model. the objective of the proposed model is to derive a comparatively simple equation which will allow to calculate the temperature of a hot bead of a thermocouple (tb) placed into the flow of hot combustion gases at a given (tfl) and being exposed to the relatively cold walls at given tw. thus, the correction temperature can be determined which is to be used when direct readings of unsheathed thermocouples which are used for temperature measurements in media with convective-radiative heat transfer. the equation was derived under the condition of equality of heat supplied to a thermocouple bead and the amount of heat, that thermocouple gave off to the walls as a result of re-radiation. heat input is realized by means of convective heat transfer, radiation of hot flue gas and by radiation of heated head burner lining towards the bead. for the approximate estimation of the reradiation effect it was decided to apply the simplified model of radiant heat transfer between the bead surface and the heated lining surface under the following conditions: a) bead surface and radiating lining surface are plane-parallel; b) the area of bead surface, that receives radiation, is equal to a half of the total area of a thermocouple bead; c) absorbing capacity of corundum is 𝜀𝑒𝑓 = 0.9; d) the temperature of the heated lining is taken equal to the temperature of the gas core tfl.then, the components of a heat balance of the bead will be: absorbed heat 𝑄𝑎𝑏𝑠 = ℎ(𝑇𝑓𝑙 − 𝑇𝑏)𝐴𝑏 + 𝐴𝑏𝐶0𝜀𝑏𝑒 [( 𝑇𝑓𝑙 100 ) 4 −( 𝑇𝑏 100 ) 4 ] 2 + + 𝐴𝑏𝐶0 [𝜀𝑓𝑙 ∞( 𝑇𝑓𝑙 100 ) 4 −𝜀𝑓𝑙,𝑤𝑎𝑙𝑙 ∞ ( 𝑇𝑏 100 ) 4 ] 𝜀 𝑓𝑙 ∞ 𝜀𝑓𝑙 + 1 𝜀𝑓𝑙,𝑤𝑎𝑙𝑙 −1 , (5) heat radiated by the thermocouple bead on the wall food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 271 𝑄𝑟𝑎𝑑 = 𝜀𝑒𝑓 𝐶0 𝐴𝑏 2 [( 𝑇𝑏 100 ) 4 − − ( 𝑇𝑤𝑎𝑙𝑙 100 ) 4 ], (6) where 𝑇𝑓𝑙 , 𝑇𝑏 , 𝑇𝑤𝑎𝑙𝑙 temperatures of gas flow, thermocouple bead and wall, respectively,  к ; h – heat transfer coefficient from gas environment to the thermocouple bead, [w/m2k]; 𝜀𝑒𝑓, 𝜀𝑓𝑙 , 𝜀𝑓𝑙,𝑤𝑎𝑙𝑙 , 𝜀𝑓𝑙,𝑤𝑎𝑙𝑙 ∞ emission factors taken for bead-lining effective, for gas flow at gas temperature, for gas flow at wall temperature and for gas flow at wall temperature at a infinite beam length, respectively [9, 11, 13, 14]; 𝐴𝑏 thermocouple bead surface area. the heat transfer coefficient was calculated for the case of an external flow of hot flue gases around a thermocouple taken from [9]; thermophysical properties were calculated by the software eneccalc3, and checked with the data base [9]: 0.54 0.33 0.58 0.362 0.03re pr 0.35re prnu    (7) the speed of the flue gases for determining the reynolds number was calculated on the basis of process modeling cfx 15 fluent code (lic.no1023420 ) and conventionally accepted as constant, w=4.5 m/s, the diameter of the thermocouple bead 005.0d m.the values of heat transfer coefficients were calculated for three different temperatures of incoming flow – flue gases core. the calculations were performed with variations of temperatures of the gas core within the limits tfl = 2200 ... 1600 k and with the temperature of cooled walls variation so that the temperature difference between the wall-flow changed recursively 200 ... 400 ... 600 k, which corresponds to the actual conditions of the experiment. thermophysical properties of combustion products, optical properties of co2 and h2o, both individually and in mixtures were defined as a result of the process modeling). convective heat transfer coefficients were calculated for each pair of the assumed combustion gas and walls temperatures, accordingly. thus derived transcendental equation of 4th degree with nonlinear coefficients was solved in mathcad software package as the point of intersection of the two functions (5 and 6). the solution was obtained in graphical and numerical forms. the results of equations solving for different cases of gas stream and wall temperatures are summarized in table 1 and shown in the graph (figure 3). table 1. summary table of thermocouple readings at different temperatures of the gas core and walls tг, k tс, k tт, k 2200 2000 2077 1800 1986 1600 1910 1400 1854 1200 1814 2000 1800 1880 1600 1792 1400 1724 1200 1677 1800 1600 1686 1400 1606 1200 1548 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 272 according to the obtained data, shown in the figure 1, at the actual 2,200 k temperature of the flow and 2000 k temperature of the wall, thermocouples will show the temperature at 123 k less than the actual gas temperature. at the same gas temperature, the difference between actual temperature and measured thermocouple will increase to 386 k at 1200 k temperature of the wall. at 1800 k of core temperature and 1600 k of wall temperature specified difference will be 114 k that is slightly smaller than the measurement of a core temperature. 3. results and discussion analyzing the obtained data, one can conclude that re-radiation plays a significant role in the measurement of temperature at the considered conditions. the graphical interpretation of mathcad solution of the developed model at a flow temperature of 1400 k and variations of wall temperature is presented in figure 4. fig. 3. calculated readings of a thermocouple installed in the flow of flue gases and emitting radiation on the channel wall (twall) at different temperatures of a flow fig. 4. calculation by the model for conditions [2, 8] 1500 1600 1700 1800 1900 2000 2100 1200 1400 1600 1800 2000 tgas =2200k tgas=2000k tgas=1800k twall tbead food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 273 each abscissa value of the lines’ intersection gives the value of thermocouple bead temperature at the flue gas temperature 1400 k and respective wall temperature. the bigger the wall temperature, the closer to the 1400 k value moves the intersection point, and respectively closer to 1400 k moves the reading of the thermocouple. to define a degree of reliability of the developed model, the data obtained from the above calculation were displayed in coordinates given in [2, 8] namely, terror difference between the gas temperature and thermocouple bead temperature as a function of wall temperature twall. the results are given in of fig. 5. fig. 5. comparison of the calculations based upon the developed model and data [2, 8] the solid line data [2, 8]. the points – calculation by the model. apparently, the data obtained from the proposed model are slightly lower than those given in [2, 8] at wall temperatures below 8500c. this can be explained by the fact that in our model it is assumed that the bead emits energy by the half of its surface that is directed down towards the ash collector, as the upper part of the bead is directed to head with burners, which emits energy to the bead. in addition, the proposed model takes into account emission and absorption of triatomic gases in the combustion products, whereas the models [2, 8] threat the gas flow as afully transparent medium. it seems possible to use the obtained data toadjust the results of a direct temperature measurement of the thermocouples located at the middle of each test section and vgp100d, and to compare them with the calculated data of process modeling by means of fluent and cfx packages. fig. 6. comparison of the results of calculation by means of cfx package with the results of measurements with a consistent refinement that based on the model of reradiation 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 t wall, c t, error , c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 274 thus, the calculation of corrective corrections held on stream temperatures according to the model, and the temperature of the wall was taken according to the measurements that were considered reliable because thermocouple of the wall, being embedded in the lining, do not participate in radiation heat transfer. thus, obtained corrections were added to the thermocouple readings because they reflect the bead temperature and not the temperature of the flow of combustion products that flows around the thermocouple bead. instead, corrections to the thermocouple readings reflect the temperature of stream that flows around beads, see equation (5, 6). as it can be seen from figure 6, based on the readings of thermocouples and proposed model of measuring, the temperature of the flow on vgp channel axis matched with the modeled results close enough (within 5%), which indicates adequacy of the proposed model and accuracy of data obtained by measurements by the thermocouples. validation of the developed 3-d model was based also on the comparison of heat losses obtained by the calorimetry of vgp standsections with the respective data calculated within the cfx model. apparently, there is a close correspondence of the results within 7.5% on sections and 2.2% on stand as a whole. table 2. comparison of heating losses on the calorimetric vgp stand sections part of the experimental set heat loss measured on the stand, w heat loss of a model, w δ, % burner 8600 9113 5.9 flange of a burner 2500 2320 7.2 diagnostic section 4300 4641 7.3 total 15400 15738 2.2 4. conclusion 3-d modeling gives a very powerful tool for the study of combustion processes of different fuels, allowing any variation of operational parameters and flow rates of components of the process. the model simulation allows to study the particulars of harmful substances appearing, and thus to prevend this. a model has been developed which allowed deriving coorection data, which when added to the direct bare thermocouple measurements present data close to those obtained by modeling. the methodology of the calculational mesh development, the set of thermal boundary conditions together with the kinetic sub models may be used for 3d modeling of gaseous fuels combustion. 5. references [1] shannon k.s., butler b.w., a review of error associated with thermocouple temperature measurement in fire environments. usda forest service, fire sciences laboratory. 2-nd international wild land fire ecology and fire management congress. november orlando, (fl. usa): 16-20, (2003), https://ams.confex.com/ams/pdfpapers/67056.pdf [2] pitts w.m., braun e., temperature uncertainties for bear-bead and aspirated thermocouple measurements in fire environments, thermal measurements: the foundation of fire standards, (pa): 15, (2002) [3] walker d., stocks b.j., thermocouple errors in forest fire research, fire technology, 4, 5864, (1968) [4] martin r., cushwa c., fire as a physical factor in wildland management, proceedings of tall timbers fire ecology conference, (1969) https://ams.confex.com/ams/pdfpapers/67056.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 pavlo zasiadko, mykola pryadko, validation of 3-dimentional models of natural gas combustion and temperature measurements, food and environment safety, volume xv, issue 3 – 2016, pag. 266 – 275 275 [5] sung chan kim, hamins a., on the temperature measurement bias and time response of an aspirated thermocouple in fire environment journal of fire sciences,no 26: 509529, (2008) [6] struk p., dietrich d., russell v., feier i., comparisons of gas-phase temperature measurements in a flame using thin-filament pyrometry and thermocouples, nasa/tm: 212096, (2003) [7] z’graggen a., friess h., steinfeld a., gas temperature measurement in thermal radiating environments using a suction thermocouple apparatus, meas. sci. technol. 18: 3329–3334, (2007) http://iopscience.iop.org/09570233/18/11/010/ [8] blevins linda g., pitts william m., modeling of bare and aspirated thermocouples in compartment fires, fire safety journal, 33: 239359, (1999) [9] grigoriev v., zorin v., theoretical fundamentals of thermal engineering, m., energyator publisher: 556, (1988) [10] smith e.j., natan g.i., omar n.h., dally b.b., comparison of thermocouple temperature measurements of simple and processing jet propane flames, 5-th asia-pacific conference on combustion, the university of adelaide, (australia): 89-92, (17-20 july 2005) http://www.adelaide.edu.au/directory/graham.nath an [12] roberts i.l., coney j.e.r., gibbs b.m., estimation of radiation losses from sheathed thermocouples, applied thermal engineering, doi: 10.1016: 14, (2011) [13] prabal talukdar, gas radiation hottel charts, http://web.iitd.ac.in/~prabal/gasradiation.pdf [14] isachenko v., osipova v., sukomel a., heat transfer, m., energy: 415, (1981) http://iopscience.iop.org/0957-0233/18/11/010/ http://iopscience.iop.org/0957-0233/18/11/010/ http://www.adelaide.edu.au/directory/graham.nathan http://www.adelaide.edu.au/directory/graham.nathan http://web.iitd.ac.in/~prabal/gas-radiation.pdf http://web.iitd.ac.in/~prabal/gas-radiation.pdf 1. introduction 4. conclusion 3-d modeling gives a very powerful tool for the study of combustion processes of different fuels, allowing any variation of operational parameters and flow rates of components of the process. the model simulation allows to study the particulars of ha... 5. references 317 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 317 320 chloro ph yll c on tent an d an tioxida nt ac tiv ity o f sun flow er o il w ith arom a tic r aw m ater ials *svitlana usatiuk1, liubov pelekhova1 1foodstuff expertise department, national university of food technologies, ukraine esmeraldo@ukr.net *corresponding author received november 18th 2014, accepted december 29th 2014 abstract: the article presents the results of the study on chlorophyll content and antioxidant activity (aoa) of sunflower oil with aromatic raw materials. dried parsley and basil were used as aromatic raw materials. sunflower oil with aromatic raw materials was obtained by extraction. dried basil and parsley were in the amount of 2 to 10 %. the chlorophyll content was quantified by the photometric method. determination of the aoa was carried out by the flourescence recovery after photobleaching (frap) method. the results show the dependence of chlorophyll content on the amount of raw materials taken for extraction. the smallest content of chlorophyll (26.5 mg/l) was found in oil with 2 % of dried basil addition. the highest content of chlorophyll (185.4 mg/l) has been found in oil with 10 % of dried parsley addition. all samples of the investigated oils have high antioxidant activity. it increases with the addition of aromatic raw materials from 2 to 10 % by 1.57 times for oils with basil and by 1.48 times for oil with parsley. keywords: chlorophyll content, antioxidant activity, aromatic raw materials, basil, parsley, sunflower oil. 1. introduction in recent decades the structure of human diet has changed significantly. the traditional foods can no longer provide human with essential nutrients due to their decreased bioavailability. an introduction to human diet of foods that are rich in polyunsaturated fatty acids, minerals, dietary fiber, vitamins and antioxidants can help satisfy the need of biologically active substances (bas). one of perspective directions is the production of vegetable oils using aromatic plant materials. it allows not only to enrich the product with bas of natural origin, but also to improve its organoleptic characteristics. these oils are used for cooking in restaurants, because they are important components of the flavoring scale of dishes [1]. we have developed the technology of vegetable oils production with increased biological value using aromatic raw materials [1]. dried basil and parsley were used as aromatic raw materials. these plants are successfully cultivated in europe and contain significant amounts of bas. furthermore, they contain chlorophyll that strengthens cell membranes, promotes the formation of connective tissue, supports the body's ph balance, improves pulmonary function, liver, kidney, thyroid, intestine, strengthens the immune system, prevents dna pathological changes, outputs the remnants of toxins and medicines, binds free radicals shows antioxidant and antibacterial properties [23]. chlorophyll is one of the most valuable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 svitlana usatiuk, liubov pelekhova, chlorophyll content and antioxidant activity of sunflower oil with aromatic raw materials, volume xiii, issue 4 – 2014, pag. 317 – 320 318 components of plants [4] and foods high in their content can prevent a number of diseases. the aim of our research was to determine the chlorophyll content and antioxidant activity of sunflower oil enriched with bas of basil and parsley. 2. materials and methods 2.1 materials oil enriched with bas of aromatic raw materials was prepared using the coldpressed sunflower oil and dried parsley and basil. fresh parsley and basil have high moisture content (about 90%). the use of fresh plants leads to increase of moisture content in the final product. it can promote the process of non-enzymatic hydrolysis of vegetable fat (accumulation of free fatty acids, increment the acid number) and early spoilage. [5-8]. sunflower oil with aromatic plant materials was obtained by extraction. the technology includes the following stages: preparation of plant material (crushing, infusion with the ethanol-water mixture), processing of oil with prepared material by mixing under vacuum (temperature of 34 to 36° c, process time 6 hours), separation of oil from the material by filtration. basil and parsley were added in the amount of 2 to 10% by weight of oil. if the addition is of 12% or more, then the final product begins to acquire a bitter taste. 2.2 chlorophyll content quantitation of chlorophyll content was carried out by the method proposed by i.h. hazzaa, v.a. vaynshteyn and t.h. chibilyaev [9]. weighed sample of oil was dissolved in chloroform. the optical density was measured at wavelength of 670 nm by cpc-2 photoelectric colorimeter. chlorophyll content (h, mg/l) was calculated by the formula: 755 10000  pd h (1) where: d – optical density; р – multiplicity of dilution of chloroform; 755 – chlorophyll specific absorption coefficient at 668 nm; 1000 – conversion of concentration in mg/l. 2.3 antioxidant activity antioxidant activity was determined by frap method. the methanol extracts of vegetable oils for the determination were obtained using the method proposed by a. szydlowska-czerniak [10]. 1 ml of fecl3 solutions (1.5 mg/l, methanol) and o-phenantroline (1 mg/l, methanol) were added 1 ml of the methanolic extract of the investigated oil. the obtained mixture volume was adjusted to 5 ml and left in the dark place for 10 minutes. the optical density was measured at wavelength of 670 nm by cpc-2 photoelectric colorimeter. the results were calculated by calibration curve using soluble vitamin e as standard sample (fig. 1). the calculation of antioxidant activity (aoa, mg/kg) based on the activity of vitamin e was carried out by the formula: м vbxа аоа s   )( (2) а,b – coefficients determined from the calibration curve; хs – optical density of the sample; v – total volume of the methanol extract, ml; м – sample weight, kg. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 svitlana usatiuk, liubov pelekhova, chlorophyll content and antioxidant activity of sunflower oil with aromatic raw materials, volume xiii, issue 4 – 2014, pag. 317 – 320 319 fig.1. аоа сalibration curve 3. results and discussion the dependence of chlorophyll content on the amount of aromatic raw materials was investigated in 5 samples of sunflower oil with basil and 5 samples of sunflower oil with parsley (fig. 2, 3). fig.2. influence of the amount of basil on chlorophyll content in sunflower oil with basil the results show the dependence of the amount of chlorophyll content on raw materials taken for extraction. fig. 3. influence of the amount of parsly on chlorophyll content of sunflower oil with parsly the smallest amount of chlorophyll (26.5 mg/l) was found in oil with 2 % of dried basil added. and the highest chlorophyll content (185.4 mg/l) has been found in oil with 10 % of dried parsley added. moreover, oils obtained with dried parsley have high chlorophyll content in comparison with oils enriched by bas of basil. the antioxidant activity of 10 samples of unrefined sunflower oil with aromatic raw materials is shown in fig. 4. fig.4. antioxidant activity of sunflour oil with aromatic raw materials food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 svitlana usatiuk, liubov pelekhova, chlorophyll content and antioxidant activity of sunflower oil with aromatic raw materials, volume xiii, issue 4 – 2014, pag. 317 – 320 320 the antioxidant activity of the oil under research has high values in the range of 100...200 mg/kg. the highest antioxidant activity was found in the sample of sunflower oil with 10 % of parsley added because of the high chlorophyll content. it should be noted that the values of the aoa of oils with basil and parsley with the same amount of plant materials added are very close, considering the difference in the content of chlorophyll. this fact can be explained by transition of basil essential oils which have high antioxidant activity to sunflower oil. 4. conclusion the use of aromatic plant materials such as dried basil and parsley as the valuable source of chlorophyll in the technology of sunflower oil increases the biological value of final product. addition of raw materials in the amount of 8...10% reaches the chlorophyll content in the finished product to 146...163 dm3 for oils with basil and to 167...185 dm3 for oil with parsley. the research results also shown a high antioxidant activity of the obtained samples. with increasing the amount of plant materials added from 2 to 10% the antioxidant activity increases of 1.57 times for oils with basil and 1.48 times for oil with parsley. thus, the significant effect of amount and type of aromatic raw materials on the antioxidant activity of spice oils has been conducted. 5. references [1] usatiuk s., pelekhova l., technology of vegetable oils of high biological value with the use of aromatic raw materials, food science, engineering and technologies. sientific works, lx, 836-839 (2013). [2] levent i̇. chlorophyll: structural properties, health benefits and its occurrence in virgin olive oils, academic food journal, 9, 26-32 (2011). [3] tychomyrova n.a. functional food technology, frantera, moscow, 213 p (2002). [4] rosu a., nistor n., miron n., popa r., ultrasound assisted extraction of photosynthetic pigments from dried dill (anethum graveolens). j. of faculty of food engineering, 11, 4, 5-9 (2012). [5] paponian v., voskanian o. analіsys of influence of various factors on the quality of fats, oil industry, 2, 10-11, (2004). [6] nekrasova t. natural antioxidants for oil and fat products, oils and fats, 4(50), 1-2, (2005). [7] tiutiunnikov b. chemistry of fats, kolos, moscow, 447 p, (1966). [8] ievtieva n. oxidative stability of commercial vegetable oils, oil industry, 5. 14-17 (2006). [9] khazza i., vainshtein v., chibiliaev t. extraction of lipophilic biologically active substances from st. john's wort by water-oil emulsions, pharmaceutical chemistry journal, т. 37, 7, 20-23 (2003). [10] szydlowska-czerniak a., dianoczki c., recseg k., karlovits g., szlyk e. determination of antioxidant capacities of vegetable oils by ferric-ion spectrophotometric methods, talanta, 76, 899-905 (2008). microsoft word 0b. contents 4_2012.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 4 – 2012 contents: 1. ultrasound-assisted extraction of photosynthetic pigments from dried dill (anethum graveolens) ana-maria rosu, denisa ileana nistor , neculai doru miron1, marcel ionel popa, ramona mihaela cojocaru 5 2. removal of antimony from water by coagulation danka barloková, ján ilavský, michal kunštek 10 3. research on the influence of temperature on the process of granulation by extrusion dmitro viktorovich rindyuk, svyatoslav yuriyovich lementar 19 4. identification and examination of some probiotic properties of lactobacillus plantarum f3 rositsa denkova, velichka yanakieva, zapryana denkova, zoltan urshev, bogdan goranov, elena sotirova 22 5. the influence of taphrina deformans (berkeley) tulasne (peach leaf curl) attack on the activity of some oxidoreductases in cultivar cardinal rodica ciobanu 30 6. some physical properties of seabuckthorn and how the packing conditions influence them mihaela jarcău 36 7. purification of cacl2 solutions using purolite s930 resin dynamic studies camelia popa, costel mironeasa 43 8. modelling of the thermophysical lactic acid aqueous solutions. density and viscosity andrei i. simion, cristina g. grigoraş, loredana e. bardaşu, adriana dabija 49 9. study on the raw cow milk hygienic parameters from different milk collection centers and different years using a multivariate analysis method silvia mironeasa, georgiana gabriela codină 59 10. the determination of flavonoids, the total polyphenols and antioxidant activity of basil seasoning micşunica rusu, roxana mihăilescu 65 11. influence of chemical composition and temperature on honey phase angle mircea oroian 72 12. investigation of radioactive contamination of bone raw material as one of the alternative sources of organic calcium l.v. peshuk, n.v. budnyk, i.i. shtyk 78 13. the influence of storage conditions upon ascorbic acid content in jonathan and golden apples marcel avramiuc, ana leahu, cristina damian, cristina-elena hretcanu 81 14. aspects concerning coagulation enzymes and different inducing parameters for milk curdling process adriana dabija, iuliana sion 87 15. author instructions 93 16. subscription information 98 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 4 – 2012 microsoft word 15 lucrare gontariu ioan _1_.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 91 pr otec tion aga inst th e pro lifer atio n o f th e co lo ra do beetle ( leptino ta rsa decem lin ea ta s ay) in th e con c ept of o rga nic c ultu re at th e potato *ioan gontariu1, ioan-cătălin enea2 1faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania,e-mail ioang@fia.usv.ro 2agricultural research and development station of suceava, b-dul 1 decembrie 1918 nr. 17 suceava, romania, e-mail catalin_i75@yahoo.com *corresponding author received 2 may 2012, accepted 5 june 2012 abstract: in intensive agricultural production application of pesticides is inevitable.within the rules of sustainable agriculture and good agricultural practice, targhet application of pesticides are necessary.modern plant protection is bazed on the balance between the pest threat for crops and intensification of chemical treatment.the present paper intends to approach the efficiency of two bioinsect powders lazer and neemazal on the viability of the pest in the conditions from suceava, the experiments were carried out at a.r.d.s. suceava in the year 2008 in field and laboratory. if we compare with the untreated witness, the egg hatching was in the ratio of 70-80% in 2-days period, on the treated lots with the two products, the hatching in the ratio of 60-70% was registered only after a few days, the “inhibitory” action of the hatching process maintaining even after the end of this process on the untreated lots. the civilized countries under the pressure of the people retired a lot of organophosphoric insecticides and carbamates from the production system, being replaced by alternatives. the reject of the pesticides is a chance for adopting biological products of fighting having as effect the result of some organic, healthy products which don’t endanger the human’s health. approximately 5% from the surface cultivated with plants is occupied by the organic agriculture. keywords: bio-insect powders, eggs, treatment, organic production 1. introduction in recent years, scientists have focused on increasing of food production to be need of the world population expanding rapidly. unfortunately, crop loss is still keep on due to plant diseases, caused by insects, plant pathogen fungi, bacteria and viruses.the colorado potato beetle (leptinotarsa decemlineata say) is the most destructive pest of potatoes, eggplants and tomatoes in the world [1]. in today, colorado potato beetle is still a destructive pest of potatoes, eggplants and tomatoes and causes an important loss in the yield of crop. the toxicities of several sesquiterpenes against colorado potato beetle has been recently reported [2-3]. a great shortcoming of the long use of insecticides is the emergence of the pests’ resistance to these substances. we have observed that this resistance is present at the insects after the continuous treatment with the same compound for 15-20 generations, against the same pest species [4]. after the intense use of synthetic insecticides, appeared a series of negative aspects like: the environment’s contamination, the toxicity on the nontarget species, human being’s health, all these got everybody’s interest for the 92 “natural” ways of control, including those for researching new sources of vegetal insecticides [5]. one possible way to reduce the high consumption of synthetic insecticides is through the application of botanical insecticides (bi), generally considered to be environmentally and medically safe [6], [7]. at present, the majority of commercially produced bis utilize the effects of plant metabolites which show acute or chronic toxicity to insects [8], [9]. their great advantage is their selective action against parasites and pest predators, as well as pollinators. plant substances acting as antifeedants are found in all the compound groups of secondary plant metabolism. however, the most effective insect feeding inhibitors come from terpenoids, alkaloids, saponins and polyphenols [10]. 2. experimental the experiences were made at the agricultural research development station suceava in 2008. it was cultivated the sante variety, the surface of an experimental lot being of 16.8 m2. for the blight were applied two treatments with curzate and antracol. the bio insecticides were applied with manual pumps of 0.5 l with delicate atomization, for an uniform dispersion of the product. the laser 0.033% and neemazal 0.33% and 0.41% and 0.5% treatments were done on the 9th of june when the process of hatching is practically inexistent. the observations regarding the hatching and an eventual larva development were made on a period of 10 days. all the places for the egg laying were counted before the treatment and after the treatment they tried to observe their effects on the eggs and of the larvae which hatched. 3. results and discussion in comparison with the non treated witness, where the hatching of the eggs in proportion of 80% happened in two days, in the treated lots with laser and neemazal bio chemicals, the hatching in proportion of 60-70% was registered only after a few days (table 1). the inhibitory action of the hatching process maintained even after 12-13 june, when this ended at the non treated lots. as a result, at the neemazal treatment, the frequency of the non hatched eggs was registered between 8 and 32% even after 78 days from the end of the hatching process on the witness lots (table 2). an extension of the hatching stage registered also after the use of laser product 0.033%. the duration of the non hatched eggs was more reduced with 1-2 days. if we analyze the dynamic of the eggs hatching not taking into consideration the non treated lots, then it results, according to the data written in the first table, that the hatching rhythms in the first stage (9-16 june) doesn’t differ significantly according to the used product. therefore, in the interval 09.06-16.06, the frequencies of the places with hatched eggs are between 2560% for the laser product, 4-64% for neemazal 0.33%, 12-68% neemazal 0.41% and between 0-75% for neemazal bio insecticide 0.5%. even if the differences between the three concentrations of neemazal product on the frequency of the hatched eggs don’t have certain significance, we can still admit the fact that a certain growth of the concentration was associated with the rise of the hatched eggs frequency. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 93 between the two types of bio insecticides, the laser product 0.033% has a remarkable capacity of reducing the adult’s population which is in the copulation period, by estimating a mortality of 80-85%. the data written in table 3 shows that laser product has a real ovicidial action, underlined even since the 11th of june, very rapid from the treatment’s execution, when the frequency of the eggs which dried was of 7%. table 1 the influence of the treatments on the eggs viability eggs non hatched hatched dried bio insecticide date % differences % differences % differences 0 1 2 3 4 5 6 7 9.06 92 mt 8 mt 11.06 20 -72 ooo 80 72xxx 16.06 0 -92ooo 100 92 xxx 19.06 0 dl 5% 27 17 dl 1% 35 24 untreated dl 0,1% 51 35 9.06 75 mt 25 mt 0 mt 11.06 64 -11 29 4 7 7 16.06 29 -46 oo 60 35xxx 11 11x 19.06 0 -75ooo 61 36xxx 39 39x dl 5% 23 16 8 dl 1% 38 23 14 laser 0,033% dl 0,1% 56 34 25 9.06 96 mt 4 mt 11.06 77 -19 23 19 16.06 36 -60 ooo 64 60xxx 19.06 32 -64 ooo 68 64xxx dl 5% 20 20 dl 1% 27 28 neemazal 0,33% dl 0,1% 41 42 9.06 88 mt 12 mt 11.06 65 -23 35 23 16.06 32 -56ooo 68 56xx 19.06 8 -80 ooo 92 80xxx dl 5% 23 30 dl 1% 34 43 neemazal 0,41% dl 0,1% 46 64 9.06 100 mt 0 mt 0 mt 11.06 71 -29 o 29 29 0 16.06 25 -75 ooo 75 75xxx 0 19.06 14 -86 ooo 82 82xxx 4 4 dl 5% 21 22 dl 1% 31 32 neemazal 0,5% dl 0,1% 44 47 94 in the eight days that followed (19.06), the ovicidal action increased by 5.5 times (table 3), what represented 39%. the same diminution effect of the larval population was also observed at neemazal 0.5% but in a smaller proportion (4%) and also pretty late (19.06) (table 3). the laser product also remarks itself regarding the anti larval action (table 3) and also by its volume and short time when happened the decimation of the larvae’s population. table 2 the evolutions of the efficiency of the tested products in 2008 (%) eggs non hatched hatched dried date used products % differences % differences % differences 0 1 2 3 4 5 6 7 untreated 92 mt 8 mt laser 0,033% 75 -17 25 17 0 neemazal 0,33% 96 4 4 -4 neemazal 0,41% 88 -4 12 4 neemazal 0,5% 100 8 0 dl 5% 34 21 dl 1% 49 30 9.06 dl 0,1% 69 43 untreated 20 mt 80 mt mt laser 0,033% 64 44xx 29 -51ooo 7 7 neemazal 0,33% 77 57xxx 23 -57ooo neemazal 0,41% 65 45xx 35 -45oo neemazal 0,5% 71 51xxx 29 -51ooo dl 5% 24 23 dl 1% 33 32 11.06 dl 0,1% 47 45 untreated 0 mt 100 mt mt laser 0,033% 29 29 60 -40oo 11 11x neemazal 0,33% 36 36x 64 -36oo neemazal 0,41% 32 32 68 -32o neemazal 0,5% 25 25 75 -25o dl 5% 33 24 dl 1% 44 34 16.06 dl 0,1% 56 48 untreated 0 mt 100 mt mt laser 0,033% 61 -39o 39 39xxx neemazal 0,33% 32 32 68 -32 neemazal 0,41% 8 8 92 -8 neemazal 0,5% 14 14 82 -18 4 4 dl 5% 37 34 8 dl 1% 54 48 14 19.06 dl 0,1% 78 68 25 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 95 so in order to register the same level of larvae eradication (25%) existent on that date (11.06) on the lots treated with laser 0.033% were necessary another 6-7 days when it was used neemazal product in doses of 0.41% and 0.5%. also in order to show the very alert rhythm of the anti larval action we can also observe the fact that in the case of using the laser product 0.033%, the total eradication registered after 4 days (from the treatment until 13.06). it is possible that the same result to be registered after 10 days (from the treatment until 20.06) in the case of the use of the bio chemical, neemazal 0.5%. the observations made also underline the fact that the emergence of the second age larvae took place on 16th of june on the non treated lots and on the 19th of june on those treated with neemazal 0.3%, while in the cases of using laser 0.033% and neemazal 0.5% bio chemicals didn’t signal their presence. 4. conclusion the laser product has a real ovicidal action, underlined even at 2 days from the treatment, when the frequency of the eggs that dried was of 7% and after 8 days they increased 5.5 times. this also has a remarkable capacity of reducing the adults’ table 3 synthesis regarding the protection capacity of the tested products against the proliferation of the colorado beetle ovicidal action (pu*) anti larval action (lm*) cumulated (pu +lm) date % differences % differences % differences laser 0,033% 9.06 0 mt 0 mt 0 mt 11.06 7 7 25 25x 32 32x 16.06 11 11x 61 61xxx 72 72xxx 19.06 39 39xxx 61 61xxx 100 100xxx dl 5% 8 17 27 dl 1% 14 26 40 dl 0,1% 25 42 65 neemazal 0,33% 19.06 0 neemazal 0,41% 9.06 0 0 mt 11.06 0 8 8 16.06 0 12 12 19.06 0 42 42xx dl 5% 20 dl 1% 32 dl 0,1% 54 neemazal 0,5% 9.06 0 mt 0 mt 0 mt 11.06 0 3 3 3 3 16.06 0 15 15 15 15 19.06 4 4 83 83xxx 87 87xxx dl 5% 24 26 dl 1% 37 39 dl 0,1% 60 63 96 population, by estimating a mortality of 80-85% for the neemazal treatment the frequency of the non hatched eggs was between 8 and 32% even after 7-8 days from the end of the hatching on the witness lots. an extension of the hatching stage also registered after the use of laser product 0.033%. the duration of the presence of the non hatched eggs was firstly reduced with 1-2 days. 5. references 1. puttler, b., long, s.h., host specificity tests of an egg parasite, edovum puttleri (hymentoptera: eulophidae), of the colorado potato beetle (leptinotarsa decemlineata) (coleoptera: chrysomelidae). proc. entomol. soc. washington 85, 383– 387p, (1983). 2. gonzalez-coloma, a., valencia, f., martin, n., hoffman, j.j., hutter, l., marco, j.a., reina, m., silphinene sesquiterpenes as model insect antifeedants. j. chem. ecol. 28, 117–129p, (2002). 3. gonzalez-coloma, a., reina, m., guadano, a., martinez-diaz, r., diaz, j.g., garcia-rodriguez, j., alva, a., grandez, m., antifeedant c20 diterpene alkaloids. chem. biodiversity 1, 1327–1335p, (2004). 4. brudea, v., biological control in integrated management of insect pests with special reference to forest ecosystems. university press „ştefan cel mare” suceava, 68p, (2007). 5. brudea, v., rîşca, i.m., tomescu, c.v., lupăştean, d., fărtăiş, l., metabolites vegetable reagents for use in integrated management of insects, university press „ştefan cel mare” suceava, 45p, (2008). 6. pavela, r., the feeding effect of polyphenolic compounds on the colorado potato beetle (leptinotarsa decemlineata say). pest technol. 1, 81–84p, (2007). 7. dayan, f.e., cantrell, c.l., duke, s.o., natural products in crop protection. bioorgan. med. chem. 17, 4022–4034p, (2009). 8. pavela, r., possibilities of botanical insecticide exploitation in plant protection. pest technol. 1, 47–52p, (2007). 9. pavela, r., insekticidni prostredek a jeho pouziti pro ochranu rostlin predhmyzem (insecticides and his using for protection of plants against insect). patent no. cz 301072 b6, vˇestnik 43, czech republic, (2009). 10. koul, o., insect antifeedants. crc press, bota racon, fl, (2005). issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume x, issue 4 15 december 2011 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro 157 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 157 161 the influence of processing by crushing and boiling on ascorbic acid content in some plant species *marcel avrămiuc1, silvia mironeasa2 1,2faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received february 15th 2015, accepted may 25 th 2015 abstract. in this work the influence of processing (crushing and boiling) on ascorbic acid content in four plant specie was searched on . the biological material was represented by: green onion (leaves and bulbs), spinach leaves, pear fruits (from vegetable farms around suceava town) and kiwi fruits (from supermarket). the ascorbic acid content was determined through a method based on reduction (by the ascorbic acid) of 2.6-dichlorphenol-indophenol to the corresponding leucoderivate. through crushing and one hour exposure to air (20-22°c), the largest reduction in ascorbic acid content was registered in pear fruits (50%), and the lowest one in spinach leaves (11.7%). as compared to fresh samples, under thermal processing (boiling) the ascorbic acid content has decreased most in pear fruits (by 52.6%) and least in spinach leaves (by 38.5%). the presence of sugar (10%) in the boiling liquid made ascorbic acid decrease less than in its absence, both in plant tissue and in the boiling fluid. keywords: ascorbic acid, green onion, spinach, pear, kiwi 1. introduction spread in fruits and vegetables, vitamin a, c, and e are bioactive compounds with antioxidant activities, which have a high antioxidant capacity [1, 2]. according to some authors [3, 4, 5, 6], the phenolic compounds are also good contributors to the total antioxidant capacity of the foods containing them, but both vitamin c, carotenoids and phenolics may be poorly absorbed and rapidly metabolized, thus limiting their antioxidant ability in vivo [7, 8]. the storage conditions, on the one hand, and processing technology, on the other hand, can influence the content of bioactive compounds and their antioxidant capacity. thus, the freezing process can cause, sometimes, significant decrease in the level of vitamin c in legumes and fruits [9]. according to ball [10], drying methods, exposing the food to air lead to the loss of vitamin c because of its oxidation. the thermal treatments are the main cause of the depletion of natural antioxidants [11]. by zia-ur-rehman et al. [12]; zhang and hamauzu [13], cooking, pasteurization and the addition of chemical preservatives guarantee vegetables and fruits safe, but bring not always desirable changes in their physical characteristics and chemical composition (ascorbic acid, phenolics, carotenoids etc.). the aim of this paper was to search the ascorbic acid content variation in four plant species during processing, to see to what extent the content of this vitamin is influenced by processing mode, by the type of plant material, or by the both. 2. experimental the biological material used in this work was represented by four plant species: green http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 marcel avramiuc, silvia mironeasa, the influence of processing by crushing and boiling on ascorbic acid content in some plant species, food and environment safety, volume xiv, issue 2 – 2015, pag. 157 – 161 158 onion (leaves and bulbs), spinach leaves, pear (peeled fruits) derived from vegetable farms around suceava town, and kiwi (peeled fruits) purchased from supermarket. the ascorbic acid evaluation was carried out in the following working variants: raw material, chopped material and left for 60 min. at room temperature, boiled material 30 min., with sugar and sugar free, and boiling liquid, with sugar and sugar free. as to boiling conditions, the ratio of vegetable material-to-water was 1:10 w/v, and the concentration of sugar used was 10%. the ascorbic acid content was determined through a method based on reduction (by the ascorbic acid) of 2.6-dichlorphenolindophenol (2.6-dcpip) to the corresponding leucoderivate. the result was expressed as mg ascorbic acid per 100 g or 100 ml (mg%) product [14, 15]. the data obtained from four replications were analyzed using statistical package for social science software, version 16.0. the correlation analyses were performed at the probability levels of 95% and 99%. the differences between mean values of ascorbic acid were tested using analysis of variance anova one-way. in order to highlight the degree of influence of different factors, such as material status, the way of processing, and interaction between them on ascorbic acid content in each studied vegetable material, or upon ascorbic acid content from boiling liquid, the factorial analysis of variance in the condition specified was applied[16]. 3. results and discussion in fig. 1 the ascorbic acid mean values from raw material (whole) and processed (by crushing), and left at room temperature for one hour are presented comparatively. as shown in the graph, in the raw plant material the highest value of ascorbic acid was recorded in spinach (30.45±0.09 mg%), followed, in order, by kiwi (23.51±0.04 mg%) and green onion (22.35±0.15 mg%), the lowest one being in pears (6.87±0.08 mg%). by crushing and exposure to air at 2022°c, the ascorbic acid content was reduced in the all four species examined, but with different percentages (fig. 1). the f test result has shown a significant main effect (p=0.000), both of the variable material type, and of the variable material status on ascorbic acid content. also, the value of f test has indicated a significant cumulative effect (p=0.000) of the factors material type and material status on ascorbic acid content. the ascorbic acid mean values in vegetable raw samples differ significantly (p=0.000) of those ones from crushed plant samples, for all kinds of analyzed materials, the material status significantly influencing the ascorbic acid content. fig. 1. the ascorbic acid mean content in raw and chopped plant material by crushing and in contact with air, the ascorbic acid content was reduced by 50% (in pears), by 47.8% (in green onion), by 39.7% (in kiwi) and by 11.7% (in spinach). according to banu et al. [9], cabbage and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 marcel avramiuc, silvia mironeasa, the influence of processing by crushing and boiling on ascorbic acid content in some plant species, food and environment safety, volume xiv, issue 2 – 2015, pag. 157 – 161 159 carrots simple cutting has lead to loss of vitamin c for up to 75%. fig. 2 shows, comparatively, the ascorbic acid mean values from unprocessed plant material and from thermally processed (cooked without sugar). fig. 2 mean values of the ascorbic acid content in sugar free cooked plant materials, as compared to the raw material the unprocessed material, regardless of its type, indicates higher mean values for ascorbic acid content, as compared with the ascorbic acid content of the material boiled sugar free. the average values of the ascorbic acid content in raw samples have differed significantly (p <0.05) from those ones in boiled sugar free samples, for all types of plant materials studied: green onion (r=0.962), spinach (r=–0.976 ), pears (r=– 0.998), kiwi (r=–0.985). as compared to fresh samples, in boiled samples the ascorbic acid content has decreased by 38.5% (in spinach), by 45.6% (in kiwi) and by 52.6% (in pears). searching a mixture of orange-carrot, torregrosa et al. [17], cited by cortés et al. [18], observed that after pasteurization the remaining concentration of vitamin c was 83% of the concentration of the untreated juice. under the same conditions (boiling), in green onion sample the ascorbic acid content has increased by 10.8%. as this percentage increase was verified by repeated measurements (over 10), it is not a human error, and the explanation could be that after boiling, substances with reducing potentials are formed. these substances (reductones), reacting with the dye (2.6-dcpip), can give a falsely high result for the vitamin c content of the sample [10]. reductones are only likely to be found in processed foods after prolonged boiling or in canned foods after standing at elevated temperatures [19, cited by 10]. in fig.3 the ascorbic acid mean values in pears and kiwi cooked with sugar, compared with unprocessed plant material are shown. fig. 3. mean values of the ascorbic acid content in pears and kiwi cooked with sugar, as compared to the raw material as seen from the graph, as compared to the raw material (unprocessed), in the samples boiled with sugar, the percentage of ascorbic acid decreases by 64.9% (in pears) and 46.3% (in kiwi). the material status (raw or boiled with sugar) and the type of material have significantly influenced (p<0.001) the ascorbic acid content in plant material. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 marcel avramiuc, silvia mironeasa, the influence of processing by crushing and boiling on ascorbic acid content in some plant species, food and environment safety, volume xiv, issue 2 – 2015, pag. 157 – 161 160 the results of anova have revealed significant differences (p<0.001) between mean values of ascorbic acid from the material processed with sugar, and the raw material for the two types of plant material. comparing the values of ascorbic acid from samples cooked in the presence of sugar, with those ones from the samples processed without sugar it observes that, reported to raw material, a greater reduction in the content of this vitamin has occurred in pear samples in the presence of sugar (64.9%) as against 52.7% (in the absence of sugar), while in kiwi samples the percentages were close (46.3% in the presence of sugar, as against 45.6% in the absence of sugar). fig. 4 renders the mean values of ascorbic acid in the boiling liquid from pear and kiwi fruits cooked with sugar, as compared to the boiling liquid from the same plant material cooked without sugar. fig. 4. mean values of the ascorbic acid content in the boiling liquid from sugar and sugar-free plant materials from fig. 4 it can be seen that in the boiling liquid with sugar, the ascorbic acid values were 14.59±0.03 mg% in kiwi, and 1.55±0.03 mg% in pears. in the boiling liquid sugar free, the ascorbic acid values were 12.5±0.04 mg% in kiwi, and 0.86±0.04 mg% in pears. the experimental version with added sugar (10%) has recorded higher values of ascorbic acid both in plant tissue and in boiling liquid as compared to sugar free version. large losses of vitamin c occur during blanching, boiling, when the water used for heat treatment is not used (mainly losses by solubilization), protective action on vitamin c having anthocyanins, sugar, starch [9]. 4. conclusions researching the influence of processing on the content of ascorbic acid in samples of plant material belonging to four species (green onion, spinach, pears and kiwi) differences between these species were found, compared to the fresh products (unprocessed) . through grinding and one hour exposure to air at 20-22°c, the largest reduction in ascorbic acid content was registered in pear fruits and the lowest one in spinach leaves. by means of thermal processing (boiling) the total content of ascorbic acid has decreased mostly in pear fruits and in spinach leaves. the presence of sugar (10%) in the boiling liquid made as ascorbic acid to decrease less than in its absence, both in plant tissue and in the boiling fluid. 5. references [1] hassimoto n.m.a., genovese m.i., lajolo f.m.– antioxidant of dietary fruits, vegetables and commercial frozen fruit pulps. journal of agricultural and food chemistry, 53, pp. 2928-2935 (2005) [2] sanchez-moreno c., plaza l., de ancos b., cano p.– nutritional characterization of commercial traditional pasteurized tomato juices: carotenoids, vitamin c and radical-scavenging capacity. food chemistry, 98, pp. 749-756 (2006) [3] dillard c.j., german j.b. – phytochemicals: nutraceuticals and human health. review. journal of science and agriculture, 80, pp. 1744-1756 (2000) [4]vinson j.a., su x., zubik l., bose p.– phenol antioxidant quantity and quality in foods: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 marcel avramiuc, silvia mironeasa, the influence of processing by crushing and boiling on ascorbic acid content in some plant species, food and environment safety, volume xiv, issue 2 – 2015, pag. 157 – 161 161 fruits. journal of agricultural and food chemistry, 49, pp. 5315-5321 (2001) [5] cano p., plaza l., sanchez-moreno c., de ancos b. – elaboración y conservación de zumos de naranja: efectos de nuevas tecnologias sobre su calidad sensorial y nutricional. alimentación, nutrición y salud, 10, pp. 108-119 (2003) [6] chaovanalikid a., wrolstad r.e. – total anthocyanins and total phenolics of fresh and processed cherries and their antioxidant properties. food chemistry and toxicology, 69, pp. 67-72 (2004) [7] gardner p.t., white t.a.c., mcphail d.b., duthie g.g. – the relative contributions of vitamin c, carotenoids and phenolics to the antioxidant potential of fruit juices. food chemistry, 68, pp. 471-474 (2000) [8] zulueta ana, esteve maria j., frasquet isabel, frigola ana – vitamin c, vitamin a, phenolic compounds and total antioxidant capacity of new fruit juice and skim milk mixture beverages marketed in spain – food chemistry, 103, pp. 1365-1374 (2007) [9] banu c., iordan m., nour v, musteaţă g. – the processing of raw materials and the loss of biologically active substances. ed. „tehnica” utm. chişinău, p. 91-93 (2003) [10] ball g.f.m.– vitamins in foods, analysis, bioavailability, and stability. crc press, taylor & francis group 6000, broken sound parkway nw, suite 300, boca raton, fl 33487-2742, p. 292-305; 369-371 (2006) [11] anese m., manzocco l., nicoli m.c., lerici c.r. – antioxidant properties of tomato juice as affected by heating. journal of the science of food and agriculture, 79, pp. 750-754 (1999) [12] zia-ur-rehman z., islam m., shah w.h. – effect of microwave and conventional cooking on insoluble dietary fibre components of vegetables, food chemistry, 80, pp. 237–240 (2003) [13] zhang d.l., hamauzu y. – phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking, food chemistry, 88, pp. 502–509 (2004) [14] artenie v., tănase elvira – practicum of general biochemistry. replication center of “al. i. cuza” university, iaşi, pp. 56-58 (1980) [15] indyk h. and konings e., eds – official methods of analysis of aoac international, 17th ed., aoac international, gaithersburg, md, pp. 45-60 (2000) [16] tabachnick b.g., fidell l.s. – using multivariate statistic (5th ed.) pearson, london, new york (2007) [17] torregrosa f, esteve m.j., frígola a., cortés c. – j. food eng., 73, pp. 339-345 (2006) [18] cortés clara , esteve j. maría, frígola ana – effect of refrigerated storage on ascorbic acid content of orange juice treated by pulsed electric fields and thermal pasteurization, european food research and technology zeitschrift für lebensmittel-untersuchung und-forschung a© springer-verlag 200710.1007/s00217-007-0766x, published online: 11 october 2007 [19] roe j.h. – ascorbic acid, in the vitamins. chemistry, physiology, pathology, methods, györgy, p. and pearson, w.n., eds., 2nd ed., vol. 7, academic press, new york, 1967, pp. 27. issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xv, issue 2 30 june 2016 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt (amariei), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 311 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2013, pag. 311 315 research on chemical composition of cow raw milk sorina ropciuc 1 1 faculty of food engineering, stefan cel mare university of suceava, romania sorina.ropciuc@fia.usv.ro *corresponding author received october 15th 2013, accepted november 17th 2013 abstract: milk is considered to be food-medicine and mythological subject which nowadays has a lot of praise,as it had in the past, praise, brought not only by ordinary people but also by scientists, doctors,, pharmacists, zootechnicians, etc. this paper was prepared for a study on the quality of cow raw milk in northern romania. the study was based on the influence of age, lactation period and the diet of cattle on some qualitative components of raw milk coming from the households. the physicochemical properties determined were density, dry substance, acidity, fat and lactose. standardized tools and methods were used. there were noticed significant variations of qualitative characteristics of raw milk in winter. it was obtained a very significantly positive correlation between dry substance and fat content, r = 0.899***. also, lactose is significantly positively correlated with the fat content, r = 0.628** but also with the dry substance content, r = 0.564*. the age and the lactation period don’t influence the variation of the physical and chemical constituents. keywords: cow milk, physicochemical characteristics 1. introduction milk is considered to be food-medicine and mythological subject which nowadays has a lot of praise, as in the past, brought not only by ordinary people but also by researchers, doctors, pharmacists, zootechnicians, etc. in recent decades, milk began to be appreciated, but also contested, being subject of dispute, especially between the alternative and allopathic medicine [1-3]. in such a range of interest and controversy, it is natural that milk should be given special attention both internationally and nationally, in terms of commercial,industrial and medical perspective [4-6]. in 2001 it was established world milk day world milk day, which romania joined in 2009. tetra pak in partnership with april, proposed the day of 20th june to be celebrated as world milk day in our country. milk is an indication of healthy eating, the government after 1989 found it necessary to implement in schools the program,’’milk and bread roll". this paper was prepared for a study on the quality of cow raw milk in northern romania. the study was based on the influence of age, lactation and the diet of cattle on the qualitative components of raw milk coming from the households [7]. 2. experimental the material used in the study was the raw milk samples from two sources from individual households in suceava. the analysis period of milk samples took place in the months january-june 2013, once a week. the samples were collected from the morning milk, an amount of 500 ml and were accompanied by a register card which had the following data: the source and milk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sorina ropciuc, research on chemical composition of cow raw milk, food and environment safety, volume xii, issue 4, 2013, pag. 311-315 312 sample number; the name of the person who brought the milk sample; the identification number of the cattle, the date and time of milk collection; the age and the lactation period of the cattle. the samples were brought to the laboratory for the qualitative determination in max. 45 minutes from the milking. with regard to hereditary and technological factors, of physiological nature of the external environment that may have an influence on milk quality, we’ll make the following comments : the cattle are not of pure breed being hybrids of black-and-white variety; in terms of the age of the cattle, the no. 6007one is 4 years old and is at the second birth and no. 0978 cattle which is 9 years old had the seventh calf. the study was conducted in the first months of lactation, the samples were taken only in the morning, being considered the first milking. from the information received it resulted that milking was done three times /day. it is worth mentioning that milk was collected from perfectly healthy animals, that were not under medical treatment and were fed with specific fodder season to which was added root plants (beet) and potatoes. physicochemical determinations were made in the chemistry laboratory with standardized apparatus and methods. the statistical data processing was done by the program xlstat 2013. 3. results and discussion figure 1 shows the variation of physicochemical quality of milk samples collected from source 1. figure 1. changes in the physical and chemical characteristics of the samples of milk taken from source 1 the samples of milk taken from source 1 show a high content of fat, the percentage of 5.3% and 5.4% in march, confirms the good quality of raw milk. the high level of dry content is another indicator of the quality which supports the hypothesis of some milk samples with high quality features. the lactose content of 7.2% and 7.3% of milk samples also show a strong potential for both processing and for the production of cream and cheese [4-6]. the fat content of the milk samples from source 1 have fluctuations both in the winter period, january-march, and in the spring-summer period. at intervals of about 3 weeks, the milk fat content recorded significant decreases. in table 1 we can see correlations between physicochemical characteristics of milk from source 1. table 1. pearson correlation matrix between the physical and chemical quality characteristics of the milk samples collected from source 1 density, g/cm3 fat, % dry substance, % acidity, °t lactose, mg% density, g / cm3 1 fat, % 0.1993 1 dry substance 0.5719 0.8998 1 acidity, °t 0.1050 0.3599 0.3017 1 lactose, mg% 0.2400 0.6285 0.5641 0.0006 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sorina ropciuc, research on chemical composition of cow raw milk, food and environment safety, volume xii, issue 4, 2013, pag. 311-315 313 the samples of milk from source 1 show physical-chemical characteristics which are positively correlated, the milk density increases in the same time with the growth of dry substance content. pearson correlation index, r =0.571* indicates significant positive correlation. it is also noted a correlation index very significantly positive between dry substance and fat content, r = 0.899 ***. lactose is significantly positively correlated with fat content, r = 0.628** and also with the dry substance content, r = 0.564*. in table 2 there are presented correlations between physicochemical characteristics of milk from source 1 with the lactation period and the age. tabel 2. pearson correlation matrix between the dependent variables and the lactation period, respectively the age of the cattle at the milk samples collected from source 1 density, g/cm3 fat, % dry substance% acidity, °t lact., mg% lactation periodg ag e density, g/cm³ 1 fat, % 0.1993 1 dry substance, % 0.5719 0.8998 1 acidity °t 0.1050 0.3599 0.3017 1 lactose, mg% 0.2400 0.6285 0.5641 0.0006 1 lactation period, months -0.1930 0.3455 0.2515 -0.4813 0.4535 1 age, year -0.1930 0.3455 0.2515 -0.4813 0.4535 1 1 pearson correlation index show a weak significant negative correlation between the period of lactation and the milk acidity in the milk samples taken from source 1. the weak significant positive influence is seen on the lactose content, r= 0.453* [4][8]. in conclusion, it is considered that the age and lactation period do not influence a lot the variation of physical and chemical constituents. the samples of milk taken from source 2 had the lowest fat content of raw milk. initially, it was suspected the forgery of the sample by adding water, but in other determinations the fat content was much lower than the other samples. by analyzing the lactose content it was discovered that the milk has an increased and constant lactose content throughout the study period. in addition, the total dry substance has higher values, the highest value being 14.9% a lot more than other milk samples analyzed [9-11]. the acidity of milk is low, milk has an acidity of 16-18 degrees thörner most of the time in the determinations that i made [7-9]. the milk collected from source 2, recorded the highest variations of density. the values of density are the highest of 1.034 following the ones of 1.033g/cm³. the lowest density value was recorded in april, 26. figure 2. shows the variation of physicochemical characteristics of milk samples collected from source 2 in table 3 we find the variation of physicochemical quality characteristics of milk from source 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sorina ropciuc, research on chemical composition of cow raw milk, food and environment safety, volume xii, issue 4, 2013, pag. 311-315 314 table 3. pearson matrix between the physico-chemical quality characteristics of the milk samples taken from source 2 density, g/cm³ fat, % dry substance, % acidity, °t lactose, mg% density, g/cm³ 1 fat, % 0.2431 1 dry substance, % 0.5035 0.7357 1 acidity, °t -0.0267 0.2077 0.1773 1 lactose, mg% 0.1972 -0.1147 -0.2207 -0.2141 1 pearson correlation index between the dry substance and the density of milk samples indicates a highly significant connection, r =0.503*. also, the fat and the dry substance correlate strongly significant positive, the correlation index r =0.735** indicates this connection. in table 4 are presented the correlations between the physicochemical characteristics of milk from source 2 with the period of lactation and the age. table 4. pearson correlation matrix between the dependent variables and the lactation period, the age of cattle at the milk sample from source 2 density, g/cm³ fat, % dry substance, % acidity, °t lactose, mg% lactating period a e density, g/cm³ 1 fat, % 0.2431 1 dry substance,, % 0.5035 0.7357 1 acidity, °t -0.0267 0.2077 0.1773 1 lactose, mg% 0.1972 0.1147 -0.2207 -0.2141 1 lactating period -0.2315 0.2270 -0.0231 0.2443 0.1141 1 age -0.2315 0.2270 -0.0231 0.2443 0.1141 1 1 the lactation period and the age don’t show any correlation with the studied variables: density, fat, lactose and dry substance. the variation of the components from the milk samples taken from source 2 could be under the influence of other factors, genetic factors or factors which are related to the animal’s individuality. 5. conclusions the study revealed significant variations in the quality characteristics of raw milk during winter in both milk samples. there were not significant variations of qualitative characteristics of raw milk during the spring-summer time, which is explained by the inclusion of green fodder in the cattle feed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2013 sorina ropciuc, research on chemical composition of cow raw milk, food and environment safety, volume xii, issue 4, 2013, pag. 311-315 315 the measurements findings reinforce the conclusion of specialty studies, according to which the qualitative factors of milk varies significantly during the period of lactation. the milk collected from source 2, recorded the largest variations of density. the density values are higher than 1.034 following the ones with a value of 1.033 g/cm³. we notice a very significant positive correlation index between dry substance and fat content, r =0.899***. lactose is significantly positively correlated with the fat content, r =0.628** and with the dry substance content, r =0.564* as well [1112]. pearson correlation index indicate a weak significant negative correlation between the period of lactation and the milk acidity in the milk samples taken from source 1. the influence weakly significant positive is also seen in the lactose content, r = 0.453*[13-14]. in conclusion, it is estimated that the age and the lactation period don’t influence a lot the variation of physical and chemical constituents. 6. references [1] clare d.a., swaisgood h.e., bioactive milk peptides: a prospectus, journal of dairy science,83 (6), 1187–1195, (2000). [2] phelan m., aherne a., fit gerald r., j., o’brien n.m.: casein-derived bioactive peptides: biological effects, industrial uses, safety aspects and regulatory status, international dairy journal, 16, 643-654, (2009). [3] ranganathan r., nicklas t. and berenson g.s.: the nutritional impact of dairy product consumption on dietary intakes of adults . journal of the american dietetic association 105(9), 1391-400, (2005). [4] petit h. v: digestion, milk production, milk composition, and blood composition of dairy cows fedformaldehyde treated flaxseed or sunflower seed, (2003). [5] kim y.k., schingoethe d.j., casper d.p., ludens f.c.: lactational response of dairy cows to increased dietary crude protein with added fat, jurnal dairy science, 1991 nov; 74(11):3891-9 (1991). [6]tidona f., criscione a., guastella a m., bioactive peptides in dairy products, italian journal of animal science, 8, 315-340, (2009). [7] enb a., abou donia m.a., abd-rabou n.s., abou-arab a.a.k. and el-senaity m.h.: chemical composition of raw milk and heavy metals behavior during processing of milk products, global veterinaria 3 (3): 268-275, (2009). [8] shakour, a.a., n.m. el-taieb and s.k. hassan: seasonal variation of some heavy metals in total suspended particulate mater in great cairo atmosphere. the 2nd international conference of environmental science and technology, egypt, 4-6 september, (2006). [9] antunac n., mioč b., ivanković a., havranek l j.: influence of stage of laction on the chemical composition and physical propreties of sheep milk, czech journal animal science, 47 (2): 80-84, (2002). [10] martin j. auldist , brian j. walsh and norman a. thomson: seasonal and lactational influences on bovine milk composition in new zealand, journal of dairy research, 65 (3), 401-411, (2004). [11] ceballos l. s., morales e. r., gloria de la torre adarve, castro j. d., martinez l.p., remedios m.: composition of goat and cow milk produced under similar conditions and analyzed by identical methodology. journal of food composition and analysis 322–329, (2009). [12] elvan ozrenk, sebnem selcuk: the effect of seasonal variation on the composition of cow milk in van province. pakistan journal of nutrition 7(1), 161-164, (2008). [13] bansal b., habib b., rebmann h. and d. chen x.: effect of seasonal variation in milk composition on dairy fouling, proceedings of international conference on heat exchanger fouling and cleaning viii, june 14-19, schladming, austria, (2009). [14] banks j.m., tamime a.y, : seasonal trends in the efficiency of recovery of milk fat and casein in cheese manufacture, journal society dairy technology 40:64–66, (1987). 141 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 141 145 relevance of nutritional intervention of canned milk to soldiers’ daily diet *natalija valeriivna ryabokon1, oksana valerjanivna kochubey-lytvynenko1, dmitro viktorovich rindyuk1 1national university of food technologies, ukraine, kiev, ryabokonnatawa@gmail.com *corresponding author received november 22rd 2015, accepted 15th april 2016 abstract: the article presents soldiers’ needs in nutrients, minerals and vitamins. drawing on the example of one of the military units of ukraine, the discrepancy of soldiers’ needs and their daily diet is dealt with, which is based on the principles focused not on quality but quantity. according to the tests made for soldiers’ needs in terms of good nutrition it is recommended to introduce canned milk (dry, condensed), which is characterized by high nutrition value and long-time term of storage under military-field conditions. keywords: canned milk, level of provision, nutrients, vitamins, military, daily diet, needs, minerals. 1. introduction the threat of losing peace and holistic functioning of ukraine as a united and independent country, pose new and pressing issues to society, which needs urgent solution. mainly, they are concerned with providing military with highly professional military education and training, new equipment, respective living conditions in the field, nutrition. in the author’s opinion, the speed and success of the task of defending the state, in general depend on the state of health of the defines, their training, resistance of organisms to extreme psychological, emotional and physical stresses. these factors greatly depend on the provision of daily military’s needs in basic nutrients. therefore, the organization of full, high-quality and balanced diet for the military is an important task for today. the diet, which is based on scientific principles specific to military work will strengthen the health and physical development of the military, raise the body's resistance to stress, positively effect on their combat readiness. 2. materials and methods analysis of the nutritional standards of servicemen, approved by the cabinet of ministers of ukraine number 426 of 29 march 2002, showed that none of the rules (combined, air, sea) cannot fully ensure daily physiological needs of the military’s body in the nutrients and energy, vitamins and minerals [1]. on the example of really existing military unit of ukraine, completed daily ration for soldiers does not stipulate detection of the level that provides nutrients, minerals and vitamins at all. the indicator, on which are oriented officers of the military unit, when are developing the diet -is total weight of food, not its qualitative composition. table 1 shows the example of menu according to rates for combined-arms ration for one of the military units of ukraine. http://www.fia.usv.ro/fiajournal http://dspace.nuft.edu.ua:8080/jspui/browse?type=author&value=kochubey-lytvynenko%2c+o. mailto:ryabokonnatawa@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 natalija valeriivna ryabokon, oksana valerjanivna kochubey-lytvynenko, dmitro viktorovich rindyuk, relevance of nutritional intervention of canned milk to military’s daily diet, food and environment safety, volume xv, issue 2 – 2016, pag. 141 – 145 142 table 1 menu according to rates for combined-arms ration for military unit of ukraine canned meat – (1), with barley porridge – (2), bread, cheese, butter – (3), tea, sugar – (4), potato salad with cucumber – (5), borshch – (6), canned meat – (7), with combined porridge – (8), sauce (9), bread, compot – (10), potato salad with cucumber (11), potato salad with cucumber (12), fry fish, with rise and sause (13), butter, sugar, tea, baking (14) d at e an d da ys o f w ee k in ge st io n n am e name of product and weight in grams per person t ot al w ei gh o f re ad y d is h r ye o r w he at b re ad f ro m fl ou r w he at b re ad f ro m f ir st g ra de fl ou r se co nd g ra de w he at fl ou r cereals meat -fish fats su ga r t ea , c of fe e d ry p ud di ng sa lt vegetables other b uc kw he at o at m ea l b ar le y ri se m ea t fi sh , h er ri ng m ar ga ri ne bu tte r oi l po ta to fe rm en te d ca bb ag e be et ca rr ot o ni on r oo t, gr ee n, to m at oe s, cu cu m be rs t om at o pa st e b ay le af pe pp er vi ne ga r m us ta rd p ow de r fi rs t g ra de fl ou r ye as t ch ee se m o n d a y , th e “ _ _ “ o f _ _ (m o n th ) 2 0 _ _ _ (y e a r ) b r e a k f a st (1) 75 (2) 5 86 5 5 5 30 8 (3) 10 0 15 0 15 15 (4) 30 0. 6 25 0 d in n e r (5) 5 90 5 5 20 10 0 (6) 12 0 90 30 5 5 50 0 (7) 5 75 44 (8) 28 49 5 5 17 3 (9) 50 50 (10) 15 0 10 0 3 10 20 25 0 su p p e r (11) 5 50 5 5 20 68 (12) 5 19 5 12 21 2 (13) 81 5 5 5 27 7 (14) 10 0 15 0 15 30 0. 6 50 0. 5 25 0 t ot al nu m be r of pr od uc ts p er da y 35 0 40 0 15 81 28 49 86 15 0 19 5 15 30 25 70 1. 2 20 25 26 0 90 30 30 30 40 6 0. 2 0. 3 1 0. 3 50 0. 5 15 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 natalija valeriivna ryabokon, oksana valerjanivna kochubey-lytvynenko, dmitro viktorovich rindyuk, relevance of nutritional intervention of canned milk to military’s daily diet, food and environment safety, volume xv, issue 2 – 2016, pag. 141 – 145 143 one of the reasons of the discrepancy between existing norms and the needs, can be called a lack of dairy products in the diet, which have high nutritional, nutritious and biological value [2]. experts are recommended to consume milk and dairy products every day for people of all ages and different groups of physical activity. and the military are no exception. recommended 30 grams of butter and 15 grams of solid rennet cheese, that are mentioned in resolution above, are insufficient to provide the body, that has, increasing level of physical activity, with necessary and valuable proteins, fats, carbohydrates and other nutrients of animal origin. however, considering the dairy products in the military’s diet, it is necessary to pay a special attention to the following aspects: availability of the product in the militaryfield conditions, easiness of preparation to consumption, transportability and expectancy of the term of storage [3]. taking into account the needs and characteristics of food consumption during military actions, the authors recommend to introduce canned milk (dry and condensed) into military’s diet. dry canned milk are products with high content of milk solids (95 ... 98.5%), that ensure their suitability for easy transportation and long-term consumption. when the degree of thickening of milkbase before drying is 3-4, then it causes high food, energy and biological value of dry canned milk compared to whole fresh milk. thus, dry milk food have a high protein (11.1 ... 37.9%); fat (12.5 ... 44.7%); carbohydrates (30.6 ... 65%); essential amino acids (9568 ... 14 237 mg per 100 g); fatty acids (11.85 ... 40.48 grams per 100 grams) content. vitamin and mineral composition of the main types of dry canned milk is characterized by containing retinol, βcarotene, thiamine, riboflavin, ascorbic acid, sodium, potassium, calcium, magnesium, phosphorus, iron [4, 5]. canned condensed milk – are high-calorie nutritional products with a shelf life about 12 months to consumption, mass fraction of protein about 7 ... 11% fat 8.5 ... 19% lactose 9 ... 14.5%; sucrose 37 ... 44%. 100g of condensed product contains about 2,600 mg of essential amino acids; 8 ... 18 g of fatty acids. chemical constitution of condensed canned milk, consists of the same macro elements and vitamins as dry milk products [4, 5, 6]. 3. results and discussion the feasibility of the introduction canned milk into military’s diet is confirmed by a comparative analysis of the value of 100 grams of canned milk with daily needs of the human body in nutrients [1]. for calculation of daily military's energy consumption ,the quantity of basal metabolism is multiplied by a factor of physical activity (2.3) for group iv activity working hard, especially hard physical labor, high and very high physical activity (table 2-4). table 2 daily requirement of men of 4 group of physical activity in proteins, fats, carbohydrates and energy age of man (years) energy [kcal.] proteins, [g] fats, [g] carbohydrates, [g] all animal 18…29 3900 107 59 10 624 30…39 3700 102 56 10 592 40…59 3500 6 53 97 560 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 natalija valeriivna ryabokon, oksana valerjanivna kochubey-lytvynenko, dmitro viktorovich rindyuk, relevance of nutritional intervention of canned milk to military’s daily diet, food and environment safety, volume xv, issue 2 – 2016, pag. 141 – 145 144 table 3 daily requirement of men of 4 group of physical activity in minerals age of man (years) са, [mg] р, [mg] мg, [mg] fe, [mg] f, [mg] zn, [mg] i, [mg] se, [mg] 18…29 1200 1200 400 15 0.75 15 0.15 70 30…39 1200 1200 400 15 0.75 15 0.15 70 40…59 1200 1200 400 15 0.75 15 0.15 70 table 4 daily requirement of men of 4 group of physical activity in vitamins age of man е, mg d, mg a, mg b1, mg b2, mg b6, mg pp, mg folate, mg в12, mg с, mg 18…29 15 2.5 1000 1.6 2.0 2.0 22 250 3 80 30…39 15 2.5 1000 1.6 2.0 2.0 22 250 3 80 40…59 15 2.5 1000 1.6 2.0 2.0 22 250 3 80 on the example of the daily needs of men aged 30 ... 39 years authors defined the integral score of canned milk for protein fat, carbohydrates, vitamins and mineral substances, which are shown in figure 1. it was determining in the products that have the greatest demand among consumers whole condensed milk with sugar and 8.5% of fat, dry whole milk with 25% of fat. fig. 1. the integral score of canned milk on the content of nutrients and minerals, vitamins. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 natalija valeriivna ryabokon, oksana valerjanivna kochubey-lytvynenko, dmitro viktorovich rindyuk, relevance of nutritional intervention of canned milk to military’s daily diet, food and environment safety, volume xv, issue 2 – 2016, pag. 141 – 145 145 4. conclusion analysis of the results shows that canned milk are able to provide military's needs in nutrients, particularly fats, calcium, phosphorus, magnesium, vitamin b2 on the high-level. in conclusion, i must say that the procedure of drawing up the daily diet for the military of ukraine needs innovation, radically opposing to the existing concepts in which the primary purpose will be balanced and quality diet for each of the military, not the mere provision of food by weight. this will allow to improve feeling of military, their health and physical fitness in general. 5. references [1]. law of ukraine "on approval norms physiological needs of the population of ukraine in the nutrients and energy": (official. text: as of december 2, 1999) / ukraine parliament. k: parliamentary publishing house, 22 p., (1999) [2]. skuryhina i.m., the chemical composition of food products. kn.2. m.: agropromizdat, 360 p., (1987) [3]. chekulaeva l.v., golubev l.v., polyanskyy k.k., storability of new milk canned / milk industry. № 5. s. 27-28. (2000) [4]. golubeva l.v., bobkova n., modern technology trends milk curds with sugar / milk industry. № 5. s. 74-75. (2006) [5]. golubeva l.v., directory technologist milk production. technology and recipe. 9. tom, сanning and drying of milk / st. petersburg: hyord, 256 p. (2005) [6]. ryabokon n. v., the balance of condensed canned milk / nv ryabokon, t.g. osmak, t. a. savchenko // food agribusiness industry. № 4. p. 15-17. (2012) 1. introduction 4. conclusion 216 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv , issue 3 2016, pag. 216 226 ştefan cel mare university of suceava, romania volume , issue 12016, pag. ... ... the influence of lupin flour addition on bread quality *georgiana gabriela codină1, andreea roxana marineac1, elena todosi-sănduleac1 1faculty of food engineering, stefan cel mare university, 13th university street, 720229, suceava, romania codina@fia.usv.ro; codinageorgiana@yahoo.com *corresponding author received 12th july 2016, accepted 23rd september 2016 abstract: in recent years there has been an increased interest in using lupine for human nutrition due to its nutritional properties and health benefits. it was used in bakery, pasta, dairy cheese, ice cream, meat products, for different purposes, and especially for its high protein content. this paper aims to identify the optimum dose of lupine that can be used in wheat bread with a high technological quality so that the customer can benefit from a quality product from all points of view. for this purpose we studied the physical changes of bread, that occur by substituting wheat flour by lupin flour in proportions of 5%,10%, 15%, 20%. the results obtained showed that with the addition of lupin, the mass of products increased the volume and porosity decreased and the textural and color parameters changed significantly. a substitution of up to 10% lupin addition leads to bread with higher mass, lower volume, firm, lower cohesiveness and elasticity as compared to the control sample and high gumminess and chewiness values as well. by using anova, the parameter values such as physical, color, crumb cell, textural and sensory characteristics did not vary significantly and therefore the changes in bread quality were not substantial. the sensorial analysis performed showed that the most appreciated bread was the one with 5% and 10% lupin addition. the most appreciated characteristics were taste, aroma, color, odor, overall appearance and overall acceptability of bread. key words: lupin, wheat flour type, bread quality 1. introduction lupin, a legume known for over 4000 years, has aroused interest not only for its nutritional composition, but also for its high adaptability on poor soils, competing with soy and other legumes [1-2]. due to high protein content, lupin was used initially as green manure and in time it became an important element in human diet [3]. studies have shown that lupin has twice more proteins as compared with other legumes consumed by humans. protein intake ranges from 28% to 48%, depending on lupin species, growing conditions and soil type [4]. globulins (α-conglutin, βconglutin, γ-conglutin) are the major storage proteins found in proportions of 80-90% in lupin seeds and prolamins and gluteins are found in smaller quantities as compared to other legumes [5]. albumins rich in amino acids are found in proportions of 15% in lupin seeds. lupin seeds have a smaller content of globulin (80%) compared with soy seeds (90%) but a much higher albumin content (20%) unlike soy seeds (10) [6]. lupin seeds are a good source of fibers, 40% are found in nucleus mass and a good source of essential amino acids. they http://www.fia.usv.ro/fiajournal mailto:codina@fia.usv.ro mailto:codinageorgiana@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 217 contain a high amount of lysine and are generally low in sulfur amino acids (methionine, cysteine) and threonine [7]. although lupin beans are not considered oilseeds, they have a considerable amount of oil, about 5-20% raw oil. in general, lupin oil is characterized by a balanced composition of fatty acids with a total of 10% of saturated fatty acids and 90% unsaturated ones, including: (18:1) 30-50% oleic, (18:2) 17-47% linoleic, (18:3) linoleic 3-11% [8]. lupin seeds are an important source of minerals. the mineral content varies depending on the variety and can reach values between 3.2 and 4.6 g/100g. the minerals present are: potassium (66-90 mg/g), calcium (15-29 mg/g), magnesium (11-20 mg/g), sodium (3-11 mg/g) [9]. lupin and lupin derivatives can be found in a wide range of foods such as meat products, fish, bread, pasta, tofu cheese or ice cream [10-16]. in bakery, they are used both as lupin protein concentrates and lupin flour to enhance the nutritional quality of the finished product [17-19]. the use of lupin flour in large quantities can affect adversely the quality of bread. this is due to the fact that it reduces the elasticity of wheat dough and weakens the gluten matrix due to its high fiber content, obtaining a product with unpleasant texture and small volume [20]. generally the addition of legume flours is associated with decreased loaf volume, the same case in lupin addition, when the proportion of lupin increases, the volume of bread decreases [21]. this is due to the decrease of gluten content, to the decrease of viscous-elastic properties of dough and to the impossibility of dough to retain gas during baking [22-23]. studies show that the volume decreases in a proportion that depends on the lupin species used, white lupin having less influence on the volume as compared to the blue lupin. the color of loaf is also influenced depending on the amount of lupin flour added. the crumb gets yellow with higher values by the increased percentage of lupin flour added, and the crust becomes darker due to maillard reaction between sugars and amino acids. research shows that substitution of wheat flour by lupin flour in amounts exceeding 10% influences negatively the quality of dough and thus affects adversely the quality of the finished product. to obtain high quality bread, several parameters must be considered such as dough kneading, dough fermentation and baking. the control of these parameters is critical to the achievement of loaves with outstanding features, specific volume and suitable textural properties. lupin-based foods draw increasingly consumers’ attention due to their possible role in preventing cardiovascular diseases and reduce blood glucose and cholesterol levels [24-29]. in recent years it has been noticed an increase in allergy cases caused by lupin, therefore, in november 2007, the labeling of lupin as potential allergen became mandatory [30]. it has been discovered that the cause of allergy is βconglutin, a major allergen contained by l. albus and l. angustifolius, types of lupin mostly used in human food. processing methods like boiling or heat treatment combined with pressure drop can completely remove allergens. this paper aims to study the influence of lupin flour addition in doses of 5%, 10%, 15% and 20% in wheat flour of 650 and 1350 types on bread quality. volume, textural parameters, color and sensory characteristics of bread from wheat-lupin composite flour were analyzed. 2. materials and methods 2.1.materials to obtain the bread samples with addition of lupin flour, the following materials were used: sweet lupin flour, 650 wheat flour type, 1350 wheat flour type, water, yeast, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 218 salt. the 650 flour used in this study presents the following characteristics: moisture content 14.5%, ash content 0.65%, wet gluten content 28%, gluten deformation index 8 mm and titratable acidity 2.3 degree of acidity. the 1350 flour used presents the following characteristics: moisture content 14.1%, ash content1.35% wet gluten content 28%, gluten deformation index 5 mm and titratable acidity 3.0 degree of acidity. the flour chemical composition was determined according to the romanian standard methods: moisture (sr en iso 712:2010), ash content (sr en iso2171:2010), wet gluten content and gluten deformation index (sr 90:2007), acidity (sr 90:2007). 2.2. preparation of composite lupin-wheat flour bread bread samples with 5%, 10%, 15%, 20% lupin flour was obtained from each type of wheat flour and two control samples as well. the quantity of yeast used was 3% of flour mass and the salt of 1.5% of flour mass. dry ingredients were blended with water with a temperature of 29-30°c for 10-15 minutes to obtain homogeneous dough. the shaped loaves were placed in trays and left to rise for 15 minutes. after rising they were placed in pre-heated oven at 130° c and steamed for 1 minute. after steaming, the temperature was raised to 180° c. baking time was of 35 minutes. 2.3. composite lupin-wheat flour bread evaluation the bread physical properties: volume, loaf volume (loaves were determined using the rapeseed displacement method), porosity, elasticity were determined according to sr 91:2007. the crumb cells were analyzed with the moticsmz–140 stereo microscope with a 20x objective to a resolution of 2048 x 1536 pixels. textural properties of bread were determined using a texture analyzer mark10-esm301, displacement of 10 mm/min, were: hardness, cohesiveness, elasticity, gumminess, chewiness (table 1). texture analysis refers mainly to the mechanical properties of the product, mostly often food products. it is associated with the sensory properties detected by humans. intense research developed a set of definitions regarding the sensory properties of products and instrumental properties that can be calculated using two cycle tests of textural profile analysis (fig. 1). texture analyzers perform those tests by applying controlled forces on the product and record its response in the form of force, deformation and time. figure 1. load diagram in two cycles used for texture profile analysis tabe1 1. primary and secondary parameters textural parameters notation (fig.3.4.) hardness h cohesiveness (a2/a1) viscosity c elasticity t2/t1 adhesiveness a3 fracturability b gumminess h·a2/a1 chewiness h·a2/a1·d2/d1 colors of bread were analyzed by konica minolta cr–700 colorimeter. this method analyzes color by recreating human color vision [32]. system cie l* a* b* is one of the most common color measurement food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 219 systems. it describes a three dimensional color space coordinates based on brightness coordinates l* and color coordinates a*, b* (fig.2). the values of l* vary from 0 pure black to 100 pure white. axis color is divided into green (a*) and red (+a*). axis b* is divided into yellow (+b*) and blue (-b*). knowing these three values l*, a*, b* can accurately describe the color in the space of color. by increasing a* and b* values and the distance from the point of interaction staining increased. the intersection of the axes is neutral gray, l*=50, a*=0, b*=0. color analysis was made using a portable colorimeter by measuring l* (brightness), a* (red/green), b* (yellow/blue), δl (difference in brightness), δa (difference red/green), δb (difference yellow/ blue), δe (color difference). figure 2. cie l*, a*, b* system [32] evaluation of sensory characteristics of bread samples was performed in a tasting session. tasters, numbering 20, were students of 4th year of the faculty of food engineering, university stefan cel mare, suceava, romania. they were presented the products and given for evaluation every sample starting with the control sample, followed by the samples with 5%, 10%, 15%, 20% lupine flour added. participants were asked to rate a set of features such as general appearance, color, taste, odor, flavor, texture and overall acceptability. the assessment was based on hedonic test. hedonic test allows not only choosing the best product but also determining the degree of preference. hedonic scale includes nine points, the first four referring to negative perceptions and the other four to positive perceptions. statistical analysis was performed using microsoft excel 2007 where we applied anova, in order to highlight the extent to which the bread quality is influenced by the addition of lupin flour. the influence of lupin flour on volume, texture parameters, color and sensory acceptability of the bread was studied. 3. results and discussion 3.1. effect of lupin addition on bread volume studies show that the substitution of wheat flour by lupin flours reduces the volume of bread. this negative effect is associated with the obtaining of poor quality products [33].by analyzing the results obtained, it has been found that the bread volume decreased with the increase of lupin flour addition at levels higher than 10% addition in the case of wheat flour type 650 substitutions and for all levels in the case of 1350 wheat flour substitution (fig. 3 and fig. 4). the decreased values of the bread volume may be due to the reduced amount of gluten from lupin-wheat composite dough which may reduce its viscous-elastic properties and the dough capacity of gas retention during baking [34]. this effect of reduced volume of composite flour bread has been reported by other researchers, too [35-38]. although volume changes occur, they are not statistically significant. regarding p value over 5% and f = 0.3453 < fcritic = 6.3882, it can be concluded that lupin flour percentage added does not influence significantly the bread volume. also, loaf volume values do not significantly vary (f = 0.4882, p = 0.747752283 > 5%). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 220 figure 3. bread volume variation figure 4. bread loaf volume variation 3.2. the influence of lupin flour addition on bread porosity regarding porosity, the values obtained were slightly lower as compared to the control samples. porosity did not exceed 97%, the lowest porosity obtained being for the 1350 flour type with 10% lupin flour addition (figure 5, 6 and 7). by applying anova, it can be said that the percentage of lupin does not affect significantly porosity (p = 0.683, f < fcritic). according to the test, porosity is influenced by the type of wheat flour (p = 0.0342, f > fcritic). other studies have concluded that porosity decreases with the addition of legume flour, the lower porosity may be an effect of low gluten content in lupin-wheat flour composite dough [39]. figure 5. porosity values of bread samples figure 6. crumb microstructure of bread samples from 650 wheat flour type with lupin addition l1 control, l2 5% lupin, l3 10% lupin, l4 15% lupin, l5 20% lupin food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 221 figure 7. crumb microstructure of bread samples from 1350 wheat flour type with lupine addition l1a control, l2a 5% lupine, l3a 10% lupine, l4a 15% lupine, l5a 20% lupine 3.3.effect of lupine flour added on elasticity of bread elasticity decreases by increasing the percentage of lupine added (fig. 8). elasticity decreased is not significant and is may be due to the low gluten content of the analyzed bread with high levels of lupine addition. in this study, the elasticity was determined after 48 h from the bread samples obtained. similar results were obtained and by other researchers [40]. fig. 8. elasticity values of bread samples 3.4. texture profile analyses table 3 shows the texture parameter values obtained. there are correlations between the percentages of lupin and texture parameter values, these correlations highlighting connections between the addition of lupin and texture parameters. following the pearson’s correlation a number of binary values of the correlation coefficient were obtained which highlights the extent to which substitution of lupin is connected to the texture parameters (table 4 and table 5). it is noticed that in the case of 650 flour bread, the correlations between the percentage of lupin and texture parameters are not very high the correlation coefficient obtained being low (table 6). strong correlation is observed between firmness, chewiness and gumminess of 1350 four bread, the regression coefficient being higher than 0.8. regarding firmness, its increase is observed with the increasing of the lupin content. cohesiveness shows a slight decrease with the increase of lupin flour addition and gumminess and chewiness increase. the results show that a substitution of wheat flour with lupin flour over 20% does not have a high influence on the textural characteristics of the finished products. other studies have found that the substitution between 5% and 10% may lead to valuable products regarding the nutritive composition without changing their textural qualities. statistically the textural parameters were not influenced by lupin flour percentage, therefore it can be concluded that bread with lupin addition is similar to wheat bread in terms of texture. values closest to control sample were recorded for both 650 and 1350 flour with a substitution of 5% and 10%. 3.5. color analysis table 8 shows the results of color analysis. statistically, by studying color parameters it has been observed that l* varies food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 222 significantly depending on the lupin flour percentage added (p = 0.00708) and the type of wheat flour (p = 0.0001). a* parameter varies significantly, being influenced by the type of wheat flour used not by the lupin flour percentage (p = 9.9762). significant variation is observed in the case of δl and δe parameters, these parameters being influenced by the type of wheat flour and the lupin flour percentage (table 2). table 2. anova test results regarding color parameters parameters f p-value fcritical l 19.24293 0.00708 6.388233 a 0.790479 0.587364161 6.388233 b 4.733412 0.080651217 6.388233 δl 19.26507 0.007064765 6.388233 δa 1.140303 0.450906 6.388233 δb 4.640253 0.083156 6.388233 δe 15.73804 0.010282 6.388233 table 3. bread texture parameter values wheat flour 650 flour 1350 flour samples control 5% lupine 10% lupine 15% lupine 20% lupine control 5% lupine 10% lupine 15% lupine 20% lupine primary texture parameters firmness 35.62 66.04 44 44 45.1 31.14 43.6 51.28 64.44 74.86 cohesivity 0.627 0.596 0.724 0.725 0.675 0.700 0.699 0.668 0.542 0.606 elasticity 0.837 0.746 0.802 0.826 0.765 0.820 0.779 0.818 0.750 0.788 secondary texture parameters gumminess 22.328 39.352 31.865 31.892 30.436 21.784 30.476 34.254 34.929 45.399 chewiness 18.686 29.360 25.556 26.354 23.272 17.856 23.748 28.016 26.199 35.763 table 4. pearson’s correlation for bread from 650 wheat flour type lupine percentage firmness cohesivenes s viscosit y elasticit y adhesivenes s gumminess chewines s lupin percentage 1 firmness -0.04298 1 cohesiveness 0.616352 -0.48038 1 elasticity -0.26026 -0.8277 0.417912 -0.56933 1 gumminess 0.228624 0.92378 9 -0.10811 0.80000 1 -0.76198 0.816702 1 chewiness 0.2449 0.84435 6 0.04797 0.82658 3 -0.60886 0.83695 0.976461211 1 table 5. pearson’s correlation for bread from 1350 wheat flour type lupine percentage firmness cohesivenes s viscosit y elasticit y adhesivenes s gummines s chewines s lupin percentage 1 firmness 0.99769 1 cohesiveness -0.79815 -0.80569 1 elasticity -0.50682 -0.55311 0.746002 0.78053 8 1 gumminess 0.958875 0.95686 2 -0.60081 -0.11675 -0.38136 -0.10237 1 chewiness 0.927189 0.918327 -0.51468 -0.00452 -0.2542 0.010091 0.990856 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 223 table 6. the correlation coefficients and regression equations based on lupine percentage parameters 650 flour 1350 flour regression equations correlation coefficients regression equations correlation coefficients firmness y = -6.6x+47.56 0.001 y = 216.5x+31.40 0.995 cohesiveness y = 0.449x+0.624 0.379 y = -0.686x+0.711 0.637 elasticity y = 0.128+0.808 0.067 y = -0.186x+0.890 0.256 gumminess y = 17.51x+29.42 0.052 y = 103.3x+23.03 0.919 chewiness y = 12.33+23.41 0.06 y = 76.52x+18.66 0.859 table 7. anova test results texture parameters f p-value fcritic firmness 0.997903 0.500787 6.388233 cohesiveness 0.184572 0.934732 6.388233 elasticity 1.393935 0.377698 6.388233 gumminess 1.975612 0.262909 6.388233 chewiness 1.591818 0.331721 6.388233 table 8. bread color values wheat flour 650 flour 1350 flour parameters 0% lupine 5% lupine 10% lupine 15% lupine 20% lupine 0% lupine 5% lupine 10% lupine 15% lupine 20% lupine l 67.6 66.75 68.13 68.89 70.39 63.06 62.74 65.32 64.72 66.4 a -1.27 -2.32 -1.81 -2.15 -2.13 1.57 0.67 0.13 0.34 -0.01 b 14.88 23.13 19.66 24.21 26.74 17.43 18.21 19.62 21.05 23.16 δl 31.78 30.93 32.31 33.06 34.56 27.24 26.92 29.5 28.9 30.58 δa -14.63 -15.67 -15.16 -15.5 -15.48 11.79 -12.68 -13.22 -13.01 -13.36 δb 0.66 8.9 5.44 9.9 12.52 3.21 3.98 5.39 6.62 8.94 δe 34.99 35.79 36.1 37.86 39.89 29.85 30.02 32.77 32.42 34.54 3.6. sensory characteristics evaluation following statistical analysis of the responses of the participants in the tasting session, it was found that in the case of 650 flour bread, the percentage of lupin flour is statistically significant (p = 0.002), the interaction between the percentage of lupin and analyzed characteristics did not influence scores (p = 0.996). regarding the 1350 flour bread, the lupin flour percentage was statistically insignificant (p = 0.697). figure 9. sensory characteristics of bread samples from 650 wheat flour type with lupin addition figure 10. sensory characteristics of bread samples from 1350 wheat flour type with lupin addition food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 224 according to the responses the most appreciated feature of the 650 flour bread was the overall appearance of the sample with 5% lupin flour addition. generally, the most appreciated 650 flour bread was the one with 10% lupin flour addition (figure 9). by analyzing the 1350 flour bread (figure 10) respondents considered as the most appreciated bread the one with 20% lupin flour addition. regarding color, overall appearance and taste, the most appreciated was the bread with 15% lupin flour addition. the consistence of samples was underappreciated as compared with control samples for both types of wheat flour used. the observations made by participants in the tasting session were related to taste, smell and aroma. they pointed out that the samples with over 10% lupin flour addition had a slight bitter taste, more pronounced in samples with 20% lupin flour, smell and flavor of cheese in samples with over 15% lupin flour. 4. conclusions lupin, a legume with a particular chemical composition, turns out to be a product with many uses in food industry. in bakery it can be primarily used as a fortifier due to its high protein content. the use of lupin flour can bring a series of changes to bread as finished product, changes that influence its quality. volume and specific volume values of the product can decrease by increasing the percentage of lupin flour addition. the textural proprieties can also be influenced, firmness increases by increasing the amounts of lupin flour addition, cohesiveness, and elasticity slightly decrease as compared with control samples and gumminess and chewiness increase. it has been shown that lupin flour can also change the color of the product obtained bread with lupin addition, leading to a more yellow crumb and brown crust. having in view all the parameters studied, it can be concluded that products with 5% and 10% lupin flour addition are the closest to the quality of the control samples. as compared to other studies, the results obtained are similar. 5. references [1]. huyghe c., white lupin (lupinus albus l.).science direct. field crops research, 53:147160, (1998) [2]. mercedes lucas m., stoddard f. l., annicchiarico p., frías j., martínezvillaluenga c., sussman d., duranti m., seger a., zander p.m., pueyo j.j., the future of lupin as a protein crop in europe, ront. plant sci. 6:705. doi: 10.3389/fpls.2015.00705, (2015) [3]. pastor-cavada, e., juan, r., pastor, j.e., alaiz, m., vioque, j.,analytical nutritional characteristics of seed proteins in six wild lupinus species from southern spain. food chemistry, 117: 466–469, (2009) [4]. bhardwaj h.l., hamama a.a, merrick l.c., genotypic and environmental effects on lupin seed composition, plant food human nutrition, 53: 1–13, (1998) [5]. gulewicz p., martínezvillaluenga c., frias j., ciesiołka d., gulewicz k., vidal-valverde c., effect of germination on the protein fraction composition of different lupin seeds. food chemistry, 107: 830– 844, (2008) [6]. segal r., lupinul o nouă sursă de proteine în alimentația umană”, ed.academica, galaţi, (2004) [7]. clark r.l., johnson s.j., sensory acceptability of foods with added lupin (lupinus angustifolius) kernel fiber using pre-set criteria. journal of food science, 67: 356–362, (2002) [8]. hamama a.a., bhardwaj h.l., phytosterols, triterpene alcohols, and phospholipids in seed oil from white lupin. journal of the american oil chemists’ society, 81: 1039–1044, (2004) http://loop.frontiersin.org/people/262709/overview http://loop.frontiersin.org/people/36624/overview http://loop.frontiersin.org/people/255521/overview food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 225 [9]. siger a., czubinski j., kachlicki p., nogala-kalucka m., antioxidant activity and phenolic content in three lupin species, journal of food composition and analysis 25(2):190197,(2012) [10]. yorgancilar m., bilgiçli n., alternative usage of lupin (lupinus albus l.) seeds. journal of food, agriculture & environment. 8 (3&4):167-169, (2010) [11]. jayasena v., leung p., nasarabbas s.m., development and quality evaluation of lupin-fortified instant noodles, prooceedings of the 12th international lupin conference, fremantle, western australia, (2008) [12]. jaisena v., nasar-abbas s.m., development and quality evaluation of highprotein and high-dietary-fiber pasta using lupin flour, journal of texture studies issn 0022-4901, (2012) [13]. herskowitz b., segal r., application of new sweet lupin derivates as alternative protein sources in meat analogs, agroalimentary processes and technologies, volume xi, no. 2, 503-508, (2005) [14]. drakos a., doxastakis g., kiosseoglou v., functional effects of lupin proteins in comminuted meat and emulsion gels. food chemistry 100, 650-655, (2007) [15]. awad r.a., salama w.m., farahat a.m., effect of lupine as cheese base substitution on technological and nutritional properties of processed cheese analogue. acta sci. pol., technol. aliment. 13(1), 55-64, (2014) [16]. asgar m.a., fazilah a., huda n., bhat r., karim a.a., nonmeat protein alternatives as meat extenders and meat analogs, comprehensive reviews in food science and food safety,9:513–529. doi: 10.1111/j.15414337.2010.00124.x, (2010) [17]. sabanis d., tzia c., effect of rice, corn and soy flour addition on characteristics of bread produced from different wheat cultivars, food bioprocess technol, 2:68–79, (2009) [18]. noorfarahzilah m., lee j. s., sharifudin m. s., mohd fadzelly a. b., hasmadi m., applications of composite flour in development of food products, international food research journal 21(6): 2061-2074, (2014) [19]. mubarak a.e., chemical, nutritional and sensory properties of bread supplemented with lupin seed (lupinus albus) products, nahrung, 45:241245.doi:10.1002/15213803(20010801)45:4<241::a id-food241>3.0.co;2-z, (2001) [20]. curic d., novotni d., skevin d., rosell c., collar c., le bail a., colicbaric i., design of a quality index for the objective evaluation the quality of bread. application to wheat breads using selected bake off technology for bread making., food research international 41-714, (2008) [21]. levent h., bilgiçli n., evaluation of physical, chemical and sensory properties of turkish flat breads (bazlama and yufka) supplemented with lupin, buckwheat and oat flours, international journal of food science and nutrition engineeing 2012,2(5):89 95doi:10.5923/j.food.20120205.04, (2012) [22]. pollard n.j., stoddard f.l., popineauy.,wrigley c.w., macritchie f., american association of cereal chemists, 79(5):662–669, (2002) [23]. abdelrahman r.,a.,mohammed i.,senge b., oscillation measurements and creep test of bread prepared from wheat-lupine fibre dough’s blends”, annual transactions of the nordic rheology society, (2012) [24]. lee y.p., mori t.a., puddey i.b., sipsas s., ackland t.r., beilin l.j., hodgson j.m., effects of lupine kernel flourenriched bread on blood pressure: a controlled intervention study. american journal of clinical nutrition, 89: 766-772, (2009) [25]. hall r.s., thomas s.j., johnson s. k., australian sweet lupin flour addition reduces the glyccemic index of a white bread breakfast without affecting palatability in healthy human volunteers. asia pacific journal of clinical nutrition, 14 (1): 91-97, (2005) [26]. lee y.p., mori t.a., puddey i.b., sipsas s., ackland t.r., beilin l.j., hodgson j.m., effects of lupin kernel flourenriched bread on blood pressure: a controlled intervention study. american journal of clinical nutrition, 89: 766-772, (2009) [27]. chavez c., de la barca c., trends in wheat technology and modification of gluten proteins for dietary treatment of coeliac disease patients, jurnal of cerial science, 52, 337-341, (2010) [28]. archer,b.j.,johnsons.k.,devere ux h.m., baxter a.l., effect of fat replacement by inulin or lupin-kernel fibre on sausage patty acceptability, post-meal perceptions of satiety and food intake in men. british journal of nutrition, 91 (04 ): 591-599, (2004) [29]. fontanari g.g., batistuti j.p., josé da cruz r., saldiva p.h.n., gomes arêas, j.a, cholesterol-lowering effect of whole lupin (lupinus albus) seed and its protein isolate. food chemistry, 132: 1521–1526, (2005) [30]. loza a., lampart-szczapa e., allergenicity of lupin proteins-a review, department of food biochemistry and analysis, faculty of food science and nutrition, polish journal of food and nutrition sciencesvol. 58, no. 3, pp. 283-287, (2008) http://www.sciencedirect.com/science/article/pii/s0308814611017602 http://www.sciencedirect.com/science/article/pii/s0308814611017602 http://www.sciencedirect.com/science/article/pii/s0308814611017602 http://www.sciencedirect.com/science/article/pii/s0308814611017602 http://www.sciencedirect.com/science/article/pii/s0308814611017602 http://www.sciencedirect.com/science/article/pii/s0308814611017602 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv , issue 3 – 2016 georgiana gabriela codină, andreea roxana marineac, elena todosi-sănduleac, the influence of lupin flour addition on bread quality, food and environment safety, volume xv, issue 3 – 2016, pag. 216 – 226 226 [31]. bordei,d.,controlul calității în industria panificației. metode de analiză, ed.academica, galați, (2007) [32]. schröder w.,schneider-häder b., instrumental sensory testing in the food industry part3: optical sensors and electronic eyes. (2005) http://www.dlg.org/about.html [33]. doxastakis g., zafiriadis i., irakli m., marlani h., tananaki c., lupin, soya and triticale addition to wheat flour doughs and their effect on rheological properties.food chemistry,77: 219–227, (2002) [34]. pollard n.j., stoddard f.l.,popineau y.,wrigley c.w., macritchie f., lupin flours as additives: dough mixing, breadmaking, emulsifying and foaming. cereal chemistry,79: 662–669, (2002) [35]. chavan j. k., kadam s. s., nutritional enrichment of barley products by supplementation with non wheat flours. crc critical.reviews in food science and nutrition,33, 180–220, (1993) [36]. sharma h. r., chauhan g. s., physical, sensory and chemical characteristics of wheat breads supplemented with fenugreek.journal of food science and technology,37,91–94, (2000) [37]. dhingra s., jood s., organoleptic and nutritional evaluation of wheat breads supplemented with soybean and barley flour.food chemistry,77, 479–488, (2001) [38]. alpaslan m., hayta m., the effects of flaxseed, soy and corn flours on the textural and sensory properties of a baked product. journal of food quality, 29, 617–627, (2006) [39]. bojňanská t., frančáková h., líšková m., tokár m., legumes – the alternative raw materials for bread production. journal of microbiology, biotechnology and food sciences 1: 876-886, (2012) [40]. correia p. m. r., gonzaga m, batista l. m, beirão-costa m. l., guiné r.p.f., development and characterization of wheat bread with lupin flour.international journal of biological,biomolecular, agricultural, food and biotechnological engineering,9(10), 923-927, (2015). http://www.dlg.org/about.html microsoft word 11 oroian de corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 72 in flu en ce o f ch em ic al com po sition a nd tem pera tu r e on hon ey phas e a ng le mircea oroian1 1 faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 25 october 2012, accepted 27 november 2012 abstract: honey is a complex material which exhibits a viscoelastic behavior under oscillatory testing. the rheological behavior of honey is influenced by moisture content, concentration and by the temperature (all the rheological parameters are decreasing with the increasing of the temperature). this article is about the influence of chemical composition (fructose, glucose, sucrose, moisture content) and temperature on the honey phase angle. the honey is meeting the codex alimentarius requirements regarding moisture content. the sum of fructose and glucose concentrations was 60 g/100 g in agreement with ce directive 2001/110/ce. the phase angle was measured at 30, 35, 40, 45 and 50 °c; the viscous component is maxim at low temperature (in the study case at 30 -45 °c). it can be observed that at 30 °c, 35 °c, 40 °c and 45 °c, the phase angle evolution is not influenced by the frequency applied; the phase angle is changing a little with the frequency. at 50 °c, the phase angle is strongly influenced by the frequency, is decreasing its value proportionally with frequency.the suitable precidtion of the phase angle with temperature and chemical composition has been observed using the 3rd grade polynomial model with variables; the model achivied has reached a regression coeffiecient (r2) 0.9915. keywords: honey, sugar, polynomial model 1. introduction honey is a natural and nutritious food used by many people all over the world since ancient times, mainly because of its significant contribution to human health. however, the quality of honeys varies dependent on the climate and environmental conditions around the foraging area of honey bees. further processing and improper storage condition also influent the quality of honeys indirectly [1]. viscosity of honey is an important factor that concerns both processing parameters of honey production (e.g. velocity of centrifuging or filtering) and its sensory properties perceived by consumers. the majority of fluid honeys shows newtonian behaviour and their viscosity strongly depends on temperature [2-4]. the viscoelasticity is the property of materials that exhibit viscous and elastic characteristics when underdoing deformation. viscous materials, like honey, resist shear flow and strain linearly with time when a stress is applied [5]. the viscoelastical parameters of a viscoelastical materials are: elastic modulus (g’), viscous modulus (g’’), complex modulus (g*), complex viscosity (η*) and phase angle (δ). the rheological properties of honey have been investigated by some authors in the last decades [2-4, 6-8], however no other papers have been reported on the phase angle determination and the influence of different factors on it. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 73 the aim of this study is to determine the phase angle of different honey types, to study the influence of the temperature on the magnitude of it, to model the chemical and temperature influence of the phase angle. 2. materials and methods 2.1. materials for the rheological behaviour of honeys samples were purchased different 6 samples of different origins (floral, polyflorals and honeydew); the samples have been purchased from the romanian market. the rheological properties of honeys can be influenced by the presence of crystals and air bubbles [8]. before measuring the rheological properties, the samples were warmed to 55 °c to dissolve the crystals, and were kept at 30 °c to eliminate the bubble airs, which could interfere during the rheological studies. 2.2. concentration (°brix) and moisture content determination the concentration of soluble solid content (°brix) and refractive index were determined using a refractometric method. we used leica mark ii plus refractometre. the moisture content was determined using the chataway table [9]. the measurement were taken in duplicate 2.3. sugar determination the determination of glucose, fructose and sucrose in honey samples was made by a hplc 10advp -shimadzu, with ridetector, according to a method described by bogdanov [9]. the compounds were separated on a alltech type alltima amino column, 250×4.6 mm i.d. and particle size 5 µm. the samples were prepared as: 5 g of honey were dissolved in water (40 ml) and transferred quantitatively into a 100 ml volumetric flask, containing 25 ml methanol and filled up to the volume with water. the solution was filtered through a 0.45 µm membrane filter and collect in sample vials. flow rate 1.3 ml/min, mobile phase: acetonitrile/water (80:20, v/v), column and detector temperature 30 °c, sample volume 10 µl. a calibration curve was made for each sugar using standard solutions of different concentrations (0.5–80 mg/ml). the linear regression factor of the calibration curves was higher than 0.9982 for all sugars. sugars were quantified by comparison of the peak area obtained with those of standard sugars. the results for each sugar were expressed as g/100 g honey. values of parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95 %. 2.4. phase angle determination (δ) phase angle determination was made using the device designed and realised at food engineering faculty, at a frequency ranged between 0.1-10 hz, and a amplitude of 2 db. 2.5. statistical analysis the statistical analysis was made using the next software packs: excel 2007 and unscrambler x 10.1. the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. 3. results and discussions in this study have been analysed 6 honey samples, each one with a different chemical composition. the phase angle was measured at 5 temperatures 30 °c, 35 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 74 °c, 40 °c, 45 °c and 50 °c). in table 1 is presented the chemical composition of the honeys samples. the honey is meeting the codex alimentarius requirements regarding moisture content the sum of fructose and glucose concentrations was 60 g/100 g in agreement with ce directive 2001/110/ce [10]. table 1 chemical composition of honey acacia dandelion honeydew polyfloral sun flower tilia moisture (g/100g) 18.6 16.9 17.7 19.2 19.3 19.0 °brix 80.2 81.9 81.4 80.0 79.9 79.8 fructose (g/100g) 47.3 40.2 35.9 35.3 38.6 43.3 glucose (g/100g) 27.6 33.2 34.5 31.3 34.8 32.4 sucrose (g/100g) 2.0 1.2 0 2.2 1.6 1.1 fructose + glucose (g/100g) 74.9 73.4 70.4 66.6 73.4 75.7 f/g 1.7 1.2 1.0 1.1 1.1 1.3 the samples were analysed at a frequency ranging 0.1 – 10 hz. in figure 1 is presented the evolution of phase angle with temperature and frequency. the phase angle ranged between 75,16 – 89,98°. the phase angle is ranging between 0 and 90°, the region between 0 – 45° corresponds to viscoelastical materials with elastic part dominant, and the region between 45-90° corresponds to viscoelastical materials with viscous part dominant. from phase angle point of view honey is a viscoelastical material with the viscous part dominant. the viscous component is maxim at low temperature (in the study case at 30 -45 °c). it can be observed that at 30 °c, 35 °c, 40 °c and 45 °c, the phase angle evolution is not influenced by the frequency applied; the phase angle is changing a little with the frequency. at 50 °c, the phase angle is strongly influenced by the frequency, is decreasing its value proportionally with frequency. 3rd grade multivariable polynomial modelling of the influence of chemical composition and temperature on phase angle of honeys the data model regarding the prediction of phase angle (δ) of honey, measured at 1 hz, in function of its chemical composition (fructose, glucose, sucrose, sugars (the difference between brix concentration –reported as dry mater – and the sum of fructose, glucose and sucrose), non-sugars components (the difference, reported to 100 g, between 100 g and the sum of moisture content and brix concentration), moisture content and temperature was been made using a 3rd grade polynomial equation with seven variables. the measured and predicted values have been compared to see the suitability of the model. the equation of the model is as given (eq. 1): where δ the phase angle predicted, b0 is a constant that fixes the response at point of the experiments, bi – regression coefficient for the linear effect terms, bij – interaction effect terms, bii – quadratic effect terms and biii – cubic effect terms. in table 2 are presented the correspondence between actual and coded values of design variables. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 75 fig. 1. phase angle variation with temperature and frequency a. acacia, b. dandelion, c. honeydew, d. polyfloral, e. sun flower, f. tilia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 76 table 2 correspondence between actual and coded values of design variables actual values of coded levels variable symbol -1 +1 temperature (°c) x1 30 50 fructose (g/100g) x2 35,29 42,85 glucose (g/100g) x3 27,65 35,36 sucrose (g/100g) x4 0 2,22 sugars (g/100g) x5 2,95 11,21 non-sugar substances (g/100g) x6 1,64 1,76 moisture (g/100g) x7 16,24 17,96 the design parameters (x1-x7) have been modeled in order to achieve the model. the model summary is presented in tabel 5.36. the coefficient of regresion of the proposed model represents 99.91 (r2 adjustat 99.82) (table 3). table 3 model summary model standard deviation r 2 r 2 adjusted p cubic 0.19 0.9915 0.9812 0.0001 fig. 2. measured vs. predicted of phase angle of honey using polynomial equations in figure 2 are plotted the predicted values versus measured values of the phase angle. it can be seen that the pairs of values are closed to the line with the equation x = y, this fact confirms the validity of the model based on chemical composition and temperature modelling of the phase angle of honey. parameter optimization the optimization of the parameters simultaneously is very important for product quality. the desirability function approach is used to optimize the multiple characteristics concurrently. in the desirability function approach, first each characteristic, yi, is converted into an food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 77 individual desirability function, di, that varies over the range (eq. 3.) (3) if the characteristic yi i is at its target, then di = 1. if the characteristic is outside an acceptable region, then di = 0. finally, the design variables can be chosen to maximize the overall desirability (eq. 4) (4) where n is the number of characteristics. when the target (t) for the characteristic y is a maximum value and the lower limit is denoted by l, (5) when the target (t) for the characteristic y is a minimum value and the upper limit is denoted by u (mo,’05). (6) where r = 1. the phase angle value would be maxim at 42.85% fructose, 30.27% glucose, 1.13% sucrose, 7.66% sugars, 16.39% moisture, 1.69% non-sugar substances and 32.1 °c respectively. 4. conclusions according to the phase angle, honey is a viscous viscoelastical material, having a viscous part much greater than the elastic part (δ > 45°). it can be observed that at 30 °c, 35 °c, 40 °c and 45 °c, the phase angle evolution is not influenced by the frequency applied; the phase angle is changing a little with the frequency. at 50 °c, the phase angle is strongly influenced by the frequency, is decreasing its value proportionally with frequency. the phase angle value would be maxim at 42.85% fructose, 30.27% glucose, 1.13% sucrose, 7.66% sugars, 16.39% moisture, 1.69% non-sugar substances and 32.1 °c respectively 5. references [1] chua, l. s., abdul-rahaman, n.-l., sarmidi, m. r., aziz, r., 2012, multi-elemental composition and physical properties of honey samples from malaysia, food chemistry, 135, 880887 [2] witczak, m., juszczak, l., galgowska, d., 2011, non-newtonian behaviour of heather honey, journal of food engineering, 104(4), 532-537 [3] oroian, m., 2012, physicochemical and rheological properties of romanian honeys, food biophysics,7(4), 296-307 [4] yoo, b., 2004, effect of temperature on dynamic rheology of korean honeys, journal of food engineering, 65, 459-463 [5] meyers, m. a., chawla, k. k., 2008, mehcanical behavior of materials, cambridge university press, uk. [6] lazaridou, a., biliaderis, c. g., becandritsos, n., sabatini, a. g., 2004, composition, thermal and rheological behaviour of selected greek honeys. journal of food engineering, 64 (1), 9-21 [7] kang, k. m., yoo, b., 2008, dynamic rheological properties of honeys at low temperatures as affected by moisture content and temperature, food science and biotechnology, 17(1), 90-94 [8] mossel, b., bhandari, b., d´arcy, b., caffin, n., 2000, use of arrhenius model to predict theological behaviour in some australian honeys, lwt – food science and technology, 33, 545-552 [9] bogdanov, s. 2002, harmonised methods of the international honey commission (swiss bee research centre, fam, liebefeld, ch3003 bern, switzerland) [10] codex standard (codex alimentarius), 2001, 12-1981, rev. 2 revised codex standard for honey 148 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 148 155 prussian blue based screen printed biosensors with improved characteristics of long term and ph stability florentina huțanu1, maria marcu2, *gheorghe gutt1 1 faculty of food engineering, ştefan cel mare university of suceava, universitatii str. 13, 720229 suceava, romania 2romanian academy, institute of physical chemistry ilie murgulescu, 202 spl. independentei, 060021, bucharest, romania g.gutt@fia.usv.ro * corresponding author received may 8th 2013, accepted june 7th 2013 abstract in this article is reported the effect of anionic surfactants aot (dioctyl sulfosuccinate) in the electrodeposition of prussian blue (pb) onto screen-printed carbon electrodes (spce). the spce/pb/aot electrode displayed a significant improvement of its electrochemical properties and of its stability compared with pb modified spce formed in absence of surfactant. the new electrodeposed pb/aot film proved excellent electroanalytic properties for glucose determination and is a promising material for assembling of biosensors. in addition, the effects of ph value, applied potential, electroactive interference and the stability of the biosensor were discussed. the applicability to food analysis was also evaluted. key words: prussian blue, surfactant anionic, modified electrode. 1. introduction the direct amperometric detection of hydrogen peroxide at conventional electrodes is possible only at 0.6 v vs. ag/agcl. at this potential, the presence of easily oxidizable compounds present in real samples (ascorbic acid, bilirubin, uric acid, etc.) can easily interfere in the measurement, being oxidized at the electrode together with hydrogen peroxide. for this reason the detection of h2o2 at potentials around 0 using electrodes modified with electrochemical modifiers, such as prussian blue (pb), has enormous advantages and applications in many fields [1-3]. the first sensors for hydrogen peroxide based on pb modified glassy carbon electrode were reported by karyakin et al [4]. screen-printed electrodes are frequently used in analytical applications because of their unique properties such as small size, low detection limit, fast response, high reproducibility, etc. [5]. screen-printed carbon electrodes (spces) are devices that are produced by printing different inks on various types of plastic or ceramic substrates. the composition of the inks used for printing on the electrodes determines the selectivity and sensitivity required for each sensor development. screen-printed electrodes are inexpensive, simple to prepare, versatile and suitable for the mass-production of disposable electrodes [6]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 149 this work aims to evaluate the possibility of using prussian blue modified screen printed electrodes substrates for the development of biosensors assembled with glucose oxidase, for the determination of glucose, in food analysis. 2. materials and methods apparatus electrochemical measurements were carried out using a autolab potentiostat/galvanostat computer controlled by the gpes software, as well as a portable palmsens potentiostat/ galvanostat controlled via the palmsenspc software. the flow injection analysis system consisted from a four-channel minipuls 3 gilson peristaltic pump fitted with tygon tubing (1.52 mm id) used for the propulsion of fluids, an injection valve (rheodyne, 7725i model) and a flow cell special for spce from dropsens, spain. the valve loop volume was 100µl. fittings and connectors were used to connect the different components of the manifold. electrodes screen-printed carbon electrodes (spces) model drp-110 purchased from dropsens (spain) were used for electrochemical measurements. reagents all chemicals from commercial sources were of analytical grade. glucose oxidase from aspergillus niger, 232 u mg, 28 mg/ml, and d-(+)glucose (97%) were obtained from sigma.7,7 ml of gluocose oxidase were dissolved in 50 mm potassium phosphate 100 mm sodium acetate, 250 mm kcl, ph = 5,5. stock solutions of glucose were prepared in water distilited and stored at 4° when not use. the stock solution of glucose was allowed to mutarotate at room temperature overnight before use. iron chloride (fecl3), potassium ferricyanide k3[fe(cn)6], hcl 37%, sodium chloride, hydrogen peroxide (30%), were purchased from sigma-aldrich. aot (dioctyl sulfo-succinate sodium salt) was from carlo erba. the 2,6-dihydroxynapthalene and 4-(2aminoethyl) aniline were from aldrich. double-distilled water was used throughout 2,6-dihydroxynapthalene and 4-(2aminoethyl)aniline were dissolved in 0.1m phosphate buffer ph 7.4. 2.1 preparation of the modified screenprinted electrodes with prussian blue chemical deposition of prussian blue prior to prussian blue modification, screenprinted electrodes (spe-c) were pretreated in the presence of 50 mm phosphate buffer in 0.1 m kcl , ph 7.4, by applying the potential of + 1.7 v versus ag/agcl for 3 minute [7]. for the chemical deposition of pb films (procedure i), two solutions were prepared. solution 1: 100 mm k3[fe(cn)6 in 10 mm hcl. solution 2: 100 mm fecl3 in 10 mm hcl. prussian blue modification of spce was then accomplished by placing 5 µl of precursor solution 1 and 5 µl of precursor solution 2 onto the working electrode area. the drop was carefully placed exclusively on the working electrode area, in order to avoid the formation of pb on the reference and counter electrodes which may increase the internal resistance of the system. the solution was left onto the electrode for 10 min and then rinsed with a few milliliters of 10 mm hcl. the electrodes were then left 90 min in the oven at 100° c to obtain a more stable and active layer of pb [7]. the pb modified electrodes were stored dry at room temperature in the darkness. an activation was performed before of the first use by applying a potential of 0.0 v during 3 min in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 150 50 mm phosphate buffer in 0.1 m kcl , ph 7.4. 2.2. electrochemical deposition of prussian blue prior to prussian blue modification, screenprinted electrodes were pretreated as in previous section. the galvanostatic and cyclic voltammetry techniques were tested for the electrochemical deposition of pb.the galvanostatic deposition (procedure ii) was made in a mixture (solution 3) of 2.5 mm k3[fe(cn)6,], 2.5 mm fecl3 and 1 mm aot prepared in 100 mm kcl and 100 mm hcl solution by applying the potential of 0.4v for 40 sec [8]. after a gentle rinsing with water, the sensor was placed in a solution of 100 mm kcl in 100 mm hcl and a number of 20 cycles, between 0.2 and 0.4 v, at a scan rate of 50 mv/s was run.the films were stabilized by keeping the electrodes at 100° for 90 min. the presence of the pb film was confirmed by performing cyclic voltammetry in 50 mm phosphate buffer, ph 7.4. the cyclic voltammetry deposition (procedure iii) was carried out in the solution 3 (20 cycles, between 0.2 and +0.4 v, at a scan rate of 50 mv/s) [9]. 3. results and discussions electrodeposition of non-conducting films on scpe/pb the spce/pb biosensors were covered with a non-conducting copolymer electrodeposited using the cyclic voltammetry technique. the copolymer was synthesized from a solution of 0.9 mm 2,6-dhn and 10 mm apea by cycling for 10 – 20 times the potential from + 0.2 v to +1.1 v with a scan rate of 5 – 10 mv/sec. 0,2 0,4 0,6 0,8 1,0 1,2 0,0 2,0x10-5 4,0x10-5 6,0x10-5 8,0x10-5 1,0x10-4 1,2x10-4 1,4x10-4 1,6x10-4 1,8x10-4 2,0x10-4 i(a ) e/v figure 1. influence of the deposition method of pb on the cyclic voltammograms recorded for the spce / pb poly(dhn – apea). the influence of the thermal stabilization by keeping the electrodes at 100° for 90 min was also studied. no evident differences between the treated and nontreated pb electrodes were observed regarding the response of the electrodes in kcl, phosphate buffer or for glucose, but the operational stability was greater improved for the electrodes stabilized via the thermal treatment. the effect of potential scan rate on the oxidation (iox) and reduction peak (ired) currents was studied for the redox couple present around 0.10 v. plotting the iox and ired vs. square root of scan rate showed a linear relationship (figure 2), the result indicating a diffusion limited process. this behaviour was observed for all the three tested methods for pb deposition. recently, we reported a new procedure for pb modification on graphite particles based on in situ chemical synthesis of pb, obtaining pbmodified electrodes with high stability even at basic ph values [10,11]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 151 0 5 10 15 20 25 -600 -400 -200 0 200 400 i( a ) v1/2 i ox =-41.1321+18.2348x r=0.9991 ired=32.3735-22.9104x r=0.9998 figure 2. variation of iox and ired vs. square root of scan rate in electrolyte solution (0.1m kcl, 0.1m hcl) the ph value of the electrolyte solution is an important parameter for h2o2 determination using the pb modified spces. the stability and sensitivity of the pb sensor may be affected by the hydroxide ions which can break the fe-(cn)-fe bonds, but also by the protons which may block the electrochemical reactivity of pb [8]. -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 -2,0x10 -5 -1,0x10 -5 0,0 1,0x10 -5 2,0x10 -5 i( na ) e /v spc e/pb /d hn /a pea/ao t pho sph ate b uffer p h 6.5 figure 3. influence of electrolyte solution ph on the response of pb modified spce to phosphate buffer ph 6.5 the ph influence on the electrochemical determination of glucose using the spce / pb was studied at ph ranging 6 7.4. (figure 3). for all the tested electrodes the highest reduction peaks were obtained for the ph 6.5. for all the following studies, as optimum electrolyte solution, was used the 50 mm phosphate buffer, ph 6.5. the selection of the optimum working potential to be applied when measuring h2o2 was studied for the next electrochemical techniques: chronoamperometry, amperometry in stirred solution and amperometry in flow injection system (fia). potentials ranging from -100 mv to +200 mv were applied in presence of a selected concentration of hydrogen peroxide (100µm). the highest signal was obtained for the potentials of -50 mv when working in chronoamperometry and of -100 mv in amperometry (for both, stirred solutions and fia). the lowest background noise and the highest signal for h2o2 measurement was recorded for the pb sensor prepared via the galvanostatic procedure. 0 100 200 300 400 500 0 500 1000 1500 2000 2500 i(n a ) h 2 0 2 m spce/pb/dhn/apea i(na)=18.58+4.51x r=0.9985 figure 4. influence of electrolyte solution ph on the response of spce / pb to 500 µm h2o2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 152 regarding the hydrogen peroxide determination, the amperometry under stirring technique gave the best results for the spce / pb sensor prepared via galvanostatic method in rapport with the background noise and with the response time. cronoamperometry technique is very advantageous due to the fact that the screen printed electrodes allow working with a very low volume of sample (100 µl), the determination is fast and reproducible, and does not require a time for working electrode polarization. according to the measurement results, the linear range was from 1 µm to 500 µm h2o2 with the linear correlation of 0.9967 for the sensor obtained by chemical deposition [12] of pb and, respectively, of 0.9985 for the sensor prepared by galvanostatic deposition. the sensitivity of the galvanostatic prepared sensors was with 50 % higher than obtained via the chemical deposition (figure 5). for both type of biosensors the detection limit was 2.5 mm 4mm concentration glucose. 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400  i ( na ) glucoza (mm) amperometry stirring spce/pb/god/dhn/apea spce/pb/dhn/apea spce/pb/dhn/apea/aot 1 mm figure 5. calibration plots for amperometric determination of (e = -50 mv/s) characterization of spce/ pb/ dhn/apea/aot 1mm biosensors the pb exhibits excellent catalytic activity for the electroreduction of glucose, but the operational stability of pb is still a matter of concern in real samples with complex matrix, such as the food samples. in order to protect the pb layer, the coverage with nonconducting films as the poly(o-aminophenol) [11] or with ionomers as nafion [9-10] was reported. in this work we report the use, for the first time, of the non-conducting monomers electrosynthesized from a mixture of 2,6dhn and apea for protection of the pb layer. the electropolymerization was performed via the cyclic voltammetry technique by cycling the potential from + 0.2 v to +1.1 v. in figure 6 one can observe that the irreversible oxidation peak at the +0.65 v present in the first three cycles disappears in the following cycles. the oxidation peak current decrease proves the formation of a non-conducting film on the surface of the spce/pb biosensor. 0,2 0,4 0,6 0,8 1,0 1,2 -2,0x10-5 0,0 2,0x10-5 4,0x10-5 6,0x10-5 8,0x10-5 1,0x10-4 1,2x10-4 1,4x10-4 i ( a ) e/v cycle 1 cycle 2 cycle 3 cycle 5 cycle 10 figure 6. cyclic voltammograms recorded during the formation of the monomer dhn – apea on the spce / pb prepared via the galvanostatic method 10 mm apea; 0.9 mm 2,6-dhn; 10 mv/s; 10 cycles food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 153 the optimization of the copolymer formation related to the hydrogen peroxide determination was performed studying the influence of the scan rate and number of cycles. in figure 7, one can observe the influence of the number of cycles used for the glucose oxidase electrodeposition on the calibration graphs obtained by chronoamperometry. 0,0 0,5 1,0 1,5 2,0 0 200 400 600 800 1000  i ( na ) glucose [mm] god/dhn/pb/scpe figure 7. influence of the number of cycles used for electrodeposition chemical on chronoamperometric calibration graphs (e = 0.1 v) in figure 7 the influence of the scan rate used during the polimer electrodeposition process on the glucose determination in chronoamperometry is presented. the scan rate has a major influence on the film porosity. a low scan rate, as 5 mv/s, lead to a polimer film less porous, which acts as a barrier especially for higher concentration of glucose 4 mm. 0 50 100 150 200 0 500 1000 1500 2000 i(n a ) h 2 o 2 m spce/pb/ without aot spce/pb/aot 1mm spce/pb/aot 2mm spce/pb/aot 5mm figure 8. influence of the scan rate used for polimer electrodeposition on chronoamperometric calibration graphs (e = -0.1 v) the influence of several aot concentration (1 mm, 2 mm and 5 mm) on the analytical performances of the pb sensor for h2o2 sensing were evaluated. the stability of the spce/pb/aot 1 mm sensor under storage condition was assessed for a period of 90 days using a solution of 50 m h2o2 by making a determination in triplicate at least twice on week. 0 1 2 3 4 0 200 400 600 800 1000 1200 i(n a ) glucoza mm spce/pb/dhn/apea/god/glut1,5% i(na)=16,62+273,02 c glucoza r=0,9998 figure 9. influence of the scan rate used for monomer spce/pb/dhn/apea/god/aot 1 mm electrodeposition on chronoamperometric calibration graphs , 50mv/s, e = 0.1 v food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 154 the optimum conditions for polimer electrodeposition were: scan rate = 10 mv/s; number of cycles = 10; potential range = 0.2 v 1.1 v. another important characteristic of the spce / pb / monomers biosensors is represented by a higher operational stability even in flow injection analysis conditions. in figures 10 is presented the fia amperogram recorded for the pb biosensor covered with the poly(dhn – apea) monomers. 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 0 200 400 600 800 1000 1200 i(n a ) glucoza (mm) amperometry in sistem fia (flow injection analysis) god/dhn/apea/pb/scpe god/dhn/apea/pb/scpe/aot 1 mm figure 10. fia amperogram recorded for the spce/pb/dhn/apea/aot 1mm biosensor (vinj=100 µl; flow rate=0.36ml/min; glucose concentration injected in duplicate = 2; 5; 10; 20; 50; 100; 200 µm) also, in fia conditions the sensitivity for glucose detection was much higher comparing with those obtained in cronoamperometric conditions. the possible interference of compounds present in beverages, such as ascorbic acid and glucose, was tested. ascorbic acid may interfere, giving a false negative signal, only if the ascorbic acid concentration is much higher than of h2o2. the spce/pb/dhn/apea/god/aot 1 mm biosensor maintained for a long period its response for glucose (94% response was retained after 60 days). real sample analysis sometimes, for the aseptic packaging of natural fruit juices, gluose is used as a chemical agent for sterilization. however, the glucose residues in higher concentration are irritative for the skin and may affect the human health. the developed spce/pb/dhn/apea/aot/god biosensor was applied for the fast and simple determination of glucose in several commercial fruit juices. the sample treatment consisted only in dilution with 50 mm phospahate buffer, ph=6.5. in figure 11. is presented the glucose concentration determined in the tested samples with the spce / pb / dhn / apea / god / aot biosensors by chronoamperometry. 0 20 40 60 80 100 0 100 200 300 400 500 i(n a ) glucose diluated apple juice 1/100) i=45.26+4.06 h 2 o 2 (m) r=0.9997 figure 11. the glucose concentration determined in the tested samples the results demonstrated that the level of gluose concentration used in tested juices preservation is very low. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 florentina hutanu, maria marcu, gheorghe gutt, prussian blue based screen printed biosensors with improved characteristics of long term and ph stability, food and environment safety, volume xii, issue 2 – 2013, pag. 148 155 155 4. conclusions in this work, it was developed a robust and cost-effective biosensor based on spce modified with pb and an electropolymerized non-conducting film for glucose determination. the experimental results showed that the polimer film remarkable improves the operational stability of the pb biosensor. the spce/pb/ polimer biosensor has an excellent electrocatalytic activity for the reduction of glucose. furthermore, introduced into a fia system, the sensor proved a great operational stability. the developed biosensor was successfully applied to the determination of glucose in commercial juices. 5. acknowledgements the authors acknowledge to the project posdru/cpp107/dmi1.5/s/78534 for financial support 6. references [1] z. li, j. chen, w. li, k. chen, l. nie, s. yao, j. electroanal. chem. 603 (2007) p. 59-66 [2] b. haghighi, h. hamidi, l. gorton, sens. actuators. b. 147 (2010) p. 270-276 [3] j. zhang, j. li, f.yang, b, zhang, x. yang, sens. actuators. b 147 (2010) p. [4] v.d. neff, j. electrochem. soc. 125 (1978) p. 886-887 [5] f. ricci, g. paleschi, j. biosens. bioelectron. sensors and biosensors preparation, optimisation and applications of prussian blue modified electrodes . in: biosens. bioelectron. (2005) vol 21, p. 389-407. [6] p. salazar, m. martin, r. roche, r.d. o’ neil, j.l gonzalez-mora, j. electrochima. acta (2010) p. 6476-648 [7] p. salzar, m. martin, r.d. o’neil, r. roche, j.l.gonzáles-mora, surfactantpromoted prussian blue-modified carbon electrodes: enhancement of electro-deposition step, stabilization, electrochemical properties and application to lactate microbiosensors for the neurosciences (2012) vol. 92, (2012) 180-189. [8] ping, j., wu, j., fan, k., yimg, y. an amperometric sensor based on prussian blue and poly(o-phenylenediamine) modified glassy carbon electrode for the determination of hydrogen peroxide in beverages. in: food chemistry (2011) vol. 126, p. 2005–2009 [9] ricci, f., amine, a., palleschi, g., moscone d. prussian blue based screen printed biosensors with improved characteristics of long-term lifetime and ph stability. in: biosens. bioelectron. 2003 vol. 18, p. 165–174. [10] de mattos, a.a., sensors for hydrogen peroxide based on prussian blue modified electrode: improvement of the opertaional stability. in: anal. sci. (2000) vol. 16, p. 795-798. [11] r. garjonyte, a. malinauskas, sens. actuators, b 46 (1998) p. 236. [12] suprun, e., evtugyn, g., budnikov, h., ricce, f., moscone, d., palleschi, g., anal bioanal chem 383, 2005, p. 597 244 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 32014, pag. 244 251 effect of soaking on the cooking quality and colour parameters of common beans (phaseolus vulgaris l.) *ana leahu1, alice iuliana rosu1 1faculty of food engineering, stefan cel mare university of suceava, universitatii str. 13, suceava, romania, analeahu@usv.ro * corresponding author received 28th august 2014, accepted 19th september 2014 abstract: the purpose of this paper was to determine the colour changes under the influence of beans soaking in sodium bicarbonate solution, followed by drying, to obtain dry beans with a reduced cooking time. two common beans varieties were stored in a cabinet at room temperature and their organoleptic properties were analyzed. the colour changing from the different treatments soaking beans samples was measured by lightness values (l*), chroma index parameter (c*) and hue angle or coloration (h*) values. the result indicated a change in hunter parameters, l* and c*. soaking in sodium bicarbonate solution produced significant reductions in cooking time for dry beans of the two varieties. the cooking time decreased from 120 min to 75 when using 5% sodium bicarbonate soaking solution for white beans, and 60 to 30 min for the red beans, respectively. keywords: dry beans, soaking effects, colour, cooking time. 1. introduction beans (phaseolus vulgaris l.) are a leguminous plant used since antiquity in human nutrition. epidemiological studies show that the biochemical composition of beans reduces and prevent the risk of several chronic and degenerative diseases such as cancer, cardiovascular diseases, diabetes, overweight and obesity. these legumes are rich in complex carbohydrates, protein, vitamins, minerals and poor in fats. according to kaloustian, j., et al., 2008, dry beans contain several phytosterols, phytosterols are known to have hypocholesterolemic properties. phytosterol potency in decreasing serum atherogenic low-density lipoprotein (ldl) cholesterol levels and thus, in protecting against cardiovascular diseases, mainly coronary heart disease, has led to the development of functional foods enriched with plant sterols [1]. carbohydrates constitute the main fraction of beans (55%–65% dry weight on average) with polysaccharides as the major constituents, and small but significant amounts of oligosaccharides (31%–76% of total sugars) [2]. dry beans are digested slowly and have low glycemic index values. legumes have a higher content in minerals than cereals. most legumes, including common beans, are consumed as a whole and as a result their mineral content is preserved [3]. beans are an important source of iron, phosphorus, magnesium, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 245 manganese, and in lesser degree, zinc, copper and calcium. beans are an excellent source of vitamins: vitamins a, c, e, k and pp, b vitamins and folic acid. according to kabagambe, e. k., et al., 2005, the importance of beans is shown by their contribution to protein, fibre and micronutrients in food; in the costa rican diet, beans contributed 11% of protein, 25% of fibre, 17% of folate, 5% of vitamin b-6, 5% of magnesium, 14% of copper, and 13% of iron [4]. the amount of folic acid can be lost from dried beans and other vegetables during the cooking and soaking process. quicksoaking beans (boiling beans for a short time and then soaking for one hour) may lead to more folate losses than a more traditional long soak [5]. julie gardenrobinson and krystle mcneal, 2013 suggest using the slow-soak method and a cooking method that prepares the beans in 150 minutes to minimize loss of natural folate content in beans. kutoš, t., et al., (2003), determine and compare soluble dietary fibre (sdf), insoluble dietary fibre (idf) and total dietary fibre (tdf) in raw, soaked and cooked beans, before and after the correction for the resistant starch—rs portion. the results confirm that processing of beans significantly affected (p≤ 0.001) all the parameters analyzed, except for water content (p≤ 0.01) [6]. the potential benefits of common beans on health are attributed to the presence of secondary metabolites such as phenolic compounds that have antioxidant properties [7]. the beans with the highest polyphenolic content are the dark, highly pigmented varieties, such as red or brown beans rather than white beans [8]. broughton, w. j., et al., 2003, reported the phytic acid (myo-inositol hexaphosphate) and its salts (phytates) represent between 54 and 82% of the phosphorous content of the bean i.e. between 0.5 and 1.6% of the seed weight [3]. midorikawa k, et al., 2001, have studied the applications of phytic acid in food, medicine and metal anti-oxidation. according to midorikawa k, et al., 2001 phytate has also been implicated in the reduction of cholesterol and other lipids due to its presence in high fibre diets [9]. beans as legumes contain some antinutritional components that may have adverse effects on human nutrition, these are mainly phenols, tannins, phytic acids and enzyme inhibitors. these components might reduce the nutritional quality of dry beans even if they are present in low concentrations. low utilization of dry beans in food has been attributed to inconvenience due to prolonged cooking and general gastrointestinal distress experienced after eating bean products. fernandes, a., c. et ales 2010, studied a systematic review of the influence of maceration (of different soaking solutions other than pure water – sodium chloride (nacl), sodium bicarbonate (nahco3) and mixed (nacl + nahco3)) on the nutritional quality of common beans (phaseolus vulgaris l.) cooked with or without the soaking water, and recommend the soaking water to be discarded as eliminating the anti-nutritional factors. this procedure appears to reduce some of the carbohydrate fraction of the beans and may reduce or maintain the contents of fibres. this method also reduced phytates, phytic acid, total phenolics and tannins. however, in the study by nergiz, cevdet, and erkan gökgöz, 2007, soaked-cooking and pressure-cooking reduced the phenols on an average of up to 77.37 and 71.17%, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 246 respectively. this reduction in the total phenols may be attributed to their solubilisation in water and heat degradation. soaking before cooking and discarding the soaking water also seems to be an effective way to reduce the amount of oligosaccharides which produce flatulence. audu, s. s. et al., 2013, studied the effect of different processing methods on nutritional composition of a lesser known crop (black turtle bean). audu, s. s. et al., 2013 reported that the processing methods showed deviations in nutrients from the raw seeds. raw protein was found to be enhanced by cooking (13.5%), roasting (4.64%) and sprouting (14.35%) methods while all the processing methods were found to reduce the contents of raw fat, fatty acids and metabolizable energy. processing (p<0.05) affected significantly the content of some minerals in phaseolus vulgaris seeds [12]. the aim of the present research is to assess the effect of cooking on surface colour of beans and the effect of soaking on the cooking quality for two varieties of beans. 2. materials and methods 2.1. plant material dry white and red beans samples, approximately 1 kg each donated by a commercial producer were grown and harvested in suceava plateau, an area of romania, during the summer of 2013, and stored at room temperature. organoleptic properties of the grains were analyzed according to the standard stas 6253-80, the beans were distinguished by colour, smell and texture and they met the standard requirements. the samples were first cleaned from broken seeds, dust and other foreign materials. each sample was divided into two portions. the first portion was used for analysis as raw. the second portion was soaked in different soaking solutions (1:5 w/v) at ambient temperature for 3 h. the soaking solutions contained 1%, 2%, 3%, 4% and 5% sodium bicarbonate in 100 ml distilled water (all percentage wt/vol). the soaked beans were then cooked (in distilled water bean: water 1/5) and dehydrated in conventional tray dryers to a final moisture content of 10-11%. 2.2. chemical analyses the determination of moisture in samples was performed according to the european standard en iso 665/2000 by using the drying process in a drying chamber at the temperature of 103 °c. total kjeldhal nitrogen was determined from which protein contents were calculated by multiplying using the factor 6.25. total ash composition was obtained by calcinations of 5g of sample at 600 °c for 240 min. content of lipids was determined in a soxhlet apparatus, extracting the lipids from 10 g of sample with petroleum ether, followed by extract evaporation to dryness and gravimetric determination according to the european standard en iso 659/1998. the water activity of beans was evaluated with the device aqua lab. total carbohydrate content of the sample was determined as total carbohydrate by difference, that is by subtracting the measured protein, fat, ash and moisture from 100 [13]. colour measurements of surface beans have been performed by using a minolta chroma meter (model cr 310, minolta camera co. ltd., japan), based on the by hunter l* (whiteness/darkness), a* food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 247 (redness/greenness), and b* (yellowness/blueness) system. the values of the hue angle or coloration (h*) of the samples were determined by the expression (1): (1) in which h* = angle of coloration or chromatic tonality colour; b*= component of the colour red-green; a*= component of the colour yellow-blue [14]; the chroma index parameter (c*), which indicates the chromaticity or colour intensity of the sample was also determined from the results of a* and b* attributes, using the following expression [14]: (2) in which c* = chroma index; b* = component of the colour red-green; a*= component of the colour yellow-blue; 3. results and discussion the moisture content of the samples was 8.00-8.10 %. these values are close to the reported values by several researchers [15,11]. the water content changed significantly due to soaking and cooking. protein contents ranged from 21.62 and 23.01 %. maribel ovando-martínez et al., 2011, reported similar protein content for the same varieties of raw beans (higher in the pinto durango beans grown under rain fed conditions 27.32 ± 0.08, and 23.14 ± 0.05 for black 8025 beans). maribel ovando-martínez et al., 2011, reported that the protein content of beans decreased after thermal processing [16]. the proportion of protein that is associated with insoluble dietary fiber may also explain the decrease of protein content in the cooked beans. content of carbohydrates of dry beans can be seen in fig 1, water activity of dry beans is illustrated in fig 2 and the chemical composition of raw and cooked beans (moisture%, protein %, lipids % and ash %) are presented in fig. 3. fig. 1. content in carbohydrates of dry beans fig. 2. water activity of dry beans food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 248 *values are in dry weigh basis fig. 3 chemical composition of dry beans myrene r. d’souza 2013, reported similar ash contents for the same varieties of raw beans cultivated in india (3.00±0.02) and the ash content was significantly decreased in all treated (soaking, cooking, roasting, autoclaving and germination) samples with significant decrease found in cooked beans. beans also have low water content, with water activity (aw) ranged from: for white beans 0.524 and up to 0.530 for red beans. paredes-lópez,1991 reported all hardening procedures (soaking in acetate buffer, ph = 4.1 at 370c for 5h, 370c 100 % relative humidity for 28 days and 31-330c 76 % relative humidity for 120 days) that enhanced water activities [15]. the effect of soaking solutions on cooking quality. soaking in sodium bicarbonate solution of different concentrations, followed by drying resulted in a significant reduction of the time for the preparation of both varieties of beans (fig. 4). fig. 4. influence of soaking solution on the cooking quality of two common beans varieties food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 249 the effect of soaking solutions on cooking time of the unprocessed beans the cooking time of the unprocessed beans was 120 minutes and 60 minutes for white and red beans, respectively (figure 1). the greatest reduction in the cooking time is in the white beans samples (37.50 %), followed by red beans (18.00%). v. schoeninger, 2013 investigated the effect of processing beans by soaking, and the bleaching salt solutions to obtain a reduced cooking time for dried beans. according to v. schoeninger, 2013, the increase in concentration of nahco3 from 0 to 4.5% reduced with 9.06 min the cooking time of the aged bean grains. the effect of sodium bicarbonate in the reduction of the cooking time of the beans has been reported in other researches. paredes-lópez, o.,1991 reported that the hardened beans by chemical and storage procedures may be remarkably softened by soaking in nacl+ nahco3 or just nahco3. the effect of soaking solutions on colour parameter of surface beans table 1 shows the parameter changes in colour according to the different proportions in sodium bicarbonate in which the studied beans were soaked. the colour parameter studied was the lightness, l*, which is a colorimetric parameter extensively used to characterize the variation of colours in foods during processing [17]. table 1 colour parameters of surface beans in control and pre-processing beans under soaking condition in sodium bicarbonate solutions followed by drying samples nahco3 (%) l* h* c* white beans raw beans (control) 122.36 -62.13 132.45 s1 1 122.06 -61.86 62.91 s2 2 117.53 -57.00 111.50 s3 3 118.03 -57.83 97.51 s4 4 121.86 -56.30 122.28 s5 5 111.10 -58.78 82.78 red beans raw beans (control) 114.80 -63.20 103.49 s1 1 110.13 -58.15 84.18 s2 2 103.80 -60.11 74.93 s3 3 105.83 -57.66 97.33 s4 4 108.60 -57.99 81.39 s5 5 103.16 -46.12 82.89 l*=luminosity; h*= colouring angle; c*= croma index; the two varieties had a reduction in the l* value when compared to the pre-processed beans (table 2). soaked white beans presented a variation between 122.06 and 111.10 whereas the control white beans presented a value of 122.36 for lightness, l*, parameter. the l* values for red beans were similar and ranged from 114.80 in control sample to 103.16 for 55 sodium bicarbonate solution soaking. there have been observed decreases also in the values of the chroma index (c*) and coloring angle (h*) of the soaked beans compared to the raw sample. however, both chroma and hue angle values of soaking beans had a small variation range, which showed that the colour differences were smaller in red food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 250 beans than those observed in white beans. the chroma parameter is an indicator of the colour intensity perceived by the human vision, the higher the value from parameter the greater the chromatic tonality of the samples. the h* parameter is understood as tonality and defines the basic coloring of the sample [14]. these results are consistent with the ones of other investigators, siddiq, m., et al., 2010 reported a beans hunter color ‘‘l’’ values differed significantly (p<0.05) across bean types, with black beans having the lowest (11.49) and red kidney beans with 21.20. there were no significant changes in the protein and lipid contents after processing the beans, indicating that the preprocessing in sodium bicarbonate solutions followed by drying may be an alternative to reduce the cooking time of the dry beans. 4. conclusions based on the results obtained, it can be concluded that the factors concentration of nahco3 presented a strong influence on the following parameters: cooking time, luminosity and colour difference of beans in relation to the control sample. soaking in sodium bicarbonate solution resulted in a 37.5 % reduction in cooking time for white beans. the protein, fat, or ash content of raw beans cooked has shown to have minimal or no effect on most of the soaking samples studied. 5. references [1]. kaloustian, j., alhanout, k., amiotcarlin, m. j., lairon, d., portugal, h., & nicolay, a. effect of water cooking on free phytosterol levels in beans and vegetables. food chemistry, 107(4), 1379-1386 (2008). [2]. campos-vega, r., oomah, b. d., loarca-piña, g., & vergaracastañeda, h. a. common beans and their non-digestible fraction: cancer inhibitory activity— an overview. foods, 2(3), 374-392(2013). [3]. broughton, w. j., hernandez, g., blair, m., beebe, s., gepts, p., & vanderleyden, j. beans (phaseolus spp.)– model food legumes. plant and soil, 252(1), 55-128 (2003). [4]. kabagambe, e. k., baylin, a., ruiznarvarez, e., siles, x., & campos, h. decreased consumption of dried mature beans is positively associated with urbanization and nonfatal acute myocardial infarction. the journal of nutrition, 135(7), 1770-1775 (2005). [5]. garden-robinson j., mcneal k., all about beans, ndsu food and nutrition, pp 116(2013). http://www.ag.ndsu.edu/pubs/yf/foods/fn1643.pdf. [6]. kutoš, t., golob, t., kač, m., & plestenjak, a. dietary fibre content of dry and processed beans. food chemistry, 80(2), 231235(2003). [7]. campos-vega, r., loarca-piña, g., & oomah, b. d. minor components of pulses and their potential impact on human health. food research international, 43(2), 461-482 (2010). [8]. madhujith t, naczk m, shahidi f, antioxidant activity of common beans (phaseolus vulgaris l), j. food lipids, 11,220 (2004). [9]. midorikawa k, murata m, oikawa s, hiraku y and kawanishi s., protective effect of phytic acid on oxidative dna damage with reference to cancer chem oprevention. biochem. biophys. res. commun. 288, 552– 557(2001). [10]. fernandes a. c., waleska n., and da costa proença r., influence of soaking on the nutritional quality of common beans (phaseolus vulgaris l.)cooked with or without the soaking water: a review."international journal of food science & technology 45.11: 22092218(2010). [11]. nergiz, cevdet, and erkan gökgöz, effects of traditional cooking methods on some antinutrients and in vitro protein digestibility of dry bean varieties (phaseolus vulgaris l.) grown in turkey. international journal of food science & technology 42.7 (2007): 868-873 (2007). [12]. audu, s. s., aremu, m. o., & lajide, l. influence of traditional processing methods on the nutritional composition of lack turtle bean food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 ana leahu, alice iuliana rosu, effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.), food and environment safety, volume xiii, issue 3 – 2014, pag. 244 – 251 251 (phaseolus vulgaris l.) grown in nigeria. international food research journal, 20(6), 32113220 (2013). [13]. myrene r. d’souza. effect of traditional processing methods on nutritional quality of field bean, advances in bioresearch, vol. 4 (3), pp. 29-33 (2013). [14]. schoeninger v., machado coelho s. r., divair christ, sampaio s. c. and bispo de almeida aj., pre-processing of aged carioca beans: soaking effect in sodium salts in the cooking and nutrition quality, journal of food, agriculture & environment, vol.11 (1), pp. 184 189 (2013). [15]. paredes-lópez, o., cárabez-trejo, a., palma-tirado, l., & reyes-moreno, c., influence of hardening procedure and soaking solution on cooking quality of common beans. plant foods for human nutrition, 41(2), 155-164 (1991). [16]. ovando-martínez, m., osorio-díaz, p., whitney, k., bello-pérez, l. a., & simsek, s., effect of the cooking on physicochemical and starch digestibility properties of two varieties of common bean (< i> phaseolus vulgaris</i> l.) grown under different water regimes. food chemistry, 129(2), 358-365(2011). [17]. leahu, a., damian c., carpiuc n., oroian m., and avramiuc m., change in colour and physicochemical quality of carrot juice mixed with other fruits. journal of agroalimentary processes and technologies 19: 241-246 (2013). [18]. siddiq, m., ravi, r., harte, j. b., & dolan, k. d. (2010). physical and functional characteristics of selected dry bean (< i> phaseolus vulgaris</i> l.) flours. lwt-food science and technology, 43(2), 232-237 (2010). [19]. stas 6253-80 seeds for consumption. 196 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 22016, pag.196 202 description of the influence of freezing on the quality of egg-white creams for pastries julya kambulova 1, * iryna sokolovska 1 1 department of bakery and confectionery goods technology, national university of food technology volodymyrska str. 68, kyiv, ukraine, 01601, sokolovska.ir@gmail.com * corresponding author received june 22th 2016, accepted june 29th 2016 abstract: egg-white creams are used for decorating pastries and have high humidity, which means short storage terms, due to fast microbiological activity. storage prolongation of creams for pastries can be reached by freezing. after freezing-defrosting organoleptic, physical, chemical and microbiological parameters, and their structural-mechanical properties in creams samples were determined. the results show that sensory and physical-chemical indicators of defrosted samples after 30 days remain unchanged in comparison with the indicators of creams before freezing; after 90 days the consistency, stability and forming ability significantly changed. so, 90 days of storage in freezer conditions is inappropriate. in the further studies of defrosted (after 30 days) samples quality indicators show that the recoverability, morbidity and plasticity significantly differ from the structural-mechanical properties of the fresh cream, which means that it is more expedient to freeze creams in composition with pastries. the study of sorption processes showed that if the storage humidity reaches 70%the samples will start to dry out. after studying the microbiological parameters, the maximum storage terms after defrosting were determined. keywords: pectin, semi-finished goods, sodium alginate, long storage, microbiological parameters. 1. introduction egg-white creams are finishing products for pastries decoration. they give them special taste and aroma. creams have a smooth structure with high concentration of air, which is saturated with mass during whipping. density of egg-white cream is 520±60 kg/m3, moisture content – 30±2 [1]. high humidity of raw egg-white creams is a favorable environment for the development of microorganisms, including pathogens. to prevent the negative impact of microorganisms different preservative agents sorbic and benzoic acids and their salts are used. however, the introduction of chemical agents is harmful to the human body, so their number is strictly regulated [2], [3]. question of the hour is the prolongation of the storage life of such products without affecting on their nutritional value. recently, manufacturers use freezing of dairy creams as a method of storage more frequently [4], [5], [6], [7]. however, detailed studies on frozen pastry with raw egg-white creams in the literature were not found. even small variations in the process can cause instability and inconsistency of production quality indicators. to prevent sedimentation and to ensure the microbiological stability of cream various technological methods are used, such as baking, boiling and addition of different http://www.fia.usv.ro/fiajournal mailto:sokolovska.ir@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 197 structurants [8], [9], [10]. biological value of creams is low, so we propose to enrich them with blackberry and sea-buckthorn puree, because these berries are rich in vitamins, minerals and dietary fibers [11], [12]. the use of sodium alginate, pectin with different degree of methoxylation and dietary fibers (berries puree) in technologies of egg-white creams promotes the stabilization of it. also, it helps to form small ice crystals both in cells and intercellular space (much smaller than in creams without structurants).therefore, the product does not "flow" while defrosting [13], [14], [15]. that’s why it was interesting to determine the effect of freezing and defrosting on the quality parameters of creams with improved recipes [16]. 2. material and methods samples of egg-white creams: with sodium alginate and low-methoxy pectin (sa and l-pectin), with sodium alginate and highmethoxy pectin (sa and h-pectin), with blackberry puree and sea-buckthorn puree were frozen to -25 °c in special plastic containers (1000 cm3). the time of absolute freezing was fixed by measuring the temperature inside samples. complete freezing of creams was reached after 6 hours. frozen cream samples were stored for 30 and 90 days at the temperature of -25 ° c. defrosting process took place at a temperature of +4...+6 ° c. creams were fully defrosted in 5 hours. sorption processes were determined on mc bean scales with precision ±0.001 mg. the structural and mechanical properties were determined with structurometer st1. results were calculated by formulas 1-3: 𝑀 = 𝐻0 − 𝐻1 𝐻0 + 𝐻1 (1) 𝑃 = 𝐻0 − 𝐻2 𝐻0 + 𝐻1 (2) 𝑅𝐴 = 1 − 𝐻0 − 𝐻2 𝐻0 (3) where m – morbidity, p – plasticity, ra – recoverability, h0 – height before pressing, h1 – general deformation, h2–elastic deformation. descriptive analysis was used to evaluate smell, taste, color and consistency of samples. about 20 g of sample from each cream were dispensed into small plastic cups and served to judges (20 trained people). the evaluation session was conducted at room temperature. drinking water was provided for mouth rising between samples. the density of cream was obtained by weighing a cup filled with 100 ml sample. microbial determination was done in compliance with lvs iso 21257-2:2008. ph was measured by az-8690 ph-meter, precision 0.01(standard method lvs iso 5542:2010). moisture content was determined by verified balance with precision ±0.001g; mass loss was determined by weighing samples on scales (lvs iso 1442: 1997). 3. results and discussion one of the most important quality parameter of pastries is sensory evaluation. the comparison of samples before and after freezing-defrosting showed that in general the indicators didn’t change. sensory evaluation of defrosted after 30 days storage samples suggest that all the indicators remain unchanged compared to indicators of creams before freezing. so, smell, color, taste are inherent with fresh creams. but some changes were noticed in consistency. the consistency of cream remains homogeneous, even fluffy, but forming ability is worse than before freezing. http://hotzone.com.ua/shop/501/desc/ph-metr-az-8690 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 198 table 1 sensory indicators of egg-white creams indicators egg-white cream with h-pectin and sa egg-white cream with l-pectin and sa sea-buckthorn cream blackberry cream color before white, correspond to its normative light orange color light viola color 30 90 taste before sweet sweet with seabuckthorn taste, little bit sour sweet with blackberry taste 30 90 aroma before correspond to egg-white cream pleasant odor of sea-buckthorn or blackberry 30 90 consistency before smooth, lush, well structured, stable, good forming ability 30 smooth, less lush, well structured, enough forming ability, stable smooth, enough lush, well structured, well forming ability, stable 90 not stable, smooth, structure is weak, forming ability is bad after 90 days all the samples retained their color, taste, and other indicators. however, the consistency significantly changed, it is not stable, not structured enough and the forming ability is very bad. results of the stability analysis of 30 days frozen creams show that creams after freezing-defrosting retain their stability for 3 days, on the 4th day the stability is 94-97%, while at freezing during 90 days the stability is already lost after 2 days, on 3rd day the stability is 89-93%. so, samples after 90 days of storage lose its consistence and are unstable after 2 days. that indicates the infeasibility of using 90-day freezing of egg-white creams. to set the feasibility of 30 days freezing physical and chemical properties were determined. the density and active acidity remained unchanged, and the humidity varies slightly in creams. for example, in a sample of the cream with l-pectin and with blackberry puree the humidity increased. moisture parameters deviate at1% and remain within the norm, because the creams with hydrocolloids can bind more water [17]. while freezing-defrosting two main processes occur: cryodenaturation of protein and partial syneresis of proteinpolysaccharide gel that impairs its quality. in creams with complex of hydrocolloids pectin and sodium alginate, they firmly bind water, preventing changes in the structure of protein molecules and preventing denaturation processes. but during long-term freezing polysaccharide frame also undergoes compression. of course, the structural-mechanical parameters in protein creams vary. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 199 table 2 physical and chemical indicators of creams after 30 days freezing indicators egg-white cream with h-pectin and sa egg-white cream with l-pectin and sa blackberry cream sea-buckthorn cream before after before after before after before after density, kg/m³(±2.9) 482 482 485 485 506 506 514 514 humidity,% (±0.75) 28 28 28 29 30 30 30 31 active acidity(±0.03) 4.8 4.8 5.3 5.3 4.7 4.7 5.1 5.1 table 3 structural-mechanical indicators of creams indicators plasticity,% (±2) morbidity,% (±1.5) recoverability,% (±3) egg-white cream with hpectin and sa before 19.8 17.1 69.9 after 25.8 29.5 60.2 egg-white cream with lpectin and sa before 14.7 18.9 62.0 after 32.1 36.8 53.1 blackberry cream before 17.0 17.8 71.8 after 25.0 30.1 61.6 sea-buckthorn cream before 26.0 28.0 63.4 after 34.3 41.1 51.4 the analysis of received data shows that in defrosted samples plasticity and morbidity significantly increase compared to samples that have not been frozen. the dependence between different types of creams remains the same as in cream samples before freezing. these changes can be associated with partial syneresis of proteinpolysaccharide complex and some cryodenaturation. as a result of this processes recoverability decreases. these results show the infeasibility of freezing creams as semi-finished goods, it is better to freeze them as a part of finished pastry. studying the sorption processes of creams after defrosting allows to give the recommendations of storage conditions and implementation of cakes and pastries with protein cream after freezing and defrosting. the isotherms show that after thawing samples have different natures of the hysteresis loop with or without puree. sorption for all samples in the area begins on monomolecular layer. thus, the value of water adsorbed is the same for all samples – about 1%. in the area of multimolecular adsorption significant changes in the nature of the samples saturation water vapor also occurs. sorption is carried out slowly; maximum value for φ = 75% ranged 2-7%.the most significant modification curves are in the area of capillary moisture the third zone. adsorption curve rises sharply. however, the greatest values φ = 100%, the amount food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 200 of adsorbed moisture of all samples is about the same and ranges from 60-68%. this means that the samples will behave almost identically during storage, slowly remove water – similar to the classic eggwhite cream. with the relative humidity φ = 75% samples of creams will dry out. a) b) c) d) fig. 1 – sorption isotherms of defrosted creams with sa and lpectin (a), with sa and hpectin (b), sea-buckthorn puree (c), blueberry puree (d) previous research showed that freezingdefrosting increases the amount of free moisture in creams, microbial activity could increase. it would determine the microbiological samples protein creams after thawing. determination was conducted for 4 days. microbiological analysis of samples of creams showed that the number of mafam, yeasts and molds are acceptable in 3 days of storage, after 3rd day (except creams with low-methoxy pectin) they do not correspond to requirements of normative documentation. these data suggest that the maximum period of storage after freezing is 3 days. during this time, the content of microorganisms in creams is within the norms stipulated in regulatory documents for cakes and pastries. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 201 table 4 microbiological indicators of creams in 1 g day of storage cream mafam sub bacteria fungi and yeast norm result norm result 1 egg-white cream with hpectin and sa 1.0×104 5.3×10² 2.1×10² 0.6×10² 100 <10 2 6.9×10² 3.8×10² 1.6×10² 30 3 8.3×10³ 4.2×10² 7.1×10² 50 4 8.9×10³ 2.3×10³ 9.9×10² 130 1 egg-white cream with lpectin and sa 1.0×104 2.1×10² 1.1×10² 1.0×10² 100 <10 2 6.6×10² 2.3×10² 1.0×10² 10 3 6.4×10³ 3.4×10² 6.8×10² 50 4 8.4×10³ 1.1×10³ 8.9×10² 90 1 blackberry cream 1.0×104 2.4×10² 1.5×10² 1.0×10² 100 10 2 2.9×10² 1.6×10² 1.8×10² 40 3 3.1×10³ 1.9×10² 7.2×10² 40 4 7.6×10³ 1.0×10³ 1.4×10³ 110 1 sea-buckthorn cream 1.0×104 2.4×10² 1.4×10² 1.5×10² 100 10 2 4.0×10² 2.4×10² 2.5×10² 40 3 4.3×10³ 8.4×10² 7.9×10² 80 4 9.6×10³ 1.2×10³ 9.7×10² 100 4. conclusions the organoleptic and physical-chemical properties are not affected, moisture is increased and stability – decreased because of partial cryodenaturation of protein, and some syneresis of gel. the optimal period of storage in frozen state of our creams is 30 days. the structure of creams corresponds to the norm, structural and mechanical properties show that creams should be frozen as a finished product but not semi finished. analyses of microbiology and stability indicators allow us to determine the maximum period of defrosted creams storage 3 days. received results of research indicate the feasibility of egg-white creams freezing as the way of storage life prolongation. 5. references [1]. jakubczyk, e., niranjan, k., transient development of whipped cream properties. journal of food engineering, 77.1: 7983, (2006) [2]. stewart, c. m., cole, m. b., schaffner, d. w., managing the risk of staphylococcal food poisoning from cream-filled baked goods to meet a food safety objective. journal of food protection, 66.7: 13101325, (2003) [3]. han, f., et al., determination of benzoic acid and sorbic acid in food products using electrokinetic flow analysis–ion pair solid phase extraction–capillary zone electrophoresis. analytica chimica acta, 618.1: 79-85, (2008) [4]. camacho, m. m., martíneznavarrete, n., chiralt, a., stability of whipped dairy creams containing locust bean food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 – 2016 julya kambulova, iryna sokolovska, description of the influence of freezing on the quality of egg-white creams for pastries, food and environment safety, volume xv, issue 2 – 2016, pag.196 – 202 202 gum/λ-carrageenan mixtures during freezing– thawing processes. food research international, 34.10: 887-894, (2001) [5]. selomulyo, v. o., zhou, w., frozen bread dough: effects of freezing storage and dough improvers. journal of cereal science, 45.1: 1-17, (2007) [6]. deng, j., et al. an improved protocol for rapid freezing of protein samples for long-term storage. acta crystallographica section d: biological crystallography, 60.1: 203-204, (2004) [7]. lewis, d. f. microstructure of frozen and dairy-based confectionery products. structure of dairy products, 236-258, (2007) [8]. van der plancken, i., van loey, a., hendrickx, m. e., foaming properties of egg white proteins affected by heat or high pressure treatment. journal of food engineering, 78.4: 1410-1426, (2007) [9]. muthukumaran, a., ratti, c., raghavan, v., g., foam-mat freeze drying of egg white and mathematical modeling part i optimization of egg white foam stability. drying technology, 26.4: 508-512, (2008) [10]. yang, x., foegeding, e. a., effects of sucrose on egg white protein and whey protein isolate foams: factors determining properties of wet and dry foams (cakes). food hydrocolloids, 24.2: 227-238, (2010) [11]. hassimotto, n. m. a., et al. physicochemical characterization and bioactive compounds of blackberry fruits (rubus sp.) grown in brazil. food science and technology (campinas), 28.3: 702-708, (2008) [12]. zeb, a., chemical and nutritional constituents of sea buckthorn juice. pakistan journal of nutrition, 3.2: 99-106, (2004) [13]. regand, a., goff, h. d., structure and ice recrystallization in frozen stabilized ice cream model systems. food hydrocolloids, 17.1: 95-102, (2003) [14]. thiebaud, m.; dumay, e. m.; cheftel, j.-c., pressure-shift freezing of o/w emulsions: influence of fructose and sodium alginate on undercooling, nucleation, freezing kinetics and ice crystal size distribution. food hydrocolloids, 16.6: 527-545, (2002) [15]. faydi, e.; andrieu, j.; laurent, p., experimental study and modelling of the ice crystal morphology of model standard ice cream. part i: direct characterization method and experimental data. journal of food engineering, 48.4: 283-291, (2001) [16]. kambulova, j., sokolovska i., studying the influence of structure forming agents on egg white foaming process. science works of nuft, 50: 113-118, (2013) [17]. smidsrød, o., molecular basis for some physical properties of alginates in the gel state. faraday discussions of the chemical society, 57: 263-274, (1974) 271 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 271 277 the quality of wastewater in the dairy industry *sonia amariei1, aspazia rusu1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania gutts@fia.usv.ro *corresponding author received july 23th 2013, accepted september 5th 2013 abstract: the paper aims a comparative study of the parameters of dairy wastewater during three years namely a comparison of wastewater parameters before entering in the treatment plant and waste water parameters after treatment in the wastewater treatment plant. the indicators used to analyze the quality of wastewater from the dairy factory were: cco-cr, cbo5, total suspensions, ph, chloride content, total nitrogen, total phosphorus, oils and fats. the purpose of monitoring is to produce information on the physico chemical waste water, on treatment technologies efficiency, focusing on the disinfection process, to determine if the waste water is adequate or not in terms of the values of the relevant parameters set by law. key words: treatment plant, wastewater, dairy industry 1. introduction monitoring the quality of discharged wastewater from the food industry is a necessary condition for all food units that produce, process, store, keep, transport and selling food. the main environmental issues in dairy industry are the high energy and water consumption, generation of waste water with a high organic content and the production and management of waste. the most important environmental problem in the dairy industry is the generation of waste water, both for their volume and as well as polluting load associated (organic basically). the dairy industry is a major source of pollution because of its effluents are usually rich in carbonaceous material, nitrogen containing (protein) shaping the decisive growth of cbo5. thus, the amount of waste water resulting from a milk factory can find values that range from 2 to 6 l / l milk processed depending on the factors such as the size and age of the plant, equipment, handling, cleaning plans. to residue obtained after processing the milk we must add leftover of antiseptics and disinfectants used to clean the milk ducts (hypochlorite, nitric acid and detergents). antiseptic substances used generates in their turn certain products(disinfection of drinking water by chlorine sometimes forms carcinogenic trihalomethanes) highly toxic (4), (6). there are microorganisms such as cryptosporidium spp. and giardia which are resistant to chlorine disinfection and in this case the most used is a chemical coagulant such as aluminum sulfate, whose use increases the metal ion content in the treated water (5), (6). the wastewater generated in a dairy factory can be classified according to three food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 272 sources of generation: processes, cleaning and cooling. table 1. the main the dairy industry effluents no effluent source characteristics observation 1 process water cleaning of equipment and installations bod5 , cod-cr, suspended solids (ss), nitrogen, detergents, oils, and fats. depending on the size and age of the plant, equipment, handling cleaning plans 2 clening solutions sterilization peroxide cleaning plans 3 cooling water for products and heat exchangers maintenance of heat exchangers hot water with suspended solids pasteurisation, sterilisation 4 sanitary waste water disinfection bod5 , cod-cr, suspended solids (ss), ammonia and detergents cleaning plans, chemical coagulants 5 water for regeneration of ions exchange resins treatment of water from wells acidity and basicity acids and bases for regeneration of ions exchange resins waters resulting from the processing of milk are characterized by a high concentration of organic material in proteins, sugars, lipids, high concentrations of suspended solids, high consumption of oxygen demand (bod5, cod-cr), high concentrations of nitrogen and phosphorus, high concentrations of oils and fats in the suspension, significant changes in ph. washing waters may also contain: sterilizing agent (1), (sodium hypochlorite), detergents, alkalis (sodium hydroxide) or acid (nitric acid, phosphoric acid) used for washing ion exchangers (2) or cip installations. 2. materials and methods the composition of the waste water is characterized by a high content of fat, lactose and protein. among its components can meet and resistant thermophilic bacteria remaining on the plates of heat exchangers and other devices that can hold milk losses and chemical treatments or electrochemical disinfection (3) that can be done that will increase the degree of pollution of water. main origin of pollution is caused losses of product in different stages of technological process (between 0.3 and 1.3% of processed). the analyzes focused on (7): 2.1. determination of oxidizable substances in water (cod-cr) / iso 6060-1996 oxidizable substances in water or chemical oxygen demand (cod-cr) are substances which oxidize both cold and hot, under the action of an oxidant. oxidizable substances in the water are oxidized by potassium dichromate in sulfuric acid medium, hot, and the excess dichromate is titrated with mohr's salt in the presence of feroinei as an indicator. 2.2. determination of biochemical oxygen demand in water (bod5) / sr 1899 / 2-2002 biochemical oxygen demand bod5 is the amount of oxygen consumed by the microorganisms in a period of time, to decompose the organic substances contained in the biochemical water. the standard time is set for 5 days at a temperature of 200c. the oxygen consumed is determined for 5 days by microorganisms in the water by the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 273 difference found between the quantity of dissolved oxygen in the water sample immediately and after 5 days of collection. determination of bod5 is made in diluted and undiluted water sample. 2.3 determination of total suspension / sr en 872-2005 they are insoluble substances in water, expressed in mg suspensii/dm3. waterinsoluble substances can remain in the suspension, the more or less time depending on the weight of the particle. colloidal substances and very light particles are permanently suspended, being in a continuous movement in the water. it is determined by separating the particles in suspension (filtration, centrifugation, sedimentation), drying and weighing them. 2.4. determination of ph / sr en iso 10523-2012 potentiometric method was used to determine ph. 2.5 determination of chlorides / sr en 9297-2001 (method mhor) chlorides from water come from salts of raw materials, from process or following an accidental pollution. chloride ion is determined by mohr method after its reaction with silver nitrate in neutral medium when form silver chloride, insoluble. end of reaction is indicated by potassium chromate. 2.6. determination of total nitrogen / sr en iso 11905/1 2003 (kjeldahl method) total nitrogen is an indicator totaling organic nitrogen in various forms (proteins and nucleic acids in various states of decay, urea, amines, etc..) and inorganic, ammonium ion nh4 +. the kjeldahl method to determine the total nitrogen is consisting in the mineralization of samples to form ammonium sulfate that releases ammonia, which is then distilled and determined by titration with hydroxide. 2.7 determination of total phosphorus / sr en iso 6878: 2005 the method of determination is based on the reaction of ammonium molybdate, that forms with phosphate ions the phosphor ammonium molybdate, which can be reduced to molybdenum blue with an organic or inorganic reducing agent. resulting blue complex is measured by spectrophotometry, absorption of solution being proportional to the concentration of phosphate ions from wastewater analyzed 2.8. determination of oils and fats / sr 7578-96 the content of extractable substances with solvents, animal and vegetable fats, hydrocarbons (mineral oil, heavy hydrocarbons), combinations with hydroxyl, carbonyl, carboxylate functions, nitrogenous compounds, insecticides, soaps, waxes, resins and tars are determined by extraction with solvents. 3. results and discussion in the three years, during which the study was conducted, they found a high levels of cod-cr, in waste water, before treatment, in the spring and summer seasons, figure 1. the high levels of cod-cr, indicates the existence of a large amount of organic matter facilitating the chemical oxidation, and requires a high consumption of oxygen. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 274 spr in g s u mmer a ut umn win ter 2010 201 1 2012 0 2 00 0 4 000 6 000 8 000 10 000 2010 2011 2012 0 50 100 150 200 250 300 350 spring summer autumn winter 2010 2011 2012 fig.1.values of bod5 from the waste water before treatment fig.2.values of bod5 in waste water after treatment after the wastewater treatment it was a small excess of permissible limit (500 mg / l) in the summer and autumn of 2010 and 2011, figure 2. spring sum m er autum n wint er 2010 2011 2012 0 2000 4000 6000 8000 10000 2010 2011 2012 0 50 100 150 200 250 300 350 spring summer autumn winter 2010 2011 2012 fig.3 bod5 values from waste water before treatment fig.4. bod5 values in waste water after treatment the highest values of bod5 in wastewater before treatment were recorded in summer season, figure 3, the maximum occurring in the summer of 2010 (8480 mg / l). the lowest values of bod5 were found in the winter season. high levels of bod5 indicates that oxidation of organic matter occurs (suspended solids, waste) because of the large number of existing bacteria. after treatment, all the recorded values were integrated into the limit of value allowed (300 mg / l), figure 4. s pringsummerautumn wi nter 2010 2012 0 5000 10000 2010 2011 2012 0 50 100 150 200 250 300 350 400 spri ng summer autumn wi nter 2010 2011 2012 a ccepted va lues fig.5. values of suspended solids content in waste water before treatment fig.6 values of suspended solids content in waste water after treatment the highest values of total suspensions content of waste water before treatment, were recorded in the spring season, the highest value reached in spring of 2011 (8140mg / l), figure 5. this is due to the presence in the waste water of a large food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 275 amount of insoluble substances, in different sizes. after treatment there was a small excess of allowed limit (350 mg / l), figure 6, in summer and autumn of 2010. spring summer autumn wi nter 2010 2012 0 5 10 2010 2011 2012 0 1 2 3 4 5 6 7 8 9 s pri ng summer autumn wi nter 2010 2011 2012 a ccepted val ues fig.7 wastewater ph values before treatment fig.8. wastewater ph values after treatment both before treatment and after treatment, the effluents had values that integrate within the range of allowed limit, 6.5 – 8.5. this is because the company uses raw materials complying with the rules of quality and to the fact that the company uses biodegradable substances for cleaning and disinfection. fig.9. values of chloride content of waste water before treatment fig.10. values of chloride content of waste water after treatment the highest values of chloride content in waste water before treatment were recorded in autumn season. the highest value was reached in autumn 2011 (710 mg / l), figure 9. after treatment there was one over exceeded of allowed limit, (500 mg / l) in autumn 2010 (570 mg / l) figure 10. 2010 2012 0 1000 2000 sprin g sum mer autum n wint er 2010 2011 2012 0 10 20 30 40 spring summer autumn winter 2010 2011 2012 fig.11. values of total nitrogen content of waste water before treatment fig.12. the values of the total nitrogen content of the waste water after treatment the highest values of total nitrogen content of the waste water before the treatment were recorded in the winter season. the highest value reached in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 276 winter of 2011 (1502 mg / l) figure 11. this may be due to excessive use of cleaning agents and disinfectants rich in nitrogen (nitric acid) or raw material that had a high content of nutrients rich in nitrogen. after wastewater treatment was a very small excess of allowed limit (30 mg / l) in the spring season in 2011 (31-33 mg / l) figure 12. spring summer autumn winter 2010 2012 0 500 2010 2011 2012 0 1 2 3 4 5 6 spring summer autumn winter 2010 2011 2012 accepted values fig.13. values of total phosphorus content of waste water before treatment fig.14.values of total phosphorus content of waste water after treatment the highest values of total phosphorus content of waste water before treatment were recorded in winter season. the highest value was reached in the winter of 2010 (425mg / l). this may be due to excessive use of cleaning agents and disinfectants rich in phosphorus (phosphoric acid) or milk has a high content of nutrients rich in phosphorus. 2010 2012 0 2000 4000 6000 spring autumn 2010 2011 2012 0 20 40 60 80 100 120 140 spring summer autumn winter 2010 2011 2012 fig.15 values of oils and fats content from waste water before treatment fig. 16. values of oil and fats content from waste water after treatment the highest values of oil and fats content from wastewater before treatment, were recorded in autumn and winter seasons. the highest value was reached in autumn 2010 (4900 mg / l), figure 15. this is due to the high content of milk fat in autumn and winter. after wastewater treatment it was a slight excess of allowed limit value (100 mg / l) in autumn and winter seasons of 2010 (120 mg / l and 105 mg / l) figure 16. 4. conclusions after performing this study the conclusions were that wastewater parameters analyzed before treatment far exceed the limit values allowed by the rules in force, which now oblige in taking measures to reduce their values. the values of parameters analyzed after treatment plant proves its effectiveness and the fact that wastewater discharged into the city sewer system is adequate. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 sonia amariei, aspazia rusu, the quality of wastewater in the dairy industry, food and environment safety, volume xii, issue 3 – 2013, pag. 271 277 277 5. references [1] ben warton, anna heitz, cynthia joll, robert kagi, a new method for calculation of the chlorine demand of natural and treated waters, wat er re s e arch 40 (2006) 2877 – 28841. [2] seok-jun seo, hongrae jeon, jae kwang lee, gha-young kim, daewook park, hideo nojima, jaeyoung lee, seung-hyeon moon, investigation on removal of hardness ions by capacitive deionization (cdi) for water softening applications, water research 44(2010) 2267 – 2275 [3] yasmine delaedta, arne daneelsa, priscilla declercka, jonas behetsa,jaak ryckeboerb, elmar petersc, frans olleviera, the impact of electrochemical disinfection on escherichia coli and legionella pneumophila in tap water, microbiological research 163 (2008) 192—199 [4] binghui zhu, zhixiong zhong, jing yao, ion chromatographic determination of trace iodate, chlorite, chlorate, bromide, bromate and nitrite in drinking water using suppressed conductivity detection and visible detection, journal of chromatography a,1118 (2006)106– 110 [5] l. nishia, a. m. s. vieiraa, m. f. vieiraa, g. f. silvab, r. bergamasco, application of hybrid process of coagulation/flocculation and membrane filtration for the removal of protozoan parasites from water, procedia engineering 42 ( 2012 ) 148 – 160 [6] toshio takayanagi, makoto ishida, julius mbuna, rim driouich, shoji motomizu, determination of bromate ion in drinking water by capillary, zone electrophoresis with direct photometric detection, journal of chromatography a, 1128 (2006) 298–302 [7] tawfik,, m. sobhey,, m. badawy,, treatment of a combined dairy and domestic wastewater in an up-flow anaerobic sludge blanket (uasb) reactor followed by activated sludge (assystem) [8] mahmoud a. el-sheikh, hazem i. saleh, joeseph r. flora, mahmoud r. abdel-ghany, biological tannery wastewater treatment using two stage uasb reactors, desalination 276 (2011) 253–259 360 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 360 364 molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage remzi cholakov1, rositsa denkova2, desislava teneva1, velichka yanakieva1, iliyan dobrev1, zapryana denkova1, zoltan urshev3 1university of food technologies, department of microbiology, inj.cholakov@gmail.com 2university of food technologies, department of biochemistry and molecular biology, rositsa_denkova@mail.bg 3lb bulgaricum ltd, laboratory of dna analysis *corresponding author received november 16th 2014, accepted december 29th 2014 abstract: yeast strain 25-g was isolated from naturally fermented cereal beverage (boza). by biochemical (api 20 c aux) and molecular-genetic (partial sequencing of the 26s rrna gene) methods, a representative of the species saccharomyces cerevisiae var. diastaticus was identified. the enzymatic profile of the strain was determined by applying a kit system api zym (biomerieux, france). its proteolytic and amylase activities were examined as well. saccharomyces cerevisiae var. diastaticus strain 25-g exhibits amylase activity, which makes it suitable for being included in the composition of starter cultures used at the production of fermented cereal foods and beverages. key words: boza, identification, sequencing, enzyme profile, amylolytic activity 1. introduction cereal foods and beverages are a major source of nutrients. boza is a traditional low-alcohol fermented cereal beverage. it is made of millet, corn, wheat, rice and others [1]. it is defined as a beverage thick in texture, light or dark beige in colour, slightly sharp or slightly sour in taste, with specific odor, which is naturally fermented by lactic acid bacteria and yeasts such as: lactobacillus fermentum, lactobacillus sanfranciscensis, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus pentosus, leuconostoc paramesenteroides, leuconostoc mesenteroides subsp. mesenteroides, leuconostoc raffinolactis, lactococcus lactis subsp. lactis, oenococcus oeni, weissella paramesenteroides, and weissella confusa and yeasts: saccharomyces cerevisiae, saccharomyces uvarum, saccharomyces carlsbergensis, candida glabrata, candida tropicalis, geotrichum candidum, and geotrichum penicilatum [1, 2, 3, 4]. the purpose of thе present study was the biochemical and molecular-genetic identification of yeast strain 25-g isolated from naturally fermented cereal beverage – boza, and determination of its enzyme profile. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 remzi cholakov, rositsa denkova, desislava teneva, velichka yanakieva, iliyan dobrev, zapryana denkova, zoltan urshev, molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage, food and environment safety, volume xiii, issue 4 – 2014, pag. 360 – 364 361 2. materials and methods 2.1. microorganisms yeast strain 25-g was isolated from naturally fermented cereal beverage (boza). 2.2. nutrient media 2.2.1. saline solution. composition (g/dm3): nacl 5. sterilization 20 minutes at 121ºc. 2.2.2. malt-agar. composition: malt extract (kamenica, bulgaria), dilluted in a ratio of 1:1 with tap water + 2% agar, ph is adjusted to 6.5 – 7.0. sterilization 25 minutes at 121ºc [5]. 2.2.3. solid medium for the determination of amylase activity. composition (g/dm3): meat extract 3, peptone 5, soluble starch 2, agar 15. ph is adjusted to 7.2 ± 0.1. sterilization 25 minutes at 121ºc. 2.2.4. solid medium for the determination of proteolytic activity. composition (g/dm3): skimmed milk 28, casein hydrolyzate 5, yeast extract – 2.5, glucose 1, agar 15. ph is adjusted to 7 ± 0.2. sterilization 25 minutes at 121ºc. 2.3. culturing and storage of the test microorganism the studied yeast strain was grown on malt-agar at 28ºc for 48 hours and was stored at 4 ± 2ºc for 2 months. 2.4. physiological methods 2.4.1. agar-diffusion method for determining the amylase activity this method comprised in determining the ability of the tested strain to hydrolyze starch. the solid medium for the determination of amylase activity was melted and poured in petri dishes (15 cm3 of the medium per petri dish). after the hardening of the medium wells with a diameter of 6 mm were made. the cellular suspension of the tested strain was pipetted into the wells. this test was performed in quadruplicates. after inoculation, the plates were cultured at 30°c for 48 hours. the results were reported as positive if there was a more turbid halo around the wells in the petri dishes. the lack of a halo was a sign of the inability of the strain to hydrolyze starch. 2.4.2. agar-diffusion method for determining the proteolytic activity this method comprised in determining the ability of the tested strain to digest milk proteins. the solid medium for the determination of proteolytic activity was melted and poured in petri dishes (15 cm3 of the medium per petri dish). after the hardening of the medium wells with a diameter of 6 mm were made. the cellular suspension of the tested strain was pipetted into the wells. this test was performed in quadruplicates. after inoculation, the plates were incubated at 30ºc for 48 hours. the results were reported as positive if there was a bright halo around the wells of the petri dishes. the absence of a halo was a sign of the inability of the strain to hydrolyze milk proteins. 2.5. biochemical methods 2.5.1. determination of the biochemical profile the system api 20 c aux (biomerieux sa, france) for identification of yeast species based on the consumption of 19 carbon sources was used for the determination of the biochemical profile of the tested strain. fresh 24-hour culture of the tested strain, developed on malt agar, was resuspended according the instructions of the manufacturer in api c resuspension medium. the honeycomb wells on the bottom of the incubation boxes were filled with sterile physiological solution. the api 20 c strips were placed in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 remzi cholakov, rositsa denkova, desislava teneva, velichka yanakieva, iliyan dobrev, zapryana denkova, zoltan urshev, molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage, food and environment safety, volume xiii, issue 4 – 2014, pag. 360 – 364 362 incubation boxes and the microtubules were inoculated with the prepared cell suspension. the sample was incubated for 48h to 72h at the optimum temperature for the studied strain. results were recorded according the change in turbidity in comparison to the control (microtubule 0). the results were processed with apiweb® identification software. 2.5.2. determination of the profile of the enzyme activity of the test cultures. the determination of the profile of enzyme activity was performed, using the test kit api zym (biomerieux, france) for semiquantitative determination of the enzyme profile of the studied strain. fresh 24-hour culture of the tested strain was centrifuged for 15 minutes at 5000 x g, the obtained biomass precipitate was washed twice and resuspended in api suspension medium. the api zym strips were placed in the incubation boxes and the microtubules were inoculated with the prepared cell suspension. the sample was incubated for 4 to 4,5 hours at 37°c. after the incubation one drop of reagent a and one drop of reagent b were pipetted into each microtubule. after 5 min staining was reported according to the color scheme described in the manufacturer's instructions. the enzyme activity was determined according to a color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 2.6. genetic methods 2.6. 1. isolation of total dna the isolation of dna was performed by the method of delley et al. [6]. 2.6.2. 26s rdna amplification and visualization all pcr reactions were performed using the pcr kit – pcr vwr in a volume of 25 µl in a progene cycler (techne, uk) according to the instructions of the manufacturer. in each pcr reaction 50 ng total dna of the tested strain and 10 pmol praimers were used. dna of the studied strain is amplified using universal primers for the 26s rdna gene nl1 (5’gcatatcaataagcggagga aaag3’) and nl4 (5’ggtccgtgtttcaagacgg-3’) [7]. the amplification program included: denaturation 95°c for 3 minutes; 40 cycles 93°c for 30 s, 55°c for 60 s, 72°c for 2 minutes; final elongation 72°c for 5 minutes. the resulting product was visualized on a 2% agarose gel stained with ethidium bromide solution (0.5 µg/ml), using an uvp documentation system (uk). 2.6.3. purification of the product of the pcr-reaction – end fragment of the 26s rdna – from tae agarose gel the purification of fragment of the 26s rdna was conducted using dnapurification kit (gfx microspintm) according to the manufacturer’s instructions. 2.6.4. partial sequencing of the 26s rrna gene. the partial sequencing of the 26s rrna gene was conducted by “macrogen europe laboratory”, netherlands, based on the method of sanger. 3. results and discussion the yeast strain 25-g was isolated from naturally fermented cereal beverage. by determining the ability of the strain to utilize the 19 carbon sources included in the kit system for rapid identification of yeasts api 20 c aux the yeast strain 25-g was identified with poor reliability: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 remzi cholakov, rositsa denkova, desislava teneva, velichka yanakieva, iliyan dobrev, zapryana denkova, zoltan urshev, molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage, food and environment safety, volume xiii, issue 4 – 2014, pag. 360 – 364 363 cryptococcus laurentii 46,1%, cryptococcus huminicula 29,5%, trichosporon mucoides 23,4%. therefore a molecular-genetic method for identification was applied sequencing of the gene for the 26s rrna. the yeast strain 25-g belongs to the species saccharomyces cerevisiae with a percentage of similarity between the partial sequence of the 26s rdna of yeast strain 25-g and the partial sequence of the 26s rdna of saccharomyces cerevisiae strain lqc 10089 99% (fig. 1). saccharomyces cerevisiae strain lqc 10089 26s ribosomal rna gene, partial sequence score expect identities gaps strand 877 bits(972) 0.0 492/496 (99%) 0/496(0%) plus/plus query 44 gctcaaatttgagtctggtaccttcggtgcccgagttgtaatttggagagggcaactttg 103 |||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||| sbjct 40 gctcaaatttgaatctggtaccttcggtgcccgagttgtaatttggagagggcaactttg 99 query 104 gggccgttccttgtctatgttccttggaacaggacgtcatagagggtgagaatcccgtgt 163 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 100 gggccgttccttgtctatgttccttggaacaggacgtcatagagggtgagaatcccgtgt 159 query 164 ggcgaggagtgcggttctttgtaaagtgccttcgaagagtcgagttgtttgggaatgcag 223 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 160 ggcgaggagtgcggttctttgtaaagtgccttcgaagagtcgagttgtttgggaatgcag 219 query 224 atctaagtgggtggtaaattccatgtaaagctaaatattggcgagagaccgatagcgaac 283 ||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||| sbjct 220 ctctaagtgggtggtaaattccatctaaagctaaatattggcgagagaccgatagcgaac 279 query 284 aagtacagtgatggaaagatgaaaagaactttgaaaagagagtgaaaaagtacgtgaaat 343 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 280 aagtacagtgatggaaagatgaaaagaactttgaaaagagagtgaaaaagtacgtgaaat 339 query 344 tgttgaaagggaagggcatttgatcagacatggtgttttgtgccctctgctccttgtggg 403 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 340 tgttgaaagggaagggcatttgatcagacatggtgttttgtgccctctgctccttgtggg 399 query 404 taggggaatctcgcatttcactgggccagcatcagttttggtggcaggataaatccatag 463 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 400 taggggaatctcgcatttcactgggccagcatcagttttggtggcaggataaatccatag 459 query 464 gaatgtagcttgcctcggtaagtattatagcctgtgggaatactgccagctgggactgag 523 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 460 gaatgtagcttgcctcggtaagtattatagcctgtgggaatactgccagctgggactgag 519 query 524 gactgcgatgtaagtc 539 |||||||| ||||||| sbjct 520 gactgcgacgtaagtc 535 fig.1 comparison of the partial sequence of the 26s rdna of yeast strain 25g and the partial sequence of the 26s rdna of saccharomyces cerevisiae strain lqc 10089. the enzyme profile of the studied strain was determined using the kit system api zym. it demonstrated leucine aminopeptidase, acid phosphatase, phosphohydrolase, α-glucosidase activity. the strain does not possess the ability to synthesize β-galactosidase, which is why it oes not develop in nutrient media containing lactose as substrate (table 1). experimental data demonstrate that the studied strain can be included in the composition of starter cultures for fermented cereal foods and beverages. the amylolytic and proteolytic activity of saccharomyces cerevisiae strain 25-g was determined by the agar-diffusion method with wells. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 4 – 2014 remzi cholakov, rositsa denkova, desislava teneva, velichka yanakieva, iliyan dobrev, zapryana denkova, zoltan urshev, molecular-genetic and biochemical characterization of saccharomyces cerevisiae strain 25-g, isolated from fermented cereal beverage, food and environment safety, volume xiii, issue 4 – 2014, pag. 360 – 364 364 table 1 enzyme profile of saccharomyces cerevisiae 25-g * the enzyme activity was determined according to a color scale from 0 (no enzyme activity) to 5 (maximum enzyme activity) the strain demonstrated significant amylolytic activity but lacks proteolytic activity (table 2). table 2. proteolytic and amylolytic activity of saccharomyces cerevisiae strain 25-g. dwell = 6mm dzone, mm strain amylolytic activity proteolytic activity saccharomyces cerevisiae 25-g 14 4. conclusion by modern physiology, biochemistry and molecular-genetic methods the newly isolated yeast strain 25-g, isolated from naturally fermented cereal beverage, was identified. it was shown that the system for rapid identification api 20 c aux does not usually have enough discriminative ability. saccharomyces cerevisiae 25-g exhibited leucine aminopeptidase, acid phosphatase, phosphohydrolase, αglucosidase and amylolytic activity. 5. references [1]. arici m., daglioglu o., boza: a lactic acid fermented cereal beverage as a traditional turkish food. food research international 18: 39– 48 (2002). [2]. hancioğlu o., karapinar m., microflora of boza, a traditional fermented turkish beverage. international j. of food micr. 35: 271– 274 (1997). [3]. todorov s.d., dicks l.m.t., screening for bacteriocins-producing lactic acid bacteria from boza, a traditional beverage from bulgaria: comparison of the bacteriocins. process biochem 41: 11–19 (2006). [4]. zorba m., hancioglu o., genç m., karapinar m., ova g., the use of starter cultures in the fermentation of boza, a traditional turkish beverage. process biochemistry 38: 1405– 1411 (2003). [5]. beshkov m., karova e., murgov i., a handbook in microbiology. hristo g. danov publishing (1970). [6]. delley m., mollet b., hottinger h., dna probe for lactobacillus delbrueckii. appl. environ. microbiol. 56: 1967–1970 (1990). [7]. kurtzman c.p., robnett c.j., identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 59 end of the large-subunit (26s) ribosomal dna gene. journal of clinical microbiology, vol. 35, 5: 1216 – 1223 (1997). impact of storage temperatures and storage on reducing sugar acumulations in sugar beet food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 39 tem pera tu r e an d time of stor a ge impact on r ed uc ing suga r acumula tions in sug a r bee t *octavian barna1, octavian baston1, aura darabă1 1biochemistry department, faculty of food science and engineering, galati, „dunărea de jos” university, 111 domneasca st., 800201 galati, octavian.barna@yahoo.com , obaston@ugal.ro, adaraba@ugal.ro *corresponding author received 19 june 2011, accepted 20 december 2011 abstract: the quality of sugar beet determines its sugar output. besides its initial quality and harvesting conditions, it is the storage conditions that influence significantly the technological quality of sugar beet. an important indicator regarding the quality of sugar beet is the content in reducing sugars. throughout the production process, the reducing sugars are some of the forming sources of coloring substances in juices and syrups. the aim of this paper is to determine the effect of storage temperature and time upon the content variation in reducing sugars of the new beethybrids adapted to the new climate conditions of europe, by analyzing statistically the correlations between storage time and temperature on the one hand, and the content variation in reducing substances on the other hand. the sugar beet was stored at different ranges of temperature of 2, 6, 10, 15 and 20 oc and determinations on the content in reducing sugars after 5, 10, 20, 30, 40, 50 and 60 daystorage time were made. the method icumsa gs 2/9-6 2007 was used to determine the content in reducing sugars. the data obtained were statistically processed using the program excel of microsoft office xp in order to get the values of correlation coefficients (r) between the content in reducing sugars and storage time depending on storage temperature. the analysis results show that there is a non-uniform variation in time of the content in reducing sugars throughout storage, with significant influence due to the heat level – storage of sugar beet. keywords: sugar beet, storage, temperature, reducing sugars. 1. introduction to obtain sugar from sugar beet is economically more advantageous than from sugar-cane as sugar beet contains by 25 % more sugar than cane [1].during storage, the chemical composition of beets changes and the amount of the recoverable sugar thus declines. sucrolitic enzymes cleave sucrose to glucose and fructose. these hexoses mainly fuel beet respirations, but a certain amount accumulates in cells [2]. besides temperature, the storage time has an important influence upon the technological quality of the sugar beet stored [3]. this one is influenced both by the sucrose content and percentage of substances preventing sugar from getting crystallized during the refining process [4]. the production of inverted sugar after harvesting is the result of sucrose hydrolysis and metabolic processes of respiration in beet roots [5]. in the first hours of storage, the quantity of inverted sugar decreases as a result of the decrease of invertase activity and consumption of the reducing sugars in metabolic processes occurring in sugar beet roots [6]. therefore, the quantity of inverted sugar increases constantly by a rate depending both on storage conditions and health state of beet. sugar beet cultivars differ among mailto:obaston:@ugal.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 40 them both in their quantity of sugar accumulated during the vegetation period and storage behavior [7]. the quantity of inverted sugar in beet is considered an indicator of its technological quality on the one hand due to the fact that the inverted sugar is obtained from sucrose degradation and on the other one some organic acids of undesirable effect are getting formed from inverted sugar processing. the new climate conditions of europe have imposed the cultivation of some new hybrids of sugar beet adapted to these ones. knowing the behaviour of these hybrids during storage and how high quality of sugar beet can be obtained after its storage is very intersting for sugar technology, because the quality of raw material affects all technological process for obtaining sugar. in the present study the behaviour of sugar beet has been studied for 60 days and this is usually the maximum storage period of sugar beet after harvest until processing. 2. experimental the beet to be analyzed represents victor hybrids of type z, soleia of type n/z and markus of type z cultivated in the county of brasov. the sugar beet has been stored together with its adherent impurities at the temperature ranges of 2, 6, 10, 15 and 20 oc and determinations on the sucrose content after storage periods of 5, 10, 20, 30, 40, 50 and 60 days have been made. the results obtained were used to determine the accumulating rate of reducing sugars in sugar beet depending on the storage temperature used. the determination of reducing sugars content was made using gs 2/9-6 2007 [8]. the information obtained was statistically processed using software excel in microsoft office 2003 suite in order to find values of correlation coefficients (r) [9] between storage time and temperature and variations of sucrose content of the sugar beet analyzed. 3. results and discussion the experimental results obtained after the samples of cultivar victor of type z have been stored at different levels of temperature which can be seen in figure 1. 0 500 1000 1500 2000 0 20 40 60 80 storage duration , days r ed uc in g su ga rs c on te nt , g/ t b ee t 2 6 10 15 20 figure 1: reducing sugar content of cultivar victor stored at different temperatures as regards the storage behavior of cultivar victor, when the heat level of 2oc is used, there is a slight decrease in the reducing sugars content displaying a limiting tendency towards the end of the storage period. when the storage temperature is scaled up to 6oc, there is a continuous increase of the parameter analyzed. at temperatures of 10, 15 and 20 oc there is a more and more significant increase of the reducing sugars content with small variations specific to each heat level. the average value of the accumulating rate of reducing sugars depending on temperature was calculated and the results are shown in figure 2. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 41 -5 0 5 10 15 20 2 6 10 15 20 temperature, c r ed uc in g su ga r ac um ul at io n, ( g/ t d) figure 2: rate of invert sugar accumulation depending on temperature during storage (cultivar victor). after having analyzed the accumulation rate of reducing sugars in the case of cultivar victor, we can notice that the maximum value of the accumulation rate in degree is reached during the heat temperature range 2-6 oc whereas the minimum one in the range 6-10 oc. for the temperature 10-15 oc and 15-20 oc, slightly lower values were registered than the maximum one. the cultivar soleia had the following storage behavior that can be seen in figure 3. 0 500 1000 1500 2000 2500 0 20 40 60 80 storage duration, days r ed uc in g su ga rs c on te nt , g / t be et 2 6 10 15 20 figure 3: reducing sugar content of cultivar soleia stored at different temperatures as a peculiarity of the cultivar soleia, this one had a similar behavior to the storage heat ranges of 6 and10oc. thus, the same as in the case of the cultivar victor, there is a decrease in the reducing sugars content after a storage period at 2oc. -5 0 5 10 15 20 25 2 6 10 15 20 temperature , c r ed uc in g s ug ar a cu m ul at io n, g/ (t d) figure 4: rate of invert sugar accumulation depending temperature during storage (cultivar soleia) the accumulation rate of reducing sugars in the case of the cultivar soleia reaches the maximum of increase in the temperature range 2-6 oc, the rest of ranges having relatively close values to the parameter analyzed. the evolution of reducing sugars content of the cultivar markus’ storage duration led to the results shown in figure 5. 0 500 1000 1500 2000 2500 3000 0 20 40 60 80 storage duration, days r ed uc in g su ga r c on te nt , g /t be et 2 6 10 15 20 figure 5: reducing sugar content of cultivar markus stored at different temperatures food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 42 in the case of the cultivar markus there is a relative slow increase of the reducing sugars content in the first 20 days of storage, followed by an acceleration of this process in the range 20 – 50 days especially in the samples stored at 10, 15 and 20 oc. the storage at 2 oc has as effect a slight decrease of the reducing sugars content together with the increase of storage duration. -10 0 10 20 30 40 2 6 10 15 20 temperature, cr ed uc in g su ga r ac um ul at io n, (g /t d) figure 6: rate of invert sugar accumulation depending on temperature during storage (cultivar markus) as can be seen from in figure 6, the accumulation rate of reducing sugars in the case of the cultivar markus reaches maximum values at the ranges 2-6 oc and 6-10 oc and minimum ones at the ranges10-15 oc and 15-20 oc. this may be a particularity of the cultivar markus making it adequate to be stored at higher temperatures. the statistical analysis led to very high values of the correlation coefficient between the reducing sugars content and storage duration depending on the storage temperature:table 1: correlation coefficient between reducing sugars content and storage duration depending on the storage temperature : t, oc victor soleia markus 2 -0.99545 -0.99814 -0.99626 6 0.99246 0.997655 0.996017 10 0.994762 0.998248 0.995082 15 0.99771 0.986814 0.995788 20 0.998926 0.991085 0.998649 the statistical analysis made shows very strong correlations of the same sense between the reducing sugars content and storage duration at the heat levels of 6. 10. 15 and 20o c. at the temperature level of 2 oc the statistical calculation shows a strong correlation of opposite sense between the parameters analyzed. 4. conclusion after having analyzed the behavior of the cultivars selected at higher temperatures than 2oc we have drawn the conclusion that the lowest accumulation rate of reducing sugars was registered in the case of the cultivar victor stored at the temperature of 10oc. this characteristic can be used in practice namely by programming batches of the cultivar victor to get into production. at the end of the processing campaign having in view their capacity of preserving their technological capacity for longer storage periods of time. in the case of shorter time storage up to 20 days. the cultivar soleia showed reduced accumulation rates. thus there is the possibility of planning their processing in the first weeks after being harvested. in the case of the 2 oc –storage. a decrease of reducing sugars level in time was registered in all the cultivars analyzed. probably due to the invertase inhibition and consumption of the initial reserve of reducing sugars during metabolic processes. these results lead to the conclusion that temperature is an important factor regarding the maintenance of sugar beet storage quality, the low temperatures being mainly recommended as closer as to the cryoscopy point of sugar beet root. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 43 1. references [1]. joshi. s.s.. datir. s.s.. pawar. m.w.. nerkar. y.s.. sucrose metabolism in different sugar beet cultivars. sugar tech. 8(1). 69-73.[ 2006). [2]. berghall. s.. eronen. l. walliander. p.. long time storage of sugar beet under cold climatic conditions. zucherindustrie.121.939-943. (1996). [3]. haagenson. d.m.. klotz. k.l..campbell.l. impact of storage temperature. storage duration. and harvest date on sugarbeet raffinose metabolism. postharvest biology and technology 49.221-228. (2008). [4]. kenter. c.. hoffman. c.. changes in the processing quality of sugar beet (beta vulgaris l.) during long –term storage under controlled conditions. international journal of food science and technology. 44. 910-917. (2009). [5]. klotz. k.l.. finger. f.l.. impact of temperature. lenght of storage and postharvest disease on sucrose catabolism in sugarbeet. postharvest biology and technology 341-9. (2004). [6]. van der poel. p.w.. schiweck. h.. schwartz.. sugar technology. beet and cane sugar manufacture. berlin:dr. albertbartens kg. (1998). [7]. martin. s.s.. narum. j.a.. chambers. k.h.. sugarbeet biochemical quality changes during pile storage. journal of sugar beet research. vol 38. no1. 2001. 35-42. [8]. icumsa methods book. international commision for uniform methods of sugar analysis. verlag dr. albert bartens kg. berlin (2007). [9]. hedges. l.v.. olkin.i.. statistical methods for meta-analysis. hartcourt brace jovanovich publishers. new york. (1985). *octavian barna1, octavian baston1, aura darabă1 microsoft word 10 albu eufrozina 15 asprilie.doc 64 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 64 72 studies regarding the pretreatment with dilute acid and enzymatic hydrolysis of wheat straws for bioethanol production *eufrozina albu1, gheorghe gutt1, mircea -adrian oroian 1 1faculty of food engineering, stefan cel mare university of suceava, romania, e.albu@fia.usv.ro, g.gutt@fia.usv.ro, m.oroian@fia.usv.ro * corresponding author received 17 january 2013, accepted 20 february 2013 abstract: in this paper there has been studied the influence of dilute acid pretreatment upon the production of reducing sugars from wheat straws during the enzymatic hydrolysis. the acid pretreatment was led at different temperatures of 120 °c, 130 °c, 160 °c and 170 °c and at different acid concentrations (4%, 1%, 0.5% and without acid) before the enzymatic hydrolysis stage. there have been selected an optimal period of time for the pretreatment of 40 minutes and a temperature of 160 °c, combined with a temperature of 48 °c for the enzymatic hydrolysis. in optimal conditions it was achieved an increasement of the reducing sugars content of 63.37% in comparison with the control sample to which no preliminary pretreatment was applied. the results obtained indicate the fact that the dilute acid pretreatment could increase the ethanol concentration at the end of the fermentation stage. in this case the maximum ethanol concentration of 1.118% vol. was obtained in 72 hours of fermentation with s. cerevisiae. keywords: lignocellulose, cellulase, dns method, reducing sugar 1. introduction bioethanol is a fuel derived from renewable sources of feedstock; it is an alternative fuel produced almost entirely from food crops. it represents an important, renewable liquid fuel for motor vehicles [1]. the bioethanol produced as a transportation fuel can help to the reduce of co2 buildup in two important ways: by relacing the use of fossil fuels, and by recycling the co2 that is released when it is combusted as fuel. an important advantage of crop-based bioethanol is its ghg benefits [2]. bioethanol can be produced from different kinds of raw materials: sucrose-containing feedstocks (e.g. sugar cane, sugar beet, sweet sorghum and fruits), starch materials (e.g. corn, wheat, rice, sweet potatoes and barley) and lignocellulosic materials (e.g. wood, straw and grasses). currently, a focus is on bioethanol production from crops, such as corn, wheat, sugar cane, as well as on highly abundant agricultural wastes [1]. the challenge in producing ethanol from cellulose is the difficulty in breaking down cellulosic matter to sugars [3]. chemical composition of lignocellulosic materials is a key factor affecting efficiency of biofuel production during conversion processes. the structural and chemical composition of lignocellulosic materials is highly variable because of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 65 genetic and environmental influences and their interactions [4]. lignocelluloses consist mainly of cellulose, hemicellulose and lignin; these components build up about 90% of dry matter in lignocelluloses, with the rest consisting of extractive and ash [5]. ethanol production from lignocellulosic biomass comprises the following main steps: hydrolysis of cellulose and hemicellulose, sugar fermentation, separation of lignin residue and, finally, recovery and purifying the ethanol to meet fuel specifications [6, 7]. pretreatment of lignocellulosic biomass is an essential step for obtaining potentially fermentable sugars in the hydrolysis step. the aim of the pretreatment is to break down the lignin structure and disrupt the crystalline structure of cellulose for enhancing enzyme accessibility to the cellulose during hydrolysis step [8]. there are several different ways of pretreating biomass depending on its type and composition, as well as the processing technology that will be applied [9]. the goal of this study was to investigate the possibilities for increasing the yield of reducing sugars and of ethanol by applying a dilute acid pretreatment in the wheat straws bioethanol production using the shf (separate hydrolysis and fermentation) procedure. it was studied the efficiency of the dilute acid pretreatment upon the lignocellulosic structure disintegration and upon the destruction of the crystalline cellulosic structure to favourize the accessibility of the enzyme in the enzymatic hydrolysis process. it was also studied the influence of the saccharification temperature upon the content of the reducing sugars. 2. experimental 2.1 wheat straws the wheat straws (named triticum aestivum) used in this study were obtained from vrancea area farmers. the wheat straws were cut into pieces with a lenght of 5 cm, dried at air and grinded in a hammer mill until they got dimensions of 2 mm. 2.2 wheat straws pretreatment in order to destroy the crystalline structure of the cellulose and to increase the lignocellulosic material susceptibility to the enzyme attack, the wheat straws were submitted to a preliminary pretreatment. so, a dilute acid pretreatment was applied to the wheat straws using sulfuric acid of concentrations between 0.5 – 4%. 5 g of wheat straws grinded in the hammer mill and 100 ml sulfuric acid were mixed and pretreated at different temperatures between 120 –170 ºc, for 30 – 60 minutes. the working data in the case of the pretreatments applied were the following: pretreatment p1: h2so4 4% at a temperature of 120 ºc, for 60 minutes; pretreatment p2: h2so4 1% at a temperature of 130 ºc, for 50 minutes; pretreatment p3: h2so4 0.5% at a temperature of 160 ºc, for 40 minutes; pretreatment p4: water, at a temperature of 170 ºc, for 30 minutes; after pretreatment, the solid fraction was separated from the liquid fraction by filtration. the wash was realized to eliminate the inhibitors resulted after the pretreatment (furfural, hmf) and the resulted glucose that inhibits the enzymatic activity of the cellulase used in the following step [10]. 2.3 methods of analysis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 66 the raw material (wheat straws) used in the study was analyzed according to the analytical procedures. the water content of the raw material was determined by drying 5g of wheat straws at 105 ºc for 2 hours in drying vials. the samples were cooled then in a desiccator weighted and introduced again at 105 ºc until a constant weight [11]. the ash content was determined by calcination of 1g of sample at 575 ºc for 3 hours. the samples were cooled in the desiccator, weighted and introduced again in the calcination oven at 575 ºc until a constant weight [12, 13]. the wheat straws cellulose content was determined by using the method described by ishtiaq et al. [11]. the lignin content was determined according to the method described by nrel [14]. the quantification of the reducing sugars resulted in the enzymatic hydrolysis phase was realized using the dns method [15]. the enzymatic hydrolysis of the solid fraction resulted after the pretreatment was realized in a buffer solution of na2hpo4 0.2 m – citric acid 0.05 m with ph = 4.8 for 72 hours at temperatures between 40 – 50 °c. the enzymatic mixture used in this stage of the study was the onozuka r 10 cellulase obtained from trichoderma viride produced by merck company. the added quantity of enzyme was established according to the initial cellulose content as being of 15 unit/g cellulose. the reducing sugars production was evaluated at periods of 12 hours. 2.4 fermentation after enzymatic hydrolysis, 1 cm3 of suspension of 10% saccharomyces cerevisiae yeast and 2 ml nutrient for the yeast that contained 0.5 g/l (nh4)2hpo4, 0.025 g/l mgso4, 0.1 m nah2po4 [16] were inoculated in each sample and the fermentation was realized at 30 °c for 72 hours in recipients with a capacity of 1l. at each recipient there were coupled bluesens gas sensors to monitor the ethanol, oxygen and co2 content during the fermentation process. 3. results and discussion 3.1 the analysis of the raw materialswheat straws the composition of the wheat straws used in this study is: 38.09% cellulose, 13.82% acid insoluble lignin, 1.78% acid soluble lignin, 92.98 % dry substance and 6.82% ash. 3.2. the production of the reducing sugars at the enzymatic hydrolysis the reducing sugars content resulted from the enzymatic hydrolysis of the solid fraction from the pretreatment in different temperature conditions is shown in the figures 1 a-d. a 4.00 5.00 6.00 7.00 8.00 9.00 10.00 12 24 36 48 60 72 enzymatic hydrolysis range, h r ed uc in g su ga rs , g /l 40°c 42°c 44°c 46°c 48°c 50°c b 3.50 4.50 5.50 6.50 7.50 8.50 9.50 12 24 36 48 60 72 enzymatic hydrolysis range, h r ed u ci n g su ga rs , g/ l 40°c 42°c 44°c 46°c 48°c 50°c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 67 c 3.50 4.50 5.50 6.50 7.50 8.50 9.50 10.50 11.50 12 24 36 48 60 72 enzyma tic hydrolysis range , h r ed uc in g s ug ar s, g /l 40°c 42°c 44°c 46°c 48°c 50°c d 3.50 4.50 5.50 6.50 7.50 8.50 9.50 12 24 36 48 60 72 enzymatic hydrolysis range, h r ed u ci ng s u g ar s, g /l 40°c 42°c 44°c 46°c 48°c 50°c figure 1. the reducing sugars content in mash during enzymatic hydrolysis: apretreatment with h2so4 4%, for 60 minutes at 120 °c, enzymatic hydrolysis with onozuka between 40-50 °c, bpretreatment with h2so4 1%, for 50 minutes at 130 °c, enzymatic hydrolysis with onozuka between 40-50 °c, c pretreatment with h2so4 0.5%, for 40 minutes at 160 °c, enzymatic hydrolysis with onozuka between 40-50 °c, dpretreatment with water, for 30 minutes at 170 °c, enzymatic hydrolysis with onozuka between 40-50 °c. figure 1-a indicates the evolution of the reducing sugars content resulted after the enzymatic hydrolysis of the wheat straws pretreated with h2so4 4% at a temperature of 120 °c for 60 minutes. the enzymatic hydrolysis was done at temperatures between 40 – 50 °c. the reducing sugars content was determined at periods of 12 hours using the dns method. it can observe an increase of the reducing sugars content after each 12 hours of enzymatic hydrolysis. the hydrolysis temperature is also an important factor that favours the reducing sugars accumulation. it can be observed that at the temperatures of enzymatic hydrolysis of 46 °c and 48 °c a higher content of reducing sugars was obtained (9.12 g/l). in figure 1-b it is presented the evolution of the reducing sugars at the enzymatic hydrolysis of the wheat straws pretreated with h2so4 1% at a 130 °c for 50 minutes. during the 72 hours of enzymatic hydrolysis a higher accumulation of reducing sugars was registered at the temperatures of 46 °c (8.83 g/l) and of 50 °c (8.92 g/l). figure 1-c shows the evolution of the reducing sugars content at the enzymatic hydrolysis of the biomass resulted after the pretreatment with h2so4 0.5 % at a temperature of 160 °c for 40 minutes. they can observe significant increases of the reducing sugar at the hydrolysis temperature of 46 °c (9.71 g/l) and 48 °c (10.92 g/l). figure 1-d indicated the reducing sugars content resulted during the enzymatic hydrolysis of the wheat straws pretreated with water at 170 °c for 30 minutes. it can observe that the temperatures of 46 °c and 48 °c led to higher quantities of reducing sugars (8.82 g/l and 8.43 g/l). it can say that the applied pretreatments with h2so4 0.5 % at 160 °c for 40 minutes combined with a enzymatic hydrolysis temperature of 46 °c and 48 °c led to the obtaining of the highest reducing sugars quantity (9.71 g/l and 10.92 g/l). in figure 2 it is presented the content of the reducing sugars after 72 hours of enzymatic hydrolysis according to the applied pretreatment and temperature of hydrolysis in comparison with the control sample (which the enzymatic hydrolysis was done without a preliminary pretreatment). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 68 1.50 3.50 5.50 7.50 9.50 11.50 40 42 44 46 48 50 enzymatic hydrolysis te mpera ture , °c r ed u ci ng s ug ar s, g /l h2so4 4%, 60 min, 120°c h2so4 1%, 50 min., 130°c h2so4 0.5%, 40 min., 160°c water, 30 min., 170°c control figure 2. the reducing sugars content after 72 hours of wheat straws enzymatic hydrolysis figure 2 indicates that the pretreatment of the lignocellulosic material plays an important role in the production of the reducing sugars facilitating the enzyme accessibility to the cellulosic layer by destroying the crystalline structure of the cellulose. in optimal conditions, it was reached an increase of the reducing sugars content of 63.37% in comparison with the control sample that was not submitted to any preliminary pretreatment. 3.3 fermentation of wheat straws mash the process of fermentation of the wheat straws mash was done separately from the enzymatic hydrolysis phase, in fermentation cells with a capacity of 1 l. during the 72 hours of fermentation, the ethanol, oxygen and co2 content were monitored with the bluesens gas sensors. the figure 3 presents the ethanol content resulted after the fermentation of the hydrolyzed mashes at temperatures between 40 – 50 °c. figure 3 indicates that the best yield of reducing sugars conversion in ethanol was evaluated in the pretreated samples with h2so4 0.5% at a hydrolysis temperature of 46 °c and 48 °c (85.12% reported to the theoretical one). also there was obtained a good yield of conversion for the sample pretreated with h2so4 4% at a hydrolysis temperature of 42 °c (81.59% reported to the theoretical one) and for the sample pretreated with h2so4 1% at a hydrolysis temperature of 50 °c (81.59% reported to the theoretical one). 0 0.2 0.4 0.6 0.8 1 1.2 40 42 44 46 48 50 enzymatic hydrolysis temperature, °c e th an ol , % vo l h2so4 4%, 120°c/60 min h2so4 1%, 130°c/50 min h2so4 0.5%, 160°c/40 min water, 170°c/30 min figure 3. the ethanol content resulted after the fermentation of the hydrolyzed mashes at temperatures between 40–50 °c 4. parameter modelling data model regarding the prediction of sugar content, sugar content after fermentation, sugar content consumed at fermentation and ethanol obtained after fermentation using lignocellulosic materials in function of different factors (pretreatment temperature, pretreatment time, sulfuric acid concentration, hydrolysis temperature and hydrolysis time, the latter factor has been used only for sugar content prediction) has been made using a 3rd grade polynomial equation with 4 or 5 variables (using design expert 6.0 trial version). the measured and predicted values have been compared to see the suitability of the model. the equation 1 of the model is:  122 1 3 1 2 10    n ji jiij n ji jiij n ji jiij n kji kjiijk n i iiii n i iii n i ii xxbxxbxxbxxxbxbxbxbba where a is the parameter predicted, b0 is a constant that fixes the response at the central point of the experiments, bi – regression coefficient for the linear effect food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 69 terms, bij – interaction effect terms, bii – quadratic effect terms and biii – cubic effect terms. the operating region and the levels of the design variables (key factors) are given in actual and coded values as it is shown in table 1. table 1 correspondence between actual and coded values of design variables actual values of coded levels design variables symbol -1 +1 pretreatment temperature, °c x1 120 170 pretreatment time, min x2 30 60 sulfuric acid concentration, % x3 0 4 hydrolysis temperature, °c x4 40 50 hydrolysis time, min x5 12 72 in table 2 are presented the predicted models for the experimental data of lingocellulose samples. the coefficients of regression were superior of 0.903, and all models were significant (p<0.01). in figure 4 a–d are presented the predictability of the proposed models for the parameters. table 2 predicted models for the experimental data of lignocelluloses samples parameter equation r2 sugar, g/l 543542541 2 54 5 2 4 2 53 2 43 2 52 2 42 2 51 2 41 3 5 3 4545343524251 41 2 5 2 454321 23.076.049.003.0 41.030.009.034.017.222.038.2 22.010.117.025.004.035.162.086.0 4.017.039.058.159.144.060.259.182.6 xxxxxxxxxxx xxxxxxxxxxxxxxx xxxxxxxxxxxxx xxxxxxxxx sugar     0.903 sugar after fermentation, g/l 2 43 2 42 2 41 3 44342 41 2 44321 02.004.003.006.009.001.0 12.003.004.029.059.034.046.0 xxxxxxxxxxx xxxxxxx onfermentatiaftersugar   0.956 sugar consumed at fermentation, g/l 2 43 2 42 2 41 3 4 434241 2 44321 68.009.546.439.1 69.015.152.046.059.11.177.515.484.7 xxxxxxx xxxxxxxxxxx onfermentatiatconsumedsugar   0.937 ethanol, %vol 2 43 2 42 2 41 3 443 4241 2 44321 01.040.039.019.005.0 01.003.008.021.005.004.030.039.0 xxxxxxxxx xxxxxxxxxethanol   0.955 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 70 figure 4. the predictability of proposed models: a –sugar content after hydrolysis, g/l, b – sugar content after fermentation, g/l, c – sugar content consumed at fermentation, g/l, d – ethanol, %vol. the optimization of parameters simultaneously is very important for product quality. the desirability function approach is used to optimize the multiple characteristics concurrently. in the desirability function approach, first each characteristics, yi, is converted into an individual desirability function, di, that varies over the range, 10  id (2) if the characteristic yi is at its target, then di = 1. if the characteristic is outside an acceptable region, then di = 0. finally, the design variables can be chosen to maximize the overall desirability:   nnxdxxddd /1 21 ... (3) where n is the number of characteristics. when the target (t) for the characteristic y is a maximum value and the lower limit is noted by, l, ly 0 id tyllt ly r         (4) ty 1 when the target (t) for the characteristic y is a minimum value and the upper limit is denoted by u [17]. ty 0 id uyttu yu r         (5) uy 1 in this study, the desirability value of n was calculated by eq. 5, while desirability values of other characteristics were calculated by eq. 4, as other authors [18]. the exponent r is referred to the weight food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 71 and specified as 1. overall desirability, which represents, the desirability of all characteristics simultaneously (d), was calculated by eq. 3. ten of lignocelluloses combinations yielding the largest d values and predicted characteristic values are given in table 3 for sugar content after enzymatic hydrolysis, and in table 4 sugar content after fermentation, sugar content consumed at fermentation, sugar content and ethanol. making tradeoffs between these parameters were possible. table 3 desirability values and predicted characteristic values of ten combinations for sugar content after enzymatic hydrolysis no. x1 x2 x3 x4 x5 sugar after enzymatic hydrolysis, g/l desirability 1 0.72 0.94 -0.58 0.11 0.43 12.21300 1.000000 2 0.98 0.03 -0.78 -0.14 0.94 11.00950 1.000000 3 -0.04 0.99 0.73 0.26 0.86 11.37950 1.000000 4 0.83 0.53 0.85 0.56 0.98 11.49220 1.000000 5 -1.00 -0.88 -0.48 1.00 -1.00 10.01700 0.877667 6 -1.00 1.00 -1.00 0.20 0.87 9.94548 0.867874 7 1.00 -0.28 1.00 0.07 1.00 9.77736 0.844843 8 -0.72 -1.00 -0.55 1.00 -1.00 9.65469 0.828040 9 -1.00 -1.00 0.37 1.00 -0.58 9.63380 0.825178 10 0.99 -0.41 1.00 0.41 1.00 9.25547 0.773352 the parameters presented in the table 3 have been predicted keeping into account that the sugar content after hydrolysis should be maximum. table 4 desirability values and predicted characteristic values of ten combinations for sugar after fermentation, sugar consumed at fermentation and ethanol no. x1 x2 x3 x4 sugar after fermentation, g/l sugar consumed at fermentation, g/l ethanol, %vol. desirability 1 0.18 1.00 0.92 0.65 0.11725 12.0294 0.624413 1 2 0.83 -0.12 -0.66 0.36 0.060924 11.6928 0.644662 1 3 0.21 0.99 1.00 -0.52 0.042227 10.7233 0.594715 1 4 -0.03 0.91 0.67 -0.15 0.127307 11.7495 0.674899 1 5 -0.08 0.91 0.54 0.25 0.103458 12.4897 0.721032 1 the parameters presented in the table 4 have been predicted keeping into account that the sugar content after fermentation should be minimum, sugar consumed at fermentation and ethanol should be maximum. according to the optimization (presented in the table 4), the highest content of ethanol (0.721032%) can be obtained in the next condition: pretreatment temperature (143 °c), pretreatment time (58.65 min), sulfuric acid concentration (3.08%) and hydrolysis temperature (46.25 °c). 5. conclusions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 eufrozina albu, gheorghe gutt, mircea -adrian oroian, s t u d i e s r e g a r d i n g t h e d i l u t e a c i d p r e t r e a t m e n t a n d e n z y m a t i c h y d r o l y s i s o f w he a t s t r a w s f o r b i o e t h an o l p r o d u c t i o n , food and environment safety, volume xii, issue 1 – 2013, pag. 64 72 72 this study has analyzed the feasibility of the diluted acid pretreatment to enhance the release of reducing sugars during the enzymatic hydrolysis process using as a lignocellulosic material the wheat straws. at the same time it was pursued an increase of the yield of ethanol by applying the hydrolysis and fermentation systems separately (shf). the pretreatment with h2so4 0.5% at 160 °c for 40 minutes increased efficiently the reducing sugars content during the enzymatic hydrolysis phase that was realized at 48 °c/72 hours (with 63.37% in comparison with the control sample that was not preliminary pretreated). therefore this study shows that the pretreatment with h2so4 0.5% at 160 °c for 40 minutes enhanced the ethanol production in the fermentation phase with s. cerevisiae. the maximum ethanol concentration of 1.118% corresponds to a yield of 85.12% from the theoretical one (0.51 g/g). 6. references [1]. balat m., production of bioethanol from lignocellulosic materials via the biochemical pathway: a review, energy convers. manag., 52, 858-875, (2011) [2]. demirbas a., producing and using bioethanol as an automotive fuel, energy source, b 2, 391-401, (2007) [3]. xu f., shi y.-c., wu x., theerarattananoon k., staggenborg s., wang d., sulfuric acid pretreatment and enzymatic hydrolysis of photoperiod sensitive sorghum for ethanol production, bioprocess biosyst. eng., 34, 485-492, (2011) [4]. lee d., owens v.n., boe a., jeranyama p., composition of herbaceous biomass feedstocks, south dakota state university publication, sgin1-07, brooking sd, (2007) [5]. dehkhoda a., concentrating lignocellulosic hydrolysates by evaporation and its fermentation by repeated fedbatch using flocculating saccharomyces cerevisiae, industrial biotechnology boras university and sekab etechnology, sweden, (2008) [6]. alvira p., tomás-pejó e., ballesteros m., negro m.j., pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review, bioresour. technol., 101, 4851-4861, (2010) [7]. gil n., ferreira s., amaral m. e., domingues f. c., duarte a. p., the influence of dilute acid pretreatment conditions on the enzymatic saccharification of erica spp. for bioethanol production, ind. crop. prod., 32, 29–35, (2010) [8]. mosier n., wyman c.e., dale b.d., elander r.t., lee y.y., holtzapple m., ladisch c.m., features of promising technologies for pretreatment of lignocellulosic biomass, bioresour. technol., 96, 673-686, (2005) [9]. kang h.-k., kim d., efficient bioconversion of rice straw to ethanol with tio2/uv pretreatment, bioprocess biosyst. eng., 35, 43-48, (2012) [10]. vintila t., dragomirescu m., croitoriu v., vintila c., barbu h., sand c., saccharification of lignocellulose with reference to miscanthus – using different cellulases, rom. biotech. lett., 4, 15, (2010) [11]. ishtiaq a., muhammad a.z., hafiz m., nasir i., bioprocessing of proximally analyzed wheat straw for enhanced cellulase production through process optimization with trichoderma viride under ssf, int. j. biol. life sci., 6:3, (2010) [12]. moore w., johnson d., procedures for the chemical analysis of wood and wood products. madison, wi: u.s. forest products laboratory, u.s. department of agriculture, (1967) [13]. sluiter a., hames b., ruiz r., scarlata c., sluiter j., templeton d., determination of ash in biomass, nrel cat task laboratory analytical procedure, (2005) [14]. sluiter a., hames b., ruiz r., scarlata c., sluiter j., templeton d., crocker d., determination of structural carbohydrates and lignin in biomass, nrel cat task laboratory analytical procedure, (2011) [15]. miller g.l., use of dinitrosalicylic acid reagent for determination of reducing sugar, anal. chem. 31, 426, (1959) [16]. senila l., gog a., senila m., varaticeanu c., roman c., silaghidumitrescu l., irimie f.-d., bioethanol from woody biomass via steam explosion and acid hydrolysis, agriculture – science and practice, 3-4, 75-76, (2010) [17]. montgomery d.c., design and analysis of experiments (6th ed.), hoboken: wiley, (2005) [18] toker o.s., dogan m., camyilmaz e., imaz e., ersöz n.b., kaya y., the effects of different gums and their interactions on the rheological properties of a dairy dessert: a mixture design approach, food bioprocess tech., (2012). 123 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 123 129 anti m ic rob i al acti vit y of the pr ob iot ic s tr ai n lact ob a cillus d elbru e cki i ss p. bulg a r ic us gb of hum an origi n aga ins t pa tho gens *rositsa denkova1, zapryana denkova2, velichka yanakieva2, svetla ilieva1 1sofia university "st. kliment ohridski", faculty of biology, department of biotechnology, rositsa_denkova@mail.bg 2university of food technology, department of microbiology, zdenkova@abv.bg * corresponding author received may 9th 2013, accepted 15th june 2013 abstract: probiotics help restore the balance of the gastrointestinal microflora, but only strains with certain properties can be included in their formulations. one of the requirements for a strain to be probiotic is to exhibit inhibitory activity against pathogens.the antimicrobial activity of the strain lactobacillus delbrueckii ssp. bulgaricus gb against the pathogens e.coli atcc 25922, e.coli atcc 8739, salmonella abony ntcc 6017, salmonella sp., staphylococcus aureus atcc 25293 and proteus vulgaris j is determined by joint cultivation at 37±1°c. lactobacillus delbrueckii ssp. bulgaricus gb inhibits the growth of the pathogens for 60 72 hours the numbers of viable cells of the pathogens are reduced. it has been shown that the changes in the proportions in the mixed populations are due to the accumulation of lactic and other organic acids which acidify the medium and change the conditions for the growth of the pathogens, leading to reduction of the number of viable cells of the pathogens. the demonstrated inhibitory activity of lactobacillus delbrueckii ssp. bulgaricus gb makes the strain potentially probiotic and after additional studies it can be included in the composition of probiotics and functional foods. keywords: joint cultivation, probiotic, e.coli, salmonella, staphylococcus, proteus 1. introduction maintaining the balance of the gastrointestinal microflora is necessary for good health. in order to restore the balance of the intestinal microflora it is neccessary to consummate food and concentrates containing beneficial lactobacilli and bifidobacteria, known as functional foods and probiotics, respectively. according to fao probiotics are live microorganisms that have beneficial effect on the host when administered in adequate amounts [1, 2]. the main components of probiotics are lactic acid bacteria (lactobacillus, enterococcus, pediococcus, lactococcus, streptococcus, leuconostoc) and bifidobacteria. they are also applied in the manufacture of probiotic foods [3, 4, 5], the largest proportion being the lactobacilli. but only lactobacilli with certain properties can be included in the composition of probiotics and probiotic foods [4, 5]: to be a part of the natural microflora in humans and animals; to be able to suppress and expel pathogenic and toxicogenic microorganisms from the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 124 biological niche; to allow industrial cultivation; to have antimicrobial activity against conditionally pathogenic, carcinogenic and pathogenic microorganisms; to have the ability to adhere to epithelial cells or cell lines; to be able to survive under the conditions in the stomach and the intestines (acidic ph in the stomach and bile) [8, 9]; to be able to reproduce in the intestinal tract; to produce antimicrobial substances; to modulate the immune response and to be safe for clinical and food applications. the studies of saxelin et al., (1996 a, b), donohue & salminen, (1996), salminen et al., (1998) [3, 4, 5, 10, 11, 12] demonstrate the safety of lactic acid bacteria and bifidobacteria and strains belonging to the genera lactobacillus, lactococcus and bifidobacterium most often are assigned with gras status. the purpose of the present paper is to determine the antimicrobial activity of a probiotic strain of human origin lactobacillus delbrueckii ssp. bulgaricus gb against the following pathogens: e.coli atcc 25922, e.coli atcc 8739, salmonella abony ntcc 6017, salmonella sp., staphylococcus aureus atcc 25293 and proteus vulgaris j. 2.materials and methods 1. media sterile skimmed milk with titrable acidity 16-18ºt. composition (g/dm3): skimmed milk powder (scharlau). sterilization 15 minutes at 118ºc. saline solution. composition (g/dm3): nacl 5. sterilization 20 minutes at 121ºc. laptg10-broth. composition (g/dm3): peptone 15, yeast extract 10; tryptone 10, glucose 10. ph is adjusted to 6.6 6.8 and tween 80 1cm3/dm3 is added. sterilization 20 minutes at 121ºc. laptg10-agar. composition (g/dm3): laptg10-broth + 2% agar. sterilization 20 minutes at 121ºc. lbg-agar. composition (g/dm3): tryptone – 10, yeast extract 5, nacl – 10, glucose – 10, agar 20. ph is adjusted to 7.5. sterilization 20 minutes at 121°c. 2. determination of the antimicrobial activity against pathogenic microorganisms to determine the antimicrobial activity of lactobacillus delbrueckii ssp. bulgaricus gb against pathogens a 48 hour culture of the lactobacillus strain is used. in the mixtures are mixed 0.5 cm3 of the suspension of lactobacillus delbrueckii ssp. bulgaricus gb, 0.5 cm3 of the suspension of the pathogen and 9 cm3 of culture medium (skimmed milk), and in the controls of the lactobacillus strain or the pathogen 9.5 cm3 culture medium (skimmed milk) is mixed with 0.5 cm3 of the suspension of lactobacillus delbrueckii ssp. bulgaricus gb or of the pathogen, respectively. joint cultivation of lactobacillus delbrueckii ssp. bulgaricus gb and each of pathogens e.coli atcc 25922, e.coli atcc 8739, salmonella abony ntcc 6017, salmonella sp., staphylococcus aureus atcc 25293 and proteus vulgaris j under static conditions in a thermostat at 37±1°c for 72 hours, taking samples at the 0th, 12th, 24th 36th, 48th, 60th and 72nd hour is conducted and the change in the titratable acidity and the concentration of viable cells of the pathogen and of lactobacillus delbrueckii ssp. bulgaricus gb is monitored. 3. processing of the results data from triplicate experiments is processed using the software ms office excel 2003 and origin pro 8.1, using statistical functions to determine the standard deviation and maximum error of assessment in the significance level of α <0,05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 125 3. results and discussion the antimicrobial activity of lactobacillus delbrueckii ssp. bulgaricus gb against the pathogens escherichia coli atcc 25922, escherichia coli atcc 8739, salmonella sp. (clinical isolate), salmonella abony ntcc 6017, staphylococcus aureus atcc 25293, proteus vulgaris j during joint and separate cultivation at 37±1ºc in skimmed milk is determined. during separate cultivation lactobacillus delbrueckii ssp. bulgaricus gb and the pathogens e.coli atcc 25922 and e.coli atcc 8739 for 12 to 24 hours at 37±1ºc accumulate high concentrations of viable cells. these high concentrations of living cells preserve lactobacillus delbrueckii ssp. bulgaricus gb in a mixed population with the pathogen. in joint cultivation of the lactobacillus strain and e.coli atcc 25922 at static conditions, an increase in the concentration of viable cells of lactobacillus delbrueckii ssp. bulgaricus gb is detected, while that of e.coli atcc 25922 starts to decrease after the first 12 hours and at the 60th hour no viable cells of the pathogen are established (fig. 1). fig. 1. survival of lactobacillus delbrueckii ssp. bulgaricus gb and e.coli atcc 25922 during separate cultivation and cultivation in a mixed population at 37±1ºc. in the study of the inhibitory activity of lactobacillus delbrueckii ssp. bulgaricus gb against e.coli atcc 8739 the concentrations of viable cells of lactobacillus delbrueckii ssp. bulgaricus gb and of the pathogen e.coli atcc 8739 increase in the first 12 hours, which is consistent with the results obtained with e.coli atcc 25922. after that the concentration of viable lactobacilli cells retains while that the pathogen is quickly reduced and at the 60th hour there are no living cells of the pathogen, similarly to the joint cultivation with e.coli atcc 25922 (fig. 1 and fig. 2). fig. 2. survival of lactobacillus delbrueckii ssp. bulgaricus gb and e.coli atcc 8739 during separate cultivation and cultivation in a mixed population at 37±1ºc. in tracking the change in the titratable acidity it is noticeable that the acidity values of both the two controls of the pathogens are significantly lower in comparison to the control of the lactobacillus strain and of that of the two mixtures (lactobacillus delbrueckii ssp.bulgaricus gb and e.coli atcc 25922; lactobacillus delbrueckii ssp. bulgaricus gb and e.coli atcc 8739) and the titratable acidity values of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 126 mixtures are lower than the values of the control of lactobacillus delbrueckii ssp.bulgaricus gb for each hour of sampling, which shows that the inhibition of the pathogen by lactobacillus delbrueckii ssp.bulgaricus gb is due to a great extent to the acidification of the medium (fig. 3). fig. 3. change of the titratable acidity of the medium during separate cultivation and cultivation in a mixed population of lactobacillus delbrueckii ssp. bulgaricus gb and e.coli atcc 8739 or e.coli atcc 25922 at 37±1ºc. during the cultivation of lactobacillus delbrueckii ssp. bulgaricus gb and salmonella sp. (clinical isolate) at 37±1°c, an increase in the concentration of viable cells of l.d.ssp.bulgaricus gb as well as of salmonella sp. during the first 12 hours is observed, after which the concentration of viable lactobacilli cells continues to increase, while that of the pathogen is reduced and by the 72nd hour no living cells of salmonella sp. are defined (fig. 4). fig. 4. survival of lactobacillus delbrueckii ssp. bulgaricus gb and salmonella sp. during separate cultivation and cultivation in a mixed population at 37±1ºc. in the joint development of salmonella abony ntcc 6017 and lactobacillus delbrueckii ssp. bulgaricus gb an increase of the cell concentration of the lactobacillus strain and of the pathogen in the first 12 hours is observed. fig. 5. survival of lactobacillus delbrueckii ssp. bulgaricus gb and salmonella abony ntcc 6017 during separate cultivation and cultivation in a mixed population at 37±1ºc. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 127 fig. 6. change in the titratable acidity of the medium during separate cultivation and cultivation in a mixed population of lactobacillus delbrueckii ssp. bulgaricus gb and salmonella abony ntcc 6017 or salmonella sp. at 37±1°c. from the 12th to the 60th hour the concentration of lactic acid bacteria continues to increase while that of salmonella abony ntcc 6017 decreases reaching 0 at the 72nd hour as in the study of the antimicrobial activity of lactobacillus delbrueckii ssp. bulgaricus gb against salmonella sp. (fig. 4 and fig. 5), which is mainly a result of the acidification of the medium, due to the accumulation of lactic acid and other organic acids (fig. 6). in the separate cultivation of lactobacillus delbrueckii ssp. bulgaricus gb and staphylococcus aureus atcc 25293 both strains accumulate high concentration of active cells. during the joint cultivation lactobacillus delbrueckii ssp. bulgaricus gb and staphylococcus aureus atcc 25293 a decrease in the concentration of the pathogen, starting after 12th hour is observed. as in the determination of the antimicrobial activity against the two strains of escherichia coli, salmonella sp. and salmonella abony ntcc 6017, the number of living cells of this pathogen is reduced completely under the action of lactobacillus delbrueckii ssp. bulgaricus gb. at the end of the process no living cells of the pathogen are detected (fig. 7), mainly due to the lowering of the ph as a result of the increase in the titratable acidity (fig. 8). fig. 7. survival of lactobacillus delbrueckii ssp. bulgaricus gb and staphylococcus aureus atcc 25293 during separate cultivation and cultivation in a mixed population at 37±1ºc. fig. 8. change in the titratable acidity of the medium during separate cultivation and cultivation in a mixed population of lactobacillus delbrueckii ssp. bulgaricus gb and staphylococcus aureus atcc 25293 at 37±1°c. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 128 in studying the antimicrobial activity of lactobacillus delbrueckii ssp. bulgaricus gb against proteus vulgaris j the concentration of viable cells of lactobacillus delbrueckii ssp. bulgaricus gb and of the pathogen increases in the first 12 hours. after that the number of living cells of the lactobacillus strain continues to grow at a slower rate, while the cell number of the pathogen is reduced, reaching 0 at the 60th hour (fig. 9). fig. 9. survival of lactobacillus delbrueckii ssp. bulgaricus gb and proteus vulgaris j during separate cultivation and cultivation in a mixed population at 37±1ºc. for each sampling the acidity of the control of the pathogen is lower than that of the control of the lactobacillus strain and that of the mixture, which again indicates that the inhibition of pathogens by lactobacilli is largely a result of the decrease of the ph of the medium, resulting from the accumulation of organic acids produced by the lactobacilli (fig. 10). fig. 10. change in the titratable acidity of the medium during separate cultivation and cultivation in a mixed population of lactobacillus delbrueckii ssp. bulgaricus gb and proteus vulgaris j at 37±1ºc. 4. conclusion the strain lactobacillus delbrueckii ssp. bulgaricus gb inhibits the growth of the pathogens e.coli atcc 25922, e.coli atcc 8739, salmonella abony ntcc 6017, salmonella sp., staphylococcus aureus atcc 25293 and proteus vulgaris j. in joint cultivation of lactobacillus delbrueckii ssp. bulgaricus gb and all of the pathogens the lactobacillus strain retains a high concentration of viable cells, while the cell number of the pathogen is reduced, the degree of reduction being strainspecific and partly due to the change in the acidity of the medium as a result of the acid production by lactobacillus delbrueckii ssp. bulgaricus gb. the antimicrobial activity against pathogens makes the tested strain a potentially probiotic one, which after further studies may be included in the composition of probiotic preparations for prophylaxis and treatment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 rositsa denkova, zapryana denkova, velichka yanakieva, svetla ilieva , antimicrobial activity of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus gb of human origin against pathogens, food and environment safety, volume xii, issue 2 – 2013, pag. 123 129 129 5. references [1] food and agriculture organization of the united nations (fao). health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. (2001) [2] food and agriculture organization of the united nations (fao). guidelines for the evaluation of probiotics in food. (2002) [3] salminen s, ouwehand ac, isolauri e, clinical applications of probiotic bacteria. int. dairy j. 8: 563-572, (1998) [4] salminen s, von wright a, current probiotics safety assured, scandinavian university press, issn 0891-060x, (1998) [5] salminen s, bouley mc, boutron-rualt mc, cummings j, franck a, gibson g, isolauri e, moreau m-c, roberfroid m, rowland i, functional food science and gastrointestinal physiology and function. br. j. nutr., suppl. 1: 147-171, (1998) [6] wolfson np, a probiotics primer. nutrition science news 4 (6): 276-280, (1999) [7] gibson gr, from probiotics to prebiotics and a healthy digestive system. j.of food science 69 (5): 141 – 143, (2004) [8] kashtan h et al., manipulation of faecal ph by dietary means. prev. med. 19 (6): 607-613, (1990) [9] segal i. et al., faecal short chain fatty acids in south african urban africans and whites. dis. colon rectum 38 (7): 732734, (1995) [10] saxelin m, rautelin h, chassy b, gorbach sl, salminen s, makela h, lactobacilli and septic infections in southern finland. clinical infectious diseases 22, 564-566, (1996a) [11] saxelin m, salminen s, the safety of commercial products with viable lactobacillus strains. infectious diseases clinical practice 5: 331-335, (1996b) [12] donohue dc, salminen s, safety assessment of probiotic bacteria. asia pac. j. clin. nutr. 5: 25-28, (1996) 108 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 2 2016, pag. 108 116 physico-chemical characteristics, antioxidant activity and mineral content of hawthorn fruits from suceava county *silvia mironeasa1, elena sănduleac todosi1, mădălina iuga1 1 faculty of food engineering, stefan cel mare university, suceava, universitatii str. 13, suceava, romania, silviam@fia.usv.ro *corresponding author received 15 th april 2016, accepted 25 th may 2016 abstract: the study of local hawthorn fruits is of particular interest as they often contain high amounts of bioactive compounds. the objective of this research was to determine the physicochemical characteristics of hawthorn ripened fruits, their color using cielab system and their antioxidant capacity by dpph method, based on scavenging 2,2-di (4-tert-octylphenil)-1picrylhydrazyl (dpph) radicals. in addition, the content in mineral elements of hawthorn fruits was determined by inductively coupled plasma mass spectrometer (icp-ms). the result showed that hawthorn fruits have a pulp / kernel ratio of 1.44. the moisture, acidity, total soluble solids content, ph, pectic substances, protein, fat, ash, carbohydrates and energy values of hawthorn fruits were found as 69.14%, 0.47%, 22.20%, 5.96, 1.01%, 3.5%, 0.80%, 1.75%, 24.81% and 123.48 kcal/100g, respectively. hawthorn fruits ripened (maturated) have a bright red color, an ic50 value of 53.47 g/ml and contain high amounts of ca (580.43 ppm), mg (278.31 ppm) and na (264.04 ppm). it is very important to evaluate the physico-chemical characteristics for the storage and processing of fresh fruits. this study revealed that hawthorn fruits from the local area should be considered a healthy product due to their bioactive compounds and are more suitable products than marmalade, jams, jelly, beverages and other food products. keywords: hawthorn fruits, chemical composition, colour, antioxidant activity, mineral elements 1. introduction hawthorn is a wild plant species from the rose family which includes over 280 species [1-4]. comparatively with north america that is the center of distribution and diversity for the genus crataegus spp., in europe and asia there have been recognized more than 60 species [5]. crataegus monogyna and crataegus laegivata is the hawthorn species found in europe, and romania, respectively. hawthorn has edible fruits of rounded, oblong or pear-shaped form, relatively small and range from orange-yellow, scarlet, red, yellow, blue, to black color. the flesh is mealy and dry, like those of rose hips [2]. crataegus monogyna plant belongs to the rosaceae family and is a small tree with thorny branches, green leaves, white flowers and bright red berries. the fruit has one woody kernel and is fully ripened in the early to midautumn [4; 6]. the color of ripe hawthorn fruits ranges from yellow, to green, red and dark purple [6; 7]. this is due to the constituents like carotenoids and anthocyanin and it is influenced by the maturity level [8]. mailto:silviam@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 109 hawthorn was used in the traditional medicine of many cultures since antiquity, especially for its cardiovascular effects [9; 10]. in addition, hawthorn berries have been used as astringent, antispasmodic, diuretic, and antiatherosclerotic agents [10]. in traditional chinese medicine, hawthorn fruits are used as a peptic agent for stimulating digestion and promoting the function of the stomach, improving blood circulation, and removing blood stasis. beneficial effects in the treatment of indigestion with epigastric distension, diarrhea, and abdominal pain, as well as amenorrhea, hypertension, and hyperlipidemia have been reported on the fruits preparations [4]. hawthorn extracts have been shown to increase myocardial contractility, reduce reperfusion arrhythmias, dilate peripheral arteries, and mildly decrease blood pressure [11-13]. these beneficial effects are attributed to the active constituents of hawthorn such as flavonoids and proanthocyanidins [4; 1416]. in actual herbal medicine, hawthorn is used for its hypotensive, cardio-tonic, coronary-dilative, and anti-arrhythmic action due to the bioactive compound such as flavonoids, organic acids, procyanidins [17]. hawthorn fruits or berries have diuretic and antidiarrheal effect [18]. also, hawthorn fruits contain compounds with antioxidant and antilipoperoxidant activity such as epicatechin, hypersoide and chlorogenic acid. various studies confirmed the potential of hawthorn fruits as a good source of compounds with free radical scavenging activity [19; 20]. some studies revealed that hawthorn berries are considered to be healthy and nutritious [21] and are consumed by children as they believe it is a good vitamin supplier [22; 23]. there are many studies that revealed the multiple health-promoting effects of hawthorn fruits on the human body. investigations on chemical composition of hawthorn fruits highlight that they are rich source of protein, fat, carbohydrates and mineral elements [6; 21; 24]. in addition, hawthorn fruits contain vitamins b1, b2, b6 and c, aminoacids, sugars, -carotene, organic acids, antioxidants, and phenolic compounds such as phenolic acids, flavones, flavonols and proanthocyanidins [1; 6; 21; 25-27]. hawthorn fruits are an excellent food source of dietary fiber and have higher pectin content than other available fruits [28]. pectin is a widely used food additive that functions as thickener, gelling agent, and food fiber human health supplement. dietary supplementation with pectin may reduce serum total cholesterol levels, decreases low density lipoprotein cholesterol, and moderates the glucose response [28]. recent studies have demonstrated that hawthorn fruits are increasingly widely used as medicine and as foodstuff due to their important role in human health and nutrition. hawthorn fruits are particularly used in herbal tea and juices production [4; 21] or to produce canned fruits, jams, jellies, alcoholic beverages and other drinks [7; 29; 30]. accordingly, hawthorn species used in this study are abundantly grown in romania and the fruits are becoming more popular. the study aims to investigate some physical and chemical characteristics of hawthorn fruits. the antioxidant activity and mineral elements of hawthorn fruits were also analyzed. this study wants to underline the importance and the potential of these hawthorn berries in order to be valorized in products such as marmalade, jams or jelly. 2. materials and methods 2.1. materials wild hawthorn fruits were collected after ripening, during the harvest season in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 110 2015 from gura humorului area, suceava county, romania. the pulp was separated from the kernel manually after the fruits were selected and washed in running water. 2.2. methods 2.2.1. physico-chemical analysis the hawthorn fruits samples were analyzed to determine the physicochemical properties according to romanian or international standards methods: moisture content and volatile matter (sr iso 1026:2008), total acidity (sr iso 750:2008), soluble solids content (sr iso 2173:2008), ph (sr iso 1842:2008), fat content (sr en iso 659:2009), protein content (sr en iso 20483:2007), total ash content (sr iso 763:2008). the pulp / kernel ratio was determined by gravimetric methods after pulp separation. the pectic substances content of berries was achieved by the gravimetric determination of calcium pectate according to the method described in the literature [31]. total carbohydrates from hawthorn fruits were calculated by difference: carbohydrates content (%) = 100 – (amount in percentage of protein, fat, moisture and ash). the total energy was calculated using the following equation: energy (kcal/100g) = 4.1 x (g protein + g carbohydrates) + 9.3 x (g fat). 2.2.2. colour analysis the colour of hawthorn fruits was measured by reflectance spectroscopy using an ocean optics spectrometer (usa). the samples were placed in 20mm thick holders and measured against a black and white background. translucency was determined by applying the kubelka– munk theory for multiple scattering to the reflection spectra [32]. colour coordinates were determined using the cielab colour system where the l* value is a measure of lightness ranging from 0 (black) to 100 (white), the a* value ranges from -100 (greenness) to +100 (redness), and the b* value ranges from -100 (blueness) to +100 (yellowness). the coordinates l*, a* and b* were obtained from r∞ between 400 and 700 nm for d65 illuminant and from 2° observer [33]. 2.2.3. dpph radical-scavening activity the antioxidant capacity of hawthorn fruits was achieved by dpph method, based on scavenging 2,2-di (4-tertoctylphenil)-1-picrylhydrazyl (dpph) radicals using a t70 uv-vis spectrophotometer (pg instruments ltd.). free radical scavenging activity was determined according to the method described by ercisli et al. (2015) with some modifications.various concentrations of hawthorn fruit extract (from 10 to 240 μl) were added to freshly prepared dpph solution (500 μl of 0.004% dpph) in methanol, and then the mixture was left to stand for 30 min in the dark. inhibition of the free radicals, dpph as a percentage, was determined by measuring the absorption at 517 nm against a blank assay. lower absorbance of the reaction mixture indicated higher free radicals scavenging activity in hawthorn fruits. the inhibition, as percentage, is calculated as the absorbance of the sample divided by that of dpph control at the same time multiplied by 100 (eq.1). i (%) = [1 – (a1/ao)] x 100 (1) where ao is the absorbance of the control reaction and a1 is the absorbance in the presence of all of the extract samples and standard. the ic50 value was determined by linear regression analysis using a calibration curve with different amounts of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 111 dpph. antioxidant activity, expressed as the ic50 is the scavenging concentration (gml-1) of the extract which quenched 50% of the initial dpph radicals under the experimental conditions given. 2.2.4. minerals content analysis the mineral elements from hawthorn fruits were determined by icp-ms technique. the method is based on ash digestion with concentrated nitric acid (65% hno3, sigma aldrich, germany) and hydrogen peroxide (30% h2o2 pure p.a, sigma aldrich, germany), sample dilution and ions quantitative determination using icp-ms equipment. the element standard solutions were prepared by diluting a stock solution of 1000 mg/l of ag, al, as, ba, be, ca, cd, co, cr, cs, cu, fe, ga, k, li, mg, mn, na, ni, pb, rb, se, sr, tl, u, v and zn. all reagents were of analytical grade. an agilent technologies 7500 series system coupled plasma-mass spectrometer with a detection limit of 10-12 was used for efficiently detecting, identifying and reliably quantifying heavy metals and trace elements from hawthorn fruits. the icp-ms parameters used for analysis have been: nebulizers 0.9 ml/min, rf power 1500 w, carrier gas 0.92 l/min, mass range 7-205 uma, integration time 0.1 s, acquisition 22.7 s. detector parameters have been: discriminator 8 mv, analogue hv 1770 v and pulse hv 1070 v. 2.2.5. statistical analysis all the analytical determinations were performed at least three times and the value reported for characteristics determined is the average  the standard deviation (s.d.) value. statistical analyses were performed on statistical software microsoft excel version 2003. 3. results and discussion 3.1. physico-chemical characteristics of hawthorn berries the mean, maximum and minimum values, as well as standard deviations are showed in table 1. the average pulp / kernel ratio of hawthorn fruits varied between 1.32 and 1.55. the pulp / kernel ratio of our study was lower than those of the study carried out in some regions from turkey [35; 36]. the moisture and volatile content of hawthorn fruits, a very important parameter when determining the physical properties of fruits, indicates a mean value of 69.14g/100g fresh weight. the moisture content found can help to suggest the stability in storage of fruits, as higher the moisture content is, more the risk of spoilage of berries. the value obtained for moisture content is close to those obtained by özcan et al. (2005) from hawthorn fruits grown in turkey (64.26 g/100g) or by erfani-moghadam et al. (2016) from hawthorn fruits grown in different geographical sites from iran (56.88 g/100g). the result for titratable acidity of the hawthorn fruits, calculated as 0.46% mean value is in agreement with the results reported by türkoğlu et al. (2005) and erfani-moghadam et al. (2016). the total soluble solid value was at mean value of 22.19%, while the ph values of hawthorn fruits (from 5.89 to 5.99) indicating are relatively less acidic than peach, apples and pears. in similar research on hawthorn fruits from different regions of turkey, balta et al. (2006), ozcan et al. (2005) and türkoğlu et al. (2005) reported that total soluble solid and ph values ranged from 11.66 to 24.00% and 3.12 to 4.09, respectively. the values obtained for ph are higher than those reported by previous mentioned studies. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 112 the pectic substances estimated as calcium pectate revealed that hawthorn fruits are a good source of pectic substances, with content varying in the ranges of 1.00 1.03 %. a study about this complex polysaccharide revealed that the pectin pentaoligosaccharide from hawthorn was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor [39]. table 1. chemical characteristics of hawthorn fruits parameter max. mean min. s.d. (±) pulp/kernel ratio (%) 1.55 1.44 1.32 0.12 moisture content (%) 70.13 69.14 68.00 1.07 acidity (%, malic acid) 0.48 0.47 0.46 0.01 total soluble solids (%) 22.90 22.20 21.23 0.86 ph 6.00 5.96 5.90 0.06 pectic substances content (%) 1.03 1.01 1.00 0.02 fat content (%) 0.98 0.80 0.61 0.19 protein content (%) 3.65 3.50 3.35 0.15 ash (%) 1.92 1.75 1.58 0.17 total carbohydrates (%) 25.60 24.81 24.03 0.78 energy (kcal/100g) 122.87 123.48 119.16 4.67 fat was the less abundant macronutrient being at mean vale of 0.80g/100g dry weight (dw) for hawthorn fruits, while the protein content was found at a mean value of 3.50g/100, in agreement with the result obtained by baros et al. (2010). the protein content was found similar to those reported by other researchers [6; 24]. ash dried hawthorn fruits falls between proteins and fat contents, with a mean vale of 1.75g/100g. similar values were found for hawthorn fruits from turkey: fat 0.87%, protein 2.48% and ash 2.28% [18]. regarding the carbohydrates content (24.81%), obtained by difference, were the most abundant macronutrient, these wild fruits can have a significant contribution to daily diets, especially during autumn and early winter. in addition, hawthorn berries presented a high energetic value (123.48 kcal/100g) but it is higher than the value reported by özcan et al. (2005) from turkey hawthorn fruits (34.02 kcal/100g). the chemical composition of hawthorn fruits is influenced by genetic factors, state of ripening, soil structure, climatic factors and other environmental factors [1]. 3.2.2. the colour the colour and appearance represent essential attributes considered by the consumer to decide the acceptability and overall quality of food [40; 41]. anthocyanins are the phytochemicals substances which can influence the red colour of hawthorn fruits [42]. also, the chemical composition and variety are some factors that can influence the colour of fruits. the cie l*a*b* values for fresh hawthorn fruits showed that the samples had a mean value of 62.12 for l*, while a* and b* were 3.30 and 3.86, respectively. the hawthorn fruits were red in colour with a slight dark red tinge, which is indicated by the positive b* value. the positive a* value with lower lightness values in hawthorn fruits shows that these fruits have darker red colour. the hue angle (eq. 2) and chrome (eq. 3) were calculated using the values of a* and b*. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 113 * * * ab a b arctanh  (2) 2*2** ab bac  (3) the results showed a chrome value of 5.08, while the hue angle value is 0,86, indicating the presence of different pigments in fruits, such as oligomeric procyanidins, according to result reported by liu et al. (2011). hawthorn fruits have a red brighter colour that may be related to their richness in anthocyanins. high clearness of fruits (lightness values) can be explained by low intensity of the phenomenon of nonenzymatic browning (reactions of maillard) due to the high content of water and the short period of storage of these samples. the duration and the temperature of storage are some factors that influence the degradation of colour and pigment. 3.2.3. dpph radical-scavening activity in dpph assay, the hawthorn fruits had the highest 1,1-diphenyl-2picrylhydrazyl radical scavenging capacity 53.47 (gml -1 ). this result revealed that the hawthorn fruits have considerable antioxidant potential that can be due to their polyphenolic compounds [25; 43]. the studied hawthorn fruits highlight a similar dpph scavenging activity with the hawthorn berries from serbia (52.04 (gml-1) studied by tadić et al. (2008). 3.2.4. minerals content the composition of major and trace mineral elements of the hawthorn fruits is given in table 2. hawthorn fruits are rich in both macroand micronutrients, providing a nutritionally significant content of minerals. calcium (ca), magnesium (mg) and sodium (na) were the predominant elements in the hawthorn berries pulp, followed by aluminum (al), barium (ba), manganese (mn), chromium (cr) and selenium (se). ca, mg and na were found also in rose fruits samples as the major elements [24]. ca has an essential role in the organism; containing substances which are required by children, pregnant and lactating women for bones and teeth development [44; 45]. mg plays an essential role in muscle relaxing along the airway to the lungs thus, allowing asthma patients to breathe easier [46]. mn is believed to support the immune system, regulates blood sugar levels and is involved in energy production and cell reproduction [47]. table 2. mineral contents of hawthorn fruits minerals values (ppm) minerals values (ppm) al 47.02 li 1.08 as 0.30 mg 278.31 ba 11.30 mn 8.82 be 5.85 na 264.04 ca 580.43 ni 3.29 cr 7.08 se 4.94 cu 3.20 tl 0.02 fe 0.77 v 0.76 ga 0.03 zn 0.87 also, essential elements for human body like cupper (cu) iron (fe) and zinc (zn) were found in hawthorn fruits. deficiency of cu can cause cardiovascular disorders as well as anemia and bone and nervous systems disorders [48]. fe is an important mineral element in the diet of pregnant women, nursing mothers, infants convulsing patients and elderly to prevent anemia and other related diseases [49]. zn, an essential trace element for protein and nucleic acid synthesis and normal body food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 114 development, is said to be vital during periods of rapid growth and during recovery from illness [50]. other elements with beneficial pharmacological properties that were found in hawthorn fruits are barium (ba), nickel (ni) and lithium (li), which have not been established as essential. for example, li was used in the treatment of manic depressive disorders [51]. hawthorn fruits are a good source of macro and micronutrients which are expected to speed up metabolic processes, improve growth and development. the content of mineral elements from hawthorn fruits is influenced by various factors such as variety, growing condition, geographical origin, soil type, storage condition, climatic conditions e.g. the analytical values of hawthorn fruits revealed the nutritional properties and mineral contents of hawthorn fruits. these results may be useful for the food industry, for many foodstuffs preparation, such as jam, jelly, etc. and for development of new products based on hawthorn fruits. 4. conclusion regarding the chemical composition, the results showed that hawthorn fruits could be considered as a potential source of bioactive compounds with high antioxidant activity, having many health benefits. hawthorn fruits are an energetic food that may be consumed such as or could be used in the manufacture of value-added products such as jelly, marmalade, jams, wine and other drink with commercial value. the knowledge of the chemical composition of this fruits will encourage the manufacturers to develop the production of hawthorn jelly on an industrial scale. the use of hawthorn fruits may also be attractive to consumers as a positive alternative to conventional fruits in jelly production. 5. references [1] gundogdu m., ozrenk k., ercisli s., kan t., kodad o., hegedus a., organic acids, sugars, vitamin c content and some pomological characteristics of eleven hawthorn species (crataegus spp) from turkey, biological research, 47: 21, (2014) [2] cseke jl., kirakosyan a., kaufman pb., warber sl., duke ja., brielmann hl., natural products from plants, in 2nd edition, crc press taylor and francis group, (2006) [3] hobbs c, foster s., hawthorn: a literature review, herbal gram, 22: 19-33, (1990) [4] chang q., zuo z., harrison, f., chow m.s.s., hawthorns: an overview of chemical, pharmacological and clinical studies, journal of clinical pharmacology, 42: 605-612, (2002) [5] sticher o., b. meier., hawthorn (crataegus): biological activity and new strategies for quality control, in phytomedicines of europe, chemistry and biological activity (acs symposium series 691), l.d. lawson and r. bauer (eds.). american chemical society, washington, d.c., pp. 241–262, (1998) [6] özcan m., mineral contents of some plants used as condiments in turkey, food chemistry, 84: 437-440, (2004) [7] unal h. g., sacilik k., drying characteristics of hawthorn fruits in a convective hot-air dryer, journal of food processing and preservation, 35(2): 272–279, (2011) [8] xiu-juan q., shan-kun x., zuo-xuan l., zhang l., dynamic chances of mineral elements and skin pigments of hawthorn fruits during its development process and their correlation, journal of fruit science, 02, (2006) [9] jorikova t., sochor j., rop o., mlcek j., balla s., szekeres l., adam v., kizek r, polyphenolic profile and biological activity of chinese hawthorn (crataegus pinnatifida bunge) fruits, molecules, 17: 14490-14509, (2012) [10] tadić vm., dobrić s., marković gm., dordević sm., arsić ia., menković nr., stević t., anti-inflammatory, gastroprotective, free-radicalscavenging, and antimicrobial activities of hawthorn berries ethanol extract, journal of agricultural and food chemistry, 56: 7700–7709, (2008) [11] von eiff m., brunner h., haegeli a., kreuter u., martina b., meier b., schaffner w., hawthorn/passionflower extract food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 115 and improvement in physical capacity of patients with dyspnoea class ii of the nyhm functional classification, acta scientiarum polonorum. hortorum cultus, 20: 47-66, (1994) [12] zhang z., chang q., zhu m., huang y., ho w. k. k., chen z. y., characterization of antioxidants present in hawthorn fruits, journal of nutritional and biochemistry, 12: 144-152, (2001) [13] weihmayr t., emst e., therapeutic effectiveness of crataegus, fortschritte der medizin, 114: 27-29, (1996) [14] edwards j. e., p. n., brown n. talent, t. a. dickinson, p. r. shipley., a review of the chemistry of the genus crataegus, phytochemistry, 79: 5–26, (2012) [15] liu p., h. kallio, b. yang., phenolic compounds in hawthorn (crataegus grayana) fruits and leaves and changes during fruits ripening, journal of agriculture and food chemistry, 59: 11141–11149, (2011) [16] cui n., k. nakamura, s. tian, h. kayahara, y. tian., polyphenolic content and physiological activities of chinese hawthorn extracts, bioscience, biotechnology and biochemistry, 70: 2948–2956, (2006) [17] khishova o. m., buzuk g. n., quantitative determination of procyanidins in hawthorn fruits, pharmaceutical chemistry journal, 40: 20-21, (2006) [18] özcan m., haciseferogullari h., marakoglu t., arslan d., hawthorn (crataegus spp.) fruit: some physical and chemical properties, journal of food engineering, 69: 409413, (2005) [19] bahorun t., greiser b., oxygen species scavenging activity of phenolic extracts from hawthorn fresh plant organs and pharmaceutical preparations, arzneimittel-forschung/drug research, 46: 1086–1089, (1996) [20] bahorun t., trotin f., antioxidant activities of crataegus monogyna extracts, planta medica, 60: 323–326, (1994) [21] barros l., carvalho a.m., ferreira i.c.f.r., comparing the composition and bioactivity of crataegus monogyna flowers and fruits used in folk medicine, phytochemical analysis, 22: 181–188, (2010) [22] carvalho am., etnobotánica del parque natural de montesinho. plantas, tradición y saber popular en un territorio del nordeste de portugal, universidad autónoma, madrid, (2005) [23] neves jm., matosa c., moutinho c., queiroz g., gomes lr., ethnopharmacological notes about ancient uses of medicinal plants in trás-osmontes (northern of portugal), journal of ethnopharmacol, 124: 270– 283, (2009) [24] demir f., özcan m., chemical and technological properties of rose (rosa canina l) fruits grown wild in turkey, journal of food engineering, 47: 333–336, (2001) [25] ljubuncic p., portnaya i., cogan u. azaizeh h., and bomzon, a. antioxidant activity of crataegus aronia aqueous extract used in traditional arab medicine in israel, journal of ethnopharmacology, 101(1–3): 153–161, (2005) [26] cao g.y., feng y.x. and qin, x.q., analysis of the chemical constituents of hawthorn fruits and their quality evaluation, yaoxue xuebao, 30: 138-143, (1995) [27] guo tj., jiao pj., hawthorn (crataegus) resources in china, hortscience, 30: 1132-1134, (1995) [28] baker r.a., reassessment of some fruit and vegetable pectin levels, journal of food science, 62(2): 229, (1997) [29] pardo de santayana m., tardio j., blanco e., carvalho am., lastra jj., san miguel e., morales r., traditional knowledge of wild edible plants used in the northwest of the iberian peninsula (spain and portugal): a comparative study, journal of ethnobiol ethnomed, 3: 27–37, (2007) [30] signorini ma., piredda m., bruschi p., plants and traditional knowledge: an ethnobotanical investigation on monte ortobene (nuoro, sardinia), journal of ethnobiol ethnomed, 5: 6–20, (2009) [31] carre m.h., haynes d., the estimation of pectin as calcium pectate and the application of this method to the determination of the soluble pectin in apples, biochemical journal, 16 (1): 60– 69, (1922) [32] hutchings j.b., food color and appearance, 2nd edition, aspen publishers, gaithersburg, (1999) [33] talens p., martinez-navarrete n., fito p.and chiralt a., changes in optical and mechanical properties during osmodehydrofreezing of kiwi fruit, innovative food and science emerging technologies, 3: 191– 199, (2001) [34] ercisli s., yanar m., sengul m., yildiz h. , topdas e. f. , taskin t., zengin y.,yilmaz k.u., physico-chemical and biological activity of hawthorn (crataegus spp. l.) fruits in turkey, acta scientiarum polonorum. hortorum cultus, 14(1): 83-93, (2015) [35] yanar m., ercisli s., yilmaz ku., sahiner h., taskin t., zengin y., akgul i., celik f., morphological and chemical diversity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 2 2016 silvia mironeasa, elena sănduleac todosi, mădălina iuga, physico-chemical characteristics, antioxidant activity and minerals content of hawthorn fruits from suceava county, volume xv, issue 2 – 2016, pag. 108 – 116 116 among hawthorn (crataegus spp.) genotypes from turkey, scientific research and essays, 6(1): 3538, (2011) [36] türkoğlu n., kazankaya a., sensoy ri., pomological characteristics of hawthorn species found in van region, journal of agricultural sciences, 15: 17-21, (2005) [37] erfani-moghadam j., mozafari m., fazeli a., genetic variation of some hawthorn species based on phenotypic characteristics and rapd marker, biotechnology & biotechnological equipment, 30(2): 247-253, (2016) [38] balta mf., celik f., turkoglu n., ozrenk k., ozgokce f., some fruit traits of hawthorn (crataegus spp.) genetic resources from malatya, turkey., research journal of agriculture and biological sciences, 2(6): 531-536, (2006) [39] zhu r., li t., dong y., liu y., li s., chen g., zhao z., jia y., pectin pentasaccharide from hawthorn (crataegus pinnatifida bunge. var. major) ameliorates disorders of cholesterol metabolism in high-fat diet fed mice, food research international, 54: 262– 268, (2013) [40] clydesdale f.m., colour measurement, in: gruenwedel d.w., whitake, j.r. (eds.), food analysis principles and technology, marcel dekker, new york., usa, (1984) [41] al-hooti s., sidhu j.s., objective color measurement of fresh date fruit and processed date products, journal of food quality, 20: 257–266, (1997) [42] heber d., bowerman s., applying science to changing dietary patterns, american institute for cancer research 11th annual research conference on diet, nutrition and cancer, (2001) [43] kostic d.a., velickovic j.m., mitic s.s., mitic m.n., randelovic s.s., phenolic content, and antioxidant and antimicrobial activities of crataegus oxyacantha l. (rosaceae) fruit extract from southeast serbia, tropical journal of pharmaceutical research, 11: 117–124, (2012) [44] margaret l., b. vickery, plant products of tropical africa, macmillan in college edition, london, (1997) [45] brody t., nutritional biochemistry, san diego, ca: academic press (1994) [46] appel l.j. nonpharmacologic therapies that reduce blood pressure. a fresh perspective, clinical cardiology, 1111-1115, (1999) [47] jørgensen s.e., fath b.d., global ecology: a derivative of encyclopedia of ecology, elsevier academic press, amsterdam, (2010) [48] mielcarz gw., howard an., williams nr., kinsman gd., moriguchi e., moriguchi y., mizushima s., yamori y., copper and zinc status as a risk factor for ischemic heart disease: a comparison between japanese in brazil and okinawa, journal of trace elements in experimental medicine, 10: 29-35, (1997) [49] oluyemi ea., akinlua aa., adenuga aa., adebayo mb., mineral content of some commonly consumed nigerian foods, journal of science focus., 11(1):153-57, (2006) [50] melaku u., clive ew., habtamon f., content of zinc, iron, calcium and their absorption inhibitors in foods commonly consumed in ethiopia, journal of food composition analysis, 18:803-817, (2005) [51] macrae r., robinso, r. k., sadler m. j., encyclopaedia of food science, food technology and nutrition, vol. 5, san diego, ca: academic press inc, (1993) food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 62 effect of different milk substitutes on rheological properties of puddings *cristina damian1 faculty of food engineering , stefan cel mare university of suceava, romania, 1cristinadamian@fia.usv.ro *corresponding author received 15 august 2012, accepted 12 september 2012 abstract: commercial pudding powders are usually composed of starch, hydrocolloids, colorings and aromas, and are intended to be dissolved in milk. the use of milk substitutes is mandatory for people suffering from lactose intolerance and milk allergies, but these ingredients have an effect on pudding structure and rheological behavior that has been little studied. the aim of the present study was to characterize the rheological behaviour of three commercial pudding powders dissolved in water, rice drink, grapefruit juice, soy drink and milk (reference sample). the three pudding powders, apparently similar in formulation, resulted in final products with large rheological differences that could be ascribed to different types and to quantity of starch present in the formulation. generally, the properties of rice drink based products were comparable to those of the water systems, while the characteristics of soy drink based puddings were similar to those of the reference sample. keywords: dairy dessert, milk fat, viscosity 1. introduction functional food contains some healthpromoting compounds beyond traditional nutrients and plays an important role in enhancing human health [1]. in the modern diet, there is a trend towards increased consumption of reduced-fat products [2]. the study of stussy, m.j., investigated the effect of non-fat chocolate pudding as a fat replacement in brownies and concludes that this modified product has potential for success in the consumer market, offering a healthier alternative to traditionally highfat brownies [3]. other studies reported replacement of sugar with stevia (a natural sweetener obtained from the leaf of stevia rebaudiana plant) [4], possibility of using full fat soy flour for replacer for whole milk flour and soybean oil as replacer for cocoa butter in chocolate manufacture [5], guar gum and gum arabic as fat replacers in low-fat iranian white cheese [6]. researches of thomson l.u. et all. examined the effect on the quality of substituting succinylated cheese whey milk protein concentrate in ice cream and instant pudding and concluded that the use of succinylated whey protein concentrate in ice cream increased viscosity and resistance to melting and reduced freezing time and overrun [7]. ares g. and all. find that addition of a new functional fibre, high-amylose maize starch caused changes in the sensory characteristics in milk puddings, an increase in manual and oral thickness and a decrease in creaminess, melting and sweetness [8]. in a study, nunes m.c. and all. find that mixed protein – polysaccharide systems, with vegetable proteins and carrageenan or gellan gum – would be good systems to develop vegetable protein desserts [9]. to offer consumers foods that are lower in fat content, several low-calorie and calorie-free fat replacers or fat mailto:cristinadamian:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 63 substitutes have been developed. these ingredients contribute fewer calories to the formulated foods without altering flavour, mouthfeel, viscosity, or other sensory properties [2]. dairy desserts (milk puddings) are especially consumed by children and elderly people throughout the world. they are generally sold in the powdered or prepacked form and composed of vanilla milk, sugar and thickeners (gum and starch) [10]. other ingredients such as cacao and fruit aromas are used in formulations to have products with different flavours, affecting consumer acceptability. regarding dairy products, many studies have been published dealing with the effect of fat content on rheological behaviour and sensory properties, mainly on flavour and texture perception [11, 12, 13]. however, only a few papers deal with the rheological characterisation of commercial dairy desserts or their corresponding model systems. the rheology of food products is very important because flow properties define food structure during industrial manufacturing or preparation in the kitchen and this is physiologically important in the mouth, stomach and intestine where the food structure is perceived and digested. in addition, the rheological properties are also important for sensory perception characteristics (e.g. portioning, scooping, dosing, filling) and nutritive characteristics (e.g. release kinetics, satiety). texturing agents are also important for flavor release because they increase viscosity which in turn impairs perception [14]. the rheological characteristics of dairy desserts mainly depend on the fat content of milk, type and concentration of hydrocolloids and their interactions [15]. the use of two or more gums in the formulation of the product is very widespread in the food industry due essentially to the synergistic effect of combined use [16]. the aim of the present study was to characterize the rheological behaviour of three commercial pudding powders dissolved in water, rice drink, grapefruit juice, soy drink and milk (reference sample). 2. materials and methods 2.1. materials three commercial puddings with vanilla, caramel and chocolate flavour were purchased from the local market. the declared ingredients list is: corn starch, vanilla flavor (contains milk), color: betacarotene (vanilla pudding); corn starch, caramel, caramel flavor, colored brown ht and curcumin (caramel pudding); corn starch, low-fat cocoa (11% cocoa butter), ethyl vanillin flavor (chocolate pudding). its approximate composition is shown in table 1. table 1. approximate composition of the comercial pudding sample studied raw material composition (%) water 59.0 proteins 10.0 carbohydrates 21.0 fats 7.2 flavours, etc. 2.8 total 100 2.2. sample preparation pudding samples were prepared as follows: 500 ml milk, rice drink, soy-drink or water were used for a bag of pudding (40 g). the contents of a bag of pudding food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 64 mix with 4 tablespoons of milk or milk substitute. the amount of milk or milk substitute boil, then removed from heat, and the mixture prepared is added. then it is boiled again on low heat, stirring gently till the composition binds. the pudding samples were made using cow milk. the cow milk analysis by lactostar 2005 funke gerber led to the physical and chemical parameters presented in tabel 2. table 2. milk properties fat, g/100g 3.30 protein, g/100 g 3.19 lactose, g/100 g 4.61 snf (solid non-fat), g/100 g 8.49 freezing point, 0c 0.5 minerals, g/100 g 0.02 conductivity, ms 0.89 boiling water for rice and soy and noncarbonated grapefruit juice were used. 2.3.determination of acidity of pudding samples acidity analysis of pudding samples was achieved by titration with naoh in the presence of phenolphthalein as indicator. 2.4. determination of rheological properties viscosity measurements were carried out on the yogurt samples at ambient temperature (250c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 0.5, 1, 2, 2.5, 4, 5 and 10 rpm with rv spindle (rv3, rv4, rv5, rv6 type). the spindle nos were used in accordance with the sample nature to get all readings within the scale [15]. the samples in 300 ml of beaker with an 8.56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain desirable temperature of 250c. first measurements were taken 2 min. after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression = k· n (1) where: – shear stress (n/m2), is the shear rate (s-1), n is the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. the shear stress is calculated using the next equation: i = k i·c (2) where: i – shear stress, (dyne/cm2) k = 0.119, this constant is for the spindle nos 2 i – torque dial, (%) c – 7.187 dyne/cm for rv viscometer the shear rate is calculated using the next equation: i = k (n)·ni (3) where: i – shear rate, (s-1) k (n) – constant, depends by the value of n ni – rotational speed, (rpm). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 65 0 10000 20000 30000 40000 50000 60000 70000 80000 0 5 10 15 20 25 v is co si ty [ cp ] shear rate [1/s] vanilla puddings vp1 (water) vp2 (rice) vp3 (grapefruit) vp4 (soy) vp5 (milk) 3. results and discussion the acidity analysis results for pudding samples are presented in table 3. table 3. acidity of pudding samples sample acidity (acidity degrees) vanilla pudding with water vp1 0.81 vanilla pudding with rice drink vp2 0.82 vanilla pudding with grapefruit juice vp3 3.53 vanilla pudding with soy drink vp4 1.98 vanilla pudding with milk (reference sample) vp5 1.76 caramel pudding with water cp1 0.32 caramel pudding with rice drink cp2 0.33 caramel pudding with grapefruit juice cp3 3.33 caramel pudding with soy drink cp4 1.87 caramel pudding with milk (reference sample) cp5 1.67 chocolate pudding with water chp1 0.82 chocolate pudding with rice drink chp2 0.84 chocolate l pudding with grapefruit juice chp3 2.11 chocolate pudding with soy drink chp4 1.87 chocolate pudding with milk (reference sample) chp5 2.09 vanilla pudding with grapefruit juice has the highest value of acidity, and rice-drink based vanilla pudding has the lowest value. puddings sample exhibit a nonnewtonian behaviour, the flow index (n) id under 1 for all the samples. the power law model is a suitable one for predicting the rheological parametersthe rheological parameters, the regression coefficient is near 1 (r2 > 0.95) – see table 2. consistency index (k) values is in the range of 3.62 – 12.93 (n.sn/m2) and flow behaviour index (n) values in the range of 0.426 – 0.706. in a study about rheological behaviour of soy protein-based puddings, lim h.s. et all found that the k and n values for soy-protein based puddings were comparable to those of the commercial puddings [17]. table 3. power law parameters for pudding samples sample n – flowindex k – consistency index (n.sn/m2) r 2 vp1 0.61 7.57 0.96 vp2 0.581 8.17 0.956 vp3 0.545 8.05 0.957 vp4 0.453 11.7 0.973 vp5 0.431 12.58 0.955 cp1 0.466 10.75 0.967 cp2 0.426 12.93 0.956 cp3 0.448 11.49 0.986 cp4 0.487 9.53 0.957 cp5 0.457 10.64 0.958 chp1 0.706 5.1 0.969 chp2 0.644 5.92 0.965 chp3 0.556 8.36 0.971 chp4 0.542 3.62 0.996 chp5 0.535 8.95 0.997 in figure 1 the viscosity of vanilla pudding with water, rice drink, grapefruit juice, soy drink and milk (reference sample) is presented. figure 1. viscosity profile of vanilla pudding with water (vp1), rice drink (vp2), grapefruit juice (vp3), soy drink (vp4) and milk (vp4) generally, the viscosity of rice drink based puddings were comparable to those of the water systems, while the viscosity of soy drink based puddings were similar to those of the reference sample. the grapefruit juice-based pudding has an intermediate behaviour between the two groups. in figures 2 and 3 the viscosity of caramel and chocolate puddings with water, rice drink, grapefruit juice, soy food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 66 drink and milk (reference sample) are presented. the influence of the type of pudding on the rheological behaviour is shown in figure 4. from the data presented in figure 4, it is observed that chocolate pudding has the lowest viscosity; caramel pudding next and vanilla pudding has the highest viscosity. this may be due to traces of milk that the composition of this pudding may contain. 4. conclusion the pudding is a complex viscous food material due to its nature and composition. rheological behaviour of pudding is influenced by milk fat. the rheological behaviour of dairy dessert is among the most crucial factors affecting consumer acceptability of such products. generally, the viscosity of rice drink based puddings were comparable to those of the water systems, while the viscosity of soy drink based puddings were similar to those of the reference sample (with milk). the grapefruit juice based pudding has an intermediate behaviour between the two groups. 5. references [1]. sun y., hayakawa s., ogawa m., izumori k., antioxidant property of custard pudding dessert containing rare hexoze, d-psicose, food control, 18, 220227, (2007) [2]. yilsay t. ö., yilmaz l., bayizit a.a., the effect of using a whey protein fat replacer on textural and sensory characteristics of low-fat vanilla ice cream, eur food res technol, 222, 171-175, (2006) [3]. stussy m.j., peterson j.k., gee d., effect of non-fat pudding as a fat replacement in brownies, journal of the american dietetic association, 95, 9, (1995) [4]. giri a., rao h.g., ramesh, v., effect of partial replacement of sugar with stevia on the quality of kulfi, j. food sci technol, (2012) [5]. pandey a., singh g., development and storage study of reduced sugar soy containing compound chocolate, j food sci technol, (2010) [6]. lashkari h., khosrowshahi a., madadlou a., alizadeh m., chemical composition and rheology of low-fat iranian white cheese incorporated figure 2. viscosity profile of caramel pudding with water (vp1), rice drink (vp2), grapefruit juice (vp3), soy drink (vp4) and milk (vp4) figure 3. viscosity profile of chocolate pudding with water (vp1), rice drink (vp2), grapefruit juice (vp3), soy drink (vp4) and milk (vp4) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 0 5 10 15 20 25 v is co si ty [ cp ] shear rate [1/s] rice-drink puddings vanillap2 (rice) caramelp2 (rice) chocolatep2 (rice) figure 4. viscosity profile of rice-drink based vanilla, caramel and chocolate puddings food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 67 with guar gum and gum arabic as fat replacers, j food sci technol, (2012) [7]. thompson l.u., reniers d.j., baker l.m., siu m., succinylated whey protein concentrates in ice cream and instant puddings, journal of dairy science, 66, 8, 1630-1637, (1983) [8]. ares g., baixauli r., sanz t., varela t., salvador a., new functional fibre in milk puddings: effect on sensory properties and consumers’ acceptability, lwt food science and technology, 42, 710-716, (2009) [9]. nunes m.c., batista p., raymundo a., alves m.m., sousa i., vegetable protein and milk puddings, colloids and surfaces b: biointerfaces, 31, 1-4, 21-29, (2003) [10]. toker o.s., dogan m., caniyilmaz e., ersöz n.m., kaya y., the effects of different gums and their interactions on the rheological properties of a dairy dessert: a mixture design approach, food bioprocess technol, (2012) [11]. mittal s., bajwa u., effect of fat and sugar substitution on the quality characteristics of low calorie milk drinks, j food sci techol, (2011) [12]. gonzáles-tomás l., bayarri s., taylor a.j., costell e., rheology, flavour release and perception of low-fat dairy desserts, international dairy journal, 18, 858-866, (2008) [13]. oroian m.a., escriche i., gutt g., viscoelastical behavior of some food materials from the spanish market, food and environment safety, 10, 4, 13-17, (2011) [14]. toker s.a., dogan m., caniyilmaz e., ersöz n.b., kaya y., the effect of different gums and their interactions on the rheological properties of a dairy dessert: a mixture design approach, food bioprocess technol, (2012) [15]. tarrega a., costell e., effect of composition on the rheological behavior of semi-solid dairy desserts, food hydrocolloids, 20, 914-922, (2006) [16]. kayacier a., dogan m., rheological properties of some gums-salep mixed solutions, journal of food engineering, 72, 261-265, (2006) [17]. lim h.s., narsimhan g., pasting and rheological behavior of soy protein-based pudding, lwt food science and technology, 39, 4, 344-350, (2006) food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 42 separation of selected pesticides by an hplc technique; performance parameters and validation *camelia popa1 1 faculty of food engineering, tefan cel mare university of suceava, romania camelia.popa@fia.usv.ro *corresponding author received 25 august 2012, accepted 14 september 2012 abstract: the aim of this work is to get the performance parameters investigated by hight performance liquid chromatography (hplc), for the separation method of 2,4-dichlorophenoxiacetic acid, 3,6-dichloro-2-methoxybenzoic acid, an organic mixture with herbicide action. the chromatographic separation with better peak shape was achieved. the retention times (tr), peak resolutions (rs), separation factors ( ), column efficiency (neff), height of theoretical plates (hetp), indicate that the mobile phases in gradient, containing acetonitrile and water with 1% acetic acid are the best for the separation of investigated components on cromatographic column c18. also it has been shown that in data conditions the methode is sensitive, precise and reproductible. keywords: hplc, environment, performance, validation 1. introduction pesticides are chemical protection tools for plants. they are obtained from one ore more biological compounds efficient.with few exception like growing regulators, the biological active ingredients are toxic. due to this toxicity are dictate good practice in dose, distribution and in use of pesticides. also pesticides pass from ground water to vegetables [1], plants and foods and finally they are accumulate in animal fat. pesticides affect the structure and immune efficiency and reduce the immunity at infection. the farmers that use the pesticides must take into acount the parameters, as follows: the configuration of marketing, the technology of conditioning, the technology to apply, the maximum limit of waste.the mixture of these two components with herbicide action are used in disproof of weed from beating cereals. 2,4dichlorophenol is the toxic component born in the of 2,4dichloro-phenoxiacetic acid manufacturing process and retrieved in the end of the mix in acceptable limit.the performance parameters of this separation method have a great importance, they reflect the correct and the exactely dosage of the components in the mixture and on ground. the presence of pesticides in environment induces the modification of the quality environment componentsground, underground and surface water, the optimization of these quntities meaning an important factor in environmental quality protection. 2. experimental chemicals and reagents the components of mobile phases: acetonitrile and water (labosi), hplc grade. acetic acid, glacial degree, 2,4diclorphenoxiacetic acid 99,9 % purity (named 2,4d acid), 3,6-dichloro-2metoxibenzoic acid 99,9% purity (named dicamba), 2,4 dichlorophenol 99% purity (named dcf) from merck. mailto:camelia.popa:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 43 instrumentation and conditions the cromatographic investigations was carry out on a varian prostar liquid cromatograph system equipped with: quaternary pump (model 9100), autosampler (model 9010), uv detector (model 9065). the data were aquired via prostar data aquisition workstation. mobile phase consists of water and acetonitrile hplc grade, injection volume: 20 µl, flow rate : 1ml/minute, : 280nm. reversed phase analysis was performed at 220c using an bondesil c18 column, 5µ (25cm, 4mm id) [2]. table 1 shows the gradient elution used. table 1 gradient of mobile phase time(minutes) b (%) c (%) 0 95 5 9 95 5 17 50 50 30 50 50 b = 1% acetic acide in hplc water; c = 1% acetic acid in hplc acetonitrile. elution order: dicamba, 2,4d acid, 2,4dcf standard preparation to get the separation parameters was used synthetic standard solution named stock solution: 0.07 g 2,4d acid, 0.025g dicamba, 5ml dcf standard solution, completed to 25 ml with (alkaline) hplc grade water. syntetic standard solution keeps the same report between the components like in the mixture with herbicide action. dcf standard solution was prepared from 0,1gdcf diluted to 25ml with (alkaline) hplc water. sample preparation a representative quantity of sample is weighed and the active ingredients are extracted with selective solvents. follow the evaporation of solvent and than active ingredients are solved and diluted to 25 ml with hplc grade water. calculations capacity factor (k’) [3] was calculated using equation (1): ot ' rt ot otrt'k (1) where: tr is the retention time of the solute t0 is the time for an unretained solute; t’r is the adjusted retention time of the solute the condition of strong separation from technical book of varian instrument is k’ 1 [3]. column selectivity ( ). the separation factor ( ) [3] was calculated using equation (2): ' 1rt ' 2rt (2) where: ' 2rt and ' 1rt are adjusted retention times for two adjacent peaks. the selectivity condition is 1 . peak resolution (rs). the peak resolution (rs) [4] was calculating using the equation (3): r s 1 2 1.18× t r = w +w (3) where: tr is the difference in retention times between the two peaks; w1 and w2 are widths of the two peaks at half of their height. the condition of separation is: rs = 1 means 98% separation; rs = 1.5 means 99.7% separation. column efficiency (neff ). the column efficiency [4] was calculated as number of theoretical plates using equation (4): 2 w ' rt54.5effn (4) food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 44 from technical book of varian instrument the efficiency condition is neff > 400. height of theoretical plates (hetp) [4] was calculated using equation (5): effn l hept (5) where: l is the length of the column (cm); neff is the effective number of theoretical plates. also, from technical book the accepted value is hetp = 0.001 ÷ 0.002 mm. standard deviation (sr) [5, 6] was calculated using equation (6): 1n n 1k 2 xkx rs (6) repeatability limit (r) [9] was calculated using equation (7): rs2tr (7) where t = 1.96, student coefficient for 95% confidence interval. 3. results and discussions for cromatographic separation of 2,4d acid, dicamba and dcf on c18 stationary phase (4,6mm, 5µm) with varying column lengths from 150 to 250 was attempted. different mobile phase composition containing water and acetonitrile with 1% acetic acid were tried. the column 250mm x 4,6mm, 5 µm showed higher elution times and good resolutions for the components of interest, respectively 21,89 seconds for dicamba, 24,60 seconds for 2,4d acid and 26,20 seconds for dcf. system suitability is shown in table 2. using equations from ,,calculations” capter and the chromatogram obtained, was calculated the performance parameters that shows the efficiency of separation in the conditios of the method. performance parameters are shown in table 3. we can see strong values for performance parameters in the conditions of the method: peak resolution, column efficiency and height of theoretical plates. the results show very good performance parameters of this separation methode.this hplc separation method of the organic mixture with herbicide action is selective, fact demonstrated by the selectivity (specificity) of the instrument/equipment and the separation conditions on chromatographic column, c18. table 2 system suitability component dicamba 24d acid dcf tr (minutes) 21,89 24,60 26,20 table 3 efficiency of separation performance parameter accepted value [4] obtained value capacity factor (k’) 1 k’dicamba = 16.6 k’2,4dacid = 17.6 k’dcf = 18.3 column selectivity ) 1 dicamba = 1.04 2,4dacid = 1.06 dcf = 1.05 peak rezolution rs 1 for 98% separation 1.5 for 99.7% separation rdicamba = 3.8 r2,4dacid = 6.5 r dcf = 4.6 column efficiency neff (number of theoretical plates) 400 neffdicamba = 258475 neff2,4dacid = 171600 neffdcf = 307000 height of theoretical plate (hetp) 0.001÷0.002mm hetpdicamba = 0.001mm hetp2,4dacid = 0.002mm hetpdcf = 0.001mm food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 45 method validation the proposed method was validated with respect to linearity, accuracy, precision, specificity, following the hp guide for hplc, ce and uv-vis spectroscopy [7]. linearity (sensitivity) and range linearity test solutions were prepared by diluting stock solution at five concentration levels of analytes concentration. the solutions were injected in triplicate and following regresion equations were found by plotting peak area versus concentration. the response is linear on area of concentration chosen if the results dont’t have a signifiant deviation from linearity, this means, an corelation coefficient bigger than 0,997 for all components. the obtained equations for regression lines are: ydicamba = 9770,32x + 15685,5; y2,4dacid = 79022x 77878,5; ydcf = 4441,75x-8114,5. the coefficient of determination (r2) obtained for regression line demonstrates the excellent relationship between peak area and components concentrations. the results are shown in table 4. precision [7] in retention times and peak area (or height) are major criterion of separation systems. the precision of the chromatographic method reported as percent of relative standard deviation (sr) was estimated by measuring repeatability on five replicate cromatograms [8]. table.4. linearity results for lc method component concentration equation for regression line r2 dicamba 0,25 2,25 mg/ml y = 9770,32 x + + 15685,5 0,998 2,4d acid 0,7-3,5 mg/ml y = 79022 x – – 77878,5 0,999 dcf 5-25 µg/ml y = 4441,75 x – – 8114,5 0,999 the relative standard deviation values (sr) and the repeatability limit (r) for retention times and areas are shown in table 5. xk – xk-1 is the difference between two individual results that must be smaller than repeability limit.the condition xk – xk-1 r, is accomplished. precision in analisys and accuracy accuracy was estimated by spinking the sample matrix of interest with a known concentration of reference material: c2,4dacid = 28.5%; cdicamba = 9.5%; c2,4dcf = 0.1% the same as the concentration of formulated herbicides. it was compared the response obtained after the extraction of analyte from the sample and injection in the column with the response of the reference material added to the pure solvent (table 6). table 5. repeatability for retention times and areas component dicamba 2,4d acid dcf parameter/run area (counts) rt (minutes) area (counts) rt (minutes) area (counts) rt (minutes) run 1 424827 21.89 2017948 24.56 1477 76 26.18 run 2 392906 22.01 1864533 24.49 1334 77 26.02 run 3 418781 22 1934687 24.53 1418 34 26.06 run 4 403003 22.05 1893372 24.6 1562 84 26.18 run 5 383215 21.97 1854117 24.56 1325 26.24 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 46 40 sr 17369 0.06 66494 0.05 9985 0.18 r 48003 0.16 183762 0.13 2759 5 0.49 xk – xk-1 r ok ok ok ok ok ok table 6 precision and accuracy of measurement run 2,4d acid (%) dicamba (%) dcf (%) 1 28.50 9.50 0.093 2 28.00 9.60 0.100 3 28.51 9.56 0.075 4 28.36 9.90 0.103 5 28.78 9.47 0.09 accuracystudent variable ( 1) 0.148 0.086 0.016 precision as sr, (%) 0.28 0.17 0.03 4. conclusions the resuls show that hplc separation method of the organic mixture with herbicide action is selective, fact demonstrated by the selectivity of the instrument/equipment and the separation conditions on chromatographic column, c18. to note, the performance parameters with strong values in the conditions of the methode: the resolution of separation, column efficiency and height of theoretical plate. also, it has been shown that in data conditions the methode is sensitive, precise and reproductible. 5. references [1]. jonhson, a.c. et al., penetration of herbicides to groundwater in an unconfined chalk aqiufer following normal soil applications, j. contam. hidrol. 53., 100-120, (2001) [2]. popa, c. et al., hplc separation methode for the content of 2,4diclhorphenol, diclhorphenoxiacetic acids and 3,6-dichloro-2 metoxibenzoic acid from an mixture with herbicide action b.i.115002b1, osim (1994) [3]. nomenclature for cromatography (iupac reccomendations 1993). pure appl chem 65. 820-870, (1993) [4]. operation book for hplc varian prostar instrument, (1994) [5]. sr env 13005:2003. guide to the expression of uncertainty in measurement. standardization association of romania, bucharest, romania [6]. chairman w. et al, eurachem/citac guide cg4-the fitness for purpose of analitycal methods. a laboratory guide to method validation and related topics, 2000. [7]. huber, l. good laboratory practice, hp publication number 12-5091-6259e, marcel dekker inc. 31-62, (1993) [8]. validation of compendial methodes, us pharmacopeia xxii, , national formulary, xii, the us pharmacopeial convention inc, rockville, md, 1990, general chapter (1225), 1710-1612 [9]. iso 3534-1:2009, statistics.vocabulary and symbols. part 1: general statistical terms and terms used in probability, standardization association of romania, bucharest, romania, (2009) 335 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 42014, pag. 335 341 in flu en ce o f citrus fibre ad dition o n textur al a nd rheolo gica l pro per ties o f yog ur t *mircea oroian1, sergiu paduret1, gheorghe gutt1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received november 18th 2014, accepted december 24th 2014 abstract: the aim of this study is to evaluate the influence of citrus fibre addition (0, 0.25, 0.5 and 1%, respectively) on the textural and rheological properties of yogurt samples (7.5% dry matter, 10% dry matter and 12.5% dry matter). the rheological properties of the samples were investigated using a brookfield viscometer, while the textural properties were measured using a mark-10 texture-meter. the textural properties analysed were: hardness (h), cohesiveness (co), springiness (s), gumminess (g) and chewiness (ch). the principal component analysis was conducted based on the dry matter content, fibre content and rheological (viscosity) and textural properties (hardness (h), cohesiveness (co), springiness (s), gumminess (g) and chewiness (ch)) using unscrambler x 10.1 (camo, norway). the two components (pc1 and pc2) explained 100% of the variations in the data set. it can be observed that the samples were placed into three main different groups based on the dry matter concentration. keywords: yogurt, citrus fibre, physical properties, principal component analysis 1. introduction yogurt, according to the codex alimentarius standard for fermented milks [1], is a form of fermented milk that contains symbiotic cultures of streptococcus thermophilus and lactobacillus delbrueckii subsp.bulgaricus that “shall be viable, active and abundant in the product to the data of minimum durability” [2]. the rheology, function and composition of ingredients are important for a food product engineer as well as quality control, and design of process equipment [3-5]. dietary fiber is a common component of food products which consists of variety of polysaccharides such as cellulose, hemicelluloses, pectin, β-glucans, gums and lignin [6] and those are taken as foods due to their beneficial effects on food nutritional properties [7]. the dietary fiber are recommended to be consumed due to their significant role in the prevention, reduction and treatment of chronic diseases such as bowel, gastrointestinal disorders, obesity, diabetes, cardiovascular disease, cancer and also promoting physiological functions including reduction in blood cholesterol level and glucose attenuation [8-10]. citrus fruits (citrus hystrix and citrus maxima) are one of the most consumed fruits all over the world. citrus fruits contain vitamins (e.g. a, b and c), minerals, dietary fibers, secondary metabolites (e.g. phenolics, flavonoids, limonoids and carotenoids) [11]. the citrus fiber addition into food products have many benefits as: in the meat product will improve the oxidative stability and prolong their shelf life by preventing the lipid peroxidation due to the presence of associated bioactive compounds food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 336 i.e.polyphenols [12] and also decrease the residual nitrite level [13,14]. the citrus fiber products have many applications because of its neutral color, taste and odor [15]. the aim of this study is to evaluate the influence of citrus fiber addition on the rheological and textural properties of yogurt. 2. materials and methods materials milk with different dry matters (7.5, 10 and 12.5% respectively), di prox 975 lactic culture and citrus fiber from enzymes &derivatescostisa, romania, were used. yogurt preparation the milk sample was pasteurized and then cold up to 42 °c prior to lactic culture inoculation. the samples were kept at 42 °c till the desired ph was achieved. the citrus fiber was added into the samples in different concentrations (0, 0.25%, 0.5% and 1.0%, respectively), mixed and then kept for 24 h at 4 °c. rheological properties measurement viscosity measurements were carried out on the yogurtsamples at ambient temperature (4 °c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 1, 2, 5, 10 , 20 and 100 rpm with rv spindle (rv3, rv4, rv5, rv6 type). the spindle nose was used in accordance with the sample nature to get all readings within the scale. the samples in 300 ml of beaker with a 8.56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain desirable temperature of 250c. first measurements were taken 2 min after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression σ = k·γn (1) where: σ – shear stress (n/m2), γ is the shear rate (s-1), n is the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. the shear stress is calculated using the next equation: τi= kτ·αi·c (2) where: τi – shear stress (dyne/cm2) kτ= 0.119, this constant is for the spindle nos 2 αi – torque dial, % c – 7,187 dyne/cm for rv viscometer the shear rate is calculated using the next equation: γi= kγ(n)·ni (3) where: γi – shear rate, s-1 kγ(n)– constant, depends by the value of n ni – rotational speed, rpm. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 337 texture properties measurement (tpa) the tpa was carried out at 4 °c with mark 10 texture analyzer (mark 10 corporation, usa) equipped with a 50 mm disc probe, the flask diameter was 70 mm. the tpa was operated at a constant speed of 150 mm/min, until a depth of 12.5 mm (the yogurt column had 25 mm). the tpa can offer a great number of texture parameters, as: hardness (h), viscosity (v), adhesion (a), cohesiveness (co), springiness (s), gumminess (g) and chewiness (ch) [16]. fig.1. texture profile [16] statistical analysis the principal component analysis has been made up using the unscrambler x 10.1 software (camo, norway). 3. results and discussions the rheological and textural parameters of the 12 samples are presented in table 1. in figure 2 is presented the viscosity evolution of a yogurt sample with shear rate. it can be observed a pseudoplastic evolution of the viscosity (the viscosity is decreasing with the increasing of the shear rate). in figure 3 is presented the texture profile of one yogurt sample. fig.2. yogurt sample rheological profile (p9) fig.3. yogurt sample texture profiles: ap4, b-p6 and cp9 -0.2 -0.1 0 0.1 0.2 0.3 0 20 40f or ce ( n ) distance (mm) a -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0 20 40 f or ce (n ) distance (mm) b -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0 20 40 fo rc e (n ) distance (mm) c food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 338 table 1. rheological and textural parameters of the yogurt samples sample s.u.% f % h co g s gu v(cp) p1 7.5 0 0.240±0.002 0.87±0.01 0.26±0.02 1.05±0.01 0.34±0.01 390±11 p2 7.5 0.25 0.220±0.002 0.88±0.06 0.24±0.01 1.00±0.02 0.21±0.02 360±12 p3 7.5 0.5 0.196±0.001 0.71±0.01 0.27±0.02 1.01±0.01 0.15±0.01 355±11 p4 7.5 1 0.202±0.005 0.84±0.02 0.23±0.01 1.40±0.03 0.39±0.03 390±14 p5 10 0 0.208±0.004 0.86±0.05 0.24±0.03 0.97±0.02 0.19±0.01 310±11 p6 10 0.25 0.196±0.010 0.78±0.01 0.25±0.01 0.99±0.01 0.20±0.04 270±14 p7 10 0.5 0.182±0.012 0.96±0.04 0.18±0.02 1.00±0.02 0.21±0.01 250±16 p8 10 1 0.186±0.013 0.85±0.01 0.21±0.01 1.24±0.02 0.24±0.02 340±14 p9 12.5 0 0.442±0.015 0.85±0.03 0.51±0.03 0.98±0.01 1.03±0.01 970±15 p10 12.5 0.25 0.334±0.014 0.90±0.01 0.37±0.01 1.01±0.02 0.53±0.02 1500±13 p11 12.5 0.5 0.352±0.012 0.91±0.02 0.38±0.04 1.18±0.02 0.82±0.02 1740±12 p12 12.5 1 0.338±0.010 0.86±0.01 0.39±0.01 1.20±0.01 0.82±0.02 1834±14 dm-dry mater, f – fibre content, h – hardness, c – cohesiveness, ggumminess, s-springiness, gu-guminess, vviscosity according to the data presented in table 1, it can be observed that: sample p9 has the highest hardness and the p7 the smallest one; in the case of cohesiveness the highest one can be observed in the case of sample p7 while p3 has the smallest one. the yogurt gumminess is strongly influenced by the total soluble content and fibre concentration (the highest gumminess can be observed in the case of sample p12). the combination of milk with 10% dry matter with 1% citrus fibre generated the yogurt with the highest elasticity. the sample with the highest chewiness was p9 which is made from milk with 12.5% dry matter without fibre addition. regarding the rheological properties it can be observed an increase in viscosity with increasing the dry matter content and citrus fiber concentrations. citrus fibers are rich in pectin products. the ph of the yogurt, pectin, casein micelles are both substantially negatively charged. depending on the position of a system in relation to the phase diagram, there is a competition between the kinetics of phase separation due to the presence of polysaccharides and the induced gelation by the enzymes. for low levels of pectin (maximum 1 %) syneresis is less in the gel and the gel strength had increased by the addition of polysaccharides. in the table 2 is presented the pearson correlation coefficients of the physicochemical properties of yogurt. it can be observed that is a strong positively correlation between the dry mater and hardness (r = 0.731**), gumminess (r = 0.674*), chewiness (r = 0.718**) and viscosity (r = 0.798**). another positively correlation can be observed between fibre content and springiness (r = 0.832**). the hardness is correlated positively with gumminess (r = 0.981**), chewiness (r = 0.954**) and viscosity (r = 0.798**). the gumminess is correlated positively with chewiness (r = 0.929**) and viscosity (r = 0.756**), while chewiness is correlated positively with viscosity (r = 0.812**). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 339 table 2. pearson correlation of physicochemical properties of yogurt dm f h c g s gu v dm 1 0.025 0.731** 0.375 0.674* -0.061 0.718** 0.798** f 1 0.204 0.053 0.200 0.832** 0.022 0.175 h 1 0.227 0.981** 0.070 0.954** 0.798** c 1 0.038 0.038 0.260 0.273 g 1 0.089 0.929** 0.756** s 1 0.174 0.186 gu 1 0.812** v 1 dm-dry mater, f – fibre content, h – hardness, c – cohesiveness, ggumminess, s-springiness, gu-guminess, vviscosity, * significant correlations at a 0.01 level, ** significant correlations at a 0.05 level the principal component analysis was conducted to evaluate the global effect of the addition of citrus fibre on physicochemical properties of yogurt, from a descriptive point of view. in the figures 4 and 5 are presented the scores and compound loadings of pca analysis performed. it was found that the two principal components (pcs) explained 100% of the variations in the data set. the pc1 explains 99% of the variability and the pc2 explains 1%. it can be observed that the samples p5, p6, p7 and p8 are forming a group; another group is formed by the samples p9, p10, p11, p12 and the last one by the samples p1, p2, p3 and p4. the main characteristic of these groups is the dry mater concentration. regarding the correlation loadings, the gumminess has the lowest influence on the scores. the samples viscosity and the dry mater (tss) have the highest influence on the loadings. fig. 4 principal component analysis – correlation loadings between rheological and textural parameters food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 340 fig. 5 principal component analysis – correlation loading between yogurt samples formulations 4. conclusions the citrus fiber addition into yogurt sample has a positively influence on the rheological and textural properties of yogurt samples. the principal component analysis conducted revealed three main groups; the sample dividing into the three main groups was based on the dry matter concentration. viscosity and dry matter content has the high influence on the pca projection, while the gumminess the lowest one. the highest positively correlation was observed between hardness and gumminess (r = 0.981**). 5. references [1]. codex stan 243-2003 codex committee on milk and milk products [2]. patro-golab, b., shamir, r., szajewska, h., yogurt for treating acute gastroenteritis in children: systematic review and meta-analysis, clinical nutrition, in press, (2014) [3]. saravos, g. g., effect of particle sizes on functional properties of dietary fibre prepared from sugarcane bagasse, food chemistry, 80(2), 221229, (1970) [4]. ofoli, r. y., morgan, r. g., steffe, j. f., a generalized rheological model for inelastic fluid foods, journal of texture studies, 18, 213230, (1987) [5]. lundberg, b., pan, x., white, a., chau, h., hotchkiss, a., rheology and composition of citrus fiber, journal of food engineering, 125, 97-104, (2014) [6]. elleuch, m., bedigian, d., roiseux, o., besbes, s., blecker, c., attia, h., dietary fiber and fiber rich by-products of food processing: characterisation, technological functionality and commercial applications: a review. food chemistry, 4, 411-421, (2011) [7]. fernandez-lopez, j., perez alvarez, j. a.,technological strategies forfunctional meat products development (pp. 41– 57). kerala: transworld research, (2008) [8]. figuerola, f., hurtado, m.l., estevez, a.m., chiffelle, i., asenjo, f.,fibre concentrates from applepomace and citrus peel as potential fibre sources for food enrichment. food chemistry, 91, 395–401, (2005). [9]. mehta, r.s., dietary fiber benefits. cereal foods world, 50(2), 66–71, (2005) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiii, issue 4 – 2014 mircea oroian, sergiu paduret, gheorghe gutt, influence of citrus fibre addition on textural and rheological properties of yogurt, volume xiii, issue 4 – 2014, pag. 335 – 341 341 [10]. marlett, j.a., dietary fibre and cardiovascular disease. in s. s. cho, m.l. dreher (eds.), handbook ofdietaryfiber (pp. 17– 30).newyork, ny:marcel dekker, (2001) [11]. roussos, p.a., phytochemicals and antioxidant capacity of orange (citrus sinensisosbeck cv. salustiana) juice produced under organic and integrated farming systemin greece. scientiahorticulturae, 129, 253–258, (2011). [12]. sayago-ayerdi, s.g., brenes, a.,goni, i., effect of grape antioxidant dietary fiber on the lipidoxidation of raw and cooked chicken hamburgers. lwt food science and technology, 42, 971–976, (2009) [13]. fernandez-gines, j. m., fernandezlopez, j., sayas-barbera, e., sendra, e., perez-alvarez, j.a..effect of storage conditions on quality characteristics of bologna sausages made with citrus fiber. journal of food science, 68, 710–715, (2003) [14]. abirami, a., nagarani, g., siddhuraju, p., 2014, measurement of functional properties and health promoting aspectsglucose retardation index of peel, pulp and peel fiber from citrus hystrix and citrus maxima, bioactive carbohydrates and dietary fibre, 4, 16-26, (2014) [15]. garau, m.c., simal, s., rosello, c., femenia, a., effect of air-drying temperature on physicochemical properties of dietary fibre and antioxidant capacity of orange (citrus aurantium v. canoneta) by-products, food chemistry, 104(3), 1014-1024, (2007) [16]. chen, l., opara, u. l., approaches to analysis and modelling texture in fresh and processed foods – a review, journal of food engineering, 119, 497-507, (2013) title …………………… 46 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 1 2016, pag. 46 54 correlations between quality of flour t-500 characteristics and bread volume *viktorija stamatovska1, tatjanakalevska1, marija menkinoska1, gjore nakov2, zora uzunoska1, lenche mitkova3 1faculty of technology and technical sciences-veles, ss.kliment ohridskiuniversity, bitola, r.macedonia stamvikima@gmail.com 2department of biotechnology and food technologies, ruse “angel kanchev” university, branch razgrad, 47aprilsko vastanie blvd., razgrad 7200, bulgaria, gore_nakov@hotmail.com 3a.d. žito vardar, mošapijade 2, 1400 veles, r.macedonia *corresponding author received august 28th 2015, accepted march 13th 2016 abstract: the quality of flour and its quality characteristics (quantity of gluten, quality of gluten, physical and chemical properties of gluten, the ability to release a gas etc.) influence the quality of flour and that of bread. if we take into consideration whether one type of flour is of quality or not, a correct answer will be given only when its rheological characteristics are well known. the aim of the present study is to determine how quality characteristics of wheat flour t-500 can influence bread volume, showing that the quality characteristics of eight different types of wheat flour t-500 are established according to the analysis result of the rheological characteristics of the kneaded dough. thus, according to the obtained results, the dependence between the examined parameters of flour (the content of moisture, the content of ash, acid level, amylographic number, moisture gluten, extensibility, extensibility resistance, the relation re/e, energy of dough) and bread volume, is determined with correlation. there is a positive correlation between certain parameters of the examined flour and bread volume. keywords: wheat flour, quality characteristics, dough, volume, bread 1. introduction the quality characteristics of the flour depend on the relation of the chemical ingredients in the flour and the quality of those ingredients. the flour which is used for the production of bread must have such a proportion and properties of proteins and starch, to be able to fix enough quantity of water when the dough is being knead. the quality of the flour is determined according to the quality of gluten and starch. [1] from technological aspect the most important proteins that are in the flour are: gliadin and glutenin. these two proteins with adding water are growing and sticking each other making gluten (stick), which is making the structure of the dough (figure 1). there are starch grains, proteins as albumin and globulin between those bubbled and sticks molecules. in this way the so called wet gluten is produced. this wet gluten which connects all the physical characteristics of gliadin and glutenin fraction in it is considered for the most responsible for the quality of the baker’s products. [2, 3, 4] http://www.fia.usv.ro/fiajournal mailto:stamvikima@gmail.com mailto:gore_nakov@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 47 fig.1. production of gluten [5] creating of the gluten is a characteristic of the wheat flour, which is considered to have the most quality gluten, while the quality of other flour gluten is different or does not exist. [2] rheology studies complex viscous-elastic systems, among which the dough is one of the most remarkable. the dough, indeed, is not a hard body, because it shows elastic properties, and is not a liquid, although it has properties of a liquid. during the rheological examination, the size of the deformation of the shape, volume or weight is measured according to the used power in a certain time interval. [6, 7] for examination of the properties and reactions of the dough for mixing and extensibility, different measurement instruments are used, whose task imitates the conditions in the production. the most famous are: instruments as registering mixers (farinograph and mixograph) and instruments for extensibility of the dough (extensograph and alveograph). [8] farinograph is an instrument for measuring physical properties of the dough and that depends on the gluten. it is used for measuring the resistance power of the dough when it is mixed. this method gives a data for the ability of the dough to absorb water, the time needed for the growth of the dough, stability of the kneaded dough, the level of softening of the dough when it is kneaded, and also data for the quality number and quality group. [1] the surface of the gotten triangle of faring graph (cm2) is determined with planimetres. for the certain surface of the triangle, in the chart according to hankoczy-u the quality number of the dough is seen, and that number is suitable for a certain quality group (a1, a2, b1, b2, c1, c2). the surface of the triangles is between 0 to 50 cm2, quality number from 100 to 0. [9, 10] the extensograph examines the physical properties of the dough and it’s reaction of fermenting and mechanical treatment. the picture, for the quality of the dough which is received as a result or a faring-graph, is completed. the above mentioned properties are registered on a diagram (extensogram). on the extensogram are shown: energy (a), extensibility power (e) as well as resistance power of the extensibility (re), that characterizes the quality of the gluten stick. from all these indicators the proportion between the resistance power of the extensibility and extensibility (re/e =k) can be calculated, and thus a data for the dough behavior in the process of production is received. [1] amylograph is a tool which is used for determination of the quality of the starch and the action of  -amilasys, and with such an examination data for the process of cauterization of the starch from which depends humidity, elasticity, porosity, look and freshness in the middle of the bread are received. [11] in this paper the received results from the rheological examination of of eight different types of wheat flour t-500 are presented and the dependence between the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 48 examined parameters of the flour and the volume of bread is determined. 2. matherials and methods the examinations are made in the laboratory in ac.”zito vardar” – veles, republic of macedonia. subject of examination are eight different types of commercial available wheat flour t-500. in every type of the flour the following parameters are determined: content of the humidity [12], content of the ash [13], acid level [14], content of the wet gluten [15] and rheological properties with: amylograph brabender, farinograph brabender duisburg and extensograph brabender (figure 2). [9, 10, 15] from each of the examined wheat flour wheat bread t-500 is produced according to the same recipe (the same percent of additive 0.5 %, yeast 3 %, salt 1.8 %). the quantity of water is changed according to the power of soaking water on the representative types of flour. the order of the basic information of the production is given in the table 1. fig. 2. amylograph brabender, farinograph brabender duisburg and extensograph brabender table 1. sequence of the main production operations whole wheat bread т-500 technological procedure time (minutes) temperature (°с) preparation of raw materials 10 room mixing of dough (i + ii speed) 9 room dough fermentation 30 room dividing the dough into pieces 3 room curvature of the pieces 2-3 room resting of the pieces 10 room finishing formation of dough 1 room finishing fermentation 38-45 70 bread baking 30 200-250 cooling of the bread 180-300 room one of the most important and used indicator for the quality of the bread is its volume. the volume of the bread is determined with measurement of the volume of the bread according to length and height (the shorter line) of the bread. the given values are multiplied and in that way it is given how much is the volume of the bread [15]. there is a correlation among the examined parameters of the and the volume of the received bread, and pearson coefficient of correlation (r) [16] is determined, and then a statistic substantiality for the level of substantiality 0.05 is determined [17]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 49 3. results and discussion the given results from the analysis of the commercial wheat flour t-500 marked as samples from 1 to 8 are shown in table 2. in table 3 the received values for pearson coefficient of correlation (r) and statistic substantiality (p <0.05) are given. from the shown results it is concluded that the examined flour content of humidity is between 12.24 % to the flour 3 and to 14.00% to the flour 5. the received values are in compliance with the rules for the minimum conditions for making operations, quality and types of flour, the way and methods for taking samples, as methods for analysis of the quality of the flour [14], according to which the relative moisture in the flour that is put in operations mustn’t be bigger than 15.00%. table 2. characteristics of wheat flour t-500 and obtained bread volume * аu amylograph units, bu farinograph units, eu – extensograph units from the given values of the content of ash in the certain samples of flour it can be determined that the samples of flour 2, 3 and 7 are remarkable for the biggest content of ash (0.55 %), and with the smallest content of ash from 0.50 % in the samples of flour 1, 4 and 5. the received values for the content of ash in all the samples are in the borders from 0.46 % to 0. 50 %, that are in compliance with the prescribed ones for the wheat flour t-500 [14] in the relation to the acid level in the examination, the received values do not exceed the allowed maximum for this type of wheat flour (level of acid to 3.00). [14] in table 2 the values for amylograph number in the amylogram units are shown. with the read values as a bаse (from 480 to 520 ) it is considered that all types of flour have a normal ability for clustering and the production of bread in optimal area, does not show friability, with color of core characteristic for that product (amylograph unit between 260 and 650). [18] quality parameter sample of wheat flour т-500 1 2 3 4 5 6 7 8 moisture (%) 12.99 13.30 12.24 13.50 14.00 13.50 13.60 13.70 ash (%) 0.50 0.55 0.55 0.50 0.50 0.52 0.55 0.52 acid level (ml) 2.00 2.20 2.40 2,50 2.30 2.30 2.40 2.10 wet gluten (%) 18 23 23 23 23 24 23 19 water absorption (%) 56.50 55.50 68.50 56.80 66.7 57.00 59.00 56.50 degree of softening (bu) 190 100 105 90 100 80 90 110 quality number 34.10 47.40 46.70 57.70 51.00 65.40 60.30 47.90 quality group c1 b2 b2 b1 b2 b1 b1 b2 extensibility (mm) 95 115 100 125 95 124 121 105 resistance to extension (eu) 40 200 230 190 270 480 280 90 resistance to extension / extensibility (re/e) 0.42 1.74 2.30 1.52 2.84 3.87 2.31 0.86 energy (cm²) 6.00 37.20 35.00 34.50 36.40 87.20 48.50 13.20 amylograph number (au) 480 480 460 460 520 500 500 520 sample of wheat bread t-500 1 2 3 4 5 6 7 8 bread volume (cm3) 2050 2400 2500 2380 2430 2610 2600 2130 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 50 table 3 correlation between the volume of the bread and the appropriate parameters of the flour and statistic substantiality the received value of the coefficient of correlation between the content of moisture, the content of ash in the flour, the acid level of the flour and amylograph number in the examined samples of flour and the volume of the bread given in the table 3 shows that there is a technical positive correlations with a weak dependence between the changeable ones. (figure 3-6). fig. 3. the dependence of the volume of bread from the content of moisture in the flour the received values for p are bigger than 0.05, so it can be considered that the received results are not statistically important for the level of substantiality of 0.05. fig. 4. the dependence of the volume of bread from the content of ash in the flour fig. 5. the dependence of the volume of bread from the content of acid level in the flour fig. 6. the dependence of the volume of bread from the amylograph number from the shown results it is seen that the content of the moisture gluten in the examined samples of flour varies from 18% (flour 1) to 24 % (flour 6). according to the division given from kaluderski, filipovic (1998) the content of gluten in the samples 1 and 8 (18 % and 19 %, appropriately) can be tagged as small (from 14 to 20 %) the content of gluten in the samples 2, 3, 4, 5 and 7 (23 %) as quality parameter coefficient of correlation r p value moisture (%) 0.26 0.53 ash (%) 0.48 0.23 acid level (ml) 0.46 0.25 amylograph number (au) 0.39 0.15 wet gluten (%) 0.53 0.17 extensibility (mm) 0.46 0.25 resistance to extension (eu) 0.61 0.11 resistance to extension / extensibility (re/e) 0.60 0.12 energy (cm²) 0.59 0.13 y = 3.025x + 964 r² = 0.152v ol um e (c m 3 ) amylograph number (au) y = 90.059x + 1243.6 r² = 0.0679v ol um e( cm 3 ) moisture (%) y = 514.1x + 1276.7 r² = 0.214v ol um e (c m 3 ) acid level (ml) y = 3805.3x + 453.23 r² = 0.2278 v ol um e (c m 3 ) ash (%) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 51 satisfactory (from 21 to 24 %) , and the content of gluten in the sample 6 ( 24 %) as good (from 24 % to 27 %). the received value for the coefficient of correlation between the content of moisture gluten in the examined examples of flour and the volume of the bread is given in the table 3 (r=0.53), and it refers to the moderate positive correlation, which means that there is a tendency with a high variable value for the content of moisture gluten in the flour to go with a high variable value for the volume of the bread and the same in reverse order. (figure 7). with increasing of the content of the moisture gluten in the flour, the volume is increased. from the received value for p (0.17) it can be considered that the result is not remarkable for p <0.05 (table 3) fig. 7. dependence of the volume of bread from the content of moisture gluten in the flour from the obtained extensorgraph and farinograph curves of the commercial wheat flours t-500, the data related to the power of water absorption, the degree of softening of the dough during kneading, extensibility and extensibility resistance, the relative number and energy of the dough, as well as the number and the quality group of the flour (table 2). the ability of water absorption of the flour is related to their strength. from the calculated values for power of absorption of water given in table 2 it is established that the flour 3 has the greatest power of water absorption (68.50 %). because more power to water absorption means stronger flour it can be concluded that this flour is stronger than the other analyzed flours. [9] in terms of the degree of softening the sample 1 (190 fu) is with highest features, indicating poor flour. in strong flours the degree of softening is very small, while in the poor is large. [2] based on the planimetric surface of the obtained farinographic curves from hankoczy table the following parameters are reported: the quality number and a quality group of the samples are determined [10]. table 2 shows that with the lowest number of quality 34.10 is the flour 1 (quality group c1), and with the highest 65.40 is the flour 6 (quality group b1). the flour samples 4 and 7 also belong in b1 quality group. the other samples according to the quality number belong to the quality group b2. the extensograms give the following parasmeters: extensibility and extensibility resistance, and the energy and the relative number is determined (table 2). the extensibility specifies the length of the extensogram curve that is extensibility of the dough i.e gluten (mm). for good quality a mean value is required, because excessive extensibility gives molten dough, which is not favorable for obtaining optimal quality product (flour 4 with extensibility 124 mm), while small length means getting short dough, which is also considered as unfavorable (1 and 5 flour with extensibility 95 mm). [1, 2] regarding the extensibility, the value of r is 0.46 (table 3). although it is technically a positive correlation, the relationship between the variables is weak (figure 8). from the resulting value of p (0.25) it can be concluded that the result is not significant for p <0.05 the extensibility resistance (resistance to extension) is expressed in eu (extensographic units) shows the amount of the extensogram after 50 mm stretching. y = 45x + 1456.3 r² = 0.2859 v ol um e (c m 3 ) wet gluten (%) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 52 fig. 8. dependence of the volume of bread from extensibility it represents the resistance that the dough gives to extension and for good quality a greater value is desirable because a great height is a sign of strong glue (flour 6 with a resistance to stretching 480 eu). [1] in terms of extensibility resistance (table 3), the correlation coefficient is r = 0.61. this is a moderate positive correlation, meaning that there is a tendency of high value variable the extensibility resistance to go higher variable value of the volume of the bread and vice versa (figure 9). fig. 9. dependence of the volume of bread from the extensibility resistance figure 9 shows that by increasing the extensibility resistance the volume of the bread increases. the resulting value p (0.11) is greater than 0.05. the result is not significant for p <0.05. energy (cm2) indicates the surface that the extensographic curve forms. that is the energy that is consumed for extension of the dough. it should be greater because it is an indicator of the volume i.e the volume of the dough. if the energy is higher, it is considered that the flour is stronger because more energy is consumed for extension (flour 6 with energy 87.20 cm²). if the surface is small we have low energy, which means that the flour is low (flour with energy 6.00 cm²). the resulting correlation coefficient between the energy consumed for extension and the volume of bread (r = 0.59), suggests a moderate positive correlation, meaning that there is a tendency of high variable value of energy to go with high variable value of volume of the bread and vice versa (figure 10). by increasing the energy increases the volume. the p-value is 0.13 (table 3). the result is not significant for p <0.05. fig. 10. dependence of the volume of bread from energy the relation re / e provides data on the behavior of the dough during processing and despite the energy gives best indication for quality. depending on the value, the yield of bread volume, the relation between the diameter and height of bread or the characterization of the physical condition of the dough during processing can be determined. if its value is greater, the dough is shorter, and if it is less, the dough is more elastic. the flour used in baking i.e. to achieve good volume of the bread the relation of 1.5 to 2.5 is considered as the best. [1, 19] y = 6.690x + 1,710.348 r² = 0.212 v ol um e (c m 3 ) extensibility (mm) y = 0.8506x + 2257 r² = 0.3737 v ol um e (c m 3 ) resistance to extension (eu) y = 4.4563x + 2280.3 r² = 0.3456v ol um e (c m 3 ) energy (cm2) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 53 based on the values of the relation re / e (table 2) it is concluded that generally, the flours subject to analyzes are distinguished by a favorable relation (1.5 to 2.5) to achieve a good volume of bread. but, the experience has shown that the most favorable relation is the relation 2.3 (flour 3 and 7). thus, with the most favorable relations are the flours 3 and 7, which is confirmed by the values obtained for the volume of the bread produced from these flours. from the table 2 it can be seen that these flours are characterized by lower extensibility and lower extensibility resistance and less energy than the flour 6, but due to favorable relative number they do not differ in terms of the volume compared to the bread produced from the flour 6. often, when the relative number is greater than 2.3 the technological process is changed, that is the time of the first growing of the dough (sample 5 and 6), and when the relative number is less than 2.3 additives are added to increase the extensibility resistance (sample 1, 2, 4 and 8). resulting correlation coefficient between the relation re / e and the volume of the bread (r=0.60), indicates moderate positive correlation which means that there is a tendency of high value variable relation re / e to go higher variable value of volume of vice versa. (figure 11). fig. 11. dependence of bread volume on the ratio re/e by increasing the energy and volume increases too. the result is statistically significant for the significance level of 0.05 (p = 0.12) (table 3). 4. conclusion the bread is thermally cooked dough, usually from wheat flour. if the flour is good and quality, then the bread made from it will be with the required quality characteristics. the analyzed eight commercial wheat flours t-500 meet the conditions prescribed in the regulations on the minimum conditions for marketing, quality and types of flour, means and methods for taking samples and the methods for analyzing the quality of flour in terms of content moisture, ash content and acid level. from performed tests it can be concluded that the sample flour 1 belongs to the quality group c1, the flour samples 4, 6 and 7 belong to group quality b1, and the other samples belong to group quality b2. for the content of wet gluten in the samples it can be concluded that flours 1 and 8 it is a small 14 to 20 %, in the samples 2, 3, 4, 5 and 7 it is satisfactory from 21 to 24 %, and the content of gluten the sample 6 is good 24 to 27 %. based on the values of pearson's correlation coefficient (r) it can be concluded that between the respective parameters of flour (moisture content, ash content, acid level, amylographic number, wet gluten content, extensibility and extensibility resistance, a relative number and energy) and the volume of bread there is a positive correlation. with the increase in the aforementioned parameters the volume increases. 5. references [1] mitevska t., jankulovska b., food technology, ministry of education and science of the republic of macedonia, skopje, (2013). [2] žeželj m., technology grain and flour: knowledge, preservation and processing of grain, faculty of technology: institute for cereal technology, novi sad, (1995.) [3] kaluðerski g., kaluðerski s., tošič b., food technology, institute for textbooks and teaching aids, belgrade, (2006). y = 100.35x + 2247.3 r² = 0.3546v ol um e (c m 3 ) resistance to extension/extensibility food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 viktorija stamatovska, tatjana kalevska, marija menkinoska, gjore nakov, zora uzunoska, lenche mitkova, correlations between quality of flour t-500 characteristics and bread volume, food and environment safety, volume xv, issue 1 – 2016, pag. 46 – 54 54 [4] wieser h., chemistry of gluten proteins, food microbiology, 24 (2): 115-119, (2007). [5] http://grist.org/food/2011-11-10-gluten-why-allthe-fuss/ mattes m., food studies: what’s up with gluten?, (2011). [6] canja m. c., lupu i. m., pădureanu v., study on rheological behavior of bakery dough 5th international conference "computational mechanics and virtual engineering " comec 2013, 2425 october 2013, brasov, romania, 383387, (2013). [7] amjid r. m., shehzad a., shehzad h., shabbir a. m., khan r. m., shoaib m., a comprehensive review on wheat flour dough rheology, pakistan journal of food sciences , 23(2): 105-123, (2013). [8] mironeasa s., gutt s., gutt g., codina g. g., rheological behavior of wheat flour during mixing and heating, annals of daaam for 2011 & proceedings of the 22nd international daaam symposium, volume 22, no. 1, 0107-0108,(2011). [9] žeželj m., technology grain and flour: processing flour, nip glas javnosti doo, belgrade, (2005). [10]http://www.sraspopovic.com/baza%20znanja% 20dokumenti/polj.i%20prehr/iv%20razred/vjezbe %20za%20analizu%20zita%20i%20brasna.pdf; chemistry and technology of grain, instructions for laboratory exercises, food technology and biotechnology, zagreb, 2010/2011. [11] hadnađev d.t., pojić m., hadnađev m., torbica a., the role of empirical rheology in flour quality control, wide spectra of quality control, dr. isin akyar (ed.), (2011). isbn: 978953-307-683-6, in tech, doi: 10.5772/24148. available from: http://www.intechopen.com/books/wide-spectra-ofquality-control/the-role-of-empiricalrheology-inflour-quality-control. [12] pajin b. (2009). exercises of technology confectionary, faculty of technology, novi sad. [13]http://www.nebraskawheat.com/wpcontent/uploads/2014/01/wheatflourtestingmetho ds.pdf; wheat and flour testing methods: a guide to understanding wheatand flour, wheat, marketing center, inc.,portland, oregon, usa, (2004). [14] regulations on minimum conditions for marketing, quality and types of flour, means and methods for taking samples and the methods for analyzing the quality of flour (official gazette, no. 24, 2014). [15] kaluðerski g., filipović n. test methods for quality of grain, flour and products. faculty of technology, novi sad, (1998). [16]http://www.socscistatistics.com/tests/pearson/d efault2.aspx; pearson correlation coefficient calculator, social science statistics. [17]http://www.socscistatistics.com/pvalues/pearso ndistribution.aspx; p value from pearson (r) calculator , social science statistics. [18] klarić, f., 2010: handbook of bakery and confectionery, library bread of life, tim zip doo zagreb ; original: albrecht t., ehrlinger, hg., willeke, e., schild e., 2009: fachkunde in lernfeldern, backer/backerin, praxis und theorie, fachbuchverlag panneberg gmbh &co.kg, han gruiten, 602, detschland. [19] stefčeska v., masin a., food technology, educational activity, skopje, (1982.) http://www.sraspopovic.com/baza%20znanja%20dokumenti/polj.i%20prehr/iv%20razred/vjezbe%20za%20analizu%20zita%20i%20brasna.pdf http://www.sraspopovic.com/baza%20znanja%20dokumenti/polj.i%20prehr/iv%20razred/vjezbe%20za%20analizu%20zita%20i%20brasna.pdf http://www.sraspopovic.com/baza%20znanja%20dokumenti/polj.i%20prehr/iv%20razred/vjezbe%20za%20analizu%20zita%20i%20brasna.pdf http://www.intechopen.com/books/wide-spectra-of-quality-control/the-role-of-empiricalrheology-in-flour-quality-control http://www.intechopen.com/books/wide-spectra-of-quality-control/the-role-of-empiricalrheology-in-flour-quality-control http://www.intechopen.com/books/wide-spectra-of-quality-control/the-role-of-empiricalrheology-in-flour-quality-control http://www.nebraskawheat.com/wp-content/uploads/2014/01/wheatflourtestingmethods.pdf http://www.nebraskawheat.com/wp-content/uploads/2014/01/wheatflourtestingmethods.pdf http://www.nebraskawheat.com/wp-content/uploads/2014/01/wheatflourtestingmethods.pdf http://www.socscistatistics.com/tests/pearson/default2.aspx http://www.socscistatistics.com/tests/pearson/default2.aspx http://www.socscistatistics.com/pvalues/pearsondistribution.aspx http://www.socscistatistics.com/pvalues/pearsondistribution.aspx there are starch grains, proteins as albumin and globulin between those bubbled and sticks molecules. in this way the so called wet gluten is produced. this wet gluten which connects all the physical characteristics of gliadin and glutenin fraction in... 5. references heat transfer in down flowing turbulent liquid films with the development of wavy structure at the regime of evaporation from the interface and with co-current steam flow 284 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv , issue 4 2016, pag. 284 298 ştefan cel mare university of suceava, romania volume , issue 12016, pag. ... ... heat transfer in down flowing turbulent evaporating liquid films with developed wavy structure and co-current steam flow valentyn petrenko1, *yaroslav zasyadko1 1national university of food technologies, thermal fluids and industrial refrigeration dept. 68, volodymyrska st.; 0160 kyiv; ukraine petrenkovp@ukr.net, *yaroslav@nuft.edu.ua *corresponding author received 2nd december 2016, accepted 29th december 2016 abstract: the result of heat transfer modeling in down flowing turbulent films with the developed wavy structure at the regime of evaporation from film interface at free falling and with co-current steam flow, a heat transfer model which takes into account a cyclic process of temperature field relaxation with the periodic mixing of film by big waves, has been presented. the temperature profiles along with the integral heat transfer coefficients (htc) were obtained as a result of approximate solutions of heat transfer differential equations of in turbulent flows, adapted thus to film flux with large surface waves, utilizing turbulent transport model by m.d.millionschykov. the results of experimental research of heat transfer processes to water and sugar solutions with concentration up to 70% in the regime of evaporation from the interphase are presented. key words: films, waves, heat transfer, temperature, turbulent, evaporation. 1. introduction the majority of existing heat transfer models applied to the flowing over the vertical surface films at wide variation of regime conditions, parameters and physical properties usually are based upon the application of the flow films’ effective film conduction. these variations include: films heated to the boiling point and those that are being heated; turbulent and laminar once and many similar. the list of correlations for heat transfer coefficients (htc) calculations in films at modes of heating and evaporation from free surface are given in works [1,2,3,4]. fundamental analysis of heat transfer and hydrodynamic processes in films was made by v.m. sobin [5]. analysis is based on integral methods of solving corresponding differential equations for stabilized laminar and turbulent flows as well as those, which develop their structure with boundary conditions of the 1,2,3 and 4 kind. nonetheless, the given analysis does not take into account the wave structure of film’s surface and contains no relevant characteristics of wave film flow. nomenclature y –normal to the heat surface coordinate; x –longitudinal to the heat surface coordinate; t –temperature; http://www.fia.usv.ro/fiajournal mailto:petrenkovp@ukr.net mailto:yaroslav@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 285   y –dimensionless transverse coordinates;   x –dimensionless longitudinal coordinates;     satw sat tt t,t ,    –dimensionless temperature;  – film thickness; vг – volumetric liquid flux; a –temperature conductivity; ta –turbulent temperature conductivity;  –cinematic viscosity coefficient; t –cinematic turbulent coefficient; satt –saturation temperature; wt – wall temperature;  –heat conduction of liquid; q – heat flow; c –heating capacity;  –liquid density; 2 –steam density;  – surface tension; wavа – wave thermal diffusivity coefficient; bwf –the frequency of the big wave’s; bwa –the amplitude of the big wave’s; bwc –the face velocity of the big wave’s; ll –the thickness of the remaining film layer between big waves; iu – liquid velocity on film interphase; u – average liquid velocity on film;     gu i – dynamic velocity; 2u − steam velocities; i –shear stress on the film interface; g –acceleration of gravity; a u a г pe v   44 –the peclet number;   v г re 4 –the reynolds number; pr the prandtl number; tpr –turbulent prandtl number; 2 2 22 2    g u we −the weber number;  –interface friction coefficient. the influence of waves on heat transfer in stabilized film was taken into account directly in the articles of e.m.vorontsov [6,7,8] by introduction of wave thermal diffusivity coefficient wavа   dy dt aacq wav , (1) with the use of average characteristic of big waves, such as speed of its transverse pulsations bu llbwbwllbwav faua  2 , (2) according to the conclusions [9], equations (1) and (2) show a good correlation with the experimental data only in terms at low flow rates and undeveloped turbulence at 800re . later the author [9] concluded that a process of heat transfer through the turbulent film is advisable to present as:   dy dt aaacq twav  , (3) the actual character of the film flow with the developed big wave structure proves that the process has a twodimensional character within the residual films layer and its cyclicity is being determine by the hydrodynamics of big wave movement. according to [10], in case of free falling films over the vertical surface the saturation of wavy movement with fully developed big wave’s structure occurs at a distance approximately 2…2.5 m from the film food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 286 forming devise. an important factor, which seriously effects a hydrodynamic film structure is the fact that big wave's contain a central swirl [11, 12]. due to this, the bulk of the liquid film is periodically mixed, which in turn causes the velocity, temperature and concentration profiles deformation. taking into account all mentioned above, the heat transfer in liquid films may be treated as a cyclic process of temperature field relaxation, which happens after each consequent passage of a big wave. this big wave due to the existence of a powerful central swirl, which transports slightly, superheated liquid from the boundary region to the external of film. according to this mechanism the peak of a big wave will be constantly fed with the slightly superheated liquid and transfer it to the wave surface, from which evaporation takes place, thus transferring heat to the steam core. the process of relaxation is stretched along the film movement until a consecutive mixture of liquid happens due to the passage of the consecutive wave. the extended analytical results of heat transfer in liquid down flowing saturated films of solutions based upon the proposed model of cyclic bulk mixing by large waves with the following temperature profile relaxation between the consecutive waves passage are presented in [13]. 2.materials and methods a direct experimentation of heat transfer in down flowing liquid films heated to the saturation temperatures with sugar solutions as model liquids has been carried out at the experimental unit with the independent formation of phases’ mass flow rates and heat flux. the main core of the experimental unit was represented by a stainless still pipe with the inside diameter of 20 mm and 1.8 m long. the experimental tube was separated into the initial 1.5 m stabilization section and 0.3 m measurement section. the down flowing of water (sugar solutions) film has been formed by means of overflowing over the tube’s upper rim. in the event of steam-liquid flow modeling, dry saturated steam has been supplied in co-current regime. the liquid falling film has been heated by dry saturated steam which was supplied into outside heating sections attached to the experimental tube. the heating chambers were designed in such a way as to provide an individual heating of the stabilization section and the experimental one. the said sections were hooked up to the individual vacuum-condensation sections which allowed for the keeping of different pressures in each chamber. such arrangement allowed also maintaining vacuum down to 0.8 bars and thus, vary the temperature head between the heating steam temperature and evaporation temperature. special probes for taking samples of liquid to determine its concentration and measurements of temperatures were positioned directly after the measurement section. a detailed description of the experimental unit is given in [4], or [13]. 3. results and discussion. the bigger is liquid flow rate in the film and the lower its viscosity the turbulence in the film appears and develops. the character of turbulence distribution within the film’s cross section and especially in films with the developed wavy structure and massive food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 287 swirl in big waves is undetermined. in this case it can be assumed that right before the passage of a big wave, the velocity profile will be developed (corresponding to the 1/7 low). the temperature profile will be close to the linear one. right after the wave passage, similarly to the laminar movement, as a result of mixing the velocity profile will be constant across the film and the temperature profile will become curved, figure 1 y u t t u u u a b 2 2 x 7 1         y uu i bwc bwc fig.1 schematics of a big wave passage on the surface of the turbulent film: velocity and temperature profile (a) before the big wave passage; (b) – after. under these conditions, taking into account, that turbulence intensifies heat transfer, when analysing temperature film fluctuation as a result of film cyclic mixing by big waves the regime 0 1     can be separated for every  between consequent big wave passage. then the equation of heat transfer in film during the period between big waves passage may be given as:   y t aa yx ty u ti                7 1 , (4) taking into account, that, 7 1 7 1 7 1 7 8 7 8                u y u y uu i , in the dimensionless form (4) can be written as                   , aa , u t7 1 7 8 , (5) replacing the left side by average value:               avu , u 7 1 1 0 7 8 , b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 288 yields:                   , a ape tav 1 4 , (6) limiting temperature curve, at which the absence of evaporation from film surface takes place, may be found from (6) at the boundary conditions 0 , ; 1 , 0   . (7) whereas, t tt pr pr a a    , the expression (6) considering (7) take the form     1 11 4 c pr pr v d pr pr dpe , t t t t av                                             , (8) where 1c – integration constant. a characteristic feature of the turbulence development in the films is supression of turbulence intensity in the layer close to the wall surface that is proved experimentally by jepsen [14], lamurell-sandal [15], and ganchev [16]. for free downflowing viscous films the turbulence viscosity profile, generally, is set layer by layer [17]; in the boundary layer– by van-drist correlation [18], in the middle layer – by reyhardt [19] , on the external film surface lamurell-sandal [15]. another form of turbulence in the film is given by m.d. millionschikov [20], according to which the laminar layer with a thickness of 87.l   exists, on whose limits 0  t , than a turbulent layer has place with a parabolic profile of turbulent viscosity in the range 1 87    .       1390 l t . , (9) where         lll ;    u . l 87 ;      u . taking into account, that the experimental data of turbulent intensity were obtained in the experiments conducted determining is completed on the inclined surfaces and at big wave’s absence [14,15,16], the received results do not represent the real picture of turbulence development in films on the vertical surfaces with developed wave structure and are not in adequate. the only the fact of turbulence suppression near the hard solid walls and interphase surface may be accepted, since a big wave contains powerful central whirl, it’s rolling on the film surface should be accompanied with the turbulent pulsation and micro-whirls penetration into the boundary layer. under these conditions, instead of layer by layer model with boundary laminar layer and turbulent core with parabolic distribution of turbulent intensity (9), [20], the simplified model of equivalent turbulence may be used. here, the turbulence develops directly from the wall, and its maximum in the central part is determined by comparison of the calculated heat transfer intensity with experimental data. under these assumptions the function of turbulence in the film should be given as: 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 289  24    m t , (10) where m – turbulence function in the center of film, which should be determined. if 1tpr , the equation (8) should be given as           122 41414 c pr d pr dpe , mm av                 , (11) and its integration, under the condition that 0 , 1 , gives          1 4 112 4 1 14 8 1 4 2                                           h pr arth hh pr arth h prln prpe , mm m mav , (12) where 22 prprh mm  the temperature is determined as the bulk mean:          1 0 7 11 0 7 8 d,d u u ,av . (13) the analytic solution of the integral (13) does not exist, but, taking into account the profile of 1/7 power, with a particular approach about the average temperature, lets replace it with a constant, then          1 0 1 0 d,d u u ,av .(14) to evaluate the correctness of replacement (13) to (14), the linear temperature profile  1 which is automodeling at ξ→∞, and power-mode  1 , which approximately corresponds to the temperature profile in the film after big wave passage may be substituted in (13) and (14) respectively. the difference in mean temperature values by (13) and (14) for the first case does not exceed 7 %, for the second – 9%. the integration of (14) gives   12 1 4 1 8 1 4                            prh pr arth hprd dpe m m m av av . (15) marking:                      2 pr 1pr 4 1 pr8 1 4 m m m h arth h pe ab    and replacing (15) yields: bbd d av av 11    , (16) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 290 with boundary conditions 0 ,   satt,t  0 ; 0av , which after integration gives:               b expav 1 . (17) substituting the derivative of (17) by  in (12), and replacing the index at  by m one receives a correlation for boundary curve, at which 0 1                                                          h pr arth hh pr arth h prln pr b exp b pe , mm m mm m 4 112 4 1 14 8 1 1 4 1 2 . (18) the coordinate m can be found out from (18) providing that at m and 1 ; the dimensionless temperature is equal to zero, in other words   0,1  m          h pr arthpe hb lnb m m 8 . (19) the region m is characterized by fact that the temperature of film surface remains constant; consequently the boundary condition (7) is changing into: 0 , 1 ; 1 , 0 , (20) the film temperature field in the period between big waves passage m develops from the boundary curve (18), at m , to the current temperature field at the distance v , at which the next mixing of the film by big wave takes place. double integration of (6) with the function of turbulent viscosity distribution (10) and boundary conditions (20) yields:     2 1 2 2 1 441 11 32 2                            h pr arth h prpr arth prpr ln pr pe , m mm mmm av (21) the derivative   d d av in equation (21) will be found by the mean bulk temperature (21) considering boundary condition (18). the average temperature of the film is:           1 0 1 0 d,d u u ,av   2 11 1 1 16                 h pr arth h pr pr pe mm m av . (22) denoting                   h pr arth h pr pr pe s mm m 1 1 1 16 , food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 291 from (22) one receives the deferential equation     0 2 11    ss av av , (23) whose solution is:         s expcav 22 1 . (24) the integration constant 2c will be found from the boundary condition (18) considering that at m , m.avav  , the average temperature m.av from the boundary curve (18) at m reads:     1 0 d,mm.av                                          h pr arth hh pr arth hpr pr pr b b exp pe mm m m m m 4 1 4 1 4 1 4 1 (25) substituting the received meaning m.av in equation (24) instead of av , we will find the integration constant 2c               s expc mm.av 2 1 2 , eventually, the expression for the mean temperature takes a form               s exp mm.avav 2 1 2 1 (26) substituting the derivative of av by ξ (21), one finally receives the expression for temperature in the film distribution in the period between big waves passage.   . h pr arth h prpr arth prpr ln prs exp s pe , m mm mmm m m.av 2 1 2 2 1 441 112 1 32 2                                                (27) the graphical interpretation of temperature field development in the film along the heating surface between the big waves passage is shown in figure 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 292 1 6 2 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1.0 1.0 7 3 8 4 5   ,  fig. 2 the dimensionless temperature profiles between big wave passage for water by (27) in the region ξ > ξm re =6780; t = 100 oc; εm = 1; гv = 0.5 10-3 s m 2 . 1 – ξ = ξm = 7.3; 2 – ξ = 15; 3 – 30; 4 – 60; 5 – 100; 6 – 150; 7 – 300; 8 – 1000 from (27) derivative of 0  on the boundary:                                        h pr arth h pr h pr s exp s pe d ,d mm mm m.av 2 22 0 1 2 1 8 (28) heat flow on the boundary:           0 0 d dtt q satw                                                  h pr arth h pr h pr s exp s pett mm mm m.av satw 2 22 1 2 1 8 (29) respectively, the mean heat flux will be found as:          v m dqq mv cp 0 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 293                                                           h pr arth hpr s exp prh prh pe hpr tt m mvmmv m mm.avm.av mmv satw 1 228 222 222 222 , (30) the heat transfer coefficient (htc) is determined as iw av tt q   , then from (30) one receives the expression:                                                         h pr arth hpr s exp prh prh pe hpr m mvmmv m mm.avm.av mmv 1 228 222 222 222 (31) the length of big waves bw respective to [18] provided that free water downflowing on the vertical pipe with a diameter of 25 mm may be approximated by the correlation  4001077120 6   re..bw at 400re , 120.bw  at 400re (32) the thickness of turbulent film free downflowing on the vertical surface according to the data [22, 23] closely complies with the velocity distribution in the film law of 1/7 according to which: 12 7312 1350 re g . /           (33) at 2060re at 2060re according to [22, 23] the film thickness fit with the expression for laminar liquid film: 3 3 g г v  (34) the function m is determined by the comparison of htc, calculated by (31), with experimental data with saturated turbulent and laminar liquid films in the mode of evaporation from interphase during the free downflowing.                        80000 re exp1 re1015.0 2 45.0 95.03    om (35) at 2060re ,   83.03 245.0 95.03 2060re1 63.0 1 exp104.0 80000 re exp1re1015.0                                       o o m      at 2060re , where s m ,.o 2 61030  . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 294 graphical interpretation of correlation (35) for sugar solutions and water is shown at figure 3. 1.0 0.8 0.6 0.4 0.2 1 2 3 4 5 6 7 1200 2400 3600 4800 6000 re em fig. 3 correlation  refm  for sugar solutions and water at ct o100 1 – water; 2 – 7sugar solution, 2 – dm = 20%; 3 – 30%; 4 – 40%; 5 – 50%; 6 – 60%; 7 – 70% a comparison of experimental data on heat transfer to sugar solutions and water films downflowing on the vertical surface to those calculated for the evaporation regime with interphase is shown in figure 4. 10 4 s m2,гv 1 2 3 4 5 6 7 4000 3000 2000 1000 4500 3500 2500 1500 500 1.2 2.4 3.6 4.8 6.0 a w кm2, 1 2 3 4 5 6 fig.4 a comparison of experimental data on heat transfer to sugar solutions and water films downflowing on the vertical surface to those calculated for the evaporation regime with interphase the lines correspond to the calculations by correlations (31, 32, 33) 1 – water, t = 100 с; 27 – sugar solution, 2 – dm = 20%; 3 – 30%; 4 – 40%; 5 – 50%; 6 – 60%; 7 – 70%. points: 1 – water, t = 100 с; 26 – sugar solution, 2 – dm = 30%; 3 – 40%; 4 – 50%; 5 – 60%; 6 – 70% a turbulent film thickness in the presence of interphase shear stress may be determined from the momentum conservation equation.                        d dug ti 11 2 , (36) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 295 using the m.d.millionschicov expression (10) for the turbulent viscosity, in which, according to [23], the constant coefficient 0.39 is changed to a function.   98041053213280 .reexp..m  at 1940re (37) 0m at 1940re in the laminar film layer, the velocity profile can be found as:   2 222                   gg u il , (38) and in the turbulent regime:               , gg mm mm ln m g n m arctg n m artcg n gg n u l l i llll l l ll li t 211 11 2 1 2 21 2 222 2 22 22                                                                                                            (39)   mmmmn ll 42 22222 . having calculated the average velocity of the liquid in the film:   ltll uuu 1         l l dudu tl 0 1 (40) where       l duu l l l 0 1 ;       1 1 1 l duu t l t ─ average velocities in the laminar and turbulent layers respectively, the thickness of the turbulent film in the presence of interphase friction is received, as u/гv . a comparison of the calculated water film thickness with the experimental data, depending on the air velocity in the tube with the diameter of 25 mm in the mode of developed turbulence is shown in the figure 5. 4 0.3 0.6 0.9 1.2 1.5 12 20 28 36 u2 m/s d, mm fig.5 a comparison of the calculated water film thickness with the experimental data, depending on the air velocity in the tube with the diameter of 25 mm in the mode of developed turbulence. points –it is data [21] water with the temperature of 25oс. d = 25 mm. water consumption 4103 vг m2/s. re = 13200. the line – is a correlation (37, 38, 39, 40) the function m in the case of interphase friction existence differs from (35) and is determined as um e . the type of multiplier to (35) ue is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 296 determined by a correlation of the measured results of heat transfer intensity in turbulent flows with the interphase friction to the calculated by (31,32,33,37,40). obtained thus formula for ue reads: 24150 15 08050 15 80 2 2 2 .. u .. u .eu              at 142 u 60 60 7020 2 2 220 2 14 721241 . . .. . u re g we.e                             at 142 u , (41) in the case of 0ue , ue =0. graphical interpretation of the correlation (41) is given in the figure 6 4 12 20 28 36 0.15 0.45 0.75 1.05 1.35 1 2 3 4 u2 m/s, ue fig. 6  2ufeu  correlation for water at ct o100 . 1 – гv = 0.2 10-3 ,m2/s; ; 2 – 0.3 10-3; 3 – 0.4 10-3; 4 – 0.5 10-3 in case of the co-current steam flow a shear stress appears on the interface, which is determined as 8 2 2 2 u i  , the value of interface friction coefficient can be determined from [11]. suppression of turbulent intensity in the film with the increasing of steam velocity above 14 m/s is explained by the significant film thickness falling as a result of interphase friction, figure 5. a comparison of the experimental data of htc to sugar solutions with concentrations up to 70% ( s/m. 2610773  ) and to water at the film downflowing on the vertical surface with the interphase shear stress with correlations (31,32,35,37,40,41) are shown in the figure 7. as it can be seen from the given plots, the correlations (31, 32, 35, 37, 40, 41) are also valid within not only the turbulent bat in viscous laminar range. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 297 a w кm2, 1000 100 0 20 30 40 2000 3000 4000 5000 6000 7000 11 3 3 4 4 5 5 6 6 2 2 u2 m/s, fig. effect of steam flow on the htc  2uf for sugar solutions and water at t = 100 oc 1,2 − water; 3, 4 – sugar solution, dm = 40%; 5, 6 – sugar solution, dm = 70%; 1, 3, 5 – гv = 0.3 10-3 ,m2/s; 2, 4, 6 – гv = 0.5 10-3 ,m2/s. lines – a calculation for (31, 32, 35, 37, 40, 41) 4. conclusions 1. big waves downflowing in the vertical channels that have a powerful central whirl which in turn periodically mixes the bulk of the film exerts a main impact on the heat transfer. 2. at the presence of big surficial waves it is advisable to utilize a developed simplified turbulence model (10) equation, which is a modification of m.d.millionschykov model [20]. thus developed model being applied, allows obtaining an analytical solution of the heat transport differential equations and eventually obtaining simplified integral process characteristics. 3. the equations (30, 31, 34, 36, 39, 40) are recommended to be used for the calculation of htc to the water and sugar solutions (up to 70% dm) at a regime of free falling and the presence of co-current steam flow, either. the equations may be applied to the films at a regime of evaporation from the interface of laminar and turbulent films within the marked above range of flow rates. 5. references [1]. gimbutis g., heat transfer in a liquid film gravitational flow, vilnius: 233, (1988) [2]. vorontsov.e.g., tananayko y.m., heat transfer in liquid films, technique k.: 196 (1972) [3]. tananayko y.m., vorontsov e.g., methods of calculation and research of film processes, technique k.: 312 (1975) [4]. riabchuk о.м., thermal and hydrodynamic processes evaporation in downstream film flows of sugar solutions, dissertation, kyiv: 167 (2013) [5]. sobin v.m., heat and mass transfer in the film flows at complicated conditions. mh science and technology: 303 (1994) [6]. vorontsov e.g., hydrodynamics and heat exchange in the watering chemical films equipment, ph.d dissertation. mm: inorganic chemistry, academy of sciences ussr: (1989) [7]. vorontsov e.g. research of average wave characteristics in the calculation of heat and mass transfer in films. heat and mass transfer, mmf-92. minsk, t.2: 43 (1992) [8]. vorontsov e.g., untersuchung mittelstatistischer charakteristiken von wellenströmungen in rieselfilmen, brennstoff wärm kraft, bd.42. no12: 730, (1990) [9]. vorontsov e.g., the thermal diffusivity of falling films. tocht, vol. 33, no 2: 117127, (1999) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2016 valentyn petrenko, yaroslav zasyadko, heat transfer in down flowing turbulent evaporating liquid films with the developed wavy structure and co-current steam flow, food and environment safety, volume xv, issue 4 – 2016, pag. 284 – 298 298 [10]. ganchev b. g., cooling of nuclear reactor of elements by film flow, energoatomizdat, moscow: 192 (1987) [11]. pryadko м.о., globa о.v., forsyuk а.v., globa v.z., film flows in tubes of apple juice evaporators, scientific works of national university of food technologies, vol. 21, no 2: 88 – 95, (2015) [12]. demekhin e.a., kalaidin e.h., rastaturin a.a., effect of wavy regimes upon the mass transfer in down flowing liquid film, termophysics and aerodynamics, vol. 12, no 2: 259-269, (2005) [13]. petrenko v.p., zasyadko y.i, heat transfer modeling downflowing laminar films the developed wavy structure with cocurrent steam flow, food and environment safety, vol.15, issue. 3: 203-215, (2016) [14]. jepsen j.c.,crosser o.k.,perry r.h. the effect of wave induced turbulence on the rate of absorption of gases in falling film. am.inst.chem. engng. journal, (1966), vol.12, no 1, pp.185 – 192. [15]. lamourelle a.p., sandal o.c.gas absorption into turbulent liquid. chem.engn.science. (1972). vol. 27, pp.1035– 1043. [16]. ganchev b.g., kozlov v.m. experimental research of a hydrodynamic liquid film structure with the free downflowing on a vertical surface. tohti, (1973). v.7, no 5, pp. 727 733. [17]. grossman g., heath m. simultaneous heat and mass transfer in absorption of gases in turbulent liquid films. int. j. heat mass transfer. (1984). vol. 27, no 12, pp. 2365 – 2376. [18]. van driest e.r. on turbulent flow near a wall. j. aeronaut. sci. (1956), vol. 23, pp. 1007– 1011. [19]. reichardt h.,angev z. math. mech. 31, 208 (1951); w. m. kays, convective heat and mass transfer, p.71. mcgraw–hill, new york ( 1966). [20]. millionschykov m.d. basic laws of turbulent flow in the boundary layer. – nuclear power, (1970), vol. 28, issue. 4, pp. 317 – 320. [21]. kulov n. n., hydrodynamics and a mass exchange in the film and disperse streams descending, dissertation, moscow, (1984), p.409 [22]. petrenko v.p., pryadko m.o., riabchuk о.м film thickness in descending circular flow with interfacial interaction. labor nuft , (2016), vol. 22, no 3, рр. 150 -156. http://enuftir.nuft.edu.ua/jspui/browse?type=author&value=globa%2c+alexander http://enuftir.nuft.edu.ua/jspui/browse?type=author&value=forsyuk%2c+andriy http://enuftir.nuft.edu.ua/jspui/browse?type=author&value=globa%2c+alexander where ; ; . with boundary conditions , ;, which after integration gives: (39) issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume x, issue 3 30 september 2011 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava http://www.fia.usv.ro/fiajournal editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates,bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius,lithuania norbert kreuzinger vienna university of technology, institute for water quality,resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic ofmoldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: indexcopernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal:@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts:@fia.usv.ro mailto:gabriela.constantinescu:@fia.usv.ro mailto:cristina.hretcanu:@fia.usv.ro 240 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 3 – 2013, pag. 240 246 determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts micşunica rusu1 1faculty of food engineering, stefan cel mare university of suceava, suceava, românia, rusu_mic@yahoo.com * corresponding author received august 11st 2013, accepted september 3th 2013 abstract: oregano is an aromatic plant used both as a condiment and for medicinal purposes, with a great number of antioxidant compounds (43). the study analyzes the main groups of antioxidant compounds – flavonoids, polyphenolcarboxilic acids and polyphenols and the antiradical capacity of this plant. the determinations are done spectrophotometrically, based on the reactions with alcl3 – for the flavonoids, with arnow reagent – for the polyphenolcarboxilic acids and folin – ciocâlteau reagent for total polyphenols. because it is very important to know the way the observed antioxidant compounds can be extracted from plants eight extractions from two samples of dried oregano – condiment and medicinal plant were performed: with water, with methanol solution 50% and ethanol, hot and cold, and two mixtures: of methanol – water – acetic acid and methanol – acetone – water – formic acid, cold. water and methanolic solution had the capacity to extract the compounds both from the condiment and the medicinal plant, the temperature influencing in a positive way the extractability of the antioxidant compounds. a valuable extract was obtained at low temperature, too, with the mixture of solvents methanol – acetone water – formic acid (s5). its scavenger activity against the free radicals, determined by the capacity of elimination of the radical 2,2-diphenyl-1-picrylhydrazyl (dpph), was very good in the majority of the extracts. at the opposite end, there was the extract with ethanol, at low temperature. keywords: flavonoids, polyphenolcarboxilic acids, total polyphenols, solvents, temperature influence 1. introduction herbs are used both with culinary and medicinal purposes, mainly due to their special antioxidant potential [1]. from the multitude of herbs with high antioxidant capacity, oregano, which is the best selling product in the world, was selected for analysis. under this name there are sold dried leaves and flowers of origanum vulgare l. subsp. hirtum or origanum onites l. or a combination of both. according to franz [2], there are at least 68 species of plants sold worldwide with the name of oregano. in kitchen, oregano has been used for a long time to spice sauces, salads, egg dishes, stew, soups, vegetable or meat courses – beef, poultry, fish [3] and also pizza, sausages, especially italian, greek, mexican recipes. it is also a good substitute for salt in products which contain tomatoes. from a medical point of view, oregano has antispasmodic action on smooth muscles, sedative effect on the central nervous system (and especially on respiratory centers), tonic and slightly astringent, due to tannins and bitter substances, according to [4], anti-aging effect (due to its components with antioxidant potential) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 241 etc., being recommended as a tincture or infusion. oregano is among the plants with a high number of antioxidants (34 were identified), according to the database of usda [5]. the main groups of compounds with antioxidant potential in oregano are the flavonoids, polyphenolcarboxilic acids and polyphenols. the main antioxidant compound identified in many studies was the rosmarinic acid [6]. the study aims to analyze the main groups of antioxidant compounds from two samples of oregano – condiment and medicinal plant – dried, and the way in which they are extracted in different solvents, in certain conditions. the type of plant used and the method of extraction of the useful compounds are very important. 2. materials and methods for the research, condiment and medicinal plant were used, bought dried. in order to analyze the way in which the type of solvent and the extraction temperature influence the extraction yields of the main compounds with antioxidant potential, eight extractions were carried out, in parallel, for oregano as condiment (k) and medicinal plant (m) – (table 1): five extracts at low temperature and three at high temperature. for this, 2 g from the air part of the dried plant were extracted, using 20 ml of solvent for each. the extractive solution was filtered through filter paper and a volume of 20 ml was obtained with the solvent for each test. filtered extractive solutions were analyzed in terms of dry weight content (d.w.) and flavonoids (f), polyphenolcarboxilic acids (ac. pf.), total polyphenols (pf) and antiradical capacity. table 1 extractions realized working conditions type of extraction extracts obtained working temperature working conditions symbol solvent extraction at cold / low temperature s1r water room temperature extraction with agitation, for 30 minutes, then filtration s2r methanol solution 50% (50:50, v/v) s3r ethanol 96 0 s4 methanol – water – acetic acid, mixture (90:9:1, v/v/v) s5 methanol– acetone – water – formic acid, mixture (40:40:19,9:0,1, v/v/v/v) extraction at hot / high temperature s1c water 85 0c extraction for 30 minutes, then filtration s2c methanol solution 50% (50:50, v/v) 65 0c s3c ethanol 96 0 70 0c a. the content of dried substance (the extracting substance) was determined on the principle of mass loss by heating in an oven, at the temperature of 103±2°c, till constant mass, of a quantity of analysed sample. b. the chemical study aimed to quantify the flavonoids, polyphenolcarboxilic acids and total polyphenols, being known that aromatic plants contribute significantly to the enhancement of the antioxidant activity [7]. b1. to determine flavonoids, the spectrophotometrical method was used, based on the reaction with alcl3. the content of flavonoids of the analysed samples was expressed in g rutoside / 100 g dry plant (d.p.). b2. the determination of polyphenolcarboxilic acids is done spectrophotometrically and it is based on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 242 colour reaction with arnow reactive, in alkaline medium. the content of polyphenolcarboxilic acids from the analysed samples is expressed in g rosmarinic acid / 100 g dry plant (d.p.). b3. the polyphenols were determined by treating the extracts with folin – ciocâlteau reactive, when a blue complex (whose colorimetry is at λ= 760 nm) is formed, using as a reference gallic acid (gae). the total content of polyphenols is expressed in g gae / 100 g d.p. and g gae / 100 g dry substance (d.w.). c. the evaluation of the antiradical action was done by measuring the capacity of elimination of the radical 2,2-diphenyl-1picrylhydrazyl (dpph), and its conversion into its reduced form, by the analysed plant extracts. the antiradical activity was expressed as a percentage. 3. results and discussion the working process was led both at room temperature and reflux temperature, with several types of extraction solvents. some of them are mentioned in and recommended by specialised literature (especially the methanol solution 50%), as well as other extraction solvents can offer information about the yields of extraction of aimed compounds in catering (water and alcohol), but also in combinations which are used in obtaining better compound extraction yields. a. in determining the extract (table 2) one can observe that most extractions were done at a high temperature, with methanol solution, for k sample (1.89 g / 100 ml) and with water, at high temperature, for m sample. the lowest values were obtained for extracts with ethanol, performed at low temperatures (0.568 g / 100 ml for the condiment and 0.24 g / 100 ml for the medicinal plant). a general analysis of all the values leads to the conclusion that the extraction at low temperature is less efficient, except for the extract s5, with a complex mixture, especially in the case of the medicinal plant. taking into account the way of obtaining the extract, the efficiency of the extraction was determined, as the ratio of dry substance from the extract and the mass of dry substance from the plant material, according to materska [8]. table 2 dry substance from the extracts used in analysis and the efficiency of the extraction no. sample (extract) dry substance from the extracts (g/ 100 ml) efficiency of the extraction (%) k m k m 1. s1r 1.322 1.01 14.46 11.07 2. s1c 1.753 1.45 19.18 15.90 3. s2r 1.230 1.29 13.46 14.14 4. s2c 1.890 1.38 20.68 15.13 5. s3r 0.568 0.24 6.21 2.63 6. s3c 1.384 0.60 15.14 6.58 7. s4 1.295 0.88 14.17 9.65 8. s5 1.285 1.40 14.06 15.35 k–spice (condiment); m–medicinal plant for both samples, the difference between the efficiency values in the case of the extraction realised with water or methanol, in the same conditions, does not rise above 3.07%, so the two solvents have almost the same capacity of extracting the compounds from both the analysed condiment and the medicinal plant. b. the concentration value in the compounds with antioxidant potential – the flavonoids (f), polyphenolcarboxilic acids (ac. pf.), total polyphenols (pf) in the extracts of the same plant material, obtained with different solvents, varies a lot (table 3). b1. when determining the flavonoids, one can observe that the extraction with water at high temperature is more complete (0.73 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 243 g rutoside / 100 g d.p. – for oregano k and 0,92 g rutoside / 100 g d.p. for oregano m). on the opposite end, the extraction with ethanol at cold is placed, in both cases the values being 8.1 and 7.7 or lower than the maximum values for oregano k, respectively m. when comparing the recorded values at the determination of the flavonoids from tinctures with the values obtained under reflux, with the same solvent, one can observe that the former are much smaller, especially in the case of water and ethanol. table 3 the quantity of antioxidant compounds solvent (sample) flavonoids (g rutoside / 100 g d.p.) polyphenolcarboxilic acids (g rosmarinic acid /100 g d.p.) total polyphenols (g gae / 100 g d.p.) pf – f – ac. pf. ((g/ 100 g d.p.) k m k m k m k m 1 2 3 4 5 6 7 8 9 s1r 0.38 0.54 0.4 0.66 3.58 5 2.8 3.8 s1c 0.73 0.92 1.18 1.9 4.72 9.13 2.81 6.31 s2r 0.4 0.8 0.71 1.85 4.56 9.33 3.45 6.68 s2c 0.58 0.85 1.02 1.91 5.39 10.27 3.79 7.51 s3r 0.09 0.12 0.09 0.17 0.78 1.81 0.6 1.52 s3c 0.64 0.29 0.92 0.62 5.15 4.44 3.59 3.53 s4 0.36 0.35 0.51 0.96 2.75 7.04 1.88 5.73 s5 0.67 0.69 0.96 2.35 4.48 11.73 2.85 8.69 thus, for water, the ratio of the content of the flavonoids from the extract obtained under reflux (s1c) and the extract obtained at room temperature (s1r) is 1.70 for oregano m and 1.91 for oregano k. this proves that by rising the temperature, the flavonoids have a better extractability. very good results were obtained also when the extraction was done with the mixture s5 (methanol – acetone – water – formic acid), these being comparable with the maximum values. comparing the two samples (k, m) one can observe higher values for the medicinal plant rather than for the condiment. the comparison between the data obtained from this study and the values from other studies is difficult, due mainly to the difference between solvents used at extraction and the reference compound used to express the results, but also due to different types of plant and extraction conditions (time, temperature etc). for example, at the extraction of the flavonoids from basil, grayer and collaborators [9] use diethyl ether and goze [3] uses hexane and dichloromethane. the most commonly used solvents are water, methanol, or the combinations of these two. there are many ways of expressing the results and some studies use as reference [10]: quercetin [3], catechin, and moreover rutoside. b2. polyphenolcarboxilic acids (table 3, columns 4, 5) are extracted better at high temperature and the solvents with higher efficacy are water (s1c), methanol (s2c) and the mixture s5. the lowest values were obtained from the extracts with alcohol 960, at low temperature (0.09 g rosmarinic acid / 100 g d.p. for oregano k and 0.17 for oregano m). in comparison, the average ratio of the values condiment / medicinal plant is 0.63 – and this shows higher values for the medicinal plant. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 244 it can also be observed that there is a correlation between the content of flavonoids and the content of polyphenolcarboxilic acids. there are limited studies on the measurement of the polyphenolcarboxilic acids in aromatic plants (the majority are related to the total content of polyphenols and flavonoids). in a study about phenolic compounds from oregano and thyme, [11] we reach the conclusion that the extracts from oregano have a high content of rosmarinic acid (0.1248 ~ 0.1546 g/100 g fresh plant), this being the main polyphenolcarboxilic acid in the analysed aromatic plant. b3. the content of polyphenols from the extracts (table 3, columns 6, 7) decreased the order: s2c > s3c > s1c > s2r > s5 > s1r > s4 > s3r, for oregano k and s5 > s2c > s2r > s1c > s4 > s1r > s3c > s3r, for oregano m. from the used solvents, methanol proved to contribute very well to recover the polyphenols from aromatic plants, both at high and low temperatures. higher values were obtained from extractions with water, at high temperature. the complex solvents extract selectively the polyphenols from plants, the values vary in the extract with methanol mixture – acetone – water – formic acid (s5) between 11.73 g gae/ 100 g d.p., for oregano m and 2.75 g gae/ 100 g d.p., for condiment, with the solvent s4. similar values were obtained by [12] for oregano (7.282 g gae /100 g), the extraction being done with a mixture of acetone – water – acetic acid (70:29,5:0,5). the content of polyphenols relative to the dry substance (fig. 1) varied from 0.78 g gae / 100 g d.w. (in the extract with ethanol, at low temperature) to 5.39 g gae / 100 g d.w (in the extract with methanol at high temperature), with an average of 4.35 g gae / 100 g d.w. for the condiment. for the medicinal plant the values are 1.81 (in the extract with ethanol, at low temperature) and 11.73 g gae / 100 g d.w. (in the extract s5), with an average of 8.15 g gae / 100 g d.w. . 0 2 4 6 8 10 12 14 k m oregano to ta l c on te nt o f p o ly ph en o ls , g g a e / 10 0 g d. w . aromatic plant min s1r s1c s2r s2c s3r s3c s4 s5 max figure 1 – the content of polyphenols for analysed condiments compared with values from the literature a comparison between the minimum values obtained by [13] – 4.351 g gae /100 g d.w., for the extract obtained with a mixture 80:20 acetone–perchloric acid 5% and the highest values obtained by [14] 10.17 g gae /100 g d.w., for the extract obtained with methanolic solution 80% demonstrates a higher capacity of extraction for water, methanol and the mixture of solvents s5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 245 taking into account the difference between total polyphenols and polyphenolcarboxilic acids and flavonoids (table 3, columns 8, 9), it can be observed that higher values are recorded for the medicinal plant (with an average, for all the extractions, of 5.47 g useful compound / 100 g d.p., compared to the condiment, with an average of 2.72 g useful compound / 100 g d.p.). so, the conclusion might be that in these aromatic plants there are significant quantities of other types of polyphenols that were not dosed. c. antiradical capacity – it can be observed that both the medicinal plant and the condiment have very high values of scavenger capacity against the free radicals (table 4). the ethanol solution is remarkable, at reflux temperature (92.43%) for the condiment and complex mixture s5 (94.09%), for the medicinal plant, but the differences from the extractions with methanol and water, at high temperature are relatively low. the two samples of oregano have similar values for the majority of the extracts, higher differences being found in the case of complex solvents (81.12%, in s4, for sample k and 22.42% in s5, for sample m). table 4 the antiradical capacity of the aromatic plants samples no. solvent (sample) antiradical capacity, % k m 1. s1r 81,16 83,80 2. s1c 86,47 85,73 3. s2r 88,09 90,37 4. s2c 90,85 91,73 5. s3r 40,12 51,41 6. s3c 92,43 89,09 7. s4 84,87 3,75 8. s5 71,67 94,09 the obtained values are consistent with those obtained in other studies: the extract obtained with methanol solution 80% showed a 83% scavenger activity against free radicals [15]; the extract with methanol solution 50% showed a 80% scavenger activity against free radicals [16]; the ethanolic extract – 99% [17]. 4. conclusions the value of concentration in compounds with antioxidant potential (flavonoids, polyphenolcarboxilic acids, total polyphenols) in the extracts of the same vegetal material, obtained with different solvents and at different temperatures varies very much, so the solvents and the working conditions influence significantly the obtained results. thus: 1. the flavonoids and the polyphenolcarboxilic acids are extracted better with water, at high temperature, but the efficiency is close to the extraction with methanol solution, at reflux temperature and with complex mixture s5. 2. the polyphenols are extracted very well with methanolic solution, at high temperature, but also with the mixture of solvents methanol – acetone – water – formic acid (s5). 3. water and methanol have the capacity of extracting all the three groups of analysed compounds both from the analysed condiment and the medicinal plant. 4. ethanol reaches valuable extractions at high temperature (hot), especially in the case of the condiment and it is less efficient at low temperature. 5. the mixtures of solvents extract selectively the target compounds, varying a lot from a sample to another. the mixture s5 was more efficient, especially in the case of medicinal plant. 6. the extraction temperature influences positively the substances extractability when it is higher. a valuable extract from the antioxidant capacity point of view may be obtained at room temperature, although the polyphenolic compounds vary a lot, in relation to the studied sample. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 3 – 2013 micşunica rusu, determination of compounds with potential antioxidant and antiradical capacity in different oregano extracts, food and environment safety, volume xii, issue 3 – 2013, pag. 240 246 246 efficiency of the solvent and the extraction temperature are confirmed by the values obtained for the total extract (the total dry substance). comparing the two samples (k and m), one can observe higher values of antioxidant compounds for the medicinal plant in comparison with the condiment. oregano – both the condiment, and the medicinal plant – have a high antiradical capacity, depending to a lesser extent on the extraction solvent, but more on the temperature. 5. references [1] zhang, l., ravipati, a. s., koyyalamudi, s. r., jeong, s., reddy, n., smith, p. t., bartlett, j., shanmugam, k., münch, d., wu, m., antioxidant and antiinflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds, j agric food chem, 2011, vol. 59, p. 12361–12367; [2] franz, ch., bauer, r., carle, r., tedesco, d., tubaro, a., zitterleglseer, k., study on the assessment of plants/herbs, plant/herb extracts and their naturally or synthetically produced components as “additives” for use in animal production, available at http://www.efsa.europa.eu /en/supporting/pub /070828.htm, 2009; [3] goze, i., alim, a., tepe, a. s., sokmen, m., sevgi, k., tepe, b., screening of the antioxidant activity of essential oil and various extracts of origanum rotundifolium boiss. from turkey, journal of med. plants research, 2009, vol. 3, p. 246-254; [4] caillet, s., yu, h., lessard, s., lamoureux, g., ajdukovic, d., lacroix, m., fenton reaction applied for screening natural antioxidants, food chemistry, 2007, vol. 100, p. 542–545; [5] duke, jim, phytochemical and ethnobotanical databases, usda, ars, national genetic resources program, 2013, available at http://www.ars-grin.gov/duke/; [6] jimenez-alvarez, d., giuffrida, f., golay, p. a., cotting, c., lardeau. a., keely, b. j., antioxidant activity of oregano, parsley, and olive mill wastewaters in bulk oils and oil-in-water emulsions enriched in fish oil, j. agric. food chem., 2008, vol. 56 (16), p. 151– 7159; [7] gülçin, i, antioxidant activity of food constituents: an overview, arch toxicol, 2012, vol. 86, p. 345–391; [8] materska, m., 2010, evaluation of the lipophilicity and stability of phenolic compounds in herbal extracts, acta sci. pol., technol. aliment, vol. 9, p. 61-69; [9] grayer, r. j., vieira, r. f., price, a. m., kite, g. c., simon, j. e., paton, a. j., characterization of cultivars within species of ocimum by exudate flavonoid profiles, biochemical systematics and ecology, 2004, vol. 32, p. 901-913; [10] haminiuk, c., maciel, g., plataoviedo, m., peralta, r., phenolic compounds in fruits – an overview, international journal of food science & technology, 2012, vol. 47, p. 2023–2044; [11] kruma, z., m. andjelkovic, r. verhe, v. kreicbergs, phenolic compounds în basil, oregano and thyme, baltic conference on food science and technology, foodbalt-2008, 3, jelgava (latvia), available at http://llufb.llu.lv/conference/foodbalt/2008/foodbal t-proceedings-2008-99-103.pdf; [12] wu, x., beecher, g. r., holden, j. m., haytowitz, d. b., gebhardt, s. e., prior, r. l. l., lipophilic and hydrophilic antioxidant capacities of common foods în the united states, j. agric. food chem., 2004, vol. 52, p. 4026-4037; [13] ninfali, p., mea, g., giorgini, s., rocchi, m., bacchiocca, m., antioxidant capacity of vegetables, spices and dressings relevant to nutrition, british journal of nutrition, 2005, vol. 93, p. 257–266; [14] shan, b., cai, y. z., sun, m., corke, h., antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents , j. agric. food chem., 2005, vol. 53 (20), pp 7749– 7759; [15] capecka, e., mareczek, a. leja, m., antioxidant activity of fresh and dry herbs of some lamiaceae species, food chemistry, 2005, vol. 93, p. 223-226; [16] cervato, g., carabelli, m., gervasio, s., cittera, a., cazzola, r., and cestaro, b., antioxidant properties of oregano (origanum vulgare) leaf extracts, journal of food biochemistry, 2000, vol. 24, p. 453-465; [17] exarchou, v., nenadis, n., tsimidou, m., gerothanassis, i. p., troganis, a. & boskou, d, antioxidant activities and phenolic composition of extracts from greek oregano greek sage, and summer savory, journal of agricultural and food chemistry, 2002, vol. 50, p. 5294–5299. 218 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 218 223 determining a sensory profile of cheese paste with spices, using quantitative descriptive analysis *rusu micşunica1 1faculty of food engineering, stefan cel mare university of suceava, suceava, romania, rusu_mic@yahoo.com *corresponding author received august 14th 2014, accepted september 19th 2014 abstract: food should nourish the body, should display sensory properties that make them be easily accepted, asked for in consumption, and at the same time it should also have a positive effect on consumer’s health. this last aspect defines best functional foods. cheese paste, using a classic recipe (by replacing paprika with oregano, thyme or basil, all dried) has been obtained and served as sandwiches. sensory analysis was performed, using quantitative descriptive analysis (qda) for the the new products obtained. the analysis was conducted by nine evaluators, who, by means of specific documents (such as questionnaire for collection of descriptors, tasting sheets) and by analyzing tasting samples, identified 85 descriptors in the first phase, then after successive filtrations 41 descriptors were kept and in the end 12 descriptors were considered to define the product characteristics. therefore, the sensory analysis focused on the size of spice particles, paste uniformity (granulometry), white uniform color, and smell of fresh cow cheese, salty or bitter taste of fresh cow cheese and butter, cheese flavors, specific spices, adhesion, persistent flavor. definitions and an order of tasting were initially established for all these descriptors. after having performed the analysis, based on averages values, a stellar diagram was obtained. it was observed that the obtained profiles for the paste with dried spices (oregano, thyme or basil) were quite similar. larger differences occurred at bitter taste and aftertaste. the new pastes differed from the original paste (with paprika) in the uniformity of color and size of the particles, which are due to the physical characteristics of the spices used. keywords: foods with functional potential, descriptors, stellar diagram 1. introduction the interest for the relationship between diet and health is a tradition for many populations. functional foods lie at the intersection of the two [1]. an item of food is considered functional if this one influences positively one or more target functions of the body, in addition to adequate nutritional effects in a way that it is relevant either by improving health and well-being/or by reducing the disease risk. functional foods must remain classic foods and must demonstrate their effects in normally consumed intakes in the diet [2]. fogliano and vitaglione [3] present three strategies that can be applied in the development of new functional foods: influencing the modification of raw materials, modifying the recipe or changing the technology. all these directions can be adopted to improve antioxidant capacity, an important aspect of the character of functional foods or catering preparations. modification of the recipe resides, most often, in the potential addition of functional foods. the aim of this study was to replace a spice paprika (total-orac 21932 μmol te /100 g; polyphenol content -tp1643 mg gae / 100 g) – from a recipe for a paste used to get sandwiches with superior antioxidant potential spices: basil (totalfood and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 micşunica rusu, determining a sensory profile of paste cheese with spices, using quantitative descriptive analysis, food and environment safety, volume xiii, issue 3 – 2014, pag. 218 – 223 219 orac 61063 μmol te / 100g; tp 4489 mg gae / 100 g), oregano (total-orac 175 295 μmol te / 100 g, tp 3789 mg gae / 100 g) and thyme (total-orac 157380 μmol te / 100 g; tp 4470 mg gae / 100 g) according to the usda database [4]. but food and dishes are chosen by consumers, most of the time, on the basis of sensory attributes. therefore, a sensory analysis for the new preparations was performed. the application of sensory analysis for functional catering preparations started from two interdependent goals: their development and characterization. characterization involves defining and framing the setting of product attributes in a predefined type or standard. it also allows the detection of faults that can occur [5]. the sensory analysis was based on quantitative descriptive analysis, which provides a complete description of all the sensory properties of a product and is one of the most complete methods to characterize important sensory attributes [6, 7]. it is a tool used at sensory analysis and requires the use of a qualified group of subjects (usually 8 to 12 assessors, or more). 2. materials and methods to perform the sensory analysis, the quantitative descriptive analysis (qda) was used. this method is based on the ability of a team of trained tasters to measure the specific attributes of a product in a reproducible manner in order to obtain a quantitative description of the product, carried out by statistical analysis. the qda approach involves the recruitment of a panel of tasters, whose task is to identify the key attributes of a certain product on an appropriate intensity scale. this panel is trained to identify correctly the values of the descriptors (attributes) of the products analyzed [5]. materials four samples of cheese paste for sandwiches were obtained and analyzed: cheese paste with paprika (pba); cheese paste with basil (pb); cheese paste with thyme (pc) and cheese paste with oregano (po). they were made (for a kg of paste) from 600 g fresh cow cheese, 150 g feta sheep cheese, 150 g butter, 100 g cream and 5 g spices [8]. the samples were prepared as described in the plug technology, ranging the spice. they were served in white plastic plates in the form of sandwich (prepared correspondingly to a portion), encoded with 3-digit numbers and served immediately after preparation to further reduce: loss of flavor, discoloration and textural changes. the tasting sheets used, contain general information on the taster, tasting descriptors set by the panel, scales for assessing the intensity of descriptors and a comment box. methods sensory profile was established by qda, a method used in many studies covering dairy products [9, 10], honey [4], chocolate [11, 12] etc. nine assessors (7 female, 2 male, aged between 42 and 58 years) were selected to participate in the study, based on interest, availability, smoker status and non-allergic to food. they have not participated in organized sensory determinations so far, still they have experience in the field of food processing. the initial training of the selected group was performed in a 40-minute meeting, when the method was presented and the tasting conditions were specified. attention was drawn to the fact that there are factors that can affect temporarily sensitivity to aromas, taste and smell food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 micşunica rusu, determining a sensory profile of paste cheese with spices, using quantitative descriptive analysis, food and environment safety, volume xiii, issue 3 – 2014, pag. 218 – 223 220 (smoking, chewing gum, mint candies and spices). therefore, the participants were advised not to eat or drink (except water) or smoke for at least one hour before the tasting session. a rinsing protocol (mineral water and unsalted bread croutons) was established as well. qda deployment was achieved in three distinct phases: a) looking for a minimum number of descriptors that provide the maximum amount of information about a particular product, and, in parallel, preparation and discussion about the tasting sheet; b) measuring the intensity of perceived sensory properties for each of the chosen descriptors; c) building, based on the quantized set of descriptors, the sensory profile of the product [13, 14]. 3. results and discussion the identification of descriptors was performed in 4 sessions of 40 minutes. the aim was for the assessors not to overlap and give a full description of the samples analyzed. in selection, a questionnaire to collect descriptors was used. out of the four sessions, in the first two, conducted individually, the assessors have tasted food prepared according to recipes, similar dishes selected in an earlier stage and working samples, and have selected a number of descriptors. the next two sessions were conducted jointly pursuing the same goal. a) in this way, 85 descriptors were selected. they were subjected to filtering. in the first stage 44 terms were removed, in order to avoid overlapping: hedonic terms (harmonious flavor, tasty, optimal, appetizing etc.), quantitative terms (strong, weak, according to.... etc.); terms that are not relevant (typical, normal). the remaining descriptors (41) were summarized in table 1, depending on the feature in question and the time they were detected. in the second phase, after a discussion, the terms to be retained for the products’ analysis were chosen. so, 12 terms agreed with by all members of the group were selected. finally the descriptive terminology of the samples included two attributes for aspect (particle size spice, paste consistency), an attribute for color (white uniform color), an attribute for odor (fresh cow cheese), four taste attributes (salty, fresh cow cheese, butter, bitter), two attributes for flavor (cheese, spice specific), a texture attribute (adhesion) and a residual taste attribute (persistent, specific for the spice used). table 1 selected attributes after first filtration moment feature before testing aspect: homogeneity; granulation; finesse; particle spice size; pasty; cream; color: white; uneven; white for the cream, green with various shades for the spices; specific cow cheese; aroma: butter; fresh cow cheese; fermented; pungent; other odors; specific of the spice; herb; during testing taste: salty; acid; spicy; harmonic; astringent; spicy; bitter; metal; sour; foreign tastes; fad; flavor: butter; fresh cow cheese; cream; other flavors; mixed; specific spice; texture: adhesion; lubricity; uniform; after testing taste: bitter; metal; salty; flavor: specific spice. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 micşunica rusu, determining a sensory profile of paste cheese with spices, using quantitative descriptive analysis, food and environment safety, volume xiii, issue 3 – 2014, pag. 218 – 223 221 in the last issue of training the order in which attributes were perceived (in the order they are shown in table 2) was established. to be sure that the attributes have the same meaning for all members of the group, a lexicon of the terms selected was drawn up (table 2). before preparing the tasting sheet it was agreed to use a scoring unstructured scale of 10 cm, with extreme anchors "missing" and "extremely strong", which means no descriptor and its intensity to measure each sensory attribute. in the last training session, preparations and attempts to obtain consistency between evaluations and tasters were reviewed. the duration of training sessions varied between 18 and 40 minutes. table 2 definitions of descriptors selected attributes definitions aspect spice particle size particle size, ranging from those that cannot be visually detected (dust) to pieces over 3 mm; paste uniformity (granulometry) paste property, given the size of the pieces of cheese and spice; color white, uniform white color intensity or strength with different tones; odor fresh cow cheese fresh cow cheese odor; taste salted the fundamental taste factor associated with a sodium chloride solution; fresh cow cheese specific taste of fresh cow cheese; butter specific taste of fresh butter; bitter taste fundamental factor associated with a caffeine solution; flavor cheese fresh cow cheese specific flavors; spice specific spice flavors used; texture adhesion textural property asseses the degree to which the food adheres to the surface of palate when it is compressed by tongue; taste remaining persistent, specific to the spice used the persistence duration of spice taste, after swallowing the sample bits. b) the proper evaluation of the evidences was done in two sessions. the two sessions were held before noon, according to the indication suggested by mason [15]. in each session four samples were presented and submitted to panelists in order to eliminate the effects of order of service as much as possible. panel members scored each attribute and stressed the intensities on the unstructured scale of 10 cm. evaluators rinsed the mouths with mineral water and unsalted croutons between assessments. breaks between tests were taken. the results were converted into numerical values. then the average of values for each evaluator was performed. in conducting the tests there were encountered some difficulties. thus, there were evaluators who used different portions of the scale to express their feeling of the same sample, and some difficulties in the interpretation of attributes were identified as well. these difficulties were mainly due to evaluators’ lack of experience in using this test food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 micşunica rusu, determining a sensory profile of paste cheese with spices, using quantitative descriptive analysis, food and environment safety, volume xiii, issue 3 – 2014, pag. 218 – 223 222 method, and to a small number of training sessions. c) assessment of results was performed using the graphical representation, using a stellar diagram. this one was drawn on the basis of average values obtained in evaluating sensory attributes of preparations by descriptive analysis (fig. 1). the intensity of attributes is plotted by the distance from center. one can notice that the profiles for new preparations are quite similar. visible differences occur aftertaste, where oregano has the highest value and bitter taste (but these differences occur at low levels). the new preparations differ more from the initial preparation in uniformity of the color and in particles size, differences due to the physical characteristics of spices (purchased in the commercially available state). 0,00 2,00 4,00 6,00 8,00 10,00 spice particle size paste uniformity (granulometry) uniform color smell of fresh cheese butter taste salted taste bitter taste fresh cheese flavor cheese flavor spice specific flavor adhesion aftertaste, spice specific pb pc po pba fig. 1. sensory profile using the stellar diagram, based on qda, for pastes analyzed the lowest intensity of fresh cheese flavor is registered by the thyme sample (which means that some substances in this spice are able to annihilate the flavor of substances that contribute to forming cheese flavor) and decreased in the following order: pb> po > pba> pc. specific spice flavor intensity decreases in the order: pc> po> pb> pba, hence the flavor of other ingredients in the recipe, which had been initially neglected (butter, salty cheese, cream), influenced the overall flavor of the product. the taste of fresh cow cheese had similar intensities in all samples, in contrast with the salty taste, whose intensity was generally higher in samples with thyme and basil and bitter taste, more prominent in samples with thyme and oregano. the results seem to suggest that the type of spice may be responsible for the perception of aromas and the tastes of other foods used in the preparation. 4. conclusions some outstanding conclusions can be drawn from this study: there is a variety of methods to obtain catering preparations with health benefits, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 micşunica rusu, determining a sensory profile of paste cheese with spices, using quantitative descriptive analysis, food and environment safety, volume xiii, issue 3 – 2014, pag. 218 – 223 223 but the simplest one is the introduction of foods with high functional potential; the qda application allows the identification and quantification of defining descriptors for a specific category of food, wherein the products are different or similar. for the new pasta analyzed, the descriptors according to which the products differ one from the other are: the aftertaste and the bitter taste; spices, even when used in small amounts, influence significantly some characteristics of preparations when annihilating or highlighting tastes, flavors, colors are introduced . 5. references [1] henry, c., functional foods, european journal of clinical nutrition, 64, 657–659, (2010); [2] diplock, a., aggett, p., et al, scientific concepts of functional foods în europe. consensus document, british journal of nutrition, 81, s1–s27, (1999); [3] fogliano, v., vitaglione, p., functional foods: planning and development, molecular nutrition and food research, 49, 256-262, (2010); [4] haytowitz, d., bhagwat, s., usda database for the oxygen radical absorbance capacity (orac) of selected foods, release 2, usda, available at http://www.ars.usda.gov/sp2userfiles/ place/12354500/data/orac/ orac_r2.pdf, (2010); [5] ciappini, m., di vito, m., gatti, m., calviño, a., development of a quantitative descriptive sensory honey analysis: application to eucalyptus and clover honeys, advance journal of food science and technology, 5(7), 829-838, (2013); [6] stone, h., sidel, j., quantitative descriptive analysis (the qda method). sensory evaluation practices, 3rd ed. elsevier academic press, san diego, ca, 215–235, (2004); [7] stone, h., bleibaum, r., thomas, h., sensory evaluation pratices, 4.ed., elsevier academic press, food science and technology. international series, london, 250-255, (2012);. [8] ***, reţetar tip pentru preparate culinare, ministerul comerţului, bucureşti, 174, (1982); [9] allgeyer, l. c., miller, m. j., lee, s.y., sensory and microbiological quality of yogurt drinks with prebiotics and probiotics, journal of dairy science, 93 (10), 4471–4479, (2010); [10] wadhwani, r., mcmahon, d. j., color of low fat cheese influences flavor perception and consumer liking, journal of dairy science, 95, 2336–2346, (2012); [11] lanza, c.m., mazzaglia, a., pagliarini, e., sensory profile of a specialty sicilian chocolate, international journal of food science, 23, 36-44, (2010); [12] lenfant, f., hartmann, c., watzke, b., breton, o., loret, c., martin, n., impact of the shape on sensory properties of individual dark chocolate pieces, food science and technology, 51, 545-552, (2013); [13] *** bs iso 13299:2003 sensory analysis — methodology — general guidance for establishing a sensory profile, iso, (2010); [14] morais, e. c., cruz, a. g., faria, j. a. f., bolini, h. m. a., prebiotic gluten-free bread: sensory profiling and drivers of liking, lwt food science and technology, 55(1), 248-254, (2014); [15] mason, r., sensory evaluation manual, university of queensland and stephen nottingham, (2002). microsoft word 7 cristina radu-rusu.doc 47 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2013, pag. 47 52 is the la ying h ens rear in g sys tem releva nt fo r tab le egg s ch em ic a l an d n utrition a l fea tu r es? cristina gabriela radu-rusu (şestaliuc)1, christelle plocus2, céline dandois2, *răzvan mihail radu-rusu1 1university of agricultural sciences and veterinary medicine, animal science faculty, 8 mihail sadoveanu alley, 700489, iasi romania, rprobios@yahoo.com 2lycée d'enseignement général et technologique agricole, btsa productions animales, areines bp 106, 41106 vendôme cedex, france * corresponding author received 10 january 2013, accepted 27 february 2013 abstract: do the changes occurred in poultry housing systems affect eggs chemical composition and dietetic parameters? to investigate this hypothesis, we used 700 isa brown hens, aged 26 weeks, randomly distributed in two groups: ec group-350 hens accommodated in furnished cages (800 cm2/hen) and fr group-350 hens, reared on deep litter, with access to paddock (9 hens/m2). same feed (corn-wheat-soymeal) was used in both groups. 100 eggs were collected/group for chemical assessments on edible compounds (yolk, albumen, whole egg), using 20 replicates per parameter and compound. analytical chemistry was performed via conventional methods, cholesterol through gaschromatography and caloricity calculated on organic matters energy basis. anova single factor was applied. 100g edible portion of whole eggs in ec group comprised 74.98% water, 1.14% ashes, 12.38% proteins, 10.45% lipids, 1.05% nfe, 174.25 kcal energy (105 kcal/egg), 397 µg cholesterol. in fr eggs, water was 75.62%, proteins and ashes didn’t significantly change, lipids and caloricity were lower (9.81%, 168.65 kcal/100 g; 101 kcal/egg) and cholesterol was 373 µg. significant differences occurred between groups for water and lipids content (p<0.05). although the dietetic value was better in free-range eggs, it must reason if this improvement could counterbalance hygienic threats that could accompany free range production. keywords: enriched cages, free range, eggs, chemical composition, dietetic value 1. introduction changes in poultry husbandry technology and housing systems, due to compliance to the fowl welfare requirements, had imposed several studies to be done, in order to know the way in which the usage of a new rearing and exploitation system could affect hens behavior, the quality of edible poultry products (meat, eggs) and the economic efficiency of production [1, 2]. related to behavior and welfare status, it was shown that laying hens generally accepts nesting in furnished cages and freedom of move, dust bathing, scratching and roosting behaviors are signs that the fowl adapt well in improved cages, the conditions being mimetic to those from the deep litter system [3]. other findings revealed that, in theory, the usage of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 cristina gabriela radu-rusu (şestaliuc), christelle plocus, celine dandois, răzvan mihail radu-rusu, is the laying hens rearing system relevant for table eggs chemical and nutritional features?, food and environment safety, volume xii, issue 1 – 2013, pag. 47 52 48 alternative rearing systems have the potential to provide satisfactory welfare conditions to laying hens, while in practice, this could not be achieved always, the main interfering factors being represented by different management, climate, design, different responses by different genotypes and interacting effects. for example there was different use of nestboxes in furnished cages by different genotypes. the design of small furnished cages also had a significant impact on dustbath use. all cage systems tend to provide a more hygienic environment with low risk of parasitic and bacterial disease and, consequently, lower probability to contaminate their edible products, including eggs [4]. when seeking a little bit more on eggs safety, as related to the conversion of production systems toward those providing better welfare conditions to hens, it could be found scientists which suggest that safety could be altered either microbiologically through contamination of internal contents with salmonella enterica serovar enteritidis (salmonella enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account [5]. therefore, it must reason clearly when choosing the husbandry system of laying hens, in order to provide a safe, but economically reliable product on the market. speaking of poultry products nutritional quality, for instant chicken meat, certain investigations [6] revealed higher lipid content and lower proportion of pufa and omega-3 fatty acids in conventional produced broilers, when compared to broilers issued from other husbandry system, such as free range or alternative farming. moreover, the alternative systems provided meat with lower cholesterol content. how about eggs quality in alternative production systems? some researchers [7] focused on the proximate composition and nutrients of the eggs issued from different husbandry systems applied in laying hens farming but mainly influenced by feed quality. thus, in free range or cage-free systems, proportion of the yolk was lower than in other systems, which differed mainly through the used diet (vegetarian diet or animal fat included or unmedicated diet). the ratio between certain fatty acids groups, such as n-6:n-3 polyunsaturated varied from 39.2 in eggs issued from naturally nested laying to 11.5 in the eggs laid by the hens fed with complete vegetarian diet (p<0.05). however, no difference was reported on the total pufa content of eggs (p > 0.05). another study run on commercial eggs [8] showed that, from nutritional point of view there were found slight significant statistic differences for proteins and saturated fatty acids, but the real variations were minimal, when eggs from conventional cage and alternative productions systems were compared. no significant differences were found in the unsaturated fatty acid groups, despite the fact that eggs used in the study were bought from market, therefore probably the hens were fed with mixed feed of different quality and compositions. other authors [9] stated that through eggs composition elasticity due to hen diet compounds, these products have immense potential to provide consumers nutritional and functional benefits, to maximize the beneficial and preventive potential. finally a question arises: in order to provide well balanced eggs for consumer needs, therefore so-called functional or designer eggs, what eggs feeding and rearing systems should be used, to better manage all involved influential factors? the present paper aims to briefly present the findings on the nutritional and dietetic quality of the eggs produced in two eu approved alternative housing systems, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 cristina gabriela radu-rusu (şestaliuc), christelle plocus, celine dandois, răzvan mihail radu-rusu, is the laying hens rearing system relevant for table eggs chemical and nutritional features?, food and environment safety, volume xii, issue 1 – 2013, pag. 47 52 49 during the peak production of laying hens, as part of a greater project that aim to assess the influence of fowl welfare friendly husbandry systems on the quality of poultry meat and table eggs in romania. 2. materials and methods the biological material was represented by the eggs produced by 700 isa brown laying hens aged 26 weeks, randomly distributed in two groups: ec group-350 hens accommodated in furnished cages (800 cm2/hen, perches and nests inside the cage) and fr group-350 hens, reared on deep litter, with access to paddock (9 hens/m2), in order to comply the welfare requirements stipulated in council dir. 74/1999/eec [10]. hens were fed an adult layer mixed feed diet, based on corn-wheat and soybean meal. both groups were fed the same diet, in order to avoid any influence of certain different feed nutrients on the ultimate nutritional value of the eggs, knowing that especially quality of lipids in animal products varies in relation to the feed composition [11]. at the end of 26th week of fowl life, 100 eggs were sampled from each group, in order to run chemical investigations. there have been performed analyses on all edible compounds of the eggs (yolk, albumen, whole egg-melange), in order to identify and quantify the values for the following parameters: water, dry mater, ash, proteins (total nitrogen matters), lipids, nitrogen free extract, gross energy and cholesterol. 50 eggs from each group have been broken, to separate yolk and albumen. after separation, yolks were mixed apart and albumens were mixed apart. the other 50 eggs/group were used to mix together yolk and albumen, in order to assess quality of whole fresh eggs. 20 repetitions (samples) were analyzed for each parameter, for each edible compound. the samples were well labelled, then were weighted and dehydrated at 60°c. the issued powder was used for analytical chemistry assessments, in order to evaluate eggs content in certain nutrients, accordingly to the analytical standards recognised internationally (humidity-sr iso 1442/1997, mineral substances -sr iso 936: 1998, total nitrogen-sr iso 937:2007, lipids-sr iso 1443:2008, fatty acids and cholesterol-gas chromatography). nitrogen free extract was calculated through difference, as follows: nfe (%) = drymatter % – minerals % (total nitrogen % + lipids %). eggs dietetic value was evaluated, besides the cholesterol level, through their caloricity, using the theoretical relation which is based on the quantity of gross energy spread by the burning of 1 g crude protein, crude fat and nitrogen free extract into a calorimeter (ge (kcal/100g) = 5.70 kcal x n%cp + 9.50kcal x n%cf + 4.2 kcal x n%nfe) [12]. most of the parameters were expressed by 100 g edible portion, while some of them, such as cholesterol and caloricity were also recalculated per one table egg (60 g), in order to better depict the nutritional involvements. collected data were subjected to statistical computation, using anova single factor algorithm, to find out any significant differences between the studied technological systems of layer husbandry. 3. results and discussion the analytical findings on the 100 yolks we studied (50 from ec group an 50 from fr group) are presented in table 1. thus, water content was higher in the eggs produced by free-range hens (+1.36%) and this difference was found as statistically significant (p<0.05). this aspect corresponded to a slight decrease of fat matters in the eggs produced in free range system, compared to those issued from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 cristina gabriela radu-rusu (şestaliuc), christelle plocus, celine dandois, răzvan mihail radu-rusu, is the laying hens rearing system relevant for table eggs chemical and nutritional features?, food and environment safety, volume xii, issue 1 – 2013, pag. 47 52 50 enriched cages (-3.59%), the differences being also found significant (p<0.05). proteins varied within 15.91±0.32% (ec eggs) – 16.08±0.03% (fr eggs), while homogeneity for this trait was good (less than 10%). differences in lipids content led to slight different values for caloricity/100 g yolk, i.e. more (361.09±7.92 kcal in eggs from cages) or less (353.09±7.63 kcal in eggs from free range system) gross energy. cholesterol was also diminished in free range eggs (1065±23.98 µg/100 g) vs. furnished cage eggs (1094±24.71 µg/100 g). table 1 chemical composition and dietetic quality of the yolks from table eggs produced in improved cages or free range systems eggs from ec group (n=20) eggs from fr group (n=20) quality parameters of the yolk mean std. error. v% mean std. error. v% water (%) 52.16a ±1.37 11.75 52.87b ±1.32 11.17 dry matter (%) 47.84 ±1.26 11.75 47.13 ±1.18 11.18 ash (%) 1.72 ±0.03 7.80 1.69 ±0.03 7.94 total nitrogen matters (%) 15.91 ±0.32 8.99 16.08 ±0.31 8.62 lipids (%) 27.08b ±0.45 7.43 26.14a ±0.39 6.67 nitrogen free extract (%) 3.13 ±0.05 7.14 3.22 ±0.04 5.56 caloricity gross energy (kcal/100g) 361.09 ±7.92 9.81 353.51 ±7.63 9.65 cholesterol (µg/100 g yolk) 1094 ±24.71 10.10 1065 ±23.98 10.07 anova: abdifferent superscripts reveal significant statistical differences (p<0.05) between groups ash content and nitrogen free extract did not vary significantly. extremely low caloricity was found in albumen mix, due to low lipidic content for this egg compartment (table 2). thus, small difference for dry matter content was detected between groups, proteins proportions of the albumens from both groups were almost similar (11.2 vs. 11.4 %), while lipids were detected at very low concentration (0.18±0.004 % in ec eggs and 0.13±0.003 % in fr eggs), hence the low energetic content (69.67-69.16 kcal/100 g albumen). cholesterol was not detectable, as expected. these facts reaffirm the exceptional dietetic features of egg white, which is in fact a reservoir of high quality proteins, packaged with small or absent amount of compounds that are not so friendly with consumers’ health issues, such as cardiovascular diseases. table 2 chemical composition and dietetic quality of the albumens from table eggs produced in improved cages or free range systems eggs from ec group (n=20) eggs from fr group (n=20) quality parameters of the albumen mean std. error. v% mean std. error. v% water (%) 86.93 ±2.12 10.91 87.14 ±2.24 11.50 dry matter (%) 13.07 ±0.32 10.92 12.86 ±0.33 11.51 ash (%) 0.71 ±0.01 6.30 0.63 ±0.01 7.10 total nitrogen matters (%) 11.2 ±0.23 9.18 11.4 ±0.21 8.24 lipids (%) 0.18 ±0.004 9.94 0.13 ±0.003 10.32 nitrogen free extract (%) 0.98 ±0.02 9.13 0.70 ±0.02 12.78 caloricity gross energy (kcal/100g) 69.67 ±1.09 7.00 69.16 ±1.02 6.60 cholesterol (µg/100 g albumen) nd* nd anova: abdifferent superscripts reveal significant statistical differences (p<0.05) between groups; *nd: not detectable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 cristina gabriela radu-rusu (şestaliuc), christelle plocus, celine dandois, răzvan mihail radu-rusu, is the laying hens rearing system relevant for table eggs chemical and nutritional features?, food and environment safety, volume xii, issue 1 – 2013, pag. 47 52 51 finally, we considered interesting to quantify the chemical compounds on whole fresh eggs (mix of edible parts – yolk and albumen), knowing that human alimentation uses mostly whole eggs. the results are presented in table 3. yolk chemical composition influenced whole egg constituants dynamics and also differentiations between the two groups. thus, water content varied between 74.98±1.08 % (enriched cage eggs) and 75.62±1.08% (free range eggs), significant differences occurring for this trait (p<0.05). protein content was found close between groups (12.38-12.56%), while lipids were 1.06% higher in cage produced eggs, than in free range (10.45±0.17 %-ec group vs. 9.81±0.19 %-fr group). therefore, caloricity was a little bit higher in those eggs issued from cages (174.25±3.26 kcal/100 g or 105.00±3.26 kcal/egg of 60 g), compared to those produced within the free-range system (174.25±3.26 kcal/100 g or 105.00±3.26 kcal/egg of 60 g). table 3 chemical composition and dietetic quality of the whole edible table eggs produced in improved cages or free range systems eggs from ec group (n=20) eggs from fr group (n=20) quality parameters of the whole edible table egg mean std. error. v% mean std. error. mean water (%) 74.98a ±1.08 6.44 75.62b ±1.08 6.39 dry matter (%) 25.02 ±0.36 6.43 24.38 ±0.35 6.38 ash (%) 1.14 ±0.02 7.85 1.09 ±0.02 8.21 total nitrogen matters (%) 12.38 ±0.21 7.59 12.56 ±0.18 6.41 lipids (%) 10.45b ±0.17 7.28 9.81a ±0.19 8.66 nitrogen free extract (%) 1.05 ±0.02 8.52 0.92 ±0.02 9.72 caloricity gross energy (kcal/100g) 174.25 ±3.26 8.37 168.65 ±2.97 7.88 caloricity gross energy (kcal/egg of 60g) 105.00 ±1.97 8.37 101.00 ±1.78 7.88 cholesterol (µg/100 g) 397 ±6.83 7.69 373 ±6.25 7.49 cholesterol (µg/egg of 60g) 238.20 ±4.10 7.69 223.80 ±3.75 7.49 anova: abdifferent superscripts reveal significant statistical differences (p<0.05) between groups cholesterol content was found at 238.2223.8 µg/egg of 60g, thus housing environment did not affect the inner cholesterol content of the egg, results quite similar to those found by other authors [13, 14]. overall, the variability of all analyzed traits was good, depicting homogeneity for the nutritional and dietetic parameters of table eggs. however, it would be interesting to use more eggs as biological material, in order to increase confidence degree of the statistical processing. the researches are still ongoing, in order to assess chemical composition and nutritional value of these products throughout the entire productive period of a commercial laying hen (20-72 weeks). moreover, the researches should be completed with fatty acids spectrum studies, knowing that are opinions among the scientists that the quality of lipids in eggs and meat (ratio ω-3:ω-6 fa) varies with the husbandry system and feeding conditions, mostly when fowl have or not access to pasture and, consequently, could lead to exacerbation of pain conditions, cardiovascular disease and probably most cancers [15]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2013 cristina gabriela radu-rusu (şestaliuc), christelle plocus, celine dandois, răzvan mihail radu-rusu, is the laying hens rearing system relevant for table eggs chemical and nutritional features?, food and environment safety, volume xii, issue 1 – 2013, pag. 47 52 52 4. conclusion most chemical components of the eggs produced in enriched cages and free range systems did not vary among the assessed samples, but the lipids and water value, thus the production system is relevant for these nutrients dynamics. all analyzed edible eggs parts or even whole fresh eggs were found to have less gross energy and less cholesterol in the samples issued from free range production system. however, the differences were not statistically significant. deeper investigations on lipids profile of table eggs could enlighten more aspects related to nutritional quality, especially the ratio between saturated: monounsaturated: polyunsaturated fatty acids that affect indeed the health status of consumers. 5. acknowledgments the authors wish to thank the romanian executive unit for financing in higher education, research, development and innovation-u.e.f.i.s.c.d.i.-c.n.c.s.i.s. (www.cncsis.ro), which supported these investigations through the research contract p.n.c.d.i. ii human resources – postdoctoral projects no. 508/2010-2012. 6. references [1]. miao z.h., glatz p.c., ru y.j., freerange poultry production a review. asianaustralasian journal of animal sciences, 18.113-132, (2005). [2]. usturoi m.g., radu-rusu r.m. alternative solutions to be used in laying hens' husbandry, bulletin of the cluj-napoca university of agricultural sciences and veterinary medicine, 62.32-36, (2006). [3]. bessei w., behaviour of laying hens in small group systems in the view of animal welfare. archiv fur geflugelkunde, 74.6-12, (2010). [4]. blokhuis h.j., van niekerk t.f., bessei w., elson a., guemene d., kjaer j.b., levrino g.a.m., nicol c.j., tauson r., weeks c.a., de weerd h.a.v., the laywel project: welfare implications of changes in production systems for laying hens. world’s poultry science journal, 63.101-114, (2007). [5]. holt p.s., davies r.h., dewulf j., gast r.k., huwe j.k., jones d.r., waltman d., willian k.r., the impact of different housing systems on egg safety and quality. poultry science, 90.251-262, (2011). [6]. aguiar a.p.s., contreras-castillo c.j., baggio s.r., vicente e., meat quality of broilers from different rearing systems. italian journal of food science, 20.213-223, (2008). [7]. cherian g., holsonbake t.b., goeger m.p., fatty acid composition and egg components of specialty eggs. poultry science, 81.30-33 (2002). [8]. hidalgo a., rossi m., clerici f., ratti s., a market study on the quality characteristics of eggs from different housing systems. food chemistry, 106.1031-1038, (2008). [9]. shapira n., every egg may have a targeted purpose: toward a differential approach to egg according to composition and functional effect. worlds poultry science journal, 66.271-284, (2010). [10]. eurlex, council directive 1999/74/ec of 19 july 1999 laying down minimum standards for the protection of laying hens, available online at http://eurlex.europa.eu/lexuriserv/lexuriserv.do?uri= oj:l:1999:203:0053:0057:en:pdf (2009). [11]. leeson s., summers j.d., commercial poultry nutrition. 3rd edition, university books; ontario; canada (2005). [12]. f.a.o., food energy – methods of analysis and conversion factors. fao food and nutrition paper, rome, 77, (2003) [13]. anderson k.e., comparison of fatty acid, cholesterol, and vitamin a and e composition in eggs from hens housed in conventional cage and range production facilities. poultry science, 90.1600-1608, (2011). [14]. shapira n., not all eggs are created equal: the effect on health depends on the composition. the canadian journal of cardiology, 27.264-268, (2011). [15]. christophersen o.a., haug a., animal products, diseases and drugs: a plea for better integration between agricultural sciences, human nutrition and human pharmacology. lipids in health and disease, 10.38, (2011). 117 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 12015, pag. 117 126 contributions to a new method for determining food gumminess *gheorghe gutt¹, sonia amariei¹, mircea adrian oroian¹, cristina elena hreţcanu1 ¹faculty of food engineering, stefan cel mare university of suceava, romania g.gutt@fiausv.ro.com *corresponding author received 7th march 2015, accepted 31st march 2015 abstract: the paper presents a method and a technique for measuring food gumminess determined on instrumental sensory way versus this texture property determined by human sensory. this purpose it is conducted a comparative study between the values obtained according to sr iso 11036/2007, with gumminess values defined on the electronic sensorial way. the researches made possible the realization of a conversion curve that allows the transition from gumminess units measured electronic instrumental, under conditions of high reproducibility, to classical gumminess units determined by a panel of 5 points. a test for assessing the gumminess, performed with a total of seventeen volunteers, strongly emphasized the subjective nature of gumminess assessment on human sensory way. in this regard it has been observed a significant influence of the color on the test, modified with high purity beta carotene and its taste, modified with commercial vanilla. key words: texture profile analysis, gumminess, resistive torque moment 1. introduction. food texture issues and secondary parameter of gumminess texture of food represents all geometrical, mechanical and surface properties of a product perceptible by touch or mechanics receptors and where appropriate, visual and auditory [1]. in turn, mechanical properties of semisolid or solid foods can be divided into five types of primary parameters and four secondary parameters [2] as follows: primary parameters are: hardness, cohesion, viscosity, elasticity, adhesion. secondary parameters are: fracturability, masticability, number of mastication, gumminess. given that instrumental sensory measurement is more performant that human sensorial assessment of primary and secondary parameters of texture, in terms of sensitivity, detection limit and precision, current trend is an advanced replacing of human assessments by more precise measurements made with electronic sensory equipment [3], [4], [5], [6]. this problematic concerns a team from the faculty of food engineering of suceava, already existing notable achievements in the promotion of modern equipment for the sensorialelectronic assessment of several primary and secondary parameters of food texture [7], [8]. in terms of secondary parameter of the texture, the gumminess, this is defined [1] as a property related to the primary parameters for hardness and cohesion of http://www.fia.usv.ro/fiajournal mailto:g.gutt@fiausv.ro.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 118 semisolid food when their hardness is reduced. food gumminess appreciation is by the effort required for decay of product inside the mouth before swallowing in a proper state. for this purpose the sample is placed in the mouth and tongue pressing the palate, human sensorial evaluated by force necessary for its disintegration. selectivity to determine gumminess is provided by a given reference scale in table 1, [1] table 1. gumminess reference scale [1] popular terms score annotation reference product sample size temperature mild intensity gumming high intensity gumming 1 40% mixture of flour with water 1 spoon room 2 45% mixture of flour with water 3 50% mixture of flour with water 4 55% mixture of flour with water 5 60% mixture of flour with water the analysis on this way of gumminess assessment has the following disadvantages: evaluation score on human sensory way contains only 5 outs units leading to poor selectivity of method; gumminess assessment by an human assessor lead to subjective errors amplified by the fact that for calibration is used flour of different types; chewing a dough of flour and water by the taster-assessor to establish the gumminess score, does not give accurate results and expected values as more than the appraisal score overlap with primary texture parameter values; cohesion, viscosity, elasticity, adhesion, and untextured taste parameter, all these parameters being simultaneously present in the dough; fig. 1. identification and quantification elements of primary and secondary parameters of texture profile analysis (tpa) performed in coordinates force time [9] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 119 since the instrumental measuring is more performant in terms of sensitivity and detection limit than human assessment of primary and secondary parameters of texture, but also because the first is objective, ensuring a high reproducibility of determinations, the current trend is to replace as more advanced human evaluations with instrumental means of measurements. in this respect, an important step was made by determining gumminess value from the texture profile analysis (tpa), represented in coordinates force f time t [3], [4], [5], [6], figure 1. within tpa, gumminess g is defined as the product of hardness h and the ratio of the area a2 of the surface under the curve of the second stage of deformation and the area a1 of the surface under the curve of the first cycle of deformation. this mode of expression leads to errors of quantification and inappropriate expressions of gumminess value in physical units. for example, the use of tpa in coordinates force time leads to the following nonsense, at least in terms of gumminess quantification and expression in physical units:      3 2 1 0 1 2 t t t dtf dtf h a a hg (1) which in physical units of expression leads to next dimensional formula:              n sf sf ng     (2) it is obvious that gumminess can not be expressed in physical units newtons. besides the disadvantages of replacing human sensory assessment [1] by instrumental methods [9], [10], [11] and the latter aspect notified under tpa [12], [13], [14] is also a reason for seeking new solutions in order to express accurately the gumminess as secondary parameter of texture. poor sensitivity and human subjectivity of appreciation and also shortcomings of instrumental sensory determination, based on tpa, of food gumminess were the basis of authors motivation for conducting experimental research from this paper. it started from the consideration that gumminess is actually an expression of the viscoelastic behavior of semisolid foods and as such may be determined by specific rheological means such as for example resistive measuring of mechanical resistive moment of a special propeller submerged in food tested or measuring the change in rotation of propeller, at constant active mechanical moment for its rotation. 2. method and procedure for advanced characterization of food texture in developing the method and procedure for determining semisolid food gumminess, the team of authors considered the definition of gumminess by standard reference [1], namely that it is a property related to primary parameters of hardness and cohesion of semisolid food when their hardness is reduced. we also considered that it is difficult for a food taster to assign values located in a score between 1 and 5 to a tested food, after he chewed in the mouth five different consistency dough, see tab.1. it was also considered that the method and recommended procedure to allow automatic electronic measurement of gumminess values and that determinations to provide a resolution of reading and a high reproducibility of measurements and the fact that these instrumental measurements can be food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 120 converted with a curve conversion in gumminess units, defined by the standard of reference [1]. for gumminess measuring it was used an experimental stand, figure 2, which comprises an especially stirrer with propeller speed automatically adjusted on electronic way so that regardless of resistive opposite of tested food, propeller speed does not deviate from setpoint. gumminess of tested food is expressed by the resistive moment value of mixing propeller, measured with a torque cell with resistive sensors at a certain speed of propeller, digital display on alphanumeric display of the mixer. fig.2. schematic diagram of the experimental stand used for determining gumminess of semisolid food and view of the propeller used in experimental research. propeller used for mixing is a particularly type, with two opposed turns, allowing the movement of food during one rotation of turns with one of the turns from the bottom up and top down with another so as to achieve an effect as close to the mastication a semisolid foods in the mouth. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 121 3. experimental for method and procedure validation and advanced characterization of food texture and achievement of conversion curve the stand described was used for experimental research and propeller with turns in opposite of figure 2 and samples of white wheat flour weighing 100 g, type 3 000, each reference sample having different concentrations of flour (different values of the evaluation score) as tab.1. measurements of resistive torque moment were performed at different speeds of propeller, figure 2, a, b, c, d, e and linear regression equations were determined, correlation factor r values ranging between 0.97% and 0 , 99%. a) b) c) d) e) fig.3. representation of resistive moment evolution according to the speed at different concentrations of flour [1] and tab.1. a. concentration of 60% flour, b-concentration of 55% flour, cconcentration of 50% flour, d-concentration of 45%, econcentration of 40% flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 122 in figure 4 is shown the cumulative representation of resistive moment evolution depending on speed at different concentrations of flour water according to [1]. on the graphical representation is highlighted a speed of 150 rpm. this speed was chosen as representative on the one hand all the time dependencies resistive momentspeed are linear to this value, on the other hand, choosing a lower speed that value would have a less reading resolution of resistive moment, while choosing a higher value would not have been possible only up to max speed. 200 rpm., because the sample with the highest concentration of flour (60%) over this speed dough separates from cylindrical glass vessel and rotates with the propeller. fig.4. graphic cumulative representation of resistive moment evolution depending on the speed at different concentrations of flour-water according to [1] and tab.2. highlighting resistive moment at 150 rpm it also watched as the evolution of stirring and flour hydration in time can lead to changes in resistive moment. testing was carried out with flour concentration of 50% (score3) obtaining the distribution in figure 5 of linear type, regression equation presenting a correlation coefficient of 0.99 which shows a good constant of mechanical resistive moment. fig.5. variation in time of resistive moment of dough with 50% flour and 150 rpm of propeller stirrer. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 123 the values of resistance moment read at speed of 150 rpm, for different degrees of gumminess, according with reference standard [1] which are based on different concentrations of flour (tab.1.), was performed resistive time conversion curve – score of gumminess in figure 6, with mathematical model describing a type polynomial equation with expression: y=2.308x3 15.93x2 + 33.66x-0.82 (3) and correlation factor r equal to 0.994. fig.6. conversion scale of resistive moment value, measured on the propeller stirrer, in gumminess units as they are defined as the reference standard [1]. all values correspond to the speed of 150 rpm. of propeller stirrer replacing in equation (1) the resistive value moment measured for some semisolid food weighing 100 grams, using a propeller stirrer as described, it can calculate gumminess score as reference standard [1] with a resolution of two decimal. stirrer interfacing with a computer and using an appropriate software allows direct display of gumminess score value. 4. testing the correspondence between human sensory analysis and instrumental sensory analysis in order to establish the correspondence between human sensory perception of gumminess with experimental values obtained by the instrument technique described above it was done a blind test for appreciation with fourteen females and three males with an age between 22 and 26 years. those seventeen persons were given a teaspoon of dough with different concentrations of flour according to table 1, to memorize the force required for it disintegration and to grant for gumminess points of appropriate panel. between two different samples of dough they rinse mouth several times with distilled water. after the test described, from the glass vessels containing five type of remained dough (corresponding to the content in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 124 flour and water), they leaked the contents by tilting the vessel horizontally and asking panelists to appreciate visual flow rheology with one of the five panel numeric values assigned by evaluating the force necessary for its disintegration by tongue. table 2 results of blind evaluation of dough gumminess without additives, of the same dough with addition of beta carotene and addition of vanilla essence, based on its free flow no. person assessment of dough with 45% flour (panel 2) assessmen t of dough with 45% flour (panel2, additivate d with beta carotene assessment of dough with 45% flour (panel2), additivated with cu vanilla 1 2 3 1 2 2 3 3 3 2 2 1 4 2 2 2 5 2 3 2 6 3 3 3 7 2 2 2 8 3 4 3 9 3 4 2 10 2 3 2 11 2 3 2 12 2 3 2 13 2 2 2 14 2 3 2 15 2 3 2 16 3 3 3 17 3 4 2 next it was used 3 lots of 0.5 kg of the dough with flour 45% (panel 2 according to [1]), concentration and panel of three lots being unknown to panelists. first lot was without additives, the second lot was additivated with 5 ml beta carotene 10% of high purity and the third group was additivated with 10 grams commercial essence of vanilla, dough preparation being done at the speed of 150 rev / min with experimental stand shown in figure 2. table 3. results of blind evaluation of dough lot gumminess according to[1] no. person assessme nt of dough with 45% flour (panel 2) assessment of dough with 45% flour (panel2), additivated with beta carotene assessment of dough with 45% flour (panel2), additivated with cu vanilla 1 2 3 1 2 2 3 2 3 2 1 1 4 2 2 1 5 2 3 1 6 3 3 2 7 2 2 2 8 3 4 2 9 3 4 2 10 2 3 2 11 2 2 2 12 2 3 4 13 2 2 1 14 2 3 2 15 2 3 2 16 2 3 1 17 3 4 2 the three lots were made two types of sensory evaluation experiments. the first experiment consisted in noting the flow rheology of three lots of dough by comparison with the flow rheology of five types of the dough made according to [1]. the result of evaluation by panelists is shown in table 2. after the first experiment of dough rheological flow evaluation each panelist received a teaspoon of dough from the three lots described above, being asked to assess the panel according to the methodology food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 125 described in [1] and to record the result in a table (table 3). obviously before each new determination mouth was rinsed several times. the test results are found in table 3. fig 7. analysis of variance type anova of values in table 2 of sensory test based on flow rheology appreciation of dough. adough assessment with 45% flour using the experimental device, b assessment of the dough with flour 45%, c assessment of dough with 45% flour (panel 2) additivated with betacarotene, d assessment of the dough with 45% flour (panel 2) additivated with vanilla an analysis of variance type anova of values after valuable component for rheological sensory appreciation is shown in figure 7, and the same analysis for assessing sensory appearance (color) and taste is shown in figure 8. in the analysis of variance shown in figure 8 is observed that gumminess measured using the proposed technique and method of human subjects show differences. from the statistic point of view there is a significant difference between measured value with the device and values measured by human subjects for dough with 45% flour (p <0.05, dough with 45% flour additivated with betacarotene (p <0.0001) and dough with 45% flour additivated with commercial vanilla essence (p <0.05). fig. 8. analysis of variance type anova of the values in the table 3 of sensorial test on visual and taste appreciation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu, contributions to a new method for determining food gumminess, food and environment safety, volume xiv, issue 1 – 2015, pag. 117 – 126 126 from the analysis it is clear that taste and color influence human sensory evaluation of food gumminess. 5. conclusions semi solid food gumminess can be determined on instrumental way by electronic measuring of mechanical resistive moment opposed by food to a special propeller with opposite step rotated by a stirrer at a speed of 150 rev / min. a parallel test conducted with volunteers to determine the gumminess on human sensory way in the purpose of comparing the results with the instrumental analysis revealed that instrumental method shows a sensitivity and a better resolution than the human sensorial analysis, differences are even higher as gumminess value is higher. subjectivity of human sensory assessment gumminess is pronounced, taste and appearance of product tested having a major influence on the verdict of panelist. gumminess determination from food texture profile analysis (tpa), does not give good results because its expression of value in physical units is incorrect. 6. references [1] xxx. sr iso 11036/2007 [2] xxx. sr iso 5492/2007 [3] brenann j.g. – food texture measurement, king r.d. – developments in food analysis techniques, applied science, essex, ik,vol. (1980) [4] figura l.o. food physics, springer verlag berlin, (2004), p.144-165 [5] mezger th., rheology handbook , vincentz verlag, hannover, (2000), p,20-36 [6] toledo r.t. fundamentals of food process engineering, van nostrand reinhold, new york (1991), p.132-161 [7] amariei s., gutt g., hreţcanu c., oroian m., a., apparatus and devices for determining food texture and advanced characterization of their behavior to mechanical stress, osim file a00322/2012 [8] amariei s., gutt g., apparatus and method for determining hard cheeses texture and degree of maturation, osim file a00323/2012 [9] malcom c., bourne, food texture and viscosity: concept and measurement, second edition, academic press san diego, (2002), p.177-189 [10] mckena m.,b., texture in food, vol.1, crc press, boca raton, boston, (2003), p. 58, 79, 102, 198, 273,308, [11] chen l., opara u., l., texture measurement approaches in fresh and processed foods, food research international 51, (2013), p.823-835 [12] dan h., kohyama k., interactive relationship bettween the mechanical properties of food and the human response during the first bite , arhive of oral biologie, 52, (2007) p.455-464 [13] nadulski r., methodological aspects of food texture measurements using tpa test , international agrophysics, 14, (2000), p.207-213 [14] rosenthal a., j., relation between instrumental and asensory measures of food texture, aspern publishers, inc, maryland, (1999), p.1-17 207 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 2 – 2013, pag. 207 213 con tri but ions to the de velo pme nt of the ma t erial ba lance o f m igra ti on pro cess of m e tal ions from the ais i3 04 stain l ess s te el i n a ce ti c ac id s ol ut ions *silviu-gabriel stroe1, gheorghe gutt1, maria poroch-seriţan1 1 faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania *silvius@fia.us.ro; g.gutt@fia.usv.ro; mariap@fia.usv.ro *corresponding author received april 8th 2013, accepted may 15th 2013 abstract: the aim of this work was the development of theoretical and real material balance by studying the diffusion phenomena of the metallic ions from aisi304 stainless steel samples in acetic acid solutions with 3%, 6% and 9% concentrations. the correlation of the quantities of substance which migrate from the metallic alloys in the food simulants through the interaction interface between the two environments can be accomplished through the materials balance developed for each component. to development the materials balance, we used the general stoichiometric equation of a chemical process. within the comparative study of the theoretical and real mass balance, we have used the experimental data obtained after the migration tests, where the variables were represented by the working parameters: the temperature of the migration testing t [°c], the exposure time t [min.] and the stirring of the corrosive environment n [rot·min-1]. the value of each parameter was varied on three levels, in accordance with the real situations met in practice. in order to express the quantitative stage of the interaction between the metallic material and the corrosive environment, at a certain moment, we have used the degree of dissolution δm of the metallic components such as mn, cr, 56fe and ni. the comparative study of the dissolution rates obtained allows to extrapolate and to elaborate in practice the optimization of the process which occurs at the interface of the two real food environments. keywords: diffusion, stainless steel, acetic acid, mass balance, dissolution rate 1. introduction the knowledge and study of chemical reactions which occur at the interface between a metallic material and a food environment, considered corrosive, play an important role in the manufacturing process of food raw materials. thus, in order to design the equipment or to optimize the processes we must know the operation conditions, such as: the nature of the corrosive environment, the temperature at which the processes occur, the duration of the contact between the two environments and the stirring of the corrosive environment. 2. materials and methods 2.1. metallic samples and corrosive environments food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 208 for the development of theoretical and real mass balance we have used the metallic samples of aisi304 stainless steel grade, the chemical composition being shown in the table 1 (according to en 100882:2005) [1]. the dimensions of the metallic samples used in the migration tests were of 40×40×1 mm. table 1. chemical composition of aisi304 stainless steel [wt %] fe c mn p s si cr ni 67 0.08 2 0.045 0.03 1 18-20 8-11 as corrosive environments, we have used solutions of ch3cooh, with the following concentrations: 3%, 6% and 9%. 2.2. experimental design the experimental design used for the migration tests in each of the three corrosive environments is presented in the table 2. tabel 2. experimental design used to perform the migration tests nr. exp. temperatura t-[°c] timp t-[min.] agitare n-[rot·min-1] 1 22 30 0 2 22 30 125 3 22 30 250 4 28 30 0 5 28 30 125 6 28 30 250 7 34 30 0 8 34 30 125 9 34 30 250 10 22 60 0 11 22 60 125 12 22 60 250 13 28 60 0 14 28 60 125 15 28 60 250 16 34 60 0 17 34 60 125 18 34 60 250 19 22 90 0 20 22 90 125 21 22 90 250 22 28 90 0 23 28 90 125 24 28 90 250 25 34 90 0 26 34 90 125 27 34 90 250 after performing migration tests of the elements from the metallic samples in the acid solutions of the corrosive environments there have been identified and dosed, using mass spectrometry and inductively coupled plasma icp-ms, the following metallic elements: mn, cr, 56fe and ni, according to the table 3. table 3. concentrations of mn, cr, 56fe and ni elements found in ch3cooh solutions, used as corrosive environments no. exp. chemical element, [mg·l-1] mn cr 56fe ni 3% 6% 9% 3% 6% 9% 3% 6% 9% 3% 6% 9% 1 0.003 0.00157 0.00122 0.004 0.005 0.001 0.990 0.32 0.08 0.0152 0.311 0.0033 2 0.003 0.00237 0.00182 0.004 0.007 0.001 0.660 0.40 0.10 0.0072 0.341 0.0058 3 0.022 0.00287 0.00222 0.077 0.012 0.003 6.120 0.54 0.26 0.0782 0.381 0.0064 4 0.003 0.00177 0.00072 0.005 0.012 0.002 0.390 0.20 0.02 0.0132 0.391 0.0056 5 0.004 0.00227 0.00102 0.009 0.015 0.004 1.170 0.28 0.04 0.0232 0.531 0.0066 6 0.005 0.00287 0.00142 0.007 0.019 0.005 0.730 0.58 0.10 0.0352 0.551 0.0071 7 0.002 0.00197 0.00082 0.005 0.014 0.002 0.760 0.36 0.02 0.0082 0.411 0.0057 8 0.003 0.00207 0.00132 0.007 0.018 0.005 0.860 0.74 0.06 0.0132 0.511 0.0060 9 0.005 0.00247 0.00182 0.019 0.022 0.007 1.040 1.22 0.28 0.0372 0.561 0.0076 10 0.003 0.01467 0.00092 0.005 0.011 0.001 0.690 0.22 0.06 0.0132 0.281 0.0038 11 0.004 0.00607 0.00122 0.005 0.013 0.002 0.400 0.60 0.14 0.0068 0.331 0.0053 12 0.041 0.00387 0.00152 0.188 0.020 0.003 9.220 1.08 0.68 0.1372 0.381 0.0066 13 0.003 0.00207 0.00112 0.007 0.014 0.008 0.880 0.22 0.18 0.0152 0.301 0.0073 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 209 14 0.005 0.00377 0.00142 0.013 0.019 0.008 1.190 0.36 0.30 0.0192 0.321 0.0091 15 0.004 0.00357 0.00162 0.010 0.020 0.009 0.720 0.50 0.34 0.0282 0.367 0.0112 16 0.002 0.00227 0.00102 0.020 0.014 0.002 0.160 0.44 0.08 0.0072 0.231 0.0072 17 0.004 0.00287 0.00132 0.012 0.019 0.004 0.960 0.54 0.18 0.0202 0.276 0.0086 18 0.007 0.00367 0.00172 0.022 0.022 0.005 2.010 0.72 0.32 0.0322 0.306 0.0101 19 0.004 0.00257 0.00082 0.007 0.012 0.003 1.090 0.76 0.02 0.0132 0.301 0.0052 20 0.003 0.00267 0.00112 0.005 0.015 0.003 0.590 0.88 0.02 0.0082 0.331 0.0057 21 0.043 0.00297 0.00142 0.238 0.020 0.004 5.320 0.96 0.10 0.2372 0.361 0.0059 22 0.004 0.00387 0.00182 0.011 0.018 0.006 0.840 0.72 0.28 0.0172 0.121 0.0071 23 0.005 0.01167 0.00372 0.014 0.031 0.009 0.870 0.88 0.88 0.0172 0.141 0.0097 24 0.008 0.01267 0.00502 0.012 0.042 0.015 1.020 1.00 1.16 0.0302 0.172 0.0131 25 0.008 0.00147 0.00172 0.012 0.013 0.008 1.230 0.20 0.44 0.0262 0.160 0.0079 26 0.016 0.00357 0.00522 0.079 0.026 0.015 4.320 0.66 1.24 0.0752 0.211 0.0088 27 0.025 0.01567 0.00972 0.108 0.039 0.036 7.920 1.04 2.28 0.1072 0.241 0.0168 3. results and discussion 3.1. theoretical material balance assuming that the reaction kinetics is known, this paper presents the theoretical and real material balances of the diffusion processes which occur between the aisi304 stainless steel grade samples and the food simulants (acetic acid solutions 3%, 6% and 9%). the correlation of the substance quantities which leave the metallic material and migrate in the food environment, at the interaction interface between the two environments, can be done by the use of the materials balance elaborated by components [2]. for the development of the material balance we have started from the stoichiometric equation of a chemical process, which can be written as (1) [2]:      m i ia n i ia aa ii 11  (1) where: ia  the stoichiometric coefficient of the reactants; ia  the stoichiometric coefficient of the reaction products; ia , ia reactants, reaction products, respectively. at the interaction interface between the food environment and the metallic material, main chemical reactions, secondary chemical reactions and electrochemical reactions occur. in order to express the quantitative stage of the interaction between the aliment and the metallic material, at a certain moment, we have used a dissolution rate of a metallic contaminant or a certain component ia  [3], [4]. this parameter is defined as dissolution rate of the metallic contaminant and is expressed by the relation: 0 0 0 0 0 0 k k i ii k k i ii k kk k a a a aa a a a aa a aa a n nn n nn n nn                (2) where: 0 ia n , 0 ka n , 0 ia n  is the composition of the initial reaction mass of the contaminant ( ia ), of a certain contaminant ( ka ), and of the reaction products ( ia ), respectively; ia n , ia n  the composition of the reaction mass at a certain moment of the contaminant ( ia ), of the reaction products ( ai ' ), respectively. the use of the dissolution rate variable for the quantitative characterization of the process allows the development of the stoichiometric calculation in a simple form food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 210 and the elaboration of the balance equation system in a form appropriate to their use for the quantitative description of the process. starting with the composition of the reaction mass at a certain moment expressed through the sizes: naa nn ... 1 ; naa nn  ...1 (3) and for each component it can be written the following balance equations [2]: kk k i ii aa a a aa nnn    00 (4) kk k i ii aa a a aa nnn     00 (5) 0 ii aa nn   (6) the total number of moles in the reaction mass, at a certain moment, is expressed by the balance equation (7): kk k ii kk k i kkiii aa a n a m a taa a m a aa k n is a m a n at nnn n a a nnnn          011001 01 1 0 1 0 1 0         (7) the equation (7) can be written also as equation (8) [2]:   k k k k ii at a t a a n a m a tt n n n nn                       1 1 0 0 0 110 (8) where: 0 11 t a a n i a m i a n n k k ii          (9) by replacing the number of moles by mass, the equation (2) can be written as equation (10): 0 0 0 0 0 0 k k i k i ii k k i k i ii k kk k a a a a a aa a a a a a aa a aa a m m m mm m m m mm m mm                (10) 3.2. real material balance in case of aisi304 stainless steel grade, used in the processing of food raw materials, the initial mass of reaction is considered to be formed by the metals presented in table 1 (according to en 10088-2:2005) [1], from the composition of metallic material and the acetic acid (ch3cooh) [5]. the acetic acid has the following properties: 105,60  molgm , 76,4apk , at 25°c. the stoichiometric equation of the chemical process from the interaction interface between the acetic acid and aisi304 stainless steel, in case of obtaining the vinegar by acetic fermentation, is the following:   ohohfefeohcniohc crohcmnohcoohcfenicrmn 2222322232 223222322242 0000 3)()( )()(2/582   (11) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 211 and the secondary reaction is:   ohhofeohfe 224323  (12) in order to express the quantitative stage of the interaction reaction between the metallic material and the aliment, at a certain moment, it is used the dissolution rate mn of a metallic contaminant (mn), expressed by the relation: 0 0 mn mnmn mn m mm   (13) the mass of each component, in a certain moment, is expressed by the equation (13) [3], [4]. the theoretical masses calculated for the contaminants mn, cr, 56fe and ni which could result from the complete dissolution of the metallic samples are presented in table 4. tabel 4. theoretical masses of the contaminants mn, cr, 56fe and ni [mg] mn cr 56fe ni 202.2 1819.8 6673.7 1011.0 based on the relation (13) we have obtained the numeric values of the dissolution rate of the mn, cr, 56fe and ni contaminants (table 5). tabel 5. the dissolution rate of the contaminants mn, cr, 56fe and ni nr. exp. δmn, [%] δcr, [%] δ56fe, [%] δni, [%] 3% 6% 9% 3% 6% 9% 3% 6% 9% 3% 6% 9% 1 0.0015 0.0008 0.0006 0.0002 0.0003 0,0055 0.0148 0.0048 0.0012 0.0015 0.0308 0.0003 2 0.0015 0.0012 0.0009 0.0002 0.0004 0,0055 0.0099 0.0060 0.0015 0.0007 0.0337 0.0006 3 0.0109 0.0014 0.0011 0.0042 0.0007 0.0002 0.0917 0.0081 0.0039 0.0077 0.0377 0.0006 4 0.0015 0.0009 0.0004 0.0003 0.0007 0.0001 0.0058 0.0030 0.0003 0.0013 0.0387 0.0006 5 0.0020 0.0011 0.0005 0.0005 0.0008 0.0002 0.0175 0.0042 0.0006 0.0023 0.0525 0.0007 6 0.0025 0.0014 0.0007 0.0004 0.0010 0.0003 0.0109 0.0087 0.0015 0.0035 0.0545 0.0007 7 0.0010 0.0010 0.0004 0.0003 0.0008 0.0001 0.0114 0.0054 0.0003 0.0008 0.0407 0.0006 8 0.0015 0.0010 0.0007 0.0004 0.0010 0.0003 0.0129 0.0111 0.0009 0.0013 0.0505 0.0006 9 0.0025 0.0012 0.0009 0.0010 0.0012 0.0004 0.0156 0.0183 0.0042 0.0037 0.0555 0.0008 10 0.0015 0.0073 0.0005 0.0003 0.0006 0,0055 0.0103 0.0033 0.0009 0.0013 0.0278 0.0004 11 0.0020 0.0030 0.0006 0.0003 0.0007 0.0001 0.0060 0.0090 0.0021 0.0007 0.0327 0.0005 12 0.0203 0.0019 0.0008 0.0103 0.0011 0.0002 0.1382 0.0162 0.0102 0.0136 0.0377 0.0007 13 0.0015 0.0010 0.0006 0.0004 0.0008 0.0004 0.0132 0.0033 0.0027 0.0015 0.0298 0.0007 14 0.0025 0.0019 0.0007 0.0007 0.0010 0.0004 0.0178 0.0054 0.0045 0.0019 0.0318 0.0009 15 0.0020 0.0018 0.0008 0.0006 0.0011 0.0005 0.0108 0.0075 0.0051 0.0028 0.0363 0.0011 16 0.0010 0.0011 0.0005 0.0011 0.0008 0.0001 0.0024 0.0066 0.0012 0.0007 0.0228 0.0007 17 0.0020 0.0014 0.0007 0.0007 0.0010 0.0002 0.0144 0.0081 0.0027 0.0020 0.0273 0.0009 18 0.0035 0.0018 0.0009 0.0012 0.0012 0.0003 0.0301 0.0108 0.0048 0.0032 0.0303 0.0010 19 0.0020 0.0013 0.0004 0.0004 0.0007 0.0002 0.0163 0.0114 0.0003 0.0013 0.0298 0.0005 20 0.0015 0.0013 0.0006 0.0003 0.0008 0.0002 0.0088 0.0132 0.0003 0.0008 0.0327 0.0006 21 0.0213 0.0015 0.0007 0.0131 0.0011 0.0002 0.0797 0.0144 0.0015 0.0235 0.0357 0.0006 22 0.0020 0.0019 0.0009 0.0006 0.0010 0.0003 0.0126 0.0108 0.0042 0.0017 0.0120 0.0007 23 0.0025 0.0058 0.0018 0.0008 0.0017 0.0005 0.0130 0.0132 0.0132 0.0017 0.0139 0.0010 24 0.0040 0.0063 0.0025 0.0007 0.0023 0.0008 0.0153 0.0150 0.0174 0.0030 0.0170 0.0013 25 0.0040 0.0007 0.0009 0.0007 0.0007 0.0004 0.0184 0.0030 0.0066 0.0026 0.0158 0.0008 26 0.0079 0.0018 0.0026 0.0043 0.0014 0.0008 0.0647 0.0099 0.0186 0.0074 0.0209 0.0009 27 0.0124 0.0078 0.0048 0.0059 0.0021 0.0020 0.1187 0.0156 0.0342 0.0106 0.0238 0.0017 after obtaining the numeric values (according to the table 5), a comparative study can be done in relation to the influence of the working parameters on the dissolution rate of the contaminants. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 212 it is known the fact that the presence of manganese in the stainless steels has the role to ensure deoxidation and to prevent the formation of iron sulphide inclusions [6]. in which concerns the behaviour of manganese (mn) in acid environments, the lowest dissolution rate (0,004%) was obtained by the 7 and 19 experiences: t34°c and 22°c, t-30 and 90 min. and stationary environments (n-0 rot·min-1). the highest dissolution rate (0.0124%) was obtained in the experience no. 27, where the working parameters were: t-34°c, t-90 min. and n-250 rot·min-1. concerning the behaviour of the chrome in acid environments, it is known the fact that this is not a chemical element to present an important migration phenomenon in food environments. due alloying chromium, the stainless steels are more resistant to corrosion [7]. the lowest dissolution rate (0.0001) can be noticed in case of experiences 4, 7, 11 and 16, where, as particularity, the corrosive environment was stationary, only in case of experience no. 11, the stirring of the environment being of 125 rot·min-1. the highest dissolution rate (0.0131) can be noticed in the case of experience no. 21, where the environment concentration was of 3% ch3cooh, t-22°c, t-90 min. and n-250 rot·min-1. regarding the behaviour of the iron to corrosion (56fe), the lowest dissolution rate were obtained in the case of experiences 4, 7, 19 and 20, where a stationary corrosive environment was used, the exception being the experience no. 20, where n-125 rot·min-1. the highest dissolution rate of iron is observed in case of experiment no. 12, where the concentration was of 3% ch3cooh, t-22°c, t-60 min. and n-250 rot·min-1. amongst all studied metallic elements within this experiment, nickel presents the highest degree of risk on human health. the world health organization (oms) recommends a maximum allowable dose of 0,005 mg/kg of body weight. oms recommended also a value of nickel for the drinking water of 0,02 mg/l [8], and the daily intake of nickel from food products is estimated to 0,15-0,7 mg/day [8]. from studying the values of the dissolution rate for ni, it is noticed that the minimum value of the degree of dissolution is met in case of the experience no.1, where solutions of 9% ch3cooh, t-22°c, t-30 min. and n 0 rot·min-1 were used. the maximum value of the dissolution rate is noticed in case of experience no. 9, where the concentration was of 6% ch3cooh, t-34°c, t-30 min. and n-250 rot·min-1. 4. conclusions the development of the theoretical and real mass balances of the migration processes of metallic acids in acid solutions, studied and presented in this paper, have had as main purpose the characterization of the migration processes from the interaction interface between acetic acid and aisi304 stainless steel grade samples through the calculation of the dissolution rate of the metallic contaminants mn, cr, 56fe and ni. by elaborating these balances, we have obtained very important information regarding the behaviour of these stainless steels in the studied experimental conditions. one can notice that minimum values of the dissolution rate of the elements mn, cr and 56fe are obtained when solutions with 3% ch3cooh concentrations were used, and the minimum dissolution rate of ni were obtained when the environment concentration was of 9% ch3cooh. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 2 – 2013 s i l v i ug a b ri e l s t r o e , g he o rg he g u t t , m a ri a po r o c h s e r iţan, c o n t r i b u t i o n s t o t h e d e ve l o p m e n t o f t h e m a t e r i a l b a l a n c e o f m i g r a t i o n p r o c e s s o f m e t a l i o n s f r o m t h e a i s i 3 0 4 s t a i n l e s s s t e e l i n a c e t i c a c i d s o l u t i o n s , f o o d a nd e n v i r o n me n t s a f e t y , v o l u me x i i , i s s u e 2 – 2 0 1 3 , pa g . 2 0 7 2 1 3 213 the maximum levels of dissolution of mn, cr and 56fe elements were obtained in case of the use of corrosive solutions with a concentration of 3% ch3cooh, and for the element ni when solutions with 6% ch3cooh concentration were used. 5. references [1]. en 10088 2 : 2005, stainless steels. technical delivery conditions for sheet/plate and strip of corrosion resisting steels for general purposes, (2005) [2].calistru c., leonti c., inorganic substances technology, ed. didactică şi pedagogică, bucureşti, (1972) [3]. gutt s., gutt gh., contributions to the mass balance and energy to electrochemical sharpening, chemistry magazine, 44(11), 972 – 977, issn 0034-7752, (1993) [4].gutt s., gutt gh., contributions to the material and energy balance sheet for the electrochemical nickel convection ultrasonic, chemistry magazine, 6(48), 521 – 531, issn 0034-7752, (1997) [5].buculei a., amariei s., poroch seriţan m., gutt g., study on the development of the material balance focused on the metal transfer between the system canlacquering and canned vegetables, international conference, modern technologies in the food industry-2012, 1-3 november, 2012, chişinău (republic of moldova), section 3, chemistry and microbiology of food, (2013) [6].ehedg european hygienic engineering & design group, materials of construction for equipment in contact with food, trends in food science & technology 18, elsevier, (2007) [7].codex alimentarius commission, doc. no. cx/fac 96/17. joint fao/who food standards programme. codex general standard for contaminants and toxins in foods. directive 91/338/eec: council directive 91/338/eec amending for the 10th time directive 76/769/eec on the approximation of the laws, regulations and administrative provisions of the member states relating to restrictions on the marketing and use of certain dangerous substances and preparations. l 186 p. 59, (1995) [8].world health organization who nickel in drinking-water. background document for development of who guidelines for drinking-water quality, geneva, world health organization, (2005). 74 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 74-83 physico-chemical properties of blends of corn oil with coriander seed oil * silvia mironeasa1, georgiana gabriela codină1 1faculty of food engineering, stefan cel mare university, suceava, romania, silviam@fia.usv.ro ; codina@fia.usv.ro *corresponding author received 15th february 2015, accepted 30th march 2015 abstract: the effects of different additions of coriander seed oil (10%, 20% and 30%) in the corn oil on the physico-chemical parameters like refractive index (ri), iodine value (iv), saponification value (sv), peroxid value (pv), acid value (av) and oil viscosity were investigated. it was found that the presence of coriander seed oil up to 30% in corn oil had an improving effect on quality parameters of the oil blends. the addition of coriander seed oil in corn oil caused a decrease in the refractive index, iodine value, saponification value and peroxid value with the increased mixing ratios (proportion) in the oil blends formulation. the viscosity of all oil samples analyzed by brookfield viscometer appeared to remain constant regardless of the shear rates tested. the results suggest that coriander seed oil can contribute to improving quality of corn oil. keywords: oil blends, corn oil, coriander seed oil 1. introduction coriander (coriandrum sativum l.) is an herbal plant of a high economic interest for the food and other industries. cultivated for both leaves and seeds, coriander is highly appreciated not only for the nutritional point of view [1, 2], but also for some medicinal benefits like hepatoprotective [3] antioxidant [4], antimutagenic [5], antianxiety [6], antidepresive [7] antitumor [8], antihyperlipidemic [9], against worms, rheumatism [10], for ameliorate insomnia, anxiety, convulsion [11], digestive stimulant [12] e.g. due to its bioactive components [13, 14], different parts of coriander plant can be used to develop new formulations as a promising functional foods. the coriander seeds are the most widely used components of the plant due to its many important constituents. it contains high levels of bioactive lipids [15] such as essential oil (linalool) and fatty oil. the content of fatty oil is around 25% of the seed while the essential oil content is usually less than 1% [16]. coriander seed oil is a triglyceride oil extracted from seeds of coriandrum sativum l. using various technique such as expelling or pressing, organic solvents extraction, supercritical fluid extraction [17], e.g. this vegetable oil, considered as a novel food ingredient, is a triglyceride oil in which the monounsaturated fatty acid petroselinic acid (cis-c18:1(n-12)) is the http://www.fia.usv.ro/fiajournal mailto:silviam@fia.usv.ro mailto:codina@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 75 main fatty acid (60-75 % of coriander seed oil) [18]. some researchers have reported that this major fatty acid is ranging from 65.7% to 84.2% and is followed by linoleic acid (c18:2) which is accounting for 12– 19%, and small quantities of oleic acid (cis-c18:1(n-9)) (8-15 %), palmitic acid (c16:0) (2-5 %) and stearic acid (c18:0) (< 1.5 %) [18, 19, 20]. the coriander seed oil is a highly promising edible oil with a high antioxidant potential [4, 21], due to it higher radical scavenging activity that has been partly attributed to its unsaponifiables (21.8 g/kg) and phospholipids (1.57%) content. in addition, coriander oil is a good source of tocols (327.47 mg/g) and tocotrienol (275.87 mg/g) [16] which inhibit the lipid peroxidation. the variations in oil composition and fatty acids of the coriander oil can be due to the geographic divergence and ecological conditions [16]. therefore, among newer sources of edible oils, the coriander seed oil may be used as potent source of valuable amounts of bioactive compounds in order to improve the quality of vegetable oils, like corn oil. oil quality is very important for both the consumers and well as an application in different industries. corn oil is extracted from the corn germ by a combination of expelling in continuous screw presses and solvent extraction of the press cake. then, crude corn oil is subject to the refining process in order to remove it free fatty acids and phospholipids content and to retain the tocopherols in the refined corn oil. the main fatty acids in corn oil is linoleic acid (c18:2) (54-60%), oleic acid (c18:1) (25-31%), palmitic acid (c16:0) (11-13%), followed by stearic acid (c18:0) 2-3% [22]. corn oil is recognized as a healthy edible oil due to its high content of the fatty acids, having beneficial effects on blood pressure, platelet aggregation, diabetes [23], atherosclerosis by prevention of the oxidation of low-density lipoproteins due to the antioxidant properties of tocopherols [24] and cholesterol-lowering properties [23, 25] due to its highest levels of unsaponifiables and phytosterols. the corn oil has wide application in food industry where this is used in frying, salad dressing, shortening, cooking, e.g. corn oil composition can affect its behaviour during frying and processing. due to it high content of polyunsaturated fatty acid (56%) [18], is not quite stable at high temperatures. also, a disadvantage of corn oil is the high linoleic acid content (4070%) [26]. the high level of this polyunsaturated fatty acid can cause a high degree of oxidation at high temperature [27]. also, during storage the quality of corn oil decreased because it undergoes hydrolysis, oxidation and polymerization [28]. therefore, the development of a more stable high product, more stable to frying and storage, with the increase of the quality parameters at a low price would be desirable. on the other hand, the demand for edible oil with a improve quality is in continually increasing due to the increased concern of the population regarding to the healthy eating. nutritional quality has been recognized for oils rich in monounsaturated fatty acids with the reducing of its polyunsaturated (like, linolenic acid) and saturated contents [29]. one way to improve the quality of corn oil is by blending with oils of high monounsaturated fatty acids contents and high antioxidants’ levels. during last years, blending of two or more vegetable oils with different characteristics have been a common permitted practice in the many countries and aiming an improving of the physicochemical parameters of the new specific products with a better quality and nutritional value at procurable prices [27, 28]. blending different types of vegetable oils lead not only to a change in fatty acids profile, but also increase the levels of bioactive lipids food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 76 and natural antioxidants in the blends and change the physico-chemical oil parameters. the refractive index, iodine value, peroxide value, saponification value and acid value can be improved in this way to get good and desirable blends. viscosity of the oil is another important parameter that must be considered for example in chemical engineering design. the rheological behaviour and flow properties of edible oils have a significant role in the food industry for the evaluation process of the equipments such as pumps, piping, e.g. [30]. the viscosity of the oil has a direct relationship with the physico-chemical oil composition, respectively with the nature of the triglycerides presents in the oil. in this way, viscosity is related to some chemical properties of the oils, such as the degree of unsaturation and the chain length of the fatty acids [31]. it has been reported that oil viscosity increased with the increased degree of saturation and decreased with the increased of the degree of unsaturation [32]. the blending of vegetable oils with various characteristics to make new products has been investigated by various researchers [15, 27, 33, 34] but, to our knowledge, the physicochemical properties of corn/ coriander oil blends in mixing ratios of 9/1, 8/2 and 7/3 have not been evaluated yet. therefore, corn oil mixed with coriander seed oil could give new oil formulation with improved characteristics. in this study the effect of blending corn oil with coriander seed oil in different mixing ratios on the physico-chemical parameters of the blends oil samples has been investigated. the studies have conducted on the following parameters: refractive index, iodine value, peroxide value, saponification value, acid value and viscosity. this research can contribute to the development of healthy blended oils with an improved quality. 2. experimental 2.1 materials corn oil and coriander seed oil were purchased from local market (suceava, romania). corn oil (co) was blended with coriander seed oil (cso) in varying proportions. the following co:cso (% w/w) blends were formulated: 100:0, 90:10, 80:20, 70:30 and 0:100. the oil blends were thoroughly mixed to form uniform blends at room temperature. all solvents and reagents used in this work were of the highest purity needed for each application.the samples was subsequently analysed. 2.2 methods of analysis the following physico-chemical parameters were carried out according to the romanian or international standard methods. all tests were performed in duplicate. refractive index the refractive index (ri) of the oil samples was determined with a abbé refractometer at 20°c by the measurement of the angle of total reflection [sr en iso 6320:2002/ac:2006]. iodine value iodine value (iv), expressed of the number of g of iodine absorbed by 100 parts by weight of the oil (g i2/100 g sample) (sr en iso 3961:2013), was determined using the hanus method. a blank was also prepared alongside the oil samples. saponification value saponification value (sv) which represents the amount of the potassium hydroxide, in mg, required for saponification of the free fatty acids and the esterified one from of a 1 g of oil sample (mg koh/g sample) was determined according to the sr en iso 3657 : 2013. a blank was also prepared alongside the oil samples. peroxide value food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 77 the peroxide value (pv) (meq/kg) was determined according to sr en iso 3960:2010, by the reaction of 2 g oil in 25 ml solvent mixture of glacial acetic acid: chloroform (3:2, v/v) with freshly prepared potassium iodide solution. the solution was placed in darkness for 5 min and was titrated with sodium thiosulfate solution (0.01n) after addition of soluble starch (1%) as indicator. the titration continued till until the blue color had just disappeared. a blank was also prepared alongside the oil samples. acid value the acid value (av), expressed in mg koh/g sample, was determined by titration with the potassium hydroxide (0.1n) of a solution of oil (10 g) in a previously neutralized solvent mixture of ethanol–ethyl ether (1:1) with phenolphthalein indicator (1% in ethanol) added [sr en iso 660 : 2009]. the titration continued until the pink coloration persists for at 1 min. viscosity a brookfield rotational viscometer (model rvdv-i prime, brookfield engineering laboratories) was used to measure the oil samples viscosity (pas) at the speed of 2.5, 5, 10, 20 and 50 rpm. a fixed volume of oil in a 600 ml beaker was used to immerse the groove on the spindle and the spindle depth was kept constant throughout the measurements. two readings were taken per sample at 30 s intervals. 2.3. statistical analysis the experimental results were expressed as means ± standard deviation (sd) of duplicate measurements. differences between means were evaluated by the analysis of variance (anova) using spss v.16.0 software. statistical significant difference was considered at p < 0.05. 3. results and discusion physicochemical characteristics refractive index (ri) refractive index of corn oil, coriander seed oil and oil blends is shown in fig. 1. the ri values for co and cso is in agreement with the values reported by different researches [18, 22, 35]. blending of cso with co has changed significantly (p < 0.5) the value of ri in co:cso blends. this decreased of ri value with the increased of the mixing ratio may be due to the decreased of the unsaturation acid content in the blends knowing that the the refractive index is the physical parameter that dependent on degree of unsaturation acid content [36]. also, the decrease of ri in the blends can be due to the increased of the monounsaturated fatty acids of blends formulation, according to ramadan and wahdan (2012) or may be due to molecular weigh, fatty acid chain length, or the degree of conjugation [37]. fig. 1. variation of refractive index (ri) in oil samples formulation: co –corn oil; cso – coriander seed oil. error bars show the variations of two determinations in terms of standard deviation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 78 iodine value (iv) comparatively with the iodine value of coriander seed oil (cso), the iodine value of corn oil (co) was higher (fig. 2) probably due to a high degree of unsaturated fatty acids from oil corn and a degree of heat treatment during corm oil processing [38]. because the iv is a measure of the degree of lipid unsaturation in oil, this high value may be an indication of a high saturation in corn oil and therefore may become more vulnerable to oxidation, influencing therefore the stability during storage. by blending co with cso in different mixing ratio was obtained a decrease in the iv with the increased ratio of coriander oil in blends, probably due to the increased content of unsaturation acid in oil samples formulation. the phenomenon of a decreased trend in iv indicates an increased in unsaturation content this fact leading to no risk on the consumer health. fig. 2. variation of iodine value (iv) in oil samples formulation: co –corn oil; cso – coriander seed oil. error bars show the variations of two determinations in terms of standard deviation. the results obtained for iv gives a reasonable quantitation of lipid unsaturation if the double bonds are not conjugated with each other or with carbonyl oxygen [39]. saponification values (sv) the sv of cso is insignificance lower comparable with the co, probably due to a high molecular weight lipids from coriander oil. the sv of the binary blends were found to be in decrease as shown in fig. 3. this trend explains the fact that with the increase level of cso ratio in formulated blends the fatty acids are not likely to formed, therefore the sv will decrease. this also indicates that, comparatively with the pure co, these blends can be stored for a long time. peroxide value (pv) the pv calculated for pure corn oil, coriander seed oil, and binary oil blends are shown in fig. 4. fig. 3. variation of saponification value (sv) in oil samples formulation: co – corn oil; cso – coriander seed oil. error bars show the variations of two determinations in terms of standard deviation. cso indicates a relatively good quality of this oil compared to the co. the pv obtained indicates an early lipid phase peroxidation probably due to the fact that peroxides are the first components of lipid oxidation. the addition of cso to the co significantly decreases (p  0.05) the pv, this showing an enhancement of the oxidative stability of oil blends. because the hydroperoxide is the primary product of lipid oxidation, the pv can be used as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 79 oxidative index during the early stage of lipid oxidation. the decrease of pv with the increase level of sco from blends may be due to the changes in the fatty acids profile, and some bioactive lipid’s such as sterols and phenolics from the seeds coriander, in agreement with other results [28] as a consequence of the changes in fatty acids and tocopherols’ profile, and minor bioactive lipids (e.g., sterols and phenolics) founded in coriander and black cumin oils. the pv of co and oil blends varied significantly (p  0.05) with the increase level of cso (90:10, 80:20 and 70:30, w/w) for the high level of cso being the most stable from the point of view of oxidation. fig. 4. variation of peroxide value (pv) in oil samples formulation: co –corn oil; cso – coriander seed oil. error bars show the variations of two determinations in terms of standard deviation. these results indicated that the natural antioxidants from coriander seed oil hindered considerably the oxidation process in oil blends formulation. acid value (av) av is a very important parameter for the assessment of oils quality, because it represents free fatty acid content due to it enzymatic activity. this parameter can be used to check the level of oxidative deterioration of the oil by enzymatic or chemical oxidation. the experimental results showed a significant decrease (p  0.05) in the av of the co when it is blended with cso (fig. 5). the greater proportion of cso from the oil blend showed the lowest av value. the higher av in co is due to the free fatty acids present in the oil and the lower level of av in cso indicating lowers levels of hydrolytic and lipolytic activities. the higher ratio of the cso blended with co led to a further decrease in the av values in the blends. by blending with coriander oil the portion of the unsaturated fatty acids increased in the blends formulated. these is probably due to the fact that these fatty acids do not take part in any chemical changes in the oil and do not interact with triacylglycerol even if they are of similar chemical composition [40]. fig. 5. variation of acid value (av) in oil samples formulation: co corn oil; cso – coriander seed oil. error bars show the variations of two determinations in terms of standard deviation. viscosity the oil samples viscosity is influenced by the triglycerides present in the pure oil, oils blends, respectively. the various arrangements of the fatty acids on the glycerol backbone of the triglyceride molecule changed the viscosity. therefore, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a b ri el a c o d in ă, ph y s i c o ch e m i c a l pr o p e rt i e s o f b l e nd s o f c or n o i l wi t h c o r i an d e r s e ed o i l, f o o d a nd e nv ir o nme nt sa f e t y, v o lu me x iv , is s ue 1 – 2 0 15 , p a g . 7 4 -83 80 this parameter is related to the chemical properties like the chain length and saturation/unsaturation [41]. the rheological behaviour of oil samples analyzed can be explained by empirical relationship of power law model (eq.1) nk  (1) where, σ is the shear stress (pa), k is the consistency coefficient (pa  sn),  is the shear rate (1/s) and exponent n is the flow behavior index (dimensionless). the empirical data obtained for oil sample formulation were converted to shear stress and shear rate followed the method described by briggs and steffe [42]. average shear stress and shear rate was calculated using eqs. 2 and 3, respectively: )c(ka   (2) nk na   (3) where, σa is the average shear stress (pa), ݇ஶ is the shear stress conversion factor (pa), c is the spring constant that depend on brookfield viscosimeter model used and α is the torque value read for the viscosimeter (%). the ݇ஶis a function of the spindle number, ߛ௔ is the shear rate (1/s), ݇ேംthe shear rate conversion factor and n is the rotational speed in rpm. values of݇ேംare as function of the spindle number and the flow behaviour index [43]. the apparent viscosity (ηa) (eq. 4) was calculated dividing eq.2 by eq. 3: a a a    (4) the results obtained for all oil samples showed a similar flow trend in which the shear stress is directly proportional to the shear rate (fig. 6) having flow indices very close to 1. fig. 6. curves of shear stress vs. shear rate of oil samples formulation at 23oc. the characteristics of the oil samples showed a newtonian flow behaviour which was adequately described by the eq. 4, indicating high determination coefficients (r2). the r2 values ranged from 0.985 to 0.0.998 for all oil samples. this behaviour can be due to their long chain molecules [44] and to the ratio saturation/unsaturation. the values of pure and oils blends viscosity is shown by the slope of each curve, varying distinctly as function on the each type of the oil samples. 4. conclusion the quality characteristics of the pure corn oil, coriander seed oils and formulated blends were evaluated through this study by different characteristics. for oil blends the physical and chemical parameters have the same tendency to decrease with the increased of mixing ratio of coriander seed oil in corn oil. the viscosity of oil blends decreased insignificantly with an increase in proportion of coriander seed oil in corn oil probably due to change of the fatty acid profile of oil blends. 0 100 200 300 400 500 600 0 2 4 6 8 10 12 14 shear rate [1/s] sh ea r st re ss [ pa ] 100_co:0_cso 90_co:10_cso 80_co:20_cso 70_co:30_cso 0_co:100_cso food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 81 this study shows that blending is a good choice by which we can obtain edible oils with desirable physico-chemical parameters, improved from the point of view nutritional and qualitative. the coriander seed oil was found to be the vegetable oil with high qualitative properties that can be use to improving quality of corn oil and may bring functional benefits to food systems. 5. references [1]. bhat s., kaushal p., kaur m., sharma h. k., coriander (coriandrum sativum l.): processing, nutritional and functional aspects, afr. j. plant sci. 8 (1), 2533, (2014) [2]. sahib n.g., anwar f., gilani a.h., hamid a.a., saari a., alkharfy k.m. coriander (coriandrum sativum l.): a potential source of high-value components for functional foods and nutraceuticalsa review, j. phytother. res. 27 (9), 1439–1456, (2013) [3]. sreelatha s., padma p.r., umadevi m., protective effects of coriandrum sativum extracts on carbon tetrachloride-induced hepatotoxicity in rats, food chem. toxicol. 47 (4), 702-708, (2009) [4]. ramadan m.f., kroh l.w., moersel, j.-t., radical scavenging activity of black cumin (nigella sativa l.), coriander (coriandrum sativum l.) and niger (guizotia abyssinica cass.) crude seed oils and oil fractions, j. agric. food chem. 51, 6961– 6969, (2003). [5]. cortes-eslava j., gomez-arroyo s., villalobos-pietrini r., antimutagenicity of coriander (coriandrum sativum) juice on the mutagenesis produced by plant metabolites of aromatic amines, j. toxicol. lett. 153, 283-292, (2004) [6]. mahendra p, bisht s., anti-anxiety activity of coriandrum sativumassessed using different experimental anxiety models, ind. j. pharmacol. 43, 574–577, (2011) [7]. kharade s.m., gumate d.s., patil v.m., kokane s.p., narikwade n.s., behavioral and biochemical studies of seeds of coriandrum sativum in various stress models of depression, int. j. curr. res. rev. 1003, 4–8, (2011) [8]. mazzio e.a., soliman k.i.a., in vitro screening for the tumoricidal properties of international medicine herbs, phytothera res. 23, 385–398, (2009) [9]. sunil c., agastian p., kumarappan c., ignacimuthu s., in vitro antioxidant, antidiabetic and antilipidemic activities of symplocos cochinchinensis (lour.) s. moore bark, j. food chem. toxicol. 50, 1547-1553, (2012). [10]. wangensteen h., samuelsen a.b., malterud k.e., antioxidant activity in extracts from coriander, food chem. 88, 293297, (2004). [11]. emamghoreishi m., heidarihamedani g.h., effect of extract and essential oil of coriandrum sativum seed against pentylenetetrazole-induced seizure, pharm. sci. 7 (2), 1-10, (2008) [12]. platel k., srinivasan k., digestive stimulant actions of spices: a myth or reality? indian j. med. res. 119, 167-179, (2004). [13]. coşkuner y., karababa e., physical properties of coriander seeds (coriandrum sativum l.), j. food eng. 80, 408-416, (2007) [14]. nejad ebrahimi s., hadian j., ranjbar h., essential oil compositions of different accessions of coriandrum sativum l. from iran, nat. prod. res. 24 (14), 12871294(2010) [15]. ramadan m.f., amer m.m.a., awad a., coriander (coriandrum sativum l.) seed oil improves plasma lipid profile in rats fed diet containing cholesterol, eur. food res. technol. 227, 1173–1182, (2008) [16]. sahib n.g., anwar f., gilani a.h., hamid a.a. saari a., alkharfy k.m., coriander (coriandrum sativum l.): a potential source of high-value components for functional foods and nutraceuticals-a review, j. phytother. res. 27 (9), 1439-1456, (2013) [17]. mhemdi h., rodier e., kechaou n., fages j., a supercritical tuneable process for the selective extraction of fats and essential oil from coriander seeds, j. food eng. 105 (4), 609-616, (2011) [18]. efsa nda panel (efsa panel on dietetic products, nutrition and allergies). scientific opinion on the safety of “coriander seed oil” as a novel food ingredient, efsa journal 11 (10), 3422, (2013) [19]. msaada k., hosni k., taarit m.b., chahed t., hammami m., marzouk b., changes in fatty acid composition of coriander (coriandrum sativum l.) fruits food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 82 during maturation, ind. crop prod. 29 (2–3), 269–274, (2009) [20]. sriti j., wannes w.a., talou t., mhamdi b., handaoui g., marzouk b., lipid fatty acidand tocol distribution of coriander fruits’different parts, ind. crop prod. 31, 294–300, (2010) [21]. wangensteen h., samuelsen a.b., malterud k.e., antioxidant activity in extracts from coriander, food chem. 88:293297, (2004) [22]. cra (corn refiners association). corn oil, 5th edition. corn refiners association, washington d.c., usa, (2006) [23]. strecker l.r., bieber m.a., maza a., grossberger t., doskoczynski w.j., corn oil. in bailey’s industrial oil and fat products, edible oil and fat products. john wiley ans sons, new york, 125-158, (1996) [24]. saldeen t., li d.y., mehta j.l., differential effects of alpha and gammatocopherol on low-density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis, j. am. coll. cardiol. 34, 1208-1215, (1999) [25]. wilson t.a., desimone a.p., romano c.a., nicolosi r.j., corn fiber oil lowers plasma cholesterol levels and increases cholesterol excretion greate than corn oil and similar to diets containing soy sterols and soy stanols in hamsters, j. nutr. biochem. 11, 443449, (2000) [26]. sakurai h., pokorný j., the development and application of novel vegetable oils tailor-made for specific human dietary needs, eur. j. lipid sci. technol. 105, 769–778, (2003) [27]. anwar f., hussain a.i., iqbal s., bhange r.m.i., enhancement of the oxidative stability of some vegetable oils by blending withmoringa oleifera oil. food chem. 103, 1181–1191, (2007) [28]. ramadan m.f., wahdan k.m.m., blending of corn oil with black cumin (nigellasativa) and coriander (coriandrum sativum) seed oils: impact on functionality, stability and radical scavenging activity, food chem. 132, 873–879, (2012) [29]. nestel p., clifton p., noakes m., effects of increasing dietary palmoleic acid compared with palmitic and oleic acids on plasma lipids of hypercholesterolemic men, j. lipid res. 35, 656–662, (1994) [30]. ahmed j., ramaswamy h.s., ngadi o., rheological characteristics of arabic gum in combination with guar and xanthan gum using response surface methodology: effect of temperature and concentration, int. j. food prop. 8, 179-192, (2005b) [31]. o’brien r.d., fats and oils, 3rd ed., crc press, taylor &francis group, 2-52, (2009) [32]. kim j., kim d.n., lee s.h., yoo s.-h, lee s., correlation of fatty acid composition of vegetable oils with rheological behaviour and oil uptake, food chem. 118, 398-402, (2010) [33]. naghshineh m., ariffin a.a., ghazali h.m., mirhosseini h., kuntom a., mohammad, a.s., effect of saturated/unsaturated fatty acid ratio on physicochemical properties of palm oleinolive oil blend, j. am. oil chem. soc. 87, 25562 (2010) [34]. li y., ma w.j., qi b.k., rokayya s., li d., wang j., feng h.x., sui x.n., jiang l.z., blending of soybean oil with selected vegetable oils: impact on oxidative stability and radical scavenging activity, asian pac. j. cancer prev. 15, 2583-2589, (2014) [35]. rudan-tasic d., klofutar c., characteristics of vegetable oils of some slovene manufacturers, acta chim. slov. 46, 511–521, (1999) [36]. hamm w., hamilton r.j., calliauw g., edible oil processin (2nd edition), john wiley & sons, ltd. (2013). [37]. gunstone f.d., vegetable oils in food technology: composition, properties and uses, blackwell publishing ltd, uk, (2002) [38]. kirk r.s., sawyer r., pearson’s composition and analysis of foods, 9th ed. longman scientific and technical england, 607-617, (1991) [39]. allen r.r., determination of unsaturation, j. am. oil chem. soc. 32, 671–674, (1955) [40]. benjumea p, agudelo j, agudelo a., basic properties of palm oil biodieseldiesel blends, fuel. 87, 2069-2075, (2008) [41]. zahir e., saeed r., abdul hameed m., yousuf a., study of physicochemical properties of edible oil and evaluation of frying oil quality by fourier transforminfrared (ft-ir) spectroscopy, arabian j. chem., available online 6 june 2014, doi:10.1016/j.arabjc.2014.05.025 [42]. briggs j.l., steffe j.f., using brookfield data and the mitschka method to evaluate power law foods, j. text. stud. 28, 517–522, (1997) [43]. mitschka l.p., simple conversion of brookfield rvt readings into viscosity functions, rheol. acta 21, 207–209, (1982) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 s i l v ia m i r o ne as a, g e o rg ia na g a br ie l a c o d in ă , ph y s i c oc h e m i c al pr o p er t i es o f b l e nd s o f c or n o i l wi t h c o r i an d e r s eed o i l, f o od a nd e nv ir o n me nt sa f e t y, v o lu me x iv , i ss ue 1 – 2 0 15 , p a g. 7 4 83 83 [44]. santos j.c.o., santos i.m.g., souza a.g., effect of heating and cooling on rheological parameters of edible vegetable oils, j. food eng. 67, 401-405, (2005) [45] sr en iso 6320:2002/ac:2006 animal and vegetable fats and oils determination of refractive index, standardization association of romania (asro), bucharest, romania [46]. sr en iso 3657:2013. animal and vegetable fats and oils determination of saponification value, standardization association of romania (asro), bucharest, romania [47]. sr en iso 3960:2010. animal and vegetable fats and oils determination of peroxide value iodometric (visual) endpoint determination, standardization association of romania (asro), bucharest, romania [48]. sr en iso 660:2009. animal and vegetable fats and oils determination of acid value and acidity, standardization association of romania (asro), bucharest, romania [49]. sr en iso 3961:2013. animal and vegetable fats and oils determination of iodine value, standardization association of romania (asro), bucharest, romania дослідження складу та антиоксидантних властивостей соєвих ізофлавонів food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 36 investigation of soy isoflavones distribution during the soy been processing tamara t.nosenko1, olena s. maksimova1 1national university of food technology, volodymirska str., 68, 1601 kyiv, ukraine, e-mail address: tnosenko@yahoo.com *corresponding author received 17 june 2011, accepted 12 october 2011 abstract: soy bean contain soy isoflavones which have an effect on different metabolic disorders in human. in this study we have analysed isomeric composition of isoflavones in soy bean and investigated distribution of this substances between different product under soy bean processing. using mass-spectroscopy and nmr analysis we have detected two main isomers in soy bean daidzin and genistin. it was shown that isoflavone fraction is extracted to alcohol solution in processing of protein concentrate. having analysed products of protein isolate processing we detected that isoflavones were extracted to alkaline solution during protein extraction. only about 17 % of isoflavones have been remaining in protein isolate. main of them were in whey water after protein precipitation. and it is possible to elicit them according to standart procedure using extraction by ethylacetate and precipitation by chloroform. taking into account our results we are proposing the scheme of soy bean processing with isoflavone concentrate producing. keywords: soy, soy isoflavones, protein concentrate, protein isolate. introduction isoflavones are the phenolic substances that are not so widely spreadible in plants but are detected only in some plant family. the highest content of isoflavones have been found in soy bean. there were 12 isomers of isoflavones. it was shown that these substances had are weakly estrogenic and anticarcinogenic effect on human [13], they could be used for prevention cardiovascular and another metabolic disorders [4-7]. in particular, epidemiological and clinical studies suggest potential chemopreventive effects for the soy isoflavone genistein against breast cancer in womens. in the same time soy isoflavones have also antioxidant properties as it was shown georgetti sr et al. [8] by the chemiluminescence method. thus soy isoflavones could be used for prevention of different human desease. commonly soy been processed for vegetable oil and different protein products and the retention of isoflavones during processing was not taking into account to nowadays. only some studies were publicated devoted to the problem of soy isoflavones in connection with protein production. jun lin et al. have shown effect of precipitating and washing temperatures on retention of isoflavones and saponins during processing of soy protein isolate [9]. authors have shown lowering of isoflavone lost in the protein isolate with reducing of processing temperature. mailto:tnosenko@yahoo.com ../../yuranos/local%20settings/temp/word_1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 37 the purpose of our study was to investigate the isomeric composition of soy isoflavones and their distribution between different by-products of soy processing. materials and methods soy been defatting.crushed soy bean were defatted in apparatus of soks-lett to contain less 1 % fat. soy cake was dryed to 9-10 % fluidity. isoflavones extraction. isoflavones were extracted from defatted soy meal using ethyl alcohol solution in the same extractor during some hour. than solvent was evaporated on the rotor evaporator and residue was treated by hot water. received solution was filtrated through paper filter to remove insoluble substances. filtrate was evaporated again and residues were extracted three time by ethylacetate. obtained extracts were combined and dried by sodium sulfate. isoflavones were precipitated by chloroform and filtrated. insoluble residue on the filter was dried in vacuum and it was a mixture of isoflavones. this mixture was analysed on the isoflavones composition. isoflavones analysis. isoflavones composition was analysed by column chromatography on silicagele with system solvents ethyl alcohol:water. eluate was analysed photometrically. massspectroscopy was used to reveal the molecular mass and the identification of isoflavone isomers. isoflavones identification. 1h nuclear magnetic resonance (nmr) spectra were measured on a bruker avance drx300 spectrometer and were used for isoflavones identification. chemical shifts obtained nmr spectra were compared with standarts. obtaining of protein concentrates. defatted soy seed was used for protein concentrate obtaining according to next procedure. defatted soy seed was mixed with ethyl alcohol solution (70 %, v/v) in relation 1:10 and exposed during 40-50 min with stirring under 45-50 °c. after this insoluble residue was precipitated by centrifugation. the supernatant (extract of soluble substances) was withdrawn and isoflavone content was analysed. pellet was dried to 8-10 % fluidity and protein and isoflavone content was analysed. obtaining of protein isolate. protein was extracted from defatted soy seed by alkaline solution (ph 8.5-9.5) under constant stirring and temperature 50-55 °c during 40-50 min, relation cake:solution was 1:10. after this insoluble residue was precipitated by centrifugation. the supernatant (protein extract) was used for isoelectric protein precipitation at ph 3.84.5. after some exposition protein pellet was separated by centrifugation (3 000 x g). protein pellet was collected and dried to 6-8 % fluidity. protein and isoflavone content was analysed in protein product and in supernatant (whey water). results and discussion our procedure of extraction of isoflavones from soy bean have given output 0.39 % isoflavone concentrate. obtained mixture of isoflavones from defatted soy seed was analysed on the isoflavone composition. the three individual substances were detected on chromatogram in a ratio 1:1:0,03 (fig.1). the holding time of these components was very close – 1.38, 1.41 and 1.53 min. mass-spectroscopy analysis gave possibility to estimate molecular mass of detected isomers. they were 417,4 and 433,4. such molecular masses correspond to daidzin and genistin respectively. we could not determine the third component due to its very insignificant content. for identification of detected isomers we have analysed nmr spectra of isoflavone mixture. aromatic part of spectrum was taken into account for this purpose (fig.2). ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 38 fig. 1. chromatogram of isoflavone extract and it was confirmed the presence of daidzin and genistin in isoflavone extract. we used defatted soy meal for processing of protein concentrate. we had proposed that isoflavone substances could extract from defatted soy meal simultaneously with other soluble substances to ethyl alcohol solution. that is why we had used obtained ethyl alcohol extract to recover isoflavones. in this case we followed the same procedure as for isoflavone extraction from defatted soy meal. we have obtained 0.39 % (from the initial mass of soy bean) output of isoflavone concentrate, that means that whole isoflavone fraction is extracted to ethyl alcohol extract in processing of protein concentrate. mass-spectroscopy and nmr analysis have confirmed the presence of two isomers of isoflavones daidzin and genistin and their hydrolysed derivatives (table 1). we have studied also isoflavone content in different product during protein isolate processing. first of all we have analysed fig. 2. 1 h nuclear magnetic resonnce (nmr) spectrum of isoflavone extract ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 39 table 1. isoflavone isomers extracted from different products of soy bean processing. mass-spectroscopy and nmr data. product molecular mass of isoflavone isomer isoflavone isomer ratio common output, % of soy bean mass soy bean 417,0 daidzin 1 0.39 433,6 genistin 1 ethyl alcohol extract from defatted soy meal 417,0 daidzin 48 0.39 433,6 genistin 42 255,3 daidzein 271,4 genistein protein concentrate not detected insoluble residue after protein extraction 417,0 daidzin 45 0.01 433,6 genistin 43 503.2 malonildaidzin 9 255,3 daidzein 271,4 genistein whey water after protein precipitation 417,0 daidzin 39 0.26 433,6 genistin 43 503.2 malonildaidzin 14 255,3 daidzein 271,4 genistein protein isolate 417,0 daidzin 46 0.05 433,6 genistin 43 503.2 malonildaidzin 7 255,3 daidzein 271,4 genistein ../../yuranos/local%20settings/temp/word_1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 40 whey water after protein precipitation. for this purpose whey water was evaporated on the rotary evaporator and residue was extracted three time by ethylacetate and obtained extracts were combined. in other respects the procedure was the same as described earlier. we have analysed the isoflavone content and composition (table 1). about 87% of whole isoflavone fraction of soy bean is containing in whey water. they contain all detectable isomers. in the same time malonildaidzin content in this fraction was higher. . extraction of soluble substances by ethyl alcohol solution soy protein extraction suspension separation protein precipitation insoluble residue ethyl alcohol extract of soluble substances drying висушу вання protein concentrate evaporation isoflavone extraction drying висушу вання isoflavone concentrate protein pellet whey water evaporation isoflavone extraction drying висушуванн я isoflavone concentrate drying protein isolate defatted soy meal fig.3 the scheme of soy bean processing with soy isoflavone producing. ../../yuranos/local%20settings/temp/word_0 ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 ../../yuranos/local%20settings/temp/word_1 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 1 – 2012 41 analysis of insoluble residue after protein extraction indicated only trace contain of isoflavone in this by-product (table 1). thus almost whole isoflavone fraction is extracted to alkaline solution together with protein our results demonstrated insignificant isoflavone content in protein isolate. it was about 17 % of total their content on soy bean. although authors of work [9] have shown that isoflavone content in protein isolate was significantly higher including whole range of temperature during processing. in agreement with our results we can propose the scheme of soy bean processing with isoflavone concentrate producing (fig.3). conclusion our results showed the presence of three main isoflavone isomers in soy beans and in by-product of their processing – daidzin, genistin and malonildaidzin. during soy processing these substances are extracted to water and alcohol solution. only about 17 % of initial content isoflavones is containing in protein isolate. we did not detect them in protein concentrate. to retain the main part of soy isoflavones it is possible to extract them from byproduct of protein processing. it is obviously that isoflavone concentrate will have higher medicinal effect than soy protein. references [1]. soy and experimental cancer: animal studies, journal of nutrition (usa), 125. 3 suppl (1995) [2]. lu l.j., anderson k.e., grady j.j., nagamani m. effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction, cancer epidemiol biomarkers prev (u.s.), 5. 1. 63-70, (1996) [3]. biological effects of isoflavones in young women: importance of the chemical composition of soyabean products, british journal of nutrition (united kingdom), 74. 4. 587-601, (1995) [4]. wagner j.d., cefalu w.t., anthony m.s., litwak k.n., zhang l., clarkson b. dietary soy protein and estrogen replacement therapy improve cardiovascular risk factors and decrease aortic cholesteryl ester content in ovariectomized cynomolgus monkeys, metabolism, 46. 6. 698-705, (1997) [5]. nestel p.j., yamashita t., sasahara t., pomeroy s., dart a., komesaroff p., owen a. abbey m. soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and prеmenopausal women, arterioscler thromb vasc biol, 17. 12. 3392-8, (1997) [6]. endocrinological response to soy protein and fiber in mildly hypercholesterolemic men, nutrition research (usa), 13. 8. 873-884, (1993) [7]. response of hormones modulating plasma cholesterol to dietary casein or soy protein in minipigs, j. nutr. (usa), 120. 11. 1387-1392, (1990) [8]. georgetti sr, casagrande r, di mambro vm, azzolini aecs, fonseca mjv. evaluation of the antioxidant activity of different flavonoids by the chemiluminescence method, aaps pharmsci, 5 (2).1208, (2003) [9]. jun lin, padmanaban g. krishnan and chunyang wang retention of isoflavones and saponins during the processing of soy protein isolates, journal of the american oil chemists' society, 83, 1. 59-63, (2006) ../../yuranos/local%20settings/temp/word_3 ../../yuranos/local%20settings/temp/word_3 ../../yuranos/local%20settings/temp/word_3 microsoft word 1 primele pagini nr 1 din 2011 23 martie.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering year x, no. 1 – 2011 university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international database: indexcopernicus journals master list. also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by editura universităţii ştefan cel mare suceava, romania. 240 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 32016, pag. 240 248 traditional leafy vegetables combinations enhance growth performance and improve health status of new zealand white x californian rabbits franck k.yao1, athanase k. otchoumou1, raïssa m.f.l. wognin1, sébastien niamke1 1laboratoire de biotechnologies, ufr biosciences, université félix houphouët-boigny, 22 bp 582, abidjan 22, côte d’ivoire. tchoumou2@yahoo.fr corresponding author received august 25th 2016, accepted september 28th 2016 abstract: fifty five rabbits (35 days old; average initial body weight: 550g) were used to assess their growth performance and health status when they were fed with leafy vegetables combinations (vigna unguiculata, abelmoschus esculentus, ipomoea batatas, solanum melongena, corchorus olitorius). ten treatments composed of 50% (dry matter) of three leafy vegetables combination and 50% of pellet were given to rabbits until 98 days of age. the control treatment contained only commercial pellets (100%). the results showed that the daily weight gain and the body weight gain did not differ between treatments. however, the final weights (1866.94-2200 g) were significantly different (p<0.05).average daily feed intake (61.55 g/day) and feed conversion ratio (2.69) of the control group were different (p< 0.05) to those obtained with the other diets. however, the daily feed intake (162.10-188.97g/day) and the feed conversion ratio (6.91-8.21) of rabbit fed with leafy vegetables combination were similar (p< 0.05). apparent coefficient of dry matter digestibility (70.52-86.21%) and of protein digestibility (86.28-91.30%) differed between treatments (p< 0.05). the digestibility of diets supplemented by mixture of corchorus olitorius, vigna unguiculata and abelmoschus esculentus, was better compared to other treatments. the blood parameters tested did not differ between treatments (p>0.05), except for low density lipoprotein which was high for diet f group and low for diet i group. this work revealed thatthe use of leafy vegetables combination facilitated the digestibility of diets contributing therefore to the improvement of growth performance and rabbits’ health status. keywords: leafy vegetables, growth, blood chemistry, haematological analysis, rabbit 1. introduction rabbit is one of the most prolific breeders among the livestock species [1]. rabbit meat is as nutritious as chicken and can serve as an excellent source of low cholesterol meat that is generally recommended to consumers [2]. in spite of having less space requirement, high production and reproduction potential of rabbit, its rearing has not got much tremendous popularity among the ivorian people. this situation can be due to fact that the rabbit meat is considered as food of rich persons in côte d’ivoire. moreover the high feeding cost has favored this situation as in most african countries [3]. the current trend in the breeding area is the utilization of vegetal raw for feeding the animals. products animal are more expensive and unavailable while the vegetal products are low expensive and available. among the vegetal raw product, the non-conventional feed as green forages are essential to reduce the feeding cost for economy production. one of the advantages of rabbit production in tropical countries is that rabbits are herbivorous animals and can be fed with forages and agricultural by-products that are not http://www.fia.usv.ro/fiajournal mailto:tchoumou2@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 241 suitable for human consumption. besides, most studies showed that the using of green forages in the rabbit feeding facilitate the digestive transit enhancing the appetite of rabbits to obtain optimum growth and good health [4-5]. the leafy vegetables as vigna unguiculata, abelmoschus esculentus, ipomoea batatas, solanum melongena and corchorus olitorius are available in abundance and under consumed by ivorian people [6-7]. they have good nutritive characteristics [8-9-10]. the leaves of these plants were efficiently used by the rabbits for best growth performances when they were associated with 50 % of pelleted diet as showed by [5]. indeed, the combination of two leaves allowed the assimilation of nutrients for the growth, which could have a beneficial effect on the quality of rabbit meat. nevertheless, the studies on concentrate diet supplemented with the combination of leafy vegetables are very scantly. therefore, the present experiment was conducted in order to investigate the effect of various combinations of three forages chosen between vigna unguiculata, abelmoschus esculentus, ipomoea batatas, solanum melongena, corchorus olitorius supplemented with concentrate diet on growth performances and health status of weaned rabbits. 2. materials and methods experimental animal and management the study has been conducted in a traditional rabbit farm in bingerville municipality (côte d’ivoire). the study area is located between 5° 21'708 "north latitude and 3° 54'639" longitude west. the animal (550 ±34g) were cross bred new zealand × california rabbits [11], which weaned at 35 days of age and were housed individually in cage in wire-netting (70 × 40 × 50 cm) raised from 80 cm to ground. the cages were arranged in stripes and under the shade of large trees (natural ventilation and daylight). the average temperature recorded during the experimental period was 29±2°c. water was provided ad libitum. the drinking and feeding bowl were made of removable metal cans stainless tied with binding wire to inner side of the cage. feeding of experimental animals five traditional leafy vegetables of côte d’ivoire as vigna unguiculata, abelmoschus esculentus, ipomoea batatas, solanum melongena and corchorus olitorius were used (table1). these forages distributed to animals were purchased daily from the market at 7am and disinfected 24 h before distribution. the forage was immersed in 10 l of water containing 2 ml of bleach lacroix®. it was then allowed to dehydrate at room temperature for 24 h [12]. a concentrate diet from ivograin® (industrial zoneyopougon-01 bp 1664 abidjan 01-ivory coast was purchased. website: www.sipra.ci) covering the nutritional needs of the growing rabbit was used as control diet (table 1). after weaning at 35 days, fifty five rabbits (550 ± 34 g) were housed individually in cages. the animals were acclimated to the experimental conditions during seven days [13]. the control diet was composed solely of concentrate, the other diets were composed of 50% forage (composed of three leaves) and 50% concentrate diet (control diet) (table 2). http://www.sipra.ci/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 242 table 1 approximate composition of leafy vegetables and concentrate diet (control diet) parameters (%) solanum melongena abelmoschus esculentus vigna unguiculata corchorus olitorius ipomoea batatas control diet dry matter 26.73 23.38 24.23 26.09 24.21 79.85 crude protein 14.04 12.31 17.20 16.84 15.32 14.75 ether extract 5.29 4.82 5.00 4.64 5.46 3.70 crude fiber 13.41 14.29 15.46 12.62 17.35 12.56 ash 11.40 9.18 9.38 9.56 11.22 9.6 source: [8-9-10] for leafy vegetables [11] for control diet. each diet has been distributed to five rabbits housed individually.during this period the animals also received prophylactic treatment with antibiotics to prevent coccidiosis. then cocciliumforte® (amprolium hydrochloride 20% and 0.2% vitamin k3) was used in the drinking water at a dose of 1g/l for three days [14]. after the acclimatized period, the rabbits were fed to satiety with different diets twice daily (9 am and 17 pm) until the age of 98 days [15]. water was provided ad libitum and renewed each morning. table 2 experiment treatments treatments leafy vegetables and pelleted diet (%) solanum melongena corchorus olitorius vigna unguiculata ipomoea batatas abelmoschus esculentus control diet a 16.66 16.66 16.66 50 b 16.66 16.66 16.66 50 c 16.66 16.66 16.66 50 d 16.66 16.66 16.66 50 e 16.66 16.66 16.66 50 f 16.66 16.66 16.66 50 g 16.66 16.66 16.66 50 h 16.66 16.66 16.66 50 i 16.66 16.66 16.66 50 j 16.66 16.66 16.66 50 control 100 growth performances weekly live weights (g) of the rabbits were determined by weighing the animals individually using an electronic scale (ohaus adventurer pro av 3102.). this was done early in the morning prior to feeding. daily feed intake (dfi) was determined by weighing the left over feed from the quantity of feed filled the previous day. data on daily feed intake (dfi)), feed conversion ratio (fcr) and daily weight gain (dwg) were also computed. daily feed intake (fi) was taken as the difference between the feed supplied and left over for each replicate per day. the rabbits were weighed on weekly basis and weight gain for each animal per week was calculated as the difference between the present weight and the weight for the previous week. the daily weight gain food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 243 (dwg) was obtained by dividing the total weight gain by the number of days. feed conversion ratio (fcr) was determined by dividing the quantity of feed consumed by the weight gained. digestibility determination the hard faeces from each cage were collected every morning before food distribution from 56th to 60th day of age [13]. faeces were weighed then packaged in labeled plastic bags and stored at -20 ° c prior laboratory analysis. for digestibility tests, all of the excreta (faeces) of each batch was first defrosted and then dried in an oven for 24 h at 80° c. half of each batch of faeces parboiled (80° c) was dried for 24 h at 103° c to determine the dry matter content [13]. the determination of the dry matter content was based on the [16] aoac (1990) method. the total protein was carried out on the sample stored at 80° c [13]. blood parameters at 88th day of age, blood samples were collected from three (3) rabbits per batch selected randomly. two samples of three (3) ml of blood from each rabbit fasted 10 hours were taken in sterile flasks (4 ml vacutainer tubes; bd-plymouth®) early morning (6:00), from the central artery of the ear with a sterile disposable syringe of 5 ml [17]. blood samples for hematological analysis were collected in flasks containing ethylene diamine tetraacetic acid (edta) and were delivered to the laboratory within 2 h of collection and promptly assayed. hematological analysis was based on mindray® bc-3200 technology [18]. the samples for biochemical analysis were centrifuged (500 rpm, 3 mm) and separated also within 2 h of collection. the resulting blood serum was stored at 4°c until assayed. biochemical parameters were determined by using a spectrophotometer (biolyzer® 100) at a wavelength of 500 nm. data analysis data were subjected to analysis of variance (anova) using spss17 software. treatment means were compared by oneway analysis of variance followed by duncan’s test. 3. results and discussion growth performances figure 1 shows the live weight of the rabbits fed with different treatments after 9 weeks of feeding. figure 1: variation of rabbit weight during 9 weeks feeding ab ab bcd bcd bcd bcd a cd d bcd abc 0 500 1000 1500 2000 2500 fi na l l iv e w ei gh t ( g) diets food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 244 the live weight of rabbits was significantly different (p>0.05). the rabbits fed with combination of corchorus olitorius, vigna unguiculata and abelmoschus esculentus (diet i) had slightly higher live weight than other treatments. while the rabbits fed with corchorus olitorius, vigna unguiculata and ipomea batatas, recorded low growth with no difference compared to those of rabbit fed with diets a, b and control diet (p<0.05). these results could be attributed to high protein content of vigna unguiculata (17.20%) and corchorus olitorius (16.84%) compared to others leafy vegetables (12-14%). table 3 presented the different responses of daily weight gain, daily feed intake and feed conversion ratio of the rabbits fed with different treatments. the daily weight gain ranged from 24.42 to 29.60 g/day and didn’t show differences (p ˂ 0.05) between treatments. these values were similar to the findings of [5] who fed rabbits with concentrate supplemented with combination of two green forages. however; the values of daily weight gain recorded in this sturdy were higher than those reported on forage-concentrate diets under most tropical conditions ranging between 5-20 g/day [19]. the feed intake (162.10-188.97g/day) and the feed conversion ratio (6.91-8.21) of rabbit fed with forage were similar (p< 0.05) but were significantly different to the control group (61.55 g/day and 2.69 respectively). this fact would suggested that the forage added in the rabbit’s feed would increased their appetite but also the feed intake to satisfy their feed requirements as reported by [4]. the increased feed intake of the rabbits for diets containing forage is understandable since forage contains high fiber, which tended to increase the total fiber content of the diet and diluted other nutrients. the present results showed that adding of forage in rabbit rations significantly (p<0.05) increased feed conversion ratio in comparison with control. high feed conversion ratio (6.918.21) of different rabbit batches fed with a combination of leaves and pelleted diet was the direct consequence of this high feed intake. furthermore, it should be noted that the diets with leafy vegetables had higher apparent coefficient of digestibility of dry matter compared to control diet. values reported about feed digestibility are in good agreement with those reported by [5]. the number of dead rabbit ranged from 1 to 2 rabbits (table 3). this could be attributed to the supplementation effect of leafy vegetables following the adaptation period. table 3 effect of forages feeding on weaned rabbit’s performance parameters (%) diets a b c d e f g h i j con-trol dwg (g/day) 25.46± 7.15a 24.42± 1.79a 28.3± 2.97 a 28.88± 4.76a 25.16± 2.21a 25.66± 7.44a 25.61± 2.40a 28.45± 2.72a 29.60± 5.09a 25.11± 5.56a 26.52± 0.62a dfi (g/day) 173.60 ±29.49 b 162.10 ±26.45 b 173.8 5±30. 0b 188.97± 43.47b 172.32 ±28.80 b 168.59 ±30.50 b 173.46± 30.89 b 172.83 ±31.43 b 172.00± 32.59 b 174.21 ±32.83 b 61.55± 17.31a fcr 7.97± 0.49 b 7.91± 0.75 b 7.02± 0.51 b 7.71± 0.22b 7.52± 0.64 b 7.52± 0.55 b 8.21± 0.46 b 7.48± 0.83 b 6.91± 0.37 b 7.44± 0.70 b 2.69± 0.38a dead rabbit 0 2 0 1 0 0 0 1 0 0 0 dfi: daily feed intake, fcr: feed conversion ratio. values are expressed as a mean value ± standard deviation (n = 5). means in the same row having different superscripts are significantly different (p < 0.05), while values in the same row with same superscript are not significantly different (p > 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 245 dry matter and protein digestibility dry matter and protein digestibility ranged from 70.52 to 86.21 % and 86.28 to 91.30 % respectively (figure 2). these parameters were significantly different (p< 0.05). the dry matter digestibility of rabbits fed with diets contained combination of leafy vegetables was higher than those obtained in rabbits fed with the control diet. protein digestibility of rabbits fed with different diets tests do not differ except diet g (lowest acdp) and i (highest acdp) (p > 0.05) (table 5). these results indicated that the forage as leafy vegetables had a positive effect on the nutrients digestibility but facilitated their assimilation. acddm: apparent coefficient of dry matter digestibility, acdp: apparent coefficient of protein digestibility. values are expressed as a mean value ± standard deviation (n = 5). means in the same row having different superscripts are significantly different (p < 0.05), while values in the same row with same superscript are not significantly different (p > 0.05). figure 2. apparent coefficient of digestibility of grower rabbits fed concentrate and mixture of leafy vegetables combinations. blood parameters blood biochemical parameters were presented in table 4. urea (0.27 0.32 g/l), creatinine t (10.15 12.47 g/l), glycaemia (0.74-0.82g/l) triglycerides (0.56-1.16 g/l) and hdl (0.49-0.57 g/l) contents did not differ between treatments (p > 0.05). the cholesterol total and ldl contents were high for rabbits fed with diet f and low for diet i respectively (p > 0.05). these results were in agreement with those observed by [5]. blood is a good indicator to determine the health of an organism. the values recorded in the present study were similar to the values reported by [20]. the identical creatinine values indicated the normal muscle metabolism as reported by [21]. it also suggested that there was no wasting or catabolism of muscle tissues as observed by [22]. the similarity of urea values is in agreement with the findings of [23] for rabbits fed with concentrate diet supplemented with leucaena leucocephala or macroptilium atropurpureum leaves. the urea values in this study indicated the good liver health. cholesterol is of animal origin and should come from some animal products used for pellets formulation. some leafy vegetables combinations especially diet i (mixture of corchorus olitorius, vigna unguiculata and abelmoschus esculentus) could contribute to reduce total cholesterol value in blood compared with the control group. blood also acted as pathological reflector of the whole body; hence haematological parameters are important in diagnosing the functional status of exposed animal to toxicants. b b bc bc b b b bc bc b a ab ab ab ab ab ab a ab b ab ab 0 20 40 60 80 100 a c d ( % ) diets acddm acdp food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 246 table 4. blood biochemical parameters of rabbit groups fed with different diets. diets parame ters a b c d e f g h i j control urea (g/l) 0.30 ±0.01 a 0.25 ±0.05 a 0.28 ±0.07 a 0.27 ±0.04 a 0.27 ±0.01 a 0.25 ±0.04 a 0.27 ±0.01 a 0.32 ±0.01 a 0.26 ±0.05 a 0.25 ±0.04a 0.27 ±0.04 a glycaemia (g/l) 0.82 ±0.02 a 0.77 ±0.06 a 0.77 ±0.03 a 0.74 ±0.05 a 0.74 ±0.04 a 0.79 ±0.01 a 0.74 ±0.01 a 0.79 ±0.01 a 0.77 ±0.01 a 0.77 ±0.11 a 0.75 ±0.06 a creatinine (mg/l) 12.11 ±0.59a 10.19 ±1.12 a 11.50 ±2.30 a 11.58 ±2.06 a 12.47 ±0.54 a 10.60 ±1.06 a 10.74 ±0.31 a 12.83 ±0.25 a 10.37 ±2.49 a 10.15 ±1.39 a 10.94 ±1.68a total cholesterol (g/l) 1.70± 0.26 ab 1.89± 0.07 ab 1.76± 0.06 ab 1.66± 0.10 ab 1.56± 0.00 ab 1.80± 0.37 b 1.48± 0.18 ab 1.74± 0.16 ab 1.63± 0.06 a 1.73± 0.08 ab 1.67± 0.05 ab hdl (g/l) 0.55± 0.04 a 0.51± 0.11 a 0.57± 0.06 a 0.51± 0.07 a 0.49± 0.01 a 0.52± 0.11 a 0.57± 0.02 a 0.53± 0.11 a 0.56± 0.08 a 0.54± 0.02 a 0.53± 0.05 a ldl (g/l) 0.97± 0.16 ab 0.88± 0.11 ab 0.98± 0.12 ab 1.07± 0.36 ab 1.04± 0.21 ab 1.15± 0.06 b 1.06± 0.06 ab 0.96± 0.00 ab 0.75± 0.10 a 1.04± 0.17 ab 0.92± 0.03 ab triglycerides (g/l) 0.88± 0.31 a 1.16± 0.11 a 0.74± 0.25 a 0.86± 0.27 a 0.56± 0.04 a 1.05± 0.52 a 0.79± 0.48 a 0.85± 0.28 a 0.95± 0.42 a 0.68± 0.04 a 1.11± 0.13 a dl: high density lipoprotein; ldl: low density lipoprotein. values are expressed as a mean value ± standard deviation (n = 5). means in the same row having different superscripts are significantly different (p < 0.05), while values in the same row with same superscript are not significantly different (p > 0.05). hematological values were found to be significantly similar (p>0.05) in all parameters measured except mchc which was high in diet a and low in diet b (p<0.05) (table 5). table 5: hematological values in rabbit’s blood diets parameters a b c d e f g h i j control erythrocytes (106/mm3) 5.91± 0.30a 5.62± 0.26a 6.34± 0.09a 5.35± 0.61a 5.68± 0.34a 6.32± 0.03a 6.23± 0.76a 5.46± 0.35a 6.19± 0.49a 5.62± 0.62a 5.71± 0.01a leukocytes (103/mm3) 4.8± 0.85a 3.65± 0.64a 4.8± 1.27a 3.9± 2.69a 4.3± 1.56a 7± 2.55a 4.15± 0.21a 6.5± 1.56a 5.8± 0.57a 4± 1.41a 5.5± 0.85a hemoglobin (g/dl) 12.7±0. 28a 12.45± 0.07a 13.95± 0.07a 12.25± 1.06a 12.55± 0.21a 13.25± 0.35a 13.2± 0.85a 12.15± 0.21a 13.15± 1.34a 12.2±1. 41a 12.25± 0.21a mcv (µm3) 67.55± 6.01a 73.8± 3.39a 70.8± 2.83a 72.3± 0.99a 72.95± 4.60a 67± 0.14a 69± 2.40a 71.2± 2.62a 68.6± 0.14a 69.75± 0.07a 71.15± 1.77a mchc (%) 31.85± 0.49b 30± 0.14a 31.05± 0.64ab 31.45± 0.92ab 30.35± 0.64ab 31± 0.71ab 30.8± 0.71ab 31.2± 0.28ab 30.95± 0.64ab 31.1±0. 14 ab 30.25± 1.20 ab mch (pg) 22.05±0.78a 22.1± 0.85a 21.9± 0.42a 22.25± 0.21a 22.05± 0.92a 20.8± 0.42a 21.2± 1.27a 22.2± 0.99a 21.15± 0.49a 21.65± 0.07a 21.4± 0.42a platelets (103/ mm3) 348.5± 41.72a 324.5± 106.77a 393± 38.18a 221.5± 217.08a 356± 151.32a 414± 21.21a 365.5± 48.79a 329± 142.84a 392.5± 102.53a 309± 94.75a 354± 101.82a mcv: mean corpuscular volume; mchc: mean corpuscular haemoglobin concentration; mch: mean corpuscular haemoglobin. values are expressed as a mean value ± standard deviation (n = 5). means in the same row having different superscripts are significantly different (p < 0.05 ), while values in the same row with same superscript are not significantly different (p > 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 247 this slight disparity in mchc results could probably be the result of the wellknown variability of haematological indicators in rabbits with regard to breedrelated and individual differences [18]. mcv, mch hb, mchc and platelets were reported to be normal [24]. these results indicated that the blood values of rabbits were not negatively affected by the ingestion of vigna unguiculata, abelmoschus esculentus, ipomoea batatas, solanum melongena and corchorus olitorius. these results suggested that the erytrogenesis was not altered. it indicated efficient oxygen transport and normal haematopoiesis [25]. the results were in agreement with findings of [26] and [27]. 4. conclusion this study showed that rabbits’ optimal growth performance can be achieved when they are fed with concentrated supplemented three leaf-vegetables. the forages combination did not have deleterious effects on blood biochemical and haematological parameters. the combination of abelmoschus esculentus, vigna unguiculata and corchorus olitorius leafy vegetables could be recommended as forage which could better support rabbit production. although, it could reduce the feeding cost, it may need further study on the quality of rabbit meat. 5. references [1]. ghosh s.k., das a, bujarbaruah k.m., das a., dhiman k.r., singh m.p. effect of breed and season on rabbit production under subtropical climate. world rabbit sciences 16: 29-33, (2008). [2]. mccroskey. r. raising rabbit in the pacific north west canadian centre for rabbit production development, surrey, bc canada, (2000) [3]. iyeghe-erakpotobor g. t., aliyu r. uguru j. effect of concentrate and forage type on performance and digestibility of growing rabbits under sub-humid tropical conditions. african journal of biotechnology 4 (20): 2004-2008, (2006). [4]. abonyi f.o., iyi e.o., machebe n.s. effects of feeding sweet potato (ipomoea batatas) leaves on growth performance and nutrient digestibility of rabbits. (2012) [5]. yao k. f., otchoumou k. a., wognin l. r., niamké l. s.. effect of combination of leafy vegetables on growth performances of rabbit oryctolagus cuniculus. j. anim. sci. adv., 5(12): 1522-1531, (2015) [6]. n’dri, m.t., kouame, g.m., konan, e., traore, d. plantes alimentaires spontanées de la région du fromager (centre-ouest de la côte d’ivoire): flore, habitats et organes consommés. sci.nat. 1: 61-70. (2008). [7]. soro, l.c., ocho-anin, atchibri, a.l., kouadio, k.k.a., kouamé, c. evaluation de la composition nutritionnelle des légumes feuilles. j. appl. biosci. 51: 3567– 3573. (2012). [8]. zoro a.f., zoué l.t., kra s.a.k., yépié a.e., niamké s.l.. an overview of nutritive potential of leafy vegetables consumed in western côte d’ivoire. pak. j. nutr. 12 (10): 949-956. (2013). [9]. acho, c.f., zoué, l.t., akpa, e.e., yapo, v.g., niamké, s.l.,. leafy vegetables consumed in southern côte d’ivoire: a source of high value nutrients. j. anim. plant sci. 20 (3): 3159-3170. (2014). [10]. oulai patricia, lessoy zoué, rose-monde mégnanou, ryta doué, sébastien niamké (2014). proximate composition and nutritive value of leafy vegetables consumed in northern côte d’ivoire. european scientific journal february 2014 edition vol.10: 212-227 [11]. martignon, m.h., combes. s., gidenne, t. digestive physiology and hindgut bacterial community of the young rabbit (oryctolagus cuniculus): effects of age and shortterm intake limitation. comparative biochemistry and physiology part a. mol. integr. physiol., 156: 156-162. (2010). [12]. kimsé m, soro d, bleyere mn, yapi j.n., fantodji a. (2013). apport d’un fourrage vert tropical, centrosema pubescens, en complément au granulé: effet sur les performances de croissance et sanitaire du lapin (oryctolagus cuniculus). int j biol chem sci 7 (3): 1234-1242 [13]. perez jm, lebas f, gidenne t, maertens l, xiccato g, parigi-bini r., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 3 – 2016 franck k.yao, athanase k. otchoumou, raïssa m.f.l. wognin, sébastien niamke, effect of traditional leafy vegetables mixture on growth performances and blood parameters of new zealand white x californian rabbits, food and environment safety, volume xv, issue 3 – 2016, pag. 240 – 248 248 dalle zotte a, cossu me, carazzolo a, villamide .j, carabano r, fraga m, ramos ma, cervera c, blas e, fernandez j, falcao e, cunha l, bengala freire j. european reference method for in vivo determination of diet digestibility in rabbits. world rabbit sciences 3: 41-43. (1995). [14]. kpodékon, m., youssao, a.k.i., koutinhouin, g.b., baba, i.l., dessou, j.m., djago, y., effet de la granulation sur les performances de croissance, l’efficacité alimentaire et la viabilité des lapereaux en condition d’élevage tropical. revue élev. méd. vét. pays trop. 62 (1): 75-80. (2009). [15]. de blas c, wiseman j. nutrition of the rabbit, 2nd edition, cab international 2010, p 334. (2010). [16]. aoac. official methods of analysis. association of official analytical chemists ed., washington dc, p 684. (1990). [17]. coulibaly fa, coulibaly a, n’guessan jd, kouame kg, djaman aj, guédé-guina f. etude des paramètres sériques biochimiques: le cas des lapins (neozelandaiscunistar) de côte d’ivoire. sci nat 4 (1): 37-43, (2007). [18]. poljičak-milas, n., i. kardumskelin, m. vuđan, t. s. marenjak, a. ballarin-perharić, z. milas. blood cell count analyses and erythrocyte morphometry in new zealand white rabbits. vet. arhiv 79, 561-571. (2009) [19]. hongthong p, kongvongxay s, ty c, preston tr. water spinach (ipomoea aquatica) and stylo 184 (stylosanthes guianensis ciat 184) as basal diets for growing rabbits. livest. res rural develop, 16 (5). (2004). http://www.cipav.org.co/lrrd/lrrd16/5/hong16034.ht m [20]. van praag e. biochemistry reference values. medi rabbit. (2004) com http://www.medirabbit.com. [21]. onifade a.a., tewe o.o. alternative tropical energy feed resourcesin rabbit diets: growth performance, diet's digestibility and blood composition. world rabbit science (1): 17-24, (1993). [22]. njidda a.a., isidahomen c.e. hematology, blood chemistry and carcass characteristics of growing rabbits fed grasshopper meal as a substitute for fish meal. pakistan vet. j., 30(1)2: 7-12. (2010). [23]. makinde o.j. growth performance, carcass yield and blood profiles of growing rabbits fed concentrate diet supplemented with white lead tree (leucaena leucocephala) or siratro (macroptilium atropurpureum) leaves in north central nigeria. trakia journal of sciences, 14(1): 80-86, (2016). [24]. mitruka b. m., rawnsley h. m. clinical biochemical and haemotological reference value in normal experimental animals. mason publishing usa inc. 86-140. (1977). [25]. blood d.c., henderson j.a., radostists o.m. veterinary medicine. london. 5th ed. beillierstindahpublishers pp. 241-249. (1979). [26]. ojebiyi o.o., shittu m.d., oladunjoye i.o. omotola o.b, olaniyi s.a. haematology, carcass and relative organ weights of growing rabbits on skip-a-day concentrate feeding regime. international journal of applied agricultural and apicultural research 9 (1&2):167-174, (2013). [27]. timibitei k.o., alikwe p. n. c., ayakurai j. i., ohimain e. i. haematological characteristics and organ weights of rabbits fed alchornea cordifolia leaf meal. intl j farm alli sci 3 (7): 754-759. (2014). http://www.cipav.org.co/lrrd/lrrd16/5/hong16034.htm http://www.cipav.org.co/lrrd/lrrd16/5/hong16034.htm 1. introduction rabbit is one of the most prolific breeders among the livestock species [1]. rabbit meat is as nutritious as chicken and can serve as an excellent source of low cholesterol meat that is generally recommended to consumers [2]. in spite of having less s... the current trend in the breeding area is the utilization of vegetal raw for feeding the animals. products animal are more expensive and unavailable while the vegetal products are low expensive and available. among the vegetal raw product, the non-con... 190 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 12015, pag. ... ... the use of texture destructive methods to assess the state of pork freshness *sergiu paduret1, gheorghe gutt1 1faculty food engineering, stefan cel mare university of suceava, romania sergiu_paduret@yahoo.com *corresponding author receive: may 25th 2015, accepted june 25th 2015 abstract: the aim of this study is to evaluate the freshness state of pork meat and for this we built an electronic device for creep test with a sensory system for measuring displacement and time. after each measurement a creep curve characterized by the penetration depth and mechanical work of compression was obtained. the color measurements was made with a usb 4000 ocean optics spectrophotometer, using the cie l*a*b* uniform color space.after 10 days of measurements while the meat was kept refrigerated at 4 °c, a linear trend between the state of meat freshness (expressed as storage days), mechanical work (r2=0.986) and penetration depth (r2=0.987) was established. a pearson correlation has also been established between meat freshness, penetration depth (r=0.994**) mechanical work, water activity and ph. keywords: creep test, freshness, mechanical work, pork, color. 1. introduction the state of food freshness is very important for consumers, retailers and also for food processors; nowadays the consumers pay more attention to food safety than food price. among the quality attributes, freshness is of special significance and can equally well be seen as a sensory, nutritional or a food safety characteristic [1]. pork represents more than 50% of the total amount of meat consumed in romania, with an annual consumption of 30-35 kg/capita and half of this meat is sold as fresh meat for culinary purposes [2]. the meat freshness can be appreciated by: smell, color, appearance and texture (elasticity, hardness under load, relaxation tests). textural characterization such as creep test can provide quick and valuable indications on the state of raw meat freshness. textural characteristics can be grouped into mechanical characteristics (hardness, cohesiveness, viscosity, elasticity, springiness, and adhesiveness); geometrical characteristics (particle size, shape, and orientation); andsurface characteristics (moisture content and fat content) [3,4]. fresh meat quality is difficult to define because it is a complexconcept determined by consumer preferences. the quality characteristics of meatare influenced by various factors such as muscle structure, chemicalcomposition, chemical environment, interaction of chemicalconstituents, postmortem (p.m.) changes in muscle tissues, stressand preslaughter effects, product handling, processing and storage,microbiological numbers and populations, etc. in particular, freshmeat quality is directly related to muscle fiber characteristics [5]. the first impression consumers have of any meat product is its color and thus color is of utmost importance. the color of meat may vary from the deep purplish-red of http://www.fia.usv.ro/fiajournal mailto:sergiu_paduret@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 sergiu paduret, gheorghe gutt, the use of texture destructive methods to assess the state of pork freshness, food and environment safety, volume xiv, issue 2 – 2015, pag. 190 – 195 191 freshly cut beef to the light gray of faded cured pork [6]. pork color can be influenced by thegenetics of the live animal, the nutritional programadministered, antemortem conditions and handling(those events and conditions prior to harvest), thevarious processes associated with harvesting the animal(stunning, dressing, and chilling), and by processing,packaging, distribution, and marketing conditions [3]. meat texture is directly related to the size of muscle fiber and the amount of connective tissue, and is partially affected by the quantity of intramuscular fat; the amount of intramuscular fat affects the meat firmness [5]. meat texture evaluation methods can be groupedinto sensory methods, instrumental methods, and indirectmethods. the most common instrumentalmethods are compression, penetration, shear, extension tests and texture profile analysis [3, 7]. the aim of this study is to assess the state of raw meat freshness by destructive methods of materials testing respectively by creep tests. in the creep test the strain is measured under a constant stress and temperature as a function of time [8, 9]. 2. matherials and methods an electronic device for creep test, figure 1, with a sensory system for measuring displacement and time was built to make this study. pork meat samples taken from pork leg with dimensions of 30x30x30 mm [10] and with a mass of about 55 g were obtained daily from bigger pieces of meat previously cut. between the testing days the meat samples were kept under refrigerated conditions at 4 °c.the determinations were carried out for 10 days until the biogenic amines appeared. fig. 1. device for determining the creep of food products a creep method with preload and a creep method without preload was used for analysis. stress time was 260 minutes and the weight stress was 1000 g. the preload consisted in sample stress with a mass of 300 g for 20 s. the moisture content of meat samples was determined by oven drying –sr iso 1442 – [11]. the fat content was determined by soxhlet method –sr iso 1444[12], and the protein content was determined by kjeldahl method – sr iso 937[13]. the color measurement of each samples was achieved with a usb 4000 ocean optics spectrophotometer, using the cie l*a*b* uniform color space, the illuminant used was d65 in reflective mode and the color measurement was made at an angle of 45 degrees to the sample. the a* axis extends from green (−a*) to red (+a*), the b* axis from blue (−b*) to yellow (+b*) and l* represents brightness (0 black and 100 white)[14]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 sergiu paduret, gheorghe gutt, the use of texture destructive methods to assess the state of pork freshness, food and environment safety, volume xiv, issue 2 – 2015, pag. 190 – 195 192 meat color difference (δe*) was calculated with the formula [15]: (1) in addition, hue angle or tone (h0) and chroma (color intensity) (c*) was calculated by the following formulas [16]: h0=tan-1 (b*/ a*) (2) c*=(a*2 +b*2)1/2 (3) statistical analysis the pearson correlation has been made up using spss 13 (usa). 3. results and discussion creep tests were also made by other scientists on dough, bread or butter to investigate the failure of soft viscoelastic solids [3]. the physico-chemical properties are presented in table 1; the ph value is in the acid region between 5.5 and 6.1, while the water activity ranged between 0.885 in the first day to 0.909 in the last day of testing. table1. meat properties meat samples protein % 21.2±0.95 fat% 6.9±0.87 moisture %wb 71.6 ±0.9 mean value ± standard deviation (n=3), wb-wet basis figure 2 shows the creep curve of meat samples stress with 1 kg, 2 kg and 3 kg for 13 minutes, and as we can see the increasing of stress leads to high penetration depth. the depth range between 3.26 mm to 11.02 mm and the mechanical work calculated as the area under the curve range between 298 and 1279 mm∙min-1 fig.2. creep curve of a meat samples stress with 1 kg, 2 kg and 3 kg. we chose for analysis weight stress of 1kg because it produced a smaller lateral flow. table 2. the characteristics of creep curves load [kg] depth [mm] mechanical work [mm∙min-1] 1 3.26 298.1 2 4.65 474.6 3 11.02 1279 figure 3 shows the creep curves on days with preload (300g for 20 s) stress with 1 kg and it can be seen a decrease of depth during the 10 days of testing, but the results of meat samples analyzed by preloading method were inconclusive, there was no correlation between the state of meat freshness and creep curves.       2*2*12*2*12*2*1 bbaalle  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 sergiu paduret, gheorghe gutt, the use of texture destructive methods to assess the state of pork freshness, food and environment safety, volume xiv, issue 2 – 2015, pag. 190 – 195 193 fig. 3. creep curves on days with preload stress with 1 kg comparing thecreep methodwith preload with the creep method without preloadwe can observe that the penetration depth increases with storagein case of second method, and also shows an increasing evolution from day to day(figure 4). fig.4. creep curves on days without preload stress with 1 kg the depth for creep test without preload ranged between 4.06 mm on the first day of determination to 8.25 mm in the tenth day, and the mechanical work of compression varies between 787 mm∙min-1 in first day to 1909mm∙min-1 on the tenth day (table 3). figure 5 and 6 show the linear evolution of mechanical work (r2=0.986) respectively the penetration depth (r2=0.987) during storage and testing. color parameters have been also measured throughout the study; meat brightness, (l*) ranges between 59.1 and 89.65, in the seventh day meat samples presented the highest brightness and in the last day the lowest brightness. a* color parameter is situated in the positive region on red-green axis, more towards red only in the first day, instead the b* color parameter is situated in the negative region on yellowblue axis, more towards yellow in the first seven day. fig. 5. the evolution of mechanical work fig. 6. the evolution of penetration depth in the last days of determination the b* parameter was situated in the positive region on yellow-blue axis, more towards blue (table 3). the color difference ranges between 1.5 in the first day to 20.58 in the seventh day of determination, if the color difference is greater than three; it means that the color difference of meat samples analyzed can be appreciated by the human eye. depth food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 sergiu paduret, gheorghe gutt, the use of texture destructive methods to assess the state of pork freshness, food and environment safety, volume xiv, issue 2 – 2015, pag. 190 – 195 194 table 3. texture,color and physico-chemical evolution of raw pork meat during 10 days of storage (without preload). storage day depth [mm] work [mm∙m in-1] l* a* b* c* h0 δe ph aw moistur e [%] 1 4.06 ±0.3 787.8 ±2.1 68.7 ±0.7 0.25 ±0.1 -2.3 ±0.1 2.31 ±0.2 -83.79 ±0.5 1.523 ±0.2 5.53 ±0.06 0.885 ±0.01 70.709 ±0.23 3 5.41 ±0.2 1129 ±1.9 81.98 ±3.1 -0.51 ±0.04 -1.665 ± 0.1 1.74 ±0.09 72.76 ±1.8 11.821 ±0.0.9 5.83 ±0.13 0.875 ±0.021 70.578 ±0.53 5 6.2 ±0.1 1316 ±1.5 67.95 ±2.1 -1.27 ±0.1 -2.4 ±0.2 2.71 ±0.2 62.02 ±1.4 17.168 ±0.5 5.52 ±0.01 0.895 ±0.01 68.492 ±0.85 7 6.91 ±0.2 1485 ±2.2 89.65 ±2.2 -4.9 ±0.21 -6.15 ±0.88 7.86±0 .6 51.45 ±1.5 20.580 ±0.7 5.74 ±0.03 0.899 ±0.02 67.36 ±0.71 9 7.67 ±0.4 1721 ±2.3 60.15 ±1.3 -1.58 ±0.1 3.66 ±0.9 3.99±0 .4 -66.56 ±0.9 11.703 ±0.5 6.1 ±0.02 0.903 ±0.02 70.013 ±0.11 10 8.25 ±0.1 1909 ±1.7 59.1 ±1 -2 ±0.06 5.2 ±0.7 5.57 ±0.3 -68.96 ±1.1 13.445 ±0.4 6.05± 0.03 0.909± 0.01 71.128± 0.32 mean value ± standard deviation (n=3). l*brightness, a*red-green axis, b*yellow-blue axis, c*color intensity, δecolor difference, h°-tone, table 4 presents the pearson correlation of the meat physico-chemical properties. in the case of creep tests without preload, a correlation between the mechanical work of compression, penetration depth and the state of raw meat freshness expressed as storage days was established. table 4. pearson correlation of meat physico-chemical properties and creep texture parameters (without preload). storage depth work moisture ph aw l a b c h δe storage 1 0.994** 0.993** -0.084 0.780* 0.891** -0.375 -0.574 0.594 0.658 -0.224 0.548 depth 1 0.998** -0.101 0.772* 0.852* 0.333 -0.578 0.575 0.634 -0.129 0.609 work 1 -0.042 0.791 0.853* 0.374 -0.538 0.619 0.611 -0.172 0.566 moisture 1 0.374 -0.158 0.591 0.734* 0.708 -0.522 -0.609 -0.737* ph 1 0.501 0.366 -0.208 0.759* 0.314 -0.357 0.143 aw 1 0.494 -0.536 0.498 0.689 -0.416 0.405 l 1 -0.464 -0.856* 0.257 0.729* 0.384 a* 1 0.297 -0.939** -0.305 -0.810* b* 1 -0.097 -0.643 -0.231 c 1 -0.012 0.621 h 1 0.650 δe 1 l*brightness, a*red-green axis, b*yellow-blue axis, c*color intensity, δecolor difference, h°-tone, awwater activity**. correlation is significant at the 0.01 level (1-tailed).*. correlation is significant at the 0.05 level (2-tailed). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 sergiu paduret, gheorghe gutt, the use of texture destructive methods to assess the state of pork freshness, food and environment safety, volume xiv, issue 2 – 2015, pag. 190 – 195 195 it can be observed that there is a strong positive correlation between penetration depth and day of storage (r=0.994**) and also between work and storage (r=0.993**), aw is correlated positively too with days of storage (r=0.891**), depth (r= 0.852*) and work (r=0.853*). the color difference of meat is negatively corelated with moisture content (r=0.737*) and a* -red-green axis (r=-0.810*). another positive correlation can be observed between moisture content and a* (r =0.734*). the ph value is correlated positively with storage (r=0.780*), depth (r =0.772*) and b*-yellow-blue axis (r=0.759*). 4. conclusion determining the state of meat freshness requires laborious operations related to spectrometric and chromatographic analytics, but given that the freshness of meat is reflected in its behavior to mechanical stress, creep methods without preload can be useful means for a rapid testing of meat freshness. another consideration is that the state of meat freshness expressed as storage days, penetration depth respectively the mechanical work of creep test without preload shows a linear trend r2 =0.987 and r2=0.986. as a general conclusion creep tests without preload can be used to assess the state of food freshness. 5. references [1]. hui y. h., handbook of meat and meat processing, second edition, crc presstaylor & francis group, new york, (2012) [2]. popescu a., study concerning the trends in pork market in romania, lucrări ştiinţifice seria zootehnie, vol. 57, university of agricultural sciences and veterinary medicine, iasi, (2012) [3]. leo m. l., terri b., feng c., coggins p., gloria m., hui h. y., handbook of meat, poultry and seafood quality, blackwell publishing, usa, (2007) [4]. xxxsr iso 11036/2007senzory analysis methodstexture profile (2007) [5]. joo s.t., kim g.d., hwang y.h., ryu y.c., control of fresh meat quality through manipulation of muscle fiber characteristics, meat science, 95, 828–836, (2013) [6]. boles j.a., pegg r., meat color, food product innovation program university of saskatchewan, montana state university and saskatchewan, (2010) [7]. bourne c.m., food texture and viscosity: concept and measurement, an elsevier science imprint, london, (2002) [8]. vithanage c. r., grimson m. j., smith b. g., wills p. r., creep test observation of viscoelastic failure of edible fats, condensed matter and materials physics conference (cmmp10), journal of physics: conference series 286 , 012008, (2011) [9]. betten, j., creep mechanics, 2nd ed., springer, aachen, (2005) [10]. gutt, g., pădureţ, s., amariei, s., plesca, m., physical and texture parameters used in the analysis of meat freshness, journal of agroalimentary prosesses and technologies, 20(3), 257-262 (2014) [11]. xxx-iso 1442 –carne şi produse din carne. determinarea umidităţii (2010) [12]. xxxsr iso 1444 carne şi produse din carne. determinarea conţinutului de grăsime liberă [13]. xxx-iso 937 –carne şi produse din carne. determinarea conţinutului de azot (2007) [14]. amsa, meat color measurement guidelines, american meat science association, illinois, usa, 61820 800-5172672 (2012) [15]. oliveira a. c. m., balaban m. o., vision system in measuring color of gulf of mexico sturgeon fillets,applied engineering in agriculture vol. 22(4): 583-587 american society of agricultural and biological engineers issn 0883−8542, (2006). [16]. alves de oliveira, e.n., costa santos, d., gomes, j. p., trinda de rocha, a. p., da silva, w. p., physicochemical stability of diet umbu-caja jams stored under ambient conditions, journal of food processing and preservation 39, 70–79 (2015) применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 4 2011 contents: 1. photocatalytic reduction of methylene blue on the heterostructural materials based on titanium dioxide with bisqinocyanine pigment igor kobasa, nataliya husyak, sonia gutt 7 2. viscoelastical behaviour of some food materials from the spanish market mircea-adrian oroian, isabel escriche, gheorghe gutt 13 3. gerodietic meat products technology enriched with calcium and phosphorus peshuk l.v., budnyk n.v., halenko о.о. 18 4. sulfur diox side utili zatio n by the treatment o f py rite chalco pyrite sul fi de conse ntra tes, combining m echa nical a nd metallother mi c proces ses v. m art iros yan, t . ag h am yan, m. s as unt s yan, a. aj vaz yan, a. z apros yan 24 5. photocatalytic degradation of methylene blue on nanostructured composites based on tio2-bi2o3 igor kobasa 30 6. investigation of soy isoflavones distribution during the soy been processing tamara t.nosenko, olena s. maksimova 36 7. new materials for photocatalytic purification of air – a review violeta vasilache, catalin popa, traian vasilache 42 8. modeling of the thermo-physical properties of aqueous sucrose solutions ii. boiling point, specific heat capacity and thermal conductivity andrei i. simion, cristina g. grigoraş, lăcrămioara rusu, adriana dabija 49 9. highlighting genetic progress in improving winter rye ioan gontariu, constantin drobotă, marinela cureleţ 57 10. studies regarding the alkaline and alkalineperoxidic pretreatment influence upon the reducing sugars quantity obtained from the wheat straws eufrozina albu (niga), alina-mihaela pşibilschi 65 11. residual chlorine in water supply systems sonia gutt (amariei), mihaela cojocariu 71 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 4 2011 12. influence of wheat flour dough hydration levels on gas production during dough fermentation and bread quality georgiana g. codină, silvia mironeasa, daniela v.voica 79 13. effect of addition of corn flakes on rheological behavior of some yogurt cristina damian, mircea-adrian oroian, andreea şmadici 84 14. advanced characterization methods for nickel and zincnickel alloy layers electrochemically deposited violeta vasilache 89 15. effect of storage on ascorbic acid content of some fruit juices nicolae carpiuc, ana leahu, ecaterina curaleţ, cristina damian 95 16. the variation of the acid ascorbic content in cynosbati fructus influenced by stationary factors sorina ropciuc, ion tănase , iuliana creţescu, giancarla velicevici 100 17. multivariate analysis in assessment relationships between milk characteristics influenced by the seasonal variations silvia mironeasa, georgiana gabriela codină 104 18. the content of carotenoid pigments in rosa canina l. fruit sorina ropciuc 108 19. study regarding the possibilities to obtain functional food from wheat flour: bread with exogenous buckwheat addition gabriela constantinescu (pop), adriana dabija, amelia buculei 112 20. the activity of -amylase and peroxidase during germination of some maize caryopses with different viabilities marcel avramiuc 118 21. author instructions 124 22. list of referees 129 23. subscription information 133 звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 19 characterist ics of three yeast strains for was tewa ter t re a tmen t *aleksander slavov1, zapryana denkova2, rositsa denkova 3 1department “environmental engineering” university of food technologies, plovdiv, bulgaria, aleksander_slavov@yahoo.com 2department “organic chemistry and microbiology” university of food technologies, plovdiv, bulgaria, zdenkova@abv.bg 3department „biotechnology”, faculty of biology, sofia university „st kliment ohridski”, sofia, bulgaria, rositsa_denkova@mail.bg *corresponding author received 20 march 2012, accepted 27 may 2012 abstract: the selection of desirable strains with certain activities that have quick adaptivity and effectively remove contaminants from water is of great importance for the technologies for biological wastewater treatment.three yeast strains are investigated. one of them was isolated from soil, and others are provided by a yeast strains’ collection and denoted as c1 and c2. the identification of the yeast strains is done using the test kit api 20 c aux (biomerieux, france) for rapid identification of yeasts. determination of the enzyme profile is obtained by applying the test kit api zym (biomerieux, france). the three yeast strains accumulate high concentrations of viable cells. they are similar in their ability to oxidize s – containing compounds (elemental s, na2s2o3) under alkaline conditions lowering the рh of the medium and to exhibit catalase activity. yeast strain y exceeds the rest in the expression of lipolytic activity. that is confirmed in the subsequent analysis of its enzyme profile through identification system api zym. through it are found and alpha –, beta – glucosidase and aminopeptidase activities. keywords: morphological, physiological and biochemical methods, api zym, api 20 c aux, candida famata 1. introduction one of the most important compounds of the planet is water [1]. the continuous decrease of potable water determines the need for different methods for its purification. technologies for biological wastewater treatment with the participation of microorganisms, including yeasts, are becoming more and more current. among the microorfanisms, participating in the wastewater treatment process are representatives of the genera candida, pichia, torulaspora, yarrowia, geotrichum, saccharomyces, cryptococcus, rhodotorula, trichosporon [2, 3, 4]. the selection of desirable strains with certain activities that have quick adaptivity and effectively remove contaminants from water is of great importance. the aim of this work is the analysis and identification of yeast strains suitable for specific applications in the field of biological wastewater treatment. 2. materials and methods 2.1. microorganisms three yeast strains, marked as y, c1, c2 are used in this study. yeast strain y is isolated from soil and strains c1 and c2 are provided by the microorganism mailto:aleksander_slavov:@yahoo.com mailto:aleksander_slavov:@yahoo.com mailto:zdenkova:@abv.bg mailto:zdenkova:@abv.bg mailto:rositsa_denkova:@mail.bg mailto:rositsa_denkova:@mail.bg food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 20 collection of the department of organic chemistry and microbiology at the university of food technologies, plovdiv, bulgaria. 2.2.nutrient media 2.2.1. malt extract medium. composition: malt extract (kamenitza, bulgaria) in 1:1 ratio with tap water (vol/vol). ph=6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.2. malt extract medium. composition: malt extract (kamenitza, bulgaria) in 1:1 ratio with tap water (vol/vol) + 2 % agar – agar (w/vol). ph=6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.3. luria – bertani glucose medium (lbg). composition (g/dm3): triptone (difco) – 10 g, yeast extract – 5 g, nacl – 10 g, glucose (scharlau) – 10 g. ph=7,5. the medium is sterilized for 25 minutes at 121ºc. 2.2.4.soy – caseine broth medium. composition (g/dm3): triptone (difco) – 17g, soy peptone (scharlau) – 3 g, nacl – 5 g, k2hpo4 – 2,5 g, glucose (scharlau) – 2,5 g. ph=7,3 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.2.5.citrate utilization medium (simons medium). composition (g/dm3): na(nh4)2po4 – 1,5 g; kh2po4 – 1 g; mgso4.7h2o – 0,2 g; na – citrate – 3 g; alcohol solution of bromthymol blue – 1%; agar – agar – 2%. the medium is sterilized for 25 minutes at 121ºc [6]. 2.2.6.gelatinase activity medium. composition (g/dm3): triptone (difco) – 10 g; yeast extract (scharlau) – 5 g; nacl – 10 g; glucose (scharlau) – 10 g; gelatine (ddr) – 250 g. рн 7,5. medium is dispensed into tubes and is sterilized for 25 min at 121ºс [6]. 2.2.7. proteolytic activity medium. composition: malt agar medium with 10 % (vol/vol) solution additive (10 cm3 milk/100 cm3 water) of skimmed milk powder (scharlau) [6]. 2.2.8.lipolytic activity medium (tween – 80 compounds hydrolysis). composition (g/dm3): peptone (scharlau) – 10 g; nacl – 5 g; cacl2 – 0,1 g; tween – 80 (merck) – 10 cm3; agar – agar – 20 g. рн 7 – 7,4. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.9. sulphur–containing compound oxidation mediua. 2.2.9.1. starckey broth medium. composition (g/dm3): elemental s – 10 g; кh2po4 – 3 g; mgso4.7h2o – 0,2 g; cacl2.2h2o – 0,2 g; (nh4)2so4 – 0,5 g; feso4 – traces; indicator – bromocresol purple. рн 8,0. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.9.2. ncl – broth medium. composition (g/dm3): elemental s – 10 g; (nh4)2so4 – 0,2 g; mgso4.7h2o – 0,5 g; cacl2.2h2o – 0,25 g; feso4 – traces; indicator – bromocresol purple. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.9.3. thiosulphate agar medium. composition (g/dm3): na2s2o3 – 5g; k2hpo4 – 0,1 g; nahco3 – 0,2 g; nh4cl – 0,1 g; agar – agar – 20 g. рн 8,0. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.10. no3– reductase activity medium. composition (g/dm3): peptone (scharlau) – 5 g; meat extract (scharlau) – 3 g; kno3 – food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 21 1 g. рн 7.0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.11. nh4+ – citrate medium (for nitrifying activity). composition (mol/dm3): na – citrate.2н2о – 9,5.10-3; nh4cl – 9,35.10-3; kh2po4 – 1,47.10-3; mgso4.7h2o – 1,62.10-4; cacl2 – 1,36.10-7; feso4.7h2o as edta – complex – 3,6.10-5. the medium is sterilized for 25 minutes at 121ºc [9]. 2.2.12. blood agar (ncipd, bulgaria). composition (g/dm3): casein hydrolysate – 14 g; nacl – 5 g; peptone – 4,5 g; yeast extract – 4,5 g; defibrinated sheep blood – 70 cm3; agar – agar – 12,5 g. ph 7,3 ± 0,2. all medium components are sterilized for 15 min at 121ºс. after cooling to 45ºс – 50ºс 70 cm3 defibrinated sheep blood is added aseptically and is poured in sterile petri dishes [8]. 2.2.13. gorodkova medium (for yeast sporulation). composition (g/dm3): peptone – 10 g; nacl – 5 g; месен meat extract – 10 g; glucose – 2,5 g; agar – agar – 20 g. рн 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.14. acetate agar medium (for yeast sporulation). composition (g/dm3): naoocch3.3h2o – 5 g; agar – agar – 20 g. ph 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.15. minimal agar medium (for yeast sporulation). composition (g/dm3): agar – agar – 20 g. рн 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.16. rice agar medium (fluka analitycal) (for pseudomycellium identification). composition (g/dm3): rice extract powder – 0,7; agar – agar – 20 g. the medium is prepared in two versions – with and without tween – 80 (merck) (1 cm3/dm3). рн 5,8 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.2.17. motility medium (ncipd, bulgaria). composition (g/dm3): peptone – 10 g, meat extract – 3 g, nacl – 5 g, agar – agar – 4 g. рн 7,4 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.3.cultivation and storage of the analyzed microorganisms. yeasts grow in malt extract medium at 30ºc in thermostat for 48 hours and are stored in a refrigerator at 4ºc for 2 months. 2.4. analytical methods. 2.4.1. morphological and cultural methods 2.4.1.1. cellular and colonial morphology. description of cellular and colonial morphology of the studied yeast strains is performed by microscopic observation the developed on malt agar single colonies of the studied strains. 2.4.1.2. identification of pseudomycellium formation ability. a part of the cultural medium of the studied strain, that has been cultivated for 48 hours in a thermostat is taken with flamed and cooled bacteriological loop and spread in touch on agar plates with rice-agar. the inoculated plates are incubated under aerobic conditions for 2-4 days at 23ºc 28ºc. a microscopic preparation is prepared from the developed colonies of the tested strain, which is examined for the presence of specific pseudomycellium or other specific formations in yeasts. the presence of vegetative cells only determines the result as negative. 2.4.1.3.identification of spore formation ability. the ability of the yeast to form spores under unfavorable environmental conditions lack of nutrients is examined. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 22 using a flamed and cooled bacteriological loop a part of the pre-developed on malt agar 48 hour colonies of the studied strain is taken and is stroked on a petri dish with minimal agar medium gorodkova agar or acetate agar. incubation conditions 20ºc 25ºc for 7 days. a microscopic preparation is prepared from the biomass and it is stained by the method of moller. spores are rubine red and vegetative bodies – blue. 2.4.1.4. determining the number of viable cells in the development of the cultures in liquid medium. sterile malt extract is inoculated under aseptic conditions with yeast suspension, 5 cm3 medium is inoculated with 1 cm3 of the inoculum of the studied cultures. after incubation in a thermostat at 30ºc for 48 h, tenfold dilution method is done and malt agar plates are inoculated. petri dishes are thermostatted at 30ºc for 48 h so that the strain would form single colonies, which are then counted. 2.4.2. physiological and biochemical methods 2.4.2.1. citrate utilization. developed in containers with 30 cm3 1:1 malt extract at 30ºc for 48h yeast cultures are centrifuged at 3000 min-1 for 10 min. the supernatant is discarded and the biomass is resuspended in 5 cm3 sterile physiological solution. the yeast inoculum is taken using a sterile bacteriological loop and it is streak-spread on petri dishes with simons medium. the inoculated plates are incubated at 30ºc for 48h. positive results are recorded in a case of a colour change of the medium from green to blue. 2.4.2.2. determination of the gelatinase activity of the tested strains. the preparation method of the yeast inoculum is analogous to that in 5.1. the suspension of the tested strain is taken with a sterile bacteriological loop and is put in point onto a pre-sterilized medium with gelatin. the tubes are incubated for 7 days at room temperature and the presence of crater melting is monitored [6]. 2.4.2.3. proteolytic activity determination using fusion agar method. the ability of the yeast strains to hydrolyze milk proteins in agar medium with milk additive is investigated. wells with d = 6 mm are made on petri dishes with medium for proteolytic activity. after inoculation, the plates are incubated at 30ºc for 48 hours. results are reported as positive in the case of a formation of a brighter halo around the wells of the plates. a lack of halo is a sign of an inability to utilize milk proteins. 2.4.2.4. determination of the lipolytic activity using fusion agar method. lipolytic activity includes the ability of the analyzed strains to hydrolyze the compounds of tween 80. wells with d = 6 mm are made on the petri dishes with agar medium. after inoculation the petri dishes are cultivated at 30ºс for 1 7 days. formation of a turbid zone around the wells, due to the precipitation of ca salts of the formed free fatty acids, indicates the presence of lipolytic activity. 2.4.2.5. analysis on the ability of the studied strains to oxidize s-containing compounds. the analysis of the oxidation of scontaining compounds includes development of the yeast strains on selective media. tubes with sterile liquid starkey medium and ncl medium "with" and "without" glucose are inoculated with 1 cm3 yeast suspension, prepared as in 5.1. with a bacteriological loop the same suspension is used to streak plates with thiosulphate media "with" and "without" glucose. the plates and tubes are cultivated in a thermostat at 30ºc for 15 days. changes in the color of the indicator bromocresol purple (from purple to yellow on starkey and thiosulphate media; and food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 23 from yellow to dark yellow or purple on ncl – broth) are reported as positive results. 2.4.2.6.determination of nitrifying activity of the studied strains. nitrifying activity includes the development of the analyzed yeasts on liquid medium with ammonium salts being the sole nitrogen source. a suspension of the tested yeast strains is prepared, as described in 5.1. 1 cm3 of it is used to inoculate tubes with 5 cm3 of ammonia acetate medium and the tubes are incubated at 30ºc for 24 hours. if the yeast strain has nitrifying activity, no3iones would be formed in the medium. their presence is determined by test strips (110,020 nitrate test merckoquant, merck). some microorganisms have the ability to reduce no3to no2-. therefore, the content of no2in the medium is determined, using a test strip (110,022 nitrate test merckoquant, merck). results are recorded as positive in the presence of no3and no2or negative when no3and no2are absent in the medium. 2.4.2.7. determination of the nitrate reductase activity of the studied strains. prepared as in 5.1., the suspensions of the studied cultures are used to inoculate medium with kno3 1 cm3 suspension is used to inoculate 5 cm3 of sterile medium for determination of nitrate reductase activity. incubation is carried out for 7 hours at 30ºc. the presence of no2in the medium is considered as a positive result. their presence is determined by test strip (110,022 nitrate test merckoquant, merck). result are recorded as positive in the presence of no2or negative in the absence of no2in the medium. 2.4.2.8.determination of the hemolytic activity of the investigated cultures. some microorganisms have the ability to utilize blood, breaking down red blood cells. depending on the mechanism of their hydrolysis there are three types of hemolytic activity. in α hemolysis iron from hemoglobin is oxidized and the colonies become dark green. in β hemolysis the erythrocytes and the hemoglobin in them are degraded, and a bright halo is formed around some of the colonies. γ – hemolysis is observed in the case of no hemoglobin hydrolysis. using a bacteriological loop inoculum is taken from the suspension of the tested yeast strain, prepared as in 5.1. and it is stroked on blood agar. the inoculated plates are cultured for 48 hours at 30ºc. the presence of hemolytic activity is recorded: in the cases of α or β – hemolysis, the result is positive, while for γ – hemolysis, it is negative. 2.4.2.9. determination of the catalase activity of the analysed cultures. catalase activity is determined by the method described in [6]. 2.4.2.10. determination of the oxidase activity of the investigated cultures. oxidase activity test includes an analysis of the suspension of the studied strains for the presence of the enzyme cytochrome oxidase. in the presence of molecular oxygen, cytochrome oxidase can reduce the number of organic substances, including reagent nadi (1 naphthol + diamine dimetilparaphenylene) with the formation of indophenole blue. test strips for oxidase activity (microbiology bactident oxidase 1.13300.0001, merck) are places in the suspension of yeasts prepared as in 5.1. after 20-60 s the result is compared to a color scale. positive result are recorded in the case of a color change of the strip from white to blue to blue violet. 2.4.2.11. determination of the profile of enzyme activity of the studied cultures. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 24 the determination of the profile of enzyme activity is performed, using the test kit api zym (biomerieux) for semiquantitative determination of the enzyme profile of the studied strain. fresh 24-hour culture of the tested strain is centrifuged for 15 minutes at 5000 g, the obtained biomass precipitate is washed twice and resuspended in api suspension medium. the api zym strips are placed in the incubation boxes and the microtubes are inoculated with the prepared cell suspension. the sample is incubated for 4 to 4,5 hours at 30° c. after the incubation one drop of reagent a and one drop of reagent b are pipetted into each microtubule. after 5 min staining result is reported according to the colour scheme described in the manufacturer’s instructions. the enzyme activity is determined according to the colour scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 2.4.2.12. determination of the biochemical profile of the investigated cultures. the system api 20 c aux (biomerieux sa, france) for identification of yeast species based on the consumption of 19 carbon sources is used for the determination of the biochemical profile of the tested cultures. fresh 24-hour culture of the tested strain, developed on malt agar, is resuspended according the instructions of the manufacturer in api c resuspension medium. the honeycomb wells on the bottom of the incubation boxes are filled with sterile physiological solution. the api 20 c strips are placed in the incubation boxes and the microtubules are inoculated with the prepared cell suspension. the sample is incubated for 48h to 72h at the optimum temperature for each of the studied strains. results are recorded according the change in turbidity in comparison to the control (microtubule 0). the results are processed with apiweb® identification software. 3. results and discussion the ability of the yeast strains to grow in three cultural media is investigated. the results represented in table 1 show that the best liquid medium for the strains’ development is malt extract (2.1.). the amount of viable cells is determined during cultivation of the investigated yeast strains on malt extract liquid medium. test results (table 2) show that each of the three yeast strains accumulate high concentration viable cells (over 1012 cfu/cm3) for 48 h cultivation at 30°с. table1 media for yeast development st ra in medium lbg (luria – bertani with glucose) me (malt extract) scb (soy – casein broth) y – abundand sludge, uniform turbidity – с1 – abundand sludge, uniform turbidity – с2 – abundand sludge, uniform turbidity – food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 25 table 2 concentration of viable yeast cells, cultivated in malt extract liquid medium yeast strains average (cfu/cm3) y 5,6.1013 c1 1,0.1013 c2 1,4.1012 the colonial characteristics of the researched strains are determined after inoculating malt agar medium (2.2) and cultivation for 48 h (table 3). cell morphology is done with coloured microscope preparations. by inoculating minimal agar media gorodkova agar, acetate agar and rice agar – it is determined, that researched yeast strains don’t form pseudomycellium and chlamidospores. experiment results, presented in table 4, show considerable similarities between the investigated yeast strains. three cultures don’t hydrolyse gelatine, don’t exhibit nitrifying and nitrate – reductase activities, are catalase – positive and oxidoreductase – negative. all of them are non – motile, without proteolysis activity. they can assimilate citrate, with the exception of yeast strain y. they cannot hydrolyze hemoglobin (γ – hemolysis). all analyzed strains don’t assimilate sulphur compounds in non – glucose medium at рн < 5 and рн 8. с2 is an exception (ncl broth without glucose). analogy between strains is also observed in s – containing compounds at рн 8 (na2s2o3 medium with glucose). as a difference from с1, y and с2 are developed in ncl broth with glucose. yeast strain y, isolated from soil, exceeds c1 and c2 in its values for the lipolytic activity. table 3 colonial characteristics and cell morphology of the investigated yeast strains strain colonial characteristics cell morphology colony description visualization cell description visualization y round colonies with wave – like ends, smooth surface, 4-5 mm in diameter, soft consistence, swelled, drop – like with plateau yeasts, ellipse shaped. with vegetative breeding, with budding, without pseudomycellium formation с1 round colonies with wave – like ends, smooth surface, 4-6 mm in diameter, soft consistence, whitish in colour yeasts, ellipse shaped, with single arrangement and make clusters, with vegetative breeding, with budding, without pseudomycellium formation с2 round colonies with wave – like ends, smooth surface, 4-6 mm in diameter, soft consistence, whitish in colour yeasts, ellipse shaped, with single arrangement and make clusters, with vegetative breeding, with budding, without pseudomycellium formation food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 26 table 4 biochemical features of the analyzed yeast strains strain y с1 с2 lipolytic activity, mm + 13,7±1,4 – – nitrifying activity – – – gelatinase activity – facultative anaerobes – facultative anaerobes – facultative anaerobes oxidation of s – containing compounds mediums with glucose na2s2o3 + growth + growth + growth starkey – – growth – growth ncl + – + mediums without glucose na2s2o3 – growth – growth – growth starkey – – growth – ncl – – + catalase activity + + + oxidoreductase actividy – – – no3 reductase activity – – – proteolytic activity 24 h – – – 48 h – – – citrate utilization – + + hemolytic activity γ γ γ motility – – – aiming at a more complete analysis of the possibilities for application of the yeast strain y in the field of wastewater treatment its enzyme profile is investigated with api zym. the performed investigations (table 5) show the presence of alkaline phosphatase, esterase, esterase-lipase, lipase, leucine – aminopeptidase, valine – aminopeptidase, cysteine – aminopeptidase, acid phosphatase, phosphohydrolase, alpha – glucosidase and beta – glucosidase. the presence of different lipolytic enzymes confirms the results from the biochemical tests for the lipolytic activities, shown in table 4. the ability of yeast strains to utilize 19 different carbon sources, included in the test kit api 20 c aux for rapid identification of yeasts, is investigated. pseudomycellium and chlamidospore formation is determined using rice agar. the results from the analyses are generalized in table 6. after data processing with the software apiweb® yeast strain y is identified as candida famata (synonym torulopsis candida [10] with reliability 99,9% (table 7). c1 is identified as candida famata 53,8%, candida lusitaniae – 22,7%, candida guillermondii – 21,6%. c2 is identified as candida famata 54,7%, candida lusitaniae – 23,1%, candida guillermondii – 22%. regardless of the lower confidential values for the identification of с1 and с2, they are undoubtedly representatives of the genus candida. other tests must be conducted in order to determine their species identification. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 27 table 5 enzyme profile of the yeast strain y enzyme activity of yeast strain y 1 control 2 alkaline phosphatase 3 3 lipase c4 1,5 4 lipase c8 0,5 5 lipase c14 0,5 6 leucine-aminopeptidase 5 7 valine-aminopeptidase 3,5 8 cysteine-aminopeptidase 3 9 trypsin 10 chymotrypsin 11 acid phosphatase 4 12 naphthol – as – bl – phosphohydrolase 1 13 α-galactosidase 14 β-galactosidase 15 β-glucuronidase 16 α-glucosidase 4 17 β-glucosidase 5 18 α-glucoseaminidase 19 α-manosidase 20 α-fucosidase table 6 ability of the investigated yeast strains to utilize the 19 carbon sources in api 20 c aux № substrate y с1 с2 1 control – – – 2 d – glucose + + + 3 glycerol + + + 4 calcium 2 – keto – gluconate + + + 5 l – arabinose – – – 6 d – xylose + + + 7 adonitol + + + 8 xylitol + + + 9 d – galactose + + + 10 inositol – + – 11 d – sorbitol + + + 12 methyl – α – d – glucopyranoside + + + 13 n – acetylglucoseamine + + + 14 d – cellobiose + + + 15 d – lactose (bovine) + – – 16 d – maltose + + + 17 d – saccharose + + + 18 d – trehalose + + + 19 d – melezitose + + + 20 d raffinose – – – 21 hyphae/pseudohyphae – – – table 7 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 28 strains, identified according to the software apiweb® strain species % of reliability y candida famata 99,9 c1 candida famata 53,8 candida lusitaniae 22,7 candida guillermondii 21,6 c2 candida famata 54,7 candida lusitaniae 23,1 candida guillermondii 22,0 4. conclusion. as a result of the conducted experimental analysеs the following more important conclusions can be drawn: 1. a yeast strain is isolated and is identified using the methods of the conventional taxonomy candida famata (synonym torulopsis candida [10]); 2. the strain has the ability to utilize s – containing compounds in the presence of glucose, lowering the рн value; 3. it has clearly defined catalase and lipolytic activities. the last one is also confirmed by the presence of wide range of enzymes with such activity. its beta – and alpha glucosidase activity, leucine –, valine – and cysteine – aminopeptidase activity, acid – and alkaline phosphatase activity, less significant – naphthol – as – bl – phosphohydrolase activity and the least is с4 –, с8 – и с14 – lipase activity are also significant. 5. references. [1] palela, m., ifrim, g., bahrim, g., the annals of the university dunarea de jos of galati fascicle vi – food technology, new series, vol. ii (xxxi), 23-30, (2008) [2] lemmel, s., heimsch, r., edwards, l., optimizing the continuous production of candida utilis and saccharomycopsis fibuliger on potato processing wastewater. applied and environmental microbiology, vol. 37, № 2. 227-232, (1979) [3] yang, q., angly, f., wang, z., zhang, h., wastewater treatment systems harbor specific and diverse yeast communities. biochemical engineering journal., vol. 58-59. 168-176, (2011) [4] yang, q., yang, m., zhang, s., lv, w., treatment of wastewater from a monosodium glutamate manufacturing plant using successive yeast and activated sludge systems. process biochemistry, vol. 40, 2483-2488, (2005) [5] beshkov, m., karova, е., murgov, i., practical handbook of microbiology. christo g. danov, (1970) [6] murgov, i., denkova, z., microbiology. nova timkompakt. 308, (2004) [7] vidyalakshmi r., sridar, r., isolation and characterization of sulphur oxidizing bacteria. journal of culture collections, vol. 5, 73-77, (2006 – 2007) [8] atlas, m., handbook of microbiological media. fourth edition. taylor and francis group, (2010) [9] papen, h., von berg, r., hinkel, i., thoene, b., rennenberg, h., heterotrophic nitrification by alcaligenes faecalis: no2-, no3-, n2o и no production in exponentially growing cultures. applied and environmental microbiology. 2068-2072 стр., (1989). [10] kreger van rij, kreger-van rij, n.j.w. (ed), the yeasts: a taxonomic study. 3rd edition. elsevier science publishers b.v., amsterdam, the netherlands, (1984) 2.1. microorganisms 2.1. microorganisms 2.1. microorganisms 2.2. nutrient media 2.2.1. malt extract medium. composition: malt extract (kamenitza, bulgaria) in 1:1 ratio with tap water (vol/vol). ph=6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.2. malt extract medium. composition: malt extract (kamenitza, bulgaria) in 1:1 ratio with tap water (vol/vol) + 2 % agar – agar (w/vol). ph=6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.3. luria – bertani glucose medium (lbg). composition (g/dm3): triptone (difco) – 10 g, yeast extract – 5 g, nacl – 10 g, glucose (scharlau) – 10 g. ph=7,5. the medium is sterilized for 25 minutes at 121ºc. 2.2.4. soy – caseine broth medium. composition (g/dm3): triptone (difco) – 17g, soy peptone (scharlau) – 3 g, nacl – 5 g, k2hpo4 – 2,5 g, glucose (scharlau) – 2,5 g. ph=7,3 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.2.5. citrate utilization medium (simons medium). composition (g/dm3): na(nh4)2po4 – 1,5 g; kh2po4 – 1 g; mgso4.7h2o – 0,2 g; na – citrate – 3 g; alcohol solution of bromthymol blue – 1%; agar – agar – 2%. the medium is sterilized for 25 minutes at 121ºc [6]. 2.2.6. gelatinase activity medium. composition (g/dm3): triptone (difco) – 10 g; yeast extract (scharlau) – 5 g; nacl – 10 g; glucose (scharlau) – 10 g; gelatine (ddr) – 250 g. рн 7,5. medium is dispensed into tubes and is sterilized for 25 min at 121ºс [6]. 2.2.7. proteolytic activity medium. composition: malt agar medium with 10 % (vol/vol) solution additive (10 cm3 milk/100 cm3 water) of skimmed milk powder (scharlau) [6]. 2.2.8. lipolytic activity medium (tween – 80 compounds hydrolysis). composition (g/dm3): peptone (scharlau) – 10 g; nacl – 5 g; cacl2 – 0,1 g; tween – 80 (merck) – 10 cm3; agar – agar – 20 g. рн 7 – 7,4. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.9. sulphur–containing compound oxidation mediua. 2.2.9.1. starckey broth medium. composition (g/dm3): elemental s – 10 g; кh2po4 – 3 g; mgso4.7h2o – 0,2 g; cacl2.2h2o – 0,2 g; (nh4)2so4 – 0,5 g; feso4 – traces; indicator – bromocresol purple. рн 8,0. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.9.2. ncl – broth medium. composition (g/dm3): elemental s – 10 g; (nh4)2so4 – 0,2 g; mgso4.7h2o – 0,5 g; cacl2.2h2o – 0,25 g; feso4 – traces; indicator – bromocresol purple. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.9.3. thiosulphate agar medium. composition (g/dm3): na2s2o3 – 5g; k2hpo4 – 0,1 g; nahco3 – 0,2 g; nh4cl – 0,1 g; agar – agar – 20 g. рн 8,0. the medium is prepared in two versions: with glucose (5 g/ dm3) and without glucose. it is sterilized at koch apparatus for 30 min on three consecutive days [7]. 2.2.10. no3– reductase activity medium. composition (g/dm3): peptone (scharlau) – 5 g; meat extract (scharlau) – 3 g; kno3 – 1 g. рн 7.0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.11. nh4+ – citrate medium (for nitrifying activity). composition (mol/dm3): na – citrate.2н2о – 9,5.10-3; nh4cl – 9,35.10-3; kh2po4 – 1,47.10-3; mgso4.7h2o – 1,62.10-4; cacl2 – 1,36.10-7; feso4.7h2o as edta – complex – 3,6.10-5. the medium is sterilized for 25 minutes at 121ºc [9]. 2.2.12. blood agar (ncipd, bulgaria). composition (g/dm3): casein hydrolysate – 14 g; nacl – 5 g; peptone – 4,5 g; yeast extract – 4,5 g; defibrinated sheep blood – 70 cm3; agar – agar – 12,5 g. ph 7,3 ± 0,2. all medium components are sterilized for 15 min at 121ºс. after cooling to 45ºс – 50ºс 70 cm3 defibrinated sheep blood is added aseptically and is poured in sterile petri dishes [8]. 2.2.13. gorodkova medium (for yeast sporulation). composition (g/dm3): peptone – 10 g; nacl – 5 g; месен meat extract – 10 g; glucose – 2,5 g; agar – agar – 20 g. рн 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [5]. 2.2.14. acetate agar medium (for yeast sporulation). composition (g/dm3): naoocch3.3h2o – 5 g; agar – agar – 20 g. ph 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.15. minimal agar medium (for yeast sporulation). composition (g/dm3): agar – agar – 20 g. рн 6,5 – 7,0. the medium is sterilized for 25 minutes at 121ºc [8]. 2.2.16. rice agar medium (fluka analitycal) (for pseudomycellium identification). composition (g/dm3): rice extract powder – 0,7; agar – agar – 20 g. the medium is prepared in two versions – with and without tween – 80 (merck) (1 cm3/dm3). рн 5,8 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.2.17. motility medium (ncipd, bulgaria). composition (g/dm3): peptone – 10 g, meat extract – 3 g, nacl – 5 g, agar – agar – 4 g. рн 7,4 ± 0,2. the medium is sterilized for 25 minutes at 121ºc. 2.3. cultivation and storage of the analyzed microorganisms. 2.4. analytical methods. 2.4.1. morphological and cultural methods 2.4.1.1. cellular and colonial morphology. description of cellular and colonial morphology of the studied yeast strains is performed by microscopic observation the developed on malt agar single colonies of the studied strains. 2.4.1.2. identification of pseudomycellium formation ability. a part of the cultural medium of the studied strain, that has been cultivated for 48 hours in a thermostat is taken with flamed and cooled bacteriological loop and spread in touch on agar plates with rice-agar. the inoculated plates are incubated under aerobic conditions for 2-4 days at 23ºc 28ºc. a microscopic preparation is prepared from the developed colonies of the tested strain, which is examined for the presence of specific pseudomycellium or other specific formations in yeasts. the presence of vegetative cells only determines the result as negative. 2.4.1.3. identification of spore formation ability. the ability of the yeast to form spores under unfavorable environmental conditions lack of nutrients is examined.using a flamed and cooled bacteriological loop a part of the pre-developed on malt agar 48 hour colonies of the studied strain is taken and is stroked on a petri dish with minimal agar medium gorodkova agar or acetate agar. incubation conditions 20ºc 25ºc for 7 days. a microscopic preparation is prepared from the biomass and it is stained by the method of moller. spores are rubine red and vegetative bodies – blue. 2.4.1.4. determining the number of viable cells in the development of the cultures in liquid medium. sterile malt extract is inoculated under aseptic conditions with yeast suspension, 5 cm3 medium is inoculated with 1 cm3 of the inoculum of the studied cultures. after incubation in a thermostat at 30ºc for 48 h, tenfold dilution method is done and malt agar plates are inoculated. petri dishes are thermostatted at 30ºc for 48 h so that the strain would form single colonies, which are then counted. 2.4.2. physiological and biochemical methods 2.4.2.1. citrate utilization. developed in containers with 30 cm3 1:1 malt extract at 30ºc for 48h yeast cultures are centrifuged at 3000 min-1 for 10 min. the supernatant is discarded and the biomass is resuspended in 5 cm3 sterile physiological solution. the yeast inoculum is taken using a sterile bacteriological loop and it is streak-spread on petri dishes with simons medium. the inoculated plates are incubated at 30ºc for 48h. positive results are recorded in a case of a colour change of the medium from green to blue. 2.4.2.2. determination of the gelatinase activity of the tested strains. 2.4.2.3. proteolytic activity determination using fusion agar method. the ability of the yeast strains to hydrolyze milk proteins in agar medium with milk additive is investigated. wells with d = 6 mm are made on petri dishes with medium for proteolytic activity. after inoculation, the plates are incubated at 30ºc for 48 hours. results are reported as positive in the case of a formation of a brighter halo around the wells of the plates. a lack of halo is a sign of an inability to utilize milk proteins. 2.4.2.4. determination of the lipolytic activity using fusion agar method. lipolytic activity includes the ability of the analyzed strains to hydrolyze the compounds of tween 80. wells with d = 6 mm are made on the petri dishes with agar medium. after inoculation the petri dishes are cultivated at 30ºс for 1 7 days. formation of a turbid zone around the wells, due to the precipitation of ca salts of the formed free fatty acids, indicates the presence of lipolytic activity. 2.4.2.5. analysis on the ability of the studied strains to oxidize s-containing compounds. the analysis of the oxidation of s-containing compounds includes development of the yeast strains on selective media. tubes with sterile liquid starkey medium and ncl medium "with" and "without" glucose are inoculated with 1 cm3 yeast suspension, prepared as in 5.1. with a bacteriological loop the same suspension is used to streak plates with thiosulphate media "with" and "without" glucose. the plates and tubes are cultivated in a thermostat at 30ºc for 15 days. changes in the color of the indicator bromocresol purple (from purple to yellow on starkey and thiosulphate media; and from yellow to dark yellow or purple on ncl – broth) are reported as positive results. 2.4.2.6. determination of nitrifying activity of the studied strains. nitrifying activity includes the development of the analyzed yeasts on liquid medium with ammonium salts being the sole nitrogen source. a suspension of the tested yeast strains is prepared, as described in 5.1. 1 cm3 of it is used to inoculate tubes with 5 cm3 of ammonia acetate medium and the tubes are incubated at 30ºc for 24 hours. if the yeast strain has nitrifying activity, no3-iones would be formed in the medium. their presence is determined by test strips (110,020 nitrate test merckoquant, merck). some microorganisms have the ability to reduce no3to no2-. therefore, the content of no2in the medium is determined, using a test strip (110,022 nitrate test merckoquant, merck). results are recorded as positive in the presence of no3and no2or negative when no3and no2are absent in the medium. 2.4.2.7. determination of the nitrate reductase activity of the studied strains. prepared as in 5.1., the suspensions of the studied cultures are used to inoculate medium with kno3 1 cm3 suspension is used to inoculate 5 cm3 of sterile medium for determination of nitrate reductase activity. incubation is carried out for 7 hours at 30ºc. the presence of no2in the medium is considered as a positive result. their presence is determined by test strip (110,022 nitrate test merckoquant, merck). result are recorded as positive in the presence of no2or negative in the absence of no2in the medium. 2.4.2.8. determination of the hemolytic activity of the investigated cultures. some microorganisms have the ability to utilize blood, breaking down red blood cells. depending on the mechanism of their hydrolysis there are three types of hemolytic activity. in α hemolysis iron from hemoglobin is oxidized and the colonies become dark green. in β hemolysis the erythrocytes and the hemoglobin in them are degraded, and a bright halo is formed around some of the colonies. γ – hemolysis is observed in the case of no hemoglobin hydrolysis. using a bacteriological loop inoculum is taken from the suspension of the tested yeast strain, prepared as in 5.1. and it is stroked on blood agar. the inoculated plates are cultured for 48 hours at 30ºc. the presence of hemolytic activity is recorded: in the cases of α or β –hemolysis, the result is positive, while for γ – hemolysis, it is negative. 2.4.2.9. determination of the catalase activity of the analysed cultures. catalase activity is determined by the method described in [6]. 2.4.2.10. determination of the oxidase activity of the investigated cultures. oxidase activity test includes an analysis of the suspension of the studied strains for the presence of the enzyme cytochrome oxidase. in the presence of molecular oxygen, cytochrome oxidase can reduce the number of organic substances, including reagent nadi (1 naphthol + diamine dimetilparaphenylene) with the formation of indophenole blue.test strips for oxidase activity (microbiology bactident oxidase 1.13300.0001, merck) are places in the suspension of yeasts prepared as in 5.1. after 20-60 s the result is compared to a color scale. positive result are recorded in the case of a color change of the strip from white to blue to blue violet. 2.4.2.11. determination of the profile of enzyme activity of the studied cultures. the determination of the profile of enzyme activity is performed, using the test kit api zym (biomerieux) for semiquantitative determination of the enzyme profile of the studied strain. fresh 24-hour culture of the tested strain is centrifuged for 15 minutes at 5000 g, the obtained biomass precipitate is washed twice and resuspended in api suspension medium. the api zym strips are placed in the incubation boxes and the microtubes are inoculated with the prepared cell suspension. the sample is incubated for 4 to 4,5 hours at 30° c. after the incubation one drop of reagent a and one drop of reagent b are pipetted into each microtubule. after 5 min staining result is reported according to the colour scheme described in the manufacturer’s instructions. the enzyme activity is determined according to the colour scale from 0 (no enzyme activity) to 5 (maximum enzyme activity). 2.4.2.12. determination of the biochemical profile of the investigated cultures. the system api 20 c aux (biomerieux sa, france) for identification of yeast species based on the consumption of 19 carbon sources is used for the determination of the biochemical profile of the tested cultures. fresh 24-hour culture of the tested strain, developed on malt agar, is resuspended according the instructions of the manufacturer in api c resuspension medium. the honeycomb wells on the bottom of the incubation boxes are filled with sterile physiological solution. the api 20 c strips are placed in the incubation boxes and the microtubules are inoculated with the prepared cell suspension. the sample is incubated for 48h to 72h at the optimum temperature for each of the studied strains. results are recorded according the change in turbidity in comparison to the control (microtubule 0). the results are processed with apiweb® identification software. manuscript guidelines template food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 18 gerodietic meat products technology enriched with calcium and phosphorus peshuk l.v. 1 , budnyk n.v. 2 , *halenko о.о. 1 1 national university of food technologies, 68, volodymyrska str., kyiv, 01601, ukraine 2 poltava university of economics and trade, 3, kovala str., poltava, 36014, ukraine *corresponding author received 22 june 2011, accepted 25 august 2011 abstract: the latest designs of national and foreign gerodietic products were analyzed. developed a technology of sausage products with bone paste. examined a microstructure of model sausage meat with different content of bone paste. determined the optimal share of bone paste in sausages. keywords: meat, bone paste, rapana seashell, quail eggshell, mussel shell, receipt, boiled sausage, gerodietic nutrition. 1. introduction at the end of the twentieth and the beginning of twenty first centuries the scientists of economically developed countries and countries that are developing became interested in gerontology (the study of the aging processes and factors which determine life expectancy of the people of advanced age, in particular the problem of nutrition in old age). 25% of citizens of ukraine are pensioners, and 20% are more than 50 years old. ukraine is on the eleventh place in the world among the countries with the share of citizens older than 65 years (belarus 23 place, russia 27). statistics shows that the percentage of people of advanced age in ukraine is 20.5%. the institute of gerontology of the academy of medical science of ukraine predicts that in 2015 it will be 22%, in 2026 26%, 2050 38% [1]. main factors which cause fast ageing are:  social: low level of income; lack of knowledge about basic characteristics of food products; insufficient level of medical aid; low level of social protection of citizens, and chronic stress;  ecological: contamination of water, soil, air, and food products;  incorrect way of life: bad habits; defective nutrition; insufficient afferent activity; incorrect work and rest regime;  infections. typical ukrainian in average consumes 20% less products that are prescribed in living wage, and 40% less than it was in 90th, the most unfavourable years of perestroika. conducted investigations in ukraine [1, 2] show that daily ration of different groups of people consists of comparatively chip high carbohydrate products (bread, macaroni products, potatoes), as a result organism lacks essential micro and macroelements, polyunsaturated fat acids, anti-oxidizing vitamins ( а, с, е). there are two ways of solving the problem. first, using biologically active additives in food. second, enrichment of traditional food products in order to raise the level of vitamins, macro and microelement content to current physiological needs of people, this will make possible to correct micronutrient deficit [3, 4, 5]. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 19 production of gerodietic products is essential in some aspects: wide application of gerodietic products in a diet of advanced age people, elderly people, and long-lived people will help to correct defects of nutrition. the usage of gerontological products is very essential in a domain of medical nutrition not only for this group of people, but also for younger age groups as preventive measures for premature ageing. citation [1, 6] shows that in spite of multiple researches on creating of high nutrition and biological value products, their assortment is insignificant. the pioneers in creating of balanced gerodietic products are: antipova l.v., bohatyriov a.n., kozlovska s.h., zaytsev а.n., kasianov h.i., samsonova м.а., hrihorov y.h., povorozniuk v.v., chebotariev d.f., nechayev а.p., lipatov n.n., pokrovskiy а.а., skurykhin к.м., uholiev а.м, ustynova а.v., kharytonov v.d., shazzo r.i. etc. the urgent problem of present time is a deficiency of calcium in daily ration that is why different scientists are looking for ways to enrich food products with calcium. leading scientists of dairy industry in ukraine [petrov а. n., kovalenko n.к., sharakhmatova т.е., 2001] developed a set of dairy gerodietic products which are not represented on market. the absence of these products is stipulated by a short expiration term (36 and 72 hours for milk and fermented milk drinks «героклат» and «лактогеровіт»); low profitability of production; low income of elderly people in ukraine; absence of state policy in a sphere of gerodietic nutrition. there was created [didukh n.а., 2008] a set of nonfermented and fermented gerodietic drinks (milk, kefir, sour milk, acidophilus milk) on milk and grain milk base, sour milk cheese and gerodietic sour cream. to enrich macaroni products with calcium scientists [koriachkina s. ya., osypova h. а., 2002] conducted a research on some components of a paste: gluconate, lactate, calcium carbonate and powder of eggshell. russian scientists [savenkova т. v., blahodatskikh v. е., dukhu т. а., shcherbakova n. а., bashkirov о. i., 2009] with the help of computer modeling developed a technology of a pastry «бисквитное» with a curd soufflé for gerodietic nutrition, and cookie on a base of functional additives [tumanova а., 2006], specifically: kelp, calcium alginate, pectin and microcrystalline cellulose. new assortment of sweet products differs from traditional products with a high content of fibre foods and vital mineral materials, iodine and calcium. meat is perspective in production of gerodietic products, as much as this is the main source of protein for human’s organism. experts [shypulyn v. i., akhtemshyna а. d., nekrasova n.n., 2009] developed a technology of production of meat foods using protein preparations based on whey. all-union research institute of meat industry (moscow) developed a biologically active mineral organic additive made of horns of a reindeer, which should be added into boiled sausage products in order to enrich them with iodine and calcium. however this preparation is not widely used due to the insufficient amount of raw materials used for its production. we are conducting researches on a possibility of using mineral additives made of mussel shells, rapana shells, and quail eggshells in a production of gerodietic products, specifically boiled sausages. to enrich meat foods scientists [faivyshevskiy м. l., 1998; khabryna к. е., 1999; ustynova а. v., 2000] suggest using natural sources of calcium: bone marrow meal, algin acid salt, bone paste. the source of bioorganic calcium compounds is dietary bone, which is gathered on meat processing plants and is not used for nutrition as a source of calcium. beliayev м.і., cherevka о.і., faivishevskiy м.l., honcharov h.і., vinokurova h.а. investigated a technology of extraction of protein, fat, bone marrow food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 20 and obtaining of meat out of bone stuff. in 2004 holovko m.p. developed a method of processing a semi-finished product out of dietary bone of the cattle. this semifinished product may be used for production of ground meat products enriched with bioorganic calcium compounds. the food industry of japan more than 20 years is using different methods of production of meat foods enriched with calcium by adding grounded animal bones into cutlet mince, schnizels, and sausage products. the food industry of usa conducts researches on creating of a protein mineral additive made of bone and bone leavings. in united kingdom dietary bone is processed by johnson-faudler method to obtain food fat, soluble protein and food phosphate [7,8]. 2. experimental when developing a technology of sausage products using bone additives, it is essential to identify its structure, structural changes of model sausage meat and final sausage products. the usage of the precise chemical, physicochemical, histological and biochemical methods allows to obtain information about quality of meat foods. microstructural researches make it possible to analyze not only the integral structure of product, but also changes which occur in separate components of the objects under investigation, it also helps to differentiate peculiarities of different tissue and cellular structures. that is why the aim of our investigation was to study the possibilities of using the paste obtained by hydrothermal hydrolysis of bones in technology of boiled sausages, investigating microstructure of model sausage meat and final sausage products [9]. for production of sausages the receipt of boiled sausage «столова» is used in accordance with дсту 4436:2005. in created receipts we substituted 5 to 20% of 1st sort beef with a dietary bone paste. the modeling of receipt components and its percentage was made with a help of a computer program віо.2. after the analysis of different kinds of receipts it was stated that the increase of amount of bone paste will not balance the amino-accids and mineral components of a product, and the minimal amount will not balance calcium and phosphorus. by modeling the maximum value of the coefficient of utilitarity of amino-accids and the ratio of protein : fat : mineral elements, we have chosen 4 receipts of sausage meat for boiled sausages (table 1). we have determined that it is not practical to use more than 40% of beef in meat products, because the cost increases, the product becames more tough and the level of digestion decreases. the concentration of beef less than 35% does not balance aminoaccid components. thus the most optimal content of bone paste is 10% . table 1 receipts of boiled sausages with different content of bone paste component the amount of bone paste, % stolova 5 10 15 20 1st sort beef 49 44 39 34 29 mild fat pork 50 50 50 50 50 dried milk 1 1 1 1 1 bone paste 5 10 15 20 the next stage was to investigate the microstructure of sausages with different amount of the bone paste. the investigation of biological tissues is more specific in comparison to native tissues, because we investigate the tissues that underwent mechanical and thermal processing [3, 5]. for histological investigations the samples of sausage products with dimensions 10х5х4 mm. and model sausage meat were fixed in 2.5% of gluteraldehyde on phosphate buffer (рн-7.4), samples were content for 24 hours in 4ºс. then pieces were washed in phosphate buffer (рн 7.3) for two hours. after this they were immersed into osmium fixator (g.m.millonig [6]) for additional fixation food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 21 for two hours. then the pieces of tissue are washed in 0,1m of phosphate buffer (рн 7.3) for one hour. the next stage is to dehydrate the tissues in spirits of different strength (50%, 70%, 80%, 90% and the absolute spirit for 10 min. in each), then goes the mixture of spirit and acetone ( 3:1, 2:1, 1:1, 1:2, 1:3 – 15 min. each). after the samples are washed, they are processed under laft method [6], and pouredwith epon 812. the cuts were made on ultramicrotome умтп-7 by aimed microtoming. before colouring, the objectplate with the cut was kept in thermostat in 45-50ºс for better fixation on the objectplate. for colouring was used the 0.1% mixture of toluidine blue. morphometric analysis was done with the light optical microscope “мбі-15”. 3. results and discussion conducted histological investigations made it possible to determine the next microstructure rates of model sausage meat and finished sausage products. histocuts of the samples of model sausage meat (figure 1) are small-grained and uniform (2) with big vacuoles (4), which are filled with fat, they are bright coloured, because fat was partially dissolved in the process of washing in spirit mixtures, but not in all the samples. sausage meat contained connecting tissues, (3) where intact muscular fibres can be find (1), this makes possible to determine the components of sausage meat (figure 1). histocuts of finished sausages (figure 2) contained coagulation layer (1), increase of vacuoles size (2), homogenous mass of glutin the product of thermal disintegration of collagen of the connecting tissue (3), which is less sensitive to histological dyes (figure 2). figure 1. microstructure of sausage meat (sample 15х40) in figures 3 and 4 are presented the images of histocuts of sausage meat and final sausages with 5% of bone paste. in the figure an insignificant increase of size of vacuoles and individual inclusions of evenly placed particles of bone paste can be seen. figure 2. microstructure of final sausage (sample 15х40) figure 3. microstructure of sausage meat with 5% of bone paste (15х40) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 22 figure 4. microstructure of final sausage with 5% of bone paste (15x40) meat sausage which contained 10% of bone paste (figure 5) has small-grained uniform structure with evenly placed particles of the bone paste and vacuoles, which are less vivid than in previous sample. final sausages have compressed coagulation layer, the fat is evenly distributed in vacuoles and in uniform protein mass (figure 6). organoleptic rates show that this sausage is juicier, has elastic consistence, is palatable, without aftertaste of bone paste. sausage meat with 15 and 20% of bone paste has soft structure with big vacuoles filled with fat (figures 7, 9). figure 5. microstructure of sausage meat with 10% of bone paste (15х40) figure 6. microstructure of final sausage with 10% of bone paste (15х40) figure 7. microstructure of sausage meat with 15% of bone paste (15x40) data on figures 7, 9 shows considerable amount of intact connecting tissue (1), which increase the amount of bone paste particles. organoleptic rates show that this sausage is soft, has specific aftertaste and watery consistency. the microstructure of finished sausage products has powdery structure and big amount of unstabilized fat and vacuoles (figures 8,10). thusly the change of the ratio of muscular and connecting proteins and adding big amount of paste to increase amount of fat causes deterioration of the quality of final sausage products [7]. figure 8. microstructure of finished meat with 15% of bone paste (15х40) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 23 figure 9. microstructure of sausage meat with 20% of bone paste (15x40) figure 10. microstructure of finished sausage with 20% of bone paste (15х40) 4. conclusion. the results of previous physicochemical and organoleptic investigations showed that bone paste can be used in the food industry. the investigation of the microstructure of sausage meat and final boiled sausages produced using model receipts, shows that addition more than 15% of bone paste to the content of the product results in powdery structure of finished sausages. it is proved that the optimal amount of the bone paste in finished product is 10%. 5. references [1]. the current state of problems gerodietic in ukraine / yg grigorov et al. / bulletin of medical sciences of ukraine 2005. №3 p. 77-89. [2]. hygienic assessment of actual nutrition and mikrostrukcher of workers of industrial enterprises of dnepropetrovsk and ways of their correction / [3]. nm denisenko, ve sugar, sa marmer et al. / / "hygienic science and practice at the turn of the century." proceedings of xiv congress of hygienists ukraine. vol ii. dnepropetrovsk: art-press. 2004. p.376-379. [4]. pilate tl sharing the principle of building a healthy nutrition diet / / proceedings of vii allrussian congress of "health nutrition of the population of russia." moscow: 12-14 november 2003. vol.2. p.407-409. [5]. yakovlev s. strengthening of health and prevention funds aging natural origin .spb.: dylya, 2006 328 p. [6]. kasyanov gi, zaporozhye aa, kovtun tv. the realization of the food feasibility study of new solutions in technology product of gerodietic destination: problems of aging 2010 № 1 p. 99. [7]. biochemical identification of meat species ed by patterson r l s, elsevier applied science publishers, london, 1985 [8]. horn d. zum nachweis pflanzhcher eiweisszubereitungen in fleischerzeugmssen mil histilogischen untersuchunssverfahren fleischwirtsch, 1987. [9]. hvulya s.i. ability histology in determining the quality and composition meat product / si wave / / technology of meat. 1996. № 5 p. 6-7. peshuk l.v., budnyk n.v., *halenko о.о. receipts of boiled sausages with different content of bone paste 267 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 267 275 fungal contamination and mycotoxins’ occurrence in peanut butters marketed in abidjan district (côte d’ivoire) zamblé a. boli 1, *lessoy t. zoue 2, rose koffi-nevry1, marina koussemon1 1laboratory of biotechnology and food microbiology, department of food science and technology (ufrsta), university nangui abrogoua, 02 bp 801, abidjan 02, côte d’ivoire. 2biotechnology laboratory, biosciences department, university félix houphouët-boigny, 22 bp 582, abidjan 22, côte d’ivoire, y.lessoy@yahoo.fr *corresponding author received september 17th 2014, accepted september 26th 2014 abstract: this study was carried out in order to assess the detection of fungi contamination and mycotoxins (aflatoxin b1 and ochratoxin a) levels in peanut butters sold in retail markets in abidjan district. samples were aseptically collected and analyzed by using agar method dilution for fungi enumeration and hplc for afb1 and ota quantification. the fungal isolates included mucor, alternaria, helmintosporium, geotrichum, fusarium, cladosporium, penicillium and aspergillus genera. the predominant fungi belonged to aspergillus genus (20.22 – 51.65 %) followed by helmintosporium (0 – 47.44 %) and penicillium (0 – 41.54%). the mycotoxigenic fungi were isolated with a frequency of 14.81; 13.95; 9.1 and 21.62 % for aspergillus versicolor, aspergillus ochraceus, aspergillus flavus and aspergillus parasiticus, respectively. mycotoxins (afb1 and ota) were detected in the marketed peanut butters. their contents varied from 0.23 to 2.49 µg/kg for afb1 and 0.53 to 2.23 µg/kg for ota . the coexistence of aflatoxins and ochratoxin a (ota) in the analyzed peanut samples was also established. the results showed that peanut butters sold in abidjan district were highly contaminated and therefore unacceptable for human nutrition. so, good manufacturing processing including sorting and good hygiene practices would help to minimize mycotoxin occurrence in order to obtain good sanitary peanut butters. keywords: peanut butter, fungal contamination, aflatoxin b1, ochratoxin a 1. introduction fungal contamination of seeds before and after harvesting remains a major problem of food safety in most parts of tropical africa. problems associated with this contamination include loss of germination, mustiness, moldy smell and mycotoxin production [1,2]. mycotoxins, secondary metabolites produced by fungi, are toxic to both animals and humans and their occurrence in the food chain may have public health effects [3]. these toxic metabolites have attracted worldwide attention due to the significant losses associated with their impact on human and animal health, and consequent national economic implications [4]. based on detailed study of the distribution of fungi in nature, the five agriculturally important toxins from fungi are aflatoxins, ochratoxin a fumonisins, zearalenone and deoxynivanelol [5]. aflatoxins are mainly produced by the fungi aspergillus flavus, aspergillus parasiticus and aspergillus nominus [6]. aflatoxins (b1, b2, g1 and g2) are very powerful hepatocarcinogen and have been classified as a class 1 human carcinogen [5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 268 the european union uses an action level of 2 µg/kg as the maximum residue limit allowed in food for human consumption [7]. ochratoxin a (ota) is produced by different species of aspergillus and penicillium, though it was first isolated from cultures of aspergillus ochraceus [2]. ota is immunosuppressive, teratogenic, genotoxic and mutagenic and iarc has classified it in-group 2b as possibly carcinogenic to human [5]. the joint expert committee on food additives (jecfa) of the who and fao set a provisional maximum intake of 100 ng/kg body weight (bw) while the scientific committee on food of the european union proposed that the maximum daily intake of ota should not exceed 3 µg/kg [8]. fumonisins produced by fusarium verticillioides and fusarium proliferatum are recently receiving increasing attention in scientific literature because they have been implicated in a number of animal diseases, such as porcine pulmonary oedema and rat liver cancer [9]. it can also cause hepatotoxicity and nephrotoxicity in many animals [10]. the united states fda has proposed a guideline of tolerance level of 2 mg/kg total fumonisins for human consumption [11]. zearalenone, an oestrogenic mycotoxin, causes problems with the reproductive organs of farm animals, especially swine and is often found together with deoxynivalenol (vomitoxin) [12]. among the food crops susceptible to contamination by mycotoxigenic fungi, peanut (arachis hypogea linn) plays an important role [13]. indeed, peanut which constitutes a major annual oilseed crop is liable to fungal contamination during handling, storage and transportation [14]. peanut seeds are eaten raw, boiled or roasted, made into butter or paste and are used for thickening soups [15]. peanut butter is traditionally made by grinding dry roasted groundnuts into a paste [14]. in côte d’ivoire, the use of marketed peanut butter in the confection of sauce is very popular among the urban population. poor harvesting practices, improper storage and less than optimal conditions during transport and marketing of peanut butter could contribute to fungal growth and mycotoxins production. therefore, the present work aimed to determine the level of mycotoxins (aflatoxin b1 and ota) and associated fungi species in peanut butters marketed in abidjan in order to explore their sanitary quality. 2. material and methods 2.1. samples collection peanut butters were collected during the period (june – october 2012) in the main markets of the 9 communes of abidjan (côte d’ivoire): abobo, adjamé, attécoubé, cocody, koumassi, marcory, port-bouet, treichville, yopougon identified as ab, ad, at, c, k, m, p, t and y. a total of 45 sellers (5 sellers per commune) were investigated and three (3) peanut butters samples (500g each) per seller were randomly and aseptically purchased. the collected peanut butters samples (135) were immediately transported in icebox (4°c) to the laboratory and aseptically mixed together to constitute a minimum of five (5) composite samples (500g each) per commune. the composite samples obtained were stored at 4°c until further analysis. 2.2. mycological analysis the isolation of fungi was carried out according to the agar dilution method [16]. ten (10) gram from each sample, were homogenized with 90 ml of buffer peptone water (aes laboratory, france) and serial decimal dilutions (10-1 to 10-4) were performed. fungal species were isolated on the semi selective dichloran rose bengal chloramphenicol (drbc) agar (biokar diagnostics, france). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 269 the medium was poured into sterile petri dish and 0.1 ml of each sample suspension was spread-plated onto the drbc agar in triplicate. the plates were incubated for 5 to 7 days at 25°c. fungal isolates were sub-cultured on malt extract and czapek yeast medium agars (oxoid, uk) and incubated for 5 to 7 days at 25°c for purification. fungi were identified by using taxonomic schemes based on microscopic observation and culture appearance including colonies colours, texture, reverse colour, hyphae arrangement, conidia shape and nature of spores [17]. for the differentiation between aspergillus parasiticus and aspergillus flavus colonies, afpa agar (oxoid, uk) supplemented with chloramphenicol, was used. the total fungal count for each plate was expressed as colony-forming units per gram of sample (cfu/g). each genus or specie identified was then expressed as percentage (%) of the total isolated fungi. 2.3. aflatoxin b1 extraction and purification aflatoxin b1 (afb1) was extracted and cleaned up following official method [18]. 25 g of peanut butter sample was mixed with 100 ml of methanol-water (80:20, v/v). the mixture was transferred to a conical flask and shaken for 30 min at 300 rpm. the extract obtained was then filtered through whatman no. 4 filter paper and diluted (1:10) in phosphate buffered saline (pbs). an aliquot (30) ml of this mixture was passed through immunoaffinity column (vicam, watertown, ma, usa). after washing with 10 ml of pbs, the column was eluted with acetonitrile (1.5 ml) at a flow rate of 0.5 ml/min. the eluate was then evaporated to dryness under a stream of nitrogen at 40ºc and the residue was dissolved in 500 μl of methanol prior to hplc analysis. 2.4. ochratoxin a extraction and purification extraction and purification of ochratoxin a were performed according official method [18]. 25 g of peanut butter sample was mixed with 100 ml of methanol-sodium hydrogenocarbonate (1:1, v/v). the mixture was transferred to a conical flask and shaken for 30 min at 300 rpm. the extract obtained was then filtered through whatman no. 4 filter paper and diluted (1:10) in phosphate buffered saline (pbs). an aliquot (20) ml of this mixture was passed through immunoaffinity column (vicam, watertown, ma, usa). after washing with 10 ml of pbs, the column was eluted with 1.5 ml of methanol-acetic acid (98:2, v/v) at a flow rate of 5 ml/min. the eluate was then evaporated to dryness under a stream of nitrogen at 40ºc and the residue was dissolved in 500 μl of methanol prior to hplc analysis. 2.5. hplc analysis final extracts were filtered through a 0.45 μm ptfe membrane and 20 μl were injected into a high performance liquid chromatography column, using a shimadzu liquid chromatograph (kyoto, japan) equipped with a fluorescence detector (exc 360 nm; em 440 nm), a c18 column (torrance, ca, usa) (4.6 × 150 mm, 4 μm) and a shim-pack pre-column (4 × 10 mm, 5 μm clc g-ods). the isocratic mobile phase consisted of methanol/water/acetonitrile (60:20:20, v/v/v) with a flow rate of 0.5 ml/min. calibration curves were prepared using standard solutions of aflatoxin b1 and ota (sigma, st louis, mo, usa). detection limits (lod) of aflatoxin b1 and ota were 0.005 and 0.05 μg/g, respectively while their quantification limits (loq) were 0.02 and 0.2 μg/g statistical analysis all analyses were carried out in triplicates and data expressed as means ± standard deviation. one way analysis of variance (anova) and duncan’s multiple food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 270 range test (dmrt) were carried out to assess significant differences between means (p<0.05) using statistica 7.1 (statsoft). 3. results and discussion the fungal count of peanut butters, collected from markets, is given in table 1. eight fungi genera were isolated from analyzed peanut butters. they included mucor, alternaria, helmintosporium, geotrichum, fusarium, cladosporium, penicillium and aspergillus genus (table 1). the predominant fungi belonged to aspergillus (20.22 – 51.65 %) included the following mycotoxigenic ones (fig. 1): aspergillus parasiticus (0 – 21.62 %), aspergillus flavus (0 – 10.81 %), aspergillus ochraceus (0 – 13.95 %) and aspergillus versicolor (0 – 14.81 %). the peanut butter samples collected from portbouet were more contaminated with helmintosporium (44.44 ± 3.00 %) and geotrichum (25.95 ± 2.00 %) while those collected from treichville were more contaminated with alternaria (31.11 ± 2.50 %) and fusarium (26.66 ± 1.00 %). the highest occurrence of mucor (30.23 ± 2.50 %) was noted for the samples collected from cocody. other studies made in kenya, benin and mali, have also revealed the occurrence of aspergillus, fusarium and penicillium in peanut butters and other peanut products [19,20,21]. the presence of these fungi in relatively large amounts is a clear indication of poor handling, e.g. poor hygiene and storage, which exposes peanuts to favorable conditions for fungal growth and mycotoxin production. indeed, aspergillus sp. mainly a. flavus and a. parasiticus are considered as the major producers of aflatoxin while a. ochraceus is involved in ota production [22]. as concern fusarium strains, they can produce mycotoxins such as fumonisins, trichothecenes and zearalenones which are associated with mycotoxicoses in humans or domestic animals. in addition, toxigenic penicillium species produce mycotoxins such as ochratoxin, citrinin, and patulin [23]. table 1 total fungi isolated from peanut butter samples sold in retail markets of abidjan. fungi isolates (%) communes investigated ab (n=5) ad (n=5) at (n=5) c (n=5) k (n=5) m (n=5) p (n=5) t (n=5) y (n=5) mucor 6.00a ±1.00 10.80b ±0.80 7.70c ±0.80 30.23d ±2.50 0.00g ±0.00 10.80b ±0.80 7.41c ±0.80 13.34e ±0.80 9.10f ±0.80 alternaria 17.00a ±1.00 21.05b ±1.00 11.11c ±0.80 0.00g ±0.00 18.29d ±1.00 0.00g ±0.00 0.00g ±0.00 31.11e ±2.50 20.45f ±1.00 helmintospor ium 6.00a ±0.00 0.00d ±0.00 13.67b ±1.00 6.98a ±1.00 0.00d ±0.00 0.00d ±0.00 44.44c ±3.00 0.00d ±0.00 7.95a ±1.00 geotrichum 0.00g ±0.00 12.63a ±1.00 15.38b ±1.00 0.00g ±0.00 0.00g ±0.00 8.12c ±1.00 25.93d ±2.00 6.67e ±1.00 3.41f ±0.00 fusarium 5.00a ±0.00 0.00d ±0.00 0.00d ±0.00 0.00d ±0.00 15.85b ±1.00 0.00d ±0.00 0.00d ±0.00 26.66c ±1.00 0.00d ±0.00 cladosporium 3.00a ±0.00 0.00d ±0.00 6.84b ±1.00 0.00d ±0.00 7.32c ±1.00 0.00d ±0.00 0.00d ±0.00 0.00d ±0.00 0.00d ±0.00 penicillium 28.00a ±1.00 33.68b ±2.50 18.80c ±1.00 39.54d ±2.00 31.71b ±2.00 32.43b ±2.00 0.00f ±0.00 0.00f ±0.00 27.27e ±2.00 aspergillus 35.00a ±2.50 22.11b ±1.00 26.50c ±1.00 23.25d ±1.00 26.83e ±1.00 48.65f ±3.00 22.22g ±2.00 22.22g ±2.00 31.82h ±2.00 data are represented as means ± sd (n=3). mean with different letters in the same row are statistically different (p < 0.05) according to duncan’s test. ab: abobo, ad: adjamé, at: attécoubé, c: cocody, k: koumassi, m: marcory, p: portbouet, t: treichville, y: yopougon. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 271 figure 1: level of mycotoxigenic aspergillus species isolated from peanut butter samples sold in retail markets of abidjan. the occurrence of other detected fungi strains such as mucor sp. can be used as indicators of potential presence of aflatoxins [24]. aflatoxins remain the most dangerous mycotoxin in the world, recognized as a cause of liver cancer and various additional important toxic effects [25]. ochratoxin a (ota) is an important nephrotoxic and nephrocarcinogenic mycotoxin and has been associated with the development of urinary tract tumours in humans [26]. the contents of afb1 and ota in the marketed peanut butters are given in table 2 and their retention times are depicted in figure 2. the retention time of afb1 was 2.53 min while that of ota was 8.99 min. there was variation (p < 0.05) in aflatoxin b1 and ochratoxin a levels among peanut butter samples ranging from 0.23 to 2.49 µg/kg for afb1 and from 0.53 to 2.23 µg/kg for ota. samples collected from attécoubé and adjamé were characterized by the highest values (2.49 and 2.23 µg/kg) of afb1 and ota, respectively. the values obtained for afb1 contents in this study are lower than those reported in other areas of the world (nigeria: 20 to 455 µg/kg; mali: 4 to 35.19; kenya: 0 to 2377.1; mozambique: 3 to 5500 µg/kg; pakistan: 24 to 800 µg/kg; and brazil: 5 to 22500 µg/kg) [27,20,21]. except the communes of attécoubé, abobo and adjamé, afb1 contents are lower than the maximum residue limit (2 µg/kg) allowed in food for human consumption [7]. these relatively lowest values of afb1 contents could be explained by the manufacturing processing of peanut butter including roasting step. thus, positive correlations were reported between loss of aflatoxins in the peanut seeds and the roasting conditions [28]. 0 5 10 15 20 25 ab ad at c k m p t y a. parasiticus a. flavus a. ochraceus a. versicolor contamination level (%) ab: abobo; ad: adjamé; at: attécoubé; c: cocody; k: koumassi; m: marcory; p: portbouet; t: treichville; y: yopougon. c om m un es food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 272 indeed, seeds dry-roasted at 140°c for 40 min resulted in 58.8 % reductions in afb1. those roasted at 150°c for 25 min resulted in 68.5 % reductions in afb1. roasting at 150°c for 30 min led to 70.0% reduction in afb1. in addition the role of processing on potential reduction of toxin in peanut butter has been underlined [29]. however, there was a negative correlation between the incidence of aspergillus spp. and the afb1 level per commune. indeed, a. flavus was not detected in peanut butters collected from attécoubé while the occurrence level of a. parasiticus was 5.98% (fig 1). moreover, the mycotoxins produced by aspergillus spp. are of greatest significance in peanuts and peanut products include aflatoxins and ochratoxin a (ota) [30]. in this study, the amounts (0.53 2.23 µg/kg) of ota detected in peanut butters are lower than the limit value (3 µg/kg) recommended by european union [7]. however consumers must pay attention to the excessive consumption of marketed peanut butters in order to avoid immunosuppressive, teratogenic, genotoxic and mutagenic effects on human [5]. table 2 aflatoxin b1 and ochratoxin a levels in peanut butter samples sold in retail markets of abidjan. communes afb1 (µg/kg) ota (µg/kg) > lod value > lod value ab (n = 5) 5 2.02a ± 0.46 5 2.13a ± 0.32 ad (n = 5) 5 1.61b ± 0.33 5 2.23a ± 0.55 at (n = 5) 5 2.49a ± 0.98 5 2.12a ± 0.25 c (n = 5) 4 1.91b ± 0.00 5 1.55b ± 0.55 k (n = 5) 4 0.93d ± 0.07 5 1.92b ± 0.18 m (n = 5) 5 1.01c ± 0.08 5 1.76c ± 0.90 p (n = 5) 3 1.08c ± 0.08 4 0.53e ± 0.05 t (n = 5) 2 0.23e ± 0.08 5 0.76d ± 0.05 y (n = 5) 5 2.20a ± 0.80 5 1.74c ± 0.41 data are represented as means ± sd (n=3). mean with different letters in the same column are statistically different (p < 0.05) according to duncan’s test. ab: abobo, ad: adjamé, at: attécoubé, c: cocody, k: koumassi, m: marcory, p: portbouet, t: treichville, y: yopougon. lod: limit of detection: 0.05 µg/g. as observed for afb1, there was a negative correlation between the incidence of a. ocharceus and the ota level per commune. the coexistence of afb1 and ota in the studied peanut butters should be taken into consideration as claimed by the european community [31]. this is particularly important in regard to possible synergism and additive effects of these mycotoxins. such co-contamination has been previously observed with other food samples such as wheat or olives [32,33]. therefore, special attention should be paid to certain critical points in the chain of processing, including sorting. furthermore, sorting can remove a major part of aflatoxin contaminated units, but levels of mycotoxins in contaminated commodities may also be reduced through food processing procedures that may involve washing, wet and dry milling, grain cleaning, dehulling, roasting, baking, frying, and extrusion cooking [34]. these methods and their impact on mycotoxin reduction were previously reviewed [35]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 273 figure 2: retention times of aflatoxin b1 (afb1) and ochratoxin a (ota) detected in peanut butter samples sold in retail markets of abidjan. 4. conclusions peanuts butters sold in abidjan are generally prepared in a traditional way and under conditions of unhealthy hygiene. the conditions of production and storage of these peanut butters and especially their exposure to the air and packaging used constitute potential sources of contamination by mycotoxigenic fungi. indeed, the presence of toxigenic fungi as aspergillus flavus, aspergillus ochraceus and aspergillus parasiticus and the detection of afb1 and ota highlight a potential public health problem concerning their consumption. therefore, the need to educate both vendors and consumers on processing, food handling procedures and personal hygiene would help to minimize fungal contamination in order to obtain good sanitary peanut butters. 5. references [1]. sauer d.b., r.a. meronuck r.a., christensen c.m. microflora. in: sauer, d.b. (ed.). storage of cereal grains and their products . american association of cereal chemists, st. paul, mn. (1992). [2]. bankole s.s., adebanjo a. mycotoxins in food in west africa: current situation and possibilities of controlling it. afr. j. biotechnol. 2. p. 254-263. (2003). 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min 0 250 500 mv detector a:ex:350nm,em:450nm b -1 b -2 g -1 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min 0.0 2.5 5.0 7.5 10.0 12.5 mv o t a afb1 ota food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 274 [3]. wu f. mycotoxin risk assessment for the purpose of setting international regulatory standards. env. sci. technol. 38. p. 4049-4055. (2004). [4]. bhat r.v., vashanti s. occurrence of aflatoxins and its economic impact on human nutrition and animal feed. agric. develop. 23. p. 50-56. (1999). [5]. iarc. some naturally occurring substances: food items and constituents, heterocyclic amines and mycotoxins. iarc monographs on evaluation of carcinogenic risk to humans, lyon, france. international agency for research on cancer 56. (1993). [6]. bradburn n., blunden g., coker r.d., jewers k. aflatoxin contamination of maize. trop. sci. 33: 418-428. (1993). [7]. fao. food and agricultural organisation. worldwide regulations for mycotoxins. fao food and nutrition paper 81. fao, rome. (2003). [8]. who. ochratoxin a-toxicological evaluation of certain food additives series. who, geneve, pp. 363-376. (1996). [9]. marasas w.f.o. fumonisins: their implications for human and animal health. nat. toxins. 3. p. 193-198. (1995). [10]. howard p.c., eppley r.m., stack m.e., warbritton a., voss k.a., lorentzen r.j., kovach r.m., bucci t.j. fumonisin b1 carcinigenicity in a two year feeding study using f344 rats and b6c3f1 mice. environ. health perspect. 109. p. 277-282. (2001). [11]. fda. fumonisin levels in human foods and animal feeds. [available on line] (www.cfsan.fda.gov/ dms/fumonbg3.html); washington, dc. (2001). [12]. atanda s.a., pessu p.o., agoda s., isong i.u., adekalu o.a., echendu m.a., falade t.c. fungi and mycotoxins in stored foods. afr. j. microbiol. res. 5. p. 4373-4382. (2011). [13]. kamika i., takoy l.l. natural occurrence of aflatoxin b1 in peanut collected from kinshasa, democratic republic of congo. food control. 22. p. 1760-1764. (2011). [14]. mutegi c.k., ngugi h.k., hendriks s.l., jones r.b. factors associated with the incidence of aspergillus section flavi and aflatoxin contamination of peanuts in the busia and homa bay districts of western kenya. plant pathol. 61. p. 1143 – 1153. (2012). [15]. campos-mondragón m.g., calderón a.m., durán-prado a., campos-reyes l.c., oliart-ros r.m., ortega-garcía j., medina-juárez l.a., angulo o. nutritional composition of new peanut (arachis hypogaea l.) cultivars. grasas y aceites. 60. p. 161 – 167. (2009). [16]. pitt j.i., hocking a.d., samson r.a., king a.d. recommended methods for mycological examination of foods. in: modern methods in food mycology. samson, r.a., hocking, a.d., pitt, j.i. and king, a.d. (eds.). elsevier, amsterdam, netherlands. p. 365-368. (1992). [17]. singh k., frisvad j.c., thrane u., mathu s.b. an illustrated manual on identification of some seed borne aspergilli, fusaria, penicillia and their mycotoxins. heller up, denmark: danish government, institute of seed pathology for developing countries. (1991). [18]. aoac. aflatoxins in corn, raw peanuts and peanut butter: immunoaffinity column (aflatest) method. aoac international. (2005). [19]. adjou s.e., yehouenou b., sossou c.m., soumanou m.m., souza c.a. occurrence of mycotoxins and associated mycoflora in peanut cake product (kulikuli) marketed in benin. afr. j. biotechnol. 11. p. 1435414360. (2012). [20]. keita c., babanaa a.h., traoré d.f., samaké a.h., dickoa f.a., faradjia k., maïga a. evaluation of the sanitary quality of peanut butters from mali: identification and quantification of aflatoxins and pathogens. sci. j. microbiol. 2. p. 150-157. (2013). [21]. ndung’u j.w., makokha a.o, onyango c.a., mutegi c.k., wagacha j.m., christie m.e. wanjoya a.k. prevalence and potential for aflatoxin contamination in groundnuts and peanut butter from farmers and traders in nairobi and nyanza provinces of kenya. j. appl. biosci. 65. p. 4922 – 4934. (2013). [22]. passone m.a., rosso l.c., ciancio a., etcheverry m. detection and quantification of aspergillus section flavi spp. in stored peanuts by real-time pcr of nor-1 gene, and effects of storage conditions on aflatoxin production. int. j. food microbiol. 138. p. 276 – 281. (2010). [23]. antonio l., antonio b., giuseppina m., antonio m., giancarlo p. epidemiology of toxigenic fungi and their associated mycotoxins for some mediterranean crops. eur. j. plant pathol. 109. p. 645 – 667. (2003). [24]. kane a., ba-diop n., diack t.s., sembene m., delobel a., guiro a.t. relationship between the condition of home-stored peanuts and aflatoxin b1 contamination. in mycotoxins and phycotoxins: advances in determination, toxicology, and exposure management, wageningen academic publishers. (2006). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon , fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire), food and environment safety, volume xiii, issue 3 – 2014, pag. 267 – 275 275 [25]. williams j.h., phillips t.d., jolly p.e., stiles j.k., jolly c.m., aggarwal d. human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. am. j. clin. nutr. 80. p. 1106–1122. (2004). [26]. bellí n., pardo e., marín s., farré g., ramos a.j., sanchis v. occurrence of ochratoxin a and toxigenic potential of fungal isolates from spanish grapes. j. sci. food agric. 84. p. 541-546. (2004). [27]. hüni k., schneider j., zanetti g., rihs t. survey of aflatoxin content in swiss concentrate for dairy cattle. j. environ. pathol. toxicol. oncol. 10. p. 160-161 (1990).[28]. ogunsanwo b.m., faboya o.o., idowu o.r., lawal o.s., bankole s.a. effect of roasting on the aflatoxin contents of nigerian peanut seeds. afr. j. biotechnol. 3. p. 451-455. (2004). [29]. siwela a.h., mukaro k.j., nziramasanga n. aflatoxin carryover during large scale peanut butter production. food nutr. sci. 2. p. 105-108. (2011). [30]. pittet a. natural occurrence of mycotoxins in foods and feeds: an updated review. rev. med. vet. 149. p. 479-492. (1998). [31]. cec. commission regulation (ec) no. 1525/98. off. j. eur. commun., l20/143, 17th july. (1998). [32]. vrabcheva t., usleber e., dietric r., martlbauer e. co-occurrence of ochratoxin a and citrinin in cereals from bulgaria villages with a history of balkan endemic nephropathy. j. agric. food chem. 48. p. 2483-2488. (2000). [33]. el-adlouni c., tozlovanu m., natman f., faid m., pfohl-leszkowicz a. preliminary data on the presence of mycotoxins (ochratoxin a, citrinin and aflatoxin b1) in black table olives ‘‘greek style” of moroccan origin. mol. nutr. food res. 50. p. 507–512. (2006). [34]. hell k., mutegi c., fandohan p. aflatoxin control and prevention strategies in maize for sub-saharan africa. 10th international working conference on stored product. protection. julius-kühn-archiv. 425. p. 534-541. (2010). [35]. fandohan p., hell k., marasas w.f. food processing to reduce mycotoxins in africa. preand postharvest management of aflatoxin in maize. in: leslie, j.f., bandyopadhyay, r., visconti, a., (eds) mycotoxins: detection methods, management, public health and agricultural trade. cabi publishing, wallingford, uk. p. 302-309. (2008). microsoft word 2 content vol 2010 nr 1 din 26 aug.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 contents: 1. doina mihăilă, the quality of the siret river in siret section similarities and differences of the results obtained for the samples collected in comon by the romanian and ukrainian 5 2. micşunica rusu , rodica segal, improving the antioxidizing capacity of catering products by adding spices. 13 3. sergey boruk, igor winkler, sonia gutt, interparticle interaction in the diluted aqueous suspensions of hydro-mica 19 4. traian zaharescu, silviu jipa, qualification of polypropylene/calcium carbonate nanocomposites to radiation degradation. 25 5. maria poroch-seriţan, gheorghe gutt, traian severin, silviugabriel stroe, synthesis and characterization of electrodeposited ni–w alloys with different levels of tungsten content. 31 6. rodica rotar, study on identifying butter faking by substitution with pork fat or with margarine. 40 7. marcel avramiuc, the influence of refrigeration time and of sugar addition on ascorbic acid content in some natural juices. 46 8. mircea-adrian oroian, , gheorghe gutt, effect of potato starch and agar on the rheological behaviour of tomato ketchup. 50 9. cristina-elena hreţcanu, ciprian-ionel hreţcanu, a linear programming model for a diet problem. 56 10. violeta vasilache, studies regarding the efficiency of a direct methanol fuel cell (dmfc). 64 11. sonia gutt , georg gutt, mariana mazareanu, study on the content of zearalenone from wheat and derivatives. 68 12. danela murariu, m. murariu, i. gontariu, „on farm” conservation of some maize races in bucovina. 73 13. cristina-elena hreţcanu, alice roşu, silviu-gabriel stroe, ana leahu, some aspects regarding the diversity of food culture and its influence on consumers’ attitudes 79 14. author instructions 85 microsoft word 4 denkova_corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 22 identification and examination of some probiotic properties of lactobacillus plantarum f3 rositsa denkova1, velichka yanakieva2, *zapryana denkova2, zoltan urshev3, bogdan goranov2, elena sotirova2 1department of biotechnology, sofia university „st. kliment ohridski”, rositsa_denkova@mail.bg 2department of microbiology, university of food technologies, zdenkova@abv.bg 3dna analyses lab, lb bulgaricum *corresponding author received 16 november 2012, accepted 10 december 2012 abstract: in order to be included in the composition of probiotic preparations each strain has to meet a number of requirements. the strain lactobacillus f3 from naturally fermented sourdough is identified as a lactobacillus plantarum strain using molecular-genetic methods (ardra and 16s rdna sequencing). some of its probiotic properties are examined: ability for industrial cultivation and survival in the model conditions of the gastro-intestinal tract. high concentrations of active cells are retained during cultivation at ph=2 + pepsin, ph=4,5 +pancreatin and ph=7 + pancreatin as well as at different concentrations of bile salts – 0,15%, 0,3%, 0,6%, 1%. the strain allows industrial cultivation with accumulation of high concentrations of viable cells. the results of the studies on some probiotic properties of lactobacillus plantarum f3 make the strain a potentially probiotic one. keywords: ardra, sequencing, probiotic, batch cultivation, pepsin, pancreatin, bile salts 1. introduction a number of factors influence negatively the interaction between intestinal microorganisms, such as stress and diet. unfortunately they lead to detrimental effects on human health. there is increasing evidence indicating that consumption of ‘probiotic’ microorganisms helps maintaining a favourable microbial profile as results of which several therapeutic benefits are observed [7]. probiotics are live microorganisms that confer a beneficial effect on the host when administered in proper amounts [4]. the beneficial effects of probiotic preparations on gastrointestinal infections, the protection of the immune system, the reduction of serum cholesterol, the improvement in inflammatory bowel disease and suppression of helicobacter pylori infection, crohn's disease, restoration of the microflora in the stomach and the intestines after antibiotic treatment; they are also characterized by anti-cancer properties, antimutagenic action, antidiarrheal properties are well known [8]. lactobacilli and bifidobacteria are a natural part of the intestinal microflora of the healthy human. they are included in the composition of probiotics and probiotic foods because of their proven health benefits to the body [6]. but not all strains of lactobacilli and bifidobacteria can be used as components of probiotics and probiotic foods, but only those that exhibit certain properties. probiotic microorganisms should be of human origin, resistant to gastric acid, bile and to the antibiotics, administered in medical practice, non-pathogenic; they should also have the potential to adhere to the gut epithelial tissue and produce antimicrobial food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 23 substances; they should allow the conduction of technological processes, in which high concentrations of viable cells are obtained as well as to allow industrial cultivation, encapsulation and freezedrying and they should remain active during storage [5]. this leads to the mandatory selection of strains of the genera lactobacillus and bifidobacterium with probiotic properties. the purpose of this paper is to identify the strain lactobacillus f3, isolated from naturally fermented sourdough, and to examine some of its technological properties – survival in the model conditions of the gastrointestinal tract and ability for industrial cultivation. 2. experimental 2.1. microorganisms the studied lactobacillus strain, lactobacillus f3, is isolated from naturally fermented sourdough. reference microorganisms: lactobacillus acidophilus dsm 20079, lactobacillus delbrueckii ssp.bulgaricus dsm 20081, lactobacillus casei ssp.casei dsm 20011, lactobacillus casei ssp.paracasei dsm 20312, lactobacillus casei ssp.rhamnosus lmg 6400, lactobacillus fermentum dsm 20052, lactobacillus helveticus dsm 20075, lactobacillus plantarum dsm 20174. 2.2. media saline solution. composition (g/dm3): nacl 5. sterilization 20 minutes at 121ºc. laptg10-broth medium. composition (g/dm3): peptone 15, yeast extract 10; tryptone 10, glucose 10. ph is adjusted to 6.6 6.8 and tween 80 1cm3/dm3 is added. sterilization 20 minutes at 121ºc. laptg10-agar. composition (g/dm3): laptg10-broth medium and 2% agar. sterilization 20 minutes at 121ºc. mrs – broth medium (scharlau) 2.3.identification isolation of total dna the isolation of dna is performed by the method of delley et al. [2]. pcr reactions and visualization all pcr reactions are performed using the pcr kit ready to gotm pcr beads (amersham biosciences), in a volume of 25 µl in a progene cycler (techne, uk). the resulting products are visualized on a 2% agarose gel stained with ethidium bromide solution (0.5 µg/ml), using an uvp documentation system (uk). 16s rdna amplification and 16s rdna ardra (amplified ribosomal dna restriction analysis) the method ardra involves enzymatic multiplication of the gene encoding the 16s rrna, using primers complementary to the conservative regions at both ends of the 16s rrna gene and the product of the multiplication is then restricted with restriction enzymes. the resulting profile is highly specific for the particular studied species. dna of the studied strain is amplified using universal primers for the 16s rdna gene fd1 and rd1 [9]. the amplification program includes: denaturation 95°c for 3 minutes, 40 cycles 93°c for 30 s, 48°c for 60 s, 72°c for 60 s, final elongation 72°c for 5 min. the resulting pcr product from the 16s rdna amplification of the tested strain is treated with the endonucleases eco ri, hae iii and alu i (boehringer mannhem gmbh, germany). reactions are carried out according to the following quantities: pcr products 10µl, enzyme solution 10 µl (1 µl of the respective enzyme, 2 µl buffer, 7 µl dh2o). incubation for 1 night at 37°c is performed. the resulting restriction products are visualized on a 2% agarose gel. 2.4. purification of the product of the pcr-reaction – 16s rdna – from taeagarose gel food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 24 the purification of 16s rdna is conducted using dna-purification kit (gfx microspintm) according to the manufacturer’s instructions: 1) sample capture. after visualizing the product of the 16s pcr-amplification reaction on a 2% agarose gel with uv light with wavelength 302 nm, the gel is visualized with uv light with wavelength 365 nm. the 16s pcr product is cut from the gel and placed in a dna-free microcentrofuge tube. through weighing the microcentrofuge tube before and after the gel fragments are put in them, the weight of the fragments is calculated and 10µl capture buffer is added to every 10mg of the gel. the microcentrofuge tube are mixed gently and incubated at 60°c for about 20 minutes until the full dissolution of the gel fragments. 2) sample binding a gfx microspintm column is labelled and placed in a collection tube and the centrofuged (shortspin) samples in the eppendorf tubes from 1) are poured in the gfx microspintm columns (no more than 600µl). the gfx microspintm columns are allowed to wet for about 60 seconds and centrofuged until the whole volume passes through the column. the liquid from the column is disposed and the gfx microspintm column is placed in the same collection tube. if a sample is more than 600µl, all the steps from the sample binding are repeated until the whole sample is eluated. 3) wash and dry 500 µl of wash buffer type 1 are poured in each gfx microspintm column, the columns are centrofuged (shortspin), the collection tubes are disposed and each gfx microspintm column is placed in a new 1,5 ml dnaase free microcentrofuge tube. 4) elution 10-50µl elution buffer type 4 or type 6 are poured in each gfx microspintm column. the column is allowed to wet at room temperature for 60 seconds and the microcentrofuge tubes with the gfx microspintm columns are centrofuged for about 60 seconds. the eluate (containing purified 16s rdna) is collected and freezed at -20°c. 2.5. dna-sequencing sequencing of the gene encoding the 16s rrna is performed by „macrogen europe laboratory”, the netherlands using the sanger method for dna-sequencing. 2.6. determination of the resistance to low ph in the presence of pepsin and to weakly alkaline ph in the presence of pancreatin [1] fresh 24 hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. the resulting sludge biomass is washed twice with pbs buffer and resuspended to the initial volume in pbs buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 buffer solution with ph = 2 containing 0,5% nacl and pepsin (at a concentration of 3.2 g/dm3) (sigma, 2,500 3,500 u / mg protein), buffer with ph = 4,5 + pancreatin and buffer with ph = 7 + pancreatin at a suitable temperature for the studied strain (37°c) for 24h. at the 0, the 2nd, the 4th and the 24th hour aliquots for the determination of the number of viable cells are taken (cfu/cm3). 2.8. determining the tolerance to bile salts [3] fresh 24 hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. the resulting sludge biomass is washed twice with pbs buffer and resuspended to the initial volume in pbs buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 of the mrs-broth medium with different concentrations of bile salts 0%, 0.15%, 0.3%, 0.6% and 1% for 24h at the optimum temperature for the strain (37°c), and aliquots for the determination of the number of viable cells (cfu/cm3) at the 0, the 2nd, the 4th, the 6th, the 8th and the 24th hour are taken. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 25 2.9. batch cultivation in a bioreactor with continuous stirring and in a thermostat at static conditions the laboratory cultural vessel (fig.1) is a cylinder with geometric volume of 2 dm3 and displacement – 1,5 dm3. figure 1. scheme of the laboratory bioreactor 1 vessel with geometric volume of 2 dm3; 2four repulse devises; 3–thermo-strength pt100; 4–heater; 5-heat exchanger for cold water ; 6– turbine stirrer; 7–ph electrode; 8–exit for co2; 9–filter; 10–peristaltic pump for ph correction; 11– reagent for ph correction – 20% koh; 12– motor; 13-control links; 14–control device "applikon". the periodic cultivation processes are conducted in mrs-broth without ph adjustment. the medium is sterilized at 118ºc for 15 min. after cooling to 39-40ºc the prepared medium in the bioreactor (mrs-broth) is inoculated with 5% (v/v) inoculum. the process of cultuvation is conducted at 37ºc, stirring speed of 100 rpm, without air supply. during the cultivation ph, eh, number of colonyforming units and tirable acidity are examined. along with the carried out periodical cultivation with constant stirring (in a bioreactor), static cultivation (in an incubator) under the same conditions is carried out as well. the number of viable cells of lactobacillus plantarum f3 is determined through appropriate tenfold dillusions of the samples and plating on coloured laptg10 – agar medium. the petri dishes are cultivated for 72 hours at 37°c until single colonies can be counted. the titratable acidity is determined using 0,1n naoh. 5 cm3 of each sample are mixed with 10 cm3dh2o and titrated with 0,1n naoh, using phenolphtalein as an indicator, until the appearance of pale pink colour, which retains for 1 minute. the value for the titratable acidity is obtained by multiplying the millilitres 0,1n naoh by the factor of the 0,1n naoh and the number 20. 3. results and discussion the strain lactobacillus f3 is isolated from naturally fermented sourdough. identification of lactobacillus f3 the identification of lactobacillus f3 is performed using ardra analysis, followed by sequencing of the gene encoding the 16s rrna. ardra analysis. as a result of the ardra analysis with the enzymes eco ri (fig.1), hae iii (fig. 2) and alu i (fig. 3) the studied strain is determined to be a representative of the species lactobacillus plantarum. dna-sequencing of lactobacillus f3 is conducted by macrogen europe laboratory, the netherlands by the method of chain termination (method of sanger). after careful comparison of the obtained sequence with the public online nucleotide blast database, the strain lactobacillus f3 is confirmed to be a lactobacillus plantarum strain (fig. 4). cooling water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 26 fig. 1. restriction profile of the 16s rdna with ecori 1. lactobacillus f3 2. lactobacillus acidophilus dsm 20079 3. lactobacillus delbrueckii ssp.bulgaricus dsm 20081 4. lactobacillus casei ssp.casei dsm 20011 5. lactobacillus casei ssp.paracasei 6. lactobacillus casei ssp.rhamnosus 7. lactobacillus fermentum dsm 20052 8. lactobacillus helveticus dsm 20075 9. lactobacillus plantarum dsm 20174 10. m fig. 2. restriction profile of the 16s rdna with haeiii 1. lactobacillus f3 2. lactobacillus acidophilus dsm 20079 3. lactobacillus delbrueckii ssp.bulgaricus dsm 20081 4. lactobacillus casei ssp.casei dsm 20011 5. lactobacillus casei ssp.paracasei 6. lactobacillus casei ssp.rhamnosus 7. lactobacillus fermentum dsm 20052 8. lactobacillus helveticus dsm 20075 9. lactobacillus plantarum dsm 20174 10. m fig. 3. restriction profile of the 16s rdna with alu i 1. lactobacillus f3 2. lactobacillus acidophilus dsm 20079 3. lactobacillus delbrueckii ssp.bulgaricus dsm 20081 4. lactobacillus casei ssp.casei dsm 20011 5. lactobacillus casei ssp.paracasei 6. lactobacillus casei ssp.rhamnosus 7. lactobacillus fermentum dsm 20052 8. lactobacillus helveticus dsm 20075 9. lactobacillus plantarum dsm 20174 10. m 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 27 > ref|nr_042394.1| lactobacillus plantarum strain nrrl b-14768 16s ribosomal rna, partial sequence length=1474 score = 1977 bits (1070), expect = 0.0 identities = 1072/1073 (99%), gaps = 0/1073 (0%) strand=plus/minus query 11 gtccaccttaggcggctggttcctaaaaggttaccccaccgactttgggtgttacaaact 70 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1467 gtccaccttaggcggctggttcctaaaaggttaccccaccgactttgggtgttacaaact 1408 query 71 ctcatggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctga 130 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1407 ctcatggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctga 1348 query 131 tccgcgattactagcgattccgacttcatgtaggcgagttgcagcctacaatccgaactg 190 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1347 tccgcgattactagcgattccgacttcatgtaggcgagttgcagcctacaatccgaactg 1288 query 191 agaatggctttaagagattagcttactctcgcgagttcgcaactcgttgtaccatccatt 250 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1287 agaatggctttaagagattagcttactctcgcgagttcgcaactcgttgtaccatccatt 1228 query 251 gtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttc 310 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1227 gtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttc 1168 query 311 ctccggtttgtcaccggcagtctcaccagagtgcccaacttaatgctggcaactgataat 370 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1167 ctccggtttgtcaccggcagtctcaccagagtgcccaacttaatgctggcaactgataat 1108 query 371 aagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaacc 430 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1107 aagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaacc 1048 query 431 atgcaccacctgtatccatgtccccgaagggaacgtctaatctcttagatttgcatagta 490 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 1047 atgcaccacctgtatccatgtccccgaagggaacgtctaatctcttagatttgcatagta 988 query 491 tgtcaagacctggtaaggttcttcgcgtagcttcgaattaaaccacatgctccaccgctt 550 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 987 tgtcaagacctggtaaggttcttcgcgtagcttcgaattaaaccacatgctccaccgctt 928 query 551 gtgcgggcccccgtcaattcctttgagtttcagccttgcggccgtactccccaggcggaa 610 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 927 gtgcgggcccccgtcaattcctttgagtttcagccttgcggccgtactccccaggcggaa 868 query 611 tgcttaatgcgttagctgcagcactgaagggcggaaaccctccaacacttagcattcatc 670 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 867 tgcttaatgcgttagctgcagcactgaagggcggaaaccctccaacacttagcattcatc 808 query 671 gtttacggtatggactaccagggtatctaatcctgtttgctacccatactttcgagcctc 730 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 807 gtttacggtatggactaccagggtatctaatcctgtttgctacccatactttcgagcctc 748 query 731 agcgtcagttacagaccagacagccgccttcgccactggtgttcttccatatatctacgc 790 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 747 agcgtcagttacagaccagacagccgccttcgccactggtgttcttccatatatctacgc 688 query 791 atttcaccgctacacatggagttccactgtcctcttctgcactcaagtttcccagtttcc 850 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 687 atttcaccgctacacatggagttccactgtcctcttctgcactcaagtttcccagtttcc 628 query 851 gatgcacttcttcggttgagccgaaggctttcacatcagacttaaaaaaccgcctgcgct 910 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 627 gatgcacttcttcggttgagccgaaggctttcacatcagacttaaaaaaccgcctgcgct 568 query 911 cgctttacgcccaataaatccggacaacgcttgccacctacgtattaccgcggctgctgg 970 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 567 cgctttacgcccaataaatccggacaacgcttgccacctacgtattaccgcggctgctgg 508 query 971 cacgtagttagccgtggctttctggttaaataccgtcaatacctgaacagttactctcag 1030 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| sbjct 507 cacgtagttagccgtggctttctggttaaataccgtcaatacctgaacagttactctcag 448 query 1031 atatgttcttctttaacaacagagttttacgaaccgaaacccttcttcactca 1083 |||||||||||||||||||||||||||||||| |||||||||||||||||||| sbjct 447 atatgttcttctttaacaacagagttttacgagccgaaacccttcttcactca 395 figure 4. comparison of the nucleotide sequences of the 16s rdna of lactobacillus f3 and the partial sequence of the 16s rdna of lactobacillus plantarum nrrl b-14768. probiotic properties of lactobacillus plantarum f3 survival in the model conditions of the gastrointestinal tract the resistance of the cells of lactobacillus plantarum f3 in the model conditions of the gastro intestinal tract is examined: survival at ph = 2 + pepsin, at ph = 4,5 + pancreatin and at ph = 7 + pancreatin. the results of the experimental studies are presented on fig. 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 28 figure 5. survival of the cells of the strain lactobacillus plantarum f3 at ph = 2 + pepsin, at ph = 4,5 + pancreatin and at ph = 7 + pancreatin. it is observed that the sensitivity of lactobacillus plantarum f3 to ph = 2 + pepsin, ph = 4,5 + pancreatin and ph = 7 + pancreatin is comparable – the reduction of the number of viable cells is about 4logn (fig. 5) by the 24th hour of cultivation. but the concentration of active cells even by the 24th hour remains high – 1,6x106cfu/cm3 at ph = 2 + pepsin, 3x105cfu/cm3 at ph = 4,5 + pancreatin, 1,1x105cfu/cm3 at ph = 7 + pancreatin, which makes the strain appropriate for incorporation in probiotics. another factor of great importance that influences the survival of probiotic strains in the gastrointestinal tract are bile salts. about three hours after ingestion of food the concentration of bile salts in the small intestine reaches about 0.3%. this requires study of the influence of different concentrations of bile salts on the survival of lactobacillus plantarum f3 in mrsbroth medium with different concentrations of bile salts, 0%, 0.15%, 0.3%, 0.6% and 1% for 24 hours of incubation. the number of viable cells of lactobacillus plantarum f3 starts decreasing since the beginning of the cultivation of the strain in mrs-broth medium with different concentrations of bile salts (fig. 6). the degree of reduction is different at the different concentrations of bile salts – it is greater at concentrations 0,6% and 1% and smaller at 0,15% and 0,3% bile salts. at 0,15% bile salts the reduction is 1,3logn and at 0,3% it is 1logn. at 0,6% bile salts the degree of reduction is considerably higher – 3logn and at 1% bile salts it is about 3,5logn. figure 6. survival of the cells of lactobacillus plantarum f3 at different concentrations of bile salts. but by the end of the experiment the concentration of viable cells remains between 1,9x105 cfu/cm3 (at 1% bile salts) and 1x107cfu/cm3 (at 0,15% bile salts), which allows the inclusion of lactobacillus plantarum f3 in the composition of probiotic preparations. batch cultivation in a bioreactor with continuous stirring and at static conditions of lactobacillus plantarum f3 the strain lactobacillus plantarum f3 is cultivated in mrs-broth at 37ºc in a laboratory bioreactor with continuous stirring and in a thermostat. it is observed that the time to reach high concentration of viable cells during cultivation in the bioreactor with continuous stirring is reduced in comparison to cultivation at static conditions (fig. 7, fig. 8). at the 6th hour the number of cells reaches 8,3x1010cfu/cm3 (fig. 7), while under static conditions, the same concentration of cells is reached at the 12th hour from the beginning of the process (fig. 8). the number of active cells of lactobacillus plantarum f3 obtained in cultivation in a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 29 bioreactor with continuous stirring and at static conditions by the 24th hour is comparable – 3x1012cfu/cm3 in the bioreactor and 7,8x1012cfu/cm3 at static conditions. the titratable acidity of the medium in the bioreactor increases from 46,2°t to 241,7°t, while at static conditions it reaches 240,1°t by the 24th hour and 248,3°t by the 48th hour. 0 6 12 18 24 8 9 10 11 12 13 0 6 12 18 24 40 80 120 160 200 240 280 0 6 12 18 24 520 560 600 640 680 720 0 6 12 18 24 3,2 3,6 4,0 4,4 4,8 5,2 5,6 log n lo g n time, h tk, ot t k , o t eh, mv e h, m v ph p h figure 7. batch cultivation of lactobacillus plantarum f3 in mrs-broth in a bioreactor with constant stirring. 0 6 12 18 24 30 36 42 48 8 9 10 11 12 13 0 6 12 18 24 30 36 42 48 40 60 80 100 120 140 160 180 200 220 240 260 log n lo g n time, h tk, ot t k , 0 t figure 8. static cultivation of lactobacillus plantarum f3 in mrs-broth the redox potential of the system starts increasing since the beginning of the batch process. it starts from +526mv and reaches +716 mv (fig. 7). the strain lactobacillus plantarum f3 allows industrial cultivation with accumulation of high concentrations of viable cells. 4. conclusion the strain lactobacillus f3 is identified as belonging to the species lactobacillus plantarum. lactobacillus plantarum f3 has the ability to survive in the model conditions of the gastro intestinal tract and allows industrial cultivation with accumulation of high concentrations of viable cells. thus, it can be defined as a potential probiotic culture, which after further research can be incorporated in the composition of probiotic preparations for treatment and prevention. 5. references [1]. charteris w., kelly p., morelli l., collins j., development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and bifidobacterium species in the upper human gastrointestinal tract. j.of appl.micr. 84 (5), pp. 759–768, 1998. [2]. delley m., mollet b., hottinger h., dna probe for lactobacillus delbrueckii. appl. environ. microbiol. 56:1967–1970, 1990 [3]. denkova z., production and application of probiotics, d.sc. thesis, 2005 [4]. kalliomaki m., salminen s., arvilommi h., kero p., koskinen p., isolauri e., probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial, lancet 357. 1076–1079, 2001 [5]. kirtzalidou e., pramateftaki p., kotsou m., kyriacou a., screening for lactobacilli with probiotic properties in the infant gut microflora, anaerobe 17. 440 – 443, 2011 [6]. marteau p., de vrese m., cellier c., schrezenmeir j. protection from gastrointestinal diseases with the use of probiotics. am. j.of clin. nutr.73 (suppl.2). 430s–436s, 2001 [7]. rybka s., kailasapathy k., the survival of culture bacteria in fresh and freeze-dried ab yoghurts, the aust. j. of dairy technology 50(2). 51–57, 1995 [8]. shah n., functional cultures and health benefits, int. dairy j. 17. 1262–1277, 2007 [9]. weisburg w., barns s., pelletier d., lane d., 16s ribosomal dna amplification for phylogenetic study. j. bacteriol.173. 697-703, 1991 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiv, issue 3 30 september 2015 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava http://www.fia.usv.ro/fiajournal editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:fiajournal@fia.usv.ro mailto:gutts@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 13 incorporation of sweet pepper extracts to improve thermal stability of vegetable oil mixtures *tatiana capcanari 1 1technical university of moldova, 168 stefan cel mare ave., md-2004 chisinau, republic of moldova, e-mail: tatiana_capcanari@mail.md *corresponding author received 7 april 2011, accepted 5 may 2011 abstract: sweet pepper is known not only for taste, aroma and flavor, but also for antioxidant effects and properties. sweet pepper contains phenolic substances such as phenolic acids, flavonoids, phenolic diterpenes and volatile oils. phenolic compounds in these vegetable materials are closely associated with their antioxidant activity and also play an important role in stabilizing lipid peroxidation. efficacy of sweet pepper extracts in stabilizing sunflower and grape seeds oils in thermal oxidation conditions has been studied. extracts were prepared in water/ethanol and oil solutions, total polyphenol content (tpc) was in the range of 25.3-59.4 mg/ml for extracts on the basis of oil and in the range of 50.01-267.2 mg/ml for extracts on the basis of etoh/h2o and antioxidant activity range in the extracts was 20-74,29%. being highest in tpc and antioxidant potential of sweet pepper extracts were added to vegetable oil mixtures. obtained samples of oils with extracts were heated at 1600c. the oxidation of the oil samples was evaluated by means of free fatty acids content, peroxide and p-anisidine value. the results of uv/vis spectroscopy confirm stabilization effect on thermal oxidation of the tested oils. this study demonstrates that natural vegetable extract such as sweet peppers can effectively inhibit the lipid oxidation of sunflower and grape seeds oil mixture in thermal oxidation conditions. © 2011 university publishing house of suceava. all rights reserved keywords: total polyphenol content, scavenging activity dpph•, antioxidants, natural vegetable extracts , green and red sweet pepper, sunflower oil, grape seed oil, free fatty acid content, peroxide value, p-anisidine value, thermal stability. 1. introduction the current development trend of the food industry is to obtain natural antioxidants, extracted from the raw materials of vegetable origin [1]. later received natural antioxidants are used to purpose of obtaining a new food products resistant to oxidation processing’s during the storage. this new and promising direction in catering is specifically created to improve nutrition and health structure and to prevent disease spread in society [2]. vegetable oils and fats are recognized as important components of our diet. they provide essential fatty acids such as αlinolenic acid (ω−3 fatty acid) and linoleic acid (ω−6 fatty acid), fat-soluble vitamins: vitamin a (retinol), vitamin e (tocopherols), vitamin k (2-methyl-1,4naphthoquinone derivatives) and vitamin d (secosteroids) and other biologically active compounds [3, 4]. during storage and frying of oils, fats and fatty foods, lipid oxidation is one of the main causes of quality deterioration. it leads to losses of nutritional value of food as well as to changes in color, texture, sensory and other physiological properties [5, 6]. in order to retard or prevent the oxidative deterioration and extend the self-life of vegetable oils, the addition of antioxidants is necessary [7, 8]. in spite of high effectiveness of synthetic antioxidants such as tert-butylhydroquinon (tbhq), their food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 14 application is restricted in several countries because of their possible toxicity and carcinogenic effects [9-12]. due to these safety concerns, there is an increasing trend among food scientists to replace these synthetic antioxidants with natural ones, which in general, are supposed to be safer. it is known that sweet pepper is characterized by biological and nutritional value due to its contents of antioxidants, including ascorbic acid, tocopherol, βcarotene, flavonoids, and phenolic acids. the highest antioxidant activity is shown by flavonoids, because their molecules contain many hydroxyl groups, which are held by neutralizing free radicals by hydrogen separation [13, 14]. the purpose of the present work was to investigate the optimum conditions of drying for sweet peppers, for which the maximum safety of polyphenols is held, and according to high scavenging activity. further on the work studied the possibility of extracts obtained from dried pepper samples and their introduction into vegetable oil in order to give thermal oxidation stability. 2. materials and methods 2.1. materials the refined and deodorized sunflower and refined grape seed oils free from antioxidant additions were purchased from a local producer in the republic of moldova. the sweet peppers of red and green color were harvested in late june 2010 in the central part of moldova. the fresh and healthy vegetables were washed, cleaned, sliced, crushed to powder, dried and used in analyses. 2.2. chemicals 1,1-diphenyl-2-picrylhydrazyl (dpph•) as free radical form (90% purity), folinciocalteu’s phenolic reagent, sodium carbonate and gallic acid were supplied by sigma-aldrich. methanol (99,8%), ethanol (99,9%), chloroform, para-anithidine, izooctane, potassium hydroxide and phenolphthalein were provided by ecochemistry (kishinev, republic of moldova). all reagents were of analytical grade. distilled water was used throughout. 2.3. drying super-high frequency and convection drying were used for vegetable drying. the shf drying was performed at 30% and 50% of magnetic intensive. the convection drying was made at 600c and 800c. 2.4. extraction dried sweet peppers were ground before extraction. the dried powder of vegetables was extracted with 70% etoh/h2o and sunflower oil for 2 h at 600c and liquid-tosolid ratio 10 ml per gram. the extracts of tasted vegetables were filtered with peppers filter and then they were used in the experiments. the extracts obtained were analyzed for the scavenging activity dpph• [15] and total polyphenol content [16] assays. absorbance measurements were recorded on a uv/vis spectrophotometer hach-lange dr5000 (germany). 2.5. sample preparation we prepared the following oil samples: sunflower oil, grape seed oil, mixture of sunflower and grape seed oils, mixture of sunflower and grape seed oils with red and green peppers extract on the basis of oil / etoh/h2o. the content of vegetable extract was 1ml extract/100 ml oil in each oil sample. the sweet peppers extracts were added directly to oils, followed by slow stirring until complete dissolution. the oil samples obtained were further exposed to the thermal oxidation test. 2.6. thermal oxidation test the test for oxidation processes was performed on the oven spt – 200 vacuum drier (germany). the oil samples were transferred in beakers, containing 30 ml of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 15 sample. the temperature was set at 160 0c. heating was carried out continuously for 25 min. the amount of primary oxidation products was determined through measuring of peroxide value (pv) and acid value (av). these were determined according to aoac official method [17]. formation of secondary oxidation products was measured as p-anisidine value [18]. the oil samples exposed to the oxidation test were analyzed using uv/vis spectroscopy [19]. 2.7. statistical analysis experimental results were means ± sd (standard deviation) of three parallel measurements and processed statistically by the method of those small squares with application of coefficient student and determination of interval of investigation [20]. 3. results and discussion 3.1. total polyphenol content and antioxidant activity dpph of sweet peppers extracts the antioxidant activity expressed as the dpph values of sweet peppers extracts tested as well as the amount of total polyphenol content of these extracts are investigated and the results obtained are shown in figure 1-4. fig.1. comparison of dpph and folin-ciocalteu assays of red sweet peppers extract on the basis of oil fig. 2. comparison of dpph and folin-ciocalteu assays of green sweet peppers extract on the basis of oil fig. 3. comparison of dpph and folin-ciocalteu assays of red sweet peppers extract on the basis of etoh/h2o fig. 4. comparison of dpph and folin-ciocalteu assays of green sweet peppers extract on the basis of etoh/h2o food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 16 the total polyphenol content in sweet peppers extracts was in the range of 25.359.4 mg/ml for oil –based extracts and in the range of 50.01-267.2 mg/ml for etoh/h2obased extracts. the highest content of total polyphenols was in accordance with the results of antioxidant function determination found in extracts of sweet peppers pre-dried by shf with magnetic intensive 50%. the lowest total polyphenol content was recorded in sweet peppers extracts dried by convection at 60 0c and 80 0c corresponding to their low antioxidant function. using the dpph assay we obtained a hierarchy of antioxidant activity ranging from 74, 29% to 20%. interestingly, the highest antioxidant function was found in the extracts of vegetable samples dried by convection at the temperature of 80 0c and by super-high frequency (shf) drying with magnetic intensive 30%. after analyzing all the aspects and factors, particularly those responsible for maximum preservation of scavenging activity of polyphenol in vegetable matter, in further research we used the shf mode of drying of sweet peppers with magnetic intensive 30%. figures 5-8 show uv/vis spectra of experimental extracts of sweet peppers. it was established, that spectra of extracts is characterized by peaks at 325 nm, which is specific to polyphenol compounds. fig. 5. uv/vis spectra of red sweet peppers extract on the basis of etoh/h2o fig. 6. uv/vis spectra of red sweet peppers extract on the basis of oil fig. 7. uv/vis spectra of green sweet peppers extract on the basis of etoh/h2o fig. 8. uv/vis spectra of green sweet peppers extract on the basis of oil 3.2. oxidative stability of sunflower and grape seed oils with addition of extracts oxidative and hydrolytic decomposition is observed in the heating process of vegetable oils. the presence and depth of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 17 the process of oil oxidation and hydrolysis is characterized by the content of free fatty acids in oils, i.e., the acid value (av). the growth of the acid value or intensity in the formation of free fatty acids in the compared oils has a linear character (table 1). when comparing the acid values of the oil group with added sweet peppers extracts, one can notice that free fatty acids are accumulated to a lesser extent in a sample containing oil-based red sweet peppers extract as an antioxidizing component and that the acid values are 0,546 mg koh/g oil. table 1. quality indices of sunflower and grape seed oils with/without addition of extracts no name of oil samples quality indices of oils tested acid value [mg koh/g oil] peroxide value [meq/kg oil] p-anisidine value [c.u.] 20±20c 160±20c 20±20c 160±20c 20±20c 160±20c 1. refined and deodorized sunflower oil 0.183±0.002 0.756±0.003 8.22±0.1 20.43±0.3 0.5294±0.0005 1.2274±0.0007 2. refined grape seed oil 0.211±0.001 0.873±0.005 8.40±0.2 20.87±0.1 0.6441±0.0003 1.4814±0.0003 3. mixture of sunflower and grape seed oils 0.192±0.001 0.774±0.002 8.27±0.1 20.57±0.3 0.5764±0.0005 1.3207±0.0003 4. mixture of sunflower and grape seed oils with addition of etoh/h2o-based red sweet peppers extract 0.178±0.002 0.722±0.003 8.02±0.1 19.76±0.7 0.4767±0.0005 1.3324±0.0003 5. mixture of sunflower and grape seed oils with addition of oil-based red sweet peppers extract 0.159±0.005 0.546±0.002 7.82±0.3 19.75±0.9 0.4024±0.0003 1.3125±0.0009 6. mixture of sunflower and grape seed oils with addition of etoh/h2o-based green sweet peppers extract 0.177±0.001 0.917±0.005 7.92±0.2 19.95±0.5 0.4951±0.0005 1.3107±0.0008 7. mixture of sunflower and grape seed oils with addition of oil-based green sweet peppers extract 0.159±0.007 0.740±0.002 7.82±0.4 19.78±0.3 0.4327±0.0007 1.2796±0.0007 table 1 illustrates the intensity of the formation processes of the primary oxidation products (peroxides) in the oils tested depending on heating temperature. peroxide compounds are known to be unstable. they decompose in the heating process with the formation of the secondary products of vegetable oil oxidation, more stable carbonyl compounds. the change in the intensity of accumulating such aldehydes like 2, 4 decadienal and 2-octenal, is compared with vegetable oils’, being expressed by the amount of the p-anisidine value. the incorporation of sweet peppers extracts in the composition of oil mixture exerts an effective influence on the processes of stabilization of the oils tested. thus, the panisidine value of the oil mixture without extracts reached 1,4814 c. u. after heating process, and this value of oils added by parsley and lovage extracts decreased and varied from 1,2796 to 1,3324c.u. respectively. 4. conclusions the results of this study indicate that the antioxidant activity of vegetable extracts depends on various factors: vegetable species, method and conditions of drying. it was established that shf mode of drying sweet peppers with magnetic intensive 30% is the most optimal from the point of view of the maximum preservation of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 18 scavenging activity of polyphenol in vegetable matter. red sweet peppers extract were the most abundant source of polyphenol compounds and showed the highest value of antioxidant activity in the group of the extracts studied. the incorporation of natural vegetable extracts into the composition of mixtures of sunflower and grape seed oils exerts an effective influence on the processes of stabilization of the oils tested; for example, it inhibits the intensity of accumulating primary and secondary oxidation products during thermal oxidation process. 5. references 1. halsted., c.h., dietary supplements and functional foods: 2 sides of a coin. american journal of clinical nutrition, vol. 77, no. 4, 1001s-1007s. american society for clinical nutrition from the department of internal medicine and nutrition, university of california, davis, (april 2003). 2. halsted., c.h., functional foods: benefits, concerns and challenges—a position paper from the american council on science and health. department of food science and human nutrition and functional foods for health program, university of illinois, urbana, il 61801. j. nutr. 132: 3772–3781, (2002). 3. whitney e. ,rolfes. sr, understanding nutrition 11th ed, california, thomson wadsworth, p.154, (2008). 4. goodhart r.s., shils, m.e., modern nutrition in health and disease 6th ed. lea and febinger. philadelphia. p. 134-138, (1980) 5. ramalho,v.c., n. jorge, antioxidant action of rosemary extract in soybean oil submitted to thermoxidation. grasas y aceites, 59 (2), p. 3841,(2008). 6. fritsch c.w., measurements of frying fat deterioration: a brief review. j. am. oil chem. soc. 58, p. 272-274,(1981). 7. azizkhani m., parvin z., effect of some natural antioxidants mixtures on margarine stability. world academy of science, enginiring and technology, 49, p. 93-96, (2009). 8. karpinska m., borowski j., danowska om., the use of natural antioxidants in ready-to-serve food. food chem. 72, p. 5-9, (2001). 9. prior r.l. absorbtion and metabolism of anthocyanins: potential health effects. in m. meskin, w.r. bidlack, a.j. davies, d.s. lewis, r.k. randolth (eds.), phytochemicals: mechanisms of action. boca raton, fl: crc: press, p. 1-19, (2004). 10. hou d.x. potential mechanism of cancer chemoprevention by anthocyanin. current advancements in molecular medicines, 3, p. 149159, (2003). 11. pocorny j., n.v. yanishlieva, m.h. gordon. antioxidants in food. boca ration: crc press, p. 324-344, 360, (2001). 12. farag r.s., badei a.z.m., el baroty g.s.a. influence of thyme and clove essential oils on cottonseed oil oxidation. journal of american oil chemists society, 66 (6), p. 800-804, (1989). 13. gorinstein s., park y.s., heo b.g, namiesnik j., leontowicz h., leontowic m., ham k.s., cho j.y., kang s.k., a comparative study of phenolic compounds and antioxidant and antiproliferative activities in frequently consumed raw vegetables. eur food res. technol, 228, p. 903–911, (2009). 14. sawa t, nakao m, akaike t, ono k, maeda h. alkylperoxyl radical-scavenging activity of various flavonoids and other phenolic compounds: implications for the anti-tumorpromoter effect of vegetables. department of microbiology, kumamoto university school of medicine, japan. j agric food chem. feb;47(2):397-402 (1999). 15. brandwilliams w., m. e. cuvelier, c. berset. use of a free-radical method to evaluate antioxidant activity, lwt-food. sci. technol. 28, p. 25-30, (1995). 16. singleton v.l., r. orthofer, r.m. lamuela-raventos,. analysis of total phenols and other oxidation substrates and antioxidants by means of folin–ciocalteu reagent, methods enzymol. 299, p. 152, (1999). 17. aoac,. official methods of analysis, 16th ed. aoac international, gaithersburg, (1999). 18. iupac. standard methods for the analysis of oils, fats and derivatives., 7th ed., method number 2.504 determination of the p-anisidine value (pa/v.), blackwell scientific publications, boston, ma and oxford, uk, (1987). 19. pretsch e., büllman c., affolter c,. structure determination of organic compounds. tables of spectral data. moscow, p.439, (2006). 20. snedecor g. w., cochran w. g,. statistical methods, 8th edition. the iowa state university press, ames, (1989). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 87 universal fluorometer based on fluorescence quenching georg gutt1, sonia gutt1, cristina hretcanu1, ana leahu1 1stefan cel mare university, faculty of food engineering, 13th university str., suceava, romania. e-mail: g.gutt@fia.usv.ro abstract. this paper describes a method and portable device meant to determine the concentration of various chemical species in solutions based on the fluorescence quenching phenomenon. the solution brought about includes the possibility of measuring the concentration of more chemical or biological species using the same optoelectronic fluorescent, requiring the change of a fluorofor capsule only when going from a chemical species to another and setting of the chemical species type analyzed. in addition to the quantitative analysis made by means of the apparatus described, assisted by computer and specialized software, it is possible to carry out a spectral scanning fluorescence in all spectral range covered by monochromatic radiation sources of led type of the device in question. it allows also carrying out simultaneously both qualitative and semiquantitative analysis of all fluorescent species in the solution tested. keywords:. fluorescence quenching, quantitative analysis, qualitative and semi-quantitative analysis. introduction in order to determine the concentration of fluorescent chemical species in liquids two processes are known. the first process, at the same time the oldest (skog and larry 1996; robinson et al. 2006; gutt, s. et.al. 2011), consists in determining photo electrically the fluorescence intensity (if ) of a species based on the difference between the intensity of incident radiation of excitation (i0) and intensity of the radiation (i) passing through a sample of a given thickness(b) on a different path than that of the incident electromagnetic monochromatic radiation: i)(iki 0f (1) the dependence between the concentration (c) of fluorescent species and fluorescence intensity (if) is determined by the lambertbeer law expressed in a synthetic form: ckif (2) where: k is a constant depending on fluorescence efficiency, thickness of the layer crossed and nature of the fluorescent species. when determining the concentration based on the measurement of fluorescence radiation intensity, the restrictions of linearity specific to lambert-beer law are valid, in this sense the equation (2) is valid only under conditions when the intensity of incident radiation (i0) is constant (skog and larry 1996: 198-203). from the constructive point of view, fluorometers are photometric equipment consisting of a monochromatic radiation source, usually a led which light emission is tuned with the fluorescence wavelength of the species investigated, a place for solution tank, a photodiode placed on a different direction than that of irradiation (usually at 900), an electronic amplifier and electronic unit. the second process, more recent, is based on the measurement of fluorescence quenching phenomenon, where the concentration of a certain chemical species (quencher) is determined by means of another chemical one (fluorofor), on the basis of the relationship which correlates time with intensity decrease of fluorescence, mailto:g.gutt:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 88 the last one not being damaged by the quencher’s concentration. this process is a means of determining the concentration of all chemical species that can act as quenchers. considering also the fact that this phenomenon is reversible, once the quencher is being removed, fluorofor’s fluorescence returns to its initial base line. an essential application based on fluorescence quenching to measure concentration is to determine free oxygen in liquids, especially in water, using portable probes. in this determination the fluorescence quenching time is measured, being related to the concentration of free oxygen (quencher) by the relation stern volmer (3). for this purpose, we used a pulsed laser diode with the radiation wavelength ranging usually in blue light as excitation source, fluorescence occurring at higher wavelength values, while in the light red field it can easily be measured by photodiodes or diode-array detectors, being possible to determine either the quenching time or phase change between excitation radiation and fluorescence radiation (quenching time can be determined accurately from the phase change). experimental the disadvantage of the existing apparatus for determining concentration, based on fluorescence quenching, is that the analysis of each species requires the use of a complete equipment (probe + electronic unit), (gutt, s. et.al. 2011; gutt, g. and gutt, s. 2011; gutt, g. and gutt, s. 2010a). the technical problem is solved by the authors’ solutions, namely they have achieved a fluorometer based on fluorescence quenching that can be used both as a portable unit and laboratory structure as well. the equipment offers the possibility to determine concentration under high accuracy conditions of more chemical species analyzed in turn or simultaneously. in the latter case the semi-quantitative analysis is possible only, having an error margin higher than the quantitative analysis of a single species. instead, the simultaneous analysis enables also a qualitative analysis, a spectral excitation of fluorescence being made by all monochromatic sources of led type from the device structure and multiplexed readout of the fluorescence quenching time. to achieve this device, a modular unit is used consisting of a probe and an optoelectronic unit, figure 1, connected by two optical fibers. the probe is of cylindrical type with metal shell and two optical fibers inside, one for transmitting the monochromatic radiation of excitation from source to sample and the other for collecting and transmitting the fluorescence radiation from sample to the electronic data processing unit. fig 1. scheme of universal fluorometer based on the principle of fluorescence quenching.3,4-optical fibers, 8fluorofor film, 14-multiple monochromatic optical source of radiation, 15-electronic multiplexer, 16-optoelectronics unit, 17-tested solution, c1, c2, c3 electrical contacts food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 89 a key element of the basic structure of fuorometer is the portable radiation source (gutt, g. and gutt, s. 2010b), formed in turn from a monochromatic optical source of radiation and multiple electronic multiplexing, both integrated into the structure of fluorometer. to facilitate the excitation of a large number of fluorescent species the monochromatic source is represented in turn by some compact removable modules, figure 2, each containing a number of the leds, emitting monochromatic radiation controlled by multiplexer fig.2. exterior view of a monochromator module. 3-optical fiber, 14-multiple monochromatic optical source of radiation, 15-electronic multiplexer, c1, c2, c3 electrical contacts each led within the module structure having the wavelength of emitted radiation tuned with a certain fluorescent chemical species (quencher). monochromator modules have leds arranged in a crown each led being aligned to an optical fiber. all optical fibers from a crown of leds come together in a single collector optical fiber to irradiate the sample with wavelength specific to the chemical species monitored. when analyzing quantitatively the concentration of a chemical species, the nature of the latter one is set from the device panel, having as effect the led lighting with the radiation wavelength tuned with the excitation wavelength value of that species. in semi-quantitative and qualitative analysis, multiple monochromatic radiation source works sequentially controlled in multiplexed operating mode, in the sense that the electronic multiplexer controls both the led lighting in turn as well as reading and management of fluorescence quenching time of each excited species a) b) fig 3. fluorometer probe equipped with hollow capsule (a) and straight capsule (b). 1–probe, 2polymer material, 3,4 – optical fibers, 5handle, 6 – hollow capsule , 7 – straight capsule, 8 – fluorofor film, 9 – optical coupler, 17 – analyzed solution. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 90 to increase the universality of the device, a module of leds, figure 3, which forms the monochromator unit at one time, can be replaced, simply by pressing hand, by another module which leds are tuned with wavelengths of other fluorescent chemical species. in order to give the possibility of using the same equipment in determining the concentration of more chemical species, different transparent hollow polymer capsules or straight ones are placed on the probe, figure 3, a chemically non-destructive fluorofor being deposited on each capsule in the outer, specific to a certain chemical species which concentration is to be determined by fluorescence quenching. a) b) c) fig 4. applications of universal fluorometer in laboratory, a) –probe held by hand, b) -probe fixed on a stand, having a hollow capsule mounted and upwards – directed cavity, c)-probe fixed on a stand, having a straight capsule steeped into a flask containing the species to be analyzed. 1-probe, 2-polymer material, 3,4optical fibers, 5-handle, 6-hollow capsule, 7straight capsule, 8-fluorofor film, 9-optical coupler, 10-stand, 11,13screws, 12-rotating head , 17-analyzed solution, 18-glass vessel, 19-glass pipette, 20thermostatic plate from the constructive point of view a fluorometer based on fluorescence quenching, in a complete structure for all applications, fig.1, fig. 2, fig.3, fig.4, consists of a cylindrical probe with metal shell 1, a filling polymer material 2, two optical fibers 3 and 4, a handle 5, a hollow capsule type 6, a straight capsule type 7, an adherent film of polymer fluorofor 8, an optical coupler 9, a rack 10, a binding screw 11, a rotating head 12, a rotationblocking screw 13, a monochromatic multiple radiation source 14, an electronic multiplexer 15, an optoelectronics unit 16, the analyzed solution containing the quencher 17 (chemical species focused on), a glass vessel used in laboratory work 18, a glass pipette 19 and a thermostatic plate of peltier type 20. results and discussion the fluorometer presented can operate both under field conditions for in situ analyses and laboratory conditions for serial analyses. specific dependencies between the intensities of radiation, quenching time and concentration of fluorescence species are given by sternvolmer equation valid for dynamic quenching of fluorofor. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 91 ck1 i i dsv 00 (3) where: i0 intensity of fluorescence of the fluorescent substance in the absence of species that causes fluorescence quenching i intensity of fluorescence of the fluorescent substance in the presence of species that causes fluorescence quenching 0 life time of excited state of fluorofor in quencher's absence life time of excited state of fluorofor in quencher's presence kdsv dynamic constant stern volmer cconcentration of species (quencher) causing flourescence quenching when operating under field conditions, the fluorometer probe is held by hand (fig. 4 a), in this way it can be equipped with either a hollow capsule 6, mounted on probe body 1, situation when a precise volume of solution 17 to be analyzed is pipette into the cavity of capsule 6 with a pipette 19, figure 4a, or with a straight capsule, situation when measurements can be made throughout the volume of tested solution, the cylindrical probe 1 being steeped into the solution 17 to be analyzed, figure 4b. it should be specified that the change of capsule type requires manual setting of the electronic unit 16, because each type of probe requires another correction factor. also, when changing capsules in order to determine another chemical species it is necessary to set properly the chemical species corresponding to the fluorofor deposited on capsule. this setting commands in multiple monochromatic optical radiation source 14 (fig. 1) the led lighting that emits on the wavelength specific to species and at the same time specific to the fluorofor film from the capsule 6 or 7. when working under laboratory conditions (fig. 4b), the cylindrical probe 1 of fluorometer is easily fixed by the binding screw 11, afterwards depending on the type of application, the rotating head 12 rotates so as the probe head should be either upward or downward (fig. 4c), then this one is screwed by the binding screw to the desired position and the rotation-blocking screw is operated. setting the probe head 1 upward is meant to series determinations. for this purpose the probe is equipped with the capsule 6 by pushing it on the probe body 1, and then determinations can be made. after each determination, the rotation-blocking screw 13 is being loosened and the probe head is rotated downward, leading to the release of the solution to be analyzed 17 from the capsule cavity 6, then it is being rinsed by bi-distilled water and the probe is brought again to its vertical position for further analysis, the accurate volume of solution analyzed 17 being provided by a glass pipette 19. setting the probe head downward is meant to the study of kinetic evolution of a certain chemical species under different conditions. for this purpose it is necessary for the straight capsule 7 to be mounted on the body of cylindrical probe 1, afterwards this one is being submerged into the vessel containing the analyzed solution 17, and the concentration evolution of the tested species over time depending on various process parameters is registered by electronic unit 16. conclusions putting the above described concept into practice has enabled us to carry out an universal portable fluorometric equipment, of low cost, which by minimum auxiliary operations and under high productivity conditions performs the functions of more independent fluorometers, each of these typically specialized on the concentration of a certain chemical species, with no compromise regarding measurement accuracy. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 92 references skoog, d.a.; leary, j.j. 1996. instrumentelle analytik, springer berlin: 198-203. robinson, j.; skelly fram, e.m et.al. 2006, undergraduate instrumental analysis, sixt edition, marcel dekker, new york: 366-376. gutt, s.; gutt, g. et.al. 2011. fluorometru universal bazat pe stingerea de fluorescenta, (in romanian), [universal fluorometer based on fluorescence quenching ] , patent proposals nr. a00163/2011, osim bucuresti. gutt, g.; gutt, s. 2011, fluorometru portabil (in romanian), [portable fluorometer], patent proposals nr.a00740/2010, osim bucuresti. gutt, g.; gutt, s. 2010. fluorometru optoelectronic portabil (in romanian), [optoelectronic portable fluorometer], patent proposals nr. a00739/2010, osim bucuresti. gutt, g.; gutt s., et.al. 2010. monocromator cu fibra optica (in romanian), [monochromator with optical fiber], patent proposals nr. a001342/2010, osim bucuresti. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 57 a thermodynamic analysis of juice pasteurization process feiza memet1 1constanta maritime university, mircea cel batran street, 104, 900663, constanta, romania, e-mail: feizamemet@yahoo.com abstract: a superior fruit or vegetable juice should be healthy and nutritious, in this purpose being heated and treated. exergy analysis is a tool able to indicate whether or not and how it is possible to design more efficient energy systems by diminishing the inefficiencies existing in the analyzed systems. this analysis is considering the quantity and quality of the energy. exergy efficiency evaluates the efficiency of a process by considering the second law of thermodynamics. being seen that the production process of juices involves a lot of stages, is concluded that a significant amount of energy is consumed. for the improvement of this process are calculated exergy efficiencies for a pasteurization unit comprised in the juice production flow chart, when inlet hot water temperature is increased from 87 to 100oc, by keeping the other parameters constant. are obtained reduced values for the exergy efficiency together with the increment of the hot water temperature at the entrance of the pasteurization unit. better values for the exergy efficiency are seen in the same operating conditions, but for a reduced mass rate of the hot water. for a decrease of about 82% of this mass rate the afferent average exergy efficiency is higher with about 80%. keywords: exergy efficiency, hot water, temperature. introduction fruit and vegetable juices are important business mostly in developed countries since they are not alcoholic drinks which supply vitamin c to the consumer. the most common fruits and vegetables used for extraction are: citric, apples, pears, apricots, strawberries, pineapples, mangoes, tomatoes, carrots and others [1]. the flow chart of production process is given bellow (see figure 1). fig. 1 raw material processing for juice. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 58 harvesting – raw materials should be picked at correct sugar concentration. storage – should avoid the bruising of raw materials from the bottom. inspection – should remove the raw materials damaged. washing – is done under water sprays and brush rollers. peeling – is done manually. juice extraction – is done by an extractor. juice formulation – consists in enriching by ingredients such as sugar, preservatives and water in order to get the right formulation. mixing – the juice is mixed in a reservoir by the help of a mixer to get a homogeneous blending of the formulated juice. bottling – the mixed juice is bottled or corked in a manual or automatic manner. pasteurization – bottled juice is heated to a specific temperature for a determined period, enough to destroy certain bacteria. some forms of pasteurization are given below[2]. flash pasteurization – consists of a plate pasteurizer with heat recovery and final product cooling. temperatures ranging between (85-95o c) for a period of time between (15-60) sec are used. in–pack pasteurization – is a saving procedure when the bottled product, being tightly closed, is immersed into a tank filled with hot water (the used temperature is about 70oc), for about 20 min. for thermal security, it is recommended a pre– immersion into a tank using a temperature of about 40oc. high–pressure pasteurization – is often met in the pasteurization of fruit juice in containers. since the process occurs as a batch operation, this pasteurization type appears to be expensive enough. cooling and packaging – the pasteurized juice is cooled and put into special cartoons to be transported to retailer. from the above described steps, it is seen that juice industry involves a considerable use of energy and materials [3]. in order to improve the efficiency of juice production process, it is important to know the losses taking place in the process and their causes. for better energy use, it is important to be aware of the quantity and quality of energy. this can be achieved by applying the laws of thermodynamics. the quality of energy is expressed through the thermodynamic function called exergy. the importance of the calculation of exergy efficiency consists in the fact that it includes the quality level of the converted energy [4]. this paper develops an exergy analysis, in steady state, for the step called “pasteurization” in the juice production process. experimental the first law of thermodynamics states that energy cannot be created nor destroyed and its quantity remains constant in all processes. the second law of thermodynamics states that energy is degraded in all processes and its quality decreases. the quality of energy is defined by specialists as the potential to transform energy into work. the potential to produce work is called exergy [5]. thus, according to the first law of thermodynamics: energy in = energy out and according to the second law of thermodynamics: energy in > energy out energy is defined by: energy = exergy + anergy the energy of high convertibility potential has high share of exergy. the anergy represents the part of the energy that can not be transformed into work. the exergy of an amount of matter is given by the amount of work resulting from a system able to bring the matter to equilibrium with the environment through food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 59 a reversible process. this system is defined by the following aspects: in the system occur only reversible processes the system is open, has a constant volume and a steady flow is entering and leaving the system the kinetic and potential energy of the matter are neglected heat can be exchanged with environment at the temperature t0 for the conversion of matter into environmental components only matter from the environment, under environmental conditions, should be used at the exit of the system, matter is in equilibrium with the environment. in principle, there are four different types of exergy: kinetic, potential, physical and chemical: chphpk exexexexex  (1) kinetic and potential exergy have the same significance like the corresponding energy terms. these components are usually neglected when analyzing industrial processes. physical exergy is the work obtainable by taking a substance through reversible physical processes from its initial state, defined by the temperature t and the pressure p, to the state given by the temperature and pressure of the environment (t0 and p0). physical exergy plays a significant role when optimizing thermal and mechanical processes. chemical exergy represents the maximum work produced when the substance is brought from the environmental state to the dead state, by a process implying heat transfer and exchange of the substances only with the environment. for the exergy calculation, it is compulsory to define temperature, pressure and chemical composition for the environment where the analyzed systems work. the exergy of an amount of matter is estimated by:    000 ssthhex  (2) in this equation “h” is the enthalpy and “s” is the entropy. one of the main equations of the exergy analysis, the exergy balance, is given below: destlossoutin exexexex  (3) exergy losses are given by exergy flowing to the surroundings, while exergy destruction indicates the loss of exergy inside the process boundaries due to irreversibility (see figure 2). the exergy efficiency is a general performance criterium; it denotes the part of the available work which is actually used [6]. in out ex ex  (4) as shown, pasteurization is a thermal treatment aiming at the destruction of pathogenic agents. pasteurized juice is a safe drink due to the fact that it has been heated long enough time to kill bacteria. also, the shelf life of the product is extended, but with minimal impact on flavour. exergy analysis is a strong tool often used to identify inefficiencies during the steps involved in the juice production process [7]. in this paper, the exergy efficiencies during pasteurization for two operation situations: hot water inlet temperature rises, for two values of the mass flow rate are calculated. in our case, hot water evacuates heat to the fruit juice, thus the temperature of hot water will decrease with c37t owh  , while the temperature of juice fruit will fig. 2 representation of exergies food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 60 increase with c48t ojf  ; weight rates are: s/kg4,3m wh  and s/kg2,2m jf  . results and discussion inlet temperature of hot water varies in the range (87÷100)o c, the other working parameters being kept at the same level. in the first case, the mass rate of the hot water is 3, 4 kg/s and in the second case decreased to 2. 8 kg/s. exergy efficiencies in both cases (see table 1) are noted. the values obtained show that there is an increase of hot water temperature at the entrance of the unit, resulting a decrease in the values of exergy efficiency. this observation is available also when the hot water mass rate is diminished. table 1. results for exergy efficiency s/kg4,3m wh  hot water inlet temperature, oc 87 90 93 95 97 100 exergy efficiency 53.82 48.74 46.35 44 42.28 38.86 s/kg8,2m  hot water inlet temperature, oc 87 90 93 95 97 100 exergy efficiency 65.31 61.14 56.73 53.21 51.32 47.08 for lower hot water mass rate, better exergy efficiencies are noted. conclusions fruit and vegetable can be kept fresher and longer by the help of pasteurization. it deals with heating the product to high temperature for a short period of time. as a result, bacteria, molds and undesirable microorganisms are eliminated. this procedure makes the juice more cost efficient. the exergy analysis method was developed in order to determine the exergy losses and potential of reducing these losses in engineering systems. the way chosen to assess the performance of the pasteurization unit analyzed in this study, to transform resources, is the exergy efficiency. when hot water temperature at the entrance of the pasteurization unit increases in the range (87-100o c), while other parameters are kept constant, a decrease for the values of exergy efficiency is obtained. an improvement of exergy efficiency values is seen when the hot water mass rate is reduced. for an increase of 82.35% in this mass rate, an average of 81.6% in the exergy efficiency is gained. references 1. philip r. ashurst, editor, production and packaging of non-carbonated fruit juices and fruit beverages, aspen publishers, inc, usa, 1999, 423 pp 2. philip r. ashurst, editor, chemistry and technology of soft drinks and fruit juices, second edition, blackwell publishing ltd, uk, 2005, 396 pp 3. li jun wang, energy efficiency and management in food processing facilities, crc press, usa, 2008, 452 pp 4. adrian bejan, george tsatsaronis, michael moran, thermal design and optimization, john wiley & sons, inc, usa, 1996, 542 pp 5. ibrahim dincer, marc a. rosen, exergy: energy, environment and sustainable development, elsevier ltd, 2007, uk, 451 pp 6. marc a. rosen, indicators for the environmental impact of waste emissions: comparison of exergy and other indicators, trans. can. soc. mech. eng. vol 33, issue 1, 2009, pp. 145-160 7. ruchira taprap, papitchaya buachote, energy and exergy analysis of fruit juice process in thailand, proc. of ecos 2007, vol ii, june 2007, padova, italy, pp. 1341-1347 contribution about nickel electrodeposition from watts bath with addition of polyvinyl pyrrolidone food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 104 multivariate analysis in assessment relationships between milk characteristics influenced by the seasonal variations silvia mironeasa1, georgiana gabriela codină1 1„ştefan cel mare” university, faculty of food engineering, 13th university street, 720229, suceava romania, e-mail address: silviam@usv.ro; codina@usv.ro *corresponding author received 22 september 2011, accepted 5 november 2011 abstract: the present research aimed to investigate the effects of seasonal variation on microbiological and technological characteristics of raw cow milk from suceava county. the cow milk samples were collected and investigated during four seasons in three years consecutively. the first period was the winter season covering the months december – january – february, the second one was the spring season covering the months march – april – may, the third was the summer season covering the months june – july – august and the fourth period was the autumn season covering the months september – october november. milk characteristics measurements were examined by multivariate data analysis, using principal component analysis (pca). the analysis of the principal components sheds light on the correlations between the characteristics of the raw cow’s milk, similarities and differences among these in four seasons. the values of the correlation coefficients indicate positive relations between the number of total germ and somatic cell count during spring and summer seasons, between protein content and solids non fat during autumn season. regarding relationships amongst milk characteristics influenced by the seasonal variation, negative correlations were obtained between fat content and somatic cell count in spring season, between lactose content and somatic cell count in winter and autumn seasons, between somatic cell count and solids non fat content in autumn season. keywords: cow milk, chemical characteristics, microbiological characteristics, seasonal variations, principal component analysis 1. introduction in suceava the dairy sector is a significant part of the local economy because stock farming is an important and traditional occupation in rural areas, especially in mountain regions. the nutritional quality associated with the technological quality of raw cow milk has great importance for human health and milk products. the milk composition varies according to certain factors, such as genetics [1], age, stage of lactation, daily variation, parity, type of diet, milking conditions [2-4] udder health [5] and season [2], [6-8]. bruhn and franke (1997), lacroix et al. (1996) have reported that percentage of fat, protein e.g. have been influenced by the seasonal variations. the fat content decreased when the environmental temperature increased [9-11]. casati et al. (1998) states that the light-to-dark ratio can also induce marked changes in milk yield and composition. region, climatic conditions and lactation periods are known as seasonal changes which have influences on the milk composition; the negative correlations were found between environmental temperature, fat and protein milk content [1213]. in this research the important county of north the romania, suceava according to milk yield and milk products and geographic location has been selected. in our study, the aim was to determine the composition of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 105 cow’s milk and to find out the effects of seasonal variation on this composition. 2. materials and methods 2.1. materials. the raw cow’s milk samples were supplied from different collecting points from suceava county. the milk was collected in three years consecutive. the research was carried out in four periods. the first period (i) was the winter season covering the months december-january-february, the second one (ii) was the spring season covering the months march-april-may, the third (iii) was the summer season covering the months junejuly-august and the fourth period (iv) was the autumn season covering the months september-october-november. collecting samples was conducted daily taking into account the season. 2.2. metods. milk quality tests were accomplished according to romanian standard methods for fat content (sr en iso 1211:2010), somatic cell counts (sr en iso 13366-3:2001) and solids-non-fat content (sr iso 6731:1996). protein and lactose content were analyzed using a infrared milk analyzer unit bentley 150 (bentley instruments inc., chaska, mn, usa) and number of total germs were done by flow cytometry count analyzer unit bactocount ibc 50 (bentley instruments inc., chaska, mn, usa). 2.3. data analysis. all analytical determinations were performed at least in triplicate. values of the characteristics are expressed as the mean ± standard deviation to a confidence interval of 95%. treatment of data was analyzed using spss software, version 16.0. multivariate exploratory technique, principal component analysis (pca) was used to highlight the correlations between the components of the raw cow’s milk, similarities and differences among biochemical and microbiological characteristics of milk from four seasons, reducing the dimension to two pcs, while keeping most of the original information found in the data. only pcs with eigenvalue larger than one were retained for further analyses. 3. results and discussion chemical and microbiological analysis. the chemical and microbiological characteristics of the samples set from four seasons are shown in table 1. it can be seen that the mean milk fat percentage was the highest during the autumn season and the lowest during the spring season. the amount of fat in milk composition was the most variable component among the milk characteristics. this variation may be due seasonal variation and lactation period. the more pronounced variation of autumn was due to outdoor grazing in summer and bar feeding in winter. the seasonal variation was due to many important differences between the feeds composition which were given to animals during these four periods. the lowest the mean value milk fat was detected during the spring season probably due to the fact that the feed diet was based on hay the herbage was not available due to long winter. according to waldner et al. (2005) the cows feeding with low fiber diets leads to an increase in milk fat levels. comparing the mean protein content detected in winter season to the mean protein content detected in summer season, the highest concentration was obtained in summer season. the protein in raw cow’s milk does not fluctuate as much as milk fat, confirming the results obtained by ozrenk and selcuk (2008). the mean lactose concentration ranged from 4.18  0.06 % to 4.31  0.03 %; the highest concentration was detected in spring season. the mean solids non fat content of the cows’ milk was 8.42  0.07 %. the highest solids non fat percentages (8.51  0.01%) was obtained during the summer season. regarding the mean value obtained for somatic cell counts (scc), the lowest mean concentration (296.33  19.85 x 103 cells · ml-1) were detected in autumn season; the highest mean value of scc (314  71.68 x 103 cells · ml-1) was detected during the spring season. the increase of scc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 106 can be due poor health condition of cow's udders [5]. a greater number of the somatic cells affects quality processing of milk, mainly has effect on cheese production [1415].the highest mean value of number of total germ (ntg) (266  6.08 x 103 no · ml-1) was detected in winter season and the lowest mean value ntg in autumn season (258.33  31.53 x 103 no · ml-1). table 1. the chemical and microbiological characteristics of cow’s milk obtained from suceava county fat (%) protein (%) lactose (%) solids non fat (%) somatic cell counts x 103 (cells · ml-1) number of total germ x 103 (no · ml-1) period i minimum 3.83 3.27 4.15 8.33 285 262 maximum 3.90 3.30 4.22 8.38 335 273 mean  sd 3.86  0.03 3.28  0.01 4.19  0.03 8.34  0.02 303.66  27.30 266  6.08 period ii minimum 3.69 3.27 4.29 8.38 236 208 maximum 3.86 3.31 4.35 8.51 377 304 mean  sd 3.80  0.09 3.29  0.02 4.31  0.03 8.44  0.06 314.00  71.68 260  48.49 period iii minimum 3.79 3.39 4.20 8.50 275 234 maximum 3.92 3.44 4.33 8.53 358 309 mean  sd 3.87  0.07 3.41  0.02 4.24  0.07 8.51  0.01 310.33  42.85 264  39.68 period iv minimum 3.78 3.30 4.12 8.30 282 226 maximum 4.15 3.45 4.25 8.45 319 289 mean  sd 3.94  0.18 3.39  0.08 4.18  0.06 8.39  0.08 296.33  19.85 258.33  31.53 general mean  sd 3.86  0,10 3.34  0.07 4.23  0,07 8.42  0.07 306.08  39.05 265.33  37.23 relationships between chemical and microbiological raw cow’s milk characteristics during the four seasons. the correlation between the results of the chemical characteristics during seasonal variation – the fat, protein, lactose, solids non fat content and microbiological characteristics – somatic cell counts and number of total germ are shown in figure 1. the first two principal components explain 83.60 % and 16.39% of the total variance. the first component (pc 1) was characterized mainly by the mean value of somatic cell count during winter period (scc i), autumn period (scc iv), number of total germs during summer (ntg iii) and autumn period (ntg iv). in respect to the first principal component pc 1, one can notice there is a very good correlation between the mean protein content (p_ii), mean values of number of total germs (ntg_ii) and somatic cell count (scc_ii) in spring season. the second component pc 2 distinguishes the mean protein content during spring (p_ii) and the mean protein content during summer seasons (p_iii) which are opposed. regarding winter season, negative correlation (r = 0.985) was obtained between mean value of lactose (l_i) and mean value of somatic cell count (scc_i). figure 1. principal component analysis of the combined chemical and microbiological characteristics of raw cow’s milk from period i, period ii, period iii and period iv. in the spring season, positive correlation was reported between the mean values of number of total germ (ntg_ii) and somatic cell count (scc_ii) (r = 0.99) and negative correlation between mean fat content (f_ii) and mean value of somatic cell count (scc_ii) (r = 0.79). positive correlation was reported between the mean values of number of total germ (ntg_iii) and somatic cell count (scc_iii) (r = 0.89) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 107 during summer season. in the autumn season, positive correlation between mean protein content (p_iv) and mean solids non fat (snf_iv) (r = 0.98) on the one hand and negative correlations between mean values of lactose content (l_iv) and somatic cell count (scc_iv) (r = 0.94), between mean values of somatic cell count (scc_iv) and solids non fat content (snf_iv) (r = 0.99) on the other hand were reported. 4. conclusions according to the results obtained in this study, the fat and the protein contents of cows’ milk have been affected importantly by the seasonal variations. principal component analysis was performed between different technological and microbiological characteristics of raw cow’s milk samples. regarding the correlations established with pca method between the characteristics of milk from the four periods, a positive correlation was obtained between number of total germ and somatic cell count during spring and summer seasons, between protein content and solids non fat during autumn season. negative correlations were obtained between lactose content and somatic cell count during winter and autumn seasons, between fat content and somatic cell count during spring season, between somatic cell count and solids non fat content during autumn season. 5. references 1. lucas a., agabriel c., martin b., ferlay a., verdier-metz i., coulon j.-b., rock e., relationships between the conditions of cow’s milk production and the contents of components of nutritional interest in raw milk farmhouse cheese, lait 86. 177–202, (2006) 2. falchero l., lombardi g., gorlier a., lonati m., odoardi m., cavallero a., variation in fatty acid composition of milk and cheese from cows grazed on two alpine pastures, dairy sci. technol., 90. 657 – 672, (2010) 3. bony j., contamin v., gousseff m., metais j., tillard e., juanes x., decruyenaere v., jcoulon .b., factors of variation of the milk composition on the reunion island, inra prod. anim. 18. 255–263, (2005) 4. salovuo h., ronkainen p., heino a., suokannas a., ryhänen e.-l., introduction of automatic milking system in finland: effect on milk quality, agricultural and food science, 14. 346-353, (2005) 5. matei s.t., groza i., andrei s., bogdan l., ciupe s. petrean a., serum metabolic parameters in healthy and subclinical mastitis cows, bulletin uasvm, veterinary medicine 67. 110-114, (2010) 6. alomirah h., al-mazeedi h., al-zenki s., al-aati t., al-otaibi j., al-batel m. sidhu j., prevalence of antimicrobial residues in milk and dairy products in the state of kuwait, journal of food quality, 30. 745-763, (2007) 7. formaggioni p., franceschi p., summer a., malacarne m., fieni s., mariani p., effects of the season and the herd on milk proteose peptone content and relationships with rennet-coagulation properties, sci. tec. latt. casearia, 53. 53–65, (2002) 8. mironeasa s., codină g.g., mironeasa c., variation analysis of cow milk composition quality depending on year, season and location in romania, bulletin uasvm, animal science and biotechnologies, 68. 225-232, (2011) 9. bruhn j.c., franke a.a., monthly variations in gross composition of california herd milks. journal of dairy science, 60. 696-700, (1997) 10. lacroix c., verret p., paquin p., regional and seasonal variations of nitrogen fractions in commingled milk. int. dairy j., 6. 947-961, (1996) 11. walstra p., wouters j.t.m., geurts t.j., dairy science and technology, second ed, crc, taylor and francis group, (2006) 12. casati m.r., cappa v., calamari l., calegari f., folli g., effects of the season on milk yield and on some milk characteristics in cows. scienza e tecnica lattiero-casearia, 49. 7-25, (1998) 13. ozrenk e., selcuk i. s., the effect of seasonal variation on the composition of cow milk in van province, pakistan journal of nutrition, 7. 161164, (2008) 14. waldner d.n., stokes s.r., jordan e.r. looper, m.l., managing milk composition: normal sources of variation, (2005). from http://www.osuextra.com 15. antunac, n., lukačhavranek, j., samardžija, d., somatske stanice i njihov utjecaj na kakvoću i preradu mlijeka, mljekarstvo, 47. 183193, (1997) 6. alomirah h., al-mazeedi h., al-zenki s., al-aati t., al-otaibi j., al-batel m. sidhu j., prevalence of antimicrobial residues in milk and dairy products in the state of kuwait, journal of food quality, 30. 745-763, (2007) food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 46 flow diffusion chamber is a new tool for vapor nucleation rate measurements * michael anisimov, elena fominykh department of chemical engineering, clarkson university, potsdam, ny 13699-5705 (usa) nanoaerosol laboratory, technological design institute of scientific instrument engineering sb ras 41 russkaya str., 630058 novosibirsk, (russia) received july 03, 2011 abstract: the quality of the vapour gas nucleation has been increased substantially in 1980th. in addition to the all known for that time experimental techniques the flow diffusion chamber (fdc) was developed. several articles devoted to fdc were published in russian scientific journals beginning 1978 [anisimov and co-workers (1978a,b), etc]. the scheme of fdc involves using the hot laminar vapor-gas flow within the colder boundaries which is similar, for example, to d. sinclair and v.k. la-mer generator of monodispersed particles. the current fdc scheme can be used for the vapor nucleation rate measurements at the total pressures from 0.3 to 3.0 atm. fdc has been reproduced in finland, usa, japan, czech republic, germany, austria, and in several research groups at other countries. a conceptual problem in vapor nucleation is its treatment as a single component problem. in general, one should consider the carrier gas-vapor nucleation as a binary nucleation. in order to test the behaviour of an experimental set-up, it is useful to have a standard system that can be measured over a range of typically employed nucleation conditions. the current state of vapor – gas nucleation rate measurements is published recently by anisimov et al. (2009) keywords: vapour-gas, nucleation rate measurements, flow diffusion chamber. 1. introduction the flow diffusion chamber (fdc) combines advantages of the static diffusion chamber to define the nucleation conditions and simplicity of the flow systems in measuring of aerosol concentration and size distribution. fdc has decoupled aerosol generation volume and aerosol detecting zone, which is useful to grow the small critical clusters up to the optically detectable particles in the residual supersaturated vapor. fdc basic scheme is partially similar to the uniform size particle generator which is designed by sinclair and la-mer (1949). anisimov and co-workers (1978a, b) have created the original scheme of the flow diffusion chamber prototype for vapor homogeneous nucleation rate measurements. the flow diffusion chamber is developing tool up to now. fdc is reproduced in several research groups of europe and north america at present. the strongest reason for all nucleation rate measuring system development is the data inconsistencies for the different devises. in general, one should consider the carrier gas-vapor nucleation as a binary nucleation. in order to test the behavior of an experimental setup, it is useful to have a standard system that can be measured over a range of typically employed nucleation conditions. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 47 2. short overview of the experimental techniques for nucleation studies presumable the first nucleation experiment was associated with measuring of the liquid and crystals supercooling which were done by fahrenheit (ostwald, 18961903). than as volmer mentioned in his monograph (volmer, 1939), the gas saturated solutions, illustrated the bubble nucleation and the critical embryos of new phase, have been introduced in nucleation science at the second half of 19-th century. figure 1. the generator scheme by sinclair and la-mer (1949) for production of monodispersed particles. systems for vapor and heterogeneous seeds generation are numbered as (1, 2, 4); vapor superheater has number (3). aitken (1888) has provided practically the first research on vapor nucleation. he has used the adiabatic expansion of water vapor in air. allen&kassner (1969) have suggested a recompression cycle to grow the generated clusters to the optically detectable sizes. that cycle was realized later in two-piston expansion chamber (strey et al., 1986). langsdorf (1939) has created the static diffusion chamber at the end of thirties. the russian scientist amelin (1948) has introduced a system where the different temperatures vapor gas streams have been jointed in the turbulent regime. first prototype of the laminar flow diffusion chamber was created (see, for example, anisimov et al., 1978; anisimov&cherevko, 1985) as an instrument for vapor homogeneous nucleation rate measurements at the end of 1970th. the quality of the experimental research has been increased substantially in 1980-th. the shock tube as a version of the expansion techniques got the considerable quality rise in the nucleation rate data (for example, peters and paikert, 1989). a shock tube represents a tube which is initially divided by diaphragm. an adiabatic expansion is initiated when the diaphragm get broken. the vapor-gas mixture from the high pressure section get vapor supersaturated under adiabatic expansion&cooling and nucleation occurs then. a shock tube (peters&paikert, 1989) and supersonic jets (kim et al., 2004) have the best chance for measuring the highest nucleation rate which is near 1011-1012 cm3s-1 now. detailed overview of the experimental techniques, which are used for the supersaturated vapor nucleation rate measurements, is published by anisimov et al. (2009). a short history of the flow diffusion chamber design is presented below. 3. flow diffusion chamber: method and results fdc scheme involves using the hot laminar vapor-gas flow within the colder boundaries which is similar, for example, scheme of the laminar part for particle growth in the generator by sinclair&lamer (1949) which is presented in fig. 1. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 48 3.1 flow diffusion chamber scheme several articles devoted to fdc first versions were published by anisimov and co-workers in scientific journals starting 1978 (anisimov et al., 1978; etc). a filtered carrier gas has been passing a vapor saturator, where gas was saturated. hot vapor-gas flow entered to a cylindrical cooler/condenser. . nucleation occurs in condenser. the present day fdc scheme is presented in fig. 2 (anisimov&hopke, 2001). gas flow laminator is used to get a fully developed laminar vapor-gas flow. figure 2. the present day block-scheme of the flow diffusion chamber by anisimov at al. (2001). that procedure makes possible to define the boundary conditions of initial vapourgas flow velocity distribution for the stationary heat-mass transfer problemaerosol concentration and particle size distribution can be measured by aerosol counter. all experimental parameters are collected by the data acquisition unit. the current fdc scheme is used at the total pressures from 0.03 to 0.30 mpa. several authors (anisimov et al., 1993; anisimov et al., 1994; wilck et al., 1998, etc) constructed and tested the laminar flow diffusion chamber at helsinki university, finland, at clarkson university in usa (anisimova et al., 2001), institute of chemical process fundamental, czech republic (brus et al., 2005), etc. 3.2 algorithms for the nucleation rate measurements algorithm for estimation of the average nucleation rates in fdc was suggested by anisimov et al., (1980). that algorithm assumes estimation of the nucleation zone and a passing time within a nucleation zone for a unit volume of a supersaturated vapor. ratio of an experimental aerosol concentration over passing time gives the average nucleation rate. the maximum value of the nucleation rate in fdc is estimating presently using an algorithm by wagner&anisimov (1993). the maximum empirical value for the nucleation rate, jmax, in diffusion chambers can be measured using an obvious relation jtheor/ntheor=jmax/nexp, where jtheor is maximum theoretical nucleation rate; ntheor and nexp are the theoretical and experimental fdc particle concentrations respectively. 3.3 fdc data re-evaluation in order to evaluate the experimental data on homogeneous nucleation rates from a laminar flow diffusion chamber experiments, a computation of the nucleation conditions is required. the influence of the used computational methodology on the derived nucleation rate curves was studied by mitrakos et al. (2008). these authors made a recalculation of published fdc experimental data for 1butanol – helium nucleation rate measurements by brus et al. (2005) using two different computational methods. the effect of total pressure on homogeneous nucleation rates of n-butanol in helium, npentanol in helium and argon using a laminar flow diffusion chamber were estimated by herrmann et al. (2009) in a result of a computer modeling. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 49 figure 3. experimental isobaric nucleation rate, j, of glycerin (1, 2) and dibutylphthalate on the nucleation temperature, t. temperatures of vapor at equilibrium are presented near each curve. 3.4 fdc using aerosol concentration was measured by nephelometer in the first experiments. two-coordinate recorder was used to write down a variation of aerosol concentration (y-axis in log-scale) versus the saturation temperature (x-axis) for case of isothermal nucleation and an aerosol concentration against a cooler wall temperature for isobaric nucleation (fig. 3). enthalpy or/and entropy of critical embryo formation can be extracted using these kind of experimental data (anisimov&cherevko, 1985). brock and co-authors designed and described a laminar coaxial flow system to study the single (brock et al.,1986)) and multicomponent vapor nucleation (brock et al., 1988). these authors compared the experimentally obtained aerosol concentrations with classical nucleation theory predictions and theoretical results by wilemski (1975) for binary nucleation. it can be mentioned that for that time only four groups [anisimov et al., 1987; brock et al., 1988; strey&wagner, 1988 (expansion chamber); okuyama et al., 1988 (turbulent flow)] measured the isothermal nucleation rates for two-vapors in a gas media systems within four or more orders of magnitude. other researchers measured critical vapor supersaturations or nucleation rate within two orders of magnitude only. nguen et al. (1987) was studied homogeneous and heterogeneous nucleation of a single vapor using a laminar flow aerosol generator. 3.5 data inconsistence results on homogeneous nucleation of n-hexanol figure 4. comparison of the n-hexanol experimental nucleation rate, j, versus vapor supersaturation ratio, s, from two piston expansion chamber (crosses) by strey et al. (1986) and fdc (dots) by anisimov – hameri kulmala (1994). (anisimov et al., 1993; anisimov et al., 1994) were compared (fig. 4) with data by strey et al. (1986). one can see that the expansion chamber data have values on four orders of magnitude higher the fdc nucleation rate results. slopes for the different measuring system isotherms are near the same. in the same fashion the recent data by brus et al. (2005) illustrate the inconsistence the static diffusion chamber and fdc data, where the fdc values are higher the static diffusion chamber results on three and half orders of magnitude. these two examples illustrate the internal inconsistence of the experimental data, which are originated from the different experimental sets. it looks like that those experimental food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 50 measurements have the uncontrollable variations of at least one physical parameter. 3.6 empirical gas pressure effect during the past two decades several research groups provided intensive research to examine the effects of pressure and carrier gas nature on homogeneous nucleation as part of an effort to get better experimental data for probing vapor to condensed phase nucleation. nontrivial results have appeared in using fdc. anisimov et.al. (1998), for example, have detected experimentally the nucleation rate surface singularity which was the result of phase transition in critical embryos which are formed from both gas and vapor molecules. articles by anisimov, et al. (1998; 2000a) can be mentioned as examples, where the phase transitions in the embryo of new phase are used as markers of a gas pressure effect to show that the gas molecules are incorporated in the condensed matter. anisimov and hopke (2001) have found the multichannel n-pentanol-sulfur hexafluoride nucleation. that is a case of several saddle points existence on the gibbs free energy surface for binary system embryo formation (each of saddle point is associated with nucleation channel, see details, anisimov and hopke, 2001). several results (for example, anisimova et al., 2001; anisimov et al., 1998b) are illustrating that high pressure flow diffusion chamber needs to be designed for the profound research of multichannel nucleation. 3.7 nucleation rate surface singularity fig. 5 illustrates the example of the experimental data where the gap in nucleation rate can be seen. gap is initiated by the phase change in critical embryos which are generated from supersaturated vapor-gas system (anisimov et al., 1998). a conceptual problem in vapor liquid nucleation is its treatment as a single component problem. in general, one should consider the carrier gas-vapor nucleation as a binary system, such as it was proved by anisimov et al. (1998, 2000a, b). another aspect of nucleation experiments is appeared when the probability of phase change in the critical embryo exists. phase change means the statistical prevail of other phase state embryos generation over the initially prevailing embryo phase state. that result was obtained on the example of glycerine vapour nucleation and condensation (anisimova et al., 2001). these authors have found bimodal aerosol particle size distribution which was initiated by two different embryo phase states. 1,3 propanediol vapor activity, log a 0.55 0.60 0.65 0.70 0.75 n uc le at io n r at e, lo g j (c m s ) 1 2 3 4 5 6 7 -3 -1 nucleation temperature, t (k) 321 323 325 328 330 332 334 336 339 341 343 345 347 350 352 figure 5. nucleation rates for 1,3 propanediol – sulphur hexafluoride system at total pressure 0.3 mpa. the gap in nucleation rate is associated with phase state change in the condensate critical embryos of binary system (anisimov et al., 2000a) anisimova et al. (2001) examined nucleation in the vicinity of the glycerine triple point using fdc and applied the particle size measurements to observe two condensed phases which are generated via two independent nucleation channels, representing two nucleation rate surfaces. the experimental scheme by anisimova et food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 51 al. (2001) with the size distribution measurements can be an effective tool for independent measurements of nucleation rates for each nucleation channel. a major problem in detection of the nucleation rate surface singularities is the random errors inherent in any experimental data (fig. 5). this problem exists even one has experimental results of high accuracy such as those of strey et al. (1995), where a nucleation rate surface singularity was missed. anisimov et al. (2000c) suggested using the mathematical condition of the continuity and monotony for fdc data analysis to find the anomalies of the nucleation rate surfaces. 4. conclusions flow diffusion chamber as an instrument for vapor homogeneous nucleation rate measurements was appeared at the end of 1970th. the flow diffusion chamber is presenting the world wide distributed tool for the vapor nucleation rate measurements at low and elevated pressure conditions from 0.03 mpa to 0.4 mpa and nucleation temperatures from -30 to 80 oc currently. fdc is permanently developing system, which can be used for single as well as multi-component vapor–gas nucleation under the relatively wide variation of nucleation conditions. fdc can be developed easily for measurements at pressures up to 20 mpa. several results (for example, anisimova et al., 2001; anisimov et al., 1998) are illustrating that high pressure flow diffusion chamber needs to be designed for the profound research of multi-channel nucleation. the introduction of one or several nucleation standard(s) is an actual current problem. the dipper understanding of the carrier-gas effects will clarify the nature of the different experimental set data inconsistencies (anisimov et al., 2009). fdc is capable tool for the vapor nucleation rate measurements as well as other tools such as the expansion & jet techniques and the static cloud chambers. 5. references aitken, j., (1888) trans. roy. soc. edinburgh, 35, 1. allen, l.b. and kassner, j.l., jr. (1969) j. colloid interface sci. 30, 8193. amelin, a.g. (1948) colloidn. zh. (russian), 10, 169-176. anisimova, l., hohke, p.k., and terry, j. (2001) j. chem. phys., 114, 9852-9855. anisimov, m.p., costrovskii, v.g., and shtein, m.s. (1978a) colloidn. zh. (russian) 40(1), 116-121. anisimov, m.p., costrovskii, v.g., and shtein, m.s. (1978b) colloidn. zh. (russian) 40(2), 317-321. anisimov, m.p., costrovskii, v.g., shtein, m.s, and mikheev, v.b. (1980) colloidn. zh. (russian) 42, 941-945. anisimov, m.p. and cherevko, a.g. (1985) j. aerosol sci., 16(2), 97-107. anisimov, m. p., hameri, k., kulmala, m., ovchinnikova, t.e. (1993) report series in aerosol sci., 23., 19-24. anisimov, m.p., hameri, k., kulmala, m. (1994) j. aerosol sci., 25(1), 23-32. anisimov, m.p., koropchak, j.a., nasibulin, a.g., and timoshina, l.v. (1998) j. chem. phys., 109(22): p. 10004-10010 anisimov, m.p., koropchak, j.a., nasibulin, a.g., and timoshina, l.v. (2000a) j. chem. phys., 112(22), 9917-9928. anisimov, m.p.; nasibulin, a.g., shandakov, s.d., (2000b) j. chem. phys. 112(5), 2348-2354. anisimov, m.p.; nasibulin, a.g., shandakov, s.d., (2000c). j. chem. phys. 112(5) 2348 2354 anisimov, m.p. and hopke, p.k. (2001) j. phys. chem. b. 105, 11817-11822 anisimov, m.p., fominykh, e.g., akimov, s.v., hopke, p.k. (2009) j. aerosol sci. 40, 733-746 brock, j.r., kuhn, p.j. and zenavi, d. (1986). j. aerosol sci., 17, 11-22. brock, j.r., kuhn, p.j. and zenavi, d. (1988). j. aerosol sci., 19, 413-424. brus, d., hyvarinen, a.. zdimal, v. and lihavainen h. (2005) j. chem. phys., 122, 214506. herrmann, e., hyvarinen, a.p., brus, d., lihavainen, h., kulmala, m. (2009) j phys chem a. feb 3. [e-pub ahead of print] food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 52 kim, y.j., wislouzil, b.e., wilemski, g., wolk, j., and strey, r. (2004). j. phys. chem. a 108, 4365-4377 langsdorf, a. (1939) rev. sci. instr. 10, 91-103. mitrakos, d., zdímal, v., brus, d., housiadas, c. (2008). j chem phys. aug 7, 129(5), 054503. nguyen, h. v., okuyama, k., mimura, t., kousaka, y., flagan, r.c., and seinfeld, j. h. (1987) j. colloid interface sci., 119, (2), 491504 okuyama, k., kousaka, y., kreidenweis, s., flagan, r.c., and seinfeld, j.h. (1988). j. chem. phys., 89(10), 6442-6453. ostwald, w. (1896-1903) lehrbuch der allgemeinen chemie. leipzig, w.engelmann. peters f. and paikert b. (1989) j. chem. phys. 91, 5672-5678. strey, r. and wagner, p.e., schmeling, t. (1986) j. chem. phys. 84(4), 2325 2335; strey, r. and wagner, p.e. (1988) j. aerosol sci., 19, 813-816. strey, r.; viisanen, y.; wagner, p.e. (1995) j. chem. phys. 103(10), 4333-4345. sinclair, d. and la-mer v.k. (1949) chem rev. 44, 245-251 volmer, m. (1939) kinetik der phasenbildung. dresden-leipzig, t. steinkopf wagner, p.e. and anisimov, m.p. (1993) j. aerosol sci., 24, suppl.1., 103-104. wilck, m., hameri, k., stratmann, f., and kulmala, m. (1998) j.aerosol sci. 29(8), 899-911. wilemski, g. (1975), j. chem. phys., 62, 37633771. 148 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 22015, pag. 148 156 prebiotic effects of inulin and acacia gum (review) *gjorenakov1, darina georgieva1, nastia ivanova1, stanka damyanova1, viktorija stamatovska2, ljupka necinova2 1department of biotechnology and food technologies, university of ruse “angel kanchev”, branch razgrad, aprilsko vastanie blvd. 47, razgrad 7200, bulgaria, gore_nakov@hotmail.com, nastiav2001@yahoo.com 2faculty of technology and technical science-veles, universityss.kliment ohridski bitola, r.macedonia, stamvikima@gmail.com *corresponding author received april 26th 2015, accepted june 16th 2015 abstract: prebiotics have great potential as agents to improve or maintain a balanced intestinal microflora to enhance health and wellbeing. they are non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract. key to this is the specificity of microbial changes.thanks to the methodological and fundamental research of microbiologists, enormous progress has been made in understanding the gut microbiota. a large number of human intervention studies have been performed that have demonstrated that dietary consumption of certain food products can result in statistically significant changes in the composition of the gut microbiota in line with the prebiotic concept. the concept prebiotics is to enhance the growth of beneficial bacteria in the lower intestine. there is much interest in increasing the numbers and activities of beneficial bacteria (bifidobacteria) in the large gut, preferably at the expense of more harmfulbacteria. the focus of this review has been to point out the prebiotic effects (bifidogenic effects) of acacia gum and inulin. some effects attributed to selected prebiotics have been proved by clinical trials, while others have been acquired on the basis of in vitro tests. keywords: prebiotics, inulin, acacia gum, prebiotic effects (bifidogenic effects) 1. introduction prebiotics are a category of nutritional compounds grouped together, not necessarily by structural similarities, but by ability to promote the growth of specific beneficial (probiotic) gut bacteria. many dietary fibers, especially soluble fibers, exhibit some prebiotic activity; however, non-fiber compounds are not precluded from being classified as prebiotics presuming they meet the requisite functional criteria [1]. gibson and roberfroid (1995) defined prebiotics as ‘‘a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health.’’ given the large number of bacterial strains present in the gastrointestinal (gi) tract, some of which are non-cultivable, the definition was revised to “a selectively fermented ingredient that allows specific changes, both in the composition and/or http://www.fia.usv.ro/fiajournal mailto:gore_nakov@hotmail.com mailto:nastiav2001@yahoo.com mailto:stamvikima@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 149 activity in the gastrointestinal microflora that confers benefits upon host well-being and health” [3]. roberfroid (2007) updated this definition in review article on prebiotics. definitions of prebiotics typically have in common an emphasis on the compound being non-digestible (and hence subject to colonic enzymatic activity and fermentation by colonic bacteria) and able to selectively stimulate the growth of one or more desirable or health-enhancing types of gut bacteria. while definitions of prebiotics do not emphasize a specific bacterial group, the number and/or activity of bifidobacteria and other lactic acidproducing bacteria must be increased for the compound to qualify as a prebiotic. either implicitly or explicitly within most definitions is the concept that the compound improve the health of the subject consuming it [1]. prebiotics are a very specific type of food. while many of the food ingredients we consume are digested immediately, prebiotics are a healthy non-digestible food ingredient. futhermore, prebiotics are heat resistant, which keep them intact during the baking process and allow them to be incorporated into every day food choices. by consuming a non-digestible ingredient, it allows for growth of bio-cultures by reaching the intestine unaffected by the digestion process. a prebiotic effect occurs when there is an increase in the activity of healthy bacteria in the human intestine. the prebiotics stimulate the growth of healthy bacteria such as bifidobacteria and lactobacilli in the gut and increase resistance to invading pathogens. this effect is induced by consuming functional foods that contain prebiotics. these foods induce metabolic activity, leading to health improvements. healthy bacteria in the intestine can combat unwanted bacteria, providing a number of health benefits [5]. a prebiotic effect has been attributed to many food components, sometimes without due consideration to the criteria required. in particular, many food oligosaccharides and polysaccharides (including dietary fibre) have been claimed to have prebiotic activity, but not all dietary carbohydrates are prebiotics [3-4]. for a food ingredient to be classified as a prebiotic it must fulfil [6] the following criteria:  neither be hydrolyzed, nor absorbed in the upper part of the gastrointestinal tract;  be selectively fermented by one or a limited number of potentially beneficial bacteria commensal to the colon, e.g. bifidobacteria and lactobacilli, which are stimulated to grow and/or become metabolically activated;  prebiotics must be able to alter the colonic microflora towards a healthier composition, for example by increasing numbers of saccharolytic species while reducing putrefactive microorganisms. inulin, oligofructose or fructooligosaccharide (fos) are the best studied prebiotics.to date, all known and suspected prebiotics are carbohydrate compounds, primarily oligosaccharides, known to resist digestion in the human small intestine and reach the colon where they are fermented by the gut microflora. studies have provided evidence that inulin and oligofructose (of), lactulose, and resistant starch (rs) meet all aspects of the definition, including the stimulation of bifidobacterium, a beneficial bacterial genus. other isolated carbohydrates and carbohydrate-containing foods, including galactooligosaccharides (gos), transgalactooligosaccharides (tos), polydextrose, wheat dextrin, acacia gum, psyllium, banana, whole grain wheat, and whole grain corn also have prebiotic effects [7]. predominance of bifidobacteria in the large intestine is essential for the prevention of many diseases and for maintaining good health. one main food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 150 strategy is the prebiotic approach – the use of selective carbohydrate substrates in the diet for the growth of indigenous bifidobacteria. to be effective, these carbohydrates must reach the colon undigested and unabsorbed in the upper gastrointestinal tract and be selectively utilized by the bacteria present there [8]. inulin and acacia gum are examples of such carbohydrates. in this review, we present an overview of the prebiotic effects (bifidogenic effects) of inulin and acacia gum. 2. inulin inulin is a non-digestible oligosaccharide that, for nutritional labelling, is classified as dietary fibre [3]. inulin is a soluble dietary fibre. inulin and oligofructose belong to a class of carbohydrates known as fructans. the main sources of inulin and oligofructose that are used in the food industry are chicory and jerusalem artichoke. they are considered as functional food ingredients since they affect physiological and biochemical processes in rats and human beings, resulting in better health and reduction in the risk of many diseases. experimental studies have shown their use as bifidogenic agents, stimulating the immune system of the body, decreasing the levels of pathogenic bacteria in the intestine, relieving constipation, decreasing the risk of osteoporosis by increasing mineral absorption, especially of calcium, reducing the risk of atherosclerosis by lowering the synthesis of triglycerides and fatty acids in the liver and decreasing their level in serum [8]. inulin is a generic term that covers all linear fructans with β (2–1) bonds, with a variable degree of polymerization [9]. this specific type of glycosidic bond gives inulin its unique structural and physiological properties. because of the beta configuration of the bonds between fructose monomers, inulin-type fructans resist enzymatic hydrolysis by human salivary and small intestinal digestive enzymes – specific for alpha-glycosidic bonds. as a result, inulin-type fructans are indigestible and are fermented in the colon [1], [4]. the "bifidogenic efect" can be defined by a specific stimulation of lactic acid bacteria. lactic acid bacteria including lactobacilli and bifidobacteria are thought to be beneficial for the host as they are associated with beneficial health effects. this group of bacteria can protect the host by inhibiting potential harmful bacteria (eg. clostridium, staphylococcus,…) through different mechanisms [10]. the bifidogenic effect of inulin and oligofructose is now well established in various studies, not only in adult participants but also in other age groups. this bifidogenic shift in the composition of the colonic microbiota is likely the basis for the impact of these prebiotic compounds on various parameters of colonic function. mainly from animal and in vitro studies and also from some human trials, there are indications, for instance, that inulin-type fructans may reduce the production of potentially toxic metabolites and may induce important immune-mediated effects [9]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 151 fig. 1: prebiotic effect of inulin consumption on the composition of colonic microbiota[11] gibson and wang (1994) confirmed the prebiotic effects of inulin and oligofructose in an in vitro study. the fermentability was compared to a range of reference carbohydrates in batch culture. bacterial growth data showed preferential fermentation by bifidobacteria while populations of escherichia coli and clostridium perfringens were maintained at relatively low levels [6]. gibson et al. (1995) studied the selective stimulation of bifidobacteria by inulin and oligofructose in a 45-day study of eight healthy male human subjects. volunteers were fed controlled diets of 15 g/d sucrose for the first 15 days followed by 15 g/d oligofructose for a further 15 days. four volunteers went on to consume 15 g/d inulin for the final 15 days of the study [6]. the studies of gibson et al. (1995) showed that oligofructose and inulin significantly modified the in vivo composition of the microbiota by stimulating the growth of bifidobacteria [8]. results from kaur and gupta (2002) have shown that ingestion of inulin compared to other sources of carbohydrate like sucrose, could significantly reduce the count of pathogenic bacteria such as bacteroids, fusobacteria and clostridia and increase the count of positive microorganisms such as bifidobacteria [14]. similar human studies in adult european, japanese and north american populations have been reported for inulin using different daily doses. it has been suggested that the beneficial effect of inulin could be due to the ability of bifidobacteria to change the colonic environment by inhibiting detrimental bacteria via the formation of bacteriocins, the successful competition for substrates or adhesion sites on the gut epithelium, and stimulation of the immune system [8]. in vitro data supporting the selective stimulation of bacterial growth by inulin has been generated in numerous studies that are summarised in table 1. this has been carried out in defined pure culture fermentation and by using a mixed faecal inocula in both batch and continuous culture [3]. inulin is naturally present in many different foods. some every day foods, such as asparagus, leek, onions, banana, wheat and garlic are sources of inulin. higher concentrations exist in herbs. dandelion root, elecampane root and chicory root all have large amounts of inulin. chicory root is the most common source of inulin due to its extremely high concentration as well as its similarities to the sugar beet. the methods used for the extraction of inulin from the chicory root are comparable to the extraction of sucrose from the sugar beet. this allows for similar equipment to be used, making it easier for chicory root producers to cultivate inulin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 152 table 1. studies made to demonstrate the in vitro selectivity of inulin in pure culture, mixed batch culture and mixed continuous culture fermentation study observations reference examining the growth of bifidobacteria on different types of oligofructose in pure culture. eight species tested as well as species of clostridium, bacteroides, enterococci and escherichia coli. linear oligofructose had more of a bifidogenic effect than greater molecularmass molecules and branched-chain varieties. bifidobacterium species showed a preference for fructans compared with glucose. gibson & wang (1994b) species of bifidobacterium (longum, breve, pseudocatenulatum, adolescentis) were tested in pure culture for their ability of fermenting oligofructose. b. adolescentis was seen to grow best and was able to metabolise both shortand longchain oligofructose. marx et al. (2000) the ability of bifidobacterium and lactobacillus to grow on mrs agar containing oligofructose was investigated. seven out of eight bifidobacteria and twelve out of sixteen lactobacilli were able to grow on agar containing oligofructose. kaplan & hutkins (2000) batch culture using faecal inocula to study fermentation of inulin, oligofructose, starch, polydextrose, fructose and pectin. bifidobacteria most increased with oligofructose and inulin whilst populations of e. coli and clostridium were maintained at relatively low levels. wang & gibson (1993) batch culture using faecal inocula to study fermentation of oligofructose, branched fructan, levan, maltodextrin. fluorescence in situ hybridisation revealed that branched fructan had the best prebiotic effect, followed by oligofructose. probert & gibson (2002) continuous culture fermentation to study fermentation of oligofructose. selective culturing showed bifidobacterium and, to a lesser extent, lactobacillius, preferred oligofructose to inulin and sucrose. bacteroides could not grow on oligofructose. gibson & wang (1994b) this dietary fibre is used as a prebiotic agent in functional foods to stimulate the growth of beneficial intestinal bacteria. it is soluble in hot water, allowing the inulin to be easily incorporated into drinks, dairy products, and baked goods [15]. because inulin, oligofructose, and fos are classified as soluble fibers they can be used as a means of increasing dietary fiber or to replace sugars or fats. depending on the taste, texture, and other attributes desired, different mixtures are considered for inclusion in food products like confectionery, fruit preparations, milk desserts, yogurt and fresh cheese, baked goods, chocolate, ice cream and sauces. inulin can also be used for the preparation of fructose syrups.in these applications they are considered to be a functional food ingredient, added to make health claims and/or persuade the consumer the product is a healthier choice than one that does not contain inulin-type prebiotics [8], [16]. 2. acacia gum acacia gum (gum) or gum arabic (codex alimentarius rome 2000) is a soluble dietary fibre obtained from the stems and branches of acacia senegal and acacia seyal. these trees are abundant in the central sudan, central africa and in west africa [17]. this product is known under different names, gum arabic (codex alimentarius rome 2000) [10]. it is composed mainly of complex food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 153 polysaccharides (95%) that consist of highly branched galactan polymers, with galactose and/or arabinose side chains, possibly terminated by rhamnose or glucuronic acid residues [17]. acacia gum has readily been used in the food industry for decades as a food additive. the joint fao/who expert committee on food additives recognizes acacia gum as a food additive (ins 414) that can be used with no specified adi. in the usa, acacia gum enjoys a gras (generally recognised as safe) classification [18]. in europe, acacia gum is also recognized as a food additive (e 414) under the “quantum satis” principle [19]. in 2001 the french administration has officially recognized ag as a dietary soluble fiber which allows the fiber content of acacia gum to be taken into account for the nutritional content labeling of fiber [10]. more than twenty studies have been performed since the late 70’s to understand the relationships between acacia gum and the colonic microflora. it is widely recognized that acacia gum induces [10]: a bifidogenic effect; a specific stimulation of scfas production; a high gut tolerance. while >80% of current production is used by the food industry for various applications (emulsification, encapsulation, coating, gum candies, etc.), gum is traditionally consumed by african and indian populations to improve digestive comfort and intestinal transit. in vitro, its fermentation is slow and supports the growth of bifidobacteria. thus, gum may be a bifidogenic soluble dietary fibre that would not induce uncomfortable intestinal side effects in the healthy consumer [17]. first studies performed in vitro showed that among different genus of bacteria from human faeces, bifidobacteria strains were able to use acacia gum for their growth [20]. various studies mention its potential as a prebiotic agent. wyatt et al. addressed the issue in one volunteer by applying 10 g gum arabic and noted an increase in the numbers of bacteroides and bifidobacterium [22]. in vitro studies showed that gum supported the growth of pure cultures of bifidobacteria strains and increased total lactic acid-producing bacteria counts in continuous cultures of human faecal microflora. in the stools of one volunteer, wyatt et al. measured an increase in the proportion of bacteria able to ferment the gum, among which species of bacteroides and bifidobacterium were found. these preliminary findings suggested that gum could be prebiotic [17]. the bifidogenic properties of acacia gum were confirmed in a single blind controlled study performed on 10 healthy volunteers consuming either acacia gum (fibregum™) at the dose of 10 g/d and 15 g/d during 10 days or sucrose as control at the same dose. concentrations of bifidobacteria, lactobacilli and total lactic acid bacteria groups were significantly increased with acacia gum at the dose of 10 g/d compared to control without affecting neutral groups as bacteroides. the bifidogenic effect was even more pronounced (+1 log) in subjects having low initial bifidobacteria count (<9.5 log). the effect was also significant at the dose of 15 g/d [17-18]. lower dose (6 g/d) of acacia gum was tested in a randomized double blind controlled study involving 96 healthy volunteers. after 1 week of consumption, 6 g/d of acacia gum (fibregum™) induced a 0.7 log increase of faecal bifidobacteria compared to initial value that was at the limit of statistical significance (p=0.09). moreover, the effect of this fiber was greater compared to fos that induced a 0.3 log increase. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 154 these experimental studies allow concluding that acacia gum has a prebiotic effect at the dose of 10 g/d and that the extent of the effect is at least equal to the effect of fos [10], [18]. due to the growing range of supplemented products, consumers are exposed to increasing amounts of prebiotics and may ingest daily doses above the threshold for induction of side effects. in vitro fermentation time for acacia gum is significantly longer than that for fos and studies have suggested a more favourable abdominal side-effect profile. replacement of a proportion of fos by acacia gum may thus attenuate the side effects of prebiotics with the additional advantage of a synergistic effect on the growth of intestinal bifidobacteria [23]. the prebiotic efficacy of acacia gum was also clinically confirmed when consumed for up to 4 weeks [22]. daily consumption of water was taken as the negative control and that of 10 g inulin as the positive control. compared with the negative control, the number of bifidobacteria and lactobacilli, 4 weeks after consumption were significantly higher for acacia gum (10g): approximately 40-fold and 6-fold difference in outgrowth. moreover, at this dose the numbers of bifidobacteria and lactobacilli were significantly higher for acacia gum than for inulin: respectively an approximately 10-fold and 7-fold difference. (graph 1) all subjects tolerated the 4 weeks of consumption of acacia gum and no significant changes were observed during the intervention period as compared to subjects who consumed the negative or positive control. fig. 2.the change in 10-logarithmic numbers of bacteria during 4 weeks of consumption of 10g acacia gum, 10g inulin or water [10] acacia gum was shown to produce a greater increase in bifidobacteria and lactobacilli than an equal dose of inulin, and resulted in fewer gastrointestinal side effects, such as gas and bloating [22] when used as food additive, acacia gum is a texturiser, thickener, stabiliser, emulsifier and coating agent. such variety of functions with a single product is unique. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 155 when used as ingredient for its health benefits, acacia gum is a dietary fibre with prebiotic effects.the health benefits of acacia gum are given by the non digestible behaviour of the high molecular weight molecule. acacia gum is a dietary fibre (more than 90 % on dry basis) with high gut tolerance and prebiotic effect. low viscosity in water system, low calorific value (1,8 – 2 kcal/g) and non-cariogenic effect explain the wide field of applications in dietary food and nutraceutical products [24]. 4. conclusion prebiotics are nondigestible food ingredients that benefit the host by selectively stimulating the growth or activity of one or a limited number of bacteria in the colon. because of their positive attributes bifidobacteria and lactobacilli are the most frequent target organisms. several researchers believe a potentially more important factor in fluencing growth of bifidobacteria, as well as other microorganisms, is the initial presence and counts of specific gut microorganisms prior to supplementation. while more research in this area is needed, it is at least possible that in order to boost the amounts of a specific bifidobacteria species or other bacteria, that species must initially be present. inulin and acacia gum have been demonstrated to be effective prebiotics. this has been shown through both invitro and in vivo assessments in different laboratories. experimental studies have shown their use as bifidogenic agents and decreasing the levels of pathogenic bacteria. because of their recognised prebiotic properties, principally the selective stimulation of colonic bifidobacteria, both inulin and acacia gum are increasingly used in new food product developments. 5. references [1] kelly g., inulin-type prebiotics – a review: part 1, alternative medicine review, volume 13, number 4, (2008). [2] gibson g.r., roberfroid m.b., dietary modulation of the human colonic microbiota: introducing the concept of prebiotics, j. nutr. 125: 1401-1412, (1995). [3] gibson g.r., probert h.m., loo j.v., rastall r.a., roberfroid m.b., dietary modulation of the human colonic microbiota: updating the concept of prebiotics. nutr res rev 17: 259-275, (2004). [4] roberfroid m. prebiotics: the concept revisited, j. nutr.,137:830s-837s, (2007) [5]http://www.prebiotic.ca/prebiotic_fibre.html. [6] kolida s., tuohy k., gibson g.r., prebiotic effects of inulin and oligofructose, british journal of nutrition, 87, suppl. 2, s193– s197, (2002). [7] slavin j., fiber and prebiotics: mechanisms and health benefits, nutrients, 5, 1417-1435, (2013). [8] kaur n., gupta a. k., applications of inulin and oligofructose in health and nutrition, j. biosci. 27 703–714, (2002). [9] meyer d., stasse-wolthuis m., the bifidogenic effect of inulin and oligofructose and its consequences for gut health, european journal of clinical nutrition, 63, 1277–1289, (2009). [10] fibregum™, colloides naturels international, www.cniworld.com (2010). [11] http://www.inspiredbyinulin.com/getinspired/healthy-food/inulin-and-weight management.html. [12] gibson g.r., wang x., enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture, fems microbiology letters, 118, 121–128, (1994). [13] gibson g. r., beatty e. r., wang x. cummings j. h., selective stimulation of bifidobacteria in the human colon by oligofructose and inulin, gastroenterology, 108, 975–982, (1995). [14] miremadi, f., shah, n. p. applications of inulin and probiotics in health and nutrition, international food research journal 19(4): 13371350 (2012). [15] http://www.prebiotic.ca/inulin.html http://www.prebiotic.ca/prebiotic_fibre.html. http://www.cniworld.com http://www.inspiredbyinulin.com/gethttp://www.prebiotic.ca/inulin.html food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 gjore nakov, darina georgieva, nastya ivanova, stanka damyanova, viktorija stamatovska, ljupka necinova, prebiotic effects of inulin and gum acacia (review), food and environment safety, volume xiv, issue 2 – 2015, pag. 148 – 156 156 [16] coussement p.a. inulin and oligofructose: safe intakes and legal status. j nutr., 129:1412s1417s, (1999). [17] cherbut c., michel c., raison v., kravtchenko t., severine m., acacia gum is a bifidogenic dietary fibre with high digestive tolerance in healthy humans, microbial ecology in health and disease, 15: 43 -50, (2003). [18] baray s., chapter 7: acacia gum, section i:soluble fibers, in: fiber ingredients: food applications and health benefits, (edited by cho s.s., samuel p.). crc press,taylor & francis group, 121-131 (2009). [19]http://www.food.gov.uk/science/additives/enu mberlist#.u43q7_l_uso. [20] salyers aa. et al. degradation of polysaccharides by intestinal bacterial enzymes. am. j. clin. nutr. 31:s128-130, (1978). [21] wyatt t.m., bayliss c.e., holcroft j.d., a change in human faecal flora in response to inclusion of gum arabic in the diet, british journal of nutrition, 55, 261–266, (1986). [22] calame w, weseler ar, viebke c, flynn c, siemensma ad. gum arabic establishes prebiotic functionality in healthy human volunteers in a dose-dependent manner, british journal of nutrition, 100, 1269–1275, (2008). [23] goetze o., fruehauf h., pohl d., giarre m., rochat f., ornstein k., menned., fried m.,thumshirn m., effect of a prebiotic mixture on intestinal comfort and general wellbeing in health, british journal of nutrition, 100, 1077– 1085, (2008). [24] http://www.treegums.org/products/gum-acacia/ http://www.food.gov.uk/science/additives/enu http://www.treegums.org/products/gum-acacia/ food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 5 analysis of chlorinated phenols in water *ján ilavský1, ján hriv ák1, danka barloková1 1department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovak republic. jan.ilavsky@stuba.sk, danka.barlokova@stuba.sk *corresponding author received 31 august 2012, accepted 5 september 2012 abstract: glass capillary gas chromatography was used for the examination of solvents (benzene, toluene, dichloromethane, trichloromethane, diethyl ether and ethyl acetate) for the extraction of chlorinated phenols (18 compounds) from water at different ph values (2.0; 4.3 and 7.3). the concentration of the individual chlorophenols in the model sample was 10 g.l-1 of water. the recoveries of the studied compounds were evaluated relative to internal standard (1-cl-n-octadecane). the highest recoveries were obtained for diethyl ether and ethyl acetate. in this case the recoveries are slight influenced by the change of ph of water. the lowest recoveries were obtained for phenol. recovery of the other chlorophenols was above 60 %. the results of the recoveries showed that this method is possible to use for routine analysis of chlorophenols in water. keywords: capillary gas chromatography, microextraction, chlorinated phenols, water analysis 1. introduction phenols are one of the most important and widely found compounds in water. they have an acidic nature and are easily extracted from water, especially from water in plants. phenols are compounds of a natural as well as an artificial origin, which are extracted with difficulties. their content decreases, particularly during vegetative periods. they get into water from waste produced by tanneries or woodworking industry. the need to monitor phenols in water arises because of their toxicity. a few mg.l-1 of phenol in industrial wastewater can poison percolating filters with activated cultures. phenols, which penetrate groundwater through infiltration from various sources, negatively affect the organoleptic properties of water in trace concentrations, especially after chloration [1]. the presence of phenols also increases the effect of polyaromatic hydrocarbons on live organisms and affects biological proportions in rivers [2]. owing to these facts, the monitoring of phenols in water is important and, at present, attracts attention (phenol and its derivates belong to a class of high priority pollutants). a photometric method based on the coloured reaction of phenols and 4aminoantipyrine [3] is used in the determining the presence of phenols in drinking and surface waters. this method is also recommended for the standard methods of water analyses in our country [4]. during its reaction with antipyrine the total phenol content is evaluated, although some substituted phenols in o-, m-, and ppositions (carboxyl, metoxyl, sulfonate or halogenide groups) can react. other phenols with a substituent in the para position do not react sufficiently. the coloured complex is measured at 460 nm (after extraction using chloroform) or 510 nm (upon direct determination). mailto:jan.ilavsky:@stuba.sk mailto:danka.barlokova:@stuba.sk food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 6 a spectrophotometric method with pnitroaniline [4] is often used. the constantly expanding spectrum of phenol-like agents in water creates the need to identify its separate components. there are various chromatographic methods, which are suitable for the analyses of trace concentrations of these components. the study [5] provides a survey of chromatographic methods applied in phenol analyses. considering velocity, sensitivity, effectiveness and multi-component character, various types of gas and liquid chromatography are applied in phenol analyses. first and foremost, the development of highly effective insulating methods and separating systems is a fundamental step in successful analyses of phenols in water through gas chromatography. the advantages of direct water dosing into the injector of a gas chromatograph are in the simplicity of the analysis, velocity, quantification and the lack of problems relating to the purification of the solvents and the pretreatment extract. on the other hand, there are problems with the rest of the salts deposited in the injector after the water evaporates, which causes impairment of the column and a low degree of sensitivity. in the direct injection of water samples, filling columns with a stationary phase or columns filled with rigid sorbent (for example, tenax gc), which withstand water vapour [6], are applied. in spite of this fact, the process of determining the presence of phenols results in peak tailing and shadow effects. during the application of the stationary phase on tenax (for example, 5% of os-138) [7], the suppression of sorptive effects, good peak symmetries, a high response by the fid detector and the sensitivity of the analysis were achieved. in standard water test methods with the direct injection of a water sample [3] are recommended in analysing phenols in water, the concentration of which exceed 1 mg.l-1. this method is recommended in determining phenol, o-, m-, and p-cresol, o-, m-, and p-chlorophenol, 2.3-, 2.4, 2.5, and 3.4-dichlorophenol. carbowax 20mtpa in the amount of 20% per chromosorb w or 5% of ffap per chromosorb w are used to fill the chromatographic columns. since the shadow effect is to be eliminated (false phenol peaks), multiple clean water injection between separate analyses of the water samples is necessary. classical separating methods, particularly various types of extractions (for example, extraction by non-ionic sorbents, annexes, thermal desorption or dissolvents), are used in removing phenols from water samples. the current determination of eleven priority pollutants in waste water (a us epa method) is based on the application of the acidification of a sample to ph 2, the extraction of dichloromethane (3 x 60 ml), drying by waterless sodium sulfate, devaporation and direct gc determination using fid detection in glass columns with a liquid se-54 phase or filling columns (1% of sp–1240 da on supelcoport). some interfering components can be removed by water extraction at ph 12, but only at the expense of partial losses of some phenols, mainly 2.4-dimethyl phenol. the limit of this method for detecting phenol ranges from 0.1 µg.l-1 to 7.4 µg.l-1 for pentachlorophenol [8]. phenols, which belong among a class of priority pollutants, may be separated and analyzed in subnanogram amounts on a capillary column coated by se-30 [9]. for chromatographic identification of separate phenols in complex mixtures, the eluting characteristics of over 50 compounds on a capillary column with superox 20 m [10] or ov-1701 [11] can be applied. phenols can be analyzed by microextraction in a glass syringe [12]. a water sample of 20 ml saturated by nacl (6 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 7 mol.l-1) at ph 2 is extracted for 15 minutes by a 0.5 ml mixture of butylacetate and hexanol. as far as phenols, cresols and xylenols are concerned, 90 % 98 % of the recovery at 5 µg.l-1 to 50 µg.l-1 of the concentration is achieved. in the study in [13] chlorophenols were extracted from an acidified water sample (0.5 mol.l-1 of h2so4) at ph 5 using three doses of toluene (50, 25, 25 ml) in a separating funnel. after thickening in a revolving vacuum evaporator, the mixed toluene extracts were analyzed through gas chromatography directly or after derivatization. in the study in [14] the recovery of 25 alkylphenols through extraction by various solvents (pentane, tetrachloromethane, trichloromethane, dichloromethane, benzene, diethyl ether) was observed. the extracts were analyzed directly by capillary gc using fid detection. a glass capillary column (50 m x 0.25 mm) with tri-2,4xylenyl phosphate liquid stationary phase were used in separating the individual components. the results indicated that the increase in dissolvent polarity resulted in an increase in the recovery of extracted alkylphenol. the best results were achieved by using diethyl ether, and the lowest for phenol and its lower alkyl derivates. in paper [15] were achieved the similar results at the extraction of chlorinated phenols by using diethyl ether. analysis acidified fraction after extracting samples of priority pollutants is describe in paper [16]. a water sample of ph 11 was extracted using dichloromethane, and the neutral and basic fractions were separated. after ph treatment to the value of 2, an acidic fracture, containing phenol, 2,4dimethyl phenol, chlorine and phenol nitroderivates, was separated. the study in [17] deals with the extraction of phenols by means of the continual distillation using water vapor or extraction. if a sample is acidified to ph 1 and salt displacement by nacl is used, this distillation method achieves about 10 % effectiveness from 0.1 to 30 mg.l-1. the whole system reaches a stable state after two hours. the phenols are extracted in a small amount of diethyl ether so another thickening for chromatographic determination is not necessary. in this method the double separation of phenols from water, extraction and distillation are applied. the limit of determination in splitless injection and using capillary columns is about 10 µg.l-1. in gc analyses of phenols and chlorinated phenols, enrichment by distillation methods is often used. even very simple distillation by means of common laboratory devices is an efficient technique in the treatment of phenols and chlorinated phenols. for example, in study [18], 50 ml of a water sample were distilled at the same time with nacl until a saturated solution was created. after distilling the first 5 ml of water, the recovery of phenols exceeding 80% were identified. there was 1 mg.l-1 of concentrated phenol in the water sample. distillation methods are applied when a spectrophotometric terminal is used for determining. recently, the most common method has been the extraction by diethyl ether or dichloromethane from an acidified solution. in the study in [19] sorption in a macroporous polymer sorbent separon se combined with thermal desorption were applied. likewise in study [20] its authors used amberlit xad and activated carbon to achieve a recovery exceeding 83% for 17 phenolic compounds to separate the phenols from the water. the above stated amberlit xad 2 and xad 4 macroreticular styrene divinylbenzene copolymers were used to separate various model samples of organic additives, including phenols, from drinking and waste water [21]. after the expulsion food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 8 of the absorbed substances by diethyl ether and the drying and evaporation of part of the diethyl ether, the extract was injected into a gas chromatograph. the recovery of phenols (after ph treatment) exceeded 80 %. phenols and chlorine phenols can be analyzed without the treatment of a sample or through derivatization, as a result of which molecular polarity and the boiling temperature increase and the parameters of gas-chromatographic separation (peak tailing, creation of hydrogen bridges, detection sensitivity) and also the separation and concentration of these substances from water improve [5, 22]. one of the methods for increasing the sensitivity of the ecd detector is the direct bromation of phenols in a water sample, the extraction of tribromine phenols and a chromatographic analysis using ecd [23]. a water sample of 300 ml is acted upon by an acidified solution of potassium bromide (10 g.l-1) for 10 minutes. after removing the excess bromine by sodium thiosulphate, the water is extracted from 10 ml of hexane, the hexane layer is dried using 2 g of waterless sodium sulfate, and the hexane extract is analyzed by gas chromatography with ecd. the injection of 5 µl (without thickening the extract), the detection limit for the phenol is 0.1 µg.l-1. phenols and chlorine phenols can be derivated directly in an alkaline water solution. acetyl phenols or acetylchlorinated phenols are created during acetylation. the acetyl derivates obtained are extracted from the water more easily than from the original substances. the study in [24, 25] describes a method, according to which 10 g of nahco3 and 0.5 ml of acetanhydride are added to 250 ml of water. the solution is shaken until the carbon dioxide starts to escape. then the created acetates are separated from the water through extraction by dichloromethane (10 to 30 ml of ch2cl2 two or three times) [24, 25] or through sorption on chromosorb 102 polymer sorbent with the following elution using carbon disulfide [26] or sorption on modified silicagel c18 and elution using benzene [25]. such methods are used deriving phenol, according to which the deriving reaction is carried out after the extraction and concentration of the phenols from the water into the extract. for example, [28] states that 3 ml of potassium carbonate solution (0.1 mol.l-1), 1 ml benzene or hexane and 50 µl acetanhydride are added to an organic extract in a test tube. after shaking, the phenols turn into acetates, and the derivates are converted to an organic phase. in the case of chlorinated phenols, chromatographic analysis of the extract is carried out by means of ecd. there are numerous variations of this method [30], and the recovery of chlorine phenol acetylation and its subsequent extraction exceeds 90%. with the exception of acetylation, the derivation of phenols and chlorinated phenols during routine analyses to ethers by means of pentafluoro benzyl bromide is recommended. the derivate obtained can be effectively analyzed by an electron capture detector. the derivation is carried out so that 100 µl of a 10% solution of sodium carbonate and 100 µl of 5% pentafluoro benzyl bromide in acetone are added to 8 ml of an acetone solution of phenols. the reactive mixture in a corked test tube is heated to 60°c for an hour. when the reaction is complete, the mixture is evaporated to 0.5 ml, 3 ml of hexane are added, and the mixture is evaporated to 0.5 ml again. after clarifying the reaction mixture in the column using silicagel or fluorisil (chlorphenol derivates eluate in the first 8 ml of the toluene-hexane mobile phase in a proportion of 25:75), the solution is injected into a chromatographic column. chlorine phenols (22 derivates) were determined by such a method [29]. a similar method of derivation is also stated in a standard method [30], which is, food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 9 except for phenols and chlorine phenols, focused on the determination of nitrophenols, too. the experimental part of this study involves the results of the observation of the recovery of sixteen chlorinated phenols by micro-extraction of water in benzene, toluene, chlorophorm, dichlorine methane, diethyl ether and ethyl acetate. 2. experimental 2.1 instrumentation the analysis was performed with the carlo erba (vega 6000) gas chromatograph (modified for usage with capillary columns) equipped with a flame ionization detector (fid) and split/ splitless capillary injector. the chromatograph was fitted with glass capillary column (50 m x 0.25 mm i.d.) coated with tri-2,4-xylenyl phosphate liquid stationary phase. the gas chromatographic conditions were as follows: the column temperature during injection 160 oc, than programmed linearly at 3 oc/min to a final temperature of 220 oc. the injection port and detector temperature were 225 oc, carrier gas hydrogen, 1.5 l samples were injected using split/splitless. chromatograms were integrated with hp 3392a (hewlettpackard) integrator. 2.2 reagents and solutions the chlorinated phenols examined were obtained from different manufactures and were generally of 98 % purity. standard mixture of this compounds were diluted in acetone (concentration of each component of 1 mg.ml-1). the purity of the internal standard (1-cl-ndodecane) was 99% (supelco, bellefonte, pa., usa). the extraction solvents (benzene, toluene, dichloromethane, trichloromethane, diethyl ether and ethyl acetate) were highly purified and checked chromatographically. 2.3 microextraction for the microextraction a simple glass extraction (volumetric) flask (1) equipped with the conical stopper (5) was used. for n-pentane and toluene separation a separator of the thin solvent layer (2) was connected to the flask, and through the side arm, 3, pure water is added until the solvent is transferred into the dry capillary, 4, to the height required. (fig. 1) [31]. figure 1 a: the glass extraction (volumetric) flask, equipped with a male joint (1) and conical stopper (5), prepared to microextraction. b: the solvent thin layer separator (2) containing side arm for water (3), capillary for extract (4) connected to the flask (1) after extraction. if dichloromethane and chloroform is used as solvent (as a heavier liquid than water) conical stopper was used for trapping the solvent (up side down). the water layer over the solvent one prevents its evaporation (or some volatile components). the extracts are easily accessible and can be injected immediately by means of a syringe into a gas chromatograph for both cases. model water samples of 0.1 litre with known contents of studied chlorophenols were acidified to ph 4.3 or 2 with concentrated hydrochloric acid, cooled to 5 oc, extracted in the presence of internal standard (1-cl-n-dodecane) by vigorous mechanical shaking for 5 minutes and food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 10 allowed to stand until the layer separator. the volumes of the organic phase added (depending on solubility) were 0,1 ml of toluene, 0,3 ml of benzene, 0,6 ml of chloroform, 2 ml of dichloromethane, 10 ml of diethyl ether and ethyl acetate. for extraction glass extraction flask equipped with the conical stopper was used. for separation of solvent (as a lighter liquid than water) a separator of the thin solvent layer was used. the extracts were injected directly by capillary gas chromatography. 3. results and discussion figure 2 shows a chromatogram of a chloroform extract of the model water sample. the concentration of the individual chlorophenols in the model samples was 10 g.l-1 of water. the extraction recoveries relative to 1-cl-n-octadecane (recovery = 100 %) are shown in table 1 – 3 (the values represent arithmetic means of tree measurements). figure 2. chromatogram of model mixture of this chlorinated phenols (at concentration of each component of 10 g.l-1 of water) in chloroform before extraction. for identification of peaks, see table 13. is = 1-cl-n-octadecane. figure 2 table 1 extraction recoveries of chlorophenols from water (at ph 2) relative to 1-cl-n-octadecane peak no. compound recovery (%) c6h6 c7h8 chcl3 ch2cl2 c4h10o c4h8o2 1 2-chlor 2.2 0 1.8 19.6 59.3 76.2 2 phenol 0 0 0 2.5 33.2 49.0 3 2,6-dichloro 16.7 6.3 21.1 63.2 93.6 96.7 4 hexachlorobenzene 85.4 98.0 94.6 98.2 97.8 98.5 a 2,5-dichloro 13.2 8.5 18.3 46.4 91.7 92.8 5 2,4-dichloro 11.6 4.6 16.7 47.4 89.1 91.3 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 11 6 2,3-dichloro 11.7 4.6 15.7 51.6 90.8 92.6 7 2,4,6-trichloro 50.3 30.7 55.9 88.7 97.6 95.0 8 2,3,6-trichloro 51.2 27.2 53.0 91.3 98.1 95.9 9 3-chlor 1.8 2.9 0.9 8.3 83.6 90.5 10 2,3,5-trichloro 42.1 29.1 49.0 72.6 96.5 95.0 b 2,3,4-trichloro 44.4 35.4 42.7 75.0 92.9 93.8 11 2,4,5-trichloro 37.0 25.8 41.1 74.1 90.3 94.5 12 2,3,5,6-tetrachloro 84.8 74.6 87.6 82.5 92.6 93.2 13 3,5-dichloro 7.3 6.1 5.4 28.3 83.6 85.6 14 3,4-dichloro 5.7 6.8 4.8 32.7 82.9 83.7 15 pentachloro 92.4 96.0 90.5 98.8 91.6 85.5 16 3,4,5-trichloro 26.8 18.5 21.7 61.0 83.0 79.8 table 2 extraction recoveries of chlorophenols from water (ph 4.3) relative to 1-cl-n-octadecane peak no. compound recovery (%) c6h6 c7h8 chcl3 ch2cl2 c4h10o c4h8o2 1 2-chlor 2.3 0 2.0 15.5 61.0 69.6 2 phenol 0 0 0 1.8 30.2 44.4 3 2,6-dichloro 17.8 6.5 21.2 50.1 87.7 93.0 4 hexachlorobenzene 86.4 96.1 93.5 92.3 94.4 95.6 a 2,5-dichloro 13.9 9.3 15.8 33.4 85.5 91.0 5 2,4-dichloro 12.4 5.2 13.4 38.7 86.2 85.1 6 2,3-dichloro 12.4 4.9 13.0 42.4 84.5 87.3 7 2,4,6-trichloro 57.0 33.9 51.1 73.4 95.9 92.8 8 2,3,6-trichloro 56.6 29.8 51.7 84.1 94. 8 92.4 9 3-chlor 1.4 3.7 1.2 6.5 80.8 86.4 10 2,3,5-trichloro 48.5 36.1 45.5 65.2 86.9 91.3 b 2,3,4-trichloro 49.8 28.8 40.3 64.1 85.3 90.0 11 2,4,5-trichloro 41.1 30.0 41.6 67.0 81.4 93.6 12 2,3,5,6-tetrachloro 89.6 73.9 88.7 79.3 87.0 90.4 13 3,5-dichloro 7.8 4.8 4.9 27.0 80.6 83.0 14 3,4-dichloro 6.0 5.6 4.5 27.5 81.1 81.9 15 pentachloro 93.7 96.7 91.2 93.4 90.3 84.5 16 3,4,5-trichloro 31.3 20.1 22.3 44.5 81.4 78.5 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 12 table 3 extraction recoveries of chlorophenols from water (ph 7.3) relative to 1-cl-n-octadecane peak no. compound recovery (%) c6h6 c7h8 chcl3 ch2cl2 c4h10o c4h8o2 1 2-chlor 2.0 0 1.2 13.3 51.8 61.1 2 phenol 0 0 0 1.8 30.1 35.6 3 2,6-dichloro 0 0 3.6 23.6 84.3 80.0 4 hexachlorobenzene 98.3 96.7 94.2 94.6 98.8 96.9 a 2,5-dichloro 6.5 7.6 4.5 25.9 79.3 82.8 5 2,4-dichloro 6.8 3.9 4.4 29.1 78.0 79.6 6 2,3-dichloro 5.6 2.7 5.2 31.5 78.6 80.4 7 2,4,6-trichloro 2.8 8.6 3.7 28.2 84.9 83.5 8 2,3,6-trichloro 3.1 7.2 3.5 34.7 86.1 84.3 9 3-chlor 1.0 1.0 0.4 6.8 72.2 70.9 10 2,3,5-trichloro 6.8 8.8 5.7 30.9 87.0 84.9 b 2,3,4-trichloro 6.6 8.2 6.3 35.8 83.1 87.3 11 2,4,5-trichloro 7.9 9.1 7.4 30.8 82.3 88.7 12 2,3,5,6-tetrachloro 1.9 6.3 6.7 37.1 85.6 85.9 13 3,5-dichloro 4.7 3.8 4.2 22.9 76.3 76.4 14 3,4-dichloro 4.8 6.0 6.3 27.3 75.8 75.8 15 pentachloro 5.3 7.2 13.9 39.8 86.7 76.6 16 3,4,5-trichloro 4.6 10.9 7.1 21.8 79.8 73.0 the influence by the change of ph value of water (ph 2.0, 4.3 and 7.3) on the recoveries chlorinated phenols from water is shown in fig. 3-8. figure 3. the extraction with benzene figure 4. the extraction with toluene food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 13 figure 5. the extraction with chloroform figure 6. the extraction with dichloromethane figure 7. the extraction with diethyl ether figure 8. the extraction with ethyl acetate table 1-3 shows that with increasing solvent polarity the extraction recoveries of chlorinated phenols increase, particularly with phenol and its lower chlorine derivatives. the recoveries of chlorinated phenols are influenced by the structure of the phenol, the length of the alkyl chain and the number of chlorine on the ring. the lowest recovery was given by phenol itself, at it shows the highest solvation of the phenols studied. on the other hand, the best recovery was obtained for pentachloro-phenols and hexachlorobenzene. the highest recovery was given by diethyl ether and ethyl acetate (recovery was above 60 %), which are thus the most suitable solvent, especially when the determination of phenol and its lower chlorine derivatives is required. they are readily available, volatile, but often require additional purification. disadvantage of this solvents are as follows: its water solubility, and its therefore not recommended for micro-extraction. the recoveries chlorinated phenols with diethyl ether and ethyl acetate are slight influenced by the change of ph value of water. the best results are given by acidified water samples (at ph value 2). in the case the next solvents, ph value of water has effect on the recoveries chlorinated phenols. table 1 may be useful for selecting extractants for the isolation of microgram amount of organic substances from water when the determination of chlorinated phenols is required. for instance, for the extraction of higher chlorinated phenols from water it is possible to use a less polar solvent (dichlormethane, toluene). the results of the recoveries showed that this method is possible to use for routine analysis of chlorinated phenols in water. 4. conclusions it result from the above considerations that the microextraction method of chlorinated phenols isolation is very rapid, simple, and economically profitable. the food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 14 results of this study give data important for analysis of these compounds in waters which may be used for their routine quantitative analysis involving microextraction and capillary gas chromatography. the recovery of sixteen chlorinated phenols from water (at different ph of water) in benzene, toluene, chloroform, dichloromethane, diethyl ether and ethyl acetate was determined. the recoveries of the studied compounds were evaluated relative to internal standard. the highest recoveries were obtained for diethyl ether and ethyl acetate. in this case the recoveries are slight influenced by the change of ph of water. the lowest recoveries were obtained for phenol. recovery of the other chlorinated phenols was above 60 %. 5. references [1] afgan b.k., belliveau p.e., larose r.h., ryan j.f.: anal. chim. acta 71, 355. (1974). [2] kalavská d., holoubek i.: analýza vôd. sntl-alfa, bratislava, pp. 165, (1987). [3] standard methods for the examination of water and wastewater, 16th edition, apha, awwa, wpcf, washington d.c., (1985). [4] horáková m., lischke p., grünwald a.: chemické a fyzikální metody analýzy vod. sntl alfa, praha, pp. 343, (1986).. [5] tesa ová e., pacáková v.: chromatographia 17, 269, (1983). [6] knuth m.l., hoglund m.d.: j. chromatogr. 285, 153. (1984). [7] bartle k.d., elstub j., novotny m., robinson r.j.: j. chromatogr. 135, 351. (1977). [8] federal register epa. u.s. environmental protection agency. washington, october 26, (1984). [9] masai o.h., gulick w.m.,jr.: j. high resolut. chromatogr. & cc. 10, 647, (1987) [10] white c.m., norman c.l.: anal. chem. 54, 1564. (1982). [11] krup ík j., repka d., benická e., hevesi t.: j. chromatogr. 448, 203. (1988). [12] korenman j.i., minasyants v.a., fokin v.n.: ž. anal. chim. 43, 1303, (1988). [13] veningerová m., uhnák j., oprchalová k.: in. hydrochémia 86, svts bratislava. pp. 389.( 1986). [14] hriv ák j., šteklá m.: j. chromatogr. 286, 353. (1984).8520[15] dietz f., traud j.: vom wasser. 51, 235. (1980). [16] averill w., purcel j.e.: chromatography newsletter 7, 13, (1979). [17] janda v., krijt k.: chem. listy 78, 768, (1984). [18] dix k.d., fritz j.s.: j. chromatogr. 408, 201. (1987). [19] popl m., voz áková z., zelinka l.: in: hydrochémia 79, svts bratislava, pp. 245, (1979). [20] chiavari g., pastorelli l.: fresen. z. anal. chem. 317, 130. (1982). [21] junk g.a. et all.: j. chromatogr. 99, 745. (1974). [22] mcintyre a.e., lester j.n.: sci. total. environ. 27, 201. (1983). [23] rennie p.j.: analyst 107, 1982, 327. [24] coutts r.t., hergesheimer e.e., possuto f.m. : j. chromatogr. 179, 291, (1979). [25] janda v., van langenhove h.: j. chromatogr. 472, 327. (1989). [26] botta d., morandi f., mantica e.: proceedings of the 2nd european symposium on analysis of organic micropollutants in water. d. riedel, dordrecht, pp. 298, (1982). [27] renberg n., lindström k.: j. chromatogr. 214, 327. (1981). [28] norén k., sjövall j.: j. chromatogr. 414, 55. (1987). [29] lee h.b., weng l.d., chan a.s.y.: j. assoc. off. anal. chem. 67, 1086. (1984). [30] method 604-phenols. fed. register epa 49, 58, (1984). [31] hriv ák j.: anal.chem. 57, 2159. (1985). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 72 influ ence o f f lat area type o n sewa ge des ig n co nd itions *štefan stanko1 1slovak university of technology, faculty of civil engineering, stefan.stanko@stuba.sk *radlinského 11, 81368 bratislava, slovakia abstract: the paper discuss about advantages and disadvantages in sewer system design in dependence on designed area composition focusing on flat areas and offer the solution, how to solve these problems, respecting the proper design conditions. the slovakia area consists from various type of surface. the choice of sewer building type depends on this type too. the west and east slovakia cover flat areas. the flat areas, without natural slope require the special approach concerning the sewer system built. this area type offers various solutions. slovak legislative recommend to keep the design principles, which are under pressure of investor – the build price decreasing. the dispute between investment cost and on the other side operational costs and operational conditions must consider the design principles. the alternative sewer system design is allowed, such as gravity and vacuum system, but it is strictly recommends to prove the validation of designed system. the very important influence on the sewer system design has the eu promises, which make a press for the fast sewer system built in slovakia, which could be decrease the quality process of designing. keywords: sewer design, pressure and underpressure system introduction the preparation process for sewer system building in slovakia depends on various factors, which have influence on quality and effective build up. the landscape of the country has important influence for the system of build up. east-slovakian lowland and west part – danube lowland, allows realize the sewage networks by various principles. the investment process needs to revalue the approaches, needs to take the consideration, that the building sewage will be operated under the municipalities, which will be want to operate it without non wanted operational costs involved by the improper design, or no adequate cheap investment. the sewer designing could be in alternatives mean gravitational, pressure or vacuum sewage system very often decides the financial conception solution. especially the flat areas allow using these alternative solutions, which very often saving the money, but the relevancy mirror could be not clear. minimal slope the present slovakia sewage state, when the greatest problem of the build is non design documentation, but the investment gaining and the fastest building process, shunts the technical, especially operational conditions on the minor side. the sewage alleviation is no discussed, only quantitative aspects are important. it concerns to the gravitational solution. the problem will be significant in the future when no only operational costs will increase, but rehabilitation and reconstruction will be necessary, too, together with the increasing financing. the present minimal slope recommended in slovakia sewage design is defined by the empirical equation (1) mailto:stefan.stanko:@stuba.sk food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 73 = 1500 (1) where d is the sewer diameter in millimeters and the minimal slope define the build-technical slope, which is possible to observe in building process and this slope have no eligible value concerning sewage alleviation, where the sewer slope we need to define regarding the amount and quality of waste water. the minimal slope were defined till few years ago and through more than 50 years by the equation (2) = 1000 (2) it was more advantage in the design process, especially when the minimal allowed sewer diameter were dn 300 mm against present recommended dn250. the historical design saved the excavation and together the investment costs and allowed more sewer capacity reservation and in the flat areas saved the number of pumping stations. the present value of minimal slope (1) is insufficient despite of slope increasing this opinion is supported by the authors stránsky d. et al. [ 2], which are interested about the question of alleviation and related problems of the odor. they recommend the minimal slopes, which are mentioned in the table 1. the columns (3) and (5) are the multiplication of the columns (1)x(2) and (1)x(4). these aspects clear the situation and we can say that there is no empirical influence between the recommended slope and sewer diameter. mentioned values of minimal slope were determined by the influence of minimal recommended transportation waste water velocity vt, at which the dry weather sewage recommended for the qhmax=0,6 m.s-1. in the case of combined sewer system, the 0,75 m.s-1 is recommended and in the case of storm water sewage this value is represented 0,75 m.s-1 where the flow is qp average flow. self-cleaning slope the en752 recommendations for the design of dewatering and sewer systems are explicitness and want to prevent the sewers against the permanent drift deposition, which increasing the risk of the flooding involving by the sewer system and follow environmental pollution. the sewers bedded in the minimal slope are drifted and we need to clean it. the ížek [1] in the 1953 advised to compute the sewer slope of the combined sewage with the exploitation of the equation for critical tangential tension near the wall of the sewer pipe by the eq. (3) = . g. r. i (n. m ) (3) where – tangential tension [pa] g – gravitational acceleration – [m.s-2] r – hydraulic radius – [m], io – slope when we put critical =4,0 pa for combined sewer system, then self-cleaning velocity is defined by eq. (4). v = 0,02. r .0,167 . n (m. s ) (4) where n – manning’s roughness coefficient. in the case of dry-weather sewage we choose the critical=2,0 pa [3]. the sewer bottom slope is equal is=0,00204/r, and the r is relative to the average dry-weather flow qb24. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 74 table 1. recommended values of minimal slopes in dependency on sewage type indicators recommended value minimal recommended slope (1) dn (2) sanitary sewage (3) x/d (4) combined sewage (5) x/d [mm] [‰] x [‰] x 250 18 4500 12 3000 300 14 4200 9 2700 400 9 3600 6 2400 500 7 3500 5 2500 600 6 3600 4 2400 800 5 4000 3 2400 computer application the design software exploitation in the flat areas is the effective tool for the alternative sewer system design. the hand calculation of sewer system take too much time, this is maybe inaccurate and very difficult. the software using offer the chance in a short time to investigate more alternatives of design, the using of more diameters of sewers and solve the conception of sewer pumping station. the department of sanitary and environmental engineering use offer the solution, through the computational system sewacad, which offer to design alternative solutions with the possibility of alternatives comparing. the using minimal diameter dn250 against dn300 in older period bring the saving the profile diameter, but not excavation and number of pumping stations. to elaborate of alternatives could clear us the effective solution, and answer the question, what is effective. for example the historical approach uses the minimal slope 3,33‰ with the profile dn300 against present minimal slope 6 ‰ together with the dn250 diameter using. it caused the lost of elevation 60 cm – 33 cm = 27 cm for every 100 meters, which represent total lost of elevation 2,7 meters per 1 km of sewer length. so we can very easy assert that we need to build twice more pumping stations against older principles. if is it effective or no, we find this answer through the alternative design evaluation, from technical and economical view, with the respecting the operational conditions and operational costs. we can say that influence of terrain composition play the important role in sewer system design. evaluation of many sewer system design in various conditions offer for us declaration, that saving profile diameters dn250 is not in every case the clever solution, but in many cases legitimate. and we can declare, that only complex alternative solution could give answer this question. fig. 1. computational example of sewer system design in the flat area (sewacad) under-pressure or vacuum sewage is the alternative way of sewage concerning only for sanitary waste waters in the locations, where the problem to build the gravitational sewer system from the technical is or investment view. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 75 this system we can use in location, where the storm water dewatering system is done and where we want to save the environment against the excavation together with the saving of building works, and where we want to save a relative financial costs. the word relative is for discussing, because investment costs saving with the excavation and profiles, which are from dn50 do dn200, against gravitational sewer system which start from dn250 are become evident, when we need to build the vacuum station with the relatively high operational costs concerning energy, which is needed for making the vacuum in the pipes. the under-pressure range is usually from 400 to 500 mm of quicksilver column, minimum 175 mm from the maximum 760 mm, which represent theoretically max. 7,5 m of water column. under-pressure 400500 mm allows overrunning the elevation differences approximately from 4 to 5 m. the main part of this sewage becomes the vacuum station, which produces the underpressure. this part is very demanding for the energy, which could cause the operational of this system economically intolerable. sewage waters are collected through the system of pipes in the collection container, from this there are transported to the wwtp. the advantage of such sewage is a minimum depth of the bedding, the ease exploitation by user and groundwater protection. its use is appropriate when we have a problem to build a gravity sewer system in places where there are no suitable geological conditions such as high groundwater level, soil inconsistency sands (slovakia záhorie). the application vacuum sewer system requires the build management base on the assumption of construction quality. the insufficiency of this type of sewage is noted by the residents in the village vajnory in slovak republic, when was implemented [6] this type of sewage, and there was published the instruction article in the local newspaper, how to exploit this sewage, and after the state is unsatisfied [5]. pressure sewage the idea of pressure sewer system started up as alternative dewatering of small residential areas, small municipalities in flat areas, resp. on the terrain with the low elevation differences with the aim to save an investment costs. the greatest influence has investors. the operational quality depends on equipment, mainly on end pressure stations, on tide gate. results from the operational experiences of pressure sewage say that this sewage alternative can be favorable. the build of pressure sewer system is suitable in spread areas, in recreational zones. from the financial demand, this type of sewage is the most competitive, considering the argument, that the pumping stations are situated on the residential owners, which are financing and operating the pressure pumping stations. from the operational company this is an advantage, but on the other side it could generate the complex problems concerning the faults cause by this pressure pumping stations. how to make a decision in eu countries every designer must consider, which type of sewer system will be designed, base on investment costs, operational costs and from the technical opportunities. in the places, where is the slope of terrain enough, the gravitational is more effective than in flat areas. we reflect the vacuum system designing in the next cases: inadequate nature slope isolated or rare build-up area no quality underbed zone of hygienic water supply protection food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 76 barriers in the way of build – other engineering networks, water streams seasonal operation touristic zones in the places where we need to eliminate excavation works the vacuum sewage design consists from the network design, longitudinal profiles designing, the separate branches designing and the location of collection manholes. the main criteria of design is the appraisal of total pressure loses on separate branches of the system, where are the losses involved by friction mixture of water-air in the velocity 6 m.s-1 and local losses which are involved by the flow direction – refractivity. the determining item is the energy demand needed for vacuum station air-pump engine and pumping stations. the specific demand of energy for airpump power drive increases with increased length of main sewer branches. [9] conclusion the built-up of sewage in flat areas is the intersection of investor interest and operator. the decision which type of sewage we will build up and how we will solve this problem absolutely belong under expert decision, and this is very incorrect, if this decision is accepted by the investor, without expert and operator discussing. the experiences show, that investment interests have the highest priority for decision and it mean the saving investment costs without interest on the operational experiences. the final problem is transferred on operational company, on users of the sewage. the start decision, which sewer type we will build up, have a longtime impact and this could be very negative in the horizon of more years, could have an impacts on lifetime of sewage, to transfer the problems into the future. eighty percent of a municipality’s problems are caused by twenty percent of its sanitary sewers and storm water drainage systems. the best way to avoid the majority of future problems is to pay greater attention to them during the design phase. only clever and expert approach of main decision, supported by expert could have an effective, optimal impact on dewatering the area from waste waters. acknowledgement the article was written with the support of the scientific grant agency – projects vega no. 1/0559/10 dealt with at the department of sanitary and environmental engineering of the slovak university of technology. references: 1. ížek, p.: hydrologie stokových sítí, stokování, 1. ást, sntl, (1961) 2. stránsky, d. et al, k.: posouzení stokových systému urbanizovaných povodí, ást iii. – ešení balastních vod a výskytu sedimentú ve stokové sítí, vodní hospodá ství, (08/2010). 3. stn 75 61 01 stokové siete a kanaliza né prípojky 4. stn en 752 stokové siete a systémy kanaliza ných potrubí mimo budov 5. gri , j.: pokyny pre ob anov k vypúš aniu odpadových vôd do podtlakových šácht a tým do podtlakovej kanalizácie vajnory, vajnorské novinky, ro ník xiv (9/2008) 6. mrva,j: vákuová kanalizácia pod válkom spôsobuje problémy, vajnorské novinky, ro ník xvi (3/2010) 7. zephyr.cit.act.edu.au/ toolboxes/ (2009) 8. www.lce.com.na/projects_ongoing.html (2010) 9. k íž, k. bareš, v. pollert, j. stránský, d. pollert, j. risk analysis of sewer system operational failures caused by unstable subsoil in: risk management of watersupply and sanitationsystems. dordrecht: springer, 2009, p. 65-72. isbn 978-90-481-2364-3, (2009) 10. hlavínek, p., perspectives of decentralized wastewater treatment for rural areas, in advanced water supply and wastewater treatment: a road to safer society and environment, isbn 978-94-007-0279-0, springer, nederland, (2011) http://www.lce.com.na/projects_ongoing.html food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 35 comparative study of methods for assessing escherichia coli species in drinking water *adriana dabija1, laura pîslaru2, cristina-gabriela constantinescu (pop)1, amelia buculei 1 1faculty of food engineering, stefan cel mare university, e-mail adriana.dabija@fia.usv.ro *corresponding author 2public health department of suceava, laura_pislaru@yahoo.com received 27 february 2011, accepted 20 april 2011 abstract: the study on drinking water quality is a priority for the health of consumers, the absence of pathogenic microorganisms is a prerequisite for compliance with eu directive. coliforms, fecal coliforms and escherichia coli are used as indicators of fecal contamination of water supplies and recreational waters.the most important bacteriological condition for drinking water is the total absence of pathogenic microorganisms. the measurement of bacteria of the coliform group has been used extensively as an indicator of water quality.given the relatively laborious methods of recognizing their presence and the inconsistent nature of that presence in water, the escherichia coli level is analysed. the purpose of this study is to analyze comparatively two methods for determining the escherichia coli species in drinking water of the drinking water supply network of suceava city and that of own resources within suceava county area. the analysis results of water samples studied by the two methods show that the membrane filtration method favours a specific growth and selective escherichia coli species, compared with the multiple-tube method. membrane filtration method is recommended for effective monitoring of drinking water contamination in terms of escherichia coli species. multiple-tube method is a method applicable to all types of water, and equipment is relatively inexpensive and less specialized. © 2011 university publishing house of suceava. all rights reserved keywords: contamination, prevention, environment, monitoring 1. introduction drinking water microbiological quality is primarily determined by using “indicator organisms”, whose presence indicates faecal contamination. the presence of the indicators is often a key in assessing potential public health risks due to pathogens and is used in drinking water quality regulations and guidelines in many countries [2, 9]. escherichia coli is the dominant flora of the large intestine having an important role in maintaining its normal physiology and in the synthesis of proteins of group b and k. if eliminated in the external environment with faeces, it gets into water, soil, food contamination etc. constant presence of escherichia coli strains in human and animal intestines and faeces have turned these bacteria into an indicator of faecal pollution of the environment, especially water [4]. the presence of escherichia coli species in water indicates recent faecal contamination. escherichia coli is a negative gram bacillus, which grows on simple medium in which glucose is the only organic constituent. it is an aerobic, facultative anaerobic that may have both respiratory and fermentative metabolism. on solid media it grows in the form of „s”-type colonies, and in liquid environment it causes uniform disturbances and slip ring on the tube wall [3, 7]. escherichia coli is a bacterium that resides in high numbers in the intestines of warmblooded animals and has proven its 36 value to detect fecal contamination in water [5]. from biochemical point of view escherichia coli ferments glucose and other carbohydrates producing acid and gas. most strains are negative oxidase, able to reduce nitrites to nitrates. there is no urease production, proteins are not broken down leading to formation of h2s, and citrate is not used as sole carbon source. lactose is broken down with acid release, and proteins lead to formation of indole with a positive methyl-red reaction [8]. contamination source is man and animal, material support of transmission being represented by water, food, hand, hospital items, teys, flies etc. the gateway varies: the digestive tract, respiratory, urinary, genital etc. the escherichia coli pathotypes responsible for extraintestinal infections are uropathogenic e. coli and meningitisassociated e. coli. escherichia coli from these pathotypes can cause hemolytic uremic syndrome, urinary tract infection, newborn meningitis and sepsis. the intestinal pathogenic escherichia coli strains belong to the pathotypes enterotoxigenic escherichia coli, enteropathogenic escherichia coli, enteroinvasive escherichia coli, enterohemorrhagic escherichia coli, enteroaggregative escherichia coli and diffusely adherent escherichia coli[1, 5]. these pathotypes have been associated with cases of mild and severe diarrhea in adults and children, mostly in developing countries and it subsequentially determines the type of contamination that is installed. prevention of contamination with escherichia coli requires compliance with collective, personal and food hygiene. thus, the most important bacteriological condition for drinking water is the total absence of pathogenic microorganisms. given the relatively laborious methods of recognizing their presence and the inconsistent nature of that presence in water, the escherichia coli level is analysed [6]. the paper presents the comparative analysis of two methods for determining the escherichia coli species in drinking water of the drinking water supply network of suceava city and that of own resources within suceava county area. 2. experimental the study used 30 water samples from suceava city’s drinking water network and around 10 water samples from own sources of drinking water within suceava county area for 6 months (january-june 2010). water sampling was carried out as follows: 250 cm3water samples to test the network and 500 cm3 fountain water samples for multiple tubes method, respectively 250 cm3 samples for membrane filter method. water sampling for microbiological diagnosis was made in sterile containers prepared in the laboratory, aiming to avoid contamination with the external environment. transport of samples was performed in insulated containers and was kept in a refrigerator at 4°c, max.24 hours. the analysis methods used complied with the requirements of sr en iso 8199/2008, sr en iso 9308-1/2004; ac2009. the study determined the estimated number of escherichia coli/100 cm3 sample by a membrane filtration method (method 1) and by multiple tube method (method 2). for membrane filtration method we used commercially available culture media: agar tergitol -7 with 2,3,5-triphenyltetrazolium chloride (ttc), tryptophan broth, kovacs reagent, oxidase reagent. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 37 fig. 1. work equipment for membrane filtration method membrane filter was examined and we counted as lactose-positive bacteria all characteristic colonies (regardless of their size) which develop a yellow colour in the membrane medium. escherichia coli suspicious colonies were counted after undergoing the procedure for confirmation: subculturing suspicious colonies on agars medium for oxidases test and on tryptophan broth medium for indole test, incubation effected at 36°c respectively 44°c for 24 h; then the confirmatory tests were performed (oxidase, indole). finally, escherichia coli colonies were counted (only those colonies that give a positive indole test (fig. 2) and negative oxidase test (fig. 3). fig 2. positive indole test a b fig.3. negative oxidase test (a – oxidazo-negative bacteria; b oxidazo-positive bacteria) confirmed colonies were counted and the results of determinations were calculated according to sr en iso 8199/2008. the results were interpreted in accordance with law no. 458/2002 and law no.311/2004. for multiple-tube method we used: sterile buffered water, lauryl broth seeding medium (simple), concentrate lauryl sulphate broth seeding medium and mcconkey confirmation medium. after incubation at 37°c for some time, the formation of acid and gas from lactose fermentation is investigated. to confirm the presence of coliforms group further tests must be carried out using another selective culture medium. to specify whether the microorganisms that fermented the lactose are coliforms and not other microbial species equipped with the same properties (lactobacilli, fungi), each vial or test-tube found positive in mcconkey test can be substituted with lauryl sulphate broth. the presence of escherichia coli is confirmed when characteristic colonies have developed: flat colonies of blue-violet metallic luster, or raised, opaque, metallic luster mucous in the middle, or having a pink or violet-blue centre. determining the number of coliform bacteria in 100 cm3 sample was carried out with tables „calculating the most probable number per 100 cm3 sample” laid down by iso 8199/1998 (e). 3. results and discussion the results of measurements performed in water samples taken from drinking water network of the city of suceava are summarized in table 1. table 1. results of determinations of escherichia coli species in drinking water samples from suceava city network crt. no. month 2010 no. escherichia coli/100 cm3 sample membrane filter method multipletubes method 1. january 25 6 16 <1 2. february 16 16 30 16 16 38 9 9 16 10 9 >18 38 3. march 16 16 15 12 4. april 16 9 2 15 8 <1 5. may 25 >18 6. june 42 10 26 8 16 4 12 2 in table 1, one can notice that by using the membrane filter method (method 1), the same samples, and the number of escherichia coli is higher in comparison with multiple-tube method (method 2). this is explained by the fact that the membrane filter method favours a specific growth and selective escherichia coli species, compared with the multiple-tube method. it can be seen that the highest microbial contamination with escherichia coli species was in february 2010 – 20% of the samples and the lowest contamination, in may 2010 to 3.33% of the samples (fig. 4). 0 5 10 15 20 n um be r po si tiv e sa m pl es january february march april may june month method 1 method 2 fig. 4. share of total number of positive samples of drinking water samples taken from suceava city network during january-june 2010 table 2. results of determinations of escherichia coli species from own drinking water samples (suceava county) crt. no. month 2010 no. escherichia coli/100 cm3 sample membrane filter method multipletubes method 1. january 56 7 26 9 2. february 26 64 45 >18 46 22 3. march 24 24 17 14 4. april 12 7 5. may 42 8 6 2 26 2 2 <1 6. june 42 8 6 2 12 26 2 2 <1 9 the results of measurements performed in water samples taken from own sources of drinking water in suceava county area during the six months (january-june 2010) are shown in table no. 2. by analyzing the data shown in table 2, we noticed a higher contamination with escherichia coli species from samples taken from own sources of drinking water compared with samples from suceava city network. it is obvious that such water does not comply with current legislation for drinking water, its consumption possibly affecting consumer health. in rural romania, organized health services are almost inexistent, the transportation to storage facilities being carried out individually by consumers. uncontrolled waste, especially from livestock (manure) contributes to microbiological and chemical pollution of groundwater and surface water in these areas. the highest microbial contamination with escherichia coli species was in june 2010 – 50% of the samples and the lowest contamination, in april 2010 – 10% of total samples analyzed (figure 5). 0 10 20 30 40 50 n um b er o f p os it iv e sa m pl es january march may month method 1 method 2 fig. 5. share of total number of positive samples of drinking water samples from own sources (suceava) suceava city network in the period analyzed food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 39 4. conclusion the study on drinking water quality is a priority for the health of consumers, the absence of pathogenic microorganisms is a prerequisite of compliance with eu directive (directive 98/83/eec). the monitoring of drinking water’s contamination with escherichia coli species was performed by two common methods: the method of membrane filtration and multiple-tube one. the analysis results of water samples studied by the two methods show that the membrane filtration method favours a specific growth and selective escherichia coli species, compared with the multipletube method. this is explained by the fact that the medium used for the membrane filter is optimal for increasing the escherichia coli species than the medium used for multiple-tube method. therefore, the following conclusion can be drawn: the membrane filtration method is recommended for effective monitoring of drinking water contamination in terms of escherichia coli species. there are still cases when the membrane filtration method has limitations, especially if water samples have a high turbidity or noncoliform bacteria. for such water it is desirable to carry out parallel tests with multiple-tube method to demonstrate applicability and comparability. multiple-tube method is a method applicable to all types of water, and the equipment is relatively inexpensive and less specialized. 5. references 1. alonso, j, et al., quantitative determination of escherichia coli in water using chrimagar e.coli, journal of microbiological methods, vol.25, 309-315, (1996) 2. barrel r.a.e., a comparison between tryptone bile agar and membrane lauryl sulphate broth for the enumeration of presumptive escherichia coli in water, water research, 26, 677-681, (1992) 3. bonadonna l., et al., comparison of methods and confirmation tests for the recovery escherichia coli in water, desalination, 213, 18-23, (2007) 4. dan v., microbiologia alimentelor, alma, galati, (2001) 5. heijnen, l., medem, g., method for rapid detection of viable escherichia coli in water using real-time nasba, water research, 43, 3124-3132, (2009) 6. renato, o., et al, genetic variability and pathogenicity potential of e. coli isolated from recreational water reservoirs, research in microbiology, 158, 420-427, (2007) 7. schets f.m., et al., escherichia coli in drinking water from private water supplies in the netherlands, water research, 39, 44854493, (2005) 8. yanez m.a., a simple and cost-effective method for the quantification of total coliforms and escherichia coli in potable water, journal of microbiological methods, 65, 608-611, (2006) 9. wang, d., fiessel, w., evaluation of media for simultaneous enumeration of total coliform and escherichia coli in drinking water supplies by membrane filtration techniques, journal of environmental science, 20, 273-277, (2008) 10. legea 458/2002 privind calitatea apei potabile, modificată şi completată de legea 311/2004 11. *** sr en iso 9308-1/2009 ”calitatea apei – identificarea şi enumerarea organismelor coliforme, organismelor coliforme termotolerante şi escherichia coli” journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 68 study on the content of zearalenone from wheat and derivatives sonia gutt 1, georg gutt1, mariana mazareanu2 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229, suceava, romania, e-mail: gutts @usv.ro 2 animal sources food safety department-suceava abstract: in food and fodder naturally contaminated with fungus there are in high concentrations seven groups of mycotoxins: aflatoxins, ochratoxin, trichothecenes, zearalenone, patulin, citrinine and acid penicilic. mycoestrogens are estrogens produced by fungi. the most important mycoestrogen is zearalenone, produced by fusarium species of fungi. zearalenone and its metabolites exert their estrogenic effects through binding to estrogen receptors[1]. zearalenone is the main phyto-oestrogen and it is the primary toxin causing infertility, abortion or other breeding problems, especially in swine. chickens fed with contaminated feed showed zearalenone residues in muscles and liver. the concentrations of zearalenone and its metabolites, αand β-zearalenol, were determined quantitatively by high-performance liquid chromatography (hplc), in placenta, in maternal liver and spleen rats, αand β-zearalenol were transferred into the foetus [2]. the paper analyzed raw materials used in the bakery industry, samples of grain, flour and bran in order to determine the content of zearalenone specific limits for zearalenone have been regulated in 2007 by the european union, ranging from 20 µg/kg for infants and small children foods to 75 µg /kg for maize flour (eu commission regulation (ec no 1126/2007). amounts of mycotoxins have been highlighted by elisa. in analyzed samples were detected exceeded limits for a number of one sample of wheat and one sample of bran, the other samples containing zearalenone below the maximum allowable limit, the analysed raw materials considered as safe for bakery. 1.introduction mycotoxins are secondary metabolites of fungi which contaminate feed and food. mycotoxins presented in food products and animal feeds are an important problem concerning food and feed safety and significant economic losses are associated with their impact on human and animal [3]. vegetable products are convenient substrates for fungi development and mycotoxins production[4]. during storage they are favorable substrates if they have a high moisture content above 80% and a temperature above 20°c. the main types of fungi producing mycotoxins are: aspergillus, penicillium, fusarium şi alternaria. it should be noted that a fung can produce more mycotoxins and that a particular mycotoxin may be produced by several fungi; it can lead to phenomena of synergism and potentiation of toxic action. zearalenone is heat-stable and is found worldwide in a number of cereal crops, such as maize, barley, oats, wheat, rice, and sorghum and also in bread. zearalenone has a chemical structure similar to steroid hormones and it is produced by fungi of the genus fusarium (f. graminearum, f. avenacum f. equisetti). zearalenone belongs to the group of phytoestrogens have an estrogenmimetic effect, but it is not a true, it is not a plant product (soy, alfalfa, clover) but a fungical one. zearalenone is a lactone of resorcylate acid, it is carcinogenic, mutagenic and genotoxic in a concentration 1.5 mm/l. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 69 zearalenone induces cell proliferation in estrogen-dependent tissues (carcinogenesis), and contributes to endometrial hyperplasia and adenocarcinoma formation. 2. 2. materials and methods a method for determination of zearalenone in cereal flour has been developed applying pressurized liquid extraction (ple) using methanol/acetonitrile (50:50 v/v) as the solvent extraction[5]. the extracted samples is analyzed with liquid chromatography coupled to mass spectrometry (lc–ms) with an electro spray ionisation interface (esi). conventional chromatographic methods are generally intensive time and capital consuming and therefore have been developed and marketed a series of rapid analysis methods mostly based on immunological principles. method used in paper is based on immunoenzymatic reactions in heterogeneous system (elisa enzymes linked immuno sorbent assay) that used for determination both of antigen and of antibody. antigenic structures, usually in the form of liquid suspension can have microbial origin (bacteria, viruses, fungi). antibodies can be represented by non-immune sera or purified polyclonal immunoglobulins. in the technique used, the specific antigen or antibody is attached to a solid support that can bind antigen or antibody by passive adsorption or covalent binding. passive adsorption of macromolecules consists of immobilization on a surface of glass or plastic.the vast majority of protein diluted (1-10 mg / ml) in an alkaline solution (ph 9.6) are adsorbed on plastic in few hours. as a solid support can be used propylene, polystyrene, polycarbonate or latex. in the tests were used microplates of polystyrene, with fully automated work advantage.covalent binding allows irreversible connections between reactants and solid support so that quantitative assessments have a maximum precision. calitatea fixării structurilor proteice pe suportul solid depinde de concentraţia proteinei, de natura suportului utilizat şi de intervenţia unor agenţi chimici de fixare. quality of protein structures are fixed on solid support depends on protein concentration, the nature of substrate used and the occurrence of chemical fixation. enzyme conjugate there should be a couple antigen enzyme or antibody enzyme, the latter being the most frequently used form. 2.1 materials the sample must be kept cold, avoid light. a quantity of 5 g of ground sample is mixed with 25 ml of methanol / water (70/30); shaked vigorously for 3 minutes; centrifuged 10 minutes at 3500 rpm. at room temperature or filter; the supernatant or filtrate are diluted 1: 7 with a buffer for diluting the sample and using 50 µl per well. reagents. it works with reagents at room temperature, prepared before use . microtitre plate. wells are coated with antibodies. conjugate solution. enzyme conjugate is concentrated and for reconstitution the enzyme conjugate is diluted 1: 11 in buffer (eg. 200 µl concentrate + 2.0 ml of buffer solution, sufficient for four wells). after use, the vial is kept at 2°c to 8°c in the dark. solution substrate / chromogen. solution substrate/chromogen is ready to use and tends to precipitate at 4°c. it brings the bottle to room temperature and kept in the dark, stirring before pipetting.. chromogen staining is an indicator of deterioration and reagents should be discarded. wash buffer. wash buffer is diluted before use. samples are needed to achieve the next steps. 1. pipette 100 µl standard dilution buffer, which is the reagents blank (wells a1 and a2). journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 70 2. pipette 50 µl standard s0 (wells b1 şi b2). 3. pipette 50 µl standard, in duplicate (s1…s6). 4. pipette 50 µl sample solution in duplicates, in the remaining wells (p1, p2 ....p40). tabel 1 order in which pipette standards and samples in microplate 1 2 3 4 5 6 7 8 9 10 11 12 a blank blank p1 p1 p9 p9 p17 p17 p25 p25 p33 p33 b s0 s0 p2 p2 p10 p10 p18 p18 p26 p26 p34 p34 c s1 s1 p3 p3 p11 p11 p19 p19 p27 p27 p35 p35 d s2 s2 p4 p4 p12 p12 p20 p20 p28 p28 p36 p36 e s3 s3 p5 p5 p13 p13 p21 p21 p29 p29 p37 p37 f s4 s4 p6 p6 p14 p14 p22 p22 p30 p30 p38 p38 g s5 s5 p7 p7 p15 p15 p23 p23 p31 p31 p39 p39 h s6 s6 p8 p8 p16 p16 p24 p24 p32 p32 p40 p40 5. add 50 µl enzyme conjugate into each well. cover the plate with aluminum foil, stir the stirrer of the microplate (60 rpm) and incubated for two hours at room temperature in the dark. 6. discard the liquid and beat with power plate face down on absorbent paper to remove traces of liquid. add 250 µl washing solution (pbs and distilled water) and discard the liquid. repeat washing step twice. 7. add 50 µl substrate and 50 µl chromogento each well. mix rotating plate and incubated 30 min at room temperature (20-25 ° c), from darkness. blue color intensity obtained is inversely proportional to the concentration of mycotoxins in the sample or standard. 8. add 100 µl stop solution to each well. mix gently rotating plate and measured at 450 nm against air blank site. not to exceed 60 min after adding stop solution. using the optical densities (od) of the standard, the calibration curve is plotted against the concentrations of other standards, and the amount of of mycotoxin in the sample is extrapolated from standard curve. results and discussion mycotoxicological samples analysed by elisa were represented by samples of wheat flour, samples of bran and samples of wheat. table 2 zearalenone concentrations for each analysed sample no. sample zearalenone ppb no. sample zearalenone ppb no sample zearalenone ppb 1 wheat 33.40 1 bran 21.12 1 wheat flour 18.12 2 wheat 42.07 2 bran 38.53 2 wheat flour 16.22 3 wheat 53.56 3 bran 58.10 3 wheat flour 19.36 4 wheat 23.60 4 bran 43.67 4 wheat flour 22.02 5 wheat 47.11 5 bran 52.13 5 wheat flour 14.21 6 wheat 44.63 6 bran 40.49 6 wheat flour 16.41 7 wheat 46.34 7 bran 44.88 7 wheat flour 11.12 8 wheat 48.19 8 bran 54.41 8 wheat flour 13.14 9 wheat 61.45 9 bran 24.62 9 wheat flour 10.84 10 wheat 111.76 10 bran 46.42 10 wheat flour 19.41 11 wheat 27.75 11 bran 53.53 11 wheat flour 52.63 12 wheat 39.39 12 bran 41.43 12 wheat flour 28.41 13 wheat 45.35 13 bran 31.11 13 wheat flour 18.74 14 wheat 68.29 14 bran 37.88 14 wheat flour 31.02 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 71 15 wheat 33.98 15 bran 51.63 15 wheat flour 24.51 16 wheat 67.22 16 bran 42.64 16 wheat flour 15.47 17 wheat 28.20 17 bran 76.87 17 wheat flour 25.74 18 wheat 82.22 18 bran 19.41 18 wheat flour 33.43 19 wheat 76.14 19 bran 32.01 19 wheat flour 57.96 20 wheat 51.13 20 bran 51.07 20 wheat flour 12.64 21 bran 48.96 22 bran 54.11 analyzing the data presented in table 2 derived from zearalenone determination it found that:  in one wheat sample, (5%) of the total number of samples, zearalenone is presented in an amount greater than the maximum limit permitted by applicable law (100 ppb);  in two wheat samples (10% of total samples), zearalenone is presented in a quantity of 76-100 ppb and falls below the maximum allowed by the applicable law;  the five wheat samples (25% of total samples), zearalenone is presented in an amount between 51-75 ppb and falls below the maximum permitted by applicable law;  the nine wheat samples (45% of total samples), zearalenone is presented in an amount between 31-50 ppb and falls below the maximum permitted by applicable law;  the three wheat samples (15% of the total number of samples), zearalenone is presented in an amount between 1-30 ppb and falls below the maximum permitted by applicable law; the frequency of wheat samples with zearalenone values is shown in fig .1 0,00% 5,00% 10,00% 15,00% 20,00% 25,00% 30,00% 35,00% 40,00% 45,00% 1-30 ppb 31-50 ppb 51-75 ppb 76100 ppb peste 101 ppb 15,00% 45,00% 25,00% 10,00% 5% fig.1. graphical representation of samples of wheat contamination with zearalenone  from the analyzing samples of bran is resulted the following values for zearalenone content  in a single sample of bran (4.54%) of the total number of samples, zearalenone is presented in an amount greater than the maximum permitted by applicable law (ppb 75);  in seven samples of bran (31.81% of total samples), zearalenone is presented in an amount of 51-75 ppb and falls below the maximum permitted by applicable law;  în 11 probe tărâţe (50% din numărul total de probe) zea este prezentă într-o cantitate cuprinsă în intervalul 31 – 50 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 72 ppb şi se încadrează sub limita maximă admisă de legislaţia în vigoare;  in eleven samples of bran (50% of the total number of samples), zearalenone is presented in an amount of 31-50 ppb and falls below the maximum permitted by legislation;  in three samples of bran (13.65% of total samples), zearalenone is presented in an amount of 1-30 ppb and falls below the maximum permitted by applicable law; 13,65% 50% 31,81% 4,54% 0,00% 5,00% 10,00% 15,00% 20,00% 25,00% 30,00% 35,00% 40,00% 45,00% 50,00% 1-30 ppb 31-50 ppb 51-75 ppb peste 76 ppb fig. 2. graphical representation of samples of bran contamination with zearalenone the results of analysis of flour samples are these:  in two samples of wheat flour (10% of total samples), zearalenone is presented in an amount of 31-50 ppb and falls below the maximum permitted by applicable law;  in eighteen samples of wheat flour (90% of total samples), zearalenone is presented in an amount of 1-30 ppb and falls below the maximum permitted by applicable law conclusions from the analysis of samples represented by wheat flour, bran and wheat using elisa method was found that a single sample of wheat, and one of bran contained zearalenone amount over the limit while the vast majority of samples (90% flour wheat, 63.65% samples of bran and 35% samples of wheat ) are below the permissible content of zearalenone and other samples are well below the maximum allowed. it can be concluded that these raw materials can be used to obtain reliable bakery food. references 1. j. fink-gremmels ∗, h. malekinejad, clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone, review, animal feed science and technology 137 (2007) 326–335 2. aksel bernhoft, gerd h. g. behrens, kristian ingebrigtsen , wenche langseth, simon berndt, placental transfer of the estrogenic mycotoxin zearalenone in rats,volume 5, issue 5, reproductive toxicology, septemberoctober 2001, pages 545-550 3. stefan d. muri , hilko van der voet , polly e. boon , jacob d. van klaveren , beat j. brüschweiler, comparison of human health risks resulting from exposure to fungicides and mycotoxins via food , food and chemical toxicology 47 (2009) 2963–2974 4. e. richard , n. heutte , l. sage , d. pottier , v. bouchart , p. lebailly , d. garon , toxigenic fungi and mycotoxins in mature corn silage, food and chemical toxicology 45 (2007) 2420–2425 5. e. pérez-torrado j. blesa, j.c. moltó, g. font, pressurized liquid extraction followed by liquid chromatography–mass spectrometry for determination of zearalenone in cereal flours, elsevier, food control 21 (2010) 399–402. microsoft word 12 shtyk corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 78 investigation on radioactive contamination of bone raw material as one of the alternative sources of organic calcium l.v. peshuk1, n.v. budnyk2, i.i. shtyk1. 1 national university of food technologies, 68, volodymyrska str., kyiv, 01601, ukraine gubena@meta.ua 2 poltava university of economics and trade, 3, kovala str., poltava, 36014, ukraine *corresponding author received 5 december 2012, accepted 14 december 2012 abstract: there were examined contents of radionuclides in bone raw material, afforded investigation results of contents of strontium– 90 and caesium– 137 in chicken and pig meat. it was determined that radionuclides contents changes wit the age of bird. key words: bone, albumen, radionuclides, strontium– 90, caesium– 137, calcium, phosphorus, mineral agents. 1. introduction among the most important problems that have to be solved by processing industry today, there is a maximum usage of slaughterhouse domestic animals and poultry products for food aims. special place in slaughterhouse products belongs to bones. during the processing of meat carcasses, bones are from 20% to 30% of the mass of carcasses. fresh, dietary bones consist of up to 50% of water, up to 15% of fat, up to 13% of albumen and up to 22% of mineral agents. in food industry bones raw material is only used as a source of albumen and fat. lately the role of fibrous and bone tissues in the process of digestion of meat products in human organism has been studied. in human organism mineral bones components has not only supportive, but also take part in exchange trophic processes. bone tissue provides with stability of calcium and phosphorus contents in blood and tissues of other mammals, by support of ph balance of organism. calcium and phosphorus play very important role in mechanism of contraction of muscles and cellular membranes. calcium ions take part in transmission of neural impulses and clotting. in this connection with that reorganization always takes place in bones. bones in too labial way take part in vital processes in human organism [1 5]. for the replenishment of calcium the person has to consume together with food and drinking water every day from 1000 to 1500 mgr of calcium in assimilative form [2, 5]. lack of calcium in organism leads to development of osteoporosis metabolism disorder, nervous disorders and other diseases. at the same time a huge territory of ukraine is polluted with radionuclides that in an alimentary way get to human organism. ecological influence of different radioactive isotopes on living tissue also greatly differs. for example, radioactive materials with half-value period of less than 8 days is not considered to be dangerous; since they keep a high level of radiation in the infected bioton just for a small period of and can be easily decreased in human organism. materials with long half-value period such as, for example, uranium-238 (4,5 billion of years) are also food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 79 almost safe because of slight radiation during the certain period of time. however, big danger is presented by radioactive strontium-90 and cesium-137,that accordingly have half-value period of 28 and 33 years. as a result of similarity of chemical qualities accordingly of calcium and potassium, they easily get into human organism, stay there and can be accumulated in such amount that can cause harm for the organism. radioactive strontium and cesium can cause chemical mutation – irreversible changes of structure of nucleic acids, albumen, lipids especially of cell membranes and appearing of active radicals. immune system is one of the accessible parts in human organism for radiation influence. as a result of weakening of immune system functioning caused by ionized radiation, leucosis and other oncologic diseases can develop. that is why in ukraine after 1986 there is a precaution against bones as a source of getting into organism radionuclides in alimentary way. it was also investigated that mammal organism assimilates calcium much better than strontium. that is why food that is rich in calcium is one of the best ways of decreasing strontium in human body [6]. 2. experimental bones are one of the most accessible and effective sources of mineral agents. bird and pig breeding is considered to be the most perspective field of cattle breeding on the territory of ukraine. that is why we conducted some research concerning cesium and strontium contents in chicken and pig bones. radionuclide structure of the investigated objects determined on scintillation spectrometers of gamma energy – types of radiation сег-001 „акп-с” and beta radiation себ-01-70. methods of investigation are standardized for strontium– 90 ми12-0,5-99, мук 2.6.1.717-98. true fault of radiation from ±10% to 50% if the inside expectancy is р=0,95. 3. results and discussion results of investigation of radionuclide contents are presented in the tables 1,2. the results of investigation affirm that experimental samples of chicken and pig bones have radionuclide contents that are below the admissible limit, so poultry and pig bones, regardless of their age, can be for food by guaranteeing radioactive safety. table 1 radionuclide contents in chicken and pig bones (n=3; р≤0,05) contents in the investigated element. bk/kg indexes chicken bones pig bones permissible level. bk/kg cesium–137 strontium–90 radium–226 potassium–40 thorium–232 15.4 < 9.5 < 9.5 < 47 < 6 18 <10 <18 <64 <10 50 200 35 50 125180 2030 table 2 contents of cesium and strontium in chicken bones ( n=3; р≤0.05) permissible levels . bk/kg age of bird. days contents of 90sr. bk/kg contents of 127cs. bk/kg 127cs 90sr 90 < 9.5 18.0 120 < 9.3 18.6 150 < 12.0 17.4 180 < 12.5 15.4 210 < 15.54 15.6 300 < 16.0 15.8 200 50 the next stage was to find out toxic elements in poultry and pig bones. the results of toxic elements contents in bones are presented in the table 3. as it can be noticed in the table. in pig and chicken bones the level of zink. copper. plumb and mercury is minimal and according to maximum permissible level is (%):3.5..5.65. 12.8..15. 10..12. 5..10. level food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 80 of cadmium and arsenic – about 40..60 %. however. neither toxic element from table 3 does not exceed limits provided by hygiene requirements of quality and safety of provision raw materials and food products [4]. that is why by taking into consideration the results of investigation of radioactive and toxic safety. and also taking into consideration literary facts [5] about high degree of assimilation of bone calcium and phosphorus by human organism. their prophylactic action. role of calcium in decreasing level of strontium. a conclusion can be made that after accurate initial control chicken and pig bones can be used for preparing bone food paste. table 3 toxic elements contents in chicken and pig bones ( n=3; р≤0.01) contents in the investigated object. mg/kg indexes chicken bones pig bones permissible level. mg/kg mercury cadmium plumb zink arsenic copper mikotoxins. aflatoxin в1 < 0.0015 < 0.02 < 0.06 3.96 0.062 0.64 0.002 < 0.003 < 0.03 < 0.05 4.26 0.062 0.75 0.003 0.03 0.05 0.5 70 0.1 5.0 0.005 for effective utilization of bone raw material and providing balanced nutrition in japan (patent №51-57681). france (patent №2505131. etc.) and the usa (patent №3873760. etc.) fresh dietary bones are processed into paste with element size 10 mkm. the paste has soft consistence of white and yellow colour and specific pleasant taste provided by cerebrum. it is absolutely digested in gastrointestinal tract and contains. %: albumen from 10 to 18. fat from 12 to 14 and mineral agents from 20 to 26 (mostly calcium. phosphorus and magnesium). bone paste can be utilized while making boiled and liver sausages. other sausages. meat bread. paste. etc [3]. by means of thermal treatment of bones and working in the electromagnetic field in the labaratory we managed to get bone paste that has similar organoleptic characteristics and chemical structure. 4. conclusion. 1. contents of radioactive strontium-90 and cesium-137 in chicken bones does not greatly depend on their age; however. concerning radioactive strontium-90 and cesium-137 chicken bones have permissible level of these radionuclides in food products. 2. taking into consideration high degree of assimilation of bone calcium and phosphorus by human organism. their effect on radiation and their prophylactic effect. presence of albumen and fat in big quantity. it is possible to utilize reduced to fragments colloidal bone raw material as a food additive for producing balanced food products including for prophylactic nutrition. 5. references [1]. lebiedieva n.а.. , bobrovskyi. а.y. , skriabin v.n. et al., anatomy and histology in meat industry/– m.: ahropromizdat. 1995. – 368 p. [2]. smoliar v.i., physiology and hygiene in nutrition. – k.: health. 2000.-336 s. [3]. rational utilization of bones abroad. -m.: tsniitti meat milkind. outline information. 1995.-40 p. [4]. hygiene standard hn 6.6.1.1-130-2006. permissible level of contents of radionuclides 127cs і 90sr in food products and drinking water. – k. 2006. [5]. fajvyshevskii m.l.. processing of dietary bones. m.: ahropromizdat. 1996. – 174 p. [6]. salavatulina r.m. rational utilization of raw material in sausage industry. – m.: ahropromizdat. 1985. – 255 p. [7]. ramad francis., basis of applied ecology. radioactive contamination. transl. from french. l.: hidrometeoizdat. 1981.-544 p. microsoft word 1 bacau rosu a m.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 5 ultrasound-assisted extraction of photosynthetic pigments from dried dill (anethum graveolens) ana-maria rosu 1, * denisa ileana nistor 1, neculai doru miron 1, marcel ionel popa 2, ramona mihaela cojocaru 1,2 1“vasile alecsandri” university of bacau, faculty of engineering, dnistor@ub.ro 2”gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, ramona_mihaela2008@yahoo.com *corresponding author received 5 november 2012, accepted 4 december 2012 abstract: extraction is an important step in studies involving the isolation of active compounds from plant materials. ultrasound assisted extraction (uae) is a simple, inexpensive and efficient technique due to shortened extraction time and reduced organic solvent consumption compared with other extraction method. chlorophyll extracts are used extensively as dye in coloring inks, resins, soaps and waxes, edible fats, cosmetics, lotions perfume. some experimental data suggest also that chlorophyll may ameliorate drug side effects, may have some anti-mutagenic and anti-carcinogenic potential and it may help protect against some toxins. the aim of this study was to determine the influence of ultrasound extraction parameters, like acetone volume (80%, 90%, 100%), ultrasounds exposure time and ratio solvent/material on the extraction of photosynthetic pigments from dill (anethum graveolens). ultraviolet spectroscopy was used to evaluate the efficiency of this extraction method and it was also used to quantitatively analyse the extracted chlorophylls and carotenoids. results show that the extraction efficiency of photosynthetic pigments from dried dill was higher when using acetone 90% comparative to the other two concentrations, because the highest level of total chlorophyll obtained was of 4,53mg/g dried dill in acetone 90% after 15 min. exposure to ultrasounds. the results suggest that uae method is an efficient technique for extraction of photosynthetic pigments. keywords: chlorophyll, carotenoids, spectrophotometric quantification 1. introduction dill is a highly versatile seasoning herb. the dill is classed among species reducing the risk of cancer [1]. its consumption also lowers the level of cholesterolaemia [2] while its components show antioxidative properties [3, 4]. besides, seasoning herbs, including the dill, enrich the main dishes with complementary compounds, such as vitamins, mineral salts, and also compounds affecting the sensory traits of food. one of the most important sensory traits is the colour. the basic pigments of seasoning leafy herbs are chlorophylls, always accompanied by carotenoids. acids, temperature, light, oxygen, and enzymes easily destroy the chlorophylls [5, 6], while carotenoids are fairly resistant to technological procedures [7]. extraction is an important step in studies involving the isolation of active compounds from plant materials. the extraction methods applied and compared in this study were: conventional extraction and ultrasound assisted extraction. ultrasound assisted extraction has been employed as an efficient food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 6 technique for plant pigments, phenolics and antioxidants extraction in the last years [8, 9]. chlorophyll is one of the valuable bioactive compounds that can be extracted from plants. chlorophylls and carotenoids concentration correlate to the photosynthetic potential of plants giving some indication of the physiological status of the plant [10]. however, the content of pigments in plants is important, not only due to the coloration and physiological function, but also due to their acknowledged roles in health [11]. the interest in new data on carotenoids in edible plants is increasing due to a more extensive use of natural compounds in the food, following the directives of european community in favor of natural rather than synthetic compounds [12]. chlorophyll provides a chelating activity which can be used in ointment treatment for pharmaceutical benefits. chlorophyll has also been investigated as source of pigments in cosmetics. because of its strong green colour and consumers demand for natural foods, chlorophyll gained importance as a food additive [13] traditional methods for analysis of photosynthetic pigments were employed based on spectroscopy and extinction coefficients that had been calculated for a range of solvents. for whole-leaf extracts these methods allowed the accurate calculation of chlorophyll a and b concentration, but were limited to a pooling of the carotene pigments to give total carotenoids content [14]. chlorophylls are highly susceptible to degradation during processing and storage [15, 16, 17]. daood, czinkotai, hoschke, and biacs [18] reported that chlorophyll a is more sensitive to degradation induced by thermal processing. the degradation process of chlorophyll a occurred faster according to canjura and schwartz [19] that studied the relative degradation of chlorophyll a with respect to b and showed too that the first degrades faster depending on temperature. the fact that chlorophyll a is more sensitive to thermal treatment was proved by the reduced chlorophyll a/b ratio. the present study was undertaken with an aim to evaluate the pigments content in dried dill. 2. experimental dill was purcheased from the local venders. the plants used in the research work were as fresh as possible and grown without using conventional pesticides and fertilizers. also the raw material was healthy without traces of yellowing. the time from purchasing to the beginning of the analyses and technological processing of the raw material did not exceed 2 h. the plant material was dried in a drier at 400c till the brittleness of the leaves. all solvent were purchased from chemical company and were of spectrophotometric grade. different concentrations of acetone (100%, 90%, 80%) were applied for choice of suitable solvent for the extraction of photosynthetic pigments from dried dill. the experiments of one step extraction of chlorophylls and carotenoids from dried dill were performed by contacting the plant material with acetone at different concentrations for 5, 10 and 15 minutes. the ultrasound assisted extraction was realized in an ultrasonic bath at fixed ultrasonic power of 200w. ultraviolet-visible spectroscopy was used to evaluate the efficiency of the extraction and also to quantitatively analyse the photosynthetic pigments. the equations used for the determination of chlorophyll a and b and total carotene concentrations are presented in table 1 [2024]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 7 table 1 simultaneous equations for the determination of chlorophyll a (ca), chlorophyll b (cb) and total carotene (c(x+c)) solvent equation for chlorophylls and total carotene concentrations (µg/ml) acetone 100% ca=11.24a661.6+2.04a644.8 cb=20.13a645+4.19a662 c(x+c)=(1000a470-1.9ca-3.14cb)/214 acetone 90% ca=11.93a664+1.93a647 cb=20.36a647+5.5a664 c(x+c)=(1000a470-1.9ca-3.14cb)/214 acetone 80% ca=12.25a664+2.55a647 cb=20.31a647+4.91a664 c(x+c)=(1000a470-1.82ca85.02cb)/198 3. results and discussion in the present study the ultrasound assised extraction technique was studied comparively with the conventional extraction. different concentrations for acetone were used to determine the suitable solvent. also the extraction tehniques were applied for 5. 10 and 15 minutes. figs. 1(a). 1(b) and 1(c) present the influence of mixtures acetone/water and extraction time on the concentration of chlorophyll a. chlorophyll b and total carotene by conventional extraction. the highest content of chlorophyll a 2.55mg/g dried dill was obtained through conventional extraction with acetone 90%. 5 10 15 ac 100% ac 90% ac 80% 0 0,5 1 1,5 2 2,5 3 c on ce n tr at io n c h l a (m g/ g d ri ed d il) extraction time figure 1(a). influence of mixtures of acetone/water and extraction time on the concentration of chlorophyll a using conventional extraction the highest content of chlorophyll b. 1.44mg/g dried dill was also obtained using acetone 90%. the extraction of total carotene through conventional method was optimum using acetone 100%. 5 10 15 ac 100% ac 90% ac 80% 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 c on ce nt ra ti o n c h l b (m g/ g dr ie d d ill ) extraction time figure 1(b). influence of mixtures of acetone/water and extraction time on the concentration of chlorophyll b using conventional extraction 5 10 15 ac 100% ac 90% ac 80% 0 1 2 3 4 5 6 7 8 c o n ce n tr at io n t o ta l ca ro te n e ( µ g /m l ex tr ac t) extraction time figure 1(c). influence of mixtures of acetone/water and extraction time on the concentration of total carotene using conventional extraction regarding the ratio of chlorophyll a versus chlorophyll b concentration in extracts the highest ratio of 1.83 was obtained using acetone 90%. the results are presented in fig. 2. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 a c 100% a c 90% a c 80% s o lv en t concentration chlor ophylls (m g/g drie d dill) chl a chl b 1,45 1,51 1,83 figure 2. concentration of individual chlorophyll and ratio of chlorophyll a/b obtained by conventional extraction food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 8 the extraction efficiency of the ultrasounds assisted method was higher then of the conventional method. for chlorophyll a the extraction using ultrasounds yielded 2.8 mg/g dried dill and for chlorophyll b 1.73 mg/g dried dill. acetone 90% was also the most efficient solvent in the case of ultrasound assisted extraction. the influence of the same extraction parameters on the concentration of photosynthetic pigments are presented in figs. 3(a). 3(b). 3(c). 5 10 15 ac 100% ac 90% ac 80% 0 0,5 1 1,5 2 2,5 3 c on ce nt ra tio n c hl a (m g/ g d ri ed d ill ) extraction time figure 3(a). influence of mixtures of acetone/water and extraction time on the concentration of chlorophyll a using ultrasounds assisted extraction 5 10 15 ac 100% ac 90% ac 80% 0 0,5 1 1,5 2 c on ce nt ra tio n c h l b (m g /g d rie d d ill ) extraction time figure 3(b). influence of mixtures of acetone/water and extraction time on the concentration of chlorophyll b using ultrasounds assisted extraction 5 10 15 ac 100% ac 90% ac 80% 0 1 2 3 4 5 6 7 8 c o nc en tr at io n to ta l ca ro te ne (µ g /m l ex tr ac t) extraction time figure 3(c). influence of mixtures of acetone/water and extraction time on the concentration of total carotene using ultrasounds assisted extraction the highest ratio of chlorophyll a versus chlorophyll b obtained was 1.61. results are presented in fig. 4. 0 1 2 3 4 5 ac 100% ac 90% ac 80% s ol ve nt concentration chlorophylls (mg/g dried dill) chl a chl b 1,41 1,53 1,61 figure 4. concentration of individual chlorophyll and ratio of chlorophyll a/b obtained by ultrasound assisted extraction 4. conclusion in terms of concentration of chlorophylls the results showed that ultrasound assisted extraction is a promising extraction technique giving the highest efficiency extraction. as conventional extraction and soxhlet extraction are not always acceptable for industrial applications due to long extraction time. large consumption of solvents and other disadvantages. ultrasound assisted extraction could be an alternative. as giving the best isolation efficiency of chlorophylls in advantageously much shorter extraction time because of the disruption of the plants cells. regardind the different concentrations of acetone used for the extraction of photosynthetic pigments. acetone 90% proved to be the most efficient both in conventional extraction and ultrasouds assisted extraction. 5. references [1]. yang. y.. huang. c. y.. peng. s. s.. li. j. carotenoid analysis of several dark-green leafy vegetables associated with a lower risk of cancers. biomedical and environmental sciences. 9. 386–392. (1996). [2]. lansky. p. s.. schilcher. h.. phillipson. j. d.. loew. d. plants that lower cholesterol. acta horticulturae. 332. 131–136 (1993). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 9 [3]. kidmose. u.. knuthsen. p.. edelenbos. m.. justesen. u.. hegelund. e. carotenoids and flavonoids in organically grown spinach (spinacia oleracea l.) genotypes after deep frozen storage. journal of the science of food and agriculture. 81. 918–923. (2001). [4]. kurilich. a. c.. juvik. j. a. quantification of carotenoid and tocopherol antioxidants in zea mays. journal of agricultural and food chemistry. 47. 1948–1955. (1999). [5]. lopez-ayerra. b.. murcia. m. a.. carmona. g. f. lipid peroxidation and chlorophyll levels in spinach during refrigerated storage and after industrial processing. food chemistry. 61. 113– 118. (1998). [6]. tonucci. l. h.. elbe. j. h. kinetics of the formation of zinc complexes of chlorophyll derivatives. journal of agricultural and food chemistry. 40. 2341–2344. (1992). [7]. granado. f.. olmedilla. b.. blanco. i.. rojas-hidalgo. e. carotenoid composition in rawand cooked spanish vegetables. journal of agricultural and food chemistry. 40. 2135–2140. (1992). [8]. annegowda h.v.. bhat r.. min-tze. l.. karim. a. a.. mansor. s. m.. influence of sonication treatments and extraction solvents on the phenolics and antioxidants in star fruits. j. food sci. technol.. doi:10.1007/s13197-011-0435-8. (2011). [9]. tiwari b.k.. donnell c.p.. cullen p.j.. effect of sonication on retention of athocyanins in blackberry juice. j. food eng. 93. 166-171. (2009). [10]. gamon j. a.. surfus. j. s.. assessing leaf pigment content with a reflectometer. new phytologist. 43. 105–117. (1999). [11]. liu y. t.. perera c. o.. suresh v.. comparison of three chosen vegetables with others from south east asia for their lutein and zeaxanthin content. food chemistry. 101. 1533– 1539. (2007). [12]. znidarcic d.. ban d.. sircelj h.. carotenoid and chlorophyll composition of commonly consumed leafy vegetables in mediterranean countries. food chemistry. 129 1164–1168. (2011). [13]. kong w. liu n.. zhang j.. yang q.. hua s.. song h.. xia c.. optimization of ultrasound-assisted extraction parameters of chlorophyll from chlorella vulgaris residue after lipid separation using response surface methodology. j. food sci technol. doi 10.1007/s13197-012-0706-z. (2012). [14]. dunn j.l.. turnbull. j.d.. robinson. s.a.. comparison of solvent regimes for the extraction of photosynthetic pigments from leaves of higher plants. functional plant biology. 31: 195-202. (2004). [15]. heaton j. w.. lencki. r. w.. marangoni a. g.. kinetic model for chlorophyll degradation in green tissue. j. agric. food chem. 44. 399-402. (1996) [16]. monreal m.. de ancos. b.. cano. m. p.. influence of critical storage temperatures on degratative pathways of pigments in green beans. j. agric. food chem. 47(1). 19-24. (1999). [17]. king v. an-erl. liu c.-f. liu y-j. chlorophyll stability in spinach dehydrated by freeze-drying and controlled low-temperature vacuum dehydration. food research international. 34. 167-175. (2001). [18]. daood. h. g.. czinkotai. b.. hoschke. a.. biacs. p.. high performance liquid chromatography of chlorophylls and carotenoids from vegetables. journal of chromatography. 472. 296–302. (1989). [19]. canjura f. l.. schwartz. s. j.. nunes. r. v.. degradation kinetics of chlorophylls and chlorophyllides. journal of food science. 56. 1639-1643. (1991). [20]. hosikian a. lim s. halim r. danquah m. k.. chlorophyll extraction from microalgae: a review on the process engineering aspects hindawi publishing corporation international journal of chemical engineering.. article id 391632. 11 pages doi:10.1155/2010/391632. (2010) [21]. porra r. j.. the chequered history of the development and use of simultaneous equations for the accurate determination of clorophylls a and b. photosynthesis research. 73. 149-155. (2002). [22]. porra r. j.. thompson w.a.. kriedemann p.e.. determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. biochimica et biophysica acta. 975. 384-394. (1986). [23]. ritchie r. j. consistent sets of spectrophotometric chlorophyll equations for acetone. methanol and ethanol solvents. photosynth res. 89. 27–41. (2006). [24]. ritchie r. j.. universal chlorophyll equations for estimating chlorophylls a. b. c. and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone. methanol. or ethanol solvents. photosynthetica. 46. 115-126. (2008). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 17 influence of some non-ionic surfactants and flocculants on stability of diluted dispersions of kaolin and hydro-mica sergey boruk1 and igor winkler1 1yu. fedkovych national university of chernivtsi, kotsiubinsky st., 2, chernivtsi, 58102, ukraine. e-mail: boruk_s@hotmail.com, igorw@ukrpost.ua abstract: the influence of some non-ionic surfactants and flocculants on aggregation and sedimentation stability of diluted dispersions of the argillaceous minerals has been investigated. this influence can be caused by some changes in physicochemical nature of the particles surface because of the surface adsorption layer formation. the substances with a branched structure of the molecules can disaggregate the dispersed phase particles, which causes a rise in the sedimentation stability of suspensions. the substances with a linear structure of molecules promote aggregation of particles, which leads to the opposite effect on the sedimentation stability.the interaction between particles is also affected by their size distribution. the wider this distribution is, the weaker the interaction between particles is. this effect is mainly caused by a smaller number of big particles, which can be packed in a volume unit and by some hydrodynamic effects, which result in flowing of smaller particles around the bigger ones with flux of liquid. the interaction between bigger and smaller particles ensures the most effective separation of disperse phase.the results can be applied to the enhancement of technologies of natural and waste waters treatment and cleaning from clay-like particles or used in the mineral raw materials refining and concentration. key words: sedimentation stability of suspension, hydrodynamic effect, mineral raw material introduction separation of finely dispersed argillaceous particles from other minerals is often required in the technology of raw materials concentration. some organic compounds can promote more effective separation through better aggregation and faster sedimentation of particles. the highest efficiency of aggregation can be reached for the mixed interaction between bigger and smaller particles when a smaller particle can be drawn into the field of attraction of a bigger one and mutual effect of its molecular, electrostatic and structurizing forces ensures formation of a more compact and stable aggregate. general stability of such an aggregate is governed by the balance of these forces [1, 2]. the characteristics of such mixed suspensions can be regulated by addition of some organic agents. experimental our investigation dealt with the following objects: 1. hydro-mica (mineral illite) with general composition к1-1,5al4[si6.5-7al14.5o20](оh)4*nh2o. this mineral can be classified as an argillaceous mica-like mineral with a layered structure and close to the hexagonal lattice. the layers are built from continuous tetrahedrons sio4. the granulometric composition of this material is represented in fig. 1, line 2. 2. kaolin with general composition al2o3*2sio2*2h2o. this mineral forms well-shaped plate-like hexagonal food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 18 crystals with well distinguished facets. all angles between facets are 1200 and thickness of plates ranged between 0.10.3 µm. the granulometric composition of this material is represented in fig. 1, line 1. 3. the selection of organic modifiers has been carried out with regard to their surface activity towards particles of the disperse phase, chemical inertness and non-toxicity. according to [4], the following substances were found to conform to the above conditions: non-ionic surfactant peg-115 purchased from the ukrainian firm “tos”. molecular weight of this agent ranged within 5000-5100 atomic mass units (a.m.u.) and its formula is ho-(ch2-ch2 -o)n –h, where n = 115. 4. non-ionic surfactant os-20 produced by the ukrainian firm “tos” with molecular weight of 1100-1200 a.m.u. and formula cnh2n-(o-ch2-ch2)m-h, where n=18; m=20. this agent can form micella with critical concentration of micella forming (ccm) = 0.2 kg/m3. 5. non-ionic surfactant twin-80 with molecular weight about 1100 a.u.m. this agent can form micella with ccm = 0.06 kg/m3 and can be characterised by this formula: where n + m = 20 6. polyethylene-oxide (peo) produced by the novosibirsk branch of the kemerovo firm “carbolite” (russia). molecular weight is about 2*106 – 3*106 a.u.m. and formula: но-[-ch2-ch2-o-]n-н. 7. polyacrylamide (paa) produced by the kalush firm “oriana” (ukraine). molecular weight is about 4*106 – 5*106 a.u.m. an effective coefficient of adhering (cad) was calculated as a slope tangent for a dependence of changes in the fine fraction concentration on the rough fraction content [4, 5] and used as a stability criterion for the interparticle interaction in diluted systems. the details of influence of organic modifiers on sedimentation stability of the suspensions have been evaluated using a coefficient of sedimentation instability (csi), which was calculated as a slope tangent for a dependence of the optical density changes during the experiment (d0 – dend) on concentration of the disperse phase particles [6]. o oh ch2 cooc17h33 h(oc2h4)no o (c2h4o)mh n ch2 ch c o nh2 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 19 results and discussion our experimental results proved that the physico-chemical properties of disperse phase particles have a significant influence on the interparticle interaction in suspensions. this interaction in kaolin suspensions (cad = 6.3*10-2) is more intense than in the suspensions with hydromica (cad = 5.3*10-2). this difference is caused by different surface hydrophily properties of kaolin and hydro-mica. surface layers of both minerals are well water-retentive because of high content of the hydroxyl groups on surface. on the other hand, this content on the surface of kaolin is higher, which causes a higher water retention value of this mineral. interparticle interaction also depends on the particles size and value of cad decreases with increase of difference between particles sizes (see fig. 2). this process can be caused by a smaller amount of bigger particles, which can be allocated in the volume unit and some hydrodynamic effects, which results in flowing of smaller particles around the bigger ones with a flux of liquid. non-ionic surfactants and flocculants actively participate in the structures forming processes. stability of argillite suspensions can be influences by addition of these compounds. a characteristic of surfactants’ and flocculants’ influence on kaolin and illite suspensions is quite similar (see tables 1 and 2). low concentrated solutions of polymers (paa and peo, concentration below 0.01 kg/m3) and surfactants (os-20 and peg-115, concentration below 0.025 kg/m3) with linear structure cause more intense particles aggregation in the suspensions of kaolin. further increase in the surface agents’ concentration causes decrease in the value of cad. such behaviour can be related to the properties of adsorbed molecules. bigger macromolecules of peo and paa have comparatively high adsorption activity and can form bridge bonds between the disperse phase particles, which results in a more active mutual flocculation (see table 1, columns 4 and 5). further increase in the polymers concentration causes formation of relatively stable spatial structures with particles and particles aggregates embedded into their nodes. this process provides a stabilizing effect on food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 20 suspensions. moreover, macromolecules of paa can effectively stabilize both suspensions of kaolin: with rough particles only or a mixture of rough and fine particles. table 1. dependence of cad and csi on concentration of non-ionic surfactants and polymers for kaolin suspensions number agent concentration cad csi (rough fraction) csi (fine fraction) csi (rough and fine fraction) i twin-80 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.063 0.018 0.012 0.02 0.021 0.022 0.022 0.22 0.098 0.081 0.076 0.071 0.076 0.079 0.082 0.083 0.108 0.107 0.106 0.105 0.102 0.1 0.099 0.098 0.158 0.09 0.096 0.101 0.103 0.105 0.106 0.107 ii os-20 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.63 0.075 0.53 0.05 0.048 0.47 0.46 0.44 0.98 0.14 0.166 0.136 0.124 0.115 0.094 0.081 0.108 0.105 0.103 0.101 0.1 0.1 0.099 0.099 0.158 0.166 0.175 0.125 0.105 0.091 0.083 0.078 iii peg-115 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.063 0.107 0.127 0.076 0.054 0.04 0.306 0.035 0.098 0.468 0.368 0.322 0.216 0.135 0.135 0.134 0.108 0.11 0.115 0.092 0.091 0.091 0.09 0.089 0.158 0.504 0.383 0.33 0.2 0.116 0.114 0.113 iv peo 0 0.005 0.01 0.025 0.05 0.075 0.1 0.15 0.2 0.063 0.079 0.091 0.117 0.139 0.119 0.092 0.087 0.85 0.098 0.555 0.12 0.115 0.11 0.1 0.098 0.097 0.096 0.108 0.099 0.098 0.096 0.094 0.094 0.093 0.093 0.092 0.158 0.63 0.179 0.158 0.156 0.155 0.154 0.146 0.14 v paa 0 0.005 0.01 0.025 0.05 0.075 0.1 0.15 0.2 0.063 0.094 0.117 0.134 0.077 0.073 0.07 0.063 0.06 0.098 0.27 0.29 0.41 0.346 0.282 0.225 0.11 0 0.108 0.089 0.076 0.052 0.015 0.005 0 0 0 0.158 0.285 0.313 0.477 0.407 0.34 0.274 0.132 0 any concentration of polymer within the experimental range provides a stabilizing effect on fine particles suspensions because they can be easily captured and embedded into spatial structures. the low-molecular surface agents peg-115 and os-20 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 21 provide a similar but weaker effect on kaolin suspensions because of the lower molecular weight of agents and some micelle formation activity of os-20 (see table 1, columns 2 and 3). table 2. dependence of cad and csi on concentration of non-ionic surfactants and polymers for illite suspensions number agent conce ntratio n cad csi (rough fraction) csi (fine fraction) csi (rough and fine fraction) i twin-80 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.053 0.028 0.029 0.036 0.037 0.038 0.039 0.039 0.254 0.209 0.198 0.194 0.196 0.200 0.198 0.201 0.105 0.103 0.101 0.102 0.098 0.095 0.096 0.095 0.364 0.282 0.262 0.250 0.249 0.248 0.242 0.234 ii os-20 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.053 0.066 0.037 0.034 0.032 0.031 0.03 0.029 0.254 0.278 0.289 0.216 0.196 0.192 0.191 0.19 0.105 0.112 0.103 0.104 0.102 0.098 0.1 0.1 0.364 0.375 0.379 0.392 0.352 0.286 0.272 0.253 iii peg115 0 0.0125 0.025 0.05 0.075 0.1 0.15 0.2 0.053 0.081 0.105 0.118 0.09 0.065 0.059 0.053 0.254 0.33 0.279 0.257 0.254 0.25 0.249 0.248 0.105 0.115 0.109 0.1 0.098 0.09 0.088 0.085 0.364 0.43 0.41 0.408 0.4 0.395 0.392 0.388 iv peo 0 0.005 0.01 0.025 0.05 0.075 0.1 0.15 0.2 0.053 0.102 0.116 0.132 0.084 0.078 0.073 0.068 0.064 0.254 0.313 0.293 0.273 0.223 0.222 0.222 0.215 0.2 0.105 0.102 0.098 0.097 0.097 0.096 0.096 0.096 0.096 0.364 0.528 0.525 0.493 0.46 0.45 0.438 0.425 0.41 v paa 0 0.005 0.01 0.025 0.05 0.075 0.1 0.15 0.2 0.053 0.092 0.111 0.111 0.082 0.065 0.059 0.055 0.052 0.254 0.348 0.318 0.192 0.121 0.085 0.048 0 0 0.105 0.082 0.064 0.031 0.011 0.003 0 0 0 0.364 0.475 0.503 0.208 0.46 0.35 0.31 0 0 low molecular weight of the non-ionic agents ensures wider concentration range of their activity but lower effect on the aggregation and sedimentation stability of suspensions. this behaviour can be related to lower adsorption activity of such food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 22 molecules and inability to form bridge bonds between particles. however, all changes in the suspensions characteristics cannot be limited to the influence of surface modification only. modifying agents also can change an associative molecular structure of the disperse medium, which results in an influence on the above mentioned characteristics. for example, a negative effect of turbulent pulsations on interparticle interaction can be diminished by the horizontally oriented adsorbed molecules of peg-115. that is why the maximal values of cad for peg115 and polymer agents are quite similar. the mechanism of modification with peg115 is similar to that of modification with polymers however; shorter molecules of peg-115 can not form a well developed and stable structure. this causes specific pattern of dependence cad – agent concentration, which passes through a maximum (see tables 1 and 2, column 3). the dependencies of sedimentation stability on concentrations of peg-115 or polymers are similar for the systems of rough only or rough and fine particles. however, in the case of systems of fine particles only these dependencies are different. low concentration of non-ionic surfactant peg-115 causes aggregation of the disperse phase particles and decrease in sedimentation stability. the increase in surfactant concentration results in some stabilization of suspension. the molecules of peg-115 promote the aggregation of particles but formation of a stable structure is impossible even for a suspension of comparatively small and light particles. an influence of os-20 on aggregation and sedimentation stability is similar to the influence of peg-115. os-20 makes a lesser influence on the changes of cad and csi because the molecules of this agent are smaller and lighter than peg-115. the aggregation effect of os-20 is very weak and the bridge bonds can not be formed by the molecules of this agent. its activity is based on the surface modification only. the effect of os-20 on sedimentation stability of the suspensions containing fine particles only causes insignificant stabilization effect because this agent is unable to ensure the particles’ aggregation in diluted argillite suspensions. the effect of twin-80 is very different from the effect of the agents with linear molecules. this agent causes lowering in cad and increase in sedimentation stability due to the better dispersion of particles. one can see a well-marked minimum in the dependence of cad on c(twin-80). the spatial structure of twin-80 molecules is responsible for such a specific effect of this agent. twin-80 acts as a dispersion promoter if its concentration is below ccm while this activity experiences some decrease for the concentrations over ccm (see tables 1 and 2, column 1). a character of all dependencies for the illite-containing systems is similar to the dependencies for the systems with kaolin (see table 2) but with a range of effective concentrations of the modifier. this range for peo and paa with kaolin suspensions is much wider comparing with the illite ones. the different granulometric composition of disperse phase and different properties of the adsorbed polymer layer can be responsible for this discrepancy. paa can form a welldeveloped and stable spatial structure, which ensures quick stabilization of the system against sedimentation. structure forming properties are less typical for peo and its higher adsorption value causes aggregation of hydro-mica even at higher peo concentration. this results in decrease in sedimentation stability of the hydro-mica suspension. decrease in cad values can be caused by prevailing interaction between rough particles at higher concentration of peo (table 1 and 2). non-ionic surfactant peg-115 shows higher activity in the suspensions of illite food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 23 and in wider range of concentrations. it can be assumed that a higher value of adsorption ensures formation of a more stable and developed spatial structure of peg-115 molecules in the illite-clay suspensions. this process results in a higher cad and lower sedimentation stability of suspensions. a range of os-20 effective concentrations for the illite suspensions is similar to a range for the kaolin ones. similar results have also been registered for twin-80. both agents act on the suspensions of illite and kaolin in a similar way (see tables 1 and 2, columns 1 and 2). conclusion it was proved that the aggregation and sedimentation stability of argillite minerals suspensions can be changed by addition of some surfactants and flocculants. those agents with linear molecules can significantly decrease the stability of diluted argillite systems. the agents with branched molecules ensure higher stability. references 1. churaev n. v. , sobolev v. d. role of the structure forces in wetting of the quartz surface with electrolytes. (2000). coll. j., 62, 278-285. 2. yeremenko b. v. et al. aggregative stability of water dispersions of yttrium oxide. (2000). coll. j., 62, 58-64. 3. surfactants. ed.: abramson a. a. (1979). chemistry. leningrad. 302 p. 4. chiganova g. a. particles aggregation in hydrosols of ultrafine diamond particles. (2000). coll. j., 62, 272-277. 5. vorobieva t. a. and voldavets i. n. influence of surfactants on the formation rate of high-molecular condensed structures. (2000). coll. j., 62, 309-312. 6. goncharuk e. v. et al. role of the structure-mechanical barrier in the mixed aqueous dispersions of hydrophilic and hydrophobic silica. (2001). ukr. j. chem., 67, 36-39. cercetări privind influenţa pretratamentului alcalin si alcalino-peroxidic asupra cantitatii de zaharuri reducatoare obtinuta food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 65 studies regarding the alkaline and alkalineperoxidic pretreatment influence upon the reducing sugars quantity obtained from the wheat straws *eufrozina albu (niga)1, alina-mihaela pşibilschi1 1 ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: e.albu@fia.usv.ro, alinap@fia.usv.ro *corresponding author received 16 august 2011, accepted 18 october 2011 abstract: the main goal of this study was the evaluation of the reducing sugars quantity obtained from the wheat straws using the enzymatic complex accellerase 1500 from genencor in different conditions of alkaline and alkaline-peroxidic pretreatment. in the first phase we observed the induced modifications for the lignocellulosic materials at the cellulasic attack by the application of some alkaline treatments upon them. at the same time, we also observed the way in which the concentration of the alkaline solution influences the results of the enzymatic attack applied afterwards to the lignocellulosic materials. we used naoh solutions at concentrations of 0,5 %, 1,0 %, 2,0 % and 4 %. from the data obtained we can conclude that the best results have been seen in the case of high concentration naoh solutions. in the second phase we tried to obtain some results with the same efficiency as in the previous phase but by applying an alkaline pretreatment coupled with a peroxidic pretreatment. for this purpose we tested several hydrogen peroxide concentrations (of 0,5 %, 1,0 %, 1,5 % and 2 %) , keeping constant the concentration of the naoh solution. the reducing sugars production was evaluated at the enzymatic hydrolysis periods of 24 and 48 h by the dns method. the maximum content of reducing sugars from the analyzed raw material was obtained in the case of the combined alkaline-peroxidic pretreatment at 100°c for 2h using naoh of 4 % concentration and h2o2 of 2,0 % concentration. keywords: lignocellulosic materials, enzymatic hydrolysis, cellulase, dns method. 1. introduction production of fuel ethanol from renewable lignocellulosic materials has been extensively studies in the last decades [1]. lignocellulosic biomass is structurally complex, composed of crystalline cellulose (source of glucose) and amorphous hemicellulose (source of pentose such as xylose and arabinose; hexose such as glucose, galactose and mannose) as its major sugar polymers [2], [3]. biochemical conversion of lignocellulosic biomass through saccharification and fermentation is a major pathway for bioethanol production [4], [5]. lignocellulosic materials to be considered for ethanol production include wood, crops from annual plants, agricultural residues (cereal straw, rice hulls, cotton waste), waste paper and municipal solid wastes [6], [7]. wheat straw is one of the most abundant crop residues in european countries with a production of 170 million tonnes and seems to be the cheapest and the most useful raw material for the ethanol production [8]. it is comprised mainly of cellulose (33-40%), hemicellulose (20-25%) and about 20% mailto:e.albu@fia.usv.ro mailto:alinap@usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 66 lignin [9]. bioethanol from these feedstocks could be an attractive alternative for disposal of these residues. an initial pretreatment stage is needed to soften the material and break down its structure to make it more susceptible to an enzymatic attack before fermentation [10]. the ideal pretreatment consists on avoiding the need for reducing the size of biomass particles, preserves the pentose fraction, limits formation of degradation compounds that inhibit growth of fermentative microorganisms, minimise energy demands and reduce costs [10]. the main goal of this study was to evaluate the reducing sugars quantity obtained from the wheat straws in different conditions of alkaline and alkaline-peroxidic pretreatment. 2. experimental 2.1. raw material the raw material used for obtaining the reducing sugars was represented by wheat straws obtained from the farmers from vrancea area. the raw material was air dried and grinded in the hammer mill at dimensions of approximately 2 mm. 2.2. the wheat straws pretreatment the alkaline pretreatment: a given quantity of lignocellulosic material (4 g) was treated in the first phase with the same volume of naoh solution (100 ml), at different concentrations. thus there were used for the straws pretreatment naoh solutions at concentrations of 0.5 %, 1 %, 2 %, and 4 %. for to realize the experiment it was used a solid/ liquid ratio of 1:25. the samples were thermostated at a temperature of 100°c, the treatment period being of 2 h. the alkalineperoxidic pretreatment in the second phase the lignocellulosic material (wheat straws) was simultaneously treated with naoh and h2o2 solutions. the naoh solution concentrations were the same as in the previous phase and the h2o2 solution concentration varied between 0.5 and 2 %. the samples were thermostated at a temperature of 100°c, for 2 h. the pretreated straws were cleaned with water (approximately 12 volumes) for to eliminate the inhibitors resulted after the pretreatment (furfural, hmf, sodium hydroxide) and the resulting glucose, that inhibits the cellulase enzymatic activity. the pretreated lignocellulosic material was suspended in buffer solution of na2hpo4 – citric acid with a ph of 5 and it was subjected to the enzymatic hydrolysis. 2.3. the enzymatic hydrolysis of the pretreated wheat straws for to check the measure in which the applied pretreatments influenced the susceptibility of the lignocellulosic materials at the enzymatic attack they were subjected to the enzymatic hydrolysis. the enzymatic hydrolysis was done by using the enzymatic complex accelerase 1500 the working method being the following: the pretreated straws were washed with distilled water until they reached a neutral ph and after they were suspended in 100 ml buffer solution of na2hpo4 0.2m – citric acid 0.1m, at a ph between 4.6 and 5.0. then the enzymatic solution was added in the dosage of 0.25 ml/g biomass. the samples were thermostated at the temperature of 50°c, and the reducing sugars content was determined after 24 and 48 h. accellerase 1500 is produced by a genetically modified strain of trichoderma reesei and represents an enzymatic complex especially formed by exoglucanases, endoglucanases, hemicellulase and beta-glucosidase. accellerase 1500 is able of efficiently hydrolyze the lignocellulosic biomass in fermentable monosaccharide. accellerase 1500 contains a high level of betaglucosidase activity in comparison with the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 67 cellulases available on the market and thus it ensures almost completely the cellobiose conversion to glucose. the benefits of the enzymatic complex accellerase 1500 in comparison with the conventional cellulases are the following: an improved saccharification performance for a variety of raw materials; ability to operate in simultaneous saccharification and fermentation (ssf) processes, two step sequential hydrolysis and fermentation (shf) processes a high beta-glucosidase activity that leads to higher saccharification and fermentation rates than for the ethanol; an improved formula for reducing the inhibition risk for the fermentation organisms. 2.4. methods of analysis the quantification of the reducing sugars after the pretreatment phases and the enzymatic hydrolysis was performed using the dns method [11]. this method tests for the presence of free carbonyl group (c=o), the so-called reducing sugars. this involves the oxidation of the aldehyde functional group (eq. 1), simultaneously, 3.5-dinitrosalicylic acid (dns) is reduced to 3-amino, 5nitrosalicylic acid (eq. 2) under alkaline conditions, whose absorbance was measured with a spectrophotometer at 575 nm. aldehyde group oxidation carboxyl group (1) 3.5-dinitrosalicylic acid reduction 3-amino, 5nitrosalicylic acid (2) with the aid of dns method one can determine the concentration of all the reducing sugars in the hydrolysis environment not only for the glucose. it is important to be aware of the presence of all the reducing sugars for the yeast has the capacity to assimilate not only the glucose but also the saccharose, maltose and maltotriose. in figure 1 it is presented the calibration curve for the reducing sugars determination by the dns method. y = 0.0006x 0.0084 r2 = 0.9974 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 concentration of reducing sugar , mg/l a b so rb an ce figure 1. calibration curve for determination of reducing sugar by dns method 3. results and discussion 3.1. the influence of the alkaline pretreatment upon the enzymatic degradation of the lignocellulosic materials the variants used in this study are: variant p1: pretreated wheat straws with naoh 0.5%; variant p2: pretreated wheat straws with naoh 1%; variant p3: pretreated wheat straws with naoh 2%; variant p4: pretreated wheat straws with naoh 4%; the alkaline pretreatment was led at a temperature of 100°c for 2 h, and the enzymatic hydrolysis was led at 50°c. the results obtained in the case of the alkaline pretreatment are presented in figure 2. we can observe that the alkaline pretreatment induces clear modifications in the release of reducing sugars. the release of reducing sugars for the enzymatic hydrolysis increases along with the increasement of naoh concentration. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 68 5. 02 7. 03 5. 16 7. 17 5. 32 7. 31 5. 39 7. 44 0 2 4 6 8 10 12 14 r ed uc in g su ga r, g /l p1 p2 p3 p4 24 h 48 h figure 2. the alkaline pretreatment and the enzymatic hydrolysis at 50°c the glucidic content is also highly influenced by the period of the enzymatic hydrolysis. in this way we can observe that in a period of 48 h of enzymatic hydrolysis the glucidic content increases with ≈ 2 g/l. 3.2. the influence of the alkalineperoxidic pretreatment upon the enzymatic degradation of the lignocellulosic materials from the data previously obtained we can say that the best results can be obtained in the case of the naoh solutions of high concentrations. but this fact brings along besides the economic issues (duet o the high costs of naoh and the using of large quantities of water for its elimination) ecological issues due to the release in the environment of the cleaning waters. in this situation we tried to obtain results with the same efficiency as for the alkaline pretreatment by applying an alkaline treatment coupled with a peroxidic one. for this purpose we tested several concentrations of hydrogen peroxide keeping constant the naoh solution concentration. 3.2.1. study 1: the variants used in this study are: variant p5: pretreated wheat straws with naoh 0.5% and h2o2 0.5 %; variant p6: pretreated wheat straws with naoh 0.5% and h2o2 1.0 %; variant p7: pretreated wheat straws with naoh 0.5% and h2o2 1.5 %; variant p8: pretreated wheat straws with naoh 0.5% and h2o2 2.0 %; the alkaline-peroxidic pretreatment was led at a temperature of 100°c for 2 h, and the enzymatic hydrolysis was led at 50°c. by the enzymatic hydrolysis of the wheat straws as presented above we obtained the results shown in figure 3. 5. 12 7. 11 5. 53 7. 52 5. 79 7. 8 5. 97 7. 99 0 2 4 6 8 10 12 14 r ed uc in g su ga r, g /l p5 p6 p7 p8 24 h 48 h figure 3. the alkaline-peroxidic pretreatment and the enzymatic hydrolysis at 50°c we can observe that the adding of h2o2 leads to the increase of reducing sugars accumulation. by comparing the values registered for the alkaline-peroxidic treated variants as well as for the naoh treated variants we observe significant increasements. the efficiency of the alkaline-peroxidic pretreatment is proportional with the h2o2 concentration. 3.2.2. study 2: the variants used in this study are: variant p9: pretreated wheat straws with naoh 1 % and h2o2 0.5 %; variant p10: pretreated wheat straws with naoh 1 % and h2o2 1.0 %; variant p11: pretreated wheat straws with naoh 1 % and h2o2 1.5 %; variant p12: pretreated wheat straws with naoh 1 % and h2o2 2.0 %; the alkaline-peroxidic pretreatment was led at a temperature of 100°c for 2 h, and the enzymatic hydrolysis was led at 50°c. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 69 by the enzymatic hydrolysis of the wheat straws as presented above we obtained the results shown in figure 4. 5. 36 7. 37 5. 83 7. 84 5. 9 7. 89 6. 03 8. 04 0 5 10 15 r ed uc in g su ga r, g /l p9 p10 p11 p12 24 h 48 h figure 4. the alkaline-peroxidic pretreatment and the enzymatic hydrolysis at 50°c the data obtained reveal an increasement of the reducing sugars accumulation when using naoh of 1 % concentration. when using h2o2 for the wheat straws treatment the increasement is accentuated along with the h2o2 concentration increasement. 3.2.3. study 3: the variants used in this study are: variant p13: pretreated wheat straws with naoh 2 % and h2o2 0.5 %; variant p14: pretreated wheat straws with naoh 2 % and h2o2 1.0 %; variant p15: pretreated wheat straws with naoh 2 % and h2o2 1.5 %; variant p16: pretreated wheat straws with naoh 2 % and h2o2 2.0 %; the alkaline-peroxidic pretreatment was led at a temperature of 100°c for 2 h, and the enzymatic hydrolysis was led at 50°c. by the enzymatic hydrolysis of the wheat straws as presented above we obtained the results shown in figure 5. 5. 75 7. 76 6. 32 8. 31 6. 48 8. 49 6. 62 8. 63 0 5 10 15 20 r ed uc in g su ga r, g/ l p13 p14 p15 p16 24 h 48 h figure 5. the alkaline-peroxidic pretreatment and the enzymatic hydrolysis at 50°c the data obtained reveal an increasement of the reducing sugars accumulation when using naoh of 2 % concentration. when using h2o2 for the wheat straws pretreatment the increasement is accentuated along with the h2o2 concentration increasement. 3.2.4. study 4: the variants used in this study are: variant p17: pretreated wheat straws with naoh 4 % and h2o2 0.5 %; variant p18: pretreated wheat straws with naoh 4 % and h2o2 1.0 %; variant p19: pretreated wheat straws with naoh 4 % and h2o2 1.5 %; variant p20: pretreated wheat straws with naoh 4 % and h2o2 2.0 %; the alkaline-peroxidic pretreatment was led at a temperature of 100°c for 2 h, and the enzymatic hydrolysis was led at 50°c. by the enzymatic hydrolysis of the wheat straws as presented above we obtained the results shown in figure 6. 5. 81 7. 82 6. 42 8. 43 6. 5 8. 52 6. 64 8. 65 0 5 10 15 20 r ed uc in g su ga r, g /l p17 p18 p19 p20 24 h 48 h figure 6. the alkaline-peroxidic pretreatment and the enzymatic hydrolysis at 50°c food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 70 it is observed that the h2o2 adding leads to the increasement of reducing sugars accumulation fact that also happens along with the naoh solution concentration increasement. 4. conclusion after the alkaline pretreatment applied to the lignocellulosic materials (wheat straws) for 2 h at 100c, from all the variants taken in consideration by this study the best results are obtained in the case of the naoh solution of 4% concentration. it can be noticed a proportional increasement of the reducing sugars accumulation with the naoh solution concentration used for the pretreatment. this is applied for a concentration of the solution up to 2%, for in the cases of higher concentrations the dependence is no longer linear. when applying the alkaline-peroxidic pretreatment for the same naoh concentration the treatment’s efficiency increases along with the used h2o2 concentration, being registered increasements of the reducing sugars accumulation ~35% higher than in the cases of the variants treated with naoh of the same concentration. we can conclude from the experiments that the optimal method for treating the wheat straws is represented by the naoh 4% and h2o2 2% variant. the enzymatic hydrolysis of the alkaline and alkaline-peroxidic pretreated wheat straws using the accellerase 1500 solution for 48 h led at an increased efficiency in comparison with the 24 h hydrolysis. the lignocellulosic biomass has the potential of becoming the key element in the future increasement of the quantity of generated bioenergy. its universal availability in high quantities as well as the fact that nowadays it is not used on a large scale are the main reasons for which the lignocellulosic biomass is considered to be one of the most promising resources for the future of bioenergy production. the pretreatments can significantly improve the reducing sugars production and subsequently the bioethanol production and they must be seen as a key for the biochemical conversion of the lignocellulosic materials. 5. references [1] eriksson t., karlsson j., tjerneld f., a model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase i (cel 7a) and endoglucanase i (cel 7b) of trichoderma reesei, appl biochem biotechnol., 101: 41-60, (2002) [2] antal m.j., allen s.g., dai x., shimizu b., tam m.s., and grönli, m., attainment of the theoretical yield of carbon from biomass, ind. eng. chem. res., 39, 4024-4031, (2000) [3] saha b.c., dien b.s. and bothast r.j., fuel ethanol production from corn fiber: current status and technical prospects, appl. biochem biotechnol, 70-72, 115-125, (1998) [4] lin y., tanaka s., ethanol fermentation from biomass resources, appl. microbiol. biotechnol. 69, 627-642, (2006) [5] wheales a.e., basso l.c., alves d.m.g. and amorim h.v., fuel ethanol after 25 years, trends biotechnol., 17, 482-487, (1999) [6] palonen h., tjerneld f., zacchi g., tenkanen m., adsorption of trichoderma reesei cbh i and eg ii and their catalytic domains on steam pretreated softwood and isolated lignin, j. biotechnol, 107: 65-72, (2004) [7] wyman ch.e., twenty years of trials, tribulations and researches progress in bioethanol technology, appl. biotech., 91-93, 5-12, (2001) [8] fang j.m., fowler p., tomkinson j., hill cas., preparation and characterization of methylated hemicelluloses from wheat straw, carbohyd polym, 47:285-93, (2002) [9] lequart c., nuzillard j.m., kurek b., debeire p., hyydrolyses of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides, carbohyd res, 319: 102-11, (1999) [10] del campo i., alegria i., zazpe m., echeverria m., echeverria i., diluted acid pretreatment of agri-food wastes for bioethanol production, industrial crops and products 24, 214221, (2006) [11] miller g.l., use of dinitrosalicylic acid reagent for determination of reducing sugar, anal. chem., 31, 426, (1959) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 43 studies regarding new way to render whey profitable adriana dabija1, gabriela constantinescu (pop) 1, ioana rebenciuc1, amelia buculei1 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii str., suceava, romania, adabija@usv.ro abstract. whey may be defined broadly, as the serum or watery part of milk remaining after the separation of curd resulting from the coagulation of milk by acid or proteolytic enzymes. whey has been used by many researchers as raw material for the alcoholic fermentation process for it contains a high lactose quantity and it is widely available. one of the most simple and economical solutions for rendering whey profitable as secondary product of cheese fabrication by cheese/ dairy manufacturers is to obtain whey drinks for it has been used for long time by many researchers as raw material for the alcoholic fermenation process. one of the whey drinks proposed is whey beer made from deproteinized whey (as a byproduct of sweet cheese fabrication). the paper presents the research results in obtaining a drink from deproteinized whey resembling beer. three samples have been experimented. the three beer samples have been analyzed from the sensorial and physicalchemical point of view as well. the second of them, containing 30% deproteinized whey has been the most appreciated. the finished product whey beer is a product with traits similar to those of normal beer: pleasant, specific smell, hop flavour, bitter-sweet pleasant taste. keywords: byproduct, deproteinized whey, whey beer, dairy industry, lactose introduction as secondary product of cheese fabrication, rendering whey valuable represents a major problem of dairy industry that needs simple and economical solutions. on the basis of cheese consumption and production details, it is estimated that approximately 9 million tones of cheese per annum is produced within the eu, giving rise to an annual whey production figure of the order of 50 million m3 [1, 2]. whey may be defined broadly, as the serum or watery part of milk remaining after the separation of curd resulting from the coagulation of milk by acid or proteolytic enzymes. the type and composition of whey in dairy plants depends mainly upon the processing technique resulting in casein removal from fluid milk [1, 3]. about 50% of total world cheese-whey production is treated and transformed into various food products, of which about 45% is used directly in liquid form, 30% in the form of powdered cheese whey, 15% as lactose and de-lactosed by-products, and the rest as cheese-whey-protein concentrates [1, 4]. the world whey production is estimated to be of 108 tons/ year. it is an important source of environment pollution. its biological treatment by the conventional aerobic process is very expensive and most of the dairy plants do not have a treating system. as a consequence, 47% of whey contaminates rivers, lakes and soil causing serious pollution problems [5]. to obtain whey drinks may be one of these solutions. whey has been used by many researchers as raw material for the alcoholic fermentation process for it contains a high lactose quantity and it is food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 44 largely available [6]. it is known that whey contains 55% of the milk nutrients: lactose, soluble proteins, lipids, mineral substances (over 50% of these being nacl and kcl), organic acids (lactic acid, citric acid), non protein nitrogenous compounds (urea and uric acid), and group b vitamins (table 1). until now non alcoholic drinks as well as alcoholic ones (slightly alcoholized with max. 1% alcohol) and drinks similar to beer and wine have been obtained from deproteinized whey. the paper presents the research results to obtain a drink from deproteinized whey resembling beer. table 1. the typical whey composition [3] whey type components sweet whey [g/l] acid whey [g/l] dry substance, of which: 63-70 63-70 lactose 46-52 44-46 protein 6-10 6-8 lipids 3 2 calcium 0.4-0.6 1.2-1.6 phosphate 1-3 2-4.5 lactate 2 6.4 materials and methods to obtain beer under lab conditions we used deproteinized sweet whey from s.c. tocar prod srl radauti company that presented the following physicochemical traits: total dry substance – 5.62%, of which: fat – 0.02%; proteins – 0.47%; lactose – 4.50%; acidity – 15.8°t; density – 1.028 g/l. in the fabrication systems we used: malt, hopped malt provided by s.c. bermas s.a. suceava, pellets hops, sugar, caramel, saccharomyces cerevisiae and saccharomyces carlsbengensis species yeast, dry raisins. the malt wortt as raw material presented the following physicochemical traits: extract content – 10°p; ph – 6.0; colour – 0.8 ml i2 0.1n/100 ml. the methods of analysis used in the experiments were the following: acidity – titrimetric method; dry substance content – refractometric method; extract content – refractometric method; ph–potentiometer method; co2 content – according to legislation. the finished productwhey beer was analyzed from the sensorial point of view using the marking scale method and from the physico-chemical point of view (colour, acidity, co2 content). the marking scale method consists in evaluating each sensorial property by comparing it with marking scales from 0 to 5 point and by obtaining the average mark from the tasters group. for this purpose we established a tasting committee formed by 7 members who had been previously trained regarding the organization of tasting meeting, its purpose and the marking system used in the tasting note papers to eradicate possible errors that can occur when appreciating the analyzed samples. on the basis of the average total mark there has been led an evaluation of the quality level of whey beer samples from the sensorial point of view using a 0 to 20 points scale. results and discussion the obtaining of whey beer under lab conditions followed the steps of industrial process. first of all the two raw materials whey and malt wortt have been analyzed from the physico-chemical point of view (quality reception) and then dosed according to the followed fabrication recipes (table 2). for colour correction caramel has been used. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 45 table 2. the fabrication recipes to obtain whey beer *saccharomyces cerevisiae species yeast; ** saccharomyces carlsbergensis species yeast after being blended, pasteurized and cooled at room temperature the samples have been inseminated with yeast and subjected to a first fermentation for 24 hours at a temperature of 20-22°c (room temperature). the prefermented whey beer has then been filtered, corked in 500 ml bottles and subjected to a final fermentation at a temperature of 5-6°c (refrigerator) for sample 2 and 20-22°c (room temperature) for sample 1 and 3. the three beer samples have then been analyzed from the sensorial and physicochemical point of view. the results of the sensorial analysis are presented in table 3. one of the beer quality characteristics, appreciated by many consumers is the foam’s height when pouring beer into glass, foam that has to persist for quite a while. for checking this, there we used 2 special glasses in which whey beer cooled at 10°c was poured and soon after the foam height was measured. the foam persistence time was measured from the pouring moment untill its total disappearance. the results of the determinations made are presented in table 4. table 3. the evaluation of quality level of the whey beer samples analyzed sample total average mark appreciation product characterizatio n sample 2 18.2 very good whey beer has positive, specific, well distinguished organoleptic traits. it does not have any obvious flaws. sample 1 12.5 sample 3 13.7 satisfactory whey beer has specific poorly distinguished traits as well as lacks and small flaws in spite of which can be situated at the admitted product standard level. table 4. checking the whey beer samples foam foam checking sample foam layer height [mm] foam stability [minutes] sample 1 45 5 sample 2 62 8 sample 3 50 6 table 5 shows the results of physicochemical analyses for whey beer samples obtained under lab conditions. table 5. the physico-chemical marks for the whey beer samples analyzed physico-chemical marks sample colour [ml i2 0,1n/100 ml] total acidity [ml naoh 1n/ 100 ml] co2 content[g/ 100 ml] sample 1 1.2 2.48 0.28 sample 2 1.3 2.3 0.45 sample 3 1.1 2.7 0.32 the fabrication recipe for 1l of finished product sample 1 sample 2 sample 3 deproteinized whey, ml 900 300 900 malt wortt, ml 600 sugar, g 100 100 100 yeast, g 2* 2* 2** pellets hops 3 3 dry raisins + + caramel + + food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 46 conclusions to obtain whey drinks represents a viable solution of its rendering valuable that can be used by cheese / dairy manufacturers in our country. this paper aimed at rendering the deproteinized whey profitable (as byproduct of sweet cheese fabrication) to obtain whey beer. of the 3 proposed samples the one that was unanimously appreciated was sample 2 that contained 30% deproteinized whey. it has been found out that lactose, the predominant component of whey does not influence the final taste of drink (the saccharomyces yeast type does not metabolize lactose which has low sweetening power). the finished product whey beer is a product with traits similar to those of normal beer: pleasant, specific smell, hop flavour, bitter-sweet pleasant taste. references 1. kosseva, m., et al. – use of immobilized biocatalysts in the processing of cheese whey, international journal of biological macromolecules 45,2009, 437-447 2. pamesar, p., et al. – bioutilisation of whey for lactic acid production, food chemistry 105, 2007, 1-14 3. jelen, p – whey processing, encylcopedia of dairy sciences, vol.4, london academic press, 2003, 2739-2751 4. koller, m. et al. – polyhydroxyalkanoate production from whey by pseudomonas hydrogenovora, bioresource technology 99, 2008, 4854-4863 5. ozmihci, s., kargi, f. – ethanol production from cheese whey powder solution in a packed bioreactor at different hydraulic residence times, biochemical engineering journal 42, 2008, 180-185 6. koutinas, a., et al. – whey valorization: a complete and novel technology development for dairy industry starter culture production, bioresource technology 100, 2009, 3734-3739 tabelelul 1 21 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xv, issue 12016, pag. 21 28 comparative study on the lipase activity from plant sources, under various conditions of ph, temperature and substrate marcel avramiuc1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received january 5th 2016, accepted march 9th 2016 abstract. the activity of the lipase from four different plant sources, under various conditions of ph, temperature and substrate, was analyzed in this paper to see which of these sources shows the highest enzyme activity. the lipase sources were represented by seeds, belonging to four plant species: sunflower, corn, pumpkin and soy, and as substrates for enzyme activity, the following refined oils were used: sunflower, pumpkin, soy bean, corn, peanut, walnut, almond and sesame. the activity of lipase was determined at 20ºc and 40ºc, at three different ph values (5.4, 7.4 and 8.2) for each temperature, and consisted in titrating (with a solution of koh 0.01 n) fatty acids released from oils by lipase, in a certain time interval. according to the experimental data, the lipases deriving from sunflower, corn, soy and pumpkin seeds, registered the highest values of activity at ph 5.4 (at 20°c or 40°c). as compared to the other three sources, the values of sunflower seed lipase activity were significantly higher (p<0.05) in the following oils: sunflower, soybean, peanut, corn and walnut. at the same ph 5.4, the corn caryopse lipase recorded the highest activity on walnut oil, at 20°c, and on corn oil, at 40°c, the pumpkin seed lipase on walnut oil, at 40°c, and the soy bean lipase on walnut, at 40°c. a high content of oleic acid, but especially of linoleic and linolenic acids within oils used as substrate, caused an increased activity of lipase in sunflower seeds. keywords: lipase, oil, seed, source, ph, temperature, substrate 1. introduction lipases act at the organic-aqueous interface, catalyzing the hydrolysis of ester-carboxylate bonds and releasing fatty acids and organic alcohols [1, 2, 3, 4]. according to some authors [5, 6], in waterrestricted environments, the reverse reaction (esterification) or even various transesterification reactions can occur. lipases are used in many sectors such as food, pharmaceutical, fine chemical, oil chemical, biodiesel and industrial detergent industries [6, 7], and they will acquire importance comparable to that of the peptidases, which currently represent 25 to 40% of industrial enzyme sales [8]. lipases are employed in food manufacturing to liberate fatty acids into food products by selective hydrolysis of the fats and oils present in many kinds of food [9]. according to some authors [6, 10, 11] ct. by [9], depending on the carbon chain length and on the degree of unsaturation, the fatty acid obtained provides the food with flavors, colors and unusual smells, playing an important role in the physical-chemical, organoleptic and nutritional properties of many products the origin of lipases can be microbial (bacterial, fungal, etc.), vegetal (oilseeds, pulses, cereals) or animal (gastric, hepatic, pancreatic, adipose tissue). according to [12], despite the extensive range of microbial lipases, the use of these enzymes on an industrial scale is still http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 22 restricted due to high production costs, favoring the search for other sources of these enzymes. seed lipases present advantages over animal and microbial lipases due to some quite interesting features such as specificity, low cost, availability and ease of purification, representing a great alternative for potential commercial exploitation as industrial enzymes [12, 13, 14, 15, 16]. considering that in recent years, the lipases from seeds arouses an increasing interest, due to the many advantages they have as compared to the microbial and animal ones, in this paper there was conducted a study of the lipases activity from four different plant sources, in terms of various ph, temperatures and substrates (oils), to see which of these sources shows increased activity and under what conditions. 2. experimental the experimental materials, used as lipase sources, and provided by suceava genebank and by suceava agricultural research and development station, have been represented by seeds (with moisture content of 10-12%), belonging to the following plant species: sunflower (helianthus annuus l., hd. rapid), corn (zea mays l., hd. f 376), pumpkin (cucurbita maxima l., local variety) and soy (glycine max l., var. turda 6114). as substrates for enzyme activity, were used the following refined oils: sunflower, pumpkin, soy bean, corn, peanut, walnut, almond and sesame, purchased from supermarkets. the lipase activity has been determined at 20ºc and 40ºc, at three different ph values (5.4, 7.4 and 8.2) for each temperature, and has consisted in titrating (with a solution of koh 0.01 n) of fatty acids released from oils by lipase, in a certain time interval [17]. in order to obtain the enzyme preparation skimmed and dried, in a glass bottle with a stopper it has mixed some seeds finely divided with two sides ether, then let stand for 2-3 hours for oil extraction, stirring periodically. it has separated ether, and they have introduced again 5 parts ether over the product partly skimmed, for one hour, after which it was separated ether. the skimmed seeds were dried in an oven with fan, at a temperature of 30°c. there were obtained defatted seeds containing lipase. for lipase activity determination, in an erlenmeyer flask were introduced: 1 g of refined oil, 2 ml phosphate buffer ph 5.4, then 1 g of defatted seeds finely ground and 3 ml of distilled water at temperature of 20ºc. the flask was closed with a stopper and it was stirred gently for 30 minutes, then they were added 15 ml of alcohol 96% (v) and 15 ml of petroleum ether (v), and the content was stirred again for 10 seconds. finally, the fatty acids present in the sample were titrated with 0.01n koh in the presence of phenolphthalein, as indicator. in the same way, it was done using phosphate buffers ph 7.4 and ph 8.2, creating each three working variants for temperature 20ºc, and 40ºc respectively. in parallel, they were done two control samples (for each working sample), consisting of the mixture of all reaction components, except the oil, which were well stirred and heated for 5 minutes in a water bath at boiling, to inactivate the enzyme. after cooling, it has added to each 1 ml oil and it has proceeded like at the investigated samples. the lipase activity (al) was expressed as fatty acid micromols (μmol), represented by oleic acid, and formed, as result of enzyme action, from a gram of product, in one minute (eq.1): tg10282 00282.0)vv( al 6 mp     (1) where: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 23 vp – the volume of koh 0.01 n used for titrating of sample where the enzyme acted (according to fatty acids released by enzyme and to those ones existing within substrate), ml; vm – the volume of koh 0.01 n used for titrating of blank sample (according to fatty acids existing within substrate), ml; 0.00282 – the oleic acid titre, according to koh 0.01 n (g/ml) 28210-6 – 1 micromols (µmol) of oleic acid; g – the product amount (g) used in experiments; t – thermostating interval (60 minutes). the data of experiments, consisting in four replicates for each determination, were statistically processed using sas version 8.02 [18]. in order to analyze the significance of differences among samples, generalized linear model analysis was carried out, and for multiple comparisons was used duncan’s multiple range test (p < 0.05). 3. results and discussion the table 1 reproduces the sunflower seeds lipase activity on the eight various substrates (oils). table 1 lipase activity mean values (±sd) of sunflower seeds on different substrates lipase source sunflower seeds refined oils sf pk sb pn cn wn al se la (μmol oleic acid/ g/min. 20°c ph = 5.4 6.4± 0.7ab* 5.16± 0.43c* 8.66± 0.72a 6.56± 0.45ab 6.66± 0.09ab 7.33± 1.08ab 5.23± 0.61c 5.33± 0.44c ph = 7.4 0.33± 0.02ef 2.5± 0.11de 0.16± 0.01ef 2.5± 0.15de 1.9± 0.22ef 4.33± 0.28c 5.66± 0.47c 4.9± 0.35c ph = 8.2 2.06± 0.31de 1.5± 0.24ef 1.33± 0.15ef 0.9± 0.08ef 1.33± 0.27ef 1.66± 0.18ef 0.56± 0.07ef 0.33± 0.04ef la (μmol oleic acid/ g/min. 40°c ph = 5.4 7± 0.9ab 5.66± 0.44c 7± 1.02ab 7.33± 0.88ab 6.33± 0.59ab 6.66± 0.76ab 5.83± 0.52c 6± 0.63c ph = 7.4 3.33± 0.57cd 3.66± 0.42cd 3± 0.39de 1.33± 0.11ef 1.16± 0.13ef 4± 0.77cd 4.33± 0.38c 2± 0.32de ph = 8.2 2.33± 0.24de 2.33± 0.48de 2.66± 0.37de 2.33± 0.31de 3± 0.42de 2.83± 0.33de 2.5± 0.27de 2.66± 0.29de sd = standard deviation; la = lipase activity; sf = sunflower; pk = pumpkin; sb = soy bean; pn = peanut; cn = corn; wn = walnut; al = almond; se = sesame; *means with different letters are statistically different (p < 0.05). at 20°c and ph 5.4 sunflower lipase had the highest activity on soybean oil, followed by activity on walnut, corn, peanuts and sunflower oils (with close values). between the activity on soybean oil, on one hand, and on walnut, corn, peanuts and sunflower oils, on the other hand, were significant differences (p < 0.05). also at 20°c, but at ph 7.4, sunflower seeds lipase had the highest activity on walnut, almond and sesame oils (with no significant differences between values), followed by activity on pumpkin and peanut oils (p < 0.05). at the same temperature, but at ph 8.2, the enzyme activity was low on all oil samples the largest one being registered on sunflower oil. at 40°c, and ph 5.4 the highest values of sunflower lipase activity were recorded on peanut, sunflower, soybean, corn and walnut oils (with close values), followed by pumpkin, almond and sesame oils (p < 0.05). at ph 7.4 the lipase had the highest activity on almond oil, followed by walnut oil, while at ph 8.2 the activity of enzyme registered low values with no significant differences between oil samples (p < 0.05). from tab. 1 it can notice that at 20°c and 40°c the lipase derived from sunflower seeds recorded the highest values of its activity at ph 5.4, and the lowest ones at ph 8.2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 24 also, one can see that the largest activity of lipase was registered on soybean oil at 20°c, and ph 5.4. the table 2 reproduces the corn caryopses lipase activity on analyzed substrates (oils). from the tab. 2 it can see that, at 20°c, and ph 5.4, the lipase from corn caryopses had the highest activity on the oils of: walnut, sunflower, soybean, peanut, sesame and on its own substrate (corn oil), where has recorded significantly higher values than on pumpkin and almond oils (p < 0.05). also at 20°c, at ph 7.4 and ph 8.2 the corn lipase has shown significantly lower activities, compared to ph 5.4. at ph 7.4 the highest activity of enzyme was recorded on almond oil, and at ph 8.2 on peanut oil (p < 0.05). table 2 lipase activity mean values (±sd) of corn caryopses on different substrates lipase source corn caryopses refined oils sf pk sb pn cn wn al se la (μmol oleic acid/ g/min. 20°c ph = 5.4 4.73± 0.51c 3.83± 0.37cd* 4.66± 0.42c* 4.56± 0.53c 4.33± 0.28c 5.33± 0.55c 3.9± 0.31cd 4.5± 0.39c ph = 7.4 0.73± 0.66fg 1.16± 0.19ef 1± 0.23ef 1.66± 0.19ef 0.9± 0.08fg 1.33± 0.12ef 3± 0.37de 0.9± 0.08fg ph = 8.2 0.73± 0.06fg 0.83± 0.08fg 1± 0.25ef 3.23± 0.34cd 1± 0.16ef 1.33± 0.2ef 0.56± 0.04fg 1.33± 0.15ef la (μmol oleic acid/ g/min. 40°c ph = 5.4 5± 0.61c 4.66± 0.39c 4± 0.41cd 4.66± 0.55c 5.33± 0.62c 3.66± 0.38cd 3.5± 0.41cd 5± 0.47c ph = 7.4 3± 0.28de 1± 0.15ef 1.66± 0.18ef 2± 0.24de 1.33± 0.17ef 1.33± 0.21ef 4.66± 0.39c 2± 0.18de ph = 8.2 0.5± 0.06fg 0.5± 0.04fg 0.33± 0.04fg 1.66± 0.22ef 0.33± 0.03fg 3.66± 0.41cd 0.83± 0.07fg 0.5± 0.04fg sd = standard deviation; la = lipase activity; sf = sunflower; pk = pumpkin; sb = soy bean; pn = peanut; cn = corn; wn = walnut; al = almond; se = sesame; *means with different letters are statistically different (p < 0.05). at 40°c the corn lipase had the highest activity also at ph 5.4, on its own substrate (corn oil) and on oils of sunflower, pumpkin, peanut and sesame (with close values), followed by oils of soybean, walnut and almond (p < 0.05). at ph 7.4 the corn lipase had the largest activity on almond oil, and at ph 8.2 on walnut oil. according to tab. 2, at 20°c and 40°c the lipase derived from corn caryopses registered the highest activity at ph 5.4, and the lowest one at ph 8.2. in the table 3 is rendered the pumpkin seeds lipase activity on analyzed substrates (oils). from the tab. 3 one can see that at 20°c, and ph 5.4, the lipase from pumpkin seeds had the highest activity on walnut and peanut oils, followed by corn, sunflower, pumpkin (own substrate), soy bean and sesame oils, with no significant differences between samples (p < 0.05). also at 20°c, but at 7.4 and ph 8.2 the lipase from pumpkin seeds has recorded significantly lower activities, compared to ph 5.4. both at ph 7.4, and at ph 8.2 the highest activity of enzyme was recorded on walnut and soybean oils (p < 0.05). at 40°c the pumpkin seeds lipase had the highest activity at ph 5.4, on walnut oil, followed by oils of: soybean, corn, sunflower, almonds and sesame (with significantly lower values, p < 0.05). at ph 7.4 the pumpkin lipase had a greater activity on sunflower and pumpkin oils, and at ph 8.2 on walnut and almond oils (p < 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 25 table 3 lipase activity mean values (±sd) of pumpkin seeds on different substrates lipase source pumpkin seeds refined oils sf pk sb pn cn wn al se la (μmol oleic acid/ g/min. 20°c ph = 5.4 4.4± 0.35c 4.33± 0.47c 4.33± 0.29c 5.23± 0.47c 4.66± 0.42c 5.33± 0.65c* 3.9± 0.41cd* 4.16± 0.49c ph = 7.4 1.73± 0.22ef 1.16± 0.18ef 2± 0.25de 1.16± 0.16ef 1.73± 0.12ef 2± 0.17de 1.66± 0.18ef 1.23± 0.1ef ph = 8.2 0.4± 0.03fg 0.56± 0.39fg 1.66± 0.17ef 0.56± 0.04fg 0.33± 0.02fg 1± 0.08ef 0.56± 0.04fg 0.33± 0.04fg la (μmol oleic acid/ g/min. 40°c ph = 5.4 4.66± 0.37c 3.66± 0.29cd 5± 0.43c 4± 0.51cd 5± 0.42c 6.33± 0.55ab 4.5± 0.37c 4.33± 0.45c ph = 7.4 2.33± 0.16de 2.33± 0.21de 1.33± 0.11ef 1.8± 0.16ef 1.33± 0.12ef 1± 0.11ef 1.33± 0.15ef 1± 0.08ef ph = 8.2 0.33± 0.02fg 0.33± 0.04fg 0.33± 0.02fg 0.66± 0.05fg 0.66± 0.06fg 1.66± 0.12ef 1.5± 0.13ef 0.98± 0.09fg sd = standard deviation; la = lipase activity; sf = sunflower; pk = pumpkin; sb = soy bean; pn = peanut; cn = corn; wn = walnut; al = almond; se = sesame; *means with different letters are statistically different (p < 0.05). analyzing the data of tab. 3, it results that the lipase from pumpkin seeds had the highest activity at ph 5.4, on walnut oil (at 40°c), and the lowest one at ph 8.2 at both temperatures. in the table 4 is reproduced the soy beans lipase activity on analyzed substrates (oils). table 4 lipase activity mean values (±sd) of soy beans on different substrates lipase source soy bean refined oils sf pk sb pn cn wn al se la (μmol oleic acid/ g/min. 20°c ph= 5.4 3.73± 0.31cd* 3.83± 0.27cd 3.33± 0.24cd 4.56± 0.39c 4± 0.28cd 4± 0.41cd 3.5± 0.36cd 3.5± 0.3cd ph= 7.4 2.4± 0.18de* 2.16± 0.22de 2± 0.15de 2± 0.14de 1.73± 0.12ef 2± 0.19de 3.33± 0.36cd 1.23± 0.09ef ph= 8.2 2.06± 0.21de 0.5± 0.04fg 0.66± 0.04fg 1.9± 0.2ef 0.33± 0.29fg 1.66± 0.18ef 0.56± 0.04 fg 0.66± 0.68fg la (μmol oleic acid/ g/min. 40°c ph= 5.4 4± 0.39cd 4.16± 0.35c 3.5± 0.29cd 3.33± 0.36cd 4.66± 0.42c 5± 0.56c 4.16± 0.51c 4± 0.3cd ph= 7.4 2.66± 0.28de 2.33± 0.19de 2.33± 0.25de 1.33± 0.1ef 1.5± 0.12ef 2± 0.22de 2.33± 0.18de 2± 0.23de ph= 8.2 3.16± 0.34cd 0.66± 0.05fg 0.83± 0.09fg 0.66± 0.04fg 0.33± 0.02fg 1± 0.09ef 0.83± 0.07fg 1± 0.12ef sd = standard deviation; la = lipase activity; sf = sunflower; pk = pumpkin; sb = soy bean; pn = peanut; cn = corn; wn = walnut; al = almond; se = sesame; *means with different letters are statistically different (p < 0.05). at 20°c and ph 5.5, the soy bean lipase had the greatest activity on peanut oil, followed by the other seven oil analyzed (including its own substrate), which registered close values, but significantly lower (p < 0.05). compared to ph 5.5, at ph 7.4 and ph 8.2 the lipase from soy bean had activities significantly reduced, with a higher value (at ph 7.4) on almond oil (p < 0.05). at 40°c the soybean lipase had the highest values at ph 5.4, on oils of: walnut, corn, pumpkin and almond, followed by oils of: sunflower, sesame, soybean and peanut, with values significantly lower (p < 0.05). at ph 7.4 the soybean enzyme had a greater activity on oils of: sunflower, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 26 pumpkin, soybean, walnut, almond and sesame, and at ph 8.2 on sunflower oil (p < 0.05). the data of tab. 4 show that the soybean lipase recorded the highest activity at ph 5.4, on peanut oil (at 20°c), on walnut, corn, pumpkin and almond oils (at 40°c), the lowest values being registered at ph 8.2, at the both temperatures. comparing the data from the four tables, one can see that lipases coming from sunflower, corn, soy and pumpkin seeds, have registered, on the eight refined oils, the highest values of activity at ph 5.4 (at 20°c or 40°c). the fig. 1 renders the evolution of lipase activity from sunflower, corn, soy and pumpkin seeds, at ph 5.4, on the eight oils. fig. 1. the evolution of lipase activity values from sunflower, corn, soy and pumpkin seeds, at ph 5.4, on the eight substrates (oils) analyzed sf = sunflower; pk = pumpkin; sb = soy bean; pn = peanut; cn = corn; wn = walnut; al = almond; se = sesame as seen from tables 1-4 and fig. 1, at ph 5.4, compared to the other three sources, the values of sunflower seeds lipase activity were higher (at 20°c or 40°c) on all eight oils analyzed, but significantly higher activities were recorded on the oils of: sunflower, soybean, peanut, corn and walnut – the highest one being on soy bean oil, at 20°c (p < 0.05). at the same ph (5.4), the corn caryopses lipase recorded its highest activity on walnut oil, at 20°c, and on corn oil, at 40°c, the pumpkin seed lipase on walnut oil, at 40°c, and the soy bean lipase on walnut, at 40°c. on their own substrate, the highest activity was registered by sunflower seed lipase, at 40°c, followed by corn caryopses lipase at 40°c, by pumpkin seed lipase at 20°c, and by soy bean lipase at 40°c (fig. 1). with some exceptions, oilseed lipases are generally more active with triacylglycerols containing short chain fatty acids [9, 15, 19]. studying the physical-chemical properties of purified sunflower seed lipase, sagiroglu and arabaci [20] observed that the enzyme showed a preference for triacylglycerols with monounsaturated fatty acids, a high temperature of 50ºc and a high ph value of 7.5. after some data published in scientific papers and some methods and standards for vegetable oils [21, 22, 23, 24, 25, 26, 27], the oleic acid content of various refined oils ranges between 17 and 67 (wt%), with higher values within oils of: peanut, sesame and pumpkin, followed by corn, sunflower, almond, soybean and walnut. according to the same sources, the linoleic acid content varies between 14 and 74 (wt%), with higher values in oils of: 0 1 2 3 4 5 6 7 8 9 10 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c 2 0 °c 4 0 °c sf pk sb pn cn wn al se substrates (oils) la (m ic ro m ol s ol ei c ac id /g /m in .) sf lipase cn lipase pk lipase sb lipase food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 27 sunflower, walnut, soybean, corn and pumpkin seeds, and lower ones in sesame, almond and peanut oils, and the linolenic acid ranges between 0.5 to 14 (wt %), being present in a higher quantity in walnut oil, followed by soybean and sunflower oils. in this paper, higher values of lipase activity in sunflower seeds, as compared to the other three sources, could be correlated with the chemical composition of the analyzed oils. thus, an increased content of linoleic and linolenic acid, but a lower content of oleic acid was the substrate (soybean oil) that sunflower seeds lipase had the highest activity at ph 5.4 and 20°c. a high content of linoleic and linolenic acid, but lower of oleic acid (walnut oil), and a high content of oleic acid but less of linoleic and linolenic acid (peanut oil), made sunflower seeds lipase to have also an increased activity, at ph 5.4 and 20°c (on walnut oil) or 40°c (on peanut oil). according to hilditch and williams [28] ct. by [9], cereal grains contain from 2 to 10% of lipids, depending on the species and variety, and about 80 to 90% of the triacylglycerol fatty acids are oleic and linoleic. corn lipase presented greater activity with the triacylglycerols containing oleic and linolenic acids, which are the main constituents of corn oil [29, 30, 31]. compared with the other oils analyzed, it seems that sunflower seeds oil has a high content of linoleic acid, average of oleic acid, and lower of linolenic acid [21, 22, 23, 24, 25, 26, 27]. 4. conclusions analyzing the activity of lipases coming from four plant sources (sunflower seeds, pumpkin, seeds, corn caryopses and soy bean), on eight refined oils (sunflower, pumpkin, soy bean, peanut, corn, walnut, almond and sesame), the highest activity of those enzymes were at ph 5.4, at 20°c or 40°c. as compared to lipase activity from pumpkin, corn and soy bean, the values of sunflower lipase activity were significantly higher at ph 5.4, on the oils of: sunflower, soybean, peanut, corn and walnut the highest activity being recorded on soy bean oil at 20°c. a high content of oleic acid, but especially of linoleic and linolenic acids within oils used as substrate, caused an increased activity of sunflower seeds lipase. of the four types of lipase analyzed, the highest activity on its own substrate was registered by sunflower seeds lipase, at 40°c. 5. acknowledgments many thanks to suceava genebank and suceava agricultural research and development station for the biological material supplied. 6. references [1] merçon f., erbes v.l., sant`anna jr. g.l., nobrega r., lipase immobilized membrane reactor applied to babassu oil hydrolysis. brazilian journal of chemical engineering, 14: 111, (1997) [2] kamimura e.s., mendieta o., sato h.h., pastore g., maugeri f., production of lipase from geotrichum sp. and adsorption studies on affinity resin, brazilian journal of chemical engineering, 16(2): 1-10, (1999) [3] leal m.c.m., cammarota m.c., freire d.m.g., sant'anna jr. g.l., hydrolytic enzymes as coadjuvants in the anaerobic treatment of dairy wastewaters, brazilian journal of chemical engineering, 19(2): 175-180, (2002) [4] pereira e.p., zanin g.m., castro h.f., immobilization and catalytic properties of lipase on chitosan for hydrolysis and esterefication reactions, brazilian journal of chemical engineering, 20: 343-355 (2003) [5] castro h.f., oliveira p.c., pereira e.b., influence of substrate partition coefficient on the performance of lipase catalyzed synthesis of citronellyl acetate by alcoholysis, brazilian journal of chemical engineering, 17: 1-4, (2000) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xv, issue 1 – 2016 marcel avramiuc, the comparative study of the lipase activity from plant sources, under various conditions of ph, temperature and substrate, food and environment safety, volume xv, issue 1 – 2016, pag. 21 28 28 [6] freire g.d.m., castilho f.l., lipases em biocatálise. in: bon et al. (org). enzimas em biotecnologia: produção, aplicação e mercado, rio de janeiro, interciência, (2008) [7] alonso f.o.m., oliveira e.b.l., dellamora-ortiz g.m., pereirameirelles f.v., improvement of lipase production at different stirring speeds and oxygen levels, brazilian journal of chemical engineering, 22: 9-18, (2005) [8] hasan f., shah a.a., hameed a., industrial applications of microbial lipases, enzyme microbial and technology, 39(2): 235-251, (2006) [9] barros m., fleuri l.f., macedo g.a., seed lipases: sources, applications and properties a review, brazilian journal of chemical engineering, 27: 15-29, (2010) [10] gandhi n.g., application of lipase, journal of the american oil chemists' society, 74(6): 621634, (1997) [11] sharma r., chisti y., banerjee u.c., production, purification, characterization, and applications of lipases, biotechnology advances, 19(8): 627-662, (2001) [12] paques f.w., macedo g.a., lipases de látex vegetais: propriedades e aplicações industriais: a review, química nova, 29(1): 93-99, (2006) [13] hellyer s.a., chandler i.c., bosley j.a., can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride?, biochemica et biophysica acta, 1440: 215-224, (1999) [14] villeneuve p., plant lipases and their applications in oils and fats modification, european journal of lipid science and technology, 105(6): 308-317 (2003) [15] enujiugha v.n., thani f.a., sanni t.m., abigor r.d., lipase activity in dormant seeds of the african oil bean (pentaclethra macrophylla benth), food chemistry, 88(3): 405410, (2004) [16] polizelli p.p., tiera m.j., bonillarodriguez g.o., effect of surfactants and polyethylene glycol on the activity and stability of a lipase from oilseeds of pachira aquatica, journal of the american oil chemists' society, 85(8): 749-753, (2008) [17] bordei d. (coord.), bahrim g., pâslaru v., gasparotti c., elisei a., banu i., ionescu l., codină g.g., controlul calitaţii în industria panificaţiei. metode de analiză, ed. academica, galaţi, pp. 280-281, (2007) [18] sas institute, sas user’s guide. statistical analysis system institute, cary, nc, (2005) [19] borgston b., brockman h.l., lipases. amsterdam, elsevier, (1984) [20] sagiroglu a., arabaci n., purification and characterization of lipase from sunflower seed, preparative biochemistry and biotechnology, 35(1): 37-51, (2005) [21] orthoefer f.t., vegetable oils. in bailey’s industrial oil and fat products, vol 1: edible oil and fat products: general applications (ed. y.h. hui), 5th edn, john wiley & sons, inc., new york, pp. 19–44, (1996) [22] aocs, physical and chemical characteristics of oils, fats and waxes, in official methods and recommended practices of the american oil chemists’ society, aocs press, champaign, il, (1997) [23] firestone d. (ed.), physical and chemical characteristics of oils, fats and waxes, aocs press, champaign, il, (1999) [24] codex alimentarius (1999) (fao/who) codex standard for named vegetable oils. codex stan 210 1999 (revision and amendments: 2003, 2005) [25] grompone a. m., sunflower oil. in vegetable oils in food technology, composition, properties and uses, (ed. gunstone f.d.), 2nd edn., john wiley & sons, ltd., publication, the atrium, southern gate, chichester, west sussex, po19 8sq, uk, p. 140, (2011) [26] kochlar prakash s., minor and speciality oils. in vegetable oils in food technology, composition, properties and uses, (ed. gunstone f.d.), 2nd edn., john wiley & sons, ltd., publication, the atrium, southern gate, chichester, west sussex, po19 8sq, uk, pp. 293294; 320-321; 329-330, (2011) [27] tong wang, soybean oil. in vegetable oils in food technology, composition, properties and uses, (ed. gunstone f.d.), 2nd edn., john wiley & sons, ltd., publication, the atrium, southern gate, chichester, west sussex, po19 8sq, uk, p. 60, (2011) [28] hilditch t.p., willians p.n., the chemical constitution of natural fats, chapman and hall, london, (1964) [29] hammer m., murphy j.b., lipase activity and in vivo triacylglycerol utilization, annual review of ecology and systematics, 24(3): 69-74, (1993) [30] huang a.h.c., lin y.h., wang s.m., characteristics and biosynthesis of seed lipases in maize and other plant species, journal of the american oil chemists' society, 65(6): 897-899, (1988) [31] lin y.h., yu c., huang a.h., substrate specificities of lipase from corn and other seeds, archives of biochemistry and biophysics, 244(1): 346-356 (1986) lipases act at the organic-aqueous interface, catalyzing the hydrolysis of ester-carboxylate bonds and releasing fatty acids and organic alcohols [1, 2, 3, 4]. применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 3 2011 contents: 1. torosyan greetings 7 2. treatment of organic pollutants on zeolites in motor transportation & auto repair enterprises n.s.torosyan, a.g.kudryavtsev, g.h.torosyan. 9 3. current vision of the nucleation rate description problem michael anisimov 16 4. geophysical ev aluatio n o f the s tability of the lands lide slo pe rolan gasparyan 25 5. modern approach to management of combined sewer owerflows p. hlavinek 30 6. in situ river water treatment with domestic materials using as sorbents g.h. torosyan, n. gh. poghosyan, a. k. gukasyan, v.a. davtyan, n.s. torosyan 40 7. flow diffusion chamber is a new tool for vapor nucleation rate measurements michael anisimov, elena fominykh 46 8. ecological aspects of sludge disposal in slovakia ivana mahríková 53 9. use of advanced oxidation processes for textile wastewater treatment – a review radka pešoutová, petr hlavínek, jana matysíková 59 10. waste disposal impacts on environment ivona škultétyová 66 11. influence of flat area type on sewage design conditions štefan stanko 72 12. the role of operating risks in interrelation with the education for sustainable development yeva torosyan 77 13. the pec as an easy-to-prepare and cheaper alternative power source g.sh. hovsepyan, a.n.kocharyan, s.h.martirosyan, g.o.torosyan 84 14. universal fluorometer based on fluorescence quenching georg gutt, sonia gutt, cristina hretcanu, ana leahu 87 15. author instructions 93 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 48 mathematical model for optimization of zinc-nickel alloy codeposition process *violeta vasilache1 1universitatea ştefan cel mare, suceava, str. universităţii, nr.13, 720229, suceava, e-mail violetav@fia.usv.ro *corresponding author received 2 april 2011, accepted 16 may 2011 abstract: in protection against corrosion domain nickel remains the principal metal for coverings, fact which is proved by the dynamics of annual global production. nickel has an important role in automotive industry and it is used together with zinc. at present the change of electroplating processes with purpose to use with maximum efficiency resources of energy and materials is being focused on. also for environment protection reasons, it is aknowledged that electroplating processes generate as few polluting substances as possible. in consequence, the processes using heavy metals (as cadmium, intensively used until now) are going to be replaced by processes using alloys. alloys with superior properties, which are cheaper and have a lower environmental impact have been promoted. the electronic industry is another domain which stimulates development of alloy electroplating processes, because many components are produced through electrochemical procedures. thorough control of processes is very important in electronic micro-components manufacturing and miniaturization. in this paper a mathematical model describing the nickel and zinc-nickel alloys electrodeposition phenomena is made. it is based on the influence of substratum through deposition and aims to calculate the partial current densities for every species, to predict composition of alloy, if the electrodeposition conditions are known. corresponding to this mathematical model a software was made to calculate and establish the composition of zinc-nickel alloy. © 2011 university publishing house of suceava. all rights reserved keywords: alloys electrodeposition, anomalous co-deposition, zinc-nickel alloys, mass transfer, electrochemical kinetic 1. introduction any optimization method implies a mathematical model which should meet the quantitative requirements of the problems. this model is based on the substratum effect and aims to calculate the partial current densities and so to give prediction regarding the quantities of metal electrodeposited and the energy involved. electrodeposition of a simple metal has also a simpler mathematical model, but the situation is different for alloys deposition. in our model the kinetic parameters are analyzed and we made the supposition that current distribution and mass transport were homogeneous on the working electrode. we also tried to determine the reaction mechanism [1], [2], [3]. figure 1. the scheme which shows partial current densities for a and b components. (a) both components under activation kinetic control, present identic tafel slopes. (b) both components present limitation of the current. (c) both components under activation control, but with different tafel slopes. (d) component a presents current limitation, component b under activation control. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 49 2. theoretical and experimental considerations 2.1. electrochemical process at cathode in our experiments a zinc-nickel alloy was deposited on gold substratum, previously deposited by sputtering on glass plates. this methode was chosen because it permits to analyze the layers with xrd and sem-edx techniques. the mechanism of electrochemical reactions which occur on the cathode surface has two steps, as matlosz described [4], [5]. zinc ions are deposited on their own substratum, on gold substratum and on nickel substratum. nickel ions are also deposited on their own substratum, on gold and zinc one. more, there are secondary reactions, zn2+ ions are combining with hydrogen to form znh+, and similar ni2+ ions are combining to hydrogen to form nih+. these intermediate species, formed in adsorption process, finally will be decomposed to metallic zinc and nickel respectively. the mechanism of electrochemical reactions could be written as follow: ni2+ + e→ ni+ads (1) ni+ads + e→ ni (2) ni + h+ + → nih+ads (3) nih+ads + h+ + 2e→ ni + h2 (4) zn2+ + e→ zn+ads (5) zn+ + e→ zn (6) zn + h+ → znh+ads (7) znh+ads + h+ + 2e→ zn + h2 (8) ni2+ and zn2+ are dissolved as metallic ions, hydrolized or not. ni+ads and zn+ads which could contain or not the group hydroxyl are adsorbed in intermediate reactions. ni and zn are metallic deposits of nickel and zinc respectively [6,7]. the kinetic of mass transfer is supposed to respect butler-volmer equation. so far the equilibrium state anodic reactions could be neglected. 2.2. determination of partial current densities for a binary alloy ab and a thicknes of deposit d, partial current density of b element is, b b b b mtm fn i   (9) here mb is mass of element b deposited in alloy, mb is atomic mass of element b, t is deposition time and nb is number of electrons implied in reaction of element b. 2.3. normal and anomalous codeposition of zinc-nickel alloys electrodeposition of zinc-nickel alloys is generally an anomalous co-deposition, after brenner’s definition, because the metal less noble, zinc is deposited preferentialy and its percent in deposit is higher than in electrolyte. anyway, normal co-deposition of zinc-nickel alloys is possible only under particular experimental conditions. the co-deposition of zincnickel alloys from different electrolytic baths was studied potentiostatically and galvanostatically, depending on different variable parameters during electrodeposition [1,2]. 2.4. mathematical modelling of zincnickel alloy co-deposition. the model of substratum effect the initial nucleation of adsorbed nickel on the electrode surface acts as a catalyser for zinc deposition, laeding to an inhibition of nickel deposition. it was also shown that pure zinc cannot be deposited from aqueous electrolyte solutions at upd (underpotential deposition), but it could be co-deposited with nickel. these phenomena can be explaine by the fact that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 50 nickel nucleation catalizes zinc deposition. at potential more negative than zinc equilibrium potential, zinc deposition rate is enough higher and inhibits nickel deposition leading to an anomalous codeposition. the alloy deposition performs a substratum effect. not only nickel affects zinc deposition, but zinc too affects nickel deposition. figure 2. the diagram of zinc-nickel alloy co-deposition figure 2 shows the diagram of the effects of different substrata during electroplating with zinc-nickel alloys. the initial electrode surface is divided into two parts. the first is corresponding to ni which is the surface covered by nickel and the second is the surface covered by zinc, zn. every surface is then divided into four parts. so, for nickel deposition, ni1 corresponds to the area of ni substratum surface covered with ni(i)ads. ni2 corresponds to the area of ni substratum surface covered with nih+ads, ni3 corresponds to the area of ni substratum surface covered with zn(i)ads. free surface ni(1-1-2-3) corresponds to the area of ni substratum surface non-covered. for zinc deposition, zn6 corresponds to the area of zn substratum surface covered with zn(i)ads. zn5 corresponds to the area of zn substratum surface covered with znh+ads. zn4 corresponds to the area of zn substratum surface covered with ni(i)ads. free surface zn(1-4-5-6) corresponds to the area of zn substratum surface non-covered. 2.5. theoretical model. general mechanism of electrode reactions a mechanism of reactions was developed as effect of substratum. this model is based on the supposition that every individual component is deposited after a two step-reaction, as matlosz [4] described. the nickel ions are deposited on their own substratum and on zinc one. zinc ions are also deposited on their own substratum and on nickel one. moreover, hydrogenated species znh+ and nih+ are strongly bonded on the electrode surface. ni(ii) will react giving nih+ads and these adsorbed species will react afterwards with the nickel deposited. zn(ii) will also react giving znh+ads and these adsorbed species will react afterwards with deposited zinc. [8] 2.6. the mass transfer effect the material balance in equilibrium state through diffusion layer for species ni(ii), zn(ii) and h+, 0<x<d, can be written 0)(  iinin (10) 0)(  iiznn (11) 0 hn (12)   hohw cck (13) supposing a constant diffusion coefficient, d, the flow of every species i, into diffusion layer is dxddcn ii / . the values accepted for diffusion coefficients are 410-10 m2s-1 for ni(ii), 5,0910-10 m2s-1 for zn(ii), and 9,310-9 m2s-1 for solvated protons, and 5,510-9 m2s-1 for hydroxid ions [3]. intermediate species, ni(i)ads, nih+ads, zn(i)ads and znh+ads exist only on the electrode surface so their concentration is equal to zero in the solution [9]. 2.7. electrochemical kinetic the charge transfer kinetic is supposed to respect butler volmer equation. far from equilibrium, the anodic reactions could be food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 51 neglected. a tafel modified expression describes the electrochemical reactions’ rate on the surface and is adapted to calculate the partial current. for example, on the first step of deposition reaction on the nickel substratum, partial current density, i11, could be written as: i11=-fk011cni2+ni(1-1-2-3)exp(-b1111) 2.8. software for predictive calculus of zinc-nickel alloy composition to simulate zinc-nickel alloy deposition, a soft-ware, which uses the calculus relationship from described mathematic model, was elaborated. the calculus stops when the difference between a calculated value and a previous one is smaller than 10-5 [9], [10], [11]. the program lines are written as follows: using system; using system.collections.generic; using system.componentmodel; using system.data; using system.drawing; using system.text; using system.windows.forms; namespace calcultetai { public partial class frmmain : form { public frmmain() { initializecomponent(); } double f, ki0, bi, etai, c0, itotal, ai, t, tetas, ch, cni, czn, tetaiinitial, tetazn, tetani; double tetaicalculat; double ci, ii; private void m_btncalculeaza_click( object sender, eventargs e ) { m_tbtetaicalculat.text = ""; try { f = double.parse( m_tbf.text ); ki0 = double.parse( m_tbki0.text ); bi = double.parse( m_tbbi.text ); etai = double.parse( m_tbetai.text ); c0 = double.parse( m_tbc0.text ); itotal = double.parse( m_tbitotal.text ); ai = double.parse( m_tbai.text ); t = double.parse( m_tbt.text ); tetas = double.parse( m_tbtetas.text ); ch = double.parse( m_tbch.text ); cni = double.parse( m_tbcni.text ); czn = double.parse( m_tbczn.text ); tetaiinitial = double.parse( m_tbtetaiinitial.text ); tetazn = double.parse( m_tbtetazn.text ); tetani = double.parse( m_tbtetani.text ); } catch { messagebox.show( "introduceti valori corecte", "atentie", messageboxbuttons.ok, messageboxicon.warning ); return; } int contor = 0; int numarbucle = 1000; tetaicalculat = tetaiinitial; do { tetaiinitial = tetaicalculat; ci = c0 * math.exp( -ki0 * ai * t ); ii = -f * ki0 * cni * czn * ch * tetazn * tetani * ( 1 tetaiinitial tetas ) * math.exp( bi * etai ); tetaicalculat = ii / itotal; contor++; if( contor > numarbucle ) break; } while( math.abs( tetaiinitial tetaicalculat ) > 1 * math.pow( 10, -5 ) ); if( contor > numarbucle ) messagebox.show( "s-a depasit numarul de bucle!", "atentie", messageboxbuttons.ok, messageboxicon.warning ); else { m_tbtetaicalculat.text = tetaicalculat.tostring(); } } private void m_btniesire_click( object sender, eventargs e ) { this.close(); food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 52 figure 3. user interface with the software for calculus of alloy composition 2.8. optimization of zinc-nickel alloy codeposition process to optimize zinc-nickel alloy electrodeposition process, the taguchitype method was used. the controlled factors will be taken as follows: solution type: (1) noted as the solution i (zinc chloride 130g/l, nickel chloride 130g/l, potassium chloride 230g/l, ph 5-6, t(°c) 24-30°c) and (2) the second solution ii respectively (zinc chloride 130g/l, nickel chloride 65g/l, potassium chloride 230g/l, ph 5-6, t(°c) 24-30°c); discharge potential: (1) -850 mv, (2) -900 mv, (3) -1000 mv, (4) -1100 mv. the current density was measured and the current efficiency was calculated. a matrix of experiment was written as follows: table 1. matrix of zinc-nickel alloy co-deposition experiment t es t n o. controlled factors measured values potenti al solu tion current (adm-2) efficienc y (%) 1 1 1 7 53 2 2 1 14 37.6 3 3 1 21 35.55 4 4 1 35 35.55 (**) 5 1 2 3 38.5 6 2 2 6 22.7 7 3 2 15 22.8 8 4 2 13 (**) measured average values 35.025 soluţia 2soluţia 1 55 50 45 40 35 30 25 20 soluţia v a lo a re a m e d ie -1100 -1000 -900 -850 potenţial interacţiunile factorilor figure 4. interaction of the factors, in this case of discharge potentials for considered experimental conditions (factorial design). as figure 4 shows (following table 1) for the discharge potential of -900 mv the influence of solution type is the highest. for -850 mv we obtained the 53% energetic efficiency for the first type solution and 38.5% for the second one, which are the highest values. the quality of electrodeposited layers was established by different techniques (optic microscope, sem-edx, xrd) [8-11] and was better also for -850 mv discharge potential. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 53 3. conclusions this model establishes a mathematical apparatus to describe zinc-nickel alloy codeposition processes, using the substratum effect model for different concentrations of electrolyte and for different applied potentials. there is a good correlation between experimental data and the prediction of this model. from analyzing the diagrams the following conclusion can be drawn: the discharge potential of -850 mv permits to obtain the best energy efficiency; at this value, the best quality was obtained, too. as regards solutions, superior results are obtained using the solution i. 4. acknowledgments this paper was supported by the project "progress and development through postdoctoral research and innovation in engineering and applied sciences– pride contract no. posdru/89/1.5/s/57083", project co-funded from european social fund through sectorial operational program human resources 2007-2013. 5. references 1. bard a.j., electrochemical methods. fundamentals and applications, john wiley and sons, new-york, 2001 2. di bari g.a., modern electroplating, fourth edition, edited by mordechay schlesinger and milan paunovic, john wiley & song, inc., 2000 3. teeratananon m., saidi k., fenouillet b., vergnes h., journnes d’electrochimic, poiters, france, june 2008 4. matlosz, m., journal electrochemistry soc., 140(1993)2272 5. soares m.e., souza c.a.c., kuri s.e., corrosion resitence of zn-ni electrodeposited alloy obtained with a controlled electrolyte flow and gelatin additive, science direct, vol.201, issue 6, dec.2006, p.2953-2959 6. schlesinger m., electrodeposition of alloys, modern electroplating, fourth edition, john wiley and sons, inc. new-york, 2000 7. brenner a, electrodeposition of alloys, vol.i, academic press, new york, 1963 8. vasilache v., gutt gh., vasilache t., rev. chim. (bucuresti), 59, nr.8, 2008, p. 915 9. vasilache v., ph. d. thesis, university stefan cel mare of suceava , 2008 10. vasilache v., gutt gh. vasilache t., studies about electrochemical plating with zincnickel alloys. the influence of potential through stoichiometric composition, revista de chimie, bucureşti, vol.59,nr.9 (2008) 11. vasilache v, gutt s, gutt gh, vasilache t, sandu i, sandu i.g. determination of the dimension of crystalline grains of thin layers of zinc-nickel alloys electrochemically deposited. metalurgia international, 2009, 14: 49-53; food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 27 study of a new coordination compounds based on cr(iii) and succinimide in aqueous solution mihaela dana tutulea (anastasiu)1, igor cretescu2, doina sibiescu2, ioan rosca2 1 faculty of food engineering, stefan cel mare university of suceava, romania, anastasiu.dana@gmail.com 2 faculty of chemical engineering and environmental protection, gheorghe asachi technical university of ia i, romania *corresponding author received 2 july 2012, accepted 3 september 2012 abstract: in this paper, the study of obtaining new coordination compounds of cr (iii) using as ligand, succinimide and the stability constants, were presented. conductometric, ph-metric and spectrophotometric studies of cr(iii)-succinimide system were carried out in aqueous medium. the “molar ratio” method was used to study the complex formation in solution and for determine the stability constants was used harvey-manning method.from experimental data resulted that the combination ratio of the ligand succinimide with central metallic atoms was: 1:1, 2:1 and respectively 3:1. keywords: ph-metry, stability constants, combination ratio 1. introduction coordination compounds play an important role in the chemical industry and in life itself; find application in qualitative as well as quantitative estimation of metal ions [1-5]. the complex ions and coordination compounds formation is influenced by many physical and chemical factors. these include environmental factors such as temperature and pressure and more important factors like the nature of the metal ion and the nature of the ligand [6]. succinimides represents an important class of organic compounds due their different applications in numerous fields, as: medicine – are used in the treatment of schizophrenia, epilepsy and depression, chemistry they can be used as inhibitors or as intermediates and reagents for the synthesis of natural and unnatural compounds [7, 8]. the transition metal ion presented in this paper, cr (iii), have the potential to form different compounds, exhibiting a wide range of colors, structures, and chemical properties. a wide range of chromim compounds based on the cr(iii) ion and different ligands such as peptides, urea, sulfates, proteins, ammonia, and organic acids, anions, acids, nucleic acids, and other macromolecules are described in the literature [9]. in aqueous medium, most chromium (iii) compounds are found as coordination complexes. the main aim of this study is to point out the formation of new compounds by the interaction of succinimide ligand with chromium (iii) ion in aqueous solution. 2. experimental all chemicals were obtained from commercial supplier and used without further purification. succinimide (99%) mailto:anastasiu.dana:@gmail.com food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 28 and crcl3·6h2o (99%) were purchased from sigma aldrich in high purity. a 10-2 m solution of succinimide (noted l) and respectively of chromium chloride was prepared by dissolving the accurate weight in bidistilated water. the coordination compounds composition was determined following the harvey— manning and respectively “molar ratio” methods. in order to determine the combination ratio of cr(iii)-succinimide it was prepared a set of 14 samples of the initial solutions, with the same concentration (10-2 m), of crcl3 and respectively of ligand (l) by mixing according to “molar ratio” method [10]. for each sample were measured the values of conductivity, ph and absorbance, respectively. a radelkis –budapest ok 109 conductometer was used for the solutions specific electric conductibility determination. the ph measurements were carried out using a ph-meter type hach one. perkin-elmer spectrum 100 spectrophotometer was used to record the spectra of chromium (iii) solution, ligand solution and respectively of chromium (iii)-succinimide complex. chromium (iii) coordination compounds were obtained according to the following equations: c4h5no2 + crcl3 + 4h2o [ cr(c4h4no2)(h2o)4]2+ + 2cl+ hcl 2c4h5no2 + crcl3 + 2h2o [ cr(c4h4no2)2(h2o)2]+ + cl+ 2hcl 3c4h5no2 + crcl3 [ cr(c4h4no2)3] + 3hcl all the experiments were performed at room temperature. 3. results and discussions the absorption spectrum of chromium (iii)-succinimide shows the maximum absorbance at 570 nm. in all instances, measurements were made at 570 nm against reagent blank. in figure 1 the absorbance values as a function the molar ratio l/cr3+ was presented. figure 1. the curve representing absorbance values of the solutions l– cr 3+, versus the molar ratio l-/cr3+ it can be observed that the complexes formation occur in three stages demonstrated by the presence of three nodal points. figure 2. the ph dependence on the molar ratio l-/cr3+ for the solution of obtained complexes from the “molar ratio” method experimental data in ph metric variant it can be pointed out that are obtained curves with change of slope at the combination ratio (ligand: central atom) 1:1, 2:1 and 3:1 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 29 respectively. these facts demonstrate that the metal ion adds to one, two or three ions from the ligand, obtaining the corresponding complexes. it was pointed out that the phs range of 3.5 4.1 corresponds to the stability of all obtained complexes. in figure 3, it can be observed that the conductivity dependence on the molar ratio l-/cr3+ presents three straight line segments with three different slopes which corresponds to the combination ratio 1:1, 2:1 and 3:1 l-/cr3+ respectively. figure 3. the conductivity variation as function of the molar ratio l-/cr3+ for the solution of obtained complexes conductivity-decrease or increase in the electrical conductance of the solution in which complex formation occurs may result due to disappearance or production of ions of comparatively higher mobility [11]. the decrease of solution conductivity values with l-/cr3+ molar ratio can be attributed to the decrease of charge of ionic species present in the solution. the formation of the three complexes in aqueous medium was demonstrated by the three presented methods. the effectiveness with which a metal ion is coordinated by a ligand, l, in aqueous solution may be assessed by determining the equilibrium constant described by, for example, an overall stability constant. in order to determine the coordination compounds stability constants, in solution, it is necessary to know the reactants concentration at the equilibrium. the stability of cr(iii) coordination compounds depends on the radius and charge of the cr3+ ion and the stabilization due to the splitting of the d orbitals [12]. based on the harvey-manning method [13] and taking in account the obtained data from molar ratio method, the stability constants were calculated. calculations were made with the use of the following equations: (1) (2) (3) where: kd = instability constant (dissociation), c = complex ion maximum concentration, at the combination ration l/m, = complex dissociation degree, n = coordination number, am = maximum absorbance at the ligand excess, from the graph constant portion which reproduce a = f (l/m), ar = stoichiometric ratio absorbance l/m, = stability constant. table 1. stability constants of the studied coordination compounds coordination compound/molar ratio stability constant (l/mol) succinimide cr3+ 1:1 0.64·105 succinimide cr3+ 2:1 0.45·107 succinimide cr3+ 3:1 4.29·1010 1 1 n n n d c n k m r m a a a 1 dk food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 30 it can be observed that the higher the value of the stability constant, the more stable the coordination compound is. 4. conclusions in the present work, three new coordination compounds with succinimide ligand were obtained in aqueous solution. the ph-metry, conductivity and uv-vis spectrometry methods relieved the formation of complexes in 1:1, 2:1 and respectively 3:1 molar ratio l/m. the stability constants pointed out that the coordination compound in the molar ratio 3:1 l/m was the most stable. 5. references [1]. ejaz, alina murtaza a., ullah khan i., synthesis and structural studies of metal complexes of alkyl and hydroxyl derivatives of pyridine, ravian forensic newsletter , p.62, (2012). [2]. satake m., mido y., satake m., chemistry of transition elements, discovery publishing house, (2010). [3]. jones c.j., thornback j., medicinal applications of coordination chemistry, royal society of chemistry, (2007). [4]. bertin i., inorganic and bio-inorganic chemistry, vol. i, encyclopedia of life support systems, p.440, (2009). [5]. hamerton i., howlin b.j., jepson p., metals and coordination compounds as modifiers for epoxy resins, coordination chemistry reviews, 224, 67–85, (2002). [6]. may e., jones m., conservation science: heritage materials, the royal society of chemistry, (2006). [7]. alvarez-gutierrez j.m., nefzi a., houghten r.a., solid phase synthesis of 1,3-disubstituted succinimides, tetrahedron letters, 41, 609–612, (2000). [8]. sharma s., kumar jain a.j., aggarwal a., singh gill n., a review: synthesis and biological activity of imides, the global journal of pharmaceutical research, 1(3), 411-421, (2012). [9]. towill l.e., shriner c.r., drury j.s., hammons a.s., james w. holleman j.w., united states enviromental protection agency, reviews of the environmental effects of pollutants: chromium, (1978). [10]. sibiescu d., the chemistry of coordinate compounds (in romanian), ed. pim, ia i, (2005). [11]. sharma r.k., coordination chemistry, discovery publishing house, (2007). [12]. kornev v. i., mikryukova g. a., coordination compounds of chromium(iii) with different complexones and citric acid in aqueous solutions, russian journal of coordination chemistry, 30 (12), 895-899, (2004). [13]. sibiescu d., synthesis and characterization of coordinate compounds (in romanian), ed. pim, ia i, (2008). microsoft word 16 bandraur et al._fia suceava_en.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 97 ca tion ic ex cha ng e ca pac ity o f pure p c2 00 fd r es in in foo d in dus tr y wa ter so ften ing proc ess bogdan bandrabur1, *liliana lazăr2, ramona-elena tataru-fărmuş2, gheorghe gutt1 1 ”stefan cel mare” university of suceava, faculty of food engineering, 13 university street, 720229 suceava, romania, bbandra@gail.com, ggut@usv.ro 2”gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of chemical engineering, 73 prof.dr.doc. d. mangeron street, 70005 iasi, romania, lillazar@ch.tuiasi.ro *corresponding author received 24 may 2012, accepted 10 june 2012 abstract: the present paper represents a preliminary study regarding the ion exchange capacity of the pure pc200fd weak acid macroporous cationic food grade resin in the water softening processes in the food industry. the ion exchange capacity was experimentally investigated in batch mode as a function of the resin dose, initial solution concentration, contact time, temperature and competitor anions corresponding to the dissolved salts that induce water hardness. more than 50 % efficiency of the cationic exchange in the presence of pure pc200fd resin was achieved in the following conditions: resin dose equal to 8 g dry resin/l, temperature of 298 k, over 250 minutes contact time. considering the ion exchange capacity of the strong acid food grade purolite c100e resin as a comparison benchmark it was concluded after the experiments that weak acid pure pc200fd resin represents a reliable alternative for water softening. keywords: cationic exchanger, food water softening, sorption, strong acid resin, weak acid resin 1. introduction water used as raw material or heat transfer agent in the technological processes specific to the food industry requires certain quality conditions to be fulfilled. the permanent water hardness induced by the calcium and magnesium dissolved salts (chlorides, sulphates, nitrates, phosphate) [1, 2] presents a special importance. the reduction of ca2+ and mg2+ ions concentrations from the water sources (softening) can be achieved using ion exchanger materials such as synthetic polymeric cationic resins in r–na or r–h form [3-7]. the ion exchange capacity of the polymeric resin can be influenced by the type of the functional group contained, that can be sulphonic groups (–so3h) strong acid cation (sac) or carboxylic groups (–cooh) weak acid cation (wac) [3-8]. the conventional water softening process consists of the exchange reaction between na+ or h+ ions and ca2+ or mg2+ cations [3, 5]. exhausted resins regeneration can be realised using sodium chloride solutions (for the na+ form) or hydrochloric acid (for the h+ form) respectively [6, 7]. in the food industrial and domestic applications the resins that do not imply acid solutions regeneration are preferred [6, 8]. nowadays various polymeric resins for water softening in the food industry are commercially available [9-12]. even so, the last year’s specialty literature lacks food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 98 scientific research referring to both the behaviour and the performance of commercial ion exchange resins in the softening process [13-18]. in this paper a preliminary study is performed on the cationic exchange capacity of the polymeric resin with weak acid functional groups sold under the brand name pure pc200fd [12]. the amounts of the ca2+ ion removal were investigated in batch stirred mode as an influence of sorption process specific parameters: resin to solution ratio (dose), initial ca2+ ions concentration, solid – liquid phase contact time, temperature, and aqueous solution nature. the cationic exchange capacity pure pc200fd resin is regarded compared to that of the strong acid cationic exchanger purolite c100e [9]. 2. experimental 2.1. material and methods weak acid cation exchanger pure pc200fd from pure co. ltd. [12] is a delivered in form of macroporous spherical gel resin beads with acrylic crosslinked with divenylbenzene matrix containing carboxyl type functional groups, in h+ form and is not regenerable. this material is similar to purolite c107e resin which can be regenerated [9, 12]. strong acid cationic exchanger purolite c100e from purolite co. [9] is a conventional gel polystyrene sulphonate ion exchanger delivered in na+ form and is regenerable. laboratory research was conducted employing the cationic exchanger resins in the delivery form, previously swollen in ultrapure water for 24 hours. experimental data interpreting and presentation was reported to the dry mass of the cationic resin. ion exchange capacity of both resins were investigated using surface water samples with total hardness of 24.5 odh (german degrees [2]) and binary solutions containing ca2+ ions. the laboratory research was performed in batch stirred mode [5, 17] at constant temperature using erlenmeyer flasks containing 50 ml solution in which the wet resin dose was introduced according to the experimental requirements. all the flasks were kept under constant agitation at 50 rpm on an orbital shacker. the all experiments were conducted at a ph 7.1, value corresponding to common natural water samples. the synthetic solutions ph value correction was performed with a sodium acetate buffer solution 0.1 m. all the experimental solutions were prepared by dissolving in ultrapure water the p.a. grade merck or sigma aldrich chemicals. 2.2. analytical methods the ca2+ and mg2+ ions concentrations respectively quantitative analysis were performed by the aid of edta volumetric methods (sr iso 6058:2008 [19], and sr iso 6059:2008 respectively [20]). an automatic titrator and specific titration indicators for water hardness analysis from a german supplier (calver® and manver® respectively) were used. the cationic exchange capacity was evaluated based on the amount of cations retained by the resin, q (expressed in mg/g dry resin) – retention capacity, calculated according to the eq. (1) [7, 17]:  0 vc c m q   (1) where: c0 is the cation concentration in the initial solution, in mg/l; c – the cation concentration in the residual solution, in mg/l; m – the amount of cationic resin on the experiment, in g dry resin; v – volume of the solution in the experiment, in l. in order to quantitatively express the calcium removal yield was calculated the sorption effectiveness percent, r (%) – removal efficiency, according to the eq. (2) [7, 17]: 0 0 c c r 100 c    (2) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 99 water hardness for the natural water and the synthetic solutions was expressed based on the ca2+ ions concentration according to the equivalence rel. (3) [2]: 1 odh = 10 mg cao/l = = 7.14 mg ca2+/l (1 odca) (3) 3. results and discussion 3.1. resin dose influence figures 1 and 2 present the cationic exchange capacity of pc200fd resin as function of the resin amount that comes in contact with a cacl2 solution and surface water respectively, for a 24 hours time. 0 10 20 30 40 50 60 0 3 6 9 12 15 18 resin dose, g dry resin/l q, m g c a2 + /g d ry r es in 0 20 40 60 80 100 r , %q r figure 1. influence of resin dose on the cationic exchange capacity of pure pc200fd resin (114 mg ca2+/l cacl2; ph = 7.1; 298 k; 24 hours). 0 10 20 30 40 50 60 0 3 6 9 12 15 18 resin dose, g dry resin/l q, m g c a2 + /g d ry re si n cacl2 natural water figure 2. comparative effect of resin dose on the cationic exchange capacity of pure pc200fd resin (114 mg ca2+/lcacl2; 151 mg ca 2+/lwater; ph = 7.1; 298 k; 24 hours). the softening process efficiency (r) in the binary system [cacl2 – h2o]aq, increases with the increase of resin dose. the amount of ca2+ retained on each mass unit of ion exchanger resin (q) decreases with the increase of the dose, indicating a decrease of the resin efficiency (figure 1). this trend was expected due to saturation level attained during a sorption proces. the same influence is valid for surface water as shown in figure 2; the cationic exchange capacity variation for the same resin dose can be explained by the different concentration of ca2+ cations in the initial solutions. following this study a dry resin dose of 8 g for 1 l of solution was chosen for further research, so that the softening process efficiency will be higher than 85 %. 3.2. initial concentration influence the cationic exchange capacity of pure pc200fd resin is influenced by the concentration of ca2+ cations in the initial solutions (figure 3). regardless of the contact time between the two phases a slight diminishing of the softening process efficiency is revealed in figure 4 for concentrations higher than 150 mg ca2+/l, corresponding to water hardness values higher than 21 odca (hard waters [2]). 0 5 10 15 20 25 30 50 100 150 200 250 initial concentration, mg ca2+/l q , m g c a2 + /g d ry r es in 24 hours 5 hours figure 3. influence of initial ca2+ concentration on the cationic exchange capacity of pure pc200fd resin (ph = 7.1; 8 g dry resin/l; 298 k). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 100 0 25 50 75 100 50 100 150 200 250 initial concentration, mg ca2+/l r , % 24 hours 5 hours figure 4. influence of initial concentration of solution on the hardness removal efficiency onto pure pc200fd resin (ph = 7.1; 8 g dry resin/l; 298 k). at higer calcium concentration, the ratio of number of moles of ca2+ in solution to the available surface area is higer and the available sites for binding is less and hence metal removal is relatively independent on the initial concentration. 3.3. contact time influence the cationic exchange capacity of pure pc200fd is positively influenced by the increase of contact time between the two phases (figure 5), until the resin saturation degree for the exchange ion is reached. the softening process efficiency is approx. 40 % after the first 180 min and more than 70 % after 15 hours. in order to achieve process efficiency higher than 50 % more than 5 h contact time is required. 3.4. temperature influence the cationic exchange capacity of pure pc200fd resin increases with temperature (figure 6) because the random motion of ca2+ was accelerated which meanwhile promoted the sorption process. considering that the temperature increase over the ambient temperature values has just slight influence on the cationic exchange capacity it makes no sense to operate the process at higher temperatures than 298 k, where softening process efficiency is higher than 80 % after 24 h. 0 25 50 75 100 0 200 400 600 800 1000 contact time, min r , % cacl2 natural water figure 5. influence of contact time on the hardness removal efficiency onto pure pc200fd resin (114 mg ca2+/lcacl2; 151 mg ca 2+/lwater; ph = 7.1; 8 g dry resin/l; 298 k). 0 10 20 30 278 298 313 333 temperature, k q, m g c a2 + / g dr y re si n cacl2 natural water figure 6. influence of initial temperature on the cationic exchange capacity of pure pc200fd resin (138 mg ca2+/lcacl2; 151 mg ca 2+/lwater; ph = 7.1; 8 g dry resin/l; 24 hours). 0 5 10 15 20 cacl2 caso4 ca(no3)2 natural water ca2+ sources q , m g c a2 + / g dr y re si n figure 7. influence of competitor anions form aqueous solution on the cationic exchange capacity of pure pc200fd resin (125 mg ca2+/laq.sol.; 151 mg ca 2+/lwater; ph = 7.1; 8 g dry resin/l; 24 hours; 298 k). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 101 3.5. competitor anions influence the cationic exchange capacity of pure pc200fd is not significantly influenced by the competitor anions that correspond to the calcium salts inducing water hardness (figure 7). a process efficiency of approx. 80 % ( 5 %) was achieved regardless of the ca2+ cation sources. 3.6. pure pc200fd resin performance versus purolite c100e considering as a benchmark the strong acid cationic resin purolite c100e the cationic exchange capacity [21, 22] of weak acid pure pc200fd resin was investigated for various concentrations of cacl2 solution corresponding to moderate and hard waters respectively (figure 8). 0 10 20 30 16 21 26 initial hardness, odca q, m g c a2 + / g dr y re si n pure pc200fd purolite c100e figure 8. cationic exchange capacity in the softening process of the polymeric resins (1 odca = 7.14 mg ca 2+/l, ph = 7.1; 8 g dry resin/l; 24 hours, 298 k). comparable results for the cationic exchange capacity can be attributed to both resins in the delivery form. pure pc200fd resin is delivered and used in the experiments in h+ form, whilst purolite c100e resin is delivered and used in na+ form. it is known that h+ form resins are more efficient than na+ [6, 7], the later being preferred in food industry because they are regenerated using nacl solutions. the fact that the producer company of pc200fd resin specifies that this is not regenerable can be a consequence of its use in h+ form. usually this h+ form resins are regenerated with hcl or h2so4, which is not so desirable for the food industry specific processes. still weak acid cation resin pure pc200fd represents a viable alternative for the water softening process, if the beneficiary has implemented a certain waste management programme to cope with the waste represented by the depleted resin. 4. conclusion the preliminary investigation of the cationic exchange capacity of the pure pc200fd resin in the water softening depending on various specific parameters of sorption process leads to the following conclusions: (1) appreciable ca2+ cations retention efficiencies can be achieved for the following conditions: (i) resin dose equal to 8 g/l (reported to the dry resin state), (ii) 25 oc working temperature; (iii) more than 200 min contact time in order to eliminate more than 50 % of the initial hardness. (2) cationic exchange capacity increases with the increase of ca2+ ions concentration in the initial solution and the increase of water hardness respectively. (3) the competitor anions that correspond to calcium salts that can be present dissolved in hard water (cl–, no3–, so42–) do not influence in a significant degree the ca2+ cations retention capacity. (4) for similar operating conditions the cationic exchange capacity of weak acid pure pc200fd resin is comparable to that of the strong acid purolite c100e resin; this performance can be assigned to the ionic form in which the two commercial cationic resins are delivered and used. because weak acid cation exchanger pure pc200fd cannot be regenerated the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 102 industrial beneficiary that uses it should also consider besides the technological performance the additional costs required by the storage, depositing and elimination of the depleted resin. it is advisable to perform an economical analysis for the use of this resin in the water softening equipment by comparison with a regenerable resin such as the strong acid cationite purolite c100e. 5. acknowledgments this research was partially supported by the project “knoweledge provocation and development through doctoral research pro-doct”, project co-funded by the european social fund in romania, under the responsibility of the managing authority for the sectorial operational programme for human resources (contract no. posdru/88/1.5/s/52946). 6. references 1. smith r., kim j.k., kleme j., handbook of water and energy management in food processing, hardcover, woodhead publishing ltd., (2008) 2. onita n., ivan e., handbook data for chemical and food industry calculus, (romanian), ed. mirton, timişoara, romania, (2006) 3. harland c.e., ion exchange. theory and practice (second ed.), royal society chemistry, cambridge, uk, (1994) 4. zagorodni a.a., ion exchange materials: properties and applications, elsevier, (2007) 5. strâmbeanu n., rus v., ursoiu i., ion exchange. theoretical principles and applications in water supply (vol. i) (romanian), ed. eurostampa, timişoara, romania, (1999) 6. strambeanu n., rus v., ion exchange. theoretical principles and applications in water supply (vol. ii) (romanian), ed. eurostampa, timişoara, romania, (1999) 7. macoveanu m., bilba d., bilba n., gavrilescu m., soreanu, g., ion exchange processes in environmental protection (romanian), matrixrom publishing house, bucharest, romania, (2002) 8. brown c.j., sheedy m., a new ion exchange process for softening high tds produced water, spe/petroleum society of cim/choa, technical paper no 78941, eco-tec inc., (2002) 9. www.purolite.com, purolite company 10. www.amberlite.com, dow chemical company 11. www.lewatit.com, lanxess company 12. www.pureresin.com, pure resin company 13. www.sciencedirect.com, keywords “water softening” or “hardness removal” 14. hoffmann h., martinola f., selective resins and special processes for softening water and solutions; a review, reactive polymers, ion exchangers, sorbents, 7(2–3), 263-272, (1988) 15. muraviev d., noguerol j., valiente m., separation and concentration of calcium and magnesium from sea water by carboxylic resins with temperature-induced selectivity, reactive & fuctional polymers, 28(2), 111-126, (1996) 16. yi w.t., yan c.y., ma p.h., removal of calcium and magnesium from lihco3 solutions for preparation of high-purity li2co3 by ion-exchange resin, desalination, 249(2), 729-735, (2009) 17. özmetin c., aydin ö., kocakerim m.m., korkmaz m., özmetin e., an empirical kinetic model for calcium removal from calcium impurity-containing saturated boric acid solution by ion exchange technology using amberlite ir–120 resin, chemical engineering journal, 148(2-3), 420-424, (2009) 18. coca m., mato s., gonzález-benito g., urueña m.á., garcía-cubero m.t., use of weak cation exchange resin lewatit s 8528 as alternative to strong ion exchange resins for calcium salt removal, journal of food engineering, 97(4), 569-573, (2010) 19. sr iso 6058:2008, water quality – determination of calcium content – edta titrimetric method 20. sr iso 6059:2008, water quality – determination of the sum of calcium and magnesium – edta titrimetric method 21. bandrabur b., tataru-fărmuş r.e., lazăr l., bulgariu l., gutt g., recherches sur les processus d’adoucissement de l’eau à l’aide de l’échangeur d’ions purolite c100e in: actes du septième colloque franco-roumain de chimie appliquée cofrroca–2012 (chief editor: ifirm, i.), ed. alma mater bacău, 238-239, (2012) 22. bandrabur b., tataru-fărmuş r.e., lazăr l., bulgariu l., gutt g., use of strong acid ion exchanger resin purolite c100e for removing permanent hardness of water – factors affecting cationic exchange capacity, scientific study & research – chemistry & chemical engineering, biotechnology, food industry, xiii, (in press), (2012) food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 16 current vision of the nucleation rate description problem michael anisimov department of chemical engineering, clarkson university, potsdam, ny 13699-5705, usa nanoaerosol laboratory, technological design institute of scientific instrument engineering sb ras 41 russkaya str., 630058 novosibirsk, russia. abstract: theoretical description and/or an experimental research of new phase embryo formation kinetics are widely presented in a problem of atmospheric aerosol dynamics. these problems involve the intensive heat-transfer devises; the natural deposits of methane or carbon dioxide, etc. nucleation is arising at aerosol generation in the vapor turbines, at vapour cooling in the heat-exchangers or/and at vapour expansion to atmosphere (any injection through piston) and so on. nucleation is common phenomenon for the broad spectrum of systems with phase transitions of the first order. critical embryo of new phase is treated as small ball (droplet) with uniform density in the classical nucleation theory (cnt) approximation. there is no warranty that the selected version of nucleation theory is good enough for the kinetics of hew phase embryos description. it is assumed that cnt has the best fit to the experimental data even cnt is distinguished among other “universal theories” by the internal consistency and simplicity of the axiomatic statements only. the experimental data are crossing usually the cnt predictions. thermodynamics consideration comes to the formal corrections to make consistent the next theory version to the next set of experimental data. it is the ordinary event when statistical mechanics is applied unreasonable for nucleation phenomenon. nucleation experiment accuracy has risen considerably during the last half of century, but it does not lead to consistent experimental results. triple points (lines, volumes etc.) or/and partial solubility of components are producing the complicate topology of the nucleation rate surfaces. that result is not understood enough by the scientific community up to now. an idea of semiempirical design of the nucleation rate surfaces over diagram of phase equilibria is generated over 10 years. phase equilibria lines are taken as a zero limit of nucleation rate. simply to say the nucleation rate surfaces arise from the phase equilibria lines. i.e. topologies of zero limits of nucleation rate exist in multiple handbooks for phase equilibria. that idea can give a considerable effect for series of nucleation problems in atmospheric aerosol dynamics. it is possible to conclude that the parametric versions of nucleation theory can be created for each set of scaled diagram of phase equilibria. design of semiempirical presentations of nucleation rate surfaces permits to create some basic set of nucleation theories, which can be used for binary and higher dimension nucleation theories. more details of the semiempirical design of nucleation rate surfaces will be presented. keywords: atmospheric nucleation, nucleation rate surfaces. 1. introduction current theories correspond to various modifications of classical nucleation theory that was completed in the 1940s (frenkel, 1975). the theoretical results look quite reasonable for low vapor nucleation rates where the droplet approximation is applicable (anisimov, 2003). however, these approaches have problems at the nanometer scale when the critical embryos contain of the order of 200 or less molecules (atoms). it appears that this quantity of molecules is near the threshold for the droplet critical embryo approximation, at least for organic vapors. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 17 figure 1. ratio of the experimental nucleation rates (jexpt) and theoretical one (jtheor) versus reciprocal nucleation temperatures (t) for n-alcohol-argon systems (strey et al., 1986). some researchers (baydakov, 1995; protsenko et al., 2006) have expressed unreasonable optimism that nucleation theory and experiment are in agreement for the case of bubble generation from the superheated liquids. at the present time, vapor-gas nucleation theory can produce values that deviate from the experimental results by up to several orders of magnitude (fladerer and strey, 2006; brus et al., 2005). figure 1 illustrates the typical relation of theoretical and experimental values of the nucleation rates on example of n-alcohol – argon systems nucleation. both problems produce difficulties in establishing one or more standard vapor/liquid nucleation systems that could be used to test vapor-gas nucleation rate measurement systems. the problem of the nucleation rate standard is more complex than simply using the npentanol-helium system as was suggested by the international workshop on nucleation in the czech republic, prague in 1995 as a candidate for a nucleation standard (anisimov et al., 2000c). the n-pentanol-helium system has unfortunately not produced sufficiently consistent data to date. the advantages and current problems of the vapor-gas nucleation experiments are discussed below and a view of the future studies is presented based on the assessment of vapor-gas/liquid nucleation experimental resutls. 2. experimental detection of the nucleation rate surface singularities another aspect of the vapor nucleation theory is associated with the wrong theory using in the vicinity of condensate phase transition (p.t.) of the first order. it is easy to show that the resulting nucleation rate surface should have a broken first derivative at the p.t. conditions. that conclusion is appeared from consideration of gibbs free energy of the critical embryo formation. such transitions can exist in the vicinity of the triple point, when the probability exists for change in phase of the forming critical embryo. figure 2. nucleation rate (j) surfaces for solid (light grey) and liquid (dark grey) phases overpressure (p) – temperature (t) phase diagram (anisimova et al., 2001). there could be a change in the dominant form of the forming embryo phase from one phase type to another such a liquid to a solid. the initial phase state of the critical embryo can exist over entire time of nucleation and embryo growth as was observed in the example of glycerin vapor nucleation and condensation (anisimova et al., 2001). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 18 they suggested that there was a difference in accommodation of molecules on surface of the two different phase states of the condensing clusters. this difference in accommodation probability led to unequal growth rates of clusters for the different phases. particle size measurements could then be used to detect the two separate phases. a clearly bimodal particle size distribution was found in the vicinity of glycerin triple point (anisimova et al., 2001). this result suggests two different embryo phases for the glycerin critical embryos and two nucleation rate surfaces simultaneously as it is shown in fig. 2. if there were phase transitions in the growing clusters, the bimodal size distribution would have been smoothed into a continuum of sizes which is not what was observed in the experimental results (anisimova et al., 2001). the gibbs free energy of the critical embryos governs the nucleation rate. in case of p.t. of condensate the first derivative of nucleation rate surface needs to split when nucleation occurs in the vicinity of phase transition of the first order in a condensed phase. in such circumstances, the conditions of continuity and monotony of the nucleation rate surface are simultaneously violated. these kinds of discontinuities have been found experimentally (anisimov et al., 1998a; 2000b). the topology of nucleation rate surfaces near a triple point is illustrated in fig. 2. the nucleation rate surface is hypothesized to arise from the pt phase equilibria. two lines for phase equilibria with vapor exist in the triple point vicinity. line, et, shows the equal nucleation rates for both phases. that line represents dynamic equilibria where a first derivative of a nucleation rate surface is figure 3. nucleation rate surface levels (higher) in nucleation temperature (t) – logarithm vapor activity (log a) axis at log j = 1; 3; 5 (anisimov et al., 1998a) and glycerin-carbon dioxide nucleation rate surface with discontinuity initiated by glycerin melting (red) and unknown p.t. near the critical line (green). broken. lines kt and tc show the boundaries of single, i.e. solid or liquid phase embryo generation, respectively. area ket illustrates nucleation rates when solid embryos prevail over droplet generation. area etch is characterized by the higher rate of droplet formation. in common case a diagram of phase equilibria involves several lines of phase equilibria and several nucleation rate surfaces are appeared. that multifold nucleation rate surface did not have the adequate theoretical consideration. multifold nucleation surface gets, as a food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 19 rule, one-fold approximation of some version of nucleation theory. theory has no chance to be proved by experiment in that way. clear discontinuities for nucleation rate surfaces for glycerin – carbon dioxide system can be seen for each data set (anisimov et al., 1998a) see, for example, fig. 3. one discontinuity can be attributed to melting of glycerin–carbon dioxide binary system. the melting temperature (tmelt) clearly increases with a rise in carbon dioxide pressure from 0.1 to 0.3 mpa. presumably the carbon dioxide concentration in solution increases proportional to the total pressure. the other disturbance is explained (anisimov et al., 1998a) by an unknown phase transition in critical embryos that appears near the critical line of the glycerine– carbon dioxide binary system. the origin of a nucleation rate surface discontinuity near critical conditions is not fully understood at this time because this type of phase transition has only been detected for the first time. a conceptual problem in vapor-gas to liquid (or solid) nucleation is its treatment of the nucleation as a single component problem instead as a two component system. the impact of a critical embryo phase change on the nucleation rate surface topology has been reported by (anisimov et al., 1998a; 2000b; 2000c). a similar problem was found by peeters et al. (2002). however, the observed jump in the water vapor nucleation rates was not reproduced in the next report from that group (holten et al., 2005). it may be that there is sufficient experimental variation in the nucleation rate measurements to prevent the detection of this kind of nucleation event. a major problem in detection of the nucleation rate surface singularities is the random error inherent in any experimental data. this problem exists even one has experimental results of relatively high accuracy. figure 4. distribution of average value of a and standard deviation, on glycerine vapor nucleation in the vicinity of critical temperature of carbon dioxide such as a = 0.01, =0.46 and a = 0.04, 0.16 at total pressure ptot=0.1 mpa for log j=3 with singularity and without it consequently (anisimov et al., 2000c). anisimov et al. (2000c) have suggested using continuity and monotonic behavior in the data analysis to find the anomalies in the nucleation rate surfaces. this approach can be illustrated by defining the nucleation rate surface by the function, f(x1, x2, ..., xn), where x1, x2, ..., xn are independent variables such as pressure or temperature. when function f has continuous partial derivatives, the surface described by the function f(x1 , x2 , . . . , xn), is continuous and monotonic. for condition f=constant, the function, f(x1, . xn) has n-1 independent variables. the nucleation rate with two variables, such as temperature, t, and vapor supersaturation, s, and constant values of all other parameters (p1...pn) can be considered. it is customary to present the nucleation rate experimental results with the a-criterion. a simple relation can be obtained along the surface section where j(t,s) = constant: 280 285 290 295 300 305 310 315 320 325 330 335 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 logj=2.0 logj=3.0 logj=4.0 glycerin co2 pt o t = 0.10 mpa t, k food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 20 1 1 1, ... , ... , ... ln lns p pn t p pn j p pn lnj lnj s = a t s t . the criterion value, a, will be equal to zero if the vapor nucleation rate surface j = j(t,s) has no singularities. because of experimental noise, the calculated values of a will include nonzero values that can be characterized by a non-dimensional standard deviation. the deviation of the a values from zero can characterize the quality of experimental results (if set of experimental values for j should represent a continuous and monotonous surface). a singularity in the nucleation rate surface (or surface for any other set of experimental results) breaks the local condition for surface continuity and monotony. figure 4 presents the application of the a criterion for glycerincarbon dioxide system (anisimov et al., 1998a). it can be seen that the a-criterion provides a sensitive tool for the detection of singularities in nucleation rate surfaces. anisimov et al. (2000a) applied the acriterion to data from strey et al. (1995). the nucleation rate surface for pentanolwater vapor on the base of strey et al. (1995) data shows the clear evidence of a nucleation rate surface singularity. this result is in contradiction to the conclusion that no effects on the nucleation rate surface topology is appeared for binary system with partial solubility of components (strey et al., 1995). 3. total pressure and carrier gas nature effects during the past two decades, several research groups have examined the effects of pressure and carrier gas composition on homogeneous nucleation to better understand the experimental data describing vapor to condensed phase nucleation. classical nucleation theory assumes that the only role of the carrier gas is to maintain the temperature equilibrium of the clusters with the ambient media. frank and hertz (1956) made the first observations of a gas-pressure effect. they found that the nucleation rate decreased as the total system pressure increased. that effect is now called a negative gas-pressure effect. the result was reproduced in several other measurements (katz et al. 1988; brus and zdimal, 2006). katz et al. (1988) found experimental variation from negative to positive gas-pressure effects. nevertheless these authors concluded that the effect is not significantly larger than the changes in nucleation rate that occur due to other uncertainties such as in thermal conductivity of the mixture. katz et al. (1988) suggested that the parameters for the heat and mass transfer calculations (diffusion coefficients, thermal conductivities, viscosities, etc.) are not sufficiently well defined and the variations of these parameters in terms of the error bars on the calculated nucleation rate values can dramatically change the gaspressure effects. clear gas-pressure effects are discussed in several other publications. anisimov and vershinin (1988; 1990) experimentally found a positive gas-pressure effect and a gas-composition effect at gas pressures from 0.10 to 0.30 mpa. fladerer and strey (2006) did attempt to measure supersaturated argon nucleation using a cryogenic nucleation pulse chamber. they concluded that growth rate of the nucleated argon droplets was too high to make permit decoupled nucleation and embryo growth. nevertheless the onset of nucleation corresponding to a nucleation rate of j=107(+/-2) cm-3s-1 at temperatures 52 < t(k) <59 and argon vapor supersaturation value near 10 was estimated. classical theory predicts nucleation rates of the order of 10-28 10-13 cm-3s-1 for these conditions. fladerer and food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 21 strey (2006) suggested that calculations based on density functional theory can only partially explain the discrepancy of 20 orders of magnitude between the experimental and theoretical nucleation rates. wedekind et al. (2008) take in to account the efficiency of thermalization and the additional work that a cluster has to spend for growing. these contributions produce the opposite trends in nucleation rates. unfortunately a scale of these effects is located within two orders in nucleation rates when discrepancy of theoretical and experimental results is over 20 orders. wedekind et al. (2008) consider their own result as qualitative because “we (wedekind et al., 2008) cannot discard the influence of other factors on the observed pressure effect.” these experiments can be provided by molecular dynamics simulations as well or/and density functional theory calculations using a lennard-jones potential. unfortunately these simulations and calculations usually fail for complex substances such as alcohols, water, etc. experimental results of different experimental schemes used for nucleation rate measurements at cryogenic temperatures are still inconsistent (fladerer and strey, 2006) because of low accuracy of the experimental data. 4. the experimental nucleation rate data inconsistency several research groups have made comparative measurements of nucleation rates (anisimov et al., 1993; 1994; wilck et al., 1998; brus et al., 2005, etc.) the results of brus et al. (2005) illustrate the inconsistency between the static diffusion chamber and an fdc for identical conditions of the same chemical system. the fdc values are higher than those from the static diffusion chamber results by three to four orders of magnitude. these examples illustrate the internal inconsistence of the experimental results that originate from different experimental systems. these inconsistencies generate the assumption that the typical nucleation vapor-gas experiments have an unrecognized and uncontrolled parameter. in the most cases, the gas is treated as an inert media to absorb the heat released from the phase transitions. inconsistencies among the experimentally measured values from the different experimental schemes are a major problem for current vapor-gas nucleation experiments. consideration of vapor-gas nucleation as a binary system is a reasonable way to resolve the data inconsistencies. it is plausible to think that different experimental systems have the inconsistent trajectories along the nucleation rate surface when the vapor-gas system is treated as binary system. several results, for example, anisimov et al. (1998a) and anisimova et al. (2001) illustrate that high pressure measuring techniques need to be designed to study multi-channel nucleation. 5. reference system for nucleation rate measurements in order to test the accuracy of an experimental system, it is important to have a standard system that can be measured over a range of nucleation conditions. the n-pentanol–helium system was proposed in prague, 1995 for such measurements. the available results from several research groups were collected and compared (anisimov et al., 2000c). they proposed a reference equation for nucleation rates of n-pentanol–helium as a practical test of any experimental measurement system for total pressures from 0.10 to 0.30 mpa. although the equation does not reproduce all of the results, the approximation is useful in its present form to provide a relative view of the different results up to time when a more accurate approximation can be food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 22 generated. the problem of a nucleation standard can only be solved when consistent results have been obtained by independent groups that use the different experimental schemes. it is believed that the vapor-gas nucleation rates should be represented by a surface instead of the single line that is used for most current presentations of the isothermal nucleation rate data in vapor-gas systems. 6. conclusions nucleation theory has no chance to be proven when multifold nucleation surface gets interpretation by some version of nucleation theory using one-surface approximation. the present day nucleation theory has no reliable experimental prove, because of the internal inconsistence of the experimental data. the pressure and carrier gas nature effects do not have reliable theoretical description and consistent experimental prove. the current recommendations for vapor-gas nucleation rate measurements can be summarized such as: vapor nucleation and cluster growth volumes should be decoupled to allow the embryos to grow before light scattering detection. because only dozens of molecules are involved in critical embryos formation for typical experimental conditions, the amount of vapor used to form the embryo is negligible compared to the total number of vapor-phase molecules and any vapor depletion problems within the nucleation volume are avoided. decomposition of embryos should be evaluated when an adiabatic recompression (allen & kassner, 1969) is applied to interrupt nucleation. theory independent algorithms are needed to estimate experimental nucleation rates based on fdc and others flow systems experimental data. homogeneous nucleation rates should be measured for sufficiently high supersaturation values to be sure that heterogeneous seeds are activated (anisimov and cherevko, 1985). impurities in the vapor substance must not exceed 0.2 % for the present time. this level of impurities shifts the nucleation rate values within one order of magnitude as shown by anisimov et al. (1987) and strey et al. (1995). this value is roughly the relative accuracy of modern nucleation rate measurements. carrier gas purity of 99.995 % and higher is sufficient and available everywhere. aerosol size distribution measurements are strongly recommended to identify the possibility of two and more channels for homogeneous and heterogeneous nucleation. continuity and monotony criteria should be applied for the detection of nucleation rate surface singularities and data quality characterization. vapor-gas systems are strongly recommended to be considered as binary nucleation systems. vapor-gas nucleation rate standards need to be developed to provide an absolute accuracy evaluation of experimental data. the problem of experimental data inconsistencies should be resolved to create a nucleation standard. nucleation rate measurements of any single component systems such as noble gases or any other single vapor in a gasfree atmosphere are very attractive for research as examples of true single component systems. the critical embryo parameters such as number of molecules of each species, enthalpy of embryo formation, excess energy, etc can be estimated using experimental data for the nucleation rates (anisimov and cherevko, 1985; anisimov et al., 1987; anisimov and taylakov, 1989). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 23 7. acknowledgments one of the authors (ma) gratefully acknowledge the travel grant ooff tthhee oorrggaanniizziinngg ccoo mmmmeettttee ooff tthhee iinntteerrnnaatt iioonnaa ll ccoonnffeerreennccee ““ggeeoo--eeccoollooggiiccaall mmoo nniittoorriinngg aanndd rriisskk oo ff aaddmmiinniissttrraattiivvee rreeggiioo nn ““ 27 – 29 september 2011,gegharkunik, marz, armenia. the present study was partially conducted under basic research russian foundation support through grant numbers of 11-03-12000-ofi-m and 11-03-00049-a. 8. references allen, l.b. & kassner, j.l., jr. (1969). the nucleation of water vapor in the absence of particulate matter and ions. journal of colloid and interface science, 30, 81 – 93. anisimov, m.p. (2003). nucleation: theory and experiment. russian chemical reviews, 72, 591 – 628. anisimov, m.p., & cherevko, a.g. (1985). gas-flow diffusion chamber for vapor nucleation studies. relations between nucleation rate, critical nucleus size and entropy of transition from a metastable into stable state. journal of aerosol science, 16, 97 – 107. anisimov, m.p. & hopke, p.k. (2001) nucleation rate surface topologies for binary system. journal of physical chemistry., 105, 11817 – 11822. anisimov, m.p. and taylakov, a.v. (1989) interface energy of critical embryos. journal of aerosol science, 20, 1063-1066. anisimov, m.p. & vershinin, s.n. (1988). spontaneous nucleation rate, size and composition of critical embryos in many component supersaturated vapor. lecture notes in physics, 309, 393 – 396. anisimov, m.p. & vershinin, s.n. (1990). dibutylphtalate nucleation rate at carbon dioxide different pressures. journal of aerosol science, 21 (1), 11 – 14. anisimov, m.p., vershinin s.n., aksenov a.a., sgonnov a.m., & semin g.l. (1987). experimental measurement of the spontaneous nucleation rates, size and a critical embryo composition in a supersaturated multicomponent vapor. colloidniy journal (russian), 49, 842 – 846. anisimov, m. p., hameri, k., kulmala, m., & ovchinnikova, t.e. (1993). homogeneous nucleation of dbf and n-hexanola in a laminar flow diffusion chamber. report series in aerosol scince, 23, 19 – 24. anisimov, m.p., hameri, k., & kulmala, m. (1994). construction and test of a laminar flow diffusion chamber: homogeneous nucleation of dfb and n-hexanol. journal of aerosol science, 25 (1), 23 – 32. anisimov, m.p., koropchak, j.a., nasibulin, a.g., & timoshina, l.v. (1998a). critical embryo phase transitions in the nucleated binary glycerin-carbon dioxide system. journal of chemical physics, 109, 10004 – 10010. anisimov, m.p. nasibulin, a.g., & shandakov, s.d. (2000a). experimental detection of nucleation rate surface singularity. journal of chemical physics, 112, 2348 – 2354. anisimov, m.p., koropchak, j.a., nasibulin, a.g., & timoshina, l.v. (2000b). 1-2 propanediol and 1-3 propanediol homogeneous nucleation rates and phase transition in new phase critical embryos. journal of chemical physics, 112, 9917 – 9928. anisimov, m.p., hopke, p.k., shandakov, s.d., & shvets, i. (2000c). n-pentanol-helium homogeneous nucleation rates. journal of chemical physics, 113, 1971 – 1975. anisimova, l., hopke, p.k., & terry, j. (2001). twochannel vapor nucleation in the vicinity of the triple point. journal of chemical physics, 114, 9852 – 9855. baydakov, v.g. (1995) superheating of cryogenics liquids. ural branch ras: ekaterinburg. brus, d., hyvarinen, a.. zdimal, v., & lihavainen h. (2005). homogeneous nucleation rate measurements of 1-butanol in helium: a comparative study of a thermal diffusion cloud chamber and a laminar flow diffusion chamber. journal of chemical physics, 122, 214506 – 21514. brus, d., zdimal, v., & stratmann, f. (2006). homogeneous nucleation rate measurements of 1-propanol in helium: the effect of carrier gas pressure. journal of chemical physics, 124, 164306 – 1634310. fladerer, a. and strey, r. (2006). homogeneous nucleation and droplet growth in supersaturated argon vapor. journal of chemical physics, 124, 164710 – 164715. frank, j. & hertz, h. (1956). messung der kritischen ubersattigung von dampfen mit der diffusionsnebelkammer. zeitschrift fur physik, 143, 559 – 590. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 24 holten, v., labetski, d., & van dongen, m.e. (2005). homogeneous nucleation of water between 200 and 240 k: new wave tube data and estimation of the tolman length. journal of chemical physics, 123, 104505 – 104509. katz, j. l. hung, c. h. & krasnopoler, m. (1988). the homogeneous nucleation of norare. proceedings of the 12-th icnaa (pp. 356 – 359). vienna (lecture notes in physics v.309. springer-verlag, berlin, 1988), 356 – 359. peeters, p., gielis, j.j., & van dongen, m.e. (2002). the nucleation behavior of supercooled water vapor in helium. journal of chemical physics, 117, 5647 – 5253. protsenko, s.p., baydakov, v.g., zhdanov, e.d., & teterin, a.s. (2006). nucleation in superheated liquids. in. metastable states and phase transitions. vol. 8, ural branch ras, ekaterinburg, 171 – 202. strey, r., viisanen, y., & wagner, p.e. (1995). measurement of the molecular content of binary nuclei. iii. use of the nucleation rate surfaces for the water-n-alcohol series. journal of chemical physics, 103, 4333 – 4345. strey, r., wagner, p.e., & schmeling, t. (1986). homogeneous nucleation rates for n-alcohol vapors measured in a two-piston expansion chamber. journal of chemical physics, 84(4), 2325 – 2335. wedekind, j.. hyvarinen, a.p., brus, d., reguera, d. (2008) unraveling the "pressure effect" in nucleation, physical review letters 101, article number: 125703. wilck, m., hameri, k., stratmann, f., & kulmala, m. (1998). determination of homogeneous nucleation rates from laminar-flow diffusion chamber data. journal of aerosol science, 29, 899 – 911. microsoft word 3 journal fia nr 1 2010 final_42-45.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 46 the influence of refrigeration time and of sugar addition on ascorbic acid content in some natural juices marcel avramiuc stefan cel mare university of suceava, faculty of food engineering, str. universităţii, no. 13, 720229, suceava, romania, e-mail: avramiucm@usv.ro abstract: following some studies concerning the main factors influencing the concentration of vitamins within food raw materials, especially ascorbic acid, this work tries to make evident the influence of the refrigeration temperature and of the sucrose addition on content of this vitamin in three types of natural juice. the biological material was represented by black currant, raspberry and quince natural juices, obtained from these fruits by means of a crushing out process. for each type of juice, samples without addition and samples with 5% and 10% sucrose addition were made up. the ascorbic acid determination was carried out from fresh juice as well as from juice kept in refrigerator, at certain time intervals (24, 48, 96 and 168 hours). the storage of these juices under refrigeration conditions has determined percentage reductions of vitamin c content of these ones. thus, after 168 hours of storage, in the three types of analyzed juices the highest loss of vitamin have been registered in samples without sucrose addition, and the least ones in samples with 10% addition. the comparison of vitamin c values in the three analyzed juices, subjected to 168 hourrefrigeration process, has emphasized that the highest loss of ascorbic acid have been in quince juice, and the least one in black currant juice. keywords: vitamin c, sucrose, black currant, raspberry, quince. introduction there are some factors which influence negatively the vitamins concentration in food raw materials, such as: high temperatures over certain values, freezing, and presence of oxygen and of some chemical substances, certain ph values etc. during food raw materials processing, the level of their vitamins can be negatively affected through some operations such as: handling-preservation after harvest of vegetable produce and of meat and aquatic produce, collecting-storage of milk, cutting chopping end/or scalding, boiling of fruits and legumes, cereals grinding, the adding of chemical substances into legumes, fruits, meat (adrian and petit, 1970; ferrando and mainguy, 1970; flanzy, 1970; scriban, 1970; ulrich and delaporte, 1970; banu et al., 2003). as for vitamin c, its concentration can be reduced with various percentages, depending on the type of processing of raw material containing this vitamin. thus, three months potatoes storage leads to loss of 50%, the sterilization, but especially vegetables boiling leads to vitamin loss between 47% and 82%. the addition of bicarbonate, used to soften some legumes, contributes to diminution of vitamin c concentration (banu et al., 2003). the storage of legumes for a year at temperatures around -10º can lead to vitamin c loss of 80–90% (selman, 1994). the addition of anthocyans, sugar and even starch seems to have a protecting action on vitamin c (banu et al., 2003). retention of ascorbic acid is better in rapid drying at high temperatures than in slower drying at lower temperatures. drying methods that expose the food to air result in losses of vitamin c due to oxidation. on the other hand, journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 47 freeze drying, which is carried out in the absence of oxygen, does not cause loss of vitamin c (ball, 2006). according to selman (1994), the inefficient blanching causes some loss of vitamin c by oxidation, as well as by leaching. as to microwave heating, the ascorbic acid content is higher in vegetables cooked by microwave heating than by conventional methods (hill, 1994). in this work, the variation of vitamin c (ascorbic acid) concentration within three types of natural juices, without and with sucrose addition, stored at +2ºc, certain time periods, has been studied. experimental the biological material was represented by natural juices of black currant, raspberry and quince, obtained from these fruits by means of a crushing out process. in order to limit the contact with air and the possibility of vitamin c oxidation, once obtained the juices were rapidly poured into plastic bottles (300 ml capacity) tightly closed, and introduced into refrigerator at +2º c. for each type of juice, samples without addition and samples with 5% and 10% sucrose were made up. the ascorbic acid determination was carried out from fresh juice as well as from juice kept in refrigerator, at certain time intervals (24, 48, 96 and 168 hours). the method was based on reduction of 2,6diclorphenolindophenol (2,6dcpip) to the leuco compound of 2,6dcpip, by ascorbic acid (artenie and tănase, 1981). results and discussion table 1 presents the values of ascorbic acid content in black currant juice. table 1. the black currant juice ascorbic acid content, with and without sugar addition, stored at certain time periods in refrigerator (2º c) ascorbic acid (mg %) juice type storage length sucrose free juice juice with 5 % sucrose juice with 10 % sucrose *0 hours 198,75 198,75 198,75 24 hours 196,52 198,04 197,13 48 hours 186,06 194,26 196,45 96 hours 165,74 170,03 175,89 168 hours 136,57 145,29 160,42 * blank sample journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 48 as seen, both in the sucrose free black currant juice and in samples with sucrose addition, the vitamin c concentration has gradually decreased once with juice keeping in refrigerator. the highest differences between samples, which have begun to come into view after 96 hours, have appeared after 168 hours of storage. the greatest value was registered by juice with 10% sucrose (19,29% vitamin loss beside blank), and the least was registered by sucrose free juice (31,29% vitamin loss beside blank). the table 2 reproduces the values of ascorbic acid content in raspberry juice. table 2. the raspberry juice ascorbic acid content, with and without sugar addition, stored at certain time periods in refrigerator (2º c) ascorbic acid (mg %) juice type storage length sucrose free juice juice with 5 % sucrose juice with 10 % sucrose *0 hours 42,00 42,00 42,00 24 hours 39,76 40,08 40,08 48 hours 35,51 38,70 39,70 96 hours 32,08 36,15 38,50 168 hours 27,14 30,45 32,65 * blank sample also in raspberry juice, reductions of vitamin c concentration can be seen, once with increase of the samples keeping length in refrigerator at +2º c. more marked then in the case of black currant juice, in raspberry juice, after 168 hours of storage, the vitamin c loss has been of 35,38%, in sucrose free juice sample, and 22,27% in juice sample with 10% sucrose. in the table 3 the values of ascorbic acid content in quince juice are given. as seen in the table, in quince juice, the vitamin c loss has been the highest as against black currant and raspberry juices. thus, after 168 hours of storage at +2º c, in sucrose free juice sample the vitamin c concentration has diminished (as against blank) with 39,47%, and in juice sample with 10% sucrose has diminished with 29,33%. table 3. the quince juice ascorbic acid content, with and without sugar addition, stored at certain time periods in refrigerator (2º c) ascorbic acid (mg %) juice type storage length sucrose free juice juice with 5 % sucrose juice with 10 % sucrose *0 hours 70.840 70.840 70.840 24 hours 68.992 68.998 69.166 48 hours 67.760 68.005 68.537 96 hours 65.296 66.108 67.214 168 hours 56.672 58.851 59.332 * blank sample journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 49 conclusions the storage under refrigeration conditions (+2º c) of some natural black currant, raspberry and quince juices, with and without sucrose addition at certain time periods, has determined percentage reductions of vitamin c content in these juices. after 168 hours of storage, in the three types of analyzed juices the highest vitamin loss have registered in sucrose free juice samples, and the least ones in juice samples with 10% sucrose. the comparison of vitamin c values in the three analyzed juices, subjected 168 hours to refrigeration process, has emphasized that the highest loss of ascorbic acid have been in quince juice, and the least one in black currant juice. references 1. j. adrien, l. petit les vitamines des céréales et leur évolution au cours des traitements technologiques. annales de la nutrition et de l’alimentation, vol. 24, nr. 1, 1970, 131. 2. v. artenie, elvira tănase practicum de biochimie generală. centrul de multiplicare al univ. “al. i. cuza” iaşi, 1981, 172. 3. g.f.m. ball vitamins in foods, analysis, bioavailability, and stability. crc press, taylor & francis group 6000, broken sound parkway nw, suite 300, boca raton, fl 33487-2742, 2006, 292305 4. c. banu, maria iordan, violeta nour, g. musteaţă, procesarea materiilor prime alimentare şi pierderile de substanţe biologic active,. edit. “tehnica” utm, chişinău, 2003, 91-92. 5. r. ferrando, r. mainguy l’évolution des vitamins en function des conditions de récolte et de conservation des denrées primaires des animaux destinés à la consommation de l’homme. annales de la nutrition et de l’alimentation, vol. 24, nr. 3, 1970. 6. m. flanzy les vitamines dans les boissons fermentées. annales de la nutrition et de l’alimentation, vol. 24, nr. 3, 1970. 7. m.a. hill vitamin retention in microwave cooking and cook chill foods. food chem., 1994, 49, 131. 8. r. scriban, les vitamines de l’orge, du malt et de la bière. annales de la nutrition et de l’alimentation, vol. 24, nr. 3, 1970. 9. j.d. selman vitamin retention during blanching of vegetables. food chem., 1994, 49, 137. 10. r. ulrich, n. delaporte l’acide ascorbique dans les fruits conservés par le froid dans l’air et en atmosphère contrôlée. annales de la nutrition et de l’alimentation, vol. 24, nr. 1, 1970. microsoft word 8 andrei simion uvab.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 49 modelling of the thermophysical lactic acid aqueous solutions. density and viscosity *andrei i. simion1, cristina g. grigoraş1, loredana e. bardaşu1, adriana dabija2 1vasile alecsandri university of bacău, department of chemical and food engineering, bacău, romania, asimion@ub.ro, cristina.grigoras@ub.ro 2ştefan cel mare university of suceava, faculty of food engineering, suceava, romania, dadianadabija@yahoo.com *corresponding author received 10 november 2012, accepted 2 december 2012 abstract: lactic acid is an industrially important product with a large and rapidly expanding market due to its attractive and valuable multi-function properties. it is widely used in various fields such as food industry, in pharmaceuticals and cosmetics etc. and knowing and predicting the evolution of its thermophysical properties at any moment may be very useful. in this paper various mathematical relations between lactic acid concentrations and temperature with density and dynamic viscosity were established. the known data were fitted in different equations in order to assess and select a suitable mathematical model. taking in consideration the level of precision and the simplicity of formulation several equations were generated for each thermophysical property. for density, two equations were formulated with average relative errors (absolute value) of 0.062% and respectively r2 = 0.9999 and average relative errors of 0.055% for intervals of temperature of 298.15 to 353.15 k and dry matter concentration range between 9.16 to 85.32%. for the dynamic viscosity an equation based on arrhenius mathematical model with average relative errors of 1.11% and an equation with other mathematical formulation with r2 = 0.9997 were generated in the same range of temperature and citric acid concentration. the obtained equations can be uploaded in computer software for storing, organizing and manipulating data available both for industrial and academic users and so facilitating the sizing and optimization calculations of various technological processes and equipments. keywords: lactic acid, thermo-physical properties, mathematical modelling 1. introduction lactic acid is an organic acid found in many products of natural origin. the first reports on isolation of lactic acid from milk can be found in as early as 1780 and the solidification by self-esterification some years later [1]. lactic acid can be obtained via chemical synthesis [2-4] or carbohydrate fermentation [5-6] with the help of microorganisms such as lactobacillus rhamosus [7], lactococcus lactis [8] or lactobacillus helveticus [9]. this last technique has a significant advantage in that by choosing a strain of microorganism able to produce only one enantiomer, an optically pure product can be obtained, whereas synthetic production results in a racemic mixture [10]. one of the most important steps of lactic acid production lies in the separation process which is needed to recover and purify the product from the fermentation broth. different methods are available to this purpose. among them, solvent extraction followed by centrifugal short path distillation [11], extraction with aliphatic amines [12] or liquid ions [13], nanofiltration [14-15], vapor-permeation assisted esterification [16], reverse osmosis downstream process [17], chromatography [18], electrodialysis [19-21], adsorption food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 50 technology [22] conducted to satisfactory results. lactic acid and its derivatives are widely used in the food [23-24], pharmaceutical leather, and textile industries [1]. recently, there has been an increased interest in lactic acid as a raw material for production of polylactic acid [25-27], a polymer used as a specialty medical and environmental-friendly biodegradable plastic as an alternative for substituting conventional plastic produced from petroleum oil. this new material can be employed as film barrier on intraabdominal adhesion formation [28], as colloidal drug delivery system [29] as membrane for periodontal guided tissue regeneration [30] etc. for proper and adequate process development it is necessary to use a lactic acid with high purity. its production and further employment depend on some of its thermophysical properties. among them density and viscosity are two of the most important ones. as for other types of products [31-32], the evolution of these lactic acid characteristics are related to parameters such temperature and solution concentrations. different data are available in literature in this field but their use is rather difficult. as consequence, this work intended to establish mathematical relations between aqueous solutions of lactic acid density and viscosity and the above mentioned parameters. relative error, anova test and correlation coefficient were used in order to verify the similarity between the experimental data and that proposed by the obtained mathematical models. 2. experimental experimental data provided by the scientific publications (tables 1 and 2) concerning the variation of aqueous lactic acid solutions density and dynamic viscosity with concentration and temperature were used as primary data for the regression analysis. table 1. variation of lactic acid aqueous solutions density with temperature and lactic acid content [1] density, ρ [kg/m3] temperature, t [k] lactic acid concentration, c [% w/w] 293.15 298.15 303.15 313.15 323.15 333.15 343.15 353.15 9.16 1019.5 1018.1 1015.8 1011.3 1006.7 1000.7 995.04 988.99 24.35 1056.7 1054.4 1051.8 1047.1 1041.4 1035.1 1029.5 1022.6 45.48 1109.8 1105.3 1101.8 1094.2 1087.0 1079.2 1072.1 1063.9 64.89 1155.2 1151.8 1147.2 1139.8 1132.0 1123.5 1115.3 1099.6 75.33 1178.6 1174.8 1170.1 1161.3 1152.6 1142.5 1134.0 1125.1 85.32 1198.9 1194.8 1190.1 1181.3 1171.8 1163.1 1153.6 1144.3 table 2. variation of lactic acid aqueous solutions dynamic viscosity with temperature and lactic acid content [1] dynamic viscosity, µ . 103 [pa . s] temperature, t [k] lactic acid concentration, c [% w/w] 298.15 303.15 313.15 323.15 333.15 343.15 353.15 9.16 1.150 1.030 0.809 0.671 0.572 0.473 0.416 24.35 1.670 1.460 1.130 0.918 0.746 0.632 0.532 45.48 3.090 2.740 2.030 1.590 1.260 1.020 0.843 64.89 6.960 6.010 4.220 3.120 2.380 1.850 1.470 75.33 13.03 10.55 7.080 4.980 3.570 2.730 2.080 85.32 28.50 22.60 13.91 9.400 6.400 4.590 3.400 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 51 microsoft excel™ 2010 software was employed for typical data integration, graphical representations and anova analysis. anova analysis tool is able to compare the experimental and the calculated data generated by the established mathematical models. complex and atypical data plotting in 2d (“vapour pressure” model, “heat capacity” model etc.) were performed with curveexpert® software and 3d representations as a surface response were fitted and analyzed in tablecurve 3d® v.4 software. thermo-physical property vs. temperature, thermo-physical property vs. lactic acid concentration in aqueous solutions were plotted and different types of regression techniques, involving the method of least squares, relative error ε (equation 1) and anova were used to reveal the best-fit equation. [%]100   calculated calculatedalexperiment data datadata  (1) 3. results and discussions 3. 1. density using microsoft excel™ 2010 spreadsheets and curveexpert® software, 8 quadratic correlations (taking in consideration the best fit and simplicity in formulation) between lactic acid concentrations c [% w/w] and density ρ [kg/m3], at constant temperature t, [k] have been established: 2 321 cacaa  (2) the a1 and a2 values are presented in table 3 and the regression coefficients r2 are greater than 0.99, thus indicating a good correlation of variables. in order to correlate a1, a2 and a3 coefficients with temperature t, [k], several models were uploaded in curveexpert® software (1st, 2nd and 3rd table 3. coefficients for equation no. 2 equation 2 coefficients temperature, t [k] a1 a2 a3 r2 293.15 994.6371 2.6771 -0.0032 0.999 298.15 994.1297 2.5713 -0.0024 0.999 303.15 992.4598 2.5202 -0.0023 0.999 313.15 989.0405 2.4335 -0.0020 0.999 323.15 984.9243 2.3689 -0.0020 0.999 333.15 980.2156 2.2684 -0.0014 0.999 343.15 975.2492 2.2243 -0.0016 0.999 353.15 969.7177 2.1592 -0.0013 0.999 degree polynomial equations, “vapor pressure” model, “heat capacity” model etc.). the best fit model is the quadratic equation with good regression coefficients (table 4). 2 321 tbtbbtcoefficien  (3) table 4. coefficients for equation no. 3 equation 3 coefficients equation 2 coefficients b1 b2 b3 r2 a1 0.0574246013 0.0003189759 -4.54e-07 0.9044 a2 11.8306041781 0.0506144371 5.8356e-05 0.9912 a3 813.3102959923 1.4872913211 -0.0029584854 0.9993 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 52 combining the equations 2 and 3 and, the final form of proposed equation model (equation 4) is: 22 131211 2 232221 2 333231 )()()( ctbtbbctbtbbtbtbb aaaaaaaaa  (4) using the relative error equation the calculated data given by the density mathematical model and the existing experimental data were compared (table 5) obtaining a final average of 0.062%. table 5. for densities of citric acid aqueous solutions the absolute value of relative errors for calculated data versus tabular data density, ρ [kg/m3] temperature, t [k] 293.15 298.15 303.15 313.15 l ac tic a ci d co nc en tr at io n, c [% w /w ] ed* cd* ε, % ed cd ε, % ed cd ε, % ed cd ε, % 9.16 1019.5 1019.1 0.044 1018.1 1017.3 0.082 1015.9 1015.3 0.051 1011.4 1011.1 0.029 24.35 1056.7 1057.8 0.103 1054.5 1055.3 0.079 1051.8 1052.6 0.076 1047.2 1047.1 0.009 45.48 1109.8 1109.5 0.022 1105.4 1106.1 0.068 1101.8 1102.6 0.073 1094.3 1095.5 0.113 64.89 1155.2 1154.7 0.046 1151.8 1150.7 0.098 1147.2 1146.6 0.053 1139.9 1138.4 0.130 75.33 1178.6 1178.1 0.042 1174.8 1173.8 0.085 1170.1 1169.5 0.052 1161.3 1160.8 0.042 85.32 1198.9 1199.8 0.081 1194.8 1195.4 0.052 1190.1 1190.9 0.070 1181.3 1181.9 0.047 average ε, % 0.057 average ε, % 0.077 average ε, % 0.062 average ε, % 0.061 temperature, t [k] 323.15 333.15 343.15 353.15 ed cd ε, % ed cd ε, % ed cd ε, % ed cd ε, % 9.16 1006.7 1006.4 0.038 1000.8 1001.2 0.040 995.0 995.5 0.043 989.0 989.3 0.032 24.35 1041.5 1041.2 0.029 1035.1 1034.9 0.019 1029.6 1028.4 0.116 1022.6 1021.5 0.106 45.48 1087.0 1088.2 0.108 1079.3 1080.7 0.137 1072.2 1073.1 0.082 1064.0 1065.2 0.117 64.89 1132.1 1130.0 0.177 1123.6 1121.6 0.088 1115.3 1113.0 0.029 1099.6 1104.3 0.064 75.33 1152.6 1152.0 0.053 1142.5 1143.1 0.050 1134.1 1134.0 0.004 1125.1 1124.9 0.022 85.32 1171.8 1172.7 0.073 1163.1 1163.3 0.019 1153.6 1153.9 0.023 1144.3 1144.3 0.003 average ε, % 0.079 average ε, % 0.058 average ε, % 0.049 average ε, % 0.057 * ed – experimental data, cd – calculated data the anova analysis was used to compare the values of density experimental and calculated data at 6 different concentrations in 8 temperatures variation. the results presented in table 6 showed that the sample p-value is 0.991263 greater than the targeted alpha 0.05 and the f crit value is larger than the f-test value and as consequence the null hypothesis is not rejected indicating that is not a statistical difference between tabular and calculated data. by plotting experimental data for aqueous lactic acid solutions in tablecurve 3d® v.4 software (figure 1) an equation for the response function was generated, chosen due to the accuracy and simplicity of formulation. the equation 5 is a simple equation, rank 33, eqn. 1033 in tablecurve 3d® v.4 library with a precision of r2 = 0.999967394, fitsderr = food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 53 1.2166463, fstat. = 20950.495. the coefficients values are presented in table 7. 3 7 2 654 321 1 ln tatataca tacaa    (5) table 6. the anova test summary lactic acid concentration, c [% w/w] summary 9.16 24.35 45.48 64.89 75.33 85.32 total experimental data count 8 8 8 8 8 8 48 sum 8056.42 8338.99 8713.71 9064.73 9239.16 9397.9 52810.91 average 1007.053 1042.374 1089.214 1133.091 1154.895 1174.738 1100.227 variance 126.4038 153.4055 271.2956 373.6544 387.4041 397.5284 3960.208 calculated data count 8 8 8 8 8 8 48 sum 8055.094 8338.821 8721.028 9059.337 9236.239 9402.199 52812.72 average 1006.887 1042.353 1090.128 1132.417 1154.53 1175.275 1100.265 variance 116.92 174.5367 260.073 337.1056 375.7415 409.9365 3956.772 anova source of variation ss* df* ms* f p-value* f crit sample 0.034015 1 0.034015 0.000121 0.991263 3.954568 columns 348403.1 5 69680.62 247.094 1.13e-48 2.323126 interaction 6.930834 5 1.386167 0.004915 0.999995 2.323126 within 23688.04 84 282.0004 * ss – sum of squares, df – degrees of freedom, ms – mean square, p-value – level of significance table 7. coefficients for equation no. 5 coefficient value coefficient value a1 1004.2716 a5 0.0032418557 a2 3.6820806 a6 -8.8833524e-07 a3 2.8476715 a7 3.4556043e-08 a4 0.000950369 r2 0.9999 0 10 20 30 40 50 60 70 80temperature, t [k] 360 350 340 330 320 310 300 lactic acid c oncen tration , c [% , w/w] 950 1000 1050 1100 1150 1200 d en si ty ,  [k g/ m 3 ] figure 1. lactic acid aqueous solutions density values plotted in tablecurve 3d and fitted with simple equation type (equation 5) with residuals 2. dynamic viscosity a common mathematical model, for fitting the viscosity values is based on the equation of arrhenius because it creates a good correlation between experimental and calculated values. tr ea e    0 (6) where: µ – dynamic viscosity [pa.s,], µ0 – water dynamic viscosity [pa.s,], ea – activation energy [kcal/mol], r – universal gas constant [1.987×10-3 kcal/mol], food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 54 t – absolute temperature [k]. taking logs of equation (6), it gets (7): tr ea 1 303.2 loglog 0    (7) and:   t r ea   loglog303.2 0 (8) by plotting in tablecurve 3d® v.4 software the results obtained from equation 8, figure 2, which make correlations between experimental data from table 1 and water viscosity an equation for the response surface was generated (equation 9). the equation 9 is a polynomial equation, rank 29, eqn. 1049 in tablecurve 3d® v.4 library with a precision of r2 = 0.9998, fitsderr = 4.6371739, fstat. = 36032. 90 80 70 60 50 40 30 20 10 lac tic a cid con cen trat ion, c [ %, w /w] 360 350 340 330 320 310 300 temperature, t [k]] -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 0 e a/ r figure 2. ea/r values plotted in tablecurve 3d and fitted with polynomial equation type (equation 9) with residuals taca tacacaa r ea    65 4 2 321 1 (9) table 8. coefficients for equation no. 9 coefficient value coefficient value a1 -230.01384 a4 0.67832811 a2 -9.7502949 a5 -0.01242596 a3 0.044803796 a6 0.0019299232 combining the equations 6 and 9 and replacing the coefficients with numeric values, the final form of proposed equation model (equation 10). ttaca tacacaa e )1(0 65 4 2 321      (10) appling the relative error equation the calculated data generated with the dynamic viscosity final equation and the existing tabular data were compared (table 9) obtaining a final average of 1.11%. table 9. for dynamic viscosities of citric acid aqueous solutions the absolute value of relative errors for calculated data versus tabular data dynamic viscosity, µ . 103 [pa . s] lactic acid concentration, c [% w/w] 9.16 24.35 45.48 t em pe ra tu re , t [k ] ed* cd* ε, % ed cd ε, % ed cd ε, % 298.15 1.15 1.16 0.83 1.67 1.68 0.39 3.09 3.14 1.66 303.15 1.03 1.02 0.62 1.46 1.47 0.35 2.74 2.69 1.77 313.15 0.81 0.82 1.45 1.13 1.15 1.98 2.03 2.04 0.53 323.15 0.67 0.67 0.32 0.92 0.92 0.51 1.59 1.58 0.64 333.15 0.57 0.56 2.45 0.75 0.76 1.51 1.26 1.26 0.24 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 55 dynamic viscosity, µ . 103 [pa . s] lactic acid concentration, c [% w/w] 9.16 24.35 45.48 t em p. , t [k ] ed* cd* ε, % ed cd ε, % ed cd ε, % 343.15 0.47 0.47 0.31 0.63 0.63 0.35 1.02 1.02 0.31 353.15 0.42 0.41 1.83 0.53 0.54 1.17 0.84 0.85 0.34 average ε, % 1.11 average ε, % 0.89 average ε, % 0.78 lactic acid concentration, c [% w/w] 64.89 75.33 85.32 ed cd ε, % ed cd ε, % ed cd ε, % 298.15 6.96 7.00 0.51 13.03 12.84 1.46 28.50 28.80 1.04 303.15 6.01 5.82 3.21 10.55 10.39 1.47 22.60 22.37 1.01 313.15 4.22 4.17 1.13 7.08 7.10 0.26 13.91 14.18 1.95 323.15 3.12 3.07 1.54 4.98 5.00 0.46 9.40 9.36 0.41 333.15 2.38 2.34 1.88 3.57 3.66 2.45 6.40 6.46 0.96 343.15 1.85 1.82 1.42 2.73 2.76 1.02 4.59 4.63 0.89 353.15 1.47 1.45 1.24 2.08 2.13 2.27 3.40 3.41 0.43 average ε, % 1.56 average ε, % 1.34 average ε, % 0.95 * ed – experimental data, cd – calculated data the anova analysis was used to compare the values of dynamic viscosity tabular and calculated data at 10 different concentrations in 4 temperatures variation. the results presented in table 10 showed that the sample p-value is 0.9997 greater than the targeted alpha 0.05 and the f crit value is larger than the f-test value and as consequence the null hypothesis is not rejected indicating that is not a statistical difference between tabular and calculated data. table 10. the anova test summary (for dynamic viscosity, µ [pa . s]) lactic acid concentration, c [% w/w] summary 9.16 24.35 45.48 64.89 75.33 85.32 total experimental data count 7 7 7 7 7 7 42.00 sum 0.005121 0.007088 0.012573 0.02601 0.04402 0.0888 0.183612 average 0.000732 0.001013 0.001796 0.003716 0.006289 0.012686 0.004371714 variance 7.76e-08 1.84e-07 7.43e-07 4.45e-06 1.73e-05 9.2e-05 3.46139e-05 calculated data count 7 7 7 7 7 7 42 sum 0.005114 0.007146 0.012579 0.025668 0.043878 0.089219 0.18360409 average 0.000731 0.001021 0.001797 0.003667 0.006268 0.012746 0.004371526 variance 8.02e-08 1.84e-07 7.51e-07 4.4e-06 1.65e-05 9.27e-05 3.47515e-05 anova source of variation ss* df* ms* f p-value* f crit sample 7.45e-13 1 7.45e-13 3.9e-08 0.999843 3.973897 columns 0.001468 5 0.000294 15.3644 2.93e-10 2.341828 interaction 2.26e-08 5 4.51e-09 0.000236 1 2.341828 within 0.001376 72 1.91e-05 * ss – sum of squares, df – degrees of freedom, ms – mean square, p-value – level of significance food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 56 by plotting directly the tabular data for the dynamic viscosity in tablecurve 3d® v.4 software an equation for the response surface was generated (figure 3). the equation 11 is a linear equation, rank 3, eqn. 1071 in tablecurve 3d® v.4 library with a precision of r2 = 0.99973191, fitsderr = 0.00010903979, fstat. = 13258.82 and the coefficients are presented in table 11. tbcbcbcb tbtbtbcbcbb    10 3 9 2 87 3 6 2 54 2 321 1  (11) table 11. coefficients for equation no. 11 coefficient value coefficient value b1 0.1095492 b6 -2.4739564e-09 b2 -4.7563422e-06 b7 -0.027799358 b3 1.9479985e-08 b8 0.00022424967 b4 -0.00092119128 b9 -6.1981778e-07 b5 2.6073585e-06 b10 0.00051576641 01 02 03 04 05 06 070 80lactic acid concentration, c [%, w/w] 360 350 340 330 320 310 300 tem per atur e, t [k] 0 0.005 0.01 0.015 0.02 0.025 0.03 d yn am ic v is co si ty , µ [p a. s] figure 3. lactic acid aqueous solutions dynamic viscosity values plotted in tablecurve 3d and fitted with linear equation type (equation 11) with residuals combining the models developed for the calculation of dynamic viscosity and density of citric acid aqueous solutions, the kinematic viscosity (ν) can be calculated using equation 12:     [m2/s] (12) 4. conclusion for density two equations were formulated with average relative errors (absolute value) of 0.062% (equation 4) and respectively r2 = 0.9999 and average relative errors of 0.055% (equation 5) for intervals of temperature of 298.15 to 353.15 k and dry matter concentration range between 9.16 to 85.32%. for the dynamic viscosity an equation based on arrhenius mathematical model with average relative errors of 1.11% (equation 10) and for a direct fitting of the experimental data in tablecurve 3d an equation with r2 = 0.9997 were generated in the same range of temperature and citric acid concentration. the proposed mathematical models can be loaded in the widespread pc software for storing, organizing and manipulating data and for targeted concentrations and temperature more precise values of the studied thermophysical properties can be found easier than using the existing experimental data in tabular form or graphic form. 5. references [1]. ren, j., lactic acid, in biodegradable poly(lactic acid): synthesis, modification, processing and applications, springer berlin heidelberg, (2011). [2]. disselkamp, r.s., harris, b.d., hart, t.r., hydroxy acetone and lactic acid synthesis from aqueous propylene glycol/hydrogen peroxide catalysis on pdblack, catalysis communications, 9. 22502252, (2008). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 57 [3]. drandarov, k., schubiger, p.a., westera, g., automated no-carrier-added synthesis of [1-11c]-labeled dand lenantiomers of lactic acid, applied radiation and isotopes, 64. 1613-1622, (2006). [4]. bjurling, p., långström, b., synthesis of 1and 3-11c-labelled l-lactic acid using multi-enzyme catalysis, journal of labelled compounds and radiopharmaceuticals, 28. 427-432, (1990). [5]. datta, r., henry, m., lactic acid: recent advances in products, processes and technologies — a review, journal of chemical technology & biotechnology, 81. 1119-1129, (2006). [6]. banner, t., fosmer, a., jessen, h., marasco, e., rush, b., veldhouse, j., de souza, m., microbial bioprocesses for industrial-scale chemical production, in biocatalysis for green chemistry and chemical process development, john wiley & sons, inc., (2011). [7]. coelho, l.f., bolner de lima, c.j., bernardo, m.p., alvarez, g.m., contiero, j., improvement of l(+)-lactic acid production from cassava wastewater by lactobacillus rhamnosus b 103, journal of the science of food and agriculture, 90. 19441950, (2010). [8]. åkerberg, c., hofvendahl, k., zacchi, g., hahn-hägerdal, b., modelling the influence of ph, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by lactococcus lactis ssp. lactis atcc 19435 in whole-wheat flour, applied microbiology and biotechnology, 49. 682-690, (1998). [9]. kulozik, u., physiological aspects of continuous lactic acid fermentations at high dilution rates, applied microbiology and biotechnology, 49. 506-510, (1998). [10]. panesar, p.s., kennedy, j.f., gandhi, d.n., bunko, k., bioutilisation of whey for lactic acid production, food chemistry, 105. 114, (2007). [11]. chen, l., zeng, a., dong, h., li, q., niu, c., a novel process for recovery and refining of l-lactic acid from fermentation broth, bioresource technology, 112. 280-284, (2012). [12]. choudhury, b., swaminathan, t., lactic acid extraction with trioctyl amine, bioprocess engineering, 19. 317-320, (1998). [13]. oliveira, f.s., araújo, j.m.m., ferreira, r., rebelo, l.p.n., marrucho, i.m., extraction of l-lactic, lmalic, and succinic acids using phosphoniumbased ionic liquids, separation and purification technology, 85. 137-146, (2012). [14]. bouchoux, a., roux-de balmann, h., lutin, f., investigation of nanofiltration as a purification step for lactic acid production processes based on conventional and bipolar electrodialysis operations, separation and purification technology, 52. 266-273, (2006). [15]. sikder, j., chakraborty, s., pal, p., drioli, e., bhattacharjee, c., purification of lactic acid from microfiltrate fermentation broth by cross-flow nanofiltration, biochemical engineering journal, 69. 130-137, (2012). [16]. khunnonkwao, p., boontawan, p., haltrich, d., maischberger, t., boontawan, a., purification of l-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification, process biochemistry, [17]. liew, m.k.h., tanaka, s., morita, m., separation and purification of lactic acid: fundamental studies on the reverse osmosis down-stream process, desalination, 101. 269277, (1995). [18]. thang, v.h., novalin, s., green biorefinery: separation of lactic acid from grass silage juice by chromatography using neutral polymeric resin, bioresource technology, 99. 4368-4379, (2008). [19]. madzingaidzo, l., danner, h., braun, r., process development and optimisation of lactic acid purification using electrodialysis, journal of biotechnology, 96. 223-239, (2002). [20]. thang, v.h., koschuh, w., kulbe, k.d., novalin, s., detailed investigation of an electrodialytic process during the separation of lactic acid from a complex mixture, journal of membrane science, 249. 173-182, (2005). [21]. wee, y.-j., yun, j.-s., lee, y.y., zeng, a.-p., ryu, h.-w., recovery of lactic acid by repeated batch electrodialysis and lactic acid production using electrodialysis wastewater, journal of bioscience and bioengineering, 99. 104-108, (2005). [22]. wu, j., hu, y., zhou, j., qian, w., lin, x., chen, y., chen, x., xie, j., bai, j., ying, h., separation of d-lactic acid from aqueous solutions based on the adsorption technology, colloids and surfaces a: physicochemical and engineering aspects, 407. 29-37, (2012). [23]. lee, c.-h., lactic acid fermented foods and their benefits in asia, food control, 8. 259269, (1997). [24]. león peláez, a.m., serna cataño, c.a., quintero yepes, e.a., gamba food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 58 villarroel, r.r., de antoni, g.l., giannuzzi, l., inhibitory activity of lactic and acetic acid on aspergillus flavus growth for food preservation, food control, 24. 177183, (2012). [25]. jung, y.k., lee, s.y., efficient production of polylactic acid and its copolymers by metabolically engineered escherichia coli, journal of biotechnology, 151. 94-101, (2011). [26]. palumbo, f.s., pitarresi, g., mandracchia, d., tripodo, g., giammona, g., new graft copolymers of hyaluronic acid and polylactic acid: synthesis and characterization, carbohydrate polymers, 66. 379-385, (2006). [27]. fang, q., hanna, m.a., rheological properties of amorphous and semicrystalline polylactic acid polymers, industrial crops and products, 10. 47-53, (1999). [28]. ersoy, e., ozturk, v., yazgan, a., ozdogan, m., gundogdu, h., effect of polylactic acid film barrier on intraabdominal adhesion formation, journal of surgical research, 147. 148-152, (2008). [29]. krause, h.j., schwarz, a., rohdewald, p., polylactic acid nanoparticles, a colloidal drug delivery system for lipophilic drugs, international journal of pharmaceutics, 27. 145-155, (1985). [30]. robert, p., mauduit, j., frank, r.m., vert, m., biocompatibility and resorbability of a polylactic acid membrane for periodontal guided tissue regeneration, biomaterials, 14. 353-358, (1993). [31]. simion, a.i., dobrovici, p., grigoraş, c.g., rusu, l., modeling of the thermophysical properties of aqueous sucrose solutions i. density and dynamic and kinematic viscosity, annals. food science and technology, 12. 225-232, (2011). [32]. imion, a.i., dobrovici, p., rusu, l., gavrilă, l., modeling of the thermophysical properties of grapes juice iii. viscosity and heat capacity, scientific study & research chemistry & chemical engineering, biotechnology, food industry, xii. 409-420, (2011). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 68 fish pollution with heavy metals *camelia popa1, petru bulai2 1 tefan cel mare university of suceava, faculty of food engineering, camelia.popa@ fia.usv.ro 2stefan cel mare university of suceava, faculty of mechanical engineering, mechatronics and management, department of technology and management, 13 universitatii street, 720229 suceava, romania bulaipetru@yahoo.com *corresponding author received 15 july 2012, accepted 20 august 2012 abstract: the aim of this work is to identify and to find the concentration of some heavy metals (cr, ni, fe, hg, pb) of different assortment of fish that we buy from markets: herring, sprat, hake, pangasius, trout, broad snout, and mackerel. heavy metal concentrations were measured using edx and uv-vis spectrometer techniques. laboratory studies showed that these fishes don’t contain ni, hg, pb. they contain only cr and fe but the concentration don’t’ exceed the maximum admissible limit from current legislation. also, heavy metals content in bone is higher than in meat. hake species do not accumulate heavy metals in meat, but they accumulate in the bone, completely. keywords: heavy metals, fish, contaminants 1. introduction in heavy metal group there are a range of chemical elements that have a great toxicity for living organisms. the toxic effect is manifested to overcome a certain threshold below which some (co, cu, fe, ni, zn) may even be essential components of proteins involved in different metabolic pathways. hevy metals are normally found in environment and may come from different pollutants, like nutrients or burning combustible waste gases [1]. heavy metal content is monitored in environment and in foods. toxicity of heavy metals is the rezult of their binding to important enzymatic systems from animal cell or their membrane [2]. heavy metals discharged into the environment eventually reach the living bodies which in return behave as a filter for them, meaning that they accumulate [3]. thus, if the food would be totally free of metal it would appear nutritional deficiencies. heavy metals are found in various concentrations in soil, water, air, vegetable or animal food, depending on various factors that determine their pollution. the good reputation of fish as healthy food has distant origins, ancient. we know that fish is good food for humans. it has beneficial properties to the development of intellect, because its constituents rich in phosphorus and lecithin which is derived from fish oil. in fish culture we meet growth hormones, antibiotics, pesticides. but the biggest concern that exists is in connection with these contaminants, which accumulate along the food chain. fishes cannot escape from the effects of these pollutants [4]. contamination with a wide range of pollutants and heavy metals in particular has become a serious problem that has threatened this important source of food: fish [5]. heavy metals enter in living organisms by ingestion, breathing and skyn contact and produces intoxications by mailto:bulaipetru:@yahoo.com food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 69 cumulative effect. they deposite in organisms in various organs, muscles and bones. depending on environmental contamination, fish can contain small or large quantities of heavy metals. accumulation depends on the species, fish age, sex, weight and its position in the food chain: an old fish will contain a larger amount of heavy metals than the yanger one grown in the same conditions. a predator fish will be contaminated in ,,pyramid” [6,7]. the aim of this work is to identify and to find the concentration of some heavy metals (cr, ni, fe, hg, pb) of different assortment of fish that we buy from markets: herring, sprat, hake, pangasius, trout, broad snout, and mackerel. 2. materials and methods materials. different fish species from markets: herring, sprat, hake, pangasius, trout, and broad snout. were performed in parallel three samples of meat and fish bones from the same type of fish and made the average of determinations. sampling. for determinations were weighted on analytical balance about 5 g of each meat sample and 2 g of each bones samples. samples were dried in porcelain crucible previously dried to constant weight at 1050c. drying and calcination was made gradually on temperature ramp, 3 hours at 6000c. after calcination samples were cooled in desiccator. to solve the metals from ash was used hno3 5% at boiling. residue was filtered on watman filter, washed with distilled water and completed to 25 ml (solution 1). determination of iron. iron were determined according to sr iso 805:1995 by spectrophotometric method [8]. principle method: fe3+ ions was reduced to fe2+ with hydroxyl amine chlorohydrin in sodium acetate made with 1,10 phenanthroline, a complex that may be measured at 510 nm. reagents and instruments: hcl for analysis, acetic acid-sodium acetate buffer for 4,4 ph, 5 g/l hydroxyl amine chlorohydrin solution, 2,5g/l 1,10 phenanthrolin solution, 10 mg/l fe2+ standard solution, uv-vis spectrophotometer uvi 1700 schimadzu model, 1 cm quartz cells. calibration curve: was draw calibration curve for 0,1-0,8 mg/l fe2+ field using feso4 7h2o certified reference material, figure1. y = 0,2001x + 0,0857 r2 = 0,9904 0 0,05 0,1 0,15 0,2 0,25 0,3 0 0,2 0,4 0,6 0,8 1 mg/l e figure 1. fe2+ calibration curve handling: a well defined part of solution 1 was transferred into 100ml volumetric flask, add 25ml buffer solution, 5ml of hydroxyl amine chlorohydrin solution and 5 ml 1,10 phenanthrolin solution and distilled water to sign. blank sample was prepared in the same way using the same volumes of reagents. the red complex was measured at 510 nm. results calculation: 100(%) d m c c p x (1) were: cx = fe2+ concentration from calibration curve; mp = sample weight (g); d = sample dilution. the report of the components from sample was found using edx technique, normalization method. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 70 3. results and discussion among of different metals analysed are classified as chemical hazards and maximum residual levels have been prescribed for human (ec 2001, fao 1983). after edx analysis of heavy metals from fish for 5 chemical elements (cr, ni, fe, hg, pb ) were identified and measured the concentrations for two elements , fe and cr, the other three elements being in too small concentrations, below the detection limit of the method. table 1 shows the results obtained for cr and fe. edx technique shows the ratio of the components in the sample. uv-vis technique shows the real percent of the component in the sample. knowing the ratio of components in edx analysis and the real concentration of iron by spectrophotometric technique was calculated the real concentration of cr. )real(crc )real(fec )edx(crc )edx(fec (2) the concentrations of heavy metals detected in the samples are given in the table 2. from this table results the following conclusions: the types of fish studied contain iron and chromium in quantifiable concentrations. the high content in heavy metals studied is contained by broad snout meat and the lowest, zero, it has meat hake. the high chromium content is found in broad snout and trout (meat). the high iron content presents sprat and broad snout (meat). heavy metals content in bone is higher than in meat. hake species do not accumulate heavy metals in meat, but they accumulate in the bone, completely. the concentrations of heavy metals studied were lower than maximum admissible levels [9,10]: cr = 12 µg/g, ni = 70 µg/g, pb = 0.3 µg/kg, hg = 1 µg/kg. table 1. calculation of fe and cr concentrations species of fish fe(µg/kg) cr (µg/kg) mackerel 0.75 0.40 sprat 1.02 0.40 hake 0 0 pangasius 0.37 0.19 trout 0.93 0.63 broad snout 1.21 0.64 trout bone 1.06 0.56 hake bone 2.86 1.71 mackerel bone 0 0 0 0,2 0,4 0,6 0,8 1 1,2 1,4 sprat mackerel hake pangasius trout broad snout species c on ce n tr at io n of f e an d c r, µ g /k g fe cr figure 2. fe and cr meat content for the studied types of fishes 4. conclusions water pollution is a major problem that threatens an important source of food: fish meat. from our studies we see that for the studied species were identified only cr and fe, the other elements being in concentrations under the detection limit of the analysis. this work also shows that heavy metals content for fish meat decreases in the order broad snout, trout, sprat, herring, hake. the high content of these heavy metals is for broad snout meat and the lowest, zero, is food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 71 for meat hake. the high cr content is found in broad snout and trout (meat). the high fe content presents sprat and broad snout (meat). heavy metals content in bone is higher than in meat and do not exceed the quality rules to date: content in pb maximum 0.3µg/kg, hg maximum 1µg/kg [10] 5. references [1]. cojocaru i., sources, process and pollution produscts, junimea publishing, ia i, (1995) [2]. pouilly m., perez t., rejas d. et al., mercury bioaccumulation patterns in fish from the itenez river basin bolivian amazon, ecotoxicology and environment safety, (83): 8-15, (2012) [3]. wakar a., levels of selected heavy metals in tuna fish, arab j. sci. eng., (31): 89-92, (2004). [4]. olaifa f.g., onwude t.e., lethal and sublethal effects of copper to the african cat fish (clarias gariepnuts), afr. j. biomed. res. (7): 65-70, (2004) [5]. velez d., montoro r., arsenic speciation in manufactured seafood products, j. food. protect. (9): 1240-1245, (1998) [6]. vinodhini r., narayanan m., bioaccumulation of heavy metals in organs of fresh water fish cyprinus carpio, j. environ. sci. tech., (5), 179-182: (2008) [7]. kensova r., celekocska o. et.al., concentration of metals in tissues of fish from the zdenka svobodova, acta vet. brno 79: 335-345, (2010) [8]. sr iso 805:1995, iron content determination. spectrophotometric methode with 1,10-phenantroline. [9]. fao, compilation of legal limits for hazardous substances in fish and fishery products , fao fishery circular 464: 5100, (1983) [10]. fda, fish and fisheries products hazards and controls guidance, centre for food safety and applied nutrition, us food and drug administration, 3-edition, (2001). microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_7-13.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 11 monitoring of contamination of the environment in bukovyna with heavy metals yarema tevtul1, olga nechyporenko1 1 yuriy fedkovych chernivtsi national university, kotsiubinsky st., 2, chernivtsi 58012, ukraine. e-mail: y.tevtul@chnu.edu.ua abstract. the main sources of soils contamination with heavy metals have been analyzed. the results of monitoring agricultural soils contamination with heavy metals and the content of metals in the plant tissues in chernivtsi region have also been registered. the average content of heavy metals in the agricultural soils of chernivtsi region complies with the corresponding background values. an average content of the following heavy metals in a 0-5 cm horizon of the agricultural soils of chernivtsi region was registered (mg/kg): 9,6 (pb), 0,26 (cd), 30,4 (cu), 30,5 (ni); 72,0 (cr); 78,3 (zn); 973 (mn). similar values for the urban soils within chernivtsi were noticed as well: 51,3 (pb), 0,3 (cd), 79,2 (cu), 25,3 (ni); 41,0 (cr); 73,4 (zn); 654 (mn). heavy metals were arranged in a sequence according to their toxicity, which also varies for different organisms and content of free ions. the dependence of the metal ions content in food and the plants growing and cooking conditions has been analyzed. some ways of heavy metals accumulation in plants are described. local areas with higher concentration of heavy metals have been identified near roads and within some gardens. no such areas were found near industrial objects. the content of heavy metals in the soils of prykarpattya and prut-dnister upland area remains below the background level, which ensures secure conditions for agricultural activities. a comparison between the heavy metals content in bukovyna and other regions of ukraine has also been made. keywords: monitoring of soils, accumulation of heavy metals, plants, human body, food chain. contamination of the environment with heavy metals compounds is very dangerous since they can get into the human trophic sequence. the more stages consists the sequence, the higher accumulation ratio of toxic agents can be reached at the end. it is known that every new stage of the sequence results in about ten times higher concentration of the toxic agents. investigation of the environmental contamination with heavy metals is quite a topical issue, which includes the study of pollutants toxic action on organisms, possible accommodation of the organisms for functioning in a polluted environment, the study of various controllable and uncontrollable parameters’ effect on the transformation of pollutants. in this way we can simulate and forecast possible results of eco-catastrophes and massive pollution of the environment and promote better natural self cleaning by using plants, which can absorb pollution agents. chemical compounds of heavy metals cause structural changes in the soils microcenosis. sensitive species are being gradually substituted by more metalresistant ones and rehabilitation of the metal-polluted soils requires fixation of heavy metals compounds in the indelible organomineral complexes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 12 heavy metals can be allocated in the following descending toxicity sequence: mercury; silver; copper; cadmium; zinc; lead; chromium; nickel; cobalt. however, this sequence can be rearranged for different organisms and depending on the nature of the acting compounds (free ions, inorganic or organic compounds) [1]. heavy metals ions oppress the functions of organisms by the ferment systems blocking, strong bonding with the sulfide groups and the cell walls crippling [1]. some microbes normally require ions of cobalt, copper, iron and others, which can be part of bioactive compounds: ferments, vitamins and pigments. these ions are important agents, which ensure normal redox processes, which occur in a cell. some ions are necessary to ensure normal structure and functioning of the microbial ribosome. micromycets, yeasts, some thionic microbes and pathogenic microorganisms are the most metal-resistant. this resistance can be caused by their ability to accumulate and fix the heavy metal ions in the cells. therefore, the intracellular content of heavy metals can become hundreds times higher comparing with the corresponding concentration in the environment. mycobacteria and actinomycetes are less metal-resistant. therefore, it is important to know the possible ways how heavy metals ingress in the human and animal trophic sequence and their concentrations in the soils and environment. in this way we can effectively counteract the metals accumulation. we briefly describe some of these ways below. some compounds of lead were used to prevent detonation in the internal combustion engines for a long time. tetraethyl lead (tel) was the most widely used detonation preventer. the fine dust of lead is formed as a result of tel decomposition inside the engine and then this dust is being released together with other combustion products. long exploitation of tel resulted in significant accumulation of various lead compounds along many roads. car-related emission of lead made about 80 % of the total anthropogenic lead emission. hard cars traffic also results in emission of some other toxic agents: compounds of cadmium, zinc, copper, oxides of nitrogen, carbon (ii), hydrocarbons and aldehydes. cadmium can be emitted as a result of car tires wearing, destruction of asphalt road cover, leakage of engine oils and, slightly, from the old and worn-out cadmium-nickel batteries with destroyed bodies. many compounds of manganese are used in engineering and medicine. the manganese (iv) oxide can be used as doping for some special steels, for discoloration of the glass mass, for production of linoleum, some dyes and lacquers. potassium permanganate can be used in medicine as a disinfectant [2]. ferrous and nonferrous metallurgy, thermoelectric power plants, mineral fertilizers and pesticides production, irrigation and melioration equipment, industrial and municipal wastes and wastewaters can also emit significant amounts of heavy metals compounds. the transport (annual emission over 28000 t), industry (about 600 t), wastewaters discharge and disposal of pesticides are the main sources of environmental pollution with heavy metals in the region of bukovyna [3]. the regional center for soils’ fertility protection and control of the production quality carries out regular control of heavy metals content in the soils of bukovyna. the analysis results have proved that this content complies with current requirements (see table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 13 table 1 total content of heavy metals ions in the agricultural soils of chernivtsi region, mg/kg higher content of lead has been identified for some spots near main roads. higher content of copper has been determined in the soil samples taken from orchards. this can be explained by the influence of plants’ treatment with some pesticides. no local areas with excessive heavy metals pollution resulting from the industrial activity have been identified. the heavy metals content in the soils of prykarpattya region and prut-dnister upland complies with the background values and can not threaten normal quality of the agricultural products [4]. it should be emphasized that the content of manganese in the soils of bukovyna is higher comparing to other metals. this is an important feature because the compounds of manganese are widely used in engineering and medicine and sometimes may have poisoning effect. manganese can often be produced from its ores and minerals: pyrolusite, manganese (iv) oxide. the technology of pyrolusite processing requires its grinding, which produces a lot of fine dust. inhalation of the dust may lead to serious poisoning. the normal content of manganese in the cell tissues is very low [2]. manganese compounds have strong protoplasmatic toxic effect and affect the central nervous system, kidneys, lungs and the blood circulation organs. even excessive throat/mouth washing with potassium permanganate solution may cause edema of vocal cords and mucous coat of mouth and throat. the ingestion of concentrated potassium permanganate solution may result in the stomach wall perforation. the ingress of manganese compounds into the urinary bladder or uterine cavity causes peritonitis [2]. the highest accumulation of manganese compounds in the human body is registered in liver. urine and digestive system are the main ways of the manganese compounds excretion. massive burns of gullet and degenerative changes in the parenchymatous organs have been reported after autopsy of the manganese pollution victims [2]. plants accumulate manganese mostly in their roots. the accumulation coefficient is 62,0÷86,0 % comparing to the content in the soil (see table 2). as seen from table 1, the distribution of manganese among parts of the corn can be represented as follows: stems contain 1,1÷4,5 %, leaves – 6,5÷30,8 %, coats – 2,1÷3,8 % and grains – 0,7÷1,4 %. therefore, we can arrange the parts in the descending line based on the content of manganese: roots>leaves>coats>steams>grains [5]. upper layer (0 ÷5 cm) pb cd cu ni cr zn mn average content in the region 9.6 0.36 30.4 30.5 72.0 78.3 973 range of concentrations within the region 3.0 ÷ 32.0 0.29 ÷ 0.82 12.0 ÷ 40.0 12.0 ÷ 50.0 32.0 ÷ 200.0 20.0 ÷ 100.0 500 ÷ 2000 average content in the city of chernivtsi 51.3 0.3 79.2 25.3 41.0 73.4 654 range of concentrations within chernivtsi 3.0 ÷ 32.0 0.29 ÷ 1.4 5.9 ÷ 40.0 12.0 ÷ 50.0 32.0 ÷ 200.0 3.6 ÷ 100.0 500 ÷ 2000 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 14 table 2 content of manganese in different parts of corn and for growing at different distances from the roads content of mn2+, g/g (for the dry weight) distance from the road, m soil root stems leaves coats grains >5 360 144 8 21 5 2 25 375 165 6 12 4 1 50 345 139 3 54 5 1 100 300 116 3 54 5 2 150 285 175 2 58 5 1 200 225 120 2 59 7 2 plants require manganese to ensure normal processes of growing and cell breathing. this element also plays an important role in photosynthesis. the ion of manganese works in many ferments such as in oxydase. it acts as a reducing agent in the process of nitrate feeding and as a strong oxidizer in the process of ammonium feeding. manganese toxicity has been reported for the plants, which grow on acid soils [6]. high concentrations of manganese affect the normal development of roots and decrease the productivity of synthesis of chlorophyll. high concentration of starch, phenolic compounds and nitrates can worsen nitrogen metabolism efficiency for the manganese-affected plants. incorrect management with dangerous industrial waste materials can bring another potential threat for plants’ growth and soils’ quality. 225,7 t of such dangerous materials have been formed at industrial units in the region of chernivtsi in 2007 [7]. this is more than it was formed in 2006 (180,4 t). there were 40,1 t or 17,8 % of the first risk class materials (threshold limit concentration – 0,1 mg/m3); 81,6 t or 36,1 % of the second class materials (0,1÷1 mg/m3) and 104 t or 46,1 % of the third class materials (1,1÷10 mg/m3) among all the wastes collected in 2007. as seen from this distribution, an amount of the most environmentally dangerous wastes was the lowest. the wastes were mainly formed in the city of chernivtsi, then in the districts of novoseytsya (12,3 t) and khotyn (11,5 t). the total amount of the most environmentally dangerous wastes is gradually decreasing. this amount was 74,2 t at the beginning of 2008 or 22,1 % lower compared to the beginning of 2007. only 17 t or 23 % of this amount is classified as the first risk class materials. again, most part (69,9 %) of the waste materials are collected in the city of chernivtsi, then in novodnistrovsk (9,5 t) and in the district of putyla (9,2 t). novodnistrovsk is an area of construction of the dnister hydropower and energy accumulation plant and the local wastes resulted from this activity. the district of putyla is located in mountain area. local anthropogenic activity mostly deals with the primary wood processing. therefore, the wastes mainly consist of wood chips and other wood processing byproducts, which can not bring any serious environment threat. all environmental dangerous materials have been removed from the storage facilities in the agricultural districts (vyzhnytsya, hertsa, kelmentsi) of the region in 2008. the decrease in amount of the environmental dangerous wastes of i-iii classes collected in various industrial storages is shown in fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 15 figure 1. changes in amount of the i-iii classes wastes collected in the region of chernivtsi in 2000 – 2007. there is a significant part of metal compounds among the waste materials collected in the chernivtsi region. the total amount of metals is estimated at 17,6 t and lead and chromium compounds constitute 86,2 % of all metal wastes. lead is present mainly in old car and other batteries. there are also 2,2 tons of old luminescent lamps, which contain mercury. such dangerous wastes can be utilized only outside the region. the changes in the amount of the most dangerous 1 class wastes are shown in fig.2. the amount of stored prohibited and old pesticides and other agricultural chemicals is continuously decreasing. in the year 2008 this amount became 18,2 times lower than in 2007. there are only 1,4 tons of these chemicals remaining in the region. figure 2. changes in amount of the 1 class wastes during 2000÷2007 years. many various factors such as growing conditions, methods of processing can influence the content of heavy metals in food. on the other hand, different plants can uptake different amounts of heavy metals from the environment. the redistribution of heavy metals among different plant organs can also influence their final content in the tissues. some cultivated plants can be placed into the following sequence according to the increasing ability to accumulate heavy metals: beans < oats and rye < barley < corn << cabbage < melons < potato < radish < onion < carrot < red beat << squash < cucumber < tomato << parsley < dill < onion leaves < spinach < lettuce [8]. there are two ways of heavy metals’ salts ingression into plants: apoplasmatic and symplasmatic. this leads in general to the tendency of decreasing of heavy metals content in the row: root > stem > leaves > seeds [8]. the size of vegetables is an important factor influencing the content of heavy metals. the larger is the vegetable, the higher is heavy metals accumulation value [8]. biocleaning of soils by planting some agricultural species is very interesting and an important field. for example, corn can actively accumulate heavy metals [5] and this plant can facilitate in the soils biocleaning. lead is mainly accumulating in roots and this tissue can accumulate up to 63,0-81,0 % of the total lead in the plant. the lead content in stem is much lower (3,8-10,1 %), in the leaves – 4,8-19,0 % and in the coats – 6,2-13,2 %. it is needful to emphasize that lead can ingress into leaves and coats mainly from the air. only traces of lead were found in the corn grains. an average concentration of lead in the corn leaves was 4,9 mg/kg [5]. this is lower than the threshold limit value for the cattle feed (5 mg/kg). contamination with lead is irreversible, i.e. this pollutant is gradually accumulating in the surface soil layer even at very low and gradual ingress. the dependence of lead food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 16 contamination on the distance from roads is nonlinear. roadside forest shelter belts can capture some amount of the metal and prevent its spreading in the nearest roadside area. on the other hand, air fluxes can spread lead-containing dust over the belts at a longer range, which causes the highest content of lead in the soils at about 100 m from roads [9]. a content of lead in 100 m from the road was found 2,2 times exceeding its limit threshold value, in 50 m – 1,2 times higher. related exceeding over the background content were 26,8 and 14,4 times. similar spatial distribution has also been determined for cadmium. its content in 100 m from the road was 1,5 mg/kg or 15 times over its limit threshold value [9]. the high content of cadmium has also been determined in the corn roots. its concentration was several times higher than in the soil. the root barrier prevents further distribution of the metal towards the vegetation and reproductive organs. that is why its content in the stem and leaves is much lower and in the grains it is only at the trace level [10]. the accumulation of copper in the plants tissues is about 34,0-42,0 of the concentration in the soil. the stem of corn accumulates 8,4-13,5 % of the total copper accumulated in the plant; leaves – 20,328,6 %; coats – 12,7-21,1 % and grains – 9,9-18,4 % [5]. copper is less labile element as compared to other ones. the most part of copper stays in the root and leave tissues until they die off and only very small part of the metal can get into new tissues. copper is a vitally needful microelement. unlikely to lead and cadmium it is required for normal plants’ growth. on the other hand, the exceeding concentrations of copper cause hard intoxications which result in chlorosis and defects of the root system. it is hard to foresee, which concentration of copper can cause toxic effect on plants. it should be remembered that the food manufactured from metals-contaminated plants can become dangerous even before having affected plants by poisoning and drop in the crop yield should be noticed. natural accumulation of heavy metals in some plants can be used for the phytorehabilitation of the contaminated soils [11, 12]. this problem has been extensively investigated by galiulin et al [11], who proposed the use of chelate compounds of etylendiamintetraacetate (edta). chen [13] proposed to use citic acid. kravets [14] reported higher mobility and accumulation ratio of the mineral pollutants for the plants exposed to the non-specific stresses (presowing irradiation, deviations from the optimal growing conditions; too dense sowing). this effect can be useful for more intense phyto-rehabilitation. biomass of the phyto-rehabilitation plants can be utilized for the bio-fuel production [15]. some non-ferrous metals can even be cost-effectively extracted from this biomass [16]. it is recommended to keep growing the food vegetables not closer than 150-200 m from the roads. highly accumulating plants are not recommended for growth on metals-contaminated soils. regional educational programs should promote more active testing of the metals content in some domestic cultivated plants in specialized laboratories. special legislation regulations should prevent the growth of food plants at dangerous distances from roads while the increase of the ecologically clean products should be encouraged and stimulated by various means. ecolabeling standards should be widely introduced. in this way a potential consumer would be able to see more adequate picture of the foods ecosafety and can make a wellgrounded decision of buying needful foods. special “green” shops, which work only with ecologically safe and clean products, can be recommended in a more distinct prospect. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 17 references 1. kushkevych i., gnatush s., gudz s. influence of heavy metals on microbial cells. bulletin of lviv university. biology. (2007) vol. 45, p. 3-28. 2. kramarenko v. toxicological chemistry. (1989). vyscha schkola, kyiv. 447 p. 3. agrochemical services of bukovyna. development and current fertility of the soils. (2004). chernivtsi. 75 p. 4. dmytruk yu. m. ecological and geochemical analysis of the soils in agroecosystems. (2006). ruta. chernivtsi. 328 p. 5. golovach o., kozlovsky v., demkiv o. pollution of the agricultural soils with heavy metals and their redistribution in the corn. bulletin of lviv university. biology. (2004). vol. 38. p. 205-211. 6. kabata-pendias a., pendias a. microelements in the soils and plants. (1989) mir. moscow. 439 p. 7. environment of bukovyna-2007. statistical volume. (2008). 144 p. 8. dubinina a., malyuk l., selyutyna g. toxic compounds in foods and methods of their determination. (2007). professional. kiev. 384 p. 9. gladkih e., details of accumulation of heavy metals in the soils depending on their allocation. bulletin of lviv university. biology. (2004). vol. 38. p. 205-211. 10. strand v., zolotareva b, lisovsky a. dependence of adding the water soluble salts of lead, cadmium and copper on on their ingress and crop yields for some agricultural plants. agrochemistry. (1991) p. 76-83. 11. galiulin r, galiulina r. phytoextraction of heavy metals from contaminated soils. agrochemistry (2003) p. 77-85 12. golovach o, demkiv o, phytorehabilitation – an effective method of cleaning heavy metals-contaminated soils. bulletin of lvivveterinary academy. (2003). vol. 5. p. 2127. 13. chen y. x., lin q., luo y. m. et al. the role of citric acid on phytoremediation of heavymetal contaminated soil. chemosphere. (2003). vol. 50. – p. 807-811. 14. kravets o., current conditions and problems of phytocleaning the soils from radionuclides and heavy metals. physiology and biochemistry of cultivated plants. (2002). vol. 34. p. 377-386. 15. shved o. ecological biotechnology. lvivska polytechnica. (2010). 424 p. lipski s. ekopaliwo z kukurydzy. kukurydza. (2002). vol. 2, p. 25-27 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 47 en vi ronm en ta l sa fety ana lys is of the proj ec ted munici pal land fi ll a r ea in a s mall city in uk ra ine alla choban1, *igor winkler1, myron rogozynskyi 2 1yu. fedkovych national university of chernivts, ukraine, igorw@ukrpost.ua 2national technical university “khpi”, chernivtsi faculty, ukraine, rmyron@ukr.net *corresponding author received 25 august 2012, accepted 2 september 2012 abstract: the influence of a relatively small dump area on the environment is analyzed on an example of a new project of the landfill in storozhinets, ukraine. the results of the analysis prove that the landfill is not expected to cause excessive contamination of the environment. however, some additional measures can lower a predicted level of contamination and allow better collection and utilization of the landfill biogas. efficiency of these measures is also discussed and simulation of the expected emission of the environment pollution agents is shown. keywords: solid municipal waste; utilization and reuse; landfill areas; environmental safety; small communities 1. introduction environment-friendly and rational reuse, utilization and/or disposal of the waste materials is an acute problem in many countries worldwide. solid municipal waste (smw) is a relatively small part of the total amount of waste materials formed by mankind but effective smw management is often problematic because of a wide variety of the components found in smw, which complicates their effective processing. land areas required for new dumps are often deficient or even unavailable. as a result, this problem is quite persistent and hard-to-resolve in many communities and countries [1-3]. daily formation of smw in the developed countries is 1-3 kg per capita, which makes 300-1000 kg of the wastes per year. complete reuse/utilization of smw is either impossible or unprofitable causing ~10 % growth in the total volume of the waste materials collected in the landfills in the usa annually [4]. the content of the harmful and environmentally dangerous components in smw is also growing. these and other related problems cause active development and implementation of new waste management technologies involving waste sorting, burning, composting and others. for instance, many of these solutions have been put into practice in france, where numerous wasteburning factories operate even in small communities. the majority of waste materials undergo preliminary sorting and a lot of waste composting facilities are engaged in the process of smw utilization, which produce vineyard fertilizers or raw materials for the biogas. deep level of reuse/recycling of smw is achieved in japan, germany and some other eu members. on the other hand, 2/3 of smw are still disposed at the landfills [5]. it should be understood, that this way of wastes disposal is a ‘timebomb’ since landfills mailto:igorw:@ukrpost.ua mailto:rmyron:@ukr.net food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 48 extend constant and growing negative influence on the environment. waste materials cause contamination of the soils and groundwater, emit noxious smell and promote growth of population of rodent and other vermin species while often selfignitions result in massive air contamination release. as a result, the waste-related legislation of many countries is a subject of thorough attention and legal requirements in this area are becoming more and more severe. establishing new landfill areas in many countries is quite complicated or even illegal at the moment. this paper deals with analysis of the current state of art in the field of operation of the landfill areas in small communities in ukraine. this issue is often shadowed by more acute problems of operation and expansion of the landfills related to big cities. that is why environmental influence of small landfills remains beyond attention, although these areas also contribute into environmental pollution processes. 2. general description of the landfill area and analysis of its environmental influence storozhynets is a typical small city (population 14500) located in the western part of ukraine (see fig. 1). local waste materials are currently disposed to an old landfill area in the city outskirt. this area has been put into regular exploitation in the 90s and has neither water proof perimeter nor any stormwater filtration equipment. as a result, it causes serious contamination of the groundwater and local authorities have made a decision to develop a new landfill and put it into regular operation. figure 1. location of storozhynets (arrowed and encircled) on the map of the western ukraine. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 49 the new landfill is expected to substitute the old one and to decrease the negative environmental impact it causes. in this paper we report results of the general analysis of the influence of the projected landfill on the local environment. the new landfill (nl) is projected to collect, thicken and store the solid municipal wastes. no reuse or another utilization of the wastes is planned. nl is supposed to collect the municipal, commercial and some industrial or construction waste materials without any toxic or radioactive agents. no chemically or epidemically unsafe materials can be disposed at the landfill. total land area of the landfill is 10 ha and projected capacity of nl is 97389 m3 of thickened wastes, which is equivalent to 263000 m3 of raw materials. with average 14.5 thousand m3 of the wastes collected in the city per year this landfill is expected to stay in service for at least 18-20 years. two trenches are projected as places for the wastes disposal. both trenches should be dug in the loamy soils with filtration coefficient 1.6 • 10-5 sm/s. projected thickness of the waste layers is under 0.3 m. technically, collection of the wastes can be performed through either “pushing in” or “pushing off” methods. the first method assumes formation of 12-20 thickened waste layers from bottom to top. a slightly sloping bank formed therefore can be up to 2 m higher than the dust-cart discharge level and then new waste collecting bank should be started nearby by pushing in new materials over the old ones. this method ensures effective natural thickening of the wastes by their own weight. the “push off” method assumes that the dust-cart discharges at the upper area with further pushing of waste materials by bulldozer down the slope. its height difference should be kept under 2.3 m. the upper edge of the slope gradually moves horizontally with every new portion of the wastes discharged until the working area limit is reached. then the newly formed waste band should be covered with at least 0.25 m of soil and the next working band can be started along the previous one. construction wastes, slag or thickened snow can be used as a temporal cover in the winter if the soil is frozen and can not be collected for the regular cover. however, no new band can be laid over this temporal cover until it is substituted with normal soil. this way the complete area of nl will be gradually filled with wastes until the total area is covered with new soil and then reclaimed agriculturally. natural anaerobic biodegradation is expected as the main way of the wastes decomposition. this process will result in formation of some amounts of gases, filtrate and insignificant heat emission. 3. environment protection measures and the efficiency analysis a special filtrate-collecting pit is projected to keep the landfill filtrate inside the working area while stormwater around it should be captured in a by-pass channel and then discharged to a nearby pond. another 2.5 m high ground bank is planned along the channel as an additional environment protection line. it is necessary to provide strict monitoring of the groundwater quality near such an environmentally dangerous object. a groundwater control well will be established 100 m away from the landfill. the well should be drilled down to 16 m as it corresponds to the groundwater depth in the area, which is 14-18 m. the landfill area is bordered on east, west and north by bushes/woodland and on south – by a small stream. protective area around such objects extends for 1 km (this is also a distance to the nearest settlement). an influence of the newly planned landfill area can be analyzed by taking into account the technology of the wastes food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 50 treatment, the location and the planning of the new area. in table 1 we summarize all groups of environmental risks related to this object. table 1 estimation of possible environmental risks related to the projected landfill area threatened component expected environmental risk expected area under threat air emission of h4, nh3, h2s, co2 into the nearsurface atmosphere within protective area (1 km) water contamination of groundwater with filtrate; pollution of the pond with the surface run-offs from the landfill within the landfill perimeter pond soil contamination of the landfill bedding soils with filtrate within the landfill perimeter forest cuttings within the new woodland (required because of fire safety) a 50 m wide area along one side of the landfill. table 2 expected emission of the air pollution agents from the landfill contamination agent expected emission g/s kg/hour t/year ch4 22.0 76.2 681.564 h2s 0.9 3.24 28.398 nh3 0.4 1.44 11.359 data of table 1 prove that the projected area will cause additional contamination of all components of the environment. biogas (50-65 % methane, 30-45 % co2, small amounts of hydrogen sulfide, mercaptans, ammonia, aldehydes, amines, etc.) is expected to become the most massive pollution agent. average emission of biogas is estimated for one ton of wastes according to [5] as 60-180 m3/ton. the results of assessment of the atmosphere contamination from the projected landfill are shown in table 2. calculated emissions of the contamination agents are insufficient to cause excessive air pollution in the area and no excessive contents of the pollution agents are expected outside the landfill sanitary protection perimeter. however, some extra measures can be taken to lower emission of the biogas. in this context some preliminary thickening of the wastes layers can result in reduced emission of the biogas. other measures are planned to protect ground waters from contamination with filtrate. some predicted parameters of the filtrate are following: bod5 – up to 4300 mg/l; cod – up to 51000 mg/l; content of detergents – up to 50 mg/l [5]. so this substance is extremely contaminated and environmentally dangerous. an average annual amount of the filtrate is estimated as ~ 117 m3/year. a special filtration/drainage system with a 10 m3 filtrate collecting pit is planned to collect this substance and protect the local groundwater. the filtrate should be collected and then pumped out for decontamination once a month. an additional anti-filtration clay screen should food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 51 also be constructed for extra protection against filtrate leakage. it seems that the landfill project has been designed according to rather outdated environment protection requirements of the 80’s-90’s [6]. some new materials, which were quite rare in 80’s-90’s but can be found in modern wastes (new polymer materials and packaging, massive amounts of carton boxes and packaging, new materials for the detergents selling, personal care materials etc.) were not taken into consideration. since no preliminary wastes sorting is currently employed and won’t be used at the new landfill, the specific influence of these wastes components becomes more important. moreover, since realization of the project has not been started yet, it can be updated and a waste sorting stage can be added to the technological scheme of waste collecting and processing. this stage would allow to separate and retrieve some secondary resources (paper, plastics, metals, glass), which can result in the decrease of the total amount of the waste materials and pay back some expenses for wastes collecting and processing. it should also be emphasized that biogas formation in the low-polymer containing waste is more effective. another remark is aimed onto possible utilization of biogas. there is a wellknown technology of establishing the vertical drillings network to collect the waste-originating biogas at landfills. then it can be burnt in electricity generators or used for heating. no surface water decontamination equipment is planned at the new landfill and this is another shortcoming of the project. as we mentioned above, new components are present in the municipal wastes today and this can cause significant chemical contamination of the stormwater run-offs which will be collected and then discharged into a local stream without any treatment. we propose to establish an additional settler and the sand-gravel filter to ensure at least some primary decontamination of the stormwater runoffs. 4. conclusion a modification of the landfill project, planning of waste sorting and grinding stage, construction of the biogas collection and utilization system can be proposed. installation of the stormwater cleaning equipment should also be considered. these steps can ensure better environmental safety of the landfill and addressing today’s wastes management trends. 5. references [1]. mcdougall f. r., white p. r, franke m. and hindle p. integrated solid waste management: a life cycle inventory, blackwell publishing company, oxford, uk. doi: 10.1002/9780470999677, p. 33-85, (2007). [2]. troschinetz a. m., mihelcic j. r. sustainable recycling of municipal solid waste in developing countries, waste management. 29, 915-923, (2009). [3]. williams p. t. waste treatment and disposal, john wiley & sons, ltd, chichester, uk. doi: 10.1002/0470012668, 375 p, (2005). [4]. reinhart d. r. and al-yousufi a. b. the impact of leachate recirculation on municipal solid waste landfill operating characteristics, waste manag. res. 14, 337346, doi: 10.1177/0734242x9601400402, (1996). [5]. korte f. (ed). environmental chemistry, mir, moscow. 396 p, (1996). [6]. stolberg f. (ed). municipal ecology, kharkiv, 560 p, (2000). microsoft word 2 cuprins nr 1_2011 23 martie.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year x, no1 2011 contents: 1. isabel guillén guillén, ángel martínez sanmartin, josé antonio gabaldón hernández, estrella núñez-delicado, rosa puchades pla, ángel maquieira catalá development of lateral flow immunoassay for rapid detection of oxytetracycline in honey samples 5 2. igor kobasа, vasyl bilogolovka, аnatoliy woloschuk atom-absorption determination of microelement composition of the human hair from different areas of chernivtsi region 13 3. sergey boruk, igor winkler influence of some non-ionic surfactants and flocculants on stability of diluted dispersions of kaolin and hydro-mica 17 4. mihaela constandache, elena condrea, steluţa radu efficacy of plant stanols/sterols in ensuring heart health 24 5. daniela giosanu, loredana elena vîjan, ionica deliu the analyse of physico-chemical parameters means to appreciate the typicity of some red wines 31 6. georgiana gabriela codină, silvia mironeasa, ana leahu predicting the organoleptic quality of some romanian beers from physical-chemical data using multivariate analysis 36 7. adriana dabija, gabriela constantinescu (pop), ioana rebenciuc, amelia buculei studies regarding new way to render whey profitable 43 8. silvia mironeasa, georgiana gabriela codină, ana leahu, costel mironeasa multivariate statistical analysis of royal feteasca wine quality from different regions of romania country 47 9. eufrozina albu (niga), alina-mihaela psibilschi influence of liquefaction temperature upon rheological properties of corn starchy mashes 53 10. feiza memet a thermodynamic analysis of juice pasteurization process 57 11. viorel iftimi life cycle management implementation of food products and services 61 12. ana leahu, georgiana gabriela codină, silvia mironeasa, alice-iuliana roşu effects of a2 phospholipase on dough rheological properties and bread characteristics 66 13. author instructions 71 microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_57-62.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 61 freshness evaluation of chicken meat using microbiota and biogenic amine index. octavian baston1, octavian barna1, aida vasile1 1 dunarea de jos university of galati, faculty of food science and engineering, 111, domeasca st., tel./fax: +40 236 460165, e-mail: octavian.baston@ugal.ro. abstract: the purpose of our study was to evaluate freshness of raw chicken meat using microbial and biogenic amines content. the objectives were to determine the variation of microbiota (total viable count, psychrotrophic and pseudomonas spp.), to study the variation of some important biogenic amines and to calculate a biogenic amine index for refrigerated chicken carcasses aerobically stored for one week. hereby, we used microbial analysis and hplc determination for the following biogenic amines: tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermine and spermidine. our determinations showed that total viable count increased in value from the first to the seventh day (the last day of storage), psychrotrophic microorganisms also increased in number and pseudomonas spp. increased from the first day to the seventh day of storage. regarding biogenic amines variation, tryptamine had a low initial content and after a week of storage the content was below 5mg/kg, β-phenylethylamine had also a small initial value and after a week of storage its value is slightly higher than 5 mg/kg, serotonin had a similar comportment with β-phenylethylamine and tyramine the same with β-phenylethylamine only that the final value was slightly higher. cadaverine and putrescine were detected beginning with the third day of storage and they had the highest values after a week refrigeration of chicken meat. after one week of refrigerated storage, spermine had a particular allure because it decreased permanently. spermidine had a very slow increase, from 4.8 mg/kg to 6 mg/kg. biogenic amines index were calculated according to the mathematical relation proposed by researchers from barcelona university. keywords: meat, freshness, microbiota, pseudomonas, biogenic amine index introduction the spoilage of refrigerated chicken meat when stored for a long period is due to the microorganisms’ action and biochemical transformations inside the product. if the refrigerating chain from producer to consumer is not ensured, or if the seller overpasses shelf life, the consumer can have an unpleasant surprise of buying an altered product. after chicken slaughter, the muscular tissue suffers irreversible physical, chemical and biochemical transformations which determine the muscle to convert into meat. the microbial spoilage processes occur later. microorganisms’ activity is slowed down by using refrigeration temperatures for meat conservation purpose. in order to obtain products with high conservation durability and to increase the refrigeration effect, it is necessary to have as less initial microbial load as possible [1]. initially, chicken meat quality was evaluated by determination of microbiological and sensorial attributes. for the identification of the early signs of meat alteration, some chemical indices were proposed: volatile nitrogen basis, composites resulted after breaking the nucleotides, volatile acidity and the biogenic amine content [2]. the biogenic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 62 amine occurrence is a consequence of the enzymatic decarboxylation of the precursor amino acids because of the microorganisms’ activities. polyamines: spermine and spermidine are natural amines produced by the body. the biogenic amines: putrescine, cadaverine, histamine, tyramine, tryptamine, βphenylethylamine can be formed when storing the chicken meat due to microorganisms’ action. the biogenic amine determination is important not only because of their toxicity but also their potential use as freshness indicators [3]. the occurrence of these amines is dependant on different factors that vary in time. the microbial population influences the profile of biogenic amines. spoilage responsible microorganisms might not have the capacity of amine forming. it is difficult to establish quality limits universally accepted based on the biogenic amine content. the above mentioned reasons are partially justifying the relative dispersal of biogenic amine values for meat, in various researches. from a practical point of view, the relative simplicity and quickness identification and quantification of the biogenic amines (compared to the microbiological measurement) besides the economical advantages (for example the quick test for determining the diamines described by hall et all. [4], are the reasons for using these substances as chemical indices for animal origin product freshness. the purposes of the study are: determination of microbiota variation from chicken meat, concerning total viable counts, psychotrophic microorganisms and pseudomonas spp at refrigerated raw chicken meat storage; evaluation of freshness of the refrigerated chicken meat using biogenic amine index. materials and methods chicken carcasses were purchased from a romanian slaughterhouse. the meat was analyzed after cooling, packaging and transportation from the plant the first day after slaughter. all the carcasses were stored aerobically, without package foil, for 7 days, at the temperature of 4±1°c in the refrigerator type electrolux enb43691s. the carcasses weight varied between 1.2÷1.5 kg. the samples were analyzed the first day when the meat was received, recorded as day 1, then at the 3rd, 5th and 7th day. the dry matter determination was made according to romanian stas 9065/3-73. pieces of raw chicken meat with skin (16 cm2 in area) were aseptically excised from carcasses and each piece was homogenized with 100 ml of saline water (0,8% nacl) by using a homogenizer model bagmixer 400. duplicate 0,1 ml aliquots of suitable dilutions of each skin homogenate were spread on the surface of nutrient agar plates. inoculated plates were incubated in atich 9082 incubator in aerobically condition at 4ºc for 14 days for psychrotrophic microorganisms and at 30ºc for 2 or 3 days for total viable count. pseudomonas were determined on centrimide-fusidin-cephaloridine agar supplemented with sr 103 for the selective isolation of pseudomonas spp. generally, after incubation at 25ºc for 1 or 2 days, and oxidase-positive colonies were enumerated. [5] after that viable colonies were counted using automatical colony counter sc6. the measurement of biogenic amines content using high performance liquid chromatography was performed according to the method proposed by food research institute from helsinki, finland [6]. the method principle is as follows: -bioactive amines are extracted from a homogenized sample with diluted perchloric acid; -an aliquot of the extract is derivated with dansyl chloride reagent; -separation and quantification of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 63 dansylated amines is performed by reversed phase liquid chromatography with ultraviolet detection at 254 nm. all the reagents used were analytic pure, for hplc use. te water used was deionised. the necessary reagents were purchased from the merck and sigmaaldrich companies. installations and equipment used for biogenic amine determination: philips 7768 food processor, homogenization device 7011s, kern 770-60 analytical balance, silent crusherm homogenization device, centrifuge eba 21, filter paper for quick filtering with 55 mm diameter, syringe filters with porosity of 0,45 µm and 13 mm diameter, heidolph reax control agitator, ultrasonic water tank aquawave tm, incubator bmt incucell 55, water deionising system easy pure rodi, filtering assembly with vacuum pump. the device for the hplc determination was a liquid chromatograph model surveyor produced by thermo electron company, configured with detector model pda plus detector, auto-sampler model autosampler plus, pump model lc pump plus and detector uv-vis. chromatography column is type bds hipersyl c18. the biogenic amines quantification: quantitative measurement was performed depending on the internal standard using peaks for each biogenic amine. the 254nm wavelength absorbance was measured and the resulted peaks were integrated with cromquest software. the concentration of each biogenic amine was expressed in mg/kg d.m. (d.m. = dry matter). our determinations refer to the following amines: tryptamine, βphenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermine and spermidine. the statistical analysis of the obtained data was made using microsoft excel features for 10 samples in each of the storage days. each sample was analyzed in triplicates. the results obtained are presented as the mean ± standard deviation (sd). the standard deviation is a measure of the dispersion of outcomes around the mean. the differences among means were determined using the method of the smallest squares and the significance level was p< 0.05. results and discussion after the chicken carcasses were refrigerated for three weeks at 4ºc, we determined the variation in time of total viable count and psychotroph count. in figure no. 1 we present the variation of refrigerated storage of total viable count. the average initial contaminations of the chicken meat were 5.11 log cfu/cm2 (cfu – colony forming unit), it increase at the third day at 5.95 log cfu/cm2 and in the 5th day of storage at 7.1 log cfu/cm2. the shelf life of carcass, as stated by the food manufacturer, was the fifth day of storage. in the 7th day of refrigeration the total viable count were 7.56 log cfu/cm2. this slow increase of microbiota is due to refrigeration temperatures that do not allow a quick spoilage of chicken carcasses and preserving the freshness for longer period of time. psychrotrophic count is characteristic for food that is preserved at refrigeration temperatures. we determined psychrotrophic microbiota after seven days of storage. the variations of microorganisms are presented in figure 2. the initial load were 4.00 log cfu/cm2, increasing permanently to 6.9 log cfu/cm2 for the 7th day of storage at refrigerated temperature. comparing to total viable count, on the first day of storage we have a difference of more than 1 log cfu/cm2, at the expiration date of chicken carcass (the 5th day), the difference was higher than 1.5 log cfu/cm2 for the total viable count. in the 7th day of storage, the difference begins to decrease, being only of 0.6 log cfu/cm2. it appears food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 64 4 5 6 7 8 9 0 2 4 6 8 storage tim e (days ) to ta l v ia bl e co un t (c fu /c m 2) figure 1. total viable count variation for refrigerated chicken meat that after the 5th day of storage, the psychrotrophic microbiota begin to prevail 3 4 5 6 7 8 0 2 4 6 8 storage time (days) p sy ch ro tr op hi c co un t (c fu /c m 2) figure 2. psychrotrophic variation for refrigerated chicken meat pseudomonas spp. is very important microbiota that spoils chicken carcasses during aerobically refrigeration storage. in figure 3 we show the variation of pseudomonas spp. from chicken carcasses until the 7th day of storage at 4ºc. as it can be noticed, the number of pseudomonas increase in time, from 3.8 log cfu/cm2 on the first day of storage to 6.4 log cfu/cm2 on the 7th day. because pseudomonas can be mesophile or psychrotrophic microorganisms, we can compare the values obtained with those of psychrotrophic biota. thus, the initial contamination of carcasses is mostly by pseudomonas spp., the trend kept until the 5th day of storage. on the7th day, besides pseudomonas spp, it appears that another biota become to increase. 3 4 5 6 7 0 2 4 6 8 storage time (days) p se ud om on as s pp . c ou nt (c fu /c m 2) figure 3. pseudomonas spp. variation for refrigerated chicken meat food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 65 biogenic amines can be an indicator of chicken meat freshness. researchers from barcelona university proposed from raw foods a biogenic amine index that has a mathematical relation of: spermidinespermine1 putrescincadaverinehystamine bai    . based on this index it can be determined allergenic risk and freshness of raw foods. our determined values for considered biogenic amines are presented in figure 4. 0 5 10 15 20 25 0 2 4 6 8 storage tim e of carcass e s at 4 grd c (days ) b io ge ni c am in es v al ue s (m g/ kg d .w .) trip fen put cad his ser tir spd spm figure 4. biogenic amines variation for refrigerated chicken meat, were: d.w.-dry weight as it can be seen, in time, the values of mostly biogenic amines increase, with one exception, of spermine which decreases. on the first day of storage, the biogenic amine content is low, spermine having the highest value: 21.5 mg/kg d.w. followed by spermidine, of 4.8 mg/kg d.w. also, putrescine and cadaverine have not been detected on the first day of storage. this is due to the fact that either our method is not sensible enough to detect smaller amounts of those biogenic amines, or they do not exist in chicken meat. tryptamine and tyramine presence is due to their action as hormones, neurotransmitters, and serotonin being one of tryptamine derivative. βphenylethylamine action as neuromodulator and neurotransmitter, its derivatives being also hormones (tyramine is in fact 4-hydroxi-phenylethylamine). spermine and spermidine are two amines involved in cellular metabolism, essential for organism growth and for development and proliferation of cells. spermine can be synthesized from spermidine. histamine is present in small amount (1.8 mg/kg d.w.). the third day of storage of raw chicken carcasses is important to be highlighted as we detected small amounts of putrescine and cadaverine. on the day of shelf life ending cadaverine and putrescine amounts are increasing, cadaverine increase being over four times, putrescine increase being of 7 times, spermine are decreasing. the spermine decrease can be due to its use by the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 66 microorganisms as nitrogen source. histamine has a particular variation because on the fifth day is slowly decreasing, after a slow increase from the first day of chicken meat refrigeration storage. all biogenic amines that we determined could be made as a result of microbiota activity on amino acids chicken carcasses. it is well known that cadaverine and putrescine are two amines with characteristic repugnant smell especially in more advanced stadium of alteration. a slight modified smell was distinguished on seventh day of storage, but we cannot be sure if this was due to cadaverine or putrescine. we calculated the bai for the chicken carcasses and we had the following results: -iab for the first day were 0.068 -iab for the third day were 0.30 -iab for the fifth day were 0.66 -iab for the seventh day were 1.37 compared with the first day we can observe that iab of the third day increased 5 times and iab for the fifth day 10 times. we can also say that fresh raw chicken meat has an iab below 1.00. conclusion an increase in total viable count number leads to spoilage, loss of freshness and shelf life of raw chicken meat. pseudomonas spp. was the prevalent microorganisms that characterized best the chicken meat spoilage and being the principal cause for freshness loss. chicken meat freshness decreases in time and this leads to quality loss and shelf life reduction. chicken freshness is good if iab is below 1.00. references 1.tofan, i., 2005, lantul frigorific al produselor alimentare perisabile (refrigerating chain of the perishable food products), ed. agir, bucuresti, p. 9, 20, 197-198. 2.halasz, a. barath, a. simon-sarkadi, l. holzapfel, w., 1994, bas and their production by microrganisms in food, trends of food sci. technol. 5, 42-49. 3.balamatsia, c.c. paleologos, ek. kontominas, mg. savvaidis, in., 2006, correlation between microbial flora, sensory changes and biogenic amines formation in fresh chicken meat stored aerobically or under modified atmosphere packaging at 4c: possible role of biogenic amines as spoilage indicators, antonie van leeuwenhoek, springerlink, 89, 9-17. 4.hall, m. sykes, pa. fairclough, dl. lucchese, lj., 1999, a test strip for diamines in tuna, journal aoac international, 82, 5, 11021108. 5.mead, g.c. and adams, b.w., 1977, a selective medium for the rapid isolation of pseudomonads associated with poultry meat spoilage, br. poult. sci., 18:661-670. 6.eerola, s., hinkkanen, r., lindorfs, e. and hirvi, t., 1993, liquid chromatographic determination of biogenic amines in dry sausages, journal aoacinternational, vol 76, nr. 3, p. 575577. microsoft word 3 journal nr 4 2010 din 8 noiembrie_121-126.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 125 grinding process of the wheat kernel with a new designed micromill cristina danciu1, ioan danciu1 1the faculty of agricultural sciences, food industry and environmental protection lucian blaga university , 5-7 ion ratiu str, sibiu, romania cristina.danciu@ulbsibiu.ro abstract: wheat is one of the most important cereal crops in the world with various end-uses: food (bread, cakes, cookies and pasta), animal feed, raw material for beer and whisky, biodegradable plastic (from wheat starch). in all these technologies, grinding has great importance and is very energy consuming. the motivation to measure energy requirements for size reduction at specified roller mill settings, led to the development of instrumented roller mills of various designs. for this research, was used a new designed micromill which can perform in the grinding process of the wheat and of the middling too, in the same conditions as in the milling industry. the adjustment of the roller characteristics can be done for each type of milling product (grain, semolina, bran). the grains are in the same time under the compression and the shearing efforts. the energy consumption is represented by one single value for one pair of rollers. this single value is significant for the comparative appreciation regarding the energy consumption in the milling proces, for different wheat cultivars or different batches, but also for different characteristics of the rollers. the micromill is designed to determine the grinding resistance of the cereals.the method has the same accuracy as the classical one and it has the advantage to be quicker and less demanding as work volume. the new designed equipment can be used in laboratory for the benefice of students as well in the milling industry. keywords: conditioning, grinding resistance, energy consumption. introduction milling wheat into flour for human consumption is a still growing up industry, represented by highly efficient processes at low economic margins. nowadays, the major issue is how to process a variable feedstock to produce a high quality product with low energy. the grinding process is the most important step in the milling system. in the grinding operation, energy is needed to break apart the bran and endosperm and to reduce the endosperm to flour. this uses about 50 % of the power connected with the milling system and results in heat generation and moisture loss in the ground material [1]. wheat milling is an energy-intensive industry because it is a wet process that produces dry products. significant amounts of energy are required to power the large motors for grinding process. opportunities exist within wheat milling plants to improve energy efficiency while maintaining or enhancing productivity. the grinding energy depends both on the properties of the grinding material and on the used machines and their work parameters. the motivation to measure energy requirements for size reduction at specified roller mill settings led to the development of instrumented roller mills food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 126 of various designs (gehle, 1965; kilborn et al., 1982; fang, 1995; pujol et al., 2000), [2], [3], [4], [5]. scanlon and dexter emphasized on the mill adjustments [6] which affect energy consumption during the grinding process. the motivation to measure energy requirments at specified roller mill settings led to the development of a micromill which can perform in the grinding process of the wheat and of the middling too, in the same conditions as in the milling industry. the adjustment of the roller characteristics can be done for each type of milling product (grain, semolina, bran). the grains are in the same time under the compression and the shearing efforts. the energy consumption is represented by one single value for one pair of rollers. this single value is significant for the comparative appreciation regarding the energy consumption in the milling process, for different wheat cultivars or different batches, but also for different characteristics of the rollers. materials and methods wheat samples table 1. quality indices of the wheat varieties the investigations were carried out on romanian winter wheat varieties (triticum aestivum, ssp. vulgare) dropia and pegasus, harvested in 2009. the preparation of the samples colected carried out according to the chessboard pattern method, after cleaning with an sadkiewicz instruments scourer.the physicochemical characteristics of the wheat were evaluated as follows: the moisture content using the sr iso 712 : 2005; the wet gluten content, protein content using the nir technique (inframatic, model 8600, perten instruments ab); vitreous kernel using the stas 6283-2/1984 (farinotom apparatus). the quality indices of the studied wheat varieties are depicted in table 1. before milling, 30 grams of each dry wheat sample was tempered overnight to reach 16 % (optimum) moisture content, wet basis; this toughens the bran and germ and softens the endosperm, making the separation of endosperm from germ and bran easier. then, the moist wheat was allowed to temper for different rest time period depending on variety. for the same rest time were prepared two paralel samples. experimental micromill for this research, was used a new designed micromill which can perform in the grinding process of the wheat and of the middling too, for the appreciation of the grain resistance (specific surface energy consumption) in the milling process, in the same conditions as in the milling industry. the adjustment of the roller characteristics can be done for each type of milling product (grain, semolina, bran). the grains are in the same time under the compression and the shearing efforts. the energy consumption is variety indicator dropia pegasus hectolitric weight [kg/hl] 71.2 77.2 vitreousness [%] 31 79 wet gluten content [%] 23.2 26.4 moisture content [%] 13.3 13.2 falling number [s] 202 277 protein content [%] 12.7 13.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 127 represented by one single value for one pair of rollers. this single value is significant for the comparative appreciation regarding the energy consumption in the milling proces, for different wheat cultivars or different batches, but also for different characteristics of the rollers: the size of the gap, roll disposition sharp-to-sharp, sharp-to-dull, dull-to-sharp or dull-to-dull, the corrugations number/cm, the differential speed ratio, profile and inclination. the appreciation of the energy consumption (kj/kg) is made by measurements of the resistant moment of the kernel, between the rollers, in the breaking process. the micromill is equipped with corrugated break rollers measuring 50 mm in length and 90 mm in diameter, using with a fast roll speed of 500 rpm, 5 corrugations/cm, 0,6 mm roll gap, with a sharp-to-sharp roll disposition and differential speed (1:2,5). to ensure the proper balance and the efficiency of the breaking operation, samples were sieved for 5 min on a test sifter from retsch gmbh, using six assortment of wire mesh sieves of 1,25 mm, 630 µm, 400 µm, 315 µm, 250 µm and 160 µm, along with a bottom pan. the experimental first–break roller micromill equipped with a computerized data acquisition system is connected to a tensometric cell to measure the resistant moment of the particles grounded between the rollers. the measurements of the resistant moment of the kernel, between the rollers, in the first breaking step lead to the appreciation of the energy consumption (kj/kg). the grinding work of the first breaking rolls is checked by sifting the ground stock on the retsch test sifter. results and discussion the rest time value obtained from each paralel samples is represented in figure 1 (for dropia) and figure 2 (for pegasus). the optimum rest time value for the conditioning of dropia variety is 5 hours. for this value it was obtained the lowest average resistant moment in the grinding process of this soft wheat variety, which it means also the lowest energy consumption. the lowest average resistant moment was obtained also for the pegasus variety (hard wheat) for 9 hours rest time in the conditioning process. dropia variety 0,000 0,500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 0 hours 13% 1hour 3hours 5 hours 7 hours res t tim e , h a ve ra ge r es is ta n t m om en t, n m figure 1. the rest time in the conditioning process of dropia variety pegasus variety 0 0,5 1 1,5 2 2,5 3 3,5 0 hours,13% 3 hours 5 hours 7 hours 9 hours 11 hours re st time , h a ve ra g e re si st an t m om en t, n m figure 2. the rest time in the conditioning process of pegasus variety the resistant moment of the particles grounded between the rollers has been obtained for each period of rest time for the moistened wheat. the results are confirming that for the lowest resistant moment value, the optimum rest time is 5 hours for dropia variety and 9 hours for pegasus wheat variety. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 128 dropia v arie ty 0 1 2 3 4 5 6 1 3 5 7 9 11 13 15 17 19 21 23 5 pulses/sec. r es is ta nt m om en t, n m 0 hours 13% 1hour 3 hours 5 hours 7hours figure 3. the average resistant moment, for different rest time values of dropia variety pegasus variety 0,000 0,500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 1 3 5 7 9 11 13 15 17 19 21 23 25 5 pulses/sec. r es is ta nt m om en t, n m 0 hours,13% 3 hours 5 hours 7 hours 9 hours 11 hours figure 4. the average resistant moment, for different rest time values of pegasus variety dropia,0,6mm 0 1 2 3 4 5 6 7 8 1 4 7 10 13 16 19 22 25 28 31 5 pulse/sec. r es is ta nt m om en t, n m dropia 16% d/d dropia 16% s/s figure 5. the resistant moment (specific surface energy consumption) for dropia variety with 0,6 mm roll gap and d/d or s/s roll disposition. dropia, 1 mm 0 1 2 3 4 5 6 7 1 3 5 7 9 11 13 15 17 19 21 23 25 27 5 pulses/s r es is ta nt m om en t, n m dropia,16%,d/d 0,6mm d/d dropia,16% 0,6mm s/s figure 6. the resistant moment (specific surface energy consumption) for dropia variety with 1 mm roll gap and d/d or s/s roll disposition food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 129 pegasus, 0,6 mm 0 1 2 3 4 5 6 7 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 5 puls es/sec. r es is ta n t m o m en t, n m pegasus 16% s/s pegasus 16% d/d figure 7. the resistant moment (specific surface energy consumption) for pegasus variety with 0,6 mm roll gap and d/d or s/s roll disposition. pegasus, 1mm 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 5 pulses/sec r es is ta n t m om en t, n m pegasus 16% d/d pegasus u 16% s/s figure 8. the resistant moment (specific surface energy consumption) for pegasus variety with 1 mm roll gap and d/d or s/s roll disposition. after the first breaking step in the wheat grinding, were obtained the curves (fig. 5,6,7,8) representing the resistant moment of the wheat kernel in the breaking process. the energy consumption is represented by the surface aria, below the resistant moment curves. for both dropia and pegasus wheat varieties, the lowest energy consumption is achieved with mill adjustment regarding the dull-to-dull (d/d) disposition, rather then sharp-to-sharp (s/s) disposition. also, the same lowest energy consumption in the first break is obtained for a maximum size of the roll gap (1 mm for the first break). the energy amount is higher when the size of the roll gap is decreasing. conclusion the assessment of the optimum parameters in the conditionning process can be made by the micromill designed to determine the grinding resistance of the cereals. the method has the same accuracy as the classical one and has the advantage to be quicker and less demanding as work volume.it is an alternative way to describe the optimum for the conditioning process and can be used in laboratory for the benefice of students as well in the milling industry. the best results for the first break, from the point of view of technological efficiency and the energy consumption were obtained for the sharp-to-sharp disposition and a roll gap related to the grinding length of the rollers. the new designed micromill can be used for the assessment of the energy consumption for the first break, in the laboratory conditions, for the benefice of the students. the micromill designed for determination of the grinding resistance of the cereals, can be used in an industrial mill plant, because it has the advantage of obtaining the cumulative compression and shearing efforts like in the industrial process. the resistant moment can be obtained also for the middling grinding process, step which can lead to the economic optimization of the cereal processing. the micromill is ideal for testing improved wheat cultivars developed in breeding programs. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 130 acknowledgments this research was supported by the national programme research council and uefiscsu, contract no. 411/20072010. references 1. posner, elieser s., hibbs, arthur n., wheat flour milling, american association of cereal chemists, st. paul, minnesota, 1997, pp. 125. 2. gehle, h. 1965. the miag “vario” rollstand: design and purpose. amer. assoc. of operative millers’ tech. bull. (may): 2861–2862. 3. kilborn, r. h., h. c. black, j. e. dexter, and d. g. martin. 1982. energy consumption during flour milling: description of two measuring systems and influence of wheat hardness on energy requirements. cereal chemistry. 59(4): 284–288. 4. fang, q. 1995. effects of physical properties of wheat and operational parameters of roller mills on size reduction. ms thesis, kansas state univ., manhattan, kansas. 5. pujol, r., c. letang, i. lempereur, m. chaurand, f. mabille, and j. abecassis. 2000. decription of a micromill with instrumentation for measuring grinding characteristics of wheat grain. cereal chemistry 77(4): 421–427. 6. scanlon, m. g., and dexter, j. e. 1986. effect of smooth roll grinding conditions on reduction of hard red spring wheat farina. cereal chem.63:431-441 microsoft word 3 journal nr 4 2010 din 8 noiembrie_32-37.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 36 method of carbon nanotubes dispersion in polymeric matrix monica murarescu1, dumitru dima1, gabriel andrei1, adrian circiumaru1 1university “dunarea de jos” of galati, domneasca street 47, romania, monica.murarescu@ugal.ro abstract: in order to obtain better homogenous composite material, different carbon nanotubes (cnts) dispersion techniques in the polymeric matrix are very well known. due to the strong interactions between cnts, the efficiency of these dispersion techniques is highly limited. the aim of this paper is to present improvement techniques considered as new step in the global dispersion process before the composite material final shape finalization. at this moment, it is very difficult to carry out a classical dispersion from technological point of view. in this paper, more than the acknowledged mechanical and ultrasonic dispersion method, the introduction of a genuine dispersion technique is proposed; it refers to an external vibrant magnetic field able to determine a vibration movement of cnts covered by a molecular fe (iii) oxide. this external vibrant magnetic field is made by using a permanent magnet involved in a rotational movement around its own axis and also interacts with the individual cnts own magnetic fields. the maintenance of a tensioned vibrating state at the individual cnt level contributes to a good dispersion state preservation and increases the connections and physical-chemical interactions hindering. the dispersion efficiency in a vibrant magnetic field was studied using the comparative methods, correlating the electronic microscopy analysis with the mechanical strength tests. with respect to the composite material obtained under these conditions, a significant quality improvement as well as a mechanical strength increase was observed. keywords: magnetic properties, composite material, polymer matrix composites. introduction carbon nanotubes (cnts) utilization in the polymeric matrix consists in the obtaining process of some unique properties as a main result of their nanometrical dimensions. their unusual structure along with a decreased density, a remarkable strength and stiffness, followed by electrical properties versatility contribute to a high interest on their use as ingenuous polymeric materials reinforcement. [1], [3] the key element of this possibility consists in the mechanic, thermic and electric properties transmission from the cnts to the polymeric composite material. hereby, there are two problems that have to be solved in order to bring substantially improvement to the polymers material properties along with carbon nanotubes addition as fillers: the interfacial connection and moreover, the optimum cnts individual dispersion in the polymeric matrix. [2] the polymer interfacial adhesion can be substantially improved by the chemical functionalization of the nanotube surface. the influence of the chemical bond between nanotubes and the matrix on the interfacial adhesion was anticipated by the molecular dynamics simulations. [1], [5] the particles having nanometrical dimensions present a large surface, with a higher size degree than conventional fillers surface. their surface area actions as the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 37 transfer interface of the strains and it is responsible in the same time for the strong and natural cnts tendency to make agglomerates. these properties efficient operation in polymers depends on their homogenous dispersion process in the matrix at the same time with the agglomerates destruction process and their wetting with polymeric substance. considering cnts distribution process in a polymeric matrix, these elements would have to be evaluated: the nanotubes length, their disorder, the volume ratio, the matrix increased thickness, the attraction between cnts themselves.[4] the usual particles dispersion in the polymeric materials seems to be difficult and finally ends with both phases separation and agglomeration phenomenon. it was demonstrated that nanoparticles’ thermodynamically stable dispersion in the polymeric liquid can be successfully obtained for the systems which lineal polymer radius of gyration is bigger than the nanoparticle radius. the dispersed nanoparticles expand the lineal polymeric chains and the primarily result is a polymer which radius of gyration increases with nanoparticles volume ratio. it was suggested the fact that this process entropic disadvantageous is balanced by an enthalpy gain due to the increased number of molecular contacts between the dispersed nanoparticles surfaces in comparison with nanoparticles surfaces in the phase separation case. [2], [5] even the dispersed state is thermodynamically stable; it is difficult to be obtained considering an inappropriate processing strategy, this one being one of the most important things referring to cnts dispersion process into lineal polymers. [2], [6] starting from a welldetermined target like nanotubes dispersion, different work techniques are proposed: ultrasonication, mechanical stirring, etc. ultrasonication has a big energy local impact but introduces small quantities of shearing forces, so that this method is appropriate only for matrix with very low thickness and small volumes. the local energy input leads to cnts breakage, decreasing their length. cnts dispersion in an adequate solvent (like: dimethylketone, styrene) represents an appropriate way for ultrasonication technique application in order to obtain cnts composite materials. in this way, it would be allowed an agglomerates separation due to the vibration energy. [3],[6] decreased agglomerates dimensions can be easily obtained by using cnts functionalization technique. mechanical stirring is a usual dispersion method of particles in the liquid systems and can be successfully used for nanoparticles dispersion. the dispersion result depends on the mixer shape and size as well as stirring speed. after an intensive cnts stirring into the resin, they present the natural tendency of agglomerating and this flocculation phenomenon experimentally observed is primarily generated by the wearing contacts as well as elastic coalescence mechanisms. [7] calendaring becomes a working obtaining way of a good dispersion state. this method is a usual well-known method of microparticles dispersion in different matrix, like: colouring agent for cosmetics and paint. a major advantage of this method, apart from the improved dispersion results would be the efficient manufacture of a diversified nanocomposites range. other methods than the above supposed techniques of some energy type introduction in cnts/polymeric matrix mixing process that would be able to realize an enthalpy/entropy optimum ratio. furthermore, a good dispersion can be anticipated and realized in this way. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 38 materials and methods in order to obtain nanocomposite materials with polymeric matrix we used an unsaturated polyesteric matrix aropoltm m105 tpb ashland olanda – rotterdam, a largely used resin at industrial level added with 1% catalyst 2ethyl-cobalt hexanoat. we used methylethyl ketone peroxide 2% as initial catalyst. multi-wall carbon nanotubes (mwcnts) were obtained from cheaptubes inc. usa, having the following characteristics: external diameter 8 – 15 nm, length 10 – 50 µm and purity over 95%. it was realized a covering process with a molecular layer of fe2o3 in accordance with a technology that represent another scientific paper aim. in order to present carbon nanotubes optimum concentration value in the polyester matrix, we considered three types of concentration: 0.10; 0.15 and 0.20%. we carried out the dispersion process considering a self-technology represented by two different types of stirring, starting with a mechanical one and followed by a ultrasonic type of stirring (fig.1). fig.1 the dispersion by ultrasonication process in polyester matrix at the end of these two different types of stirring, a dispersion process in a vibrant magnetic field (fig.2) took place. we made two experimental series coded with a and b using these three types of concentration for carbon nanotubes covered by a molecular layer of fe2o3. the samples coded with b made by using three different types of concentration are different from the samples coded with a due to the fact that the dispersion technology contains an extra-phase represented by a supplementary dispersion in a vibrant magnetic field (fig.2). fig.2 the dispersion process of cnts in polyester matrix in a vibrant magnetic field the samples made in accordance with standards en 63, astm d790-81, nft 57-105 or nft 51-001 from a dimensional and 3 point flexural test point of view were moulded in rubber matrices that were previously made-up by flush cutting procedure. after the samples were extracted from the matrices, they were dimensionally and chemically stabilized using a thermal treatment in the oven at 278k for 8 hours. for each experiment we used 10 samples for a statistically interpretation. the samples were fixed at 3 points flexural test on a testing machine “win testtm analysis – testometric materials testing machines, england”. the machine working parameters are: working speed – 2,500 mm/min, flexural load – 1,000n, span – 32,000mm. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 39 results and discussion the testing results were plotted and shown in the following shape (fig.3). the experimental data at 3 points flexural test for the two series coded a and b are schematically presented in table 1. it is easier to understand in this way the experimental data interpretation in order to justify the anticipated effect of an external vibrant magnetic field at carbon nanotubes dispersion technology. 3 point fle xural te s t 0 50 100 150 200 250 300 350 400 450 0 0,5 1 1,1 1,5 2 2,5 3 3,5 strain(% ) fo rc e( n ) fig.3 three points flexural test. the variation of deformation plot (%) depending on the applied force (n) table 1 bending modulus values sample bending strength @ break (n/mm²) bending modulus (n/mm²) transv. rupture strength (n/mm²) a0,10% 103.90 4168.64 103.90 b0,10% 105.04 4728.29 105.09 a0,15% 105.25 4305.62 105.45 b0,15% 109.24 4500.66 109.61 a0,20% 110.40 4605.21 110.44 b0,20% 111.50 4805.25 112.50 it was observed a bending modulus and other mechanical parameters increasing with carbon nanotubes concentration increasing. moreover, at the same concentration values it was observed an increasing at b series in comparison with a series that demonstrates the vibrant magnetic field efficiency in the dispersion process of carbon nanotubes in polyester matrix. the increasing variation of mechanical parameters at 3 point flexural test is presented in table 2. the highest value for bending modulus is observed at the concentration of 0.1%. this conclusion is explained by the fact that the vibrant magnetic field efficiency is quantified when the gaps between nanotubes clusters are large. table 2 mechanical parameters variation at 3 points flexural test for the same version of b series in comparison with a series conc.( %) bending strength variation (%) bending modulus variation (%) transv. rupture strength variation (%) 0.10 1.08 11.83 1.13 0.15 3.65 4.30 3.79 0.20 0.99 4.16 1.83 at the highest concentration values, that means 0.15% and 0.20%, the same parameter variation maintains quasiconstant; the explanation would be that gaps decreasing have important impacts on the vibrant magnetic field efficiency concerning the dispersion process of carbon nanotubes covered by a molecular layer of fe2o3. sem analysis (fig.4) confirms the experimental data obtained at 3 points flexural test; an improved carbon nanotubes distribution at b series in comparison with a series was observed. in the samples of 0.10% and 0.15% from a series we observed a stronger agglomeration in comparison with the sample of 0.20% of the same set. this aspect is possible due to the equilibrium established by the attractive forces energy between the nanoparticles and the dispersion forces energy for 0.20% samples. in this case we made a comparison on the adverse energetic state for the dispersion forces considering the samples of 0.10% and 0.15% concentration. at b series, considering all food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 40 concentration values, a superior distribution at carbon nanotubes was observed. a0,1% a0,15% a0,2% fig.4 sem analysis for a series samples of 0,10%, 0,15% and 0,20% concentration without the vibrant magnetic field presence b0,1% b0,15% b0,2% fig. 5 sem analysis for b series samples of 0,10%, 0,15% and 0,20% concentration in a vibrant magnetic field presence food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 41 from sem analyses made by using quanta™ 200 scanning electron microscope (2006) we noticed that the non-agglomerated state of the particles from b series in comparison with a series demonstrates the fact that the enthalpic gain from the external vibrant magnetic field leads to the bond breakage between the nanoparticles participating in the clusters formation. this phenomenon is better observed at decreased concentration values of carbon nanotubes covered by a molecular layer of fe (iii) oxide. this is a consequence probably due to the vibrant magnetic field energy. it would be an interesting topic to be focused on, but considering the technological reasons we analyzed only a single type of magnetic stirrer. conclusions the experimental data and the physical analysis confirm the theory of an optimum equilibrium existence between the dispersed system enthalpy and entropy. the mechanical, ultrasonic and external electro-magnetic field energy that is induced in the dispersed system due to carbon nanotubes covered with a molecular layer of fe (iii) oxide in the same time contribute at this target. this aspect is reflected by sem analysis of the samples in / without a vibrant magnetic field and also by the flexural modulus and other mechanical parameters increasing resulted from three points flexural test. in conclusion, we demonstrated that another technological step introduction in the dispersion process of carbon nanotubes covered with a molecular layer of fe (iii) oxide allows an improved distribution in the polyester matrix. this supplementary stage in the technological dispersion process is responsible for the mechanical properties improvement and also the final product quality represented by nanocomposite material. acknowledgements this work was supported by cncsis – uefiscsu, project number pnii – idei 519/2008 9/2008. references 1. z. guo, k. lei, y.li, h.wai, s.prikhodko, h. thomas hahn, fabrication and characterization of iron oxide nanoparticles reinforced vinyl-ester resin nanocomposites, composites science and technology 68 (2008) 1513–1520; 2. m. e. mackay, a. tuteja, p. m. duxbury,c. j. hawker, b. van horn, z. guan, general strategies for nanoparticle dispersion, science 24 march 2006, vol. 311. no. 5768, pp. 1740 – 1743; 3. s.cooper, c.fleischer, m.duffy, a.wagner, polymeric composite including nanoparticle filler, int.ci.c08k 9/04, 2006; 4. s.asgari, composite materials containing carbon nanoparticles, provisional applications no.60/643.842, ian.2005; 5. abraham,“nanoceramics,nanotubes and nanocomposites paving the way for nanotechnology revolution-a review of the industry and markets,” surfaces and interfaces in nanostructured materials and trends in liga, miniaturization, and nanoscale materials, materials processing and manufacturing division fifth global symposium, ed. m. mukhopadhyay et al. (warrendale, pa: tms, 2004), pp. 247-269. 6. g.desie, l.vanmaele, g.deroover, method to improve the quality of dispersion formulations, pub. no.: us 2005/0235740 a1; 7. gou j.h., minaie b., wang b., liang z.y., zhang c., “computational and experimental study of interfacial bonding of swnt reinforced epoxy composites” computational materials science, 2005,31 (3-4) 225-236. microsoft word 3 journal nr 4 2010 din 8 noiembrie_99-103.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 103 dietary fibres role in manugacturing bread with low content of glucides monica dinu1, gabriela constantinescu (pop) 2 1sc baneasa sa, str. nicolae g. caramfil., bucuresti, romania, e-mail: gabi_dinu2005@yahoo.com 2 university „ stefan cel mare” of suceava, food engineering faculty abstract nowadays the food producers meet the demands of the consumers that want food products with an important role in health preservation. thus, the bread producers focused their attention towards the researches of the functional food. a functional role it is attributed especially to the soluble fibres, that help controlling the level of glucose and lipids in the blood. therefore, the practical and theoretical researches were made in order to obtain bread with a low content of glucides, and comply with legal requirements. the aim of this paper is to prove that adding soluble fibers in bread products it is possible to obtain functional food with glucides low content, with good sensorial characteristics, fated consumers with different diseases. it was make a study on bread made with added insoluble (bran) and soluble fibres (inulin), in different amounts. inulin was obtained from chicory and helianthus tuberosus (topinambur). it was analyzed the percent of inulin retrieved in the final product relatively to the initial supplementation, the percent of protein and glucides. sensorial tests were performed on the products containing inulin and compared to a free inulin control sample. it was calculated the energetically value and it was estimated the intake of the fibres in comparison with the recommended daily dose, without passing over the economical aspect. based on results that were obtained, it is recommended the use of 3% inulin (irrespective of source) related to the flour content. keywords: insoluble fibres, soluble fibres, functional food, bran, inulin, chicory. 1. introduction the tendency of the great corporation is to satisfy both, the consumer’s demands, and also their desires to maintain health. thus, the notion of functional foods had appeared, especially created for pretentious consumers, which want to maintain a healthy lifestyle. [1] functional foods in bread making industry were appeared through adding soluble fibers supplementary, witch have prebiotic effects. [2,3] the common know soluble fiber is inulin, which has like obtaining sources chicory and helianthus tuberosus (topinambur). adding inulin in bread products we could observe a technological improves like: considerable volume increase, an equable porosity, easiness slices up, freshness maintaining during the shelf life without microbial modification. [4,5,6] looking the nutritional aspect it was observed there is an improvement of proteins and soluble fibers (inulin) content and a decrease of glucides content without taste affected. starting with these facts we want to present the bread products making on s.c. baneasa s.a., which meet the demands of the consumers. 2. materials and methods materials in order to obtain some available experimental data we used like materials:  dietetic flour/total grinding, from dobrogea cultivar, with alveograph characteristics: w 120, p/l 2,28, and pharinograph characteristics: development 2’30”, stability 5’30”, softening 90 uf;  black flour, from dobrogea cultivar, with alveograph characteristics: w 128, p/l 1,9, and pharinograph characteristics: development 2’30”, stability 5’30”, softening 80 uf; food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 104  wheat bran  soluble fiber (inulin) from chicory (provided by enzymes &derivates s.a. romania) and from helianthus tuberosus (extract 12.5% provided by s.c. hofigal export import s.a., romania ) in different percents;  vital gluten , provided by enzymes &derivates s.a. romania  compacted fresh yeast (saccharomyces cerevisiae), provided by s.c. rompak s.a. pascani, romania with 32.5% dry matter and 46.54% protein content (n x 6.25)  iodated salt  water samples preparation the frame recipes which are used in order to obtain bread products with fibers added are shown in table1. table 1 frame recipes for 100 kg final products braed with bran and inulin topinambur extract 12.5% ingredients bread with bran witness ( m ) chicory p1 p2 p3 p4 graham dietetic flour 5 dietetic flour/total grinding 37 37 37 37 black flour 40 yeast 2 1.8 1.8 1.8 1.8 vital gluten 0.7 13 13 13 13 bran 7 13 13 13 13 inulin 0 2 2 2.5 3 iodated salt 1 1 1 1 1 work method for samples preparation we used the direct monophazic method, pan baking. kneading time was 15 minutes in spiral mixer. the dough temperature was 26° c. the dough’s were allowed to rise for 40-55 minutes at 34° c and, after degassing and moulding by hand, for 15 minutes at 34° c. the bread was subsequently baked for 30 minutes at 230° c. after baking, the samples were cooling 6-8 hours in controlled atmosphere (uv lamps). in order to be exam from sensorial point of view (after 24 hours), the samples were sliced for packed in plastic bags. the bran and inulin samples were added to the baking formula during the mixing stage. bread volume was determined after 24 hours of cooling by means of rape seeds. the microbial charge was determinate after 24, 48 and 72 hours after cooling. methods of analyses the analytical flour quality was determined according to the international standard methods. we use a chopin alveograph (aacc/no.54-30a, icc121, and iso 5530/4) to determine the relationship between elasticity of the dough and rising power in order to analyze the rheological characteristics of tested flours. inulin determination was made means spectrophotometer method validate by s.c. hofigal export import s.a. ntg (total number of germs) was determinate according to stas iso 4832:1992 and microbial charge 9yeasts and molds) according to sr iso 7954:2001. for sensorial analyze we used the scoring method validated by researchers from university “stefan cel mare” of suceava, food engineering faculty food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 105 3. results and discussions physical – chemical analyses the results are shown in table 2. in case of samples with inulin (p1, p2, p3, and p4), we could observe improve of volume against the witness (m1). also we notice there aren’t major humidity differences between samples and manufacture standards. the retrieve inulin percent report to 100 grams final product increase significant with increase of inulin percent added. the protein and insoluble fibers percent remain relatively constant report to 100 grams final product. table 2 physical – chemical analyses sample humidity [%] volume [cm3/100g] proteins [g/100g prod] inulin [g/100g prod] lipids [g/100g prod] glucides [g/100g prod] m 51.6 525 8.3 0.05 1.1 41.4 p1 50.1 556 12.5 2.1 1.8 27.6 p2 50 570 14.3 2.3 0.67 26.73 p3 50 570 16 2.7 0.72 24.58 p4 49.8 567 18.3 3.2 0.86 21.84 if we analyze the data recording in table 2, we could observe that once with the increase of inulin added percent decline the glucides percent in all samples. this aspect became very important from nutritional point of view, and we could recommend the obtaining bread in diet of diabetic persons. microbiological analyses if we compare the obtaining results, shown in table 3, with legal admitted limited according to oms 975, we concluded that our breads are safety for human consume. table 3 microbiological analyses sample yeasts and moulds spores / g oms spores / g bacillus subtilis spores / g oms spores/ g m 0 absent p1 0 absent p2 0 absent p3 0 absent p4 0 max 1000 absent max 10 the dietetic fibres analyses and nutritional characteristics analyzing the data recording in table 4, we could observe that for each inulin added percent, the retrieve inulin percent rise with 0.2 – 0.5 grams. in case of adding soluble fibres, the energetic value decline through decline of glucides percent and increase of proteins and fibres content. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 106 table 4 nutritional analyses breads with inulin nutritional information’s/ 100 g final product bread with bran m p1 p2 p3 p4 energetically value, kcal 203 185 179 180 181 proteins, g 8.3 12.5 14.3 16 18.3 glucides, g 41.4 27.6 26.73 24.58 21.84 lipids, g 1.1 1.8 0.67 0.72 0.86 fibers, g 3.2 6 6 6 6 inulin, g 0 2.1 2.3 2.7 3.2 energetic values of samples (p1, p2, p3, and p4) with inulin added are with almost 11% lower against the witness (m1). for this reason we could recommend this kind of bread in diet of all persons which are interested in obesity control. sensorial analyze we applied the scoring method, with a 5 points scale, 24 hour after baking. the selected peoples fills the analyze sheets, we pick out five fundamentally characteristics (table 5), which are graphic represented in figure 1. the recording data were statistical process means anova method. table 5 sensorial analyze medium score ( 11 peoples) nr crt sensorial characteristic m p1 p2 p3 p4 1. color of crumb 2.6 2.4 2.2 2.6 2.2 2. softness 4.8 4.4 4.2 4.4 4.2 3. equable of pores dimensions 1.2 1.6 1.6 1.2 2 4. fundamentally taste ( sweet) 3 3.2 4.1 4 3.8 5. residual (little fibers through teeth) 4.3 4.2 4 3.9 3.8 0 1 2 3 4 5 m p1 p2p3 p4 color of crumb softness equable of pores dimensions fundamentally taste( sweet) residual (little fibers through teeth) figure 1:sensorial analyze the price analyze the price analyze is shown in table 6. from economical considerate we recommend selecting the 3% percent of inulin added report to flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 107 table 6 the influence of inulin adding on expenses with raw materials, for1 kg final product ingredient price per unit [lei] consume per unit m [kg/kg val [lei] consume per unit p1 [kg/kg val [lei] consume per unit p2 [kg/kg val [lei] consume per unit p3 [kg/kg val [lei] consume per unit p4 [kg/kg val [lei] graham flour 9400 0.05 470 0 0 0 0 0 0 0 0 total grinding flour 10500 0 0 0.37 3885 0.37 3885 0.37 3885 0.37 3885 black flour 9400 0.4 3760 0 0 0 0 0 0 0 0 yeast 23000 0.02 460 0.018 414 0.018 414 0.018 414 0.018 414 gluten 52000 0.007 364 0.13 6760 0.13 6760 0.13 6760 0.13 6760 bran 2300 0.07 161 0.13 299 0.13 299 0.13 299 0.13 299 inulin from chicory 115500 0 0 0,02 2310 0 0 0 0 0 0 inulin from topinambur 100000 0 0 0 0 0.020 2000 0.025 2500 0.03 3000 salt 7000 0.01 70 0.01 70 0.01 70 0.01 70 0.01 70 total 5285 13738 13428 13928 14428 profit[%] 26.8 10.63 11.15 10.31 9.49 4. conclusions  the results obtaining in this study prove that inulin adding (like soluble fibre) do not affect the sensorial characteristics of bread products;  from the point of view of physical – chemical properties we could observe an volume improve of bread;  the microbiological parameters are under the legal limits admitted, which demonstrate that the final products are safety for human consume;  in case of modified of soluble fibres adding percent, it was observe differences in retrieved amounts in final product, indifferent which was the inulin source;  if we made a price analyze, we observe when we use native inulin, with helianthus tuberosus (topinambur) like source the raw material expenses are small, and the profit is with almost 1% bigger that the products obtained with import inulin ( 2% addition). the profits begin to lower with increase of percent of inulin addition. at 2,5% addition of topinambur inulin we could obtain almost the same profit like in case of adding 2% inulin from chicory;  taking into consideration that inulin (dietetic fibre) have prebiotic role, the adding could be considerate like a viable alternative for obtain functional bread products. references 1. georgescu, a., 2004, noi ingrediente functionale utilizabile in panificatie, bimp, vol 15, nr4., pag. 24-30 2. giurea, a.m., 2001, studiu privind fibrele alimentare: definitie, constituienti, implicatii nutritive si fiziologice, bimp, vol 12, nr. 3-4, pag. 60-73 3. georgescu, a., 2001, fibrele alimentare. studii biochimice si tehnologice, bimp, vol 12, nr. 1, pag. 66-112 4. coman, m., 2004, fibre dietetice, bimp, vol 15, nr 1-2, pag. 86-92 5. segal, r., 2003, prebioticele – ingrediente functionale in dezvoltarea produselor cerealiere, bimp, vol 14, nr. 2, pag.6-9 6. bantea, v., 2007, tendinte noi privind unele aspecte teoretice a inulinei in calitate de remediu pentru bolnavii de diabet zaharat, bimp, vol 18, nr 2-3, pag 65-70. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 70 ways of optimizing the nutritional value of dietetic pastry products *iuliana cioarbă 1, ana batariuc 2 1ştefan cel mare university of suceava, faculty of food engineering, universităţii street, number 13, postal code 720229 suceava, romania, iulianac@usv.ro *corresponding author 2 dsvsa suceava, scurta street, number 2, postal code 720223 suceava, romania, any_b03@yahoo.com received 20 february 2011, accepted 19 may 2011 abstract: a balanced nutrition means a diet which contains necessary nutrients in proper proportions for our body's health. the lately led studies prove that the persons whose diets are poor in vegetable fibers are more exposed to degenerative diseases such as: constipation, colitis, and diabetes or colon cancer. the general concern for the development of functional foods has led to reconsidering the use of dietary fibres in food production and thus researches on their physiological role for human organism had been initiated. the vegetable fibers represent an important compound of the diet for they don’t fatten and they ensure a good digestion. the purpose of this paper was to find some ways to improve the nutritional value of a dietetic pastry product based on sweeteners. for accomplishing this, the water used in preparing the fibre-rich product was replaced by carrot juice. thus, a general improvement of the nutritive value could be noticed, but with a slight growth of the energetic value of the final product, a growth that was determined also by the use of some caloric sweeteners. good knowledge of the nutritional value of food is a basic condition in order to be able to appreciate their quality level and nutrition specialists should be interested in optimizing it. © 2011 university publishing house of suceava. all rights reserved keywords: diet, fiber, nutritive value, sweetener. 1. introduction nutrition is the science of dietary. it refers to the food digestion, nutritional needs, and food science and food behavior [1]. a correct diet ensures a normal and harmonious development of the growing organism; it ensures health and resistance at the harmful environment factors and a high intellectual and physical capacity as well as a prolonged life expectancy [2]. when the organism receives all the nutrient substances (especially those unsynthesizable ones) in the correct quantities for its needs the diet is “proper”. in this case it constitutes one of the main ways that can influence favorably the health state of the individual or of the collectivity from many aspects: the harmonious growth and development of the youngsters and youth, the maintenance in the normal limits of some somatic parameters (body weight, height etc.), physiologic parameters (the normal functioning of the body systems) and biochemical parameters (the maintenance in normal concentrations of the blood and tissues constituents in children and adults, the enhancement of the working ability, the adaptation and resistance to the environment unfavorable factors ) [3]. the nutritional mistakes can aggravate a series of diseases of the digestive system and of the attached organs: gastritis, ulcers, entero-colitis, chronic constipation, diseases of the biliary ways, chronic pancreatitis, cancer etc. most of the technological processing used in the modern food industry determine a diminishing of the nutritive substances content of the raw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 71 materials subjected to the processing and most of the resulting food stuff has a high energetic density on low basis of active biological compounds [4]. presented in an attractive way from the sensory point of view they influence the human’s diet behavior as they select products from the first shelf according to their appealing nature without thinking of their nutritive qualities. under the conditions of globalization of the food stuff market, consumers’ attention is driven by the new food stuff products, different from those which they were used to and those with insufficiently explained labels like: "dietetic food"', "light food", "fast food", "food supplements", "functional food" [5]. the rediscovering of the diet influence upon the human health led to the appearance of the functional foods as foods with prevention and improvement properties upon the different non contagious chronic diseases (ncd) such as the type 2 diabetes, obesity, cardio diseases, different forms of cancer, diseases that have a higher and higher incidence and strongly contribute to a high mortality ratio. the general preoccupation for the functional foods development generated the need of studying and utilization of new food ingredients with a role in the maintenance and improvement of the health state [6]. thus, the using of nutritional fibers was reconsidered in the food production and several studies regarding their physiological role in the human body were initiated [7]. consequently, cereal fibers are important for the health of the human body and play an important part in the good functioning of the digestive system, of the vascular system but also in treating diabetes and controlling weight [8]. the specialists recommend a daily average consumption of 20 – 35 gr of fibers but people generally consume only 9 – 12 gr daily if not even less. a poor fiber diet can cause a series of unpleasant things starting with the bloating sensation, flatulence, hemorrhoids, varicosity and even heart and colon cancer. the prevention as well as the control of diabetes is based on the medical examination but mostly on the careful daily diet of the patient. the three basically diet principles: sugars, fats and proteins must be found in certain ratios in the normal diet ratios that are modified in the case of the diabetes mellitus or in the case of the predisposition for this disease. in the case of diabetes mellitus the diet fibers especially of those insoluble can improve the blood glucose control decreasing the hyper insulin and the plasmatic concentrations of the lipids in type 2 diabetes facts that confer an ideal cardiovascular protection [9]. the previous research has shown that bran reduces the endothelium inflammation and improves its functioning. the diabetics are generally advised to consume lots of integral cereals and choose brown rice in favor of white rice. the nutritional value of bakery products is increased by the enhancement with substances that they do not normally contain in a sufficient ratio or lack them totally [10]. the protean enhancement can be made by adding during the fabrication process protean rich substances especially those with high lysine content [11]. bran and especially bran particles of flour (which are richer in lysine than the grain endosperm) can have an important role in increasing the lysine content [12]. to use them as addition it is necessary to process them in the fermentative way or to increase the human assimilation degree of the proteins contained. being very rich in the vitamins b group (b1, b2, b3, b5, b6, b8, b9), in amino acids and minerals (sodium, potassium, magnesium, calcium etc.), enzymes, minor elements (90% of them are found in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 72 bran) and vegetal fibers, wheat bran has numerous medical properties: they improve the digestion, are emollients especially for the stomach and intestines, laxative, nutritional, re-comforting, re mineralizing, general tonics, help with decreasing the cholesterol and the fat deposits from certain organs. they are recommended in diseases such as: the endocrine diseases (regulate the glands due to their high mineral, amino acid and vitamin content), adenophaty, anemia, cancer, the different dermatosis, diabetes mellitus, epilepsy, obesity, neuro muscular diseases, neurological, cardio, liver ones, the convalescence period and most of the hypo or hyper diseases as bran contributes in regulating the entire body functions [13]. even though they are not a nutrient the vegetal fibers represent an important component of the diet for they don’t fatten and they ensure a good digestion. this type of dietetic pastry was made for being introduced in human diet as the studies that have been led lately show that the humans whose diet is poor in vegetal fibers are more affected by the degenerative diseases such as constipation, colitis, and diabetes or colon cancer. the fabrication recipe for the dietetic pastry products (for 100 kg finished product) is presented in the following table: table 1 the fabrication recipe for the dietetic pastry products ingredients quantity (kg) flour type 650 41.95 dietetic flour 41.95 wheat bran 35.96 sun flower oil 1.90 sodium bicarbonate 0.87 ammonium bicarbonate 0.87 salt 0.73 water 62.92 sweetener 5.5 2. materials and method: organoleptic examination [14] the principle of the method: the organoleptic examination consists in evaluating the organoleptic characteristics of the products with the aid of the sensory organs and it is led upon the whole or sliced product (halves or slices). humidity’s determination [15] the principle of the method: the sample is dried at the temperature of 103 ± 2ºc, for 40 minutes, afterwards the mass loss is being calculated. acidity’s determination [16] the principle of the method: the water extract of the analyzed sample is titrated with a sodium hydroxide solution of 0.1n in the presence of phenolphthalein as indicator. total sugar determination by the iodine-metric method (schoorl method) [16] the principle of the method: an alkaline solution of cupric salt is reduced at heat with the aid of a reducing sugar from the sample analyzed. the resulting cupric oxide is titrated indirectly with a sodium thiosulphate solution. fats determination [16] the principle of the method: the fat is extracted from the sample with petroleum ether or ethyl ether in the soxhlet device, and then it is evaporated and dried till constant weight and then weighed. protean substances determination [17] the principle of the method: the mineralization of the organic substances with h2so4 in the presence of a catalyst. the alkalinization of the reaction product followed by the freed ammonia distillation and titration. we calculate the nitrogen content and multiply the result with a conventional food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 73 factor to obtain the raw protean content. this multiplication factor is 6,25. 3. results and discussion after having made the product according to the established recipe we performed the organoleptic examination, determined acidity, fat content, proteins and sugar content as well as humidity in the product (in comparison with its version) by replacing water in the fabrication recipe by carrot juice. in each of the two versions of the dietetic pastry products (with water and carrot juice) three types of different sweeteners were introduced: saccharine and cyclamate sweetener (clio) which contains 20,82% sodium cyclamate and 5,58% saccharine with an energetic value of 0 calories; dextrose, saccharine and acesulfame k sweetener (dolcificante) with an energetic value of 360 kcal/530 kj per 100 g; pure fructose (fruttil) with an energetic value of 400 kcal/1700 kj per 100 g. 6 samples were obtained: 1 – with clio sweetener; 2 – with dolcificante sweetener; 3 – with fruttil sweetener; 4 – with clio sweetener and carrot juice; 5 –with dolcificante sweetener and carrot juice; 6 – with fruttil sweetener and carrot juice. we made two samples, one with water (7) and one with carrot juice (8) in which we did not introduce any type of sweetener and they served as reference samples. initially the first six samples were subjected to an organoleptic examination regarding appearance, form, consistency, color, flavor and taste. we distributed individual sensory analysis papers to 25 examiners with ages between 21 and 52 to see which of the six samples got the highest mark. the marks were centralized in a sensory analysis centralization paper. following the marks obtained by the six samples we can assess that the replacing of water by carrot juice was appreciated by the examiners, carrot juice bringing the highest marks especially regarding color, flavor and taste. after the organoleptic examination of the six samples along with the reference samples the physico-chemical determinations were made. from this point of view the characteristics determined were humidity, acidity, protean content, fat content and total sugar content. from the humidity point of view some difference between the reference samples and those prepared with water, the humidity content (u = 20.13%) was a little higher than in the samples prepared with carrot juice (u = 18.86%) was noticed. 20.13 22.44 24.54 19.34 22.17 24.33 20.63 18.86 0 5 10 15 20 25 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 humidity, % fig. 3. the humidity’s variation for the dietetic pastry products regarding the influence of the sweetener used we observed a humidity increase in the order clio→dolcificante→ fruttil, both in the cases where the water was used and where the carrot juice was used. the humidity increase in this order is explained by the fact that in the same order the sweetener’s hygroscopicity also increases. the increase is of 10,29% (clio→dolcificante), and of 8.55% (dolcificante→fruttil) in the case of the series prepared with water and of 12.76% (clio→dolcificante), and 8.87% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 74 (dolcificante→fruttil) for the series prepared with carrot juice. in comparison with the reference sample in the case of carrot juice we saw a humidity increase with 22.48% when using the fruttil sweetener. the pastry products acidity is of maximum 3 degrees of acidity. the acidity values of the dietetic pastry products are given in the following graphic representation. 2.1 2.6 2.1 3.1 2.8 3.2 2.4 3.05 0 0.5 1 1.5 2 2.5 3 3.5 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 acidity, degrees of acidity fig. 4. the acidity’s variation for the dietetic pastry products first of all we noticed an increase of acidity (over 3 degrees of acidity established for pastry products) in the case of the samples prepared with carrot juice. in the case of the samples prepared with water, acidity is between the limits imposed. an increase of 8.34% was seen in the sample 2 (dolcificante) in comparison with the reference sample 7, while the other two samples (1 and 3) had their acidity below the value of the reference sample. in the case of the samples prepared with carrot juice we saw an over limit of the limit imposed in the cases of sample 4 (clio and carrot juice) and 6 (fruttil and carrot juice), an over linit with 1.64%, and 4.92% in comparison with the reference sample 8. we have to compare the two reference samples we can say that there is an acidity increase with 27.09% when replacing the water by carrot juice. regarding the protein content of the eight samples we noticed slight differences between the samples prepared with water and those prepared with carrot juice. 9.69 9.49 9.43 10.69 10.12 10.05 9.51 10.19 8.5 9 9.5 10 10.5 11 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 proteins, % fig. 5. the proteins content variation in dietetic pastry products the carrot juice did not significantly alter the proteins content of the product, the difference between water reference sample and carrot juice sample being of 6.67%. there was as well noticed an increase in the cases of the samples where clio sweetener was added, the difference between sample 1 (clio) and sample 4 (clio and carrot juice) being of 9.35%. regarding the total sugar content we saw many differences determined both by the sweeteners and the carrot juice used. first of all we saw a difference between the sample prepared with water and those prepared with the carrot juice in the way that they have increased the total sugar content. this thing was also well noticed in the reference samples. the total sugar content of the reference sample 8 prepared with carrot juice is with 68.36% higher food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 75 than that of reference sample 7 prepared with water. 5.18 7.37 12.05 10.58 11.61 13.46 3.11 9.83 0 2 4 6 8 10 12 14 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 total sugar, % fig. 6. the total sugar content variation for the dietetic pastry products another aspect noticed is an increase of the total sugar in the order of the energetic addition of the sweeteners used fact seen in the samples with water as well as in the ones with carrot juice. the order of the total sugar increase is sample 1→ sample 2→ sample 3, and sample 4→ sample 5→ sample 6, as the energetic addition brought by the mixture dextrose+saccharine+acesulfame k is higher than that brought by cyclamate+saccharine, and that brought by fructose is higher than that brought by the mixture dextrose+saccharine+acesulfame k. in comparison with sample 1 the total sugar content of sample 3 increased with 57.01%, and in comparison with sample 2 with 29.71%. the total sugar content of sample 6 increased with 21.39% in comparison with sample 4, and in comparison with sample 5 with 13.74%. the fat content of the pastry products must be as high as 35%. the carrot juice used instead of water in the samples 4, 5 and 6 having fat content of 0.3g/100 g, did not influence significantly the fat content of the finished product, the differences being slight. 23.45 24.27 22.72 23.89 24.73 23.51 23.37 24.16 21.5 22 22.5 23 23.5 24 24.5 25 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 fat, % fig. 7. the fat content variation for the dietetic pastry products the reference sample 8 with carrot juice had a fat content with 3.27% higher than the reference sample with water 7. we also observed an increased content in the samples 2 and 5 where the dolcificante sweetener was introduced. sample 2 had a fat content with 3.86% higher than the reference sample 7 and sample 5 with 2.36% higher than reference sample 8. we must say that all the samples had a fat content in the limit imposed for the pastry products. 270.53 289.92 290.4 301.09 309.49 305.63 260.81 297.52 220 240 260 280 300 320 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 energetic value, kcal fig. 8. the energetic value variation for the dietetic pastry products the energetic value (expressed in kilocalories or kilojoules/ 100g of product) is the one that determines the food quantitative appearance, the covering of the daily individual energetic intake and being given by the calorigen trofins: sugars, fats and proteins. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 76 1121.04 1184.61 1205.8 1245.52 1284.42 1269.54 1079.23 1234.26 950 1000 1050 1100 1150 1200 1250 1300 p 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 energetic value, kj fig. 9. the energetic value variation in kj for the dietetic pastry products from the energetic value point of view we noticed an increase in the products containing dextrose or fructose as well as a higher value in the products containing carrot juice in comparison with those containing water. the samples 1, 2 and 3 had an energetic value of 3.73%, 11.17% and 11.35% higher than the reference sample 7. in the same way the samples 4, 5 and 6 had an energetic value of 1.2%, 18.67% and 17.19% higher than the reference sample 8. this thing was due in both cases to the calorigen sweetener adding. the use of carrot juice brought an energetic adding to the samples, increasing the energetic value by 12.33% in the case of the reference sample 8 prepared with carrot juice in comparison with he reference sample 7 prepared with water. 4. conclusion to determine the nutritional value of a food product supposes to emphasize the ratio between the daily necessary of nutritive substances and the substances given by a unit of product (usually 100g). when we talk about the nutritional value of a product we usually refer to: the psycho-sensorial value given by the organoleptic and aesthetic value; the biological value given by the essential amino acids, vitamins and mineral substances; the energetic valuethe number of kilocalories resulted by the body burnings of sugars, fats and protides; the hygienic-sanitary value given by the ratio between the useful and worthless substances and the absence of impurities and some harmful substances. from the psycho-sensorial value point of view the dietetic pastry products prepared with carrot juice had an organoleptic and aesthetic value superior to that of the products prepared with water. carotene in carrot juice brings a plus of color to the product enhancing its appearance. the additions of vitamins (vitamins a, b, c), mineral salts and elements like iron, magnesium, calcium, potassium phosphorus, sodium, of carotene and asparagine leads to the increase of the biological value. the carrot juice addition modifies the energetic value of the products in the sense of increasing it up to 12.33%. the products have a high hygienic-sanitary value being rich in useful substances, lacking impurities, harmful substances, non nutritional substances and microbiological contaminants. regarding the sweeteners used we have noticed that first of all they influence the energetic value of the products producing its increase when they are calorigen, leading to the increase of the total sugar content and influencing the products’ humidity according to their hygroscopicity. when a diet is scientifically made we have to know the nutritive value of the food products, this fact being a basic condition for appreciating their quality level, their general enhancement, being a preoccupation for the specialists in this field. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 77 5. references 1. segal, r., principiile nutriţiei, editura academica, galaţi, 2002; 2. ionete, e., ghid naţional de bune practici pentru siguranţa alimentelor. produse de patiserie şi cofetărie, editura uranus, bucureşti, 2006; 3. rădulescu, e., alimentaţie inteligentă, editura ,,viaţă şi sănătate”, bucureşti, 2003; 4. leonte mihai, tehnologii, utilaje, reţete si controlul calităţii in industria de panificaţie, patiserie cofetărie, biscuiţi si paste făinoase, editura millenium, piatra neamţ, 2004; 5. costin, c.m, segal, r., alimente funcţionale, ed. academica, galaţi, 1999; 6. banu, c., biotehnologii în industria alimentară, editura tehnică, bucureşti, 2000; 7. dabija adriana, tulbure monica, valorificarea produselor secundare din industria berii, editura pim, iaşi, 2010; 8. răducanu, a.e., cercetări privind utilizarea fibrelor alimentare în produse de panificaţie – teză de doctorat, usamv, bucureşti, 2008; 9. bordei despina, tehnologia moderna a panificaţiei, editura agir, bucureşti, 2005; 10. moldoveanu, gh., tehnologia panificaţiei, editura tehnică, bucureşti, 1992; 11. moldoveanu, gh., procedee moderne folosite la controlul proceselor tehnologice în industria morăritului, editura tehnică, bucureşti, 1994; 12. moraru, c., tehnologii moderne în industria morăritului, editura tehnică, bucureşti, 1988 13. giurgiu, e., tărâţele de grâu, revista formula as, nr. 713/2006; 14. bordei, d. ş.a., controlul calităţii în industria panificaţiei. metode de analiză, editura academica, galaţi, 2007; 15. *** sr iso 712:2005 cereale şi produse cerealiere. determinarea umidităţii. metodă practică de referinţă. 16. *** sr 91:2007 pâine şi produse proaspete de patiserie. metode de analiză; 17. *** sr en iso 20483:2007 cereale şi leguminoase. determinarea conţinutului de azot şi calculul conţinutului de proteină brută – metoda kjeldhal. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 35 use of some clay matrices in biotechnology of acid dairy products alisa vasilica arus1, ana maria georgescu 1, iuliana mihaela lazar 1, ileana denisa nistor 1, neculai doru miron 1, azzouz abdelkrim 2, andrea – eliza farkas 1, ramona mihaela cojocaru 1 1food engineering department, „vasile alecsandri” university of bacau, arusalisa@yahoo.com 2département de chimie, université du québec à montréal abstract: in this paper we analyze the use of some clay matrices in the cultivation process of lactic acid bacteria, in dairy industry. the object of this paper is the use of anionic clays hydrotalcite type, with food purity grade, that are synthesized in laboratory, and can be conditioned in porous tablets or pellets form. these are introduced into the culture medium and help optimal growth parameters achievement of lactic acid bacteria by temporary retention of lactic acid. after multiplication and cellular growth, the tablet or pellet insoluble clays, can be removed, washed and recovered. the lifetime (using) of such materials and devices is practically unlimited. according to this study, the cultivation process of lactic acid bacteria includes the immersion in culture medium of a hydrotalcite clay matrix, with anions exchange role, cultivated through continuous cycle of hydrotalcite matrices immersion, removing and recovering, the lactic anion is continuously fixed, achieving at the same time the proton neutralization of lactic acid, with an aqueous phase, separated from the culture medium, with subsequent extraction and separation of lactic acid. the advantages of the proposed process are: about four time decrease of achieved period of lactic bacteria cultivation phase; 2 3 time decrease of final cultures productivity growth; increase with 50-70% rate of bacteria growth in fermentative process; decrease with 30-60% of lactic bacteria cost for industrial fabrication of acid dairy products. keywords: hydrotalcites, lactic fermentation, lactic cultures, lactic acid introduction the growing process for the production of lactic acid is, generally, the same for all of these products, based on the classical method, varying only the conditions of control phase fermentation culture medium depending on the desired culture microbiological component to be selected [1]. the disadvantages of these methods of cultivating lactic bacteria are the difficulty of maintaining the culture medium composition optimum and stable. the composition is changing as microorganisms eat nutrients and after metabolic formation of new substances. the classical growing process of lactic bacteria which serves as the nearest solution consists in sterilization (pasteurization) of milk at the temperature of 63…72ºc, for 0,5 h, cooling milk to the seeding temperature (40...48ºc) for 0,25…0,5 h, adding the milk to seed powder culture, cultivation of lactic bacteria at temperature 36…40ºc and their separation [2]. technologically the growing of lactic acid bacteria is inhibited by two processes: 1) gradual increase of v ph in the culture food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 36 medium, followed by lactose conversion into lactic acid; 2) accumulation of lactic groups. these two processes combined have a bad influence on cellular growth and development. in known processes the lactic acid anion is not removed, the principal inhibitor of cellular development. thus, the classic drawbacks are large losses of bacteria in the fermentative process and low productivity of lactic bacteria, which leads to reduced growth rate of bacteria and growth in the cost of selected final cultures. therefore, according to statistical laboratory data, growth of lactic bacteria from initial concentration of 5…50 cells/ cm3 up to final concentration of 20…30 millions cells/ cm3 takes 8…12h. we propose to solve this problem, by developing a lactic bacteria cultivation technology, to ensure their growth rate during the fermentative process and to reduce considerable the cost of final selected cultures. materials and methods the problem presented above can be solved by making a cultivating lactic bacteria procedure, which includes the preparation of the culture medium, containing sterilized and cooled milk to the seeding temperature, addition of the culture medium prepared by lactic acid bacteria, cultivation and their separation. lactic acid bacteria are used for seeding increase on a nutrient medium consisting of yogurt: fresh pasteurized cow milk, volumetrically taken 1:10 and powdered, which is added by agitation in the culture medium at constant temperature; then as the first choice the culture medium is immobilized in the porous matrix. lactic bacteria cultivation is achieved at the temperature of 36…40ºc for 2…3 hours with the elimination of lactic acid formed by continuous elution of the mobile aqueous phase, which is separated from matrix by the immobilized culture medium and after separation the lactic acid from aqueous phase returns to the culture medium. the cultivation process of bacteria under the second variant includes the introduction in the culture medium of an alkaline agent with food purity, insoluble, which in cultivation time sets continuously the lactic acid formed in the culture medium to achieve continuous cycle entry, discharge and regeneration of basic agent and extraction and separation of lactic acid. the cultivating lactic bacteria according to the third variant includes immersion in the culture medium of a solid insoluble matrix of anion exchange resin, filled with groups of ohexchangeable, r+ohtype, during the cultivation period by continuous immersion cycle, discharge and regeneration of resins into a solution of sodium hydroxide, lactic anion is fixed continuously and with free ohgroup to achieve neutralization of the proton which is made simultaneously with the formation of aqueous phase, which is separated from the culture medium by extraction and subsequent separation of lactic acid. in all these variants, the fermentation processes lasted for 2…3 hours. the common element for all the variants of lactic bacteria cultivation processes is milk sterilization (pasteurization) at the temperature of 63…72ºc for 0,5 h, cooling milk to the seeding temperature (40…48ºc) for 0,25…0,5 h, addition in milk of the culture medium seeding powder, their cultivations for 2…3 h by continuous agitation at the temperature of 36…40ºc and lactic bacteria separation. as initial products we used fresh milk and one of the traditional culture used in milk industry, such as: lactobacillus bulgaricus (for milk fermentation), lactobacillus acidophilus (for acid fermentation of milk), lactobacillus longum (for milk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 37 coagulation), lactobacillus casei (for fermentation and maturation of cheese) etc. culture medium composition includes: yogurt, buttermilk or another medium in which lactic bacteria were grown – 1 ml; pasteurized milk or cow milk fresh pasteurized – 10 ml; lactic cultures specific to the group listed above. for the first variant of proposed processes, as initial products we used fresh milk, water and one of the cultures listed above. as porous matrices we used solid matrixes from organic polymers and mineral categories, for example montmorillonite clay type, or semi porous matrix of cellulose and gels categories. for the second variant we used as base agent: a) a disperse aqueous phase, formed by an organic substance from chelates’ category, dissolved in comestible liquid fat, from vegetables oil class and animal fat. the continuous fixation of lactic acid is made by organization of a relative movement between disperse phase and culture medium, and before evacuation is made, the separation of disperse phase from culture medium takes place. b) an insoluble solid matrix, for example a clay from the category used in medicine such as gastric bandages, which is regenerated by washing and impregnation with a sodium hydroxide solution. the carrying out of the process of cultivating lactic bacteria on the proposed variations is conditioned by three essential factors: 1) attachment velocity of the lactate group; 2) elimination and regeneration speed of the lactate group; 3) attachment capacity of the lactate group. lactic acid separation from de regeneration medium or from aqueous solution was achieved by a known process, for example, by treating with calcium hydroxide to obtain calcium lactate. after filling the precipitate, the liquid was decanted and the sediment was passed through the filter. the liquid was treated with sulfuric acid until the reaction became acid for the calcium decomposition. after the filtration of calcium, sulfate was formed; the diluted acid solution concentration was of approximately 50%, thus obtaining a concentrated solution named „ lactic acid for technical use”. by keeping the concentration of lactic acid at low pressure until the concentration reaches 50...80% we obtained raw lactic acid, which can be used in industry, being purified by re-crystallization of calcium lactate and subsequent treatment with sulfuric acid, resulting free lactic acid. given the complexity of the technical solutions adopted and proposed in this article, we made a statistical study of the process of cultivating lactic bacteria using a factorial program, which took into account the individual effect of all parameters, and their interactions can potentially be synergistic. such a study required a small number of experimental attempts made in short time and with negligible costs. the experimental testing methodology includes the performance of two series of experiments. in the first of them we carried out a lactic bacteria cultivation process. for seeding the pasteurized milk (producer sc prolabac sa bacau, acidity 14…16ºt and 1.8% fat) or fresh pasteurized cow milk we used bacterial cultures grown in yogurt and buttermilk (producer mamy’s dairy sibiu) medium, the volumetric proportion of „yogurt/ pasteurized milk” or “buttermilk/ fresh pasteurized cow milk ” was 1:10. in “buttermilk/fresh pasteurized cow milk” case the composition of milk-culture medium contains: buttermilk mamy’s lactate, sibiu food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 38 – 1 ml; fresh pasteurized cow milk – 10 ml; specific lactic cultures listed above. the fresh pasteurized cow milk composition (energy value 64 kcal/ 100g) was: nutrients – 100 g; fats – 2 g; carbohydrates – 3,9 g; proteins – 3,2 g; calcium – 125 mg; dried substances – 11 g. after some time, the lactic bacteria started to develop, the lactic acid formed in pursuing one of the proposed variants of the process of cultivating lactic bacteria was partially and gradually removed from milk culture – fermentation i medium. under the same conditions of temperature and acidity, we determined the rate of growth and development of the lactic bacteria population that do not depend on the milk-culture medium used, and thus we presented below the results for both milkculture media. we studied three alternative ways of achieving continuous elution process of the lactic acid. 1. the culture medium was immobilized in a fix bed, the conditions of the lactic acid were not disturbed by any of the phases, the milk-culture medium maintaining constant its composition 2. the culture medium was continuously dialyzed through a dialysis membrane 3. immobilization of milk-culture medium in a solid or semi-solid matrix and continuous elution of the lactic acid in the aqueous mobile phase. we have noticed that the first way raises the problem of excessive consumption of culture medium and the necessity of recycling, which leads to the increasing of technology spending. the major inconvenience of the second procedure consisted in very small values of efficiency, because of the relatively slow kinetics of the diffusion process of lactic acid during passage through the membrane. the main problem arises from the concern to ensure a good transfer of the lactic acid into the mobile aqueous phase that must be separated. the third alternative method proved to be the most judicious that was adopted as optimal technical solution for the first variant of the proposed process of formation of lactic acid bacteria. the problem was reduced by creating a porous matrix of organic polymers, cellulose, gels and mineral polymers type montmorillonite, in which the pore size allows the development of bacteria mass. in the second series of experiments, yeast (grown culture medium), obtained in the fermentation process we used clot pasteurized milk (producer sc prolabac sa)fermentation ii, to determine the degree of efficiency of the lactic ferment obtained, which is estimated in range of the increase in acidity of inoculated milk in fermentation process ii. in this variant the culture fluid is not fixed, but has a mobile fluid phase. as insoluble alkaline agent we used a liquid dispersed phase, which comes across milk-culture medium, and before discharge it was separated from the culture medium. the testing of various lactic acid binding agents proved that in this purpose the organic substances of chelate type (a complexing agents class) that have a great affinity for acids are better suited. these compounds, before being added to the culture medium, were dissolved in a fat food fluid (vegetable oil or animal fat). the fermentation period depends on the dose of reducing agent of lactic acid concentration, which in this variant was dosed by varying the amount of insoluble alkaline agent. as a criterion to start the fermentation period, we chose the initial concentration of cells in culture medium – food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 39 milk 20 cells/ m3, and as a finalization criterion of the fermentation period we chose the final concentration of bacterial population of 25 million cells/ m3. after selection of lactic bacteria grown for each sample from fermentation i, we used in fermentation ii, the efficiency of the culture bacteria grown in fermentation i testing at 360c for 7 hours and determining the average speed increase of acidity of milk. alternatively, cultivation of lactic bacteria was carried out by stirring at the constant temperature of 37 c. in this variant the technical solution proposed supposes that the culture medium remains fluid and the lactic acid formed is fixed on some movable or immovable matrices (surfaces) with basic character. surfaces were chosen to satisfy the following conditions: have sufficient physical adsorption capacity for each contact cycle in milk-culture medium have no cation exchange properties, otherwise, the lactate group will not be evacuated anion exchanger to allow elimination of lactate group to have a renewable surface to have a long life to present inertia in relation to other constituents of culture medium to have a low cost. such a surface can be achieved relatively easily, for example a classical anion resin type. it was found an advantageous variant of such a varied hydrocalcit similar to that used for the treatment of stomach hyperacidity (clay, used in medicine as gastric bandages). the surface can be easily made in laboratory according to standard procedure. hydrocalcits are also much cheaper than ion exchange resins. the fermentation period depends on the dose reducing agent concentration of lactic acid; which in this variant was assayed by the amount of hydrocalcit. as criteria for stating fermentation period we chose the initial concentration in milk-culture medium 20 cells / m3 and quality criteria for finalization fermentation period, we chose the final concentration of bacteria’s population of 25 millions cells/ m3. after the selection of grown lactic bacteria for each sample from fermentation i, these ones were used in second fermentation of efficiency testing of growth culture in first fermentation, achieved at 36ºc for 7 h and determining medium growing velocity of milk acidity. results and discussion the results of the three different methods of cultivating lactic acid bacteria consist of: reduction of four times the period of implementation of lactic acid bacteria growing phase; increase of 2…3 times the growth productivity of the final culture increased with 50…70% the growing process rate of bacterial fermentation; reduction with 30…60% of lactic bacteria cost for industrial production of lactic-acid products (buttermilk, kefir, sour milk, cheese etc) depending on the procedure used; use of cheap, affordable, virtually unlimited lifetime materials, which do not affect the properties of dairy and food products and are not a health risk for consumers either; obtain supplementary lactic acid an additional valuable product for food, chemical, leather industries and medicine. conclusion the result obtained is due to the following issues: the lactic acid inhibiting cell growth and proliferation of lactic acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 40 bacteria, used to obtain cheese and lactic acid-products was removed from the culture medium; contrary to known methods, which do not eliminate lactic anion, the main inhibitor of cell growth, the technology proposed expects the fixation of this ion by anion-exchange materials; the lactic acid elimination which represents the main reason of the present study, is achieved by using insoluble materials with a high food purity grade and with basic characteristics; insoluble material technologies and technological interest, which correspond to the above, can be obtained by ion exchange resins; insoluble material of technological interest, corresponding to the above mentioned requirements, can be also obtained on anionic clay of hydrocalcit, having a similarly structure with gastric bandages used in hyperacidity treatment of people; anionic clay of hydrocalcit type can be synthesized in laboratory based on calcium, magnesium carbonate and sodium aluminates, after a 6 h of maturation period; anionic clays can be conditioned under pills form or as a porous plate that is inserted into the culture medium for lactic acid removal; after multiplication and cell growth, the pills or insoluble clay plates can be extracted, washed and regenerated with naoh; life (use) of such materials and devices is virtually unlimited. references: 1. chintescu g. îndrumător pentru tehnologia produselor lactate, bucureşti, ed. tehnică, 1982, p. 175-177 2. constantin i. milică, biotehnologiile viitorului, iaşi, ed. ion ionescu de la brad, 1999, p. 232 – 233. 3. nistor d.i., azzouz a., miron n.d ingineria proceselor chimice şi biochimice, ed. tehnica-info, chişinău 2006; 4. nistor i.d., jinescu c., azzouz a., ingineria proceselor biotehnologice şi alimentare, vol. i, ed. alma-mater, bacău, 2008. 5. nistor i.d., jinescu c., azzouz a., ingineria proceselor biotehnologice şi alimentare, vol. ii, ed. alma-mater, bacău, 2008. microsoft word 3 journal nr 4 2010 din 8 noiembrie_57-62.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 61 effect of some natural herbs incorporation in sunflower oils on its resistance at frying temperatures bianca furdui1, rodica-mihaela dinică1, mariana lupoae 2*, romică creţu1 1dunărea de jos university of galati, faculty of sciences, chemistry department, domnească 111, galati, 800201, romania, rodinica@ugal.ro, bfurdui@ugal.ro 2ovidius university constanta, faculty of natural and agricultural sciences, street mamaia no. 124, 900527, constanţa, romania, mariana_lupoaie@yahoo.com abstract: sunflower oil is an excellent source of essential fatty acids necessary for human body. extending the frying life of oil is of commercial and economic importance. therefore, improving the thermal stability of cooking oils could provide considerable savings to the food processors. the present work aims at evaluating thermal resistance of sunflower oil after the incorporation of three species of natural herbs, from salvia genus and allium ursinum, used in food as condiments or under other forms. by this treatment, we seek to preserve almost unchanged the oil quality during thermal treatment, by increasing the level of antioxidants from oil. for this study, four different frying temperatures i.e. 110, 150, 180 and 200ºc were applied for 30 minutes to sunflower oil before and after the addition of plants. at 110 ºc we have also made a kinetic study on samples stability in time. official aoac methods were used to determine free fatty acids content (ffa) and peroxide value (pv) of all samples during heating. the quality parameters of sunflower seed oil were improved by treatment with either salvia officinalis., salvia sclarea or allium ursinum (wild garlic). salvia and wild garlic exhibited a comparable high ability in reducing peroxide value (pv), probably due to their high content of polyphenols and flavonoids. in conclusion, the incorporation of salvia and wild garlic into sunflower oil helped improve its thermal resistance and stability, which sustains their use in order to extend frying life of oils during food processing. keywords: sunflower oil; salvia; wild garlic; heat treatment; pv; ffa. introduction oils used in the home and oils used commercially require significantly different properties. perhaps most importantly, commercial oils need to withstand intense heat and frying for longer periods of time. during the frying process, a number of changes take place in fats and oils, depending on the type of oil used and the food fried [1,2]. so complex thermolytic and oxidative reactions occur, leading to the formation of new compounds such as diacylglycerols, monoacylglycerols, free fatty acids (ffas), monomers, polymers, and so on, which are harmful to the human body [3,4]. however, the most interesting transformations are ffa content, viscosity and colour change of the vegetable cooking oil as well as formation and decomposition of hydro-peroxides and polymerization via complex free radical processes at elevated temperatures above 160ºc [5]. extending the frying life of oil is of commercial and economic importance. therefore, improving the thermal stability of cooking oils could provide considerable savings to the food processors. in order to food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 62 increase stability, many healthy oils must be hydrogenated for commercial use, a process that adds unhealthy transfats. therefore, the present work aims at evaluating the thermal resistance of sunflower seed oil before and after treatment with three kinds of aromatic plants, from salvia and allium families, used in alimentation as condiments or under other forms. by this treatment, we believed that the quality of frying oils can be kept almost constant throughout heat treatment, by increasing of oil’s antioxidants content. the choice of the sunflower oil has been determined by its very frequent use in home and food industry. sunflower (helianthus annuus) is one of the four major annual oilseed crops produced in the world [6]. being an excellent source of the essential fatty acids required by the human body, sunflower oil is among the healthiest vegetable oils available. the linoleic acid is one of its essential unsaturated fatty acids [7]. also, sunflower oil is an important source of vitamin e (45 mg tocoferol/100g). aromatic plants have been used since ancient times in food flavorings, pharmaceuticals, cosmetics and perfumery. essential oils or some of their constituents such as polyphenols, flavonoids and carotenoids present biological activities, including antimicrobial and antioxidant properties [8,9]. since antiquity, salvia species have been well known plants and widely used as folk medicines with antibacterial, antituberculosis, antiviral, cytotoxic, cardiovascular, liver protective and other properties [10-15]. sage is also used to preserve foods, especially meat and cheese, due to its antioxidant properties, as well as being employed as a spice for flavoring. phytochemical investigations have shown that salvia species are mainly rich in diterpenoids and triterpenoids (ursolic acid, oleanic acid) as well as in flavonoids and other phenolic compounds (tannins, cholorogenic, p-cumaric, cafeic and nicotinic acids) [16-20]. allium is the largest and most important representative genus of the alliaceae family and comprises 450 species, widely distributed in the northern hemisphere. besides the well known garlic (allium sativum l.) and onion (allium cepa l.), several other species are widely grown for culinary use, such as leek (allium porrum l.), scallion (allium fistulosum l.), shallot (allium ascalonicum hort.), wild garlic (allium ursinum l.), chive (allium schoenoprasum l.) etc.. allium species are a rich source of phytonutrients, useful for the treatment or prevention of a number of diseases, including cancer, coronary heart disease, obesity, hypercholesterolemia, diabetes type 2, hypertension, cataract and disturbances of the gastrointestinal tract (e.g. colic pain, flatulent colic and dyspepsia)[21]. due to its composition rich in volatile oils, minerals and oligoelements, flavonoids, polyphenols, vitamins (c and b complex), sulfur compounds, the wild garlic (allium ursinum) has garlic’s properties but stronger curative power. experimental considering their interesting properties, we wanted in particular to test the effect of salvia officinalis, salvia sclarea and allium ursinum plants, which are widely used as condiments in salads in most of the countries of the balkan peninsula. in order to test the contribution of natural herbs to the thermal resistance of sunflower seed oil, the evolution of peroxide value (pv) and free fatty acids content (ffa) during heat treatments, was investigated before and after oil aromatization with the three species of plants. we studied the evolution in time of those parameters at a determined frying temperature, and the evolution at different food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 63 frying temperatures for a constant period of time, too. we used sunflower oil directly from a technological flow, without added antioxidants. plants were bought from local market (salvia off. and allium ursinum) or harvested from the botanical garden of galati (salvia sc.). all chemicals used were of analytical grade. oil treatment with natural herbs the aerial part of plants dried in air, mortar and sieved to 630 m, was incorporated into sunflower oil at 2% (w/v). after ultrasonic stirring for 30 minutes, samples were kept in refrigerator for 7 days. after this treatment, the undesirable colour observed in the mixture was removed by an additional decolorizing step, using cameroun clay (50 m). thermal stability evaluation in order to evaluate the effect of sunflower oil treatment with an aromatic herb on its thermal resistance, both untreated oil and treated oils were heated for 30 min at 110, 150, 180, and 200° c, respectively. also, a kinetic evaluation of samples was made at 110°c. peroxide value determination the peroxide value (pv) was determined by using the aoac method (aoac, 1999). about 1 g of oil was weighed into a 250 ml iodometric flask. previously prepared acetic acid–chloroform (1:2) solution (6 ml) and saturated potassium iodide (1 ml) were added. after 3 min. stirring and 5 min. rest, the mixture was titrated with 0.01 n na2s2o3 until yellow colour is almost gone. approximately, 5 drops of 1% starch solution was added, and titration was continued with shaking vigorously to release all iodine from chcl3 layer, until the blue colour just disappeared. in parallel, a blank sample is similarly treated. pv was calculated by using the following equation: pv=s·n·1000/mp (meq na2s2o3/kg oil) where, s is the ml na2s2o3 (blank corrected), n is the normality of na2s2o3 solution and mp is the mass of oil sample (g). ffa determination ffa content was determined in triplicate, by the titration method of aoac (aoac, 1999). about 1 g of oil was weighed into a 250 ml flask. 10 ml benzene: alcohol (1:1) mixture and 3-4 drops of 1% phenolphthalein, as indicator, were added. the mixture was titrated with 0.1 n naoh with vigorous shaking until permanent faint pink colour appeared and persisted at least for 1 min. the ffa value was calculated according to the following equation: sample] naoh/g [mg v/m 3.9998 pai . where mp is the mass of the oil test portion (g), and v the volume of naoh consumed (ml). results and discussion peroxide formation is a major concern from the point of view of rancidity and toxicology in fried oils. food lipid oxidation products such as peroxides, malonaldehyde, and several cholesterol oxidation products are reported to cause atherosclerosis and coronary heart disease [22]. the evolution in time of pv (meq. of peroxide per kg of sample) of the sunflower oil samples during heat treatment at the 110°c is shown in fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 64 0 5 10 15 20 p er ox id e va lu es 0' 30' 60' 90' time (min) original sunflower oil sunflower oil with salvia sc. sunflower oil with salvia off. sunflower oil with wild garlic fig.1. effect of salvia and wild garlic addition to sunflower oil on time evolution of peroxides (pv) formed after exposure at 110°c temperature the change in pv of the untreated and treated sunflower oil during heat treatment, at different frying temperatures is shown in fig. 2. 0 2 4 6 8 10 12 14 16 p er ox id e va lu es 25 110 150 180 200 heating temperatures (celsius degrees) original sunflower oil sunflower oil with salvia sc. sunflower oil with salvia off. sunflower oil with wild garlic fig.2. effect of salvia and wild garlic addition to sunflower seed oil on peroxides (pv) formed after exposure to different frying temperatures. the obtained results showed a significant difference between pv of treated (sunflower oil with salvia sc., salvia off. and with allium ursinum) and pv of untreated (original sunflower oil) samples during heating. although the same temperatures were applied to all samples, the amount of peroxides found in untreated sample was higher than that of peroxides contained in treated samples. however, no big differences were observed between pv of treated oil samples. the peroxides values increased in 90 minutes from 7 to 20 meq/kg for original sunflower oil, from 6.25 to 12 meq/kg for sunflower with salvia sc., from 6 to 13 meq/kg for sunflower with salvia off. and from 6.5 to 12.5 meq/kg for sample with allium ursinum, after heating at 110°c. as the temperature was raised from 25 to 200°c respectively, pv for original sunflower oil increased from 7 to 16 meq/kg and for treated oils the increase was smaller, about from 6 to 12 meq/kg. another important indicator of oil deterioration during heating is the ffa content. during frying, at elevated temperatures (160–180°c), in the presence of air and moisture, free fatty acids resulted from the hydrolysis of triacylglycerols as well as from further decomposition of hydroperoxides. the released fatty acids are more susceptible to thermal oxidation at frying temperatures. the oxidized products of fatty acids give the off-flavors and odors (hydrolytic rancidity) to the frying medium and fried foods. therefore, controlling the level of ffa within a reasonable range would prevent the breakdown of fats. in the present study, the ffa content increased from 1.2 to 2.6 (mgnaoh/g sample) in the original sunflower oil, from 1.2 to 1.5 (mgnaoh/g sample) sunflower with salvia sc. and salvia off. and from 1.2 to 1.45 (mgnaoh/g sample) in the sunflower oil with wild garlic, by heating from 25 to 200 °c, respectively, as shown in fig. 3. 0 0.5 1 1.5 2 2.5 3 fr ee f at ty a ci d s (m g n ao h /g sa m pl e) 25 110 150 180 200 hea ting tempera ture s (ce lsius degree ) original sunflower oil sunflower oil with salvia sc. sunflower oil with salvia off. sunflower oil with wild garlic fig.3. effect of salvia and wild garlic addition to sunflower seed oil on free fatty acids (ffa) formed after exposure to frying temperatures. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 65 the analysis of results shown in fig. 1 revealed the existence of a significant difference between treated and untreated samples during heating. however, it did not show any difference between sunflower oil with salvia species and sunflower oil with wild garlic samples. conclusions in the present work we have studied the stabilizing effect of aromatizing plants from salvia and allium ursinum (wild garlic) species added to sunflower oil, during heat treatments. the obtained results showed that the quality parameters of sunflower oil following exposure to frying temperatures were improved by the addition of natural herbs. in fact, though the pv and ffa increased with temperature for all samples, however, the evolution of studied parameters in treated oil was found to be slower than the one observed in untreated oil. so, sunflower oil with salvia and wild garlic samples exhibited relatively reduced peroxides values and ffa contents, following heat treatment, comparatively with the original sunflower oil. in conclusion, our studies have proved that these plants (salvia officinalis, salvia sclarea and allium ursinum) used in alimentation for their aromatizing and curative properties, exhibit also good properties as antioxidants and/or free radical scavengers. thus, their incorporation into sunflower oil helped improve its thermal resistance and stability, which support their use to control lipid oxidation during food processing. this may be due to the abundance of natural antioxidants (such as polyphenols and flavonoids) present both in salvia and wild garlic which were transferred into sunflower seed oil following its treatment with natural herbs. in fact, these natural components can react with free radicals of the frying oil, acting synergistically as free radical scavengers and/or contributed to the protection of tocopherols, susceptible of heat-induced loss, particularly to the protection of -tocopherols, the main antioxidant contained in original sunflower seed. the obtained results comply with other studies regarding the effect of natural herbs on oil stabilization [23]. further studies will be carried out to understand better the factors influencing antioxidant activity of those aromatizing plants. references 1. r. demir, m. bas-han, a study on the investigation of fatty acids in pulps and seeds of olea europae l. (hırhali) and olea europae l. (zoncuk), biochemical archives, 14, 1998, 187– 191; 2. r. demir, b. otludil, investigation of the saturated and unsaturated fatty-acids in the internodes and leaves of vitis-vinifera l. (mazruma), biochemical archives, 13(4), 1997, 223–228; 3. w. l clark, g. w. serbia, safety aspects of frying fats and oils, food technology, 45(2), 1991, 84–86, 88–89, 94; 4. p. j. white, an overview of methods for measuring changes in deep-fat frying oils, food technology, 45(8), 1991, 75–80; 5. m. m. blumenthal, a new look at the chemistry and physics of deep fat frying, food technology, 45(2), 1991, 68–71; 6. k. m schmidt, sunflower oil processing manual. bismarck, north dakota: national sunflower association, 1991; 7. j. m. bourre, m. dumont, m. piciotti, g. pascal, g. durand, les acides gras du cerveau; nature, role, origine, leur importance alimentaire, in congres international chevreul pour lţetude des corps gras, proceeding, angers, 1989, 49–81; 8. a. loo, h. richard, nature, origine et proprietes des epices et des aromates bruts, in h. richard (ed.), epices et aromates, paris, tec. & doc. lavoisier, 1992, 18–22; 9. v. prakash, leafy spices, boca raton, fl: crc press inc, 1990; 10. a. ulubelen, s. öksüz, g. topçu, a. c. gören, w. voelter, antibacterial diterpenes from the roots of salvia blepharochlaena, journal of natural products, 64, 2001, 549–551; food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 66 11. a. ulubelen, g. topçu, h. lotter, h. wagner, c. eris, triterpenoids from the aerial parts of salvia montbretii, phytochemistry, 36, 1994, 413–416; 12. g. topçu, e. a. altiner, ş. gozcu, b. halfon, j. m. pezzuto, d. g. i. kingston, studies on diand triterpenoids from salvia staminea with cytotoxic activity, planta medica, 69, 2003, 464–467; 13. a. ulubelen, cardioactive and antibacterial terpenoids from some salvia species, phytochemistry, 64, 2003, 395–399; 14. a. ulubelen, h. birman, s. oksüz, g. topçu, u. kolak, a. barla, cardioactive diterpenes from salvia eriophora, planta medica, 68, 2002, 818–821; 15. l. zhou, z. zuo, m. s. s. chow, danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. therapeutic reviews/herbal medicine, the journal of clinical pharmacology 45, 2005, 1345–1359; 16. g. topçu, a. ulubelen, structure elucidation of organic compounds from natural sources using 1d and 2d nmr techniques, journal of molecular structure, 834–836, 2007, 57–73; 17. g. topçu, z. türkmen, j. k. schilling, d. g. i. kingston, highly hydroxylated triterpenes from salvia kronenburgii., journal of natural products, 67, 2004, 118–121; 18. g. topçu, n. tan, a. ulubelen, d. sun, w. h. watson, terpenoids and flavonoids from the aerial parts of salvia candidissima, phytochemistry, 40, 1995, 501–504; 19. y. lu, l. y. foo, polyphenolics of salvia – a review, phytochemistry, 59, 2002, 117–140; 20. volkan yeşilyurt, belkıs halfon, mehmet öztürk, gülaçtı topçu, antioxidant potential and phenolic constituents of salvia cedronella, food chemistry 108, 2008, 31–39; 21. virginia lanzotti, review. the analysis of onion and garlic, j. chromatogr. a, 1112, 2006, 3– 22; 22. r. subramanian, k. e. nandini, p. m. sheila, a. g. gopalakrishna, k. s. m. s. raghavarao, m. nakajima et al., membrane processing of used frying oils, journal of the american oil chemistssociety., 77(3), 2000, 323–328; 23. meriem bensmira, bo jiang, consolate nsabimana, tang jian, effect of lavender and thyme incorporation in sunflower seed oil on its resistance to frying temperatures, food research international 40, 2007, 341–346. 250 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 250 255 technological properties of white brined cheese produced from organic certified goat milk *aleksandar saveski1, tatjana kalevska1, dragan damjanovski1, viktorija stamatovska1, elena joshevska2 1faculty of technology and technical science-veles, st.kliment ohridski university, bitola dimitar vlahov nn str., 1400 veles, r. macedonia; 2faculty of biotechnical science, st. kliment ohridski university, bitola partizanska nn str., 7000 bitola, r. macedonia *corresponding author saveskia@yahoo.com received september 5th 2015, accepted september 25th 2015 abstract: the research has been conducted on organic goat milk, certified by eu equivalent regulations reg. 834/2007 and on two types of white brined cheese using two types of lyophilized starter cultures. a combined mesophilic and thermofilic starter culture (lactococcuslactisssp.lactis, lactococcuslactis ssp. cremoris, streptococcus termophilusi lactobacillus bulgaricus) was used for the first type of cheese and a thermofilic starter culture for the second type of cheese. the results showed that the use of starter cultures in cheese production did not affect negatively the cheese components. sensory panel showed that the highest average sensory evaluation points were recorded in cheeses made with mesophilic and thermofilic starter culture, whereas other starter culture combinations had been affected less in regard to taste or appearance. keywords: organic, goat milk, white brined cheese, starter culture, acidity, sensory analysis 1. introduction over the last years the production of organic food has reached an expansion in the world, thus changing the consumers’ habits and the number of those who decide to buy organic products is increasing, including the dairy products. compared to the conventional production, the organic one complies with the legal standards and regulations for control of each phase of the production process, which contribute to the production of safe products of animal origin. the organic system of production is made according to determined standards which do not allow the use of antibiotics, coccidiostats, medicinal apparatus, growth stimulators, genetic modifications, hormones and other chemical substances in the whole production process [10]. for dairy products, cow milk is used most of the time, but there is always a reason to change our eating habits. goat milk is considered one of the healthiest food, its chemical composition being similar to human milk and has specific taste and smell, thus being unacceptable for the consumers [1,2]. it is believed that goat was the first animal humans used for the production of milk. the ancient greeks and romans drank goat milk, because they believed it was very healthy [2]. since ancient times goat milk has been used all over the world not only as a food, but also in the treatment of many diseases such as: bronchitis, allergies, by providing better immunity, treating and strengthening of the lungs. b http://www.fia.usv.ro/fiajournal mailto:saveskia@yahoo.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska,production and characteristics of white soft marinated cheese from organic certified goat milk, food and environment safety, volume xiv, issue 3 – 2015, pag. 250 – 255 251 vitamins of the goat milk are beneficial for the nervous system, it has higher concentration of calcium, being important in building strong bones and keeping normal blood pressure [1]. the nutritional content of goat milk ranks it as the most balanced dairy product used in the production of cheese. there are 400 varieties of goat cheese and due to their specific taste and nutritional value, they have higher price on the market. the characteristic smell of goat milk is given by the higher quantity of capric, caprylic and caproic acids. [3]. the characteristics and quality of the cheese depends mostly on the quality of the milk, i.e. its physicalchemical composition. the most variable component of goat milk is the fat content which depends mostly on the stadium of lactation (2-8%). the content of milk fat at the beginning of the lactation is 3.34%, in the middle of lactation 2.73% and at the end 4.58% [4]. the proteins from goat milk are more digestible than those of cow milk and the absorption of amino acids is more efficient [5]. goat milk protein content is 3 to 4.5%. goat milk has less lactose ranging from 4.3 to 4.8% compared to cow milk, giving it advantage among the lactose intolerant human population [6]. 2. matherials and methods the analysis of the chemical composition of goat milk was made in the study, where the content of the milk fat, also proteins, lactose and dry matter were dewtermined by infrared analyzing machine milcoscan in accordance with the idf 141c:2000 standard.the total solid, fat, raw protein, ash were also determined according to aoac (2009) and ph was determined by ph meter metler –toledo. the sensory evaluation of the two types of goat cheese is made by method of points, a quantitative descriptive method for sensory analysis [7,8]. the samples of the two types of cheese were coded with letter a and b. for each of the cheese characteristics profile: cross-section, consistency, colour, smell and taste, a coefficient of importance was determined. the sensory characteristics of the goat cheese are valued with marks from 1 to 5 multiplied by the coefficient of importance, and their collection is expressed in (%) from the maximal possible quality. the balanced middle value, i.e. the balanced general value of the quality of the goat cheese is made by dividing the maximal possible quality with the summation of the importance coefficient (σ=20) [7]. the technology of goat cheese production: one hundred (100) liters of goat milk collected from the same lot is used for the production of the two types of cheese.  the pasteurization of goat milk for the two types of cheese is made at 720с for 15/sec.  cooling of milk to temperature of 350с for inoculation of starter cultures  inoculation of starter culture frc-75 which consists of combination of mesophilic and thermophilic milkacidic bacteria (lactococcus lactisssp.lactis, lactococcus lactis ssp.cremoris, streptococcus termophilus, and lactobacillus bulgaricus) and maturing of the milk for 30 minutes.  addition of solid cacl2 previously dissolved in water in the amount of 10 gr/100 liters of milk.  curding of milk on temperature of 340с with rennetenzyme (chrhansen) in quantity which will provide the curding of the milk in 60 minutes.  the curd was then cut into small cubes (2x2x2 cm). after draining, salt at 2% (w/v) was mixed with the curds.  the curd was poured into small clean wooden moulds lined with cheese cloth and pressed for 5 hours. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska,production and characteristics of white soft marinated cheese from organic certified goat milk, food and environment safety, volume xiv, issue 3 – 2015, pag. 250 – 255 252  cutting of the cheese in moulds with dimensions of 11 х 8 cm.  addition of brine (16% nacl) 12ºc for 12 hours)  the packaging of the cheese in plastic cans which are filled in 10% brine.  the ripening of cheese is 30 days on 180с.  lagering of cheese until sale (2-4 0с). cheese а: starter culture with the combination of mesophilic and thermophilic milk-acid bacteria is used (lactococcus lactisssp.lactis, lactococcus lactis ssp.cremoris, streptococcus termophilus and lactobacillus bulgaricus) and maturing of milk for 30 minutes. cheese b: inoculation of cultivated thermophilic culture (str.thermophilus and lactobacillus delbruecki, sub. bulgaricus). 3. results and discussion the mean data of organic goat milk samples for the physical-chemical composition and ph are presented in (table 1). table 1. physical-chemical composition of organic goat milk parametres value (%) milk fat 5.14 proteins 3.28 dry nofat matter 8.28 lactose 4.34 minerals 0.65 ph value 6.61 from the results of table 1 we can determine that the content of milk fat in the goat milk used to make the two types of cheese is of 5.14%, proteins of 8.28%, lactose is of 4.34%, minerals of 0.65%, and dnm of 8.28%.ph value of the goat milk is in the range of the standard milk of 6.61%. considering the fact that the research was conducted in autumn, the goat milk is higher in dry matter, so it has a higher content of milk fat and proteins. these findings were in agreement with those reported by other authors [4,6]. 3.1 determining the dynamics of ph value of cheese goat cheese has characteristic ph value which indicates the degree of conversion of lactose into milk sugar. the ph value of the curd and the two types of cheese (a and b) during ripening are shown in table 2. table 2. ph value of goat cheese (types а and b) phvalue а b coagulum 6.15 6.04 after cutting 5.07 4.72 after salting 4.91 4.60 15 th of rippening 4.50 4.48 30ty day of rippening 3.85 3.90 from the results it can be seen that ph value of the cheese a coagulum is of 6.15 and it is higher as compared to the ph value of cheese b coagulum with 6.04. after the cheese is cut and salted a significantly lower ph value of cheese b compared to cheese a was determined, showing greater activity of cultivated thermophilic starter culture compared to the combined starter culture. after thirty (30) days, at the end of the ripening process, a continuous decrease of the ph value is registered in both types of cheese (figure 1). the differences in ph values in the two types of cheeses are due to the different starter cultures used in the production technology. during the ripening of cheese, complex biochemical processes take place and they change the structure of the cheese components which determine the sensory evaluation, giving characteristic taste, smell and texture. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska,production and characteristics of white soft marinated cheese from organic certified goat milk, food and environment safety, volume xiv, issue 3 – 2015, pag. 250 – 255 253 fig.1. dynamics of ph over the ripening period 3.2.chemical composition of goat cheese during rippening, the three main milk components (lactose, proteins and fat) take part with different percentage. the modifications start by transforming the lactose into milk acid which is more common in the first days of ripening. the biggest changes during the rippening of goat cheese are in the proteins and less in the milk fat. the average values of the chemical composition of the two types of goat cheese a and b produced by certified organic goat milk are presented in (table 3). fat content in cheese a is 22.92%, and 23% in cheese b. the protein content in cheese a is of 14.09%, and of 13.98% in cheese b, respectively. the water content of cheese a is of 52.12% which is by 2.02% higher than that of cheese b. from the data we can determine that the starter cultures used with different microbiological content influence the process of cheese rippening with repercussions on the quality of goat cheese. table 3. chemical composition of goat cheese (types a and b) parameters (%) а b water 52.12 50.10 dry matter 47.89 45.90 fat 22.92 23.00 proteins 14.09 13.98 nacl 3.05 3.00 3.3 sensory evaluation of goat cheese the sensory characteristics of goat cheese: smell, taste, cross-section, consistency, colour, profile are important parameters which influence the quality of the product, including the consumers’ product choice [9]. the mean sensory scores of white soft brined cheese samples are presented in table 4. table 4. sensory evaluation of goat cheese (types a and b) sensory characteristics a b profile 5.00 5.00 cross-section 4.75 4.88 consistency/texture 5.00 4.50 colour 4.88 4.88 smell 4.88 4.10 taste 4.63 3.88 average value 4.85 4.56 we can see from the sensory scores that the general suitability of cheese a has got an average grade of 4.85, while the general suitability of cheese b has got average grade of 4.56 (fig. 2). the profile of the two types of cheese is excellent and has got an average grade of 5 for both types. the consistency i.e. the texture of cheese a is standard hard with grade 5, while 0 1 2 3 4 5 6 7 coagulum cheese a cheese b after cutting after salted 15th 30ty ph va lu es rippening period food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska,production and characteristics of white soft marinated cheese from organic certified goat milk, food and environment safety, volume xiv, issue 3 – 2015, pag. 250 – 255 254 cheese b is harder and has got a grade of 4.5. fig. 2 sensory scores of goat cheese samples the cross-section of the two types of cheese was normal, with very few small holes, which are more common in cheese a. the cross-section of cheese a has got a grade of 4.75 and cheese b has 4.88. the colour of the two types of cheese is significantly white with a grade of 4.88. cheese samples showed characteristic smell of white brined cheese. higher amounts of caproic and caprylic acid in cheese seem to contribute to the development of piquant smell and taste. the taste of cheese a has got a grade of 4.63, while cheese b registered a slight deviationfrom the normal taste, getting a grade of 3.88. from the results (fig.2) we can determine that goat cheese a has better marks for the sensory characteristics as compared to cheese b, and we can conclude that the different starter cultures used for the production of the two types of cheese (a and b) influence the sensory evaluation of the cheese. 4.conclusion from the nutritional point of view goat milk is a more balanced product compared to cow milk, since it has all the necessary nutrients for a healthy body. the increased production of goat milk is the result of several attributes that makes it be by far a superior choice. goat milk is less allergenic, naturally homogenized, easier to digest, lactose intolerant friendly, and biochemically and thermodynamically superior to cow milk. the purpose of our research was to determine the differences of the two types of goat cheese made from milk from the same lot with the same physical and chemical composition, emphasizing that the variations in the physical and chemical parameters of the two types of goat cheese are not due to the physical or chemical composition of the goat milk. the differences of ph values of the two types of goat cheese are the result of the different starter cultures used in the process of cheese production. according to the given scores for the sensory evaluation the total suitability of goat cheese a had best results with average of 4.85 compared to goat cheese b which had mark of 4.56. the taste, consistency and smell are better marked in cheese a as compared to cheese b. the results from our research show that the starter cultures used in the production process of goat cheese influence the differentiation of milk component during the rippening of cheese; the biggest difference seen in the sensory characteristics of the goat cheese gives its specific quality. 5. references [1] presilski s, presilska n: nekonvencionalni izvori na mleko, fakultet za biotehnick inauki, univerzitet sv. kliment 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 1 2 3 4 5 6 5 4.75 5 4.88 4.88 4.63 5 4.88 4.5 4.88 4.1 3.88 cheese a cheese b profile cross-section consistency food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska,production and characteristics of white soft marinated cheese from organic certified goat milk, food and environment safety, volume xiv, issue 3 – 2015, pag. 250 – 255 255 ohridski bitola, fackultet za biotehnicki nauki, (2006) [2] presilski s.: proizvodstvo na sirenje i puter, fakultet za biotehnicki nauki, univerzitet sv. kliment ohridski bitola, (2004) [3] babayan v. k.: medium chain length fatty acid esters and their medical and nutritional application. j. am. oil chem. soc., 59, 49a-51a, (1981) [4] antunac, n., samarźija, d., lukać havranek, j., pavić, v., mioć, b.: effects of stage and number of lactation on the chemical composition of goat milk. czech journal of animal science, 46(12), 548-553, (2001) [5] park y. x.: nutrient and mineral composition of commercial us goat milk yogurts.small ruminant research, 13, 63-70, (1994) [6] haenletn, g.f.w., (1992.): role of goat meat and milk in human nutrition. v international conference on goats:. indian council of agricultural research, new delhi. 575-580, (1992) [7] kocoski lj.interna skripta za senzorna analiza na hranata, fakultet za biotehnicki nauki, univerzitetsv. kliment ohridski bitola (2010) [8] dehlholmc.: descriptive sensory evaluations, comparison and applicability of novel rapid methodologies, phd thesis, (2012). [9] popov-raljić j., radovanović r.: senzorna analiza u funkciji urvrdivanja bezbednosti i kvaliteta prehrambenih proizvoda, savremena poljoprivredavol. 56, 5, pp. 142–149, novi sad, (2007) [10] european organic regulations (ec) no 834/2007, 889/2008 and 1235/2008, ifoam eu group, brussels 2012. microsoft word 3 journal fia nr 1 2010 final_21-26.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 25 qualification of polypropylene/calcium carbonate nanocomposites to radiation degradation traian zaharescu1, silviu jipa1,2 1 incdie-icpe ca, 313 splaiul unirii, p. o. box 149, bucharest 030138, romania 2 faculty of sciences, “valachia” university, 18-22 unirii av. târgovişte 130082, romania abstract: this paper describes the stability of irradiated polypropylene containing calcium carbonate nanoparticles. the main goal of this investigation is the characterization of additive effect on the polymer matrix, which is degraded in two different environments (air and water). the stability qualification of isotactic polypropylene modified with caco3 nanoparticles were investigated by oxygen uptake at 190c. the concentrations of filler are 0, 5, 10, 15, 20 and 25 % (w/w). two types of covered calcium carbonate nanoparticles were used in the preparation of compounds three irradiation doses (5, 15 and 25 kgy) were applied. the efficient protection of stabilizers that are present in the as-prepared formulations was emphasized by proper values of the kinetic parameters obtained for oxidation. the contribution of caco3 nanoparticles to the oxidative process of i-pp is discussed. the technological applications of ipp/carbonate nanoparticles requires the decision on the temperature regime in connection with filler loading to satisfy simultaneously the manufacture conditions and the improved life of products. keywords: polypropylene, carbonate nanoparticles, radiochemical stability introduction a large number of studies on the preparation and characterization of polymer/nanoparticles matrix were reported [1-10], because of their excellent functional properties. the presence of nanoscale inorganic fillers improves mechanical, thermal and gas barrier characteristics [11-15]. several promising formulations of polymer hybrids were proposed for satisfying various requirements for a large number of applications. however, the problem of thermal stability of polymer composites is of a great interest being directly related to the material durability. the topic of thermal degradation has received a special attention due to the strong influence of the service conditions on the behavior of composites. the characterization of material depreciation by heat provides useful information for storage, processing and long-term use, namely the life of products. ever using well-elaborated technologies of preparation, the stress factors act on any time. recent papers [16, 17] have emphasized the phenomenological analysis of degradation in connection with various testing methods. the most applied procedures for depicting the thermal behavior of polymer composites were dsc and tga [4, 18-20]. these papers have reported the difference between unloaded and polymer composites, which is caused by the modification in surface energy between the microphases, which would exist in the tested material. for obtaining a high rate and advanced degradation, ionizing radiation may be used. data obtained from accelerated ageing testing prove the capacity of material to resist for a certain period to the action of vigorous energetic agents. the durability of any material under high energy-radiation exposure journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 26 depends on many features through which the chemical nature of basic material and the sample formulation determine the kinetic behavior during degradation [2123]. effects of irradiation on polypropylene have been extensively studied. the most of them have emphasized the degradation of this polyolefin under radiochemical processing [21, 24-30] or its crosslinking in the presence of suitable additional monomer [23, 31, 32]. other assays on radiation resistance of polypropylene have investigated the effects of stabilization additives (antioxidants), which delay the start of oxidation and mitigate the rate of destruction [33, 34], the radiolysis of polymer substrate causes the free radical formation, which is followed by oxidation reactions [35]. the rate of oxygen consumption and the absorbent properties of calcium carbonate nanoparticles seem to influence the state of degradation of irradiated polypropylene [36]. this paper describes the stability of irradiated polypropylene containing calcium carbonate nanoparticles. the main goal of this investigation is the characterization of additive effect on the polymer matrix, which is degraded in two different environments (air and water). experimental isotactic polypropylene, a commercial grade material, was supplied by hmc polymers co., ltd (rayong, thailand) as moplen cs-42 hexp type. its initial characteristics were presented in a previous paper [37]. the compounding had concerned two types of caco3 nanoparticles (average size: 40 nm). the samples containing uncoated filler received mark a, while i-pp samples having carbonate particles superficially modified with stearic acid were placed in category b. the process of sample preparation was described earlier [37]. the reference (pristine material) and five different ipp/carbonate filler formulations (5, 10, 15, 25 and 25 % w/w) were prepared as thin sheets. the exposure to -rays for control and modified i-pp sheets was performed in an irradiator gammator m-38-2 (usa) provided with a 137cs source. this step was carried out in air at room temperature. two radiolysis surroundings (air and distilled water) were used in order to describe the oxidation resistance of material under these two common circumstances. for this investigation, a low value of dose rate (0.4 kgy/h) was used, which ensures an accelerated degradation. three total doses, namely 5, 15 and 25 kgy were applied; higher irradiation doses were avoided due to the brittleness of polypropylene sheets accompanying high energy treatment in oxidizing medium. the oxygen uptake measurements were accomplish with a laboratory equipment, which was previously described [38]. the experimental conditions were chosen in order to obtain convenient values for kinetic parameters: 1900c for thermal degradation, normal pressure of oxidation environment, air as testing medium. the selected temperature seems to be somewhat higher, but the supplied polypropylene (control and modified polymer) presented high thermal stability due to the presence of antioxidants. thin films were cut in small pieces having around 20 mg each. they were placed in round aluminum trays to be thermally oxidized in an electrically heated oven at constant temperature. irradiated specimens were investigated for their thermal oxidability soon as they were withdrawn from exposure room. the main kinetic parameters of oxidation: oxidation induction time and oxidation rates were determined from the dependencies of consumed oxygen on thermal degradation time. from these curves (sigmoidal type) induction period journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 27 was assessed by the crossing point of drawing the tangent from the propagation part with ox axis; the rate of oxidation was determined on the propagation step, where it attends the maximum value. results and discussion radiation processing of polymers is a useful tool for the check of material resistance under hard service conditions and provides a conclusive picture on the manner through which the material follows standard recommendations. for composite, the accelerated tests offer suitable conclusions concerning the effects of additives and fillers on the long term usage. ionizing radiation causes the split of weaker bonds. polypropylene is subjected to radiation degradation due to the presence of tertiary carbons in macromolecule backbone. oxygen which preexists in virgin material or is diffused into polymer bulk during irradiation promotes oxidation by the reactions with free radicals. final radiolysis products will be spread in the polymer mass in correlation with material crystallinity and the size of molecules. in the case of nanocomposites, the filler particles can influence the progress of oxidation. calcium carbonate nanoparticles modify the behavior of basic material because of their large specific surface and the homogenous dispersion in polymer. the decrease in mechanical properties was reported earlier [36], but it would be accompanied by the change in thermal stability. in the case of coated particles, it would be expected that the covering film will influence the progress of oxidation. isotactic polypropylene filled with calcium carbonate nanoparticles presents slight differences in the values of oxidation induction time, while the propagation of oxidation looks somewhat unlike (figure 1). the initial amount of dissolved oxygen is about the same for all samples, but, the o2 diffusion into polymer during uptake measurements depends on the filler consistency. the higher the concentration of nanoparticles, the faster the oxidation degradation (tables 1 and 2), namely, the thermal stability is determined by the homogeneity spreading of carbonate particle in polymer. the exposure of i-pp samples to radiation brings about the decrease in the both kinetic parameters: oxidation induction time and oxidation rate. the nature of degradation environment determines different values of kinetic parameters for the advance in oxidation. in figure 2 the dependencies of consumed oxygen on time is presented for samples containing coated caco3 nanoparticles, which were irradiated at 25 kgy in the both oxidative media. 0 50 100 150 200 250 300 350 0 20 40 60 80 100 120 o xy ge n up ta ke ( m m ol o 2/ g) time (min) figure 1. the dependencies of oxygen uptake on degradation time for unirradiated samples consisting of i-pp and caco3 nanoparticles coated with stearic acid. (□) control; (■) 5 % loading; (●) 25 % loading. 0 50 100 150 200 250 300 350 400 0 20 40 60 80 100 120 o xy ge n up ta ke ( m m ol o 2/ g) time (min) figure 2. time dependencies of oxygen uptake for irradiated at 25 kgy i-pp samples/25% caco3 coated nanoparticles. (○) control; ( ) 10 % loading; (◊) 25 % loading. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 28 table 1. the values of kinetic parameters of oxidation for radiolyzed nanostructured i-pp (environment: air) experimental conditions i-pp/uncoated caco3 particles i-pp/stearic acid coated caco3 particles filler concentration (%) dose (kgy) induction period (min) oxidation rate (mol o2.g -1.s-1). 104 induction period (min) oxidation rate (mol o2.g -1.s-1). 104 0 52 6.33 110 5.63 5 60 7.06 118 5.84 15 61 7.41 96 5.99 0 25 48 8.77 78 6.53 0 62 6.53 168 5.46 5 71 6.84 170 5.82 15 75 7.11 154 6.61 5 25 70 7.93 131 7 0 85 7.23 199 6.48 5 96 7.58 161 6.82 15 113 7.84 125 7.09 10 25 148 8.03 112 7.22 0 172 7.22 160 6.61 5 155 7.51 148 6.84 15 149 7.85 133 7.27 15 25 137 8.09 129 7.17 0 160 7.61 183 7.38 5 145 7.9 146 7.55 15 120 8.37 119 7.93 20 25 90 8.72 89 8.14 table 2. the values of kinetic parameters of oxidation for radiolyzed nanostructured i-pp (environment: water) experimental conditions i-pp/uncoated caco3 particles i-pp/stearic acid coated caco3 particles filler concentration (%) dose (kgy) induction period (min) oxidation rate (mol o2.g -1.s-1). 104 induction period (min) oxidation rate (mol o2.g -1.s-1). 104 0 52 6.33 110 5.63 5 65 7.06 78 5.95 15 70 7.72 70 6.41 0 25 59 7.97 58 6.88 0 62 6.53 168 5.46 5 86 7.24 157 6.34 15 84 8.15 132 6.92 5 25 80 8.43 109 7.70 0 85 7.23 122 6.48 5 96 7.68 104 7.26 15 101 8.44 90 7.65 10 25 88 9.23 81 8.14 0 172 7.22 160 6.61 5 148 7.91 140 7.43 15 130 8.45 128 7.68 15 25 120 9.49 119 8.93 0 160 7.61 183 7.38 5 139 8.30 130 7.69 15 132 8.96 109 8.34 20 25 114 9.82 71 8.61 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 29 during radiochemical ageing of studied systems, the formed intermediates are spread in the bulk of polymer, closer of further from carbonate nanoparticles. hear that was transferred to samples increases the rate of diffusion of radicals through the polypropylene molecules attending filler particles on which they would be adsorbed. on the first step of thermal oxidation (oxygen uptake measurements), the oxygen consumption decrease with carbonate loading. it means that the precursors of oxygenated final products are efficiently scavenged by particles. on the propagation step of oxidation, when the rate of oxidative degradation increases with carbonate concentration, the desorption of radiolysis products increases the oxygen uptake and the oxidation occurs fast. the radiolysis of water generates various intermediated, especially radicals (h., ho., ho2.), which initiate the superficial degradation of samples. the higher concentration of carbonate nanoparticles brings about an increase in oxidation induction time. the difference between the coated and pristine filler consists of the presumable penetration of organic shell by water intermediates, followed by their remove from stearic acid covering. this behavior was exhibited by all formulations of ipp/nanocarbonate specimens. the advanced exposure of materials to the action of x-rays promotes a larger degradation, due to the greater abundance of radical intermediates. their depletion will require higher oxygen amount starting from the beginning of thermal measurements. however, the longer induction times are presented by the polymer samples containing carbonate loading higher than 15 %. the radiation ageing accelerates at higher rates the pristine polypropylene in comparison with the same material loaded with calcium carbonate nanoparticles; the coating shell ameliorates the thermal resistance of host material. conclusion the polypropylene compounds with calcium carbonate nanoparticles exhibits favorable kinetic parameters (induction time and process rate) during accelerated oxidation. the presence of these small particles modifies the interphase diffusion of radiolysis intermediates, which would react with molecular oxygen. the thermal strength of polypropylene is improved the efficiency of carbonate nanoparticles in isotactic polypropylene on retardation of oxidation is a great advantage for prolongation of the product life, which are subjected to the action of various hazards. the technological applications of ipp/carbonate nanoparticles requires the decision on the temperature regime in connection with filler loading to satisfy simultaneously the manufacture conditions and the improved life of products. acknowledgment the authors express his gratitude to international atomic energy agency (iaea), vienna, division of physical and chemical section, for the contract ro 12704/2004, which represented the financial support for the present investigations. this project follows the policy of iaea on new radiation procedure concerning the amelioration of durability and usage of polymer. references 1. r. a. andrievski, j. mater. sci. 2003, 38, 1367. 2. t. s. ellis, j. s. d’angelo, j. appl. polym. sci. 2003, 90, 1639. 3. w. jie, l. yubao, c. weiqun, z. yi, j. mater. sci. 2003, 38, 3303. 4. m. zanetti, p. bracco, l. costa, polym. degrad. stabil. 2004, 85, 657. 5. w. h. ruan, m. q. zhang, m. z. rong, k. friedrich, j. mater. sci. 2004, 39, 3475. 6. n. m. mallikarjuna, a. venkataraman, t. m. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 30 aminabavi, j. appl. polym. sci. 2004, 94, 2551. 7. m. c. g. rocha, a. h. m. f. t. silva, f. m. b. coutinho, a. l. n. silva, polym. testing 2005, 24, 1049. 8. m. diagni, m. guèye, l. vidal, a. tadjani, polym. degrad. stabil. 2005, 89, 418. 9. s. morlat-therias, b. mailhot, j-l. gardette, c. da silva, b. haidar, a. vidal, polym. degrad. stabil. 2005, 90, 78. 10. t. wan, f. feng, y-c. wang, polym. bull. 2006, 56, 413. 11. j. w. gilman, appl. clay sci. 1999, 15, 31. 12. n. haegawa, h. okamoto, m. kato, a. usuki, appl. polym. sci. 2000, 78, 1918. 13. r. s. sinha, m. okamoto, prog. polym. sci. 2003, 28, 1 14. m. patel, p. r. morrel, j. j. murphy, a. s. maxwell, polym. degrad. stabil. 2006, 91, 406. 15. j. zhang, d. d. jiang, c. a. wilkie, polym. degrad. stabil. 2006, 91, 298. 16. j. pospíšil, z. horák, j. pilař, n. c. billingham, h. zweifel, s. nešpůrek, polym. degrad. stabil. 2003, 82, 145. 17. j. pospíšil, j. pilař, n. c. billingham, a. marek, z. horák, s. nešpůrek, polym. degrad. stabil. 2006, 91, 417. 18. f. bertini, m. canetti, g. audisio, g. costa, l. falqui, polym. degrad. stabil. 2006, 91, 600. 19. f. g. r. filho, t. j. a. mélo, m. s. rabello, s. m. l. silva, polym. degrad. stabil. 2005, 89, 383. 20. z. dobkowski, polym. degrad. stabil. 2006, 89, 488. 21. t. czvikovszki, h. hargitai, radiat. phys. chem. 1999, 55, 727. 22. h. n. dahlan, m. d. khairul zaman, a. ibrahim, radiat. phys. chem. 2002, 64, 429. 23. j. gao, y. lu, g. wei, x. zhang, y. liu, j. qian, j. appl. polym. sci. 2002, 85, 1758. 24 d. j. carlsson, j. lacoste, polym. degrad. stabil. 1991, 32, 377 25. p. gijsman, j. hennekens, polym. degrad. stabil. 1993, 42, 95 26. b. a. gorelik, i. v. kolganova, l. matisovarychlá, g. i. listvojb, a. m. drabkina, g. a. gorelik, polym. degrad. stabil. 1993, 42, 263. 27. m. szadkowska-nicze, j. mayer, t. szreder, a. faucinato, radiat. phys. chem. 1999, 54, 193. 28. m. denac, v. musil, i. šmit, f. ranogajec, polym. degrad. stabil. 2003, 82, 263. 29. h. f. naguib, r. o. aly, m. w. sabaa, s. m. mokhtar, polym. testing 2003, 22, 825. 30. t. savasaki et al, radiat. phys. chem. 1998, 31, 877. 31. r. a. jones, j. i. cail, r. f. t. stepto, i. m. ward, macromolecules 2000, 33, 7337. 32. z. stojanović, z, kačarević-popović, s. galović, d. miličević, e. suljovrujić, polym. degrad. stabil. 2005, 87, 279. 33. a. shamshad, a. a. basfar, radiat. phys. chem. 2002, 57, 447. 34. t. zaharescu, s. jipa, r. setnescu, c. santos, b. gigante, l. m. gorghiu, i. mihalcea, c. podină, polym. bull. 2002, 49, 289. 35. j. s. j. stags, polym. degrad. stabil. 2004, 85, 759. 36. t. zaharescu, m. kaci, r. setnescu, s. jipa, n. touati, polym. bull. 2006, 56, 405. 37. t. zaharescu, polym. testing 2001, 20, 3. 38. r. dangtungee, j. yun, p. supaphol, polym. testing 2005, 24, 2. stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume x, issue 2 30 june 2011 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: indexcopernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiv, issue 1 31 march 2015 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava http://www.fia.usv.ro/fiajournal editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:fiajournal@fia.usv.ro mailto:gutts@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 22 oxidative stability of iron enriched oil –in-water emulsions diana curchi1, rodica sturza1 1 technical university of moldova; 168 stefan cel mare ave., md-2004 chisinau, republic of moldova; sturzar@yandex.ru abstract : in the present work the oxidative stability of sunflower oil-in-water emulsions has been researched on. proteins can prevent or promote lipid oxidation by various mechanisms depending on their physicochemical characteristics. sodium caseinate (nacas) can prevent lipid oxidation, essentially by chelating metals ions, but other mechanisms such as free-radical scavenging may also be involved. conjugated dienes (cd) were evaluated (primary products of lipid oxidation), and the volatile compounds: pentane, hexanal, 1-octen-3-ol, and 2-(e)-octenal were selected as markers of secondary products of lipid oxidation (gc/spme). the affinity of milk proteins to bind free iron ion is the first factor that controls the transfer to oil/water interface. all the soluble iron that could be present in the emulsions was likely bound by caseins at ph = 6.5, the oxidative stability of nacas-stabilized emulsions greatly depends on metal availability. the chelating properties of nacas and electrostatic interactions favored positioning of the metal ions at the interface, a key place to initiate the oxidation reactions. a change in ph modifies the complex formation between metallic ions and proteins: hydrogen ions compete with metallic ions to bind the protein. at low ph, the anionic groups of aminoacyl residues tend to become protonated. as a consequence, their affinity for cations decreases, and therefore reduces their complexation with the protein.the presence of ferrous ion influences the oxidation rate. the ability of the complex to retain iron ion and to avoid “free” ferrous ion is very important factor to be controlled. keywords: caseinate; iron; lipid oxidation introduction lipid oxidation is a major cause of food quality deterioration. it leads to the development of off-flavors (rancidity) and potentially toxic compounds. the main rules of lipid oxidation in oils are known and recent studies have focused on lipid oxidation in oil-in-water (o/w) emulsions [1]. in many foods, the lipid phase is dispersed as oil droplets in an aqueous matrix. oxidation phenomena in emulsions mimic what happens in food products. lipid oxidation is favoured because the droplets present a large contact surface between oxidizing fatty material and the watersoluble chemical compounds, namely, oxygen and ions of transition metals, which participate in the initiation and propagation of oxidation reactions [2,3]. proteins are generally regarded as safe food ingredients and they are widely used as emulsifiers in food products [4]. many proteins also exert antioxidant activity when added in the aqueous phase of o/w emulsions stabilized by small surfactants such as phospholipids. the antioxidant activity of milk proteins was demonstrated in various studies [5-6]. in particular, caseins inhibited formation of lipid hydroperoxides and hexanal in brij stabilized corn oil emulsions. the ability of casein phosphorylated groups to chelate pro-oxidant metals ions may explain this inhibition, but free-radical-scavenging activity could also be involved [7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 23 in emulsified systems and in other model systems, some proteins generally considered as antioxidant were pro-oxidant under specific conditions. numerous examples showed that proteins can behave as prooxidant, depending on physicochemical conditions of the systems [8-10]. sodium caseinate (nacas) is a mixture of disordered proteins of relatively low molecular weights. they can prevent lipid oxidation, essentially by chelating metals ions, but other mechanisms such as free-radical scavenging may also be involved. as their hydrophobic and hydrophilic residues are clustered into large and separate domains, caseins adsorb to the oil-water interfaces through their hydrophobic domain, whereas the hydrophilic domain forms a tail protruding in the aqueous phase (27). the aim of this paper is to study the oxidative stability of iron enriched o/w emulsions stabilized by proteins such as nacas is related to the availability of metal ions and protein properties. materials and methods materials. commercial sunflower oil was stripped of tocopherols, monoand diacylglycerides, and free fatty acids using adsorption chromatography on an alumina column. powdered nacas (92.7% purity) sodium azide (nan3, purity ≥99%); hexanal (98%), 1-octen-3-ol (98%), 2-(e)-octenal (94%), 1.10-phenantroline monohydrate (99%), hydroquinone (99%), and ammonium iron (ii) sulfate hexahydrate [fe(nh4)2(so4)2. 6h2o] (>99%) were purchased from aldrich. methods. preparation, characterization and storage of emulsions. the day before emulsion preparation, nacas solutions (20 g/l; 0.4 g /l nan3; ph adjusted to 6.5) were prepared and stirred overnight at +4 ºc to entirely dissolve the proteins without foam formation. the solutions were equilibrated at room temperature just before use and their ph was adjusted to 6.5 by addition of either naoh or hcl (1 mol /l). o/w emulsions were prepared with 30 vol % sunflower oil and 70 vol % protein solutions. the two phases were premixed for 2 min at 8 000 rpm using rotor-stator homogenizer.the coarse emulsions were then homogenized for 4 min at 35 bar through a one-stage low-pressure valve homogenizer. the size distribution of the oil droplets in the emulsions was measured immediately after homogenization with a laser light scattering instrument. it was daily checked to monitor emulsion stability and reported as volume-surface mean diameter ([d3,2]; μm). the charge of the emulsion droplets (zeta potential, ζ; mv) was also measured after homogenization. aliquots (1.5 ml) of emulsions were distributed in 20-ml headspace vials sealed with teflon/silicon septa and aluminum crimp caps. the vials were rotated in the dark at 30 ºc, 20 rpm. ph of the samples was measured with a ph-150 ma. with an electrode suitable for measurements in emulsions. measurement of lipid oxidation to evaluate the formation of primary products of lipid oxidation in the emulsions, conjugated dienes (cd) were evaluated according to the method described by lethuaut et al. [2]. aliquots of emulsions were diluted in 2-propanol to obtain lipid concentrations of 250 mg /l. the solutions were centrifuged for 4 min at 5600 rpm. the absorbance of the supernatants was measured at 233 nm using a uv-vis spectrophotometer (unicam, uk).the reference cell contained water in 2-propanol in the same proportions as in samples. the results were expressed in millimoles of conjugated dienes per kilogram of oil (mmol cd kg/ oil) at 233 nm [11]. four volatile compounds: pentane, hexanal, 1-octen-3-ol, and 2-(e)-octenal, were food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 24 selected as markers of secondary products of lipid oxidation. they were analyzed by gas chromatography (gc) of the volatile compounds sampled in the headspace of the vials equilibrated at 30 ºc, with a solidphase micro-extraction (spme) fiber. all tests have been carried out using the shimadzu gc system coupled with a single quadrupole mass-spectrometer gcmsqp2010 plus equipped with the threedimensional automated system for the injection of samples aoc-5000 (gcmsqp2010 plusxaoc-5000). for the identification, the general library of nist-5 mass spectra was used. the accuracy of displacement has been verified according to the library of covatz retention indices (mlri). the analysis of the experimental data was carried out with the gc/ms solution software (shimadzu), which contains the scan/sim options (fast automated scan/sim type (fasst); creation of automatic sim (scan/sim) table (coast). iron-binding properties of the proteins. to evaluate the ability of the proteins to bind soluble iron, mixtures of the protein solution and known amounts of ferrous iron were filtrated through cutoff filters and the unbound metal was quantified [12]. ferrous iron was added to nacas (1 g/ l, ph adjusted to 6.5) at concentrations ranging from 1.0 to 5.1 mg /l. after one night at room temperature, protein solutions were filtered through disposable cutoff filters to remove proteins and protein-bound iron. the filtrate contains only the free iron. free fe2+ reacts with o-phenantroline to give a colored complex that is quantified by measurement of solution absorbance at 508 nm. one volume of ophenantroline reagent (2.5 mm phenantroline, 18.2 mm hydroquinone in ethanol/water; 50/50; v/v) was added to one volume of the filtrate. the solution absorbance was read immediately at 508nm against a blank. the concentration of free iron was determined from the calibration curve built with ammonium iron (ii) sulfate hexahydrate solutions (1-10 mg/ l). it was then subtracted from the concentration of added iron to obtain the concentration of bound iron. the experiment was also performed with pure water as a control. experimental design and data treatment. all analyses were made in triplicate. the statistical analyses were carried out by using the statistical package statistica for windows. results: physicochemical characterization of emulsions. the freshly prepared emulsions had very similar and monomodal droplet size distributions. for nacas-stabilized emulsions, [d3,2] was 1.32 ± 0.12 μm. the droplet size distributions of the emulsions largely overlap each other and oil-water interface areas (6/d3,2]) are very close. consequently, differences in oxidative stability of the nacas-stabilized emulsions could not be attributed to differences in droplet size and interfacial surface area [2]. the droplet size distributions of the emulsions remained constant even after 10 days of storage, and no phase separation or creaming was observed, showing that the emulsions were physically stable and not destabilized during the rotating agitation at 30 ºc. nacas-stabilized oil droplets were negatively charged (-81.2 (5.1 mv), as revealed by ζ-potential measurements. the initial ph of the emulsions was slightly above the ph of the protein solutions (6.5): it was 6.66 ±0.02 for nacas-stabilized emulsions. during aging, the ph of the nacasstabilized emulsions remained stable for the first 8 h, respectively, then decreased rapidly to finally decrease slowly after 24 h aging (figure 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 25 oxidative stability of emulsions content of conjugated dienes increased slowly during the first 8 hours of aging, then increase sharply to a plateau when around 230 -240 mmol rooh / kg oil after 24 h. (figure 2). this level remained constant even for a long time of storage (10 days). twenty volatile compounds were identified as secondary products of oxidation in the headspace of the oxidizing emulsions. pentane, hexanal, 1-octen-3-ol, and 2-(e)octenal were chosen as markers of lipid oxidation. pentane and hexanal were the major compounds produced and were often used as indexes of lipid oxidation in oils or fatty materials containing high level of linoleic acid (3, 6, 13, 30-32). 1-octen-3-ol, 2-(e)-octenal, and hexanal are involved, among others, in the development of offflavors during lipid oxidation (14, 33). these four volatile compounds were even detected (from 0.1 to 10 ng) in the headspace of the fresh emulsions equilibrated for 15 min at 30 ºc before spme-gc measurements. quantities of each volatile compound detected in the headspace of the emulsions were plotted versus time (figure 3). the amount of hexanal desorbed from the spme fiber increased rapidly during the first 24 h and then continued to increase slowly even after 10 days. pentane quantity increased linearly as a function of time throughout incubation. 2-(e)-octenal amount remained very low during the first 16 h and then increased progressively during aging. the amount of 1-octen-3-ol increased slowly during the first 8 h, then increased sharply until 24 h, and finally remained constant. 0 5 10 15 20 25 30 0 8 16 24 32 40 48 56 time (h) v ol at il e co m p ou n ds , n g hexanal pentan 1-octen-3-ol 2-(e)-octenal 0 1 2 3 4 5 6 1,04 2,12 3,03 4,17 5,12 total iron, mg/l d et ec te d io n, m g/ l free iron bound iron figure 3. evolution of volatile compounds: pentane, hexanal, 1-octen-3-ol and 2-(e)-octenal in the headspace of 30% sunflower oil emulsions stabilized by nacas figure 4. retention of iron (ferrous sulfate) by solution of nacas 5,6 5,8 6 6,2 6,4 6,6 6,8 0 8 16 32 40 48 56 time (h) p h 0 50 100 150 200 250 300 0 8 16 24 32 40 48 56 time (h) c on ju ga te d di en es ,m m ol /k g oi l figure 1. evolution of ph during aging at 30 ºc of 30% sunflower oil emulsions stabilized by sodium caseinate figure 2. formation of conjugated dienes during aging of 30% sunflower oil emulsions stabilized by sodium caseinate food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 26 iron-binding properties of proteins when 0.5-5.0 mg/l iron was added to the 1 g /l nacas solution, nearly all the metal was bound by the proteins (figure 4). the difference between bound iron and total added iron accounted for experimental losses as shown by the values obtained on control. this result is in accordance with the previous studies that demonstrated the efficient iron-chelating properties of caseins [10,11]. indeed, the total iron-binding capacity of caseins was not reached in the present study, but the results show that all the soluble iron that could be present in the emulsions was likely bound by caseins. proteins can prevent or promote lipid oxidation by various mechanisms depending on their physicochemical characteristics [12]. in emulsions, lipid oxidation is greatly influenced by electrostatic interactions between ions of transition metals and droplet surface but interfacial film thickness, ph, or the presence of solutes in the aqueous phase may also interfere [13,14]. a better knowledge of the factors that determine the development of lipid oxidation in the emulsions where proteins constitute the stabilizing interface can lead to an optimized use of proteins as food ingredients. among these factors, the interactions that can take place between the metal ions and proteins, either at the interface or in the aqueous phase of the emulsions, and their influence on emulsion chemical stability should be investigated. these results demonstrate that the oxidative stability of protein-stabilized emulsions both depends on the interactions of proteins with transition metal ions and on the antioxidant properties of the emulsifying proteins. at ph = 6.5, that is above the pi of the proteins, the oxidative stability of nacas-stabilized emulsions greatly depends on metal availability. the chelating properties of nacas and electrostatic interactions favoured the positioning of the metal ions at interface, a key place to initiate the oxidation reactions [15]. the location of these compounds in emulsions, and more generally in food products, is a critical factor that should be controlled to improve their oxidative stability. references 1. frankel, e. n. free radical oxidation. in lipid oxidation; the oily press ltd: dundee, scotland, 1998; pp 13-22. 2. lethuaut, l.; genot, c. effect of droplet size on lipid oxidation rates of oil-in-water emulsions stabilized by protein. j. am. oil chem. soc. 2002, 79, 425-430. 3. dickinson, e. les colloides alimentaires; dickinson, e., ed.; masson: paris, 1996. 4. diaz m., dunn c. m., mcclements d. j., decker, e. a. use of aseinophosphopeptides as natural antioxidants in oil-in-water emulsions. j. agric. food chem. 2003, 51, 2365-2370. 5. rival, s. g.; boeriu, c. g.; wichers, h. j. caseins and casein hydrolysates. 2. antioxidative properties and relevance to lipoxygenase inhibition. j. agric. food chem. 2001, 49, 295-302. 6. tong, l. m.; sasaki, s.; mcclements, d. j.; decker, e. a. mechanisms of the antioxidant activity of a high molecular weight fraction of whey. j. agric. food chem. 2000, 48, 1473-1478. 7. jacobsen, c. sensory impact of lipid oxidation in complex food systems. fett/lipid 1999, 101, 484-492. 8. lu, c. l.; baker, r. c. effect of ph and food ingredients on the stability of egg yolk phospholipids and the metal-chelator antioxidant activity of phosvitin. j. food sci. 1987, 52, 313-316. 9. pryor, w. a.; castle, l. chemical methods for the detection of lipid hydroperoxydes. in oxygen radicals in biological systems; packer, l., ed.; academic press: orlando, fl, 1984; pp 293-295. 10. mei, l.; mcclements, d. j.; wu, j.; decker, e. a. iron-catalyzed lipid oxidation in emulsion as affected by surfactant, ph and nacl. food chem. 1998, 61, 307-312. 11. van ruth, s. m.; roozen, j. p.; posthumus, m. a.; jansen, f. j.h. m. volatile composition of sunflower oil-in-water emulsions during initial lipid oxidation: influence of ph. j. agric. food chem. 1999, 47, 4365-4369. 12. guerin-dubiard, c.; anton, m.; dhenegarcia, a.; martinet, v.; brule, g. hen egg and fish egg phosvitin: composition and iron binding properties. eur. food res. technol. 2002, 214, 460-464. 13. silvestre, m. p. c.; chaiyasit, w.; brannan, r. g.; mcclements, d. j.; decker, e. a. ability of surfactant headgroup size to alter lipid and antioxidant oxidation in oil-in-water emulsions. j. agric.food chem. 2000, 48, 2057-2061. 14. genot, c.; meynier, a.; riaublanc, a.; chobert, j.-m. protein alterations due to lipid oxidation in multiphase systems. in lipid oxidation pathways; kamaleldin, a., ed.; aocs press: champaign, il, 2003; pp 265292. 15. villiere, a.; le viau, m. ; bronnec, i. ; moreau,n. ; genot, c. oxidative stability of bovine serum albuminand sodium caseinate-stabilized emulsions depends on metal availability. j. agric. food chem. 2005, 53, 1514-1520 microsoft word 3 journal nr 4 2010 din 8 noiembrie_12-20.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 16 some metals (fe, mn, zn, cu, pb, cd) contents in vegetables from a nonpolluted plain area of cenad-banat (romania) ioan gogoasa1, gabriela oprea3, monica harmanescu2, teodor ioan trasca1, adrian rivis1 and iosif gergen1 1 usamvbfaculty of food technology, calea aradului nr. 119, timişoara, ro-300645, românia, email: igergen@yahoo.com 2 usamvbfaculty of agriculture, calea aradului nr. 119, timişoara, ro-300645, românia 3 nord university, baia mare, str. dr. victor babes nr. 62a, baia mare, ro 430083, românia abstract: this paper presents original studies about the occurrence of some heavy metals (fe, mn, zn, cu, pb and cd) in soil and different vegetables (pepper, cucumbers, tomatoes. eggplants, green beans, cauliflower, cabbage, dill-leaves, parsley-leaves and roots, celery-leaves and roots, onion, garlic and potatoes) cultivated in nonpolluted plain area in cenad-banat county. the accurate determination of toxic heavy metals in vegetables is of importance because of the toxicity of these elements and their compounds. total heavy metals in vegetable samples were analyzed after wet digestion with mineral acid, and in the soils they were analyzed after digestion in aqua regia. all metals were analyzed using high-resolution continuum source spectrometer contraa-300, analytikjena in the acetylene flame (99.99 % purity). in comparison with official imposed limited the average values for heavy metals contents in soil are in the normally interval for zn, cu and pb, for cd the limit is exceeded, but under warning threshold. the smaller content of total heavy metals (around 2.5 ppm) was identified in cucumbers, cauliflower and cabbage. in pepper, tomatoes, eggplants, green beans, onion and potatoes the total content of heavy metals are between 3-6 ppm. in root of carrot, celery and parsley the total content is between 3-7 ppm with maximum in parsley root. the highest values were identified in leaves of parsley, dill, celery and garlic. for all soil and vegetables samples the average concentrations of all analyzed metals are lower than the limits established by the present romanian legislation. key words: heavy metals, toxicity, soil, plants, faas introduction the main route of entry for these toxic elements is the food chain with soil, vegetables, plant feed and livestock all being important ecological strata. inhalation can also bee a significant pathway. the accessibility of metalmicroelements for plants depends of soil reaction, organic matter content, mineral colloids, soil humidity and microbiological activity. organic matter, especially humus compounds can form organo-metallic compounds with high mobility in soil solution and availability for plants [1,2]. the essential metal microelements for plants and animals: fe, mn, cu, zn, mo, se, can will bee toxic in high quantity. other metal microelements: hg, cd, cr, ni, pb, is very toxic for green plants and for animals, too. these metal microelements can affect the biological and biochemical processes in plants: nutrition, photosynthesis and respiration, yield of crops. toxicity degree of heavy metals was estimated in function of critical concentration for a normal development of plant (table 1). from these data we can see food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 17 that hg, cr, cd and pb have the highest toxicity for plant [3]. zn and cu in trace quantities are very important for plants and animals metabolism. they are building component of many enzymes. the importance of zn for enzymes was discovered early this century (1986). the biochemistry of zn includes an account of the genetic disorder acrodematitis enterophathica. this was previously fatal for all babies born with it, but can now bee cured with doses of zn. excessive consumption of alcohol damages the liver and those suffering cirrhosis have lower levels of zn than normal in that vital organ. again if the damage is not severe then zn supplements can restore the liver's function. levels of zn are highest in prostate, muscles, kidney and liver; semen is particularly rich in zn. in human nutrition is the daily intake is of about 15 mg, almost the same as fe [4]. table 1. critical concentration of heavy metals in plants [3] metal cr hg cd co pb cu ti ni zn mn fe critical concentration (ppm) 1 2 5 10 10 15 20 20 150 150 200 copper is another essential metal for humans but is never lacking in human diet because we only need 2 mg of cu per day. like zn, copper is required for enzymes especially by those which govern respiration. one disease directly related to cu is wilson's disease, a genetic disorder in which sufferers accumulate cu in tissues causing damage to liver and brain [4]. in high quantities the two metals have toxic effects on plants and animals. the adjoining of other heavy metals, cd and pb, affects especially the enzymes with thio (-sh) group [4, 5]. cd is not an essential metal for plant and animal. at very low concentration (0.0001m) is very toxic for germination and primary development of plants. at old plants the toxic effect is low [6]. pb is also a strong toxic for germination and primary development of plants. at some concentration (0.0001m) pb is more toxic than cd, for young plants. at 4-200 ppm cd in plant tissue, for 22 plant species, the loss of yields was more 10% [3]. the high concentration of cd and pb in soil and culture medium is favorable for their accumulation in plants, more in roots than in leafs [6]. in plants tissue heavy metals have negative influence of the intern transport of major nutrition elements. cd and pb have an inhibitory effect over nitrogen assimilation enzyme [3]. in the presence of high level of cd and pb, the content of protein, calcium and phosphorus in grain of cereals is loss [7]. these metals can influence negatively the photosynthesis (hill reaction), pigment content, o2 development, electron transport and oxidative phosphorilation. at high concentration these metals can reduce the reaction from calvin cycle at c3 plants and at c4 plants can reduce the carboxilation of phospho-enol piruvic acid. cd and pb have an inhibitory effect over the cellular respiration at mitochondrial level. they reduce the reaction of krebs cycle and glucose oxidation. they reduce the plasticity and elasticity of cellular wall and have an inhibitory effect on cellular division. at high concentration they have a mutagenic effect [3]. the plants, which have high tolerance at cd, cu, zn and pb, can synthesize protein rich in cystein. this protein can make chelates with metallic ions. [5]. lead and cadmium are toxic metals that accumulate in human tissues. lead poisoning is of particular concern in children because it results in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 18 neurobehavioral defects. cadmium is carcinogenic when inhaled. the kidney appears to be the sensitive organ following chronic oral exposure. cadmium affects the resorption function of the proximal tubules, causing an increase in the urinary excretion of low-molecular-weight known as tubular proteinuria [8]. in animal tissue cd and pb have an inhibitory effect over the thiolo-group of enzymes. they interact with other trace elements, cu, zn and fe and have a toxic effect on langerhans cells from pancreas and on the cells from kidneys and testicles. pb has a toxic effect on liver and the mobility of spermatozoid especially. the absorption of heavy metals has generally digestive way, followed by different tissue penetrations. the high concentration of heavy metals was identified in kidneys and liver. the excretion is made in general by faecal and urine. the intoxication with these heavy metals can have acute or chronic form. frequently the visible clinical signs of diseases appear in 2-3 days and are represented by depression and anorexia [9]. because of high toxicity grade of these metals, their concentrations in soil and food are restricted by legislation. the studied area, within the village cenad is located in the north of banat county, in a plain region settled on the left part of mures river valley. this area is far away from the industrial zone and has an old tradition in vegetable production. also the climatic and soil conditions are favourable for ecological vegetables production. materials and methods reagents and solutions the stock standard solutions (1000 mg/l) were analytical grade purchased from riedel de haen (germany). the working solutions were prepared by diluting the stock solutions to appropriate volumes. the hydrochloric acid 32%, nitric acid 65% and h2o2 25% solutions used were of ultra pure grade, purchased from merck (germany). all reagents were of analytical-reagent grade and all solutions were prepared using deionised water. soil samples preparation for soils, 6 samples were sampled with agrochemical steel probe, from 0-20 cm deep, within the cultivated area. one mixed sample was made from 10 single samples. the soil samples were kept in plastic bags. in laboratory the soil samples were dried in clean air, ground and passed through a 2mm sieve to remove roots, stones and other large particles. the removal out of total metals content from soil to solution, in the purpose of dosage, can be made by wet proceeding which consists in treating the soil sample with mixture of mineral acids (hcl, hno3, 3:1 ratio) [10,11]. the soils were analyzed for total contents of heavy metals after digestion in aqua regia. 10ml mixture of nitric acid and hydrochloric acid (3:1) was added to 5.0 g of soil samples and heated on a hot plate. then, the residue was treated with 1n nitric acid, centrifuged and finally we added 25 ml in quoted flask. the mobile forms of zn, cu, pb and cd were extracted from soil with a na2edta 0.1 n solution buffered at ph=7, at 1/5 soil-solution ratio [11,12]. for the determination, standard working solutions were prepared with the same extraction solution. vegetable samples preparation heavy metals in vegetable samples were analyzed after wet digestion with mineral acid [13]. for the vegetables’ analysis we selected 100 g (or 3 pieces of bulky vegetables) from different location until 1000 g for one sample. after having been washed with deionised water the vegetables were homogenized by mincing. after homogenization, 100 g materials was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 19 transferred into a quartz capsule, dried at 1050 c and after cooling the samples were homogenized by mincing. 1g of each dry sample was submitted to digestion with 10 ml pure hno3 and 10 ml h2o2 at 170 0c in a digesting device digestion system 61007 digester, provided by tecator. after complete digestion the sample solutions were filtered, made up to 50 ml with deionised water. this solution was used for heavy metal zn, cu, pb and cd analysis by fass in air/acetylene flame using an aqueous standard calibration curve. all analyses were made in triplicate and the mean values were reported. sample analysis the most data referring to the abundance of these metals in soil, water and food were obtained by atomic absorption spectrometry methods, which suit best for routine determination. atomic absorption spectrometry in air-acetylene flame presents a sufficient sensibility for the direct determination in the acid solution resulted after digestion [14-16]. the analysis of heavy metals (zn, cu, pb, cd) was made by contraa-300, analytik-jena device, by flame atomic absorption spectrometry (fass) in air/acetylene flame. the device working parameters are presented in table 2. table 2 the working parameters for spectrometer contraa 300, analytik-jena nr.crt . element wave length (nm) acetylene flow (l/h) air flow (l/h) concentration range (mg/l) correlation coefficient 1 zn 213.9 60 400 0.010-1.600 0.991 2 cu 324.8 60 400 0.025-3.000 0.975 3 pb 217.0 60 400 0.025-5.000 0.980 4 cd 228.8 60 400 0.015-2.000 0.992 all metals were analyzed using highresolution continuum source spectrometer contraa-300, analytik-jena and the acetylene was of 99.99 % purity. the characteristics of working parameters and metal calibration are presented in table 2. under the optimum established parameters, the standard calibration curves for metals were constructed by plotting absorbency against concentration. in a definite range of each metal, good linearity was observed (table 2). results and discussion the experimental data of heavy metal total content in soil around the village cenad are presented in table 3. we can observe a large variation of content, especially for cu and cd. in comparison with the official imposed limited, presented in table 4, the average values for heavy metals contents in soil are in the normally interval for zn, cu and pb, for cd the limit is exceeded, but under warning threshold. our experimental data are in agreement with literature data, this hill area have acid soils with small content of clay and mineral underground poor in heavy metals [17-19]. in this area other sources of pollution with heavy metals are absent. table 3 heavy metals content, in total forms, in the soil horizon 0-20 cm, in village cenad area metal (mg/kg) zn cu pb cd minimum values 75.0 25.5 15.6 1.00 maximum values 101 40.0 29.0 3.00 average values 81.00 34.5 21.0 1.85 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 20 table 4 excerpt from romanian agriculture ministry ordinance 756/1997, standard values for trace elements in soil [20] experimental values mg/kg zn cu pb cd normal values 100 20 20 1 warning threshold 300 100 50 3 intervention threshold 600 200 100 5 table 5 heavy metals content mobile formsof the soil horizon 0-20 cm in village cenad area zn cu pb cd minimum values 1.87 2.07 1.68 0.05 maximum values 4.38 4.10 3.05 0.20 average values 4.10 3.25 2.17 0.12 table 6 maximum accepted limits for some heavy metals in soil, mobile forms in edta-ammonium acetate (lăcătuşu, 1995) [23] the phytoavailability of trace elements, from soil to plant, is a key factor controlling the quality of plant food and it correlates best with the concentration (total and mobile forms) of elements [3]. for all investigated trace elements the concentrations in soil, both total and mobile form, are below toxic limits. so this area is favourable to ecological vegetables production. the experimental data for heavy metals content in vegetables are presented in table 7. similar to soil, the limits of variation in heavy metals contents are very large. in comparison with legal limits presented in table 8 we can observe that in general the experimental average values are below these limits. in all analyzed vegetables, the average zn content is included between 1.20-21.0 ppm, with maximum value 21.0 ppm in parsley leaves. in general we can observe that in leaf vegetables (dill, parsley, and celery) the content of all heavy metal are high. for these vegetables in our legislation the maximum limits are absent (table 8). the average cu content in the analyzed vegetables is included between 0.111.50 ppm, with maximum content in garlic (1.50 ppm). this value is below the accepted limit (5.0 ppm). the average pb content in the analyzed vegetables is between 0.07-0.46 ppm, also below legal limit (0.5 ppm), with maximum value in dill leaves (0.46 ppm). in humans, approximately 50% of the pb intake is through food which more than half originates from plants [21]. the rest is inhaled and absorbed depending on the particle size of the pb-contaminated dust. children however, absorb 50% of ingested pb compared to adults who absorb 1015%, making pb ingested through hand-tomouth behaviour or food an exposure source of significant concern [22]. metal (mg/kg dry matter) specification zn cu pb cd maximum limit accepted 43.00 8.00 18.00 1.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 21 the cd average content in the analyzed vegetables are low (0.02-0.11 ppm) also below the legal limit (0.1 ppm or 0.2 ppm). maximum value was identified in celery and parsley leaves. cadmium accumulates gradually in the kidney, especially the renal cortex. animals over 10 years old showed concentrations of up to 40 mg/kg dry matter, whereas young animals’ kidneys showed only trace amounts of 0.5 mg/kg [21]. the liver is the other target organ with levels of 0.08 mg/kg in young animals and up to 10 mg/kg in older ones. in our study smaller content of total heavy metals (around 2.5 ppm) was identified in cucumbers, cauliflower and cabbage. in peppers, tomatoes, eggplants, green beans, onion and potatoes the total content of heavy metals is between 3-6 ppm. in the root of carrots, celery and parsley the total content is between 3-7 ppm with maximum in parsley root. the highest values were identified in the leaves of parsley, dill, celery and garlic. the high content in root is associated with high content in leaves. anke and coworkers obtained the same result [24]; in parsley and lettuce they have identified more pb (0.81ppm) than in cucumbers (0.16 ppm) and potatoes (0.12 ppm) table 7 heavy metals content (mg/kg, ppm) in some vegetables cultivated within village cenad area zn cu pb cd .metals/ vegetables m in im um v al ue s m ax im um v al ue s a ve ra ge v al ue s m in im um v al ue s m ax im um v al ue s a ve ra ge v al ue s m in im um v al ue s m ax im um v al ue s a ve ra ge v al ue s m in im um v al ue s m ax im um v al ue s a ve ra ge v al ue s pepper 3. 0 0 9. 6 5 6. 4 0 0. 0 5 0. 3 1 0. 1 1 0. 0 5 0. 3 3 0. 1 1 0. 0 1 0. 0 5 0. 0 2 cucumbers 1. 0 0 4. 0 0 2. 1 1 0. 0 7 0. 5 1 0. 2 2 0. 0 1 0. 2 5 0. 0 8 0. 0 1 0. 0 5 0. 0 2 tomatoes 0. 5 0 5. 0 0 2. 9 1 0. 0 5 0. 3 1 0. 1 2 0. 0 5 0. 4 0 0. 1 2 0. 0 1 0. 0 8 0. 0 3 eggplants 2. 5 1 5. 2 0 4. 0 4 0. 2 5 1. 6 9 0. 8 1 0. 0 1 0. 1 5 0. 0 7 0. 0 1 0. 0 5 0. 0 2 green beans 2. 20 7. 10 4. 33 0. 20 2. 10 0. 85 0. 10 0. 50 0. 23 0. 05 0. 15 0. 09 cauliflower 0 .5 0 2. 5 0 1. 7 0 0. 1 0 1. 0 0 0. 5 1 0. 0 5 0. 4 0 0. 0 8 0. 0 1 0. 0 5 0. 0 2 cabbage 0. 5 0 3. 0 0 1. 2 0 0. 2 0 1. 8 2 0. 5 7 0. 1 0 0. 9 5 0. 3 6 0. 0 1 0. 0 5 0. 0 2 dill leaves 5. 50 12 .3 8. 40 0. 35 2. 65 1. 12 0. 28 0. 50 0. 46 0. 01 0. 10 0. 06 parsley leaves 10 .5 32 .0 21 .0 0. 50 3. 00 1. 40 0. 30 0. 50 0. 40 0. 05 0. 15 0. 08 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 22 celery leaves 4. 50 9. 70 6. 50 0. 50 3. 10 1. 40 0. 26 0. 50 0. 38 0. 05 0. 18 0. 11 potatoes 1. 10 4. 20 2. 10 0. 10 2. 00 0. 71 0. 05 0. 30 0. 11 0. 01 0. 08 0. 05 onion 2. 10 7. 10 4. 60 0. 10 2. 00 0. 75 0. 05 0. 38 0. 18 0. 01 0. 07 0. 03 garlic 7. 50 16 .0 11 .0 0. 50 3. 90 1. 50 0. 05 0. 40 0. 21 0. 01 0. 10 0. 04 carrots root 1. 05 3. 10 2. 10 0. 05 0. 31 0. 19 0. 21 0. 50 0. 41 0. 01 0. 05 0. 03 parsley root 2. 90 9. 80 5. 90 0. 50 2. 90 1. 10 0. 18 0. 46 0. 39 0. 01 0. 10 0. 04 celery – root 2. 10 5. 70 3. 10 0. 10 0. 95 0. 47 0. 15 0. 31 0. 25 0. 01 0. 10 0. 04 table 8 excerpt from the ministry of public health ordinance no 975/1998, maximum accepted limits for heavy metal in foods [25] metal (mg/kg fresh product, ppm) food zn cu pb cd fresh or deep frozen vegetables except for leaf vegetables 15 5.0 0.5 0.1 leaf vegetables 0.5 0.2 fresh or deep – frozen fruits 5.0 5.0 0.5 0.05 these values of heavy metals concentrations are close to literature data for similar soil and regional condition [26] and smaller than the values identified in polluted area [27]. in humans, about one-third of the total cd burden originates from animal products and twothirds from plants [21].the fao/who advises provisional tolerable weekly intakes (ptwi) of 25 and 7 µg of pb and cd, respectively, per kg of body weight, corresponding to maximum tolerable daily intakes of 3.6 and 1 µg of pb and cd, respectively. the mean daily intake per kg of body weight in a recent 24-hours total diet study conducted in the netherlands, was 0.25 µg pb (range 0.05 to 2.17 µg) and 0.15 µg cd (range 0.05 to 0.43 µg). these values were greatly below the limits corresponding to the results of other recent studies [22]. in march 2001 the new commission regulation (ec) no.466/2001 in relation to the permitted concentration of pb and cd in foodstuffs has come into force. the maximum levels are 0.1, 0.5, 0.5 and 0.1-0.3 mg/kg wet weight for pb in meat, liver, kidney and vegetables, respectively. the corresponding figures for cd are 0.05, 0.5, 1.0 and 0.05-0.3 mg/kg wet weight. vegetable consumption is very important for human nutrition. however vegetables polluted with high content of heavy metals can be the main way of entry for toxic metals in humans. for a healthy nutrition it is very important to know these contents. our data come to complete the knowledge in this domain. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 23 conclusions this paper presents some original studies concerning fe, mn, zn, cu, pb and cd, distribution in different vegetables (peppers, cucumbers, tomatoes. eggplants, green beans, cauliflower, cabbage, dillleaves, parsley-leaves and roots, celeryleaves and roots, carrots-roots, onion, garlic and potatoes) from a plain nonpolluted area of cenad-banat (romania). for all analyzed metals in vegetable samples, the average values are below the legal limits. the legal limit for zn (15 ppm) is exceeded in garlic and parsley leaves maximum values. the limit for cu (5 ppm) and cd (0.2 ppm) is not exceeded but the limit for pb (0.5 ppm) is reached by green beans, carrot root and leaves of parsley, dill and celery and exceeded by cabbage maximum values. for the other vegetables also the maximum values are below the legal limits. in this area the soil and climatic conditions, agricultural practice, are favourable to ecological vegetables production. the identified contents in heavy metals are in general below the legal limits of investigated toxic metals. acknowledgements this study was partially financed by the grant nr: 52157/1.10.2008 (cisppa) from the romanian ministry of education, research and innovation. references 1. kabata – pendias a. & pendias h., trace elements in soils and plants, 3rd edition, crc press, boca raton, fl., 2001, p.365. 2. băjescu i., chiriac a. distribuţia microelementelor în solurile din romania; implicaţii în agricultură, ed. ceres, bucureşti, 1984, p.156, 3. kastori, r., heavy metal in the environment, ed. novi sad, 1997, p. 95. 4. gârban z., biochimie, tratat comprehensiv vol. i, ed. a ii-a, ed. didact. si ped. bucureşti, 1999, p.575-579. 5. grill e., winnacker e. l., zenk m.h., science, 230, 1985, p.674-676. 6. kiss a.s., galbacs z., galbacs g., proceedings of the 2nd international symposium on metal elements in environment, medicine and biology, timişoara, 1996, ed. eurobit, timisoara,1997, p. 287. 7. avram, n., medrea, n., jurnal of medical veterinary, 1, nr. 1, 1994, p. 4. 8. who (1992): cadmium. environmental health criteria 134. international programme on chemical safety. world health organization, geneva ; who (1995): inorganic lead . environmental health criteria 165. international programme on chemical safety. world health organization, geneva 9. trif a., toxicologie, lito iat, timişoara, 1993, p. 192. 10. jeffery p.g.: metode chimice de analiză a rocilor, ed. tehnică, bucureşti, 1983, p. 29-31. 11. borlan z., hera c., metode de apreciere a stării de fertilitate a solului în vederea folosirii raţionale a îngraşămintelor, ed. ceres, bucureşti, 1973, p. 269-323. 12. lăcătuşu r., ştiinţa solului, vol. xxix, nr. 2,1995, p.69-80. 13. gergen i., metode chimice si fizicochimice in controlul calitatii produselor agroalimentare vegetale, ed. orizonturi universitare, timisoara, 2003, pag. 63-70. 14. saulea m., stoica a. i.., baiulescu g.e., marinescu d., ionică m., rev. chim.(bucuresti), 55, nr.5, 2004, p.301303. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 24 15. chirilă e., dobrinaş s., coatu v., rev. chim. (bucuresti), 55, nr.6, 2004, p.379-385. 16. birghilă s., dobrinaş s., matei n., magearu v., popesc v., soceanu a., rev,chim.(bucuresti), 55, nr.9, 2004, p. 683-685. 17. gergen i., gogoaşă i., ursulescu m., man e., metal elements in environment, medicine and biology, publishing house eurobit, timişoara, vol. iv, 2000, p.151154. 18. ianoş gh., analele univ. de vest timişoara, seria geografie, vol. vii, 1997, p.81-86. 19. ianoş gh., goian m., solurile banatului-evoluţie şi caracterizare agrochimică, ed. mirton, timişoara, 1995, p. 100-150. 20. xxx – ordinul ministerului apelor pădurii şi protecţiei mediului nr. 756/1997 valori de referinţă pentru urme de elemente chimice în sol. 21. hapke, h.-j., metal accumulation in the food chain and load of feed and food, in: merian, e. (ed) (1991): metal and their compounds in the environment. occurrence, analysis and biological relevance. weinheim, 469-479 22. willecke-wetstein, c., fischer, a.b., penkov, d., bainova, a., georgieva, r., stanislavova, l., dinev, n., bacon, j.r., huzior-balajewicz, a. proc. of 21.workshop 2002, macro and trace elements, friedrich schiller university jena, p.1169-1181. 23. lăcătuşu r., kovacsovics b., alexandrescu a.: lucrările celei de a xx-a conf. naţ. de ştiinţa solului, timişoara, 27-31 viii 1985, nr. 23b, p.327 (publ. 1987). 24. anke m., grün m., müller r., schäfer u., seifert m., proceedings of the 6th int. symp. on metal elements in environment, medicine and biology, vi, 2004, p.5-26. 25. xxx – ordinul ministerului sănătăţii nr. 975/1998. limite maxime de arsen şi metale grele în alimente, 26. gogoaşă i., jianu i. gergen i., lucrarile ştiintifice zilele academice timişene ediţia a vii-a, 24-25 mai 2001, ed. eurostampa, 2001, p.81-87. 27. vlad m., caseanu e., porr p.j. and uza g., 19.arbeistagung "mengen und spurenelemente" friedrich – schiller univ., jena, 1999, p.874-879. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 44 study on the parameters’ variation in fruits drying process gabriel – dănuţ mocanu1, oana – viorela nistor1, elisabeta botez1, doina – georgeta andronoiu1 1 dunărea de jos university – galaţi, faculty food science and engineering, domnească street, no. 111, 800201, phone + 40 336 130 185, fax + 40 236 460 165, galaţi, romania danut.mocanu@ugal.ro abstract. the aim of the study was the pursuing of the parameters’ variation of fruits drying process.as materials we use fruits with hard texture (apple, pear, quince) and as methods we used the conventional drying (with air convection) and unconventional drying (with microwave and infrared radiation). we determined the variation of the following parameters : mass, product’s core temperature, humidity, drying time. we used hydration as an estimation method of the drying products’ texture modification. the conventional drying one was done at 100°c for 120 minute. the microwave treatment was performed at 30% from oven maximum power – 900 w, in this case the drying period depending on each kind of fruit, so the thermic treatment lasted different intervals (30 minutes apple, 36 minutes pear and 20 minutes quince). the ir drying depends also on fruit type, as at 70°c the drying time for fruits was variable (apple 49 minutes, quince 25 minutes and 73 minutes for pear). the experimental determinations pointed out that the drying duration is sensitivly decreasing as compared to the conventional drying process (~1/3) for the same content of moisture. also, we found out that the quince texture is the hardest one leading to an easier drying and hydration process. the pear had very high water content and is a very rich source of carbohydrates, which are the principal responsible for the changes happened during the drying treatment, resulting in some colour transformation. keywords: air convection, microwave, infrared radiations, fruit drying, texture. 1.introduction drying is one of the oldest and most costeffective means of preservation of grains, crops and foods in all varieties. from both the energy and environmental point of view and in order to feed the growing population, it is important that drying technology is improved to reduce spoilage and enhance quality of the products. much has been accomplished over the past decades as far as understanding and development of drying technologies are concerned for food and agro-products (mujumdar, 2000; askari et. al., 2004). dried vegetables and fruits are an important sector in the ingredient market. most vegetables and fruits are generally dried convectively with heated air. hot-air dried vegetables are often difficult to rehydrate because of case-hardening and shrinkage occurring during the drying process. the consumer demand has increased for processed products that keep more of their original characteristics. major disadvantage of hot air drying of food is low energy efficiency and lengthy drying time during the falling rate period. prolonged exposure to elevated drying temperature may result in substantial degradation in quality attributes, such as food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 45 colour, nutrients and flavour, severe shrinkage also reduces bulk density and rehydration capacity (feng et al., 1998; askari et. al., 2004). the fact that fruits and vegetables collapse during dehydration has been established before (lozano et al., 1980; askari et. al., 2004). these researchers concluded that the slow and difficult rehydration of dehydrated apples is explained by the development of locked–in pores caused by cellular collapse during dehydration. cellular shrinkage during air drying was faster than the bulk shrinkage from full moisture to a moisture content of 1.5% (w/w). after this point, the shrinkage coefficient was converged. the result of the uneven shrinkage was an increased cellular porosity. this can be explained by a three– dimensional rearrangement of tissue due to the cellular collapse (askari et. al., 2004). the use of microwave energy and infrared radiation has been of growing interest over the years (askari et. al., 2004; swasdisevi et al., 2007). microwave heating is an important technique in industrial drying and food processing because it has several advantages over convective heating, for example, fast heating, high energy efficiency, and uniform moisture distribution in products. microwaves are electromagnetic waves in the frequency range 300 mhz to 300 ghz (equivalent to a wavelength of 1-0.01 m) generated by a magnetron type vacuum tube. electromagnetic energy at 915 and 2450 mhz can be absorbed by water containing materials or other substances, such as carbon and some organics, and converted to heat. because waves can penetrate directly into the material, heating is volumetric (from inside out) and provides fast and uniform heating throughout the entire product. the quick energy absorption by water molecules causes rapid water evaporation (resulting in higher drying rates of food), creating an outward flux of rapidly escaping vapour. in addition to improving the drying rate, this outward flux can help to prevent the shrinkage of tissue structure, which prevails in most conventional air drying techniques. hence better rehydration characteristics may be expected in microwave dried products (khraisheh et al., 1997; prabhanjan, 1995; quezada & bórquez, 2005). some researchers reported that using microwave reduces drying time (25-90%) and applying energy at lower level improves quality of final products, such as color, rehydration capacity, density and porosity (prabhanjan et al., 1994; funebo et al., 2000; askari et. al., 2004). the use of infrared radiation technology in dehydrating foods has several advantages as follows: decreased drying time, high energy efficiency, high quality product, uniform temperature in the product and reduced necessity for air flow across the product (swasdisevi et al., 2007). infrared (ir) radiation is the part of the electromagnetic spectrum that is predominantly responsible for the heating effect of the sun. infrared radiation is found between the visible light and radio waves (figure 1), and can be divided into three different categories, namely, nearinfrared radiation (nir), mid-infrared radiation (mir), and far-infrared radiation (fir) (ranjan et al., 2002; seyhun et al., 2009) .several researchers have applied far infrared radiation (fir) drying technique successfully to many food products, e.g., potato (afzal & abe, 1998), barley (afzal & abe, 2000), and rice (abe & afzal, 1997). mongpraneet et al. (2002) examined the drying behavior of the leaf parts of welsh onion undergoing combined far infrared and vacuum drying. the results showed that the radiation intensity food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 46 levels dramatically influenced the drying rate and the dried product qualities. figure 1. electromagnetic wave spectrum (krishnamurthy et al., 2008) mongpraneet at al. (2004) later determined the energy consumption in far infrared drying of onion. from their experiments, less than half of the energy input was utilized for evaporating water from the onion. approximately 73-99% of the energy input, depending on the dryer configulation, was converted into radiant energy. the efficiency decreased with increasing distance between the heater source and drying materials (swasdisevi et al., 2007). the objective of this investigation was to observe the effect of drying method on the structural changes of some fruits (pears, apples and quinces). 2. materials and methods 2.1 materials pears, apples and quinces purchased from a supermarket were washed under running cold water, and then the excess was removed from pears surface using paper towels. fruits were then peeled, portioned and sized in the cube-shaped samples of 10 mm side. 2.2. methods the following parameters were analyzed:  internal temperature of product with an amprobe tpp1-c1 pocket thermometer immersion probe;  product mass using a digital balance partner as110/c/2, with an accuracy of ± 0.001 g;  heat treatment using warm air convection in a memmert unb 400 oven;  heat treatment using a samsung microwave, 900 w power;  heat treatment using infrared radiations in a and 4714 balance. the hydration method was used in order to determine the modification of the dried fruits texture. hydration of dried fruits is used to bring back the initial state of the fruits. hydration of fruits strictly depends on water content, air humidity and storage duration. 3. results and discussion 3.1. heat treatment using warm air convection the internal temperature of fruits (figure 2) increases rapidly in the first 30 minutes (the highest values for this parameter were registered in pears at 50.5 ºc and in apples at 48.1 ºc), followed by a stabilisation of internal temperature and, in the end of the process, by a decrease of internal temperature, probably due to the accelerated rate of water evaporation at the fruits surface. at the end of the heat treatment with warm air convection the maximum internal temperature was reached by pears at (38 ºc) as against quinces at (37.2 ºc). in figure 3 one can observe that the weight of the fruits treated by warm air convection decreases. after 30 minutes of heat food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 47 treatment the weight of pears was 5.8 g and the weight of quinces was 5.3 g. 15 25 35 45 55 0 10 20 30 40 50 60 time, min temperature, 0c pears apples quince figure 2. variation of internal temperature function of time during heat treatment using warm air convection 2 3,5 5 6,5 8 9,5 11 0 10 20 30 40 50 60 time, min weight, g pears apples quince figure 3. the evolution of fruits weight during heat treatment with warm air convection after 60 minutes of heat treatment with warm air convection the lower value of weight was registrated for quince (2.17g), and the highest for apples (2.81g). this means that thermic treatmentby warm air convection leads to the elimination of a high quantity of water from the product. at hydratation of fruits dried by warm air convection (figure 4) one can observe that pears and apples require the longest hydratation time and this means that this type of treatment affected the cappilary structure of these fruits. this fenomenon was not observed in quinces. 1,5 3,5 5,5 7,5 9,5 0 10 20 30 40 50 60 time, min weight, g pears apples quince figure 4. evolution of warm air convectin dried fruits weight during hydratation 3.2. heat treatment using microwaves in the case of microwaves drying process (figure 5), using 30% of power, one can observe that the internal temperature increases in 2-10 minutes for all the fruits. after 10 minutes treatment the highest value of internal temperature was registred in pears (76.2 ºc) and the lowest one was registred in quinces (60.8ºc). these values for internal temperature are not desirable because they produce the degradation of vitamins. in the first part of microwave heat treatment an increase of water evaporation rate is noticed and may be explained by the temperature increase. 15 32 49 66 83 1 4 7 10 13 16 19 22 time, min temperature, 0 c pears apples quince figure 5. variation of internal temperature function of time during heat treatment using microwaves food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 48 after 15 minutes of microwaves treatment the internal temperature begings to stabilize, the highest value of this parameter being registered in pears (75.4 ºc). 0 2 4 6 8 10 0 3 6 9 12 15 18 21 time, min weight, g pears apples quince figure 6. evolution of fruits weight during heat treatment using microwaves in figure 6 one can observe that the weight of microwaves dried fruits decreases. after 10 minutes of treatment the following values were registered: 2.41g for pears, 3g for apples and 1,63 grams for quince. in the case of microwaves heat treatment, fruits hydratation (figure 7) requires a shorter time, as compared to the warm air convection treatment. 0 2 4 6 8 10 0 10 20 30 40 50 60 time, min weight, g pears apples quince figure 7. evolution of microwaves dried fruits weight during hydratation of the three analysed ranges of fruits, apples required a shorter hydratation time (30 minutes) as compared to pears (52.5 minutes). this can be explained by a negatttive influence of microwaves on fruits texture. 3.3. heat treatment with infrared radiations the highest content of evaporated water was registered in apples (after 9.5 minutes humidity was 27.9%). for pears and quince the loss humidity was 19% and 18.6% respectively. 0 15 30 45 60 75 90 0 3,5 7 10,5 14 17,5 21 24,5 time, min humidity, % pears apples quince figure 8. variation of humidity loss function of time during infrared radiations heat treatment variation of evaporated water quantity function of time is liniar for all the fruits (r2 values varying between 0.9866 for pears and 0.997 for quinces). 3 4,5 6 7,5 9 10,5 0 3,5 7 10,5 14 17,5 21 24,5 time, min weight, g pears apples quince figure 9. evolution of fruits weight during heat treatment using infrared radiations evolution of fruits weight during infrared radiation treatment is the same as during warm air convection treatment and microwaves treatment. after 9.5 minutes of treatment the weight of pears was 6.37g, the weight of apples was 5.92g and the weight of quince was 6.72g. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 49 2 3,5 5 6,5 8 9,5 0 10 20 30 40 50 60 time, min weight, g pears apples quince figure 10. evolution of infrared radiation dried fruits weight during hydratation in the case of infrared radiations treated fruits (figure 10) one can observe that the shortest hydratation time was required by quinces (37.5 min) while pears required 52.5 min for hydratation. 4. conclusions we appreciated that unconventional heat treatment (microwaves and infrared radiations) assures constant water elimination as compared to warm air convection treatment, fact that does not determine major textural modifications in the analyzed fruits. accelerated drying interval of the first period in conventional drying, which affects the quality of dried product is replaced in unconventional drying by gentle moisture elimination. fruits that are most suitable for drying using both conventional and unconventional heat treatment are quinces, while pears were the most difficult ones to get dried. references 1. abe, t., afzal, t.m., 1997. thin-layer infrared radiation drying of rough rice. j. agr. eng. res., 67, 289-297. 2. afzal, t.m., abe, t., 1998. diffusion in potato during far infrared radiation drying. j. food eng., 37: 353-365. 3. afzal, t.m., and abe, t., 2000. simulation of moisture changes in barley during far infrared radiation drying. com.& electro. agri., 26, 137-145. 4. askari, r.g., djomeh, z.e., ali mousavi, s.m., 2004. effect of drying method on microstructural changes of apples slices. proceedings of the 14th international drying symposium, vol. b, 1435 – 1441. 5. feng, h. & tang , j., 1998. microwave finish drying of diced apples in a spouted bed. journal of food science, vol. 63 , no. 4, 679-683. 6. funebo, t., ohlsson t., 1998. microwaveassisted air dehydration of apple and mushroom, journal of food engineering, vol. 38, 353-367. 7. khraisheh, m.a.m., cooper, t.j r., magee, t.r.a., 1997. shrinkage characteristic of potatoes dehydrated under combined microwave and convective air conditions. drying technology international, 15, 1003-1022. 8. krishnamurthy, k., khurana, h.k., jun, s., irudayaraj, j., demirci, a., 2008. infrared heating in food processing: an overview. comprehensive reviews in food science and food safety, vol. 7. 2 – 13. 9. mongpraneet, s, abe, t., tsurusaki, t., 2002. accelerated drying of welsh onion by far infrared radiation under vacuum conditions. j. food eng., 55, 147-156. 10. mongpraneet, s, abe, t. and tsurusaki, t. 2004. kinematic model for a far infrared vacuum dryer. drying tech., 22(7), 16751693. 11. mujumdar, a.s., 2000. drying technology in agricultural and food science, science publishers inc., plymoth, united kingdom. 12. quezada, p.a., bórquez, r.m., 2005. combined osmotic and microwave / vacuum dehydration of food. journal of food processing and preservation, 20, 1 – 9. 13. prabhanjan, d.g., ramaswamy, h.s., raghavan, g. s. v., 1995. microwaveassisted convective air drying of thin layer carrots. journal of food engineering, 25, 283-293. 14. ranjan, r., irudayaraj, j., jun, s., 2002. simulation of infrared drying process. drying technology 20, 363–379. 15. seyhun, n., ramaswamy, h., sumnu, g., sahin, s., ahmed, j., 2009. comparison and modeling of microwave tempering and infrared assisted microwave tempering of frozen potato puree. journal of food engineering, 92, 339–344. 16. swasdisevi ,t., devahastin, s., ngamchum, r., soponronnarit, s., 2007. optimization of a drying process using infraredvacuum drying of cavendish banana slices. songklanakarin j. sci. technol., 29(3), 809-816. звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 42 new ma ter ia ls for ph oto ca ta lytic pu r ific a tion o f air – a r ev iew *violeta vasilache1, catalin popa2, traian vasilache3 1stefan cel mare university of suceava, universitatii street 13, suceava, romania e-mail: violetav@fia.usv.ro 2universitatea tehnica cluj napoca, catalin.popa@stm.utcluj.ro 3vasile alecsandri university of bacau, calea marasesti 157, bacau, romania e-mail: traian.vasilache@ub.ro *corresponding author received 17 september 2011, accepted 20 october 2011 abstract: photocatalytic processes became to be used for water and air depollution. titanium dioxide (tio2) as photocatalyst is activated by electromagnetic waves from uv-vis range of spectrum and is used for degradation of some volatile organic compounds (vocs) and nasty smells removal. the quality of air, equally indoor and/or outdoor is a serious environmental problem. different methods are used for improving the air quality and photocatalysis as an advanced oxidizing process, could be a promising solution. using different techniques, new materials were developed by scientists from many countries, proving a real interest for these new environmental friendly technologies. this study presents last developments regarding new photocatalytic materials which have to work not only in uv (like pure titanium dioxide) but also in visible range of electromagnetic spectrum. also these new photocatalysts have to be able to oxidize different types of organic and/or inorganic molecules and diminishing the non-desiderated co-products from occurring reactions. to achieve new materials with superior photocatalytic properties there are used different procedures in order to combine and control the precursory components. the objective of the study was the understanding of the tio2-photocatalysis phenomena including gaseous and adsorbed phase mechanisms. this review presents some achievements in domain. keywords: photocatalysis, air depollution, vocs removal, nitrogen oxides degradation 1. introduction the quality of air, equally indoor and/or outdoor is a serious environmental problem. different methods are used for improving the air quality and photocatalysis as an advanced oxidizing process, could be a promising solution. initially photocatalysis processes were developed in direction of applications for water treatment, to degrade some specific pollutants or dyes. during the last decade, appear new directions, as far as concern air depollution, because photocatalytic processes were able to oxidize most of common vocs (volatile organic compounds). this study presents last developments regarding new photocatalytic materials which have to work not only in uv (like pure titanium dioxide) but also in visible range of electromagnetic spectrum. also these new photocatalysts have to be able to oxidize different types of organic and/or mailto:violetav@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 43 inorganic molecules and diminishing the non-desiderated co-products from occurring reactions. to achieve new materials with superior photocatalytic properties there are used different procedures in order to combine and control the precursory components. 2. theoretical approach photocatalytical reactions occur at the interface between catalyst and reaction medium in condition of irradiation with waves with electromagnetic field from uv and visible range of the spectrum. photocatalysts are semiconductors with a band gap between 1.4 and 3.6 ev. incident photons having energy more than the band gap energy are absorbed and as a fact, electrons jump from valence band in conduction band, and subsequently appear pairs of electron-hole particles. this pairs of particles exhibit oxidative and reductive properties, promoting redox reactions. the process follows few steps as described below: 1. semiconductors’ photo-excitation with em energy equal or higher then gap energy; 2. generation of the electron-hole pairs; 3. separation of electrons and holes, the rate of the process necessarily to be higher than the rate of the inverse process, although the photocatalyst is deactivated; 4. adsorption of the pollutants on the catalyst surface; 5. redox reactions on the photocatalyst surface occurring between electrons, gaps and molecules; the processes of this stage are determinants for final products formation; 6. desorption of the products from the catalyst surface. the efficiency of photocatalysis is closed by the rate of use of em energy and by reduction of the percent of recombination processes. the type, the structure and the morphology of the photocatalyst are very important properties determining the degradation of organic pollutants, destruction of bacteria and viruses, decomposition of dyes or synthesis of some compounds. titanium dioxide is the most used photocatalyst due to the fact it has some specific properties like a great capability for producing hydroxyl radicals, could be activated by solar irradiation, it is stable (even for extreme values of ph), it has low cost and could be prepared in laboratory as powder or thin films. also, it has an acceptable economic cost and it is environmental friendly. the processes occurring at tio2 surface could be briefly described by following equations: generation of electron-hole pairs 2tio2 + hν  tio2 (e-) + tio2 (h+) (1) recombination of electron-hole pairs: tio2 (e-) + tio2 (h+)  2tio2 + (heat, light) (2) surface reactions: tio2 (h+) + red1  ox1+ + tio2 (3) tio2 (h+) + h2o  oh* + h+ + tio2 (4) tio2 (h+) + ho oh* + tio2 (5) tio2 (e-) + o2  o2-* + tio2 (6) o2-* + h+  ho2* (7) 2ho2*  o2 + h2o2 (8) tio2 (e-) + ox2  red2+ tio2 (9) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 44 degradation of different adsorbed substances, like volatile organic compounds, nitrogen oxides, bacteria, dyes, etc. the processes are illustrated in figure 1. figure 1. processes occurring on the tio2 photocatalyst surface 3. recent studies in air depollution and vocs removal using photocatalysis promoted by titanium dioxide we mention general studies about photocatalysis and applications in air depollution from publications covering last years. b. ohtani (sapporo, japan) pointed to what we know and what we don’t know in present about photocatalysis [19]. it is a comprehensive study for documentation, a help for researchers. j.-m. herrmann (lyon, france) realized a study about fundamentals and misconceptions regarding photocatalysis, a very useful material which points to some important theoretical and practical aspects of the processes[12]. m. hunger and co. (enschede, holland) presented a comprehensive study about photocatalytic degradation of air pollutants [14]. no was used for degradation rate measurements. also it was described in brief an upcoming project in a dutch city. this idea is applicable in next future and could help cities to be more comfortable. figure 2. an hypothetical ecological town with self-cleaning surfaces based on photocatalysis the program of „the 14th international conference on tio2 photocatalysis: fundamentals and applications (tio2-14) the conference center niagara falls, new york, usa october 5-8, 2009” proved to be significant to point the newest achievements and tendencies in domain [29]. useful for present stage documentation is the work presented by a. selloni (department of chemistry, princeton university, usa), at workshop on nanoscience for solar energy conversion, 2008 and called „materials-related aspects in tio2 based photocatalysis: insights from first principles simulations” [21]. the study of different methods in tio2 photodegradation mechanism and gaseous and tio2-adsorbed phases was performed by p.-a. deveau and co. (france) [7]. the development of photocatalysis processes offers a significant number of perspectives especially in gaseous phase depollution. it is proved that the photo-oxidizing properties of photocatalyst (tio2) activated by uv plays an important role in the degradation of volatile organic compounds (voc). heterogeneous photocatalysis is based on the absorption of uv radiations by tio2. this phenomenon leads to the degradation and the oxidation of the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 45 compounds, according to a mechanism that associates the pollutant’s adsorption on the photocatalyst and radical degradation reactions. the objective of the study was the understanding of the tio2photocatalysis phenomena including gaseous and adsorbed phase mechanisms. results obtained with three different apparatus were compared; gaseous phases are analyzed and mechanisms at the gaseous phase/photocatalyst interface are identified. knowing the parameter is important for the design and construction of industrial size reactors that aim to purify the atmosphere. regarding vocs (volatile organic compounds) degradation, nasty smells removal, new materials with photacatalytic properties and new types of reactors, some recent studies are briefly pointed as follow. m.m. ballari and co. (eindhoven, holland) focused their work on no and no2 degradation by photocatalytically active concrete[3]. the application of photocatalytic concrete containing tio2 in urban streets is a method to improve the air quality in highly polluted areas. by using this technology it is possible to degrade a wide range of air contaminants, like nitric oxide (no) and nitrogen dioxide (no2), mainly emitted by automobiles. in their work paper, the photocatalytic degradation of no and no2 was experimentally studied, and the atmospheric reactions involving nitrogen oxides and solar radiation were analyzed as well. in addition, the influence of different system parameters, such as inlet pollutant concentration, relative humidity, and irradiance was investigated in detail. peter c.k. vesborg and co. (lyngby, denmark) studied gas-phase photocatalysis in μ-reactors [24]. they oxidized co and methanol and demonstrate that the system exhibits great versatility as far as concern photocatalysts, illumination source and target reaction. photocatalytic reaction intensification using monolithic supports designed by stereolithography was studied by m. furman and co. (nancy, france) [10]. they designed an original photochemical reactor for polluted air treatment. photocatalysis of gas-phase toluene using tio2/sio2 composites was studied by c. akly and co. (university of florida, usa) [2]. the performances provided by this novel catalyst are suitable for large-scale applications. a. strini and l. schiavi (italy) studied toluene degradation in air using a cementitious material with photocatalytic properties, in low irradiance uv [22]. there was used a stirred flow reactor. four pollutants were tested. they discovered a non-linear behavior of photoactivity at low irradiance. c. aguia and co. (porto, portugal) performed a study regarding the influence of photocatalytic paint components against the photooxidation of no capability [1]. a high quality vinyl paint was used, modified with tio2 degussa p25. it was concluded that the paint matrix plays an important role on the photocatalytic activity, because tio2 absorbs uv light in competition with other components; extenders, such as caco3 impair photoactivity, more in the case water is involved in mixing the paint components; also organic components affect the performance of paint during a transient time of 100-250 h. the study performed by j. chen and c.-s. poon from hong kong polytechnic university pointed to photocatalytic activity of tio2 in modified concrete materials and the influence of glass cullets used as aggregates [5]. no degradation was the measure of photoactivity. the glass cullets enhanced the photocatalytic activity of concrete, but a loss of 20% in photoactivity of surface layers was observed after 56 days curing. another study of the same authors focused on fundamentals and applications of photocatalytic construction and building food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 46 materials [6]. h. huang and d. ye (guangzhou, china) studied oxidation of gas-phase toluene in a combination process of photocatalysis downstream the non-thermal plasma reactor [13]. they investigated multiple synergies of o3/uv/tio2 system. a good conversion rate for toluene was achieved. d. kibanova and co. (mexico, usa) studied the synthesis and photocatalytic activity of small-sized tio2 supported on hectorite and kaolinite [16]. photocatalytic performance of these new composites was evaluated with atr-ftir following the oxidation of adsorbed toluene and dlimonene, two air pollutants. in either case, the photocatalytic removal was a success. h. wang and co. (jiangsu university, zhenjiang, china) analyzed tio2 photocatalysis in a pulsed discharge system for phenol degradation [26]. the photocatalyst prepared with eight times dipping process proved a higher photocatalytic activity than other tested photocatalysts. also h. wang and co. followed enhanced generation of oxidative species for phenol degradation in a discharge plasma system coupled with tio2 photocatalysis [27]. m. faure and co. (nancy university, france) compared different types of reactors used for air treatment [9]. they studied decomposition of different vocs (volatile organic compounds) in diverse conditions (like gas transit time through reactor, relative humidity, temperature). the aim of the study was to systematize the necessaries data for proper design of photoreaction systems. a novel photocatalytic monolith reactor for multiphase heterogeneous photocatalysis was designed and studied by p. du and co. (delft, holland) [8]. this was so-called internally illuminated monolith reactor (iimr). the results were discussed for different substances which were oxidized and a comparison with other types of reactors was made. c2h2 oxidation by plasma/tio2 combination and influence of the porosity against photocatalytic mechanism and plasma exposure were studied by o. guaitella and co. (lyon, france) [11]. the synergy plasma/photocatalysis is well known and could be a convenient solution for oxidation of acetylene. benoit boulinguiez and co. (france, algeria) studied photocatalytic degradation of ammonia and butyric acid in a plugflow reactor and they moderated degradation kinetic with contribution of mass transfer [4]. those substances were chosen because they are air pollutants and odorous contaminants. they found that chemical degradation kinetics follows the langmuir–hinshelwood model. reduction of nasty smells through photocatalytic oxidation was studied by g. vincent and o. zahraa [25]. they successfully performed degradation of 2butanone and methyl-ethyl-ketone (mek), two vocs which could cause olfactory discomfort. acetaldehyde results as byproduct in both cases. h.-h. ou and s.-l. lo (taiwan) studied phocatalysis of gaseous trichloroethylene (tce) and the effect of oxygen and relative humidity on the generation of dichloroacetyl chloride and phosgene [20]. f. thevenet and co. (lyon, france) studied oxidation of acetylene by photocatalysis coupled with dielectric barrier discharge [23]. vocs removal from air requires oxidative processes. a special reactor has been designed and interactions between photocatalysis and non-thermal plasma were investigated. acetylene has been selected as model molecule in order to evaluate oxidation efficiency. removal of vocs by photocatalysis process using adsorption enhanced tio2sio2 catalyst was the interest of l. zou and co. (australia) [28]. vocs exist in both the indoor and outdoor environment. some of them are toxic and carcinogenic to human health. in the experiment toluene food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 47 was used like voc indicator. the catalyst was synthesized using a sol-gel technique. toluene was successfully removed from air but there were detected some suspected intermediates or aliphatic hydrocarbons and co, too. the preparation of porous nano-tio2 with high activity and the discussion of the cooperation photocatalysis mechanism were performed by y. jiang and co. (china) [15]. a new preparation method was used. the researchers proved relations between the crystalloid and the size of tio2 nanograins, depending also by the technical conditions in which the process occurred. t. morikawa and co. (toyota, japan) created a new photocatalyst using titanium dioxide doped with nitrogen (tio2-xnx) which yields a high reactivity under visible light irradiation [18]. this material exhibits a photodecomposition rate for gaseous acetaldehyde 5 times higher than simple tio2, and also exhibits significant antibacterial properties. it works under visible light (below 520 nm) and it has production low costs. m. mohseni (vancouver, canada) studied gas phase trichloroethylene (tce) photooxidation through tio2 photocatalysis [17]. even tce oxidation was successfully achieved; small quantities of phosgene and dichloroacetyle chloride (dcac) were produced as by-products. 3. conclusions photocatalysis offers new perspectives in many fields of technology and industry. it is a clean and environmental friendly process. there were developed procedures for purifying water and for dyes degradation. now air depollution is the new trend of researches in the domain of applications of photocatalysis. some pollutants, like nitrogen oxides and volatile organic compounds were successfully degraded by photochemical methods. the achievement results could promote new commercial applications. 4. acknowledgments this paper was supported by the project "progress and development through postdoctoral research and innovation in engineering and applied sciences– pride contract no. posdru/89/1.5/s/57083", project co-funded from european social fund through sectorial operational program human resources 2007-2013. 5. references [1] aguia, c., angelo, j., madeira, l.m., mendes, a., influence of photocatalytic paint components on the photoactivity of p25 towards no abatement, catalysis today, 151. 77–83, (2010) [2] akly, c., chadik, p.a., mazyck, d.w., photocatalysis of gas-phase toluene using silica– titania composites: performance of a novel catalyst immobilization technique suitable for large-scale applications, applied catalysis b: environmental, 99. 329–335, (2010) [3] ballari, m.m., yu, q.l., brouwers, h.j.h., experimental study of the no and no2 degradation by photocatalytically active concrete, catalysis today (2010) [4] boulinguiez, b., bouzaza, a., merabet, s., wolbert, d., photocatalytic degradation of ammonia and butyric acid in plugflow reactor: degradation kinetic modeling with contribution of mass transfer, journal of photochemistry and photobiology a: chemistry, 200. 254–261, (2008) [5] chen, j., poon, c.-s., photocatalytic construction and building materials: from fundamentals to applications, building and environment, 44. 1899–1906, (2009) [6] chen, j., poon, c.-s., photocatalytic activity of titanium dioxide modified concrete materials – influence of utilizing recycled glass cullets as aggregates, journal of environmental management 90. 3436–3442, (2009) [7] deveau, p.-a., arsac, f., thivel, p.-x., ferronato, c., delpech, f., chovelon, j.-m., kaluzny, p., monnet, c., different methods in tio2 photodegradation mechanism studies: gaseous and tio2-adsorbed phases, journal of hazardous materials 144. 692–697, (2007) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 48 [8] du, p., carneiro, j.t., moulijn, j.a., mul, g., a novel photocatalytic monolith reactor for multiphase heterogeneous photocatalysis, applied catalysis a: general, 334. 119–128, (2008) [9] faure, m., furman, m., corbel, s., carre, m.c., gerardin, f., zahraa, o., caracterisation de reacteurs photocatalytiques utilises pour le traitment de l’air, séminaire de l’ecole doctorale rp2e «ingénierie des ressources, procédés, produits et environnement», nancy (2008) – isbn 2-9518564-6-6 [10] furman, m., corbel, s., wild, g., zahraa, o., photocatalytic reaction intensification using monolithic supports designed by stereolithography, chemical engineering and processing, 49. 35–41, (2010) [11] guaitella, o., thevenet, f., puzenat, e., guillard, c., rousseau, a., c2h2 oxidation by plasma/tio2 combination: influence of the porosity, and photocatalytic mechanisms under plasma exposure, applied catalysis b: environmental, 80. 296–305, (2008) [12] herrmann, j.-m., fundamentals and misconceptions in photocatalysis, journal of photochemistry and photobiology a: chemistry, 216. 85–93, (2010) [13] huang, h., ye, d., combination of photocatalysis downstream the non-thermal plasma reactor for oxidation of gas-phase toluene, journal of hazardous materials, 171. 535–541, (2009) [14] hunger, m., hüsken, g., brouwers, h.j.h., photocatalytic degradation of air pollutants — from modeling to large scale application, cement and concrete research, 40. 313–320, (2010) [15] jiang, y., zhang, p., liu, z., xu, f., the preparation of porous nano-tio2 with high activity and the discussion of the cooperation photocatalysis mechanism, materials chemistry and physics, 99. 498–504, (2006) [16] kibanova, d., trejo, m., destaillats, h., cervini-silva, j., synthesis of hectorite–tio2 and kaolinite–tio2 nanocomposites with photocatalytic activity for the degradation of model air pollutants, applied clay science, 42. 563–568, (2009) [17] mohseni, m., gas phase trichloroethylene (tce) photooxidation and byproduct formation: photolysis vs. titania/silica based photocatalysis, chemosphere, 59. 335–34, (2005) [18] morikawa, t., asahi, r., ohwaki, t., aoki, k., suzuki, k., taga, y., visible-light photocatalyst – nitrogen-doped titanium dioxide, r&d review of toyota crdl, 40. 3, (2005) [19] ohtani, b., photocatalysis a to z -what we know and what we don’t know in a scientific sense, journal of photochemistry and photobiology c: photochemistry reviews (2010) [20] ou, h.h., lo, s.-l., photocatalysis of gaseous trichloroethylene (tce) over tio2: the effect of oxygen and relative humidity on the generation of dichloroacetyl chloride (dcac) and phosgene, journal of hazardous materials, 146. 302–308, (2007) [21] selloni, a., materials-related aspects in tio2 based photocatalysis: insights from first principles simulations, workshop on nanoscience for solar energy conversion, department of chemistry, princeton university, (2008) [22] strini, a., schiavi, l., low irradiance toluene degradation activity of a cementitious photocatalytic material measured at constant pollutant concentration by a successive approximation method, applied catalysis b: environmental, (2010) [23] thevenet, f., guaitella, o., puzenat, e., herrmann, j.-m., rousseau, a., guillard, c., oxidation of acetylene by photocatalysis coupled with dielectric barrier discharge, catalysis today, 122. 186–194, (2007) [24] vesborg, p.c.k., olsen, j.l., henriksen, t.r., chorkendorff, i., hansen, o., gas-phase photocatalysis in μreactors, chemical engineering journal, 160. 738– 741, (2010) [25] vincent, g., zahraa, o., reduction de la nuisance olfactive par oxidation photocatalitique, séminaire de l’ecole doctorale rp2e «ingénierie des ressources, procédés, produits et environnement», nancy – isbn 2-9518564-5-8, france, (2007) [26] wang, h., chu, j., ou, h., zhao, r., han, j., analysis of tio2 photocatalysis in a pulsed discharge system for phenol degradation, journal of electrostatics, 67. 886–889, (2009) [27] wang, h., li, j., quan, x., wu, y., enhanced generation of oxidative species and phenol degradation in a discharge plasma system coupled with tio2 photocatalysis, applied catalysis b: environmental, 83. 72–77, (2008) [28] zou, l., luo, y., hooper, m., hu, e., removal of vocs by photocatalysis process using adsorption enhanced tio2–sio2 catalyst, chemical engineering and processing, 45. 959–964, (2006) [29] ********** the 14th international conference on tio2 photocatalysis: fundamentals and applications (tio2-14), the conference center niagara falls, new york, usa, october 5-8, 2009 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 61 life cycle management implementation of food products and services viorel iftimi1 1s.c. iso project suceava e-mail: viorel_iftimi@yahoo.com abstract: the aim of this paper is to develop integrated management systems by implementing, in an ims, the lifecycle management of products and services (lca) under a management system, already existing quality – environment – osh. the first part of the article contains a brief summary of the data in the literature on the principles and stages of implementing an integrated management system in an organization. it continues with the implementation of lca in a comprehensive way: the final product for example apples. this includes a series of processes analyzed such as: apple cultivation, collection, transportation in regional centers, packaging, sales in retail markets and hypermarkets, plus related servants involved in the manufacture of packaging, washing machines, recycling, etc. in the second part of this article, we focus our interest on meeting the requirements of reference standards sr en iso 14040 series on lca management with sr en iso 9001 quality management system. it further shows how the system design and system procedures are provided with mandatory operational procedures as well as the forms required by the sr en iso 14040 series standards. the approach takes into account the evolution of induced systemic process of economic globalization that makes it difficult to enter a business in one of the production chain of a product demanded by the market. keywords: management system, apple cultivation, collection, transportation, packaging, sales introduction frequently, the integrated management system implementation includes the reference standards of quality, environmental and occupational health and safety, with certification completed by a recognized and accredited certification body. jm juran trilogy was postulated as follows: planning, control and improvement of product quality and process carried out by the juran institute which insists on effective implementation (efficiency and effectiveness) of these standards, without putting emphasis on certification. our goal is to implement these integrated systems into small and medium-sized companies with emphasis on the integration of food in the flow of firms offering final product on the shelf. we firstly implemented the reference standard sr en iso 9001:2008. it introduces order and predictability in the management of companies / organizations. the standard requires clear determination of system processes, clear targets (by policy) and development of procedures for specific processes. compulsory rules have been established to check the documents and records management policy and commitment, market position, control of human, material and authority, to achieve control of products / services and how to monitor the products / services which do not comply with the auditing processes and analysis management. the certification aims at ruling the 3-d system: documents (the existence of documentation), describes (documentation is known and implemented), shows (as food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 62 practical action + records). it monitors the implementation of the pdca cycle (plan perform check act) and the ability to continuously improve the system. the implementation of the referential sr en iso 14001:2005 environmental management has been imposed from two converging directions: efficiency (seen as loss of energy and materials) and environmental impact and pollution (penalties, image, notes).[1] the referential meets the main requirements of iso 9001 and brings minimal identification of environmental aspects and environmental impacts assessment, emergency preparedness and response capacity, and management and disposal. the implementation of the referential sr ohsas 18001:2007 occupational health and safety management was subsequently required two-fold: osh (occupational safety and health) and ohs (occupational health and safety), with specific approaches and nuances. and this includes the referential main requirements of iso 9001 and brings still minimal hazard identification, risk assessment and control, emergency preparation and response capacity ssm approach. one way of integrating the last two referential standards is by implementing them as a starting point with clause 6.4.4 work environment. lca life cycle assessment (life cycle assessment), as shown in sr en iso 14040 standard itself, is a technique for assessing environmental aspects and potential impacts associated with a product, by: developing an inventory of relevant input and output elements of a systemproduct; evaluating the potential environmental impacts associated with those inputs and outputs; interpreting the results of inventory analysis and assessment phases of the impacts in relation to the objectives of the study. lca studies the environmental aspects and potential impacts of the product life cycle (cradle-to-grave) from raw material acquisition, continuing with the production, use and post-use. [2] general categories of environmental impacts, which are required to be taken into consideration include resource use, human health and environmental consequences. note that lca introduces the concept of &quot;system-product&quot; and analyzes a stream that includes several processes which basically are small / medium with its integrated management systems. for example, in figure 1, we present a transport stream of apples packed in corrugated cartons. the paper for corrugated cardboard logging kraft paper production corrugated cardboard production (34% kraft, 64% testliner) t t manufacture of cartons 500 km ship + 400 km truck intermediate mounting boxes large distributors organized market distribution channels rest t apple co t central supply t wholesale distributor 50 km (20% of cases) 400 km 50 km distribution end of life cardboard waste for recycling 70% household waste pathway 30% t apple co t wholesale t retail distributor 200 km 150 km distribution end of life cardboard waste for recycling 6% household waste pathway 94% waste paper / cardboard logging t 300 km truck t mounting location 20% 50 km 80% 50 km (20% of cases) figure 1. phases of lca food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 63 these concepts and standards are fully assimilated and implemented in the european union (en) and national default (sr). on the romanian market of all the major certification bodies operating certification are the credentials interconnected through mutual. experimental integrated management system requirements include implementation of quality management standards, environmental and occupational health and safety. we assume that firms have already implemented an integrated system and want to extend the scope of certification, at which point they will have to demonstrate that the revised documentation comply with the standards. typically, the implementation of the systems on the skeleton of sr en iso 9001:2008 based on the premise that this system was initially implemented (at least for 3 years), and having already documented and implemented procedures for the system that are imposed and other standards, in equivalent terms, as presented in table 1. table 1 mandatory procedures related to the terms of referential standards clauses of iso procedure 9001 1400 1800 ps 4.2.3 document control 4.2.3 4.4.5 4.4.5 ps 4.2.4 control of records 4.2.4 4.5.4 4.5.4 ps 8.2.2 internal audit 8.2.2 4.5.5 4.5.5 ps 8.3 control of 8.3 4.4.7 4.4.7 ps 8.5.2 corrective action 8.5.2 ps 8.5.3 preventive action 8.5.3 4.5.3 4.5.3. 2 po 5.6 management review 5.6 4.6 4.6 po 6.2.2 competence, training and awareness 6.2.2 4.4.2 4.4.2 po-7.2 customer related processes 7.2 po-7.3 design 7.3 po-7.4 supply 7.4 po-7.5 production and service 7.5 4.4.6 4.4.6 po 7.6 control of monitoring and measuring devices 7.6 4.5.1 4.5.1 these procedures are mandatory system standards required by the terms of the three referential standards. we recall that any certification audit is required, whereas mandatory, internal audits and report require last analysis management. implementation of environmental management requirements and standards of occupational health and safety procedures require a series of compulsory measures. see table 2. table 2 introduced additional procedures referential standards 14001 and 18001 iso clauses procedure 9001 14001 1800 ps-4.3.1 identifying environmental aspects and environmental impacts 5.2 7.2.1 7.2.2 4.3.1 4.3.1 po 5.5.3 internal 5.5.3 4.4.3 4.4.3 po 4.4.7 emergency preparedness and response capacity 8.3 4.4.7 4.4.7 po-6.4-1 hazard identification, risk assessment and control 5.2 7.2.1 4.3.1 4.3.1 ps-4.4.6 management and waste disposal 7.5 po 4.3.2 legal and other 5.2 4.4.6 4.3.2 4.4.6 4.3.2 these system procedures are specific referential standards iso 14001 and iso 18001 and there is a minimal scheme that can add forms, work instructions or operating procedures, and specific activities of the office. the process begins with establishing processes on form f-4.1 smi-a process map and this one contains the procedure and necessary records. the authors are at your disposal with the necessary advice from the experience of over 70 implementations and certification systems with more than seven internationally recognized certification bodies. „green” concepts have brought a new set of standards sr en iso 14040 series environmental management life cycle assessment principles and framework (life cycle assessment) or abbreviated lca. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 64 these standards promote a new philosophy of product design thought from the beginning, taking into account the consumption of raw materials, manufacturing, its life cycle until the end of life (preferably by recycling). [4] the whole process includes defining the purpose, scope and inventory analysis, life cycle impact assessment, life cycle interpretation, all in a data format of the documentation set. given the technical rule of these various standards supporting soft-ware sites have emerged. the sr en iso 14040 principle states within the framework of life cycle assessment the following: in clause 4.1 key features of lca stipulates that there „is no single method for conducting lca studies. organisations should have flexibility to implement virtually lca as set forth in this international standard, based on specific application and user requirements.” by this, we postulate that there are no systems or soft-ware solutions universal each system is unique and has its specificity, but must comply with referential standards. clause 4.2 phases of a lca studies the main phases of lca in figure 2. [5] figure 2. phases of lca it further establishes the general methodological framework, reporting and how to perform critical analysis. sr en iso 14041 defines the purpose, scope and inventory analysis in detailing steps, the working and structure of reports produced. all provisions of the standard should be applied, as it introduces the concept of system-product. [3] sr en iso 14042 life cycle impact assessment lcia is stage 3 of the lca and assessment process aims to assess the results of the analysis of life cycle inventory (lci) of a system-product. sr en iso 14043 life cycle interpretation concerns the interpretation of data following the next steps required: overview of life cycle interpretation identification of significant issues assessment conclusions and recommendations reporting other investigations iso-ts 14048 documentation format of the data structure of tables detailing is even used in developing the lca studies. in this perspective, practical experience in implementations and the system require the records presented in table 3. table 3 procedures and forms brought by 14040 additional reference standards document f-3-1 terms and definitions po-6.4-40 lca – principles and framework (sr en iso 14040) po-6.4-40-1 lca – define the purpose, scope and inventory analysis (sr en iso 14041) f-6.4-40-1-1 input form for carrying supplies f-6.4-40-1-2 data sheet for internal transport f-6.4-40-1-3 data sheet for the unit process f-6.4-40-1-4 centralize data sheet for the life cycle inventory analysis po-6.4-40-2 lca – life cycle impact assessment (sr en iso 14042) po-6.4-40-3 lca – life cycle interpretation (sr en iso 14043) f-6.4-40-3-a1 structuring data for input and output lci life cycle stages f-6.4-40-3-a2 percentage contributions of input and output of the lci in the life cycle stages f-6.4-40-3-a3 ranking of input and output of the lci to stages of life cycle f-6.4-40-3-a4 matrix structure of processes define the purpose and scope inventory analysis impact assessme nt inter pret atio n direct applications: improving product development and strategic planning process of making public policymarketing other food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 65 grouped into groups f-6.4-40-3-a5 classification by degree of influence of input and output data of lci arranged in groups of processes f-6.4-40-3-a6 marking the anomalies and unexpected results of input and output processes in groups f-6.4-40-3-a7 structuring the class outcome indicator (gwp) than life cycle stages f-6.4-40-3-a8 structuring the category indicator results (gwp) than life cycle stages, expressed in percentages f-6.4-40-3-a9 content verification completeness f-6.4-40-3-a10 verification sensitiveness to allocation rulesf-6.4-40-3-a11 checking the data uncertainty applied to sensitiveness f-6.4-40-3-a12 verification sensitiveness applied to the characterization data f-6.4-40-3-a13 the results of consistency checks po-6.4-40-8 lca format of the data documentation (sr iso-ts 14048) f-6.4-40-8-a1 process f-6.4-40-8-a2 modeling and validation f-6.4-40-8-a3 administrative information f-6.4-40-8-b1 process f-6.4-40-8-b2 elements of input / output elements f-6.4-40-8-b3 modeling and validation f-6.4-40-8-b4 administrative information conclusion in recent years there has been accumulated a large volume of communications on lca studies, and almost each of them claims fairness. to speak a common language we should relate strictly to the implementation of the reference standards sr en iso 14040 series and procedures and records required by them. it is good to make implementation a consultant may, by curriculum vitae, evidence that leads directly to people and businesses. vision is needed for overall management, linking the action system product assembly and parts (sub-systems) components. lca requires that analysis be made by independent experts, preferably external, to ensure objectivity. objectivity ensures correct conclusions that will lead to finding that waiver decisions on economic and environmental grounds, a number of other systems a product which actually represents the chain of companies and business, technical facilities, capital and workers with families and local communities. [6] [7] [8] there are no acknolwedged and acredited certification bodies of referential standards sr en iso 14040 series, still their application can solve the problem out – by developing a dialogue between stakeholders and consulting firms, creating regional accreditation bodies for our benefit. the authors may provide available databases and software forms and useful information. abbreviations ims integrated management systems lca life cycle assessment sr romanian standard en european norm iso international organization for standardization ps system procedure po operational procedure f form references 1.declaraţia de mediu egger – http://www.egger.com/pdf/zf_epd_egger_dpr _en_2009_kurzfassung.pdf 2. coles r, mcdowell d., kirwan m.j., food packaging technology, blackwell publishing ltd, 2003 3. analyse du cycle de vie des caisses en bois, carton ondulé et plastique pour pommes – synthèse : version finale (l045-s4) préparée par ecobilan 4. ademe, evaluation des impacts environnmentaux des sacs de caisse carrefour, rapport prepare pour carrefour, 20045.iosip alina şi elena bobu , analiza impactului de mediu al produselor şi proceselor prin evaluarea ciclului de viaţă (lca) , revista de celuloză şi hârtie nr.1 din 2010 6.urbaneco, eco arhitectura, aplicatii tehnologice in ecologie, http://ggrreenn. ecosapiens.ro/ecoarhitectura-aplicatii-tehnologice -inecologie/ 7.v.i. adomnicăi, la frontiera comună a judeţului suceava cu regiunea cernăuţi, proiect european de mediu , 11.05.2006| 8. p. bulzan , ungurii fac focul cu lemne din arad, observator, 23 iul 2008 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 19 ph eno lic con ten t an d a ntio xid an t ac tiv ity in m illing fr ac tions o f ry e cristian serea1, *octavian barna 1 1biochemistry department, faculty of food science and engineering, galati, „dunărea de jos ” university, 111 domneasca st., 800201 galati, cristian_serea@yahoo.com;octavian.barna@yahoo.com *corresponding author received 3 april 2011, accepted 10 may 2011 abstract: phenol compounds are a class of plants’ metabolites with role in protecting against ultra violet radiations and pathogen agents. rye contains phenol compounds that have raised the specialists’ interest especially due to their antioxidant action with benefic effects in reducing the incidence of cancer and cardiovascular diseases. antioxidants are not homogeneously distributed in the rye grain. the aim of this paper is to determine the distribution of phenol compounds in the fractions resulted from grinding the rye cultivars gown in our country. three rye cultivars of the 2008 harvest were used. the samples were ground by the pilot mill buhler and the fractions resulted were submitted to the extraction method. the extracts were filtered and analyzed further on to determine the total phenol content and total antioxidant capacity. total phenol content was determined using the method folin –ciocâlteu in all the fractions resulted from grinding. total antioxidant capacity was spectrophotometrically determined on phenol extracts by the method dpph. the results obtained did not show notable differences between the cultivars analyzed as regards the total phenol content, complying with the limits specified by the literature in the field. as for the distribution on milling fractions, the quantity of total phenols is higher as the fractions contain more milling material coming from the aleuronic layer and bran. when determining the antiradical capacity we noticed that the reducing capacity increases as the fraction ground contains a higher percentage of aleuronic tissue. © 2011 university publishing house of suceava. all rights reserved keywords: phenolic content, antioxidant activity, rye, milling fractions 1. introduction phenol compounds are a class of plants’ metabolites with role in protecting against ultra violet radiations and pathogen agents [1]. the primary sources of naturally available antioxidants are whole cereals, fruits and vegetables [2]. wheat contains phenol compounds that have raised the specialists’ interest especially due to their antioxidant action with benefic effects in reducing the incidence of cancer and cardiovascular diseases [3]. antioxidants are a group of small molecular weight – phytochemicals that can be especially found in produces of vegetal origin. these ones include carotenoids, tocopherols, lignans and phenolic acids. these antioxidant components can prevent the oxidation of some enzymes and dna by different mechanisms. the antioxidants from food play an important role as protecting factor. it has been scientifically proved that they reduce the incidence risk of cancer and cardiovascular diseases [2]. vitamin c, vitamin e, phenolic acids, phytates and phytoestrogenes have been acknowledged as potential factors in preventing the incidence risk of cancer and cardiovascular diseases [4]. the antioxidants in cereals and cereal products are already known but their potential food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 20 contribution to health by diet has been essentially ignored. their capacity to fight against free radicals makes them being of special interest at this research moment in the field of foods which can provide certain solutions regarding the intake of antioxidants in consumers’ diet [5]. studies have shown that wheat antioxidants can directly react with the oxygen reactive species such as hydroxyl radicals or singlet oxygen molecules, diminishing their attack upon biological molecules [6]. rye antioxidants can also form chelating complexes with transitive metals in order to reduce the availability of these ones as catalyzers to produce free radicals [7]. by investigating significant levels of natural antioxidants in cereals, the research results in the field have shown that antioxidants are not homogenously distributed in grains [8]. the antioxidant activity of different fractions in cereals resulted from the milling process has little been studied and it is useful in the manufacturing of functional foods with important role in ensuring consumers’ health condition. 2. experimental the cereal samples to be analyzed are from romania, the harvest of 2008, recommended by the national institute of agricultural researchdevelopment fundulea (cultivar gloria, suceveana and orizont) for their resistance to new climate conditions from our country and to the attack of specific pests. the preparation of rye samples for grinding: the samples were brought to a moisture of 13,5% 24 hours before grinding and about 30 minutes before grinding to a moisture of 15%. the moisture content was determined by the help of the electronic humidometer t1. the samples were ground in the pilot mill buhler. the seven fractions of cereals obtained (three from milling, three from grist and bran) symbolized as m1, m2, m3, şr1, şr2, şr3, and t, were weighed by the analytical balance mettler toledo. 2.5 g from each sample were taken and put into recipients on which 64% ethanol solution was poured. the samples were placed into an ultrasound bath of sonica 2200 type at the temperature of 60 o c, for 25 minutes. after that, the extracts from the cereal fractions were vacuum filtered to get the clear watery extract, without any impurity, ready for further determinations. the extracts were further filtered and analyzed to determine the total phenol content and oxidizing capacity. all the reagents used were of analytical purity and all tests were made in duplicates. the method folin-ciocâlteu was used to determine the total phenol content [9]. the extracts were diluted to the proportion of 1:3 by ultra-pure water obtained by the help of the water ultra purifying system tka smart 2 pure, then 1 ml of diluted extract sample was transferred to a test tube containing 5 ml of folinciocâlteu 1/10 solution into water. then 4 ml of sodium carbonate 7.5 % (w/v) solution was added to neutralize. this operation was repeated for all the fractions of cereals analyzed. the test tubes were maintained at room temperature for 60 minutes; afterwards the absorbance was measured at the wavelength of 765 nm by the spectrophotometer jasco model v 530, using ultra-pure water as control sample. the total phenol content was expressed in equivalents of gallic acid ( gae) in g/100 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 21 g material, using a standard curve of gallic acid, with concentrations varying between 0 -50 µg/ml ( pearson correlation coefficient: r²= 0.9917) accordingly to the standardized method iso 14502-1[ 93]. determination of reducing activity was made using the method dpph [10]. dilutions of 1: 100 of ultra pure water for each sample analyzed were made. 200µl of sample or standard were taken and introduced into eppendorf tubes and 1.4 ml dpph solution 80 µmol/100 ml was added. the control sample consists of 200 µl ethanol plus 1.4 ml dpph solution. the samples were centrifuged at 15000 rpm by the universal 320r centrifuge, for 10 minutes, at the temperature of 18 o c in order to get some homogeneity and remove possible impurities left in. the absorbance of samples is read at the minute 0 and minute 30. for the quantitative determination, a calibrating plot was made, represented by the intensity variation of absorption peak of dpph at 517 nm under different concentrations of trolox (6hydroxy 2,5,7,8 tetramethylecroman-2-carboxylic acid, a synthetic analogue of vitamin e). the comparative analysis of samples was made by calculating the antiradical activity [% radpph], standing for the relative decrease of absorbance in the samples analyzed. the absorbance inhibition percentage of dpph solution was calculated using the following equation: % radpph = [abst0 min abst30 min] / abst0 min × 100 (1) where abst0 min is the absorbance pph at the moment zero and abst30 min is the absorbance dpph after 30 de minutes of incubation. statistical analysis the program excell of microsoft office 2003 was used to determine the correlation coefficient [11] between total content of antioxidants and the antioxidizing activity of the cultivars analyzed. 3. results and discussion the results of experimental data regarding the total phenol content are presented in the graph of figure 1. the total phenol content varies between 0,2140 and 0,9527 µg gae./ml extract. 0 0,2 0,4 0,6 0,8 1 1,2 m1 m2 m3 sr1 sr2 sr3 t milling fractions to ta l p he no lic s (µ g g a e / m l ex tr ac t) gloria suceveana orizont figure 1:total phenolics content in milling fractions the total phenolic content of the cultivars gloria and orizont registered near values in the milling fractions resulted from breaking and grinding, the cultivar suceveana having a higher content of phenol compounds. it has been observed that in the case of fractions resulted from grinding, the cultivar gloria has the lowest content of phenol substances and in the case of grists the lowest polyphenol content is registered by the cultivar orizont. in all the three cultivars analyzed the total polyphenol quantity increases slightly as the fractions contain more ground material resulting from the aleuronic layer. a significant increase of the total polyphenol content in bran is registered as compared with the rest of fractions obtained from grinding. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 22 comparing the total polyphenol content in bran we can see that the cultivar orizont had the minimum values, the cultivar gloria the medium ones and the cultivar suceveana the maximum ones. the antioxidant activity of the three cultivars analyzed can be seen in the graph of figure 2. as a whole the fractions ground from the rye cultivar orizont have a higher reducing activity than those corresponding to the cultivars suceveana şi gloria. all three cultivars analyzed show in the fractions resulting from milling [m1, m2, m3] a more reduced antiradical activity as compared with the values obtained from the grists and bran, fact explained by the higher percentage of starch poor in antioxidants. the maximum values of antiradical activity were registered in bran, as it was expected, the minimum ones corresponding to the cultivar gloria, the medium ones to suceveana and the maximum to orizont. the calculation of correlation coefficient between total content of antioxidants and antioxidant activity of the cultivars analyzed led to values of r =0,993864 for the cultivar gloria and r=0,895667 for suceveana and r = 0,950117 for orizont. these values show a strong correlation in the same sense of the parameters analyzed in the case of all the cultivars analyzed. 0 10 20 30 40 50 60 m1 m2 m3 sr1 sr2 sr3 t milling fractions a nt io xi da nt a ct iv ity (µ g g a e /m l e xt ra ct ) gloria suceveana orizont figure 2 antioxidant activity in milling fractions 4. conclusion in the rye cultivars analyzed, the total polyphenol quantity had relatively near values in the cultivars gloria and orizont, suceveana registering some higher values. it has also been noticed that the bran had the highest percentage of total polyphenols, and the fractions resulted from milling had the lowest one due to the higher content of endosperm. the different values of total phenol content obtained from breaking can be explained as well by the presence of their different proportions of bran, aleuronic layer and germs. in the fractions processed the endosperm having a reduced content of polyphenols decreases the share of antioxidant substances present in bran and consequently the total phenol content of whole cereals is lower, as compared with the bran fraction taken individually. the quantity of polyphenols found in the cultivars analyzed subscribes to the limits of the literature in the field. as regards the antioxidant activity, even if we expected that there would be a direct proportionality between the total phenol content and antioxidant activity, we can see that the antioxidant activity of the fractions of the cultivar suceveana hold the second position though its has registered a food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 23 maximum polyphenol content. it is obvious that the antioxidant activity of the romanian rye cultivars analyzed registers comparable values to those cited in the literature of the field at world level. the study shows that the processing degree should be kept at a minimum level in order to maintain high the value of bioactive compounds in cereals. the results bring some additional confirmation on the location of phenol compounds in external layers of cereal grains. 5. references 1. alvarez-jubete,l., wijngard, h., arendt,b., gallagner,e.,polyphenol composition and vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as as affected by sprouting and baking, food chemistry 119 770-778, (2010) 2. moure, a., cruz, j.m.,franco, d., dominguez, j.m.,natural antioxidants from residual sources, food chemistry vol 72, issue 2, feb, 145-171, (2001) 3. andreasen, m.f., landbo,a., k., lars, p., christensen,l.p, hansen,a., meyer, a.s., antioxidant effects of phenolic rye (secale cereale l.)extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins, j. agric. food chem., , 49 [ 8], pp 4090–4096, (2001) 4. halliwel, b., antioxidants in human health and disease, annual review of nutrition,vol. 16: 33-50 (1996) 5. baublis, a.j., lu, ch, fergus, m., clydestale, m.f., decker, e.a., potential of wheat-based breakfast cereals as a source of dietary antioxidants, journal of the american college of nutrition, vol. 19, no. 90003, 308s311s (2000) 6. chandrika, m., fereidon, s., antioxidant and free radical scavenging activities of whole wheat and milling fractions, food chemistry 101 1151–1157,(2007) 7.rice evans, c.a., miller, n.j., paganga, g., structure antioxidant activity relationships of flavonoids and phenolic acids.free radical biology and medicine, 20, 933–956, (1996) 8.lloyd, b.j., siebenmorgen, t.j., beers, k.w.,effects of commercial processing on antioxidants in rice bran, cereal chemistry, volume 77, number 5,pages 551-55, (2000) 9.singleton , et all., current protocols in food analytical chemistry i1.1.1-i1.1.8, by andrew l. waterhouse university of california, (2002) 10.molyneux, p., the use of the stable free radical diphenylpicrylhydrazyl (dpph) for estimting antioxidant activity. songklanakarin j. sci. technol., v. 26, n. 2, p. 211-219. (2004) 11. hedges, l.v., olkin, i., statistical methods for meta-analysis, hartcourt brace jovanovich publishers, new york, (1985) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 108 the content of carotenoid pigments in rosa canina l. fruit *sorina ropciuc university stefan cel mare suceava -romania e-mail: sorinaropciuc@yahoo.com *corresponding author received 12 august 2011, accepted 15 november 2011 abstract: carotenoid pigments represent the integral vegetable carotenoids. they are the most widespread of all vegetable pigments. in the animal kingdom, these pigments come from the foods with a plant origin. carotenoids are yellow, orange, red, purple, etc. colored pigments. in the plant body it is found in free state or in combination with holoprotoids and carbohydrates (carotenoprotoids, carotenoid glycosides). carotenoid pigments are spread in all plant organs with or without chlorophyll (leaves, fruits, stems, bulbs, seeds, etc.). carotenes are the precursors of retinols, that belong to the class of carotinids – the unsaturated pigments largly widespread in all the plants found in association with chlorophyll, in algae and microorganisms. the ingested carotenoids are absorbed through the intestinal wall in the presence of lipases and bile salts; the absorption is accompanied by oxidation reactions that form trans complete structures. carotenoids are absorbed especially by the liver in the form of higher fatty acids. vegetable pigments give the flavor, the taste and the color of the plant products. they can be part of the constitution of redox coenzymes, some of them are protectors of the enzymes and of the enzymatic activity, adjusting the redox potential of the biochemical environment, others are directly or indirectly involved in the process of photosynthesis. keywords: color substances, enzymatic protectors, the retinol’s precusors 1. introduction the carotenoids are lipid-soluble substances with 40 carbon atoms, disposed in a polyenic chain with 18 carbon atoms and 9 double conjugated bonds. at one end of the polyenic chain is grafted a β-ionone cycle and at the other end a α-ionone cycle or an open γ-ionone cycle (pseudo-ionone). depending on the nature of the ionone cycle, there aare three types of carotenoids – α, β, γ. [1] among the vegetable materials with high content of carotenoid pigments are: the carrot, the pepper, the apricots, the tomatoes, the sour cherries, the rose hip fruits. [2] the fruits of rosa canina l. are found abundantly in our country, the rose hip has an extended growth area, but the fruits are not processed in terms of carotenoid pigments. the content of carotinids increases gradually in the rose hip fruit beginning with the shaping phase of the fruit until the full ripeness stage, when it reaches maximum value. along with aging, the fruits soften, depreciate qualitatively, it decreases the content of useful substances and implicitly the content of carotenoid substances. [3] there occur interconversions between different types of carotenoids and the proportion between the content of hydrocarbonic carotenoids modifies. [1] the paper presents the extraction method and the content of total carotenoids from the fruits of rosa canina l. these pigments can be used as a natural dye in food, pharmacy and cosmetic industry, because they are natural and nontoxic. [3] the content of carotenoids varies with the content of vitamin c, with the altitude and with their position in the field. mailto:sorinaropciuc@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 109 datorită poluării cu noxe chimice, cu praf, apar fenomene de deteriorare rapidă a fructelor de măceș, dimensiuni reduse și desprinderi de pe ramuri deteriorare rapidă la atingere. [4] due to the pollution with chemical emissions, with dust, there occur quick deterioration phenomena of the rose hip fruits, small sizes, easy branches detachments and quick deterioration to the touch. [4]. from the category of fotochemical oxidants, the strongest are the ozone (o3) and the peroxyacetyl nitrates (pan). they form as a result of the disturbance of the photolytic cycle of nitrogen diocide and the presence in atmosphere of the hydrocarbons from the combustion processes on the mobile sources (vehicles). [5] 2. experimental material and method the analysed material are the fruits of rosa canina l., collected from the spontaneous flora at full maturity from the marked bushes on the route suceava, pătrăuţi, dărmăneşti, costâna, părhăuţi, todireşti, cajvana, arbore, solca, clit, marginea, rădăuţi, suceviţa, palma. figure 1. the map of resorts the principle of the method. the dry vegetable material is triturated with a mix of anhydrous sodium sulphate and calcium oxid, that retain the colored substances except the carotenes. in order to prevent the dissolution of the carotenes in the acid medium, we add an anhydrous sodium carbonate. the carotenes from the triturated mix are extracted using acetone and perol ether. we measure the color intensity of the etheric solution of carotenes using the spectrophotometer with the wavelength of 450nm (blue filter). the content of carotenoid pigments is calculated according to formula (1). mg carotene%= pe vae   2 1 100 (1) where: e1-the extinction of the investigated sample; e2 the extinction of the standard solution; athe number mg of carotene in a ml of standard solution; vthe volume of carotene solution, ml; pthe weight of the analysed material. the ph of the soil was determined using the potentiometric method with a ph-metre wtw, inolab1, in a aqueous solution according to sr 718413:2001. [6] 3. results and discussions for the determination we used rose hip fruits collected from the spontaneous during the full ripeness stage from 42 existing biotops within the chosen resorts. the results are correlated both with the chemical composition of the fruits and with the pedographic factors, with the location of the bushes to the pollution sources. when we compare the obtained results we notice the fact that the rose hip from the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 110 resorts found on the edge of the roads or on countryside roads with heavy vehicles traffic the content of pigments is reduced. the other resorts are next to people’s houses or on the way out of the city. the mobile pollution sources are the one that affect the vegetation with polycyclic aromatic hydrocarbons through the incomplete combustion of fuel, like gas or gasoline, as well of some solid fuel used in domestic heating installations. the content of carotenoid pigments correlate very well with the soil ph r2=0,3283. the regression straight line and the dispersal of the individual values is shown in figure 1. the content values of the carotenoid pigments orient themselves along a curve and we distinguish hereby a logarithmic regression. the correlation with the altitude of the carotenoid pigments is insignificant. the value of the regression is way below the significant value of 0,5. the values of the carotenoids are scattered, r2 equaling 0,1367. the graphic representation of the correlation with the altitude is shown in figure 2. table 1. the measured values of the content of carotenoid pigments using the spectrophotometre resorts the content of water in rose hip fruits soil ph carotenoid pigments in rose hip fruits u% mg% s1 suceava 16 7.48 13.42 s2 pătrăuţi 14.61 4.99 16.75 s3 dărmăneşti 16 7.12 52.52 s4 costâna 14.83 6.15 17.11 s5 părhăuţi 15.69 5.4 15.76 s6 todireşti 13.93 5.38 19.76 s7 cajvana 14 4.34 9,37 s8 arbore 13.58 6.38 36,53 s9 solca 12.57 6.04 21.64 s10 clit 12.5 5.17 37.32 s11 marginea 14 7.29 46.95 s12 rădăuţi 10 6.56 39.63 s13 suceviţa 10.75 7.31 62.47 s14 palma 11.31 6.33 13.26 the correlative bond in the form of a logarithmic regression y = 58.823ln(x) 77.268 r2 = 0.3283 0 10 20 30 40 50 60 70 2 4 6 8 soil ph ca ro te no id p ig m en ts , m g% analiza regresiei log. (analiza regresiei) figure2. the graphic representation of the correlative bond in the form of a logarithmic regression food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 111 the correletive bond between pigments and altitude y = 1.4945x + 17.541 r2 = 0.1367 0 10 20 30 40 50 60 70 344 343 292 327 368 411 407 426 378 418 460 388 550 1080 altitude, m cr ot en oi d pi gm en ts , m g% caroten mg linear ( caroten mg) figure 3. the correlative bond between pigments and altitude 4.conclusions the results of the determinations highlight the fact that the carotenoid pigments are found in quantities between 9,37mg% in cajvana resort and 62,47mg% in suceviţa. the different values are due partially to the postion of the bushes around houses, infields, roads with heavy traffic that pollute with dangerous chemical emissions and with aromatic polycyclic hidrocarbons. the bushes located in the vicinity of infields can be polluted with pesticides and insecticides, organic pollutants used in agricultural treatments. the dust, the microscopic particles hinder the process of photosynthesis, and as the result, the leaves of the plants fall down earlier or the plants are underdeveloped. fruits come off easily and fall off the stems. the content of carotenoid pigments correlate very well with the soil acidity. good results were registred at a ph betweeb 4 and 8. this value indicates an acid to a slightly alkaline ph. there is a type of behaviour in the case of some plants, that intensify their color when the soil has acid values. the fruits with low content of carotenoid pigments have been collected from bushes that presented early defoliation, fact that indicates pollution and sediments on the surface of the leaves, all this blocking the plant’s photosynthesis. 5. references 1. şerban m. roşu n., biologically active substances, romanian academy publishing house, bucharest, 298299 (1992) 2. neamţu g tămaş v., carotenoid pigments and metabolites, vol. i . 32 (1986) 3. goodwint.w., chemistry and biochemistry of plant pigments, acad. press london & new york 129 (1980) 4. ivănescu l. toma c., the influence of atmospheric pollution on the plants’ structure, „andrei şaguna” foundation publishing, 365, 122( 2002) 5. artenie v tănase e., practicum of general biochemistry, „alexandru ioan cuza” university publishing, iaşi 266-268, 1981 6. sr 718413:2001 soil analysis methods determination of ph, standardization association’s of romania, bucharest figure2. the graphic representation of the correlative bond in the form of a logarithmic regression food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 77 the role of operating risks in interrelation with the education for sustainable development ye. g. torosyan european regional educational academy, yerevan, armenia, yevatorosyan@yahoo.com abstract: the risks of the modern society provide the basis for the process of decision-making also in the field of education. the importance of risk regulation systems, it is necessary to analyse the ways the public is engaged in education system related with environmental study processes. interrelation between risks and the education for sustainable development should be considered for educational system in armenia. the sustainable development under the conditions of risk society today is quite extensively investigated. according to the studies conducted all over the world for the evaluation of ecological awareness in many developed and developing countries the level of awareness of the issues of environmental protection, as well as the perception of ecological risks has been growing gradually during the last ten years. in armenia there are no many data available regarding ecological awareness of the population and the perception of ecological risks. the main global problem is sustainable development, that main part is protection of an environment. the influence of the human factor or anthropogenous influence on an environment becomes the basic part in problem of protection of an environment. necessity of for education for steady development here is shown, as necessities for modern educational system, is especial in higher education. it is necessary to note, that the education for sustainable development is an educational novelty, which continues the successful introduction in global educational system. ecological education and education for sustainable development are in close interrelation. the education for sustainable development not only is closely connected to ecological education, and also occured from it. the bases of education for sustainable development assume reception of fundamental knowledge on natural sciences, understanding for inter discipline connections and global processes, modern practical researches with application of new and newest devices and apparatus, in the present paper the attempts of introduction of ecological education in higher education system in armenia with close interrelation with education for sustainable development are resulted. keywords: the risks , the sustainable development, ecological education, 1. introduction globalization and industrial development of the states create fast changes in an environment and opportunity of occurrence of new principles for definition of tasks of a society. moreover, growth of the extreme climatic phenomena create sharp situation on a global scale. in the present paper the idea develops that in above mentioned circumstances there is a necessity of complete spiritual and physical formation of a human individual for an environment. this can reach by development of complex educational system. in this aspect the special value is received with development of all forms of education in interrelation with ecological thinking of the pupils. 2. the higher education system in former soviet countries the modern period of development of armenia (after the collapse of the soviet system) especially difficultly proceeds in education – the major mechanism social mailto:yevatorosyan:@yahoo.com 78 and cultural heritage. the role of education system is defined in a present stage of development of armenia by the tasks of its transition to democratic and lawful state, to market economy. it will enable to overcome backlog of the country in economic and social development. the purpose of education varies from narrow professionalism education and development of the person. it changes the representation about the forms of university training, about a nature of knowledge, place and role of teachers and another considerably. one of the important reasons of long crisis in economy on a post the soviet space is the fall of a level and ability to a competition at a global level of education system. the soviet educational system had been considered as the best in the world under many factors. at 1960 70 years for ussr characteristic became growing demand for higher education. and is not casual, that the students went here not only from the socialist countries and third world, but also from many west countries. it was considered, that the high level of education system was one of few factors, which allowed to be proud to the soviet system. the level of education system have allowed ussr also to be one of supercountries. however system of education, as well as much another events in former ussr, was beginning to hand over to a position at the end of 80-s'. at the first, the quantity of higher schools so has increased, the government, in generally communist party, decided that more higher education means more social mobility. in this case the state assignation on higher education were not ripening behind growing costs for the system of higher education developed in logic of the centralized planned economy. the most part of the budget left on the salary, the consequence that arose deficiency of means on service, purchase of the literature for libraries, equipment. moreover, the instability of teaching structure was increased in the beginning of 1990 years, when many of professors have begun to change sphere of the activity. one of other reasons was, that the state began interested less for education qualifications and on the foreground was put forward reception of the diploma, piece of a paper. especially, the managing elite has made all for his generation for reception not only the diploma of higher education, but also even of a doctor's degree and professorial rank. as the present directly working persons in the field of higher education system, we would like to note, that this tendency remains unfortunately, moreover on our sight prospers. other reasons here are latent, and most important, that some of present leaders have passed on this ways. the opportunity of soviet system democratization has appeared also in education. alongside with it there was a necessity of integration with the international system of education. the processes of integration have begun to develop in many directions, that has appeared useful to a developing of educational process on more modern level. for armenia by this major factor is also maintenance of activity of educational institutions in abroad for the armenians, with the purpose of preservation of the armenian nationality. though this question was decided and in the soviet time, but it carried more political character. and moreover question on self-management of higher schools has risen. more financial independence for universities means less state control. since 90 years the post-soviet system of higher education began to reformer the administration and management structure and revised the curricula, accepted the internationally adopted 2 or 3-level model of university education. the difficulties have begun here, as many of institution send by copying by western, most of all of american system of higher education, instead of the changes have connected from the substantial party of his educational process. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 79 there were two basic models of higher education in the world practice. 1. the orientation of preparation of the experts, professionals was characteristic for the german school. here student studied all for mastering by the necessary sum of knowledge, skills and skills. 2. other, liberal model, assumed the importance of development of the person. the soviet system basing on the first model mainly has gone on a way of revealing of the educational purposes, definition of the contents and principles of organization of educational process. it carried out the order of the state assuming mass manufacture of the experts for system. it also has entered into the contradiction with volume of the information and increased quantity of disciplines. there was a characteristic individualization of education with the minimal set of disciplines for the present time, with study of bases of scientific knowledge, preparation of the expert of a narrow structure within the framework of the advanced branch development of manufacture. it was began with a definite risk factor in former socialism countries the organization of private education, originally in higher education system was. at 1990 some of communist rulers countries was embracing private education. the east european countries as poland, romania with demand for higher education growing began to make buildings and rooms for private universities. now there is hundred thousands students are learning in private universities, which provide more high level of education and opportunities for realization of educational process, than official bodies. the same took place and in the asian countries. for example, malaysia so successfully has applied system of private universities, students of such educational institutions surpass now in quantity of the students of public institutes. the last, for example, have lavished funds on malay and islamic studies departments for nationalist reasons, despite little demand for their graduates. such situation was also in former ussr, when many persons studied at party higher schools on ideological bases. moreover, there it had aspired the persons, who basically did not differ in other educational institutions and went for reception " communist " of higher education. all of them were basis for higher level employers not for party only, but also as state and government rulers. in our opinion given graduates made faster the collapse of the soviet system. the post-soviet system of education also have begun to occur private higher educational institutions in the same time, which are taking place outside of a field of scheduled system of the state. the state began to provide an opportunity of training in state and private educational institutions. it should become the form of realization of principles of market economy in social and spiritual spheres of life of a society. one of the first became of the, largest private educational institution of armenia, one of best on former ussr. the private education system has revealed new opportunities of all system education and stimulated development of its not budget forms and inside state higher institutions. the chiefs of these universities are guided on market, inquiries of the employers. 3. ecological education sustainable development courses during changes there is a necessity of introduction of subjects, completely new to our higher school, educational disciplines of the increased level for the purposes of postgraduated education. now we shall consider what new educational programs such are entered in educational space former ussr, in particular, which are directly connected to democratic trends anywhere and everywhere. moreover, a major direction program is convergention. nowadays different higher schools carry out various educational modules and disciplines, 80 specialists under the all-european circuits. it promotes also movement of the teachers and students on europe, promotes attraction of a labor in the european market of work. in this paper we represent two programs according to our scientific interests. in a modern society there are all preconditions to promote education of the person. thus it is necessary to take into account integrity spiritual and physical formation of the individual in a natural environment. for this it can reach by development of ecological thinking. in this aspect the special value is received with development of ecological education. with this purpose in many private high schools, including at university hrachya acharyan, general ecological formation education of the students widely takes root. moreover at university all is done for development of ecological thinking of the students not of humanitarian directions. the importance of ecological education is marked also law of republic armenia " about ecological education and formation " from november 20, 2001. there it is marked, that the ecological education is continuous process directed to the person with the purpose of its orientation in an environment and nature of use. globalization and development of postindustrial countries with his problems result in sharp and fast changes in a society and to occurrence of new principles in definition of tasks of a community. the main global problem is a sustainable development. the basic making of sustainable development is a environmental protection. the influence of human amplifies on an environment, anthropogenic factor becomes as principal. at the international level, the developed nations must be prepared to provide help to the third world if sustainable development is to be successful. education for sustainable development is a dynamic concept that utilizes all aspects of public awareness, education and training to create or enhance an understanding of the linkages among the issues of sustainable development and to develop the knowledge, skills, perspectives and values which will empower people of all ages to assume responsibility for creating and enjoying a sustainable future. the realization of principles of ecological policy and its program purposes requires the wide and organized actions in the field of the education. the similar approach should provide to the nearest future specialists in wide involving in process of product realization, in creative process of making the new goods and the end prosperity in modern life. the modern approach to sustainable development is increasingly multi-faced, with a need to integrate a wide range of elements. it has become people centred with local strategies being developed to meet local problems, for example, as education. the urgency of ecological-educational programs is caused by a place of chemical manufactures in modern life. today commodity nomenclature of chemicals consists more than of thousands. thus many from received target and collateral substances represent danger for industrial personals, for consumers and in generally for environment. the aim of this program is training the students of the last course in ba level of chemical technologies and engineering ecology department of seua. this type of course will ensure and create conditions for perception of scientific and technical achievement by the students not only in narrow local space, but also in wider geographical, global scale. the global scale it is a level of the international community, that is connected to development of the international institutes and documents accepted by them. the priority of ecological imperative will promote an orientation in a choice of possible directions turning in a major condition socially ecological policy. on food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 81 the other hand, it is known that if the company or other economical organization invests in knowledge, which increase productivity of investments of the physical or human capital or raises a common level of participation of the businessman, the gain of productivity conducts an achievable due to this to growth of economy as a whole. the beginning of 21 centuries has shown that the mankind leaves on a new coil of challenges and uncertainties. the growth of the extreme weather phenomena all over the world precisely testifies. that the change of a climate is not the skeptical forecast and the reality. this call requires resolute measures. the realization of ideas and projects can made at taking into consideration of balance between requirements present and vital prospects of the future. the stability of human development is something greater the continuation of ecological policy by different means. it is strategy of modernization in which to cover huge innovation potential for economy, environment and society. the students of the chemical technology and environmental engineering department of seua pass some of ecological disciplines: general ecology, engineering ecology, microbiology and microbial growth, toxicology, air pollution and control, water quality, waste water treatment, solid waste disposal, monitoring. the students, the future experts in the field of chemical technologies basic organic synthesis, electrochemical manufactures, manufacture of a glass an silicates, the biotechnology and pharmacy, should be acquaint with the newest ecologically pure technologies, with reorientation on sharp reduction of resources and pollution of an environment. the students should understand that with limiting care it is necessary to carry out industrialization. the criterion of sd is considered such economic attitude to an environment and natural ecosystem that allows keeping them within the limits of economic capacity. the given course will allow acquainting the future specialists with global problems facing by mankind in the 21-th century. it will present to the students of the tendency of change of an environment on a global scale. acquaintance with principles of sd will allow the future engineers to take possession of methods of management, scientific, technical and economic levels connected with researches in the field of protection of an environment and feedback, and also with legal-political norms in researched area. a rate propose also acquaintance of the young experts with the circumstance of republic armenia before a world public in the field of protection of an environment with organizations of different levels responsible in the sanction of the above mentioned tasks. the realization of the program on study of a course < sustainable development> provides with the pedagogical staff having wide experience of activity in social sphere regularly engaged in scientific and pedagogical activity. the lectures of proposed discipline have a scientific degree also. effective means for solving the task of sd program has become: science and methods of seminars and conferences, holding discussions, issuing of specialized literature on sd, collections of scientific themes end method developments for ecological, political, economical and low problems. book popularizing science and explaining the surrounding world. for to days of armenian educational system the most important thing is mass and accessible educational technologies and techniques, able to solve the problem of joining the students to social intellectual potential. the basic preparation of the program provides with access of each student to library funds and databases. it is necessary to organize the student’s visits in special libraries of the foreign representations in ra, firms, factories. the realization of practical employment at the modern enterprises succeeded in realization of 82 the programs of environmental protection is supposed. there are supposed also acquaintances of students with the practice work of national assembly and constitutional court of ra, with the practice work of government agencies that are responsible for organization and sanction of questions in sphere of human development and environmental protection. the passage of a course should be accompanied by display of the didactic materials the pictures, tables, video films, clips and others. education for sustainable development has two major thrusts. promotion and improvement of basic education: the quality of basic education must improve to focus on imparting knowledge, skills, values and perspectives throughout a lifetime that encourage and support citizens to lead sustainable lives. reorienting existing education programs: rethinking and revising education to include more principles, knowledge, skills, perspectives and values related to sustainability in each of the three realms – social, environmental, and economic – is important to our current and future societies. this should be done in a holistic and interdisciplinary manner. the best chance of success of education for sustainable development lies not in a separate program but in embedding its vision within other initiatives. conclusions the traditional predisposition of armenians to education as the integral part of our mentality is not crossed out by complexities of life, and on the contrary, gives itself to know every time, when in it there is a public need. the problems of education excite all society. the armenian citizens of should receive such knowledge, which are necessary not only today, but also for a future. for small and rather poor natural resources of armenia is important the development of high technologies, for which are necessary the competent experts. the final purpose of spent reforms in sphere of higher education – is became of such system, which would allow the country to be entered in a new social and economic context of development of the country. on the schedule of day a number of reforms with definition of the status of establishments of higher education, which should receive the large autonomy in view of the requirements of the market. the role of the state should change considerably. the state should become the original chief of educational policy. it should trace processes proceeding in a society and education, to coordinate an efforts, to carry out an estimation and accreditation, to render to universities information services. more farreaching reforms are needed. central control of higher education stops universities from competing for the best students, professors. professors and other higher education system employees had been paid by performance. the universities should be run like industrial companies, that mean it`s necessary the competition among the educational organizations. for example, american universities are fiercely competitive. they struggle for talented staff and students, for donations and results. “ fund-raising efforts at the best-organized universities start even students have graduated. star professors attract star salaries”. for small republic as armenia there we need no more universities, here we need a competitive edge, when better universities may be essential. the influence of state for higher education system should be hade same for state and for private universities. it` s not be forgotten, that government and ministry responsible for educational program, for license and accreditation and not more. the realization of reforms should pass not by copying of this or that system. for example, praising american system nor is deprived of defects, is not flawless “ the diversity which makes the system so dynamic also leaves it vulnerable to abuse. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 83 in the humanities, intellectual fashion seems bizarrely distant from the real world. many bad ideas – notably political correctness – started life as american campus fads. and budget pressures squeeze the system when times are tough. this year, the axe has fallen hard on california's public universities. yet for all that, the numbers going into american higher education continue to rise, and the average tuition fee in an american university is around $4,500—some $1,000 less than the proposed maximum to be charged in england. fees in the california state system, even after two steep recent rises compelled by leaner budgets, are less than $3,000, and a third of the income from them goes into grants for students who cannot afford even that” / the economist, print edition, jan, 22nd 2004/. 6. references literature 1. beck, u. ecological politics in an age of risk, polity press, cambridge. (1995). 2. bruntland, g. (ed.). our common future: the world commission on environment and development, oxford university press, oxford. ( 1987) . 3. diesendorf, m. and hamilton, c. (eds.). human ecology, human economy, allen and unwin, st. leonards, new south wales. ( 1997). 4. ekberg, m. the parameters of the risk society // a review and exploration,current sociology. ( 2007). microsoft word 13 vasilacheart_fia_full paper corect.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 80 studies regarding the incidence of bacteria from listeria genus in fish meat *violeta vasilache, constantin ciotau stefan cel mare university of suceava, universitatii street 13, suceava, romania e-mail adress: violetav@fia.usv.ro *corresponding author received 25 may 2012, accepted 15 june 2012 abstract: listeria is a bacterial genus whose species are gram positive bacilli and it contains seven representatives. joseph lister was an english surgeon who was one of the first in the field of sterile surgery and the name listeria is given in his honor. sources of listeria are soil, contaminated water, animals and vegetables. because bacteria from listeria monocytogenes specie are very dangerous for human health, with a mortality rate of 20% for infected patients, a serious attention has to be accorded in consumption of those foods able to transmit microorganisms. this article presents a study about the incidence of listeria in two species of fish which were tested to prove presence or absence of listeria monocytogenes, because fish meat is one of the foods susceptible to transmit bacteria and to induce grave intoxication. from fresh water fish carp was selected and as a representative of ocean fish, mackerel respectively. different methods for detection were described and discussed. those methods are legally accepted in european and romanian normative for food consumption security as food microbiology and specific rules for microbiological analyzes. oxford, palcam and camp are test used for detection and confirmation of listeria species of bacteria. also apilisteria tests were used for confirmation the presence or absence of different listeria species in tested samples. agar with sheep blood medium was inseminated with samples of fish meat because this is the proper medium for bacterial colonies development. the reason is the fact that bacteria colonies formed dark colored rings rounded by a black haloes, because in their metabolic process bacteria produce hydrolysis of aesculin (a glycoside contained in agar medium). the results of analyzes for those two species of fish meat do not confirm the presence of listeria monocytogenes, but other species of listeria (listeria welshimeri and listeria inocua), fortunately not pathogenic, were present. keywords: pathogenicity, listeria monocytogenes, fish meat, infection 1. introduction bacteria from listeria genus and listeria monocytogenes especially provide a high level of pathogenicity, being responsible by a lot of alimentary toxicinfections and other dangerous diseases, even female abortion. many sources of listeria monocytogenes exist in nature, like environment, animals, people [1-7]. from environment, listeria monocytogenes was mainly isolated from soil, water and vegetables in decomposition. weis detected listeria monocytogenes in 21% of samples (from 779 samples of soil and plants). the high capacity of bacteria to survive in difficult conditions permits to explain infections which occur from sources contaminated long time before the reference moment. it was noted that in wastewater dispersed on farm land, bacteria remain active till eight weeks, what could explain human infections in new scotland – canada in 1981. the pathogenicity of listeria monocytogenes is proved by the fact that food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 81 almost 20% of infected patients died. even the reported cases of listeriosis diminished in developed countries, researchers found varied modes of transmission of bacteria through diverse foods [1,2,5,7,8]. 2. materials and methods there were selected two species of fish, a carp representing a fresh water fish and a mackerel representing an ocean fish respectively. the samples were analyzed in the laboratory for food safety of dsvsa suceava. figure 1. an image of laboratory with a carp fish and culture media for listeria monocytogenes from fresh water fish carp was selected and as a representative of ocean fish, mackerel respectively. different methods for detection were described and discussed. those methods are legally accepted in european and romanian normative for food consumption security as food microbiology and specific rules for microbiological analyzes [9-24]. figure 2. an image of laboratory with a mackerel fish and culture media for listeria monocytogenes preparing of samples followed sr en iso 6887-4/2005 regarding food microbiology and specific rules for microbiological analyzes and initial suspensions and decimal dilutions preparing followed sr en iso 68871/2002. fish samples investigation was performed through the method of analyze for detection and confirmation of listeria monocytogenes implemented and actualized in romania by sr en iso 11290-1/2000 with amendment a1/2005 regarding isolation media and hemolysis test. [21-23]. analyzes evolved following next stages: primary enrichment. in 225 ml liquid media with low concentration of selective agents of semi-fraser bouillon, insemination of 25 ml of sample followed by 24 h incubation at 30°c was performed. secondary enrichment. from obtained culture 0.1 ml were transferred in each test tube with 10 ml fraser bouillon, followed by incubation for 48 h at 35°c or 37°c. corrugation and identification. from obtained cultures samples were passed in two isolation media (agar oxford and agar palcam), with 24 – 48 h incubation at 35°c. in parallel, plates inseminated with positive control tell-tale (reference stem of listeria monocytogenes atcc 13932) were incubated in similar conditions [3,5,8]. confirmation. 3. results and discussions examination of final obtained cultures confirmed that oxford and palcam media were highly selective for inhibition of associated flora [3,5,8]. on control tell-tale listeria monocytogenes formed dark colored colonies, rounded by a black haloes, food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 82 because in their metabolic process bacteria produce hydrolysis of aesculin (contained in agar medium) (see figures 3 and 4). figure 3. aspect of colonies of listeria monocytogenes from positive control tell-tale sample on palcam selective medium, after 24 hours in thermostatic conditions figure 4. aspect of colonies of listeria monocytogenes on oxford selective medium, after 24 hours in thermostatic conditions confirmation for listeria monocytogenes was realized by hemolysis test and camp tests. because listeria generates hemolysis on agar with sheep blood, this could be used to confirm the presence or absence of bacteria. so, listeria monocytogenes stem from control tell-tale sample produced small colonies rounded by small but clear zone of hemolysis, which is characteristic. figure 5. aspect of colonies of listeria monocytogenes on an agar with sheep blood medium another reaction, gentle or accentuated could be highlighted with a camp test. this test consists in insemination of agar surface with staphylococcus aureus and rhodococcus equi on vertical parallel lines (knurling). then were knurled horizontal parallel lines with listeria monocytogenes, listeria innocua and listeria ivanovii, but horizontal lines did not intersect vertical lines, a distance of 1 – 2 mm remaining. incubation time was 24 h at 37°c. image in figure 6 presents a positive reaction, consisting in an arrow form at virtual intersection of the lines. figure 6. camp test – control tell-tale plate (+) the test results show tha absence of listeria monocytogenes and listeria ivanovii, but a possible contamination with listeria inocua or other species of listeria genus. another confirmation was achieved using api-listeria tests, containing dehydrated substrates. inseminated and incubated for 18 – 24 h at 35 – 37°c, the color change could signalize the presence or absence of bacteria. so, listeria inocua and listeria welshimeri respectively were confirmed for analyzed samples. figure 7. listeria monocytogenes identified on api-listeria galleries food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 83 figure 8. listeria welshimeri identified on apilisteria galleries figure 9. listeria inocua identified on api listeria galleries all results of tests are summarized in table 1. table 1. distribution of species of listeria genus bacteria examined product num ber of sampl es listeria monocyto genes listeria inocua listeria welshim eri probe 1. fish meat from carp specie 5 absent absent 2 probe 2. fish meat from mackerel specie 5 absent 2 1 4. conclusions analyzes of those two species of fish with five samples each infirm the presence of listeria monocytogenes. carp fish meat contain listeria welshimeri at two samples from five and mackerel fish meat contain listeria inocua at two samples and listeria welshimeri at one sample from five, respectively. because listeria inocua and listeria welshimeri are not pathogenic, conclusion is that tested fish could be approved for consumption. 5. references [1]. banu, c. & col., 2007, food treaty. food technology, publisher asab, bucureşti [2]. bercea i. & col., 1981, infectious animal diseases, pedagogical publishing , bucureşti [3]. bărzoi d., apostu s., 2002, microbiology of food, publisher risoprint, cluj napoca [4]. bărzoi d. & col., 1999, food poisoning, deacon publishing house, coresi, bucureşti [5]. bondoc i., şindilar e., 2002, quality control of veterinary and food sanitation, publishing house, ion ionescu de la brad, iaşi [6]. ciotău c., 2010, veterinary control of raw materials alimentary, suceava university press [7]. rotaru o., mihaiu m., 2004, veterinary hygiene of foodstuffs, vol.i., publisher risoprint, cluj –napoca. [8]. stănescu v. apostu s., 2010, hygiene, food safety inspection and animal.vol.ii. fish and fishing products and avacultură, publisher risoprint, cluj – napoca. [9]. ioancea l., kathrein i., 1989, condiţionarea şi valorificarea superioară a materiilor prime animale în scopuri alimentare, editura ceres, bucureşti [10]. kouba m., 2002, qualite des produits d,origine animale, inra, prod. alim., p.161169 [11]. robin l.t., churchill, hung lee, j. christopher hall, detection of listeria monocytogenes and the toxin listeriolysin in food, journal of microbiological methods 64 (2006) 141–170 [12]. okutani a., okada y., yamamoto s., igimi s., overview of listeria monocytogenes contamination in japan, international journal of food microbiology, 93 (2004) 131– 140 [13]. duodu s., holst-jensen a., skjerdal t., cappelier j-m., france pilet m., loncarevic s., influence of storage temperature on gene expression and virulence potential of listeria food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 84 monocytogenes strains grown in a salmon matrix, food microbiology, 27 (2010) 795801 [14]. de cesare a., mioni r., manfreda g., prevalence of listeria monocytogenes in fresh and fermented italian sausages and ribotyping of contaminating strains, international journal of food microbiology, 120 (2007) 124–130 [15]. orsi r. h., den bakker h. c., wiedmann m., listeria monocytogenes lineages: genomics, evolution, ecology, and phenotypic characteristics, international journal of medical microbiology, 301 (2011) 79–96 [16]. corcoran d., clancy d., o’mahony m. l., grant k., hyland e., shanaghy n., whyte p., mclauchlin j., moloney a., se´amus fanning, comparison of listeria monocytogenes strain types in irish smoked salmon and other foods, int. j. hyg. environ.health, 209 (2006) 527–534 [17]. chang y., gu w., lynne mclandsborough, low concentration of ethylenediaminetetraacetic acid (edta) affects biofilm formation of listeria monocytogenes by inhibiting its initial adherence, food microbiology, 29 (2012) 10-17 [18]. zunabovic m., domig j.k., kneifel w., practical relevance of methodologies for detecting and tracing of listeriamonocytogenes in ready-to-eat foods and manufacture environments – a review, lwtfood science and technology, 44 (2011) 351-362 [19]. ryser e.t., listeria monocytogenes, encyclopedia of dairy sciences, p.16501655 [20]. conter m., paludi d., zanardi, ghidini e.s., vergara a., ianieri a., characterization of antimicrobial resistance of foodborne listeria monocytogenes, international journal of food microbiology, 128 (2009) 497–500 [21]. ***order ansvsa, no. 13/2005 veterinary food safety standard that establishes rules for the sampling of animal products for laboratory examination. [22]. *** regulation (ec) nr.2073/2005 the european parliament and council amended by regulation (ec) nr.1441/2007 of on microbiological criteria for foods. [23]. ***sr en iso 11290-1/a 1:2005, detection methods for listeria monocytogenes. amendment 1. changing the isolation media and the haemolysis test and inclusion of precision. microsoft word 0a. first pages.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xi, issue 4 15 december 2012 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved microsoft word 3 journal nr 4 2010 din 8 noiembrie_81-85.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 85 quantitative determination of polyphenol compounds from raw extracts of allium, alliara and urtica genus. a comparative study rodica dinică1, mariana lupoae2, bianca furdui1, dragomir coprean2 1dunărea de jos university galaţi, chemistry department, faculty of science, street domnească no. 111, 800201, galaţi, rodinica@ugal.ro, bfurdui@ugal.ro 2ovidius university constanta, faculty of natural and agricultural sciences, street mamaia no. 124, 900527 ,constanţa, mariana_lupoaie@yahoo.com abstract: flavonoids are a large family of plant secondary metabolites, one of the largest groups of natural compounds known, principally recognized for their health-promoting properties in human diets. most flavonoids outperform well-known antioxidants, such as ascorbate (vitamin c) and αtocopherol (vitamin e), in vitro antioxidant assays because of their strong capacity to donate electrons or hydrogen atoms. flavonoids have been demonstrated to accumulate with oxidative stress during abiotic and biotic environmental assaults. there are many foods that contain flavonoids and phenolic acids, besides these being dietary plants. this paper presents the spectroanalytical profile of the flavonoids and polyphenolic compouns from organs of allium, brassicaceae and urticaceae genus raw hydroalcoholic extracts. the quantitative analysis of the examined chemical compounds showed that 70% ethanol solution was the best solvent used in order to obtain the highest polyphenolic content. the content of phenol compounds was determined colourimetrically with the folin–ciocalteu (fc) reagent and was expressed in gallic acid equivalents (gae). the flavonoid contents was determined using a method based on the formation of complex flavonoidaluminium and was expressed in quercetin equivalents (qe). the results show that the plants may be potent sources of natural antioxidants. keywords: allium ursinum, alliaria petiolata, urtica dioica, polyphenols, flavonoids introduction in recent years much attention has been devoted to natural antioxidant and their association with health benefits1. plants are potential sources of natural antioxidants. it produces various antioxidative compounds to counteract reactive oxygen species (ros) in order to survive2. phenol compounds are responsible for major organoleptic characteristics of plant particularly colour and taste properties. they are also reported to contribute to the health benefits associated with consumption of diets high in fruits and vegetables or plant-derived beverages. innumerable studies have been devoted to polyphenols, their occurrence in plants and their effects on quality of life. however, plant polyphenol composition is still poorly understood. furthermore, polyphenols are highly reactive compounds and good substrates for various enzymes, including polyphenoloxidases, peroxidases, glycosidases, and esterases. they undergo numerous enzymatic and chemical reactions during post harvest food storage and processing. although the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 86 occurrence of such reactions and their roles in the development or degradation of food quality is well documented, the structures of the resulting products are still poorly understood and their concentrations in food are usually unknown. plant polyphenols comprise a great diversity of compounds, among which flavonoids and several classes of nonflavonoids are usually distinguished3. the latter (figure 1) are mostly rather simple molecules, such as phenolic acids (which are subdivided into benzoic acids and hydroxycinnamic acids, based on c1c6 and c3-c6 skeletons, respectively) and stilbenes, but also include complex molecules derived from them (eg, stilbene oligomers, gallotannins, ellagitannins, and lignins). the flavonoids ( figure 2) have a common nucleus consisting of 2 phenolic rings and an oxygenated heterocycle. they are divided into several groups differing in the oxidation state of the heterocyclic pyran ring (eg, anthocyanins, flavonols, and flavanols). more than 4000 flavonoids have been identified in plants, and the list is constantly growing4. this is because of the occurrence of numerous substitution patterns in which primary substitutes (eg, hydroxyl, methoxyl, or glycosyl groups) can themselves be substituted (eg, additionally glycosylated or acylated), sometimes yielding highly complex structures. moreover, flavanols are also encountered as oligomers and polymers, referred to as condensed tannins or proanthocyanidins. cooh oh oh ho gallic acid (g) o g g g g g o o o o o g g g g g tannic acid o o o o o o co oc oc co oc oh ohho ho ho ho ho ho oh oh ho oh oh oh oh gallotannins (pentagalloiyglucose) figure 1. chemical structures of the main classes of nonflavonoid polyphenols oh o oh ohho oh o o oh ohho oh gucose-ramnose quercetin rutinflavonols figure 2. chemical structures of the main classes of flavonoids plant polyphenol composition is highly variable both qualitatively and quantitatively; some of the compounds are ubiquitous, whereas others are restricted to specific families or species (eg, isoflavones in legumes). polyphenol diversity in fruits5 and in plant foods6 has been described in excellent reviews. within a single species, large variations may also occur, particularly because of genetic factors, environmental conditions, and growth or maturation stages. recently, there are numerous methods that have been developed to evaluate the polyphenol content of complex mixtures such as plant extracts7and to identify all possible mechanisms characterizing an antioxidant activity8,9. the aim of this study is to evaluate the total phenolic and flavonoid content of some romanian plants which are in relationship with antioxidative activity and of the plants. according to our knowledge, there are very few data regarding the potential antioxidant properties related to phenolic and flavonoid fractions of allium, alliaria and urtica plants, which are widely used as salads and foods in most of the countries of the balkan peninsula. with respect to this, in the study we present our investigations on the total phenol and flavonoids content of the mentioned above plant extracts. materials and methods plant material. the biological material analyzed in the present paper was collected from the north dobrogea (luncavita forest) and is made from the following vegetal products: folium of allium ursinum (wild garlic), alliaria petiolata (garlic food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 87 mustard) and urtica dioica (nettle). the harvesting was made when the leaves grew until maturity before the bloom of the plants and were macroscopic determined in botanical garden laboratory of galati. chemicals. the chemicals were purchased from sigma co. folin–ciocalteu (fc) reagent was purchased from merck (germany). all other chemicals and reagents were of analytical grade. extracts preparation. the ground air-dried immature plants (5 g), were extracted using a method of extraction with ultrasounds with 70% ethanol for 24 h, at room temperature. after the extraction, the extracts were collected and filtered. to remove chlorophylic pigments, ethanolic extract is subject to repeated extraction with petroleum ether until disappearance of its green colour. ethanolic phase obtained after extraction is used to determine flavonoids and polyphenols, the volume being adjusted to 100 ml with cold 70% ethanol. analysis of total phenolic content the total polyphenol content (tpc) of the extracts was determined by spectrophotometry, using gallic acid as standard, according to the method described by the international organization for standardization (iso) 14502-110,11. briefly, 1.0 ml of the diluted sample extract was transferred into triplicate to separate tubes containing 5.0 ml of a 1/10 dilution of folin-ciocalteu’s reagent in water. then, 4.0 ml of a sodium carbonate solution (7.5% w/v) was added. the tubes were then allowed to stand at room temperature for 60 min before absorbance at 765 nm (in a uv– vis spectrophotometer) was measured against water. the tpc was expressed as gallic acid equivalents (gae) in mg/100 g material. the concentration of polyphenols in samples was derived from a standard curve of gallic acid ranging from 10 to 50 µg/ml (figure 1, pearson’s correlation coefficient: r2 : 0.9988). the determination of gallic acid concentration y = 0.0082x + 0.0891 r2 = 0.9988 0 0.1 0.2 0.3 0.4 0.5 0.6 0 10 20 30 40 50 60 conce ntration (mg/ml) ab so rb an ce gallic acid standard curve urtica lyophillis ed extract urt ica dry plant extract alliaria dry plant extract alliaria lyophillis ed ext ract allium dry plant extract allium lyophillis ed extract liniară ( gallic acid s tandard curve) figure 3 standard curve of gallic acid and concentration in polyphenols of analyzed extracts estimation of total flavonoid content measurement of total flavonoid content in the investigated extracts was determined spectrophotometrically12 using a method based on the formation of complex flavonoid-aluminium with the maximum absorbtivity at 430 nm. the aqueous dilutions of samples, in the amount of 1 ml, were separately mixed with 1 ml of 2% alcl3. after incubation at room temperature for 30 min, the absorbance of the reaction mixtures was measured at 430 nm. the flavonoids content was expressed as quercetin equivalents (qe) in mg/100 g material, by using a standard graph. (figure 4, pearson’s correlation coefficient: r2 : 0.9732) the determination of flavonoid concentration y = 0.008x + 0.0311r2 = 0.9732 0 0.1 0.2 0.3 0.4 0.5 0 10 20 30 40 50 60 concentration (mg/ml) ab so rb an ce standard cur ve quercetin urtica dry plant extr act urtica lyophilised extract alliaria lyophillised extract alliaria dry plant extract allium lyophillised extract allium dry plant extract liniară (standar d curve quercetin) figure 4 standard curve of quercetin and concentration in flavonoid of analyzed extracts food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 88 results and discussion although most antioxidant activities from plant sources are derived from phenolictype compounds, their effects are not always correlated with the presence of large quantities of phenolics. therefore, both sets of data on phenolic and flavonoid compounds need to be examined together. with respect to this, the investigated plant extracts were analysed for total phenolic and flavonoid contents. total polyphenol(tpc)expressed as gallic acid equivalents (gae) and flavonoid content (fc)expressed as quercetin equivalents (qe) 0 200 400 600 800 1000 1 2 3 4 5 6 analyse d plant % ( m g/ 10 0g d ry pl an t) t pc fc figure 5-the total polyphenol content and flavonoid content of analyzed plants legend: allium ursinum (1-dp, 2-lp), alliaria petiolata (3-dp, 4-lp), urtica dioica (5-dp,6-lp) dp-dry plant; lp-lyophilised plant polyphenols content. the folin-ciocalteu assay is one of the oldest methods developed to determine the content of total phenols. in this work, the total polyphenol content of 3 samples of food plants, belonging to the romanian autochthonous flora was analyzed. the amount of total phenolics varied widely in plant materials and ranged from to 265 mg to 594 mg gae/100g dried plant (fig.5). the results are presented in figures 3 . as shown in figure 3, the total polyphenol content in plants was found higher in air dried plant than in lyophilized one. our experiments show that the wild garlic is the richest in polyphenol compounds. total flavonoid contents. furthermore, the results obtained from the evaluation of total flavonoid content also indicate great variations (fig.4). in the wild garlic plant, the content of quercetin equivalents was notably higher than it was in nettle and mustard garlic. also, the total flavonoid content in plants was found higher in air dried plant than lyophilized plant. the decreases of total phenolic and flavonoid contents in lyophilized plants are most probably caused by the solubility of compounds in the water removed by lyophilization . conclusion in this work, a comparison between the total polyphenol and flavonoid content of 3 autochthonous plants was made (fig.5). the total polyphenol content and the flavonoid content are both parameters of quality in plants regarding their biological properties, and both assays should be applied for the antioxidant capacity studies. in conclusion, of all the local plants extract analysed, the wild garlic (allium ursinum) showed the highest yield of tpc and fc. the lyophilized plant extracts content of polyphenols are lower than air dried plant extracts. our studies shows that romanian plants may be potent sources of natural antioxidants because the total phenolic content had positive correlation with antioxidant capacity12,13. according to the results obtained, the plants from the autochthonous flora are of very good quality. references 1. a arnous, dp makris, p. kefalas, effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. j. agric. food. chem. 2001, 49: 5736-5742. 2. huda-faujan, n., noriham, a., norrakiah, a. s.and babji, a. s., antioxidant activity of plants methanolic extracts containing phenolic compounds, african journal of biotechnology 2009, vol. 8 (3), pp. 484-489, 3. jb. harborne, methods in plant biochemistry. i. plant phenolics. london: academic press, 1989. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 89 4. jb harborne, ca williams, advances in flavonoid research since 1992. phytochemistry 2000, 55:481–504. 5. jj macheix, a fleuriet, j. billot fruit phenolics. boca raton, fl: crc press, 1990. 6. f shahidi, m. naczk food phenolics: sources, chemistry, effects, and applications. lancaster, pa: technomics publishing co, 1995. 7. m anatolovich, pd prenzler, e patsalides, s mcdonald, k robards, methods for testing antioxidant activity. the analyst, 2002, 127: 183-198. 8. en frankel, as meyer, the problems of using one dimensional methods to evaluate multifunctional foods and biological antioxidants. 9. s. gorinstein, z. jastrzebski, h. leontowicz, m. leontowicz, j. namiesnik, k. najman et col, comparative control of the bioactivity of some frequently consumed vegetables subjected to different processing conditions, food control, 2009, 20, 407–413 10. j. sci. food. agric., 2000, 80: 1925-1941, iso 14502-1: 2005. determination of substances characteristic of green and blck tea. part 1: content of total polyphenols in tea. colorimetric method using folin-ciocalteu reagent. 11. v. l singleton,. r orthofer,. lamuela-ravento´s, r. m. analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. method enzymol. 1999, 299, 152–178. 12. z. jastrzebski, h. leontowicz, m. leontowicz, j. namiesnik, z. zachwieja, h. barton and all, the bioactivity of processed garlic (allium sativum l.) as shown in vitro and in vivo studies on rats, food and chemical toxicology, 2007, 45, 1626–1633 13. j. yang, j. guoa, j. yuan, in vitro antioxidant properties of rutin, lwt, 2008, 41, 1060–1066 advanced characterization methods for nickel and zinc-nickel alloy layers electrochemically deposited food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 89 advanced characterization methods for nickel and zincnickel alloy layers electrochemically deposited *violeta vasilache stefan cel mare university of suceava, faculty of food engineering. 13 th university street, 720229, romania, e-mail adress: violetav@usv.ro *corresponding author received 12 august 2011, accepted 5 november 2011 abstract. nickel and zinc-nickel alloys were electrochemically deposited. the layers were studied with scanning electron microscope (sem), composition was revealed through studies of energy dispersive of x-rays (edx) and x-ray diffraction (xrd), the reflectance was studied by reflectance spectroscopy and the hardness with a vickers indenter device. nickel electroplating has a great commercial and industrial importance and offers a very good quality finish of surfaces. this importance is emphasized by increasing of annual global consumption of nickel, which goes to 100.000 tones. the applications of electroplating could be derived in three categories: decorative, functional and electroforming. in ordinary coating the zinc remains the principal metal for industrial applications, especially for coverage of steel products. in the last two decades it was registered an important increasing of application of electrodeposited alloys due to the market demand for products with high quality coatings. on the first place there are the machine buildings industry and the aerospace industry, also those for electrical components and for fixing devices. because of fact that consumption of heavy metals has to be reduced year by year, until the total elimination, there are searching for new technologies and one of the most promised of them is that of alloys electrochemical coatings with zn-ni, zn-co and zn-fe. keywords: nickel, zinc-nickel alloys, sem, edx, and xrd techniques, reflectance, vickers hardness 1. introduction nickel electroplating has a great commercial and industrial importance and offers a very good quality finish of surfaces. this importance is emphasized by increasing of annual global consumption of nickel, which goes to 100.000 tones. the applications of electroplating could be derived in three categories: decorative, functional and electroforming. in ordinary coating the zinc remains the principal metal for industrial applications, especially for coverage of steel products. in the last two decades it was registered an important increasing of application of electrodeposited alloys due to the market demand for products with high quality coatings. on the first place there are the machine buildings industry and the aerospace industry, also those for electrical components and for fixing devices. because of fact that consumption of heavy metals has to be reduced year by year, until the total elimination, there are searching for new technologies and one of the most promised of them is that of alloys electrochemical coatings with zn-ni, znco and zn-fe.[1,2,3] the greatest automobile companies are in a continuous course for prolonging the warranty period, so their products are better and better year by year. the majority of the european factories have been changed the specifications regard the mailto:violetav@usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 90 improvement of the performances of the electrodeposited layer. those new economic politics are based just on the using of alloys like zn-ni, zn-co and zn-fe [4,5,6,7]. out of the machine building industry there are interesting in the using of electrodeposited alloys in the field of industry for defense. the state department of usa is asking for eliminate during the follow years of the old technologies with new technologies based on the zinc-nickel alloys [11,12,13,16]. 2. experimental electrodeposition of nickel was performed at incdfm bucharest-magurele institute, in the electrochemistry department. a watts bath was used, having the composition: niso4.6h2o, 120g/l; nicl2.6h2o, 35g/l şi h3bo3, 35g/l. we worked at different temperatures (4060°c). the experimental device used to realize nickel deposition is composed by a potentiostat-galvanostat voltalab 40 including voltamaster 4 software, a thermostated electrolyze cell with a thermostate lauda 003, magnetic shaker and thermometer to control temperature. as reference electrode it was used calomel electrode and the contra-electrode was made by electrolytic nickel. the work mode respected the next steps in the designing of the experiments. primary the plates of copper were cut and the thickness was measured with micrometer. then it followed a mechanical processing of the surfaces (like polish) with emery paper and with felt. the solutions were prepared following the recipe described as above mentioned (merk reactive substances were used). the copper plates were degreased with chloride acid (5%, temperature 65c), washed, dried and weighing. before proceeds to depose nickel there were drawn the polarization curves to establish the range of values for discharging of ions in solution. during the deposition there were recorded the values for current density. the electrochemical reactions on the cathode could be written generally: ni2+ + 2e→ ni, (1) but the proposed mechanism is by next type: ni2+ + h2o → (nioh)+ + h+ (2) (nioh)+ + e→ (nioh)ads (3) (nioh)ads + (nioh)+ + 3e→ 2ni + 2oh (4) this mechanism was proposed after the study of inductive impedance loops with the method of electrochemical impedance spectroscopy, but all steps was not identified and elucidate yet. the anomalous co-deposition of thin films of zinc-nickel alloys was performed electrochemically. in the way to obtain the desired properties it was necessary to search the influence of electrodeposition conditions (co-deposition potential, the bath’s composition, the temperature for deposition, mechanical stirring) through the structure, morphology, composition and optical properties, transport properties and magnetic properties of layers. the next recipes were used to prepare the low acid electrolyte for electrodeposition of zinc-nickel alloys: the first solution: zinc chloride 130g/l, nickel chloride 130g/l, potassium chloride 230g/l, ph 5-6, t(°c) 24-30°c; the second solution: zinc chloride 130g/l, nickel chloride 65g/l, potassium chloride 230g/l, ph 5-6, t(°c) 24-30°c. as working electrode it was used a glass plate with a deposed gold layer, made by sputtering method (a hummer 6 installation was used). the ph was maintained at a level between 5 and 6 naturally without adding acids, because the salts used were chlorides which through electrolytic dissociation have acid character (excepting kcl, which comes from a strong acid and a strong base). the work temperature was between 24°c and food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 91 30°c. as reference electrode it was used the calomel electrode directly immersed in the electrolyze cell. the chemical reactions which occur on the cathode follow two steps, as there were described by matlosz. zinc ions are deposited on their own substrate, on the gold substrate and on the nickel substrate. also, nickel ions are deposited on their own substrate, on the used gold substrate and on the zinc substrate. in addition it has to take account the secondary reactions when ions zn2+ combine with hydrogen to form znh+, in the same way ions ni+ combine with hydrogen to form nih+. these intermediate species, formed in the process of adsorption, will decompose finally to form metallic zn and metallic ni respectively. the electrochemical reactions which occur could be written as follow: ni2+ + e→ ni+ads (5) ni+ads + e→ ni (6) ni + h+ + → nih+ads (7) nih+ads + h+ + 2e→ ni + h2 (8) zn2+ + e→ zn+ads (9) zn+ + e→ zn (10) zn + h+ → znh+ads (11) znh+ads + h+ + 2e→ zn + h2 (12) ni2+ and zn2+ are dissolved as metallic ions, hydrolyzed or not. ni+ads and zn+ads which could or not to contain hydroxyl group are monovalent adsorbed in intermediate reactions. ni and zn are the metallic deposits of nickel and zinc respectively. 3. results and discussion the nickel layers electrodeposited were analized at incdfm bucuresti-magurele using scanning electron mycroscopy type zeiss evo 20. figure 1 presents a sem image of a sample of nickel electrodeposited at -700 mv potential, working temperature 65c. in the first image which has the resolution 10620x it can be observed the steps of electrocrystallization, and also the micro-pores produced by hydrogen evolution. in the image having the resolution 58450x, it can be seen a micro-pore in the right-downcorner. both images present a uniform covered surface, a good quality of the deposited layer [14,15]. one set of measurements of the obtained samples are reflectance measurements. brightness is a characteristic which depends by human eyes sensibility, so an objective physics parameter is reflectance. in order to measure this we used an ocean optics spectrometer, doted with spectra suite soft, in stefan cel mare university, suceava. the reflectance is defined as a percent (%rλ) relatively on the reflectance of a standard reference suface: %100 dr ds r% × x x = λλ λλ λ (13) where sλintensity of sample at light length λ; dλdarkness intensity at light length λ; rλreference intensity at light length λ. the first necessary step when it has to do reflectance measurements (generally for all types of measurements) is calibration of devices. in our case we used a mirror from spectrometer auxiliaries and we chose the value 100%. for the next example we considered two samples of nickel electrodeposited on copper figure 1. sem images of the nickel electrodeposited layer from a watts bath with addition of pvp at -700 mv potential, 65c temperature, deposition time 10 minutes, with magnetic shaking of the electrolyte (sem-zeiss evo 20 device) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 92 substrate, both in the same conditions, excepting temperature, one of them performed at 45c and the other at 65c. as it could see, the reflectance for the lowest temperature is higher. figure 2. graph chart recorded for a sample of nickel electrodeposited on copper substrate at -900 mv potential, temperature 65c, time 2 minutes for all samples of nickel electrodeposited on copper substrate at -900mv potential and temperatures from 45c to 65c, we measured reflectance for all visible domains. corresponding to wave length of 550 nm, we draw the next chart for dependence reflectance versus temperature: 0 5 10 15 20 25 45 50 55 60 65 t(°c) re f% figure 3. reflectance vs. temperature these results are similar those obtained by other researchers and it is easy to see that the best reflectance (and brightness, of course) is obtained for the lowest temperature. for higher than 60c temperatures the brightness increases again, but economic efficiency of the process is lowest, because it is necessary to spend heat for increasing the temperature of the electrochemical cell. another type of investigations we’ve made there were sem-edx analyzes performed on al.i. cuza university – iasi.. the investigation was performed by means of a sem vega ii lsh scanning electronic microscope manufactured by tescan for the czech republic, coupled with an edx quantax qx2 detector manufactured by roentec germany. table 1 composition of a sample of nickel electrodeposited on copper substrate, from a watts bath at -750mv potential, 60c, temperature, sodium lauryl-sulfate and saccharine agents added element [norm. wt.-%] [norm. at.-%] error in % nickel 72,98950 70,04394 1,95537 copper 24,72697 21,91705 0,687472 carbon 7,39e-09 3,47e-08 0,025 oxygen 2,283528 8,039002 0,535272 100 100 figure 4. edx pattern for a nickel sample electrodeposited on copper substrate at -750 mv and 60ºc analyzing the x-ray diffraction specter of a zinc-nickel alloy electrodeposited on gold substrate, there were found the next elements (table 2) the pick corresponding to (111) plane of nickel was found at an angle of 2=4436, and those corresponding to (111) plane of zinc at an angle of 2=3812. this analyze confirms too, the formation of zinc-nickel alloy. the deposited metals crystallize in cubic with centered faces. so, for nickel, the values for axes are (in ångstrom) a=3,52380, b=3,52380 and c=3,52380 with all angles equals of 90, and for zinc the dimensions are a=b=c=2,4730, with the same angles of 90. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 93 table 2 identified elements after x-ray diffraction analyze for a sample of zinc-nickel alloy electrodeposited at -1100 mv potential and 30°c temperature identified element dhkl (å) crystalin plane zn cfc-2,4730 (111) au cfc-4,07860 (222) kcl cfc-6,29170 (222) zn5(oh)8cl2.h2o rombic6,3400;6,3400;23,6600 (111) ni cfc-3,52380 (111) ni cfc-3,52380 (220) (ni(oh)2)(niooh) rombic3,0710;3,0710;23,2000 (222) 0 20 40 60 80 100 0 5 10 15 20 25 30 35 40 45 solutia ii solutia i int en sia ta te a d e d ifr ac tie (u nit .ar b.) 2-theta(grade) 200 111 200 111 220 003 111 200 311 222 aliaje zn-ni-p2p4 figure 5. x-ray diffractogramme recorded for a zn-ni alloy co-deposited on gold substrate a) from solution i, b) form solution ii another type of measurements was those of micro-hardness. the experiments were performed in stefan cel mare university – suceava. for different conditions we found the next results for vickers microhardness: table 3 results of micro-hardness measurements for some samples of nickel electrodeposited on copper substrate sample deposi tion potenti al (mv) tempe rature (c) force (n) hv 26 700 60 0.050 62.1 27 750 60 0.050 71.2 28 800 60 0.050 76.6 29 850 60 0.050 78.6 30 900 60 0.050 68.7 0 20 40 60 80 100 700 750 800 850 900 mv h v figure 6. micro-hardness vs. deposition potential it was observed that micro-harness increases directly with deposition potential, but when it is surpassing a limit, will follow a decrease of the microhardness. this is again a result confirmed by other studies. figure 7. photo of hardness indent for nickel deposited on copper substrate from a watts bath with addition of pvp, at 1000 mv potential and 65c temperature (shimadzu hmv) figure 8. photo of hardness indent for nickel deposited on copper substrate from a watts bath with addition of pvp, at -900 mv potential and 65c temperature (shimadzu hmv) 4. conclusion the deposited ni and zn-ni alloys are important in technique. to obtain the stoichiometric composition it has to follow carefully the values of the physical and chemical parameters. the quality of the deposed could be controlled through the electrolyte food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 94 concentration, discharge potential and the working temperature. the analyses semedx and xrd confirm that the zincnickel alloys were formed. also it is confirmed that the percents of those two metals in the deposed alloy depend of working conditions. conditions to obtain desired properties like hardness and brightness were established. 5. acknowledgment this paper was supported by the project "progress and development through postdoctoral research and innovation in engineering and applied sciences– pride contract no. posdru/89/1.5/s/57083", project co-funded from european social fund through sectorial operational program human resources 2007-2013. 6. references 1. bajat, j.b., maksimovic, m.d., radovic, g.r., electrochemical deposition and characterization of zinc-nickel alloys deposited by direct and pulse current, j. serbian chemical society. 67. 625-634, (2002). 2. bard, a., j., electrochemical methods. fundamentals and applications, john wiley and sons, new-york, 830p, (2001) 3. di bari, g., electrodeposition of nickel, modern electroplating, fourth edition, john wiley and sons, inc. new-york, (2000) 4. cui, c., q., lee, j., k., electrochemistry acta 40. 1653-1659, (1996) 5. firoiu, c., tehnologia proceselor electrochimice, editura didactică şi pedagogică, bucureşti, (1983) 6. grünwald, e., mureşan, l., vermeşan, g., vermeşan, h., culiuc, a., tratat de galvanotehnică, editura casa cărţii de ştiinţă, clujnapoca, 709p, (2005) 7. holm, m., o,kofe, t., journal of applied electrochemistry, 30. 1125-1132, (2000) 8. mohanty, u., s., tripathy, b., c., das, s., c., mistra, v., n., metallurgical and materials transactions b, 36. 737-743, (2005) 9. mitoşeriu, o., iticescu, c., cârâc, g., revista de chimie, bucureşti, 55. 525529, (2004) 10. vasilache v., gutt s., gutt g., vasilache t. studies about magneto-optic kerr effect on electrodeposited nickel layers, revista de chimie, bucureşti, 61. 471-474, 2010 11. paunovič, m., schlesinger, m., and weil, r., fundamental considerations, modern electroplating, fourth edition, john wiley and sons, inc. new-york, (2000) 12. tripathy, b., c., singh, p., muir, d., m., das, s., c., journal of applied electrochemistry, 31. 301-305, (2001) 13. soares, m.e., souza, c.a.c., kuri, s.e., corrosion resitence of zn-ni electrodeposited alloy obtained with a controlled electrolyte flow and gelatin additive, science direct, 201.2953-2959, (2006) 14. vasilache, v., gutt, gh., vasilache, t., electrochemical researches about influence of the additives of watts’s s solutions on throwing power and brightness, revista de chimie, bucureşti, 59. 912-919, 2008 15. vasilache v., gutt gh., vasilache t, studies about electrochemical plating with zinc nickel alloysthe influence of potential through stoichiometric composition, revista de chimie, bucureşti, 59. 1005-1009, (2008) 16. vasilache v., gutt s., gutt g., vasilache t, sandu i., sandu g.i. determination of the dimension of cristalline grains of thin layers of zinc nickel alloys electrochemically deposited, metalurgia international, 3.49-53, (2009) 17. vasilache t., gutt s., sandu i., vasilache v., gutt g., risca m., sandu a.v., electrochemical mechanism of nickel and zinc-nickel alloy electrodeposition, recent patents on corrosion science, 2. 1-5, (2010) 18. vasilache v., popa c., benta m., mathematic model for optimization of zinc-nickel alloy co-deposition process, tehnomus, new technologies and products in machine manufacturing technologies, journal, 18.257-261, (2011). 5. acknowledgment удк food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 7 photocatalytic reduction of methylene blue on the heterostructural materials based on titanium dioxide with bisqinocyanine pigment *igor kobasa1, nataliya husyak1 , sonia gutt2 1 yuriy fedkovych national university of chernivtsi, kotsiybynsky st., 2, chernivtsi 58012, ukraine. e-mail: imk-11@hotmail.com 2 stefan cel mare university of suceava, university street nr. 13, 720 229 suceava, romania *corresponding author received 12 june 2011, accepted 5 november 2011 abstract. new light sensitive heterostructures working through all visible light range and containing a semiconducting material (tio2), a bisquinocyanine pigment-sensitizer, and a polyepoxypropylcarbazole polymer, which protects the material from dissolution have been developed. our results prove that such heterostructures can be used as active photocatalysts. dependence of their photoactivity on the quantitative composition has been established and energy characteristics of the electronic processes occurring at the light absorption have been analyzed. we have also proposed mechanism explaining photocatalytic activity of these complex materials. key words: titanium dioxide, bisquinocyanine pigment, heterostructures, photocatalytic activity, reaction mechanism introduction selective sensitizing of photo catalytic systems promotes its activity within some wavelength range and this is one of important fields in the photo catalytic systems design and development. some photocatalytic systems can be suitable for use in the visible and infrared solar energy transformation and accumulation equipment, which is very promising direction [1-7]. previous investigations proved that systems combining photoactive oxide (tio2) with pigment-sensitizer or other long wave sensitizing compound can be effectively used as photo catalysts [8-14]. some systems can be applied in the wastewater and water decontamination technologies. here we describe a method of design the solid and structurally oriented photosensitive hetero structures (the blocks). needful amount of the pigment should be applied on the semiconductor surface and then protected from dissolution by the polymer film, which does not prevent any component to take part in the electron transferring processes occurring on the interfacial bound. heterostructures of this class have been synthesized in [1, 8] and showed good efficiency in the photo catalytic water decomposition process. obviously, we should determine if this method can be used as a universal tool for synthesis of other photo catalytic materials for other redox processes. it is also interesting to investigate if other classes of pigments and semiconductors can be used as source materials for the photocatalytic blocks. we have synthesized a series of new heterostructures based on titanium dioxide and a bisquinocyanine class pigment. then activity of the heterostructures has been measured in the test photocatalytic reaction of methylene blue (mb) reduction. mailto:imk-11@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 8 experimental we used pyrogenic titanium dioxide to obtain a photosensitive heterostructure f/p/tio2. titanium dioxide was produced through the high temperature hydrolysis of ticl4 vapour in the air-hydrogen flame and the specific surface area of the material was ssp = 50 m2/g. bromide n-[α-(3coumarinylecarbo) 1,3,3 trimethyl-2indolinyliden) propenile] (4-quinolile)(1,3,3trimethyl–2–indolinyliden) trimethyncyanine was used as a pigment (p) and the polymer film was formed using polyepoxypropylcarbazole (f). hc ch ch n c co ch oo n ch3 ch3 h3c ch n ch3 h3c ch3 + br the heterostructures were formed from the pigment and semiconductor (p/tio2) using following operations. tio2 suspension was mixed with the alcohol solution of the pigment at the room temperature. then this mixture was remained until the solvent evaporated completely. then we applied a benzene solution of the pigment, which formed protective film. spectrophotometer sf-46 was used to record absorption spectra of the solutions while the diffusion reflection spectra of the heterostructures of p/tio2 and f/p/tio2 were recorded at spectrophotometer perkin elmer lambda bio 40 with integrating sphere. then the kubelka-munk method [15] was engaged to transform the latter spectra into the absorption spectra using parameter (1 – r2)/2r, where r means % of the diffusion reflection at the given wavelength. photocatalytic activity of the heterostructures was determined through mb discolouring rate. photocatalytic reaction of mb discolouring also involves formaldehyde and ethanol as reducing agents [12]. activity of the heterostructures was calculated by the formula: pa = (с0·1000)/(τ1/2·s·m), where pa means specific photocatalytic activity (mg/ml·min·m2), c0 – initial concentration of the pigment (mg/ml), τ1/2 – time of semi-discolouring (min), s – specific surface area (m2/g), and m – weight of the sample. results and discussion absorption spectra of p/tio2 and f/p/tio2 heterostructures (calculated by kubelka-munk method from diffusion reflection spectra) are shown in fig. 1 together with the absorption spectra of an alcohol solution of the pure pigment. comparison of the spectra shows that association processes become more intense as a result of applying the pigment on the solid substrate (similar result was reported in [16]). this process results in a change of intensity of the narrow bands with λmax = 446 nm (lg ε = 4,52) and λmax = 632 nm (lg ε = 5,02), which widen and almost join into the single band extending over a significant part of the visible light region. polymer component of the heterostructure gives no influence on the pigment association ratio and pattern of the band. this component slightly changes the band intensity only. we found that the homogeneous alcohol solution of the pigment undergoes photo discolouring under the visible light irradiation. this phenomenon is caused by reduction of the pigment in the electron-donoring media and this is a typical process for many pigments [1214]. investigation of oxidation and reduction of some bisquinocyanine pigments resulted in the following redox potentials for our pigment: ./  pp e = + 0,38 v and ./  pp e = 0, 24 v (all the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 9 potentials are given here and below regarding the normal hydrogen electrode potential). we used these values as references to compare the redox properties of the pigment in its basic state. redox potentials of the excited pigment molecules were calculated from the basic state values. therefore, we calculated the redox potentials for the singlet excited state after absorption of light (λmax = 632 nm, е* = 1,96 v). using equations * .. //* eee pppp   and * .. //* eee pppp   [17] we obtain the following values: ./*  pp e = 1,58 v and ./*  pp e = + 1,72 v. comparing these values to electrochemical potential of ethanol (e = 0,15 v [18]), free radical ch3ċhoh (е = 1,25 v [19]), ./  pp e , and ./  pp e we can conclude that the following reactions are thermodynamically allowed and can take place in the solution at the visible light irradiation: p* + p → p + p , p + p → p2+ p , p + c2h5oh → p + ch3ċhoh + н+, p + ch3ċhoh→ p + ch3cho + h+, p + ch3ċhoh → p2+ ch3cho + h+. some other reactions can also take place. each of these reactions contributes to the process of the pigment discolouring. adding of tio2 or f/p/tio2 suspension and active stirring of the solution accelerates the process in several times. settling of the suspended particles stops after the stirring discontinued and the reaction rate returns to its initial value. this proves that visible light-inactive titanium dioxide can catalyze the photoreduction only if there are pigment molecules adsorbed on tio2 surface. a character of the heterostructures influence on photoreduction of mb depends on wavelength of the irradiation light. the visible light can be absorbed by both pigment-sensitizer of the heterostructure and dissolved mb. table 1 dependence of the heterostructures photocatalytic activity on the pigment concentration at various wavelength light irradiation pigment content (mg/g) photocatalytic activity, (mg/ml·min·m2) 310 < λ < 400 (nm) λ > 400 (nm) 0 1,6·10-1 0 0,019 8,5·10-2 1,7·10-2 0,094 2,7·10-2 1,4·10-1 0,188 1,0·10-2 2,6·10-1 0,94 6,4·10-3 1,1·10-1 1,88 4,1·10-3 4,6·10-3 therefore, photoreduction of mb runs under the visible light irradiation through the photocatalytic action of the heterostructures. our results proved that the photoreduction is almost completely inactive if a system does not contain f/p/tio2 or contains only tio2. photocatalytic activity of a system containing heterostructures initially rises as concentration of the pigment increases. then it reaches the maximum value and further portions of the pigment cause decrease in the photocatalytic activity (see table 1). uv-light irradiation is being absorbed mainly by tio2 particles and results in a different kind of relation where nonmodified source material shows the highest activity level (see table 1, experiment 1), which smoothly decreases as concentration of the pigment rises (see table 1, experiments 2-6). the investigation proved that thickness of the polymer film deposited on the heterostructure does not provide significant influence on its photocatalytic activity until this thickness remains within some range. a content of 0,2 mg/g of the pigment in the f/p/tio2 material is the optimal and does not influence any electron transferring processes on the solid/solution interface. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 10 figure 1. absorption spectra: a – alcohol solution of the pigment; b – alcohol solution of the heterostructures p/tio2 (1) and f/p/tio2 (2) on other hand, such a content of the polymer prevents dissolution of the pigment. raise in the polymer concentration to 1,8 mg/g causes decrease of 20 % in the photocatalytic activity. we have built energy transformation diagrams for the photocatalytic systems based on f/p/tio2 heterostructures and schemes of various light-generated electron transferring processes. information related to location of tio2 conductivity and valence bands (есв = 0,54 v, еvb = + 2,66 v at рн = 7 [20]) and redox potentials of normal and excited molecules of the pigment ( ./  mbmb e =+0,011 v [20]) and polyepoxypropylcarbazole ( ./  ff e =+1,28 v [1]) has been used to build the diagrams and schemes. as seen from fig. 2, the process of an electron injection to the conductivity band of tio2 followed by its transferring to mb molecule can occur at photoexcitation of the pigment by the light radiation with λ = 632 nm. both processes should be rather fast because of quite a significant potentials differences || ./* 1 cb pp eee   = 1,04 v and || ./ 2  mbmb cb eee = 0,55 v. a molecule of the pigment-sensitizer p is oxidized to the cation-radical p+ and redox potential of this process is ./  pp e = 0,38 v. this cation-radical should easily react with a strong electron donoring agents such as formaldehyde (е = 1,08 v at рн = 14 and е = 0,06 v at рн = 1,0 [21]) or ethanol (е = 0,15 v). these processes are thermodynamically advantageous since their energy gaps are rather wide and they should run fast ensuring reduction of the source form of the pigment-sensitizer. in fact, we did not observe any colour change at irradiation of f/p/tio2 heterostructures while mbrelated light absorption decreased. therefore, we can conclude that sensitizing of the semiconducting food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 11 photocatalyst to the visible light and photocatalytic reduction of mb are caused by the electron transferring processes described above. figure 2. energy transformation diagram of a photocatalytic system based on the heterostructure containing of tio2 and bromide n-[α (3-coumarinylecarbo)-1,3,3trimethyl-2-indolinyliden) propenile] (4quinolile) (1,3,3-trimethyl – 2 – indolinyliden) trimethyncyanine and scheme of the electron transferring processes at the visible light irradiation as supposed from this description, photocatalytic activity of a sample should rise at increase of concentration of the pigment-sensitizer ensuring more effective absorption of the light. one can see (table 1) that experimental results prove this assumption. on other hand, lower activity of the samples with higher concentration of the pigment can be caused by lower photosensitivity of the associates, which form at such concentration. analysis of the energetic diagrams shows that transferring of excited electrons on the pigment and electron-hole recombination involving the pigment molecules are the key processes in the photoexcitation of the heterostructures with tio2-absorbable light (λ<400 nm). the higher is the pigment concentration, the lower is probability of the direct transferring of an electron from the conductivity band to mb molecule. then probability of the recombination will rise causing decrease of the heterostructures photocatalytic activity. experimental data (see table 1) completely confirm this assumption. conclusion new heterostructures consisting of a semiconductor, pigment-sensitizer, and polymer material have been designed. analysis of our results proves that combination of such three parts can be used as a general method to develop various light sensitive materials using various source materials, which can be applied in various fields including solar energy conservation and transformation, water and wastewater decontamination, etc. references 1. krykov a. i., kuchmy s.ya., pokhodenko v.d. molecular design in photocatalysis: physico-chemical principles of the highly effective photocatalytic redox systems. theoret. and exper. chem. 30 141 (1994). 2. ciping chen, xiaoyan qi, benmao zhou. photosensitization of colloidal tio2 with food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 12 a cyanine dye. j. photochem. and photobiology a. 109 155 (1997). 3. maira a. j. et al. fourier transform infrared study of the performance of nanostructured tio2 particles for the photocatalytic oxidation of gaseous toluene. journal of catalysis. 202 413 (2001). 4. houas a., lachheb h., ksibi m., elaloui e., herrman j.-m. photocatalytic degradation pathway of methylene blue in water. appl. cat. b. 31 145 (2001). 5. nagaveni k. et al. solarphotocatalytic degradation of dyes: high activity of combustion synthesized nano tio2. appl. cat. b. 48 83 (2004). 6. сhung-hsin wu. photodegradation of toluic acid isomers by uv/tio2. chemosphere 59 601 (2004). 7. rajeshwar k. et al. heterogeneous photocatalytic degradation of organics contminants over titanium dioxide: a review of fundamentals, progress and problems j. photochem and photobiology c: photochemistry reviews 9 171 (2008). 8. kuchmiy s.y., korzhak a.v., guba n.f., kulik s.v., krykov a.i. sensitizing of cadmium sulfide with cyanine dyes in the photocatalytic hydrogen synthesis. theoret. and exper. chem. 31 309 (1995). 9. pokhodenko v.d., guba n., krykov a., kushmiy s., korzhak a. physico-chemical aspects of the application of “semiconductorconducting polymer dye” type heterostructures in photocatalytic systems for light energy conversion. functional materials 5 387 (1998). 10. kobasa i.m. novel light-sensitive materials with photocatalytic activity: heterostructures containing cadmium sulfide, cyanine pigment and polyepoxypropylcarbazole. functional materials 10 276 (2003). 11. boschloo g., hӓggman l., hagfeldt a. quantification of the effect of 4-tert-butylpyridine addion to j-/j3redox electrolytes in dye-sensitized nanostructured tio2 solar cells. j. phys. chem. b. 110 13144 (2006). 12. kobasa i.m., kondratyva. sensitizing of semiconduction photocatalysts by cyanine pigment with two conjugated chromophores. polish j. chem. 82 1639 (2008). 13. kobasa i.m., kondratyva. i.v., gnatiuk y.i. photocatalytic reduction of methylene blue by formaldehyde with titanium dioxide and cadmium sulfide sensitized with (1phenyl-5,6-benzoquinolin-2)-2,4dihydroxistyrileiodide theoret. and exper. chem. 44 40 (2008). 14. kobasa i., kondratyeva i., odosiy l. tio2/biscyanine and cds/biscyanine heterostructures – influence of the structural composition on the photocatalytic activity. can. j. chem. 88(7) 659 (2010). 15. w.n. delgass, c.l. haller, r. kellerman, j.h. lunsford spectroscopy in heterogeneous catalysis, academic press n.y., san francisco, london, 1979. – 341 p. 16. terenin a.n. photonica of dyes molecules, nauka, leningrad (1967) (in russian). 17. kogan i.m. chemistry of dyes, goskhemisdat, moscow (1956) (in russian). 18. dobosh d. electrochemical constantes, mir, moscow (1980) (in russian). 19. pokhodenko v.d., beloded a. a., koshechko v. g. redox reactions of the free radicals. naukova dumka, kyiv (1977) (in russian). 20. bedja i., hotchandani s., kamat v. photoelectrochemistry of quantized tungsten trioxide colloids: electron storage, electrochromic, and photoelectrochromic effects. j. phys. chem. 97 (42) 11064 (1993). 21. stepanova l. i., azirko v. n., sobol n. s. a method of the palladium-free dielectrics activation before their metallization byelorussian univ. bulletin. series 2 (1) 14 (1997). journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 5 the quality of the siret river in siret section similarities and differences of the results obtained for the samples collected in comon by the romanian and ukrainian doina mihăilă water system management, suceava, romania, e-mail doina_mihaila@yahoo.com abstract: this article describes the evolution of water quality of siret river at the entrance in romania for a 5 years period. the correlation of the determined physical, chemical and biological indicators offer an overall image of their season, annual and multi-annual variation but also their ranging into quality classes. the comparative study of the results obtained by the romanian and the ukrainian side for the samples collected in common, highlights the similarities and differences between the methods and methodologies used in analyzing water quality of the two countries. keywords: analysis, indicators, concentration. introduction assessment of water quality into the siret river in romania is achieved by monitoring surface water quality. the purpose of monitoring is to know the effect of the pollution sources and cleaning, in order to take the immediate and future measures for the protection of water resources and for the checking of the measures which have been taken. water quality analysis is monitored by physical, chemical and biological analysis on certain points and with a certain frequency. types of the monitoring programs and number of sections monitored by the sga suceava in the water quality laboratory (directiva 2000/60/ce) are presented in table. 1 determination of water quality in terms of physical and chemical indicators is correlated with the biological analyses, the environmental conditions having a major influence on benthic fauna, thus obtaining a complete picture of it (ord. 161/2006). table 1 monitoring programs and monitoring section number number of sections monitored monitoring programs river lake groundwater waste water surveillance 13 13 155 operational 15 4 23 46 drinking 7 2 8 reference 4 1 ichthyofauna 23 13 vulnerable areas 3 38 protection of habitats and species 2 5 international conventions 1 the monitoring of siret river at the border is carried out jointly with the ukrainian side based on a cooperation protocol, which provides water sampling from the siret river at the border, in order to establish quantities of existing pollutants in the river, at the transit of the two countries. [1] we used the data base of physical, journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 6 chemical and biological indicators for the period 2004-2008 obtained from the archives of suceava water management system, in order to make a classification on quality classes. [2] the framing in quality classes was done under the order 161/2006 concerning the chemical elements and the chemical and physical-chemical quality standards in surface water. the results and conclusions we reached are set out below experimental basic methods for studying the evolution of river water quality during a 5 year period were: the processing of experimental data, analysis, graphical methods of data analysis, analytical comparison. [3-8] it was considered a mediation of multiannual values to see the progress in a period exceeding one year, to eliminate the inherent variations due to a complex overlapping of hydrological, physical, chemical, biological and technical factors. 1. physical-chemical analyses 1.1 ph measure the activity of hydrogen ions (sr iso 10523/1997), the normal range of variation of ph is between 6,5-8,5 ph units. values in the last 5 years fall within the range noted, however, the 2008 average of 8.12 ph units is slightly higher than during previous years. the values recorded (fig. 1) have no dramatic developments, the range of values generally ranging between 7 and 8.3 ph units. it is noted that most values are higher than 8, at the upper part of the range, which means a trend of slight alkaline water. the minimum value of ph was 6.8 and it was registered in may 2004 and the maximum value was registered also in2004 but in november the same year, having the largest amplitude. comparatively, in the graphical representation it can be observed that the values determined by ukrainian side are consistently higher, but they do not exceed 6,5 6,9 7,3 7,7 8,1 8,5 i f m a m i i a s o n d p h units 2008 2007 2006 2005 2004 monthly average fig. 1 annual evolution of ph values on section siret siret (2004-2008) the maximum permitted by the applicable law (fig. 2). 7,5 8 8,5 i f m a m i i a s o n d ph units rom ania ucraina fig. 2 comparative evolution of ph values on section siret siret (2008) 1.2 dissolved oxygen as a dissolved gas, oxygen behaves as such: with increasing temperature in the summer, the concentration decreases, then increase during the cold period of the year. the amount of dissolved oxygen in water varies according to atmospheric pressure, water temperature, the content of mineral salts and organic substances (sr en 25814 iso 5814/ -1999). this concave variation is unchanged; the minimum values determined are invariably recorded each year in the hot season as seen in fig.3. even in summer, the minimum concentrations recorded (7.93 mg/l o september 2007) included the river in class ii, which means water with a good journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 7 ecological status. the variations in time of the values for dissolved oxygen are dependent on: the amount of oxygen resulting from photosynthesis oxygen consumption of organisms in the ecosystem temperature, depth and stratification of water load of organic substances of water loads strong reducing agents of water 7 8 9 10 11 12 13 14 15 i f m a m i i a s o n d class i mgo/l 2008 2007 2006 2005 2004 class i monthly average fig. 3 annual dissolved oxygen regime on section siret siret (2004-2008) fig. 4 shows the dependence of dissolved oxygen on the content of organic matter and temperature: with the arrival of the warm season the amount of organic matter present in water increases due to weaker amount of dissolved oxygen. 2 4 6 8 10 12 14 i f m a m i i a s o n d mgo/l monthly ave rage dissolved oxygen monthly ave rage ccocr fig. 4 comparative evolution of the monthly averages for dissolved oxygen and cod the siret siret section 2008 for the samples collected in common we have: average values for 2008:romania 10,53 mg/l, ucraina 9,82mg/l, maximum for 2008 romania 13,19mg/l, ucraina 13,09mg/l, minimum 2008 romania 8,33mg/, ucraina 6,08mg/l (trufaş 2003) class ii 5 7 9 11 13 15 i f m a m i i a s o n class i mgo/l romania ucraina fig. 5 comparative evolution of the concentration of dissolved oxygen section siret siret (2008) the differences in evolution of the dissolved oxygen appear at the minimum values recorded of the two countries, is generally due to samplings techniques and transportations. 1.3 chemical oxygen demand (cod) cod puts into evidence the amount of organic matter present in water oxidable with k2cr2o7 (iso 15705:2002). the cod (quantity of organic matter) does not exceed the limits of class ii. high concentrations are generally reported in june, july, august. during the cold of the year when the activity of micro organisms is reduced, flow rates are relatively constant, the amount of organic matter is low, and codr variations are insignificant. in the studied period, the maxims values do not exceed class ii , which means a good ecological status (mănescu 1994). graphical representation of mean monthly values of the studied period (fig. 6) shows that in warm months registers a slight increase, and the maximum values do not exceed class ii, therefore showing a good ecological state. monthly peaks have a similar running of the minima, the background thermal and flow variations. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 8 fig. 6 the average change, monthly minima and maxims on cod values section siret siret (2004-2008) 1.4 biochemical oxygen demand (bod5) bod5 basic element in the correlation between the amount of organic matter existing in the water and biological activity of micro organisms (sr en 18991/2003), the maximum values was recorded in june, july, august. the mean in the period under review shows an increase in june, july, august and a decrease in winter. class i 0 2 4 6 8 10 i f m a m i i a s o n d class ii mgo/l class iii 2008 2007 2006 2005 2004 monthly averages clas s iii fig. 7 evolution bod5 concentration on section siret siret (2004-2008) it is observed (fig. 7) so that during the summer months high temperatures and intense biological activity had put their mark on the bod5 values meaning an increase value and significantly lower values in winter. during the studied period, there is a similarity between the evolution of average cod (fig. 6) and bod5 (fig. 7), in both cases the maximum values were recorded during the summer months, once with an increased amount of organic substances in water. clas s ii 0 1 2 3 4 5 i f m a m i i a s o n d class i mg o/l romania ucraina fig. 8 comparative evolution of the concentration of bod5 in section siret siret (2008) it can be observed the increase of maximums in the warm period in june, july, august generally. the highest value was determined in 2006, reaching a maximum value of 8.04 mg/l, which has put the river (ord.161/2006) in ivth quality class (fig. 7). in 2008 the results obtained by the two countries have a similar trend, the difference from the results recorded in february and march does not change the class of the river (fig 8). 1.5 ammonium nitrogen (n-nh4) variations concentration of ammonium ion in the studied period, are not dependent by temperature regime as clas s i 0 0,1 0,2 0,3 0,4 0,5 0,6 i f m a m i i a s o n d class i mg n nh4/l 2008 2007 2006 2005 2004 mon thly aver age fig. 9 evolution of the concentration of n-nh4 by section siret siret (2004-2008) shown and do not exceed the limit value of 0.4 mg n/l for class i. the maximum recorded value was 0.498 mg n / l, which means one ii class good ecological status. it is interesting the comparison journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 9 between the values determined by the two laboratories since they use two different methods (fig.10). 0 0,1 0,2 0,3 0,4 i f m a m i i a s o n d class i mg n nh4 / l romania ucraina class i fig. 10 comparative evolution of the concentration of n-nh4 in section siret siret (2008) ukrainian part determined the ion ammonium content by treating the sample with nessler reagent (stas 6328/85) and this method is not so sensitive for low concentrations, and the romanian side had determined the ion ammonium content by the reaction of ammonium ions content salicylate and hypo-chlorite in the presence of sodium nitroprusiat (sr iso 71501/2001) (the method using nessler reagent is repealed due to its toxicity and because the method with the nitroprusiat obtain better performance at low levels). 1.6 nitrogen from nitrates and nitrites (n no2, şi n – no3) the presence of nitrites in water is generally due to the existence of insufficiently treated sewage (mănescu, 1994). regarding the n-no2 concentration variation, in october 2005 it was recorded a maximum value of 0.072 mg / l n which includes the river in class iv, the maximum frequency values during the studied period being up to the concentration of 0.03 mg n/l. when referring to n-no3 concentration, it can be observed a slight increase during cold, season plateau of variation is generally up to 2 mg / l. class ii 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 i f m a m i i a s o n d c l ass iiimg n-no2 / l 2008 2007 2006 2005 2004 month l y ave rage fig. 11 evolution of the concentration of n-no2 by section siret siret (2004-2008) class ii 0 1 2 3 4 i f m a m i i a s o n d class i mg nno3/l maximum monthly minimum monthly average monthly class i class i fig. 12 the average change, monthly minima and maxims on n-no3 by section siret siret (2004-2008) class ii 0 0,01 0,02 0,03 i f m a m i i a s o n d mg n-no2/l romania ucraina fig. 13 comparative evolution of concentration n-no2 by siret siret section – 2008 a single maximum of 3.42 mg / l was recorded in february 2007, and then the river is included in class iii (ord.161/2006). when referring to concentration n-no2 there are sensitivity differences between the used methods. for nitrates (fig.14) it is showed a similar trend (no significant differences) of journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 10 class i 0 1 2 3 i f m a m i i a s o n d mg n-no3/l class ii romania ucraina fig. 14 comparative evolution of n-no3 in section siret siret (2008) nitrates concentration recorded by the two laboratories, since the principle of determining the methods used is identical. 1.7 total mineral nitrogen, total phosphorus for total mineral nitrogen, the quality class determined according to values is of maximum ii, the maximum concentration recorded being 1.28 mg n/l. class i 0 0,15 i f m a m i i a s o n d mg p/l 2008 2007 2006 2005 2004 monthly average fig. 15 evolution of the concentration of p in section siret siret (2004-2008) for the samples collected in common, the variability of determined values falls within ± 0.29 mg n/l, the large differences are recorded in november and october. determined values for total p did not exceed class ii (0.4 mg p/l) generally the variation values ranging up to 0.15 mgp/l (class i). it is noted that during the monitored table 2 comparative values of total mineral n concentration in 2008 monthly romania mg n/l ucraina mg n/l january. 1.112 1.219 february 1.13 1.379 march 0.82 0.084 april 1.076 1.119 may. 1.284 1.559 june 0.748 0.81 july 0.71 0.599 august 0.634 0.779 september 1.275 1.03 october 0.708 0.989 november 0.753 1.039 december 0.811 0.999 period only during september 2006 it was recorded a value of 0,183 mg p/l, which included the river in class ii (fig.15). analyzing the values for phosphorus from orthophosphate, from both laboratories (fig. 16), it can be observed that concentrations do not exceed class ii (ord161/2006). class i 0 0,15 0,3 i m m i s n class ii m g p-po 4/l romania ucraina fig. 16 comparative evolution of the concentration of p-po4 in section siret siret (2008) we can differentiate the fact that the values obtained by the romanian side are constant in class i and those obtained by the ukrainian side had higher values but also with different trend. in this case, the class of quality obtained by the two laboratories is different, but the ecological status of the river is good. 1.8 iron the general area of variation of the concentration of iron is frequently included journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 11 between 0 and 0.3 mgfe / l, which corresponds to quality class i. class i 0 0,1 0,2 0,3 0,4 0,5 i f m a m i i a s o n d class ii mg fe/l max min class i monthly average fig. 17 evolution maxims, minima and average monthly concentration of fe on siret section siret (2004-2008) fig. 17 shows that values exceeding class i are very rarely recorded: thus we have a maximum of 0.48 mg / l in march 2005 and another value of 0.34 mg fe / l in may 2008, which implies a class ii of the river on the analysis of daily samples. monthly average values indicate a decrease of the iron concentrations in general during the months of july, august, september, higher values being recorded during the cold months of the year: january, february, march, december (archive sga suceava). the trend of the values obtained by the two countries in iron determination is similar, with no major differences. 1.10 other metals among the metals determined by the romanian part we mention: beryllium, boron, aluminum, titanium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, molybdenum, silver, cadmium, tin, antimony, tellurium, barium, mercury , cadmium, titanium, lead, uranium. the only element which was overrun is tin found, the value determined is 4.9 mg / l and the limit is 2.2 mg/l (ord.161/2006). 1.11 priority and priority hazardous substances the indicators analyzed include the determination of chlorobenzenes group (pentachlorobenzene, trichlorobenzenes, hexachlorobenzene, endosulfan,) pesticides (alachlor, aldrin, lindan, isodrin, dieldrin, endrin) herbicides, insecticides and fungicides with n and p (chlorpyrifos, chlorfenvinphos, trifluralin, atrazine, simazine, benzo(k)fluoranthene, benzo(a)pyrene, benzo(b)fluoranthene, benzene, anthracene, naphthalene, polyaromatic hydrocarbons, lead, mercury, nickel, benzo(g,h,i)perylene, indeno(1,2,3cd)pyrene. the values obtained for these determinations by atomic emission spectrometry technique with inductively coupled plasma, there are not exceed ances of the allowed concentration limits 2. biological determination in order to determine the factors who modify the water quality, the biological methods are based on the relationships between organisms and abiotic conditions. the living creatures who lived in those waters form two distinct types of biocoenosis the plankton, less represented by organisms and the benthic organisms, abundant, varied. the benthic biocoenosis is represented by benthic algae and benthic macroinvertebrates (antonescu 1963). table 3 classes of surface water quality by saprobic index (ord.161/2006) saprobic index contamination quality class ecological status <1,8 contamination absent, weak i very good <2.3 moderate pollution ii good <2.7 moderate to critical pollution iii moderate <3.2 strong contamination iv poor >3.2 strong contamination v bad among the analysed bioindicators, the benthic macroinvertebrates of rivers have specific characteristics depending on the characteristic of the substrata, water velocity, water chemistry and the river journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 12 benthos are poorer than in streams benthos. phytobenthos is represented by benthic autotroph algae. their major role is the synthesis of organic substances from water and are the basis of most food chains in the water. after the identification of these organisms we are making an assessment of water quality, due to rapid response of organisms to changes in environmental conditions, calculating the saprobic index. in according with order 161/2006 and saprobic index we establish following water quality class (table 4): table 4 quality classes depending on siret -siret section biomarkers (2004-2008) indicator 2004 2005 2006 2007 2008 river siret – siret quality class plankton ii ii ii ii ii zoobentos ii ii ii ii ii benthic algae ii ii ii depending of biological analysis in the section examined we distinguish a high diversity of organisms, the number and diversity is close related with the characteristic of the substrate and the water chemistry. 1 the planktonic biocoenosis is reprezented by taxonomic groups: bacillariophyceae, euglenophyta şi clorophyceae 2 the benthic algae are represented by taxonomic groups: bacillariophyceae, cyanophyceae 3 the zoobenthos is represented by taxonomic group: ephemeroptera, trichoptera, diptera (sga suceava archive) results and discussion the data presented in this material can be concluded: the general physical-chemical indicators make the ranking of the river in class ii according to the annual average (20042008), (order 161/2006 art. 6. 1 letter a), concentrations determined from metal and priority hazardous substances do not show value that exceeds the allowed limits, biomarkers values are in close correlation with the physical, chemical, including the river in grade ii. environmental status of the river is good for the samples collected in common with the ukrainian side, the differences arising between the analytical results are due to different sensitivity of different used methods and equipment. conclusions the conclusion is that for those 5 monitored years, there were not changes in the quality class of the river, because industrial activity upstream did not increase in intensity amid economic stagnation chernivtsi region of ukraine, and due to increased capacity selfpurification generated large flows of this river. references 1. c. antonescu, biologia apelor, editura didactică şi pedagogică bucureşti, 1963 2. s. mănescu, m. cucu, mona ligia diaconescu, chimia sanitară a mediului, editura medicală bucureşti, 1994 3. v. trufaş, c. trufaş, hidrochimie ediţia a ii a, editura agora călăraşi, 2003 4. ***buletine de analiză a apei pentru râul siret secţiunea siret din perioada 2004-2008, arhiva sistemului de gospodărire a apelor suceava 5. ***directiva cadru privind apa 2000/60/ce 6. ***geografia româniei, i, geografie fizică, edit. academiei române, bucureşti, 1983 7. ***ordinul 161/2006 pentru aprobarea normativului privind clasificarea calităţii apelor de suprafaţă în vederea stabilirii stării ecologice a corpurilor de apa 8. *** iso 15705:2002, sr en 25814 iso 5814/1999, sr en 1899-1/2003,sr iso 10523/ 1997, sr iso 8466–1/1999, sr iso 7150-1/2001, sr en 26777:2006, sr iso 7890-3/2000, sr iso 7890-1/1998, sr en iso 6878/2005, sr iso 6439:2001 /c91:2006, iso 15705:2002, sr iso 6332-19 microsoft word 3 journal fia nr 1 2010 final_69-74.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 73 „on farm” conservation of some maize races in bucovina d. murariu1, m. murariu2, i. gontariu3 1 suceava genebank, b-dul 1 decembrie 1918, nr. 17, suceava, e-mail: dmurariu@suceava.astral.ro 2a.r.d.s. suceava b-dul 1 decembrie 1918 nr. 17 suceava e-mail:marius_murariu2005@yahoo.com 3 ştefan cel mare university of suceava, faculty of food engineering, 13 universitaţii st., 720229, suceava, romania e-mail: ioang@usv.ro abstract: submontane and mountain area is a very heterogeneous eco-geographic space, where maize during about 360 years, underwent an ample diversification process, under the pressure of natural evolution factors and selection carried out by man-forming locale maize populations, genetically distinct and well adapted to local ecological conditions. it is obvious that this long period has favored an active genetic process of population formation, stopped in 1957-1962 by very quick introduction in romania, of more than 90% of maize cultivated area of corn hybrid seeds [1]. at present people cultivate maize locale landraces only in submontane and mountain areas of the carpathian mountain countries. these old varieties have some valuable features (e.g. earliness, resistance to cold and prolificacy, high nutritive elements content) [2]. the study of 17 maize accessions coming from bucovina, for evaluation of genetic erosion level during 30 years, were accomplished, through utilization of biometrical descriptors. the accession collected in 2002, has the higher values at the descriptors: plant height, insertion height of the main ear, and panicle length then the values of descriptors of the hanganesc race. the analyzed maize accessions are coming from 4 villages: brosteni, vama, vatra moldovitei and frumosu. these villages are situated at very different altitudes, from 529m to 639m. in these locations two races were identified: hanganesc and moldovenesc. key words: eco-geographic space, locale landraces, genetic erosion, biometrical descriptors introduction it is recognized that a collection value is given by the level of its utilization not by the number of the accessions. although the sharing of benefits through their redistribution is important, more direct ways of making benefits are needed, such a wider evaluation enhancement and utilization of local germless. in romania the local pgr are utilized, directly-through the conservation and utilization of local varieties by farmers from the mountain areas, and indirectly through their utilization in the plant breeding programs. in the sub mountain and mountain areas maize old landraces can meet to 800-900 m elevation. here maize represents a traditional food for people and an important animal fodder where during about 350 years, underwent an ample diversification process, under the pressure of evolution natural factors and selection carried out by man-forming locale maize populations, genetically distinct and well adapted to local ecological conditions. it is obvious that this long period has journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 74 favored an active genetic process of population formation, stopped in 19571962 by the very quick introduction, of more than 90% of maize cultivated area of corn hybrid seeds [1]. at present people cultivate maize locale landraces only in submontane and mountain areas of the carpathian mountain countries. these old varieties have some valuable features (e.g. earliness, resistance to cold and prolificacy) [2]. the maize breeding programs by heterozis capitalization between the inbred lines leaded to the realization of some maize performance hybrids, but the initial germplasm sources represented by the old varieties were neglected. being convinced that a collection value is given by the level of its utilization the maize resources could be utilized, both directly through the conservation of farmer’s locale maize varieties and indirectlyin an intermediate stage when are used by plant breeders to develop new hybrids or to broaden the base of plant maize breeding regional program for the cold and wet areas [3]. materials and methods the study of 17 accessions of maize landraces coming from bucovina mainly for evaluation of their genetic erosion was accomplished through utilization of the biometrical descriptors (plant height, panicle length, number of braches on category i, number of braches on category ii, total number of the leaves, number of the leaves of the lower ear, insertion height of the main ear, maximum diameter of the stem, ear length, number of kernel rows, grain length, 1000 kernel weight). the analyzed maize landraces accessions coming from 4 villages (broşteni, vama, vatra moldoviţei şi frumosu county suceava) situated at different altitudes, from 442 m to 639 m. in the targeted villages two maize races were identified: hanganesc and moldovenesc. the 17 maize landraces were collected during 30 years (1970 – 2000). in 2007 all accessions were sown and studied in the experimental field of the suceava gene bank. results and disscussion all data were analyzed separately, as follows: 1. the accessions originated from broşteni area the studied accessions were collected in the years: 1970, 1971, 1990 and 1995. all accessions belong to the race hăngănesc. table no. 1 shows the values of descriptors which describe the race hanaganesc in comparison to values which we obtained in the experimental field in 2007 at the four accessions which belong to the same race, collected in different years, coming from brosteni, county suceava. table 1 the middle values of the descriptors which determine the specific traits of the hanaganesc race at 4 maize landraces coming from village brosteni, county suceava accessions name descriptors broşte ni 244 broste ni 247 brost eni 9 broste ni 14 collecting data the specific traits of the hanganesc race 1970 1971 1990 1995 plant height (cm) 132-172 151.0 175.7 185.2 201.1 panicle length (cm) 25.5 27.5 29 29.4 33.1 no. of branches on category i 8.8 8 7.66 15.1 9.1 no. of branches on category ii 1.7 1.2 1 2.7 2 total numbers of the leaves 8.3 6.5 7.83 9.5 9.1 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 75 number of the leaves of the lower ear 4 3.5 4 5.3 4.9 insertion height of the main ear (cm) 44.0 43.5 51.66 69.9 69.4 maximum diameter of the stem (mm) 14.3 14.20 13.96 16.13 16.33 ear length (cm) 11.5 12.15 11.15 12.93 12.70 no. of kernel rows 12-18 13 16.3 17.2 15.8 grain length (mm) 7.8 7.83 8.14 9.39 8.87 grain width (mm) 6.2 6.42 6.63 6.64 7.2 1000 kernel weight (g) 150-191.6 150 170 176 212 if we analyze the data from the above table we can say that the accession collected in 1995 shows higher values in comparison to the accession collected in 1970. after 25 years (1970-1995), the values of plant and ear descriptors grow up. through modification of these traits, the accession collected in 1995 has values of biometrical descriptors very different in comparison to the values of descriptors of the hanaganesc race. these differences demonstrate the fact that the genetic erosion was manifested at high level in this village, explained by the fact that this village is situated at 600 m altitude, and the hybrids are not cultivated in this region. 2. the accessions originated from frumosu area the 6 accessions were collected in the 6 different years (1971, 1972, 1973, 1988, 1995, and 2002). also, all accessions belong to hanganesc race like the samples originated from brosteni village. the next table presents values which describe the hanganesc race in comparison to the values of registered descriptors at 6 local maize landraces which belong to the same race, coming from frumosu village, county suceava. table 2 the middle values of the descriptors which determine the specific traits of the hanganesc race at 6 maize landraces coming from village brosteni, county suceava accession name descriptors frum osu 161 frum osu 406 frum osu 162 frum osu 15 frum osu 26 frum osu 32 collecting data the specific traits of the hanganesc race 1971 1972 1973 1988 1995 2002 plant height (cm) 132-172 149.7 175.1 153.7 169.5 192.1 215.7 panicle length (cm) 25.5 25.6 31.8 26.7 32.8 34.1 36 no. of branches on category i 8.8 10.2 11.5 8.2 9.1 6.2 10.7 no. of branches on category ii 1.7 0.6 2.5 1.8 1.7 0.3 0.8 total numbers of the leaves 8.3 7.9 8 7.7 8.6 8.1 8.2 number of the leaves of the lower ear 4 3.5 4.1 3.5 3.4 3.2 4.8 insertion height of the main ear (cm) 44.0 41.5 50.7 35.9 48.7 54.3 61.4 maximum diameter of the stem (mm) 14.3 13.95 18.45 16.64 16.64 17.75 19.67 ear length (cm) 11.5 11.43 13.13 9.85 12.53 12.77 12.30 no. of kernel rows 12-18 16.8 14.8 15.8 14.00 18.00 17.25 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 76 grain length (mm) 7.8 8.53 9.08 8.39 9.23 8.77 8.5 grain width (mm) 6.2 6.83 7.61 7.11 8.04 7.25 7.01 1000 kernel weight (g) 150-191.6 162 195 197 200 208 204 the data from table 2 show that the accessions collected in the years 1995 and 2002 have higher values of descriptors: plant height and panicle length, in comparison to values of the same descriptors registered at samples collected in 1971, 1972 and 1973. after 30 years the values of plant, ear and kernel descriptors experienced a slight increase in comparison to values of same descriptors at samples collected in the year 1971. according to the obtained results, it is noted that the sample collected in 2002 has slightly increased values in comparison to values of same descriptors at samples collected in 1972 and 1973, which shows that the area frumosu had a genetic erosion of this race, but not as strong as in the area broşteni. 3.the accessions originated from vatra moldovitei area the analyzed accessions were collected in years 1972, 1985 and 1989. all accession belongs to the moldovenesc race. the below table shows the descriptors which describe the moldovenesc race in comparison to the values registered at three maize accessions which belong to the same race coming from vatra moldovitei village table 3 the middle values of the descriptors which determine the specific traits of the moldovenesc race at 3 maize landraces coming from village brosteni, county suceava accession name descriptors the specific traits of the moldovenesc race vatra mold. 423 vatra mold. 2 vatra mold. 427 collecting data 1972 1985 1989 plant height (cm) 185.6 171.3 160.5 165.5 panicle length (cm) 28.9 30.12 27.4 28.4 no. of branches on category i 15.7 8.25 4.5 8.7 no. of branches on category ii 0.9 0.6 0.6 0.37 total numbers of the leaves 8.7 8.0 7.0 7.76 number of the leaves of the lower ear 4.8 3.2 3.1 3.7 insertion height of the main ear (cm) 46.4 46.1 35.6 38.2 maximum diameter of the stem (mm) 17.4 16.03 15.63 17.56 ear length (cm) 16.7 14.73 10.79 12.73 no. of kernel rows 14.2 15.5 14.2 13.8 grain length (mm) 8.2 7.36 8.13 8.01 grain width (mm) 7.1 7.52 7.64 6.61 1000 kernel weight (g) 220 206 178 188 the data from above table emphasize the following aspects: the maize accession collected in the year 1972 had similar traits with the moldovenesc race, but, after 17 years, the traits of this race modified very significantly, the grain length and grain width are lower than of the moldovenesc race, and the 1000 kernel weight of this accession is similar to 1000 kernel weight of the hanganesc race. after 17 years is it possible to make an impurification of the moldovenesc race with the other two races: hăngănesc and cincantin, because the plant height, insertion height of the main ear and ear length have the lower values than the journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 77 values of the same descriptors of moldovenesc race. in future, due to modification of these characteristics it is possible for this race to disappear from this village, being replaced by sub race hăngănesc/moldovenesc 4. the accessions originated from vama area the analyzed maize landraces were collected in 4 different years (1972, 1985, 1988, and 2002). like the samples originating from broşteni and frumosu, all studied accessions belong to hanganesc race. table 4 emphasizes all descriptors which describe the hanganesc race in comparison to the values of descriptors at the four maize landraces which belong to the same race, originated from vama village, county table 4 the middle values of the descriptors which determine the specific traits of the hanganesc race at 4 maize landraces coming from vama village, county suceava accession name descriptors the specific traits of the hanganesc race vama 404 vama 10 vama 19 vama 1 collecting data 1972 1985 1988 2002 plant height (cm) 132-172 156.75 108.2 169.66 181.4 panicle length (cm) 25.5 26.75 28.57 28.0 31.7 no. of branches on category i 8.8 8.5 3.87 5.60 6.4 no. of branches on category ii 1.7 1.6 1.56 1.7 1.6 total numbers of the leaves 8.3 6.62 7.57 7.33 7.9 number of the leaves of the lower ear 4 2.8 3.7 3.66 3.8 insertion height of the main ear (cm) 44.0 37.5 39.7 42.3 51.9 maximum diameter of the stem (mm) 14.3 17.5 14.9 19.1 15.79 ear length (cm) 11.5 11.9 12.03 12.5 11.83 no. of kernel rows 12-18 16.5 16.85 15.3 18.4 grain length (mm) 7.8 7.9 8.1 8.06 8.13 grain width (mm) 6.2 6.8 6.6 7.1 6.8 1000 kernel weight (g) 150-191.6 180 160 188 180 the data recorded in the above table highlight the following aspects: the accession collected in 2002, has higher values of the descriptors: plant height, insertion height of the main ear, and panicle length than the values of descriptors of the hanganesc race. after 30 years the descriptors: ear length, grain length, grain width and 1000 kernel weight have the same values. the accession collected in year 2002 shows the typical traits of the hanganesc race. also this accession is similar to the accession collected in 1972 conclusions by analyzing the genetic erosion of maize local landraces, one can draw the following conclusions: 1. the characteristics of race hanganesc have changed strongly in the area broşteni to a lesser extent in the area frumosu whereas in the area of vama the characteristics remained unchanged after 30 years from the first collections, especially the descriptors regarding grain and ear. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 78 2. the race moldovenesc underwent significant changes in the area vatra moldovitei, at present is in danger of extinction and will soon be replaced by the sub race hanganesc/ moldovenec. references 1. cabulea i., has i., copandean a., 1998 actual status of maize genetic resources from transilvania. report of the workshop” conservation and utilization of plant genetic resources for food and agriculture”, 24-25 march, 1998, suceava, romania, pag. 2627. 2. cristea m., 1978rasele de porumb din romania. edit. academiei romane, bucuresti; 3. murariu d., 1998the aspects concerning the precocity to some maize landraces conserved in suceava genebank. report of the workshop” conservation and utilization of plant genetic resources for food and agriculture”, 24-25 march, 1998, suceava, romania, pag. 39-43 microsoft word 13 avramiuc recorectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 81 the influence of storage conditions upon ascorbic acid content in jonathan and golden apples *marcel avramiuc1, ana leahu1, cristina damian1, cristina-elena hretcanu1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received 5 november 2012, accepted 28 november 2012 abstract: in this work it has searched the influence of storage conditions upon ascorbic acid content, in jonathan and golden apples. the study of ascorbic acid content and ph evolutin for 16 weeks has been carried out using, as biological material, apple samples from jonathan and golden varieties, coming from the orchards in the area of fălticeni. the apples were kept in containers of glass and wood, constituting, for each sample, variants of experiences, in the presence and in the absence of oxygen, at following thermal thresholds: 4°c, 8°c and 15°c. in order to keep the apples in the presence of oxygen, they were placed in wooden boxes. to store apples in an oxygen-free environment, it used glass containers where the fruits were introduced along with a small candle lit, and then the lid of the container was tightly closed. the extinguish of the candle has confirmed that the oxygen in the container was used. the chemical investigations have been carried out on freshly harvested material (week 0) and then every two weeks, for a total of 16 weeks. at the end of the analysed interval (after 16 weeks of storage), the ascorbic acid content has registered different rates of diminution, depending on storage temperature and length, as well as on variety (sucrose concentration and ph). the lower degradation of ascorbic acid in apples from golden variety, during storage, is due both to higher sucrose content of those ones, and to higher ph values compared to apples from jonathan variety. keywords: ascorbic acid, apples, oxygen, ph, storage, temperature. 1. introduction the industrial processing and cooking of food raw materials such as: cutting, grinding, blanching, scalding, boiling, addition of chemicals etc. can negatively influence the quality of these products, resulting in significant loss of hydroand fat-soluble vitamins [1, 2, 3, 4, 5, 6]. the addition of anthocians, sugars and even starch seems to have a protecting action on vitamin c [6]. the drying methods, exposing the food to air, lead to the loss of vitamin c because of oxidation. but, the freeze drying, which is carried out in the absence of oxygen, does not cause loss of vitamin c [7]. the storage conditions can also influence the level of some raw material vitamines causing, sometimes, significant decrease in the concentration of these biocatalysers within finished product [6]. in some juices the ascorbic acid degradation is predominantly aerobic (due to the presence of dissolved oxygen), but in absence of oxygen the ascorbic acid decomposition continues anaerobicaly, being especially influenced by temperature [8]. the loss of acid ascorbic is fast in the early stage of fruits juice storage, coincident with the consumption of dissolved oxygen, and then becomes gradual [7]. in the case of ascorbic acid (vit. c) the reduction of this vitamine in vegetable products is caused by the presence and activity of ascorbic oxydase enzyme, which has a large spread in plants, catalysing the oxidation of ascorbic acid to dehydroascorbic acid [9]. the mean storage length of fruits, in modern conditions, may be: days in strawberries and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 82 cherries, 1-2 weeks in blueberries, currants, up to a month at apricots, watermelons, melons, peaches, up to 1-2 months at plums, 3-4 months at quince, up to 5-6 months at pears, 3-8 months at apples, 8-13 months at nuts in shell [10]. in this work it has studied the influence of temperature and oxygen upon ascorbic acid content of some apple samples, belonging to jonathan and golden varieties, stored for 16 weeks. 2. experimental the research has been carried out using, as biological material, apples from jonathan and golden varieties, coming from the orchards in the area of fălticeni. it has studied the evolution of ascorbic acid content and ph for 16 weeks. the apples were kept in containers of glass and wood, constituting, for each sample, variants of experiences, in the presence and in the absence of oxygen, at following thermal thresholds: 4°c, 8°c and 15°c. in order to keep the apples in the presence of oxygen, they were placed in wooden boxes. to store apples in an oxygen-free environment, it used glass containers where the fruits were introduced along with a small candle lit, and then the lid of the container was tightly closed. the extinguish of the candle has confirmed that the oxygen in the container was used. the chemical investigations have been carried out on freshly harvested material (week 0) and then every two weeks, for a total of 16 weeks. the ascorbic acid content of apples was determined through a method based on reduction by the ascorbic acid of 2.6dichlorphenol-indophenol (2.6-dcfif) to the corresponding leucoderivate [11]. the dosing of sucrose in apples was based on evaluation of reducing sugars before and after inverting [12]. the ph values were determined with a digital ph-meter type hanna. 3. results and discussion table 1 plays the acid ascorbic values of jonathan apples during storage of samples under certain conditions. table 1 ascorbic acid values in apples from jonathan variety +o2 * = storage in the presence of oxygen; –o2** = storage in the absence of oxygen determination ascorbic acid (mg/100g) storage temperature t=4°c t=8°c t=15°c test variants +o2 * –o2** +o2 –o2 +o2 –o2 week 0 11.08 week ii 9.90 11.08 9.90 9.90 8.31 7.04 week iv 7.92 9.50 7.20 7.28 7.22 6.01 week vi 6.33 6.41 6.17 7.20 6.01 4.03 week viii 6.30 5.54 5.14 5.94 5.14 3.90 week x 5.14 4.75 3.72 5.46 4.19 3.90 week xii 4.52 4.56 3.16 4.03 4.03 3.72 week xiv 3.16 3.32 3.18 3.70 3.29 3.30 week xvi 2.29 2.61 1.98 2.05 1.48 1.80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 83 as seen in the table 1, at temperature of 4°c in the presence of oxygen, the ascorbic acid content in apples of jonathan variety has recorded different discounts, depending on the length of storage. thus, the greatest reductions of acid ascorbic concentration were registered in the ranges iiiv and iv-vi (20%), followed by ranges x-xii and viii-x (12-18.4%). at the same temperature of storage, but in the absence of oxygen, the greatest reduction in the content of ascorbic acid was in the range iv-vi weeks (32.5%), followed by range xii-xiv (27.2%). at the end of the analysed interval (after 16 weeks of storage) the ascorbic acid content has decreased by 79.3%, compared to the blank (week 0), in apples stored at 4°c in the presence of oxygen, and by 76.4%, compared to blank, in apples kept at the same temperature, but in the absence of oxygen. at temperature of 8°c, in the presence of oxygen, the largest reduction of ascorbic acid content was within the range xiv-xvi weeks (37.73%), followed by ranges ii-iv and viii-x weeks (27.2%). at temperature of 8°c, but in the absence of oxygen, the biggest reductions of the ascorbic acid content were within the interval xiv-xvi weeks (44.6%), followed by intervals ii-iv and x-xii weeks (over 26%). at the end of the analysed interval (after 16 weeks of storage) the ascorbic acid content has decreased by 82.1%, compared to the blank (week 0), in apples stored at 8°c in the presence of oxygen, and by 81.5%, compared to the blank, in apples kept, at the same temperature, but in the absence of oxygen. at temperature of 15°c, in the presence of oxygen, the greatest reduction of the ascorbic acid content was in the range xiv-xvi weeks (55%), followed, in order, by the range 0-ii (25%) and the ranges xii-xiv weeks (18.36%) and ii-iv weeks (13%). at the same temperature, but in the absence of oxygen, the biggest reductions in the levels of ascorbic acid content were within intervals xiv-xvi (45.45%), 0-ii (36.5%) and iv-vi (33%). at the end of the analysed interval (after 16 weeks of storage) the ascorbic acid content has decreased by 86.6%, compared to the blank (week 0), in apples stored at 15°c, in the presence of oxygen, and by 83.75%, compared to the blank, in apples kept, at the same temperature, in the absence of oxygen. compared to week 0 (fresh sample), the ph of the jonathan apple has recorded an increase with 13% in the 8th week and with 7% at the end of the test (after 16 weeks). table 2 plays the acid ascorbic values of golden apples during storage of samples under certain conditions. as seen in the table 2, at temperature of 4°c, in the presence of oxygen, the ascorbic acid content in apples of golden variety has recorded the largest decreases within intervals xii-xiv and xiv-xvi weeks (20.3-21.6%), followed, in order, by intervals ii-iv, iv-vi and vi-viii (12.53-14.45%). at the same temperature of storage, but in the absence of oxygen, the greatest reduction of the ascorbic acid content was in ranges iv-vi and xiv-xvi weeks (32.8-34%), followed by range xii-xiv (11.32%). at the end of the analysed interval (after 16 weeks of storage), the ascorbic acid content has decreased by 72.63%, compared to the blank (week 0), in apples stored at 4°c in the presence of oxygen, and by 68.16%, compared to the blank, in apples kept, at the same temperature, lack of oxygen. at temperature of 8°c, in the presence of oxygen, the largest reduction of ascorbic acid content was within range viii-x weeks (30%), followed by ranges xii-xiv (26.77) and x-xii (20.75%). at temperature of 8°c, but in the absence of oxygen, the biggest reductions of the ascorbic acid content were within range xiv-xvi (29.72%), followed, in order, by ranges x-xii (22.4%), viii-x and xii-xiv (over 20%). at the end of the analysed interval (after 16 weeks of storage), the ascorbic acid content has decreased by 76.31%, compared to the blank (week 0, in apples stored at 8°c in the presence of oxygen, and by 74.12%, compared to the blank, in apples, kept at the same temperature, but in the absence of oxygen. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 84 at temperature of 15°c, in the presence of oxygen, the largest reduction of ascorbic acid content was within range xiv-xvi weeks (36.82%), followed by range x-xii (34.4%) and ranges xii-xiv, viii-x and 0-ii (16-19%). at the same temperature, but in the absence of oxygen, the largest decreases of ascorbic acid content were in the range x-xii (32.3%), followed by range xiv-xvi (27%), and the ranges 0-ii and viii-x (20-25%). at the end of the analysed interval (after 16 weeks of storage), the ascorbic acid content has decreased by 82.58%, compared to the blank (week 0), in apples stored at 15°c in the presence of oxygen, and by 79.10%, compared to the blank, in apples kept, at the same temperature, in the absence of oxygen. compared to week 0 (fresh sample), the ph of the golden apples has recorded an increase by 7% in the weeks viii-xii, and by 10% at the end of the test (after 16 weeks). table 2 ascorbic acid values in apples from golden variety +o2 = storage in the presence of oxygen; –o2 = storage in the absence of oxygen the acid ascorbic content in potato tubers, stored 90 days under house cellar conditions, has decreased by over 60% in tubers stored in wooden boxes, and by 20% in tubers stored in tightly closed glass jars [13]. comparing the data from the two tables, it notice that, under the same conditions of storage, the apples of golden variety have recorded a lower reduction of ascorbic acid content than apples of jonathan variety. the differences between the two varieties, as to sucrose content and ph values, could explain the difference as to ascorbic acid reduction during storage. after 168 hours storage of some natural juices, under refrigeration conditions, the highest vitamin c losses have registered in sucrose free juice samples, and the least ones in juice samples with 10% sucrose [14, 15]. from the figure no. 1 it can remark a very closed corelation between the ascorbic acid variation (mg/100g) and the storage time. the table no. 3 renders the regression function for the variation of ascorbic acid values in apples from jonathan and golden variety. determination ascorbic acid (mg/100g) storage temperature t=4°c t=8°c t=15°c test variants +o2 –o2 +o2 –o2 +o2 –o2 week 0 10.05 week ii 9.50 9.58 9.50 9.58 8.07 8.07 week iv 8.31 9.42 8.23 8.57 7.52 7.99 week vi 7.12 6.33 8.02 8.20 6.33 7.21 week viii 6.09 6.01 7.20 7.51 6.09 6.01 week x 5.30 5.59 5.06 5.98 5.06 4.51 week xii 4.40 5.30 4.01 4.64 3.32 3.05 week xiv 3.45 4.70 3.25 3.70 2.77 2.88 week xvi 2.75 3.10 2.38 2.60 1.75 2.10 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 85 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 2 4 6 8 10 12 14 16 18 week a sc or bi c ac id (m g/ 10 0g ) j4+ j4g4+ g4j8+ j8g8+ g8j15+ j15g15+ g15 figure 1. the variation of the ascorbic acid content in function of week storage g = golden variety, j = jonathan variety 4, 8, 15 coresponds to the storage temperature of t=4°c, t=8°c, t=15°c, respectively + and coresponds to the storage in the presence of oxygen (+o2), respectively to the storage in the absence of oxygen (–o2) table 3 regression function for the variation of ascorbic acid values depending of the storage time in apples from jonathan and golden variety apples variety test variants, storage temperature regression function r 2 +o2 , t=4°c y = -0.0022x 3 + 0.0652x2 1.0435x + 11.272 r2 = 0.9888 -o2 , t=4°c y = 0.0016x 3 0.0184x2 0.6534x + 11.627 r2 = 0.9615 +o2 , t=8°c y = -0.0006x 3 + 0.0426x2 1.095x + 11.325 r2 = 0.9873 -o2 , t=8°c y = -0.0026x 3 + 0.0738x2 1.0732x + 11.233 r2 = 0.9831 +o2 , t=15°c y = -0.0044x3 + 0.1226x2 1.4334x + 11.009 r 2 = 0.9936 jo na th an v ar ie ty -o2 , t=15°c y = -0.0077x 3 + 0.2224x2 2.1676x + 10.98 r2 = 0.9884 +o2 , t=4°c y = 0.0008x 3 0.0147x2 0.4252x + 10.171 r2 = 0.9977 -o2 , t=4°c y = -0.0003x 3 + 0.0148x2 0.5923x + 10.423 r2 = 0.9302 +o2 , t=8°c y = 0.0019x 3 0.0524x2 0.1178x + 9.9411 r2 = 0.9861 -o2 , t=8°c y = 0.0008x 3 0.0335x2 0.143x + 9.9786 r2 = 0.9936 +o2 , t=15°c y = -0.0014x 3 + 0.0341x2 0.7068x + 9.8068 r2 = 0.9846 g ol de n va ri et y -o2 , t=15°c y = 0.0014x 3 0.0318x2 0.3325x + 9.6602 r2 = 0.9779 +o2 = storage in the presence of oxygen; –o2 = storage in the absence of oxygen according to banu et al. (2003), the oxidation of vitamin c depends on ph values, being very fast at ph<4,5. it seems that the lower degradation of ascorbic acid in apples from golden variety during storage is due both to higher sucrose content of those ones (4.56%), and to higher ph values (6.20-6.82), compared to apples from jonathan variety (sucrose=2.37%, ph = 4.4-5.2), as seen in the figure no. 2. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 w eek 0 w eek 4 w eek 8w eek 12 w eek 16 ph of jonathan variety ph of golden variety figure 2. the variation of ph values depending of the storage time in apples from jonathan and golden variety food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 86 4. conclusions the storage of apples from jonathan and golden varieties, under certain conditions of temperature and aeration, has influenced the content of ascorbic acid. at the end of the analysed interval (after 16 weeks of storage), the ascorbic acid content has registered different rates of diminution, depending on storage temperature and length, as well as on variety (sucrose concentration and ph). in jonathan variety samples, the greatest decrease of ascorbic acid content has registered after 16 weeks of storage at 15°c: by 86.6% (compared to the blank week 0) in apples stored in the presence of oxygen, and by 83.75% in apples kept in the absence of oxygen. in golden variety samples the greatest decrease of ascorbic acid content has registered after 16 weeks of storage at 15°c: by 82.58% (compared to the blank week 0) in apples stored in the presence of oxygen, and by 79.10% in apples kept in the absence of oxygen. under the same conditions of storage, the apples of golden variety have recorded a lower reduction of ascorbic acid content than apples of jonathan variety. it seems that the lower degradation of ascorbic acid in apples from golden variety, during storage, is due both to higher sucrose content of those ones, and to higher ph values compared to apples from jonathan variety. 5. references [1] chow y, louarme l. , bonazzi c., nicolas j., billaud c, apple polyphenoloxidase inactivation during heating in the presence of ascorbic acid and chlorogenic acid, food chemistry 129, 761– 767, 2011 [2] adrien, j., petit, l., les vitamines des céréales et leur évolution au cours des traitements technologiques. annales de la nutrition et de l’alimentation, vol. 24, nr. 1, p. 131., 1970 [3] lin, y. l., li, s. j., zhu, y., bingol, g., pan, z., & mchugh, t. h. heat and mass transfer of apple slices under simultaneous infrared dry blanching and dehydration process, drying technology, 27, 1051–1059, 2009 [4] ferrando r., mainguy r,l’évolution des vitamins en function des conditions de récolte et de conservation des denrées primaires des animaux destinés à la consommation de l’homme. annales de la nutrition et de l’alimentation, vol. 24, no.3., 1970 [5] ulrich r., delaporte n., l’acide ascorbique dans les fruits conservés par le froid dans l’air et en atmosphère contrôlée. annales de la nutrition et de l’alimentation, vol. 24, no 1, 1970 [6] banu c., iordan m., nour v, musteaţă g., the processing of raw materials and the loss of biologically active substances. ed. „tehnica” utm. chişinău, p. 91-93,2003 [7] ball g.f.m., vitamins in foods, analysis, bioavailability, and stability. crc press, taylor & francis group 6000, broken sound parkway nw, suite 300, boca raton, fl 33487-2742, p. 292-305, 2006 [8] kennedy, j.f., rivera, z.s., lloyd, l.l., warner, f.p., and jumel, k., l-ascorbic acid stability in aseptically processed orange juice in tetrabrik cartons and the effect of oxygen, food chem., 45, 327, 1992 [9] neamţu, g., food biochemistry. ed. ceres, bucureşti, p. 266., 1997 [10] beceanu, d., chira, a., paşca, i., fruits, vegetables and flowers methods for keeping prologation of fresh. ed. m.a.s.t., bucureşti, p. 9-15, 2003 [11] official methods of analysis of aoac international, indyk, h. and konings, e., eds., 17th ed., aoac international, gaithersburg, md, p. 45-60., 2000 [12] sr 110-5:1995 [13] avramiuc, m., fărtăiş, l., herghelegiu ioana, 2008 the ascorbic acid content variation in potato tubers, during storage under house cellar conditions. cercetări agronomice în moldova, vol. xli, no. 4 (136),2008 [14] avramiuc, m., fărtăiş, l. – the influence of refrigeration length and of sugar adition on ascorbic acid content in some natural juices. an. st. ale univ. "al. i. cuza" din iaşi,, gen. şi biol. mol., tom x, fasc. 2, p. 35-38, 2009 [15] avramiuc mthe influence of refrigeration time and of sugar addition on ascorbic acid content in some natural juices. journal food and environment safety of the suceava university – food engineering, year ix, no.1 2010, p. 46-49, 2010 food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 9 treatment of organic pollutants on zeolites in motor transportation & auto repair enterprises n.s.torosyan1 a.g.kudryavtsev1, g.h.torosyan2 1 csto institute, yerevan, armenia, yerevan, armenia, 2. state engineering university of armenia, department of chemical technology & environmental engineering, yerevan, armenia, email:gagiktorosyan@seua.am abstract: the objective of the present study was increasing the adsorption activities of zeolites for organic pollutants. for this purpose it was realize the using of modified by quaternary ammonium salts natural zeolite. natural zeolites and them modified analogs can be made a renewable resource. the advantages of natural by-products in comparison with other sorbents are their low cost, availability of extraction, operational flexibility and control and others. motor transportation and auto repairs station wastewaters include typical sewage from the petrochemical and petrol using industries, of course in smaller amounts. adsorption from wastewater with organic pollutants involves concentration of the solute on the surface. here, it has been had adsorption and desorption process together which will attain an equilibrium state. we used freundlich model for description of the adsorption data for activated carbon. keywords: natural zeolites, modified zeolites, quaternary ammionium salt (quat), adsorption, adsorbent, wastewater treatment, organic solution. introduction organic compounds are major pollution problem in soil and groundwater. their presence in water can create a hazard to public health and the environment. soil and groundwater pollution from small enterprise which have a relation with petroleum and this type of other products is becoming an important topic of interest as a result of the increasing public and regulatory concerns worldwide with problems of groundwater contamination and resultant impact on human health and ecological systems. nowadays for wastewater treatment in small enterprise wide application have found fine layer clarifier, in which height of upholding makes 100mm and less. the cost of such clarifier is pleased high and frequently comes nearer to cost usual clarifier. therefore at the decision of a question of waste water treatment follows not only to find rational height of upholding, but also the appropriate component, which promotes fast upholding, moreover for sorption of petroleum. last are in interaction in a flow of water, that results them agglomeration is especial at low meaning turbulence of a flow. with the purpose of the sanction of a question of wastewater treatment from mentioned enterprises by us it is offered to apply clarifier with natural zeolites and them modified with quaternary ammonium salts analogs. taking into account that fact, that our small enterprises have no while the appropriate means, we offer the elementary technological circuit of clearing of drains. so, we offer to make special holes with the boxes, filled in them by zeolites, which further are used in building industry. motor transportation and auto repairs enterprises wastewater sources and characteristics: for motor transportation and auto repair enterprises wastewater needs primary and tertiary treatments. primary treatment includes screening the wastewater for removing the suspended large and small particles settling and skimming the oil from the top layer in the wastewater in the setting smaller tanks. following the primary treatment the wastewater undergoes a tertiary treatment food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 10 which is mostly a physical process. in this case, the physical treatment includes adsorption by natural zeolites and them modified analogs. zeolites is used in water treatment plants for the removal of odors and tastes [1-3]. natural zeolites and them modified analogs on the other hand, can be made a renewable resource. the advantages of natural by-products in comparison with other sorbents are their low cost, availability of extraction, operational flexibility and control and others. the effectiveness of natural zeolites and them modified analogs for the removal of organics by adsorption also is enhanced by its large surface area. the characteristics of wastewater discharges form location to location depending upon the population and industrial sector served namely, land uses, groundwater levels and degree of separation between storm water and sanitary wastes. motor transportation and auto repairs station wastewaters include typical sewage from the petrochemical and petrol using industries, of course in smaller amounts. this type of wastewater usually is characterized by dark gray color and disagreeable odor. chemically this wastewater composed of organic compounds as hydrocarbons, phenols, aniline and other aromatics. materials and methods zeolites occur in nature in specific kinds of rocks. zeolite rich rocks are widespread in northern part of armenia, occurring in very extended geological formations. the zeolite types are exclusively clinopilolite in idjevan / northern-east of armenia / and mordenite in shirak / northern-west of armenia/.the mineral composition for clinopilolite from idjevan was generally clinpotilolite with tufas. this zeolite had an external surface area around 15 m2/g. the zeolite was crushed and sieved to different grain size – 0.4 to 1.5 mm for laboratory batch and column experiments. here are we used the quaternary ammonium salts on the basis of n, n-dimethyl-nethanolamine [ 5 ]. from all of series of quat in this paper there is used n, n-dimethyl-nhexadecyl-n-2-oxyethlammonium bromide, which activity it`s possible to compare with the known quat as n, n, n-trimethyl-nhexadecyl bromide (hdtma) [ 4 ]. in other case n, n-dimethyl-n-hexadecyl-n-2oxyethlammonium bromide more thermal and basic stable than hdtma [ 5 ]. the quat used in the laboratory experiments was produced by treating 100 g of clinoptiololite with 300 ml of a 0,01m quat or amine solution in water and shaking 24 h. the modified zeolites was rinsed with purified water 2 times and made air-dried. the final n, n-dimethyl-n-hexadecyl-n-2oxyethlammonium bromide loading was 6 gr (15,0 mmol) on 100 gr zeolite and monoethanolamine loading was 6 gr (100 mmol) on 100 gr zeolite. the xrd patterns for sample with n, ndimethyl-n-hexadecyl-n-2-oxyethlammonium bromide show four bragg peaks which indicating the long range order of these material. sorption of btex, phenol and aniline by zeolites proceedes in the unbuffered solutions ( ph – 7,0 ). sorption isotherm were prepared for benzene, phenol, anilin using different zeolites. 1 gr air dried zeolite was put in the into glass with 10 ml of benzene, phenol, aniline solutions in water ( with the maximum solution in water for benzene, for phenol, aniline 0,1 n). the samples were shaken at 25oc ( room temperature) for 2 days a shaker table. after that was determined the rested amount of solute in water by refractometry, liquid chromatography and uv spectrometry analysis. liquid chromatography is passed on helch / higher-effective liquid chromatography, detector waters 486, controller warers 600s, pump, waters 626, colon 250x4mm, si-100 c 18, p 150 bar, v 1ml/m, mobile phase acetonitryl-water (50:50), detector uv-254). uv spectrometry is passed on uv-specord spectrometer. technological decision primary treatment: this treatment involves settling clarifier, there settleable organic materials settle out and are pumped away, while oil floats to the top and is skimmed off. tertiary treatment: after primary treatment the above small enterprises wastewater is subjected to tertiary treatment. this process food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 11 includes adsorption of organic pollutants on sorbents. for treatment used natural zeolites and them modified analogs have in the powder form at 15– 25 mm size putting in the form of a bed. sorbent ( zeolite ) restoration: for this purpose it has been passed methylation or ozonation of sorpted organic compounds / see experiment/. the other advantages of zeolites sieves is a catalytic activities in organic synthesis as catalysts. zeolites can be used us useful catalysts for many chemical conversion, for instance in alkylation & oxidation reactions. this possibilities make a opportunity for zeolites reduction and them application as regenerative adsorbents in wastewater treatment processes. ozonation of aromatic compounds containing wastewater ozonation destroys toxics in industrial wastewater by breaking down molecular structures into simpler components organic molecules or such as water and carbon. the process is based on the discovery that organics will oxidize in water at relatively low temperatures as long as oxygen is present and the proper operating pressure is maintained. the research indicates that aromatic compounds will be oxidized under these conditions. the processes in which ozone is involved basically two reaction pathways are of importance: 1. ozone molecules will directly oxidize the organic compounds with an initial addition step, 2. ozone dissociated by the reaction with hydroxyl and amino ions or hydroperoxyl anions the objective of this study is to investigate the natural zeolites to be used as adsorbent in the simultaneous adsorption and ozonation processes under sequencing batch reactor operation to treat wastewater containing phenol. ozonization of the organics adsorbed o n zeolite: for carrying out of experiments (diameter of 10 sm, height of 50 sm) loaded 25 ml of a mix into a reactor zeolite-water a solution of aniline after adsorption, further a mix barbotated an ozono-air mix. ozono-air mixed moved from the generator of ozone developed for biomedical appointments. the ozone maintenance in an initial mix made 500 mg/m3. for the purpose of revealing of the most active sorbent we study adsorption of organics from water solutions according to this technique. results & discussion zeolites expose simultaneously hydroxyl groups, coordinatively unsaturated cations, as lewis acid sites, and lewis basic sites, anions, as o-2 ions, on their surface. to retard the movement of organic species, materials with high affinity for organic molecules needed. it has been found recently that the surfactant modified (smz), a type of inexpensive cation exchanger, can fulfill this requirement [ 4 ]. the maximum loading of hexadecyltrimethylammonium (hdtma) salts ( with chloride, bromide or sulfate anion) can increase the sorption capacity of organic compounds with diminished the hydrophilic (water) substances sorption to the minimum. it`s known also, that the removal percentages for organobentonites to treat organic compounds from water were positively correlated to the length of alkyl chains of quats [6 ]. at our opinion to make some of efforts to have with long chains alkyl group the additional hydroxyl groups and lewis basic sites, anions, as o-2 ions, appears a possibilities to increase acid-basic properties of zeolites. one of the practice way for this problem decision is the modification of zeolites and its surface, so much the more with the organic compounds containing oh-groups as monoethanolamine and its derivatives. this study evaluated the use of monoethanolamine and n, n-dimethyl-nhexadecyl-n-2-oxyethlammonium bromide, as modified agents for zeolite surface. the objective were determined the sorptive capacity of modified zeolites for organic compounds sorption from water solutions which results of research are in a processing stage. here was investigated the removal of organic compounds – btex, aniline, phenol sorption process from waste water by means of food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 12 modified clinoptilolite with ethanolamine and them ammonuim salts. the analysis data for the clinoptilolite as sorbent for organic compounds from water system at a constant temperature were produced by uv spectrometry and liquid chromatography. on superficial groups of hydro group and atoms of oxygen of zeolites processes of formation of chemical bonds owing to molecular interactions with groups of hydroxyl / for phenol/ and amino groups / for aniline / proceed. here is specially investigated the aniline retain on zeolites. by the irspectroscopic method it is confirmed, that at zeolite modifying by monoethanolamine here is a considerable change of properties of its surface. ir-spectroscopic research shows in the 1400-3700 cm-1 region for clinoptilolite ( under treatments at 400oc) and aniline clinoptilolite samples after sorption equilibrium at room temperature and heating in vacuum. the free electron pair on nitrogen is less able to hold proton aniline is a weak organic base than aliphatic amines. aniline also an amphiprotic compound, it can react as proton acceptor ( in general ) and as donor. along with the valence and deformation with the specific frequencies belonging to the zeolite (3600, 3450, 1630 cm-1), in zeolite structure absorption peak, which can be carried to valence and deformation vibrations in oh and n-bonds monoethanolamine (3750, 2230, 2150, 1050 cm-1) are observed. the new absorption peaks testifying to formation of intermediate connections are found out also: 3600, 3050, 2710, 2600 sm-1. the absorption peak of 3600 sm-1 corresponds to energy of formation of hydrogen bond o … . the sorption of aniline on the modified sorbent of 3600 and 1050 sm-1 disappear, that testifies to occurrence instead of them additional coordination bonds between aniline and ohgroups. an absorption peaks of 3050 sm-1 be conditional also can it is carried to … bonds, but weaker. probably, therefore this connection is not realized with aniline, but it collapses at the subsequent regeneration of zeolite. occurrence of absorption peaks in a spectrum recycled after sorption proves of 1050 sm-1 to zeolite aniline, that, first, really, given connection has been realized in superficial intra-complex with of aniline. previously the sorption of aniline on zeolites, for instance, sorption on montmorillonite was investigated by israelien group of scientists [ 7 ]. they were identified various type of association between aniline and zeolite surface by ftir. aniline had been looked as selecting the better host for adsorption modes as a posterior in situ aniline polymerization. the aniline adsorption on to dehydrated nay zeolite was investigated also by ftir spectroscopy method [ 8 ]. the analysis data for the clinoptilolite as sorbent for organic compounds from water system at a constant temperature were produced by uv spectrometry and liquid chromatography. previously it was investigated the adsorptive activities of zeolites at removal of phenol from tetrachloromethan solution / cci4 / organic solvent which is more phenol on the geometrical sizes [ 3 ]. it has appeared that phenol removal it is better on zsm-5 modified by et4n+br. the max removal capability of phenol on zeolites is presented at the table 1. table 1. absorption of phenol from a cci4-solution on sorbents / 1 g phenol in 10 ml cci4 / temperature 20oc of measurements n/n sorbent the max removal capability g phenol/ 1g sorbent 1.* armenian natural zeolite – mordenite 0,16 2.* armenian natural zeolite – clinoptilolite 0,21 3.* clinoptilolit modified by salts of ba 0,27 4.* zsm-5 modified by et4n+br 1 0,31 5. clinoptilolite modified by monoethanolamine 0.30 6. clinoptilolite modified by n,n-dimethyl-nhexadecyl-n-2-oxyethy-lammonium bromide 0.32 *) zeolite from the family zsm-5 with hydrophobic characteristics from the reference [ 3 ] food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 13 the measurements were carried out for phenolic water solutions in concentration limits from 0,1 up to 0,45 mol/l. it was earlier established, that the sorption in these limits grows and has linear dependence on factor of refraction in correction with the results of uv spectrum [ 3 ]. it was determined quantity of sorpted phenol by the graphic dependence concentration-molar refraction. the amount of absorbed phenol is increasing in dependence of concentration of solutions. more active has appeared clinoptilolite modified by n,n-dimethyl-n-hexadecyl-n-2oxyethylammonium bromide. all experiments were performed at the room temperature. solutions were constantly stirred for one hours. after filtration quantity of adsorbed phenol was determined by uv-vis spectrometry. the amounts of adsorbted pollutants were calculated from the differences between the amount of pollutants added and that remaining in the final equilibrium solution.in the table 2 presented the results for phenol sorption from water solutions. table 2 the sorption of phenol from a water solution on sorbents / temperature 20oc, duration 4 hours / concentration of phenol in water solution nd 20 initial nd 20after sorption on clinoptilolite modified by monoethanolamine g phenol. on 1 g sorbent nd 20after sorption on clinoptilolite modified by n,ndimethyl-n-hexadecyl-n-2oxyethylammonium bromide / g phenol. on 1 g sorbent nd 20after sorption on h-clinoptilolite* / g phenol. on 1 g sorbent nd 20after sorption on zsm-5* g phenol. on 1 g sorbent 0,05 1,3324 full sorption full sorption full sorption full sorption 0,10 1,3328 full sorption full sorption 1,3314/0,075 full sorption 0,15 1,3342 1,3321/0,094 full sorption 1,3322/0,085 full sorption 0,20 1,3355 1,3320/0,107 1,3315/0,170 1,3329/0,103 1,3315/0,165 0,30 1,3371 1,3325/0,117 1,3317/0,247 1,3345/0,118 1,3318/0,245 *) from the reference [ 3 ] table 3 the sorption of aniline from a water solution on sorbents / an aniline concentration 0,01m, temperature 20oc,/** n natural supporters nd 20 after sorption time, hours aniline sorption, % 1. h-mordenite* 1,3291 4 75 2. h-clinoptilolite* 1,3289 4 100 3. clinoptilolite* 1,3290 4 98 4. clinoptilolite modified by monoethanolamine 1.3289 3 100 5. clinoptilolite modified by n, ndimethyl-n-hexadecyl-n-2oxyethlammonium bromide 1.3289 2 100 *) from the reference [ 3 ] **) nd 20 for initial aniline solution is 1,3300, for water nd 20 is 1,3289 food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 14 the theoretical analysis of various types of isotherms of adsorption allows to receive a lot of helpful information on the adsorption mechanism. adsorptive isotherms received of aniline adsorption from water solutions on zeolite it is possible to classify as type l3 [ 7 ]. this testifies that in this case influence of adsorption of solvent is expressed not brightly. the type of an isotherm of adsorption confirms that fact, that adsorption proceeds in a monolayer. on the basis of the received data re calculated adsorption isotherms in co-ordinates of the equations of freundlich and langmuir. the deduced constants of the equations of the analysis of sorptived parameters, on calculated by equations of freundlich and langmuir has shown, that freundlich's equation describes character of isotherms aniline sorption on zeolites is better. it proves to be true coincidence of experimental and settlement data (table 5.), that is not observed at application of langmuir equation. the obtained data confirm that freundlich's equation is applied for porous adsorbents. table 4 freundlich and langmuir parameters sorbent freundlich`s equation langmuir`s equation (calculated) (experimental) k clinoptilolite modified by n, n-dimethyl-nhexadecyl-n-2oxyethlammonium bromide 0.60 0.5 2.2 0.045 500 h-clinoptilolite 0.38 0.45 0.31 0.017 556 fig.1 the adsorption istherm clinoptilolite modified by n, n-dimethyl-n-hexadecyl-n-2oxyethlammonium bromide / rhombic symbol / and h-clinoptilolite / square symbol / 0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04 0,045 0,05 0 0,002 0,004 0,006 0,008 0,01 0,012 a g/g mol/l food and environment safety journal of faculty of food engineering, tefan cel mare university – suceava volume x, issue 3 2011 15 higher sorptive activity appears for the clinoptilolite modified by n, n-dimethyl-nhexadecyl-n-2-oxyethlammonium bromide. it can explained that large cationic surfactant molecules have a strong affinity for zeolite surface and replace positively charged inorganic counterions that neutralize the negative surface charge of the zeolite.the surfactant molecules impart hydrophobic properties to the zeolite surface organic compounds [ 7 ] is proportional to a specific surface hydroxyl group. here is established, that sorption of aromatic compounds from solutions on the modified zeolites by monoethanolamine and n, n-dimethyl-nhexadecyl-n-2-oxyethlammonium bromide occurs by means of formation of superficial connections with modifiers: in the beginning the modifier is adsorpted on a surface of grains of the natural not modified zeolite covered with groups sioh, . then monoethanolamine co-operate with a structural lattice of zeolites, being fixed on a zeolite surface. then n, n-dimethyl-n-hexadecyl-n2-oxyethlammonium bromine co-operate with a structural lattice of zeolites, being fixed on a zeolite surface. during the sorption of aniline, phenol on modified zeolite appears, that oh-group of ethanolamine in zeolites forms a bonds with amino or hydroxyl groups of adsorptives. the kinetic researches of the sorption processes were spent basically on modeling solutions with initial concentration of aromatic connections of 0.01 mol/l. always we have full sorption for aromatic compounds. conclusion it has been found advantageous to go on with the researches in organic pollutants – btex, phenols, aniline make up a significant percentage of petroleum products sorption by armenian natural zeolites and them modified analogs. in particular, such sorbents can be used in the poor countries for sewage treatment from small enterprises. it is experimentally established, that sorption of organic compounds by the natural zeolites modified with n,n-dimethyl-n-hexadecyl-n2-oxyethylammonium bromide higher effectual, it is satisfactory described by friendlich's known equation. references: 1. donald w.breck zeolite molecular sieves: structure, chemistry & use. a wileyinterscience publication, john wiley & sons, new york, london, sydney, toronta, (1974) p.782. 2. c.collela, porous materials in environmentally friendly processes, in studies in surface science and catalysis, 1999, vol. 125, p.641655,. 3. g.torosyan, d.hovhannisyan, s.shahinyan, h.sargsyan armenian zeolites & its possibilities in industrial & municipal waste water cleaning. ecological journal of armenia, 2002, vol 1, no 1, pp.93-96. 4. karapanagioti, h.k., d.a. sabatini, and r.s. bowman. partitioning of hydrophobic organic chemicals (hoc) into anionic and cationic surfactant-modified sorbents. water res. 2005, .39:699-709. 5. nazaretyan a.kh., prazyan anna s., prazyan anush s., torosyan g.h., synthesis of quaternary ammonium salts, bulletin of seua, collection of scientific & metodical papers, 2009p. , volum 1,123-128. 6. lizhoung zhu, yimin li, jianying zhang, sorption of organobentonites to some organic pollutants in water, environ.sci.technology, 1997, 31 (5), 14071410,. 7. yarif s., heller l., kaufherr n, effect of acidity in motmorillonite inter layers on sorption of aniline derivatives. clays and clay minerals, 1969, 17, 301, 8. marcos .b.gomez costa, oscar a.anunziata, aniline adsorption onto na-almcm-41 and nay zeolite as host materials, enpromer, 2th mercosur congress on chemical engineering, 4th mercorus congress on process systems engineering, p.1-10, 2005, villagerio das pedras, club med, rio de janiero august 2005, 14-18,. microsoft word 5 ciobanu articol 1 corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 30 the influence of taphrina deformans (berkeley) tulasne (peach leaf curl) attack on the activity of some oxidoreductases in cultivar cardinal *rodica ciobanu1 1alexandru ioan cuza university, faculty of biology, iaşi, romania ciobanu.rodica1981@yahoo.com *corresponding author received 18 october 2012, accepted 10 december 2012 abstract: taphrina deformans (berk.) tul. is the causal agent of peach leaf curl, a fungal disease which in present is widespreaded in all places where peaches and other related species are cultivated. in this paper are presented the result concerning the influence of the attack of fungus taphrina deformans on the activity of catalase (ec 1.11.1.6), peroxidase (ec 1.11.1.7) and polyphenoloxidase (ec 1.10.3.1) compared with the activity of this oxidative enzymes in healthy leaves at cultivar cardinal. the determination of enzymes activity were carried out at 4.05.2009, 18.05.2009, 25.05.2009, 1.06.2009 and 8.06.2009. catalase activity was higher in diseased leaves at the beginning of the infection and decreased with the evolution of disease simptoms. peroxidase and polyphenoloxidase activities were found higher in diseased leaves when compared with enzymes activity from healthy leaves, at all dates of the determination. polyphenoloxidase dicreased with leaves age, but remained considerably grater in infected tissues. this study suggest that the accumulation of reactive oxygen species in peach tissues, as a response at fungus attack, increased activities of catalase, peroxidase and polyphenoloxidase, this enzymes beeing involved in host defence mechanisms. keywords: catalase, peroxidase, polyphenoloxidase, persica vulgaris, diseased leaves, healthy leaves 1. introduction taphrina deformans (berk.) tul. is a parasitic fungus that causes the disease named „peach leaf curl” and it affects peaches (persica vulgaris mill.) but also almonds, apricots and necatarines. the disease was observed for the firt time in england in 1821 [1]. in romania, the presence of taphrina deformans was mentionated for the first time in 1929 by traian săvulescu [2]. in present this disease is widely distributed in all the countries where peaches are cultivated. peach leaf curl affects especially leaves but it also can affect sprouts, twigs and rarely flowers and fruits. the leaves infected by taphrina deformans became red, thickened and curled as they develop and eventually become brown and fall prematurely. the disease occurrs in springtime when the temperature is oscillateing between 15°c and 20°c (optimum of 18 °c) and the weather is wet in late february until early march [3]. the attack of taphrina deformans, if untreated, can affect the quality and quantity of the crop and lead to the death of trees in a few years. cardinal is a peach cultivar (cv.) obtained in 1941 at fort valley, georgia (sua) by seflpollination of cv. halehaven. in our contry this peach cultivar was introduced in 1962; it’s a cultivar with an average vigour and hight resistance at frost and drought but susceptible at taphrina deformans and other peach pathogens such food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 31 as fusicoccus amygdali and pseudomonas mors-prunorum [4]. 2. materials and methods the material used in this study was represented by fresh healthy leaves (as control) and leaves infected by fungus taphrina deformans collected from cv. cardinal cultivated in didactic orchard „vasile adamachi” iaşi. the vegetal material was collected starting to the beginning of may until early june in 2009. the determination of enzymes activity was carried out in the day of collecting. catalase activity was estimated using the iodometric method with sodium thiosulfate; this method is measureing the cantity of hydrogen peroxide remained after the intrreruption of catalase activity on it [5]. orto-dianisidine method was used for estimateing of peroxidase activity; the method is measureing the optic density of ortodianisidine (as hydrogen donor) oxidation product with the participation of peroxide under the action of peroxidase [5]. the results were expressed in peroxidase units (up) and it represent the quantity of peroxidase which decompose 1µmol of peroxide in one minute in optimum conditions. determination of polyphenoloxidase activity, a copper containing enzyme, was performed using the method with pyrocatechol described by ermakov quoted by roşu crăiţa maria [6]. the results were expresses in polyphenoloxidase units (u ppo). polyphenoloxidase is a ubiquitous enzyme involved in the oxidation of endogenous phenols and it is responsible for browning in plants. 3. results and discussion in fig. 1 are presented the results concerning catalase activity in healthy leaves and in leaves infected by taphrina deformans. in healthy leaves catalase activity registered the highest value 218,1818 u c/g/min at 25.05.2009, followed in decreasing order by the next values: 130,3167 u c/g/min (1.06.2009), 112,0043 u c/g/min (8.06.2009), 76,5957 u c/g/min (4.05.2009) and 71,3286 u c/g/min (18.05.2009). in the leaves attacked by taphrina deformans catalase activity recorded the highest value -171,4285 u c/g/min at 25.05.2009 and it was followed in decreasing order by the values registered at: 18.05.2009 (142,2924 u c/g/min), 4.05.2009 (120,9302 u c/g/min), 1.06.2009 (119,502 u c/g/min), 8.06.2009 (107,8632 u c/g/min). catalase activity recorded the maximum value in healthy and diseased leaves at 25.05.2009. from analyseing the dynamics of catalase activity at cv. cardinal it has been found that catalase is increasing its enzymatic activity in diseased leaves at the beginning of the fungal infection 4.05.2009 (diseased/healthy = 1,6953), 18.05.2009 (d/h= 1,8577)but with the evolution of the disease this oxidoreductase is recording a decreasing activity in diseased leaves when compared with the activity from healthy leaves: 25.05.2009 (d/h=0,7857), 1.06.2009 (d/h=0,9170), 8.06.2009 (d/h=0,9630). at the appearance of the first simptoms of disease, catalase activity is considerably higher in the leaves infected with taphrina deformans comparatively with the enzymatic activity from the healthy leaves, this situation can be explain by the role of catalase which is well known that it occurs in regulateing the hydrogen peroxide content resulted from intense methabolic proceses and to stop the toxic effect of this product upon the plant tissues [7]; as the infection progress, and peach leaves are aging, catalase activity decrease in diseased leaves, the situation can be correlate with the intensification of peroxidase activity observed in the diseased tissues of cv. cardinal. at the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 32 beginning of fungus attack, catalase is more active in diseased leaves due to the higher amount of hydrogen peroxide, then the enzyme remains without its specific oxidation substrate because of the peroxidase activation which can be active at a smaller amount of hydrogen peroxide. from the catalase reaction we can say that it is involved, beside other enzymes, in the host defence mechanismes agains the pathogen. the results obtain at cv. cardinal in 2009 are similar with those observed by roşu crăiţa maria in the case of foliar attack of cercospora beticola at sugar beet [6]; the same dynamics in catalase activity was observed and by elźbieta kużniak et maria skłodowska at foliar infection of lycopersicon esculentum with botrytis cinerea [8]. similar result concerning the dynamics of catalase and peroxidase activity as in the present experimental study were mentionat also by arun et al. at pennisetum glaucum plants infected with sclerospora gaminicola [9], by mahmoud et al. in the case of the attack of botrytis fabae at vicia faba [10] and by garcia-limones et al. who observed an intensification of catalase and other oxidative enzymes in the case of the attack of fusarium oxysporum f. sp. ciceris produced at cicer arietinum [11]. 0 50 100 150 200 250 300 u c /g /m in 4.05.2009 18.05.2008 25.05.2009 1.06.2009 8.06.2009 healthy leaves diseased leaves figure 1 catalase activity in healthy leaves and in leaves infected by taphrina deformans at cv. cardinal the result concerning peroxidase activity in healthy leaves and in leaves infected by taphrina deformans, are presented in fig. 2, from which it can be observed that in healthy leaves the highest value of peroxidase activity -1,0024 up/g min was recorded at 25.05.2009, followed in decreasing order by next values: 0,965 up/g min (4.05.2009), 0,8487 up/g min (1.06.2009), 0,8125 up/g min (18.05.2009) şi 0,6765 up/g min (8.06.2009). in diseased leaves peroxidase activity had the highest value-1,44419 up/g min at 8.06.2009, followed in decreasing order by the next values: 1,3704 up/g min (1.06.2009), 1,088 up/g min (18.05.2009) and a minimum value 0,5517 up/g min was recorded at 4.05.2009. the d/h (diseased/healthy) ratio of peroxidase activity is subunit at the beginning of the attack 4.05.20090,5717 and with the intensification of the attack it take place a intensification of enzymatic activity in diseased leaves when compared with the activity recorded in healthy leaves, the ratio d/h had values higher then the control (healty leaves): d/h=1,339 at 18.05.2009; d/h=1,1002 (25.05.2009), d/h= 1,6147 (1.06.2009) and d/h= 2,1314 (8.06.2009). this increasing of peroxidase activity at one time with the intensification of the fungus attack demonstrates the rol played by this enzyme in the defensive mechanisms of peach against oxidative stress caused by taphrina deformans attack. in 2009 at cv. cardinal it take place an intensification of the peroxidase activity at one time with the evolution of taphrina deformans infection in compare with the activity from healthy leaves; this intensification of the activity in diseased leaves take place because peroxidase is involved together with catalase in reduction of harmful effect of reactive oxygen species resulted as an effect of increasing of metabolic processes due to fungal infection, but also to peroxidase capacity to reduce small amounts of hydrogen peroxide from plant tissues, because peroxidase can act only in this food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 33 special conditions; the results obtained in this experiment can be compared with those observed in the case of wheat infected by different pathogenic fungi by johnson et cunningham and flott et al. in the case of puccinia recondita attack [12, 13], by diani et al. in infection with septoria tritici [14], by patykowsky et al. in erysiphe graminis attack [15], by kerby et somerville at barley infected with erysiphe graminis f.sp. horedi [16]; the same dynamic of peroxidase activity was observed by grzelinska et sierakowska at tomatoes plants infected with verticiliium albo-atrum [17] and by reuveni et ferreira who observed an increasing in peroxidase activity from tomatoes leaves infected with verticillium dahliae [18]; by reuveni et bothma at cucumis melo infected by sphaerotheca fuliginea [19], catalase and peroxidase are two defensive enzymes which act synergistically and degradate the hydrogen peroxide to help to protect plant tissues against its toxic effect. 0.0 0.5 1.0 1.5 2.0 2.5 u p / g m in . 4.05.2009 18.05.2008 25.05.2009 1.06.2009 8.06.2009 healthy leaves diseased leaves figure 2 peroxidase activity in healthy leaves and in leaves infected by taphrina deformans at cv. cardinal in fig. 3 are presented the results of the researches concerning the polyphenoloxidase activity in healthy and diseased leaves at cv. cardinal. the maximum polyphenoloxidase activity in healthy leaves, occurred at 18.05.2009 (4,2551 u ppo/g/min), followed in decreasing order by the values recorded at: 1.06.2009 (3,6176 u ppo/g/min ), 8.06.2009 (3,4072 u ppo/g/min), 4.05.2009 (2,9285 u ppo/g/min) and at 25.05.2009 was recorded the smallest value2,7961 u ppo/g/min. polyphenoloxidase activity from diseased leaves recorded the highest value at 4.05.2009 9,1780 u ppo/g/min, followed in decreasing order by next values: 5,2439 u ppo/g/min (18.05.2009), 3,7319 u ppo/g/min (25.05.2009), 3,7313 u ppo/g/min (1.06.2009) and the smallest value3,6949 u ppo/g/min was recorded at 8.06.2009. the dynamics of polyphenoloxidase activity recorded in diseased leaves at cv. cardinal, in 2009, showed higher values compared with those observed in healthy leaves, the ratio diseased/healthy had the following values: d/h=3,1339 (4.05.2008), d/h=1,2323 (18.05.2009), d/h=1,3346 (25.05.2009), d/h=1,0314 (1.06.2009) and d/h=1,0844 at 8.06.2009. polyphenoloxidase activity was higher in diseased leaves at all periods of the study when compared with the activity recorded in healthy leaves. the higher activity of this oxidoreductare in diseased peach leaves is probably due to its role in the oxidation of phenolic compunds (which are toxic for the fungi) to quinones which are more reactive and more toxic to the pathogen. by increasing its polyphenoloxidase activity the host plant attempts to limit or to stop the pathogen penetration. the increased activity of polyphenoloxidase is accompanied at cv. cardinal by an increased activity of peroxidase, this indicates the role of this two enzymes in disease resistance of peaches. in diseased leaves polyphenoloxidase activity is decreasing with the leaves age; in healthy leaves the enzyme activity recorded specific variation to one date to another and remains lower then the activity from attacked tissues. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 34 the results obtained in this study are confirmed by those mentionated by elfiki et al. in the case of sugar beet leaves infected by uromyces betae and they point out the role played by catalase, peroxidase and polyphenoloxidase which are implicated in defensive mechanisms of host plant against the fungal pathogen [20]; the results are similar with those also observed by tyagi et al. at some wheat varieties with different degrees of susceptibility at the attack of alternaria triticina [21]; increasing activities of polyphenoloxidase and peroxidase in response of the attack of alternaria tenuis on rosa centifolia leaves were observed by khatun et al. (2009) [22]; arun et al. observed the same dynamics of this enzymes activity at pennisetum glaucum plants infected by fungus sclerospora graminicola [9]. 0 2 4 6 8 10 u p p o /g /m in 4.05.2009 18.05.2009 22.05.2009 1.06.2009 8.06.2009 healthy leaves diseased leaves figure 3 polyphenoloxidase activity in healthy leaves and in leaves infected by taphrina deformans at cv. cardinal 4. conclusion the results recorded in this study showed that catalase, peroxidase and plyphenoloxidase activity was influenced by wheather contition, by the presence or the absence of taphrina deformans attack and by the stage of infection. peroxidase activity was higher in diseased leaves then in the healthy ones; the researches indicated that catalase and peroxidase act synergistically to help to protect peach leaves against taphrina deformans infection. results revealed that in cv. cardinal polyphenoloxidase activity was higher in diseased leaves in all study periods; polyphenoloxidase activity in diseased leaves is decreasing at one time with the intensification of the attack of the pathogenic agent, probably due to the leaves ageing. this study confirmed that catalase, peroxidase and polyphenoloxidase work together to protect plant tissues against oxidative stress induced by the attack of taphrina deformans on persica vulgaris leaves. 5. references [1]. eftimiu panca, contribution to cytological study of exoascaceae (contribution á l’étude cytologique des exoascées), le botaniste 18. 1-154, (1927) [2]. săvulescu tr., the phytosanitary situation in romania in the years 1929-1930, anal. icar, iii, 3-16-17, (1931) [3]. agarwala r. k., arora k.n., singh a., effect of temperature and humidity variation on the development of peach leaf curl in mid hills and its control, indian phytopath., 19. 308-309, (1966) [4]. ivascu antonia, hoza d., peach varieties catalog (catalogul soiurilor de piersic), medro, .31p, (2003) [5]. cojocaru d. c., practical enzymology (enzimologie practică), tehnopress, iaşi, (2005) [6]. roşu crăiţa-maria, biochemical and physiological mechanisms of sugar beet’s resistance to mycoses (mecanisme biochimice şi fiziologice ale rezistenţei sfeclei pentru zahăr la micoze), tehnopress, iaşi, (2007) [7]. bolwell gp, wojtaszek p., mechanisms for the generation of reactive oxygen species in plant defence-a broad perspective, physiol. mol. plant pathol. 55. 347-366, (1997) [8]. kużniak elźbieta, skłodowska maria, fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants, planta, 222 (1). 192-200, (2005) [9]. arun k., mali p.c., manga v.k., changes of some phenolic compounds and enzyme activities on infected pearl millet caused by sclerospora graminicola, international journal of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 35 plant physiology and biochemistry, 2 (1). 6-10, (2010) [10]. mahmoud yehia a.g., soad m. abu el souod, saeid alsokari, abd-elwahb ismaeil, magid attia, mohsen k. ebrahim, recent approaches for controlling brown spot disease of faba bean in egypt, acad. j. biolog. sci., 3(1): 4153, (2011) [11]. garcia-limones carmen, hervas ana, navas-cortes j. a., jimenez-diaz r. m., tena m., induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (cicer arietinum l.) and fusarium oxysporum f. sp.ciceris, physiological and molecular plant pathology, 61(6). 325-337, (2003) [12]. johnson l.b., cunningham b.a., peroxidase activity in healthy and leaf-rust infected wheat leaves, phytochemistry, 11. 547-551, (1972) [13]. flott, b.e., moerschbacher b.m., reisner h., peroxidase isoenzymes patterns of resistant and susceptible wheat leaves following stem rust infection, new phytol., 111. 413–421, (1989) [14]. diani z., ouarraqi e.m., aissam s., hsissou d., modafar c.e., induction of early oxidative events in soft wheat leaves inoculated with septoria tritici and their relationship to resistance of maroccan cultivars, int. j. agric. biol, 11(4). 351-359, (2009) [15]. patykowski j., urabanek h., kaczorowska t., peroxidase activity in leaves of wheat cultivars differing in resistance to erysiphe graminis dc., j. phytopathol., 122. 126– 134, (1988) [16]. kerby k., somerville s., enhancement of specific intercellular peroxidase following inoculation of barley with erysiphe graminis f.sp. hordei, mol. plant. pathol., 35. 323–337, (1989) [17]. grzelinska a., sierakowska j., effect of infection with verticillium albo-atrum and agents interacting with dn a on the activity of peroxidase and phenylalanine ammonia lyase, phytopath. z., 84. 271-298, (1975) [18]. reuveni r., ferreira j. f., the relationship between peroxidase activity and the resistance of tomatoes (lycopersicum esculentum) to verticillium dahliae, journal of phytopathology, 112 (3). 193–197, (1985) [19]. reuveni r., bothma g. c., the relationship between peroxidase activity and resistance to sphaerotheca fuliginea in melons, journal of phytopathology, 114 (3). 260-267, (1985) [20]. el-fiki, a.i.i., mohamed, f.g., elmansoub, m. m.a., studied on sugar beet rust disease in egypt: disease control and some vital activities of infected sugar beet plant grown under greenhouse conditions, eleventh congress of phytopathology, giza, egypt, 25-39, (2007) [21]. tyagi m., kayastha m.a., sinha b., the role of peroxidase and polyphenol oxidase isozymes in wheat resistance to alternaria triticina, biologia plantarum, 43 (4). 559-562, (2000) [22]. khatun s., bandyopadhyay p.k., chatterjee n.c., phenols with their oxidizing enzymes in defence against black spot of rose (rosa centifolia), asian j. exp. sci., 23 (1). 249252, (2009) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 54 life cycle assessment new direction in addressing systemic processes in food industry *viorel iftimi1 1s.c. iso project suceava e-mail viorel_iftimi@yahoo.com *corresponding author received 22 january 2011, accepted 8 may 2011 abstract: the aim of this paper is to present family standards sr en iso 14040 life cycle assessment as a new approach to food industry processes from design and production to display on shelves and removal / disposal of waste (including packaging). these standards introduce the concept of “system – product” and “cradle-to-grave” to the study of environmental aspects and potential impacts of the product life cycle. the first part of the article contains an overview of lca standards requirements. i presented the five standards that deal with this problem including their principles and some explanatory diagrams. the five standards are sr en iso 14040 principles and framework, sr en iso 14041 define the purpose, scope and inventory analysis, sr en iso 14042 life cycle impact assessment, sr en iso 14043 life cycle interpretation, sr iso-ts 14048 format of the data documentation. their name contains the phrase “environmental management, life cycle assessment”, focusing on environmental standards. the second part presents some practical ways to apply the lca standards in food industry. the implications of implementing these standards may bring about the risk for small firms in the food industry to be blocked by brokers and lose their identity. © 2011 university publishing house of suceava. all rights reserved key words: system – product, cradle-to-grave, environmental management 1. introduction lca life cycle assessment is a relatively new standard governed by the terms sr en iso 14040 environmental management life cycle assessment principles and framework. the approach starts from the environmental management standards and aims to interpret and evaluate inputs, outputs and potential environmental impacts of a “system-product” throughout its life cycle. if all other iso standards concerned with the system and processes of a firm that produces a product / service, analyze the new standards until the entire system from design to disposal / recycling of waste, with all related issues “cradle-to-grave” approach. key features of an lca are: systematically and adequately addresses the environmental aspects of systems-product, from raw material acquisition to post-use; -degree of detail and deployment time of a lca study may vary in a wide range; -scope, assumptions, description of data quality, methodologies and results are transparent lca studies; -provision is determined depending on the intended application of lca study, to comply with privacy and ownership issues; -lca-methodology is open to embrace new scientific discoveries and improvements in technology – bat; -lca is used to make comparative statements further presented to the public; -commercial activities are complex because there is no scientific basis for reducing lca results to a single overall score or number; -there is no single method for conducting lca studies but organizations should have flexibility to implement lca practice as stipulated in this international standard. phases of a lca study are presented in figure 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 55 figure 1 phases of lca scope of the study aims according to standard terms: -system-product-functions or, for comparative studies, the eia systems; -functional unit; -system-product that is studied; -product system boundaries; -allocation procedures; -type of impact and methodology for impact assessment and subsequent interpretation will be used; -data requirements; -assumptions; -limitations; -requirements relating to quality of original data; -type of critical analysis, if any; -the type and format of the report called for the study. reporting the results of lca must be impartial and complete. objective reporting is made to a third party; the requirements of the standard should cover at least the following aspects: a) general aspects; b) defining the purpose and scope; c) life cycle inventory analysis; d) life cycle impact assessment; e) life cycle interpretation; f) critical analysis. critical analysis is essential and it can be made by an independent internal expert lca study or by an external expert analysis that will give an analysis statement. sr en iso 14041 standard definition of the purpose, scope and inventory analysis establishes requirements and procedures for setting up and training the purpose and scope definition for a life cycle assessment (lca) and for the development, interpretation and reporting cycle inventory analysis life (icv). the product-system is detailed here and the final product is indicated next to intermediates, auxiliary inputs, uncontrolled emissions, data quality, sensitivity analysis, uncertainty analysis, etc. for life cycle inventory analysis, the “systemproduct” which is composed of “units of product” must be defined more clearly. it is essential that function, functional unit and reference flow to be clearly defined and to determine the boundaries of the original system. inventory analysis is made according to the logical scheme of figure 2. figure 2simplified procedures for the analysis of inventory 2. experimental the standard model detailed report of the study. in the annexe, examples of data collection sheets can be found. sr en iso 14042 life cycle impact assessment constitutes the third stage of the process lca and define the purpose and scope inventory analysis impact assessment inter preta tion direct applications: -improving product development -strategic planning -process of making public policy -marketing other define the purpose and scope plugs revised data collection analysis data collection sheets preparing data collection data collection data collected data validation date validated data association process unit validated data process unit reporting of the functional unit date validated functional unit data aggregation calculated inventory review of system limits allocation and recycling additional data or process units required final inventory food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 56 is to assess the life cycle inventory analysis results of a “system-product” to better understand their environmental significance it prepares the stage for life-cycle interpretation. the data obtained are used: -to identify opportunities to improve product and system-to help determine their priorities; -to characterize or evaluate the performance (benchmark) of a system-produced and its units in the long run; -to make relative comparisons between systemsbased product category indicators selected; to indicate environmental problems for which other techniques can provide complementary environmental data and information useful for decision makers. lcia phase elements are illustrated in figure 3. figure 3 elements of lcia phase sr en iso 14043 2002 lca life cycle interpretation provide requirements and recommendations for conducting life cycle interpretation in lca and lci studies. identification and structuring of information are suggested in figure 4. figure 4 relations between elements of the interpretation phase of lca and other phases the standard annexes find several examples of forms for the interpretation of lca. iso-ts 14048 data documentation format provide a framework and clear documentation of data requirements for life cycle inventory analysis (lci). data documenting a process is illustrated in figure 5. figure 5general knowledge about the data documentation format must haves selection of impact categories, category indicators and models to characterize distribution of lcia results (classification) calculation of category indicators results (characterization) category indicators results (lcia profile) optional elements class size calculation results indicators compared with reference information (normalization) group pond analyzes data weighting the life cycle assessment define the purpose and scope inventory analysis impact assessmen interpretation phase 1. identify worse problems 2. evaluation by: -check the completeness -sensitivity verification -checking inconsequence -oher tests 3. conclusions, recommendations and tests 3. apply direct -product development -improvement of strategic planning, -marketing, -public policy-making -other documentation of process data process modeling and validation administrative information description of the process elements of input and output elements food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 57 standard forms offer models that describe processes, input-output elements, modelling and validation, etc. being explained in the annexes. 3. results and discussion in practice, this new approach to the food industry is extremely important. for the first time we see that a company that manufactures, for example traditional products cheese, cooked pork, smoked trout, etc. to be integrated into a “system – product” including product packaging, storage, demonstrating a raw material traceability (cows, pigs, fish tank, etc.) with food, medical tests, etc. needs to have control of the transport machinery (accreditation, washing, disinfection, maintenance, ...), intermediaries and wholesale deposits democrats, shelf presentation, labelling, etc. [1] packages involve chemical triple laminated polyethylene film, low permeability to co2 aluminium foil, cans technologies, insulating varnishes, technological lines for manufacturing stainless steel, etc. [2] [3] [4] all they bring their own carbon footprint and loads of pollutants with cumulative lca studies. [5] [6] so we see when it comes to a chain of companies – each one with its own management systems implemented and their own lca studies, that there is a possibility and opportunity to join a system that produces something marketable. if we implement these standards management system in that company, there will have its own carbon footprint, tables and other pollutants, and will be able to access a viable globalized system. this is the only chance. [7] [8] if the manufacturing company can not provide this lca study, it will not have access to supermarkets. although the product is good looking and with a price planes. [9] [10] then, an intermediate properly packed product will emerge, organize and monitor the labeling, transport, storage, marketing, distribution, sale, and keep most gain with low price to the vendor. the next step will be to require amounts of increasingly large and globalized market to meet increasing profits. small traditional producers are unable to finance an increase in capacity implies: an average production line automation with an increase in raw material supply base (cows, pigs, fish), that would increase demand for feed (by increasing the number of farms in the area), etc. they are attracted by the appearance of the equipment maintenance companies, sales of agricultural machinery factories, production recovery profiles, steel and mining industry refreshing transport or ship or train. [11] this means jobs, development, living standards but also structuring and strengthening the brand country. as these approaches have not solved out all these problems in appealing to the best suppliers more environmentally friendly, from china ,germany, poland, etc. this is due to globalization. taking control of all traditional product information broker will record it. [12] to implement lca in companies in bukovina and romania, we offer a soft ware application that contains all the forms required by the standards of sr en iso 14040. i also created a framework for promoting these concepts and supporting information in an ngo bukovinean society for management j.m. juran. we can provide a lca certification of management systems from a regional certification body regarding the access to this organized framework based on local expertise in the implementation of lca . in this way we can impose on the european market its own certification. 4. conclusion integration of companies from various parts of the world in production and distribution of ordinary products (salad, fruit, flowers, etc..) is a globalized phenomenon. of course, transportation is no longer a problem, technology on a bed of closedcircuit water, organic loading is widely controlled. disputes of gm food technology have social implications in employment in overcrowded areas. to gain access to these disputes the only way is to implement lca management systems. so we have the technical data and even the possibility of participating in the dialogue provided by the policy in the field. . food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 58 5. abbreviations ims integrated management systems lca life cycle assessment sr romanian standard en european norm iso – international standardization organization icv cycle inventory analysis life 6. references 1. a.m. erg, m. johansson, g. finnveden, a.jonsson screening environmental life cycle assessment of printed, web based and tablet e-paper newspaper 2007 2. y. kobayashi, h. kobayashi, a. hongu, k. sanehira a practical method for quantifying eco-efficiency using eco-design supporttools 3. commission du codex alimentarius codex alimentarius etiquetage des denrées alimentairestextes complets programme mixte fao/oms sur les normes alimentaires 4. etiquetage des denrees direction générale de la concurrence, de la consommation et de la répression des fraudes -http://www.minefe.gouv.fr/directions_ services/dgccrf/documentation/fiches_pratiques/fiches/ etiquetage_alimentaire.htm 5. fridge: socio-economic impact assessment of the proposed plastic bag regulations life cycle assessment of paper and plastic checkout carrier bags 6. guidelines for social life cycle assessment of products belgian federal public planning service sustainable development copyright © united nations environment programme, 2009 7. s. joshi, j. madison product environmental life-cycle assessment using input-output techniques college michigan state university east lansing, mi usa 8. r. p. henson, a. b. culaba, g. a. mendoza evaluating environmental performance of pulp and paper manufacturing using the analytic hierarchy process and life-cycle assessment 9. opinia comisiei ştiinţifice asupra aditivilor alimentari, aromelor, materialelor şi accesoriilor utilizate în prelucrarea alimentelor (afc) recomandări pentru întocmirea unui dosar pentru evaluarea de către efsa a siguranţei procesului de reciclare în producţia de materiale plastice reciclate utilizate în materiale şi obiecte ce vin în contact cu produsele alimentare, jurnalul efsa (2008) 717 10. guidelines for social life cycle assessment of products belgian federal public planning service sustainable development copyright © united nations environment programme, 2009 11. k.a. barnes, c. r. sinclair, d.h. watson chemical migration and food contact materials – crc press boca raton boston new york washington, dc woodhead publishing limited cambridge, england 12. y.kobayashi, h. kobayashi, a. hongu, k. sanehira a practical method for quantifying eco-efficiency using eco-design support tools http://mitpress.mit.edu/jie 2005 by the massachusetts institute of technology and yale university food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 53 eco log ica l aspec ts o f slud ge dis posa l in sl ovak ia *ivana mahríková1 1slovak university of technology, faculty of civil engineering, ivana.mahrikova@stuba.sk *radlinského 11, 81368 bratislava, slovakia abstract: there are monitored “ great boom” by building of new sewage systems in slovakia in last years, especially in areas, where was low connection to the public sewage systems. there is coming great amount of finances for building a new sewage system in slovakia. but at first we have to find the general idea how to build this system with the view of technical and economical relations. quantitative production of sludge from urban waste water treatment as well as the issue of their contamination has been continuously monitored in slovakia since 1998. basic principles, together with the monitoring, have been derived from the concepts of management of sludge from municipal wastewater treatment plants. controlled land application of sludge was stipulated as the principal method of handling sludge. this process has not only been selected for being relatively the cheapest way for the final disposal of sludge, but is assumed to be the most acceptable option in the slovak conditions, as well. keywords: sludge treatment, sludge disposal, environment versus sludge, legislation in eu and slovakia, sewage sludge applications. sludge definition and characteristics. sludge is a disposal from urban waste water treatment. we define is as a disperse system, which consists from the suspended, colloid matters. the main part creates suspended matters with characteristics concentration 5-50 g.l-1. the sludge consistence is liquid till greasy. sludge creates from 1 till 2% of waste water volume and contains 50-80% of primary pollution. the sludge as waste product from various steps of waste water treatment is non stabile, it is the reason of the next sludge treatment, before it´s using as a lateral stuff. one of the most important sludge character is hygienic aspect. waste water treatment sludge is a waste product, which comes into being in the process of waste water treatment and so underlies under waste low. this act allowed to be each stuff, which has at least one dangerous property to the dangerous waste. the producer of this waste is obligatory disposal the sludge as a dangerous waste. lot of sludge produced on the wwtp has one dangerous characteristic. it is infectivity. infectivity is caused by pathogen microorganism. their amount depends from the geographic, climate and demographic factors. the main source of pathogen microorganism is excrement of ill people or animals. majority of these microorganisms is disposal by the treating process, but without perfect sludge hygienic inspection, the small amount stays still in the sludge. by the using of sludge in agriculture can expose these pathogenic germs healthy of living organisms. by the pathogenic organisms which occurs in the waste waters belong: • viruses • germs (salmonella, escherichia coli) • protozoas • lateral worms mailto:ivana.mahrikova:@stuba.sk food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 54 sludge often consist toxic chemical matters, which we can classify as a dangerous waste. there are some organic matters and heavy metals. the source of heavy metals is cadmium, chrome, copper, mercury, lead and zinc. we can find this matters into the industrial waste waters from the metal and leader industry, from the wet and dry deposition. table 1. waste water sludge composition ww sludge composition primary sludge activated sludge % solid matters % solid matters organic matters 60 80 62 90 carbon matters 25 35 38 47 nitrogenous matters 3 4 3 9 hydrogen matters 3 5 5 7 cellulose 3,8 7 fats 7 35 5 14 albuminous 19 25 19 56 lignin 5,8 1 legislation on sewage sludge management at european level the european waste catalogue classifies the sewage sludge as a waste. the current european standard for waste is directive 2008/98/ec of 19 november on waste and repealing certain directives, more commonly known as the waste framework directive (wastefd). the adoption of this directive has repealed its predecessors: directive 75/442/eec, of 15 july on waste. directive 91/156/eec, of 18 march on waste. directive 2006/12/ec, of 5 april on waste. the european community adopted in 1975 the directive 75/442/eec of 15 july on waste, thinking especially that the disparity of provisions in the ms could create competition distortions in a common market and pretending, at the same time, to protect human health and the environment by establishing a community system for waste management. three years later, with the same purpose was adopted directive 78/319/eec of 20 march concerning hazardous waste. subsequently, it was adopted the directive 91/156/eec of 18 march amending directive 75/442/eec on waste. in this case, the council of the european communities considered necessary both ensure the removal and recovery of waste and take measures aimed to limit the production of waste. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 55 in this line, directive 91/156/eec binds ms to promote the prevention, reduction, recovery and use of waste as a source of energy. a few months later, in order to adapt the provisions of directive 91/156/eec on waste, the council adopted directive 91/689/eec of 12 december on hazardous waste. hence, the directive states that the "sludge from wastewater treatment facilities" and "untreated sewage sludge or unusable in agriculture" may be considered hazardous waste if they have any of the properties listed in annex iii of the directive. as a result of the disparity between the ms legislation concerning the removal and recovery of waste, the commission adopted the directive 2006/12/ec of 5 april on waste. it defines the concept of waste and sets out a clear hierarchy for their management. one of the highlights is the importance that assigns to the recovery of waste in general and therefore, also to sewage sludge recovery. given the difficulties for the implementation of directive 2006/12 due inter alia to the lack of specificity in any of its terms, the commission states that it was necessary to review such directive in order to clarify key concepts. the result was the adoption of the directive 2008/98/ec of 22 november on waste and repealing certain directives (wastefd). taking into account the novel definition of waste proposed by the wastefd, if sewage sludge is properly managed would be considered as a useful product instead a waste. moreover, wastefd updates the hierarchy in waste management and redefines the concept of recovery. except prevention and elimination, all other operations are considered as recovery ones. in the case of sewage sludge, it is evident that recycling and other types of recovery are the points of interest. according the directive, these operations must be favored by the ms through the adoption of national plans of waste management. directive 1986/278/eec, of 12 june 1986 on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture the directive 1986/278/eec seeks to encourage the use of sewage sludge in agriculture and to regulate its use in such a way as to prevent harmful effects on soil, vegetation, animals and people. to this end, it prohibits the use of untreated sludge on agricultural land unless it is injected or incorporated into the soil. the directive specifies rules for the sampling and analysis of sludge and soils. it sets out requirements for the keeping of detailed records of the quantities of sludge produced, the quantities used in agriculture, the composition and properties of the sludge, the type of treatment and the sites where the sludge is used. limit values for concentrations of heavy metals in sewage sludge intended for agricultural use and in sludge-treated soils are provided. the european commission is currently assessing whether this directive should be reviewed – and if so, the extent of this review. fig. 1. hierarchy of sludge management (directive 2008/98/ec) legislation on sewage sludge management at state level , sludge disposal in slovakia. today are in slovakia valid two acts according to the sewage sludge management. • act no. 231/2000 coll., of the waste food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 56 • act no. 188/2000 coll. on application of waste water sludge into soil. there are the methods of controlled application of sludge into soil are used: • direct application of sludge into soil as of the act no.188/2003 coll. on application of waste water sludge into soil • application as of the act no. 136/2000 coll. in wording of act no.555/2004 coll. on fertilizers, for example, as compost or soil-growing medium. in this case the product is subject to certification. overview of sludge production from urban wastewater treatment for wastewater treatment plant and carried out handling them in the time period of 2001 – 2009, as shown in figure 2. fig. 2 sewage sludge production in slovakia pressure sewage in the context of increasing requirements for wastewater treatment the implementation of council directive 91/271 eec on urban waste water, the increase of sludge production by about 2040% is to be expected in the near future. it is mainly the addition of sludge from small sewage treatment plants without significant involvement of industrial waste water, so a certain degree of contamination of sludge can be expected, which corresponds to the requirements of the process, limiting its application to the soil. [3] methods of sewage sludge disposal and hygienic inspection the most using process of sewage sludge disposal in slovakia is application into the soil. the controlled application must be done according to the valid legislation (act no.188/2003 coll. and act no. 136/2000 coll.). hygienic inspection is the technological process of sludge treatment, which is used in the case, when the sludge properties are not fulfilled the request of valid slovak legislation. the main part of waste, microorganisms and viruses proceed after treatment into the sludge. by hygienic inspection improved the hygienic and sanitary properties of the sludge. we can use for it various methods as a pasteurisation, aerobical, thermophilic stabilisation, chemical methods-with using cao, by gamma radiation or irradiation and incineration pasteurisation pasteurisation is a process, when the sludge is heated during relative short time on a temperature 70°c. process of pasteurisation was established in switzerland. the goal of it is elimination of germs and viruses especially of salmonella. pasteurisation no replaced process of sludge stabilisation. it has to be combined with some other process of stabilisation. the process of pasteurisation is high effective by killing of various types of viruses especially on entherogerms and salmonella. it is no effective by thermo tolerant types of life forms as a certain types of viruses and spores. irradiation there are only marginal experiences of irradiation of sewage sludge in the europe. by the irradiation are most reduced salmonella and entherogerms. by the operational point of view is interesting, that the irradiated sludge has better 45.000 50.000 55.000 60.000 20 01 20 03 20 05 20 07 20 09 sludge production solids t/year sludge production solids t/year food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 57 dewatering properties as a no irradiated sludge. aerobical thermophilic stabilisation aerobical thermophilic stabilisation was developed for the sewage sludge stabilisation. high level of aeration reinitiate by biological processes in such intensity, that the warmth generated by the process keeps the temperatures needed for the disinfection. we know two concepts of aerobical thermophilic stabilisation: aeration by clear oxygen aeration by air oxygen by the using of clear oxygen, are the average temperatures about 60-80°c. the disinfection effect will be comparable with the pasteurisation and we can reach also better results then by pasteurisation. when is the process supplied with oxygen from the air, the reached temperature is 4060°c. in this temperature interval is the process of disinfection reduced and there is needed longer time for deactivation of pathogens. to find the optimal relation between aeration and temperature is not easy. composting composting is a process, which is depending on aerobical reduction of organic matter by thermophilic germs. sludge is mixing with filling mass, what served to the increase of porosity for better aeration, for reduction of content of humidity and improve of relationship between c:n. there are often combined all three functions in one product. for example: straw, wood peel or household waste. there can be used also no degradable material as a plastic. it was developed many types of composting processes, so is really difficult to define average data for requested disinfection effect. generally we can speak about two basic processes: composting in composting lagoon composting in bioreactor by composting in composting lagoon is the sewage sludge in a role of filling mass. for requested sanitary effect is necessary to achieve the critical temperature for requested time distance. for elimination of salmonella in summer period is the critical time established on 6-7 weeks. lime treatment lime is often added into the swage sludge before dewatering. that caused increase of ph level, it can reached value from 9-13 in dependency of lime supply and sludge characteristics. vegetative germ cells (coliform germs or salmonella) are fast reduced to nothing by value of ph from 9 till 10. according to strauch and berg, ph level has to be higher than 11,5 for reaching of requested efficiency. by these ph values will be destroyed most of viruses. destruction of the viruses is not effected directly by ph effect, but by unlocking of free ammonia by ph level around 12. complete destruction can be reached by adding of quicklime into the dewatered sludge. at this moment the temperature increase till 70-80 °c and all pathogens died very fast. the ain destruction factor is temperature. incineration incineration is one of the most effective process of sewage sludge treatment and sanitation. by the still growing amount of sludge it is a perspective solution for sludge disposal. but the process of sewage sludge incineration is not easy to realise in really conditions. the dewatered sludge contents about 75% of water. the complication is also high value of heavy metals in sludge. it can cause creation of vapours by higher incineration temperatures. one of the perspective methods of sewage sludge incineration was founded fluid layer. the fluid layer create thermal homogeneous environment, which food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 58 is reach on oxygen with strong abrasive influence on the sludge elements. the thermic degradation has a lot of advantages. at first it is volume reduction about 87%. the ash from the incinerated sludge is inorganic and mostly is it sterile matter. so we can use it in building industry. the heavy metals are strong bounded on sorbate and so they are not washed up. the gas emissions are deep under allowed limits. also the process of thermic degradation is friendly to the environment. by the treatment on wwtp we can skip the sludge treatment from the technology of waste water treatment and replace it with thermic sludge degradation. conclusions wastewater treatment involves the generation of large volumes of sludge and other waste which management in an economical and environmental acceptable way has become a matter of increasing importance during the last few years. while the technologies and processes to reduce sludge generation are being widely studied, contributions relative to economic aspects are much more limited. however, when wwtps operators face to the implementation of these technologies, not only technical aspects must be considered but also influence on environment regarded. technical solution, economical aspect and environment friendly, that are the three highlights, which have to bee in balance. we can see, that is possible to use various methods for sludge disposal. but our goal is to choose the best alternative from the technical, environmental and last and but not least economical point of view. each wwtp has its own sludge treatment and also specific condition for sewage sludge disposal and hygienic inspection. we have to find the optimal solution for the people and for the environment. acknowledgement the article was written with the support of the scientific grant agency – projects vega no. 1/0559/10 dealt with at the department of sanitary and environmental engineering of the slovak university of technology. references: 1. mahríková, i.: operation of sludge tanks in small and mid-sized waste water treatment plants. in: proceedings of arw, advanced water supply and wastewater treatment: a road to safer society and environment, published by springer, netherland 2011, pp. 163-171 2. mahríková i.: doktorandská práca optimalizácia prevádzky kalojemov ma malých a stredných komunálnych ov, bratislava 2009 3. stn 75 6402 malé istiarne odpadových vôd, 1999 4. šumná j., drahovskaá d., kozáková, k.: k stratégii nakladania s kalmi z istiarní odpadových vôd. zborník z konferencie kaly a odpady 2008, s.25-28 5. stanko, š., kriš, j., škultétyová, i.: environmental impact of waste-water reuse. in: 31st international geographical congress : resumes/tunis, 12.-15.8.2008. , 2008. s. 378-379 6. stanko, š.: combined versus separated sewer system in slovakia. in wmhe 2009 : eleventh international symposium on water management and hydralic engineering. vol.i. ohrid, macedonia, 1.-5.9.2009., skopje : university ss.cyril and methodius, 2009. isbn 978-9989-2469-6-8. s. 515-522 7. hlavínek p.: perspectives of decentrazed waste water treatment of rural areas. in: proceedings of arw, advanced water supply and wastewater treatment: a road to safer society and environment, published by springer, netherland 2011, pp. 75-87 8. senante molinos , m.: technoeconomical efficiency, cost modeling and economic valuation of environmental benefits from waste awter treatment, doctoral thesis, valencia 2011 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 25 geophysical evaluation of the stability of the landslide slope rolan gasparyan institute of geophysics and engineering seismology of the national academy of sciences (armenia), rolangas@rambler.ru abstract: some peculiarities of radiometrical and electrometrical methods for landslide slope study are considered. taking as a basis the theory of slope abyssal creepage, the mechanism of formation of emanation anomalies within the limits of an active landslide is suggested. it is noted that all the three preparation phases of the abyssal creepage process are characterized by the extreme values of radioactive emanation field. the revealed statistic intercomunication between ground geoelectrical and physical-mechanical properties makes it possible to carry out effective calculation of landslide slope stability reserve. keywords: landslide, geodynamics, radiometry, geoelectricity. 1. introduction among exogeneous geological processes (egp) in armenia landslides belong to the most developed and dangerous ones. according to data of modern research more than three thousand landslides are revealed on the territory of armenia. study of these landslides began in 1926 [1]. as it is known, well-founded assessment of landslide hazard requires solution of a number of problems concerning the slope structure, composition, state, properties, occurrence conditions, etc. solution of these problems together with data about climatic, hydrogeological and seismic conditions of the region, materials concerning human activities and history of landslide development represent necessary basis for calculation of the landslide slope stability. traditional methods of engineering-geological investigations are not always capable of answering the above-mentioned questions with the required fullness. in this respect application of geophysical methods discovers additional possibilities. they make it possible to investigate in detail great areas, which is inaccessible for other types of geological and geomechanical research. specifically, measurable parameters of geophysical fields automatically take into account geological and hidrogeological peculiarities of landslide slopes, which at times can't be identified singly. finally, possibilities of routine observations infinitely increase, as geophysical measurings can recur any number of times without disturbing natural conditions [2], [3], [5]. in the suggested work we'll try to present some characteristic peculiarities of landslide hazard assessment according to radiometrical and electrometrical data. 2. radiometrical research at present numerous examples of application of radiomertical methods in the sphere of seismology and engineering geodynamics indicate their comparatively high informative character on one hand, and availability of a number of variable theoretical and methodical problems on the other hand [4], [7]. the most sensitive mailto:rolangas:@rambler.ru food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 26 method among radiometrical ones, reacting to the change of fields of rock mass stress and deformations, is the emanation method. the results of investigation of landslides in armenia prove existance of certain connection between the changes of landslide stressed-deformed state and variations of radioactive field. let's consider in brief the mechanism of formation of emanation anomalies in the active landslide zone [3]. on the basis of the theory of slope abyssal creepage, developed by ter-stepanyan g.i. [8]. one may suggest that in the slope upper steady part, at some deapth, the tangential stresses concentration occurs. the creepage takes place in the closed zone in which the ground stressed state is evaluated by the coefficient of mobilized shearing strength tg the value of which is defined by the relation of tangential and reducted stresses. note that 0tg is some threshold value of the mobilized shear strength with which longtime deformation do not occur yet and tg is the mobilized shear strength value when there is ground shear. in consequence of the ground mass slow movement, in the creepage zone landslide cracks are formed, thus surrounding this zone (figure 1). the zone itself is the deformed part of the continuous environment. upward and downward of the creepage zone, respectively, the undeformed moving slope part and the undeformed non-moving slope part are located. as a rule, the abyssal creepage is considered as a preparatory stage for landslides proper which is superimposed on the general tendency of the geological landslide development. such a distribution of stresses and creepage is called hearth. the creepage zone is located along the sliding surface de (figure1) along which the creepage ground displacement occurs. owing to this the stressed state changes, that is the front part of the creepage zone is compressed and the back part is dilatated. so, in the creepage process potential sliding surface sections are involved. figure 1: emanation concentration (i) and value distribution of mobilized shear strength coefficient (ii) along the potential slip surface. the emanation concentration increase is conditioned by arising in the hearth zone slope of creepage where small tension, fractures form which promote the free emanation displacement to the soil layer (in this case in the creepage zone the coefficients of rock diffusion d and emanating an increase). it is known from the emanation method theory that the emanation concentration in the ground air mainly depends on the quantity of radioactive elements being present in the ground. for infinite halfspace this dependency is discribed by the following formula [6] 1 ra 13 rn n107,3n (1) where rnn radon content in ground pores; ran radium content in ground; ground density; emanating coefficient; porosity. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 27 as it is evident from formula (1) that emanation concentration besides the quantity of radioelements depends also on petrophysical properties and rock emanating coefficient. assuming that it is easy to note for the concrete rock types constn ra ; that rnn will depend on ,, only. these very parameters undergo the largest changes in different geodynamic zones (landslides, karst, tectonic disturbances etc.), thus influencing essentially the displacement process and the emanation concentration process in rocks. without pretending to the unambiguity of such an interpretation of the rock emanating process in geodynamic zones, at the sections of sliding process development particularly, one notes that by the experimental investigation results all the three preparatory phases of the abyssal creepage process are characterized by the extreme values of the emanation concentration (figure 1, table). table emanation concentration of different phases of sliding slopes abyssal creepage creepage phases coefficient of mobilized shear strength emanati on concent ration 1. absence of longtime deformations (phase of stiffness) 0tgtg0 back ground 2. long-time deformations (phase of creepage) tgtgtg 0 max 3. shear occurs (phase of plasticity) tgtg min figure 2. results of the geophysical prospecting of the landslide slope (armenia, jajour pass). a – coefficient of radon variations; b – emanation survey profile; c – geological section; d – geoelectric section figure 2 presents the results of field experimental research of one of the active landslides in northern armenia. the analysis of these data testify the fact that the middle level of coefficient of radon variation in the steady part of slope food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 28 is higher than in the area of active landslide manifestation (figure 2a). the fact is explained by the change of the strain condition of massive of the rocks on the plateau is taken place weaker. the interleaving intervals of extreme values of radon over all the slope are causedl by the various degree of jointing of landslide blocks and activity of the process. the zone of increase in variation coefficient in the steady part of the slope (interval between points 460-480) coincides with the region of genetic joints. the fresh joints of fishweir were formed further on the very zone by which the new blocks of landslide massive were torn away. the foregoing statement is obviously connected with the formationi of creepage focal zone of the landslide slope stable part, where abrupt change of values of ground diffusion and emanating coefficients has taken place subsequently, in the main ledge zone fresh fissures have been formed, which are the direct sign of the given landslide advancing character. in the presence of emanation survey data and some compressional ground properties one may preliminarily evaluate the stressed-deformed state of a sliding slope using the following formula [2]: 112211 a}x)]q1[ln(da{ (2) }1)]q1[ln(xd{ae 212110 (3) where -emanation concentration per volume unit of pore space; q-emanation apportionment velocity into pores per volume unit of environment; the decay constant emanation; d diffusion coefficient; x depth of air sumple collection; a coefficient of ground compressibility; factor, for sandyclaycey rocks it changes from 76,0 to 43,0 ; a value characterizing ground compressional properties; stress conditioned by rock pressure; 0e modulus of total deformation. values ,q and d for concrete ground types are quasiconstant. compressional properties ( a and a ) are determined experimentally. 3. electrometrical research the slope stability coefficient ( sk ) is the main and, as a matter of fact, the only qualitative indicator, determining the peculiarities of landslide process development. numerous practical examples of application of the sk calculation methods, based on consideration of a landslide slope idealized model, give only approximate notion of the stability coefficient value. it is explained by the fact that calculation parameters of the stability coefficient, such as angle of inner friction, cohesion power, density, etc., are determined by relatively small number of examples, which are not representative for description of the whole landslide. besides, between the existing rock resistance in the landslide, shear force and the results of sample laboratory tests essential difference is observed owing to the display of wide-spreading effects, etc. the universal character of electrometrical methods in determining the calculation parameters (geometrical and physicalmechanical) of slope stability is their indisputable merit. we have studied the stastistic connections between the electric resistance ( s ) and specific cohesion, the angle of inner friction and the landslide ground density. 22lg19,1c s (4) 95,248,1lg s (5) s015,003,2 (6) where c specific cohesion; angle of inner friction; density; s seeming specific electric resistance of ground. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 29 for sandy-clayey ground (if dampness is constant) statistic connection (4, 5, 6) between the above-mentioned parameters are quite satisfactorily motivated by physical prerequisites. undoubtedly, for each concrete case (if necessary) correction of digital coefficients, included in these formulas, is required. conclusions summarizing the foregoing statement, the following principal conclusions can be drawn. 1. the main geometrical and geophysical parameters of landslide slopes are determined and their calculation models are constructed. 2. geodynamic zones of landslide slopes (compression and strain zones) are characterized by the extreme values of radioactive emanation field. 3. the increase of subsoil emanation concentration in the landslide front part is caused by formation of the creepage zone, where free emanation is squeezed out to the surface. 4. the proposed analytical formulas and statistic intercommunication make it possible to assess the stressed -deformed state and the stability coefficient of landslide slopes. references 1. avagyan a.a., jadoyan r.b. the principles of the base system of data about exogeneous geological processes on the territory of armenia. collection of scientific works. ra nas publishing house, giumry, 463-467 ,(1998) 2. bondarenko v.m., et al. new methods of engineering geophysics, ed., nedra, moscow, 22, (1983) 3. gasparyan r. k. about concentration relation of ground emanation with stresseddeformed state of rock massive.. ed., all-union institute of scientific and thecnical information, moscow, 14 p, (1987). 4. gasparyan r. k on some reason of radioactive emanation concentration variation in seismoactive regions. proc. 3th school-seminar geodynamics of caucasus, yerevan, tex., 116-119, (1986) 5. gasparyan r.k., gazaryan g.o. geophysical methods of stress-deformation control ed..acad.sci.ussr, novosibirsk, 68-72, (1985 ) 6. ogilvy a.a. fundamentals of engineering geophysics.. ed., nedra.moscow,501p, (1990) 7. novikov g.f., kapkov y.n. radioactive methods of prospecting, ed., nedra, leningrad, 759 p, (1965) 8. ter-stepanyan g.i. new methods of landslide study .ed., acad. sci. arm. ssr., yerevan, 151 p, (1978). 212 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 212 217 development of operational quality control method for meat products olha lyubchyk1, mykola mykyjchuk1, *mariуa vorobets2 1national university “lviv polytechnic”, department of metrology, standardization and certification, 12, s.bandery str., lviv, ukraine, 79013 2yury fedkovych national university of chernivtsi, ukraine, m.vorobets@chnu.edu.ua * corresponding author received march 14th 2015, accepted june 17th 2015 abstract: assortment of finished products of meat in the consumer market in ukraine is quite wide, but a large number of products are very varied in quality. today quality control of meat and meat products is very important. doing assessing of a quality of meat, the importance is focused on meat organoleptic characteristics which shall identify the most characteristic features of a particular type of meat for the consumer and classify them. the following distinguishing features of meat freshness are: the number of fibers, medium size, color, edge definition fibers. a method for operational quality control for meat products by customers in the points of product sale based on the optical methods using modern digital optoelectronic has been developed. in the article are described problems that shall be solved to implement optical method of operational identification of a meat type. the operational analysis method allows getting fast results and assesses the structure of most types of meat products, which gives reason to recommend it for quality control and detect adulteration of meat products. keywords: pattern recognition, adulteration, microstructural analysis, meat products, operational quality control 1. introduction today a consumer has an opportunity to choose a product from a wide range of goods with the same (or similar) consumer properties, but here he encounters another problem – how to evaluate a product quality with the optimal properties out of this range. under these conditions of commodity sufficiency the consumer should be provided with as much information as possible to enable him to identify the product properly and buy the one quality product that will satisfy his needs to the fullest extent. an important factor in assurance of the required quality of the food products is the availability of the objective information about the quality parameters obtained by measurements using appropriate methods and techniques. the correct choice of the measurement methods and techniques is essential to ensure the information adequacy on the actual level of the food product quality. in determining the quality of meat the most typical features of a particular type of meat shall be identified for the consumer, classified, for example, using the pattern identification theory and up-to-date programming units. the operational impact on the production process of meat and meat products shall be an objective of the modern process organization in order to minimize the consumer and environmental risks. http://www.fia.usv.ro/fiajournal mailto:m.vorobets@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 olha lyubchyk, mykola mykyjchuk, mariуa vorobets, development of operational quality control method for meat products, food and environment safety, volume xiv, issue 2 – 2015, pag. 212 – 217 213 2. materials and methods the range and sales of meat products have significantly increased over the recent years. in the meat market, being in the steady demand of consumers, its various types are available and it is sometimes hard to the customer to choose a quality product out of this variety. increase in the production rates and volumes of output of the meat industry requires improvement of existing and development of new production processes, which ensure efficient use of raw material resources, increase in the output and improvement in the product quality. the following parameters play the main role in assessing the quality of meat: • contents of ingredients used by the body for biological synthesis and covering the metabolic cost; • organoleptic characteristics (appearance, odor, color, texture); • absence of toxic substances and pathogens. according to law of ukraine “on the safety and quality of food products” all food products getting to the consumer market shall pass the safety check during the compulsory certification. it is practically impossible to detect adulteration using conventional methods, because one has to check not only the type and grade of products, but also to carry out their identification by ingredients making it possible to identify and verify the adequacy of the information specified on the label, compliance with the formulation, type and name of the product [1]. enzyme immunoassay methods, chromatographic methods and pcr diagnostics are used most of all. they are highly sensitive, accurate and fast. however, they are difficult to adapt to such complicated multicomponent systems as meat products and with all the advantages of these methods they are not able to detect substitution of meat with other animal ingredients – lacteal gland, liver, vascular glands and other by-products. application of chemical, physicochemical and biochemical methods provides information about the energy value of meat products [1]. the existing control methods shall be improved towards their cheapening and approaching the point of sale to increase the reliability and efficiency of identification of the meat quality. 3. results and discussion the range of end products of meat is quite wide in the consumer market in ukraine, but a large number of products vary in quality. as studies show [2], in many cases while producing food items from meat, producers substitute meat with the phytogenic products, including soy protein, introduce low value additives, which were not provided by formula, and often use tainted meat or reused materials. quality control of meat and meat products is the most topical issue today. the control methods in ukraine are obsolete and do not enable to determine all modern adulterations. forthis purpose many european countries such as germany, austria, and russia apply microstructure analysis of meat products based on the principles of histopathological and histochemical examinations. method of microstructural analysis is a methodological approach to the study of short-term cognitive and executive actions. it is assumed that in each time interval after receiving instructions for the execution of action – specific input data conversion happens due to inclusion of a particular functional unit [3]. meat products at various stages of processing treatment, as well as finished products retain their morphological features. therefore, using microstructure analysis of raw materials, semi-finished or finished products, we can determine the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 olha lyubchyk, mykola mykyjchuk, mariуa vorobets, development of operational quality control method for meat products, food and environment safety, volume xiv, issue 2 – 2015, pag. 212 – 217 214 presence of certain types of tissues, organs, spices, and low-grade additives [2, 4–6]. the microstructure analysis makes it possible not only to detect adulteration but also to control the compliance of the ingredients of the meat products with the approved formula [7]. the conclusion on the meat freshness is drawn based on the test results [8], according to the distinguishing features shown in table 1. table 1 organoleptic indicators of meat of different grade of freshness indicator characteristic feature of meat fresh doubtful freshness tainted color and appearance of product drying crust is pale pink or pale red wet in places, slightly sticky, darkened very dried, covered with grayish-brown slime or mold state of fat beef has a white, yellowish or yellow color; firm texture, crumbles when squeezing pork has a white or pale pink color, soft, elastic chicken has a pale yellow or yellow color has a grayish-matte shade, slightly sticks to fingers chicken has a pale yellow or yellow color has a grayish-matte shade, smears when squeezing. pork fat can be covered with a bit of mold. the smell is rancid (expresses bitterness). chicken fat has a pale white color with a gray shade muscles in the cut slightly wet, do not leave wet spots on filter paper; color is common to this type of meat wet, leave wet spots on filter paper, are slightly sticky; have a dark red color wet, leave wet spots on filter paper, are sticky; have a redbrown color texture the meat is dense and elastic in the cut; flexible, when pressing with a finger a dent appears, which is quickly recovered the meat is less dense in the cut, when pressing with a finger a dent appears, which is slowly recovered the meat is flabby in the cut, when pressing with a finger a dent appears, which is not recovered odor peculiar, specific to each type of fresh meat slightly acid with a hint of mustiness acid or musty or slightly odorous state of tendon tendons are elastic, dense, articular surfaces are smooth, shiny tendons are less dense, have a dull white color. articular surfaces are slightly covered with slime tendons are softened, have a grayish color. articular surfaces are covered with slime the choice of the characteristic features was made based on the study results specified in [1, 2] and on their control availability using simple optical devices (digital cameras). for the application of the proposed approach for the operational control of the meat quality, it is necessary to identify the most characteristic features of a particular type of meat for the consumer and classify them, for example, using pattern recognition theory and up-to-date programming units. a pattern recognition system involves the following three aspects [9]: data acquisition and preprocessing; data representation; decision making. pattern recognition presents one of the most significant challenges for scientists and engineers, and many different approaches have been proposed [10]. within the artificial intelligence theory pattern recognition is included in the broader science discipline – computerassisted instruction theory, which purpose is to develop algorithm design methods able to learn. machine pattern recognition mainly has two basic methods: statistics food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 olha lyubchyk, mykola mykyjchuk, mariуa vorobets, development of operational quality control method for meat products, food and environment safety, volume xiv, issue 2 – 2015, pag. 212 – 217 215 pattern recognition and structure pattern recognition [11]. pattern recognition is widely applicable and is used in the creation of all computer systems having intellectual functions, i.e. functions related to decision making instead of a human: computer-aided diagnosis, criminalistics expert examination, information search and data mining, etc. each image is a set of numbers that describe the properties and are called attributes. ordered set of attributes of an object is called feature vectors. feature vector is a point in the space of features. the classifier or the decision rule is a function that assigns a class to the feature vector pattern, to which it belongs. the problem of pattern recognition can be divided into a number of subtasks. 1. generation of signs measurement or calculation of numerical features that characterize the object. 2. selecting features identifying the most informative features for classification (both early signs, and their functions can be included in this set). 3. building the classifier formation of the decision rule, under which the classification is made. 4. evaluation of the classification quality calculation of the rate of classification accuracy (accuracy, sensitivity, specificity, first and second kind errors). let us introduce the notation and formulate a mathematical classification problem. we use the following classification problem model [12]: ω – a set of recognition objects (pattern space). ω: ωω – recognition object (pattern). g (ω): ω →m, m={1, 2, ..., m} – indicator function that breaks up the pattern space into ω to m of disjoined classes ω1, ω2, ..., ωm. indicator function is unknown to the observer. x – space of observations perceived by the observer (feature space). x (ω): ω → x – function that assigns to each object ω point x (ω) in the feature space. vector x (ω) is the pattern of the object perceived by the observer. in the feature space disjoint set of points is determined ki∩x, i=1, 2 ..., m, corresponding samples of one class. gˆ(x): x → m – decision rule – estimation for g(ω) based on x(ω), that is gˆ(x) = gˆ(x(ω)). let (xj)=x(ω j), j=1, 2 ..., n – the information about function g(ω) and x(ω) be available the observer but most of these features are unknown to the observer. then (gj, xj), j=1, 2 ..., n – there are many precedents. the problem is to construct such decision rule gˆ(x) to make the recognition with the minimum number of errors. the usual case to consider the feature space as euclidean of x = r1. the quality of decision rule is measured with the frequency of proper solutions. usually it is estimated by providing a certain probability measure to a set of objects ω. then the problem is written as min p{gˆ(x(ω)) ≠ g(ω)}. the following problems shall be solved to implement optical method of operational identification of a type of meat: 1) determine the characteristic features of a particular type of meat based on a number of pictures of different types of meat; 2) create a mathematical model of types of meat that will consider its characteristic features (virtual standards of types of meat); 3) develop algorithms for operational identification, which based on the pattern recognition theory will quickly identify a type of meat and its level of quality by a consumer himself. operational quality control method for meat products was developed based on the analysis made, shown in figure 1. at the first stage we determine identification characteristics of different food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 olha lyubchyk, mykola mykyjchuk, mariуa vorobets, development of operational quality control method for meat products, food and environment safety, volume xiv, issue 2 – 2015, pag. 212 – 217 216 types of meat: number of fibers, average fiber size, color, and edge definition of fibers. the color of meat is one of the main quality indicators, estimated by a consumer, by which a marketable appearance of products is judged, as well as some chemical transformations, which may take place in the meat. muscular tissue is the main edible part, which consists of separate long thin fibers covered with a thin translucent membrane. the basis of connective tissue is collagen and elastin fibers. there are following types of connective tissue depending on their ratio and location: loose, dense, elastin and reticular. fig.1. algorithm of the operational quality control for meat products in order to identify the type of the meat samples, one should determine their statistical characteristics of the meat features, such as mathematical expectation and dispersion. according to the study it is necessary to create a classifier of types of meat, which shall include a database with statistical characteristics of meat features classification. this classification is shown in table 2. its statistical characteristics are determined based on the results of estimate of the meat classification features and comparison is made by the features being in the classifier. the meat under examination is identified when comparing the characteristics and probability estimate is made by the decision rule. choice of classification features of a sample of meat determination of statistical characteristics of a sample of meat construction of the classifier of types of meat measurement of classification features of a sample of meat determination of statistical characteristics of a sample of meat identification of the type of meat (by the decision rule) comparison of the meat characteristics by the classification features estimate of the identification probability food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 olha lyubchyk, mykola mykyjchuk, mariуa vorobets, development of operational quality control method for meat products, food and environment safety, volume xiv, issue 2 – 2015, pag. 212 – 217 217 table 2 meat features classification features pork beef chicken number of fibers nc nт nк average fiber size dс dт dк color pale-pink to red pale-pink to dark red pale pink to pink edge definition of fibers ŋс ŋт ŋк 4. conclusions adulteration of meat not only has an effect on the lower quality of the finished products, but it may be a hazardous factor for consumers’ health. operational analysis method enables not to conduct additional analyses to obtain characteristics of the sample under examination. this enables to use this method in different operating conditions and facilitates the analysis. use of operational analysis enables to identify adulteration, establish a grade of freshness and meat composition, its substitution with various ingredients. today there is an urgent need for means of quality control of meat products in the places of distribution. these facilities must meet two basic requirements: to provide a sufficient level reliability of information on the status of quality meat and have a low price that will contribute to mass use. 5. references [1]. kotsyumbas i.y., kotsyumbas g.i., schebentovska o.m., examination of semifinished meat and meat-vegetable products using microstructure method, recommended practice. lviv: afisha, 80, (2011). [2]. kotsyumbas g.i., bisyuk i.u., kotsyumbas i.y., schebentovska o.m., rudyk g.v., mysiv o.v., kozak m.v., microstructure study of raw materials in ground meat, recommended practice, lviv: afisha, 48, (2006). [3]. zinchenko v.p., image and activities, institute of practical psychology, voronezh: npo modek, 608, (1997). [4]. adutskevich v.a., belousov a.a., microstructural indicators of meat aging and taint, xvii european congress of workers of meat industry, england: 142, (1971). [5]. beem r., pleva b., microscopy of meat and raw animal materials, moscow: 336, (1964). [6]. goreglyad h.s., veterinary and sanitary examination with the basics of processing technology of animal products, saint-petersburg: koloc, 584, (1981). [7]. tinyakov g.g., histology of meat animals, moscow: food industry, 460, (1967). [8]. http://medbib.in.ua/opredelenie-stepenisvejesti.html, 1/2015. [9]. jayanta kumar basu, debnath bhattacharyya, tai-hoon kim, use of artificial neural network in pattern recognition, international journal of software engineering and its applications, vol. 4, 2, 23-34, (2010). [10]. devroye l., gyorfi l., lugosi g. a., probabilistic theory of pattern recognition, new york: springer-verlag, (1996). [11]. youguo pi, wenzhi liao, mingyou liu, jianping lu, pattern recognition techniques, technology and applications, theory of cognitive pattern recognition, vienna: in-tech, 432-462, (2008). [12]. mestetsky l.m., mathematical methods of pattern identification. course of lectures, moscow: faculty cmc of msu, department of mmp, 85, (2004). http://medbib.in.ua/opredelenie-stepenifood and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 52 copper ions concentration using ion exchange resins *petru bulai1, camelia popa2 1 faculty of mechanical engineering, mechatronics and management, tefan cel mare university of suceava, romania, bulaipetru@yahoo.com 2 faculty of food engineering, tefan cel mare university of suceava, romania, camelia.popa@ fia.usv.ro *corresponding author received 2 september 2012, accepted 17 september 2012 abstract: the paper present the recovery process of copper ions adsorbed onto purolite s930 chelating resin. the recovery process was investigated using hydrochloric acid, sulfuric acid and disodium salt of ethylenediaminetetraacetic acid solutions. experiments were carrying out using different resin dose in the range 1 25 g dried exhausted resin/l and varying the concentration of regeneration solution in the range 0.1 2 eq./l for hydrochloric and sulfuric acid, and in the range 0.1 0.2 eq/l for disodium salt of ethylenediaminetetraacetic acid. keywords: recovery, concentration, ion exchange resins 1. introduction the heavy metals are non-biodegradable in the nature, present bioaccumulation tendency in environment and toxicity to most of life forms [1,2]. when they are found in water in soluble form, heavy metal ions present a high mobility increasing the risk of contamination of large adjacent areas. therefore the presence of heavy metals in the environment is still a major concern for nature health. the most important source of heavy metals for environmental is the industrial wastewater [3]. to remove the heavy metals from wastewater can be used different treatment techniques such as chemical precipitation, coagulation– flocculation, flotation, membrane filtration processes (ultrafiltration, nanofiltration and reverse osmosis), electrochemical treatment techniques (electro-dialysis, membrane electrolysis, electroextraction and electrochemical precipitation) and sorption treatment techniques (ion exchange, adsorption and biosorption) [1, 3-7]. all these methods have been studied extensively and can be successfully applied in industry. despite of these possibilities, the industry still uses mainly precipitation. by using precipitation process does not completely eliminate the problem of heavy metals from wastewater. the sludges that result in heavy metals precipitation process need to be treated and stored under special conditions. recovery of the precipitated metal ions is laborious and expensive. instead ion exchange methods are ecological as purification methods and may be used both for organic and inorganic compounds [6]. retention of metal ions from wastewater by ion exchange resin process has a number of advantages over other methods. one of these advantage is the easiness in recovery process. recovery of metal ion retained on resin can be concentrated and recovered in the form of various salts according to the substance used for regeneration of resin: acids (hcl, h2so4, hno3), bases (naoh) or salts (nacl, disodium salt of edta). another mailto:bulaipetru:@yahoo.com food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 53 advantage is the purity of the solution obtained from regeneration. this solution may contains metal salt and acids, bases or salts, depending on regeneration solution used. these solutions can be reused in process, or used to obtain solid metal by electrochemical methods or other products. 2. materials and methods the chelating resin used in the experiments was s930 obtained from purolite international limited (hounslow, uk). table 1 presents the main physical and chemical properties of the resin. table 1. proprieties of the chelating resin * polymer matrix structure macroporous styrene divinylbenzene functional groups iminodiacetic acid ionic form (as shipped) na+ ph range (operating): h+ form, na+ form 2 6; 6 11 maximum operating temperature 70ºc particle size range + 1.0mm <10%, -0.3mm <1% total exchange capacity 1.9 meq/ml * manufacturer supplied. the conversion of the sodium form of the resin into hydrogen form was done with 10% hcl solution, followed by washing with distilled water until the ph of the effluent dropped to neutrality. then, the resin has been dried at 60 ºc using an oven. exhausted resin was obtained using 50.0 ml of cu2+ with initial concentration c0 = 300 mg cu2+/l that were added to erlenmeyer flask already containing 0.05 g of dry resin in h form. the initial phi = 5 of the solution was adjusted by using diluted solutions of h2so4. the flasks where mechanically shacked at several fixed temperatures and at the rate of 120 cycles min 1 using orbital shaking incubator gfl 3031. after equilibrium (24 hours), the resin and solution were separated by filtration and the copper content of the solution was measured. the resin was washed with distilled water and then dried using an oven. further the resin was used in the regeneration and desorption experience concentration of cu (ii) in solution was determined by spectrophotometric method with rubeanic acid ( =395 nm, linear range 1-4 mg cu /l) using a hach dr/2000 spectrophotometer. concentration ratio was calculated using equation 1. o r d c c c (1) where, rc is concentration ratio, 0c is copper concentration in solution used in the resin exhausted process (eq./l) and dc is copper concentration in regeneration solution after resin regeneration process. recovery percent meaning the percent of copper recovery in the regeneration solution from the copper retained onto ion exchange resin was calculated using equation 2. * *100 *p d a q r c v (2) where, pr is recovery percent in %, a the resin dose, g/l, q amount of cu (ii) retained on resin, mg/g, dc is copper concentration in regeneration solution after resin regeneration process, and v the volume of regeneration solution. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 54 3. results and discussion 3.1. desorption using hydrochloric acid to study the resin regeneration process a range of resin dose from 1 to 25 g dried exhausted resin/l. in the regeneration experiments the concentration hydrochloric acid solutions were varying in the range 0.1 2 eq./l. figure 1 present copper concentration ratio using hydrochloric acid solution for regeneration. using a concentration regeneration solution of 1eq hcl/l concentration ratio increase from 0.54 for o resin dose of 1 g dried exhausted resin/l to 12.21 for a resin dose of 25 dried exhausted resin/l. using a concentration of 2eq. hcl/l the concentration ratio increase from 0.54 for o resin dose of 1 g dried exhausted resin/l to 13.40 for a resin dose of 25 dried exhausted resin/l. figure 1. copper concentration ratio varying dried exhausted resin dose figure 2. copper recovery percent using a regeneration solution of 2eq hcl/l and varying dried exhausted resin dose figure 2 illustrate the influence of dried exhausted resin dose onto copper recovery percent using a regeneration solution of 2eq hcl/l. using the same volume of regeneration solution and the same concentration of hydrochloric acid the percent of recovered copper decrease 100% for a dry exhausted resin above 5 g/l. 3.2. desorption using sulfuric acid in the regeneration experiments using sulfuric acid the concentration of regeneration solution was varying in the range 0.1 to 2 eq h2so4/l and the dry exhausted resin was varying in the range 1 to 25 g/l. figure 3 present the influence of dry exhausted resin dose onto copper concentration ratio using sulfuric acid solution for regeneration. figure 3. copper concentration ratio using a regeneration solution of h2so4 and varying dried exhausted resin dose using a concentration of 2eq. hcl/l the concentration ratio increase from 0.53 for o resin dose of 1 g dried exhausted resin/l to 12.67 for a resin dose of 25 dried exhausted resin/l. the concentration ratios obtained using hydrochloric acid solution for regeneration were higher up to 6 percents by values obtained using sulfuric acid solution with the same concentration for regeneration. figure 2 illustrate the influence of dried exhausted resin dose food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 55 onto copper recovery percent using a regeneration solution of 2eq h2so4/l. figure 4. copper recovery percent using a regeneration solution of 2eq h2so4/l and varying dried exhausted resin dose using the same volume of regeneration solution and the same concentration of sulfuric acid, 2eq./l, the percent of recovered copper decrease for a dry exhausted resin above 3 g/l. 3.2. desorption using disodium edta in the regeneration experiments using disodium edta, the concentration of regeneration solution was varying in the range 0.1 to 0.2 meq.disodium edta/l and the dry exhausted resin was varying in the range 12.5 to 37.5 g/l (figure 5). figure 5. copper concentration ratio using a regeneration solution of disodium edta and varying dried exhausted resin dose using a concentration of 0.2 meq. disodium edta/l the concentration ratio increase from 2.15 for o resin dose of 12.5 g dried exhausted resin/l to 9.11 for a resin dose of 25 dried exhausted resin/l. 4. conclusion the highest rate of regeneration, 13,40, was obtained using hydrochloric acid solution of 2eq/l and a high dose of dried exhausted resin. good rates of regeneration was also obtained using sulfuric acid solution of 2eq/l or 0.2meq disodium salt of ethylenediaminetetraacetic acid/l and a high dose of dried exhausted resin. 5. references [1]. kurniawan t. a., chana g.y.s., loa w., babel s., physico–chemical treatment techniques for wastewater laden with heavy metals, chemical engineering journal, 118, 83– 98, (2006). [2]. bulai p., balan c., bilba d., macoveanu m., study of the copper (ii) removal from aqueous solutions by chelating resin purolite s930, environmental engineering and management journal, 8, 213-218, (2009). [3]. polat h., erdogan d., heavy metal removal from waste waters by ion flotation, journal of hazardous material., 148, 267–273, (2007). [4]. smara a., delimi r., poinsignon c., sandeaux j., electroextraction of heavy metals from diluted solutions by a process combining ion-exchange resins and membranes, separation and purification technology, 44, 271–277, (2005). [5]. bernat x., fortuny a., stüber f., bengoa c., fabregat a., font j., recovery of iron (iii) from aqueous streams by ultrafiltration, desalination, 221, 413–418, (2008). [6]. popa c., bulai p., macoveanu m., the study of iron(ii) removal from 34% calcium chloride solutions by chelating resin purolite s930, environmental engineering and management journal, 9, 651-658, (2010). [7]. juang r.s., shiau r.c., metal removal from aqueous solutions using chitosan-enhanced membrane filtration, journal of membrane science, 165, 159–167, (2000). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 contents: 1. risk to human health caused by non conditional drinking water borys skip, sonia gutt , štefan stanko 5 2. incorporation of sweet pepper extracts to improve thermal stability of vegetable oil mixtures tatiana capcanari 13 3. phe no lic co nt e nt and ant io xida nt act iv it y in milli ng fr act io ns o f ry e cristian serea, octavian barna 19 4. rheological, textural, color and physico-chemical properties of some yogurt products from the spanish market mircea-adrian oroian, isabel escriche, gheorghe gutt 24 5. quality evaluation of services provided by the restaurant “president” from mangalia by analyzing customer’s satisfaction regarding the waiter’s taking order mihaela constandache, elena condrea, anghelina dudan 30 6. comparative study of methods for assessing escherichia coli species in drinking water adriana dabija, laura pîslaru, cristina-gabriela constantinescu (pop), amelia buculei 35 7. the sunflower seeds lipase activity on various substrata under the influence of some outer and inner factors marcel avramiuc 40 8. impact of temperature and storage time on sucrose content in sugar beet octavian barna, octavian baston, aura darabă 44 9. mathematical model for optimization of zinc-nickel alloy co-deposition process violeta vasilache 48 10. life cycle assessment new direction in addressing systemic processes in food industry viorel iftimi 54 11. the variation of annual rainfall in the suceava plateau ioan gontariu , ion tănasă 59 12. correlation analyses between some technolgical parameters of some romanian wheat varieties grown in the suceava area ana leahu, georgiana gabriela codină, silvia mironeasa, cristina damian 65 13. ways of optimizing the nutritional value of dietetic pastry products iuliana cioarbă , ana batariuc 70 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 14. analysis of toxic and trace metal contaminants in bottled water by using atomic absorption spectrometry gholamreza jahed khaniki, mansoor ghaderpoori, mohammad hadi dehghani, shahrokh nazmara 78 15. influence of some surfactants and flocculants on interparticle interactions in aqueous suspensions of hydro-mica sergey boruk, igor winkler, sonia gutt 84 16. the effect of rural settlements on water quality in northern suceava plateau andrei-emil briciu, dinu oprea-gancevici 88 17. author instructions 97 181 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 181 189 study on the influence of samples’ dimensions on the analysis of smoked cheese texture properties *elena sănduleac (todosi sănduleac)1, gheorghe gutt1, andreea ianovici (ianovici iordache)1 1faculty of food engineering, stefan cel mare university of suceava, romania sanduleacelena@yahoo.com, g.gutt@fia.usv.ro, andreea_iordache88@yahoo.com *corresponding author received april 17th 2015, accepted june 17th 2015 abstract. one of the main objectives of this study is to determine the optimal geometry of the probe in order to provide an advanced sensitivity of testing and also to ensure a high reproducibility of data and to establish the influence of the sample’s dimensions on the analysis results of texture parameters. in the study on the modification of the texture parameters values depending on the sample dimension, the force-displacement curves were plotted for smoked cheese, which was kept refrigerated at the temperatures between 6-8°c. regarding the type of texture profiling test the penetration test was carried on utilizing probes with different geometries and sizes, and the cheese samples were of different sizes. six different types of penetrating devices were used: artificial finger, cone, ball, compression disk, v-knife and blunt knife. the cheese samples were cut into a parallelepiped shape with sides varying from 20 mm, 25 mm, 30 mm, 35 mm to 40 mm and different thicknesses: 7 mm, 9 mm, 11 mm, 13 mm, 15 mm, 17 mm. the samples were mechanically tested with the penetrating devices resulting force-displacement curves. key words: cheese, curve slope, force-displacement curve, sample size, sensitivity, texture 1. introduction smoked cheese belongs to the category of cheese with scalded paste made from whole cow's milk or normalized, pasteurized cow's milk by adding selected lactic cultures and rennet coagulation, based on the technological process of blanching the curd, smoking with bio hardwood resin and followed by its maturation. cheese texture may be defined as a composite of sensory attributes resulting from a combination of physical properties perceived by the sense of sight and touch [1]. a number of factors, both compositional and process parameters, are known to influence texture of cheese [2]. a good knowledge of textural properties is important for all the stakeholders in the food chain, for manufacturers, processing, marketing and consumers. texture was defined by a series of authors, but the definition given by the sr iso 11036:2007 standard is the most representative because it best highlights the texture notion of a food product: „texture represents all mechanical, geometrical and surface properties of a food product, discernible through touch or mechanical receptors and where appropriate, through visual and auditory receptors”[13]. many scientists, engineers and technologists have made contributions in the mechanical properties evaluation to understand the texture subjectively [4], while scientists of materials have developed rheology and fracture http://www.fia.usv.ro/fiajournal mailto:sanduleacelena@yahoo.com, mailto:g.gutt@fia.usv.ro, mailto:andreea_iordache88@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 182 mechanics for understanding material properties of food products [5]. the most recent reviews, some researchers [6], [7] have examined specific issues related to food texture, including sensory and instrumental measurements. to overcome the limitations of human sensory perception of food in terms of texture, instrumental methods were developed [8] and a great effort has been made in improving measurement tools and techniques for a significant prediction of textural properties [9], [10]. because human sensory perception of texture largely depends on the mechanical behavior of food, which will determine the dynamics of breakdown during mastication [11], most of the objective measuring research are based on the mechanical/rheological properties of food the types of experiments can be classified as fundamental, empirical and imitative methods [12]. penetration test for texture determination measures the force required for a device to penetrate instrumentally a food product or a raw food material. penetration of food products causes irreversible or reversible deformation. puncture tests are some of the simplest techniques for measuring the texture properties widely used [12]. one of the main objectives of this study is to determine the optimal geometry of the probe in order to provide an advanced sensitivity of testing and also to ensure a high reproductibility of data and to establish the influence of the sample’s dimensions on the analysis’ results of texture parameters. 2. materials and methods we took cheese samples with rectangular shape with a square base with l=20mm and different heights (7mm, 9mm, 11mm, 13mm, 15mm and 17mm).afterwards, noting the slope of the sensitivity curve, it was chosen an optimum thickness of 15mm and it was modified the square side (cuboid base) 25mm, 30mm, 35mm and 40mm respectively, in order to highlight the sample size influence on texture profile analysis results. it was used the mark 10 texturometer, at the rate of application of 10 mm/min, a mesur gauge specific software for data interpretation and also a serie of other penetrating devices designed and obtained in the food safety research laboratory of food engineering faculty of suceava. for the purpose of determining the highest sensitivity (noted with „s”) the tangent of the α angle was used corresponding to the curves of various penetrators with the abscissa (penetration depthh) or mechanical work: )1( h f tgs   )2( max 0  f fdhw it was used penetrating devices with various shapes and dimensions (artificial finger, ball, compression disk, cone, blunt knife and v knife) such as the following: fig. 1. penetrating devices: aartificial finger, b-ball, c-compression disk, d-cone, e-blunt knife, fv knife food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 183 3. results and discussion fig. 2. families force-penetration curves made with different samples of cheese penetrating with dimensions 20x20 mm square side and the thickness of samples to: a) 7 mm, b) 9 mm, c) 11 mm, d) 13 mm, e) 15 mm, f)17 mm. figure 2 shows force-displacement curves on a penetration depth of 5 mm using various penetrating devices having different shapes: ball, cone, blunt knife, v knife, artificial finger, compression disk and the smoked cheese samples were cut into parallelipipedic shape with the square side l=20x20 mm and different thickness (7 mm, 9 mm, 11 mm, 13 mm, 15 mm, 17 mm). force-displacement curves shows that the highest sensitivity, given by the force/displacement ratio, is when the thickness of the sample measures 15 mm. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 184 fig. 3. families force-penetration curves made with different penetrating the cheese samples with a thickness of 15 mm with sides: a-20 mm, b-25 mm, c-30 mm, d35 mm, e-40 mm figure 3 shows force-displacement curves on a penetration depth of 5 mm using various penetrating devices having different shapes: ball, cone, blunt knife, v knife, artificial finger, compression disk and the thickness of the smoked cheese samples was 15 mm and its sides 20 mm, 25 mm, 30 mm, 35 mm, 40 mm. the highest sensitivity is in the case of the sample with the side of 35 mm. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 185 fig. 4. families force-displacement curve for samples with thickness of: 7 mm, 9 mm, 11 mm, 13 mm, 15 mm, 17 mm and the side of the square of 20x20 mm made with different penetrating devices: a-ball, bcone, c-blunt knife, dv knife, eartificial finger, f-compression disk figure 4 shows the sensitivity variation of the method of textural determination properties (hardness) using the penetration method with different penetrating devices: ball, cone, blunt knife, v knife, artificial finger, compression disk. the highest sensitivity was obtained when using the artificial finger as a penetrating device. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 186 fig. 5. families force-displacement curve for samples of cheese with 15 mm thickness and sides of 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, made with different penetrating devices: aball, bcone, cblunt knife, dknife v, eartificial finger, fcompression disk figure 5 presents the influence of the cheese sample’s side size over the sensitivity variation using the penetration method with penetrating devices having different geometries. the highest sensitivity was obtained when the sample’s dimensions where of 15 mm thickness and sides of 35 mm, utilizing the artificial finger as a penetrating device. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 187 table 1. mechanical work variation depending on thickness of the samples with the base remaining constant using different penetrating devices, displacement 5mm penetrating devices sample side [mm] sample tickness [mm] mechanical work [n*mm] cone 20 7 11.96 9 17.19 11 19.99 13 14.3 15 16.1 17 16.5 blunt knife 20 7 22.24 9 21.23 11 25.89 13 24.32 15 22.08 17 24.65 vknife 20 7 39.07 9 35.19 11 32.62 13 33.81 15 35.4 17 37.05 artificial finger 20 7 52.7 9 49.7 11 36.1 13 42.9 15 75.6 17 63.5 compression disk 20 7 51.9 9 49.7 11 48.8 13 60.1 15 60.8 17 58.2 ball 20 7 16.97 9 28.36 11 22.63 13 19.87 15 23.11 17 22.32 table 1 lists the mechanical work as integral under the curve of the load on the depth of 5mm: )3( max 0  f fdhw obtained after penetrating the cheese samples having parallelepiped shape with square base and base side 20 mm. the thickness of the samples was modified and mechanical tests were conducted using penetrating devices of different shapes and geometries. it resulted that the maximum value of the mechanical work was recorded in case of the sample with 15 mm thickness and utilizing the artificial finger as a penetrating device with l=75, 6n*m. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 188 table 2. mechanical work variation depending on the sample side with thickness remaining constant using different penetrating devices, displacement 5mm penetrating devices sample tickness [mm] sample side [mm] mechanical work [n*mm] cone 15 20 16.1 25 9.6 30 12.8 35 14.9 40 14 blunt knife 15 20 22.08 25 21.7 30 26 35 26.3 40 25.3 vknife 15 20 35.4 25 43.1 30 39.4 35 39.5 40 38.8 artificial finger 15 20 75.6 25 93.2 30 70.7 35 103 40 94.8 compression disk 15 20 60.8 25 78.9 30 76.4 35 82.6 40 97.3 ball 15 20 23.11 25 19.4 30 20.9 35 24.5 40 21.1 in table 2 are registered mechanical work values obtained from tests by penetration with six different devices, changing the side of the parallelepiped and keeping the 15 mm thickness. the maximum value of the mechanical work in this case was recorded when the artificial finger was used as a penetrator and the square side of the parallelepiped of 35 mm, l=103 n*m. 4. conclusions one of the criteria for choosing a certain analysis method is represented by sensitivity. the aim of this study was to establish the sensitivity of the method for determining the texture properties of smoked cheese (hardness) through the slope of force-displacement curves, on a default penetration depth of 5 mm, as well as from the values of mechanical work, which represents the area under the forcedisplacement curve. sensitivity is influenced by the sample’s dimensions and by the geometry of the penetrating device. by using a large number of penetrators, having various geometries and dimensions, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 elena sănduleac (todosi sănduleac), gheorghe gutt, andreea ianovici (ianovici iordache), study on the influence of sample's dimension on the analysis of smoked cheese texture properties, food and environment safety, volume xiv, issue 2 – 2015, pag. 181 – 189 189 it was possible to conduct a study on choosing the optimal type of penetrator for hard cheese food-type. considering the experimental results, high anisotropy of cheese and also literature recommendations regarding the need for a more uniform axial distribution of charging units from mechanical testing, the most recommended penetrator for making the texture profile using the penetration method is the artificial finger and the optimal dimension of the sample is a parallelipiped with a square base having l=35 mm and height 15 mm. 5. references [1]. gehan a.m. hussein, samah m. shalaby, microstructure and textural properties of kareish cheese manufactured by various ways, annals of agricultural science, 59(1): 25-31, (2014). [2]. wium h, pedersen p.s., qvist k.b., effect of the coagulation conditions on the microstructure and the large deformation properties of fat-free feta cheese made from ultrafiltered milk, food hydrocoll, 17, 287-296: (2003). [3]. dan h., hayakawa f., kohyama k., modulation of biting procedures induced by the sensory evaluation of cheese hardness with different definitions, appetite, 50: 158-166, (2008). [4]. saeleaw m., schleining g. a review, crispness in dry foods and quality measurements based on acoustic-mechanical destructive techniques, journal of food engineering, 105: 387-399, (2011b). [5]. rogers n.r., drake m.a., daubert c.r., mcmahon d.j., bletsch t.k., foegeding e.a., the effect of aging on low-fat, reduced-fat, and full-fat cheddar cheese texture. journal of dairy science, 92: 4756-4772, (2009). [6]. szczesniak a.s., texture is a sensory property, food quality and preference, 13: 215225, (2002). [7]. de wijk r.a., janssen a.m., prinz j.f., oral movements and the perception of semisolid foods, physiology & behavior, 104: 423-428, (2011). [8]. costa f., cappellin l., longhi s., guerra w., magnago p., porro d., soukoulis c., salvi s., velasco r., biasioli f., gasperi f., assessment of apple (malus×domestica borkh.) fruit texture by a combined acoustic-mechanical profiling strategy, postharvest biology and technology, 61: 21-28, (2011). [9]. oraguzie n., alspach p., volz r., whitworth c., ranatunga c., weskett r., harker r., postharvest assessment of fruit quality parameters in apple using both instruments and an expert panel, postharvest biology and technology, 52: 279-287, (2009). [10]. zdunek a., cybulsk j., konopacka d., rutkowski k., new contact acoustic emission detector for texture evaluation of apples, journal of food engineering, 99: 83-91, (2010a). [11]. foegeding e.a., çakir e., koç h., using dairy ingredients to alter texture of foods: implications based on oral processing considerations, international dairy journal, 20: 562-570, (2010). [12]. bourne m. c., food texture and viscosity (2nd ed). new york: academic press, (2002). [13]. sr iso 11036:2007, sensory analysis methodology -texture profile. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 47 multivariate statistical analysis of royal feteasca wine quality from different regions of romania country silvia mironeasa1, georgiana gabriela codină1, ana leahu1, costel mironeasa2 1stefan cel mare university, faculty of food engineering, 13th university street, 720229, suceava – romania, e-mail : silviam@usv.ro; codina@usv.ro; leahu@usv.ro 2 ştefan cel mare university, faculty of mechanical engineering, mechatronic and management, suceava – romania 13th university street, 720229, suceava – romania, e-mail: costel@fim.usv.ro abstract: the objective of this study is to establish a relation between the physico-chemical parameters and the sensorial characteristics of the royal feteasca wine from eight viticulture regions from romania. the correlation between the physico-chemical parameters of wine and the sensorial characteristics were analyzed using the principal component analysis (pca) method. the analysis of variation and the principal component analysis showed that there were significant correlations between the wines from the hills of banat viticulture region – the winegrowing center recas, the hills of dobrogea viticulture region – the winegrowing center murfatlar and the danube’s terraces viticulture region – the winegrowing center fetesti. the wines from these viticulture regions were the best renowned (the silver medal) both sensory – using the sensorial analysis of the wines, with denotation in the penalty points, and physicochemical through parameters – density, alcoholic concentration, total acidity, volatile acidity and total dry extract. the multivariate analysis of the data show significant differences between various variables that were used for the quality assessment of wine. significant direct correlations were obtained between taste-quality, taste–intensity, odourintensity and clarity (r = 0.800, r = 0.775, respectively r = 0.800) and reverted correlations between odour–quality and wine density and volatile acidity (r = 0.712, r = 0.771), correlation is significant at the 0.05 level. keywords: romanian wines, principal component analysis, physico-chemical parameters, sensory evaluation introduction royal feteasca is an assortment of grapes originating from transylvania, 7-8 decades ago and cultivated in the majority of romanian vineyards. the wines obtained from this assortment have the following characteristics: moderate alcoholic concentration, light, balanced, restful, but their main characteristic remains the flavour, a very interesting floral flavour [1]. thanks to its valuable characteristics, the wines are largely spread in plantations, being cultivated in almost all vineyards from the country. the royal feteasca wines produced in the vineyard from tarnave, in general from transylvania, have a special quality [2]. the quality of the wine is determined both by the physico-chemical parameters and sensorial characteristics. the main physico-chemical parameters which are computed usually for evaluating the quality of wine are: density, alcoholic concentration, total acidity, volatile acidity, content of total sugars, dry irreducible extract etc. sensory analysis involves a series of techniques designed to study wine attributes, how they are perceived and how they relate to features such as chemical, varietal, regional or stylistic origin. that is, these are the primarily research tools. these procedures include techniques such as discrimination testing, descriptive sensory analysis, timeintensity analysis and charm analysis. as in all tests, specific instructions on how the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 48 procedure is to be conducted and clear indications of what is expected are essential if the desired results are to be obtained [3]. in romania, wine results are usually reported to the consumer in the form of a specific type of medal, i.e. gold, silver or bronze according to sr 13461:2001. descriptive analysis transcends quality and differences in measurements by determining sensory attributes and physico-chemical parameters that differ from royal feteasca wines were studied. from our knowledge, there have been made few studies using multivariate analysis to characterize the impact of physico-chemical parameters on sensory attributes of wines. therefore, we conducted a study in which sensory attributes of wines from different eight viticulture regions from romania were evaluated by the method of grading through penalty points. the obtained data were correlated to the physico-chemical parameters which were computed using the principal component analysis (pca). materials and methods wines-study material. eight commercial royal feteasca wines (harvest 2008) were provided by different vineyards, abbreviated as shown in table 1. physico-chemical analysis. the physicochemical parameters of wines were determined according to romanian standard methods: density (stas 6182/8-71), alcoholic concentration (stas 6182/6-70), total acidity (sr 6182-1:2008), volatile acidit y (sr 61822:2008), sugars (sr 6182-18:2009) and total dry extract (stas 6182/9-80). all analytical determinations were performed at least in triplicate. values of different parameters were expressed as the mean of all measurements. table 1. the source of the studied royal feteasca wines viticulture region vineyard winegro wing center abbreviation moldovia's hills husi husi a transylvania plateau tarnave jidvei b banat hills recas c moldovia's hills cotnari cotnari d dobrogea hills murfatlar murfatlar e crisana and maramures hills silvaniei ratesti f muntenia and oltenia hills dealu mare valea calugarea sca g danube’s terraces ostrov fetesti h sensory analysis. sensory testing has been carried out, in conformity with the method described by sr 13461:2001 which mentions a sensorial analysis of the wines by scoring them with penalty points. in order to use this type of sensorial analysis, a 13 member panel was selected from available students and personnel at “stefan cel mare” university, faculty of food engineering, (five men and eight women, aged between 21 and 50 years). the majority of the judges had participated in previous sensory tests, but only a few had extensive wine tasting experience. all wines were presented in coded (according sr iso 6658:2007) standard, tulip-shaped, clear 250 ml wine glasses and evaluated at 10-12oc in the sensory laboratory of the food engineering faculty from suceava. a 50 ml sample of wine was poured into each glass and covered with a watch glass at least for 30 min prior to testing. distilled water was provided for cleaning the palate between wines. the organoleptic properties of wine, measured trough this sensorial method of analysis, are: clearness of wine, intensity and quality of odour, intensity and quality of its taste, colour and harmony of the whole product. the sensorial impression, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 49 individually determined for every organoleptic property, is quantified by 4 penalty coefficients as it follows: excellent: 0; very good: 12 = 1; good: 22 = 4; acceptable: 32 = 9; eliminated: –. this penalty coefficients are established by combining a consecutive logarithmic method with 5 correlated factors of importance: 1. clarity; 2. odour-quality; 3. taste-intensity; 4. taste-quality; 5. harmonycolour. the penalty points are calculated, resulting in the total penalty score. using the total penalty scores, which were assigned to every wine type by the members of tasting board, a panel score range is determined, representing the only score that can be found at the middle of the individual wine scores, as sorted in ascending order. the connection established between the qualitative graduations, median penalty points and award is represented in table 2. table 2. quality scoring ranges used to allocate quality designations (sr 13461:2001) qualitative graduations median penalty points award excellent 0...6 gold medal very good 7...8 silver medal good 9...11 bronze medal acceptable 12...48 honorary degree acceptable 49...108 degree of participation eliminated  109 data analysis. in order to make the correlation analysis between the two variables: one is the effect (the dependent one), the other is the cause (the independent one) the bivariate correlation was used. the selected method to analyze the interdependence between all the variables involved in the wine quality evaluation is the principal component analysis (pca). pca is a multivariate analysis technique that is intended to reduce the number of variables by preserving as much as possible the variance of original data, resulting in a smaller set of variables. in this way, new variables (principal components) are determined, expressed as linear combination of original variables that have both no correlation in between and maximum variance. the graphical representations visualize the relations between variables, while allowing the detection of possible groups of variables. pca is in fact a powerful method for summarizing variations in many variables of wine quality, since it is able to project the principal variation onto a few principal components. factors extracted are retained if they have an eigenvalue   1(kaiser's criterion) because they bring a lot more information than the initial variables. the principal components are pairs of uncorrelated variables: the first principal component has maximum variance while the second component has a variance as high as possible, but less than the value of the first component. samples and variables were compared and interpreted through two dimensional bi-plots that visually represented the results of the pca [4]. bi-plots show the position of explanatory variables and identify how the variables relate to both the principal components and other input. the goal of the principal component analysis is to establish groups of similar samples and significant variables and use those to distinguish among the studied cases. the principal component analysis was performed using spss on the covariance matrix of the variables involved, in order to illustrate the relationships between sensory characteristics and physico-chemical parameters of wine. results and discussion physico-chemical parameters. table 3 presents the physico-chemical parameters of royal feteasca type of wine from all the eight wine regions of romania. all the values shown in table 3 are means of the three determinations. the variation limits of organoleptic characteristics which are used to evaluation wine quality are shown in table 4. the alcoholic concentration is one of the most important parameters defining wine quality. considering the analyzed samples, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 50 the alcoholic concentration did not have significant variations between 10.92% v/v (sample f) and 12.45% v/v (sample d). although sample a has a higher alcohol content (12.19% v/v.) it was less appreciated comparing with the other analyzed samples, probably due to the lowest dry extract (25.19 g/l) and sugar content (8.0 g/l). table 3 physico-chemical parameters royal feteasca wine samples range physico-chemical parameters abbreviated name mean min max cv % wine density at 20oc, [g/cm3] w_d 0.99 0.9937 0.9993 0.19 alcoholic concentration, [% v/v] a_c 11.65 10.92 12.28 4.64 total acidity, [g/l sulphuric acid] t_a 4.51 4 5.22 9.48 volatile acidity, [g/l sulphuric acid] v_a 0.48 0.2 0.88 40.94 sugar, [g/l] s 8.89 7.7 10.45 11.11 total dry extract, [g/l] t_d_e 31.10 25.19 39.55 16.96 table 4 organoleptic characteristics used to assess the royal feteasca wine samples range organoleptic characteristics abbreviated name min max clarity cl 0 1 odour intensity o_i 0 4 odour quality o_q 0 8 taste intensity t_i 2 18 taste quality t_q 0 12 harmony colour h_c 3 12 the density of wine depends on the content of extractive substances and alcohol, but also on temperature. the highest value of density was found at the sample h – the wine originating from danube’s terraces region. the amount of remaining unfermented sugars is in small and variable quantities and has an important contribution in identifying the wine types. this has an important role in the taste sensorial assessment of wine and contributes to its completion. the content in sugars gives the type of wine and has a direct relation with the geographical position of the wine region [5]. the samples with the highest sugar content (h = 10.45 g/l; e = 9.8 g/l) were appreciated fairly well by the taster (second place) considering the odourintensity, odour – quality, taste – intensity, taste-quality parameters. this result is due to the fact that a higher content of sugars highlights and exacerbates the flavour, providing the wine with better taste. another parameter, acidity, provides physico-chemical stability to wine and gives its bright colour and fresh taste. total acidity and volatile acidity are directly connected to its sensorial assessment. the acidity sour effect can be attenuated by the total sugar content. the wine extract is crucial for its sensorial assessment. it gives corpulence, fullness, amplitude, generosity to wine. a higher of solid content gives wine greater consistency. a wine with high alcoholic strength has higher extract content because it is made from concentrated musts that are rich both in sugars and other compounds. a significant association between total dry extract content and alcohol content has been found by comparing all the samples. a major correlation exists between total dry extract and the organoleptical parameters of taste-intensity and taste – quality, probably due to the higher sugar content in some of these wines. this correlation makes difficult to properly assess the corpulence of wine. for example: although samples d and g had a higher solid food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 51 content (37.8 g/l and 31.33 g/l), they were less appreciated than sample c (29.26 g/l) because of the fact that they had a lower sugar content and higher acidity. this specific balance had a significant influence over the organoleptical assessment of taste-intensity and taste-quality parameters. although, compared to the other samples analyzed, sample c is characterized by a low alcohol content (11.0% v/v) and a moderate content of total dry extract (29.26 g/l), it won the second place in consumer preferences (silver medal), perhaps due to a combination of relatively high sugar content (9.1 g/l) and lowest value of total acidity (4.1 g/l sulfuric acid). its low acidity has created a sweet taste sensation, more intense compared with samples e, h, which although having a higher sugar content (9.8 g/l and 10.45 g/l) were rated as less sweet thanks to their higher acidity (4.73 g/l sulfuric acid, i.e. 5.22 g/l sulfuric acid). the correlation between the results of tsensory testing and the analysis of physicochemical parameters obtained for royal feteasca wine coming from all the eight wine regions of romania are represented in figure 1(a). the first two principal components explain 97.63% of the total variance (pc1 = 92.91% and pc2 = 4.72%). concerning the first principal component, pc1, a very good correlation between the wines from regions h and c (r = 0.990), h and e (r = 0.997) was found and correlation coefficients are significant at the 0.01 level. wines from regions e, h, c and d are strongly associated with the first principal component pc1. compared to the second principal component, pc2, the quality of wine coming from region a is opposed to that of the wines coming from wine regions e, h and c. the second principal component is strongly associated with the quality of wine from region a, characterized by a high value of clarity (cl) variable. (a) (b) figure 1. (a) score plot pc1 and pc2 showing interrelationships among royal feteasc wines from eight regions of romania; (b) correlation loading plot of pc1 and pc2, showing interrelationships among sensory characteristics and physico-chemical parameters pca loadings of the physico-chemical parameters and the variables used in sensory wine evaluation are represented in figure 1 (b). the two principal components represented here 64.83% and 15.09% of the total variance. the plot of pc1 vs. pc2 loadings shows, along the pc1 axis, a close association between the total acidity (t_a), density (w_d), total dry extract (t_d_e) and volatile acidity (v_a), variables strongly associated with this specific axis. in other words, pc1 extends along the maximum variance of these variables. in conclusion we can say that the total acidity, density, dry extract, total and volatile acidity parameters have a distinctive role in assessing the royal feteasca wine from regions c, h and e. another variable, opposed to the total dry extract parameter (t_d_e), but nevertheless contributing to pc1 is odour quality variable (o_q). pc2 distinguishes between clarity (cl) and sugar content (s). clarity (cl) shows food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 52 a positive effect on taste-quality (t_q), taste – intensity (t_i) and a negative one on total acidity (t_a), density (w_d), total dry extract (t_d_e). compared to the second principal component pc2, the intensity odour (o_i) variable is placed at the top while quality (o_q) variable is placed at the bottom. pc2 clearly has something to do with discriminating between the specific variables of sensory analysis. compared to the second principal component, odour quality (o_q), taste quality (t_q), taste intensity (t_i), clarity (cl) and odour intensity (o_i) variables, are located on the left graph and the harmony colour (h_c) variable is on the right. this leads to the observation that the contribution of the first variable to the appreciation of wine is less important than the one of the right. the taste quality variable (t_q) is opposed to alcoholic concentration (a_c). significant direct correlations were obtained between density and volatile acidity (r = 0.835), respectively total dry extract (r = 0.941), and correlation coefficients are significant at the 0.01 level. wine density and odour quality is inversely correlated, r = 0.712, given the significance level of 0.05. total acidity is directly correlated with total dry extract, r = 0.869 (significant correlation coefficient at the 0.01 level), also with wine density, r = 0.769 (significant correlation coefficient at the 0.05 level). volatile acidity is directly correlated with total dry extract, r = 0.807 and inversely correlated with odour quality, r = 0.771 (significant correlation coefficients at the 0.05 level). a very good correlation was obtained between the clarity and odourintensity (r = 0.800), taste intensity (r = 0.775), respectively taste-quality (r = 0.800), (significant correlation coefficients at the 0.05 level). this shows a relatively deterministic connection. conclusions the principal component analysis of the data set shows a high association between some physico-chemical parameters (density, total acidity, volatile acidity, total dry extract) and some of the sensorial characteristics of wine (odor quality, taste-quality and panel score range). the correlation established with pca between the determined physico-chemical parameters and the results obtained by sensory assessment has highlighted the best correlation. this is between density, total acidity, respectively volatile acidity on one part and taste intensity, taste-quality, odour intensity, odour quality respectively clarity characteristics on the other. wines from regions c, e and h have been best appreciated, receiving a silver medal, while physico-chemical parameters (density, total acidity, volatile acidity, total dry extract) had a key role in the assessment. references 1. stoian, v., 2006. marea carte a degustării vinurilor, editura artprint, bucureşti. 2. macici, m., 1996. vinurile româniei, editura alcor edimpex srl, bucure;ti. 3. jackson, r.s., 2009. wine tasting. a professional handbook, 2nd ed. 4. gabriel, k.r. 1971. the biplot graphic display of matrices with application to principal component analysis. biometrika 58, 453–467. 5. ţârdea, c., 2007. chimia şi analiza vinului, editura „ion ionescu de la brad”, iaşi. 6. sr 13461:2001. wine. senzory analysis, standardization association of romania, bucharest, romania. sr iso 6658. sensory analyses. methodology. general guidance, standardization association of romania, bucharest, romania. 7. sr iso 11035. sensory analyses. identification and selection of descriptors for establishing a sensory profile by a multidimensional approach, standardization association of romania, bucharest, romania. 8. stas 6182/8-71. wine. determination of density, standardization association of romania, bucharest, romania. stas 6182/6-70. wine. determination of alcohol ethyl content, standardization association of romania, bucharest, romania. 9. sr 6182-1:2008. wine. part 1: determination of total acidity, standardization association of romania, bucharest, romania. 10. sr 6182-2:2008. wine. part 2: determination of volatile acidity, standardization association of romania, bucharest, romania. 11. sr 6182-18:2009. wine. part 18: determination of reducing sugar, standardization association of romania, bucharest, romania. 12. stas 6182/9-80. wine. determination of total dry extract, standardization association of romania, bucharest, romania. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 15 probiotic properties of lactobacillus acidophilus z10, isolated from naturally fermented sourdough *rositsa denkova1, svetla ilieva1, donka dimbareva2, zapryana denkova2 1sofia university “st. kliment ohridski”, department “biotechnology”, rositsa_denkova@mail.bg 2university of food technologies, department “microbiology”, zdenkova@abv.bg *corresponding author received 6 august 2012, accepted 7 september 2012 abstract: the strains included in the composition of probiotic preparations have to possess a number of probiotic properties including the ability to survive and reproduce in the conditions in the gastrointestinal tract and antibiotic resistance. the resistance of lactobacillus acidophilus z10, isolated from naturally fermented sourdough, to gastric and pancreatic juices is examined by incubation of the strain in mrs-broth medium with ph=2, containing pepsin; ph=4.5, containing pancreatin and ph=7, containing pancreatin, as well as incubation in mrs-broth medium, containing different concentrations of bile salts. the profile of antibiotic resistance is determined by the disc diffusion method. the cells of lactobacillus acidophilus z10 are resistant to the model conditions of the gastrointestinal tract. the profile of antibiotic resistance of lactobacillus acidophilus z10 against 20 of the most commonly applied antibiotics in medical practice is examined and the strain is resistant to most of them. the resistance of the strain to most of the antibiotics included in the study together with its resistance to the model conditions of the gastrointestinal tract makes lactobacillus acidophilus z10 a potentially probiotic strain. key words: lactobacillus, probiotic, gastric juice, pancreatic juice, bile salts, antibiotic resistance 1. introduction probiotics are live microorganisms that confer a beneficial effect on the host when administered in proper amounts [1, 2]. their beneficial effects on gastrointestinal infections, the reduction of serum cholesterol, the protection of the immune system, anti-cancer properties, antimutagenic action, anti-diarrheal properties, the improvement in inflammatory bowel disease and suppression of helicobacter pylori infection, crohn's disease, restoration of the microflora in the stomach and the intestines after antibiotic treatment, etc. are proven by addition of selected strains to food products [3, 4, 5, 6]. lactobacilli and bifidobacteria are normal components of the healthy human intestinal microflora. they are included in the composition of probiotics and probiotic foods because of their proven health effects on the body [7, 8, 9]. they are the main organisms that maintain the balance of the gastrointestinal microflora [10]. not all strains of lactobacilli and bifidobacteria can be used as components of probiotics and probiotic foods, but only those that are of human origin, nonpathogenic, resistant to gastric acid, bile and to the antibiotics, administered in medical practice; they should also have the potential to adhere to the gut epithelial tissue and produce antimicrobial substances; they should allow the mailto:rositsa_denkova:@mail.bg mailto:zdenkova:@abv.bg food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 16 conduction of technological processes, in which high concentrations of viable cells are obtained as well as to allow industrial cultivation, encapsulation and freezedrying and they should remain active during storage [11, 12]. this requires the mandatory selection of strains of the genera lactobacillus and bifidobacterium with probiotic properties. moreover, the concentration of viable cells of microorganisms in the composition of probiotics should exceed 1 million per gram [13] in order for the preparation to exhibit a therapeutic and prophylactic effect. the survival of probiotic bacteria in the gastrointestinal tract, and their translocational and colonizational properties and the destruction of their active components are essential for the realization of their preventive role. different probiotic strains react differently in different parts of the gastrointestinal tract some strains are killed very quickly in the stomach, while others pass through the entire gastrointestinal tract at high concentrations [14, 15, 16, 17, 18, 19, 20, 21, 22]. the purpose of the present paper is to examine some of the probiotic properties of the strain lactobacillus acidophilus z10 isolated from naturally fermented sourdough: determination of the profile of antibiotic resistance, determination of the resistance of the strain to the model conditions of gastric and pancreatic juice, as well as to elevated concentrations of bile salts. 2. materials and methods microorganisms the studied strain lacobacillus acidophilus z10 is isolated from naturally fermented sourdough. nutrient media mrs – broth medium (medium of de man, rogosa & sharpe). composition (g/dm3): peptone from casein 10 g; yeast extract 4 g; meat extract 8 g; glucose 20 g; k2hpo4 2 g; sodium acetate 5 g; diammonium citrate 2 g; mgso4 0.2 g; mnso4 0.04 g; tween 80-1 ml; ph = 6.5. the medium is sterilized for 15 min at 118ºc. mrs – agar medium. composition (g/dm3): mrs broth +2% agar. the medium is sterilized for 15 min at 118ºc. laptg10-agar medium. composition (g/dm3): laptg10-broth medium + 2% agar. the medium is sterilized for 20 minutes at 121ºc. saline. composition (g/dm3): nacl 5 g; distilled water 1l. sterilization for 20 min at 121ºc. cultivation and storage of the studied microorganism the studied strain is cultivated in a liquid medium (mrs-broth) and on agar medium (mrs-agar) at 37ºc. it is isolated from a single colony and is cultivated in mrsbroth medium for 24 hours. the strain is stored as a stock-culture in mrs-broth with 20% v/v glycerol at -20ºc. determination of the profile of antibiotic resistance the profile of antibiotic resistance is determined by the disk diffusion method of bauer, kirby et al. [23]. fresh 24-hour culture of the tested strain is used to inoculate the plates with laptg10-agar. standard discs impregnated with antibiotics are placed in the plates. the plates are incubated for 48 hours at optimum temperature. the diameters (in mm) of the sterile zones formed around each of the antibiotic discs are recorded. then they are subjected to the following designations: r resistant (zone < 8 mm), food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 17 sr intermediately sensitive (zone 8-16 mm), s sensitive (zone > 16 mm). determination of the resistance to low ph in the presence of pepsin and to weakly alkaline ph in the presence of pancreatin [24] fresh 24 hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. the resulting sludge biomass is washed twice with pbs buffer and resuspended to the initial volume in pbs buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 buffer solution with ph = 2 containing 0,5% nacl and pepsin (at a concentration of 3.2 g/dm3) (sigma, 2,500 3,500 u / mg protein), buffer with ph = 4,5 + pancreatin and buffer with ph = 7 + pancreatin at a suitable temperature for the studied strain (37°c) for 24h. at the 0, the 2nd, the 4th and the 24th hour aliquots for the determination of the number of viable cells are taken (cfu/cm3). determination of the tolerance to bile salts (method modified by denkova z., 2005 [22]) fresh 24 hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. the resulting sludge biomass is washed twice with pbs buffer and resuspended to the initial volume in pbs buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 of the mrsbroth medium with different concentrations of bile salts 0%, 0.15%, 0.3%, 0.6% and 1% for 24h at the optimum temperature for the strain (37°c), and aliquots for the determination of the number of viable cells (cfu/cm3) at the 0, the 2nd, the 4th, the 6th, the 8th and the 24th hour are taken. 3. results and discussion a series of tests are conducted in order to determine the probiotic potential of the strain lactobacillus acidophilus z10 with optimum temperature 37° . in vitro determination of the ability of lactobacillus acidophilus z10 to survive in conditions simulating the various departments of the gastrointestinal tract the resistance of the cells of lactobacillus acidophilus z10 in model conditions of the gastro intestinal tract ph = 2 + pepsin, ph = 4,5 + pancreatin and ph = 7 + pancreatin is examined. in a parallel experiment the tolerance of this strain to different concentrations of bile salts is tested. the results of the experimental studies are presented on fig. 1 and fig. 2. fig. 1. survival of the cells of the strain lactobacillus acidophilus z10 in acidic ph (ph = 2) + pepsin, ph = 4,5 + pancreatin and ph = 7 + pancreatin. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 18 higher sensitivity to low ph = 2 + pepsin than to ph = 4,5 + pancreatin and ph = 7 + pancreatin is observed (fig. 1). by the 24th hour of cultivation of the strain at ph=2 + pepsin the concentration of viable cells decreases by 7logn. for 24 hour incubation at ph = 4,5 + pancreatin the reduction in the number of viable cells is 2.8 logn, while at ph = 7 + pancreatin – 2.4 logn. another important factor that influences the survival of probiotic strains in the intestinal tract are bile salts. it is known that about three hours after ingestion of food the concentration of bile salts in the small intestine reaches about 0.3%. this requires a study on the influence of different concentrations of bile salts on the development of lactobacillus acidophilus z10. it is conducted by incubation of lactobacillus acidophilus z10 in mrsbroth medium with different concentrations of bile salts, 0%, 0.15%, 0.3%, 0.6% and 1% for 24 hours of incubation (fig. 2). fig. 2. survival of the cells of lactobacillus acidophilus z10 at different concentrations of bile salts. the experimental data presented on fig. 2 show that in the first four hours of the incubation of lactobacillus acidophilus z10 in the presence of bile salts the number of viable cells is retained. this retention is a result of the development of naturally sustainable branches, which is consistent with the test of luria and delbruk. after the fourth hour, a reduction of the number of viable cells in varying degrees depending on the concentration of bile salts is observed. 1.103cfu/cm3 active cells are defined at 1% bile salts in the medium at the 24th hour. antibiotic resistance of lactobacillus acidophilus z10 20 of the most commonly used antibiotics in medical practice antibiotics with different mechanisms of action are selected and the sensitivity of lactobacillus acidophilus z10 towards them is tested. the results of the studies using the agar diffusion method [23] for 24 hours are summarized in table. 1. lactobacillus acidophilus z10 is sensitive to three of the antibiotics from the group of the inhibitors of the synthesis of the cell walls, azlocillin, piperacillin and vancomycin, and is resistant to the other 5 antibiotics in this group. the strain is food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 19 resistant to 4 out of 10 antibiotics that inhibit protein synthesis, but it demonstrates sensitivity towards lincomycin, chloramphenicol and erythromycin, and intermediate sensitivity towards tetracycline, doxycycline and amikacin. lactobacillus acidophilus z10 is resistant to the antibiotics inhibiting the synthesis of dna and/or cell division (table 1). the strain is resistant to most of the antibiotics included in the study. table 1. antibiotic resistance of lactobacillus acidophilus z10 legend: r-resistant, sr – intermediate sensitivity (zone 7-16 mm), s sensitive (zone> 16 mm) # mechanism ofaction antibiotic concentration lactobacillus acidophilus z10 1 in hi bi to r o f t he s yn th es is o f t he c el l w al ls penicillin p 10 e/disc r 2 azlocillin az 75 µg/disc s 3 piperacillin p 100 µg/disc s 4 ampicillin a 10 µg/disc r 5 oxacillin o 1 µg/disc r 6 amoxicillin ax 25 µg/disc r 7 vancomycin v 30 µg/disc s 8 cefamandole cm 30 µg/disc r 9 in hi bi to r o f t he p ro te in s yn th es is tetracycline t 30 µg/disc sr 10 doxycycline d 30 µg/disc sr 11 gentamicin g 10 µg/disc r 12 kanamycin k 30 µg/disc r 13 tobramycin tb 10 µg/disc r 14 amikacin am 30 µg/disc sr 15 rifampin r 5 µg/disc r 16 lincomycin l 15 µg/disc s 17 chloramphenicol c 30 µg/disc s 18 erythromycin e 15 µg/disc s 19 in hi bi to r o f th e sy nt he si s of d n a an d/ or c el l di vi si on nalidixic acid nx 30 µg/disc r 20 ciprofloxacin cp 5 µg/disc r food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 20 1. conclusion lactobacillus acidophilus z10 has the ability to survive in the model conditions of the gastro intestinal tract and is resistant to most of the antibiotics applied in medical practice. thus, it can be defined as a potential probiotic culture. 5. references [1] kalliomaki m., salminen s., arvilommi h., kero p., koskinen p., isolauri e. probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial. lancet 357: 1076-1079, (2001) [2] brown a. c., valiere a. probiotics and medical nutrition therapy. nutr. clin. care 7: 5668, (2004) [3] agerholm-larsen l., raben a., haulrik n., hansen a. s., manders m., astrup a. effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. eur. j. clin. nutr. 54: 288-297, (2000) [4] nomoto k. (2005). review prevention of infections by probiotics. j. biosci. bioeng.100: 583592, (2005) [5] imasse k., tanaka a., tokunaga k., sugano h., ishida h., takahashi s. lactobacillus reuteri tablets suppress helicobacter pylori infectionda doubleblind randomised placebocontrolled cross-over clinical study kansenshogaku zasshi. j. jpn. assoc. infect. dis. 81: 387-393, (2007) [6] shah n. p. functional cultures and health benefits. int. dairy j. 17: 1262-1277, (2007) [7] hirayama k., rafter j. the role of probiotic bacteria in cancer prevention. microbes infect. 2: 681-686, (2000) [8] isolauri e. probiotics in human disease. american journal of clinical nutrition, 73 (6): 1142s-1146, (2001) [9] marteau p. r., de vrese m., cellier c. j., schrezenmeir j. protection from gastrointestinal diseases with the use of probiotics. american journal of clinical nutrition 73 (suppl. 2): 430s-436s, (2001) [10] rybka s., kailasapathy k. the survival of culture bacteria in fresh and freeze-dried ab yoghurts. the australian journal of dairy technology 50 (2): 51-57, (1995) [11] mitsuoka t. the human gastrointestinal tract. in: wood bjb, editor. the lactic acid bacteria. vol.1, gaithersburg, md, usa: aspen publishers inc.: 69-114 p, (1999) [12] kirtzalidou e., pramateftaki p., kotsou m., kyriacou a. screening for lactobacilli with probiotic properties in the infant gut microflora. anaerobe 17: 440-443, (2011) [13] donald j., brown d. probiotics and the intestinal ecosystem. let's live, november, 45-47, (1993) [14] pochart p., mavtean p., bouhnik y., goderel i., bourlioux p., rambrand, j. c. survival of bififdobacteria ingested via fermented milk during their passage through the human small intestine: an in vivo study using intestinal perfusion. am. j. clin. nutr. , 55, 78-80, (1992) [15] nielsen e. m., schlundt j., gunvig a., jacobsen b. l. epithelial mucus and lumen subpopulations of escherichia coli in the large intestine of conventional and gnotobiotic rats. microbial. ecol. health dis. 7, 263-273, (1994) [16] alander m., korpela r., saxelin m., vilponen-salmela t., mattilasandholten t., von wright a. recovery of lactobacillus rhamnosus gg from human colonic biopsies. lett. appl. microbiol. 24, 361364, (1997) [17] donohue d. c., salminen s. safety assessment of probiotic bacteria. asia pac. j. clin. nutr. 5, 25-28, (1996) [18] donohue d. c., salminen s., marteau p. safety of probiotic bacteria. in: salminen s., a.von wright (eds.), lactic acid bacteria. "marcel dekker" inc, new york, 369384, (1998) [19] adams m. r. safety of industrial lactic acid bacteria. j. biotechnol., 68, 171-178, (1999) [20] saarela m., mogensen g. probiotic bacteria: safety, functional and technological properties. journal of biotechnology, 84, 197-215, (2000) [21] nikolova, d. "probiotic and biotechnological characteristics of strains from the genus lactobacillus" phd thesis, (2010) [22] denkova z. "obtaining and application of probiotics" d.sc. thesis, (2005) [23] bauer a.w., kirby w.m., sherris j.c., turck m. antibiotic susceptibility testing by a standardized single disk method. american journal of clinical pathology 36, 49-52, (1966) [24] charteris w.p., kelly p.m., morelli l., collins j.k. development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and bifidobacterium species in the upper human gastrointestinal tract, journal of applied microbiology 84 (5), pp. 759–768, (1998) microsoft word 17 mihaela jarcau articol.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 103 some physical propreties of bilberries and how the packing conditions influence them *mihaela jarcău1 1faculty of food engineering, stefan cel mare university od suceava, romania mjarcau@yahoo.com *corresponding author received 26 may 2012, accepted 8 june 2012 abstract: i am interested in the way the physical proprieties of the bilberry (vaccinium myrtillus) beans are influenced by the storage conditions. i have analised the dimensions (l,w,t), geometric mean diameter, sphericity, porosity, volume, and unit mass before and after storing in the refrigerator at a temperature of +40c. the beans have been packed in different packages and i noticed that the geometrical dimensions have varied so the average length, width, thickness and geometric mean diameter of seed ranged from: for sample 1 plastic box with lid – 8.18 to 7.73mm, 7.72 to 7.23mm, 6.13 to 5.48mm, 7.273 to 6.74mm; for sample 2 glass package – 7.93 to 7.20mm, 7.54 to 7.07mm, 6.06 to 5.42mm and 7.12 to 6.49mm; for sample 3 double paper wrapper –8.82 to 7.44mm, 8.21 to 7.04mm, 6.66 to 5.19mm and 7.84 to 6.15mm; for sample 4 -plastic bag – 8.14 to 6.35mm, 7.82 to 6.43mm, 5.98 to 4.61mm and 7.24 to 5.72mm. after 5 days in refrigerator the average of unit mass of bilberries ranges from 0.437g to 0.433g. the objective of this study was to investigate the dependence between the packaging and the physical proprieties of bilberry, namely, size dimensions, bulk density, true density and porosity. this information is important to design equipment for aeration, storage and for omptimizing the equipment design for harvesting, handling, storing and so forth. keywords: bilberry, physical properties 1. introduction the bilberry is a small size shrub that will grow up to 30-50 cm in length and can be found in the mountain areas, alpine pastures or in rocky places up to an altitude of 2500 m. fruits should occupy an important place in our alimentation because they contain a lot of biologically active substances that have beneficial effects on human health as antioxidants, anticancerogens, antimutagens and antibacterial compounds [1-4]. extracts of bilberries (vaccinium myrtillus) are used in gastroenteritis disease [3]. anthocyamins and other polyphenolic compounds present in bilberries probably promote human health [5]. bilberries have received special attention due to their history in folk medicinal uses. in 16th century, the bilberries were used for treating biliary disorders, scurvy coughs and lung tuberculosis [6]. clinical trials have demonstrated the benefits of bilberries in the inhibition of cancer growth [7]. lately the physical proprieties of different seeds have been studied and observed the way they vary in humidity conditions, for example the fenugreeck seed [8-10]. having considered the beneficial proprieties of the bilberries in the treatment of different conditions but also in the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 104 prevention of diseases i believe that it is important to analyse the way the physical propreties vary during storage depending on the type of the package. nomenclature dg-geometric mean diameter, mm l-length, mm m-unit mass of the seed, g m100-100 seed mass, g r2-determination coefficient t-thickness, mm v-single seed volume, mm3 w-width, mm ρb-bulk density, g/cm3 ρt –true density, g/cm3 ε-porosity, % φ-sfericity, % the objective of this study was to investigate the dependence between the packaging and the physical proprieties of bilberry, namely, size dimensions, bulk density, kernel density and porosity. this information is important to design equipment for aeration, storage and for omptimizing the equipment design for harvesting, handling, storing and so forth. 2. materials and methods processes 2.1. sample preparation the bilberries used in this study were obtained from a local market in suceava, city of romania. the samples were cleaned manually to remove all the leaves, the green beans or other foreign bodies. (the selected beans have been packed in different materials). i have slected 10 beans, randomly, for which i calculated l, t, w, using a caliper with a precision of 0.01mm, the beans were packed in: blastic boxes with lids, glass with paper lids, double paper wrapping, paper bag, wrapped in paper and introduced in a plastic bag (sample 1 plastic box with lid, sample 2 glass package, sample 3 double paper wrapper, sample 4 plastic bag). 2.2. dimensions, sphericity, mass, volume and surface area. the samples were kept at 277k (+4oc) in a refrigerator for 5 days. ten replications of each test were made for each bag. the geometric mean diameter dg and sphericity φ of bilberries was calculated by using the following relationship (mohsenin, 1970): 1/3 (1) (2) 2.3. one hundread seed weight and the unit mass to obtain the unit mass of the bilberry, the mass of 100 beans were measured with an electronic balance with an accuracy of 0.01g. the volume and seed surface was calculated using the following relationship (jain, 1997) (3) (4) (5) 2.4. bulk and true density the bulk density is the ratio of mass sample of the beans to its total volume. it was determined by filling a food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 105 1000 ml container with bilberries from a height of about 15 cm, triking the top level and then weighing the contents [9,10]. the true density was determined using water displacement method. the bilberries were used to displace water in a measuring cylinder after their masses had been measured. the true density was found as an average of the ratio of their masses to the volume of water displaced by bilberries. 2.5. porosity the porosity is the fraction of space in the bulk seeds that is not occupied by the seeds. the porosity ε of bulk bilberries was calculated using the following relationship: (6) where ρt is true density in kg/m3 and ρb is bulk density in kg/m3 3. results and discussion 3.1. seed size about 50 % of the beans have a length ranging from 8.15 to 8.86 mm, about 57.5 % a width ranging from 7.02 to 7.95, about 55 % a thinckness ranging from 6.0 to 6.97 and about 77.5 % frouits geometric mean diameter ranging from 7.01 to 7.98 ( fig. 1). figure 1. a frequency distribution of bilberry seed length (mm) figure 1. b frequency distribution of bilberry seed width (mm) figure1. c frequency distribution of bilberry seed thickness (mm) figure 1 d. frequency distribution of bilberry seed geometric mean diameter the dimensions of the bilberry beans packed in different materials before and after 5 days of storage are present in tabel 1 and 2. table 1 dimensions of the packed bilberry beans sample l(mm) w(mm) t(mm) dg(mm) 1 8.18 7.715 6.125 7.270 2 7.93 7.535 6.064 7.120 3 8.82 8.212 6.663 7.840 4 8.14 7.817 5.979 7.247 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 106 table 2 dimensions of the packed bilberry beans sample l(mm) w(mm) t(mm) dg(mm) 1 7.73 7.229 5.483 6.74 2 7.20 7.071 5.422 6.50 3 7.44 7.0375 5.19 6.51 4 6.35 6.433 4.612 5.72 as shown in the table 3, the dimensions of the bilberries have decreased according to the packaging used: table 3 dimensions of the packed bilberry beans after storage sample 1 5.5 6.3 10.5 7.3 2 9.2 6.2 10.6 8.7 3 15.6 10.6 22.1 17 4 22 8.7 22.9 21.1 in figure 2 can be observed that the bilberry dimensions from sample 4 (plastic bag) have the largest decrease in size, and the smallest variation in size is in sample 1 (plastic box with lid). figure 2 a l(%) relative variation for every sample figure 2 b. t(%) variation for every sample figure 2. c t(%) variation for every sample figure 2 d dg(%) variation for every sample 3.2. sphericity the values of sphericity were calculated with eq (2) and the results obtained are presented in fig. 3. about 67.25 % of the fruits have sfericity ranging from 80 to 99.9 mm3 before storage. about 90 % of the bilberries – sample 1have sphericity ranging from 80 to 89.9 mm3 after 5 days; 70 % from sample 2 have sphericity ranging from 90 to 99.9 mm3 ; 62.5% from sample 3 have sphericity ranging from 80 to 89.9 mm3; 60% from sample 4 have sphericity ranging from 80 to 89.9 mm3. figure 3 φ variation for every sample food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 107 3.3. one hundread bilberries weght the mass of 100 bilberries before storage is: ; ; and after storage ; ; 3.4. bulk and true density the average of bulk density and true density of bilberries before storage is 0.57 g/cm3 , 1.02 g/cm3; and after storage 0.58 g/cm3, 1.06 g/cm3. 3.5. porosity the value of porosity were calculated with eq (6) by using the data on bulk and true densities of bilberries and the results obtained are presented in fig? the average of porosity was 0.44% before and 0.45 % after 5 days of storage. 4. conclusion the following conclusions are veiled from the investigation of some physical propreties of bilberries from romania before and after storaging the in a refrigerator for 5 days at +4oc in different packages. 1. the average length, width, thickness, geometric mean diameter and unit mass of the bilberries ranged from 8.27 to 7.18 mm, 7.82 to6.94 mm, 6.21 to 5.18 mm, 7.37 to 6.37 and 0.44 to0.43 g in sample 1,2,3,4. 2. spheicity, volume of bilberries were varied from 89.21 to 89.17 mm3, 0.43 to 0.41cm3. 3. the true and the bulk density varied from 1.02 to 1.06 g/cm3 and 0.57 to 0.58 g/cm3 4. it is found that the analised physical quantities that vary most are form sample 4 (plastic box with lid); and the least in sample 1(plastic bag). 5. references [1]. awad, m. a. (2000). flavonoid and chlorogenic acid levels in apple fruit: characterization of variation. scientia horticulturae , 83. [2]. hakkinen, s. h. (1999). screening of selected flavonids and phenolic acid in 10 berries. food research international , 32. [3]. bobrowska e, grzesik, a. j.-k. (2008). leaching of cadmium and lead from dried fruits and fruit teas to infusions and decoctions. joornal of food composition and analysis 21 , 326 [4]. rauha, j. p. (2000). antimicrobial effects of finnish plant extracts containing flavonoids and other phenolic compunds. inernational journal of food microbiology , 3-12. [5]. frankel, e. n. (1999). food antioxidants and phztochemicals: present and future perspectives. fett lipid, 101 , 450-455. [6]. valentova, k. u. (2007). cytoprotective effect of a bilberry extract against oxidative damage of rat hepatocytes. food chemistry , 912-917. [7]. zhao, c. g. (2004). effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth. journal of agricultural anf food chemistry, 52 , 6122-6128. [8]. ebubekir, a. e. (2005). some physical prperties of fenugreek (trigonella foenum graceum l.) seeds. journal of food engineering 71 , 37-43. [9]. deshpande, s. b. (1993). physical properties of soybean seeds. journal of agricultural engineering research, 56 , 89 92. [10]. yalcin, c. e. (2007). physical properties of coriander seeds (coriandrum sativum l.). journal of food engineering 80 , 408 416. microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_14-18.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 18 mixtures with gradient of mobile phases utilized in hplc separations of 2.4-dinitrophenylhidrazones provided by inferior carbonyl compounds gheorghe zgherea1, cristina stoian1, sandu peretz2 1university “dunarea de jos” of galati, department of chemistry, 111 domneasca street, 800201 galati, romania, gzgherea@chem.ugal.ro, cstoian@ugal.ro, 2 institute of physical chemistry “i. g. murgulescu”, department of colloids, 202 spl. independentei, 060021, bucharest, romania, peretz@icf.ro abstract. many mixtures of small quantities of carbonyl compounds are present in foods, concerning sensorial qualities (aroma and fragrance). the inferior carbonyl compounds (c2-c4, boiling point <100°c) – mono and dicarbonyl – can be identified and their concentrations can be measured, after being separated by distillation on water bath. they are transferred into a strongly acid solution of 2.4dinitrophenylhidrazine (2.4-dnph), generating a mixture of insoluble 2.4-dinitrophenylhidrazones (2.4-dnph-ones). the 2.4-dnph-ones are organic compounds with weak polarity, solids, crystallized, yellow and water insoluble, but soluble in organic solvents. the mixture of 2.4-dnphones may be separated by liquid chromatography, using hplc the reverse phase mechanism [1-3]. this paper contains experimental and theoretical considerations on the means of separation through liquid chromatography of two models and a natural mixture containing 2.4-dnph-ones provided by inferior carbonyl compounds; to obtain decisive results, in the model mixtures 2.4-dnph-ones provided by carbonyl compounds having three (acetone and propanal) and four atoms of carbon (isobutylaldehyde) were introduced. keywords: acetaldehyde, diacetyl, 2.4-dinitrophenylhidrazone, reverse phase, low polarity, gradient of mobile phase introduction in many cases, for the foods obtained by fermentation, it is very important to know the concentration of diacetyl and acetaldehyde. according to literature, beer contains diacetyl and acetaldehyde in the ratio 1:100, in mass units. the interest on diacetyl concentration requires especially analyticcal conditions. it is possible to solve that problem by hplc for the mixtures of 2.4-dnph-ones provided by inferior carbonyl compounds. hplc can make a good separation only for the model mixtures of 2.4-dnph-ones; the molecules of inferior carbonyl compounds have properly physical and chemical behavior, assuring a good separation [4]. the difficulty appears for the natural mixtures concerning acetaldehyde and diacetyl; the two carbonyl compounds – and 2.4dnph-ones – have similar physical behavior. in addition, their mass ratio creates great problems in liquid-chromatographic separation. to have a very good analytical performance, it is necessary to use liquid chromatography separation with gradient of mobile phase [5]; in addition, separation columns with gradient of stationary phase are used in this paper. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 19 materials and methods solvents and mixtures of mobile phases the acetonitrile was utilized to solve the pure 2.4-dnph-ones. for liquidchromatographic separations, two similar mixtures with gradient of mobile phase, according with the programs abbreviated mgmpi and mgmpii, containing bidistillated water (2 s·cm-1) and methanol pro chromatography (merck) were utilized. the model mixtures of 2.4-dnph-ones the pure 2.4-dnph-ones – yellow powders – were obtained in our laboratory, using a strongly acid solution of 2.4-dnph and chemical pure carbonyl compound; by synthesizing: 2.4-dnphaa (acetaldehyde), 2.4-dnphd (diacetyl), 2.4-dnpha (acetone) and 2.4-dnphiba (isobutylaldehyde). in acetonitrile (merck, pro liquid chromatography), 5·10-4 m solutions were obtained. by controlled mixing we obtained two model mixtures, abbreviated mmi and mmii, considered as approximate models of natural mixtures; the ratio between the quantities 2.4-dnphaa and 2.4dnphd is higher to one, in each mixture. the beer’s mixtures of 2.4-dnph-ones the carbonyl compounds from beer were separated by distillation on water bath and transferred into a strongly acid solution of 2.4-dnph; the precipitates form a natural mixture of 2.4-dnph-ones. they are isolated by filtration, washed with pure water, dried and solved in acetonitrile. apparatus for the separations we utilized pye unicam philips liquid chromatograph, equipped with: an installation for degasing of mobile phase (by refluxing) [7], gradient programmer for mobile phase (type lcxpd, able to mix two different liquids), separation columns with gradient of stationary phase, installation for column thermostat control, electronic integrator (type dp101, spectra-physics) and potentiometer recorder (type pm8251, philips). the mixing program of liquids has nine segments of time, g=1-9; each timing segment has independent dimension of 0-99 minutes. every moment of analytical separation, the value of b, the percent of the second component in the mixture of mobile phase (a + b=100%), is ntkb % (1) where: t – dimension of timing segment (minutes), k – slope of curve (describes the evolution of b value on the t segment), n – exponent, with values 0.0-9.9 (describes the geometry of mixing curve). conditions of chromatographic separations sample volume: 10 l; separation column: l = 25 cm, = 4.6 mm; stationary phase: spherisorb 5 ods, with gradient of stationary phase; 37.5ºc, the temperature of separation column: lc uv detector, = 365 nm; flow rate: 1ml·min-1; eluate: a controlled mixture of a-methanol and b-water, accordingly to two programs – mgmpi and mgmpii – achieved by the lc-xpd chromatographic module. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 20 the liquid-chromatographic separations both categories of liquid-chromatographic separations were carried out on the same column, at the same temperature, changing only the program of mixing liquids in binary mixture of mobile phase. results and discussion the model mixtures (mmi and mmii) and a natural mixture from beer were separated by liquid-chromatography, using a mechanism of reverse phase. the mixtures of mobile phases have the same initially composition, with 45% water. in this instance, the 2.4-dnphaa will be eluted before the 2.4-dnphd. in this way we guaranteed the maximum difference between the values of retention times for the mentioned 2.4-dnph-ones and a preliminary separation of the 2.4-dnph-ones provided by aliphatic carbonyl compounds with three and four atoms of carbon. the model mixture mmi contains four 2.4dnph-ones, in the following ratio of volume: 2.4-dnphaa: 2.4-dnphd: 2.4-dnpha: 2.4-dnphiba = 2:1:1:1. the first program of binary mobile phase, mgmpi (32 minutes, figure 1), contains four time segments; figure 2 shows the chromatogram of model mixture mmi, obtained with mgmpi, with the retention times (seconds, in brackets). according to mgmpi, the separation begins with a mobile phase having 45% water; the mixture of mobile phase has a higher polarity. this mixture is hold during the first time segment, g = 1 (t = 12 minutes), assuring a better resolution between the peak of 2.4-dnphaa (844 s) and the peak of 2.4-dnphd (918 s). the weak polar molecules of two 2.4-dnphones are strongly retained at the no polar stationary phase. therefore, the longitudinal diffusion of concentrated zone is higher. simultaneously, in the stationary phase 2.4-dnph-ones provided by acetone and isobuthanal are strongly retained. on the second timing segment, g = 2 (t = 10 min, n = 0.1), the percent of water will be reduced at 20%, thus the polarity of mobile phase subsides; the 2.4-dnphaa and 2.4-dnphd (more soluble in organic solvent) diffuse in the mobile phase and will be transported to the end of separation figure 2. the chromatogram of model figure 1. the program of mgmpi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 21 column. on the third timing segment, g = 3 (t = 5 min, n = 1), the percent of water is constant (20%), to assure the best separation of 2.4-dnpha and 2.4-dnphiba. the last timing segment, g = 9 (t = 5 min, n = 5.5), is meant to recondition stationary phase, for a new separation; the intermediate timing segments, g = 4-8, are inactive. the second program of binary mobile phase, mgmpii (40 minutes, figure 3), contains four time segments too; figure 4 shows the chromatogram of model mixture mmii, obtained with mgmpii. the model mixture mmii contains the same 2.4-dnph-ones as mmi, in the ratio of volume: 2.4-dnfhaa: 2.4-dnfhd:2.4-dnfha: 2.4-dnfhiba = 4:1:5:5. as in the previous case, the first timing segment, g = 1 (t = 15 min), the mixing program assures an eluate with 45% water, for the best separation between 2.4dnphaa and 2.4-dnphd; the polarity of mobile phase is higher (the molecules of 2.4-dnph-ones are strongly retained on the slow polar stationary phase). the second timing segment, g = 2 (t = 10 min, n = 9.9), the percent of water is reduced at 20%; this value is constant and during the third timing segment, g = 3 (t = 15 min). subside of mobile phase polarity produces a desorptive process in the stationary phase. the timing segments, g = 4-8 have not got a specific content. in the last timing segment, g = 9 (t = 5 min, n = 0.1) the initial mixture of mobile phase is quickly rebuilt; at cessation, the analytical system is completely ready for a new separation. by comparing the chromatograms from figures 2 and 4 we have drawn the following conclusions: the mgmpi assures a better separation for the 2.4-dnphaa and 2.4-dnphd; for the 2.4-dnpha and 2.4-dnphiba, the resolutions being comparable. figure 5 shoes a chromatogram of a natural mixture of 2.4-dnph-ones, for the inferior carbonyl compounds of beer. the liquid-chromatographic separation is on the same column and the eluting program is mgmpi. the peaks for 2.4-dnphaa and 2.4-dnphd are in the central zone of chromatogram. figure 4. the chromatogram of model mixture abbreviated mmii figure 3. the program of mgmpii food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 22 figure 5. the chromatogram of natural mixture of 2.4-dnfh-ones using the external standard quantitative method, for the chromatographic peak of 2.4-dnphaa, we obtained 14.75 mg·l-1 as concentration of acetaldehyde in beer; the value complies with literature data [6], assuring a higher satisfaction to the analyst operator. using the same quantitative method, the surface of chromatographic peak of 2.4dnphd gives a value of 2.5 mg·l-1 as concentration of diacetyl in beer; this value is higher, bringing no satisfaction to the analyst operator. in beer, the normal value of diacetyl concentration is 0.01-0.2 mg·l1; 0.15 mg·l-1 is the threshold value [6]. conclusions on the basis of these experiments, the following conclusions may be drawn: 1. we established two optimal programs for providing mixtures of binary eluate; according to them, two mixtures of binary phase that assure liquid-chromatographic separation, with a good resolution are obtained for the etalon mixtures of 2.4dnph-ones provided by the aliphatic carbonyl compounds with a number of 2-4 atoms of carbon. 2. the binary mixtures of mobile phase, obtained by the programs mgmpi and mgmpii, assure a better separation of model mixtures which contain derivate compounds of inferior carbonyl compounds; in these model mixtures the ratio between 2.4-dnphaa and 2.4-dnphd is higher than one (in mass units), but very low. 3. in the case of natural mixtures of 2.4dnph-ones, similarly with model mixtures, the binary mixtures of mobile phase offer only partially analytical satisfaction. thus, the figure 5 shows obviously the dominant peak of 2.4-dnphaa. by using the surface value for quantitative appreciation by external standard method, we obtained experimental values accordingly to literature, namely 15 mg·l-1 acetaldehyde [6]. in the same chromatogram, the peak of 2.4-dnphd is the second, but it is on the tailing peak of the first one. as for the natural mixture of 2.4-dnph-ones one may notice the following aspect: if literature offers real concentration values for the two carbonyl compounds, the accuracy of analytical system will be justified by high value, ~100, of the ratio between the quantities of acetaldehyde and diacetyl. in this instance, the low peak of 2.4-dnphd appears as a tailing peak on the high peak of 2.4dnphaa, thus, its surface is higher than the normal one; the mistake value of surface chromatographic peak became a source of mistake for concentration value. 4. each chromatogram – figures 2, 4, and 5 – contains any peaks with lower values of retention time; it is the peaks for solvent. references 1. simion gocan, cromatografie de înaltă performantă, partea a ii-a, cromatografia de lichide pe coloane, editura risoprint, clujnapoca, romania, 2002, 173-183 pp 2. douglas a. skoog, principles of instrumental analysis, third edition, saunders college publishing, 1985, 801-815 pp 3. candin liteanu; simion gocan; t. hodisan; h. nascu, cromatografia de lichide, editura stiintifică, bucuresti, romania, 1974, 156157 pp 4. radu bacaloglu; carol csunderlik; livius cotarcă; hanshorst glatt, structura si proprietătile compusilor organici, vol i, editura tehnică, bucuresti, romania, 1985, 116-127 pp 5. gort s.m., hogendoorn e.a.; dijkman e.; van zoonen p.; r. hoogerbrugge, the optimisation of step-gradient elution conditions in liquid chromatography, chromatographia, vol. 42, no ½, ian 96, 17-24 pp 6. bieranalyse 9.11 bestimmung der gärungsneben produkte in bier, 246-250 pp 7. gheorghe zgherea, installation to continuously degassation by refluxing of mobile phases for hplc, annals of west university of timisoara, romania, series chemistry 12 (3) (2003) 1157-1160 pp microsoft word 2 content vol 2010 nr 2 din 12 oct final.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 contents: 1. gheorghe gutt, yarema tevtul, andrei gutt, alina psibilschi research and achievements for new electrochemical biosensors 5 2. yarema tevtul, olga nechyporenko monitoring of contamination of the environment in bukovyna with heavy metals 11 3. gheorghe zgherea, cristina stoian, sandu peretz mixtures with gradient of mobile phases utilized in hplc separations of 2.4dinitrophenylhidrazones provided by inferior carbonyl compounds 18 4. alina-petronela haller benefits of multilateral liberalization for the countries which are exporting light industry products in a knowledge based society 23 5. gabriela pop, adriana dabija, amelia buculei high-fiber wheat bread produced with fermented bran 30 6. alisa vasilica arus, ana maria georgescu, iuliana mihaela lazar, ileana denisa nistor, neculai doru miron, azzouz abdelkrim , andrea – eliza farkas , ramona mihaela cojocaru use of some clay matrices in biotechnology of acid dairy products 35 7. boris skip, sergiy zelinskyi dispersion and assessment of formaldehyde emissions by automobiles and their influence on the air quality 41 8. elisabeta botez,constantina sălceanu, oana – viorela nistor, aura darabă concerns on the development of ecumenical tourism in bucovina 47 9. silvia mironeasa, costel mironeasa, georgiana-gabriela codină evaluation of mineral element content in grape seed and defatted grape seed 53 10. octavian baston, octavian barna, aida vasile freshness evaluation of chicken meat using microbiota and biogenic amine index 61 11. enuţa iorga, monica catană, luminiţa catană, mioara negoiţă, alina bâlea, gabriela lilios influence of fertilization treatments on nitrates content of some vegetable species cultivated in the field 67 12. mircea-adrian oroian, gheorghe gutt influence of total soluble content, starter culture and time period on rheological behaviour of cultured buttermilk 73 13. constantin drobotă , ioan gontariu genetic resources and the progress reached by winter wheat breeding 79 14. mathew shiju a review on the effect of fungi on the wheat grain under post harvest storage ecology 87 15. author instructions 97 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 31 removal of humic substances in water by coagulation *danka barloková1, ján ilavský1 1department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovakia danka.barlokova@stuba.sk, jan.ilavsky@stuba.sk, *corresponding author received 2 september 2012, accepted 15 september 2012 abstract: basic characteristics of humic substances and their negative influence on water quality and its treatment are described in the paper. humic substances are unwanted therefore their removal is necessary. this can be done by coagulation which plays an important role in surface water treatment. this statement was confirmed in laboratory experiments. in order to remove humic substances from surface water of the water reservoir in hri ová coagulation was used. as the coagulation reagent either ferric sulfate or aluminium sulfate was used as well. an optimum coagulant dosage was soughed. a treatment with ferric sulfate as well as aluminium sulfate can be sufficient if the acceptable limit for humic substances is 2.5 mg/l, our experiments showed. to reach a reduction of humic substance contents below its level of 1 mg/l dosage of 16 mg/l of 1% solution fe2(so4)3 (prefloc), which is the equivalent of 4,47 mg/l fe3+ was sufficient. keywords: water treatment, humic substances, coagulation, drinking water, water analysis 1. introduction humic substances (mainly humic acids and fulvic acids) are present in almost all natural waters, which often represent a major proportion of organic pollution (in literature known as the natural organic matter nom). humic substances (hs) are complex high molecular weight organic compounds, with aromatic-aliphatic character, containing carbon, oxygen, hydrogen and nitrogen. relative molecular mass of humic substances ranges from a few hundred to tens of thousands [1]. the elementary composition of humic substances, which are most commonly found in natural waters is shown in table 1 [2]. table 1 elemental composition of humic substances present in natural waters humic composition (%) substances c o h n fulvic acid 43-52 42-51 3.3-6.0 1.0-6.0 humic acid 52-62 30-39 2.5-5.8 2.6-5.1 humic substances contain on average about 50% of organic carbon, followed by oxygen, hydrogen and nitrogen. representation of oxygen in fulvic acids is significantly higher than in humic acids. the humic substances are characterized by the presence of carboxylic acid, the hydroxy (phenol, alcohol) and also methoxy and carbonyl groups. these groups are bound to the nucleus, and the aliphatic side chains too. the average composition of functional groups of humic substances (with molecular weight in the range 1200 – 1600) is shown in table 2 [3]. table 2 average composition of functional groups in humic substances functional group humic acid fulvic acid cooh 4.4 8.1 oh 3.3 3.9 oh (r) 1.9 4.0 c o 1.2 1.4 o – ch3 0.3 0.4 mailto:danka.barlokova:@stuba.sk mailto:jan.ilavsky:@stuba.sk food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 32 the structure of humic substances could not be fully identified. these substances are attributed different, essentially hypothetical formulas, which is to capture the particular position of cooh and oh in the molecule, which significantly influences the properties of humic substances [4, 5]. the negative effect of humic substances on water quality and its treatment can be summarized as follows [2,3]: increase the intensity of the color of water increase the acidity of water affect the biochemical stability influence the formation of metal complexes influence the formation of thm in water chlorination the presence of humic substances in waters is showed of their colouring, for example, fulvic acids are dependent on the concentration of light yellow to yellowishbrown color, humic acids are dark brown. because of their origin it is the natural color of water. generally, the concentration of humic substances 2.5 mg.l-1 corresponds to the color of water about 20 mg.l-1 pt. however, color changes considerably with the ph of water (the colorful solutions with higher ph). in addition, the color of the water depends on the composition of humic substances, on the size of disperse particles, etc., which may be different in various surface waters. therefore, it cannot get universal linear relationship between the concentration of humic substances and water color. humic substances are characterized by complexing properties [6,7,8]. formation of complex is one of the causes leaching of heavy metals from soil humic substances (increasing their content in water) and cause increased concentrations of fe and mn in peat waters. from a technological point of view, humic substances cause problems in water treatment [9,10], for example, increase consumption of coagulant and disinfectants, but mainly act as precursors of halogenated compounds. only high molecular humic acids can be removed from the water with coagulation, but for fulvic acids with low molecular weight (molecular mass to 500) is efficiency of removing smaller ones and some fraction cannot be removed at all.this is due to the size of molecules or particles. larger molecules eventually particles are easily separated by coagulation, sedimentation and filtration processes in comparison with small molecules and particles. with increasing concentration of humic substances with increasing ph, the size of molecules or particles decreases, what makes worse the effect of separation. the best effect is achieved in the treatment of humic waters by clarification earlier in the acidic range (ph 4-6), which formed a large and well separable units. aggressive properties of humic waters have negative effect for metals and building materials (mortar and concrete), which cause not only acidity but also their ability to form complexes, in case of mortar and concrete to the aggressive leaching, which tends to increase the solubility of caco3 and cao. important precursors of organohalogen compounds in water are mainly fulvic acids and humic acids. experimentally, it was confirmed that the fulvic acids occurs in approximately 60% more organochlorine compounds in comparison with humic acids [11]. fulvic also have greater water solubility than humic acids, therefore natural water contains an average of 87% fulvic acids. to prevent the formation of chlorinated hydrocarbons it is necessary to reduce the content of humic substances in water or change the method of disinfection. due to these characteristics, the humic substances in drinking water and process water are unwanted. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 33 in some industries, these substances (e.g. textile, paper) can impair the quality of the product color. the determination of humic substances in drinking water is not present in slovak government regulation no.496/2010 on drinking water. it is based on the value of the cod-mn, absorbance (a254), or color of water. exceeding the limit values is the reason for the decision to determination of humic substances. in the older standard for drinking water (stn 757111) humic substances were limited to value of 2.5 mg.l-1. the excess value of 2.5 mg.l-1 indicates the possible presence of thm in water provided by chlorine. in the requirements for the quality of raw water for treatment of drinking water (stn 757214) humic substances are included in the cut-off value for the color of water (20 mg.l-1 pt). the lowest concentration of humic substances found in groundwater (up to 0.1 mg.l-1). in year 1996, concentrations of humic substances in czech drinking water was ranged from about 0.04 mg.l-1 up to 6.77 mg.l-1, the average value 0.63 mg.l-1 [12]. the surface water concentration of the order of units mg.l-1. in the waters of peat humic substances concentration fluctuates over a wide range, typically in the tens mg.l-1. in some standing water can be determined the concentration of humic substances up to 500 mg.l-1 [13]. tab. 3 concentration of humic substances (hs) in the raw water (rw) and treated water (tw) from water treatment plant (wtp) wtp inlet of rw outlet of tw hs (mg/l) codmn (mg/l) hs (mg/l) codmn (mg/l) stariná 4.5 2.08 2.9 1.76 hri ová 4.9 4.72 4.7 1.92 klenovec 3.3 2.32 3.6 1.76 málinec 4.5 2.08 2.9 1.76 for the determination of the humic acids are recommended spectrophotometry methods in the visible zone at a wavelength of 420 nm or in the zone of ultraviolet light at a wavelength of 254 nm.we used spectrophotometric method, which uses extraction of humic substances at low ph in pentanol and subsequent reextraction of pentanol solution of naoh [14,15]. the conversion of absorbance to concentration is using either an empirical coefficient (valid for peat water in slovakia), or even commercial standard (aldrich, merck). currently is used concentrating on various sorbents (xad, sephadex-deae) followed by desorption with naoh solution. after removal of the inorganic forms of carbon is determined value of doc (toc). the concentration of humic substances is obtained by multiplying the doc mg.l-1 in two, because humic substances contain about 50% carbon. in literature [16] is present relation for humic substances cod-cr doc. indirect determination can be used for water, where in the organic matter dominate humic substances. then content of humic substances is calculated by the concentration of cod-mn, whose value is multiplied by an empirical factor of about 1.2 (valid for peat water in slovakia). complicated determination of humic substances is the reason not to apply the results to their weight, but to the amount of organic carbon which is contained in them. taking into consideration that humic substances contain about 50% organic carbon, is recommended to express the concentration of humic substances as doc (toc) in mg.l-1 [17]. 2. experimental the experimental part of this work aime consists in removing the humic substances from water by coagulation. there were compared two coagulants, aluminum food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 34 sulphate (kemwater) and ferric sulphate prefloc (kemifloc) to find the optimal dose for reduction of humic substances in surface water from water reservoir hri ová. 2.1. coagulation test for the coagulation test was used device with five mixers with adjustable speed and high-speed mixing. into five flasks were added per liter of raw water. after adding a coagulant (1% solution) followed by 3 minutes rapid mixing (180 rpm) and 10 minutes slow mixing (40 rpm). after mixing followed sedimentation of sample, which took 1 hour and then filtration through filter paper. thus prepared sample was analyzed. the following parameters were monitored: ph, anc4,5, fe or al concentration, turbidity, color, toc, and cod-mn concentration of humic substances. results of measurements are shown in the table 4. 2.2 determination of humic substances into separating flask 500 ml was added the 250 ml samples of water and 5 ml of concentrated h2so4. after mixing, into banks was added 25 ml pentanol. after intense hand-shaking during 30 seconds and the separation of the layers (after two hours), was removed the aqueous layer. into the separation flask was added 25 ml 0.5% solution of naoh. content of the flask was mixed (10 seconds). after 10 minutes part of the sample (about 3-4 ml) was took away (to clean the stem separating funnel) and the clear solution was added into a spectrophotometric cell (length 1 cm) and then the absorbance at a wavelength of 420 nm was measured. from the measured absorbance was read the absorbance of the blank of distilled water, which was made with the same procedure. 3. results and discussion coagulation is an important process in the treatment of surface water. on the present surface water treatment is possible without the addition of chemical compounds only in sporadic cases. the advantage of coagulation is that addition of coagulants effects not only to colloidal and fine dispersion substances but to all others suspended solids which form flakes with high ability to settle. iron, respectively aluminum salts are the most widely used coagulants. required dose of coagulant varies with the quality of raw water. the optimum dose of coagulant is possible to determine in the laboratory with coagulation test. the results of coagulation tests aimed at removing of the humic substances from surface water hri ová dam (wtp hri ová) are listed in tables 1 and 2. the effectiveness of coagulation was monitored for two coagulants, aluminum sulphate and ferric sulphate. on the basis of the results shown in tables 4 and 5 it can be concluded that the ferric sulphate coagulant is more suitable for removing humic substances from water as aluminum sulphate (there are not wide differences between the used coagulants). these results should be confirmed by long-term monitoring, specially monitoring of changes water quality during the seasons. the highest efficiency of coagulation, respectively lowest value of humic substances was reached at a dose of coagulant 16 mg.l-1 (1% solution), i.e. 4.47 mg.l-1 converted to fe3+, or 2.52 mg.l-1 calculated for al3+. the comparison of the removal efficiency of humic substances by different coagulants (fe3+, al3+) is shown in fig. 1. humic concentration of the raw water before coagulation was 5.44 mg/l. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 35 table 4 the results of coagulation tests removal of humic substances with fe2(so4)3 the dose coagulant [mg fe3+/l] ph acn4,.5 [mmol/l] fe [mg/l] hs [mg/l] codmn [mg/l] toc [mg/l] ftu [zf] color [mg/l] 0 6.52 0.534 0.08 5.44 2.56 3.4 3.0 11.0 3.35 5.90 0.276 0.06 1.22 1.92 2.5 < 2.0 9.0 3.91 5.54 0.241 0.05 1.08 1.76 2.4 < 2.0 7.0 4.47 5.05 0.076 0.08 0.89 1.28 1.9 < 2.0 5.0 5.03 4.62 0.034 0.08 1.46 1.26 1.8 < 2.0 4.0 5.58 3.91 0.0 0.06 1.63 1.38 2.2 < 2.0 4.0 table 5 the results of coagulation tests removal of humic substances with al2(so4)3 the dose coagulant [mg al3+/l] ph knk4,.5 [mmol/l] al [mg/l] hs [mg/l] codmn [mg/l] toc [mg/l] ftu [zf] color [mg/l] 0 6.67 0.548 0.01 5.44 2.56 3.5 3.0 11.0 1.89 6.60 0.445 0.02 3.26 2.40 3.3 < 2.0 6.0 2.21 6.48 0.342 0.02 2.28 2.24 3.2 < 2.0 6.0 2.52 6.43 0.310 0.02 1.43 2.02 3.1 < 2.0 4.0 3.00 6.82 0.457 0.02 1.67 2.08 3.2 < 2.0 5.0 3.15 6.90 0.532 0.02 1.92 2.16 3.2 < 2.0 6.0 figure 1. the removal of humic substances from water by coagulation because the influence of ferric sulfate, respectively oxide reduces the ph value, it is necessary to adjust the ph after coagulation. determination cod-mn and toc conversion confirmed the relationship between these parameters and humic substances in the literature. 4. conclusion laboratory tests with surface water from wtp hri ová showed that coagulation using ferric sulfate (alum also) is the type of water sufficient method to remove humic substances from water (if we limit the comparison to humic substances 2.5 mg.l-1). already dose of 16 mg.l-1 of 1% solution fe2(so4)3 (prefloc), which is the equivalent of 4.47 mg.l-1 fe3+ is sufficient for reduction of humic substances below 1 mg.l-1. by the influence of iron sulphate to a reduction in ph should be adjusted ph of water after coagulation. 5. acknowledgement this work was supported by the financial support of project vega 1/1243/12 provided by slovak grant agency. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 36 we would like to thank the company hydrotechnologia bratislava for assistance in the coagulation experiments. 6. references [1] liao w.: structural charakterization of aquatic humic material. environ.sci.tech., 16,403,(1982). [2] schnitzer m., khan s.u.: humic substances in the environment. m.dekker, new york, p. 327, (1972). [3] žá ek l.: chemical and technological methods of water treatment (in czech). noel publischer 2000, p. 271, (1999). [4] stumm w., morgan j.j.: aquatic chemistry. 3. ed., wiley, new york, p. 1022, (1996). [5] stevenson, f.j.: humus chemistry: genesis, composition, reactions. new york, john wiley & sons, p. 512, (1982). [6] gamble d.s., schnitzer m.: the chemistry of fulvic acid and its reactions with metal ions. in: singer p.c. (editor): trace metals and metal-organic interactions in natural water. ann arbor sci. publ., ann arbor, mich., p. 380, (1974). [7] nriagu j.o., coker r.d.: trace metals in humic and fulvic acids from lake ontario sediments. environ. sci. technol., 14, 443, (1990). [8] snoeying v.l., jenkins d.: water chemistry. wiley, new york, p. 463, (1980). [9] žá ek l.: treatment of humic waters by clarification (in czech). work and study. vúv tgm praha, issues 118, (1991). [10] žá ek l., šorm j.: using spectrophotometric methods for the assessment of organic pollution in the water treatment process. specialized publications (in czech) vúv no.16, szn praha (1987). [11] grünwald a., janda v., fišar p., bížová j., štastný b.: evaluation of thm formation potential in humic waters (in czech). in: proceedings of the conference voda zlín 2002, zlín, p. 67-71, (2002). [12] kratzer k., kožíšek f.: health effects of drinking water contamination. expert report for 1996 (in czech). national institute of public health, prague 1997. [13] chalupa j.: proceedings of všcht praha, technol. vody 7(1), 17-57, (1963). [14] stn 83 0520-29 (1978) physico-chemical analysis of drinking water. determination of humic substances (in slovak). [15] stn 83 0530-35 (1979) chemical and physical analysis of surface water. determination of humic substances (in slovak). [16] pitter p.: hydrochemie 4th edition, institute of chemical technology press, praha, p. 568, (2009). [17] standard methods for the examination of water and wastewater. 5510 aquatic humic substances. 19th. edition, amer. publ. health assoc., washington 1995. microsoft word fia journal no 1_2011_23 martie_20-26.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 24 efficacy of plant stanols/sterols in ensuring heart health mihaela constandache1, elena condrea 2, steluţa radu3 1„dimitrie cantemir” christian university, faculty of touristic and commercial management constanta, 90a dezrobirii street, 900234 constanta, romania, e-mail micky_eve68@yahoo.com 2„ovidius” university, faculty of economics, 1 university alley, constanta, romania 3 agricultural science and veterinary medicine university of iasi, faculty of agriculture, 3-5 mihail sadoveanu street, 700490 iasi, romania abstract: in all civilized countries the first cause of mortality and morbidity is a cardiovascular disease. cardiovascular diseases are a group of degenerative diseases of heart and blood circulatory system including heart disease, peripheral artery disease and stroke. to reduce ldl-cholesterol level two significant areas of functional foods development are based on the one hand on plant sterols and stanol esters use and respectivel,y on the other hand on soy proteins use. more recent studies have shown plant sterols’ and stanols’ (hydrogenated derivatives of sterols) capacity to reduce ldlcholesterol under certain conditions. recent technological advances have provided plant sterols and respectively stanols extractions and esterification opportunity and then their solubilization in matrix of fatty food has become possible so easily by their incorporation in food at effective levels. dobrogea group with raisio finland introduced on the romanian market the first bread with added plant stanols esters showing that after 14 days only of regular consumption of this product the level of serum cholesterol decreased. keywords: functional food, plant sterols, health, heart, ldl-cholesterol, benecol product introduction in all civilized countries the first cause of mortality and morbidity is a cardiovascular disease. in romania, statistics shows a significant increase in the incidence of these diseases that are the main cause of death caused by cancerous disease. most romanians suffer from diseases with various forms of cardiovascular disorders. a romanian of five has high blood pressure, while 17% have severe impairment of peripherial arteries caused by artherosclerosis. of these, more than half are obese [1]. studies show that the diet of most people is based on carbohydrates and fat and contains an excess of salt. besides food, other causes and more other characteristic factors of our times should be incriminated such as: physical inactivity, stress, smoking, pollution, etc., heredity occupying the last position in the list [2]. passclaim and fufose activities and revised some aspects of target functions available through diet could influence the health and welfare of the body. the concept was tested and evaluated to provide an initial characterization of the scientific basis to support innovation and development of functional foods and any claims related to. cardiovascular diseases are a group of degenerative heart diseases and blood circulatory system including coronary disease, peripherial artery disease and stroke. coronary disease is a serious problem in industrialized states and increasingly more acute problem in developing countries and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 25 countries in transition. predominant clinical manifestations are pectoris angina (chest pain), myocardial infarction (heart attack) and congestive heart failure. reduction and especially prevention of obesity and cardiovascular diseases is made by diet with a lower intake of carbohydrates, lipids and salt, respectively with a high level of fiber, specific attributes of hipocaloric diet food, gives positive results [1]. there is strong evidence that cardiovascular risk reduction is possible through the consumption of fruit and vegetables, fish and fish oils rich in eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), linoleic acid and potassium rich foods, together with appropriate physical activity and moderate alcohol consumption. docosahexaenoic acid (dha) is a very important omega-3 fatty acid in brain development, in ensuring the maintenance of visual acuity and brain function for all age groups. eicosapentaenoic acid (epa) falls into the same class of omega-3 acids that are vital for cardiac function and general health maintaining [3]. obesity and excessive alcohol consumption increase the risk of cardiovascular disease. a low risk is found in the consumption of products containing α-linolenic acid, olei acid, fiber, plant sterols and stanols. a reduced risk was found for products rich in flavonoids [4]. two significant areas of development of functional foods are based on the use of plant sterol and stanol esters, on the one hand and soy protein on the other hand respectively, to reduce ldl-cholesterol levels. sterols are natural constituents of plants and common crops such as soybeans and maize, having a role similar to cholesterol in the body. for 50 years it has been acknowledged that sterols interact with cholesterol in the intestinal tract leading to reduced cholesterol absorption and significantly reduce blood cholesterol levels respectively. more recent studies have shown that plant sterols and stanols (hydrogenated derivatives of sterols) reduce ldl-cholesterol under certain conditions. these substances are naturally found in the diet, but in much lower concentrations than those necessary to achieve an effect. however, recent technological advances have enabled the extraction and esterification of plant sterols and stanols respectively, and their solubilizing in the matrix of fatty foods makes it possible so easily to get them incorporated in foods at effective levels [5]. materials and methods demonstrating the effectiveness of bioactive compounds is critical in building a solid scientific basis to show the functional foods health claims. in assessing the effectiveness of plants stanols and sterols on ensuring the heart health we applied the process developed by the us institute of food technology to address critical issues in the design, development and marketing of functional foods (fig. 1). thus, after having identified the potential bioactive components, plants stanols and sterols respectively (stage 1) some evaluation was made for efficacy and safety (steps 2 and 3). when selecting food vector for these bioactive compounds (stage 4), the characteristics of food, ingredients, nature and destination were taken into account. an independent assessment of compliance with regulatory aspects (step 5) ensures the accuracy of health claim, which must be properly communicated to consumers (step 6). finally, by monitoring the product on market, there could be confirmed the findings from the market pre-evaluation (step 7). demonstrating the effectiveness of bioactive compounds is essential in building a solid scientific basis necessary food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 26 to render evident the contribution of functional foods. figure 1. process of design, development and marketing of functional foods. source: ift, 2005 the ability of identifying and quantifying key components of functional foods is an important first step in determining their effectiveness. nutrients and bioactive substances must be stable in set in food to become functional at the time of consumption. tests of long-term stability assessment are used to assess the efficacy of bioactive compounds in commercial products. bioavailability of bioactive compounds, essential to the pursuit of their functional effects is influenced by physical and chemical form, the overall effect of diet, effect of food processing technology and environment factors [6]. to ensure the health benefit, bioactive substance must be consumed in adequate amounts. the increase or decrease of bioactive components consumption has an effect on efficacy and safety of food. the impact assessment on safety and effectiveness requires quantitative knowledge on the consumption of target population, potential increased consumptions, population segments that present special risks or benefits. the food vector selection depends on their acceptability, stability and bioavailibility of bioactive compounds in food, consumption and lifestyle practices. once the health claim is scientifically validated, this information must be communicated to consumers. if they are not informed about the possible beneficial effects induced by food, few of them will know the benefits of these products, and manufacturers will be less motivated to develop other functional foods. this communication must submit significant links between the attributes of functional food and the consequence of their consumption on health care needs. specific regulations should allow manufacturers to characterize accurately the health benefits of functional foods and scientific support of these claims. all parties involved must ensure that the message is correct and easily understood by consumers. the results obtained in the first five stages form the basis of messages to consumers, to convey the benefits of functional foods consumption. supervision of the functional food on market refers to the process of obtaining information on functional ingredients effects induced after having released the product on the market. this information step 1: identify the relationship between food component and health benefits step 2: demonstrating the effectiveness and determine apropiate sizes to ensure the intended contributions step 3: demonstrating the effectiveness and food safety levels step 5: the existance of sufficient scientific evidence to demonstrate the effectiveness step 6: communicating the benefits to consumers step 4: choosing the most appropiate food vector for the bioactive component step 7: management of market confirmation of efficacy and food safety food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 27 was obtained by monitoring the consumption patterns and the consumer’s impact can complement the results obtained during market pre-evaluation, such as food safety and effectiveness of bioactive compounds, and any adverse effects (complaints) were not been identified in market pre-testing. results and discussion phytosterols, widely distributed in the vegetal area, significantly reduce blood ldl-cholesterol and therefore, the risk of cardiovascular disease. structurally, phytosterols are close to cholesterol. scientific plausibility of phytosterols’ benefits is well understood. phytosterols compete with cholesterol for incorporation sterol into micelles of intestinal lumen, interfering with both intestinal absorption of cholesterol ingested and the endogenous, secreted in the intestinal lumen [7]. scientific evidence indicates that phytosterols affect proteins that form the membrane structure [8]. action mechanisms of plant sterols and stanols are not fully clarified. however, two separate and sequential mechanisms seem to inhibit the cholesterol absorption with plant stanol. the first mechanism occurs in the intestinal lumen, while stanols replaced cholesterol from mixed micelles. the second mechanism occurs in the epithelial cells of the intestinal wall. the stanols plant that captures these cells activates specific protein carriers, which pump a part of cholesterol from the epithelial cells in the intestinal lumen [9]. this reduction in cholesterol influx reduces cholesterol availability for incorporation into ldl particles [10]. interference of cholesterol intestinal absorption has been demonstrated in both animal studies and in humans [11, 12]. clinical trials using double-isotope method showed that the intake of plant stanols reduces cholesterol absorption by 50% to 80%. decreased cholesterol absorption occurs in both exogenous and endogenous cholesterol. this explains the effectiveness of cholesterol reduction by plant stanols even when consumed in a low cholesterol diet. vanstone et allia (2002) have established the parity of the two families of compounds (stanols and sterols) in lowering ldl-cholesterol and free nonesterified stanols and sterols equivalence in reducing cholesterol. it is shown that free and esterified sterols and stanols act similarly in lowering serum cholesterol levels and interfering with intestinal absorption of cholesterol [13]. table 1 presents the fda and efsa conclusions regarding data supporting the stanols/ sterols esters health claim and coronary diseases. in october 2000 fda approved a health claim for certain foods and dietary supplements containing stanols or sterols esters, and later on, has expanded the health claim for additional food and other free forms and mixtures of stanols and sterols. in may 2010, some results of evaluation of the applications submitted and data presented by the european food safety authority stated that a causal relationship between the consumption of foods containing plant sterols and plant stanols esters and lower cholesterol level has been established, and the european commission by the ce regulation no. 384/2010 has approved the health claim that "it was found that plant stanols esters reduce blood cholesterol; high cholesterol is a risk factor for coronary heart disease"[14,15, 16]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 28 table 1. plant sterols and stanols esters and coronary heart diseases hill criteria scientifically evidence 1. how strong the association is: strong in the most intervention studies conducted on subjects who had moderate to high cholesterol levels (total cholesterol <300 mg/dl, plant sterols esters have significantly reduced the serum total cholesterol and ldlcholesterol. 2. consistency of association observed: association was observed by various people, places, times and different circumstances four studies indicated a close correlation between the consumption of plant sterols and reduced serum cholesterol levels in hypercholesterolemia subjects. the results of three studies support the effect of plant sterols to reduce cholesterol levels among subjects who had normal cholesterol levels. two studies have shown a relationship between the consumption of plant stanols esters and reduced serum cholesterol levels in hypercholesterolemia subjects who consumed stanols esters as part of a low cholesterol and saturated fat diet. eight studies have shown a relationship between the consumption of plant stanols and reduced levels of total cholesterol or ldl – cholesterol in hypercholesterolemia subjects’ case. 3. association specificity: an association is obvious for specific causality. given the variation both in content and foods containing plant sterols and sterols esters, which have been the subject of these studies, the response of serum cholesterol levels appear to be consistent and substantial. given the variability in content and sterols food transporters used in these studies, cholesterol levels, the answer seems coherent and substantial. 4. biological gradient: evidence of dose response variations. plant sterols may be more effective than previously thought, used in small doses. researchers have found that reducing serum cholesterol level is even greater as the daily intake of plant stanols esters is higher. a minimum consumption of 0.8 g/day free plant sterols esters showed a substantial reduction of serum total cholesterol and ldlcholesterol levels. 5. plausibility: the association is biologically plausible long ago it was shown that plant sterols (β-sitosterols and its compounds) prevent the cholesterol absorption by blocking cholesterol absorption in the intestine. 6. consistency reasons: the association is consistent with current knowledge on the disease / biomarkers known to be associated with fda and efsa concluded that it is generally accepted that serum total and ldl cholesterol are major risk factors of cardiovascular disease and dietary factors that influence serum cholesterol levels affect the risk of developing cardiovascular disease. source: isli, 2002 rye bread benecol dobrogea is the only product in romania with the optimal dose of plant stanols esters, supported ingredients in europe for over 50 clinical studies. this product is the best choice for reducing cholesterol with a healthy diet. benecol is the name of food ingredient with plant stanols, which lower cholesterol and is now found on the market in romania. clinical studies show that daily consumption of benecol products for two consecutive weeks reduced total cholesterol by 10% and ldl-cholesterol by 15% and these values remain low for a period of 12 months if products containing plant stanols esters are daily consumed [17]. plant stanols esters of benecol product reduce total cholesterol by 10% and the combined effect of nutritional recommended fatty acids and plant stanols esters of benecol reduced cholesterol level by 15%. blood cholesterol concentration is the sum food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 29 of their synthesis, absorption and excretion. decrease intestinal absorption of cholesterol by 50-80% is possible when the plant stanols are included in food. in response, the liver increases the cholesterol synthesis from its precursors. despite the increased level of cholesterol synthesis, the final result is a decrease of plasma ldl cholesterol. hdl cholesterol concentrations remain unchanged. plant stanols esters’ efficacy in reducing blood cholesterol has been confirmed by double-blind, randomized and placebo controlled clinical studies. the effect of cholesterol reduction was seen at subjects with high and normal cholesterol levels, patients with cardiovascular disease, patients with i or ii degree of diabetes, subjects in europe, north america, australia and asia, in case of combination with typical western diet, with a low-fat diet or a vegetarian diet [17]. the lasting effect of cholesterol-lowering with plant stanols esters was demonstrated by two double-blind controlled clinical studies. one study lasted 12 months and was conducted on subjects with mild hypercholesterolemia. this study involved 153 subjects with moderate hypercholesterolemia who changed margarine typically consumed for rapeseed oil margarine with or without the addition of 24 g / day plant stanols esters. the intervention began with a dose of 2.6 g of sitostanol per day and continued for six months. after six months the amount of sitostanol ingested was reduced for half of the subjects at 1.8 g while the other half continued with 2.6g. in the first subgroup ldl-cholesterol was reduced by 15.2%, respectively 14% compared with that receiving placebo margarine. statistically there were no significant differences between the two doses studied [17]. similar results were obtained, for example, in a study on diabetic patients: dressing for salads with plant sterols esters added reduced cholesterol in the first weeks of consumption, an effect that was attenuated during eight weeks. furthermore, in another study lasting 52 weeks made on margarine added by sterol esters there was a reduction of only 4-6% of total cholesterol and ldl-cholesterol, for a daily dose of 1.6g of sterols. clearly, more studies are needed to assess long-term efficacy of sterols. studies were made using stanols esters both for men’s and women’s diet and the effects were similar in both cases. in contrast, the effectiveness of cholesterol reduction when using sterol esters was not as consistent as in the case when studying plant stanol esters. the efficacy and safety of consuming products enriched with plant stanol esters has been shown in clinical trials made both on adult subjects and children with familial hypercholesterolemia (fh). an initial study concluded that a partial replacement of usual dietary fats by margarine enriched with plant stanols esters is a treatment of hypercholesterolemia, safe and effective in children with fh. a significant cholesterol lowering effect was demonstrated recently in a study where children with fh consumed yoghurt enriched with plant stanols esters. in a study made on patients suffering from heterozygous familial hypercholesterolemia, a serum cholesterol decrease by 11-20%, after a consumption of spreads containing plant stanols esters alone or in conjunction with statin-based medication has been observed. the study was performed on 11 patients suffering from hypercholesterolemia, noninsulin dependents and mellitus diabetes studying cholesterol absorption and metabolism such as kinetic ldlcholesterol and hdl-cholesterol in a plant stanols esters (3 g/day) treatment [17]. total cholesterol and ldl-cholesterol decreased significantly between 6 and 9%, in that order. vldl serum cholesterol values and lipoprotein b decreased food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 30 significantly between 6 and 12%, while hdl-cholesterol increased by 11% for consumption of plant stanols esters. conclusion one risk factor in developing coronary heart disease is high cholesterol level. stanols increase cholesterol-lowering effect, with recommended changes in diet. several clinical studies have shown that benecol effectively reduces total cholesterol level by 10% and respectively ldl-cholesterol level by 15% when products are consumed as part of a recommended diet, at least for 14 days. dobrogea benecol bread consumption is recommended to any age group or different present or future pathologies that the patient may be faced with as a prevention or curative way, being fully supported by the romanian medical comunity. all these analyses suggest that for every 1% decrease in total cholesterol, there is a 2% decrease in the incidence of cardiovascular disease. these results can be obtained firstly by a change of lifestyle, and secondly by drug therapy. references 1. costin, c.m., segal, r., special nutrition food – food and health, academica publisher, bucharest, 2001; 2. louis-sylvestre, j., repas on grignotage? la difference n’est ni quantitative, ni temporelle: elle est physiologique, chole-doc, ian-febr 2000; 3. ***. 2009. omega-3 health claims, the authority on seafood, http://sin.seafish.org); 4. banu, c. (coord)., food for health, asab publisher, bucharest, 2009; 5. howlett, j., functional foods: from science to health and claims, international life science institute, brussels, belgium, 2008, (http://europe.ilsi.org/nr/rdonlyres/97c50d2f8ddb-415f-ac6ce20ed1b5e7d1/0/functionalfoods2008.pdf); 6. ift, expert report, functional foods: opportunities and challenges, institute of food technologists, chicago, il., 2005, (http://members.ift.org/nr/rdonlyres/20b9ebdd93b9-4b1b-b37b3cf15066e439/0/finalreport.pdf); 7. jones, p.j., raeini-sarjaz, m., ntanios, f.y., vanstone, c.a., fena, j.y., parsons, w.e., modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters, j. lipid res. 41: 697-705, 2000; 8. chen, h.c., molecular mechanism of sterol absorption, j. nutr., 131:2603-2605, 2001; 9. miettinen, t.a., vuoristo, m., nissinen, m., jarvinen, h.j., gylling, h., serum, biliary and fecal cholesterol and plan sterols in colectomized patients before and during consumtion of stanola ester margarine, am. j. clin nutr. 71:1095-1102, 2000; 10. hallikainen, m.a., sarkkinen, e.s., uusitupa, m.i., plant stanol esters affect serum cholesterol concentrations of hypercholesterolemic men and women in a dose-dependent manner, j. nutr. 130: 767-776, 2000; 11. jones, p.j., raeini-sarjaz, m., plant sterols and their derivates: the current spread of results, nutr. rev. 59: 21-24, 2001; 12. ostlund, r.e. jr., phytosterols in human nutrition, annu. rev. nutr. 22: 533-549, 2002; 13. normen, l., dutta, p., lia, a., anderson, h., soy sterol esters and b-sitostanol ester as inhibitors of cholesterol absorption in human small bowel, am. j. clin. nutr. 71:908-913, 2000; 14. ***, regulation (ec) no 1924/2006 of the european parliament and of the council of 20 december 2006 on nutrition and health claims made on foods; 15. ***, regulation (eu) no. 384/2010 of the commision on 5 may 2010 on approval and denial authorization of certain health claims on food and reduce the risk of disease development and child health; 16. ilsi, functional foods-scientific and global perspectives. pp. 7-10. ilsi europe series summary of a symposium hold in octombre 2001. international life science institute press, washington, dc, 2002; 17. ***, www.dobrogeabenecol.ro microsoft word 9 r_mironeasa_corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 59 study on the raw cow milk hygienic parameters from different milk collection centers and different years using a multivariate analysis method *silvia mironeasa1, georgiana gabriela codină1 1 faculty of food engineering, ştefan cel mare university of suceava, romania, silviam@fia.usv.ro; codina@fia.usv.ro *corresponding author received 15 october 2012, accepted 25 november 2012 abstract: the present study was performed in order to monitor the hygienic quality of raw cow milk from different milk collection centers of the suceava county. the raw cow milk samples were collected from three milk collection centers, a, b and c, during four seasons, in two years consecutively, 2009 and 2010. the hygienic quality of cow milk from the point of view of somatic cell counts (scc) and number of total germs (ntg) of each sample were analyzed. according to the results obtained by factorial analysis of variance (anova) comparing the hygienic quality of the raw cow milk analyzed in 2009 and 2010 years, we concluded that the highest quality was obtained in 2010, in all milk collection centers. each year had significant effect on scc (p < 0.01), as well as on ntg (p < 0.01) in bulk milk. regarding the seasonal influence on hygienic quality of raw cow milk, the higher mean values of ntg and scc were found in the summer season, while the lowest mean values of them were found in the spring and winter seasons respectively. all these mean values obtained are in accordance with the regulation 853/2004/ce. in addition, the milk collection centers did not have significant effect on the hygienic quality of raw cow milk, from the point of view of the scc and ntg mean values. keywords: raw cow milk, hygienic quality, milk collection centers, multivariate analysis 1. introduction the assessment of the hygienic parameters in raw cow milk, in terms of somatic cells counts (scc) and number of total germs (ntg) is an essential stage in the quality control process. in romania, the interest for the process of monitoring the quality of raw cow milk has increased among milk producers and processors in order to comply with the european community legislations establi-shed after 2004 (reg. ce 853/2004) which prohibits the collecting of cow milk that exceeds the value for somatic cell count of 400.000 no/ml and the value for number of total germs of 100.000 no/ml [1]. knowledge of the hygienic quality of raw cow milk has great importance for human and animal health, and therefore for milk products. cow milk is a product that contains even from the milking stage a number of germs and it is a very good medium for the development of many spoilage and pathogenic microorganisms [2]. after its ejection from the udder, the milk may be contaminated from different sources such as microflora presented in the teat skin, milking equipment and milker’s hand, water, milking environment, milking storage (at which it minimal temperature is 6oc according to ce 853/2004), e.g. the hygienic quality of raw cow milk can be influenced by various factors. such factors are the animal breed and genotype [3], animal health [4] (mammary gland health in particular [5, 6]), stage of lactation, season [7, 8, 9] microbiological food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 60 contamination [10], milking region [10, 11], milking conditions [8, 12, 13, 14] hygienic conditions of handling, transport, equipment for milk storage. bacterial contamination of raw cow milk can provide from varies sources such as air, soil, milking equipment, feed and grass [15, 16]. somatic cell counts (scc) and the number of total germs (ntg) in raw milk are an important criteria in evaluating the hygienic quality of milk. the somatic cells are a common component of milk, but if their number increases over the limit values, they affect the quality of the processed milk [17]. the scc is an indicator of the cow’s milk health state, an increase of this value indicates a poor health condition of the cow's udders or a degree of glandular alteration at the bovine mammary gland [18]. a high number of ntg in raw cow milk indicates a high microbial charge and therefore unsafe milk for consumers and milk processors. the hygienic quality of cow’s milk is an important parameter for milk processors and it must comply with the european community legislations. therefore the main objective of this manuscript was to analyzes by a statistical approach the variation of somatic cell counts (scc) and number of total germs (ntg) from three milk collection centers from suceava county during four season, in two consecutively years and to explain and analyze the possible causes of this variations. 2. materials and methods 2.1. materials the samples of raw cow milk were supplied from three milk collection centers, symbolized in this manuscript a, b and c from suceava county. the study was carried out in four periods corresponding to the four seasons of the year. the first period corresponding to the winter season covered the months december-january-february, the second one corresponding to the spring season covered the months march-april-may, the third corresponding to the summer season covered the months june-july-august and the fourth period corresponding to the autumn season covered the months september-october-november. collecting samples from a, b and c milk collection centers was conducted daily, during a period of two consecutive years, 2009 and 2010, respectively. 2.2. methods milk quality tests were accomplished according to romanian standard method for somatic cell counts (sr en iso 13366-3:2001) by infrared spectrophotometery using a bentley somacount 150 (bentley instruments inc., chaska, mn). the number of total germs was determined by flow cytometry count analyzer unit bactocount ibc 50 (bentley instruments inc., chaska, mn, usa). 2.3. statistical analysis the collected data was analyzed using factorial analysis of variance (anova) with the statistical package for social science (v. 16, spss inc., chicago, il, usa). the general linear model (glm) procedure was applied and the least significant difference (lsd) method for comparison of mean values of the characteristics was studied. a 2  4  3 factorial experiment for the type of years, seasons and milk collection centers, was conducted to evaluate the effect of each treatment. a 5% significance level was used throughout the study. 3. results and discussion the results obtained emphasize the mean variation of somatic cells count food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 61 (scc) and number of total germ (ntg) values with the seasons, milk collection centers and the collecting years on the one hand and the interaction effect between the factors (season, milk collection center, year) on the other hand. factorial anova revealed significant main effects for season and year on scc and statistically significant interaction effects, at different levels, between factors. it can be noted that the mean values of somatic cells count in the raw cow milk is lower from all milk collection centers in the year 2010 compared to 2009 (figure 1), the mean difference being significant at p < 0.01. in terms of comparison between milk collection centers a, b and c, the highest mean values for somatic cells count are recorded in b and c centers, while the lowest mean values is recorded in a center which does not exceed the value of 400 x 103 cell/ml for proper milk, according to the eu legislations, in all centers. the most significant decrease in somatic cells counts from a milk collection center, in the year 2010 is probably due to the animal health, mammary gland health in this area, the absence of subclinical mastitis and to the high natural resistance of the cows to mammary infections. figure 2 shows the results of the number of the total germs variation for the raw cow milk, from a, b and c collection centers during the two years. in our study, the year factor had a significant effect on the ntg (p < 0.01) which has been proven. the effect of milk collection centers did not have a significant impact on the mean values of ntg. in this study it was found that ntg values are comparatively higher in milk samples from the year 2009 and lower in the milk samples from the year 2010. the decreasing mean values of ntg from milk samples may be due to the hygienic maintenance during milking and cleanliness. according to parekh and subhash (2008), the use of clean milking and transport equipments contributed to the good hygienic quality of produced milk. in the b milk collection center we noticed a lower number of total germs comparatively with the values from a and c centers. this difference may be due to poorer hygiene and other sanitary conditions during milking, storage and transporting when compared with the conditions from the b milk collection center. however, we have not found a relativly significant difference between the mean values of ntg from the milk collection centers that were analyzed. figure 1. variation of somatic cells counts (scc) in milk collection centers from the two years consecutively figure 2. variation of number of total germs (ntg) in milk collection centers from the two years consecutively food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 62 a significant variation in the quality of raw cow milk, from the point of view of somatic cells count and the number of total germ values has been found during the two years of analysis in all milk collection centers used in our study. when comparing the mean values of scc noticed in the year 2009 with the mean values noticed in the year 2010, a decrease in scc values was noticed in 2010, in each season (figure 3). the highest mean values of scc were noticed during summer season, while the lowest mean values were noticed during winter season. somatic cells count was significantly (p < 0.05) higher in summer season comparatively to winter season (figure 3) which it may be related to the increasing temperatures in the environment and therefore to a more intense microbial multiplication. these results are in agreement to the results obtained by auldist (1998), adesiyun et al. (1995) who have analyzed the effects of seasons on the hygienic quality of raw cow milk. the high level of somatic cells counts during summer season may be related to the animal health from this period and can be due poor health condition of the cow’s udders [20, 22]. by applying the control measures on animal health, an increase of the milk quality parameters in the year 2010 was noticed, when compared with the year 2009. therefore, the raw cow milk parameters were filed in a proper milk category according to the ue legislation. the results obtained for the number of total germs (figure 4) show a significant influence of the factors season and year and also a significant interaction between them. regarding the ntg values, in the summer season, the highest mean values were noticed, whereas in the spring season the lowest values were noticed, with lower mean values in the year 2010 when compared to the values from the year 2009. season variations had a mjor impact on (p < 0.05) the number of total germs from cow milk. the mean values for ntg noticed in all seasons show that the raw cow milk analyzed was classified in a proper milk category according to the ue legislation. figure 3. variation of somatic cells counts (scc) during seasons from the two years consecutively figure 4. variation of number of total germs (ntg) during seasons from the two years consecutively 4. conclusions this study shows that different results were obtained for hygienic quality of raw cow milk from three milk collection centers during four seasons in two consecutively years. the obtained results indicate the fact that a decrease of the number of total germs and of the somatic cells count leads to an increase of the hygienic quality of milk, positively affecting the milk and milk products quality. the decreased values of somatic cell count (scc) and the number of total food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 63 germ (ntg) in the raw milk samples that were collected in the suceava county from the analyzed milk collection centers, shows that the proper hygiene practices in milking and collecting the milk are applied. the principal hygiene practices are: taking action in order to establish proper sanitation procedures during the milking process, the high degree of farmers education, the interest of the farmers for a decrease of the microbiological charge due to the fact that this influences the price of the milk, e.g. therefore the practices and regulations such as food safety management systems, which only follow established standards, have been properly used and have facilitated the production of cow milk of high quality and safety. 5. references [1]. european commission, regulation (ec) no. 853/2004 of the european parliament and of the council of 29 april 2004 laying down specific hygiene rules for food of animal origin. off. j. eur. commun. l 139, 55 -205, (2004) [2]. frank j.f., milk and dairy products. in p. doyle, r. beuchat, & j. montville (eds.), food microbiology – fundamentals and frontiers. washington dc: asm press, (2009) [3]. lucas a., agabriel c., martin b., ferlay a., verdier-metz i., coulon j.b., rock e., relationships between the conditions of cow’s milk production and the contents of components of nutritional interest in raw milk farmhouse cheese, lait, 86, 177–202, (2006) [4]. mubarack h.m., doss a., dhanabalan r., balachander s., microbial quality of raw milk samples collected from different villages of coimbatore district, tamilnadu, south india, indian journal of science and technology, 3 (1), 61-63, (2010) [5]. haenlein g.f.w., nutritional value of dairy products of ewe and goat milk. in production and utilization of ewe and goat milk, proceedings of the idf/greek national committee of idf/cirval seminar, brussels, belgium, international dairy federation, 159–177, (1996) [6]. othmane m.h., carriedo j.a., de la fuente l.f., san primitivio f., factors affecting testday milk composition in dairy ewes and relationships amongst various milk components, j. dairy res., 69, 53–62, (2002) [7]. mironeasa s., codină g.g., multivariate analysis in assessment relationships between milk characteristics influenced by the seasonal variations, food and environment safety, x (4), 104-107, (2011) [8]. van nieuwenhove p. c., oliszewski r., gonzález n.s., fatty acid composition and conjugated linoleic acid content of cow and goat cheeses from northwest argentina, journal of food quality, 32, 303-314, (2009) [9] alomirah h., al-mazeedi h., alzenki s., al-aati t., al-otaibi j., al-batel m., sidhu j., prevalence of antimicrobial residues in milk and dairy products in the state of kuwait, journal of food quality, 30, 745-763, (2007) [10]. gaucher i., boubellouta t., beaucher e., piot m., gaucheron f., dufour e., investigation of the effects of season, milking region, sterilisation process and storage conditions on milk and uht milk physico-chemical characteristics, a multidimensional statistical approach, dairy sci. technol., 88, 291–312, (2008) [11]. mironeasa s., codină g.g., mironeasa c., variation analysis of cow milk composition quality depending on year, season and location in romania, bulletin uasvm animal science and biotechnologies, 68 (1-2), 225-232, (2011) [12]. falchero l., lombardi g., gorlier a., lonati m., odoardi m., cavallero a., variation in fatty acid composition of milk and cheese from cows grazed on two alpine pastures, dairy sci. technol., 90, 657–672, (2010) [13]. bony j., contamin v., gousseff m., metais j., tillard e., juanes x., decruyenaere v., coulon j.b., factors of variation of the milk composition on the reunion island. inra prod. anim., 18, 255–263, (2005) [14]. salovuo h., ronkainen p., heino a., suokannas a., ryhänen e.l., introduction of automatic milking system in finland: effect on milk quality. agricultural and food science, 14, 346-353, (2005) [15]. torkar k.g., teger s.g., the microbiological quality of raw milk after introducing the two day’s milk collecting food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 64 system, acta agri. slovenica, 92 (1), 61 – 74, (2008) [16]. angulo f.j., lejeune j.t., rajalaschultz p.j., unpasteurized milk: a continued public health threat, clinical infectious diseases, 48 (1), 93-100, (2009) [17]. antunac n., lukač havranek j., samardžija d., somatske stanice i njihov utjecaj na kakvoću i preradu mlijeka, mljekarstvo, 47, 183-193, (1997) [18]. sharif a., umer m., muhammad, g., mastitis control in dairy production, journal of agriculture & social sciences, 5 (3), 102-105, (2009) [19] parekh t.s., subhash r., molecular and bacteriological examination of milk from different milch animals with special references to coliforms, curr. res. bacteriol., 1 (2), 56-63, (2008) [20] auldist m. j., hubble i. b., effects of mastitis on raw milk and dairy products. the australian journal of dairy technology, 53, 28-36, (1998) [21] adesiyun a.a., webb l., rahman s., microbiological quality of raw cow’s milk at collection centres in trinidad. journal of food protection, 58 (2), 139-146, (1995) [22] matei s.t., groza i., andrei s., bogdan l., ciupe s., petrean a., serum metabolic parameters in healthy and subclinical mastitis cows. bulletin uasvm, veterinary medicine, 67 (1), 110-114, (2010) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 71 residual chlorine in water supply systems *sonia gutt (amariei)1 mihaela cojocariu1 stefan cel mare university, faculty of food engineering, universitatii st.13, suceava, 720 229, romania, e-mail: gutts@fia.usv.ro, myhaela_coj@yahoo.com *corresponding author received 2 august 2011, accepted 25 september 2011 abstract water disinfection is practiced in surface water, groundwater in fissured soils, karst or poorly filtered. this study determined the efficiency of chlorination of water plants treating surface water (river suceava) and accumulations of water. disinfection of water may have undesirable effects by persistence in drinking water of substances used to treat it or their byproducts, such as chlorophenols or trihalomethanes (case of chlorination) that aldehydes, phenols and carboxylic acids ( treatment with ozone). the analysis results show that chlorine values depend greatly on temperature, contact time, ph and oxidisability. like other studied parameters chlorine shows an increase in concentration for warm summer period, due to the requirement for chlorine in water. in the study in three years to monitor the chlorine was found that it tends toward a slight increase due to application of chlorine in water and rehabilitation works for the city water network. physical-chemical quality parameters of water distributed by the two plants that treat surface water (dragomirna and mihoveni) were within the limits allowed under the law. key words: water disinfection, contact time, ph, organic matter content, temperature 1. introduction water disinfection with chlorine, played and still plays an important role in reducing diseases spread by water. chlorine is a strong oxidant which produces in water several substances including hypochlorous acid (hoci) and hypochlorite ion (oci). both substances kill microorganisms by attacking the cell wall lipids and changes cell membrane permeability[2], (at concentrations of 0.2 mg / l) [7], by destroying the enzymes and nucleic acids (at doses of 40.3 mg / l ) [7]. hocl and ocl concentration values vary depending on the ph level. if the ph is too high, the hocl concentration is not enough, ideally, the ph level should be between 7 and 8, value of 7.4 is the ideal ph. in slightly alkaline water (ph = 7.5), chlorine is found in equal proportions in the two forms. the more alkaline becomes water (high ph) more increases the hypochlorite formed and vice versa, the more acide becomes water ph (ph<7) more increases the amount of hypochlorous acid formed. in terms of power to destroy bacteria, hypochlorous acid is more effective. hypochlorous acid and hypochlorite ion are strong oxidizers for organic and inorganic substances contained in water [4]. once it is introduced into water, chlorine (hocl and clo-) oxidizes some minerals (salts of iron, magnesium, nitrites, etc.) resulting chlorides and oxidised compounds. cantitatea de clor consumată în aceste reacţii constituie cererea de clor imediată. the amount of chlorine consumed in these reactions are immediate chlorine demand. after immediate chlorine demand, the remaining amount of chlorine acts on ammonia (when it is present in water), leading to the formation of chloramines [4] (bound active chlorine). between hypochlorous acid (hocl) and ammonia following reactions are possible[11]: nh3 + hoci h2o + nh2ci nh3 + 2hoci 2h2o + nhci2 mailto:gutts@fia.usv.ro mailto:myhaela_coj@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 72 nh3 + 3hoci 3h2o + nci3 further introduction of chlorine is called in the literature the breaking point and will cause the oxidation of chloramines (which is desirable). 4nh2cl + 3cl2 + h2o n2 + n2o + 10hcl continuing the addition of chlorine over the breaking point it will produce a proportional increase in free active chlorine, used for the disinfection. if water containing natural organic substances (humic acid) or artificial (pesticides, phenols), chlorine may cause certain organic compounds, with unpleasant taste and smell or even dangerous, suspected to be carcinogens [5] [6], as trihalomethanes, chlorophenols or influence premature births [2]. residual chlorine can be present in two forms namely hypochlorous acid or hypochlorite, which is called free chlorine and chloramine which is called bound chlorine. the amount of free residual chlorine and bound chlorine is total chlorine. 2. materials and methods the presence of residual chlorine in water disinfection is an important health subject indicating on the one hand that was introduced a sufficient quantity of chlorine to ensure disinfection, and on the other side shows the water distribution system integrity. free residual chlorine is more valuable than bound chlorine because it is much more sensitive. for the analysis was used the method with metiloranj [10], that molecular chlorine discolores metilorange solution proportional to its concentration. 2.1. reagents: 0.46 ‰ metiloranj solution, ); sulfuric acid solution (h2so4) 1:3 , solution of potassium bromide (kbr) 1%. all reagents were acquired from merck; (darmstadt, germany). standardization of metilorange solution is achieved so that 1 ml metiloranj to be by discolored by 0.1 mg of chlorine. metiloranj, working solution is obtained from metiloranj solution prepared as above, diluted 10 times(1 ml of this solution is discolored 0.01 mg cl). parallel is a done a blank using doubly distilled water. the quantity of metiloranj used in the blank is subtracted from the amount of metiloranj used in the sample. 3. results and discussion using the method described, were monitored between 2008 may 2011, water stations dragomirna, mihoveni, radauti and berchişeşti in which raw water is disinfected with chlorine. figure 1. variation of chlorine for a period of one year figure 1. variation of chlorine for a period of one year variaţia clorului în decursul unui an 0,2 0,25 0,3 0,35 0,4 0,45 0,5 0,55 iarna primavara vara toamna perioada anului co nc en tra tia d e c lo r ( m g/ l) dragomirna mihoveni rădăuţi berchişeşti chlorine concentration on year winter spring summer autumn c hl or in e co nc en tra tio n (m g/ l) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 73 minimum chlorine is achieved in winter since the activity of microorganisms in the winter is very low, chlorine concentration is less, and the more temperatures rise, increases the solubility of chlorine in water, but also the chlorine demand to destroy pathogens. surface waters of dragomirna and mihoveni require higher chlorine demand to those of ground waters as berchişeşti and radauti. consumul de clor în decurs de trei ani 0,32 0,4 0,47 0,54 0,42 0,48 0,52 0,55 0,39 0,32 0,37 0,35 0,36 0,4 0,43 0,5 0,25 0,3 0,35 0,4 0,45 0,5 0,55 0,6 2008 2009 2010 2011 perioada (ani) c on ce nt ra ţia d e cl or (m g/ l) dragomirna mihoveni rădăuţi berchişeşti figure 2. variation in total chlorine for a period of three years waters of the three stations were monitored between 2008 may 2011. figure no.2 shows a constant concentration in radauti areas, unlike the other three stations that have an upward trend, growth due to the requirement of chlorine in water, due to water hyperchlorination to achieve rehabilitation programs suceava, replacement of water pipelines. variaţia clorului în funcţie de punctul de pre le v are a probe i 0,57 0,5 0,47 0,3 0,1 0,63 0,57 0,5 0,4 0,2 0,4 0,36 0,3 0,25 0,15 0,55 0,5 0,45 0,38 0,24 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 puţul colector ieşirea din staţie primul consumator mijlocul reţelei capat de reţea punctul de pre le v are a probe i c o n ce n tr aţ ia d e cl o r (m g /l) dragomirna mihoveni rădăuţi berchişeşti figure 3 . total chlorine variation depending on the sampling point chlorine concentration on year c hl or in e co nc en tra tio n (m g/ l) c hl or in e co nc en tra tio n (m g/ l) period (years) sampling point pit collector leaving the station first consumer middle network end network food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 74 from figure 3 it can be seen retention of chlorine in water, residual chlorine decreasing as the water is away from the shaft collector, where there is the dosage of chlorine in water. chlorine retention is dependent on the length of water network and the number of pathogenic microorganisms in water. . variaţia clorului în 24 ore 0,2 0,25 0,3 0,35 0,4 0,45 0,5 0,55 0,6 0,65 0,7 1 2 3 4 5 6 7 8 10 12 13 14 15 16 17 18 19 20 21 22 23 24 ora la care s-a făcut analiza c o n c e n tr a ţi a d e c lo r (m g /l ) dragomirna mihoveni radauti berchisesti figure 4. variation of total chlorine in the course of the day if the length of the network is too high and it does not keep the need of chlorine in water and the pressure in the pipeline, chlorination points and pressure boosting can be made. chlorine concentration was monitored over 24 hours, chlorine demand changing in hour to hour, depending on time of day. temperature has an important role in the solubility of chlorine in water, since decreases water temperature, the chlorine is harder dissolved in water. since in the same diagram we can not compare these parameters due to different units of measurement, we will analyze in turn. the content of organic substances in water influence the demand of chlorine, so there are concerns for its reduction by different methods, which results in decreased demand for chlorine. figure 5.variation of organic matter content in between january and december time the analysis was done c on ce nt ra tio n (m g/ l) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 75 the diagram oxidabilităţii shows an upward trend of analysed values in the range from february to july, a flat from july to august and a decrease in augustdecember, increasing of organic matter in water due to the growth of microorganisms during the summer. figure 6. temperature variation between january-december usually, deep water has a constant temperature throughout the year with little changes of values in summer. figure 7. variation of ph in between january and december water ph also belongs to the category of parameters that influence the chlorine in water and shows high values in summer and values approaching neutral value of 7. drinking water legislation (law 458/2002) provides a ph in the range 6.5 to 7.5. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 76 figure 8. variation of total chlorine between january and december figures 5, 6, 7, shows that chlorine values depend largely of temperature and ph oxidisability. like other parameters studied chlorine shows an increase in concentration during the warm summer months, due to increased activity of microorganisms. from the same water samples taken at different time intervals 10, 20, 30 and 45 minutes, 1 hour, 12 hours and 24 hours were determined free and bound chlorine. figure 9. variation of free and bound chlorine in 24 hours as can be seen from figure 9, free residual chlorine (hocl, hcl), decreases over time, and free residual chlorine must be at least 80% of total chlorine. bound chlorine as chloramines and other chlorine compounds is inversely proportional to the free chlorine and its concentration increases with the passage of hours which thus explains the taste and odor in drinking water. if the free residual chlorine must be 80% of total chlorine it can see the water after 12 hours water no longer meets the above condition. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 77 varitaţia clorului total în 24 ore 0,53 0,5 0,49 0,44 0,41 0,4 0,36 0,3 0,35 0,4 0,45 0,5 0,55 0,1 0,2 0,3 0,45 1 12 24 timpul (ore) c o n ce n ta tia (m g /l) clorul total figure 10. variation of total chlorine in 24 hours in the same water sample 4. conclusions this study determined the effectiveness of chlorination stations dealing with surface water (river suceava) and accumulation of water. disinfection of water may have undesirable effects by persistence in drinking water of substances used to treat it or their by-product [1], as chlorophenols, or trihalomethanes (if chlorination) that aldehydes, phenols and carboxylic acids (for ozone using). as disinfection methods, their effectiveness decreases in the following order: cl gaseous >o3 > uv > cl2o > hclo > clo> chloramines because o3 and uv are very expensive and requires a smaller volume and flow of water and a very high price the method of disinfection with chlorine is the most used. chlorine is effective due to its retention in water, destroys pathogens throughout the network of water distribution of a large volumes of surface and groundwater, with flow rates and variable pressures from one station to another. in the study for the three years to motorized chlorine was found that it tends toward a slight increase due to the demand of chlorine in water. relative to sampling, chlorine concentration decreases as it moves away from the treatment plant so at the end of the network the chlorine concentration is 0.1 mg/l. within 24 hours the chlorine demand varies greatly depending on the population water demand which is lower during the night between 23-5 and higher between 6-22. annually, the requirement for chlorine is variable increasing in summer and decreasing during autumn and spring. temperature and ph have an effective contribution to the chlorine in the water as could be determined from diagrams. physical-chemical parameters of water distributed by the two plants that treat surface water (dragomirna and mihoveni) were within the limits allowed under the law. concentration of organic substances, an important indicator for water chlorination, was within acceptable limits (5 mg o2 / l). water disinfection with chlorine generates chlorine compounds presented in water supplied by the four stations. c on ce nt ra tio n (m g/ l) total chlorine time (hours) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 78 5. references 1. akbar mehrsheikha, marian bleekea, stephan brosillons, alain laplanches, pascal roche, investigation of the mechanism of chlorination of glyphosate and glycine in water, water reasearch, 40 (2006) 3003 – 3014 2. meng-huot phea, manuel dossotb, he´ le`ne guilloteaua, jean-claude blocka, nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria, water research 39 (2005) 3618–3628 3. chun-yuh yang, drinking water chlorination and adverse birth outcomes in taiwan, toxicology, 198 (2004) 249–254 4. wontae leea,, paul westerhoffb, formation of organic chloramines during water disinfection – chlorination versus chloramination, water research 4 3 ( 2 0 0 9 ) 2 2 3 3 – 2 2 3 9 5. jeffrey w.a. charrois,1, s. e. hrudey, breakpoint chlorination and free-chlorine contact time: implications for drinking water nnitrosodimethylamine concentrations, wat er research 41 ( 2007 ) 674 – 682 6.christelle lagay, manuel j. rodriguez, rehan sadiq, jean b.serodes, patrik lavallois, francois proulx, spatial variations of human health risk associated with exposure to chlorination by-products occurring in drinking water, journal of environmental management,volume 92, issue 3, march 2011, pages 892-901 7. anastasia d. nikolaou, spyros k. golfinopoulos, george b. arhonditsis, vassilis kolovoyiannis, themistokles d. lekkas, modeling the formation of chlorination by-products in river waters with different quality, chemosphere 55 (2004) 409–420. 8. luigi rizzo, annamaria di gennaro, marialuisa gallo, vincenzo belgiorno, coagulation/chlorination of surface water a comparison between chitosan and metal salts, separation and purification technology, volume 62, issue 1, 1 august 2008, pages 79-85. 9. fei gea, lizhong zhua, hairong chena, effects of ph on the chlorination process of phenols in drinking water, journal of hazardous materials b133 (2006) 99–105 10. manescu s., cucu m. „chimia sanitara a mediului”, editura medicala, bucuresti, 1978; p.150-157 11. negoiu d. „tratat de chimie anorganica”, vol. ii, editura tehnica, bucuresti, 1972 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%236871%232011%23999079996%232832784%23fla%23&_cdi=6871&_pubtype=j&view=c&_auth=y&_acct=c000062632&_version=1&_urlversion=0&_userid=4257031&md5=31faed19ac7b36a5f618036673a9e672 http://www.sciencedirect.com/science/journal/13835866 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235284%232008%23999379998%23691961%23fla%23&_cdi=5284&_pubtype=j&view=c&_auth=y&_acct=c000062632&_version=1&_urlversion=0&_userid=4257031&md5=d75654f1bba3c0297c85f1a39712d69e http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235284%232008%23999379998%23691961%23fla%23&_cdi=5284&_pubtype=j&view=c&_auth=y&_acct=c000062632&_version=1&_urlversion=0&_userid=4257031&md5=d75654f1bba3c0297c85f1a39712d69e food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 66 an obtaining method for carbon nanotubes with modified surface dumitru dima1, monica murarescu1, gabriel andrei1, simona chiculita1 1dunarea de jos university of galati , domneasca street 47, romania, monica.murarescu@ugal.ro abstract: it is well-known the fact that carbon nanotubes (cnts) contain carbon atoms in different hybridization states (sp3 and sp2), that generates complex structures (diamond and graphite respectively). these multiple connections generate strong interaction of forces between particular cnts, that means van der waals forces and also π-π type those immediate result in the agglomerates obtaining and, singularizing the composite materials, the first effect is clusters generation process. the physical-chemical properties of nanocomposite materials are influenced by the appropriate cnts dispersion obtaining in the matrix. this fact is efficiently obtained when the interactions energy between cnts themselves is lower than the dispersion energy cumulated with the interactions energy between cnts and the matrix. the dispersion energy considers cnts blending techniques inside the matrix and the interactions energy between cnts and the matrix supposes the chemical bond obtaining and physical-chemical interactions between cnts and the matrix. the aim of this paper is to present some techniques of oxidative chemical treatment in order to obtain a cnts functionalized surface as well as some techniques of coating with ceramic materials molecular layers in order to increase the chemical bond energy and the physical-chemical interactions between cnts and the matrix. we used simple treatment methods of cnts in acidic solutions (hno3 – h2so4) and basic solutions (nh3) having hydrogen peroxide as the oxidant agent (h2o2 30%). cnts coating process with ceramic materials was made by using the precipitation method of their components (fe2o3) from the supersaturated aqueous solutions. keywords: nanoparticles, functionalization, ceramic materials, multi wall carbon nanotubes (mwcnts). introduction an increased cnt – matrix interfacial bond can be obtained due to nanocomposites cracking resistance improvement that represents one of the most important aspects ensuring the shear stress transfer to the reinforcement. the use of this functionalization process in order to obtain an improved connection between matrix and nanotubes was largely presented by frankland and co. they showed that the mechanical properties improvement can be obtained if less than 1% of carbon atoms from cnts make reactive bridges to the matrix. a great discovery in cnts reinforcing composites development was made by cnts chemical functionalization with multifunctional amines. fig.1 cnts functionalization process [1] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 67 an oxidative treatment on cnts was made in order to obtain the carboxylic groups attached to their surfaces. this carboxylic group obtaining process is combined with cnts ends opening (fig.1). this phenomenon would allow a direct connection between different nanotubes ends, process that facilitates the mechanical reinforcement. in the next step, the carboxylic groups would react with the multifunctional amines in order to make chemical links (ionic or covalent in this case) due to the acid – base reaction. once the resin is added, the free amino functions on cnts surface would react with macromolecules contributing to the new links obtaining process that improves the matrix – nanotube interface. excepting the direct chemical functionalization, another simple method of cnts and matrix polarity assuming is the use of surfactants. the advantage of this procedure is the physical adhesion that is not affecting the cnts structural quality. the functional groups covalent integration is always reported to the structural modifications of the graphitic layers. [2] the surfactants use is a general method that represents some licensed procedure base [3], [4], [5]. surfactants can make the cnts dispersion into polymeric matrix, so that the attraction forces between them are surpassed. the dispersion mechanism is made by the multiple interactions between surfactants and the two main components of the nanocomposite, which are: the matrix and cnts. the conjugated polymers of the main resin can be physically linked by cnts, so that being used for cnts – matrix compatibility improvement. anyway, the covalent bond is stronger than the physical interactions and the assumptions anticipate an insignificant influence on cnts mechanical performance. thus, the final conclusion is that cnts surface chemical functionalization process is a key point in cnts / polymer composite development. materials and methods the aim of this paper is the presentation of carbon nanotubes treatment methods for their compatibilization with the polymeric matrix in order to obtain an efficient dispersion. [10], [11] the method consists in carbon nanotubes dispersion insurance through chemical processes of surface functionalization due to a controlled oxidative attack, followed by a coupling process of the chemical compatible groups with the polymeric matrix. it is also used a covering method with a particles molecular layer obtained after a crystallization process from a saturated solution. [8] the modified nanotubes after this process are more efficiently dispersed by a mechanical, ultrasonic and vibrant magnetic field stirring in the polymeric matrix. [12] we applied the oxidative method using chemical agents like: hno3, h2so4 and h2o2 as well as functionalization agents, like: nh3, in order to break the c-c bonds from the multi-wall carbon nanotubes surfaces and to create new functional groups covalently bonded with them. it was used three functionalization methods of the multi-wall carbon nanotubes surfaces. the first surface functionalization method is made by using a mixture of hno3: h2so4 = 1:3 (hno3 65% and h2so4 98%) for 24 hours at 295k. it follows a step to step washing process of the functionalized nanotubes with bidistilled water till ph = 1 because above this ph value the carbon nanotubes free sedimentation from the solution can not be efficiently made, so that another separation technique should be applied. under these conditions, nanotubes’ cleaning from the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 68 solution lasts 10 minutes. the fair liquid is separated and follows a step to step washing process of the functionalized nanotubes with bidistilled water till ph = 6. under these conditions, nanotubes separation from the solution is efficiently rmade by a centrifugation process at 6000rpm. in order to obtain sustainable drying, we applied a step to step washing process of the functionalized nanotubes using anhydrous ethylic alcohol. it follows an oven drying of 8 hours at 378k and finally the dry milling process. the second surface functionalization method consists in carbon nanotubes functionalization achievement by using a mixture of h2o2: nh3 = 1:4 (h2o2 30% and nh3 25%) for 48 hours at 295k, followed by step to step washing process of the functionalized nanotubes with bidistilled water till ph = 8 because above this ph value the carbon nanotubes free sedimentation from the solution can not be efficiently made, so that another separation technique should be applied. it follows the nanotubes’ separation from the solution that lasts about 15 minutes, a step to step washing process of the functionalized nanotubes with bidistilled water till ph = 7, nanotubes centrifugation from the solution at 6000rpm. the step to step washing process of the functionalized carbon nanotubes using anhydrous ethylic alcohol contributes to the fast drying process efficiently ended by an oven drying for 8 hours at 378k. finally, the obtained powder is dry milled. another method of modifying carbon nanotubes surface for an increased compatibility with the polymeric matrix consists in covering technique with a molecular layer of fe2o3 [6], [7]. the first step of this method is represented by carbon nanotubes dispersion using a solution of 1% sodium dodecyl sulphate (sds) as surfactant agent followed by ultrasonication process for 10 minutes with bandelin hd3200 electronic gmbh & kg berlin germany as presented in fig.2, having 40% amplitude. subsequently, a solution of fecl3 1mol/l was quantitative added under a magnetic stirring for 5 minutes and the resulted solution is ultrasonicated for 10 minutes using the same ultrasounds generator, at the same parameters. fig.2 cnts suspension ultrasonication using bandelin hd3200 as generator after that, a solution of nh3 1mol/l till ph = 8.5 was quantitatively added, followed by the same ultrasonication process. the final stage consists in a step to step washing process of the nanotubes covered by a molecular layer of fe2o3 particles using bidistilled water till ph = 5.5, followed by a centrifugation process of the nanotubes from the solution at 6000rpm, the final washing with anhydrous ethylic alcohol, the oven drying for 8 hours at 443k and finally the dry milling process. results and discussion the obtained materials complying with these methods presented above were analyzed in order to show the modifications appeared in comparison with the initial cnts. for this reason, we used analysis techniques of the functional groups from cnts surface level as the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 69 oxidative decoupling result of c-c bonds localized at mwcnts external tube level. the un-oxidized inner side tubes maintain the mwcnts initial properties and the functionalized or covered with a molecular layer of fe2o3 external tube makes a better compatibility between matrix and nanotubes. fe2o3 molecular layer has an important function to interpose to the adhesion forces between carbon nanotubes creating a coating while the deposit is forming on their surface. the nanotubes’ analysis by x ray fluorescence presents the primary modifications due to the chemical treatments of surface modifications (table 1). table 1 xrf analysis in order to present the modifications suffered by cnts surfaces after these three different treatments applied, we performed an xrf analysis with niton xlt 793 thermo fischer scientific usa and the results were presented in table nr. 1. it can be observed that considering the samples covered with a molecular layer of fe2o3 (coded with f3), fe concentration is about 5.52 %. this covering layer represents a consistent deposit of fe (iii) oxide enough for generating in the composite material structure some magnetic properties utilized also in the obtaining step represented by the dispersion process in a vibrant magnetic field as well as subsequent at the general properties of the composite material. [9] referring to ni and co presence, the variations are not very important meaning that in the functionalization and covering process there are not losses or accumulations due to the chemical reagents. it does not happen the same thing at mn, resulting in an increased value about four times for the samples covered by a molecular layer of fe2o3. considering cr value, this one maintains quasi-constant referring to the second method of functionalization and decreases at a moiety in the first functionalization method as well as in the covering process with a molecular layer of fe2o3. titanium and k present a spectacular increase in f3, this value representing a result of the chemical reagents influence. at calcium there are no significant variations excepting the functionalized samples by method 1 or 2 as a result of frequently washings as well as chemical reagents attack. moreover, the nanotubes were analyzed referring to their behaviour in the polymeric matrix, so that we analyzed their concentration influence on the rheological properties. we analyzed the influence of pure carbon nanotubes (coded cnt-pure) concentration and modified by functionalization and covering process (coded cnt-f1, cnt-f2 and cnt-f3 respectively). the aim of this study was to determine the optimum value of carbon nanotubes concentration in the polyester resin in order to correspond to the technological requests for a nanocomposite material obtaining process. cnts optimum concentration value in polymeric matrix can be determined using a viscosity variation study in relation to nanotubes concentration from the polymeric resin, in accordance with scientific literature. cnt elem. (ppm) pure f1 f2 f3 fe 1120.15 640.67 1093.34 55167.66 ni 7185.23 4612.44 6775.87 4109.67 co 157.55 126.15 160.15 27.85 mn 47.47 35.67 55.61 244.58 cr 1302.86 593.27 1242.42 610.21 v 424.23 38.16 300.37 437.52 ti 1027.25 41.11 755.16 3277.15 ca 1901.15 507.16 672.23 1255.14 k 225.16 353.85 281.93 2889.62 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 70 experimentally the optimum concentration value is determined at an important viscosity increasing moment, after that a constant value is registered or even a decreasing value is possible. the experimental values are synthetically presented in fig.3. viscosity variation with cnts concentration -0,2 0 0,2 0,4 0,6 0,8 1 0,05 0.10 0,15 0,2 0,25 %cnt δη cnt pure cnt-f1 cnt-f2 cnt-f3 fig.3 viscosity variation depending on cnts concentration it can be observed that cnt-f1 and cntf2 respectively present wide equal variations and their plots are similar and the experimental viscosity variation data are quite higher than cnt-pure data. this can be explained by carbon nanotubes dispersion improving at cnt-f1 and cntf2 respectively in polyester matrix at the same concentration and under technological experimental conditions. this result is an expected one, considering the increased energy value when between the polymeric matrix and the functional groups the strong covalent bond is present. [13] in the cnt-f3 case, fe2o3 presence can determine contradictory effects, which mean: van der waals and π-π interactions decline between carbon nanotubes due to oxide layers “arrow effect” that interpose themselves and, the most important effect would be the magnetically interactions that are responsible for clusters generation in the matrix added with covered nanotubes. that is the reason because new dispersion energy must be introduced in order to block clusters formation; this target is technological realizable due to an external vibrant magnetic field introduction. [14] conclusion considering the experimental data analysis and processing, an optimum concentration value about 0.15% cnt in cnt-f1, cntf2 and cnt-f3 systems respectively was obtained, excepting cnt-pure system which optimum value was registered at 0.20% cnt, according to the scientific literature data concerning nanoparticles suspensions. acknowledgements this work was supported by cncsis – uefiscsu, project number pnii – idei 519/2008 9/2008. references 1. m. e. mackay, a. tuteja, p. m. duxbury,c. j. hawker, b. van horn, z. guan, general strategies for nanoparticle dispersion, science 24 march 2006, vol. 311. no. 5768, pp. 1740 – 1743; 2. s. bose, a. r bhattacharyya, r. a. khare, a. r. kulkarni, t. umasankar patro, tunning the dispersion of multiwall carbon nanotubes in co-continuous polymer blends: a generic approach, nanotechnology 19 (2008) 335704. 3. b. duk yang, k. h. yoon, k. w. chung, dispersion effect of nanoparticles on the conjugated polymer–inorganic nanocomposites, materials chemistry and physics 83 (2004) 334– 339; 4. z. guo, k. lei, y.li, h.wai, s.prikhodko, h. thomas hahn, fabrication and characterization of iron oxide nanoparticles reinforced vinyl-ester resin nanocomposites, composites science and technology 68 (2008) 1513–1520; 5. j.r.baran, b.j.gabrio, j.s.stefely, s.w.stein, t.e.wood, stabilized particle dispersions containing surface-modified inorganic nanoparticles, us 2008/0268062 a1, oct.30, 2008; food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 71 6. a. sundaresan, c. n. r. rao, ferromagnetism as a universal feature of inorganic nanoparticles, nano today (2009) 4, 96-106; 7. gou j.h., minaie b., wang b., liang z.y., zhang c., “computational and experimental study of interfacial bonding of swnt reinforced epoxy composites” computational materials science, 2005,31 (3-4) 225-236; 8. wong m.h., paramsothy m., ren y., xu x.j., li s., liao k., “physical interactions at carbon nanotube-polymer interface”, polymer, 2003, 44 7757-7764; 9. b. fiedler, f. h. gojny, m. h. g. wichmann, m. c. m. nolte, k. schulte, fundamental aspects of nano-reinforced composites, composites science and technology 66 (2006) 3115–3125; 10. g.desie, l.vanmaele, g.deroover, method to improve the quality of dispersion formulations, pub. no.: us 2005/0235740 a1; 11. s.stankovich, s.t. nguyen, r.s.ruoff, stable dispersions of polymer-coated graphitic nanoplatelets, provisional applications no.60/738.334, nov., 2005; 12. s.asgari, composite materials containing carbon nanoparticles, provisional applications no.60/643.842, ian.2005; 13. i.j.kim, h.joo, d.w.jeong, s.k.kang, thermoplastic nanocomposite resin composite materials, pub. no.: us 2007/0049678 a1; 14. t. abraham, “nanoceramics, nanotubes and nanocomposites paving the way for nanotechnology revolution-a review of the industry and markets,” surfaces and interfaces in nanostructured materials and trends in liga, miniaturization, and nanoscale materials, materials processing and manufacturing division fifth global symposium, ed. m. mukhopadhyay et al. (warrendale, pa: tms, 2004), pp. 247-269. microsoft word 2 barlokova corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 10 removal of antimony from water by coagulation *danka barloková1, ján ilavský1, michal kunštek 1 1department of sanitary and environmental engineering, faculty of civil engineering of the slovak university of technology, radlinského 11, 813 68 bratislava, slovakia jan.ilavsky@stuba.sk , danka.barlokova@stuba.sk, michal.kunštek@stuba.sk *corresponding author received 10 november 2012, accepted 11 december 2012 abstract: increased pollution of water resources leads to a deterioration of surface water and groundwater quality, and it initiates the application of various methods for water treatment. the enactment of the slovak technical standard 75 7111 for drinking water in 1998 resulted in a reduction in heavy metal concentrations and, for the first time, defined the limit concentrations for some heavy metals (as, sb), respectively. based on this fact some water resources in slovakia became unsuitable for further use and require appropriate treatment. the aim of this work was to investigate the effectiveness of coagulation to remove antimony from the surface water of bukovec water tank and groundwater source of dúbrava, determine the optimum dose of coagulant and optimize the coagulation process. ferric sulphate was used (prefloc) as coagulation reagent. the results showed that the dose of 11 mg.l-1 of fe3+ into the surface water of bukovec is sufficient to reduce the antimony below the limit value of 5 g.l-1. to the groundwater from the site dúbrava be added more than 30 mg.l-1 of fe3+ to reach the limit for drinking water. keywords: water treatment, removal of antimony, coagulation, ferric sulphate, water analysis 1. introduction antimony (sb), atomic number 51, molecular weight 121.75 is a brittle silverwhite metal that is found in the earth's crust as the chemically bound state and minerals. antimony is released into the environment from natural sources and from industry, while the anthropogenic emission into the atmosphere exceeds the natural resources emission. antimony is a standard component of coal and oil. industrial plants, emissions from car exhausts and the burning of fossil fuels are the main sources of antimony in the air. the emissions are getting into the atmosphere also from factories by melting the ore and by incineration of the municipal waste. then they are transported from the air into the soil, lakes, rivers and bottom sediments. major part of antimony ends up in the soil where the antimony is strongly bounded to particles containing iron, manganese or aluminum. from the soil is released into the food chain. at lower concentration, antimony could be found in some lakes and rivers, and even in drinking water. at the present time, it is very difficult to distinguish the anthropogenic pollution of the waters from the natural background. antimony is a toxic heavy metal with effects similar to arsenic and lead. the intoxication by antimony is not as much severe as it is in the case of arsenic because the compounds of antimony are absorbed slowly. antimony is an inhibitor for some enzymes, has an effect on the metabolism of proteins and carbohydrates, and causes a failure of glycogen production in kidneys. its ability of accumulation in the bodies of organisms is low. findings on health aspects related to the occurrence of some food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 11 heavy metals in drinking water are summarized in publication [1,2]. till now, the world health organization (who) and institutes dealing with the monitoring of carcinogenic effects have not classified antimony as a carcinogen. the limit concentration of antimony in drinking water in slovakia is 5 µg.l-1 [3]. this limit value is in accordance with the who recommendations [4] and the eu directive [5]. antimony is presented in water as sb3-, sb0, sb3+ and sb5+ (sb3+ is ten times more toxic than sb5+), depending on the ph of the water, the oxidation-reduction potential (sb3+/sb5+ ratio) and the oxygen content. the most common form is antimonate – oxyanion (h2sbo4)and (hsbo4)2or it can be present as antimonite (h3sbo3). the organic form of antimony is very rare, especially in drinking water [6,7]. concentrations in natural waters not polluted by anthropogenic activity are in a range from tens of ng.l-1 up to 1µg.l-1. the threshold limit value (sb = 5 µg.l-1) has been exceeded in slovakia (figure 1), for instance, in the locality of košice and its surroundings (zlatá idka, the bukovec water reservoir), the low tatras in the locality of dúbrava (liptovský mikuláš), spišsko-gemerské rudohorie (čučma, poproč) and in the little carpathians (pernek). today, it is very difficult to distinguish between anthropogenic and natural pollution caused by antimony. figure 1 areas of antimony in slovakia there are several technological methods of heavy metals removal, which are used in water treatment: precipitation (clarification), ion exchange, membrane technologies, adsorption, electrochemical processes and recently also biological methods [8-20]. the most common method for the removal of heavy metals is water clarification – the precipitation of metal hydroxides and carbonates. this process is based on the dosing of appropriate agents (iron and aluminum salt, lime, sodium carbonate, sodium hydroxide and sulphates) to obtain the optimum ph value of the solution in which an insoluble solid phase of precipitated heavy metal hydroxides or carbonates is formed. the effectiveness of the precipitation depends on the type of contaminant, its concentration, water composition and the type of agent. precipitation seems to be an ideal solution for the treatment of water containing heavy metals, provided that the process is not limited by certain effects that are reducing the effectiveness of coagulation. for example, the efficiency of precipitation is lower at a higher concentration of metals in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 12 water. if the solution is too diluted, the precipitation will be too slow. the precipitation is also influenced by the ph value. hydroxides are especially very sensitive to this parameter, and they are not effective enough in acid areas. in addition, the presence of other salts (ions) in water has an adverse effect on the precipitation process. the disadvantages of precipitation are the addition of other chemicals to the treatment process and the high production of sludge that should be processed and stored under specific conditions. in comparison with the other metal removal methods the advantage of precipitation is its relatively low cost. the coagulants used in this process are easily available. precipitation can be used for a wide range of metals, and an acceptable level of effectiveness is achieved through its proper operation. ion exchange is based on the mutual exchange of ions with the same charge between an ion exchanger (an exchangeable ion) and the treated water (captured ion). the ion exchanger is a material capable of the reverse stoichiometric exchange of cations or anions in a condition of electroneutrality. the advantage of the ion exchange process is the relatively low cost compared to the other methods. the method is tried and tested, and all the components required for its operation are commercially available. it is possible to remove undesirable metals from water using the cation exchanger in a wide range up to the µg/l level. the disadvantage of ion exchangers is that they disrupt the ion exchange due to the high competitiveness of some ions (selenium, fluorine, nitrates and sulphates) to finding a place in the ion exchanger. in addition, these ions reduce the efficiency due to suspended and organic substances, which may cause fouling of the ion exchanger filter. it is not possible to use the ion exchange method in the treatment of water with a high concentration of metals. moreover, this method is sensitive to the ph value of the treated water and water quality (alkalinity, concentration of competing ions). the need to dispose of the regenerative agent used and the ion exchanger are also among the disadvantages of this material. adsorption processes are based on the adsorption of contaminants on the surface of an adsorption material. the molecules of the contaminant pass from the water environment to the solid adsorbent. it is possible to use activated alumina, activated carbon or new adsorption materials such as granular hydroxide or ferric hydroxide (gfh, cfh12, cfh18, bayoxide e33) etc., for removing heavy metals. both filtration sand and zeolite modified by higher manganese oxides (mno2), whether in a reactor with a fluidized bed or filter, have significant adsorption properties for the removal of antimony and arsenic. efficiency of heavy metal removal by adsorption material depends on the ph of the water, oxidation-reduction potential of a given metal in the water, concentration of substances in the water that have a potential to affect (interfere with) adsorption or modify adsorbent surface loading, concentration of substances and colloid particles that can physically block the entry into the particle and the access to grains of adsorption media, respectively., specific surface area and distribution of pores of adsorption material, hydraulic properties of filtration media in treatment (filtration rate, the empty bed contact timeebct, the filter medium height). membrane methods belong to a group of diffuse processes in which the selective properties of membranes are used (thin semipermeable films, the thickness of whose walls range from 0.05 to 2.0 mm) to eliminate contaminants from water. depending on the type of membrane (structure and driving force), it is possible to divide these processes into food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 13 microfiltration, ultrafiltration, nanofiltration and reverse osmosis. today, electrochemical methods are not commonly used in the treatment of water and wastewater. these methods are still in the process of development, but it is important to note that they may become very useful for the removal of metals from water in the future. biological methods are based on the production of a special microbial culture capable of using heavy metals dissolved in water as a substratum for further microbial growth. the most of the water treatment processes described in terms of removal of the antimony are only in the experimental stage, and the antimony removal possibilities of some of these technologies has not been proved sufficiently. the literature relating to water treatment deals more often with the arsenic removal, which presence is relatively more frequent in the water. by water treatment processes effectiveness evaluation it has been documented that the conventional technological water treatment processes such as coagulation, iron and manganese removal or water softening to elimination of dissolved arsenic as5+ could be very effective [19]. in the case of coagulation the removal efficiency of arsenic is particularly influenced by ph value, while the ph decrease from 7,4 to 6,8 increased the arsenic removal efficiency from 30% to 70%. throughout the coagulation by ferric coagulants there was reached a higher arsenic removal efficiency than it was reached by aluminous coagulants. in technologies focused on iron and manganese oxidation the removal efficiency of the dissolved forms of arsenic was particularly depending on the fe(oh)3 coagulum formation and in the case of the optimal conditions was the arsenic removal efficiency higher than 75%. by the water treatment aimed at the water softening the striking reduction of dissolved arsenic was not detected and the high efficiencies (from 60% to 95%) were reached by mn(oh)2 coagulation what is related to the as adsorption on the hydroxide particles. the aim of this work was to investigate the effectiveness of coagulation of antimony removal from the surface water from bukovec water tank and groundwater source from dúbrava, determine the optimum dose of coagulant and optimize the coagulation process. as coagulation reagent was used ferric sulphate (prefloc). 2. experimental 2.1 chemical analysis of raw water table 1 shows the quality comparison of monitored natural waters, the concentration of antimony in surface water ranged from 23 to 29 µg.l-1, and groundwater range was 58 to 67 µg.l-1. table 1 quality comparison of monitored natural waters parameter unit bukovec dúbrava ph 7.34 7.10 conductivity ms/m 9.7 21.8 color mg/l 6 3 turbidity ftu 1 1 anc4.5 mmol/l 0.943 3.122 bnc8.3 mmol/l 0.094 0.378 clmg/l 6.82 5.94 no3 mg/l 8.83 6.67 so4 2mg/l 19.23 24.89 fmg/l 0.14 0.09 po4 3mg/l 0.03 0.01 mn mg/l 0.026 0.001 fe mg/l 0.05 0.01 ca2+ mg/l 16.59 38.47 mg2+ mg/l 5.30 15.62 codmn mg/l 0.61 0.48 toc mg/l 1.7 1.2 solutes mg/l 225 160 ca+mg mmol/l 0.632 1.602 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 14 table 1 shows the difference in some parameters, for example in ph, anc4,5 (alkalinity), bnc8,3 (acidity), in the contents of ca+mg, in the concentration of solute, conductivity, etc. these parameters significantly affect the coagulation process. 2.2 coagulation test the coagulation test is a common laboratory procedure used to determine the optimum operating conditions for water treatment procedure. a jar test simulates the coagulation and flocculation processes. coagulation is the process by which colloidal particles and very fine solid suspensions initially presented in the water are combined into larger agglomerates that can be separated via sedimentation, floccula-tion, filtration, centrifugation or other separation methods. coagulation is commonly achieved by adding different types of chemicals (coagulants) to the wastewater to promote destabilization of the colloid dispersion and consequently to agglomerate the resulting individual colloidal particles [20]. for coagulation test the device with five mixers adjustable for slow and fast mixing was used (figure 2). figure 2 scheme of laboratory coagulation device one liter of natural water was added into 5 beakers. after the addition of coagulation agent (1% solution prefloc) followed 3 minutes of fast mixing (180rpm) and 10 minutes of slow stirring (40 rpm). after sedimentation (1 hour) and filtration using filter paper, the sample was analyzed. ph, anc4,5 and the concentration of iron and antimony was determined. 3. results of coagulation experiments with water from wt bukovec the results obtained by wt bukovec sample analysis are shown in table 2. concentration of antimony in the raw water (rw) before coagulation test was 29.4 µg.l-1. coagulation using ferric sulphate (prefloc) is a sufficient method for removing the antimony from the water what results from the measurements. the dose of 40 mg.l-1 of 1 % fe2(so4)3 which represents 11,2 mg.l-1 fe3+ is already sufficient amount for antimony content decrease below the limiting value 5 µg.l-1, which is set up by slovakian drinking water legislation no. 496/2010 (collection of laws). because of ph decreasing by impact of ferric sulphate the ph adjustment after the coagulation is needed, e.g. by lime addition. 4. results of coagulation experiments with groundwater from water source dúbrava the second group of laboratory tests were performed with groundwater from the locality of dúbrava. the concentration of antimony in water before coagulation tests was 66.8 g.l-1. the table 3 shows the results of coagulation tests with 1% solution of ferric sulphate (prefloc). according the results mentioned in table 3 it is obvious that by this coagulation test was achieved reduction of antimony to the desired value of 5 µg.l-1 as much as about 150 mg.l-1 of 1% fe2(so4)3, which is the equivalent around 42 mg.l-1 of fe3+. the disadvantage of this process is the increased fe content in the treated water and low ph, which requires additional water treatment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 15 table 2 the results of coagulation tests removing sb from surface water bukovec bukovec 1 % prefloc [mg/l] coagulant dose [mg fe3+/l] ph anc4,5 [mmol/l] fe [mg/l] sb [µg/l] sv 0 6.91 0.825 0.05 29.4 32.2 9 5.98 0.340 0.03 7.9 35.8 10 5.72 0.204 0.03 5.4 39.4 11 5.48 0.131 0.03 5.0 42.9 12 5.12 0.079 0.04 3.8 46.5 13 4.67 0.038 0.06 2.2 table 3 the results of coagulation tests removing sb from groundwater in dúbrava dúbrava 1 % prefloc [mg/l] coagulant dose [mg fe3+/l] ph anc4,5 [mmol/l] fe [mg/l] sb [µg/l] sv 0 7,40 3.088 0.01 66.8 71.6 20.0 6.41 1.716 0.04 25.3 107.4 30.0 5.90 0.926 0.08 10.2 143.2 40.0 5.52 0.515 0.11 6.5 179.0 50.0 4.10 0 0.24 3.9 214.8 60.0 3.20 0 1.32 0.67 in order to optimize coagulation and effort to reduce the amount of coagulant, which would also reduce the operating costs of the treated water the effect of slow mixing times, respectively rotational speed of slow mixing to produce a flakes was investigated. these experiments have shown that a 15 minute slow mixing (at 40 rpm), respecttively 10-minute slow mixing at 20 rpm are the most effective in removing antimony from the water by coagulation. not by the dose of 30 mg/l fe3+, nor by the time of mixing adjusting or nor by changing the speed of slow stirring the value 5 gl-1 was attained, which represents the limit for antimony in drinking water. by adjusting the ph of water with addition lime (calcium hydroxide dosing in the range 10 to 40 mg/l) prior to dosing coagulant has not been achieved more effective coagulation to remove antimony from water. therefore, ph adjustment and coagulant dosing should be done together, respectively after coagulation. the new coagulation tests of groundwater from the site dúbrava have been performed, whereby the additional parameters were monitored (turbidity and color). obtained results are shown in the table 4. the result shown in table 9 indicates the change in water quality led to the optimum coagulant dosing reduce. already the dose 107 mg/l of 1% of fe2(so4)3, which is the equivalent of 30 mg/l fe3+ suffice to reduce the antimony below the limit value of 5 µg/l, which is given by the slovak government regulation 496/2010 for drinking water. the disadvantage of this process is the increased fe content in the treated water and the low ph, which require additional water treatment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 16 table 4 the results of coagulation tests removing sb dúbrava 1 % prefloc [mg/l] coagulant dose [mg fe3+/l] ph turbidity [ftu] color [mg/l pt] anc4,.5 [mmol/l] fe [mg/l] sb [µg/l] 0 7.62 2 10 3.432 0.08 62.0 71.6 20.0 6.08 1 8 1.052 0.09 9.2 89.5 25.0 5.56 1 9 0.396 0.17 5.3 107.4 30.0 4.83 1 10 0.172 0.22 < 3.0 125.3 35.0 3.48 1 12 0 0.44 < 3.0 143.2 40.0 3.02 2 13 0 1.76 < 3.0 note : < 3.0 = under the detection limit the increased value of ph and anc4,5, respectively lower amount of antimony in the raw water had a significant influence on the coagulation results (to decrease the dose of coagulant). by determining the optimal coagulant dosage is necessary to take into account not only the electric charge of dispersed particles and coagulant, but also their morphology (the shape and size) and dynamic properties. when the coagulant is overdosed, it leads to a significant reduction in its effectiveness. it is therefore necessary to achieve such a state that the water was indented as little flakes whose size is less than 125 microns. just this negative phenomenon was observed during our experiments, a maximum indentation micro-flakes in water after adding 50 60 mg fe3+ to 1 liter of water. at the optimal course of coagulation there are mostly elongated heavier mainly extended flakes in the sediment. the most accurate reproduction (representation) of the flakes was in the sediment, which was obtained at a coagulant dose 60 mg/l of fe3+ (figure 3). the decline of these flakes onto the sediment depends primarily on the size of precipitated dispersed particles. in the table 5 and 6 there is shown the size, shape and the total number of flakes obtained by coagulation. figure 3 elongated shape of flakes in the sediment table 5 number and shape of flakes in the indented water the shape of flakes dúbrava 1 % prefloc [mg/l] coagulant dose [mg fe3+/l] round elongated <125 µm 125 – 250 µm >250 µm number of flakes sv 0 0 0 0 0 0 107.4 30.0 35 65 155 40 85 380 143.2 40.0 22 50 165 35 92 364 161.1 45.0 25 60 195 110 90 480 179.0 50.0 5 18 350 140 70 583 214.8 60.0 3 3 425 46 8 485 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 17 table 6 number and shape of flakes in the sediment the shape of flakes dúbrava 1 % prefloc [mg/l] coagulant dose [mg fe3+/l] round elongated <125 µm 125 – 250 µm >250 µm number of flakes sv 0 0 0 0 0 0 107.4 30.0 25 75 200 96 80 576 143.2 40.0 15 45 250 140 70 520 161.1 45.0 15 148 200 185 163 711 179.0 50.0 49 200 236 198 146 829 214.8 60.0 94 355 126 105 300 980 the inorganic particles have penetrated into the filtered water (the dominant were bright and dark crystal formations) which were a bit fewer presented there than they were presented in the raw water. (fig. 4). figure 4 non-coagulated the bright and dark crystal formations in flaked 5. conclusion performed laboratory tests of surface water from water tank bukovec and also the tests of groundwater originated in the spring dúbrava have shown that the antimony content could be decreased at the values limited by slovak government regulation no. 496/2010 for drinking water throughout the application of ferric sulphate. the disadvantage of this method usage is the high coagulant dose requirement. this disadvantage is particularly and significantly being shown in the water treatment stations in which only the antimony value does not match the legislation. this disadvantage is not so striking in antimony removal from surface water where the coagulant addition is needed for the increased turbidity removal, oxidability and insoluble substances removal. according to the coagulation test results the single-stage water treatment is sufficient for the decreasing the antimony content, i.e. dosing of ferric sulphate, 10 minutes of slow mixing, 40 rpm. optimal dose of coagulant is important. we have evaluated that the dose 11 mg.l-1 of fe3+ is sufficient for antimony content decrease below the limit value at 5 µg.l-1 from the surface water bukovec. for the achievement of the limit value there must be added more than a 30 mg.l-1 of fe3+ to the dúbrava undeground water. 6. acknowledgement this work was supported by the financial support of project apvv-0379-07 and vega 1/1243/12 provided by slovak grant agency. 7. references [1]. water quality and treatment. a handbook of community water suppliers. awwa, 1990. [2]. usepa. antimony: an environ-mental and health effects assessment. washington, dc, us environmental protection agency, drinking water office, 1984. [3]. regulation of the government of the slovak republic no. 496/2010 on drinking water, 2010. [4]. who. guidelines for drinking-water quality. 4th edition. who, geneva, 2011. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 18 [5]. council directive 98/83/ec, quality of water intended for human consumption, 1998. [6]. pitter p. hydrochemie, fourth ed., institute of chemical technology press, praha, 2009 (in czech). [7]. who. antimony in drinking-water. background document for preparation of who guidelines for drinking-water quality. who, 2003. [8]. bellack e. arsenic removal from potable water. j. awwa 63, 454, (1971). [9]. gannon k., wilson d.j. removal of antimony from aqueous system. sep. sci. technol. 21, 475, (1986). [10]. macphee m.j., charles g.e., cornwell d.a. treatment of arsenic residual from drinking water removal processes. (epa/600/r-11/090), 2001. [11]. kang m, kamei t., magara y. comparing polyaluminium chloride and ferric chloride for antimony removal. water res. 37, 4171, (2003). [12]. mohan d., pittman ch. u. jr. arsenic removal from water-wastewater using adsorbents a critical review. j. of hazard. mater. 142 (1-2), 1, (2007). [13]. guo x., wu z. and he m. removal of antimony (v) and antimony (iii) from drinking water by coagulation–flocculation–sedimentation (cfs). water res. 43 (17), 4327, (2009). [14]. dzul erosa m.s., höll w.h. removal of selenium and antimony species from aqueous solutions by means of a weakly basic ion exchanger. loureiro j.m. and kartel m.t (eds.), combined and hybrid adsorbents, springer, 287–292, (2006). [15]. kang m, kawasaki m., tamada s., kamei t., magara y. effect of ph on the removal of arsenic and antimony using reverse osmosis membranes. desalination 131 (1-3), 293, (2000). [16]. nguyen v.l., chen w.h., young t., darby j. effect of interferences on the breakthrough of arsenic: rapid small scale column tests. water res. 45 (14), 4069, (2011). [17]. zeng h., arashiro m., giammar d. effect of water chemistry and flow rate on arsenate removal by adsorption to an ironbased sorbent. water res. 42 (18), 4629, (2008). [18]. driehaus w., jekel m., hildebrandt u. granular ferric hydroxide – a new adsorbent for the removal of arsenic from natural water. j. water supply: research and technology-aqua 47, 30-35, (1998). [19]. mcneill l.s., edwards m.: soluble arsenic removal at water treatment plants. jour. awwa 29 (4), 105, (1995). [20]. bratby j. coagulation and flocculation in water and wastewater treatment. iwa publishing, london, p. 407, (2006). microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_83-92.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 87 a review on the effect of fungi on the wheat grain under post harvest storage ecology mathew shiju1 1ministry of higher education, department of natural sciences, aksum university, ethiopia contact no.: (+251) 920126043; e-mail: shijumathew_biotech@yahoo.com abstract: the review paper is aimed to assess the effect of fungi on the wheat grain under the post harvest storage system. it has been observed that very high percentage of wheat kernels has been lost under post harvest storage. the grain quality greatly depends upon the storage conditions. the storage requirement violation can result in grain deterioration. the biotic and abiotic factors influence the various physical and chemical properties of wheat grains under post harvest storage. the fungal ecology present in the stored grain has an important role in spoilage and the production of mycotoxins. the mycoflora of stored wheat grains predominantly consisted of ubiquitous mould genera aspergillus, alternaria, cladosporium, fusarium, mucor, rhizopus and penicillium possibly because of their omnipresence, capacity to grow on all possible substrates and a wide range of temperature and humidity. the most frequent species observed on the stored wheat grains of aspergillus were a. niger, a. fumigatus, alternaria alternata, fusarium moniliformis, rhizopus arrhizus and a few pencillium species. among these almost all have the capacity to produce mycotoxin which can contaminate and cause spoilage. the various physical, microbial and biochemical analyses of post harvest storage wheat grain is an essential component to evaluate the grain quality in various indian wheat varieties and also important for facilitating the minimal post harvest food grain loss. keywords: loss, biotic factor, abiotic factor, mycotoxin, infestation. 1.1 introduction the grain production varies from year to year and hence the grains should be stored strategically from years of overproduction for the use in year of under production. stored grains can have losses in both quantity and quality. the wheat grain after harvest is influenced by a wide variety of biotic and abiotic factors and has been studied as a stored grain ecosystem. losses occur when the grain is attacked by microorganisms and particularly by fungi. the grain losses in quantity and quality can be under the form of depletion in seed viability, hardness, colour, size and shape, grain weight and various biochemical parameters, protein, carbohydrate and vitamins under post harvest storages. the presence of mycotoxins in grains is traditionally regarded as an indicator of poor storage conditions. mycotoxins may already be present in grains coming into storage or may be produced as a result of poor storage only if there is sufficient moisture. conversely, not all moulds that grow in stored commodities produce mycotoxins. the metabolic activity of these pioneer species raises the moisture content of the grain, which may allow growth of mycotoxigenic species and ultimately, the formation of mycotoxins. alternaria, aspergillus and penicillium can act as pre or post-harvest pathogens of grain and may form mycotoxins. 1.1.1 the origin and classification of wheat wheat belongs to the genus triticum of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 88 grass family, poaceae. this genus is originated in the tropical south west asia, where it occurs in wild as well as in cultivated forms. wheat is well adapted to harsh environments and is mostly grown on wind swept areas that are too dry and too cold for the more tropically inclined rice and corn, which do best at intermediate temperature levels. wheat was first grown in the united states in 1602 on an island of the massachusetts coast. man has been dependant upon the wheat plant for thousands of years. wheat genetics is more complicated than that of most other domesticated species. some wheat species are diploid, with two sets of chromosomes, but many are stable polyploids, with four sets of chromosomes (tetraploid) or six (hexaploid) set of chromosomes [1-9]. globally, wheat is the most-produced food among the cereal crops after rice. table 1.1 shows the taxonomic status of the genus triticum . the development of a modern classification depended on the discovery, in the 1918s, of the fact that wheat was classified according to three ploidy levels [10-16]. the classification of van slageren (1994) is probably the most widely used genetic-based classification at present. most species of wheat can be described in latin binomials, e.g., triticum aestivum, rather than the trinomials necessary in the genetic system, e.g., triticum aestivum subsp. aestivum. both approaches are equally valid and both are widely used [17]. table 1.1: the taxonomical status of triticum kingdom plantae plants subkingdom tracheobionta vascular plants superdivision spermatophyta seed plants division magnoliophyta flowering plants class liliopsida monocotyledons subclass commelinidae order cyperales family poaceae grass family genus species triticum aestivum wheat common wheat wheat (the triticum spp.) is cultivated worldwide. globally, wheat is the mostproduced food among the cereal crops after rice. wheat grain is a staple food used to make flour for leavened, flat and steamed breads; cookies, cakes, breakfast cereal, pasta, noodles; and for fermentation to make beer, alcohol, vodka or even biofuel. durum is the most commonly used to make pasta. the most common wheat is the hexaploid wheat and includes spelt, modern bread wheat and soft wheat used for cookies and cakes. wheat is grown to a limited extent as a forage crop for livestock, and the straw can be used as fodder for livestock or as a construction material for roofing thatch. although wheat supplies much of the world's dietary protein and food supply, as many as one in every 100 to 200 people suffers from coeliac disease, a condition which results from an immune system response to a protein found in wheat: gluten [18-20]. grain fed to livestock whole or coarsely ground. starch is used for pastes and sizing textiles. straw is turned into mats, carpets, baskets, and used for packing material, cattle bedding, and paper manufacturing. some wheat is cut for hay. wheat grown for grain crop is also used for pasture before the stems elongate and as a temporary pasturage; it is nutritious and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 89 palatable. 1.1.2 the effect of stored wheat grains under post harvest storage the spoilage of grain in storage is brought about by two variables, the biotic and abiotic [21]. wheats are attacked by many fungi and other organisms. some cultivars are resistant to the various rusts, smuts, and virus diseases. the most important fungal diseases of wheats are the extension agents which are concerning diseases in an area before growing wheat. also cvs should be selected for growing as resistant to various diseases. the biotic variables are different types of insects, mites, rodents, birds and microorganisms, i.e., bacteria and fungi. the abiotic variables include temperature and moisture content of the grains; the latter in grains increases due to seepage of moisture from soil or from cemented floor [22]. the micro flora of cereal grains normally includes fungi, bacteria and actinomycetes [23-25]. a number of fungi have been reported to be associated with cereal grains and their milling fraction [26-27]. the majority of mycotoxins are chemically tough, resistant to temperature, conditions of storage and processing technologies. therefore, detoxication is not always effective. based on the stages at which invasion and growth occur, the fungi associated with grains can conveniently be divided into two groups i.e., field fungi and storage fungi [28]. spoilage of stored grain by fungi is determined by a range of factors which can be classified into four main groups including (a) intrinsic nutritional factors, (b) extrinsic factors (c) processing factors and (d) implicit microbial factors. the factors produce fungal colonization within the stored grains [29-30]. these reviews examined some important mycotoxins and the post-harvest control strategies which have been developed for effective management to minimize entry of mycotoxins into the food chain. in some cases, pre-harvest decisions can significantly impact the capability for subsequent post-harvest control. the propagates of mould fungi, frequently found in grain and its surroundings, start to develop when favourable conditions emerge. temperature and substratum moisture are the most significant factors that determine the intensity of micromycete development and grain damage. 1.1.2.1 the effect of fungi on the odour and discolouration of the stored wheat grains the fungal contamination of the grains is responsible for emitting undesirable odours [31] found that under high moisture condition cereals gave a musty odour. the dark colouration of germs of the wheat grains are due to the damage caused by fungi [32]. this is discoloration of the germ end of the wheat grain. in the commercial grading system, black point involves the germ end only and not extended into the grain crease. if more than half the grain is damaged and well into the crease, it is classified as smudge. it has been also observed that if the wheat grains, with moisture content of 13% or above is kept long enough at a temperature of 350c to 400c, the grains turn brown even in the absence of storage fungi [33]. grain itself and the microbial contaminants respire slowly when stored dry. however, if the water availability is increased to 15-19% moisture content (=0.75-0.85 water activity (aw), wheat) spoilage fungi, particularly aspergillus and penicillium species grow, resulting in a significant increase in respiratory activity [34]. this can result in an increase in temperature and sometimes spontaneous heating from the colonization by a succession of fungi resulting in colonization by thermophilic fungi and actinomycetes [35-37]. the quantitative grain contamination with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 90 micromycete propagules was established by a number of methods given by various scientists [38-44]. the grain colour changes by some micromycetes produced and contaminate it with poisonous substances including mycotoxins [45-52]. 1.1.3 the presence of micro flora contaminating the stored wheat grains mould contamination of food and feed is a constantly occurring phenomenon but the attitude towards it has been far from consistent [53]. it has been found that fungal damage of grain is caused in various ways. they reduce the grain viability, produce odour, grain discoloration and depletion in food added value (fav) by the increase in fav and also produce toxins injurious to the health of consumers. the fungi are responsible for emitting of undesirable odour from stored grains [54]. a number of studies have been reported the loss of dry matter content and visible moulding of stored wheat grains [55-62]. this could be considered as a subjective index of the safe storability of grain. there are problems with the use of visible moulding as a criterion of deterioration [63-66]. wheat quality loss has been measured and models developed based on germination rates, visible mould growth or respiration of grain and microorganisms. the microscopic growth may be a more effective measurement of initial colonization than visible moulding [67-70] an extensive micro flora has been found to be associated with stored wheat grains [7183]. earlier in the laboratory a number of cereals have been screened with respect to micro flora associated in storage grains [84-87]. the effect of microbial infestation on decrease in the germination of grains is now well known. it has been established in other cereals and especially in wheat. discolouration of wheat is a common occurrence. during the course of their development breakdown and utilization of the constituents of the seeds occur [88-89]. the dark germs of grains are known to be due to damage by storage fungi [90-94]. the high moisture and temperature conditions for cereals gave a musty odour due to fungi, instead of bacteria [95]. 1.1.4 fungal infestation in post harvest stored wheat grains the wheat grains come in association with the fungi from the time of grain maturity and also at the time of storage. some of these fungi are in intimate association and are present as dormant mycelium under the pericarp or dormant spores on the surface of the kernel. however, there is a number of fungi which are only superficially associated with stored grains. mould growth in grains may cause deleterious changes in addition to the formation of mycotoxins. many spoilage fungi cause loss of germination in seed grains, discolouration and darkening of the grains, reduction in protein content, musty odours, and changes in fatty acid profiles and other constituents of the grains. mould development may also encourage mite and insect infestation. the association of fungi with cereal grains starts from the field itself. shortly after the grain reaches to maximum size, the lemma and palea protecting it are pushed apart exposing the grain to infection by fungi [96] and their extensive studies have been carried out in the laboratory on these aspects [97-99]. poor post harvest management can lead to rapid deterioration in grain quality, severely decreasing the germinability and nutritional value of stored grains. fungal activity can cause undesirable effects in grains including discolouration, contribute to heating and losses in nutritional value, produce off-odours, losses in germinability, deterioration in baking and milling quality, and can result in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 91 contamination by mycotoxins [100-102]. a number of studies have been reported on the loss of dry matter content and visible moulding of stored wheat grains [103110]. this could be considered as a subjective index of the safe storability of grain. there are problems with the use of visible moulding as a criterion of deterioration [111-114]. fungi seldom occur on grains in isolation, but usually as a mixed consortium of bacteria, yeasts and filamentous fungi. it is thus inevitable that interspecific and intraspecific interactions will occur depending on the nutritional status of the grain and the prevailing environmental conditions. wheat quality loss has been measured and models developed based on germination rates, visible mould growth or respiration of grain and microorganisms [114-118] the microscopic growth may be a more effective measurement of initial colonization than visible moulding. some attempts have also been made to relate dry matter losses to actual calorific losses due to the activity of mycotoxigenic moulds. 1.1.5 decrease in nutritional value as due to myctoxin production in stored wheat grains during storage, the grains undergo some biochemical changes like the increase in fatty acids. the increase in fatty acid value is chiefly due to storage fungi and not due to the activity of seeds [119]. the stored grain will have quantitative and qualitative losses. weight loss during storage (not due to a loss of moisture) is a measure of food loss but the latter may be proportionately larger owing to selective feeding by the pests. rodents and moth larvae may preferentially attack the germ of the grain thus removing a large percentage of the protein and vitamin content, whereas weevils feeding mainly on the endosperm will reduce the carbohydrate content. many pests may eat the bran of cereals reducing vitamins such as thiamin [120123] the quantity and quality of endosperm proteins are the major factors responsible for baking quality, and nutritional value of wheat [140-142]. various seed storage proteins in wheat are classified upon their solubility in different solvents, such as water, saline solution, 70% aqueous ethanol and diluted acid or alkali solutions and are named as albumins, globulins, gliadins, and glutenins, respectively [126128]. the other storage factors such as moisture and fungal infection also lead to changes in vitamin content. in beans in particular, loss of protein is very important where there is infestation, as up to 25% of the dry matter may be raw protein. fungi growth leads to reduced nutritional and technical quality of cereal grains [130-132]. grain storage conditions affect its quality due to high percentage of mycotoxins [133-137]. wheat grains are also rich in pantothenic acid, riboflavin and some minerals, sugars etc [138]. the composition of wheat grain and flour is given in table 1.2. table 1.2: composition of wheat grain and flour [139] parameter grain (%) flour (%) moisture 9 18 13 15.5 starch 60-68 65-70 protein 8-17 8-15 cellulose 2-2.5 trace fat 1.5-2 0.8-1.5 sugars 2-3 1.5-2 mineral matters 1.5-2 3-6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 92 storage temperature affects the keeping quality of wheat. increase in temperature accelerates the rate of respiration until it is limited by oxygen supply; build up of carbon dioxide concentration, exhaustion of substrate, and thermal inactivation of essential enzymes. heat damages the gluten proteins and even discolours the kernels of the stored hard red winter wheat with moisture contents of 10.95% in sealed half gallon glass jars at -10c and 240c for two years [140-142]. the grain losses in quantity and quality can be under the form of depletion as mentioned above like that of seed viability, hardness, colour, size and shape, grain weight and various biochemical parameters, protein, carbohydrate and vitamins under post harvest storages. the storage fungi damage the grains in several ways; they reduce the germinability, produce undesirable odour and kernel discoloration, decrease the food value and also produce toxins injurious to the health of consumers. the effect of mycotoxigenic fungi in post harvested stored wheat grains must be examined in the context of the ecology as a whole in order to understand the dominance of certain species of fungi under certain environmental conditions. interactions between these fungi and other contaminants are complex and are significantly affected by the prevailing and changing environmental factors. as a conclusion the mycoflora of stored wheat grains predominantly consisted of ubiquitous mould genera aspergillus, alternaria, cladosporium, fusarium, mucor, rhizopus and penicillium possibly because of their omnipresence, capacity to grow on all possible substrates and a wide range of temperature and humidity. the most frequent species observed in the stored wheat grains of aspergillus were a. niger, a. fumigatus, alternaria alternata, fusarium moniliformis, rhizopus arrhizus and a few pencillium species and some of the fungi have the capacity to produce mycotoxin which can contaminate and cause spoilage. references 1. u.s. department of agriculture appropriations (1957). hearings of 84th congress. 2nd day session in 1956. united states congress. pp 242. 2. smith, j.e. and albert, e. (1995). handbook of weed management systems. marcel dekker. pp. 411. 3. belderok, b., hans, m. and dingena a.d. (2000). bread-making quality of wheat. springer. pp3. 4. palmer, j. j. (2001). how to brew. defenestrative pub co. pp. 233. 5. neill, r. (2002). booze: the drinks bible for the 21st century. octopus publishing group cassell illustrated. pp.112. 6. cauvain, a., stanley, p. and cauvain, p. (2002). bread making. crc press. pp 540. 7. colledge, s., conolly, j. and shennan, s. (2002). archaeobotantical evidence for the spread of farming in the eastern mediterranean. curr. anthropol. 45:s35-s58. 8. u.s. department of agriculture (2003). annual world production summary, grains, http://www.usda.gov/agriculture crop production. 9. hancock and james, f. (2004). plant evolution and the origin of crop species. cabi publishing. 10. magan, n. (1993a). early detection of fungi in stored grain. int. j. biodeterior. biodegradat. 32:145160. 11. magan, n. (1993b). use of sulphur dioxide to control fungi in stored grain. in: proceedings of int. conference on controlled atmosphere and fumigation in grain storages (s. navarro and e. donahaye, eds.). capital press, jerusalem, israel. pp. 163-171 12. kabarity, a. (1966). on the origin of the new cultivated wheat ii. cytogenetical studies on the karyotypes of some t. macha varieties. beitr. biol. pflanzen. 42: 339-346. 13. morris, r. and sears, e.r. (1967). the cytogenetics of wheat and its relatives. in: wheat and wheat improvement. american society of agronomy monograph (k.s. quisenberry and l.p. reitz, eds.). madison, wi. pp 18-97. 14. bowman, c.m., bonnard, g. and dyer, t.a. (1983). chloroplast dna variations between species of triticum and aegilops. location of the variation on the chloroplast genome and its relevance to the inheritance and classification of the cytoplasm. theor. appl. genetics 65: 247-262. 15. kerby, k. and kuspira, j. (1987). the phylogeny of the polyploidy wheat triticum aestivatum (bread wheat) and triticum turgidum (macaroni wheat) genome. 29: 722-737. 16. breiman, a. and graura, d. (1990). wheat evolution. israel j. plant sci. 43: 85-98. 17. waines, j.g. and barnhart, d. (1992). biosystematic research in aegilops and triticum. hereditas 116: 207-212. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 93 18. hill, i. d., horvath, k. and fasano, a. (1995). epidemiology of celiac disease. am. j. gastroenterol. 90: 163-164. 19. fasano, a. berti, i. and gerarduzzi, t. (2007). "prevalence of celiac disease in at-risk and notat-risk groups in the united states: a large multicenter study". arch. int. med. 163: 286-292. 20. presutti, j., (2007). celiac disease. am. family physician 76: 196-1802. 21. white, n.d.g., sinha, r.n. and muir, w.e. (1982). intergranular carbon dioxide as an indicator of biological activity associated with the spoilage of stored wheat. j. can. agric. eng. 24: 35-42. 22. pingale, s.v. (1970). prevention and losses in storage. bull. grain technol. 8: 3-13. 23. james, n., wilson, j. and stark, e. (1946). the microflora of stored wheat. can. j. res. 24: 224-233. 24. graves, r.r., rogers, r.f., lyons, a.j. jr. and hesseltine, c.w. (1967). bacterial and actinomycetes flora of kansas – nebraska and pacific northwest wheat and wheat flour. cereal chem. 44: 288299. 25. mehrothra, b.s. and basu, m. (1975). survey of microorganisms associated with cereal grain and their milling fraction in india. j. imported wheat int. biodegradation bulletin 11. 26. mehrothra, b.s. and kunwar, i. k. (1980). survey of microorganisms associated with cereal grain and their milling fraction in india ii. indigenous wheat int. biodegradation bull. issn0020 6164 spring. 27. wicklow, d. t. (1995). the mycology of stored grain: an ecological perspective. in: stored grain ecosystems (d. s. jayas, n. d. g. white and w. e. muir, eds.). new york, marcel dekker, pp. 197-249. 28. christensen, c.m. and kaufmann, h.h. (1969). grain storage the role of fungi in quality loss. university of minnesoto press, minn u.s.a., pp. 153. 29. wallace, h.a.h. (1973). fungi and other organisms associated with stored grains in grain storage part of a system (r.n. sinha and w.e. muir, eds.). avi publishing co., connecticut sec. ii capt. 4 : 71-78. 30. wallace, h.a.h. and sinha, r.n. (1981). causal factors operative in distributional patterns and abundance of fungi: a multivariate study. in: the fungal communityits organisation and role in ecosystems (d.t. wicklow and g.c. carroll, eds.). marcell dekker inc., new york. pp. 233-247. 31. schroeder, h.w. and halick, j.v. (1963). effect of moisture content, humidity and length of storage on maintenance of quality in rough rice. phytopathol. 54: 858-862. 32. golumbic, c. and laundani, h. (1966). storage and warehousing. year book of agriculture (u.s. department of agriculture) pp. 139-152. 33. goldblatt, l.a. (1969). aflatoxin scientific background control and implications. academic press, new york. pp. 472. 34. petersson, s. and schnurer, h. (1995). biocontrol of mold growth in high-moisture wheat stored under airtight conditions by pichia anomala, pichia guilliermondii, and saccharomyces cerevisiae. appl. environ. microbiol. pp. 1027-1032. 35. fleurat-lessard, f. (2002). qualitative reasoning and integrated management of the quality of stored grain: a promising new approach. j. stored product res. 38: 191-218. 36. magan, n., sanchis, v. and aldred, d. (2004). role of spoilage fungi in seed deterioration. chapter 28, in: fungal biotechnology in agricultural, food and environmetal applications (d.k.aurora, ed.). marcell dekker. pp. 311-323. 37. shuey, w.c. (1960). a wheat sizing technique for predicting flour milling yield. cereal sci. today 5: 71-72 38. ellis, m. b. (1971). dematiaceous hyphomycetes. kew: commonwealth mycological institute. pp. 608. 39. ellis, m. b. (1976). more dematiaceous hyphomycetes. kew: commonwealth mycological institute. pp. 785. 40. domsch, k. h., gams, w. and anderson, t. (1980). compendium of soil fungi. london: academic press. vol.1. pp. 405. 41. gerlach, w., and nirenberg, h. (1982). the genus fusarium: a pictorial atlas. berlin–dahlem: institute of microbiology. pp 406. 42. ramirez, c. (1982). manual and atlas of the penicillia. elsevier, amsterdam. pp. 874. 43. lugauskas, a., bridziuviene, d., levinskaite, l., paskevicius, a., peciulyte d., repeckiene, j., salina, o. and varnaite, r. (1997). mikrobiologiniai medziagų pazeidimai. vilnius: valstiecių laikrastis. pp 469. 44. lugauskas, a., paskevicius, a. and repeckienė, j. (2002). pathological microorganism application vilnius pp. 434. 45. scot, p.m. (1994). penicillium and aspergillus toxins. in: mycotoxins in grain compounds other than aflatoxins (j. o. miller and h. l. trewnholm, eds.). eagan press, st. paul, minesota, usa. pp 261-268. 46. moss, m. o. (1991). mycology of cereal grain and cereal products. in: cereal grain: mycotoxins, fungi and quality in drying and storage (j. chelkowski, ed.). elsevier, amsterdam–london–new york–tokyo. pp. 2351. 47. lacey, l. and magan, n.(1991). fungi in cereal grains: their occurrence and water and temperature relationships. in: cereal grain: mycotoxins, fungi and quality in drying and storage (j. chelkowski, ed.). elsevier, amsterdam–london– new york–tokyo. pp. 77118. 48. kuzmiene, g., pazarauskiene, j. and sirtautaite, s. (1991). augalininkystės produktų laikymas ir perdirbimas. vilnius: mokslas. pp.381. 49. marin, s., sanchis, v., ramos, a.g. and magan, n. (1998a). environmental factors, interspecific interactions, and niche overlap between fusarium moniliforme and f. proliferatum and fusarium graminearum, aspergillus and penicillium spp. isolated from maize. mycol. res. 102: 831-837. 50. abramson, d. (1998). mycotoxin formation and environmental factors. in: mycotoxins in agriculture and food safety (k. k. sinha and d. bhatnagar, eds.). new york: marcel dekker inc. pp. 255-277. 51. singh, h. singh, n. kaur, l., and saxena, s.k. (2001). effect of sprouting conditions on functional and dynamic rheological properties of wheat. j. fd. eng. 47: 23-29 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 94 52. zvicevicius, e., raila, a., novosinskas, h. and krasauskas, a. (2006). mycotoxin producents in the grain layer. j. ecologia. 3: 105-111. 53. forgacs, j. (1965). stachybotrys toxicosis and moulds corn toxicosem. in mycotoxins in food stuffs. (g.n. wogans, ed.). m.l.t. press cambridge, massachusetts. pp. 87104. 54. f. a. o. (1966). grain of life. pp. 193. 55. kreyger, j. (1972). drying and storing grains, seeds and pulses in temperate climates. institute for storage and processing of agricultural products, bulletin 205, wageningen, the netherlands. 56. seitz, l.m., sauer, d.b., mohr, h.e. (1982). storage of high moisture corn: fungal growth and dry matter loss. cereal chem. 59: 100-105. 57. white, n.d.g., sinha, r.n. and muir, w.e. (1982). intergranular carbon dioxide as an indicator of biological activity associated with the spoilage of stored wheat. j. can. agric. eng. 24: 35-42. 58. hofstein, r. s., droby, e., chalutz, c., wilson and frilender, b. (1990). scaling-up the production for application of an antagonist-from basic research to r & d, in: biological control of postharvest diseases of fruits and vegetables, workshop proceedings. agricultural research service document ars-92. u.s. department of agriculture, washington, d.c. pp. 197210. 59. hamer, a., lacey, j. and magan, n. (1991). use of an automatic electrolytic respirometer to study respiration of stored grain. in: proceedings of 5th int. working conference of stored product protection, (f. fleurat-lessard and p. ducom eds.). bordeaux, france, imprimerie dumedoc, 1: 321-329. 60. krasauskas, a., steponaviciene a., railiene, m., lugauskas, a., raila, a. and raudoniene v. (2005). impact of environmental conditions on the spread of micromycetes in grain during its harvesting and storage. bot. lithuanica. 11: 101109. 61. waines, j.g. and barnhart, d. (1992). biosystematic research in aegilops and triticum. hereditas 116: 207-212 63. singh, h. singh, n. kaur, l., and saxena, s.k. (2001). effect of sprouting conditions on functional and dynamic rheological properties of wheat. j. fd. eng. 47: 23-29. 64. leelavathi, k., vetrimani, r. and haridas-rao, p. (1990). changes in the functional characteristics of wheat during soaking and subsequent germination. j. fd. sci. technol. 27: 349-354. 64. wosniak, w. and styk, w. (1996). internal damage to wheat grain as a result of wetting and drying. j. drying technol. 14: 349-365. 65. lacey, j., hamer, a. and magan, n. (1997). respiration of wheat grain in different environments. (e.j. donahaye and s.navaro eds.). proc. int. symp. on controlled atmospheres and fumigation (caf), caspit press, jerusalem, israel. pp. 113-122. 66. abramson, d. (1997). toxicans of genus penicillium. in: handbook of plant and fungal toxicants (j.p.f. d’mello, eds.). crc press, boca raton, fl. pp. 303-317. 67. anjum, f.m. and walker, c.e. (1991). review on the significance of starch and protein to wheat kernel hardness. j. sci. fd. agric. 56: 1-13. 68. armolik, n., dickson, j.g., dickson, a.d. (1956). deterioration of barley in storage by microorganisms. phytopathol. 46: 457-461. 69. autran, j.c., hamer, r.j., plijter, j.j. and pogna, n.e. (1997). exploring and improving the industrial use of wheats. cereal fd. world 42: 216-227. 70. bakutis b. (2004). microrganism grown on seeds. kaunas: terra publication. pp. 81. 71. baliukoniene, v., bakutis, b. and stankevicius, h. (2003). mycological and mycotoxicological evaluation of grain. ann. agric. environ. med. 10: 223-227. 73. basu (1974). studies of microorganisms associated with stored wheat and its flour in india. research paper presentation at university of lucknow, india. 74. berg, s.o. (1947). is the degree of grittiness of wheat flour mainly a varietal character? cereal chem. 24: 274-283. 75. brook, p.j. and white, e.p. (1966). fungus toxins affecting mammals. ann. rev. phytopathol. 4: 171-194. 76. byers, m., miffin, b.j. and smith, s.j. (1983). a quantitative comparison of the extraction of protein fractions from wheat grain by different solvents and of the polypeptide and amino acid composition of the alcohol solution proteins. j. sci. fd. agric. 34: 447-462. 77. chelkowski, j. (1991). mycological quality of mixed feeds and ingredients. in: cereal grain, mycotoxins, fungi and quality in drying and storage, (j. chelkowski, ed.): elsevier, amsterdam, london, new york, tokyo, pp. 217-227. 78. christensen, c.m. (1955). grain storage studies 21: viability and moldiness of commercial wheat in relation to the incidence of germ damage. cereal chem. 32: 507-518. 79. christensen, c.m. (1956). deterioration of stored grains by moulds. wallerstein lab. commun. 19 : 31-48. 80. christensen, c.m. and linko, p. (1963). moisture content of hard red winter wheat as determined by meter and by oven drying and influence of small differences in moisture content upon subsequent deterioration of the grain in storage. cereal chem. 40: 129-137. 81. christensen, c.m., olafson, j.h. and geddes, w.f. (1949). grain storage studies 8: relation of moulds in moist stored cotton seed to increase production of carbon dioxide, fatty acids and heat. cereal chem. 26 : 106-129. 82. ciegler, a. (1978). fungi that produce mycotoxins conditions and occurrence, mycopathologia 65: 5-11. 83. coskuntuna, l., koç, f. and coskun, f. (2008). effect of chemical content and physical characteristics on nutritional value of wheat varieties collected from thrace region. pak. j. nutrition 7: 218221. issn 1680-5194 84. drochner, w. and lauber, u. (2001). occurrence of the three important fusarium toxins deoxynivalenol, nivalenol and zearalenon in grains in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 95 central europe and effects in farm animals. proc. soc. of nutrition physiol. pp 163-168 85. duggeli, m. (1904). the mycological florainfection. zentr. microbial infection 12: 602-614. 86. eriksen, g.s. and aleksander, j. (1998). fusarium toxins in cereals – a risk assessment. tema nord. nordic council of ministers, copenhagen, denmark, 10-165. 87. field, r.w. and king, t.h. (1962). influence of storage fungi on the deterioration of corn in storage a review. lowa acad. sci. 51: 265-280. 88. finney, k.f. and barmore, m.a. (1948). loaf volume and protein content of hard red winter and spring wheats. cereal chem. 25: 291-312. 89. gooding, m.j. and davis, w.p. (1997). wheat production and utilization, systems, quality and the environment. book cab international. 90. halverson, j. and zeleny, l. (1988). criteria of wheat quality. chapter 2. in: wheat: chemistry and technology, vol. 1. (y. pomeranz, ed.). am. assoc. cereal chem., inc., st. paul, mn. pp. 15. 91. hasan, h.a.h. (1999). mycoflora and changes of safflower, wheat and faba bean seed quality during the storage. rostl. vyr. 45: 85-91. 92. hesseltine, c. w. (1968). flour and wheat: research on their microbiological flora, bakers dig. 42: 40-42, 66. 93. hesseltine, c. w. and graves, r.r. (1968). microbiology of flours. econ. bot. 20: 156-168. 94. hocking, a.d. (2003). stored grain in australia 2003. proceedings of the australian postharvest technical conference, canberra. csiro stored grain research laboratory, canberra. pp. 55-58. 95. hoseney, r.c. (1987). wheat hardness. cereal fd. world 32: 320-322. 96. humpisch, g. (2001). gesundheitsvorsorge im getreidelager. neue landwirtschaft. vol. 2. pp. 4448. 97. inagaski, n. and ikeda, m. (1959). studies on the fungi isolated from foods ii. identification of penicillia and aspergilli from flour. bull. nat. hygenic lab. tokyo 77: 347-366. 98. jayas, d. s. (1995). stored-grain ecosystems. new york: dekker, pp. 757. 99. jones, d.b. and gersdorff, c.e.f. (1941). the effect of storage on the protein of wheat, white flour, and whole wheat flour. cereal chem. 18: 417. 100. jonsson, n., petterson, h. and schnurer, j. (2000). study of the relationship between storage conditions and the growth of moulds and production of ochratoxin a in grain 24 preliminary results. proc. eur. agricul. eng. sym. warwick university, u.k. pp.44. 101. jorgensen, k. and rasmusen, g. (1999). ochratoxin in danish cereals 19861992 and daily intake by danish population. fd. additives contaminants. 13: 95-104. 102. kentjones, d.w. and amos, a. j. (1930). preliminary studies in bacteriology of wheat and flour. analyst 55: 248-268. 103. koeksel, h., atli, a., oezkaya, h. and demir, z. (1993). comparison of physical properties of wheat and nir spectroscopy hardness value for prediction of semolina yield. j. agr. forest. 17: 821-830. 104. kurasova, v.v., kostin, v.v. and salovskaja (1945). methodolgy of isolating vertanary microrganism, pp. 194 -195. 105. langseth, w., stenwing, h., sogn, l. and mo, e. (1993). growth of moulds and production of mycotoxins in wheat during drying and storage. acta agric. scand. [b] soil plant sci. 1993, 43, 32-37 106. lugauskas, a. (1997). microbiological book for research: vilnius, 35-390. 106. machacek, j.e. and greaney, f.j. (1938). the block point of kernel smudge disease of cereals. can. j. res. 16: 84-113. 107. magan, n., hope, r., cairns, v. and aldred, d. (2003). post-harvest fungal ecology: impact of fungal growth and mycotoxin accumulation in stored grain. european j. plant pathol. 109: 723-730. 108. mehrothra, d.k. (1974). studies on some microrganisms associated with rice in storage. paper presentation abstract at university of lucknow. 109. milner, y. and geddes, w.f. (1946). grain storage studies of influence of certain moulds inhibitors on respiration of moist wheat. cereal chem. 24 : 507517. 110. milner, y., christensen, c. m. and gedess, w.f. (1947). grain storage studies 6: wheat respiration in relation to moisture content, mould growth, chemical deterioration and heating. cereal chem. 24: 182-199. 111. mis, a. and grundas, s. (2002). wheat grain hardness and quality. int. agrophysics 16: 283-288. 112. mohammadkhani, a. (2005). study of pentosans (non starch polysaccharides), in durum wheat and its relation to the quality of protein and grain hardness index (h.i.). pak. j. nutri. 4: 208-209. 113. orth, r.a. and shellenberger, j.a. (1988). origin, production and utilization of wheat. in: wheat: chemistry and technology (y. pomeranz, ed.). vol. i, aacc, st. paul, mn, pp.1-14. 114. palni, i.k. (1975). studies on the microflora associated with wheat grains and various milling fraction. paper presentation. abstract, university of lucknow. 115. papavizas, g.c. and chritensen, c.m. (1960). grain storage studies 29: effect of invasion by individual species and mixture of aspergillus upon germination and development of discoloured germs in wheat, cereal chem. 27: 197-230. 116. pelhate, j. (1968). inventaire de la mycoflora des bles de conservation (inventory of mycoflora of stored wheat) bull. soc. mycol. france 84: 127-143. 117. poisson, j. and guilbot, m.a. (1956). mycoflora des farines franscaises. menuerie, france. pp 42-52. 118. pomeranz, y. and williams, p.c. (1990). wheat hardness: its genetic, structural, and biochemical background, measurement, and significance. in: advances in cereal science and technology (y. pomeranz ed.). american association of cereal chemists, st. paul, mn. 10: 471-548. 119. preston k.r., dexter j.e. and kruger j.e. (1978). relationship of exoproteolytic and endoproteolytic activity to storage protein hydrolysis in germinating durum and hard red spring wheat. cereal chem. 55: 877-888. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 96 120. samson, r.a. and van reenen, h. (1988). introduction to foodborne fungi. 3rd ed. centraalbureau voor schimmelcutures, baar. delff. institute of the royal netherland academy of arts and sciences. 121. sankaran, r. (1976). mould problems in the storage of food grains. symposium ‘stratergies on storage, handling and processing of food grains in the present context'. bangalore chapter of association of food scientists and technologists, india. 122. sankaran, r., sehgal, d. d., parihar, d. b. and nath, h. (1976). cereal grain fungi and grain quality. def. sci. 26: 161. 123. sankaran, r., mattada, r. r. and thangamani. (1975). mycoflora and changes of wheat seed quality during storage. itzd. fd. packer 29: 1. 124. schnurer, j. and jonsson, a. (1992). ergosterol levels and mould colony forming units in swedish grains of food and food grade. acta agric. scand. [b] soil plant sci. 42: 240-245. 125. shuey, w.c. and gilles, k.a. (1969). experimental milling of sized wheat. northwest mill. 276: 9. 126. singh, h.b., anderson, e. and pal, b.p. (1957). studies in the genetics of triticum vavilovii jacob. agron. j. 49: 4-11. 127. smirnova, a., kostrova, o. (1989). mikrobiologija zerna i produktov jego pererabotk. pp.195. 128. tatham, a.s., and shewry, p.r. (1985). the conformation of wheat glutenin proteins. the secondary structure and thermal stabilities of glutein and gliadins. j. cereal sci. 3: 103-113. 129. tuite, j.f. and christensen, c.m. (1957). grain storage studies 24: moisture content of wheat seed in relation to invasion of seed by species of the aspergillus glaucus group and the effect of invasion upon germination of the seed. j. phytopathol. 47: 323-327. 130. turnbull, k.m. and rahman, s. (2002). endosperm texture in wheat. j. cereal sci., 36: 327-337. 131. wall, j.s. (1979). the role of wheat proteins in determining baking quality. in: recent advances in the biochemistry of cereals, (d.l. laidman, and r.w.wynjones, eds.). phytochem. soc. eur. symp. ser. no. 16, academic press london ny and san fransico. pp. 275311. 132. wicklow, d. t. (1995). the mycology of stored grain: an ecological perspective. in: stored grain ecosystems (d. s. jayas, n. d. g. white and w. e. muir, eds.). new york, marcel dekker, pp. 197-249. 133. worzella, w.w. (1942). inhe ritance and interrelationship of components of quality, cold resistance, and morphological characters in wheat hybrids. j. agric. res. 65: 501-522. 134. wosniak, w. (2001). effect of moisture treatment of wheat grains on technological hardness (in polish). acta agrophysics 46: 227-233. 135. wrigley, c.w. and bietz, j.a. (1988). protein s and amino acids. in: wheat: chemistry and technology (y. pomeranz, ed.). american association of cereal chemists, saint paul, mn, 1: 159-275. 136. magan, n. (1993a). early detection of fungi in stored grain. int. j. biodeterior. biodegradat. 32:145160. 137. magan, n. (1993b). use of sulphur dioxide to control fungi in stored grain. in: proceedings of int. conference on controlled atmosphere and fumigation in grain storages (s. navarro and e. donahaye, eds.). capital press, jerusalem, israel. pp. 163-171 138. orth, r.a. and shellenberger, j.a. (1988). origin, production and utilization of wheat. in: wheat: chemistry and technology (y. pomeranz, ed.). vol. i, aacc, st. paul, mn, pp.1-14. 139. gooding, m.j. and davis, w.p. (1997). wheat production and utilization, systems, quality and the environment. book cab international. 140. finney, k.f. and barmore, m.a. (1948). loaf volume and protein content of hard red winter and spring wheats. cereal chem. 25: 291-312. 141. pomeranz, y. and williams, p.c. (1990). wheat hardness: its genetic, structural, and biochemical background, measurement, and significance. in: advances in cereal science and technology (y. pomeranz ed.). american association of 142. halverson, j. and zeleny, l. (1988). criteria of wheat quality. chapter 2. in: wheat: chemistry and technology, vol. 1. (y. pomeranz, ed.). am. assoc. cereal chem., inc., st. paul, mn. pp. 15. microsoft word 3 journal nr 4 2010 din 8 noiembrie_38-43.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 42 quantification of carotenoids and chlorophyll leaf pigments from autochthones dietary mariana lupoae 1, bianca furdui2, rodica dinică2, dragomir coprean1 1 ovidius university constanta, faculty of natural and agricultural sciences, street mamaia no. 124, 900527 ,constanţa , mariana_lupoaie@yahoo.com, dcoprean@ovidius.ro 2 dunărea de jos university galaţi, faculty of science, street domnească no. 47 ,800008, galaţi, rodinica@ugal.ro bfurdui@ugal.ro abstract: chlorophylls are the preponderant photosynthetic pigments of the verdant tissues of vascular plants, liverworts, and various algae. carotenoids are essential for the survival of photosynthetic organisms. they function as light-harvesting molecules and provide photoprotection. information gathered from the screening of secondary plant metabolites is vital for the accurate determination of the dietary intake of these micro-nutrients, and in the development of comprehensive food tables. determination of basal levels is also necessary for the rational engineering of healthpromoting phytochemicals in food crops. in addition this approach can also be applied to the routine screening of products to determine metabolic differences between varieties and cultivars, as well as between genetically modified and the corresponding non-genetically modified tissue. beta -carotene accounts for more than 90% of total carotenoids in vegetables. in human beings, beta-carotene not only serves as valuable source of vitamin a, but also serves as a potent antioxidant, scavenging free radicals and quenching singlet oxygen. by this latter property, beta-carotene is understood to reduce the risk of development of certain types of cancer this study therefore is aimed at determining the beta-carotene and chlorophyll contents of same selected autochthones plants (allium ursinum, alliaria petiolata, urtica dioica) from macin mountains harvested on spontaneous flora. photosynthetic pigments of investigated plants were extracted from leaves using appropriate solvents. the pigment quantification of individual plants was investigated by spectrophotometric analysis. the levels of carotenoids, and chlorophyll varied in each plant and the results were similar to the previously results reported in the literature. keywords: allium ursinum, alliaria petiolata, urtica dioica introduction carotenoids are natural pigments which are synthesized by plants and are responsible for the bright colors of various fruits and vegetables. carotenoids are the most ubiquitous and widespread pigments which are characteristic for organisms of all taxa[1]. ability of carotenoids in modifying structure, properties, and stability of cell membranes, and thus affecting molecular processes associated with these membranes, may be an important aspect of their possible beneficial effects on human health [2]. some carotenoids, including β-carotene (fig.1), quench highly reactive singlet oxygen under certain conditions and can block free radical-mediated reactions. in epidemiological researches, the intake of carotenoid-rich fruits and vegetables has been correlated with protection from some food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 43 forms of cancer. analogically, serum βcarotene levels have been associated with a decreased chance of developing lung cancer. it must be stressed, however, that these epidemiological associations do not show cause and effect. in this regard, longterm intervention trials with beta-carotene supplements are in progress. whatever the results of these trials, carotenoids clearly show biological actions in animals distinct from their function as precursors of vitamin a[3]. chlorophyll is the pigment that gives plants and algae their green color. plants use chlorophyll to trap light needed for photosynthesis [4]. chlorophyll a and b differ from one to another through the radical put in position three. chlorophyll a contains one methylic radical (ch3), and chlorophyll b one radical cho (fig.2). -caroten fig.1-β-carotene chemical structure β-carotene is under the violet cristal form following ever the chlorophylla and is soluble in organic solvents. through oxidative enzimatic hydrolysis, βcarotene transforms in two molecules of vitamine a. the average content of carotenoids from vegetables and fruits is different on species between 6,0 and 24,0 mg/100g dry product of the carrot and 0,1 mg/100g dry product of the plums tree. carotenoids pigments are spread in all of the plant˙s section with or without chlorophyll (leaves, fruits, stems, bulbs, seeds, etc.). however, the contend in carotenoids pigments depends of the species but also the influence of the enviromental conditions is very important[7]. the present paper consists in the determination of the contend of β-carotene and chlorophyll from three species of plantsallium ursinum(fam. alliaceae), alliaria petiolata(fam. brassicaceae), urtica dioica(fam. urticaceae) –harvested from spontaneous flora of the macin mountains. fig. 2-chlorophyll a and b[5,6] some representative examples indentified on the area are stored in the herbarium of botanical garden galati and of the pharmacy and medicine faculty of “dunarea de jos” university galati. materials and methods methods of the determination and extraction, for chlorophyll and β-carotene, used, are in according with the official methods aoac [8,9,10]. for the extraction and for the spectrophotometric determination of clorophyll from the biological material harvested (folium) was used 1 g dry vegetal product from every plant. the cold mortar was done with quart sand and the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 44 extraction was done with 85% acetone. the extinction was determined at 660 and 642nm. the calculation of the totale clorophyll concentration and of the clorophyll a and b(mg/l) is done with the help of the next relationships: (1) total chlorophyll = (7.12 a660.0 + 16.8 a642) fd (2) chlorophyll a = (9.93 a660.0 0.777 a642) fd (3) chlorophyll b = (17.6 a642 2.81 a660.0) fd for the calculation of the chlorophyll concentration in mg/100g plant, we remind of the acetone extract volume obtained from vegetal material and of the partition from filtrate solution submissived to the extraction with ether. for the separation and spectrophotometrical determination of the β-carotene was used extract from 1 g vegetal product removed with ether brew of petrol-benzene (v:v=1:1). qualitative determination of the β-carotene was put in evidence with the help of the uv-vis spectrometry. spectrometrical analysis was realized with the uv-vis double beam pc spectrometric and scanned with the auto device cell uvd-3200, in comparison with specter β-carotene absolute merck provenience. the separation of the carotenoidic pigments by the chlorophyll and xantophyll pigments, consists into a pass through an adsorbtion column, with al2o3 (4 – 7 cm height). obtained carotenoidic extract was measured at 436 nm. the concentration in β-carotene (cx) of the analyzed samples was derived from a standard curve of β carotene, ranging from 2 to µg/ml (pearson˙s correlation coefficient: r2:0,9913). the contain in mg carotine/100g vegetal poduct is determined with the help of the relationship [11]: 10-1 where: cx – concentration in carotene of the analyzed samples,removed from the standard curve(µg/ml); fd – the factor of dilution applied on the analyzed samples, in order to frame their absorbance; vex – the volume of obtained extract(ml); m – the mass of the analyzed sample(g). results and discussions the results obtained from the study are presented in tables 1 and 2. the chlorophylic extracts were diluated with anhidrous ether in raport 1:2, for obtaining of some optime values of the absorbance, at the wavelength used(660 nm and 642nm). after the obtaining of the results phisycochimical analysys, we observed the following: the graphic representation with the auto device cell uvd-3200, is between 400 and 500 nm (fig. 3, fig. 4), values wich appart of the uv-vis spectrum of the β carotene standard absolute (merck). fig. 3-spectrometry uv-vis β carotene absolute (merck) ethalon food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 45 fig. 4-spectrometry uv-vis β-carotene of allium ursinum the values obtained of analyzed samples can be observed at standard curve characteristical β carotene with the values r2=0,9913 (fig.5). new researches concerning the contains of chlorophyll and carotenoids from allium genus put in evidence valoric differences that can be compared with the obtained results(by us). so, the highest content of investigated pigments was obserwed in leaves of wild garlic(allium ursinum) : 999mg/100g for carotenoids and 287 mg/100g for chlorophyll a, and 135mg/100g for chlorophyll b [12]. the contain of chlorophyll a, determined by us from wild garlic is higher so of 374,95mg/100g from the dried plant on natural way but the contend of chlorophyll b was lower (104,77mg/100g). it must be observed that like in all freeze samples from three species, the values of the chlorophyll a and b were lowed in comparison with the dry samples by natural way. also, the greatest contain, of the total chlorophyll, can be found on the urtica dioica species (2604.4mg/100g dry plants and 2291.94mg/100g lyophilised plants). the lowest values are in alliaria petiolata cases. fig.5-β-carotene determination of samples legend:  carotene standard curve  sample analyzed table 1 determination of chlorophyll content (a and b) on samples(fd=1:2) legend:dp-dry plant; lp-lyophilised plant urtica dioica allium ursinum alliaria petiolata samples dp lp dp lp dp lp a660 0.641 0.581 0.217 0.201 0.175 0.164 a642 0.282 0.241 0.112 0.110 0.093 0.081 total chlorophyll (mg/l) 18.603 16.371 3.4266 2.2254 2.8084 2.4921 chlorophyll a (mg/l) 12.292 ±0,002 11,1642 ±0,004 2.6782 ±0,006 2.0201 ±0,002 1.6655 ±0,003 1.4871 ±0,00 chlorophyll b (mg/l) 6.634 ±0,001 5.218 ±0,002 0.7484 ±0,005 0.2053 ±0,004 1.1429 ±0,001 1.005 ±0,001 total chlorophyll (mg/100g plant) 2604.4 2291.94 479.72 480.02 395.64 351.63 chlorophyll a (mg/100g plant) 1720.9 ±0,02 1562.9 ±0,02 374.95 ±0,06 283.47 ±0,01 233.71 ±0,05 206.41 ±0,02 chlorophyll b (mg/100g plant) 928.76 ±0,05 730.52 ±0,04 104.77 ±0,02 196.55 ±0,00 161.93 ±0,06 145.49 ±0,03 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 46 tabel 2 determination of β carotene content by samples(fd=1:20) legend:dp-dry plant; lp-lyophilised plant urtica dioica allium ursinum alliaria petiolata samples dp lp dp lp dp lp a (λ= 436 nm) 0.036 0.079 0.125 0.136 0.090 0.115 conc. (µg/ml) β-carotene 2.89 6.53 10.43 11.36 7.46 9.58 vex (ml) 158 80 24 24 25 25 mg β-caroten % 913.24 ±0,01 1044.8 ±0.02 500.64 ±0.06 545,28 ±0,04 373 ±0.01 479 ±0.02 the β carotene contain determinate in present paper is highered in case of urtica dioica species (1044,8 mg/100g lyophilised sample) and lowered at alliaria petiolata species (373g/100g dry plant). however, we must do an specification about lyophilised samples wich have superior values in comparison with the ones on natural way. allium ursinum(wild garlic), alliaria petiolata (garlic mustard) and urtica dioica (common nettle) are spread on large areas in macin mountains zones, in special near the rivers. the growing of the plants is conditioned by the trophicity of the soil but also the atmospheric humidity[13]. this plants are used by the humans in the early spring to prepare salads or another types of foods. the harvested leaves in optime periods can have good effects to the human organism, this was proved and through thir contining of βcarotene with antioxidant effect.[14,15]. conclusions the highest contend in total chlorophyll (a and b) from the three analyzed plants can be found in common nettle. the lyophilised samples have high values in comparison with the dry samples on natural way only in case of determination of β-carotene. our results indicates that leaves of urtica dioica, allium ursinum and alliaria petiolata could be used as potential sources of natural untoxic antioxidants in food and pharmaceutical industries. references 1. d.i. corol , i.i. dorobantu, n. toma, r.nitu, diversity of biological functions of carotenoids, roumanian biotechnological letters, vol. 8, no.1, 2003, p. 1067-1074 2. g. britton, – faseb j., 9(1), 1995,p. 551558. 3. a. bendich, j.a. olson, – faseb j., 3(1), 1989,p. 927-932 . 4. ck matthews, van holde ke. biochemistry, 2nd ed. menlo park: the benjamin/cummings publishing company, 1996. 5. dl. sudakin, dietary aflatoxin exposure and chemoprevention of cancer: a clinical review. j toxicol clin toxicol,41(2), (pubmed), 2003,p.195204. 6. rh. dashwood, the importance of using pure chemicals in (anti) mutagenicity studies: chlorophyllin as a case in point. mutat res. (pubmed);381(2), 1997,p.283-286. 7. gavril neamţu, biochimie ecologica, editura dacia, cluj napoca, 1983, p.13-55. 8. aoac 1999 official method 941.15, carotene in fresh plant, spectrofotometric method. 9. aoac 1999 official method 938.04, cartenoids in macaroni products. 10. aoac ,vitamins and other nutrient official methods of analysis. association of official food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 47 analytical chemists. inc. arlington, virginia, 1984, p. 884-847. 11. r. dinică, t. florea, b. furdui, r. creţu, chimie organică . sinteză şi analiză funcţională , editura academică galaţi , 2009, p. 200, 221-223. 12. dubravka stajner, ilona szollosi varga, an evaluation of the antioxidant abilities of allium species, acta biologica szegediensis, vol.47(1-4), 2003,p. 103-106. 13. v. ciocârlan , flora ilustrată a româniei – pteridophyta et spermatophyta , editura ceres bucureşti, 2000, p. 919-925. 14. g.n.gayathri, kalpana platel, jamuna prakash, k. srinivasan, influence of antioxidant spices on the retention of β-carotene in vegetables during domestic cooking processes, food chemistry 84, 2004,p.35-43. 15. j.m. herdan m. georgiuca , a. meghea, antioxidanţi , editura tehnică bucureşti , 1995. microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_63-68.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 67 influence of fertilization treatments on nitrates content of some vegetable species cultivated in the field enuţa iorga1, monica catană1, luminiţa catană1, mioara negoiţă1, alina bâlea1, gabriela lilios2 1national institute of research&development for food bioresources – iba bucharest, 021102, bucharest 2, 6 dinu vintila street, romania, e-mail: enutai@yahoo.com 2ovidius university, 900527, constanţa, 124 mamaia street, romania, e-mail: liliosgabriela@yahoo.com abstract: the existing amount of nitrates in plant at a given time is the result of balance between the amount absorbed and used in protein-genesis. a main cause of nitrate accumulation in vegetables is the use of nitrogenous fertilizers on crop land. high concentration of nitrates in plants (especially in vegetables) means a hazard for human and animal body, for two reasons: possibility of methemoglobin appearance at children and nitrates conversion to nitrites in saliva and formation of cancerigen nitrosamines in the intestinal tract. this paper presents the results of researches made in order to establish the influence of fertilization treatments on nitrates content of some vegetable species, cultivated in the field: egg plants, green peppers, bell peppers and carrots. in the culture technology of these vegetables, the following fertilization variants were applied (fertilizer being ammonium nitrate with 33% nitrogen): v1 = 0 kg nitrogen/ha; v2 = 100 kg nitrogen/ha; v3 = 200 kg nitrogen/ha; v4 = 400 kg nitrogen/ha; v5 = 800 kg nitrogen/ha. in order to establish the accumulation potential of nitrates in vegetables cultivated in the field, an enzymatic method was used. when no fertilizer was added in the culture, bell peppers had the minimum value of the nitrates content (11.53 mg no3-/kg), and carrots the maximum one (90.42 mg no3-/kg). in the case of the highest fertilization level, carrots had the highest nitrates content (391.20 mg no3/kg), and bell peppers the lowest one (63.08 mg no3-/kg). key words: egg plants, green peppers, bell peppers, carrots, nitrates introduction achieving a sustainable soil fertility reintegration involves the mineral, plant and animal in its natural flow. under natural conditions, soil organic matter is maintained in terms of intake of animal and vegetable, devoid of life, which falls on the surface of the land mass of dead roots, soil microorganisms that die, all incorporated by biological cycles characteristic of biocenoses [1]. fertilizer application is made on the basis of careful analysis for each crop separately and for each type of soil, taking into account that their use in uncontrolled quantities can pollute soil, groundwater and end products. when determining the dose of fertilizer, the following aspects should be taken into consideration: nutrients in the soil, the degree of assurance of water plants, pre-plant, the planned production on cultivated plot [2]. naturally, between nitrates and nitrites in soil, water and plants equilibrium is established, which can be disrupted by intensive use in agriculture of natural organic fertilizers and of those synthetic nitrates, especially. their degradation products enrich the soil and they can be accumulated in the cultivated plants until deleterious levels for consumers. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 68 vegetables and fruits can also accumulate nitrogen as nitrates and nitrites form. mineral nitrogen amount in tissues of vegetable and fruit is higher in the case of species at which nitrates reductions is made in leaves and when light intensity and temperature are lower [3]. the factors determining the nitrates accumulation in cultivated horticultural products within protected environments (greenhouses, solar) or in the open field are [4]:  genetical potential of species  high doses of mineral fertilizers with nitric nitrogen, applied to cultures  low temperature  reduced light intensity  maintenance system of soil experimental we carried out a study to determine the influence of fertilization treatments on nitrates content of some vegetable species, cultivated in open field: egg plants (zaraza variety), green peppers (buzău 10 variety), bell peppers (splendens variety), and carrots (nabuco variety). the samples were supplied by the vegetable research development plant for horticulture buzau. in the culture technology of these vegetables, the following fertilization variants were applied (fertilizer being ammonium nitrate with 33% nitrogen): v1 = 0 kg nitrogen/ha; v2 = 100 kg nitrogen/ha; v3 = 200 kg nitrogen/ha; v4 = 400 kg nitrogen/ha; v5 = 800 kg nitrogen/ha. for each fertilization variant, we made 4 repetitions. to determine the accumulation potential of nitrates in vegetables cultivated in open field, an enzymatic method was used. in this method, nitrate is reduced by the reduced nicotinamide adenine dinucleotide (nadph), to nitrite, in the presence of nitrate-reductase (nr) [5]: nr no3+nadph + h+ no2+ nadp+ + h2o the amount of oxidized nadph is stoechiometrically equal to the nitrate one. decreasing of nadph amount is measured by absorbance at 340 = ג nm. figure 1. kit for nitrates determination through enzymatic method results and discussion in the case of experimental variant v1 (without fertilizer addition), the nitrates content of egg plants is in the range 41.15 – 44.55 mg no3-/kg. the average value of accumulation potential of nitrates, in this case is 42.61 mg no3-/kg. in the case of fertilizer application, the nitrates content of egg plants increases with fertilizer dose (ammonium nitrate with 33% nitrogen). thus, in the case of fertilization variant v2 (100 kg nitrogen/ha) the egg plants have the nitrates content in the range 81.95 – 84.67 mg no3-/kg, and the average accumulation potential of nitrates is 83.22 mg no3-/kg, with 95.30% much higher than in the case when no fertilizer is applied. in the case of fertilization variant v3 (200 kg nitrogen/ha) the egg plants have a nitrates content in the range 137.59 – 143.25 mg no3-/kg, and the average accumulation potential of nitrates is 140.56 mg no3-/kg, approximately 3.3 times higher than in the case when no fertilizer is applied. the application of fertilizer dose of 400 kg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 69 nitrogen/ha (fertilization variant v4), determines an increase of 4.83 times of the average accumulation potential of nitrates in egg plants cultivated under these conditions, in comparison with those nonfertilized ones. in the case of this fertilization variant, egg plants have nitrates content in the range 203.55 – 209.23 mg no3-/kg, and the average accumulation potential of nitrates is 206 mg no3-/kg. the use within culture technology of a fertilizer dose of 800 kg nitrogen/ha (fertilization variant v5), determines an increase of 7.11 times of the average accumulation potential of nitrates within the egg plants cultivated under these conditions, in comparison with those nonfertilized ones. in the case of this fertilization variant, the egg plants have nitrates content in the range 295.37 – 308.19 mg no3-/kg, and the average accumulation potential of nitrates is 303.05 mg no3-/kg. according to the achieved results, egg plants are a vegetable species with an average accumulation potential of nitrates. 0 50 100 150 200 250 300 350 n itr at es (m g n o 3/k g) v1 v2 v3 v4 v5 fe rtilization variants acumulation potential of nitrates in eggplants cultivated in field, depending on fertilization level nitrates content (mg v1 0 kg nitrogen/ha v2 100 kg nitrogen/ha v3 200 kg nitrogen/ha v 4 400 kg nitrogen/ha v 5 800 kg nitrogen/ha figure 2. accumulation potential of nitrates in egg plants cultivated in the field, depending on fertilization level between the fertilization level with nitrogen of egg plants culture and accumulation potential of nitrates in egg plants, a linear correlation is established, described by the equation y = 0.3216x + 58.607, linearity coefficient (r2) being 0.9732. correlation between fertilization level with nitrogen and accumulation potential of nitrates in egg plants, cultivated in the field y = 0,3216x + 58,607 r2 = 0,9732 0 50 100 150 200 250 300 350 0 200 400 600 800 1000 fertilization variants n it ra te s (m g n o 3/k g) figure 3. correlation between fertilization levels with nitrogen and accumulation potential of nitrates in egg plants, cultivated in the field in the case of experimental variant v1 (without fertilizer addition), the nitrates content of green peppers is in the range 19.8 – 21.57mg no3-/kg. and the average value of accumulation potential of nitrates, in this case, is 20.79 mg no3-/kg. in the case when fertilizer is applied, the nitrates content of green peppers increases with fertilizer dose (ammonium nitrate with 33% nitrogen). thus, in the case of fertilization variant v2 (100 kg nitrogen/ha) green peppers have the nitrates content in the range 32.28 – 33.05 mg no3-/kg, and average accumulation potential of nitrates is 32.58 mg no3-/kg, with 56.71% much higher than in the case when no fertilizer is applied. in the case of fertilization variant v3 (200 kg nitrogen/ha) green peppers have the nitrates content in the range 46.24 – 49.77 mg no3/kg, and the average accumulation potential of nitrates is 48.02 mg no3-/kg, approximately 2.31 times higher than in case when no fertilizer is applied. the application of fertilizer dose of 400 kg nitrogen/ha (fertilization variant v4), determines an increase 3.23 times of the average accumulation potential of nitrates in green peppers under these conditions, in comparison with those non-fertilized ones. in the case of this fertilization variant green peppers have nitrates content in the range 65.74 – 68.80 mg no3-/kg, and the average accumulation potential of nitrates is 67.14 mg no3-/kg. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 70 0 20 40 60 80 100 n it ra te s (m g n o 3/k g) v1 v2 v3 v4 v5 fertilization variants acumulation potential of nitrates in green peppers cultivated in field, depending on fertilization level nitrates content (mg v1 0 kg nitrogen/ha v2 100 kg nitrogen/ha v3 200 kg nitrogen/ha v 4 400 kg nitrogen/ha v 5 800 kg nitrogen/ha figure 4. accumulation potential of nitrates in green peppers cultivated in the field, depending on fertilization level the use within the culture technology of fertilizer dose of 800 kg nitrogen/ha (fertilization variant v5), determines an increase 4.59 times of the average accumulation potential of nitrates in green peppers cultivated under these conditions, in comparison with those non-fertilized ones. in the case of this fertilization variant, the achieved green peppers have nitrates content in the range 93.56 – 98.34 mg no3/kg, and the average accumulation potential of nitrates is 95.41 mg no3-/kg. between the fertilization level with nitrogen of green peppers culture in field and accumulation potential of nitrates in green peppers, a linear correlation is established, described by the equation y = 0.0922x + 25.14, linearity coefficient (r2) being 0.9769. according to the achieved results, green peppers are a vegetable species with low accumulation potential of nitrates. correlation be twee n fertilization le ve l with nitrogen and accumulation potential of nitrates in gre en pe ppers , cultivate d in the field y = 0,0922x + 25,14 r2 = 0,9769 0 20 40 60 80 100 120 0 100 200 300 400 500 600 700 800 900 fe rtiliza tion va riants n itr at es (m g n o 3/k g) figure 5. correlation between fertilization level with nitrogen and accumulation potential of nitrates in green peppers, cultivated in the field within the fertilization variant v1 (without fertilizer addition), the nitrates content of bell peppers is in the range 11.53 – 14.04 mg no3-/kg. the average value of the accumulation potential of nitrates in this case is 12.63 mg no3-/kg. when fertilization is applied, nitrates content of bell peppers increases once with increasing of fertilizer dose (ammonium nitrate with 33% nitrogen). thus, in the case of fertilization variant v2 (100 kg nitrogen/ha) bell peppers have a nitrates content in the range 17.03 – 19.25 mg no3/kg, and the average accumulation potential of nitrates is 18.17 mg no3-/kg, with 43.86% much higher than in the case when no fertilizer is applied. within the fertilization variant v3 (200 kg nitrogen/ha) bell peppers have a nitrates content in the range 27.10 – 29.35 mg no3/kg, and the average accumulation potential of nitrates is 28.08 mg no3-/kg, of 2.22 times higher, than in the case when no fertilizer is applied. the application of a fertilizer dose of 400 kg nitrogen/ha (fertilization variant v4) determines an increase of 3.22 times of the average accumulation potential of nitrates in bell peppers cultivated under these conditions, in comparison with those nonfertilized ones. in the case of this fertilization variant bell peppers have nitrates content in the range 39.67 – 41.55 mg no3-/kg, and the average accumulation potential of nitrates is 40.67 mg no3-/kg. the use in the culture technology of a fertilizer dose of 800 kg nitrogen/ha (fertilization variant v5) determines an increase of 4.88 times of the average accumulation potential of nitrates in bell peppers cultivated under these conditions, in comparison with non-fertilized ones. in the case of this fertilization variant, bell peppers have the nitrates content in the range 60.85 – 63.08 mg no3-/kg, and the average accumulation potential of nitrates is 61.70 mg no3-/kg. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 71 0 10 20 30 40 50 60 70 n it ra te s (m g n o 3/k g ) v1 v2 v3 v4 v5 fertilization variants acumulation potential of nitrates in bell peppers cultivated in field, depending on fertilization level nitrates content (mg v1 0 kg nitrogen/ha v2 100 kg nitrogen/ha v3 200 kg nitrogen/ha v 4 400 kg nitrogen/ha v 5 800 kg nitrogen/ha figure 6. accumulation potential of nitrates in bell peppers cultivated in the field, depending on fertilization level between the fertilization level with nitrogen of culture of bell peppers in the field and accumulation potential of nitrates in bell peppers, a linear correlation is established, described by the equation y = 0.0617x + 13.736, linearity coefficient (r2) being 0.9901. correlation betwe en fertilization leve l with nitrogen and accumulation potential of nitrate s in be ll pe ppe rs , cultivated in the fie ld y = 0,0617x + 13,736 r2 = 0,9901 0 10 20 30 40 50 60 70 0 100 200 300 400 500 600 700 800 900 fertilization va riants n itr at es (m g n o 3/k g) figure 7. correlation between the fertilization level with nitrogen and accumulation potential of nitrates in bell peppers, cultivated in the field according to the obtained results, bell peppers are a vegetable species with low potential of nitrates accumulation. within the fertilization variant v1 (without fertilizer addition), the nitrates content of carrots is in the range 90.18 – 91.88 mg no3-/kg. the average value of the accumulation potential of nitrates in this case is of 90.85 mg no3-/kg. when fertilizer is applied, the nitrates content of carrots increases once with increasing the fertilizer dose (ammonium nitrate with 33% nitrogen). thus, in the case of fertilization variant v2 (100 kg nitrogen/ha) carrots have a nitrates content in the range 159.55 – 161.93 mg no3-/kg, and the average accumulation potential of nitrates is 160.98 mg no3-/kg, with 77.19% much higher than in the case when no fertilizer is applied. within the fertilization variant v3 (200 kg nitrogen/ha) carrots have a nitrates content in the range 199.85 – 201.85 mg no3-/kg, and the average accumulation potential of nitrates is 200.83 mg no3-/kg, of 2.21 times higher than in the case when no fertilizer is applied. the application of a fertilizer dose of 400 kg nitrogen/ha (fertilization variant v4) determines an increase of 2.87 times of the average accumulation potential of nitrates in carrots cultivated under these conditions, in comparison with those non-fertilized ones. in the case of this fertilization variant carrots have nitrates content in the range 260.53 – 261.44 mg no3-/kg, and the average accumulation potential of nitrates is 260.62 mg no3-/kg. the use in the culture technology of a fertilizer dose of 800 kg nitrogen/ha (fertilization variant v5) determines an increase of 4.3 times of the average accumulation potential of nitrates in carrots cultivated under these conditions, in comparison with non-fertilized ones. in the case of this fertilization variant, carrots have the nitrates content in the range 389.72 – 391.50 mg no3-/kg, and the average accumulation potential of nitrates is 390.49 mg no3-/kg. between the fertilization level with nitrogen of culture of carrots in field and accumulation potential of nitrates in carrots a linear correlation is established, described by the equation y = 0.3544x + 114.42, linearity coefficient (r2) being 0.9806. according to the obtained results we have drawn the conclusion that carrots are a vegetable species with an average accumulation potential of nitrates. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 72 0 100 200 300 400 n itr at es ( m g n o 3/k g ) v1 v2 v3 v4 v5 fertilization variants acumulation potential of nitrates in carrots cultivated in field, depending on fertilization level nitrates content (mg no3v1 0 kg nitrogen/ha v2 100 kg nitrogen/ha v3 200 kg nitrogen/ha v 4 400 kg nitrogen/ha v 5 800 kg nitrogen/ha figure 8. accumulation potential of nitrates in carrots cultivated in the field, depending on fertilization level correlation betwee n fe rtilization level with nitroge n and accumulation pote ntial of nitrates in carrots, cultivated in the fie ld y = 0,3544x + 114,42 r2 = 0,9806 0 50 100 150 200 250 300 350 400 450 0 200 400 600 800 1000 fertilization variants n itr at es (m g n o 3/k g) figure 9. correlation between the fertilization level with nitrogen and accumulation potential of nitrates in carrots, cultivated in the field conclusions 1. egg plant samples achieved within culture in the field, through application of 5 fertilization variants, have an accumulation potential of nitrates in the range: 42.61 mg no3-/kg – 303.05 mg no3-/kg. the minimum value of nitrates content is for non-fertilized egg plants, and the maximum value is for fertilized egg plants with 800 kg nitrogen/ha. 2. green pepper samples achieved within culture in the field, through application of 5 fertilization variants, have an accumulation potential of nitrates in the range: 20.79 mg no3-/kg – 95.41 mg no3-/kg. the minimum value of nitrates content is for non-fertilized green peppers, and the maximum value is for fertilized green peppers with 800 kg nitrogen/ha. 3. bell pepper samples obtained in the field culture, through application of 5 fertilization variants, have an accumulation potential of nitrates in the range: 12.63 mg no3-/kg – 61.70 mg no3-/kg. the minimum value of nitrates content is for non-fertilized bell peppers, and the maximum value is for fertilized bell peppers with 800 kg nitrogen/ha. 4. carrot samples obtained in the field culture, through application of 5 fertilization variants, have an accumulation potential of nitrates in the range: 90.85 mg no3-/kg – 390.49 mg no3-/kg. the minimum value of nitrates content is for non-fertilized carrots, and the maximum value is for fertilized carrots with 800 kg nitrogen/ha. acknowledgments the experiments were performed within the contract no. 51-050/18.09.2007, financed through programme 4 “partnerships in priority s&t domains” 2007 – 2013 – national centre for projects management. references 1. luchian v., legumicultură generală şi specială, ed. elisavaros, bucureşti, 2007 2. borlan z., dorneanu a., nutriţia minerală a plantelor în sisteme durabile de protecţie vegetală şi cu referire specială la fertilizarea cu fosfor, ed. europa nova, bucureşti, 1999 3. gherghi a., burzo i., miruna bibicu, liana mărgineanu, liliana bădulescu and all, biochimia şi fiziologia legumelor şi fructelor, ed. academiei române, bucureşti, 2001 4. bibicu m., cercetări metodologice privind determinarea nitraţilor şi nitriţilor din ţesuturi vegetale şi nivelului de acumulare în produsele horticole, bucureşti, 1994 5. *** protocol de lucru pentru metoda enzimatică uv de determinare a nitraţilor din produse alimentare, r – biopharm, 2008 microsoft word 3 journal nr 4 2010 din 8 noiembrie_127-134.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 131 lingocellulosics: convenient sorbents for waste water treatment from phenol and fufural g.h.torosyan1, ghazi aidan2, d.n.hovhannisyan3 1 state engineering university of armenia, department of chemical technology & environmental engineering, yerevan, armenia, email: gagiktorosyan@seua.am 2 institute of geological sciences of academy of sciences of armenia, yerevan 3 yerevan state university of architecture & construction, yerevan, armenia & european regional educational academy, yerevan, armenia abstract.in this paper it has been introduced phenol and furfural sorption from water solutions as more important substances for chemical synthesis and as solvent in specially for oil refining solvent and the results of research in the field of the using of natural materials – as agricultural by-products, activated carbon from them as sorbents and as promising materials for waste water treatment from phenol and furfural. the advantages of agricultural by-products in comparison with other sorbents are their low cost, availability of extraction and others. phenol and furfural exist in significant percentage of petroleum products as solvent and refining agent. this organic compound entering in soil and groundwater system are considered such a serious problem is that all have some acute and long term toxic effects. apricot stones based acid-activated carbon has higher sorptive activity. the sorption activity of this sorbent as well as comparable with the commercial carbon carbonsorb-ab. keywords: natural materials, water solution, phenol, furfural, oil products, agricultural by-product, fruit stones and based on them activated carbon, adsorption, adsorbent, wastewater treatment,. introduction organic compounds can be major pollution problem in soil and groundwater. their presence in water can create a hazard to public health and the environment. soil and groundwater pollution from enterprises which have a relation with petroleum and this type of other products is becoming an important topic of interest as a result of the increasing public and regulatory concerns worldwide with problems of groundwater contamination and resultant impact on human health and ecological systems. phenols, furfural make up a significant percentage of petroleum products. this organic compounds entering in soil and groundwater system are considered such a serious problem is that all have some acute and long term toxic effects. taking into account that fact, that some of enterprises have no while the appropriate means, here is offer the elementary technological circuit of clearing of drains, for instance special holes with the boxes, filled in them by activated carbon. waste water here flows down which further are removed and are burnt in furnaces. the offered circuit already has worked at several stations on sale of combustible materials. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 132 one of the best method for wastewater treatment from organic pollutants is an application of inorganic and organic adsorbents. absorption of organic compounds from water solutions with application of sorbents on the basis of a waste of agricultural products – soft lingocellulosic precursors as shells of nuts and fruit stones had been investigated. the increasing demand for sorptive materials as ecological problems simulated the intensive study of lingocellulosic precursors because they are considered to be cheep sorbents and also source for activated carbon obtaining [1-5]. activated carbon has been utilized as chemical sieves, gas absorbers, odor control and filters in water and wastewater treatment processes. the technological stability of lingocellulosic precursors is determined first of all by such characteristics as mineral and chemical composition, sorption ability and then mechanical, physical and following from them filtering properties [5 ]. high performance lingocellulosic materials with uniform densities, durability in adverse environment and high strength can be produced by using as bonding agents the important issues for adsorption processes. the other sites the wide distribution, renewability and recyclability of lingocellulosics can be expend the market for low-cost materials. the advantages of lingocellulosic precursors in comparison with other sorbents are their reserves in agricultural developing countries as armenia, a unique complex of technological properties as well as their natural origin, possibilities of they modification in activated, and utilization [5]. the application of lingocellulosic precursors in the processes of water preparation has been scientifically approved according to the all-round evaluation of physical-chemical, technological properties of agricultural by products developed. it has been established that the lingocellulosic precursors and activated carbon are necessary to remove metal ions from water. it have been established the regularities of extraction processes of ions of iron, magnesium, calcium, zinc, copper, nickel, cobalt, lead and ammonium from water. the dependence of efficiency and mechanism of sorption of components from water, filtering parameters, length of contact liquid and solid phases ratio and other factors are obtained. in present paper it has been investigated the phenols and furfural sorption from water solution. that compounds are discharged in open reservoirs, they destroy the microflora and have negative effect on human health. the major way to diminish the discharge of phenols, also furfural dissolved in water is strong purification and reuse. experimental part the using agricultural by-product in present research the abundance and availability of agricultural by-products / the using byproducts in this investigation apricot, peach stones and based on them activated carbon / make them good candidates as precursors for activated carbons. it has been exists rare literature on the use of agricultural by-product and based on them activated carbon for wastewater treatment processes. the samples used in this study consisted of: (a) four experimental carbons, namely 1. row peach stones breaking, 2. peach seeds based acid-activated carbon, 3. row apricot stones breaking, 4. apricot seeds based acid-activated carbon. one commercial carbons, namely carbonsorb-ab food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 133 this carbon was selected as a control for this experiment as they have found to possess the desirable physical and chemical characteristics and was extensively used in municipal wastewater treatment plants. a process for treating wastewater realized through the use of powdered natural lignocellulosic materials, which is can be responsible: 1. for physically removing of colloidal and suspended volatile solids through adsorption; 2. for adsorbing of organic substances and elements that interfere with biological processes, thus serving to reduce their contact with and exposure to activated sludge organisms effecting wastewater treatment functions; 3. for providing fixed surfaces in activated sludge wastewater treatment bioreactors for bacteria and other organisms. adsorption of phenol from water solutions: polluted by phenol water had passed through column filled with adsorbents. phenol had taken from water, remaining in limits of adsorption column. the treated water leaves a column for direct use or further treatment. the higher adsorptivity shows apricot stones based acid-activated carbon the linear dependence between concentration of phenol in a water solution and appropriate molar refraction is preset at 20oc. the measurements were carried out in concentration limits from 0,01 up to 0,05 mol/l. it was earlier established, that the sorption in these limits grows and has linear dependence on molar refraction. from graphic dependence is determined amount of adsorpted phenol. the results are given in the table 2. it is necessary to note, that partial sorption of water /1-2ml from 10ml of a solution for 4 hours sorption of a solution on sorbents / takes place. it’s visible from the given tables with increase of concentration of solutions the amount of absorbed phenol is increased. adsorption of furfural from water solutions: on 1 gr of sorbent added on 100ml solutions of furfural in water. the mix was carefully shaken up within 4 hours. the measurements of molar refraction of a solution were carried out before and after sorption. on a difference of concentration of an organic solution expected amount of adsorpted furfural.here it has been check the results by liquid chromatography and uv spectrum dates also. the results are given in the table 3. properties peach and apricot stones the both of them were obtained from the garden in ararat valley. elemental analysis of stones and activated carbon from them were determined using a “ elemental analyzer”. chemical composition of the using materials is given in table 1. activated carbon can contain some impurities depending on the type of using stones. in this case it should be clean before the using, it make here. also, the row materials and the activated carbon should contain as small ash as possible. adsorption equilibria. adsorption from wastewater with organic pollutants involves concentration of the solute on the surface. here, it has been had adsorption and desorption process together which will attain an equilibrium state. we used freundlich model for description of the adsorption data for zeolites and activated carbon. it`s known, that the freundlich adsorption equation is the most widely food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 134 used mathematical description for aqueous systems. we use here the freundlich equation is written in logarithmic form. where, x amount of organic adsorbted m the weight of adsorbent ce the solute equilibrium concentration k, 1/n constants characteristic of the system. bulk density is an important criterion for consideration in the designing of adsorption towers for use in pilot plant studies as well as large commercial applications. for activated carbon, for instance, the adsorption rate is influenced by carbon particle size, which again depends on the bulk density of the granular activated carbon. during municipal wastewater treatment the residence time of the wastewater in the column containing granular activated carbon is affected by the bulk density (ρ) as shown by the equation: where, rs carbon usage rate (lb/ft2 min) ρ bulk density of the gac (lb/ft3) a adsorber cross-section area (lb/ft2) ν linear flow rate (gallons/min/ft) r residence time (min.), and t processing time (perrich, 1981). attrition or hardness measures the mechanical strength and determines zeolites and activated carbon (agricultural by-products ) ability to withstand normal handling operations. methods: the measurement of physical & mechanical properties for investigated sorbents a. measurement of physical properties of sorbents total surface area (m2/g/) the total surface area of the activated carbons was determined by the method pendyal et al. (1999) using micromeritics gemini 2375 surface area analyzer. the total surface area was measured by nitrogen adsorption at 77°k using 15 point bet. b. bulk density (g/m3) for agricultural by-products and activated carbon from them bulk density was measured using the method of ahmedna et al. (1997), which consisted of placing a known weight of activated carbon of 10-30 mesh size carbon in a 25 ml cylinder to a specified volume and tapping the cylinder for at least 1-2 min and measuring the volume of carbon. the bulk density was measured as: c. attrition/ hardness (%) the attrition of the samples was measured using wet attrition method described by toles et al. (2000). one gram of granular activated carbon of 10-30 mesh was added to 100 ml of acetate buffer (0.07 m sodium acetate and 0.03 m acetic acid, ph 4.8) in a 150 ml beaker. the solution was stirred at 500 rpm for 24 h using variomag electronic ruhrer multipoint hp 15 stirrer (daytona beach, fl) with a 1/2 inch stir bar for agitation. the solution was then filtered through 50 mesh screen and the retained carbon was thoroughly washed and dried at 90°c under vacuum for 4 h and weighed. the % attrition was measured as: ecn k m x log 1 loglog  t48.7 ra r s   (g) sampledry packed of volume (g) sampledry ofweight ) (g/m3density bulk  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 135 a/ adsorption of phenol and furfural from water solutions on sorbents. d. waste water treatment from organic impurities by activated carbon and agricultural by-products liquid chromatography is passed on helch / higher-effective liquid chromatography, detector waters 486, controller warers 600s, pump, waters 626, colon 250x4mm, si-100 c 18, p 150 bar, v 1ml/m, mobile phase acetonitrylwater (50:50), detector uv-254). uv spectrometry is passed on uvspecord spectrometr result and discussion adsorption by agricultural by products is preferred because of its have low sensitivity to flow fluctuations and exhibits greater flexibility. the advantages of agricultural by-products in comparison with other sorbents are their low cost, availability of extraction, operational flexibility and control and others. the effectiveness of agricultural by-products adsorption also is enhanced made activated carbon for the removal of organics by its large surface area resulting in higher separation efficiency by activated carbons. the pore structures of breaking apricot and peach seeds, also activated carbon from them were determined as follows: 1. for mesopores the diameter range 50…2nm areas and volumes determined by n2 adsorption technique at – 195oc, 2. for micropores the diameter range less 2nm areas and volumes were determined by co2 adsorption measurements at 0oc by the application of dubinin-radushkevich equation. 3. here was not determined any measurement for macroporosity, because of it`s known that for activated carbon surface areas consists in generally with microporose ( around 90% ) and 610% only mesopores with small amount of macroporose. activated carbon is also used in water treatment plants for the removal of odors and tastes. agricultural by products and activated carbons from them, on the other hand, can be made a renewable resource. preliminary studies on cost estimation have shown that agricultural by-products can be manufactured for as $ 0,03 per 1kg and agricultural byproduct based activated carbons – $0,14 per 1 kg. in the present study have describe the using of some fruits / apricot, peach / stones and based on them activated carbon. the technological scheme of use of such sorbents includes in itself set of difficulties. it have been solved a question with additional application of elastic bags with small holes impervious the filled sorbents. adsorption from wastewater with organic pollutants involves concentration of the solute on the surface. here, it has been had adsorption and desorbtion process together which will attain an equilibrium state. here is used the dubinin-radushkevich model for description of the adsorption data. it`s known that activated carbon surface area has a nonpolar nature, but during the treatment process with some of chemicals, as acids for instance, this sorbents surface took slightly polar character, which is in future can be responsible for hydrophobic influence of this surface. in this case, as row stone materials and more activated carbon can be used for organic molecule sorbents [ 5 ].. it should be noted also it`s known, that activated carbon obtained from fruit stones can favorably compared with other activated carbons used in industry with 100* (g) weight initial (g) weight final (g) weight initial (%)attrition  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 136 respect of they properties here, as it is possible is take place the physical adsorption which is held on the surface of stones or activated carbon, which have not active sites on the surface, by weak van der waals forces. the measurements for furfural in a water solution were carried out in concentration 0,01m. the furfural content in water before and after the sorption made a correction by liquid chromatography and uv spectrum also. the results are similarly, apricot stones based acidactivated carbon has higher sorptive activity. it also followed expect, as this sorbent has higher physical indicators. the sorption activity of mentioned sorbent as well as comparable with the commercial carbon carbonsorb-ab. from economical point of view the using of breaking stones can be effective. the using of stones also can be useful for future them treatment as burning agent in furnace. table 1. physical properties of experimental sorbents physical properties row peach stones breaking row apricot stones breaking peach stones based acid-activated carbon apricot stones based acidactivated carbon surface area ( m2/g) 220 250 620 710 bulk density (g/m3) 0.41 0.46 attrition (%) 11.3 8.60 elemental analysis results ( % ) c h o ash 48.0 6.0 46.0 50.0 5.0 45.0 74.0 2.5 18.0 5.5 85.0 2.0 8.0 5.0 table 2. the sorption of phenol from water (0,05m)* solution on sorbents / temperature 20oc, duration 4 hours / n= sorbent nd 20after sorption sorption g phenol./ g sorbent 1. row peach stones breaking 1.3321 0.0227 2. row apricot stones breaking / crushing/ 1.3322 0.0219 3. peach stones based acidactivated carbon 1.3315 0.0289 4. apricot stones based acidactivated carbon full sorption 0.0395 5. carbonsorb-ab full sorption 0.0390 *nd 20 for initial phenol solution is1,3324 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 137 table 3. the adsorption of furfural 100 ml 0,01m furfural on agricultural by-products and activated carbon /1g/ / temperature 20oc, duration 4 hours /* n= sorbent the adsorption mg/g the adsorption % 1. row peach stones breaking 0.12 12.8 2. row apricot stones breaking 0.14 15.0 3. peach stones based acidactivated carbon 0.29 25.0 4. apricot stones based acidactivated carbon 0.32 34.0 technological decision during the flood it`s known that during the flooding treatment and dispersal component is a trench system and it has inspection ports, the service provider may be able to pump water from the trenches to help the soil dry and aerate. however here it is necessary to take into account the following factors: 1 if the onsite wastewater treatment system has electrical components, an ability to restart the system will depend on the flood elevation. 2. if the flood water covered only the tanks and the components in the tanks, it`s possible to restart the system without further evaluation of the components. 3. if the flood water covered components located on the ground surface (air pumps, panels), the system should be inspected for determine whether it is safe to restart electrical service and use the system. alongside with this, a case of flooding naturally exists other difficulties for extraction sorption systems as usual cleaning river waters by cascade system. however, sorption materials can serve as a part of ground and can be removed after end of flooding. with the purpose of the technological decision of a problem it is offered to create special space along coast of the river on achievement of former levels of a surface of pollution to arrange dams with mobile walls filled by adsorbents. the protective system for a river stream will consist of a primary treatment system and also secondary treatment where protective walls filled with proposed sorbents are established. some walls with forward pools are desinged to create an opportunity for replacement of a sorbent. additionally, sorption materials can be placed on the floodplain, e.g., in ponds with mobile walls or elastic bags with small holes impervious filled by adsorbents, and removed after the flood. there would be a primary treatment system and a secondary treatment where the containers filled with activated carbon or agricultural by products from them are established. conclusion it has been found advantageous to go on with the researches in organic pollutants food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 138 sorption by agricultural by-products and based on them activated carbons. it has been offered the convenient method for successfully sorption phenols, furfural and probably other organic substances as btex / benzene, ethyl benzene, toluene and xylenes /, pesticides from water. the treated natural materials can be propose for using as sorbents and as promising materials for waste water treatment from small enterprises. acknowledgements this work was supported by a grant from the ministry of higher education and sciences republic of armenia. we gratefully acknowledge to d-r vartan martirosyan from institute of general & inorganic chemistry of nas armenia for his kind help to obtained activated carbon and comments. references: 1. g.h.torosyan, g.f.galstyan, a.r.alexanyan, v.a.davtyan, v.g.martirosyan, j.s.sarkizova, a.a. kazinyan. the use of agricultural by-products as a sorbent for removal organic compounds from water solutions, annals of high schools of the russian federation, a series chemistry and chemical technology, 2008, v.51, n.12, pp.99-101, 2. gurina n., bardakhivska k., enterosorbents as a remedy for organism detoxication, dovkillja ta zdorov, n 3, p. 64-66, 2007. 3. sych n.v. dogwood stone processing into high porous sorption materials. ecothechnology and resource saving, 2008, no.3, p.54-57 4. sych n.v., volynets v.p.trofimenko s.i., kovtun m.f.tsyba n.n., mironyuk t.i. obtaining and efficiency evaluation (estimation) of activated carbons from coffee residue. ecothechnology and resource saving, 2009, no.3, p.50-53./ . 5. ahmedna m., marshall w.e. and rao r.m. ( 2000), bioresource technology. 71 (2), 113123. microsoft word 3 journal nr 4 2010 din 8 noiembrie_104-107.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 108 studies on plant biomass in various stages of degradation in various stages oradation studies on plant biomass in various stages of aurora bunea1, rodica dinică 2*, bianca furdui2, mariana lupoae 3 1 institute for research and development of aquatic ecology, fisheries and aquaculture galati,.portului 54, galaţi, 800211 2dunărea de jos university of galati, faculty of sciences, chemistry department, domnească 111, galati, 800201, romania, rodinica@ugal.ro, bfurdui@ugal.ro 3ovidius university constanta, faculty of natural and agricultural sciences, street mamaia no. 124, 900527 ,constanţa, mariana_lupoaie@yahoo.com abstract: lignocellulosic residues from wood, grass, agricultural, forestry wastes and municipal solid wastes are particularly abundant in nature and have a potential for bioconversion. due to their abundance and renewability, there has been a great deal of interest in utilizing this biomass for the production and recovery of many value-added products. accumulation of lignocellulosic materials in large quantities in places where agricultural residues present a disposal problem results not only in deterioration of the environment but also in loss of potentially valuable material that can be used in paper manufacture, biomass fuel production, composting, human and animal feed among others. several novel markets for lignocellulosic residues have been identified recently. the use of fungi in low cost bioremediation projects might be attractive given their lignocelluloses hydrolysis enzyme machinery. this paper presents the evolution of the lignocelluloses degradation from vegetal residues (sawdust, rose leaves, rushes leaves and corn leaves) during five months, in aerobic and anaerobic conditions. the degradation process gives small molecules like polyphenols. we used folincicâlteu method for evaluation of lignine amount that is transformed in corresponding polyphenols. it was observed a growth of transformed amount along this period. therefore, we can conclude that the lignocellulosic degradation occurred. this increase in degradability could have important implications in the evaluation of the composting process. keywords: lignocelluloses, biodegradation, polyphenol. introduction lignin has great importance for plants. it has evolutionary significance as it has played a major role in the emergence of land plants from the aquatic plants. it forms a major integral cell wall component encrusting cellulose in all vascular plants including herbaceous species. it plays an important role in conferring rigidity, strength, resistance to pathogen ingress and water impermeability to the polysaccharideprotein matrix of the cell wall1,2. lignin, present in all vascular plants, represents on an average 25% of the terrestrial plant biomass. it is a complex phenolic heteropolymer resulting from the oxidative polymerization of three types of hydroxy cinnamyl alcohols termed monolignols3. the notion of lignin does not reflect a substance with well defined chemical structure similar to the other macromolecular natural products as cellulose, starch or protein, but rather refers to a group of chemically related polymer combinations between them. the elemental chemical composition of lignin has been determined yet by gay lussac and t. thenard4,5. the research on the breakdown products of lignin has established that its food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 109 basic structural unit is phenylpropane group, c6-c3. phenyl propane structural(fig.1) unit contains a hydroxyphenol group in para position and one or two methoxy groups in 3.5 positions of the core benzene. ho h3co c c c 1 23 4 5 6    figure1.phenyl propane structure biodegradation is the phenomenon which a substance is decomposed naturally by means of microorganisms. the biodegradation is the key feature that allows organic substances both natural cleansing process and biological treatment, and refers to the ability of organic matter to chemically change from the action of microorganisms6. the biochemical degradation of polymeric compounds in plant biomass composition is achieved through the action of many enzymes including amylase, cellulase, protease, keratinase, lipase, etc. these compounds with complex structure must be available for enzymes biodegradation to achieve a degree of conversion of biomass into biogas as high. the chemical changes produced in the biodegradation of lignin is complicated, first because the action of each individual organism, and secondly because most of the links in lignin are not hydrolysable. difficult biodegradability of lignin in comparison with the most other biological molecules depends on molecular dense texture, which prevents entry of large enzyme molecule and that this polymer is formed by polyaddition, not by polycondensation7,8. the most important biological degradation process is aerobic degradation of organic matter, degradation which uses the microorganism in a rich oxygen and nutrients environment. the removal of organic substances is performed through abiotic degradation under the action of photo-chemical processes in the atmosphere or under the processes of hydrolysis; biological degradation in the absence of oxygen is known as anaerobic degradation. polyphenols are organic compounds which contain two or more hydroxyl groups linked to an aromatic radical. in this category of substances are: pigments (coloring matter) and tannins. a lot of studies of natural phenolic compounds are of particular interest9. these substances, which for centuries are used as active principles of medicinal plant remedies, are used in technical textiles as natural dyes, the preparation of inks, leather tanning10. they are widespread in nature, especially flora. this group of natural compounds, plant pigments, tannins, lignin, requires a complex investigation as in terms of their physiological functions in plants, and for their structure, biosynthesis, various properties and usability. materials and methods the knowledge of quantitative and qualitative structure of the main polyphenolic compounds found in plant debris as a first stage requires developing working methodology for decomposing plant debris and concentrates the main polyphenolic fractions. the works began by collecting plant material and necessary preconditioning dilutions made for the separation of polyphenolic compounds. plant material biological materials so taken were leaves of corn, leaves of rushes, leaves of rose and sawdust. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 110 sample preparation after being harvest the leaves were dried and then weighed. the plants were put at degradation:10 g in 300 ml distilled water. it were placed two parallel samples of the same biological material: a sample container to degrade in aerobic conditions and the other in anaerobic conditions. the prepared samples were left at room temperature 20 ° c for 30 days to degrade before making the first analysis. analysis of total phenolic content the total polyphenol content (tpc) of the extracts was determined by spectrophotometry, using gallic acid as standard, according to the method described by the international organization for standardization (iso) 14502-19,10,11. briefly, 1.0 ml of the diluted sample extract was transferred in duplicate to separate tubes containing 5.0 ml of a 1/10 dilution of folin-ciocalteu’s reagent in water. then, 4.0 ml of a sodium carbonate solution (7.5% w/v) was added. the tubes were then allowed to stand at room temperature for 60 min before absorbance at 765 nm (in a uv– vis spectrophotometer) was measured against water. the tpc was expressed as gallic acid equivalents (gae) in g/100 g material. the concentration of polyphenols in samples was derived from a standard curve of gallic acid ranging from 10 to 50 µg/ml (pearson’s correlation coefficient: r2 0.9986). all measurements were carried out in for replicates. using the standard curve for gallic acid it were determined concentrations expressed in mg polyphenols and polyphenols calculation was done using the formula: 100x10x)l/mg(xfxc )g(m 10x)ml(vs spolyphenol% 3 3    vs-volume solution prepared from the product; mmass produced vegetable; f-dilution factor; csample concentration from standard curve results and discussion the temperature solutions were kept constant around 20°c unregistered sudden changes during experiments. a. ph determinations the degree of acidity or alkalinity is one of the most important indices of solutions that occur in nature chemical degradation processes, being given by the concentration of hydrogen ions. the ph solutions were measured by multiparameter consort c 862. ph variation in ae robic and anaerobic condditions 4 5 6 7 8 9 13ian 4-feb 25-f eb 18-m ar 12-ap r 4-m ay time [u ph ] corn leaves aerobic degradat ion corn leaves anaerobic degradat ion rushes leaves aerobic degradat ion rushes leaves anaerobic degradat ion rose leaves aerobic degradat ion rose leaves anaerobic degradat ion sawdust aerobic degradat ion sawdust anaerobic degradat ion figure 2. ph determination of studied plants from figure 2 is evident that all solutions obtained by anaerobic degradation have a ph acid lower than ph of the solutions obtained from aerobic degradation. it is also evident that the ph of the solutions increases steadily with the advancing of biodegradation process. b. total polyphenols contents this part of the study proposed to investigate the dynamics of the phenolic compounds in a sawdust, rose leaves, rushes leaves and corn leaves. the determination of polyphenol content was made by colorimetric method with folin-ciocâlteau reagent. in the following figure we represented the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 111 concentration of polyphenols for all the investigated samples during the five months in aerobic and anaerobic conditions. total polyphenol content in analysed samples 0 10 20 30 40 50 60 1 2 3 4 5 6 sample in time c on ce nt ra tio n corn leaves aerob corn leaves anaerob rushes leaves aerob" rushes leaves anaerob rose leaves aerob rose leaves anaerob saw dust aerob saw dust anaerob figure 3. tpc during five month legend: 1-january, 13th, 2-february, 4th, 3-february, 25th, 4-march, 18th, 5-april,12th 6-may, 4th all the experiments shows a steady growth in concentrations of polyphenols degradation both aerobic and anaerobic degradation of analysed plants. the concentration of polyphenols in solutions resulting from aerobic degradation is higher than the concentrations of solutions resulting from anaerobic degradation. conclusions lignocellulosic biomass has the potential to become a key element in future growth of the quantity of bioenergy generated. its universal availability in large quantities and that is little used at present are reasons for lignocellulosic biomass is regarded as one of the most promising resources for future production of bioenergy. our experiments have shown that the ph of the solutions degraded anaerobically, is under acid range varying with the stage of decomposition and degradation from 4.452 to 6.692 uph. also, the ph of aerobically degraded solutions is higher than the solutions degraded anaerobically. the total polyphenol content varies according to species and depending on type of biodegradation : aerobic or anaerobic. the solutions maintained in anaerobic conditions have a lower content of polyphenols than those under aerobic process. references 1. c. sánchez, “lignocellulosic residues: biodegradation and bioconversion by fungi”, biotechnology advances, no 27 , 2009, 185194 2. r. p. de vries, jaap visser, “aspergillus enzymes involved in degradation of plant cell wall polysaccharides”, microbiology and molecular biology reviews, no 4, vol 65, 2001, 497-552; 3. a. demirbas, “green energy and technology – biofuels”, ed. springer, verlag london, 2009; 4. d. cullen, kersten p. j., „enzymology and molecular biology of lignin degradation” , springer-verlag: 13: 1996, 249-273; 5. y. godliving, s. mtui „recent advances in pretreatment of lignocellulosic wastes and production of value added products”, african journal of biotechnology vol. 8 (8), 2009, 13981415; 6. t. javora , w. buchberger, i. tanzcos, „determination of lignin degradation compounds in different wood digestion solutions by capillary electrophoresis” anall.chim.acta, 484, 181-187, 2003; 7. e. moira, k. henderson, „fungal metabolism of certain aromatic compounds related to lignin”j . gen. microbiol26, (1961), 155-165; 8. m. ohkuma, y. maeda, t. johjima, k. toshiaki, „lignin degradation and roles of white rot fungi: study on an efficient symbiotic system in fungus-growing termites and its application to bioremediation”: focused on ecomolecular science research, riken review no. 4, ; 2001; 9. j. sci. food. agric., 80: 1925-1941, iso 145021: 2005. determination of substances characteristic of green and blck tea. part 1: content of total polyphenols in tea. colorimetric method using folin-ciocalteu reagent, 2000; 10. v. singleton, r. orthofer, lamuelaravento´s, r. m. analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. method enzymol., 299, 1999, 152–178; 11. t. florea, b. furdui, r.m. dinică, r. creţu, „chimie organică, sinteză şi analiză funcţională”; ed. academica, 2009 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 5 lifetime of freezing activated water igor winkler1 , antonia andrusyak1 yuriy fedkovych national university of chernivtsi, kotsiybynsky st., 2, chernivtsi 58012, ukraine. igorw@ukrpost.ua abstract. special properties of the “activated” water have been reported in numerous papers. water activation can be achieved either through applying electromagnetic field, through consequential freezing-melting of the water or using some other methods. freezing-melting activation is the simplest yet still effective method of activation. activated water ensures better growing of some plants and can be helpful in order to prevent negative effects of unsatisfactory environment conditions. on the other hand, mention should be made that the activation effect is limited by some time frames, which depend on many factors. activated water can lose its special properties during quite short period of time and then it turns into regular water, which does not reveal any special characteristics. this work reports the results of investigation of the activated water lifetime, which has been determined through measurements of the activated water electroconductivity in comparison to conductivity of the regular one. this method assumes that some nano-sized clusters can keep the ice crystal structure in the freshly melted state for some period of time, which should influence electro conductivity of the melted water. the approximated lifetime of the activated water was found equal to about 3,5 hours at the temperature of 180c, 2 hours – at 23 0c and immeasurably short at 28 0c. this result proves that any special properties of the activated water can be expected only within rather very short time after melting. then the activated water loses its special features and turns into regular one. keywords: ice nano-associates; melted water; electro conductivity measurements introduction there are many publications reporting specific properties of water after so-called “activation” [1-3]. this water exhibits anomalous electro conductivity, different viscosity value and other specific differences. there is reference on the weak but still detectable sound generation, which occurs in the process of water activation [4]. activated water also exhibits special biological activity. there are references reporting its stimulating effect on the growth of some agricultural cultures, antimicrobial activity and others [5-7]. specific properties and action of the activated water is caused by its specific composition and structure. it is considered that this water can have nano-sized structures, which bond together some amount of the water molecules and form relatively strictly associated structural components. these components can save the structure of solid ice and represent the elements of the ice preserved in liquid water. there are many available methods of water activation: freezing-melting, electrochemical activation, activation by ultrasound and others [6, 8, 9]. the freezing-melting method is the simplest one but the most effective is the water activation method. the application of the melted water ensured better growth of some plants and faster rehabilitation of the injured animal tissues. there are also some reports on the wholesome effect of the melted water in the human organism [6, 7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 6 however, there is also a question of duration of the above mentioned effects. the ice-like complexes existing in water can not stay forever and should have some life time. since they cause specific properties of the activated water, destruction of the complexes should result in transformation of the activated water into a normal liquid and gradual disappearance of its specific properties. the aim of this work was to investigate the life time of ice-like complexes formed in melted water and to estimate possible duration of its specific properties [10]. the presence of these complexes should influence the water electroconductivity. in other words, detection of the anomalous electroconductivity would indicate presence of the nanocomplexes and gradual return back to the normal conductivity would indicate decomposition of the complexes and transformation of the activated water into normal one. the complexes should change electroconductivity of the water since the well known grotgus mechanism of electrons or ions movement in water assumes some gain in the distance covered by the travelling charged particle because of its “jumps” between molecules (relayrace ions movement). the larger the size of a particle is or the complex charge is jumping between, the higher the distance gain is and, consequently t the changes in electroconductivity. thus, the conductivity of melted water should be higher than the conductivity of the regular one. therefore, by studying temporal changes in water electroconductivity we can determine the life time of ice-like complexes existing in the freezing-meeting activated water. experimental results and discussion a regular alternating current electroconductivity measurer r-5066 together with a temperature-controlled cell has been used for the conductivity measurements. the device measured the electric resistance of water, which has been transformed into specific conductivity using the formula r 1  , (1) where r is the electric resistance (ohm) and  is the specific electroconductivity (si). table 1 measured values of electric resistance and calculation of electroconductivity for melted and regular water samples sample electric resistance (melted water), ohm electroconductivit y (melted water), si electric resistance (regular water), ohm electroconductivity (regular water), si 1 4.35.104 2.30.10-5 1.34.105 7.46.10-6 2 4.31.104 2.32.10-5 1.32.105 7.58.10-6 3 4.28.104 2.34.10-5 1.28.105 7.81.10-6 4 4.43.104 2.26.10-5 1.23.105 8.13.10-6 5 4.21.104 2.38.10-5 1.25.105 8.00.10-6 6 1.49.104 6.71.10-5 1.43.105 6.99.10-6 7 1.87.104 5.35.10-5 1.37.105 7.30.10-6 8 1.81.104 5.52.10-5 1.33.105 7.52.10-6 9 1.79.104 5.59.10-5 1.52.105 6.58.10-6 10 1.95.104 5.13.10-5 1.72.105 5.81.10-6 11 1.89.104 5.29.10-5 1.68.105 5.95.10-6 12 2.65.104 3.77.10-5 1.68.105 5.95.10-6 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 7 13 3.66.104 2.73.10-5 1.49.105 6.71.10-6 14 3.48.104 2.87.10-5 1.51.105 6.62.10-6 15 4.44.104 2.25.10-5 1.54.105 6.49.10-6 averaged 3.11.104 3.79.10-5 1.45.105 6.99.10-6 average deviation 1.11.104 1.45.10-5 1.36.104 6.45.10-7 freshly distilled water and the same cell were used throughout all experiments. some part of the water has been frozen and another part left liquid. then some pieces of ice were spilled off, melted, placed into a thermo controlled cell until the temperature stabilized and then the electroconductivity values have been measured. similar procedure has been applied to determine the electroconductivity of the regular water sample. all the experiments have been carried out at 18 0c. table 1 shows the results of a series of electroconductivity measurements. the data in table 1 clearly outlines that electroconductivity of the melted water was about 5 times higher as compared to the value of the regular one. it is needful to emphasize that there are several processes, which contribute to electroconductivity of water. the first one is the dissociation of water:   ohohoh 322 , (2) which takes place even in distilled water and results in the formation of some amount of hydroxonium and hydroxide ions. they can travel between the water molecules according to the relay-race mechanism and contribute to the total conductivity. the second one is the presence of some dissolved admixtures, for example carbon dioxide, which can form dissociable molecules. this compound can be absorbed from the air and also contribute to total conductivity: 2 2 2 3 32 2 3 (3)h o co h hco co       the dissolution of air oxygen also can influence total electroconductivity. figure 1. temporal changes in electric resistance of the melted water as against the regular water resistance at the temperature 18 0c. electric resistance of the melted water 50,00 55,00 60,00 65,00 70,00 75,00 80,00 85,00 90,00 95,00 100,00 0 50 100 150 200 250 300 time , min r es is ta n ce a s % r el at ed to th e re si st an ce o f th e re gu la r w at er food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 8 the concentration of the above mentioned compounds in freshly distilled water is very low, almost negligible but it gradually increases as water takes contact with air. different water examples had different contacting time and this is the reason for some scattering in the values of electroconductivity from table 1. on the other hand, both examples, frozen-melted and regular one had similar contacting time for every experimental series, which ensured similar concentration of the dissolved compounds. therefore, only various conditions for the relay-race charges transfer can explain the difference in frozen-melted and regular electroconductivity values. figures 1 and 2 prove that the resistance of melted water is increasing with time and becomes almost equal to the resistance of the regular one in about 3.5 hours at 18 0c and in about 1.5-2 hours at 23 0c. the electric resistance of melted water has become practically equal to the resistance of the regular one during the time of thermostabilization of the sample at 28 0c (~ 15-20 min). that is why data for 28 0c are not graphically presented here. the above data can be another confirmation for the assumption of influence of ice-like nano-clusters presence in the melted water on its resistance (or electroconductivity). the higher the temperature is, the faster these clusters are being decomposed because of the brownian movement, which brings the resistance back to the level of regular water. taking into account the very fast decomposition of ice-like clusters at 28 0c we can suppose that at higher temperatures such structures do not survive at all and decompose just after the melting process. electric resistance of the melted water 38,00 48,00 58,00 68,00 78,00 88,00 98,00 0 50 100 150 200 time, min r es is ta nc e as % r el at ed to th e re si st an ce o f th e re g ul ar w at er figure 2. the same as in figure 1 but at the temperature 23 0c food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 9 conclusions the electroconductivity of melted water samples significantly differs from the values of those ones which have not been frozen and melted. taking into account the grotgus model of relay-race charges movement in water these results can be explained by remainders of solid ice-like associated with structural clusters, which can be found even in liquid water some time after its melting. gradual decrease in electroconductivity values of the melted and regular water in the course of time occurred after melting. the higher the water temperature is, the faster these values equalize. this fact indicates the role of the brownian thermal movement of molecules, which destabilizes and destroys the ice-like clusters. therefore, there are quite distinct lifetime frames for the activated water, which can be used to recover biological objects after ecological stresses. activated water loses almost all special ice-like clusters within 3.5 hours at the temperature of 18 0c and within 1.5-2 hours at 23 0c. higher temperature leads to a very fast deactivation of the melted water, which transforms into a regular liquid. references 1. n. l. lavrik. specific physico-chemical properties of the melted water. (2008). chemistry for sustainable development, 16, 331-339. 2. g. n. zatsepina. physical properties and structure of water. (1987). moscow state univ., moscow. 170 p. 3. a. n. smirnov, v. b. lapshin, a. v. balyshev, i. m. lebedev, a. v. syroeshkin. (2004). supermolecular complexes in water. researched in russia. http://zhurnal.ape.relarn.ru/articles/2004/038.p df 4. d. m. kuznetsov, a. n. smirnov, a. v. syroeshkin. acoustic emission at the phase transformations in water. (2008). russian j. chem. 52. 114-121. 5. v. i. prilutsky, v. m. bahir. electrochemically activated water: anomalous properties and mechanism of bioactivity. (1997). medical tech. institute. moscow. 232 p. 6. method and device for producing activated liquids and methods of use thereof. usa patent 6022479. published 8 feb 2000. 7. i. v. smirnov, v. i. vysotskii, a. a. kornilova. introduction to the biophysics of activated water. (2005). universal publishers. usa. 164 p. 8. yu. m. barabash. dynamics of water systems under influence of weak electromagnetic irradiation. (2000). nauka. moscow. 285 p. 9. v. i. klassen. physical activation of water and its use in industry. (1985). chem. industry. 5. 85-92. 10. o. f. mohammed, d. pines, j. dreyer, e. pines, e. t. j. nibbering. sequential proton transfer through water bridges in acidbase reactions. (2005). science. 310. 83-86. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 78 analysis of toxic and trace metal contaminants in bottled water by using atomic absorption spectrometry *gholamreza jahed khaniki 1, mansoor ghaderpoori1, mohammad hadi dehghani 1, shahrokh nazmara 1 1department of environmental health engineering , school of public health, tehran university of medical sciences, tehran, poorsina street, tehran, iran, p.o.box:14155-6446, e-mail: ghjahed@tums.ac.ir *corresponding author received 12 april 2011, accepted 10 june 2011 abstract: heavy metals are toxic elements for human and they may enter the human body through polluted water. consumption of bottled water is a proper choice when no safe water is available or water treatment is difficult, but the safety of bottled water is important and it is necessary to monitor toxic and trace metal contaminants. this study was conducted for the measurement and determination concentrations of toxic metals such as cadmium (cd), chromium (cr) and lead (pb) in bottled water. a descriptiveanalytical and cross-sectional study was conducted for the determination of cd, cr and pb in thirteen brands of bottled water available at the tehran stores. after sampling, the toxic trace elements were extracted and then their contents (or concentrations) were determined using in bottled water samples by using atomic absorption spectrometry. the results showed that mean concentrations of cd, cr and pb in examined samples of bottled water were 1.5± 0.34, 1.06± 0.72 and 3.18±0.44 µg/l, respectively. all of the examined samples had these trace elements in concentration below who guidelines, epa standards and iranian drinking water standards. it can be may be assumed that there is no basic public health problem by consumption of examined bottled water in regarding these toxic metals. © 2011 university publishing house of suceava. all rights reserved keywords: heavy metals, cadmium, chromium, lead, bottled water, drinking water standards 1. introduction. consumption of bottled water is a proper choice when no safe water is available or water treatment is difficult. use of bottled water has considerably increased around the world in recent years [1]. nowadays, bottled water is being used in many places due to its availability, low cost, better quality and taste. water is a vital material for human being and it is one of the main sources of trace elements intake in the body. thus, its quality considerably affects our health. trace elements such as cobalt, copper, iron, manganese, molybdenum, selenium and zinc can be classified as essential elements for human life while some other trace elements (silver, mercury, aluminum, arsenic, cadmium, chromium, lead and nickel) are potentially toxic [2,3,4]. many inorganic elements have a dual role in body physiologies and they are essential in low concentrations but toxic in high amounts [5]. epidemiological studies in recent years have indicated that there are relations between content of trace elements in drinking water with some kidney and heart disorders and also different types of cancer [6]. cadmium life time in bones is thirty years and this element is a carcinogen. also, lead is a neurotoxin and it is responsible for many toxic effects in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 79 human body [7]. the polluting of bottled water can arise from filling by contaminated water or leakage raw materials through packaging to water in to bottles. according to many studies, toxic metals may release from pet (poly ethylene tere-phthalate) into the water bottle [6,7,8].usually, water bottles are kept at room temperature and a large number of consumers may keep many water bottles for necessary cases that it may cause the increased risk of leakage of metals from bottle wall into stored water [7]. the aim of this study was to analyse and evaluate content of toxic trace metals including cadmium (cd), chromium (cr) and lead (pb) in domestic brands of bottled water available at the tehran market by using atomic absorption spectrometry and to compare our results with international guidelines and iranian standards. 2. materials and methods 2.1. sample collection 26 samples of 1.5l bottled water of thirteen different commercial domestic brands were randomly collected from shops and supermarkets of tehran city, iran. two samples with different production dates of each brand were collected. the bottles were sent to laboratory and they were preserved at 4°c until analyses (not more than a week). the commercial brands of bottled water were damavand, kooh dasht, damash, zam zam, nestle, polur, kooh rang, hobab, bidestan, dasani, siva, vata and lubon. 2.2. metals analysis using standard methods [9], concentration of cadmium, chromium and lead was measured by atomic absorption spectrometer varian techtron (model 1200). the measurements were done at a specific wave length for each element (cadmium at 288.8, chromium at 357.9 and lead at 217 nm) and the instrument was calibrated by distilled water and also by the standard solutions. to achieve a suitable detection limit for desired elements, all standard and sample solutions were concentrated twenty times by means of a hot plate to evaporat. the mean absorbance produced by the standards (corrected for the standard blank) was plotted vs the concentration of the analyte in the sample to produce an external calibration curve. the concentration of the analyte in the sample was calculated from the following equation: [m]= (c × v)/sv; where [m] is the concentration (µg/l) of in original sample, c is the concentration of metal in the analytical sample as calculated from standard curve in units of µg/l, v is the volume of the analytical sample in units of ml and sv is the volume of the sample employed to concentrate in units of ml. detection limit was about 0.1µg/l after concentration. all the recoveries of the metals studied were over 95%. 2.3. statistical analysis for the analyzing data, spss 11.5 and descriptive statistics (mean standard deviation) and analytical statistics (anova and tukey with post hoc method) were used. a probability level of p<0.05 was considered statistically significant. 3. results and discussion the results show that the mean concentration of cadmium, chromium and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 80 lead in bottled water in tehran was 1.5± 0.34, 1.06± 0.72 and 3.18±0.44µg/l respectively (table 1). anova test showed that mean concentrations of these trace elements is significantly different in all the samples (p<0.5).also, tukey test with post hoc method showed that there is a significant difference in concentrations of cadmium, chromium and lead in some samples (p< 0.5). table 1. standard deviation and mean for trace metal concentrations in bottled water samples brand cadmium ∗ mean(±sd) chromium∗ mean(±sd) lead∗ mean(±sd) damavand nd 0.88 ±0.02 5.1 ±0.64 kooh dasht 1.7 ±0.24 0.5 ±0.11 1.85 ±0.61 damash 0.43 ±0.15 1.32 ±0.6 1.85 ±0.62 zam zam 1.72 ±0.44 2.22 ±0.67 3.7 ±1.28 nestle 1.7 ±0.34 1.4 ±0.56 nd polur 1.72 ±0.84 1.8 ±0.56 7.55 ±0.78 kooh rang nd 0.7 ±0.28 2.77 ±0.92 hobab 1.72 ±0.44 0.9 ±0.18 5.55 ±3.23 bidestan 8.05 ±0.78 2.1 ±0.14 6.8 ±0.85 dasani 0.85 ±0.2 0.92 ±0.6 1.85 ±0.61 siva 1.4 ±0.78 0.5 ±0.11 0.49 ±0.01 vata nd 0.15 ±0.05 0.4 ±0.56 lubon 0.45 ±0.59 0.38 ±0.16 0.45 ±0.28 total 1.55 ±0.34 1.06 ±0.72 3.18 ±0.44 iranian standards 10 50 10 who guideline 3 50 10 epa standard 5 100 15 ∗ means are given in µg/l nd: not detectable concentrations of cadmium, chromium and lead in different brands are compared in fig.1. fig 1. comparisons of toxic metals analysis in different brands of bottled water. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 81 in the examined bottles, mean concentration of cadmium in the brand of bidestan (8.05µg/l) is significantly different from the other brands (p< 0.5) and its concentration is lower than iranian drinking water standard (<10 µg/l) [10]. this is due to different reasons such as type of source of bottled water or long period of water storage in bottles. some studies reported that there is probability of increase of cadmium contamination due to the long period of water storage in bottles. they referred that cadmium concentration in some brands was above who guidelines and epa drinking water standards (3 and 5 µg/l, respectively) [11,12]. comparisons of mean concentrations of toxic metals in bottled water obtained from tehran market and other countries is shown in table 2. in present study, cadmium concentration in examined samples is higher than those of other countries. maximum concentration of chromium was found in zam zam brand (2.22µg/l). the mean concentration of chromium in the brands of zam zam and bidestan is significantly different from other brands. this can be caused by different types of source of bottled water but the concentration of chromium is lower than iranian drinking water standards (50 µg/l) [10]. chromium concentration in these samples is lower than in the samples from greece and kuwait. also, these concentrations are below who guidelines and epa drinking water standards. table 2. comparisons of mean concentrations of toxic metals in bottled water obtained from tehran market and other countries country name number of brands cadmium∗ chromium∗ lead∗ reference turkey 15 < 0.001 0.0056 < 0.001 [13] kuwait 6 0.013 2.95 0.04 [14] greece 4 2.6 0.02 [4] india 6 < 0.05 < 0.05 [15] croatia 18 0.016 0.31 0.091 [2] greece 16 0.291 0.101 [5] canada 34 0.2 5.3 [7] iran 13 1.52 1.06 3.18 [present study] ∗ means are given in µg/l concentration of lead in brand polur is significantly different from other brands. this can be caused by different reasons such as type of source of bottled water or long period of water storage in bottles. according to the studies of pip [7], ehp [6] and shotyk and krachler [8,16], there is a probability of dissolution of metals from the bottles during long storage. in present research, concentration of lead was lower than who guidelines and epa and iranian drinking water standards (lower than 10 µg/l) [10]. also, the highest concentrations of cadmium, chromium and lead is determined in brands of bidestan, zam zam and polur, respectively (fig.1). by comparison of the mean concentration of heavy metals in samples, the maximum concentration in these metals was attributed to lead (3.18±0.44 µg/l) and it is similar to pip [7] and [17]. pip [7] reported that 40 brands of imported and local bottled drinking water available in manitoba, canada was studied and it was reported that concentration of lead was more than 5 µg/l and from examined bottle water samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 82 typically, all metals are somewhat soluble in water and excess concentrations of any metals can affect human health. toxic metals can accumulate in body and can be brought into body by water, air and food sources and even low concentration of these metals can be important because of accumulation. since bottled water is usually stored in polyethylene terephthalate (pet), plastic bottles or glass bottles, the releasing of metals from bottle to inside water can occur. studies in different countries had shown this problem [8,16,18,19]. although the low concentrations of these metals are measured in these bottles which are lower than drinking water guidelines and standards but, due to their cumulative and toxic effects, they are important in point view of health. thus, they are in concern for public consumptions. therefore, monitoring of these waters usually is necessary to cause the probable concerns. 4. conclusion as there is growing use of bottled water, therefore monitoring of its quality is necessary. the study shows that most distributed bottled water brands in tehran have a good quality considering toxic trace elements and meet drinking water guidelines and standards. but more studies are necessary for determination other toxic metals such as aluminum, mercury, arsenic. 5. references 1. salvato, j., magardy, n., agardy, f., environmental engineering sanitation. john wiley and sons publication. 5 ed, pp. 469-47, 2005. 2.fiket, z., roje, v., mikax, n., kniewalk, g., determination of arsenic and other trace elements in bottled waters by high resolution inductively coupled plasma mass spectrometry. ccacaa, 80: 91-10, 2007. 3. misund, a., frengstad, b., siewers, u., reimann, c., variation of 66 elements in european bottled mineral waters. the science of the total environment, 243-244:21-41, 1999. 4. soupioni, m.j., symmeopoulos, b.d, papaefthymious, h.v., determination of trace elements in bottled water in greece by instrumental and radiochemical neutron activation analyses. journal of radio analytical and nuclear chemistry, 268: 441-444, 2006. 5. who., guideline values for drinking water quality. 2ed, geneva, 1993. 6.ehp., forum: is bottled water better? environmental health perspectives, 103: 322-32, 1995. 7. pip, e., survey of bottled drinking water available in manitoba, canada. environmental health perspective, 108: 863-866, 2000. 8. shotyk, w., krachler, m., contamination of bottled waters with antimony leaching from polyethylene terephthalate(pet) increases upon storage. environmental sciences and technology, 41: 1560-1563, 2007. 9. eaton, a.d., clesceri, l.s., rice, e.w., standard methods for the examination of water and wastewater, american water works association (awwa) washington d. c, 21ed, pp. 5-50, 2005. 10. isiri., physical and chemical properties of drinking water: institute of standards and industrial research of iran (isiri). no. 1053, 5ed, 1997. 11.epa., drinking water standards; office of drinking water, us environmental protection agency washington dc, 2003. 12. who., guideline for drinking water quality; world health organization: geneva, 2004. 13. baba, a., erees, f.s., cam, h.s., an assessment of the quality of various bottled mineral water marketed in turkey. environmental monitoring & assessment, 139: 277-285, 2008. 14. khalefa, a., mohammed, a., hamad, a., comparative study of potable and mineral waters available in the state of kuwait. desalination, 123: 253-264, 1999. 15. babiji, p., shashikiran, n., reddy, s.v., comparative evaluation of trace elements and residual bacterial content of different brands of bottled waters. journal of indian society of pedodontics and preventive dentistry, 22: 201-204, 2004. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 83 16. shotyk, w., krachler, m., lead in bottled water: contamination from glass and comparison with pristine groundwater. environmental science and technology, 41: 3508-351, 2007. 17. dabeka, r.w., conacher, h.b.s., lawrence, j.f., newsome, w.h., mckenzie, a., wanger, h.p., chadha, r.k.h., pepper, k., survey of bottled drinking waters sold in canada for chlorate, bromide, bromate, lead, cadmium, and other trace elements. food addatives & contaminants, 19: 21-72, 2002. 18. shotyk, w., krachler, m., chen, b., contamination of canadian and european bottled waters with antimony from pet containers. journal of environmental monitoring, 8: 288-292, 2006. 19. westerhoff, p.m., prapaipong, p., shock, e., hillaireau, a., antimony leaching polyethylene terephthalate(pet) plastic used for bottled drinking water. water research, 42: 551556, 2008. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 5 development of lateral flow immunoassay for rapid detection of oxytetracycline in honey samples isabel guillén guillén 1 , ángel martínez sanmartin 1 , josé antonio gabaldón hernández 1,2 , estrella núñez-delicado 2 , rosa puchades pla, 3 ángel maquieira catalá 3 1departamento de seguridad alimentaria. centro tecnológico nacional de la conserva y alimentación, c/ concordia s/n, 30500 molina de segura. murcia. e-mail: gabaldon@ctnc.es, jagabaldon@pdi.ucam.edu, guillenguillen@hotmail.com 2dpto. de ciencia y tecnología de alimentos. universidad católica san antonio de murcia. avenida de los jerónimos s/n. 30107 guadalupe. murcia. españa. 3instituto universitario de reconocimiento molecular y desarrollo tecnológico. universidad politécnica de valencia. camino de vera s/n 46071. valencia. abstract: a lateral flow immunoassay (lfia) was developed in the competitive reaction format and applied to test residues of the antimicrobial oxyteracycline (otc) in honey samples. to prepare the assay test, a nitrocellulose membrane was stripped with hapten otc3 conjugate to ovoalbumin egg protein (ova-otc3) and goat anti-rabbit antisera (gar) as capture and control reagents, respectively. polyclonal antisera against oxyteracycline was conjugated to colloidal gold nanoparticles and used as the detection reagent. the visual detection limit (cut-off value) of the oxyteracycline lfia was 20 ng/g, and qualitative results were obtained within 10 min without expensive equipment. the assay was evaluated with otc spiked honey samples from different geographical origin (n = 25). the results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (lc-ms), indicating that the lfia test might be used as a semiquantitative method for the determination of oxyteracycline. the system was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. keywords: immunoassay, lfia, oxytetracycline, honey introduction since their discovery in the mid-1900s, tetracyclines (tcs) have been widely used as therapeutic agents in human and veterinary medicine. these drugs also have particular applications as growth promoters in animal husbandry because of their broad-spectrum activity against a variety of gram-positive and gram-negative microorganisms –inhibiting the protein synthesis by binding to the small ribosomal subunit at the a site which binds with the aminoacyl trna-, and low cost [1,2]. honeybees are subject to a number of diseases that affect their brood, with two of the most serious being the larval bacterial diseases american foulbrood (caused by spore-forming paenibacillus larvae) or european foulbrood (caused by melissococcus pluton) [3,4]; highly contagious and destructive diseases that affects honeybees [5,6]. in beekeeping, drugs known to be effective against these diseases are oxytetracycline (otc) and sulfathiazole, in use for a long time. the use of drug residues to treat bacterial diseases of honeybees cannot be ignored because their presence in the environment is a big concern in many countries. interference with foodstuffs may lead to the production of food, which is harmful, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 6 having implications in nutrition and safety including the possibility of allergic reactions or resistance. in fact, the widespread overuse of antibiotics in raising animals for food has helped create "superbugs", bacteria resistant to these drugs. these drugs certainly kill bacteria, but in doing so, they drive the process which selects in favour of resistant strains [7]. no maximum residue levels (mrls) for tetracyclines in honey were set in the european union, which means that if present, must be below the limit of quantitation (loq) of the analytical method used [8]. since loqs differ between laboratories, some countries within the european union have established action limits or tolerated levels, ranging from 20 to 50 ng/g, referring to the sum of all substances within the tc group or 10 ng/g for one, like otc. several methods have been reported for the determination of tetracyclines using a variety of techniques, including chromatography or microbiological growth inhibition [9]. the microbial inhibition tests are cheap and easy to perform, but require 2–3 days for microbe growth, sometimes being non-specific and not sensitive enough for residue monitoring. among the chromatographic techniques, high performance liquid chromatography (hplc) is the most used for the determination of multiple tc residues in honey including otc [10–13]. it is the reference method, being very suitable for confirmation but not for screening purposes. a rapid, sensitive and specific assay would be interesting to detect positive tc samples in routine analysis, which then can be confirmed by reference methods. immunoassays can dramatically reduce the number of analyses required to characterize food samples for drug residue contamination. therefore, during the past decades, a variety of elisas have been developed, each for multi-tcs screening in foods [14,15] or specific for an individual tc [16,17]. nowadays, there is a need for more cost effective, field portable assay systems that can be conducted by users that are as close to the source of contamination as possible. most of them are basically designed as visual test that require only low-cost instrumentation and offer an advantage of speed, essential to accept or reject goods on-site [18,19]. in the present work, we describe the development and evaluation of a prototype rapid lateral-flow immunochromatographic assay (lfia) test kit for on-site testing for otc residues in honey samples, using colloidal gold nanoparticles as the detection reagent. materials and methods chemicals tetracycline hydrate (tc), 7chlortetracycline monohydrochloride (ctc), oxytetracycline dehydrate (otc), anhydrotetracycline hydrochloride (atc), demeclocycline hydrochloride (dmc), doxycycline hyclate (dxc), daunorubicin hydrochloride (dr), methacycline hydrochloride (mc), minocycline hydrochloride (mnc) and rolitetracycline (rtc) were purchased from fluka–sigma– aldrich química (madrid, spain). analytical grade solvents were provided by scharlab (barcelona, spain). goat antirabbit immunoglobulins (gar) were purchased from sigma (madrid, spain). all other reagents used were analytical grade. nitrocellulose membrane cnpcss12 was from advanced microdevices pvt. ltd. (ambala cantt, india). the sample pad, the conjugate release pad and the absorbent pad were from schleider & schuell gmbh (dassel, germany). plastic backing was from estok plastics (nj, usa) and the plastic housing was supplied by acon biotech (hangzhou, china). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 7 apparatus isoflow reagent dispenser was from imagene technology (hanover, germany) and cm4000 guillotine cutting module used to prepare test strips was from biodot inc. (irvine, ca, usa). the centrifuge (hereaus multifuge 3 s-r) was from vwr international eurolab s.l. (madrid, spain). particle size was carried out by transmission electron microscope s3700n, from hitachi high-technologies europe gmbh (krefeld, germany). the optical density was established spectrophotometrically in a shimadzu model uv-1063 spectrophotometer from izasa s.a. (barcelona, spain). preparation of colloidal gold nanometer colloidal gold was prepared according to the procedure described by frens [20]. basically, l00 ml of 0.01% chlorauric acid solution (in milli-q purified water) was heated to boiling, and then 2.0 ml solution of 1% trisodium citrate was added under constant stirring. once the colour of the solution changed from blue to dark red within 2 min, approximately, it was boiled for another 15 min. the obtained gold colloidal suspension supplemented with 0.05% (m/v) of sodium azide was stored at 4 ºc in a dark-coloured bottle until use. with the scan between 500 and 600 nm, there is only one maximum absorbent wavelength: 525 nm. the particles obtained were checked by transmission electron microscope, showing that the average diameter of these particles of uniform size was 40 nm. gold labelling of anti-otc sera before conjugation with the colloidal gold, the optimal ph value and antibody concentration were determined to obtain the best sensitivity by checkboard titration. by gentle stirring, 10 ml of colloidal gold solution was adjusted to ph 7.0 with 0.1 m k2co3 or 0.1 m hcl, and then 50 µg of protein a purified polyclonal antisera otc3-i was added by drop wise. after incubation at room temperature for 15 min, 3 ml of 5% bsa solution was added, and stirring was continued for another 15 min. the mixture was centrifuged at 12000 rpm for 15 min, and the precipitate of the goldlabeled antibodies was resuspended in 5 ml of dilution buffer (0.01 m pbs, containing 1% sucrose and 0.5% triton100, ph 7.2) and stored at 4 °c. gold labelled otc3-i (detection reagent) was sprayed onto a conjugate pad (0.5 µl per cm2, glass fiber membrane) and then dried for 1 h at 37 °c. immobilization of capture reagents isoflow reagent dispenser was used to put two lines on the nitrocellulose membrane strips (25 mm width 88 m large). the dispensed volumes were both 1 µl/cm. after dispensation, the nitrocellulose membrane was dried for 12 h at 37 °c and stored under dry conditions at room temperature until use. the lfia device for the detection of otc was a single-antigen direct immunoassay. the device consists of a plastic support to which a nitrocellulose membrane (thickness, 15 ± 1 µm) is mounted. ova-otc3 was striped in the “test line” position (0.4 mg/ml), while the goat anti rabbit igg was striped in the “control line” position (1 mg/ml). gold particles (40 nm) individually conjugated to otc3-i was dispensed onto a conjugate pad. the conjugate pad was then affixed to the test strip by overlapping the nitrocellulose membrane at its proximal end; the addition of a sample pad completed the assembly by overlapping onto the conjugate pad (fig. 1). devices that could be used to analyze 100 µl of honey solution were produced. test procedure the assay was based on the competitive reaction theory (see figure 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 8 figure 1. schematic diagram of lateral-flow immunochromatographic assay when honey diluted sample (100 µl) is placed in the “sample port” at “s” on the device, it rapidly went through to the conjugate pad, and the gold-conjugated otc3-i was then solubilised. after that, the gold-conjugated otc3-i migrates down to the nitrocellulose membrane by capillary action. at the test “t” line, the gold-conjugated otc3-i binds to immobilized ova-otc3, forming a red line. the excess of gold-conjugated otc3i migrated farther and fab region of rabbit polyclonal sera was trapped by the goat anti-rabbit immunoglobulins to form the control line “c”. if the sample contains higher amount of oxytetracycline (otc) upper than 20 µg/kg-, they bind the goldconjugated otc3-i on the conjugate pad, blocking all recognition sites of otc3-i sera. as a result, the gold-conjugated otc3-i-otc does not bind to immobilized ova-otc3, obtaining only a red line in c position. when the sample was free of otc or the concentration was lower than 20 µg/kg, it competed with the immobilized ova-otc3 (test line) to bind the limited amount of gold-conjugated otc3-i. if the concentration of the antibody is sufficient, the gold can be visualized as a red line at “t” position. the intensity of the test line would be inversely proportional to otc present in the sample. the colour formation for both reactions is complete after 5 to 10 min. analysis of honey samples twenty five samples from different sources (tc free as assessed by lc-ms) were stored in a dark and dry place. before analysis, a mix of all honey samples were spiked with otc or cross related compounds and diluted 1/20 (w/v) in pbst at ph 7.5. the solution was vigorously agitated until honey was completely dissolved (around 5 min). finally, 100 µl of the solution is placed in the sample port of the lfia using a plastic mini pasteur pipette. for confirmation purposes, three grams of honey were exactly weighted into 50 ml polyethylene centrifuge tube. then, 15 ml of mcllvaine buffer (ph 4.0) with 0.10 mol/l na2edta were added and the mixture was vortexed until the honey dissolved completely. ocatadecyl spe cartridges (varian iberica, spain) were conditioned with 5 ml of methanol and 5 ml of mcllvaine buffer (ph 4.0) containing 0.10 mol/l na2edta. after condition, 5 ml of sample were allowed to pass through the cartridge followed by 2.5 ml of mcllvaine buffer (ph 4.0): methanol (85:15 v/v) and 2.5 ml of water. the cartridge was dried for 2 min by aspiration and another washing step with 2.5 ml acetonitrile was done. cartridge was dried again and analytes were eluted with 3.0 ml of ethyl acetate: methanol (75:25 v/v). plastic support nitrocellulose membrane “c” control line gar igg “t” test line ova-otc3 absorbent pad conjugate pad gold otc3-i sample pad flow “s” sample port plastic support nitrocellulose membrane “c” control line gar igg “t” test line ova-otc3 absorbent pad conjugate pad gold otc3-i sample pad flow “s” sample port food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 9 the elution mixture was evaporated until dryness under a gentle nitrogen flow in a water bath (30–35 ºc) and the residue was dissolved in 1 ml methanol: water (15:85 v/v). the final extracts were filtered through 0.45 mm syringe filters (millipore, spain) and injected onto the hplc. all honey samples were also analysed by hplc-esi-ms in an agilent 1100 series lc/msd ion trap (agilent technologies, waldbronn, germany). the separation was achieved on a mediterranean c18 (15.0 cm × 2.1 mm i.d., particle size 3.5 microns) purchased from teknokroma (barcelona, spain). a binary mobile phase with gradient elution was used. ultra-pure water with 0.1% formic acid and acetonitrile were used as solvent a and solvent b, respectively. the gradient starts with 5% b, increases to 90% in 35 min, and then returns to the initial composition for 10 min to condition the column for next injection. the flow-rate was 0.4 ml/min, the column temperature was set to 25 ºc and the injection volume was 20 µl. the mass spectrometer acquired data in quantitative multiple reaction monitoring (mrm) mode. for otc, two different characteristic fragmentation reactions m/z 444 and m/z 426 were monitored in the selected reaction monitoring (srm) mode using a dwell time of 0.1 s results and discussion researchers are aware that nanometer colloidal gold particles have been gradually applied in immunoassay, biosensor, bioidentification, gene therapy, arrays and dna computation [21]. because immunoglobulins could be labelled with gold instead of enzyme, the substrate is therefore not needed in the reacting system. the strength of colour shows that it is closely related to the size determined by the amount of trisodium citrateand quality of the colloidal gold particles. previous experience in gold assays has shown us that a 40 nm particle is the best for the strip assays. these results have also been described in the literature [22]. smaller particles give fewer signals because of how the gold scatters light and larger particles tend to migrate slowly, generate a purple blue colour, and are difficult to work with (stability). also, membranes with a pore size of 15 microns are preferred to for both flow rate and reactivity, since if larger pore sizes are used the flow rate is usually too fast for reactions to take place at low sensitivities and if smaller pore sizes are used a test is difficult to finish in 5-10 minutes. the concentrations of immunoreagents were optimized to satisfy the following criteria: the appearance of a clear pink colour on the test lines for negative samples within a reasonably short time (up to 10 min); the difference between positive and negative samples could be easily distinguished with the naked eye; minimum immunoreagent consumption. for this purpose, experiments similar to the “checkerboard titration” in elisa were performed for otc test. several dilutions of ova-otc3 conjugate coated on the membrane against different amounts of colloidal gold labelled otc3-i and control line (gar), from 0.05 mg/ml to 2.5 mg/ml were investigated, using a blank honey sample and samples containing between 5 ng/g otc and 100 ng/g otc. combinations satisfying the above mentioned criteria were selected for further experiments. in accordance with the competitive immunoassay principle, lower amounts of coating conjugates and gold labelled sera resulted in an increase in the assay sensitivity. however, under certain concentrations of the immunoreagents, the colour of the control lines was too faint for visual evaluation. as a result, concentrations of 0.8, 0.4, and 1.0 mg/ml were selected, respectively for gold labelled otc3-i, ova-otc3 and goat anti rabbit. for the determination of the visual limit of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 10 detection, a control honey, previously shown to have no detectable otc (lcms), was fortified by otc standard (100 mg/g) at nine concentrations and provided to a panel of 22 trained referees as blind samples, with a protocol assay (as described in analysis of honey samples section) and sufficient number of lfia, for naked eye evaluation. the results are shown in table 1. table 1. detection results of blind samples by lfia and lc-ms sample otc added (ng/g) otc found (ng/g) lfia otc found (ng/g) lc-ms 1 0 0/66* n.d. 2 5 0/66 n.d. 3 50 66/66 48.6 ± 4.7 4 15 5/66 17.0 ± 3.6 5 3 0/66 n.d. 6 20 66/66 21.5 ± 3.0 7 35 66/66 32.3 ± 2.4 8 10 15/66 9.8 ± 2.2 9 30 66/66 33.2 ± 4.1 *visual reading (n positive/n analyzed). all samples were tested by triplicate (n=3). as can be seen in figure 2, referees indicated -as positivethe smallest amount of otc that resulted in no colour development at the test line, which was considered as the cut-off value. in this sense, the visual detection limit (vdl) was selected as the point corresponding to a concentration threshold that ensures 95 % of positive responses [23]. from the data supported by referrers (naked-eye determination), the lateral-flow immunochromatographic assay yielded a positive result at 20 ng/g otc (100 % of positive responses), while equivocal result was obtained at 10 and 15 ng/g otc, since 15 test were interpreted as positive and 51 as negative for 10 ng/g (77% of positive responses) and 5 positive versus 61 negative for 15 ng/g (92% of positive responses). the results from the visual evaluation of the lateral-flow tests were in good agreement with the amounts spiked (table 1) and determined by lc-ms, demonstrating the practical applicability of the developed assay. ii t c i s 1 2 3 4 5 6 7 8 9 ii t c i s t c i s 1 2 3 4 5 6 7 8 9 figure 2. lateral-flow immunoassay of spiked honey samples. otc concentrations (from left to right). in panel i: 0 ng/g (s-sample well; t-test line; c-control line); in panel ii: 5; 50; 15; 3; 20; 35; 10 and 30 ng/g (strips are out of the plastic housing). upper line is the control line; bottom line is the test line. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 11 the specificity of the otc method was evaluated in comparison to other analogue compounds: tc, ctc, atc, dmc, dxc, dr, mc, mnd and rtc. stock solutions of each sulphonamide (100 mg/l) and a mix containing all of them (except otc), at the same concentration, were prepared in dmso and stored at 4 ºc and proper diluted with phosphate buffered saline (pbs; 100 mmol/l sodium phosphate, 137 mmol/l nacl, 2.7 mmol/l kcl, ph 7.5), containing 0.05% tween 20; yielding concentrations ranging from 0.01 to 50 mg/l. from a control honey free of tetracyiclines (previously tested by lcms), different aliquots (1 g) were fortified by adding 20 ml of standards diluted in pbs-t solution of different related compounds. positive or negative results from the lfia, three replicates for each honey concentration and cross-reactant were scored, after 100 µl of the corresponding solution was placed in the sample port of the lfia using a plastic mini pasteur pipette. two clear bands were observed in the test and control lines test strip, even though these compounds were present at a high level. each analogue compound was found not cross-reacted when tested at concentrations up to 50 mg/g, only tetracycline (tc) have cross reacted at concentrations upper than 70 ng/g. this fact indicates that the developed technique had a high specificity towards otc. few attempts have been made to develop sensitive immunoassays, mainly in elisa format, for tcs in different matrices such as milk, kidney, fish and honey, with sensitivities ranging from 0.048 to 150 ng/g [24]. a very sensitive lfia named tetrasensor, was developed and validated for tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey with detection limits between 6 and 12 ng/g for each compound [25]. however, the current status of the available immunoassays for a single sulphonamide, such as otc, is scarce. in the present work, we describe a lfia which has a clear limit of detection at 20 ng/g otc. the assay can be used with small volumes (100 µl) of diluted honey. the assay was shown to have 100% diagnostic sensitivity and good specificity for the detection of otc in honey. existing elisa and other assays for stz tests are laboratory based, require sample preparation, and are relatively slow compared to the lfia described here, for the first time. this result supports the establishment of 20 ng/g as vdl of the lfia that will be in compliance with the further eu minimum required performance limit (mrpl) of 20 ng/g for otc in honey, as proposed by the european federation of honey packers and distributors (feedm). the lfia could be stored at room temperature for at least 6 months or more, and the kit could be delivered and used at ambient temperature (data not shown). another advantage is cost, where the gold conjugate used is considerably less expensive than enzyme or fluorescent conjugates with easy handling and simple to perform. for the reasons mentioned, the test lends itself better for making rapid large scale screening of honey samples for otc. the otc-lfia has an immediate application as a rapid analytical tool to screen field samples in situ. on the whole, the lfia is versatile and can be adapted for fast detection of other analytes of interest, which could be an advantage to take decisions about foods (accept or reject) in a short time. by switching the antibodies or antigen and making small adjustments to the chemistry of the strip format, the same test design may be used for many applications. conclusions a rapid lateral-flow immunechromatographic device with a colloidal food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 12 gold-polyclonal probe was developed and optimized for the detection of oxytetracycline in honey samples. coupled with a simple and fast sample preparation method, the assay could be accomplished within 10 min without the need of any equipment. the visual detection limit was 20 ng/g, and the developed technique had a high specificity toward otc, since it recognises tc when is present in honey at concentrations ≥ 70 ng/g. the results from visual evaluations of the lateral-flow tests of spiked honey samples proved the reliability of the assay. this qualitative one-step test based on the visual evaluation of results appears to have practical advantages such as rapidity and simplicity, over existing conventional immunoassay formats. the proposed analytical system has no equivalence in the market and could be used in the honey sector to carry out onsite screening for otc at the beginning of the food chain to improve/facility commercial trade. acknowledgements this work was supported by a grant from ministry of education and science of spain. cit-060000-2007-57. references 1. s. sczesny, h. nau, g. hamscher, j. agric. food chem. 51, (2003) 697-102. 2. u. koesukwiwat, s. jayanta, n. leepipatpiboon, j. chromatogr. a 1149, (2007), 102-107. 3. e.c. mullen, am. bee j. 140, (2000), 300306. 4. p. van westendorp, proceedings of the canadian association of professional apiculturalists, jan. 2001. moncton, nb, 2001. 1822. 5. m. heyndrickx, k. vandemeulebroecke, b. hoste, p. janssen, k. kersters, p. de vos, n.a. logan, n. ali, r.c.w. berkeley, int. j. syst. bacteriol. 46, (1996), 270-275. [6] h. shimanuki, in: r.a. morse, k. flottum (eds.), honey bee pests, predators, & diseases, third ed., a.i. root co, medina, oh, 1997, p. 33, ch. 3. 7. h.c., wegener. curr. opin. microbiol. 6, (2003), 439-445. 8. commission of the european communities, council regulation eec 2377/90. off. j. l. 224, (1990), 1. 9. j. kurtittu; s. lönberg; m. virta; m. karp. j. agric. food. chem. 48, (2000), 33723377. 10. a. carrasco-pancorbo; s. casadoterrones; a. segura-carretero; a. fernández-gutiérrez. journal of chromatography a, 1195, (2008), 107–116. 11. k. fujita; h. ito; m. ishihara; s. inukai; h. tanaka; m. taniguchi. journal of the food hygienics society of japan. 49, (2008), 196-203. 12. y.a. hammel; r. mohamed; e. gremaud; m.h. lebreton; p.a. guy. journal of chromatography a. 1177, (2008), 58– 76. 13. p. viñas; n. balsalobre; c. lópezerroz; m. hernandez-córdoba. journal of chromatography a, 1022, (2004), 125-129. 14. r-biopharm (2001) operator’s manual ridascreen tetracycline. article no r3501. rbiopharm, darmstadt. 15. n. pastor-navarro; s. morais; a. maquieira; r. puchades. anal. chim. acta, 594, (2007), 211-218. 16. y. zhang; s. lu; w. liu; c. zhao; r. xi. j. agric. food chem. 55, (2007), 211-218. 17. m. jeon.; i. rhee paeng. anal chim acta. 626, (2008), 180-185. 18. j.a. gabaldon; a. maquieira, r. puchades. intern. j. environ. anal. chem. 82, (2001), 133-144. 19. j.a. gabaldon; a. maquieira, r. puchades. intern. j. environ. anal. chem. 82, (2001), 145-155. 20. g. frens, nat. phys. sci. 241, (1973), 20-26. 21. y.p. deng; h.q. zhao; l. jiang. china basic sci. 9, (2000), 11-17. 22. w.b. shim; z.y. yang; y.y kim; j.g. choi; j.h. je; s.j. kang; a.y. kolosova; s.a. eremin; d.k. chung. j. agric. food chem. 54, (2006), 9728-9734. 23. l.r. althaus, in: estudio sobre los métodos de detección de inhibidores en leche de oveja de raza manchega (ph. d. dissertation, universidad politécnica de valencia eds., valencia 1999). 24. n. pastor-navarro; a. maquieira; r. puchades. anal. bioanal. chem. 395, (2009), 907–920. 25. w. reybroeck; s. ooghe; h. de brabander; e. daeseleire. j. agric. food chem. 55, (20079), 8359-8366 microsoft word 3 journal nr 4 2010 din 8 noiembrie_63-69.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 67 comparative studies of composite coatings with ceo2 and nd2o3 in copper matrix by electroless plating catalina iticescu1, monica murarescu1 1dunarea de jos university of galati, domneasca street 47, romania, monica.murarescu@ugal.ro abstract: the study presents the influence of ceo2 and nd2o3 particles as dispersed phase in copper coatings. these coatings were obtained by electroless deposition from copper sulphate electrolyte bath. advantages of electroless plating include excellent uniformity, bulk processing capability and ability to produce unique catalytic coatings. the main disadvantage of electroless plating method is the fact that thin layers are obtained and the working time is long enough. the concentration of ceo2 particles was between 5-50g/l. similar samples were made using the same concentration of nd2o3 particles in bath solution. many factors, like the nature of electrolyte and concentration of the dispersed phase in the electrolyte, methods of codeposition contributed to the obtaining of qualitative coatings. the embedded particle of dispersed phase in the copper matrix determines changes of surface coatings and also of their properties. morphology is an important property of electrodeposited composite coatings and was studied by scanning electron microscopy (sem) and edax analysis. the mechanical properties of the composite coatings were investigated. the vickers microhardness was measured in cross section. the results are compared with copper coatings without oxide particles to understand the effects of dispersed phase on the properties of composite coatings. the obtained composite coatings properties are increased in comparison with the metal copper coatings. keywords: electroless, composite coatings, electrolyte bath. introduction composite coatings consist of a metal matrix with small particles (or fibres) like a dispersed second phase in the matrix. these composites are developed for a large scale of applications. composite coatings have been developed primarily for their physical and chemical properties because they combine the properties of the metal matrix and dispersed phases: high temperature strength, good resistance to thermal fatigue, good shock resistance, high thermal conductivity and low coefficients of thermal expansion. metal matrix composite coatings have been attracted considerable attention because of their relatively low cost and the simple manufacture facility in the case of codeposition in an electroplating bath. composite coatings can be obtained by the following methods: vapor phase chemical deposition (cvd), vapor phase physical deposition (pvd), electrodeposition, electroless. in this paper are discussed the easiest methods, like: chemical codeposition and electrochemical codeposition. the composite coatings deposition from solutions without an external current source, that means a chemical reduction of the substances being in ionic state, was studies since 1969 [1]. the electroless deposition is a simple method usually used for metallic layer depositions on metallic support, with a low price, for extended surfaces. it is specially used for the obtaining of thin layers, with usability in electronic and electrotechnical industry (for circuit and multicip modules obtaining) [2]. pure copper and copper composite coatings can be deposited by wet chemical plating techniques such as electroplating and electroless plating. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 68 these techniques have the advantage of low cost of tools and materials, low processing temperature, high quality material and high throughput of the process. it has a very high selectivity, it uses a very thin seed layer, and it has excellent step coverage and good trench filling capability that does not need any electrical contacting of wafers during deposition. it was used the electroless plating method both for obtaining pure metal coatings and efficient composites that need simple installations and accessible reactives. in the same time, the structural properties of these obtained coatings are improved because of a bigger uniformity found at the deposited layers than obtained through electrochemical procedures [3]. materials and methods the installation utilized for chemical depositions is simple and the metal covering is deposited by a simple metallic support immersion in electrolyte. the electrolyte for deposition is composed of soluble metal salt, reducing agent and additives such as complexing agents, buffers, bath stabilizers and rate promoters. the copper electroless plating has been prepared using an electrolyte with the chemical composition: cuso4·5h2o30g/l; na2co3 anh.-12g/l; na and k tartrat 150g/l, naoh-50g/l, edta 6g/l [7]. the formic aldehyde (37%) was used as reducing agent and its amount was 20 g/l hcho. the ph of the electrolyte was 13, 50 at the temperature of 20-250c [7]. carbon steel was used as metallic support for copper deposition. in the electrolyte there were added particles of nd2o3 with a concentration between 5-50g/l or ceo2 particles with a concentration between 550g/l. therefore, the plating bath is capable of plating receptive surface, including plating tank and equipment surfaces. to maintain a constant plating rate, high control of ph and temperature are necessary. metal and reducing agent concentration must also be kept at optimal specified levels [4]. deposition of copper was obtained on carbon steel as metallic support with an effective area of 25 cm2. the samples were cleaned mechanically by polishing with abrasive papers, organically (with acetone) and chemical degreased (at 70-800c for 5 minutes in alkaline solution), washed with hot distilled water and finally, for the activation of surface, treated with hcl 15% (2 minutes) [5]. in this study we used formic aldehyde as reducer. the copper chemical deposition can be performed from alkaline solutions only, and reacts according to the chemical equation below: cu2+ + 2hcho + 40h- cu + 2hcoo+ + h2 + 2h2o molecular hydrogen occurs from the reaction, the cu / h2 ratio being 1 [6]. copper chemical deposits from electrolyte with and without particles in suspension were tested by optical microscopy in cross section. surface morphology and composition were studied by scanning electron microscopy (sem) with an x-ray analyzer (edxs) from leo instruments. the results are compared with pure copper coatings to understand the effects of particles dispersed on the microstructure and properties of composite coatings during the electroless crystallization. results and discussion advantages of electroless plating include excellent uniformity, bulk processing capability and ability to produce unique catalytic coatings. the main disadvantage of electroless plating method is the fact that it was obtained thin layers and the working time is long [7], [8]. by this food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 69 consideration, the obtaining of composite layers by electroless plating methods was less studied. another consideration consist the instability of solutions of depositions and low deposition speed that interaction create a bigger activity of copper reducing reaction represent another reason. they are thus suitable methods for low-cost processing. electroless plating occurs simply by immersion of the samples in a plating bath. no rectifiers, batteries or anodes are involved. the essential elements of the solution are soluble metal salt, and reducing agent, additives such as complexing agents, buffers, bath stabilisers and rate promoters. electroless plating is an autocatalytic process; that means that metal deposition serves to catalyse the reaction [9], [10]. a. structural aspects the structure of copper coatings was investigated by light microscopy and scanning electron microscopy (sem) in cross section. thin layers of copper deposits between 3-18 µm were obtained. fig.1. optical microscopy for copper and copper composite deposition the layer thickness depends on the immersion time of samples in solution. the aspect of layers is not completely uniform but this can be explained by destruction of metallic support during the preparation of samples for optical analyses (fig. 1). composites coatings structure is modified by the presence of nd2o3 and ceo2 particles in copper matrix. sem micrographs of composite layers were obtained by chemical deposition. an nd2o3 particles agglomeration with their concentration increase was observed. the copper matrix structure is modified, the particles presence conduce to the surface polishing that was also demonstrated by the roughness measurements. the presence of nd2o3 and ceo2 particle affects the mechanism of the copper reduction by electroless plating. when the nd2o3 particles are added in solution, the particles of oxide were embedded in the copper deposits in a satisfactory amount. the results of the investigations confirm that nd2o3 was embedded into copper matrix by electroless plating. the edx analysis in point of nd2o3 also indicated the presence of particles in copper matrix (fig.2, fig.3). from experimental date it was observed a growth amount of nd2o3 in deposit with the amount of oxide in the electroless solution. fig.2. edax analysis (in point) on nd2o3 particles included in copper matrix food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 70 table 1. composite coatings cu/ ceo2 obtained from the electrolyte containing 5g/l ceo2 when ceo2 was added into solution, the particles of oxide were included in the copper deposits in a very small amount. the results of the investigations have shown that cerium oxide was included into copper matrix by electroless plating. the edax analysis in point of ceo2 also indicated the presence of copper. (fig.4) 0 10 20 30 40 50 5 10 15 20 25 30 35 n d 2 o 3 em be dd ed in la ye r, % nd2o3 in electrolyte, g/l fig. 3 nd2o3 amount content (percentage by weight) in the copper coatings fig.4 edax analysis on ceo2 particles included in copper matrix the standard analysis realized in point by x ray tests referring to ceo2 inclusion percentage in the composite coatings went to the following results: table 2. composite coatings cu/ ceo2 obtained from the electrolyte containing 10g/l ceo2 element atom (%) weight( (%) error (±) norm (%) cu 9.41 2.67 0.69 10.26 ce 30.48 19.05 0.76 73.25 o 60.10 4.29 0.23 16.49 total: 100.00 26.00 100.00 composites coatings structure is modified by the presence of particles oxides in copper matrix. sem micrographs of composite layers obtained by chemical deposition are showed in fig.5, fig.6 and fig.7. (a) (b) fig.5. sem micrographics: a) cu; b) cu-nd2o3 (1 g/l in electrolyte) elemen t atom (%) weight( (%) error (±) norm (%) cu 41.92 9.73 1.09 43.19 ce 20.74 10.61 0.64 47.12 o 37.34 2.18 0.16 9.69 total: 100.0 22.52 100.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 71 the copper matrix structure is modified, the particles presence conduce to the surface polishing that was also demonstrated by the roughness measurements. (a) (b) fig.6 sem micrographics: a) cu-nd2o3 (5 g/l in electrolyte); b) cu-nd2o3 (10 g/l in electrolyte) the presence of nd2o3 and ceo2 particles affects the mechanism of the copper reduction by electroless plating. when nd2o3 is added in solution, the particles of oxide were embedded in the copper deposits in a satisfactory amount. the results of the investigations confirm that nd2o3 and ceo2 particles were embedded into copper matrix by electroless plating. more studies are necessary to understand the phenomena occurring during the electroless plating in order to find the optimal parameters for deposits with better properties such as corrosion resistance and good wear resistance. (a) (b) (c) fig.7 sem analysis on the surface for the deposits cu-ceo2: a) metallic cu; b) 10g/l ceo2; c) 50g/l ceo2 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 72 b. microhardness the vickers microhardness (hv0.025) of coatings in copper matrix was studied. the presence of dispersed particles in the electrolyte and in coatings modifies the microhardness of layers and can be correlated to the amount of dispersed phase in the deposit. the content of particles in the electrolyte influences the microhardness of the coatings. the vickers microhardness (hv0.025) of coatings in copper matrix was studied. the presence of dispersed phases in the electrolyte and in coatings modifies the microhardness of layers and can be correlated with the amount of dispersed phase in the deposit. figure 8 shows the results obtained for copper coatings with ceo2 particles in electrolyte. the amount of ceo2 in the electrolyte influences the microhardness of the coatings. the microhardness of the coatings obtained with 5g/l and 10 g/l ceo2 in electrolyte has increased smaller in comparison with that of the copper deposits without particles in the electrolyte. a decreasing in the microhardness of the layers obtained with 50 g/l ceo2 in the electrolyte was observed. 0 10 20 30 40 50 106 108 110 112 114 116 118 120 h v 0, 02 5 ceo2, g/l fig.8. microhardness variation of copper coatings with ceo2 particle in electrolyte the dispersed phase content in a larger amount of electrolyte modifies the metal electroless deposition process. considering that neodinium oxide is a hard one, it would be expected that the composite layers hardness to increase a lot but this is not happening. it was observed a slow microhardness increase at the samples obtained by electrodeposition, due to the small inclusion degree. for a large microhardness increase, the inclusion degree should be for at least 15%. even a slowly microhardness decrease can be observed at the samples obtained by electroless. this behaviour can be explained both by the structural changes due to the oxide particles presence in copper matrix and the structural differences determined by the inclusion method. table 3. microhardness of layers in matrix of copper with and without nd2o3 particles in the electrolyte disp. phase conc. of dp, (g/l) hv min. hv max. hvme diu stand ard deviat ion 98 115 108 6.3 nd2o3 5 102 118 110 5.4 nd2o3 10 104 121 113 4.6 nd2o3 50 115 134 125 5.3 conclusion 1. the experiments were performed to study the obtaining of copper coatings matrix with and without ceo2 and nd2o3 particles from a copper sulphate electrolyte with formaldehyde as reducing agent. 2. the dispersed phase of ceo2 and nd2o3 was included in copper layers but in a very small amount. 3. the layers and the properties of coatings presented differences and this fact depended on the amount of particles oxide in electrolyte. 4. the composite layers properties are improved but not in a spectacular way, probably due to the small amount of included particles. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 73 references 1. f.pearlstein, d.baudraud, k.parker, electroless deposition of metals. from the aesf illustrated lectures series. metals park, ohio: american society for metals (asm international), 1987 2. electroless plating, ece4803: electronic packaging substrate fabrication, www.geocities.com/collegepark/den1314/ele ctrolessplating, 2003 3. d. baudrand, electroless processes in aesf surface finishing shop guide. orlando:american, electoplaters and surface finishers society, 1995 4. iticescu, c., cârâc, g., mitoseriu, o. (2005), bulletin of the polytechnic institute of iassy li (lv), 149-154. 5. cârâc, g., benea, l., iticescu, c., lampke, th., steinhäuser, s., wielage, b. (2004) surface engineering 20(5), 353-359. 6. searson, p.c. (2002), journal of the electrochemical society 147(7), 2571-2575. 7. schlesinger, m. and paunovic m. (2000), modern electroplating, fourth edition, john wiley & sons, new york. 8. shrestha, n. k., sakurada, k., masuko, m., saji, t. (2001), surface and coatings technology 140, 175. 9. henry, j.r. (2007), metal finishing 105, issue 10, 350-360. 10. aithal, r. k., yenamandra, s., gunasekaran, r.a., coane, p., varahramyan, k. (2006), materials chemistry and physics 98, 95–102. microsoft word 3 journal fia nr 1 2010 final_46-51.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 50 effect of potato starch and agar on the rheological behaviour of tomato ketchup mircea-adrian oroian1, gheorghe gutt1 1ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: m.oroian@usv.ro, g.gutt@usv.ro abstract: two different type of hydrocolloids (potato starch and agar) were added, at different levels (for agar 0, 0,25, 0,5 and 1 g/100 g (w/w), and for potato starch 0, 0,5, 1, 2, 4g/100 g (w/w)), to a tomato ketchup which was a homemade one, having total soluble solid (tss) content of 10 g/100 g (w/w). the intrinsic viscosity of the tomato ketchup is leading from the pectic substances which normal occurs in fruits. the effect of these two hydrocolloids used, was investigated by a rotational viscometer, brookfield viscometer (brookfield engineering inc, model rvdv i prime) at 6 rpm with rv spindles. the brookfield viscometer dv i prime with disk spindles represents an easily and cheap method for rheological characterization of non-newtonian fluids, in this case of tomato ketchup. both biopolymers increased the viscosity of tomato ketchup; however, the agar generated the maximum increase of the apparent viscosity of tomato sausage. the highest viscosity was obtained for the sample where 1 g agar / 100 g tomato ketchup was added. the tomato ketchup viscosity of the samples with potato starch has a linear evolution with the increasing of the concentration, while the viscosity of the sample with agar increases exponentially with the increasing of agar concentration. keywords: hydrocolloids, pseudo-plastic, viscosity, viscometer introduction: rheology is now well established as the science of the deformation and flow of matter: it is the study of the manner in which materials respond to applied stress or strain. [1] food rheology is the study of the manner in which food materials respond to an applied stress and strain. the food rheology has many applications in the fields of food acceptability, food processing and food handling. determination of rheological properties of foodstuffs provides an instrumental quality control of raw material prior to processing, of intermediate products during manufacturing, and on finished goods after production. [2] a gel is a solid-in-liquid colloid in which the solid phase forms a network structure that immobilizes the liquid and produces solid-like properties. gelation arises either from chemical cross-linking through polymer-polymer interactions. the macromolecular substances responsible for network formation in food systems are primarily polysaccharides and proteins. [3] hydrocolloids are water-soluble, high molecular weight polysaccharides that serve a variety of functions in food systems, such as enhancing viscosity, creating gel-structures, formation of a film, control of crystallization, inhibition of syneresis, improving texture, encapsulation of flavours and lengthening the physical stability, etc. [4] agar is a cell wall component of seaweeds such as gelidiaceae or gracilariadeae belonging to the red algae family, and is extracted with boiling water and dried. this extract is composed of two kinds of journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 51 straight chain polysaccharides with galactose as the basic skeleton; one is based on the neutral polysaccharide “agarose” which governs the gelling ability of agar, the other is the acidic polysaccharide “agaropectin” which includes ionic groups such as sulfate, pyruvate and methoxy groups. agar swells in cold water but is not soluble. the agar dissolves in boiling water to become an aqueous solution, and forms a gel when it is cooled. it is widely accepted that agarose molecules take a random coil conformation in aqueous solution at higher temperatures while they form double helices at lower temperatures, and the aggregation of helices leads to the gel formation. [5] starch is a common place material of great nutritional and industrial importance. starch occurs in most green plants as minute granules in the leaves, stems, roots, fruits and seeds [6]. in these plants, starch traditionally has been considered as a reserve material, stored for the future use of the plant. starch chain structure: amylose. chemical and biochemical evidence indicated that the main chains of starch are composed of d glucose units, linked by α-l,4 bonds. amylose consists entirely of α-l,4-1inked glucose units. most commercial starches (e.g., potato, corn) contain about 25% amylose, the remainder being amylopectin. molecular-weight measurements indicate that amylose contains several hundreds or thousands of glucose units. branching in starch: arnylopectin. it is well established that al,6-1inks constitute the major, if not exclusive, branching linkages. these α-l,6 branch links comprise about 4 to 5% of the total number of linkages in amylopectin.[7] tomato is one of the most important vegetable products and is mainly marketed as a processed product, i.e. pastes, concentrates ketchup, salsa, etc. viscosity is one of the most important quality parameters of such tomato products. knowledge of the rheological properties of fluid and semisolid foodstuffs is important in the design of flow processes in quality control, in storage and processing stability measurements. [4] consistency/viscosity of ketchup is an important attribute from the engineering and consumer viewpoints. therefore, reliable and accurate rheological data are necessary for designing and optimization of various unit operations (pumping, mixing, heating, etc.), and ensuring product acceptability since the products with improper consistency may be graded as unacceptable, or sold at lower price.[4] 2. materials and methods 2.1. materials the tomato ketchup with 10% tss was obtained using a homemade recipe, with ingredients typically used in ketchup preparation (salt, sugar, onion, garlic and spices). agar and potato starch were provided from romanian market. table 1 recipe used for tomato ketchup preparation (for 1 kg) ingredients tomato paste, g 794 sugar, g 50 salt, g 10 onion, g 50 garlic, g 5 spices, g 1 2.2. preparation of tomato ketchup to the tomato ketchup, obtained using a homemade recipe, was added agar (0,25, 0,5, 1 g/ 100g) and potato starch (0,5, 1, 4g/ 100 g). the samples were stirred for 5 min at 200 rpm with a stirrer with paddle impeller. the samples were heated at the gel formation temperature (350c for agar, and 630c for starch) for 10 minutes on a water bath. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 52 table 2 agar characteristics moisture % 7.5 ph 1,5% solution 6.6 gelation point, 0c 35 melting point, 0c 87 ash, % 3.14 table 3 potato starch characteristics moisture, % 18-20% ph solution 5-8 ash, % 0-0.5 gel formation temperature, 0c 63 each ketchup sample was then immediately poured into the glass jar, while still hot, sealed with screw caps, and then stored at room temperature (20–220c) for 24 h before being analyzed. 2.3. determination of rheological properties of ketchup viscosity measurements were carried out on the ketchup samples at room temperature (200c), with a brookfield viscometer (brookfield engineering inc, model rvdv i prime) at 6 rpm with rv spindles (rv2, rv3, rv4, rv5, rv6, rv7). fig.1. rv disk spindles the samples in 300 ml of beaker with a 8,56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain the desirable temperature of 250c. first measurements were taken 2 min after the spindle was immersed into each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. 3. results and discussion the pectins present in tomato have the ability to form gel and they influence the rheological properties of tomato ketchups. according to mitschka, p., steffe, j.& daubert, c. [8, 9] the brookfield viscometer with disk spindles represents a good method to determine non-newtonian fluid properties. the gel formation is caused by the galacturonic acid chains associations obtained and stabilized by the noncovalent bonds: hydrogen bounds and ionic bounds.[2] the agar has a gel formation temperature lower than the other gums. the agarose is responsible for the gel formation because of the changes in the agarose conformation from the initial linear structure to a double helices formed from left-handed threefold helices.[2] starch gelatinization is a process that breaks down the intermolecular bonds of good method to determine non-newtonian fluid properties. the intrinsic viscosity of the tomato ketchup is leading from the pectic substances which normal occurs in fruits. pectins are complex carbohydrate molecules, used especially as gelling agents in food industry. y = 5.7661x-0.0559 r2 = 0.6181 3 3 .5 4 4 .5 5 5 .5 6 0 5 0 100 15 0 2 0 0 2 5 0 3 0 0 shear time (s) a pp ar en t vi sc os it y (p a s) fig.2. tomato ketchup viscosity journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 53 viscosity functions data showed that all ketchups under examination were nonnewtonian fluids, (the tomato ketchup without any hydrocolloids has a flow behaviour index n = 0,32) which was indicative of the pseudo-plastic shear thinning nature of tomato ketchups. the two hydrocolloids increased the apparent viscosity of the tomato ketchup. the agar added to the sample obtained the higher apparent viscosity of the sample at small concentration. y = 9.5927x -0. 0531 r 2 = 0.7006 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0 50 100 150 200 250 300 shear time (s) a pp ar en t v is co si ty (p a s) fig.3. tomato ketchup viscosity with 0,5% potato starch y = 15.104x-0.0644 r2 = 0.8185 6 8 10 12 14 16 0 50 100 150 200 250 300 shear time (s) a pp ar en t v is co si ty (p a s) fig.4. tomato ketchup viscosity with 1% potato starch y = 22.981x -0.052 r 2 = 0.4944 5 10 15 20 25 0 50 100 150 200 250 300 shear time (s ) a pp ar en t v is co si ty (p a s) fig.5. tomato ketchup viscosity with 4 % potato starch table 4. tomato ketchup viscosity sample apparent viscosity pa s tomato ketchup 4,43 tomato ketchup 0,25% agar 11,38 tomato ketchup 0,5% agar 61,51 tomato ketchup 0,1% agar 326,02 tomato ketchup 0,5% potato starch 7,47 tomato ketchup 1% potato starch 11,17 tomato ketchup 4% potato starch 18,01 y = 15.053x -0.0598 r 2 = 0.5776 6 8 10 12 14 16 0 50 100 150 200 250 300 shear time (s) a pp ar en t v is co si ty ( pa s ) fig.6. tomato ketchup viscosity with 0,25% agar y = 90.688x-0 .08 32 r2 = 0.614 30 40 50 60 70 80 90 0 50 100 150 200 250 300 shear time (s) a pp ar en t v is co si ty (p a s) fig.7. tomato ketchup viscosity with 0,5% agar y = 286.93x 0.0269 r 2 = 0.4808 200 250 300 350 400 0 50 100 150 200 250 300 shear time (s ) a pp ar en t v is co si ty (p a s) . fig.8. tomato ketchup viscosity with 1% agar journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 54 y = 3.1443x + 5.9466 r 2 = 0.9329 0 5 10 15 20 0 1 2 3 4 5 potato s tarch concentration g/100 g tomato ketchup a pp ar en t v is co si ty (p a s) fig.9. influence of potato starch concentration on the tomato ketchup viscosity the tomato ketchup viscosity of the samples with potato starch have a linear evolution with the increasing of the concentration (fig.9), while the viscosity of the sample with agar increased exponentially with the increasing of agar concentration (fig.10). all samples with agar or with potato starch decrease their viscosity with the shear time, having a pseudo-plastic shear thinning behaviour, characteristic for the tomato ketchup (fig.3-7), the sample with 1% agar seems to have a pseudo-plastic shear thickening behaviour (fig.8). the influence of agar is quite different from the potato starch, at 0,25% agar the consistency is like a paste, at 0,5% agar the tomato ketchup appears like a gel, and at 1% agar the tomato ketchup appears like a firm gel which rives in slices. all the samples with potato starch appear like a paste, the sample with 4% potato starch has the highest viscosity of all the samples with potato starch. conclusions the present study showed that all tested hydrocolloids (agar and potato starch) can be used to improve viscosity of tomato ketchups. the brookfield viscometer dv i prime with disk spindles represents an easily and cheap method for rheological characterization of non-newtonian fluids, in this case of tomato ketchup. the addition of agar generated the highest viscosity range (at 1% agar added to tomato ketchup) but the tomato ketchup structure appears like a gel and not like a y = 4.6285e4.3 986 x r2 = 0.9803 0 50 100 150 200 250 300 350 400 0 0.2 0.4 0.6 0.8 1 1.2 agar concentration g/100 g tomato ketchup a pp ar en t v is co si ty (p a s) fig. 10. influence of agar concentration on the tomato ketchup viscosity paste. for the industrial obtaining of tomato ketchup the addition of 0,25% agar will lead to a good consistency of tomato ketchup, the sample with 1% agar has an unpleasant aspect and it rives in slices. on the other hand, the addition of potato starch do not generate the same viscosity (the sample with 1% potato ketchup has the same viscosity with the sample with 0,25% agar), all the samples (with 0,5%, 1%, 4%) appear like a paste (having the structure of a tomato ketchup but with different consistency). the potato starch represents a cheap method of improving the rheology behaviour of tomato ketchup. acknowledgment this paper was supported by the project "knowledge provocation and development through doctoral research pro-doct contract no. posdru/88/1.5/s/52946 ", project co-funded by the european social fund through sectorial operational program human resources 2007-2013. references 1. steffe, j., daubert, c., bioprocessing pipelines: rgeology and analysis, freeman press. usa, 2006 2. banu, c. aditivi şi ingrediente pentru industria alimentară,editura tehnică, bucureşti, 2000 3. tabilo-munizaga,g., barbosacánovas, g. rheology for the food industry, journal of food engineering, 67, 147-156, elsevier, 2004 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 55 4. sahin, h., ozdemir, f., effect of some hydrocolloids on the rheological properties of different formulated ketchups, food hydrocolloids, 18, elsevier, 2004 5. uzuhashi, y., nishinari, k., physicschemical properties of agar and its utilization in food and related industry foods & food ingredients journal of japan., vol. 208, no.10, japan, 2003 6. baden-huizen, n. p. the biogenesis of starch granules in higher plants. appletoncentury-crofts, new york. p. 27-37, 1969 7. french, d., journal of animal science chemical and physical properties of starch, vol. 3, no. 4, standford university libraries highwire press, usa, 1973 8. mitschka, p. a simple conversion of brookfield r.v.t. readings into viscosity functions, rheologica acta, 21, 207-209, springerlink, 1982 9. steffe, j., daubert, c., bioprocessing pipelines: rgeology and analysis, freeman press. usa, 2006 microsoft word fia journal no 1_2011_23 martie_62-66.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 66 effects of a2 phospholipase on dough rheological properties and bread characteristics ana leahu1, georgiana gabriela codină1, silvia mironeasa1, alice-iuliana roşu 1“stefan cel mare” university, faculty of food engineering, 13th universitatii street, suceava romania; e-mail: analeahu@usv.ro; codina@usv.ro; silviam@usv.ro; alicer@usv.ro abstract: flour lipids, though representing 2% of flour mass, play an important technological role because they interact with proteins and starch in dough, influencing the rheological properties of dough, bread quality and its freshness. the properties of lipids and particularly of phospholipids are given by the structural and functional particularities of molecules. it is considered that phospholipids are amphipathic substances because there is no polarity in the core of molecule, and because the extremities have opposite poles. the paper presents some experimental results obtained regarding the effects of exogenous phospholipase (a2) used in different quantities on the rheological properties of dough and bread quality. the rheological property of flours were determined on mixolab, as well as by using a chopin alveograph and the effect upon the bread quality was determined by baking tests. the results obtained on mixolab indicate an increase of dough stability, a clear reduction of c1 value and a greater difference of the points c5-c4 with the addition of a2 phospholipase. from the alveographic point of view an increase of dough strength was noted along with higher phospholipase a2 content. from the technological point of view, the best results have been obtained for a dose of 3000 u/100 kg flour added to flour. keywords: phospholipase, mixolab, alveograph, baking test introduction it has been demonstrated that lipid fractions may be involved in the glutenice complex either through hydrophilic bonds or hydrophobic interactions. as far as starch-lipid interactions are concerned, the lipid fractions appear as inclusions in the matrix of amylase polyglucans or are chemically tied to carbohydrates. this is why it is considered that, the hydrophiliclipophilic balance (hlb) and the content of fat acids are important criteria for establishing the probability of appearance of interactions such as lipids-proteins, lipids-starch while preparing flours in the process of bread making [1]. a phosholipase is an enzyme that converts phospholipids into fatty acids and other lipophilic substances [2]. it is known that phospholipids contain in their molecules glycerol, fatty acids, phosphoric acid and preferential nitrogenous bases, reason for which they are amphophilic properties [3]. depending on the type of polyalcohol in their structure glycerol, inositol or sphingosine, phospholipids have been classified as follows: glycerophospholipids, inosiphospholipids and sphingphospholipids. as fatty acids, phospholipids contain in their molecules both saturated and unsaturated fatty acids: palmitic, stearic, lignoceric, nervonic, linoleic, arachidonic etc. usually, in the phospholipid molecule we can only find a single rest of phosphoric acid, scarcely two. colin, ethanolamine and serine are predominant as nitrogenous bases in phospholipids [4]. the extremity that contains the rest of phosphoric acid and the nitrogenous base forms the hydrophilic part of the molecule food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 67 due to the existent polar groups, whereas the acyl groups resulted from the fatty acids form the hydrophobic part. due to the bipolar molecular structure table 1, the phospholipids participate equally in hydrophobic interactions and in forming hydrogen bonds [5] table 1. fosfolipids polarity, on hlb base [5] phospholipids hlb phosphatidyl choline 6 phosphatidyl ethanolamine 7 lysophosphatidyl choline 7 non-polar lipids lysophosphatidyl ethanolamine 8 phosphatidyl inozitol 11 polar lipids phosphatidyl seryne 12 depending on the phospholipases’ action on phospholipids (see figure 2), these enzymes are divided into four groups as follows: phospholipases a1 (pla1) which specifically eliminate fatty acid from the terminal α position of phospholipids; phospholipases a2 (pla2) which specifically eliminate fatty acid from the central β position of phospholipids thus forming the lysophospholipids; phospholipases b (plb) which eliminate two fatty acids from the phospholipids’ molecule in positions α and β corresponding to phospholipase a1 and a2; it is actually thought that phospholipase b is a mixture of phospholipases a1 and a2; phospholipases c (plc) also named lecithinases c or glycerophosphatase, hydrolyze the esteric bond between glycerol and phosphoric acid from phospholipids; phospholipases d (pld) also named lecithinases d, hydrolyze the esteric bond between the phosphoric acid and the nitrogenous base (coline, ethanol-amine etc.) from phospholipids; depending on the nitrogenous base from phospholipids, phospholipases d are also named colinophosphatases. phospholipase a2 facilitates conversion of lecithines into lysolecithines (the main reaction components) through unbinding the unsaturated fatty acid in position c-2 of lecithines and the growth of content of free fatty acids, mainly of linoleic acid [6]. figure 1. phospholipases acts on phospholipids [6] the lysolecithines’ molecules are more polar (higher hlb) than the molecules of initial lecithines, having a better ability to interact with certain flour components in dough while preparing semi-manufactured goods. it also appears that lysolecithin is an emulsifier with great capacity of acting on the rheological properties of dough with effects in terms of increasing loaf volume [7]. besides forming lysolecithine, phospholipases (a1, a2, and b) lead to an increase of content of free fatty acids, mainly linoleic acid. unsaturated fatty acids formed this way can become substratum for enzymatic oxidation, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 68 catalyzed by lipoxygenase or for the chemical oxidation, with positive effects on flour growth. materials and methods the study was carried out on flours for bread making of good quality. commercial white flour 650 type, obtained from the last (2007) crops’ milling was used – abbreviated m. as flour improvers lysomax in different doses (doses of 1000 u/100 kg – p1 abbreviated, 2000u/100kg – p2 abbreviated, 3000u/kg p3 abbreviated and 4000 u/kg – p4 abbreviated flour added to the flour) was used. lysomax is a commercial product supplied by enzymes & derivates romania ltd. which comprised true phospholipase a2 from bacterial non-gm strain of streptomyces violaceoruber with an enzymatic activity of 500 pla2 units/grams (minimum). control flour was analyzed by performing romanian standards methods: ash content (sr en iso 2171:2002), wet gluten content (sr en iso 21415-1:2007), gluten deformation (sr 90:2007) and falling number (sr en iso 3093:2007). the determined values for physico-chemical properties are mentioned as follows: ash content 0.65 %, wet gluten content 26 %, gluten deformation 4 mm, and falling number 342 s. the rheological properties of flours were determined on mixolab, as well as by using a chopin alveograph according to sr iso 5530-4:2005. bread quality characteristics are determined after the baking tests have been made (sr 91:2007 ref.). results and discussion dough rheological behaviour, prepared according to the working scheme, is appreciated on the basis o f determinations made using mixolab device (table 2) and chopin alveograph (table 3). all values shown are the means of three times analyses ± standard deviation ( x s.d.). table 2. the parameters resulted on the mixolab for dough obtained from control flour supplemented with different doses of lysomax characteristics/samples m p1 p2 p3 p4 water absorption (%) 58% maximum consistency during phase 1 (c1, n·m) 1.12 0.02 1.09 0.02 1.05 0.01 1.09 0.02 1.11 0.01 maximum consistency during phase 2 (c2, n·m) 0.35 0.01 0.34 0.02 0.31 0.02 0.30 0.01 0.29 0.02 maximum consistency during phase 3 (c3, n·m ) 1.52 0.02 1.54 0.02 1.57 0.02 1.60 0.02 1.63 0.02 maximum consistency during phase 4 (c4, n·m) 1.08 0.02 1.10 0.01 1.14 0.02 1.18 0.02 1.22 0.01 maximum consistency during phase 5 (c5, n·m) 1.37 0.02 1.44 0.01 1.54 0.02 1.64 0.02 1.73 0.01 stability (min) 6.54 0.04 6.63 0.05 7.04 0.04 7.44 0.04 7.53 0.03 difference of the points (c5 c4, n·m) 0.29 0.34 0.4 0.46 0.51 taking into consideration the rheological behaviour of different lysomax dose addition tests, comparatively to control sample, expressed on the basis of mixolab results, a decrease of maximum consistency during phase 1 (c1, n·m) was noticed. dough stability increase was registered as well while kneading the samples where a smaller quantity of lysomax was added in. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 69 table 3. the parameters resulted on the alveograph for dough obtained from control flour supplemented with different doses of lysomax samples maximum pressure (mm h2o) extensibility (mm) deformation energy (10-4 j) alveograph ratio p/l m 100 2 72 2 268 2 1.38 p1 104 2 74 2 282 2 1.40 p2 112 2 77 2 310 2 1.45 p3 110 1 79 1 315 2 1.39 p4 110 1 81 1 325 2 1.35 also maximum consistency during phase 2 (c2, n·m) has decreased with 17.14% by lysomax addition comparatively to control sample. this is understandable, because through the action of phospholipase a2 on the phospholipid substrate, they help to change the polarity of molecules, facilitating the transformation of lecithins into lizolecitines, as well the increase in free fatty acids content. the reaction compounds formed by their high content of hydrophilic groups and lipophilic groups can form cross-links between the granular starch with hydrophilic surface and gluten, also between gliadin and glutenin, forming true lipoproteic complexes between starch, gluten and other hydrophobic compounds. depending on the complexity of bonds, they can form cross-linked components, which compete in the compactness and stability of dough, formation of gas penetration-resistant film. regarding the parameters of phases 3, 4 and 5, we observed that, by adding some lysomax, a clear change of dough behaviour in the gelling process. lizolecitines form complexes with amylose and amylopectin, slowing the post-baking starch recrystallisation. this slowdown is reflected by a decrease of degree of bread ageing, showed by a c4 c5 difference with 75.8 % for one dose of 4000u/100 kg lysomax flour added. from the alveographic point of view, though the control sample has higher extensibility, by lysomax addition, the effect upon gluten strengthening is obvious from the dough resistance increase. lysomax showed also an increase of dough tenacity with 10% for p4 sample, with increasing effect of mechanical effect, but without significant modification in the ratio of alveograph curve configuration. the increase of baking strength (energy of deformation) is explainable, because in the process of kneading, due to the absorption of oxygen, free fatty acid oxidation resulted from the action of phospolypases a2 on phospolypides is promoted. this influences positively the enzymatic oxidation of dough rheological properties, which means higher energy consumption in kneading, but also a dough that gives bread higher volume after the process of baking. the technological effects of lysomax improver were obtained by baking tests assessment. the variation registered for the loaf volume values are shown in figure 4. according to the samples shown in figure 4, all improved flour samples, regardless of additive type and dose used, lead to the obtaining of higher volume breads than the blank sample. all samples showed also an improvement of crumb characteristics such as crust aspect, crumb texture, porosity and elasticity as well as freshness. the effects of lysomax on dough rheological properties were obvious, in relation to the method of analysis used. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 70 340 350 360 370 380 390 400 410 420 430 440 450 m p1 p2 p3 p4 samples lo af v ol um e, c m 3/ 10 0g figure 4. loaf volume values of the samples analyzed conclusion in flours of good breadmaking potential, the addition of phospolipase a2 promotes a substantial improvement in therms of physical specific feature of bread (significant increase of volume by 16 %) for an added dose of 3000u/lysomax in the baking process. these improvements are a consequence of improving rheological properties of dough, to strengthen the glutenice network, resulting in higher dough strength on demand and increasing the p parameter. at mixolab, the addition of lysomax into flour led mainly to a decrease in maximum consistency during phase 2 and a slight increase in its stability. an increase in the difference of points c4-c5, which shows a reduction of starch gelling can be seen as well. references 1. pyler, e.j., baking science&technology, third edition, usa, sosland publishing co., 1988. 2. pâslaru v., bordei d., influence of lysomax phospholipase on bread’s rheological properties, annals of the suceava university, 2008, 6 (1):182-186. 3. sîrbu a., pâslaru v., effect on lysomax formulation on rheological behaviour of doughs, journal of agroalimentary processes and technologies, 2006, 12 (1), p. 199-208. 4. segal r., biochimie, galati, editura academica, 2006. 5. giurea a.m., lipidele din cereale şi făină, bul. inform. pt. industriile de morărit şi panificaţie, 2002, 13(1), p. 4 36. 6. pâslaru v., using phospholipases in bread making. technological and biochemical aspects, doctoral degree, galati, 2008. 7. cauvin s., zoung, l., baking problems solved, england, woodhead publishing ltd., 2004 microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_43-48.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 47 concerns on the development of ecumenical tourism in bucovina elisabeta botez1,constantina sălceanu1, oana – viorela nistor1, aura darabă1 1 bioingineering department, food science and engineering faculty, „dunarea de jos” university, 111 domneasca street, 800201, galati, phone/fax +40 236 460165, elisabeta.botez@ugal.ro abstract: the purpose of the study was to investigate the possibilities of the ecumenical tourism in northern moldavian monasteries’ area. the monasteries of putna, sucevita, moldovita, humor and voronet are great touristic attractions of the beautiful bucovina lands and being visited by a large number of foreign and romanian tourists. ecumenical tourism is a matter that already exists for centuries. it keeps some of the characteristics of pilgrimage, but it has greatly evolved over years. today, ecumenical tourism asks for a higher level of culture and conditions for tourists. thus, if in this context the tourists could assure their own necessities, they would be able to appreciate all the cultural objectives as well as the architecture, constructions, values, spiritual connotation and art. there are only two programs in romania for this kind of tourism: “know bucovina” and “easter in bucovina”, both promoted by anat and by the council of suceava county. the accommodation facilities for the monasteries’ visitors have been studied as well as transportation and advertising points of view in order to enlarge the sizes of the actual tourism which converge to a higher number of tourists. in order to improve the touristic activity it was developed a ten-question questionnaire which had been distributed to a sample of 200 tourists. key words: pilgrimage, “know bucovina”, “easter in bucovina”, questionnaire 1. introduction tourism is defined as “the word to describe the activity of traveling” (mill, 1990) or “tourism includes any activity that concerns the temporary movement on short term of people towards destinations far from their daily places and habits, along with the activities developed during their staying” (middleton, 1993). tourism means hiking or organizing and developing pleasure and recreation trips. starting from these definitions and from the ending of the word, “-ism”, we can conclude that it is “an action or process” (theobald, 1994). bucovina is one of the most attractive and visited touristic regions from romania. this area, well known nowadays worldwide, was awarded in 1975 with the international prize “pomme d’or”, awarded by the international federation of journalists and tourism writers. the ancient architectural monuments with internal and external frescoes from this part of the country were listed by unesco as monuments of universal art. (http://www.turisminbucovina.ro) types of tourism: a. cultural and ecumenical tourism: if we talk about the main touristic motivation from bucovina, the starting point is represented by the unique and wonderful monasteries, subscribed to unesco’s patrimony: moldovita, sucevita, humor, voronet, putna, arbore. the patrimony also enlists the museums, the ethnographic collection “ion gramada” and the black ceramic of marginea (pottery: expo sales show). this type of tourism can be developed without considering the fact that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 48 the region is on the land of bucovina monasteries. voronet monastery it is an historical architectural monument, the best representation of the moldavian architectural style. it was built between 26.05 – 14.09.1488 by stephen the great. voronet became famous all around the world for its exterior paintings: doomsday, ieseu’s tree, st. john the new’s prayer for the dead. another peculiarity is the usage of a certain type of blue, so called “blue of voronet”. this color was made by locals including in the composition “lazurit” paint. humor monastery it is a valuable medieval architectural monument, built in 1530 by the great chancellor teodor bubuiog. the first element that became famous was the open veranda with its unique exterior painting. these were made in 1536 by toma from suceava. the main piece that caught the eye was “constantinople siege”. red is the dominant color and the founder’s portrait is settled inside the crypt. moldovita monastery it was built in 1532 and painted in 1537. it is one of the most valuable medieval romanian art monuments from petru rares reign. the monastery appears to be a fortress composed from the actual church, the wall surrounding it with three massive towers on each side and the royal residency. the painting is of a great artistic value, similar with the one from voronet, being special through the figure humanization tendency and their presentation in movement. green and blue are combined in a very original way, being similar with the grass green and the blue sky. sucevita monastery built at the end of the 16th century, it is the most fortified monastery complex from moldavia. the inner yard was made to hide movilesti family and their huge fortune. in the exterior painting, red and green are the main colors. the monastery’s museum shelters embroideries, silver pieces, popular paintings, manuscripts, ancient books. these treasures confirm the cultural role that this monastery had. putna monastery the most important monument of stephen the great is situated at 80.5 km from pojorata. its construction started in 1466 but it was burnt in 1484. the renovation was made by the founder in 1498. the monastery’s church shelters the graves of the prince, his two wives and his sons. the museum holds embroideries, fabrics, silver pieces, valuable manuscripts, authentic medieval manuscripts. suceava citadel the central part, “musatin fort”, belongs to petru musat the1st. he was the one that gave up using scheia. alexander the good over-fortified scheia and the one who made it legendary was stephen the great. he built the exterior walls, the defensive ditch and the main entry. well defended, it was able to withhold the ottoman siege from 1476, when mahomed the second had to admit himself defeated. (http://www.romanianmonasteries.org/ro/b ucovina) b. the hunting tourism its destination is for hunters and fishermen. there is a rich hunting fund: bears, boars, mountain and birch rooster, stags and dears, rabbits etc. also, the fishing fund has its resources: trout, dace, barbell, boisten. the main species from the mountain area is the carpathian stag. c. hiking – it has as an advantage: beautiful marked routes (rarau – giumalau, calimani, rodnei etc.). it reminds us of the old “tartars’ way” that ends in carlibaba. these routes can be run by foot or riding a local’s horse. d. recreation tourism has all the means to be put in practice, considering the positive effects of the climate and the beautiful scenery. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 49 e. pleasure tourism: river rafting, paragliding, paintball, mountain biking and climbing, archery, riding classes, long walks through the mountains and practicing winter sports. the purpose of the study was to investigate the possibilities of the ecumenical tourism in northern moldavian monasteries’ area. 2. materials and methods: 2.1. materials visiting bucovina monasteries. collecting data from the talks with the monks. interviewing the tourists. a questionnaire regarding the infrastructure quality and the local ecumenical tourism. the questionnaire was made of 10 items, multiple choices. the questionnaire 1. which is the best known ecumenical area from romania? a) moldova b) bucovina c) dobrogea d) muntenia 2. would you be interested in the ecumenical tourism in romania? a) yes b) no c) may be d) i don’t know 3. what do you think would be the main obstacles for a tourist not to visit bucovina monasteries? a) the lack of transportation b) the lack of accommodation place into the monasteries or in the neighborhood c) the impossibility of ensuring a meal during the day 4. would you like to receive a meal during the monasteries visiting? a) yes b) no c) may be d) i don’t know 5. would you be excited that the offered meal to contain a specific monk menu? a) yes b) no c) may be d) i don’t know 6. would you like to be accommodated to the monasteries? a) yes b) no c) may be d) i don’t know 7. what measures should anat take? a) the organization of a touristic tour of the monasteries b) the assuring of the roads’ infrastructure to the monasteries gates c) the monasteries and the places of worship promotion 8. which do you think is the efficient method to promote the monasteries? a) by mass-media b) by churches c) by authorities from national and international information programs d) by tourism agencies 9. how do you think bucovina monasteries should improve their facilities? a) by eu funds b) by the city councils c) by the monasteries own funds d) from sponsorships 10. would it be good for the monasteries to apply to european funds in order to improve their facilities? a) yes b) no c) may be d) i don’t know 2.2. methods graphical data interpretation – pie graphics. swot analysis based on the comparison between the data collected on the spot and the solutions adopted for improving ecumenical tourism. swot analysis is the most important managerial technique used for comparing and solving the improvement strategies of a certain situation. the analysis starts with enumerating strong and weak points concerning the actual state of the objective. swot is an acronym, the letters coming from: strengths: strong points of the location, the resources it had, its advantages, distinctive components of the personnel, managerial activities. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 50 weaknesses: weak points, vulnerable areas, poor resources. opportunities: the ways to improve the purpose, ways to exploit the strengths and eliminate the weaknesses. threats: points of danger for achieving the goal, negative aspects coming from faulty resource management, generally, any type of risk. 3. results and discussion 3.1. questionnaire graphic interpretation: we interviewed a number of 200 persons, 75% women and 25% men. from these, 45% were undergraduates, 40% graduates, 10% postgraduates and 5% had gymnasium studies. the interview had been attained at sucevita and voronet monasteries and the assigned time for this purpose was 3 days. the interpretation of the principal questions from the questionnaire is presented in the following graphics, as the area with the highest ecumenical potential is illustrated in figure 1. figure 1 the area with the highest ecumenical potential the conclusion was that 93,33% from the interviewed people indicated bucovina as the area with the highest ecumenical potential, 21,11% moldavia, 1,11% dobrogea and 1,11% muntenia. the interest for the ecumenical tourism is remarked in figure 2. figure 2 interest for ecumenical tourism concerning the interest for ecumenical tourism, 80% answered “yes”, 8,88% “maybe”, 5,55% “i don’t know” and only 2,22% said “no”. the figure 3 presents the possible obstacles in monastery visiting. what do you think would be the main obstacles for a tourist not to visit bucovina monasteries? lack of transportation lack of accomodation not having a meal insured during the visit figure 3 possible obstacles in monastery visiting at this question, 85.55% considered that the biggest problem for the ecumenical tourism in bucovina is the lack of means of transportation. at present, the access is possible only by car. the second problem for 40% of the tourists is the lack of accommodation inside the monasteries (only putna disposes of 120 places for accommodation). only 5.55% considered that not insuring one meal per day when visiting a monastery is a problem. when they had to choose between a meal during the visit and accommodation, 55.55% chose the meal and 63.33% chose the accommodation. figure 4 presents the possible measures the area with the biggest ecumenical potential bucovina moldova dobrogea muntenia would you be interested in the ecumenical tourism in romania? yes may be i don't know no food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 51 that have to be taken by anat for the development of bucovina ecumenical tourism. figure 4 measures that have to be taken by anat concerning the measures that anat has to take, 56,66% thought that there should be some funds allocated for the infrastructure, 48,88% considered that the promotion of the monasteries is necessary and 15,55% would organize a tour of the monasteries. at the last question, 63,33% answered that mass-media advertising would be the most efficient, 38,88% chose promoting through national/international information programs, 31,11% would promote only inside the churches and 5,55% would use tourism agencies. the funds related to the improving of the accommodation inside the monasteries are represented in figure 5. figure 5 improving accommodation inside monasteries from the interviewed people 64,44% considered that the monasteries from bucovina should insure themselves with funds for developing ecumenical tourism, european funds, 52,22% chose sponsorships, 21,11% public money given by the authorities and only 3,33% chose monasteries’ own funds. the possibility of applying for european funds/ european grants is the subject of figure 6. figure 6 the possibility of applying for european funds/ european grants when answering about bucovina monasteries applying for european funds, the interviewed people 56,66% said “yes”, 25,55% “maybe”, 12,22% “i don’t know” and 2,22% thought that there is no need of european funds. 4. conclusions applying swot analysis demonstrated its efficiency in establishing the common interest of sustainable development regarding the ecumenical tourism in bucovina. this analysis has conclusive results when having a significant number of subjects with different interests and different ways of achieving the final goal. the main criterion is that all of them have to participate at the follow up and implementing possible solutions. from the visit and the talks with the monasteries’ management, it has been concluded that their involvement in promoting and improving the conditions is minimal. the monks consider that promoting the monasteries is not a task for them to do. how do you think bucovina monasteries should improve their facilities? using european funds with the help of city council using monasteries funds from sponsorships would it be good for the monasteries to apply to european funds in order to improve their facilities? yes may be i don't know no what measures should anat takes? organizing a monasteries' tour insuring transportation promoting the monasteries food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 52 table 1 swot analysis interpretation strenghts weaknesses receiving ‘pomme d’or’ international award in 1975, given by the international federation of journalists and tourism writers having the possibility to visit unique places (putna, sucevita, moldovita, humor, voronet) practicing hunting, recreation and pleasure tourism as well as hiking the possibility of visiting any time of the year history and unique customs the ‘1000 churches’ memorial houses and museums reduced number of national/international information programs defective promotion of the monasteries lack of transportation lack of accommodation inside the monasteries (excepting putna) not having a meal included during the visit limited possibilities of applying for european funds opportunities threats restoring historical monuments, especially the monasteries encouraging new forms of tourism (e.g.: scientific, adventure etc.) phare funds including agro-touristic mansions in the national network of tourism agencies developing some resorts: campulung moldovenesc, cacica, botus (ethnographical resort) expansions and renovation of infrastructure no trademark for ‘bucovina’ as a brand defacement of art and architecture monuments faulty collaboration between local councils analyzing the present situation, it has been discovered that arbore church and voronet monastery can not be the beneficiaries of a rehabilitation program from phare funds, because of the neglect of the city council and suceava and radauti archiepiscopate. a phare program, started in 2003, worthing 1,5 € millions, having as main beneficiary suceava city council, should have already modernized 13 monasteries and churches so far, some of them belonging to unesco patrimony. this project was a failure because of the common neglect. although the solutions for reviving the ecumenical tourism are pretty complicated at first sight, they can be solved from a source of european funding, regio projects, espon projects etc. (http://www.afaceripublice.ro/investigatie) 5. references 1.http://www.romanianmonasteries.org/ro/bucovina 2.http://www.turismbucovina.ro/ghid%20bucovinea n.htm 3. http://www.turisminbucovina.ro 4.http://www.scritube.com/geografie/turism/etim ologia-cuvantuluiecoturis133522615.php 5. s.c.e., sarino, tourism principles, quote (mill, 1990), (middleton, 1993), 2009 6. t., filipas, analiza strategica swot a unei intreprinderi/organizatii, 2005 7. c., băbăiţă, impactul factorilor organizaþionali asupra satisfacþiei consumatorului de servicii turistice, 2006 8. agenţia de monitorizare a presei – active watch, transparenţa fondurilor europene în românia – studiu de caz, 2009 9. http://www.afaceripublice.ro/investigatie 10. commission of the european communities, basic orientations for the sustainability of european tourism, communication from the commission, brussels, 2003 11. commission of the european communities, a renewed eu tourism policy: towards a stronger partnership for european tourism, communication from the commission, brussels, 2006 12. a., holden, environment and tourism, routledge, london, 2000 13. a., ispas, imaginea româniei ca destinaţie turistică, în revista convorbiri economice, nr. 4/aprilie 2007, p.48-53. 14. www.wto, tourism vision 2020 15. www.mturism.ro/studii/studii internaţionale food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 24 rheological, textural, color and physico-chemical properties of some yogurt products from the spanish market *mircea-adrian oroian 1, isabel escriche 2, gheorghe gutt1 1,2ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1m.oroian@usv.ro, 1g.gutt@usv.ro *corresponding author 2universitat politecnica de valencia, food technology department, camino de vera s/n, valencia, spain, e-mail iescriche@tal.upv.es received 27 april 2011, accepted 20 may 2011 abstract: yogurt is nowadays one of the most consumed fermented milk products in the world due to its benefits for the human body. the purpose of this work is to study the rheological, textural and color aspects of different types of yogurts from the spanish market. for this study, two types of yogurt, blank yogurt and fruit yogurt respectively were selected; five samples for each type, each one with different concentration of total soluble content, fat content, protein and sugar content were analyzed. the study shows the existence of some correlations between some physico-chemical and textural parameters, so the highest adhesiveness and cohesiveness are reached by the sample with 10% fats, while the tension parameter is the highest for the sample with the lowest concentration of sugars, and the highest firmness is reached in the sample with the lowest concentration of fats. the color parameters are influenced by the presence of the food colorants, and their nature. the pca made provides some explanation on the two components of 100% of the total variance and it was observed that the 2 types of yogurt are situated in different groups. the analysis of variance (anova) conducted revealed that considering that a higher value of f-ratio means a more noticeable effect of the factor (type of yogurt) in a variable, the parameters tone and a* were more distinguished by the type of yogurt. © 2011 university publishing house of suceava. all rights reserved keywords: yogurt, viscosity, consistency, luminosity, water activity 1. introduction yogurt is produced by a fermentation process during which a weak protein gel develops due to a decrease in the ph of the milk. the ph of the milk is decreased due to the conversion of lactose to lactic acid by the fermentation culture bacteria. in the liquid milk, casein micelles are present as individual units. as the ph reaches ph 5.0, the casein micelles are partially broken down and become linked to each other under the form of aggregates and chains forming part of a three-dimensional protein matrix in which the liquid phase of the milk is immobilized. this gel structure contributes substantially due to the overall texture and organoleptic properties of yogurt and gives rise to shear and time dependent viscosity [1]. texture is one of the most important attributes used by consumers to assess food quality. texture is one of the main characters that define the quality of yogurt. the most frequent defects related to yogurt texture that may lead to consumer rejection food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 25 are apparent viscosity variations and the occurrence of syneresis [2]. yogurt rheological characterization is required for product and process development and to ensure consumer acceptability [3]. this characterization can be made using either instrumental or sensory measurements. the firmness of yogurt and the viscosity of just-stirred gel are greatly influenced by the amount of heat treatment the yogurt mix receives. heating unfolds the globular whey proteins and exposes sulphydryl groups, which react with other sulphydryl groups and disulfides and induce linkages and protein-casein aggregates [4, 5]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions. the binding of β-lacto globulin to the casein micelle seems to be responsible for the increase of gel strength [6, 7, and 8]. the purpose of this paper is to make a statistical analysis of the yogurt samples from the spanish market keeping into account physico-chemical, rheological, textural and color parameters using anova and pca analysis, and to notice which parameters make the difference. 2. materials and methods 2.1. materials for this study eight samples of yogurt were purchased from the spanish market: sample 1 –hacendado griego, sample 2 – hacendado cremoso natural, sample 3 – hacendado yogur natural, sample 4 – hacendado sin grasa, sample 5 – danone densa, sample 6 –hacendado fresa 0% grasa, sample 7 –activia con fresa, sample 8 – hacendado yogur cremoso con fresa. the samples were divided into two main categories: natural yogurt (blank samples) and fruit yogurt, respectively. the samples had different physical chemical composition (different concentrations of sugar content, fat content and protein content, respectively). 2.2.chemical analysis yogurts were analyzed for protein by the kjeldahl method (sr iso 17837:2009), fat by gravimetric method (sr en iso 1736:2009). 2.3. color measurement the color measurement was achieved with a konica minolta spectrophotometer cm 3500 d (konica minolta business technologies, inc. japan). the samples were fitted in plastic flask with 3.8 cm height and 6 cm diameter. to measure the reflected light, a diagram of 8 mm is opened. the reflection specters are registered and the cielab parameters are calculated at a 100 angle, using the d65 light: l*(luminosity, between 0[black], at 100 [white]), a*(+a*[red], -a* [green]), b*(+b*[yellow], b*[blue]) and h* [tone]. the color intensity c* is calculated with: c*=(a2+b2)0.5 (1) 2.4. texture yogurt samples were carefully scooped into acrylic cylindrical containers (70mm diameter 80mm height) with the help of a spatula to a depth of 50 mm. yogurt texture measurements were carried out with a stable micro systems texturometer model ta-xtplus (texture technologies corp., white plains, new york) with a 5 kg load cell. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 26 a combination of back extrusion and texture profile analysis (tpa) was used. a 35mm diameter solid rod (a/be35) was thrust into the cylindrical containers holding the test sample, so that the fluid flew upwards through the concentric annular space. all determinations were carried out in duplicates. the positive area represents the consistency; the positive force represents the firmness, while the negative force and negative area represents the cohesiveness and the index of viscosity, respectively. 2.5. rheological measurement viscosity measurements were carried out on the yogurt samples at room temperature (200c), with haake rheostress 1 rheometer, with control thermo bath, with a coaxial cylinder system. all the data were transformed using linear regression with the herschkel bulkley model: nk   0 (2) where: τshear stress, pa, τ0 – coefficient, k – consistency, pa·sn, γ – shear rate, s-1, n – flow index. 2.6. water activity the water activity of the yogurt samples was performed using the fast lab 2 water activity meter. 2.7. ph measurement ph measurements were made with a metler toledo ph.-meter, at 200c. 2.7. statistical analysis an analysis of variance (anova) (α=0.05) with least significant difference (lsd) test using statgraphics plus 5.1 was made for the data of physico-chemical, rheological, textural and color parameters. in addition to this, the data were analyzed by using multivariate techniques, applying the software unscramble version 10.1 (camo process as, oslo, norway, 2005). the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. a principal components analysis (pca) was applied to describe the relation among the chemical, rheological, textural and color parameters. 3. results and discussion the samples submitted to this study presented a fat level between 0.1 and 10%, respectively, proteins between 3.3 and 5.2% respectively and sugar 3.90-17.10%. the ph value was in the acid region, while the water activity was ranged between 0.98-0.99. the yogurt is a complex non-newtonian fluid with a great quantity of water which influences the rheological and textural behavior. the different concentration of proteins, fats, sugars generated different rheological and textural data set. the rheological data were transformed using the herschkel bulkley model, suitable for this type of fluid (r2 of the model was between 0.95-0.99). the viscosity ranged between 0.35 and 0.90 pa·s, respectively. the sample with the highest content of fat exhibited the highest viscosity, while the samples with the highest content of sugars presented the lowest level of viscosity. the samples of fruit yogurt seem to have the same viscosity due to the presence of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 27 fig.1. textural profile of blank yogurt samples: black line sample 1, dark blue line sample 2, red line sample 3 and blue line sample 4 hydrocolloids used to stabilize fruit in the yogurt. the flow index, n, has an interesting evolution; it ranges between 0.04 and 0.6, placing yogurt in the class of the pseudo plastic fluids. the sample with the highest content of fats and fat free samples presented the lowest flow index, while the fruit ones presented the highest flow index. the consistency index, k, is greater for the samples with a lot of fats or without fats, while the samples with fruits presented the lowest index. the texture is a property of the foods that always is related to a chemical physical system that is a colloidal system formed by two non-miscible phases. in the figures 1 and 2 the textural grams of the yogurt samples subjected to the study are presented. fig.2 textural profile of fruit yogurt samples, dark blue line sample 5, blue line sample 6, red line sample 7 and green line sample 8 it can be seen that in the case of blank samples, each one having a different textural behavior, while in the case of fruit yogurt there are three samples which do not have great differences in their behavior. that phenomenon could be generated by the presence of the same hydrocolloid in the composition of yogurt. the textural parameters of the yogurt samples analyzed ranged in a large spectrum; adhesiveness from 0.05 to 0.35 g, cohesiveness from 0.79 to 3.90 g, tension from 0.01 to 0.05 g and firmness from 0.06 to 0.31 g. it seems that there are some correlations between some physico-chemical parameters and textural ones, so the highest adhesiveness and cohesiveness is reached by the sample with 10% fats, while the tension parameter is the highest in the sample with the lowest concentration of sugars, and the highest firmness is got in the sample with the lowest concentration of fats. the yogurt color was measured in the sense of lightness, red-green axis a*, yellow-blue axis b*, tone and color intensity. the lightness l*(91.15) of the black samples are influenced by the content of fats, so the high lightness is achieved by the samples with the high percentage of fats, while the rest of the samples have the same level of this parameter. in the case of the fruit yogurt samples, the yogurt with 0.1% fat has the lowest level of lightness, while the fruit samples which have in their compositions anthocyans have the highest level of lightness (81.79). the samples with fruit have lower lightness than the blank yogurt due to the presence of food colorants (carmine and anthocyans). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 28 table.1 physicochemical, rheological, textural and color anova of the yogurt samples yogurt type f ratio blank (sd) fruit (sd) physicochemical parameters proteins [g/100g] 3.95a(0.57) 4.5a(0.60) 4.58ns fats [g/100 g] 4.1a (3.70) 1.82a (1.14) 2.53ns sugars g/100 g] 5.8b (4.44) 12.05a (4.90) 7.82* fruits [g/ 100g] 0b (0) 7.35a (3.83) 29.4*** ph 4.21a (0.08) 4.26a (0.04) 2.08ns aw 0.87a (0.30) 0.98a (0) 0.9ns rheological parameters viscosity [pa·s] 0.51a (0.23) 0.54a (0.14) 0.11ns n, flow index 0.18a (0.1) 0.37a (0.16) 2.82ns k, consistency index [ 166.53a (40.2) 16.23a (4.49) 4.59ns color parameters l* (luminosity) 89.9a (3.87) 78.64b(3.11) 99.08*** a* (red-green axis) -2.14b (0.51) 9.29a (2.3) 187.14*** b* (yellow-blue axis) 8.12a (2.14) 4.55b (1.96) 22.71*** c*(color intensity) 8.4b (1.35) 10.58a (1.70) 10.14** h*(tone) 104.58a (31.37) 27.26b (13.24) 263.13*** texture parameters adhesiveness 0.22a (0.13) 0.17a (0.04) 1.39ns cohesiveness 2.74a (1.36) 1.84a (0.45) 3.09ns tension 0.018a (0.02) 0.006a (0) 2.67ns firmness 0.19a (0.09) 0.07b (0.02) 12.33** note: ns not significant (p<0.05), * p>0.05, ** p>0.01, *** p>0.001 fig.3 pca scores of yogurt samples fig.4 pca loadings of the yogurt samples in the case of a*, the blank samples are situated in the negative region (more towards green), while the fruit sample on the positive region (more towards red) due to the presence of red colorants. all the b* samples are situated in the positive region (more towards blue). the tone of the samples is higher for the blank samples without colorants, while color intensity is higher for the colored samples. a pca was conducted to evaluate the global effect of the type of yogurt (blank, fruit) on the physical chemical, textural, rheological and color, from a descriptive point of view. figure 3 shows the sample scores and compound loadings of the pca analysis performed. it was found, that two principal components (pcs) explained 100% of the total variance in the data set. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 29 the pc1 explains 95% of the total variance and the pc2 explains 5%, respectively. there are two main differentiated groups of samples on the plot: one representing the fruit yogurt samples and the other one the blank samples. the loadings of each parameter on the principal components show that the grouping of different type is primarily influenced by certain parameters. in the central ellipse the parameters (tension, fat content, adhesiveness, ph, aw, protein content, viscosity, sugar content, flow index, firmness) could be seen which influence less the differential of the samples, while in the outer ellipse the parameters (consistency index, cohesiveness, luminosity, fruit content, a*, b*, tone, color intensity) with a higher influence. in order to know if the differences observed among different yogurt types for each parameter are statistically significant, an analysis of variance (anova) with one factor (type) was carried out (table 1). it is known that if p-value from the anova analysis is equal or superior to 0.05 there are not significant differences among types. taking these differences into account for the physico-chemical, rheological, textural and color parameter and considering that a higher value of f-ratio means a more noticeable effect of the factor (type of yogurt) in a variable, tone and a* were the parameters more affected by the type of yogurt. 4. conclusion the yogurt is a complex viscous food material due to the complexity of its nature and composition. rheological behavior of yogurt is influenced by sugars, fats and water. it seems that fats and sugars have a different influence on the flow index; the fats reduced the index, while the sugars increased it. the study revealed the existence of some correlations between some physicochemical parameters and textural parameters, so sugars and fats influenced more. the color parameters are influenced by the presence of the food from fruit addition, e.g. anthocyans, and its nature. 5. acknowledgment this paper was supported by the project "knowledge provocation and development through doctoral research pro-doct contract no. posdru/88/1.5/s/52946 ", project co-funded from european social fund through sectorial operational program human resources 2007-2013. 6. references 1. o donnell, h.j., butler, f., time-dependent viscosity of stirred yogurt. part i: couette flow, journal of food engineering, volume 51(3), p. 249254, (2002) 2. kroger, m. quality of yogurt. journal of dairy science 59(2): 344-350. (1975) 3. benezech, t., maingonnat, j.f. characterization of the rheological properties of yogurt-a review. journal of food engineering. 21, 447-472. (1994) 4. sawyer wh. complex between –lacto globulin and γ-casein. a review. journal of dairy science 52:1347–55. (1969) 5. kinsella h. milk proteins: physio-chemical and functional properties. crc crit rev food sci nutr 21(3):197. (1994) 6. bonomi f, iametti s, palgliarini e, peri c. a spectrofluorometric approach to estimation of the surface hydrophobicity modifications in milk proteins upon thermal treatment. milchwissenschaft 43:281–5. (1998) 7. mottar j, bassier a, joniau m, baert j. effect of heat-induced association of whey proteins and casein micelles on yogurt texture. journal of dairy science 72(9):2247–56. (1989) 8. bonomi f, iametti s. real-time monitoring of the surface hydrophobicity changes associated with isothermal treatment of milk and milk protein fractions. milchwissenschaft 46:71–4, (1991). microsoft word 3 journal nr 4 2010 din 8 noiembrie_21-26.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 25 assessment of milk allergens into foodstuffs mihaela begea1, elena bălăuţă1, alexandrina sirbu2 1 ica research&development, bucharest, 202 /s6 splaiul independentei st., romania, ela_begea@yahoo.com 2 constantin brancoveanu university, fmmae ramnicu valcea, 39 nicolae balcescu bld, valcea county, romania, sirbu.alexandrina@rdslink.ro abstract. allergens represent specific substances, such as certain proteins, that are able to give an immune reply very dangerous in the case of the sensitive individual. food allergens are normally harmless for the non-allergic individuals; but for some people these allergies result in severe symptoms, reasons for which these substances have engendered food safety issues. allergens represent biological hazards for animals and people as well. it is obvious necessary to detect the presence of allergens in agri-food raw materials, food products and fodder for their entire monitoring of the food and feed chain, also taking into account the major financial losses generated by contamination. food producers must protect allergic people using clear labelling for their food products. as regards the food allergens, in the eu legislation there are no limits specified in the legislation, only the mention of their likely presence on the labels is compulsory. the paper shows the incidence of certain compounds with potential allergen activity in foodstuffs containing milk by using the s-elisa veratox method. we tested milk allergens from a few types of food matrix containing undeclared milk or its derivatives (wafers with cream, instant coffee, sauces). the results indicated different degrees of contamination with potentially allergenic proteins depending on the food matrix type. keywords: food allergens, food safety, milk protein, elisa, label introduction 1. food allergens allergens are specific substances, such as pollen, food or drugs, which introduced into the body through the digestive, air etc.., produce pathological manifestations caused by hypersensitivity reactions of the immune system. generally speaking, the substances with allergenic potential in food are certain proteins or glycoproteins [1]. in fact, foods contain many proteins, but only a small fraction is allergens. although the structural properties of proteins that cause allergic reactions have not been characterized completely, known food allergens in general have molecular weights between 10 and 70 kda, and are stable molecules in the sense that they are resistant to processing, cooking, and digestion. in the case of allergic individuals, these substances stimulate the immune response by inducing the production of allergen-specific ige. [2] experience shows that the immune response can be initiated by eating a food, which contains traces of that allergen. the amount of allergen that can generate immune response varies from one allergic individual to another one. it seems that food matrix can influence the responses to individual proteins; consequently, the food matrix must be taken into account when developing models for allergenic potential assessment [3]. moreover, any type of food is food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 26 potentially allergenic, but the majority of food allergies are caused by a small group of foods, such as: cows' milk, ground peanuts and tree nuts (almonds type, pistachio etc.), wheat, vegetables, eggs, fish and other seafood, soybean and other ingredients that contain protein derived from these food groups (over 90% of allergic reactions are induced by array of protein compounds of these types of foods). over 170 foodstuffs have been identified as having potentially allergenic compounds in composition, fruits included (strawberries, plums, etc.), sesame seeds, sunflower seeds, poppy seeds, mollusks, peas, lentils, beans, with its different varieties. besides protein allergens, foods may contain also haptens, which are substances (small molecules), different from the proteins that may become allergens only when attached to a large carrier such as a protein. [4] world’s statistics figures show that approximately 4% of adults and 8% of the children are allergic to different foods. the adverse effects induced by food allergens, even when they are ingested in very small amounts, range from rash and tingle to anaphylactic shock and in severe cases to death. for this reason, assessment of food allergens became part of risk assessment in the framework of food safety management system. since 2005, according to eu directive 2003/89/eg of 10 november 2003, in the eu countries all food products must comply with the changed labelling directive. in that way, the allergen ingredients must be declared on food labels, since they can induce allergic reactions. a list of 12 allergens, namely gluten containing grains (e.g. rye, wheat, barley), shellfish, eggs, fish, peanuts, soy, milk, nuts, celery, mustard, sesame seed, sulphur dioxide and sulphites in concentrations over 10 mg so2/kg, must be compulsory mentioned on the food label. [5] laws regarding the labelling of food allergens in the u.s. came into force in january 2006, as following the regulations of the act on food labelling and consumer protection (falcpa) of 2004. in this sense, it requires that food manufacturers use in their plants such processes that reduce or even eliminate cross-contact among the non-allergenic foods and food allergens. the u.s. federal food, drug, and cosmetic act was amended and the document imposes new requirements for the labelling of foods containing major food allergens. [6, 7] therefore, it is necessary to test compounds potentially allergenic food for any of the following purposes:  it is necessary for their identification and assessment; food manufacturers are required to protect allergic people through a clear labelling of their products, with the complete list of the food ingredients;  it is a measure that an ingredient potentially allergen, which is not mentioned on the foodstuff’s label should not accidentally have entered into product, saying it does not contain any;  it is a tool for identification of the cross contamination sources among allergens and non-allergic products during food processing. 2. milk allergens milk allergy is food allergy, which has as main symptoms gastrointestinal, dermatological and respiratory ones. these can be skin rash, hives, vomiting, and gastric distress such as diarrhoea, constipation, stomach pain or flatulence, or other clinical disorders. the symptoms may occur within a few minutes after exposure to immediate reactions, or after hours or even after several days in delayed reactions. although the symptoms and treatment are similar to milk allergy and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 27 milk protein intolerance, is not the same disease. in general terms, milk allergy is one of the most popular food allergies. moreover, høst (2002) expressed that milk allergy is the most common food allergy in early childhood, and it affects approximately 2…3% of infants in developed countries. the prognosis of cow's milk allergy is with a remission rate of around 85-90% of the affected children once they surpass 3 years of age, but there is the risk to develop adverse reactions to other foods, later in childhood. [8] cow's milk has 3.2% protein composition, consisting of:  80% caseins ( s1-, s2-, βand κ-) (main part of coagulum/ curd),  20% lactoserum (β-lactoglobulin, -lactalbumin, bovine serum albumin) (main whey protein). milk basic proteins provide nutritionally important benefits if used as a food ingredient for dairy products, as well as in other foods. sometimes, milk or its derivates may be present like food contaminant of raw materials or certain processed foodstuffs. regarding the cow’s milk allergenicity, there are studies on milk proteins identified as allergens and their epitopes were characterized. the results indicated that most milk proteins, even proteins present in low concentrations, are potential allergens, but no particular structure or function is associated with milk allergenicity [9]. in fact, molecules of bovine milk proteins have numerous epitopes, which are located in hydrophobic parts of the molecules where they are inaccessible for ige antibodies in the native conformation of the proteins but become available after digestion. [10] as examples of allergenic compounds we can mention -s1-s2-caseins, lactoglobulin, -lactalbumin, immunoglobulin, and lactoferrin in milk. referring to the milk allergens, lactoglobulin reactivity is lower, but through the heat treatment of milk, it interacts with lactose, which makes its reactivity increase. allergenic factors generally occur in the early stages of heating, when premelanoidins are formed, compounds involved in these phenomena. it is considered that the casein products are responsible for allergies in adulthood, while β-lactoglobulin is mainly responsible for children's allergies to milk. other authors, based on molecular characteristics and expected exposure of milk basic proteins, expresses opinions according to which protein components in milk are unlikely to present any increased risk of allergy for milk allergic subjects or of cross-reactivity for other allergic subjects. [11] however, food products containing milk proteins (caseins, lactoglobulins etc.) need to be labelled as containing milk as a caution to warn milk allergic subjects of the potential risk of allergic reactions. in that way the food labelling directives in the us and the european union are clear, as long as it has not been demonstrated that milk proteins are free from milk allergens. in conclusion, it is recommended to evaluate the presence of caseins and βlactoglobulin in various foods with the aim to prevent food safety issues and for a suitable labelling of foodstuffs. materials and methods the purpose of this study was to survey the natural occurrence of the milk allergens in food products declared as not containing milk or milk derivates. different food matrices were tested, namely cream wafer, instant coffee, and dressings/ sauces. for this study 17 samples were assessed. the testing of presence of milk allergens was performed by using the neogen veratox kits (quantitative milk allergen test kits, code 8470). the allergens test is food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 28 based on the sandwich elisa (s-elisa) technique. [12] the milk residues are extracted from samples by using a buffered salt solution (pbs) through stirring into a water bath at preset temperature of 600c. the extracted milk residues thus obtained are collected and then added into the capture antibody wells and incubate. the unlinked residues are then washed and the linked ones are treated in a second stage with a detector antibody linked to the specific enzyme. the detector antibody is bound to the residue already linked by capture antibody. after a second washing step, the specific substrate is added and the substrate reacts with the bound enzyme conjugate to produce coloured reaction. then the reagent stops ass the colour reaction is added and the test wells with standards and samples are analyzed. the results are read by using a microwell reader with a filter at 650 nm as optical density, in equipment stat fax. type 321 plus, from awareness technology inc., sua. based on the optical densities curves, milk allergens concentration is calculated using a software neogen type log/logit. the equipment and apparatus required to perform these experiments is the following: analytical balance, accurately weighing 0.0001 g; reader veratox state neogen fax 303 plus, 650 nm filter, mill for grinding solid samples; unimax orbital shaker 1010, equipped with thermostatic chamber (1000 heidolph incubator) or water bath and thermostatic mixing at 60 ˚ c ± 1 ˚ c; timer. the performance characteristics for neogen veratox kits (quantitative milk allergen test kits, cod 8470) are the following:  range of quantitation 2.5 to 25 ppm  standard deviation (std) – 0.1  uncertainty (ua) – 8.3  repeatability, r – 0.1  relative standard deviation (std) rel, % 0.3. criterion for acceptance/ rejection of the results is the following: curve correlation coefficient r ≥ -0.980. if the curve correlation coefficient is r <-0.980, the analysis is repeated. the general procedure to determine allergens in milk is briefly shown in figure 1. figure 1. procedure for the determination of milk allergens the reagents used in the kits contained specific solutions (standard solutions of different concentrations, extraction solutions, wash buffers, substrate solutions, conjugate solutions, solutions to stop), powdered concentrated solvent extraction of pbs 10 mm, concentrated washing buffer pbs-tween, extraction additive, deionised or distilled water. whole milk powder nist, code rm 8435 was used as reference material. but the samples used for determination of the food allergens are end-products, not ingredients. for data interpretation we used analytical software spss for windows. sampling preparation of the extraction solution (pbs) extraction (5 g / 5 ml sample) filtration / separation analysis itself food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 29 the analysis method using elisa kits represents a viable alternative to hplc and fluorimetric methods for the determination of allergens, which are more expensive methods and require much more time working and more sophisticated equipment as well. certain specific conditions are imposed in order to determine the milk allergens in foodstuffs, namely:  milk allergens must be extracted separately from those for other food allergens, like peanuts or egg residues; reagents are specific for each target food allergen, too;  although partially digested milk proteins may be undetectable by elisa analysis, the residues of allergenic proteins could be active;  cross contamination must be avoided. results and discussion the samples evaluated during experiments consist of different food matrices, as follows: 1. instant coffee assortments, 2. dressings – vinaigrette sauces, and 3. wafer with lemon and chocolate cream. the analysis samples were taken from the foodstuffs available in stores. the overall results indicated lower values for milk allergens present in the abovementioned samples and were registered in instant coffee, while the highest values were registered in cream wafers. as it is shown in figure 1 and table 1, the majority of values obtained in determining milk allergens from coffee varied between 0 and 1 ppm, with an average around 0.745 ppm. exception made the eighth sample, namely instant coffee c-3, in which a higher concentration of 2.3 ppm milk allergen was registered, as well as the seventh sample, instant coffee c-2 that was free from milk allergen. case number 1110987654321 2.5 2.0 1.5 1.0 .5 0.0 -.5 high_cof low_coff coffee figure 1. low-high chart of set of coffee samples table 1. statistics of coffee set 11 .00 2.30 .7455 .5854 11 coffee valid n (listwise) n minimum maximum mean std. deviation descriptive statistics the results obtained from the analysis of vinaigrette sauces with milk contaminants are listed in the table 2. figures indicated a contamination with more than 1 ppm milk allergen for each sample. table 2. quantitative determination of milk allergens in vinaigrette sauces no. matrix results (ppm) 1 vinaigrette sauce natural -1 1.0 2 vinaigrette sauce natural -2 1.4 the highest contamination with milk allergen was registered in cream wafers. for the set of samples the minimum value was 15.3 ppm, while the maximum one was 57.4 ppm, and the mean reached 38.875 ppm (see also figure 2). generally speaking, the wafers with chocolate cream had a higher content of milk allergens in comparison with lemon ones. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 30 high 1.001.001.001.00 70 60 50 40 30 20 10 0 ref wafer figure 2. graph for set of cream wafers samples conclusion the testing of milk proteins occurrence in foodstuffs ensures food manufacturers that a potentially unsafe/allergenic ingredient, unspecified on the label is not really found in those foods. in this purpose, some types of foodstuffs declared as not containing milk or its derivates were tested for milk allergens. consequently, several samples of instant coffee, dressings and cream wafers were assessed by using veratox elisa method for determining the total milk allergens. after analysis performing, there was solely a coffee sample free from milk allergen. the lowest values for milk allergen natural occurred in samples were registered in coffee samples, in general less than 1 ppm. the analyzed vinaigrette sauces samples were characterized by a maximum contamination of 1.4 ppm milk allergens. also the data showed an allergen milk contamination at maximum level in the case of wafers samples. although the prevention of crosscontamination with food allergens is a major component of haccp system, our results indicate that there is plenty of foodstuffs in stores that could be contaminated by food allergens, which are not described on the label goods in accordance with the law. references 1. gary a. bannon, what makes a food protein an allergen?, curr. allergy asthma reports, 2004, 4, 43–46 2. samuel b. lehrer, w. elliott horner, gerald reese, steven taylor, why are some proteins allergenic? implications for biotechnology, food science and nutrition, 1996, 36(6), 553–564 3. frank van wijk, the effect of the food matrix on in vivo immune responses to purified peanut allergens, toxicological sci., 2005, 86(2), 333–341 4. haptens(http://medical.dictionary.thefreedictio nary.com/haptens), last accessed on: august 2010 5. directive 2003/89/ec of the european parliament and of the council of 10 november 2003 amending directive 2000/13/ec as regards indication of the ingredients present in foodstuffs, official journal of the european union, 2003, l 308/15-18 6. federal food, drug, and cosmetic act (http://www.fda.gov/regulatoryinformation/legislati on/federalfooddrugandcosmeticactfdcact/default.ht m) last accessed on: august 2010 7. food allergen labeling and consumer protection act of 2004 (falcpa) (http://www.fda.gov/food/labelingnutrition/food allergenslabeling/guidancecomplianceregulatory information/ucm106890.htm) last accessed on: sept 2010 8. arne høst, frequency of cow's milk allergy in childhood, ann allergy asthma immunol., 2002, 89(6 suppl. 1), 33-7 9. jean-michel wal, bovine milk allergenicity, ann. allergy asthma inmunol., 2004, 93 (5 suppl. 3), s2-s11 10. jean-michel wal, structure and function of milk allergens, allergy, 2001, 56, 35–38 11. richard e. goodman, et al, assessment of the potential allergenicity of a milk basic protein fraction, food chem. toxicol., 2007, 45(10), 17871794 12. veratox elisa method for determining the total milk allergens food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 27 determination of dioxins and furans from eggs and oils, through high resolution gas chromatography in combination with high resolution mass spectrometry monica catană1, luminiţa catană1, mioara negoiţă1, enuţa iorga1, alina bâlea1, gabriela lilios2 1national institute of research&development for food bioresources – iba bucharest, 021102, bucharest 2, 6 dinu vintila street, romania, e-mail: mona.catana@bioresurse.ro 2ovidius university, 900527, constanţa, 124 mamaia street, romania, e-mail: liliosgabriela@yahoo.com abstract. dioxins (polychlorinated dibenzo-p-dioxins – pcdd) and furans (polychlorinated dibenzofurans – pcdf) represent a group of chemical substances with high toxicological potential, which are persistent within environment and which can be accumulated within organisms through food chain. in this paper we present the results of the performed researches for dioxins and furans determination in eggs and oils, through high resolution gas chromatography in combination with high resolution mass spectrometry. egg and oil samples available in commerce were analyzed. within the performed experiments, some steps were taken: sample preparation for testing, fat extraction, extract clean-up and concentration, separation, identification and quantification of different native compounds of dioxins and furans. fat extraction from eggs was achieved in many more steps with organic solvents. extracts cleanup was achieved on multiple columns, using different absorption materials (silica gel, aluminium oxide, florisil). concentration of cleaned extract was achieved under nitrogen flow, at 40ºc and pressure 5 psi.separation, identification and quantification of different compound pcdds/pcdfs were achieved by complex equipment: a system of two high resolution gas chromatographs coupled with high resolution mass spectrometer. in the case of analysed oil samples, no item of the native congener of dioxins and furans was detected. the total concentration of dioxins and furans in the analysed egg samples was expressed in toxic equivalents (teq) and was in the range: 0.0216 – 0.034 pg whopcdd/pcdf-teq/g fat, being under maximum allowed limit by regulation of the european commission 1881/19 of december 2006 (3 pg who-pcdd/pcdf-teq/g fat). keywords: high toxicological potential, furans, fat extraction introduction dioxins (polychlorinated dibenzo-pdioxins – pcdd) and furans (polychlorinated dibenzofurans – pcdf) represent a group of chemical substances with high toxicological potential, which are persistent within environment and which can be accumulated within organisms through food chain. decomposition of dioxins in the external environment is extremely slow, so that dioxins can be accumulated in food chain, animals having in their bodies (through bioaccumulation) higher concentrations (hundreds and thousands times) than plants, water and soil [1]. dioxins have 75 congeners, of which 7 are the most toxic, and furans have 135 congeners with variable toxicity. among these, the compound with the highest toxicity is: 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (2,3,7,8-tcdd) [2]. because of their extreme toxicity, dioxins and furans, at european and international level, are given special attention on the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 28 monitoring of these contaminant concentrations, both in environment (water, air, soil) and in foods. analytical methods for determination of dioxins and furans in foods use high resolution gas chromatography in combination with high resolution mass spectrometry, present high sensitivity and selectivity. the first method for determination of dioxins and furans through high resolution gas chromatography in combination with high resolution mass spectrometry, was elaborated by u.s. environmental protection agency office of waterengineering and analysis division, under the coordination of professor dr. william a. telliard in 1994 [3]. in this paper we present the results of the performed researches for dioxins and furans determination in eggs and oils, through high resolution gas chromatography in combination with high resolution mass spectrometry. egg and oil samples available in commerce were analyzed. experimental we determined the contamination degree by dioxins and furans of eggs and oils, through high resolution gas chromatography in combination with high resolution mass spectrometry, within the performed experiments. we analyzed samples available in commerce. within the performed experiments more steps, were taken: preparation of test sample, fat extraction, extract cleanup and concentration, separation, identification and quantification of different compound pcdds/pcdfs [3]. fat extraction from eggs was achieved in many more steps with organic solvents (ethyl alcohol hplc grade (99.7%, v/v), diethylether pico grade, n–hexane pico grade). extracts cleanup was achieved on multiple columns, using different absorption materials (acid silica gel, aluminium oxide, florisil activated with ultra pure water). the concentration of cleaned extract was achieved under nitrogen flow, at 40ºc and pressure 5 psi, about 15-18 minutes. in the case of oil samples, cleanup and, subsequently concentration of cleaned extract were achieved through the same procedures, as in the case of egg samples. separation, identification and quantification of different compound pcdds/pcdfs were achieved by a complex equipment: system of two high resolution gas chromatographs in combination with high resolution mass spectrometer (capillary column 5% phenyl – 95% dimethylpolysiloxane, 5ms, l = 30 m, di = 0.25 mm, thickness film = 0.1 µm; carrier gas = he 6.0, flow of carrier gas = 15 ml/min; high resolution mass spectrometer – ionization type = ei+; ionization energy = 30–50 ev; resolution = 10,000; source temperature = 260ºc). in order to achieve calibration curves of those 17 native congener of dioxins and furans from the analyzed egg samples, we used standard solutions s1, s2, s3, s4, s5 (solutions certified bcr – 614, lgc promochem, wesel, germany). we also used the following internal standards: standards for verification of extraction efficiency–s6, recovery standards–s8, quantification standards–s7. results and discussion within the performed experiments, we made a calibration curve for each interest compound, thus obtaining 17 calibration curves. the compounds for which the calibration curves were made are the following: 2378-tetracdd, 12378pentacdd, 123678-hexacdd, 123789hexacdd, 123478-hexacdd, 1234678heptacdd, octacdd, 2378-tetracdf, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 29 23478-pentacdf, 12378-pentacdf, 1234678-heptacdf, 123789-hexacdf, 123478-hexacdf, 234678-hexacdf, 123678-hexacdf, 1234789-heptacdf, octacdf. the calibration curve is used to calculate relative response factor for each congener of interest. relative response factors are used with 13c12-labeled dioxins and furans congeners, which are added into sample, in order to determine the mass of native congeners of interest through isotope dilution. figures 1 and 2 show the calibration curves for 2378-tetracdd and 12378-pentacdf. r at io o f a re as 1 2 3 4 5 6 7 8 specified amount 10 20 30 40 50 60 70 80 2378-tcdd (linear fit) stddev = 0.026 rsd = 1.233 c f = 0.99994 1 y = ax + b (a=0.100 b=0.015) specified amounts figure 1. calibration curve for 2378-tetracdd the relative response factor for each congener is defined and calculated by the following equation (1): nll lnn caa caa rrf    )( )( 21 21 (1) where: rrf – relative response factor of the native cdd/cdf compound against the labelled cdd/cdf compound (a1n+a2n) – the areas of the two strongest ions (m/z) in the molecular ion cluster, for native cdd/cdf compound in the standard solution (a1l+a2l) – the areas of the two strongest ions (m/z) in the molecular ion cluster, for the labelled cdd/cdf compound in the standard solution cn – the concentration of the native compound in the calibration standard cl – the concentration of the labelled compound in the calibration standard r at io o f a re as 5 10 15 20 25 30 35 40 sp ecified amount 50 100 150 200 250 300 350 400 12378 -pecdf (linear fit) stddev = 0.06 6 rsd = 0.636 cf = 0.999985 y = ax + b (a=0 .099 b =6.92 8e-003) specified amounts figure 2. calibration curve for 12378pentacdf isotope dilution quantization we added a known amount of labelled compounds (surrogates) to every sample prior to extraction and correction for recovery of the pcdds/pcdfs because the native and their labelled analogues exhibit similar effects upon extraction, concentration, and gas chromatography. using the surrogate responses from the sample run, and the rrf values, recovery corrected concentrations of pcdds/pcdfs are calculated directly. the concentration of the native congener i, in sample is calculated using the following equation (2): rrfexaexa csexaexa c ll nn ex    )[( ])[( 21 21 1 (2) where: cex – the concentration of the native cdd/cdf in extract (a1exn + a2exn) – the areas of the two strongest ions (m/z) in the molecular ion cluster for the native cdd/cdf surrogate compound in the sample extract (a1exl + a2exl) – the areas of the two strongest ions (m/z) in the molecular ion cluster for the labelled cdd/cdf surrogate compound in the samples extract food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 30 cs1 – concentration of the labelled compound in the sample extract rrf – relative response factor, response factor of unlabelled relative to the 13c12labeled internal standard recovery ratios of the internal standards are calculated using the equation (3):     100 )21()21( )21()21(     nnll nnll exaexaaa aaexaexa r (3) where: (a1exl +a2exl) – the areas of the two strongest ions (m/z) in the molecular ion cluster for the labelled cdd/cdf compound in the sample extract (a1n + a2n) – the areas of the two strongest ions (m/z) in the molecular cluster of the performance (recovery) internal standard in the standard injection (a1l + a2l) – the areas of the two strongest ions (m/z) in the molecular ion cluster for the labelled cdd/cdf compound in the samples extract (a1exn +a2exn) – the areas of the two strongest ions (m/z) in the molecular ion cluster of the performance (recovery) internal standard in the sample injection the concentration of the native congener i, in sample is corrected by recovery factor. as food samples contain, in generally, complex mixtures of different congeners of dioxins, the concept – toxic equivalency factors (tef) was developed, in order to facilitate the evaluation of their risk on human body. toxic equivalency factors (tef) to evaluate human risk were established by specialists in the field, based on the conclusions of the world health organization, at stockholm, sweden, on 15-18 of june 1997 (table 1). table 1. toxic equivalency factors of cdd/pcdf no. congener pcdd/pcdf tef (who 1997) 1. 2378-tetracdd 1.0 2. 12378-pentacdd 1.0 3. 123478-hexacdd 0.1 4. 123678-hexacdd 0.1 5. 123789-hexacdd 0.1 6. 1234678-heptacdd 0.01 7. octacdd 0.0001 8. 2378-tetracdf 0.1 9. 12378-pentacdf 0.05 10. 23478-pentacdf 0.5 11. 123478-hexacdf 0.1 12. 123678-hexacdf 0.1 13. 123789-hexacdf 0.1 14. 234678-hexacdf 0.1 15. 1234678-heptacdf 0.01 16. 1234789-heptacdf 0.01 17. octacdf 0.0001 the concentrations of each native congener of dioxins and furans in each analyzed egg sample, are multiplied through own toxic equivalency factor and, then, are summarized, giving the total concentration of dioxins and furans, expressed in toxic equivalents (teq). ).( tefxconcteq i  (4) where: teq – toxic equivalents conc. xi – concentration of the native congener i, of dioxins or furans tef – toxic equivalency factor in the case of the analyzed egg samples, total concentration of dioxins and furans was also expressed in the following forms: upper bound who teq (a), in pg/g fat "superior limit of teq" (teq-value calculated by including the full value of the loq for non detected congeners) lower bound who teq (a), in pg/g fat "inferior limit of teq" (teq-value calculated by including quantified congeners only) in the case of those 6 analyzed egg samples, the only detectable native congener was 2378-tcdf, its concentration being in the range 0.22 – 0.34 pg/g fat (figure 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 31 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 23 78 -t c d f , p g /g f at i ii iii iv v vi egg s am ple concentration of 2378-tcdf in eggs 2378tcdf(pg/g fat) figure 3. concentration of 2378-tcdf in eggs in the case of those 6 analyzed egg samples, the total concentration of dioxins and furans, expressed in toxic equivalents (who-teq(a)) also varied in the range: 0.0216 – 0.034 pg who-pcdd/pcdfteq/g fat, being under the maximum allowed limit by regulations of the european commission 1881/19 december 2006 (3 pg who-pcdd/pcdf-teq/g fat). the "superior limit of teq" (upper bound who teq (a)), was in the range 0.1627 – 0.2191 pg who-pcdd/pcdfteq/g fat, being under the maximum allowed limit by regulations of the european commission 1881/19 december 2006 (3 pg who-pcdd/pcdf-teq/g fat). figure 4 shows the total concentration of dioxins and furans for each analyzed egg sample, expressed in toxic equivalents (teq (a)), as well as upper bound who teq (a), lower bound who teq (a) respectively. u pp er b ou nd w h o te q (a ) w h o te q (a ) lo w er b ound w h o te q (a ) i ii iii iv v vi 0 0,05 0,1 0,15 0,2 0,25 pg / g fa t s ample total con ce n trati on of di oxi n s an d fu ran s i n e ggs i ii iii iv v v i figure 4. total concentration of dioxins and furans in eggs when determining dioxins and furans in egg samples, the average recovery factors of the used internal standards are in the following ranges:  83.67% 88.67% (in the case of internal standards used for control of extraction efficiency-s6)  56.83% 99% (in the case of quantification standards-s7)  100% (in the case of recovery standards–s8) within the performed experiments, the detection limit of the native congeners of dioxins and furans varied in the range 0.0231 – 0.3734 pg/g fat. the minimum value of this range is the detection limit of congener 1234678-hpcdf, and the maximum one represents the detection limit of congener ocdf. also, in the case of those 6 egg samples, the detection limit of the compound with the highest toxicity among dioxins and furans, 2, 3, 7, 8tcdd, is in the range: 0.0395 – 0.0478 pg/g fat. for all the native congeners of dioxins and furans, the detection limit was calculated at a “signal-to-noise ratio” (s/n = 2.5). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 32 also, within the performed experiments, the limit of quantification of the native congeners of dioxins and furans varied in the range 0.0924 – 1.4936 pg/g fat. the minimum value of this range is the limit of quantification of the congener 1234678hpcdf, and the maximum one represents limit of quantification of the congener ocdf. also, in the case of those 6 egg samples, the limit of quantification of the compound with the highest toxicity among dioxins and furans, 2,3,7,8-tcdd, is in the range: 0.158 – 0.1912 pg/g fat. for all the native congeners of dioxins and furans, the detection limit was calculated at a “signal-to-noise ratio” (s/n = 10). in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, none of the native congeners of dioxins and furans was detected (so, there are under the limit of detection lod). therefore, for all these samples, the total concentration of dioxins and furans, expressed in toxic equivalents (teq), was 0. in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, the "superior limit of teq" (upper bound who teq (a)), was in the range 0.15550 – 0.1801 pg who-pcdd/pcdfteq/g fat, being below the maximum allowed limit by regulations of the european commission 1881/19 december 2006 (0.75 pg who-pcdd/pcdf-teq/g fat). when determining dioxins and furans in samples of sun flower oil, with addition of 5% olive oil, the average recovery factors of the used internal standards are in the following ranges:  60.50% 91.00% (in the case of quantification standards-s7)  100% (in the case of recovery standards–s8) in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, the detection limit of the native congeners of dioxins and furans was in the range 0.0314 – 0.283 pg/g fat. the minimum value of this range is the detection limit of congener 1234678hpcdf, and the maximum one represents the detection limit of congener ocdf. the detection limit of the most toxic compound among dioxins and furans, 2,3,7,8-tcdd is in the range: 0.0366 – 0.0512 pg/g fat. in the case of all native congeners of dioxins and furans, the detection limit was calculated at a “signal-to-noise ratio” (s/n = 2.5). in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, the limit of quantification of the native congeners of dioxins and furans was in the range 0.1257 – 1.1321 pg/g fat. the minimum value of this range is the limit of detection of congener 1234678-hpcdf, and the maximum one represents the limit of detection of congener ocdf. the limit of quantification of the most toxic compound among dioxins and furans, 2,3,7,8-tcdd, is in the range: 0.1465 – 0.2048 pg/g fat. for all native congeners of dioxins and furans, the limit of detection was calculated at a “signal-to-noise ratio” (s/n = 10). conclusions 1. in this paper we presented some performed experiments to determine dioxins and furans in eggs and oils, using high resolution gas chromatography in combination with high resolution mass spectrometry. 2. in the case of those 6 analyzed egg samples, the only native congener detectable was 2378-tcdf, which concentration was in the range 0.22 – 0.34 pg/g fat. 3. in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, none of the native congeners of dioxins food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 33 and furans was detected (so, they are below the limit of detection lod). 4. in the case of those 6 analyzed egg samples, the total concentration of dioxins and furans, expressed in toxic equivalents (who-teq(a)), was in the range: 0.0216 – 0.034 pg who-pcdd/pcdf-teq/g fat, being below the maximum allowed limit by regulations of the european commission 1881/19 december 2006 (3 pg who-pcdd/pcdf-teq/g fat). 4. in the case of those analyzed egg samples, within the performed experiments, the limit of detection of the native congeners of dioxins and furans was in the range 0.0231 – 0.3734 pg/g fat. the minimum value of this range is the limit of detection of the congener 1234678hpcdf, and the maximum one represents the limit of detection of the congener ocdf. 5. in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, the limit of detection of the native congeners of dioxins and furans was in the range 0.0314 – 0.283 pg/g fat. the minimum value of this range is the limit of detection of congener 1234678-hpcdf, and the maximum one represents the limit of detection of congener ocdf. 6. in the case of those 6 analyzed egg samples, within the performed experiments, the limit of quantification of the native congeners of dioxins and furans was in the range 0.0924 – 1.4936 pg/g fat. the minimum value of this range is the limit of quantification of congener 1234678-hpcdf, and the maximum one represents the limit of quantification of congener ocdf. 7. in the case of those 8 analyzed samples of sun flower oil, with addition of 5% olive oil, the quantification limit of the native congeners of dioxins and furans was in the range 0.1257 – 1.1321 pg/g fat. the minimum value of this range is the limit of detection of congener 1234678-hpcdf, and the maximum one represents the limit of detection of congener ocdf. acknowledgments the experiments were performed within the contract no. 51-050/18.09.2007, financed by the programme 4 “partnerships in priority s&t domains” 2007 – 2013 – national centre for projects management. references 1. otles s., yildiz h.dioxin in food and human health, electronic journal environmental agricultural and food chemistry, 2 (5), [593-608], issn 1579-4377, 2003 2. bernard a., hermans c., broeckaert f. et al. food contamination by pcbs and dioxins. nature sept. 16, 401:231-232, 1999 3. telliard w.a. et al., method 1613 tetra through octa-chlorinated dioxins and furans by isotope dilution hrgc/hrms, october, u.s. environmental protection agency office of water engineering and analysis division (4303) 401 m street s.w. washington, d.c. 20460, 1994 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 40 the sunflower seeds lipase activity on various substrata under the influence of some outer and inner factors *marcel avramiuc1 1„stefan cel mare” university of suceava, faculty of food engineering, str. universităţii, no. 13, 720229, suceava, românia, e-mail: avramiucm@fia.usv.ro *corresponding author received 20 february 2011, accepted 25 april 2011 abstract: in this work the sunflower seeds lipase activity on its own substratum (sunflower oil), as well as on other substrata, of 7 plant species (pumpkin, soy bean, sesame, almond, maize, walnut and peanut), at various temperatures and ph values, has been analyzed. the experiment materials consist of dried sunflower seeds degreased (with petroleum ether), used as enzyme (lipase) source, and refined oils from 7 plant seeds above mentioned – as substratum for enzyme. the lipase activity was determined at 20ºc and 40ºc as well as at 3 ph values (5.5; 7.4 and 9.5). the method principle consists in titrating fatty acids (released from oils by enzyme, in a certain time interval) with a solution of koh 0.01n. the determination of the lipase activity from sunflower seeds, at 20°c, 40°c and various ph, has shown distinct values of the enzyme activity depending on substratum nature, temperature and ph values. the enzyme activity on various substrata has registered: at 20°c the highest values in oils of: soy and walnut (ph 5.5), walnut and peanut (ph 7.4), sunflower and walnut (ph 9.5); at 40°c the highest values in oils of: peanut, soy and sunflower (ph 5.5), walnut and maize (ph 7.4), maize and walnut (ph 9.5). the comparative analysis of the sunflower seeds lipase activity on various substrata has shown, both at 20°c and at 40ºc, the highest values at ph 5.5, and the lowest ones at ph 9.5. © 2011 university publishing house of suceava. all rights reserved keywords: lipase, substratum, ph, oil, seeds. 1. introduction from the technological point of view, lipolytic enzymes, which are hydrolases involved in lipids’ metabolism and demoting, make a controlled hydrolysis (favorable) of fats in foods during maturation, or an uncontrolled hydrolysis of fats (harmful) leading to foods’ spoilage and occurrence of a pronounced rancid taste and odor 1. due to their short outgrowth cycle, microorganisms are used to obtain lipolytic enzymes and some scientific works have analyzed some aspects related to lipases isolated from candida antarctica 2, 3, 4 or candida rugosa 5, 6. in the last decades, the vegetable lipases isolated in plants belonging to some families such as euphorbiaceae 7, 8, 9, 10, brassicaceae 11, caricaceae 12 were used in various scientific research studies. in this work the activity of the lipase in sunflower seeds on their own substratum (sunflower oil), and on food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 41 various substrata, at different temperatures and ph values, has been analyzed. 2. experimental the experiment materials consist of dried sunflower seeds, degreased (with petroleum ether) used as lipase source, and refined oils of: sunflower, pumpkin, soy bean, almond, maize, walnut and peanut – as substratum for enzyme. the sunflower seeds have been obtained from suceava gene bank collection and the refined oils from supermarkets. the lipase activity has been determined at 20ºc, 40ºc and at 3 ph values (5.5, 7.4 and 9.5) and consisted (as principle) in titrating (with a solution of koh 0.01 n) fatty acids released from oils by enzymes, in a certain time interval 13. the lipase activity was expressed by fatty acid micro molls, represented by oleic acid, formed (as result of enzyme action) from a gram of product, in a minute. the data, consisted in 4 replicates for each sample (determination), were statistically processed, using the mean values and standard deviations. 3. results and discussion figure 1 shows the comparative evolution of lipase activity in sunflower seeds of the 8 oil types, at 20°c temperature and at the 3 ph values. at ph 5.5 the highest values were registered on soy bean oil, followed, in order, by walnut, maize, peanut and sunflower oils. the lowest ones were registered in sesame, almond and pumpkin oils. at ph 7.4 the highest values of lipase activity were registered, in order, in walnut, peanut and maize oils, and the lowest ones in sunflower and soy bean oils. at ph 9.5 there were not so high differences between samples as before, the highest value being registered by sunflower oil and the lowest ones by sesame and almond oils. except for the ph 9.5, where on the same substratum (sunflower oil) the lipase has registered the greatest value, whereas for the other analyzed ph, the enzyme has registered superior values on other substrata, for example, in soy bean, maize, walnut and peanut oils (at ph 5.5), and in pumpkin, sesame, almond, maize, walnut and peanut oils (at ph 7.4). at ph 5.5 the highest values were registered, in the following order: in peanut, soy bean and sunflower oils, and the lowest ones in almond and pumpkin oils. at ph 7.4 the highest values of lipase activity were registered, in order, in walnut and maize oils, and the lowest ones in almond and sesame oils. at ph 9.5 there were not so high differences between samples as in the case of ph 5.5 and 7.4, the highest values being registered by maize and walnut oils, and the lowest ones by sunflower, pumpkin and peanut oils. as can be seen in this figure, for all ph values analyzed, the enzyme has registered superior values on other substrata than on its own one, as follows: on peanut oil (at ph 5.5), on pumpkin, maize, walnut (at ph 7.4) and on soy, sesame, almond, maize and walnut (at ph 9.5). comparing the lipase activity in the two thermal thresholds analyzed (20ºc and 40ºc) it can be observed that, except for the ph 9.5, at ph 5.5 and 7.4, superior values were registered at 20ºc. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 42 fig. 1. the lipase activity of sunflower seeds on various substrata (oils), and various ph values, at 20ºc fig. 2 reproduces the comparative evolution of lipase activity of sunflower seeds in the 8 oils types, at 40°c temperature and at the 3 ph values. fig. 2. the lipase activity of sunflower seeds on various substrata (oils), and various ph values, at 40ºc 6.4 5.16 8.66 5.33 5.23 6.66 7.23 6.56 0.33 2.5 0.16 2.5 1.9 4.33 5.66 4.9 2.06 1.5 1.33 0.33 0.56 1.33 1.66 0.9 0 1 2 3 4 5 6 7 8 9 10 sunflower pumpkin soya sesame almond maize walnut peanut ph 5.5 ph 7.4 ph 9.5 7 5.66 7 6 5.83 6.33 6.66 7.83 3.33 3.66 3 1.33 1.16 4 4.33 2 2.33 2.33 2.66 2.66 2.5 3 2.83 2.33 0 1 2 3 4 5 6 7 8 9 sunflower pumpkin soya sesame almond maize walnut peanut ph 5.5 ph 7.4 ph 9.5 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 43 4. conclusions the determination of the lipase activity of sunflower seeds, at 20°c, 40°c and various ph, has shown distinct values of the enzyme activity depending on the substratum nature, temperature and ph values. the enzyme activity on various substrata has registered: at 20°c the highest values in oils of: soy and walnut (ph 5.5), walnut and peanut (ph 7.4), sunflower and walnut (ph 9.5); at 40°c the highest values in oils of: peanut, soy and sunflower (ph 5.5), walnut and maize (ph 7.4), maize and walnut (ph 9.5). comparing the lipase activity in two thermal thresholds (20° and 40°c) it can be observed that, except for the ph 9.5, at ph 5.5 and 7.4, superior values were registered at 20°c. the comparative analysis of the sunflower seeds lipase activity on various substrata has shown, both at 20°c and at 40ºc, the highest values at ph 5.5, and the lowest ones at ph 9.5. 5. references 1. bârnescu r. et al. acţiunea lipolitică şi proteolitică a unor preparate din specii de aspergillus asupra deşeurilor de piele. microbiologie industrială şi biotehnologie. iaşi, 1986. 2. anderson e.m., larsson k.m., kirk o. one biocatalyst-many applications: the use of candida antarctica b-lipase in organic synthesis, biocatal biotransform 16 (1998), 181–204. 3. kirk o., christensen m.w. lipases from candida antarctica: unique biocatalysts from a unique origin, org process res dev 6 (2002), 446– 451. 4. domínguez de maría p., carbonioerlemans c., tuin b., bargeman g., a. van der meer, r. van gemert biotechnological applications of candida antarctica lipase a: state-of-the-art, j mol catal b enzym 37 (2005), 36–46. 5. akoh c.c., lee g.c., shaw j.f. protein engineering and applications of candida rugosa lipase isoforms, lipids 39 (2004), 513–526. 6. domínguez de maría p., sánchezmontero j.m., sinisterra j.v., alcántara a.r. understanding candida rugosa lipases: an overview, biotechnol adv 24 (2006), pp. 180–196. 7. giordani r., moulin a., verger r. tributyroylglycerol hydrolase activity in carica papaya and other latices, phytochemistry 30 (1991), 1069-1072. 8. moulin a., teissere m., bernard c., pieroni g. lipases of the euphorbiaceae family: purification of a lipase from euphorbia characias latex and structure-function relationships with the b chain of ricin, proc natl acad sci 91 (1994), 11328– 11332. 9. palocci c., soro s., cernia e., fiorillo f., belsito c., monacelli b. et al. lipolytic isoenzymes from euphorbia latex, plant sci 165 (2003), 577–582. 10. villeneuve p., turon f., caro y., escoffier r., baréa b., barouth b. et al. lipase-catalyzed synthesis of canola phytosterols oleate esters as cholesterol lowering agents, enzyme microb technol 37 (2005), 150–155. 11. hills m.j., kiewitt i., mukherjee k.d. lipase from brassica napus l. discriminates against cis-4 and cis-6 unsaturated fatty acids and secondary and tertiary alcohols, biochim biophys acta 1042 (1990), 237–240. 12. dhuique-mayer c., villarreal l., caro y., ruales j., villeneuve p., pina m. lipase activity in alcoholysis and esterification reactions of crude latex from babaco fruit (carica pentagona), oléagineux corps gras lipides 10 (2003), 232–234. 13. bordei d (coord.)., et al. controlul calităţii în industria panificaţiei. metode de analize. ed. academica, galaţi, 2007; 206 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 206 211 the use of plant materials in culinary products out of yeasted dough *oksana arpul1, tetiana sylchuk1, vira kulinich1, olena usatiuk1 1molecular and avantgarde gastronomy, national university of food technologies, ukraine kseniya arp@mail.ru *corresponding author received january 6th 2015, accepted june 16th 2015 abstract: the article deals with the technology of garlic donuts (pampushkas) enriched with schavnat (hybrid of spinach dock or patience dock and tien shan sorrel) as a source of plant protein. the influence of schavnat on the technological process and the quality of pampushkas was determined. the organoleptic properties and the physical and chemical properties such as titrated acidity, texture, specific volume, dough fermentation property and viscoplastic properties were investigated. we obtained a «special pampushkas with garlic» with a high content in plant proteins which could be recommended for balancing the nutrient composition of population diet. keywords: schavnat, schavnat powder, yeast dough, garlic donut, pampushka, protein, culinary products. 1. introduction all over the world millions of people do not get enough protein. protein and amino acid deficiencies negatively affect the state of health, human performance and longevity. the ways of protein deficiency recovery are the search of new plant sources of food protein and the development of methods to use them for fortification of mass-consumption products. bakery products, unlike other foods, are the daily products, so it is possible to influence on the diet and human health by regulation of their chemical composition [1, 2]. among a large number of protein raw materials schavnat as a culture of new generation deserves attention. schavnat is the species hybrid of spinach dock or patience dock and tien shan sorrel. this new long-term culture was selected in the department of new cultures of m. m. gryshko national botanic garden (ukraine). it contains a large amount of essential nutrients such as ascorbic acid, beta-carotene, minerals, plant protein etc. schavnat is first among the vegetable plants for the content of protein and vitamins in the early phases. this culture is suitable for complex use as food, feed, energy, medicinal and plant for technical use. schavnat as early spring culture is characterized by high ecological plasticity, winter and frost hardiness, productivity, food, feed, technical and medicinal properties in different climatic and geographical zones. its high food and feed properties are noted in the periods of growth, shooting and budformation. during this period the overground mass (12…18 % on a dry matter) contains about 30...40 % of http://www.fia.usv.ro/fiajournal mailto:arp@mail.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk, the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 2 – 2015, pag. 206 211 207 protein, 700 mg% of ascorbic acid and 60 mg% of carotene. the content of nitrogenfree extractive substances is between 35 and 55 %, among them about 6...20 % of sugars. the number of lipids in the green mass is negligible (about 2.8...6.2 %) [3,4]. the use of such plant material as schavnat in the technologies of culinary products out of yeast dough is timely and topical [5]. 2. materials and methods 2.1 materials in the laboratory of the department of molecular and avantgarde gastronomy of national university of food technologies (ukraine) the culinary products out of yeast dough such as garlic donuts (pampushkas) were baked. dry schavnat was added in the amount of 0.5…1.0 and 1.5 % of the flour weight for fortification of the products by plant protein. the moisture content of the yeasted dough was 42 %. the fermentation time of dough was 60 min at the temperature of proofer about 30 ± 2 °с. pampushkas were baked at the temperature of 210 to 220 ° c with sprinkling of the baking oven. the weight of dough piece was about 30±1 g each. 2.2 methods the influence of schavnat on the indicators of the technological process and the quality of finished products was investigated. after baking the organoleptic properties and the physical and chemical properties such as titrated acidity, texture, specific volume and moisture content of the finished culinary products were determined. the protein content of schavnat and the culinary products was determined by the modified kjeldahl method. modification of the method was in determination of the nitrogen content directly in the solution of the ashing sample (without pre-distillation) with using indirect hypochlorite and iodometric titration. the mineralizing of the sample was made by the kjeldahl method with the mixture of potassium sulphate and copper (ii) sulphate. peroxide of hydrogen was used for catalyzation the ashing. the titrated acidity of semi-finished product (dough) was determined by the titration method. the viscoplastic properties of dough were investigated by the degree of dough running and the dynamic of specific volume of dough during fermentation. the dough running was determined by the method based on the changes in the diameter of the dough balls with the weigh of 100 g during fermentation at the temperature of 30 ° c for 180 min. the dough portion with the weigh of 50 g was put in the graduated cylinder with a capacity of 250 cm3 and then was put in the thermostat with the temperature of 30 ° c for determination of the dynamic of specific volume. the start value of the specific volume and the changes during fermentation were fixed [6, 7, 8]. 2.3 technology of pampushkas enriched by schavnat the technology of «special pampushkas with garlic» is presented in fig. 1. 3. results and discussion the research of the granulometric composition of dry schavnat was shown that the optimal size is 0.4…0.5 mm for the good organoleptic indicators and minimal protein loss (fig. 1). fig. 1. dependence of protein content from fractional composition of schavnat powder 23,3 % 34,3 % 30,6 % fraction – 0.7…1.0 mm fraction – 0.6 mm fraction – 0.4…0.5mm food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk, the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 2 – 2015, pag. 206 211 208 fig. 2. the technological scheme of «special pampushkas with garlic» vegetable oil garlic salt water peeling pulping heating t=100…110 °c cooling t=18…20°c addition grinding gradual blending mixing «garlic sauce» baking to golden crust τ =15…20 min, t=210…220 °c addition of sauce sales pampushky with garlic water salt sugar «lviv» pressed yeast schavnat powder (w=12.4%) wheat flour vegetable oil heating t=35…40°c dilution dilution dilution bolting moistening τ=5…7 min addition mixing of dough to homogeneous consistency τ=7…8 min fermentation τ=60min, t=35…40°c dough piece forming, weight =30±1 g fermentation of dough piece τ=15…20 min, t=35…40 °c yeast dough food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk, the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 2 – 2015, pag. 206 211 209 the protein content is directly proportional to the particle size of fractions. thus, the fraction of schavnat with the particle size of 0.4...0.5 mm contains the most amount of protein (34.3%), and the fraction with the largest particle size of 0.7...1.0 mm contains only 23.3% of protein. the schavnat fraction with the smallest particle size should be used for enrichment of pampushkas by protein. the protein content was determined in the finished products with the addition of 0.5%, 1.0% and 1.5% of schavnat powder (fit. 3). the protein content of pampushkas is directly proportional to the additive of schavnat powder. fig. 3. the protein content in the finished product (pampushkas) with the additive of schavnat powder the finished products with the addition of 0.5% of schavnat powder contained 7.71% of protein, with the addition of 1.0% – 7.98% of protein, with the addition of 1.5% – 11.44% of protein. the research results of organoleptic and technological indicators of the finished products with the additive of schavnat were presented in table. 1. the organoleptic quality of pampushkas did not change significantly with the addition of schavnat in the amount from 0.5 to 1.0% compared to the control, but the colour and surface crust, texture and elasticity were slightly worse with the addition of schavnat in the amount of 1.5%. it was noted the pleasant slightly acid flavour of the finished products. the schavnat additive decreased the specific volume and texture of the finished products (fit. 4). thus, it was established that pampushkas with the addition of schavnat powder in the amount from 3,5 to 13% had the decreased specific volume compared to the control sample (without schavnat). and the texture indicator of pampushkas has been decreasing with the dosage of schavnat powder from 1.05 to 9.17%. fig.4. dynamic of specific volume of dough with the additive of schavnat powder the research results were shown that the start value of titrated acidity was increased slightly with the using of schavnat products (fig. 5). fig. 5. titrated acidity of dough with the additive of schavnat powder 0,00 2,00 4,00 6,00 8,00 10,00 12,00 control (without schavnat) 0.50% 1.00% 1.50% samples under investigation pr ot ei n, % 35 45 55 65 75 85 95 105 115 0 30 60 90 fermentation time, min sp ec ifi c vo lu m e, c m 3 control (without schavnat) 0.50% 1.00% 1.50% 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 control (without schavnat) 0.50% 1.00% 1.50% titrated acidity, degree: a ci tid y, d eg re e start value final value food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk, the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 2 – 2015, pag. 206 211 210 table 1 the influence of schavnat on the technological process and the quality of pampushkas indicator control dosage of schavnat in the dough, % of flour weight 0.5 1.0 1.5 1 2 3 4 5 dough mixing time, min 8 8 8 8 fermentation time, min 60 60 60 60 proofing time, min 30 30 30 30 baking time, min 25 25 25 25 titrated acidity, degree start value final value 2.2 2.4 2.7 2.9 3.2 3.4 3.6 4.0 finished products – pampushkas titrated acidity, degree 2.0 2.6 2.8 3.4 texture indicator, % 80 79 78 76 specific volume, cm3 3.07 2.89 2.72 2.61 crusts colour golden brown surface crust smooth rough texture medium, uniform compacted elasticity medium medium, compacted flavour inherent to pampushkas sour flavour with increasing the dosage of schavnat powder the dough fermentation property was investigated by the floating dough ball (fig. 6). fit. 6. investigation of dough fermentation property with schavnat powder the research results were shown that the incensement of the additive of schavnat powder decreased the dough fermentation property, but within the established norms for bakery products. the dynamic of dough ball running was investigated for establishing the nature of the impact of schavnat on viscoplastic properties of semi-finished products (fig. 7). fig. 7. investigation of viscoplastic properties of dough with schavnat powder the dough ball running describes the state of protein and proteinase complex during autolysis. it was found that schavnat improved the viscoplastic properties of dough. the addition of dry schavnat increased the water absorbing capacity and reduced the ability to dough running during fermentation. 0 2 4 6 8 10 12 14 16 18 20 control (without schavnat) 0.50% 1.00% 1.50% samples under investigation fe rm en ta tio n pr op er ty , m in 60 70 80 90 100 110 120 130 0 30 60 90 fermentation time, min d ia m et er o f d ou gh b al l, m m control (without schavnat) 0.50% 1.00% 1.50% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk, the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 2 – 2015, pag. 206 211 211 4. conclusion the fortification of food products by schavnat is appropriate and relevant to the development of new products with a high content of plant protein. it was proposed the technology of «special pampushkas with garlic» that could be recommended as a garnish for soups, such as borsch or rassolnik. such fortified pampushkas could balance the food diet by protein of plant origin. 5. references [1] marina n.v., novoselova g.n., shavnin s.a. products with increased biological value out of non-traditional plant raw materials, news of the samara scientific center of the russian academy of sciences, vol. 12, 1(8), 2079-2082 (2010). [2] constandache m. the influence of fortification of bread with exogenes proteins on the protein digestibility, journal of agroalimentary processes and technologies, vol. xiii, 2, 461-466 (2007). [3] raxmetov d.b., raxmetova s.o. schavnat: vegetables, food, and phytofuel, grain, 3, 62-71 (2011). [4] bazhaj-zhezherun s.a., raxmetov d.b. schavnat is a promising raw material for the production of health products, new ideas in food science – new food products for industry: international scientific conference devoted to the 130th anniversary of the national university of food technologies, 167 (2014). [5] yermolenko m., arpul o., sylchuk t. the use of alternative raw materials at restaurants, scientific achievements of young people – solving the problems of human nutrition in the xxi century: the program and materials of 80 international scientific conference of young scientists and students, part iii, 473-475 (2014). [6] maría e. matos, teresa sanz, cristina m. rosell. establishing the function of proteins on the rheological and quality properties of rice based gluten free muffins, food hydrocolloids, vol. 35, 3, 150-158 (2014). [7] solina m., johnson r.l., frank b. whitfield, effects of soy protein isolate, acidhydrolysed vegetable protein and glucose on the volatile components of extruded wheat starch, food chemistry, vol. 104, 4, 1522-1538 (2010). [8] drobot v.i., arsenyeva l.yu., bilyk o.a. etc. laboratory workshop on technology of bakery and macaroni production. kyiv: center of educational literature, 341 (2006). microsoft word 0a. first pages.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xi, issue 3 30 september 2012 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia sorin sabau tokai university, faculty of biological science, sapporo, japan alice roşu ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. postala address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro copyright © university publishing house of suceava. all right reserved title …………………… 245 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 245 249 separation and quantification of biogenic amines in bananas by high performance liquid chromatography *veronica tanasa, daniela moise, maria stanca chemistry and biochemistry analysis laboratory, institute of research and development for industrialization and marketing of horticultural products horting, 1a intrarea binelui, 4 district, bucharest, romania e-mail: vero.tanasa@yahoo.co.uk *corresponding author received july 13th 2015, accepted september 29th 2015 abstract: this paper reports separation and quantification of biogenic amines in bananas by high performance liquid chromatography. fresh and preserved samples (seven days of storage at refrigeration temperature, at room temperature respectively) were analyzed. we obtained the separation of all nine biogenic amines (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermidine, spermine) with good resolution, including good resolution of serotonin and internal standard (1,7diaminoheptane). mean biogenic amines levels of 10.344 mg/kg (tryptamine) to 61.502 mg/kg (putrescine) were found in fresh bananas. after seven days of storage at room temperature, 2-phenylethylamine decreases significantly. after seven days of storage at refrigeration temperature, tryptamine increases significantly, whereas phenylethylamine decreases significantly. keywords:2-phenylethylamine, bananas, biogenic amines, cadaverine, histamine, hplc, putrescine, spermidine, spermine, serotonin, tryptamine, tyramine 1. introduction biogenic amines are natural low molecular weight organic bases of aliphatic, aromatic or heterocyclic structure derived from decarboxylation of the corresponding amino acids. biogenic amines are present in small quantities in some products such as fruits, vegetables, milk etc. polyamines such as putrescine, spermidine and spermine have been found in all higher plants and are involved in important physiological processes, such as fruit growth and development [1cited by 2]. free biogenic amines in fruits and vegetables shape the typical and characteristic taste of mature foods and are precursors of certain aroma compounds [3 cited by 4]. biogenic amines can be found in relatively large amounts in some fermented/ matured foodstuffs. also biogenic amines can be used as indicators of quality and freshness, especially for animal raw material and food, as food spoilage is accompanied by increased content of biogenic amines. biogenic amines exert physiological and toxicological effects. some aromatic amines (tyramine, tryptamine, and 2phenylethylamine) show a vasoconstrictor action while others (histamine and serotonin) present a vasodilator effect. despite of low levels of biogenic amines in fruits, vegetables and fruit juices [5, 2, 6, 7 8], quantification of biogenic amines is important due to the existence of risk consumer categories. these include both patients treated with painkillers and drugs used for the treatment of stress and http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 veronica tanasa, daniela moise, maria stanca,separation and cuantification of biogenic amines in bananas by high performance liquid chromatography, food and environment safety, volume xiv, issue 3 – 2015, pag. 245 – 249 246 depression, acting as a blocker of monoamine oxidase the main route of biogenic amines detoxification [9], and young children and infants, as they are early feed with bananas, at 5 6 months of age. through our research we aimed at the separation and quantification of biogenic amines in bananas by high performance liquid chromatography, since few data are reported about this topic [10, 11, 7, 12]. 2. materials and methods 2.1. samples bananas were obtained from romanian retail stores and were analyzed fresh and after preservation: seven days of storage at refrigeration temperature, respectively at room temperature. 2.2. reagents and standards tryptamine (trp), 2-phenylethylamine (phe), putrescine (put) , cadaverine (cad), histamine (his), serotonin (ser), tyramine (tyr), spermidine (spd), spermine (spm) and internal standard 1,7 diaminoheptane were purchased from sigma-aldrich. stock solutions of biogenic amines and internal standard (1 mg/ml) were prepared, which were kept in the refrigerator for a month. these solutions were used for the preparation of standard solutions (100μg/ml) used for calibration curves. all other reagents used were p.a. grade and solvents were hplc grade. 2.3. the analytical procedure has been adapted in the chemistry and biochemistry laboratory of the horting institute [13] following a previously published protocol [14]. in our case chromatographic separation was performed using a bds hypersil c18 column (250 * 4.6 mm), 5μ particle size (thermo electron corporation). the mobile phase consisted of ammonium acetate 0.1 mol / l (solvent a) and acetonitrile (solvent b). we modified the gradient elution program proposed by eerola et al. (1993) [14] (table 1). the mobile phase flow rate was of 1ml/min and the column temperature was set at 400c. dansylated derivatives of biogenic amines were uv detected at 254 nm. the control, data acquisition and processing software used chromquest 4.2. (thermofinnigan). the results were statistically processed with student test using graphpad prism (version 5.00, graphpad software inc., san diego, 2007). the differences p <0.05 were considered significant. 3. results and discussion by modifying the gradient elution programme proposed by eerola et al. (1993) [14] we found that resolution of serotonin and the internal standard was good (fig. 1). eerola et al. (1993) [14] asserted that the overlapping of serotonin and internal standard peaks did not interfere with the quantitation because the samples investigated contained no serotonin or only very small amounts. all biogenic amines investigated were present in fresh samples, but putrescine was predominant (table 2). kalac & krausova (2005) [6] and okamoto (1997) [5] reported that the putrescine content was predominant in grapefruit and apple, and orange respectively. cirilio et al. (2003) [2] reported that the predominant amines in green coffee were serotonin and putrescine. putrescine concentration in fresh samples (61.502 mg/kg) was comparable with the values reported by kalac & krausova (2005) [6] in grapefruit and fresh green pepper (62.1 respectively 70 mg/kg). moret et al. (2005) [4] carried out a survey on free biogenic amine contents in fresh vegetable products and reported that putrescine was one of the most represented amines. adao & et al. (2005) [11] found a putrescine content around 0.75mg/100g in bananas. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 veronica tanasa, daniela moise, maria stanca,separation and cuantification of biogenic amines in bananas by high performance liquid chromatography, food and environment safety, volume xiv, issue 3 – 2015, pag. 245 – 249 247 fig. 1. chromatographic separation of dansyl derivatives of biogenic amines (2 μg/ml) by gradient elution fig. 2. chromatogram of biogenic amines extracted from bananas after 7 days of storage at room temperature minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 m a u -250 0 250 500 750 1000 1250 1500 1750 2000 2250 m a u -250 0 250 500 750 1000 1250 1500 1750 2000 2250 t ri p ta m in a 8 .3 2 0 1 1 0 2 8 9 0 f en il et il a m in a 1 0 .8 6 2 1 4 4 0 7 3 7 p u tr es ce in a 1 2 .3 4 8 3 8 6 8 3 6 7 c a d a v er in a 1 4 .3 3 5 3 3 2 9 0 0 8 h is ta m in a 1 5 .5 4 7 3 3 8 8 4 5 9 2 0 .0 9 2 7 1 0 4 5 1 ,7 d ia m in o h ep ta n 2 1 .0 7 3 1 1 5 7 6 5 3 s er o to n in a 2 1 .7 5 8 1 7 0 4 7 8 0 t ir a m in a 2 5 .2 6 8 2 4 2 7 5 2 1 2 5 .7 9 8 1 9 6 2 1s p er m id in a 2 6 .3 8 0 3 0 1 4 6 8 7 2 6 .6 2 0 1 7 3 2 5 2 8 .6 7 8 3 1 4 8 0s p er m in a 2 8 .9 8 5 2 1 7 7 9 3 8 2 9 .4 1 3 1 3 9 6 9 pda plus-254nm name retention time area minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 m a u 0 500 1000 1500 2000 2500 3000 m a u 0 500 1000 1500 2000 2500 3000 7 .2 4 5 1 4 3 1 5 t r ip ta m in a 7 .6 4 2 4 0 6 9 3 9 .3 5 8 2 9 9 7 9 .7 9 2 2 2 5 7 4 f e n il e ti la m in a 1 0 .1 6 5 8 0 6 7 5 p u tr e sc e in a 1 0 .9 7 3 1 1 0 7 8 6 3 1 1 .7 6 0 1 0 0 3 3 7 1 2 .0 9 0 6 5 1 1 3 c a d a v e r in a 1 2 .9 6 3 3 0 4 9 2 0 h is ta m in a 1 3 .8 1 2 3 0 6 5 2 2 1 4 .3 1 0 4 7 6 4 9 7 1 ,7 d ia m in o h e p ta n 1 8 .6 7 8 7 7 6 1 0 0 s e r o to n in a 1 9 .5 4 7 4 5 5 2 8 5 2 0 .8 7 3 4 6 3 4 2 1 .6 7 0 5 8 4 1 0 2 2 .8 0 3 5 7 5 8 1 2 3 .3 3 2 3 1 8 1 9 3 t ir a m in a 2 4 .0 2 5 1 0 7 4 7 9 2 4 .4 7 2 2 5 1 4 2 5 .0 9 5 3 1 2 5 6 6 s p e r m id in a 2 5 .6 4 7 2 2 7 6 3 4 2 5 .8 9 0 3 6 3 9 6 2 6 .6 1 5 2 6 0 7 3 6 2 7 .1 1 8 8 1 5 9 4 2 7 .9 3 0 1 6 2 5 7 1 1 2 8 .3 5 0 2 1 5 6 5 5 s p e r m in a 2 8 .7 2 3 4 7 7 0 1 2 9 .0 3 3 2 0 0 2 3 9 2 9 .2 6 8 4 1 0 3 6 9 2 9 .7 3 7 7 1 6 6 pda plus-254nm name retention time area food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 veronica tanasa, daniela moise, maria stanca,separation and cuantification of biogenic amines in bananas by high performance liquid chromatography, food and environment safety, volume xiv, issue 3 – 2015, pag. 245 – 249 248 table 1. gradient elution programme for the separation of biogenic amines in bananas table 2. mean biogenic amines levels (mg/kg) in bananas trp phe put cad his ser tyr spd spm fresh samples 10.344 ±0.346 39.42 ±6.091 61.502 ±16.91 32.313 ±4.455 17.991 ±8.71 25.379 ±5.664 14.831 ±0.839 23.303 ±1.199 19.164 ±3.218 samples preserved 7 days at room temperature 13.759 ±5.652 12.595 ±5.464 p<0.05 51.381 ±27.605 24.784 ±10.268 25.271 ±7.355 25.265 ±19.756 11.537 ±7.567 18.154 ±8.125 6.084 ±3.486 samples preserved 7 days at refrigeration temperature 17.971 ±1.126 p<0.05 14.300 ±2.621 p<0.05 57.082 ±16.214 29.859 ±1.199 28.053 ±1.352 21.663 ±4.966 13.978 ±0.207 22.436 ±5.418 9.104 ±5.430 histamine (17.991 mg/kg) was detected in fresh samples. the presence of histamine was not detected in bananas by lavizzari et al. (2006) [7], but g.g. mohamed et al. (2009) [12] reported histamine levels of 0– 33.10 mg/100g in seven banana samples. the tiramine concentrations in fresh samples (14.831 mg/kg) were in agreement with those values obtained by udenfriend et al. (1959) [10] in bananas (0.7 mg/100g). we detected tryptamine, 2phenylethylamine, cadaverine in fresh samples, but these biogenic amines have not been detected in bananas by lavizzari et al. (2006) [7]. mean serotonin levels of 25.379 mg/kg were found; lower amounts of serotonin were found by lavizzari et al. (2006) [7], adao& et al. (2005) [11] in bananas and cirilio et al. (2003) [2] in green coffee (11.5 mg/kg, around 1.25 mg/100g respectively 1.13 mg/100g ). mean spermidine and spermine levels of 23.303 respectively 19.164 mg/kg were found. adao & et al. (2005) [11] found a spermidine content around 1.10 mg/100g in bananas and spermine was present at very low level. cirilio et al. (2003) [2] also reported prevalence of spermidine to spermine in green coffee with mean levels of 0.60 mg/100g, 0.44 mg/100g respectively. moret et al. (2005) [4] reported prevalence of spermidine to spermine in fresh vegetables (0.4–4.5 mg/100 g, a maximum of 1.1 mg/100 g respectively). the samples analyzed after seven days at room temperature showed significant lower 2-phenylethylamine content (table 2) and no differences were found for the other biogenic amine levels, while putrescine was the most represented amine. the samples analyzed after seven days of refrigeration showed a statistical significant higher tryptamine content and a lower phenylethylamine content, and no differences were found for the others biogenic amine levels, while putrescine was the most represented amine (table 2). it is widely known that some aromatic biogenic amines (tyramine, tryptamine, 2phenylethylamine) exert a vasoconstrictor effect, and the quantification of these biogenic amines is important, especially due to the existence of risk consumer categories. with regard to biogenic amines' content time (min) gradient flow rate (ml/min) wavelength (nm) column pressure (bar) column temperature (ºc) tray temperature (ºc) sample volume (µl) ammonium acetate (a) (%) acetonitrile (b) (%) 0.01 40 60 1.00 254 minim 70 40 7 20 15 40 60 20 30 70 25 5 95 30 40 60 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 veronica tanasa, daniela moise, maria stanca,separation and cuantification of biogenic amines in bananas by high performance liquid chromatography, food and environment safety, volume xiv, issue 3 – 2015, pag. 245 – 249 249 modification during refrigeration, moret et al. (2005) [4] reported that vegetables analyzed after a 3-week refrigeration period showed a lower spermidine content and minor differences were found for concentrations of other amines. further investigation on more samples is required. 4. conclusion a) we obtained the separation of all nine biogenic amines from bananas by hplc with good resolution, including good resolution of serotonin and internal standard (1,7 diaminoheptane). b) mean biogenic amines levels of 10.344 mg/kg (tryptamine) to 61.502 mg/kg (putrescine) were found in fresh bananas. c) after seven days of bananas storage at refrigeration temperature, their content in 2-phenylethylamin decreased significantly, while their tryptamine content increased significantly. d) after seven days of storage at room temperature, 2-phenylethylamine decreased significantly in bananas. 5. acknowledgments we would like to thank the romanian ministry of education and scientific research national authority for scientific research (ancs), for supporting this work under the excellence research program, ceex iv project contract no. 115/10.08.2006. all the authors declare no conflict of interest. 6. references [1]. esti, m., volpe, g., massignan, l., compagnone, d., la notte, e., palleschi, g. determination of amines in fresh and modified atmosphere packaged fruits using electrochemical biosensors. journal of agricultural and food chemistry, 46, 4233–4237 (1998). [2].cirilio, m.p.g., coelho, a.f.s., araujo, c.m., goncalves, f.r.b., nogueira, f.d., gloria, m.b.a. profile and levels of bioactive amines in green and roasted coffee. food chemistry, 82, 397-402 (2003). [3]. askar, a., treptow, h. biogene amine in fleisch-producten. ernahrung/nutrition, 13, 425–429 (1989). [4]. moret, s., smela, d.,populin, t., conte, l.s., a survey on free biogenic amine content of fresh and preserved vegetables. food chemistry, 89, 355– 361 (2005). [5]. okamoto, a., sugi, e., koizumi, y., yanagida, f., udaka, s. polyamine content of ordinary foodstuffs and various fermented foods. bioscience, biotechnology and biochemistry, 61 (9) , 1582-1584 (1997). [6]. kalac, p., krizek, m., pelikanova, t., langova, m., veskrna, o. contents of polyamines in selected foods. food chemistry, 90, 561-564 (2005). [7]. lavizzari, t., veciana-nogues, m. t., bover-cid, s., marine-font, a., vidalcarou, m. c. improved method for the determination of biogenic amines and polyamines in vegetable products by ion-pair highperformance liquid chromatography. journal of chromatography a, 1129, 67-72 (2006). [8]. saaid, m., saad, b., hashim, n.h., ali, a.s.m., saleh, m.i. determination of biogenic amines in selected malaysian food. food chemistry, 113(4), 1356-1362 (2009). [9]. bardocz, s. polyamines in food and their consequences for food quality and human health. trends in food science & tehnology, 6, 341-346 (1995). [10]. udenfriend, s., lovenberg, w., sjoderma, a. physiologically active amines in common fruits and vegetables. archives of biochemistry and biophysics, 85, 487–490 (1959). [11]. adao, r. c., gloria m. b. a. bioactive amines and carbohydrate changes during ripening of ‘prata’ banana (musa acuminata · m. balbisiana). food chemistry, 90, 705–711 (2005). [12]. mohamed g.g., el-hameed a.k., eldin a.m., el-din l.a. high performance liquid chromatography, thin layer chromatography and spectrophotometric studies on the removal of biogenic amines from some egyptian foods using organic, inorganic and natural compounds. the journal of toxicological sciences, 35 (2), 175-187 (2010). [13]. baston, o., stroia, a. l., moise, d., barna, o. validation of a hplc method able to measure biogenic amines in chicken meat. the annals of the university dunarea de jos of galati. fascicle vi – food technology, new series year ii (xxxi), 44-50 (2008). [14]. eerola, s., hinkkanen, r., lindfors, e., hirvi, t. liquid chromatographic determination of biogenic amines in dry sausages. journal of aoac international, 76 (3), 575-577 (1993). food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 56 researches on the stability and shelf-life of canned tomatoes *amelia buculei1 1faculty of food engineering, tefan cel mare university of suceava, romania, ameliab@fia.usv.ro *corresponding author received 2 august 2012, accepted 7 september 2012 abstract: there is a series of internal and external factors in the food chain that influence the shelflife of the packaging systemfood stuff: the selection and quality of the raw materials, the fabrication network, the processing and conservation techniques, the packaging, storage and distribution for the consumers. the analyze of the long term stability of the lacquer layer in the case of the tin canned tomatoes with different lacquers (3 systems of epoxy phenolic lacquer) was accomplished to compare the protection abilities of the lacquers and the extension of the shelf-life. the analyze was done initially and along the storage and focused upon: the characteristics of the inner lacquer layer; the sensorial and physico-chemical characteristics of the product as well as the metal content (aas). the results obtained were the following: the porosity of the lacquer layer on the empty cans as well as on the tested cans during the storage of 1 and respectively 2 years did not suffer any modifications. during the two analyzed periods of storage the sensorial properties and the physico-chemical ones of the product „tomatoes paste”, including the heavy metals, differ according to the protection lacquer type of the cans: for the cans with white lacquer there are no modifications while for the cans with yellow lacquer, for both situations after 2 years of storage there are significant sensorial modifications due to the aggressiveness of the product content. keywords: cans, lacquer layer, storage, inner layer, outer layer 1. introduction a very common material for food packaging is steel, in the form of metallic containers (cans). the steel cans are generally obtained by mechanical deformation from tin plated steel sheets and coated with an organic lacquer in order to increase the corrosion protection. [1] the corrosion degradation of the packaging must be carefully controlled, not only because the packaging integrity must be preserved, but also in order to avoid any significant contamination of the food or drink, compromising the flavour. [1,2] at a high temperature (sterilization) the resins that are part of the protection lacquers can decompose into components with toxic potential and can migrate from the package into the food stuff.[3,4] testing migration from food contact materials (fcm) in just four liquid simulants covering the whole range of foods is an ingenious concept, as it greatly simplifies compliance testing.[5] according to the ue directive 82/711eec, the four simulants are: simulant awater for the liquid foods, with a ph higher than 4.5; simulant b -3% acetic acid in water for the acidic liquid foods with a ph lower than 4.5; simulant c -10% aqueousethanol for the alcoholic products and simulantul d – olive oil for the fat foods[6]. the protection degree of the lacquers applied on the inner and outer surface of a can is a fundamental property for its commercial life that is determined according to the physico-chemical traits of the polymer, its application conditions, mailto:ameliab:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 57 its compatibility with the food stuff and the metal support [7,8]. in order to increase the coating performance and the food compatibility, new organic coatings are under development with very high protective properties, with the final aim to increase the shelf life of the product.[9,10] both chemical and physical characteristics of the coating and metal substrate may influence the adhesion properties of the organic paint. it is well known that the higher adhesion is possible when the coating reacts (chemically and/or mechanically) with surface of metal. [11] 2. experimental the characteristics of the lacquer layer were analyzed after oven thermostating at 50 c, for a month (30 days) and respectively 2 months (60 days) in shelf storage conditions after 12 months. the analyzing of the inner appearance of the lacquer layer of a representative set of opened cans was done piece by piece and it was compared with the conditions from the product standards. the porosity of the lacquer layer was also analyzed piece by piece for the entire set of opened cans (the average values being presented in the following tables). for the ensemble metallic materiallacquering system the following tests were done: 1. the drying time 2. the determination of the weight of the dry lacquer layer is done by the gravimetric method: the vial of 10 x 5 cm is weighted then introduced in a solution mixture of 8% nacl and 12% i butyl glycol and after the lacquer layer removal the vial is weighted again. by difference and reporting to m2 the dry film is determined [g/m2]. 3. the lacquering appearance 4. the adhesion before and after the sterilization is realized with the aid of a tessor adhesive band with a surface of 1 cm (1 x 1 mm), figure 4.2. 5. the porosity of the lacquer applied on the can and the possible scratches on the body or on the bottom of the can were tested with the aid of a porosimeter sencon si 9000 plus, formed by two parts: 1) a special device ; 2) the measurement device. the test consist in measuring a continuous electric flow established between the metal of the can and an electrolyte contained by the can as the effect of a constant potential difference existing between the can and an electrode placed in the electrolyte. the intensity of the flow is directly proportional with the lacquer porous areas and the possible scratches. the electrolyte must be at a room temperature of 17-23 °c. the lacquer quantity applied and the lacquer technology was established by the metallic packages manufacturer according to the technical specifications of the lacquers and tins used. the lacquer layer porosity was determined both on the empty cans and also on the cans tested in the presented storage conditions. the analysis was done with the porosimeter according to a control method of the welded can. the results regarding the bisphenol a content (bpa) obtained by the gas chromatography coupled with the mass spectroscopy (gc-ms) method are presented in table 5. the gc-ms method allows the accurate identification and dosage of bisphenol a (bpa), according to the mass fragment 213, characteristic for that. on the basis of the measured areas from the spectrograms can be calculated with a great accuracy the bpa concentrations. the values determined by this method are strictly individualized for bpa. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 58 due to all this things the gc-ms method is the most indicated for the dosage of bisphenol a content. 3. results and discussion the initial characteristics of the lacquer layer were tested according to the analyzing standards and presented values corresponding to: the layer sterilization resistance in the b, c, d and e solutions (stas 1687/1-1981); the adhesion of the lacquer layer before and after the sterilization (sr iso 2409:1995); the lacquer quantity applied (weight/m2). table 1. the initial characteristics of the lacquer layer – vegetable products cans characteristics tomatoes paste/white lacquer tomatoes paste/ yellow lacquer 1 tomatoes paste/ yellow lacquer 2 the layer sterilization resistance: solution b solution c solution d solution e lacquer layer appearance: -appropriate appropriate -appropriate appropriate lacquer layer appearance: -appropriate appropriate -appropriate appropriate lacquer layer appearance: -appropriate appropriate -appropriate appropriate weight/m2 (dry lacquer) 8.2 – 8.36 10.76 – 12. 04 15.76 – 16.08 layer adhesion: -before sterilization -after sterilization good good good good good good table 2. the inner appearance of the can – vegetable products cans characteristics storage conditions initially after 1 month at500c 12 months of shelf storage 24 months inner appearance of the can (lacquer layer): tomatoes paste/ white lacquer the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. inner appearance of the can (lacquer layer): tomatoes paste/ yellow lacquer 1 the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. the canlacquer layer exfoliated in points on the wrinkles and areas with a surface 1 2 cm2, nearby the welding area the canlacquer layer exfoliated in frequent points on the wrinkles and areas with a surface 2-3 cm2, nearby the welding area the canlacquer layer exfoliated in scarce points and areas with a surface of 1cm2, nearby the welding area inner appearance of the can (lacquer layer): tomatoes paste/ yellow lacquer 2 the lid and metal can don’t present black spots or rust, exfoliations or wrinkles of the lacquer layer. the canlacquer layer exfoliated in points on the wrinkles and areas with a surface 0.5 cm2, nearby the welding area the canlacquer layer exfoliated in frequent points on the wrinkles and areas with a surface 2-3 cm2, nearby the welding area the canlacquer layer exfoliated in scarce points and areas with a surface of 1cm2, nearby the welding area food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 59 the analysis of the tomatoes paste cans after the oven thermostating at 50 c indicated a degradation of the layer in the case of the yellow lacquer applied in one or two layers. the degradation was more intense after two months in comparison with one month of thermostating. the analysis of the cans after one year of shelf storage presents exfoliations in different points and areas situated nearby the welding area these being more intense for the cans with one layer of yellow lacquer. in both lacquering cases with this type of lacquer after one year of shelf storage the layer degradations are less intense than those seen after one month of thermostating. table 3. the verifying of the lacquer layer porosity – vegetable products cans characteristics storage conditions initially after 1 month at500c 12 months of shelf storage 24 months layer porosity. ma: tomatoes paste/ white lacquer 43.3 43.5 44.5 42.8 layer porosity. ma: tomatoes paste/ yellow lacquer 1 71.9 72.2 73.5 72.8 layer porosity. ma: tomatoes paste/ yellow lacquer 2 51.9 52.8 57.9 53.0 the results subscribed to the following technological standards: max. 112 ma – vegetable cans; max. 89 ma – meat in natural juice cans. after the oven thermostating at 50 c: for one month and respectively for two months the characteristics of the white lacquer layer and that of the yellow lacquer layer for the product tomatoes paste do not suffer significant modifications. the same thing is available after one year of shelf storage. table 4. the verifying of the lacquer layer porosity – vegetable products cans no. name / source chemicalnature extraction environment extraction conditions/ extraction ratio global migration, [ppm] metals release, [ppm] pb cd 0 1 2 3 4 7 8 9 1 gold he 1526-13 1 layer ici packaging coatings epoxyphe nolic distilled water 1h, 121oc/ 1:1 7.75 3 % acetic acid sol 1h, 121oc/ 1:1 9.75 0.012 0.000 isooctane 48h, t.c./ 1:1 4.75 2 gold he 1526-13 2 layers ici packaging coatings epoxyphe nolic distilled water 1h, 121oc/ 1:1 9.0 3 % acetic acid sol 1h, 121oc/ 1:1 11.5 0.019 0.000 isooctane 48h, t.c./ 1:1 5.5 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 60 3 pl 1333-16 grace darex gmbh epoxyphe nolic distilled water 1h, 121oc/ 1:1 9.5 3 % acetic acid sol 1h, 121oc/ 1:1 16.5 0.015 <0.006 isooctane 48h, t.c./ 1:1 7.0 4 vitalure 344 cod 16-4344 l ici packaging coatings epoxyphe nolic distilled water 1h, 121oc/ 1:1 11.5 3 % acetic acid sol 1h, 121oc/ 1:1 24.0 0.014 0.000 isooctane 48h, t.c./ 1:1 5.25 5 vitalure 334 cod n 49234 ici packaging coatings epoxyphe nolic distilled water 1h, 121oc/ 1:1 19.0 3 % acetic acid sol 1h, 121oc/ 1:1 26.75 0.048 <0.001 isooctane 48h, t.c./ 1:1 9.0 6 gz 036 spt polonia epoxyphe nolic distilled water 1h, 121oc/ 1:1 10.5 3 % acetic acid sol 1h, 121oc/ 1:1 18.25 0.007 0.001 isooctane 48h, t.c./ 1:1 6.5 7 sulf resistent lacquer l 3312 policolor romania epoxyphe nolic distilled water 1h, 121oc/ 1:1 21.0 3 % acetic acid sol 1h, 121oc/ 1:1 29.75 0.024 <0.006 isooctane 48h, t.c./ 1:1 6.5 8 acid resistant lacquer l 3311 policolor romania epoxyphe nolic distilled water 1h, 121oc/ 1:1 16.5 3 % acetic acid sol 1h, 121oc/ 1:1 18.25 0.029 <0.006 isooctane 48h, t.c./ 1:1 table 5. the bisphenol content in the protection lacquers for the food cans no. name / source chemicalnature extraction environment extraction conditions/ extraction ratio bisphenol content, [ppm] gc ms 1 he 1526-13 gold 1 layer ici packaging coatings epoxyphenolic distilled water 1h, 121oc/ 1:1 2 he 1526-13 gold 2 layers ici packaging coatings epoxyphenolic distilled water 1h, 121oc/ 1:1 1.08 3 pl 1333-16 grace darex gmbh epoxyphenolic distilled water 1h, 121oc/ 1:1 0.3 4 pl 1014-69 gold grace darex gmbh epoxy-modified distilled water 1h, 121oc/ 1:1 0.85 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume xi, issue 3 – 2012 61 5 vitalure 344 cod 16-4344 l ici packaging coatings epoxyphenolic distilled water 1h, 121oc/ 1:1 > 3.0 6 vitalure 334 cod n 49234 ici packaging coatings epoxyphenolic distilled water 1h, 121oc/ 1:1 0.15 7 gz 036 spt polonia epoxyphenolic distilled water 1h, 121oc/ 1:1 0.19 8 sulf resistent lacquer l 3312 policolor romania epoxyphenolic distilled water 1h, 121oc/ 1:1 0.02 the values obtained for the bpa presented in table 5 are the following: below the value of 1 ppm, for the chromatographic method gc-ms, for all the epoxyphenolic analyzed lacquers with the exception of the two layered he 152613, lacquer; extremely high values obtained for the vilature 344 lacquer, for both methods not recommended for the hygienic sanitary approval. 4. conclusion on the basis of the studies presented we can conclude the fllowings: the values obtained for the global migration of the components in all the extraction environments are situated below the admitted value of 60 ppm; the heavy metals release are below the admitted values : the pb are way below 0,1 ppm, and the cd is situated below the detection value of the device of 0,006 ppm; the uv fluorescence indicated the absence of the condensed polynuclear aromatic hydrocarbons; there was no detection of the following components: epichlorohydrin in the epoxyphenolic lacquers, acetaldehyde in the modified polyester lacquer and amines in the modified epoxy aminic lacquer. 5. references [1]. f. deflorian., s. rossi., m. d. c. vadillo., m. fedel., electrochemical characterisation of protective organic coatings for food packaging, j appl electrochem, 39:2151–2157, doi 10.1007/s10800-009-9818-1, (2009) [2]. bernardo pem, dos santos jlc, costa ng (2005) prog org coat 54:34 [3]. marcus p, mansfeld f (eds) (2006) analytical methods in corrosion science and engineering. taylor and francis, london [4]. begley, t. (2006). from migration to exposure: fda experience. lecture held at the closing conference of the eu project foodmigrosure. baveno, italy, september 27/28, 2006. [5]. koni grob., the future of simulants in compliance testing regarding the migration from food contact materials into food, food control 19, 263–268, (2008) [6]. barilli f, fragni r, gelati s, montanari a (2003) prog org coat [7]. b. ramezanzadeh., m.m. attar., an evaluation of the corrosion resistance and adhesion properties of an epoxynanocomposite on a hot-dip galvanized steel (hdg) treated by different kinds of conversion coatings, surface & coatings technology, doi: 10.1016/j.surfcoat.2011.04.001, (2011) [8]. robertson gl (2006) food packaging: principles and practice. crc press/taylor and francis, london [9]. bonora pl, deflorian f, fedrizzi l (1996) electrochim acta 41:1073 [10]. mojica j, garcia e, rodriguez fj, genesca´ j (2001) prog org coat 42:218 [11]. rohwerder m, turcu f (2007) electrochim acta 53:290 microsoft word 3 journal fia nr 1 2010 final_75-80.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 79 some aspects regarding the diversity of food culture and its influence on consumers’ attitudes cristina-elena hreţcanu1, alice roşu1, silviu-gabriel stroe1, ana leahu1 1stefan cel mare university of suceava, faculty of food engineering, romania, e-mail: cristinah@usv.ro abstract: our main aim in this paper is to explore how food consumption leads to a deeper understanding of societies, its culture and beliefs. the significance of “diversity” of the food products refers to social, cultural and spatial variability and variety as major characteristics of food globalization. the great diversity of food and beverage products forms a part of regional or national cultural identity. when it comes to the diversity of food culture, one should take into consideration some of the following important aspects: a). consumption of food, including gastronomic habits, determined by cultural and social conditions as well as by political and historical contexts; b). food identity markers in terms of ethnic, religious and class identification factors; c). globalization of food, seen as an economic and cultural process; d). highly processed food as against genuine home made food; and the list of enumeration remains open. depending on locale, multiple food producers may specialize in specific types of food characteristic of a given cultural or ethnic tradition. as a product of globalisation we commonly consumme nowadays some kinds of products like coffee and tea. food products have played a major part in the social and religious life of human groups, beeing much more than a source of nutrition. key words: gastronomic habits, identity markers, product of globalisation introduction the definition of food and its ways of consumption are determined by social and cultural conditions and contexts [1]. for human, the consumption of food is affected by many factors such as social positions income, social traditions and religious beliefs. thus, the culture, lifestyle and economic conditions can make a development of local food traditions. in the last decade, a variety of new foods are transplanted from other places and the spread of cuisines across the globe is understood as a way to clarify economic and cultural processes of globalization. as a result of the expansion of the globalization effect, every society has its own particular diet affected by many factors such as social traditions, geographical position and historical context, population, religious beliefs and social class. [2-3]. food functions as an “intimate frontier between self and other, local and foreign, past and present” [4]. as an important part of any celebrate special occasions all over the world like christmas, new year, weddings and birthdays food can unite community bonds and helps to maintain a common identity among a group of persons. materials and methods gastronomic habits become the markers of cultural continuity, difference and assimilation. a). our starting point in studding the journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 80 diversity of food culture might be the different ways of food consumption affected by factors such as income, social traditions, religious beliefs, social positions and so on. then, from another perspective the importance of food in understanding human culture lies precisely in its infinite variability. we can describe, for examples, some aspects regarding french cuisine, italian cuisine and japanese cuisine respectively, as follows: french cuisine is extremely diverse, with only the chinese having similar variety in their food. this variety is supported by the french passion for good food in all its forms, france's extraordinary range of different geographies and climates which support the local production of all types of ingredients, and france's long and varied history. in many ways, an understanding of the culture of french food and recipes is an understanding of france itself. [5] italian cuisine is noted for its regional diversity, abundance of difference in taste, and is known to be one of the most popular in the world, with influences abroad. ingredients and dishes vary by region. many dishes that were once regional, however, have proliferated with variations throughout the country. each area has its own specialties, primarily at regional level, but also at provincial level. the differences can come from a bordering country (such as france or austria), whether a region is close to the sea or the mountains, and economics. italian cuisine is also seasonal with priority placed on the use of fresh produce.[6] japanese cuisine has developed over the centuries as a result of many political and social changes. the cuisine eventually changed with the advent of the medieval age which ushered in a shedding of elitism with the age of shogun rule. in the early modern era massive changes took place that introduced non-japanese cultures, most notably western culture, to japan. the modern term "japanese cuisine" means traditional-style japanese food, similar to that already existing before the end of national seclusion in 1868. in a broader sense of the word, it could also include foods whose ingredients or cooking methods were subsequently introduced from abroad, but which have been developed by japanese who made them their own. japanese cuisine is known for its emphasis on seasonality of food, quality of ingredients and presentation.[7-8] b) as for the second above mentioned aspect we have chosen to deal with the importance of diversity of food culture from the religious point of view, focusing on the spiritual values that food has acquired in food religious rituals and in different religions. for many faiths including christianity, judaism, islam, hinduism and buddhism, these rituals have become central due to the essential role played by food in cultural practices and religious beliefs which is complex and varies among individuals and communities. however, the regulations governing food and drink differ from one to the next, including some faiths that do not advocate any restrictions. for example, the ritual of communion is regularly celebrated by christians. this involves eating bread and drinking wine (or substitutes) to represent the body and blood of jesus christ. catholics, for example, could find a bond between each other and a mark of difference from protestants by substituting fish for meat on fridays. numerous cakes, puddings, pies, and pastries are reserved throughout europe for special occasions. in all these cases, the special food serves to mark the special occasion, emphasizing its spiritual significance. further on we give some examples of traditional christmas foods in different journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 81 countries of the world, differing from one country to the other, depending on many cultural factors and significance [9-10]: in france black and white pudding, this is sausage containing blood; in germany gingerbread biscuits and liqueur chocolates; in russia a feast of 12 different dishes, representing christ’s disciples. c) the third aspect looked upon in this paper is food globalization as most of the foods that people commonly eat today are the products of this global phenomenon. it is primarily due to the interconnectivity between nations and regions, namely social, political, historical and cultural relationships and bonds established between peoples and ethnic communities. but globalization began centuries before the term came into use. historically, trade in food and agriculture has been a key driver of globalization [11]. the collected data presented in table 1 give us some examples of beverage products that have a significant impact on our life at present at global level. table 1: some examples of the globalization results regarding food spreading type of product historical description spreading description coffee [12] 8th c. ethiopia—legend of the dancing goats; 900s coffee first appears in writing, by arab physician, rhazes; 1710 french invent infusion method of brewing coffee; 1820 caffeine (c8h10n4o2) was isolated from green coffee beans; 1900 hills bros. invent vacuum-packed coffee can. by 1500 muslim pilgrims spread coffee to egypt, turkey and persia; 1536 coffee (called “mocha”) was exported through city of mocha in yemen; 1600s india begins cultivation from seeds smuggled in by a muslim; 1650–1690 coffeehouses open in england, germany, vienna, venice and paris; 1699 dutch transplant trees to java in indonesia; 1727 coffee smuggled into brazil; by 1900 americans drink 50% of the coffee in the world. tea [13] 2737 b.c. the second emperor of china, shen nung, discovers tea; 648-749 japanese monk gyoki plants the first tea bushes in buddhist temple gardens; 1211 japanese buddhist abbot eisai writes the first japanese tea book of tea sanitation; 1657 the first tea is sold as a health beverage in london, england; 1702-1714 during queen anne’s reign, tea drinking thrives in british coffeehouses; 1866 over 90 percent of britain's tea is still imported from china; 1909 thomas lipton begins blending and packaging his tea at new york. 479 turkish traders bargain for tea on the border of mongolia; 593 buddhism and tea journey from china to japan; 1610 tea reaches europe, carried by the dutch from bantam, java. the portuguese created trade routes to china. from lisbon the east india company transported the tea to holland, france and germany; late 1600s russia and china sign a treaty that brings the tea trade across mongolia and siberia; 1650 the dutch introduce several teas and tea traditions to new amsterdam (which later becomes new york); 1840 – 1850 the first tea plants, imports from china and india, are cultivated on a trial basis in sri lanka; 1910 sumatra, indonesia becomes a cultivator and exporter of tea followed by kenya and parts of africa. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 82 d) highly processed food as against genuine home made food might be related to food globalization, as they go to some extent hand in hand. fast food chains are more accessible during the standard lunch break than personal home which are usually too far away to return to for a shared meal. food, as we know it today, in microwaveable, flash frozen, snacks, junk food or fast food has gone wrong and lost somehow its basic identity. sometimes people consider their meals around the clock: for example, mcdonald’s seems to be the same everywhere, but according to watson and colleagues [14], the locals of beijing, seoul, tokyo and other asian cities, changed the famous burger company, its food and its menu. mcdonald’s in japan offers rice and shrimp. fast food is now an integral part of the japanese life, but in addition to the usual fare, other “local” specialties are available. [15] modern commercial fast food is often highly processed and prepared in an industrial fashion, i.e., on a large scale with standard ingredients, standardized cooking and production methods. also, eating is one form of creative activity in which subjects are allowed to make choices. [16] it is usually rapidly served a fashion manner which minimizes cost: in cartons, bags or in plastic wrapping. results and discussion taking into consideration a detailed list of dishes and the total cost of the amount for one week food consumption for families from 12 different countries [17], we focused on consumers’ preferences, paying attention to shelflife data of food and beverages for human consumption, expenditure on food and beverages, including the prices paid. as we can remark in the figure 1 the enormous differences regarding the cost of the alimentation is intimately influenced by the income. figure 1: total amount for one week food consumption in a family one can easily notice that there are some disparities between the consumption of certain food items usually related to whether or not a product can be supplied locally. food and beverages are amongst the journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 83 most important consumption items, satisfying the basic physiological needs of hunger and thirst and forming one of the most recurrent expenditure items for the majority of european union households. diets are sometimes considered as signs of ethnic groups and people select their diet from food items available in their geographical region. the influence of the conditions determined by the geographical position and historical influence of the country, religion or cultural as well as the means of production which leads to the development of different dietary habits. great attention should also be paid to the influence of the conditions determined by the geographical position and historical influence of the country, religion or cultural as well as the means of production which lead to the development of different dietary habits in the european countries in question. when purchasing food, european consumers take into consideration, first of all, the quality and secondly the price. using the policies on food safety and hygiene, animal and plant health, nutritional information, labeling, welfare regulations and, also, restrictions on pesticide residues, the european union works to safeguard food quality. using the information from eurostat pocketbooks: food from farm to fork statistics [18], consumption of cereals (excluding rice) and meat are presented in the figure 2 and figure 3: average per capita apparent consumption (for the eu country which sends details regarding these topics): conclusion diversity of food culture leads obviously to a deeper understanding of societies, its culture and beliefs where the gastronomic habits become markers of cultural continuity, difference and assimilation. one can notice that, as a result of the expansion of the globalization effect, every society has its own particular diet affected by many factors such as social traditions, geographical position and historical context, population, religious beliefs and social class. food globalization as a significant cultural phenomenon is due to the interconnectivity between nations and regions, namely social, political, historical and cultural relationships and bonds established among peoples. and not in the last, when it comes to food culture, people should primarily have in view the policies on food safety and hygiene, animal and plant health, nutritional information and labeling and welfare regulations and also restrictions on pesticide residues promoted by the european union that works to safeguard food quality. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 84 acknowledgments this paper was presented in the grundtvig workshop: “challenges in food safety and food quality control„ , 17-22 may 2010, suceava, romania, project 2009-1ro1-gru1303339, ref. no. gru – 09 grat-20-usv, funded by the european union’s grundtvig programme. this paper reflects the views only of the authors. references 1. ferraro g., introduction to cultural anthropology. 3rd edition. belmont: west/ wadsworth, 1998. http://www.betterhealth.vic.gov.au/bhcv2/bhcart icles.nsf/pages/food_and_celebrations?open 2. locher j. l., cuisine and globalization: homogeneity, heterogeneity and beyond. in globalization: critical concepts in sociology, ed. robertson roland and kathleen e. white, london and new york: routledge, vol. 6, 243260, 2003. 3. j hum ecol, ghaderpour yaghob, the ecological and cultural bases of nutrition and dietary, habits of iranian kurds: a case study of bukan rahim farrokhnia, 29(1):39-46, 2010. 4. stoller, p. embodying colonial memories. american anthropologist 96 (3), 634 – 648, 1994. 5. mennell s., all manners of food: eating and taste in england and france from the middle ages to the present, 2nd ed.. chicago: university of illinois press, 1996. 6. italian cuisine. encyclopædia britannica. 2010. encyclopædia britannica, //www.britannica.com/ebchecked/topic/718430 /italian-cuisine online. 20 jul. 2010 7. a day in the life: seasonal foods, the japan forum newsletter no.14 september 1999, http://www.tjf.or.jp/eng/content/japaneseculture/ pdf/ge09shun.pdf online. 20 jul. 2010 8. hiroko shimbo, the japanese kitchen, harvard common press, (2001) 9. fox r., food and eating: an anthropological perspective, the holy meal http://www.sirc.org/publik/food_and_eating_1 1.html online. 20 jul. 2010 10.www.betterhealth.vic.gov.au/bhcv2/bhcpdf. nsf/bypdf/food_and_celebrations/$file/food _and_celebrations.pdf online. 20 jul. 2010 11. kiple, kenneth f. 2007. a movable feast: ten millennia of food globalization. cambridge: cambridge university press. 12. civitello l., cuisine and culture: a history of food & people. john wiley & sons, pag.66, 2003. 13. mccoy e., walker g. f., coffee and tea, g.s. haley company, inc., 1998, www.inpursuitoftea.com, online. 20 jul. 2010 14. watson, james. l. , golden arches east: mcdonald's in east asia. ed. watson james. stanford: stanford university press, 1997. 15. dwyer e., risako i., japan. in “teen life in asia”, ed. judith j. slater. westport, ct: greenwood press, 2004. 16. rouse c., hoskins j., purity, soul food, and sunni islam: explorations at the intersection of consumption and resistance”. cultural anthropology 19 (2), 226-249, 2004. 17. menzel p., d’aluisio f., hungry planet: what the world eats. napa, ca: material world press. 2005, http://www.npr.org/templates/story/story.php?st oryid=5005952 online. 20 jul. 2010 18. pocketbooks: food: from farm to fork statistics http://epp.eurostat.ec.europa.eu/cache/ity_off pub/ks-30-08-339/en/ks-30-08-339-en.pdf online. 20 jul. 2010 microsoft word 14 dabija adriana_2 articol cu citari.doc food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 85 a study establishing an optimum malting diagram *adriana dabija1 1faculty of food engineering, stefan cel mare university od suceava, romania adriana.dabija@fia.usv.ro *corresponding author received 20 april 2012, accepted 21 may 2012 abstract: malt is the main raw material used in beer manufacturing. this is why numerous investigations have been performed to improve its specifications. the quality of the malt used depends mostly on the quality of barley, of brewing barley from which it is obtained, as well as from the way the malting process was conducted. micromalting constitutes a step through which every new variety of barley must pass. in general, one could say that malt has superior biotechnological qualities if it is easily processable, if it has a rich enzyme equipment, ensures high efficiency and if the resulting beer wort ferments well. the raw material used in the experiments was barley originating from england, which was first analyzed from organoleptic and physico-chemical points of view, then subjected to a micromalting process using four different technologies. the quality indices as determined by chemical analysis of brewing barley confirmed that it can be used as raw material in the manufacturing of malt for the brewing industry. finally an option to set optimum malting technology, establishing technological parameters and working diagrams for each stage of malt production. the technological version of malting by resteeping which presented higher values for most physical and chemical indicators determined for the malt-finished product. keywords: brewing barley, micromalting, biotechnological characteristics, malting losses 1. introduction malt, considered by experts "the heart of beer", is the essential raw material used in beer manufacturing. the modern malting and brewing industry makes use of a wide range of technical, biochemical, microbiological and genetic inventions [1, 2]. the basic processes in order to obtain malt have remained the same over time, with changes in technology, science and techniques enabling the shortening of production cycles and ensuring a constant quality of malt produced [3, 4]. the main purpose of malting is the accumulation of enzymes and the partial hydrolysis of macromolecular substances of the grain. administering on an industrial scale the malting process aims not only to obtain a high quality malt, but takes into account the economic aspect, namely to reduce the process’ time and to achieve a high productivity [5, 6, 7]. the main factors influencing the quality of malt are: barley used as raw material, malting technology adopted and malting equipment used [8, 9]. this paper proposes, starting from a given raw material, an optimal way of malting, as well as determining the main technological parameters and working diagrams for each basic operation for obtaining malt 2. experimental the raw material used in the experiments was barley originating from england, the harvest of 2011. for analysis we used two average samples of barley from the same batch, taken as standards. the results of organoleptic and physicochemical analysis for the two barley food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 86 samples studied are presented in tables 1 and 2. table 1 organoleptic analysis results for barley samples characteristics sample 1 sample 2 aspect typically, coated with fine wrinkles typically, coated with fine wrinkles colour pale yellow, <5% grains showing black spots or peaks, <10% grain of matte colour pale yellow, <2% grains showing black spots or peaks, <6% grain of matte colour smell fresh straw, no hint of mould fresh straw, no hint of mould taste sweet, characteristic for fresh cereal sweet, characteristic for fresh cereal table 2 physicochemical analysis results for barley samples characteristics sample 1 sample 2 hectolitre weight 65.8 65.5 mass of 1000 grains 41.5 42.2 uniformity (assortment) 85 88 foreign body. total. out of which: 3.2 2.8 inert mineral body; 0.1 0.2 inert organic body; 0.1 0.1 weeds’’ seeds; 0.6 0.5 embrio-less seeds and broken grains; 2.0 1.5 empty seeds. 0.4 0.5 germinative energy 82 82 germinative capacity 86 85 water sensitivity 37 40 moisture. % 13.4 13.8 proteins. % 10.8 10.5 starch as percentage dry weight 61.5 62 evaluating the quality of the malt obtained through the experimental malting versions was done via a physico-chemical analysis of beer wort obtained in the laboratory by the "kongress" method. using the following methods: humidity oven drying method; glassines with grain cutter; yield in extract method kongress; colour iodometric method; ph – potentiometric method; hartong index kongress method; kolbach index windisch-kolbach method. 3. results and discussions all malting versions were applied with the purpose of obtaining blond malt. as this type of malt can be produced on a larger scale than other types of malt (brown malt. special malts). 3.1. malting process using constant temperature germination steeping in this first version of malting used the chart below (fig.1): 13,6% 44% 40%38% 32%30% 0 5 10 15 20 25 30 35 40 45 50 0 4 18 20 34 35 steeping period [h] b ar le y m oi st ur e [% ] figure 1. steeping diagram for first experimental version wet steeping for 4 to 6 hours to a moisture content of 30%; food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 87 dried steeping for 14 ÷ 6 p.m. when grain moisture increased slightly to 31 ÷ 32% due to water adhering to the beans. during this period co2 adsorption was performed at first regularly and then continuously; wet steeping until it reached 38% humidity for 2 to 4 hours; dried steeping for 14 ÷ 18 hours of temporary adsorption of co2. barley moisture reaching is 40% in the end; wet steeping for 1 ÷ 2 hours. final moisture of 44 ÷ 46%. then barley was switched to germination. thus the total of 35 ÷ 48 hours of steeping. steeping wet lasted 7 to 12 hours. so approx. 25% of the duration of steeping. steeping temperature was 12 ÷ 15°c. germination process applied in this version used a constant temperature of 15°c. the process of germination was 7 days. because the drying process under laboratory conditions does not influence decisively malt quality obtained. for all experimental versions the same drying diagram was used to obtain blond malt. in preparing this diagram was intended to obtain pale malt with high enzyme activity. normal humidity and shorter final kilning operation which lasted on average 20 to 22 hours (fig.2). 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 14 16 18 20 21 kilning period [h] k iln in g te m pe ra tu re [0 c ] figure 2. kilning diagram for all experimental versions 3.2. malting process with high germination temperatures the steeping diagram in this version of malting was the following: wet steeping to a moisture content of 40%. steeping temperature of 11°c; dried steeping followed by sprinkled wet steeping. increasing temperature to for 14 ÷ 6 p.m. when grain moisture increased slightly to 15°c for 20 hours. until grain moisture reaches 43%; dried steeping followed by sprinkled wet steeping at 15°c for 12 hours until a final moisture of 46%. from steeping. barley was passed for germination 6 to 7 days. aeration of germinating green malt was made daily and malt humidity decreased. to achieve the desired humidity level at the end of germination (44 ÷ 45%). from the third day of germination. green malt was moistened by spraying with water. 3.3. malting process with decreasing germination temperatures steeping diagram adopted in this version was as follows: wet steeping for 6 to 8 hours. with strong aeration. steeping temperature of 18°c. until grain moisture reached 36 ÷ 38%; dried steeping for 24 hours with ventilation to remove co2; wet steeping up to a final moisture of 46 ÷ 48%. steeping temperature of 20°c. then barley was sent to germinate. the literature shows that the germination process with decreasing temperature is a modern malting process based on the fact that germination begins as early as steeping phase [9]. the barley introduced to germinate had a temperature of 18°c. with 30% seeds germinated (sprouted). after steeping. the maximum humidity reached 48%. with a drop in temperature to levels of 12 to 13°c. on the fourth day of germination. 3.4. malting process with resteeping in the literature this process is presented as a modern malting process based on repeated steeping of green malt plants even in germination fixtures. noteworthy is the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 88 fact that the malt manufactured by this process has rootlets less developed but is rich in enzymes and malting losses can be reduced to only 5 to 6% for dry substance. the whole process of steeping germination takes only 6 to 7 days. the steeping process used the steeping diagram presented in the first experimental version. except that steeping was considered completed when grain moisture reached 40%. the barley grains with this moisture were transferred to germination and maintained for approx. 60 hours at 17°c. during which the barley began to germinate quickly and uniformly. at this point resteeping was applied. namely the introduction of water with temperature of 17°c. after a period of 24 hours barley reached a moisture content of 50 ÷ 52%. thus. the embryo was inactivated. but malt modification continued by enzymatic activity which maintained due to the high moisture of the germinating grains. simultaneously. resteeping determined the rootlets losses to decrease. mainly due to low temperatures for germination. 3.5. evaluating the quality of malt produced by the adopted experimental versions malt obtained in the four experimental variants was analyzed in laboratory conditions. knowing that only physicochemical analysis are objective and decisive for assessing the biotechnological qualities necessary for brewing. sensory analysis results are presented in table 3. malt obtained in all experimental variants corresponds from sensorial point of view to blond malt for brewing. physico-chemical analysis results for malt are presented in table 4. table 3 organoleptic analysis results for malt – finished product experimental version characteristic v1 v2 v3 v4 aspect uniform sized and shaped grains uniform sized and shaped grains uniform sized and shaped grains uniform sized and shaped grains colour pale yellow pale yellow. < 6% grey coloured grains pale yellow.< 4% grey coloured grains pale yellow smell characteristic. no hint of mould characteristic. no hint of mould characteristic. no hint of mould characteristic. no hint of mould taste pleasant. sweet pleasant. sweet pleasant. sweet pleasant. sweet table 4 physico-chemical analysis results for malt – finished product experimental version quality index v1 v2 v3 v4 moisture. % 4.0 4.0 4.3 4.1 friabilimeter value. % 87.5 85 87.5 93 friable grains. % 85 83 86 90 semivitreous grains. % 10 13 11 4 vitreous grains. % 5 4 3 6 colour. unit. ebc 3.4 2.9 3.1 3.0 extract % based on dry weight 82.5 82.8 83 82.9 saccharification rate. min. 15 12 15 10 filtration rate. min. 55 53 45 40 ph of wort 5.7 5.90 5.8 5.6 hartong index 4.0 4.2 3.9 5.6 kolbach index 34.8 37.4 39.6 42.5 extract difference % based on dry weight 1.4 1.7 1.0 0.7 amylolitic capacity. 0wk 255 230 265 305 malting losses. % s.u. 12.0 14.2 12.8 8.5 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 89 malt moisture. it is known that malt moisture is of great importance if one takes into account the gain of extract that is made from high water content. but due to high water content. malt quality suffers during storage. losing its flavour. its degree of solubility changes during storage. causing difficulties in milling and leads to some beers with lower taste quality and colloidal stability. glassines malt. all malt samples analyzed have values close of this indicator gives information on the degree of malt modification but only malt obtained with version 4 of malting with resteeping corresponds to quality standards. with a value of 7%. malt colour. malt obtained in all experimental versions matches the type of blond malt. the palest malt was obtained by process of malting with decreasing temperatures to germination version 2. this quality indicator does not provide reliable information about future colour of beer. but the malt analysis gives us information on the type of malt. saccharification rate. this indicator shows similar values falling within the standard limits for all four samples analyzed. filtration rate. general observation that emerges from comparison of this indicator is that none of the samples analyzed has reached or exceeded the recommended maximum ebc rules. namely 60 minutes. from which malt is considered insufficiently soluble. malt modification is best obtained by malting with resteeping. experimental version 4. ph of wort value. influences enzymatic decomposition processes and determines protein solubilization. the bitter substances of hops and the boiling wort colour. there is dependence between the ph of the wort and beer produced from it. beers with high ph values are subjected to physicochemical disturbances due to insufficient coagulation of proteins at boiling phase. ph value on malt samples analyzed is approximately constant. which shows that the technological process for obtaining malt went normally. without variation of process parameters in the four experimental versions. hartong index. this index is a summary of physical and chemical methods of assessing the quality of malt and gives information on how the main malting operations were conducted. assessment of malt modification according to hartong index after ebc criteria shows that: malt obtained by malting versions 1. 2 and 3 is slightly weathered. with a low enzyme output; malt obtained by malting with resteeping. version 4. is well weathered. rich in enzymes and appropriate for producing beers with good stability. malt extract yield varies between 82.5% (version 1) and 83% (version 3). so they have very close. high values. this index summing the total of soluble substances. kolbach index. it is known that the quality indicator is a measure of proteolytic disintegration of malt and is a hint on proteolytic enzymes content of malt. analyzing the data shown in table 5 we can say that: malt obtained by versiont 1 of malting is an insufficient disaggregated malt; malt obtained by malting versions 2 and 3 is a well weathered malt; malt obtained by version 4 of malting is a highly disaggregated malt. yield difference between fine and coarse milling is an important criterion for malt modification. as well as for hemicelluloses and enzymatic capacity of malt. all samples analyzed showed a good malt cytolytic modification. one presents the best value obtained by malting with resteeping version 4. the largest diastatic power was obtained by malting version 4. this malt will lead to wort with high degree of fermentation. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 2 – 2012 90 with regard to malting losses. the lowest values were recorded for version 4. malting process with resteeping. which can be explained by reducing losses due to grains forming rootlets and limit breathing. from the pooled analysis presented from blond malt assessment in laboratory conditions. it can be observed that. of the four versions of malting applied. the best results. as reflected in indices determined were obtained by applying the malting process with resteeping version four. 4. conclusions administering the malting process is generally aimed at achieving quality malt with superior biotechnological characteristics. with minimum costs of production and an increased productivity. through a rational management of technological parameters during the malting process. these objectives can be achieved. the raw material used was barley. the origin of 2011 harvest england. the main physical and chemical quality indices of two samples taken from the same batch of barley were determined that they meet the technical requirements for manufacturing quality malt for beer industry. data from the literature provided the malting methods that were suitable for analysis of barley’s technological qualities. parameters were established for each technological operation and diagrams were drawn for four different malting versions. malt finished product was analyzed from organoleptic and physico-chemical points of view. based on the results presented. the paper showed that malt obtained by version 4 malting with resteeping – has higher values for most indicators determined. which recommends it as an optimal option for malting the barley samples taken. also. the lowest losses were obtained for version 4. and so from an economical point of view. malting process with resteeping is most advantageous. a verification of the optimal manufacturing conditions established under laboratory conditions is required. completing the work charts is advisable to be carried out only after going through a malting cycle inside a factory. 5. references [1]. banu c.. et al.. the science and technology of malt and beer (tratat de ştiinţa şi tehnologia malţului şi a berii). vol. i. editura agir. bucureşti. (2000) [2]. brrigs d.e.. malting and brewing science. chapman et hall. london (1995) [3]. kunze w.. technology brewing and malting. vlb. berlin. (1996) [4]. moll m.. beers and coolers. editura lavoisier. paris. (1991) [5]. bamforth c.w.. barclay a.h.p.. malting technology and the uses of malt. barley: chemistry and technology american association of cereal chemists. st.paul. mn usa (1993) [6]. berzescu p.. et al.. technology brewing and malting (tehnologia berii şi a malţului). editura ceres. bucureşti. (1981) [7]. fix g.. principles of brewing science. boulder: brewers publications. (1999) [8]. jones b.. endoproteases of barley and malt. journal of cereal science. 42. 139+156 (2005) [9]. stroia i.. factors determining malt quality (factori care determină calitatea malţului). universitatea politehnică bucureşti. (1998) microsoft word 3 journal fia nr 1 2010 final_9-14.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 13 improving the antioxidizing capacity of catering products by adding spices micşunica rusu 1, rodica segal2 1economic college of piatra neamţ, 16 alexandru lăpuşneanu st., piatra neamţ, romania e-mail: rusu_mic@yahoo.com 2dunarea de jos galati university, faculty of food science and engineering, 47 domneasca st., galati, romania abstract: biologically active compounds are chemical compounds of food with an essential role in the good development of the human body. to this category belong: proteins, fatty acids monoor polyunsaturated, dietary fibers, vitamins, mineral salts, bioantioxidants, etc. these compounds are spread into a large variety of foods, but especially of vegetal origin. in this way, spices and aromatic herbs can represent important sources of antioxidants for the body. these can be found in different catering products but their quantities are limited by changes in sensory characteristics that are not always agreed by consumers. the study intends to assess the antioxidant potential of three spices (basil, thyme and oregano) and the acceptance by consumers of some catering products with the content of antioxidants improved by addition of these spices. to investigate the acceptability of new spreads by consumers a preference test was used, of 9-point hedonic scale, verbal anchors as those devoted. the preparation has been tasted by 106 consumers aged between 14 and 52 years (14-17 years, 18-20 years, more than 20 years). the medium scores were attained 6,19 for oregano dish, 6,88 for the thyme dish and for the one with basil, placed between „like moderately” and „like slightly”. the degree of sympathy has evolved differently between groups. for consumers under 16 years with basil product had the best scores, followed by thyme. for other consumers, the preparation with thyme had higher score, followed by basil. key words: bioactive compounds, basil, thyme, oregano, orac, sensory analysis introduction antioxidants have gained a reputation of “miracle health component”, by 2007 being published over 45,000 scientific articles about natural antioxidants [1]. a bioantioxidant is a substance in food that significantly decreases the adverse effects of free radicals on normal physiological functions in humans demonstrated that free radicals are involved in the occurrence of many diseases and aging [2]. to assess the antioxidant capacity, a number of methods are used, the most commonly used being orac, trap, teac, frap, cuprac, dpph. the best known method for determining and expressing the antioxidant capacity is orac (oxygen radical absorbance capacity). it enjoys special advertising and also databases for total antioxidant capacity with most records are increasingly using this method, the most popular database at this time is the antioxidant capacity of the united states department of agriculture usda [3]. according to their origin, there are endogenous and exogenous antioxidants. endogenous antioxidants play a crucial role in maintaining optimal health-related cellular functions, thus influencing the well-being of the body [4]. endogenous antioxidants available to counteract free radicals are the body type enzymatic journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 14 (glutathione peroxidase, catalase and superoxide dismutase etc.) and nonenzymatic (vitamins e and c; thiol antioxidants: glutathione, lipoic acid; melatonin; carotenoids; bioflavonoids; etc.). for proper functioning of the body, external support of antioxidants is needed, they are recommended to be taken from diet. the main role of the diet is to provide sufficient nutrients to meet metabolic requirements of an individual to give the consumer a good feeling, satisfaction and wealth but also to maintain a healthy and optimal development and reduce risk of disease [5]. these roles are fulfilled by the antioxidants present in preparations from the diet. fruits, vegetables, grain products are considered good sources of antioxidants, due to their high content in vitamins, minerals, bioflavonoids, high components antiradicalic potential [6]. aromatic herbs represent a reservoir of phenolic compounds concentrated in just a few grams of material and can represent one of the simplest ways to increase the phenolic content and antioxidant capacity of the daily diet, with possible health benefits [7]. there are many research studies on the influence of dietary flavonoids on consumer health, many of them referring to the effects on the circulatory system. some of the conclusions of the studies from the first period are: (a) foods rich in flavonoids have a beneficial effect on flow-mediated dilation, blood pressure, lipid profile, which are relevant indicators of cardiovascular health [8]; (b) many food flavonoids proved to be beneficial in atherosclerosis, in platelet aggregation and vascular reactivity; (c) flavonoids may induce neutral lipid hydrolysis from lipid stores through cyclic nucleotide phosphodiesterase (pde) inhibition in adipose tissue and liver [9]; (d) cocoa polyphenols reduce blood pressure [10]. flavonoids also play other beneficial roles in the body: they enhance memory and concentration power, increase efficiency of vitamin c, prevent blood clots, maintain proper functioning of the immune system, etc. [11]. a recent study by french researchers on extracts of 30 plants has shown that there is a significant relationship between total phenolic compounds and antioxidant capacity, implying that these phenolic compounds have a major contribution to the antioxidant properties of plants analyzed [12]. thus, the addition of spices and herbs to meals, in a percentage consistent with the sensory properties, can cause a significant increase in total antioxidant capacity. this study proposes the introduction of spices of high antioxidant value (according to orac values of data taken from the usda) in preparations catering assessing consumer acceptability of new products and coverage of the recommended daily dose of antioxidants (the recommended dietary allowance rdaax ) by new preparations. the selected spices were basil, thyme and oregano, and as the matrix-product, the cheese spread. the reasons for the selection of spices refer to the antioxidant capacity, and in addition: the basil [hydrophilic-orac: (horac)– 64439 µmol te/100g, lipophilicorac (l-orac),– 3114 µmol te/100g, total-orac 67553 µmol te/100g, total phenolics (tp) 4489 mg gae/100g, as the usda] is one of the most popular plants, being considered a ”bridge” between medicine, food and tradition; thyme (h-orac 27426 µmol te/100g, total-orac 27426 µmol te/100g, pt 1734 mg gae/100g, for fresh thyme and total-orac 391800 µmol te/100g for dried thyme) is a spice that grows well in our country conditions and it is spread in romanian traditional recipes, being used both fresh and dried; oregano (h-orac 183141 µmol journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 15 te/100g, l-orac 16988 µmol te/100g, total-orac 200129 µmol te/100g, pt 7282 mg gae/100 g) a special spice with antioxidant properties (as orac value), less used in traditional romanian, but frequently used in mediterranean cuisine. materials ad methods dry spices purchased from different manufacturing companies were used. before being used, a rough division was made. for the chosen matrix – product, namely cheese spread, the basic recipe is taken over by stere stavrositu [13] and is shown in table 1. in the original recipe for cheese paste, the classic spice – paprika was replaced by new spices, in the same amount. the working stages were respected:  preparatory operations: bread is browned on the stove or grill, cheese is washed, the two types of cheese are passed through the sieve, butter is rubbed, spices are sliced;  getting cheese paste: mix all ingredients, mixing until it mixes;  preparing the spreads: cheese paste is stretched on slices of bread with the toothed knife. table 1 preparation recipe – cheese spread material um quantity for 40 portions white bread slices 80 cheese paste kg 1 sheep cheese kg 0.150 fresh cow cheese kg 0.600 butter kg 0.150 cream kg 0.100 sweet paprika kg 0.020 to investigate the acceptability of new spreads by consumers, we used the sensory analysis, a preference test, of 9-point hedonic scale, verbal anchors as those devoted. [14]. in the preparatory phase samples were encoded (by numbers) and when analyzing, the purpose of the activity, conduct and document structure were presented to participants. tasting sheet implied included: scoring scale, appropriate verbal anchors (like extremely, like very much, like moderately, like slightly, neither like nor dislike, dislike slightly, dislike moderately, dislike very much, dislike extremely), codes for the three samples (combinations of numbers), instructions for the tasters and a special rubric for comments. the instructions on the form (taster's tasks) have two functions: first, to describe the test mechanism, then to encourage the freedom of reply: “you will be given multiple servings of food for consumption and asked to tell about each, how much you like or dislike. use the scale to indicate your attitude, by marking (x) the score that best describes your feelings about food. remember that you judge. you alone can say what you like. nobody knows if these foods should be considered good, bad or indifferent. an honest personal opinion of you will help us decide. take a glass of water after you finish each sample and then wait for the next sample.” to assess the degree of coverage rdaax values in the usda database, expressed in orac units were used. results and discussion the preparation has been tasted by 120 consumers aged between 14 and 52 years (14-17 years, 18-20 years, more than 20 years). 14 of them say they do not like cheese, they wanted to participate in the test, but those records were not taken into account. the distribution of responses, when using the hedonic scale, when tasting the three preparations is shown in figure 1. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 16 for each sample the average value, standard deviation, percent responses “dislike” were calculated (table 2). by characterizing the products, the following ratings were obtained :  for cheese spreads with oregano: 6,19 (“like slightly”);  for cheese spreads with thyme: 6,88 (“like slightly”);  for cheese spreads with basil: 7,25 (“like moderately”). 1 2 3 4 5 6 7 8 9 oregano thyme 0 5 10 15 20 25 30 35 40 45 50 fr eq u en cy o f sc o re s core oregano basil thyme fig. 1 distribution of responses when using hedonic scale table 2 statistical values for sensory analysis cheese spread paste with statistical parameters age category oregano thyme basil 14-16 70 70 70 17-20 18 18 18 >20 18 18 18 total of answers total 106 106 106 total score 656 729 769 14-16 5.96 6.47 7.17 17-20 6.61 7.67 7.44 >20 6.67 7.67 7.39 average value total 6.19 6.88 7.25 14-16 6.50 11.12 8.80 17-20 2.50 2.65 3.43 >20 2.55 3.04 3.46 standard deviation total 7.68 10.09 9.86 14-16 17.14 10 4.28 17-20 0 0 0 >20 0 0 0 percentage responses to “dislike” total 11.32 6.60 2.83 the degree of sympathy has evolved differently between groups. for consumers under 16 years with basil product had the best scores, followed by thyme. for other consumers, the preparation with thyme had higher score, followed by basil. less than 5% of consumers 14-16 years group (and none of the other groups) reported that “they didn't like” the preparation with basil, compared with more than 17%, declaring that they didn't like the product with oregano. varied distributions of responses and high standard deviations resulting from all the products are typical. they do not necessarily indicate the lack of precision of the method, but it reflects the fact that normally there are large differences among people, about feelings related to food analysis. of data analysis there was also found a greater inconstancy in options for age group 14 – 16 years. also, in the same category were included those who declared that they do not consume cheese (they do not like it). we can say that all the three products can be placed in consumption, but especially those with basil. considering rdaax 3000 orac units journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 17 (µmol te/100g), the recommended minimum value of cao and prior [15], we determined the percentage that can be met by consuming a portion of preparations analyzed. the comparison includes the spreads with paprika (figure 2). 65,30 33,35 11,26 2,99 0,00 20,00 40,00 60,00 80,00 100,00 120,00 thyme oregano basil paprika condiment % r d a ax % coverage by rdaax fig. 2 – the percentage of coverage by the rda of antioxidants spices used to obtain various types of spreads the obtained spreads which normally cover 4-5% of energy, can cover up to 1/3 (for oregano), of daily requirement of antioxidants. the highest value is for thyme, oregano and basil followed. to increase the effectiveness of this approach for safe food preparation, preparations frequently used should be chosen and those which ideally combine with good sources of antioxidants. conclusions 1. in the case of spreads with cheese paste, the matrix preparation chosen to introduce the spices accepts, in terms of sensory analysis, a large number of recipes and a significant percent represent those which include different spices, resulting in preparation of a high antioxidizing capacity; 2. the results obtained when tasting the products recommend the basil paste cheese spreads as a preparation that can be successful, followed close enough by the thyme preparation ; 3. a simple sandwich can cover more than 1/3 of daily requirement of antioxidants for a day. references 1. caillet, stéphanem, yu, hanling, et al., fenton reaction applied for screening natural antioxidants, food chemistry, vol. 100, 2007, p. 542–545 2. valdez, l., et al., free radical chemistry in biological systems, biol res, vol. 33, 2000, p. 6570 3. http://www.ars.usda.gov/ 4. segal, rodica, costin, g..m., alimente funcţionale, ed. academica, galaţi, 1999, p. 34-36 5. harnly, james, doherty, robert, et al., flavonoid content of u.s. fruits, vegetables, and nuts, j. agric. food chem., vol. 54,2006, p. 9966– 9977 6. zheng, w., wang, s.y., antioxidant activity and phenolic compounds în selected herbs, j agric food chem, vol. 49, 2001, p. 5165–5170 7. ninfali, paolino, mea, gloria, et al., antioxidant capacity of vegetables, spices and journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 18 dressings relevant to nutrition, british journal of nutrition, vol. 93, 2005, p. 257–266 8. geleijnse, johanna, hollman, peter, flavonoids and cardiovascular health: which compounds, what mechanisms?, american journal of clinical nutrition, vol. 88, 2008, p. 12-13 9. peluso, michael, flavonoids attenuate cardiovascular disease, inhibit phosphodiesterase, and modulate lipid homeostasis în adipose tissue and liver, experimental biology and medicine, vol. 231, 2006, p.1287-1299 10. taubert, d., roesen, r., schömig, e., effect of cocoa and tea intake on blood pressure; a meta-analysis, arch intern med, vol. 167, 2007, p. 626–34 11. rahman, khalid, studies on free radicals, antioxidants, and co-factors, clin. interv. aging, vol. 2, 2007, p. 219–236 12. dudonne, s., vitrac, x., et al., comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using dpph, abts, frap, sod, and orac assays, j. agric. food chem., vol.57, 2009, p. 1768–1774 13. stavrositu, stere, reţetar de preparate culinare: produse de cofetarie, patiserie si arta serviciilor în restaurante pentru turismul intern şi internaţional, ed. arta serviciilor în turism prof. stere stavrositu, constanţa, 2007, p. 579 14. http://www.sensorysociety.org/ 15. prior, r. l., cao, g. h., analysis of botanicals and dietary supplements for antioxidant capacity: a review, j. aoac int, vol. 83 (4), 2000, p. 950-956 233 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, stefan cel mare university of suceava, romania volume xiv, issue 2 – 2015, pag. 233 240 mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments *silviu-gabriel stroe1 1faculty of food engineering, stefan cel mare university of suceava, 13 universitatii street, 720229, suceava, romania, silvius@fia.usv.ro *corresponding author received may 16th 2014, accepted june 7th 2014 abstract: the purpose of this research was to study the mathematical modelling of dependence between testing parameters and some metallic elements diffused into simulated acidic environments. diffusion processes occurring at the contact between aisi 304 stainless steel samples and simulated acidic environments were analyzed to fulfill this goal. in order to process the experimental data by statistical and mathematical methods, the following steps were taken: diffusion testing of cr, mn, 56fe and ni elements from aisi304 stainless steel samples into solutions with concentration of 3%, 6% and 9% acetic acid; chemical analysis of corrosive solutions using mass spectrometry and inductively coupled plasma method (icp-ms); the results were processed using anova method and thus resulting the mathematical models that describe the dependence between variables. a polynomial model with independent variables was used to obtain the mathematical models: temperature of acidic simulated solutions (x1), testing time (x2), stirring grade of environment (x3) and the dependent variables (response function): y1 concentration of chromium (cr), y2 concentration of manganese (mn), y3 concentration of iron (56fe) and y4 concentration of nickel (ni) found in corrosive environments. after having made the analysis of variance anova, a model having the lowest p value (critical probability) for all variables was chosen. the regression coefficient was determined to verify the validity of each mathematical model. keywords: stainless steel, simulated environments, diffusion, anova method, mathematical model. 1. introduction it is known that food raw materials have their own natural metal content. in case when there are additions to this natural content, the diffusion from metal surfaces coming into direct contact can cause serious health problems or a change unwanted organoleptic characteristics of the finished product. these metals are found in absolutely all foodstuffs in lower or higher concentrations, depending on various circumstances [1]. a mathematical model that describes the physical processes which occur at the contact of some material from the food processing chain is considered a very important tool that can replace, at least partially, the experimental investigations which are expensive and require longer time. the models thus obtained aimed at complying with the conformity assessment of specific regulations concerning migration limits and at describing the time variation of the http://www.fia.usv.ro/fiajournal mailto:silvius@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 3-2 4 0 234 values of parameters. having in view the advantages of this tool, in recent years, numerous studies have been undertaken for this purpose [2-7]. the research described in the literature highlights a relatively limited approach of the diffusion systems consisting of materials intended to come into contact with foodstufs. firstly, it is useful to present a schematic illustration of the mathematical formulation stages of this problem and the finding of adequate solutions is shown in figure 1 [8]. fig. 1. the problem formulating stages and finding solutions [8] the general mathematical model solutions thus obtained provide a quantitative and qualitative perspective on how the parameters affect the phenomenon; further research could be focused on the application of these basic equations [8]. the aim of this work was to study the mathematical modelling of dependence between the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic solutions and the testing parameters. similar researches have been conducted for advanced characterization behavior of aisi321 stainless steel samples in acidic environments [4]. 2. matherials and methods 2.1. metallic samples in this research metallic samples made of aisi304 stainless steel grade were used. the samples sizes were of 400.5 x 400.5 x 1 mm and they were established by the ministerial decree of 21.03.1973, which stipulates that the ratio of exposed surface of the stainless steel samples and solution volume should be between 0.5 ... 2 [9]. the chemical composition of metallic samples (according to the sr en 100882:2005 romanian standard) is shown in table 1. table 1 chemical composition of aisi304 stainless steel samples (wt %) fe c mn p s si cr ni 67 0.07 1-2 0.045 0.03 0-1 17-19 8-10.5 a system μscan (manufactured by nanofocus germany) was used to determine roughness of the metallic samples. measurement and calculation of usual surface parameters were made according to din en iso 4287 and din en iso 4288 standards. the surface mean roughness of aisi304 stainless steel samples was = 0.5988750.0125 . 2.2. corrosive environments all solutions were freshly prepared with quality analytical chemical reagents. given the fact that acidic environments are one of the most aggressive environments in the food processing industry, within the experiment we used 3%, 6% and 9% ch3cooh in double distilled water (according to italian d. m. of 21-03-1973) [9]. glacial acetic acid (sigma-alorich, germany) was used to prepare the experimental solutions. 2.3. testing method to cover a domain as broad as that of using aisi304 stainless steel, three testing solutions solving mathematizatio n initial assumption s problem formulation identificatio n phenomenon food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 3-2 4 0 235 parameters were chosen. to determine the variation levels of testing parameters, the usual values encountered in the processing industry were considered. the levels of the three parameters used in the experiment are shown in table 2. table 2 variation levels of testing parameters levels parameters minimum level central level maximum level temperature solution, [°c] 22 28 32 testing time, [min] 30 60 90 stirring environment, [r/min] 0 125 250 the samples were kept in the oven to make the experiments in stationary environment and at temperatures different from the room temperature one (22°c). a magnetic heat stirrer heidolph mr hei-tec (heidolph, germany) was used to make the experiments at the temperature of 28°c, 34°c respectively and in stirred environment. 2.4. chemical analysis of corrosive environments inductively coupled plasma mass spectrometry (icp-ms) was used to analyze the chemical composition of corrosive environments, both before having used them in the corrosion tests, and after having made them, taking into consideration the adavantages of this method as compared to other analytical ones. 2.5. mathematical modelling of the constituents’ concentrations anova method, known as the variance analysis was appealed to in order to find a mathematical model as accurate as possible which describe the diffusion phenomena. this technique was chosen due to the fact that it is highly recommended when studying a larger number of levels of independent variables, providing higher accuracy of the effect that the independent variables have upon the dependent ones as well as of the their joint effect. having in view that some practical values (minimum values) of responses are to be determined, it is necessary to establish some interdependences capable of describing both the nature and the extent of the influences taken into consideration (see also [4]). to develop the predictive-mathematical models the design expert software (trial version) was used. in order to obtain the mathematical models a polynomial model with independent variables was used: acidic simulated solutions temperature (x1), testing time (x2), stirring grade of environment (x3) and the dependent variables (response function): y1 concentration of chromium (cr), y2 concentration of manganese (mn), y3 concentration of iron (56fe) and y4 concentration of nickel (ni) found in corrosive environments. after having made the analysis of variance anova, a model having the lowest p value (critical probability) for all variables was chosen. the regression coefficient was determined to verify the validity of each mathematical model. 3. results and discussion the chemical analysis of corrosive environments was intended to determine the concentration of cr, mn, fe and ni elements migrated from aisi304 stainless steel samples. 3.1. mathematical modelling of metallic constituent concentrations migrated into 3% ch3cooh solutions the statistical summary of mathematical models is shown in table 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 324 0 236 table 3 summary statistic of mathematical models proposed for the dependent variables dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.0070 quintic 8.59 0.9967 0.9783 y2 0.0190 quintic 1.71 0.9964 0.9765 y3 0.0490 quintic 323.04 0.9910 0.9300 y4 0.0099 quintic 7.82 0.9964 0.9765 the mathematical model that defines the variation of chromium concentration (dependent variable) depending on the testing parameters (independent variables) is shown in equation 1. = 7.96 + 3.83 ∙ x − 3.72 ∙ x − 1.28 ∙ x + 3.06 ∙ x + 6.06 ∙ x + 3.06 ∙ x + 17.75 ∙ x ∙ x − 45.25 ∙ x ∙ x − 0.25 ∙ x ∙ x + 25.08 ∙ x ∙ x + 48.92 ∙ x ∙ x + 15.25 ∙ x ∙ x + 9.58 ∙ x ∙ x − 9.50 ∙ x ∙ x ∙ x + 5.42 ∙ x ∙ x + 30.25 ∙ x ∙ x ∙ x + 43.42 ∙ x ∙ x + 21.00 ∙ x ∙ x ∙ x − 26.25 ∙ x ∙ x ∙ x − 9.58 ∙ x ∙ x (1) in order to check the validity of the mathematical model proposed, the experimental values vs model values were plotted (figure 2). fig. 2. validity check of the y1 (chromium concentration) mathematical model the mathematical model of the y2 response function is shown in equation 2. = 4.04 − 0.59 ∙ x − 0.35 ∙ x2 + 1.39 ∙ x + 0.38 ∙ x + 0.68 ∙ x2 + 0.18 ∙ x + 3.18 ∙ x ∙ x2 − 8.20 ∙ x ∙ x + 0.30 ∙ x2 ∙ x + 4.18 ∙ x ∙ x2 + 8.98 ∙ x ∙ x + 4.24 ∙ x ∙ x2 − 7.88 ∙ x ∙ x − 0.47 ∙ x2 ∙ x + 1.95 ∙ x2 ∙ x − 0.63 ∙ x ∙ x2 ∙ x + 0.83 ∙ x ∙ x2 + 4.02 ∙ x ∙ x2 ∙ x + 8.35 ∙ x ∙ x + 3.43 ∙ x ∙ x2 ∙ x − 2.73 ∙ x ∙ x2 ∙ x − 0.40 ∙ x2 ∙ x (2) the validity of the y2 mathematical model proposed was plotted by experimental values vs model values (figure 3). fig.3. validity check of the y2 (manganese concentration) mathematical model the mathematical model of the y3 response function is shown in equation 3. = 394.07 − 88.33 ∙ x − 1.67 ∙ x2 + 116.67 ∙ x − 56.11 ∙ x + 123.89 ∙ x2 − 1.11 ∙ x + 437.50 ∙ x ∙ x2 − 887.50 ∙ x ∙ x + 0.002 ∙ x2 ∙ x + 372.50 ∙ x ∙ x2 + 1012.50 ∙ x ∙ x + 637.50 ∙ x ∙ x2 − 762.50 ∙ x ∙ x − 115 ∙ x2 ∙ x + 55 ∙ x2 ∙ x + 430 ∙ x ∙ x2 ∙ x + 194.17 ∙ x ∙ x2 + 305 ∙ x ∙ x2 ∙ x + 1019.17 ∙ x ∙ x + 772.5 ∙ x ∙ x2 ∙ x + 52.50 ∙ x ∙ x2 ∙ x − 253.3 ∙ x2 ∙ x (3) the validity of the y3 mathematical model proposed was plotted by experimental values vs model values (figure 4). r² = 0.9967 0.00 50.00 100.00 150.00 200.00 250.00 0.00 50.00 100.00 150.00 200.00 250.00m od el v al ue , c r [p pb ] experimental value, cr [ppb] r² = 0.996 0.00 10.00 20.00 30.00 40.00 50.00 0.00 10.00 20.00 30.00 40.00 50.00 m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 324 0 237 fig. 4. validity check of the y3 (iron concentration) mathematical model the mathematical model of the y4 response function is shown in equation 4. = 20.44 ∙ x − 8.39 ∙ x2 + 2.00 ∙ x − 7.56 ∙ x − 0.86 ∙ x2 + 0.64 ∙ x + 15.25 ∙ x ∙ x2 − 24.75 ∙ x ∙ x − 2.25 ∙ x2 ∙ x + 26.83 ∙ x ∙ x2 + 37.50 ∙ x ∙ x + 8.60 ∙ x ∙ x2 − 37.40 ∙ x ∙ x + 9.00 ∙ x2 ∙ x + 10.83 ∙ x2 ∙ x − 13.63 ∙ x ∙ x2 ∙ x + 14.23 ∙ x ∙ x2 + 28.88 ∙ x ∙ x2 ∙ x + 34.23 ∙ x ∙ x + 2.63 ∙ x ∙ x2 ∙ x − 23.88 ∙ x ∙ x2 ∙ x + 4.03 ∙ x2 ∙ x (4) the validity of the y4 mathematical model proposed was plotted by experimental values vs model values (figure 5). fig. 5. validity check of the y4 (nickel concentration) mathematical model 3.2. mathematical modelling of metallic constituent concentrations migrated into 6% ch3cooh solutions statistical summary of mathematical models to describe the found dependent variables cr, mn, 56fe and ni is shown in table 4. table 4 summary statistic of mathematical models proposed for the dependent variables dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.0400 cubic 2.67 0.9618 0.9006 y2 0.0480 quintic 0.19 0.9999 0.9978 y3 0.0015 quintic 17.25 0.9979 0.9866 y4 0.0018 quintic 27.76 0.9890 0.9553 the mathematical models that define the studied response functions (dependent variables) depending on testing parameters (independent variables) are presented in the following equations. = 19.67 + 2.50 ∙ x + 7,72 ∙ x + 4,28 ∙ x − 4.33 ∙ x + 2.00 ∙ x + 0.17 ∙ x + 0.083 ∙ x ∙ x 1.50 ∙ x ∙ x + 3.00 ∙ x ∙ x − 3.58 ∙ x ∙ x − 0.67 ∙ x ∙ x + 3.25 ∙ x ∙ x − 1.00 ∙ x ∙ x + 2.83 ∙ x ∙ x − 0.33 ∙ x ∙ x + 2.13 ∙ x ∙ x ∙ x (5) the validity of the y1 mathematical model proposed was plotted by experimental values vs model values (figure 6). fig. 6. validity check of the y1 (chromium concentration) mathematical model the mathematical model of the y2 (mn concentration) response function is shown in equation 6. r² = 0.9964 0.00 50.00 100.00 150.00 200.00 250.00 0.00 50.00 100.00 150.00 200.00 250.00 m od el v al ue , n i [ pp b] experimental value, ni [ppb] r² = 0.9965 0.00 10.00 20.00 30.00 40.00 0.00 10.00 20.00 30.00 40.00m od el v al ue , c r [p pb ] experimental value, cr [ppb] r² = 0.996 0.00 10.00 20.00 30.00 40.00 50.00 0.00 10.00 20.00 30.00 40.00 50.00 m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 324 0 238 = 3.87 − 1.60 ∙ x + 4.70 ∙ x + 0.75 ∙ x + 0.54 ∙ x + 3.04 ∙ x − 1.11 ∙ x + 0.30 ∙ x ∙ x + 3.05 ∙ x ∙ x + 1.92 ∙ x ∙ x − 4. 25 ∙ x ∙ x − 3.10 ∙ x ∙ x + 1.75 ∙ x ∙ x − 1.55 ∙ x ∙ x + 1. 72 ∙ x ∙ x − 1.73 ∙ x ∙ x + 1.83 ∙ x ∙ x ∙ x − 4.77 ∙ x ∙ x − 0.32 ∙ x ∙ x ∙ x + 2.83 ∙ x ∙ x − 1.42 ∙ x ∙ x ∙ x + 1.15 ∙ x ∙ x ∙ x − 0.49 ∙ x ∙ x + 2.68 ∙ x ∙ x ∙ x + 3.00 ∙ x ∙ x ∙ x + 2.85 ∙ x ∙ x ∙ x (6) the validity of the y2 mathematical model proposed was plotted by experimental values vs model values (figure 7). fig. 7. validity check of the y2 (manganese concentration) mathematical model the mathematical model of the y3 (56fe concentration) response function is shown in equation 7. = 167.41 − 16.11 ∙ x + 143.33 ∙ x + 80.00 ∙ x + 123.89 ∙ x + 128.89 ∙ x + 18.89 ∙ x − 70 ∙ x ∙ x − 72.50 ∙ x ∙ x − 12.50 ∙ x ∙ x − 90 ∙ x ∙ x + 57.50 ∙ x ∙ x + 31.67 ∙ x ∙ x − 0.83 ∙ x ∙ x − 2.50 ∙ x ∙ x − 22.50 ∙ x ∙ x + 0.002 ∙ x ∙ x ∙ x − 88.33 ∙ x ∙ x + 10 ∙ x ∙ x ∙ x − 5.83 ∙ x ∙ x + 152.50 ∙ x ∙ x ∙ x − 5.00 ∙ x ∙ x ∙ x − 5.83 ∙ x ∙ x (7) the validity of the y3 mathematical model proposed was plotted by experimental values vs model values (figure 8). fig. 8. validity check of the y3 (iron concentration) mathematical model the mathematical model of the y4 (ni concentration) response function is shown in equation 8. = 311.44 − 24.67 ∙ x − 181.50 ∙ x + 41.17 ∙ x + 7.50 ∙ x + 24.33 ∙ x − 8.83 ∙ x − 69.25 ∙ x ∙ x + 6.33 ∙ x ∙ x − 15.67 ∙ x ∙ x + 97.25 ∙ x ∙ x − 1.50 ∙ x ∙ x + 35.75 ∙ x ∙ x − 8.00 ∙ x ∙ x + 7.50 ∙ x ∙ x + 12.50 ∙ x ∙ x − 7.38 ∙ x ∙ x ∙ x (8) the validity of the y4 mathematical model proposed was plotted by experimental values vs model values (figure 9). fig. 9. validity check of the y4 (nickel concentration) mathematical model 3.3. mathematical modelling of metallic constituent concentrations migrated into 9% ch3cooh solutions statistical summary of mathematical models to describe the found dependent variable cr, mn, 56fe and ni is shown in table 5. r² = 0.9981 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0.00 5.00 10.00m od el v al ue , m n [p pb ] experimental value, mn [ppb] r² = 0.9958 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0 500 1000 1500m od el v al ue ,5 6 f e [p pb ] experimental value, 56fe [ppb] r² = 0.9623 0.00 5.00 10.00 15.00 20.00 2.00 7.00 12.00 17.00 m od el v al ue , n i [ pp b] experimental value, ni [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 324 0 239 table 5 summary statistic of mathematical models proposed for the dependent variables dependent variable p model standard deviation [σ] r-squared [r2] adjusted r-squared [r2 adjusted] y1 0.010 quintic 1.06 0.9965 0.9771 y2 0.020 quintic 0.21 0.9981 0.9876 y3 0.035 quintic 41.82 0.9960 0.9730 y4 0.040 cubic 0.89 0.9823 0.9220 the mathematical models that define the studied response functions are presented in the following equations. = 8.67 + 0.33 ∙ x + 2.17 ∙ x + 0.17 ∙ x − 6.00 ∙ x − 2.50 ∙ x − 0.50 ∙ x + 2.00 ∙ x ∙ x + 0.25 ∙ x ∙ x + 1.50 ∙ x ∙ x + 1.00 ∙ x ∙ x + 1.25 ∙ x ∙ x + 4.00 ∙ x ∙ x + 0.75 ∙ x ∙ x + 3.00 ∙ x ∙ x + 6.00 ∙ x ∙ x + 3.00 ∙ x ∙ x ∙ x + 6.00 ∙ x ∙ x + 1.25 ∙ x ∙ x ∙ x + 0.75 ∙ x ∙ x + 3.50 ∙ x ∙ x ∙ x + 2.00 ∙ x ∙ x ∙ x + 1.50 ∙ x ∙ x (9) the validity of the y1 mathematical model proposed was plotted by experimental values vs model values (figure 10). fig. 10. validity check of the y1 (chromium concentration) mathematical model the mathematical model of the y2 (mn concentration) response function is shown in equation 10. = 1.46 − 0.00005 ∙ x + 1.21 ∙ x + 0.13 ∙ x − 0.22 ∙ x + 0.88 ∙ x − 0.12 ∙ x + 1.15 ∙ x ∙ x + 0.025 ∙ x ∙ x + 0.63 ∙ x ∙ x − 0.33 ∙ x ∙ x + 0.26 ∙ x ∙ x + 0.93 ∙ x ∙ x + 0.11 ∙ x ∙ x + 0.91 ∙ x ∙ x + 0.042 ∙ x ∙ x + 0.93 ∙ x ∙ x ∙ x + 0.25 ∙ x ∙ x + 0.20 ∙ x ∙ x ∙ x + 0.17 ∙ x ∙ x + 0.90 ∙ x ∙ x ∙ x + 0.10 ∙ x ∙ x ∙ x + 0.025 ∙ x ∙ x (10) the validity of the y2 mathematical model proposed was plotted by experimental values vs model values (figure 11). fig. 11. validity check of the y2 (manganese concentration) mathematical model the mathematical model of the y3 (56fe concentration) response function is shown in equation 11. = 147.78 − 18.89 ∙ x + 190.00 ∙ x + 57.78 ∙ x − 66.67 ∙ x + 83.33 ∙ x − 16.67 ∙ x + 157.50 ∙ x ∙ x − 47.50 ∙ x ∙ x + 100.00 ∙ x ∙ x − 42.50 ∙ x ∙ x + 40.83 ∙ x ∙ x + 180.83 ∙ x ∙ x − 9.17 ∙ x ∙ x + 53.33 ∙ x ∙ x − 15.00 ∙ x ∙ x + 105.00 ∙ x ∙ x ∙ x + 12.50 ∙ x ∙ x − 7.50 ∙ x ∙ x ∙ x + 77.50 ∙ x ∙ x + 162.50 ∙ x ∙ x ∙ x + 7.50 ∙ x ∙ x ∙ x − 20.00 ∙ x ∙ x (11) the validity of the y3 mathematical model proposed was plotted by experimental values vs model values (figure 12). r² = 0.9965 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 0.00 10.00 20.00 30.00 40.00 m od el v al ue , c r [p pb ] experimental value, cr [ppb] r² = 0.9981 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 0.00 2.00 4.00 6.00 8.00 10.00 m od el v al ue , m n [p pb ] experimental value, mn [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceavavolume xiv, issue 2 – 2015 s i l v i ug ab r ie l s t r oe , mathematical modelling of the constituents’ concentrations of aisi 304 stainless steel samples that diffuse into simulated acidic environments, fo od a nd e nvir o nme nt sa f e t y, v o l u me x iv , is s ue 2 – 20 1 5, p a g 2 3 324 0 240 fig. 12. validity check of the y3 (iron concentration) mathematical model the mathematical model of the y4 (ni concentration) response function is shown in equation 12. = 8.44 + 1.09 ∙ x + 1.28 ∙ x + 1.74 ∙ x − 1.49 ∙ x − 0.23 ∙ x + 0.37 ∙ x + 1.07 ∙ x ∙ x + 0.59 ∙ x ∙ x + 0.76 ∙ x ∙ x − 0.47 ∙ x ∙ x − 0.21 ∙ x ∙ x + 0.0083 ∙ x ∙ x + 0.91 ∙ x ∙ x + 0.24 ∙ x ∙ x + 0.72 ∙ x ∙ x + 1.18 ∙ x ∙ x ∙ x (12) the validity of the y4 mathematical model proposed was been plotted by experimental values vs model values (figure 13). fig. 13. validity check of the y4 (nickel concentration) mathematical model 4. conclusions icp-ms method and anova method were used to establish the relationships between migration test parameter values and cr, mn, 56fe and ni concentrations found in solutions. besides the fact that these instrumental analysis techniques provide the checking of mathematical models’ validity for each dependent variable, we can also draw the conclusion that they are highly performing and accurate instruments that can be used in estimating the values of objective function (see also [4]). 5. references [1]. barnes, k. a., sinclair, c. r., watson, d.h., chemical migration and food contact materials, woodhead publishing limited, isbn-13: 978-1-84569-029-8, england, (2007); [2]. sanches silva a., cruz j. m., sendor garcia r., franz r., paseiro losada, p., kinetic migration studies from packaging films into meat products, sciencedirect, meat science 77, 238-245, (2007); [3]. soojin j., irudayaraj j.m., food processing operations modeling design and analysis, second edition, crc presstaylor&francis group, 1-2, (2009); [4]. stroe s. g., gutt g., statistical study of the dependence between concentration of metallic elements migrated from stainless steel grade aisi321 and working parameters, food and environment safety, faculty of food engineering, volume 12, issue 2, (2013); [5]. tehrany e. a., desobry s., partition coefficient of migrants in food stimulants/polymers systems, sciencedirect, food chemistry 101, 17141718, (2007); [6]. amani s. alturiqi, lamia a. albedair, the egyptian journal of aquatic research, sciencedirect, volume 38, issue 1, 45– 49, (2012); [7]. kamal j. elnabris, shareef k. muzyed, nizam m. el-ashgar, heavy metal concentrations in some commercially important fishes and their contribution to heavy metals exposure in palestinian people of gaza strip (palestine), journal of the assoc. of arab universities for basic and applied sciences, volume 13, issue 1, april 2013, pages 44–51, (2013); [8]. datta a. k., biological and bioenvironmentat heat and mass transfer, cornell university ithaca, new york, marcel dekker inc., isbn: 0-8247-0775-3, 11-12, (2002); [9]. d.m. 21-03-1973, italian law text, decreto ministeriale del 21/03/1973 disciplina igienica degli imballaggi, recipienti, utensili, destinati a venire in contatto con le sostanze alimentari o con sostanze d'uso personale, (1973); r² = 0.9958 0.00 500.00 1000.00 1500.00 0 500 1000 1500m od el v al ue ,5 6 f e [p pb ] experimental value, 56fe [ppb] r² = 0.9623 0.00 5.00 10.00 15.00 20.00 2.00 7.00 12.00 17.00m od el v al ue , n i[ pp b] experimental value, ni [ppb] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 59 the variation of annual rainfall in the suceava plateau *ioan gontariu1 , ion tănasă2 1faculty of food engineering, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania, e-mail ioang@fia.usv.ro *corresponding author 2ph.d. candidate, stefan cel mare university, street. universitatii no. 13, 720229, suceava, romania, e-mail ion_tanasa.meteo@yahoo.com received 22 february 2011, accepted 6 may 2011 abstract: the atmospherical rainfall (resulted from the condesation and crystallization of water vapours which fall from the clouds in liquid, solid or mixed form) represents one of the most important climatic elements, with noticeable influence on the natural environment of a certain region. rainfall has an important impact on the environment of terrestrial atmosphere (which is an important link in the water’s nature circuit) and contributes to keeping the soil humid. after all, life wouldn’t have been possible without water, a key element in the formation of terrestrial atmosphere, a real protection against ultraviolet radiations and against strong termic amplitudes. besides influencing all the other climatic elements, water plays an important part in all earth’s covers by continously modifying the relief (so it is an external factor of modelation), the configuration of hydrographical network and being involved in the formation and development of vegetation, fauna and soils (the biosphere). like all the other elements, atmospherical rainfall could be seen as either a resource (link in the water’s circuit in nature) or a hazard (when its extremes are beyond normality and bring serious damage to society). besides pluviometric differences, there is also an alternation of humid sectors which are positioned over the higher areas of the relief with drier sectors, sequence well distinguished on the direction north-west – south-east. meanwhile, the slopes experiencing the change in masses of air from west and north-west, being more humid, perceive bigger quantities of precipitations while the southern ones are drier, this also because of the foehn phenomenon. © 2011 university publishing house of suceava. all rights reserved key words: climatical hazards, termic amplitudes, water vapours, terrestrial atmosphere 1. introduction the plateau of suceava is positioned in the central and northern part of moldova, between the eastern carpathians and moldova’s plain, being the highest part of the moldova’s plateau and partially overlaping hystorical bucovina [1]. the dynamic factors of the climate are the ones which record substantial changes from year to year, contributing mostly in the unperiodical variation of rainfall. the various aspects of weather, continously changing, are determined by the position of the baric centers in relation to the studied habitat, the existing dorsals of the two anticyclones, azoric and euroasian; cause in the north of our country the change in cold air in north and north-est [2]. the mountainous chain of the eastern carpathians is a real obstacle against the movement of air masses to suceava’s plateau but also against the eastern and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 60 north-eastern ones, increasing their existence above this habitat [3]. the plateau is characterized by relatively abundent precipitations (between 500mm and 700 mm) and relatively moderated temperatures (between 7.5°c and 9.5°c). the most frequent pluviometrical or baric climatic hazards of termic nature are: early frosts in autumn or late frosts in spring (harmful to agricultural crop and plants), blizzards (from 9 to 14 days each year), white frost (from 10 to 12 days a year), glazed frost (9 to 12 days a year) and fog (30-50 days annually), each representing a specific phenomenon for the cold seasons of the year [4]. during the warm season, there are storms and showers, sometimes rich in precipitations, other times accompanied by hail (once or twice a year). the extraordinary variability of this climatic aspect from the habitat of suceava’s plateau points out remarkable pluviometrical differences during different temporal entities (annually, biannually, monthly and daily speaking), strongly related to the general or local circulation of the atmosphere (the physical, geographical factors, regional and local factors) and of the solar radiation [5]. 2. experimental the database used in this material looks into the annual sums of precipitation from 1992-2010 (suceava and roman) and 1961-2010 (radauti, falticeni and cotnari). meaningful reasonings were based on documentation of specialty (see bibliography) and the main used processes were observation, statistical and mathematical analysis, comparation and also several graphical methods [6]. 3. results and discussion the annual average sum of atmospheric precipitations from suceava’s plateau is approximately 580 mm (the average of annual sums from 1961 to 2009, information provided by the 5 main meteorological centers). as we have analyzed the pluviometric repartition from the area to be studied (figure 1), we can observe meaningful differences, of over 100mm between the southern habitat and the northern one; in roman an annual average quantity of 523,9mm while in radauti 634,8mm (figure 2a) is registered. taking into account the pluviometric stations, these differences increase even more, from 500mm in south (498.6 mm in upper muncel) to over 700mm in the northwestern part (737.5mm in solca). the atmospheric precipitations gradually decrease from north-west to south–east, once the relief looses altitude (figure 2b) and once the masses of humid air, of oceanic and atlantic origin, are less frequent (including a slight foehnization). also, the relief is largely open to east which facilitates the entrance of continental drier and warmer masses of air (of lower humidity). figure 1. the territorial repartition of the annual quantities of precipitations in suceava’s plateau food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 61 634.8 620.1 608.0 587.3 531.2 544.3 523.9 389 352 348 289 248 230 216 0 100 200 300 400 500 600 700 rădăuţi suceava fălticeni dolhas ca cotnari paş cani roman mm 0 100 200 300 400 500 600 mpma altitudinea a. b. figure 2. the variation of the annual average quantities of atmospheric precipitations, reported to the altitude from the meteorological centers (a) and pluviometric stations (b) from suceava (19612010) besides pluviometric differences, there is also an alternation of humid sectors which are positioned over the higher areas of the relief with drier sectors, sequence well distinguished on the direction north-west – south-east. meanwhile, the slopes experiencing the change in masses of air from west and north-west, being more humid, perceive bigger quantities of precipitations while the southern ones are drier, this also because of the foehn phenomenon. analyzing the annual quantities of precipitations from 1961 to 2010, recorded at the meteorological centers from suceava’s plateau, we can observe a special variation in time of this climatic element, sometimes with opposite aspects. the data covering a longer period of time (before 1961), indicate an even higher variation than in the period studied, due to some years in which extremely different quantities were recorded (higher or lower, like in suceava where the annual extremes were 330mm in 1946 and 1021.3mm in 1933, or 346.7mm in 1986 and 883.2mm in 2008). the annual sums of precipitations from suceava’s plateau have had bigger or smaller variations, situated between the extreme values reported by the 5 meteorological centers (table 1): radauti – 352.8mm in 1986 (56% from normality); suceava – 330.0mm in 1946 (57%) and 1021,3mm in 1933 (173%); falticeni – 369.2mm in 1986 (61%) and 819,1mm in 1991 (137%); cotnari – 313.5mm in 1986 (60 %) and 825,1mm in 1991 (158%); roman – 299.0mm in 1973 (58%) and 945.6mm in 1991 (183%). table 1. the biggest and smallest annual sums of precipitations, exceptions and differences between them in the suceava’s plateau (1961-2010). precipitations radauti suceava falticeni cotnari roman medium 634.8 620.1 608.0 531.2 523.9 minimum 352.8 346.7 365.3 313.5 299.0 year 1986 1986 1986 1986 1973 maximum 914.5 883.2 819.1 825.1 945.6 year (ii) 2005, 2010 2008 1991 1991 1991 deviation-282.0 -273.4 -242.7 -217.7 -224.9 % 56 56 60 59 57 deviation + 279.7 263.1 211.1 293.9 421.7 % 144 142 135 155 180 amplitude 561.7 -536.5 -453.8 -511.6 -646.6 the graphical representations of the evolution in time of the quantity of precipitations show us some very anfractuous lines, oscillating from one side of the mean to the other. apparently, they do not obey any rule, registering general tendencies of soft increase in the analyzed time (figure 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 62 y = 0,6326x + 613,3 r2 = 0,0056 y = 1,3905x + 580,86 r2 = 0,0255 y = 1,2619x + 572,07 r2 = 0,0254 y = 1,5111x + 491,96 r2 = 0,0359 y = 1,523x + 481,35 r2 = 0,0353 200 300 400 500 600 700 800 900 1000 1961 1965 1969 1973 1977 1981 1985 1989 1993 1997 2001 2005 2009 mm rădăuţi s uceava fălticeni cotnari roman trend rădăuţi trend s uceava trend fălticeni trend cotnari trend roman figure 3.the evolution, variation and tendencies of annual atmospheric precipitations in suceava. in comparison with other climatic elements (temperature, pressure, humidity, nebulosity), the variation in the annul sums of precipitation is much more highlighted. in the same time, the parallelism of the evolution of these sums is not that much noticeable, sometimes the sums intersect and other times they slowly move away one from another. the general tendency of annual precipitations from 1961 to 2010 was to easily increase, the coefficients of the regression equation (y) being positive (between 0.8741 in radauti and 1.9067 in roman). a more detailed analysis could determine smaller intervals of time with tendencies of a more accentuated increase between 1961-1972 and 1998-2008, but also of stagnation, between 1972-1979 and 1981-1998. on each station these periods are much more accurately registered. the spreading of the annual values of precipitations is at minimum in roman (0.053) and at maximum in falticeni (0.0369), from 1961 to 2009, becoming even larger for a longer period of time we analyzed (between 0.067 in roman and 0.0748 in suceava). the calculus and the evolution of the precipitations’ deviation from normality (the multiannual average) from all the meteorological centers in the period 19612010 or beyond it (figure 4) in suceava and roman, point out some interesting aspects. in radauti, some atmospherical precipitations have had the biggest variation from all suceava’s plateau; the minimum was of -282.0mm(1986) and the maximum 279.7 mm (2005, 2010); for a larger period of observation (1955-2008), minimum negative exception becomes 286.9mm (1986) and the positive maximum exception changed to 577.9mm (in 1955 (when there was a maximum of 1217.6 mm per year). in suceava, the negative minimum was of –263.1mm (1986) and the positive maximum was of 263.1mm (in 2008, year known for the biggest floods ever experienced in this area); between 1922 and 2008, the variation increased, being comprised in the total precipitations of 330mm (1946 the drought of the century), from a negative of -249.1mm to 1021mm in 1933 and a positive maximum change of 442.1mm. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 63 mm y = 1.6063x 96.929 r 2 = 0.0921 -300 -150 0 150 300 450 19 22 19 26 19 30 19 34 19 38 19 42 19 46 19 50 19 54 19 58 19 62 19 66 19 70 19 74 19 78 19 82 19 86 19 90 19 94 19 98 20 02 20 06 20 10 abate ri plu vi om e tri ce an u a le la s u ce ava tre n d mo bil (5 an i ) tre n d lin ia r a. mm y = 0.53 06 x 26 .4 72 r2 = 0.014 -250 -100 50 200 350 19 22 19 26 19 30 19 34 19 38 19 42 19 46 19 50 19 54 19 58 19 62 19 66 19 70 19 74 19 78 19 82 19 86 19 90 19 94 19 98 20 02 20 06 20 10 abate ri plu vio me trice a n u ale la rom an lin ia ră (abate ri plu vi om e tri ce an u al e la rom an ) tre n d m obi (5 an i) b. figure 4.the long run evolution of annual sums of precipitations from 1922 to 2009 in suceava (a) and roman (b) in roman, the variations of precipitations were registered between 299.0mm (1973) and 945.6mm (1991), with departures between -219.5 and 427mm (calculated for the period 19612009) or between 212.8 and 433.8mm (1986-2009). analyzing the frequency of the annual precipitations’ sums (figure 5), we can see that the annual sums of precipitations between 500mm and 600mm are the most numerous as percentage in suceava’s plateau, of 29%, with an assurance higher than 73%. on stations, the situations do not always obey this rule. in suceava and falticeni, years with precipitations between 600mm and 700mm are the most frequent (38%), while annual sums between 500mm and 600mm are in proportions of only 23-25%. so, the most frequent annual quantities of precipitations are between 500mm-700mm in radauti (61%), suceava (61%), in falticeni (63%) and 400-600mm in cotnari (56%) and roman (62%). lower frequencies were registered by the sums of precipitations between 700800mm (4-17%) and the minimum, the very large, ranging from 800-900mm and over 900mm (0-2%). between 1961 and 2009, there was just one case of precipitations which registered less than 300mm (roman) and there were not more precipitations than 1000mm. in radauti (where the maximum annual quantity of precipitations occured), in 40% of the years were registered precipitations of 500mm-600mm and in the others below 400mm and above 900mm (2%). 1 4 11 28 30 21 7 1 5 16 44 74 95 102 y = -58,861ln(x) + 119,83 r2 = 0,8945 0 20 40 60 80 100 120 <400mm 400-500 500-600 600-700 700-800 800-900 >900mm % % po d. s uce ve i asigu rare tl og_asi gura re figure 5. probability and assurance rate on intervals of 100mm for annual sums of precipitations in suceava’s plateau. in most of the cases, years with precipitations in surplus, close to normal in deficit, are grouped in shorter or longer periods. certain years have had annual quantities much more below average (1887, 1896, 1964 and 1973); the smallest sums of precipitations occurred due to atmospheric anticyclonic circulation, with changes resulting in warm masses of air [7]. in other situations, annual sums were beyond the multiannual average, thanks to food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 64 the predominant cyclonic activity (1933, 1943, 1974, 1981, 1985, 1988 and 1991). these large quantities of precipitations were generated by rain and abundant showers during the warm season and transition periods (they characterize a relatively humid region, rich in precipitations, which combined with the thermal regime and evaporation (both low), assure a normal development of spontaneous vegetation and agricultural crops, because of the periods of drought and dryness [8]. also, this phenomenon occurs in autumn months, when the vegetative cycle of plants is over or almost finalized and is more frequent in the south-eastern part of the studied area. from the pluviometric point of view, in the plateau of suceava rainy periods are more frequent than the droughty ones [9]. 4. conclusion ◊ the small quantities of precipitations in the habitat of suceava city have had a great variation in time, the pluviometric excess and deficit fallowing one another in many different intervals of time. ◊ taking into account the frequency of the annual precipitations’ sums we can see that the annual sums of precipitations between 500 mm and 600 mm are the most numerous as percentage in suceava’s plateau, of 29%, with an assurance higher than 73%. ◊ this variation is increasing, as naturally, with the expanding of the periods we have analyzed and it fits numerically the extreme quantities of precipitations: annually (330mm-1946 and 1021mm-1933), seasonally (69mm-19351936, 311mm-1987-1988 in the cold season, 173mm–1976 and 694mm-1974 in the warm season), monthly (0mm in november 1926, february or december 1931 and 253mm july 2003), daily (between days without precipitations and the maximum of 85.5mm registered july 8th, 1967). 5. references 1. popp n., iosep i., paulencu d. 1973 judeţul suceava, editura academiei r.s. românia, bucureşti. 2. călinescu gh., călinescu niculina, soare elena, 1994 caracteristici şi tendinţe ale precipitaţiilor maxime căzute în diferite intervale de timp în moldova, lucr. sem. geogr. „dimitrie cantemir”, iaşi. 3. apăvăloae m., apostol l., pîrvulescu i., 1984 topoclimatele oraşului suceava, buletin ştiinţific, i.i.s. suceava. 4. apostol l., 2000 precipitaţiile atmosferice din subcarpaţii moldovei, editura universităţii “ştefan cel mare”, suceava. 5. filipov f., lupaşcu angela, răducu daniela, beldiman brânduşa elena, 2001 influenţa măsurilor ameliorative asupra însuşirilor morfologice fizice şi chimice ale solurilor cu exces de umiditate din podişul sucevei, iaşi. 6. ţîştea d., neacşa o., sabău a., sîrbu v., călinescu n., 1974 studiul parametrilor climatici locali pentru stabilirea condiţiilor de restaurare şi conservare a picturilor murale ale unor monumente istorice din bucovina, studii de climatologie, vol. 1, i.m.h., bucureşti. 7. slavic gh., 1977 podişul sucevei studiu climatologic, rezumatul tezei de doctorat, iaşi. 8. erhan elena, 1986 fenomenul de grindină în podişul moldovei, analele ştiinţifice ale universităţii „al. i. cuza”, din iaşi, secţ. ii b geol.geogr., tom xxxii. 9. topor n., 1963 ani ploioşi şi secetoşi în r.p.r., i.m., bucureşti. *** (1962, 1966) „clima r.p.r.”, institutul meteorologic, vol. i şi ii, bucureşti. *** (1983) „geografia româniei i. geografia fizică”, vol. i, bucureşti. *** (1989) „geografia româniei iv. geografia fizică”, vol. iv, bucureşti. *** (1984) „un secol de la înfiinţarea serviciului meteorologic al româniei”, i.n.m. bucureşti. *** (1972 -2001) arhiva de date climatologice a staţiei meteorologice suceava, centrul meteorologic regional moldova food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 13 viscoelastical behaviour of some food materials from the spanish market *mircea-adrian oroian1, isabel escriche2, gheorghe gutt3 1,3ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1m.oroian@usv.ro, 3g.gutt@usv.ro 2universitat politecnica de valencia, food technology department, camino de vera s/n, valencia, spain, e mail iescriche@tal.upv.es *corresponding author received 19 april 2011, accepted 5 august 2011 abstract: the yogurt presents a weak viscoelastical component due to the presence of the casein in its structure. these casein gels can be clearly distinguished from polymer gels that are formed by long macromolecules with a considerable conformational flexibility between crosslinks. on the macroscopic scale, particle gels are more brittle than polymer gels, and they tend to have a shorter linear elastic region and smaller fracture strain. the measurements of the viscoelastic parameters of weak gels must be done at very small deformations and the stress and the strain involved in the measurements may frequently be below the limits of accuracy of conventional rheological instruments. the purpose of this work is to observe the viscoelastical behavior of some yogurts from the spanish market. for this study where picked two types of yogurt, simple yogurt and yogurt with fruit, respectively; four samples for each type, each one with different concentration of soluble content, fat content, protein and sugar content. the pca realised obtained an explanation of the 2 components of 100%, and it was observed that yogurts are divided in two different groups, influenced by the physical chemical composition. the analysis of variance (anova) conducted revealed that considering that a higher value of f-ratio means a more noticeable effect of the factor (type of yogurt) in a variable, g’ was the parameters more distinguished by the type of yogurt. keywords: yogurt, viscosity, consistency, luminosity, water activity 1. introduction viscoelasticity is the property of materials that exhibit both viscous and elastic characteristics when undergoing deformation. viscous materials, like honey, resist shear flow and strain linearly with time when a stress is applied. elastic materials strain instantaneously when stretched and just as quickly return to their original state once the stress is removed. viscoelastic materials have elements of both of these properties and, as such, exhibit time dependent strain. whereas elasticity is usually the result of bond stretching along crystallographic planes in an ordered solid, viscosity is the result of the diffusion of atoms or molecules inside an amorphous material [1] when a stress is applied to a viscoelastic material such as a polymer, parts of the long polymer chain change position. this movement or rearrangement is called creep. polymers remain a solid material even when these parts of their chains are rearranging in order to accompany the stress, and as this occurs, it creates a back stress in the material. when the back stress is the same magnitude as the applied stress, the material no longer creeps. when the original stress is taken away, the accumulated back stresses will cause the mailto:m.oroian@usv.ro mailto:g.gutt@usv.ro mailto:iescriche@tal.upv.es http://en.wikipedia.org/wiki/viscosity http://en.wikipedia.org/wiki/elasticity_%28physics%29 http://en.wikipedia.org/wiki/deformation_%28engineering%29 http://en.wikipedia.org/wiki/shear_flow http://en.wikipedia.org/wiki/strain_%28materials_science%29 http://en.wikipedia.org/wiki/stress_%28physics%29 http://en.wikipedia.org/wiki/amorphous http://en.wikipedia.org/wiki/polymer http://en.wikipedia.org/wiki/creep_%28deformation%29 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 14 polymer to return to its original form. the material creeps, which gives the prefix visco-, and the material fully recovers, which gives the suffix –elasticity [2]. protein particle gels, which consist of a continuous three-dimensional network of flocculated particles, have attracted constantly increasing attention in the last decade. this interest reflects in part, the technological importance of these systems including a variety of soft food colloids (yoghurt, cheese, etc.) [3, 4]. protein particle gels are normally formed by the generation of crosslinks or other attractive interactions between particles in a suspension induced by a number of different processes (enzyme action, ph lowering, heating, etc.) and followed by the transformation of a liquid suspension to a gel. these gels can be clearly distinguished from polymer gels that are formed by long macromolecules with a considerable conformational flexibility between crosslinks. on the macroscopic scale, particle gels are more brittle than polymer gels, and they tend to have a shorter linear elastic region and smaller fracture strain. the weakness of the gel network is particularly obvious around the point of sol–gel transition, thus creating complications for the application of macroscopic mechanical methods. therefore, the measurements of the viscoelastic parameters of weak gels must be done at very small deformations and the stress and the strain involved in the measurements may frequently be below the limits of accuracy of conventional rheological instruments [4, 5]. the purpose of this paper is to make a statistical analysis of the yogurt samples from the spanish market keeping in count the physical chemical and viscoelastical parameters using anova and pca analysis, and to note which the parameters which distinguish are. 2. materials and methods 2.1. materials for this study were picked 8 samples of yogurt from the spanish market. the samples were divided in two main categories: natural yogurt (blank samples) and fruit yogurt, respectively. the samples had different physical chemical composition (different concentrations of sugar content, fat content and protein content, respectively). 2.2. chemical analysis yogurts were analyzed for protein by the kjeldahl method, fat by gerber method and total solids by oven drying. 2.3. water activity the water activity of the yogurt samples was performed using the fast lab 2 water activity meter. 2.4. ph measurement ph measurements were realized with a metler toledo ph.-meter, at 200c. 2.5. viscoelastic behaviour rheological measurements were carried out in a controlled stress rheometer rs1 (thermo haake, germany), using a parallel cone plate geometry (35 mm diameter; and a angle of 20), and a sample temperature of 50c. a batch of each composition was prepared and at least two measurements were performed on each batch, using a fresh sample for each measurement. after loading the sample, a waiting period of 5 min was used to allow the sample to recover itself and to reach the desired temperature. in order to determine the linear viscoelastic region, stress sweeps were run at 1hz first at different shear stress. then, the frequency sweeps were performed over the range f = 0.1 -10 hz, the shear stress was τ = 0.05 pa and the values of the storage modulus (g’), loss modulus (g’’), loss tangent angle (tan food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 15 δ) and complex viscosity (g*) were registered as a function of frequency using the rheowin job software. 2.7. statistical analysis an analysis of variance (anova) (α=0.05) with least significant difference (lsd) test using statgraphics plus 5.1 were performed on the data from physical chemical and viscoelastical parameters. in addition to this, the data were analyzed by using multivariate techniques, applying the software unscramble version 10.1 (camo process as, oslo, norway, 2005). the variables were weighted with the inverse of the standard deviation of all objects in order to compensate for the different scales of the variables. a principal components analysis (pca) was applied to describe the relation among the physical chemical and viscoelastical parameters. 3. results and discussion the samples submitted to this study presented a level of fats between 0 and 10%, respectively, proteins between 3.3 and 5.2% respectively and sugar 3.9017.10%. the ph value was in the acid region, while the water activity was ranged between 0.98-0.99. yogurt samples presents a viscoelastical behaviour, but due to the weakness of the gel strength (casein chain macromolecule) the shear stress needed to achieve the linear viscoelastical region is small (τ = 0.05 pa). the samples present a phase angle between 13.67 to 20.73 so the yogurt is a viscoelastical fluid but with the elastical component bigger than the viscous component. the complex viscosity is strongly influenced by the frequency applied (increasing the frequency the viscosity is decreasing due to the pseudo plastic fig. 1. rheogram of yogurt sample (blue line g’, red line g”, green line η* tab.1 physical chemical, and viscoelastical parameters anova of the yogurt samples yogurt type f ratio simple fruit physical chemical parameters proteins 3.95a 4.5a 4.58ns fats 4.1a 1.82a 2.53ns sugars 5.8b 12.05a 7.82* fruits 0b 7.35a 29.4*** ph 4.21a 4.26a 2.08ns aw 0.87a 0.98a 0.9ns viscoelastic parameters g’ 658.4a 261.64a 3.51ns g” 178.05a 79.95a 2.67ns g* 682.1a 175.3b 5.05* |η*| 108.56a 126.65a 0.11* δ 15.59a 15.71a 0.01ns note: ns not significant (p<0.05), * p>0.05, ** p>0.01, *** p>0.001 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 16 behaviour of the yogurt), while the elastic and viscous component are linear, they increase with the increasing of the frequency but not in the same magnitude with the decreasing of the complex viscosity (fig.1). a pca was conducted to evaluate the global effect of the type of yogurt (blank, fruit) on the physical chemical, textural, rheological and color, from a descriptive point of view. figure 2 shows the sample scores and compound loadings of the pca analysis performed. it was found, that two principal components (pcs) explained 100% of the variations in the data set. fig.2. pca scores of yogurt samples the pc1 explains 95% of variability, and the pc2 explains 5%. there are two main differentiated groups of samples on the plot, each group have quiet the same physical chemical composition (concentration of fats, fruits, proteins and sugar). the pc2 differentiate the two groups (fig 2). the loadings of each parameter on the principal components show that the grouping of the different type is primarily influenced by certain parameters. in the central ellipse could be seen the parameters which influence more the differential of the samples fig.3. pca loadings of the yogurt sample (fruit, fats, proteins, fase angle) while in the outer ellipse the parameters which a lower influence (sugar, complex viscosity, g’, g’’, g*). the viscoelastical parameter which influence more the differential of the two groups in the phase angel (fig3.).in order to know if the observed differences among the different yogurt type for the each parameter are statistically significant, an analysis of variance (anova) with one factor (type) was carried out (table 1). it is known that if p-value from the anova analysis is equal or superior to 0.05 there are not significant differences among type. taking this into account differences for the physical chemical and viscoelastical parameters. considering that a higher value of f-ratio means a more noticeable effect of the factor (type of yogurt) in a variable, g’ is the viscoelastical parameter more affected by the type of yogurt. 4. conclusion the yogurt is a weak viscoelastical fluid for which linear viscoelastical behaviour achieving is needed a small shear stress. the elastic component is bigger than the viscous component (confirmed by the phase angle). the pca divide the samples in 2 major groups, but they are divided keeping in count the physical composition and not by food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 17 the type (blank or fruit yogurt). the anova finds that the g’ is the parameter which distinguish better the two type of yogurts. 5. acknowledgment this paper was supported by the project "knowledge provocation and development through doctoral research pro-doct contract no. posdru/88/1.5/s/52946 ", project co-funded from european social fund through sectoral operational program human resources 2007-2013. 6. references [1] meyers m., chawlam k. mechanical behavior of materials, upper saddle river, nj: prentice hall (1999) [2] mccrum n.g., buckley c.p., bucknell c.b. principles of polymer engineering, oxford university press, new york (2003) [3] dickinson e., an introduction to food colloids, oxford university press, oxford, (1992). [4] dickinson e., chem. ind. 1, oxford university press, (1990) [5] smyth c., kudryashov e.d., buckin v., high-frequency shear and volume viscoelastic moduli of casein particle gel, colloids and surfaces a: physicochemical and engineering aspects 183– 185 517–526, (2001) *mircea-adrian oroian1, isabel escriche2, gheorghe gutt3 1,3ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1m.oroian@usv.ro, 3g.gutt@usv.ro 2universitat politecnica de valencia, food technology department, camino de vera s/n, valencia, spain, e mail iescriche@tal.upv.es 4. conclusion the yogurt is a weak viscoelastical fluid for which linear viscoelastical behaviour achieving is needed a small shear stress. the elastic component is bigger than the viscous component (confirmed by the phase angle). the pca divide the samples in 2 major groups, but they are divided keeping in count the physical composition and not by the type (blank or fruit yogurt). the anova finds that the g’ is the parameter which distinguish better the two type of yogurts. 5. acknowledgment 6. references звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 24 sulfur dioxside utilization by the treatment of pyrite chalcopyrite sulfide consentrates, combining mechanical and metallothermic processes *v. martirosyan1, t. aghamyan2, m. sasuntsyan3, a. ajvazyan4, a.zaprosyan5 1 v. martirosyan, e-mailv.a.martirosyan@mail.ru, 2t. aghamyan, e-mailajvazyanaa@rambler.ru 3 m. sasuntsyan, e-mail msasuntsyan@mail.ru , 4a. ajvazyan, e-mail timulik@mail.ru 5 a. zaprosyan, e-mail zaprossian@gmail.com *corresponding author received 22 july 2011, accepted 20september 2011 abstract: in the research a new non-conventional tec hnology for the treatment of local pyritechalcopyrite sulfide concentrates is suggested allowing to extract copper and iron from the concentrates by an energy-saving, efficient and ecologically friendly method. for this purpose the reactivity of pyrite-chalcopyrite sulfide concentrates was enhanced for further intensification of metallurgical processes and obtaining of the copper-iron ligatures by direct aluminothermic reduction of activated concentrates. with the object of increasing the chemical activity of pyrite-chalcopyrite concentrates, they were subjected to preliminary mechanochemical activation in the vibratory mill. fine grinding was carried out in air, water and applying other combined methods. in all cases mechanochemically activated copper and iron sulfides underwent deep chemical transformations forming metal sulfates, hydrosulfates, and partly oxides. thus, mechanical activation of sulfides led to the same effect that takes place during the low-temperature roasting in the furnace. then the mechanochemically activated (ma) concentrate was reduced by aluminothermy. the combination of mechanochemistry with the metallothermy allowed to perform a unique reduction process with more efficiency, and to obtain modified copper-iron ligatures bypassing the roasting processes common for the traditional metallurgy that lead to formation of volatile sulfur oxides. . the structure of the obtained ligature was also examined. metallographic analysis shows that it depends on the duration of mechanochemical activation. it is characterized by the microheterogenous nature, is more dispersed and presents a mechanical mixture of iron and copper (47% fe, 53% cu). keywords: mechanochemical, copper concentrate, iron concentrate, sulfate, grinding, planetary mill, aluminothermic reduction. 1. introduction in the area of industrial progress of the republic of armenia the issues of developing materials with required properties and advanced technologies for their production are of special interest. particularly, great attention has attracted to alloys with new structures, that exhibit high strength, durability and corrosion resistance in different aggressive media. among these alloys are iron bronzes, which are doped by copper-iron ligatures. the production of such ligatures by traditional methods is a complex and energy-consuming process that requires expensive equipments. taking into account the demand for iron bronzes and high content of copper in concentrates obtained in ra, it is advisable to organize such local production. for this production kapan pyrite-chalcopyrite sulfide mailto:v.a.martirosyan@mail.ru mailto:ajvazyanaa@rambler.ru mailto:msasuntsyan@mail.ru mailto:timulik@mail.ru mailto:zaprossian@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 25 concentrate with the copper content from 18 to 20% may be used as a raw material. these concentrates currently are exported, while organization of the local production for complex and ultimate processing of raw materials would provide the most economical benefits. on the other hand, copper production by the traditional technology is a complex process requiring expensive equipments and is associated with some difficulties. so far the problems related to the recycling of so2 and iron extraction from final tailings (with iron content up to 38%) remain insoluble. from this point of view it is vital to develop new alternative technologies for the pyrite-chalcopyrite sulfide concentrates, which will make possible to extract with minimal economic costs not only copper, but also iron up to the metallic state in the form of ligatures, bypassing the roasting process. thereby, it is of prime interest to develop a new approach for metallothermic reduction of sulfide concentrates combined with the preliminary mechanochemical activation, which promotes of obtaining fundamentally new modified products. based on the foregoing, the development of a modern technology of obtaining copper-iron ligatures from the pyrite 2. experimental investigations were carried out on the copper concentrate from kapan (in south af armenia) deposit. average chemical composition of the concentrate is as follows: cu-27.2%; fe-24.1%: s-29.1%; 80.4%, the rest are sio2 and non-mineral materials. mineralogically, kapan deposit's composition is as follows: chalcopyrite (cufes2) – 60%; chalcozin (cu2s) chalcocite – 8%; pyrite (fes2) – 5%; tenorite (cuo) – 4%; bornite (cu5fes4) – 3%. as it could be seen, the concentrate mainly is composed of pyrite and chalcopyrite, that's why they are called of the pyrite-chalcopyrite type. pyrite occurs in free form. vibration planetary mill (2474 rpm) was used for grinding (pretreatment) of the chalcopyrite concentrate (down to –0.1 + 0.74 mm in size). around 50 g sample was thus vibro-treated. mill ball diameter is 0.8 cm, weight: 1750 gr. both dry and wet (in aqueous media) grinding was used. solid to liquid (s:l) ratio was at 1:1.5 for wet grinding, duration: 30, 60, 90, 120 min. the sample obtained after the experiments was filtered for the quantitative determination of copper and iron in the solution, both by the atomic-adsorption and photo-calorimetric methods. x-ray diffraction (xrd) examination with monochromatic cukα radiation (dron2 diffractometer) was performed. scanning electron microscope (sem) vega ts 5130mm, tescan, czech republic, microanalysis system inca energy 300, oxford instruments, uk7 and energy dispersive x-ray microanalizer were used for metallographic investigations. in addition, current inspections of initial mixtures and final products were realized by a “neophot-32” microscope (germany). 3. results and discussion x-ray diffraction patters of initial concentrate as well as the pretreated samples (dry and wet activated) are presented in fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 26 fig. 1. diffraction patterns of the copper concentrate from kapan deposit 1initial concentrate; 2dry activated; 3 wet activated. duration: 1 h. as it follows from the figure (fig. 1, curve 1), there are strong reflexes pertaining to 3.12, 3.03, 1.85 å, which are characteristic of chalcopyrite. one could discern also reflexes of pyrite: d=3.14, 2.71, 2.21, 1.637 å. khalcozin-characteristic reflexes are: d=3.39, 2.40, 1.969, 1.87, 1.695 å, while the 2.72, 1.937, 1.876, 1.258 å reflexes pertain to bornite. 3.31, 3.10 å patterns of the initials are also present in the x-ray plots, the quantity of which, according to their intensities, are not high. the basic lines coincide with standards. thus, the x-ray and the mineralogical data coincide. pyrite-chalcopyrite concentrate's characteristics undergo steep changes after the mechanical treatment. new reflexes are appeared after dry mechanical treatment, 1 h. the lines: d =2.53; 1.596, 1.696 å are characteristic of magnetite; the lines: d=4.85, 3.44, 3.12, 2.52 å are characteristic of feso4; and 5.11, 3.68, 3.13, 3.09, 2.84, 1.985, 1.897 å are characteristic of fe2(so4)2. for some cases, these reflexes coincide with iron oxide reflexes. wet treatment produces reflexes which are characteristic of sulphate crystal-hydrates: 4fe2(so4)35fe2o327h2o, (d=5.11, 3.13, 3.09, 2.55, 1.987, 1.838 å); feso4h2o (d=3.99, 3.44, 3.12 å) and cuso45h2o (d=5.48, 4.73, 3.99, 3.71, 2.75 å): on the other hand, characteristic lines of chalcopyrite and of other minerals gradually tend to decrease. the intensity of characteristic line's at 3.03 å is reduced turning primarily into d=3.08 å, which is characteristic of fe2(so4)3. so42ions are present in the activated samples in considerable quantities (so42ions were determined by bacl2). here are some phase transitions of chalcopyrite due to mechanical activation: dry: cufes2  30 min cufes2+feso4+ +cuso4  60 min cufes2+4fe2(so4)35fe2o3+cuso4+fe3o4 wet:cufes2  30 min feso4h2o+cuso4 5h2o+cufes2  60 min fe(so4)35h2o327h2o + cuso45h2o + +fe3o4 combined treatment (60 min): 4fe(so4)35fe2o3+cufes2+cuso4+5h2o +fe3o4 during wet activation in water, the structures of chalcopyrite and chalkozine are disintegrated, as follows from intensity reduction of d=1.870 and 1.855 å lines characteristic of cufes2. new phase is emerged: 4fe(so4)35fe2o327h2o, along with cuso45h2o phase being present in considerable quantities. this new phase is a result of consistent crystallization (the sharp peaks at d=3.12 and 5.08 å lines). hence, effective activation already holds right in the initial phase of activation. further grinding makes 4fe(so4)35fe2o327h2o phase more crystalline, which is confirmed by the sharp reflex of 3.08 as well as 4.73 line being characteristic of cuso45 h2o. the interstitial plane distances of 4fe2so45fe2o32h2o and chalcopyrite are the same equaling to 3.08 å. actually, cufes2 is transformed into cu9fe9s16. thus, chalcopyrite losses most of the sulphur during mechanical treatment. from above said it follows that dry and wet activation (1 h) causes mechanicalfood and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 27 chemical changes, and chalcopyrite-pyrite concentrate diffraction pattern undergoes deep changes. chalcopyrite crystal structure becomes amorphous after dry grinding which later re-crystallizes in hexagonal trigonal lattice structure of the following: cufes2 (2.04 å), up to oxidized: cuso4, feso4, fe2o3(2.68 å) and fe3o4 (2.74, 2.53, 1.691): thus, mechanochemical activation experiments showed that preliminary mechanochemical activation process in the vibratory mill of kapan (armenia) pyritechalcopyrite sulfide concentrates is similar to the low-temperature roasting of minerals. in both cases the process is accompanied by the transformation of sulfides into sulfates, hydrosulfates and oxides. investigations have been carried out aimed at direct, out-of-furnace aluminothermic reduction of previously mechanochemically activated concentrates. preliminary thermodynamic calculations showed that aluminothermic reduction of iron and copper sulfates are possible, and these reactions are characterized by high thermal effect and by negative values of g. 3feso4+10al=3fe+al2s3+4al2o3, g = -4431.76 + 2.72t kj/mol 3cuso4 + 10al = 3cu + al2s3 + 4al2o3, g = -4894.34 – 0.36t kj/mol most probably the following reaction takes place as well: al2s3+3cao=3cas+al2o3, g = -693.86 – 0.59t kj/mol therefore, one can assume that due to the mechanochemical activation, the transformation of sulfides into sulfates promotes their aluminothermic reduction and extraction of sulfur in the form of cas. aluminothermic reduction was carried out with the samples prepared from the mixture of copper concentrate, aluminum powder, copper shavings, cao and nano3[7,8].as a result metallic and slag phases were obtained, which were easily separated from each other after the cooling. the dependence of the extraction degree of the metallic phase from the amount of reducer was studied at fixed content of other components and at various milling durations. it has been found that the degree of metallic phase extraction strongly depends on the particle size of the reducer and its quantity. in the case of 60 minutes of milling duration and theoretically necessary amount of the reducer, the reduction proceeds with low efficiency and partly separation between the metal and slag phases takes place. besides, the metallic phase contains sulfur traces. at the same milling conditions and at 120% excess of the aluminum in the initial mixture the reduction proceed violently resulting in formation of a metallic phase where traces of sulfur were also detected. in all cases the metallic phase (with the yield of 81%) represented a mixture of iron and copper. this alloy was porous because of significant sulfur content. to increase the yield of the metallic phase by the increasing of aluminum amount in the initial mixture is not reasonable, because this leads to an increase of the aluminum content in the final product (up to 1.0-1.5%) and the specific heat of the process as well. to decrease the specific heat of the reaction and to avoid the presence of sulfur in the alloy, the dependence of the metallic phase extraction degree from the amount of the fusible additive, cao, was studied at the same composition of initial mixture and 60 minutes milling duration. the best results were obtained at 30% content of cao. in this case the metallic phase and the slag were separated easily, resulting in growth of the alloy yield up to 90-92% and low sulfur content (0.01%). thus, the addition of quicklime promotes the transition of sulfur into the slag. the dependence of the extraction degree of the metal phase from the content of nano3 was also studied. as a result the following improved optimum composition of the initial mixture was food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 28 established: copper concentrate 50%, aluminum powder 13%, nano3 5%, copper shavings 2%, cao 30%. at this composition and 90 minutes of milling duration the extraction degree for the ligature reaches about 98.9% with low content of sulfur (0.01%). the metallic phase obtained was subjected to chemical analysis, resulting in copper and iron contents of 53% and 47%, respectively, without traces of sulfur and aluminum. metallographic analysis has shown that increase in the milling degree does not change chemical composition of the alloy, while an influence on the structure and particle size distribution is significantly higher. as it follows from the structure of nonactivated samples (fig. 2.1) the metallic phase is distributed irregularly in the form fig. 2. microstructures of the metallic phases obtained at different milling conditions and nonactivated initial mixture (x 400): non-activated (1), samples activated for 15 min (2), 30 min (3) and 60 min (4). of agglomerations. the microstructure of the metallic phase obtained after 15 min activation (fig. 2.2) differs significantly. here metal particles are larger hypothetically as a result of agglomeration caused by strong surface forces. activation during 30 minutes (fig. 2.3) allows to obtain metal particles with a smaller size and more uniform distribution, and 60 minutes activation leads to the formation of a more fine-grained microheterogeneous structure (fig. 2.4). thus, comprehensive studies prove the fundamental possibilities of producing the copper-iron ligatures by the direct aluminothermic or silicathermic reduction of mechanochemically activated copper sulfide concentrates. the modified alloy obtained by such a method can be used as alloying agent in the production of different copper alloys and bronzes. 4. conclusion the behavior of kapan copper concentrate was investigated during fine grinding in the vibratory mill in water or in air for durations of 15, 30 and 60 minutes. it was shown that both the separate minerals and the copper concentrate undergo deep chemical changes in these environments during fine grinding. in addition, milling in the air for 60 minutes leads to the formation of copper and iron sulfates, and iron oxides (fe2o3 and fe3o4) in the case of iron. hydrosulfates were obtained in water environment at the milling duration of 60 minutes. it was shown that the formation of sulfates and oxides during the mechanochemical activation partly replaces the roasting process, making the further reduction process of sulfides more vigorous. technical parameters were selected for the aluminothermic reduction of copper concentrate and structure investigations of the obtained alloys were carried out. metallographic analysis has shown that the structure of depending on the activation duration a more dispersed and microheterogeneous alloy is obtained. a technology was developed for the production of copper-iron ligatures by a direct out-of-furnace aluminothermic reduction of the kapan copper concentrate. this energy-saving technology helps also food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 29 to solve some environmental issues, including the extraction of so2 and can be easily applied in factories operating in yerevan, where ferroalloys are producing by similar technologies. 6. references [1]. web references: 1. kulebakin v.g.,transformation of sulfides during activation, novosibirsk. nauka publishing house. 209 p., (1983) (in russian). [2]. kulebakin v.g, yusupov t.s., on some peculiarities of fine grinded chalcopyrite,novosibirsk. v. 10, release 305, 219–223 pp., (1976) (in russian). [3]. avakumov ye.g, boldirev v.v, kosobutski i.i., mechanochemical activation of solid-state reactions, proceedings of the academy of sciences of the ussr. series: chemical sciences. release 4, 45–50 pp. (in russian). [4]. golosov s.i., molchanov v.i., centrifugal planetary mill: its technical possibilities and use in geological survey. in the book: physical-chemical changes of the minerals during super fine grinding. novosibirsk, 5–25 pp., (1966) (in russian). [5]. urakayev f., shevchenko v., boldirev v., proceedings of russianacademy of sciences. v. 377, n1, 69-71 pp., (2001) (in russian). [6]. boldirev v.v., tsibulia s.v., cherepanova s.v., et al, proceedings of russian academy of sciences, v. 361, n6, 784789 pp., (1998) (in russian). [7]. martirosyan v.h.,agamyan t.s.,sasunthyanm.e.,the influence of mechanochemical activation on the reactivity of iron and copper sulfides, bulletin of nas ra and seua. tgseries, may-august 2006, vol.lix, no.2, pp.318-323. [8]. martirosyan v.h., ghukasyan j.g.,agamyan t.s., shmavonyanm.sh.,zakaryank.r., the study of possibilities ofdirect aluminothermic reduction ofcoppersulfide concentrates. collected papers of the “mining and metallurgical institute” cjsc, yerevan. x-ray diffraction patters of initial concentrate as well as the pretreated samples (dry and wet activated) are presented in fig. 1. as it follows from the figure (fig. 1, curve 1), there are strong reflexes pertaining to 3.12, 3.03, 1.85 å, which are characteristic of chalcopyrite. one could discern also reflexes of pyrite: d=3.14, 2.71, 2.21, 1.637 å. khalcozin-characteristic ref... 3.31, 3.10 å patterns of the initials are also present in the x-ray plots, the quantity of which, according to their intensities, are not high. the basic lines coincide with standards. thus, the x-ray and the mineralogical data coincide. pyrite-chalcopyrite concentrate's characteristics undergo steep changes after the mechanical treatment. new reflexes are appeared after dry mechanical treatment, 1 h. the lines: d =2.53; 1.596, 1.696 å are characteristic of magnetite; the lines: d=4.8... wet treatment produces reflexes which are characteristic of sulphate crystal-hydrates: 4fe2(so4)3(5fe2o3(27h2o, (d=5.11, 3.13, 3.09, 2.55, 1.987, 1.838 å); feso4(h2o (d=3.99, 3.44, 3.12 å) and cuso4(5h2o (d=5.48, 4.73, 3.99, 3.71, 2.75 å): on the other hand, characteristic lines of chalcopyrite and of other minerals gradually tend to decrease. the intensity of characteristic line's at 3.03 å is reduced turning primarily into d=3.08 å, which is characteristic of fe2(so4)3. so42ions a... here are some phase transitions of chalcopyrite due to mechanical activation: dry: cufes2cufes2+feso4+ +cuso4 cufes2+4fe2(so4)3(5fe2o3+cuso4+fe3o4 wet:cufes2feso4(h2o+cuso4( (5h2o+cufes2 fe(so4)3(5h2o3(27h2o + cuso4(5h2o + +fe3o4 combined treatment (60 min): 4fe(so4)3(5fe2o3+cufes2+cuso4+5h2o+fe3o4 during wet activation in water, the structures of chalcopyrite and chalkozine are disintegrated, as follows from intensity reduction of d=1.870 and 1.855 å lines characteristic of cufes2. new phase is emerged: 4fe(so4)3(5fe2o3(27h2o, along with cuso4... the interstitial plane distances of 4fe2so4(5fe2o3(2h2o and chalcopyrite are the same equaling to 3.08 å. actually, cufes2 is transformed into cu9fe9s16. thus, chalcopyrite losses most of the sulphur during mechanical treatment. from above said it follows that dry and wet activation (1 h) causes mechanical-chemical changes, and chalcopyrite-pyrite concentrate diffraction pattern undergoes deep changes. chalcopyrite crystal structure becomes amorphous after dry grinding which ... thus, mechanochemical activation experiments showed that preliminary mechanochemical activation process in the vibratory mill of kapan (armenia) pyrite-chalcopyrite sulfide concentrates is similar to the low-temperature roasting of minerals. in both c... 3feso4+10al=3fe+al2s3+4al2o3, (g = -4431.76 + 2.72t kj/mol 3cuso4 + 10al = 3cu + al2s3 + 4al2o3, (g = -4894.34 – 0.36t kj/mol most probably the following reaction takes place as well: al2s3+3cao=3cas+al2o3, (g = -693.86 – 0.59t kj/mol therefore, one can assume that due to the mechanochemical activation, the transformation of sulfides into sulfates promotes their aluminothermic reduction and extraction of sulfur in the form of cas. aluminothermic reduction was carried out with the samples prepared from the mixture of copper concentrate, aluminum powder, copper shavings, cao and nano3[7,8].as a result metallic and slag phases were obtained, which were easily separated from each o... as it follows from the structure of non-activated samples (fig. 2.1) the metallic phase is distributed irregularly in the form of agglomerations. the microstructure of the metallic phase obtained after 15 min activation (fig. 2.2) differs significantly. here metal particles are larger hypothetically as a result of agglomeration caused by strong surface forces. activation duri... thus, comprehensive studies prove the fundamental possibilities of producing the copper-iron ligatures by the direct aluminothermic or silicathermic reduction of mechanochemically activated copper sulfide concentrates. the modified alloy obtained by s... the behavior of kapan copper concentrate was investigated during fine grinding in the vibratory mill in water or in air for durations of 15, 30 and 60 minutes. it was shown that both the separate minerals and the copper concentrate undergo deep chemic... technical parameters were selected for the aluminothermic reduction of copper concentrate and structure investigations of the obtained alloys were carried out. metallographic analysis has shown that the structure of depending on the activation duratio... a technology was developed for the production of copper-iron ligatures by a direct out-of-furnace aluminothermic reduction of the kapan copper concentrate. this energy-saving technology helps also to solve some environmental issues, including the extr... food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 21 experiments concerning physico-chemical and microbiological control of bakery yeast industrial production *adriana dabija1, oana elena buzatu2 1faculty of food engineering, stefan cel mare university od suceava, romania 2s.c. rompak s.r.l. pa cani, romania adriana.dabija@fia.usv.ro *corresponding author received 25 june 2012, accepted 30 july 2012 abstract: biotechnological characteristics of bakery yeast can be affected by deficiencies attributed to raw materials (especially molasses), technological process and certain microbiological causes. compliance with process parameters and standards of hygiene throughout the manufacturing process of the yeast, using appropriate raw materials and taking appropriate action to prevent contamination of yeast lead to sensory and biotechnological qualities of yeasts that are suitable for use in bakery and confectionery industry . during the process of yeast manufacturing, concomitant with the multiplication of the cells that belong to the pure culture, other microorganisms can develop in the different phases of technological flow, thus increasing the contaminations degree of the finished product and causing a reduction of biotechnological qualities of yeast. so as to prevent the multiplication of contaminated microorganisms, it is imposed a severe microbiological control on the production stages through the study of hygiene degree and the detection of contaminants that can proceed from various sources. this paper aims to determine the biotechnological qualities and the extent of contamination of bakery yeast for four industrial batches of finished product. the importance of monitoring physical, chemical and microbiological parameters in each technological phase of the industrial production of bakery yeast was highlighted. keywords: monitoring, biotechnological qualities, storage-ability 1. introduction yeast is used in bakery industry as biological loosener and flavor-maker in bread. yeast is used for centuries, but industrial-scale production began in 1850. today the total amount of yeast produced annually is in the tens of millions of tons, and the benefits are estimated at several billion dollars [1, 2]. technology of baking yeast in our country is to multiply a pure culture of yeast using diluted leaven and five successive stages of multiplication, in the laboratory and industrial system respectively: phase i called yeast inoculums, phase ii yeast pre-culture, phase iii yeast culture, phase iv yeast starter, phase v commercial yeast. to obtain a quality yeast biomass, every stage of the technological process is monitored from physico-chemical and microbiological points of view. during the process of making bakery yeast, simultaneously with the multiplying yeast cells can develop other microorganisms that increase the degree of contamination of the final product and reduces yeast biotechnological qualities and storageability [3-6]. the paper presents a monitoring of physico-chemical and microbiological mailto:adriana.dabija:@fia.usv.ro food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 22 parameters for the industrial production of bakery yeast for four batches, from receipt of raw materials and ending with finished product, highlighting relevant aspects for obtaining bakery yeasts with constant biotechnological qualities [7 10]. 2. experimental monitoring the technological process of obtaining bakery yeast was performed in s.c. rompak s.r.l. pa cani in april 2012. pure culture used in the project was a strain of saccharomyces cerevisae obtained from the center for biotechnology pak, a part of pakmaya yeast factory from izmit, turkey. physicochemical control for all phases of fermentation and for the final product was performed both in the fermentation department’s laboratory and in the central laboratory of s.c. rompak s.r.l. pa cani. the monitored physico-chemical parameters and the apparatus used are shown in table 1. table 1 physico-chemical parameters and apparatus physico-chemical characteristics apparatus mash concentration (°bllg) thermosacharometer ph phmeter temperature electrical thermometers pressure manometer molasses and air flow rate flow-meter alcohol determination alcoholmeters yeast concentration determination centrifuge percentage of budding and dead cells determination microscope yeast biomass determination 27% (kg) vacuum pump, analytical balance fermentative capacity (cm3 co2/h) fermentograph sja yeast dry substance thermo balance monitoring frequency of bllg level, ph and yeast milk and diluted molasses’ temperature is that of every two hours, starting at zero fermentation hours, while molasses and air flow is monitored hourly. alcohol determination from fermenting leaven is done hourly, starting from the first hour of fermentation by two methods: physical (with alcoholmeter) and chemical method. determination of yeast concentration is the amount of yeast in suspension volume, percentage having a significant role because it provides an important clue to the normal multiplication of yeast cells and the total quantity of yeast. determination of yeast for phases ii and iii is at the end of the fermentation process and for phases iv and v of the monitoring frequency is two hours from the first hour of fermentation. study of biomass accumulation during yeast multiplying is a current analysis and consists in determining the amount of biomass by centrifuging a sample of leaven, the result expressed in g yeast / l leaven. the analysis was performed for all phases of the manufacturing process, being determined the initial concentration of inoculums in the growing medium and the final accumulation of biomass in fermented leaven. fermentative ability of yeast biomass was estimated using fermentograph at the end of each technological stage, respectively the finished product. physiological state of the yeast cells at different stages of the growing period was assessed by determining the percentage of budding cells and dead cells. microbiological control at each fermentation stage and the final product food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 23 was performed daily in the microbiology laboratory. samples were collected from a sterile environment (beginning of fermentation) and from the yeast suspension (end of fermentation) and the number of cfu of total bacteria, coliforms bacteria, atypical yeasts, and escherichia coli were determined. the same analyzes were performed for bakery yeast finished product (500g block). the method used is the cultural, using culture mediums of european standards: pca nurturing environment for all bacteria, tbx medium for growing escherichia coli; vrbl nurturing environment for coliforms bacteria; lysine medium for cultivation of yeasts and moulds. table 2 monitored microbiological characteristics and utilized apparatus microbiological charactaristics apparatus coliforms bacteria ufc/g total bacteria ufc/g atypical yeasts ufc/g escherichia coli ufc/g microbiological hood, sterile pipette, petri plate, colonies counter num tor colonii, thermostat 3. results and discussions 3.1.physico-chemical control at manufacturing stages the results for main parameters’ monitoring at each manufacturing stage are shown in tables 3, 4, 5, and 6. table 3 technological parameters per manufacturing phase – batch 111 multiplication phase mash concentration [°bllg] temperature [°c] ph alcohol content [%] multiplication period [h] phase i 10÷11 32÷34 5.6÷4.8 0÷2.85 24 phase ii 8.9÷3.7 33÷34 5.4÷4.7 0.05÷2.75 13 phase iii 2.7÷4.7 33÷34 4.6÷5.2 0.12÷2.9 16 phase iv 1.7÷10.3 31÷35 4.17÷6.24 0.59÷0.15 19 phase v 2.2÷10.5 30÷32 3.24÷7.53 0.09÷0 16 table 4 technological parameters per manufacturing phase – batch 168 multiplication phase mash concentration [°bllg] temperature [°c] ph alcohol content [%] multiplication period [h] phase i 10÷11 32÷34 5.6÷4.9 0÷3.5 24 phase ii 9.2÷3.8 32÷34 5.4÷4.7 0÷2.7 13 phase iii 2.9÷5.1 33÷34 4.55÷5.3 0.21÷2.3 16 phase iv 1.8÷10.5 31÷35 4.29÷6.08 0.72÷0.26 19 phase v 2.2÷11.3 31÷32 3.34÷7.83 0.04÷0 16 table 5 technological parameters per manufacturing phase – batch 172 multiplication phase mash concentration [°bllg] temperature [°c] ph alcohol content [%] multiplication period [h] phase i 10÷11 32÷34 5.6÷4.9 0÷3.4 24 phase ii 9.8÷3.9 33÷34 5.7÷4.75 0÷2.65 13 phase iii 2.6÷4.8 33÷34 5.2÷4.6 0.14÷2.5 16 phase iv 1.7÷10.9 30.7÷35 4.23÷6.5 0.62÷0.24 19 phase v 2÷10.8 30.5÷32 3.39÷7.67 0.08÷0 16 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 24 table 6 technological parameters per manufacturing phase – batch 183 multiplication phase mash concentration [°bllg] temperature [°c] ph alcohol content [%] multiplication period [h] phase i 10÷11 32÷34 5.6÷4.7 0÷2.95 24 phase ii 9.2÷3.5 33÷34 5.5÷4.6 0÷2.45 13 phase iii 2.8÷5.3 33÷34 4.6÷5.4 0.20÷2.7 16 phase iv 1.9÷10.8 31÷34.5 4.16÷6.25 0.41÷0.22 19 phase v 2÷11.4 30÷32 3÷7.7 0.08÷0 16 the company's central laboratory makes very important determination, namely, the accumulation of biomass per multiplying phase of yeast, an indicator which shows if the process was conducted in pre-established technological parameters. the data presented in figure 1., for example, show that the amount of yeast biomass accumulated in the culture medium increased with 864% (from 25.7 g / l to 222.21 g / l) in only 64 hours (2 days and 16 hours). figure 1. biomass accumulation per manufacturing phases a very important biotechnological feature of bakery yeast end product is the fermentation power, which is determined in the factoring by using a sja fermentograph. this method is based on the fact that yeast fermentation power is the time required by certain amount of yeast to develop 450 cm3 co2, under conditions determined by fermentograph apparatus. all four batches analyzed showed a fermentation power corresponding to the quality management systems limits implemented in society (table7). table 7 bakery yeast’s fermentation power at different stages of technological process yeast batch fermen tation power [cm3 co2 / h] manufacturing phase yeast blockii iii iv v 111 540 660 680 700 790 168 690 660 710 690 760 172 670 660 700 680 750 183 620 650 680 660 740 3.2. microbiological control for manufacturing phases a major source of contamination can be pure laboratory culture or culture resulting from phase ii, under the conditions that the regime of nutrient medium sterilization, hygienic conditions at sowing and cultivation are not complied with, or when there is no efficient air filtering. microbiological control was determined using counting chambers for yeast cell concentration and percentage of cells autolysates, by suspending the cells in citrate methylene blue. in general, according to the microbiological control conducted, the culture of yeast used was appropriate from microbiological point of view, without contamination by foreign microorganisms and having a high percentage of viable cells. for each stage of the technological multiplication process, a microbiological control was achieved for food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 25 the four physico-chemically monitored batches. results of the microbiological examination conducted are summarized in tables 8, 9, 10 and 11. table 8 results of microbiological control – batch 111 multiplication phase total bacteria [cfu/g] total coliforms bacteria [cfu /g] escherichia coli [cfu /g] atypical yeast [cfu /g] phase ii phase iii phase iv 2.90 x 102 starter 6.40 x 102 1 phase v 3.70 x 102 yeast milk 1.04 x 103 4.10 x 10 7.00 yeast block 4.10 x 103 5.60 x 102 3.00 x 10 table 9 results of microbiological control – batch 168 multiplication phase total bacteria[cfu /g] total coliforms bacteria [cfu /g] escherichia coli [cfu /g] atypical yeast [cfu /g] phase ii phase iii phase iv 1.10 x 102 starter 2.00 x 102 1.00 phase v 4.00 x 102 yeast milk 3.00 x 102 yeast block 8.20 x103 1.00 x 10 table 10 results of microbiological control – batch 172 multiplication phase total bacteria[cfu /g] total coliforms bacteria [cfu /g] escherichia coli [cfu /g] atypical yeast [cfu /g] phase ii phase iii 3.00 x102 phase iv 9.00 starter 1.00 x 102 1.00 phase v 2.00 x 10 yeast milk 3.00 x 10 yeast block 1.95 x 104 5.00 table 11 results of microbiological control – batch 183 multiplication phase total bacteria[cfu /g] total coliforms bacteria [cfu /g] escherichia coli [cfu /g] atypical yeast [cfu /g] phases ii and iii phase iv 1.60 x 102 starter 2.50 x 102 1.00 phase v 8.70 x 102 yeast milk 2.00 x 10 yeast block 7.40 x 103 food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 26 it is observed that, in general, in the first three phases of multiplication no contamination occurs (except batch no.172 phase three, having a bacterial count of 3.00 x102 cfu / g the degree of bacterial contamination is reduced, taking into account the fact that in the yeast block a 2 ‰ contamination (two bacterial cells allowed tp 1000 yeast cells) is accepted (auerman, 1995). yeast comes in blocks (500 g), which retain its technological properties up to 40 days, depending on the quality of yeast and storage conditions (optimum at 0 ÷ 0÷40c, relative air humidity 65 ÷ 70%). yeasts with a high degree of purity do not contain putrefaction bacteria or atypical yeasts, and can be stored up to 3 months; good quality yeasts must not contain bacteria of putrefaction in excess of 0.1 ÷ 0.2% (1 g yeast containing 5•109÷1010 cells, of which may be allowed 100 to 1000 bacterial contaminants). 4. conclusions bakery yeast is a cell biomass of the species saccharomyces cerevisiae, with particular importance for the bakery industry. the industrial manufacturing of bakery yeast requires a physical, chemical and microbiological rigorous control, covering all phases of production. to obtain quality yeast is necessary to use high quality raw materials with appropriate physico-chemical and microbiological characteristics, in accordance with a strict observance of technological process and good working and hygienic practices (gmp and ghp). following measurements it was found that saccharomyces cerevisiae yeast culture is used as inoculum is a microbiologically appropriate culture. by studying the biomass accumulation during multiplication of yeast in each manufacturing phase, it was observed a good multiplication of cells, which reach values of 108 ÷ 1010/cm3 environment. main biotechnological property for evaluating bakery yeast quality is the leavening power/ability. bakery yeast finished product is the result of efforts made to avoid contamination with foreign microorganisms, the quality of which is conditioned by the degree of microbiological purity posed by delivery in bakery industry; out of the four analyzed batches, one batch presented contamination microorganisms (bacteria) in phase iii of multiplication, microorganisms that can cause a decrease of biotechnological properties during storage. 5. references [1]. dan v., microbiology of food (microbiologia alimentelor),editura alma, gala i, (2001) [2]. spencer j., spencer d.m., yeast technology, berlin, springer-verlag, (1990) [3]. dabija a., biotechnology for industrial manufacturing yeast of high enzymatic activity, phd thesis, gala i, (2000) [4]. iserentant d., yeast handling: the key to optimal performance in fermentation, cerevisia and biotechnology, 3, 35-39, (1994)duenassanchez, r., et al., increased biomass production of industrial bakers’ yeasts by overexpression of hap4 gen, nternational journal of food microbiology, volume 143, issue 3, 150-160, (2010) [6]. john, e., yeast biotechnology, the yeast, p.21-44, (2011) [7]. piper, p., resistance of yeasts to weak organic acid food preservatives, advances in applied microbiology, volume 77, 97-113, (2011) [8]. gassent-ramirez, j., et al., genomic stability of saccharomyces cerevisiae baker’s yeasts, systematic and applied microbiology, volume 22, issue 3, 329-340, (1999) [9]. attfield, p., bel, p., genetic improvement of baker’s yeast, applied mycology and biotechnology, 213-240, (2003) [10] zimmermann, f.k., entian, k.d. , yeast sugar metabolism, technomic publishing co. inc., pennsylvania, usa, (1997) звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 112 study regarding the possibilities to obtain functional food from wheat flour: bread with exogenous buckwheat addition *gabriela constantinescu (pop) 1, adriana dabija1, amelia buculei1 1faculty of food engineering, stefan cel mare university, e-mail: gabriela.constantinescu@fia.usv.ro *corresponding author received 12 august 2011, accepted 5 october 2011 abstract: this paper presents the beneficial role of the addition of buckwheat in bakery products. it highlights aspects of buckwheat as food and drug use and possible use as a flour or buckwheat groats in bakery products. the research paper presented to obtain bread with added buckwheat flour the purpose of the bread samples were made several mixtures of wheat flour with different proportions of buckwheat flour (5%, 10%, 15%), after the flour samples were subjected to laboratory testing where absorption and wet gluten were seen in the difference in the behavior of the flour mixture. also, it was made determination of technological properties through alveograph curves, in order to make recommendation for different usages. in the laboratory control sample and the samples under study were made with the same formulation and the same treatment technology. once made bread samples were also subjected samples of flour mixture, followed study tests the core porosity and elasticity and also to check the acidity during the technological process of hygienically. bread made with buckwheat flour 5% is the bread with the best organoleptic properties and physico-chemical. bread is a normal appearance, glossy shell, corresponding smell, taste sweet. the core is uniform with a very good elasticity, optimal porosity and acidity normal bread so obtained can be considered a very good bread. keywords: alveograph courves, technological properties, bakery products 1. introduction in the production of bread can be used in addition to wheat flour and other raw materials that can help improve the nutritional value of finished products. buckwheat is a plant that is within the boundary between food and medicine. as food, particularly because energy intake is higher cereal classics. in terms of treatment, apart from energy intake particularly rich in routine is a good protector of vascular fragility [1]. diets that contain buckwheat have led to a lower risk of developing cholesterol and blood pressure. china's population consumes large amounts of buckwheat (100 g / day). when researchers tested blood lipids over 800 people in china, found that buckwheat flour has contributed significantly to lower cholesterol, lower density lipoproteins from cholesterol (related to cardiovascular disease)[2]. buckwheat can help to cure diabetes. a single dose of buckwheat seeds can lower blood glucose levels by 12-19% after 90120 minutes of administration time. component in buckwheat responsible for lowering blood glucose appears to be chiro-inositol, a compound that has shown through several tests both in animals and humans, it has an important role in glucose metabolism and cell. [3] buckwheat also prevents gallstones, improves circulation, lowers blood mailto:gabriela.constantinescu@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 113 pressure and has a significant energy value. buckwheat contains no gluten. because of this, is a very valuable food for people with gluten intolerance (celiac disease sufferers). buckwheat prevent the formation of bladder stones. eating foods high in insoluble fiber, as buckwheat, helps women to be protected by the formation of bladder stones, according to a study published in american journal of gastroenterology. buckwheat groats contain carbohydrates (68-72%), protein (10-13%), fat (2%). the composition of the protein falls lysine and methionine. [2] buckwheat is rich in vitamins b. it is indicated in liver disease, atherosclerosis, diabetes and other diseases that requires increasing the amount of lipotropic substances in the diet. their nutritional value depends on the variety and how to obtain them. by removing the membrane decreases the content of vitamins, mineral salts, cellulose, and assimilation of carbohydrates and protein improves. groats contain many carbohydrates (6577%), including starch (55-74%), protein (7-13%) lower value, a small amount of fat (0.6 to 6%) [1]. these foods, particularly oat groats, buckwheat, millet, barley, serve as important source of vitamins b, pp, magnesium, phosphorus, potassium. oat groats, buckwheat, millet are rich in lipotropic substances. they are easily digestible meal groats, oat flour, flour made from cereal diet. oat groats, buckwheat, barley, barley contain increased amounts of cellulose. buckwheat flour is used best when mixed with wheat or rye buckwheat the generally recommended for pregnant women, people with overload syndromes, diabetic shoes, paddy rice mixed with wheat flour to replace the yeast dough [3]. buckwheat contains also magnesium which helps relax blood vessels improving circulation and distribution of nutrients and resulting in decreased blood pressure perfect combination for a healthy cardiovascular system. nutrients in buckwheat help maintain blood sugar. in a test comparing the effect on blood sugar bran bread made from buckwheat and one made from wheat flour showed that buckwheat bran decreased blood glucose and insulin. [2] this paper presents research to obtain their bakery products with added buckwheat flour. 2. experimental in order to obtain some available experimental data, wheat flour obtaining from flamura 85 wheat variety grinding in chopin laboratory mill was used like control sample. the analytical flours’ obtained quality (table 1) was determined in accordance with the international standard methods (ash content – icc104/1, wet gluten – icc105/2, protein content – icc106/2, hydration capacity with pharinograph icc115/1). the moisture content of the wheat flour and bran were determined by oven drying at 1300c for 1 hour. table 1 analytical parameters of control flour moisture % ash % wet gluten % protein % hydration capacity % 13.42 0.68 31.1 13.3 62.6 buckwheat flour was provided by local producers. like raw material i used also compacted fresh yeast (saccharomyces cerevisiae) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 114 from s.c. rompak, pascani, with 32.5% dry matter and 46.54% protein content (n x 6.25). the recipe used for making breads is shown in table 2. table 2 recipe for bread with buckwheat flour ingredients and technological regime um quantities raw materials and ingredients wheat flour grams 500 buckwheat flour grams fresh yeast grams 8 salt grams 7 water cm3 270 to 300 technological parameters kneading time min 10-15 proffer time min 20-25 temperature of blanks 0c 30-32 baking time min 30-35 baking temperature min 240-260 dough was prepared using a straight dough method. after baking, the samples were cooling 6-8 hours in controlled atmosphere (uv lamps). in order to be scoring (after 24 hours), and to be examined from microbiological point of view (after 72 hours), the samples were sliced for packed in plastic bags microbiological quality of bread with buckwheat, over 3 days at room temperature was evaluated according with sr en iso 4832/2006 quality standard. also, it was made determination of technological properties through alveograph curves, in order to make recommendation for different usages. a chopin alveoconsistograph was used for determination of resistance of deformation (tenacity) p, dough extensibility, l, the value of p/l, and the mixing energy w according with the international standard sr iso 5530 – 4.the experiments are made in the research laboratory of “ştefan cel mare” university of suceava, faculty of food engineering. 3. results and discussion in order to evaluate the influence of buckwheat on the technological characteristics of flour it was made the following samples: witness sample only control flour; p1 control flour with 5 % buckwheat; p2control flour with 10 % buckwheat; p3control flour with 15 % buckwheat. after establishing the flour mixtures, they were subjected to laboratory analysis: hydration capacity, wet gluten content and acidity were seen in the difference in the behavior of mixtures of flour (table 3). the results of these tests led to the following conclusions:  samples increases with increasing acidity addition of buckwheat flour;  when content is high and buckwheat flour hydration capacity is greater;  wet gluten content decreases depending on the proportion of buckwheat flour in the sample, the lowest value with a sample with the highest amount of added buckwheat flour (15%). table 3 the technological properties of flour mixing samples characteristic flour mixing samples control p1 (5%) p2 (10%) p1 (15%) acidity, acidity grades/100 g 1.3 1.4 1,5 1,68 wet gluten, [%] 28 25 23 20 hydratation capacity, [%] 62.6 63.11 63.79 64.25 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 115 for trace possible correlations between the physical-chemical characteristics and the technological behavior of the obtained flours, we studied the rheological parameters of the four samples (figure 1). with alveoconsistograph it were measuredthe following rheological parameters: p tenacity (maximum pressure required for the deformation of the sample), l (or g) extensibility (length of the curve), w baking strength (surface area of the curve) and p/l -configuration ratio of the curve. the results are measured or calculated from the five curves (for each sample) obtained to the alveolink. however, if one of the curves is far away from the 4 others, in particular following premature rupture of the bubble, it will not be taken into account when expressing the results. a. control sample b. p1 5% c. p2 10% d. p3 15% figure 1 alveograph curves for flour mixture samples research showed that between physicchemical parameters of flour and dough alveographic parameters a number of significant correlations are established, suggesting that achieving a predictive model of alveographic characteristics based on physics-chemical properties of flour is possible. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 116 control sample and p1 (5%) samples have very good rheological properties: a w (kneading energy) between 123-241, which results in obtaining high quality flour, with very good tenacity and extensibility and could be used to manufacture a quality bread. p2 (10%) has good rheological properties (with a w like118-136) which leads to obtaining good bakery products. p3 (15%) has weaker rheological properties and could be used only for simple biscuits or cakes and cake tops (fluid dough’s). it was adopted three options for obtaining work in the laboratory of bread: 5%, 10%, 15% of buckwheat flour added in relation to wheat flour used in the formulation. the finished product was analyzed in terms of sensory and physico-chemical (table 4). the results of these tests are shown in the table above where it is observed that the acidity is low enough that the core porosity ranges between 25 and 35%. humidity and elasticity and core differs depending on the proportion of buckwheat flour under trial. sample p1, bread made with buckwheat flour 5% is the bread with the best organoleptic properties and physicochemical. bread is a normal appearance, glosy shell, corresponding smell, taste sweet. the core is uniform with a very good elasticity, optimal porosity and acidity normal bread so obtained can be considered a very good bread. table 4 the physico-chemical analysis of the packed bread samples characteristic bread samples control p1 (5%) p2 (10%) p1 (15%) height/ dimension ratio 0.62 0.64 0.64 0.67 volume, cm3/100g 300 295 320 310 porosity, [%] 35 35 30 25 elasticity, [%] 78 83.33 63.66 55.55 acidity, acidity grades/100 g 0,6 0.9 1.4 1.6 humidity, [%] 69.46 70.73 78.78 69.02 4. conclusion bread wheat flour with different proportions of buckwheat, was a new attempt to highlight the difference between wheat bread and buckwheat bread. it had produced several samples with different proportions of wheat-buckwheat flour, comparing these samples with a blank (a sample made from wheat flour obtaining from flamura 85 wheat variety grinding in chopin laboratory mill). it were made the following samples: witness sample only control flour; p1 control flour with 5 % buckwheat; p2 control flour with 10 % buckwheat; p3 control flour with 15 % buckwheat. after establishing the flour mixtures, they were subjected to laboratory analysis: hydration capacity, wet gluten content and acidity were seen in the difference in the behavior of mixtures of flour. the results of these show that the buckwheat adition led to increase the acidity and flour hydration capacity. wet gluten content decreases depending on the proportion of buckwheat flour in the sample, the lowest value with a sample with the highest amount of added buckwheat flour (15%). once made bread samples were also subjected samples of flour mixture, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 117 followed study tests the core porosity and elasticity and also to check the acidity during the technological process of hygienically. the results of these show that the bread made with buckwheat flour 5% is the bread with the best organoleptic properties and physico-chemical. bread is a normal appearance, glossy shell, corresponding smell, taste sweet. the core is uniform with a very good elasticity, optimal porosity and acidity normal bread so obtained can be considered a very good bread. 5. acknowledgments this paper is published by cnmp grant nr. 52-132 “reduce of contaminate with mycotoxinnes through cereals reducerea contaminarii cu micotoxine pe filiera cerealelor channel in order to obtain bakery products with fibres higher content safety for consumtion”– fibresig, fonds 6. references [1]. krkošková b., mrázová, z., prophylactic components of buckwheat, food research international, volume 38, issue 5, 561-568, (2005) [2]. sun-lim kim, sung-kook kim, cheol-ho par, introduction and nutritional evaluation of buckwheat sprouts as a new vegetable food research international, volume 37, issue 4, 319-327, (2004) [3]. li-yun lin hsiu-man liu, yawen yu a, sheng-dun lin jengleun mau , quality and antioxidant property of buckwheat enhanced wheat bread, food chemistry , 112,987-981, (2009) [4]. aacc. (1988). approved methods. st. paul, mn, usa: the american association of cereal chemists. [5]. aoac (1990). official methods of analysis (15th ed.). washington, dc, usa: association of official analytical chemists. http://www.sciencedirect.com/science?_ob=redirecturl&_method=outwardlink&_partnername=27983&_origin=article&_zone=art_page&_linktype=scopusauthordocuments&_targeturl=http%3a%2f%2fwww.scopus.com%2fscopus%2finward%2fauthor.url%3fpartnerid%3d10%26rel%3d3.0.0%26sortfield%3dcited%26sortorder%3dasc%26author%3dmr%25c3%25a1zov%25c3%25a1,%2520zuzana%26authorid%3d8321049100%26md5%3de2b3a10de7913304c656591e5d60b40b&_acct=c000062632&_version=1&_userid=4257031&md5=8ecc435694e3950e6efe5339a1e45913 http://www.sciencedirect.com/science/article/pii/s0963996904000183?_alid=1858715101&_rdoc=5&_fmt=high&_origin=search&_docanchor=&_ct=3347&_zone=rslt_list_item&md5=ce8a5a8483854c0b6ddf0b9852a0dce0 http://www.sciencedirect.com/science/article/pii/s0963996904000183?_alid=1858715101&_rdoc=5&_fmt=high&_origin=search&_docanchor=&_ct=3347&_zone=rslt_list_item&md5=ce8a5a8483854c0b6ddf0b9852a0dce0 http://www.sciencedirect.com/science/article/pii/s0963996904000183?_alid=1858715101&_rdoc=5&_fmt=high&_origin=search&_docanchor=&_ct=3347&_zone=rslt_list_item&md5=ce8a5a8483854c0b6ddf0b9852a0dce0 microsoft word 1 primele pagini nr 4 din 2010.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering year ix, no. 4 – 2010 university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international database: indexcopernicus journals master list. also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by editura universităţii ştefan cel mare suceava, romania. microsoft word fia journal no 1_2011_23 martie_27-31.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 31 the analyse of physico-chemical parameters means to appreciate the typicity of some red wines daniela giosanu1, loredana elena vîjan1, ionica deliu1 1university of pitesti, faculty of science, pitesti, romania, e-mail giosanu@yahoo.com abstract. depending on soil, weather characteritics (temperature and precipitations) and varieties, every wine has its own fingerprint. this fingerprint helps to establish the authenticity of wines. at the same time, chemical analyses of wine quality are important in establishing their composition parameters. we intend to set down some characteristics for several varieties of romanian wines produced in the valea calugareasca region. in this paper we present our results for two wines: merlot (2006) and feteasca neagra (2007). to reach this goal, we used different analytical methods: optical methods (refractometry, polarimetry and colourimetry), spectrometer methods (uv-vis), electrochemical methods (potentiometry). the preliminary examination of samples includes colour analysis and microbiological stability. mention should be made that the studied wines are stabilized and did not contain viable yeast cells. using the cie – lab – 76 method we objectively determined the colour of wine: fetească neagră – 2007 is red brick, and merlot – 2006 is intense red. then, for each wine we determined: minimum alcohol content (alcoholmeter titer), relative density, total acidity, contents of methanol, glycerol, acetaldehyde, so2 and anthocyanins. the characteristics obtained from chemical analysis are normal for these wines, but do not present the typicity of the valea calugareasca area. finally, using the data from all the studied wines, we intend to create an analytical database for wines produced in this region. keywords: cie – lab – 76, fingerprint, analytical database, authenticity introduction wine analysis is a laborious and expensive work. as the analytical results should be useful, they must express the real physicochemical parameters of wine composition, based on the establishing of wine quality. the typicity and authenticity of wine are basic attributes that generate the wine quality. the criteria for checking the authenticity of red wines are: the anthocyanins’ spectrum, the fingerprint of amino acids, the wine’s alcohol and water isotopic composition6. the features of variety due to the vineyard and production technology give the wine typicity. in the preliminary analysis of the studied samples, we took into account the colour of wines (objectively determined, using colour space) and their microbiological stability. yeasts are microorganisms with a considerable influence on quality and value of wine1,2. yeasts guide the alcoholic fermentation and contribute to achievement of the typicality of wine savour and fragrance. in the winemaking process, yeasts may lead to the depreciation of wine quality and consequently the decrease of wine value, after alcoholic fermentation. the development conditions determine the useful or harmful nature of wine microorganisms and this is the main problem for the oenologist3. the wine microbiota could contribute to wine quality with the final products of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 32 alcoholic fermentation. the optical method is one of the favorite techniques for the inspection of wine quality, because it is fast, simple and versatile4. to establish the quality of wine, the chemical analysis can provide information about the natural character and the grapes origin only, without the presence of any fraud or impurity. in this paper we studied the characteristics of different red wines from valea calugareasca area, using different analytical methods: optical, spectrometric and electrochemical ones. in fact, our goal was to set down the fingerprint for each of these wines and finally, using all our data about another romanian wines, to get a database and establish their authenticity and eliminate fraud. materials and methods our studies were made on many sorts of wines, from different areas, but in this paper we present just only two of them: merlot (2006) and feteasca neagra (2007) – valea calugareasca area, important vine area for red wine production. we started the wine analysis with an important stage: preliminary examination of samples. this includes colour analysis and microbiological stability. usually, shade and intensity of wine colour are calculated by optical methods5. but cie established that colour could be exactly defined by chromatically parameters: lightness, chromaticity and purity. thus, we used the spectrophotometrical method in tri-stimulus coordinates (cie – lab – 76) for colour analysis as parameter of wine quality. the absorption spectra were measured on a perkin elmer lambda 25 uv-vis spectrophotometer, with double fascicle. basing on transmittance values, we determined the colour coordinates (x, y) for every sort of red wine. the microbiological control was achieved by cultivation of wine decimal dilutions on solid medium ypg (yeast extract peptone glucose) at 28ºc for 24 hours. this step was part of indirect technique for the determination of number of viable cells (cfu/ml), to assess the yeasts’ microbiota of the investigated wines. then, a very large set of chemical analyses were performed. for each wine we determined: minimum alcohol content (alcoholmeter titer), relative density, total acidity, contents of methanol, glycerol, acetaldehyde, so2 and anthocyanins. the alcoholmeter titer, one of the most important parameters of wine quality, was determined by indirect method, (pycnometer method), after prior separation of alcohol from wine by distillation. acidity provides physico-chemical stability of wine, gives colour, brightness and freshness of taste. to characterize acidity of wine the following types of acidity are taken into consideration: total acidity, volatile acidity, fixed acidity and ionic/ real acidity of wine. the determination of total acidity was made by titration with bromothymol blue. the method consists of wine sample titration (acid neutralization) with a solution of sodium hydroxide in the presence of bromothymol blue, after prior removal of carbon dioxide. the evaluation of total acidity in mechiv./liter is the most appropriate, because it is an unitary mode to quantify acids and substances with acid reaction in wine. to determine the fraction of volatile acidity, it is separated from wine by steam stripping of volatile acids in wine. once separated from wine, it is determined from the distillate obtained by titration with an alkaline solution of sodium hydroxide in the presence of phenolphthalein as indicator. sulfur dioxide and sorbic acid, resulting from distillate and not being part of the volatile acidity of wine, are determined separately and they are subtracted from volatile acidity. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 33 the dosage of metatartric acid from wine was made by the precipitation with cadmium acetate. usually, citric acid in wine is determined by spectrophotometer method using acetic anhydride. the method is based on the reaction of citric acid in wine by acetic anhydride in basic medium; a compound with a maximum absorbance at 363 nm is formed. the total remaining unfermented sugars in wine were evaluated by refractometric method, by measuring the percentage of soluble solids or refractive index, after prior removal of alcohol and volatile compounds from wine (which changes the refractive index value). sulphur dioxide is the only antiseptic allowed in wine conservation. the determination of free so2, combined so2 and total so2 is rapidly made by using iodometric oxidation. the anthocyanins are visible phenolic compounds (pigments), which are getting accumulated in grapes and give red colour to wine. they represent 38 % of the total phenolic compounds present in wine. the quantitative determination of anthocyanins is made by visible spectrophotometry and is based on the change of anthocyanins colour depending on ph. we measured the absorbance variation of anthocyanins colour at two ph values, 0.6 and 3.5, and compared with distilled water. the measurements were made at 520 nm, the absorbance of the samples being proportional to the anthocyanins content. the methanol determination is made after its separation from wine (by distillation), by spectrophotometric dosage. the methanol from the wine distillate is oxidized to formaldehyde with potassium permanganate, acidified with phosphoric acid. the aldehyde amount is proportional to the methanol content in wine. then, the formaldehyde is dosed with chromotropyc acid (colour reaction), colour intensity being measured by spectrophotometer at 575 nm. the determination of polyhydric alcohols in wine was made using volumetric method, by oxidation with periodic acid. the method involves the prior defecation of wine, followed by separation of glycerol and butilenglycol by extraction with different solvents. the separated glycerol is cold oxidized by periodate potassium in excess, to formaldehyde and formic acid. then, the excess periodic acid is titrated with sodium thiosulfate. acetaldehyde is the compound that represented 90% of the total of wine aldehydes. its determination was made by volumetric method, acetaldehyde being separated from wine (by distillation) and fixed in a combination aldehydo-sulfur. results and discussion the preliminary analysis shows that the studied wines are stabilized and do not contain viable yeast cells. thus alcoholic fermentation will not continue in the studied wines. for colour analysis we studied the transmittance spectra. these spectra of different sorts of red wine from valea calugareasca area are presented in figure 1. figure 1. the transmittance spectra for feteasca neagra and merlot using the data from these spectra, we calculated the colour coordinates of wine (x, y) and its brightness. based on the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 34 dominant wavelength (λ), we objectively determined the colour of wine (table 1). we noticed that the colour of fetească neagră – 2007 is red brick, while the colour of merlot – 2006 is intense red. the sample of feteasca neagra had a lighter colour due to its faster evolution. to establish the parameters of composition defining wines quality and detect any falsification of them, we carried out some analyses on key components in wine. thus, for each wine we determined: alcoholmeter titer, relative density, total acidity, contents of methanol, glycerol, acetaldehyde, so2 and antocyanins. table 1. the colour coordinates and brightness for red wine (valea calugareasca area) to determine the methanol or citric acid content in wines, we traced the calibration curves shown in figures 2 and 3. the absorbance of wine samples was reported in the calibration curve. the concentration of methanol, (expressed in mg/l of wine diluted with water to 5% vol. alcohol) and the content of citric acid were determined respectively. the characteristics of samples, obtained by chemical analysis, are presented in table 2. the values of the alcoholmeter titer (>10.5% volume) show that the two wines can be placed in the category of superior wines. we have noticed very low volatile acidity and normal values for total acidity and fixed acidity. figure 2. the calibration curve for methanol figure 3. the calibration curve for citric acid the content of citric acid in wine merlot (2006) fits within acceptable limits (up to 1g/l) to correct defficient acidity of wine, while feteasca neagra (2007) samples show an unusual value for a quality wine produced in the valea calugareasca area. the presence of free metatartric acid was not detected in the two red wines from valea calugareasca area, as demonstrated by the absence of any lamellar precipitate in the centrifuge tube. in addition, the values of total sugar are normal in semidry wines whereas the anthocyanins content correlated with the value of the colouring intensity comply with the normal range of red wines. the colour coordinat es x y y (brigh tness), % domin ant λ, nm merlot 2006 0.3331 0.3155 70.86 597 feteasca neagra 2007 0.3069 0.3774 94.30 585 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 35 table 2. chemical characteristics of some red wines (valea calugareasca area) conclusions the cie-lab 76 method facilitates understanding of the correspondence between visual impression of wine colour and numerical expression of chromatic parameters of wines. the chromatic characteristics are normal for wines from this area and these years. the microbiological stability of the studied wines demonstrates a good makewine process. the characteristics obtained from chemical analysis are normal for these wines, but do not present the typicity of the valea calugareasca area. this might be due to the technological process. it is necessary to carry out more experiments, on several kinds of wines from this area, produced in different years, to perform a database that contains the fingerprint for each authentic wine from valea calugareasca. references 1. g. h. fleet, yeast interactions and wine flavor, int. j. food microbiol. 86, 2003,pp.11–22. 2. g. h. fleet, wine. food microbiology: fundamentals and frontiers, 3rd edition, doylemp & beuchat lr eds., asm press, washington, dc, 2007, pp. 863–890. 3. a. popa, d. popa and f. dragomir, microbiologie oenologică. ed. universitaria, craiova, 2007; 4. d. giosanu and i. deliu, the chromatically and microbiological charactersistics of some romanian red wines. annals of food science and technology. 10(2), 2009, p.482-485. 5. i. niskanen, j. mutanen, p. lehtonen, j. räty, k. e. peiponen and t. jaaskelainen, optical sensor for inspection of color, turbidity and refractive index spectra of red wine, oiv'09 the 7th general assembly of the international organisation of vine and wine, june 29 july 3, 2009, zagreb, croatia. 6. c. ţârdea. chimia şi analiza vinului, ed. ion ionescu de la brad, iaşi, 2007, p.32-40. analysis merlot 2006 feteasca neagra 2007 alcoholmeter titter total, % 11 10.5 total acidity, mechiv/liter g/l tartric acid g/l sulphuric acid 60 4.5 2.94 74 5.55 3.63 volatile acidity, mechiv/liter g/l sulphuric acid g/l acid acetic 2.5 0.12 0.15 3.6 0.18 0.22 fixed acidity, mechiv/liter g/l sulphuric acid g/l tartric acid 57.5 2.82 4.31 70.4 3.45 5.28 citric acid, g/l 0.87 1.2 ionic acidity/real ph 3.76 3.75 total so2, mg/l 192 214 total sugar, g/l 10 7.9 antocyanins, mg/l 109 241 glycerol, g/l 12 8.3 methanol, mg/l 25 360 acetaldehyde, mg/l 120 155 microsoft word 14 dabija recorectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 87 aspects concerning coagulation enzymes and different inducing parameters for milk curdling process *adriana dabija1, iuliana sion2 1faculty of food engineering, stefan cel mare university of suceava, romania 2d.s.v.s.a. bacau, romania adriana.dabija@fia.usv.ro *corresponding author received 20 september 2012, accepted 12 december 2012 abstract: cheese making is signing up together all word researcher efforts to find brand new sources of animal protein. one of main sequences in cheese fabrication, the coagulation, could be made even through acidification or biocatalysis. the clotting of milk by enzymes is a key passage in cheese making that, markedly, could affect the characteristics of produced cheese. they differ both on their origin: animal, vegetable, microbial and recombinant from genetically modified microorganism, and their physical state, liquid, powder or paste. the coagulation enzymes used in the cheese industry for milk clotting, being the oldest known application of enzymes in food industry. this paper presents some aspects from a comparative study between different useful types of coagulation enzymes: one of them is from animal origin, that consists pepsine and chymosin and the others two are microbial origin enzymatic prepared, one from bacillus subtilis and the other one from aspergillus niger var.awamori. there was also studied various agents of influence and the raw material used was cow milk and sheep milk. milk coagulation properties could vary greatly among animal species (cattle, buffalo, heep, and goat), among breeds within species, and among individuals. variations occur in coagulation time, extent of curd firming, and development of syneresis. keywords: clotting enzymes, cheese, coagulation activity 1. introduction in the last decade, the fraction of total milk destined to cheese production has increased by about 10% in the european union, and it is now slightly higher than 50% in the former continent and slightly lower in the latter. an increase in the amount of milk used to manufacture cheese has been reported in other european countries, oceania, and latin america, and the amount used is much lower in asia and africa. milk coagulation properties vary greatly among ruminant species, among breeds within species, and among individuals. variations occur in coagulation time, extent of curd firming, and development of syneresis [1,2]. milk coagulation properties receive much attention in dairy science and industry, mainly because the amount of milk used to manufacture cheese is growing worldwide, and several works have confirmed the importance of milk coagulation properties in terms of cheese processing, yield, and quality [1,3,4]. milk proteins and their genetic variants strongly affect milk coagulation properties [1,3]. nowdays, milk clotting properties receive a lot of attention, not only in dairy science but also in other fields of industry, mainly because the amount of milk used in cheese making processes, and also from the point of view of cheese processing, yield, and quality [5]. the coagulation represents one of the capital steps in cheese making that, markedly, could affect the characteristics of produced cheese. there are known two modalities of obtaining: by means of acids, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 88 resulting in soft curd, with low calcium content and high acidity (isoelectrodeposition); by means of coagulation enzymes, so called biocatalysis. generally, for main types of cheese, it is known mixing types of coagulation. enzymatic coagulation could uses clotting enzymes animal, vegetable or microbial origin by different physical state, liquid, powder or paste (table 1). is is very important knowing the nature of coagulation enzymes, that could have a distinct influence through cheese processing. the animal and microbial enzymes are actually, the most spent. the main influence parameters for curdling process are presented forward: the temperature, optimal in the range 40 41° c and 25-42 ° c in practice; the quantity of calcium salts, that may influence the coagulation time and also the quality of curdle; time of coagulation growth with calcium salts decline and also it would have a flossy structure; the level of milk acidity the coagulation rate raises up in the same time with ponderate augmenting of acidity; quantity of coagulation enzyme determines the rate of the process, in certain limits; milk chemical composition, a high dry matter content, respectively, reffers by enzyme quantity augmentation in order to maintain a certain period of time for coagulation and a normal consistency for the curd; the preliminary heating treatment for milk could extend the time of coagulation; milk homogenization could decrease the coagulation time, because of aggregation degree casein augmentation. [6,7] table 1 sources for coagulation enzymes [2, 6, 7, 8, 10] origin enzymes animal ruminant species calf billy goat lamb adult cattle single gastric species pig birds chicken rennine + pepsine 88-94% 6-12% pepsine + rennine 90-94% 6-10% pepsine pepsine vegetable fig (juice) pineapple (stem) artichoke carica papaya pumpkin, watermelon calotropis procera cynara cardunculus flowers ficine bromelline papaine callotropine moulds rhizomucor miehei rhizomucor pusillus cryphonectria parasitica aspergillus niger v. awamori thermomucur protease protease protease „genetic”rennine yeasts kluyveromyces marxianus v. lactis „genetic”rennine bacteria escherichia coli bacillus subtilis „genetic”rennine food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 89 in this study were used three different origin types of coagulation enzymes: one of animal extraction (consist of pepsine and rennine); two microbial type curd: o enzyme prepared from bacillus subtiliss biomass (rennine); o enzyme prepared from aspergillus niger var. awamori (rennine); the most used clot derives from the stomach of unweaned calves. it is available as liquid or powder form. in many countries, where sheep and goat breeding is largely diffused, is largely common the use of lamb or kid rennet paste [6]. the active principle consists of chymosin 8894% and pepsin 6-12% [7]. the specific milk-curdling enzyme present in animal clot is the chymosin (ec 3.4.23.4), that is an acid protease enzyme. apart from this, a different number of generic proteases, such as pepsin a (ec 3.4.23.1), gastricsin (or pepsin b, or pepsin c) (ec 3.4.23.3) are also present. milk-clotting enzymes, other than rennet, are called coagulants and are represented by fermentation produced chymosin, which is 100% calf chymosin produced by recombinant dna technology involving aspergillus niger, kluyveromyces lactis or escherichia coli and by different microbial coagulants, especially the ones from rhizomucor miehei, rhizomucor pusillus and cryphonectria parasitica, thermomucor [8]. the most important difference between calf rennet and lamb or kid rennet prepared is the presence of lipolytic enzymes that are denatured during the activation process of chymosin and pepsin zymogens. there are few aspects with significant influence through clotting activity, as forward: small quantity of enzyme; the presence of sunlight or artificial light; intense shaking of solution and foam presence; overheating, 60°c; ph = 6.6-7.4. microbial origin rennet, generally, could have intensified proteolytic activity, relative with animal origin rennet, that have bring forward cheese making. they have also revealed higher thermal stability, beside animal or vegetable origin enzyme prepared, but sensorial qualities are constantly under the performances achieved by animal origin enzymes [9, 10]. there are requested some general aspects concerning purity, antibiotic residues absence, inocuity and also a higher level for coagulation activity, for enzymatic prepared. the coagulation capacity is a ratio between enzymatic prepared volume and milk volume in the time of coagulation, in standard conditions [7]. the experimental study consist of comparison between three types of clotting enzymes for cheese making and also the aspects with significant influence to the coagulation process. 2. experimental we have studied three rennet type, delivered from a local milk producer: p1 – microbial origin coagulation enzymes, from bacillus subtilis biomass, that contains chymosine; p2 – animal origin rennet (lamb abomasums), that contain chymosine; p3 – clotting enzymes from submerged fermentation of a vegetable substrate with aspergillius niger var. awamori. the coagulation process has been studied upon two milk samples, their characteristics being related in table 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 90 table 2 physico-chemical parameters for milk samples physical-chemical characteristics cow-milk (l1) sheep-milk (l2) fat content, % 4.02 8.01 protein, % 3.28 4.85 acidity, °t 17 22 ph 6.6 6.2 the physical-chemical characteristics for two milk samples were achieved both by lactoscope method and verified by classical methods (gerber, kjeldahl, titration). the coagulation activity was determined by soxhlet method for all three types of rennet and then the influence of rennet quantity and the coagulation temperature upon the process were studied. 3. results and discussions 3.1. the method for coagulation capacity determination the definition for coagulation activity is the milk volume that can be clotted by an enzyme solution volume, at 35°c, in 40 minutes (2400 seconds). the final moment can be appreciated visually. we have taken 1 g from each type of rennet and then we’re putting in 100 cm3 calibrated flask. in 250 cm3 erlenmeyer we have introduced 100 cm3 milk, heated at 35°c, and then was added rapidly and vigorously shaking 1 cm3 rennet solution 1%. the time between the moment of rennet adding and the flocks’ apparition was measured by chronometer. the coagulation power for rennet powder was determined by forward formula: p = tm v   24001 (1) where: p – unknown coagulation power for rennet, unities per gram; v1 – milk quantity tacked in the study, in cm3; 2400 – theoretically time for coagulation, in seconds; m – rennet quantity for determination, in grams (0,01g for powder rennet); t – coagulation time, in seconds. the results for coagulation power determination by soxhlet method are synthesized in table 3. table 3 coagulation activity for clotting enzyme prepared used in the experiment coagulation activity[cm3/g], clot type cow milk [l1] sheep milk [l2] p1 115942 120852 p2 45282 58624 p3 126316 138434 from table 3 it can be observed that the higher coagulation capacity is, in the case of cow-milk, for enzymatic prepared from aspergillius niger var. awamori (p3) as 2.79 times higher than animal origin rennet (p2), and 1.09 times higher than enzymatic rennet from bacillus subtilis (p1). the results for sheep milk are the same: coagulation power for p3 is 2.36 times higher than for p2, respective 1.15 times higher than p1(bacillus subtilis). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 91 the differences have been kept significantly higher for all three types of enzymes, with milk assortments, the advantage being developed for sheep milk. 3.2. the influence of rennet quantity about time coagulation in the same quantity of milk (50 cm3), with constant temperature 35°c, we have introduced various quantities of rennet solution 1% and then the time was measured between the moment for adding rennet solution and the time for coagulation starting. in table 4 are presented the results for two samples of milk. table 4 the influence of rennet quantity about time coagulation coagulation time [min.] p1 p2 p3 nr. crt. rennet solution, 1 % [cm3] l1 l2 l1 l2 l1 l2 1 1 120 115 215 188 109 97 2 2 70 59 130 120 65 49 3 3 66 46 98 81 37 29 4 4 48 35 80 65 32 26 5 5 37 28 72 50 28 23 the conclusion is that, time for coagulation decrease with rennet quantity augmentation, if it is maintained the same temperature for all three types of rennet. 3.3. the temperature influence on milk coagulation the coagulation process it takes a higher or lower duration time, for the same conditions of acidity, concentration and percent of rennet solution depend on milk temperature for the coagulation. we have taken 5 samples of milk of 50 cm3 that have been heated to: 25°c, 30°c, 35°c, 40°c and 45°c. in each of them it has been added 5 cm3 rennet solution 1% and then has been kept in water bath, for constant temperature. and then we have measured the duration time between the moment of adding rennet solution and the beginning of coagulation. table 5 consists of results for the temperature influence about coagulation time. table .5 the temperature influence on coagulation coagulation time [min.] p1 p2 p3 nr. crt. temperature [°c] l1 l2 l1 l2 l1 l2 1 25 124 110 270 248 86 72 2 30 118 98 237 210 51 44 3 35 37 32 72 58 28 22 4 40 45 40 87 63 27 20 5 45 57 48 128 64 28 25 the results for two samples of milk: cow-milk and sheep-milk and for three types of rennet from various origin extraction reveals that we food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 92 4. conclusions cheese making requires milk coagulation and development of syneresis. it can be achieved by acidification and/or enzymatic way. the conclusions from this study can be synthesized as: coagulation power depends on rennet type, so that microbial origin rennet is more powerful than one of animal origin; the most important aspects for coagulation process are temperature of the milk and the rennet quantity; as the rennet quantity increase as long the time for coagulation decrease; if we didn’t respect the optimal temperature with rennet type, it could vary the starting moment for coagulation process; it has been determined the coagulation capacity for three kinds of coagulation enzymes and also the influence of rennet quantity and coagulation temperature about milk coagulation. the coagulation power depends with rennet origin (extraction): microbial enzymatic preparat (a.niger) >microbial enzymatic prepared (b.subtilis)> animal origin chymosin. 5. references [1]. bittante, g., penasa, m., cecchinato, a., genetic and modelling of milk coagulation properties, journal of dairy science, vol.95, no.12, 6843-6870 (2012) [2]. adetungi, v.o., et al., production of wara, a west african cheese using lemon juice as a coagulant, lwt 41, 331-337 (2008) [3]. bittante, g., modelling rennet coagulation time and curd firmness of milk, journal of dairy science, vol.94, no.12, 5821-5832 (2011) [4]. leitner, g., merin, u., silanikove, n., effects of glandular bacterial infection and stage of lactation on milk clotting parameters: comparison among cows, goats and sheep, international dairy journal, 21, 279-285 (2011) [5]. calvo, m.v., et al., effect of a hygienized rennet paste and a defined strain starter on proteolysis, texture and sensory properties of semi-hard goat cheese, food chemistry, 102, 917-924 (2007) [6]. addis, m., piredda, g., piresi, a., the use of lamb rennet paste in traditional sheep milk cheese production, small ruminant research, 79, 2-10 (2008) [7]. costin g.m., cheese making science and engineering , editura academica, galati (2003) [8]. merheb-dini, c., use of a new milk-clotting protease from thermomucor indicae-seudaticae n31 as coagulant and changes during ripening of prato cheese, food chemistry, 130, 859-865 (2012) [9]. bittante, g., cantiero, b., cecchinato, a., prolonged observation and modelling of milk coagulation, curd firming, and syneresis, international dairy journal, 12 (2012) [10]. pino, a., et al., proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet, food research international, 42, 324-330 (2009) 162 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 162 170 influence of temperature on the physical properties of vegetable oils *mircea oroian1, gheorge gutt1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 15th february 2015, accepted 9th april 2015 abstract. the aim of this study is to investigate the influence of temperature (20 80°c) on the physical properties of vegetable oils and to establish some correlations between them. in this study were studied eight types of vegetable oils as: olive oil, corn oil, sunflower oil, grape seeds oil, squash oil, hazelnut oil, peanut oil and sesame oil. the temperature influences negatively almost all the physical parameters measured (exception isentropic compressibility and rao´s molar sound function). a master curve of the correlation between the experimental data of oil samples density, surface tension and ultrasound velocity was achieved (r2 = 0.996). the correlation between surface tension and viscosity was made using pelofsky, modified pelofsky, exponential and polynomial models, the second one fits better the experimental data. keywords: density, oils, surface tension, temperature, ultrasonic parameters, viscosity 1. introduction the physical properties of fluid are different in nature but whose values need to be known for a wide variety of industrial and physicochemical processes. surface tension affects important stages in such production processes as catalysis, adsorption, distillation and extraction. viscosity is important in processes involving a flow of fluids extensively [1]. ultrasonic velocity is usually correlated with density fluctuations of pure liquid; thereby it is closely related to compressibility, which is a response function of the density to the mechanical pressure [2]. one of the models used for the correlation of surface tension was proposed by pelofsky [3]. the pelofsky model can be applied for the organic and inorganic phases of pure and mixed components. the model involves two adjustable coefficients, whose values may depend in the temperature range being considered [1]. the influence of the temperature on ultrasonic velocity was studied [4], however the correlation between different physical properties of vegetable oils have not been reported to the authors knowledge. the aim of this study is to study the influence of temperature on the physical properties of vegetable oils (density, viscosity, ultrasonic parameters and surface tension) and to establish some correlations equations between them. 2. experimental materials for this study were analyzed eight different types of vegetable oils as: olive oil, corn oil, http://www.fia.usv.ro/fiajournal mailto:m.oroian@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 163 sunflower oil, grape seeds oil, squash oil, hazelnut oil, peanut oil and sesame oil. they were purchased from the local market of romania. density measurements density (ρ) of the oils samples was measured using pycnometer with an accuracy 10−4 gm/cc. the calibration of pycnometer was made with ultrapure water. temperature was kept constant within ±0.1 °c using pid controller and circulating water using thermo static-fluid bath. the density of the eight samples of oils was measured at 20, 30, 40, 50, 60, 70 and 80 °c. the values of parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95 %. ultrasonic velocity measurement the ultrasonic speed measurement was carried out using a flow detector usm 35 x (ge measurement and control, usa) with a dual-element (tr) probe working at 4 mhz. the measurements were carried out at 20, 30, 40, 50, 60, 70 and 80 °c. the values of parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95 %. viscosity measurement viscosity measurements were carried out on the oil samples at different temperatures (20, 30, 40, 50, 60, 70 and 80 °c), with hoppler viscometer using glass ball, with temperature controlled water bath. the oil sample was allowed to reach the desired temperature 20 min. each measurement was taken in duplicate. the values of parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95 %. surface tension determination the surface tension was computed using the auerbach´s equation [5]: (1) where u is the ultrasonic velocity (m/s), σ is the surface tension in n/m and ρ is the density in kg/m3. therefore, for the calculation of the surface tension, first we measured the ultrasonic velocity and density and later on by using the auerbach equation we were able to compute the surface tension. prediction accuracy the mean relative deviation modulus, d, was used to verify the suitability of model for experimental data: (2) where n is the total number of data in the number of data in the sample. subscript exp. and cal. denote experimental and calculated values, respectively. x represents density, ρ, or viscosity, η, surface tension, σ, and ultrasonic velocity, u. statistical analysis the parameters were fitted to the proposed equation using spss trial version and excel 2007. 3. results and discusion effect of temperature on density the measured densities of oils samples from 20 – 80 °c in function of temperature are presented in figure 1. the density of oils samples was smaller than water density in all the samples; it ranged (20 80 °c) between 866.9533 – 925.1260 kg/m3. the density is slightly influenced by temperature; the values of the oils density decreased with the increasing of temperature. the evolution of density with temperature shows a small change of the density with the change of the temperature of the solution. the evolution of the density with temperature was subjected to linear regression to see its prediction using the eq. 3: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 164 (3) where ρ – density (kg/m3), a and b are constants, and t – temperature (°c). fig. 1. density of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the prediction of the equation 3 is in good agreement with the experimental values. all the oils’ densities were good predicted using this equation, all the regression coefficients ranged between 0.991-0.999. the values of a ranged between 919.8 and 938.5, while b ranged between -0.662 and -0.676 respectively. effect of temperature on viscosity the viscosity measurements were made at 20 – 80 °c. in order to obtain the fitting results, the viscosity data were plotted into two different ways: viscosity, η, versus temperature, t. the plot is presented in figure 2. viscosity is a temperature function, it decreases with the increasing of temperature. correlations were made to allow the prediction of viscosity of the oils samples. the correlations of viscosity were as a function of temperature using polynomial fitting by means of the experimental data. the following expression was used for the regression equations of the experimental data: (4) where η is the viscosity in pa·s, t is the temperature in °c, and e, f, g are fitting parameters. fig. 2. viscosity of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the regression coefficients ranged between 0.959 and 0.987. the parameters values of equation 5 ranged between: e 29.803 and 116.646, f -5.391 and -1.317 and g 0.015 and 0.062, respectively. the parameters values of equation 6 ranged between: h 315.9 and 19778, i -500.7 and -8.131 and j 0.052-3.170, respectively. the mean relative deviation modulus between the prediction and experimental values and average ranged between 1.161 and 5.514%. effect of temperature on ultrasonic parameters the ultrasonic velocity ranged between 13011489 m/s. in order to obtain the ultrasonic velocity data of oils samples at any temperatures, the experimental data were expressed in a form of ultrasonic velocity, u, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 165 versus temperature, t. these results are presented in figure 3. it can be observed that the ultrasonic velocity is linear with regard to temperature. the ultrasonic velocities of the oil samples decreased linearly as the temperature increased. fig. 3. ultrasonic velocity of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the ultrasonic velocities evolution with temperature shows a small change of the ultrasonic velocity with the change of the temperature. the evolution of the ultrasonic velocity with temperature was subjected to linear regression to see its prediction using the eq. 5: (5) where u – ultrasonic velocity (m/s), f and g are constants, and t – temperature (°c). the prediction of the equation 5 is in good agreement with the experimental values. all the oil densities were good predicted using the equation, all the regression coefficients ranged between 0.996-0.998. the mean relative deviation modulus between the prediction and experimental values and average ranged between 0.014 and 0.031%. isentropic compressibility (ks) has been calculated from the equation of newton laplace: (6) where ρ – density (kg/m3) and u ultrasonic velocity in oils. the isentropic compressibility evolution of oil samples is presented in figure 4. the isentropic compressibility is increasing with the temperature. the evolution of the ks with temperature was fitted using a polynomial model (eq. 7): (7) where ks – isentropic compressibility (pa -1), h, i, and j are constants, and t – temperature (°c). fig. 4. isentropic compressibility (ks) of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the prediction of the equation 15 is in good agreement with the experimental values. all the oils’ isentropic compressibility were good predicted using this equation, all the regression coefficients were 0.999. the parameters of equation 15 are presented in table 1. the intermolecular free length (lf) is determined using the jacobson’s relation: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 166 (8) where k is the temperature jacobson’s constant given by the relation k = (90.875+0.375)·10-8. the intemolecular free lenght of oil evolution with temperature is presented in figure 5. table 1 correlation coefficients between isentropic compressibility and temperature h i j r2 d% corn oil 7.14·10-15 1.85·10-12 4.72·10-10 0.999 0.010 sunflower oil 6.90·10-15 1.83·10-12 4.63·10-10 0.999 0.009 squash oil 5.59·10-15 1.90·10-12 4.60·10-10 0.999 0.013 grape seed oil 8.92·10-15 1.53·10-12 4.53·10-10 0.999 0.012 sesame oil 8.33·10-15 1.76·10-12 4.80·10-10 0.999 0.015 olive oil 4.68·10-10 1.20·10-12 1.22·10-14 0.999 0.016 ground nut oil 1.26·10-14 1.26·10-12 4.74·10-10 0.999 0.014 peanut oil 6.07·10-15 1.91·10-12 4.61·10-10 0.999 0.010 the experimental data were fitted to the equation 9: (9) where lf is the intermolecular free length of oils (m), k, l and m are fitting parameters, and t is the temperature (ºc). the regression fitting parameters and regression coefficients are presented in table 2. the regression coefficients are closed to 1 for all the oil samples. fig. 5. intermolecular free length of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the specific impedance (z) is given by the formula: (10) in figure 6 is presented the specific impedance of oils in function of temperature. the parameters is sensitive with the increasing of the temperature. fig. 6. the specific impedance (z) of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil it appears that the evolution of the impedance can be fitted to an linear model: (11) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 167 where z is the specific impedance (kg/m2/s), n and o fitting parameters, t – is the temperature (ºc). the regression coefficients are closed to 1 (table 3), while the relative deviation modulus between the prediction and experimental values and average ranged between 0.564 – 1.821%. table 2 correlation coefficients between intermolecular free length and temperature oil k l m r2 d% corn oil 3.57· 10-16 1.15·10-13 2.05·10-11 0.999 0.010 sunflower oil 2.97· 10-16 1.18·10-13 2.03·10-11 0.999 0.009 squash oil 2.97· 10-16 1.18·10-13 2.02·10-11 0.999 0.012 grape seed oil 4.04· 10-16 1.06·10-13 2.01·10-11 0.999 0.008 sesame oil 3.45· 10-16 1.17·10-13 2.06·10-11 0.999 0.013 olive oil 3.92· 10-16 1.07·10-13 2.03·10-11 0.999 0.011 ground nut oil 4.16· 10-16 1.07·10-13 2.04·10-11 0.999 0.012 peanut oil 2.26· 10-16 1.26·10-13 2.00·10-11 0.999 0.008 table 3 correlation coefficients between specific impedance and temperature n o r2 d% corn oil 1·106 -3193 0,999 1.012 sunflower oil 1·106 -3213 0,999 1.556 squash oil 1·106 -3164 0,999 0.978 grape seed oil 1·106 -3275 0,999 1.245 sesame oil 1·106 -3136 0,999 1.782 olive oil 1·106 -3191 0,999 1.821 ground nut oil 1·106 -3197 0,999 0.564 peanut oil 1·106 -3190 0,999 1.249 rao’s molar sound function (r) [6] was calculated with the expression: (12) fig. 7. rao’s molar sound function of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the evolution of rao´s molar sound is presented in figure 7. the parameter is temperature dependent, is increasing with the increasing of the temperature. the experimental data were fitted to the equation13: (13) where r – rao´s molar sound function of oils (m3/mol), p, r and s are fitting parameters and t is the temperature (ºc). in table 4 are presented the parameters of equation 13. the regression coefficients ranged between 0.985 and 0.994, while the relative deviation modulus between the prediction and experimental values and average ranged between 0.012 – 0.078 %. effect of temperature on surface tension. the oil’s surface tension (computed using the auerbach relation) ranged between 0.0259 – 0.0334 n/m. a linear evolution of the surface tension evolution with temperature was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 168 observed (fig. 7). the evolution of the surface tension with temperature was subjected to linear regression to see its prediction using the eq. 14: (14) where σ – surface tension in n/m, x and y are constants, and t – temperature (°c). table 4 correlation coefficients between rao´s molar sound function and temperature oil p r s r2 d% corn oil -1.151·10-8 2.881·10-6 0.012 0.994 0.012 sunflower oil -6.781·10-9 2.422·10-6 0.012 0.985 0.056 squash oil -1.311·10-8 3.249·10-6 0.012 0.995 0.078 grape seed oil -1.388·10-8 3.149·10-6 0.012 0.989 0.045 sesame oil -9.536·10-9 2.781·10-6 0.012 0.985 0.082 olive oil -2.382·10-8 4.295·10-6 0.012 0.994 0.021 ground nut oil -2.038·10-8 3.906·10-6 0.012 0.993 0.064 peanut oil -9.614·10-9 2.829·10-6 0.013 0.990 0.049 figure 8 shows that from the relation between surface tension of oil samples and temperature. the relation between the surface tensions of the honey [2] and potassium citrate and its concentration has a similar evolution [7]. all regression coefficients were higher than 0.998. the values of x ranged between 0.034 and 0.036, while the p was 0.001 for all the oil samples. the mean relative deviation modulus between the prediction and experimental values and average ranged between 0.016 and 0.032%. fig. 8. surface tension of vegetable oils in function of temperature: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil correlations between density, ultrasonic velocity and surface tension singh & singh [8] proposed a linear relation between the ultrasonic velocity, density and surface tension as: (15) where u – ultrasonic velocity (m/s), σ – surface tension (n/m), ρ density (kg/m3), ψ and ξ constants. fig. 9. correlation between ultrasonic velocity, density and surface tension of vegetable oils: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil the experimental data of oils (density, viscosity and surface tension) have been subjected to linear regression to assess the applicability of the equation and to compute food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 169 the two constants. the ultrasonic velocity ( ) density and surface tension ( for all the oil samples analyzed are plotted in figure 9; it can be observed that the evolution of parameters is a linear one (the regression coefficients is r2 = 0.996), ψ = 1.500 and ξ = -9.198. the equation is a perfect tool for predicting the correlation of the three parameters irrespective of the oil origin. correlation between surface tension and viscosity the surface tension and the viscosity are two important properties from the thermophysical point of view for bulk, binary and ternary mixtures of polar and non polar liquids. however, it is interesting to correlate two important thermophysical properties (surface tension and viscosity) of oils so that we can enhance our knowledge about functional dependence of thermophysical properties. for the correlation between surface tension and viscosity we checked the fitness of the experimental values with 4 models: pelofsky model, a modified pelofsky model, an exponential model and a polynomial model. pelofsky [3] proposed a linear relationship between surface tension and viscosity as: (16) where σ – surface tension (n/m), lna and b are fitting coefficients and η – viscosity (pa·s). table 5 correlation coefficients between surface tension and viscosity according pelofsky model oil lna b r2 d% corn oil -3.390 -0.001 0.972 0.176 sunflower oil -3.386 0.001 0.984 0.105 squash oil -3.397 0.001 0.962 0.101 grape seed oil -3.367 0.001 0.971 0.070 sesame oil -3.423 0.001 0.966 0.074 olive oil -3.388 0.001 0.965 0.089 ground nut oil -3.401 0.001 0.972 0.056 peanut oil -3.406 0.001 0.953 0.094 in table 5 are presented the fitting coefficients, regression coefficients and the mean relative deviation modulus. the regression coefficients ranged between 0.953 and 0.984, while the mean relative deviation modulus ranged between 0.056 and 0.176%. we propose that the surface tension and viscosity of oil samples can be fitted using an modified pelofsky model as it is presented in equation 17: (17) where ln c and d are substance dependent constants, independent of the thermodynamic state of the system, and is the universal exponent. the exponent = 0.30 is used in eq. 17 to fit the experimental viscosity and surface tension data of oil samples. the characteristic exponent conveniently linearises the plots of ln σ versus oil viscosity, as well as providing the best fit with high accuracy. figure 10 presents the correlation between the ln σ and η-0.3, it can be observed the linearised form of the correlation between the two parameters. fig. 10. correlation between surface tension and viscositymodified pelofsky model: corn oil, sunflower oil, squash oil, grape seed oil, sesame oil, olive oil, ground nut oil, peanut oil food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 mircea oroian, gheorge gutt, influence of temperature on the physical properties of vegetable oils, food and environment safety, volume xiv, issue 2 – 2015, pag. 162 – 170 170 table 6 presents the parameters of the equations 17; it can be observed that the regression coefficients (r2) are close to 1. table 6 correlation coefficients between surface tension and viscosity according to modified pelofsky model lnc d r2 d% corn oil -3.099 -0.102 0.985 0.166 sunflower oil -3.103 -0.096 0.996 0.099 squash oil -3.132 -0.087 0.997 0.095 grape seed oil -3.093 -0.091 0.999 0.066 sesame oil -3.158 -0.088 0.998 0.070 olive oil -3.124 -0.094 0.998 0.084 ground nut oil -3.136 -0.093 0.999 0.053 peanut oil -3.145 -0.091 0.997 0.089 4. conclusions the influence of the temperature upon the physical properties of vegetable oils has been achieved at 20 – 80 ºc. the temperature decreased almost all the physical properties of vegetable oils (exception isentropic compressibility and rao´s molar sound function). a very good regression coefficients (r2 = 0.996) was observed between the density, ultrasonic velocity and surface tension. the modified pelofsky model predict better than exponential, polynomial and pelofsky models the correlation between surface tension and viscosity. references [1] li, x., tian, j., mulero, a., empirical correlation of the surface tension versus the viscosity for saturated normal liquids, fluid phase equilibria, 352: 54-63, (2013) [2] oroian, m., measurement, prediction and correlation of density, viscosity, surface tension and ultrasonic velocity of different honey types at different temperatures, journal of food engineering, 119: 167172, (2013) [3] pelofsky, a.h., surface tension-viscosity relation for liquid. journal of chemical & engineering data, 11(3): 394–397, (1966). [4] sidek, h.a.a., chow, s.p., shaari, a.h., senin, h.b., ultrasonic studies of palm oil and other vegetables oils, elais, 8(1): 37-44, (1996). [5] auerbach, n., oberflächenspannung und schallgeschwindigkeit. experientia, 4: 473–474, (1948). [6] vigaureux, p., ultrasonics. london: chapman & hall [chapter vi], (1952). [7] lu, j.-g., hua, a.-c., xu, z.-w., fan, f., cheng, l., lin, f., measurement and prediction of densities, viscosities and surface tension for aqueous solutions of potassium citrate. fluid phase equilibria, 327: 9–13, (2012). [8] singh, m.p., singh, r.k., correlation between ultrasonic velocity, surface tension, density and viscosity of ionic liquids. fluid phase equilibria, 304: 1–6, (2011). microsoft word 1 primele pagini nr 3 din 2010.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering year ix, no. 3 – 2010 university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international database: indexcopernicus journals master list. also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by editura universităţii ştefan cel mare suceava, romania. звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 95 effect o f s tor ag e on asco rbic ac id c on tent of s ome fr u it j u ic es nicolae carpiuc1, ana leahu2, ecaterina curaleţ3, *cristina damian4 1-4ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, românia, e-mail: 4cristinadamian@fia.usv.ro *corresponding author received 15 july 2011, accepted 2 october 2011 abstract: the effect of storage on ascorbic acid content of some fruit juices was evaluated. the preparation of fruit juices, the applied technologies, seeks to extract and preserve in them the most valuable substances in fruit. this will provide food value and organoleptic characteristics, as they were in the raw fruit. therefore, fruit juices and fruits are called liquids. fruit juices and fresh fruits were selected randomly from supermarkets, selected by smell, shape and color. fruits were selected at different stages of maturity, and fruit juices to different periods of validity. fresh fruits were squeezed by hand with a mini plastic juicer. the laboratory method chosen is the dosage of ascorbic acid with 2.6 diclorfenolindofenol. once opened conventional juices lose potential vitamin. compared to bottled natural juices, natural juice, freshly squeezed, has the advantage that it contains no preservatives. as long as the juice will be consumed fresh, it will keep its properties and nutrients. keywords: storage, fruit juices, ascorbic acid 1. introduction fruit and vegetables contain many antioxidant compounds, including phenolic compounds, carotenoids, anthocyanins and tocopherols. fruit peels are especially rich in polyphenolic compounds, flavonoids, ascorbic acid and many other biologically active components having positive influences on health. [1] the antioxidant activity of fruit and vegetables is assumed to be of greatest importance in combating a number of degenerative diseases. when the level of reactive oxygen species (ros) exceeds the antioxidant capacity of the cell, the intracellular redox homeostasis is altered, and the resulting oxidative stress may destroy all major classes of biomolecules in the vicinity of their source, including lipids, protein and dna, with concomitant changes in structure and function. [2] fruits have a short life, due in part to a high respiration rate and a rapid ripening process. to extend the shelf life of fruits different preservation methods have been developed including canning, freezing, drying and packing in controlled atmospheres. however, the processing can change the concentration of nutrients. any loss of nutrients in fruits and vegetables will depend on the type, temperature and time of processing and storage conditions. the concentration of vitamin c can be reduced with various percentages, depending on the type of processing of raw material containing this vitamin. [3] the inefficient blanching causes some loss of vitamin c by oxidation, as well as by leaching. the storage of vegetables for a mailto:4cristinadamian@fia.usv.ro 96 year at a temperature around -100c can lead to vitamin c loss of 80-90%. [4] the addition of bicarbonate, used to soften some vegetables, contribute to diminution of vitamin c concentration. the addition of anthocyans, sugar and even starch seems to have a protecting action on vitamin c. [5] retention of ascorbic acid is better in rapid drying at high temperatures than in slower drying at lower temperatures. drying methods that expose the food to air result in losses of vitamin c due to oxidation. the freeze drying, which is carried out in the absence of oxygen, does not cause loss of vitamin c. [6] as to microwave heating, the ascorbic acid content is higher in vegetables cooked by microwave heating than by conventional methods. [7] some preservation methods are also believed to be responsible for depleting antioxidants naturally occurring in food, with a subsequent decrease in the lower health protecting capacity of fresh foods. juice is the liquid that is naturally contained in fruit or vegetables tissues. [8] the aim of this study was to evaluate the effect of storage on ascorbic acid content of fruit juices. 2. materials and methods the preparation of fruit juices, the applied technologies, seeks to extract and preserve in them the most valuable substances in fruit. this will provide food value and organoleptic characteristics, as were the fruit raw. therefore, fruit juices and fruits are called liquids. fruit juices and fresh fruits were selected randomly from supermarkets, selected by smell, shape and color. fruits were selected at different stages of maturity, and fruit juices to different periods of validity. fresh fruits were squeezed by hand with a mini plastic juicer. the laboratory method is chosen by 2,6-diclorfenolindofenol ascorbic acid dosage. natural can not be associated with any processed product obtained from a natural product on which there was an intervention in various physical or chemical activities. so, no juice, even the one freshly squeezed, can be called natural 100%, as long as the fruit has occurred (physically) by squeezing. 3. results and discussion six juices were selected on the market, whose values of ph are listed in ‘table 1’. the juices are natural (samples 1, 2), conventional (samples 3, 4) and organic (samples 5, 6). table 1 ph values of juice samples no sample ph 1 freshly squeezed orange juice 4 2 freshly squeezed grapefruit juice 3 3 orange juice tymbark 4.5 4 grapefruit juice tymbark 3.5 5 orange juice biotta 4 6 blackcurrant juice biotta 3.5 freshly squeezed grapefruit juice is the acid juice of the review, followed by grapefruit juice brand tymbark conventional, organic blackcurrant juice then biotta. grapefruit juice is more acidic than the orange or the kiwi, whether the juice is natural, organic or conventional. freshly squeezed juice can be associated with any processed product, it was obtained from a natural product on which there was an intervention in various physical or chemical activities. conventional juices have the following characteristics, which are mandatory under standard: appearance of homogeneous liquid, clear to opalescent, without suspended sediment or impurities in the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 97 raw materials used, color, smooth texture, fragrant, aromatic, characteristic odor of fermented fruit, the mold, pleasant taste, sweet or sweet-sour fruit characteristic, without taste. 14 4244 14 36 32 13 33 29 0 10 20 30 40 50 mg a.a./100g day 1 day 5 day 7 prigat kiwi orangetymbark grapefruittymbark figure 1. the evolution of ascorbic acid in conventional nectar the data presented in figure 1 indicates that the juice prigat kiwi has a constant content of vitamin c, the largest differences being the orange juice and grapefruit juice tymbark. freshly squeezed orange juice is made from 100% fruit juice (100% juice) by squeezing them. the juice is not subject to dehydration (not remove water from fruit juice) and is always kept cold. the shelf life is very short the juice should be consumed in 10 days of production. figure 2. ascorbic acid in some freshly squeezed juices figure 2 shows the vitamin c content of freshly squeezed juices, orange, grapefruit and kiwi. kiwi juice contains only 22.176% ascorbic acid, compared with grapefruit juice containing 32.4%. grapefruit juice has a very high food value, characterized by: good taste, fruity and refreshing, increased sugar content directly assimilated and organic acids, significant proportions of minerals, enzymes and vitamins, especially ascorbic acid. biotta organic orange juice is rich in vitamin c. biotta produces this 100% natural orange juice, fresh, preserving the original flavor and taste. very important to maintain a high nutritional value is the quality of processing methods and retention of natural purity and orange juice is biotta quality and taste of freshly pressed orange juice. biotta blackcurrant juice is made from organic and fresh blackcurrant. black currants are rich in vitamin c and flavor of this juice is enhanced with an infusion of rose petals tea. biotta uses light pasteurization methods, thus keeping large amounts of vital nutrients. the juice is then heated and cooled quickly; the process is repeated as many times as possible. oxygen is removed thus preventing oxidation and loss of vitamins. an important role for maintaining the content of vitamin c is tightly closed glass container with 100% waterproof against air. glass protects best biotta juicestaking unwanted elements (eg oxygen) on the outside, thereby achieving a high retention of vitamins. another enemy of vitamin c is the uv rays that can destroy vitamins, but the products are packed in cartons biotta closed for transport and storage. organic juices are consumed liquid to quench thirst and also have to produce a cooling effect to combat the sensation of heat. they are produced in the water, to be a more pleasant and refreshing mix of substances that print pleasant taste and aroma, beautiful color. lately the industry has passed the introduction of substances into drinks 98 necessary for man: vitamins, iron, lecithin, honey, caffeine, phosphorus, sodium, potassium, etc. the study allowed the observation of the evolution of ascorbic acid content of juice and freshly squeezed unconventional juices. figure 3. evolution of ascorbic acid in organic and conventional fruit juices in terms of conventional juices notice that: juice made of kiwi has fared fairly constant ascorbic acid, not very large losses 14.4 on day 1 to day 7 13.68; decreasing the amount of ascorbic acid per 100 g of product is evident in orange juice and grapefruit nectar, compared to 100 g product; although the blackcurrant juice biotta organic content is rich enough in ascorbic acid per 100 g product its evolution is constant; in general, organic juice biotta, which is rich in vitamin c, shows a steady evolution in time. conventional juices, once opened, lose the potential vitamin, unlike the green ones. the data presented in figure 3 show that tymbark juices containing ascorbic acid, rich on the first days after opening, lose vitamin potential after only five, seven days while biotta organic juice ascorbic acid levels during the 7 days is maintained approximately constant. as shown in figure 4, the content of vitamin c in orange juice freshly squeezed (30.096% compared to 100 g) is lower than that of conventional orange juice tymbark and juice biotta organic oranges. conventional juices are treated with ascorbic acid, and the presence of vitamin c raises the nutritional value of juices. figure 4. evolution of ascorbic acid in fresh squeezed juices, conventional and organic the antioxidant activity aims to prevent undue influence of atmospheric oxygen in contact with juice, respectively oxidizable substances therefrom, arising mainly from fruit crushing time, and until the last, that of bottling and pasteurization of juice. to choose a healthy product one must know the difference between different types of juices. the first category would be freshly squeezed juices such as grapefruit or orange ones. these are the only ones that actually can be called natural because they have gone through other processing improvements/conservation than fruit juice extraction. compared to bottled juices, natural juice, freshly squeezed, has the advantage that it contains no preservatives. as long as the juice will be consumed fresh, it will keep its properties and nutrients. another option is the most concentrated juice. in their case, the fruit is squeezed and then heated. the water content evaporates and results in a thick concentrate, which is pasteurization. reintroduce water before packing, and loss of vitamin c is low. pasteurized juices have a shelf life of two times that of fresh juice. pasteurization destroys an insignificant amount of vitamin c. 14 4244 35 56 14 363233 55 13 332932 54 0 10 20 30 40 50 60 day 1 day 5 day 7 m g a.a./100g kiwi prigat orange tymbark grapefruit tymbark orange biotta black currant biotta k 30 42 35 0 10 20 30 40 50 m g a.a./100g jui ce o r ang e f r eshl y sq ueez ed o r ang e j ui ce t ymb ar k o r ang e j ui ce b i o t t a food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 99 4. conclusion among the factors that influence the quality and quantity of unwanted flavors and colors of juices, the most important are the phenomena of oxidation, heat and handling. to preserve flavor and color of material it would be necessary to process fruits and juices at low temperatures and away from air. these conditions are practically difficult. the study allowed the observation of differences in the evolution of ascorbic acid in juices, organic juices and conventional juice. evolution of ascorbic acid in organic juice was constant compared to conventional juices where vitamin c content decreased within seven days by about 20%. once opened juices lose potential conventional vitamins. significant differences exist between conventional and organic juices. thus, conventional tymbark juices containing ascorbic acid on the first day after opening lose vitamin potential after only five, seven days while biotta organic juices have a fairly constant level of the vitamin c. in terms of vitamins, nothing can fully replace eating the fruit itself. fresh juice, squeezed and consumed immediately only has a higher content of vitamins than the pasteurized one (although there is a loss of vitamins and spinning). compared to bottled juices, natural juice, freshly squeezed, has the advantage that it contains no preservatives. as long as juice will be consumed fresh, it will keep its properties and nutrients. juice with a high content of fruit 100%, pasteurized or aseptic can be considered a saving variant; in the sense that it retains the taste and most of the vitamins of the fruit is done (pasteurization destroy an insignificant amount of vitamin c). 5. references 1. kim h., moon j. y., kim h., lee d.-s., cho m., choi h.-k., kim y. s., mossaddik a., cho s. c., antioxidant and antiproliferative activities of mango (mangifera indica l.) flesh and peel, food chemistry 121: 429-436, (2010) 2. jiménez a. m., martínez-tomé m., egea i., romojaro f., murcia m. a., effect of industrial processing and storage on antioxidant activity of apricot (prunus armeniaca v. bulida), eur food res technol, 227: 125-134, (2008) 3. avramiuc, m., the influence of refrigeration time and of sugar addition on ascorbic acid content in some natural juices, journal food and environmental safety of the suceava university – food engineering, year ix, no1, (2010) 4. selman, j. d., vitamin retention during blanching of vegetables, food chem., 49, 137, (1994) 5. banu, c., iordan, m., nour, v., musteață, g., procesarea materiilor prime alimentare și pierderile de substanțe biologic active, ed. “tehnică”, chișinău, 91-92, (2003) 6. ball, g. f. m., vitamins in foods, analysis, bioavailability and stability, crc press, taylor & francis group 6000, broken sound parkway nw, suite 300, boca raton, fl 33487-2742, 292-305, (2006) 7.hill, m. a., vitamin retention in microwave cooking and cook – chill foods, food chem., 49, 131, (1994) 8.nistor, o.v., popa, e., botez, e., constantin, o.e., researches on the evolution of concentrated fruit juices quality at storage, food and environmental safety journal of faculty of food engineering, ștefan cel mare university – suceava, year ix, no3, (2010) six juices were selected on the market, whose values of ph are listed in ‘table 1’. the juices are natural (samples 1, 2), conventional (samples 3, 4) and organic (samples 5, 6). freshly squeezed grapefruit juice is the acid juice of the review, followed by grapefruit juice brand tymbark conventional, organic blackcurrant juice then biotta. grapefruit juice is more acidic than the orange or the kiwi, whether the juice is natur... conventional juices have the following characteristics, which are mandatory under standard: appearance of homogeneous liquid, clear to opalescent, without suspended sediment or impurities in the raw materials used, color, smooth texture, fragrant, aro... figure 1. the evolution of ascorbic acid in conventional nectar figure 2. ascorbic acid in some freshly squeezed juices biotta organic orange juice is rich in vitamin c. biotta produces this 100% natural orange juice, fresh, preserving the original flavor and taste. very important to maintain a high nutritional value is the quality of processing methods and retention o... biotta blackcurrant juice is made from organic and fresh blackcurrant. black currants are rich in vitamin c and flavor of this juice is enhanced with an infusion of rose petals tea. biotta uses light pasteurization methods, thus keeping large amounts of vital nutrients. the juice is then heated and cooled quickly; the process is repeated as many times as possible. oxygen is removed thus preventing oxidation and loss of vitamins. ... the study allowed the observation of the evolution of ascorbic acid content of juice and freshly squeezed unconventional juices. figure 3. evolution of ascorbic acid in organic and conventional fruit juices in terms of conventional juices notice that: juice made of kiwi has fared fairly constant ascorbic acid, not very large losses 14.4 on day 1 to day 7 13.68; decreasing the amount of ascorbic acid per 100 g of product is evident in orange juice and grapefruit nectar, compared to 100 g product; although the blackcurrant juice biotta organic content is rich enough in ascorbic acid per 100 g product its evolution is constant; in general, organic juice biotta, which is rich in vitamin c, shows a steady evolution in time. conventional juices, once opened, lose the potential vitamin, unlike the green ones. the data presented in figure 3 show that tymbark juices containing ascorbic acid, rich on the first days after opening, lose vitamin potential after only five, seven... as shown in figure 4, the content of vitamin c in orange juice freshly squeezed (30.096% compared to 100 g) is lower than that of conventional orange juice tymbark and juice biotta organic oranges. conventional juices are treated with ascorbic acid, and the presence of vitamin c raises the nutritional value of juices. figure 4. evolution of ascorbic acid in fresh squeezed juices, conventional and organic the antioxidant activity aims to prevent undue influence of atmospheric oxygen in contact with juice, respectively oxidizable substances therefrom, arising mainly from fruit crushing time, and until the last, that of bottling and pasteurization of jui... among the factors that influence the quality and quantity of unwanted flavors and colors of juices, the most important are the phenomena of oxidation, heat and handling. to preserve flavor and color of material it would be necessary to process fruits ... microsoft word 6 mihaela jarcau corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 36 some physical properties of seabuckthorn and how the packing conditions influence them *mihaela jarcău1 1faculty of food engineering, stefan cel mare university, suceava, romania, mjarcau@ yahoo.com *corresponding author received 15 october 2012, accepted 7 november 2012 abstract: i paid attention to the way how the packaging can influence the physical properties of seabuckthorn fruits (hippoephae rhamnoides l.) during storage. the physical properties of the seabuckthorn are important in designing the equipments, in packing, as well as in higher work efficiency and identifying of the best package, in order to decrease product loss. i have analysed the physical dimensions such as length l (mm), width w (mm), thickness t (mm), geometric mean diameter dg (mm), sphericity φ ( %), porosity ε (%), volume v (mm3), mass m (g), bulk density ρb (g/cm3), true density ρtr (g/cm3) before and after storage in a refrigerator for 5 days at a temperature of +4oc. the fruits were packed in four different packages: samples 1 paper and plastic (paper bag in a plastic bag); sample 2 glass jar; sample 3 paper (paper bag); sample 4 plastic box. the dimensions of the sea buckthorn fruits measured by three perpendicular directions before storage had values between : for t minimum – 5.05mm, maximum 7.6 mm, for w – minimum 5.06 mm, maximum 9.87 mm, for l – minimum 6.74 mm, maximum 9.91 mm. after the 5 days of storage at a temperature of +40c, the dimensions of the sea buckthorn fruits suffered different modifications that depended on the nature of the package used for each sample. a greater variation has been observed in t and w, compared to l. keywords: seabuckthorn, hippoephae rhamnoides l, berries, physical properties. 1. introduction the seabuckthorn (hippoephae rhamnoides l.) is a shrub from eleagnacee family that can find in nature but also in cultures. seabuckthorn stands out through its exceptional capacity of adaptation in different soil and climate conditions, making it easier to grow, and to spread in the lands. seabuckthorn has been used for many years, not only for its therapeutical properties, but also in profilactic purpose. seabuckthorn has won the attention of many research teams because of its nutritional, medical and cosmetic properties. seabuckthorn is a thorny shrub, with falling leaves, widespread in europe and asia, being found also in romania, where it grows spontaneous in the subcarphatic regions of moldova and muntenia, starting with the upper basin of the siret to olt. because of its nutritional and medical properties, it is homegrown in a large number of parts of the world 1,2. it is a cold and drought resistant plant, and it is considered to be a good source for a large number of bioactive substances, like: vitamins (a, c, e, k, riboflavin, folic acid), carotenoids, fitosterols (ergosterol, stigmasterol, lanosterol, amyrins), organic acids (malic and oxalic acid) polyunsaturated fatty acids, and some essential aminoacids 3, 4, 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 37 this plant has been used in all nordic countries, baltic region and asia as a human and veterinary medical drug 6,7. seabuckthorn has been used in the traditional oriental system as a drug for treating skin deseases, astma, gastric ulcer and pulmonary deseases. recent research of the seabuckthorn have reported that it can be used also as an antioxidant, imunomodulator, antiaterogen, anti-stress, hepatoprotector, radioprotector and tissue regenerator 8,9,10. the active substances from seabuckthorn oil are anticancerous and antitumor, inhibing the development of cancerous cells, increasing the immunity, reducing the secondary effects of chemotheraphy and treating stomach, pulmonary and rectal cancer. also it stops internal hemmoragies, destroys intestinal parasites, slows the aging process and generally acts like a tonic in stress11,12,3,13. given the properties of the berries in preventing illnesses but also in treating some conditions, i have determined some physical properties of the seabuckthorn fruits and the way they are influenced by the packaging and storage. i have acorded the same atention to the bilberry, analising the behavior during the storage period, and in different packaging (plastic box with lid, jar package, double paper wrapper, plastic bag) 14. although many studies have considered the benefical effects of the sea buckthorn on the human organism, there is a limited number of them that describe the bioactivity of it compared to the fitochimical composition. the objective of this study was to investigate in which way the package influences the physical proprieties of seabucktorn fruits l (mm), w (mm), t (mm), dg(mm), φ( %), ε,(%), v(mm3), m(g), ρb (kg/m3), ρtr (kg/m3). all these informations are important for designing the equipment, storage and packing to increase the work efficiency and decrease product loss. nomenclature dg-geometric mean diameter, mm l-length, mm m-unit mass of the seed, g m100-100 seed mass, g sseed surface, mm2 t-thickness, mm v-single seed volume, mm3 w-width, mm ρb-bulk density, g/cm3 ρt –true density, g/cm3 ε-porosity, % φ-sfericity, % 2. materials and methods 2.1 sample preparation the analysed seabuckthorn fruits have been bought from a market in fălticeni, romania. the fruits have been washed, all the impurities removed, leaves, any other foreigh bodies and dried. i have randomly selected 10 seabuckthorn fruits, using an electronic caliper with a precision of 0.01mm, i have measured the three major perpendicular dimensions of the fruits namely length l, width w and thickness t, after which i have packed them in: samples: 1. paper and plastic (paper bag in a plastic bag) 2. jar 3. paper (paper bag) 4. plastic box food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 38 the samples were stored for 5 days in a refrigerator at +40 c and remesured. 2.2 geometric mean diameter, sphericity, volume and surface area the geometric mean diameter dg and sphericity of seabuckthorn was calculated using the following relationship 15: dg = (lwt)1/3 (1); φ = [(lwt)1/3/l] x 100 (2); for volume and seed surface was calculated by using the following relationship 16: (3) (4) where b is: (5) 2.3. one hundread fruits weight and the unit mass to obtain the unit mass of the seabuckthorn, the mass of 100 fruits were measured with an electronic balance with an accuracy of 0.01g. 2.4. bulk and true density the bulk density is the ratio of mass sample of the seed of seabuckthorn to its total volume. it was determined by filling a 1000 ml container with seabuckthorn from a height of about 15 cm, triking the top level and then weighing the contents 17. the true density was determined using water displacement method. the seabuckthorn were used to displace water in a measuring cylinder after their masses had been measured. the true density was found as an average of the ratio of their masses to the volume of water displaced by seabuckthorn 17. 2.5. porosity the porosity is the fraction of space in the bulk fruits that is not occupied by the fruits18. the porosity ε of bulk seabuckthorn was calculated using the following relationship 14: (6) where ρt is true density in g/cm3 and ρb is bulk density in g/cm3 3. results and discussion 3.1. seed size the dimensions of the sea buckthorn fruits measured in three perpendicular directions, before storage had the values between two intervals: l – minimum 5.05 mm, maximum 7.6 mm, w minimum 5.06, maximum 9.87 mm, t – minimum 6.74 mm, maximum 9.91 mm for the data analysis i have used anova. in table 1 are the medium values and the dimension variance of the sea buckthorn l(mm), w(mm), t(mm) before storage, for every test, in table 2 are the medium values and dimension variance of the same dimensions after 5 days of storage at a temperature of +40c. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 39 tabel 1 medium values and the dimension variance of the sea buckthorn l(mm), w(mm), t(mm) before storage tabel 2 the medium values and dimension variance of the same dimensions after 5 days of storage at a temperature of +40c. t (mm) l (mm) w(mm) sa m pl e a v e r a g e v a r ia n c e a v e r a g e v a r ia n c e a v e r a g e v a r ia n c e 1 6.09 0.41 5.97 0.21 7.96 0.66 2 6.04 0.60 5.99 0.30 7.95 0.38 3 6.10 0.13 5.87 0.23 7.59 0.08 4 6.45 0.21 6.40 0.43 7.88 0.05 from the graphical representations of the relative variations of l(%), w(%) and t(%), represented in figure 1-3, we can compare the way how the storage influences the physical properties of the sea buckthorn fruits. figure 1 t(%) relative variation for every sample figure 2 w(%) relative variation for every sample figure 3 l(%) relative variation for every sample 3.2 geometric mean diameter, sphericity, volume and surface area to determinate the geometric diameter of the sea buckthorn fruits we have used eq (1). the relative variations for each sample, after five days of storage are presented in figure 4. figure 4 dg(%) relative variation for every sample t (mm) l (mm) w(mm) sa m pl e a v e r a g e v a r ia n c e a v e r a g e v a r ia n c e a v e r a g e v a r ia n c e 1 5.90 0.25 5.70 0.39 7.84 0.63 2 5.99 0.62 6.02 0.59 8.10 0.68 3 5.84 0.45 5.91 0.32 5.91 0.35 4 5.92 0.46 6.00 0.34 6.00 0.21 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 40 as seen in figure 4, the greatest variation of the geometric mean diameter is 8.8% and it is in sample 4 (plastic box) and the smallest relative variation of the geometric mean diameter has been registered for sample 2 (jar), only 0.4%. to calculate sphericity, volume and surface sea buckthorn berries we used eq (2), (3) and (4). the values obtained for each sample before storage are shown in table 3, and the values obtained for each sample after the 5 days of refrigeration, are presented in table 4. tabel 3 sphericity, volume and surface sea buckthorn berries obtained for each sample before storage sample s (mm2) v(mm3) φ (%) 1 121.95 123.50 83.33 2 123.59 125.99 82.34 3 121.74 124.44 85.67 4 142.85 157.42 86.35 tabel 4 sphericity, volume and surface sea buckthorn berries obtained after the 5 days of refrigeration sample s (mm2) v(mm3) φ (%) 1 114.19 111.44 82.16 2 121.11 122.24 82.30 3 114.69 114.02 85.47 4 118.40 120.19 87.37 3.3. one hundread seabuckthorn weight the average of mass of 100 seabuckthorn before storage was: , and after storage: for sample 1was 19.1g, for sample 2 was 22.1g, for sample 3 was 20.1g, and for sample 4 was 23.0g figure 5 relative variation for every sample as shown in figure 5, the largest relative variation of sea buckthorn fruits weight is 12% and this is shown in sample 2, and the lowest relative variation of sea buckthorn fruits weight was recorded for sample 4, only 4.17%. 3.4. bulk and true density the relative variation of bulk density, for each sample, is presented in figure 7 and the relative variation of true density for each sample, is presented in figure 6. figură 6 ρt(%) relative variation for every sample figure 7 ρb(%) relative variation for every sample i have observed great variations for the bulk density and for the true density for sample 2 and small variations in sample 3. so, the highest variations are for sample 2: the relative variation of true density of the sea buckthorn fruits was 6.90%, and the relative variation for bulk density was 9.38%. 3.5. porosity the value of porosity were calculated with eq (6) by using the data on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 41 bulk and true densities of seabuckthorn and the results obtained are presented in figure 8. figure 8 ε (%) relative variation for every sample no significant variation of the porosity in none of the samples analyzed has been seen. 4. conclusion after the measurements made on physical properties of sea buckthorn fruits from fălticeni area, romania, before and after their storage for a period of 5 days at a temperature of 4 ° c in various packages, we drew the following conclusions: 1. the dimensions of the sea buckthorn fruits measured by three perpendicular directions before storage had values between : for t minimum – 5.05 mm, maximum 7.6 mm, for w – minimum 5.06 mm, maximum 9.87 mm, for l – minimum 6.74 mm, maximum 9.91 mm; 2. after the 5 days of storage at a temperature of +40c, the dimensions of the sea buckthorn fruits suffer different modifications, that depend on the nature of the package used for each sample in particular: the relative variation of thickness is maximum for sample 2 (14.08%) and minimum for sample 4 ( 9.9%); the relative variation of width is maximum for sample 4 (17.65%) and minimum for sample 1 (8.3%); the relative variation of lenght is maximum for sample 1 (10.08 %) and minimum for sample 2 (5.94%); the relative variation of geometric mean diameter is maximum for sample 4 (8.40%), and minimum for sample 2 (0.4%); the relative variation of mass is maximum for sample 2 (12%), and minimum for sample 4 (4.17%); the relative variation of bulk density is maximum for sample 2 (9.38%), and minimum for sample 4 (0.32%); the relative variation of true density is maximum for sample 2, of 6.90%, and minimum for sample 4 4.17%. 3. a greater variation has been observed in t and w, compared to l. 4. no considerable variations of density have been observed in the sample 3 and sample 4. 5. the physical dimensions of the sea buckthorn fruits varied the least for the fruits in sample 4 (plastic box) and these physical dimensions varied the most for the fruits in sample 2 (jar). 5. references 1 rousi, a. (1971). the genus hippophae l., a taxonomic study. annals botanica fennici 8 , 177–227. 2 li, t. (2003). taxonomy, natural distribution and botany. у t. b. li, sea buckthorn (hippophae rhamnoides l.): production and utilization. (сс. pp. 7–11.). ottawa: nrc research press. 3 beveridge, t. l. (1999). seabuckthorn products: manufacture and composition. journal of agricultural and food chemistry, 47 . 4 yang, b. k. (2001). fatty acid composition of lipids in sea buckthorn (hippophae rhamnoides l.) berries of different origins. journal of agriculture and food chemistry 49 , 1939–1947. 5 pintea, a. m. (2001.). polar lipids and fatty acid distribution in carotenoprotein complexes extracted from seabuckthorn fruit. phytochemical analysis 12, , 293–298. 6 yang, b. k. (2000). effect of dietary supplementation with seabuckthorn (hippophae rhamnoides)seed and pulp oils on the fatty acid composition of skin glycerophospholipids of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 42 patients with atopic dermatitis. journal of nutritional biochemistry 11 , 338–340. 7 dhyani, d. m. (2010). endorsing the declining indigenous ethnobotanical knowledge system of seabuckthorn in central himalaya, india. journal of ethnopharmacology 127 , 329–334. 8 suleyman, h. d. (2001). antiulcerogenic effect of hippophae rhamnoides. phytotherapy research 33 , 77–81. 9 upadhyay, n. k. (2010). antioxidant, cytoprotective and antibacterial effects of sea buckthorn (hippophae rhamnoides l.) leaves. food and chemical toxicology 48 , 3443–3448. 10 suryakumar geetha, g. a. (2011). medicinal and therapeutic potential of sea buckthorn (hippophae rhamnoides l). journal of ethnopharmacology, 138 , 268– 278. 11 rodriguez-meizoso, i. m. (2006). subcritical water extraction of nutraceuticals with antioxidant activity from oregano.chemical and functional characterization. journal of pharmaceutical and biomedical analysis, 41 , 1560 1565. 12 upendra, k. s. (2008). microwaveassisted efficient extraction of different parts of hippophae rhamnoides for the comparative evaluation of antioxidant activity and quantification of its phenolic constituents by reverse-phase high performance liquid chromatography (rp-hplc). journal of agricultural food chemistry, 56 , 374 379. 13 m.s. yogendra kumar, r. d. (2011). subcritical water extraction of antioxidant compounds from seabuckthorn(hippophae rhamnoides) leaves for the comparative evaluation of antioxidant activity. food chemistry, 127. 14 jarcău, m. (2012). some physical proprieties of bilberries and how the packing conditions influence them. food and environment saftey . 15 mohsenin, n. n. (1970). physical properties of plant and anima lmaterials. new york: gordon and breach science publishers. 16 jain, r. k. (1997). physical properties of pearl millet. journal of agricultural engineering research, 54 . 17 deshpande, s. b. (1993). physical properties of soybean seeds. journal of agricultural engineering research, 56 , 89 92. 18 thompson, r. a. (1967). porosity determination of grains and seeds with air comparison pycnometer. transaction of the asae, 10 , 693–696. microsoft word 3 rindyuc corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 19 research on the influence of temperature on the process of granulation by extrusion *dmitro viktorovich rindyuk1, svyatoslav yuriyovich lementar1 1national university of food technologies, ukraine, kiev, rel_dv@ukr.net *corresponding author received 10 november 2012, accepted 11 december 2012 abstract: the process of extrusion pelletizing particulate materials, such as sunflower and buckwheat husk, oak and pine chips, wheat straw and bran has been considered. it was investigated the effect of temperature of raw materials, compacting pressure and the diameter of the press-matrix on quality of fuel pellets. keywords: temperature, particulate materials, biofuels, pelleting. 1. introduction production of solid biofuels is an urgent requirement for fuel and energy complex of any developed country in the world, and therefore there is a requirement for research that would allow discovering the peculiarities of the process with granulation of fuel pellets. in article [1] the problem of determining the optimal technical and technological parameters of the process of granulation extrusion was treated and was got the disperse of materials obtained depending influence of the main parameters of unit pressed pellet (fig. 1) on the quality of the final product – the pellet. regardless of the hardware design at process of granulation significant influence on the formation of granules of a material which has been granulated: raising of the temperature increases the density of granules, and also reduces energy consumption in the process of pressing [2]. as you know, the basic constituents of raw materials for solid biofuels are cellulose, hemicellulose and lignin. lignin as an amorphous polymer is a kind of binding between cellulose fibrils, providing strength and rigidity of the cell wall (if cellulose with properties corresponding fixture, the lignin, which has a high compressive strength concrete). at low temperature processes (up to 160 ° c) reaction hydrolytic decomposition of carbohydrate content and partial impolymerization of lignin to form low molecular weight fragments are predominant. rising of the temperature of the process increases the degree of degradation of wood carbohydrates, and yet with lignin impolymerization reactions begins to compete re-polymerization his reaction. therefore, attached to change of the temperature of the process to 150-170 ° c of lignin in wood decreaseswith increasing temperature and process of lignin increases markedly, reaching 33-36% [3-7]. figure 1. scheme of node compression screw extruder-granulator food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 20 the aim of this work is to study the effect of temperature on the material quality (density) of the final product (pellets) considering structural and technological parameters of the equipment for its compaction during pressing extrusion. 2. experimental to solve this problem a multifactorial experiment is proposed and to develop mathematical and statistical model of dependence of density fuel pellets produced from sunflower and buckwheat hulls, oak and pine shavings, wheat straw and bran from raw temperature, pressure and pressing channel diameter pressmatrix. ),,( dptf where ρ density of pellets, kg/m3; t temperature materials, ° c; p pressure compression, mpa; d diameter of the press matrix. factorial experiment of second order, which is used to describe non-linear objects, in our case, is represented by polynomial [8]: .2310 2 29 2 183217326 3152143322110 xbxbxbxxxbxxb xxbxxbxbxbxbby   table 1 intervals of variation intervals of variation x1 (t, °c) x2 (p, mpa) x3 (d, м) zero level 100 175 0,024 step of variation 80 125 0,016 the lower level 20 50 0,008 the upper level 180 300 0,04 in fig. 2 a scheme of pilot plant for studying the influence of temperature on the process of materials’ sealing is represented. figure 2. scheme of pilot plant for studying the effect of temperature on the process of sealing materials 1 autotransformer 2 electric heating coil, 3 punch, 4 press matrix, 5 – potentiometer, 6 thermocouple, 7 – example. pressing is performing by using of a hydraulic press to press the matrix 4 with varying diameter punch 8–40 mm at a speed of pressing 0,005 m/s. temperature pressmatrix regulate by changing the voltage filed in the electric spiral 2 of isolated press-matrix 4. the temperature of press-matrix is measured by thermocouple 6, potentiometer connected to the direct current 5. pressure was measured by a manometer. the experiment was conducted for the following types of materials: sunflower and buckwheat husk, oak and pine chips, wheat straw and bran. the analysis of the represented dependences showed that with increasing of temperature of the raw materials from 20 to 160 ° c increasing the density of obtained granules is observed. this is due to the intensification of the process of dissolution of lignin with increasing temperature. it should be noted that a further increasing in temperature of material over 160 ° c is not appropriate because it does a little effect on increasing the density of granules, and thus leads to unnecessary energy costs. also it is experimentally confirmed that the increase in compacting pressure and decreasing of diameter of press-matrix provides density increase granules. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 21 table 2 the results of mathematical and statistical analysis of experiments number type of raw regression equation 1. husk of sunflower .31,24144500462,002887,0375,59375,10 0017,031,81153567,27184,76743,155 222 dptdpdt ptdpt   2. buckwheat of husk .813,9013200852,00206,00773,0734,7 535,130042,0391,4229571,4787,4358,113 222 dptdptdp dtptdpt   3. shavings of pine .156,20903500605,002107,0 00289,0875,8148175,3379,5741,293 222 dpt ptdpt   4. chips of oak .969,21766700404,00238,04375,5301,8 00114,0453,61596715,2654,6882,201 222 dptdpdt ptdpt   5. wheat straw .156,2010350069,002585,0328,9 00265,0813,7828792,3598,6176,35 222 dptdt ptdpt   6. wheat bran .656,9047200643,00118,0 00333,05,2612385,3446,2271,330 222 dpt ptdpt   a) b) c) figure 3. the dependence of the density of granules from: a) diameter of d press matrix and temperature t of raw materials;b) compacting pressure p and temperature t of raw materials;c) compacting pressure p and diameter d press matrix. 4. conclusions the obtained results should be used as a guideline at the organization of the technological process of granulation of dispersed materials by extrusion and at construction at related equipment. 5. references [1] rindyuk d., lementar s., the method of determination of the optimal parameters of dispersed materials granulation through consolidation, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava, volume xi, issue 2, 113. 15 -18, 2012 [2] klassen n., grishayev i., shomin i., basic techniques of granulation, moscow, chemistry, 272 р, 1982 [3] bogolitsyn k., skrebets t., environmentally sound technologies chemical processing, the 5th international forum «aims for future of engineering science»: proceeding, paris, 627. 475-478, 2004 [4] burov a., beygelman t., lukanina a., organic-solvent process delignication , boom. industry, № 2, 141. 15-16, 1989 [5] obolensky a., practical work on the chemistry of wood and cellulose, moscow, wood industry, 441 р, 1965 [6] skrebets t., bogolitsyn k., verbitskaya d., change the properties dioksanlignin after alkaline treatment in the presence of ethanol, forestry journal, № 3, 167. 106-110, 2004 [7] dahlmann g., solvent pulping the no-odor approach to chemical pulping, intern. symposium «papfor-92": proc. reports, 458. 297-315, 1992 [8] fedorov v., theory of optimal experiments (planning regression experiments), moscow, nauka, 312 p, 1971 microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_1-6.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 5 research and achievements for new electrochemical biosensors gheorghe gutt1, yarema tevtul2, andrei gutt1, alina psibilschi1 1 faculty of food engineering, stefan cel mare university, street. universitatii no.13, 720229, suceava, romania, g.gutt@usv.ro 2yuriy fedkovych national university of chernivtsi, kotsiubynsky st., 2, chernivtsi, 58012 ukraine abstract. the paper presents results and research of a team involved in instrumental analysis from faculty of food engineering of suceava university in biosensors field, for food, health and environment issues. starting from previous achievements were developed electrochemical performance biosensors, extensive use, for analysis both in situ as well as in the laboratory, mainly in pursuing the universal use of such equipment in all reactions using as catalyst type oxidase enzymes. another aim was to increase sensitivity of measurement and accuracy of such equipment, also by proposed solutions were removed single-use kits, commonly used in the realization of biosensors by using watertight vials containing oxidase which is enough for hundreds of tests. combined biosensor described in this paper has the great advantage of using in the amperometric and conductometric methods only their advantages since the two electrochemical methods are complementary, also the dosing system of oxidazes is simple and accurate ensuring a good reproducibility of experimental data. by the avant-garde research and achievements of the team itis opened the way for development of new types of biosensors. keywords: amperometric and conductometric biosensor, universal biosensor, dual biosensor introduction. biosensors are biological-selective electronic integrated systems, consisting of a biological active receiver, a transducer and an electronic amplification, processing and display data. biological active receptor provides specific analytical information enabling recognition of certain biological or chemical species in a complex mixture that consists matter under review, followed by quantitative or semiquantitative determination of it. following the interference of biological active receptor and analytes resulted changes of raport between reactants and products of the reaction that causes in turn, depending on active biological used systems, proportional evolution of physico-chemical measurements values such as: electric charge, absorption / photon emission, temperature, refractive index, layer thickness, values that are converted by transducers type amperometric, ph meters, photometers, thermal, refractive, piezo oscillator, surface plasmon resonance, in proportional electrical signals to the concentration of the species sought in the matter under review. basic applications for biosensors are in medicine, environmental quality control issue, pharmaceutical industry and cosmetics, food industry and in biotechnology. experimental measurement with biosensors are simple and require no specialized advanced knowledge but only the user browsing of short successive stages in time. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 6 new methods for achieving electrochemical amperometric biosensors amperometric biosensors are used for the detection of reaction products or metabolites that oxidizes or shall slightly reduces. amperometric method consists in measuring the electrical current of a miniature electrolysis cell electrolysis (this is a biosensors system), fed at a constant voltage. according to faraday's law, a mass m of a chemical species discharged from an electrode of an electrochemical cell at a time t is proportional to current i of electrolysis and with electrochemical constant k: tikm  (1) currently, one of the most popular applications in biosensors amperometric detection is a portable glucose biosensors used to determine in situ blood sugar for diabetics and athletes. glucose sensor has as an active biological material a glucosoxidase enzyme used as biocatalysts in oxidation reaction of glucose with oxygen in the air with obtaining gluconolactone and hydrogen peroxide (h2o2 -hydrogen peroxide) as products of reaction. biologically active receptor for rapid analysis of blood is in the form of single-use plastic strips that has at one end two miniature plate electrodes between which is deposited glucoseoxidase in a form of dry gel that containes a conductive polymer and at the other end the strip has electrical pine connection with the electronic system. when submitting a drop of blood on the area covered by the glucoseoxidase the folowing reaction occurs: 222cos ohtonegluconolacoeglud aseglucozoxid   (2) hydrogen peroxide is highly reactive and breaks down at the electrodes of electrochemical cell, current intensity of electrolysis is proportional to the amount decomposed and proportional to the amount of glucose in analysed blood . such biosensors systems can be used for determining concentration of phenols, polyphenols, flavonoids and for the development of substances with antioxidant activity [3], [4] below are listed some typical examples of biosensors that use different enzymes from oxidase type, as a catalyst, the result being also hydrogen peroxide obtaining: 222 ohecolestenonocolesterol oxidasecolesterol   (3) 2232 ohnhteoxiglutaraoglutamate idaseglutamatoxd    (4) 22 aselactatoxid ohpiruvatolactate   2 (5) 222 ohcoateacetilfosfhpopiruvat dasepiruvatoxi 4   (6) the concerns of analytical instrumental group from the faculty of food engineering of suceava university focused on improving the amperometric food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 7 biosensors. research focused on the following directions: 1. achievement a conductometric biosensor as a superior alternative to amperometric biosensors 2. design and implementation a conductivity-amperometric biosensor combined with concurrent analysis by both methods, biosensor that can be used in reactions 1-6 using oxidase enzyme as catalyst 3. extending limits of concentrations of a new type of biosensors from maximum 500mg/dl, as they are encountered in blood, to limit of 20% glucose, the latter concentration being specific to analytical food, particular applications in fermentative processes 4. elimination of chemical kit for single use from typical biosensors research have focused initially only on glucose biosensors [1], [2], [4], [6] and were mainly intended to find an alternative to low reproducibility of typical portable biosensors used to determine blood glucose this low reproducibility is due to electrolysis phenomenology, electrolysis current, so the indication of glucometer, depending on the addition of hydrogen peroxide concentration and other parameters relating primarily to ionic transport phenomena that lead to advanced electric polarization at electrodes that alter current yield. very promising results obtained in experimental research [1], [2] have confirmed that using conductometric method with frequency of 5 khz eliminates the phenomenon of polarization and also allows determination both of glucose low concentration, as they are present in blood, as well as the concentration at high levels up to 20% , specific to analitical food. using conductometric method presents also a disadvantage, namely that the value of conductivity is an adder for all electrolytical species of blood. in this issue is shown that using amperometric method, in which electrolysis current is also adder, enables reducing the error by limiting electrolysis voltage of hydrogen peroxide at the nernst voltage. to combine the advantages of both methods and to eliminate the disadvantages of each of them the team has developed a combined biosensor type conductometric amperometric [3], [4] for the clinical laboratory and analytical food in addition to those mentioned this biosensor has a great advantage of not working with expensive kits of single-use but with glucoseoxidase and pure liquid colesteroloxidase which is precisely dosed[5], [6] using the equipment designed by collective starting from the results of theoretical and applied research has been developed a portable dual biosensor [7], [8] that allows concurrent determination of both glucose and cholesterol from a single drop of blood, but it is not the subject of this paper. contributions to conception, design and implementation of combined electrochemical biosensor with superior performance research team has designed and developed a prototype of enzyme biosensor that uses oxidase type enzymes as a catalyst in liquid form, biosensors being capable of rapid determinations and in situ of chemical or biological concentration of species without using of single-use biological kits [4 ], [4]. the advantages of using this measurement system are: obtaining an universal and reliable biosensor a long-term usable in all types of reactions catalyzed by enzymes type oxidase eliminating single-use kits and thereby significantly lowering of cost price of the analysis combined use of both amperometric food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 8 and conductometric method in the same determinations, it ensure a high a precision of measurements fig.1.amperometric and conductometric scheme of measurement principle with enzyme biosensor. 2 acquisition electronics unit, data processing and display, 3-amperometric electronic unit, 4conductometric electronic unit, 10silicone hose, 14,15-platinum electrodes, 17-cup, 21pipette or manually electronic unit. in figures 1.3 are represented elements of principle, construction and function of combined biosensors [4]. fig.2.sectional view of enzyme biosensors. 1-body 2electronics unit of acquisition, data processing and display, 3-electronic amperometric unit, 4-conductometric electronic unit, 5-keyboard, 6plastic capsule, ooxidase solution, 7-bolt, 8-marker, p-drill, f-strip, c-con, 9-nut, 10-silicone hose, 11supported eccentric cylinder l-conical seat, 12ratchet, 13-bar 14.15-platinum electrodes, 16-con, 17-cup, 18.19electric contacts, e-slot 20-ratchet ball, 21pipette or manually hopper. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 9 the procedure to work with biosensors is the folowing: it is maximum screwed up 7 cav screw then returned device to 1800 over normal working position, place the cap with oxidase in cavity of the screw 7 and after it is screwed by hand until it is sensitive mechanical strength which means that strip of capsule 6 was pierced by the knife p of the marker 8 and capsule neck 6 sealing made with the marker 8, then returned again biosensors in working position and screw up removable cup 17 on rod 13, it is running several full rotations to the right of the cylinder 11 eccentric supported until on the perforated cone 16 it is a drop of oxidase after these operations the cup 17 is screwed and it is executed a full rotation of eccentric cylinder to dosing, by peristaltic system, of required volume for oxidase in cup 17, with the stop of cylinder 11 eccentric supported on full shuttered position of silicone hose, position indicated by arrow signs on the click swing button but also by the ball's ratchet 12. it follows the dosage in cup 17 of the prescribed volume for examined species by a dosing pipette 21 or manually hoppers after the device is keeped upright and digital display is intended. triggering of catalyzed reaction lead to the emergence of first quantities of hydrogen peroxide, a threshold of its concentration causes initiation amperometric and conductometric measurements, these are alternatively carrying by an automatic electronic switching. fig.3.exterior front and side of enzyme biosensors, 1-body, 2electronic unit acquisition, data processing and display, 3-amperometric electronic unit, 4conductometric electronic unit, 5-keyboard, 6plastic capsules, ooxidase solution, 7-bolt, 8-marker, p-drill, f-strip, c-con, 9-nut, 10-silicone hose, 11-cylinder eccentric supported, l-conical seat, 13-rod 14.15 platinum electrodes, 16-con, 17-cup, 21pipette or manually hoppers. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 10 after 10 seconds is displayed concentration value resulting from the average of dozens of amperometric and conductometric measurements statistically processed by biosensors microprocessor. the volume of oxidase capsule reaches hundreds of determinations, between two determinations with the same oxidase is not required than washing under running water of cup 17. on oxidase changing the whole route is washed using for this purpose a capsule with double distilled water which is fixed, as already was described, on the body 1 of biosensors and run more full rotation of cylinder 11 supported eccentric supported for pumping water through the circuit. in some situations it is necessary to replace the silicone hose 10, these cases occur in specific applications where the volume of dosing oxidase is different from the previous application. this requires replacement of silicone hose with another with an greater inside diameter, that smaller, the operations arising in a few seconds. the replacement of silicone hose 10 is required and its wastage in compression zone-stretching as a result of numerous pumping of eccentric supported cylinder 11. at hose replacement it unwind the nut on the opposite side of the actuating button of cylinder 11, after which it is extracted from the body 1 of biosensors, it follows the return with 1800 of the device from the working position, is unscrew the screw 7, extract of capsule 6 and marker 8 from its slot, full unscrew of nut 9, extraction of silicone hose on the marker 8, followed by extraction of the con 16 from rod 13, retrieval of hose 10 from cone 16, at the mounting of new siliconic hose operations are repeated in reverse of of the dismantling of 10 silicone hose. conclusions using both amperometric and conductometric principle it made possible the achievement of combined performance biosensor that combines the advantages of both electrochemical methods of measurement. devising a simple and accurate dosing system for catalytic enzymes type oxidase and its implementation on the biosensor allowed elimination of single-use kits that determine the decisive price of analysis. at the same time other design and construction solutions embedded in the architecture of biosensors allow its universal use in all catalyzed reactions by oxidase theoretical and applied research carried out allowed the design of new biosensors one being a dual portable biosensor that allows co-determination both of glucose and cholesterol in blood using for this purpose a single drop of blood. this type of biosensor is not the subject of this work, research and prototype implementation are still pending. references 1. gutt g., sumusche i., gutt a., amperometric biosensors for glucose determination, anals of the suceava universityfood engineering, year vii, no. 1, 2009, p 5-11 2. gutt g., gutt a., sumusche i., research on achievement an conductometric enzyme biosensor, anals of the suceava university-food engineering, year vii, no. 1, 2009, p 11-18 3. gutt s., gutt g, gutt a.biosenzor , propunere de inventie , dosar osim a/00285/2008 4. gutt a, gutt s., gutt g, biosenzor enzimatic, propunere de inventie , dosar osim a/00857/2009 5. gutt s., gutt g, gutt a, biosenzor miniatural, propunere de inventie , dosar osim a/00 854/2009 6. gutt a, gutt s., gutt g, biosenzor de glucoza, propunere de inventie , dosar osim, a/00848/2009 7. gutt s., gutt g, gutt a, -biosenzor de laborator pentru glucoză şi colesterol, propunere de inventie , dosar osim, a/00161/2010 8. gutt s., gutt g, gutt a, psibilschi a. biosenzor portabil pentru glucoză şi colesterol , propunere de inventie , dosar osim, a/00162/2010 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 50 the present state of researches regarding electroplating problems (review) violeta vasilache ştefan cel mare university of suceava, str. universităţii, nr.13, 720229, violetav@usv.ro abstract. during the last years researches regarding galvanic electroplating problematic had an accelerated development due to environment protection reasons, because more of the present technologies will have to be changed by new ones of minor environmental impact. for example, in machine building industry, heavy metals (as cadmium) are replaced by new materials with superior properties and non-toxic. alloys and composite materials are a convenient alternative. one of the greatly developed galvanic-technique branches is represented by functional electroplating under very hard working conditions. human activity is extended in all places of the planet, so metals have to work under very corrosive conditions. decorative electroplating represents another branch in quick development. there are new and unexpected uses for electroplating materials. for example deposits of silk nickel have a silky aspect, a uniform appearance, corrosion resistance etc. galvanic electroplatings with nickel are used for fabrication of smaller and smaller mechanical micro-components. to control the quality of these micro-components is necessary to understand the relations between variables of the process and resulting properties. the influence of additives is intensively studied because these ones permit to obtain high quality deposits. keywords: galvanic electroplatings, nickel layers electrodeposited, nickel electroplating mechanism, nickel electro crystallization actual directions in researches of nickel and nickel alloy electroplating 1. actual directions in researches of nickel electroplating at chagsha university, china [4] was performed a new bath for nickel electroplating based on a solution with citrate. tri-sodium citrate was used as complexion and blotter agent. there were compared blotting capacity of tri-sodium citrate and boric acid. the effects were investigated under different conditions and for different compositions of the bath. xrd analysis shows the finest crystalline structure as compared to watts bath. at technique university dresda, germany, sub-micro-crystalline nickel (smc) electrodeposited was studied, through a method in ped (pulse electrodeposition) without additives. fresh deposited layer was deformed at room temperature and for different amplitudes of plastic deformation forces and it was found that the interactions of dislocations with grain boundaries are responsible by diminution of internal stress observed on smc ped fresh prepared nickel [5]. in china, at shanghai university, nickel was deposited from sulfamate bath for different current densities. it was studied corrosion behaviour in nal solutions using potentio-dynamics polarization and electrochemical impedance spectroscopyeis. the best corrosion resistance was obtained at 0.05 a/cm2 current density. the corrosion potential and breaking potential food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 51 decreased with the increasing of current density used in electrodeposition. in china, at shanghai university, electrodeposited nickel was prepared from a sulphamate bath at different current densities. based on the microstructure, the corrosion behaviour of the electrodeposited nickel in 3.5% nacl solution was studied using potentiodynamic polarization and electrochemical impedance spectroscopy (eis). the electrodeposits with the best corrosion resistance are obtained at 0.05 a/cm2 current density. the corrosion potential and breakdown potential decrease with increasing current density used to prepare electrodeposits [6]. at university of nanjing, china, bright nickel was produced by a new technique with spherical hard particles filling between electrodes. the related theories and structure of the bright deposit were studied. it was found that the hard particles could increase the brightness of nickel deposits by perturbing the process of crystal nucleation and polishing the cathode surface during electrodepositing [6]. at košice, slovakia and brno, czehch republic, electrodeposition of nickel on a paraffin impregnated electrode (pige) has been studied by cyclic voltammetry (cv) and chronoamperometry from chloride and sulphate electrolytes. the differences in mechanism of nickel deposition from these solutions have been observed. the experimental results obtained from cyclic voltammetry were evaluated by elimination voltammetry with linear scan (evls). the results indicated two basic differences between the nickel deposition process in both electrolytes: (i) in chloride electrolyte the initial stage is ni2+ reduction, while in sulphate electrolyte it is the proton reduction; (ii) in choride electrolyte the charge carrying species is nicl+, while in sulphate solutions it is nioh+ [7]. at sao paolo university (brasil) the electrodeposition of nickel on to platinum substrate in a watts bath in the presence of glycerol, mannitol or sorbitol has been studied. the presence of polyalcohols in the electrolytic solution proved to be significant since deterioration of the bath contents during operation was not observed [8]. in denmark, at lyngby, the influence of current density and the temperature on the microstructure and hardness of nickel layers electrodeposited from an additivefree sulphamate bath were investigated [9]. 2. actual directions about establishing nickel electroplating mechanism a group of researchers from košiceslovakia and leics, great britain have studied ternary alloys of nickel with copper and cobalt. special attention was given to the mechanism of electrodeposition process [10]. the mechanism is the following one: ni2+ + x→ nix+ (1) nix+ + e→ nixads (2) nixads + e→ ni+x (3) the anion xhas been variously assumed to be oh-, so42or cl-. by comparing the experimentally determined kinetic parameters to those calculated for the various rate-determining steps and ranges of coverage, they deduced that if a reaction mechanism of the general type (1)-(3) is applied in a watts bath (consisting of niso4 + nacl + h3bo3): (i) the anion x must be the chloride and (ii) the ratedetermining step was the reaction (2), what means the first-electron transfer step [10]. some studies on the electrochemical deposition of nickel have indicated that the nickel monohydroxide ion, nioh+, is an important species in the charge transfer steps in aqueous non-buffered solutions: ni2+ + h2o ↔ ni(oh)+ + h+ (4) ni(oh)+ + e+ → ni(oh)ads (5) ni(oh)ads + ni2+ + 2e→ ni + ni(oh)ads (6) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 52 ni(oh)ads + e→ ni + oh (7) where ni(oh)ads represents the active intermediate, which may also be a chloride containing complex. two one-electron metal reduction reactions take place in succession, giving rise to two clearly identifiable peaks in the cyclic voltammograms. the hydrogen evolution also occurred under different extents in the same potential region. the electrolytic deposition of nickel from a watts bath onto a paraffin impregnated graphite electrode (pige) at ph 2, 3 and 4 was studied, also from the chloride electrolyte. cyclic voltammetry and elimination voltammetry with a linear scan were applied. the results indicated three steps in the deposition mechanism from the watts electrolyte solution: (i) a chemical reaction preceding an electrochemical reaction, (ii) the occurrence of surface reactions with the adsorption of intermediates onto the pige and (iii) a reaction of the electro active substance transported to the electrode by diffusion. taking these results into account the reaction mechanism proposed was: ni2+ + h2o ↔ ni(oh)aq+ + h+ (8) ni(oh)aq+ ↔ ni(oh)ads+ (9) ni(oh)ads+ + e→ nioh (10) nioh + h+ + e→ ni + h2o (11) the adsorption of chloride anions on pige was detected from the chloride electrolyte. the elimination voltammetry indicated the importance of a kinetically controlled adsorption/ desorption process in the nickel deposition mechanism. following the distribution diagram, the particle most likely to be electro active at the start of electro reduction is nicl+ [10]. similar results were obtained by ji and cooper [11]. they determined the nickel speciation in aqueous chloride solutions over a broad range of concentration and ph, and clarified the role of boric acid in nickel electrodeposition. they found that in concentrated nicl2 solution the predominant nickel species in the acidic region were ni2+ and nicl+ and in a concentrated mixed sulphate-containing, ni2+, nicland niso4 were important. the concentration of the nioh+ species was negligible until the nicl2 concentration was lowered to 10-3 mol/dm3. the reduction of ni(ii) was one of the first steps in the mechanism proposed for nickel deposition by gomez et al.[12]: ni(ii)+e-→ni(i) (12) followed by one more steps that lead to the deposition. this simple scheme may apply to potentials close to those just after the start of the deposition (until the maximum potential of the reduction peak) and may explain the compact, uniform deposits obtained. at more negative potentials, the first step was maintained but was followed by a possible disproportion reaction of ni(i): ni(i)+ni(i)→ni(0)+ni(ii) (13) moreover, under these conditions a simultaneous reaction may occur between ni(i) and h2o: ni(i)+h2o→nioh(i)+h (14) the reaction (14) explains the production of hydrogen during the nickel deposition. using voltammetric and potentiostatic methods, gomez et al. [12] studied the initial stages of the deposition of nickel onto vitreous carbon from an aqueous chloride solution. the morphology of the deposit was observed by scanning electron and optical microscopy. the mechanism of the ni2+ reduction from acid sulphate, chloride and watts electrolytes has also been extensively studied by epelboin, wiart et al.[13]. in an impedance study of nickel deposition, they observed that the electrode kinetics were dependent on the type of anion. in chloride electrolytes, slow electrode activation with cathodic polarization was predominant. in sulphate solutions, the low-frequency capacitive feature, favoured by a ph decrease, appeared to result from food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 53 interactions between the nickel and hydrogen discharges. an interpretation was proposed in which the ad-ion niads+ acts as both a reaction intermediate and a catalyst, associated with a propagating kink site, and where the adsorbed species hads*, generated by the presence of niads+, inhibited the hydrogen evolution. it was concluded that the active area is closely connected to the extent of coverage by adsorbates. further impedance measurements showed that the electrolyte composition influenced the kinetics of nickel electro crystallization. the following mechanism was suggested for electrolytes of ph 2-4. 2 (h+ + e) → h2 (15) ni2+ + e→ niads+ (16) niads+ + e→ ni (17) niads+ + h+ + e→ niads+ + hads* (18) 2hads* → h2 (19) niads+ + hads* + e→ ni +hincl (20) an electrochemical impedance spectroscopic (eis) study of metal deposition by wiart [14] showed that the inhibition of the charge transfer was caused by adsorbates (hydrogen, anions and additive molecules) or by an interfacial layer. various examples (cu, ag, ni, zn) illustrated these situations. the electrodeposition from a low concentration of nickel onto vitreous carbon at ph 3 and 5 with different anions has been studied by proud and műller using eis. they observed an adsorption process starting at potentials far removed from the potential corresponding to the deposition process. this process occurred more rapidly in chloride systems at ph 5 and more slowly in sulphate systems at ph 3 indicating that the adsorbing species was dependent primarily upon the ph of the system and secondly upon the anion. an overall mechanism was proposed based on the work of wiart. reaction: ni(oh)aq+ ↔ ni(oh)ads+ (21) was the predominant step for the initiation of deposition at ph 5. the next step was: ni(oh)ads+ + e→ ni(oh)ads (22) in the ph 3 system, the first step was the direct discharge of the hydroxylated complex: ni(oh)+ + e→ ni(oh)ads (23) nickel deposition then took place via the steps: ni(oh)ads + ni2+ + 2e→ ni + ni(oh)ads 24) ni(oh)ads + e→ ni + oh (25) protons were discharged simultaneously with nickel deposition: h+ + e→ hads (26) 2hads → h2 (27) ni(oh)ads + hads + e→ ni + hincl + oh (28) the adsorbed hydrogen (hads) was responsible for the passivation observed at the lower potentials at ph 5. at higher potentials this intermediate tended to evolve gaseous hydrogen or produce hydrogen-containing forms of nickel. the second passivation process occurred through the precipitation of a passivating coverage of the hydroxylated nickel species: ni2+ + 2oh→ ni(oh)2 (29) this passivation was eventually broken by the relative acceleration of other processes or the potential dependence of the reaction: ni(oh)2 + 2e→ ni + 2oh (30) eis was used by holm and o’keefe [15] to characterize the deposition of nickel onto stainless steel cathodes from non-buffered acid sulphate electrolytes at ph from 2 to 3,5. the impedance spectra showed features, which were related to both the quality of the deposit and possible shifts in the deposition mechanism. the spectra for good quality nickel deposits consisted of a single, high frequency loop. as the deposit quality worsened, a second loop appeared at lower frequencies. the characteristics of this loop indicated the formation of a passivating nickel hydroxide layer, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 54 possibly resulting from the onset of diffusion control. the second loop was related to the presence of an oxidized nickel film which can form if the hydrogen ion concentration is low. from the kinetic measurements during the electrodeposition of nickel, it was found that the rate of this discharge reaction was controlled by ion transport through the double layer. it had also been established experimentally that the rate-determining stage of the overall reaction was the single electron discharge of nioh+ to niohads. this offered the possibility of studying the time dependence of the current or potential during the transitional processes of nickel nucleation and growth. nickel nucleation onto glassy carbon substrates has been extensively studied. bozhkov et al. [16] determined the concentration of nickel ad-atoms at the surface during the initial stages of nickel deposition onto glassy carbon substrates from the watts electrolyte using both galvanostatic and potentiostatic pulse methods. they found that nickel ad-atoms carried a partial positive charge corresponding to about 25% of the total charge of the ion in the bulk of the solution. 3. recent studies about establishing models for nickel electrodeposition processes abyaneh and co-workers [17] proposed a model for the electro crystallisation processes, following the application of a pre-pulse method. the transient equations derived on this basis were shown to closely fit the behaviour of the recorded pre-pulse transients for the electro crystallisation of nickel onto a vitreous carbon electrode from watts-type baths. the kinetic information about the initial nucleation and growth of the nickel deposit was obtained by analyzing the experimental current-time transients. nucleation rate constants were obtained over a range of deposition potentials [18]. trevisan-souteyrand et al. [19] also their stepwise computer model to be applicable to the potentiostatic current-time transient response of the nucleation of hemispherical centres and the radial growth mechanism. particular attention was devoted to the effects of the ohm drop on the transient responses and on the number of growing centres. computed values were compared to experimental transient curves and to transmission electron microscopy (tem) micrographs obtained in the case of nickel electro crystallization onto vitreous carbon. jensen et al. [20] investigated the process of nickel electrochemical deposition from watts-type electrolytes under the influence of high frequency ultrasound. an improvement in the distribution of the deposited nickel in millimetre-sized groove-features on the cathode surface was observed. lemaire et al. [21] focused their work on nickel electrodeposition from an eutectic licl-kcl melt, which is the most frequently used molten salt in industrial processes. a discrete fourier transform (dft) analysis was carried out of the interfacial processes which occurred in the first steps of electrodeposition. by means of molecular modelling techniques, the most stable complexes were determined, and their interaction with the nickel cathode during electrodeposition process was studied. the results showed that complexes with more than four chlorine atoms were not stable. a computational model for nickel electrodeposition from acid solutions (ph 1) was presented by lantelme et al. [22]. the growth of the nickel film prepared by electrodeposition was described by saitou et al. [23]. two kinds of growth rate were measured using columnar photo-resists food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 55 formed on indium-tin glass plates. the nickel film surface was analysed by atomic force microscopy. the ratio of the mean growth rate at the edge to that of the nickel layer indicated the presence of anisotropy between the up and down steps of the incorporation probabilities of the ad-atoms. experimental results suggested that the nickel growth process had a dynamic scaling property. 4. actual studies about nucleation, growth and structure of electrodeposits the structural and magnetic properties of nickel films growth by electrodeposition from sulphate solutions onto gaas surfaces have been studied by evans et al. [24]. in-plane x-ray diffractometry was used to study the nickel growth. the results showed that both the preferred growth relationship of growing film and the magnetic properties were strongly dependent on the substrate orientation. according to amblard et al. [25], the structure of the ni electrodeposits grown on oriented substrates resulted in a competition between an epitaxial growth process and a non-epitaxial growth initiated by a substrate-independent nucleation. this independent nucleation was a necessary step prior to the progressive development of a definite fibre texture in thicker deposits. both processes were investigated separately on two kinds of cathodic substrates: single crystals and amorphous carbon. for an amorphous substrate, multi-twinned particles with a roughly hemispherical shape were generated by independent nucleation. several competitive growth processes contributed to the whole current when the substrate was a low-index plane of a single crystal (cu or ni). models were also proposed which accounted for the experimental current-time transients. the same group discussed the quantitative x-ray diffraction analysis of the fibre texture exhibited by the nickel electrodeposits obtained from a watts bath. they investigated the preferred orientations exhibited by the nickel samples, which had been characterized by well-defined conditions of both preparation and x-ray diffraction analysis. experimental results described the quantitative modification of four different orientations – namely [110], [211], [100] and [210] – vs two relevant parameters; the ph of bulk solution and [100] orientation exhibited the character of a rather free growth, unlike the three other orientations. these were found to be associated with a definite chemical species ni(oh)2, hads or gaseous h2 which disturbed ni electro crystallization. correia et al. [26] used chronoamperommetry to study the ni and co electro crystallization onto carbon vitreous substrate and onto gold substrate from diluted chloride baths. scanning electronic microscopy and atomic force microscopy were used to view the surface morphology and particularly growth centres. 5. actual researches regarding influence of additives in the electrolyte solution previous research showed that the addition of a modifier, such as boric acid, enhanced the electro winning from high quality nickel deposits under a broad range of electrolyte parameters. inorganic additives showed little influence on the current efficiency. increasing either the nickel concentration or the electrolyte temperature improved the current efficiency, while decreasing the ph significantly reduced the current efficiency. the additives and operating parameters had a complex relationship in food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 56 terms of their influence on the deposition mechanism. mockute and bernotiene [27] examined the interaction of additives with the cathode during nickel electrodeposition in the watts electrolyte. the interplay of saccharin, 2-butyne-1,4-diol and phthalimide, were studied by the determination of the consumption rates of cathodic reaction products, and the incorporation of sulphur and carbon in the electrodeposits. a synergistic effect of the additives was observed. the aromatic compounds increased additive adsorption by carbonyl group, and 2-butyne-1,4-diol increased the adsorption of saccharin by sulphonyl group. in another study, the same authors examined the reaction mechanism of some benzenesulphobamide and saccharin derivatives during nickel electrodeposition in the watts electrolyte. it was found that the methyl group of oand ptoluenesulphonamides and n-methylsaccharin increased the rates of the consumption of additives, mainly by the acceleration of desulphurization reactions. the triple bond in the n-(2-butyn-4-ol) saccharin derivative complicated the reaction mechanism. the effect of cd2+ ions on the current efficiency, surface morphology and crystallographic orientation of the electrodeposited nickel from sulphate solutions has been studied by mohanty et al. [28]. their results indicated that cd2+ ions did not have a significant effect on the current efficiency but caused a noticeable change in the surface morphology and deposit quality. x-ray diffractometry was used to study the crystallographic orientation of the electrodeposited nickel. they also examined the effect of pyridine and its derivatives on the electrodeposition of nickel from aqueous sulphate solutions onto nickel and stainless steel substrates. the results indicated that the presence of additives did not have a significant effect on current efficiency, but changed the surface morphology of the deposits. the electrochemical reactions occurring during the deposition of nickel were examined by cyclic and linear sweep voltammetry techniques. kinetic parameters, such as tafel slope, transfer coefficient and exchange current density, were determined. froment and wiart [14] also studied the effect of different organic inhibitors, especially 2-butyne-1,4-diol on nickel deposits from a watts electrolyte on the basis of differential interferometry. the inhibiting action of an organic additive was characterized by a movement of the current density/cathode potential curve towards more negative potentials. the maximum amplitude was determined of the microrelief of a deposit. using impedance measurements, the process was also investigated in strongly acidic chloride and sulphate electrolytes and in electrolytes containing two additives: 2-butyne-1,4-diol and sodium benzenesulphonate. the additives had a more pronounced inhibiting effect in chloride than in sulphate solutions. in chloride electrolytes, the inductive low-frequency effect was observed at more negative potentials than in additive-free electrolytes. with the addition of sodium benzenesulphonate in watts or in chloride electrolytes, both an inductive and a capacitive low-frequency feature were present. these observations were interpreted on the basis of the specific effects of the anions. in sulphate electrolytes, the model involved the interaction between adsorbed hydrogen strongly bonded to the surface and the intermediate ad-ions niads+. in chloride electrolytes, the model were based on the slow desorption of an adsorbed anionic species. kuzeci et al. [29] studied the effect of organic commercial extractors lix841, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 57 cyanex272, d2ehpa, versatic10 and tbp with or without mg2+ ions, through different parameters of nickel electrodeposition on stainless steel substrate from aqueous sulphate solutions. there were not significant modification in current efficiency in presence of these additives, but there were observed changes in deposits morphology and in crystal orientation even when deposits were brightly, smooth and coherent. the changes also were observed in cathodic polarization behaviour in presence of these additives. nickel is often recovered by hydrogen reduction and electrodeposition from industrial waste and wastewaters. the quality of produced metal is strongly influenced by metallic and organic impurities, even at trace level. the impurities come from initial materials or are introduced in recuperation process. it was detected the effect of impurities in nickel electrodeposition process. organic reagents used for extraction from solvents are the major contamination source. these are adsorbed usually in active coal or other solid materials. the presence of lix64n in bath, even in low concentration, produces petting, fissures and deposits black coloured. also the authors noted a decreasing in current efficiency with increasing in lix64n concentration. also, bright and smooth nickel deposits were obtained in presence of d2ehpa, at high values of current efficiency (96%), but concentration has not exceeded 50 mg/l. lin and co-workers [30] examined the influence of ammonium ions on the texture and structure of ni deposits plated from a sulphamate bath onto a copper plate. the detailed microstructure of the nickel deposits was characterized using planeview and cross-sectional tem. the results indicated that the presence of ammonium ions in the bath created harder nickel deposits. the internal stress of the deposits also increased markedly when 100 ppm of ammonium ions was added. references 1. bard, a., j., electrochemical methods. fundamentals and applications, john wiley and sons, new-york, 2001 2. badea, t., popa, m., v., nicola, m., ştiinţa şi ingineria coroziunii, editura academiei române, bucureşti, 2002 3. brenner, a., electrodeposition of alloys, vol.i, academic press, new york, 1963 4. yang, z., zhang, z., leang, w., ling, k, zhang, j., trans.nonferros met.soc.,china, 16(2006)209-216 5. holm, m., o,kofe, t., journal of applied electrochemistry, 30 (2000) 1125-1132 6. zhao, h., liu, l., zhu,j., tang, y., hu, w., materials letters, vol. 61(2007)1605-1608 7. oriňáková, r., strečková, m., trnková, l., rozik, r., gálová, m., journal of electroanalytical chemistry 594(2006)152-159 8. giz, m., j., machado, l., ticianelli, e., a., gonzalez, e., r., ecl.quim., sao paulo, 28(2003)21 9. jensen, a., d., pocwiardowski, p., persson, p., hultmann, l., moller, p., chemical physics letters 368(2003)732-737 10. oriňáková, r., turoňová, a., kladeková, d., gálová, m., smith, r., journal of applied electrochemistry (2006) 36957-972 11. ji, j., cooper, w., c., dreisinger, d., b., peters, e., journal of applied elecrochemistry, 25(1995) 624 12. gómez, e., műller, c., pround, g., w., vallès e., journal of applied electrochemistry, 22(1992)872 13. epelboin, e., joussellin, m., wiart, j., journal electroanal.chem. ,119(1981)61 14. wiart, r., electrochim. acta, 35(1990)1587 15. holm, m., o,kofe, t., journal of applied electrochemistry, 30 (2000) 1125-1132 16. bozhkov, c., tzvetkova, c., rashkov, st., butniok, a., journal eelectroanal. chem., 296(1990)453 17. abyaneh, m., fleischmann, m., journal of electroanalytical chemistry, 530(2002)89 18. abyaneh, m., journal of electroanalytical chemistry, 530(2002)82 19. trevisan-souteyrand, e., maurin, g., mercier, d., journal of electroanalytical chemistry, 161(1984)17 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 58 20. jensen, a., d., pocwiardowski, p., persson, p., hultmann,l., moller,p., chemical physics letters 368(2003)732-737 21. lemaire, g., hebant, p., picard, s., g., journal mod. struct., 419(1997)1 22. lantelme, f., seghiouer, a., derja, a., journal of applied electrochemistry, 28(1998)907 23. saitou, m., oshikawa, w., makabe, a., journal of physics and chemistry of solids 63 (2002) 1685-1689 24. evans, p., scheck, c., schad,r., zaugari, g., journal of magnetism and magnetic materials, vol. 260(2003)467-472 25. amblard, j., froment, m., maurin, g., trevisan, e., electrochim.acta, 28(1983)909 26. correia, n., a., machado, s., avaca, l., a., journal of electroanalytical chemistry, 488(2000)110 27. mockute, d., bernotiene, g., vilkaite, r., surface & coatings technology, 160 (2002) 152-157 28. mohanty, u., s., tripathy, b., c., singh, p., das, s., c., mistra, v., n., journal of applied electrochemistry (2005) 35-545-54 29. kuzeci, e., kammel, r., gogia, k., journal of applied electrochemistry, 24(2004)730736 30. lin, c., s., hsu, p., chang, l., chen, c., h., journal of applied electrochemistry, 31(2001)925-933 31. rasmussen, a., moler, p., somers, m., a., j., surface & coatings technology, 200(2006)6037-6046 32. vasilache v., gutt gh. vasilache t., studies about electrochemical plating with zinc nickel alloys the influence of potential through stoichiometric composition, revista de chimie, 59(2008)9, 10051009; 2008 33. vasilache v., gutt gh. vasilache t., electrochemical researches about influence of the aditives of watts s solutions on throwing power and brightness, revista de chimie, bucureşti, 59,(2008)8, 912-919, 2008 34. vasilache, v., gutt, s., gutt g., vasilache t, filote c., sandu i, studies of hardness for the electrodeposited nickel from watts baths with addition of polyvinyl pyrrolidone (pvp), revue roumaine de chimie, 2009, 54(3), 245-248 35. vasilache v., gutt g., vasilache t, sandu i., studies concerning nickel electrodeposition from watts bath with addition of polyvinylpyrolidone (pvp), revista de chimie, bucureşti, 60(2009)1, pag.15-19 36. vasilache v., gutt s., gutt g., vasilache t, sandu i., sandu g.i., ditermination of the dimension of cristalline grains of thin layers of zinc nickel alloys electrochemically deposited, metalurgia international, vol.xiv(2009), no.3, p.49-53 37. vasilache t., gutt s., sandu i., vasilache v., gutt g., risca m., sandu a.v., electrochemical mechanism of nickel and zinc-nickel alloy electrodeposition, recent patents on corrosion science 2010, 2, 1-5 pg., issn: 18776108 contribution about nickel electrodeposition from watts bath with addition of polyvinyl pyrrolidone food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 79 influence of wheat flour dough hydration levels on gas production during dough fermentation and bread quality *georgiana g. codină1, silvia mironeasa1, daniela v.voica2 1* stefan cel mare university, faculty of food engineering, codina@fia.usv.ro, silviam@fia.usv.ro 2rompan, breadmaking and flour products industry, daniela_voica@hotmail.com *corresponding author received 2 august 2011, accepted 25 september 2011 abstract: the purpose of this study was to investigate the influence of different dough hydration levels on gas production during dough fermentation and on the quality of the finished bakery products. experiments were performed using like row materials wheat flour with an average quality for breadmaking and a compressed yeast saccharomyces cerevisiae type. the hydration level of dough analyzed was equal to the wheat flour water absorption optimum value determined by a farinograph device and values lower and higher than the 10% of this. a fermentograph sja device was used to analyze gas production during 10, 20, 30, 45, 60, 75 and 90 minutes fermentation times. like finished bakery products investigated in this study was loaf volume, height, length, height/ length ratio and bread humidity. a series of graphical representation was made using a statistica 6.0 software between dough gas production at 90 minutes of fermentation time, the different hydration levels used and some parameters of the bakery products obtained. once with the increase of water content in dough, the fermentative activity of yeast and the quality of bread obtained through baking tests is stimulated. so, the gas production level increase in all fermentation times analyzed, the bread loaf volume is enhanced, the parameters height and height/ length ratio decrease and the bread parameters length and dough humidity increase. key words: wheat flour, fermentation time, water content, gas production, bread quality 1. introduction for a saccharomyces cerevisiae type with a given biochemical composition, the conditions of the dough medium such as temperature, osmotic pressure, ph, level of water, sugar, salt, affect the fermentation rate of yeast. generally, dough’s with low water content take longer to ferment as compared to that’s with high water content. with additional water, the soluble solids are diluted and the osmotic pressure on the yeast cells is reduced. this causes an increase in yeast activity and the overall of fermentation rate [1]. although numerous factors have a bearing on the fermentative activity of yeast, we should remember that the baker judges this activity by the rising of the dough. this is the result of the force exerted by the increase in internal pressure (impermeability plus carbon dioxide production) and the resistance of the dough to deformation [2]. so, the carbon dioxide causes the gluten proteins to stretch, and some escape, but most of the gas is retained and is trapped within the matrix. this process is known as dough leavening [3]. we can say that the gas production is the most important effect of fermentation process because it creates the foam like structure of dough that is the prerequisite for a rapid heat flow through the dough’s. heat applied to mailto:daniela_voica@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 80 dough’s permits the gas and water vapor to be expelled from the dough while starch gelatinization causes the frothy structure of the dough to set, thus producing the bread crumb [4]. various methods based on monitoring gas production have been used to characterize the physical properties of dough fermentation process [5-8]. fermentograph sja (sweden) or advanced type rheofermentomenter (chopin, france) can describe this dough behavior [9]. the objective of this study was to study the influence of dough hydration level on the yeast fermentative activity during 90 minutes of fermentation time and bread quality. 2. materials and methods 2.1. materials. commercial wheat flour (harvest 2008) was milled on an experimental buhler mill from s.c. vel pitar s.a. valcea. flour quality tests were accomplished according to romanian standard methods: ash content (sr en iso 2171:2002), moisture content (sr en iso 711:1999), wet gluten content (sr en iso 21415-1:2007), gluten deformation (sr 90/2007), protein content (sr en iso 20483:2007), fat content (sr 90/2007), sugar content (sr 90/2007), falling number (sr en iso 3093:2007) and the viscoelastic parameters, specific for the farinograph method (sr iso 5530-1:1999) and for the alveograph method (sr iso 5530-4:2005). the yeast used in this study was saccharomyces cerevisiae type made by s.c. rompak s.a romania in compressed yeast commercial form. yeast quality tests were accomplished according to romanian standard methods: humidity (stas 98579), protein content (sr iso 1871:2002), yeast dough gas leavening ability release using a fermentograph sja (sweden). the dough fermentation behavior was analyzed at different hydration levels using the sja fermentograph (sweden). the amount of water used in dough was equal to the wheat flour water absorption optimum value determined by a farinograph device and values lower and higher than the 10% of this. the dough for the fermentograph test is prepared in farinograph (brabender, germany). after 5 minutes of mixing the dough sample is placed into the temperate fermentograph chamber at 35˚c. fermentograph plotter registers the changes in the increase volume of gas production at different fermentation time (10, 20, 30, 45, 60, 75 and 90 minutes). bread recipe comprised flour, water (accord. different wa, %), 2% compressed yeast (% flour basis) and 1.5% salt (% flour basis). baking tests were done and bread characteristics (loaf volume, height, length and ratio height/length) were analyzed according romanian standard method (stas 91:1983). 2.2. data analysis. all determinations were performed at least in triplicate. values of parameters were expressed as the mean ± standard deviation to a confidence interval for mean of 95%. treatment of data was analyzed using statistica 6.0 software. 3. results and discussion 3.1. analytical characteristics. the characteristics of the flour samples are shown in table 1. according to the table 1 data the flour used like row material has a good to average quality for bread making. the compressed yeast sample used has the humidity of 67.48%, protein content of 40% and the gas leavening ability during 60 minutes, 120 minutes and total gas release of 740/1190/1930. the yeast fermentative activity expressed by the gas production obtained with the fermentograph sja device on different dough hydration levels is shown in table 2. by changing the dough hydration level its consistency changes, affecting the bakery yeast fermentation rate. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 81 table 1 physicochemical and rheological properties of flours analytical characteristics mean value ± std. deviation rheological characteristics mean value ± std. deviation parameter parameter ash content (%) 0.65 ± 0.02 water absorption (%) 57.1 ± 0.5 moisture content (%) 14.6 ± 0.06 development time (min) 1.7 ± 0.08 wet gluten (%) 27.2 ± 0.4 stability (min) 2.4 ± 0.05 gluten deformation (mm) 6 ± 1 weakening (b.u.) 83 ± 0.7 protein content (%) 11.46 ± 0.22 tenacity (mm h2o) 65 ±0.2 falling number (sec.) 354 ± 2 extensibility (mm) 62 ± 0.8 baking strength (10-4 j) 121 ± 1.2 configuration ratio 1.05 ± 0.05 comparing the samples with 51.4% water content to the samples with 62.8% and 57.1% water content, the highest values of gas production was obtained for the samples with a level of 51.4% water content for all the fermentation times taken into account. so, the highest quantity of dough gas production was obtained when the level of water content in dough increase. these results are in agreement with those obtained by potus j. et al. (1994). this is to be explained because the increase of environment humidity leads to an increase in enzyme mobility and the possibility to react with the substratum. as a consequence, sugar hydrolysis intensifies and leads to an increase of the quantity of fermentable sugars which are metabolized by yeasts that form carbon dioxide. from the point of view of bread characteristics (table 3) obtained from wheat flour dough with different hydration levels, an improvement in the bread quality is to be noticed in proportion with the increase of the water content used for bread making. table 2 gas production (cm3) during dough fermentation time at different dough hydration levels (mean value ± std. deviation) water content (%) fermentation time (min) 10 20 30 45 60 75 90 51.4 70 ± 1 185 ± 1.2 325 ± 1.3 530 ± 1 735 ± 2 1025 ± 2.2 1270 ± 2 57.1 80 ± 1.4 195± 1.3 335 ± 1.9 560 ± 2 785 ± 2.2 1125 ± 1 1410 ± 2 62.8 100 ± 0.9 205 ± 1.7 370 ± 1.9 595 ± 1.2 825 ± 2 1157 ± 2.5 1433 ± 2.2 table 3 bread characteristics at different dough hydration levels characteristics abbreviation range mean ± std. deviation minimum maximum loaf volume (cm3) v 312.00 370.00 341.33 ± 20.00 height (cm) h 5.10 5.50 5.30 ± 0.20 length (cm) l 11.50 14.10 13.03 ± 1.36 height/length h/l 0.36 0.48 0.47 ± 0.06 bread humidity (%) dh 41.08 44.83 42.98 ± 1.87 water content (%) wa 51.40 62.80 57.10 ± 5.70 gas production (cm3) gp 1270.00 1433.00 1371.00 ± 88.22 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 82 this is to be explained because an increase in dough water content leads to an increase of the quantity of starch hydrolyzed in the fermentation process. this leads to an increase of maltose quantity and implicitly to an increase of the quantity of gas production. it is known that the bread loaf volume can be directly related to the quantity of sugars in the flour, including those sugars produced from starch by diastatic action [10]. as the flour taken into account presents an optimum water content of 57.1%, we can state notice that reaching a level of water content of 62.8% does not worsen the rheological properties of the dough significantly. as a consequence, a higher loaf volume of the bread obtained will be the result of a higher quantity of gas relieved in dough capable to retain the gas produced. it was found that using a low amount of water content leads to bakery products with low loaf volume and humidity. loaf volume of the bakery products increase gradually with the increase of water content in dough respectively with the decrease of dough consistency which is consistent with the gas production at different hydration levels of dough as it shown in figure 1. figure 1. variation of loaf volume (v) with gas production (gp) and water content (wa): spatial representation as the water content used in dough will be increase the bakery products result will have higher loaf volume and humidity as it shown in figure 2. figure 2. variation of bread humidity (u) with gas production (gp) and water content (wa): spatial representation it should be taking into account that bread humidity should not be very high because it could have negative influence on bakery products preservation. the h/l ratio variation is shown in figure 3. figure 3. variation of height/length (h/l) ratio with gas production (gp) and water content (wa): spatial representation it could be noticed that its value decreases with the increase of the amount of water used in dough which it could be explained by an increased of the dough length as it shown in figure 4. from the point of view of dough height this presents a maximum to an optimum wheat flour hydration level as is shown in figure 5. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 83 figure 4. variation of length (l) with gas production (gp) and water content (wa): spatial representation figure 5. variation of height (h) with gas production (gp) and water content (wa): spatial representation 4. conclusions the water content in the dough influences both the fermentative activity of yeast and the quality of the finish bakery products. the carbon dioxide relieved during dough fermentation presents higher values in samples with 57.1% and 62.8% water content than in samples with 51.4% water content due to the increase of the quantity of free water in the system, which ensures the mobility of molecules, necessary for the development of biochemical processes in the dough. regarding the volume of the samples obtained, the best results were registered for the 62.8 % dough water content. also, the bread parameters height, length, height/length ratio and bread humidity vary with the increase of dough hydration level in the way of the decrease of bread parameters height and height/length ratio and the increase of bread parameters length and dough humidity. 5. references 1. potus j., poiffait a., drapron, r., influence of dough-making conditions on the concentration of individual sugars and their utilization fermentation, cereal chem., 71. 505-508, (1994) 2. poitrenaud b., yeast in handbook of food science, technology and engineering, vol. ii, editated y.h.hui, usa, chapter 69, (2006) 3. hutkins r.w., microbiology and technology of fermented foods, blackwell publishing, chapter 8, p.281, (2006) 4. meuser f., valentin m., fermented dough in bread production, in handbook of food and beverage fermentation technology, editated y.h.hui, usa, chapter 42, (2004) 5. shuey w.c., practical instruments for rheological measurements of wheat products. cereal chem., 52. 42-81, (1975) 6. rubenthales g.l., finney p.l., demaray, d.e., finney, k.f., gasograph: design, construction, and reproductibility of a sensity 12-channel gas recording instrument, cereal chem., 57. 212-216, (1980) 7. mert b., a new instrumental setup for determination of small amplitude viscoelastic properties of dough during fermentation, eu. food res technol., 227. 151-157, (2007) 8. ktenioudaki a., butler f., gallagher e., dough characteristics of irish wheat varieties ii. aeration profile and baking quality, lwt-food sci technol., 44. 602-610, (2011) 9. svec i., hruskova m., wheat flour fermentation study, czech j. food science, 22. 17-23, (2004) 10. henry r.j., saini h.s., characterization of cereal sugars and oligosaccharides. cereal chem., 66. 362-365, (1989) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 5 risk to human health caused by non conditional drinking water *borys skip1, sonia gutt2, štefan stanko3 1 chernivtsi national university by yuriy fedkovich, chemistry department, ukraine e-mail: b.skip@chnu.edu.ua, *corresponding author 2 suceava university, food engineering faculty, romania e-mail: gutts@usv.ro 3 slovak university of technology, department of sanitary and environmental engineering bratislava, slovakia e-mail: stefan.stanko@stuba.sk received 12 october 2010, accepted 5 april 2011 abstract: drinking water quality is one of the key factors that may cause harmful effects to health, especially during lasting continuous consumption. this report analyzes information on current state of some basic health factors for chernivtsi citizens depending on their place of living and consuming drinking water from local wells. non quality contact medium risk analysis and some peculiarities of this function application for problem of kinetic modelling are under discussion as well. the analysis of risk increase effects during natural nitrification process on the basis of two kinetic models of the nitrification cycle is studied. the analysis is focused on two cases: when enzymes concentrations are constant and when nitrifying bacteria grow. the numerical solution of kinetic model and geoinformatical analysis are used to explain possible causes of high risk values and oncological diseases occurrences that may be caused by nitrate contamination. the question: why nitrates pollution level correlates with oncological pathology is under discussion of the study. an attempt to forecast health quality factors for citizens taking into consideration nitrogen intermediates is made. a simple kinetic model of immune status of people under chemical pollution of drinking water is considered as well. a comparison of the results of simulation with real statistic illnesses data and contaminant dispersion is the subject of the study. © 2011 university publishing house of suceava. all rights reserved keywords: drinking water quality, risk assessment, mathematical model, illnesses, nitrite, nitrate, kinetic, nitrification cycle. 1. introduction health risk assessment is one of the elements for methodology of risk analysis that pre-empts risk assessment, determines risk and informs about risk factors. from the point of view of science health risk assessment is a successive, systemic consideration of all the aspects of a factor or factors’ influence being analysed on human health including admissible levels of influence. most typically in practice while making risk assessment is obtaining and generalizing data about possible influence of human environment factors on health necessary and sufficient for hygienic argumentation of optimized, substantiated managerial decisions to eliminate, decrease or taking pre-emptive actions against environmental pollution, environmental objects monitoring optimization [1]. among typical practical tasks is to work out methods and strategy of regulation measures, by comparing and ranking degrees of manifestation of human environment influence factors, formulation and adoption of more reliable and safer levels of influence and hygienic standards, monitoring direct and indirect indicators of human health and health risks. thus, in such a way it is possible to improve the system of hygienic regulation and its harmonization with internationally established principles, criteria and methods of establishing safe levels of chemical substances influencing human health. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 6 2. materials and methods modern methodology for comparative risk assessment for human health is based on risk factors connected with contact environments. as a rule, comparative assessment precedes conducting in-depth studies of risk assessment and is done on the basis of the data about possible adverse effects of chemical substances. on the previous stage it is useful to apply a conceptual model of the area that is an aggregate of ties between the objects of the system under study. the objective of the present paper is to make an attempt to calculate risk factors, draw a comparison of computational methodologies and provide explanation of the influence on health based on example of using surface waters of poor quality for consumption needs by population. and to explain possible reasons of correlation of cancer disease rate with the level of nitrate pollution. in a previous paper (skip and nakonechny, 2010, in press) a poor correlation between the quality of drinking water of decentralized water supply (nitrate contents) and cancer sickness rate, in particular general pathology, and that of digestive apparatus and abdominal cavity localized similarly to distribution of surface waters contamination by nitrates is made. the area under study is characterized by the absence of industrial companies and other overland runoffs except those of private households. that is why the number of toxic substances in water is limited to health protection unit, decree #383 “drinking water” [2] with a list of substances to be under control that are monitored. among these substances we can single out typical ones among carcinogens [3-5] according to their maximum admissible concentration (mac) in ground water (e.g. some pesticides, surfactants) or known and probable human carcinogens [6] (ammonium, nitrates, nitrites). it is worth mentioning that some pesticides such as ddt have not been used for at least ten years, and according to water samples from wells these substances are not found. that is why to establish the area distribution according to this pollutant is not possible. thus, the main factors of carcinogenic risk are nitrogen-containing compounds in concentrations that get closer to or exceed their mac and also new synthetic organic substances (surfactants, pesticides) that are not included in the list that regulates drinking water in ukraine. but such an analysis of the situation needs detailed chemical analysis of organic substances in drinking water. due to the lack of technical and financial possibility to carry out such studies their analysis is beyond the objective of the present paper. that is why we shall dwell on the previous methodology of nitrate pollution. the well known nitrification chain of ammonia nitrogen with enzyme catalysis can be described by the following reactions: where e1, e2, [e1nh4+], [e2no2–] – enzymes and corresponding enzymesubstrate compounds. in ground waters after discharge of ammonium nitrogen under favourable conditions and in the presence of nitrifying bacteria, nitrification reactions take place. model eq. (1)–(4) is the simplest one that accounts for fermentation kinetics. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 7 it does not take into account the reproduction of nitrifying bacteria. as model calibration (establishing constants of appropriate reactions speeds for the process eq.(1)–(4) we used experimental data [7]. the nitrifying bacteria reproduction can be calculated by the malthus equation as for ground waters substrate contents for nitrification is relatively low, intra-specific competition of the bacteria can be ignored. as regards the most adequate description of bacteria reproduction kinetics, we can presume in a separate study that the rate of bacteria reproduction will depend on substrate concentration. as a rule, massive discharge of ammonia nitrogen of faecal origin happens during thawing snow, heavy precipitation, and filtration of waste water from settlers and septic tanks [8]. that is why such a process will be of periodic, stochastic character and, as a result, risk assessment is problematic in terms of current exposition and dose as a function dependent on time and concentration averaging function. 3. results and discussion for risk assessment [1] and different ways of averaging concentration and the rate of polluted water penetration into the body during nitrification, a mathematical model of fermentative process eq.(1) – (4) was used, the results of modelling being shown in fig. 1 (solid lines). by comparing the field data and modelling results, we can see that the model, taking into account monod kinetics, satisfactorily describes the dynamics of nitrification. from the analysing field data [7], (markers in fig. 1) we can see that nitrification process lasts roughly about fifty days. we can see in fig. 1 that the secondary contamination in practice requires quite a long time and nitrite ions are found whether nitrate contamination is of a permanent nature. comparing mac values for three forms of nitrogen mac 5.0 4nh  , mac 1.0 2no  , mac 50 3no  mg/l (directive 80/778/ eu [9]), we can see that the most toxic are nitrites and ammonia ions. when calculating mac values for the above mentioned elements we used reference values. as we can see from fig. 2a, excess of mac for ammonia ions and nitrates (as a result of its presence in consumed media) within time framing up to 5 days from the emergence of ammonia nitrogen according to kinetic fig. 1 – is quite high. а) b) figure 1. nitrification kinetics. solid lines — model results of concentration, dots and markers – experimental data. (а) without and (b) taking into account nitrifying bacteria reproduction food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 8 the aggregate pollution on higher levels than mac 1 is determined by nitrite and ammonia ions and lasts for fifty five days. as nitrification is accompanied by the process of reproduction of the appropriate bacteria from which enzyme concentration depends in water, the components of nitrification process safety assessment will be more accurate taking into account the process of nitrifying bacteria reproduction. as it turned out from the analysis of modelling outcome, this process is sensitive to the influence of nitrifying bacteria depending on speed of behaviour and duration of secondary (nitrite) pollution. this sensitivity is manifested especially in the decrease of time of massive secondary water pollution (fig.1b) as a result of nitrobacter and nitrosonomas in comparison with the model (fig. 1a). actually the population of these micro organisms is responsible for widely known fact of self-purification of water objects from nitrites and its speed. the presence of toxic components in water and their considerable influence on the body takes (according to nitrites) ≈ 55 days according to the model data and ≈ 50 days according to the experimental data, and health effects in this period of time are substantial. it is worth mentioning that surpass of mac (fig. 1b) of no3– for model situation is not observed (0.014 mac), and of no2– and nh4+ in accordance with model outcome, is 2.8 and 1.4 mac respectively or in correlation 3 2 4 3 2 4 : : 1:200:100no no nh no no nh c c c mpc mpc mpc        it is worth mentioning that mac а) b) figure 2. water pollution level. (а) without and (b) taking into account nitrifying bacteria reproduction а) b) figure 3. pollution substance hazard quotient while consuming water with dynamic concentration of nitrogen forms that contaminate water according to model (1)-(4). averaging intake of pollutant substance to digestive tract is one day. intake rate ir(t) = const. (а) without taking into account bacteria reproduction, and (b) taking into account bacteria reproduction. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 9 exceeding (fig. 2b) for no3– for model situation is not observed (table 1 line 5), components of nitrification process concentration at the point of time corresponding to the maximum of nitrites have the correlation no3–: nh4+: no2– = 1:467:2.1·105. as we can see by comparing fig. 1a and 1b, taking into account nitrifying bacteria reproduction, it describes ammonia nitrogen transition to nitrate more realistically in the nitrification model. major water pollution will be caused by aggregate ammonia and nitrite contamination at the level of 6.4 of the part of mac. the level of pollution will be at the limit of designation that is a negligibly small value. but hazard quotient calculated by using the average of concentration will be incorrect for the model after fifty day time (fig. 3b), as after 50 days neither ammonia nor nitrite nitrogen in water in essential concentrations will be observed (fig. 1b and 2b). by applying equations (5) and (6) we can see that concentration changes in time and hazard quotient has different form in time frame up to 50 days. but taking into account the concentration changes of all the forms of nitrogen during nitrification enables us to calculate more accurately the hazard quotient. such an approach to hazard quotient in practice is somewhat inconvenient: first, it is next to impossible in most cases to establish the onset of ammonia ions getting into ground water. taking into account the change in concentration in time implies constant monitoring of water object that is possible only in automatic stations. in most cases specific hazard quotients are calculated for averaging concentrations as discrete quantities but for complex integral assessment of hazard quotient and use of risk function for modelling pollution impact on population health consuming such coupling medium, a continuous function is needed. according to [1] health risk may be assessed by consumed dose as:   2 1 )()( t t dttirtcdose , (5) c(t) – concentration and ir(t) – intake rate of intake of contaminant substance during time t2–t1.or: 365   atw edefvc dose , (6) under conditions c(t) and ir(t) = const. ed= t2–t1, v – volume of consumed medium (water), ef – rate of event of consumption, w – weight, at – exposition time extrapolation. taking into account the substance toxicity that is determined by its mac or reference dose then hazard quotient may be calculated: hq=dose/rfd. (7) the analysis of correlation (6) and fig. 3 indicate constant increase of consumed nitrates dose and absence of dose decrease with the absence or decrease of pollution level for nitrites and ammonia after fifty days from the onset of nitrification process. without taking into account the nitrate table 1 correlation of inorganic nitrogen forms during nitrification process №   4 4 nh nh mpc c   2 2 no no mpc c   3 3 no c no mpc c  1 0.4 1 0.0025 1 2 0.5 5.9 0.0016 6.4 3 0.15 0.84 0.004 1 4 0.87 5.6 9·10-3 6.4 5 0.014 6.4 3·10-5 6.4 6 0.35 6 0.004 6.3 lines no. 1,2 – stoichiometric calculations. lines no. 3,4 – model data without taking into account bacteria reproduction. lines no. 5 – model data when taking into account bacteria reproduction. lines no. 6 – experimental data [7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 10 consumption by biota, the dependency of hazard quotient of each of the nitrification process components on time, shown in fig. 3, is depicted by bold curves. by analysing the data received, it turned out that ammonia nitrogen hazard quotient increases steadily in time. nitrite and nitrate forms of nitrogen have similar tendencies that unlike the dynamics of concentrations fig. 1, are of monotonous increasing nature. the hazard quotient values of ammonia ions and nitrites do not exceed the values of 2.3·10-5 and 1.4·10-4 respectively. the hazard quotient caused by nitrites which value is 1.3·10-6 is considerably lower than ammonia and nitrite hazard quotients. thus, the risk caused by no3– is considerably insignificant compared with nh4+ and no2– risks. as in real practical activity in ukraine the quality control of decentralised water supply is made for no3– concentration, then no2– and nh4+ concentration may reach dangerous, including carcinogenic nature, values; as a result correlation of most conservative component of nitrification process no3– , there is a cancer rate observed in practice (skip and nakonechny, 2010. in press.). aggregate hazard quotient may be calculated for the situation with ground water contamination by household nitrogen containing discharges, e.g. for a situation mentioned in (skip and nakonechny, 2010. in press.). a weak correlation between high content of nitrates and oncological pathology is observed. but nitrates at present do not belong to carcinogens or potential carcinogenic substances. that is why oncological pathology may be caused by nitrite ions causing secondary water contamination in the nitrification process. there is a logical question: how do ion no3– concentrations correspond to ions nh4+ and no2– concentration values on similar or lower than mac levels? to answer the question we shall use the procedure of minimization for calculating these values. in this way we obtained the correlation of concentrations (table 1 line 2) that corresponded to lower or equal mac values for three forms of nitrogen at the moment of maximum nitrite concentration. it follows that out of the three forms of nitrogen during natural nitrification process, decisive contribution to toxic and possibly carcinogenic risks will be caused by intermediate form of nitrogen – nitrites, and toxic – by a combination of ammonia and nitrite ions, and toxic risk – by ammonia ions and nitrites. though in literature [10, 11] there is no clear view about cancerogenity of nitrates and nitrites on human body, from the point of view of chemistry, nitrosation reaction in a human body is possible. and thus nitrosation products – nitrosamine are carcinogenic. moreover, transferring the experiments’ results on animals is not always adequate for the effects observed in a human being. that is why the information about the absence of carcinogenic effect in lab mice, rats etc. may not coincide with epidemiological studies for the inhabitants of the locality in question. in such cases it is obviously necessary to use the results of statistical observations of the ground water pollution level and epidemiological data as most reliable source of information about the influence of separate substances on the disease rate. of course, in practice in ground water there is a great number of organic, including nitrogen-containing substances. but having a systemic monitoring of sources of water supply in terms of quantitative and qualitative analyses of contamination and applying modern multivariate analysis or neural networks one can with sufficient reliability establish the cause and effects of the relationship dose-effect as such, and effects of synergism. it is worth mentioning that consumed dose calculation is sensitive to time of averaging food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 11 concentration of polluting substances. if the time of averaging concentration, e.g. takes up 146 days (maximum model time), then for contamination that took place about thirty days, the averaging will lead to the case when toxic effect (exceeding the average concentration of 1 mac) will not be observed. alternative way of pollution level assessment may be made using stoichiometric nitrification reaction. as can be seen from figure (1)–(3), 1 mol of nitrate ions is formed by 1 mol of nitrite ions and 1 mol of ammonia ions. if the water pollution level of nitrates reaches 9 mac, e.g. for ground water (b.skip and k.nakonechny, 2010, in press.), then it will be      3 3 3 3109 no no no mr mac  mol (the hypothesis is that we have 1 liter of water in natural setting). this number of nitrates formed from an equivalent quantity of nitrites, and exceeding water pollution in ammonia will make 3.261 10 3 4 43       nh nhno mac mr and nitrites 3338 10 3 3 23       no nono mac mr times. the approximate water toxicity may be assessed as 4.8% and 0.2% for values ld50 respectively for nitrites and ammonia ions for a grown-up. when nitrate pollution level is 1 mac, the concentration of ammonia and nitrate forms will be 29 and 371 mac respectively. thus, the controlling of pollution level of drinking water only by nitrate level according to health protection unit, decree #383 “drinking water” [2] or directive 80/778/eec [9] does not require conformity to standard. but such an approach in health protection unit, decree #383 “drinking water” [2] to drinking water quality assessment of decentralized water supply causes dangerous situation in terms of health risks, including carcinogenic ones. apparently while assessing safety of drinking water consumption we should depart from allowable levels of water pollution by nitrites and ammonia ions. the calculations show that the water pollution level during nitrification process with interconnected nitrogen forms has the values from table 1 line 1. but we have to mention that stoichiometric calculations do not allow for length of nitrification process and dilution by ground waters. that is why at any moment components of nitrification reaction concentration will be lower and calculated by nitrification kinetics and process variances in aquifer. exceeding admissible level of drinking water contamination by inorganic nitrogen forms value   3 3 no c no mpc c or increase over the period of time less than fifty-five days larger than 0.004 that demonstrates exceeding admissible pollution level of drinking water by nitrites or ammonia in the past (according to model data) in areas of aquifer that is adjacent to pollution sources, should be avoided. referring back to experimental values of nitrates concentrations in wells, we can see that there are areas where the correlations of nitrate content and oncological disease rate exceed 9 mac. thus, water pollution level of ammonia ions and nitrites had to exceed permissible level of 261 and 3338 times respectively. insignificant discrepancy of values received by stoichiometric calculations (table 1 line 2) and kinetic model (table 1 lines 4 and 5) is evidently stipulated by not taking into account the dispersion of substances in aquifer, nitrification kinetics, kinetics of nitrifying bacteria reproduction, and difficulty to keep to the purity of experiment in real ecosystem with a great number of sources and asynchronous inflow of nitrogen-containing substances. this is corroborated by insignificantly understated outcomes between model values of nitrites concentrations with food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 12 experimental ones, fig. 1, and similar concentrations [7] ammonia and nitrite ions concentrations. the most probable reason of such discrepancy is a repeated inflow of ammonia ions. but despite the above mentioned drawbacks, the values of relative concentrations of nitrate ions in water may be used as an indicator of the water pollution level during nitrification process in aquifer by ammonia ions and nitrites. for sources of decentralized water supply from ground water, it is necessary to ensure in appropriate regulatory documents to make tests on the availability of nitrites and ammonia ions in water samples as mandatory with a frequency not exceeding half of massive nitrite pollution duration ~25 days to discover real state of affairs by nitrite and ammonia water pollutions and to prevent possible oncological effects. 4. conclusions therefore while establishing standard values for ammonia and nitrite nitrogen forms it is necessary to comply with the calculated values (table 1). the simplest is the stoichiometric calculation. otherwise, the risk of oncological diseases onset will remain at a significant level and toxic poisoning by nitrites and ammonia ions is possible. as nh4+ and no3– do not belong to apparent carcinogens, but nitrification process indirectly causes carcinogenic effect by means of secondary pollution by nitrites, that is why it is reasonable to consider ammonia ions as potential carcinogens or substances that potentially cause carcinogens formation for ground water and ground water supply. another alternative possibility is to consider carcinogens nitrification process with the above mentioned mac. 5. references: 1. guidelines for ecological risk assessment http://www.epa.gov/ncea/ last accessed 14 oct 2010. 2. dsanpin №383 (186/1940) state santitary rules and norms. drinking water. hygienic requirements for water quality of centralized potable water supply. 3. integrated risk information system. a-z list of substances. http://cfpub.epa.gov/ncea/iris/index.cfm?fuseaction =iris.showsubstancelist last accessed 14 oct 2010. 4. the pollution information site. carcinogens. http://www.scorecard.org/healtheffects/chemicals.tcl?short_hazard_name=cancer&a ll_p=t last accessed 14 oct 2010. 5. report on carcinogens; u.s. department of health and human services, public health service, national toxicology program. http://ntp.niehs.nih.gov/?objectid=72016262bdb7-ceba-fa60e922b18c2540 last accessed 14 oct 2010. 6. known and probable human carcinogens http://www.cancer.org/cancer/cancercauses/other carcinogens/generalinformationaboutcarcinogens/ known-and-probable-human-carcinogens last accessed 14 oct 2010. 7. m. straskraba and a. h. gnauck. freshwater ecosystems, modelling and simulation. elsevier science publishers, amsterdam and veb gustav fischer verlag, jena, 1985, 309 pp. 8. š. stanko reuse of waste water in slovakia, water supply sustainability. in risk management of water supply and sanitation systems impaired by operational failures, natural disasters and war conflicts:dordrecht: springer science-business media, 2009. – pp. 233-240. 9. council directive 80/778/eec of 15 july 1980 relating to the quality of water intended for human consumption // oj l 229, 30/08/1980 p. 0011 – 0029. 10. j.s. griesenbeck, j.d. brender, j.r. at al. maternal characteristics associated with the dietary intake of nitrates, nitrites, and nitrosamines in women of child-bearing age: a cross-sectional study environmental health 2010, 9:10 11. ingested nitrate and nitrite and cyanobacterial peptide toxins. iarc monographs on the evaluation of carcinogenic risks to humans. volume 94 (2010), 448 p. 223 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 22015, pag. 223 232 strategic alliances between firms – a model of competitive strategy in food industry *ciprian ionel hretcanu1, cristina elena hretcanu2 1faculty of economics and public administration, stefan cel mare university of suceava, romania, 1faculty of food engineering, stefan cel mare university of suceava, romania, ciprian_hretcanu @yahoo.com, cristina.hretcanu @fia.usv.ro *corresponding author received april 17th 2015, accepted june 25th 2015 abstract: a statistical analysis on food engineering companies from the north eastern of romania was made in order to emphasize the fact that alliances are clear examples for companies to achieve more important and stronger market presence. today, alliances are a fact of life for business, a substantial part of current operations as well as a future strategy. among other existing solutions, creating strategic alliances (through various types of cooperation, collaboration and partnership) is a solution for business firms to get competitive advantages. strategic alliances have become increasingly more and more important in the global economy and nowadays they are a kind of competitive weapon for survival in the business world. even if there have been made many alliances between business organizations, a "magic formula" for creating them has not been found yet, because each alliance has its own particularities and its specificity. since the foreign literature and romanian literature on strategic alliances, clusters, networking and other forms of cooperation / collaboration between firms are extremely broad, we shall present a synthesis and give a definition of strategic alliances, by focusing on the key reasons for creating strategic alliances. keywords: strategic alliances, advantages and disadvantages of strategic alliances, reasons of strategic alliances 1. introduction the current global competition induces a permanent pressure for new alliances, acquisitions or mergers between large multinational corporations in various fields [1]. in this context, the strategic vision of big corporations must necessarily take into account cultural differences that separate countries in the world to succeed in various foreign markets. in other words, only big companies build their "initial strategic framework", afterwards making appeal to a cycle of strategic management process (various distinct strategies are blended, changed or adapted quickly if the market requires etc.); we understand that the only element of stability in the global context is only this initial strategic framework, further on any adjustment or modification being even recommended [2]. companies have used strategic alliances as a key source of competitive advantage, and many have formed alliances worldwide with the objective of increasing the economic benefits of the parties involved. strategic alliances or strategic partnerships are a suitable option in situations where competitive advantage can be achieved together, not separately [3]. 2. materials and methods 2.1. strategic alliances to define clearly the terms used in this article various books and articles, which are specified in the references, were consulted. a questionnaire was applied to a total of 31 food companies, becoming a case study. http://www.fia.usv.ro/fiajournal mailto:@yahoo.com, mailto:@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 224 currently, the companies admit that both competition and cooperation are necessary to ensure optimal growth in a very restless world economy. strategic alliances are now considered to be one of the most powerful mechanisms for combining competition and cooperation and for industrial restructuring at global level. organizational alliances are favorite forms under the conditions in which the company has to be connected permanently to pulse standing market, the price mechanism remaining important, the risk of information leakage is not disturbed, the financial risks are high, the resources are limited and flexibility is very important. the term ”alliance” refers to a particular type of relationship between firms, and becomes an ”umbrella” for this cooperation, collaboration or other activities between those entities [4]. currently there are several forms of cooperation agreements between companies, organizations or enterprises and the terminology describing these agreements is far from uniform and consistent in the literature where we find various terms such as ”joint ventures”, ”collaboration contracts”, ”partnerships”, ”business alliances”, ”strategic alliances”, or simply "alliances" , "strategic coalition" [5] or "strategic network" [6]. it is very confusing for those who want to understand the field of application, characterization and distinguish between the different ways in which companies collaborate and therefore, in a general definition, we can designate practically as business alliances (or simply alliances) all forms of cooperation relations between different companies involving joint contributions and property and a common control. we consider a strategic alliance as a strategic association, collaboration or union or as a strategic pact. strategic alliances have been defined so far in the economic literature from different perspectives and as a result there are many definitions of this kind of collaboration. according to our own opinion, we can define strategic alliances as arrangements, agreements, collaboration, business or partnerships through which two or more partners, even if they are or can be competitors or potential competitors in the market, who want to cooperate with each other in mutual benefit to support or strengthen the competitive advantages of participating companies. we can also consider that an alliance is a “marriage” between partner companies and at the time when partners do not understand each other, then a “divorce” takes place, actually the dissolution of the alliance. even simpler, a strategic alliance is sometimes referred to as a "partnership", which gives organizations / business units the chance to unite forces in view of a beneficial mutual opportunity and support of competitive advantages [7]. alliances are created in order to achieve the objectives of the partners involved in. according to the experiences of the past 15-20 years it can be concluded that those companies that have a "strategic alliance", without any coherent and clear "alliance strategy" are almost sure to end up in failure [8]. the difference is not only of semantics. a strategic alliance refers to an agreement and a specific organization or a contract; an alliance strategy is much broader and deeper and includes four elements: a business strategy which shapes the design of the alliance; a dynamic view to guide the management of each alliance; a clear approach to manage the alliance firms; an organizational infrastructure to build and support the ability of alliance. the experience and the research made over the last few years show that there is no universal method of establishing strategic alliances, the methods applied in a case are not fit in another one, each strategic alliance having its specific characteristics. strategic alliances are considered to be intermediate in the relations between firms, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 225 being located between ordinary transactions and mergers between companies. strategic alliances are agreements between companies that remain independent and are often in competition. therefore, it is argued that the definition of strategic alliance involves some important issues that can be listed as follows [9]: a) strategic alliance is a cooperative strategy that needs a good partner to make a developing partnership; b) organizational resources and capabilities are shared, whereas the new ones are acquired and developed by a well-managed strategic alliance; c) participating firms pursue common objectives and create a process of adding value to improve their competitive advantage by creating competitive advantages of cooperation. the great diversity of alliances existing so far shows that strategic alliances are ideal tools to combine cooperation and competition in corporate strategies. the models of cooperation and competition can be classified into the following category [10]: to cooperate, then to compete: when companies are not ready to compete in a particular area, first they cooperate with competitors for short-term goals; then cooperating companies can compete with each other, once they have got their competence or performed a common standard; to cooperate while competing: companies can continue to compete while they cooperate in certain fields; to cooperate with each other and compete with others: companies can conclude cooperation agreements with third parties to compete. 2.2. reasons and contexts for involvement in alliances it should be mentioned that the formation or development of strategic alliances cannot be achieved overnight, as it involves several steps:  the decision of adopting a business alliance strategy;  selection of the right partner, depending on the reasons for which the alliance is formed;  negotiations based on each party’s needs;  establishing the partnership form and implicitly the management one, to ensure benefits for all the parties involved. the firm’s decision to adopt a strategy of alliance must be a decision based on the orientation of the company strategy [11], and according to various studies conducted on several companies it was found that strategic alliances are very important in the success of the participating companies. if companies cannot buy new technologies fast enough, it is essential for them to work together [12]. the second step in creating an alliance, partner selection criteria can be summarized as follows:  sharing of unique skills with the partner (skills and knowledge related to the alliance: the specific goals and objectives) [13][14];  resources including tangible and intangible assets, such as technical, managerial, financial and reputation [15];  co-specialized assets and access to such assets (including complementary resources and capabilities [13], [15], [16];  access to international or local markets [17];  alignment of partners’ compatibility including the cultural side, their strategic fit and the desire to contribute to the alliance [17];  inter-organizational learning potential to increase town skills [18]; in other words, we should have in view that for creating a successful alliance the following 10 rules must be obeyed [8] that we can call the “decalogue of successful food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 226 alliances” or “the 10 commandments of successful alliances”: 1. clear strategic goals alliances are never an end in themselves, they provide tools to achieve business strategy; 2. suitable partner a partner with compatible goals and complementary capabilities; 3. specialization the allocation of tasks and responsibilities in alliances should be made in a way that allows each party to do what they know best; 4. creating incentives for cooperation – working together does not happen automatically, especially when partners were previously rivals; 5. reducing conflicts between partners the scope of the alliance and partners’ roles should focus on avoiding the competition against each other in the market; 6. sharing information ongoing communication develops trust and maintains joint projects on target; 7. staff exchange irrespective of the alliance, contact staff and site visits are essential to maintain communication and trust; 8. operation with long-term horizons mutual tolerance in short-term conflict resolution is enhanced by the expectation of long-term gains; 9. development of joint projects successful cooperation in a single project can be helpful for partners on „stormy weather” in less successful joint projects; 10. flexibility – alliances are dynamic and open relationships which must evolve with their environment and look for new opportunities. the diversity of involvement reasons in a strategic alliance can be divided into four distinct categories [19]: organizational reasons (different types of learning and incorporating tacit collective and integrated skills; restructuring; performance improvement; acquisition of distribution facilities; recreation and supply extent in order to adapt to environmental changes; complementarity of goods and services to markets; legitimation.), economic reasons (finding a market; cost and resource sharing; risk reduction and risk diversification; achievement of scale economies; cospecialization.), strategic reasons (achievement of vertical integration; achievement of a competitive advantage; diversification into new businesses; access to new technologies; technological convergence; research and development; development of new products and technologies; cooperation with potential rivals or competitors’ disadvantage; monitoring trends in the industry) and political reasons (development of technical standards; overcoming of legal or regulatory barriers). by analyzing the reasons for the formation of strategic alliances we can say that alliances are created [20]: • to reduce competition, because more and more competition in most industries occurs between different strategic alliances, between firms and not in isolation this means that former competitors cooperate in a strategic alliance and thus they act against others who have already joined; • to acquire dimensional savings in production and marketing, since by pooling their efforts in the manufacture of components, assembly and marketing of products, companies can achieve economies of scale which are not feasible in terms of production volume of each company if taken separately; • to reduce disparities in terms of technical and production skills, as partners learn from each other about the conduct of joint research, the sharing of technologies; • to gain access to other markets or to learn from alliance partners. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 227 2.3. case study a case study was conducted on 31 companies in the food industry in the north east of romania, companies that run in the field of processing meat and meat products, milk and dairy products or bread making, as follows: 58% of the companies surveyed are enterprises with a number of employees ranging between 50 249, 32% of them with a number of employees between 10-49 and 10% have fewer than 9 employees. the aim of our research was to establish the influence of alliances, partnerships or collaborations between companies that are active in the food industry on their income and survival in the global market today. the objectives proposed in this research aim at: setting the number of business alliances, partnerships or collaborations made by business organizations and the percentage of annual turnover derived from these alliances; determining the main reasons for which companies get engaged in a business strategic alliance, partnership or collaboration; identifying the key elements influencing the results of a strategic alliance; setting elements that determine and influence the performance of an alliance or partnership; having in view the fierce competition of the global marketplace, companies can survive by making a larger number of alliances, partnerships or collaborations; over 50% of their turnover comes from these alliances; determining the reason for which a firm is involved in a business alliance, by facing competition in the market. 3. results and discussion according to the survey made, we have found out that 26% of companies are not involved in any strategic alliance business, 13% of them have already made alliances, 6% are part of 2-3 alliances and 55% of these companies are part of more than 4 alliances, partnerships and collaboration business, aspect graphically shown in the figure 1. fig.1 company’s involvement in business strategic alliances from the data analysis it is obvious that our first hypothesis is confirmed since almost three quarters of the companies analyzed (74%) have made at least one partnership, collaboration or a business alliance. considering the reasons for which a company gets involved in an alliance, partnership or collaboration we found the data shown in the figure 2. fig.2 reasons for which a company got involved in an alliance, partnership or collaboration from the above figure we remark that: 32% of them have made an alliance to deal with competition on the market (v1 in the 26% 13% 6% 55% without alliances 1 alliance 2-3 alliances more than 4 alliances v1 32% v4 13% v3 26% v2 29% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 228 graph), 29% of them to gain access to other markets or to learn from alliance partners (v2), 26% to reduce disparities in terms of technology and production (v3), 13% to capitalize scale economies in production and marketing (v4) and all. from the analysis of time when the company has become part of an alliance, a partnership or a collaborative form, one can notice that 91% of business organizations are involved for more than 4 years in such collaboration and 9% of firms for 2-3 years only. these data are shwon in the figure 3. fig. 3 how long the company has been engaged in a strategic alliance by studying the situation in which the company was part of a strategic alliance, partnership or collaboration that ceased to exist or did not exist at that moment, we found that 78% of the companies surveyed did not display such a situation and 3 % of them came across at least once with such a case, these issues being shown graphically in the figure 4. it should be mentioned that 19% of the companies surveyed preferred not to answer to this question. fig. 4 company has been part of a strategic alliance which does not exist at present another objective of our research was to determine the percentage value of the annual turnover of the companies studied resulted from business alliances which includes those organizations or companies. by analyzing the data obtained, we found that the annual turnover of 26% of the companies derives in the proportion of 11% to 30% from the alliances created, 52% of companies have less than 10% of their turnover being influenced by their alliances, and 9% of companies have the annual turnover in the proportion 31-49% deriving from the alliances created. it should be mentioned that in the case of 4% of the organizations studied, over 50% of their annual turnover is given by the alliances made as part of that organization, and 9% of companies did not want to specify any answer to this question. a graphical representation of the influence of alliances on the annual turnover of the organizations is given in the figure 5. fig.5 proportions of annual turnover derived from the alliances made to find out what the main elements influencing significantly every form of business alliance are, the following aspects are highlighted: selecting a compatible partner and a focus for setting the stage for communication based on mutual trust has the biggest influence for micro-enterprises (companies with 1-9 employees) and medium businesses (firms with 50-250 9% 91% less than 1 year 2-3 years more than 4 years 3% 78% 19% yes no i do not know / no answer 52% 26% 9% 4% 9% less than 10% 11-30% 31-49% more than 50% i do not know / no answer food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 229 employees); special attention should be given to critical information to maintain partners’ competitiveness being of the same strategic importance for all companies; firms with 10-49 employees have the lowest quotations on those elements that influence business alliances. these aspects are graphically presented in the figure 6, where: v1 selecting a compatible partner and a focus on setting a communication stage based on mutual trust; v2 choosing a partner whose resources and skills complement each other; v3 striving to learn more and faster about the partner’s technology and management used; v4 special attention to critical information so as not to be disclosed to the strategic partner in order to maintain competitiveness; v5 subsequent management of cooperative alliance. respondents were asked to specify the degree to which each option is fulfilled, ticking on a scale of 1 to 5 points (where 1 point represents “a very small influence” and 5 points represent “a very large influence”). means of scores were compared by using one-way analysis of variance (anova) and fisher (lsd) test, in order to assess significant differences among samples. all analyses were performed by using the software program xlstat™ (addinsoft©, u.s.a.). the variance analysis did not show significant differences among average scores given by firms with different number of employees (figure 6). in general, the average scores fell between 3 and 4.66 points in the scale for firms with 1-9 or 50-250 employees, respectively and between 2.1 and 3.6 points in the scale for firms with 10 49 employees. the variant v1 obtained the best scores. thus, “selecting a compatible partner and a focus on setting a communication stage based on mutual trust” has “a large influence” in creating strategic alliances. means with different superscript letters are significantly different (fisher (lsd) / analysis of the differences between the categories with a confidence interval of 95%) fig.6 elements that influence strategic alliances 4,33a 3,33a,b 3,67a,b 3,00a,b 3,33 a,b 3,60a,b 2,10b 2,10b 2,90a,b 2,40b 4,17a 3,28a,b 3,33a,b 3,28a,b 3,39a,b 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 average of v1 average of v2 average of v3 average of v4 average of v5 1-9 employees 10-49 emplyees 50-250 employees food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 230 in the figure 7, principal component analysis (pca) of data regarding the scores for the main elements influencing significantly every form of business alliance was computed. two principal components (pcs) accounting for 86.24% of the variation of scores can be observed in the figure 7. fig.7 pca for scores of elements that influence strategic alliances the first factor (f1) explains 66.07 % of the total variance with significant parameters v1, v5 and v3 and the second factor (f2) explains 20.17% of the total variance with significant parameters v2 and v4. by means of the next question “what are the reasons for performing an alliance or a partnership”, with the following items: v1 alliance or partnership provides improved access to technologies for the companies involved; v2 alliance increases the sales market and / or entering new markets; v3 alliance determines economies of scale and on this basis a reduction of costs; v4 competitive advantages are gained by pooling global resources; v5 improved access to new knowledge by identifying new distribution channels, new technologies or know-how; v6 access to capital is facilitated, respondents were asked to specify on a scale of 1 point (for “a very small”) to 5 points (“a very large”) the grade of influence. means of scores were compared by using one-way analysis of variance (anova) and fisher (lsd) average test. these aspects are shown graphically in the figure 8. means with different superscript letters are significantly different ( fisher (lsd) / analysis of the differences between the categories with a confidence interval of 95%) fig.8 elements rendering business alliances performing v1 v2 v3 v4 v5 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 f2 (2 0, 17 % ) f1 (66,07 %) variables (axes f1 and f2: 86,24 %) 4,33a 3,67a,b 3,33a,b,c 3,00a,b,c,d 2,00c,d,e 1,33e 3,10a,b,c 3,20 a,b,c 3,30a,b,c 2,90a,b,c,d,e 3,30a,b,c 1,40d,e 3,83a,b 4,06a,b 3,39a,b,c 3,39a,b,c 3,33a,b,c 2,67b,c,d,e 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 average of v1 average of v2 average of v3 average of v4 average of v5 average of v6 1-9 employees 10-49 employees 50 250 employees food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 231 every manager and company representative’s views were analyzed to identify the basic elements influencing the performance of any kind of collaboration and the following can be mentioned: firms with 1-9 employees consider that an alliance performance partnership improves access to new technologies for the companies involved; organizations with 10-49 employees consider that an alliance performance is equally due to several factors, such as: improved access of the alliance or partnership to new knowledge by identifying new distribution channels, new technologies or know how, improved access to technologies for the companies involved and the alliance benefits from increasing market outlets and / or from entering new markets; companies with 50250 employees believe that the main aspect determining the performance of an alliance resides in its increases regarding the sales market and / or entering new markets. the variance analysis showed significant difference (p ≥ 0.05) among average scores for “elements rendering business alliances performing”, given by firms with different number of employees (figure 8) and “alliance or partnership provides improved access to technologies for the companies involved” has “a large influence” in business alliances performing. in the figure 9, principal component analysis (pca) of data regarding the scores for the main elements influencing significantly every form of business alliance was computed. two principal components (pcs) accounting for 82.58% of the variation of the scores can be observed in the figure 9. the first factor (f1) explains 69.67 % of the total variance with significant parameters v2 and v4 and the second factor (f2) explains 12.91% with significant parameters v6 and v3. fig.9 pca for scores of elements rendering business alliances performing 4. conclusions strategic alliances regardless of their forms they can exist are being questioned by some researchers, considering that the secret of a real competitive advantage is the competition itself [21]. however, strategic alliances are a product of globalization that can respond best in certain situations to emerging challenges. alliances are a way to reap the rewards team effort and gains from forming strategic alliances appear to be substantial. therefore, we conclude that the creation of alliances brings significant profit as well as other benefits: increase in competition, increase of the need to operate on a global scale, rapid change of market and industry convergence in many markets. it is those who make appeal to such forms of cooperation type to decide whether to take this step and to assess long-term benefits that may arise from it. 5. references [1]. mockler r., multinational strategic management, ed. economică, bucurești, 2001, p.36. v1 v2 v3 v4 v5 v6 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 f2 (1 2, 91 % ) f1 (69,67 %) variables (axes f1 and f2: 82,58 %) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 ciprian ionel hretcanu, cristina elena hretcanu, strategic alliances between firms – a model of competitive strategy in food industry, food and environment safety, volume xiv, issue 2 – 2015, pag. 223 – 232 232 [2]. burciu a.(coord.), introduction into management, ed. economică, bucurești, 2008, p.206. [3]. faulkner d., elements of competitive strategy, ed. teora, bucurești, 2000, p.84. [4]. faulkner d., de rond m., cooperative strategy: economic business and organizational issues, oxford university press, oxford, uk, 2001, pp3-5. [5]. porter m., fuller m., coalitions and global strategy, michael porter (eds), competition in global industries, boston, harvard business school press, 1986, pp.315-344. [6]. jarillo c., on strategic networks, strategic management journal, vol. 9, no. 1, 1988, pp.3141. [7]. wei l. et al., a study on the collaborative innovation in alliance based on the knowledge embeddedness perspective, research and development management, china, 2007 [8]. gomes-casseres b., alliance strategy: managing beyond the alliance, criticaleye publications ltd., iunie august 2004 [9]. najmaei a., sadeghinejad z., competitive strategic alliances through knowledge value chain, international review of business research papers vol. 5 no. 3 april 2009, pp.297-310. [10]. culpan r., multinational strategic alliances, international business press, new york, 1993. [11]. weber k., chathoth p., strategic alliances, chapter 18 in handbook of hospitality marketing management, elsevier ltd., oxford, 2008, p.504. [12]. kelly m., schaan j.l., jonacas h., managing alliance relationships: key challenges in the early stages of colaborations, r & d management 32/1, 2002, p.11. [13]. yoshino m., rangan s., strategic alliance: an entrepreneurial approach to globalisation. boston, harvard business school press, 1995. [14]. martin a., abramov m., partner selection and trust building in west european– russian joint ventures. international studies management organization, 27(1), 1997, pp.19-37. [15]. klein s., chekitan d., partner selection in market-driven strategic alliance. south african journal of business management, 28(3), 1997, pp.97-106. [16]. dyer j., singh h., relational view: cooperative strategy and sources of interorganizational competitive advantage. academy of management review, 23(4), 1980, pp.660-679. [17]. child j., david faulkner, strategies of cooperation: managing alliances, networks, and joint ventures, oxford university press, 2008. [18]. kale p. et.al., learning and protection of proprietary assets in strategic alliances: building relational capital. strategic management journal, 21, 2000, pp.217-237. [19]. todeva e., knoke d., strategic alliances and models of collaboration, in management decision, vol.30(1), 2005, pp.122-149. [20]. wheelen t., hunger d., strategic management and business policy, 12th edition, new jersey: pearson education, 2006, p.158. [21] porter m. , europe’s companies after 1992: don’t collaborate, compete, the economist, 9 june 1990. microsoft word 1 primele pagini nr 1 din 2010 26 august.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering year ix, no.1 – 2010 university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey dirk flottman technical university of aalen, germany georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany cristina-gabriela pop ştefan cel mare university of suceava, romania regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international database: indexcopernicus journals master list. also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by editura universităţii ştefan cel mare suceava, romania. microsoft word 3 journal nr 4 2010 din 8 noiembrie_108-113.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 112 intense light pulses effect on fungal burden of mustard and black pepper elena alexandra oniciuc1, liliana gîtin1, sorin ciortan2 nicoleta maftei (aron)1, anca nicolau1 1faculty of food science and engineering, bioengineering dept., university dunarea de jos of galati, romania 2faculty of mechanical engineering, mechanical design and graphics dept., university dunarea de jos of galati, romania 111 domneasca street, 800201, galati, romania oniciuc_elena@yahoo.com abstract: mustard seeds and black pepper berries were used as test materials in an experiment that aimed to put the basis of a decontamination procedure for spices and condiments using a non-thermal minimal processing method based on intense light pulses. the method wants to be a better solution to actual decontamination procedures of spices, food ingredients that have to fulfill the safety requirements regarding fungal burden and mycotoxin content. the need for the development of innovative technologies for the production of high quality spices is widely recognized by different authorities involved in food safety. the tests were performed on an installation prototype consisting of a vibratory sieve (800 rpm) and ifp 800 flash lamp discharging in xenon gas. different energetic densities (0.170 j/cm2, 0.783 j/cm2 and 1.393 j/cm2) and different pulse regimes (10·10-3 s, 20·10-3 s and 30·10-3 s) were used in order to establish the most suitable regime for fungal decontamination. the experimental results showed that it is possible to obtain 100% fungal decontamination of mustard seeds by setting appropriate regimes that take into consideration the initial fungal burden, the seeds quantity per sieve squared centimeter, the energetic density and the pulse regime. for black pepper berries, contamination was significantly reduced (1.7 log), but total fungal decontamination was not achieved. comparatively to mustard seeds, black pepper berries display a similar roughness but a different undulation, the last one being the potential responsible for mould spores protection against light pulses. key words: moulds, decontamination, mustard, black pepper, intense light pulses introduction most spices contain a high number of microorganisms among which moulds represent a significant part. having been dried material from plant origin, spices are commonly heavily contaminated with xerophilic storage moulds [1]. the most frequent fungal contaminants of spices are species from the genera aspergillus and penicillium [2]. some species that belong to these genera are known as potential producers of different toxic substances such as aflatoxins, ochratoxins and sterigmatocystine, i.e. mycotoxins that exhibit toxic, mutagenic, teratogenic and carcinogenic effects in humans and animals [3]. mycotoxins synthesized by moulds in spices pose an important health risk in the end-products, being so resistant (degrade at 265oc) that cannot be eliminated during food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 113 food processing. for example, the contamination of processed meat with aflatoxin was shown to correlate with the addition of spices to fresh meat [4]. to minimize the hygiene and health risks of moulds presence on spices, the european spice association (esa) has set up quality minima (qm), establishing requirements on the minimum quality for spices entering the eu market. in some respects, the quality minima are additional to legislation, in other respects, they further define legal requirements. thus, esa stipulates for spices a content of 105 yeast and moulds per gram as target and 106 yeast and moulds per gram as absolute maximum [5]. only few conventional technologies, such fumigation and irradiation, are efficient for spices sanitation. however, applications such as the fumigation with ethylene oxide are restricted and even banned by law in the european union [6], while gamma irradiation of spices (10 kgy) is allowed by the european legislation, but frighten consumers who are afraid of formation of low-molecular-weight volatile or nonvolatile radiolysis products that are potentially harmful and may migrate into the food and impair food flavor. modern technologies as high hydrostatic pressures ranging from 100 mpa to 1000 mpa are not efficient on products with water activities below 0.66 as spices are. as a consequence, there is a need for the development of innovative technologies for the production of high quality spices. while lilie and coworkers [7] propose as novel decontamination method of spices a modification of the well-known vacuumsteam-vacuum (vsv) procedures characterized by a rapid evacuation after a short-steam treatment, other solutions should be taken into discussions. in this respect, the present paper aims to put the basis of a new decontamination procedure for spices and condiments using a nonthermal minimal processing method based on intense light pulses. materials and methods materials two spices were used in the experiments: mustard seeds and black pepper berries. mustard seeds packed in 100 g bags by sc orlando import export 2001 ltd., a company that is certified ifs and iso 9001/2000, were sampled from a local food store. all sampled bags belonged to the same lot (297 014 lot 12), but even this, the content of the bags was mixed in order to obtain a homogenous laboratory sample, which was then kept in a sterile plastic bag at room temperature. black pepper berries were kindly provided by interservices sa, a company that provide insurance related services, case handling and assistance in shipping and trading of goods. a part of these berries were artificially contaminated with spores of penicillium camemberti, in order to obtain a higher fungal contamination. equipments the tests were performed on a prototype of an installation consisting of a vibratory sieve (400 rpm and 800 rpm) driven by an electric motor with eccentric, and an ifp 800 flash lamp discharging in xenon gas (figure 1). the distance between the flash lamp and sample was set at 0.10 m. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 114 figure 1. experimental installation for irradiation of condiments with intense light pulses 1 flash lamp; 2 protective enclosure; 3 tray for seeds; 4 vibrating platter; 5 support site; 6 – platter holder (rigid), 7 engine support (elastic), 8 electric motor, 9 platter support (elastic), 10 – eccentric. methods seeds/berries treatments amounts of 20 g of mustard seeds or black pepper berries were aseptically taken, placed on the vibrating sieve and submitted to ilp treatments. the speed of the vibrating sieve was set at 800 rpm, in order to made 1600 flights per minute and to ensure seeds and berries exposure to light pulses on all sides. different energetic densities (0.170 j/cm2, 0.783 j/cm2 and 1.393 j/cm2) and different pulse regimes (10·10-3s, 20·10-3s and 30·10-3s) were used in order to establish the most suitable regime for fungal decontamination. tests were performed in duplicates. microbiological analysis total fungal counts were performed according to sr iso 21527-2:2009 (microbiology of food and fodder. horizontal method for enumeration of yeasts and molds. part 2: colony count technique in products with less than or equal to aw 0.95). rose bengal (scharlau chemie, spain) was used as culture medium. after inoculation, the petri dishes were incubated at 25°c for 5 days. the genus of the isolated moulds was determined based on the characteristics of the colonies and the microscopic aspect of the mycelium. results and discussion mustard seeds and black pepper berries used in these experiments had similar fungal contamination levels (103 per gram), and displayed similarities regarding the quality of their microbiota (figure 2 and figure 3), whether considering the presence of aspergillus and penicillium genera as toxicogenic moulds. representatives of penicillium genus were present in proportion of 26% on mustard seeds and 20% on black pepper berries, while representatives of aspergillus genus were present in proportion of 26% on mustard seeds and 79% on black pepper berries. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 115 26% 26% 13% 35% penicillium aspergillius rhizopus others figure 2. initial fungal microbiota of mustard seeds 20% 79% 1% penicillium aspergillius mucor figure 3. initial fungal microbiota of black pepper berries the initial level of the existing fungal microbiota on mustard seeds (1.1∙103 cfu∙g-1) was gradually decreased during ilp treatments. a treatment consisted in applying a certain number of pulses per 10-3 second at different tensions. the results obtained for 10·10-3 s and 30·10-3 s at 700 v, 1500 v and 2000 v are presented in figure 4a and figure 4b. it is noticed that when tension increases, the fungal burden of the seeds decreases. the decontamination effect is more powerful when a higher number of pulses are applied. at a tension of 2000 v during 10·10-3 s destroyed 90 % of the fungal population, while 30 pulses per 10-3 second destroyed 96 % of the fungal population. 0 200 400 600 800 1000 1200 1400 c o ntro l s a m ple 700 1500 2000 source tension, v n um be r of c ol on ie s, lo g c fu xg -1 figure 4a. evolution of the fungi level on mustard seeds as result of ilp treatments at the regime of impulse 10∙10-3s 0 200 400 600 800 1000 1200 1400 c o ntro l s a m ple 700 1500 2000 source t ension, v n um be r of c ol on ie s, lo g c fu xg -1 figure 4b. evolution of the fungi level on mustard seeds as result of ilp treatments at the regime of impulse 30∙10-3s total fungal decontamination of mustard seeds is possible to be achieved by setting appropriate ilp regimes. the initial level of the existing fungal microbiota on black pepper berries (6.5∙103 cfu∙g-1) was not significantly decreased, despite the fact that different ilp treatments were tested and their intensity was gradually increased (700 v / 10·10-3s, 700 v / 30·10-3s, 1500 v / 10·10-3s, 1500 v / 30·10-3s). in all these cases the fungal contamination level was not decreased beside 103 cfu∙g-1, which means that less 1 log decreasing of fungal burden was obtained (figure 5). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 116 1 10 100 1000 10000 control sample 700 v 10x10-3 700 v 30x10-3 1500 v 10x10-3 1500 v 30 x10-3 treatment variants n um be r of c ol on ie s, lo g c fu x g-1 figure 5. evolution of the fungi level on naturally contaminated black pepper berries as result of ilp treatments the experiments were repeated on black pepper berries that were artificially contaminated with penicillium camemberti spores in order to obtain a fungal burden above the limit indicated by esa (european spicy association) as target value (105 cfu∙g-1). the results of ilp treatments on these artificially contaminated black pepper berries (8∙106 cfu∙g-1 initial fungal burden) is shown in figure 6. it was necessary a heavy treatment (2000 v / 30·10-3 s) to reduce the fungal burden with almost 1.7 log. 1 10 100 1000 10000 100000 1000000 10000000 control sample 700 v 10x10-3 700 v 30x10-3 2000 v 10x10-3 2000 v 30 x10-3 treatment variants n um be r o f c ol on ie s, lo g c fu xg -1 figure 6. evolution of the fungi level on artificially contaminated black pepper berries as result of ilp treatments to reduce the fungal burden of black pepper berries it is necessary to use much intense ilp treatments that in the case of mustard seeds. it is possible that the surface roughness of the black pepper berries to exert a protection against light pulses for the mould spores present on their surface. conclusions an installation consisting of a vibratory sieve (400 rpm and 800 rpm and an ifp 800 flash lamp discharging in xenon gas was successfully tested for fungal decontamination of mustard seeds and black pepper berries. the treatments using intense light pulses succeeded to totally reduce the fungal burden of mustard seeds and to reduce the fungal burden of black pepper berries below the target value indicated by the european spicy association. the study demonstrated that ilp treatments could be a solution to actual decontamination procedures of spices, responding in this way to the need of developing innovative technologies for the production of high quality spices. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 117 references: 1. romagnoli, b., menna, v., gruppioni, n., bergamini, c. 2007. aflatoxins in spices, aromatic herbs, herbs – teas and medicinal plants marketed in italy. food control, 18, 697-701 2. šariã, l. ã., škrinjar, m. m. 2008. share of aflatoxicogenic moulds from genera aspergillus and penicilllium in mycopopulations isolated from spices from meat processing industry, matica srpska proceedings for natural sciences, 114, 115—122 3. heperkan, d., ermis, o.c., 2004. mycotoxins in spices in meeting the mycotoxin menace (editors: d. barug, h. van egmond, r. lopez-garcia, t. van osenbruggen and a. visconti), wageningen academic publishers, p.206 4. hampikyan, h., baris bingol e., colak, h., aydin, a. 2009. the evaluation of microbiological profile of some spices used in turkish meat industry, journal of food, agriculture & environment, 7 (3&4): 111-115. 5.***2004, european spice association quality minima document, page 9 (http://www.esaspices.org/content/pdfs/esaqualityminimadocum ent191104.pdf) 6. schweiggert, u., carle r., schieber, a. 2007. conventional and alternative processes for spice production – a review, trends in food science & technology, 18 (5): 260-268 7. lilie, m., hein, s., wilhelm, p. mueller, u. 2007, decontamination of spices by combining mechanical and thermal effects an alternative approach for quality retention, international journal of food science & technology, 42 (2): 190-193(4) food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 37 pseudomonas sp. and aeromonas sp. selection for treatment of goose down washing wastewater aleksander slavov1, zapryana denkova2, mima hadjikinova1, nikolai mihalkov3 1department “environmental engineering” university of food technologies, plovdiv, bulgaria, aleksander_slavov@yahoo.com , mimi_uht@abv.bg 2department “microbiology” university of food technologies, plovdiv, bulgaria, zdenkova@abv.bg 3”water projects” ltd, plovdiv, bulgaria vodniproekti@abv.bg *corresponding author received 28 august 2012, accepted 4 september 2012 abstract: 10 bacterial strains, isolated from municipal wastewater and active sludge that belong to aeromonas sp. (3) and pseudomonas sp. (7), were investigated. strains were analyzed for their ability to treat pre-centrifuged and diluted in different ratios goose down washing wastewater. it is shown that aeromonas sp. exhibit higher purification degree of goose down washing wastewater compared to the strains of pseudomonas sp. only at a dilution of 1:1, and the highest purification degree is determined for strains aeromonas 2 and aeromonas 2as with 64,94% each. at a dilution of goose down washing wastewater 1:5 the highest purification degree is at a strain pseudomonas 1 – 86,49%, and at a dilution of goose down washing wastewater 1:10 pseudomonas 3as – 87,27 %. keywords: aeromonas sp., pseudomonas sp., goose down washing wastewater, purification degree 1. introduction from poultry processing as by-product large quantities feathers are produced 7% of live weight [1]. utilization of feathers include using of various technologies – composting, in food industry after hydrolysis of keratin [2, 3, 4, 5, 6], as adsorbent of heavy metals [7], filling material for clothes and bags [8], in composite wood mdf [9]. obtaining quality feather include its preliminary treatment with nacl and hcl solutions, followed by washing it with detergents and, if necessary discoloration. as a result large quantities of wastewater with high pollution levels are produced. biological methods have application in wastewater treatment from food industry, and many of them are based on the specific action of particularly selected bacterial strains. the aim of this work is the selection of aeromonas sp. and pseudomonas sp. strains for treatment of goose down washing wastewater. 2.materials and methods 2.1.microorganisms in this work are used 10 strains of bacteria. bacterial strains aeromonas sp. aeromonas 2 are isolated from municipal wastewater, while aeromonas 1as, aeromonas 2as – from activated sludge. bacterial strains pseudomonas sp. pseudomonas 1, pseudomonas 3, pseudomonas 4 were isolated from municipal wastewater, while pseudomonas 1as, pseudomonas 2as, pseudomonas 3as, pseudomonas 4as – from activated sludge. mailto:aleksander_slavov:@yahoo.com mailto:mimi_uht:@abv.bg mailto:zdenkova:@abv.bg mailto:vodniproekti:@abv.bg food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 38 2.2.nutrient mediums 2.2.1.luria – bertany glucose agar medium (lbg) with composition (g/dm3): triptone (difco) – 10 g, yeast extract – 5 g, nacl – 10 g, glucose (scharlau) – 10 g; agar – 20 g. ph=7,5. the medium is sterilized for 25 minutes at 121º c. 2.2.2.glutamate-starch-phenol red agar medium (gsp) with composition (g/dm3): sodium glutamate 10 g, starch (soluble) 20 g, kh2po4 2 g, mgso4, phenol red 0,36 g, agar 12 g. ph 7,2 ± 0,2. all ingredients are dissolved and medium is sterilized for 15 min at 121° c. cool to 4550° c and aseptically add to it 100 iu/ml penicillin g, and if necessary, 10 g/ml pymaricyn. the resulting composition is mixed thoroughly and dispensed in sterile petri dishes. 2.3.wastewater: in the experiments goose down washing wastewater to slaughterhouse is used. wastewater is precentrifuged at 3000 min-1 for 10 min to remove insoluble substances and fat and diluted with non-sterile distilled water in ratios 1:1, 1:5 and 1:10. 2.4.cultivation and storage of microorganisms. isolated strains are grown in lbg agar medium at 30º c in thermostat for 48 h and stored in a refrigerator at 4º ± 2° c for 2 weeks. 2.5.analytical methods. 2.5.1.isolation of aeromonas sp. pseudomonas sp. strains. isolation of strains of aeromonas sp. and pseudomonas sp. was carried out by seeding samples of municipal wastewater, and active sludge on gsp agar medium and their subsequent development at 30° ± 2° c in a thermostat for 24 h. colored in yellow colonies belong to aeromonas sp., while red relate to pseudomonas sp. 2.5.2.development of isolated strains of bacteria in the diluted wastewater. with biomass from developed on lbg agar medium at 30° ± 2° c for 48 h bacterial strains 5 cm3 of diluted wastewater is inoculated. wastewater samples with investigated cultures are cultivated for 72 h at 30° ± 2° c. 2.5.3.purification degree wastewater determination using the permanganate oxidizability method. developed for 72 h at 30° ± 2° c in wastewater cultures are centrifuged at 3000 min-1 for 10 min. the resulting supernatant is analyzed for permanganate oxidizability in accordance with standard bs 17.1.4.1679. 100 cm3 sample or lower volume brought to 100 cm3 with distilled water is placed in a 250-300 cm3 flask, which has added pumice. 5 cm3 h2so4 (1+2) and 20 cm3 0,01 n kmno4 solution are added. the composition is heated so as to boil for no more than 5 min and boiling for 10 min. to the hot solution 20 cm3 0,01 n solution of hooc-cooh are added. hot discoloured solution is titrated with 0,01 n kmno4 solution. the temperature of the solution during titration should not be below 80° c. if the boiling solution is discolored or brown precipitates are formed, the determination is repeated with a smaller sample size. during titration should not spend more than 12 cm3, and not less than 4 cm3 of kmno4 solution for diluted sample. similarly, a blank is done with wastewater. oxidizability (x) in mg/dm3 oxygen is given by the formula: v nba x 8000.).( , where – vkmno4, spent for titration of the sample, cm3; b – vkmno4, spent for titration of blank, cm3; n – exact normality of kmno4 solution; food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 39 v – volume of sample taken for analysis, cm3. similarly oxidizability of a control sample of analyzed wastewater at the corresponding dilution of 1:1, 1:5 and 1:10 is determined. purification degree pd, in %, is determined using the formula: ,%100 s sc pd , where xc – oxidizability of control sample, mg/dm3 oxygen; xs – oxidizability of sample, mg/dm3 oxygen. 3. results and discussion. in a set of experiments the ability of isolated aeromonas sp. and pseudomonas sp. strains to break down pollutants in washing goose down wastewater, diluted with distilled water in different ratios, is investigated. summarized results are presented in fig. 1, fig. 2 and fig. 3. experimental data show an average level of treatment for all strains of aeromonas sp. and pseudomonas sp. at wastewater dilution 1:1, 1:5 and 1:10, respectively. 54,11 35,53 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p ur ifi ca ti on d eg re e, % aeromonas sp. 1:1 pseudomonas sp. 1:1 figure 1. average purification degree of goose down washing wastewater, diluted in ratio 1:1 with aeromonas sp. and pseudomonas sp. it is noteworthy that with increasing dilution increases the degree of purification as in aeromonas sp., and in pseudomonas sp. exceptions are strains aeromonas sp. with the degree of dilution 1:5 (fig. 2). strains aeromonas sp. purify wastewater better than the strains pseudomonas sp. when diluted 1:1 with 18,58% (fig. 1), while at 1:5 and 1:10 dilutions of the strains pseudomonas sp. show a higher level of treatment with 34,86% (fig. 2) and 29,73% (fig. 3). 39,91 74,77 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p ur ifi ca tio n de gr ee , % aeromonas sp. 1:5 pseudomonas sp. 1:5 figure 2. average purification degree of goose down washing wastewater, diluted in ratio 1:5 with aeromonas sp. and pseudomonas sp. 55,21 84,95 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p u ri fic at io n de gr ee , % 1aeromonas sp. 1:10 pseudomonas sp. 1:10 figure 3. average purification degree of goose down washing wastewater, diluted in ratio 1:10 with aeromonas sp. and pseudomonas sp. a comparative characteristic between strains of aeromonas sp. and pseudomonas sp. for the purification degree of goose down wastewater at different dilution ratios is done. results from these studies are presented in figure 4, figure 5 and figure 6. the experimental data represented in figure 4 show that with purification degree over 50% are strains food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 40 aeromonas 2, aeromonas 2as, pseudomonas 3, pseudomonas 2as and the highest is the degree at aeromonas 2 and aeromonas 2as – 64,94%. strain pseudomonas 3 exceeds the other three pseudomonas sp. with the purification degree 59, 09 %. 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p ur if ic ai o n d eg re e, % aeromonas 2 (1:1) aeromonas 1 s (1:1) aeromonas 2 s (1:1) pseudomonas 1 (1:1) pseudomonas 3 (1:1) pseudomonas 4 (1:1) pseudomonas 1 s (1:1) pseudomonas 2 s (1:1) pseudomonas 3 s (1:1) pseudomonas 4 s (1:1) figure 4. purification degree of goose down washing wastewater, diluted in ratio 1:1 with aeromonas sp. and pseudomonas sp. 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p ur ifi ca tio n d eg re e, % aeromonas 2 (1:5) aeromonas 1 s (1:5) aeromonas 2 s (1:5) pseudomonas 1 (1:5) pseudomonas 3 (1:5) pseudomonas 4 (1:5) pseudomonas 1 s (1:5) pseudomonas 2 s (1:5) pseudomonas 3 s (1:5) pseudomonas 4 s (1:5) figure 5. purification degree of goose down washing wastewater, diluted in ratio 1:5 with aeromonas sp. and pseudomonas sp. at a dilution ratio 1:5 (figure 5) strain pseudomonas 1 shows the highest degree of purification – 86,49%, followed by strain pseudomonas 3 – 79,48 %. third place is shared by strains pseudomonas 4 and pseudomonas 3as with average value 78,70 %. from aeromonas sp. with the highest purification degree is strain aeromonas 2 with 54,23%. at 1:10 dilution ratio (figure 6) the highest purification degree has strain pseudomonas 3as – 87,27%, followed by pseudomonas 3 and pseudomonas 1 with 87,19% and 87,01%, respectively. purification degree of aeromonas sp. strains reached only 57,95% of aeromonas 2as, followed by aeromonas 2 and aeromonas 1as with 56,92% and 50,77%, respectively. food and environment safety journal of faculty of food engineering, tefan cel mareuniversity suceava volume xi, issue 3 – 2012 41 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 p u ri fic at io n d eg re e, % aeromonas 2 (1:10) aeromonas 1 s (1:10) aeromonas 2 s (1:10) pseudomonas 1 (1:10) pseudomonas 3 (1:10) pseudomonas 4 (1:10) pseudomonas 1 s (1:10) pseudomonas 2 s (1:10) pseudomonas 3 s (1:10) pseudomonas 4 s (1:10) figure 6. purification degree of goose down washing wastewater, diluted in ratio 1:10 with aeromonas sp. and pseudomonas sp. 4. conclusion. as a result of investigated analys s on goose down washing wastewater treatment the following more important conclusions can be made: 1.the more dilution of goose down washing wastewater is done, the greater is its purification degree. exception is made for strains aeromonas sp. with dilution factor of the effluent 1:5; 2.strains aeromonas sp. exhibit higher purification degree of goose down washing wastewater compared to pseudomonas sp. strains only at a dilution factor 1:1. 3.at a dilution factor of goose down washing wastewater 1:1 the highest purification degree is determined at strains aeromonas 2 and aeromonas 2as with 64,94%, at dilution ratio 1:5 – at strain pseudomonas 1 – 86,49% and at dilution factor 1:10 strain pseudomonas 3as – 87,27%. 5. references. [1] lortscher, l. l., g. f. sachsel, d. wilkelmy jr. and r. b. filbert jr., processing poultry by products in poultry slaughter plants. u.s. department of agriculture marketing research department, no. 181, washington, dc, (1957) [2] nagal, s. jain, p. c., feather degradation by strains of bacillus isolated from decomposing feathers. brazilian journal of microbiology, vol. 41, 196-200, (2010) [3] wrzesniewska-tosik, k. adamiec, j., biocomposites with a content of keratin from chicken feathers. fibres and textiles in eastern europe, vol. 15 (1), 106-112, (2007) [4] fuji, t. li, d., preparation and properties of protein films and particles from chicken feather. journal of biological macromolecules, vol. 8 (2), 48-55, (2008) [5] kim, j.-d., preliminary characterization of keratinolytic enzyme of aspergillus flavus k-03 and its potential in biodegradation of keratin wastes, mycobiology, vol. 31 (4), 209-213, (2003) [6] kumar, d. j. m., priya, p., balasundari, s. n., devi, g. s. d. n., rebecca, a. i. n. kalaichelvan, p. t., production and optimization of feather protein hydrolyzate from bacillus sp. mptk6 and its antioxidant potential. middle-east journal of scientific research, vol. 11 (7), 900-907, (2012) [7] sun p., liu z.-t., liu z.-w., chemically modified chicken feather as sorbent for removing toxic chromium(vi) ions. industrial and engineering chemistry research, vol. 48 (14), 6882-6889, (2009) [8] choban, a. winkler, i., featherprocessing wastewater: composition, influence on the natural water objects and decontamination technologies. croatian journal of food technology, biotechnology and nutrition, vol. 6 (1-2), 58-62, (2011) [9] winandy, j. e., muehl, j. h., micales, j. a., raina, a. schmidt, w., potential of chicken feather fibre in wood mdf composites. proceedings paper ecocomp 2003. queen mary, university of london, 1-6, (2003) microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_19-25.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 23 benefits of multilateral liberalization for the countries which are exporting light industry products in a knowledge based society alina-petronela haller phd. senior researcher iii ices “gh. zane" iassy branch of romanian academy hallalina@yahoo.com abstract:the evolution of international economic processes, interdependencies between countries, between production and goods movements under the incidence and the impact of scientific and technical progress required, in the middle of the 20th century, the institutionalization of the economic liberalization process by laying the foundations of gatt (general agreement for tariffs and trade transformed later in wto world trade organization). initially, gatt's rules were applied only for goods trade but gradually it had broadened its sphere of activity. based on interpretative approach, in this paper we analyze the degree in which gatt wto activity can solve the business problems of the countries which are exporting industrial light products (we are talking especially about developing countries). wto multilateral agreements concern among others agriculture, textiles, food products and industrial standards. in this paper, we want to show that exporting countries of light products can enjoy many advantages due to the signing of agreements in a multilateral frame. this type of benefits are directly reflected in the economic growth and development of the society based on knowledge. keywords: economic liberalization, knowledge society, export, light industry products, economic and social growth, development. methods and methodology the theme is dwelt upon from a conceptual standpoint, on the basis of varied, direct and indirect methods, like analysis, synthesis, observation. introduction the multilateral commercial system’s tendency towards development is determined by the evolution of the international economic processes, of the interdependencies between the production and the circulation of goods within the international division of labour, under the incidence and influence of the technicalscientific progress. under these circumstances, the commercial policies promoted by different states acquire new forms, sizes and values. after world war ii, many economists believed in the possibility of a restructuring crisis, regarding the world expansion as a key for the “free world’s” power1, so that the united states came with a project of reconstruction of the 1 irwin douglas, free trade under fire, princeton university press, oxford, 2003, p. 34. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 24 international trade based upon the suppression of the preferential regional agreements(following the commonwealth’s model) and upon multilateral negotiations.2 the measures the americans foresaw were relying on the abolishing of trade discrimination, on the control of the depreciation policy, on the liberty of the capital mobility, on the free convertibility of currency, on the multilateralization of exchanges and payments. for them, peace and the return to the liberty of trade do not exclude each other but, on the contrary, they presume each other. thus, the world states started a wide action, aiming at liberalizing the multilateral trade exchanges in order to reduce economic and social gaps between the industrialized and the industrializing countries3. results and discussion the first major effort to adopt the rules meant to organize the international trade relationships was made, as we mentioned above, by some countries immediately after world war ii, when emerged the necessity to create an organism that should watch, control and get directly involved in the development of the international trade. these efforts led to the 1948 adoption of the general agreement on tariffs and trade (gatt), whose rules were applied to the international trade of goods. in time, the gatt text has been modified, to include new provisions and especially to deal with the developing countries’ trade problems. moreover, a number of subsidiary agreements were adopted, detailing some of the main provisions of gatt. gatt and subsidiary agreements were revised and improved over several 2 teulon frederic, comerţ internaţional, editura institutului european, iaşi, 1996, p. 43 3 haller a.p., exporturile factor de dezvoltare şi creştere economică, editura performantica, iaşi, 2008, p. 41 rounds of negotiations, the most important one being the uruguay round, from 1986 to 1994. one of the results of the uruguayround was the creation of the wto (world trade organization), on 1st of january 1995. gatt, under whose auspices these negotiations were started, was included in the wto. wto is responsible for the control of the multilateral trade system, which has gradually evolved over the last decades and it also represents a forum for the continuation of negotiations on the liberalization of trade with goods and services, by abolishing barriers and elaborating new rules in the trade related fields. the wto agreements stipulate a common mechanism to regulate the disputes by which the members defend their rights and regulate the divergences between them. currently, only few countries are not part of the wto system, which were in the past planned economies, and some of the developing countries. the countries with a transition economy regard wto as a structure that offers important opportunities to get integrated in the world economy and to improve the national legal basis in the economic and commercial fields. wto represents the institutional-legal basis of the multilateral trade system, being the only international organization that deals with the global rules of trade between countries4. the organization particularly focuses on the necessity to make special efforts to support the participation of the developing countries and especially of ldcs in the raising of the world trade5. the wto agreements are voluminous and complex, referring to agriculture, textiles 4 dinuţă i., relaţii economice multilaterale, editura scrisul românesc , craiova, 1995, p.54 5 dinuţă i., relaţii economice multilaterale, editura scrisul românesc , craiova, 1995, p. 56 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 25 and clothing industry, bank system, industrial standards, intellectual property and many other fields. at the basis of these documents there are principles representing the foundation of the multilateral trade system: a) mutual and unconditional granting of the most favoured nation clause. according to the wto agreements, the states are not allowed to show discrimination towards their trade partners. if a partner was offered a special favour (like a smaller customs tax for a product), this will be applied to all the other wto members. this principle is the first gatt article, being a priority in gats (general agreement on trade in services, art. ii) and in trips (agreement on traderelated aspects of intellectual property rights, art. iv). yet, the gatt rules allow for the customs taxes and other barriers standing in the way of trade to be preferentially reduced within the regional arrangements. the reduction of customs taxes, applicable in the relations between the members within regional arrangements should not be extended upon other countries. the regional preferential arrangements represent an important exception to the rule of the most favoured nation clause. such arrangements could borrow the customs unions or the free trade areas. b) the granting of the national treatment clause, a fact that involves equal attitude towards the import and the domestic goods, after the import goods’ penetration on the market. the same is true in the case of the services made by foreign and national agents, in the case of national and foreign trademarks, of the copyright and of patents. this principle is provided in the three main agreements of the wto, though it is differently approached in each of them. c) the elimination of quantitative restrictions and their application among the member states only in the exceptional situations commonly admitted, on an undiscriminating basis, given that the protection of national economy in front of the foreign competition is made only by means of customs taxes. an exception allows the countries facing difficulties related to the balance of payments to restrict imports in order to safeguard the external financial position. this exception grants a bigger flexibility to developing countries as far as the utilization of the quantitative restrictions in imports are concerned, if these restrictions are necessary to prevent a serious downward slope of the currency reserves. d) the interdiction of disloyal competition, represented by the application of subventions and dumping prices in export. the non-discriminatory rules – the most favoured nation clause and the national treatment – are elaborated in such a manner as to ensure loyal conditions for trade. many of the wto agreements play the role of supporting loyal competition (in agriculture, for instance, or intellectual property, services). e) the utilization of a methodology of customs evaluation that could reflect the real value of goods, in order to fulfil the correct functioning of the customs taxes resulted from the multilateral negotiations. f) the fulfilling of obligations. within wto, if the states agree to open their markets for goods and services, they make some mandatory commitments, which can be modified after negotiations with the trade partners, involving the payment of some compensation for the partners’ prejudice. g) the parties’ engagement to negotiate at a multilateral level the reduction or the abolishing of customs taxes and of other hinders in the way of the international trade. h) encouraging the development and the economic reform. wto offers multiple benefits, some of them well-known, others less obvious. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 26 objective of this system based on rules is to ensure that the markets stay open and to avoid the interruption of access by sudden and arbitrary imposition of restrictions upon imports. in some developing countries, the business communities are still not fully aware of the advantages of wto trade system. yet, the multilateral trade system does not only offer benefits to the industrial producers and to enterprises, but it also creates rights in their favour6. these advantages would be: a) it contributes to maintaining international security. particularly, security is a result of the application of the trade system’s most important principles. b) it allows constructive solutions for disputes. it offers effective modalities to peacefully and constructively settle the disagreements pertaining to trade activities. moreover, trade means several possibilities of disagreements. the fact that the countries can resort to wto to regulate their dispute is a substantial contribution to the soothing the international tensions from the trade area. c) trade stimulates economic growth, representing a potentiality to create workplaces. practice shows that the diminution of the number of trade obstacles positively influence the number the workplaces. a well-planned policy could make easier the employment problems. d) free trade reduces costs. the studies on the impact of protectionism and of free trade demonstrated that protectionism is expensive, determining the price raising. the multilateral trade system aims at reducing trade barriers through the commonly agreed rules and bases upon the principle of non-discrimination. the result – a reduction of production costs and a 6 hoekman bernard, the political economy of the world trading system, oxford univesity press, 2001, p. 87 reduction of prices for finished articles and services. e) trade contributes to income raising. the analyses and evaluation of the impact had by the uruguay round (and therefore of the wto foundation) or by the creation of the eu common market demonstrated that these processes ended up with considerable supplementary incomes, thus stimulating the processes of economic development. trade involves challenges and some risks – domestic producers must offer products as qualitative as the imported ones, but not everyone succeeds. the fact that supplementary incomes are generated, demonstrates the existence of the necessary resources for the governments to redistribute the available benefits, for instance, to help the economic agents become more competitive. f) the system encourages the governing. the governments often use the multilateral trade framework as internal restriction useful in elaborating economic policies, arguing that they could not proceed differently, not to contradict the wto agreements. for business, engaging entails a greater certitude and clarity in the trade conditions, and for governments, it means more discipline and correctness. the advantages for the exporters of goods and services are: a) the security of access. in goods trade, almost all the customs taxes of the developed countries and a big part of the developing ones were consolidated within wto against their rise. the consolidation ensures the fact that the improved access on the market, obtained by tariff reductions included in the lists of concessions for each country, should not be interrupted by sudden tax rises or the imposition of other restrictions by the importing countries. in the field of services, the countries engaged not to restrict the access of products beyond the conditions and limitations mentioned in their national lists. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 27 b) the stability of access. the system also ensures the stability of access on the export markets, requiring all the states to apply a uniform set of rules, elaborated within different agreements. the countries have the obligation to ensure conformity to the pertinent agreement provisions for the rules regarding the value determination in customs, the inspection of products with a view to establishing the mandatory standards of quality or the emission of import licenses. there are frequent situations when companies are forces to acquire from import raw materials, intermediary products and necessary services for the export production. the basic rule, requiring for the exports to be allowed without further restrictions, after the payment of taxes and the obligation to ensure conformity of the other national rules applied in customs with uniform rules within the agreements, facilitates imports. the exporting industries are thus granted a certain guarantee to procure the necessities without delay and at competitive prices. furthermore, the tariff consolidations serve to assuring importers that import costs will not be raise by increased customs taxes. the whole gatt system is based on four basic rules: 1. the protection of national industry by customs taxes. though gatt aims at gradually liberalizing the trade, it admits that the member countries could be in the situation to protect their domestic production in front of the foreign competition. yet, it asks the countries to ensure protection through customs taxes. the utilization of quantitative restrictions is forbidden, with the exception of a limited number of situations. 2. ceiling of tariffs. the countries are required, every time this is possible, to reduce or to eliminate the protection of the domestic production by reducing customs taxes and removing the other trade barriers within multilateral trade negotiations. at the same time, the rule provides the ceiling of reduced tariffs, that is the exclusion of their subsequent raising. 3. the most favoured country clause. this important gatt rule refers to the principle of non-discrimination. thus, if a member state grants to another country any tariff or any advantage for certain products, it must immediately and unconditionally extend it on the similar products of other countries. this obligation is applied both to imports, and to exports. if a countries takes taxes for export towards a certain destination, it must take the same tax for the export to all destinations. there are derogations to it as well. the exchanges between the general agreements of trade members submitted to preferential customs taxes or are exempted from these taxes are one of the exceptions. another one is the generalized system of preferences granted to the developing countries by the developed ones. 4. national treatment. according to this principle, an imported product that passed the frontier after having paid the customs taxes and other taxes benefits by a treatment equal to the one applied to local products. gatt also includes annexes that refer to agriculture, textile industry, state trade, standard products, subventions and actions against the dumping practices. the countries engage in categories of merchandise, referring to the reduction and limitation of customs tariffs for the import of products. in certain cases, tariffs are reduced to zero, in other cases, they are just limited. therefore, engagements are taken to wto, for a product to be applied one rate that will not be raised in the future. the uruguay round offers some advantages for the developing countries. on the one hand, many of these states resisted, especially at the end of negotiations, the american demands, that their domestic policies, the industry of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 28 services, the foreign investments and the industrial property should become an object of negotiations within gatt. on the other hand, the perspective of a significant reduction of agricultural protection and for a big share of the textiles and clothing industry, offers the possibility of real gains for many of the developing countries. the final act of the uruguay round reflects a numbers of compromises between the developed countries’ requirements and the developing countries’ possibilities7. for instance, in the case of trips, the licensing of patented investments is allowed, but under certain conditions, including the compensations for the patent owner. this is a significant compromise of the american standpoint, mainly shared by the ue and japan, that the mandatory licensing will not be allowed for a period of twenty years of patent protection. in the case of trims, the final act shortly characterizes the demands for the technological transfer that violates the trade rules and allows significant scope for investments with a view to make sure that the foreign companies will contribute in the host-country’s developing. again, usa made big pressures for a more restrictive variant as far as the investments measures are concerned. while the general provisions of the final act reflect the developing countries’ interests in several fields, the tendency was not to grant them wider exceptions. in certain cases, the developing countries can benefit by a longer period to follow, at the domestic level, the rules, but the result of the uruguay round demonstrates that they would be required to make mutual arrangements of trade liberalization8. 7 jackson john, word trading system law and policy of international economic relations, princeton university press, oxford, 2003, p. 144 8 jackson john, word trading system law and policy of international economic relations, princeton university press, oxford, 2003, p. 145 the agreement on agriculture allows the developing countries to reduce protection and internal support for a period of ten years. article 15 stipulates that the normal period of implementation for the developing countries should be six years. the effects of liberalization established in the agreement for agriculture on the developing countries are different. like winters suggests, the “exporters of products from the temperate areas, whose prices are the most affected by the liberalization of industry, have direct and strong interests in starting protection; their gains will significantly raise”. the importers will be submitted to less desirable consequences. the fact that the liberalization of commerce in agriculture can have negative outcomes as well, especially for the developed countries, is taken into consideration in the decision regarding the measures taken for possible consequences on the reform programmes in the less developed countries, importers of farm products. the ministers of commerce should make sure that the ldcs benefit by sufficient quantities of food products and technical assistance to develop their own farm sectors. when the agreement on textiles and clothing industry comes into force, the members are to remove the restrictions based upon the multifiber agreement or on the bilateral agreements upon the products summing up at least 16% of the total amount of their imports in the following groups of goods: fibres, textile processed products, ready-made clothes. considering the restrictions remaining after this initial phase of liberalization, over three years, the members have to remove the restrictions upon the products for a supplementary percentage of 17% of the total import amount in the terms of the 1990s. the elimination of restrictions for products holding at least 18% of the total food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 29 imports amount is necessary after seven years. all restrictions remained will be eliminated over ten years since the agreement’s enforcement. these agreements of liberalization are only applied to the restrictions imposed within the multifiber agreement or the bilateral arrangements beyond the legal framework of gatt. the agreement on the technical barriers stipulates that the developing countries will be offered, upon request, technical assistance and consultancy by other members, in order to facilitate the process of technical standardization. they will occur under the “commonly agreed terms and conditions”9. the agreement on the subventions forbids the subsidizing of exports after a transition period of eight years (art. 27/1). once a country reached the “competitiveness of export” for a certain product, it must give up subventions (art. 27/4). competitiveness of export supposes reaching the 3.25% of the world trade for two consecutive years (art. 27/5). conclusions we could first of all conclude that the wto enforcing represents a particularly important moment, but not important enough to ensure the effectiveness of the new multilateral trade system. from the practice of the first functioning months of the organization, one could infer that a permanent “wakeful state” is necessary for all member states, meant to ensure the functioning of all engagements of this system in the requested rhythm. secondly, the efforts meant to establish the trade multilateralism and the tendencies to solve the major problems in restricted circles should also be continued. the processes of globalization and liberalization determined the 9 mansfield e, world trade organisation – facts and policies, inc white lains, new york, 1999, p. 107 transformation of the international trade into a powerful engine of growth and into an important mechanism for the integration of countries in the global economy. a big number of developing countries took advantage from the existing opportunities, obtaining a rapid growth of their economies. yet, not all states were able to benefit from these opportunities. there is a real risk for them to be still marginalized. at the same time, it is well known that their integration and full participation in the global economy significantly intensifies widening of international exchanges. undoubtedly, the wto developing countries can effectively capitalize on the market the results of their own farm and industrial activities, especially the food and textile industry ones. presently, wto is an organization disposing of a multilateral trade legal and institutional framework, which makes the globalization process, with all of its minuses and pluses, an unavoidable one, becoming a natural phase of the general process of political, economic and cultural development. acknowledgements: this paper is supported by the sectorial operational programme human resources development (sop hrd), financed by the european social fund and by the romanian government under the contract number posdru id 56815” references: 1. dinuţă i, relaţii economice multilaterale, editura scrisul românesc, craiova, 1995 2. haller a.p., exporturile factor de dezvoltare şi creştere economică, editura performantica, iaşi, 2008 3. hoekman bernard, the political economy of the world trading system, oxford univesity press, 2001; 4. irwin douglas, free trade under fire, princeton university press, oxford, 2003; 5. mansfield e, world trade organisation – facts and policies, inc white lains, new york, 1999; 6. teulon frederic, comerţ internaţional, editura institutului european, iaşi, 1996 food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 30 m odern a ppro ach to m an ag eme nt of c om bin ed s ew er o we r flo ws p. hlavinek1 1brno university of technology, faculty of civil engineering, institute of municipal water management zizkova 17, 602 00 brno, czech republic, e-mail: hlavinek.p@fce.vutbr.cz abstract: combined sewer overflows (csos), which are build on combined sewerage network to diminish amount of wastewater carried to wastewater treatment plant (wwtp), loud receiving waters with considerable amount of mixture of sewage and storm water and pollution which they carry. receiving waters are polluted and their ecological status is affected. the aim of stormwater management in the combined sewerage network during rain event is to keep csos overflows in sustainable limits. it is generally necessary to minimize the total emissions from csos and wwtp´s. the paper presents basic results of a critical state-of-the-art review on impacts of combined sewer overflows on receiving waters. paper also describes the potential of technologies for csos treatment. today the conventional measures for reducing cso pollutant load are well known, however the stochastic behavior of rainfall and the uncertainties in land use development as well as the limited knowledge on efficiency of cso treatment yield the most difficult conditions for design. therefore, special attention should be paid to efficiency and cost-effectiveness of applied measures. paper bring the integrated view of present knowledge on cso treatment measures especially considering the current demands outgoing from the implementation of eu framework directive (wfd). keywords: combined sewer overflows, urban river, ecological condition,emission limit, imission limit 1. introduction combined system overflows (csos) constructed in the combined sewerage for technical and water management reasons in order to restrict the influent to a wastewater treatment plant (wwtp) during a rain event ensure intermittent discharging of considerable volumes of a mixture of wastewater and storm water, and the pollution contained in the water, into water courses. this strongly overloads the receiving bodies of water and their ecological condition is influenced. the objective of handling the storm water flows in sewage systems is to maintain the shock effects on the receiving bodies of water caused by the csos within tolerable limits. at the same time, it is necessary to generally minimise the total emissions from the csos and the wwtps. the modern approach is built upon the fact that the simulation of rainfall/runoff processes in an urban river catchment area is the current status of knowledge in the field of municipal drainage. minimum demands on managing rainwater run-off from the combined sewerage are therefore not based on the requirements for the installation of csos any more, but they are based on the objective of conveying a certain proportion of pollutants on a yearly average in the flow to the biological stage of the wwtp during rain events. 2. legislation and methodological approaches in the eu the legal framework of the european union in the field of water management and the environment is based on the framework directive 2000/60/ec establishing a framework for community action in the field of water policy, mailto:hlavinek.p:@fce.vutbr.cz food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 31 requesting the ec member countries to restrict the pollution of bodies of water caused by diffusion and point sources and to achieve a good chemical and ecological condition of all bodies of water where it is economically and socially feasible by 2015. the framework directive also lists the main pollutants the presence of which must be restricted or fully prevented from. combined system overflows are a source of a number of these substances (e.g. suspended solids, substances contributing to eutrophication and substances with adverse impacts on oxygen balance). to control the discharges from the point or diffusion sources of pollution into surface water, the directive requires the so-called „joint approach" using restriction of pollution at the source by determining emission limits and environmental quality standards. 3. ecological condition of water courses the overflows of the mixture of wastewater and storm water from the csos cause significant disturbance to the water courses and endanger their ecological condition and compromise demands on their use. the ecological condition of the water courses is put at risk by the mass and physical disturbances that are very complex and act simultaneously. the mass disturbances are caused by a number of substances of various origins as the overflowing water is a mixture of polluted surface runoff, sewage water and industrial wastewater, re-suspended sediments and eroded biofilm in the sewerage. easily degradable organic substances are decomposed by heterotrophic bacteria consuming oxygen dissolved in water. oxygen disappears not only from water but also from the bottom sediments in small water courses. lack of oxygen endangers mainly sensitive fish species. repeated organic pollution transfer lead to transformation of the trophic conditions of the ecosystem from autotrophic to heterotrophic, resulting in stronger decomposition processes and further oxygen content reduction. the composition of the food chain societies changes from the previously diverse biocenosis into monotonous biocenosis consisting of several destruents tolerant to oxygen concentrations (krej í et al., 2004). nutrients (nitrogen and phosphorus) transferred to the receiving water of water through the storm water overflows may contribute to eutrophication of the slow flowing reaches of water courses and reservoirs and associated night oxygen deficit. in the total nutrient balance in the river basin, the csos are sometimes less important sources than surface sources, diffuse sources and wwtp effluents; however, with the rising number of the wwtps where nutrients are removed, and with the rising efficiency of these processes and improved agricultural processes, the importance of the combined system overflows as a source of nutrients keeps increasing (in particular, in large agglomerations). serious problems are caused by the prevailing form of nitrogen in wastewater, being ammonium (nh4+) dissociating in the receiving body of water at higher temperatures and ph into toxic ammonia (nh3) endangering fish species in lower development stages. the csos overflows contain a high share of suspended solids of organic and mineral origin with a number of negative effects on the aquatic biocenosis (krejci et al., 2004). suspended solids cause water turbidity, reduce light transmission for plant species and cause physiological stress to fish. the settled substances cause mud silting and silting at the river bed restricting the exchange processes between flowing water and water in the sediment pores, which is negatively reflected in the oxygen supply at the river bed. the decay of the organic share of suspended solids at the river bed causes further oxygen content decrease or even anaerobic conditions. suspended solids absorb heavy metals and organic pollutants accumulated in slow flowing food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 32 reaches and are harmful to organisms that are in contact and penetrate to the supply chain. physical disturbance results in changes of the living space in the water course which may lead to the disappearance of some of the aquatic societies. the most important physical disturbance is hydraulic stress, and, to a lesser extent, short-term temperature changes. the shock storm water discharges from sewerage change, especially in small water courses, the hydraulic conditions over in a very short time. high flow velocities result in the removal of the aquatic organisms from their living space. in extreme cases, there occurs a large-scale movement of the bed and transport of sediments and extensive washing out, damage or death loss of a major part of the population. flood waves volumes caused by runoffs from urban areas and their flow rate maxima and frequencies are much higher compared to previous runoffs from the natural environment to which the aquatic organisms are adopted. too frequent population losses then result in major unspecified impoverished of the aquatic fauna. on the other hand, high flow rates carry away fine sediments and the river bed gets silted, which is accompanied by the decrease in the chronic effects of substances adsorbed on the sediment (krej í et al., 2004). a short-term temperature increase as a result of hot storm water discharges in the summer has an influence on minor water courses where this can cause reduced dissolvability of oxygen in water and accelerated microbial processes resulting in further oxygen losses. increased temperature also boosts toxicity of some substances (krej í et al., 2004). the disturbance of some demands on using the water courses is related to its disturbed ecological condition as this is conditioned by it (e.g. fishing). the aesthetic condition is often disturbed, too, by objects in wastewater (e.g. toilette paper, sanitary napkins) and hygienic disturbance by pathogenic organisms endangering the use of the water course as a source of potable water and a bathing place (krej í et al., 2002). the disturbances of the water courses by cso overflows have various duration (krej í et al., 2002). short-term (acute) effects of the increased concentration of toxic substances (in particular, ammonia), oxygen deficit, turbidity, increased temperature and hydraulic stress lasts for up to several hours after the overflow and they usually intensify each other. delayed effects occur for several days up to weeks after the overflows and include, in particular, oxygen deficit in water and river bed sediments and disturbance of the hygienic condition by pathogenic organisms. long-term effects of cso overflows are a result of cumulative effects of a series of events. this includes river bed silting by suspended solids, oxygen deficit in the sediment, chronic toxicity of heavy metals and organic pollutants, eutrophication by nutrients and changed morphological structure of the water stream as a result of erosion. the potential endangering of the water courses differs in relation to the size of the river basin and type of the water course. generally speaking, the most endangered streams are minor water courses where the ratio of water discharged from sewerage during rain events to the flow rate in the river is substantially higher than in the streams with sufficiently high water content. as regards minor water courses in middle highlands, the hydraulic disturbance is more serious than mass disturbance, as given the high gradient of the river beds, even a relatively small increase in the flow rate may result in the removal of organisms and erosion. compared to this, water streams in lowlands and bloated streams have a low velocity and therefore the hydraulic effects food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 33 give way to the mass effects (bwkmaterialien 1, 2003). the flow velocity in the water course has a major impact on the duration of pollution and settling of suspended solids. the slower the water flows, the longer the duration of the adverse effects of pollution transfer on biocenosis. fast flowing and cold streams in middle highlands are not so much affected by the organic pollution as the lowland water courses, as the substances are transported faster, and aeration and oxygen transfer through the water table is faster thanks to increased turbulence. the critical oxygen deficit does not occur immediately downstream the cso, but further downstream, especially in reaches with low reaeration. in minor water streams the suspended solids are transported faster downstream but the river bed may be locally silted. in slow flowing reaches there occurs significant settling of fine particles and organic suspended solids from the cso, which may lead to anaerobic conditions at the river bed associated with nutrient release and eutrophication, or, as the case may be, heavy metal re-mobilisation. ammonia toxicity endangers river courses with limy subsoil or reaches with a higher temperature as a result of missing shading or slow flowing eutrophicated river reaches in lowlands with increased ph (bwk-materialien 1, 2003). morphological quality of the csos has a high potential for ensuring re-settling of disturbed reaches by organisms from the undisturbed river reaches upstream or from protected spaces in the area of disturbance and therefore it is able to compensate for the cso overflow effects relatively fast. if the interconnection with undisturbed river sections or other water streams is not provided and heterogenic habitats serving as protective spaces are lost, the resettlement potential decreases (krej í et al., 2002). 4. protection of the receiving body of water from the emission point of view the objective of protecting the receiving body of water from the emission point of view is to make sure that a certain share of pollution contained in the mixture of wastewater and storm water conveyed through the combined sewerage is conveyed to the biological stage of a wwtp in the average yearly balance. this protects the receiving body of water mainly from the built-up pollution and its long-term effects. emission criteria are defined both for the whole urban river catchment area drained by a combined sewage system as well as for the individual combined system overflows. whereas the emission criteria for the whole urbanised catchment areas are directive, the criteria for the individual structures are only recommendatory. this approach makes it possible not to have to construct individual structures if the river catchment area meets the prescribed pollutant removal proportion and if it also meets the pollution criteria. as regards the average yearly balance, a minimum proportion of storm water flows and the relevant dissolved pollution and suspended solids carried by the combined sewerage must be conveyed during rain events to the biological stage of the wwtp. dissolved pollution is understood as, for example, n-nh4 and a major proportion of cod, bod5, nt and pt. these so-called „minimum efficiencies“ of removal do not concern the specific structures but the whole catchment area of the combined sewerage irrespective of whether the emissions are discharges into one or several receiving bodies of water. differentiation of the emission criteria for various size categories of wwtps reflects the efficiency of pollutant removal at the wwtp, and the density of sewerage connection rate in urbanised catchment areas. as regards larger size wwtp, the government regulation no. food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 34 61/2003 sb. as amended, provides a higher pollutant removal efficiency and therefore it makes sense to convey stronger pollutions to the plants. a higher density of inhabitants is usually in the catchment areas of large wwtps rather than small wwtps. a lower required efficiency of conveying substances to small wwtps prevents from the disproportionally high specific retention volumes in scarcely populated areas and lower wastewater treatment efficiency. the calculation of the actual efficiency of storm water flows and pollutants in sewerage conveyed to the wwtp is conducted by means of a simulation of rainfall/run-off processes in the catchment areas using calibrated hydrological or hydrodynamical models of a multi-year rain series (preferably a minimum of 10 years). the efficiency of conveying substances during rainfalls to the wwtp may be increased by a number of diverse measures that allow for substantial flexibility in the planning process. this includes, for example, storm water infiltration, retention, increased influent to the wwtp, real time sewerage flow control and mechanical pre-treatment of water flowing through storm water overflows into the receiving body of water. the most efficient method of reducing the total emissions in sewerage using one or multiple measures and ensuring an optimal cost/benefit ratio in a specific case must be searched for by using long-term simulation of the rainfall/run-off processes in the urban catchment areas (kabelkova et al, 2010). 5. protection of the receiving body of water from the pollution point of view in specific cases it is necessary to tighten up the emission criteria based on the actual effects of overflows from the csos in the receiving body of water , i.e. pollution. the pollution protection of the receiving body of water respects the parameters of the specific water course and concentrates on the hydraulic disturbance of the receiving body of water, acute ammonia toxicity, oxygen deficit and suspended solids. for these key indicators, the target values of the receiving body of water are defined during wet flows (pollution criteria). the pollution protection also includes hygienic pollution and aesthetic disturbance of the receiving body of water as a result of cso overflows, for which, however, the key indicators have no target values for rain events. hydraulic disturbance the cso must not have significant adverse hydraulic effects on the biocenosis of the water stream. their effects depend on the water course morphology, in particular the volume of protective spaces and the river bed stability. negative ecological effects can be expected when the number of the river bed erosion events as a result of increased storm water flows is more than double compared to the natural condition; as regards water courses with disturbed morphology, the disturbance may be caused by a series of minor events. suitable measures to reduce the hydraulic disturbance of the water courses by cso and storm water discharges from separate sewerage are represented by reducing storm water discharges from urban river basin by reducing the size of impermeable surfaces or their disconnection from the sewerage and retention tanks and storm water infiltration preferably in the area of the storm water discharge retention in the sewerage or storm water tanks, potentially equipped with runoff control in the sewer system in order to optimise the use of retention spaces and ensure even utilisation of the wwtp capacity and reduction in the tangential tension directly in the water course. broadening of the river bed must be defined with respect to the depth so as not to disturb the ecological condition of food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 35 the water stream. it is necessary to examine potential negative effects of the changed morphology of the water course. acute ammonia toxicity sewage water contains high concentration of ammoniated nitrogen which passes through the csos to the receiving bodies of water. non-dissociated ammonia nh3 is formed from the nh4+ ions at higher ph and temperature values and it is toxic mainly to fish at lower development stages. the longer the effect of ammonia, the lower the concentration the fish can handle. to exclude acute toxicity of ammonia, the n-nh3 concentration in the water stream may not be exceeded even for as short time (for a period of 1 hour) over 0.1 mg/l in salmon water and 0.2 mg/l in carp water (max. permissible recurrence of 1 year). what can be considered as a measure to reduce acute ammonia toxicity in the water course is sewage water retention and controlled discharges into the sewerage, increased throttled flow to the wwtp (lower n-nh4 concentrations in separated water) and increased retention volume in the sewer, separated water pre-treatment, e.g. outlet to an earth filter. however, given the considerable space demands, this measure is usually confined to little urbanised areas. natural vegetation shading results in reduced temperatures and ph fluctuation in the water course. this affects the dissociation balance of ammoniated nitrogen and reduces the proportion of toxic non-dissociated ammonia to the total ammoniated nitrogen. oxygen deficit the organic substances transfer through the csos and their subsequent decomposition may result in considerable oxygen deficit in the water course or its sediments. salmon fish is the most sensitive to the lack of oxygen. in order to ensure sufficient protection of biocenosis, the oxygen concentration in the river water as a result of the csos may not drop below 5 mg/l. at this concentration it is expected that there will not be anaerobic conditions even at the uppermost sediment level. the calculation of oxygen concentration in the receiving body of water after the separation is subject to considerable uncertainties even if the simulation programmes are used. oxygen deficit in the water course is usually caused by a combination of organic dry weather loading, eutrophication, and cso overflows. it is therefore recommended that the water course should be examined and dissolved oxygen concentrations should be measured. the lowest values occur at night and early morning when there is still no photosynthetic oxygen production. improvement of the situation usually calls for a combination of measures enhancing their efficiency. potential measures to reduce the oxygen deficit include a reduction of other sources of mass loading in the water courses than the csos, reduction in the number of overflows and pollution transfers from the csos by means of measures taken in the river basin and the sewerage. it also includes the reduction in impermeable surfaces in the catchment areas connected to the sewerage, increased influent to the wwtp, more frequent sediment removal from the sewer system. suspended solids suspended solids transfer from the csos to the water course may result in shortterm and long-term disturbance (turbidity, river bed silting, oxygen deficit at the bottom etc.). the determination of the pollution criteria for separated water is not possible given the current status of knowledge. the informative value of the pollution criteria to determine the critical cases is the concentration of suspended solids in the water course downstream the overflow amounting to 50 mg/l. a guideline when exceeding this value is the ratio between the number of inhabitants in food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 36 the river catchment area (pe) and q 347 in the water course. the exceeding is likely at pe/q347 > 25 pe/(l/s), potentially at pe q 347 > 15 pe/(l/s), if there are sediments in the sewers. if the recommended ratios are exceeded, it is necessary to make an onsite examination and, potentially, propose measures to retain suspended solids. suitable measures to reduce the load caused by suspended solids are partly identical with measures to reduce oxygen deficit, i.e. a reduction in the number of overflows and pollution transfer from the csos in the catchment area and the sewer system, in particular regular cleaning of the sewerage in sections with low gradients and separated water pre-treatment (e.g., earth filter). hygienic pollution the overflows contain a significant volume of pathogenic microorganisms (bacteria, viruses) causing hygienic pollution to the receiving body of water endangering its recreational use. indicative organisms of faecal pollution are usually escherichia coli and intestinal enterococca. in the separated water, concentrations of 104-107 ktj/100 ml of escherichia coli are expected. although the pathogenic microorganisms in water do not propagate and, on the contrary, die relatively fast, several days after the overflow the target values of bathing water quality set out by directive 2006/7/ec may be exceeded. bathing water should not receive water from the csos or the overflow frequency should at least be reduced as much as possible. a potential solution might be a temporary ban on bathing. aesthetic disturbance waste and objects coming from the sewerage (e.g. sanitary protection trapped by the vegetation) are visible for a long time even after overflowing through the csos. they normally do not affect the water quality. there is no limit defined for the aesthetic disturbance. experience shows that csos with the maximum frequency of overflows of 5-10x/rcsos usually cause only small aesthetic problems. as regards river courses with increased requirements for protection against aesthetic disturbance, the following is recommended to capture the floating objects: installation of screens and sieves in the combined system overflows and scum boards. however, to trap the drifted objects, these are more efficient in respect of front overflows than side overflows. construction of plug flow tanks is suitable for short distances in the catchment areas. in this case, the plug flow tanks are more suitable than otherwise generally recommended retention tanks retaining the first flush. once filled up, the mixture of dry weather and wet weather flows through the combined sewerage is diverted upstream the tank directly into the receiving body of water. on the contrary, the plug flow tank carries the whole overflow which is mechanically pre-treated and gross impurities are trapped. another possibility is to install vortex separators with a special protection of the spillway with sieves that can trap floating and drifted objects and, after a rain event, they can also ensure automatic cleaning. along with the technical measures it is also necessary to address the method of cleaning and removing the entrapped objects while respecting all related regulations.(kabelkova et al, 2010) 5. checking the measures efficiency the efficiency of the adopted measures must be checked in terms of emissions in the sewer system and pollution and its effect on the receiving body of water. once the measures are taken, measurements should be conducted to recalibrate the simulation model and to perform numerical evaluation of fulfilling the emission and pollution criteria. checking is also performed by field examination. once the measures are put in practice, food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 37 detailed information should be gathered, regular checks may be performed using more simple methods. to document the function and operation of the overflows to the water courses (tanks, csos, vortex separators etc.) and to verify the calculation assumptions it is appropriate to ensure continuous measurement of the most important structures or to calculate the frequency of filling up the tank, overflow duration, outflow through the throttling route downstream the cso or the vortex separator and running time of the mechanical equipment. during the pollution check it is necessary to carry out a physical-chemical and biological-ecological survey of the water course in the reaches upstream and downstream the csos outlets. priority is given to a simple check of visible signs of disturbance. as regards large outlets, the ecological condition of the water course is assessed based on the structure of the macrozoobenthos society, potentially diatom. the survey is performed at the minimum flow rate and usually indicates long-term effects of the discharge. to determine the immediate effects, it is necessary to measure chemical and hygienic indicators. given the high demands, this is usually carried out only for potable water and bathing water. (kabelkova et al, 2010) 5. tools and methods the evaluation of the effects of csos on the receiving body of water should be a standard part of projects focusing on drainage master plans. therefore, before commencing the work, a decision should be made on the applied simulation tools given the specific tasks, extent of schematic presentation of the urbanised catchment area and available data. the assessment of fulfilling the emission criteria may be performed using the outputs of the long-term simulation of the rainfall/run-off process using an urbanised catchment area model usually developed as part of the drainage master plan. by using the same simulation it is possible to make an assessment of the hydraulic disturbance of the receiving body of water. the assessment of the ammonia toxicity must be based on a model containing particle transport. to speed up the calculations and to ensure flexibility when assessing the alternatives it is advisable to use a hydrological model with gross schematising of the urbanised catchment area. the assessment of other pollution criteria does not require the simulation of the rainfall/run-off process in the urbanised catchment area. the models must be calibrated and verified and a sensitivity analysis of the results of the calculation focusing on the most uncertain input data is recommended. the most accurate information in the current disturbance of the water course ecosystem caused by an urban drainage system can be obtained based on a biological-ecological survey. the assessment using the biological-ecological survey is meaningful in the existing outlets or as a check of the efficiency of the measures taken. the survey encompasses the assessment of the visible signs of disturbing the water courses and societies of benthic invertebrates. its integral part is represented by assessing the water quality and the hydromorphological condition of the water course, which affects its biological condition. the assessment of the visible signs of disturbing the water courses can be used separately for a fast indicative assessment of the status of the water course and it also provides a series of additional information when evaluating the overall ecological condition. it is carried out along with the assessment of the morphological condition of the water course in the reaches upstream and food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 38 downstream the outlets. the data of the long-term water quality in the water stream is used as additional information to assess its biological condition and to assess the fulfilment of the pollution criteria of the combined system overflows. the assessment of the morphological condition of the water course and the potential of re-settlement in the disturbed reaches provide for important supporting information to assess the structure of the macrozoobenthos societies as it enables differentiation between the effects of degraded water course morphology on the biological condition of the water course and the effects of storm water separators. it is also used to differentiate the hydraulic loadibility of the water course. the objective of assessing the morphological condition is to determine the deviation from the natural condition. what is assessed is the changeability of the depth and width of the river channel including the flow characteristics, character and diversity of the river bed and its structure, character of the banks, width and structure of the bank zone and longitudinal passability of the stream defined by the presence of obstacles in the stream. to assess and classify the morphological condition of the water courses, a number of methodologies are available assessing the main aspects with various levels of detail. (kabelkova et al, 2010) 3. conclusions and recommendations in the first place, it is necessary to evaluate the fulfilment of the emission criteria. however, the measures needed in the event of a potential failure to ensure the prescribed minimum efficiency of dissolved and suspended pollution at the wwtp are taken only after assessing the pollution in the water course so as to enable potential coordination of the measures. measures proposed to fulfil the pollution criteria only on the basis of a numerical evaluation may not be economically feasible given the inherent safety of the evaluation even if more detailed levels of calculation are applied, and therefore, biological-ecological assessment of the water course is recommended as it enables better target orientation. the biological-ecological survey and continuous measurements of structures identified as critical is recommended to commence after determining the actual condition prior to the implementation of the proposed measures. the biological-ecological survey determines both the ecological condition of the water course in the reference profile upstream the assessed urbanised catchment area or the municipal drainage discharge point, and it identifies causes, extent and distance of the disturbance. if the ecological condition of the water course downstream the cso is good (directive 2000/60/ec), no measures need to be taken at a slight disturbance compared to the reference. the specific proposed measures must be taken to determine the causes of the problem and while respecting the local conditions. the measures may be taken in the urbanised catchment area, in the sewer system or in the natural catchment area and in the water course. priority is given to measures at the source, i.e. the urbanised catchment area aimed at eliminating the cause of the problem before taking remedial measures. as regards morphologically degraded water courses, it is also necessary to consider the possibility of their revitalisation which may be more efficient for improving their biological condition than measures taken in the municipal drainage system (krej í et al., 2002). similarly, with respect to rivers with a very low water quality in the urban catchment areas it is necessary to take measures in the urban drainage system and reduce the pollution transfer at more elevated food and environment safety journal of faculty of food engineering, tefancelmareuniversity suceava volume x, issue 3 2011 39 localities. the procedure when selecting the measures includes first the selection of suitable measures for a more detail survey, determination of costs and subsequent consideration of further criteria such as uncertainties, operating reliability etc. the measures are ranked according to the priorities in relation to measures in the catchment areas upstream and according to the severity of the water course disturbance by the urban drainage. a good ecological condition of the water course or a good ecological potential (with respect to strongly affected bodies of water) (directive 2000/60/ec) in the evaluated urban catchment areas is envisaged in the near future. 6. references 1.bwk-materialien 1 (2003): begleitbandzudem bwk-merkblatt 3. 2.kabelkova et al, 2010: metodickáp íru kaposuzovánídeš ovýchodd lo va jednotnýchstokovýchsystém v urbanizovanýchúzemích, sfžp 2010, praha 3. krej í a kol. (2002): odvodn níurbanizovanýchúzemí koncep níp ístup (eds. p. hlavínek a e. zeman), noel 2000, brno 4. krej í et al.(2004)gewässerbelastungendurch abwasserauskanalisationenbeiregenwetter, projekt storm: abwassereinleitungenauskanalisationenbeiregenw etter, eawag und buwal. 5.sm rniceevropskéhoparlamentu a rady 2000/60/es zedne 23. íjna 2000, ustavujícírámec pro innostspole enství v oblastivodnípolitiky. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 72 dielectrophoretic devices for separating foodborne pathogens iuliana mihaela lazar1, arcadie sobetkii2, ileana denisa nistor1, neculai doru miron1, marius stamate1, gabriel octavian lazar1 1vasile alecsandri university of bacau, calea marasesti 157, 600115 bacau, romania, ilazar@ub.ro 2mgm star construct srl, str. pincota 7b, bucuresti, romania abstract. today an increased interest in bioparticle separation research field is shown. microtechnology and nanotechnology opens new perspectives in food quality analysis such as biomems (micro-electro-mechanical-systems) devices for simultaneous detection of microorganism in food products with improved sensitivity and velocity [1]. diseases caused by different foodborne pathogens such as bacteria, viruses, fungi, parasites, etc. have been a serious problem. classical microbiological methods are taking a long time to confirm results for a particular pathogen organism. at present numerous rapid methods are being studied, for example polymerase chain reaction (pcr) [2], enzyme linked immunosorbent assay (elisa) [3], dielectrophoresis (dep) [4,5], dielectrophoresis combined with elisa, dielectrophoresis combined with electro rotation (rot), travelling wave dielectrophoresis, etc. dielectrophoresis is a method of manipulation of a micro particle in an electric field gradient which results from the interfacial polarization [6]. using low voltages and temperatures below 39 degrees allows us no permanent damage to the cells. theoretical modelling of behaviour in electric field is very important for the applications we need. electrode arrangement from biomems and channel geometries affects abilities to separate foodborne pathogens. this article describes the results obtained by our research team for manipulating bacteria with a specific electrode type of dep. keywords: bioparticle, dielectrophoresis, separation introduction the ability to accurately control and handle micrometer and nanometer scale particles is intensively studied in recent years especially as a capture method in different fields, including the food industry. [7] one of the methods used for this purpose is dielectrophoresis dep. dielectrophoresis movement consists in a bioparticle capture caused by an electric field applied. dielectrophoretic force depends on the electrical properties, geometrical and morphological factors and environmental bioparticle of suspension and the applied electric field characteristics (frequency and intensity). it may be highlighted both in continuous current (dc) [8] and in alternating current (ac) [9-14] because dielectrophoretic force does not depend on the electric field polarity [9]. in literature the most commonly used is the alternating electric field [10-14]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 73 dielectrophoresis as electrokinetic phenomenon that utilizes an asymmetric electric field to sort different bioparticle like nano-pathogen agents was performed using a micro fabricated device [11]. separation was possible through operation of the characteristically properties differences between normal and infected cells. sorting experiments were placed inside a microchip made from different microelectrodes patterned on a glass substrate. in this study we establish the experimental arrangement for capture lactic bacteria with dielectrophoretic forces. this method is not destructive, which is a great advantage. theory the basic law which describes the forces acting on charged particles in electric field: ( ) ( )f qe r r qe r        where q = q+ = qis the dipole electric charge and r  = d is the distance between charges. the behavior of a dipole in a variable electric field is described in figure 1 and the movement of particles under the effect of the resultant force in a uniform phase (a,b) and variable phase (c) is described in figure 2. fig.1 dipole placed in irregular electric field [16] in the nonlinear medium as biological medium, the dependence between electric field e and polarization p has the following form: p = e + e + e +... ,1 2 2 3   size called molecule polarizability, is a physical quantity numerically equal to the induced dipole moment in the molecule by an electric field intensity unit. fig. 2 (a) uniform electric field resultant force acting on the particle is zero. (b) a particle is under a dep force because of gradient electric field intensity. (c) a particle is under a dep force because of a gradient electric field phase [14] cases: a. if α does not depend on the direction of electric field orientation, the molecule is isotropic in terms of electric polarization. polarization vector is in this case:     p = np = n e = nql , n is the number of molecule, q is charge molecule and l is the distance between positive center charge distribution and negative center charge distribution; b. if α polarizability molecule varies with electric field orientation, the molecule behaves an anisotropic induced dipole moment and the electric field inducing a relationship tensor form: i ik kp = e cu (i,k =1,2,3) , ik is the components polarizability tensor molecule; c. in anisotropic media with different properties after different directions: food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 74 k 0 ki ip = e , cu (i,k=1,2,3=x,y,z) ,  is the electric susceptibility tensor components of the environment: 11 12 13 21 22 23 31 23 33 xx xy xz ik yx yy yz zx zy zz                                       for charged particles and electric field polarized in the absence of magnetic field, the force has two components, electrostatic force and dielectrophoretic force. ( )tot es deff f f qe p e           if there is a gradient electric field, then it will cause a net shift of the charged particles. the dielectrophoretic force must overcome brownian motion. this relationship establishes a requirement on the minimum particle size that can be manipulated, so high values of gradient electric field are necessary to manipulate the order of nano-size particles. next we express dielectrophoretic force (dep) in a three dimensional configuration. in an ac electric field, e(t) is a harmonic function of time and the force can be write as: where γ is the geometric factor of the particles, εm is the dielectric constant of suspending medium, and fcm is the clausius–mossotti factor. the terms re(fcm) and im(fcm) refer to the real and imaginary parts of fcm [8]. the dep force has two major terms: the first term represents the “classical dep force” the second term represents the “travelling-wave dep force”. for a spherical particle the re[fcm] is determined by taking the real component of the complex form of the clausius– mossotti factor: * * * * 1 22 2 1 p m p m p m p m cm sferic p mp m p m p m j f j                                  where *p and * m are the conjugate complex dielectric permitivities of the particle and medium, respectively and σp is the conductivity of the particle, σm is the conductivity of the medium and ω is the angular frequency of the applied electric field [15]. results and discussion we aimed to study the behaviour of lactic acid bacteria, particularly lactobacillus and streptococcus species. for this purpose we used a starter culture yoghurt (yo-mix 495 lyo 100 dcu, danisco, sassenage, france)[12,13,16]. after the standard procedure, the suspension of bacteria is put under the microscope. we follow the behaviour of bacteria under the influence of electric field. the dep force allows particles to move independently of their charge in an inhomogeneous electric field applied to the micro device (chamber electrode). materials constituting the dielectrophoretic chamber electrode are complex (for example: pt pd au mo cr al). it is very difficult to choose the material which the electrodes are made from. electrode geometry, their number and distance between them are important parameters in choosing the experimental set-up. to avoid the complexity of the manufacturing process, only one material is usually used to manufacture electrodes. nowadays different combinations of materials are used. the selection of materials for manufacture of electrodes depends on the desired destination, ionic species involved, environmental impact of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 75 materials and their suitability to manufacture in our study we established to generate a variable electrical field and to serve as dep electrodes for capturing bacterial cells to the electrode surface. we performed an experimental set-up composed of alternating signal source, a dielectrophoretic chamber made as a result of collaboration between umf bucharest, “vasile alecsandri" university of bacau and mgm star construct srl. several electrodes images are shown in figure 3(a, b, c). fig. 3a dielectrophoretic chamber with “teeth” electrodes. this image is made at biophysics department umf bucharest fig. 3b dielectrophoretic chamber with linear electrodes. this image is made at biophysics department umf bucharest fig. 3c dielectrophoretic chamber with castellated electrodes. this image is made at biophysics department umf bucharest in this study the electrodes of dielectrophoretic chamber were made from chromium, with a thickness of about one quarter micron. it is very difficult to produce technologically the space between electrodes, which must provide electrical isolation on the one hand, and on the other hand must have a good regular dielectrophoretic effect. we made different types of electrodes. images are viewed with optical microscopy at umf bucharest and afm microscope at “vasile alecsandri" university of bacau. afm applications in cell biology can be classified into several broad categories: imaging as shown in figure 4, micromanipulation studies, material property measurements and binding force measurements. in this study we are using afm to view the lactic bacteria behaviour. fig.4 scheme of an afm coupled with an inverted optical microscope [17] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 76 imaging of living cells has been coupled with controlled culture systems, enabling the possibility of continuous, long-term imaging. an important feature of afm is the possibility of studying dynamic processes at high spatial resolution [17]. the afm images of a micro channel electrode which has handled the bacteria are presented in figure 5 a, b: fig. 5a the image of a micro channel with 0.29 µm thickness fig. 5b the image of a micro channel with 0.25 µm thickness figure 6 shows the experimental set-up accomplished for capture lactic bacteria with dep force and figure 7, the dielectrophoretic chamber. fig.6a experimental set-up fig.6b dielectrophoretic chamber conclusions the first steps in a dep experimental setup were established for lactic acid bacteria, particularly lactobacillus and streptococcus species. we determined the optimal geometric configuration for the electrodes between which a maximum electric field gradient is applied. we made an experimental arrangement which allows the variation of frequency and voltage applied to study the influence of this pulse of electric current on bacteria. a number of investigations by optical microscopy lactic bacteria migration were observed and the first results are encouraging, but working with so many variable parameters is a difficult task. in the future we intend to establish the experimental conditions in which lactic bacteria capture is achieved by positive dielectrophoresis (dep); the method allows concentration of lactic bacteria in a sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 77 acknowledgments the author would especially like to thank the entire team of teachers from “biophysics and cell biotechnology”, master in medicine, “carol davila” university of medicine and pharmacy of bucharest for initiating and guiding into cell biotechnology. references 1. hua yang, huaping li, xiuping jiang, detection of foodborne pathogens using bioconjugated nanomaterials, microfluidics and nanofluidics, volume 5, number 5, 2008, pages 571-583, 2. martin c. easter, rapid microbiological methods in the farmaceutical industry, publisher crc press informa ltd registered in england and wales registered number: 1072954 registered office: mortimer house, 37-41 mortimer street, london w1t 3jh, uk, taylor & francis elibrary, 2005. 3. m. castellarnau, a. errachid, c. madrid, a. juarez, j. samitier, dielectrophoresis as a tool to characterize and differentiate isogenic mutants of escherichia coli, biophysical journal, volume 91, issue 10, 2006, pages 3937-3945 4. liju yang, dielectrophoresis assisted immunecapture and detection of foodborne pathogenic bacteria in biochips, talanta, volume 80, issue 2, 2009, pages 551-558 5. héctor moncada-hernández & blanca h. lapizco-encinas, simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach, analytical and bioanalytical chemistry volume 396, number 5, 2010, pages 1805-1816 6. hui-sung moon & hee taek im & ahmi choi & hyo-il jung, real-time detection of food-borne bacterial adenosine triphosphate (atp) using dielectrophoretic force and a bioluminescence sensor, microchimica acta, volume 170, numbers 3-4, 2010, pages 283-288 7. ben greenebaum, frank barnes, bioengineering and biophysical aspects of electromagnetic fields, series: handbook of biological effects of electromagnetic fields, crc press taylor & francis group, third edition, 2006 8. frank barnes “interaction of dc and extremly lowfrequency fields with biological materials and systems," chapter 5 in the crc handbook on biological effects of electromagnetic fields, 3rd edition edited by frank barnes and ben greenebaum , 2006, pages 115-152 9. r. pething, biodetection using microphysiometry tools based on electrokinetic phenomena, defense against technologies: detection technologies, implementation strategies and commercial opportunities, volumul 1, 2006, pages 129–142 10.yuejun kang, barbaros cetin, zhemin wu, dongqing li, continous particle separation with localized ac-dielectrophoresis using embedded electrodes and an insulating hurdle, electrochimica acta vol.54 , 2009, pages 1715-1720 11. liju yang, padmapriya p. banada nigannad r. chatni, kwan seop lim, arun k. bhunia, michael r. ladisch, rashid bashir, a multifunctional micro-fluidic system for dielectrophoretic concentration coupled with immuno-capture of low numbers of listeria monocytogenes, birck nanotechnology center, birck and ncn publications, purdue libraries, 2006, http://docs.lib.purdue.edu/nanopub/290/ 12. walid m. el-sharoud, prevalence and survival of campylobacter in egyptian dairy products, food research international, volume 42, issues 5-6, 2009, pages 622-626 13. harley prescott, laboratory exercises in microbiology, 5th ed, the mcgraw-hill companies, 2002 14. c. zhang, k. khoshmanesh, a. mithell, k. kalantar-zadeh, dielectrophoresis for manipulation of micro/nano particles in microfluidic systemsanalytical and bioanalytical chemistry, volume 396, number 1, 2009, pages 401-420, 15. carlos f. gonzaleza and vincent t. remcho, fabrication and evaluation of a ratchet type dielectrophoretic device for particle analysis, journal of chromatography a, 1216 , 2009, pages 9063–9070 16. product description yo-mix 495 lyo 100 dcu, danisco, www.danisco.com 17. andrea alessandrini and paolo facci, afm versatile tool in biophysics, measurement science and technology volume 16, number 6, 2005, pages r65-r92 microsoft word 1 primele pagini nr 2 din 2010 5 octombrie.doc stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering year ix, no.2 – 2010 university publishing house of suceava editor-in-chief: sonia gutt ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey marija bodroža-solarov university novi sad, institute for food technology, serbia dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), national technological centre of the canning and food industry (ctc) molina de segura (murcia), spain georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli süleyman demirel university, isparta, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain feiza memet maritime university of constanţa, romania christian neususs technical university of aalen, germany cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania regina proskuviene vilnius pedagogical university, lithuania alice roşu ştefan cel mare university of suceava, romania rodica rotar ştefan cel mare university of suceava, romania osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia charles e. walker kansas state university, u.s.a. igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international database: indexcopernicus journals master list. also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal is made by niculai moroşan. cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by editura universităţii ştefan cel mare suceava, romania. microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_69-74.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 73 influence of total soluble content, starter culture and time period on rheological behaviour of cultured buttermilk mircea-adrian oroian1, gheorghe gutt1 1 ştefan cel mare university of suceava, faculty of food engineering, universităţii street, 720229, suceava, romania, e-mail: m.oroian@usv.ro, g.gutt@usv.ro abstract: casein gels are part of the dairy products, hard or soft products, as cheese and yogurt, cultured buttermilk, and their rheological behaviour can be measured by various instrumental techniques. for this study we used three types of milk (7.5%, 10%, and 12.5% respectively tss) and starter culture (lactococcus lactis subsp. lactis and lactococcus lactis subsp. cremoris) in different concentrations (2 g/100 l, 3 g/100 l, 4 g/100 l). the rheological properties of the cultured buttermilk obtained from milk were investigated by a brookfield rv pro ii+ viscometer at different shear rates, during the time period keeping (from the 1st day – the obtaining day, till the 20th day – the last day when the product is good for human consumption). the samples with the highest tss (total soluble content) present the highest viscosity, while the samples with 7.5% tss content present the smallest viscosity. during the rheological tests, the samples with 3 g/100 l starter culture presented the best rheological properties (the samples had the highest viscosity), while the samples with 4 g/100 l had the smallest viscosity due to the proteolysis generated by the starter culture activity. the flow index behaviour was influenced more by the tss content rather than by the starter culture dose. it can be seen that the change in the apparent viscosity was not linear with solids concentration, where a 5% (from 7.5% to 12.5%) increase in the solids concentration led to triplicate in the apparent viscosity. keywords: viscometer, shear stress, shear rate, flow index 1. introduction rheology is the study of deformation and flow of matter. the science of rheology grew considerably due to research work done on synthetic polymers and their solutions in different solvents that in turn was necessary due to the many uses of the polymers in day-to-day and industrial applications. nevertheless, because of the biological nature of foods, food rheology offers many unique opportunities of study. many foods are composed mainly of biopolymers and aqueous solutions containing dissolved sugar and ions. the former ones are large molecules, often called macromolecules, such as proteins, polysaccharides, and lipids from a wide range of plant and animal sources. [1] rheological properties are based on flow deformation responses of foods when subjected to normal and tangential stresses [2]. in food research, the term is often used interchangeably with texture, which refers to the flow, deformation, and disintegration of a sample under force. strictly speaking, texture relates to solid foods, and viscosity—the tendency to resist flow— relates to fluid foods. food can exhibit both solid and liquid characteristics, and rheology can identify the properties of such foods [3]. typically, milk is fortified with dairy ingredients to produce a milk base which is then submitted to a drastic heat treatment, which results in a high level of thermal denaturation of the whey proteins and their partial fixation on the casein micelles. as a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 74 consequence, aggregation is promoted, giving stronger gels and decreasing the extent of acidification required to allow association. finally, the lactic acid production during the fermentation step results in the destabilization of the micellar system and associated gelation of the proteins. as the isoelectric point of denatured proteins (ph 5.2), and casein (ph 4.6) are reached, low-energy bonds, mainly hydrophobic, are progressively established between the proteins [4]. casein gels are responsible for many rheological properties of cheese, yogurt, buttermilk, cream and other dairy products that gel, stretch, and fracture. rheological studies are performed as a quality control method in dairy plants and as a technique for scientists to study the structure of the product [5]. the dairy products (yogurt, cultured buttermilk, sana cultured buttermilk) presents the thixotropy phenomena. thixotropy is the property of certain gels or fluids that are thick (viscous) under normal conditions, but flow (become thin, less viscous) over time when shaken, agitated, or otherwise stressed. thixotropy should be defined as: the continuous decrease of viscosity with time when flow is applied to a sample that has been previously at rest and the subsequent recovery of viscosity in time when the flow is discontinued. this is consistent with the iupac terminology [6]. it should be noted that various general scientific dictionaries and encyclopedias still give different definitions, often more closely in line with freundlich's original definition [7]. the essential elements of the definition used nowadays are that: it is based on viscosity; it implies a time-dependent decrease of the viscosity induced by flow; the effect is reversible when the flow is decreased or arrested [8]. the members of lactococcus gen are gram positive lactic bacteria, having a coccus form or swerved form from it, which can be oval. the cells, which can be associated in pairs or chains, have a diameter between 0,5-1,5 µm and do not present mobility. lactococcus lactis lactis and lactococcus lactis cremoris are mesopilic, and are characterised by their potential to produce lactic acid. they have the capacity to ferment lactose under 0,5% and have a reduce acidotolerance (are inhibited at ph<4.5) [9]. 2. materials and methods 2.1. materials milk with a tss 7,5 %, 10%, 12,5% reconstitute, lactococcus lactis subsp. lactis and subsp. cremoris pure starter culture (enzymes & derivates s.a. costisa neamt); orbital shaker; thermostat; brookfield viscometer model rvdv ii pro, with disk spindle, rv2 type. tab. 1. milk properties 7,5% 10% 12,5% fat, g/100g 2.1 2.8 3.5 protein, g/100 g 1.92 2.56 3.2 sugar, g/100g 2.7 3.6 4.5 ash, % 0.43 0.57 0.72 acidity, 0t 18 18 18 2.2. sample preparation all the samples were made using reconstituted milk. the starter culture is extracted from the freezer and thawed. the milk was heated at 90 0c for 5 min, afterwards the samples are cold to 28 0c. the starter culture was inoculated at 28 0c (2 g, 3 g and respectively 4 g starter culture /100 l milk with 7.5%, 10% and respectively 12.5% tss). for a good activation of the starter culture, the samples are shaken for 15 min on a orbital shaker at 150 rpm. the starter culture was inoculated at 28 0c. all the samples were maintained at 28 0c for 12 h in the thermostat. the samples were kept in the refrigerator at 4 0c for 20 days. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 75 2.3. determination of rheological properties of cultured buttermilk viscosity measurements were carried out on the cultured buttermilk samples at room temperature (200c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 0,5; 1; 2; 2,5; 4; 5, 10 and 20 rpm with rv spindle (rv2). the spindle nos (spindle nos: 2) was used in accordance with the sample nature to get all readings within the scale. the samples in 300 ml of beaker with a 8,56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain the desirable temperature of 250c. first measurements were taken 2 min after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression σ=k·γn, where σ – shear stress (n/m2), γ is the shear rate (s-1), n is the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. 3. results and discussion viscosity data showed that all samples under examination were non-newtonian fluids, since the values for the flow behaviour index, n were under 1, which was indicative of the thixotropy nature of culture buttermilk. the power law equation was found to be an adequate model to describe the flow behaviour of the samples in this study. the r2 values of the equations log shear rate versus log rotational speed were found to vary from 0.950 to 0.998. the flow index behaviour (n) of the power law model varied between 0.208 to 0.255. although n does not have a strong dependence on the concentration of starter culture, while the tss concentration has a great impact on the index flow behaviour. according to mitschka, p., steffe, j.& daubert, c. [10, 11] the brookfield viscometer with disk spindles represents a good method to determine non-newtonian fluid properties. one of the manifestations of thixotropy is the hysteresis loop. the hysteresis technique was introduced by green and weltmann [12]. it consists in systematically increasing and decreasing the shear rate between zero and a maximum value. fig.1. hysteresis loop of culture buttermilk the change can be a continuous ramp or a series of small steps. when the transient data are plotted as shear stress versus shear rate, a thixotropic sample will describe a hysteresis loop (fig.1) because the stress will lag behind the shear rate [8]. the gap between the two signals leads from the breakdown of the casein chain during the strain application. shear rate [s-1] sh ea r s tr es s 1 0 · [ dy ne ·c m 2 ] ] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 76 the empirical data were converted to shear rate and shear stress. the shear stress is calculated using the next equation: τi=kτ·αi·c [1] fig. 2. changes in the viscosity in cultured buttermilk (1st day) where τi – shear stress [dyne/cm2] kτ= 0.119, this constant is for the spindle nos 2 αi – torque dial, % c – 7,187 dyne/cm for rv viscometer the shear rate is calculated using the next equation: γi = kγ(n)·ni [2] γi – shear rate, s-1 kγ(n) – constant, depends by the value of n ni – rotational speed, rpm the influence of tss on the rheology of culture buttermilk is a positive one, the viscosity increases with the increasing of tss percent, due to the quantity of protein in the sample. the samples with 12.5% tss had the highest viscosity, while the samples with 7.5% tss the lowest viscosity (fig. 2), the shear stress had the same evolution like the viscosity did. the flow index behaviour varies between 0.211 and 0.255 due to the increasing of protein content of the samples. it can be seen that the change in the apparent viscosity was not linear with solids concentration, the same observation was obtained by hazim et al. [13], where a 5% (from 7.5% to 12.5%) increase in the solids concentration led to triplicate in the apparent viscosity (from 11.14 to 30.05 pa·s at 0.5 rpm). this is an indication that the control of the solids concentration is an important quality factor and it may affect the final acceptance of cultured buttermilk by the consumer. the changes of the viscosity with the starter culture dose is not a linear one, the increasing of the dose from 2 g to 3 g fig. 3 changes in the apparent viscosity with shear rate in cultured buttermilk: a. 7.5% tss; b. 10% tss; c. 12.5% tss: 2 g starter culture / 100 l milk; 3g starter culture/100 l milk; 4 g starter culture / 100 l milk *the apparent viscosity measuring were made for the 1st day a pp ar en t v is co si ty [p a· s] a pp ar en t v is co si ty [p a· s] a pp ar en t v is co si ty [p a· s] shear rate [s-1] shear rate [s-1] shear rate [s-1] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 77 increased the viscosity and the shear rate (tab.2, fig.4), but from 3 g to 4 g the viscosity and the shear rate decreased. the maximum of rheological behaviour ( viscosity, shear rates, flow index) is observed at 3 g starter culture as is presented by the producers on the starter culture description sheet. the decreasing of the rheological properties can be generated by the activity of the cells of lactococcus lactis subsp. lactis and subsp. cremoris, which is a power full proteolysis activity. the starter culture activity increased form the 1st day to the 20th day, and the viability tab. 2. changes in the rheological properties of the cultured buttermilk prepared in three different formulations sample flow index [n]* apparent viscosity [pa·s]** 2 g/100 l 0.211 8.49 3 g/100 l 0.216 11.14 7.5% tss 4 g/100 l 0.208 9.57 2 g/100 l 0.229 15.41 3 g/100 l 0.234 18.89 10 % tss 4 g/100 l 0.227 17.13 2 g/100 l 0.238 27.07 3 g/100 l 0.255 30.05 12.5% tss 4 g/100 l 0.235 23.53 *the flow index is for the samples with 3 g/100l starter culture, in the 1st day **the apparent viscosity was measured at 0.5 rpm of the cell increased in all the samples and led to the decreasing of shear stress for all the samples due to the break of the casein chains by the activity of cells (fig.4). conclusions the rheological properties of cultered buttermilk are influeced by the tss, starter culture and time period keeping, leading to the conclusions that the samples with 12.5% tss and 3 g starter culture/100 l milk have the best rheology (viscosity and shear stress functions are the highest for these samples). the brookfield viscometer model rvdv ii pro+, represents a cheap method to determine rheological properties of nonnewtonian fluids, providing information about viscosity, flow index behaviour, shear rate and shear stress of fluids. the flow index behaviour (n) varies between 0.211 and 0.255, the variation is greater with the increasing of the tss content compared with the starter culture dose. it can be seen that the change in the apparent viscosity was not linear with solids concentration, where a 5% (from 7.5% to 12.5%) increase in the solids fig. 4 changes in the shear stress with the shear rate in the cultured buttermilk: 2 g starter culture /100 l milk 1st day; 3g starter culture/100 l milk 1st day; 4 g starter culture /100l milk 1st day; 2 g starter culture /100 l milk 10th day; 3g starter culture /100 l milk 10th day; 4 g starter culture / 100 l milk 10th day; 2 g starter culture /100 l milk 20th day ; 3g starter culture /100 l milk 20th day ; 4 g starter culture / 100 l milk 20th day shear rate [s-1] shear rate [s-1] shear rate [s-1] sh ea r s tr es s [d yn e/ cm 2 ] sh ea r s tr es s [d yn e/ cm 2 ] sh ea r s tr es s [d yn e/ cm 2 ] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 78 concentration led to triplicate in the apparent viscosity. this is an indication that the control of the solids concentration is an important quality factor and it may affect the final acceptance of cultured buttermilk by the consumer. acknowledgment this paper was supported by the project "knowledge provocation and development through doctoral research pro-doct contract no. posdru/88/1.5/s/52946 ", project co-funded by european social fund through sectorial operational program human resources 2007-2013. references 1. rao, m.a. phase transitions, food texture and structure, in texture in food, volume 1: semi-solid food, ed. b.m.mckenna, pp. 36-62, woodhead publishing ltd., cambridge, uk. 2. rao, m.a., rheology of fluid and semisolid foods, principles and applications, second edition, food engineering series, springer, 2007 3. giese, j.. measuring physical properties of food. food technol. (1995), 19:54–63. 4. lucey, j. a., & singh, h.. formation and physical properties of acid milk gels: a review. food research international (1998), 30(7), 529– 542. 5. tunick, m.h. rheology of dairy foods the gel, stretch, and fracture, journal dairy science 83:1892-189 6. iupac. compendium of chemical terminology, electronic version http://goldbook. iupac.org. w06691.html. 7. barnes ha. j non-newton fluid mech 1997;70:1. 8. mewis, j., wagner, n.j. thixotropy, advances in colloid and interface science 147-148 (2009) 214-227 9. costin, g.m., produse lactate fermentate, editura academica, 2005 10. mitschka, p. a simple conversion of brookfield r.v.t. readings into viscosity functions, rheologica acta, 21, 207-209, springerlink, 1982 11. steffe, j., daubert, c., bioprocessing pipelines: rheology and analysis, freeman press. usa, 2006 12. green h, weltmann rn. ind eng chem anal ed 1943;15:201 13. hazim, a.m., basim, a.-j., ali, al-s., effect of solids concentration on the rheology of labneh (concentrated yougurt) produced from sheep milk, journal of food engineering 61 (2004) 347352 196 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 196 205 biochemical characterization of microbial populations involved in loh-djiboua cocoa’s fermentation in cote d’ivoire liliane m. kouame 1 , *ginette g. doue 1, niamke j. adom1, honore g. ouattara1 , sébastien l. niamké 1 1laboratoire de biotechnologies, filière biochimie-microbiologie, unité de formation et de recherche en biosciences, université félix houphouët-boigny, cocody, abidjan-côte d’ivoire. 22 bp 582 abidjan 22. doueginette @ yahoo.fr, *corresponding author received may 26th 2015, accepted june 26th 2015 abstract: comprehensive report on biochemistry characteristics of microorganisms involved in lohdjiboua cocoa fermentation is made in order to highlight diversities that occur among them. microbial growth monitored showed that yeasts, lab and bacillus were initially present, while aab emerged after 12 h. throughout the process the aab population load was higher than other microorganisms with rates between (7.76 7.39) log cfu / g of beans while yeast, lab, aab and bacillus reached a maximum of load (7.28 8.82) log cfu/g of beans, under fermentative conditions of temperature (28 – 36 °c) with a peak at 45 °c (24 60 h) and ph ranged between 4.5 – 8. the 24 best producing yeasts pectolytic enzymes resulting from 267 isolates presented four fermentatives profiles. among the 173 bacillus isolates were obtained 45.08% pectinolytic activity streams with high, medium and low production level, 37.05% citrate lyase activity strains and 70.02% power acidifying strains. homofermentative type (92.83%) largely dominated the heterofermentative type (7.72%) among the 210 strains of lab isolated. all lab isolated showed ability to ferment glucose while only 2 not ferment fructose and 9 not ferment sucrose. 76.19% proved to be able to assimilate citrate. the 166 isolates of aab with clear acidifying power were constituted into 2 genera mainly acetobacter (80.76%) and gluconobacter (19.24%). this study emphasized the clear dominance of aab population mainly constituted of acetobacter during the growth dynamic of microbial groups involved in loh-djiboua cocoa fermentation and showed the wide variety in activities and population among these different groups. keywords: indigenous microflora, biochemistry characteristics, growth dynamic, ivorian beans fermentation 1. introduction fermentation of cocoa beans is the first step in chocolate-making chain, followed by drying and roasting. this step which is crucial for ensuring desired characteristics cocoa flavor and aroma, is one of the most important process leading to commercial cocoa and chocolate quality [1, 2] during the cocoa beans fermentation, the mucilaginous sugary pulp surrounding the bean is removed by the action of various microbial species indigenously present in the cocoa beans such as yeasts, lactic acid bacteria (lab), acetic acid bacteria (aab) and bacillus sp [3]. microbial fermentation induces numerous chemical reactions leading to a deep modification of the biochemical characteristics of beans [4]. early in this process, yeasts are favored under anaerobic conditions, high sugar http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 197 content and low ph. they produce ethanol and present pectinolytic activity. the reduced oxygen availability, combined with increased temperature and ph, favor the development of lab, which consume citric acid and ferment sugar to lactic acid, acetic acid, ethanol and mannitol. the increased aeration, due to pectin degradation, promotes the development of aab, which oxidize ethanol into acetic acid, in a highly exothermic process [3, 4, 6]. ethanol and acetic acid diffuse deep into the beans and combine with the temperature, trigger the activation of endogenous enzymes, mainly proteolytic enzymes but also aminopeptidase, invertase, polyphenol oxidase and glycosidase [7]. this leads in serial reactions responsible for final quality of the fermented beans and chocolate [3]. although the entire physiological role of these microbial groups involved in cocoa fermentation is not well-defined, it’s clear that any fermentation cannot be correctly processed without the micro flora. several studies have investigated the microbial diversity of spontaneous cocoa pulp fermentation in different countries such as ghana [8], brazil [9], malaysia and trinidad [10], côte d’ivoire [4], but very few studies are focus on the diversity of this indigenous microflora in each area of cocoa producing countries. geographic location is known to have an influence on the composition and characteristic of microbial consortium responsible for cocoa fermentation [3] since changes in the local climatic conditions influence the sequence of microorganisms involved in this fermentation. in côte d’ivoire, there are different cocoa producing regions with certainly different qualities. the characterization of the microbial flora involved in cocoa fermentation in each region may allow well knowing their diversity and establishing their cartography. the region of loh-djiboua (5° 40′ n 5° 30′ w), a part of main growing areas of cocoa in côte d’ivoire which recorded for the 2013-2014 campaign a production of 110.000 tons of cocoa beans [11], has been so far no studies concerning microbial diversity involved in cocoa fermentation in this western region. yet these studies could be used to carry out the cartography of ivorian cocoa microbial flora. the aim of study is to establish a comprehensive report on the biochemistry characteristic and to investigate some functional properties of the microbial groups involved in cocoa fermentation in loh-djiboua. this could help us to control this process and improve quality of product. 2. matherials and methods 2.1. fermentation conditions and sampling: spontaneous cocoa bean heap fermentation was performed at the cnf (national floristic center) of the university félix houphouet boigny. cocoa pods constituted of mixed genotypes (foraster, trinitario and criollo cultivars) were harvested from lohdjiboua (geographic coordonates 5° 40′ n 5° 30′ w), a western province of côte d’ivoire. beans were removed from pods and mass about 50 kg fermented traditionally by heap fermentation on banana leaves for six days. samples of fermenting cocoa bean about 200 g were taken directly at 15 cm depth on the fermenting mass according to a fixed time schedule, notably at the start (0 h) and each 12 h of fermentation. each sample was transferred into sterile plastic bag for microbiological analysis. the ph and temperature were also regularly recorded at the same depth on the fermenting heap, with ph-meter and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 198 thermometer (hanna instruments, europe, romania). 2.2. isolation and enumeration fermenting samples (25 g) were aseptically mixed with 225 ml of 0.1% saline-peptone water (oxoid, basingstoke, united kingdom) in a stomacher bag (seward, worthington, united kingdom) and manually shaken for 5 min to give an uniform suspension of the cocoa pulp material. after appropriate dilution in sterile saline, 0.1 ml samples from each dilution were spread inoculated onto duplicate plates of different agar media. yeasts were enumerated on malt yeast peptone glucose agar [12, 13]. lab were isolated by plating the fresh fermented samples on three selective agar media; mrs, mse and m17 agar (all from oxoid, basingstoke, hampshire, uk), supplemented with 50 pg/ml of nystatin to inhibit fungal growth. aab were enumerated on potato medium [14]. nutrient agar (merck) supplemented with 0.1 % nystatin to inhibit the growth of fungal was used as a general medium for bacillus sp. the culture was incubated at 30°c for 48 h. following incubation, the number of colony-forming units (expressed as cfu per gram cocoa pulp bean mass) was recorded, and each colony type was morphologically characterized and counted. yeasts, lab and aab were identified according conventional biochemical identifications keys. identification of bacillus sp isolates were done using conventional colonial morphology, gram staining and biochemical reactions according to bergey’s manual of systems bacteriology. isolated strains of lab were stored in mrs buffer medium, aab in luria bertani medium, yeasts and bacillus sp on the same isolation medium at -80 °c supplemented with 20 % (v/v) glycerol in eppendorf tubes for further studies. 2.3. biochemical characterization of microbial from loh-djiboua’s cocoa beans fermentation 2.3.1. yeasts population yeasts strains isolated were analyzed for their ability to produce pectinolytic enzymes and catabolize carbohydrates containing in cocoa pulp pectinolytic strains were screened according to [4, 15] methods. the carbon metabolism of pectinolytic yeasts strain study was carried out by[16] method. the carbohydrates tested were d-glucose, maltose, d-fructose, sucrose, lactose, and d-galactose. cultures were incubated at 30 ºc for 48 h to 3 weeks. the presence of gas in durham tubes indicates that the isolates ferment carbohydrates. 2.3.2. lab population lab strains isolated were analyzed for their ability to produce acid by catabolizing the main sugar and for their ability to catabolize citric acid containing in cocoa pulp. the carbon metabolism of bacterial strains was evaluated by [17] method with slight modification. this study was performed in a modified mrs medium containing the appropriate carbohydrate at 2 % as sole carbon source, 1.7 % agar and supplemented with 0.005 % of bromocresol purple. the carbohydrates tested were glucose, fructose and sucrose that are known to be the sugars contained in the cocoa pulp [18]. each strain was cultivated by central sting in the medium and then incubated at 30 °c for 72 hour in anaerobic conditions. the capacity of strains to metabolize the carbon source is assessed by the presence of colony in the tube and the change of medium color due to ph lowering, comparatively to the negative control. the fermentative type food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 199 was determined by ability of strains to produce gas from carbon source. indeed, the presence of gas at the bottom of the tube accompanied by yellow zone indicates heterofermentative lab type while homofermentative lab form are characterized by the absence of gas on the tube presenting yellow zone. lab strains presenting citrate lyase activity were also screened according to [19] method. after incubation, the colonies which appear blue on the medium are those able to metabolize citrate. 2.3.3. aab population aab strains isolated were analyzed for their ability to produce acid in solid medium and by identification of their main genera involved in loh-djiboua’s cocoa beans fermentation. acidification capacity in solid medium of bacterial strains was evaluated as described by [6] method. acid production in solid medium was monitored by formation of yellow zone around the spot. acidification capacity of strains was evaluated by measuring of the yellow zone diameter. the isolates were identified up to genera (acetobacter and gluconobacter) using over oxidation capacity test of [6] method. 2.3.4. bacillus sp population bacillus sp strains isolated were screened for their ability to produce pectinolytic enzymes, acid in liquid medium and to catabolize citric acid containing in cocoa pulp. pectinolytic activity of strains were screened according to [4, 14] method. bacillus sp strains presenting citrate lyase activity were also screened by [19] method. acidify capacity of bacillus sp strains was evaluated by [20] method. a negative control was prepared in the same conditions and not inoculated with the microbial culture. acid production was monitored by formation of yellow area in the tube with or not production of gas and acidify capacity was analyzed by evaluating in a visual scale. 3. results and discussion 3.1. change in ph, temperature and dynamic of the microorganism during cocoa bean fermentation analysis of heap spontaneous fermentation condition indicates that the ph of fermentation heap ranged from 4.5 at the beginning to 8.0 at the end of the fermentation process, while the temperature ranged from 28 to 36 °c with a peak at 45 °c within 24 -60 h (figures 1a and 1 b). the same profile of temperature and ph variation has been regularly recorded in cocoa fermenting mass in côte d’ivoire [4, 6] and other countries [3 ,5] indicating that the increase of both parameters constitutes an inherent property of cocoa fermentation worldwide. moreover, ph continuously increasing during the fermentation became alkaline at the end of the process. several authors [5, 6, 21] also reported an alkaline ph (8.5, 7.9 respectively) at the end of spontaneous cocoa fermentation in côte d’ivoire. this result confirms the particularity of fermentation conditions of ivorian cocoa since alkaline ph has not been yet reported in other country [3, 5, 22]. we could not explain the reason for why, sometimes the ph became alkaline in cocoa fermentation but we find that loh-djiboua cocoa fermentation conditions remain the same as those of the country. so this particularity of fermentation of loh-djiboua cocoa beans provided an ecosystem that selected for successional growth of various species of yeasts, lab, aab and bacillus the dynamic of microorganism’s population obtained from numeration during fermentation is shown in figure 2. in general, simultaneous growth of yeasts, lab, aab and bacillus took place with an food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 200 undetectable level of bacterial load for aab at the beginning (figure 2) since no colony corresponding to ab was identified on the plate at this time, in contrast to the other microorganisms. fig. 1. evolution of ph (a) and temperature (b) during lôh-djiboua’s cocoa beans fermentation. error bar indicate standard deviations between three replicates these microorganisms are present throughout the fermentation process with some particularities. indeed, populations of yeasts, lab and bacillus sp are present from the beginning of the process (5.56 to 5.96) log cfu/g of beans, unlike aab population which appears after 12 h of fermentation. then, the microbial population of yeasts, lab and aab rapidly increased to their maxima (7.58 to 8.82) log cfu/g of beans, at 12 h and 72 h of time respectively and subsequently decreased in two stages for lab and for yeasts, gradually until reaching undetectable levels at the end of fermentation. fig. 2: microbial growth dynamic during lôh-djiboua cocoa beans fermentation bacillus sp unlike the other microbial genera, recording a different growth dynamic insofar after a rapid increase over the first 12 h, these bacteria recorded a slow increase until the end of fermentation with high values around 7.70 log (cfu / g of beans). moreover, we can also noticed that aab population recorded the highest load than that of other microorganisms between 24 and 96 h of fermentation with rates ranged (7.76 7.39 log cfu / g of beans) comparatively to the other food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 201 microbial types. the presence of these microorganisms from the beginning on the fermentation heaps would be due to an accidental inoculation of fermentation heaps by a variety of microorganisms from the pod surface, knives, labors hands, fermentary [23, 24] while the lack of aab observed during the same time, can be explained by their undetectable low rates. furthermore difference in growth dynamics recorded by bacillus sp strains isolated is due to their inherent nature which allowed them able to withstand drastic environmental conditions and show great adaptability. finally regarding aab, it also should be noted that the same growth pattern was observed with maximum population in mexico [1] and in ghana [8]. but on the other hand, as aab population isolated was consistently larger than the other microbial groups, this differs from most other fermentations where yeasts or lab were the dominants microorganisms in bahia [24] and in ghana [25]. this could, in certain extent, explain differences observed in cocoa quality between these countries and be a particularity of loh djiboua cocoa fermentation leading to well fermented beans. 3.2. biochemical characterization of yeast strains isolated search of pectinolytic activity of yeasts isolated from loh-djiboua cocoa fermentation revealed that only 10.48% of the 267 yeasts isolated were able to produce pectinolytic enzymes. although the proportion of yeast strains pectinolytic activity is lower than that reported by [2] with 17.56%, the study has revealed a wide level of production with large, medium and small producers. this result suggested that some of yeasts isolates from loh-djiboua cocoa fermentation would participate in aeration of the fermentation heaps by producing these pectinolytic enzymes. aeration conditions necessary for growth of aab are in part created by pectinolytic enzymes which break down the pectin responsible for the viscosity and stickiness of cocoa pulp [4, 22]. so this wide level of pectinolytic activity obtained by yeast strains during loh-djiboua cocoa fermentation enable strengthen the elimination of the pulp and thereby accelerate this process. this ensuring thus permanently quality commercial of lohdjiboua cocoa’s beans.hence, the best pectinolytic yeasts isolated (24 strains) based on halo diameter were further studied for their specificity. their carbon metabolism was analyzed. the strains showed a large carbon metabolism profile (3) with capacity to ferment glucose, fructose, sucrose and galactose among the six carbohydrates tested (table 1). table 1: fermentative profile of pectinolytic yeasts strains isolated from lôh-djiboua profiles sugars number of isolates glucose fructose sucrose maltose lactose galactose 1 + + + 14 2 + + 8 3 + + + 2 nb: (+): gaz production (-): not gaz production this suggested a great diversity of species among yeast streams isolated. it is known that, yeasts present one of the wide microbial diversity in which strains can behave very differently. this difference in carbon metabolism is due to the transport system associated to the membrane which exerts selection of carbohydrate to enter into the yeast cells [26]. generally, the pulp of cocoa fermentation contains mainly food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 202 glucose, fructose and sucrose which are to be fermented into ethanol and at term, into acetic acid [3]. these reactions are essential for specific colour, flavour and aroma of the cocoa beans and chocolate [27]. from this point of view, with their large carbon metabolism profile, the best pectinolytic yeasts isolates should keep entirely their enzyme production capacity due to their adaptability and contributed to the maintain of the quality of loh-djiboua cocoa beans. 3.3. biochemical characterization of aab strains isolated the study of biochemical characteristics of aab revealed that 166 aab strains isolated are grouped into two mainly genera acetobacter (134 strains, 80.72%) and gluconobacter (32 strains, 19.24%) with a clear dominance of acetobacter genus. and it is known that acetobacter genus can degrade the acetic acid produced into co2 and h2o when there is a high level of dissolved oxygen and no ethanol in the medium [28] which is not desirable. however, this dominance would be an advantage for loh djiboua cocoa fermentation, since it may enable to obtain quality beans and chocolate due to ability of acetobacter genus to degrade lactic acid into acetoin and co2 [1, 29, 30]. indeed, lactic acid product degradation could help reduced the load of lactic acid beans after fermentation thereby contributing to the achievement of quality beans while the presence of acetoin, a chocolate precursor aroma, would provide a good quality chocolate. in addition, the study of the acidification capacity of these acetic bacteria strains revealed between the 166 isolates a large diversity in production level of acetic acid with strong (14 strains, 8.33%) medium (97 strains, 57.73%) and low (56 strains, 33.33%) producers in which medium producers load is the most important. this large diversity of production level associated with the dominance of acetic acid bacteria population on the other microbial strains of fermentation could lead to an important production of acetic acid even if the best acidification strains isolated from loh djiboua cocoa fermentation showed less important capacity of acetic acid production [6]. acidification is one of the most relevant properties in cocoa fermentation since it influences greatly the quality of fermented bean and chocolate [3]. the production of acetic acid during cocoa fermentation allows the development of chocolate flavor and aroma [31, 32]. hence, the strong acidification of lohdjiboua cocoa beans, which is a desired parameter for improvement of fermentation, should ensure the production of high quality beans market. 3.4. biochemical characterization of lab strains isolated regarding lab study, biochemical and morphological identification of strains isolated, revealed the presence of lactococci (33 strains) and lactobacilli (177 strains) in ivorian cocoa spontaneous fermentation. evaluation of lab strains fermentative type further showed that all the lactococci stains isolated were homofermentative whereas for lactobacilli strains isolated with 162 strains (91.73%) homofermentative and 15 strains heterofermentative. in spite of this fermentative type diversity observed on these two main groups of lactococci and lactobacilli strains isolated, the lab population is characterized by a large dominance of homofermentative type (162 strains, 92.83%) on the heterofermentative type (14 strains, 7.17%) as it is reported about in ghana [8] nigeria [13] and cote d’ivoire [21]. homofermentative lab strains are known to convert sugars almost exclusively into lactic acid and then produce more lactic acid than food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 203 heterofermentative strains [33]. as aab and particularly acetobacter genus remain in a high percentage of the total microbial population in this region, high concentrations of lactic acid product will be transformed [1, 29, 30] leading to well fermented beans and quality chocolate. concerning main sugars carbon metabolism, among 210 lab strains isolated all were able to metabolize glucose (100%) unlike fructose (99.04%) and sucrose (95.75%). these results are consistent [21] highlighting among these strains a wide diversity in their capacity to metabolize these main sugars. indeed during fermentation, glucose and fructose were used by lab to be converted into lactic acid, acetic acid, ethanol, and mannitol. sucrose inversion took place in the beans due to cotyledon invertase activity [7] and/or induced acid hydrolysis as a result of acetic acid penetration into the beans upon fermentation. glucose was preferentially fermented above fructose following sucrose hydrolysis [8]. in light of this particularity, growth and thus activity of these strains isolated during fermentation should not be limited by a possible depletion in one of these carbon sources. one the other hand, 76.19% of the lab strains isolates presented the capacity to catabolize citric acid. citrate metabolism constitutes, an important and particular property, since lab is not usually able to utilize citric acid as carbon source [21]. this finding confirms the results concerning lab strains isolated from agboville in cote d’ivoire [21]. citric acid is the compound responsible for the initial ph of cocoa pulp before fermentation processing [9, 24, 34]. the degradation of this acid in the first stage of fermentation allows the raise of the ph favorable for the development of many bacterial groups [3]. 3.5. biochemical characterization of bacillus sp strains isolated biochemical characterization of bacillus sp strains isolated from loh-djiboua cocoa fermentation showed that among 170 isolates 44.32% of strains presented pectinolytic activity with strong (19.23%), medium (19.23%) and low (61.53%) producers. 37.05% of the isolates were able to metabolize citric acid while about 70% of them expressed acidification capacity. these results suggested that bacillus strains involved in cocoa fermentation are able to express a wide diversity of useful activities for successful cocoa fermentation as they are able to survive under fermentation conditions. this emphasis the important role that bacillus sp may play with these capacities in the remaining time of cocoa fermentation as no useful role has been clearly attribute to them in cocoa fermentation [35], ensuring to maintain the merchantability of loh-djiboua cocoa beans and derivate chocolat. 4. conclusions the current emergence sequence of microorganisms with yeasts, lab, aab and bacillus has been observed during loh-djiboua cocoa fermentation with a clear dominance of aab population mainly constituted of acetobacter on other microbial groups. biochemical characterization of each microbial group emphasized the wide variety in activities and population among these different groups. however, other biochemical or molecular methods are necessary to better apprehend this diversity. 5. acknowledgments this work was supported by a ph.d. grant to the first author and this research was supported by the ascad project. references food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 204 [1]. pereira g.v.m., magalhães k.t., almeida e.g., coelho i.s., schwan r. f., spontaneous cocoa bean fermentation carried out in a novel-design stainless steel tank: influence on the dynamics of microbial populations and physical– chemical properties, int. j. food microbiol., 161. 121-133, (2013) [2]. samagaci l., ouattara h.g., goualié b.g., niamke s.l., growth capacity of yeasts potential starter strains under cocoa fermentation stress conditions in ivory coast, emirates j. food agric., 26. 861-870, (2014). [3]. schwan r.f., wheals a.e., the microbiology of cocoa fermentation and its role in chocolate quality, crit rev food sci nutr, 44. 205– 221, (2004) [4]. ouattara h. g., ban-koffi l., karou g.t., sangare a., niamke s.l., diopoh j.k., implication of bacillus sp. in the production of pectinolytic enzymes during cocoa fermentation, world j. microbiol. biotechnol., 24. 1753-1760, (2008) [5]. guehi s. t., dabone s., ban-koffi, kra d.k., zaouli g.i. effect of turning beans and fermentation method on the acidity and physical quality of raw cocoa beans, adv. j. food sci. technol., 2. 163-171, (2010) [6]. yao, w., ouattara h.g., goualie b., soumahoro s., niamke s., analysis of some functional properties of acetic acid bacteria involved in ivorian cocoa fermentation, j. appl. biosci., 75:6282–6290, (2014) [7]. hansen c.e., del olmo m., burri c., enzyme activities in cocoa beans during fermentation, j. sci. food agric., 77. 273–281, (1998) [8]. camus n., de winter t., verbrugger k., cleenwerck i., vandamme p., takrama j.s., vancanneyt m., de vuyst l., dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in ghana, appl. environ. microbiol, 73. 1809-1824, (2007) [9]. lefeber t, janssens m, moens f, gobert w, de vuyst l, interesting starter culture strains for controlled cocoa bean fermentation revealed by simulated cocoa pulp fermentations of cocoa-specific lactic acid bacteria, app. environ. microbiol., 77. 6694–6698, (2011) [10]. carr j.g., davies p.a., cocoa fermentation in ghana and malaysia: part 2. further microbial methods and results. bristol: university of bristol, (1980) [11]. anonymous 1, revue de presse du lundi 25 aout 2014, http://www.commerce.gouv.ci/fichier/rp-25-aout2014.pd, (2014) [12]. lefeber t., papalexandratou z., gobert w., camu n., de vuyst l., on-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavor of chocolates produced thereof, food microbiolo., 3. 379-392, (2012) [13]. kostinek m.l., ban koffi l., ottahatikpo m., teniola d., schillinger u., holzapfel w.h., franz c.m.a.p., diversity of predominant lactic acid bacteria associated with cocoa fermentation in nigeria, curr. microbiol., 56. 306-314, (2008) [14]. duthathai f., pathom-aree, w., application of chemical dyes as color indicator for selective isolation of acetic acid bacteria, res. j. microbiol., 2. 885-888, (2007) [15]. soares m.m.c.n., da silva r., gomes e., screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by bacillus sp., revista de microbiologia, 30. 299-303, (1999) [16]. wickerham l.j., taxonomy of yeasts, vs. dept. agric washinton. d. c. tech. bull., 1029, 156, (1951) [17]. dicks l.m., van vuuren, h.j.j., microbiology methods, 6. 273-275, (1987) [18]. afoakwa e.o., kongor j.e., takrama j.f., budu a.s., changes in acidification, sugars and mineral composition of cocoa pulp during fermentation of pulp preconditioned cocoa (theobroma cacao) beans, int. food res. j., 20. 1215-1222, (2013). [19]. kempler g.m., mckay l.l. improved medium for detection of citrate-fermenting streptococcus lactis subsp. diacetylactis. applied and environmental microbiology, 39:926–927, (1980) [20]. aydin y.a., aksoy n. d., isolation of cellulose producing bacteria from wastes of vinegar fermentation. proceedings of the world congress on engineering and computer (2009) [21]. ouattara h.d., ouattara g.h., goualie b.g., kouame l.m., niamke s.l., biochemical and functional properties of lactic acid bacteria isolated from ivorian cocoa fermenting beans, j. appl. biosci., 77. 6489-6499, (2014) [22]. schwan r.f., cocoa fermentation conducted with a defined microbial cocktail inoculums, appl. environ. microbiol., 64. 14771483, (1998) [23]. grimaldi j., the possibilities of improving techniques of pod breaking and fermentation in the traditional process of the preparation of cocoa. café cacao the, 22. 303-316, (1978) http://www.commerce.gouv.ci/fichier/rp-25-aoutfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 liliane m. kouame, ginette g. doue, niamke j. adom, honore g. ouattara, sébastien l. niamké, biochemical characterization of microbial populations involved in loh-djiboua cocoa's fermentation in coted d'ivoire, food and environment safety, volume xiv, issue 2 – 2015, pag.196 – 205 205 [24]. thompson s.s., miller k.b., lopez a.s., cocoa and coffee, food microbiolfundamentals and frontiers, 35. 721-733, (2001) [25]. passos f.m.l., silva do, lopez a., ferreira c.l.l.f., guimaraes w.v., characterization and distribution of lactic acid bacteria from traditional cocoa bean fermentations in bahia, j. food sci., 49. 205–208, (1984) [26]. perez, m., luyten, k., michel, r., riou, c., blondin, b., analysis of saccharomyces cerevisiae hexose carrier expression during wine fermentation: both lowand high-affinity hxt transporters are expressed, fems, yeast res., 5. 351-361, (2005) [27]. rohsius c., andersson m., niemenak n., sukha d., lieberei, r., la qualité de la fermentation et ses rapports avec la structure de la testa et les processus de transport. 15ème conférence internationale sur la recherche cacaoyère présenté par le programme national cacao de l’iniap, équateur. (2009) [28]. du toit w.j., pretorius i.s., the occurrence, control and esoteric effect of acetic acid bacteria in winemaking. annals microbiol, 52. 155– 179, (2002) [29]. penia m.t.a., suryani l., affifa h., improvement of cocoa beans fermentation by starter addition. journal of medical and bioengineering, 2. 274-278, (2013) [30]. de vuyst l., lefeber t., papalexandratou z., camu n., the functional role of lactic acid bacteria in cocoa bean fermentation. in: mozzi, f., raya, r.r.,vignolo, g.m. (eds.), biotechnology of lactic acid bacteria: novel applications. wiley-blackwell, ames, pp. 301e326, (2010) [31]. biehl b, voigt j, heinrichs h, senjuk v, bytof g, ph-dependent enzymatic formation of oligopeptides and amino acids, the aroma precursors in raw cocoa beans. in: lafforest, j. (ed.), proceedings in xith international cocoa research conference. cocoa producers’ alliance, yamassoukro, ivory coast, pp. 717722, (1993) [32]. de brito e.s., pezoa garcia n.h., gallao m.i., cortelazzo a.l., fevereiro p.s. braga m.r., structural and chemical changes in cocoa (theobroma cocoa l.) during fermentation drying and roastin, j. sci. food agric., 81. 281288, (2000) [33]. raimbiault m., importance des bactéries lactiques dans les fermentations du manioc. in transformation alimentaire du manioc. t. agbor egbe, a. brauman, d. griffon, s. trèche (éd orstom), (1995) [34]. pettipher g.l., analysis of cocoa pulp and the formulation of a standardized artificial cocoa pulp medium. j. sci. food agric., 37. 297–309, (1986) [35]. ardhana m., fleet g., the microbial ecology of cocoa bean fermentations in indonesia, int. j. food microbiol, 9. 87-99, (2003) microsoft word fia journal no 1_2011_23 martie_9-12.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 13 atom-absorption determination of microelement composition of the human hair from different areas of chernivtsi region igor kobasа1, vasyl bilogolovka1, аnatoliy woloschuk1 1yuriy fedkovych national university of chernivtsi, kotsiybynsky st., 2, chernivtsi 58012, ukraine. e-mail: imk-11@hotmail.com abstract. an atom-absorption microelement analysis of the human hair taken from people living in different areas of chernivtsi region (ukraine) has been performed. the microcontent of zinc, iron, manganese and copper was determined in relation to the examinees’ residence place, sex and age. a comparative analysis of the hair microcomposition proved that concentration of copper and iron in the men’s hair of all age groups was higher than in the corresponding women’s hair while concentration of manganese and zinc in the men’s hair was lower. an average content of zn, fe, mn and cu in men’s hair remains within the corresponding normal ranges. exceeding concentrations were determined for the men from novodnistrovsk (zn), novoselytsya district (fe and cu) and chernivtsi (mn). an average content of fe and cu in the women’s hair remains within the normal ranges. exceeding concentrations of zn were reported for the samples from putyla district while in some samples from novodnistrovsk this parameter was lower than the mean value. concentration of mn exceeded limits for all the women hair samples from chernivtsi. significant exceeding in the concentration of zn and deficiency of cu has been found in the samples from the first age group (1725 years). samples of the fourth age group (50-83 years) have shown deficiency of zn and some excess of mn and fe. key words: microelement composition, hair, pollution, environment introduction human’s hair can be used as an indicator of the environment ecological safety and can provide integral information related to the microelemental balance of the human organism [1, 2]. hair is one of the metabolically active tissues and can be used as an informative object for various investigations. the unique structure and composition of hair depends on the regional, sexual, racial and group parameters of each given person and can also be influenced by different external factors (temperature, humidity, acidity of drinking water, etc.) [3]. once a substance takes part in the human metabolism, it leaves traces in hair, remaining in the hair during its lifetime. this feature can be used for a retrospective analysis of an examinee’s life conditions and health status [4]. the analysis of hair should be performed using modern precise analytical equipment and can be useful for a wide variety of ecological, hygienic and clinical investigations. hair analysis can be useful for historical retrospective investigations, which can disclose information related to the past and present microelements contents in the human body and environment. biological, medical, chemical-ecological and criminalistic investigation of the hair is a comparatively new field of research. the macroand microelement analysis of hair can provide accurate information about metabolic conditions of the examinee. the mineral composition of hair is mainly caused by external factors. a balance between processes of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 14 mineralization (accumulation of some mineral compounds in hair), demineralization and transmineralization (transfer of the mineral compounds within the human’s body) is governed by nourishment conditions, physical activity and environment pollution. therefore, human hair can be used as an indicator of the long-term internal and external influence of the environment pollution on the human organism. experiment 37 hair samples have been collected from various examinees from chernivtsi region (districts of putila and novoselytsya, cities of novodnistrovsk and chernivtsi). all the samples were classified by age and sex of the examinees. the first group included children of both sexes from 4 to 16 years, the second – men and women from 17 to 29 years, the third – men and women of 30-49 years and fourth – men and women of 50-83 years. all the samples were 2-3 cm long, taken from non-dyed hair and then stored at room temperature without any additional conservation. preliminary preparation of the samples was carried out according to [5]. the hair was rinsed with distilled water to remove surface pollutants, dried at room temperature during 24 hours and weighed. then a 0.3 g sample was placed in the crucible and burnt to ash in the muffle burner at 550 0c during 5 hours. then the incinerated sample was boiled with nitric acid until transparence of the mixture and evaporated to almost dry condition. the evaporation residue then was placed in a volumetric flask of 25 ml with 1m nitric of acid solution. an atom-absorption spectrophotometer kas-120-m1 has been used to perform measurements of the mineral compounds contents. all the samples were injected in the atomizator as solutions. the concentration of copper has been measured at a wavelength of 324.8 nm; fe – at 248.3; mn – 279.5 and zn – at 213.9 [6]. results and discussion the results of atom-absorption analysis on metals contents in the hair samples are shown in figures 1-4. all the figures show averaged contents of elements. as seen in fig. 1, some significant exceeding of zn content (up to 1.5 times) has been registered for the samples from the district of putyla. the content of zn in the women hair from chernivtsi and district of novoselytsya is higher than in the men hair but it remains under the threshold limit. as seen in fig. 2, the content of fe in the men hair is slightly higher than in the women but both parameters stay under the threshold limit value. concentration of fe is almost similar for the samples from all residence places. the content of mn in the women hair was much higher than in the men hair (see fig. 3). the content of mn in the samples from novodnistrovsk was the lowest. an average content of cu in the men hair samples from chernivtsi and novoselytsya district were higher comparing to the corresponding women hair samples but they all remained within a range of normal contents of cu (see fig. 4). as seen from the above-mentioned results, concentrations of fe and cu remained within corresponding normal ranges with small deviations from the mean values. the concentrations of zn in men hair were more stable than those in women hair and also stayed within their normal range except samples from the district of putyla. an average concentration of mn in the women hair exceeded normal values for the samples from chernivtsi and the districts of novoselytsya and putyla. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 15 0 10 0 20 0 30 0 40 0 1 2 3 4 с , m g/ g me n wome n fig. 1. content of zn in the hair of men and women for various residence places: 1 – chernivtsi; 2 – district of novoselytsya; 3 – novodnistrovsk; 4 – district of putyla. an average normal content of zn in the hair ranged from 100 to 250 g/g. 0 5 0 1 00 1 50 2 00 2 50 1 2 3 4 c , m g/ g me n wome n fig. 2. content of fe in the hair of men and women for various residence places: 1 – chernivtsi; 2 – district of novoselytsya; 3 – novodnistrovsk; 4 – district of putyla. an average normal content of fe in the hair ranged from 192 to 230 g/g. 0.0 1.0 2.0 3.0 4.0 1 2 3 4 c , m g/ g men women fig. 3. content of mn in the hair of men and women for various residence places: 1 – chernivtsi; 2 – district of novoselytsya; 3 – novodnistrovsk; 4 – district of putyla. an average normal content of mn in the hair ranged from 0.1 to 2.0 g/g. 0. 0 5. 0 10. 0 15. 0 20. 0 25. 0 1 2 3 4 c , m g/ g men wom en fig. 4. content of cu in the hair of men and women for various residence places: 1 – chernivtsi; 2 – district of novoselytsya; 3 – novodnistrovsk; 4 – district of putyla. an average normal content of mn in the hair ranged from 10 to 28 g/g. another series of bar charts represents the distribution of microelements concentrations in age groups. as seen from figures 5-8, the concentration of zn reaches the highest level in the hair of men and women of 1725 years and this concentration is 1.5 times higher than the corresponding normal level. it is known [7] that concentrations of zn and fe are interconnected since assimilation of zn is complicated by cu, mn and fe. this result has also been proved by our experiments (see fig. 5 and 6, especially the results for age group iv). on the other hand, deficiency of cu can be seen in the age group ii (fig. 8) and this can be caused by quite a high concentration of zn (see fig. 5). concentration of mn (fig. 7) in the first age group is also very high and its value is almost twice higher than the normal limit. concentrations of zn, fe and cu for this age group are within the normal limits. concentration of fe remains within the normal range for all age groups. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 16 0 100 200 300 400 g r oup i g roup ii g roup iii g r oup iv c , m g/ g c once ntr at ion of zn fig. 5. concentration of zn in the hair samples for different age groups 0 50 100 150 200 250 g roup i g roup ii g r oup iii g r oup iv c , m g/ g conc entr at ion of fe fig. 6. concentration of fe in the hair samples for different age groups 0.0 1.0 2.0 3.0 4.0 g roup i g r oup ii g r oup iii g roup iv c , m g/ g c onc entration of m n fig. 7. concentration of mn in the hair samples for different age groups 14 15 16 17 18 19 20 group i group ii group iii group iv c , m g/ g concentration of cu fig. 8. concentration of cu in the hair samples for different age groups conclusion an investigation on the microelements’ content and distribution in the human hair from examinees living in different areas of the chernivtsi region has been performed. deviations in the microelement composition of the hair samples from different locations can be explained by different biogeochemical conditions in the examinees’ residence places, different health conditions of the participants, sexual differences in the metabolic processes and individual differences in the living conditions of each participant. references 1. k. a. bushtueva and i. s. sluchanko. methods and criteria of assessment health conditions for the population of environmentally polluted areas. (1979). medicine, moscow. 167 p. 2. a. v. gudkov, v. n. bagryantsev and l. m. isachkova. relation between an average children sickness rate and content of the heavy metals in hair. in: infection pathology in the primorsky region. (1994). dal’, vladivostok. p. 94-95. 3. s. s. nikolaeva and v. p. dubinskaya. chemical elements and their influence on the human health. (2001). mir, moscow. 243 p. 4. yu. v. pavlov. chemical elements and their influence on the human organism vital functions. (2000). naukova dumka. kyiv. 142 p. 5. p. clanet and s. m. de antonio. (1982). clin.chem. 28. 2050. 6. f. o. chmilenko and t. m. derkach. methods of atom spectroscopy and atom-absorption spectral analysis. (2002). univ. of dnipropetrovsk, dnipropetrovsk. 120 p. 7. a. v. skalny and i. a. rudakov. bioelements in medicine. (2004). mir, moscow. 254 p. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 59 use of a dv an ced oxida tion process es fo r tex tile wastewater treatment – a review *radka pešoutová1, petr hlavínek2, jana matysíková 3 1crea hydro&energy, cornet@creacz.com 2aqua procon s.r.o, petr.hlavinek@aquaprocon.cz 3aqua procon s.r.o *corresponding author abstract: recently, an increasing application of various aops for textile wastewater treatment has been observed for its advantages to conventional treatment methods. aops provide satisfactory result of textile effluent treatment. in this paper, on overview of use of ozone and ozone combined with other aops (o3/h2o2, o3/uv, o3/us) for effluent decolouration and bod/toc elimination is given. keywords: o3, o3/h2o2, o3/uv, o3/us, dye mineralization, effluent decolouration 1. introduction the shortage of water resources around the world increases the demands on the use of secondary sources, such as wastewater. in this perspective water recycling and re-use of treated effluent in high water consuming industrial sectors seem be to be a viable alternative to save valuable resources [1]. advanced wastewater treatment by ozone and other oxidation technologies has a number of advantages compared to other technologies. although some oxidation processes – use of ozone for disinfection of drinking water – have been known since the 19th century, the rapid development and application of advanced oxidation processes (aops) is a matter of development in the last decades. aops found application mainly in the oxidation of complex organic compounds which are difficult or impossible to degrade into simpler organic compounds by biological treatment. aops are based on generation and use of powerful but relatively nonselective highly reactive oxidizing species (primarily the hydroxyl radical oh• having high oxidation potential and low selectivity). these radicals are able to oxidize compounds that are not degradable by conventional oxidizing agents as oxygen o2, ozone o3 or chlorine cl2 [2]. the fact that the oh• radical attack is characterized by low selectivity is an important factor for a broad application of aops in a wide range of technologies. to make the advantages of advanced wastewater treatment by small and medium enterprises, further cross cutting is needed. the proposed project aop4water aims to use aops in combination with biological treatment to define better and cost-efficient wastewater treatment. the project proposes use of ozone and use of ozone in combination with ultrasound (us), ozone with hydrogen peroxide, ozone with uv light. in the first stage of the project identification of main water uses and associated water quality and quantity requirements have been performed to evaluate the actual water uses in the different factories of the target industrial sector and to prepare water flow mass balance analysis for the chosen factories. this paper aims to provide an mailto:cornet:@creacz.com mailto:petr.hlavinek:@aquaprocon.cz food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 60 overview of recent use of ozone and selected aop in textile industry. 2. textile industry textile industry is very water intensive. water is used through the whole textile production from cleaning of raw material to many washing through textile production or as a principal medium in various processes. processes in the textile industry can be divided into mechanical and finishing operations. during mechanical operations fabrics are manufactured of fibre by spinning, weaving and knitting. sizing is one of the technological steps of weaving of cotton and its blends. in terms of water consumption mechanical operations are not very water demanding. the second process is finishing (wet processing) providing the textiles with the main functional characteristics (appearance, feel, absorbency, softness, water repellence, crease resistance). during finishing a significant amount of water is consumed and also a significant amount of waste and pollution are produced. according to the material composition these processes are used desizing, scouring, bleaching, mercerization, carbonizing, fulling, washing, dyeing, finishing. 2.1 wastewater management in textile industry the main environmental issues arising from textile manufacturing regard primarily emissions to water and air and energy consumption. however, the major environmental concern in textile industry is the amount of water being discharged as well as its chemical content and load. water is the principal medium for removing impurities, applying dyes and finishing agents, and for steam generation. the primary water consumption reaches 80–100 m3/ton of finished textile and wastewater discharge 115–175 kg of cod/ton of finished textile; a large range of organic chemicals, low biodegradability, colour, and salinity in discharged water are also of environmental concern [3]. major pollutants specifically found in textile wastewater are suspended solids, highly recalcitrant chemical oxygen demand, dyes giving intense colour and other soluble substances [4]. pollution load and content is related to the processes used. a large percentage of the total emission load from textile industry activities is attributable to substances that are already on the raw material before it enters the finishing mill (e.g. impurities and associated materials for natural fibres, preparation agents, spinning lubricants, sizing agents, etc.). all these substances are usually removed from the fibre during the pretreatment process before colouring and finishing [5]. secondly, significant amounts of water are consumed during wet processing. bulk of water is utilised in washing at the end of each process. dying is another important source of pollution in textile industry wastewaters containing dyes and other chemical compounds mainly from dyeing and finishing processes. polluting substances can originate from the dyes themselves (e.g. aquatic toxicity, metals, colour), auxiliaries contained in the dye formulation (e.g. dispersing agents, anti-foaming agents, etc.), basic chemicals and auxiliaries used in dyeing processes (e.g. alkali, salts, reducing and oxidising agents, etc.) and residual contaminants present on the fibre (e.g. residues of pesticides on wool, spin finishes on synthetic fibres) [5]. however, strong colouration of the textile industry wastewater is of the major environmental concern and if not treated properly it can cause serious problems in the environment. 2.2. use of ozone for textile industry wastewater treatment textile industry produces large quantities of highly coloured effluent, which is food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 61 generally toxic and resistant to destruct by conventional treatment methods. low biodegradability of many fibre acid, direct and reactive dyes allows them to pass untreated through the sewerage works due to their high water solubility and relatively low molecular weight [6]. important advantage of use aop and their combinations is the destructive character of aop to conventional such as activated carbon adsorption, coagulation, flocculation or precipitation. besides not satisfactory treatment result, sludge is created when conventional methods are used causing another source of problems [7]. ozonation is relatively effective in reducing colouration of various dye origins and toxic effects of textile effluents; the main environmental concern related textile wastewater effluent discharge. dissolved in water ozone reacts with many organic compounds in two different ways: by direct oxidation as molecular ozone or by indirect reaction through formation of secondary oxidant like hydroxyl radical [2]. use of ozone for wastewater treatment in textile industry to remove colouration and lower toxicity has been documented in several studies; however sufficient effect on bod/toc removal was not confirmed. baban et al. [8] applied ozone (co3=18.5 mg/l; input rate) to treat remaining composite wastewater (spent dye baths and first and second rinses) from woollen textile finishing industries. the results indicated that 40 min ozonation of biologically treated wastewater yielded almost colourless effluent with a decolourization efficiency of around 98– 99% and with a corresponding ozone absorption rate of 58.0 mg/l. biological treatment followed by 10 min ozone oxidation reduced the overall toxicity significantly (92%); however ozonation has a slight effect on cod removal [8]. similar results have been achieved in study of selchuk [9]. the study confirmed almost complete colour absorbances (over 98%) in 20 min ozone contact time, while cod removal (37%) was very low and almost stable in 30 min ozonation period. the toxicity of wastewater reduced after colour degradation by 85% at the transferred ozone concentration of 82.3 mg l 1 [9]. commercial dyes used in textile industry can be classified according to their chemical composition (azo, antrachinone, sulphur, triphenilmethane, indigoid, phtalocyanine, etc.) or according to their application class (acid, direct, disperse, metal complex, chrome, reactive, etc.) [5]. azo-dyes are the most commercially used dyes and comprise of a nitrogen double bonds (–n=n–), and when attached to molecules they become monoazo, diazo or polyazo dyes. azo dyes, with the exception of few simply structured dyes, resist biodegradation under aerobic conditions. on the other hand, azo bond is vulnerable to reductive cleavage and its degradation products include colourless aromatic amines which are known to be toxic and potentially carcinogenic [10]. the study of wang et al. [11] documented significant effect on colour removal and increased biodegradability and cod, toc reductions about 40% and 25% for 6 h ozonation; however, short-term ozonation led to a formation of highly toxic byproducts. similar results for decolouration, cod and toc removal were achieved by chu et al. [12] on practical textile wastewater treatment. use of ozone for decolouration textile industry effluent was also documented for reactive dyes that are widely used for dyeing of cotton [13-15]. the latter two studies [14-15] also investigated the possibility of recycling of ozone treated effluent. ozonation provided satisfactory decolouration after a relatively short period ozonation (for example 10 minutes) [15]. ozonation also proved to be efficient for colour removal caused by dispersed dyes (complete decolorization in food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 62 1 min at the ozone dose of 50 mg dm-3, provided the colloidal and suspended particles were previously removed from the wastewater); however, removal of cod was not sufficient to reach discharge standards [16]. the main factors affecting ozonation performance are ph, the nature and concentration of oxidisable organics, applied ozone dose, competition between the target compound and biodegradable by-products, the presence of oxidant scavengers, and the efficiency of ozone mass transfer [17-18]. although increased ozone doses has a positive effect on decolourisation of acid dye solutions effluents and the efficiency of the treatment increases with higher ph (ph range 5–9), an adverse effect of buffered solutions was reported [19]. increase of the mass transfer rate of ozone and enhancement of efficiency of the ozonation process was achieved by using a microbubble generator that enabled high intensity microbubble solution, utilization of almost all input ozone and faster decolourization and organic reduction [12]. 2.3. use of ozone and hydrogen peroxide for textile industry wastewater treatment hydrogen peroxide is relatively inexpensive, readily available chemical oxidant that accelerates decomposition of ozone and enhances formation of hydroxyl radical. the addition of both hydrogen peroxide and ozone to wastewater accelerates decomposition of ozone and enhances production of hydroxyl radical [20]. h2o2 acts as a catalyst and accelerates the decomposition of ozone to hydroxyl radical [21]. higher ph positively influences dissociation of h2o2 into ho2ions that initiate decomposition of ozone more effectively than oh• [22]. rapid and complete decolourization of textile industry effluent can be achieved; however this combination like use of ozone alone is not able to bring complete mineralisation either [23]. kurbus et al. [24] tested performance of h2o2/o3 to remove colour, cod and toc for several vinylsulphone reactive dyes solutions in a pilot scale plant. the study showed that decolourisation took a long time (90 min) than other combinations tested; cod removal obtained after decolourisation was satisfactory (about 300 mgl-1 from the initial dye concentrations around 1 500 mgl-1). low performance of the combination was explained by ph; alkaline conditions enable reaction of hydrogen peroxide with naoh resulting in formation of sodium peroxide (na2o2) and decreasing concentration of hydrogen peroxide for formation of hydroxyl radicals [24]. 2.4. ozone and ultrasound (us) sonochemical reactions are induced upon high-intensity acoustic irradiation of liquids at frequencies that produce cavitation (typically in the range 20–1000 khz). cavitation refers to formation, growth, and implosive collapse of gasor vapour-filled cavities (bubbles) in a liquid matrix. collapse of the cavities produces localized high-temperature (about 5 000°c) and high-pressure (about 50 mpa) hotspots [10]. during collapse, water vapour and gas trapped inside the bubble are fragmented and dissociate into a variety of radicals, including hydroxyl. when ozone is introduced into a sonolysis system, the ultrasonic irradiation enhances the mass transfer, dispersion, and solubility of o3, and it enhances the production of additional oh• [25]. recently, attention has been given to the use of ultrasound in combination with ozone for textile industry wastewater treatment. the studies proved that combination of ozone with ultrasound is effective for removal of colour, cod and toc [16, 10]. use of ultrasound alone for removal of toc and colour caused by azo dyes appeared to be rather inefficient food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 63 [10]. similar results were achieved in the another study [28] proving that combination was more effective form mineralization of reactive dye c.i. reactive blue than use of ozone or ultrasound alone. combination of ultrasound with ozone or ultrasound with ozone and uv provides better results in terms of dye degradation and effluent decolourization. the study of tezcanli-guyer and ince [26] compared performance of various aop (ozone, ultrasound and uv) on degradation of azo dyes. the study showed that ozone in combination with ultrasound (520 khz) has similar performance like combination of ozone with uv for decolouration of textile wastewater, while combination of ozone, ultrasound and uv was more than 25 % than each. the synergic effect of the latter combination was attributed to enhanced ozone diffusion by mechanical effects of ultrasound, and photolysis of ultrasound-generated h2o2 to produce hydroxyl radicals [26]. 2.5. ozone with uv light uv-based advanced oxidation processes are based on formation of hydroxyl radicals oh• through the direct ozonation and photolysis reactions and hydroxyl radical oxidation [28]. ozone combined with uv light demonstrated to be effective mean for textile wastewater treatment. azbar et al. [29] studied efficiency of several aop combinations for colour and cod for a polyester and acetate fibre dyeing effluent. the study showed that combination of ozone and uv light reached cod removal of 94% and colour removal of 93%. however, among the aops tested (o3, o3/uv, h2o2/uv and o3/h2o2/uv), the combination of ozone, uv and peroxide appeared to be the most efficient in terms of cod and colour removal with a 99% and 96% [29]. decolouration effect of uv/o3 combination was successfully tested on six azo dyes effluent; more than 95% of colour was removed in less than 11.5 minutes for all six azo dyes (initial dye concentration 20.0 mg dm-3, oxygen flow rate 6.0 dm3min-1) [30]. the study showed dependence of the chemical structure of the dye on the efficiency of the process. the more azo bonds (–n=n–) the more difficult the process of decolourization is [30]. ozone combined with uv also proved to be more efficient for bod elimination. efficiency of bod elimination on biologically treated textile industry effluent was almost six times higher for ozone combined with uv after 30 minutes contact time [31]. 3. conclusions this review confirmed that aop have a significant potential for textile industry wastewater treatment. large quantities of colours and other contaminants present in textile wastewaters pose significant demands on its treatment. as single treatment method cannot effectively remove all contaminants from wastewaters, preliminary laboratory tests have to be carried out before selecting final treatment method considering that the efficiency of aop is compound specific and thus have an effect on efficiency of the treatment. the actual laboratory testing textile industry wastewaters in relation to cost-effectiveness of the treatment will be performed in the next stage of the aop4water project. 4. acknowledgements the financial support provided by a grant of the ministry of industry and trade of the czech republic via operational programme industry and enterprise (opie) is greatly acknowledged. 5. references [1] stanko š., kriš j., škultétyová i., environmental impact of waste-water reuse, in: food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 64 31st international geographical congress: resumes/tunis, 12.-15.8.2008. 378-379 (2008) [2] metcalf eddy, inc. wastewater engeneering, treatment and reuse. 4th edition. new york: mc graw hill. water reuse, 1819 p, (2003) isbn 0-07-112250-8 [3] savin i., butnaru r., wastewater characteristics in textile finishing mills. environmental engineering and management journal [online], 7 (6). (2008) [accessed on 2011-06-07]. available from: <http://omicron.ch.tuiasi.ro/eemj/pdfs/vol7/no 6/42_irina savin.pdf> [4] oller i., malato s., sánchez-pérez j.a., combination of advanced oxidation processes and biological treatments for wastewater decontamination—a review, science of the total environment [online], article in press, corrected proof, (2010) [cit. 2011-05-16]. available from <http://www.sciencedirect.com/science/article/p ii/s0048969710009344> [5] european commission, integrated pollution prevention and control (ippc): reference document on best available techniques for the textiles industry [online]. brussel: european commission. (2003) [accessed on 2011-06-07]. available from: <ftp://ftp.jrc.es/pub/eippcb/doc/txt_bref_0703.p df> [6] reife a. freeman s., environmental chemistry of dyes and pigments, john wiley & sons, canada (1996) [7] mahríková i., operation of sludge tanks in small and mid-sized waste water treatment plants, in: hlavinek et a., advanced water supply and wastewater treatment: a road to safer society and environment, springer. 163171 (2011) [8] baban a., yediler a., lienert d., kemerdere n., kettrup a., ozonation of high strength segregated effluents from a woollen textile dyeing and finishing plant, dyes and pigments, 58 (2). 93-98 (2003) [9] selchuk h., decolorization and detoxification of textile wastewater by ozonation and coagulation processes, dyes and pigments, 64 (3). 217-222, (2005) [10] vajnhandl s., le marechal a.m., case study of the sonochemical decolouration of textile azo dye reactive black 5, journal of hazardous materials, 141 (1). 329-335 (2007) [11] wang c., yediler a., lienert d., wang z., kettrup a., ozonation of an azo dye c.i. remazol black 5 and toxicological assessment of its oxidation products, chemosphere, 52 (7). 1225-1232 (2003) [12] chu l.; xing, x.;yu a.; sun x.; jurcik b. enhanced treatment of practical wastewater by microbubble ozonation. process safety and environmental protection, 86 (5). 389 – 393 (2008) [13] wu j., doan h., upreti s., decolorization of aqueous textile reactive dye by ozone, chemical engineering journal, 142 (2). 156-160 (2008) [14] senthilkumar m., muthukumar m., studies on the possibility of recycling reactive dye bath effluent after decolouration using ozone, dyes and pigments, 72 (2). 251-255 (2007) [15] colindres p., yee-madeira h., reguera e., removal of reactive black 5 from aqueous solution by ozone for water reuse in textile dyeing processes, desalination, 258 (1-3). 154-158 (2010) [16] szpyrkowicz l., juzzolino c., kaul s.n., a comparative study on oxidation of disperse dyes by electrochemical process, ozone, hypochlorite and fenton reagent, water research, 35 (9). 2129-2136 (2001) [17] alvares a.b.c., diaper c., parsons s.a., partial oxidation by ozone to remove recalcitrance from wastewaters – a review, environmental technology. 22 (4). 409427(19), (2001) [18] sevimli m.f., sarikaya h. z., ozone treatment of textile effluents and dyes: effect of applied ozone dose, ph and dye concentration, journal of chemical technology and biotechnology, 77. 842–850, (2002) [19] soares o.s.g.p., orfao j.j.m., portela d., vieira a., pereira m.f.r., ozonation of textile effluents and dye solutions under continuous operation: influence of operating parameters, journal of hazardous materials, 137 (3). 1664-1673 (2006) [20] al-kdasi a., iris a., saed k., guan c.t., treatment of textile wastewater by advanced oxidation processes – a review, global nest: the int. j., vol. 6, no. 3. s. 222230, (2004) [21] staehlin j., hoigne j., decomposition of ozone in water: rate of initiation by hydroxide ions and hydrogen peroxide, environmental science and technology, 16. 676-681, (1982) [22] glaze w. h., wun kang j., advanced oxidation processes. description of a kinetic model for the oxidation of hazardous materials in aqueous media with ozone and hydrogen peroxide in a semibatch reactor, ind. eng. chem. res., 28 (11). 1573–1580 (1989) [23] arslan i., akmehmet balcioglu i. & tuhkanen t., advanced oxidation of synthetic dyehouse effluent by o3, h2o2/o3 and http://omicron.ch.tuiasi.ro/eemj/pdfs/vol7/no http://www.sciencedirect.com/science/article/p ftp://:@ftp.jrc.es/pub/eippcb/doc/txt_bref_0703.p food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 65 h2o2/uv processes, environmental technology, 20(9). 921 – 931, (1999) [24] kurbus t., le marechal a.m., voncina d.b. comparison of h2o2/uv, h2o2/o3 and h2o2/fe2+ processes for the decolorisation of vinylsulphone reactive dyes. dyes and pigments, 58 (3). 245-252 (2003) [25] mason t.j., pétrier c., ultrasound processes in parsons, s. (eds.) advanced oxidation processes for water and wastewater treatment, iwa publishing, 356 p, (2004) [26] tezcanli-guyer g., ince n.h., individual and combined effects of ultrasound, ozone and uv irradiation: a case study with textile dyes, ultrasonics, 42 (1-9). 603-609 (2004) [27] he z., lin l., song s., xia m., xu l., ying h., chen j., mineralization of c.i. reactive blue 19 by ozonation combined with sonolysis: performance optimization and degradation mechanism, separation and purification technology, 62 (2). 376-381 (2008) [28] stefan m. i., uv photolysis: background in parsons, s. (eds.) advanced oxidation processes for water and wastewater treatment, iwa publishing, 356 p, (2004) [29] azbar n., yonar t., kestioglu k., comparison of various advanced oxidation processes and chemical treatment methods for cod and color removal from a polyester and acetate fiber dyeing effluent, chemosphere, 55 (1). 35-43 (2004) [30] shu h.-y., chang m.-c., decolorization effects of six azo dyes by o3, uv/o3 and uv/h2o2 processes. dyes and pigments, 65 (1). 25-31 (2005) [31] bes-pia a., mendoza-roca j. a., roig-alcover l., iborra-clar a., iborra-clar m.i., alcaina-miranda m.i. comparison between nanofiltration and ozonation of biologically treated textile wastewater for its reuse in the industry, desalination, 157 (1-3). 81-86 (2003) microsoft word 3 journal nr 4 2010 din 8 noiembrie_44-50.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 48 the influence of cold conventional storage on fruits quality oana – viorela nistor1, mihai ţâmpău1, elisabeta botez1 1bioingineering department, food science and engineering faculty, „dunarea de jos” university, 111 domneasca street, 800201, galati, phone/fax +40 236 460165, oana.nistor@ugal.ro abstract: to determine the most suitable method of cold fruits preservation, we used three types of fruits: pome fruits (apples, pears and quinces), citrus (oranges) and exotic fruits (kiwi and bananas). the fruits were stored, using a household refrigerator, operating at refrigeration temperature (5˚c) and freezing temperature (-10˚c). the storage was made in two variants: bulk and packed fruits (using ldpe bags) for a total period of 14 days. temperature measurements were made with a two day frequency, inside the storage space and in the fruits’ core. to determine the water losses during evaporation, the fruits were weighted, before and after the storage. during the refrigeration of the bulk fruits, the quince, the orange and the banana registered a constant evolution, while the behaviour of the same packed fruits was irregular. the evolution of bulk fruits represented by quince, orange and banana, during freezing period, was regular while for the packed ones such as banana, apple and orange we registered the best results. the results lead to the conclusion that the refrigeration storage of the bulk pome fruits had a lower quantity of moisture losses (2.5-4.1%) comparing to the exotic fruits (6.5-21.3%). in packed fruits case the lowest quantities of moisture losses were registered in the pome fruits (0.6-1.1 %). to draw a final conclusion, orange and apple presents the best evolution during the conventional cold preservation, while the behaviour of banana and kiwi is not proper for this kind of storage. key words: fruits preservation, refrigeration, freezing, pome fruits, bulk, packed, ldpe bags 1. introduction deterioration of fruits and vegetables during storage depends largely on temperature. one way to slow down this change and so increase the length of time fruits and vegetables can be stored, is by lowering the temperature to an appropriate level. it must be remembered that if the temperature is too low the produce will be damaged and also that as soon as the produce leaves the cold store, deterioration starts again and often at a faster rate [1]. all fruits and vegetables have a “critical temperature” below which undesirable and irreversible reactions or “chill damage”' takes place. the storage temperature always has to be above this critical temperature. one has to be careful that even though the thermostat is set at a temperature above the critical temperature, the thermostatic oscillation in temperature does not result in storage temperature falling below the critical temperature. even 0.5°c below the critical temperature can result in chill damage. table 1 gives the critical temperatures for various fruits and vegetables. [1] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 49 table 1. the critical temperatures for various fruits ([2]) fruit type temperature, °c relative humidity, % maximum storage time recommended apple 0-4 90-95 2-6 month orange 0-4 85-90 3-4 month pear 0 90-95 2-5 month the biological factors involved in postharvest deterioration of fruits are: respiration, ethylene production, transpiration or water loss, physiological disorders physical damage, pathological breakdown. [2-4] ethylene production rates, which depend on the fruit (table 2), generally increase with maturity at harvest, physical injuries, disease incidence, increased temperatures up to 30°c, and water stress. on the other hand, ethylene production rates by fresh fruits are reduced by storage at low temperature and by reduced o2 (less than 8%) and elevated co2 (above 1%) levels in the storage environment around the commodity. table 2. classification of fruits according to their ethylene production ([2-4]) ethylene production rate fruits very low cherry, citrus fruits, grape, jujube, strawberry, pomegranate low blueberry, cranberry, olive, persimmon, pineapple, raspberry, tamarillo moderate banana, fig, guava, mango, plantain high apple, apricot, avocado (ripe), nectarine, papaya, peach, pear, plum very high cherimoya, passion fruit temperature is the most important environmental factor that influences the deterioration rate of harvested fruits. for each increase of 10°c (18°f) above the optimum temperature, the rate of deterioration increases by twoor threefold. exposure to undesirable temperatures results in many physiological disorders as mentioned above. temperature also influences how ethylene, reduced oxygen, and elevated carbon dioxide levels affect the commodity. the growth rate of pathogens is greatly influenced by temperature and some pathogens, such as rhizopus rot, are sensitive to low temperatures. thus, cooling of commodities below 5°c immediately after harvest can greatly reduce rhizopus rot incidence. [2-5]. table 3. classification of fruits according to their optimum storage temperatures and potential storage life optimum storage temperatures potential storage life (weeks) 0-2°c 3-5°c 12-14°c <2 apricot, bush berries, strawberry, fig cantaloupe, ripe avocado cherimoya, guava, pineapple 2-4 cherry, nectarine, peach, plum tangerine and mandarin, carambola avocado, banana, mango 4-6 grape, tamarillo orange, pomegranate, kumqut grapefruit, lime, pummelo >6 apple (nonchilling sensitive cultivars), pear, cranberry, kiwifruit apple (chilling sensitive cultivars) lemon food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 50 relative humidity the rate of water loss from fruits depends upon the vapor pressure deficit between the commodity and the surrounding ambient air, which is influenced by temperature and relative humidity. air circulation rate and velocity can influence the uniformity of temperature and relative humidity in a given environment and consequently rate of the water loss from the commodity [25]. produce is usually cooled to its longterm storage temperature in special facilities designed to rapidly remove produce heat. forced-air cooling is the most widely adaptable method and is commonly used for many fruits, fruit-type vegetables and cut flowers [6-10]. hydrocooling uses water as the cooling medium and is less widely used than forced-air cooling because some products do not tolerate water contact, and it requires the use of water-resistant packaging. it is commonly used for root, stem and flowertype vegetables, melons and some tree fruits [6-10]. vacuumand water spray vacuum-cooling are usually reserved for crops, such as leafy vegetables, that release water vapor rapidly allowing them to be quickly cooled. cooling times are at least 24 h and can be much longer if produce is not packaged correctly or no provision is made to allow airflow past boxes. it is used for a few commodities, such as citrus and ca-stored apples that can have acceptable, although not optimal, quality without use of rapid cooling. fruit are commonly classified by growing region as follows: temperate-zone (pome fruits: apple, asian pear (nashi), european pear, quince, stone fruits: apricot, cherry, nectarine, peach, plum, small fruits and berries: grape (european and american types), strawberry, raspberry, blueberry, blackberry, cranberry), subtropical (citrus fruits: grapefruit, lemon, lime, orange, pummelo, tangerine, and mandarin, noncitrus fruits: avocado, cherimoya, fig, kiwifruit, olive, pomegranate), and tropical (major tropical fruits: banana, mango, papaya, pineapple, minor tropical fruits: carambola, cashew apple, durian, guava, longan, lychee, mangosteen, passion fruit, rambutan, sapota, tamarind). growing region and environmental conditions specific to each region significantly affect fruit quality. [2-5] the aim of the study was the chosen of the most suitable method of cold fruits preservation for three types of fruits: pome fruits (apples, pears and quinces), citrus (oranges) and exotic fruits (kiwi and bananas). the fruits were stored, using a home refrigerator, at refrigeration (5˚c) and freezing temperature (-10˚c). the storage was made in two variants: bulk and packed fruits (using ldpe bags) for a total period of 14 days. 2. materials and methods 2.1. raw materials: fruits (pome fruits apples, pears and quinces, citrus oranges and exotic fruits kiwis and bananas), ldpe (low density polyethylene) bags. 2.2. apparatus/equipments: mercury thermometer, core product thermometer type amprobe tpp1-c1, refrigerator zanussi a enery class, scale owa laborator. 2.3. methods the fruits were stored, using a home refrigerator, at refrigeration (5˚c) and freezing temperature (-10˚c). the storage was made in two variants: bulk and packed fruits (using ldpe bags) for a total period of 14 days. as main analyses we studied: the temperature in fruits’core, the cooling rate, water losses and the final aspect of the fruits. the temperature measurements were made with a two day frequency, inside the storage space and in the fruits’ core. to determine the water losses during evaporation, the fruits were weighted, before and after the storage. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 51 the water evaporation was calculated as a difference between the initial fruit’s weight and the final one. mw = mif mff where, mw – water mass; mif initial mass of the fruit; mff – final mass of the fruit. also it was determined the cooling rate for the refrigeration storage with a 3 hours frequency in a 15 hours interval. to express the cooling rate it was used the newton's law of cooling states that the rate of change of the temperature of an object is proportional to the difference between its own temperature and the ambient temperature. this equation is of interest for either positive or negative values of the constant r. in fact r=k, and another case in which r=-k. this is a differential equation which describes cooling slope. 3. results and discussion to determine the rate of cooling is directly related to the temperature difference between the cooling medium and the product. so the medium temperature was 5°c and the time interval was 15 hours fragmented into 3 hours sections. in the figure 1. is represented the cooling rate of the studied fruits at refrigeration temperature. first, when the temperature of the fruit is the same with the room temperature, the temperatures drops rapidly, after the point of cooling shock (registered after 9 hours of refrigeration 4.5°c, an almost similar temperature for all the fruits being under the medium temperature 5°c). 3 5 7 9 3 5 7 9 11 13 15 time, hours te m pe ra tu re , 0 c quince apple pear kiwi orange banana figure 1. the cooling rate of the bulk fruits at refrigeration for the core temperature determinations there were obtained the variation of each fruit variety as a particularity depending on the chemical composition and anatomical structure [11-15]. the core temperature variation of each studied fruit is presented in figure 2. 2 3 4 5 2 4 6 8 10 12 14 time, days te m pe ra tu re , 0 c apple pear quince orange banana kiwi figure 2. the variation of bulk fruits’ core temperature at refrigeration from this graphic it can be drawn that the orange, the quince and the banana had a constant variation, even if their anatomical structure is not similar. this can be explained as a relation between the peel presence for the citric and for the exotic fruit. referring to quince structure, the core temperature variation can be constant as an influence of the rich fiber structure, pectic substances and sugar (8,5%) against the water natural contain which is the main responsible for the temperature variation. the bigger temperature variation was observed for the apple (decreasing temperature reaching 2.9°c) and kiwi (increasing after reaching the 3.0°c). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 52 the water losses variation during the refrigeration storage of the bulk fruits were centralized in the following graphic. 0 100 200 300 apple pear quince orange banana kiwi fr u its weight, g final weight initial weight figure 3. the water losses variation during refrigeration of bulk fruits the water losses produced by the storage at refrigeration temperature preservation are lower for the pome fruits (2.5-4.1%) than for the citric and exotics (6.5-21.3%). correlating these technological aspects with the final aspect of the products, the exotic fruits represented by kiwi and banana had become unfit for consumption. the lowest quantity of moisture which was lost by the pome fruits can be justified by the homogeneous structure of these fruits. in figure 4 is presented the core temperature variation of the packed fruits in ldpe bags. it had been chose the ldpe bags because the material is considered an excellent barrier for steam and water, but not so good for gases. also because is a very used material in food industry and also for economic criteria being cheaper comparing to other types of packages. the allure of the temperatures curves is more similar than the other from the bulk fruits, but it can be seemed that during the refrigeration of the bulk fruits, the quince, the orange and the banana registered a constant evolution, while the behavior of the same packed fruits was irregular. 2 3 4 5 6 2 4 6 8 10 12 14 time, days t em pe ra tu re , 0 c apple pear quince orange banana kiwi figure 4. the variation of packed fruits’ core temperature at refrigeration in comparison with the bulk fruit the packed fruits as banana, apple and orange were registered the best results; this is a positive indicator in use of the ldpe bags. in the following graphic is presented the water losses variation during refrigeration of packed fruits. 0 50 100 150 200 250 apple pear quince orange banana kiw i f ru it we ight,g final w eight initial w eight figure 5. the water losses variation during refrigeration of packed fruits the ldpe package is a very good protection for the water elimination, as it can be seemed all kinds of fruits presented a lower quantity of moisture content than the bulk fruits. the significant losses were diminishing since 10% for kiwi, 9% for orange and 17% for banana. for the freezing storage preservation of the bulk and packed fruits there were lead the same analyses as for refrigeration. the storage temperature was -10°c for 14 days. besides the refrigeration process, at freezing there were made two kinds of weight determination, before the thawing food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 53 and after it. the thawing temperature is the room temperature (21°c). in figure 6 is represented the core temperature variation of the freeze fruits. -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 tim e, days t em pe ra tu re , 0 c apple pear quince orange banana kiwi figure 6 the core temperature variation of freeze bulk fruits from the graphic it can be seemed that the biggest variation of the core temperature at freezing temperature is assigned to apple. kiwi and orange had a constant variation during the freezing period. the following graphic is presenting the water losses during the storage process at freezing temperatures for the bulk fruits. 0 100 200 300 ap ple pe ar qu inc e or an ge ba na na kiw i fruits w ei g h t, g initial w eight weight bef ore thaw ing weight af ter thaw ing figure 7. the water losses variation during freezing of bulk fruits it can be observed that the evolution of the quantity of moisture lost from the product is very similar for apple and pear in all three phases (freeze, before thawing and after thawing). the quince had hard texture and for this consideration it retained a big quantity of water (~20%) after thawing, by hydrating the fibers and the pectin. even the banana had a big content of starch (29.6%) or sugars (glucose – 4.7%, fructose – 8.6% and sucrose – 13.7%) after the thawing it didn’t received a proportional quantity of water, this fact may be put on the protective thick peel which didn’t allow the mass transfer. banana is the only fruit which is registering a water loss after thawing. figure 8 presents the core temperature variation of freeze packed fruits. -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 time, days t em pe ra tu re , 0 c a pple pear quince orange banana kiw i figure 8. the core temperature variation of freeze packed fruits the allure of the temperature curves is constant, almost similar for the pome fruits. the minimum temperature inflexion is detected after 10 days of storage for the majority of the studied fruits. -50 50 150 250 350 ap ple pe ar qu inc e or an ge ba na na ki wi fruits w ei g ht , g initial w eight weight before thaw ing weight after thaw ing figure 9. the water losses variation during freezing of packed fruits the water losses variation is lower than in the bulk fruits; almost the weight food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 54 increasing is minimal (~3 g) for all the fruits. these aspects can be justified by the ldpe bags protection. 4. conclusions as a final conclusion apples and oranges are both suitable to refrigeration and freezing storage both in bulk as in packed state. due to the natural wax protection layer the apple complies with this kind of preservation methods. the exotic fruits represented by banana and kiwi did not comply with this kind of cold preservation, being improper for later consumption. after refrigeration the exotic fruits had been dehydrated, while after thawing the tissue structure was soaked. for the cooling rate the product is considered ‘half cool’ when its temperature drops to half the difference between its initial temperature and the cooling medium temperature. after another half-cooling period the product is ‘three-quarters’ cool. the product is usually finished at ‘seven-eighths’ or ‘fifteen-sixteenths’ cool. the cooling time predictions can be made by equations presented by thompson et al. (1998) or a graphical method by sargent et al. (1988). 5. references 1. fao (food and agriculture organization of the united nations fruit and vegetable processing), fao agricultural services bulletin 119,1995 2. a.a., kader, fruit maturity, ripening, and quality relationships, acta hortic:485, 1999, 203– 208 3. a.a., kader, postharvest technology of horticultural crop, 3rd ed. university of california, division of agriculture and natural resources, publication no. 3311, 2002 4. a.a., kader, a summary of ca requirements and recommendation for fruits other than pome fruits, acta hortic. 600, 2003, 737–740 5. a.a., kader, d., zagory, e.l., kerbel, modified atmosphere packaging of fruits and vegetables. crc, crit. rev. food sci. nutr. 28(1), 1989, 1–30 6. c.a., davis, pre-cooling and storage, facilities james f. thompson, department of biological & agricultural engineering university of california, 2004 7. ashrae (american society of heating, refrigerating and air-conditioning engineers), handbook series, atlanta ga, 1999 8. g.s.v., raghavan, c., vigneault, y., gariépy, n.r., markarian, p. alvo, refrigerated and controlled/modified atmosphere storage, 2006 9. f.b., abeles, p.w., morgan, m.e., saltveit jr., ethylene in plant biology. 2nd ed. academic press, san diego, 1992 10. b.r., champ, e., highley, g.i., johnson, postharvest handling of tropical fruits. international conference proceeding, the australian centre for international agricultural research, canberra, australia, 1994 11. http://postharvest.ucdavis.edu: university of california postharvest research and information center 12. http://www.uckac.edu/postharv: university of california kearney agricultural center 13. http://postharvest.ifas.ufl.edu: university of florida postharvest group. 14. http://www.nutrition.gov: gateway to u.s. government information on human nutrition and nutritive value of foods. 15.http://www.nal.usda.gov/fnic/foodcomp: composition of foods звіт з ндр 29-81 за 2007 – 2009 р food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 49 modeling of the thermo-physical properties of aqueous sucrose solutions ii. boiling po in t, s pecific heat ca pa city an d th erma l con duc tiv ity andrei i. simion1, cristina g. grigoraş1, lăcrămioara rusu 1, *adriana dabija2 1“vasile alecsandri” university of bacău, department of chemical and food engineering, mărăşeşti no 157, bacău 600115, romania 2“ştefan cel mare” university of suceava, faculty of food engineering, universităţii no 13, suceava 720229, romania *corresponding author received 10 september 2011, accepted 25 october 2011 abstract: the aim of this study was to establish mathematical relations between temperature or pressure and sucrose concentration with boiling point, specific heat capacity and thermal conductivity of aqueous sucrose solutions. in order to assess and select a suitable mathematical model the known data were fitted in different equations. two equations were generated for each thermo-physical properties, taking in consideration the level of precision and simplicity of formulation. for boiling point of aqueous sucrose solutions two equations with an average of relative errors of 0.22% and regression coefficients greater than 0.999 were generated, for ranges of 5 – 90% in sucrose concentration and pressure of 0.123.105 – 1.105 pa. for specific heat capacity two equations were formulated with average relative errors of 0.002% and r2 = 0.9994 for intervals of temperature of 0 to 100 °c and sucrose concentration of 0 to 90% and for thermal conductivity were generated two equation with average relative errors of 0.004% and r2 = 0.9992 for a range of temperature of 0 to 80 °c and sucrose concentration between 0 and 60%. the obtained equations can be loaded in computer software available both for industrial and academic users and so facilitating the sizing and optimization calculations of various technological equipment and processes. keywords: sucrose, aqueous solutions, mathematical models 1. introduction aqueous solutions of carbohydrates have been studied for many years due to their both scientific and practical importance (molecular biology and biochemistry, food chemistry and technology, sugar industry etc.). due to the common availability of sucrose and the ease of its purification, the system sucrose water was the subject of many physicochemical studies. the fact that dilute sucrose solutions could be used as a model system to demonstrate the validity of fundamental laws of physical chemistry and chemical thermodynamics additionally stimulated the popularity of these studies [1]. the increasing practical interest in sucrose solutions caused a great demand for predictive methods of determining its physical properties such as boiling point, heat capacity and thermal conductivity. the boiling point can be directly correlated to the chemical structure of the molecule and reflects the strength of the intermolecular forces (among other forces food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 50 present) that hold the sucrose molecules together. the stronger the intermolecular forces, the more tightly the atoms will be held together and, therefore, the boiling point is higher [2]. heat capacity and thermal conductivity are two of the most fundamental thermodynamic properties of liquid substances and they are closely related to their other physical and chemical properties. they are intimately related to the temperature dependence of fundamental thermodynamic functions [3]. these properties may be relatively easily determined in the laboratory with great accuracy; and they are of key importance for linking thermodynamics with microscopic fluid structure and dynamics but having predictive models can be a useful adjunct. the accumulated data have led to the development of both empirical and phenomenological correlations, and many such correlations have been incorporated into design and analysis methods [4]. most correlations, however, are applicable to only a relatively narrow range of conditions. the purpose of this study is, therefore, to develop correlations by critically evaluating and regressing various types of experimental data available from the literature. 2. experimental tabular data (table 1, 2 and 3) concerning the variation of aqueous sucrose solutions boiling point, thermal conductivity and specific heat capacity with sucrose concentration and temperature were used as primary data for the regression analysis. microsoft excel™ 2007 spreadsheets, curveexpert® and tablecurve 3d® v.4 software were used to establish the equations. the tabular data were plotted in temperature – thermo-physical property, sucrose content – thermo-physical property coordinates and different regression techniques, involving the method of least squares were used to reveal the best-fit equation. table 1 boiling point bp [°c] of aqueous sucrose solutions as a function of the mass fraction x [%] pressure p [pa] boiling point, bp [°c] sucrose concentration, x w [%] pressure p.105, pa 0.123 0.199 0.311 0.473 0.7 1 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 50.05 50.12 50.17 50.26 50.39 50.52 50.69 50.80 51.01 51.32 51.70 52.30 52.80 53.65 55.05 56.80 60.05 60.10 60.18 60.27 60.40 60.54 60.71 60.85 61.10 61.40 61.82 62.45 63.00 63.90 65.40 67.30 70.00 70.05 70.11 70.18 70.28 70.42 70.55 70.73 70.90 71.18 71.52 71.94 72.60 73.20 74.18 75.80 77.85 80.75 86.00 80.06 80.11 80.19 80.28 80.43 80.57 80.76 80.95 81.25 81.61 82.06 82.75 83.40 84.46 86.20 88.35 91.50 97.20 90.06 90.12 90.19 90.29 90.44 90.58 90.78 91.00 91.32 91.72 92.18 92.90 93.60 94.75 96.60 98.90 102.25 108.40 100.06 100.12 100.20 100.30 100.45 100.60 100.80 101.05 101.40 101.80 102.30 103.05 103.80 105.05 107.00 109.40 113.00 119.60 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 51 table 2 thermal conductivity [w/(m . k)] of aqueous sucrose solutions as a function of the mass fraction x [%] and temperature t, [°c][5, 6] sucrose concentration, x w [%] thermal conductivity, [w/(m . k)] at temperatures t, [°c] 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 0.565 0.544 0.505 0.473 0.443 0.413 0.383 0.583 0.551 0.52 0.488 0.457 0.391 0.384 0.599 0.566 0.535 0.501 0.47 0.437 0.405 0.614 0.581 0.548 0.514 0.48 0.449 0.415 0.628 0.594 0.56 0.526 0.492 0.458 0.419 0.641 0.607 0.572 0.536 0.502 0.467 0.432 0.652 0.617 0.588 0.547 0.512 0.477 0.441 0.663 0.628 0.592 0.555 0.519 0.484 0.449 0.672 0.636 0.6 0.563 0.526 0.491 0.455 table 3 specific heat capacity cp [j/(kg . k)] of aqueous sucrose solutions as a function of the mass fraction x [%] and temperature t, [°c][7] temperature, t [°c] specific heat capacity cp [j/(kg . k)] at sucrose concentration, x [%] 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 100 3936 3936 3936 3977 3977 3977 3977 3977 3977 4019 4016 3684 3684 3726 3726 3726 3726 3726 3810 3810 3810 3852 3433 3475 3475 3517 3517 3559 3559 3601 3601 3643 3643 3182 3224 3224 3266 3308 3349 3349 3391 3433 3475 3475 2931 2973 3014 3056 3098 3140 3140 3182 3224 3266 3308 2680 2721 2763 2805 2847 2889 2973 3014 3056 3098 3140 2428 2470 2554 2596 2638 2680 2763 2805 2847 2889 2973 2177 2219 2303 2345 2428 2470 2554 2596 2680 2721 2763 1926 2010 2052 2135 2219 2261 2345 2386 2470 2554 2596 3936 3936 3936 3977 3977 3977 3977 3977 3977 4019 4016 3. results and discussion 3.1 boiling point using microsoft excel™ 2007 spreadsheets and curveexpert® software, a modified hoerl model correlation between pressure p and boiling point, at constant aqueous sucrose concentration x, [%] has been established:   cpp pbab /1  (1) for ranges of sucrose concentration between 5 and 85%, the coefficients of the modified hoerl model equations a, b and c are presented in table 4. the regression coefficients r2 are greater than 0.99, thus indicating a good correlation of variables. table 4 coefficients for equation (1) sucrose conc., x [%] a b c r 2 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 101.596 101.644 101.728 101.973 101.973 102.308 102.112 102.562 102.917 103.313 103.791 104.521 105.264 106.475 108.405 110.793 114.586 121.375 0.98196 0.98211 0.98204 0.98214 0.98214 0.98236 0.98226 0.98234 0.98231 0.98251 0.98273 0.98304 0.98320 0.98373 0.98422 0.98466 0.98295 0.98210 0.26788 0.26803 0.26762 0.26712 0.26712 0.26670 0.26697 0.26675 0.26629 0.26609 0.26558 0.26462 0.26411 0.26404 0.26228 0.25944 0.25192 0.24535 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 52 in order to correlate a, b and c coefficients with sucrose mass concentration, more models were used in curveexpert® software (1st, 2nd and 3rd degree polynomial equations, “vapor pressure” model, “heat capacity” model etc.). the best fit model is a 3rd degree polynomial equation for the a coefficient and quadratic equations for the b and c coefficients (table 5). 32 dtctbtatcoefficien a  (2) 2, ctbtatscoefficien cb  table 5 coefficients for equation (2) coeff. a b c a 101 0.9823 0.2672 b 0.1128961704 -3.00e-05 6.00e-05 c -3.820845e-03 7.004e-07 -2.00e-06 d 4.88158e-05 r2 0.994 0.970 0.932 combining the equations (1) and (2) and replacing the coefficients with numeric values, the final form of proposed equation model (eq. 3) is:   )3( )9823.0053077( )1011128961704.0 038208452.30588158.4( )2672.0056062( /12 23 2      exe p p p xexe x xexeb to quantify the deviation of calculated densities from tabular data, the relative error equation was use and its values are presented in table 6: [%]100   tabular calculatedtabular    (4) the average of the induced relative errors for the proposed models is 0.22% for the proposed mathematical model. by plotting the tabular data for aqueous sucrose solutions boiling point in tablecurve 3d® v.4 software (fig. 1) an equation for the response function was generated, chosen due to the relative simplicity of formulation and regression coefficient (eq. 5). the coefficients of the fitted polynomial equation are presented in table 7. table 6 the induced relative errors for the proposed model (3) for boiling point of aqueous sucrose solutions relative errors, ε [%] sucrose concentration, x w [%] pressure p.105, pa 0.123 0.199 0.311 0.473 0.7 1 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 0.00422 0.06781 0.10495 0.03253 0.11270 0.21509 0.31780 0.18847 0.10589 0.03310 0.12519 0.11667 0.59176 0.72748 0.21195 0.50231 0.24345 0.40210 0.42817 0.39174 0.27933 0.17721 0.08829 0.14724 0.15940 0.25959 0.36936 0.38017 0.79412 0.95659 0.52791 0.10705 1.44869 0.03757 0.21822 0.29134 0.26487 0.16100 0.08711 0.00604 0.00229 0.02143 0.02919 0.16137 0.18842 0.55981 0.70410 0.31942 0.24715 1.45175 4.84629 0.12326 0.09753 0.17967 0.18448 0.08590 0.00734 0.08982 0.10788 0.14693 0.10964 0.00217 0.04117 0.38047 0.51124 0.16027 0.29716 1.45218 4.71030 0.17590 0.05329 0.16393 0.17006 0.08578 0.01369 0.08762 0.13819 0.19018 0.18603 0.06926 0.02193 0.29154 0.40029 0.08526 0.33774 1.40415 4.55567 0.02649 0.21966 0.33292 0.34955 0.27582 0.19789 0.10193 0.0239 0.04984 0.03455 0.05520 0.10741 0.39870 0.47823 0.19893 0.15364 1.19791 4.26999 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 53 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 pre ssu re, [ 10^ 5 p a] 01 02 03 04 05 06 070 80 sucrose concentration, [%] 50 50 60 60 70 70 80 80 90 90 100 100 110 110 120 120 b oi lin g p oi nt , [ [° c ] b oi lin g p oi nt , [ [° c ] boiling point rank 31 eqn 157010504 lnz=a+blnx+c/x0.5+dy2+ey2lny+fy2.5+gy3 r2=0.99958643 df adj r2=0.99955659 fitstderr=0.38092776 fstat=39477.482 a=4.7356452 b=0.20791255 c=-0.1303382 d=-0.00037254489 e=0.00044369357 f=-0.00026010431 g=9.6176697e-06 figure 1. aqueous sucrose solutions boiling point values plotted in tablecurve 3d and fitted polynomial equation (5) with residuals. )5( lnlnln 35.2 225.0 gxfx xexdxpcpbabp   table 7 coefficients for equation (5) coeff. value coeff. value a b c d 4.7356452 0.20791255 -0.1303382 -0.0003725448 e f g 0.00044369357 -0.00026010431 9.6176697e-06 the equation (5) is a polynomial equation, rank 31, eqn. 157010504 in tablecurve 3d® v.4 library with r2 = 0.99958643, r2adj = 0.9995565, fitsderr = 0.0003725 and fstat. = 39477.482. 3.2 specific heat capacity using microsoft excel™ 2007 spreadsheets linear correlation between temperature t, [k] and specific heat capacity, at constant aqueous sucrose concentration x, [%] has been established: btac p  (6) for ranges of temperature between 273 and 373 k, the values of the a and b coefficients and regression coefficients, r2 of each linear equation are presented in table 8. in order to correlate a and b coefficients with sucrose concentration, more models were used in microsoft excel™ 2007. the best fit model is also a linear equation. bxatscoefficien ba , (7) table 8 coefficients for equation (6) sucrose conc., x [%] a b r2 10 20 30 40 50 60 70 80 90 3722.12 3234.75 2869.25 2339.64 1952.12 1371.81 984.973 511.335 93.2872 0.777273 1.603636 2.100000 3.079091 3.619091 4.755455 5.293636 6.088182 6.734545 0.813 0.864 0.975 0.985 0.991 0.995 0.994 0.996 0.997 table 9 coefficients for equation (7) coeff. a b a b 4182.8620454545 0.0045707071 -45.7032772727 0.0755772727 combining the equations (6) and (7) and replacing the coefficients with numeric values, the final form of proposed equation model (eq. 8) is: t xc p x)075577.000457070.0( )70327.4586.4182(   (8) to quantify the deviation of calculated densities from tabular data, the relative error equation was use and its values are presented in table 10. the average of the induced relative errors for the proposed models is 0.002% for the proposed mathematical model. by plotting the tabular data for aqueous sucrose solutions specific heat capacity in tablecurve 3d® v.4 software (fig. 2) an equation for the response function was generated (eq. 9). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 54 table 10 the induced relative errors for the proposed model (8) for specific heat capacity of aqueous sucrose solutions temperature, t [k] relative errors, ε [%] sucrose concentration, x [%] 0 10 20 30 40 50 60 70 80 90 273 283 293 303 313 323 333 343 353 363 373 0.066 0.127 0.320 0.523 0.331 0.140 0.051 0.242 0.433 0.427 0.163 0.035 0.376 0.348 0.058 0.465 0.872 1.279 0.556 0.158 0.240 0.459 0.029 0.584 0.070 0.479 0.167 0.377 0.261 0.277 0.354 0.180 0.444 0.023 0.386 0.553 0.187 0.170 0.488 0.416 0.065 0.277 0.611 0.260 0.014 0.154 0.257 0.390 0.519 0.645 0.560 0.422 0.287 0.156 0.028 0.005 0.157 0.277 0.394 0.507 0.617 0.699 0.544 0.425 0.310 0.198 0.048 0.490 0.742 0.308 0.112 0.519 0.584 0.185 0.202 0.578 0.482 0.067 0.900 0.153 0.639 0.309 0.445 0.489 0.231 0.652 0.074 0.743 0.091 0.706 0.579 0.143 0.856 0.313 0.378 0.763 0.092 0.535 0.478 0.066 0.127 0.320 0.523 0.331 0.140 0.051 0.242 0.433 0.427 0.163 the equation (9) is a polynomial equation, rank 50, eqn. 302 in tablecurve 3d® v.4 library with r2 = 0.99944977, r2adj = 0.9994138, fitsderr = 13.701972, fstat. = 33785.237. 27028 029030 031032 033034 035036 0370 temperature, [k] 90 80 70 60 50 40 30 20 sucro se co ncen tratio n, [% ] 1500 1500 2000 2000 2500 2500 3000 3000 3500 3500 4000 4000 4500 4500 h ea t c ap ac ity , [ j/ kg k ] h ea t c ap ac ity , [ j/ kg k ] heat capacity rank 50 eqn 302 z=a+blnx+cy+d(lnx)^2+ey^2+fylnx r 2̂=0.99944977 df adj r 2̂=0.99941388 fitstderr=13.701972 fstat=33785.237 a=25132.56 b=-7267.3551 c=-161.21331 d=630.05763 e=-0.0010015742 f=24.255336 figure 2. aqueous sucrose solutions specific heat capacity values plotted in tablecurve 3d and fitted polynomial equation (9) with residuals. tfx extdcxtbac p ln )(lnln 22   (9) the coefficients of the fitted polynomial equation are presented in table 11. table 11 coefficients for equation (5) coeff. value coeff. value a b c 25132.56 -7267.3551 -161.21331 d e f 630.05763 -0.0010015742 24.255336 3.3 thermal conductivity using microsoft excel™ 2007 spreadsheets linear correlation between aqueous sucrose concentration x, [%] and specific heat capacity, at constant temperature t, [k] has been established: bxac p  (10) the a and b values for the fitted linear equation are presented in table 12. btatscoefficien ba , (11) table 12 coefficients for equation (10) temperature, t [k] a b r 2 273 283 293 303 313 323 333 343 353 0.568357 0.584857 0.598821 0.613964 0.628893 0.641393 0.653679 0.663179 0.672036 0.00311 0.00330 0.00323 0.00332 0.00345 0.00349 0.00353 0.00358 0.00363 0.997 0.998 0.999 0.999 0.999 1.000 0.999 0.999 1.000 in order to correlate a and b coefficients with temperature, more models were used in microsoft excel™ 2007. the best fit model is also a linear equation (table 13). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 55 table 13 coefficients for equation (11) coeff. a b a b 0.2145613296 -0.0014797877 0.0013113690 -0.0000061488 combining the equations (10) and (11) and replacing the coefficients with numeric values, the final form of proposed equation model (eq. 12) is: )12(06t)6.1488e70.00147978( 90.00131136960.21456132( x t   to quantify the deviation of calculated densities from tabular data, the relative error equation was use and its values are presented in table 15. the average of the induced relative errors for the proposed models is 0.004% for the proposed mathematical model. 60 50 40 30 20 10sucrose concentration, [%] 270 280 290 300 310 320 330 340 350 temp eratu re, [k ] 0.35 0.35 0.4 0.4 0.45 0.45 0.5 0.5 0.55 0.55 0.6 0.6 0.65 0.65 0.7 0.7 t he rm al c on du ct iv ity , [ w /m k ] t he rm al c on du ct iv ity , [ w /m k ] thermal conductivity rank 17 eqn 316 z=a+bx+c/y+dx^2+e/y^2+fx/y+gx^3+h/y^3+ix/y^2+jx^2/y r 2̂=0.99940883 df adj r 2̂=0.99929515 fitstderr=0.0019651459 fstat=9955.5537 a=-0.84385948 b=-0.0051670655 c=1602.2814 d=2.3609905e-06 e=-528571.74 f=0.45271455 g=3.7037037e-08 h=53598501 i=42.375474 j=-0.0020785223 figure 3. aqueous sucrose solutions thermal conductivity values plotted in tablecurve 3d and fitted polynomial equation (13) with residuals. by plotting the tabular data for aqueous sucrose solutions thermal conductivity in tablecurve 3d® v.4 software (fig. 3) an equation for the response function was generated chosen due to the best regression coefficient (eq. 13). tjxtixthtfx tedxtcbxa //// // 223 22   (13) the coefficients of the fitted polynomial equation are presented in table 14. table 14 coefficients for equation (13) coeff. value coeff. value a b c d e -0.84385948 -0.0051670655 1602.2814 2.3609905e-06 -528571.74 f g h i j 0.45271455 3.7037037e-08 53598501 42.375474 -0.0020785223 the equation (13) is a polynomial equation, rank 17, eqn. 316 in tablecurve 3d® v.4 library with r2 = 0.999440883, r2adj = 0.999295, fitsderr = 0.00196514, fstat. = 9955.553. 4. conclusions two equations were generated for each thermo-physical properties, taking in consideration the level of precision and simplicity of formulation. table 15 the induced relative errors for the proposed model (12) for thermal conductivity of aqueous sucrose solutions temperature, t [k] relative errors, ε [%] sucrose concentration, x [%] 0 10 20 30 40 50 60 273 283 293 303 313 323 333 343 353 1.339 0.459 0.035 0.341 0.475 0.447 0.115 0.205 0.815 0.555 0.450 0.004 0.434 0.509 0.581 0.166 0.075 0.782 0.871 0.246 0.343 0.539 0.548 0.557 1.241 0.099 0.745 1.017 0.222 0.130 0.463 0.591 0.344 0.288 0.306 0.882 0.729 0.026 0.524 0.377 0.641 0.498 0.361 0.348 1.038 0.398 0.875 0.521 0.944 0.698 0.462 0.444 0.190 0.806 0.016 2.210 0.763 0.883 0.420 0.421 0.316 0.007 0.760 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 56 for boiling point of aqueous sucrose solutions two equations with an average of relative errors of 0.22% and regression coefficients greater than 0.999 were generated, for ranges of 5 – 90% in sucrose concentration and pressure of 0.123.105 – 1.105 pa. for specific heat capacity two equations were formulated with average relative errors of 0.002% and r2 = 0.9994 for intervals of temperature of 0 to 100 °c and sucrose concentration of 0 to 90% and for thermal conductivity were generated two equation with average relative errors of 0.004% and r2 = 0.9992 for a range of temperature of 0 to 80 °c and sucrose concentration between 0 and 60%. 5. references 1. starzak, m., mathlouthi, m., temperature dependence of water activity in aqueous solutions of sucrose, food chemistry, 96 (3), 346-370, (2006) 2. li, q., chen, x., hu, z., quantitative structure–property relationship studies for estimating boiling points of alcohols using calculated molecular descriptors with radial basis function neural networks, chemometrics and intelligent laboratory systems, 72 (1), 93-100, (2004) 3. wilhelm, e., heat capacities: introduction, concepts and selected applications in: heat capacities liquids, solutions and vapours (editors: wilhelm, e., letcher, t.m.), the royal society of chemistry, england, 1-27, (2010) 4. bubník, z., henke, s., kadlec, p., hinková, a., pour, v., database of the properties of sucrose, sucrose solution and food, journal of food engineering, 77 (3), 399-405, (2006) 5. iliescu g., vasile c., caracteristici termofizice ale produselor alimentare, ed. tehnica, bucuresti, 109-103 p (1982) 6. macovei v.m., culegere de caracteristici termofizice pentru biotehnologie şi industrie alimentară, ed. alma, galaţi, 199-200 p, (2000) 7. mathlouthi m., reiser p., sucrose, properties and applications, chapman & hall, london, 208 p, (1995) 8. simion, a.i., dobrovici, p.e., rusu, l., gavrilă, l., modeling of the thermophysical properties of grapes juice i. thermal conductivity and thermal diffusivity, annals of food science and technology, issn 20652828, x (2), 363-368, (2009) 9. simion, a.i., grigoraş, c., rusu, l., gavrilă, l., modeling of the thermophysical properties of grapes juice ii. boiling point and density, studii şi cercetări ştiinţifice: chimie şi inginerie chimică – biotehnologii industrie alimentară, issn 1582540x, x (4), 365-374, (2009) 10. simion a.i., dobrovici p.e., grigoras c.g., rusu l., modeling of the thermo-physical properties of aqueous sucrose solutions i. density and dynamic and kinematic viscosity, annals of food science and technology, issn 20652828, in press, (2011) title …………………… 241 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 241 244 decontamination of toluene pollution in water using raw walnuts shells *igor winkler1,2, ivan kuvila1 1institute of biology, chemistry and bioresources, yu fedkovych national university of chernivtsi, 2 kotsyubynsky str., chernivtsi, 58012, ukraine 2medical chemistry department, bukovyna medical university,2 thetralna sq., chernivtsi, 58002, ukraine i.winkler@chnu.edu.ua *corresponding author received july 20th 2015, accepted september 15th 2015 abstract: raw untreated walnut shells were tested as an adsorbent for the extraction of toluene pollution from water systems, and high cleaning efficiency (up to 28 % of the specific toluene extraction) was determined by the direct uv-photometry measurement of the residue toluene concentration. the adsorption efficiency of the shells is higher than that of some widely distributed carbon-based adsorbents. extended use of shells is recommended since this material is very cheap and easily available as waste in some food processing factories. further utilization of the used shells through burning is proposed as an environment friendly solution. keywords:toluene; walnut shells; uv-photometry; wastewater decontamination 1. introduction decontamination of water and wastewater is still a very topical problem since billions tons of insufficiently treated and untreated effluents are discharged systematically and washed-off into various water bodies [1, 2]. many kinds of water treatment technologies can be used in order to decontaminate water and remove toxicants from effluents: mechanical treatment methods, biotreatment, oxidation decomposition, adsorption, evaporation, etc. it is well known that the organic pollutants, especially aromatic compounds, are considered as quite persistent pollution agents, which are very difficult to remove, extract or decompose [3-6]. on one hand, this problem is grounded on a wide variety of various aromatic compounds used in many fields of the everyday activities. they are used extensively as engine oils and grease components, in various mixtures for construction and so on. on the other hand, oxidation and/or decomposition of most aromatic compounds are a very slow process while their biodecomposition is also low effective. finally, it should be mentioned that the majority of the aromatic compounds are considered very highly toxic agents with a distinct ability to cumulate along the trophic chains. that is why rather strict limitations are set for their content in wastewater to be discharged while development of new effective solutions for the extraction of the organic compounds remains a topical issue. various porous materials of plant nature become more and more popular as possible adsorbents for such class of pollution agents because such materials are quite cheap, easily available and can exhibit http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 igor winkler, ivan kuvila, decontamination of toluene pollution in water using raw walnuts shells, food and environment safety, volume xiv, issue 3 – 2015, pag. 241 244 242 rather high efficiency. it has been reported [7] that the oil products content in water can be decreased under its maximum permissible level using the carbonized walnut shells after their preliminary thermotreatment at 450-600 0c in the air or at 550-600 0c in nitrogen. such carbonized material represents mostly mineralized and highly porous substrate containing 5-10 wt % of sio2 and 80-90 wt % of carbon. modification of the carbonized shells using acids is another way to increase their porosity [8]. it should be emphasized that carbon is the main adsorption substrate for most organic, especially aromatic compounds and, therefore, the content of carbon in the water cleaning material is the key factor and it should be kept high. it should also be mentioned that the air bubbles can be fixed firmly on the carbon surface during its contact with wastewater, which results in the total or partial deactivation of the adsorbent surface [9]. that is why deaeration of water is a required pretreatment stage in the adsorptive extraction of the oil products. the aim of our investigation was to assess the efficiency of removing toluene from water by the raw untreated walnut shells. this material is also easily available and does not require any additional treatment, which keeps its potential cost very low. practically, in many cases this material can be obtained for free. on the other hand, potential utilization of the used shells after oil products adsorption is also very simple since this material is combustible and it can be burned directly in any solid fuel equipment independently or as an admixture to the main fuel (coal, wood, etc.). high combustion temperature would ensure complete decomposition of the adsorbed aromatic compounds to environmentally safe combustion products. 2. materials and methods all model water/toluene systems were made using the distilled water and pure toluene with concentration 0.2 ml/l. then some amount of the preliminary dried and roughly ground shells (to the particles size 2-5 mm) was added to the system and the toluene adsorption was determined as a ratio between its initial and final concentration in the system. the contact time was of 24 hours. the concentrations of toluene in water were determined using the modified uvphotometry method [10], which implies the measuring of the light absorbance of the system at the wave length of 258 nm. the method has to be modified since our preliminary investigation proved that some unidentified and optically active substance is released to water from the shells. as a result, the shell itself results in some decrease in the absorbance of the system at 258 nm even without toluene. a series of the blank experiments was carried out in order to take this effect into account and the absorbance decrease values in the blank experiments are shown in table 1. then the calibration diagram was built on the basis of data of table 1 and the ‘blank’ values were subtracted from the corresponding results obtained in further measurements where the tolueneconsisting systems were used. table 1. decrease in the absorbance for the water/walnut shells without toluene (blank experiments) walnut sample weight, g absorbance (*) average absorbance for all three series series 1 series 2 series 3 0.49 0.075 0.08 0.072 0.0757 0.98 0.139 0.142 0.136 0.139 1.52 0.162 0.163 0.167 0.164 3.02 0.28 0.27 0.279 0.276 (*) absorbance of the clear distilled water was taken as the reference point. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 igor winkler, ivan kuvila, decontamination of toluene pollution in water using raw walnuts shells, food and environment safety, volume xiv, issue 3 – 2015, pag. 241 244 243 it should be emphasized that the absorbance values can be used in our experiments instead of the toluene concentrations because these parameters are in linear dependence and we can consider the relative decrease in the toluene concentrations instead of finding their absolute values. in other words. in the framework of our investigation it is sufficient to determine that the concentration of toluene has decreased for instance by 50 % instead of finding its initial and final values (for instance. 0.18 and 0.09 ml/l). 3. results and discussion the initial absorbance of the water/toluene systems and the final absorbance after the addition of some amount of shells to the system are shown in table 2. additionally. the percent of the absorbance decrease is shown in the right column of the table. as seen from table 2. walnut shells is a quite effective adsorbent. which is capable to adsorb (in terms of 1 g of the adsorbent) up to 27 % of toluene (0.5 g of the shells); 28 % (1 g); 18.7 % (1.5 g) and 11.3 % (3 g). therefore. the highest specific adsorption of toluene has been determined for the system containing 1 g of the shells. it should be noted that this adsorption performance value can be considered as quite high since it is better than the efficiency of various carbon-clay compositions and comparable with the efficiency of the widely used activated carbon [11]. taking into account the data of this experiment. we can conclude that the walnut shells exhibit good adsorption properties and can be effective for decontamination of water and wastewater system through extraction of toluene and other aromatic toxicants. besides. it was found that in case of activated carbon. low amount of adsorbent can cause an adverse effect of ‘bridging’ the toluene into the aqueous phase [11. 12]. which results in an unexpected raise in the pollutant’s concentration. the shells do not exhibit such an effect. which makes this adsorbent safer than the activated carbon. even an insufficient amount of shells would not cause any unwanted increase in the pollutant’s concentration in water. 4. conclusion walnut shells can be used as an inexpensive and easily available material for adsorption removal of the organic aromatic pollutants in water or wastewater. table 2. absorbance of the experimental water/toluene systems with various walnut shells samples sample weigh. g initial absorbance final absorbance (*) percent of absorbance decrease (**) (1) (2) (3) (4) 0.5 0.228 0.197 13.5 1.0 0.235 0.170 28.0 1.5 0.196 0.142 28.1 3.0 0.217 0.144 33.9 (*) – all values here are calculated as a result of subtraction of the experimental absorbance from the corresponding value of the blank experiment (see table 1); (**) – all values here are calculated by the formula %100* )2( )3()2( value valuevalue  food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 igor winkler, ivan kuvila, decontamination of toluene pollution in water using raw walnuts shells, food and environment safety, volume xiv, issue 3 – 2015, pag. 241 244 244 within the framework of this investigation. this material has shown the highest specific adsorption performance 28 % of the initial toluene has been extracted from the system containing 0.2 ml/l of the substance. higher amounts of the adsorbent provided higher total extraction of toluene. but the specific adsorption efficiency of the shells was lower for the systems with more than 1 g of the shells per liter. it is expected that the most effective adsorbent amount will vary depending on the initial concentration of toluene in water. 5. references [1] singh k. p.. mohan d.. sinha s.. dalvani r.. impact assessment of treated/untreated wastewater toxicants discharged by sewage treatment plants on health. agricultural and environmental quality in the wastewater disposal area. chemosphere. 55(2): 227-255. (2004) [2] wichelns d.. qadirm. policy and institutional determinants of wastewater use in agriculture. in: wastewater. p. ii. springer netherlands: 93-112. doi: 10.1007/978-94-0179545-6_ (2015) [3] farrington. j.w.. takada h.. persistent organic pollutants (pops). polycyclicaromatic hydrocarbons (pahs). and plastics: examples of the status. trend. and cycling oforganic chemicals of environmental concern in the ocean.oceanography 27(1):196–213. doi: http://dx.doi.org/10.5670/oceanog.2014.23 (2014). [4] lohmann r.. belkin i. m.. organic pollutants and fronts across the atlantic ovean: a review. progress in oceanography. 128: 172-184. (2014). [5] prokes m.. vrana b.. komprodova k.. klanova j.. annual dynamics of persistent organic pollutants in various aquatic matrices: a case study in the morava river in zlín district. czech republic. j. soils and sediments. 14(10): 1738-1752. (2014). [6] kanzari f. et al.. distributions and sources of persistent organic pollutants (aliphatic hydrocarbons. pahs. pcbs and pesticides) in surface sediments of an industrialized urban river (huveaune). france. sci. total environ.. 478: 141151. (2014). [7] russian federation patent 2031849. russian federation inventions bull. #9. (1995). [8] nabatkin a. n.. khlebnikov v. n.. use of the sorbents for elimination of the oil spills. oil industry. 11: 61-69. (2000) (in russian). [9] temirkhanov b. a. et al. efficiency assessment for some adsorbents in the cleaning of the surface water from oil and some oil products. environ. protection in the oil industr. 5: 22-23. (2005) (in russian). [10] winkler i.. agapova n. determination of water pollution by the oil products through uv photometry. environ. monitor. and assessment. 168: 115-119 (2010). [11] winkler i.. diychuk v..adsorptionoftoluenefromthemixedwater−toluen esystemonmedicinalactivatedcarbon.canadian j. chem.. 92(5): 392-396. (2014). [12] winkler i.. bodnaryuk e.. boruk s. concentration dynamics of toluene in an open aqueous phase containing the free toluene phase. noosfera. 8: 125-128. (2013). 171 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 171 180 assessment of environmental impacts of municipal solid waste management in iasi, romania *cristina ghinea1,2, andreea spranceana1, diana elena comanita1,3, gabriela constantinescu2, maria gavrilescu1,4 1“gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof. dr. doc. dimitrie mangeron street, 700050 iasi, romania 2stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania, cbghinea@yahoo.com 3“alexandru ioan cuza” university of iasi, 11 carol i street, 700506 iasi, romania 4academy of romanian scientists, 54 splaiul independentei, ro-050094 bucharest, romania *corresponding author received may 15th 2015, accepted june 16th 2015 abstract: the aim of this paper was to evaluate from environmental point of view the municipal solid waste management (mswm) from iasi, romania considering two different systems existent in 2008 and 2013, respectively. therefore, in 2008 the mswm system included temporary storage of waste in containers, collection and transport and finally landfilling in the old landfill at tomesti. after 2009 the mswm system was improved: a new landfill at tutora was developed according to the legislation, which also included leachate collection and treatment and biogas collection systems; and a sorting and composting plants were opened. in this study we have applied life cycle assessment (lca) methodology for the evaluation of these two mswm systems. after analyzing all the results it can be concluded that the environmental impacts of the mswm system existent in 2013 are lower compared to the environmental impacts of system from 2008, but they are still negative impacts and an improvement of mswm system is urgently required. keywords: abiotic depletion, climate change, environment, life cycle assessment, solid waste 1. introduction the amount of solid waste generated in cities over the years increased due to a continuous welfare improving and changes in the life style. application of environmental, institutional, financial, economic and social tools is required to guarantee a sustainable waste management [1-4]. different tools for environmental impacts assessment were developed but one of the most commonly used is lifecycle assessment (lca) [5]. during the 1990s lca was developed rapidly and become a useful tool in decision-making processes and system performance documentation [6]. the result of an lca study expresses the performance of the total system life cycle as well as for single life cycle stages. up to now, for environmental assessment of the municipal solid waste treatment and disposal, several models based on lca methodology were developed and applied [7-11]. in the situation when municipal solid waste management systems in different communities are characterized by a quite low level of recycling and recovery of materials and is limited to only some http://www.fia.usv.ro/fiajournal mailto:cbghinea@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 172 activities: temporary storage, collection and transport of mixed waste directly to the landfill site located outside the city, these tools allow the results modeling and scenario simulation based on integrated waste management and lca concepts [1213]. in this paper we have applied one lca tool (gabi software) to evaluate the environmental impacts of municipal solid waste management (mswm) system in iasi, romania. for this study we have considered two mswm systems: 1) the mswm system existent in 2008 and 2) the mswm system existent in 2013, both from iasi. in order to achieve this aim, all the specific steps in an lca studies were covered: goal setting, establishment of system boundaries, functional units setting and inventory analysis. 2. waste management in iasi, romania iasi county is a part of the region 1 north east (established by law no. 315/2004 on regional development in romania), which also includes botosani, suceava, neamt, bacau and vaslui counties. iasi area is 5475.58 km2, representing 2.3% of the country, being the 23rd largest county in romania [14]. in 2005 iasi county had 813943 inhabitants, population increasing in 2008 to 826552 inhabitants with a density of 151 inhabitants per km2. in 2013 population number was 849670 from which 404611 in the urban area. iasi relief morphology is characterized by two major relief units: a high unit (in the west and south) with an average altitude of 300350 meters and another plain-looking lower (in north, north-east) with an average altitude of 100-150 m. plateau area is represented by the suceava plateau and central plateau moldovan. plain area is represented by the plain of moldavia, namely through its subdivision jijia-bahlui plain, which occupies almost half of the county [14]. iasi territory belongs to the temperate zone continental pronounced, under atlantic anticyclone and the influence of euro-asian. the average annual temperature of air is between 8°c 9°c in the west and south, and between 9°c 10°c in the north and northeast, declining with increasing in altitude. main economic activities in iasi are represented by trade and transactions properties. in iasi county there are concerns about the development of organic farming by using bio-fertilizer and organic fertilizers. they were distributed over an area 16100 ha, representing 6.27% of the arable land [14 17]. municipal solid waste include the waste generated and collected (mixed or selective), as well as uncollected waste. collected waste represents the waste generated which are collected by sanitation services. the increase of the amount of municipal waste generated is closely related to population growth and higher consumption of population [14]. the waste composition consists in organic waste, which represents the fraction with the major percentage (approximately 40%) followed by paper and cardboard, plastic, glass, metals. the municipal solid waste indicators for iasi, region ne and romania are illustrated in fig. 1. 2.1. mswm system in 2008 municipal solid waste management system existent in 2008 included temporary storage in containers, collection and transport and finally landfilling (in the old landfill) without treatment of leachate and biogas collection (fig. 2). 2.2. mswm system in 2013 municipal solid waste management system in iasi, in 2013 besides landfilling included also sorting and composting (fig. 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 173 fig. 1. municipal waste generation indicators [15, 17, 18] fig. 2. municipal solid waste management in 2008, iasi, romania fig. 3. municipal solid waste management in 2013, iasi, romania 3. life cycle assessment of municipal solid waste management system 3.1. goal the goal of this study was to evaluate from environmental point of view two different municipal solid waste management systems existent in iasi, romania in 2008 and 2013 and to compare the environmental impacts of these systems. the functional unit is represented by the amount of solid waste that was generated in iasi, in these two years. 3.2. inventory analysis in the inventory analysis phase are collected, calculated and estimated all the inputs and outputs for each process included in mswm systems considered for the evaluation. for assessment of a waste management system is necessary the knowledge of the waste amounts generated, fractions (fig. 4) and elemental composition of waste fractions. for the evaluation of temporary storage process we considered the number of containers and the annual quantity of material required for containers fabrication. 0 50 100 150 200 250 300 350 400 450 kg/inh/year 2003 2004 2005 2006 2007 ye ar municipal waste generation indicators iasi region north-east romania food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 174 fig. 4. waste generated in 2008 and 2013 the following data were considered for 2013: 310 metal containers (4 m3); 4248 metal containers (1.1 m3); 244 plastic containers (1.1 m3); 10904 plastic bins of 240 l and 2146 plastic bins of 120 l. in order to calculate the amount of material, we considered the following: 1 container 4 m3 from metal has 463.6 kg metal and 9.1 kg plastic; 1 container of 1.1 m3 metal has 127.5 kg metals and 2.5 kg plastic; 1 container of 1.1 m3 plastic has 51 kg plastic and 1.3 kg iron; 1 bin of 240 l has 13.8 kg plastic and 0.2 kg metal [19]. the necessary data for collection and transport processes include: number of vehicle and loading capacity, transport distance, fuel consumption and emissions resulted from fuel consumption. iasi had 30 vehicles in 2008 and 36 vehicles in 2013, for waste collection and transportation. diesel consumption is estimated to be 30 l/100 km for truck of 18 m3 and 25 l/100 km for truck of 12 m3 [20]. emissions from the combustion of diesel fuel used to transport waste are shown in table 1. tutora sorting station has an area of 1622 m2, a 51,000 tons/year capacity and 2 sorting bands by 15 m each. there are sorted paper, cardboard, glass, plastic, metals which are sent for recycling; biodegradable waste are sent to the composting station and street and mixed waste are sent to landfilling. table 1 emissions from burning diesel fuel used for waste transportation emissions 2008 (kg) 2013 (kg) co2 121211 146167 co 513 619 nox 1428 1723 n2o 3.05 3.6 pm10 65.8 79.3 ch4 6.7 8.1 so2 192 232 hydrocarbons 136 164 consumption or resources used for sorting and pre-cleaning of recyclable materials are given by [21]. the composting station from tutora covers an area of 2,100 m2 and has a capacity of 95,000 tons/year. the emission factors for composting are provided by [21]. the inputs for landfilling process consists of the amounts of waste fractions landfilled and fuel consumption, while the outputs are: emissions from fuel consumption, landfill gas and leachate. most of them are calculated based on the equations presented by [21]. the data related with leachate from tomesti landfill are provided by [22]. 0% 20% 40% 60% 80% 100% p a pe r p la st ic g la ss m et al s w oo d b u lk y w as te w as te p ar ks an d g ar d en s w as te fr o m m ar ke ts s tre e t w as te o th er ty p es 2013 2008 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 175 3.3. impact assessment and interpretation the overall goal of impact assessment phase is to connect each life cycle inventory results to the corresponding environmental impacts [23-24]. the impact categories such as: abiotic depletion potential (adp), acidification potential (ap), – eutrophication potential (ep), global warming potential (gwp), ozone depletion potential (odp), photochemical ozone creation potential (pocp) and others can be evaluated in the life cycle impact assessment phase [7, 9, 21, 25]. in our case we have applied gabi software to obtain the environmental impacts of the municipal solid waste management systems from 2008 and 2013 in iasi, romania. in this paper we presented the results obtained by considering two lca methods recipe (figs. 5-6) and cml 2001 (figs. 7-10), which are included among others in gabi software. the values obtained for each category of impact in different measurement units are normalized (pe – person equivalents) so that the environmental impact categories to be compared and illustrated on a single graphic. all impact categories determined showed positive values which mean negative impacts on the environment. according to the recipe method all the impacts categories (agricultural land occupation alo; climate change ecosystems cce; climate change human health cchh; fossil depletion fd; particulate matter formation pmf) have positive values for both mswm systems (negative impacts on the environment) decreasing in the following order: for 2008 fd>cchh>pmf>cce>alo and for 2013 fd>pmf>cchh>cce>alo (figs. 5-6). it can be observed that fossil depletion (fd) has the highest value for both systems, followed by climate change human health (cchh) in the case of mswm system from 2008 and particulate matter formation (pmf) impact category for mswm system from 2013. fossil fuel depletion (fd) refers to a group of resources that contain hydrocarbons such as methane or non-volatile materials like anthracite coal [26]. in our case this indicator has highest value due to the use of diesel (that contains hydrocarbons with 12-20 carbon atoms) which is used both in collection and transportation of solid waste and in waste treatment processes. climate change – damage to human health (cchh) has direct effects (heat waves, air pollution and aeroallergens) and some infectious diseases, malnutrition and others as indirect effects. in 2008 the mswm system included only landfilling of solid waste as treatment/elimination process. the cchh impact category has high value because all the amount of waste was landfilled in a non compliant landfill at tomesti, without leachate (a gas-liquidsolid phase which may contain unwanted and toxic chemicals) collection and treatment and also without biogas (which contains mainly ch4 and co2, both greenhouse gases) collection. analysis of leachate composition at tomesti landfill and the environmental components quality indicators are presented by [22]. in the case of mswm system from 2013 the cchh has a lower value compared to 2008 because of the decreased amount of waste landfilled and the fact that landfilling of waste is performed in a compliant landfill at tutora (with leachate collection and treatment). particulate matter formation (pmf) particulate matter with a diameter of less than 10 μm (pm 10) causes health problems, they can be formed in air from emissions of sulphur dioxide (so2), ammonia (nh3), and nitrogen oxides (nox) among others [26]. in this case the pmf impact category has highest value due to the combustion of diesel used but also because of other emissions. another indicator that has positive value (fig. 5) is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 176 climate change damage to ecosystem diversity (cce). species loss is related to climate change, an important role in the extinction of species plays the temperature. in the calculation of this indicator temperature factor and a damage factor are combined [26]. according to the cml 2001 method both mswm systems negative impacts on the environment (impacts categories have positive values) decreasing in the following order: for 2008 gwp>adp>ap> pocp>ep>odp and for 2013 ap>gwp>ep>adp>pocp>odp (figs. 7-8). fig. 5. environmental impacts of mswm system in 2008 (recipe method) fig. 6. environmental impacts of mswm system in 2013 (recipe method) for the comparison there were chosen global warming potential expressed in kg co2 -eq. and abiotic depletion potential (kg sb-eq.). abiotic depletion covers all natural resources, while the “depletion” depends on ultimate reserves and rates of extraction of a given resource. depletion of abiotic resources occurs by the decrease of availability of the total reserve of potential functions of resources [27]. fig. 7. environmental impact of mswm system in 2008 (cml method) fig. 8. environmental impact of mswm system in 2013 (cml method) this impact category applied to waste management assessment allows for accounting positive aspects of the recovery/recycling of waste [13, 20, 28]. climate change involves environmental mechanisms that affect both the human health and the environment [28]. climate change is caused by the emission of greenhouse gases like ch4, co2, halocarbons, nitrogen oxides, non-methane volatile organic compounds which are substances with the ability to absorb infrared radiation from the earth [28]. some of the greenhouse gases (ghg), such as water vapour, co2, o3, ch4 and n2o, besides being emitted through human activities occur naturally [29]. some gases like the fluorinated gases only occur due to 0.00e+00 1.00e+02 2.00e+02 3.00e+02 4.00e+02 5.00e+02 6.00e+02 pe a lo c c e c c h h fd p m f impact categories recipe 0 .00e+00 1 .00e+02 2 .00e+02 3 .00e+02 4 .00e+02 5 .00e+02 6 .00e+02 7 .00e+02 pe a lo c c e c c h h fd p m f impact categories recipe 0.00e+00 5.00e-04 1.00e-03 1.50e-03 2.00e-03 2.50e-03 pe adp ap ep gwp odp pocp impact categories cml2001_ dec.07 0.00e+00 1.00e-04 2.00e-04 3.00e-04 4.00e-04 5.00e-04 6.00e-04 7.00e-04 8.00e-04 pe adp ap ep gwp odp pocp impact cat egories cml 2001_dec 07 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 177 human activities [29]. a greenhouse gas can be more or less potent regarding to how much contributes to global warming and affects climate change. the contribution to the global warming depends on greenhouse gas properties such as atmospheric lifetime, the degree of infrared absorption and the spectral location of its absorbing wavelengths [29]. according with kyoto protocol emissions of four greenhouse gases: carbon dioxide (co2), methane (ch4), nitrous oxide (n2o) and sulphur hexaflouride (sf6), and two groups of ghg: hydrofluorocarbons (hfcs) and perfluorocarbons (pfcs) must be reduced [29]. the global warming potential (gwp) represents a comparative index based on the radiative forcing of a unit mass of a given greenhouse gas. the relative capacity of each greenhouse gas to absorb heat is compared by gwp [29]. ghg emissions from the municipal solid waste management system may be divided into the following categories: direct emissions, which originate from waste management activities such as methane from landfills and co2 emissions from transport, incineration and recycling plants; avoided emissions, which represent the life-cycle benefits from resource recovery (using waste as a secondary material or energy source) and replacing the use of virgin materials or fuels [30]. when the waste management practices are analyzed, the following particular aspects can be found:  the ghg emission, especially co2 from waste collection and transport result from the use of fuel by the vehicles;  recycling can produce substantial ghg emission savings;  composting is an aerobic process with energy consuming and implies co2 emission and carbon storage in soil by the application of compost;  anaerobic treatment of solid waste generates significant quantities of biogas, as well as ch4 and n2o emission;  incineration is mainly used to minimize the volume of solid waste to be disposed in landfills and to obtain energy;  nitrous oxide (n2o) and carbon dioxide (co2) are the main emissions from incineration that contributes to climate change;  biogas produced from landfilling of solid waste contains mainly methane and carbon dioxide, but it is considered that only ch4 emission is relevant to the global warming [3135]. global warming potential has high values because of the higher content of organic waste that were landfilled. the organic wastes are degraded through a series of consecutive reactions, which take place in the body of landfill, as follows: the large molecules (biopolymers like carbohydrates, proteins, fats) are broken down into simpler molecules; the simpler molecules (monomers like sugars, amino acids etc.) are biodegraded in intermediates such as fatty acids, alcohols; the intermediate compounds are degraded under anaerobic conditions in acetates and hydrogen; the acetates and hydrogen will then form biogas (ch4 and co2) [36]. fig. 9. abiotic depletion potential – comparison 2008 and 2013 0.00e+00 5.00e+06 1.00e+07 1.50e+07 2.00e+07 2.50e+07 3.00e+07 kg s be ch iv . mswm_2008 mswm_2013 cml 2001_dec 07_adp food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 178 ch4 and co2 are the main constituents of the biogas. because the biogas is not collected in the non-compliant landfill (fig. 10, mswm_2008), it will reach the atmosphere as a contributor to global warming potential. fig. 10. global warming potential or climate change – comparison 2008 and 2013 4. conclusions in this paper two mswm systems were evaluated from the environmental point of view with two methods (recipe and cml2001) included in lca software. after the assessment it can be concluded that both mswm systems have negative impacts on the environment, mentioning that mswm system from 2008 has higher impacts as compared to the system existenting in 2013. the impacts categories with higher values are fossil depletion (values obtained by applying recipe) global warming potential from the assessments of mswm system existent in 2008 and acidification potential obtained by evaluating mswm system from 2013 with cml2001 method. it can be concluded that an improvement of mswm system is urgently required; it can be considered implementation of other treatment methods given the amount of waste generated and its composition. also a better separate waste collection on waste fractions followed by the recycling may lead considerably to a decrease in environmental impacts. 5. acknowledgements this work was supported by the grant of the romanian national authority for scientific research, cncs – uefiscdi, project number pn-ii-id-pce-2011-30559, contract 265/2011, gabi education: software and database for life cycle engineering, pe international gmbh and by the strategic grant posdru/159/1.5/s/133652, co-financed by the european social fund within the sectorial operational program human resources development 2007 – 2013. 6. references [1]. ghinea c., gavrilescu m., models for sustainable waste management, the bulletin of the polytechnic institute from iasi, chemistry and chemical engineering section, lvi (lx): 21-36, (2010) [2]. rathi s., alternative approaches for better municipal solid waste management in mumbai, india, waste management, 26: 1192-1200, (2006) [3]. schiopu a.m., gavrilescu m., options for the treatment and management of municipal landfill leachate: common and specific issues, clean – soil, air, water, 38: 1101-1110 (2010) [4]. schiopu a.m., gavrilescu m., municipal solid waste landfilling and treatment of resulting liquid effluents, environmental engineering and management journal, 9: 9931019, (2010) [5]. ekvall t., assefa g., bjorklund a., eriksson o., finnveden g., what lifecycle assessment does and does not do in assessments of waste management, waste management, 27: 989-996, (2007) [6]. glavic p., lukman r., review of sustainability terms and their definitions, journal of cleaner production, 15: 1875-1885, (2007) [7]. banar m., cokaygil z., ozkan a., life cycle assessment of solid waste management options for eskisehir, turkey, waste management, 29: 54-62, (2009) [8]. frioriksson g.b., johnsen l.t., bajarnadottir h.j., sletnes l.h., guidelines for the use of lca in the waste management sector, technical report, nordtest project no. 1537-01 (2002) 0.00e+00 2.00e+09 4.00e+09 6.00e+09 8.00e+09 1.00e+10 1.20e+10 kg c o 2 -e ch iv . mswm_2008 mswm_2013 gwp 100 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 179 [9]. ghinea c., gavrilescu m., decision support models for solid waste management – an overview, environmental engineering and management journal, 9: 869-880, (2010) [10]. tsilemou k., panagiotakopoulos d., solid waste management systems assessment: the municipality’s point-of-view, environmental engineering and management journal, 6: 51-57, (2007) [11]. winkler j., bilitewski b., comparative evaluation of life cycle assessment models for solid waste management, waste management, 27: 1021-1031 (2007) [12]. mcdougall f., white p., franke m., hindle p., integrated solid waste management: a life cycle inventory, 2nd edition, blackwell science (2001) [13]. pe international, handbook for life cycle assessment (lca) using the gabi education software package, germany, (2009) [14]. doba a., nistorescu m., pastravanu l., petruc i., simionescu i., strategic plan for integrated waste management in iasi county, (in romanian), online at: http://www.scribd.com/doc/13769639/planuljudetean-de-gestionare-al-deseurilor, (2008) [15]. iasi county council, long-term investment plan for the period 2008-2038 on integrated waste management, online at: http://www.icc.ro/activitate/mediu/masterp/master plan.pdf, (2009) [16]. ins iasi county statistics, county statistics, online at: http://www.iasi.insse.ro /main.php?id=373, (2011) [17]. rwmp ne, regional waste management plan, region 1 ne, ministry of environment and water management, bucharest, romania, (2006) [18]. nwmp, national waste management plan, ministry of environment and water management, bucharest, romania, (2004) [19]. ecofriends, waste containers, online at: http://www.ecofriends.ro/, (2013) [20]. den boer (szpadt) e., den boer j., jager j., rodrigo j., meneses m., castells f., schanne l., the use of life cycle assessment tool for the development of integrated waste management strategies for cities and regions with rapid growing economies lcaiwm, deliverable report on d3.1 and d3.2: environmental sustainability criteria and indicators for waste management (work package 3), technische universitaet darmstadt (tud), darmstadt, (2005) [21]. ghinea c., petraru m., bressers h., gavrilescu m., environmental evaluation of waste management scenarios – significance of the boundaries, journal of environmental engineering and landscape management, 20: 7685, (2012) [22]. schiopu a.m., ghinea c., municipal solid waste management and treatment of effluents resulting from their landfilling, environmental engineering and management journal, 12: 16991719, (2013) [23]. iso, environmental management – life cycle assessment – principles and framework (iso 14040: 2006). european standard en iso 14040. the international organization for standardization, geneva, switzerland, (2006) [24]. jolliet o., margni m., charles r., humbert s., payet j., rebitzer g., rosenbaum r., impact 2002+: a new life cycle impact assessment methodology, international journal of life cycle assessment, 8: 324-330, (2003) [25]. ghinea c., petraru m., simion i., sobariu d., bressers h.th.a, gavrilescu m., life cycle assessment of waste management and recycled paper systems, environmental engineering and management journal, 13: 2073-2085, (2014) [26]. goedkoop m., heijungs r., huijbregts m., de schryver a., struijs j., van zelm r., recipe 2008 a life cycle impact assessment method which comprises harmonised category indicators at the midpoint and the endpoint level, first edition, ministerie van vrom, den haag, (2013) [27]. van oers l., de koning a., guinee j.b., huppes g., abiotic resource depletion in lca, road and hydraulic engineering institute, netherlands, (2002) [28]. jrc european commission, framework and requirements for lcia models and indicators, joint research centre institute for environment and sustainability, ilcd handbook, (2010) [29]. ags, the complexity of climate change mechanisms aspects to be considered in abatement strategy planning, ags pathways report, göteborg, sweden, (2010) [30]. eea, waste opportunities past and future climate benefits from better municipal waste management in europe, european environmental agency report, copenhagen, issn 1725-9177, (2011) [31]. calbro p.s., greenhouse gases emission from municipal waste management: the role of separate collection, waste management, 29: 21782187, (2009) [32]. chen t.c., lin c.-f., greenhouse gases emissions from waste management practices using life cycle inventory model, journal of hazardous materials, 155: 23-31, (2008) http://www.scribd.com/doc/13769639/planulhttp://www.icc.ro/activitate/mediu/masterp/master http://www.iasi.insse.ro http://www.ecofriends.ro/, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 cristina ghinea, andreea spranceana, diana elena comanita, gabriela constantinescu, maria gavrilescu, assessment of environmental impacts from municipal solid waste management iasi, romania, food and environment safety, volume xiv, issue 2 – 2015, pag. 171 – 180 180 [33]. ghinea c., petraru m., gavrilescu m., bressers h., waste management and recovery of fibres – the impact on climate change, igs-sense conference resilient societies governing risk and vulnerability for water, energy and climate change, 19 21 octombrie 2011, universitatea twente, enschede, olanda, online at: http://www.utwente.nl/igs/conference/2011_resilien t_societies/papers%20and%20presentations%20cl imate%20change/postersession_paper%20gh inea_igs_conference.pdf, (2011) [34]. iswa, waste and climate change iswa white paper aka, international solid waste association, aka print, aarhus, denmark, (2009) [35]. us epa, solid waste management and greenhouse gases, a life-cycle assessment of emissions and sinks, 3rd edition, united states environmental protection agency, online at: http://www.epa.gov/climatechange/wycd/waste/do wnloads/fullreport.pdf, (2006) [36]. sundqvist j.o., life cycles assessments and solid waste–guidelines for solid waste treatment and disposal in lca, final report, swedish environmental protection agency, (1999). http://www.utwente.nl/igs/conference/2011_resilien http://www.epa.gov/climatechange/wycd/waste/do food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 84 the pec as an easy-to-prepare and cheaper alternative power source g.sh. hovsepyan1, a.n.kocharyan2, s.h.martirosyan2, g.o.torosyan2 1institute of general and inorganic chemistry of nas of ra, 10 arghutian street, 10 tel. 564029, gohar2007@mail.ru 2state engineering university of armenia, terian 105 abstract: porous cdse photoelectrochemical converter is fabricated and tested using highly porous substrate of metallic cd. following mean pec characteristics obtained: efficiency: 5%. on the other hand, cdse semiconductor developed on the highly porous (60%) cadmium substrate showed some 2% of efficiency with high dark current value only. it was shown that the light-sensitive phase is monoclinic 2 corresponding to cd0.53se0.47 formula determined by the x-ray, sem and elementary analysis tool methods. keywords: cdse pec, porous semiconductor electrode, semiconductor film 1. introduction coming from the worldwide harsh ecological situation, nowadays more and more attention induce alternative power sources, an example of which is the photoelectrochemical converter (pec). semiconductor solar converters are widely known power sources capable directly to convert light energy into electrical one. the unique properties of cadmiumchalcogenide compounds (cds; cdse; cdte and their mixed alloys), such as direct transition, optimal bandgap width (1.35-1.85 ev) etc., attracted immense interest for the use in pec. on the other hand, electrochemically fabricated semiconductor films are salient by their easeof-technology and low-cost virtues. besides, electrochemically fabricated semi-conductor layers can be produced with any given surface area and form, as they are polymorphic and usable in pecs. conventionally, cdse semiconductor films are obtained by the potentiostatic tool method, which has two inherent drawbacks: first, it is impossible to contain the layer thickness as the cadmium ions are co-deposited at diffusion limiting current density values; and secondly, the devised pec with cdse semiconductor exhibited poor reproducibility and below-par light conversion efficiency. it was possible to produce cdse semiconductor light-sensitive electrodes by the precipitation of selenium on the predeveloped porous cadmium carrier galvanostatically [1]. on the other hand, substitution of ti-metal or ni-metal used as conventional substrates with cd-metal improves both the cdse pec output characteristics (over 4.5% with practically no dark current) and the reproducibility as well, as it was shown in [2] . developed technology permits to obtain highly porous semiconductor electrode predominantly having open pores. it is known that porosity increases pec efficiency and, at the same time, permits to reduce pec cell power losses incurred within the cell (re the [2]). cd0.53se0.47 phase formation was confirmed by sem and elemental analysis methods. 2. experimental electrochemical workstation 600 brand potentiostat (ch instruments, usa) has mailto:gohar2007:@mail.ru food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 85 been used both for the development of cdse films and for cutting the main photoelectrochemical parameters. platinum wires served as reference and counter electrodes. collimated 100 w halogen lamp was the source of light at 65 mw/cm2 intensity. the used technique is described elsewhere, for example in [3]. experiments were carried out in the quartz cell. on fig 1 the typical volt-ampere characteristic is presented from which one can determine main pec characteristics: open circuit voltage, voc; short circuiting current density, isc; dark current, and fill factor, vm*im/voc*isc. figure 1. n-type semiconductor typical voltampere curve redox electrolyte has the following formulae: naoh : s : na2s = 1 : 1 : 1 in m. the calculated thickness of cdse coating was circa 0.5 m. redox electrolyte has the following formulae: naoh : s : na2s = 1 : 1 : 1 in m. rotating disc electrode (dia is 5 mm at 800 rpm) was used for non-porous cdse film obtainment. se bath formula is 4 mm seo2, ph = 2.2 t = 90 0c, t = 4 min, j= 1.2 ma/cm2. porous cdse were prepared according to the technology developed for the fabrication of pasted cd electrodes based on polyvinyl butyral (pvb) binder [4]. porosity of the electrodes was determined by the bet method and lies within 55–65%. further development of the se layer on the surface – and partially within the pores of the cd electrode – was performed as is described in our previous works [4,5]. scanning electron microscopy (sem) images were taken on vega tescan instrument. 3. results and discussion on figure 2 the typical volt-ampere characteristic is presented for the flat (nonporous) cdse pec obtained by the rotating disc electrode method. figure 2. rde output in polysulfide redox electrolyte please note that this figure curve is reversal of the fig. 1. for the rde case we have practically no dark current as one could easily see from the curve pattern depicted on fig. 2. on fig. 3 the same plot for the porous pec is provided. figure 3. porous cdse output in polysulfide redox electrolyte here, as opposed to rde experiments, we have increased dark current and lower output. actually, this is a result of poor se deposition inside the pores of the cd-metal voc vm o + im isc +i food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 86 substrate serving as active short-circuiting centers of the pec. this is hard-toovercome technological issue with regard to porous systems and our further investigations are focused on the use of forced convection to cover the inner pores of cd substrate with cdse layer. on figure 3 the electron-microscope images of semiconductor cdse porous film are presented. a b c figure 4. sem images of the semiconductor cdse porous film magnifications: a. 10220x, b. 3950x, c. 1000x. as fig. 3c shows, the produced porous layers are homogenous with even distribution of the binder (pvb) throughout the porous structure. according to fig. 3a, the diameter of the main pores is ca 2 µm. the results of an elementary analysis are presented in the table. table the results of semiconductor cdse electrode elementary analysis element weight% atomic% o 13.38 43.23 na 4.55 10.23 s 3.20 5.15 se 26.36 17.25 cd 52.51 24.14 as it follows from the table, the mass content of sulfur is 3.2% which inculcates into the electrode structure due to presence of free cd on the surface of the electrode to produce cds. this is a non-direct proof of the presence of metallic phase on the cdse porous structure which is the main culprit of deterioration of the characteristics’ of the porous pec. on the other hand, the mass ration of cd and se in the semiconductor is 21.2/19.2, respectively if we exclude se in-electrode structure invasion. this result corresponds to cd0.53se0.47 which is close to of lightsensitive monoclinic 2 phase composition. conclusion experiments show that it is possible to develop cdse light-sensitive phase electrochemically corresponding to monoclinic 2 phase on the highly-porous substrate. higher porosity not only increases the light response of the pec due to band bending of the cdse semiconductor, but the porous structure could lead to pec overall output power enhancement thanks to reduced voltage drop in the electrolyte. this is because, in this pec configuration, the counter electrode could be positioned directly behind the semiconductor electrode as it has open-ended pores with redox ions’ shorter run path in between the lightsensitive and counter electrode. references 1. martirosyan s., hovsepyan g., zourabyan p. method of binary chaleogenide semiconductor film fabrication. patent #139. industrial property #4, 2003. 2. martirosyan s. streamlining cdse/cds pec characteristics using cd metal collector.solar energy materials and solar cells. elsevier, 70, p. 115 (2001) 3. bouroushian m., loizos z., spyrellis n., maurin g. thin solid films. 101, (1993) 4. martirosyan s., hovsepyan g., kocharyan h. porous cdse films in pecs third international conference of renewable and pure energy. – yerevan 2008, june 27-29. 5. martirosyan s., kocharyan h. porous cdse electrodes fabrication international conference: enikolopov's readings.2006. p.98 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 105 near infrared spectroscopy – an alternative to determine the crude fiber content of forages monica hărmănescu1, alexandru moisuc1, iosif gergen2 1 banat’s university of agricultural sciences and veterinary medicine, faculty of agriculture, timisoara, calea aradului nr. 119, ro-300645, romania, monicaharmanescu@yahoo.com 2 banat’s university of agricultural sciences and veterinary medicine, faculty of food technology, timisoara, calea aradului nr. 119, ro-300645, romania. abstract: in our days nir spectroscopy represent a promising alternative to the chemical methods for crude fiber contents of forages. the main objective of this study was to obtain a nir calibration model for prediction this parameter of forages harvested in june 2009 from hill permanent grassland (grădinari, caraş-severin district). the experimental field was organized in ten experimental trials fertilized organic, mineral, and organo-mineral. the floristic composition of forages from this period was determined gravimetrically. from poaceae were present festuca rupicola and calamagrostis epigejos. fabaceae family was represented by trifolium repens and lathyrus pratensis. from other botanical family: rosa canina, filipendula vulgaris, galium verum and inula britanica. like input data for nir calibration were used the results for this qualitative parameter by chemical method and the reflectance values from 150 nir spectra for all analysed samples. partial last square (pls) regression was used to obtain the “nir total fiber” model, implemented in panorama program (version 3, labcognition, 2009). the statistical parameters (r2=0.80; rmsec=2.73) and the differences between references and predicted values situated in range 0.03 and 9.24% suggest a medium quality of calibration model, but it is promising to use it to predict the crude fiber contents of forages from grassland in this period of year using higher number of samples for calibration. key words: forages quality, complex fertilizers, plsnir model, grassland. introduction since romania becomes part of european union the national authorities must adjust the legislation to those european also regarding the quality and safety of the food. the 150/2004 low from our country transpose partially the ce 178/2002 rule, which establish the most important recommendations on the qualities of row matter, from vegetal and animal origin, destined to obtain the food [8]. the security and quality of food with animal’s origin must be discussed in direct correlation with the requests for forages quality used like animal’s feed. the forages from permanent grassland represent the cheaper source of feed for ruminants. fiber content of these forages, alongside of protein, lipids, minerals and vitamins, represents one of the most important parameter which characterize the forages quality [3]. ruminants have the capacity to digest and use crude fiber like source of nutrients [2]. for example the cellulose content of forages must be in their ratio between 2325% [1], representing the key in intestinal transit, in stimulation of satiety sensation [2], like energetic source in animal’s metabolism [6]. the chemically determination of this parameter request a high reagents consumption, qualified human resources to perform the operation, a long time to obtain the results [7]. an important food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 106 alternative for these chemical models is represented in our days by nir spectrometry, a non-destructive method which allows obtaining very fast the final results (appreciatively 3 minutes), without reagents consumption, medium pollution and samples destroying [4, 5]. experimental the forages were harvested in june 2009 from hill permanent grassland (grădinari, caraş-severin district). the soil of permanent grassland was calcic luvisol and the annual average temperature around 10.4oc. the experimental field was organized in ten fertilized trials in randomized plots, in multiple stage blocks with five replications. it was used for mineral fertilization: 15:15:15 npk complex, ammonium nitrate, potassium salt, superphosphat). like organic fertilizer was chose fermented sheep manure. the fermented sheep manure was applied at each two years, even the mineral fertilizers yearly. the fertilization process was made during the period 2003-2008. the ten trials were: v1-unfertilized trial, v2-20 t/ha sheep manure, v3-40 t/ha sheep manure, v4-60t/ha sheep manure, v5-20 t/ha sheep manure + 50p2o5(kg/ha), v6-20 t/ha sheep manure + 50p2o5 (kg/ha) + 50 k2o (kg/ha), v720 t/ha sheep manure + 50 p2o5 (kg/ha) + 50 k2o (kg/ha) + 50n(kg/ha), v8-100 n (kg/ha) + 50 p2o5 (kg/ha) + 50 k2o (kg/ha), v9-150 n (kg/ha) + 50p2o5(kg/ha) + 50 k2o (kg/ha), v10 (100+100)n (kg/ha) + 50 p2o5 (kg/ha) + 50k2o(kg/ha). the floristic composition of forages from the ten trials for this period of year was determined gravimetrically. from poaceae familly dominant was festuca rupicola (varied between 16.00 – 52.00%), followed by calamagrostis epigejos (5.00-13.00%). fabaceae family was represented mainly by trifolium repens (dominant) and lathyrus pratensis. from other botanical family were present rosa canina (7.0018.00%), filipendula vulgaris (3.009.00%), galium verum (3.00-7.00%) and inula britanica (5.00%). nir calibration model was obtained by pls (partial last square) regression, implemented in panorama software (variant 3, labcognition, 2009). like input data were selected the chemical data for crude fiber content, determined by jaoac 962.09/1990 [7] method (samples are sequentially refluxed in dilute base followed by dilute acid), and the reflectance values from 150 nir spectra. the v670 spectrophotometer by abblejasco was the instrument used to scan the spectra in the range 800-2500 nm, and than was selected with panorama software three spectral ranges favourable to perform the calibration model for crude fiber determination. these spectral ranges were specific for the overtones of fundamental frequencies of oh bound, characteristic for fiber compounds (table 1). table 1. calibration data for the „nir-cf” model with 3 spectral ranges no. selected spectral ranges number of wavelengths 1 [1282.5 .. 1433.5] 303 2 [1542.0 .. 1949.0] 815 3 [2263.0 .. 2356.0] 187 „nir-cf” nir-crude fiber model food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 107 for all the grounded dried samples the chemical results and nir spectra were obtained in triplicate. results and discussion statistical parameters for “nir-cf” model with the three selected spectral ranges are presented in table 2: table 2. statistical parameters for „nir-cf” model with three selected spectral ranges r2 0.80 rmsec 2.73 sd 4.75 these parameters suggest a medium quality of “nir-cf” model, but were better than the case when were used the entire spectral domain (r2 = 0.7355, rmsec = 3.28, sd = 4.39). the quality of nir calibration model is underlined also in the graphical presentation of prediction for crude fiber by „nir-cf” model with three spectral ranges (figure 1) and also by the differences between the chemical results and those predicted for control samples. the control samples were harvested in the same period of year and grassland and conditioned in the same manner with those used to perform the „nir-cf” calibration model (table 3). table 3. the results of crude fiber (%) prediction for the control samples forages (june2009) by „nircf” calibration model with 3 spectral ranges crude fiber (%) control sample’s name real (chemical method) predicted (nir model) differences between real predicted 101a 32.10 34.11 -2.01 101b 43.99 34.75 9.24 101c 18.85 24.24 -5.39 101d 31.06 26.92 4.14 102a 34.57 34.38 0.19 102b 24.89 27.08 -2.19 102c 25.46 23.27 2.19 102d 28.25 26.31 1.94 103a 36.45 36.35 0.10 103b 32.08 32.28 -0.20 103c 21.07 23.42 -2.35 103d 28.22 28.68 -0.46 104a 38.85 34.97 3.88 104b 32.98 30.26 2.72 104c 20.72 23.36 -2.64 104d 30.82 26.35 4.47 105a 35.17 34.70 0.47 105b 20.73 29.55 -8.82 105c 24.33 25.05 -0.72 105d 32.08 30.91 1.17 106a 33.01 33.91 -0.90 106b 22.60 27.64 -5.04 106c 24.74 23.06 1.68 106d 31.02 26.77 4.25 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 108 107a 36.41 36.30 0.11 107b 29.78 31.26 -1.48 107c 20.03 24.18 -4.15 107d 31.62 29.87 1.75 108a 36.44 37.69 -1.25 108c 22.42 24.18 -1.76 108d 29.55 30.21 -0.66 109a 33.75 38.03 -4.28 109b 28.22 30.83 -2.61 109c 24.37 24.34 0.03 109d 36.07 35.46 0.61 figure 1. prediction of crude fiber by the „nir-cf” model with 3 selected spectral ranges the differences between values obtained by chemical method and those predicted by „nir-cf” model were situated between 0.03 and 9.24%. almost 31.43% from these values were under 1.00%; 40.00% in range 1.01-3.00%; 17.14% between 3.015.00%; and 11.43% in range 5.01-9.24%. these results indicate a medium quality of performed calibration model, but encourage us to continue these researches using a high number of samples to characterize better the concentrations of this qualitative parameter of forages from the permanent grassland conclusion the pls regression model “nir-cf” with three selected spectral ranges for the determination of crude fiber of forages harvested in june 2009 had a medium quality. but this model promised to be used with success to determine routinely this parameter for the samples harvested in this period of year from the permanent grassland after the enrichment with a higher number of forages samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 109 acknowledgement the authors are grateful to cncsis uefiscsu (romania) for financial support (pd project / 28 sept.2010: on the applications of spectroscopic and chromatographic methods to establish the effects pf fertilisation on the quality of forages from grasslands). references 1. drinceanu, d., 1994, alimentatia animalelor, ed. eurobit, p: 16-17. 2. georgescu, gh., mărginean, gh., petcu, m., 2007, cartea producătorului şi procesatorului de lapte, vol. 2, editura ceres, bucureşti, isbn: 978-973-40-0773-8. 3. linskens h.f, jackson j.f., 1989, plant fibers. modern methods of plant analysis, vol. 10, springer-verlag berlin heidelberg, germania. 4. mcclure w.f., 1992, making light work: advances in near-infrared spectroscopy, eds. murray i., and cowe i.a., vch publishers, new york, p.4-7. 5. sugiyama j., mcclure w.f., hana m., 1992, in advances in near-infrared spectroscopy, eds. murray i., and cowe i.a., vch publishers, new york, p.61-66. 6. van soest, p. j., 1994, nutritional ecology of the ruminant, 2nd ed. cornell university press, ithaca, ny. 7. *** jaoac official methods of analysis, 1990, 962.09 – fiber (crude) in animal feed. ceramic fiber filter method, edited by herlich kenneth, 15 edition, publshed y association of official analytical chemists, arlington, virginia, sua. 8. www.immromania.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 – 2011 84 influence of some surfactants and flocculants on interparticle interactions in aqueous suspensions of hydro-mica *sergey boruk1, igor winkler1 , sonia gutt2 1 yuriy fedkovych national university of chernivtsi, kotsiybynsky st., 2, chernivtsi 58012, ukraine. e-mail: boruk_s@hotmail.com * corresponding author 2 stefan cel mare university of suceava, university street nr. 13, 720 229 suceava, romania received 3 january 2011, accepted 16 may 2011 abstract. an influence of composition of nonionic surfactant twin-80, and combined effect of the water-soluble surfactant polyethylene-oxide (peo) and flocculant polyacrylamide (paa) on interpartcile interaction in the water suspensions of hydro-mica with electrolytes have been investigated. ions of electrolyte do not cause any changes in a character of the surfactants’ influence while can result in significant changes in the concentration range for their effective action. these changes can be induced by adsorption of the electrolytes ions on the surface of the dispersed phase particles. following consequence can be formed from the three kinds of electrolytes according to their influence on the interparticle interaction: k+ mg2+ na+. a combination of the cation charge and radius is a key factor governing its activity. a character of the surfactants’ effect undergoes dramatic changes because of the micelle-forming processes occurring in presence of the electrolytes. combined application of peo and paa results in more intense interpartcile interaction in the system. results of this investigation can be applied to various technologies for separation of the dispersed phase from disperse media (treatment of some natural waters and wastewaters, refining and concentration of the mineral ores and other source materials, mixtures separation, etc.). © 2011 university publishing house of suceava. all rights reserved key words: surfactants; electrolytes; interparticle interactions; disperse system 1. introduction an intensity of interparticle interaction should be regulated within some intervals for many technological applications related to production and refining (concentration) of the highly concentrated suspensions. some organic modifiers can change a composition of the particles surface and influence a structure of the disperse system [1, 2]. such agents hold much promise for regulation of interparticle interactions. various ion-soluble substances can be found in many industrial suspensions, which results in changes in properties of surface of the dispersed phase particles and characteristics of the disperse media. therefore, details of influence of electrolytes on interparticle interaction in the disperse systems are of significant interest for investigation. 2. experimental our investigations dealt with aqueous suspensions of hydro-mica (mineral illite). this is a layered mineral with general composition к1-1,5al4[si6.5-7al1-4.5o20](оh)4•nh2o, which can be classified as a mica and has close to hexagonal crystals with continuous layers of the sio4 tetrahedrons [3]. granulometric composition of this mineral is represented in the fig. 1. selection of the modifiers has been realized in respect to the following parameters: surface activity towards dispersed phase particles; no chemical interaction between the modifier and surface of the particle; availability and applicability of the modifying agent. following substances were found [4] conforming to the above conditions: food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 – 2011 85 1. nonionic surfactant twin-80 made by the firm “tos” (russia). atomic weight of this substance is about 1100 atomic mass units (a.m.u.) and formula: where n+m=20. this substance can form micelle with critical constant of micelle forming (ccm) = 0.06 kg/m3. 2. polymer polyethyleneoxide made by the firm “carbolite” (russia). its atomic weight is about (2-3)•106 a.u.m. and chemical formula но-[-ch2-ch2-o-]n-н. 3. polymer polyacrylamide made by the firm “oriana” (ukraine). its atomic weight is about (4-6)•106 a.u.m. and formula: solutions of electrolytes kcl, nacl and mgcl2 with equal ionic strength have been used as disperse media. 3. results and discussion an einstein’s equation  = 0 exp(), (1) can be used to calculate viscosity of such systems. here  and 0 denote viscosity of the suspension and disperse media respectively;  is a parameter, which describes intensity of the interparticle interaction. a wide variety of particles with different sizes were present in the source mineral suspension (see fig. 1). there were also some coarse-dispersed particles, which can sedimentate in the aqueous media very fast. that is why all viscosity measurements have been carried out using a rotation viscosimeter “rheotest-2” working at a high shifting velocity. this mode enables to keep the coarsedispersed particles suspended. the parameter  in (1) depends on the dimensionless parameter , which can be calculated as a ratio between the shifting velocities  = d/dmax, where dmax is the maximal shifting velocity of the viscosimeter.  can be calculated as  =  ()-m. since  and m stay constant for each individual measurement, a value of  depends solely on . in such manner an intensity of the interparticle interaction can be evaluated through reaction of the system on the external mechanical disturbance [5, 6]. 0 5 10 15 20 0 20 40 60 80 d, mm % figure 1. granulometric composition of the hydro-mica suspensions an influence of electrolytes on the interparticle interaction is caused by adsorption of the electrolyte ions on the dispersed phase particles, which changes properties of the phase. the particles charge is a key factor governing bonding strength between functional groups of the surfactants and polymers with the dispersed phase particles. on other hand, changes in the particles electrokinetic potential induced by the electrolytes can affect a character and magnitude of effect of the organic substances on intensity of the interparticle interaction. following ‘aggregation activity’ sequence can be formed from the electrolytes under this study according to their effect on the n ch2 ch c o nh2 o oh ch2 cooc17h33 h(oc2h4)no o (c2h4o)mh food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 – 2011 86 interparticle interaction in the hydro-mica suspensions: kcl (m = 4,45) > mgcl2 (m = 4.0) > > nacl (m = 3,9). (2) figure 2. dependence of a value of parameter m in the hydro-mica suspensions on concentrations of surfactants and flocculants in aqueous solutions of nacl (a); kcl (b) and mgcl2 (c). ionic strength of all the solutions i = 1. admixtures: twin-80 (1); paa (2); twin-80 + paa (c=0.01 kg/m3) (3); twin-80 + paa (c=0.05 kg/m3) (4). figure 3. dependence of a value of parameter m in the hydro-mica suspensions on concentrations of the polymers in aqueous solutions of: nacl (a); kcl (b) and mgcl2 (c). ionic strength of all the solutions i = 1. admixtures: peo (1); peo + paa (c=0.01 kg/m3) (2); peo + paa (c=0.05 kg/m3) (3). higher efficiency of potassium ions is caused by their bigger radius, which results in a deeper perturbation in a structure of the surface adsorbed water layer. hydration ratio of the potassium ion is lower than that of sodium and magnesium and the surface adsorbed water layer disruption by potassium is comparatively significant, which promotes more intense interaction between the particles of hydro-mica. a value of m for the magnesium ions is smaller than value for the sodium ions and higher charge of the magnesium ions should be taken into consideration to account for stronger disruption of the boundary water level on the particles. this layer plays an important role in the structural forces of repulsion and its disruption promotes better aggregation of the dispersed phase particles. electrolytes do not change a principal character of influence of paa in the hydromica suspensions. a concentration range of the effective structures formation in the suspensions can slightly widen (see lines 2 in fig. 2). it is known [7] that the paa concentration range of the effective structures formation in the water suspensions is quite narrow. however, this range undergoes significant widening in the solutions of the electrolytes under investigation. for example, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 – 2011 87 concentration dependencies either flatten out (fig. 2a, line 2) or pass through a maximum (fig. 2b and 2c, line 2) near the concentration 0.08-0.1 kg/m3. a structures formation influence of peo undergoes intensification in presence of the electrolytes (see figs. 3, line 1). it can be assumed that a spatial conformation of the adsorbed macromolecules of peo can be changed by the electrolytes, which results in more intense interparticle interaction. the sequence (2) remains unchanged even in presence of peo. however, a maximum in the dependence of m on peo concentration in the system with ions of manganese is shifted towards higher concentrations of peo (figs. 3, line 1). this effect can be caused by formation of the coordination bond between an ion of manganese and oxygen from the molecule of peo. this bond weakens efficiency of the peo effect. a value of m decreases as concentration of peo raises that can be caused by better aggregation in the system and decreasing of amount of the structural elements in a volume unit of the disperse system. a character of the influence of the nonionic surfactant twin-80 undergoes dramatic changes in presence of the electrolytes. a characteristic minimum related to this surfactant has not been registered in a graph m versus concentration in presence of the electrolytes (see figs. 2, line 1). this can be caused by formation of the surfactant micelles under the effect of the electrolytes and decrease in the disperse efficiency of twin-80, which does not provide any effect on the interparticle interaction in hydro-mica suspensions containing the electrolytes. the sequence (2) also remains unchanged in presence of this surfactant. an intensity of the interparticle interaction and values of m rise as a result of combined application of peo and paa to the electrolyte-containing systems. this process also manifests itself through a shift of a peak in the diagram m vs concentration of peo (figs 3, lines 2-3). this peak shifts towards higher concentrations in presence of potassium and sodium ions and in the opposite direction in presence of magnesium ions. combined application of twin-80 and paa to the electrolyte-containing systems does not cause any significant changes in the relation m – surface active agents concentration, which remains almost similar to the relation for the solitary application of paa (figs 2, lines 34). however, values of m for the combined application are slightly higher than for the solitary ones because of more intense interparticle interaction in the first case. 4. conclusion the electrolytes do not result any significant changes in a character and intensity of the highmolecular surfactants and flocculants effect on the interparticle interaction in the hydro-mica suspensions. on other hand, significant changes in activity of the low-molecular surfactants have been registered after adding an electrolyte to the disperse system. this effect is caused by changes in the ccm value because of the electrolytes action. radius and charge of the electrolyte ion govern an intensity of its effect. three electrolyte ions can be placed in the following sequence according to the decreasing activity: potassium-magnesium-sodium. 5. references 1. churaev n. v., sobolev v. d. contribution of the structural forces in the processes of quartz humectation by electrolytes solutions. (2000). coll. j., 62. 278-285. 2. goncharuk e. v. et al. role of the structuremechanical barrier in the mixed aqueous dispersions of hydrophilic and hydrophobic silica. (2001). ukr. j. chem., 67, 36-39. 3. tarasevich yu. i. composition and surface chemical properties of the layered silicates. (1988). naukova dumka. kiev. 248 p 4. surfactants. ed.: abramson a. a. (1979). chemistry. leningrad. 302 p 5. slipeniuk t. s. et. al. mutual flocculation in suspensions. (1996). ukr. j. chem., 62, 97-100. 6. gamera a. v. et. al. interaction between various sized particles in the hydro-suspensions of an artificial diamond. (1999). superhard mater., 2, 59-62. 7. boruk s., winkler i. influence of some nonionic surfactants and flocculants on stability of diluted dispersions of kaolin and hydro–mica. (2010). proceedings of the international conference biotechnologies, present and perspectives. suceava. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 30 quality evaluation of services provided by the restaurant “president” from mangalia by analyzing customer’s satisfaction regarding the waiter’s taking order *mihaela constandache1, elena condrea2, anghelina dudan3 1„dimitrie cantemir” christian university, faculty of touristic and commercial management constanta, 90a dezrobirii street, 900234 constanta, romania, e-mail micky_eve68@yahoo.com * corresponding author 2„ovidius” university, faculty of economics, 1 university alley, constanta, romania 3„carol i” commercial college, 15 decebal street, 900665 constanta, romania received 2 december 2010, accepted 6 april 2011 abstract: service features assessment of their quality has certain characteristics. the quality of services and hence the satisfaction degree of consumers’ needs are determined by the qualifications, competence, discipline of personnel serving, but also by the organizing capacity of the sector. for consumers the serving time is a quality aspect of live, an element that indicates the satisfaction degree of consumers’ needs. to assess the satisfaction degree of president restaurant customers regarding the takeover order by waiters we used a specific quality management tool that allows comparison of trial results with the requirements set out above and identification of the extent to which the process has good results and to which point there are deviations to the limits set in requirements. after the selection of analyzed process and variables respectively, the time (in minutes) needed to take order we proceeded to collect relevant data during the period 1 to 2 may 2010, when 54 observations were made. the scheduled requirement was that making order should be performed within no more than 10 minutes from a list-menu presentation. the results showed a relatively large number of observations that have not met the target. measures are necessary to reorganize the serving activity in order to reduce the waiter’s taking order time so that they fall within specified limits. © 2011 university publishing house of suceava. all rights reserved keywords: taking order time, customer’s satisfaction, histogram, services quality 1. introduction catering and tourism businesses release a specific product, especially that of industrial and retail trade specific food and non-food sector, a more complex goods and services. the quantity, quality and various combinations of good and services ensures a certain product specificity and within it each unit of product [1]. thus, for a catering unit, a product may include a first level of analysis, providing food services. at the second level of product analysis, all foods and food goods of a full meal and dining services are required by the process according to the type of establishment (restaurant, bar, cafeteria, fast food, etc.) and the category that is assigned to the unit requested by consumer [2]. the management of public catering unit is to ensure proper aggregation of components produced, so that the unit provides a product to be distinguished from other types of units (keeping the unit production of those specific foods, maintaining the quality of preparation and serving drinks, etc.); so after a certain period the product’s image in top management is confused with the image of unity that offers it. for this purpose the unit management uses a complex set of continuous control means of the activities for each group of the unit, coordinates food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 31 sections activity (components of the retail sector – reception, bar, buffet, kitchen, serving staff) in order to ensure prompt serving and consistently higher quality level. the level of service and hence the degree of satisfaction of consumer needs are determined by qualifications, competence, discipline of the personnel serving, but also by the organization quality of the sector. establishing an optimal degree of work tasks to be entirely achieved during work, in terms of ensuring a prompt and good quality serving, is one criterion by which the management in determining the staff’s needs, size of work teams, work schedule and duties for each worker should be carried out . given the large consumers’ fluctuations in public catering establishments in different moments of the day, the waiters serving are faced with a number of problems in terms of rational organization, scientific and efficient work. this requires the use of work teams organized on variations of graphs; for example, hourly gradual chart (workers come to work slowly, so this working band is minimum in low activity periods and maximum in those of peak), totalizing monthly schedules (allow differentiation of daily working hours in condition to achieve the legal number of working hours during the month), double brigade charts (homogeneous brigades alternating on different days) and incomplete working day schedules (for workers employed at peak times). services features’ assessment of their quality has certain characteristics. firstly, production is simultaneous with the delivery service. this prevents the achievement of service quality control before it reaches the customer to have the opportunity to reject those features that do not meet the standards required. if these do not meet the standards, the customer receives a defective service, and usually is not possible to rectify it. therefore, quality must be assessed continuously throughout the process, including design phase. this principle is already applied in industrial production because it is less expensive to avoid manufacturing defects through control process than to reject products already completed [3]. secondly, in services’ sector the quality control subject is not the physical dimensions but another type of features such as service integrality, time, lead time. on the other hand, a consequence of defining quality in terms of meeting the customers’ needs and expectations is placing the customer satisfaction assessment in a central point to assess the real quality (achieved or actual quality). it is not only to verify compliance with quality standards, but also to understand to which point the customer feels satisfied with the product or service received. this, moreover important in any business, is especially relevant in services because they involve many subjective factors related to customers’ satisfaction. the criteria that should be considered in service assessment, are the criteria established by customers. therefore an organization interested in quality will able to: o identify its customers’ needs and expectations; o assess the perception they have about the service and about its various components; o identify their errors and causes; o act to improve quality. knowing the customers’ expectations is essential, both for quality planning and for quality assessment. in fact, there is satisfaction when the service perception will be at least equal to expectations. the success of this correlation even if the perception would exceed expectations is essential for the quality of service acquisition. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 32 a service as a whole can not be considered favorably in terms of a quantitative component failure. at the same time, the quantitative component itself can not only leave the customer a neutral impression. for example, favorable comments about a clean restaurant or quick service are rare while a slow service, a spotted tablecloth or an unrelieved table often leads to customers’ complaints. 2. materials and methods the staff – customer relationship, staff behavior, their attitude, how to act and react is the key criteria for assessing the quality of services as a whole, elements that generate customer satisfaction. serving time is the time elapsed between the time of the service requested by consumer dining and the beginning of consumption "[2]. serving time has multiple meanings, both for enterprise and consumer. for enterprise its reduction is an important way to increase labor productivity of serving staff (during a period when more customers are being served), so a means by which the company carries out its planned sales volume. also, the company gets extra security in achieving its planed objectives because by completing a short time serving, a permanent consumer demand for its products and services is registered. for consumers the serving time is a qualitative aspect of life, an element that indicates the satisfaction degree of their needs. therefore, there are sufficient arguments for the enterprise to propose time serving as research object and to find the optimal solution since unlimited higher of serving staff is not an optimal solution because it generates pay expenses and uneconomic service facilities. careful studies should be made in order to optimize the serving time and the sales sector management determine the adequate form of sales (classical, self serving), reduce the optimal need of sales staff, prepare work schedule of band staff. to assess the degree of president restaurant customers’ satisfaction regarding the waiters’ taking order time a specifically quality management tool, a histogram, was used. this tool allowed the comparison of trial results with the requirements outlined above and identification of the extent to which the process produces good results and to what point there are deviations to the limits set in the requirements. for a frequency histogram development the following stages were taken: a) data collecting and recording: to ensure the sample authenticity, its size is calculated. for this statistical population effectiveness should be known. between 1 and 2 may 2010 at president hotel were accommodated 56 tourists who had lunch at the restaurant. our sample size is calculated for a non exhaustive survey: ne=t2 x p x (1-p) / δω2 ne – the sample size for a non exhaustive survey; t coefficient corresponding to the probability guarantee of outcome; p – the sample proportion that has the feature (p=0.5); δω acceptable error limit [4]. considering the probability of guaranteeing the outcome of p = 95%, t = 1.96, p = 0.5 the proportions of the sample components and the error limits on δω = + / 3%, we calculate ne = 1067 persons. then we calculate the final size of the sample corresponding to exhaustive survey: nf = n x ne / (n + ne), were n – effectiveness of statistical population investigated. nf = 56 x 1067 / (56 + 1067) = 59752/1123 = 53.2 ~ 54 persons. after selection of the analyzed process variables, respectively time (in minutes) needed to take command, we proceeded to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 33 collect relevant data corresponding to the staying period, 1 to 2 may 2010, when 54 observations were made (table 1). table 1. time (minutes) required by the waiter taking order 4 6 11 13 5 2 8 13 10 20 6 9 18 17 4 9 3 7 23 5 10 2 7 5 15 8 5 11 4 9 2 5 12 29 18 15 4 16 24 22 3 19 6 12 25 13 7 8 11 15 7 3 26 2 the scheduled requirement was that order making should not take more than 10 minutes from a list-menu presentation. b) determining the rank of data sets: the achievement gap between the maximum and minimum data sets. c) setting the number of intervals and their amplitude: the number of intervals (k) is determined according to the number of available data. 10 intervals were used. to calculate the magnitude intervals (h) the rank was divided by the number of selected intervals. d) determining the interval limits: this allows final grouping of data. we took into account that the extreme values within each interval can create confusion about which they belong to. therefore it is necessary to clarify the limits very well. we calculated the starting point of the first interval by applying the formula: starting point = minimum value – ½ starting from here, we summed amplitude value range (h) to obtain the next interval limits. e) table of frequencies: records the intervals limit values by calculating the elements belonging to each class, noting them down in the "verification column" and accounting the total observations for each interval in the frequency column. f) draw the histogram by drawing vertical bars starting at each interval. 3. results and discussion among president restaurant customers during 1 to 2 may 2010, 54 observations were made (table 1). the data sets rank was then calculated by making the difference between the maximum and minimum values recorded: maximum value: l = 29; minimum value: s = 2; rank (l – s) = 27. the number of intervals (k) was determined according to the number of available data. 10 intervals were used. to calculate the intervals magnitude (h) the rank was divided by the selected number of intervals rounding the result of the greater whole. (h) = (l – s)/k = 27/10 =2.7 = 3 the starting point of the first period is calculated by: starting point = minimum value – ½ = 2 – ½ = 1.5 table 2. the frequency table no. intervals check frequency 1 2 5 ||||||||||| 11 2 5 8 |||||||||||| 12 3 8 11 |||||||| 8 4 11 14 |||||||| 8 5 14 17 |||| 4 6 17 20 |||| 4 7 20 23 || 2 8 23 26 ||| 3 9 26 29 | 1 10 29 32 | 1 n = 54 then we draw the frequency table (table 2) recording the intervals of limit values, calculating the elements belonging to each class and accounting the total observations for each interval in the frequency column. the histogram of quality process analyzed is shown in figure 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue 2 2011 34 the requirement was set that the waiter’s taking order should be performed in less than 10 minutes after the customer’s arrival in. figure 1. histogram of the process quality by analyzing the histogram, one can see that a large number of observations to the right of the additional line (which marks the limit requirement), did not meet this goal. therefore, the efforts should be geared to move the histogram to the left so that data from the extreme to be within the specified limits. one of the reasons leading to these results might be that, while the questionnaires were applied to, even at the beginning of the summer season (1-2 may 2010) the unit has a staff shortage which means that the waiting time has not complied with the customer’s expectations. nor the waiter’s professionalism was at high level since the staff was supplemented by workers without special experience. 4. conclusions establishing an optimal degree of work tasks to be entirely achieved during work, in terms of ensuring a prompt and good quality serving, is one criterion by which the management in determining the staff’s needs, size of work teams, work schedule and duties for each worker should be carried out . optimizing human resources management in the president restaurant will depend on the success of the following activities: • approaching human resources policy at the company level in an integrated system with other aspects of society management activity, the basic condition for ensuring positive results; • flexibility of organizational structure and adapting to current stage of society development; • attracting necessary qualified staff suitably to the requirements of providing services; • creating conditions for maximum use of training, experienced and skilled staff; • professional training to maintain high service standards; • providing cooperation atmosphere based on interpersonal and interprofessional relationships that stimulate employees to produce positive results at the firm level; • motivate employees to better cover the staff needs and attract those with best experience. 5. references 1. micu, a., commerce economy, didactic and pedagogical publisher, bucharest, 2005; 2. alexandrescu, m., public food units management, lecture notes, 2006; 3. simionescu, g., product and services quality study, niculescu abc publisher, bucharest, 2005; 4. catoiu, i., balan, c., onete, b., popescu, i.c., veghes, c., methods and technics used in marketing surveys – aplications, uranus publisher, bucharest, 1999 quality assesement 0 2 4 6 8 10 12 14 2 5 8 11 14 17 20 23 26 29 intervals f r e q u e n c y food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 44 impact of temperature and storage time on sucrose content in sugar beet *octavian barna1, octavian baston1, aura darabă1 1biochemistry department, faculty of food science and engineering, galati, „dunărea de jos ” university, 111 domneasca st., 800201 galati, e-mail octavian.barna@yahoo.com *corresponding author received 27 january 2011, accepted 2 may 2011 abstract: during storage the chemical composition of sugar beets changes as harvested sugar is lost due to respiration by the living beet root. the efficiency of sugar manufacturing depends largely on the quality of the raw beet material. good processing quality is characterized by a combination of high sucrose concentration and low concentration of non sucrose substances that impair white sugar recovery. long –term storage of sugar beet is a solution to extend the processing period of sugar factories. the sugar companies consider prolonging the processing campaign in order to reduce the fixed costs of their factories. in future, the storage period will thus be extended, too, and the amount of stored beets will increase. the aim of this study is to determine the temperature and storage time influence on the variation of sucrose content of new sugar beet hybrids adapted to the new climate conditions of europe. sugar beet has been stored at the temperatures of 2, 6, 10, 15 and 20 oc and analyses of the sucrose content after 5, 10, 20, 30, 40, 50 and 60 day-storage periods have been made. all along storage the sucrose content decreased in inverse ratio to the storage temperature and direct ratio to storage period having specific variations for all cultivars analyzed. © 2011 university publishing house of suceava. all rights reserved keywords: sugar beet, storage, temperature, sucrose content. 1. introduction facing the changes in the eu sugar regime which will fall the price for sugar, the sugar industry has to reduce the cost of production by all possible means. prolonging the processing and storage period is an option to reduce fixed costs of sugar factories [1]. in doing so, it is crucial to optimize storage conditions. even if sugar beet is harvested in autumn, by storage its processing period extension is ensured in sugar factories and in winter as well until the end of december. the quality of sugar beet which is to be processed after having been stored is extremely important, its parameters influencing directly the sugar productivity of the factory. the main change in the chemical composition of sugar beet is the decrease in its sucrose mass. the respiration process usually accounts for 50 to 80 % of the total sucrose loss during storage [2]. the remainder is the result of the sucrose conversion into invert sugar, raffinose and other organic compounds. additional sugar losses occur via microbiological activity in cuts and especially in bruises [3].sucrose catabolism in sugar beet is mainly due to the activities of acid and alkaline invertase and sucrose synthetase. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 45 these enzymes reduce the yield of extractable sucrose from the sugar beet crop directly by their ability to degrade sucrose and indirectly by producing invert sugars, which increase the loss of sugar during storage [4]. it is known that the most important effect on the change of sugar beet quality in the period after harvest is exerted by temperature [5]. the new climate conditions of europe have imposed the cultivation of some new hybrids of sugar beet adapted to these ones. knowing the behavior of these hybrids during storage and how high quality of sugar beet can be obtained after its storage is very useful. the behavior of sugar beet has been studied for 60 days as this is usually the maximum storage period of sugar beet after harvest until processing. 2. materials and methods the beet subject to analysis is of cultivars victor z type, solea n/z type and markus z type, cultivated in the county of brasov. it has been stored together with its adherent impurities at the temperatures of 2, 6, 10, 15 and 20 oc and analyses of the sucrose content after 5, 10, 20, 30, 40, 50 and 60 daystorage periods have been made. determination of sucrose was made using polarimetric method icumsa method gs6-1 [6]. the information obtained was statistically processed using software excel in microsoft office 2003 suite in order to find values of correlation coefficients (r) between storage time and temperature and variations of sucrose content of the sugar beet analyzed. the bi-factorial analysis anova has been used to determine whether the temperature variation and storage period influence significantly the experiment in view. 3. results and discussion during storage, considerable changes in beet quality occurred. in the first days of storage, a significant decrease in sugar content is noticed as a result of a more intense respiration due to sugar beet root’s adaptation to the new life conditions in all types analyzed proportionally with the storage temperature. the variation of sucrose content depending on storage period and temperature for victor type z can be seen in the graph of figure 1. figure 1. sugar content as a function of temperature and duration storage for victor z type sugar beet following the graph in figure 1 one can notice that there is a stabilizing tendency of sucrose content for the end of storage interval when using the temperature levels of 10, 15 and 20 oc. this phenomenon might be explained by slowing down the biochemical processes subject to analysis and reaching a relative equilibrium state in the metabolism of the sugar beet root after 50-day storage at temperatures of 15 and 20 c. figure 2 shows the variation of sucrose content of solea type n/z. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 46 figure 2. sugar content as a function of temperature and duration storage for solea n/z type sugar beet in the case of solea type n/z there is a clear separation on temperature groups of variation curves of sucrose content. the first group consists of the curves corresponding to the temperatures of 2, 6 and 10 oc, being characterized by a smaller slope whereas the second corresponding to the temperatures of 15 and 20 oc has a more pronounced slope. the curves’ grouping shows that the metabolism change of solea type n/z has the critical temperature limit close to the value of 10 oc. the variation slope of curves is relatively constant throughout the storage period for all temperature levels analyzed. figure 3. sugar content as a function of temperature and duration storage for markus z type sugar beet in the case of markus type z there is some emphasis of the slope of variation curve after a 40-day storage period, especially in the case of storing at the temperatures of 15 and 20 oc. this fact implies a favorable storage period for this hybrid with a shorter period of 40 days in order to avoid fast depreciation of sugar beet at these higher temperatures. the graph in figure 4 shows the data resulted from the calculus of average losses in sucrose figure 4. daily sugar losses as a function of beet temperature during 60 days storage by analyzing the graph of figure 4 one can see that average sucrose losses of the hybrids analyzed vary on storage temperature. thus, in the interval 2-10 oc the biggest sucrose losses are registered by the hybrid markus, followed by solea and viktor. at the temperature of 15oc solea and viktor have the same average of daily sucrose losses per tone, being exceeded by viktor. at the superior limit of the temperatures tested, that is 20 oc, markus registered the biggest losses of sucrose, followed by viktor and solea in the last place. for all three hybrids analyzed the bifactorial statistical calculus anova led to the values of f exceeded fcr and to the conclusion that temperature variation and storage period influence significantly with a probability of 95% the experiment made. the statistical analysis made shows very strong food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 47 correlations of the same sense between storage temperature variation and sucrose losses registered. the values of correlation coefficients between sucrose losses determined and storage temperature are given in table 1. table 1. correlation coefficients between sucrose losses and variation of storage temperature nr. crt. hybrid r 1 viktor 0.995504 2 solea 0.996183 3 markus 0.995627 the highest value of correlation coefficient is registered by the hybrid solea, followed by markus, viktor being the last one. 4. conclusions storage duration and temperature have significant effects on the changes in sugar content of sugar beet. at low temperature, changes in beet quality could be kept at minimum. we noticed that for all cultivars analyzed the storage at 2-10 oc led to relatively reduced losses of sucrose throughout the storage period in view. besides temperature the storage period has also a significant influence on sucrose losses of sugar beet. as a result of graphs’ analysis, a decrease of sucrose content is not uniform throughout storage. thus, in the first 5 days of storage the decrease is higher due to respiration phenomenon and adaptation of sugar beet to new life conditions. then a period when sucrose losses are relatively constant depending on storage temperature follows. great differences occur at the storage temperatures of 15 and 20 oc, especially towards the end of storage period. thus the cultivar viktor has a tendency of relative decrease of daily sucrose losses after 50-day storage period whereas the cultivar markus shows a tendency of increasing them after 40-day storage period. the different behavior of sugar beet cultivars at storage might be the consequence of different enzyme activity. further research is necessary to examine whether genetic variability in the activity of sucrolytic enzymes of sugar beet exists. this could be the basis for the selection of cultivars with better storability 5. references 1.kenter, c., hoffman, c., changes in the processing quality of sugar beet (beta vulgaris l.) during long –term storage under controlled conditions, international journal of food science and technology, 44, 910-917, (2009). 2. wyse, r.e., dexter, s.t., source of recoverable sugar losses in several sugar beet varietes during storage, journal of the american society of sugar beet technologists, 16, 390398, (1971). 3. imura, e.,nakayama.,hayasaka, m.,saito, h.,kanzawa, k.,michiba, m., relation between mechanical damage and storability in sugar beets. influence of harvesting equipments on mechanical damage and storability .proceedings of the sugar beet research association , 29 , 154-160. (1987). 4. joshi, s.s., pawar, m.m., datir,s.s., more, d.b., physiological studies and sucrose metabolism during root development in three sugar beet cultivars, sugar technology, 7(4):150-153, (2005). 5.van der poel, p.w., schiweck, h., schwartz, t., sugar technology. beet and cane sugar manufacture. berlin: dr. albert bartens kg, (1998). 6. icumsa methods book, international commission for uniform methods of sugar analysis, (2007). microsoft word 2 cuprins nr 4 revista fia.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 contents: 1. yarema tevtul, t. filipchuk, sonia gutt, silviu stroe electrochemical modification of graphite surface 5 2. ioan gogoasa, gabriela oprea, monica harmanescu, teodor ioan trasca, adrian rivis, iosif gergen some metals (fe, mn, zn, cu, pb, cd) contents in vegetables from a nonpolluted plain area of cenad-banat (romania) 16 3. mihaela begea, elena bălăuţă, alexandrina sirbu assessment of milk allergens into foodstuffs 25 4. cristian logofatu, daniela cioboata, mariana ionescu, gabriel mustatea, amelia buculei computerized system for closure control of packages in food industry 31 5. monica murarescu, dumitru dima, gabriel andrei, adrian circiumaru method of carbon nanotubes dispersion in polymeric matrix 36 6. mariana lupoae, bianca furdui, rodica dinică, dragomir coprean quantification of carotenoids and chlorophyll leaf pigments from autochthones dietary 42 7. oana – viorela nistor, mihai ţâmpău, elisabeta botez the influence of cold conventional storage on fruits quality 48 8. maria cioroi, lucian tudor miron, gabriela rapeanu, nicoleta stanciuc, elena postolache, constanta vicol study on free radical scavenging and total polyphenols of some romanian wines 55 9. bianca furdui, rodica-mihaela dinică, mariana lupoae, romică creţu effect of some natural herbs incorporation in sunflower oils on its resistance at frying temperatures 61 10. catalina iticescu, monica murarescu comparative studies of composite coatings with ceo2 and nd2o3 in copper matrix by electroless plating 67 11. mioara negoiţă, monica catană, luminiţa catană, enuţa iorga, gabriela lilios, alina bâlea, valentin ionescu iron fortification of bread and baked goods as against nutritional deficiencies of population 74 12. octavian baston, octavian barna biogenic amines content of romanian market mackerel 80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 13. rodica dinică, mariana lupoae, bianca furdui, dragomir coprean quantitative determination of polyphenol compounds from raw extracts of allium, alliara and urtica genus. a comparative study 85 14. monica catană, luminiţa catană, mioara negoiţă, enuţa iorga, alina bâlea, gabriela lilios validation of an enzymatic method for nitrates determination from vegetable products 90 15. mirela calu, petru alexe electronic nose fast method for apples discrimination to determine optimum harvest moment 97 16. monica dinu, gabriela constantinescu (pop) dietary fibres role in manugacturing bread with low content of glucides 103 17. aurora bunea, rodica dinică, bianca furdui, mariana lupoae studies on plant biomass in various stages of degradation in various stages oradation studies on plant biomass in various stages of 108 18. elena alexandra oniciuc, liliana gîtin, sorin ciortan, nicoleta maftei (aron), anca nicolau intense light pulses effect on fungal burden of mustard and black pepper 112 19. nijolė kazlauskienė, milda zita vosylienė, renata karpovič, edvinas taujanskis sublethal effects of nickel to rainbow trout oncorhynchus mykiss biological parameters 118 20. cristina danciu, ioan danciu, grinding process of the wheat kernel with a new designed micromill 125 21. g.h.torosyan, aidan ghazi, d.n.hovhannisyan lingocellulosics convenient sorbents for waste water treatment from phenol and fufural 131 22. author instructions 139 microsoft word 0b cuprins 6_09_2017 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2– 2017 contents: 1 quality evaluation of wheat-pumpkin-golden flaxseed composite bread georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosi-sănduleac 61–70 2 investigation on lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant olena grek, olena krasulya, alla petryna 71 76 3 3-dimensional simulation and feasibility study of biomass/coal co-combustion burner nataliya dunayevska, yaroslav zasiadko, pavlo zasiadko, taras schudlo 77 91 4 the influence of slow thawing on evolution of some biochemical compounds in frozen fishes marcel avramiuc 92 –97 5 honeydew honey adulteration: e-tongue and physicochemical analyses sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei 98 103 6 studyon the acceptability of yoghurt with carrot juice ana leahu, cristina e. hretcanu 104 110 7 low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk 111 116 8 effect of plant protein isolates on the structural – mechanical properties of wheat dough valeriy makhynko, vira drobot, tatjana golikova 117 122 author instructions i v subscription information vi microsoft word 2 cuprins nr 3 revista 28 oct final.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no3 2010 contents: 1. igor winkler, antonia andrusyak, lifetime of freezing activated water 5 2. alina-petronela haller, romanian trade liberalization of food products in terms of export benefits in the knowledge society 10 3. marian petre, alexandru teodorescu, biotechnology of edible mushrooms cultivation on vine and winery wastes 17 4. diana curchi, rodica sturza, oxidative stability of iron enriched oil-in-water emulsions 22 5. monica catană, luminiţa catană, mioara negoiţă, enuţa iorga, alina bâlea, gabriela lilios, determination of dioxins and furans from eggs and oils, through high resolution gas chromatography in combination with high resolution mass spectrometry 27 6. loredana dumitraşcu, alina ardelean and nicoleta stănciuc, the influence of processing and medium composition on the thiol availability of whey protein concentrate 34 7. iosif gergen, despina maria bordean, monica harmanescu, florina radu, marilena miclau, selectivity of calix [6]arene-coated piezoelectric quartz crystal sensor for the detection of some organic amine in gas flow 40 8. gabriel – dănuţ mocanu, oana – viorela nistor, elisabeta botez, doina – georgeta andronoiu, study on the parameters’ variation in fruits drying process 44 9. violeta vasilache, the present state of researches regarding electroplating problems (review) 50 10. andrei sturza, olga deseatnicova, optimization of the extraction of polyphenols from grape seeds 59 11. dumitru dima, monica murarescu, gabriel andrei, simona chiculita, an obtaining method for carbon nanotubes with modified surface 66 12. iuliana mihaela lazar, arcadie sobetkii, ileana denisa nistor, neculai doru miron, marius stamate, gabriel octavian lazar, dielectrophoretic devices for separating foodborne pathogens 72 13. luminiţa catană, monica catană, mioara negoiţă, enuţa iorga, gabriela lilios, fruit-based concentrated products, iron fortified, intended for prevention and diet therapy of iron deficiencies of vulnerable population groups 78 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no3 2010 14. oana viorela nistor, elena popa, elisabeta botez, oana emilia constantin, researches on the evolution of concentrated fruit juices quality at storage 84 15. mihaela mardare (chirilă), gheorghe zgherea, gabriel murariu, constantin mateescu, iosif gergen, gabriela constantinescu (pop), effect of tq processing on cell wall in relation to firmness of carrot tissue – part i. the tissue model 90 16. cristina popovici, tatiana capcanari, olga zagarschih, olga deseatnicova, rodica sturza antioxidant activity of plant extracts containing polyphenol compounds 97 17. monica hărmănescu, alexandru moisuc, iosif gergen near infrared spectroscopy – an alternative to determine the crude fiber content of forages 105 18. gintaras svecevičius1, nijolė kazlauskienė1 use of fish behavioral responses in identifying sublethal exposure to pollutants 110 19. author instructions 114 microsoft word 3 journal fia nr 1 2010 final_15-20.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 19 interparticle interaction in the diluted aqueous suspensions of hydro-mica sergey boruk1, igor winkler1 and sonia gutt2 1yuriy fedkovich chernivtsi national university, kotsiubinsky st., 2, chernivtsi, 5 8102, ukraine. e-mail: boruk_s@hotmail.com 2ştefan cel mare university, universităţii st., 13, suceava, 720229, romania abstract: an influence of some compositions containing water soluble polymers on the interparticle interaction in the aqueous suspensions of hydro-mica has been investigated. surface modification of the particles preceding adding a flocculation agent to the system causes much higher intensity of the interparticle interaction in the suspension, which results in the particle aggregation and decreasing in the sedimentation stability of the systems. results of this investigation can be used in some cleaning technologies to remove highly dispersed pollutants from some natural water and wastewater, raw mineral materials refining and separation process. keywords: water soluble polymer, flocculation agent, sedimentation stability introduction highly dispersed clay-like particles often should be removed in course of the mineral raw materials refining or in the wastewater treatment technologies. some organic substances can promote better aggregation of the particles, lower sedimentation stability of the system and, therefore, increased efficiency of the clay particles removing. mutual aggregation of small and coarse particles ensures the most effective removing [1, 2]. a smaller particle can get into the sphere of molecular attraction of a coarse one and then the both particles form a contacted associate. stability of the associate depends on the mutual effect of the molecular, electrostatic and structuring forces. various organic additives can significantly influence interparticle interactions in the disperse systems. for instance, sedimentation stability of the systems can be regulated through changes in concentration and type of the admixtures. experimental this investigation deals with hydro-mica – a clay-like material with general formula к1-1,5al4[si6.5-7al14.5o20](оh)4*nh2o. this mineral can be classified as mica with a layered structure and close to hexagonal lattice [3]. granulometric composition of the material has been determined using a laser granulometer “granulometer-750” (see fig. 1). journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 20 such requirements as availability, inertness in the surface interaction with hydro-mica and lower toxicity were taken into account in selection of the organic additives. easy practical applicability of the additives has also been considered. hence, following two substances have been selected basing on the data from [4]: 1. polyethyleneoxide (peo) made by novosibirsk branch of the firm “karbolit” (russia). formula of peo can be shown as но-[-ch2ch2-o-]n-н and it approximate molecular weight is about (23).106 a.m.u. 2. polyacrylamide (paa) made by the firm “oriana” (kalush, ukarine). approximate molecular weight is about (5-6).106 a.m.u. and formula is an effective coefficient of adhering (cef) was calculated as a tangent of slope of the dependence of the small fraction concentration on concentration of the coarse fraction and used as a criterion of the interparticle interaction in diluted (up to 3 kg/m3) suspensions [5]. a coefficient of sedimentation instability (csi) was calculated as a tangent of slope of the dependence of changes in the optic density (d0–dc) during the experiment on concentration of the dispersed phase and used to determine a character of the additives effect on sedimentation stability [6]. a product of coefficients mβ from the einstein equation was used to characterize intensity of the contacted interaction in the highly concentrated systems. these coefficients describe functional relationship of the parameter α in the equation with shifting forces ratio [7, 8]: )exp(0   , where: η, η0 – viscosity of suspension and disperse medium respectively; α – a parameter, which describes interparticle interaction. viscosity of the system has been investigated using a rotation viscosimeter “rheotest-2” at high shifting speed values. this condition ensured suspension state of the particles. parameter α was found dependent on the dimensionless parameter γ, which can be calculated through the shifting n ch2 ch c o nh2 0 5 10 15 0 20 40 60 80 d, µm w ei gh t % fig. 1. granulometric composition of the hydro-mica suspensions journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 21 speeds ratio: γ = d/dmax, where dmax is the maximal shifting speed and d – given shifting speed. then α can be obtained as m )( [4, 9]. parameters β and m are constant in each individual experiment and α depends on the shifting speed. therefore, an intensity of the interparticle interaction can be characterized as a systems’ response to an external disturbance. it is known [1, 4] that some highmolecular additives (for instance, peo) can modify surface of the disperse phase particles, which results in the primary structuring of the system. the structuring significantly influences the bonding force between paa functional groups and the particles. that is why it was needful to determine how preliminary modification of the disperse phase particles using peo can influence the system’s stability in presence of the flocculation agent (paa). first modification stage implied modification of the solid phase particles with peo then we used paa. concentration of the flocculant was kept constant through all the experiments and concentration of the modifier was variable. results and discussion it is found that preliminary modification of the hydro-mica particles with peo (cpeo = 0.01 kg/m3) causes rise in cef value comparing to the value after modification with individual paa. aggregation of the particles results in the decrease of sedimentation stability of the suspensions (see fig. 2). a range of the peo concentrations ensuring effective flocculation is narrowed if a system contains paa. low concentration of peo (under 0.02 kg/m3) causes formation of the particles associates, which decreases aggregation and sedimentation stability and increases absolute values of cef and csi. adding of paa causes intense flocculation of the particles associates, which reveals itself through intense clarification of the suspensions containing only coarse or a mixture of coarse and small particles (fig. 2). basing on the results of this investigation we can recommend a composition of peo and paa for clarification of various natural waters, wastewaters and some technological solutions from the hydrophilic suspended particles. previously aggregated small particles can cohere with the coarse ones forming big floccules, which rapidly precipitate. this process of precipitation can be registered through decrease of optical density of the suspensions. another effect of the preliminary modification of the particles is reducing of the sediment’s volume. this phenomenon can be caused by some hydrophobisation of the clay particles surface, which reduces amount of the liquid captured between structural components of the floccules. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 22 fig. 2. dependence of effective coefficient of adhering cef (a) and coefficient of sedimentation instability csi (b) for the hydro-mica suspensions on concentration of peo at cpaa = 0.01 kg/m 3. 1 – suspension of the coarse particles; 2 – suspension of the small particles; 3 – suspension of the both particles. results of this investigation have been applied to improve the technology of clarification of the clay-containing wastewater from the mineral fertilizers production. data of table 1 prove that joint adding of peo and paa ensures 1.5-2.3 faster separation of the suspensions. cpeo : cpaa = 1 : 100 is the most effective ratio of the components concentration [10]. table 1 influence of paa : peo ratio on the clay-salt suspensions clarification rate reagent concentration, g/kgsolid phase ratio сpaaа : сpeo clarification rate, m/h paa peo 0.36 0.0009 400 3.00 paa peo 0.36 0.0018 200 4.61 paa peo 0.36 0.0036 100 5.62 paa peo 0.36 0.09 40 3.60 paa peo 0.36 0.018 20 3.21 paa peo 0.36 0.022 16 2.86 the suspensions become more unstable and a spatial structure forms in the disperse phase as a result of raise in the peo concentration. this process also causes decrease in the values of cef and csi. raise in the paa concentration to 0.02 kg/m3 also causes decrease in cef and csi and formation of a spatial structure (or strengthening of the previously formed one) (see fig. 3). joint adding of peo and paa ensures stronger interparticle interaction in the concentrated suspensions, which can be described through a product of two rheological constants m and β (see fig. 4). this product shows strong rise at initial journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 23 increase of the additives concentration (up to 0.005 kg/m3) followed by leveling and flattening of the dependence at further increase of the concentration. a pattern of the concentration dependence is similar for the dependence, which describes an individual effect of peo. only values of the mβ product are different for these two cases. on other hand, joint adding of the polymers additives results in substantially stronger interparticle interaction. the “joint” mβ values are higher than the individual ones. this fact proves that exactly peo governs a character of the interparticle interactions in the highly concentrated systems nevertheless another strong flocculant paa is also present in the system. conclusion a value of the interpartcile interaction can be substantially intensified through the surface modification of the disperse phase particles prior to adding a flocculant. this process ensures lower aggregation and sedimentation stability of the system. fig. 3. dependence of effective coefficient of adhering cef (a) and coefficient of sedimentation instability csi (b) for the hydro-mica suspensions on concentration of peo at cpaa = 0.05 kg/m 3. 1 – suspension of the coarse particles; 2 – suspension of the small particles; 3 – suspension of the both particles. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 24 moreover, a modification of the hydrophilic surface of the particles with water soluble polymers and surface-active additives results in hydrophobisation of the surface, faster sedimentation of the particles and their aggregations and reduction in volume of the sediment because of decreased in amount of the liquid captured between structural elements of the floccules. results of this work can be utilized in enhancement of the water treatment technologies applied to the cleaning of some natural waters, technological wastewaters as well as in some raw mineral materials refining and separation processes. references 1. a. k. zapolsky and a. a. baran. coagulants and flocculants for the water treatment processes. leningrad: “khimiya”, 1987. – 208 p. 2. kh. m. alexandrovich, et. al. physical chemistry of the selective floatation of potassium salts. minsk: “nauka i tekhnika”, 1983. – 272 p. 3. surface-active substances. (ed.: a. a. abramson). leningrad: “khimiya”, 1979. – 302 p. 4. t. s. slypenyuk, v. p. rudi. coll. j., 1987. – vol. 49. – p. 372-375. 5. d. p. salo, f. d. ovcharenko, n. n. kruglitsky. highly dispersed materials in pharmacy and medicine. kiev: “naukova dumka”, 1969. – 224 p. 6. v. v. nechyporuk, t. s. slypenyuk, s. d. boruk. coll. j., 2000. – vol. 52. – p. 97-100. 7. yu. g. frolov. course of colloid chemistry. moscow: “khimiya”, 1982. – 400 p. 8. yu. g. frolov, n. a. shabanova, s. i. molodchikova. coll. j., 1983. – vol. 45. – p. 970-974. 9. t. s. slypenyuk, v. p. rudi. chem. and chem. tech., 1987. – vol. 30. – p. 57-60. 10. ussr certificate of authorship 1719016 (c 02 f 1/56). a method of the suspensions clarification. (t. s. slipenyuk et.al.). fig. 4. dependence of m for the hydro-mica suspensions on concentration of the additives: peo + 0.01 kg/m3 paa (1); peo + 0.05 kg/m3 paa (2); twin-80 + 0.01 kg/m3 paa (3); twin-80 + 0.05 kg/m3 paa (4). microsoft word 3 journal nr 4 2010 din 8 noiembrie_86-92.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 90 validation of an enzymatic method for nitrates determination from vegetable products monica catană1, luminiţa catană1, mioara negoiţă1, enuţa iorga1, alina bâlea1, gabriela lilios2 1national institute of research&development for food bioresources – iba bucharest, 021102, bucharest 2, 6 dinu vintila street, romania, e-mail: mona.catana@bioresurse.ro 2ovidius university, 900527, constanţa, 124 mamaia street, romania, e-mail: liliosgabriela@yahoo.com abstract: nitrates and nitrites are natural components of soil, derived from mineralization of nitrogenous organic substances of vegetable and animal origin. plants absorb nitrogen from soil, mainly as nitrates or ammonium forms. this paper presents the results of researches carried out to validate an enzymatic method for nitrates determination in vegetable products. within this method, the nitrate is reduced by the reduced nicotinamide adenine dinucleotide phosphate (nadph) to nitrite, in the presence of nitrate-reductase (nr). in the case of the enzymatic method for nitrates determination in vegetable products, the calibration curve achieved with 9 standard levels of nitrate ions (with three rehearsals for each one), in the range of concentrations 6 mg/l – 30 mg/l, beginning from origin and the linearity coefficient (r2), is 0.9999. for 10 vegetable samples grown in open field (egg plants, bell peppers, green peppers and onion) the average nitrate concentration of vegetable species varied in the range 27.91 mg no3-/kg – 82.85 mg no3-/kg, and repeatability was in the range 1.33 mg no3-/kg – 1.50 mg no3-/kg. the determination of nitrates in a cabbage sample, by three analysts, led to an average nitrate concentration of 107.42 mg no3-/kg, relative standard deviation rsd(r), and the determined concentration is 0.81 %, and reproducibility is 2.45 mg no3-/kg. the detection limit is 0.15 mg/l and quantification limit is 0.30 mg/l. in the case of onion sample grown in open field, the nitrates concentration was 55.5 mg no3-/kg, and uncertainty was 0.68 mg no3-/kg. key words: linearity, repeatability, reproducibility, limit of detection, limit of quantification introduction in growing and development of horticulture-wine-growing plants, the improvement of soil fertilization state and achievement of high and constant crops, organic or mineral fertilizers are applied in the soil [1]. nitrates and nitrites are natural components of the soil from nitrogen mineralization of organic substances of plant and animal origin. nitrogen mineralization takes place primarily through the existing micro-organisms in soil. in areas with temperate climate, this process is carried out with maximum intensity in hot season [2]. plants absorb nitrogen from soil, mainly as nitrates or ammonium forms. vegetables and fruits can accumulate also nitrogen, as nitrate and nitrite forms. the mineral nitrogen amount in tissues of vegetable and fruit is much more in the case of species which the nitrates’ food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 91 reduction happens in leaves and when light intensity and temperature are lower [3]. high concentration of nitrates in plants (especially in vegetables) means a hazard for human and animal body, for two reasons: the possibility of methemoglobin appearance at children and nitrates conversion to nitrites in saliva and formation of cancerigen nitrozamines in the intestinal tract [2]. in a normal diet, 54% of nitrates content comes through vegetables consumption. in the daily ingestion of vegetable products, especially in the case of vegans, the percentage can be much more, 75 – 80% [2]. taking into consideration the cancerigen potential of nitrosamines, the high content of nitrates in vegetable products, presents a potential hazard. in order to determine nitrates within vegetable products, the following methods can be used:  spectrophotometer methods  high performance liquid chromatography method, using uv detector  high performance ion chromatography, using electrical conductivity detector  potentiometer method with selectively electrode  enzymatic method this paper presents the results of the performed researches carried out to validate an enzymatic method for nitrates determination in vegetable products. this enzymatic method presents no risks for the analyst, eliminating the danger of using metallic cadmium and α – naphthyl amine (cancerous substance, which is one of the components of colour reagent) represent within classical spectrophotometer method. experimental this paper presents the results of the performed researches carried out to validate an enzymatic method for nitrates determination in vegetable products. within this method, the nitrate is reduced by the reduced nicotinamide adenine dinucleotide phosphate (nadph) to nitrite, in the presence of nitrate-reductase (nr) [4]. nr no3+ nadph + h+ no2+ nadp+ + h2o the amount of oxidized nadph is stoechiometrically equal to the nitrate one. the decrease of nadph amount is measured through absorbance at 340 = ג nm. figure 1. kit for nitrates determination through enzymatic method the objective of validation is to demonstrate that a defined analytic system leads to the obtaining of some precious and reproducible results, for a given property. in order to validate the method for nitrates determination in vegetable products, the following performance criteria were established:  linearity  accuracy  precision (repeatability and reproducibility) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 92  sensitivity (limit of detection, limit of quantification)  measurement uncertainty in order to validate the enzymatic method for nitrates determination in vegetable products, we used as matrices the following vegetable species, cultivated in open field: egg plants, bell peppers, green peppers, cabbage and onion. results and discussion linearity is defined as the ability of method to provide results proportional with level of measuring. calibration curve has to be straight and to start from origin. any deviations that appear must be known. in order to accomplish the calibration curve, minimum five concentrations are necessary, with three rehearsals for each of them. linearity coefficient (r2) has to be of minimum 0.999, lower values may be accepted if there is a justification for this. in the case of the enzymatic method for nitrates determination in vegetable products, we achieved a calibration curve with 9 levels of nitrate ions standard (with three rehearsals for each one), in the concentrations range 6 mg/l – 30 mg/l, as following:  level 1 – 6 mg/l  level 2 – 9 mg/l  level 3 – 12 mg/l  level 4 – 15 mg/l  level 5 – 18 mg/l  level 6 – 21 mg/l  level 7 – 24 mg/l  level 8 – 27 mg/l  level 9 – 30 mg/l the achieved calibration curve starts from origin and linearity coefficient (r2) is 0.9999. calibration curve of ion nitrates y = 0,0335x 0,0054 r2 = 0,9999 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 5 10 15 20 25 30 35 concentration of ion nitrares (mg no3-/l) a b so rb an ce linear figure 2. calibration curve of ion nitrates the equation of calibration curve is the following: y = 0.0335 x – 0.0054 (1) of which: y = absorbance measured at λ = 340 nm x = concentration of nitrate ions, in mg no3-/l by equation (1), knowing absorbance value, we can directly calculate the concentration of nitrate ions in a test sample, using the following formula: 0335.0 0054.0  y x (2) accuracy means the ability to make an analysis with low difference between real value and founded value. in the case of enzymatic method for determination of nitrate ions in vegetable products, accuracy is expressed through recovery. recovery is calculated by the equation (3): recovery (%) = (cd/ct) x 100 (3) of which: ct – theoretical concentration of nitrate ion, in mg/l cd – determined concentration of nitrate ion, in mg/l in order to determine recovery, in the case of enzymatic method for determination of nitrate ion, we used 9 prepared solutions as in the case of the achievement of calibration curve. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 93 the concentration of nitrate ions of those 9 nitrate solutions was determined in two ways:  calculated by equation (2)  using the enzymatic method the concentrations of nitrate ions of those 9 solutions, using the enzymatic method, were calculated by formula (4): cnitrate = (1.01xv x mw / ε x d x v )xδa in [mg no3-/l sample solution (4) of which: 1.01 – correction factor of final volume of sample v – final volume of sample (3.05 ml) mw – molecular weight of nitrate ion (62 g/mol) ε – extinction coefficient of nadph (at λ = 340 nm, ε = 6.3 l x mol-1 x cm-1) d – length of optical way (1 cm) v – sample volume, in ml δa – difference between variance of sample absorbance and blank absorbance difference of absorbance for sample  (a1 – a2)sample – 2x(a2 – a3)sample difference of absorbance for blank  (a1 – a2)blank – 2x(a2 – a3)blank anitrate  asample – ablank (5) changing these elements, we have obtained: v a x nitrat   316.30 (6) the determination of concentrations of nitrate ions of 9 solutions of kalium nitrate was made by the formula (2) and recovery was in the range: 98.67 % 100.23 %. the determination of concentrations of nitrate ions of those 9 solutions of kalium nitrate was made by the formula (6), using the protocol of the enzymatic method, recovery was in the range: 97.00 % 102.67 %. precision is reverberated by the concordance degree between independent analytical results obtained under specified conditions. precision is a measure of results dispersion and is determined in conditions of repeatability and reproducibility. repeatability (r) – value below which the absolute difference between results of the two separated tests, achieved on the same test object, the same apparatus, the same executant and in the same short time spell, is below the specified probability limits (as a rule 95%). the limit of repeatability is calculated by the equation (7): r = 2.8 x sd(r) (7) of which: r – limit of repeatability; sd(r) – standard deviation, calculated in the case of results obtained in repeatability conditions. relative standard deviation, rsd(r), obtained from the achieved results in repeatability conditions is calculated by equation (8): rsd(r) = (sd(r)/ x )x 100 (8) of which: sd(r) – standard deviation, calculated in case of the obtained results in repeatability conditions; x – arithmetic average of determined concentrations in repeatability conditions. reproducibility (r) – value below which the absolute difference between results of a single test, obtained for an identical material, in two or more laboratories, by different operators, on different apparatus, is below the specified probability limits (as a rule 95 %). relative standard deviation, rsd(r), obtained from the achieved results in reproducibility conditions is calculated by the equation (9): rsd(r) = (sd(r)/ x )x 100 (9) of which: food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 94 sd(r) – standard deviation, calculated from the obtained results in reproducibility conditions; x – arithmetic average of the obtained results in reproducibility conditions. standard deviation – measure dispersal tendency of data and is calculated by equation (10): 1 )( 2     n xx s id (10) of which: sd – standard deviation; xi – result of measurement; x – arithmetic average of measurement results; n – number of tests. in order to determine repeatability in the case of enzymatic method for determination of nitrates in vegetable products, we analysed 10 parallel samples of vegetables cultivated in field (egg plants, bell peppers, green peppers and onion) and we calculated for each vegetable species the following:  average concentration of nitrates  value of relative standard deviation rsd(r), in repeatability conditions  limit of repeatability in the case of the 10 parallel samples of egg plants analysed to determine nitrates content, the average concentration of nitrates is 65.98 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 0.77%, and the limit of repeatability is 1.43 mg no3-/kg. in the case of the 10 parallel samples of bell peppers analysed to determine nitrates content, the average concentration of nitrates is 27.91 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 1.90%, and limit of repeatability is 1.49 mg no3-/kg. in the case of 10 parallel samples of green peppers analysed to determine nitrates content, the average concentration of nitrates is 30.85 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 1.54%, and limit of repeatability is 1.33 mg no3-/kg. in the case of 10 parallel samples of onion analysed to determine nitrates content, the average concentration of nitrates is 82.85 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 0.65%, and limit of repeatability is 1.50 mg no3-/kg. in order to determine intralaboratory reproducibility in the case of the enzymatic method used in the determination of nitrates in vegetable products, we analysed by three analysts (analyst a – 3 parallel samples, analyst b – 3 parallel samples, analyst c – 3 parallel samples) one cabbage sample, carrots and onion and we calculated:  average concentration of nitrates  value of relative standard deviation rsd(r), in reproducibility conditions  limit of reproducibility in the case of the analysed cabbage sample by analysts for determination of nitrates content (each analyst performed 3 parallel samples), the average nitrates concentration is 107.42 mg no3-/kg, and relative standard deviation rsd(r), for determined concentration is 0.81% and limit of reproducibility is 2.45 mg no3/kg. in the case of the analysed carrots sample by analysts for determination of nitrates content (each analyst performed 3 parallel samples), the average nitrates concentration is 92.27 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 1.24%, and limit of reproducibility is 3.20 mg no3-/kg. in the case of the analysed onion sample by analysts for determination of nitrates content (each analyst performed 3 parallel samples), the average nitrates concentration is 71.88 mg no3-/kg, relative standard deviation rsd(r), for determined food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 95 concentration is 1.22%, and limit of reproducibility is 2.45 mg no3-/kg. limit of detection of the method is 0.15 mg/l and corresponds to a difference of absorbance between sample and blank (anitrate) of 0.010 (absorbances being measured at λ = 340 nm) and a sample maximum volume v = 2 ml. limit of quantification of the method is 0.30 mg/l and corresponds to a difference of absorbance between sample and blank (anitrate) of 0.020 (absorbances being measured at λ = 340 nm) and a sample maximum volume v = 2 ml. uncertainty is a parameter associated with the result of measurement that characterises the dispersion of the values that could reasonably be attributed to the measurand. in order to estimate uncertainty the following steps should be taken:  specification of measurand  identification of the significant sources of uncertainty  estimation of combined standard uncertainty  estimation of extended standard uncertainty combined uncertainty is calculated with equation (11): 22 4 2 3 2 2 2 1 ..... nc uuuuuu  (11) of which: uc – combined uncertainty u1, u2, u3, u4, .....un – component uncertainties of the budget of uncertainties. extended uncertainty is calculated by equation (12): u = k x uc (12) of which: u – extended uncertainty uc – combined uncertainty k – covering factor, for a confidence level of 95%, k = 2 measurand = concentration of nitrates in vegetable products, expressed in mg no3/kg. in the case of determination of nitrates in vegetable products, through enzymatic method, significant sources of uncertainty are:  measurement of volume of aqueous extract of sample  weighting of sample  measurement of absorbance of aqueous extract of sample relative standard uncertainty, within enzymatic method of nitrates determination, is calculated by equation (13) [5]: 2.221 )()() 2 ( )( a u m uu x xu absmc   (13) of which: uc1 – combined standard uncertainty at measurement of the aqueous extract of sample um – combined standard uncertainty at weighting of sample uabs. combined standard uncertainty at measurement of the aqueous extract of sample m – weight of analysed sample, in g δa – difference between variance of absorbance of sample and blank x – nitrates concentration of sample, in mg/kg relative s tandard uncertainty ,at nitates determ ination, in the cas e of onion sam ple 0 0,002 0,004 0,006 0,008 v m ∆a u(x)/x relative s tandard uncertainty series1 figure 3. relative standard uncertainty, at nitrates determination, in the case of onion sample food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 96 in the case of the analysed onion sample, by changing the suitable data, we obtained: 0062.0 )(  x xu x = 55.5 mg no3-/kg sample 34.0)( xu mg no3-/kg sample u(x) = k x u(x) of which: u(x) – extended uncertainty k – covering factor for a confidence level of 95%, k = 2 u(x) = 0.68 mg no3-/kg sample therefore, result can be expressed in this way: x = 55.5 ± 0.68 mg no3-/kg sample conclusion 1. this paper presents the results of the performed researches carried out to validate an enzymatic method for nitrates determination in vegetable products. 2. in the case of the enzymatic method for nitrates determination in vegetable products, the calibration curve achieved with 9 standard levels of nitrate ions (with three rehearsals for each one), in the range of concentrations 6 mg/l – 30 mg/l, starts from origin, and the linearity coefficient (r2) is 0.9999. 3. for 10 vegetable samples grown in open field (egg plants, bell peppers, green peppers and onion) the average nitrate concentration of vegetable species varied in the range 27.91 mg no3-/kg – 82.85 mg no3-/kg, and repeatability was in the range 1.33 mg no3-/kg – 1.50 mg no3-/kg. 4. the determination of nitrates in a cabbage sample, by three analysts, led to an average nitrate concentration of 107.42 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 0.81%, and reproducibility is 2.45 mg no3/kg. 5. in the case of carrots sample analysed by analysts to determine nitrates content (each analyst performed 3 parallel samples), average nitrates concentration is 92.27 mg no3-/kg, relative standard deviation rsd(r), for determined concentration is 1.24%, and limit of reproducibility is 3.20 mg no3-/kg. 6. the detection limit of method is 0.15 mg/l and corresponds to a difference of absorbance between sample and blank (anitrate) of 0.010 (absorbances being measured at λ = 340 nm) and a maximum volume of sample v = 2 ml. 7. the quantification limit of method is 0.30 mg/l and corresponds to a difference of absorbance between sample and blank (anitrate) of 0.020 (absorbances being measured at λ = 340 nm) and a maximum volume of sample v = 2 ml. 8. in the case of onion sample grown in open field, nitrates concentration was 55.5 mg no3-/kg, and uncertainty was 0.68 mg no3-/kg. acknowledgments the experiments were performed within the contract no. 51-050/18.09.2007, financed through programme 4 “partnerships in priority s&t domains” 2007 – 2013 – national centre for projects management. references 1. luchian viorica, legumicultură generală şi specială, ed. elisavaros, bucureşti, 2007 2. bibicu miruna, cercetări metodologice privind determinarea nitraţilor şi nitriţilor din ţesuturi vegetale şi nivelului de acumulare în produsele horticole, bucureşti, 1994 3. gherghi a., burzo i., miruna bibicu, liana mărgineanu, liliana bădulescu, şi colab., biochimia şi fiziologia legumelor şi fructelor, ed. academiei române, bucureşti, 2001 4. .*** protocol de lucru pentru metoda enzimatică uv de determinare a nitraţilor din produse alimentare, r – biopharm, 2008 5. *** citac / eurachem guide quantifying uncertainty in analytical measurement, 2000 issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvi, issue 3 30 september 2017 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 34 the influence of processing and medium composition on the thiol availability of whey protein concentrate loredana dumitraşcu1, alina ardelean1 and nicoleta stănciuc1 dunărea de jos university of galaţi, faculty of food science and engineering, 111 domneasca street, 800201, galati, romania, nicoleta.sava@ugal.ro abstract. whey protein concentrates (wpc) play an important role in the development of functional foods with an increase in global demand. whey proteins are highly soluble over a broad range of ph, a property that is important in their application as foaming, emulsifying, gelling, and water-binding agents in various types of food products. denaturation of whey proteins is critical in modifying the functional behavior of dairy products. a knowledge of the thermal behavior of wpc occurring in different medium conditions, might allow to choose the most appropriate technology and to manipulate the thermal regime for a particular process or/and products, so that the final product possesses the optimal desirable properties. the objective of this study was to investigate the heat induced changes in whey protein concentrate solution at ph 7.5 and 6.6 in salt conditions (cacl2) on thiol availability. the results presented in this paper demonstrate that heat-induced changes in wpc greatly influence their degree in surface sh exposure. the exposure was more pronounced at higher temperatures. however, above 80°c, the formation of disulfide bonds seems to be favored, resulting in a lower level of surface reactive sh groups after prolonged heating compared with the lower treatment temperatures. the increase was related to the intensity of applied temperature-time combination and medium composition. the thermal denaturation of wpc is regarded as a complex reaction involving several unfolding steps and ending in aggregation reactions. keywords: heat-treatment, sulfhydryl groups, functional properties introduction whey proteins are currently seen as a commodity product in food industry. they are largely used as emulsifiers, texture enhancers and healthy ingredients [1]. the functional properties of whey proteins may be classified into three main groups: (a) hydration, dependent upon protein-water interactions which have an important bearing on wetability, swelling, adhesion, dispersibility, solubility, viscosity, water absorption and water holding; (b) interfacial, including surface tension, emulsification and foaming characteristics; and (c) aggregation and gelation, which are related to protein-protein interactions [2]. heat treatments are important steps in both production of whey protein concentrates and processing of food products. the commonly used heat treatments are preheating, pasteurization, and sterilization [3]. the functionality of the whey proteins is strongly influenced by the processing conditions and can be modified by thermal treatment either reversibly or irreversibly. the main components of whey proteins are β-lactoglobulin and α-lactalbumin. when the whey proteins are heated denaturation of β-lactoglobulin occurs. this protein contains two intra-molecular disulphide bonds and one free sh-group (sh121). during heat treatment these thiol groups are responsible for changes in the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 35 structure and thus in functionality of whey proteins. knowledge in the thermal behaviour of wpc occurring in different medium conditions, might allow to choose the most appropriate technology and to manipulate the thermal regime for a particular process or/and products, so that the final product possesses the optimal desirable properties. materials and methods wpc was purchased from kuk romania. the chemicals 5,5’-dithiobis-(2nitrobenzoic acid) (dtnb), sodium dodecylsulfate (sds) were obtained from sigma chemicals (st. louis, mo, usa). all solvents and chemical reagents were of analytical grade. isothermal treatment of wpc solutions 5 ml of wpc solutions (2.5% in distilled water at two ph values: 7.5 and 6.6 with or whitout 0.002 m cacl2) were heated in glass tubes in a thermostatically controlled water bath at constant temperatures between 70 and 85°c for 0 to 30 min. after the heat treatment, the samples were immediately transferred to ice-cold water to prevent further denaturation. the samples were stored at 40c over night. thiol availability the exposed sh groups were determined as described by sava et al. (2005) [4]. the (un) heated samples (50 μl) were diluted with appropriate buffer solution (standard solution at ph 8.0 for exposed sh groups. dtnb reagent (10 µl) was added to the (un)treated samples. the absorbance at 412 nm was measured against a reagent blank after 15 min at 20°c. the sh groups (μmol/g protein) were calculated with the equation 1: μmol sh/g protein=   13600c d0a1a   (1) where a1 is the absorbance of (un)heated sample at 412 nm, a0 is the absorbance of the blank , d the dilution factor, 13 600 the molar extinction coefficient, and c the protein concentration (mg/ml). results and discussion the influence of ph in the first sets of experiments, the samples were heat-treated at two different phvalues: 6.6. and 7.5. figure 1 shows the extent of sh groups exposure at ph 6.6. as it can be seen, the maximum exposure was reached after 20 minutes of keeping at 850c. figure 1. the extent of sh groups exposure (%) after heat-treatment of wpc solutions at ph 6.6 figure 2 shows the heat-induced changes in the degree of sh groups exposure at ph 7.5. figure 2. the extent of sh groups exposure (%) after heat-treatment of wpc solutions at ph 7.5 heating the samples at ph 7.5 caused the exposure of free sh groups, the maximum extent was measured at 850c after 10 minutes of keeping. 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 he ating time, min % e xp os ur e 60 65 70 75 80 85 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 hea ting time, min % e xp os ur e 60 65 70 75 80 85 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 36 at both ph values studied, the increase of keeping time resulted in a gradual increase in the extent of free sh groups exposure. these heat-induced changes in free-sh complied with observations made by sava et al., (2005) [4]. one can see from figure 3, that at ph 7.5 the extent of denaturation, expressed as exposure of sh groups is considerable lower as compared to ph 6.6. the reactivity of free thiol group in βlactoglobulin is strongly dependent on ph. β-lactoglobulin undergoes ph-dependent conformational changes in the ph range of 6.5 to ph 7.5, the so called n→r or tanford transition [5]. these changes are associated with an anomalous carboxylate residue, which has a pka of 7.3 instead of 4.5, and which was tentatively identified as glu89 [6]. according to this theory, the dimmer starts to dissociate, followed by critical conformational changes of the molecule with the exposure of hydrophobic groups and reactive free sulfhydryl group, known as the tanford transition [7]. figure 3 shows the comparative analysis of % exposure at 600c and 850c at both ph values. a) b) figure 3. the degree of free sh groups exposure at 600c (a) and 850c (b) under sub-denaturation condition, the degree of exposure was considerable higher at ph 6.6 (figure 3, a) when compared to ph 7.5. it seems that when protein thiol availability is measured close to the isoelectric point of proteins, the isoelectric precipitation is stimulated. this affirmation is sustained by the increase in the turbidity and decrease of solubility of the samples at both ph values studied (data not shown). when the temperature is increased, the free sh groups become more accessible for interaction with dtnb at both ph values. as it was mentioned earlier, the heating of whey proteins lead firstly to the denaturation of the β-lactoglobulin. this denaturation process involves a rearrangement of the tertiary structure so that the free thiol group from cysteine 121, in the native state buried within the protein molecule, now becomes accessible. this activated thiol group can subsequently react with disulfide bonds that are also present in β-lactoglobulin or in other whey proteins such as α-lactalbumin in an exchange (or propagation) reaction, or can react with another thiol group to form a disulfide bond (termination reaction), as it was explained by roefs and de kruif (1994) [8]. 0 10 20 30 40 50 60 70 80 90 100 % e xp o su re 0 5 10 20 30 he ating time , min ph 6.6 ph 7.5 0 5 10 15 20 25 % e xp os ur e 0 5 10 20 30 he ating time , min ph 6.6 ph 7.5 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 37 influence of salt the presence of calcium enhances the heat-induced aggregation of wpc [9]. the formation of whey protein aggregates can be affected by the addition of salts. in this study, the solutions at ph 6.6 and 7.5 were heat-treated in the presence of 0.002 m cacl2. figure 4 shows the heat-induced changes in free-sh groups exposure at ph 6.6 in the presence of 0.002 m cacl2. 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 he ating time, min % e xp os ur e 60 65 70 75 80 85 figure 4. the extent of sh groups exposure (%) after heat-treatment of wpc solutions at ph 6.6 in the presence of 0.002 m cacl2 despite the intensity of heat treatment, the exposure of free sh groups remained high, the maximum extent of denaturation was reached after 20 minutes at 850c (91.5% ± 0.9). figure 5 shows the extent of wpc denaturation at ph 7.5 in the presence of the same concentration of salt. 0 10 20 30 40 50 60 70 80 0 5 10 15 20 25 30 hea ting time, min % e xp os ur e 60 65 70 75 80 85 figure 5. the extent of sh groups exposure (%) after heat-treatment of wpc solutions at ph 7.5 in the presence of 0.002 m cacl2 the increase in sh groups exposure was dependent on the intensity of applied thermal treatment and the duration of the treatment, and consequently, for both sets of experiments, was significantly pronounced at higher temperature. the heating at ph 7.5 in the presence of salt leads to a lower extent of exposure, the maximum extent was reached at 850c after 10 minutes of heating. it can also be seen from figure 5 that under more severe treatment conditions, the content of free exposed sh groups decrease, probably because the electrostatic interactions are favoured. figure 6 shows the degree of free sh groups exposure at ph 6.6 at 600c (a) and 850c (b) in the presence of 0.002 m cacl2. the addition of salt at 600c caused no denaturation in the first 10 minutes of heating, with a slight increase in exposure after 20-30 minutes of keeping. (a) (b) figure 6. the degree of free sh groups exposure at ph 6.6 after heat treatment at 600c (a) and 850c (b) 0 5 10 15 20 25 30 % exposure 0 5 10 20 30 he at in g t im e, m in 0.002 m cacl2 0 m cacl2 0 10 20 30 40 50 60 70 80 90 100 % exposure 0 5 10 20 30 he at in g t im e, m in 0.002 m cacl2 0 m cacl2 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 38 the presence of calcium under more severe heat treatment conditions did not have any influence on the free sh exposure for both ph values studied. there was a significant difference in the degree of exposure when samples at ph 7.5 were heated at 600c in the presence of cacl2 (figure 7). the degree of exposure was almost 11 times higher after 30 minutes of keeping in the presence of salt. figure 7. the degree of free sh groups exposure at ph 7.5 after heat treatment at 600c with and without salt addition this observation indicates that thermal denaturation of wpc as measured by the changes in surface sh groups exposure involves 2 steps: an unfolding step (60 to 75°c) and an aggregation one (75 to 85°c), that mostly follows unfolding. it has been suggested that three effects, or a combination of them, might be responsible for calcium-induced protein aggregation [10]. the first phenomenon is related to intermolecular cross-linking of adjacent negatively charged or carboxylic groups by the formation of protein-ca2+protein complexes [11]. the second phenomenon is the intra-molecular electrostatic shielding of negative charges on the protein [12]. monovalent and divalent cations both screen electrostatic interactions between charged protein molecules although the effect is greater with divalent cations [13]. the third phenomenon is an ion-induced conformational change, which leads to altered hydrophobic interactions and aggregation at elevated temperatures [9]. conclusions the results presented in this paper demonstrate that heat-induced changes in wpc greatly influence their degree in surface sh exposure. the increase was related to the intensity of applied temperature-time combination and medium composition. the exposure of surface to sh groups was more pronounced at ph 6.6, especially in a higher temperature range. these behaviours may be explained by the fact that in lower temperature range the proteins firstly unfold, as shown by the time-and temperature-dependent increases in the surface sh groups and then aggregates at higher temperature. at ph 6.6, in a lower temperature range, the addition of cacl2 did not have any influence on the degree of exposure at the surface of molecules. at ph 7.5, the calcium ions had significantly higher effect on the sh groups exposure to the temperature range of 60 to 750c. the exposure was more pronounced at higher temperatures. however, above 80°c, the formation of disulfide bonds seems to be favoured, resulting in a lower level of surface reactive sh groups after prolonged heating compared to the lower treatment temperatures. the thermal denaturation of wpc is regarded as a complex reaction involving several unfolding steps and leading to aggregation reactions. it should be mentioned that the phenomena taking place in the present model system do not necessarily predict the behaviour of wpc in commercial dairy foods, since the presence of other components (sugars, fats, other proteins, etc.) could modify the observed behaviour. further research is underway to optimize the conditions of thermal treatment, in 0 5 10 15 20 25 30 % e xp os ur e 0 5 10 20 30 heating time , min 0.002 m cacl2 0 m cacl2 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 39 order to obtain enhanced functional properties related to protein-water and protein-protein interactions. acknowledgment the authors acknowledge financial support from the national university research council (nurc, pn-ii-id-pce-2008-2, idea, id 517) romania. the work of alina ardelean was supported by the project sop hrd – eficient 61445/2009. bioaliment research platform is also acknowledged for providing material support. references 1. floris, r., bodnár, i., weinbreck, f., alting, a.c., dynamic rearrangement of disulfide bridges influences solubility of whey protein coatings, international dairy journal, 2008, volume 18, issue 5, pg. 566-573; 2. kresic, g., lelas, v, herceg, z., rezerk, a. effects of high pressure on functionality of whey protein concentrate and whey protein isolate, lait 2006, 86, 303–315; 3. de wit, j. n., klarenbeek g., effects of various heat treatments on structure and solubility of whey proteins. journal of dairy science 1984, 67:2701–2710. 4. sava, n., i. van der plancken, claeys, w., hendrickx, m. the kinetics of heat-induced structural changes of β-lactoglobulin, journal of dairy science 2005, 88, 1646-1653. 5. hambling sg, mcalpine as, sawyer l.. β-lactoglobulin. in: fox pf, ed. advanced dairy chemistry: 1. proteins. london: elsevier applied science, 1994, pp 141–190. 6. brownlow s, cabral jhm, cooper r, flower dr, yewdall sj, polikarpov i, north act, sawyer l. bovine βlactoglobulin at 1.8 å resolution— still an enigmatic lipocalin. structure, 1997, 5:481–495. 7. iametti, s., de gregori, b., vecchio, g. bonomi, f., modifications occur at different structural levels during the heat denaturation of βlactoglobulin, european journal of biochemistry, 1996, 237, 106-112; 8. roefs s.p.f.m. and de kruif c.g. a model for the denaturation and aggregation of βlactoglobulin, european journal of biochemistry, 1994, 226: 883-889; 9. mounsey, j.s., o’kennedy, b.t., conditions limiting the influence of thiol– disulphide interchange reactions on the heatinduced aggregation kinetics of β-lactoglobulin, international dairy journal, 2007, 17, 1034–1042; 10. simons, j.-w. f. a., kosters, h. a., visschers, r. w., de jong, h. h. j., role of calcium as a trigger in thermal β-lactoglobulin aggregation. archives of biochemistry and biophysics, 2002, 406, 143–152. 11. hongsprabhas, p., the structure of coldset whey protein isolate gels prepared with ca2+, lebensmittel-wissenschaft und-technologie, 1999, 32, 196–202 12. hongsprabhas, p., barbut, s., structure-forming processes in ca2+-induced whey protein isolate gelation, international dairy journal, 1997, 7, 827–834. 13. veerman, c., baptist, h., sagis, l. m. c., van linden, e., a new multistep ca2+induced cold gelation process for βlactoglobulin, journal of agriculture and food chemistry, 2003, 51, 3880–3885. применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 contents: 1. research on obtaining high β-glucans content from different sources of yeast by harnessing their biologically active potential ionuț avrămia, sonia amariei 190 195 2. quinoa as a substitute for semolina: some aspects and problems of introduction sergiy boruk, igor winkler, olga romanovska, olga gerych 196 201 3. the interaction between beans hulls and food dye laura carmen apostol 202 208 4. enrichment of pasta products using beetroot péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri 209 215 5. perspectives of utilization of nano-dispersive materials based on sio2, tio2 and sio2–tio2 for wine fining anastasiia sachko, igor kobasa, olesya moysyura 216 221 6. determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2ethylhexyl) phthalate (dehp) in soft plastic toys and the first survey of the bulgarian market valentina christova-bagdassarian, julieta tishkova, anton tachev 222 233 7. estimation of heavy metal levels in green leafy vegetables purchased from suceava ancuța elena prisacaru, laura carmen apostol, sorina ropciuc 234 238 8. the influence of regular and genetically modified soybeans on postnatal development of rats n. n. omelchenko, g. v. dronik, і. а.winkler, м. s. rogozynskyi, v. а. kucheriava 239 244 9. sensory evaluation of goat milk cheese under different freezing conditions eugenia mihaela pricop 245 249 10. supplementation of wheat flour with soy flour, sensory and physico-chemical evaluation of fortified biscuits flavia pop, zorica voșgan, lucia mihalescu 250 255 11. technology of fresh herbs storage using hydrogel and antioxidant composition olesia priss, viktoria yevlash 256 261 12. research of quality indicators of curd products on basis of protein-herbal clots olena grek, alla tymchuk, larisa chubenko, kira ovsiienko 262 268 13. sustainable packaging solutions for organic fresh berries elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa 269 275 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 14. antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties mădălina iuga, sorina ropciuc, silvia mironeasa 276 281 15. recyclable porous materials for the uptake of bisphenol a rodica sturza, mihai melenciuc, denisa nistor, farida boudissa, thiziri terkani, nou-houda hadj abdelkader, azzouz abdelkrim 282 – 286 16. study on the kinetics of biomass blends dewatering as a stage of thermal degradation yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra 287 295 17. author instructions i v 18. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 10 romanian trade liberalization of food products in terms of export benefits in the knowledge society alina-petronela haller 1 1 phd. senior researcher iii, ices “gh. zane” iassy branch of romanian academy hallalina@yahoo.com abstract. the aim of this work is to demonstrate that the export of food products is an important factor of economic growth and development, regardless of the economic situation, especially in terms of recession mainly in the situation of romania, without claiming to consider that exports are the only “engine” capable to restore the economic balance at both national and international level. the current recession of the global economy brings new challenges and each country must be responsible to overcome it. romania has to use all its advantages and all the market opportunities to find the best solutions which can help the economic recovery. in this paper, we want to show that exports are a “trampoline” for the national economy, especially in the case of food products exports, because romania has a relatively high agricultural potential, able to support the light food industry – a traditional economic sector. by creating some brand names recognized in other countries and by the orientation on profitable niches on the foreign markets, the export of romanian food products will be a stimulating factor of economic growth and development in a knowledge based society. the methods used in this study are theoretical, based on analysis, observations, synthesis, induction and deduction. keywords: export, international trade, economic growth, development, economic liberalization, knowledge society. introduction prior to the world economy’s entering recession nothing seemed to be able to perturb in any way the right course of action in the economic activities. the more economically developed countries were following, untroubled, the path of the economic-social growth, the developing ones seemed to catch up, every day, with the industrialized countries, and together they were trying to focus their attention on material, financial and human resources to solve the problems of the poorer states and the global issues as a whole. though there were “voices” announcing a significant change approaching, nobody believed, in the euphoria of an unprecedented dynamism, that the economy would fall. the unavoidable occurred and, one by one, the world states made officially public the fact they were entering the recession, intensifying, at the same time, their efforts to find recovery solutions in times as short as possible, with risks and costs as low as possible. the measures each country proposed are strictly correlated to their own economic potential, without neglecting the current reality, a reality called “regionalization and globalization”, and wearing a label characterized by “liberalization”, under all of its forms: the economic, the social, the cultural one, giving birth to a new type of society, a mainly knowledge-based one, that alvin toffler expected to take shape in a more or less definite future, and called “society of presumption”. when we say economic food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 11 liberalization, we are first of all talking about the commercial one, that is to say export and import. thereof, we are convinced that any country that wishes to follow the path of economic growth and development, romania included, should emphasize the external exchanges and render the exportation an asset capable of propel it among the powerful states. the issue of liberalization is not a new one, but it grows more and more important. international trade started to be theorized since the mercantilist period, but, with the beginnings of the economic liberalism, there is no major economist or school of thought that has not theorized the economic liberalization and its importance in the national and international economies. material and method the methods i used in this approach are interpretative, specific to fundamental research, of both deductive and inductive type. i used, as research methods, both observations, seizing the economic reality in different countries, and comparisons, that i introduced into a descriptive approach. results and discussion romanian export, after 1990, suffered some structure and value-related transformations, with implications at the economic and social level. in a more and more globalized and interdependent world, export is one of the major factors of economic growth and development, the romanian case included. there is no doubt that effective export can induce multiple advantages, at both an economic and social level, through the absorption of unemployment, the adjustment of the current account balance and of the balance of trade, debt decreasing, gdp’s rise and the rise of the life standard. the paradigms referring to the foreign trade and export in general, and to the romanian one in particular, over the last two decades, have changed, rendering obvious the priority position it occupies within the economic and commercial policies, as well as in the policies of economic growth and development. after 1990, the start of the transition to the market economy was rather difficult for romania, and the perturbing outcomes of the structural disequilibrium grew deeper and deeper. the beginning of the transition period to the market economy was influenced by the process of liberalization of the commercial transactions abroad by eliminating the state monopoly, so that in a short while most of the restraints of trade were lifted, the non-tariff barriers were significantly reduced and the level of the tariff ones was much diminished. to achieve positive rates of growth and economic development generally involves reforming and reconstructing economy by focusing on the factors that determine these processes, among which export holds a very important place. after 1990, the reconstruction of the romanian economy and the structural modernization under stability and increasing efficiency conditions were not possible beyond the import of the lacking production factors (raw materials, power, competitive techniques and technologies on the international market) and of commodities and services, and the new structure of the foreign trade entailed current account and trade imbalances. a fundamental condition to rejoin the ascendant economic trend has been that of covering import with the export of competitive products on the foreign markets, particularly under the present circumstances of recession, with consequences both at the economic and at social level. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 12 in the romanian economy, there are market deficiencies rendering quite difficult the improvement of the structure of external trade, and the exceeding of the economic recession: the high energyconsuming nature of the industrial production, the high degree of obsolescence of some techniques and technologies of production, the ineffectiveness of marketing in international relations, the legal framework not yet sufficiently adapted to the demands of the external market, the regression of some important segments of the local production as a result of some weakly protectionist commercial policies. currently, the world economy faces the most powerful economic recession since the great depression (’29-’33), and the romanian economy cannot elude it. the recession renders even more serious the problems existing on the labour market (the high unemployment rate), the ones related to the decrease of incomes, consumption, production, and to the rise of inflation. the limits of the domestic market determine a change of paradigm as for the economic recovery modalities, one of the best solutions turning out to be, together with the stimulation of foreign and domestic investments, the export, as its implications are so very important, complex and beneficent, at both the economic and social level. the current economic situation comes with new challenges for the romanian economy: we cannot speak any longer exclusively about an inadequate structure of export and import, about low competitiveness of local products or about an inferior value of export compared to that of import; once the world economy entered the recession, we assist at the closing or narrowing of some export markets. confronted with such reality, characterized by high incertitude and risks, the preoccupations and theories that found their applicability until recently are now outdated. the objectives of foreign trade are difficult to reach and, furthermore, following the example of some other countries, including members of the european union, there is a choice between liberalization and commercial protectionism; the temptation is to theoretically opt for the latter strategy, which apparently brings forth bigger economic advantages in the descendent periods of the economic cycle. it is not only that the eu member status does not allow the consolidation of the protectionist practices, but these are not a valid solution for the exceeding of recession and the resuming of economic growth. we argue that economic recovery will only be possible, from the external trade standpoint, through trade liberalization, particularly through exportation, even if rendering it effective is the most difficult way – it is also the safer one towards economic-social growth, development and progress. we do not intend to minimize the role played by other factors in economic growth, like investments, involving employment creation and progress engendering. we must remember that in times of economic recession, the best option is orientation towards the domestic resources and potential, and now the investment resources are low, whichever the type we have in view (domestic or foreign). the problems of the romanian economy, resembling those of other countries (the developing ones), became common to all the world’s states once the international recession started; getting over them needs changes of the paradigm and new solutions meant to revive the market dynamism. romanian export is strongly oriented towards the eu market, in its turn affected by the world economic recession and confronted with the significant reduction of the demand with direct impact upon the romanian export. under these food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 13 circumstances, it is important to find new outlets, which does not mean to give up the present ones, but to make an effort to enlarge the export markets. to find new market niches should not turn into an objective in itself (the “export at any price” type), but one must take into consideration the current structure of the romanian export and its degree of competitiveness. the orientation towards those markets where the romanian products will be demanded becomes necessary, and they will be highly competitive, compared to the similar already existing supply. in this context, we refer not only to new possible outlets, but also to a possible return to once traditional markets, with a view to promoting romanian production at a level that might bring forth multiple benefits (gains from the foreign trade activity, the optimal capitalization of the labour force and therefore the absorption of unemployment, the effective capitalization of the domestic resources, etc.). as for the romanian export, its image over the last two decades is that of a status-quo, especially from the structural standpoint, with effects upon the value level, far from the real potentialities of the romanian economy and from the necessities. the export is supported especially by the products of light industry whose prices relatively reduced on the international market, and the main competitive advantage remains the cheap labour force, while import is dominated by high technology products, whose prices are big, thus leading to a significant trade imbalance. an easily observable paradox in the structure of foreign trade is the important weight in the import of some products for which romania has always had a high production potentiality, like for instance the agribusiness products. a revaluation of the structure of the foreign trade should become a priority, in order to balance, from the value’s standpoint, export and import, given that “a favourable balance, considering that it is not very big, will turn out to be extremely stimulating, while an unfavourable one could soon lead to a condition of persistent economic depression” (keynes j. m., 2009, p. 409). to change the structure of the external trade by increasing the weight of products with high added value and increasing the weight of those with low market value in the import must become an objective, but equally important is the enhancement of competitiveness through quality for the romanian products. depending on the export market, we have the alternative to sell products competitive by their price, by their quality or by their price and quality. the last variant is priority, as romania’s main commercial partners are the ue states, and the economic recession renders the acquisition decisions more difficult to make, even within those economies that everybody knows for their consumerist nature under economic boom. presently, romania has the great opportunity to make the food industry efficient, to enter external markets with products that were, before, appreciated and sold well. the economic recession deeply affects all economies, but each country should learn something out if it. romania should change its foreign trade strategy, use its agricultural potentialities and emphasise again the domestic food industry so that, by domestic production, it could meet the domestic consumption and sell on the foreign markets price and quality competitive products. the opportunities are to be found as well in the fact that the new types of food represent a real concern everywhere in the world. as long as more and more people, especially in the developed states, look for “healthy” products that they cannot find on their own markets, the romanian economy could offer them and enter new niches where it food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 14 might establish romanian brands from the food category. the main factor of economic growth has not been, starting with 2003, the export, nor the investments (it would have influenced the rise of export to a greater extent, as well as the reduction of the value gap between export and import), but domestic consumption. circumstantially, the stimulation of consumption could help the economic growth. on the long run, the effects are not the expected ones, especially under recession conditions. as long as the rise of the aggregate demand involves a rise of the incomes, especially through crediting, and in periods of crisis the financial solvency is significantly diminished, it is impossible for the recovery to be achieved through consumption, given that the economy is not in a situation of quasi-complete occupation of labour force. in such circumstance, priorities must be revaluated. domestic consumption cannot be any more the engine of economic growth, because of the unemployment, of the income and investment reductions. in their turn, the latter will be insufficient, compared to the necessities of economic growth. the small domestic financial resources, meant for investments, are added to the foreign ones1, less and less numerous, because of the problems that the investors are confronted with on other markets, as well as because of the high country risk that transforms romania into an unattractive destination. when the investments and the domestic consumption lose their capacity to support economic growth, the most valid alternative remains export which, under the conditions of 1 direct foreign investments offer advantages on the short and medium run. on the long run, there are risks associated with them (profit repatriation, industry changes of place, leaving behind unemployed, etc.), which are the object of other debates. effectiveness, can ensure the economic dynamism on the long run, from inside to outside. when making the export effective, an important advantage, besides the stimulation of production, the reduction of the unemployment rate, the raising of incomes, the adjustment of the balance of trade and the reduction of the state’s efforts to cover the deficit, the economic growth rate, etc., is the reduction of the dependence upon the outside world, a quite important and necessary aspect. a country is all the more developed as the tertiary sector holds a bigger part in the gdp and as this is more effective; we should also consider that the effectiveness of the sector of services is reflected in the balance of services too. romania makes great efforts to develop the tertiary sector, but, with no intention to minimize the importance of services, we believe the emphasis should rather be placed on the development of the activities which are characteristic for the primary and secondary sectors, where the romanian economic potentialities are higher and where the national economy has had a long tradition. let us not forget that we have entered, for some years, a new phase of economic and social evolution, based upon knowledge. in a knowledge based economy and society, any activity must be motivated by our know-how. rather than making huge efforts to develop much too costly activities or activities we have just been learning how to make, we should improve our knowledge and excel in the traditional fields, like agriculture, food industry, textile industry, chemical industry, etc., branches that could bring forth remarkable economic and social advantages. the export is the surplus production in relation to the necessities of the domestic market, so exportation implies, first of all, from a theoretical point of view, meeting the domestic demand for a certain product food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 15 and utilizing the surplus on the tertiary markets. the most complex relationship with the international trade is fulfilled by the rationalized or integrated production at an international level (miron d., p. 156). for the romanian economy, to achieve supplementary production also means creating new workplaces. given the low engineering extent of the romanian economy, the substantial reduction of investments, both internal and direct foreign ones, as well as the difficulties that the entrepreneurs face as far as the possibility to acquire an investment credit (the risk to acquire an investment credit is quite high in periods of recession, both for banks and for investors), together with the incertitude concerning the possibility to amortize the investments, accompany the decision to raise the agricultural and industrial production, in order to sell it on the foreign markets, on the short run, with the creation of workplaces and the pertaining unemployment decrease. the export rise has a significant impact on the labour market, under the circumstances of a massive increasing of the unemployment rate and of an economic growth that makes it difficult to absorb (if not impossible, on the short run). the higher the working active population’s weight (in the sense that they have a workplace) is, the higher the number of the taxpayers to the state budget is. the income rise, when the expenses are consistently kept up, will reduce the budgetary deficit and ease the authorities’ efforts to cover it or it will allow a rise of the expenses for the investments creating workplaces and value added. the relation export-unemployment does not have implications exclusively on the state’s incomes, but also on the population’s incomes. any rise in the population’s incomes is reflected, according to keynes’ fundamental psychological law, in the rise of the consumption. any rise of consumption, in its turn, will unavoidably attract the rise of the supply. as long as the supply rise will be supported by the domestic production, and not by import, the economy will get a further impetus. in the long run, the raising of the production for exportation will stimulate investments, given that competitiveness by price, based on the abundant and cheap labour force, will not be an eternal economic advantage of romania, especially on the markets of the developed countries. to stay on the foreign markets, and to enter new market niches, the exporters will have to take measures meant to improve their supply and even innovate in order to discover novel products for the foreign consumers (these can very well be traditional products). undoubtedly, the export and investments are the main factors lying at the basis of economic growth and development, being interlinked and interdependent, as long as the export will be an economic policy priority. export, once stimulated, leads, sooner or later, to the investment rise, and investments allow the raising of surplus production in relation to the domestic necessities, a surplus that must be capitalized on the tertiary markets or can be exclusively destined to external sale. according to michael porter’s theory of the competitive advantage, foreign trade is a source of benefits for the country that, comparing to its partners, exchange products which are not necessarily abundant, but are made by strong companies, which managed to assimilate technical progress. starting from this theory, it is easy to deduce that any country’s objectives of growth and development can be reached when the priority, at the level of the economic policy, is the investments as well as the export. if we look at the example of the countries with a tradition of export, like japan, germany or the newly industrialized countries in asia, we notice food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 16 that the development of production for exportation is not made in the detriment of the domestic market. given the demonstrated advantages of export, the foreign trade activity will surely be the barometer to announce the economic recovery, as this is the main link between the domestic and the international economy. under the present circumstances, the romanian producers can opt to revive their activities through exportation, considering that the domestic market cannot represent a solid basis anymore, having been affected by the major decrease of the purchasing power, and of internal demand. conclusions the export, a component of the activity of foreign trade, is tightly related to the economic growth and development, evolving from a mere trading action to a particularly complex process, institutionalized at the national and international level. the economic rise and development of many countries, more or less industrialized, are supported by exportation as long as they are adequate ones, focusing on quality, productivity and costs. generally, export leads to the maximum of economic advantage (rising of competitiveness, of incomes, of investments, of life standard; the decreasing of external debts, of unemployment, of dependence upon outside world), to economic and social progress, through its multiplying effect. the “japanese miracle” is the most illustrative example for the confirmation of the outcomes of export promotion strategy. combining labour force with the financial capital, adequate technology and a good export strategy, one can get the satisfactory formula to achieve macroeconomic objectives, romania included. we should meditate upon yielding the acquisition of commodities from outside, commodities that could be made, in significant quantities of high quality, on the domestic market. this entails, at the level of the economic policy, measures meant to support the domestic producers, an effort to improve the price/quality ratio (the most difficult and costly issue, probably), measure to regain the romanian consumers’ trust in domestic products and the directing of attention towards traditional branches that made us known: agriculture and food industry, without neglecting the other branches of the light industry. given the multitude of advantages of export, this should become a priority among the national economic policies, keeping thus romania, by these means, from becoming a simple market for the production of other countries, fully dependent upon them. acknowledgement: this paper is supported by the sectorial operational programme human resources development (sop hrd), financed from the european social fund and by the romanian government under the contract number posdru id 56815” references 1. amartya s., dezvoltarea ca libertate, editura economica, bucureşti, 2004; 2. berinde m., regionalism şi multinaţionalism în comerţul internaţional, editura universitaţii oradea, 2004; 3. ciupangea c., fundamentele teoretice ale comerţului internaţional, editura economica, bucureşti, 2001; 4. haller, a., p., exporturile factor de dezvoltare şi creştere economică, editura performantica, iaşi, 2008; 5. keynes, j., m., teoria generală a ocupării forţei de muncă, a dobânzii şi a banilor, editura publica, bucureşti, 2009; 6. miron, d., comerţ internaţional, editura ase, bucureşti, 2003; 7. olaru l., promovara exportului, componentă esentială a strategiei naţionale de comerţ exterior, editura economică, bucureşti, 2005. microsoft word 3 journal nr 4 2010 din 8 noiembrie_70-75.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 74 iron fortification of bread and baked goods as against nutritional deficiencies of population mioara negoiţă1, monica catană1, luminiţa catană1, enuţa iorga1, gabriela lilios2, alina bâlea1, valentin ionescu1 1national institute of research&development for food bioresources – iba bucharest, 021102, bucharest 2, 6 dinu vintila street, romania, e-mail: mioaranegoita@yahoo.com 2ovidius university, 900527, constanţa, 124 mamaia street, romania, e-mail: liliosgabriela@yahoo.com abstract: in romania, according to the studies achieved by u.n.i.c.e.f., health and family ministry, institute of mother and child care “alfred rusescu”, about 50% of 2-year aged children and about 30% of those 5-year aged have ferriprive anaemia (determined by iron deficiency). also, according to the same studies about 25% of pregnant women and about 32% of those who suckle, have iron deficiencies and ferriprive anaemia. in this paper some results of the performed researches on the achievement of bread and some baked goods fortified with iron (rolls with sun flower and sesame seeds and poppy sticks) are presented. as fortification agents, ferrous sulfate, ferrous lactate and ferrous gluconate, and the fortification levels were 20 mg fe/kg flour, 40 mg fe/kg flour, 60 mg fe/kg flour, 80 mg fe/kg flour were used. taking into consideration the phytase role in phytates hydrolysis and increasing of iron bioavailability into human body, in bread and baked goods composition some standardised fungal phytase was added. also, because the ascorbic acid is a promoter of the iron absorption into human body, it was used in composition of the achieved products. bread and baked goods fortified with iron were analysed from sensorial, physic-chemical and microbiological point of views. the used fortification agents did not modify the product sensorial characteristics (appearance, colour, taste and smell), in comparison with control sample (bread and baked goods non-fortified with iron). the iron content of the achieved baked goods varied in the range 3.08 – 7.53 mg fe/100g. key words: iron, content, bread, roll, poppy stick introduction in romania, according to the studies achieved by u.n.i.c.e.f., health and family ministry, institute of mother and child care “alfred russescu”, about 50% of 2-year aged children and about 30% of those 5-year aged have ferriprive anaemia (determined by iron deficiency). also, according to the same studies about 25% of pregnant women and about 32% of those who suckle, have iron deficiencies and ferriprive anaemia [1]. the enrichment of food products is an essential element of nutrition strategies for correction of micro-nutrient deficiencies. in generally, fortification of food products is acceptable from the social point of view and it is not necessary to change food habits, it does not modify the qualitative characteristics of the product, it can be easy introduced and it has nutritional advantages for task groups, it is safe from the nutritional point of view and it is economical [2]. because bakery products have a special place within the nutrition of population in our country, it is obviously that all of these have a special interest in iron fortification. the iron compounds used for fortification food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 75 have to have a relative high bioavailability grade, have to be stable and do not cause adversely sensorial modification of products [3]. at the international level, important researches on the development of iron fortification technologies of food products [4] have been carried out. in this paper the results of the performed researches on the achievement of bread and some baked goods fortified with iron (rolls with sun flower and sesame seeds and poppy sticks) are presented. as fortification agents, ferrous sulphate, ferrous lactate and ferrous gluconate, and the fortification levels were 20 – 80 mg fe/kg flour were used. experimental the experiments to achieve iron fortified food products were performed within the micro-production pilot plant of the national institute of research&development for food bioresources – iba bucharest. in order to obtain food products fortified with iron, more experimental variants were made, the variable factors being: iron fortification agent iron fortification level ascorbic acid fortification level as fortification agents of the achieved products, we used ferrous sulphate, ferrous lactate and ferrous gluconate. in the case of baked goods, the fortification levels were of 20 mg fe/kg flour, 40 mg fe/kg flour, 60 mg fe/kg flour, 80 mg fe/kg flour. to achieve bakery products fortified with iron, the following raw materials and materials were used: wheat flour 650, wheat flour 1250, powder milk, margarine, oil, whole egg powder, yeast, sun flower, sesame and poppy seeds, salt, standardized fungal phytase, ascorbic acid, ferrous sulphate, ferrous gluconate, ferrous lactate, polypropylene bags. figure. 1 flour type 650 flour type 1250 to achieve bakery products fortified with iron a bi-phasic method was used, because this method assures higher content of soluble iron, bio-available in human body. the iron content of the fortified products with iron, achieved within the national institute of research&development for food bioresources – iba bucharest, was determined through atomic absorption spectrometry (λ = 248.3 nm), from samples mineralised on dry way. an atomic absorption spectrometer aanalyst 400, with a lamp with cathode for fe was used. two samples for each iron fortified product were analyzed and the arithmetic average of iron content was calculated. results and discussion fortification of food products is legislated through regulation (ec) no. 1925/2006 of european parliament and council, on 20 december 2006. this document specifies the following: requirements concerning the addition of vitamins and minerals, restrictions concerning addition of vitamins and minerals and vitamins and minerals sources which can be added in food products. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 76 in the case of wheat flour fortification, in order to achieve bakery products with high nutritional value, two aspects should be taken into consideration. the first one refers to the establishment of an iron level low enough, thus through consumption of some important bread quantities and it does not imply risks for adversely effects in the human body (gastro-intestinal undesired effects, especially). at the same time, the iron level in wheat flour has to be high enough, so that the following nutritional benefits should be met: prevention and diet-therapy of iron deficiencies, of vulnerable population groups. in this sense, the tolerable superior limit of iron intake is used, that is the biggest daily iron intake which determines no risk or adversely effects on the health of population majority. food and nutrition board in u.s.a. has established in 2002 a tolerable superior limit of 40 mg iron in the case of patients with ages until 13 years and 45 mg iron, in the case of those with ages higher than 14 years, respectively. the tolerable superior limit of 45 mg iron is applied also in the case of pregnant women and of those who suckle. as critical final point of iron intake, the moment of gastro-intestinal disorders appearance has been chosen. flour quantity daily consumed by a person is very much variable according to food habits. for a caloric intake of 2000 kcal/day to be achieved exclusively from flour, a person has to consume 540 g flour/day. taking into consideration this aspect and the tolerable superior limit of iron intake (45 mg/day), nutritionists established the safe maximum limit of iron in flour at 83 mg fe/kg. within the national institute of research&development for food bioresources – iba bucharest, at laboratory level, three bakery products, fortified with iron were made:  "iron fortified bread"  "roll with sun flower and sesame seeds, fortified with iron"  "poppy stick fortified with iron" taking into consideration the safe maximum limit of iron in flour (83 mg fe/kg), within the performed experiments, the following fortification levels with iron were used: 20 mg fe/kg flour, 40 mg fe/kg flour, 60 mg fe/kg flour, 80 mg fe/kg flour. considering the phytase role in hydrolysis of phytates in increasing iron bioavailability in human body, in the composition of bakery products standardized fungal phytase (0.1 g/kg flour) was added. also, because ascorbic acid is a promoter of iron absorption in human body, it was used in the composition of bakery products fortified with iron (0.25 g/kg flour). for each bakery product, fortified with iron, we made alongside control sample (bakery product unfortified with iron), 12 experimental variants (3 iron fortification agents, 4 fortification levels with iron and ascorbic acid). these ones were analysed from the sensorial, biochemical and microbiological point of views. the sensorial analysis of bakery products fortified with iron showed that in the case of all experimental variants, the used fortification agents (ferrous sulphate, ferrous lactate, ferrous gluconate) do not lead to modification of the sensorial characteristics (appearance, colour, taste and smell), in comparison with the control sample. thus, bakery products fortified with iron comply with the provisions of the sp 3232 – 97 „white bread”, sp 1489 – 97 „bakery products from wheat flour”. in the case of those 12 experimental variants, the volume of iron fortified bread is in the range 362 – 392 cm3/100g, and acidity is in the range 0.97 – 1.20 degrees, being below the provisions of sp 3232 – 97 „white bread”. also, within the achieved experimental variants, the iron content of iron fortified bread varied in the range 3.02 – 7.45 mg fe/100g. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 77 applying an american method used within the american institute of baking (kansas), the iron fortified breads were given scores depending on the sensorial characteristics such as: volume, lateral cracks, crust colour, crumb colour, porosity, texture and flavour. using this method for sensorial analysis, iron fortified breads achieved within experiments were given scores higher than 94 points. score of iron fortified bread 96 98 98 95 96 97 97 96 95 95 95 94 99 control p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11 p12 score of iron fortified bread p1-p4 – ferrous sulfate p5p8 – ferrous lactate p9 p12 – ferrous gluconate figure 2. score of iron fortified bread the maximum score was obtained in the case of experimental variant p12 (bread with white flour type 650, fortification level 80 mg/kg flour, fortification agent – ferrous gluconate). figure 3. iron fortified bread p12 figure 4. iron fortified bread p12 0 1 2 3 4 5 6 7 8 ir on (m g/ 10 0g ) c v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 v11 v12 variation of iron content in"iron fortified bread" product iron (mg/100g) v1 v4 ferrous s ulf ate v5v8 ferro us la ctate v9v12 ferrous gl uco nate figure 5. variation of iron content in "iron fortified bread" product in the case of 12 experimental variants, the volume of rolls with sun flower and sesame seeds, fortified with iron is in the range 350 – 389 cm3/100 g, and acidity is in the range 1.42 1.56 degrees, complying with the provisions of the sp 1489 – 97 „bakery products from wheat flour”. also, within experimental variants achieved, the iron content of rolls with sun flower and sesame seeds, fortified with iron, is in the range 3.20 – 7.53 mg fe/100 g. figure 6. roll with sun flower and sesame seeds, fortified with iron – p7 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 78 figure 7. roll with sun flower and sesame seeds, fortified with iron – p7 0 1 2 3 4 5 6 7 8 iro n, m g /1 00 g c v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 v11 v12 variation of iron content in "roll with sun flower and sesame seeds, fortified with iron" product iron (mg/100g) v1v4 fe rrou s sul phate v5v8 fe rrou s lactate v9v12 fe rrous gl uconate figure 8. variation of iron content in "roll with sun flower and sesame seeds, fortified with iron" product in the case of those 12 experimental variants, the volume of poppy sticks fortified with iron, is in the range 309 – 390 cm3/100 g, and acidity is in the range 1.67 1.92 degrees, complying with the provisions of the sp 1489 – 97 „bakery products from wheat flour”. also, within experimental variants achieved, the iron content of poppy sticks fortified with iron is in the range 3.08 – 7.50 mg fe/100 g. figure 9. poppy stick fortified with iron (ferrous sulfate) figure 10. poppy stick fortified with iron 0 1 2 3 4 5 6 7 8 ir on (m g/ 10 0 g) c v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 v 11 v 12 variation of iron content in "poppy stick fortified with iron"product iron (mg/100g) v1v4 fe rrous sulfate v5v8 fe rrous l actate v9v12 ferrous gluconate figure 11. variation of iron content in "poppy stick fortified with iron" product bread and baked goods fortified with iron can be used for prevention and diet therapy of iron deficiencies of vulnerable groups within population (children, teenagers, pregnant women). conclusion 1. in the national institute of research&development for food bioresources – iba bucharest three bakery products, fortified with iron "iron fortified bread", "roll with sun flower and sesame seeds, fortified with iron", "poppy stick fortified with iron" were made. 2. the use of fortification agents has not determined modification of sensorial characteristics (appearance, colour, taste and smell), of baked goods fortified with iron, in comparison with those of control products. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 79 3. the iron content of the achieved baked goods varied in the range 3.02 – 7.53 mg fe/100g. 4. bread and baked goods fortified with iron, can be used for prevention and diet therapy of iron deficiencies of vulnerable groups within population (children, teenagers, pregnant women). acknowledgments the experiments were performed within the contract no. 51-092/18.09.2007, financed through programme 4 “partnerships in priority s&t domains” 2007 – 2013 – national centre for projects management. references 1. unicef – românia, statusul nutritional al femeii gravide, al copiilor cu vârstă sub cinci ani, al şcolarilor cu vârste de 5-7 ani, vol. i, ii, iii, editura marlink, bucureşti, 2006 2. marin v. şi colab., fortificarea făinii, ghid pentru industria de morărit, editura uranus, bucureşti, 2007 3. ciobanu corina, cercetări privind influenţa compoziţiei şi a factorilor tehnologici asupra biodisponibilităţii fierului din produse alimentare fortificate, teză de doctorat în ştiinţe tehnice, chişinău, 2007 4. mehansho, h., iron fortification technology development: new approaches, j. nutr. 136, p. 1059-1063, 2006 21 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017, pag. 21 28 assessment of environmental impact of food waste: a case study apple fruits *cristina ghinea1 1faculty of food engineering, stefan cel mare university of suceava, romania cristina.ghinea@fia.usv.ro *corresponding author received february 5th 2017, accepted march 27th 2017 abstract. the aim of this paper was to evaluate the environmental impacts (ei) of the apple supply chain from the ne region, romania and to calculate the ei from apple waste landfilling. the evaluation was performed by applying life cycle assessment (lca) methodology. in the first phase the apple supply chain was investigated and the environmental impacts were calculated and interpreted, while in the second phase the evaluation of apple waste landfilling was performed. the results showed that the apple production phase has the highest contribution to all the impact categories studied (acidification potential (ap), eutrophication potential (ep), global warming potential (gwp), human toxicity potential (htp) and photochemical ozone creation potential (pocp) etc.). transportation and storage stages contribute mainly to pocp and gwp, while landfilling contributes especially to ap, gwp and pocp. keywords: apple, environment, food waste, landfilling, life cycle assessment 1. introduction the management of food loss and waste is a critical aspect for sustainable development and represents a sensitive issue at global level [1]. food waste composition includes waste consisting of parts or entire products such as meat, dairy, cereal, vegetables, fruits and others (fig. 1). these are the main products from consumer basket which can be wasted during different life cycle stage of these products. apples represent 3% of the overall consumer basket in the eu which also includes dairy products (25%), meat (14%), cereal (7%), crop-based products (7%), vegetables (13%), beverages (32%), pre-prepared meals (0.5%) etc. [2]. the food can be loss (which is equal with food wastage) avoidable and unavoidable (such as seeds) during the primary production stage (edible products left in the field and others), transport and storage (for example product damaged by machineries, storage at a wrong temperature etc.), processing (food damaged by inappropriate packaging), distribution (expired food, unsold food, rejected food after quality controls etc.) and consumption (unavoidable loss, food not eaten or others) [2, 3]. the food waste hierarchy was developed in order to prioritize efforts to reduce food waste (from the last favorable to the most favorable option) (fig. 2) [4, 5]. the environmental impacts of food waste disposal were evaluated and discussed in various studies, for example: -notarnicola et al. [2] investigated the environmental impacts of the most representative food types consumed in the eu-27 in 2010 and showed that food is http://www.fia.usv.ro/fiajournal mailto:cristina.ghinea@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 22 wasted in both the agricultural/industrial and domestic phases; fig. 1. solid waste fractions fig. 2. food wastage hierarchy corrado et al. [3] provided a preliminary analysis of food loss by lca modeling, and suggested a definition for food loss, discussed the strengths and weaknesses of various approaches for food loss in the supply chain and provided some recommendations for lca practitioners; salemdeeb et al. [5] studied food waste utilization for animal feed and compared the environmental and health impacts of different technologies such as composting, anaerobic digestion, dry pig feed and wet pig feed. in this study the production, consumption and loss of apple fruits were investigated and evaluated from the environmental point of view. the method chosen for elimination of apple waste was landfilling. 2. production, consumption and waste of apples apple tree is one of the most common fruit tree cultivated in europe and covers 450000 ha. the area cultivated with apple in romania represents 11% of the total production area of the eu [6, 7]. total area planted with apple trees in 2012 in romania was 51225.7 ha from which 6149.9 ha in ne region [8]. the apple production in eu is ranked differently from the area covered by apple trees: poland harvested a quarter of the eu's apple production, followed by italy (19.2 %), france (15.5 %), germany (7.7 %) and romania only 3.6 % [7]. the areas cultivated in romania with apple orchards and apple productions in 2012 in ne region are illustrated in fig. 3. fruit cultivation has a long tradition in romania and holds an important place in agriculture. the main species of fruit trees cultivated are: apple, pear, apricot, peach and nectarine. in our country about 100 varieties of apple grow, the most important ones being: jonathan and golden delicious (69.5%) followed by starkrimson, idared, florina, parmen gold, etc. [8]. apple food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 23 consumption values per capita in eu in 2015 were: austria (53 kg), slovenia (36 kg), netherlands (34 kg), hungary (31 kg), luxembourg (26 kg), croatia (25 kg), portugal (24 kg) and romania (23.5 kg) [9]. fig. 4 illustrates the average annual consumption per capita in romania for fruits and apples [10]. fig. 3. areas cultivated in romania with apple orchards and apple production in 2012 in ne region fig. 4. average annual consumption per capita fruits along with vegetables are recommended in daily consumption (400800 g/day), in europe average fruits consumption accounts for 166 g/day, but the consumption varies from a region to another [9]. according to [11] one medium apple with skin (192 g) provides 95 kcal, 19 g total sugars, 4 g dietary fiber and 195 mg k. [12] investigated various type of apples cultivated in romania and established the following contents for jonathan apple: 14.29% dry matter total content, 85.71 % water content, 9.84% sugar, 7.66% vitamin c/ 100 g fruit and ash content 2.38%. apple loss and waste through the supply chain can be: field loss (5-25%), grading loss (5-25%), storage loss (3-4%), packing loss (3-8%) and retail waste (2-3%) [13]. 3. life cycle assessment 3.1. methodology, system boundaries, functional unit life cycle assessment which is a standardized methodology was selected and applied to calculate and interpret the environmental impacts generated by apple fruit production, transport, processing, consumption and waste disposal. the modeling was performed with gabi software which is an instrument that includes lca methodology and allows environmental impacts calculation of complex process [14, 15]. the system boundaries considered in this evaluation are illustrated in fig. 5. the functional units considered are the amount of apple produced in 2012 in the 0 10 20 30 40 50 60 70 80 90 kg 2010 2011 2012 2013 2014 2015 av e rage annual consumption pe r capita fruits apples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 24 ne region of romania (for the supply chain, in the first part of the study) and the quantity of food waste resulted from distribution and consumption stages (for landfilling). fig. 5. system boundaries 3.2. inventory analysis in the inventory analysis phase the inputs and outputs for each stage included in the supply chain were established and calculated. the amount of apple produced in 2012 was of 84400 t. the apple losses considered are: the field loss 6%, transport loss 3%, processing loss 13% and consumption loss 6%. in 2012, 60876 t of apple were consumed in the ne region of romania. quantities of organic and mineral fertilizers were calculated based on the literature data. [2] considered the following amounts of fertilizers used in apple production: n 62 kg/ha, p2o5 4 kg/ha, k2o 47 kg/ha, lime fertilizer 52 kg/ha. emissions to air can be n2o (direct and indirect emissions from n fertilizers) 1 kg/ha, nh3 from n fertilizers 7.5 kg/ha, co2 from fertilizers 43.3 kg/ha, while emissions to water no3 from n fertilizers 82.4 kg/ha, p from fertilizers 0.1 kg/ha and emissions to soil: pesticides (chlorpyrifos 0.35 kg/ha, glyphosate 0.28 kg/ha, mancozeb 1.5 kg/ha, mineral oil 1.6 kg/ha sulfur 1.68 kg/ha) according to [2]. the total amount of diesel consumed for the production phase was calculated knowing the quantities of diesel used for plowing (4.3 l/ha), spraying herbicides food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 25 (1.6 l/ha), phytosanitary treatments (38.4 l/ha), orchard maintenance (70.8 l/ha), technological transport (44 l/ha) etc. [16]. emissions in air (co2, co, nox, n2o, pm10, ch4, so2 and hydrocarbons) from fuel consumption were calculated based on the emission resulted from burning 1 kg of diesel. the electricity consumed for apple sorting considered was 0.5 kwh/t [17]. also, cardboard packaging and plastic films amounts were calculated. 3.3. life cycle impact assessment, results and discussion all the data obtained in the inventory step were introduced in gabi software, where the potential environmental impacts are calculated based on plan, process and the inputs and outputs related to the system. impact categories like acidification potential (ap), eutrophication potential (ep), global warming potential (gwp), human toxicity potential (htp) and photochemical ozone creation potential (pocp) can be analyzed and calculated with gabi tool [14]. emissions of nitrogen oxides (nox), sulphur dioxide (so2) and ammonia (nh3) contribute to acidification. in lca the acidification potential (ap) is given in kg sulphur dioxide equivalents (so2 equivalents). emissions of nox, nh3, p and n contribute to eutrophication potential which is expressed in kg 34po equivalents in lca. the substances which may contribute to the gwp are: co2, co, ch4, n2o, cfcs etc. in lca the gwp is expressed in kg co2 equivalents. human toxicity potential is related with voc, particles, heavy metals, pops, nox, so2 emissions and can be expressed in kg dcb equivalents. volatile organic compounds (vocs) and carbon monoxide (co) are degraded in reactions with nitrogen oxides (nox) initiated by sunlight in the lower atmosphere which leads to photochemical ozone formation (pocp) expressed in lca as kg c2h5equivalents [18, 19]. after evaluation, the results were obtained by several impact assessment methods. this paper presents and focus on the results obtained with cml 2001-jan. 2016, recipe 1.08 and ubp (ecological scarcity) methods. the values obtained for each category of impact in different measurement units were normalized in order to compare and illustrate on a single graphic the environmental impact categories. fig. 6 show the normalized values (in pe = person equivalents) obtained for the following impact categories ap, ep, gwp, htp and pocp. it can be observed that all the impact categories have positive values which mean negative impacts on the environment. ap has the highest value followed by pocp, ep and htp, the lowest value being recorded for gwp. acidifying substances such as so2 and nox which contribute to the acidification potential come mainly from the burning of fossil fuels [20]. in this study the emission of substances which contribute to ap are due to diesel burning used for plowing, treatments, transport and distribution etc. emissions with a slower contribution to ap come from n fertilizers application. fig. 6. environmental impacts of apple fruit supply chain 0.00e+00 2.00e+08 4.00e+08 6.00e+08 8.00e+08 1.00e+09 1.20e+09 1.40e+09 1.60e+09 1.80e+09 2.00e+09 pe ap ep gwp htp pocp impa ct categories cml 2001jan.2016 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 26 fig. 7 presents the contribution of production, distribution, transport and storage phases and also diesel and electricity consumption to the ap, ep, gwp, htp and pocp. it can be observed that apple production phase has the highest contribution to the all impact categories (almost 90 % for ap and ep; 42% for htp). fig. 7. contribution to the environmental impacts of the production, distribution, transport and storage phases and diesel, electricity consumption diesel influence mainly the values of htp (50%), pocp (21%) and in a lower degree gwp, ap and ep. emission from transportation and storage contribute mainly to pocp (16.5%) and gwp (13.2%), having a lower contribution to the other impact categories. fig. 8 shows the normalized values obtained for climate change impact category with recipe method, based on three cultural perspectives: egalitarian (e), hierarchist (h), individualist (i). egalitarian principle considers long term aspects based on precautionary principle thinking, individualist principle is based on short term and is characterized by optimism aspects that technology can avoid many problems in future while hierarchist is based on consensus model often considered the default model [21]. from fig. 8 it can be observed that all the values for climate change impact categories are positive which means negative impacts on the environment. the highest values are registered for climate change impact on human health. also, fig. 8 shows that the hierarchist perspective provides middle values compared with the other two perspectives. fig. 9 presents the contribution of phases from apple supply chain to the environmental impacts according to ubp ecological scarcity method. production and diesel contributes to water pollution, while the amount of pesticides that enter the soil is influenced entirely by the production phase. the main air pollutants come from production, transport and storage phases. production phase contributes to the global warming in proportion of 75%, followed by transport and storage (13%). landfilling of apple waste amounts resulted mainly from distribution and consumption was also evaluated. the amount of leachate and biogas which may result from this type of food waste were calculated, also the emission in air, water and soil were estimated. after the calculation of environmental impacts was observed that the emissions from landfilling contribute mainly to ap, gwp and pocp (fig. 10). fig. 8. impact on climate change of apple fruit supply chain (ccheclimate change ecosystems; cchhclimate change human health) 0% 20% 40% 60% 80% 100% ap ep gw p htp pocp production diesel electricity distribution transport and storage 0.00e+00 5.00e+02 1.00e+03 1.50e+03 2.00e+03 2.50e+03 pe e h i recipe, endpoint cche cchh food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 27 fig. 9. contribution of phases from apple supply chain to the environmental impacts according to ubp ecological scarcity method fig. 10. environmental impacts of food waste landfilling 4. conclusions in this paper the environmental impacts of apple supply chain were investigated. life cycle assessment methodology was applied for environmental impacts calculation. the results obtained showed that the production phase has the highest impacts on the environment followed by transport and storage phases. substance emission during the apple supply chain contributes mainly to acidification potential. the amounts of apple loss during the chain were also calculated. landfilling was considered the elimination method for apple waste. in the next study different scenarios for apple loss/waste treatment will be developed (considering that this type of waste can be used to feed animals, can be composted or disposed in landfills) and evaluated and compared from the environmental point of view. 5. acknowledgments this paper was performed with the support of gabi education life cycle assessment software. 6. references [1]. ghinea c., gavrilescu m., impact of food waste on climate change, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava, xiv, (4): 340 – 344, (2015). [2]. notarnicola b., tassielli g., renzulli p.a., castellani v., sala s., environmental impacts of food consumption in europe, journal of cleaner production, 140: 753765, (2017). [3]. corrado s., ardente f., sala s., saouter e., modelling of food loss within life cycle assessment: from current practice towards a systematisation, journal of cleaner production, 140: 847-859, (2017). [4]. papargyropoulou e., lozano r., steinberger k., wright j., ujang n., bin z., the food waste hierarchy as a framework for the management of food surplus and food waste, journal of cleaner production, 76: 106-115, (2014). [5]. salemdeeb r., zu ermgassen e.k.h.j., kim m.h., balmford a., altabbaa a., environmental and health impacts of using food waste as animal feed: a comparative analysis of food waste management options, journal of cleaner production, 140: 871-880, (2017). [6]. badiu d., arion f.h., muresan i.c., lile r., mitre v., evaluation of economic efficiency of apple orchard investments, sustainability, 7: 10521-1053, (2015). [7]. eurostat, fruit and vegetable sector in the eu – a statistical overview, (2016), on line at: http://ec.europa.eu/eurostat/statisticsexplained/index.php/the_fruit_and_vegetable_sect or_in_the_eu_-_a_statistical_overview. [8]. ins, production potential of the orchards and vineyards for the production of table grapes in 2012, press release, nr. 233 of september 30, 2013, national institute of statistics, (2013). 0% 20% 40% 60% 80% 100% global warming main air pollutants pesticides into soil water pollutants ubp, 2013 production diesel electricity distribution transport and storage 3.60e-07 3.80e-07 4.00e-07 4.20e-07 4.40e-07 4.60e-07 4.80e-07 5.00e-07 pe ap gwp pocp impact categories cml 2001jan. 2016 http://ec.europa.eu/eurostat/statisticsfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, volume xvi, issue 1 – 2017, pag. 21 – 28 28 [9]. popescu a., some considerations on the dynamics of fruit and apple production and consumption in romania in the period 2007-2014, scientific papers series management, economic engineering in agriculture and rural development, 16: 267-276, (2016). [10]. ins, consumption per capita in romania for fruits and apples, national institute of statistics (2017). [11]. o’neil c.e., nicklas t.a., fulgoni v.l., consumption of apples is associated with a better diet quality and reduced risk of obesity in children: national health and nutrition examination survey (nhanes) 2003–2010, nutrition journal, 14:48, (2015). [12]. campeanu g., neata g., darjanschi g., chemical composition of the fruits of several apple cultivars growth as biological crop, not. bot. hort. agrobot. cluj, 37: 161-16, (2009). [13]. wrap, fruit and vegetable resource maps. mapping fruit and vegetable waste through the retail and wholesale supply chain, (2011). [14]. ghinea c., petraru m., bressers h.th. a., gavrilescu m., environmental evaluation of waste management scenarios – significance of the boundaries, journal of environmental engineering and landscape management, 20(1): 76-85, (2012). [15]. pe international, handbook for life cycle assessment (lca) using the gabi education software package, germany, (2009). [16]. cdep, specific consumption of diesel for mechanical works, (2006), on line at: www.cdep.ro/proiecte/2006/600/20/6/ax2.pdf. [17]. inma, technology for mechanization and technical equipment for conditioning and calibration apples fruit for semi-subsistence farms, national institute of research and development for machines and installations for agriculture and food industry, (2014). [18]. ghinea c., gavrilescu m., decision support models for solid waste management – an overview, environmental engineering and management journal, 9: 869-880, (2010). [19]. ghinea c., waste management models and their application to sustainable management of recyclable waste, phd thesis, gheorghe asachi technical university of iasi, romania, (2012) [20]. eea, europe's environment the dobris assessment, chapter 31, european environment agency, (1995). [21]. recipe mid/endpoint method, version 1.08 december (2012). http://www.cdep.ro/proiecte/2006/600/20/6/ax2.pdf. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 53 influence of liquefaction temperature upon rheological properties of corn starchy mashes eufrozina albu (niga)1, alina-mihaela psibilschi1 1 stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229, suceava, romania, e-mail: e.albu@fia.usv.ro, alinap@fia.usv.ro abstract: in this paper we studied the effect of liquefaction temperature of the corn starchy mashes upon their rheological properties. for the study two types of corn grind starchy mashes respectively one of fine grind with particles of <0.5 mm and one of rougher grind with particles of > 0.5 mm have been made. the enzymatic package, used in obtaining the two types of starchy mashes, consists of three enzyme types: spezyme® (liquefaction enzyme), stargen 001 (α-amylase and amiloglucosydase for the starch hydrolysis) and fermgen (protease). for the two types of starchy mashes we tested different liquefaction temperatures of 40ºc, 50ºc, 60ºc and 70ºc. the rheological properties of starchy mashes have been analyzed by the viscometer brookfield rv pro ii+ during their obtaining in two phases: before and after the starchy mashes liquefaction. from the study carried out we observed that the mashes obtained from fine corn grind, the liquefaction temperature of 60ºc is more efficient whereas more reduced viscosity being obtained in comparison with the other liquefaction temperature of 40ºc, 50ºc and 70ºc. for the mashes obtained from rough corn grind we observed the fact that the liquefaction temperature of 50ºc is more efficient, more reduced viscosity being obtained in comparison with the other liquefaction temperatures of 40ºc, 60ºc and 70ºc. keywords: viscosity, enzyme, fine grind, rougher grind. introduction the utilization of biomass as the starting material for the production of biofuels has received considerable interest in recent years. starchy and cellulosic materials of plant origin are the most abundant utilizable biomass resources. starchy biomass has to be hydrolyzed either by enzymatic or acid hydrolysis to release fermentable sugars. the enzymatic hydrolysis of starchy material for ethanol production via fermentation consists of two or three steps and requires improvement if it is to make efficient production at low cost [1]. besides the particular traits, the fermentation processes have specific rheological behaviours and rheological characteristics modification in time. knowing the way of flowing of these liquids as well as its evolution during the fermentation time represents an essential condition for obtaining the optimum parameters of the process. the rheological characteristics of the fermentation liquids have a decisive influence upon the biosynthesis process performances by controlling mass and heat transfer, the conditions under which the mixing is made as well as the further separation operations [2]. experimental starch biomass in the fuel bioethanol industry, starch is mainly provided by grains (corn, wheat, or barley). corn, which is the dominant feedstock in the starch-to-ethanol industry worldwide, is composed of 60 to 70% starch. [1] starch, by its two components, has four types of bonds in its structure: α1.4 glycosidic inside amylose and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 54 amylopectin macro-molecules, α-1.4 marginal glycosidic, α-1.3 glycosidic and α-1.6 glycosidic [3]. starch complete hydrolysis into glucose requires enzymes for all four types of bonds [4]. the raw material used for this study is the starchy biomass made from corn mash. enzymes and microorganisms the hydrolysis of starchy raw materials can be done by acids or enzymes, but due to several advantages of enzymatic hydrolysis, it is the preferred choice for industrial applications [5]. enzyme hydrolysis is carried out in two stages [6]: the first stage is called “liquefaction”; the second stage is called “saccharification” the liquefaction enzymes are represented by α-amilase which divides the α-1.4 cross links from the amylose and the amylopectin. the α-amylase is an endohydrolase which acts randomly in the inner side of the amylose chains having as result linear dextrins and even small glucose quantities. in the case of amylopectin the α-amylase forms linear dextrins and branched dextrins with medium and small molecular mass [7]. spezyme® enzyme contains a thermostable starch hydrolyzing α-amylase that is produced by a genetically modified strain of bacillus licheniformis. the endoamylase in spezyme® enzyme hydrolyzes α-1.4 glycosidic bonds to quickly reduce the viscosity of gelatinized starch, producing soluble dextrins and oligosaccharides under a variety of process conditions [8]. recommended conditions [8]: solids 30 to 36% optimal ph 5.7 to 5.8 optimal temperature 83 to 85ºc liquefaction time 90 to 140 minutes dosage recommendation 0.20 to 0.24 kg/mt ds. spezyme® enzyme provides the following benefits to ethanol producers [8]: quick viscosity reduction allowing for higher solids liquefaction ph’s as low as 5.2 process flexibility improved performance at low slurry temperatures for the saccharifying of liquefied mashes, glucoamylase is utilized and this is an endohydrolase which devides the α1.4, α-1.6 and α-1.3 glucosidic cross links from the amylose and the amylopectin [7]. stargentm 001 enzyme contains aspergillus kawachi alpha amylase expressed in trichoderma reesei and glucoamylase from aspergillus niger that word synergistically to hydrolyze granular starch substrate to glucose [9]. the endo-activity, alpha amylase and exo-activity, glucoamylase, in stargentm 001 enzyme catalyzes the complete hydrolysis of granular starch under a variety of alcohol fermentation conditions. recommended conditions [9]: solids 12 to 38% optimal ph 3.0 to 4.5 dosage recommendation 1.0 to 2.5 kg /mt ds. fermgen™ is an acid proteolytic enzyme characterized by its ability to hydrolyze proteins under low ph conditions. the fungal protease is obtained by controlled fermentation of a genetically modified selected strain of trichoderma reesei [10]. recommended conditions: solids 26 to 38% optimal ph 3.5 to 5.0 optimal temperature 28 to 35ºc dosage recommendation 0.1 kg/mt food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 55 study on corn mash viscosity to study the influence of liquefaction temperature upon the rheological properties of corn starchy mashes we used two types of grind. for the first type we used fine corn grind with particles of < 0.5 mm and for the second type we used rougher corn grind with particles of > 0.5 mm. the rheological properties of starchy mashes have been analyzed by the viscometer brookfield rv pro ii+ during their obtaining in two phases: before and after the starchy mashes liquefaction. the two types of mashes were obtained by mixing the grind with water in 1:3 ratio. the mixture obtained was treated by enzymes in two stages: liquefaction and saccharifying. the liquefaction was performed at different temperatures of 40ºc, 50ºc, 60ºc and 70ºc, at a ph of 5.8 for 120 minutes by adding 0.22 kg/mt ds spezyme enzyme. the liquefied mash was saccharified at 34°c, at a ph of 4.5 for 60 minutes by adding 2 kg/mt ds stargen 001enzyme. the saccharified mash was treated at a ph of 4.5 with 0.1 kg/mt fermgen enzyme, to which was added yeast at a ratio of 3 g/l and it was than subjected to fermentation for obtaining bio ethanol fuel. the study was carried out at two different rotation speeds, both 50 rpm and 100 rpm, using ha4 spindle and specific temperature phase. the mashes obtained after previous described method were fermented in bluesens bioreactors with a capacity of one liter which monitoring free oxygen, of leaven ethanol content from mash and free carbon dioxide. the mashes fermented were distilled with a rotoevaporator volume of 500 ml. the analysis of obtained distillate was made by a shimadzu gas chromatograph with flame ionization detector. results and discussion the experimental results obtained for the fine grind mash are presented in table 1. the experimental results obtained for the rougher grind mash are presented in table 2. table 1. the fine grind mashes viscosity according to the liquefaction temperature viscosity before liquefaction, cp viscosity after liquefaction, cp nr. crt. temperature, °c 50 rpm 100 rpm 50 rpm 100 rpm 1. 40 4.97 5.66 4.77 5.48 2. 50 4.97 5.66 4.07 4.4 3. 60 4.97 5.66 3.14 3.88 4. 70 4.97 5.66 3.2 4.01 table 2. the rougher grind mashes viscosity according to the liquefaction temperature viscosity before liquefaction, cp viscosity after liquefaction, cp nr. crt. temperature, °c 50 rpm 100 rpm 50 rpm 100 rpm 1. 40 5.4 6.35 3.85 4.6 2. 50 5.4 6.35 3.97 4.54 3. 60 5.4 6.35 5.28 5.49 4. 70 5.4 6.35 5.41 5.28 in figure 1 and 2 the viscosity values obtained for the two types of mashes at the four liquefaction temperatures, at the two rotation speeds are compared. figure 3 shows the content of ethanol at the end of fermentation for the two types of mashes. the content of ethanol determined by chromatography of the distillate obtained from fermented mashes is shown in figure 4. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 56 5. 48 4. 6 4. 4 4. 54 3. 88 5. 49 4. 01 5. 28 0 1 2 3 4 5 6 v is co si ty , cp 40 50 60 70 t emperature,ºc fin e g rin d 100rpm rou gh er gri nd 100 rpm figure 1. the corn mashes viscosity variation according to the liquefaction temperature at 100 rpm 4. 77 3. 85 4. 07 3. 97 3. 14 5. 22 3. 2 5. 41 0 1 2 3 4 5 6 v is co si ty , c p 40 50 60 70 temperature,ºc fine grind 50rpm rougher grind 50rpm figure 2. the corn mashes viscosity variation according to the liquefaction temperature at 50 rpm figure 3. ethanol content of mashes at the end of the fermentation figure 4. ethanol content of the distillate obtained from fermented mashe conclusions from the study carried out we observed that the mashes obtained from fine corn grind at the liquefaction temperature of 60ºc is more efficient and a more reduced viscosity resulted in comparison with the other liquefaction temperature of 40ºc, 50ºc and 70ºc. for the mashes obtained from rough corn grind we observed the fact that the liquefaction temperature of 50ºc is more efficient and more reduced viscosity resulted in comparison with the other liquefaction temperatures of 40ºc, 60ºc and 70ºc. from figures 3 and 4 one may notice that a higher content in ethanol at 60ºc for the mashes obtained from fine grind and at 50ºc for mashes obtained from rougher grind is registered. references 1. gnansounou, e., dauriat, a. ethanol fuel from biomass: a review, journal of scientific & industrial research, 2005, 64: 809–821. 2. oniscu, c., galaction, a-i., caşcaval, d., comportarea reologică a lichidelor de fermentaţie, romanian biotechnology letter 1996, 1(2), 139. 3. jurcoane, st., tratat de biotehnologie, editura tehnica, bucuresti, 2004, p.360-364. 4. gutt, s., rotar, r., niga, e., psibilschi, a., study on enzyme hydrolysis to obtain bioethanol, annals of the suceava university, food engineering section, year vi, nr. 1-2007, pg.170-174. 5. subhash u nair, sumitra ramachandran, ashok pandey, bioetahnol from starchy biomass, handbook of plant/based biofuels, crc press, 2008. 6. gutt, s., gutt, g., factors influencing the fermentation process and ethanol yield, romanian biotechnological letters, vol. 14, no. 5, 2009, pg. 4648-4657. 7. banu, c., bioalcoolul combustibilul viitorului, editura agir, bucuresti, 2006. 8. spezyme®, alpha-amylase for dry grind ethanol production, genencor, danisco us inc. 2009. 9. stargentm 001, granular starch hydrolyzing enzyme for ethanol production, genencor, danisco us inc. 2005. 10. fermgentm, acid fungal protease enzyme for ethanol production, genencor, danisco us inc. 2006. 7. 71 6. 25 6. 2 7. 64 8 .2 8 7. 11 8. 02 7. 23 0 1 2 3 4 5 6 7 8 9 e th an ol , % vo l 40 50 60 70 temperature, °c fine grind rougher grind 14 0. 51 12 2. 81 12 9. 42 13 1. 66 16 4. 02 12 5 15 2. 2 12 9. 43 0 20 40 60 80 100 120 140 160 180 e th an ol , m g/ m l 40 50 60 70 temperature,°c fine grind rougher grind microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_49-56.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 53 evaluation of mineral element content in grape seed and defatted grape seed silvia mironeasa1, costel mironeasa2, georgiana gabriela codină1 1ştefan cel mare university, faculty of food engineering, 13th university street, 720229, suceava – romania, e-mail address: silviam@usv.ro 2 ştefan cel mare” university, faculty of mechanical engineering, mechatronic and management, suceava – romania 13th university street, 720229, suceava – romania, e-mail address: costel@fim.usv.ro abstract: a study of the grape seed and of the defatted grape seed has been made in order to evaluate the content of mineral elements, with the purpose of investigating the possibility of using them for human/animal consumption. the research was made individually for each grape seed sample, defatted grape seed respectively. the total ash content was determined according to sr iso 2171:2009, as a percent of dry substance. the determination of mineral content was performed by the energy dispersive spectrometer, using röntgen radiation, shimadzu type (edx 900hs) with high resolution and sensibility. the results obtained show that the grape seed and defatted grape seed contain macro elements such as calcium (ca), phosphorus (p), potassium (k) and sulphur (s) and trace elements such as: zinc (zn), copper (cu) and manganese (mn). the seeds of the studied grape varieties and the defatted grape seed have proven to be good sources of mineral elements. both varieties of seeds can be considered an important source used for fortifying aliments in order to obtain food products with higher nutritional characteristics. at the same time, these products can be valued to obtain new products which result in diminishing waste. the data regarding the mineral content of the grape seeds are useful for the formulation of new diets and food processing techniques. keywords: cakes, x-ray, fluorescence spectrometer introduction the study of the physical and mechanical properties of grape seeds, including the chemical composition and obtaining new products through the active natural components are the source of many researches. the grape seed contains: oil, protein, cellulose, mineral elements and other components, with benefits for the human health [1]. in larger or smaller quantities, such as salts (cations and ions), especially in the solid part of the grape–peel, seeds, the cellular walls of the core, soluble elements from the soil can be found [2]. mineral elements play an important role in the human metabolism and the interest for investigating them is rising [3, 4]. although, they do not produce energy, the mineral elements have an important role in many activities of the human body [5, 6] and are necessary in order to maintain certain chemo-physical processes which are essential for life. mineral elements represent inorganic components which are vital because they contribute to the normal development of vital activities and to the development of organism. as ions are linked to vitamins, minerals play important functional roles of metabolic nature, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 54 activating numerous enzymatic systems, of chemo-physical nature, controlling the ph, electrical neutrality, and the gradients of the chemical potential [7]. the mineral substances from vegetables and fruit help the ossification of the skeleton, growth, maintaining different functions of the internal secretion glands and the forming of blood. the mineral elements are a part of the chemical composition of enzymes, pigments, nucleic acids, peptic substances. these have an important role in nutrition because they are necessary in the development of the metabolic processes in the human body. no matter of quantity existing in the human body, mineral elements are essential because the body cannot synthesize or replace them. for this reason, they must be consumed together with nourishment [8]. in accordance with the quantity that exists in the organism, mineral elements are classified into three categories: macro elements, microelements and ultra microelements. macro elements include: calcium, phosphorus, sodium and chloride and microelements include iron, copper, cobalt, potassium, magnesium, iodine, zinc, manganese, molybdenum, fluoride, chromium, selenium and sulphur [6]. macro-minerals are necessary for the organism in larger quantity than 100 mg/day and micro-minerals, in smaller quantities than 100 mg/day [9]. the lack and the excess of macro elements produce metabolic disorders. trace elements are essential in the maturation, activation and functions of host defence mechanism [10; 11]. essential components of the enzymatic systems, biological and of structural portions of the biologically active components [12] are used by the immune system in order to face infectious agents [13, 14]. ultra micro elements can be found as traces and include boron, silicon, arsenic, nickel, cadmium, lead, tin, lithium, vanadium, uranium, radium, thorium and a part of these have an essential metabolic effect. as regards cadmium, lead, tin, lithium and vanadium there is not enough evidence that could indicate their essential role in the organism [15]. the diversity of mineral elements which exist in the seed constitutes an important source of macro and microelements which are necessary in covering the needs of the human body [16] and contributes to the increase of food value of the products obtained from these. an exception is made by the contaminating materials (lead, cadmium), which can appear in seeds due to the pollution of soil, water, in contact with processing equipment and are toxic for the organism. analyzed macro elements and their role in the organism calcium (ca) is present in the organism in larger quantities than other mineral substances. together with phosphor, calcium contributes to the formation of the skeleton and teeth and insures their growth. ca is important in the formation and consolidation of the cellular walls, and maintains the structure and permeability of the membrane, activating the enzymes [17]. it facilitates the normal activity of the neuromuscular system, of the heart, it intervenes in the blood coagulation process, it increases the water in protoplasm and the possibility of synthesizing cellular albumin. ca decreases the size of allergic processes, it increases the resistance of organism to infectious diseases, activates an important number of metabolic processes, stimulates the enzymatic equipment of the organism, regulates energetic processes and tissue regeneration, participates in the stimulation of gastric secretion, the use of iron and the absorption of vitamin b12 [18]. potassium (k) is an essential nutrient for the life and the human health. in the organism, k participates in the water circulation process, intervenes in the muscular and myocardial contraction, participates in the protein synthesis and it is a diuretic. potassium ions participate in the forming of acetylcholine and in the transmission of nervous excitation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 55 to the muscles. the increase of k in the organism will result in lower sodium and it emphasizes its elimination because there is a conflict between the potassium and sodium metabolism. the lack or insufficiency of k determines the growth of enzymes which distorts the nutritional balance, especially through the collapse of the phosphorylation process [8]. this maintains the acid-basic balance of the living cell, activating a series of enzymes. the components of k influence the tissue colloids. through the reduction of the hydration level of proteins from tissues, these determine the elimination of liquid from the organism [7]. phosphorus (p) is a part of the nucleic amino acids structure, of the phospholipids and participates in the division and multiplication process of cells. in living matter, phosphorous has an important role in growth. in medicine, p is used to treat rickets, functional disorder of the nerve system [19]. p is a partner of the majority of oxidation stages of sugar and lipids. these are a part of adenosine triphosphate (atp) structure, made of a high quantity of energy, which plays an essential role in the release of energy from food products and supplies the energy necessary for the organism to function. moreover, p has an important role in the activity of vitamins from group b, the majority of these become active only after combining with phosphoric acid [7]. sulphur (s) is present in all the cells of the organism. it is a component of: amino acids with sulphur, vitamin b1, biotin, coenzyme a and the insulin hormone. this mineral element has an important role in the process of substance exchange of the organism and especially in detoxification. s participates in expiration, has a favourable action in a balanced diet. through the bridges s-s, sulphur is one of the major elements of the protein tertiary structure, and the radicals -sh are involved in the activity of some enzymes. s represents an integrated part of the food protein. analyzed micro elements and their role in the organism iron (fe) is a component of vegetal and animal organisms and it participates in metabolic oxidation. this element is a part of the chemical composition of some enzymes and participates in the substance exchange of the organism; it is an oxygen carrier and plays an important role in cellular respiration. fe balances the blood forming processes. the value of a food product as a fe source is influenced more by the chemical state of this micro element than the total content of fe. solubility, slight ionization and the fe balance state are properties which determine the absorption of fe. ascorbic acid, sulphide groups and other reduction substances, and normal gastric acidity facilitate the absorption of integrated iron [7]. zinc (zn) participates in the metabolism of sugar and, especially, of protein, it stimulates the activity of some enzymes, and is involved in digestion. also, zn has a specific role in the metabolism of essential fatty acids [7]. in the human organism, zn is a part of insulin, stimulating its secretion, of some enzymes, it takes part in the synthesis of vitamins c, d and p [20], it facilitates the normal absorption and the vitamin action, especially those in the b1 complex and provitamin a. zn participates in the biosynthesis of nucleic acids and of proteins while being an essential nutrient for the growth and development of children. zn influences the memory and the behaviour of the individual. zn has an important role in the metabolism of sugar and lipids influencing the concentration of cholesterol. one of the first effects of zn deficiency is loss of appetite which is established very early. along with this, growth stops and the individual can even experience weight loss. copper (cu) is present in all the fruits in variable quantity and has an essential role in the carbohydrate metabolism. in the human organism, cu participates in the formation of haemoglobin and of erythrocytes, facilitating the absorption of fe. manganese (mn) together with iron, zinc and copper stimulates the activity of some food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 56 enzymes, takes part in the synthesis of some vitamins, the synthesis of proteins, lipids and carbohydrates. at the same time, mn participates in the vitamin activity, improves the activity of the central nervous system and contributes to the forming of urea and of blood, of milk, intervenes in the development of the skeleton; also in the processes of reproduction contributes at maintaining the production rhythm of sexual hormones. the lack of mn can delay the child’s growth; it can lead to deafness and blindness, and in adults it can produce ear noises or hearing loss. cobalt (co) is found in the organism in the smallest quantity, but it has a very intensive activity. it is a component of vitamin b12 and it participates in the proper functioning of red blood cells. recent studies have provided information on the biochemistry and bioinorganic chemistry of several proteins containing cobalt in a form other than that in the corrin ring of vitamin b12 [21]. the level of cobalt in the organism is dependent on the hexogen, greater than in the case of other nutrients, considering that there are no tissues that can accumulate it directly. nickel (ni) takes part in blood formation and plays an important biological role, generally similar to that of co. ni activates several enzymes [7] and is involved in preserving the integrity of cell membranes [18]. a comprehensive synthesis of the influence of minerals on the organism has been presented by soetan [17]. materials and methods seeds – study material. the grape seeds come from grapes picked in 2009, from different geographical regions of the country, during the wine making season. the seed of the grape varieties have been separated from the dry grape pomace (mixtures of white varieties of aligoté and frăguţă b) and from wet grape pomace (white nohan, frăguţă a) which were dried in open air at that moment, at temperatures between 20oc and 25oc. the separation was made by eliminating the impurities from seeds by sifting and free falling in air. the seed were minced mechanically as powder. the defatted grape seed was also a study material in the present paper. chemical analysis. determination of ash and mineral content. in order to remove carbon, every 2 g of powdered seeds of each variety were weighed and put into a crucible and then incinerated in oven at 550 celsius degrees for 6 hours. the total ash content, determined according to sr iso 2171:2009, is the percentage of dry matter. the determination of mineral content, from each seed sample, and defatted grape seed respectively, was performed by an energy dispersive spectrometer using röntgen radiation, shimadzu edx type 900hs, of high sensitivity and resolution. before the analysis, the homogenization of the samples was taken into consideration so that the radiation could penetrate each cell of the sample. analytical methods. all analytical determinations were performed at least three times and the value of each determined parameter is the average of the three values, which is added with ± the standard deviation values. the veracities of the obtained results and the ones presented in the paper are confirmed at the signification level of 0,95. results and discussion the results obtained at the determination of the total mineral elements (in %, dry matter basis) from the grape seed, and from the defatted grape seed respectively are given in table 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 57 table 1 the total mineral elements from grape seed and defatted grape seed total mineral elements grape varieties grape seeds [% s.u.] defatted grape seed [% s.u.] white variety mix 3.18 2.73 aligoté 3.35 2.84 hybrid variety nohan white 2.14 1.80 hybrid variety frăguţă a 3.20 2.86 hybrid variety frăguţă b 3.28 2.80 the way in which the content of mineral elements from the grape seed varies in report with the defatted grape seed for the white mix variety is given in the graphic from figure 1. for the white mix varieties, a larger concentration of calcium in seeds is obtained (55.74%) than from the defatted grape seed (49.51%), and the other predominant mineral elements, k, p and s are found in larger concentration in the defatted grape seed than in seeds. also, in the defatted grape seed from the white mix variety the element ti (0,090%±0,006) is present. ti is non-toxic even in larger quantities and has no natural role in the human organism. a quantity of 0,8 milligrams is ingested by humans on a daily basis, but the majority passes through the organism without being absorbed [22]. the content of mineral elements from the grape seed and defatted grape seed, from the aligoté variety is given in figure 2. in the case of seeds from the aligoté variety, the mineral elements ca, k, s are found in larger quantities in the defatted grape seed than in seeds; only p is in a smaller percentage in the defatted grape seed (13.12%) than in the seeds (16.632%). the concentration of ca from the defatted grape seed (53.477%) does not differ significantly from the concentration of ca in grape seed (54.11%). the variation of mineral elements from the grape seed and defatted grape seed of nohan variety is given in figure 3. the predominant mineral elements ca, k, s from the seeds of nohan hybrid variety can be found in smaller concentration than in the defatted grape seed. in seeds, just p is found in large concentration (18,853%) than in the defatted grape seed (14,364%). the content of mineral elements from grape seed and defatted grape seed for fraguţă hybrid variety is presented in figure 4 and for fraguţă b in figure 5. 55.74 49.251 26.447 28.374 15.346 19.139 2.088 2.34 0.253 0.289 0 0.332 0.123 0.11 0.069 0.074 0 0.09 0 10 20 30 40 50 60 % s .u . ca k p s mn fe zn cu ti grape seed defatted grape seed figure 1. the content variety of mineral elements from grapes and defatted grape seed for the white variety mix food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 58 53.47754.11 27.403 29.913 16.632 13.12 1.942 2.53 0.314 0.227 0.149 0.115 0.0820.083 0 10 20 30 40 50 60 % s .u . ca k p s mn zn cu grape seed defatted grape seed figure 2. the content of mineral elements form seeds and defatted grape seed – aligoté variety 55.058 56.89 23.655 25.699 18.853 14.364 1.759 2.419 0 0.297 0.246 0.206 0.07 0.048 0.054 0.078 0 10 20 30 40 50 60 % s .u . ca k p s mn fe zn cu grape seed defatted grape seed figure 3. the content of mineral elements form seeds and defatted grape seed – nohan hybrid variety 52.153 53.285 23.918 22.389 21.263 19.202 2.247 2.557 0 1.904 0.236 0.272 0 0.23 0.082 0.059 0.1 0.088 0 0.014 0 10 20 30 40 50 60 % s .u . ca k p s sc mn fe zn cu ni grape seed defatted grape seed figure 4. the content of mineral elements form seeds and defatted grape seed – frăguţa a hybrid variety food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 59 52.697 50.912 23.051 22.298 21.676 21.512 2.238 2.778 0 1.725 0.236 0.329 0 0.284 0.097 0.069 0.068 0.058 0 0.035 0 10 20 30 40 50 60 % s .u . ca k p s sc mn fe zn cu co grape seed defatted grape seed figure 5. the content of mineral elements form seeds and defatted grape seed – frăguţa b hybrid variety when compared with the other varieties varieties from vrancea, in the hybrid variety fraguţă – variety from the suceava county, a new element appears – scandium (sc). in the defatted grape seed which results after the extraction of oil, the nickel (ni) cation contamination can be found in concentration of 0,014%. from the comparative analysis of the content of mineral elements of the fraguţă hybrid variety, it can be observed that seeds gathered from the dry pomace – fraguţă b have a larger concentration of ca (52,697%) when compared to the seeds gathered from the wet pomace – fraguţă a (52,153%). moreover, in the seeds gathered from the dry pomace, ni is not present but a new element appears co. generally, the chemical composition of the mineral elements of the tested samples is comparable with the one presented in the specialty literature [23, 24]. from the analysis of the obtained results for the mineral content, it can be observed that in the grape seed and also in the defatted grape seed, there is a predominant quantity, in the specified order, of calcium, potassium, phosphor and sulphur. it has been found that cations such as arsenic, cadmium, selenium, mercury, etc. do not exist. the only cation that appears is nickel, in the case of the defatted grape seed from the frăguţă a hybrid variety, variety gathered from humid marc. its occurrence may be due to the contact with processing machinery. its concentration is in the allowed limits. conclusion the results of the mineral elements show that all the studied grape seed variety and the defatted grape seed contain considerable quantities of mineral elements: calcium, potassium and phosphorus which can be used to improve the nutritional value of the human diet. the grape seed and the defatted grape seed, due to their high content of mineral elements, can constitute an alternative in the manufacture of products which ensure benefits for health, following the acceptance of the idea that diet plays a decisive role in the prevention and treatment of disease. such products, in addition to having appropriate nutritional effects they improve health and/or reduce the risk of disease also have components which are derived from natural ingredients that provide additional health benefits. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 60 references 1. mironeasa, s., leahu, a., codină, g.g., stroe, s.g. and mironeasa, c. grape seed: physico-chemical, structural characteristics and oil content, journal of agroalimentary processes and technologies 2010, 16 (1), 1-6. 2. pomohaci, n., stoian, v., gheorghiţă, m., sîrghi, c., cotea, v. v. and nămoloşanu, i., oenologie vol. i, prelucrarea strugurilor şi producerea vinurilor, editura ceres, bucureşti, 2000. 3. pelus, e., arnaud, j., ducros, v., faure, h., favier, a. and roussel, a.m., trace element (cu, zn, fe, mn, se) intakes of a group of french men using the duplicate diet technique. int. j. food sci. nutr. 1994, 45, 63 –70. 4. fennema, o.r., food chemistry. new york: marcel dekker, 2000. 5. malhotra, v.k., biochemistry for students, tenth edition, jaypee brothers medical publishers (p) ltd, new delhi, india, 1998. 6. eruvbetine, d., canine nutrition and health. a paper presented at the seminar organized by kensington pharmaceuticals nig. ltd., lagos on august 21, 2003. 7. segal, r., principiile nutriţiei, galaţi, editura academica, 2002. 8. jamba, a. and carabulea, b. tehnologia păstrării şi industrializării produselor horticole, chişinău, editura cartea moldovei, 2002. 9. murray, r.k., granner, d.k., mayes, p.a. and rodwell, v.w., harper’s biochemistry, 25th edition, mcgraw-hill, health profession division, usa, 2000. 10. bendich, a., antioxidant micronutrients and immune responses, ann. n.y. acad. sci., 1990, 587, 180. 11. romero, r., intra-uterine infection, premature birth and the fetal inflammatory response syndrome. j. nutr. 2003, 33, 1668-1673. 12. arinola, o.g., nwozo, s.o., ajiboye, j.a. and oniye, a.h. evaluation of trace elements and total antioxidant status in nigerian cassava processors, pak. j. nutr., 2008, 7(6), 770-772. 13. spallholz, e.j., boylan, m.l. and larsen, s.h., advances in understanding selenium’s role in the immune system, ann. n.y. acad. sci., 1990, 587, 123-139. 14. sherman, r.a., influence of iron on immunity and disease resistance, ann. n.y. acad. sci., 1990, 587, 123-139. 15. albion research notes. a compilation of vital research updates on human nutrition, 5: 2, albion laboratories, inc. may, 1996. 16. faur, v. and goian, m. fitoterapie şi fitoprotecţie alimentară, editura dacia, europa nova, lugoj, 1998. 17. soetan, k. o., olaiya, c. o. and oyewole o. e., the importance of mineral elements for humans, domestic animals and plants: a review. african journal of food science, 2010, 4(5), 200-222. 18. dumbravă, d.g., contribuţii la studiul, izolarea şi purificarea pigmenţilor carotenoidici din produsele naturale. teze de doctorat ale upt, seria 4, nr. 11, editura politehnica, 2008. 19. segal, b., cotrău, m. and segal, r., factori de protecţie prezenţi în produsele alimentare, editura junimea, iaşi, 1996. 20. ţârdea, c., chimia şi analiza vinului, editura „ion ionescu de la brad”, iaşi, 2007. 21. kobayashi, m. and shimizu, s., 1999, cobalt proteins, european journal of biochemistry, 1999, 261 (1), 1–9. 22. emsley, j., "titanium". nature's building blocks: an a-z guide to the elements. oxford, england, uk: oxford university press, 2001. 23. gurin, a.a., razrabotka tehnologhii proizvodstva ăkstaktov, konţentratov i vin iz sladkoi vinogradnoi vîjimki. avtoreferat kand. disertaţii, moskva, 1991. 24. gasanov, d.h.a., razrabotka tehnologhii proizvodstva kormovogo belkovogo producta iz vinogradnîh bîjimok. ealta, avtoreferat kad.diss, 1990. 269 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 269 275 new challenges in the dairy wastewater treatment alla choban1, *igor winkler1,2 1institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine,. a.choban@chnu.edu.ua, 2medical chemistry department, bukovyna medical university, 2 thetralna sq., chernivtsi, 58002, ukraine i.winkler@chnu.edu.ua *corresponding author received july 31st 2015, accepted september 25th 2015 abstract: the efficiencies of three different dairy wastewater treatment technologies have been analyzed and compared on example of several milk, cheese and butter making facilities. it is shown that the standalone mechanical treatment is completely inefficient while the required clarification cannot be ensured by regular aerobic bio-treatment also because of rather unstable ph value in the wastewater. the modified landfill bio-treatment method involving seeding the spots with chlorella is discussed as more stable, effective and less expensive alternative technology for the dairy wastewater treatment. keywords: dairy; dairy wastewater treatment; bio-treatment efficiency; chlorella 1. introduction milk treatment is one of the leading branches in nowadays food processing industry. over 210 dairies are currently functioning in ukraine while the domestic market demand for various milk products is increasing continuously for about 2.7 % annually since 2008 [1, 2]. any dairy produces some industrial wastewater with rather complex and hard to process composition including residual milk components, proteins, milk sugars, butterfat, detergents and other agents [3, 4]. massive formation and discharge of such complex wastewater can seriously disturb the conditions of the receiving water body and provoke water blooming, fish suffocation and general decrease in the water quality. on the other hand, the problem of the dairy wastewater cleaning seems very common for many countries since this branch is widely distributed in many countries across europe. that is why various aspects of the dairy wastewater treatment and reuse stay in focus of extensive investigations. main efforts in this field are given to the problems of wastewater treatment efficiency and better utilization of the wastes and byproducts formed in the course of milk processing. these materials cannot be discharged directly to water bodies or even to the general purpose sewage network without some preliminary treatment. moreover, some milk treatment reagents can shift ph of the wastewater either towards acidic or alkaline reaction, which decreases bio treatment efficiency and causes the need in additional wastewater treatment stages. in the light of the above, this paper deals with the analysis of the dairy wastewater treatment technologies efficiency on example of some milk processing lines. our extended analysis will involve butter and cheese productions as well as drinking http://www.fia.usv.ro/fiajournal mailto:a.choban@chnu.edu.ua, mailto:i.winkler@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 270 milk and fermented milk products technologies. 2. general survey of the dairy wastewater composition and efficiency of the traditional wastewater processing technologies this is well known that the dairy wastewater composition is quite complex since it contains raw milk remainders, milk products, milk transformation products and byproducts, detergents and many other components. the dairy wastewaters are collected after various production stages including main technological operations, side operations, equipment and production shops cleaning, etc. the amount and composition of the resulting wastewater can vary widely depending on the plant’s capacity, specialization, source materials used, technologies implemented and wastewater treatment methods. the following plants were involved in this investigation: private jsc “monastyrsky milk plant” (1); llc “molochni dary” (2); llc “galievo butter plant” (3); jsc “molochnyi kray” (4) first three companies produce mainly cheese and butter while the last one specializes in drinking milk and fermented milk products manufacturing. as an example, the milk processing plant (4) wastewater composition is shown in table 1. unlike the above, cheese production lines cause formation of the lactose-enriched serum with low proteins content. buttermaking plants cause formation of the buttermilk wastewater containing much lactose and proteins but depleted with fats. both serum and buttermilk effluents result in serious contamination of the wastewater. as an example, the cheese and butter production plant (2) wastewater composition is shown in table 2. as seen from comparison of tables 1 and 2, the cheese/butter making wastewater contamination level is lower than that of the milk/fermented milk products making facilities. this situation is caused by the churn-milk catching equipment that operated at the latter plants as the wastewater pretreatment stage. table 1. wastewater composition parameters for the dairy jsc molochnyi kray № wastewater composition parameter wastewater quality parameters, mg/l (except ph) input to wastewater treatment line projected values after treatment real values after treatment 1 2 3 4 5 1 рн 5.6 6.5 6.2 2 suspensions 500 15 6.1 3 butterfat 25 0 0 4 cod 2400 75 132.1 5 bod5 1600 15 62.3 6 ammonium nitrogen n-nh4 + 42.8 0.39 0. 16 7 nitrates, no34 18 15.6 8 phosphates, ро4375 1,0 20,5 9 nitrites, no2 1.2 0.8 0.31 10 general iron 9.5 0.2 0.1 11 surfactants 0.9 0.1 1.1 12 chlorides, cl700 280 79.8 13 sulfates, so4 2 300 85 112.3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 271 table 2. wastewater composition parameters for the dairy llc molochni dary № wastewater composition parameter value, mg/l (except ph) sump input sump output 1 2 3 4 1 рн 8.73 8.31 2 cod 286.7 148.5 3 bodfull 183.2 102.0 4 bod5 130.5 78.2 5 suspensions 142.3 68 6 ammonium nitrogen, n-nh4+ 2.8 4,1 7 nitrites, no20 0 8 nitrates, no3 8.2 6.8 9 chlorides, cl 412 417 10 sulfates, so4298 96 11 phosphates, po4 38.3 5.6 12 dry residue 860 720 13 fats 32 12 14 surfactants 1.2 0.8 15 phenols 0 0 this situation is caused by the churn-milk catching equipment that operated at the latter plants as the wastewater pretreatment stage. churn milk-effluents are caught and collected by this equipment and then used for the cattle feeding mixtures making. on the other hand, bod5 must be lowered to 15 mg/l before the wastewater can be discharged. this is the limit efficiency value for the biotreatment technologies [5], and we shall analyze various options related to application of this technology at some diaries. efficiency of the mechanical standalone treatment technology this technology is implemented at plants (1) and (2). the production capacities of the plants are 11000 (1) and 400 (2) tons of milk per year. butter is the main product of both plants. plant (1) is equipped with the gravityflowing sewage system collecting all wastewater and transporting it to the multisectional mechanical water treatment settler. after mechanical cleaning, the wastewater is discharged to the nearby river dobrodivka. composition of this treated wastewater is shown in table 3. table 3. composition of the plant (1) wastewater after mechanical treatment № wastewater quality parameter value, mg/l 1 cod 489.6 2 bodfull 186 3 bod5 143 4 suspensions 126 5 ammonium nitrogen, n-nh4 + 0 6 nitrites, no21.0 7 nitrates, no38.7 8 chlorides, cl183 9 sulfates, so4268 10 phosphates, po43 2.83 11 dry residue 786 12 oil products 0.26 13 phenols 0 14 surfactants 0.32 15 рн 7.03 16 fats 6.8 as seen from table 3, mechanical treatment is unable to ensure required wastewater quality. for instance, bod5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 272 value remains 9.5 times higher than the maximum permissible level [5]. similarly, low efficiency of the standalone mechanical wastewater treatment can be seen for plant (2) where the wastewater is collected and then treated mechanically in the two-sectioned settler (see table 2). an efficiency of the combined dairy wastewater treatment technology, which includes the biotreatment stage, is analyzed in the next section. efficiency analysis for the combined dairy wastewater treatment technology this technology is realized at plant (4) where the physico-chemical and biological treatment technologies are combined with mechanical cleaning. an average wastewater treatment capacity of the plant (4) is ca. 6 m3/day. the technology includes the following stages (fig. 1). first stage involves preliminary treatment in the fat-trap (position 1, fig. 1). then, the wastewater is collected in the pump-sewage station (position 2) and pumped periodically to the equalizing tank (position 3) where some additional reagents (alkali to keep required ph value and to facilitate better removing of lactose; iron chloride to intensify clarification of water, and flocculant to ensure formation of easy-to-remove flocks) are being added for better cleaning. water contamination level is decreasing after adding the above mentioned reagents and as a result of the anaerobic oxidation of the organic pollutants by the developing biocenose. the suspended particles are being aggregated and form the sediment, which adsorbs additionally some solute species. besides, some new soluble forms of organic substances and the products of their decomposition can also appear in the system. if the ph level is appropriate, the ammonium nitrogen is transferring gradually into the nitrite and nitrate forms by eutrophication. all these processes result in decrease in the wastewater cod value. as mentioned above, alkali, iron chloride and flocculant are used for chemical treatment of the wastewater. these reagents should be added to the main supplying pipe of equalizer. a pipe (position 5) is used to transport required amounts of the caustic soda solution from the storage tank (position 4) to the equalizer pipe. iron chloride is fed from the dissolution tank (position 6) using another pipe (position 7). some amounts of insoluble hydroxides fe(oh)3 and ca(oh)2 are formed as a result of interaction between the two above reagents and the wastewater components. a solution of flocculant is stored in a separate tank (position 8) and should be added to the mixture using a pump (position 9) to ensure aggregation of the hydroxides flocks into floccules. finally, cod and bodfull decrease by 50 % and the suspended particles content drops by 70-80 % as a result of these treatment procedures. following this physico-chemical treatment, the wastewater is directed to the aeration tank-settler (position 10), where the processes of fats, surfactants, proteins and carbohydrates decomposition and organic pollutants oxidation occur. besides, the transfer of the ammonium nitrogen into the nitrate form also continues at this stage. all these biochemical transformations are performed by activated sludge, bacteria and protozoa species present in the system as immobilized or floating forms, which consume oxygen pumped in by the air compressor (position 11). finally, excessive activated sludge should be separated from the treated water in the same settler (position 10). final decontamination is exercised using the sodium hypochlorite solution, which is prepared and added to the mixture using the dissolution tank (position 12) and the drop-dosing feeding equipment (position food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 273 13). this stage should be included in the flowchart in order to remove excessive pathogenic microbes usually present in the dairy wastewater. after complete treatment, the wastewater is discharged to river siret. fig. 1. the flowchart of the wastewater processing stages at the dairy (4) it is expected that the final treated wastewater composition will comply with the values indicated in column 4, table 1 as it is required by the ukrainian legislation norms set for the wastewater to be discharged in the open water bodies. however, real wastewater cleaning efficiency is much lower, which causes excessive concentrations of some pollution agents (see column 5, table 1). for instance, real bod5 of the treated wastewater is 62.3 mg/l while the limit value is 15 mg/l; phosphates content is 20 mg/l against 1,0 mg/l, surfactants content is 1,1 mg/l against 0,1 mg/l. this proves low efficiency of the combined wastewater treatment technology with typical biotreatment stage as the main tool of water cleaning. in our opinion, this situation is caused mainly by principal inability to keep the ph value within the range 6.5-8.5, which is required to ensure the full biotreatment efficiency. the alkali dosing equipment cannot respond to small deviations in the wastewater ph level and either overdose or underdose the alkali reagent causing too alkaline or too acidic reaction. these factors result in swelling of activated sludge and decrease in its efficiency. in this context, we consider the chlorellabased cleaning technologies as a more appropriate option for the dairy wastewater treatment. this method is less expensive and more reliable for the wastewaters with unstable ph value. 3. results and discussion of the chlorella-based dairy wastewater treatment technologies efficiency chlorella is a microscopic unicellular green alga, which is a 2-10 μm immobile bubble without flagella. it is a quite undemanding organism with a very simple life cycle which exhibits fast and massive reproduction. this alga can be applied in many directions such as wastewater treatment, organic wastes recycling, phycotechnologies and others. an intense interest towards this alga is fed additionally by its composition: 40 % or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 274 more of important proteins, 20 % lipids, 35 % carbohydrates and about 10 % of ash residue in terms of dry weight. besides, it contains some amounts of vitamins b, c and k. one of chlorella components, ‘clorelin’ can exhibit some antibiotic activity. in some countries the chlorella biomass is used as a food component after it passes special treatment [6]. it should be noted that the application of chlorella to the acid winery wastewater treatment is quite effective [7]. our previous investigation [8] proved also that this technology can be applied to treatment of the acid juice-making factory wastewater. as seen from the comparative analysis of plant (3) wastewater treatment efficiency, the same technology is highly effective at dairy as well. table 4. comparison of the real and projected wastewater biotreatment efficiency for the plant (3) * the values in this column represent the percentage of the contamination agent concentration remained after treatment by chlorella compared to the projected concentration values (taken as 100 %) table 5. composition of the treated wastewater after the chlorella-enhanced biotreatment at the landfill spots wastewater quality parameter concentration, mg/l spot 1 spot 3 spot 5 average 1 2 3 4 5 cod 35.8 48 96 59.9 bod5 11.5 12.0 18.0 13.8 ammonium nitrogen 1.5 1.5 2.0 1.67 nitrites 0.001 0.004 0.06 0.022 nitrates 0.8 1 2.5 1.43 plant (3) wastewater is collected by the local sewage system and then it self-flows to the pumping station, which transports it to the biotreatment landfill area. there are wastewater quality parameter concentration, mg/l real values (after the chlorella-seeded landfill) projected values (without chlorella) remaining contamination*, % 1 2 3 4 cod 62.8 80.0 78.5 bod5 14.2 15.0 94.6 ammonium nitrogen 1.42 1.56 91 nitrites 0.22 3.12 7.05 nitrates 2.96 45.0 6.6 suspensions 13.6 15.0 90.6 chlorides 92.7 350.0 26.5 sulfates 42.3 500 8.5 phosphates 1.78 3.5 50.8 dry residue 782 1000 78.2 oil products not determined 0.3 0 surfactants not determined 0.5 0 phenols not determined 0.001 0 iron 0.26 0.3 86.7 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 alla choban, igor winkler, new challenges in the dairy wastewater treatment, food and environment safety, volume xiv, issue 3 – 2015, pag. 269 – 275 275 5 landfill spots, and each of them is seeded with chlorella spores each year before the summer season, which ensures significantly higher efficiency of the wastewater decontamination (see table 4). it should be emphasized that according to the specifics of the technological process, the treated dairy wastewater is discharged periodically, after completion of its biotreatment at each landfill spot. as seen from table 5, the biotreatment process has enough time to complete till the end of the warm season at all landfill spots and the outcoming discharge composition meets all requirements set for the biotreated wastewater. 4. conclusion neither plain mechanical wastewater cleaning nor typical aerobic biotreatment can ensure the required clarification of the dairy wastewater. the former option is ineffective because of massive organic contamination of such wastewater, while the latter is too sensitive to accidental ph deviations outside the range 6.5-8.5, which appear quite often at most dairies. an efficiency of the typical aerobic biotreatment can be increased to some extent by using the automatic ph control/alkali dosing system capable of self-adjusting to possible changes in the wastewater ph. however, it would require rather high expenses and wouldn’t be completely secured from irregular acidic or alkaline jumps. in our opinion, the chlorella-based landfill biotreatment is a more appropriate solution. this technology remains effective within a wider ph range 4.5-7.5 and no additional wastewater treatment equipment is required. the need for additional land resources to establish the landfill spots is the only problem expected for this technology. however, the total landfill spots area is comparatively small and no serious obstacles are expected in the course of its introduction. 5. references [1] shubravska o.v., sokolovska t.v., development of the milk and milk products market: global trends and domestic prospects, economics and prognosis, 2: 80-93, (2007) (in uktrainian) [2] http://www.milkua.info [3] nguen l.m., organic matter composition, microbial biomass and microbial activity in gravelbed constructed wetlands treating farm dairy wastewaters, ecol. eng., 16(2): 199-221, (2000) [4] healy n.g., rodgers m., mulqueen j., treatment of dairy wastewater using constructed wetlands and intermittent sand filters, bioresource tech., 98(12): 2268-2281, (2007). [5] zapolsky n.a., mishkovaklimenko n. a., astrelin i. m., physicochemical principles of the wastewater treatment technologies. kiev: libra, (2000) (in ukrainian). [6] andreeva v.m., soil and aerofilic green algae (chlorophyta: tetrasporales, chlorococcales, chlorosarcinales). s.-petersburg: nauka, 351 p., (1998) (in russian). [7] shepherd h.l., grismer m.e., tchobanoglous g., treatment of strength winery wastewater using a subsurface-flow constructed wetland, water env. res., 73: 394-403, (2001). [8] choban a., winkler i. a case study of juice-making factory wastewater treatment and possible ways of its optimization, food and env. safety, 13(3): 218-224: (2013). http://www.milkua.info photocatalytic degradation of methylene blue on nanostructured composites based on tio2-bi2o3 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 30 photocatalytic degradation of methylene blue on nanostructured composites based on tio2-bi2o3 *igor kobasa department of analytical chemistry, chernivtsi national university, kotsiubinsky st., 2, chernivtsi 58012, ukraine. e-mail: imk-11@hotmail.com *corresponding author received 7 september 2011, accepted 15 november 2011 abstract. oxide systems based on mixtures tio2-bi2o3 with various quantitative and phase compositions have been synthesized and their light absorption spectra were analyzed. these systems can reveal the quantum dimensional effects and exhibit photocatalytic activity in the reaction of photodegradation of methylene blue (mb). a complex investigation of the structure, spectral and photochemical properties of the oxide systems proved that low concentration of the dope (0.01-0.1 wt % of bi(iii)) causes rise in the light absorption value and higher intensity of the mb photodegradation. further rise in the bi(iii) concentration results in formation of the amorphous bi2o3 and decrease in the photocatalytic activity. an energy analysis of the oxide system has been provided and possible mechanism for the components interaction was proposed. keywords: photocatalytic activity; tio2-bi2o3; methylene blue; nanostructured composites, dye photodegradation 1. introduction titanium dioxide and tio2-based oxide mixtures, are known as semiconductors, which reveal wide range of important and useful electrophysical and physico-chemical (photochemical, catalytic, photocatalytic) characteristics. these features promise many useful applications for such tio2 system [1-7]. for instance, they can be used to synthesize new products [8, 9], for neutralization of hazardous wastes and industrial gas emissions [10-12], photocatalytic and photoelectrochemical solar energy transformation [13-15], and for systems for information storage and transmission etc. [1618]. photocatalytic activity can be enhanced through development of new compositions, which include a semiconductor-photocatalyst and some additional compounds (another semiconductors, ions-modifiers, metal nanoparticles, etc.). such composition ensures more effective separation of the light-generated charges, which can be transported from the photocatalyst to the substrate. this process reduces efficiency of the recombination. therefore, a system tio2-bi2o3, seems promising for potential applications in the water decontamination technologies, in the vis-induced oxidation processes [19]. activity of this system in degradation of 4-chlorophenol is higher than activity of the pure titania or p25 under the sunlight [20]. however, a generally recognized theory of an influence of the bismuth admixture on various properties of tio2, which determine the potential of application of titania as a functional material (catalyst, photocatalyst, energy convertor etc.) is not developed yet. 2. experimental we synthesized some tio2-bi2o3 samples using one of the following methods. mailto:imk-11@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 31 first method supposed synthesis of metal hydroxides at the room temperature with further thermal processing and transformation of the hydroxides into oxides. basic flowsheet of this method includes following stages: deposition of a metal hydroxide through adding of ammonia solution (5-10 %) to solution of the corresponding metal salt. jelly-like deposit of the hydroxide forms after this operation, then it should be separated from the solution and washed to remove unreacted compounds. then the deposit undergoes drying, heating to 600 0c to remove remainders of ammonia, calcination for formation and structuring of oxides (at 600 0c for about 6 hours), and, finally, the oxide products should be ground. another method supposed high temperature synthesis of the product and it was engaged to obtain some other tio2-bi2o3 systems. method of the pyrogenic synthesis of tio2 [21] was modified according to the following flowsheet. source materials should be heated previously then ticl4 undergoes the high temperature hydrolysis and tio2 forms after this operation. at the beginning, all parent components (air, hydrogen and titanium tetrachloride) are mixed together in the combustion chamber at 70-100 0c and piped to the reactor. this mixture burns in the reactor and forms water. temperature of the hydrogen-oxygen flame is high (700-1100 0c) and just-formed water causes hydrolysis, which runs in the burning zone. a solution of bi(no3)3 had been injected to the cooling zone of the reactor (t = 600-800 0c) through an additional sprayer. a highly disperse particles of tio2-bi2o3 were formed in the cooling zone of the reactor as a result of these processes. we have synthesized tio2-bi2o3 samples containing 0.01; 0.1; 1.0; 5.0; and 10.0 mass % of bi2o3. an atom-absorption spectroscopy was used to determine contents of bi2o3. photocatalytic activity (pa) of the samples has been determined in the characteristic reaction of methylene blue (mb) reduction to its leucoform [22]. electroconductivity (σ) of the samples has been determined using a direct current method and specific surface area was measured by bet method with low temperature adsorption of argon. 3. results and discussion as it is seen from the data of tab. 1, the samples with 0,1 mass % of bi2o3 exhibit the highest level of photocatalytic activity in redox reaction. besides that, pyrogenically synthesized samples exhibit much higher activity comparing to the samples, which were synthesized through low-temperature liquid phase deposition (see results 2, 3, 8, 9). activity of the samples lowers at the rise of bismuth content and the samples with 10,0 mass % of bi2o3 exhibit same level of photocatalytic activity as non-doped titanium dioxide. pure bismuth oxide without dope of tio2 (see tab. 1, example 7) exhibits very low level of photocatalytic activity in the redox reaction. this fact proved through four hours exposition of such a sample to uv radiation, which did not result any change in optical density of the methylene blue solution. analyzing data of tab. 1, one can see that electroconductivity and photocatalytic activity of the samples are cymbatic while their pa does not exhibit such dependencies on the specific surface area. for instance, we have synthesized a 0,01 mass % doped sample with sspc 2,5 times lesser than sspc of the non-doped sample. however, pa and σ of the doped sample were 5,8; and 2,8 times correspondingly higher than the values of the non-doped sample. dependence of pa on σ becomes even more obvious if we compare data for samples 3 and 6. they have the same specific area surface but various content of the dope (0,1 mass % for the sample 3 and 10,0 mass % for the sample 6) and σ. as a result, pa of the sample 6 substantially differs from the values of the sample 3. therefore, comparison between values of σ and activity of the samples proves that quantity of free electrons, which present in the material, mainly governs its activity. the latter characteristic does not substantially depend on sspc. of course, it influences the activity but, in this case, not significantly. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 32 table 1 photocatalytic (pa) activity, specific surface area (sspc) and electroconductivity (σ) of tio2-bi2o3 systems number bi2o3 mass % sspc, m2/g pa (mg/(ml min m2)) σ, s/cm 1 0 15 8,3·10-1 9,63·10-7 2 0,01 6 4,8 2,65·10-6 3 0,1 20 5,8 7,95·10-6 4 1,0 8 5,4 3,22·10-6 5 5,0 24 4,7 2,55·10-6 6 10,0 20 9,0·10-1 9,85·10-7 7 99,99 9 1,0·10-4 1,82·10-7 8* 0,01 130 6,5 9,12·10-6 9* 0,1 55 7,3 9,48·10-6 * flame hydrolysis synthesized samples figure 1. absorption spectra for tio2-bi2o3 systems. the spectra derived by kubelka-munk method from spectra of diffusion reflection (r, %) for: (1) – anatase with rutile admixtures; (2) – (6) co-deposition products containing mass % of bi2o3: 0,01 (2); 0,1 (3); 1,0 (4); 5,0 (5); 10,0 (6) and pure rutile (7) the kubelka-munk method [23] of calculation of absorption spectra from the data of diffusive reflection spectra can bring important information about characteristics of tio2-bi2o3 systems. as it is seen from fig. 1 (see spectra 2 and 3), position of the absorption bands for 0,01 mass % and 0,1 mass % doped samples almost coincide with the position of non-doped tio2 band (see spectrum 1) while absorption intensity of the doped bands is higher. the most active sample (spectrum 3) also exhibits the highest rise of the absorption coefficient and, consequently, energy of the oscillator, which determines probability of the electron jump to the conduction band. rise in bismuth content over 1,0 mass % causes significant spectral changes. a new absorption band with absorption maximum near 340 nm appears for the samples containing over 1,0 mass % of the dope. absorption level of this band gradually raises as bismuth content rises to 5,0 and 10,0 mass % (see fig. 1, spectra 4-6). in our opinion, these changes can be caused by formation of the new bismuth oxide phase. to verify this assumption, we recorded diffusion food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 33 reflection spectra for two bi2o3 samples with various purity grades and derived absorption spectra from the previous ones. as seen from fig. 2, a sample graded as “pure” generates spectrum, which is close to the shape of samples 4-6 spectra (see fig. 1). however, there is a difference between them. bismuth oxide samples spectra are bounded from the long wave edge at 438442 nm (see fig. 2) and these wavelengths correspond to eg = 2,81-2,83 ev, which is very close to eg = 2,80 ev [24]. as contrary to these values, the doped samples spectra (dope content is from 1,0, to 10,0 mass %) are bounded from the long wave edge at 403-405 nm (see fig. 1), which corresponds to eg = 3,07 ev. this fact gives us ground to suppose influence of quantum dimensional effects for bi2o3 particles for the samples 46. this assumption was verified and proved through determination of the particles size range using scanning electronic microscope zeiss evo 50xvp. particles sizes were ranged from 10 to 20 nm for the samples 46. alternative explanation involving rutilerelated long wave bands for the samples 4-6 should not be accepted because of the following. our rutile samples (purity grade is “very pure”) spectra have another shape: there is a minimum at 310 nm and their long wave edge is bounded at 412 nm (see fig. 1), which corresponds to eg = 3,01 ev. this value is in agreement with the reference value eg = 3,02 ev [1]. then we investigated energetic parameters of tio2-bi2o3 system basing on previous spectral data. this investigation stage was aimed to finding connection between changes in the photocatalytic activity depending on the dope content. as reported in [24], conductivity band of the regular (without any quantum dimensional effects) bismuth oxide corresponds to ecb = -4,83 ev (referring to the absolute potential scale, which starts from the zero point in vacuum). potential of the normal hydrogen electrode is e0 = -4,50 ev in this scale, therefore, one can recalculate the above ecb into the normal electrochemical potential scale as ecb = +0,33 v (referring to the normal hydrogen electrode). figure 2. absorption spectra for bi2o3 materials: “pure” graded (1) and “pure for analysis” graded (2). the spectra derived from diffusion reflection spectra (r, %) by kubelkamunk method the band-gap potential eg = 2,80 ev and the valence band potential evb = +3,13 v should also be taken into consideration. further, we analyze influence of the quantum dimensional effects on the above potentials. there is a difference (see spectra in fig. 1) between the bandgap width for the regular (eg) and nanosized (eg(nano)) particles of bismuth oxide obtained in experiments 4-6 and this difference is δeσ = eσ(nano) – eσ = 3,07 – 2,8 = 0,27 ev. theory of the quantum dimensional effects shows that both bands (conductivity and valence) shift because of the band-gap change. an “excessive” δeσ distributes between the two bands inversely as to effective mass of electrons and holes (m*eand m*h+). it has been found25 that the conductivity band of n-type semiconductors is a subject of more substantial shift comparing to the valence band shift. we did not have any information regarding actual m*eand m*h+ and assumed the ratio between food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 34 contributions to ecb and evb as 3:1. this assumption proceeded to values of ecb = +0,13 v and evb = +3,20 v. we recalculated ecb = -4,12 ev24 to the electrochemical potential scale and obtained ecb = -0,29 v in order to find position of the allowed energy bands. then this potential was compared to eg = 3,20 ev, which resulted evb = 2,91 v. an energy diagram for titanium oxide and both regular and nano-sized (with quantum dimensional effects) bismuth oxide is shown in fig. 3. a dye reduction potential is also shown in the diagram. since potential of an electron injection to the conductivity band of the nano-sized bismuth oxide ecb is more positive than potential of an electron transfer from photoexcited titanium oxide to the dye emb/mb.-, which results its reduction, both processes can be considered as competitive. consequently, effect of an electron injection process increases as bismuth oxide content rises, which should promote decrease of pa. figure 3. an energy diagram of titanium and bismuth oxides based system and scheme of the electron transfers at mb reduction indeed, data from tab. 1 proves decrease of the photocatalytic activity for more than 6 times as bi2o3 content increases from 1,0 to 10,0 mass % (samples 4-6). further decrease of pa can be caused by increase of the particles size, which leads to gradual disappearance of the quantum dimensional effects and widening of the energetic gap δe = ecb(tio2)–ecb (bi2o3), which governs driving force of the process. 4. conclusion results of investigation of the structure, electric and photocatalytic properties, thermodynamic analysis of the energy characteristics of the system tio2-bi2o3 proved that this material can reveal quantum-dimensional effects and promote photoreduction of methylene blue. 5. references [1].m. grätzel (ed), (1986). energetic resources through a prism of photochemistry and catalysis, mir, moscow. [2].m. fox, m. t. dulay, heterogeneous photocatalysis. (1993). chem. rev. 193 341357. [3].m. r. hoffmann, s.t. martin, w. choi, and d.v. bahnemann., environmental applications of semiconductor photocatalysis. (1995). chem. rev. 195 69-96. [4].a. mills and le hunte s., an overview of semiconductor photocatalysis. (1997). j. photochem. and photobiol. a: chem. 108 135. [5].a. hagfeldt and m. grätzel, molecular photovoltaics (2000). acc. chem. res. 33 269275. [6].a. fujishima, x. zhang, d. a. tryk, tio2 photocatalysis and related surface phenomena. surface science reports. (2008). 63 515-582. [7].m. grätzel, conversion of sunlight to electric power by nanocrystalline dye-sensitized solar cells. (2004). j. of photochemistry and photobiology a: chem. 164 3-14. [8].k.t. ranjit, t.k. varadarajan, b.j. visanathan, photocatalytic reduction of dinitrogen to ammonia over noble-metalloaded tio2. (1996). j. photocem. and photobiol. a: chem. 96 181-185. [9].m. anpo, h. yamashita, y. ichihashi et al., photocatalytic reduction of co2 with h2o on titanium oxides anchored within micropores of zeolites: effect of the structure on the active sites and the addition of pt. (1997). j. phys. chem. b. 101 2632-2636. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 35 [10]. c. nasr, k. vinodgopal, l. fisher et al., environmental photochemistry on semiconductor surfaces. visible light induced degradation of a textile diazo dye, naphtol blue black, on tio2 nanoparticles. (1996). j. phys. chem. 100 84368442. [11]. i. poulios, m. kositzi, a. kouras, photocatalytic decomposition of triclopyr over aqueous semiconductor suspensions. (1998). j. photocem. and photobiol. a: chem. 115 175-183. [12]. wang chao, ao yanhui, wang peifang, hou jun, qian jin, zhang songhe, preparation, characterization, photocatalytic properties of titania hollow sphere doped with cerium (2010). j. hazard. mater., 178(1-3) 517-522. [13]. a.j. bard, m.a. fox, artificial photosynthesis: solar splitting of water to hydrogen and oxygen. (1995). acc. chem. res. 28 141-145. [14]. m. ashokkumar, an overview on semiconductor particulate systems for photoproduction of hydrogen. (1998). internat. j. hydrogen energy. 23 427-438. [15]. g.r. bamwenda, s. tsubota, t. nakamura, m. haruta, photoassisted hydrogen production from a water-ethanol solution: a comparison of activities of au—tio2 and pt—tio2. (1995). j. photocem. and photobiol. a: chem. 89 177-189. [16]. sahyun m.r.v., serpone n., primary events in the photocatalytic deposition of silver on nanoparticles tio2 (1997). langmuir. 13 50825088. [17]. ch. wang, ch. liu, et al., spectral characteristics and photosensitization effect on tio2 of fluorescein in aot riversed micelles (1998). j. colloid interface sci. 197 126-132. [18]. i. kobasa, i. kondratyeva, l. odosiy, tio2/biscyanine and cds/biscyanine heterostructures: influence of the structural composition on the photocatalytic activity. (2010). canadian j. of chemistry. 88(7) 659666. [19]. y. bessekhouad, d. robert, j. v. weber, photocatalytic activity of cu2o/tio2, bi2o3/tio2 and znmn2o4/tio2 heterojunctions. (2005). catalysis today, 101 315-321. [20]. xu jing-jing, chen min-dong, fu degang, preparation of bismuth oxide/titania composite particles and their photocatalytic activity to degradation of 4-chlorophenol. (2011). trans. nonferrous met. soc. china. 21 340-345. [21]. i. m. kobasa, j. w. strus and m. a. kovbasa, usa patent wo/2009/052510. method of surface modifying titania using metal. (2009) [22]. i. m. kobasa, semi-conductive materials based on the titanium dioxide doped with zinc: catalytic activity for copper deposition and effect of uv-irradiation. (2004). polish j. chem. 78 553-560. [23]. w.n. delgass, c.l. haller, r. kellerman, j.h. lunsford, (1979). spectroscopy in heterogeneous catalysis. academic press, n.y., san francisco, london. [24]. y. xu., m. a. a. schoonen, the absolute energy positions of conduction and valence bands of selected semiconducting minerals. (2000). american mineralogist. 85 543-556. [25]. a. i. kryukov, s. ya. kuchmii, v. d. pokhodenko, energetics of electron processes in semiconductor photocatalytic systems. (2000). theoret. and exper. chem. 36 63-81. 1. introduction 2. experimental 3. results and discussion journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 56 a linear programming model for a diet problem cristina-elena hreţcanu1, ciprian-ionel hreţcanu2 1stefan cel mare university of suceava, food engineering faculty, romania, e-mail: cristinah@usv.ro 2 ciprian porumbescu college of art, suceava, romania abstract: in this paper we solve a diet problem which has the goal to find an optimal combination of proposed foods on the condition that the daily nutritional requirements of a person are satisfied. first of all, we find the nutrient content of our proposed menu which a person generally consumes, throughout an entire day, 1 or 2 portions of some given foods (yogurt, cereals, rye bread , feta cheese, coffee, chicken soup, baked salmon, boiled potatoes, lemon juice, dry wine, ice with milk and vanilla, spaghetti in tomato sauce with cheese, apple juice). secondly, we search for quantities of all specified items of the same menu as the diet amount of calories is 2200 kcal / a day, respectively 2500 kcal / a day. finally, we search for quantities of the same proposed menu, using linear programming techniques, as the diet contains minimum amount of calories. the mathematical model of the problem is formulated as a linear program where the objective function is the total amount of calories for the proposed menu, on the condition that the constraints regarding the amounts of protein, vitamins, minerals, fats, dietary fiber consumed throughout an entire day are satisfied. we solve this problem by using the sover tool in ms excel and we describe all the steps involved in solving the linear programming model in the context of the menu proposed by us. key words: daily nutritional requirements, solver in ms excel, mathematical optimization introduction linear programming (lp) is a technique for solving optimisation problems which involves the optimization of a linear objective function, subject to the linear equality or inequality constraints of decision variables. a mathematical optimization model consists of an objective function and a set of constraints in the form of a system of equations or inequalities. the process of variable selection requires multiple reiteration before a satisfactory objective function is developed. linear programming deals with a class of optimization problems, where both the objective function to be optimized and all the constraints, are linear in terms of the decision variables. lp problems are usually solved by the simplex method, originally developed by dantzig in 1948, using methods from numerical linear algebra. linear programming is being successfully applied to problems of design of diets, conservation of resources, economic growth prediction, transportation systems. the first computer-based menu planner, optimizing menu for nutritional adequacy and budgeted food cost was built in 1964 by balintfy who applied linear programming techniques [1]. today there is a variety of software packages to solve optimization problems such as: lindo, winqsb, what'sbest for solving nonlinear and linear problems. also, microsoft excel can provide a fast way to solve linear problems, using solver. in our paper, we propose a computer-based method using the solver tool from microsoft excel for planning an optimal menu with respect to the daily nutritional requirements of a person. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 57 materials and methods a linear programming problem can be defined as a problem of optimizing (maximizing or minimizing) a linear function subject to linear constraints [2] . a feasible vector which the objective function gets the value is called optimal. a feasible optimal problem is said to be unbounded if the objective function can assume arbitrarily large positive (resp. negative) values at feasible vectors; otherwise, it is said to be bounded. the value of a bounded feasible minimum (resp, maximum) problem is the minimum (resp. maximum) value of the objective function as the variables range over the constraint set. any linear program consists of four parts: a set of decision variables, the parameters, the objective function, and a set of constraints. to solve a linear programming problem, we must find an objective function that minimizes, maximizes or achieves a specific goal (a feasibility problem), respectively, while variables satisfy the constraints of the model. a classical diet problem [3] has to supply the required nutrients at minimum cost, for m different types of food, f1, . . . , fm, that supply varying quantities of the k nutrients, n1, . . . ,nk , that are essential to good health. in this paper we replaced the minimum cost of a given diet by a minimum amount of calories. we find the nutrient content of a proposed diet, on the condition that the daily nutritional requirements of a person (the amounts of protein, vitamins, minerals, fats and dietary fiber) are satisfied. for m different types of food, f1, . . . , fm, nutrients: n1, . . . ,nk , let ci be the amount of calories per unit of food fi,, let lj be the minimum daily requirement of nutrient nj ; let uj be the maximum daily allowance of nutrient nj; and let aij be units of nutrient nj contained in one unit of food fi mathematically, the problem can be stated as follows: if xi is the number of units of food fi , then the objective function of this diet is: 1 2 1 1 2 2( , ,..., ) ... (1) t m m mf x x x c x c x c x x c     where we use the notation:    1 2 1 2... ... t t m mx x x x and c c c c  the amount of nutrient nj contained in this menu is: 1 1 2 2 ... (2) t j j j mj mn a x a x a x x a      for j = 1, . . . , k, where 111 1 ... ... ... ... ... k m mk aa a a a            is the matrix of amount of nutrients from the menu. we may consider the diet if the minimum and respectively the maximum daily requirements are met, as follows: 1 1 2 2 ... (3) j j j mj m jl a x a x a x u     for j = 1, . . . , k, where lj , uj denote the lower and the upper daily requirements of nutrients. because we can not have a negative amount of food , ee must have the constraints: x1 ≥ 0, x2 ≥ 0, . . . , xm ≥ 0 (4). our problem is to minimize the function (1) subject to the constraint (3) and (4) and the goal is to find and compare feasible solutions until no better solution can be found. solutions are feasible if they satisfy all the problem constraints that are defined by dietary recommendations and guidelines [4]. for the first proposed quantities of the menu, we suppose that a person eat throughout an entire day the following dishes: yogurt made with whole milk, breakfast white cereals, bread of rye, feta cheese, coffee – espresso, home made chicken soup, baked salmon, boiled potatoes, lemon juice, bread of rye journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 58 (reduced calorie), dry wine, ice with milk and vanilla (in cone), spaghetti in tomato sauce with cheese, apple juice (bottled). we used a food database usda national nutrient database for standard reference [4]. for the component of the proposed menu, values of nutrients are listed for: calories, protein, total fat, saturated, cholesterol, carbohydrate, total dietary fiber, some minerals (such as: calcium, iron, potassium and sodium) and vitamins (vitamin a, thiamin, riboflavin, niacin, ascorbic acid and vitamin c). the nutrients of this menu are specified in the table 1, as follows: table 1 nutrient content of proposed foods [4] amount of feeds x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 x14 nutrients apple juice, bottled boiled potatoes bread of rye, reduced calorie bread of rye, un toasted breakfast cereals white cheese feta chicke n soup home prepar ed coffee espresso dry wine ice with milk and vanilla, in cone lemon juice salmon baked spaghetti in tomato sauce with cheese yogurt made with whole milk calories (kcal) 117 116 47 83 145 75 86 5 130 164 12 184 192 139 protein (g) 0.01 2 2 3 3 4 6 0.1 0.1 4 0.01 23 6 8 total fat (g) 0.1 0.1 1 1 0.4 6 3 0.2 0 6 0 9 2 7 saturated fatty acids (g) 0 0 0.1 0.2 0.1 4.2 0.8 0.1 0 3.5 0 1.6 0.7 4.8 cholesterol(mg) 0 0 0 0 0 25 7 0 0 28 0 74 8 29 carbohydrate (g) 29 27 9 15 31 1 8 1 4 24 4 0 39 11 total dietary fiber (g) 0.2 2.4 2.8 1.9 0.5 0 0 0 0 0.1 0.2 0 7.8 0 calcium (mg) 17 11 17 23 0 140 7 1 8 153 3 6 40 274 iron mg) 0.9 0.4 0.7 0.9 1.5 0.2 0.5 0.1 0.2 0.2 0.001 0.5 2.8 0.1 potasium (mg) 295 443 23 53 53 18 252 69 95 169 58 319 305 351 sodium (mg) 7 7 93 211 0 316 343 8 9 92 0,001 56 963 105 vitamin a(re)g 0 0 0 0,1 0 36 0 0 0 52 1 54 58 68 thiamin (mg) 0.05 0.13 0.14 0.14 0.24 0.04 0.08 0.001 0.02 0.05 0.01 0.18 0.35 0.07 riboflavi n (mg) 0.04 0.03 0.11 0.11 0.15 0.24 0.2 0.11 0.02 0.26 0.001 0.15 0.28 0.32 niacin (mg) 0.2 1.8 1.2 1.2 2 0.3 3.8 3.1 0.2 0.3 0.001 5.7 4.5 0.2 ascorbic acid(mg) 2 10 0.001 0.001 0 0 0.001 0.001 0 1 22 0 10 1 daily values have been established by the food and drug administration, [4], as references to help consumers use information on food labels to plan a healthy overall diet. food energy is reported as calories (as a unit of measure for the amount of energy furnished by protein, fat, and carbohydrate). the official journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 59 unit of measurement for food energy is kilocalories (kcal). the daily values are depending on what we eat: 2200 kcal / a day or 2500 kcal / a day. table 2 recommended daily values for fat, sodium and cholesterol for a diet of: 2200 kcal/ a day or 2500 kcal / a day [4] quantity of calories nr. crt. nutrient recommended for 2200 kcal 2500 kcal 1 total fat less than 73 80 2 saturated fat less than 22 25 3 cholesterol less than 300 300 4 sodium less than 2400 2400 it is well known that 2,200 kcal level is for moderately active women (of 20-60 years old), teenager girls, and sedentary men, whereas 2,500 calories is the target level for men (of 20-60 years old), teenager boys and active women. many older adults, children and sedentary women need fewer than 2,200 calories a day and may want to select target levels based on 1,600 calories a day [4]. table 3 contains nutritional requirements for a menu of: 2200 kcal / a day or, respectively, 2500 kcal / a day table 3 nutrient contents of foods daily values for: 2000 kcal or 2500 kcal [4] nr. crt. recommended values for 2200 kcal 2500 kcal 1 protein (g) 50 63 2 carbohydrate (g) 330 375 3 total dietary fiber (g) 27 30 4 calcium (mg) 1000 1000 5 iron (mg) 10 15 6 potasium (mg) 3500 3500 7 vitamin a (mg re*) 800 1000 8 thiamin (mg) 1.1 1.2 9 riboflavin (mg) 1.1 1.3 10 niacin (mg) 14 16 11 ascorbic acid (mg) 75 90 * retinol equivalents the mathematical model of our proposed diet problem is formulated as a linear program, which has the objective function coresponding to the total amount of calories: f(x1,…,x14) = 117 x1 + 116 x2 + 47 x3 + 83 x4 +145 x5 + 75 x6 +86 x7 + 5 x8 + 130 x9 +164x10 +12x11 +184x12 +192 x13 +139 x14 (5) at the daily-menu level, there are constraints that need to be satisfied: protein (g): 50 ≤0,01. x1+2. x2 +2. x3 +3. x4 +3. x5 +4. x6 +6. x7 +0,1. x8 +0,1. x9 +4. x10 +0,01. x11 +23. x12 +6. x13 +8 x14≤ 65 (6) total fat (g): 0,1 x1 +0,1 x2 + 1 x3 +1 x4 +0,4 x5 +6 x6 +3 x7 +0,2 x8 +0 x9 +6 x10 +0 x11 +9 x12 +2 x13 +7 x14≤ 80 (7) saturated fatty acids (g): 0. x1 +0. x2 +0,1. x3 +0,2. x4 +0,1. x5 +4,2. x6 +0,8. x7 +0,1. x8 +0. x9 +3,5. x10 +0. x11 +1,6. x12 +0,7. x13 +4,8 x14 ≤ 25 (8) cholesterol(mg): 0. x1 +0. x2 +0. x3 +0. x4 +0. x5 +25. x6 +7. x7 +0. x8 +0. x9 +28. x10 +0. x11 +74. x12 +8. x13 +29 x14 ≤ 300 (9) carbohydrate (g): 300 ≤ 29. x1 +27. x2 +9. x3 +15. x4 +31. x5 +1. x6 +8. x7 +1. x8 +4. x9 +24. x10 +4. x11 +0. x12 +39. x13 +11 x14 ≤375 (10) total dietary fiber (g) : 27 ≤ 0,2 x1 +2,4 x2 +2,8 x3 +1,9 x4 +0,5 x5 +0. x6 +0. x7 +0. x8 +0. x9 +0,1. x10 +0,2 x11 +0. x12 +7,8. x13 +0 x14 (11) calcium (mg) : 1000≤ 17. x1 +11. x2 +17. x3 +23. x4 +0. x5 +140. x6 +7. x7 +1. x8 +8. x9 +153. x10 +3. x11 +6. x12 +40. x13 +274 x14 ≤1300 (12) iron (mg) : 10 ≤ 0,9x1+0,4x2+0,7 x3+0,9 x4+1,5 x5 + 0,2x6+0,5x7+0,1 x8+0,2x9 +0,2 x10 +0,001 x11 +0,5 x12 +2,8 x13 +0,1x14 ≤15 (13) potasium (mg) : 295x1+443x2 +23x3 +53x4 +53x5 +18x6 + 252x7 +69x8 +95x9 +169x10 +58 x11 + 319x12 +305 x13 +351x14 =3500 (14) journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 60 sodium (mg) : 7x1 +7x2 +93x3 +211x4 +0.x5 +316x6 +343 x7 +8x8 +9x9 +92 x10 +0,001. x11 +56x12 +963x13 +105 x14≤ 2400 (15) vitamin a (re) g: 800 ≤ 0. x1 +0. x2 +0. x3 +0,1. x4 +0. x5 +36. x6 +0. x7 +0. x8 +0. x9 +52. x10 +1. x11 +54. x12 +58. x13 +68 x14 (16) thiamin (mg) : 1.1 ≤ 0,05x1 +0,13. x2 +0,14.x3 +0,14.x4 +0,24.x5 +0,04.x6 +0,08.x7 +0,001.x8 +0,02.x9 +0,05.x10 +0,01.x11 +0,18.x12 +0,35.x13 +0,07x14 ≤ 1,4 (17) riboflavin (mg) : 1.1 ≤ 0,04. x1 +0,03. x2 +0,11. x3 +0,11. x4 +0,15. x5 +0,24. x6 +0,2. x7 +0,11. x8 +0,02. x9 +0,26. x10 +0,001. x11 +0,15. x12 +0,28. x13 +0,32 x14 ≤ 1,4 (18) niacin (mg) : 14 ≤ 0,2x1+1,8x2 +1,2x3 +1,2x4 +2.x5 + 0,3x6 +3,8x7 +3,1x8 +0,2x9 +0,3x10 + 0,001x11+5,7x12 +4,5x13 +0,2 x14 ≤18 (19) ascorbic acid (mg) : 75 ≤ 2. x1 +10. x2 +0,001. x3 +0,001. x4 +0. x5 +0. x6 +0,001. x7 +0,001. x8 +0. x9 + x10 +22. x11 +0. x12 +10. x13 +1 x14≤ 90 (20) with x1 ≥ 0, x2 ≥ 0, . . . , x14 ≥ 0 (21) where x1, x2, x3, x4, x5, x6, x7, x8, x9, x10, x11, x12, x13, x14 are amounts of foods given in the table 1. the minimum and respectively the maximum daily requirements are given in table 2 and table 3 respectively. to avoid the illogically diet (too much quantity of a type of food), or if we want to have a minimum quantity of a given food, we introduce some more inequalities regarding the maximum amount of some foods: x4 ≥0,5 , x5≤1, x7 ≥1, x8 ≥1, x10≤2, x14≤2. any specific nutritional requirements can be simply incorporated into the planning and a suitable solution will be computed [5]. the microsoft excel spreadsheet data for the problems is shown in figures 1 for solving the linear programs for the proposed diet problem. the data include the ingredients, the nutrient contents of feed amount (to be solved for) and the ingredient restrictions diet as we can show in the figure 1: figure 1. inputs and results for the diet linear program solved by solver in microsoft excel journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 61 results and discussion the mathematical model of the problem is formulated as a linear program where the objective function coresponds to the total amount of calories. at the daily-menu level, there are constraints that need to be satisfied, regarding the amounts of protein, vitamins, minerals, fats, dietary fiber, etc. a linear program excel workbook was developed using solver tool. first of all, we find the nutrient content of our proposed diet, on the condition that a person consumes throughout an entire day 1 or 2 portions of some given foods. in this situation we obtain a diet of 1687kcal. secondly, we search for the nutrient composition of the same diet such as the diet amount of calories is 2200 kcal, respectively 2500 kcal. to obtain these, we must put in the solver parameters windows (figure 1) at “value of” the given amount of calories (2200 kcal and 2500 kcal, respectively). thus, solver tool can find the quantities of every given food so that the constraints are satisfied, on the condition that the total amount of calories is 2000 and respectively 2500 kcal. finally, we search for the quantities of the proposed food, using linear programming techniques, so that the diet contains a minimum amount of calories. the results of determinations regarding numbers of units of portions and consumed amount (g) in these four situations are given in the table 4 : table 4. units of portion of food consumed and consumed amount (g) obtained using solver in ms excel type of diet= diet 1 of 1687kcal diet 2 of 2200 kcal diet 3 of 2500 kcal diet 4 of 1851 kcal m ea ls food description units of portion of food consumed consumed amount (g) units of portion of food consumed consumed amount (g) units of portion of food consumed consumed amount (g) units of portion of food consumed consumed amount (g) yougurt made with whole milk, plain 1.0 227 1.6 357.1 1.8 407.8 2.0 454.0 breakfast cereals white 1.0 242 1.8 445.5 2.2 528.9 1.0 242.0 bread of rye, untoasted 1.0 32 1.0 32.4 1.1 36.5 0.5 16.0 cheese feta 1.0 28 0.7 20.6 0.7 20.8 1.4 39.7 b re ak fa st cofee -expreso 1,0 60 1.0 60.7 1.0 61.2 1.0 60.0 chicken soup home prepared 1.0 240 0.7 177.1 0.8 194.4 1.0 240.0 salmon baked 1.0 85 1.9 165.1 2.1 181.9 0.3 22.3 boiled potatoes 1.0 135 1.7 223.7 1.9 260.6 2.7 361.7 lemon juice 1.0 47 1.1 50.3 1.1 51.6 2.3 108.0 bread of rye, reduced calorie 1.0 23 1.1 24.5 1.1 26.4 4.7 108.5 dry wine 1.0 103 1.7 178.6 2.0 210.1 0.0 0.0 l un ch ice milk, soft, vanilla, in cone 1.0 103 1.7 179.9 2.0 209.8 2.0 206.0 spaghetti in tomato sauce with cheese 2.0 272 1.0 132.9 1.0 139.9 0.6 87.8 d in ne r apple juice, bottled 1.0 248 1.7 412.4 1.9 480.8 1.1 273.1 journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 62 we describe all steps involved in solving the linear programming model in the context of our proposed diet problem, as follows: after setting-up the cell formulas in the spreadsheet, we can use the solver dialogue box (as you can see in figure 1). the target cell $g$16 (which contain the total amount of protein from the proposed diet) represents the objective of the linear program. we can adjust the changing cells $c$2:$c$15, which contains the portion of food consumed, to optimize the target cell. constraints are restrictions that we placed on the changing cells and these are given in subject to constraints. in our model, all changing cells must be nonnegative because the amount produced of each product is nonnegative. after we input all the information in the dialogue boxes, we can press the solve button to obtain the solution of our diet problem. solver searches over all feasible solutions and finds the feasible solution that has the "best" target cell value, the smallest for minimum optimization. we find the quantities of food so that the diet has the minimum amount of calories which is 1851 kcal / a day, on the condition that restrictions of the daily nutritional requirements of a person are satisfied. from the figure 2 to the figure 10 we can observe the variation of the amounts of protein, vitamins, minerals, fats, dietary fiber, depending on the diet type, comparatively to the recommended values for 2200kcal / a day or 2500 kcal / day, respectively figure 2. variation of the quantity of protein (g) depending on the diet type figure 4. variation of totally fat quantity (g) and saturated fatty acids (g), depending on the diet type figure 3. variation of the quantity of cholesterol (mg) depending on the diet type figure 5. variation of the quantity of carbohydrate (g) and, respectively, dietary fiber (g), depending on the diet type journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 63 figure 6. variation of the quantity of calcium (mg) potasium (mg) and sodium (mg) figure 7. variation of the quantity of iron (mg), depending on the diet type figure 8. variation of the quantity of vitamin a (re) (g), depending on the diet type figure 9. variation of the amount of thiamin, riboflavin, niacin (mg), depending on the diet type figure 10. variation of the amount of ascorbic acid (mg) depending on the diet type conclusion a practical model of a linear programming applied to a dietary situation was constructed. the procedure described was adapted to a diet problem, where the variables are food items, the restrictions are nutritional requirements and the objective function is the amount of calories of the diet. using solver in microsoft excel we can provide a fast way to solve this diet problem and to observe if the daily nutritional requirements of a person are satisfied. acknowledgments this paper was presented in the grundtvig workshop: “challenges in food safety and food quality control„ , 17-22 may 2010, suceava, romania, project 2009-1ro1-gru1303339, ref. no. gru – 09 grat-20-usv, funded by the european union’s grundtvig programme. this paper reflects the views only of the authors. references 1. j. f. raffensperger, b. koroušić seljak, computer-based dietary menu planning, journal of food composition and analysis, vol.22 (5) 2009, pag. 414-420 2. g. dantzig, linear programming and extensions, princeton university press, new jersey, 1963, pag 6-12 3. n. darmon, e.l. ferguson, a. briend, the least-cost low-carbohydrate diet is expensive, nutrition research, vol. 28(1), 2008 4. us department of agriculture, agricultural research service, usda national nutrient database for standard reference nutritive value of foods, home and garden bulletin (hg-72), (2002) www.ars.usda.gov/services/docs.htm?docid=6282 5. d. sklan, i. dariel, diet planning for humans using mixed-integer linear programming, british journal of nutrition 70, (1993), pag. 27-35. microsoft word 8 mircea oroian_corectat.docx 54 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 1 – 2017, pag. 54 60 im pac t o f ad ulter atio n w ith g lu cos e, fruc tos e a nd hyd rolys ed in ulin s yr u p on ho ney ph ys icoch em ica l pr operties sorina ropciuc1, *mircea oroian1, vlad olariu1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 15th february 2017, accepted 17th march 2017 abstract: the aim of this study is to evaluate the influence of the adulteration with glucose, fructose, hydrolysed inulin syrup on honey physico-chemical properties (ph, aw, electrical conductivity (ec), water activity and colour parameters (l*, a*, b*, chroma)) of three honey samples of different botanical origins (acacia, tilia and polyfloral). the honeys were adulterated in different percentages (10%, 20%, 30%, 40% and 50% respectively) with glucose, fructose and hydrolysed inulin syrup. the moisture content of all the three samples did not exceed the maximum allowable level of 20% established by the european commission. the physico-chemical parameters (ph, aw, electrical conductivity (ec), water activity and colour parameters (l*, a*, b*, chroma)) of the analysed honeys are in agreement with other studies reported in the international scientific literature. the physicochemical parameters prediction, in function of the botanical origin, adulteration agent and adulteration agent percentage have been made using the analysis of variance (anova). according to the anova it was observed that in the case of l*, ph and electrical conductivity (ec) there is a good correlation (r2>0.90) between the parameters and the botanical origin, adulteration agent and adulteration agent percentages. keywords: honey, physico-chemical parameters, adulteration, anova 1. introduction honey is a natural product produced by honey bees (apismelifera l.) from various secretions of plants [1]. the act of deliberately adulteration degrades the quality of food meant to sale, either by the addition or exchange of low-grade materials or by the elimination of various important chemical compounds is termed as “food adulteration” [2]. adulterated honey samples are labelled as natural pure honey and have the same price like original ones. normally, honey adulterations do not have an impact on the human health. the identification of the adulterated honey is an important process and it is difficult to identify this fact by producers, consumers, retailers and for the authorities too [3], [4]. the honeys have been adulterated over the last years with different agents such as: starch syrup [4], fructose and glucose [5], high fructose corn syrup (hfcs) [6], sugar syrups [7], glucose [8] and maltose syrup [9]. the aim of this study is to evaluate the influence of the adulteration with glucose, fructose, hydrolysed inulin syrup on honey physico-chemical properties (ph, aw, electrical conductivity (ec) and colour parameters (l*, a*, b*, chroma)) of three samples of honey of different botanical origins (acacia, tilia and polyfloral). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 55 2. materials and methods materials 3 honey samples of three different botanical origins (acacia, tilia and polyfloral) were purchased from local beekeepers from the suceava county. the honey samples have been adulterated with glucose, fructose and hydrolysed inulin syrups in different percentages 10%, 20%, 30%, 40% and 50%, respectively. physico-chemical properties the physico-chemical properties (ph, aw, electrical conductivity, colour parameters (l*, a*, b*, chroma and hue angle) were determined accordingly to the harmonised methods of the international honey commission [10]. statistical analysis statistical analysis was performed using a unscrambler x 10.1 (camo norway) software system. the data corresponding to each variable were analyzed by onefactor analysis of variance (anova). 3. results and discussion in this study the influence of the adulteration agents (glucose, fructose and hydrolysed inulin syrup) on the physicochemical parameters (colour (l*, a*, b*, c*, hue angle), ph, aw and electrical conductivity) of three difference honeys (acacia, tilia and polyfloral) was evaluated. the experimental data were subjected to anova in order to establish if the honey type, adulteration agent or adulteration agent percentage are influencing significantly the physico-chemical parameters and if these parameters can be used in the identification of honey adulteration. table 1 presents the physicochemical parameters of the authentic honey analysed. table 1. physico-chemical parameters of authentic honeys parameter acacia polyfloral tilia l* 48.96 37.90 36.13 a* 0.20 0.30 0.10 b* 7.90 3.40 0.18 c* 1.33 3.40 1.86 hue angle -85.33 85.10 -87.70 ph 4.11 3.72 3.89 aw 0.801 0.806 0.764 ec (µs/cm) 59.40 207.10 413.50 honey colour depends on the content of phenolic compounds, pollen and minerals. in the present study acacia honey has exhibited the highest colour purity (table 1), aspect highlighted by kadar et al. [11]for acacia honey samples from romania and spain. honey polyfloral presented the most pronounced yellow components, indicated by high values of b* (table 1) and chromaticity angle h. water is the second important parameter of honey. the water activity represents a proportional unit with the free water in the food products. a lower level of water activity than 0.60 is specific to microbiological stable products. the values of the water activity measured in this study are in agreement with those reported by other scientists [12], [13]. the ph of the honeys is an important parameter for the extraction and keeping of the products, because it influences texture, stability and shelf life [14]. the honeys’ ph ranged between 3.72 and 4.57 in the same range with those reported in other papers in the case of romanian honeys [15]. the electrical conductivity is influenced by the botanical origin, ash content, organic acids concentration and some complex sugars and polyols. the electrical conductivity was in the same range with those reported by other scientists in the case of romanian honeys [16]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 56 influence of honey type the influence of honey type on honey physico-chemical parameters after the adulteration is shown in table 2. the a*,ph and ec are not influenced by the addition of agents (p values are higher than 0.05). in the case of the other parameters, the honey types influence the evolution of the parameters (l*, b*, c*, hue angle and aw). in the case of the electrical conductivity there can be observed higher values because the adulteration agents (glucose and hydrolysed inulin syrup) were prepared into acidified water (ph lower than 2). table 2. influence of honey type on physico-chemical parameters of adulterated honey honey type parameter acacia tilia polyfloral f-value l* 52.23(51.16-53.30)a 39.55(38.48-40.62)b 39.7(38.7-40.8)b 202.8*** a* 0.96(0.60-1.32)ab 0.88(0.52-1.24)b 1.37(1.02-1.73)a 2.39ns b* -3.90(-5.68 -2.13)c 1.46(-0.31-3.23)b 5.51(3.74-7.28)a 31.39*** c* 5.56(4.40-6.68)a 2.69(1.57-3.82)b 5.26(4.12-6.38)a 8.73*** hue angle -60.64(-87.7—33.5)c -4.43(-31.5-22.6)b 72.9(45.8-100.1)a 27.19*** ph 3.46(3.31-3.61)a 3.33(3.18-3.48)ab 3.21(3.06-3.65)b 3.13ns aw 0.80(0.80-0.81)a 0.79(0.79-0.80)b 0.81(0.80-0.81)a 9.24*** ec (µs/cm) 425.84(186.3-665.3)a 501.95(262.4-741.47)a 480.5(241.0-720.1)a 0.12ns influence of adulteration agent the evolution of the physico-chemical parameters of adulterated honeys according to the adulteration agent used (glucose, fructose and hydrolysed inulin syrup) is shown in table 3. there can be observed that the addition of glucose, fructose or hydrolysed inulin syrup do not influence significantly the b* and hue angle, while in the case of c*, ph and electrical conductivity there can be observed a significant influence (p < 0.001). the addition of glucose and hydrolysed inulin syrup in the honey decreases the values of ph and increases the values of the ec because the solutions are prepared at lower ph values (ph lower than 2). influence of adulteration agent percentage the evolution of the physico-chemical parameters of the adulterated honeys in function of the adulteration agent percentage is shown in table 4. in the case of l* there can be observed that the parameter values increase because the addition of the adulterated agent (which has l* values higher than honey) increases the luminosity. the addition of the adulteration agent influences significantly the evolution of the parameter. in the case of the a* and c* values there can be observed the same evolution like in the case of l* because of the same conditions, but there it cannot be observed a significant difference in function of the percentage of the adulteration agent (p > 0.05). the water activity of the honey is influenced significantly by the addition of glucose, fructose or hydrolysed inulin syrup because these compounds influence the magnitude of the aw [13]. the electrical conductivity increases with the increase of the percentage of sugars added because they are prepared at ph lower than 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 57 table 3. influence of adulteration agent on honey physicochemical parameters adulteration agent parameter glucose fructose hydrolysed inulin syrup p-value l* 44.1(42.9-45.2)b 44.05(42.9-45.2)ab 44.83(43.6-45.9)a 4,33* a* 0.66(0.33-0.99)b 1.27(0.89-1.66)a 1.28(0.90-1.67)a 4,07* b* 0.67(-0.94-2.28)a 2.23(0.32-4.13)a 0.16(-1.74-2.06)a 1,53ns c* 2.85(1.83-3.87)b 5.33(4.12-6.53)a 5.33(4.13-6.54)a 6,76*** hue angle -13.1(-37.7-11.4)a 19.7(-9.4-48.7)a 1.33(-27.7-30.4)a 1,53ns ph 3.98(3.84-4.11)a 4.01(3.85-4.17)a 3.98(3.89-4.11)b 236,9*** aw 0.81(0.80-0.81)a 0.80(0.79-0.81)b 0.80(0.80-0.81)ab 3,48* ec (µs/cm) 274.0(143.1-291.1)b 263.74(193.1-320.6)b 1270.5(1013-1527)a 34,87*** table 4. influence of adulterated agent percentage on honey physico-chemical parameters adulteration agent percentage parameter 0 10 20 30 40 50 p-value l* 42.17(39.644.7)c 42.4(41.043.8)c 43.2(41.844.6)bc 43.8(42.445.2)bc 44.3(42.945.7)b 47.0(45.648.5)a 5,91*** a* 0.61(-0.231.46)a 1.12(0.661.58)a 1.22(0.761.68)a 1.33(0.871.79)a 1.42(0.961.87)a 1.53(1.062.01)a 0,54ns b* 4.71(0.528.91)b -0.08(2.632.18)ab 0.12(2.152.40)ab 0.61(-1.662.88)ab -0.02(-2.292.25)ab 0.78(-1.493.06)a 0,96ns c* 3.85(1.196.50)a 4.68(3.246.12)a 4.01(2.575.45)a 5.27(3.836.71)a 5.28(3.846.72)a 5.94(4.507.38)a 0,74ns hue angle -13.6(-77.650.4)a -1.68)36.4133.04)a -0.80(35.533.9)a 7.86(-26.842.6)a 12.8(-25.8 – 47.5)a 21.2(-33.555.9)a 0,31ns ph 3.26(2.913.62)a 3.52(3.323.71)a 3.31(3.113.50)a 3.31(3.113.50)a 3.31(3.113.50)a 3.32(3.133.51)a 0,80ns aw 0.78(0.770.79)d 0.80(0.790.80)c 0.80(0.800.81)bc 0.80(0.800.81)b 0.80(0.800.81)ab 0.80(0.800.81)a 10,55*** ec (µs/cm) 93.33(59.4113.5)f 124.0(57.9286)e 252.6(36501)d 429.8(541352)c 613.6(341921)b 907.7(312920)a 3.31* parameter prediction based on the parameter values (experimental values), the evolution of the parameter based on honey type, adulteration agent and adulteration agent percentage using the anova method has been predicted. the regression coefficients of the models obtained are shown in the table 5. based on the information data presented in the table 5, the parameters which are well predicted by the honey type, adulteration agent and adulteration agent percentage are: l*, ph and electrical conductivity. the experimental vs. predicted values of l* and ph and electrical conductivity (ec) are shown in the figs. 1-3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 58 table 5. statistical parameters of the linear models for the physico-chemical parameters l* a* b* c* hue angle ph aw electrical conductivity r2 0.990 0.583 0.663 0.480 0.823 0.920 0.649 0.975 f 147.788 2.001 2.805 1.320 6.636 16.401 2.637 54.645 pr> f < 0.0001 0.154 0.068 0.333 0.004 < 0.0001 0.080 < 0.0001 fig. 1. predicted vs. experimental data for l* according to the anova fig. 2. predicted vs. experimental data for ph according to the anova food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 59 fig. 3. predicted vs. experimental data for ph according to the anova 4. conclusions the honey analysed were of three different botanical origins (acacia, tilia and polyfloral) and their physico-chemical parameters were in agreement with the international literature. the adulteration agents influence strongly the evolution of the physico-chemical parameters. the anova analysis was a good method for the prediction of l*, ph and electrical conductivity (ec). acknowledgement this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs – uefiscdi, project number pn-ii-rute-2014-4-0110. 5. references [1]. official journal of the european communities, directive 2001/77/ec, communities, 6: 33–40, (2001). [2]. siddiqui a. j., musharraf s.g., choudhary m. i., rahman a., application of analytical methods in authentication and adulteration of honey, food chemistry, 217: 687– 698, (2017). [3]. li s., shan y., zhu x., zhang x., ling g., detection of honey adulteration by high fructose corn syrup and maltose syrup using raman spectroscopy, j. food compos. anal., 28(1): 69– 74, (2012). [4]. wang s., guo q., wang l., lin l., shi h., cao h., cao b., detection of honey adulteration with starch syrup by high performance liquid chromatography, food chem., 172: 669–674, (2015). [5]. yilmaz m. t., tatlisu n., toker o.s., karaman s., dertli e., sagdi o., arici m., steady, dynamic and creep rheological analysis as a novel approach to detect honey adulteration by fructose and saccharose syrups: correlations with hplc-rid results, food res. int., 64: 634–646, (2014). [6]. çinar s. b., ekşi a., coşkun i., carbon isotope ratio (13c/12c) of pine honey and detection of hfcs adulteration, food chem., 157: 10–13, (2014). [7]. mosun t., detection of adulteration in honey samples added various sugar syrups with 13 c/ 12 c isotope ratio analysis method, food chem., 138: 1629–1632, (2013). [8]. downey g., fouratier v., kelly j. d., detection of honey adulteration by addition of fructose and glucose using near infrared transflectance spectroscopy, j. near infrared spectrosc., 11(6): 447–456, (2003). [9]. li s., shan y., zhu x., zhang x., ling g., detection of honey adulteration by high fructose corn syrup and maltose syrup using raman spectroscopy, j. food compos. anal., 28(1): 69– food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 sorina ropciuc, mircea oroian, vlad olariu, impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties, food and environment safety, volume xvi, issue 1 – 2017, pag. 54– 60 60 74, (2012). [10] bogdanov s. et al., honey quality and international regulatory standards: review by the international honey commission, bee world, 80(2): 61–69, 1999. [11]. kadar m., juan-borras m., carot j. m., domenech e., escriche i., volatile fraction composition and physicochemical parameters as tools for the differentiation of lemon blossom honey and orange blossom honey, j. sci. food agric., 91(15): 2768–2776, (2011). [12]. marghitas l. a., dezmirean d. s., pocol c. b., ilea m., bobis o., gergen i., the development of a biochemical profile of acacia honey by identifying biochemical determinants of its quality, not. bot. horti agrobot. cluj-napoca, 38(2): 84–90, (2010). [13]. chirife j., zamora m. c., motto a., the correlation between water activity and % moisture in honey: fundamental aspects and application to argentine honeys, journal of food engineering, 72(3): 287–292, (2006). [14]. gomes t., feás x., iglesias a., estevinho l. m., study of organic honey from the northeast of portugal, molecules, 16(7): 5374– 5386, (2011). [15]. oroian m., physicochemical and rheological properties of romanian honeys, food biophys., 7(4), (2012). [16]. oroian m., amariei s., leahu a., gutt g., multi-element composition of honey as a suitable tool for its authenticity analysis, polish j. food nutr. sci., 65(2), (2015). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 65 correlation analyses between some technolgical parameters of some romanian wheat varieties grown in the suceava area *ana leahu1, georgiana gabriela codină1, silvia mironeasa1, cristina damian1 1“stefan cel mare” university, faculty of food engineering, 13th university street, suceava, romania; e-mail: analeahu@usv.ro *corresponding author received 22 october 2010, accepted 6 february 2011 abstract: the wheat technological properties are mainly given by the particularities of gluten structure, by protein compounds respectively. the research in the area of gluten was made to identify the gluten protein fractions complex and find out the ratios between those fractions. the establishment of protein fractions of glutenines and gliadines are considered as mainly variables for the bakery potential of grains. flour quality, proteins content and the ratio between glutenines and gliadines influence to a great extent the rheological properties of dough and the quality of bakery products implicitly. this paper presents the research results following the selection and characterization, according to quality indexes, of eight wheat varieties, grown in the suceava area. the objectives of this paper are to study the correlations between some technological parameters (acidity, ph, wet gluten, protein content) from the selected wheat varieties and identify their gluten proteins (gliadines and glutenines). from the statistical analysis significant direct correlations were obtained between raw protein and gliadines (r = 0.649), glutenines (r = 0.720), between wet gluten and glutenines (r = 0.741), gliadines (r = 0.704) and between gliadines and glutenines (r = 0.876). © 2011 university publishing house of suceava. all rights reserved keywords: wheat, gliadines, glutenines, statistical analysis 1. introduction wheat has always been considered an important source of food during the history of mankind as wheat caryopses satisfy to a great extent the nutritional demands of humans. wheat is by far the most important cereal in bread making, though in some parts of the world the use of rye is quite substantial. it is known that the gluten-generating proteins are responsible for the majority of differences occurring in the quality indices of bakery wheat flour. although the varieties harvested at the agricultural research station from suceava are improved by a medium value of 14% for gluten proteins, they react differently in bakery. flour quality, protein content and the ratio glutenines/gliadines influence significantly the rheological properties of dough [1]. gluten proteins are largely insoluble in water or dilute in salt solutions. two functionally distinct groups of gluten proteins can be distinguished: monomeric gliadines and polymeric (extractable and non-extractable) glutenines. gliadines and glutenines are usually found in more or less equal amounts in wheat. gliadines represent a highly polymorphic group of monomeric gluten proteins with molecular weights varying between 30000 and 80000. glutenines are a heterogeneous mixture of polymers with molecular weights varying from about 80000 to several millions. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 66 quantity and composition give the nutritional quality of grain protein. protein accumulation in grain depends on several factors, such as wheat species, variety, weather conditions, natural soil fertility and nitrogen fertilizer doses used. among these factors, climate conditions have a particularly important role, in dry and hot climates, the accumulation of protein in grain is favored, and on the other hand, during training and shorter grain filling, ripening is hastened. the gliadine and glutenine fractions from wheat represent 80 – 85 % from the endosperm proteins and those fractions form together the gluten. usually both gluten fractions represent – in average – approximately half from the total amount of proteins from wheat. for a good wheat variety, the ratio gliadine/glutenine was found with values of 54.5/45.5. in order to explain the significance of this ratio, some suggestions were made that gliadines act as plastifying agent against gluten [2]. the main compounds of gluten are the glutenines with big molar weight which open themselves and act together, building a net. the glutenines are polymeric proteins with a various number of subunits, not necessarily the same (about 25), intermolecular bounded with disulphide bridges, hydrogen bridges, hydrophobic interactions and other covalent bindings [3]. the gliadines were less studied than the glutenines probably because the research was focused on the dough’s rheology (and more precisely on the elasticity of gluten) associated with glutenines. if glutenines are associated with elasticity, the gliadines are associated with viscosity and plasticity of the dough. all gliadines act in the dough by increasing its plasticity, decreasing the kneading time, resistance and elasticity. the largest part of wheat grain consists of carbohydrates (mostly starch) and this makes variation of grain yield, as well as variation of other grain components concentration, strongly associated with carbohydrates accumulation [4]. this negative correlation is caused by the fact that the most of yield is represented by carbohydrates (starch) and so the genotypes or the conditions that encourage the obtaining of high yields are bound to the capacity to accumulate higher amounts of starch in the grain, and that dilutes the protein content [5]. 2. materials and methods materials. the seeds’ material used was represented by eight wheat grain varieties: gasparom, magistral, enesco, dropia, flamura 85, drobeta, voronet, sv 207100, coming from scda suceava experimental farms (harvested in 2008). methods. flour quality tests were accomplished according to romanian or international standard methods: ash content [6], moisture content [7], and wet gluten content [8]. the gliadine and glutenine content was determined, in conformity with the method described by [9] which separated them, based on the solubility of different solvents through an excessive extraction. the total nitrogen (%), raw protein (%) and gliadines and glutenines content were determined according to kjeldahl method and consisted of sample mineralization by heating with 96% sulfuric acid in the presence of a catalyst. following desegregation of proteins and other compounds containing nitrogen, free ammonium ions that react with sulfuric acid to form ammonium sulfate acid, were used. ammonia released by strong alkalinization with naoh 30% was distilled and then titrated with 0,1n h 3 bo 3 in the presence of phenolphthalein. in this work, the titratable acidity, expressed as degrees (˚t), according to standard 90/1988, and the ph values, according to standard 8201/82 were determined. data analysis. statistics 6.0 (stat soft inc., usa) was used for data analyzing. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 67 3. results and discussion the table 1 shows the values of chemical parameters in grain belonging to eight wheat varieties. as can be seen, there were small differences between samples, especially between values of wet gluten, protein and water content. table 1 chemical parameters for wheat variety analyzed (mean  standard deviation of analyses performed in triplicate) wheat variety water [%] ph acidity [%] wet gluten [%] ash [%] raw protein [%] protein class gliadines [%] % of grain protein total glutenines [%] % of grain protein total gasparom 13.650.05 6.20.1 4.80.1 27.80.1 2.190.01 14.850.09 5.800.1 39.2 6.490.03 43.9 magistral 13.700.1 6.60.1 4.20.1 28.80.1 2.20.01 15.200.1 6.010.05 39.6 6.700.12 44.1 enesco 13.900.1 6.50.1 3.90.1 26.60.1 2.180.02 14.600.1 5.490.03 37.6 6.230.03 42.7 dropia 13.500.1 6.70.1 4.60.1 28.90.1 2.140.02 15.300.1 5.830.04 38.1 6.550.05 42.8 flamura 85 13.200.1 6.80.1 4.10.1 28.10.1 2.310.03 14.900.1 5.660.03 38.0 6.420.02 43.1 drobeta 13.630.08 6.20.1 4.70.1 27.90.3 2.210.01 13.950.15 5.550.05 39.78 6.030.02 43.22 voronet 14.080.08 6.30.1 4.60.1 28.30.5 2.250.01 13.950.15 5.350.05 38.35 6.090.04 43.65 sv 207100 14.150.05 6.40.1 4.40.1 27.90.3 2.290.01 13.700.1 5.450.05 39.56 6.050.05 44.16 the gluten proteins (between 80 and 85% of wheat protein total) are the major storage proteins of wheat. gluten proteins are found in the endosperm of mature wheat grain where they form a continuous matrix around the starch granules. the first factor is the gliadine/glutenine ratio of gluten proteins. this is a direct consequence of the fact that, within the visco-elastic gluten protein network of wheat flour doughs, gliadines and glutenines play a different role. the study of statistical links between the technological parameters of the analyzed sample of wheat indicates significant direct correlations: between raw protein and gliadines (r = 0.649), glutenines (r = 0.720), between wet gluten and glutenines (r = 0.741), gliadines (r = 0.704) and between gliadines and glutenines (r = 0.876). by analyzing the obtained data, it can be concluded that all the eight types are of superior quality. the content of raw protein has very good values with an average of 14.53, varying between 13.70, the minimum value and 15.30 the maximum value. likewise, the content of wet gluten has very good values, with an average value of 28.03, a minimum of 26.60 and a maximum of 28.90. the positive correlation of raw protein and gliadine and glutenines respectively shows that an increase of raw protein will lead to an increase of the content of gliadines and glutenines. likewise, the increase of wet gluten will lead to the increase of the content of glutenine and gliadine. figure 1 shows a graphical representation of the variation of wet gluten depending on glutenines and gliadines for all the eight types of wheat; and figure 2 shows the variation of raw protein depending on glutenines and gliadines. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 68 figure 1.the graphical representation of addiction wet gluten = f (glutenines, gliadines): a) spatial representation; b) representation of the dimension line. the graphical representation of the values of wet gluten in relation with the glutenines and the gliadines are given in figure 1. the representation shows two bounds for the maximum value for wet gluten depending on glutenines. the first bound is reached when the gliadines have the value of  5.60% and the glutenines reach the value of  6.23%, and  6.01 for the gliadines and  6.38 for glutenines. the second bound is reached when the content of gliadines is 5.45% and the glutenines reach the value of  6.36%,  6.68% for glutenines and  6.01% gliadines. figure 2.the graphical representation of addiction crude protein = f (glutenines, gliadines): a) spatial representation; b) representation of the dimension line. glutenines gliadines glutenines wet gluten (%) gliadines glutenines gliadines glutenines crude protein (%) gliadines food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume x, issue 2 2011 69 figure 2 shows that the raw protein parameter can have two values for the minimum bound depending on the value of gliadines and glutenines parameters. the first minimum bound is obtained when the gliadines reach the value of  5.6% and glutenines reach the value of  6.23%, gliadines  6.01% and glutenines  6.60%. the second minimum bound is obtained when the gliadines represent 5.45% and glutenines  6.35%, gliadines represent  5.73% and glutenines  6.7%. likewise, it can be observed that at the value of  5.60% for gliadines and of  6.35% for glutenines, there is an inverse relation: the content of wet gluten is minimal while raw crude protein has a maximum value. the same of inverse relation is observed as well for the coordinates represented by the pair of points 5.45% for gliadines and  6.35% of glutenines. maximum values of  30% for wet gluten and of  13.7% for content of raw protein are obtained for the above mentioned values for gliadines and glutenines. the variation of wet gluten and raw protein depending on the content of gliadines and glutenines can be correlated easier using the equations of statistical models. 4. conclusions the analysis of the obtained data shows that the percentage of gliadine has a maximum value of 39.78% of the total content of proteins and the percent of glutenine has a maximum value of 44.16% of the total content of proteins. of all the types of grain considered, it can be remarked that the best type from the point of view of the gliadines/glutenine ratio is the type magistral, created at s.c.d.a. suceava; similar results were obtained also for the type gasparom. the significant direct correlations between the technological parameters of types of grain indicate that an increase of raw protein will lead to an increase of gliadines and glutenines. likewise, the increase of the content of wet gluten will trigger the increase of the content of glutenines and gliadines. in the case of variation of raw protein depending on the two components, the angle of dependence between gliadines and glutenines is higher than the angle of dependence between gliadines and glutenines in the case of variation of wet gluten depending on gliadines and glutenines. 5. references 1. sapirstein h.d., fu b.x., intercultivar variation in the quantity of monomeric proteins, soluble and insoluble glutenin, and residue protein in wheat flour and relationship to breadmaking quality, cereal chemistry, 75:500-507, 1998. 2. bordei d., teodorescu f., toma m., ştiinţa şi tehnologia panificaţiei, editura agir, bucureşti, 2000. 3. sârbu a., proteinele glutenice din grâu, editura agir, bucureşti, 2000. 4. triboi, e., branlard, g., landry, j., environment and husbandry effects on the content and composition of proteins in wheat. aspects of applied biology, 25: 149-158, 1990. 5. mustatea, p., saulescu, n.n., ittu, gh., simion g., rezultate in ameliorarea calitatii de panificatie a graului comun de toamna la i.c.d.a. fundulea. ii. relatia dintre continutul de proteine si productia de boabe. probleme de genetica teoretica si aplicata, xxxvii (1-2): 13-20, 2005. 6. aacc. method 08-21. in approved methods of the american association of cereal chemists, 10th edition; the association: st. paul, mn, 2000. 7. icc standards no: 202. in methods of the international association for cereal science and technology, 2th supplement, vienna, austria, 1991. 8. icc standards no: 155. in methods of the international association for cereal science and technology, 4th supplement, vienna, austria, 1994. 9. bordei d., controlul calităţii în industria panificaţiei. metode de analiză. editura academicăgalaţi, 2007. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 100 the variation of the acid ascorbic content in cynosbati fructus influenced by stationary factors *sorina ropciuc1, ion tănase 2 , iuliana creţescu3, giancarla velicevici4 1 university “ştefan cel mare suceava” -romania e-mail: sorinaropciuc@yahoo.com 2 university “ştefan cel mare suceava” -romania e-mail : ion_ tanase.meteo@yahoo.com 3banat’s university of agricultural sciences and veterinary medicine timişoara – romania e-mail: iuliana.cretescu@gmail.com 4banat’s university of agricultural sciences and veterinary medicine timişoara – romania e-mail: giancarlavely2000@yahoo.com *corresponding author received 15 october 2011, accepted 12 november 2011 abstract: cynosbati fructus is the false fruit of rosa canina l. (rosaceae), also called in romanian rosehip. rosehips contain as active ingredients 500-1000% vitamin c, 600-10.000 mg% carotenoids, pectins, dextrins, vitamins b2, e, pp, flavones (rutozyd, hyperozyd), carbohydrates, organic acids (citric, malic), tannins, volatile oil (linalol, geraniol, citronelol, nerol), vanillin, triterpenic saponosids, beta-sitosterol, fat substances (lecitin, glycerides of fat acids in seeds), minerals (potassium, calcium, magnesium, iron). in terms of precipitations and tempreature the specialized literature situates the rosehip in the category of xerophyte species with low humidity requirements and that do not bear the soils with stagnant water. water requirement is mainly in the spring months before flowering and early august. high atmosphric humidity has a negative influence on the vitamin c content. the measurements made on atmospheric humiduty, including precipitations, air temperature, geographical exposition, relief slope revealed the fact that rosehip grows well even on wet soils, with northern and northwestern exposition, the result consisting in fruit with qn important content of vitamin c. keywords: humidity, slope exposition, gradient relief, chemical compostion 1. introduction the area of rosehip includes southern and central europe, reaching even southern scandinavia. in our country there is the most widespread of the many species of rosa, common in all areas, starting from the black sea up to altitudes of approx. 1200 m, in shrubberies, forest outskirts, thinned forests, ditches. [1] it grows on eubasic-mezobasic soils, dried to moist (xerophyte-mesophilic), often compact soils. [2] rosehip fruits are rich in vitamins. the data recorded in the world over time shows the fact that dry rosehip contain ascorbic acid between 0.1% , 0.5% and 1.0%, some varieties even up to 9%. [3-4] environmental components that perform actions on the growth and fruit composition are considered living conditions because they condition metabolic activities, reducing or increasing precisely the effects of the ecological factors. this would be the case of certain constituents of the physical and geographical environment, such as heat, light, humidity, acidity, depth, texture, soil structure, etc. the area of a certain species is the expression of its adaptation to a certain complex of physical and geographical conditions. [5] the components of the resort – known as „stationary factors” – affect differently the plant life, i.e. directly, the way the light, mailto:sorinaropciuc@yahoo.com mailto:tanase.meteo@yahoo.com mailto:iuliana.cretescu@gmail.com mailto:giancarlavely2000@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 101 the heat, the water and the nutrients act and indirectly, the way the relief, the texture and the soil structure act, only changing values and the regime of the ecological factors, functioning as their determinants. they were also called indirect ecological factors or „phisical and geographical conditions”. in order to make the difference between them and the ecological factors, the term „ecological determinants of the resort” was poposed. [6-7] 2. materials and methods the material of the study is the rosehip fruit is collected from rosa canina l. in the spontaneous flora, natural dried due to the sensitivity of the ascorbic acid due to light and heat. the fruits are harvested at full ripeness from the shrubs studied over four years (2007-2010) on the route that includes the following resorts: suceava-pătrăuţidărmăneşti-costâna-părhăuţi-todireşticajvana-arbore-solca-clit-marginearădăuţi-suceviţa-palma. at each resort we took three research points or three bushes similar in size. the plant material was shred up to the stage of powder and used in determination. for the determination of ascorbic acid content we used the method of titration with iodine solution. we have determined thr morphometric parameters (slope, exposition) by computer processing the altitude terrain models, using the arcgis program and the topographic analyst module. the average values of rainfall and temperature were processed accoring to the information given by the weather station in suceava. we processed the data using various exel applications. 3. results and discussion analyzing the ascorbic acid content of rosehip fruit preserved by natural drying we obtained values ranging from 374.12 mg/100g and 663 mg/100g rosehip powder. the analyses show a significant relationship between vitamin c content values on years of study (see figure 1) the relationship betwen the content of vitamin c over the years 2009 2010 y = 0.6878x + 161.8 r2 = 0.6764 400 450 500 550 600 650 400 500 600 700 vitamin c the year 2009 mg/100g v it am in c t h e y ea r 2 0 1 0 m g /1 0 0 g c2010 linear (c2010) figure 1. the relationship between the content of vitamin c over the years 2009-2010 the values of the morphometric parameters have been presented in the following table 1. table1. the values of the morphometric parameters according to the topografic analyst module resorts altitude exposition slope m n-s, e-v ° suceava 344 n 8.7 pătrăuţi 343 s-e 2.3 ărmăneşti 292 n-e 0.8 costâna 327 e 2.6 părhăuţi 368 s-v 5.2 todireşti 411 s-e 1 cajvana 407 v 0.8 arbore 426 s-v 4.5 solca 378 n-e 0.8 clit 418 e 3.5 marginea 460 e 1 rădăuţi 388 n-v 0.1 suceviţa 550 s-v 0.9 palma 1080 s-v 13,4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 102 table2. the values of the temperatures and rainfall on months and years of study monthly and annual average temperatures in rădăuţi year jan feb mar apr ma y jun jul aug sep oct nov dec annual average 2007 3.4 -0.4 5.5 8.7 16.0 18.9 20.9 19.1 13.0 8.0 0.1 -3.4 9.2 2008 -3.4 1.2 4.3 9.1 13.7 18.1 18.4 19.4 12.6 9.0 3.4 0.1 8.8 2009 -3.3 -1.4 1.9 10.1 14.2 17.6 20.3 18.7 14.9 8.2 5.2 -2.3 8.7 2010 -7.6 -3.8 2.5 9.0 14.9 17.7 20.5 20.6 12.8 5.2 7.1 -4.0 7.9 average 2007-2010 -2.7 -1.1 3.6 9.2 14.7 18.1 20.0 19.5 13.3 7.6 4.0 -2.4 8.6 monthly and annual average temperatures in suceava year jan feb mar apr may jun jul aug sep oct nov dec annual averge 2007 3.6 -0.3 5.9 9.2 16.3 19.9 21.5 19.7 13.6 8.7 0.9 -2.7 9.7 2008 -2.9 1.3 4.9 9.6 13.8 18.3 18.7 20.0 13.0 9.7 3.8 0.6 9.2 2009 -2.5 -1.0 2.3 10.8 14.7 17.7 20.4 18.9 15.7 8.7 5.5 -2.3 9.1 2010 -7.0 -3.5 2.6 9.4 15.1 18.0 21.1 21.4 13.6 5.9 8.0 -3.8 8.4 average 2007-2010 -2.2 -0.9 3.9 9.8 15.0 18.5 20.4 20.0 14.0 8.3 4.6 -2.1 9.1 monthly and annual precipitations in rădăuţi year jan feb mar apr may jun jul aug sep oct nov dec annual amount 2007 10.8 31.3 51.1 28.8 83.8 61.9 85.8 102.0 71.8 92.8 56.7 32.2 709.0 2008 13.6 23.7 18.0 121.8 84.0 73.6 271.8 93.0 72.4 51.8 8.6 28.9 861.2 2009 31.3 17.5 23.8 18.0 90.4 114.1 73.4 33.8 8.4 82.4 19.8 40.6 553.5 2010 28.5 39.5 27.9 38.4 138.2 282.4 129.4 47.8 91.2 42.8 17.3 31.1 914.5 average amount 2007-2010 21.1 28.0 30.2 51.8 99.1 133.0 140.1 69.2 61.0 67.5 25.6 33.2 759.6 monthly and annual precipitations in suceava year jan feb mar apr may jun jul aug sep oct nov dec annual amount 2007 10.1 45.2 41.8 32.9 53.9 30.7 105.8 135.1 67.3 87.0 43.4 34.3 687.5 2008 5.7 23.9 15.9 135.2 91.5 99.2 297.8 72.3 60.4 43.4 8.0 29.9 883.2 2009 45.3 18.9 29.2 8.4 82.2 154.8 120.8 23.7 20.1 62.9 18.7 37.1 622.1 2010 31.4 35.5 28.5 32.0 152.7 226.5 112.1 72.3 64.3 43.1 38.0 33.1 869.5 average amount 2007-2010 23.1 30.9 28.9 52.1 95.1 127.8 159.1 75.9 53.0 59.1 27.0 33.6 765.6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 103 15,9 16,1 15,2 16,0 470,5 338,1 716,6 434,1 15,0 15,3 15,6 15,9 16,2 16,5 2007 2008 2009 2010 temperatura aerului oc 300 350 400 450 500 550 600 650 700 750 precipitatii (mm) vitamin c content mg/100g temperatura s1 suceava s2 pătrăuţi s3 lunca sucevei s4 costâna s5 părhăuţi s6 todireşti s7 cajvana s8 arbore s9 solca s10 clit s11 marginea s12 rădăuţi s13 suceviţa s14 palma precipitaţii figure2. the correlation between the air temperatures, precipitations and the quantity of vitamin c the graphic analysis highlights the large amount of vitamin c in marginea in 2008, when there is recorded the highest value of rainfall and low temperatures. also by comparison with data obtained from the topographic analysis it shows that the resort marginea doessn’t have slopes with southern orientation, as it is found in the specialized literature as a species with preference for sunny slopes. 4. conclusions from the obtained data we found out that rosehip has a good development on moist soils as well, with eastern and southwestern exposition, shaded, with very good productivity and valuable chemical composition. it developes itself pretty well in any type of soil, however it has a preference for fine-grained soils. in the future there can be developed a tool to assess the indicators based on topographic, soil variables, and correlate them with the chemical composition of the species. based on the output there could be derived the indicators of the variability of the species rosa canina l. the vitamin c content correlates significantly iin 2 years time, a fact that is represented in the analysis of the correlation in figure 1. 5. references 1.bojor o., alexan m. herbs from a to z, ulpia traian publishing 2.bruneton j. (1993) pharmacognosie, phytochimie, plantes médicinales, lavoisier tec&doc, paris, 36 3. prodan i., buia al (1964) – romanias illustrated small flora – agrosylvicultural publishing house, bucharest 4.vadova, v.a. pliner, v.a accumulation of vitamina c and p in wild rose hips. try vessoyuz nauch issledovatel vitamin inst. 119-21 5. arsenescu-popa adina, mladin paulina, popescu h – (2007) biometric and pharmaceutical fitochemistry data in the croped fruit of cynosbati fructus,the medical craiova, 2007, in press 6. v. stănescu, (1979) – dendrology, bucharest, didactic and pedagogic publishing 7. v. stănescu, a. negruţiu, gh. văcaru (1971). – the guide for practical works in dendrology, politechnical institute braşov issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvii, issue 1 31 march 2018 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 300 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag.300 -305 study on antimicrobial characteristics of spices’ composition *s. teterina1, n. yushchenko1, u.kuzmyk1, i. khlystun1 1national university of food technologies, kiev, ukraine uly1083@yandex.ru *corresponding author received june 12nd 2015, accepted july 19th 2015 abstract. in order to produce safe, high-quality milk and cultured milk foods with an extended shelf life it is important to inhibit the development of microorganisms. the research aims to study the effects of previously developed spice aromatic composition on test cultures of microorganisms (samples of contaminating microflora), such as enterobactercloaceae, micrococcus albus, bacillus subtilis, endomyceslactis. the spice composition containsginger, cinnamon,cloves, curcuma, sumac, anise, black pepper and sweetpepper, cardamom, fenugreek, nutmeg andbadiane (staranise).it is proved that all spicy compositions showed evident antimicrobial characteristics against the testing cultures. it has been established that all compositions of spices demonstrated expressed antimicrobial characteristics with respect to the typical representatives of the contaminating microflora of fermented milk products. thus, developed compositions of spices can be recommended for using in receipts of fermented milk products without additional processing. the discovered bacteriostatic effect of spices compositions will provide deceleration of undesirable processes promoting the increase of products’ stability during storage. keywords: antimicrobial characteristics, fermented milk products, microbiology of dairy products, spices 1. introduction application of natural ingredients in food is becoming increasingly important. customers are interested in natural ingredients since synthetic additives may be harmful. spicy aromatic substances represent a wonderful alternative. spices are promising ingredients not only in cooking, but they also can be used as enhancing components in formulas of manufactured foods .the application of spicy aromatic substances as components of milk product formulas is limited, thus the development of new kinds of milk products with spicy ingredients is currently an urgent task. spices are complex biologically active composition that not only impart vivid taste and scent to food, but also promote digestion, have bactericidal and antioxidant properties, and actuate metabolism. they contain significant amounts of biologically active substances, such as volatile oils, vitamins, polyphenol, catechine, microand macro-elements. thanks to volatile oils, spices canin hibit emergence and development of bacteria, thus promoting increased storage period of products. biological activity of volatile oil depends on its composition and main components. for instance, phenols and terpenoids display antiseptic action; monoterpenoids have antiseptic and http://www.fia.usv.ro/fiajournal mailto:uly1083@yandex.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 s. teterina, n.yushchenko, u.kuzmyk, i. khlystun, studyofantimicrobial characteristics of spices composition, , food and environment safety, volume xiv, issue 3 – 2015, pag. 300 – 305 301 bactericidal action; sesquiterpenoids and diterpenoids have fungicidal action, whereas aldehydes have antiviral action. therefore, it is quite important to study germicidal qualities of spices applied in the manufacturing technologies of cultured milk products[1]. the spices compositions developed earlier by the authors were used as study subjects and include:  compositionno.1 – sweet pepper, ginger, cinnamon in proportion 1:1:1;  compositionno.2 – cloves, sweet pepper, ginger in proportion 0.75:1:1;  compositionno.3 – ginger, curcuma, sumacin proportion 1:1:8;  compositionno.4 – anise, cloves, ginger, black pepperin proportion 1:0.75:1:1;  compositionno.5 – sweet pepper, ginger, cardamon,fenugreek in proportion1:1:0.75:1.25;  compositionno.6 – anise, ginger, nutmeg, black pepperin proportion 1:1:1:1;  compositionno.7 – badiane, ginger, curcuma, sumacin proportion 1:1:1:8;  compositionno.8 – ginger, sumacin proportion 1:8[2]. constituents (or components) of composition were selected following not only organoleptic indicators of obtained products, but also their antimicrobial properties based on information given in source of literature [1,3,4]. we took into consideration the detailed characteristics of each component of given compositions: ginger (zingiber)is a well-known antiseptic, which activates metabolism, helps to normalize functioning of cardiovascular system, improve immunity, organism resistance to seasonal viral diseases. the content of essential oil in ginger is 3%, main component of which is ginger, zingiberene. this spice contains almost all the necessary for the human body such as amino acids, vitamins a, в1, в2, с, mineral substances – zinc, sodium, potassium, iron, calcium, phosphor etc [1,3]. curcumacontains starch, curcumine colorant (0.6%, dissolved in fats and alcohol, does not dissolve in water),aromatic essential oil (1.55.5%), main component of which is zingiberene, tumeron. curcuma contains vitamins k, сand vitamins of group в, as well as calcium, magnesium, iodine, phosphor and iron[1,3]. cinnamon has a very tender aroma, due to its content of (4-10%) eugenol, phellandrene. the taste is sweetish, a bit burning, warming. its basic components arecinnamicaldehyd 6576% (80%). cinnamon shows antiseptic effect and improves digestion[1,3]. clavusis a perfect antiseptic, its content of essential oil is up to 22% and eugenol is the basic component. clavus contains vitamins а, е, к, сand vitamins of group в, as well as microand macro-elements[1,3]. sweet pepperas a spicy combines aromas of clavus, cinnamon, nutmeg and black pepper and as medicine it is used for therapy of stomach diseases. its content of essential oil is up to 5% and eugenol is thebasic component[1,3]. black рepper has got its aroma due to the essential oil which content is up 15%. basic components of this oil arealkaloid piperine, terpenes and sesquiterpenes, depentenes. black рepper has general strengthening, bactericidal, anti-inflammatory effect. it contains vitamins of group в and а, с, е, к, as well as magnesium, calcium, potassium, phosphor, iron, zinc etc[1,3]. sumac contains a number of acids, vitaminс, apple, citric, tartaric, amber, maleic, fumaric, ascorbic acids. sumac also contains volatile oils, aldehydes, terpenoids, and fatty oils. sumac leaves contain a number of tanning substances, basic part of which is tannin, and the rest – derivatives of gallic acid[1,3]. anise. essential oil – anethol is used in medicine, perfumery, and liquor and spirit production, food industry. anise fruit contains2-3% of essential oil, 4-23% of fatty oil, 18% proteins, 3-5% sugar, furfurol, caffeic and chologenic acids and other wholesome substances. anise essential oil contains 80-90%of anethol, estragon food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 s. teterina, n.yushchenko, u.kuzmyk, i. khlystun, studyofantimicrobial characteristics of spices composition, , food and environment safety, volume xiv, issue 3 – 2015, pag. 300 – 305 302 10%,anise aldehyde and anise alcohol [1,3]. badiane (staranise) contains polysaccharides, resins, essential oil (5-10%), tanines, terpenes, vitamins, microandmacro-elements and other biologically active substances. badiane has anti-inflammatory, general strengthening effect, stimulates functioning of digestive system [1,3]. cardamomis recommended for improving appetite, having a general strengthening effect. its content of essential oil is up to 10% and the basic components are borneol, cineol, sabinene andcitrene. cardamom contains vitamins group в and vitamin с, micro andmacro-elements(iron, zinc, potassium, calcium, magnesium, phosphor)[1,3]. fenugreek contains small amounts of alkaloid trigonelline, nicotinic acid, steroid sapogenin, flavonoids, glair and bitter substances, essential oils (main component coumarine), proteins, tanines, vitamins, phosphor, iron [1,3]. nutmeg has stimulating effect. it contains vitamins а, с, в1, в2, в3, в6, в9, в4andmicroand macro-elements. its content of essential oil is up to 15% (the basic components are dipentene, borneol)[1,3]. milk and dairy products microflora is conditionally divided into the following groups: 1) microorganisms instrumental in the technology of milk and milk products. useful lactobacilli are applied in the manufacturing of various milk products. instances of such lactobacilli are homofermentative and heterofermentative streptococci and bacilli, which facilitate lactic fermentation. they are applied in the manufacturing of cultured milk products and cheeses, as well as in forage siloing, leavening of vegetables and fruit[5,6,7]. 2) microbeswhich are harmful to the technology and cause deterioration of milk and milk products. in case they are present in milk, they develop taste, scent and consistence defects and their sanitary characteristics deteriorate. the consumption of such products can be harmful and cause disturbance of the function of food system. such harmful microbes are micrococcus, colibacillus, hay bacillus, proteus, and fluorescent bacteria[5,6,7]. 3) pathogenicmicroorganisms which are dangerous to human/animal health. they do not alter the composition and properties of milk and milk products, but they act as causative agents of human/animal infectious diseases. when contaminating milk, some bacteria (staphylococcus, proteus) can secrete toxins which cause food poisoning [5,6,7]. the characteristics of some contaminants in the production of milk products are examined as follows: micrococcus is immovable and does not produce spores. during reproduction in milk it secretes rennet and lactic acid. certain types decompose fat, producing bitter taste of milk. in addition to sour taste, clot also has a bitter taste which is caused by an increasing amount of peptones [5,6,7]. enterobacter is a genus of facultative anaerobic, rod-shaped bacteria. it is a part of a normal intestinal microflora of humans and animals, thus it is used as a bacterial indicator of sanitary quality of soil, water and foods; it is considered an opportunistic pathogen bacterium. in milk products, acid is produced as a result of fermenting lactose; other carbohydrates and alcohols can also be fermented with or without gas production. when consuming food products containing such bacteria, consumers with weakened immunity may develop serious diseases[5,6,7]. bacillus is a genus of gram-positive aerobic soil bacteria, and, as all species of the genus, it can produce endospores. it grows quickly in milk and can cause severe protein disintegration producing methane, indole, hydrogen sulfide, mercaptans and other musty-smelling substances. when growing in milk and milk products it produces protein disintegration products food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 s. teterina, n.yushchenko, u.kuzmyk, i. khlystun, studyofantimicrobial characteristics of spices composition, , food and environment safety, volume xiv, issue 3 – 2015, pag. 300 – 305 303 and toxic substances. striking examples of the genus are hay and potato bacillus, which develop quickly in milk and disintegrate protein producing albumin, peptides, amino acids and ammonia and imparting a bitter taste to the milk. after a long time in milk it can accumulate harmful substances[5,6,7]. endomyces is an aerobe; it reproduces in cultivated milk products, leavened vegetables, malt and on damp walls. when developing in milk and milk products it produces protein disintegration products and toxic substances. such bacteria travel into milk using particles of forage and soil [5,6,7]. 2. materials and methods the research aimedto study the effects of spicy aromatic composition on microorganisms which are species of contaminating microbiota characteristic of production of cultivated milk products. previous researches based on cultured cheese have shown that during holding cultured milk products, which contained 2.75-11% of spice composition, there were no significant alterations of organoleptic or technological quality characteristics[8]. during the first 3 days of holding, the actual acidity characteristic did not change. during the next 5 days of holding there was a gradual decrease in the actual acidity characteristic, an average from 4.5 to 4.2 ph units accordingly. during the holding period a test sample showed a decrease of the actual acidity characteristic from 4.4 to 3.8 ph units. the organoleptic properties did no undergo any changes during the abovementioned time. it may be assumed that the applied spice composition have shown sufficient germicidal action. therefore, a decision was made to look into the influence of spice composition on pure growth of microorganisms that are typical contaminants of cultured milk products. the following typical contaminants of cultured milk products were used as test cultures: enterobactercloaceae, micrococcus albus, bacillus subtilis, endomyceslactis. pure growth of bacteria and yeast, used in the research, are kept in the collection of viable cultures of microorganisms in the department of biotechnology and microbiology of the national university of food technologies. in order to determine germicidal properties of the tested spice composition, suspensions of twenty-four-hour test cultures, cultivated in agar growth medium (meat peptone (mpa) for bacteria and wort agar for yeast at the temperature of 30с) were prepared. the number of viable cells was calculated by the koh method (colony-forming units/sm3). then suspensions from each test culture were moved into 8 vials (putting 10 sm3 of each suspension) and 0.1 g of each tested spice composition was added into the vials; the produced substances were kept at the optimal temperature for growth (30 с) for 2 hours. after the exposure, the number of living cells was calculated by the koh method. the microorganism survival rate was expressed by a percentage ratio between the number of viable microorganisms in exposed samples and the number of viable microorganisms in the original suspension. 3. results and discussion the number of cells in the original suspensions: 8.9·105 – enterobactercloaceae, 9.3·105 – micrococcus albus, 1.2·106 – bacillus subtilis, and 7.4·104 – endomyceslactis. figures 1 and 2 illustrate the survival rate of cells of the cultures tested. based on the data given in figure 1, all tested spice compositionshave shown significant germicidal properties against the test cultures of bacteria. however, the germicidal action of composition against food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 s. teterina, n.yushchenko, u.kuzmyk, i. khlystun, studyofantimicrobial characteristics of spices composition, , food and environment safety, volume xiv, issue 3 – 2015, pag. 300 – 305 304 different cultures was not identical, e.g. the composition no.6 showed themaximal germicidal properties against the cultures of enterobactercloaceae, the degree of survival of which was 3.3%, whereas its germicidal properties against cultures of micrococcus albus were only third highest. the most stable germicidal properties against the inhibition of alltested cultures of bacteria were demonstrated by the composition no.1 and no.2: the degree of survival of bacterial cultures was 7.234.8%. from the analysis of results, illustrated by the diagram below (figure 2), it is clear that all spice compositions have displayed germicidal properties against the cultures of endomyceslactis. compositions no. 3, 4, 6, 7 and 8 demonstrated stronger inhibitory action; notably the application of compositionno.6 resulted in a 9% survival rate. fig.1.antimicrobial effect of spices on bacteria test-cultures fig.2.antimicrobial effect of spices against the test-culture endomyces lactis. 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 su rv iv al , % compositions enterobacter cloaceae micrococcus albus bacillus subtilis 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 su rv iv al , % compositions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 s. teterina, n.yushchenko, u.kuzmyk, i. khlystun, studyofantimicrobial characteristics of spices composition, , food and environment safety, volume xiv, issue 3 – 2015, pag. 300 – 305 305 4. conclusion the conducted research demonstrated germicidal action of spice composition against test cultures of microorganisms which cause deterioration of milk products. the spice composition that displayed the strongest germicidal properties contained anise, cloves, ginger, black pepper and nutmeg, due to their higher levels of volatile oils and phenol compounds. notably, the spice compositions were introduced into suspensions in amounts, specified by the formulations of cultured milk products. therefore, the applications of the developed spice composition in the technology of cultured milk products will result in bacteriostatic effect, thus increasing the storage periods of produced milk products and stabilizing the physicochemical, organoleptic and microbiological properties of the products during holding. 5. references [1]. dudchenko l., some aspects of using aromatherapy in doctor’s practice, journal, 3:716,(2007) [2]. yushchenko n., kuzmyk u., spices for new cheese products, journal food industry, no.6:23-26, (2011) [3]. pekli f., aromatology, medicine: 129259(2001) [4]. tsypriian v, matasar і,slobodkin v, hygiene of nutrition with basics of nutrition science;1:508-517, (2007) [5]. biloruska y., basics of microbiology, sanitary and hygiene, study guide, 10-128, (2003) [6]. vekirchyk k., microbiology with basics of virology, textbook,130-210, (2001) [7]. shulha n., mlechko l., vital activity of microorganisms in dairy products,study guide, 142, (2010) [8]. yushchenko n., kuzmyk u., substantiating of storage termof paste like fermented milk products with spices, ukrainian food journal,no.1: 34-37,(2012). sasfsadfasdfasfasdfafasfdasad food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 118 the activity of -amylase and peroxidase during germination of some maize caryopses with different viabilities *marcel avramiuc stefan cel mare university of suceava, faculty of food engineering, str. universităţii, no. 13, 720229, suceava, românia, e-mail: avramiucm@fia.usv.ro *corresponding author received 2 october 2011, accepted 15 november 2011 abstract. the paper examines, during pregermination and germination (after 48 and 96 hours), the activity of amylase and peroxidase in endospermum of maize caryopses with different viabilities (germination percentages) to emphasize what is the relationship between maize viability and activity of these enzymes, which belong to two different classes: oxidoreductases and hydrolases. the biological material, used in this work, was represented by six samples of maize caryopses belonging to a local variety, whose germination percentages (g%) were between 34% and 95%. the data derived from experiences show that samples with g ≥ 70% registered after 48 hours or after 96 hours of germination, growth between 18% and 242% of the activity of α-amylase, compared with the period before germination (0 h). at samples with g ≤ 70% the α-amylase activity registered either increases with maximum 18% o reductions between 1% and 24%. the peroxidase activity has risen after 48 and 96 hours of germination, versus run-up (0 hours), also in the samples with fg ≥ 70%, but there was smaller (8% to 107%). in trials with fg ≤ 70% the enzyme activity has recorded either increases between 12% and 18%, or fall between 8% and 32%. corelaţiile între viabilitate şi activitatea peroxidazei, respectiv a amilazei cariopselor de porumb analizate au arătat că doar valorile activităţii peroxidazei în timpul germinaţiei au fost în relaţie directă cu viabilitatea cariopselor. correlation between caryopses viability and activity of peroxidase, respectively -amylase, have shown that only the peroxidase activity values during germination were into a direct relationship with caryopses viability. the caryopses structural and functional damages, shown, indirectly, through their viability reduction, have influenced the both enzymes, leading to the decrease of their activity in samples with low germinations. keywords: -amylase, peroxidase, caryopse, sample, germination. 1. introduction within the plants world, the beginning of a new vital cycle is marked of germination, physiological and biochemical complex process influencing the future plant body growth and development. under appropriate environmental conditions (temperature, humidity, ph, etc.), the substances stored in seed reserve tissues, suffer, under the influence of different enzymes, major transformations, qualitative and quantitative, evidenced by the emergence of biologically active compounds that positively affects cell activity and its equilibrium (1). the vitamins e şi c biosynthesis, superoxid-dismutase, catalase and peroxidase activation are evidence of the antioxidant potential of germinated seeds, which are used as protective foods in nutrition (2). the germination process, through the study of seed compounds, on the one hand, and the enzymes involved in their transformations, on the other hand, can offer precious advice to farmers on some seed features and of the future plant (viability, productivity etc.). this paper examines, during seeds pregermination and germination, the activity of peroxidase and -amilase in reserve tissue of some maize caryopses with different viabilities to highlight what is the relationship between viability and activity of these enzymes, which belong to two different classes: oxidoreductases and hydrolases. mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 119 2. experimental the biological material used in this work, was represented by six samples of maize caryopses, belonging to a local variety, deriving from the last 10 years production. both the new samples and the older ones (with smaller viabilities) have been stored without controlled temperature and humidity. the samples were encoded, having specified the germination capacity values in parentheses (g%), as follows: pm (g = 95%); p1 (g = 80%); p2 (g = 56%); p3 (g = 70%); p4 (g = 34%); p5 (g = 85%). to determine the germination capacity (g%), it have been used 4 repetitions of 50 seeds for each sample (3, 4). it have been used petri plates of glass with special filter paper. the germination medium was distilled water that has soaked the filter paper, and temperature of 25°c. the maximum duration of the test was 4 days (96 hours). the -amilase activity was determined using the noelting-bernfeld method (5), based on free maltose forming through hydrolysis of starch solution 2% by α-amylase extracted from the sample to be analysed. the calculation of the results was done by means of a standard curve, and the -amilase activity was expressed in micromols (μm) of maltose content, formed under the action of the enzyme, from 1 g of seed (flour). determination of peroxidase activity was performed with a colorimetric method (5). the enzyme extract was obtained through grinding of 0,1-0,5 g of caryopses with a buffer solution m/15 of nah2po4 and na2hpo4, with ph=6,7, followed by centrifugation. the activity dosing was made colorimetrically at 430 nm, using a pyrogalol solution (189 mg in 25 ml distilled water), hydrogen peroxide 0,15%, distilled water, phosphate buffer solution of sodium and enzyme extract. the peroxidase activity was calculated by the reaction speed, and was expressed in conventional units at 1 g of seeds (flour). during germination, the -amylase and peroxidase activity dosages were made from caryopses reserve tissue, after excision of new appeared formations. the data of experiments (consisting in 4 replicates for each determination) were statistically processed, using the mean values and standard deviations. 3. results and discussion table 1 reproduces the -amylase activity mean values of maize samples before germination (0 hours) and after 48 and 96 hours from seed germination start. table 1 the activity of -amylase during germination of maize samples samples g (%)  -amylase activity (m maltose / g dm) 0 hours* 48 hours* 96 hours* pm 95 32.80 30.50 84.25 p1 80 20.55 70.30 18.50 p2 56 30.80 23.35 25.82 p3 70 25.69 45.68 14.51 p4 34 26.45 26.20 30.85 p5 85 24.58 49.46 16.42 * = germination processs duration food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 120 the determination of -amylase activity before germination has evidenced values between 20.55 m maltose/g dm at p1 sample (g = 80%) and 32.80 m maltose /g dm at pm sample (g = 95%). the caryopse samples p3 (70%), p4 (g = 34%) and p5 (g = 85%) have registered close values (24.58 – 26.45 m maltose/g dm) of -amylase activity, while the p2 sample (g = 56%) had an enzyme activity close to pm sample (g = 95%). after 48 hours of germination, the enzyme activity values have vacillated between 23.35 m maltose/g dm at sample p2 (g = 56%) and 70.30 m maltose/g dm la proba p1 (g = 80%). as against the period before germination (0 hours), the enzyme activity values after 48 hours of germination have registered rises with 242% at p1 sample (g = 80%), with 101% at p5 sample (g = 85%), with 78% at p3 sample (70%) or reductions with 24% at p2 (fg = 56%), with 7% at pm (g = 95%) and with 1% at p4 (fg = 34%). after 96 hours of germination, the enzyme activity values have vacillated between 14.51 m maltose/g dm at p3 sample (g = 70%) and 84.25 m maltose /g dm at pm sample (g = 95%). compared with the period before germination (0 h), the enzyme activity has grown with 176% at pm sample (g = 95%), with 18% at p4 sample (g = 34%) and reductions with 43% at p3 sample (g =70%), with 33% at p5 sample (g = 85%) and with 16% at p2 sample (g = 56%). as against the enzyme activity after 48 hours, the -amylase activity values after 96 hours of germination have risen with 176% at pm, with 18% at p4, and with 10% at p2. in the same conditions, the -amylase activity has reduced with 74% at p1, with 68% at p3 and with 67% at p5. an analysis of the data in table 1 shows that samples with fg ≥ 70% (pm, p1, p3 and p5) have registered either after 48 hours or after 96 hours of germination, growth between 18% and 242% of the α-amylase activity, compared with the period before germination (0 h). in samples with fg ≤ 70% (p2 and p4) the enzyme activity has recorded either increases with maximum 18% (p4) or reductions between 1% (p4) and 24% (p2). the viability (germination) reduction of some seeds is accompanied by reducing of the synthesis capacity of the hydrolitic enzymes (6). during germination of barley aged seeds the -amylase activity was lower by 50-70% compared to the control group, represented by young seeds (6). figure 1 reproduces the correlations between the values of caryopses germination (g%) and the values of -amylase activity, determined before (0 hours) and after 48 and 96 hours of germination. as seen in graph, between germination (g%) and -amylase activity at 0, 48 and 96 hours of germination, one could establish positive correlations, but insignificant, after 48 hours (r2 = 0,1235), and 96 hours (r2 = 0,084) of germination. y = 0,0044x + 26,502 r2 = 0,0005 y = 0,2794x + 18,524 r2 = 0,1235 y = 0,3479x + 8,3749 r2 = 0,084 0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 100 germination (%) am yla se ac tiv ity (u m m alt os e/g .d m ) 0 h 48 h 96 h linear (0 h) linear (48 h) linear (96 h) fig. 1 linear regression for the correlation between germination (%) and amylase activity (μm maltose/g. dm) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 121 researching the dynamics of starch content and -amylase activity during germination of maize caryopses with different viabilities, porochseritan and avramiuc (2002) have found the greatest values of enzyme activity after 24, 48, 72 or 96 hours of germination in samples with g > 80%. calculating the correlation between changes in the levels of starch and -amylase activity during seed germination, the same authors have argued that when the viability (g%) decreased the enzymatic activity efficiency decreased too, leading to the reduction of the caryopses capacity of using this reserve sugar. the table 2 reproduces the peroxidase activity mean values of maize samples, before germination (0 hours) as well as during germination, after 48 and 96 hours. table 2 the activity of the peroxidase during germination of maize samples samples g (%) peroxidase activity (ui/g.dm) 0 hours* 48 hours* 96 hours* pm 95 15.56 20.03 31.98 p1 80 12.46 21.02 24.45 p2 56 14.40 16.55 15.83 p3 70 20.58 19.03 28.25 p4 34 13.84 9.34 12.70 p5 85 17.65 19,14 34.85 * = germination processs duration the determination of peroxidase activity before germination shows values between 12.35 iu/g. dm at p1 (g = 80%) and 20.58 iu/g. dm at p3 (fg = 70%). the caryopses samples p4 (34%), p2 (fg = 56%), pm (95%) and p5 (85%) have registered close values of peroxidase activity (13,84 – 17,65 iu/g. dm) after 48 hours of germination, the enzyme activity values have vacillated between 9,34 iu/g. dm at p4 (g = 34%) and 21.02 iu/g. dm at p1 (g = 80%). at the same germination interval, four samples (p1, p3, p5 and pm) have recorded very close values of this enzyme activity. as against the period before germination (0 hours), the enzyme activity values after 48 hours of germination have registered rises with 66% at p1 sample (g = 80%), with 27% at pm sample (g = 95%), with 18% at p2 sample (g = 56%), and with 8% at p5 sample (g = 85%). the samples p4 and p3 have registered reductions with 32% and 7%. after 96 hours of germination, the peroxidase activity values have vacillated between 12.70 ui/g.dm at p4 sample (g = 34%) and 34.85 ui/g.dm at p5 sample (g = 85%). compared with the period before germination (0 h), the enzyme activity has grown with 107% at pm sample (g = 95%), with 97% at p5 sample (g = 85%) and p1 sample (g = 80%), with 37% at p3 sample (g = 70%) and with 12% at p2 sample (g = 56%). p4 sample has registered a reduction of activity with 8%. as against the enzyme activity after 48 hours, the peroxidase activity values after 96 hours of germination have risen with 63% at pm, with 82% at p5, with 48% at p3, with 36% at p4, and with 18% at p1. the only one reduction of the peroxidase activity (with 4%) has been registrated in p2 sample. the analysis of the data in table 2 shows that samples with fg ≥ 70% (pm, p1, p3 and p5) have recorded increases in activity of peroxidase after 48 and 96 hours of germination, compared to the period before food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 122 germination (0 hours), but there were smaller (8% to 107%). in samples with fg ≤ 70% (p2 and p4) the enzyme activity has recorded either increases between 12% and 18% (p2) or fall between 8% and 32% (p4). fig. 2 reproduces the correlations between the values of caryopses germination (g%) and the values of peroxidase activity, determined before (0 hours) and after 48 and 96 hours of germination. as can be seen in the graph, between germination (g%) and peroxidase activity at 0, 48 and 96 hours of germination one could establish positive correlations, significant for 48 hours (r2 = 0,8255) and 96 hours (r2 = 0,8319) of germination. y = 0,0331x + 13,392 r2 = 0,0601 y = 0,1713x + 5,4477 r2 = 0,8255 y = 0,3667x 0,8978 r2 = 0,8319 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 germination (%) pe ro xid as e a cit ivi ty (u i/g .d m ) 0h 48h 96h linear (0h) linear (48h) linear (96h) fig. 2. linear regression for the correlation between germination (%) and peroxidase activity (ui/g. dm) researching the peroxidase activity in caryopses of 4 samples of maize (with germination from 0 to 95%), avramiuc (2004) found on pregermination period small differences between enzyme values. after 48 and 96 hours of germination the differences between samples have emphasized, the peroxidase activity values placing on a ascending path (apart from the sample with g = 0%), in direct correlation with gemination values of the analysed.samples. computing the correlations between germination values and enzyme activity at 0, 48 and 96 hours of germination, the author has found direct correlations (significant and very significant) between germination capacity (g%) and peroxidase activity only at 48 and 96 hours of germination. 4. conclusions the research of the -amylase and peroxidase activity of some samples of maize caryopses with different viabilities in pregermination (0 hours) and at 48, and 96 hours of germination, has revealed certain differences between samples depending on caryopses viability values. the study of -amylase has shown that samples of cariopse with germination capacity ≥ 70% have registered, either after 48 hours or after 96 hours of germination, increases between 18% and 242% of the enzyme activity, compared with the period before germination (0 h). in samples with fg ≤ 70% the -amylase activity has recorded either increases with maximum 18% or reductions between 1% and 24%. the analysis of peroxidase showed that the maize samples with germination capacity ≥ 70% have registered rises between 8% and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume x, issue no.4 2011 123 107% of this enzyme activity after 48 and 96 hours of germination compared to period before germination (0 hours). in samples with fg ≤ 70% the enzyme activity has recorded either increases between 12% and 18%, or fall between 8% and 32%. the germination process has stimulated the activity of the both enzymes (-amylase – from hydrolase class, and peroxidase – from oxidoreductase class) in samples of caryopses with germination capacity ≥ 70%, after 48, respectively 96 hours of germination, compared to pregermination period. in these samples of caryopses, -amylase has registered, at the same time intervals, more increases of its activity as against peroxidase. the correlations between viability (g%) and the peroxidase, respectivelly -amylase activity, in analysed maize caryopses, have shown that only peroxidase activity values (during germination) have been in direct relationship with caryopses vibility. the caryopses structural and functional damages, shown, indirectly, through their viability reduction, have influenced the both enzymes, leading to the decrease of their activity in samples with low germinations. 5. references 1. segal r., segal b., teodoru v., gheorghe v., 1982 valoarea nutritivă a produselor agroalimentare. ed. ceres, bucureşti. 2. segal r., fulea v., 1998 acţiunea antioxidantă a grâului germinat, „noutăţi în microbiologie şi biotehnologie”. ed. corson, iaşi, 96-104. 3. anghel g., raianu maria, matei c., bucurescu n., rădulescu i., anganu i., velea c., 1959 determinarea calităţii seminţelor. ed. acad. r.p.r., bucureşti. 4. ellis r.h., hong t.d., roberts e.h., 1985 handbook of seed technology for genebanks. international board for plant genetic resources, vol. ii, rome. 5. artenie v., tănase elvira, 1981 practicum de biochimie generală. centrul de multiplicare al univ. “al. i. cuza” iaşi, 138-140. 6. anderson j.d., 1973 metabolic changes associated with senescence. seed sci. techn., i, 401-416. 7. avramiuc m., 2004 research on seed exudate indices and peroxidase activity in some maize and broad bean seeds. analele universităţii “ştefan cel mare” suceava, anul iii, nr.2-2004, 5-8, issn 1583-2295. 8. poroch-seriţan maria, avramiuc m., 2002 dinamica conţinutului de amidon şi a activităţii -amilazei în timpul germinaţiei unor cariopse de porumb. analele universităţii “ştefan cel mare” suceava, secţ. ing. alim., anul i, nr.2-2002, 14-17, issn 1583-2295. the activity of (-amylase and peroxidase during germination of some maize caryopses with different viabilities *marcel avramiuc 1. introduction 2. experimental 4. conclusions 5. references microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_26-30.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 30 high-fiber wheat bread produced with fermented bran gabriela pop1, adriana dabija1, amelia buculei1 1 ştefan cel mare university of suceava, faculty of food engineering, str. universitatii, no. 13, 720229, suceava, romania,, e-mail: gabriela.pop@usv.ro abstract: dietary fiber is a common and important ingredient of a new generation of healthy food products demanded more each day by the consumer. one of the main sources of dietary fibre is cereals and cereal products. this study is dedicated to investigate the application of new sources of dietary fiber such as fermented bran for increasing the nutritional value of wheat bread. the objectives of the present investigation were to compare the effects of spontaneous fermentation of the bran fraction and fermentation with added yeast or added yeast and lactic aceid bacteria (lactobacillus delbrueckii subsp.bulgaricus) on the quality of wheat bread supplemented with bran. prefermentation with yeast and lactic acid bacteria had the greatest effect on the general quality of obtainig breads: improved the loaf volume, crumb struture and the shelf-life of bread supplemented with bran. fermentation of wheat bran with yeast or with yeast and lactic acid bacteria improved the loaf volume, crumb structure and shelf-life of bread supplemented with bran. even though we used bran instead flour for the fermentation, the results are similar to the effect of sourdough on bread quality. spontaneous fermentation of the bran fraction did not have the same positive effects on bread quality. keywords: yeast, lactic acid bacteria, fermentation introduction cereals (wheat, rye, oat, corn, sorghum, millet and rice) have been an all times and will always be the most important plant group for the human existence and activity. some researchers estimated, even from the beginning of this century, the potential advantages of food fibres in preventing some chronic affection. for a healthy person, the recommended fibre ratio is placed at medium value of 30grams fibres/day, half of which must derive from cereals. due to the main humankind concerns regarding the increase of population health state in general and obesity control in particular, we may say that the development of some cereal and pseudocereal processing technologies becomes of fundamental importance. the make of no refined based – products is highly necessary especially that lately it has been demonstrated that weight increase is directly related to food ratio from refined cereals [1]. this show how important is for us to make the difference between the products of whole grain and those of refined cereals to help weight control. one way to obtain high-fiber wheat bread is to add bran in the bread formulas. [6] a major disadvantage could be the fact that when we used bran in order to supplement the wheat bread, ussualy we could observe a wakenes of structure and baking quality of wheat dough and, also, a decrease of volume and elasticity of the crumb. the effect has been atributed to the dilution of gluten wich would affest the gas-holding food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 31 capacity of the dough [2] the increased concentration of insoluble and soluble cell wall material may also have detrimental effects on dough structure. bran particles can machanically disrupt the structure of the gluten network. the baking quality of bran could be influence also by the size of particle or by the chemical components like soluble pentosane and ß-glucan. [4] as the specific volume of bread is one of the important quality characteristics, differnt bran pretreatmens have been used to improve the volume of breads supplemented with bran. the aim of the present study was to improve the quality of wheat bread supplemented with bran by prefermentation of the bread fraction. we compared the effects of spontaneous fermentation and fermentation with yeast or with yeast and lactic acid bacteria on the quality and the crumb structure of bread supplemented with 20% wheat bran. materials and methods in order to obtain some available experimental data we used commercial white wheat flour and wheat bran, provided by local producers. the chemical composition of the material is given in table 1. the analytical flour quality was determined according to the international standard methods (ash content – icc104/1, wet gluten – icc105/2, protein content – icc106/2). the moisture content of the wheat flour and bran were determined by oven drying at 1300c for 1 hour. [3] table 1. analytical parameters of the raw materials component wheat flour wheat bran 80% flour +20% bran moisture 13.42 14.3 13.86 protein (n x 6,25 8.5 8.2 8.5 total dietary fiber 2.7 52.0 12.6 total pentosan 1.9 26.0 7.3 starch 78.8 12.0 64.2 wet gluten 27.11 27.05 ash 0.49 0.54 0.51 starch content was determined by megazyme colorimetric method and total dietary fiber byenzymatic-gravimetric method of douglas. [3] the starter culture of lactobacillus delbrueckii subsp.bulgaricus (jointec) was obtained from s.c.liliput s.a. constanţa. instant active dry yeast from rompak s.a. pascani, was used for bran pretreatment and baking. in order to obtain some available experimental data our breads we use formulas which are given in table 2. table 2. bread formulas component, g control bread bread with bran addition wheat flour 500 400 wheat bran 100 yeast 7.5 7.5 salt 7.5 7.5 sugar 7.5 7.5 fat 7.5 7.5 water 345 400 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 32 the doughs were mixed with a spiral mixer for 8 min. after a floor time of 20 min at 280c, the dough was divided into 400g loaves. the loaves were proofed in pans (55 min at 350c) and baked at 2000c for 25 min. [5] after baking, the samples were cooled 6-8 hours in controlled atmosphere (uv lamps). in order to be scoring (after 24 hours), the samples were sliced for packed in plastic bags. crumb structure was determined after 24 hours of cooling by means of a stereomicroscope in order to obtain the fermented bran we used an adapted method (m.salmenkalliomarttila, k.katina, k.autio, 2001): 100g bran was mixed with 350 g of water and dry micoroganisms (1.25g of yeast ore 1.25g of yeast and 0.162 of l. bulgaricus jointec) in a large bowl; the bowl was covered with aluminum foil and incubated in temperature room. after tretment, the ph an total titrable acidity (tta) were measured. the tta was expressed as cm3 of 0.1 m naoh/10g of bran suspension. the experiments are made in the research laboratory of “ştefan cel mare” university of suceava, faculty of food engineering. results and discussions in order to obtain some available data we made the following samples: p1: bread with 20% bran without pretreatment p2: bread with spontaneus bran fermentation, 4hour p3: bread with spontaneus bran fermentation, 16hour p4: bread with prefermentation bran with yeast, 4hour p5: bread with prefermentation bran with yeast and lactic bacteria, 16 hour p6: control bread without bran  effect of bran fermentation on the loaves volume baking experiments showed that wheat bran at a substitution level of 20% decreased loaf volume by 19% in comparision to white control bread (figure 1) figure 1. effect of bran fermentation on loaf volume off breads supplemented as it could see in figure 1, bran treated by short fermentation with yeast (p4) and long fermentation with yeast and lactic acid bacteria (p5) improved the specific bread volume over untreated bran by 10-15%. the 4 hour spontaneous fermentation of bran (p2) slightly improved specific volume, whereas the 16-hours spontaneous fermentation (p3)had no effect on bred volume. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 sp ec if ic v ol um e, m g/ g p1 p2 p3 p4 p5 p6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 33  effect of bran fermentation on the ph and tta of bran-water slurries as it could see in table 3, bran possessed a good buffering capacity, that’s why the pretreatment had only a slight effect on the ph of the bran slurries. table 3. effect of bran fermentation on the ph and tta of bran-water slurries p2 p3 p4 p5 yeast no no yes yes lactobacillus delbrueki subsp.bulgaricus no no yes yes fermentation time, hour 4 16 4 16 temperature, 0c 28 25 28 25 initial ph 6.6 6.6 6.6 6.6 final ph 6.6 6.5 6.6 5.8 initial tta 3.3 3.3 3.3 3.3 final tta 3.3 4.4 5.9 8.7 the bran fermented by lactic acid bacteria and yeast was the most acidic (ph=5.8, tta=8.7), whereas spontaneous fermentation of the bran for 16 hours had a smaller effect, ph= 6.5 tta = 4.4. wheat sourdoughs usally have value of ph=3.5 to 4.5 and tta values from 8 to 22, depending on the ash content of the flour and the starter culture used. higher ash content of the flour gives more acidic ph and higher tta values. if we compare the ph and tta values presented in the literature we can see that the lactic acid bacteria-yeast fermented bran was not very acidic. addition of baker’s yeast to the sourdough slow the souring. bran contains a much higher number of microbes than flour. so we could notice that adding starter cultures to the bran instead of spontaneous souring is advisable for the production of bread with an even repeatable quality.  effect of bran fermentation on the crumb firming. in order to analyze the crumb firmness it were made tests on breads from each of the dough with voland –stevens crumb firmness analyzer in baking day and after each half of next three days.the results are given in figure 2. 0 100 200 300 400 500 600 0 0.5 1 1.5 2 2.5 3 3.5 storage time, days h ar d ne ss /g p6 p5 p4 p3 p2 p1 figure 2. effect of bran fermentation on crumb firmness of bread supplemented food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 34 four– hours fermentation with yeast (p4) and 4-hours spontaneous fermentation of the bran (p2) slowed the crumb firming slightly during three days of storage in comparision with white control bread (p6). the bread baking with bran treted by 4hours fermentation with yeast (p4) was slightly more firm than white control bread (p6) after storage for three days, but the firming rate (the changes in firmness from day0 to day 3) was much smaller. the 16-hours fermentation with yeast and lactoc acid bacteria (p5) improved the crumb softness and keeping qualities most. on the day of baking, all breads supplemented with bran were firmer than the white control bread (p6), but after storage for only one day, the crumb firmnes of the white control bread (p6) was the same as that of the bread baking with fermented bran. after 3 days of storage the bread baking with yeast and lactic acid bacteria (p5) was softer (20%) than the white control bread (p6). conclusions fermentation of wheat bran with yeast or with yeast and lactic acid bacteria improved the loaf volume, crumb structure and shelf-life of bread supplemented with bran. even though we used bran instead flour for the fermentation, the results are similar to the effect of sourdough on bread quality. this a very interesting result especialy if we take into consideration the fact that we used lactobacillus delbrueckii subsp.bulgaricus instead of lactobacillus brevis (usualy used for flour fermentation in bakery). endogenous enzymes of flour, especialy amylases and proteases, as well as the enzymes produced by yeast and lactobacillus delbrueckii subsp.bulgaricus during fermentation seem to have a positive effect on the dough properties and the structure of bread. acid produced during the fermentation lower the ph level of the dough, thereby affecting the enzyme activity and gluten characteristics. if we use bran fermented for repalcing 20% of flour and by optimizing the baking process it is possible to produce good consumer quality wheat bread containing up to 10% dietary fiber. references 1.c.s lai, a.b davis and r.c. hoseney, production of whole wheat bread with good loaf volume. 1989, cereal chemistry, 66:224-227 2. d.rogers, variety whole grain ingredients, international whole grains seminar, 2006, aib international, manhattan, ks, usa 3. american association of cereal chemists, 2000, approved methods of the aacc, 10th ed. method 74-09. the association: st.paul, mn, u.s.a 4. s. hegenbart, understanding enzymes function in bakery foods,2006, cereals chemistry 5. w. atwell, wheat flour, egan press handbooks series, st. paul, minnesota, 2006, usa, 6. p. pyler, baking science & technology, 3rd edition, vol. 1 sosland publishing co., 1998, kansas city mo pp. 30-32, 132-151. microsoft word 3 journal fia nr 1 2010 final_27-35.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 31 synthesis and characterization of electrodeposited ni–w alloys with different levels of tungsten content maria poroch-seriţan1, gheorghe gutt1, traian severin2, silviu-gabriel stroe1 1„ştefan cel mare” university of suceava, faculty of food engineering, str. universitatii, no. 13, 720229, suceava, romania, phone: +40 230 216147, int. 298, fax: +40 230 520267, e-mail: mariap@usv.ro, g.gutt@usv.ro 2„ştefan cel mare” university of suceava, faculty of mechanical engineering, mechatronics and management, str. universitatii, no. 13, 720229, suceava, romania, phone: +40 230 216147, int. 298, fax: +40 230 523743, email: severintraian@fim.usv.ro abstract: tungsten alloys are known for their excellent mechanical and tribological properties. ni-w alloys exhibit enhanced properties such as corrosion resistance, wear resistance and catalytic activity for h2, useful in practical applications. pure tungsten cannot be electrodeposited from aqueous electrolytes, but can be codeposited with iron group elements such as nickel to form an alloy. the objective of the current work was to study the effect of bath chemistry and current density on chemical composition, microstructure and properties of ni-w alloys deposited from citrate-containing baths, without stirring of the bath solution on copper substrate. the investigations include characterization of complex formation by uv-vis spectrometry. the absorption spectrum of solutions with nickel in composition is similar and it shows the presence of four peaks. the current efficiency is increased with increased concentration of ni2+ ions in bath. the layer thickness of the deposition obtained in niw-1 bath is low, but increase with the current density. the homogeneous hardness of the ni-w deposit may be attributed mainly to three factors: layer thickness, the deposition structure and deposit composition. all these tree factors are current density dependent. the micrograph of niw coating, studied by scanning electron microscopy (sem), shows the presence of cracks on a relatively uniform surface and the electron diffraction spectroscopy (eds) analysis confirmed that ni and w are uniformly distributed on the surface. keywords: electrodeposited ni-w alloys, current density, chemical composition, microstructure, properties introduction the interest in electrodeposition of nickel– tungsten (ni–w) alloys has increased in recent years due their excellent corrosion resistance [1], [2], [3], wear resistance, high hardness and magnetic properties [4], [5]. lowe et al. [6] found that the hardness of ni–w alloys is three times higher than that of pure electrodeposited ni. in addition, the hardness is found to increase with increase in w content in the alloy as reported by singh et al. [7]. it is well known that tungsten cannot be electrodeposited from an aqueous solution of sodium tungstate (na2wo4) or any other soluble compound containing this element. however, if a suitable nickel compound such as nickel sulphate (niso4) is added to the plating bath, induced codeposition can take place, forming ni–w alloys. a similar phenomenon is observed during electrodeposition of w, mo or re with ni, co or fe, as well as in electroless deposition of ni–p or co–p alloys. the tungsten content of the alloy is usually in the range of 5–25 at. % (13–50 wt. %). one of the commercial plating solutions journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 32 (enloy® ni-500 from enthone), for example, yields 35–40 wt. % tungsten in the deposit [8]. in order to further improve the tribological properties and thermal stability of the coating, it is sometimes desirable to increase its tungsten content. unfortunately, this has been found difficult, even when thewo42− ion in solution is in large excess compared to the ni2+ ion [9]. one possible way to increase the tungsten concentration in the alloy is to apply periodic current reverse pulse plating, which may also increase the throwing power and the deposition rate, as well as improve the properties of the deposit (e.g., reduce residual stresses and porosity, refine the grain size, improve wear and corrosion resistance, etc.) [10], [11]. another route has recently been suggested by gileadi and co-workers [9], [12], [13], [14], [15], who developed a novel plating bath for codeposition of ni–w alloys with high tungsten content. by removing the nh3 from the bath and using citrate (c6h5o7)3− as an addendum ligature, the tungsten content of the alloy could be increased to 50 at. % (76 wt. %). the working hypotheses in the above studies are that (a) the tungstate/citrate complex forms a complex with nickel citrate in the bulk of the solution or on the surface; (b) this ternary complex, of the type [(ni)(hwo4)(cit)]2−, is the precursor for the deposition of the ni–w alloy; (c) nickel can also be deposited from its complexes with either citrate or nh3. the overall reaction of the ternary complex requires eight electrons and can be written as: [(ni)(hwo4)(cit)]2− +8e− +3h2o → niw + 7(oh)− +cit3− (1) in the previous work of gileadi and coworkers, a rotating gold cylinder served as the working electrode. such working electrodes, however, are impractical for industrial use. furthermore, it is well known that codeposition of nickel and tungsten is very sensitive to changes in operating conditions. the objective of the current work is to study the effect of bath chemistry and current density on chemical composition, microstructure, thickness and hardness of nickel – tungsten alloys deposited from citrate-containing baths, without stirring of the bath solution on copper substrate. materials and methods preparation of niw alloys nickel–tungsten alloys were electroplated from aqueous solutions containing niso4·6h2o (nickel sulphate hexahydrate, sigma aldrich) and na2wo4·2h2o (sodium tungstate dehydrate, sigma aldrich) as the electro-active species, and c6h5na3o7·2h2o (tri-sodium citrate dehydrate, sigma aldrich) as complexing agent (table 1). all reagents were dissolved in bi-distilled water (conductivity 3.406 µs·cm-1). the ph was measured by means of ph/mv/°c meter from cole parmer and adjusted to a value of 8.0 through additions of h2so4 and naoh solutions. table 1. the chemical composition of electrolytic baths for alloys ni-w electroplating bath niso4·6h2o [g·l-1] na2wo4·2h2o [g·l-1] c6h5na3o7·2h2o [g·l-1] niw-1 2.6286 131.944 147.00 niw-2 13.143 131.944 147.00 niw-3 26.286 131.944 147.00 in this work, the home made hull electrochemical cell was used with a 250 ml capacity. the current intensity was calculated with the hull mac intyre equation:  lii log240,51019,5  (1) where: i current density, a·dm-2; i current intensity in cell circuit, a; journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 33 l – distance along the cathode from the point of maximum current densities, cm. the cell was equipped with a cathode (made of 99.98 % purity copper) and a carbon anode. the cathode dimensions are of 8.0 cm (height), 9.8 cm (length), 0.1 cm (with) while the dimensions of anode were of 6.0 cm (height), 1.0 cm (length), 1.0 cm (with). the immersion surface of cathode was of 0.6 dm2 and respectively the anodic immersion surface was of 0.045 dm2. before electrodeposition, the electrode surface was polished with emery paper (320–1000 grain size), then was had with distilled water, thoroughly degreased with acetone, activated with 20% h2so4, washed once more with distilled water and immerse in the bath solution. the above described cell was connected with electroplating regime to the gwinstek gpr-1810hd power supply, having a digital control of current and voltage. for each experiment 1800 coulomb of electricity were used. the temperature of electrolyte solution was kept constant at the value of 25 °c. the electrodeposition was carried without stirring the bath solution. current efficiency the plating current efficiency (ce) for deposition of each alloy was calculated according to the following equation:     ti eqwmeqnim ce    21 /%/% (2) where: m is the deposit weight (g), ni% and w% (wt.%) is the percent of nickel and tungstate in deposits, respectively, eq1 and eq2 is the electrochemical equivalent (g·c-1) of ni2+ and w6+, respectively, i is the deposition current (a), and t is the deposition time (s). the percent of nickel and tungstate in deposit for the entire cathodic surface of hull cell was calculated as the average of three determinations for each of the ten areas (previously the cathodic surface was divided into ten areas with different current densities). characterization techniques absorption spectrum of electrolytes were recorded with a miniature spectrometer (hr 4000 cg-uv-nir, ocean optics inc., dunedin, fl), using 1-cm cells. a tungsten halogen light source (uv-vis-nir light source dh-2000, mikropack) was used for this study. the light from light source dh-2000 was conveyed to the sample through two optical fibres (qp400-2sr/bx, ocean optics inc., dunedin, fl). the morphology of the deposits after drying was observed with a scanning electron microscope (sem, vega ii lmu tescan, czech republic). the attached energy dispersive spectroscopy (eds, bruker axs microanalysis gmbh probe) was used to determine the approximate composition of the alloy. each of the ten areas was measured in three different locations to confirm uniformity. the thickness of metallic layer was measured by means of a positector 6000 – defesko analyzer, based on nondestructive physical method. seven local values of cathode thickness were measured in order to determine the average thickness of metallic layer, u (µm). the micro-hardness of metallic cover, w (hv), was measured using the shimadzu, hmv – 2t, micro-hardness analyzer. the loading weight was of 490.3 mn. results and discussion composition of the actual alloy ni-w bath figure 1 shows a typical spectrum of reagents in the electrolyte composition of alloy ni-w bath for three different nickel sulphate content of deposition bath in wavelength range from 300 to 1100 nm. it may be noted that the spectra obtained for solutions of 131.944 g·l-1 na2wo4·2h2o and (131.944 g·l-1 na2wo4·2h2o + 147 g·l-1 c2h5na3o7· 2h2o) mixture have no absorption peaks. the absorption spectrum journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 34 of the deposition baths containing nickel: niw-1 bath, 26.286 g·l-1 niso4·6h2o, (26.286 g·l-1 niso4· 6h2o + 147 g·l-1 c2h5na3o7·2h2o) mixture, niw-2 bath, niw-3 bath are similar and it shows the presence of four peaks in the following wavelength ranges: 350 – 450 nm, 600 – 700 nm, 700 – 750 nm and 1000 -1100 nm. the 4 absorption spectrum of figure 1 is the specific absorption spectrum of nickel sulphate solution, which due to d-d electronic transitions the ni2+ ion shows characteristic absorption bands in the visible spectral region with absorption maxima at 398.660 nm and 720 nm. for spectra of 5 and 3, 6, 7, respectively, due to presence of 147 g·l-1 c2h5na3o7·2h2o and (147 g·l-1 c2h5na3o7·2h2o + 131.944 g·l-1 na2wo4·2h2o) mixture, respectively in the electrolyte distinct changes in the ni(ii) absorption spectrum can be observed. the higher absorption values at all wavelengths due to complex formation of ni2+ ions with tri-sodium citrate dehydrate or mixture of tri-sodium citrate dehydrate and sodium tungstate dehydrate have also been described in the literature [16]. it was reported [17] that at ph 4.5 nickel citrate complexes, as well as uncomplexed nickel ions (nih2cit+, nihcit, nicit-), all coexist. the most predominant species is a mixture of nih2cit+ and nihcit. on the other hand, tungstate forms soluble complexes with citrate ions and at ph 4.5 the chemistry of tungstate ions is rather complex. poly-ions are formed and it is not clear how these poly-ions interact with citrate. figure 1. overlapping spectra of uv vis nir range for the following solutions: 1 – 131.944 g·l-1 na2wo4 · 2h2o, 2 – 131.944 g·l -1 na2wo4 · 2h2o + 147 g·l -1 c2h5na3o7 · 2h2o, 3 – niw-1 bath, 4 – 26.286 g·l -1 niso4 · 6h2o, 5 – 26.286 g·l -1 niso4 · 6h2o + 147 g·l -1 c2h5na3o7 · 2h2o, 6 – niw-2 bath, 7 – niw-3 bath nevertheless the equilibrium constants of different complexes of tungstate with citrate, of the type [(wo4)(h)n(cit)]5-n are given in the literature and, at ph 4.5, the complex with n=2 is predominant. this is confirmed by the recent data published by younes et al. [9] who studied the abundance of [(wo4)(h)n(cit)]5-n complexes as a function of ph (in the range of 2.0–12.0). this result confirms the increase in w% with increasing ph. the tungsten can only be codeposited (together with nickel) from this ternary complex. the existence of a ternary complex containing nickel and tungsten explains the observation that, although w is only deposited with ni (by discharge of the ternary complex), a parallel route for deposition of ni from its complex with citrate exists, leading to high ni-content in the alloy. cathodic current efficiency and composition of ni–w alloys in most cases studied the cathodic current efficiency (ce) of ni–w alloys journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 35 codeposited from citrate-containing baths is less than 100% denoting simultaneous hydrogen evolution. the effect of bath composition on the overall cathodic current efficiency for the alloy deposition as well as on the tungsten content in the deposits w% (wt. %) were analyzed and the results are given in figure 2. figure 2 illustrates the influence of increasing ni2+ ion content in the bath on the cathodic current efficiency of the alloy codeposition as well as on w% in the deposit. in all cases the concentrations of the sodium tungstate dehydrate and trisodium citrate dehydrate in the bath were 131.944 g·l-1 and 147.00 g·l-1, respectively. at low ni2+ ion concentration, the w% in the deposit is low (about 8%); however, with further increase in ni2+ ion concentration, the w content in the deposit increases and reaches a maximum value of about 12%. this confirms the induced codeposition of w in the presence of ni. 0 2 4 6 8 10 12 14 2,6286 13,143 26,286 niso4 concentration [gl-1] c at ho di c cu rr en t e ffi ci en cy [% ] 0 0,05 0,1 0,15 0,2 0,25 w [w t.% ] i n th e de po si t w [wt.%] current efficiency [%] figure 2. effect of niso4 concentration on the ce% and on w% of ni–w alloy codeposited from niw solution (i=1 a, ph = 8, t = 30 min., 25 °c). for comparison, younes and gileadi [9] reported a maximum cathodic current efficiency of 11% and a maximal tungsten content of approximate 67 at. % in two different baths containing different concentrations of na2wo4. an increase in the bath concentration of ni2+ has also been reported to cause a dramatic increase in the ce [13, 18]. furthermore, it has been reported that an increase in the concentration of ni2+ results in an increase in the rate of both ni and w deposition [19, 20], but the tungsten content in the alloy decreases [15]. the effect of current density on composition of ni–w alloys and on deposit properties for investigating the effect of current density on the composition of ni–w alloys from citrate-containing baths at ph of 8, the hull cell test was carried out. the results on the appearance of deposits are shown in figure 3. for the niw-1 bath, zones marked with 1 and 2, zones with low current density (0 and 0.1 a·dm-2 respectively), don’t have any deposition. the depositions achieved in niw-2 bath and niw-3 are shiny for the all density current range from 0 to 5.10 a·dm-2. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 36 a) b) c) figure 3. the appearance of hull cell panels plated from following baths for alloys ni-w electroplating: a) niw-1 bath, b) niw-2 bath, c) niw-3 bath (i=1 a, ph = 8, t = 30 min., 25 °c) figure 4 illustrates the influence of applied current density on the thickness and on the w content in the deposit. the layer thickness of the deposition obtained in niw-1 bath is extremely low (0.25 µm), but constant. for the niw-2 and niw-3 baths, the deposition layer thickness increase with the current density till 1.5 µm and 2.00 µm respectively. tungsten content of the deposits is influenced by the current density (figure 4). for the deposit obtained in ni-w-1 bath it can be observed that the tungsten content slightly increases with the current density increases. a different behaviour could be observed for the depositions achieved in niw-2 and niw-3 baths, where the highest w content is obtained for the lower current density. the effect of current density in the ni–w system has already been studied in the literature. brenner et al. [21] observed a significant increase in tungsten content with increasing current density in ammonia–citrate bath. yamasaki et al. [22] reported a similar trend. atanassov et al. [23] noted a linear increase in tungsten content with increasing current density when vigorous stirring was applied. on the other hand, a maximum was observed at 50 – 70 ma·cm-2 in the absence of stirring. at current densities higher than 20 ma·cm-2, the ce was 15 – 40% higher in the stirred bath, where hydrogen evolution was less pronounced, compared to an unstirred bath 0 0,5 1 1,5 2 2,5 3 0 0,1 0,36 0,67 1,02 1,43 1,94 2,6 3,53 5,1 current density [adm-2] t h ic kn es s [u m ] 0 2 4 6 8 10 12 14 16 18 20 w [ w t. % ] in t he d ep o si t niw-1 niw-2 niw-3 niw-1 niw-2 niw-3 figure 4. the influence of current density on thickness and the w content in the deposit of niw alloy figure 5 represents hardness as a function of the current. the vickers hardness of asdeposited layer is about hv98. the homogeneous hardness of the ni-w deposit may be attributed mainly to three factors: layer thickness, deposit structure and composition. all these three factors are current density dependent. higher the w content and the layer thickness are, higher the deposit hardness is [24]. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 37 0 20 40 60 80 100 120 0 0,1 0,36 0,67 1,02 1,43 1,94 2,6 3,53 5,1 current density [adm-2] h ar dn es s [h v ] 0 2 4 6 8 10 12 14 16 18 20 w [w t.% ] i n th e de po si t niw-1 niw-2 niw-3 niw-1 niw-2 niw-3 figure 5. the influence of current density on hardness and the w content in the deposit of niw alloy surface morphology the surface morphology of the asdeposited samples and their approximate chemical composition were studied by sem and eds, respectively. figure 6 presents typical sem images. a) b) c) d) journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 38 e) f) figure 6. scanning electron microscope secondary electron images and surface mapping images for niw coatings in: a) b) niw-1 bath, i = 1.43 a·dm-1, ph = 8, t = 30 min., 25 °c, c) d) niw-2 bath, i = 3.53 a·dm-1, ph = 8, t = 30 min., 25 °c, e) f) niw-3 bath, i = 5.10 a·dm-1, ph = 8, t = 30 min., 25 °c the micrograph of niw coating (figures 6a, c, e) shows the presence of cracks on a relatively uniform surface, and no grains of any type could be observed even at high magnifications. because the deposited layer is very thin, the final deposit will fallow the rugosity and the surface defects of the original substrate. energy dispersive spectrometry (eds) was further used in order to determine the chemical composition of the coatings and to investigate the distribution of the elements on the deposition surface (graphs are not shown here). all the eds spectra showed the characteristic peaks for ni and w elements. the surface mapping of ni and w confirmed a very uniform distribution of the elements on the surfaces of all the coatings. conclusions the following conclusions were drawn and discussed with respect to a comprehensive literature survey on electrodeposition of ni–w alloys: 1) the absorption spectrum of ni containing solutions: niw-1 bath, 26.286 g·l-1 niso4·6h2o, (26.286 g·l-1 niso4·6h2o + 147 g·l-1 c2h5na3o7 · 2h2o) mixture, niw-2 bath, niw-3 bath are similar and it shows the presence of four peaks corresponding to the wavelength ranges: 350 – 450 nm, 600 – 700 nm, 700 – 750 nm and 1000 -1100 nm. 2) the current efficiency is increased with increased concentration of ni2+ ions in bath. 3) the layer thickness of the deposition obtained in niw-1 bath is extremely low (0.25 µm), but constant. for the niw-2 and niw-3 baths, the deposition layer thicknesses increase with the current density till 1.5 µm and 2.00 µm respectively for a 5.10 a·dm-2 current density. 4) the homogeneous hardness of the ni-w deposit may be attributed mainly to three factors: layer thickness, the deposition structure and deposit composition. all these tree factors are current density dependent. 5) the micrograph of niw coating shows the presence of cracks on a relatively uniform surface and the electron diffraction spectroscopy (eds) analysis confirmed that ni and w are uniformly distributed on the surface. references 1. m. obradovic, j. stevanovic, a. despic, r. stevanovic and j. stoch, j. serb. chem. soc. 66 (2001) 899. 2. m. obradovic, j. stevanovic, a.r. despic and r. stevanovic, j. serb. chem. soc. 64 (1999) 245. journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 39 3. d.r. gabe, j. appl. electrochem. 27 (1997) 908. 4. t. yamasaki, p. schlossmacher, k. ehrlich and y. oginoi, nano-struct. mater. 10 (1998) 375. 5. t. yamasaki, scripta materialia 44(8–9) (2001) 1497. 6. h. lowe, w. ehrfeld and j. diebel, proc. spie 3223 (1997) 168. 7. v.b. singh, l.c. singh and p.k. tikoo, j. electrochem. soc. 127 (1980) 590. 8. pwa 36975: electroplated niw—thin deposit (enloy ni-500). http://www.enthone.com. 9. o. younes, e. gileadi, electrochem. solid-state lett. 3 (12) (2000) 543. 10. l. namburi, electrodeposition of niw alloys into deep recess, m.sc. thesis, louisiana state university, december 2001. 11. m. donten, z. stojek, h. cesiulis, j. electrochem. soc. 150 (2) (2003) 95. 12. o. younes, l. zhu, y. rosenberg, y. shachamdiamand, e. gileadi, langmuir 17 (2001) 8270. 13. o. younes, e. gileadi, j. electrochem. soc. 149 (2) (2002) 100. 14. l. zhu, o. younes, n. ashkenasy, y. shachamdiamand, e. gileadi, appl. surf. sci. 200 (1–4) (2002) 1. 15. o. younes-metzler, l. zhu, e. gileadi, electrochim. acta 48 (18) (2003) 2551. 16. j. aikaitė, o. gylienė, o. nivinskienė, formation of insoluble polinuclear ni(ii)-citrate complexes in alkaline solutions, chemija (vilnius), 2003, t. 14, nr.3. 17. m.a.m. ibrahim, s.s. abd el rehim, s.m. abd el wahaab and m.m. dankeria, plat. surf. finish. 86 (1999) 69. 18. h. cesiulis, a. baltutiene, m. donten, m.l. donten, z. stojek, j. solid state electrochem. 6 (4) (2002) 237. 19. m. donten, z. stojek, h. cesiulis, j. electrochem. soc. 150 (2) (2003) 95. 20. w.h. safranek, the properties of electrodeposited metals and alloys, 2nd ed., american electroplaters and surface finishers society, orlando, fl, 1986, p. 348. 21. a. brenner, p.s. burkhead, e. seegmiller, j. res. natl. bur. stand. 39 (1947) 351. 22. t. yamasaki, r. tomohira, y. ogino, p. schloßmacher, k. ehrlich, plat. surf. finish. 87 (5) (2000) 148. 23. n. atanassov, k. gencheva, m. bratoeva, plat. surf. finish. 84 (2) (1997) 67. 24. c. firoiu, tehnologia proceselor electrochimice, editura didactică şi pedagogică, bucureşti, 1983 microsoft word 5 mircea oroian.docx 98 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2 – 2017, pag. 98 103 hon eyd ew h on ey adu ltera tion : e-to ngu e a nd ph ys icochem ic al a na ly ses sorina ropciuc1, *mircea oroian1, sergiu pădureț1, amelia buculei1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 15thapril 2017, accepted 27th june 2017 abstract: the aim of this study is to evaluate the usefulness of the e-tongue and physico-chemical analyses (ph, electrical conductivity (ec), colour parameters (l*, a*, b*, chroma and hue angle)) for honeydew honey adulteration detection. for this purpose the honeydew honeys have been adulterated with inverted sugar and malt wort in different percentages (5, 10%, 20%, 30%, 40% and 50% respectively). the e-tongue used consisted of three electrodes: the reference electrode (ag/agcl), counter electrode (glassy carbon electrode rod gc) and working electrodes (ag and au). the measurement has been made using the cyclic voltammetry. the addition of malt wort and inverted sugar influenced all the physico-chemical and e-tongue parameters. the addition of malt wort decreased the magnitude of the colour parameters (l*, a* and b*) and ph, and increased free acidity and electrical conductivity. the addition of inverted sugar increased the colour parameters’ values and free acidity and decreased the electrical conductivity and ph. according to the principal component analysis (pca) the samples with 5% and 10% inverted sugar and 5% malt wort are placed near to the authentic samples, so we can conclude that the samples have physico-chemical and etongue parameters closed to the authentic honeydew honey. keywords: honey, physico-chemical parameters, e-tongue, adulteration, pca. 1. introduction honeydew honey is defined by the european commission council directive 2001/110/ce [1] as a food obtained from secretions of living parts of plants or from excretions of plant-sucking insects. honeybees collect honeydew honey from the green parts of plant and at the same time, with honeydew, they may collect other attached structures, e.g. hyphae or fungal spores of plant pathogens and microalgae [2], [3]. honeydew honeys contains different concentrations of carbohydrates and smaller amounts of proteins, minerals, vitamins, amino acids, phenolic acids [4]. adulterated honeys are not just a issue for consumers only, but they raise problematic aspects for the authorities and retailers as well [5], [6]. the honey samples have been adulterated with many substances, which replace the main compounds (e.g. glucose and fructose) with industrial compounds obtained from cereals, sugar beet etc. [6]– [10]. the aim of this study is to evaluate the influence of the adulteration with inverted sugar and malt wort on honey physicochemical properties (ph, electrical conductivity (ec) and colour parameters (l*, a*, b*, chroma)) on honeydew honeys and to check the usefulness of the e-tongue food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei, ho n ey d e w h o n ey a d u l t er a t i o n : et o n g u e a n d p h y s i c o c h e mi c a l a n a l y s es , food and environment safety, volume xvi, issue 2 – 2017, pag. 98 – 103 99 and physico-chemical analyses for the adulteration detection. 2. materials and methods materials the honeydew honey has been purchased from local beekeepers located into suceava county, in the north-east part of romania. physicochemical parameters physico-chemical properties the physicochemical properties (ph, electrical conductivity, colour parameters (l*, a*, b*, chroma and hue angle) were determined accordingly to the harmonised methods of the international honey commission [11]. e-tongue electrodes the electrodes used for the electrochemical measurement were: reference electrode (ag/agcl), counter electrode (glassy carbon electrode rod) and working electrodes (ag and au) (metrohm, germany). measurement system and experimental procedure – cyclic voltammetry the measurement system consists in a pgstat 204 with fra32m module (metrohm, germany), coupled to the electrodes presented above. the experimental data were recorded by using a nova 2.0 software (metrohm, germany). the voltage has been set between – 1v to + 1 v, with a scan rate of 0.5 v/s for the electrodes, and the frequency was set at 100 hz. the electrodes were immersed in a vessel with 50 ml of honey solution (40 g honey completed to 200 ml with deionized water). to reach the electrochemical balance, the experimental data acquisition was made 10 minutes after the electrode has been placed into the honey solution. the experimental procedure was set for 45 s (1664 readings). all the samples were warmed to 25 °c before the experiment. to control the baseline drifts of the metallic electrodes, the electrodes were placed into a buffer solution at ph 7 before starting the measurements. the final values of current intensity were obtained subtracting their potential values from that of the buffer solution. each experiment was made in triplicates. statistical analysis statistical analysis (principal component analysis and linear discriminant analysis) was performed using unscrambler x 10.1 software system (camo, norway). 3. results and discussion in this study the influence of inverted sugar and malt wort on the physicochemical parameters (ph, aw, electrical conductivity, colour parameters (l*, a*, b*, chroma and hue angle)) of honeydew honey was investigated. two adulteration agents has been used for two different reasons: the inverted sugar has been added into the honeydew honey because it is the cheap adulteration agent on the market (it can be made at home by just boiling a sugar solution with some citric acid) and the malt wort has been added into the honeydew honey because it has the same colour and appearance as the honeydew honey, respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei, ho n ey d e w h o n ey a d u l t er a t i o n : et o n g u e a n d p h y s i c o c h e mi c a l a n a l y s es , food and environment safety, volume xvi, issue 2 – 2017, pag. 98 – 103 100 fig. 1. the evolution of physico-chemical parameters in function of adulteration agent (rhombus –inverted sugar, square malt wort) and adulteration agent percentage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei, ho n ey d e w h o n ey a d u l t er a t i o n : et o n g u e a n d p h y s i c o c h e mi c a l a n a l y s es , food and environment safety, volume xvi, issue 2 – 2017, pag. 98 – 103 101 the evolution of the physico-chemical parameters with the adulteration agent type and percentage is shown in figure 1. in the case of the l* it can be observed that the addition of inverted sugar is increasing the parameter values for the adulteration agent as the addition percentage is increasing, while in the case of malt wort addition the parameter is decreasing, respectively. inverted sugar has a higher l* value than the honeydew honey, while the l* value for the malt wort is lower. however, the addition of inverted sugar or malt wort changes the l* value up to 6.1%, which is not normally observed by the human eye. the other two colour components (a* and b*) are negatively influenced by the addition of malt wort and positively by the addition of inverted sugar. the two adulteration agents (inverted sugar and malt wort) have a positively impact on free acidity. in the case of malt wort the influence on free acidity is due to the presence of different acids which are being produced during the malting process, while in the case of the inverted sugar the acidity is given by the citric acid used for the hydrolysis process. the ph decreases with the increase of both adulteration agents in honey, the decrease of ph is due to the presence of acids in the malt wort and inverted sugar. the electrical conductivity of the honeydew honey is in agreement with the literature, being higher than 800 µs/cm [11]. the malt wort addition in the honey increased significantly the electrical conductivity (the malt wort has high content of minerals), while the inverted sugar decreased the electrical conductivity. e-tongue the maximum positive and negative voltage observed using the cyclic voltammetry with two different working electrodes (au and ag) are presented in the table 2. as it can be observed, the addition of inverted sugar is decreasing the au+ and ag+ and decreasing the agand aubecause of the samples’ acidity. in the case of malt wort adulterated honey samples, the addition of the agent is increasing the ag+ and au+, and decreasing the agand audue to the high content of minerals and acids. table1. the evolution of current with the adulteration agent on the two working electrodes current (v) sample ag+ agau+ au honey 0.000985 -0.00054 9.39e-06 -4.4e-05 h 5%is 0.000893 -0.00054 9.1e-06 -6.6e-05 h10%is 0.000873 -0.00054 8.8e-06 -9.4e-05 h20%is 0.000808 -0.00052 8.61e-06 -0.00014 h30%is 0.000753 -0.00049 8.49e-06 -0.00017 h40%is 0.000736 -0.0005 8.33e-06 -0.00019 h50%is 0.000726 -0.00051 8.18e-06 -0.00021 h5%m 0.000981 -0.00059 8.7e-06 -5.1e-05 h10%m 0.001018 -0.00058 8.93e-06 -5.2e-05 h20%m 0.001103 -0.00051 9.55e-06 -5.1e-05 h30%m 0.001205 -0.00054 1.01e-05 -5.3e-05 h40%m 0.001271 -0.00055 1.05e-05 -5.7e-05 h50%m 0.001361 -0.00058 1.11e-05 -5.6e-05 principal component analysis the principal component analysis has been conducted to evaluate the influence of adulteration agent using the physicochemical and e-tongue on honey based on a descriptive point of view. as it can be observed in the fig. 1, the honey samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei, ho n ey d e w h o n ey a d u l t er a t i o n : et o n g u e a n d p h y s i c o c h e mi c a l a n a l y s es , food and environment safety, volume xvi, issue 2 – 2017, pag. 98 – 103 102 adulterated with 5%is, 10% is and 5% m, are very closed to the authentic honey, this aspect being explained by the fact that adulteration in small percentages does not change significantly the physico-chemical and e-tongue parameters. the samples with high concentration of malt wort are placed in a single dial; so according to the pca the samples with high concentration of malt wort have the same characteristics. in the case of inverted sugar, the honey with 50% inverted sugar is placed in a different dial due to its chemical composition. according to the principal component loadings, the colour parameters are correlated with au+, the ph with ag+ and electrical conductivity with au-. the electrical conductivity and free acidity are negatively correlated. p c -2 ( 0% ) fig. 2. principal component analysis scores – h – honeydew, isinverted sugar, m – malt worth pc-1 (100%) -1 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 p c -2 ( 0% ) -1 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 l* a* b* free acidi ph ec ag+ agau+ au fig. 3. principal component analysis -loadings food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 sorina ropciuc, mircea oroian, sergiu paduret, amelia buculei, ho n ey d e w h o n ey a d u l t er a t i o n : et o n g u e a n d p h y s i c o c h e mi c a l a n a l y s es , food and environment safety, volume xvi, issue 2 – 2017, pag. 98 – 103 103 4. conclusions the adulteration of honeydew with inverted sugar and malt wort influenced significantly the physico-chemical and etongue parameters. according to the principal component analysis (pca) the samples with 5% and 10% inverted sugar and 5% malt wort are placed near to the authentic samples, so we can conclude that samples display physico-chemical and etongue parameters closed to the authentic honeydew honey. acknowledgement this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs – uefiscdi, project number pn-ii-rute-2014-4-0110. 5. references [1]. e. commission, commission decision 2002/657/ec implementing council directive 96/23/ec concerning the performance of analytical methods and the interpretation of results, off. j. eur. union, vol. l221, no. 23 may 1996, pp. 8–36, (2002). [2]. escuredo o., fernándezgonzález m., seijo m c., differentiation of blossom honey and honeydew honey from northwest spain, agriculture, 2:25–37, (2012). [3]. özkök a., bruce d., sorkun k., total phenolic acid and total flavonoid content of turkish pine honeydew honey, j. apiproduct apimedical sci., 2:65–71, (2010). [4]. seraglios. k. t. et al., development and validation of a lc-esi-ms/ms method for the determination of phenolic compounds in honeydew honeys with the diluted-and-shoot approach, food res. int., 87: 60–67, (2016). [5]. li s., shan y., zhu x., zhang x., ling g., detection of honey adulteration by high fructose corn syrup and maltose syrup using raman spectroscopy, j. food compos. anal., 28:69–74, (2012). [6]. wang s. et al., detection of honey adulteration with starch syrup by high performance liquid chromatography, food chem., 172:669–674, (2015). [7]. yilmazm. t. et al., steady, dynamic and creep rheological analysis as a novel approach to detect honey adulteration by fructose and saccharose syrups: correlations with hplc-rid results, food res. int., 64:634–646, (2014). [8]. çinar s. b., ekşi a., coşkun i., carbon isotope ratio (13c/12c) of pine honey and detection of hfcs adulteration, food chem., 157:10–13, (2014). [9]. tosun m., detection of adulteration in honey samples added various sugar syrups with 13 c/ 12 c isotope ratio analysis method, food chem., 138:1629–1632, (2013). [10]. downey g., fouratier v., kelly j. d., detection of honey adulteration by addition of fructose and glucose using near infrared transflectance spectroscopy, j. near infrared spectrosc., 11: 447–456, (2003). [11]. bogdanov s., et al., honey quality and international regulatory standards: review by the international honey commission, bee world, 80:61–69, (1999). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 84 researches on the evolution of concentrated fruit juices quality at storage oana – viorela nistor1, elena popa1, elisabeta botez1, oana emilia constantin1 1 bioingineering department, food science and engineering faculty, „dunarea de jos” university, 111 domneasca street, 800201, galati, phone/fax +40 236 460165, oana.nistor @ugal.ro abstract. the purpose of the study was to compare some concentration methods of clear fruit juices without added chemicals, foaming agents, clarification. for this study we chose three apples varieties (idared, golden delicious and red delicious) and they were processed into clear juice using a robot type fruit squeezer. apple juices were concentrated in a rotavapor type concentrator at 40°c temperature and 175 mbar pressure from almost 12% dry matter to almost 25%. the samples were examined over a period of 30 days shelf life at refrigeration temperature. the physical and chemical determinations were: acidity, ph and dry matter variation. we also made for juice the following microbiological determinations: total number of yeasts and moulds, mesophilic aerobic bacteria and osmofile yeasts. the three types of apple juice concentrates have shown stability all over the storage period at refrigeration temperatures (6-8°c), their aspect being determined by the acidity variation of the products. we also noticed that the dry matter variations in the three types of concentrated juices are constant during the storage period. the difference between the values of the dry matter is made by the apple varieties, as follows: for golden delicious is higher (26.3%) than the one for idared which is the lowest (25%). after the microbiological analyses have been made we reached the conclusion that the concentrated apple juice of idared variety has the greater loading number of microorganisms ( mesophilic aerobic bacteria 13 x106). keywords: idared, golden delicious, red delicious, concentration, clear juice 1. introduction apple juice is a fruit juice manufactured by the maceration and pressing of apples. the resulting expelled juice may be further treated by enzymatic and centrifugal clarification to remove the starch and pectin, which holds fine particulate in suspension, and then pasteurised for packaging in glass, metal or aseptic processing system containers, or further treated by dehydration processes to a concentrate. apple juice may also be sold in an untreated state. due to the complex and costly equipment required to extract and clarify juice from apples in large volume, apple juice is normally commercially produced. in the united states, unfiltered fresh apple juice is produced by smaller operations in areas of high apple production, in the form of non-clarified apple cider. apple juice is one of the most common fruit juices in the world; the world production is led by china, followed by poland, germany and the united states. (usda foreign agricultural service, 2004-2005). 100% apple juice concentrate is produced from mature apples to retain the characteristic flavour, colour, and freshness of the whole fruit. the product food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 85 contains no added sugars, acid, colour, preservatives, or other foreign material. the predominant varieties processed are: red delicious, golden delicious, fuji, gala and granny smith. single variety concentrates and custom blends are available upon request. each lot is guaranteed to be manufactured in accordance with usda, fda, and other generally recognized regulatory agencies.(c. huffman, 2007) fumaric acid is not considered as the natural constituent of freshly prepared apple juice without heat treatment. however, fumaric acid content slightly increases due to malic acid dehydration during processing when heat treatment such as evaporation and/or pasteurization is applied (lee and wrolstad 1988). it has been reported that the level of fumaric acid in well-prepared apple juice usually does not exceed 3,0 mg (junge and spadinger 1982). therefore, higher contents of fumaric acid in apple juices may be due to adulteration by the addition of synthetic malic acid which contains fumaric acid as a minor contaminant, excessive heating, microbial spoilage of juice or an intermediate or processing of decayed fruits (evans et al. 1983, zyren and elkins 1985, kvasnicka and voldrich 2000). several comprehensive reviews were published that discussed the chemical composition of authentic single strength apple juice (mattick and moyer, 1983; withy et al., 1978; lee and wrolstad, 1988) and commercially produced apple juice concentrate (elkins et al., 1996). various factors such as cultivar, growing region, climate, cultivar practices, harvest maturity (drake and eisele, 1997), storage atmosphere (drake and eisele, 1994), storage condition and processing (spanos et al., 1990; wrolstad et al., 1989) are known to affect the chemical composition of apple juice and apple juice concentrate. also, many of the juice compositional studies were limited to common or commercial varieties of apples which were developed to meet various marketing schemes and customer acceptance for sweetness, acidity, colour, and texture (way and mclellan, 1989). the compositional data on apple juice reported in this study are unique in that the fresh fruit was obtained from a local plot in selah, wa that contained over 400 varieties on 85 semi-dwarf orchard trees (prater, 1996). scion wood for grafting was collected from major growing regions of the world over a 15-year period. this included new and ‘‘antique’’, old, varieties—many of which are not easily available. there was sufficient fruit formed in the fall of 1997 to investigate the compositional characteristics of 175 varieties. care was taken to maximize cooling and minimize treatment times to maintain the quality integrity of the samples. the compositional information can be used in conjunction with existing databases to better describe acceptable attribute ranges for authentic apple juice and in the development of commercial apple varieties that would target specific consumer requirements. the purpose of the study was the comparison between some concentration methods of clear fruit juices without added chemicals, foaming agents, clarification. for this study we chose three apples varieties: idared, golden delicious and red delicious. idared apple is medium/large, has a round flat shape. the ratio of 1/4 3/4 some red stripes on a yellow inaccurate, it is soft, rather than hard and smooth and has glossy appearance. the colour inside is white. when the fruit is ripe,it is crispy, juicy and very sweet, but it can be gummed and tasteless. to be very colourful, it should stay for a long time to be collected. it remains very good in the tree until late autumn. golden delicious flavour is outstanding and golden delicious apples are kept long. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 86 it is characterized by fine texture, greenyellow skin hiding inside a crispy, sweet and juicy fruit. some people think that golden delicious variety is "lime yellow", red delicious variety of popular, but there is only similarity between the two in terms of their name. the golden delicious is an independent variety as sweet as honey. the apples of this variety are perfect for snacks or picnics and ideal for salads, pies, jams and other dishes that requires cut slices. the red delicious is one of the most popular types of apples, and one of the most cultivated species of apples. although similar in name red delicious and golden delicious are two totally different species. there are many similarities between them: both species have been discovered in america in the late 19th century, both in need of warm climates and both are species of fresh apples. table 1. physico-chemical composition of apples nutritional qualities for 100 g product proteins 0.4 g carbohydrates 0.1 g fats 11.8 g fibers 1.8 g calories 4.7 kcal vacuum concentration operation evaporation by using a rotary evaporator is the most commonly used method to separate solvents. vacuum evaporators are considered class function because by lowering the pressure above a bulk liquid lowers the boiling points of the component liquids in it. generally, the component liquids of interest in applications of rotary evaporation are research solvents that one desires to remove from a sample after an extraction, for instance, following natural product isolation or a step in an organic synthesis. the use of a "rotavap" therefore allows liquid solvents to be removed without excessive heating of what are often complex and sensitive solvent-solute combinations. rotary evaporation is the most often and conveniently applied to separate "low boiling" solvents such as n-hexane or ethyl acetate from compounds which are solid at room temperature and pressure. however, careful application also allows removal of a solvent from a sample containing a liquid compound if there is minimal co-evaporation (azeotropic behaviour), and a sufficient difference in boiling points at the chosen temperature and reduced pressure. the characteristics of the concentrated apple juice are presented in table 2. table 2 the characteristics of the concentrated apple juice characteristics values acidity 0.16 – 1.27 (g malic acid) ph 3.3 – 4.5 dry weight 20 – 40% in sensorial terms the concentrated apple juice must have characteristic flavour of apple, to be palatable and to have the characteristic odour of the apples. the microbiological determinations may characterize the concentrated apple juice regarding the free microorganisms in product. in this order it we determined the total number of yeasts and moulds, mesophilic aerobic bacteria and osmofile yeasts. the concentrated apple juice must not contain any other microorganisms than mesophilic aerobic bacteria. the purpose of the study was the comparison between concentration methods of clear fruit juices without added chemicals, foaming agents, clarification. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 87 2. materials and methods 2.1 materials 2.1.1. raw materials: apples idared, golden delicious, red delicious. 2.1.2. auxiliary materials: knife, fruit squeezer, erlenmeyer and berzelius glasses, cylinders, naoh 0.1 n, burettes, rod, dropper, petri plates, tube, culture medium (mma for yeasts and moulds and bca for bacteria). 2.1.3. instalation/equipment: evaporator (rotavapor type concentrator), ph-meter, refractometer, incubator. 2.2. methods 2.2.1. the method of obtaining concentrated apple juice apples were washed and squeezed to obtain apple juice. the juice was filtered to obtain a clear juice and it was stored for 24 h at refrigeration temperature to allow the natural enzymatic clearing. the clear juice was concentrated using a rotavapor type concentrator at a temperature of 40°c and a pressure of 175 mbar. the concentration had the purpose to increase the value of the dry matter from 12% to 25%. the samples were examined over a period of 30 days shelf life at refrigeration temperature with a 5 days periodicity. the physical and chemical determinations were: acidity, ph and dry matter variation. we also made microbiological determinations on juice such as: the total number of yeasts and moulds, mesophilic aerobic bacteria and osmofile yeasts. 2.2.2. the physical and chemical determinations the titratable acidity: we used the standard method for titratable acidity determination according to stas 5952-71. ph determination: we used a ph-meter called ph-meter iq scientific. dry matter: we used the refractometer method according to stas 5956-71 for soluble substances determination. 2.2.3. microbiological determinations the total number of yeasts and moulds: the sample was distributed in petri plates. we used a mma specific medium incubated at 37 °c. the colonies formed were counted. mesophilic aerobic bacteria: the sample was distributed in petri plates. we added a specific medium pca (plate count agar) and they were incubated at 25°c. the colonies formed were counted. osmofile yeasts: are microorganisms which can live and develop in sugar/salt rich medium, being the principal responsible for food alteration. the determinations are based on the growth study of the osmofile yeasts on a specific medium pca (plate count agar) or hyperglucidic, incubated at 37°c. the colonies formed were counted. 3. results and discussion the concentrated apple juices from the three apple varieties (idared, golden delicious and red delicious) were stored at refrigeration temperature (6-8°c) for 30 days. the determination had been made with a 5 day frequency. the results obtained for the titratable acidity variation for the three apple varieties were presented in figure 1. 0.4 0.6 0.8 1 0 5 10 15 20 storage time [days] a ci di ty [m al ic a ci d gr ad e] go lden idared red delicio s figure 1. the titrable acidity variation for the three apple varieties food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 88 all three varieties of apples had an acidity expressed in g malic acid /100 ml concentrated juice situated in normal limits (0.15-1.27 g malic acid). the acidity depends on the apple varieties, so that a greater amount of malic acid had the golden apple juice (0.871 g malic acid/100 ml product). malic acid was lower in the concentrated apple juice red delicious (0.494 g malic acid/100 ml product). in the next graphic (figure 2) the ph variation during storage for idared, golden delicious and red delicious is presented. 3.5 3.7 3.9 4.1 4.3 4.5 0 5 10 15 20 storage time [days] ph [p h u ni ts ] golden idared red delicios figure 2. the ph for the three varieties of apples the ph varied inversely proportional in comparison with the malic acid. so, a high ph (4.5) is corresponding to the concentrated apple juice of red delicious apple and the concentrated apple juice from idared had a lower ph (4.3). the concentrated apple juice golden had values between the other two varieties of juices (3.9). in the following graphic (figure 3) the dry matter evolution of the concentrated apple juices is shown. by concentration we doubled dry matters of the concentrated apples juices. from a value of 12% we got 24 26%. the concentrated apples juice which has high dry matter was golden (26.3%) and that obtained from idared presented lower values of dry matter throughout determinations (25%). 24.5 25.5 26.5 0 5 10 15 20 storage time [days] d ry m at te r [% ] golden idared red delicios figure 3. the dry matter for the three varieties of concentrated apple juices 4. conclusions juice is the liquid that is naturally contained in fruit or vegetable tissue. juice is prepared by mechanically squeezing or macerating of fresh fruits or vegetables flesh without the application of heat or solvents. the concentrated apple juice obtained from the three apple varieties introduced and performed within normal limits throughout refrigerated storage for all the physical and chemical specific characteristics. during the 30 days of refrigeration (6-8°c) the concentrated apple juice had good storage stability. the malic acid content was in the proper limits (0,15-1,27 g malic acid), but it was varying depending on apples varieties. the golden apple juice had greater amount of malic acid, whereas the red delicious one presented a lower content of malic acid. the ph varied inversely proportional in comparison with the evolution of the malic acid. the difference between the values of the dry matter is also an important agent depending on the apple varieties, as for golden delicious is higher (26.3%) than the one for idared which is the lowest (25%). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 89 after the microbiological analyses have been made we can draw the conclusion that the concentrated apple juice from idared variety has the greater loading number of microorganisms (mesophilic aerobic bacteria – 13 cfu/ml). both other studied types did not show any microorganism loading. 5. references 1. usda foreign agricultural service. world apple juice situation. 2004-2005. 2. s.r., drake, t.a., eisele, influence of harvest date and controlled atmosphere storage delay on the colour and quality of ‘delicious’ apples stored in a purge-type controlled-atmosphere environment, horticultural techniques 4 (3) 1994, 260–263 3. s.r., drake, t.a., eisele, carbohydrate and acid contents of gala apples and bartlett pears from regular and controlled atmosphere storage. journal of agricultural and food chemistry 47, 1999, 3181–3184 4. h.j., bielig, h.j., hofsommer, the importance of the amino acid spectrum in apple juices. flussiges obst 49, 1982, 50–56 5. s.r., drake, t.a., eisele, quality of ‘gala’ apples as influenced by harvest maturity, storage atmosphere and concomitant storage with ‘bartlett’ pears. journal of food quality 20, 1997, 41–51 6. e.r., elkins, a., matthys, r., lyon, c.j., huang, characterization of commercially produced apple juice concentrate. journal of food composition and analysis 9, 1996, 43–56 7. r.h., evans, a.w., van soestbergen, k.a., ristow, evaluation of apple juice authenticity by organic acid analysis. journal of the association of official analytical chemists 66, 1983, 1517–1519 8. l.r., mattick, j.c., moyer, composition of apple juice. journal of the association of official analytical chemists 66, 1983, 1251–1255 9. r.e., wrolstad, d.a., heatherbell, g.a., spanos, r.w., durst, j., hsu, b.m., yorgey, processing and storage influences on the chemical composition and quality of apple, pear, and grape juice concentrates, 1989 10. g.a., spanos, r.e., wrolstad, d.a., heatherbell, influence of processing and storage on the phenolic composition of apple juice. journal of agricultural and food chemistry 38, 1990, 1572–1579 11. g.a., spanos, r.e., wrolstad, phenolics of apple, pear, and white grape juices and their changes with processing and storage—a review. journal of agricultural and food chemistry 40, 1992 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 84 effect o f a dd ition o f c orn fla k es on rh e o log ic al behav io r o f some y og u rt *cristina damian1, mircea-adrian oroian2, andreea şmadici3 1-3 ştefan cel mare university of suceava, faculty of food engineering, 13 universităţii street, 720229, suceava, romania, e-mail: 1cristinadamian@fia.usv.ro *corresponding author received 20 september 2011, accepted 2 november 2011 abstract: yogurt is one of the most consumed fermented milk product in the world due to its benefits for the human body. the purpose of this work is to study the effect of addition of some corn flakes on rheological behavior of different types of yogurts. for this study, the yogurt samples were obtained in the laboratory using the cultures starter diprox yba 986. the effect of addition of corn flakes in yoghurt was investigated by a rotational viscometer, brookfield viscometer (brookfield engineering inc, model rv – dv i prime) with rv spindles. the brookfield viscometer dv i prime with disk spindles represents an easily and cheap method for rheological characterization of non-newtonian fluids, in this case of yoghurt. keywords: yogurt, viscosity, viscometer, corn flakes 1. introduction yogurt is produced by a fermentation process during which a weak protein gel develops due to a decrease in the ph of the milk. the ph of the milk is decreased due to the conversion of lactose to acid lactic by the fermentation culture bacteria. in the liquid milk, casein micelles are presented as individual units. as the ph reaches ph 5.0, the casein micelles are partially broken down and become linked to each other under the form of aggregated and chains forming part of a three-dimensional protein matrix in which the liquid phase of the milk is immobilized. this gel structure contributes substantially due to the overall texture and organoleptic properties of yoghurt and gives rise to shear and time dependent viscosity [1]. the most frequent defects related to yogurt texture that may lead to consumer rejection are apparent viscosity variations and the occurrence of syneresis [2]. yogurt rheological characterization is required for product and process development and to ensure consumer acceptability [3]. this characterization can be made using either instrumental or sensory measurements. the firmness of yogurt and the viscosity of juststirred gel are greatly influenced by the amount of heat treatment the yogurt mix receives. heating unfolds the globular whey proteins and exposes sulphydryl groups, which react with other sulphydryl groups and disulfides and induce linkages and protein-casein aggregates [4-5]. the gel strength of yogurt is related to the cumulative effects of the chemical interactions. the binding of δ-lacto globulin to the casein micelle seems to be responsible for the increase of gel strength [6, 7, and 8]. mailto:1cristinadamian@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 85 cereals constitute the staple food of the human race. in many countries, they are mainstay of life and form the single largest component in their daily diet. ready-to-eat (rte) cereals are processed grain formulation suitable for human consumption without requiring further processing or cooking. corn flakes are possible the most common form of breakfast cereals [9]. in the final step, the cereal is treated to restore vitamins lost through cooking and is often coated with sweet flavourings to make it more attractive [10]. toasting is an important processing step for the manufacture of breakfast cereals that dictates the attributes of rte corn flakes that are usually characterised by their unique crispness and maintaining the integrity while consuming with milk. these attributes are critical for the acceptance of the product by the consumers [11]. 2. materials and methods 2.1. materials uht milk, lactobacillus bulgaricus and streptococcus thermophilus pure starter culture di prox 986 provided by enzymes & derivates, piatra neamț, românia, corn flakes from the market; orbital shaker; thermostat; brookfield viscometer model rvdv ii pro, with disk spindle, rv3, rv4, rv5, rv6 type. 2.2. sample preparation the yogurt samples were made using uht milk, having the physical and chemical parameters in tabel 1. tabel 1. milk properties 10,5% fat, g/100g 3 protein, g/100 g 3 sugar, g/100g 4.5 ash, % 0.72 acidity, 0t 18 300 ml milk was inoculated using 0.015 g starter culture. after inoculation with starter culture, the sample were homogenised with an orbital shaker for 15 min at 100 rpm. after shaking the sample were thermostated at 420c for 6 hours. the corn flakes were added in the next concentration to the yogurt sample: 0, 1.5 g (s1), 2 g (s2), 2.5 g (s3), 3 g (s4). 2.3. determination of rheological properties viscosity measurements were carried out on the yogurt samples at ambient temperature (250c), with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 2,5; 5; 10; 20; 50; and 100 rpm with rv spindle (rv3, rv4, rv5, rv6 type). the spindle nos was used in accordance with the sample nature to get all readings within the scale [12]. the samples in 300 ml of beaker with a 8,56 cm diameter (according to the brookfield requests) were kept in a thermostatically controlled water bath for about 10 min before measurements in order to attain desirable temperature of 250c. first measurements were taken 2 min after the spindle was immersed in each sample, so as to allow thermal equilibrium in the sample, and to eliminate the effect of immediate time dependence. all data were then taken after 40 s in each sample. each measurement was duplicated on the sample. the obtained empirical data were converted using the mitschka relationships to shear rate and shear stress. the shear rate versus shear stress data were interpreted using the power law expression σ = k·γn (1) where: σ – shear stress (n/m2), γ is the shear rate (s-1), n is the flow behaviour index, k is the consistency index (nsn/m2). the values for the flow behaviour index n, were obtained from plots of log shear stress versus log rotational speed; the slope of the line (if the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 86 dependence is sufficiently close to a linear one) is simply equal to the flow index of the fluid, n. the shear stress is calculated using the next equation: τi = kτ·αi·c (2) where: τi – shear stress (dyne/cm2) kτ = 0.119, this constant is for the spindle nos 2 αi – torque dial, % c – 7,187 dyne/cm for rv viscometer the shear rate is calculated using the next equation: γi = kγ(n)·ni (3) where: γi – shear rate, s-1 kγ(n) – constant, depends by the value of n ni – rotational speed, rpm. 3. results and discussion the yogurt samples exhibited a non-newtonian behaviour, the flow index (n) is under 1 for all the samples. the power law model is a suitable one for predicting the rheological parameters, the regression coefficient is near 1 (r2>0.95) – see table 2. in general, addition of corn flakes increases the viscosity of yogurt in proportion to the dose added. this is due to its high starch corn flakes. it achieved a stabilization of aqueous dispersions whose continuous phase is the water and the dispersed one is solid or liquid and tends to separate. through addition of starch, the viscosity is increased and is yogurt reduces the tendency of destabilization by separation of components. table 2. power law parameters of yogurt samples sample n – flow index k – consistency index (nsn/m2) r2 s1 0.903 4010 0.986 s2 0.843 6420 0.959 s3 0.567 24340 0.970 s4 0.357 32100 0.985 the starch improved the rheological properties of the corn flakes yogurt samples, due to the water-bind capacity, high molecular weight and due to the forming of the casein-starch system, the water-bind capacity. various studies have reported the existence of phase separated networks in casein–polysaccharide mixtures [13, 14], and the amylopectin-casein system in particular [15], but not in the samples with low levels of amylopectin added. depending on the weight ratio of the two biopolymers in the mixture, as well as their relative affinities for water, a casein–starch system may either have the protein or the polysaccharide-rich domain forming the continuous phase, with the second component dispersed as the discontinuous phase. as a result, only a fraction of the total water content will be available to one biopolymer phase (e.g. casein), with theremaining water being bound by the second phase (e.g.starch) [16]. all viscosity samples increased with the increasing of corn flakes quantity, as shown in figure 1. the flow index decreased with the increasing of corn flakes doze, while the consistency index decreased with the increasing of the doze. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 87 4. conclusion the yogurt is a complex viscous food material due to the complexity of its nature and composition. rheological behavior of yogurt is influenced by sugars, fats and water. the brookfield viscometer dv i prime with disk spindles represents an easily and cheap method for rheological characterization of nonnewtonian fluids, in this case of yogurt. in the case of yogurt samples obtained in the laboratory, the addition of corn flakes increases the viscosity, proportional to the doses added. this is due to its high starch corn flakes, which carries stable aqueous dispersions whose continuous phase is and the dispersed one is solid or liquid and tends to separate. by means of starch addition, yogurt viscosity is increased and reduces the tendency to destabilize the separation of components. 5. references [1].oroian m. a., escriche i., gutt g., rheological, textural color and physico-chemical properties of some yoghurt products from the spanish market, food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, year x, no 2, 24 p, (2011) [2].kroger, m. quality of yogurt. journal of dairy science 59(2): 344-350. (1975) [3].benezech, t., maingonnat, j.f. characterization of the rheological properties of yogurt-a review. journal of food engineering. 21, 447-472. (1994) [4].sawyer wh. complex between –lacto globulin and γ-casein. a review. journal of dairy science 52:1347–55. (1969) [5].kinsella h. milk proteins: physiochemical and functional properties. crc crit rev food sci nutr 21(3):197. (1994) figure 1. viscosity profile of yogurt sample with corn flakes (cross 1.5 g corn flakes, triangle 2 g corn flakes, square 2.5 g corn flakes, rhombus 3 g corn flakes) food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 88 [6].bonomi f, iametti s, palgliarini e, peri c. a spectrofluorometric approach to estimation of the surface hydrophobicity modifications in milk proteins upon thermal treatment. milchwissenschaft 43:281–5, (1998) [7]. mottar j, bassier a, joniau m, baert j. effect of heat-induced association of whey proteins and casein micelles on yogurt texture. journal of dairy science 72(9):2247– 56, (1989) [8]. bonomi f, iametti s. real-time monitoring of the surface hydrophobicity changes associated with isothermal treatment of milk and milk protein fractions. milchwissenschaft 46:71–4, (1991) [9]. fast, r. b., manufacturing technology of ready-to-eat cereals, in: fast, r. b., caldwell, e. f., (eds.), breakfast cereals and how they are made, american association of cereals chemists, st. paul, minnesota, usa, pp. 15-42, (1990) [10]. anon., encyclopedia britanica ultimate reference, suit 2005 dvd, encyclopedia britanica, uk, (2005) [11]. sumithra, b., bhattacharya, s., toasting of corn flakes: product characteristics as a function of processing conditions, journal of food engineering, 88, 419-428, (2008) [12].http://www.brookfieldengineering.com/products/ accessories/spindles/rv_ha_hb_spindles.asp (accessed at 7.09.2011) [13].bourriot, garnier, & doublier, phase separation, rheology and microstructure of micellar casein–guar gum mixtures. food hydrocolloids, 13, 43–49, (1999) [14]. schorsch, clark, jones, &norton, behavior of milk protein/polysaccharide systems in high sucrose. colloids and surfaces b, 12, 317–329, (1999) [15]. de bont, van kempen, & vreeker, phase separation in milk protein and amylopectin mixtures. food hydrocolloids, 16, 127–138, (2002) [16]. shiu-kin chan p., chen j., ettelaie r., law z., alevisopoulos s., day e., smith s., study of the shear and extensional rheology of casein, waxy maize starch and their mixtures, food hydrocolloids 21 716–725, (2007) 1-3 ştefan cel mare university of suceava, faculty of food engineering, 13 tabel 1. milk properties where: 4. conclusion the brookfield viscometer dv i prime with disk spindles represents an easily and cheap method for rheological characterization of non-newtonian fluids, in this case of yogurt. in the case of yogurt samples obtained in the laboratory, the addition of corn flakes increases the viscosity, proportional to the doses added. this is due to its high starch corn flakes, which carries stable aqueous dispersions whose continuous phase... 5. references figure 1. viscosity profile of yogurt sample with corn flakes (cross 1.5 g corn flakes, triangle 2 g corn flakes, square 2.5 g corn flakes, rhombus 3 g corn flakes) применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 contents: 1 effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenonahoussi, mohamed soumanou, dominique c.k. sohounhloue 123 134 2 comparative study of adsorption activity of activated charcoal and silica gel in case of water / toluene mixtures igor winkler, kateryna bodnaryuk 135 139 3 research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk 140 146 4 microbial characteristics of egg-white creams with reduced sugar nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova 147 152 5 study on the influence of heat treatments on the antioxidant properties of ginger amalia cosmovici, sonia amariei 153 159 6 the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people antonella dorohovych, mykola petrenko 160 – 166 7 water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour olexandr rozhno, vira yurchak, tatjana golikova 167 179 8 biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov 180 189 author instructions i v subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 title …………………… 140 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 140 146 research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder *mariia ianchyk¹, oleksandra niemirich¹, oksana vasheka¹, olena yanchyk² ¹national university of food technologies, kyiv, ukraine ²vinnytsia trade and economic institute of knteu *corresponding author: mariia_ianchyk@ukr.net received 2nd july 2017, accepted 25th september 2017 abstract: the article is devoted to research of model systems of a new finishing confectionery semifinished product which contains sugar fondant, plant powder, butter and surfactant citric acid ester. a derivatographic analysis of the model systems of a confectionery semi-finished product was carried out and the distribution of moisture connection forms was investigated. types of moisture connections were determined in terms of the classification system of rehbinder p.a. which is based on a magnitude of connection energy of water with material. the results of microstructure researches of model systems are presented in the article. it has been found that particles of banana powder do not act as crystallization centers and therefore do not exert impact on the process of grain formation of sucrose in fondant mass. the expediency of surfactant use in the production of a new confectionery semi-finished product with banana powder is proved. keywords: finishing semi-finished products, plant powder, fondant mass, surfactant, grain formations. 1. introduction in the 21st century, a topical issue of the world level is considerable prevalence of chronic non-communicable diseases (ncd), which causes global social and economic losses of the population in many countries of the world, including ukraine. risk factors that cause by ncd are known and widespread among the population. simultaneous reduction of the frequency and levels of several common risk factors contributes to the reduction of the prevalence of the main ncd. in this regard, world health organization (who) has proposed an integrated prevention program for non-communicable diseases across the country. one of the main directions of disease prevention is healthy eating, which suggests that diets should consist of a variety of products of predominantly plant origin. today, the nutrients lack of the plant origin in nutrition of the population has become a serious problem, being connected with a sharp decrease in energy consumption and a change in diet, which does not provide the formed physiological needs in a number of irreplaceable nutrients. consumption of confectionery products plays a significant role in full nutrition of various age groups of the population, especially children. they are in steady demand, first of all, thanks to refined taste properties. value of confectionery products in food is caused by the high energy value which is provided by the considerable content of sugars, and in some products by content of fats, but their nutrition value is limited [1]. therefore, the use of plant raw materials for enrichment this category of products is relevant today. high moisture content in plant raw materials is the reason of their instability during storage, because of bacterial, enzymatic http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 141 and chemical damage. drying at low temperatures is the most rational way of preservation, as in dried products microbiological processes are slowed down, and the composition of nutritious and biologically valuable substances remains close to natural [2, 3]. therefore, in order to reduce the energy value and increase the nutritional value of confectionery products, it is expedient to use plant powders which are concentrates of biologically active substances [4]. in this regard, after having taken into consideration some modern studies in the field of finishing semi-finished products, we came to the conclusion of making some universal confectionery semi-finished products which will considerably reduce time of technological process with production, enlarge the range of products, have increased nutrition value and improve organoleptic properties. for enrichment, sugar fondant was chosen. banana powder was chosen as material for enrichment of fondant, provided by the company "naturex ag", switzerland (conclusion of the state sanitary and epidemiological service no. 05.03.02-03 / 125796 dated 27.12.2011), which has a moisture mass fraction of 5% and a dispersion of 10-20 microns. this powder has high organoleptic properties, namely, intense taste and aroma. banana powder is a source of dietary fiber, organic acids, potassium and magnesium [5,6]. addition of butter will make the basis of a semi-finished product more plastic: mechanical influence, on its further use, will carry out much easier as it receives the necessary form when being decorated. research results of confectionery semifinished products showed that for the best structural and mechanical properties it is important to add the surfactant when adding the powder-butter mix, since it allows holding the corresponding consistency under the influence of temperature. citric acid ester and mono diglycerides (е 472 c) – are the surfactants added in an obligatory amount of 0.5% from the weight of a semifinished product that help keep uniform structure with multiple thermal and mechanical influence [7, 8]. the addition of plant powder, butter and surfactant influences significantly the structural and mechanical properties of a ready-made product and changes its rheological properties. the considerable influence on formation of structural characteristics and storage terms has to comply with the condition of water phase [9]. due to addition in its composition of plant powder and butter, the amount of moisture in a semi-finished product increases, thus making possible the premature development of microorganisms and reduction its storage shelf life. therefore, the purpose of research was to study redistribution of the moisture connection forms in model systems of a confectionery semi-finished product with banana powder by the method of thermogravimetric analysis. 2. materials and methods to do the research aimed at, the following samples were used: a control sample consisting of sugar fondant with moisture content of 11.2% (sample a); sugar fondant with addition of banana powder in amount of 18% to prescription weight with the general moisture content of 12.6% (sample b); sugar fondant with mixture addition of powder and butter (moisture content of 16.6%) to prepare preliminary mixing of butter (20%) with banana powder (18%) to homogeneous mass (sample c); sugar fondant with mixture of powder and butter and additionn of surfactant in number of 0,8% to prescription weight with moisture content of 11.2% (sample d) [11]. the refood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 142 search of redistribution of the moisture connection forms was conducted by method of the thermogravimetric analysis, which at the same time allows taking measurements of temperature in samples, change in weight and speed. curves were taken on a derivatograph of paulie-erden system q-1500 d on air with the growth rate of temperature of 5 °c/min. the weight of samples was of 197-205 mg. derivatographic curves were reviewed: the differential-thermal analysis (dta), thermogravimetric (tg), differential thermogravimetric (dtg) and temperature curve (t). on receival of derivatograms with the subsequent processing by means of the mathematical program peakfit 4.12, the curve dta on gauss's curves was displayed and temperature peaks and intervals of its removal with various types of connections were determined. the tg curve determined the amount of moisture removed in percentages. the microstructure was investigated by means of analytical scanning electron microscope jeol jsm-6060la with a 600time increase. 3. results and discussion the method of the thermogravimetric analysis provides derivatograms of the experimental samples of semi-finished products. in order to identify moisture with different connection forms and establish their role in formation of consistency and structure of finished products, according to the method proposed by rashevskaya t.o. [10], the received curves of dta were decomposed in simpler components, based on regularities of normal curves distribution of gauss. figure 1 shows the mathematically processed derivative curves of experimental types of fondant masses. quantitative distribution of different moisture connection forms with material, and also temperature intervals and peaks of its removal are shown in table 1. the derivatogram of the control sample (fig. 1a) shows that the removal of weakly bound osmotic moisture takes place in two stages: in a wide temperature interval with a clear peak at 69 º c and diffuse peak at 78 ºс, which is well manifested during the mathematical processing of the curve. the total amount of weakly bound moisture in the structure of the product is 10.7%. the temperature peaks of 97 ºс and 143 ºс in the range 92147 ºс indicate the removal of the strongly bound moisture of the polyand monomolecular layers respectively. the analysis of the derivatogram of the semi-finished product enriched with banana powder (fig. 1b), indicates the redistribution of the moisture connection forms with the components of the product and a significant increase in the weakly bound osmotic moisture. in comparison with control sample, its relative quantity grows in structure by 2.2 times, and removal happens at higher temperatures than in the control sample and in three stages with peaks of 69 °c, 80 °c and 88 °c. removal of strongly bound moisture takes place discretely at temperatures over 100 °c, which indicates high energy of connexions between components of a product and its water phase. the relative amount of strongly bound moisture in comparison with control sample decreases by 1.2 times. the obtained research results indicate that in the structure of the enriched fondant mass, as a result of swelling processes and interaction between water phase and components of banana powder, the quantity of a disperse phase grows substantially and the surface of interphase interaction infood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 143 creases. such processes promote formation of additional coagulative connection and structure plasticization of the enriched fondant mass respectively. the following figures show derivatograms of enriched fondant mass with plant powder and butter without surfactant (fig. 1c) and with surfactant (fig. 1d). the nature of the dta curves of the experimental samples, the similarity of the peaks and the temperature intervals of the moisture removal and its quantitative distribution between various forms of bonding indicates the similarity of the interaction processes of the water phase with the components of the enriched fondant masses described above. in sample c, the amount of weakly bound moisture is increased by 1.5 times as compared with the sample b and by 3.3 times as compared with the sample a (control). it is caused by the addition of butter to the model system, which contains a significant amount of weakly bound moisture, part of which is absorbed by banana powder. in sample d, this moisture content is reduced by 1.3 times as compared with the previous sample, which allows us to conclude that the surfactant is able not only to hold the fat phase of the semi-finished product, but it also contributes partly to the redistribution of moisture to the side of the strongly bound. at the next stage, the microstructure of experimental species of confectionary semi-finished products with an increase of 200 and 500 times was studied. the received pictures of a microstructure are given in the figure 2. 62 78 97 143 dta tg t 50 100 a tg dta 69 80 88 125 137 50 100 t b tg dta t 50 100 51 65 79 94 101 119 137 c 50 100 t dta tg d 60 71 87 98 108 122 146 fig. 1. derivatograms of confectionery semi-finished products model systems food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 144 table 1. the moisture connection forms of confectionery semi-finished products model systems forms connection of moisture sample a sample b sample c sample d r e la ti v e m o is tu r e c o n te n t, % removal temperature, ˚c r e la ti v e m o is tu r e c o n te n t, % removal temperature, ˚c r e la ti v e m o is tu r e c o n te n t, % removal temperature, ˚c r e la ti v e m o is tu r e c o n te n t, % removal temperature, ˚c p e a k in te r v a l p e a k in te r v a l p e a k in te r v a l p e a k in te r v a l weakly bound moisture 10.7 23.7 34.8 25.9 mechanical osmotic 6.3 4.4 62 78 47-70 71-91 11.1 8.7 7.9 69 80 88 55-71 72-83 84-99 15.0 9.0 10.8 65 79 94 56-72 73-86 87-92 9.8 4.5 11.6 60 71 87 50-69 70-75 76-96 strongly bound moisture 89.3 72.3 65.2 74.1 poly molecular 34.8 97 92-120 43.7 125 100-126 25.9 22.9 101 119 93-115 116-123 23.2 4.5 98 108 97-104 105-110 mono molecular 54.5 143 121-147 28.6 137 127-146 16.4 137 124-146 38.4 8.0 122 146 111-138 139-149 during the research of the model systems microstructure, it is shown that in sugar fondant prevails homogeneous crystal phase. fondant mass is presented by separate crystals up to 10 microns in size, evenly distributed over the entire mass (fig. 2a). at addition of banana powder, there is uneven, namely, conglomerate distribution in research system. it was found that particles of powder do not act as centers of crystallization during maturing and therefore do not exert an impact on the process of sucrose crystal formation in fondant mass (fig. 2b). the uniform distribution of powder in model system is reached by preliminary receiving powder and butter mix (fig. 2c). by microstructural research it is shown that the crystals of fondant are surrounded by a fatty phase of butter, and the bulked-up particles of powder do not form groups. the surfactant introduction promotes the receiving of homogeneous polyphase systems that is confirmed by uniform plastic structure (fig. 2d). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 145 a b c d fig. 2. microstructure of confectionery semi-finished products model systems 4. conclusions consequently, according to the results of studies on the redistribution of the moisture connection forms, it has been established that the addition of banana powder and butter contributes to the increase of osmotic moisture, which influences negatively the process of storage of confectionery semi-finished products. the addition of surfactant acts in the opposite direction and contributes to the increase of strongly bound moisture, in particular monomolecular one. the study of microstructure has shown that banana powder does not affect the crystal formation of the fondant mass, but some uneven distribution of particles in the research system is observed. the addition of butter and surfactant helps to obtain a homogeneous polyphase system. 5. references [1]. annunziata a., vecchio r., functional foods development in the european market: a consumer perspective, journal of functional foods, 3 (3): 223–228, (2011). [2]. dolinsky a., maletskaya k., snezhkin y., fruit and vegetable powders production technology on the bases of spray and convective drying methods, drying technology: an international journal, 10 may: 265, (2007). [3]. milner j.a., functional foods and health: a us perspective, british journal nutrition, 88(l.2): 151–158, (2002). [4]. ianchyk m., niemirich a.. theoretical aspects enrichment of fondant masses nontraditional plant materials, scientific messenger of vinnitsa national agrarian university, 1 (89): 168-173, (2015). [5]. ianchyk m., niemirich a., gavrysh a., yanchyk o., study of functional and technological properties of plant powders for use in confectionery industry, food science and technology, 10(4): 31-36, (2016). [6]. kremnev o., borovsky v., snezhkin y., method of production powders from liquid and paste-like products, ussr, n 947985. – 1981. http://www.sciencedirect.com/science/journal/17564646/3/3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 mariia ianchyk, oleksandra niemirich, oksana vasheka, olena yanchyk, research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder, food and environment safety, volume xvi, issue 3 – 2017, pag. 140-146 146 [7]. ianchyk m., niemirich a., technology of confectionery semifinished with bananas and carrots, scientific messenger of lviv national university of veterinary medicine and biotechnologies named after s.z. gzhytskyj , т.18, 2 (68): 130-133, (2016). [8]. kaur s., das m., functional foods: an overview, food science biotechnology, 20 (4): 27-35, (2011). [9]. bratu m., stoica a., mihail b., the influence of additives on preparing the fondant, the annals of “valahia” university of targovişte. 7 (1): 5-9, (2007). [10]. rashevskaya t., gulyi i., pryadko n., simachina g., the role of water phase in fоrmation of microstructure of butter with red beet powder additive, water management in the desing and distribution of quality foods: international simposium, 30 may – 4 jun.: 202-203, (1998). [11]. ianchyk m., niemirich a., definition indicators of quality of the confectionery semifinished product with powders from banana and carrot, ukrainian journal of food science, 4 (1): 76-84, (2016). research on microstructure and redistribution of the moisture connection forms in the model systems of confectionery semi-finished product with banana powder *mariia ianchyk¹, oleksandra niemirich¹, oksana vasheka¹, olena yanchyk² ¹national university of food technologies, kyiv, ukraine *corresponding author: mariia_ianchyk@ukr.net abstract: the article is devoted to research of model systems of a new finishing confectionery semi-finished product which contains sugar fondant, plant powder, butter and surfactant citric acid ester. in the 21st century, a topical issue of the world level is considerable prevalence of chronic non-communicable diseases (ncd), which causes global social and economic losses of the population in many countries of the world, including ukraine. risk factors that cause by ncd are known and widespread among the population. simultaneous reduction of the frequency and levels of several common risk factors contributes to the reduction of the prevalence of the main ncd. in this regard, world health organization (who) has proposed an integrated prevention program for non-communicable diseases across the country. one of the main directions of disease prevention is healthy eating, which suggests that diets should consis... microsoft word 7 corectat-popa camelia.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 43 purification of cacl2 solutions using purolite s930 resin dynamic studies *camelia popa1, costel mironeasa2 1 faculty of food engineering, ştefan cel mare university of suceava, romania, kamelia_popa@yahoo.com 2 faculty of mechanical, mechatronic and management engineering, ştefan cel mare university of suceava, romania, costel@fim.usv.ro *corresponding author received 15 november 2012, accepted 12 december 2012 abstract: resin purolite s930 is used with numerous applications in refining salt solutions concerning transition metals. this work presents the influence of experimental conditions(?) to remove fe(ii) from 34% cacl2 solutions using chelating resin purolite s930 in dynamic regime and also the experimental conditions about desorbtion efficiency. the resuls show that under circumstances ( t=220c, volume of resin = 10,2 ml, layer heihgt=13 cm, ph=2.3), usable breakthrough capacity decrease with the increasing of feeding flow and increasing of the concentration of fe(ii) in feeding solution. the volume of refined solution decreases with the increasing of fe(ii) concentration in feeding solution. usable capacity for sorbtion in dynamic regime is 197 mg fe(ii)/g. desorbtion effiency increase with height of resin bad and hcl concentration. increasing the concentration of hcl from 2n to 10% and resin dose from 2 grames to 4 grames desorbtion effiency increases from 50 to 520 times. keywords: dynamic, purolite, capacity, desorbtion 1. introduction one of the most used techniques to remove heavy metals from solutions is based on ion exchange process. there were tested different typs of polymers who operate in different conditions to obtain maximum sorption capacity [1]. resin purolite s930 is used with numerous applications in refining the salt solutions concerning transition metals, for purification of organic and anorganic chemical solutions from heavy metals [2]. the percent of fe(ii) removal from 34% cacl2 solution by ion exchange in static regime depends on process variables, such as initial solution ph, initial metal ion concentration, metal/resin ratio, contact time and temperature [3]. in this study, the purolite s930 resin with iminodiacetic acid (ida) functional groups was used to remove fe(ii) ions from synthetic 34% calcium chloride solution, in dynamic regime. in literature technical data, the value of capacity for different types of resins is around 200 mg/g [4]. table 1. capacity for different types of resins metal resin capacity (mg/g) reference fe(iii) amberlite irc-50 200 fe(iii) amberlite irc-76 235.2 fe(iii) dowex mac-3 182.9 fe(iii) duolite c433 231.5 fe(iii) duolite c436 216.5 fe(iii) amberlite irc-86 225.9 p.a. riveros 2004 [7] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 44 2. materials and methods 2.1. materials. the chelating resin used in the experiments was s930 obtained from purolite international limited (hounslow. uk). the main physical and chemical properties of the resin are presented in table 2. table 2. characteristic proprieties of the chelating resin polymer matrix structure macroporous styrene divinylbenzene functional groups iminodiacetic acid ionic form (as shipped) na+ ph range (operating): h+ form; na+ form 2 6; 6 11 maximum operating temperature 70ºc particle size range + 1.0mm < 10%. 0.3mm < 1% total exchange capacity ≥ 1.9 meq/ml * manufacturer supplied. the conversion of the sodium form of the resin into hydrogen form was made with 10% hcl solution. followed by washing with distilled water until the ph of the effluent dropped to neutrality. accordingly to the manufacturer supplied. the resin has been dried using an oven at 60 ºc. to avoid thermal destruction of functional groups. the calcium chloride 34 % solution with iron was prepared using cacl2 analyticalreagent grade and distilled water. in this solution was added 2g/l fe(ii) solution to obtain 100 200 mg fe(ii)/l in 34% cacl2 solution. the stock solution of iron (2g fe(ii)/l) was prepared from analyticalreagent grade iron sulphate (feso4 ∙ 7h2o) in distilled water and hydrochloric acid. analytical-reagent grade. 2.2. sorbtion experiments. to find exchange capacity in dynamic regime was performed a classic installation [5]. like in figure 1. formed by an glass column (6) filled with exchange resin. solution storage tank (1). thermostat (8). magnetic stirrer (4). flowmeter (5) intermediate vessel for feeding. overflow (2). intermediate vessel for collect tap (3). eluate collection vessel (7). control valve (9.10). the values of column parameters are predicted as a function of flow rate and bed hight. in all the studies was used the same column (10 mm inner diameter). synthetic calcium chloride solution containing fe(ii) in controlled concentration and maintained under stirring was fed from the top of the column. flow was adjusted with control valves and intermediate vessels wich helps to maintain a constant liquid level in the column resin. figure 1. experimental installation for the sorbtion study. dynamic regime the content of iron for solutions was determined using a spectrophotometric method with 1.10 phenantroline and hydroxilaminochlorohidrat (λ = 510nm) and hach dr/2000 spectrophotometer (düsseldorf. germany) [6.7]. for plotting calibration curve was used feso4 ∙ 7h2o reference certified material from merck (darmstadt. germany). because in the presence of dissolved o2 a part of fe(ii) oxidizes to fe(iii). in all experiments was measured the concentration of total iron as fe(ii) ions. for reproductible results. the experiments were conducted in three replicates. the studies have followed the influence of parameters like flow and the concentration of influent solution on exchange capacity in dynamic regime. ph was kept at constant value of 2.3 due the solubility causes and operating ph range for resin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 45 so. fe(ii) precipitates at ph = 4 12 depending on his concentration. fe(iii) derived from the oxidation of fe(ii) in the presence of oxygene precipitates at ph around 2.5 and operating range for purolite resin is between 2 and 6. in experiments flow was varied between 0.5 and 2 ml/minute. the range of fe(ii) concentration in influent solution was between 100 and 200 mg fe(ii)/l. work temperature was 22oc. 3. results and discussions 3.1. the influence of feeding flow against exchange capacity. to find usable capacity exchange of purolite s930 resin was made experiments using 34% cacl2 solutions with 200 mg/l iron content. resin dose 4g (10.2 ml) varying power flow of the column between 0.5 and 2 ml/minute. experimental conditions are shown in table 3. the breakthrough time of the column and usable capacity of the resin at breakthrough are shown in table 4. the breakthrough time was determined as the time of operation of the column after its effluent concentration has a concentration in fe (ii) lower than 10 mg/l. according with stas 2073-75. calcium chloride. from table 4 it can see the decreasing volume of treated solution and breakthrough time with the increasing of feeding flow. plotting the dependence between the usable capacity breakthroughs and feeding flow (figure 2). it results the increasing of usable capacity breakthrough of purolite s930 resin with the increasing of feeding flow. as expected. time to exhaustion the resin is the time after which the concentration in fe(ii) of the eluate is equal to the concentration in the initial solution. table 5. 0 2 4 6 8 10 12 0 0.5 1 1.5 2 2.5 feeding flow, ml/min u sa bl e ca pa ci ty b re ak th ro ug h, m g/ g figure 2. variation of usable breakthrough capacity with feeding flow. figures 3 and 4 show the variation of concentration in the column effluent depending on the volume passed through the column. and respectively the variation of concentration in the column effluent. depending on operation time. table 3. experimental conditions in the study of the influence of flow on exchange capacity nr. phi t ( ºc) co mg fe(ii)/l resin volume (ml) layer heihgt (cm) time (h) feeding flow (ml/min) 1 0.5 2 1.0 3 2.3 22 200 10.2 13 0 60 2.0 table 4 usable breakthrough capacity of purolite s930 resin for sorbtion of fe (ii) from 34%cacl2 solution nr. feeding flow (ml/min) breakthrought time (min) purged solution volume (ml) usable capacity breakthrough (mg/g) 1 0.5 469 220.85 10.8 2 1.0 57 54 2.6 3 2.0 25 44 2.1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 46 table 5. usable exhaustion capacity of purolite s930 resin for sorbtion of fe (ii) from 34%cacl2 solution nr. feeding flow (ml/min) solution volume passed through column (ml) exhaustion time (h) usable capacity exhaustion (mg/g) 2 1.0 1957 32 179 -10 0 10 20 30 40 50 60 0 100 200 300 400 500 solution volume, ml c e, m g/ l flow 0.5 ml/min flow 1.0 ml/min flow 2.0 ml/min figure 3. variation of the effluent concentration depending of volume passed -10 0 10 20 30 40 50 60 0 200 400 600 800 1000 time, min c e, m g/ l flow 0.5 ml/min flow 1.0 ml/min flow 2.0 ml/miin figure 4. variation of the effluent concentration depending of operating time. 3.2 influence of initial concentration of the solution on the exchange capacity. to study the influence of initial concentration in sorbtion of fe(ii) ions on purolite s930 resin in dynamic regime. the experiments was made on the same column using 4 g of resin (10.2 ml). power flow 2 ml/minute and variable concentration for fe(ii). like in table 6. breakthrought times of the column and usable capacity to breakdown are presented in table 7 table 7 shows the decrease of usable capacity to breakthrought with the increasing of the initial feeding concentration (co). also for 4g resin dose and feeding flow of 2 ml/minute it observed the decreasing of refined solution volume and breakthrought times with the increasing of concentration of fe(ii) in feeding solution. plotting the breakthrought usable capacity function of initial concentration of iron in initial feeding solution observe it’s increasing with the decrease of fe(ii) concentration in feeding solution. figure 5. variation of column effluent concentration in time is plotted in figure 6. table 6. experimental conditions in the study of the influence of initial concentration of fe(ii) in cacl2 solution. on usable capacity nr. exp. phi t (ºc) feeding flow (ml/min) resin volume (ml) layer heihgt (cm) time (h) co (mg fe(ii)/l) 1 100 2 150 3 2.3 22 2 10.2 13 25 30 200 table 7. experimental conditions in the influence of initial concentration of fe(ii) in cacl2 solution. on usable capacity nr. exp. co (mg fe(ii)/l) breakthrought time (min) volume of refined solution (ml) usable capacity breakthrough (mg/g) 1 100 208 387 197 2 150 28 55 2.7 3 200 25 44 2.16 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 3 – 2012 47 0 2 4 6 8 10 12 14 16 18 20 80 100 120 140 160 180 200 220 c0, mgfe(ii)/l q us ab le , m g/ g figure 5. variation of breakthrought usable capacity function the concentration of fe(ii) 0 25 50 0 100 200 300 400 500 600 time, min c e, m g/ l 200 ppm 150ppm 100 ppm figure 6. variation of effluent concentration in time 3.3. desorbtion efficiency. for the study of desorbtion efficiency the experiments were performed in the same column using hcl 2n and 10%. feeding flow rate 0.3 ml/minute. figure 7 and 8 show the variation of eluent concentration as function of regeneration solution for 2g and 4 g of resin. as seen in table 8 and 9. increasing the resine dose and the concentration of the regeneration solution. the degree of concentration for iron have superior results: concentration degree increases from 50 to 520 times. maximum concentration from eluate increases from 9.86 g/l to 104 g/l. concentration peak from eluate also increases from 24.6 g/l to 172 g/l. -5 0 5 10 15 20 25 30 0 20 40 60 80 100 120 rege ne r ation s olution volume , m l c e, g f e( ii) /l figure 7. variation of eluent concentration with solution regeneration volume. 2 g resin dose. flow 0.3ml/minute. hcl 2n. 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 60 regeneration solution volume, ml c e, g fe (i i)/ l figure 8. variation of eluent concentration with solution regeneration volume. 4 g resin dose. flow 0.3ml/minute. hcl 10%. table 8. degree of fe(ii) concentration in eluate. resin dose 2g. hcl 2n resin dose (g) feeding flow. ml/min peak concentration of eluted iron. g fe(ii)/l maximum concentration of eluate. g fe(ii)/l degree of iron concentration 2 0.3 24.6 9.86 50 table 9. degree of fe(ii) concentration in eluate. resin dose 2g. hcl 10% resin dose (g) feeding flow. ml/min peak concentration of eluted iron. g fe(ii)/l maximum concentration of eluate. g fe(ii)/l degree of iron concentration 4 0.3 172 104 520 food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume xi, issue 3 – 2012 48 4. conclusions dynamic column experiments show that the resin is able to selectively remove iron from 34% calcium chloride solutions. the resuls show that under circumstances (t=220c. volume of resin = 10.2 ml. layer heihgt=13 cm. ph=2.3) usable breakthrough capacity decrease with the increasing of feeding flow and increasing of the concentration of fe(ii) in feeding solution. the volume of refined solution decrease with the increasing of fe(ii) in feeding solution. usable capacity for sorbtion in dynamic regime is 197 mg fe(ii)/g. according to data from technical literature [4] and close with the value determined in batch process [3]. efficiency of desorbtion increases with height of resin bad (dose) and hcl concentration. 5. references [1]. duran a.. soylak m.. tuncel s.. poly(vinyl pyridine-poly ethylene glycol metacrylate-ethylene glycol dimetacrylate) beads for heavy metal removal. journal of hazardous materials. 155. 114-120. (2008). [2]. belsten m.. chirică m.. les resins exhangeurs d’ions. purolite international guide. (2004) [3]. popa c.. bulai p.. macoveanu m.. the study of iron (ii) removal from 34% calcium chloride solutions by chelating resin purolite s930. environmental engineering and management journal. 9. 651-658. (2010). [4]. xue s.s., gula, m.j., harvey, j.t., horwitz, e.p., control of iron in copper electrolyte streams with a new monophosphonic/sulphonic acid resin. miner. metall. process. 18 (3), 133–137, (2001) [5]. popa g.. moldoveanu s.. quantitative chemical analysis using organic reagents. technical press. bucharest. (1969). [6]. *** sr iso 6332:1996. water quality. determination of iron. 1.10 phenantroline metode. [7]. riveros p.a.. the extraction of fe(iii) using cation exhange carboxylic resins. hidrometallurgy. 72. 279-290. (2004). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 110 use of fish behavioral responses in identifying sublethal exposure to pollutants gintaras svecevičius1, nijolė kazlauskienė1 1 institute of ecology of nature research center, akademijos 2, lt-08412 vilnius-21, lithuania, gintaras.svecevicius@takas.lt abstract. in present the controlling of pollution of aquatic environment is performed basically by use of analytical investigations. however, biological control of effluent toxicity before effluents are discharged into receiving waters is still seldom used. therefore, the search into suitable, easy available and maintainable test-objects, their rapid, sensitive, and non-specific test-functions which provide information not only about lethal but also about sublethal effects of pollutants is needed. fishes are considered to be among the best test-objects. alterations in a number of fish behavioral responses are sensitive indicators of sublethal exposure to aquatic pollutants. these are no standardized procedures yet. laboratory tests were conducted on rainbow trout oncorhynchus mykiss. behavioral responses, such as: leaving the nest, response to external stimuli and breathing rate in larvae as well as detectionavoidance, locomotor activity, gill ventilation frequency, and coughing rate in juveniles and adults were investigated. obtained data showed that fish behavioral responses can be successfully applied in solving not only theoretical but also practical problems of aquatic toxicology, such as bioassay testing for hazard assessment of separate toxicants and their mixtures, industrial and municipal wastewaters as well as natural inland waters containing complexes of pollutants of different chemical origin. keywords: rainbow trout; behavior; aquatic pollutants; toxicity introduction at present the controlling of pollution of aquatic environment is performed basically by use of analytical investigations. however, the evaluation of effluent quality by physical-chemical measurements does not provide information regarding the possible synergetic or antagonistic effects that complex effluents may have on aquatic life. therefore it is necessary to perform biological control of effluent toxicity, before effluents are discharged into receiving waters. for rapid determination of wastewater toxicity level, bioassay testing is used. bioassay testing is defined as a rapid experimental evaluation of water toxicity level by use of biological objects and processes [1]. test-objects must be easily available and maintainable. their testfunctions must be sensitive not only to acutely toxic effluent levels, but also provide information about sublethal concentrations. fishes are considered to be among the best test-objects. alterations in fish behavioral responses are sensitive indicators of sublethal exposure to aquatic pollutants [2– 5]. these are no standardized procedures yet. the aims of the present study were: (1) to generalize data on behavioral responses of rainbow trout at different stages of food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 111 development (larvae, juveniles, adults) under the effect of aquatic pollutants, (2) to perform comparative analysis of their sensitivity to the effect of pollution, and (3) to evaluate suitability of the responses studied for needs of bioassay testing of the polluted waters for hazard assessment. materials and methods the tests were performed on rainbow trout oncorhynchus mykiss at different stages of development (larvae, juveniles, adults). test fish were obtained from hatchery and acclimated to laboratory conditions. the effect of separate toxicants, their mixtures as well as various natural and wastewaters was investigated. a number of fish behavioral responses (leaving the nest, response to external stimuli, gill ventilation frequency in larvae, detection and avoidance, locomotor activity as well as gill ventilation frequency and coughing rate in juveniles and adults) were studied. deep-well water was used for dilution. average hardness of the water was approximately 284 mg/l as caco3, alkalinity was 244 mg/l as hco3ˉ, ph was from 7.9 to 8.1, temperature was maintained at 10.5 to 11.5c, and oxygen concentration was maintained within the range 8 10 mg/l. results and discussion the data obtained showed that the sensitivity of fish behavioral responses to pollution varies significantly depending on fish stage of development and chemical origin of the pollutant or test water studied (table 1). table 1 behavioral responses of rainbow trout to pollutants and test waters test-object pollutant or test water sensitive response reference larvae heavy metal model mixture (cu, zn, ni, cr, fe) leaving the nest; response to external stimuli [6] larvae drūkšiai lake water; ignalina nuclear power plant wastewater gill ventilation frequency [7] larvae ammonia gill ventilation frequency [8] larvae heavy metal model mixture (cu, zn, ni, cr, pb, cd, mn) leaving the nest; response to external stimuli [9] larvae heavy metal model mixture (cu, zn) gill ventilation frequency [10] larvae heavy fuel oil gill ventilation frequency [11] larvae treated wastewater gill ventilation frequency [12] juveniles zinc avoidance [13] juveniles kairiai dump filtrate coughing rate [14] juveniles baltic sea water after oil spill in būtingė terminal in 2001 coughing rate [15] juveniles heavy metal model mixture (cu, zn, ni, cr, fe) avoidance [16] juveniles heavy metal model mixture (cu, zn, ni, cr, pb, cd, mn) locomotor activity [17] juveniles nemunas river and curonian lagoon water coughing rate [18] juveniles hexavalent chromium avoidance; locomotor activity, [19, 20] juveniles heavy fuel oil avoidance [11] adults heavy metal model mixture (cu, zn, ni, cr, fe) coughing rate; gill ventilation frequency [21] adults ammonia coughing rate; gill ventilation frequency [8] adults heavy metal binary mixture (cu, zn) gill ventilation frequency [10] adults heavy fuel oil coughing rate; gill ventilation frequency [11] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 112 overall, the most sensitive rainbow trout behavioral responses in the tests using separate toxicants or their mixtures were found to be avoidance and locomotor activity in juveniles. low sublethal concentrations of pollutants corresponding to 0.00006 – 0.006 parts of their 96-hour lc50 values can be detected by use of these responses. [17, 19, 20]. respiratory responses (coughing rate and gill ventilation frequency) were found to be less sensitive. by use of these responses such effective levels as 0.001 – 0.07 parts of 96-hour lc50 values can be estimated [17, 20, 11]. in spite of that behavioral data indicates that they are much more sensitive than many fish biochemical–physiological parameters [8]. meanwhile, the least sensitive responses have been gill ventilation frequency, leaving the nest and response to external stimuli in larvae. they can be used only to detect nearly lethal (0.5 – 0.7 parts of 96-hour lc50 value) pollutant concentrations [6]. although the literature on various types of fish behavioral responses to pollutants is abundant they are still seldom used as ecotoxicological tools in the dayto-day management monitoring of the industrial wastewaters and/or toxicity of the aquatic environment. conclusion determination of water-quality criteria is based on conventional standard acute and chronic testing procedures. fish behavioral tests seem sensitive, rapid, ecologically significant, and they could be easily standardized. consequently, in solving practical problems of aquatic toxicology, such as bioassay testing, fish behavioral responses can be successfully applied for hazard assessment of industrial wastewaters as well as natural inland waters containing separate pollutants of different chemical origin or their complexes. acknowledgements reserach council of lithuania project no. mip-58/2010 for finance support. references 1. b.a. flerov. ecological and physiological aspects of toxicology in fresh-water animals. nauka, leningrad, 1989, 144 pp. (in russian). 2. j.d. giattina, r.r. garton. a review of the preference-avoidance responses of fish to contaminants. res rev, vol.87, 1983, p. 43-90. 3. e.e. little, s.e. finger. swimming behavior as an indicator of sublethal toxicity in fish. environ toxicol chem, vol. 9 1990, p. 13-19. 4. e. scherer. behavioural responses as indicators of environmental alterations: approches, results, developments. j appl ichthyol, vol. 8, 1992, p. 122-131. 5. a.s. kane, j.d. salierno, s.k. brewer. fish models in behavioral toxicology: automated techniques, updates and perspectives. in: g.k. ostrander (ed.) methods in aquatic toxicology. volume 2. lewis publishers, boca raton, fl, 2005, p. 559-590. 6. n. kazlauskienė, a. burba, g. svecevičius. reactions of hydrobionts on the effect of mixture of five galvanic heavy metals. ekologija, vol 4, 1995, p.56 -59. 7. n. kazlauskienė, ž. čepulienė. ecotoxicity of drūkšiai lake waters assessed by biotesting with rainbow trout spawn and larvae. proc latvian acad sci, vol. 52, 1998, p. 126-129. 8. m.z. vosylienė, g. svecevičius, n. kazlauskienė. toxic effects of ammonia to rainbow trout oncorhynchus mykiss at all stages of development. in: g. rupp, m.d. white (ed.) fish physiology, toxicology, and water quality. proceedings of the seventh international symposium. tallinn, estonia, 2003, p. 159-170. 9. n. kazlauskienė. classification and possible consequences of the sublethal effects of heavy metal model mixture in fish at early ontogenesis. in: m. anke et al. (ed.) macro and trace elements. 22 workshop 2004. friedrich schiller university, jena, germany, 2004, p. 11721177. 10. n. kazlauskienė, m.z. vosylienė. characteristic features of the effect of cu and zn mixtures on rainbow trout oncorhynchus mykiss in food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 113 ontogenesis. polish j of environ stud, vol. 17, 2008, p. 291-293. 11. n. kazlauskienė, g. svecevičius, l. petrauskienė, m.z. vosylienė. behavioural responses of medicinal leech and rainbow trout exposed to crude oil and heavy fuel oil in ontogenesis. polish j of environ stud, vol. 19, 2010, p. 429-433. 12. m.z. vosylienė, n. kazlauskienė, j. baršienė, l. andreikėnaitė, a. milukaitė, e. taujanskis. ecotoxicity and genotoxicity relating to fish in wastewaters discharged from the vilnius treatment plant. water sci technol, vol. 62, 2010, p. 859-865. 13. g. svecevičius. the use of fish behavioralrespiratory responses in identifying sublethal toxicity of zinc. ekologija, vol. 2: 2002, p. 9-12. 14. g. svecevičius. evaluation of toxicity of the kairiai dump filtrate by use of rapid behavioral responses of juvenile rainbow trout oncorhynchus mykiss. in: protection and control of water bodies. proceedings of international scientific conference, kaunas, lithuania, 2004, p. 208-213. (in lithuanian) 15. g. svecevičius. estimation of the baltic sea water toxicity level after oil spill in būtingė terminal in 2001 by use of fish behavioral bioassay. in: usa – baltic international symposium “advances in marine environmental research, monitoring & technologies”. abstracts. klaipėda, lithuania, 2004, p. 138-139. 16. g. svecevičius. use of fish behavioralrespiratory responses in identifying sublethal toxicity of heavy metal mixture. in: m. anke et al. (ed.) macro and trace elements. 22. workshop 2004. friedrich schiller university, jena, germany, 2004, p. 94-99. 17. g. svecevičius. behavioral responses of rainbow trout oncorhynchus mykiss to sublethal toxicity of a model mixture of heavy metals. bull environ contam toxicol, vol. 74, 2005, p. 845-852. 18. g. svecevičius, m.z. vosylienė. curonian lagoon water and bottom sediment toxicity assessment using fish bioassays. abstracts of the 2nd international conference on coastal lagoons “european lagoons and their watersheds: function and biodiversity”, klaipėda, lithuania, 2005, p. 18. 19. g. svecevičius. avoidance response of rainbow trout oncorhynchus mykiss to hexavalent chromium solutions. bull environ contam toxicol, vol. 79, 2007, p. 596-600. 20. g. svecevičius. use of behavioral responses of rainbow trout oncorhynchus mykiss in identifying sublethal exposure to hexavalent chromium. bull environ contam toxicol, vol. 82, 2009, p. 564-568. 21. m.z. vosylienė, g. svecevičius, s. šyvienė. toxicity of factory wastewaters and heavy metal solutions to rainbow trout. in: r.v. thurston (ed.) environmental studies in the nemunas river basin, lithuania. us epa /600/r94/155, washington dc, 1994, p. 107-114. microsoft word 3 fia journal 2010 -2 din 12 oct 2010 final_75-82.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 79 genetic resources and the progress reached by winter wheat breeding constantin drobotă 1 , ioan gontariu2 1 agricultural research and development station of suceava, b-dul 1 decembrie 1918 no.15, suceava, e-mail: constantin_drobota@yahoo.com 2 stefan cel mare university of suceava, faculty of food engineering, 13 universitaţii st., 720229, suceava, romania, e-mail: ioang@usv.ro abstract: this paper shows the contribution in the development of the winter wheat crop, from the beginning of the breeding activity, through the testing and identification of the most adequate autochthonous and foreign wheat varieties, and later, through creating, introducing and extending in production of eight varieties: bucovina, suceava 84, aniversar, gaşparom, magistral, esenţial, drobeta and voroneţ. the cultivars aniversar, gaşparom, magistral and esenţial were cultivated during period 1996-2005. these cultivars registered an average yield of 6367 kg/ha, reaching a yield rise of 197% in comparison with the average yield registered during the period 1951-1955. thus, during the last 60 years an ascending dynamic progress, materialized through an yearly increasing rate up to 1,4% was registered, in comparison with the average yield obtained at the standard variety flamura 85, during 1996-2005. the superiority of the eight varieties created at the agricultural and development research station of suceava, from the productivity and yield stability point of view, was statistically demonstrated, through the distribution of the correlations and variability of productions, in comparison with the standard varieties from the above mentioned periods. an important progress concerning the physiological breeding characteristics such as: winter, foliar diseases and lodging resistances, was registered, too. keywords: average yield, ecological factors, agricultural areas introduction: the presence of some risk ecological factors with a limitative action not only on the quantity but also on the yields quality has determined the fact that during the 60 years period since the foundation of a.r.d.s. suceava, a correct strategy of the way of organization and development of the breeding researches for winter wheat should be objectively approached. this affirmation is also sustained through the results that have been registered [1, 2, 3, 6, 7]. the agricultural increasing rate on the more poor lands in the agricultural areas with a wet and cold climate, from the north and north west of moldova have amplified the necessity to create some new varieties that are superior from both the quality and the quantity point of view to the varieties cultivated in time, as the wheat cultures behave risks because of the biotic and abiotic factors, registering frequently big yield fluctuations [5, 6, 9]. in connection with that, at the a.r.d.s. of suceava, a research activity sustained in the breeding field, following initial selection and identification of the most appropriate varieties for winter wheat, in our country and abroad, having in mind the introduction of them in the culture and replacing from the cultivated variety the one with poorer production performances, has been carried on, since its foundation in 1946 [1, 4, 7, 12]. the research organized on the basis of a national experimentation system of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 80 wheat varieties has developed only after 1950, increasing after 1970, when we initiated the special works of breeding in the creation of new varieties and wheat lines more valuable and better adapted to the ecological and cultural conditions in moldova and in the northern part of the country [1, 2, 6]. through the development of breeding researches, superior wheat genotypes have been created, this being one of the safest ways and efficient ones to increase productivity, stability and yield quality. having this in mind, at present the appreciations made by the best researchers in the field are unanimously accepted, as they estimate a continuous genetic progress for the wheat varieties created in our country, dimensioned to a annual rate of up to 1, 4 % and even more for other agronomical characters, with a direct influence on the stability of yield [8, 9, 10, 11, 12]. this work aims at appreciating the contribution brought to wheat culture, initially through the testing and identification of the most appropriated national and foreign wheat varieties and after that through the creation, introduction and extension in the production of the varieties obtained at a.r.d.s. of suceava in 60 years since its foundation. biological material and research method the breeding working method used so far at the a.r.d.s. of suceava in the last 60 years has contributed in the first half of the period to the testing and generalization in the culture of the most valorous autochthonous and foreign wheat varieties, and in the second half of the period to creation of the 8 wheat varieties and also to obtaining of a valuable breeding material, well adapted to specific conditions of culture in the north part of the country, that offers the possibility of creating in the future new more performing genotypes. the value dimensioning of the superiority of the varieties created is presented during three significantly periods of time as follows: 1. period 1946-1975 highlights the difference in behavior of varieties and the wheat lines verified at suceava having in consideration the aspect of physiological, quality and production features. 2. period 1976-1995 presents the productive value of the varieties bucovina, suceava 84 and aniversar, cultivated in this period of time, comparing with the fundulea 29 used as a standard in the northern areas. 3. period 1996-2005 highlights the gaşparom, magistral, esenţial, drobeta and voroneţ varieties, in comparison with witness variety fundulea 4 used in this period of time. for the appreciation of the experimented varieties value, from the point of view of the productive potential, the obtained results have been used, during the experimentation years, in the comparative national crop, comparing it with the witness varieties with the greatest spread in production: fundulea 29 and fundulea 4. the relation between the results of the varieties created in suceava and those of the standard variety fundulea 4 in the period 1996-2005 has been described through the calculation of the linear regressions and the establishment of the production has been characterized through the determination of the variances(s2).the results of the phenological observations made during the vegetation are presented in the work through smaller values, and the interpretation of the production data has been made through statistic methods of variance analyses the experimentation has been carried out in comparative crops, executed at the beginning after the method of plots position in latin rectangle (with 5 replications and 10 m2 the cropped food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 81 surfaces of one plot) and then in the balanced grating with reiteration of the basis scheme (6 replications and 7 m2 cropped plot) over the years the experiments have been placed after potato, in the leached chernozem soil, reached in humus and total nitric, middle provided with phosphor and mobile kalium, not being necessary the phase fertilization in autumn, as the potato has received larger quantities of chemical fertilizer. in spring, before the beginning of vegetation period, 60-70 kg n s.a. /ha as ammonium nitrate or nitro-limestone have been administered. the sowing has been carried out, in the majority of the experimental years, in the optimum epoch for this area being assured 550 germinated grains/ m2. for the creation of the 8 winter wheat varieties to a.r.d.s. of suceava, with a genealogy and method obtained being presented in short terms, valuable genetic resources have been used, most of them being adapted to the northern and west northern part of moldova (table 1). table 1 the genealogy and obtaining method of the winter wheat varieties created at the a.r.d.s. of suceava variety parental forms cross year homologation year. obtaining method bucovina f.53-67 x f. 117-67 1968 1979 suceava 84 bezostaia 1 x f.208 – 65 1969 1984 aniversar lovrin 11 x f.53 – 67 1970 1986 magistral 1502w4-1/154i1-1//aniversar/roxana 1986 1998 gaşparom sv. 9710-79 x fundulea 4 1984 1998 esenţial sv. 2946 – 86 x sv. 9710 – 79 1988 2001 drobeta sv. 9710 – 79 x f. 4141 – w 1 1991 2003 voroneţ sv. 9710 – 79 x progres 1994 2004 the individual selection (pedigree method) climatic conditions, appreciated from the point of view of the annual precipitation quantity and the way of distribution of these in the course of vegetation, have been favorable in the majority of the experimentation years. not only in the autumn period but also in the winter period, the climatic conditions have not influenced negatively the vegetation of plants , so that on spring arrival the crops have been closed, having a normal density. in the majority of the years the regime of the rains has been richer in the intense vegetation period, may – june, being an advantage for the growths and development of plants, exception making the years: 1996, 1999, 2000, 2003 and 2004, when a humidity deficit of 116.3 mm has been registered (table 2). table 2 the precipitation regimes at ards of suceava during vegetation periods (1996 – 2005) precipitation per years, mm: period 1995/ 1996 1996/ 1997 1997/ 1998 1998/ 1999 1999/ 2000 2000/ 2001 2001/ 2002 2002/ 2003 2003/ 2004 2004/ 2005 multiyear average august 51.7 136.0 88.1 21.1 68.2 39.1 73.2 112.3 58.0 52.9 69.7 septemberoct. 95.0 150.6 100.8 186.5 92.0 81.9 191.0 108.3 74.4 65.1 79.2 november 65.5 27.1 21.2 30.5 22.6 10.8 63.5 47.0 10.9 39.5 30.4 dec. march 126.5 84.5 104.4 68.3 77.3 89.1 76.6 67.8 90.8 101.4 92.8 april 38.3 52.8 63.0 87.6 50.9 60.5 6.3 10.8 19.0 100.6 45.6 may – june 93.3 153.2 262.9 90.1 54.0 123.9 119.0 46.4 61.6 225.4 163.2 july 53.8 111.9 101.6 65.8 115.0 91.4 238.0 252.5 160.7 45.3 89.3 total 524.1 716.1 742.0 549.9 480.0 496.7 767.6 645.1 475.4 630.2 570.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 82 the normal calculation of 61 years the wet and cold regime in the vegetation period has positively influenced the appearance and development of foliar diseases attack and contributed in the same time to the amplification and sometimes early falling of plants from the most sensitive varieties . under the report of thermal regime, in the majority of the experimental years , values relatively close to the multiannual average have been registered , without inducing negative influences over the plants vegetation. results and discussion a valuable contribution in the agricultural progress in bucovina is brought to the plants breeding research field, beginning with the foundation of the ards in 1946 through verifications of different varieties and wheat populations’ behavior from the western part of suceava plateau area, the research activities being carried out on the basis of a national experimentation system of wheat varieties which have been developed only after 1950 . it can be appreciated that winter wheat , and the other plants taken into consideration in the area has registered an ascending line . beginning with the year 1946 and until 2005 , during six decades , a number of 6391 varieties and wheat lines of perspective, have been verified, from the point of view of production and vegetation behavior of which 1750 autochthonous and foreign varieties. the most valuable of these varieties have been introduced and generalized in production , occupying at the level of different periods numerous surfaces. the yield quality from the point of view of the physical aspects of production it is easy to see that one thousand kernel weight of the experimented varieties during 1970 -1975, has varied greatly from one variety to another, ranging values from 33,8g to 42,4 g (table 3). it has been noticed that from this point of view the lovrin 10, aurora and kaukaz varieties have registered the highest values for one thousand kernel weight (39,9 g –42,4 g) table 3 the yield quality of wheat varieties and lines which were experimented during period 1970 -1975 at ards of suceava gluten from 100g flour variety or line one thousan d kernel weight -g total protein % from d.s. n x 5,7 wet dry pelshenk e index farinog raf note bread volum e -cm3 bread weigh t -g lovrin 10 42.4 15.4 32.9 10.3 59 45 508 152 aurora 41.0 15.3 31.1 10.4 81 45 518 156 kaukaz 39.9 16.1 33.6 10.9 75 46 531 154 brucker 6111 35.5 15.4 31.6 10.4 94 48 530 151 t. 195 65 33.8 14.0 27.2 9.6 135 50 514 153 dacia 36.8 16.1 31.6 10.9 101 49 540 153 favorit 33.9 16.2 32.9 11.4 107 59 547 154 bezostaia1-st. 33.8 14.4 29.0 9.8 114 51 496 151 according to protein content, represented through values of 14,0-16,2%, it has been noticed for the favorit , dacia and kaukaz varieties, to have higher content of wet food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 83 and dry gluten. from the point of view of gluten quality, which bread making features depend on ultimately, for the favorit, bezostaia 1, dacia, brucker 6111 varieties and for inbred line t. 195 – 65, the following aspects have been analyzed: the highest value for pelshenke index, of 94 – 135, farinograf note, up to 48, the bread volume calculated at 100 g flour between 496 and 547 cm3, and its weight up to 151 g the yield quality from the point of view of the quality features we can notice that between the experimented varieties in the period 19911995 there are no differences that could surprise us, these displaying good qualities of milling and bread making features (table 4). according to the protein content, represented by average values from 12.2 % at transilvania 1 and turda 81, up to 13.8 %, in the varieties aniversar, fundulea 29, dropia and bezostaia 1, the gluten content has registered higher values up to 33.3 % at wet gluten and 10.8 % for the dried one. taking into consideration this aspect we have noticed that, through the increased gluten content, the varieties bucovina, aniversar, suceava 84, apullum, ariesan, bezostaia 1 and dropia have registered values up to 30% at wet gluten and 10.8% dried gluten. from the point of view of the gluten quality, which finally determines the bread making features of flour, we can observe that higher gluten content varieties had also the higher glutenin index, from 54.5 at bucovina, up to 60.5 at ariesan. the bread making features value of the experimented varieties, appreciated through bread volume and weight, from 100g of flour , demonstrates that from this point of view the advanced varieties are mostly varieties with good bread making features qualities, the bread volume registering from 441 cm3 in the variety apullum, up to 506 cm3 at dropia, and their weight varying between 139 g at variety turda 81 and 144 g at varieties dropia, aniversar and fundulea 4. table 4. quality traits of the wheat varieties experimented at ards of suceava (1991-1995) % gluten bread from 100 g flour curre nt no. variety % protein wet dry glutenic index volume -cm3 weight –g 1 fundulea 29 13.8 28.4 9.7 52.8 477 143 2 fundulea 4 12.4 25.3 8.5 54.4 443 144 3 flamura 85 13.6 28.1 9.5 54.2 453 142 4 rapid 13.6 29.0 9.5 58.6 457 143 5 dropia 13.8 32.1 10.2 59.6 506 144 6 bucovina 13.1 30.9 9.3 54.5 476 142 7 suceava 84 12.8 33.0 9.5 54.7 480 143 8 aniversar 13.8 33.3 10.1 55.3 479 144 9 moldova 83 12.8 29.4 9.2 53.0 495 140 10 turda 81 12.2 25.2 8.3 49.6 489 139 11 apullum 13.4 30.2 9.5 50.2 441 142 12 arieşan 13.3 30.5 9.4 60.5 437 140 13 transilvania 1 12.2 26.9 8.6 53.9 502 141 14 f. 308 13.2 28.5 9.6 52.5 495 141 15 bezostaia 1 13.7 33.3 10.8 57.1 496 141 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 84 the yield quality the physical features of production, at the varieties created at ards of suceava between 1996-2005, are highlighted by bigger and heavy grains, fact that confers them higher values not only for one thousand kernel weight, most often registered between 45-50 g, and the test weight had values between 75-80 kg. taking into consideration the bread making features, the gasparom, magistral, esential, drobeta and voronet varieties frame themselves among the crops with superior qualities, proving also to be competitive in comparison to the actual requests regarding this matter (table 5). the chemical content of grains frequently registers high values, superior to the flamura standard, not only for the protein content from 13.8 % to 14.2 %, but also for the wet gluten content (28.4 – 32.4 %) and dried gluten (9.5 – 10.5 %). the gluten indicator between 85.0 and 97.9 ml, superior to the flamura 85, proves in a special way that the quantity and quality values of the gluten from protein for the newly varieties created at ards of suceava between 1996 and 2005, is obviously due to the lower deformation indicator level (6 – 7 mm as against to 8 mm at standard). table 5 the main indicators for bread making features quality for the created and experimented varieties at ards of suceava during period 1996-2005 in comparison with the flamura 85 variety. crt no. specification gasparom magistral esential drobeta voronet flamura 85 1 protein content (%) 14.2 13.9 13.8 13.8 14.1 13.6 2 wet gluten (%) 30.2 32.4 28.5 28.4 29.8 28.1 3 dried gluten (%) 10.5 9.8 9.8 9.5 9.5 9.5 4 glutenic index (ml) 85.0 89.6 95.8 97.9 91.8 84.2 5 the deformation index (mm) 7 6 6 7 6 8 farinograma 6 hydration capacity (%) 66.3 62.8 61.2 62.4 61.8 61.2 7 developing (min.) 2.1 2.1 2.3 2.5 2.5 2.1 8 stability (min) 2.3 2.2 2.4 2.6 2.6 2.1 9 elasticity (uv) 130 128 126 133 131 125 10 power 56.8 57.7 59 56.6 57.5 50.5 11 value index 68.4 67.4 70.8 65.2 67.4 61.8 12 bread volume (cm3) 510 512 510 505 510 500 13 bread weight (g) 145 145 145 143 145 142 value group a2 –b1 a1 –b1 a2 –b1 a2 –b1 a1 –b1 b1 –b2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 85 6367 kg/ha5740 kg/ha 4650 kg/ha 3410 kg/ha2680 kg/ha2145 kg/ha 1000 kg/ha 0 1000 2000 3000 4000 5000 6000 7000 1946-1950 1951-1955 1956-1965 1966-1975 1976-1985 1986-1995 1996-2005 fig. 1 the genetic progress made by winter wheat, in the period of time 1946-2005, through utilization in the crop of the most valuable varieties, under crop conditions offered by ards of suceava analyzing the evolution of the genetic progress registered by wheat through the utilization in the crop production of the most valuable varieties, during the 60 years since the foundation of the ards of suceava, we can notice that there is a continuous increase, quantified by average productions from 2145 kg/ha, during the period 1951 – 1955, to 6367 kg/ha, during the period 1996 – 2005, which in relative values means an increase of 197 % (fig.1). the genetic progress reflects especially the increase of production capacity, registered especially in the last 30 years (1976-2005), when as a consequence of intensification of creating new varieties, wheat productivity has increased a lot, reaching an average level of 4650 kg/ha, during the period 1976-1985, up to 6367 kg/ha during the period 1996 – 2005 conclusions  the research activities carried out between 1946-2005 at ards of suceava, initially in the direction of identifying varieties and more valuable lines and afterwards creating new varieties and wheat lines more productive and better adapted to crops conditions from the north moldova , have contributed to the rational zoning of the best varieties, at the introduction and generalization in the crop and through this to progressive increase of production.  through the researches carried out over time at ards of suceava, 8 common winter wheat varieties have been created (bucovina, suceava 84, aniversar, gasparom, magistral, esential, drobeta and voronet) more productive and better adapted to the specific crop conditions for the wet and cold climatic areas.  the global analyses of the production performances have proved that, through the creation and utilization in the crop of these genotypes, in time, dynamic increasing progress has been registered, dimensioned through an annual increasing rate of production, in comparison with the annual average yield of the standard variety flamura 85, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no2 2010 86 of up to 1.4 % during the period 19962005  significant progresses concerning the breeding of the physiological features and quality features have been also registered: the winter resistance and resistance to cryptogrammic diseases attack has been increased from average forms to resistant forms; the lodging resistance has been improved, through the shortening of height and the thickening of stem, from low to very good; concerning the quality features, the progress obtained places the new varieties, created at ards of suceava, among the wheat varieties with superior bread making features (classes a1-b1 and a2-b1). the winter wheat varieties that may be found at present in moldova, especially in the northern and west-northern part of moldova, are autochthonous varieties with multiple adaptability qualities to the ecological conditions, and until the creation of some more performing varieties , we strongly recommend the following ones: gasparom, magistral, esential, drobeta and voronet. references 1. gaşpăr, i., 1968 – soiuri şi linii de grâu de toamnă de perspectivă pentru cultură în podişul sucevei. probleme agricole, nr. 8; 2. gaşpăr, i., 1976 – soiuri şi linii de grâu şi secară valoroase şi de perspectivă pentru judeţul suceava. volum omagial, 1946 – 1976: 47-61; 3. gaşpăr,i., 1984 – soiul de grâu de toamnă suceava 84. cercetări agronomice în moldova, vol. 3 (67): 39-44; 4. gaşpăr, i., 1987 – caracterizarea bioproductivă a soiului de grâu „aniversar”, creat la staţiunea agricolă suceava. cercetări agronomice în moldova, anul xx, vol. 4 (80):37-42; 5. gaşpăr,i., 1993 – rezultate experimentale privind comportarea unor soiuri de grâu, secară şi triticale la s.c.a. suceava. cercetări agronomice în moldova, anul xxvii, vol. 3-4 (102): 31-38; 6. gaşpăr,i., zamă, e., 1996 – rezultate experimentale privind comportarea unor soiuri şi linii de grâu de perspectivă în condiţiile de cultură din podişul sucevei. volum omagial, 1946-1996: 21-36; 7. gaşpăr, i.,cristea, m., popovici,d., 1996 – strategii şi obiective în ameliorarea plantelor la staţiunea suceava. lucrări ştiinţificevolum omagial, 1946-1996. s.c.agris-red. rev. agricole s.a.: 11-21; 8. gaşpăr, i., zamă, e., popescu, constanţa, murariu, m., 2000 – gaşparom şi magistral – soiuri noi de grâu de toamnă, create la staţiunea suceava. analele i.c.c.p.t. fundulea, vol lxvii: 49-64; 9. gaşpăr, i., murariu, m., zamă, e., 2002 – progresul realizat în ameliorarea grâului de toamnă la s.c.a. suceava în ultimele trei decenii, cercetări de genetică vegetală şi animală, vol.vii: 59-69. sensory evaluation of goat milk cheese under different freezing conditions 256 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 256 261 technology of fresh herbs storage using hydrogel and antioxidant composition *olesia priss1, viktoria yevlash2 1tavria state agrotechnological university, melitopol, ukraine, olesyapriss@gmail.com 2kharkiv state university of food technology and trade, kharkiv, ukraine, evlashvv@gmail.com *corresponding author received 14th october 2017, accepted 19th december 2017 abstract: there is a stable consumer demand for fresh culinary herbs. also, the greenery contains a large number of valuable phytonutrients. despite high efficiency and increasing annual production of fresh herbs, the problem of preserving their quality in the post-harvest period remains unresolved. because of the high specific surface area of evaporation, in the green crops droop quickly, they lose their marketable quality, and, as a result, the level of profitability of greenery production in general is being reduced. it is necessary to use new effective approaches to leafy greens storage in order to reduce product losses during transportation and storage. for example, agrarian hydrogel can be used for storage of greenery. hydrogel is an acrylic potassium polymer that is non-toxic and has a high environmental standard. the hydrogel granules can absorb up to 250 times more moisture than their weight. we propose the following procedure as the method of greenery preservation: the greens are packed in bundles and put in sticks in polyethylene bags with a fastener, pre-filled with hydrogel solutions. the storage temperature is maintained optimally for each species of fresh herbs, the relative humidity is 95 ± 3%. usage of the proposed method allows obtaining environment-friendly products, preserving their high biological value and increasing the shelf life. the accumulation of peroxide products, which cause physiological disorders, is inhibited as the result of such storage. the use of hydrogel reduces the natural loss of mass by 10% as compared with the control. duration of greenery storage increases by 30 days. keywords: storage, сulinary herbs, leafy greens, hydrogel, antioxidants, marketable quality, ascorbicacid, chlorophylls, carotenoids. 1. introduction green herbs are rich with phytonutrients [1, 2]. the availability and universalism of leafy greens usage stimulates the high consumer demand all around the year [3, 4]. widespread commercialization of freshculinary herbs has been restricted due to high perishability and arelatively short shelf-life [5]. however, there is a need for the effective storage of herbs in order to maintain uninterrupted supply of these products to the consumers. there is a large number of scientific works devoted to the problem of greenery storage due to the complexity of request [6, 7]. the greatest problem in the storage of culinary herbs and leafy greens is the large surface of evaporation, which leads to the rapid loss of mass [6, 8]. moisture losses in vegetables during storage affect negatively the normal course of metabolic processes. as a result of dehydration, reduction of turgor and texture changes occur [9]. the naturally rapid metabolism of fresh herbs and leafy greens accelerates during the post-harvest period, leading to the quick loss of marketable quality [6]. most often, the problem of mass loss is solved by packing greenery in polymer materials [9–11]. perforated film allows reducing weight losses and the intensity of http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olesia priss, viktoria yevlash, technology of fresh herbs storage using hydrogel and antioxidant composition, food and environment safety, volume xvi, issue 4 – 2017, pag. 256 – 261 257 breathing, also prolonging the shelf life. however, the use of film materials often does not solve the problem of products yellowing. approach of storagethat relies on usage of hydrogel-and-antioxidant-based nutrient mediumcontributes positively to the preservation of marketable quality and biological value of green herbs [12]. method of parsley storage using nutrient medium based on the agrarian hydrogel and antioxidants allows extending shelf life of greens without any significant loss of quality and biological value [13]. 2. materials and methods the research was conducted in 2013–2016. for storage greens of parsley (oscar cultivar), dill (allihator cultivar) and spinach (matador cultivar) were chosen. greenery was grown in the south of the region of zaporizhzhia in the open ground. herbs collected in the second decade of september were used for the study. greens were cut in the morning in dry, clear weather. the parsley and dill greens with stems of at least 10 cm length were harvested for storage. the spinach with at least 2cm long peas was selected for experiment. the greens were packed in bundles of 120–150g and put in sticks in 80 × 30 mm polyethylene bags pre-filled with nutrient solutions of hydrogel and antioxidants. hydrogel isa special polymer in granules, which can absorb up to 250 times more moisture than their own weight, and then release it to the plants as the necessity arises. the composition with antioxidant activity was injected to the solution of hydrogel in order to prevent the loss of nutrients in greenery [12]. the antioxidant composition contains butylated hydroxytoluen, a highly active synthetic antioxidant common in food industry, lecithin, a natural antioxidant and emulsifier, and chlorophyllin, an extraction from eucalypt leaves that contains chlorophylls a and b andpossesses antiseptic properties. such packed bundles were put into boxes lined with a polyethylene film (thickness of 60 microns). storage temperature of dill and parsley greens was 1 ± 0.5 °с and 0 ± 0.5 °с for spinach. relative humidity was held at 95 ± 3%. the herbs that were kept in the refrigerator under the same conditions were taken as control. the output of marketable product after storage was determined in accordance with the requirements of the state standards of ukraine and recommendations from researches on the storage and processing of crop production [14]. determination of pigment content. the contents of chlorophyll and carotenoids were determined spectrophotometrically – at 644 and 662 nm for chlorophyll, and additionally at 440.5 nm for determination of carotenoids – from an extract in acetone [15]. for this study, samples of 150– 200 mg were rubbed with mgco3 and 5 cm3 of 100% acetone.the extract was poured through a glass pore filter into a bunsen flask, quantitatively transferred to a volumetric flask of 25 cm3 and adjusted to the mark with pure acetone. the obtained acetone extract contained the mixture of green and yellow pigments. the optical density (d) of the mixture was quantified at three wavelengths –662, 644 and 440.5 nm –with the spectrophotometer. the concentration of pigments derived from the measurements with following equations: x(chla) = 9.784 d662 – 0.990 d644; x(chlb) = 21.426 d644 – 4.650 d662; x(chla+chlb)=5.134 d662 + 20.436 d644; x(carotenoids)=4.695 d440.5 –0.268 x(chla+chlb). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olesia priss, viktoria yevlash, technology of fresh herbs storage using hydrogel and antioxidant composition, food and environment safety, volume xvi, issue 4 – 2017, pag. 256 – 261 258 the content of pigments (a) in the plant material was calculated by the formula: а = x·v/н·100, where x is the pigments concentration, mg·dm-3; v – the volume of extract, ml (25 cm3); h – the sample of plant material, g (0.1–0.2 g). determination of vitamin c. the iodometric method was used to determine the vitamin c content [16]. the method is based on the measurement of reducing ability of ascorbic acid. specifically, ascorbic acid reduces the potassium iodide to free iodine while oxidation. this iodine concentration is then determined by the colour reactionwith starch. the samples (2.0–10.0 g) were weighed and levigated with a small amount of water in a porcelain mortar. the substance then was quantitatively transferred to a volumetric flask of 100 cm3, adjusted to full volume and filtered. for titration 1 cm3 of 2% hcl, 0.5 cm3 of 1% potassium iodine solution, 2 cm3 of 0.5% starch solution and 6–7 cm3 of distilled water were added to 10 cm3 of the extract. titration with 0.01 n solution of potassium iodide was conducted until the stable blue colour appeared. a control titration containing 10 cm3 of water instead of the extract was conducted simultaneously. vitamin c content was quantified basing on the volume of 0.01 n potassium iodide added, with a conversion factor of 0.8806 mg/cm3. all determinations were executed three times and then the mean values of all results were calculated. 3. results and discussion duration of green herbs storage fluctuated slightly throughout years of research and reached 20…40 days in average depending on the species (table 1). using a nutrient medium with antioxidants allows extending the shelf life of the greenery for more than twice (to prolong it at least for 30 days). such results can be achieved first of all due to the absence of fading. in studied variants of greens the reduction of mass losses down to 4...4.5-fold in dill and spinach, and to 40-foldin parsley is observed (table 2). the quality of products stored with the use of hydrogel and antioxidants remained fairly high while the shelf life was extended. thus, at the end of storage period, the output of standard production reached 88.67...93.65% depending on the type of herb (natural loss of mass was taken into account (table 3). table1 green herbs storage duration, days herb control with nutrients parsley 40 80 dill 18 48 spinach 22 55 table2 natural weight loss(n=5) herb experiment lossofmass, %, м ± m parsleyafter40 da ys control 15.98±1.49 withnutrient s 0.35±0.21 dill after18 days control 25.32±2.09 with nutrients 6.33±0.76 spinach after 22 days control 16.44±1.28 with nutrients 3.59±0.46 table 3 quantity of standard products (n=5) herb experiment marketable quality, %, м ± m parsley control 80.84±1.06 with nutrients 93.65±0.44 dill control 69.30±1.10 with nutrients 88.67±1.05 spinach control 79.98±1.07 with nutrients 90.65±1.01 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olesia priss, viktoria yevlash, technology of fresh herbs storage using hydrogel and antioxidant composition, food and environment safety, volume xvi, issue 4 – 2017, pag. 256 – 261 259 such a high yield of standard production is provided not only by the reduction of mass losses, but also by the reduction of the amount of yellowed herbs. transformation of pigment singreenery is one of the markers of metabolic processes during its storage –amount of chlorophylls and carotenoids decreases during storage. decomposition of chlorophylls and carotenoids was slower in leafy greens, which were stored with a nutrient medium based on hydrogel and antioxidants. the content of chlorophylls in the control groups in the end of storage decreased by15...40% of the initial amount. in contrast, the content of chlorophylls in greenery, which was stored using hydrogel and antioxidants, was 15...52% higher than in control group (fig. 1, 2, 3). the proposed method allows maintaining a higher content of vitamin c (fig. 4-6). content of vitamin c was gradually decreasing during storage. in samples that were stored without usage of hydrogel ascorbic acid was decomposing more intensively and at the end of storage its content dropped to 17...72 % from the initial amount depending on the variety. fig. 1. changes of chlorophylls content inparsley during storage: 1 before storage; 2 control group after 40 days; 3 greenery in nutrition medium after 40 days; 4 greenery in nutrition medium after 80 days. fig. 2. changes of chlorophylls content indill during storage: 1 before storage; 2 control group after 18 days; 3 greenery in nutrition medium after 18 days; 4 greenery in nutrition medium after 48 days. fig. 3. changes of chlorophylls contentinspinach during storage: 1 -beforestorage; 2 control group after 22 days; 3 greeneryin nutritionmedium after 22 days; 4 greeneryin nutritionmedium after 55 days. the vitamin c amount was 13.7...40.1% higher in the experimental specimen with the addition of antioxidant compositions than in control. fig. 4. changes ofvitamin c content in parsley during storage: 1 before storage; 2 control group after 40 days; 3 greenery in nutrition medium after 40 days; 4 greenery in nutrition medium after 80 days. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olesia priss, viktoria yevlash, technology of fresh herbs storage using hydrogel and antioxidant composition, food and environment safety, volume xvi, issue 4 – 2017, pag. 256 – 261 260 fig. 5. changes ofvitamin c contentindill during storage: 1 before storage; 2 control group after 18 days; 3 greenery in nutrition medium after 18 days; 4 greenery in nutrition medium after 48 days. fig. 6. changes of vitamin c contentinspinach during storage: 1 before storage; 2 control group after 22 days; 3 greenery in nutrition medium after 22 days; 4 greenery in nutrition medium after 55 days. 4. conclusions hydrogel and antioxidants used in storage procedure can extend the shelf life of greenery for 48-80 days, and allow increasing the yield of marketable production to 80...90% after greenery storage. this method of storage allows stabilization of the biological value of greenery. level of preservation in experimental variants at the end of storage is higher than that in control groups for 2.2...70.7 % for vitamin c and for3.7... 15.5% for chlorophylls. 5. references [1]. chohan m., forster-wilkins g., opara e. i., determination of the antioxidant capacity of culinary herbs subjected to various cooking and storage processes using the abts*+ radical cation assay, plant foods for human nutrition, 63(2): 47–52, (2008). [2]. peter k. v. (ed.), handbook of herbs and spices, elsevier, isbn: 978-0-85709-040-9 (2012). [3]. francis g.a., gallone a., nychas g.j., sofos j.n., colelli g., amodio m. l. et al., factors affecting quality and safety of fresh-cut produce, critical reviews in food science and nutrition, 52(7): 595-610 (2012). [4]. charles d. j., antioxidant properties of spices, herbs and other sources, new york: springer, isbn13 9781461443094 (2013). [5]. cantwell m.i., reid m.s., postharvest physiology and handling of fresh culinary herbs, journal of herbs, spices & medicinal plants, 1(3): 93-127, (1993). [6]. aharoni n., dvir o., chalupowicz d., aharon z., coping with postharvest physiology of fresh culinary herbs,in international symposium on medicinal and aromatic plants 344: 69–78 (march 1993). [7]. ihab m. hekal, chaochen, xiaolingdong, methods for preserving fresh produce, us patent 7851002 b2(2010). [8]. martin-diana a.b., rico d., barry-ryan c., frias j. m., mulcahy j., henehan g.t., calcium lactate washing treatments for salad-cut iceberg lettuce: effect of temperature and concentration on quality retention parameters, food research international, 38(7): 729–740, (2005). [9]. caleb o., mahajan p., al-said f., opara u., modified atmosphere packaging technology of fresh and fresh-cut produce and the microbial consequences: a review, food and bioprocess technology, 6(2): 303–329, (2013). [10]. brar j.k., rai d.r., singh a., kaur n., biochemical and physiological changes in fenugreek (trigonellafoenum-graecum l.) leaves during storage under modified atmosphere packaging, journal of food science and technology, 50(4): 696–704, (2013). [11]. sakaldaş m., aslim a. ş., kuzucu c. ö., kaynaş k., the effects of modified atmosphere packaging and storage temperature on quality and biochemical properties of dill (anethumgraveolens) leaves, journal of food, agriculture & environment, 8(3&4): 21-25, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olesia priss, viktoria yevlash, technology of fresh herbs storage using hydrogel and antioxidant composition, food and environment safety, volume xvi, issue 4 – 2017, pag. 256 – 261 261 [12]. kalytka v.v., priss o. p., kulyk a.s., zhukova v.f., method of preparation of green vegetable storage, uа patent 85031 u201305153; appl. 22.04.2013; publ. 11.11.2013, vol. 21 (2013). [13]. kulyk a.s., improving storage technologies of parsley with using of agrarian hydrogel and antioxidant composition, doctoral thesis, kyiv, 22 (2016). [14]. skaletska l. f., podpryatov g.i., zavadska a.v., introduction to the scientific research of storage and processing of plant products, nau, kyiv, 202 p.(2006). [15]. musienko m.m., parshikova t.v., slavniy p.s., spectrophotometric methods in practice, physiology, biochemistry and ecology of plants, fitosotssotsentr, kyiv, 200 р.(2001). [16]. najchenko v.m., practice recommendations on technology of storage and processing of fruits and vegetables with the basics of commodity, fada ltd, kyiv, 211 р. (2001). technology of fresh herbs storage using hydrogel and antioxidant composition *corresponding author 1. introduction microsoft word fia journal no 1_2011_23 martie_32-38.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 36 predicting the organoleptic quality of some romanian beers from physical-chemical data using multivariate analysis georgiana gabriela codină1, silvia mironeasa1, ana leahu1 1„stefan cel mare” university, faculty of food engineering, 13th university street, 720229, suceava romania, e-mail address: codina@usv.ro; silviam@usv.ro; leahu@usv.ro abstract: two categories of pale lager beer produced in romania, of which twelve samples are common pale lager beer type and eight samples are superior pale lager beer type were studied to evaluate their quality through physical-chemical (apparent extract, original extract, alcohol concentration, ph, turbidity, viscosity, colour, acidity, bitterness value) and sensorial (appearance, colour, odour, taste, gas release, foam stability) characteristics. beer characteristics measurements were examined by multivariate data analysis, using principal component analysis (pca). the analysis of the principal components sheds light on the correlations between variables as well as on differences between the samples analyzed. the values of correlation coefficients indicate important direct statistical relations between the variables taste and alcohol concentration for all the 20 samples of beer analyzed. for the samples belonging to the category of common pale lager beer type, the values of correlation coefficients show that there are reverse statistical connections between the bitterness value and characteristics used in the sensorial analysis (p  0.05). for this type of beer, skol was mostly appreciated, while for the superior pale lager beer category ursus premium was considered the best, both types of beer being regarded as very good quality beers according to the sensorial evaluation methodology described by sr 13355-1:1997. keywords: principal component analysis, physical-chemical parameters, sensory property, pale beer introduction the quality of beer depends mostly on the composition and quality of raw materials used [1]. its chemical composition, brewing and technological process are highly determinative for the beer quality which embodies both its sensorial characteristics and physical chemical ones. brewers focus primarily on chemical and physical properties as percentage of original wort, amount of alcohol, degree of fermentation, ph-level, degree of bitterness, colour, etc., to compare various sorts of beer. chemical and physical methods of analysis can provide a great deal of information about beer quality but are unable to render it with the greatest precision. this is mainly due to the fact that the consumer is not interested in the beer composition but in its sensorial characteristics as for they the latin saying “de gustibus non est disputandum” works for the best in this situation. for this reason, most large-scale breweries used different sensory methods of quality between different types of beers to underline certain differences of quality between various types of beer. most of the interest has shifted to factors affecting the changes in beer aroma and taste, as beer flavour is regarded as the most important quality parameter of the product [3]. beer sensorial parameters are evaluated in many ways. in romania the most important one is quality assessment using a 20-points system according to sr 13355-1:1997 where beer is appreciated on certain qualities with regards to its comparative food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 37 ranking and value. since a beer sample is characterized by many different chemical, physical and sensory parameters (variables) it is difficult to obtain a comprehensive overview of its quality. therefore, it might be useful to reduce the number of variables to describe beer. therefore, the main objective of this study is to use a multivariate statistical method, namely principal component analysis (pca) [4], to analyze the relationships between physical, chemical and sensory properties of two types of beer (common pale lager beer and superior pale lager beer) both produced in romania. the pca method makes it possible to distinguish beer samples and also to identify the most important variables in a multivariate data matrix. materials and methods beers. twenty beers from the romanian market were chosen for this study. they represented beer samples easily available to consumers in the off-premise chain. they covered according to sr 4230: 2004 two categories of beer: common pale lager beer (twelve samples) and superior pale lager beer (eight samples). the commercially available beers included in the study are listed in table 1. sample abbreviations used in biplots, tables and text are also reported. table 1. identification of the 20 beers studied common pale lager beer abbreviated name superior pale lager beer abbreviated name skol skol bergenbier bergenbier bucegi bucegi azuga azuga ciucas ciucas ursus premium ursus_p harghita harghita golden brau golden_b noroc noroc timisoreana timisoreana senior senior ursus ursus coroana moldovei coroana_m burger burger coroana premium pils coroana_pp bermas bermas zimbru premium pils zimbru_pp zimbru zimbru caimani caimani suceava suceava physical and chemical analysis. the physico -chemical parameters of beer were determined according to the romanian standard methods at s.c. bermas s.a. suceava: apparent extract (sr 133555:2005), original extract (sr 13355-5:2005), alcohol concentration (sr 13355-3:2005), ph (stas 6182/72), colour (sr 13355-7:2005), acidity (sr 13355-6:2000) bitterness value (sr 13355-9:2003), turbidity (ebc method) and viscosity (ebc method). all analytical determinations were performed at least in triplicate. the values of different parameters were expressed as the mean. sensory analysis. sensory testing has been carried out, in conformity with the method described by sr 13355-1:1997 which evaluates the organoleptic properties of beer, using a 20-points scale system. a panel of 15 judges consisting of students and staff from “stefan cel mare” university, faculty of food engineering were selected on the basis of interest and availability. the judges evaluated the 20 commercial beers in duplicate in 14 sessions over a two-week period. at each session, three beers chosen from the two categories of beers were presented in coded according to sr iso 6658:2007, standard, tulip-shaped, clear 330 ml beer glasses. all beers were evaluated at 12-140c in the sensory laboratory of food engineering faculty, suceava. a 50 ml of each beer sample was poured in glasses, covered with a watch glass food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 38 to preserve volatile compounds and presented with nearly the same level of foam. panelists received distilled water for palate cleansing between samples and dry unsalted breadsticks to avoid cross-contamination among samples. appearance, colour, smell, taste, gas release, foam stability and overall impression were tested. each organoleptic property was evaluated using a 5-point scale. based on their evaluation and on the weight factor (appearance 0.6; colour 0.8; smell 0.2; taste 1.4; gas release 0.6; foam stability 0.4) we have obtained the total score of beer which placed beer on different levels of quality as follwos: 18.1 ÷ 20 – very good beer; 15.1 ÷ 18 good beer; 12.1 ÷ 15 ordinary beer; 7.1 ÷ 12 poor beer but commercially acceptable; 0 ÷ 7 unacceptable. data analysis. all statistical analyses were performed using statistical package for the social sciences (spss). principal component analysis (pca) was carried out for the physical and chemical data for the set of 20 beers and a pca was also made on the combined sensory and instrumental data. results and discussion physical and chemical analysis. to illustrate the physical-chemical characteristics of the sample set, the mean, range, and variation coefficient are given in table 2 and table 3 respectively. the quality of beer samples complied with the romanian standard for pale lager beer products of common and superior type. all values given are means of three determinations. table 2 physico-chemical characteristics of common pale lager beer samples range physical-chemical parameters abbreviated name mean min max cv % apparent extract, op ae 1.7 0.1 2.3 33.37 original extract, op oe 9.91 6.67 10.67 10.82 alcohol concentration, % (v/v) alc 4.15 3.3 4.6 10.70 ph ph 4.36 3.88 4.57 4.70 turbidity, ebc units tu 0.53 0.3 0.75 27.71 viscosity, mpa·s vi 1.37 1.18 1.5 6.12 color, ebc co 6.58 5.0 9.6 21.70 acidity, ml naoh 1n/100 ml beer ac 1.79 1.5 2.5 19.54 bitterness value, be bv 19 16.5 23 9.38 table 3 physico-chemical characteristics of superior pale lager beer samples range physical-chemical parameters abbreviated name mean min max cv % apparent extract, op ae 1.8 1.5 2.1 11.50 original extract, op oe 10.87 10.42 11.52 3.45 alcohol concentration, % (v/v) alc 4.68 4.3 5.2 6.97 ph ph 4.40 4.21 4.6 3.27 turbidity, unit. ebc tu 0.45 0.2 0.63 30.98 viscosity, mpa·s vi 1.37 1.3 1.42 2.97 colour, ebc co 7.28 4.2 8.7 21.42 acidity, ml naoh 1n/100 ml beer ac 1.82 1.4 2.7 21.86 bitterness value, be bv 21.5 18 23.5 9.04 different quantities of alcohol for various sorts of beer analyzed result from the chemical reaction of transforming sugar into alcohol, carbon dioxide and water. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 39 the higher the quantity of fermented wort, the higher the quantity of alcohol is. therefore, there is a direct correlation between the alcohol content, apparent extract and original extract of the samples analyzed. thus, the correlation factors r = 0.791 (for common pale lager beer) and r = 0.946 (for superior pale lager beer), significant for p = 0.01 between alcohol and the original extract, show a very strong association. a significant correlation factor between the original and apparent extract was obtained r = 0.786 at a level p = 0.01, for all 20 beer samples analyzed. for the first type of beer analyzed (common pale lager beer), the variation limits of original extract content, apparent extract and alcohol content are shown in table 2, while for the second type of beer analyzed (superior pale beer), in table 3. there is a direct connection between beer acidity and ph. these variables depend on respecting a proper hygiene throughout the technologic process, starting from brewing and ending with bottling. the variation limits of acidity and ph of the samples of common pale lager beer and those of superior pale lager beer are shown in tables 2 and 3 respectively. beer colour has values varying between 5 and 9.6 ebc for common pale lager beer and between 4.2 and 8.7 ebc for superior pale lager beer. its value depends on various factors such as: raw material quality (barley used for making malt), the quality of malt produced, brewing process, pasteurizing process etc. bitterness value depends on the quality of the hops used and quantity of its α-acids. for the analyzed types, there are variations in values probably due to the fact that different types of hops are used. the samples analyzed have shown bitterness values that vary between 16.5 and 23.5 units be. sensory characteristics. the variation limits of the organoleptic characteristics obtained in the analysis of common pale lager beer samples are given in table 4 and those for superior pale lager beer samples in table 5. table 4 the sensorial characteristics for common pale lager beer samples range sensorial characteristics abbreviated name min max appearance a_p 3 5 colour c_o 3 4.9 odour o_d 3.8 5 taste t_a 2.8 4.9 gas release g_r 3.8 5 foam stability f_s 3.2 5 table 5 sensorial characteristics for superior pale lager beer samples range sensorial characteristics abbreviated name min max appearance a_p 3.3 4.8 colour c_o 3.4 4.8 odour o_d 3.7 5 taste t_a 2.8 4.8 gas release g_r 4.1 5 foam stability f_s 3.9 5 for the first category of beer analyzed (common pale lager beer), the largest score was registered in skol beer (19.7 points), followed by ciucas, calimani (beers 18.6 points) and harghita (18.3 points). these types of beer have been regarded as very food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 40 good. at a lower level (the good beer category) the analysis has placed suceava, coroana premium pils, coroana moldovei, zimbru premium pils, coroana moldovei, and zimbru premium pils. the rest of the sorts of beer analyzed have been placed in the category of ordinary beer (12.1 ÷ 15 points). for the second category of beer analyzed (superior pale lager beer), the highest score was registered by ursus premium (19.3 points), which was the only one to be placed in the category of a very good beer. timisoreana, bergenbier, bermas, golden brau, ursus have been placed in the category of good beer, while burger and azuga were regarded as ordinary beers. sensory attributes combined with physical and chemical parameters. the correlation between the results of sensory testing and physico-chemical parameters obtained for common pale lager beer samples are represented in figure 1a. the first two principal components explain 99.41% of the total variance (pc1 = 97.87% and pc2 = 1.54%). in respect to the first principal component pc1, one can notice that there is a very good correlation between the beers skol, coroana_m, harghita, coroana_pp, calimani, senior, ciucas and suceava. these beers are strongly associated with the first component pc1. the second principal component, pc2 is strongly correlated to the quality of zimbru beer which is characterized by high value of the original extract (oe). pca loadings of the physico-chemical parameters and variables used in sensorial quality evaluation are represented in figure 1b. the two plots represented here have 51.92% and 26.17% of the total variance. (a) (b) figure 1a. principal component analysis of the combined physico-chemical and sensory parameters from common pale lager beer samples; the 12 common pale lager beers, with their commercial names being plotted on the first two principal components. figure 1b. principal component analysis of the combined chemical and sensory data from superior pale lager beer samples: the 9 chemical and 6 sensorial parameters and panel score range are plotted on the first two principal components the first component pc1 underlines the difference between bitterness value (bv) and appearance (a_p). the closeness of the acidity variable to the centre of pc shows that it is not useful to describe the differences between the 12 samples from the category of common pale lager beer. the second component pc2 distinguishes the variables apparent extract (ae) and turbidity (tu), which are opposed. weighted heavily on the second pc was apparent extract (ae). as regards the second main component pc2, the parameters bitterness value (bv), colour (co), apparent extract (ae) and turbidity (tu) are placed at the left of graph showing that these contribute to a larger extent in the evaluation of beers in comparison with the variables at the right. the plot of pc1 vs. pc2 loadings food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 41 shows, alongside with the pc1 component, a close association between gas release (g_r), foam stability (f_s), odour (o_d), appearance (a_p), colour (c_o) and taste (t_a) which reflect highly significant correlation coefficients. the variables mentioned indicate a strong correlation with this component, which can be characterized as a function of the organoleptic characteristics. these characteristics have an essential role in the appreciation of common pale lager beer samples skol, coroana_m, harghita, coroana_pp, calimani, senior, ciucas and suceava, figure 2a. (a) (b) figure 2a. principal component analysis of the combined physico-chemical and sensory parameters from superior pale lager beer samples; the 8 superior blonde beer, with their commercial names are plotted on the first two principal components figure 2b. principal component analysis of the combined chemical and sensory data from superior pale lager beer samples: the 9 chemical and 6 sensorial parameter and panel score range are plotted on the first two principal components there is a significant correlation between the original extract variable (oe) and viscosity (vi) (r = 0.790), (r = 0.769), alcohol concentration (alc) (r = 0.791), which are important coefficients for a level p = 0.01. these variables have a major role in the evaluation of beers such as zimbru, zimbru_pp, noroc and bucegi. the placement of zimbru, zimbru_pp, and noroc in the space of principal components shows that these beers have a high level of original extract (o_e). for all the samples in the category of common pale lager beer, significant reverse correlations have been obtained between bitterness value and taste (r = 0.660), odour (r = 0.697), gas release (r = 0.620) and foam stability (r = -0.671), for a level of 0.05. the correlation between the results of sensory testing and physico-chemical parameters obtained for the 8 beer samples from the first category are represented in figure 2a. the first two principal components explain 99.48% of the total variance (pc1 = 97.16% and pc2=2.32%). as regards the principal component pc1, very good correlation between the beers burger, azuga, timişoara and bermas can be noticed. these brands are closely associated with the first principal component. the second principal component is strongly correlated to ursus beer, which is characterized by high value of variable original extract (oe). pca loadings of the physicochemical parameters and variables used in the sensorial evaluation are represented in figure 2b. the two plots represented here 49.69% and 34.10% of the total variance. the first component pc1 underlines the opposition between turbidity (tu) and colour (co). closeness of viscosity (vi) to the centre of pc shows that this variable is not useful to describe the differences between beers. as for the second component pc2, the original extract parameter (oe) is placed the highest while food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year x, no1 2011 42 the parameter bitterness value (bv) is placed the lowest. the position of original extract in the space of main components is associated with a high level of original extract (oe) for ursus beer. the characteristics that contribute to a large extent in the evaluation of beers are the following parameters: alcohol concentration (aic), turbidity (tu), appearance (a_p), foam stability (f_s) and taste (t_a). conclusions principal component analysis of the physico-chemical and sensorial data revealed the first pc to be a function of sensorial data and the second pc was a function of physico chemical parameters. all the beers clustered together by those physico-chemical analyses have been identified as being related to sensorial attributes. when principal component analysis was performed on the combined sensory and physico-chemical data, the clustering of 20 beers irrespective of the beer type was also seen in the pc space. the sensorial characteristics used in the organoleptic analysis appearance, colour, odour, taste, gas release and foam stability have a decisive role in identifying samples from the category of common pale lager beer. to identify samples of the superior pale lager beer category, the following characteristics such as appearance, foam stability and taste had a major contribution. regarding the correlations between physico-chemical parameters and sensorial characteristics for all the samples analyzed, irrespective of the category they belong to, a direct significant correlation was obtained between alcohol concentration and taste. for all the samples in the category of common ale beer, significant reverse correlations were obtained between bitterness value and almost all the sensorial characteristics used: taste, odour, gas release and foam stability. sensory evaluation of the tested types of beer could reveal that physico-chemical parameters are responsible for sensorial attributes and important interaction occuring among these parameters. acknowledgements the authors thank s.c. bermas s.a. for technical support. references 1. yonkova, g., georgieva, n., ginova, t. and terzi, a., biochemical processes by mashing and characterization of the fermentation of feed barley during brewing, journal of the university of chemical technology and metallurgy, 2007, 42 (4), 407-412. 2. banu, c. (ed.), the science and technology of malt and beer, bucureşti, agir press, 2000. 3. bart, v., hedwing, n., verachtert, h. and derdelinckx, g., the chemistry of beer aging – a critical review, food chemistry, 2006, 95 (3), 357-381. 4. gacula, m.c. (ed.), descriptive sensory analysis in practice, u.s.a., food&nutrition press, 1997. 5. sr 13355-1:1997, beer. analysis methods. sensorial analysis, standardization association of romania (asro), bucharest, romania. 6. sr 4230:2004, beer, standardization association of romania (asro), bucharest, romania. 7. sr 13355-6:2000, beer. analysis methods. determination of total acidity, standardization association of romania, bucharest, romania. 8. sr 13355-7:2005, beer. analysis methods. color determination, standardization association of romania, bucharest, romania. 9. sr 13355-9:2003, beer. analysis methods. determination of bitterness of beer, standardization association of romania, bucharest, romania. 10. sr 13355-3:2005, beer. analysis methods. determination of alcoholic concentration, standardization association of romania, bucharest, romania. 11. sr 13355-5:2005, beer. analysis methods. extract determination, standardization association of romania, bucharest, romania. 12. sr iso 6658, sensory analyses-methodologygeneral guidance, standardization association of romania, bucharest, romania. 13. sr iso 11035, sensory analyses-identification and selection of descriptors for establishing a sensory profile by a multidimensional approach, standardization association of romania, bucharest, romania. microsoft word 3 journal nr 4 2010 din 8 noiembrie_51-56.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 55 study on free radical scavenging and total polyphenols of some romanian wines maria cioroi1, lucian tudor miron1, gabriela rapeanu1, nicoleta stanciuc1, elena postolache1, constanta vicol1 1dunarea de jos university, 111 domneasca street, 800201, galati maria.cioroi@ugal.ro abstract: phenolic compounds contribute to organoleptic characteristics such as colour, astringency and bitterness of grapes and wines. some relevant analytical parameters of the wines produced by r & d station for viticulture and wine bujoru and regional laboratory for quality control and hygiene wine odobesti, are given in this paper. free radical scavenging activity was determined for several red and white wines from different grape varieties cultivated in the south-east of romania vineyards region. the free radical scavenging activity of the wine samples was analysed by using the 2,2,-diphenyl-1-picrylhydrazyl (dpph) assay. the absorbance was read at 515 nm. the amount of polyphenols in selected wines was investigated by means of uv–vis methods according to the folin–ciocalteu colorimetric method. the mean concentration of total polyphenols (tps) content of the analyzed red wines was 639.5205 mg/l gallic acid equivalent and for the white wines was 180.5233 mg/l gallic acid equivalent. the investigated red wines showed antioxidant behaviour in the range of 89.85% to 76.60%. the free radical scavenging activity of the wines was correlated to total polyphenol compounds content. the polyphenolic compounds content in the investigated wines contributes to their antioxidant activity. the hypothesis of a protective effect of red wine is supported by obtained results which show that the red wines with higher amounts of polyphenols possess higher antioxidant properties. keyword: antioxidant capacity, dpph, gallic acid, red wines, white wines, tg bujor, odobesti introduction polyphenols are an important group of secondary plant compounds. they play an important role in the plant itself as protective agents against fungus attack and uv irradiation. according to several epidemiological studies they have positive effects on human health: they decrease the incidence of coronary heart disease, reduce platelet aggregation, and provide antioxidative and anti-carcinogenic protection [1, 2]. from a winemaking perspective, polyphenols serve as antioxidants, colouring components (especially anthocyanins in red wine), and contribute to the mouthfeel and bitterness of wine [3, 4]. phenolic compounds represent one of the most important quality parameters of grapes and wines since they contribute to organoleptic characteristics such as colour, astringency and bitterness. recent studies indicate that consumption of a small amount of red wine on a regular basis reduces the risk of coronary heart disease and atherosclerosis, and this benefit is attributed to the antioxidant properties of the polyphenol compounds [5]. several studies suggest that some phenolic compounds, when ingested at high concentrations, may exhibit certain roles in food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 56 carcinogenicity, genotoxicity, thyroid toxicity and can be connected with oestrogenic activity [6, 7]. the aim of this study was to determine the free radical scavenging activity of several wines produced in south-east of romania vineyards and to correlate this with the total polyphenolic content. materials and methods 2.1. chemicals 2,2-diphenyl-1-picrylhydrazyl (dpph) in free radical form was obtained from sigma chemical co. (st. louis, mo). folin– ciocalteu reagent, gallic acid, quercetin and ethanol were purchased from merck co. (germany). all reagents were analytical grade. 2.2. wine samples the wines were produced from different grape varieties grown in the south-east of romania. thirty-three wines were supplied by r&d station for viticulture and wine bujoru and regional laboratory for quality control and hygiene wine odobesti. 2.3. determination of total phenolic content the total phenol content in selected wine samples was determined spectrophotometrically according to the folin–ciocalteu colorimetric method [8] (singleton & rossi, 1965) using gallic acid as a standard polyphenol: 0.1 ml of wine was mixed with 7.9 ml distilled water and 0.5 ml of folin–ciocalteu reagent. after 1 min, 1.5 ml of 20% na2co3 was added. the absorbance was measured after 120 min at 760 nm. the concentration of the total phenolic compounds in the wines was expressed as gallic acid equivalents (mg/l). the results in every assay were obtained from three parallel determinations. 2.4. free radical scavenging activity the free radical scavenging activity of the wine samples was analyzed by using the 2,2,-diphenyl-1-picrylhydrazyl (dpph) assay [9,10,11] the antioxidant assay is based on the measurement of the loss of dpph colour by the change of absorbance at 515 nm caused by the reaction of dpph with the tested sample. the reaction was monitored by a uv/vis spectrophotometer. a volume of 0.1 ml of wine appropriate diluted with distilled water and 3.9 ml of freshly prepared dpph (0.025 g/l) in ethanol was introduced and put into a small vat at room temperature. after 60 min incubation period at room temperature, the absorbance was read as against the blank at 515 nm. the determinations were performed in triplicate. 2.5. statistical analysis all the measurements were carried out in triplicate, and presented as mean values. the direction and magnitude of the correlation between the variables was quantified by the correlation factor r2. the correlations were considered statistically significant, if the p-value was less than 0.05. results and discussion 1. general characterizations of wines some relevant analytical parameters of the wines produced by r & d station for viticulture and wine bujoru and regional laboratory for quality control and hygiene wine odobesti, are given in table 1. the wines differ in many analytical parameters such as the content of alcohol, total extract, total and free so2, reducing sugar, total acidity (as tartaric acid), volatile acidity (as acetic acid) and specific weight. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 57 table 1. some relevant analytical parameters in some romanian wines wine specific weight (g/cm3) alcohol content (vol%) total acidity (tartaric acid) (g/l) volatile acidity (acetic acid) (g/l) total extract (g/l) reducing sugar (g/l) total so2 (mg/l) free so2 (mg/l babeasca neagra (veritas panciu, 2009) 0.9925 14.73 8.23 2.22 29.6 4.22 103.4 6.63 cabernet sauvignon (vincon vrancea, 2007) 0.9919 14.04 5.27 0.66 26.15 1.73 25.25 4.59 merlot (vincon vrancea, 2007) 0.9916 14.15 6.09 0.66 25.66 1.73 8.04 9.95 babeasca neagra (veritas panciu, 2007) 0.9905 14.92 6.37 0.64 25.0 0.23 28.02 5.34 cabernet sauvignon (murfatlar, 2007) 0.9932 12.18 4.57 0.59 23.9 0.72 60.59 6.63 pinot gris (veritas panciu, 2007) 0.9885 15.43 5.42 0.54 21.4 0.14 69.66 5.36 cabernet sauvignon (vincon vrancea, 2009) 0.9908 15.48 5.79 0.80 27.5 0.76 160.3 19.38 cabernet sauvignon (vincon vrancea, 2008) 0.9955 12.53 10.02 0.56 31.0 1.11 20.41 5.10 pinot gris (veritas panciu, 2009) 0.9892 16.60 5.5 1.20 37.7 11.48 191.75 7.14 merlot (vincon vrancea, 2009) 0.9900 14.88 6.09 4.47 23.7 1.21 321.7 23.46 babeasca neagra (veritas panciu, 2008) 0.9891 15.01 6.14 0.58 21.6 0.16 19.13 4.08 merlot (vincon vrancea, 2008) 0.9929 12.45 10.82 4.47 24.1 0.36 135.99 10.71 pinot gris (veritas panciu, 2008) 0.9892 14.23 5.42 1.13 19.8 0.16 33.1 5.61 cabernet sauvignon (murfatlar, 2008) 0.9936 11.28 7.56 1.8 22.3 0.1 37.42 3.83 aligote (vin club, 2008) 0.9889 13.01 4.41 0.54 15.2 0.15 66.74 3.83 galbena de odobesti (scdvv, 2008) 0.9909 12.10 7.56 1.06 17.8 0.21 80.27 4.59 cabernet sauvignon (vincon vrancea, 2009) 0.9953 12.23 6.52 3.64 29.6 0.76 160.3 23.97 muscat ottonel – 2009 dealurile bujorului 0.9892 13.0 5.5 0.39 23.8 14.0 84 20 băbească gri -2009 dealurile bujorului 0.9898 10.3 5.9 0.60 23.3 34.0 248 55 băbească neagră – 2009 dealurile bujorului 0.9892 14.2 6.1 0.54 23.4 3.8 128 35 merlot 2009 dealurile bujorului 0.9968 14.1 6.3 0.60 28.9 2.9 76 29 fetească neagră2008 dealurile bujorului 0.9890 11.9 6.4 0.58 28.4 2.6 96 22 cabernet sauvignon – 2008, dealurile bujorului 0.9889 12.2 5.8 0.45 25.2 1.6 85 30 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 58 2. total polyphenols the concentration of the total phenolic compounds in the wines was expressed as gallic acid equivalents (mg/l). the equation of standard curve was y = 0.4317x – 0.024 and r2 = 0.9934. the total polyphenols content of selected wines, obtained using the folin–ciocalteu reagent, is presented in table 2. the mean concentration of the total polyphenols (tps) content of the red wines was 639.5205 mg/l gallic acid equivalent. the total polyphenols content on the white wines was 180.5233 mg/l gallic acid equivalent. table 2. the total polyphenols content of selected wines, obtained using the folin–ciocalteu reagent no wines tp (mg gallic acid equivalent/l) 1. babeasca neagra (veritas panciu, 2009) 448.5692 2. cabernet sauvignon (vincon vrancea, 2007) 616.396 3. merlot (vincon vrancea, 2007) 845.321 4. aligote (vin club, 2007) 160.8662 5. babeasca neagra (veritas panciu, 2007) 256.7672 6. cabernet sauvignon (murfatlar, 2007) 517.4014 7. pinot gris (veritas panciu, 2007) 409.8995 8. cabernet sauvignon (vincon vrancea, 2009) 696.0557 9. cabernet sauvignon (vincon vrancea, 2008) 544.4702 10. pinot gris (veritas panciu, 2009) 372.0031 11. merlot (vincon vrancea, 2009) 620.263 12. babeasca neagra (veritas panciu, 2008) 353.4416 13. feteasca regala (vin club, 2009) 252.9002 14. merlot (vincon vrancea, 2008) 501.9335 15. pinot gris (veritas panciu, 2008) 516.628 16. cabernet sauvignon (murfatlar, 2008) 623.3565 17. feteasca regala (vin club, 2008) 366.5893 18. aligote (vin club, 2008) 146.9451 19. galbena de odobesti (scdvv, 2008) 208.0433 20. feteasca regala (vin club,2007) 160.8662 21. galbena de odobesti (scdvv, 2007) 343.3875 22. cabernet sauvignon (vincon vrancea, 2009) 583.9134 23. aligote (vin club, 2009) 137.6643 24. galbena de odobesti (scdvv,2009) 126.0634 25. sarba (dealurile bujorului, 2009) 600.1547 26. muscat ottonel (dealurile bujorului, 2009) 209.5901 27. babeasca gri (dealurile bujorului, 2009) 344.9343 28. babeasca neagra (dealurile bujorului, 2009) 478.7316 29. feteasca regala (dealurile bujorului, 2009) 397.5251 30. feteasca alba (dealurile bujorului, 2009) 161.6396 31. merlot (dealurile bujorului, 2009) 1474.091 32. feteasca neagra (dealurile bujorului, 2009) 1139.985 33. cabernet sauvignon (dealurile bujorului,2009) 1089.714 the maximum values of pt was 1474.091 mg/l of gallic acid equivalent on the red wine merlot (scdvv bujoru, 2009) and minimum value on galbena de odobesti (scdvv, 2009) was 126.0634 mg/l gae. the total polyphenols content of the red wines samples analyzed is in range of the levels quoted in the literature for wines grown in different countries such as in the czech republic 874–2262 mg/l gae [12], spain 1010–2446 mg/l gae [13]. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 59 the grape phenolic content and also the total polyphenol compounds on wines could be affected by the numerous viticulture and wine-making factors. the phenol composition has an important role in stabilizing lipid oxidation and is associated with antioxidant activity. 3. antioxidant capacity the free radical scavenging activity of the wine samples was analyzed by using the 2.2,-diphenyl-1-picrylhydrazyl (dpph) assay. wine is a complex mixture of polyphenols and the antioxidative capacity can be expressed as the amount of wine needed to quench a certain amount of dpph radical. the decrease in the absorbance at 515 nm is taken as a measure of the extent of radical-scavenging. the inhibition of dpph in percent (i %) of each wine sample was calculated from the decrease of absorbance according to the relationship: 100(%)    ablank asampleablank i where ablank is the absorbance of control reaction (ethanol–water with dpph radical) and asample is the absorbance of the tested wine sample. quercetine was used as standard polyphenol in antioxidant capacity assay. the equation of standard curve was y = 7.0028x – 0.1399 with value r2 = 0.9935. all the wine samples showed a different radical scavenging activity after incubation with a free dpph radical solution. the values of i% for red wines are in the range between 89.85 (pinot gris veritas panciu, 2008) .and 76.6 (merlot bujoru, 2009). also for white wines inhibition (i%) was in the range from 12.96 (galbena de odobesti scdvv, 2009) to 35.29 (galbena of odobesti scdvv, 2008) the percentage of dpph radical scavenging activity was plotted as against the total phenolic content of the wine samples in fig. 1. for the investigated wines, the correlation between antioxidant activity and the total phenolic content was r2 = 0.4781, (sd = 0.025, n = 33). 0 10 20 30 40 50 60 70 80 90 100 0 500 1000 1500 2000 tp(mg/l gae) in hi bi tio n( % ) figure 1. the percentage of dpph radical scavenging activity plotted against total phenolic content of the tested wine samples the polyphenol compounds content in the investigated wines contributes to their antioxidant activity. since structural features of phenolic compounds are responsible for the antioxidant activity, the antioxidant activities of the tested wines may be related to their total phenols content. the different antioxidant activity of tested wines is dependent especially on the grape varieties. conclusion the present work regarding the free radical scavenging and total polyphenols of some romanian wines some general has drawn the following conclusions: food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 60 according to the obtained results, the free radical scavenging ability of romanian wines is correlated to total polyphenol content. high amounts of polyphenols and significant antioxidant activity were observed in the red wines produced from vine varieties from the south-east romanian regions. the significant differences in the antioxidant activity between the analyzed wines were confirmed. it is important to determine which group of phenolic compounds is most significant in antioxidant activities of wines. this aspect will be a subject of our next research activity. acknowledgements the research was supported by the bilateral cooperation: romania and cyprus, acyrowine study on antioxidant and allergens in authentic cypriot and romanian wines (4141/19.05.2010). the authors acknowledge financial support from the national university research council (nurc, pn-ii-id-pce-2008-2, idea, id 517) romania. references 1. de lange, d. w., van golde, p. h., scholman, w. l. g., kraaijenhagen, r. j., akkerman, j. w. n., & van de wiel, a. (2003). red wine and red wine polyphenolic compounds but not alcohol inhibit adp-induced platelet aggregation. european journal of internal medicine, 14, 361–366. 2. stoclet, j. c., chataigneau, t., ndiaye, m., oak, m. h., el bedoui, j., chataigneau, m., & schini-kerth, v. b. (2004). vascular protection by dietary polyphenols. european journal of pharmacology, 500, 299–313. 3. pour nikfardjam, m. s., la´szlo´ , gy., & dietrich, h. (2003). polyphenols and antioxidative capacity in hungarian tokaj wines. mitteilungen klosterneuburg, 53(5-6), 159–165. 4. vidal, s., francis, l., noble, a., kwiatkowski, m., cheynier, v., & waters, e. (2004). taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine. analytica chimica acta, 513, 57–65. 5. youdim, k., mcdonald, j., kalt, w., joseph, j. (2002). potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and antiinflammatory insults. journal of nutritional biochemistry, 13(5), 282–288. 6. lila, m. a. (2004). anthocyanins and humans health: in vitro investigative approach. journal of biomedicine and biotechnology, 5, 306–313. 7. meiers, s., kmeny, d., weyand, u., gastpar, r., von angere, e., marko, d. (2001). the anthocyanidins cyanidin and delphinidin are potent inhibitors of the epidermal growth-factor receptor. journal of agriculture and food chemistry, 49, 958–962. 8. singleton, v. l., rossi, j. a. (1965). colorimetry of total phenolics with phosphor– molybdic–phosphotungstic acid reagents. american journal of enology and viticulture, 16, 144–158. 9. lachman, j., sulc, m., schilla, m. (2007). comparison of the total antioxidant status of bohemian wines during the wine-making process. food chemistry, 103, 802–807. 10. turkoglu, a., duru, m., mecan, n., kivrak, i., gezer, k. (2007). antioxidant and antimicrobial activities od laetiporus sulphurous (bull) murrill. food chemistry, 101, 267–273. 11. villano, d., fernandez-pachon, m. s., troncoso, a. m., & garcia-parrilla, m. c. (2006). influence of enological practices on the antioxidant activity of wines. food chemistry, 95, 394–404. 12. stratil, p., kuban, v., fojtova, j. (2008). comparison of the phenolic content and total antioxidant activity in wines as determined by spectrophotometric methods. czech journal of food sciences, 26(4), 242–253. 13. sanchez-moreno, j., larrauri, j. a., sauracalixto, f. (1999). free radical scavenging capacity of selected red, rose and white wines. journal of agriculture and food chemistry, 79, 1301–1304. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 40 in situ river water treatment with domestic materials using as sorbents g.h. torosyan1, n. gh. poghosyan2, a. k. gukasyan2, v.a. davtyan3, n.s. torosyan4 1 state engineering university of armenia, department of chemical technology & environmental engineering, yerevan, armenia, email: gagiktorosyan@seua.am 2 gegharkunik regional administration ( marzpetaran), gavar, armenia 3 “nairit plant” cjpc, 4 csto institute, yerevan, armenia, yerevan, armenia abstract: argichi river is the deepest river running into the lake sevan. the river waters irrigate areas under crops of more of villages of gegarkunik region in armenia. the river argichi is a habitat for reproduction brought in the red book of fauna of armenia for fishes. hence, for the purpose of maintenance of safety of fauna of the rivers, it is necessary to treated river water. this research includes a situation of sewage in river argichi, type and quantity of polluted materials and products, the attempts to present of the sewage treatment systems. and the development of technology for sorbent with polysorption properties with application of natural materials widespread on territories of armenia. the results of purposeful researches are submitted in the field solutions and waste water of application natural zeolites as sorbents for removal of organic substances from water. high level of adsorption of organic substances natural zeolites is provided with their initial saturation by ions of calcium and magnesia. it has been found advantageous to organic compounds sorption by natural zeolites. the advantages of natural zeolite in comparison with other sorbents are technological stability, low cost, availability, filtering properties. thus, the sequence of operations in sorption will be the following: the first is sorption of metals and ammonia, the second is sorption of organic substances. keywords: the argichi river, sevan, zeolite, modified zeolite, sorption, ammonia, organic substances, introduction the argichi river has been the deepest river running into the lake sevan and remains an important irrigate areas under crops of more of villages of gegarkunik region in armenia. the water situation in river argichi and the type and quantity of polluted materials and products depend from many factor. the argichi waters pollutes with metal ions, organic pollutants as petrol products and pesticides. this study includes waste the attempts to present the wastewater treatment systems. one of the best method for wastewater treatment from pollutants is an application of inorganic and organic adsorbents. current treatment processes usually involve also biological degradation or chemical oxidation of the waste organics. nowadays natural zeolites are focused in applications in the sector of wastewater decontamination, for instance for removal of ammonium from municipal sewages and abatement of radionuclides from effluents of nuclear power plants. there are many reasons for zeolites using in mentioned fields: good selectivity for many toxic cations and harmful compounds , as adsorbents organic compounds. zeolites occur in nature in specific kinds of rocks. zeolite rich rocks are widespread in northern part of armenia, occurring in very extended geological formations. the zeolite types are exclusively clinopilolite in idjevan / northern-east of armenia / and mordenite in shirak / northern-west of armenia/. taking advantage of their high zeolite content, high cation exchange capacity and selectivity many mailto:gagiktorosyan:@seua.am food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 41 agricultural applications of armenian zeolites have been proposed recently. nowadays some of armenian natural zeolites were characterized from the point of view of chemical composition, type of structure and chemical, thermal and radiation resistance. the ammonium in water and wastewater can be toxic to the aquatic life and should be removed to prevent environmental protection. several processes are currently used for the removal of ammonium from aqueous solutions. ion exchange on natural zeolites is among the methods most commonly employed. materials and methods nowadays natural zeolites are focused in applications in the sector of wastewater decontamination, for instance for removal of ammonium from municipal sewages and abatement of radionuclides from effluents of nuclear power plants [1-4]. there are many reasons for zeolites using in mentioned fields: good selectivity for many toxic cations and harmful compounds [ 1-2], as adsorbents organic compounds [ 5-7]. zeolite rich rocks are widespread in northern part of armenia, occurring in very extended geological formations. the zeolite types are exclusively clinopilolite in idjevan / northern-east of armenia / and mordenite in shirak / northern-west of armenia/. taking advantage of their high zeolite content, high cation exchange capacity and selectivity many agricultural applications of armenian zeolites have been proposed recently. nowdays some of armenian natural zeolites were characterized from the point of view of chemical composition, type of structure and chemical, thermal and radiation resistance. armenian natural zeolites mordenite and clinoptilolite were dehydrated at 400oc in vacuum ( 0.1 mm) for 3-4 hours. clinoptilolite and mordenite were prepared by heated pile method, using 0,5 and 1 n solution of caci2. adsorptive isotherms were determined for the equilibrium status of water containing organic compounds in concentrations of 0.0025 to 0.070 mol/l on the zeolite sorbents. adsorption was carried out at a temperature 27.0oc, the average temperature in argichi river around of sevan in spring and autumn. the removal of the pollutants realized for each one separately by two type : 1) the precisely weighed portions of sorbents are brought in to the certain volumes of petroleum substances in water, which initial concentration vary. the mix is carefully shaken up during 6 h. 2) polluted by hydrocarbons water passes through a column filled with adsorbents. hydrocarbons are taken from water, remaining in limits of adsorption column. the treated water leaves a column for direct use or further treatment. further test is settled. the quantity of the besieged substance on zeolites is determined by the precipitated organic fraction in the filtered solution by the methods of uv spectroscopy, highly effective liquid chromatography and refractometry. example for phenol sorption. the linear dependence between concentration of phenol in a water solution and appropriate molar refraction is preset at 20oc. the measurements were carried out in concentration limits from 0,05 up to 0,3 mol/l. it was earlier established, that the sorption in these limits grows and has linear dependence on molar refraction. from graphic dependence is determined amount of adsorpted phenol [5-7]. the results are given in the tables 1-3. zeolites as materials well known for they ability to remove ammonia from waste water preferentially [ 8 -12 ]. one of the goals of this work is to investigate ion exchange of ammonium ( nh4)+ on zeolites obtained from mentioned here deposits in armenia. the ion exchange capacity of natural zeolites, especially from armenian deposits, offers the possibility of their application to the purification of ammonia-contaminated water. example for ammonia sorption. after sorption of ammonia vapour produced by 1 n nh3 solution by natural zeolites and its treated forms for 10 days at room temperature the adsorption data were plotted. the ammonia adsorption by zeolites was investigated by ir spectroscopy. the results are given in the table 4. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 42 results and discussion argichi river water mainly is polluted by oil, fat, detergents, ammonia, metals as vanadium, manganese, selenium and others. adsorption from wastewater with organic pollutants involves concentration of the solute on the surface. here, it has been had adsorption and desorption process together which will attain an equilibrium state. we used dubinin-radushkevich model for description of the adsorption data. in the present work the results of purposeful researches are submitted in the field solutions and waste water of application natural zeolites as sorbents for removal of organic substances. ammonia, metal ions from water. it is necessary to note, that partial sorption of water /1-2ml from 10ml of a solution for 4 hours sorption of a solution on sorbents / takes place. here is investigated each polluted ions, molecules separately. as organic compounds here is presented the results for benzene, phenol and aniline removal results. it was established that with increase of concentration of solutions the amount of absorbed phenol is increased. fairly active has modifided clinoptilolite by caci2 and mgci2. the h-form of mordenite shows activity, where, on all probability, the formation of hydrogen connections takes place. here is obtained the same results for aniline sorption too. the h-form of mordenite and clinoptilolite show activity, where, on all probability, the formation of hydrogen connections takes place. the sorption of water-soluble oil products are compared between different samples of natural zeolites. high level of adsorption of organic substances natural zeolites is provided with their initial saturation by ions of calcium and magnesia. the present is confirmed also processed by calcium chloride zeolites. zeolites expose simultaneously hydroxyl groups, coordinatively unsaturated cations, as lewis acid sites, and lewis basic sites, anions, as o-2 ions, on their surface. in this case some of efforts to have additional hydroxyl groups and mentioned sites, appears a possibilities to increase acid-basic properties of zeolites. here was investigated the removal of organic compounds – benzene, aniline, phenol sorption process from waste water by means of natural zeolites. on superficial groups of hydro group and atoms of oxygen of zeolites processes of formation of chemical bonds owing to molecular interactions with groups of hydroxyl / for phenol/ and amino groups / for aniline / proceed. here is specially investigated the aniline retain on zeolites. by the irspectroscopic method it is confirmed, that at zeolite modifying by monoethanolamine here is a considerable change of properties of its surface. ir-spectroscopic researches were spent on zeolite of the noemberyan / eastnorth part in armenia / deposit. as result ir shows in the 1400-3700 cm-1 region for clinoptilolite ( under treatments at 400oc) and aniline -clinoptilolite samples after sorption equilibrium at room temperature and heating in vacuum. the free electron pair on nitrogen is less able to hold proton aniline is a weak organic base than aliphatic amines. aniline also an amphiprotic compound, it can react as proton acceptor ( in general ) and as donor. along with the valence and deformation with the specific frequencies belonging to the zeolite (3600, 3450, 1630 cm-1), in zeolite structure absorption peak, which can be carried to valence and deformation vibrations in oh and n-bonds monoethanolamine (3750, 2230, 2150, 1050 cm-1) are observed. the new absorption peaks testifying to formation of intermediate connections are found out also: 3600, 3050, 2710, 2600 sm-1. the absorption peak of 3600 sm-1 corresponds to energy of formation of hydrogen bond o . it has been investigated the phenol and other aromatic sorbtion from water solution. phenols and aromatics / benzene, toluene, xylenes and others/ are discharged in open reservoirs, they destroy the microflora and have negative effect on human health. the major way to diminish the discharge of phenols dissolved in water is strong purification and reuse. the methods are offered for successfully sorption of phenols and other aromatics from waste water in natural and modified zeolites the given method can be applied at rather low initial concentration of phenols and aromatics. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 43 the oil products content in water before and after the sorption was estimated by gravimetric method, after there was made a correction by liquid chromatography. here was investigated also adsorption capacities of zeolites after sorption ammonia ans calcium or manganese from them chlorieds water solution. table 1. the sorption of phenol from a water solution on sorbents / temperature 20oc, duration 4 hours / c on ce nt ra tio n of ph en ol in w at er so lu tio n n d 20 in iti al n d 20 af te r s or pt io n on c lin op til ol ite / gp he no l./ g so rp tio n n d 20 af te r s or pt io n on m or de ni te g ph en ol ./g so rp tio n n d 20 af te r s or pt io n on h -m or de ni te g ph en ol /g so rp tio n n d 20 af te r s or pt io n on o n cl in op til ol ite m od if ie d g ph en ol /g so rp tio n 0,05 1,3324 1,3320/ 0,0236 full sorption full sorption 0,10 1,3328 1,3320/ 0,0470 1,3314/ 0,0752 full sorption 0,15 1,3342 1,3325/ 0,0710 1,3338/ 0,0019 1,3322/ 0,0846 full sorption 0,20 1,3355 1,3335/ 0,0940 1,3346/ 0,0047 1,3329/ 0,1034 full sorption 0,30 1,3371 1,3350/ 0,1034 1,3361/ 0,0063 1,3345/ 0,1175 table 2. distribution coefficient of the oil products ka ( mg/g ) during the sorption on clinoptilolite and others. sorbent ka / light organicb ka/ heavy organic clinoptilolite-tuff 50 300 clinoptilolite-treatied with cacl2 160 540 clinoptiloliteafter treated with ammonia 220 580 b) conditions t= 2 days, concentration of oil product in water – 70 mg/l table 3. sorption of the oil from water solutions on sorbents sorbent weight of sorpted oil product/ mg b % of sorpted oil product clinoptilolite-tuff 20 30 clinoptilolite-treatied with cacl2 32 45 clinoptiloliteafter treated with ammonia 40 55 food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 44 b) oil product concentration in water 70 mg/l, 100 gr sorbent, v water – 1000 ml the ammonium in water and wastewater can be toxic to the aquatic life and should be removed to prevent environmental protection. several processes are currently used for the removal of ammonium from aqueous solutions. ion exchange on natural zeolites is among the methods most commonly employed. zeolite as material well known for they ability removes ammonia from waste water preferentially [1,2]. one of the goals of this work is to investigate ion exchange of ammonium ( nh4)+ on zeolites obtained from mentioned here deposits in armenia. the ion exchange capacity of natural zeolites, especially from armenian deposits, offers the possibility of their application to the purification of ammonia-contaminated water. table 4. the adsorption of nh3 [g] on the zeolites /10g/ n zeolite 1day 3day 5day 7day 10day 1. mordenite 10.38 10.51 10.62 10.70 10.76 2. mordenite treated by 0,5n caci2 10.86 11.02 11.15 11.22 11.38 3. mordenite treated by 1n caci2 11.14 11.71 12.13 12.46 12.98 4. clinoptilolite modified by 1n caci2 11,76 12,04 12,32 12,54 12,98 at first the ability of mordenite is compared to purify river waste water with normal river gravel. the tested zeolite had superiority over gravel in the oxidation of ammonium ions through nitrite ions into nitrate ions. the decrease in the amount of ammonium ions resulted not only exchange with ca2+ mainly in the zeolites, but also the microbial oxidation on the surface of the zeolites. thus, a long-term decrease in the amount of ammonium ions may be possible. this observation suggest that mordenite seems to promote purification with the help of microbial oxidation, with frequent exchange between ca2+ and nh4+ at the zeolite surface. the same capacity is allowed to treated sewage from metal ions. in this research is presented the results for manganese ion removal from water. the analysis data for the clinoptilolite as sorbent for organic compounds from water system at a constant temperature were produced by uv spectrometry and liquid chromatography. proposal method for river water treatment we use during the flood, whish is happen any times in argichi the service provider treating floodwaters must take into account the following: 1. if the onsite wastewater treatment system has electrical components, the ability to restart the system will depend on the flood elevation. 2. if the floodwater only covers the tanks and the components in the tanks, it is possible to restart the system without further evaluation. 3. if the floodwater covers components located on the ground surface (air pumps, panels), the system should be inspected to determine whether it is safe to restart the electrical service and use the system. additionally, sorption materials can be placed on the floodplain, e.g., in ponds with mobile walls filled by adsorbents, and removed after the flood. there would be a primary treatment system and a secondary treatment where the containers filled with zeolite . conclusions food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 45 it has been shown that armenian zeolites have polysorption capacity and can be used for river water treatment. advantages of natural zeolite in comparison with other sorbents are that as technological stability, the low price, availability, filtering properties. it has been found, that the sequence of operations in sorption will follow: 1. the sorption of metals and ammonia, 2. the sorption of organic substances. this sequence can allowed to make cascade from permeable barriers from armenian domestic zeolites for river water treatment. references 1. donald w.breck zeolite molecular sieves: structure, chemistry & use. a wileyinterscience publication, john wiley & sons, new york, london, sydney, toronta, (1974) p.782. 2. c.collela, porous materials in environmentally friendly processes, in studies in surface science and catalysis, 1999, vol. 125, p.641655,. 3. zeolites and mesoporous materials at the dawn of the 21st century, proceedings of the 13th international zeolite conference, montpellier, france,8-13 july, 2001, edited by a galarneau, f.di renzo, f.fajula, j.verdine. series studies in surface science and catalysis, adisory editors: b.delmon and j.t.jates, vol.135.and recent research repotrts for 13th izc. 4. a. dyer, a., an introduction to zeolite molecular sieves, john wiley & sons, chichester, uk, 1988, p. 83. 5. torosyan g.h., water supply emergency for lake sevan ,r.n.hull and al (eds) strategies to enhance environmental security in transition country, springer,.2007pp,.239247, 6. torosyan g., isakov a.,.aleksanyan a,.zakaryan m,use of natural materiales for municipal wastewater treatment, y.sharon at al (eds) water supply in emergency situations, springer, 2007, 133-138, 7. torosyan g., harutyunyan s., removing chlorine-containing organic compounds in the environment after floods, / j.anthony a.jones at al (eds) threats to global water security, springer, 2009. 253-256, microsoft word 3 journal nr 4 2010 din 8 noiembrie_27-31.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 31 computerized system for closure control of packages in food industry cristian logofatu1, daniela cioboata1, mariana ionescu2, gabriel mustatea2, amelia buculei3 1incdmtm, 6-8 pantelimon road, bucharest, cristilogofatu@yahoo.com 2 institute of food bioresources bucharest 3 stefan cel mare universitysuceava abstract: canned food and beverages, currently, comprise a total area of approx. 59 billion cans per year, of which 23 billion cans for food. they represent 18% of total global annual 321 billion cans. in north america, annually, over 32 billion steel cans are produced and sent to the use of packaging [1]. the quality of a food container is determined by its ability to protect the product it contains from chemical deterioration or microbiological spoilage [2]. in the area of industrial applications the problem of closure control for packages used in the food industry is very important as canned products should resist long time (2-3 years), and the consequences of a package defect (content alteration) are not immediately detectable; occurring after a long time and may become extremely dangerous for consumer’s health. one of the can integrity and leakage detection investigation methods is the seam projector measuring system. the computerized system presented was designed for closure control of packages used in the food industry. it contains an integrated system of computing and control which shall be used for a total control of the encasing of the canned product. thus, there shall be measured the encasing parameters and the dimensional and shape parameters of the packages subject to the sterilization process to find out the defects that may lead in time to the alteration of the content. keywords: package encasing, sterilization, canned food introduction the increase in the national economy competitiveness in producing high quality products in compliance with the international standards and especially, the alignment of quality at technical performances thereof to the requirements imposed by the european union is one of the main targets. according to the data furnished by the sanitary veterinary national authority for food safety (ansvsa), more than 500 units of the food industry could be closed, unless they are modernized and restructured, so that to comply with the european regulations. the late data of ansvsa reveals that only 20% are according to the eu regulations, while 37%, although not complying with the conditions of the european community could carry on their activities by investments.[1] the immediate consequences of such situation could be summarized as follows: • a serious risk people’s food safety; • weak presence of the romanian canned products on the european market though romania has a high agricultural potential (vegetables, fruits), and the danube delta represents a huge piscicultural capacity (canned fish); food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 32 • immediate shut-off of all trade agents not complying with the quality regulations of the european union; • cost price increase of the products, if, due to market requisites, control equipment for the food industry packages are to be imported; • neglecting the scientific capacity of the romanian researchers and specialists. the computerized system for controlling package encasing in the food industry, dimensionally verify the packages, according to the european union regulations, furnishing information on certain parameters that may influence the quality of the canned product and decides if it has any potential risk for consumer’s food safety. the geometrical parameters of the package are extremely important, as the quality of the encasing of such food stuff depends on them. the encasing is one of the most important stages in the technology for canned product manufacturing, being decisive for the conservations interval, and implicitly food safety of such product. considering the long life canned products should have (2-3 years) and the fact that the defects (content alteration)are not immediately detectable, as they appear after a long time and being extremely dangerous for consumer’s health, a special attention should be granted the control of canned stuff packages. [2] scientific and technical description from the point of view of the technical and constructive complexity, at the end of the project an integrated system of computing and control shall be realized which shall be used for a total control of the encasing of the canned product. thus, there shall be measured the encasing parameters and the dimensional and shape parameters of the packages subject to the sterilization process to find out defects that may lead in time to the alteration of the content. from a constructive point of view, the computerized system for the control of package encasing in the food industry is designed modular and has the following functional structure, schematically represented in the following image: figure 1: computerized system for the control of package encasing in the food industry ● the equipment for the checking of packages is made of a set of devices provided with flexible basing systems (pos.6), adjustable to various types of packages (3), with measuring systems provided with inductive transducers (2), (4) highly viable and high-precision systems for the displacement and positioning (1). the equipment is provided with an electronic block (5) for data acquisition, control and monitoring provided with dedicated measuring software, making possible a complex analysis of the parameters that may influence upon food preservation safety, accurately indicating the occurrence of the defect and preventing its perpetuation. there may be measured, stored, processed and recorded information related to the geometrical dimensions of the packages that may influence the encasing, such as: at deviation from the nominal quotas of the height and diameter, deviations from smoothness and ovality of the encasing hole, geometry of the higher part of the package, etc. all these data are sent to the central computer unit (11), where they are food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 33 stored and may be edited in complete measurements bulletins. ● the equipment for checking package encasing is an integrated system of opticalelectronic control, containing a high resolution video camera (7) and a telecentric lighting system (9). the camera takes over the real image of the encasing (welt) of the controlled package (8) and sends it to the electronic block (13).this block display the image on a lcd screen (12), measures the encasing parameters and calculates the real union. the real union represents the synthesis of all composing elements of the encasing, is expressed in percentages of total union and consists in the welt portion where sealing effectively takes place, by gripping the rubber seal between the hook of the body and that of the tap. a welt is correctly formed when the real union has a value above 50%. below the mentioned value, the welt may be suspected of leakage. the results of the measurements are displayed on the electronic block and are sent to the main computer. ● the main computer (11) correlated the measurements directly on the package, to those obtained after encasing and identifies the elements that could cause a defect. the measurements bulletin is typed by the editing system of the computer (10) to the complete data of the measurements, including the lot, change, date and time. all these operations are made automatically, with no intervention of the human factor (operator). there is a possibility of printing the real image of the welt for a complex analysis of the material and sealing after the encasing process. all measured date shall be stored for monitoring the entire process in time. thus, there shall be statistically monitored the evolution of such elements in time and the appropriate measures can be taken as soon as there may be found out deviations from the interval considered normal. the values thus obtained may furnish accurate indices as to the causes having generated an inappropriate encasing. the main computer may be connected (if the production line allows it) directly to the control system of the encasing line (see fig.), with the possibility of identifying any problems that may occur at encasing, out of which we specify: the inadequate functioning of the encasing devices (decentration of the head and of the encasing pan, non-parallelism of the surfaces, mismatch of the compression resort pressure, position of the encasing rolls in relation to the head of the device), incorrect adjustment, wear or mismatch o the device rolls, etc. performance and quality parameters the level of performance and quality parameters of the suggested solution can be found in the technical characteristics aimed at by the new product: the resolution of the measuring system for the linear quotas: 0,005mm; resolution of the system for the measuring of shape and surface deviations: 0,01mm; accuracy of measurement: ± 0,01mm; optical enlargement on the lcd screen: 30x; parameters measured at encasing: welt thickness and height, tap depth, body hook, tap hook, outer reinforcement and inner indentation (in line with the body union), as well as encasing percentage; parameters measured for packages: diameter and height on the encasing quota, deviation from surface smoothness of the surface on which encasing is made, circularity (ovality) deviation of the surface on which encasing takes place; encasing parameters of the taps: outer diameter, tap height, edge height, area „elongated metal”; working field: this equipment may control: food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 34 metallic canned products (stas 1687/81, sr iso 3004/92, sr iso 10653/96): ø73mm and ø99mm; taps for metallic tins with dimensions: ø73mm and ø99mm. metallic taps for steel (twist-off, pt, etc.) and aluminum (omnia, keller, etc.) recipients. the most used of them are omnia with the following variants p(pasteurization) and s(sterilization) with the dimensions: ø56mm, ø68mm and ø83mm. due to the high flexibility provided by the equipment, it may be used for checking an important sort of packages for the food industry. there should be underlined the fact that when designing the equipment, there shall be considered the possibility of permanently adjusting the measuring accuracy and field to the demands of the future beneficiaries. there shall also be considered the future evolution of the field, by the occurrence of new typo-dimensions of packages. degree of novelty and complexity the idea of this project proposal occurred because of the many requests which were received from certain trade agents of the food industry. they are interested in acquiring some systems measuring the encasing, but also the dimensional and shape deviations of the packages that may influence on their encasing. accurate control systems were requested where the operator’s influence be manifest as little as possible to avoid any possibility of occurring objective or subjective errors. for this reason, this project proposes the creation of a computer-assisted system, to carry out the control and monitoring of the technological process for the encasing of packages and to automatically issue the measurement bulletin with all the identification data on it, with no intervention of the operator. project contribution to the settlement of certain high complexity problems, in the area of industrial applications, is that it settles the problem of closure control for packages used in the food industry. this issue is very important as canned products should resist long time (2-3 years), and the consequences of a package defect (content alteration) are not immediately detectable; occurring after a long time and may become extremely dangerous for consumer’s health. by mathematical models and dedicated measuring software to be worked out, the project directly contributes to the development of new s/t field, determining its research character at the border of scientific and technical border. from this point of view, we should mention: the use of dimensional measurements, and computer-assisted deviations in industrial applications and transfer of the decisional factor of the control operation, from man to computer. the degree of novelty and originality of this project is represented by the creation for the first time of a computerized system for the control of package encasing to be integrated in the manufacturing technology of trade agents producing packages for the food industry and meat, fish, vegetable and fruit canned products, in order to comply with the quality demands set by the european union. on international level, there have been produced devices checking both packages, and their encasing, but no informational system has been produced so far directly correlating the two operations which are essential for the canned product safety. another objective sustaining the high degree of novelty and originality on international level of the project is represented by the working out of dedicated, flexible software, so that the measuring diagram may contain as many data as required by the user of the equipment. this original software shall be food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 35 designed in a modular (reconfigurable) way as reusable elements. the hardware and software components shall be the basis for the subsequent development, in order to refit the systems and to adjust them to the new types of packages to appear in the future. for this reason, we may say that the project has a high degree of novelty and originality, falling within the major tendencies manifested on international level. conclusions the implementation of the project shall have a significant technical, economic, scientific, and social impact. the technical-economic impact consists in: the creation of a new, sealable product demanded by the market; the creation of a new product and the implementation of modern technologies for control in the production processes; increase of consumer’s food safety; economic growth in regional plan, including in the developed areas, but with a significant potential for the food industry; increase of the turnover and profit by the significant increase of the productivity, quality and viability; increase of economic competitiveness by economic relaunching of the field-related trade agents; the scientific impact shall be emphasized by: increase of the personal capacity of research development; training young researchers with experience in complex researchdevelopment projects; scientific knowledge obtainable after the completion of the project tat shall be easily usable in other projects; developing a system of acquisitions and data processing of high velocity; use of highly accurate mechanical elements; development of algorithms for the control of products and processes in the food industry. the social impact of implementing the project in the mechanical processing fields shall be significant as well: boosting the regional economic growth by providing trade units with modern equipment; increase of the canned stuff production with a positive impact on the raw material producers; providing an important data basis for the romanian technical education; establishing better labour and life conditions by reducing the risk of occurrence of accidents at the working place; reducing the level of noise in the canned stuff production units. acknowledgements the financial support of "parteneriate" program of national management program centre, by means of project no. 51052/2007 for achieving this contribution is gratefully acknowledged references 1. the canmaker online, 2006 2. u.s. food & drug administration, center for food safety & applied nutrition, bacteriological analytical manual online, 2001 microsoft word 3 journal nr 4 2010 din 8 noiembrie_93-98.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 97 electronic nose fast method for apples discrimination to determine optimum harvest moment mirela calu1, petru alexe1, 1 dunarea de jos university, galati, romania, mirelacalu@yahoo.com abstract: appes should harvested before it is fully ripe, but after it has time to mature. this varies according to cultivar and different apples varieties will often ripen at different times over a threemonth period. sugars are major soluble solid in fruit juice and therefore soluble solids are often used as an estimate of sugar content. as the fruit mature, the starch changes to sugar and the aroma and flavors develops, when is generating volatile compounds. the perception of volatile compounds by the human nose is of great importance in evaluating quality of foods; therefore, similar principle as the human nose, the electronic nose, was used. an electronic nose (e-nose) was used to classify apple samples based on their smell. seven varieties of apples from romania, golden, starkrimson, jonathan, gala were from reghin region and pinova, fuji, golden,too, from insuratei area, were used. all the samples were analyzed using the e-nose fox 4000 with 18 metal oxide coated or uncoated sensors. the resulting e-nose intensities were analyzed by principal component analysis (pca), discriminant factor analysis (dfa) and statistical quality control (sqc), which resulted in grouping the used varieties of apples or in grouping the types of samples (peel, homogenate or diluted homogenate from the same apple). the obtained results indicated that e-nose could discriminate successfully among varieties of apples (% of variance >> 90; percentage of recognition ≈ 100 %). choosing proper harvest moment for apples give us posibility of long term preservation in good quality conditions. key words: multivariate statistics (pca, dfa, sqc), correlating maturity indices, response surface. introduction apples are the most widely consumed fruits in romania. romania is a leading producer of fresh fruits and vegetables, topping the 6th place among the biggest european producers of fruit, after france, spain, poland, italy and germany, which limits export opportunities in this country, according to a release of the ministry of agriculture,forestry and rural. apple flavors and essences are food ingredients that emit a vast array of aromatic volatile gases, which contribute to the sensorial quality of foods in which they are incorporated. the headspace atmosphere of different flavors and essences are distinct both qualitatively and quantitatively. [1] volatile (often aroma) compounds are traditionally analyzed by gas chromatography (gc), gc analysis with flame ionisation (fid), gc mass spectrometry (gc-ms), and gc olfactory (gco) methods (lebrun et al., 2008), which involve very expensive equipment, timeand labor intensive steps, methods development, sample preparation, separation of specific volatile compounds using appropiate chromatographic columns, and chromathogram interpretation; but, a major challenge for the fruits and vegetables industry is to replace time-consuming laboratory analyses, used in process and control quality monitoring, with new application techniques that are fast, precise and accurate. e-nose technology represents a food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 98 possible alternative to volatile measurement, at least in some applications. [3,4] these are multi-sensor arrays designed to measure headspace volatiles. each sensor type has a greater or lesser affinity for a particular chemical class or group of compounds. using chemometric techniques and multivariate statistical analysis, it is possible to distinguish among groups of samples, and possibly identify individual sample components. [5] several studies have investigated the use of e-nose type sensors for apple quality evaluation. a non-destructive evaluation of apple maturity has been measured by means of e-nose. [6] evaluation of the feasibility of detecting differences between volatile gases from intact apples and apple juice extracts from different cultivars using e-nose was studied.[7] were realized a comparative analysis of apple aroma by a tin-oxide gas sensor array device. [8] other researchers like developed a multisensor data fusion model to integrate two volatile detection instruments (enose and znose) for apple defect detection, a new method throw which they could differentiate undamaged from deteriorated apples using enose and znose and an ann-integrated enose and znose system for apple quality evaluation. [9,10,11] the objective of this study was to develop a methodology for discriminating apples from two regions of romania, regarding optimal harvesting period determination. in order to achieve this goal we focused our efforts on fizico-chemical and sensory (instrumental) analysis. as far as the sensory approach is concerned, were elaborated “fingerprints” (↔ e-nose intensities) of the apples analyzed using an e-nose fox 4000, that were further used for mutivariate statistics analysis (pca, dfa and sqc). materials and methods 1. experimental set-up golden, starkrimson, jonathan, and gala varieties are the most in romania while pinova, fuji are varieties less cultivated by growers across the country. prior to testing, all the samples were kept at room air temperature (23°c) for about 12 hours to reach ambient air temperature. average samples from each of the analyzed varieties were made. three types of samples were analyzed using an e-nose fox4000: pare of apples, homogenized pare and pulp of apples and diluted homogenates to 25%. 2 grams of each sample were weighted into 10 ml vials (to ensure the repetability 3 vials for each kind of sample were used). the samples were heated to 50°c for about 3 minutes. 1500 µl of headspace was injected at 2000 µl/s. the signal aquisition lasted 2 minutes. the resulting e-nose intensities were analyzed by pca, dfa and sqc. 2. optimum harvest moment choosing this important stage for horti field was done by three maturity indices correlating: firmness (f), soluble solids(s.s.) and starch iodine index (si). firmness of the apples was evaluated using a electronic penetrometer penefel dft 14, with a plunger of 11 mm. soluble solids were determined using an abbe refractometer model 2 waj optika and starch iodine index was determinated with o solution of iodine. fruits testings for maturity were starts three weeks before harvesting period expectations. maturity indices was fallowed in dynamics once at every five days in preharvest period. correlating those three indices help us to accomplish specific charts for each apples type offering us oportunity to deduce food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 99 optimal harvesting moment for each apples variety. results and discussion in order to evaluate the capability of a commercial electronic nose to discriminate varieties of apples from different regions of romania, the resulting e-nose intensities of the analyzed apples were grouped on varieties of apples and types of sample (pare of apples, homogenized pare and pulp of apples and diluted homogenates to 25%). best results was obtained for pare sample. because of the big number of figures obtained from multivariate statistics (pca, dfa and sqc; ≈ 12 for each group) only two of them are shown below. for the others are presented in table 1, the % of variance and the percentages of recognition obtained. table 1 % of variance and the percentage of recognition for some of the groups mentioned at the begining of the discussion % of variance percentage of recognition group pare homogenate diluted homogenate pare homogenate diluted homogenate 1 91 89 87 2 91 89 100 100 3 97 94 92 100 100 4 98 96 94 100 100 100 5 98 88 100 100 100 6 97 95 100 100 7 92 100 the obtained results indicated that e-nose could discriminate successfully among varieties of apples:  12 of 16 pca ploted graphs were having a % of variance >> 90;  13 of 13 dfa ploted graphs were having a percentage of recognition = 100 %. in figure 1 and figure 2 are presented the graph and the chart of principal component analysis and respectively statistical quality control analysis for toate grupurile (pare samples). the pc on x-axis (figure 1), represents 99,15% of the global information given by the instrument analysis, with an discrimination index of 91 % of variance. it can be easily observed that the golden pare samples have well distinct profiles. the golden samples from insuratei and reghin, are located on the same axis but on different positions meaning that golden from reghin should be harvested before golden from insuratei. on the same chart for pinova variety (on the right) we can observed that harvesting period was delayed; for jonathan is the optimal period. starkrimson and fuji varieties are closer like positions and harvesting period is more delayed comparing with the others apples varieties (on the left) in comparison with gala which is in climacteric stage, already (consumption maturity ). all those were confirmed by sqc. green field is acceptability area where fuji variety was considered sample references by the equipment (figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 100 figure 1: pca analyse for pare sample figure 2: sqc analyse for pare sample in september in september each country has developed its own standard procedure to determine the right time to pick apples. various indices have been devised, but because there are considerable variations in a wide number of quality parameters each year, considerable practical experience is needed when determining best harvest data. in our determinations were correlate firmness, soluble solids and grade hydrolysis(si) of the starch for obtaining optimum harvest moment. because of the big size of figures obtained from representing this point only one of them are shown below (figure 3). for the others are presented in table 2, the co-ordinates of the optimum harvest moment. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 101 al, % = 733,2527-91,7768*x+3,8231*y > 80 < 80 < 70 < 60 < 50 < 40 < 30 < 20 fig.3-graphic of correlate for jonathan apples table 1 typical values to determine optimum harvest moment m(optimum harvest moment) variety of apple fimness, n/cm2 soluble solids, % grade hydrolysis of the starch, % of surface transverse section of the apple gala 9.425 14.375 35% starkrimson 9.568 13.160 40% fuji 8.795 15.320 45% pinova 8.500 12.075 40% golden reghin 8.878 13.230 35% golden insuratei 8.626 13.875 35% conclusions we used response surface metodology to establish independent variable efect (firmness, soluble solids ) against optimum harvest moment considered to be dependent variable. results indicate that electronic nose may be successfully applied as rapid method for discriminating apples, regarding optimum harvest moment. rapid methods usually refer to methods that take minutes to get a result. in our study, under the mentioned experimental conditions, the e-nose intensities were available for statistics after approximately 20 minutes of analysis. compared to traditional methods, multivariate analysis combined with food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 102 modern instrumental techniques (eg, enose) often give new and better insight into complex problems by measuring a greater number of chemical compounds at once. these methods are attractive due to their inherent features of versatility, flexibility, effectiveness, and richness of information. acknowledgement the authors are grateful to institute of food bioresources and prof. dr. ing. burzo ioan from usamv bucharest for the offered support for this study. references 1. marrazzo, w.n., heinemann, p.h., saftner, r.a., crassweller, r.e., leblanc, e. (2005a): electronic nose chemical sensor versus gas chromatography: a feasibility study for the differentiation of apple flavors and essences. asae vol. 48, 5: 2003-2006 2. lebrun, m., plotto, a., goodner, k., ducamp, m.n., baldwin, e. (2008): discrimination of mango fruit maturity by volatiles using the electronic nose and gas chromatography. postharv. biol. tech. vol. 48: 122-131 3. luykx, d.m.a.m., van ruth s.m. (2008): an overview of analytical methods for determining the geographical origin of food products. f. chem. vol. 107: 897-911 4. peris, m.., escuder-gilabert, l. (2009): a 21st century technique for food control: electronic noses. anal. chim. acta vol. 638: 1-15 5. zoecklein, b. (2009): electronic nose application for the grape and wine industry. enology notes147: 1-14 6. pathange, l.p., mallikarjunan, p., marini, r.p., o’keefe, s., vaughan, d. (2006): non-destructive evaluation of apple maturity using an electronic nose system. j. food eng. vol. 77: 1018-1023 7. marrazzo, w.n., heinemann, p.h., crassweller, r.e., leblanc, e. (2005b): electronic nose chemical sensor feasibility study for the differentiation of apple cultivars. asae vol. 48, 5: 19952002 8. xiaobo, z., jiewen, z. (2008): comparative analyses of apple aroma by a tin-oxide gas sensor array device and gc/ms. f. chem. vol. 107: 120-128 9. li, c., heinemann, p.h., sherry, r.. (2007a): neural network and bayesian network fusion models to fuse electronic nose and surface acoustic wave sensor data for apple defect detection. sens. actuators b vol. 125: 301-310 10. .li, c., heinemann, p.h., irudayaraj, j. (2007b): detection of apple deterioration using an electronic nose and znose. asabe vol. 50, 4: 1417-1425 11. li, c., heinemann, p.h. (2007c): annintegrated electronic nose and znose system for apple quality evaluation. asabe vol. 50, 6: 1-10 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 59 optimization of the extraction of polyphenols from grape seeds andrei sturza1, olga deseatnicova1 1technical university of moldova; 168 stefan cel mare ave., md-2004 chisinau, republic of moldova andrei.sturza@yahoo.com abstract. in the present work the extractability of polyphenols from grape seed has been studied. in winemaking, the degree of use of grapes as a material for the production of wine or juice reaches the maximum value of 80%, leading to the formation of considerable amounts of secondary products (marc, stalks, yeast, tartaric acid salts). regarding the chemical composition, secondary winemaking products represent a valuable bonus material for the manufacture of a wide variety of products with an important role in the national economy. the determination of the quantity of the total amount of polyphenols with the help of folin-ciocalteu method in a stirred reactor at laboratory scale was made. different values of temperature, time and concentration of the reagent were applied during the extractions to search for the optimal parameters where the extractability will be the most efficient in quantity and quality. a pulsed electric field was applied on the raw material to study its effect and check its fiability. the effect of the extractability was influenced by the parameters of the applied electric field, due to different values of intensity of the electric field, the number and the duration of impulse. the greater amount of extracted polyphenols was obtained at high temperatures, 30 minutes of extraction, with a concentration of ethanol of 40 %. the grape seed represents a valuable material which contains precious components and therefore the study of extractability is interesting from both scientific and economic point of view. keywords: solid-liquid extraction, grape seed, polyphenols introduction a particular interest in the technology of waste treatment is the rational use of grape seed. depending on variety, the bay contains up to 8.0% of seeds, which are not currently exploited3. each year the primary wine making factories produce huge volumes of marc, for which is necessary to separate the seeds for drying and further processing. for several years, the wine tannins have diversified and are subject to many and varied uses. besides the conventional tannin of galls, new tannins appeared from oak, chestnut trees, exotic fruits and, recently, grape seed. the chemical composition and properties of various tannins are not the same, especially the grape tannins that can a priori be considered "endogenous" to the wine compared to others, extracts of exotic fruits or trees. the constituents of grape are numbered by hundreds. this fruit is characterized by its richness in qualitative and quantitative phenol constituents localized mainly in the solid parts (seeds, skins, stalks), with the exception of grape varietals called dyers which also contain in the pulp. the phenol compounds of wine have many properties: • anthocyanins, red pigments, are responsible for wine color • tannins are responsible for the properties that give wines the characteristic taste of astringency4 the phenol compounds are food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 60 able to polymerize and combine with many substances (polysaccharides, proteins, other polyphenols ...)5. these condensed forms to which they operate have a major role in the quality of wines: • the taste properties of tannins depend on their state of combination that provides them different taste characters); • the combinations tannins-anthocyanins (ta) is sought for the stability of the color of wine; from grape marc, distilleries recover, in addition to alcohol, seeds for the manufacture of edible oil, marc anthocyanin colorants for mainly for food industry, seeds of white grapes to extract oligomers proanthocyanidols for pharmaceutical industry (drugs against cardio-vascular diseases). materials and methods as raw material we used dry seeds of red grapes from moldova. the grape is a berry consisting of skins (7-11% by weight), seeds (2-6%) and juicy and fleshy pulp (80-85%). the quantity and types of polyphenols existing in grape seeds may change and are considerably influenced by the extraction process, the variety and storage of used seeds. solid-liquid extraction dry seeds of red grapes from moldova were used for the experiments. the extracts were prepared using water-ethanol mixtures as solvents (ethanol concentration of 20, 40, 50, 60, 80 and 95% by volume). the experiments were performed in a water bath in a conventional batch, which eliminates the external resistance to mass transfer6.uncrushed seeds samples (3 g) were extracted by dissolving the appropriate volume (22.5 ml) at different temperatures up to 65 ± 2 ° c for different periods. after the extraction, the dry extract was obtained by evaporating the solvent on the water bath and the entire extraction yield was determined by weight differences. the results are expressed as mass fraction (total solid extract magnesium gram). the determination of tannins preparation of samples for analysis: the grape seed extract (0.5 ml) was placed in the 50 ml volumetric flask and was dissolved in deionized water (approximately 25 ml), the folin-ciocalteu reagent (2.5 ml) and sodium carbonate solution (10 ml of 2% m/v) were added and then the sample was filled with water to 50 ml. the mixture was shaken thoroughly for 30 minutes and the formed blue color was measured at 750 nanometers using a spectrophotometer unicam. due to relatively short linear part of absorbance values of standard curve obtained may extend between the absorbance of 0.210 at 0.340. if the absorbance is not in this field, the sample should be diluted or concentrated for it to be measured7. the measurement was performed using a blank reagent solution without extract. the results are calculated by the following analytic function: where mt total tannin mass, ct (mol/liter) the total concentration of tannins was reported on the calibration curve, mt – tannins molar mass (gram/mol), vext volume of the extract (ml). when the absorbance values fall without assigned support, the mixture was further diluted and then the elution volumes were taken into consideration [4]. each sample was measured three times. the folin-ciocalteu phenol reagent and tannic acid (m = 1701.2 gram/mol) used as standard to determine the total phenol compounds were purchased from sigma-aldrich (darmstadt, germany). ethanol and other chemicals were of analytical grade. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 61 identification and determination of phenol compounds all tests have been carried out using the shimadzu gc system coupled with a single quadrupole mass-spectrometer gcms-qp2010 equipped with the threedimensional automated system for the injection of samples aoc-5000 (gcmsqp2010 plusxaoc-5000). for the identification, the general library of nist-5 mass spectra was used. the accuracy of displacement has been verified according to the library of covatz retention indices (mlri). the analysis of the experimental data was carried out with the gc/ms solution software (shimadzu), which contains the scan/sim options (fast automated scan/sim type (fasst); creation of automatic sim (scan/sim) table (coast). research on the influence of extractions number on the extraction capacity of tannins the use of grape seed tannins for extraction is very important because grape seeds are byproducts of winemaking that are not used practically. numerous studies have shown that grape seeds are rich in polyphenols that can be extracted and used in different fields. the solid / liquid extraction is a transfer of material between a solid phase material to extract, and a liquid phase, the extraction solvent. extraction is an operation used to remove old plants and certain organs of animals, food products, pharmaceutical or incense, for production of beverages, drugs or perfumes. the manufacturers use this particular separation process for extracting plant products by water, alcohols and organic solvents and chlorinated solvents. in thermodynamics, the solid is a homogeneous mixture at equilibrium is to say that in the absence of an external disturbance, it shall not be changed as opposed to treating a heterogeneous mixture can be divided by filtration, decantation or centrifugation, for example. in solid / liquid extraction, this disturbance is a heat exchange with the external and mechanical, coupled with the input of third body, the solvent. following this disturbance, the solid will be more balanced and solid + solvent system will evolve towards a new equilibrium by mass transfer. the art of extraction is the knowledge of parameters influencing the nature and kinetics of mass transfer of solid to the solvent. to determine the maximum extractable was used 3 grams of basic equipment-whole seeds that were placed in an aqueous solution of 50% ethanol. the extraction was done for one hour at a temperature of 65 º c. after the first extraction the seeds were reused and placed in a newly prepared solution of 50% ethanol. this was done four times in the same way to achieve a solution with a minimal amount of tannins. the solutions with the extract were filtered in a buchner and each filtrate was collected in a dark glass container. the extract was analyzed by folinciocalteu method for determining the concentration and mass of tannins in each filtrate. so we conducted the analysis of the conductivity of materials using a circuit forms a galvanic cell and a digital multimeter. from the results we can observe the variation of the total content of each polyphenol extraction. it is obvious that the content of polyphenols is extracted during the initial extraction and concentration of tannins decreased substantially after the second extraction. as a result we can say that it is reasonable to make two extractions for extracting the maximum amount. many bio-ingredients derived from plants are of high value chemicals with additional applications in food, cosmetics and pharmaceutical sectors. to satisfy a growing demand for a shorter time and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 62 reduced cost in developing manufacturing processes, a systematic process is required to produce alternatives extraction process that transformed plants harvested in the desired products. many factors affect the extraction and should be optimized for maximum recoveries particle size of the sample, type of solvents, ph of the extraction time and extraction temperature, number of extraction and degradation of compounds during extraction. among these factors, the type of solvent is most important because it can affect the quantity, biological activity and the types of compounds extracted. the thermodynamic principles on the solubility of structurally complex materials were not studied because of the complexity of molecular interactions involved. hildebrand theory of regular solutions and concepts of the hansen solubility parameter (hsp) provide a useful correlation for the solubility parameter of the solvent and solute. the hildebrand solubility parameter is a numeric value that indicates the relative solvency behavior of a specific solvent. it is derived from the cohesive energy density of the solvent, which in turn is derived from the heat of vaporization: 2/1         m v m coh h v rth v e  where: (1) ecoh is the molar cohesive energy (2) vm molar volume (3) hv – vaporization enthalpy (4) r ideal gas constant (5) ttemperature the concept of hsp has brought all the cohesive energy (ecoh ) by the amount of energy required to overcome the nuclear forces of dispersion forces between permanent dipoles of adjacent molecules (dipolar interaction) and to break the hydrogen bonds ( exchange of electrons, proton donor / acceptor) between the molecules ( h ) 2222 hpdt   (2) where δt (total solubility parameter) should be identical to δh. defined the difference between the solubility parameters of solute i and solvent j indicates an affinity: 222 , )()()(4 j h i h j p i p j d i dji   (3) the parameters follow the rule that if the δδij is smaller, the affinity is high between the solute and the liquid. this method is often applied to pure chemicals with welldefined chemical structures. recently, the concept of hsp has been extended to interpret the relationships involved in the miscibility of polymer-solvent, adsorption on solid surfaces, as well as for the solubility of components useful to plants in various solvents pure. this study is extracted from red grapes (vitis vinifera l.), whose seeds are an important source of biologically active compounds such as polyphenols (also known as proanthocyanidins and condensed tannins) that act as strong antioxidants. the seeds are the parts richest in polyphenols, and a grape full beam is the part where polyphenols are very difficult to be extracted. the objective of this work was to experimentally determine the optimal operating conditions for a more complete recovery phenol grape seed. we determined the total mass of tannins extracted from four extractions is 124.2mg tannins from 3g base material (figure 1). so we extracted 4.14% of the total mass. figure 1. the effect of multiple extraction steps on the amount of tannin extracts food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 63 we also measured the total extracted mass by drying the filtrate which was obtained and the total mass of extract that can also contain mucilage. to see the change in total mass and the mass of extracted tannin the weights of the extracts are reported to 100 g and are shown in figure 2. 0 5 10 15 20 25 30 35 40 45 m as se , m g/ g p ro du it i ii iii iv nr d'extraction mas se totale extraite mas se tanins extraits figure 2. representation of the total mass extracted from a mass of tannins extracted according to the number of extraction so we can conclude that with the tannins extracted in essential quantities of other compounds. and the ratio of the total extract and extract tannins is 50%. the total extract obtained after 4 extractions is 0.08g per 100g of seeds and extract tannins is 0.04g per 100g of seeds. variation of parameters of tannin extraction the solid / liquid extraction is influenced by many factors. the selection of raw materials, choice of solvent, use of the method, equipment performance is crucial. the implementation of a solid / liquid extraction is linked to the state of the solid and the solute, the nature of solvent, temperature and dispersion phases. the nature and size of the particles have a direct influence on the rate of extraction. indeed, if the solute is distributed throughout the grain, the first solvent will dissolve the solute at the surface will then have to progress within the grain to dissolve the solute particles. research on the influence of solvent on the extraction capacity of tannins the most commonly used solvents are: water, ethanol, acetone, ethyl acetate, hexane and dichloromethane, regardless of the physical structure of plants and nature (flower, fruit), but they must be of suitable purity so that no remaining fraction of high boiling point. to find the optimal concentration of solvent extractions were made extraction of tannins with different concentrations of alcohol. we prepare solvents with 0, 20, 40, 50, 60, 80.96 percent of ethanol. we prepared samples of seeds for each solvent extraction was placed at 65 º c for 30 minutes with the agitator, after each extraction being filtered and prepared for measurement. by measuring all the samples we concluded that the solvent with the concentration 50% ethanol is the most optimal for the extraction. influence of solvent concentration on the degree of extraction of tannins the best extraction of polyphenols in the compound solvent (50: 50 ethanol / water) is due to synergism between these two solvents. on the other hand, in pure ethanol (96%), extraction of total dry matter is more complete. but the rate of dietary fiber, insoluble in water, in this case is considerably higher. in the compound solvent (50: 50-60: 40 ethanol / water) rate of polyphenol extracts ranged from 32-40 mg / 3 g of dry matter (figure 3). 29,4 20,6 20,5 39,4 32,1 11,1 2,0 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0 m as se t an in s, m g 0 20 40 50 60 80 96 concentration du solv ant, % figure 3. the influence of the concentration of solvent (ethanol) on the amount of tannin extracts this degree of extraction after the first cycle, a total of 30 min is optimum as food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 64 allows extracting the most soluble fraction of tannins. research on the influence of temperature on the extraction capacity of tannins the temperature is a factor that strongly influences the degree of extraction of tannins from grape seeds. the rise in temperature has a positive effect because it increases solubility and facilitates the diffusion of the solute and solvent viscosity decreases. however, an excessive rise in temperature can also facilitate the dissolution of harmful compounds or a deterioration of compounds extracted. to have a temperature dependence with the mass of tannin, extractions were performed at different temperatures 25 º c, 45 º c and 65 º c. after measuring the samples we can conclude that the rise in temperature has a positive influence on increasing the amount of tannin extracts. 0,0 10,0 20,0 30,0 40,0 50,0 60,0 m as se d e ta ni ns , m g 25°c 45°c 65°c température d'extraction 15 min 30 min 45 min 60 min figure 4. the effect of temperature on the amount of tannin extracts it is obvious that increasing the temperature of the solvent and extraction time have a positive influence on the rate of polyphenol extracts. the duration of the extraction has a great influence on the amount of tannin extracts. we observe that increasing the extraction time has a direct influence on the proportional amount of tannin extracts. it is very difficult to evaluate separately the influence of temperature and extraction time. this gives a higher concentration at 65 ° c for one hour of extraction. but it is important to take into account the fact that you can not get the same results for a longer duration and temperature decreased because we observed that at 65 º c for 15 minutes you get twice as 45c and 45 min or so for a period of 15 min at 45 ° c extracted twice for 60 minutes at 25 º c. so the temperature plays a key role. in order not to destroy the antioxidant tannins may be considered maximum duration of 60 minutes at 45 º c when the mass reached 11.57 g of tannins extracted from 100 g of seeds. this result is equivalent with the result obtained at 65 ° c for 15 minutes when extracted 12.19% of the tannins. identification and quantification of extracted phenol compounds the identification and quantification of phenol extracts was performed by gas chromatography coupled with mass spectrometer (gc / ms). the results are presented in table 14. the experimental data obtained show the presence of 10 phenol compounds, which have been identified, including: vanillin, cinnamic acid, p-hydroxybenzoic acid, floretic acid , vannilic acid, gallic acid, pcoumaric acid, caffeic acid, resveratrol, quercetin. the other seven peaks were not identified, and their contribution was taken into account. the presence of catechin and epicatechin, as the presence of polyphenols and their derived conjugates esters have not been highlighted by this study. also, a sizeable rate of resveratrol (0.14 0.01) was documented in the extracts of grape seed. the predominant compounds, in descending order, are: vannilic acid (1.24 0.25), caffeic acid (0.55 0.08), gallic acid (0.52 0.15) . food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 65 conclusions 1. a study concerning the influence of the number of fractional extraction on the extraction rate of wine-tannins was performed. it was established that two extraction steps are used to extract 80-85% of the total the quantity of tannins, which make ineffective the following steps. with the extracted tannins an important quantity of other compounds are extracted. the ratio of the total extract and tannin extract is 50%. 2. the study on the composition influence of the extracting on the extraction rate of wine-tannins showed that the extraction is more complete in the case of ethanol / water mixture (50:50) due the synergism between these two solvents. in pure ethanol (96%), extraction of total dry material is more complete. but the rate of wine-tannins extracts in this case is considerably lower.3. the extraction time greatly influences the rate of extraction of wine-tannins: 2.2 to 7.5 mg tannins / g 3 seed (25 º c) of 5.4 to 34.8 mg tannins / g 3 seed (45 º c) from 39.4 to 50.4 mg tannins / g 3 seed (65 º c). another important factor is the temperature of extraction. under the same conditions, the extraction is 7-8 times higher at a temperature of 65 º c as compared to room temperature. 4. the identification and quantification of the extracted phenol compounds helped to highlight the presence of 10 phenol compounds, including predominant compounds (mg / 3g seeds): vannilic acid (1.24  0.25); caffeic acid (0.55  0.08), gallic acid (0.52  0.15). also, a sizeable rate of resveratrol (0.14  0.01) was documented. this composition reflects the high biological activity of wine-tannins from grape seed. references 1. urîtu d, parasca p., »schema tehnologică perfecţionată de obţinere a seminţelor din tescovina de struguri » agricultura moldovei nr.3.2000,pag.12. 2. parasca p.,urîtu d., dubosaru n, « procedeu de obţinere a taninei şi produselor secundare insoţitoare din seminţe de struguri » brevet de invenţie nr.8969 bopi nr. 11(1997) 3. haslam e., 1994 the cup that cheers : polyphenols and astringency. sci lect. pap. ser., 30,15 4. ribereau-layon p., stonestreet e., 1966 le dosage des anthocyanes dans le seins rouges. bull. soc.chim. 9, 2649-2652 5. ribereau-layon p., stonestreet e., 1966 le dosage des tanins du vin rouge et détermination de leurs tructure. chimie anal., 48, 188-192 6. ribereau-gayon p., 1970 dosage des composés phénoliques totaux des vins rouges. chim. anal., 52, --627-631 7. lapornik, b., prosek, m., & wondra, a. g. (2005). comparison of extracts prepared from plant by-products using different solvents and extraction time. journal of food engineering, 71, 214–222 8. glories y, 1978 recherches sur la matière colorante des vins rouges. thèse de doctorat ès sciences, université de bordeaux ii 234 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. pag. 234 238 estimation of heavy metal levels in green leafy vegetables purchased from suceava * ancuța elena prisacaru 1 , laura carmen apostol 1 , sorina ropciuc 1 1”stefan cel mare” university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania; ancuta.prisacaru@fia.usv.ro *corresponding author received 14th october 2017, accepted 17th december 2017 abstract: in the present study the levels of five important heavy metals were identified in vegetable samples purchased from suceava local markets. the concentrations of cadmium (cd), lead (pb), iron (fe), zinc (zn) and copper (cu) were analysed using a mass spectrometer with inductively coupled plasma (icp-ms) from the instrumental analysis laboratory within the faculty of food engineering suceava. the mean levels of heavy metals examined in spinach (spinacia oleracea), ramsons (allium ursinum), lettuce (lactuca sativa), orache (atriplex hortensis) and nettle (urtica dioica) were found to be in the order: fe (13.52 µg/g) > cu (4.83 µg/g) > zn (3.623 µg/g) > cd (1.890 µg/g)> pb (0.290 µg/g). the highest concentration of heavy metal was identified in the case of fe (51.333 µg/g in ramsons), whereas the lowest amount was identified for pb (0.227 µg/g orache). the estimated daily intake for cd is above 60 µg/kg b.w./day. the levels of the other metals are lower than the safe limits predicted by the fao/who. keywords: heavy metals, green leafy vegetables, daily intake 1. introduction green leafy vegetables are an important ingredient of human diet that contains essential nutrients like vitamins, minerals, dietary fiber and anti oxidants [1]. leaves from different plant species such as perennial and annuals are consumed especially in rural areas and there has been observed an increased trend of the consumption among the urban community. green leafy vegetables are an economic source to ensure the micronutrient intake. rapid industrialization and the use of natural resources have increased the accumulation of toxic substances like heavy metals in the soil. heavy metal contamination is a major environmental problem because they are harmful to humans [2]. plants accumulate these toxic substances in their edible parts [3]. zn, cu, and fe are essential for various biological activities within human body, but elevated concentrations can have bad consequences on people [4, 5]. pb and cd are non essential and toxic elements associated with many chronic diseases [6, 7]. this study aimed the determination of the concentration of cd, pb, fe, zn, and cu in five different types of green leafy vegetables collected randomly from two markets sites located in suceava in order to estimate daily intake of heavy metals through consumption. 2. materials and methods 2.1. materials green leafy vegetables were purchased from two markets sites from suceava city. the vegetables were spinach (spinacia oleracea), ramsons (allium ursinum), http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 4 – 2017 ancuța elena prisacaru, laura carmen apostol, sorina ropciuc, heavy metal levels in selected green leafy vegetables obtained from suceava market sites, food and environment safety, volume xvi, issue 4 – 2017, pag. 234 – 238 235 lettuce (lactuca sativa), orache (atriplex hortensis) and nettle (urtica dioica), collected in clean polyethylene bags and brought to the laboratory for the analysis. green leafy vegetables samples were washed with distilled water to remove soil and dirt. 2.2. methods moisture determination in samples was performed according to the european standard en iso 665/2000 by the drying process in a drying chamber at the temperature of 103 °c [8]. total ash composition was determined by calcinations of 5g of sample at 600 °c for 240 min (sr iso 763: 2008) [9]. for the determination of heavy metal concentration 3 grams of green leafy vegetables were mineralized in an electric furnace at a temperature of 600°c, for 6 hours. the resulted ash was transferred into a 25 ml volumetric flask, where it was dissolved by adding a mixture of nitric acid 65% and deionized water. all solutions were prepared with reagent grade chemicals and ultra-pure water (18 mω cm). nitric acid was purchased from sigma aldrich. the determination of five heavy metals was performed in a mass spectrometer with inductively coupled plasma (icp-ms) agilent technologies 7500 series (agilent, usa). calculation of results concentration (c) of the heavy metals in samples is expressed in µg/g sample and is calculated using the formula (eq. 1): (1) (1) where: a concentration value measured by the device, [ppb]; v volume of acid dissolving the sample, [ml]; m mass of sample mineralized, [g]. 2.3. data analysis all analyses were carried out in triplicates with replication. the statistical data processing was done by the program xlstat 2013. 2.4. daily intake of heavy metals from vegetables daily intake of heavy metals was calculated according to the equation 2 [10]: dim = dvc x vmc (2) where: dim = daily intake of metals; dvc = daily vegetable consumption; vmc = mean vegetable metal concentrations (mg/day, fresh weight). daily vegetable consumption was 98 g of vegetables per person per day as set by fao/who (1999), for heavy metal intake based on body weight for an average adult (60 kg body weight) [11]. 3. results and discussion samples were always analyzed in triplicates. moisture and ash content of the samples are shown in figure 1. from the data presented we can observe that lettuce has recorded the highest moisture and ash content (94.44%, respectively 2.82%). the lowest level of moisture and ash content was obtained in the case of nettle (86.81%, respectively 2.44%). the metal content from studied vegetables from the market sites of suceava are listed in table 1. among the five leafy vegetables studied, spinach recorded highest concentrations of cu and zn which are 7.50 µg/g and 4.233 µg/g, respectively. ramsons recorded highest level of fe which is 59.333 µg/g, lettuce recorded highest level of pb, 0.318 µg/g and spinach the highest concentration of cd (2.117 µg/g). similarly, orache recorded the lowest mean levels of pb (0.227 µg/g) and zn (2.667 µg/g). the mean levels of the metals examined in nettle, orache, lettuce, ramsons and spinach samples were found to be in the order: fe food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 4 – 2017 ancuța elena prisacaru, laura carmen apostol, sorina ropciuc, heavy metal levels in selected green leafy vegetables obtained from suceava market sites, food and environment safety, volume xvi, issue 4 – 2017, pag. 234 – 238 236 > cu > zn > cd > pb. regarding plant ability to retain pb we can notice that the lowest level was obtained in the case of orache, followed by nettle, ramsons, spinach and lettuce. in the case of cd retention the hierarchy in increasing order is as follows: orache, ramsons, lettuce, nettle and spinach. these trends suggest that nettle, orache, lettuce, ramsons and spinach have a high retention capacity for fe followed by cu and then zn, cd and pb. fig. 1 moisture and ash content of green leafy vegetables table 1 concentrations of heavy metals (µg/g) in green leafy vegetables plant sample pb cd fe zn cu nettle 0.300 ± 0.0632 2.083 ± 0.0753 2.283 ±0.0983 3.767 ±0.0516 3.550 ±0.0837 orache 0.227 ±0.0137 1.550 ±0.1049 1.433 ±0.1033 2.667 ±0.0816 7.367 ±0.0816 lettuce 0.318 ±0.0117 2.050 ±0.1049 2.400 ±0.1414 4.033 ±0.1862 3.000 ±0.1414 ramsons 0.302 ±0.7091 1.650 ±0.9316 59.333 ±1.7512 3.417 ±0.3793 2.733 ±1.1009 spinach 0.302 ±0.0643 2.117 ±0.3189 2.150 ±0.2429 4.233 ±0.2422 7.500±0.2757 mean ± sd 0.290 ±0.1724 1.890 ±0.3071 13.520 ±0.4674 3.623 ±0.1882 4.830 ±0.3367 range 0.227-0.381 1.550-2.117 1.433-59.333 2.667-4.233 2.733-7.500 fao/who safe limit (2001) a 0.300 0.2 425.00 99.40 73.00 sd = standard deviation; source: a = adu et al. (2012) [11] the mean concentrations in increasing order of the metals in spinach are found to be in the following order: pb > cd > fe > zn > cu. this indicates that spinach has a high retention capacity for pb followed by cd, fe and then zn and cu. it could be observed that majority of studied vegetables have higher concentration of toxic elements (pb and cd) than cu, zn and fe. this study showed that, in the plants, pb content exceeded the permissible limit in the case of lettuce, ramsons and spinach. pb is a toxic element for the plants and human body and high levels can be attributed to pollutants from irrigation water, farm soil and traffic [12]. cd levels detected in all vegetable samples examined in this study were above safe limits. in the case of spinach determined values for all the heavy metals were bellow the results obtained in other studies [13, 14]. related health risks are usually expressed as provisional tolerance daily intake. fao/who (1999) have set a limit for the heavy metal intake based on the body weight for an average adult, 60 kg body weight. the average diet per person per day of vegetables is 98 g. if the mean levels of pb (0.290 µg/g), cd (1.890 µg/g), fe (13.520 µg/g), zn (3.623 µg/g) and cu (4.830 µg/g) found in this study are consumed daily, the contribution of heavy metal intake for an average adult from the vegetable diets were calculated and presented in table 2. it can be concluded that our estimated daily intakes for heavy metals studied are above those reported by the fao/who in food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 4 – 2017 ancuța elena prisacaru, laura carmen apostol, sorina ropciuc, heavy metal levels in selected green leafy vegetables obtained from suceava market sites, food and environment safety, volume xvi, issue 4 – 2017, pag. 234 – 238 237 the case of zinc and copper, which had set a limit for heavy metal intake based on body weight for an average adult (60 kg body weight) for pb, cd, zn and cu. table 2 estimation of heavy metal intake through consumption of green leafy vegetables heavy metal mean conc. (µg/g) daily intake (µg/day) who/fao limit (µg)b pb 0.290 28.42 214 cd 1.890 185.22 60 fe 13.520 1324.96 zn 3.623 355.054 60000 (60 mg) cu 4.830 473.34 3000 (3 mg) source: b = elbagermi et al. (2012) [15] principal component analysis was carried out according to the mineral concentrations, moisture content and ash in different samples green leafy vegetables and to determine the significant differences among vegetables. the correlation value of the parameters measured in selected green leafy vegetables is presented in table 3. the highest negatively correlation has been observed in the case of total moisture with the level of cd (r = -0.488) this can be explained as follows: when the product moisture content is low, the content of cd increases. table 3 correlation matrix for the variables measured in green leafy vegetables variables pb cd fe zn cu moisture (%) ash (%) pb 1 0.721 0.201 0.883 -0.634 -0.252 0.257 cd 0.721 1 -0.488 0.918 -0.115 -0.488 -0.155 fe 0.201 -0.488 1 -0.174 -0.499 0.091 0.315 zn 0.883 0.918 -0.174 1 -0.220 -0.340 0.149 cu -0.634 -0.115 -0.499 -0.220 1 0.017 -0.167 moisture (%) -0.252 -0.488 0.091 -0.340 0.017 1 0.824 ash (%) 0.257 -0.155 0.315 0.149 -0.167 0.824 1 significance level alpha=0,05 fig. 2. principal component analysis of samples food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 4 – 2017 ancuța elena prisacaru, laura carmen apostol, sorina ropciuc, heavy metal levels in selected green leafy vegetables obtained from suceava market sites, food and environment safety, volume xvi, issue 4 – 2017, pag. 234 – 238 238 the influence of chemical composition of the main component analysis is presented in the figure 2. principal component 1 (pc1) explained 57.19% of variance, while component 2 (pc2) explains 34.64% of variation, the overall percentage of variation of the two main components being 91.82%. pca grouped the studied parameters according to their chemical composition. in the first quadrant there are ramsons, in the second orache, in the third there are grouped nettle and spinach and in the fourth lettuce. 4. conclusions the results reported confirm that leafy green vegetables purchased from the market sites of suceava contained substantial amounts of heavy metals. levels of the metals are found to be below the safe limits prescribed by the fao/who. these are important results as heavy metal intake through the consumption of vegetables may possibly cause numerous health hazards in people. 5. references [1]. gupta s., gowri b.s., lakshmi a. j., prakash j., retention of nutrients in green leafy vegetables on dehydration, journal of food science and technology, 50(5): 918-925, (2013). [2]. gratao p.l., polle a., lea p.j, azevedo r.a., making the life of heavy metalstressed plants a little easier, journal of function of plant biology, 32: 481-494, (2005). [3]. olajire a.a., ayodele e.t., study of atmospheric pollution levels by trace elements analysis of tree bark and leaves, bull. chem. soc. ethiopia., 17: 11-17, (2003). [4]. buculei a., gutt g., amariei s., dabija a., constantinescu g., study regarding the tin and iron migration from metallic cans into foodstuff during storage, journal of agroalimentary processes and technologies, 18(4): 299-303, (2012). [5]. sharma r. k., agrawal m., biological effects of heavy metals: an overview. journal of environmental biology, 26(2): 301-313, (2005). [6]. chen y., wu p., shao y., ying y., health risk assessment of heavy metals in vegetables grown around battery production area, scientia agricola, 71(2): 126-132, (2014). [7]. amariei s., gutt g., oroian, m., bodnar a., study on toxic metal levels in commercial marine organisms from romanian market, analele universitatii" ovidius" constantaseria chimie, 25(2): 59-64, (2014). [8]. standard en iso 665/2000, determination of moisture and volatile matter content. [9]. standard sr iso 763/2008, produse din fructe și legume. determinarea cenușii insolubile în acid clorhidric. [10]. cui y.j., zhu y.g., zhai r.h., chen d.y., huang y.z., qiu y., liang j.z. , transfer of metals from soil to vegetables in an area near a smelter in nanning, china, environment international, 30(6): 785-791, (2004). [11]. fao/who, joint expert committee on food additives, “summary and conclusions”, in proceedings of the 53rd meeting of joint fao/who expert committee on food additives, rome, italy, (1999). [12]. adu a.a., aderinola o.j., kusemiju v., heavy metals concentration in garden lettuce (lactuca sativa l.) grown along badagry expressway, lagos, transnat. j. sci. technol., 2(7):115-130, (2012). [13]. qui x.x., huang d.f., cai s.x., chen f., ren z.g., cai y.c., investigation on vegetables pollution and pollution sources and its control in fozhou, fujin province, fujian j. agric. sci., 15: 16-21, (2000). [14]. singh s., kumar m., heavy metal load in soil, water and vegetables in periurban delhi, environment monitoring and assessment, 120, 179191, (2006). [15]. elbagermi m.a., edwards h.g.m., alajtal a.i., monitoring of heavy metal content in fruits and vegetables collected from population and market sites in the misurata area of libya, intl. scholarly res., (2012). microsoft word 0 first page 2_2018.docx stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvii, issue 2 30 june 2018 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania microsoft word 3 journal nr 4 2010 din 8 noiembrie_114-120.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 118 sublethal effects of nickel to rainbow trout oncorhynchus mykiss biological parameters nijolė kazlauskienė1, milda zita vosylienė1, renata karpovič1, edvinas taujanskis1 1institute of ecology of nature research center, akademijos 2, vilnius-21, lt-08412, lithuania kazlauskiene.nijole@gmail.com abstract. the aim of this study was to assess sublethal effects of nickel on rainbow trout juveniles evaluating alterations in fish biological parameters behavioral responses, hematological, morphological parameters and physiological indices. the study of the responses of rainbow trout to different concentrations of nickel demonstrated their different effect on fish organism. feeding behavior of test-fish showed their adaptation to lower nickel concentrations (0.1 mg/l, 0.2 mg/l). higher concentration of nickel (0.4 mg/l) had affected fish feeding irreversibly. there was no significant effect on fish ventilation frequency during exposure to 0.1 mg ni/l concentration. ventilation frequency decreased during exposure to 0.2 mg ni/l, and 0.4 mg ni/l, which leaded to increase in coughing rate. hematological indices of fish were affected during exposure to 0.2 mg ni/l, and 0.4 mg ni/l concentrations. erythrocyte, leucocyte count, haematocrit level and haemoglobin concentration had decreased significantly at 0.2 mg ni/l, and 0.4 mg ni/l concentrations. no significant changes in glucose concentration were found during the exposure to all nickel concentrations tested. present investigation confirmed different sensitivity of the morphophysiological parameters studied, the most sensitive ones were: gill somatic > liver somatic = spleen somatic indices. this study showed that nickel even at low concentrations can affect biological parameters of fish. this phenomenon can lead to revision of maximum-permissible-concentration of nickel in ambient water. keywords: fish, nickel, sublethal effects, biological parameters introduction environmental pollutants such as heavy metals, pesticides, and organic compounds pose serious risks to many aquatic organisms. nickel as a water-soluble metal is easily absorbed and accumulated by the aquatic organisms resulting in metalinduced disturbances in the structure and function of various tissues and organs [1, 2, 3]. the data on nickel toxicity to fish is very scarce, however impact of this metal was partially investigated for other freshwater organisms [4]. the metabolic role of nickel in aquatic organisms is poorly known, however, recent studies of chowndhury [5] had shown that nickel maintained homeostasis of rainbow trout (oncorhynchus mykiss), while high concentrations of ni2+ are toxic and disturbed fish homeostasis [6]. the ionic form a bivalent nickel (ni2+) was found to disturb the gills ventilation of fish [7]. the majority of studies on nickel toxicity had been carried out only with high concentrations of this metal in order to determine the lc50 of this pollutant, while in unpolluted waters ni2+ concentrations food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 119 rarely exceed 0.001 0.2 mg/l ranges [4, 8]. in acute exposures nickel caused decreasing locomotors activity [9]. remarkable changes in the behavior of fish exposed to lethal and sublethal concentrations of nickel were observed (10, 11]. many scientists consider that the many toxicants disrupt complex fish behaviors, such as predator avoidance, reproductive, and social behaviors [12]. many behavioral test involving predator avoidance, feeding behavior, learning, social interactions, and a variety of locomotors behaviors show promise but have been insufficiently studied to judge their sensitivity or utility [13]. changes in certain fish behaviors, especially cough rate and avoidance reactions, are very sensitive indicators of sublethal exposure to metals [13]. according to little [10] behavioral measurements may be useful indicators of sublehal contamination due to concentrations even being lower than those that affect growth. behavioral changes usually occur much earlier than mortality. after exposure to sublethal concentration of metals (nickel, copper and zinc) some behavioral changes in fish were observed due to stress, such as mucus like secretion over gills, excessive excretion, and increased distance between gills and operculum [14]. stress response of the fish organism is an integrated reaction with behavioral, neural, hormonal and physiological elements all combined together to provide fish with the best possible chance of survival [15]. physiological effects of toxicants in the literature include disruption of sensory, hormonal, neurological, and metabolic systems, which are likely to have profound implications for many fish behaviors [12]. however, little toxicological research has sought to integrate the behavioral effects of toxicants with physiological processes. those studies that take this multidisciplinary approach add important insight into possible mechanisms of behavioral alteration [12]. the aim of this study was to assess nickel (ni2+) sublethal effects on rainbow trout juveniles evaluating alterations in fish biological parameters behavioral responses, haematological, morphological parameters and physiological indices. materials and methods exposure of fish for the toxicity studies was performed at the laboratory of ecology and physiology of hydrobionts (nature research center institute of ecology). rainbow trout (oncorhynchus mykiss) juveniles were obtained from the žeimena hatchery (lithuania). the toxicity tests were undertaken under semi-static conditions. deep well water of high quality was used for storing control fish. control water and ni2+ in aquaria were renewed on alternant days. the temperature of water in the aquaria was maintained at 10 ± 0.5oc. average hardness of water was approximately 250 mg/l as caco3, dissolved oxygen concentration and ph were not less than 7 mg/l and 7.9–8.1, respectively. during these studies, juvenile fish were fed with commercial dana feed fish food ad lib. studies with juvenile were performed in two replications (n = 7 in a group). the concentrations of ni introduced into the aquarium water were 0.1; 0.2; 0.4 mg/l. ni2+ concentration 0.2 mg/l is accepted as the maximumpermissible-concentration in water bodies according to the european parliament and council directive 2000/60/ec. long-term (10 days) toxicity tests of ni2+ were conducted using juvenile rainbow trout (oncorhynchus mykiss) according to iso 10229:1994 [16]. the average total length of test fish was 150 ± 10 mm and the total weight was 40 ± 2 g (mean  sem). for rainbow trout juvenile nutritional and behavioral responses were recorded. nutrition responses were measured every food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 120 day for each test fish individually and were estimated in %: 0% not ate; 50% ate a half food; 100% ate the all food. the following behavioral responses of juvenile rainbow trout were investigated: two types of fish behavioral-respiratory responses – gill ventilation frequency (gvf) and coughing rate (cor) in counts per minute [17]. gill ventilation frequency (counts/min) and coughing rate (counts/min) of juvenile fish were measured during 3-min periods for each test fish individually and the mean value was calculated. morphological parameters studied were: mean body weight (q, g; q, g), mean body length (l, cm; l, cm), weight of spleen, gills, liver (g). morphophysiological parameters evaluated: spleen-somatic index (ssi), gill-somatic index (gsi), liver-somatic index (lsi). tissue-somatic indices were calculated using the formula [18]. for each fish, the erythrocyte count (rbc, 106 mm-3), the haemoglobin concentration (hb, g l-1), the haematocrit level (hct, l l-1) the leucocyte counts (wbc, 103 mm-3) were determined using routine methods [19]. glucose concentration (mmol/l) was determined with „eksan-g“ [20]. differences between the measured characteristics were tested by student’s ttest (p < 0.05) using the programme graphpad instat (usa). results and discussion feeding behavior. feeding behavior of testfish showed their adaptation to lower nickel concentrations (0.1 mg/l, 0.2 mg/l). sublethal concentrations of ni2+ after the first days of exposure caused a stressful state fish feeding behavior reduced in all tested ni2+ concentrations. mid-term examination at 0.1 and 0.2 mg/l ni2+ concentrations demonstrated that fish feeding behavior returned at the control level. feeding behavioral assessment showed the fish organism's adaptability to permissible pollutant concentration in water bodies. higher concentration of nickel (0.4 mg/l) had affected fish feeding irreversibly. comparison our data obtained with those of other authors revealed not only similarities but also differences. atchison and coauthors [13] investigated the feeding behavior of various fish, including rainbow trout, exposed to different toxicants (cu, zn, ni, pb) and found that during the first days of exposure to sublethal concentrations the feeding behavior has been disturbed. meanwhile vosylienė and kazlauskienė [21] study with rainbow trout juveniles, 10 days exposed to mixture of five heavy metals (cu, zn, ni, cr, fe) showed that at the first days the feeding behavior of the fish did not changed. midway through the study 50-60% of the fish stopped eating. meanwhile at the end of the study feeding behavior of test-fish gradually restored. respiratory parameters. the gill ventilation frequency (gvf) of juvenile exposed to 0.2; 0.4 mg/l concentrations of ni2+ was significantly (p ≤ 0.01) lowered (59.6 ± 4.3; 61.5 ± 2.8 counts/min) as compared to the controls (72.0 ± 1.0 counts/min) from the initiation of exposure till the conclusion of the tests (table 1). the significantly (p < 0.0001) decreased coughing rate was found in the groups of juveniles exposed to 0.2; 0.4 mg/l concentrations of ni2+ for 10 days (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 121 table 1 the effect of ni2+ for gill ventilation frequency and coughing rate of the juvenile rainbow trout (oncorhynchus mykiss) (x  se). concentration, mg/l gvf, counts/min cor, counts/min control 72.0 ± 1.0 0.2 ± 0.7 0.1 70.2 ± 2.6 0.6 ± 0.3 0.2 59.6 ± 4.3* 3.3 ± 0.9* 0.4 61.5 ± 2.8* 6.3 ± 0.6* * different from the control (p ≤ 0.01). in summary, we may suggest that respiratory parameters are among the most sensitive biological indicators of heavy metals effects. the respiratory-frequency fluctuations we consider to result from the difference in toxicant concentrations. as well as, the data obtained coincide with vosylienė and kazlauskienė [21] conducted studies on the toxicity of model mixture of seven heavy metals (cu, zn, cd, ni, cr, cd, fe) to fish. sublethal concentrations of the mixture induced the ranges in fish respiratory frequency, which increased or decreased, while the “coughing” rate increased. fish “coughing” rate is also considered as a one of the most sensitive indicators, which alterations are among the first signs of intoxication and usually are observed at very low concentrations of toxicants when other indicators are not registered [17]. the data suggest that 0.2 mg/l and a 0.4 mg/l ni2+ concentration adversely affect fish respiratory system, causing a significant decrease in respiratory frequency and the increase in “coughing” rate. haematological parameters. changes in haematological parameters of rainbow trout blood are presented in the table 2. statistically significant decrease in erythrocyte count was observed at 0.2 mg/l and 0.4 mg/l ni2+ concentrations. table 2 the effect of ni2+ for haematological parameters of the juvenile rainbow trout (oncorhynchus mykiss) (x  se). haematological parameters eritr., 1x10-6 mm-3 hb, g/l hct, l/l leucoc., 1x10-3 mm-3 glucose mmol/l control 1.06 ± 0.04 73.6 ± 1.5 0.31 ± 0.01 18.4 ± 1.2 2.27 ± 0.15 0.1 mg/l 1.08 ± 0.03 68.5 ± 3.5 0.33 ± 0.02 17.6± 1.4 1.93 ± 0.13 0.2 mg/l 0.92 ± 0.06* 64.5 ± 3.1* 0.18 ± 0.01* 16.1 ± 1.6* 2.04 ± 0.13 0.4 mg/l 0.83 ± 0.06* 62.9 ± 2.8* 0.21 ± 0.02* 14.6 ± 1.1* 2.09 ± 0.11 * different from the control (p ≤ 0.01). changes in erythrocyte count in blood of fish showed a sensitivity of this parameter to ni2+ exposure. haemoglobin concentration in metal-affected fish decreased as compared with controls, and significant changes were observed at the 0.2 mg/l and 0.4 mg/l concentrations of ni2+. haemoglobin concentration decrease can be attributed to the drop in erythrocyte count in blood. studies on the effect of heavy metals (cu, zn, cd) on red blood cell count changes demonstrated direct proportional alterations in haemoglobin concentration [22, 23, 24]. significant changes in haematocrit also were determined in the blood of fish at 0.2 mg/l and 0.4 mg/l food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 122 concentrations of ni2+. the decrease in haematocrit level is associated with reducing of red blood cells counts and haemoglobin concentrations. pane and coauthors [25] studies on effects of high ni2+ concentrations (11-16 mg/l) on rainbow trout showed the significant increase in haematocrit level accompanied by an elevation in hemoglobin concentration. at 0.2 and 0.4 mg/l ni2+ concentrations significant decrease in leucocytes count in blood of rainbow trout was recorded. such reducing in leucocytes count at higher metal concentrations indicated disturbance in homeostasis and immune functions in fish. similar data were obtained affecting rainbow trout with sublethal concentrations of cu and zn [26, 27, 28, and 29]. no significant changes were observed in glucose concentration in blood of fish after exposure to 0.1 mg/l; 0.2 mg/l and 0.4 mg/l concentrations of ni2+ (table 2). morphophysiological parameters. all tested concentrations of ni2+ caused significant decrease in fish liver weight and at 0.4 mg/l ni2+ significant reducing in the liver somatic index (lsi) was calculated (table 3, 4). these results are confirmed by our previous data [21]. the alterations in liver weight could be explained by detoxifying function of the liver, which, furthermore, regulates excretion of heavy metals from the organism [30]. according to lowe-jindu and niimi [31] the liver weight changes may also be associated with liver enzyme synthesis and carbohydrate metabolism changes. changes in liver size and changes in the lsi are sometimes associated with liver glycogen level changes [32]. liver weight and lsi decrease show that nickel caused adverse changes in the liver structure. table 3 the effect of ni2+ for morphological parameters of the juvenile rainbow trout (oncorhynchus mykiss) (x  se). concentration, mass, g mg/l liver spleen gills control 0.57 ± 0.05 0.07 ± 0.007 1.22 ± 0.10 0.1 0.47 ± 0.04* 0.06 ± 0.006 1.41 ± 0.10 0.2 0.46 ± 0.05* 0.08 ± 0.015 1.48 ± 0.07* 0.4 0.45 ± 0.05* 0.09 ±0.007* 1.58 ± 0.09* * different from the control (p ≤ 0.01). spleen weight and ssi significantly increased only at 0.4 mg/l ni2+concentration (table 3, 4). spleen enlargement and ssi increase respectively could be induced by the stress state of the fish after exposure to toxicant. table 4 the effect of ni2+ for morphophysiological parameters of the juvenile rainbow trout (oncorhynchus mykiss) (x ± se). concentration, somatic indices mg/l lsi ssi gsi control 1.13 ± 0.09 0.18 ± 0.01 3.00 ± 0.14 0.1 1.00 ± 0.01 0.17 ± 0.03 2.67 ± 0.21 0.2 1.15 ± 0.16 0.19 ± 0.01 3.86 ± 0.29* 0.4 1.05 ± 0.20* 0.23 ± 0.02* 3.89 ± 0.34* * different from the control (p ≤ 0.01). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 123 gill weight was mostly influenced by 0.4 mg/l, while gsi was significantly elevated by 0.2 and 0.4 mg/l ni2+ concentrations (table 3, 4). according to pane and coauthor [33], high (16 mg/l) nickel concentration caused the gill epithelial cell necrosis, secondary lamella increase, and disorders in gas and ion metabolism. according to the sensitivity to nickel the morphophysiological parameters may be arranged in the following sequence: gill somatic > liver somatic = spleen somatic indices. results obtained by pane [25, 33], and leonard [34] showed that gills were mostly affected by nickel as compared to other organs and tissues. in this study the toxicity of maximumpermissible-concentration of nickel (0.2 mg/l) in water bodies [35] was investigated. under this directive, this concentration should not cause any noticeable changes in fish organism. however, our study demonstrated that this concentration can exert toxic effects upon all studied biological (feeding behavior, respiratory, morphophysiological, haematological) parameters of fish. we consider, after more detailed studies, the revision of the nickel "safe" standards for aquatic life will be proposed. conclusions  feeding behavior of test-fish showed adaptation of organisms to lower nickel concentrations (0.1 mg/l, 0.2 mg/l). higher concentration of nickel (0.4 mg/l) had affected fish feeding behavior irreversibly.  no significant effect on fish gill ventilation frequency during exposure to 0.1 mg ni/l concentration was observed. gill ventilation frequency decreased during fish exposure to 0.2 mg ni/l, and 0.4 mg ni/l, in contrast, the increase in “coughing” rate was registered.  haematological indices of fish were affected of 0.2 mg ni/l, and 0.4 mg ni/l concentrations. erythrocyte, leucocyte count, haematocrit level and haemoglobin concentration had decreased significantly at 0.2 mg ni/l, and 0.4 mg ni/l concentrations. no significant effect on glucose concentration during the exposure to all test concentrations of nickel was determined.  investigation confirmed different sensitivity of the morphophysiological parameters studied to nickel. gill somatic index was the most sensitive parameter to nickel toxic effect.  this study showed that nickel even at low concentrations can affect biological parameters of fish. this phenomenon can lead to revision of maximum-permissibleconcentration of nickel in ambient water. acknowledgements this work was funded by research council of lithuania, project no. mip58/2010. references 1. m. e. deleebeeck, a. c. schampheleare, c. r. janssen. ecotoxicology ant environmental safety. 67, 2006, 113. 2. j. s. meyer, w. j. adams, k. w. brix, s. n. luoma, d. r. mount. setac press. pensacola. 2005, 15-18. 3. m. e. deleebeeck, a. c. schampheleare, d. g.. heijerick, t. a. bossuyt, c. r. janssen. ecotoxicology and environmental safety. 70, 2008, 6778. 4. h. sigel, a. sigel. nickel and its role in biology. university of toronto, canada. 23, 2006, 13-18. 5. m. j. chowndhury, c. bucking, c. m. wood. environmental science technology. 42, 2008, 13591364. 6. a. farkas, j. salanki, a. specziar. arch. of environ. contam. and toxicol. 43(2), 2002, 236-243. 7. i. javed. ijp science. 2, 2003. 326-331. 8. a. i. ololade, o. oginni. the internet journal of veterinary medicine. 6(1), 2009, 67 75. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 124 9. c. kienle, h.-r. köhler, j. filser and a. gerhardt. environmental pollution. 152(3), 2008, 612-620. 10. e. e. little, j. f. fairchild and a. delonay. j. fisheries society symposium. 14, 1993, 67. 11. h. f. alkahem. j. univ. kuwait (sei.). 1994, 21. 12. r. scott and k. a. sloman. environmental pollution. 152(3), 2008. 612-620. 13. g. j. atchison, m. g. henry and m. b sandheinrich. aquatic toxicology, 68(4), 2004, 369-392. 14. r. p. khunyakari, v. tare, r. n. sharma. j. environ. biol. 22(2), 2001, 141-4. 15. a. d. pickering. fish. res. 17, 1993. 3550. 16. iso 10229:1994. 17. g.. svecevičius. bull. environ. contam. toxicol. 74(5), 2005, 845. 18. d. e. facey, v. s. blazer, m. m. gasper, c. l. turcotte. j. aquat. anim. health. 17, 2005, 263–266. 19. z. svobodova, b. vykusova. diagnostics, prevention and therapy of fish diseases and intoxications. vodnany, chechoslovakia, 1991, 270. 20. j. kulys, v. laurinavičius, m. pesleckienė, m. gurevičienė. biotechnol. appl. biochem. 11, 1989, 149-154. 21. m. vosylienė, n. kazlauskienė. acta zoologica lituanica. 9(2), 1999, 56-70. 22. j. h. j. van vuren, m. van der merwe, h. h. du preez. ecotox. environm. safety. 29, 1994, 187199. 23. h. s. singh, t. v. reddy. ecotoxicol. environ. saf. 20, 1990, 30-35. 24. z. svobodova, b. vykusova, j. machova. sublethal chronic effects of pollutants on freshwater fish. lugano, 1994, 39-55. 25. e. f. pane, j. g.. richards, c. m. wood. aquatic toxicology. 63, 2003, 6582. 26. n. kazlauskienė, p. stasiūnaitė. acta zoologica lituanica. 9(2), 1999, 47-55. 27. b. m. dethloff, h. c. bailey. environ. toxicol. chem. 17(9), 1998, 1807 – 1814. 28. m. z. vosylienė., ekologija. 3, 1996, 12– 18. 29. m. z. vosylienė. acta zoologica lituanica. 9(2), 1999, 76 82. 30. m. witeska, b. jezierska. metal toxicity to fish. review on fish biology and fisheries. 11(3), 2001, 279. 31. l. lowe-jindi, a. j. niimi. arch. environ. contam. toxicol. 54, 1984, 302 -308. 32. c. haux, b. norberg. biochemistry and molecular biology. 81, 1985. 275281. 33. e. f. pane, a. haque, c. m. wood. aquatic toxicology. 69, 2004, 124. 34. e. m. leonard, s. m. nadella, c. bucking, . c. m. wood. aquatic toxicology. 93, 2009, 205216. 35. european parliament and council directive 2000/60/ec. применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancel mareuniversity suceava volume xiv, issue 3 – 2015 contents: 1 decontamination of toluene pollution in water using raw walnuts shells igor winkler, ivan kuvila 241 – 244 2 separation and quantification of biogenic amines in bananas by high performance liquid chromatography veronica tanasa, daniela moise, maria stanca 245 249 3 production and characteristics of white soft marinated cheese from organic certified goat milk aleksandar saveski, tatjana kalevska, dragan damjanovski, viktorija stamatovska, elena josevska 250 255 4 effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire constant acho , lessoy zoue, niamkey adom and sébastien niamke 256 – 268 5 new challenges in the dairy wastewater treatment alla choban , igor winkler 269 – 275 6 assessment of potential contamination and health risk associated with metals in drinking waters from copsa mica region maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu 276 281 7 study of chemical composition influence on the rheological and textural properties of various types of animal and vegetal pâté elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe 282 292 8 sensory evaluation of lamb’s meat according to conventional and organic breeding systems tatjana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski 293 299 9 study on antimicrobial characteristics of spices’ composition s. teterina, n. yushchenko, u.kuzmyk, i. khlystun 300 305 10 the nitrogen compound content of some natural mineral waters from bukovina, romania, versus their bottled form anca-mihaela sidor 306 309 11 sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies for optimum formulation by the mixture experimental design silvia mironeasa,georgiana gabriela codină 310 319 12 physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil bernard assielou , edmond ahipo due, djary michel koffi, patrice kouame 320 326 food and environment safety journal of faculty of food engineering, ştefancel mareuniversity suceava volume xiv, issue 3 – 2015 13 the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice anca-mihaela sidor 327 330 author instructions i v subscription information vi 218 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 2 2015, pag. 218 222 study on flour quality assessment designed to obtain biscuits * adriana dabija1, anca magda păiuş 1faculty of food engineering, stefan cel mare university of suceava, romania adriana.dabija@fia.usv.ro *corresponding author received may 15th 2015, accepted june 15th 2015 abstract: the main aim of this study was to research the influence of wheat flour on biscuits’ manufacturing. flour, the main ingredient in most biscuits, does not bring much flavour, except perhaps where bran is included. it does contribute strongly to the baked texture, hardness and shape of biscuits. the nature of these effects differs for different biscuits related to the enrichment with fat and sugar and to the way in which the dough has been mixed. wheat flour is the ingredient responsible for quality in confectionary including biscuits. among the many indicators of quality of the wheat flour, the following are considered as having technological importance: the content and the quality of gluten, the fineness of the grinding, and the water absorption. most biscuits can be made from flour that has low quantity of protein and gluten which is weak and extensible. unfortunately, for biscuit makers, nearly all the research on flour quality by rheological testing has been done with simple flour/water/salt systems which are not easy to relate to biscuit recipes. but, it is useful to know what can be measured by using a brabender farinograph and extensograph. thus, flour with a protein level of less than 9% is best and levels of more than 9.5% often create processing problems. but, to a certain extent, the quality of the gluten can be adjusted by additives and processing techniques. keywords: raw materials, dough, rheological properties, proteins quality 1. introduction biscuits are mainly made from flour, sugar and fat. biscuits are manufactured in many shapes and sizes, and they can be covered with chocolate, containing fat-based fillers or other pleasantly fragrant fillers. there is a very extensive assortment of biscuits due to many raw and auxiliary materials which are used, with different proportions of raw materials and technological processes applied [1]. raw and auxiliary materials used in the manufacture of biscuits give their taste, flavour and appearance; physico-chemical transformations of raw and auxiliary materials that occur during the technological process also help to improve the final product characteristics [2, 3]. the flour, the main raw material used has an important role in achieving high quality endproducts. the influence of the flour on biscuits’ quality depends on the assortment of biscuits, determined by the total flour weight. if flour has a lower share in the total mass of product, then flour influence on the product quality is proportionally lower [4] permanent development of assortments, along with the progress in manufacturing technology of several products, requires the use of flour with differentiated physicalchemical and technological properties. the flour quality is nowadays one of the fundamental problems for bakery and pastry industry. this is due to advanced mechanization and, especially, automated processes that do not allow easy modification of established operating parameters. therefore, to obtain good quality products in superior economic conditions, the flour should have reasonably constant characteristics and appropriate manufacturing requirements of each item or group of products. the flour used for making biscuits is, generally white wheat flour (can be used oat http://www.fia.usv.ro/fiajournal mailto:adriana.dabija@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 adriana dabija, anca magda păiuş, study on flour quality assessment designed to obtain biscuits, food and environment safety, volume xiv, issue 2 – 2015, pag. 218 – 222 219 flour, corn flour, etc.), with a minimum content of 27% gluten. depending on the type of desired biscuit, a higher or lower content of gluten is imposed. the biscuits require flour with medium gluten content. if flour with high gluten content is used, the final products will strongly deform during baking, the surface will be rough and covered in blisters. tender biscuits are prepared from flour with weak gluten, because a high content of gluten leads to considerably hard biscuits. to reduce the high protein gluten content of flour, starch can be used from potatoes or corn, which reduces elasticity and increases the plasticity of the dough. flour granularity should be in the range 50-150μ, a higher granularity determines an obtainment of products with reduced volume and porosity [5, 6, 7]. biscuits belonging to various groups of varieties require appropriate quality flours for each respective group. the most common index that characterizes the quality of biscuit flour is gluten protein content. the flour for the manufacture of biscuits differs from that used by bakers due to its protein content and gluten quality content [8, 9]. wheat flours used to make the biscuits must have a low content of 9-10% protein, low gluten content and plastic properties given (table 1). table 1. selection criteria of biscuits flour [10] indicator of flour quality quality indicator for assortment groups gluten biscuits sugary biscuits crackers wet gluten,% 25 35 24 34 26 36 strain index, mm 15 25 5 25 5 15 dry gluten, % 8.5 11.5 8 11 9 12 proteins,% 11 13 10 12 11.5 13.5 usual flours used for biscuits manufacturing have extensible gluten, and for crackers it is recommended a mixture of 75% strong flour and 25% weak flour. for laminated biscuits is recommended flour richer in gluten, while for biscuits with sugar content of about 40% and about 24% fat, are recommended even stronger flours. many researchers recommend weak flour for biscuits manufacture. in the case of sugary biscuits which are characterized by a high friability, flour should form a small amount of gluten (less than 20%) and poor quality. studying the rheological characteristics of biscuit dough, it was considered appropriate amounts of biscuit flour those contained in table 2. table 2 quality characteristics of the main types of biscuits [10,11] quality indicator recommended levels for gluten biscuits sugary biscuits crackers farinogram the stability of the dough, min dough softening, ub power, uc 5 – 8 60 – 120 40 – 50 5 – 11 50 – 120 40 60 6 – 11 30 – 60 50-60 extensogram the ratio r / e (135 min) area, cm2 0.2 – 0.1 5 – 50 0,2 – 0.25 5 – 90 1.0 – 2.5 30 90 the paper presents summary results obtained from the analysis of wheat flour from different origins, used as raw material in the manufacture of biscuits, with the destination of its use in formulation of the different types of biscuits. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 adriana dabija, anca magda păiuş, study on flour quality assessment designed to obtain biscuits, food and environment safety, volume xiv, issue 2 – 2015, pag. 218 – 222 220 2. materials and methods to study the quality of flour used for preparing biscuits, six samples of flour from a leading producer of biscuits, crackers, sticks and pretzels were analyzed. analyses were performed in the laboratory of the centre for training and bakery, belonging to sc rompak srl pascani. thus, the six samples of flour received from the various mills of biscuit manufacturers were analyzed using a pharinograph, a brabender extensograf and apparatus for the determination of the falling number. table 3 methods and equipment used for flour analysis quality indicator determination method equipment used wet gluten, % stas 90-88 strain index mm stas 90-88 falling index sr iso 3093/2007 falling number fariongrame sr iso 5530-1/1999 farinograf brabender extensogram sr iso 5530-2/ 1999 extensograf brabender in technological terms the assessment technique of flour behaviour during dough stage presents interest. during this phase, all intrinsic properties of the flour, the dough behaviour constituting a summary of all interactions that occur between the various components of the flour are expressed. 3. results and discussion for wheat flour, protein substances content, wet gluten quantity and properties are very important indicators in terms of technological properties. there is an optimum of the protein content, flour gluten respectively, as well as the rheological properties of the gluten. technological properties required for the manufacture of flour biscuits varies by group of assortments. for gluten biscuits and crackers requiring dough with good elastic properties, the flour should have the following technological properties: a high capacity to form and retain gas which requires adequate quantity and quality of gluten, good hydration capacity and ownership to form light dough. for sugary biscuits, dough is indicated to be brittle, therefore no special conditions are imposed on its elasticity and technological properties required from flour are hydration and assimilation capacity to form plastic dough with low elasticity that is easily processed, does not shrink and therefore the product keeps its printed form. the flour quality requirements for obtaining biscuits are almost opposite intended as compared with those of flour for bread. biscuit flour is weak flour with low protein content and should have a small grain starch. a high falling number is not an advantage in this technology, but a low falling number can positively influence the obtainment of biscuits with optimal characteristics. as regards biscuit dough, it must be characterized by elasticity and not by resistance. the results of analysis of 6 samples of flour made of the above mentioned methods are shown in table 4. all the six samples of flour have an average content of the gluten-forming proteins, being characterized by different qualities. samples s4 and s6 have a poor quality gluten because during wash was quickly formed a linked mass which had a low consistency and elasticity, high extensibility and low tensile strength. left to stand after washing, the rheological properties worsened. samples s2, s3 and s5 have average gluten quality and after wash formed a bounded mass which was sufficiently elastic, with average extensibility and deformation and, ranking in terms of its rheological properties of gluten in an intermediate position between strong and weak gluten. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 adriana dabija, anca magda păiuş, study on flour quality assessment designed to obtain biscuits, food and environment safety, volume xiv, issue 2 – 2015, pag. 218 – 222 221 on the other hand, sample 1 has strong gluten, after washing are obtained small particles, separated, which gradually become a homogeneous-bounded mass. it has high elasticity, low extensibility and high tensile strength. left to rest and very little has changed in its rheological characteristics. regarding the development of the dough, it can be seen that the dough for samples s1, s3 and s5 grew slower compared to the other 3 samples. stability is a measure of the tolerance of the flour to mixing, and strong flours have high stability, therefore the stability of the sample s1 is the largest, whereas the samples s4 and s6 have little stability; stability during mixing of the dough is strongly influenced by the protein quality (deformation index). the degree of softening (uf) is the difference measured between the centre of curvature consistency at the end of the dough development curve centre and 12 minutes after this point. a high degree of softening is associated with poor quality flour, even if flour hydration capacity is high. increased wetting is an important indicator of proteolytic degradation of wheat. after analysis there is a great variability of this parameter quite understandable because the degree of softening is strongly influenced by the type of grinding and physical-chemical indicators of wheat. of the six samples of flour samples s2, s4 and s6 have the highest softening index. in terms of dough energy, it can be seen that the energies for samples s1, s3 and s5 have a value above 50 u.b thus we can classify them as good quality flours, optimal for baking. s4 sample is characterized by a low amount of energy even since the first measurements, a figure which drops to 0. the other 2 samples (s2 and s6) have an average energy which decreases during the technological flow. in practice, biscuit dough strength and elasticity depends on the amount of water used in the dough and mixing time, its strength and elasticity being directly proportional to the increase of mixing time. the ratio resistance / extensibility are above par for samples s1, s3 and s5 and below par for samples s2, s4, s6. table 4. the analysis results of the flour sample sample analysed s1 s2 s3 s4 s5 s6 physical-chemical indicators wet gluten, % 26 27.2 25.6 28 26.4 26 strain index mm 3 6 5.5 10 7.5 10 falling index 439 385 408 436 459 343 rheological indicators hydration capacity,% 58.6 58.7 57.4 61.8 60 61.6 dough development, min 2.2 2.0 2.3 2.0 2.5 1.7 stability, min 11.9 8.1 10.7 1.9 8.4 1.4 wetting index, fu 26 54 25 195 59 168 45 – 90 – 135 min ratio r / e 1.6 – 2.0 – 1.9 0.9 – 0.8 – 0.8 1.1 – 1.3 – 1.4 0.5 – 0.3 – 0 1.11.3 – 1.1 0.9 – 0.8 – 0.4 extensibility, mm 150 – 140 – 129 169 – 158 – 149 173 – 181 – 155 186 – 140 – 0 157 – 152 – 141 177 – 178 – 201 energy cm2 64 – 63 – 49 46 – 35 – 27 65 – 75 – 63 29 – 11 – 0 52 – 51 – 35 49 – 41 – 23 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2015 adriana dabija, anca magda păiuş, study on flour quality assessment designed to obtain biscuits, food and environment safety, volume xiv, issue 2 – 2015, pag. 218 – 222 222 4. conclusion the dough used to make biscuits must be able to take shape with little effort and without subsequent deformation of the cut pieces, for this purpose the flour intended for the manufacture of biscuits must have a sufficiently low content of low quality protein and high extensibility. therefore, according to the results of the analysis, the samples s4 and s6 are the best for obtaining biscuits. these 2 samples are characterized by an average content of gluten-forming proteins, a ratio r / e below par and a small amount of energy that decreases during the technological flow. although other samples of flour do not possess optimal characteristics for this technology, they can still be used in manufacturing of biscuits. very often it happens in practice that the raw materials (wheat) do not possess optimal properties for biscuit manufacture. therefore, in order to obtain quality products it is necessary to control the plasticity of the dough by changing the quality of the gluten. this may be achieved by exogenous proteases. to improve the quality of flour for biscuits’ manufacture, may be used combinations of proteases with amylase and cysteine, depending on the quality parameters of flour or can be used flour mixtures in various proportions. for example, for the flour samples analyzed it is recommended to use the mixture of flour, between the sample 1 (10%) and sample 4 (90% of the recipe) in biscuits’ manufacturing. 5. references [1]. hooda s., jood s., organoleptic and nutritional evaluation of wheat biscuits supplemented with untreated and treated fenugreek flour, food chemistry, 90: 427-435, (2005) [2]. sudha m.l., et al., influence of fibre from different cereals on the rheological characteristics of wheat flour dough and on biscuit quality, food chemistry, 100: 1365–1370, (2007) [3]. seevaratnam v., et al., studies on the preparation of biscuits incorporated with potato flour, world journal of dairy & food sciences, 7 (1): 79-84, (2012) [4]. maache-rezzoug z., et al., effect of principal ingredients on rheological behaviour of biscuit dough and on quality of biscuits, journal of food engineering, 35: 23–42, (1998) [5]. singh b., et al., studies on the development of high-protein biscuits from composite flours, plant foods for human nutrition, 43: 181-189, (1993) [6]. sai manohar r., haridas rao, r., interrelationship between rheological characteristics of dough and quality of biscuits; use of elastic recovery of dough to predict biscuit quality, food research international, 35: 9807– 813, (2002) [7]. sai manohar r., haridas rao r., effect of emulsifiers, fat level and type on the rheological characteristics of biscuit dough and quality of biscuits, journal of the science of food and agriculture, 79: 1223–1231, (1999) [8]. nandeesh k., et al., effect of differently treated wheat bran on rheology, microstructure and quality characteristics of soft dough biscuits, journal of food processing and preservation, 35: 179–200, (2011) [9]. manley d., manley’s technology of biscuits, crackers and cookies, woodhead publishing limited, cambridge, (2011) [10]. bordei d., the quality and marketing of wheat flour, academica publishing, galati, (2001) [11]. edwards w.p., the science of bakery products, the royal society of chemistry, (2007) 202 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 202 208 the interaction between beans hulls and food dye * laura carmen apostol 1,2 1”stefan cel mare” university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania 2”gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof.dr.docent d. mangeron str., 700050 iasi, romania *corresponding author: laura.apostol@fia.usv.ro received 15th october 2017, accepted 19th december 2017 abstract: in this study, a local agro – waste, namely beans hulls, was used to identify the interactions between it and the xanthene food dye red no. 3. batch experiments were made iin order to identify if the dye can be adsorbet by the hull components. thus, the investigation started with the influence of different parameters on bean-hull absorption capacity: ph, contact time, initial dye concentration, and temperature. the amount of dye sorbed was found to vary with the initial dye concentration and contact time. the process is favorable at low acid ph (5) value and the optimum sorbent dosage was found to be 20 g l-1 sorbent. keywords: beans hull, red no. 3, sorption parameters. 1. introduction effluents from industries like food, cosmetic and pharmaceutical contain different kinds of synthetic dyes. coal-tar dyes can have hazardous effects on the living systems because of their carcinogenicity and toxic nature. due to their synthetic origins and aromatic structures, with by-products biologically non-degradable, it is rather difficult to treat these effluents (i.e. red no. 3). therefore, the removal of dyes from aqueous solution becomes environmentally necessary. out of the processes applied for the removal of dyes, sorption is often one of the effective methods because it produces an effluent that is free of harmful substances that can appear in the case of biodegradation and photodegradation i.e. [1, 2]. red no. 3, also known as erythrosine, is an organoiodine compound, obtained from fluorone. it is the disodium salt of 2,4,5,7tetraiodofluorescein used for food coloring [2]. different studies in view of eliminating red no. 3 from aqueous solutions have been done considering degradation methods and the researchers supposed that the intermediates formed can be toxic [36]. some examples of adsorbents used for red no. 3 removal are feathers [7], deoiled mustard [8], montmorillonite [9], bottom ash and de-oiled soya [10]. other unconventional biosorbents used for different dye classes’ removal are hazelnut shells [11], nut shells [12], pumpkin seed hull [13] or bagasse pith [14]. in this study, the agro – waste beans hull (bh) was used as potential biosorbent for red no. 3 removal from aqueous solutions because it has a good efficiency when tested for the sorption of lead ions [15] and acid red 57 decolorization [16]. batch experiments were conducted for the study of the influence of some parameters on dye adsorption efficiency, such as: http://www.fia.usv.ro/fiajournal mailto:laura.apostol@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 203 initial solution ph, initial dye concentration, process temperature and the biosorbent concentration. the results were correlated with thermodynamic parameter (free energy of adsorption (δgº), enthalpy (δhº), and entropy (δsº)). 2. materials and methods 2.1. materials the research performed in this section brings information on a representative xanthene dye, (red no. 3) removal from aqueous solutions by agro-waste (bean hull), in batch mode. adsorbate the acid dye used in this study, red no. 3, was purchased from sigma–aldrich (c.i. acid red 51, 45430). a stock solution of 1000 mg l-1 was prepared by dissolving an accurately weighed amount of red no. 3 in 500 ml distilled water. for the experimental assays, dilution of the stock was done to achieve the desired concentration, always when necessary. absorbent phaseolus vulgaris l. used in the study was purchased from a local farm. the byproduct is an herbaceous annual plant botanically classified as a dicotyledonous. dried pods, no seeds of bean contain aminoacids (arginine, asparagines, tryptophan, tyrosine, lysine, betaine, etc.), vitamin c, salicylic acid, phosphoric acid, minerals [17, 18, 19] (table 1). before being used as sorbents, the by-product was washed several times with distilled water to remove any adhering dirt and dried at 40ºc for 24h. the dried sample was crushed using a retsch gm 200 laboratory mill. no other chemical or physical treatments were used prior to adsorption experiments. finally, they were sieved and classified. the less 3 mm particles size of beans hulls were used in the experiments study. the sorbent was stored in plastic boxes. the point of zero charge (phpzc) of bh was determined by immersion technique and potentiometric mass titrations (pmt) [20]. table 1. chemical composition of bean hull (adapted after duke [17]) compound amount of compound water (%) 58.30 proteine (%) 7.40 carbohydrates (%) 29.80 fates (%) 1.00 fibers (%) 1.91 ash (%) 1.63 calcium (mg/100g) 50 phosphorus (mg/100g) 160 iron (mg/100g) 2.60 thiamine (mg/100g) 0.34 riboflavin (mg/100g) 0.19 ascorbic acid (mg/100g) 27 carotene (mg/100g) 0.057 2.2. batch sorption study batch experiments were performed in erlenmayer flasks, containing red no. 3 solution at various concentrations (10, -400 mg l-1) and different amount of sorbents (10 30 g l-1). the flasks were incubated at room temperature (25±1ºc) and 150 rpm in an isothermal shaker (ika ks 4000 ic) for 24 h until the dye removal reached equilibrium. all the experiments were carried-out in duplicates and at the natural ph of the solution (ph = 5.6).the factors affecting dye adsorption efficiency were investigated: initial dye concentration, sorbent amount, ph, temperature and contact time. the effect of ph on the color removal efficiency was analyzed over the ph range of 4 to 10. the ph study was carried out using britton–robinson buffer solution. 2.3. analysis methods the solution containing different dye concentrations was determined spectrophotometrically at the maximum absorption wavelength. red no. 3 shows food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 204 an intense absorption peak in the visible region at 524 nm which corresponds to the maximum absorption peak of the red no. 3 monomer. the ph solution was measured using a hanna ph meter provided with a combined glass electrode (model hanna hi1053b). the amount of dye uptake per unit mass of sorbent at time t, (qt, mg g -1) and at equilibrium (qe, mg g -1) was calculated using eqs. (1) and (2). the sorption efficiency was calculated applying eq. (3): v m cc q ti t *   (1) v m ceci * q e   (2) 100* (%) r ce ceci   (3) where: ci is the initial dye concentration (mg l-1); ct and ce, the concentration of dye at time t and at equilibrium (mg l-1); m, the amount of dried sorbent used (g); v, the volume of the solution (l). 3. study on the factors influencing the red no. 3 sorption process 3.1. determination of point of zero charge (phpzc) the data obtained using immersion techniques (it) for phpzc determination are plotted in the fig.1 a. the experimental curves corresponding to potentiometric mass titrations method (pmt) technique are presented in the fig. 1b. the results obtained by applying it and pmt are in concordance. the point zero charge determines the surface charge of the sorbent at a given ph and its knowledge offers information’s about the possible electrostatic interactions between sorbent and various chemical species [21]. the phpzc determined value for bh was 4.6. the obtained data indicated that the bh surface is mostly acidic and this could be a favorable property for red no. 3 adsorption at ph<6 when the sorbent is positively charged. -3 0 3 6 0 2 4 6 8 10 12 phi [ph] a) bh 0 2 4 6 8 10 12 14 0 1 2 3 4 5 ml hno3 p h blank 10 g/l 20 g/l 30 g/l b) fig. 1. point of zero charge of bh determined using a) immersion techniques and b) potentiometric mass titrations method 3.2. effect of ph on dye uptake to analyze the effect of ph on the sorption capacity of bh on the amount of dye removal, the experiments were carried out at different initial solution ph values varying from 4 to 10 (fig. 2). for a ph less than 5, red no. 3 in contact with sorbent reacts with the buffer solution and the absorbance of the solution decreased very much due to the strong acidity of the medium that conducts to dye precipitation [22]. the effect of ph on the amount of dye sorbed and process efficiency, respectively can be observed in the fig.2. the minimum amount of red no. 3 sorption was achieved at ph 10, value that increased at 0.72 mg g-1, with the decrease of ph until 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 205 as in the case of psh, in the study conducted by apostol et al. [13] the removal of red no. 3 using bh occurs mainly by ion-exchange reaction because at ph values coresponding to acid domain the surface of the adsorbent is positively charged (high h+ concentration), so the electrostatic attraction between the adsorbent and the adsorbate (which is an anionic dye) is enhanced. 5 6 7 8 9 10 0.0 0.2 0.4 0.6 0.8 1.0 ph q e ( m g g -1 ) q e (mg g -1 ) r (%) 50 60 70 80 90 100 r ( % ) fig. 2. effect of ph on the red no. 3 uptake onto bh (ci=20 mg l -1 ; csorbent=20 g l -1 ) 3.3. effect of sorbents concentration on sorption efficiency the influence of bh concentration on red no. 3 adsorption was conducted at an initial dye concentration of 20 mg l-1. in solution, the concentration of bh varied between 5 and 50 g l-1. fig. 3 shows the results of red no. 3 sorption, at the equilibrium for various sorbent dosages. the percentage of dye removed starting with 20 g l-1 of bh concentration become almost constant, the curve approaching plateau (fig. 3). the increase in the sorption efficiency of red no. 3 with the bh dosage can be explained by a high surface area and the availability of more adsorption sites [23, 24]. at sorbent dosage higher than 20g l-1 the red no. 3 concentration in the two phases (bh surface and the solution) come to equilibrium with each other [25]. 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 10 q bh (mg g -1 ) r bh (%) m (g) q ( m g g -1 ) 0 20 40 60 80 100 r (% ) fig. 3. effect of amount of sorbents on red no. 3 removal (ci = 20 mg l -1 ; ph = 5.6) 3.4. effect of initial dye concentration on uptake capacity of the sorbent the contact time influence on the sorption of red no. 3 on bh was investigated at the ambiental temperature using initial concentrations between 10 and 30 mg l-1. for 50 and 100 mg l-1 red no. 3 uptakes were evaluated at various temperature (20, 30 40 and 50°c). the results show that the amount of red no. 3 adsorbed increased with contact time for the concentrations tested at ambiental temperature in the shaker. the apparent equilibrium is achieved at around 5h after experiment started. the increasing of initial red no. 3 concentration raises the q value from 0.4 to 1.2 mg g-1 (fig. 4). figs. 5a and 5b present the results obtained for the assay with 50 mg l-1 and 100 mg l1 red no. 3 at different temperatures. it can be observed that, when a solution of 50 mg l-1 red no. 3 was used at different temperature the dye uptake at equilibrium shows differences as compared to the assay conducted with 100 mg l-1. using 50 mg l-1 red no. 3 initial concentration, at 20°c the dye uptake is around 1.3 mg g-1, while at 50°c the dye uptake is around 1.7 mg g1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 206 in the case of 100 mg l-1 initial dye concentration at 20°c the dye uptake is around 1.7 mg g-1, while at 50°c dye uptake is around 2.2 mg g-1. 0 5 10 15 20 25 0,0 0,3 0,6 0,9 1,2 1,5 10 mg l -1 20 mg l -1 30 mg l -1 q t (m g g -1 ) time (h) fig. 4. effect of contact time for different initial concentration of red no. 3 3.5. thermodynamic parameters thermodynamic parameters obtained from the slope and intercept (fig. 6) of van’t hoff plots are shown in the table 2. the negative values of ∆g° demonstrate that red no. 3 sorption is spontaneous for the temperature range evaluated. the positive values of enthalpy’s change (δhº) shows that the sorption process is endothermic, increased temperature leads to a higher red no. 3 amount uptake at equilibrium. the positive values of ∆s° achieved in this case is in concordance with the study conducted using pumpkins seeds hulls as sorbent by apostol et al. [13] and reveals the affinity of red no. 3 for its uptake by bh. 3.1x10 -3 3.2x10 -3 3.3x10 -3 3.4x10 -3 10.0 10.5 11.0 11.5 12.0 ln k c t -1 (k) fig. 6. plot of ln kl vs.t −1 for the removal of red no. 3 by bh 0 5 10 15 20 25 0,0 0,5 1,0 1,5 2,0 2,5 q t (m g g -1 ) time (h) 293 k 303 k 313 k 323 k 0 5 10 15 20 25 0,0 0,5 1,0 1,5 2,0 2,5 3,0 q t (m g g -1 ) time (h) 293 k 303 k 313 k 323 k a) b) fig. 5. effect of contact time for (a) 50 mg l -1 and (b) 100 mg l -1 red no. 3 at different temperature food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 207 table 2. thermodynamic parameters for the sorption of red no. 3 on bh temperature (k) kl (l mmol -1 ) thermodynamic parameter δgº (kj mol -1 ) δhº (kj mol -1 ) δsº (j mol -1 k -1 ) 293 0.04068 -22.97 30.44 182.3 303 0.05705 -24.79 313 0.06103 -26.61 323 0.1465 -28.44 4. conclusions the analyses made in this study indicated that red no. 3 can be attached by beans hulls molecules. the amount of dye sorbed was found to vary with the initial dye concentration and contact time besides the main parameters influencing the process: low acid ph (5) value and at 20 g l-1 sorbent dosage. thermodynamic parameters obtained from the slope and intercept of van’t hoff plots showed negative values of ∆g° and demonstrate that adsorption is highly favorable and spontaneous; the positive values of enthalpy change (δhº) stand for the endothermic nature of the processes. the positive values of ∆s° obtained for red no. 3 bh system reveal the affinity of the dye for the adsorbent. in these conditions bh is effective for red no. 3 sequestration from aqueous solutions since a wide range of concentrations may be applied in batch system (5-400 mg l-1). 5. acknowledgments the work is supported by the romanian national authority for scientific research, cncs-uefiscdi, project number pn-iiip4-idpce-2016-0683, contract 65/2017. 6. references [1]. crini g., non-conventional low-cost adsorbents for dye removal: a review, bioresource technology, 97: 1061–1085, (2006). [2]. apostol l.c., gavrilescu m., bean husk used for erythrosine b removal, proceedings of the 14th international symposium on cellulose chemistry and technology, 208-213, (2010). [3]. hasnat m.a., uddin m.m., sameda a.j.f., alama s.s., hossain s., adsorption and photocatalytic decolorization of a synthetic dye erythrosine on anatase tio2 and zno surfaces, journal of hazardous materials, 147: 471–477, (2007). [4]. jain r., sikarwar s., semiconductormediated photocatalyzed degradation of erythrosine dye from wastewater using tio2 catalyst, environmental technology, 31: 1403-10, (2010). [5]. jain r., bhargave m., sharma n., electrochemical degradation of erythrosine in pharmaceuticals and food product industrial effluent, journal of scientific and industrial research, 64: 191-197, (2005). [6]. uddin m.m., hasnat m.a., samed a.j.f., majumdar r.k., influence of tio2 and znophotocatalysts on adsorption and degradation behaviour of erythrosine, dyes and pigments, 75: 207 – 212, (2007). [7]. gupta v.k., mittal a., kurup l., mittal j., adsorption of a hazardous dye, erythrosine, over hen feathers, journal of colloid and interface science, 304: 52–57, (2006). [8]. jain r., sikarwar s., adsorptive removal of erythrosine dye onto activated low-cost de-oiled mustard, journal of hazardous materials, 164: 627–633, (2009). [9]. rytwo g. huterer-harari r., dultz s., gonen y., adsorption of fast green and erythrosin-b to montmorillonite modified with crystal violet, journal of thermal analysis and calorimetry, 84: 225–231, (2006). [10]. mittal a., mittal j., kurup l., singh a.k., process development for the removal and recovery of hazardous dye erythrosine from wastewater by waste materials—bottom ash and de-oiled soya as adsorbents, journal of hazardous materials, 138: 95 – 105, (2006). [11]. ferrero f., dye removal by low cost adsorbents: hazelnut shells in comparison with wood sawdust, journal of hazardous materials, 142: 144-152, (2007). [12]. de oliveira brito s.m., carvalho andrade h.m., soares l.f., de azevedo r.p., brazil nut shells as a new biosorbent to remove methylene blue and indigo carmine from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 laura carmen apostol, the interaction of beans hulls with food dye, food and environment safety, volume xvi, issue 4 – 2017, pag. 202 – 208 208 aqueous solutions, journal of hazardous materials, 174: 84 – 92, (2010). [13]. apostol l.c., ghinea c, alves m., gavrilescu m., removal of erythrosine b dye from water effluents using crop waste pumpkin seed hulls as adsorbent, desalination and water treatment, 57: 22585-22608, (2016). [14]. gad h.m., el-sayed a.a., activated carbon from agricultural by-products for the removal of rhodamine-b from aqueous solution, journal of hazardous materials, 168: 1070-1081, (2009). [15]. özcan a.s., tunali s., akar t., özcan a., sorption of lead(ii) ions onto waste biomass of phaseolus vulgaris l.: estimation of the equilibrium, kinetic and thermodynamic parameters, desalination, 244: 188–198, (2009). [16]. tunali s., ozcan a., kaynak z., ozcan a.s., akar t., utilization of the phaseolus vulgaris l. waste biomass for decolorization of the textile dye acid red 57: determination of equilibrium, kinetic and thermodynamic parameters, journal of environmental science and health part a, 42: 591600, (2007). [17]. duke james a., handbook of energy crops, on line at: https://www.hort.purdue.edu/ newcrop/duke_energy/dukeindex.html, (1983). [18]. aremu m.o., olaofe o., akintayo t.e., a comparative study on the chemical and amino acid composition of some nigerian underutilized legume flours, pakistan journal of nutrition, 5: 34-38, (2006). [19]. mcgoodwin m., the physiology of higher plants. an outline, on line at: www.mcgoodwin.net/pages/plantphysuw425.pdf, (2008). [20]. fiol n., villaescusa i., determination of sorbent point zero charge: usefulness in sorption studies, environmental chemistry letters, 7: 79-84, (2009). [21]. gadd g.m., sorption: critical review of scientific rationale, environmental importance and significance for pollution treatment, journal of chemical technology and biotechnology, 84: 13– 28, (2009). [22]. colombini m., wu c.y., a food dye, erythrosine b, increases membrane permeability to calcium and other ions, biochimica et biophysica acta (bba) – biomembranes, 648: 49-54, (1981). [23]. gong r., zhong k., hu y., chen j., zhu g., thermochemical esterifying citric acid onto lignocellulose for enhancing methylene blue sorption capacity of rice straw, journal of environmental management, 88: 875–880, (2008). [24]. malakootian m., moosazadeh m., yousefi n., fatehizadeh a., (2011), fluoride removal from aqueous solution by pumice: case study on kuhbonan water, african journal of environmental science and technology, 5: 299306, (2011). [25]. jaikumar v., sathish kumar k.d., gnana p., sorption of acid dyes using spent brewery grains: characterization and modeling, international journal of applied science and engineering, 7: 115-125, (2009). microsoft word 0b cuprins 1_2018.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 contents: 1. elaboration of a method to determine the dry white wine falsification with sucrose igor winkler, antonia vitvitska 5 8 2. heat transter at evaporative concentration of down flowing in vertical pipes solutions in annular regimes valentin petrenko , yaroslav zasiadko 9 19 3. pork supply chain, consumption and risk factors for infections of consumers in the abidjan district (côte d'ivoire) koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke 20 31 4. antioxidant capacity of local berries in complex food products violina popovici, rodica sturza 32 36 5. influence of thermal treatment the basalt tufa on its phase composition and sorption capacity volodymyr diichuk, iryna diichuk, igor kobasa 37 40 6. edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova 41 47 7. walnut shells bleaching using oxidizing and reducing agents eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac 48 52 8. comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water muhammad haroon, muhammad vaqas 53 58 9. the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy aliona ghendov-moșanu 59 65 10. effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau” mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea 66 73 11. use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets valentina bantea-zagareanu 74 80 12. basalt tufa as a bactericide filler for some packaging materials igor kobasa, mariya vorobets, larysa аrsenieva 81 86 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 13. water chemical analysis of wells in three settlements of suceava plateau, romania andrei-emil briciu, luciana-alexandra costan (briciu), vasile adrian ionesei, iulian ciprian gîza, constantin mihai șchiopu 87 96 14. study on total phenolic content in some romanian forest mushroom species, before and after heat treatment marcel avramiuc 97 102 15. author instructions i v 16. subscription information vi issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvi, issue 1 31 march 2017 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 contents: 1. the use o f pla n t ma t eri als in culin a ry p rodu cts ou t of y ea st ed d oug h oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk 1 6 2. yogurts enriched with pea protein zapryana denkova, emil filipov, bogdan goranov, iliyan dobrev, velichka yanakieva 7 14 3. specifics of air conditioning in the germination process of malt serhyi but 15 20 4. screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke 21 -29 5. technological properties of sonochemical treated reconstituted milk olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova 30 36 6. biochemical and molecular-genetic characterization of a strain isolated from a thermal healing spring in starozagorski mineralni bani, stara zagora region, bulgaria nedyalka vylcheva-zhekova, zapryana denkova, radosveta nikolova 37 44 7. contributions to achievement a composite material for advanced electromagnetic shielding of living and work spaces second part quantitative aspects cristia n flo rin al ex iuc, gh eo rgh e gutt, so n ia a mari ei 45 52 8. nan osi zed impl an ts o f th e i ons ag +, cu + a nd cu 2 + i nt o the su r fa ce la y e r of p oly cry st alline cds pho t oca taly st fo r th e r ea cti on o f oxida ti on of iod id e i ons igor kobasa, lyubomyra odosiy 53 57 9. comparison of aluminum sorption using inorganic and organic natural sorbents danka barloková, ján ilavský, eleonóra franková, tomáš molnár 58 66 10. the influence of storage temperature and time on total phenolic content in a soft wheat cultivar marcel avrămiuc 67 73 11. physico-chemical properties of blends of corn oil with coriander seed oil silvia mi rone asa , geo rg ian a g ab ri ela cod i nă 74 83 12. influence of hydrocolloids in oil-in-water emulsions during storage of food oksana lugovska, vasilij sidor 84 88 13. nanodispersed composite antimicrobial materials based on calcium hydroxylpatite igor kobasa, galyna bilokopyta, larysa arsenieva 89 92 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 14. application of the infrared semiconductors interference filters for optical sensors in express spectroscopy of organic materials volodumur strebezhev, viktor strebezhev, george vorobets, ruslan hurjui, roman rogov 93 – 100 15. blending of sunflower oil with grape seed oil: impact on physico-chemical parameters and radical scavenging activity georgiana gabriela codină, maria poroch-seriţan, silvia mironeasa 101 107 16. cont ributi ons t o a me th od and a n ew t e chn ique for det ermi na ti on of so me imp ort ant me cha nica l p rop er ti es o f m assiv e wo od t ex tu re ştefa n beşl iu, gh eo r gh e gutt, son ia a ma r ie i 108 116 17. contributions to a new method for determining food gumminess gheorghe gutt, sonia amariei, mircea adrian oroian, cristina-elena hreţcanu 117 126 18. author instructions i – v 19. subscription information vi food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 66 was te dis posa l im pacts o n en vironm ent *ivona škultétyová1 1slovak university of technology, faculty of civil engineering, ivona.skultetyova@stuba.sk *radlinského 11, 81368 bratislava, slovakia abstract: historical trends in waste generation show an increase in the quantities of waste generated for most countries and it is clear that the trend will continue. the average european now creates more than half a tone of municipal solid waste each year. treatment methods are used to reduce the amount of residual waste for disposal, and to achieve one or more of the following goals -reduce the potential environmental impacts of the waste, separate and recovery materials or energy, reduce transport costs, reduce the volume of landfill needed, reduce the minimise overall costs the treatment and disposal of solid waste involves a range of processes including landfill, incineration, composting, pyrolysis/gasification, etc., all of which may result in emissions to the environment. residents near undesirable facilities are often highly concerned about the facility’s impact on people’s health, environment and property values. keywords:, landfill, landfill gas, leachate, pollution, waste introduction we are now in an age where the aspects of public and occupational health and safety have been joined by policy needs on environmental protection, resource consumption and sustainability. the design, management, and control of waste facilities, e.g. landfills, has changed considerably in recent years in response to a better understanding of the pollution potential of wastes and of more effective means of control. landfills landfills have been identified as one of the major threats to groundwater resources. landfill represents the largest route for the disposal of waste the modern landfill site is an advanced treatment and disposal option designed and managed as an engineering project in which the waste is degraded to a stabilised product and the product leachate is treated to minimise pollution (see table 1) to the environment and the landfill gas is recovered for energy [3]. a common landfill classification system for reflects the type of waste each receives. there are landfills for hazardous wastes, municipal wastes and inert wastes. areas near landfills have a greater possibility of groundwater contamination because of the potential pollution source of leachate originating from the nearby site. such contamination of groundwater resource poses a substantial risk to local resource user and to the natural environment [6]. mailto:ivona.skultetyova:@stuba.sk food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 67 table 1. sanitary landfilling: matrix of health impacts: biophysical environment nature of stressor impact on environment affected area control measures standards or recommendations fire smoke/dust deposits plume dispersion covering application of provincial laws and regulations concerning landfill site management explosion destruction site and perimeter biogas management, leakproof site biogas greenhouse effect global collection and burning none ch4, co2 greenhouse effect global none international commitments to counter the greenhouse effect vocs air pollution local and regional collection and destruction benzene: 15 mg/m3 (e.g. tumor producing effects) dichloromethane: 2200 mg/m3 trichloroethylene: 82 mg/m3 leachate bod, cod1; heavy metals, organic compounds, and microorganisms all pollutants: disturbance of aquatic life, pollution of surface and ground water local and in receiving environment (down-stream) or in water table all pollutants: collection and processing in the water treatment plant bod: none for metals, organic compounds, and microorganisms: e.g. drinking water guidelines n/a n/a n/a n/a n/a noise unhealthy conditions vicinity and community all these nuisances: buffer zone; litter fence; daily covering; reduction in waste handling operations leq 2 55 dba (day) and 45 dba (night) (who guidelines) odors unhealthy conditions vicinity and community none airborne waste aesthetics and unhealthy conditions air/ground perimeter none vermin unhealthy conditions local none 1 cod = chemical oxygen demand 2 leq = equivalent sound pressure level food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 68 municipal solid waste is a bioreactive type of waste, which undergoes biodegradation in the landfill site. the processes of degradation of bioreactive waste in landfills take many years to complete and involve not only biological processes but also inter-related physical and chemical processes. figure 1. shows the major stages of municipal solid waste degradation in landfills [4]. figure 1. major stages of bioreactive waste degradation in landfills biological processes the wide varieties of fill components that can be broken down biologically constitute the biodegradable organic fraction of msw. more than half of household waste is organic. this degrades gradually through five stages within a landfill: aerobic hydrolysis, in which micro-organisms convert some carbohydrates to simple sugars (such as glucose), carbon dioxide and water; hydrolysis and fermentation, when carbohydrates, lipids and proteins are broken down and fermented yielding volatile acids, acetate, carbon dioxide hydrogen and inorganic salts; acetogenesis, where bacteria turn soluble acids to carbon dioxide and hydrogen. these, with carbohydrates are also transformed into acetic acid; methanogenesis, in which bacteria convert acetic acid to methane and carbon dioxide. finally, conditions may become aerobic again as the landfill becomes more stabilized [4]. chemical processes two general types of chemical reactions take place within landfilled waste. obviously, the extent of the oxidation reactions is rather limited, inasmuch as the reactions depend upon the presence of oxygen trapped in the fill when the fill was made. ferrous metals are the components likely to be most affected [2]. secondly, acid-metal reactions, due to the presence of organic acids and carbon dioxide. these processes mobilise metallic ions and salts which are potential pollutants. however, once methane generation is established the landfill becomes less acid and metals (especially mercury, lead and cadmium) are generally retained (as relatively immobile sulphides). physical processes compaction of waste has a strong bearing on its behavior. this process begins at the collection stage, takes place at the landfill site and continues within the landfill itself. the effects of water as it passes through the landfill also have a profound influence on the long-term behavior of the waste, dissolving soluble materials and transporting unreacted matter. the absorption of dissolved pollutants (by, for example, cellulose-based matter within a landfill), helps retain materials, at least before saturation occurs. finally, food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 69 adsorption is an important factor within a landfill, as wastes become bonded to the surface of other materials [5]. pollution from landfills a waste disposal facility must guarantee adequate control over the two main types of pollution — leachate and landfill gas. when landfill gas is collected some liquid (condensate) is also collected. leachate is generated as a result of moisture entry into a landfill, either as rain, snow melt, and run-on or as moisture in the waste itself. a typical landfill design includes run-on control and a final cover to minimise moisture flow into the waste. the two most significant components of leachate are organic chemicals and heavy metals. these may be conserved in the landfill in the short term, but through biochemical processes can be mobilised and released. organic chemicals are present as soluble decomposition products (e.g. organic acids). they are also present as organic chemicals (e.g. benzene, toluene, dioxins, halogenated aliphatics, pesticides, pcbs and organophosphates) discarded in the waste. heavy metals, such as mercury, chromium, nickel, lead, cadmium, copper and zinc, are often found in landfill leachate. discharges depend mainly on the acidity and the rates of flow of leachate. many heavy metals come from the non-regulated hazardous waste fractions from households and businesses. no engineering design can guarantee total containment, and some migration of leachate to contaminate groundwater supplies may occur. policymakers generally demand quality control systems and the use of comprehensive environmental impact and risk analysis techniques. leachate management systems tend to be one of the three following types: on-site treatment (generally some form of aeration tank system), disposal to sewerage systems, or transport off-site for treatment elsewhere. leachate the water percolating through landfills is called leachate. it is a complex, highly variable mixture, consisting of various organic and inorganic compounds and microorganisms. leachate is generated by precipitation or by other moisture that enters the landfill from the breakdown of organic matter or from ground water (water table). it is generally characterized by a strong odor and dark brown color and contains high levels of pollutants, creating a bod of 5000 mg/l, compared to 100200 mg/l for typical municipal wastewater. the average absorptive capacity of household waste in landfill is 8.5 cm of liquid per meter of thickness. inorganic pollutants consist essentially of heavy metals, generally present at low concentrations, with the exception of iron and manganese, which are usually present in the form of more or less insoluble metal salts. some particularly toxic metals can pose a risk if they infiltrate ground water aquifers used for drinking water [1]. organic compounds come from many domestic products: disinfectants, deodorants, cleaning agents, pesticides, furniture cleaners and waxes, cosmetics, soaps and shampoos, dyes, paints and varnishes, medications, etc. landfills contain thousands of organic compounds. however, the following compounds are universally found or are found in larger concentrations in leachate: benzene (solvents, dyes, pesticides, detergents); vinyl chloride (manufacturing of various products); dichloromethane (solvents, liquid refrigerant, use by the pharmaceutical industry); food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 70 tetrachloroethylene (dry-cleaning solvent, inks, rubber solutions, paint solvents); carbon tetrachloride (aerosols, dry cleaning); toluene (solvents, dyes, pharmaceutical manufacturing); 1,1,1-trichloroethane (cosmetics, aerosols); and xylene (dyes, pharmaceutical products, insecticides, solvents, resins, varnish, and polyester products). the main sources of pathogens are facial tissues, pet feces, diapers, paper towels, and food waste. wastewater treatment sludge, contaminated industrial waste, and biomedical wastes can also be transported to certain sanitary landfills, thus increasing the number of microorganisms. the presence of vermin (rats, wild animals, birds, and insects) also contributes to increasing the pathogen load. leachate contains bacteria (including coliforms, fecal coliform bacteria, and pseudomonas aeruginosa and aeromonas hydrophila); viruses (particularly the hepatitis a and norwalk group viruses); and protozoan parasites (such as giardia lamblia and cryptosporidium parvum). landfill gas the various gases produced at landfill sites are collectively known as biogas. it is formed by the microbial decomposition of the organic matter in the waste. the process can last several decades and occurs in several stages: hydrolysis of organic matter into basic molecules (amino acids, sugars, fatty acids, etc.) and simple molecules or atoms (nh3, hydrogen, co2, etc.); acetogenesis of the simple molecules or atoms (conversion to acetic acid); and methanogenesis (conversion of acetic acid to ch4 and co2). the composition of a mature biogas is almost always constant and consists essentially of equal parts of ch4 and co2 (47% each). it also contains gaseous nitrogen (close to 4%) as well as several dozen compounds in concentrations of less than 1%. the principal classes are: aromatic hydrocarbons, including vocs (e.g., benzene, toluene, xylene); halogenated hydrocarbons (e.g., vinyl chloride, dichloromethane, chloroform); sulphide compounds (e.g., h2s, mercaptans); various alcohols (e.g., methanol, isopropanol); and other substances, such as acetone, ethane, and propane. the accumulation of ch4 in confined spaces or enclosed structures can result in asphyxia, explosions, and fires, which may cause injury or loss of life. the risk of ch4 gas explosions is highest at ambient concentrations of between 5% and 15%. underground migration of biogas (lateral migration) can result in its infiltration into buildings and can cause explosions or asphyxia in confined spaces. a number of polluting gases can have a variety of impacts on health [3, 4]. co2 and ch4 are greenhouse gases partially responsible for global warming. a number of organic compounds are toxic, including several vocs, which can cause health problems following chronic exposure. these include, for example, aplastic anemia; teratogenic and fetotoxic effects; damage to the liver, lungs, and kidneys; nervous system damage; and various cancers, such as leukemia and myelomas. it is important to note, however, that these effects are associated with high concentrations, which are not necessarily found in proximity to landfills. those at greatest risk are landfill workers, particularly operators of heavy equipment used to compact the waste. food and environment safety journal of faculty of food engineering, tefan cel mare university suceava volume x, issue 3 2011 71 there are some health impacts in the table 1. conclusion to prevent off-site migration of leachate and landfill gas, the landfill site must be naturally impermeable (clay soil) or a watertight geomembrane barrier and gas collected system must be installed. the leachate must be collected by means of a network of perforated piping and transported to a treatment plant similar to that used to treat urban wastewater. both physicochemical treatment (oil removal system, filters, coagulation, precipitation, etc.) and biological treatment (biofilters, activated sludge, etc.) are generally required, given the very high levels of contaminants in landfill leachate. the local hydrogeology and the containment technologies and impermeabilization systems employed are therefore key factors in terms of environmental and health risks. an uncontrolled above-grade landfill located near a stream poses considerable risks to water quality. acknowledgement the paper has been written with the support of the grant research task vega no. 1/0559/10 dealt with at the department of sanitary and environmental engineering of the faculty of civil engineering of the university of technology bratislava references 1. hlavínek, p.: perspectives of decentralized wastewater treatment for rural areas. in: advanced water supply and wastewater treatment: a road to safer society and environment, isbn 978-94-007-0279-0, springer, niderland, 2011 2. k íž, k., bareš, v., pollert, j., stránský, d., pollert, j.: risk analysis of sewer system operational failures caused by unstable subsoil in: risk management of watersupply and sanitationsystems. dordrecht: springer, 2009, p. 65-72. isbn 978-90-4812364-3 3. williams, p. t.: emissions from solid waste management activities in: environmental science and technology, environmental and health impact of solid waste management activities, hester r.e. and harrison r.m. (eds.), the royal society of chemistry, 2002, no. 18,. 141-170 4. williams, p. t.: waste treatment and disposal, john wiley & sons ltd., chichester, 1998 5. hall, d. h., drury, d., gronow, j. r., rosevear, a., pollard, s. j. t., smith, r.: estimating pollutant removal requirements for landfills in the uk: i. benchmark study and characteristics of waste treatment technologies. in: environmental technology, vol 27, no 12, 2006, pp.1309-1321 6. suman mor, khaiwal ravindra, r. p. dahiya, a. chandra: leachate characterization and assessment of groundwater pollution near municipal solid waste landfill site. in: environmental monitoring and assessment volume 118, numbers 1-3, 435456, 2006 microsoft word 10 rusu_m_usv_articol_corectat.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 65 the determination of flavonoids, the total polyphenols and antioxidant activity of basil seasoning *micşunica rusu1, roxana mihăilescu2 1faculty of food engineering, stefan cel mare university of suceava, romania, rusu_mic@yahoo.com 3s.c. ccppm plantavorel s.a. piatra neamţ, romania *corresponding author received 25 october 2012, accepted 21 november 2012 abstract: the study aims to determine quantitative and qualitative of the flavonoids, total polyphenols and the antioxidant capacity of the dried basil used in food. the investigations were made on extracts obtained at cold or hot, in water, solutions of methanol, ethanol and complex solvents (methanol water acetic acid, methanol – acetone – water – formic acid). the determination on more types of extracts give us information about the availability of bioactive compounds in different conditions, different by temperature, ph, and lypoor hydrophilic character. the obtained data demonstrate that the bioactive substances analysed are very well represented in basil, and this can explain the observed remarkable antioxidant capacity and interest in the presence of this plant in creation of some aliments with a high biological potential. keywords: antioxidants, spices, thin layer chromatography, dpph 1. introduction oxidative stress, which is caused by free radicals, is considered the cause of many diseases of today civilization: cardiovascular, diabetes, various cancers and aging processes [1]. one of the ways to prevent pathologies mentioned above is an appropriate intake of antioxidants. antioxidants are chemical compounds that protect cells from the harmful effects of reactive oxygen species (ros) [2]. good sources of antioxidants are fruit and vegetables, whole grains, because of the intake of vitamins, minerals, bioflavonoids, components with high antiradical potential [3]. the spices are also a reservoir of concentrated phenolic compounds with antioxidant proprieties, such as polyphenols. concentrated in only a few grams of material, they can represent the simplest ways to increase the phenolic content and the antioxidant capacity of the daily diet with possible health benefits [4]. in this context, i did experimental studies on basil to determine the amount of phenolic compounds (polyphenols and flavonoids) and to evaluate the antioxidant activity of the spice, given the relationship between the total phenolic compounds and antioxidant activity, as evidenced in numerous studies for other foods [1,5]. basil (ocimum basilicum), one of the most popular plants, a “bridge” between medicine, gastronomy and tradition, is an annual plant of the labiatae family, with a taste / flavour of sweet – peppery, spicy – hot. it has various uses both to obtain culinary products or flavoring and seasoning (salads, sauces, fish dishes in mediterranean cuisine and asian cuisine especially) and in medicine (being used as a remedy in many diseases: intestinal colic, intestinal bloating, vomiting, flu, colds, dry acute and chronic bronchitis, headache, stomach ulcer, urinary tract food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 66 infections, anorexia, diarrhea, colitis fermentation [6]. 2. materials and methods for comparison, quantitative and qualitative chemical study was performed on extracts from two samples of basil: a working evidence (sample k) obtained by purchasing it from the market and a blank sample (sample l) obtained from the warehouse of company plantavorel piatra neamt. because the way meaning is done decisively influences the result and gives a more complete picture of the analyzed compounds content, there were varied extraction conditions, working both at room temperature and a reflux temperature, with several types of solvent extraction, experienced and recommended in the literature:  with water, cold (s1r) and hot (s1c);  methanolic solution (50:50 v/v) cold (s2r) and hot (s2c);  with ethanol 960, cold (s3r) and hot (s3c);  with moisture of methanol water acetic acid (90:9:1, v/v/v), cold (s4);  with mixture of methanol – acetone – water – formic acid (40:40:19,9:0,1, v/v/v/v), in cold conditions (s5). thus, there were analyzed eight samples for each type of spice: five extracted in cold conditions and three in hot conditions. these were obtained through the collapse of the vegetative material with the corresponding solvent. for this, 2 g of each aerial part of the dry vegetative part was extracted with 20 ml solvent (the report vegetative product / solvent is 1:10) in hot conditions, at the reflux temperature and in cold conditions, at the room temperature. the extractive solutions were filtrated and analyzed concerning the total content of the flavons (experimented in the rutoside) and total polyphenols expressed in gallic acid) and the antioxidant capacity. for the qualitative analyze of the active principals from the obtained extracts, using the method of thin layer chromatography (tlc) [7, 8]. the reference solutions were rutoside – chlorogenic acid caffeic acid (e1) and rosmarinic acid (e2). the chemical quantitative study aimed the quantification through spectrophotometry of the flavonoids and polyphenols from the analyzed samples, knowing the fact that for the spices those essentially contribute to the antioxidant activity [9]. it was used in visible and ultraviolet spectrophotometry. 2.1. the quantitative determination of the flavonoids is based on their property to form, in the presence of the cation al3+, intern complexes, coloured in vivid yellow, which measure the absorbance at λ= 430 nm. the content of total flavons was expressed in g rutoside / 100 g dried vegetative product (g/100g dvp). 2.2. the determining of the content of total polyphenols was realized through the spectrophotometric method, using the folin reactive: polyphenolic substances react with folin reagent (phosphowolframic acid in sodium carbonate medium) forming a bluecoloured complex colorimetry at λ= 760 nm. the content of total polyphenols was expressed in gallic acid. 2.3. the determination of the antiradicalic capacity was made though the determination of the capacity of neutralisation of the radical 2,2 diphenyl –1picrilhidrazil (dpph) and its transformation in its reduced form by the analyzed vegetative extracts. the results were expressed in inhibition percents of the dpph. there were used to make these determinations the following: spectrophotometers uv-vis cary 50, cecil 2020, cintra 101, v-550 jasco, applicator: camag linomat iv; hptlc plates silica gel g60f254 merck (100 x 100, 200 x 100). there was used various types of reactive: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 67  for tlc: developer: formic acid ethyl methyl ketone ethyl acetate (4:4:1:1); reagent of identification: diphenylborinate methanolic solution of β-ethylamine (np) and polyethylene glycol 400 (peg); reference solutions: rutoside – clorogenic acid – caffeic acid, rosmarinic acid;  for quantitative determination of flavonoids: ethyl alcohol 50%, v/v, 10% sodium acetate (chemical company sa) 2,5% aluminum chloride;  for determination of total polyphenols: folin reagent, sodium carbonate solution 20%, gallic acid monohydrate;  for determining the antiradicalic capacity: 2,2 diphenyl – 1 picrilhidrazil (dpph) from fluka, quercetol dihydrate (roth) absolute methanol, dimethyl sulfoxide (dmso); all chemicals used were analytically pure. 3. results and discussion 3.1. determinations by tlc chromatograms were viewed in four stages: three stages to identify the presence of flavonoids and polyphenols (figure no. 1) and a time to identify the antioxidant capacity (figure no. 2):  view at 254 nm before spraying with identification reagent; the purpose of this analysis is to identify the presence of antioxidant compounds of polyphenols type; view at 254 nm before spraying with identification reagent (np+ peg) view at 366 nm before spraying with identification reagent (np+ peg) view at 366 nm after spraying with identification reagent (np+ peg) sample l s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c sample k s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c figure no. 1 – the aspect of the plates and the spots at viewing on thin layer chromatography (tlc) for the flavonoids ad polyphenols in extracts with basil food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 68  view at 366 nm before spraying with identification reagent;  view at 366 nm after spraying with identification reagent, there are highlighted, depending on position and size of spots, the main types of flavonoids;  view after spraying with dpph reagent in order to identify the antioxidant capacity. chemical qualitative study conducted by tlc has shown that regardless the extraction solvent and extraction conditions (temperature, extraction technique), the extract obtained solutions were a mixture of phenolic substances (flavonoids and polyphenols), responsible for the antioxidant activity of plant products studied. thus:  the occurrence of grey spots – close to samples examined the chromatoplate viewed at 254 nm before spraying with identification reagent, indicating the presence of flavonoid compounds;  the occurrence of blue spots on the chromatoplate viewed at 366 nm before spraying with identification reagent, indicating the presence of polyphenol carboxylic acids.  after viewing at 366 nm and spraying with identification reagent are found rutoside, chlorogenic acid and caffeic acid in accordance with standards used. beside the characteristic spot of the chlorogenic acid, one can find an orange spot that probably belongs to lutein derivatives. immediately under the characteristic spot of the caffeic acid, we see a blue fluorescent spot with lower intensity which belongs to all polyphenol carboxylic acids. the spots characteristic to flavonoid compounds and polyphenol carboxylic acids suffer a fading action after being sprayed with a solution of dpph, indicating a consumption of this radical compound by antioxidants in the extracts and their antiradicalic effect. sample l sample k s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c s1 r s2 r s3 r s4 s5 e1 e2 s1 c s2 c s3 c figure no. 2 – the aspect of the plates and the spots at viewing on thin layer chromatography (css) for identify the antioxidant capacity comparing the aspect of the correspondent chromatoplates for samples l and k there is established that:  the content of flavonoid compounds in the sample k is lower than the sample l, although the amount and type of polyphenol carboxylic acids are comparable;  the antioxidant activity is attributed mostly mainly to polyphenol carboxylic acids;  the extracts obtained with water, 95% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 69 alcohol respectively, by stirring at room temperature are significantly lower than those obtained using the same solvent extraction, but at reflux temperature. the optimal extraction of active principles with antioxidant activity is provided with a mixture of methanol / water (50:50), at reflux temperature. 3.2. spectrophotometric determinations the values obtained at the spectrophotometry dosing of the flavonoids from the samples of spices are various (table no. 1). table no. 1 the quantitative determination of the flavonoids from basil extracts flavonoids (g / 100 g dvp) nr solvent (sample) k l 1. s1r 0,09 0,19 2. s1c 0,28 0,40 3. s2r 0,23 0,21 4. s2c 0,43 0,51 5. s3r 0,12 0,08 6. s3c 0,24 0,07 7. s4 0,23 0,13 8. s5 0,26 0,03 firstly, one can observe that for sample k the extraction in hot conditions with methanolic solution is the most complete. on the contrary, the extraction in cold conditions with ethanol the value is 4 times less than the maximum (0,43 g / 100 g dvp). the content of flavonoids decreased in extracts in the order: methanol, hot > water, hot > mixture of methanol – acetone – water – formic acid > ethanol, hot > methanol, cold and mixed water – methanol – acetic acid > ethanol, cold > water, cold. at the sample l a different order is observed for lower values. if we compare the values recorded in the determination of flavonoids in tinctures (cold extraction) this will indicate that they are much lower than extracts made at reflux temperature. this is evidence that by increasing the temperature, the extractability of flavonoids is better. modest results were obtained at the extraction with complex solvents (s4, s5). comparing the two samples, the blank sample presents superior values for the majority of the types of the extractions. these facts are due to the fact that when purchasing the materials, in the case of the company plantavorel piatra neamt one mainly takes into consideration the bioactive principles and in the case of the materials for obtaining the commercial spices, the accent is based on the sensorial characteristics (mainly on the taste). values obtained from spectrophotometric determination of total polyphenols in the two samples have a similar pattern (figure no. 3). s1r s1c s2r s2c s3r s3c s4 s5 sample k sample m 0 1 2 3 4 5 6 g g a e / 1 00 g e xtract sample k sample m figure no. 3 –the quantitative determination of polyphenols from basil extracts food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 70 the concentration of total polyphenols are in accordance with those in the literature, with one exception: the extract in cold conditions, with ethanol (table no. 2). table no. 2 comparison between the content of polyphenols of the analyzed samples and the data in the literature the content of polyphenols of the analyzed samples (g/100 g dvp) values from literature (g/100 g dvp) sample minimum maximum minimum maximum k 0,31 5,98 l 0,98 4,68 0,740 [10] 6,550 [2] one can observe the wide range of polyphenols content of basil in the date from literature (the difference between the maximum and minimum values is of 5.81 g/100 g dvp). this is because the fact that the analyzes were made on different species of plants, some of them used as spices (due mainly to their intake of volatile oils), some of them in medicinal purposes (because of their bioactive complex), the way of extracting them being different, too. from the analysis there resulted the fact that the sample obtained in hot conditions with methanolic solution was most effective in „working” with dpph radicals (table no. 3). the values obtained in determining the antiradicalic capacity are sensible for many of extraction achieved. table no. 3 the antioxidant activity of basil extracts, determined through dpph method % antiradicalic activity towards the dpph nr solvent (sample) k l 1. s1r 63,55 59,95 2. s1c 88,70 89,17 3. s2r 82,40 90,76 4. s2c 89,48 89,27 5. s3r 12,01 14,94 6. s3c 53,23 91,90 7. s4 4,72 75,1 8. s5 75,35 8,14 is is also noted that there were obtained high values of antiradicalic capacity for extracts wit complex solvents, that is extracts in cold conditions, although the values obtained for polyphenolic compounds have, mostly modest values. 4. conclusions from the obtained results we can conclude:  the extract obtained solutions were a mixture of phenolic substances (flavonoids and polyphenols), as demonstrated by qualitative chemical study performed by tlc and by quantitative measurements;  flavonoids are extracted more efficiently heat (methanol, and water);  methanol (and comparable, water), hot, achieved good extraction of polyphenols, both working sample and the blank;  the antioxidant activity is attributed mostly mainly to polyphenol carboxylic acids (according to qualitative analysis, the most effective "sweeping" radical dpph hot extracts were performed with methanol and water solution;  comparing the two samples (k and l), the reference sample presents superior values for the majority of the types of the extractions, so when choosing the materials for obtaining spices one should pay attention to their content in chemical compounds that gives them a high antioxidant potential. in this way it is possible to increase the dietary intake of antioxidants with beneficial effects for health; analysed bioactive compounds are very well represented in the basil, but their highlight requires the choice of optimal conditions for extraction. 5. references [1]. cai t., wang y., et al. ., a review of antioxidant activity studies using electron food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xi, issue 4 – 2012 71 paramagnetic resonance methods[j], progress in chemistry, vol.23(9), 2011, p. 1959-1972 [2]. javanmardi, j., et al., antioxidant activity and total phenolic content of iranian ocimum accessions, food chemistry, vol. 83, 2003, p. 547550 [3]. chan, e., lim, y, chong, l., tan j., wong s., antioxidant properties of tropical and temperate herbal teas, j food compos anal, vol. 23(2), 2010, p. 185-189. [4]. chan, e., et al, rosemary and sage outperformed six other culinary herbs in antioxidant and antibacterial properties, international journal of biotechnology for wellness industries, vol. 1, 2012, p. 142-151 [5]. zheng, w., wang, s.y., antioxidant activity and phenolic compounds în selected herbs, j agric food chem, vol. 49, 2001, p. 5165–5170 [6]. pettinari, a., effect of polyphenolic compounds on the proteolytic activities of constitutive and immuno-proteasomes, antioxid redox signal, vol. 8, 2006, p. 121-129 [7]. * * *, farmacopeea romana, 10th ed., ed. medicală, bucuresti, 1998 [8]. ***, european pharmacopoeia, 7th ed., council of europe, strasbourg, 2011 [9]. hossain, m., barry-ryan c., martin-diana a., brunton, n., effect of drying method on the antioxidant capacity of six lamiaceae herbs, food chem, vol. 123(1), 2010, p. 85-91 [10]. proestos, c., et al., rp-hplc analysis of the phenolic compounds of plant extracts. investigation of their antioxidant capacity and antimicrobial activity, journal of agricultural and food chemistry, vol. 53, 2005, 1190-1195 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 17 biotechnology of edible mushrooms cultivation on vine and winery wastes marian petre1, alexandru teodorescu1 1university of pitesti, faculty of sciences, romania corresponding author address: 1 targul din vale street, pitesti, arges county, 110040, marian_petre_ro@yahoo.com abstract. every year, in romania huge amounts of wine and vine wastes cause serious environmental damages in vineyards as well as nearby winery factories, for instance, by their burning on the soil surface or their incorporation into soil matrix. the optimal and efficient way to solve these problems is to recycle these biomass wastes as main ingredients in nutritive composts preparation that could be used for edible mushrooms cultivation. in this respect, the main aim of this work was to establish the best biotechnology of winery and vine wastes recycling by using them as appropriate growth substrata for edible and medicinal mushrooms. according to this purpose, two mushroom species of basidiomycetes, namely lentinula edodes as well as pleurotus ostreatus were used as pure mushroom cultures in experiments. the experiments of inoculum preparation were set up under the following conditions: constant temperature, 23°c; agitation speed, 90-120 rev min -1; ph level, 5.0– 6.0. all mycelia mushroom cultures were incubated for 120–168 h. in the next stage of experiments, the culture composts for mushroom growing were prepared from the lignocellulose wastes as vine cuttings and marc of grapes in order to be used as substrata in mycelia development and fruit body formation. the tested culture variants were monitored continuously to keep constant the temperature during the incubation as well as air humidity, air pressure and a balanced ration of molecular oxygen and carbon dioxide. in every mushroom culture cycle all the physical and chemical parameters that could influence the mycelia growing as well as fruit body formation of l. edodes and p. ostreatus were compared to the same fungal cultures that were grown on poplar logs used as control samples. keywords: biomass, composts, edible mushrooms introduction the agricultural works as well as the industrial activities related to wine crops and wine processing have generally been characterized by huge formation of wide range of waste products. many of these lignocellulose wastes cause serious environmental pollution effects, if they are allowed to accumulate in the vineyards or much worse to be burned on the soil [1, 2]. the solid substrate fermentation of plant wastes from agro-food industry is one of the most challenging and technically demanding biotechnologies known to humankind [3-5]. the major group of fungi to degrade cellulose and lignocellulose are the edible mushrooms of basidiomycetes class [6-9]. the main aim of this work was to find out the best biotechnology of recycling winery and vineyard wastes by using them as a growing source for edible mushrooms and, last but not least, to protect the vineyard ecosystems [9-12]. taking into consideration that most of the edible mushrooms species requires a specific micro-environment including complex nutrients, the influence of all physical and chemical factors upon fungal food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 18 biomass production and mushroom fruit bodies formation has been studied by testing new biotechnological procedures [7-12]. materials and methods according to the main purposes of this work, two fungal species of basidiomycetes group, namely lentinula edodes (berkeley) pegler (folk name: shiitake) as well as pleurotus ostreatus (jacquin ex fries) kummer (folk name: oyster mushroom) were used as pure mushroom cultures isolated by authors from the natural environment and now being preserved in the local collection of the university of pitesti. the stock cultures were maintained on malt-extract agar (mea) samples (20% malt extract, 2% yeast extract, and 20% agar-agar). samples were incubated at 25°c for 120-168 h and stored at 4°c. the pure mushroom cultures were expanded by growing in 250-ml flasks containing 100 ml of liquid malt-extract medium at 23°c on rotary shaker incubators at 110 rev. min -1 for 72-120 h. to prepare the inoculum for the spawn cultures of l. edodes and p. ostreatus the pure mushroom cultures were inoculated into 100 ml of liquid malt-yeast extract culture medium with 3-5% (v/v) and then maintained at 23-25°c in 250 ml rotary shake flasks. the experiments of inoculum preparation were set up under the following conditions: constant temperature, 25°c; agitation speed, 90-120 rev. min -1; initial ph, 5.5– 6.5. all the seed mushroom cultures were incubated for 120–168 h. after that, the seed cultures of these mushroom species were inoculated into liquid culture media (20% malt extract, 10% wheat bran, 3% yeast extract, 1% peptone) at ph 6.5 previously distributed into rotary shake flasks of 1.000 ml. during the incubation time period, all the spawn cultures were maintained in special culture rooms, designed for optimal incubation at 250c. three variants of culture compost made from marc grapes and vineyard cuttings in the following ratios: 1:1, 1:2, 1:4 (w/w) ere prepared. the vine and winery wastes were mechanically pre-treated by using an electric grinding device to breakdown the lignin and cellulose structures in order to make them more susceptible to the enzyme actions [10-12]. all the culture compost variants made from ground vineyard and winery wastes were transferred into 1.000 ml glass jars and disinfected by steam sterilization at 1200 c for 60 min. after the jars filled with composts have been chilled, they were inoculated with liquid spawn already prepared. each culture compost variant for mushroom growing was inoculated using liquid spawn having the age of 72–220 h and the volume size ranging between 3–9% (v/w). during the period of time of 18–20 d after this inoculation, all the mushroom cultures had developed a significant mycelia biomass on the culture substrata made from vineyard cuttings and marc of grapes [10-12]. results and discussion according to the registered results of the performed experiments the optimal laboratory-scale biotechnology for edible mushroom cultivation on composts made from marc of grapes and vineyard cuttings was established. as it is shown in fig. 1, two technological flows were carried out simultaneously until the first common stages of the inoculation of composts with liquid mushroom spawn followed by the mushroom fruit body formation. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 19 fig. 1. scheme of laboratory-scale biotechnology for edible mushroom production by recycling winery and vineyard wastes series of three cultivation cycles were made and the effects induced by some additional ingredients as carbon sources (xylose, sucrose, maltose, glucose) upon the mycelia growing during each cultivation period were investigated. as it could be noticed in figure 2, each carbon source was added to the basal composts at a concentration level of 5% (w/w) and the incubation time period lasted for 168-288 h [12-14]. maltose, as one of all mostly tested carbon sources, had shown the highest influence upon the mycelia growing and fresh fungal biomass production about of 28–35g%. g lu co se m al to se su cr os e x yl os e p. o l.e p. o l.e. p. o l.e.p. ol.e. 0 10 20 30 40 50 60 70 80 90 100 f un ga l b io m as s w ei gh t (g % ) carbon sources (5% w/w) fig. 2. comparative effects of carbon sources upon mycelia growing of p. ostreatus (p.o.) and l. edodes (l.e.) pure mushroom cultures inoculum preparation and growing on culture media addition of carbon, nitrogen and mineral sources to the compost variants growing of liquid mushroom spawn in nutritive media steam sterilization of the filled jars transfer of each compost variant to 1000 ml jars inoculation of the filled jars with liquid mushroom spawn expanding of pure mushroom cultures by growing in liquid media spawn growing on the composts made from winery and vineyard wastes mushroom fruit body formation and development mushroom fruit bodies cropping mechanical pre-treatment of winery and vineyard wastes by grounding food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 20 the effects of nitrogen sources were noticed and registered as they are shown in figure 3. w he at b ra n p ep to ne m al t e xt ra ct t ry pt on e y ea st e xt ra ct l.e.p. ol.e. p. o p. o l.e.p. o l.e.p. o 0 10 20 30 40 50 60 70 80 90 100 f un ga l b io m as s w ei gh t ( g% ) nitrogen sources (3% w/w) l.e. fig. 3. comparative effects of nitrogen sources upon mycelia growing of p. ostreatus (p.o.) and l. eodes (l.e.) from all tested nitrogen sources (wheat bran, peptone, malt extract, tryptone and yeast extract), only the wheat bran was the most efficient upon the mycelia growing and fungal biomass producing at 35-40 g% fresh fungal biomass weight, being closely followed by the malt extract at 25–30 g%. peptone, tryptone and yeast extract are also well known as nitrogen sources for fungal biomass synthesis but their efficiency in these experiments was relatively lower than the mycelia growing and fungal biomass production induced by the wheat bran added as natural organic nitrogen sources [15-17]. among the various mineral sources examined, such as: caco3, caso4, k2hpo4, mgso4 · 5h2o, kh2po4, the first one, caco3, had the best mycelia growing yielded. in this case, the fungal biomass production was registered at 28-32 g% and for this reason the most appropriate mineral source was caco3 (fig.4). c ac o 3 c as o 4 k h 2p o 4 m gs o 4• 5h 2o k 2h p o 4 l.e.p. o l.e.p. o l.e.p. op. ol.e. p. o 0 10 20 30 40 50 60 70 80 90 100 f un ga l b io m as s w ei gh t ( g% ) mineral sources (1% w/w) l.e. fig. 4. comparative effects of mineral sources upon mycelia growing of p. ostreatus (p. o.) and l. edodes (l.e.) all the experiments were carried out for 288 h at 25°c with the initial ph 6.5 and data are the means of triple determinations carried out on the variants of composts made from vineyard cuttings and marc of grapes in the ratio 1:4. similar observations were made by stamets (1993), during the experiments concerning other techniques of mushroom cultivation as well as other researchers [1517]. also, other tested mineral sources, such as mgso4 · 5h2o have shown an optimal influence upon the fungal biomass growing [17-18]. the mineral sources k2hpo4 and kh2po4 as essential phosphates could improve the ph level through their buffering action, but they were less appropriate for mycelia growing in submerged as well as in surface cultures of mushrooms. the whole period of mushroom growing from the inoculation to the fruit body formation lasted between 30–60 d, depending on each fungal species used in the experiments. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 21 during the whole period of fruit body formation, the culture parameters were set up and maintained at the following levels, depending on each mushroom species: air temperature, 15–170c; the air flow volume, 5–6m3/h; air flow speed, 0.2–0.3 m/s; the relative moisture content, 80– 85%, light intensity, 500–1.000 luces for 8–10 h/d. the final fruit body production of these mushroom species used in the experiments was registered between 1.5 – 2.8 kg relative to 10 kg of composts made from vineyard and winery wastes. conclusions 1. the registered data revealed that by applying this biotechnology, the winery and vineyard wastes could be recycled as useful raw materials for culture compost preparation to get edible mushrooms. 2. maltose, as one of all mostly tested carbon sources, had shown the highest influence upon the mycelia growing and fresh fungal biomass production about of 28–35 g%; 3. among the five nitrogen sources examined, wheat bran was the most efficient one upon the mycelia growing and fungal biomass production of l. edodes and p. ostreatus, at 35-40 g% fresh fungal biomass weight, closely followed by malt extract at 25–30 g%. 4. caco3 was registered as the best mineral source, yielding the best mycelia growing as well as fungal biomass production at 28-32 g%. 5. the final fruit body productions of these two mushroom species were registered between 1.5–2.8 kg relative to 10 kg of composts made from vineyard and winery wastes. acknowledgements this work was supported by the contract no. 51-002/2007, in the framework of national research program pn ii, granted by the romanian ministry of education and research. references 1. p. beguin and j.p. aubert, fems microbiol. rev., 13, 25 – 58 (1994). 2. m.j. carlile and s.c. watkinson, “fungi and biotechnology”, in the fungi, edited by m.j. carlile and s.c. watkinson, academic press, london, 1996, pp. 214221 3. d.s. chahal, “biological disposal of lignocellulosic wastes and alleviation of their toxic effluents” in biological degradation and bioremediation of toxic chemicals, edited by g.r. chaudry, chapman & hall, london, 1994, pp. 347-356. 4. d.s. chahal and j.m. hachey, am. chem. soc. symp., 433, 304 – 310 (1990). 5. s.t. chang and w.a. hayes, the biology and cultivation of edible mushrooms, academic press, new york, 1978, pp. 114-143. 6. r.t. lamar, j.a. glaser, t.k. kirk, “white rot fungi in the treatment of hazardous chemicals and wastes”, in frontiers in industrial mycology, edited by g.f. leatham, chapman & hall, new york, 1992, pp. 127-143. 7. j.g. leahy and r.r. colwell, microbial rev., 54, 305–315 (1990). 8. a. moser, acta biotechnology 12, 2-6 (1994). 9. m. petre, biotechnology for microbial degradation and conversion of plant constituents, ed. didactica si pedagogica, bucuresti, 2002, pp. 140-214 10. m. petre, c. bejan, e. visoiu, i. tita, a. olteanu, intl. j. of med. mush., 9(3), 241-243 (2007). 11. m. petre, v. petre, journal of environmental protection and ecology, 9(1), 88-97 (2008). 12. m. petre, a. teodorescu, g. dicu, intl. j. of med. mush. 7(3), 444-446 (2005). 13. m. ropars, r. marchal, j. pourquie, j.p., vandercasteele, biores. technol., 42, 197-203 (1992). 14. j.e. smith, biotechnology, third edition, cambridge university press, 1998, pp. 95-110. 15. p. stamets, growing gourmet and medicinal mushrooms, ten speed press, berkeley, toronto, 1993, pp. 390-400. 16. w. verstraete and e. top, holistic environmental biotechnology, cambridge university press, 1992, pp. 120-128. 17. m. wainwright, an introduction to fungal biotechnology, wiley-chichester, 1992, pp. 97-115. 18. g. zarnea, tratat de microbiologie generala, vol 5, ed. academiei, bucuresti, 1994, pp. 93-120. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 40 selectivity of calix [6]arene-coated piezoelectric quartz crystal sensor for the detection of some organic amine in gas flow iosif gergen1, despina maria bordean1, monica harmanescu3, florina radu1, marilena miclau2 1. usamvb-faculty of food technology, timisoara, calea ardului 119, ro 300645, igergen@yahoo.com 2. incemc (institutul national de c-d pentru electrochimie si materiale condensate), timisoara 3. usamvb-agriculture faculty, timisoara abstract: in this study, we developed calixarene-coated pqc sensors for the determination of organic amines in gas faze and study the selectivity of this coated sensor for normal and isoform of propyl and butyl amines. p-tert-butylcalix[6]arene compound was applied as piezoelectric quartz crystal (pqc) sensory coatings for the selective detection of volatile organic in gas phase. the at-cut quartz crystals used in this study (stanford research systems) having a diameter of 25.4 mm and a thickness of 0.33 mm. an oscillator circuit providing an alternating voltage will produce a fundamental frequency of 5 mhz for this particular crystal diameter and thickness. about 10.0 l of analyte was injected into the mixing chamber with an approximately volume of 250 ml. the measuring chamber with coated pqc has a volume of approximately 5 ml. the concentration of amine sample in mixing chamber and respectively in gas flow was around 40 ppm. al experiments were conducted at 30oc. the complexation based on the host–guest interaction between the calixarene coating and the analyte makes the coated pqc sensors sensitive to organic amines and results in quasi-irreversible frequency changes. a pqc sensor coated with a p-tert-butylcalix[6]arene exhibits different selectivity for amines in gas phase, in the order: iso-propylamine< iso-butylamine<<n-butylamine. the relative selectivity of the analytes is explained by the cavity inclusion of analyte molecule, complete or partial, or by steric hindrance interaction. the application of pqc sensors to gas faze has provided new analytical possibilities for the food control, environmental science, biotechnology and medicine. keywords: food quality, volatile amines, calixarene, sensitive membranes introduction the detection of food quality and freshness is of increasing interest both for the consumer and food industry. amines, especially volatile amines, are the principal compounds formed by the activity of bacterial amino acid decarboxylase during the degradation processes of proteins, which have a carcinogenic effect on human body and can be used to indicate bacterial contamination, respectively food quality and freshness. gas chromatography methods are the best for control and monitoring the quality and freshness of food. but these methods usually require long time and pre-treatment processes which are not suitable to perform an in situ analysis and field application [1]. developments in quartz crystal microbalance (qcm) sensor technology has progressed in the area of gas phase analysis since the first report in 1964, where king [2] used a qcm as a gas chromatograph absorption detector. since then, a series of reports of other detection schemes for different gas phase analytes have appeared in the literature. the change food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 41 of resonance frequency (f in hz) related to the mass (m in g) deposited on the crystal surface of area (a in cm2) and the fundamental frequency (f0 in mhz) of the pqc can be calculated from sauerbrey’s equation [3]: f=−2.26×10−6×f0 2 ×(m/a) (1) the theoretical detection limit of an oscillating quartz crystal is reported to be as low as 10-12 g (if f0 = 10 mhz) for coating materials according to sauerbrey’s equation. piezoelectric quartz crystal (pqcs) with appropriate coating materials can interact effectively with specific analytes. to improve the sensitivity and selectivity of pqc sensors, lately macrocyclics, e.g., crown ethers, cryptands, cavitants, cyclodextrins and calixarenes, have been used as coatings for gas detection. simple dispersion forces and dipole–dipole interactions lead to sensor signals with poor selectivity but good reversibility. hydrogen bonding (biochemistry) and steric interactions (key– lock principle) between the sensor coating and the analyte result in an increased signal intensity but it is quasiirreversible. the application of pqc sensors to gas faze has provided new analytical possibilities for the food control, environmental science, biotechnology and medicine [4]. in this study, we developed calixarenecoated pqc sensors for the determination of organic amines in gas faze and studied the selectivity of this coated sensor for normal and isoform of propyl and butyl amines. experimental quartz crystal microbalance the at-cut quartz crystals used in this study were purchased from stanford research systems (sunnyvale, california) [5] having a diameter of 25.4 mm and a thickness of 0.33 mm. they were patterned with two concentric gold-on-chrome electrodes having a wrap around geometry that allows both ground and radio frequency q connections to be made to one side. application of a voltage to the two electrodes produces a strain in the surface of the qcm along the cut of the crystal. an oscillator circuit providing an alternating voltage will produce a fundamental frequency of 5 mhz for this particular crystal diameter and thickness. the qcm200 system is a stand-alone instrument with a built-in frequency counter and resistance meter. it includes controller, crystal oscillator electronics, crystal holder, and quartz crystals. series resonance frequency and resistance are measured and displayed directly on the front panel, and there is an analogue output proportional to the relative frequency to interface directly with potentiostats. in addition, the qcm200 has an rs-232 interface and comes with both windows and mac software providing real-time display, analysis and storage of your qcm data. figure 1 depicts the experimental setup of the pqc sensor detection system with the assembled computer interface. the frequency of the vibrating crystal was measured with a qcm 200 (stanford research systems) type frequency counter connected to a microcomputer system. about 10.0 l of analyte was injected into the mixing chamber with an approximately volume of 250 ml. the measuring chamber with coated pqc has a volume of approximately 5 ml. the concentration of amine sample in mixing chamber and respectively in gas flow was around 40 ppm. all experiments were conducted at 30oc. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 42 computer qcm 200 m sample injection mixture chamber flow meter waste colection n2 flow n2 carrier gas coated quartz crystal chamber figure 1. experimental set-up for the calixarenecoated pqc sensor and amines. coatings the coating solutions were prepared by dissolving 20 mg of the calixarene in 5 ml of chloroform. the concentrations of the resulting solutions were about 4 mg/ ml. superior sides of the crystals were coated with the calixarene solution via the dropping method with a micro-syringe. an aliquot of 3.0 l of calixarene was dropped on to superior side of the quartz crystals. a thin membrane was obtained after solvent evaporation and dried with a hot air blower for approximately 30 s. reagents the analytes iso-propylamine (98%), nbutylamine (99%), iso-butylamine (98.5%), and pure solvents were all of analytical-reagent or chemically pure grade (sigma aldrich). the boiling point and molecular mass is presented in table 1. pure n2 was used as carrier gas. table 1. boiling point and molecular mass of amines nr. compound bp (0c) mw 1 iso-propylamine 33 59.11 2 iso-butylamine 68-69 73.14 3 n-butylamine 78 73.14 sensing material, calixarenes (ca) are cyclic oligomers containing large-scaled molecular cavities, dimensions of which depend on the number of aryl fragments (usually 4, 6, 8) in macro-cycle (figure 2). various functional groups can be bound to upper and lower rim. it is known that calixarenes can form “host–guest” complexes. calixarene molecule is as a “host”, while metal ions and different organic compounds are as “guests”. varying the number of aryl fragments and functional groups one can easily manage with sensitivity and selectivity of calixarene films [6]. especially the cavity of calix[6]arene (2.1-2.8 å cavity diameter) is sufficiently large to accommodate organic amine guests, whereas that of calix-[4]arene is too small [7]. it is probable that the close correspondence between the relative magnitudes of the organic amine (ionic diameter of ammonium ion = 2.96 å) and the calixarene cavity diameters is an important factor in determining the complexation interactions between the calixarene and the amine guests [8]. figure 2. general structures of the p-rcalix[n] arene [7]; r=tert-butyl, ri= oh and n=3 for p-tert-butylcalix[6]arene. results and discussion the response curves for calixarene coated pqc sensor detection systems for 40 ppm of 3 organic amines at 30 °c in the measuring chamber are shown in figure 3 and absolute area values of measurement in table 2. figure 3. the response characteristics of pqc sensors coated with calyx[6]arene to 40 ppm of 3 organic amines. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 43 table 2. absolute response for three amines detected in n2 flow with the pqc sensor the response characteristics of pqc sensors coated with p-tertbutylcalyx[6]arene to 40 ppm of organic amines decrease in the order n-butylamine>>iso-butylamine>isopropylamine. this is attributed to the extra lypophilic stabilized interaction between the host (calixarene) and guest (amines) molecules. organic amines are incorporated in the cavity of calixarene (ca) to form endo-ca complex and the complex involves hydrogen bonding with inclusion of the non-polar moiety of the organic amines in the ca cavity. on the basis of the data in figure 3, it can be seen that n-butylamine have remarkable higher responses and iso-butylamine and isopropylamine have relative lower responses. this is due to the bulkiness of their nonpolar moieties that cannot be incorporated into the cavity of ca, while n butylamine, can be incorporated into the cavity of ca to form hydrogen bonding [4]. in similar experiment but with acetate derivatized calixarene (ri = acetate), wang et al [4] obtain similar results for linear long chain amines (n-butylamine, n-hexylamine, noctylamine and dodecylamine). it is concluded that a long alkyl group is another important factor controlling the response of organic amines. conclusion in this study, we investigated p-tertbutylcalix[6]arene-coated pqc sensors for the detection of organic amines in gas flow. the relative sensitivity of amines is based on host–guest complexation between the organic amine and the calixarene. stronger responses were observed for nbutylamine molecules which are easy included in the calyx[6]arene cavity. the response of the amines with a bulky group (iso-forms) is weak, since the bulky group hinders the amine from being included in the cavity. acknowledgement the authors are grateful to the cncsis romania for financial support (grant pncd ii prog. 4 – nr. 102/01.10.2007, new α-quartz structure compounds performat piezoelectric sensor for food quality and safety, senzali). references 1. guang l., junbao z., xingfa m., yu s., jun f., gang w.; development of qcm trimethylamine sensor based on water soluble polyaniline, sensors, 2007, 2378-2388. 2. king, w.h. piezoelectric sorption detector, anal. chem.,1964, 36, 1735-39 3. sauerbrey, g. verwendung von schwingquarzen zur wagung diinner schichten und zur mikrowiigung, z. phys., 1959, 155,206. 4. chun w., xi-wen h., chen l.x. : a piezoelectric quartz crystal sensor array self assembled calixarene bilayers for detection of volatile organic amine in gas, talanta, 2002,57, 1181–1188. 5. stanford research system, qcm200,quartz crystal microbalance, digital controller qcm25, 5 mhz crystal oscillator, operation and service manual, , revision 2.1(6/2005) 6. koshets i.a., z.i. kazantseva, yu.m. shirshov, s.a. cherenok, v.i. kalchenko, calixarene films as sensitive coatings for qcm-based gas sensors, sensors and actuators b 106, 2005, 177–181. 7. ursale t.n., i. silaghi-dumitrescu, e.-j. popovici, a. ursale, n. popovici; new materials based on phosphorilated calix[n]arene, journal of optoelectronics and advanced materials vol. 6, no. 1, march 2004, p. 307 – 313. 8. chen l.x. , xi-wen he, xu-bo hu, hua xu; calixarene-coated piezoelectric quartz crystal sensor for the detection of organic amine in liquids, analyst, 1999, 124, 1787–1790. amines (number of determina tion) iso propyl amine (4) isobutyl amine (4) n-butyl amine (2) area, sqm average values 118 222 1090 cercetări privind obținerea unui conținut cât mai ridicat de β-glucani din diferite surse de drojdii pentru valorificarea potențialului biologic activ 190 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 190 195 research on obtaining high β-glucans content from different sources of yeast by harnessing their biologically active potential ionuț avrămia 1 , *sonia amariei 1 1faculty of food engineering, ștefan cel mare university of suceava, universitatii 13, ro 720229 suceava, romania *corresponding author received 9th october 2017, accepted 29th november 2017 abstract: isolated polysaccharides from different natural sources have gained a real interest from the scientific community due to biologically active effects on body functions such as: lipid metabolism correction, immune stimulating, glycemic control in type ii diabetes, antitumor activity, etc. a category of these polysaccharides is β-glucans, β-d-glucose polymers produced by various organisms such as bacteria, yeasts, algae and plants. this paper presents the experimental results obtained from the analysis of four types of yeast available on the market in order to select the one with the highest content of β-glucan, which can be later exploited in various fields: medical, pharmaceutical, food or cosmetics. according to the experimental data, the highest level of β-glucans content is represented by beer yeast, 21.49% higher than bakery yeast and 36.36% higher than wine yeast. keywords: β-glucan, yeast, immunity system, food supplement. 1. introduction β-glucans are widely spread biopolymers of glucose in many prokaryotic and eukaryotic organisms. although they derived from different sources of yeast, fungi, algae, mold, oats or barley, βglucans exhibit some biologically active features depending on their origin. thus, some authors state that β-glucans isolated from yeast saccharomyces cerevisiae have the highest biological effect [1]. immunomodulation induced by β-glucans has been demonstrated both in vitro and in vivo in numerous studies on animals and humans in a wide range of tumors of breast, lung and gastrointestinal cancers [1]. β-glucans are formed at several cellular structures and are predominantly found in the cell wall of the yeast. in the s. cerevisiae yeast cell wall structure is 1525% of the dry matter of the cell [2] and 25-50% by volume based on a calculation carried out at the electronic microscope [3]. the cell wall consists of three layers: glucanic, mannanic and lipidic, and the percentage in these compounds varies from one strain to another and depends largely on cultivation conditions including: nutrition, oxygenation, temperature and ph of the nutrient medium [4]. table 1. average cell wall content [8] component mass (% s.u.) (1→3)-β-d-glucan 50-55 (1→6)-β-d-glucan 5-10 (1→4)-α-(1→3)-β-d-glucan 3-7 manoprotein complex 35-40 chitin 2 http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 ionuț avrămia, sonia amariei, research on obtaining high β-glucans content from different sources of yeast harnessing their biologically active potential, food and environment safety, volume xvi, issue 4 – 2017, pag. 190 – 195 191 table 1 shows that glucans represent the major components of the cell wall in the proportion of about 60% of the dry matter, which is favorable in the subsequent isolation processes. from the literature it was found that the three dimensional structure of the glucan is essential for the biologically active effect on the body. thus, some authors consider the triplehelix conformation to be the most active [1, 5]. β-1,3 glucan forms a microfibrilar mesh, visible through electron microscopy, to the internal surface of the cell wall. it has a molecular weight of 240 kda and a maximum fiber length of 600 nm [3]. the greatest part of the β-1,3 glucan has a helical conformation on the basis of in vitro studies demonstrated and confirmed by nuclear magnetic resonance of intact solid state yeast cells [3]. thus, the helical structure consists of three hydrogen chains (triple helix conformation). the chains are separated at a distance of 1.56 nm with a 0.6 nm period between the fibers, as a result defining six units of β-dglucopyranose each helical structure [6]. β-1,6 glucan is a highly branched polysaccharide that binds each component of the cell wall [3]. in contrast to the microfibrilar structure of β-1,3 glucan, in the saccharomyces cerevisiae yeast cell the polymer is amorphous in structure, bonded in β-1,6 position, shorter and acts as a flexible glue to form crosslinks with β1,3 glucan, chitin and manoproteins [7]. the main challenge in the process of isolating glucan is to remove impurities such as mannoproteins and lipids without a significant loss of biologically active compounds. the american food and drug administration (fda) has named s.cerevisiae β-glucans with the "generally recognized as safe" (food and drug administration, 1997) and may be used as ingredients in the food industry but not as additives [6]. the european food safety authority (efsa) has issued an opinion that yeast βglucans are a "safe food ingredient" that can be used as a dietary supplement at a concentration of up to 375 mg/day and in foods for "special nutritional use"at doses up to 600 mg/day [8, 9]. however, these do not exclude the possibility of adverse reactions from impurities resulting from isolation processes (in rare cases the accumulated proteins can cause allergies) [1]. research on glucans focuses primarily on two similar medical domains. the most recent and important area of research is the stimulation of β-glucans immunity by binding to dectin-1 and tolllike (tlr) receptors [10, 11]. the other field of study is anti-tumor activity mediated by macrophage cells [10]. specifically, β-glucans do not directly attack infected cells, or agents that cause infections, but they modulate the host's defense system by stimulating the production of precursor cells, which are distributed by the blood stream in various lymphoid organs of the body. regarding the second field of study, some reports have shown that triple helix conformation plays a decisive role in the antitumor activity and cytokine secretion [10]. moreover, in vitro studies have shown that the introduction of sulfate groups in the structure of β-glucan increases the inhibitory activity against the proliferation of sarcoma-180 tumor cells [10, 12]. in the two main areas of use (immunomodulation and antitumor activity), β-glucans also find medical applications in: development of new polyvalent vaccines, prevention of infections, reduction of cholesterol, glycemic control, wound healing, potential for healing of digestive ulcers and proved to have a prebiotic effect [8, 13, 14]; and in the food industry as: thickening agents, water retention, oil binders or stabilizers of some emulsions without affecting taste and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 ionuț avrămia, sonia amariei, research on obtaining high β-glucans content from different sources of yeast harnessing their biologically active potential, food and environment safety, volume xvi, issue 4 – 2017, pag. 190 – 195 192 smell [15, 16]. adjustment of glycemic levels is explained by the rheological ability of β-glucans to increase the viscosity of the solutions, molecular weight dependent properties, solubility and concentration of the glucose polymer [17] that reduces glycemic response by complexing with ingested carbohydrates leading to slow release of carbohydrates in the blood [18]. with a strong topical effect, creams containing β-glucans stimulate skin macrophages that are considered to be the first barrier to cellular defense against infectious agents [14]. this property is especially beneficial in treating wounds in people with diabetes who have a condition called diabetic neuropathy as the wounds heal heavily, have a high susceptibility to developing infections and skin ulcers. beyond preparations for human use, there are also some examples of using glucan as antiviral agents in plants such as: protecting numerous tobacco species against mosaic virus invasion or against the virus that causes the black ring in tomatoes. these plants can be treated by injection or spraying with the glucanic polymer [19]. 2. materials and methods materials. four different types of active yeasts of the species saccharomyces cerevisiae available on the market were used in this study. of these, two types of baker's yeast (noted as: sample 1 and sample 2) were chosen from a native producer and import yeast, a yeast brew species (sample 3), and a wine yeast strain (sample 4). all other chemicals used in this research were of analytical grade and freshly prepared. methods. isolation of β-glucans from yeast cells of s. cerevisiae was carried out in two steps: 1. destruction of the cell walls followed by centrifugation to separate the insoluble cytoplasmic compounds; 2. the proper extraction of the cell suspension using the alkaline-acid method patented by james et al. (1989) and modified by usatii and chiseliţă, (2015) [5, 20]. determination of β-glucans content was performed gravimetrically. induction of autolysis. the destruction of cell walls was accomplished by induced autolysis. thus, the mixture of 10 g of yeast (30% v/v) and 10 ml of sterile water was subjected to heat treatment at 55° c for 8 hours. the obtained autolysate was centrifuged at 3500 r.p.m. for 10 minutes to separate the suspension component from the yeast extract. it is also known from the bibliographic study that autolysis proceeds under optimal conditions at a temperature of 50° c [21]. extraction and isolation of β-glucan. the cell disintegration sediment is treated with 50 ml of 1n naoh for 1 hour on the water bath at 90±5°c. after alkaline treatment, the cell walls are separated and treated with 0.5 n acetic acid in volume 1: 5 at 75±5°c for 1 hour to remove glycogen. the deposit was washed twice with distilled water, centrifuged at 3500 r.p.m. for 10 minutes and dry in the oven at 50 ± 5°c. the resulting product consists of a mixture of β-1,3 and β-1,6 glucans. 3. results and discussion for the presentation of glucan content from different yeast brands, a table has been compiled that includes the amount of polysaccharides of bioactive interest as a percentage and weight of the yeast active biomass. the results are shown in the table 2. the results of analyzes of the four types of active yeasts obtained from different producers, table 2, showed that the maximum percentage of β-glucans content food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 ionuț avrămia, sonia amariei, research on obtaining high β-glucans content from different sources of yeast harnessing their biologically active potential, food and environment safety, volume xvi, issue 4 – 2017, pag. 190 – 195 193 was identified in the brewery yeast (15.91%) followed by the two bakery yeast samples (12.49%) respectively (12.10%) and finally the lowest concentration (5.78%) was identified in the wine yeast. the variations in β-glucan content from the four samples can be explained mainly by the diversity of yeast strains subjected to isolation but it should not be neglected and the influence of growth factors on the carbohydrate accumulation in yeast biomass. in order to evaluate the yield of the glucan from the four yeast sources, we have chosen to refer to the highest level of polysaccharides obtained from the extraction. the figure 1 shows the percentage of glucan isolate from samples. table 2 content in β-glucans relative to active levurian biomass. sample number biotech purpose active yeast mass (g) βglucans mass (g) glucan extract (%) 1. bakery 10 1.2106 12.10 2. bakery 10 1.2491 12.49 3. brewery 10 1.5913 15.91 4. winery 10 0.5786 5.78 fig. 1. influence of yeast strains on β-glucan accumulation. the results show that 21.5% more glucans are found in the beer yeast than in the baking yeast (sample 2) and 63.68% more than in the wine yeast (sample 4). no significant variation in β-glucans content (12.49% versus 12.10%) was observed between the two yeast samples that indicates regularities of analyzed strains. the present research on the isolation of bioactive compounds from the wall of yeast saccharomyces cerevisiae by the method of alkaline-acid are consistent with other studies carried out by different authors in particular conditions, and by optimizing the culture medium from strains patented as producing β-glucans end to obtain a maximum of up to 29.47% ± 0.52% β-glucans [22]. similar research on three different types of baking yeast reveals a maximum content of 7.35% βglucans [23], compared to 12.49% obtained in this paper. similar studies in the yeast resulting from the fermentation process by spray drying shows a content of 23.59% ± 0.34% of the total cell mass [15]. the composition of the β-glucans of the yeast study examined in this work is limited to 15.91% that can be explained by replacing the drying process to isolate the glucan or by using other yeast strains. also, similar results on the level of βglucans were recorded in the strain saccharomyces cerevisiae cnmn-y-20 (approx. 20%) using the same autolysis and extraction methods [5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 ionuț avrămia, sonia amariei, research on obtaining high β-glucans content from different sources of yeast harnessing their biologically active potential, food and environment safety, volume xvi, issue 4 – 2017, pag. 190 – 195 194 maximum values were also obtained in s. cerevisiae cnmn-y-18 (18.54%) and s. cerevisiae cnmn-y-19 (17.61%) [24]. as noted, the literature shows that under normal conditions and using the alkali-acid method, a maximum level of yeast glucan content from yeasts available on the market are up to 15-20% of the active mass. fig.2. β-glucan mixture from bakery yeast(increased to 1.50kx.) the sem microscopic image of the β-1,3 and β-1,6 glucans mixture, fig. 2, the microfibrilar structure of β-1,3 glucan and the highly branched polymer with binary branching of β-1,6 glucan, with high binding capacity of other components, demonstrates the behavior of this mixture of polymers in liquids and their importance in the rheology of the mixtures in which they are used. 4. conclusions the screening of the four available yeast sources revealed a varied level of βglucans content. the results confirm the hypothesis of other authors that glucan is found in varying proportions depending on the strain of the species. the study conducted shows that the highest level of β-glucan possesses the beer yeast (15.91%), with 21.49% more than the baker's yeast. the problem of triple helix conformation (known to produce biologically active effects) and the aggressive chemical method for the extraction of glucan is solved by using the thermal drying process at 55° c with the reconfiguration of the structure. the extraction method seems to be appropriate for the large-scale production of β-glucans from yeast biomass. unlike other sources of glucans (oat, barley, rye and wheat), yeast isolates are not toxic in terms of aflatoxin contamination. although it is impossible to define the biological activity of β-glucans isolated from four samples without carrying out clinical tests and immunological tests, they can be put into relation with other data presented in the scientific literature. the yeast selection study with the highest content in bioactive compounds allows for future the development of glucan supplements from levurian compounds resulting in fermentative processes. 5. references [1]. petravic-tominac v., zechnerkrpan v., grba s., srecec s., panjkotakrbavcic i., vidovic l., biological effects of yeast β-glucans, agriculturae conspectus scientificus, 75 (4): 149-158, (2010). [2]. waites m., morgan n., rockey j., higton g., industrial microbiology: an introduction, school of applied science, south bank university, london, uk, (2001). [3]. lipke p., ovalle r., cell wall architecture in yeast: new structure and new challenges, minireview, journal of bacteriology, 180 (15): 3735-3740, (1998). [4]. stewart g.g., russell i., brewer’s yeast, brewing science & technology series iii (1998): 28-46, (1998). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 ionuț avrămia, sonia amariei, research on obtaining high β-glucans content from different sources of yeast harnessing their biologically active potential, food and environment safety, volume xvi, issue 4 – 2017, pag. 190 – 195 195 [5]. usatîi a., chiselița n., metode de extragere din levuri a β-glucanilor și proprietățile lor fizico-chimice, buletinul academiei de ştiinţe a moldovei (methods of extraction in yeasts of βglucans and their physicochemical properties, bulletin of the academy of sciences of moldova), 1 (325): 153-159, (2015). [6]. zekovic d.b., kwiatkowski s., vrvic m. m., jakovlievic d., moran c., natural and modified (1→3)-β-d-glucans in health promotion and disease alleviation, critical reviews in biotechnology, 25 (4): 205-230, (2005). [7]. lessage g., bussey h., cell wall assembly in saccharomyces cerevisiae, microbiology and molecular biology reviews, 70 (2): 317-343, (2006). [8]. kwiatkowski s., kwiatkowski s.e., yeast (saccharomyces cerevisiae) glucan polysaccharides – occurrence, separation and application in food, feed and health industries, intech, the complex world of polysaccharides, (2): 47-70, (2012). [9]. european food safety authority, scientific opinion on the safety of ‘yeast betaglucans’ as a novel food ingredient, efsa journal, 9 (5): 2137, (2011). [10]. batbayar s., lee d.h., kim h.w., immunomodulation of fungal b-glucan in host defense signaling by dectin-1, biomol ther, 20 (5), 433-445, (2012). [11]. stier h., ebbeskotte v., gruenwald j., immune-modulatory effects of dietary yeast beta-1,3/1,6-d-glucan, nutrition journal, 13:38, doi:10.1186/1475-2891-13-38, (2014). [12]. xiaohua w., lina z., physicochemical properties and antitumor activities for sulfated derivatives of lentinan, carbohydrate research, 344 (16): 2209-2216, (2009). [13]. rafael o., africa g., martinlomas m., penades s., preparation of multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines, carbohydrate research, 342 (3-4): 448-459, (2007). [14]. mason r., what is beta glucan, editura safe goods, isbn no. 1-884820-66-2, (2011). [15]. thammakiti s., suphantharika m., phaesuwan t., verduyn c., preparation of spent brewer’s yeast β-glucans for potential application in the food industry, international journal of food science and technology, 39 (1): 21-29, (2004). [16]. petravic-tominac v., zechnerkrpan v., berkovic k., galovic p., herceg z., srecec s., spoljaric i., rheological properties, water-holding and oilbinding capacities of particulate b-glucans isolated from spent brewer’s yeast by three different procedures, food technology and biotechnology, 49 (1): 56–64, (2011). [17]. kwong m., the glycemic response elicited by oat β-glucan solutions and hard gel varying in physiochemical properties and food form, master of science, department of nutritional sciences university of toronto, (2012). [18]. silva v.o., lobato r.v., andrade e.f., macedo c.g., napimoga j.t.c., napimoga m.h., et.al., β-glucans (saccharomyces cereviseae) reduce glucose levels and attenuate alveolar bone loss in diabetic rats with periodontal disease, plos one, 10 (8): e0134742, (2015). [19]. blair p. et.al., biopolymers: making materials nature’s way-background paper, office of technology assessment, ota-bp-e-102, (1993). [20]. james s., rha c., sinskey a., glucan compositions and process for preparation thereof, brevet de invenție us4810646 a (patent us4810646 a), massachusetts institute of technology, (1989). [21]. javmen a., grigiskis s., gliebute r., β-glucan extraction from saccharomyces cerevisiae yeast using actinomyces rutgersensis 88 yeast lyzing enzymatic complex, biologija, 58 (2): 51-59, (2012). [22]. chiselița n., usatîi a., molodoi e., efremova n., fulga l., borisova t., optimizarea matematică a mediului de cultură pentru producerea β-glucanilor la tulpina saccharomyces cerevisiae cnmn-y-20, revista științifică a universității de stat din moldova (mathematical optimization of the culture medium for the production of β-glucans in the saccharomyces cerevisiae strain cnmn-y-20, scientific review of the state university of moldova), nr.6 (66), (2013). [23]. many j.n., vizhi k., analysis of different extraction methods on the yield and recovery of β-glucan from baker’s yeast (saccharomyces cerevisiae), international journal of innovative science, engineering & technology, 1 (6): 268271, (2014). [24]. usatîi a., molodoi e., chiseliţa n., chiseliţa o., efremova n., borisova t., fulga l., tulpini de drojdii – surse valoroase pentru obţinerea β-glucanilor şi mananilor, buletinul academiei de ştiinţe a moldovei (yeast strains valuable sources for obtaining β-glucans and mannan, bulletin of the academy of sciences of moldova), 3 (315): 128-134, (2011). https://dx.doi.org/10.1186%2f1475-2891-13-38 evidenţierea progresului genetic în ameliorarea secarei de toamnă, food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 57 highlighting genetic progress in improving winter rye *ioan gontariu1, constantin drobotă2, marinela cureleţ1 1 ştefan cel mare university of suceava, faculty of food engineering, 13 universitaţii st., 720229, suceava, romania, e-mail: ioang@fia.usv.ro, marinelac@fia.usv.ro 2agricultural research and development station of suceava, b-dul 1 decembrie 1918 nr.15, suceava, e-mail: constantin_drobota@yahoo.com *corresponding author received 10 february 2011, accepted 25 july 2011 abstract this paper highlights the contribution of winter rye culture, initially by testing and identifying the most suitable local populations and foreign varieties, and subsequently through the creation, introduction and widespread in the production of gloria, orizont, ergo and suceveana varieties, obtained at a.r.d.s. suceava. during the 1951-1955 stage, certain local landraces and the cultivar petkus registered an average yield on 1300 kg/ha, and during period 1970-1975 the average yield of the cultivars danae and dankovskie zlote grows at the 3160 kg/ha. the cultivars danae, dankovskie zlote and ianoşi during period 1976-1985 registered an average yield on 4207 kg/ha, and during 1986-1995 the cultivars gloria, ergo and orizont which were created at the a.r.d.s. of suceava had an average yield on 4980 kg/ha. the cultivar suceveana was cultivated during 1996-2005, with an average yield on 5075 kg/ha. the superiority of the four cultivars: gloria, orizont, ergo and suceveana from the productivity and yield stability point of view ,was statistically demonstrated through the distribution of the correlations and variance of productions in comparison with the standard cultivars from the above periods. the superior values of the quality traits recommend the utilization of the rye cultivars which were created both for bread making feature and for fodder users. key words: cultivars, breeding, yield, quality, resistance. 1. introduction although there has lately been registered a considerable restraint of the cultivated surfaces, the autumn rye crops still marks a wider spreading, not only in the submountaneous and hilly areas , but also in the areas with a wet and cold climate, where there still can be found podzolic and podzolite soils, with an increased acidity and a reduced natural fertility, and in the areas with sandy land, from the south and western part of the country, with a short and irregular pluviometrical regime [1]. the restraint of the cultivated areas has happened because of the involution of the researched in the domains of genetic and breeding, as a follow up of non-financing of the agricultural research, and of the profound changes too, that have come to recently in the structure of the territorial fond, that determined the productive potential of the rye to be decreased in comparison with the one of the wheat and barley [2]. thus, in our country, the rye advanced cultivars sorts, is reduced in comparison with the other cereals [3]. because the level of the rye productions is frequently affected by the decreased resistance to lodging, as a follow up to the still high enough height of the species, by the sensibility to the diseases attack, especially to the snow mould, that presents a great danger for the cultures , in the wet and colder areas and also the insufficient resistance to the seed germination in the spike, there has appeared the necessity of food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 58 creating new cultivars, superior from the agronomic point of view in comparison with the cultivated varieties in the past time [4]. the production components are closely related to their genetic stability. the components with high genetic stability are: the grain’s weight, the ear’s density, the number of small parts from an ear [5]. the ear’s length and the number of grains from an ear present a medium stability and the number of fertile brothers, the grains weight from the ears, the number of grains from the ear and the number of grains from the plant presents a low stability [6]. through the development of the genetic and breeding researched there have been created not only superior rye cultivars, resistant to lodging, to diseases and less favorable climatic conditions that could face the culture on fields with a reduced fertility and following perfection technologies, and also new lines with a genetic determinism dominant for the short height of the straw [7]. this paper aims at appreciating the contribution brought to the rye culture, initially through the testing and identification of the most appropriated local populations and foreign cultivars and afterwards through the creation, introduction and extension in the production of the obtained cultivars at a.r.d.s. of suceava. 2. biologic material and research method the breeding work carried out so far for the autumn rye have had a great contribution to the identification of the local populations and the foreign cultivars, to the creation of autochthon lines and the extension in the culture of the most valuable of these, but, in the second half to the creation of four cultivars, and also to the obtaining of a more valuable breeding material, with a shortened height, well adapted to the specific conditions of the rye culture, that can be used for the creation of more advanced genotypes. • the evaluation of the superiority of the cultivars and the lines presents itself during three significant periods of time that are: 1. 1946-1975 period highlights the experimental results of the cultivars and the rye lines verified at a.r.d.s. of suceava, from the point of view of the physiological features, quality features and productivity features in comparison with the petkus cultivar; 2. 1976-1995 period highlights the productive value not only of the gloria cultivar but also of the sv. 2546-78 and carpaţi lines, created in this period and that were to become cultivars, in comparison with the dankovskie zlote cultivar used as a standard in this period ; 3. 1996-2005 periods presents the value of the gloria, orizont, ergo, and suceveana cultivars but also the achievements in the direction of creating short straw forms, in comparison with the standard orizont, witch is the tallest. 4. the cultivars and the experimented lines have been appreciated during time, under the aspect of physiological features, quality and production features, using the results obtained in the comparative crops, during the experimental years, in comparison with the standards petkus and dankovskie zlote, that had the greatest spread in the production, but also with the orizont cultivar, created in suceava, even though it has not occupied significant areas, is the national cultivar with the greatest height of plants. the correlation between the results of the created cultivars in suceava and the ones of the food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 59 standard cultivars dankovskie zlote and orizont in the periods 19861995 and 1996-2005 has been highlighted through the calculation of the linear regressions, and the stability of the productions has been marked through the determination of the variances (s2). the phenological observations made during the vegetation over the main physiological features are presented in the work through medium values and the interpretation of the production data has been carried out through statistic methods of variance analyses [8]. the experimentation has been carried out in comparative crops, executed at the beginning, after the method of plots position in latin rectangle (with 5 replications and 10 m2 the cropped surfaces of one plots) and then in the simple grating (3 replications and 7 m2 cropped plot). in majority of the experimented years the cultures have been placed after potatoes, in a leached chernozem soil, rich in humus and total nitric, provided with phosphor and mobile kalium, not being necessary the fazial fertilization in autumn, as the potato has received larger quantities of chemical fertilizer. in spring, before the beginning of vegetation period, there have been administered 40-70 kg n s.a. /ha as ammonium nitrate or nitrocalcar. the sowing has been carried out, in the big majority of the experimental years, in the optimum epoch for this area being assured on 500 germinated grains/ m2[9]. for creation at a.r.d.s. of suceava of the four cultivars and 5 more valuable lines, with a genealogy and obtaining method , that will be briefly presented, there has been used valuable genetic resources, combining not only genes from the european germplasm, included in the hybrid population through the polish an german cultivars, of high productivity, and also different ecological genes, from the national germplasm fond, included through the inbred lines characterized by a short height and an increased resistance to falling and diseases (table 1). table 1 genealogy and obtaining method of cultivars and inbred lines created at a.r.d.s. of suceava cultivar or line parental forms cross year homologate d year selection method gloria canorus x danae 1974 1983 selection on families orizont (l.c.4415 x danae)xpetkuser kurzstroh 1975 1988 mass selection ergo pecuro x (dankovskie zlote x danae) 1974 1988 selection on families suceveana (dankovskie zlote x sv.623-83) x lad 185) 1988 1996 selection on families sv. 90 bl (orizont x borellus) x borellus 1988 selection on families sv. 21 90 (ensi x ergo) x e.m. – 1 pollinated 1986 mass selection sv.4 2 90 (ergo x borellus)x dankovskie zlote 1987 selection on families sv. 90 – em em 1 – repeated open pollinated 1986 mass selection sv. 90 – cp ciulpan – repeated open pollinated 1986 mass selection 3. results and discussions the breeding researches for the winter rye begun after year 1950, in short time, after the foundation of the ards of suceava, through the checking of the behavior of the different local population and foreign cultivars in the crops conditions from the western part of the suceava plateau have been a precursory stage for the systemized organization and the amplifying of the breeding work for the rye, that have been developed after 1970, appreciating that, once with these, the crop of the winter rye, and of the other plants from this area, have registered on an ascendant trajectory. in this paper we present the dynamic of the cultivars number and the experimented lines during 6 decades, so that, beginning food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 60 with year 1946 until 2005, there have been checked, in comparative crops, from the point of the vegetation behaviour and productivity views a number of 3116 cultivars and rye lines, from which, 759 national and foreign cultivars. the most valuable among these cultivars have been introduced and generalized in the production, occupying significant surfaces at the level of the three distinct periods. (table 2). table 2 the volume and dynamic of the breeding researches at the winter rye during period 1946 – 2005 in ards of suceava the research specification and obtained results period: total 1946 2005 1946 1975 1976 1995 1996 2005 the study of the cultivars and lines in the comparative crops 368 2 153 595 3116 cultivars from which : lines 57 510 192 759 311 1643 403 2357 the cultivars and inbred lines considered valuables local populations petkus breeded petkus harrach visa danae dankovskie zlote dankovskie zlote danae ianoşi pluto gloria orizont ergo sv. 4 2 – 90 sv. 92 – a sv. 92 – r sv. 185 – 90 gloria orizont ergo suceveana sv. 90 – bl sv. 21 – 90 sv. 90 – em sv. 921 – 97 sv. 90 – cp sv. m. 72 – 98 sv. 200 s –99 sv. 300 s – 99 from which: introduced and generalized in crop production petkus breaded petkus danae dankovskie zlote danae dankovskie zlote ianoşi gloria orizont ergo gloria orizont ergo suceveana 1. 1946-1975 period has been characterized through the experimentation of a number of up to 368 local populations, native and foreign cultivars and inbred lines, from which some inbred lines created at ards of suceava, have been highlighted, from the point of view of the physiological and productivity features. the quality of the production concerning the physic and chemical features of the production we can notice that this represents different values from a genotype to another. so, the hectoliter mass has registered middle values, from 64.8 kg at cultivar harkovskaia 60 to 69.5 kg at line sv. 25-65 and one thousand kernel weight oscilated between 29.1 g at panterne cultivar and 33.2 g at dankovskie zlote cultivar. differences between the cultivars and the experimented lines have been noticed also concerning the chemical features, these having values for the protein content comprised between 11, 5% at danae cultivar and 13.5% at line sv 32-66 and for starch content, between 58.2% at petkus cultivar and 61.4% at harkovskaia 60 cultivar (table 3). food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 61 table 3 the physic and chemical traits of production for the rye cultivars and lines experimented during 1970 -1975 at ards of suceava nr. crt. cultivar or line test weight(g) one thousand kernel weight at dry substance: protein content (n x 5,7) % starch content % 1 petkus st 67.2 30.5 13.4 58.2 2 danae 66.3 30.8 11.5 61.3 3 harkovskaia 60 64.8 30.9 11.7 61.4 4 desneanka 2 65.2 30.4 13.4 58.3 5 panterne 67.7 29.1 12.5 60.6 6 dankovskie zlote 67.9 33.2 12.8 60.8 7 sv. 11 – 65 69.3 30.8 13.2 59.1 8 sv. 25 – 65 69.5 31.2 12.8 60.1 9 sv. 17 – 65 68.8 31.4 13.4 58.5 10 sv. 5 – 65 68.6 31.0 12.9 58.8 11 sv. 3 – 65 69.2 31.3 12.7 60.2 12 sv. 6 – 65 69.3 30.5 12.3 60.4 13 sv. 32 – 66 67.4 29.9 13.5 58.3 2. 1976-1995 period emphasized the value of the cultivars and the lines created in suceava during this time, from the point of view of the physiological, quality and production traits in comparison with the standard cultivar dankovskie zlote. the yield quality concerning the quality features of the production we can notice that between the cultivars and the experimented lines during 1981-1985, there have been differences (table 4). table 4 the quality traits of the production for the rye cultivars and lines experimented during 1981–1985 at ards of suceava nr. crt. cultivar or line hectoliter mass kg one thousand kernel weight g at dry substances: % protein content % starch content 1 dankovskie zlote – st. 67.4 34.3 13.1 61.5 2 danae 66.2 35.1 12.5 60.3 3 ianoşi 67.8 34.9 11.5 62.5 4 voshod 1 67.5 34.7 12.0 65.7 5 pluto 68.7 33.9 12.2 64.3 6 golubka 65.3 36.2 12.4 61.2 7 dorna 67.9 35.3 12.8 61.0 8 prima 67.6 35.6 12.3 62.0 9 dragomirna 68.8 34.5 11.7 62.5 10 gloria 68.7 36.6 13.4 62.1 11 sv.2546–78 68.5 35.4 12.8 64.2 12 carpaţi 69.6 33.2 12.7 61.4 the test weight has registered in this period medium values from 65.3 kg at golubka cultivar to 69.6 kg at line carpaţi and one thousand kernel weight between 33.2 g at line carpaţi and 36.6 g at line sv. 2546–78. from the point of view of the chemical traits, also, there are differences between the cultivars and the experimented lines, concerning the protein and starch contents. the brute protein content, reported to the dried substance, has oscillated in relatively large limits, from 11.5 % at cultivar ianoşi to 13.4 % at cultivar gloria, and starch content registered values between 60.3 % food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 62 at cultivar danae and 65.7 % at cultivar voshod 1. 3. 1996-2005 period is characterized by progresses registered in the direction of creating new forms with short straw, with a big productivity and improved resistance to the plant lodging the yield quality from the point of view of the physics and chemical features of the production we can notice that the new genotypes experimented during 1996-2005, have proved different values (table5) table 5 the physic and chemical traits of the production for the rye cultivars and lines experimented during 1996 – 2005, at ards of suceava nr. crt. cultivar or line hectoliter mass kg one thousand kernel weight g at dry substances: % protein content % starch content 1 orizont – mt. 69.8 42.1 12.4 60.2 2 gloria 71.5 40.6 12.7 60.9 3 ergo 72.2 38.8 12.1 61.2 4 suceveana 69.5 42.5 13.3 61.5 5 dankovskie zlote 68.3 41.0 12.9 59.9 6 amando 70.2 39.8 12.4 60.4 7 sv. 90 – bl. 69.1 42.7 12.9 62.5 8 sv.21 – 90 68.1 43.0 11.9 63.1 9 sv. 90 – em 71.2 38.9 12.6 62.2 10 sv. 90 – cp 71.0 39.1 13.5 61.1 11 sv. m 72 – 98 71.1 39.2 12.6 62.5 12 sv. 921 – 97 69.5 42.8 12.2 64.2 13 sv. im – 98 70.4 40.0 12.5 59.6 14 sv. 200 s – 99 71.0 39.8 12.9 60.6 15 sv. 300 s – 99 70.5 40.5 13.1 65.1 in this period the tests weight has registered, function by climatic and crop conditions, the higher middle values, from 68.1 kg at line sv. 21 – 90 to 72.2 kg at ergo cultivar and one thousand kernel weight has oscillated between 38.8 g at ergo cultivar and 42.8 g at line sv. 921 – 97. regarding the chemical features, the cultivars and the experimented lines have been differenced for the protein content between 11 9 % at line sv. 21 – 90 and 13.5 % at line sv. 90 –cp, and for starch content, between 59.9 % at dankovskie zlote cultivar and 65.1 % at line sv. 300 s – 99. this work reveals the fact that at ards of suceava there have been made sustained researches on a long period of time in the domain of the winter rye breeding, over the great cultivars and diversified ones. these researches have aimed at the creation of a valuable material in the direction of shortening the straw for this plant, together with the more detailed acknowledgement of the behavior of the cultivars and the lines in the conditions of the crop production [10]. so,there have been highlighted the most valuable and better adapted genotypes, which, through their introduction and generalization in the crops have contributed to the continuous spreading of the production in the northern and north-western part of moldavia. food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 63 50754980 4207 3160 2018 1300 1000 0 1000 2000 3000 4000 5000 6000 1946-1950 1951-1955 1956-1965 1966-1975 1976-1985 1986-1995 1996-2005 p ro du ct io n (k g/ ha ) fig. 1 the genetic progress realized for the winter rye, in period of time 1946-2005, through utilization in the crop of the most valuable cultivars, in the crop conditions offered by ards of suceava. the genetic progress reflects especially in the increase of the production capacity, registered especially in the last 40 years (1966-2005) , when as a follow to the intensification of the creation of new cultivars works, the rye productivity has greatly increased, from a middle level of 3160 kg/ha during period 1966-1975, until 5075 kg/ha during period 1996-2005. 4. conclusions ►at ards of suceava, during 1946– 2005 have been carried out researches, initially in the direction of identification of foreign cultivars and more valuable and afterwards for the creation of new cultivars and rye lines with a short height, more productive and better adapted to the crop conditions from the north of moldavia, that have contributed to the introduction, rational arrangement and generalization in the crop of the best cultivars of rye and from this to the progressive increase of the yield capacity. through utilization in the crop culture of the most valuable rye cultivars has registered a genetic progress, that, reported to the period 1951–1955, can be dimensioned during period 1996-2005 through an yield increase on 290 %. ► on the basis of the researches carried out during time at ards of suceava there have been created 4 winter rye cultivars: gloria, orizont, ergo and suceveana, with an increased productive potential, resistant to crop lodging and better adapted to the specific crop conditions for the wet and cold areas. ►there have been registered, also, obvious achievements concerning the breeding of the physiological and the quality traits: the wintering resistance and diseases attack resistance, especially to the snow mould has been improved from sensitive forms to resistant forms; the crop lodging resistance has been increased, from very weak to very good, through the creation of cultivars and food and environment safety journal of faculty of food engineering, ştefan cel mareuniversity suceava volume x, issue 4 2011 64 lines with short plants height and the thick straw; the superior values of the quality traits recommend the using of the rye cultivars created at ards of suceava, not only for the bread making features, but also fodder. ► the winter rye cultivars that find themselves at this moment in the variety cultivated in romania, unlike the northern and north western part of moldavia, but also in the areas with podzolic and sandy soils, are valuable cultivars, with a large ecological adaptability, and until the creation of better new genotypes we recommend in the future the cultivation of the gloria, orizont, ergo and suceveana cultivars. 5. references [1]. gaşpăr, i., 1976 –valuable varieties and lines of perspective wheat and rye for suceava. volume anniversary 19461976:47-61. [2]. gaşpăr, i., 1973 gaşpăr, i., 1973 – research on behavior in culture of rye varieties of global range. volume anniversary, 1946 – 1971 : 91 – 99. [3]. gaşpăr i., 1984 – morpho-productive characterization of the new variety of gloria rye.moldova agronomic research , vol. 1 (65): 51-54; [4]. gaşpăr, i., 1976 research on behavior in culture of perspective rye varieties . volume anniversary1946-1976: 63 – 71; [5]. nedelea g., moisuc a., 1985 – heredity components of ear production of winter wheat .scientufic papers i.a.t., vol. xx: 54-60. [6]. madoşă e., nedelea g., ciulcă s., avădanei c., 2001 – heredity studies of the numbers of grains in the ear of wheat.biotechnology and biodiversity. agroprint publicher , timişoara:51-54. [7]. gaşpăr i., zamă e., huţanu livia, 1983 –gaşpăr i., zamă e., huţanu livia, 1983 experimental results with varieties and new lines in suceava plateau. moldova agronomic research vol. 4 (64): 39-43; [8]. tronsmo a. m. 1993 resistance to winter stress factors in half-sib families of dactylis glomerata, tested in a controlled environment. acta agric scand ;43: 89-96. [9]. tanino k.k., mckersie b.d., 1985 injury within the crown of winter wheat seedlings after freezing and icing stress, can. j. bot. 63; 432–435. [10].moore j.c., 1994 impact of agricultural practices on soil food web structure: theory and application. agric. ecosyst. environ. 51, 239–247. 2. biologic material and research method genealogy and obtaining method of cultivars and inbred lines created at a.r.d.s. of suceava 3. results and discussions the volume and dynamic of the breeding researches at the winter rye during period 1946 – 2005 in ards of suceava the physic and chemical traits of the production for the rye cultivars and lines experimented during 1996 – 2005, at ards of suceava sensory evaluation 276 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4-2017, pag. 276 281 antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties mădălina iuga 1 , sorina ropciuc 1 , silvia mironeasa* 1 1faculty of food engineering, stefan cel mare university, suceava, universitatii str. 13, suceava, romania, silviam@fia.usv.ro *corresponding author received 19th october 2017, accepted 8th december 2017 abstract: grape seeds and peels are valuable by-products from wine production industry that can be valorized in food production. both grape seeds and peels have a great amount of antioxidants and polyphenolic compounds. the aim of this study was to investigate the total phenolic content of red and white grape seeds and peels and to establish the optimal solvent concentration for highest extraction yield. for this purpose, folin ciocâlteu method was used to achieve the phenols content and the antioxidant activity was estimated using 2,2 – diphenyl-1-1picrylhydrazyl (dpph) reagent. the total phenolic content ranged from 81.13 mg gae/g for red peels, 93.47 mg gae/g for white peels, to 128.47 mg gae/g for red seeds and 164.70 mg gae/g for white seeds. the one-way anova method was used to see if there are differences between the antioxidant activities depending on the solvent concentration. the results showed that the methanol concentration significantly (p < 0.05) influences the antioxidants extraction. the inhibition percent ic50 ranged from 0,24 to 4,37 μg/ml for white peels, from 3,12 to 6,29 μg/ml for red peels, from 5,53 to 5,90 μg/ml for white seeds and from 4,59 to 6,14 μg/ml for red seeds. this study highlighted the possibility to use grape seeds and peels as food ingredients or natural antioxidant to extend the shelf life of food, especial of lipids and lipidcontaining foods because of their high antioxidant activity and total phenolic content. key words: grape seeds, grape peels, antioxidant activity, total phenolic content. 1. introduction grapes, one of the most cultivated fruits in the world, are used in high proportion (80%) in wine-making production [1]. wine industry has an adverse environmental impact because of residual phenols of by-products resulted from grapes processing. relative high amounts of phenolic compounds in waste are a problem for flora and fauna from discharge zones due to their inhibition of germination properties [2]. grape pomace generated in winemaking process is a valuable by-product for oil extraction and some active compounds, such as dietary fiber, polyphenols, anthocyanins, flavonols and resveratrol [3, 4]. the wine-making grape pomace includes as main components peels and seeds. the polyphenols and other compounds with antioxidant activity are present in grape peels and seeds. the quantitative and qualitative distribution of polyphenolic compounds in grape peels and seeds depends on grape variety, harvesting conditions, geographic location of the culture, soil, vinicultural techniques, winemaking process, etc. [5]. many studies showed that the phenolic profile is related to the fruit maturity level: it increases with the ripening level increase [6]. during the winemaking process, part of the phenolic compounds in grapes is http://www.fia.usv.ro/fiajournal mailto:silviam@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 mădălina iuga, sorina ropciuc, silvia mironeasa, antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties, volume xvi, issue 4 – 2017, pag. 276 – 281 277 transferred to wine, but a high amount still remains in the grape by-products, mainly in the grape pomace. more than 70% of phenols from grapes rest in pomace [5]. the phenolic compounds are present in plants and have many biological functions. the researchers revealed hundreds of polyphenols in edible plants [7]. in wine pomace there are flavonoid compounds such as anthocyanins, flavonols, flavan-3ol, tannins and non-flavonoid substances, especially resveratrol [8] and phenolic acids [9]. in red pomace there are many antochianyns, while in white pomace the flavonols are in higher quantity [10]. the total polyphenolic content of grape pomace can be influenced by the differences between white and red wine making processes [11]. in grape peels there are elagic acid, resveratrol, myricetin, quercetin, kaempferol, epigalocatechine. the results showed by many researches revealed the fact that the phenolic content is higher in red peels than in white peels because the monomeric anthocyanins are absent from white peels [10]. in grape seeds catechin, epicatechin, malic acid and galocatechin are the most important phenols. the phenolic content of grape seeds is greater than of grape peels [12]. the most important operation for phenols recovery from grape seeds and peels is the extraction. various solvents including ethanol, methanol, ethyl ether, etc. were used by researchers for the phenols extraction. also, various techniques, such as ultra-sonication, stirring and shaking were applied. the most used solvent is methanol or methanol/water mixtures [13]. a higher total phenolic content gives a better antioxidant activity [14, 15]. the inclusion of antioxidants is a method to prolong shelf life of food products while they add extra health benefits. polyphenolic compounds exhibits health benefits due to its cardioprotective and anti-inflammatory effects, anti-ulcer and anti-carcinogenic activity [16]. there are many researches about the phenolic compounds and antioxidant activity from grape peels and seeds, but there are very few reports that compare the total phenolic content distribution between peels and seeds among different varieties. the aim of this study was to determine the antioxidant activity and the total phenolic content of grape seeds and peels from two wine pomace varieties, red and white. this study wants to underline the importance of grape seeds and peels with respect their potential as a source of natural antioxidants and as an alternative to the reuse of byproducts from winemaking process. 2. materials and methods 2.1. materials grape seeds and peels from red and white wine grape pomace provided by the viticulture center jaristea, odobesti ecosystem were used in this study. grape seeds and peels were separated manually from dried grape pomace and ground in a speed grinder to obtain grape seeds and grape peels flours. as reagents of analytical grade, standards of gallic acid, ascorbic acid, methanol, calcium carbonate, 2,2 – diphenyl-11picrylhydrazyl (dpph) reagent and folin-ciocâlteu phenol reagent purchased from sigma-aldrich and merck were used. 2.2. methods 2.2.1. antioxidant activity the antioxidant activity was achieved by dpph method. the extracts were prepared according to xuetao and collaborators method [17], with some modifications. 4 g of sample was extracted with 15 ml of solvent (methanol: distillated water, 0:100, 60:40, 70:30 and 80:20). for this purpose, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 mădălina iuga, sorina ropciuc, silvia mironeasa, antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties, volume xvi, issue 4 – 2017, pag. 276 – 281 278 the mixes were ultra-sonicated at 40 ºc, at 25 hz for 30 minutes. then, they were centrifuged at 3000 rpm for 5 minutes. the supernatant was collected and the residue was mixed with 15 ml solvent and centrifuged again. this operation was repeated 3 times. the supernatants were collected and used for analysis. each extract was diluted to 50%, 40%, 30%, 20% and 10% with the corresponding solvent (methanol: distillated water, 0:100, 60:40, 70:30 and 80:20). 2 ml of sample was mixed with 1 ml of dpph reagent and the absorbance at 517 nm wavelength was read immediately by using uv-vis-nir spectrophotometer (shimadzu corporation, japan). a blank sample was prepared for each solvent. the free radicals inhibition percent ic50 was calculated using eq.1 [18]: ic50 = [1-(a517sample-a517 blank] x 100 (1) where a517 blank is the absorbance value of the control sample and a517sample is the absorbance value in the presence of the extract sample and standard. 2.2.2. total phenolic content the phenolic content of grape seeds and peels were determined using the folin ciocâlteu method. 2 g of samples were mixed with 4 ml solvent (methanol: distillated water, 70:30) and shacked for 5 minutes on the orbital shaker. then, the mixes were centrifuged at 6000 rpm for 10 minutes. the supernatant was collected and the residue was extracted again with 4 ml solvent, as it is described above. the two supernatants were mixed and used for determination. 0.5 ml of extract was mixed with 0.5 ml of folin ciocâlteu reagent and 3 ml of na2co3 20%. the mixture was diluted with distillate water to final volume of 10 ml. the samples were allowed to stand for 30 minutes and the absorbance was read at 750 nm by using uv-vis-nir spectrophotometer (shimadzu corporation, japan). a blank sample was prepared with distillate water. each determination was made in triplicate. the calibration curve was made with different gallic acid concentrations. the results were expressed as milligrams of gallic acid equivalents (gae) per g of extract (mg gae / g). 2.2.3. statistical analysis the results were expressed as the mean values  s.d. of three parallel measurements. one-way anova method from microsoft excel 2007 was used to test the effect of variation factors – different samples on variable ic50. a difference was considered statistically significant at p  0.05. 3. results and discussion 3.1. antioxidant activity figure 1 shows the total phenols content, measured as absorbance at 517 nm, for the white and red grape peels extracts in water and methanol mixture at different proportion and at various extracts concentration. statistical analysis of the data shows that the extraction solvent had a positive effect (p < 0.05) on the antioxidant activity by increasing it with concentration increase. according to the results obtained, there were significant differences (p < 0.05) between the total phenolic content of each seed and peel extract. white grape pomace had higher solvent extraction yield than the red varieties. the total phenolic content for the white and red grape seeds extracts in water and methanol mixtures at different proportions (0:100, 60:40, 70:30 and 80:20, methanol: distillated water) and at various extracts concentration is showed in figure 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 mădălina iuga, sorina ropciuc, silvia mironeasa, antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties, volume xvi, issue 4 – 2017, pag. 276 – 281 279 a) b) fig. 1. absorbance curves for extracts diluted with aqueous methanol solutions (0:100, 60:40, 70:30 and 80:20, methanol: distillated water) from: a) white peels; b) red peels the results obtained highlight that the optimal mixture solvent for white grape peels and seeds is methanol/water in ratio 80:20, while the antioxidants from red peels and seeds are best extracted in water (0:100). the parameter used to estimate the antioxidant activity is ic50 and it represents the antioxidant amount required to decrease the initial dpph concentration by 50%. low ic50 values indicated a high antioxidant activity [19]. as it is shown in figure 3, grape peels antioxidants are best extracted with lower methanol concentrations as compared to grape seeds. a) b) fig. 2. absorbance curves for extracts diluted with aqueous methanol solutions (0:100, 60:40, 70:30 and 80:20, methanol: distillated water) from: a) white seeds; b) red seeds fig. 3. ic50 values for grape seeds and peels extracts the fact that the aqueous extracts from white peels and white seeds had the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 mădălina iuga, sorina ropciuc, silvia mironeasa, antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties, volume xvi, issue 4 – 2017, pag. 276 – 281 280 highest antioxidant activity underlines the possibility to use these by-products as natural additives in food products. 3.2. total phenolic content the variation of total phenolic contents of grape seeds and peels from white and red grape varieties are showed in figure 4. fig.4. total phenolics content in grape peels and seeds from red and white varieties regarding the total phenolic content, white peels have a higher amount of polyphenols than red peels, while white seeds have higher polyphenolic content than the red ones (figure 3). the results revealed that grape seeds have more polyphenols than grape peels, in according to the results obtained in other studies [12, 20]. the main interest in phenolic compounds from grape byproducts lies in their antioxidant capacity. 4. conclusion solvent concentration is an important parameter that influences significantly the antioxidants extraction and consequently the antioxidant activity. the results for total phenolic content of grape seeds and peels confirmed other specialist’s hypothesis: grape seeds are richer in polyphenols than grape peels. in conclusion, grape seeds and peels are a natural source of phenolic compounds with high antioxidant activity. these byproducts can be valorized in food industry as a source of natural phenols with the antioxidant power similar to that of synthetic antioxidants. 5. acknowledgements this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs/cccdi – uefiscdi, project number pn-iii-p2-2.1-bg-2016-0136, within pncdi iii. 6. references [1] zhu f., du b., zheng l. and li j., advance on the bioactivity and potential applications of dietary fibre from grape pomace, food chemistry, 186: 207-212 (2015) [2] bonilla f., mayen m., merida j. and medina m., extraction of phenolic compounds from red grape marc for use as food lipid antioxidants, food chemistry, 66(2):209-215, (1999) [3] yu j. m., and ahmedna m., functional components of grape pomace: their composition, biological properties and potential applications, international journal of food science and technology, 48 : 221–237, (2013) [4] deng q., penner m. h., zhao y. y. , chemical composition of dietary fiber and polyphenols of five different varieties of wine grape pomace skins, food research international, 44: 2712–2720, (2011) [5] lafka t.i., sinanoglou v. and lazos, e.s., on the extraction and antioxidant activity of phenolic compounds from winery wastes, food chemistry, 104(3) : 1206-1214, (2007). [6] prior r. l., cao g., martin a., sofic e., mcewen j., o’brien c., lischner n., ehlenfeldt m., kalt w., krewer g., mainland c. m., antioxidant capacity as influenced by total phenolic and anthocynin content, maturity and variety of vaccinium species, journal of agriculture and food chemistry, 46: 2686-2693, (1998) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 mădălina iuga, sorina ropciuc, silvia mironeasa, antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties, volume xvi, issue 4 – 2017, pag. 276 – 281 281 [7] manach c., scalbert a., morand c., remesy c., jimenez l., polyphenols: food sources and bioavailability, the american journal of clinical nutrition., 79(5) : 727-747, (2004) [8] moreno-arribas m. v., polo m. c., wine chemistry and biochemistry vol. 233, springer, new york, (2009) [9] ramirez-lopez l. m., dewitt c. a., analysis of phenolic compounds in commercial dried grape pomace by high-performance liquid chromatography electrospray ionization mass spectrometry, food science and nutrition, 2(5) : 470-477. (2014) [10] mironeasa s., valorization of secondary products from wine making, publishing house performantica, iasi, romania, (2017) [11] cerda-carrasco a., lopez-solic r. nunes-kalasic h., pena-neira a., obreque-slier e., phenolic composition and antioxidant capacity of pomaces from four grape varieties (vitis vinifera l.), journal of the science of food and agriculture, 95: 1521–1527, (2015) [12] pinelo m., rubilar m., jerez m., sineiro j., and núñez m. j., effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace, journal of agricultural and food chemistry, 53(6): 2111-2117, (2005) [13] khalil f., forough e., mehdi h., mehrdad f., rahim m., determination of phenolic compounds content and antioxidant activity in skin, pulp, seed, cane and leaf of five native grape cultivars in west azerbaijan province, iran, food chemistry, 199: 847–855, (2016) [14] velioglu y. s., mazza g., gao l., oomah b. d., antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. journal of agriculture and food chemistry, 46 : 4113-4117, (1998) [15] moyer r. a., hummer k. e., finn c. e., frei b., and wrolstad r. e., anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes. journal of agriculture and food chemistry, 59 : 519-525, (2002). [16] terra x., valls j., vitrac x., mérrillon j.m., arola l., ardèvol a., bladé c., fernández-larrea j., pujadas g., salvadó j. and blay m., grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated raw 264.7 macrophages by inhibiting nfkb signaling pathway, journal of agricultural and food chemistry, 55(11):4357-4365, (2007) [17] xuetao c., mao x., huang h., wang t., zhuo q., miao j., wenyuan g., effects of drying processes on starch-related physicochemical properties, bioactive components and antioxidant properties of yam flours, food chemistry, 224 : 224–232, (2017) [18] iora s.r.f., maciel g.m., zielinski a.f., da silva m.v., pontes p.v., haminiuk c., granato g., evaluation of the bioactive compounds and the antioxidant capacity of grape pomace, international journal of food science and technology, 1-8, (2014) [19] zhao x., zhu h., zhang g., tang w., effect of superfine grinding on the physicochemical properties and antioxidant activity of red grape pomace powders, powder technology, 286 : 838– 844, (2015) [20] shi j., yu j., pohorly j., young j.c., bryan m. and wu y., optimization of the extraction of polyphenols from grape seed meal by aqueous ethanol solution, journal of food, agriculture and environment, 1(2) :42, (2003) http://ajcn.nutrition.org/ http://ajcn.nutrition.org/ 48 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1 2018, pag. 48 52 walnut shells bleaching using oxidizing and reducing agents * eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac faculty of food technology, technical university of moldova, 168 stefan cel mare blvd., md 2004 chisinau republic of moldova, *eugenia.boaghi@toap.utm.md received 17 th october 2017, 19th march 2018 abstract: walnuts with dark-colored shells, with blackish brown spots that are more or less punctuated, have poor consumer acceptability. to improve the appearance and the commercial quality of shell nuts, certain bleaching processes are applied by means of several oxidizing and reducing agents. walnuts juglans regia l, variety calarasi and cogalniceanu, harvest 2015, exploitation iargara, moldova were analyzed. the determination of chromatic characteristics according to cielab and effects of the reaction parameters on the color of the whitened shell by the method of the response surfaces of the color variables l*, a* and b* was made. walnuts bleaching was performed with oxidative bleaching agents (hydrogen peroxide, calcium hypochlorite, sodium perborate and okorn 12) and reductants (sodium dithionite and sulphurous anhydride) at different concentrations, ph values and temperatures. based on the analysis of the effects of the independent whitening parameters (the ph of the environment, the concentration of bleaching agents and the temperature of the bleaching medium) and their interactive effects on the chromatic profile and the whitening process of the nut shell it was found that the oxidative whitening agents are more effective. taking into account the whitening activityof the agents and their toxic emissions, the whitening of walnuts can be done with sodium perborate(cperbora t=5%, τ=60 min, t=60 0 c, ph=10), okoron 12 (cokoron12 =5%, τ=90 min, t=60 0 c, ph=10) and with hydrogen peroxide (ch2o2=10%, cnaoh =2.2 %, t=60 0 c, 90 min). keywords: walnts, bleaching, chromatic parameters, oxidizing and reducing agents 1. introduction walnuts are grown across the world [1]. dark shell colored and stained walnuts have poor consumer acceptability [2]. moldova ranks sixth among world exporters of walnuts, after the united states, mexic and china, being the third among the european exporters of shelled nuts, after france and germany, with an amount that worths $84.8 million dolars. [3]. in recent years, due to the adoption of the national program for the walnut culture development until 2020, to the more active involvement of state and nongovernmental institutions are increasingly emphasized the paths of further walnut culture development. during the harvesting of the walnuts, the wood peels are colored both by intact and crushed green, which is rich in tannins. besides tannins, the green bark is also very rich in hydrogeldone glucoside, which, upon oxidation, releases juglone, causing the endocarp to stain. the quality requirements for exporting walnuts require a clear, attractive product that can be obtained by using various oxidizing and reducing agents during the controlled washing and drying process. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac, walnut shells bleaching using oxidizing and reducing agents, food and environment safety, volume xvii, issue 1 – 2018, pag. 48 – 52 49 both oxidizing and reductive agents are used for bleaching cellulosic products like walnut shell. data on walnuts bleaching with oxidative agents was presented in previous studies [4]. the bleaching activity of the agents is due to the hydrogen peroxide (hp)contained in their structure. when bleaching with hydrogen peroxide, direct action on whitening occurs due to peroxide ion (ho2 -) resulting from the hydrolytic dissociation of peroxide. these ions are trained in reduction or oxidation reactions of the chromogenic lignin groups, thus contributing to the desired color change [5, 6-11]. 2. materials and methods walnut fruits, variety calarasi, were collected from local walnut plantation, iargara, moldova. all chemicals used for experiments were at least analytical grade. the walnut were cleaned to remove trash and damaged, sunburned, and broken walnuts. for walnut bleaching were selected oxidising agents: h2o2, okoron12, na2b2o4(oh)4 and ca(clo)2 (concentrations in the neighborhood of 1 10% being found satisfactory). the process was performed in the temperature range 20-600c and at the ph range 3 to 10 (ph adjusted with hcl or naoh to the desired value). prepared walnut samples were immersed in the solution of bleaching agent being agitated to insure complete contact, after which the walnuts were withdrawn from the solution and allowed to dry. walnuts bleaching was performed at different concentrations, ph values (3, 7, and 10) and temperatures (20, 40, 60 ° c). color changes were judged after the total color difference between the initial (control) and bleached samples, calculated using the formula: e* = (l*2 + a*2 + b*2 )1/2 3. results and discussion previous research on walnut shell whitening is limited and provides the whitening only with solutions of hydrogen peroxide or various combinations of it with oxalic acid or sodium hydroxide and chlorine compounds [12 14]. the first question when performing experiences, was whether they are effective or not for bleaching walnuts and in how much time a 1 to 10% concentration of bleaching agents will manifest its effect. below are the results for walnuts bleached with sodium perborate and okoron 12 that have proven to be the best oxidative bleaching agents. in table 3 are presented the obtained results for the walnuts bleached with sulfur dioxide. table 1. the effect of sodium perborate concentration, ph and temperature on chromatic parameters of walnuts shell agent c, % t, min δe na2b2o4(oh)4 1% 10 21.26 30 21.96 5% 10 22.49 30 23.19 agent ph t, min δe na2b2o4(oh)4 (5%) 3 10 42.03 30 33.92 7 10 41.55 30 35.05 10 10 45.19 30 42.97 agent t, 0c t, min δe na2b2o4(oh)4 (5%) 20 10 41.04 30 42.67 40 10 44.45 30 55.17 60 10 48.73 30 73.90 the results show that the brightness of the walnut shell increases with the increase of the agent concentration, the yellow shade decreases (the value of the component b https://www.hindawi.com/journals/ijd/2009/313845/#b2 https://www.hindawi.com/journals/ijd/2009/313845/#b3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac, walnut shells bleaching using oxidizing and reducing agents, food and environment safety, volume xvii, issue 1 – 2018, pag. 48 – 52 50 increases). the maximum brightness value is reached after 30 minutes of bleaching. in all cases, for each bleacing agent, bleaching rate increases significantly with increasing the temperature of the mediu m from 20 to 60 ° c. the sodium perborate bleaching mechanism is similar to the hydrogen peroxide bleaching mechanism, but the perborate alkalinity is higher than hydrogen peroxide and has more significant effects on the delignification and bleaching process at the same active oxygen concentration. table 2. the effect of okoron 12 concentration, ph and temperature on chromatic parameters of walnuts shell agent c, % t, min δe okoron 12 1% 10 5.64 30 6.32 5% 10 3.76 30 11.47 agent ph t, min δe okoron 12 (5%) 3 10 7.27 30 9.38 7 10 9.44 30 10.91 10 10 15.52 30 19.54 agent t, 0c t, min δe okoron 12 (5%) 20 10 22.71 30 25.61 40 10 25.03 30 34.19 60 10 31.39 30 34.63 the effectiveness of whitening, as can be seen from the results presented in table 4, largely depends on the ph value of the medium, which determines the decomposition of the hydrosulfite, but also the browning caused by the basic medium too strong. table 3. the effect of sulfur dioxide concentration, ph and temperature on chromatic parameters of walnuts shell agent c, % t, min δe so2 1% 10 2.96 30 4.22 5% 10 3.14 30 9.51 agent ph t, min δe so2(5%) 3 10 12.47 30 14.45 7 10 6.72 30 4.17 10 10 4.41 30 7.78 agent t, 0c t, min δe so2 (5%) 20 10 3.09 30 7.12 40 10 12.63 30 20.77 60 10 24.30 30 30.22 table 4. the effect of sodium dithionite concentration, ph and temperature on chromatic parameters of walnuts shell agent c, % t, min δe na2s2o4 1% 10 4.64 30 11.33 5% 10 12.95 30 13.76 agent ph t, min δe na2s2o4 (5%) 3 10 5.84 30 6.67 7 10 10.79 30 16.77 10 10 9.91 30 15.49 agent t, 0c t, min δe na2s2o4 (5%) 20 10 12.95 30 13.76 40 10 18.72 30 29.48 60 10 27.32 30 35.88 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac, walnut shells bleaching using oxidizing and reducing agents, food and environment safety, volume xvii, issue 1 – 2018, pag. 48 – 52 51 whitening of walnuts is relatively low at ph≤5,0 and is caused by accelerated decomposition of hydrosulfite. at ph values greater than 6.0, the hydrosulphite is stable and the bleaching process is more pronounced. at the same time, with the further increase of ph, the formation of chromophores, which are little attacked by hydrosulfite, is amplified. results with a similar trend have been obtained with other whitening agents: hidrogen peroxide and calcium hypochlorite. it has been found that oxidative bleaching agents are more effective and that the bleaching process depends on the concentration of the agents, the temperature and the ph of the medium and the duration of retention of the walnuts in the bleaching medium. after the whitening process of the walnut shell, the agents studied form the following series: na2b2o4(oh)4 >okoron 12>h2o2 > ca (clo)2> na2s2o4 > so2. the obtained results are correlating with others researcher’s data that mention that the effectiveness of the whitening process depends on the concentration, temperature, time, environment ph. all these factors must keep in balance to achieve the desired degree of bleaching [16, 17]. taking into account the bleaching activity of the agents and the toxic emissions produced by them, whitening of the walnut can be done with sodium perborate (%na2(h4b2o8)=5%, 60 min, t=60 0c, ph=10), okoron 12 (%okoron 12 =5%, 90 min, t=600c, ph=10) and hydrogen peroxide (%h2o2=10%, % naoh =2,2 %, t=600c, 90 min). 5. conclusions colour is one of the most important quality attributes influencing consumer food choices, perceptions and purchase behaviour. colour measurement and analysis is therefore important in postharvest handling and biopro-cessing to optimize the quality and value of food. there were quantified chromatic characteristics of walnuts in shells by cielab system and conducted experimental studies on theyr bleaching with oxidizing and reductive agents. taking into account the whitening activityof the agents and the toxic emissions produced by them, whitening of the walnuts can be done with sodium perborate (cperborat=5%, τ=60 min, t=60 0c, ph=10), okoron 12 (cokoron =5%, τ=90 min, t=600c, ph=10) and with hydrogen peroxide (ch2o2=10%, cnaoh =2,2 %, t=600c, 90 min). 6. references [1]. labuckas d.o., maestri d.m., perello m., martinez m.l., lamarque a.l., phenolics from walnut (juglans regia l.) kernels: antioxidant activity and interactions with proteins, food chemistry, 107, (2008). pp. 607-612. [2]. ramos d.e., walnut production manual, university of california, division of agriculture and natural resources, oakland, california, publication 3373, (1998). [3]. foreign agricultural service, tree nuts: world markets and trade, (2017). [4]. boaghi e., impact of treatment with oxidative bleaching agents on walnut (juglans regia l.) shell chromatic parameters. ukrainian food journal volume 5, issue 4 2016, issn 2304–974x, (2016), p.644. [5]. yarborough d.k., the safety and efficacy of tooth bleaching: a review of the literature 1988– 1990, compendium of continuing education in dentistry, 12(3), (1991), pp. 191–196. [6]. dahl j.e., pallesen u., tooth bleaching—a critical review of the biological aspects, critical reviews in oral biology and medicine, 14(4), (2003), pp. 292–304. [7]. robert i., lazau r., dyes and pigments, 105, (2014), p. 152. [8]. pop m., lupea a.x., popa s., gruescu c., int. j. of food prop., 13(4), (2010), p. 771. [9]. driscoll r.h., madamba, p.s., modeling the browning kinetics of garlic, food australia, 46, (1994), pp. 66–71. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac, walnut shells bleaching using oxidizing and reducing agents, food and environment safety, volume xvii, issue 1 – 2018, pag. 48 – 52 52 [10]. moss j.r., otten l., a relationship between color development and moisture content during roasting of peanut, canadian institute of food science and technology journal, 22, (1989), pp. 34–39. [11]. salvador a., sanz t., fiszman s.m., changes in color and texture and their relationship with eating quality during storage of two different dessert bananas, postharvest biology and technology, 43, (2007), pp. 319–325. [12]. kristiansson l., chemical bleaching of wood and its aging: an investigation of mahogany, walnut, rosewood, padauk and purpleheart. bachelor thesis. linköping university, (2012), 24 p.. [13]. suess h.u. (2010), pulp bleaching today, walter de gruyter, berlin. [14]. milovidova l.a., komarova g.v., koroleva t.a., otbelka tselliulozy, agtu, arkhangelsk, (2005). [15]. u.s. forest service research note fpl, v. 165, the laboratory, 1967, minesota university, 8 october 2009 [16]. uysal b, atar m and özçifçi a., the effects of wood bleaching chemicals on the bending strength of wood. tr j agric for 23:615-619, (1999). [17]. singh rp., principles of pulp bleaching. the bleaching of pulp. 3rd ed., atlanta,ga tappi press, (1979), p. 17. * eugenia boaghi, vladislav resitca, pavel tatarov, jorj ciumac *eugenia.boaghi@toap.utm.md keywords: walnts, bleaching, chromatic parameters, oxidizing and reducing agents 1. introduction microsoft word 3 journal nr 4 2010 din 8 noiembrie_76-80.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 80 biogenic amines content of romanian market mackerel octavian baston1, octavian barna1 1dunarea de jos university of galati, faculty of food science and engineering, 111, domeasca st., tel./fax: +40 236 460165, e-mail: octavian.baston@ugal.ro. abstract in romania we eat only imported mackerel (scomber scombrus) because we do not have this kind of fish locally. in our paper we studied samples of frozen and smoked mackerel from romanian market terms of biogenic amines content. the purpose was to find if the samples had low or high quantities of biogenic amines, because it is well known that amines such as: histamine and tyramine when exceeding some values are bad for human health, especially in scombroid fishes. biogenic amines from mackerel samples were determined by hplc (high pressure liquid chromatography) and the amines traced were: tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermine and spermidine. in frozen mackerel samples we detected small amounts of histamine, cadaverine, spermidine and putrescin. only one sample of the frozen mackerel showed clearly the incipient state of spoilage. the biogenic amines from smoked mackerel samples were higher than those found in frozen mackerel. in smoked samples we have not detected serotonin, and only one sample had phenylethylamine. this sample also showed an incipient state of spoilage. histamine and tyramine values were higher in smoked mackerel samples than in the frozen ones. the quantities found of those two amines in the analyzed samples do not cause health problems to people health. keywords: spoilage, fish meat, histamine, tyramine, scombroid poison, biogenic amines. introduction scombroid fish poisoning (scombrotoxism, scombroid ichthyotoxicosis) is a foodrelated illness typically associated with the consumption of fish. most of the published literature suggests that symptoms are related to the ingestion of biogenic amines, especially histamine; others, like putrescine and cadaverine, may potentate toxicity. although scombroid poisoning is more common in nations with a warm water fishing industry, the illness is worldwide in scope. the most commonly implicated fish species are scombroid dark meat fish (eg, tuna, mackerel, skipjack, bonito, and marlin) and nonscombroid species, such as mahi-mahi (dolphinfish), amber jack, sardine, yellowtail, herring, and bluefish. although rare, cases of whitefish scombrotoxism also have been reported. [1] cadaverine, putrescine and histamine can be produced postmortem from decarboxilation of correspondant free aminoacids in mackerel: histidinehistamine, ornithine-putrescine, lysinecadaverine. those are biogenic amines that influence negatively the consumer’s health and sensorial characteristics of mackerel. other amines that can be found in mackerel are: tyramine, tryptamine, 2phenylethylamine. also polyamines: spermine and spermidine can be found in mackerel. the decarboxilation process takes place in two biochemical processes: -by endogenous decarboxylase enzymes naturally occurring in mackerel and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 81 -by exogenous enzymes released by microorganisms associated with the mackerel. hystamine is the most studied biogenic amine in mackerel meat. this amine is associated with the illness called scombrotoxicosis. the content of histamine must be higher than 300 mg, otherwise the intoxication do not occur [2]. in fresh mackerel, the content of biogenic amines varies in function of the anatomic part studied, of muscle type and of processing after catch. intestinal wall samples contain high amine levels than muscles [3]. in red muscle mackerel histamine and histidine were produced in larger amount as compared to white muscle. the effect of evisceration on biogenic amines production is inconsistently reported in literature. the rates of cadaverine and putrescine production can be ranked as follows: whole ungutted fish> fillets from whole ungutted fish and fillets from gutted fish> whole gutted fish [4]. this generalized description is heavily dependent on the extrinsic factors such as harvesting method and procedures for transportation, processing and retailing. after hot smoking of frozen mackerel the study made by zotos et al. [5] showed that a significant increase in histamnine formation in previously frozen mackerel was solely due to temperature and reduction in smoking process. so, it was demonstrated that is very important to control temperature and time in the smoking process. our objectives were to find out the biogenic amines content of some market samples of mackerel (scomber scombrus) and if those values found represent a threat to consumer’s health. materials and methods we went to a hypermarket and bought frozen mackerel, with head, guts and tail, packaged in plastic bag and gutted smoked mackerel headless, gutted and without tail, packaged individually in vacuum. frozen mackerel were put in refrigerated box and then transported to our laboratory where it was analyzed. the smoked mackerel was transported normally, with no special preservation techniques. all the samples were from the same producer. after we had the samples in our laboratory, we checked the shelf life term and all the samples were at the second third period of validity term. the samples processing was made as follows: we cut transversally three pieces of mackerel, first near the head, the second in the fish middle and the third near to fish tail. the width of the fish cut was of 2 cm. after that we deboned the fish and, if necessary we eliminated the guts. the measurement of biogenic amines content using high performance liquid chromatography was performed according to the method proposed by food research institute from helsinki, finland [6]. all the reagents used were analytic pure, for hplc use. the water used was deionised. the necessary reagents were purchased from the merck and sigma-aldrich companies. installations and equipment used for biogenic amine determination: philips 7768 food processor, homogenisation device 7011s, kern 77060 analytical balance, silent crusherm homogenisation device, centrifuge eba 21, filter paper for quick filtering with 55 mm diameter, syringe filters with porosity of 0,45 µm and 13 mm diameter, heidolph reax control agitator, ultrasonic water tank aquawave tm, incubator bmt incucell 55, water deionising system easy pure rodi, filtering assembly with vacuum pump. the device for the hplc determination was a liquid chromatograph model surveyor produced by thermo electron company, configured with detector model pda plus detector, auto-sampler model autosampler food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 82 plus, pump model lc pump plus and detector uv-vis. chromatography column of type bds hipersyl c18. the biogenic amines quantification: quantitative measurement was performed depending on the internal standard using peaks for each biogenic amine. the 254nm wavelength absorbance was measured and the resulted peaks were integrated with cromquest software. the concentration of each biogenic amine was expressed in mg/kg. the method principle is as follows: -bioactive amines are extracted from a homogenized sample with diluted perchloric acid; -an aliquot of the extract is derivatised with dansyl chloride reagent; -separation and quantification of dansylated amines is performed by reversed phase liquid chromatography with ultraviolet detection at 254 nm. the values obtained for biogenic amines content for each sample were done in triplicates. our determinations refer to the following amines: tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermine and spermidine. results and discussion in order to know exactly if romanian market mackerel samples have biogenic amines content and to what extent we analyzed frozen mackerel and smoked mackerel, and the data obtained are shown in table 1 and table 2. in the case of table 1 it is clear that phenylethylamine and serotonin were below the detection concentration in all the samples analyzed. table 1. biogenic amines content of frozen mackerel samples biogenic amines (mg/kg) sample number trypt fen put cad hist ser tyr spd spm 1 nd nd 3.7 nd 1.1 nd nd 1.3 0 2 nd nd 6.2 nd nd nd 12.1 0 nd 3 nd nd 9.6 nd nd nd 30.5 1.8 nd 4 nd nd nd nd nd nd 7.4 0 nd 5 nd nd 7.5 nd nd nd nd 1.5 nd 6 0 nd 2.2 1.3 1.7 nd 42.6 2.3 nd 7 nd nd 5 nd 2.6 nd 15.5 1 0 8 0 nd nd 0 nd nd nd 0 0 9 0 nd nd nd 0 nd 7.7 2.1 nd 10 nd nd 9 0 2.4 nd 13.9 5.5 nd where: trypt-tryptamine, fen-phenylethylamine, put-putrescine, cad-cadaverine, hist-histamine, ser-serotonin, tyr-tyramine, spd-spermidine, and spm-spermine. nd-not detected. the values presented in table 1 are an average of three determinations for each sample. tryptamine and spermine had some samples with 0mg/kg while the rest of samples had concentration below the detection limit. also, below the detection limit we found in descending number cadaverine, histamine putrescin and tyramine content in frozen mackerel samples. it is important to highlight that for tyramine we have the highest quantities of all the biogenic amines. tyramine maximum value of all the samples analyzed was 42.6mg/kg. it is possible that high values of tyramine to be in the fishes that were unfrozen during transportation or manipulation, which means that mackerel food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 83 cold chain, was interrupted. another possible cause might be the fact that the fish was caught in hot weather (in summer) and was not frozen as quickly as possible. another possibility was that the ungutted mackerel before freezing were hurt and bacteria from intestine to action at fish meat. tyramine is a naturally occurring amino acid that forms from the breakdown of protein in food as it ages. though tyramine helps regulate blood pressure, it can also affect the human body in many different, negative ways. it has been known to trigger headaches and severe migraines. in our case tyramine do not exceed 50 mg/kg and theoretically do not threaten the human health because is in low amount. strong [7] has shown that 1 mg tyramine can trigger an attack in one particular individual while provocation tests made by hanington et al.[8] used up to 100mg tyramine. so, we cannot say that peoples that eat the fishes that contain tyramine will get sick and have headaches or high blood pressure. the symptoms vary from individual to individual, because some of them tolerate this biogenic amine better than other. frozen mackerel samples had histamine content between nd to 2.6mg/kg. we can say that 2.6mg/kg is not a high value that threatens the human health. histamine is a toxic agent implicated in human scombroid poisoning. in hystamine intoxication the symptoms are at the beginning the following: facial flushing/sweating, dizziness, nausea, and headache, tachycardia, followed by rush, diarrhoea and abdominal cramps. jarisch and wantke who used 50 mg/kg of histamine for predisposed individuals observed all these symptoms. [9]. also, the commission regulation (ec) no 2073/2005 (oj l338, p1, 22/12/2005) of 15 november 2005 on microbiological criteria for foodstuffs specify that for fishery products from fish species associated with a high amount of histidine, the histamine content should be between 100 and 200 mg/kg. in frozen mackerel studied samples the inferior limit is wide off putrescin give us an interesting view. we know that cadaverine and putrescin appear in altered fishes. the presence of those biogenic amines, even in small quantities (cadaverine maximum quantity is 1.3 mg/kg and putrescin maximum quantity is 9.6 mg/kg) without exceeding10 mg/kg, shows us that fishes were in incipient state of spoilage. it is well known that fishes that are caught in warm weather and with guts inside spoil faster. spermidine content of mackerel varies from 0 to 5.5 mg/kg. of our samples, it appears that sample 6 have five biogenic amines and characterizes the spoiled sample. smoked mackerel samples do not contain serotonin at all. spermine is not present because it has many values of nd and some of 0 mg/kg. table 2. biogenic amines content of smoked mackerel samples biogenic amines (mg/kg) sample number trypt fen put cad hist ser tyr spd spm 1 12.9 nd nd nd nd nd 0 1 0 2 20 22 43.8 37.5 35.9 nd 19 5.8 nd 3 7.8 nd 2.5 nd nd nd 8.5 4.6 nd 4 nd nd 16 nd nd nd 4.1 7.2 nd 5 nd nd 13.3 7.4 1.2 nd 2.4 3.4 nd 6 0 nd nd 5.8 11.6 nd 1.9 3.2 nd 7 3 nd 5.6 1.1 nd nd 4.7 2.9 nd 8 0 nd 4 nd 6.8 nd nd 3.9 nd 9 2.2 nd 7.8 2.6 14.9 nd 8.6 6 0 10 1.4 nd 1.7 15.4 7.9 nd 5.3 2.8 0 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no. 4 2010 84 tryptamine is present in eight of the ten samples analyzed. tryptamine values for smoked mackerel samples are between nd to 20 mg/kg. on human body, tryptamine has a similar effect like vasoactive amine tyramine. tyramine is the carboxylated form of tryptophan. this is due to the microorganism action on amino acid tryptophan, and so, it is a product of certain spoilage microorganisms. very interesting is the presence of phenylethylamine in sample number two. also, it is in large amount. phenylethylamine is created by foods breaking down or spoiling. putrescin and cadaverine are found in many samples. also are histamine and tyramine. their presence clearly indicates the mackerel spoilage. because for the most of samples the values are relatively small, we can say that the mackerel was already before the smoking treatment in incipient spoilage state. sample number two is especially the most spoiled of all analyzed samples for smoked mackerel. making a comparison between frozen and smoked mackerel, we can say that smoked mackerel has higher content of biogenic amines. this is probably due to the fact that the mackerel was imported in frozen state, it had to be thawed and possibly ungutted before smoking, and those operations influenced the microbiota activity in mackerel leading to biogenic amines formation in higher quantities than in the frozen mackerel. conclusion the romanian studies on mackerel samples showed clearly that smoked mackerel had more biogenic amines than frozen mackerel. the amount of biogenic amines found in frozen samples showed that some of the mackerel samples were in incipient state of spoilage. we have also have found that the majority of smoked mackerel was in incipient state of spoilage. the smoked mackerel samples had higher content of biogenic amines comparing with frozen mackerel ones. histamine and tyramine content for frozen and smoked samples do not cause health problems, maybe with one exception, but for healthy individuals they surely do not cause any health problems. references 1.john d patrick, toxicity, scombroid, http://emedicine.medscape.com/article/818338overview. 2.george j. flick and l. ankenman granata, 2005, toxins in food,chapter: biogenic amines in food, crc press, editors: waldemar m. dabrowski, zizislaw e. sikorski, florida, usa, p. 123. 3.gloria, m, daeschel, m.a, craven, c, hilenbrand jr, k.s., 1999, histamine and other biogenic amines in albacore tuna, j. aqu, food prod. technol., 8, 54. 4.haaland, h, arnesen, e and njaa, l.r., 1990, amino-acid composition of whole mackerel (scomber scombrus) stored anaerobically at 20degrees cand at 2-degrees c, int. j. food sci. technol., 5, 42. 5.zotos, a., hole, m. and smith g., 1995, the effect of frozen storage of mackerel (scomber scombrus) on its quality when hot smoked, j. sci. food agric., 67, 43 6.eerola, s., hinkkanen, r., lindorfs, e. and hurvi, t., 2001, liquid chromatographic determination of biogenic amines in dry sausages, journal aoac international, 76, 3, 575-577. 7.strong, f.c., 2000, why do some dietary migraine patients claim they get headaches from placebos?, clin exper allergy 30 (5), 739-743. 8. hanington e, harper am., 1968, the role of tyramine in the aetiology of migraine and related studies on the cerebral and extracerebral circulations. headache 8:84-97. 9. jarisch r, wantke f., 1996, wines and headache. a mini-review. int arch allergy immunol;110: 7–12. microsoft word 3 journal fia nr 1 2010 final_60-63.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 64 studies regarding the efficiency of a direct methanol fuel cell (dmfc) violeta vasilache stefan cel mare university of suceava, faculty of food engineering. 13 th university street, 720229, romania, e-mail adress: violetav@usv.ro abstract: combustion cells are devices which transform chemical energy into electric and thermal energy. there could be an acceptable solution to the problem of alternative sources of energy, for a lot of practical applications. dmfc combustion cell (dmfc – direct methanol fuel cell) uses methanol, instead of hydrogen, so it works safely. more, the storage of combustible does not represent a difficult problem, like the hydrogen storage presents. a reforming device is not necessary, because the cell itself transforms methanol into hydrogen protons, free electrons and co2. the absence of reforming device make this type of cell more adapted for automotive applications where the objective is to have a simple source of electric power. a polymeric membrane which could conduct protons is used as electrolyte. the technology dmfc (direct methanol fuel cell) is in fact a variant of pemfc (polymer electrolyte membrane fuel cell) technology, which was developed having hydrogen as combustible. using chemical reactions, the efficiency was calculated theoretically to find the possibilities of increasing it. thermodynamic considerations were also used to complete the efficiency study. finally, using scientific data, a calculus was performed. keywords: methanol fuel cell, material and energy balance, thermodynamic introduction (actual state of research) surampidi and co. have studied the effect of temperature and of the methanol concentration through efficiency in a dmfc. the performances were measured at 30ºc, 60ºc and 90°c respectively, and the results prove an increase of performances with increasing the temperature. they worked with concentrations of 0,5, 2,0 and 4,0 m; higher voltage was obtained for 2,0 m concentration when the current density was higher too. optimal concentration was between 0,5 and 2,0 m. jung and co. studied the effect of working temperature for the range 60-120°c and of the methanol concentration for the range 0,54,0 m. the methanol flow on anode was 9 ml/min and the pure oxygen flow was 105 sccm. the results also confirmed the fact that performances of the cell are increasing with temperature and the optimal concentration was 2,5 m. nakagawa and xiu studied a cell in the temperatures range from 30ºc to 100°c and the effect of proportion of oxidant gas (air and pure oxygen respectively)[1,2,3,4]. recent studies at los alamos national laboratory (lanl) focalized on possible applications of dmfc as principal and auxiliary power source for vehicles and as a portable power source for the range 1100 w. the conditions in exploitation force some restrictions, so working temperatures have not to be higher than 100°c and the consumption of precious metals has to be reduced at minimum. for portable power sources the temperature should not exceed 80°c, but the power demands are reduced, journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 65 fact that imposes a decreasing of consumption of noble metals [5,6]. theory, studies, results and discussion function of a dmfc is based on next phases: phase1. those two gases, oxygen (air) and methanol, maintained in two separated tanks – oxygen on the cathode side and methanol on anode side, flow from tank to catalyzer. phase2. methanol (ch3oh) reacts with water forming carbon dioxide and hydrogen. at catalyzer side, hydrogen is decomposed into two atoms of h+ (protons) and every atom yields its electron in this process[7,8]. phase3. protons pass through electrolyte (polymeric membrane capable to conduct protons) and displace to cathode. phase4. electrons migrate from anode to cathode and generate an electric current (electric power). phase5. at contact with cathode, four electrons always recombine with an oxygen molecule. phase6. just formed oxygen ions have a negative electric charge, and furthermore react with protons forming water. the chemical reactions occurring are: at anode side: ch3oh + h2o → co2 + 6h+ + 6e (oxidation) (1) at cathode side: 3/2o2 + 6h+ + 6e→ 3h2o (reduction) (2) total reaction: ch3oh + 3/2o2 → co2 + 2h2o (3) the efficiency of a combustion cell can be measured by measuring the potential curve v(i). generally the next relation should be taken into consideration: v(i)=ε0 – ηact(i) – ηohm(i) – ηconc(i), (4) where ε0 is a constant term which represents the value of potential difference of the cell in open circuit (i=0); ηact(i) is a logarithmic term (activation overpotential), which represents a lose connected with the activation energy for starting electrochemical reactions, important for lower values of the current density; ηohm(i) is a linear term, representing potential drop due to the ohm resistance of the electrolyte, electrodes and conductors; ηconc(i) is an exponential term (diffusion or concentration over-potential), which represents the lose due to reactants reactivity variation on interface, important for high values of current density. figure.1. image of a dmfc: water, methanol and protons change membrane (pem) for a thermal engine the maximum theoretic efficiency is that of a carnot cycle: ηc=1 – t2/t1, (5) where t1 and t2 are absolute temperatures of hot source and cold source respectively; for combustion cell, maximum efficiency is expressed by the ratio between free energy gibbs (δg) and the variation of free enthalpy (δh) in electrochemical reaction: ηpc= δg/ δh . (6) the global conversion efficiency of a combustion cell is superior to that of a journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 66 thermal engine. thermodynamics of transformations chemical energy – electric energy give: – δg = lutil, δg = δh – tδs (7) the work-function of the electrical charge is giving by relation: lutil = nfδe or –δg = nfδe (8) where, n=number of electrons participating in reaction, e = e.m.f. (v) (electromotive force), f = faraday’s constant. the heat produced is given by the relation: q = tδs = δh – δg. (9) for a general chemical reaction:        ba dc ba dc rtgg ln0  (10) which could be written for an ideal gas,     i i reactpart prodpart rtgg     .. .. ln0 (11) where δg0 is gibbs free energy in standard conditions (p=760mmhg, t=25c°c), νi are stoichiometric coefficients of reaction. assuming that: – δg = nfδe, on obtains:      i i partprod partreact rtee   . . ln0 (12) for standard conditions it follows, e = e0. in irreversible thermodynamics there are loses due to: 1. ohmic polarization – due to resistance phenomena when ions pass through electrolyte and electrons pass through interface. 2. concentration polarization – due to quickly consumption of reactants on electrodes surface and appearing of concentration gradients. 3. activation polarization – refers to surpass of a initial threshold potential δvact similar with the case of concentration polarization. the efficiency is defined after the relation: lhv elel e e nflhv nfl lhv l  (13) lhv represents the heat obtained by combustion of a mol of combustible (j/mol combustible). elhv represents the electric equivalent of caloric inferior power of the combustible. then the potential difference of the cell is: vc=e – δvohm – δvconc – δvact . (14) here, δvohm represents potential drop due to electric resistance of the cell, δvconc potential drop due to concentration variation and δvact potential drop due to initial activation processes. subsequent sources of losing are bonded by the way of using combustible and air. inh consh comb m m u , , 2 2 or (15) inohch consohch comb m m u , , 3 3 . (16) here ucomb is the ratio which describes the efficiency of combustible utilization, and m are masses of combustibles consumed, and initial masses respectively. it follows that: lhv combc e uv  . (17) if one particularizes electrochemical oxidation of methanol, it follows assuming that a=1, b=3/2, c=1 and d=2,         232 1 3 2 2 1 20 ln oohch ohco rtgg  (18) fact which diminishes δg comparing with δg0, and consequently a decreasing of efficiency. as a general rule, valid for combustion cells, the removal of formed water should be ensured, because it induces a decrease of methanol solution concentration. in this case, of direct methanol fuel cell, it works with totally immersed electrodes, which simplify the problem from the mechanical point of view. ideal condition consists of complete penetration of electrolyte into porosity of journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 67 the electrode. it is pointed to obtain a higher percent of volume porosity, poresfree. the dimension of particles have to be chosen to obtain a good contact through them and with support layer, in order to facilitate electronic conductibility in all electrode volume [9,10,11]. the reaction rate in the pores of the electrode, in every point, is dependent by interface potential, current density which flows through interface electrodeelectrolyte; by the concentration of reactant species which arrive in pores, and by nature of electro-catalyzer deposited on electrode. these local conditions are depended by mass transfer of reactant species and by current transport to a specified point and by this point to another interface. so, to make a complete characterization of the porous electrode it is necessary to know the distribution of reaction in all electrode volume. a calculus example is presented here: global reaction: ch3oh + 3/2 o2 → co2 + 2h2o for ideal conditions: kmol/kg32m oh3ch = ; kmol/kg32m 2o = on anode: ch3oh + h2o → co2 + 6h+ + 6e so, every ch3oh molecule produces 6e-. 1kmol ch3oh : 6enq a ••= ; q = 5,78208 . 108c total produced energy is (u=1,21v) w = q.u = 6,99....108j or, w = 194,3 kwh for real conditions: u = 0,82 v (from literature) 5% lost by intermediate reactions, so the efficiency, %95oltanme  c10...492,595,06enq 8a =•••= kwh125j105,4 82,010...492,5uqw 8 8 =•= ==•= %64 kwh3,194 kwh125 w w ideal real energetic ===η references 1. h. dohle, j. divisek and r. jung, process engineering of the direct methanol fuel cell, j. power sources 86 (2000), pp. 469–477 2. h. dohle, h. schmitz, t. bewer, j. mergel and d. stolten, development of a compact 500w class direct methanol fuel cell stack, j. power sources 106 (2002), pp. 313–322 3. d.h. jung, c.h. lee, c.s. kim and d.r. shin, performance of a direct methanol polymer electrolyte fuel cell, j. power sources 71 (1998), pp. 169–173. 4. muscalu, s., platon,v., pile de combustie, editura tehnica, 1989 5. s.r. narayanan, a. kindle, b. jeffriesnakamura, w. chun, h. frank, m. smart, t.i. valdez, s. surampudi, g. halpert, j. kosek and c. cropley:, recent advances in pem liquid-feed direct methanol fuel cells, ann. battery conf. appl. adv. 11 (1996), p. 113. 6. n. nakagawa and y. xiu, performance of a direct methanol fuel cell operated at atmospheric pressure, j. power sources 118 (2003), pp. 248–255. 7. a.k. shukla, p.a. christensen, a.j. dickinson and a. hamnett:, a liquid-feed solid polymer electrolyte direct methanol fuel cell operating at near ambient conditions, j. power sources 76 (1998), pp. 54–59. 8. a.k. shukla, c.l. jackson, k. scott and g. murgia, a solid-polymer electrolyte direct methanol fuel cell with a mixed reactant and air anode, j. power sources 111 (2002), pp. 43–51. 9. s. surampidi, s.r. narayanan, e. vamos, h. frank, g. halpert, a. laconti, j. kosek, g.k. surya prakash and g.a. olah, advances in direct oxidation methanol fuel cells, j. power sources 47 (1994), pp. 377–385. 10. t.i. valdez, s.r. narayanan, recent studies on methanol crossover in liquid-feed direct methanol fuel cells, proceedings of the 194th meeting of the electrochemical society, boston, november 1998. 11. veziroglu, t., n., international journal of hydrogen energy, 2000, 1143 29 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017, pag. 29 33 oxidative stability of refined sunflower oil at room temperature and during conventional frying *gorica pavlovska1 , *natasha shurkova2 , *vezirka jankuloska1 1faculty of technology and technical sciences, university st. kliment ohridski bitola, dimitarvlahov 57, veles, r macedonia, 2f.i. blagojgjorev jsc, veles, r macedonia gorica.pavlovska@uklo.edu.mk; natasha.shurkova@gmail.com; vezirka.jankuloska@uklo.edu.mk *corresponding author received january 30th 2017, accepted march15th 2017 abstract: the aim of this stady was to assess the oxidative stability of refined sunflower oil both at room temperature and during conventional frying in a pan. for this purpose, the refined sunflower oil was heated for 10 minutes in a fryer pan. under these conditions, the wheeler method was used to determine the peroxide number. oven test was used to determine the oil stability. peroxide number of the oil fried from 0 to 10 minutes at a temperature from 27ºc to 200ºc continuously increases from 0.357 mmol/kg to 6.4762 mmol/kg. peroxide number of the oil analyzed by the oven test increased from 2.41 mmol/kg for 24 hours to the value of 16.67 mmol/kg for 96 hours. the oil that has been fried for 10 minutes and reached a temperature of 200ºc should not be used as food. the oil which is kept in an open bottle at room temperature can be used up to 24 days. keywords: sunflower oil, peroxide number, oven test, frying 1. introduction sunflower oil is one of the most used oils. the first oil produced from sunflower was patented in england in 1716. sunflower seeds contain 40-60% oil and it depends on the type, location and method of cultivation of the sunflower [1]. sunflower oil contains essential fatty acids, polyphenols, phosphatides and vitamins a, d, e and k [2-4]. among the fatty acids, the most represented are linolenic acid 5065% and oleic acid 30-40% [5]. the oil that is obtained by pressing is called cold pressed oil, while the oil obtained by extraction is refined oil. cold pressed oils have greater antioxidant activity due to higher content of polyphenols, but they are less stable and the oxidized more easily [6]. oils oxidize after longer standing, at a temperature higher than that of room temperature and in case of frying [7-9]. the oxidation of products depends on temperature, availability of oxygen, surface oil exposed to oxygen, light, and the presence of metals [9-10]. the most important factors for the oxidative stability of oils are: contact with oxygen, temperature, metals as catalysts and light. when the temperature increases by 10ºc the oxidation of products increases doubly [11-12]. oxidation of fats and oils that are present in food products today are a major problem in food technology. oxidation products in oil are reduced if antioxidants are added. today, aromatic herbs, fruits and vegetables are used as natural antioxidants, being added to oil and food with high content in fats and oils [13-16]. oxidation products in fats and oils are toxic and harmful to human health. experiments on animals have shown a decline in nerve cells, memory and attention [17]. they are carcinogenic, http://www.fia.usv.ro/fiajournal mailto:gorica.pavlovska@uklo.edu.mk; mailto:natasha.shurkova@gmail.com; mailto:vezirka.jankuloska@uklo.edu.mk food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 1 – 2017 gorica pavlovska, natasha shurkova, vezirka jankuloska, oxidative stability of refined sunfloweroil at room temperature and during conventional frying, food and environment safety, volume xvi, issue 1 – 2017, pag. 29 – 33 30 hepatotoxic and can contribute to the development of alzheimer and parkinson disease [18-19]. there are several physical and chemical methods for the determination of the oxidation products of fats and oils. peroxides and hydroperoxides which are the main oxidation products in oil are characterized by the peroxide number. the type of oxidation products is determined by various instrumental methods [20]. the stability of oils and fats except of external factors depends on the type and composition of oils and fats.the methods used to determine the stability are based on the accelerated oxidation of sample under the influence of one or more factors that accelerate this process. in practice, most applications are methods in which the oxidation is accelerated by increasing the temperature. the mostly used methods for determining the stability of fats and oils are: oven test, swift test and the rancimat test [21-22]. the purpose of this study is to evaluate the stability of sunflower oil "kristal" at room temperature and to determine the oxidation products occurred during conventional frying of oil in a pan. 2. materials and methods the refined sunflower oil "kristal"was analyzed. 10 bottles of 1l packages were purchased from supermarkets in macedonia. for each experiment oil from each bottle is analyzed individually. the results from our experiments are mean of 10 measurements of oil from 10 different bottles. the stability of oil is determined by oven test at a temperature of 63ºc for 4 days (96 hours). the oil temperature was maintained constant using a drying oven instrumentaria st-05. every 24 hours, a sample was taken for analysis and the peroxide number was determined by the method of wheeler [23]. oven test is performed to determine how long the oil can be stored in an open bottle at room temperature. it has been proven that one day of oven test corresponds to a real stability of the oil of 6 to 12 days at room temperature [24]. to see how the oil gets oxidized during conventional frying in a pan, the following analysis was conducted. 100 ml oil was placed in a pan with a diameter of 24 cm and heat on hot plate at half of its maximum power of 2 500 w. the sample is heated for 10 minutes. after 2, 4, 6, 8 and 10 minutes the temperature of the oil is measured and the peroxide number is determined by the method of wheeler. the temperature is measured with barbecue high thermometer tbt 08h which has a range of -50ос to +300ос, with a tolerance of +/1°c. all chemicals for the determination of peroxide value were of analytical grade and purchased from merck (germany) and sigma (usa). 3. results and discussion the results of the determination of the oven test are given in fig. 1. fig. 1. peroxide number in the analyzed oil with oven test after 24 hours the peroxide number is 2.41 mmol/kg and it is within the allowed limits according to the rules of the republic of macedonia [25]. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 1 – 2017 gorica pavlovska, natasha shurkova, vezirka jankuloska, oxidative stability of refined sunfloweroil at room temperature and during conventional frying, food and environment safety, volume xvi, issue 1 – 2017, pag. 29 – 33 31 peroxide number after 48 hours is 5.567 mmol/kg, which is above the maximum value (5.00 mmol/kg) according to the official journal of r. macedonia no.127/12. if 48 hours in drying oven at temperature of 63ос corresponds to maximum 24 days at room temperature, that means that after 24 days an open bottle of oil stored at room temperature (about 25°c) has a large amount of oxidation products and it is not recommended for use. after 72 hours in a drying oven at a temperature of 63ос or 36 days at room temperature, peroxide number of the oil is 10.635 mmol/kg and it is two times higher than the maximum which is allowed. as expected, the oxidation increases constantly, and the peroxide number increases considerably. after 96 hours at 63ос or 48 days at room temperature, the peroxide number is 16.67mmol/kg. this value is more than three times higher than the maximum allowedand cooking oil in this case is too harmful to use. the second experiment was made to determine the oxidation products in the oil during frying. for this purpose, the temperature is measured and the peroxide number of oil after 2, 4, 6, 8 and 10 minutes is determined. the dependence of the peroxide number on the heating time is shown in fig. 2, and its dependence on the temperature is shown in fig. 3. fig. 2. influence of time on the peroxide number the initial value of the peroxide number is 0.357mmol/kg and it increases as the heating time is increased. after just two minutes of heating it increases to the 0.5376 mmol/kg, after 4 minutes it is of 0.6558 mmol/kg, and after 6 minutes it is of 0.9375 mmol/kg. in the next 2 minutes there is a rapid increase of peroxide number to 4.1202mmol/kg. however, all these values of peroxide number are smaller than the maximum allowed values of 5.00mmol/kg according to the official journal of r. macedonia no.127/12 [25]. when the oil is heated for 10 minutes the peroxide number exceeds the maximum value and is 6.4762 mmol/kg. this means that the prepared food, in which the oil is heated more than 10 minutes at this temperature, contains a high concentration of oxidation products and it is not recommended for use. when the oil is heated its temperature increases. the initial oil temperature is 27°c and then it increases continually. after 2 minutes the oil temperature is 66°c, after 4 minutes 91°c, after 6 minutes 127°c, after 8 minutes 147°c, and after 10 minutes, it reaches even 200°c. at this temperature the oil starts smoking and this oil, as shown by peroxide number is not suitable for use (fig. 3). fig. 3. influence of temperature on the peroxide number food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 1 – 2017 gorica pavlovska, natasha shurkova, vezirka jankuloska, oxidative stability of refined sunfloweroil at room temperature and during conventional frying, food and environment safety, volume xvi, issue 1 – 2017, pag. 29 – 33 32 4. conclusions the results of our study show that the stability of refined sunflower oil “kristal” evaluated on the base of oven test is significantly affected during storage in a drying oven at a temperature of 63ос. after 48 hours, the peroxide number of the oil corresponds to that of the oil stored at room temperature not exceeding 24 days. for oil samples kept in a drying oven at a temperature of 63ос, for 72 and 96 hours the peroxide number was two, respectively three times higher than the allowed upper limit.our data highlight that the cooking oil “kristal” can be safely used up to 24 days, if it is kept open at room temperature.as regards the peroxide number of the fried oil samples, the obtained results reveal that in the frying time there were recorded increased values of peroxide number with the increasing of temperatures from 27ос to 200ос. when the oil is fried for 8 minutes the peroxide numbers were in acceptable range while the oil fried for 10 minutes contains peroxide amounts over the allowed value. 5. references [1]. dimič e., cold pressed oils, faculty of technology, novi sad, (2005). [2]. dimič e., quality control of cold pressed oil, acta periodica technologica, 31: 165-174, (2000). [3]. talal e., jiang j., yuanfa l., chemical refining of sunflower oil: effect on oil stability, total tocopherol, free fatty acids and colour, international journal of engineering science and technology, 5: 449-454, (2013). [4]. tasan m., demirci m., total and individual tocopherol contents of sunflower oil at different stepsofrefining, europian food research and technology, 220: 251–254, (2005). [5]. jamieson j., baughman w., the chemical composition of sunflower oil, journal of the american chemical society, 44: 2952–2957, (1922). [6]. kostadinovic-velickovska s., mitrev s., caracterization of fatty acid profile, polyphenolic content and antioxidationt activity of cold pressed and refined edible oils from macedonia, journal of food chemistry and nutrition, 01: 16-21, (2013). [7]. maniaki b., szmigielski m., piekarski w., markowska a., physicochemical changes of post-frying sunflower oil, international agrophysics, 23: 243-248, (2009). [8]. wasowicz e., gramza a., marzanna h., jelec h., korczak j., maiecka m., mildner-szkudlarz s., rudzicska r., samotyja u., zawirskawojtasiak r., oxidation of lipids in food, polish journal of food nutrition sciences, 13: 87– 100, (2004). [9]. sadoudi r., ammouche a., ali ahmed d., thermal oxidative alteration of sunflower oil, african journal of food sciences, 8: 116-121, (2014). [10]. crapiste g., brevedan m., carelli a., oxidation of sunflower oil during storage, journal of the american oil chemists society, 76: 1437-1443, (1999). [11]. berger k. g., practical measures to minimize rancidity in processing and storage, 3rd edition. j.c. in rancidity in foods, allen and r.j. hamilton, chapman and hall, london, 68-83, (1994). [12]. farhoosh r., hoseini-yazdi s. z., shelf-life prediction of olive oils using empirical models developed at low and high temperatures, food chemistry, 141: 557-565, (2013). [13]. al-dalain s. al-fraihat a., al kassasbeh e., effect of aromatic plant essential oils on oxidative stability of sunflower oil during heating and storage, pakistan journal of nutrition, 10: 864-870, (2011). [14]. usatiuk s., pelekhova l., chlorophyll content and antioxidant activity of sunflower oil with aromatic raw materials, food and environment safety, 13: 317-320, (2014). [15]. codina g.g., poroch-seritan m., mironeasa s., blending of sunflower oil with grape seed oil: impact on physicochemical parameters and radical scavenging activity, food and environment safety, 14: 101-107, (2015). [16]. capcanari t., incorporation of sweet pepper extracts to improve thermal stability of vegetable oil mixtures, food and environment safety, 10: 13-19, (2011). [17]. zarate j., goicoechea e.., pascual j., echevarria e., guillen m.d., a study of the toxic effect of oxidized sunflower oil containing 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal on cortical trka receptor expression in rats, nutritional neuroscience, 12: 249-259, (2009). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 1 – 2017 gorica pavlovska, natasha shurkova, vezirka jankuloska, oxidative stability of refined sunfloweroil at room temperature and during conventional frying, food and environment safety, volume xvi, issue 1 – 2017, pag. 29 – 33 33 [18]. barrera g., oxidative stress and lipid peroxidation products in cancer progression and therapy, international scholarly research notices: oncology, 2012: 1-21, (2012). [19]. farooqui t., farooqui a., lipidmediated oxidative stress and inflammation in the pathogenesis of parkinson's disease, parkinson's disease, 2011: 1-9, (2011). [20]. dieffenbacher a., pocklington w.d., standard methods for the analysis of oils, fats and derivatives, 1st supplement to the 7th edition, blackwell scientific publications, oxford, (1992). [21]. grettie d.p., newton r.c., susceptibility of fats to oxidative rancidity, oil and fat industry, 8: 291-294, (1931). [22]. joyner n.t., mcntyre j.e., the oven test as an index of keeping quality, oil and soap, 15: 184-186, (1938). [23]. aocs cd 8-53, official methods and recommended practices of the american oil chemists’ society method cd 8-53, (1996). [24]. dimič e., turkulov j.,quality control in the technology of edible oils, faculty of technology, novi sad, 135-137, (2000). [25]. official journal of r. macedonia. rulebook on requirements regarding the quality of vegetable oils and vegetable fats, margarine, mayonnaise and related products of r. macedonia, 127, (2012). microsoft word 1 codina_corectat_lucrare_fia_rev_2.docx 61 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 22017, pag. 61 70 quality evaluation of wheat-pumpkin-golden flaxseed composite bread georgiana gabriela codină1*, denisa atudorei 1, ana cimpoi1, silvia mironeasa1, elena todosi-sănduleac1 1faculty of food engineering, ştefan cel mare university, adress, str. universităţii nr. 13, 720229, suceava, românia *corresponding author: codina@fia.usv.ro; codinageorgiana@yahoo.com received april 3rd 2017, accepted 24th june 2017 abstract: the purpose of this study was to optimize the level of wheat, pumpkin seed (psf) and golden flaxseed flour (gfs) that can be used in order to obtain high quality bread. the independent variables levels used were between 90 and 95 % for wheat flour and between 2.5% and 7.5% for pumpkin seed and golden flaxseed flour. the quality parameters analyzed were the following: loaf volume, porosity, elasticity and bread crumb structure. the mixture experiment design was used for optimization. special quadratic mixture models were obtained for all the dependent variables. the optimum mixture levels were of 92.43% for wheat flour, 5.06% for pumkin seed flour and 2.51% for golden seed flour. the values of these flours in terms of loaf volume of bread, porosity and elasticity were of 422 cm3/100g, 76.15%, and 92.82%, respectively. the textural properties (hardness, cohesiveness, adhesiveness, viscosity, elasticity, gumminess, chewiness) were analyzed for the control sample and the optimum bread sample obtained with psf and gfs addition. for the last one mentioned hardness, elasticity, gumminess and chewiness increase with 25.03%, 7.31%, 23.41%, 25.77% while the cohesiveness value decreases with 1.47%. keywords: loaf volume, porosity, elasticity, crumb structure, textural properties. 1. introduction bread is a well known and widely used food all around the world. wheat is the most common grain used to obtain bread, but it can also be obtained from rye, oats, corn and other grain varieties [1]. the bread making performance obtained from wheat flour is related to its protein quantity and quality. wheat is the only cereal capable of forming gluten, a viscoelastic mass which stands out from all other commercially available vegetable proteins. in most baking formulation, wheat flour is the predominant ingredient. other ingredients are used especially in order to improve the quality of the finished bakery product [2, 3]. apart from gluten, wheat flour also contains many other components such as: non-starch polysaccharides, lipids, e.g. which are important in terms of the quality of the finished bakery products [4]. bread making producers are trying to improve the nutritional quality of bread by using various additions in order to satisfy the consumers’ demand. also different bakery products are made especially for people suffering from different diseases such as celiac disease, obesity, diabetes, e.g. different oilseeds may be used in the bakery products due to their high content in essential amino acids, especially lysine which will improve the nitrogen balance of bread and therefore will increase the coefficient of amino acids assimilation. these seeds can be excellent sources of calcium, iron and also good sources of vitamins from the b complex [5]. from a chemical point of view, oilseeds are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 62 characterized by a high level of fat (>20%) which can have beneficial effects in bread making [6]. in this research, the aim was to investigate and scientifically explain the mechanism by which the use of two oilseeds flours namely pumpkin seed and golden flaxseed added in wheat flour may affect the baking quality results. pumpkin and flaxseed flour are healthy ingredients used in bakery products nowadays in order to provide them with different minerals and vitamins. thus, flaxseeds present a high mineral content of p, mg, k, na, fe, cu, mn and zn [7]. flaxseeds are a rich source of mammalian lignanan α-linolenic acid (ala) and it has been shown that it is protective in the early promotion stage of carcinogenesis [8]. the antioxidant activities of flaxseed lignin secoisolariciresinol diglycoside (sdg) and its mammalian lignan metabolites, enterodiol (ed) and enterolactone (el) were evaluated in both lipid and aqueous in vitro model systems [9]. due to the flaxseed functional components, it presents health benefits in the case of cardiovascular disease, atherosclerosis and neurological disorders [10]. pumpkin seeds have special properties due to their high content in proteins 37.80– 45.40%, oil 25.20–37.00%, dietary fibers 16.84-24.02%, minerals 4.59% and b vitamins [11]. due to it high content in these elements they are being regarded valuable for the food industry [12]. therefore pumpkin seeds are valued from a nutritional standpoint, in part at least, for its high protein content and the useful amount of the essential fatty acid, linoleic acid which it provides [13]. the four dominant fatty acids are palmitic, stearic, oleic and linoleic acids, these ones making up almost 98% of the total amount of fatty acids in pumpkin seeds [14]. to our knowledge, there has not been published any work on the optimum pumpkin and golden flaxseed addition that may be used in wheat flour in order to obtain high quality bread by using the mixture experiment design. the aim of this study was to evaluate the bread physical characteristics (loaf volume, porosity, elasticity) of bread samples baked with different mixtures made from wheat flour, pumpkin seed flour and golden flaxseed flour. both the pumpkin seed and golden flaxseed flour contain a high level of fats, proteins and minerals. however, pumpkin seed presents a higher content in protein than flaxseed which presents a lower amount of fats than pumpkin seed. both types of flours present a high lysine content which exist in a lower amount in wheat flour suggesting its ability to be incorporated in bakery products, especially bread. regarding its fiber content, in flaxseeds it is significantly higher than in pumpkin seeds and therefore, sustainable to be incorporated in white wheat flour which contains a low amount of fibers due to its high extraction rate [15], [16]. 2. materials and methods materials a commercial wheat flour of 650 type (harvest of 2015) was used as a base for wheat-pumpkin-golden flaxseed flour mixtures. wheat flour was analyzed according to the international or romanian standard methods: gluten deformation index, moisture content, wet gluten content, ash content and falling number index (sr 90:2007, icc methods 110/1, 106/2, 104/2 and 107/1 respectively). golden flaxseed and pumpkin seed grains were ground in a domestic blender and the flour obtained was analyzed in terms of its chemical characteristics moisture, protein, ash according to international methods (icc methods 110/1, 105/2, 104/2) and oil content (sr en iso 659-2009) as well. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 63 baking process and bread analysis the bread recipe reported to the 100 g wheat-pumpkin-golden flaxseed composite flour mass consisted of dried yeast (3%), sodium chloride (1.5%), pumpkin seed flour (2.5-7.5%), golden flaxseed flour (2.5-7.5%) and water (56.3%) according to wheat flour hydration capacity. we chose this level for golden and pumpkin seed flour addition based on our previous studies [15], [16], the best quality bread being obtained up to a level of 10% pumpkin seed and golden flaxseed flour addition in wheat flour. the dough was kneaded for 15 minutes, rested for 5 minutes, and then dough pieces were modeled and proofed at 30oc for 60 min and 85% rh and baked for 30 min in an electrical bakery convection oven with steam production, ventilation and humidification (caboto pf8004d, italy). after the bread samples were cooled (approximativaly 1 h after baking) they were evaluated in terms of loaf volume, porosity, elasticity according to the srromanian standard method 91: 2007 and crumb structure using the motic smz–140 stereo microscope with the 20x objective to a resolution of 2048 x 1536 pixels. for the optimum bread sample obtained the textural properties (hardness, cohesiveness, adhesiveness, viscosity, elasticity, gumminess, chewiness) using an electronic texture analyzer mark-10-esm301 were determined. the crumb structure and the textural properties of the bread samples were prepared and processed according to [17], [18]. design of the experiment in order to obtain an optimum bread quality from the technological point of view the mixture experiment design with the statistical state-ease design expert 10.0.0 software package (trial version) was used. this statistical software from statease inc. is currently used to perform the design of experiments (doe). it is very used for comparative tests, optimization, mixture designs, and combined designs, e.g. it provides matrices of many factors on which statistical significance are established with the analysis of variance (anova). anova is very useful because it determines the impact of the independent variables on the dependent ones in a regression analysis. it allows us to identify the factors which have contributed to the data set's variability from a statistical point of view. the option mixture-optimal was used from the stateease design expert software program menu. the experimental plan that was used for this study was mixture design, v. simplex lattice. the method is very effective having in view that the effects of several factors can be studied simultaneously. this allows us to optimize the bread recipe relying on statistical design of experiments depending on the technological characteristics analyzed. this method allowed us to determine the optimum combinations of the three different types of flour used (wheat flour, golden flaxseed and pumpkin seed) in order to obtain bread with the best physical characteristics [19]. for this purpose, we introduced the limit restriction of each component in the mixture design for each three types of flour used. the lower and upper quantity set up was the 2.5% and 7.5 % respectively for golden flaxseed flour and pumpkin seed flour reported to the 100 g wheat-pumpkin-golden flaxseed composite flour. the combinations of the three types of flours (x1, x2, x3) were evaluated by changing their levels (proportions) simultaneously and maintaining the total level (proportions) constant (x1 + x2 + x3 = 1). we analyzed 9 different combinations of the mix flours and a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 64 control sample made only from wheat flour. the experimental design used in terms of pseudo components and real levels is shown in table 1. table 1. experimental design for ternary mixture of wheat flour, golden flaxseed flour and pumpkin flour in pseudo components and in real levels component proportion in the composite flour pseudo components real levels (%) formulation wheat flour pumpkin seed flour golden flaxseed flour wheat flour pumpkin seed flour golden flaxseed flour m 1.000 0.000 0.000 0.950 0.025 0.025 p1 0.000 0.500 0.500 0.900 0.050 0.050 p2 0.000 0.000 1.000 0.900 0.025 0.075 p3 0.165 0.667 0.167 0.908 0.058 0.033 p4 0.667 0.167 0.167 0.933 0.033 0.033 p5 0.500 0.500 0.000 0.925 0.050 0.025 p6 0.000 1.000 0.000 0.900 0.075 0.025 p7 0.167 0.167 0.667 0.908 0.033 0.058 p8 0.333 0.333 0.333 0.917 0.042 0.042 p9 0.500 0.500 0.500 0.925 0.025 0.050 this study contained 3 factors (independent variables) and 3 responses (dependent variables). characteristics such as: loaf volume, porosity and elasticity were selected as the response variables. the response is a function of component proportions. thus, for the characteristics analyzed, the appropriate model obtained was a special quadratic mixture model in the form of equation (1) which may be converted in the equation (2) [20]: ji ji iji i i xxxxf   )( (1) by using the identity 1 ix model (1) can be converted to a canonical homogeneous quadratic model as follows: ji ji iji i ii xxxxf    2)( (2) in the simple mixture experiment, the response (in our case loaf volume, porosity, elasticity) depends on the relative proportions of the components (the proportion/level for the three independent variables used in this study: x1 wheat flour, x2pumpkin seed flour flour and x3 golden flaxseed flour). the amounts of components, measured in weights (grams) add up to a common total [21]. the β1, β2, β3 are the linear terms of the model and the β12, β13, β23 are the interaction terms of the variables x1and x2, x1and x3, and x1, x2 and x3. 3. results and discussion physico-chemical characteristics for wheat, pumpkin seed and golden flaxseed flours are shown in table 2. according to the data shown for wheat flour it is of a very good quality for bread making but with a low alpha-amylase activity due to its high falling number value [2]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 65 table 2 the physico-chemical characteristics of wheat, pumpkin seed and golden flaxseed flours characteristics wheat flour (wf) golden flaxseed flour (gfs) pumpkin seed flour (psf) moisture (%) 14.5 5.6 5.4 ash (%) 0.65 3.41 5.48 protein (%) 12.6 20.85 33.58 fat (%) 1.5 41.12 29.26 gluten deformation (mm) 8 falling number (s) 380 the best-fit models for loaf volume, porosity and elasticity were special quadratic mixture ones. table 3 shows the equations obtained for the response variables with the indication of the significantly regression coefficients expressed in terms of coded values, r2 and adj.-r2 obtained by the analysis of variance (anova). table 3 effects of formulation factors, expressed as their corresponding coefficients in the predictive models for bread quality characteristics: loaf volume, porosity, elasticity parameters factorsa loaf volume (cm3/100g) porosity (%) elasticity (%) wf 331.47* 77.94 87.90 psf 418.47* 74.40 90.90 gfs 304.47* 75.45 82.90 wf x psf 183.86 0.10 13.59 wf x gfs -56.14 -20.63* 9.59 psf x gfs -231.14 -0.43 11.59 wf2 x psf x gfs 467.82* -97.67 -171.53 wf x psf 2 x gfs -7236.18* -185.86 -567.53 wf x psf x gfs2 6344.82 250.42 332.47 r2 0.99 0.99 0.97 adj.-r2 0.97 0.93 0.74 *p < 0.01. awf, wheat flour (g/100g); psf, pumpkin seed flour (g/100g); gfs, golden seed flour (g/100g); r2, adj.-r2 are measures of fit of the model. anova results for bread loaf volume highlight that the f-test value of 38.74 is not a significant one. for the loaf volume model all the independent variables used are significant ones (p  0.01). the positive value of the terms from the special quadratic mixture model of wheat flour, pumpkin seed flour, golden flaxseed flour and the interaction between wheat flour and pumpkin seed flour show that the loaf volume values increased due to its addition. the contour surface of bread loaf volume as a simultaneous function of wf, psf and gfs dose adition in bread recipe is shown in fig. 1a, emphasizing that the most positive effect on loaf volume value is provided by wheat flour and pumpkin seed flour addition. similar results were obtained by meral and dogan (2013) on bread with golden flaxseed flour addition where the loaf volume increases up to 15% gfs addition [18]. also ptitchkina et al. (1998) concluded that the addition of pumpkin pulp flour up to 10% addition increases the loaf volume of the bread samples [23]. for porosity the coefficient of determination (r2) of the model was 0.99, implying that only 1% of the total variation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 66 was not explained by the model. the adjusted determination coefficient (adj r2) was of 0.93, indicating high significance of the obtained model. table 2 shows clearly that the cross product coefficients (wf x gfs) were significant, with a p value (p < 0.01). the other term coefficient for the response variable porosity was non significant (p > 0.05). fig. 1b shows the interaction between wheat flour, pumpkin seed flour and golden flaxseed flour. for elasticity the anova results highlight that the quadratic terms of the model were not statistically significant. however, the quadratic mixture model is statistically significant at a determination coefficient of r2 = 0.97 showing that the model could be used to explain more than 95 % of the variability in the response. the positive effect of these independent variables on the response showed that the bread elasticity increases with the increased level of wheat flour, pumpkin seed flour and golden flaxseed flour addition. the contour surface of bread elasticity and contour graphs as simultaneous function of wf, psf and gfs addition is shown in fig. 1c. a) b) c) fig. 1. the contour surface plots of: a) bread loaf volume affected by the levels of wheat flour, pumpkin seed flour and golden flaxseed flour; b) bread porosity affected by the levels of wheat flour, pumkin seed flour and golden flaxseed flour; c). bread elasticity affected by the levels of wheat flour, pumpkin seed flour and golden flaxseed flour. the numerical optimization technique of the statistical state-ease design expert 10.0.0 software package (trial version) was used for simultaneous optimization of the multiple responses analyzed: loaf volume, porosity and elasticity. for each factor and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 67 response, the desired goals were chosen namely for the response variables the maximum goal was used. the overlay plot (fig. 2) shows the interactive effect of the independent variables (wheat flour, pumpkin seed flour, golden flaxseed flour) on loaf volume, porosity and elasticity values of breads. the mixture experiment design suggested the optimal solution for the composition of bread ingredients with maximum desirability (d = 0.983) for 92.43% wheat flour, 5.06% pumpkin seed flour and 2.5% golden seed flour. due to the fact that d value is closed to 1, it means that our solution is a good one to predict the best bread quality for the independent variables chosen. the flour levels in terms of loaf volume of bread, porosity and elasticity were predicted to be 422 cm3/100g, 76.15%, and 92.82%, respectively. fig. 2. the over plot of the mixture experiment design used for bread quality evaluation fig. 3 shows the bread crumb structure for the samples obtained with different combinations of mix flours as indicated in the table 1. as we can see, together with the increase level of psf and gfs addition, more and larger cells can be noticed maybe due to the decrease of the gluten content, a low content not being capable of retaining the gases formed during fermentation. the textural characteristics of bread samples (the control sample and the optimum sample suggested by mixture experiment design) are shown in table 4. it can be noticed that for the optimum sample hardness, elasticity, gumminess and chewiness increased whereas the cohesiveness value decreased. these results are in agreement with those obtained by conforti and davis (2006) who found that by flaxseed addition the hardness value increases [20]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 68 fig. 3. crumb structure of bread samples with wheat flour, golden flaxseed flour, pumpkin seed flour at various levels (m-p9) according to the table 1 table 4 textural parameters of bread samples textural parameters control sample optimum sample hardness (n) 16.22  0.22 20.28 0.20 cohesiveness 0.69  0.11 0.68 0.09 elasticity 0.82  0.15 0.88 0.11 gumminess (n) 11.19  0.17 13.81 0.14 chewiness (n) 9.62  0.09 12.1 0.10 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 69 4. conclusions the mixture experiment design was successfully applied to optimize the quality of breads obtained from different mixtures of wheat-pumpkin-golden flaxseed flours. with the increase level of psf and gfs the bread quality characteristics are improved namely loaf volume, porosity and elasticity. the optimal solution for the independent variables was obtained at a high desirability (d = 0.983) indicating the adequacy of the method used. the optimum values obtained for the wf, psf and gfs levels were 92.43%, 5.06% and 2.5% for which the bread loaf volume, porosity and elasticity were of 422 cm3/100g, 76.15%, and 92.82%. 5. acknowledgments this work was supported by a grant of the romanian national authority for scientific research and innovation, cncs – uefiscdi, project number pn-ii-rute-2014-4-0214. 6. references [1]. cozzolino c. a., rovera c., anchisia c., savoldellia s., campanella g., muroni e., meloni m., farrisa s., preservation of bread-made museum collections by coating technology, journal of cultural heritage, (2017). [2]. codină g.g., rheological properties of wheat flour dough, agir publishing house, bucharest (2010). [3]. dewettinck k., van bockstaele f., ku¨ne b., van de walle d., courtens t.m., gellynck x., nutritional value of bread: influence of processing, food interaction and consumer perception, journal of cereal science, 48 (2): 243-257, (2008). [4]. goesaert h., brijs k., veraverbeke w.s., courtin c.m., gebruers k., delcour j.a., wheat flour constituents: how they impact bread quality, and how to impact their functionality, trends in food science & technology, 16:12-30, (2005). [5]. sarwar m.f., sarwar m.h., sarwar m., qadri n.a., moghal s., the role of oilseeds nutrition in human health: a critical review, journal of cereals and oilseeds, 4 (8): 97100, (2013). [6]. moure a., sineiro j., dominguez h., parajó j.c., functionality of oilseed protein products: a review, food research international, 39, 945-963, (2006). [7]. goyal a, sharma v, upadhyay n, gill s, sihag m., flax and flaxseed oil: an ancient medicine & modern functional food, journal of food science and technology, 51:16331653, (2014). [8]. thompson l.u. rickard s. e., orcheson l. j. seidl m.m., flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis, carcinogenesis, 17(6): 1373-1376, (1996). [9]. kitts d.d. , yuan y.v., wijewickreme a.n.,, thompson l.u., antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone, molecular and cellular biochemistry, 202: 91–100 (1999) . [10]. goyal a., sharma v., upadhyay n., gill s., sihag m., flax and flaxseed oil: an ancient medicine & modern functional food, journal of food science and technology, 51: 16331653 (2014). [11]. el-soukkary f.a.h., 2001. evaluation of pumpkin seed products for bread fortification, plant foods for human nutrition, 56: 365–384, (2001). [12]. patel s., pumpkin (cucurbita sp.) seeds as nutraceutic: a review on status quo and scopes, mediterranean journal of nutrition and metabolism, 183-189, 6 (3): (2013). [13]. glew r.h., glew r.s., l.-t. chuang, huang y.-s., millson m., constans d., vanderjagt d.j., amino acid, mineral and fatty acid content of pumpkin seeds (cucurbita spp) and cyperusesculentus nuts in the republic of niger, plant foods for human nutrition, 61 (2): 49-54, (2006). [14]. murkovic m., hillebrand a., winkler j., leitner e., pfannhauser w., variability of fatty acid content in pumpkin seeds, zeitschrift für lebensmittel-untersuchung und forschung, 203 (3): 216-219, (1995). [15]. codină g.g., mironeasa s., gutt g., todosi-sănduleac e., influence of the golden flaxseed addition on bread quality of wheat flour with a very good quality for bread making, international conference modern technologies in the food industry, 20th october 22th october, chișinău, conference proceeding, p. 151-157, isbn 978-9975-87-138-9 (2016). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 2 – 2017 georgiana gabriela codină, denisa atudorei, ana cimpoi, silvia mironeasa, elena todosisănduleac, quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, volume xiv, issue 2 – 2017, pag. 61 – 70 70 [16]. mironeasa s., codină g.g., mironeasa c., effects of the pumpkin seed addition on bread quality of wheat flour with a very good quality for bread making, international conference modern technologies in the food industry, 20th october 22th october, chișinău, conference proceeding, p. 229-237, isbn 9789975-87-138-, (2016). [17]. codină g.g., marineac a.r., todosisănduleac e., the influence of lupin flour addition on bread quality, food and environment safety, 15 (3): 216-226, (2016). [18]. codină g.g., franciuc s.g., todosisănduleac e., studies on the influence of quinoa flour addition on bread quality, food and environment safety, 15 (2): 165-174, (2016). [19]. montgomery, d. c., design and analysis of experiments, 6th edition, john wiley & sons, new jersey, (2005). [20]. hiron j., macak t., mixture design for food packaging in a modified atmosphere, agricultural economics, 61 (9):393-399, (2015). [21]. antony j., improving the manufacturing process quality using design of experiments: a case study, international journal of operations and production management, 21: 812–822 (2001). [22]. meral r., dogan i.s., quality and antioxidant activity of bread fortified with flaxseed, italian journal of food science, 25: 51-56, (2013). [23]. ptitchkina n.m., novokreschonova l.v., piskunova g.v., morris e.r., large enhancements in loaf volume and organoleptic acceptability of wheat bread by small additions of pumpkin powder: possible role of acetylated pectin in stabilizing gascell structure, food hydrocolloids, 12, 333-337, (1998). [24]. conforti f.d., davis s.f., the effect of soya flour and flaxseed as a partial replacement for bread flour in yeast bread, international journal of food science technology, 41, 95–101, (2006). 66 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 12018, pag. 66 73 effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau” *mirela ardelean 1 , aurel ardelean 1 , ioan don 2,3 , andrei lobiuc 4 marian burducea 5 1„vasile goldiş” western university from arad, plant biotechnology, institute of life science, romania e-mail: mirela.ardelean1@yahoo.com 2 department of engineering and computer science, faculty of economics, computer science and engineering, “vasile goldiş” western university of arad, liviu rebreanu 86, 310426, arad, romania 3 “pavel covaci” university botanical garden of macea, 317210, macea village, arad county, romania 4”stefan cel mare” university, faculty of food engineering, universitatii str. 13, suceava, romania 5„alexandru ioan cuza” university of iasi, romania, faculty of biology, carol i bd., iasi, romania received 9th january 2018, 23 th march 2018 abstract: the aim of the experiment was to compare the effect of light emitting diodes led and fluorescent lamps (fl)d as sources of light on ,,in vitro” growth and development of sweet basil (ocimum basilicum l. cultivar ,,aromat de buzau”). sweet basil is an important medicinal and aromatic plant used in aromatherapy, cosmetics, perfume and food products in fresh or dried form. a major difficulty in the use of lamiaceae species for pharmaceutical purposes is the individual variability, due to genetic and biochemical heterogeneity. in vitro, micropropagation is an effective mean for rapid multiplication of species in which it is necessary to obtain a high progeny uniformity. therefore, the interest in using these techniques for rapid and large-scale propagation of medicinal and aromatic plants has been significantly increasing. basil seedlings were grown under four monochromatic lights irradiated with blue, green, yellow, red leds with peak wavelengths of 470, 500, 525 and 660 nm.basil plants produced greater fresh herbage mass as well as shoot height under fluorescent lamps used as control after 60 days of light treatment. however, the total leaf area of lateral shoots was the largest one under red light. the total phenolic content of basil plants was significantly higher under blue led illumination as compared to the rest of the treatments. also, leds increased the amount of flavonoids compared to fl light. our study demonstrates that leds did not affect growth characteristics and increased the total phenolic content as compared to conventional fluorescent. keywords: ocimum basilicum l. cultivar ,,aromat de buzau”, light emitting diodes – led, phenols, flavonoids, antioxidant capacity. 1. introduction plant development can be influenced by modulating light quantity and quality, as well as the photoperiod [1], the response of plants being recorde at chemical, morphological, and physiological level. plant growth equipment (phytotrons) usually rely on conventional light emissions such as fluorescent tubes (especially cool-white) often used in combination with additional sodium and/or http://www.fia.usv.ro/fiajournal mailto:mirela.ardelean1@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 67 incandescent sources, in order to obtain a complex light spectrum, closer to outdoors conditions [2]. nevertheless, such light equipments present disadvantages, such as high energy requirement, short lifetime, and heat production. more recently, the use of light emitting diodes (leds) for plant cultivation in controlled conditions has appeared as an efficient, low-cost alternative technology [3]. leds are well suited for plant growth equipment, having reduced weight andvolume and long lifespan (approximately 100,000 hours). moreover, led lighting achieves major energy consumption reduction due to emission of very narrow wavelengths. in two varieties of lettuce [4], the results showed that blue leds are more efficient for bioactive substances production. for both varieties, increases of vitamin c and carbohydrates amounts were obtained using led with 40% blue light and 60% red light. furthermore, combining red and blue leds proved effective for photosynthesis considering the higher chlorophyll content in plants grown with leds light compared with plants grown with ne source. research indicate that both light quantity and quality influence plant morphology. regarding radishes, soybean and wheat grown with leds with different percentages of blue light, the results showed that blue light did not influence total dry weight, blue light from warm white leds led to augmented stem elongation and leaf growth, while blue light from cool white leds led to more compact plants. in the present, a viable option to reducing costs for commercial micropropagation is represented by replacements for cultivation mediums needed for commencing or subcultivation of vitroplantlets. sweet basil (ocimum basilicum l.) is a india native specie [5] and is also encountered in tropical and temperate regions of asia. the genus ocimum is distributed in asia, africa and central america [6] and is grown in several european countries such as italy, france and spain. in turkey, basil is grown in eastern anatolian area, used as a medicinal and aromatic plant. sweet basil presents large intrapsecific variations on a morphological level [7, 8] and also regarding the composition of essential oil [9] as well as other bioactive substances [10]. basil is a highly appreciated medicinal and aromatic specie mainly as a result of the high content in essential oils obtained from leaves and flowers [11], containing phenolics [12] and of the presence of other non-volatile compounds such as flavonoids and anthocyanins [13]. in vitro propagation of medicinal species using tissue culture techniques provides an effective way of optimized production of secondary metabolites from in vitro induced callus or whole plants. although an important culinary and medicinal plant, few studies have been conducted on basil growth using leds as a light source. in the same time, there is no extensive information regarding the relationship between light captured by basil and physiological mechanisms underlying the secondary metabolism production under the influence of different light quality. secondary metabolites are produced to allow plants to overcome stressful conditions. in tissues such as stems and leaves, secondary metabolite synthesis can be influenced by environmental, physiological, biochemical and genetic factors [14, 15], light having a major influence [16]. one of the main groups of secondary metabolites is represented by phenolics. this group is one of the most ubiquitous class of secondary compounds food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 68 in plants and indicates high metabolic plasticity, enabling plants to adapt to biotic and abiotic environmental influences [14]. their concentration varies with the season and with the stage of growth [14]. phenolics are substances exhibiting free radical scavenging capacity, as well as inhibitory activity against microorganisms and insects [17]. physiological changes are triggered by exposure to varying light quality [18]. red light is the wavelength affecting biomass production and organ elongation through the phytochrome photoreceptor [19]. blue light also influences photomorphogenic responses (such as leaf flattening and compact appearance) through phototropins and cryptochromes acting independently and/or synergically with phytochromes [20]. the present paper hypothesizes that precultivating basil seedlings under light with varying wavelengths would differently influence morphological and chemical properties. in addition, different light emitting diodes spectra would induce varied morphological and phytochemical reactions among different basil cultivars. the objective of this research was (1) to investigate the effect of led sources on morpho-chemical characteristics of ,,aromat de buzau” basil cultivar in vitro and (2) to determine the optimum wavelength for basil growth. 2. materials and methods 2.1. plant material, growth conditions and light treatments the culture substrate used in the in vitro cultures experiments consisted of basal medium (mb) murashige skoog (1962) (ms) agar medium, which consisted of macronutrients, fe edta and trace elements, mineral mix according to the original recipe, but with increased addition of vitamins: pyridoxine hcl, nicotinic acid and thiamine hcl (1 ml / l each of the original recipe to where indicated 0,5 mg / l), to which was added m-inositol 100 mg / l, sucrose 30 g / l and agar agar 10 g / l; this basic medium (mb) were added as growth regulators 0,5 mg / l iba (indolyl butyric acid) and 0,5 mg / l ba (benzyladenine). before autoclaving of the culture medium, the ph value was adjusted to 5,5 with hcl or naoh, depending on the basicity or acidity of the final medium. for autoclaving, 15 ml of the medium were placed into the clear glass culture containers that were temperature resistant, 8 cm height and 4 cm diameter. after portioning the culture medium, the culture containers were filled with aluminium foil. sterilization of the containers and culture media was performed by autoclaving at 121°c for 21 minutes. the plant material used for the initiation of vitro cultures was the meristematic apexesof basil plants ,,aromat de buzau” cultivar with a length of about 1 cm and 23 leaf primordia, harvested from seedlings regenerated from zygotic embryos that have sprouted from 30 days old seeds that were germinated on a septic substrate consisting of peat mixed with perlite. after cooling of the culture medium, was performed the inoculation of explants on culture medium and then the inoculated containers were transferred into growth chamber that were placed on racks exposed to a temperature ranged from 23°c ± 2°c, in the light regime, 20°c ± 2°c, during darkness and a photoperiod of 16 h light / 24h. fluorescent tubes emitting coloured light (osram company) were used as a control, length 590 mm, ø 26 mm, 120 lux light intensity and leds emitting colored light with different wavelengths: blue (465 nm), yellow (590 nm) and red (650 nm) from osram company. 2.2. quantification of phenols, flavonoids, antioxidant capacity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 69 extracts were prepared by macerating 5 g of ground fresh plant in 95 ml of distilled water or 30% w/v ethanol for 24 hours. total phenolic content was assessed using the folin ciocalteu reagent method, by spectrophotometric readings at 760 nm of the colour of incubated extracts [26]. total flavonoid content was determined according to the method described in makri in 2008, by evaluating the absorbance at 510 nm of extracts reacted with 5% nano2 and 10% alcl3. the free radical scavenging capacity was assessed through the dpph method where the relatively stable free radical can be reduced by electron-rich radical scavengers from medicinal plant extracts[27], measuring the decolouration of dpph solution reacted with extracts at 515 nm for 3 hours. 2.3. statistical analysis the statistical analyses conducted were represented by analyses of variance among treatments and the tukey test at p<0.05, the results being expressed as means and standard errors. 3. results and discussions 3.1. morphological and developmental measurements number of leaves, shoot height, root length, measurements were conducted during the two months experimental period (60 days). specifically, shoot height was measured on a weekly basis with the first measure taking place 7 days after inoculation. after 60 days experimental period 10 randomly selected seedlings were sampled. morphological characteristics such as leaf number, shoot height, root length were measured. at day 60, leaf number was not affected by the different light treatments, with fl (control) and leds forming around 4 and 5 leaves respectively (table 1). shoot height was variably affected by the different light treatments. seedlings grown under red light of leds exhibited significantly higher shoots compared to control and others leds treatments. table 1. morphological indices of ocimumbasilicum l. grown at different led colors after 60 days. indices control blue light red light yellow light green light stem height (cm) 5.92b±0.05 4.08a±0.08 6.06b±0.07 5.08b±0.09 5.82b±0.06 root length (cm) 6.94b±0.06 5.1a±0.02 7.2b±0.08 5.4a±0.04 7.12b±0.06 number of leaves 4.6b±0.21 5.4b±0.21 4.2b±0.17 3.4a±0.21 3.6b±0.21 (average of 5 measurements ± standard error, different letters between rows represent significant statistical differences). regarding the root length, significant differences were found between red light that exhibited the highest average value and blue light treatment (figure 1 and figure 3). after 60 days of vitroculture, if comparing vitroseedlings in terms of growth, we can see that the red light and yellow stimulates increase in length of vitroseedlings and blue light produce small and stocky and even slowing growth of plants (figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 70 fig. 1. morphological aspects of in vitro seedlings basil after 30 days of cultureunder red lighttreatment and blue light treatment. fig. 2. morphological aspects of in vitro seedlings basil after 60 days of cultureunder white fluorescent tubes (control) and light emitting diodes treatments(red, yellow, blue). fig. 3. morphological responses of in vitro seedlings basil after 60 days of cultureunder blue lighttreatment (a) and red light treatment(b). 3.2. phytochemical characteristics the research on the basil in vitro plants, cultivated on murashige-skoog medium culture exposed 30 days in light fluorescent tubes and leds revealed that the lighting regimestimulated antioxidant compounds production. as can be seen in figure 6, blue light treatment determines the highest amount of polyphenols, followed by the quantity produced in vitro plants grown under yellow light. regarding assimilatory pigment, vitroseedlings grown in colored light food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 71 produced by leds contained the highest amount of assimilating pigments, especially those illuminated with blue light, followed by those illuminated with yellow and red light. high yield using red light treatment is reported [22], and described in other species such as lycopersicum esculentum [23]. the amount of carotenoids is related to green and blue light (0.4 to 0.48 mg/g), these pigments absorbing mainly at these wavelengths [24]. in this study, the antioxidative proprieties of basil were measured spectrophotometrically by dpph assay. this method is preferred due to its simplicity, convenience and time-saving properties [25]. the extract, functioning as an antioxidant, reacts with dpph (1diphenyl-2-picrylhydrazyl), resulting in the formation of 1-diphenyl-2-picrylhydrazine. almost all variants of vitroseedlings (two months old leaf and stem) showed considerable antioxidant activity. nevertheless, among all the extracts assessed, the extract with the highest antioxidant activity was that of the leaf and stems illuminated with red light, (22,36% higher) than that of vitroplants illuminated with others colours of light (figure 6), especialy with blue light (14,86%). antioxidant capacity in plants is known to be variable, according to specie but also to the origin of the samples within the same specie [28]. however, considering the potential uses of antioxidant extracts in food applications [29,30], production of raw material with enhanced free radical scavenging activity is highly desirable. fig. 4. total phenolic content of basil extracts “aromat de buzau” cultivated under different color led lights. the mean difference is significant at the 0,05 level compared to control. fig. 5. total flavonoid content of basil extracts “aromat de buzau” cultivated under different color led lights. the mean difference is significant at the 0,05 level compared to control. fig. 6. free radical scavenging capacity of basil extracts “aromat de buzau” cultivated under different color led lights. the mean difference is significant at the 0,05 level compared to control. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 72 4. conclusions the results from the present study clearly suggested that basilplants may serve as an excellent source of antioxidants. these findings are also beneficial in providing a useful benchmark in determining the optimum colour of light from this species at its best, most promising antioxidative effects. the results of this study would be used to give guidance on light colour sources design for basil cultivation in a controlled environment. in addition, it may serve as a basis for even more extensive researches to be done on this species with the focus of interest directed towards its phytomedicinal values, hence would be incorporated into healthpromoting supplementary foods and pharmaceutical preparations. 5. acknowledgements this research work was carried out with the support of „vasile goldiş” western university from arad, plant biotechnology, institute of life science, romania and also was financed from project cernesim (the environmental science studies center for the north-east developing region) project nr. 257/28.09.2010, smis/cnmr code 13984/901. 6. references [1]. astolfi, s., zuchi, s., adequate s supply protects barley plants from adverse effects of salinity stress by increasing thiol contents. acta physiol plant (in press) doi: 10 1007/ s11738-0121060-5,(2012). [2]. bubenheim, dl., bugbee, b., salisbury, fb., radiation in controlled environments: influence of lamp type and filter material. j amer soc hort sci 113:468-474, (1988). [3]. yeh, n., chung, jp., high-brightness ledsenergy efficient lighting sources and their potential in indoor plant cultivation. renew sust energ rev 13:2175-2180, (2009). [4]. panter, e. , pele, m., draghici, me., postamentel m.,influence of illumination with leds on some biochemical compounds rev.chim.(bucharest)67, no. 6 (2016). [5]. viera, r.f., and j.e. simon, chemical characterization of basil (ocimum spp.) found in the markets and used in the traditional medicine in brazil. econ. bot. 54(2): 207-216, (2000). [6]. darrah, h.h., the cultivated basil. buckeye printing co., independence, mo., (1998). [7]. simon, j., e. morales, m.r. phippen, w.b. vieira, r.f.z. hao basil: a source of aroma compounds and a popular culinary and ornamental herb. in: janick, j. (ed), perspecctives on new crops and new uses. ashs press, alexandria v.a. (1999). [8]. labra, m., m. miele, b. ledda, f. grassi, m. mazzei, and m. sala. morphological characterization, essential oil composition and dna genotyping of ocimum basilicum l. cultivars. plant sci. 167(4): 725-731, (2004). [9]. sifola, m.i., and g. barbieri, growth, yield, and essential oil contentof three cultivars of basil grown under different levels of nitrogen in the field. sci. hort. 108: 408-413 (2006). [10]. nguyen, p.m., e.m. kwee, and e.d. niemeyer,potassium rate alters the antioxidant capacity and phenolic concentration of basil (ocimum basilicum l.) leaves. food chem. 123(4): 1235– 1241, (2010). [11]. nurzyńska-wierdak, r., b. borowski, k. dzida, g. zawiślak, and r. kowalski,essential oil composition of sweet basil cultivars as affected by nitrogen and potassium fertilization. turk. j. agric. for. 37(4): 427-436, (2013). [12]. simon, j.e., j. quinn, and r.g. murray, basil: a source of essential oils. in: janick, j., and j.e. simon (eds.), advances in new crops. timber press, portland, or. 484-489 (1990). [13]. phippen, w.b., and j.e. simon,anthocyanins in basil. j. agr. food chem. 46(5): 1734–1738, (1998). [14]. wink m., functions and biotechnology of plant secondary metabolites.annual plant reviews, 3, wiley-blackwell, oxford, (2010). [15]. zhao, j., davis, l.c., verpoorte r., elicitor signal transduction leading to production of plant secondary metabolites, biotechnol. adv., 23: 283-333, (2005). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 mirela ardelean, aurel ardelean, ioan don, andrei lobiuc, marian burducea, effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73 73 [16]. kopsell, d.a., sams c.e., increases in shoot tissue pigments, glucosinolates, and mineral elements in sprouting broccoli after exposure to short-duration blue light from light emitting diodes, j. am. soc. hortic. sci., 138: 31-37, (2013). [17]. lattanzio v., cardinali a., role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects. f. imperato (ed.), phytochemistry: advances in research, research signpost, trivandrum, india,37:23-67,(2006). [18].samuolienė, g., brazaitytė a., sirtautas, r., viršilė, a., sakalauskienė s., led illumination affects bioactive compounds in romaine baby leaf lettuce. j. sci. food agric., 93: 3286-3291, (2013). [19]. sager, j.c. mcfarlane j.c. radiation r.w. langhans, t.w. tibbits (eds.), plant growth chamber handbook, north central region research publication, iowa state university press, 1-29, (1997). [20]. kozuka t., suetsugu, n., wada, m., nagatani a., antagonistic regulation of leaf flattening by phytochrome and phototropin in arabidopsis thaliana, plant cell physiol., 54: 6979, (2013). [21]. murashige, t., skoog, f., a revised medium for rapid growth and bioassays with tabaco tissue culture. physiol. plant, 15: 473-497, (1962). [22]. darko, e., heydarizadeh, p., schoefs b., sabzalian m.r., photosynthesis under artificial light: the shift in primary and secondary metabolism. phil. trans. r.sc. b. 369: 2013243, (1962). [23]. suyanto, h., rupiasih n. n., dewi, h., influence of light wavelengths on growth of tomato. jurnal bumi lestari (journal of environment), volume 12 no. 2, (2012). [24]. govindjee, r., rabinowitch, e., thomas m.b., inhibition of fotosynthesis in certain algae by extreme red light.biophys j.1, 917, (1960). [25]. molyneux, p., the use of free radical diphenylpicryl-hydrayl (dpph) for estimating antioxidant activity. songklanarin j. sci. technol., 26:211-219, (2004). [26]. makri, o., kintzios, s.,. ocimum sp. (basil) botany, cultivation, pharmaceuticalproperties, and biotechnology. j. herbs spices med. plants 13, 123–150, (1962). [27]. herald t.j., gadgil p., tilley m., high-throughput micro plate assays for screening flavonoid content and dpph-scavening activity in sorghum brand and flour, journal of the science of food and agriculture, 92 (11), 2326-2331, (2012). [28]. leahu a., damian c., oroian m., miclescu v., ropciuc s., variation in content of antioxidant and free radical scavenging activity of basil (ocimum basilicum), dill (anethum graveolens) and parsley (petroselinum sativum), food and environment safety journal, 12(4), 347353, (2013). [29] cosmovici a., amariei s., study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety journal, 16(3), 153-159 (2017). [30] amariei (gutt) s., sanduleac (todosi-sanduleac) e., ciornei (ștefăroi) s., comparative study of oxidative stability for different types of vegetable oils, food and environment safety journal, 12(2), 156-160 (2013). microsoft word 3 journal nr 4 2010 din 8 noiembrie_1-11.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 5 electrochemical modification of graphite surface yarema tevtul1, t. filipchuk1, sonia gutt2 and silviu stroe2 1yuriy fedkovych national university of chernivtsi, kotsiubynsky st., 2, chernivtsi, 58012 ukraine 2stefan cel mare university of suceava, university st., 13, suceava, 720 229 romania abstract: thermodynamic and electrochemical parameters of intercalation and deintercalation of hydrosulfate ions into the graphite substrate have been investigated using electrodes after preliminary intercalation and without this stage and involving methods of the cyclic voltammetry, electronic microscopy and general principles of the irreversible thermodynamics. potentials of the intercalation and deintercalation starting points, potentials of the anodic and cathodic maximums have been determined and changes in the free gibbs energy for the intercalation and deintercalation processes, changes in the enthalpy and enthropy of these processes, diffusion and migration fluxes of the hydrosulfate ions were calculated using the experimental data. criteria of schtakelberg, tomesh and shevchik and measurements of an amount of electricity required for the processes were used to evaluate the reversibility ratio of intercalation and deintercalation. it was found that raise in the intercalation-deintercalation cycles number results in increase in the limit anodic current value. this increase may be caused by partial decomposition of the graphite substrate structure and faster intercalation of the hydrosulfate ions during next cycles. evaluation of the diffusion and migration fluxes of hydrosulfate ions has been made using an electronic microscopy. thermodynamic moving forces of the fluxes have been analyzed. the higher is the concentration of sulfuric acid, the higher is the surface concentration of sulfur. a value of the flux of the electrochemical intercalation of hydrosulfate ions from a 1 m solution of sulfuric acid is about 42 times higher comparing to the physical intercalation flux value. a contribution of the latter flux into the total intercalation flux is only 3.8 % from the electrochemical intercalation flux contribution. keywords: intercalation; sulfuric acid; electrochemical; parameters; diffusion; ions migration introduction graphite is a material with numerous applicable physico-chemical properties, which can be used in various production branches. atoms and molecules of various substances can be embedded into graphite and change its properties. graphite can interact with many acids: nitric, hydrofluoric, phosphoric, trifluoracetic and chloric if a strong oxidizing agent is present. graphite hydrosulphate was synthesized by p. schaufheitel and became the first known chemical compound of graphite [1]. now this compound is used in technologies of the graphite sealers production [2]. some graphite modification products can be used as a source or raw material in various technological processes. for example, this material is used as a source for production of thermally expanded graphite. graphite food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 6 can be modified with chemical, physical and electrochemical methods. intercalated graphite usually reveals increased electroconductivity. for example, conductivity of the intercalated c24cl is higher than conductivity of gold. intercalated carbon nanomaterials can be used as electrodes in the processes of electrochemical production of hydrogen. intercalated graphite products are widely used in various applications such as lithium-ion batteries (as an electrode material), synthetic conductors, catalysts for some petrochemical processes, lubricants (especially for use in chemically aggressive media), membranes, fire-protection coverings, etc [4]. thermotreatment of the intercalated graphite products results in destruction of the embedded sulphuric acid, which causes significant (300-500 times) expansion of the crystallites and formation of flexible and tortuous particles of the thermoexpanded graphite [1]. all useful characteristics of the regular graphite are retained in this product, which also has higher surface area can be treated mechanically (pressed, rolled) without any additional adhesion agent [5-7]. plain (nonreinforced) thermo-expanded graphite materials can endure heating up to 500-550 0c in the air [8, 9], up to 650 0c in the steam and 3000 0c in the inert medium and stay mechanically durable at freezing to 240 0c [10]. this material is chemically inert and retains high electroand thermoconductivity [8]. the thermo-expanded graphite can be used as adsorbent for collection of the spilled oil products [11, 12] and as source material for manufacturing of membranes and filters [13]. thermo-expanded carbon can adsorb inorganic compounds same as organics. for example, it can be used for adsorption of gold and copper ions [14]. an american company union carbide has patented a special foil of the thermoexpanded graphite, which can be used as a sealer. graphite sealers and stuffing blocks are very durable, they wear-resisting and inert in many chemically aggressive media [4]. some products of the graphite intercalation can be used as a fireproof expandable dye-coating for the electric cables. in the case of fire or excessive heating it expands and forms a strong shell preventing further burning. this coating protects the substrate against heating up to 500 0c and can be applied to some metal constructions [1]. an electrochemical method of intercalation and synthesis of the embedded products (ep) is more favourable than pure chemical modification. the first method is more ecologically safe; it requires low concentrated acids and can be realized in a well-controlled regime, which ensures production of ep with predefined composition and purity. the electrochemical intercalation primary requires such acids: h2so4, hno3, hclo4, h2seo4, cf3cooh, hcooh. most experimental results of the carbon materials oxidizing relate to the compact and wellstructured materials. some results related to the kinetics and mechanism of anodic processes occurring on the dispersed graphite electrodes in 15-60 % solutions of hno3 are also available [15]. it was found that thermo-expandable graphite compounds can be synthesized only within some region of the anode potentials. therefore, investigation of synthesis and possible applications of the intercalated graphite products seems very topical and interesting however, information related to specific details of intercalation and deintercalation of hydrosulphate ions is insufficient. our work was aimed onto investigation of the above mentioned processes and analysis of properties of the hydrosulphateembedded carbon materials. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 7 experimental the hydrosulphate ions intercalation into the graphite substrate has been investigated for the aqueous solution of sulphuric acid in the range 1-15 m/l. the results of the intercalation were analyzed using a cyclic voltammetry (cva) and electron microscopy. the following graphite samples were used as source materials for electrodes: 1)crucible graphite (referred hereafter as ge-1 electrode); 2)bsp-20as/high brand graphite made by the japanese company “chuetsu graphite works co. ltd.” (ge-2 electrode). a suspension containing 90 % of the source graphite and 10 % of the adhesive (polyvinylidenchloride in 2methylpyroliden) has been prepared and then the graphite composition was deposited on the platinum substrate. a silver chloride electrode in the saturated solution of kcl and mercury sulphate electrode in the 1 m solution of h2so4 were used as reference electrodes. all experiments were carried out using a standard electrode cell yase-2. anodic polarization was ranged from 0 to 1200 mv. upper limit of the polarization was set according to previous investigation of the working electrolytes decomposition potentials on the graphite electrodes. cathodic part of the cva curve was inversed to the anodic and ranged from +1200 to 0 mv. temperatures of the working solutions were from 293 to 323 k and the cva potential change speeds were 0,005-0,100 v/s (for the anodic part) and 0,02-0,10 v/s (for the cathodic part). the potentials of the intercalation and deintercalation starting points (φstart(а) and φstart(c)) and potentials of the highest cathodic and anodic currents (φmax(а) and φmax(c)) were determined using cva (see fig. 1). these parameters made possible to calculate changes in the gibbs free energy for the intercalation (gstart(а)) and deintercalation (gstart(c)) starting points, changes for the points of the highest speed of intercalation (gmax(а)) and deintercalation (gmax(c)) and enthalpy (н) and entropy (s) changes for these processes. indexes a and c mean anodic process (intercalation) or cathodic process (deintercalation) respectively. the concentrations of carbon, sulphur and oxygen in the surface layer of the modified graphite materials were found and fluxes of the physical and electrochemical transport of hydrosulphate ions towards the carbon matrix were calculated. results and discussion an electrochemical system for the hydrosulphate intercalation/deintercalation has been designed according to the following scheme: me | c (hso4–) | h2so4, h2o ¦ ¦ h2o, kcl, agcl | ag | me (1) the thermodynamic parameters of the systems were calculated using relations – ∆g = n∙f∙e, ∆s = – (∂∆g/∂t)p, ∆h = ∆g + t∙∆s, where ∆g is gibbs free energy change, n – quantity of electrons acting in the elementary electrochemical stage, f – faraday’s constant, e – e.m.f, ∆s – change of entropy, t – temperature and ∆h – change of enthalpy for the resulting electrochemical process [16]. carbon electrodes with preliminary intercalated hydrosulphate ions were used to determine thermodynamic parameters of the circuit (1). an inter-phase potential c (hso4–) | h2so4, h2o was considered equilibrium. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 8 figure 1. a typical cva curve for intercalation/deintercalation processes for electrodes ge-1, hydrosulphate ions, potential change speed 2∙10-2 v/s, 3 m solution of sulphuric acid and mercury sulphate reference electrode. a schtakelberg’s method has been employed to assess a reversibility ratio of the intercalation/deintercalation processes. a tilt angle of a tangent to the cva curve in the point 2 1φ can be calculated using this method through the relation: ,i а n d id d 2 1         where n means quantity of electrons participating in the intercalation. a value of the parameter “a” from relation above should be equal to 100.7 mv for the single-electron process at 293 k. an experimental value of “a” for the system with ge-2 electrode and concentration of sulphuric acid 3 m/l was ≈ 101 mv. the criterion of tomesh states that a difference between φ3/4 і φ1/4 for the reversible voltammetry curves recorded at 298 k should be 56 mv [17]. in our case this difference was 60 mv. a difference between the cathodic and anodic peak potentials for a reversible process occurring at the solid electrodes should be equal to the theoretical value calculated through the shevchik relation [17] ∆φrv = φa – φc = 2,22(rt/nf), where φa and φc denote peak potentials for the anodic and cathodic parts of the cva curve. a theoretical value of ∆φrv should be 80.7 mv at 293 k. our experimental results gave ∆φrv = 70 mv. a quantity of electricity spent for the intercalation/deintercalation processes (q) has been determined in order to evaluate their reversibility [18]. since qint=0,9 qdeint for both ge-1 and ge-2 electrodes these processes can be considered as reversible. preliminary experiments with nonintercalated electrodes proved that previous intercalation/deintercalation cycles can influence parameters of next intercalations (see fig. 2). for example, previous cycles result in higher value of the limit anodic current, which can be caused by defects in the graphite substrate remained after previous embeddings. new anions can faster embed into defective spots of the substrate during next intercalations. higher speed of the potential change results in small positive shifting of φmax(а). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 9 figure 2. consecutive cva curves for intercalation/deintercalation processes on ge-1 electrode and 1 m solution of sulphuric acid with silver chloride reference electrode. absolute values of the intercalation enthalpy change for the electrode ge-2, which underwent preliminary intercalation (under the following conditions: с(h2so4) = 3 m/l, mercury sulphate reference electrode, intercalation current іint = 35 mа/sm2, intercalation time 10 min) and decrease at the raise of temperature according to the formula: нint = – 0,0434 · т + 52,873. deintercalation of the same electrode under similar conditions gives the following relation for the enthalpy changes: нdeint = 0.4394 · т + 0,147. previous intercalation causes decrease in the intercalation potential φint at next intercalation and in absolute changes in the free gibbs energy of the intercalation gint. an absolute value of the intercalation enthalpy |–нint| raises. intercalation of graphite in the aqueous solutions has been extensively investigated by many authors but results of different works are quite controversial [19-22]. authors of [21] reported better intercalation results that can be achieved in the concentrated solutions while no intercalation occurred for a 10 % solution of sulphuric acid. on the other hand, successful intercalation of graphite in the 0.1 m solutions of h2so4 has been reported in [22]. this concentration corresponds to a quite diluted 0.98 wt % solution. a completely reversible intercalation in the 4 m (~ 31.8 wt %) solution of sulphuric acid has been reported in the same reference. good agreement was achieved only for the results [19-22] related to the highly concentrated systems containing 60 wt % of the acid or more. no data of composition of the graphite intercalation products in the diluted solutions are available. two maximums can be found on the cva curves recorded for the diluted solutions. one of them appears on the anodic and another one – on the cathodic (see fig. 1). two maximums in the anodic part and another two in the cathodic have been recorded during intercalation in the 10 m solution of h2so4 (see fig. 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 10 figure 3. cva curve for the 10 m solution of h2so4. a method of the polarization diagrams in the aqueous solutions of h2so4 [23] has been employed to investigate destruction of the graphite electrodes. following processes can occur at the graphite destruction:  4hsoсх + h2o →    нос х + 42soн (2)  n424 )soh2)((hsoс х →   n424 )soh)((2hsoс х + н + (3) an intercalation current density consists of two components: diffusive current and the current, which depends on the particles transportation at formation of the oxide compounds. process (3) causes decrease in the electric resistance because of transformation of the graphite-embedded h2so4 molecule into an electroactive ion 4hso [20]. the maximal corrosion current determined in our experiments was found insignificant comparing with the total intercalation current. this proves that the process of intercalation can run in the diluted solutions of sulphuric acid. a change in the decomposition energy δu = n∙f∙φ of the graphite intercalation compounds (gic) for various concentrations of the acid has been calculated in order to evaluate thermodynamic possibility of the decomposition. current consumption for the side processes decreases with decrease of the acid concentration for c(h2so4) = 10-15 m. this result is in a good agreement with the reference [24]. the maximal corrosion current has been recorded for the 10 m solution. a contribution of the corrosion current into the total process current value remains insignificant even in the diluted solutions. cva and the electron microscopy with edx – shimadzu 900 hs, icp ms agilent – sua were engaged to determine surface content of sulphur and oxygen after the 30 min long amperostatic intercalation with current density 40 ma/sm2. working area of the electrode was 0.25 sm2. the spectral characteristics of the ge-1 surface before intercalation are represented in fig. 4 and table 1. fig. 5 and table 2 represent results related to the same sample after intercalation. table 1 spectral investigation of the surface composition of a graphite sample before intercalation el an series unn. [wt.%] c norm. [wt.%] c atom. [at.%] error [%] c 6 k-series 99.47 99.48 99.81 0.3 s 16 k-series 0.45 0.45 0.17 0.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 11 figure 4. surface map of a graphite sample before intercalation. table 2 spectral investigation of the surface composition of a graphite sample after intercalation in the 5 m solution of h2so4. el an series unn. [wt.%] c norm. [wt.%] c atom. [at.%] error [%] c 6 k-series 26.08 26.08 33.35 8.3 o 8 k-series 65.57 65.58 62.95 20.9 s 16 k-series 7.63 7.63 3.66 0.3 figure 5. surface map of a graphite sample after intercalation in the 5 m solution of h2so4. surface atomic concentrations of carbon, sulphur and oxygen are shown in fig. 6. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 12 figure 6. dependence of the surface concentrations of carbon, sulfur and oxygen on concentration of sulfuric acid during electrochemical intercalation. the interface gradients of chemical and electric potentials should exist on the boundary graphite surface | sulphuric acid solution in order to ensure embedding of hydrosulphate ions into the graphite matrix. only physical intercalation can run if a gradient of the hydrosulphate ions chemical potential is the only gradient existing in the system. carbonium cations can be formed in the system if electric potential also exists and these cations facilitate faster intercalation of hydrosulphate anions into the matrix. an aggregated effect of both gradients can be characterized through the difference in the electrochemical potentials of hydrosulphate ion (∆µ~i) on the interphase boundary graphite | solution of sulphuric acid: ∆µ~i = ∆µi + ∆ψ , where ∆µi and ∆ψ mean interphase differences in the chemical and electric potentials of the ions respectively. physical intercalation has been carried out using solutions of sulphuric acid with concentrations 1.0; 3.0; 5.0; 10.0 and 15.0 m during 60 min. electrochemical intercalation has been carried out using similar solutions with intercalation current 10 ma and samples area 0.25 sm2 during 30 min. table 3 hydrosulphate ions intercalation conditions. № graphite sample number, c(h2so4) – concentration of sulphuric acid, t – intercalation time, iint – intercalation current. contents of elements in wt. % denoted as c(x), where x means carbon с(с), sulphur с(s) and oxygen с(o). relative error of determination (%) is shown in the square brackets № type of intercalation с(h2so4) mol/l t, min iint, ma с(с) с(s) с(о) с(о)/с(s) 5 electrochemical 1.0 30 10 27.85 [9.6] 4.77 [0.2] 65.92 [21.8] 13,8 6 electrochemical 10.0 30 10 17.22 [6.3] 9.61 [0.4] 70.80 [23.0] 7.4 7 electrochemical 15.0 30 10 94.86 [1.3] 8 physical 1.0 60 – 20.50 [6.3] 0.22 [0.0] 61.43 [20.7] 279.2 9 physical 3.0 60 – 25.11 [7.7] 0.30 [0.0] 58.26 [20.2] 194.2 10 physical 5.0 60 – 22.72 [7.0] 0.52 [0.0] 63.02 [21.0] 121.2 11 physical 10.0 60 – 25.33 [7.8] 0.73 [0.1] 58.59 [20.4] 80.3 12 physical 15.0 60 – 24.38 [7.6] 1.55 [0.1] 62.15 [21.0] 40.1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 13 the spectral parameters have been recorded for small selected parts of the electrode surface only. nevertheless, even these results prove that higher concentration of sulphuric acid during physical intercalation results in higher surface concentration of sulphur on the electrode (see fig. 7). figure 7. influence of concentration of sulphuric acid (c(h2so4), m/l) on the surface content of sulphur (c(s), wt %) after physical intercalation. increased concentration of h2so4 during electrochemical intercalation also results in higher surface content of sulphur (fig. 8). figure 8. same as figure 7 but for the electrochemical intercalation. relatively high surface content of oxygen can be caused by formation of the carbon oxide compounds in the surface layer of graphite as a result of reaction between embedded hydrosulphate ion and water molecule:  4hsoс х + h2o →    нос х + 42soн (4) the raise in concentration of h2so4 from 1.0 to 15.0 m/l agrees with the increase in the surface content of sulphur and decrease of the ratio c(o)/c(s). this can be caused by retardation in the reactions (4) rate with increasing of concentration of sulphur acid. the fluxes of the electrochemical and physical intercalation have been evaluated basing on the spectral investigation of the electrodes surface. the evaluation has been made through determination of a mass of the embedded sulphur after a 30 min process on the 0.25 sm2 electrode area and with intercalation current 10 ma. the evaluation results are represented in table 4 and fig. 9. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 14 table 4 fluxes of the hydrosulphate ions intercalation into the graphite matrix. j means total flux of the ions and j’ – the specific flux on a unit surface area per unit of time (g/min sm2) intercalation conditions sample time, min c(s), g c(s), for 30 min j’ j 1 м sulphuric acid electrochem 5 30 4.77 4.77 4.77 0.636 physical 8 60 0.22 0.11 0.11 0.015 10 м sulphuric acid electrochem 6 30 9.61 9.61 9.61 1.28 physical 11 60 0.73 0.365 0.365 0.049 figure 9. electrochemical (1 and 3) and physical (2 and 4) fluxes (g/min·sm2) of the hydrosulphate ions intercalation for the 1 m (1 and 2) and 10 m (3 and 4) sulphuric acid. an electrochemical intercalation flux value was 0.636 g/min·sm2 and a physical flux value was 0.015 g/min·sm2 for the 1 m sulphuric acid. corresponding fluxes for the 10 m solution were 1.28 and 0.049 g/min·sm2. conclusion the processes of intercalation and deintercalation, which run under the above mentioned experimental conditions, can be classified as reversible. a contribution of the current caused by the corrosive destruction of the graphite matrix is insignificant. as concentration of sulphuric acid is increasing from 1.0 to 10.0 m/l, the physical intercalation flux raises from 0.015 to 0.049 g/min·sm2 and the electrochemical raises from 0.636 to 1.28 g/min·sm2. this change in h2so4 concentration also causes raise in the “physical intercalation” bringing contribution to the total flux of anions from 2.3 to 3.8 %. references 1. www.sealur.ru/pdf/usefl/reports/grafit.pdf 2. a. ubbelode, f lewis. graphite and its crystal compounds. (1965). mir, moscow. 256 p. 3. n. globa et al. carbon nanosized materials as elements for electrochemical collection of hydrogen. (2008). chernivtsi uni. sci. bulletin. 401. 20-22. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava year ix, no. 4 2010 15 4. a. fialkov. carbon and carbon based interlayer and composite compounds. (1997). aspect-press, moscow. 718 p. 5. k. mahorin, a. kozhan, v. veselov. natural graphite heaving after treatment with sulphuric acid. (1985). chem. tech. 2. 3-6. 6. a. yaroshenko, m. savoskin. highquality heaving compounds of intercalated graphite. (1995). j. appl. chem. 68. 1302-1306. 7. a. antonov et. al. investigation of formation of some powdery materials without polymer adhesives. (1984). chem. technique and technology. 1. 114-117. 8. k. mahorin et. al. analysis of derivatograms of oxidized and expanded graphite. (1990). chem. tech. 3. 44-47. 9. m. kalashnikova. a derivatographic study of the details of thermoexpanded graphite. (2001). bull. of perm uni. 7. 82-91. 10. m. belova et.al. sealers of thermoexpanded graphite: conditions for safe use in the liquid and gaseous oxygen. (2006). armature construction. 41. 70-75. 11. m. savoskin et.al. sorption of industrial oil with expanded graphite. (2003). j. appl. chem. 76. 936-938. 12. a. samoilov et al. formation of a consolidated layer of the carbon sorbent/oil at oil collection from the emergency spill spots. (2004). j. appl. chem. 77. 328-334. 13. r. avrbe et. al. strengthening of the selfpressed thermoexpanded graphite with pyrocarbon. (1996). j. appl. chem. 69. 2068-2070. 14. www.secna.ru 15. a. yakovlev. scientific and technological basis of electrochemical synthesis of thermoexpandable graphite compounds in the nitric acid electrolytes. (2006). d. sc. thesis abstract. saratov. 40 p. 16. e. kovalchuk and o. reshetnyak. physical chemistry. (2007). publishing centre of lviv univ., lviv, 800 p. 17. yu. delimarsky and e. skobets. polarography on the solid electrodes. (1970). technique, kiev, 220 p. 18. ya. tevtul et al. thermodynamical and electrochemical parameters of intercalation of graphite materials (2006). ukr. j. chem. 72. 88-91. 19. f. kang et al. electrochemical synthesis of sulfate graphite intercalation compounds with different electrolyte concentrations (1996). j. phys. chem. solids. 57. 883-888. 20. a. metrot and j. e. fischer. chargetransfer reactions during anodic oxidation of graphite in h2so4 (1981). synth. met. 3. 201-207. 21. o. shornikova et al. intercalation of graphite in the systems h2so4 – r (r – h2o, c2h5oh, c2h5cooh). (2005). inorg. mater. 41. 162-169. 22. f. beck and h. krohn. reversible electrochemical intercalation of anions in graphite electrodes. (1983). j. electrochem. society. 574 594. 23. v. scorceletti. theoretical electrochemistry. (1959). state chemistry publishers. 608 p. 24. t. filipchuk et al. graphite surface corrosion at intercalates formation in sulphuric acid. (2008). proceed. xvii ukr. conf. of inorg. chem. lviv. 271. effect of plant protein isolates to the structural – mechanical properties of wheat dough 167 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 32017, pag. 167 179 water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour olexandr rozhno 1 , vira yurchak 1 , * tatjana golikova 2 , 1educational-scientific institute of food technologies, national university of food technologies, volodymyrska str. 68, kyiv, ukraine, rozno777@gmail.com 2technology of nutrition and restaurant business dept., national university of food technologies, volodymyrska str. 68, kyiv, ukraine, tatjana.golikova12@gmail.com *corresponding author received 14th august 2017, accepted accepted 27th september 2017 abstract: authors have developed macaroni products made from fine meal corn flour by structure forming additives of different nature and without them. the correlation between water of different bond forms in macaroni dough with different structural forming additives and without them has been investigated. results show that water of macro and microcapillaries is prevalent in the corn dough – 39.40 54.69 % of overall amount of water. osmotically bound water amounts 18.75 – 28.04 %, adsorbically bound water -18.49 – 23.13 % of overall amount of water. the absorbtion capability of the macaroni products and amount of adsorbed water has been determined. the micropore structure of these samples was characterized. the correlation between structures of macaroni products, both amount of adsorbed moisture and energy of sorption were proven. the amount of monomolecular layer’s moisture for gluten-free corn macaroni products is significantly higher – in 1.2 – 1.5 times – when compared to the wheat macaroni products. due to this fact, corn samples obtained higher energy of moisture sorption. the correlation between structural characteristics of the macaroni samples and their quality was shown. keywords: gluten-free macaroni products, water bond forms, absortion capability, structural characteristics, absorbtion energy. 1. introduction one of the main tasks of food industry is to provide dietetic foods. dietetic nutrition is an important factor and it plays an essential part in the complex treatment of different deseases. dietetic food is a special food that differs from traditional food by chemical content and low caloric value. over years a number of metabolic deseases (in particular, celiac desease) has emerged in the population. the celiac – genetic desease involves intolerance to some proteins. the only effective and safe method of celiac treatment is strict dietary intervention during life time that excludes gluten. gluten is a group of proteins that takes part in forming of wheat dough naming gliadine. gliadine is an innocuous substance, but it causes allergy or recrudescence at people with genetic predelection for celiac desease [1]. derived products from corn, rice and buckwheat are often used as gluten-free raw materials in the production of bread, pastries and macaroni products. corn flour is the most relevant raw material to obtain macaroni products in ukraine because corn does not contain gluten and it is one of the http://www.fia.usv.ro/fiajournal mailto:rozno777@gmail.com mailto:tatjana.golikova12@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 168 widely spread cereal crops on this territory. corn contains higher amount of cellulose, polyunsaturated fatty acids of ω-3 and ω-6 groups, important for the organism, minerals such as ferrum, selenium, folic acid, tokoferol, biotine, β-carotene and others when compared with wheat [2]. gluten-free macaroni products are not produced in ukraine and they are provided by the import. the production of macaroni products is closely connected with the role of gluten in forming the structure of dough and ready products. gluten-free flour does not contain proteins that form gluten, thus the making of macaroni products from corn flour raises some difficulties. some researchers believe that the structure forming of gluten-free macaroni products may be provided by starch gelatinization of raw materials and use of other structure forming additives [3]. most of the publications concerning the production of gluten-free macaroni products focus on their quality, providing little information about technology, in particular the colloidal processes in dough, kneading, drying processes and storage. for example, the european patent ep 0792109 в1 stipulates the method of production of corn macaroni products by means of additional operation of flour scalding [4]. according to this method corn flour is scalded completely or partially before kneading and drying. this flour is mixed with water and formed one more time. still this method has not come into common use obviously because of the additional preparing operations. also, publications do not contain data on the main quality indexes of macaroni products. under the supervision of lucia padalino [5] some investigation on the effect of different hydrocolloids on the chemical content and quality of gluten-free spaghetti made from corn and oat flour was carried out. pectin, agar, carbulose, helan gum and others were used as structure forming additives. the results show that most of hydrocolloids increase the quality of macaroni products and structuralmechanical properties of dough. investigations on the technological processes are not approached in this paper. authors have developed macaroni products made from corn fine meal flour by using structure forming ingredients – xanthan gum, carboximethylcellulose (cmc) [6, 8], gelatin and dry egg white [7, 8], as well as without use of structure-forming ingredients – with addition of extruded corn fine meal flour [9, 10]. the dosage of structure forming additives, amount of added extruded or scalded flour that provide the best quality of products by strength, cooking properties in particular amount of dry matter passed into cooking water have been investigated. forms of water bonds in dough determine the reological properties, kinetics of drying process and quality of macaroni products [11]. microstructure effects on the quality indexes of macaroni products that, therefore, will have an impact on the sorption properties of products and changes during its storage. determination of energy and forms of water bond in dough as well as investigation on the sorption properties of corn flour help to clarify the mechanism of influence of main raw materials and structure forming additives on the quality indexes and technological processes of its production and storage. 2. matherials and methods flour corn fine meal flour and corn extruded flour were used (see table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 169 table 1 quality indexes of the corn flour indexes samples of flour fine meal corn flour extruded corn flour coarseness, % >264 µ 7.0 48.6 >219≤264 µ 53.1 32.7 >195≤219 µ 28.3 18.7 >165≤195 µ 9.9 – >115≤165 µ 0.8 – <115 µ 0.9 – average diameter of the particles, µ 106.40 115.30 homogenity of the particles, units of device. 0.59 0.66 water absorption, % 260 320 content of the carotene pigments, mg/100 g 0.339 0.733 amount of amine nitrogen, mg % per 100g (proteolytic activity) 140 420 autolytic activity, % per dry matter 8.9 9.1 acidity, grade after production after 3 months 3.2 3.9 3.3 3.3 fat content, % per dry matter 2.9 2.6 structure forming additives structure forming additives of carbohydrate origin – xanthan gum 0.7% of the weight of flour, carboximethyl cellulose – 0.3% of the weight of flour were used. this dosage has been installed as optimal. additives have been added in colloidal solutions. structure forming additives of protein origin – dry egg white (dew) in disoxidated kind – 5% of the weight of flour, gelatin – in colloidal solution – 1 % of the weight of flour were used. the extruded corn flour was used to make macaroni samples without use of structure forming additives (20 % of the weight of mix of corn fine meal flour and extruded flour). scalding of part of flour – 10% of the total weight – was also used. part of corn fine meal flour was replaced by the extruded corn flour. macaroni samples made from wheat baking flour were used like control samples. all dough samples were prepared with moisture content 36% in the laboratory press makma-m. products had a short noodle shape. water bond forms forms of water bond were investigated by means of derivatograph q-1000 in temperature interval 20 – 200oc with speed of heating of samples 1g mass – 1.25°с/min. authors have used the method of interpretation of derivatograms [12, 13], that helps to determine temperature intervals of different water bond forms due to the analysis of graph of temperature changes (ta) and mass of the dough during heating (tg), and its derivates food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 170 (dta and dtg). by the dots of dta curve knee we have marked v of main intervals: i – free moisture (temperature till 40…60°с), ii – moisture of macroand microcappillars (till 110°с), iii – osmotically bond moisture (till 120°с). osmotically bond moisture has low bonding energy although the mechanism of drying is related to the removal of water from macroand microcappillars, releasing cappillars for migration of osmotically bound water; iv – adsorbically bound water (till 145oc) and v – chemically bound water (higher than 145oc). energy of activation energy of activation during water removal was determined due to the arrenius formula by the obtained triangle, by the tangents of tip angle of hypotenuse that was created in coordinates lnδt – 1/t: e = r·b/a, where r – universal gas constatnt 0.0083 kj/moll·к; a – length of adjacent side of triangle (on axis 1/t), mm; b – length of opposite side (on axis lnδt), mm [14]. sorption properties sorption properties of dry macaroni samples were determined by means of vacuum-statistic method by the mc-ben apparatus [15]. the amount of water of monomolecular layer, polimolecular layer and capillary bound water (water of macro and microcappillars) were determined by the obtained isotherms of sorptiondesorption [15]. determination of the amount was carried out taking into account knee points at adsorption graph in diapasons р/рs 0.05…0.36; 0.36…0.74; 0.74…1.0 respectively. as the authors have proved [15], the interaction in adsorbic layers (lateral interaction) takes place in case of р/рs ˃ 0.3. this interaction just determines the shape of adsorption isotherm in the segment of polimolecular water. cappilar sweating in pores may appear at high pressure in porous adsorbents, so the water found is of hygroscopic state. data of square, diameter, porous volume and energy of absorption have been obtained by means of isotherms processing for the macaroni products with and without use of structure forming additives [15]. 3. results and discussion water bond forms the derivatograms obtained of the samples under analysis are shown in figure 1. the results of description of derivatograms of samples made from corn dough with structure forming additives of carbohydrate and protein origin are shown in table 2. the results show that a small amount of unbound water – 1.15-1.53% of overall moisture mass is lost in i temperature interval in the wheat dough, for both samples of dough made from corn flour with xanthan, gelatin and dry egg white. thus, corn dough with cmc contains 4.48 % of overall unbound moisture content in half-ready product. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 171 fig. 1 – derivatograms of macaroni dough samples made from flour: а) wheat; b) corn with addition of xanthan; c) corn with addition of cmc; d) corn with gelatin; e) corn with dry egg white; f) corn with addition of extruded flour; g) corn with part of scalded corn fine meal flour. table 2 results of description of derivatograms of samples made from corn dough with structure forming additives of carbohydrate and protein origin dough samples made from flour temperature interval, °c moisture loss, mg moisture loss, w, % energy of activation, kj/mole % of sample mass % of overall moisture mass wheat (control) і. 20-34 4.15 0.41 1.15 4.06 іі. 34-110 143.33 14.33 39.81 ііі. 110-120 86.42 8.64 24.00 iv. 120-145 105.21 10.52 29.23 v. 145-179 20.90 2.09 5.80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 172 corn with xanthan gum (0.7 %) і. 20-44 5.50 0.55 1.53 5.53 іі. 44-97 141.85 14.18 39.40 ііі. 97-118 100.94 10.09 28.04 iv. 118-140 79.06 7.91 21.96 v. 140-157 32.65 3.27 9.07 corn with cmc (0.3 %) і. 20-60 16.12 1.61 4.48 4.74 іі. 60-100 145.07 14.51 40.30 ііі. 100-117 85.97 8.60 23.88 iv. 117-144 83.28 8.33 23.13 v. 144-177 29.55 2.96 8.21 corn with gelatin (1.0 %) і. 20-42 5.16 0.52 1.43 7.93 іі. 42-105 196.89 19.69 54,69 ііі. 105-120 73.30 7.33 20,36 iv. 120-145 66.57 6.66 18,49 v. 145-177 18.07 1.81 5,02 corn with dry egg white (5.0 %) і. 20-41 5.49 0.55 1,52 7.19 іі. 41-104 175.61 17.56 48,78 ііі. 104-120 93.84 9.38 26,07 iv. 120-145 68.60 6.86 19,05 v. 145-163 16.46 1.65 4,57 all unbound water from big pores and meshes of these samples of dough is removed in this interval. with the increasing of temperature up to 110oc (ii interval) the water from macroand microcapillars removes from all dough samples. amount of macroand microcappillar water in the wheat dough and corn dough samples with xanthan and cmc is almost the same – 39.81%, 39.40% and 40.30% of overall amount of water respectively. weight percentage of water of macroand micro-cappillars in corn dough with protein structure forming additives is slightly higher when compared to the other samples and amounts 54.69% in dough with gelatine and 48.78% in dough with dew respectively. the amount of osmotically bound moisture (iii diapasone) in the corn dough samples is 20.36 – 28.04% of the total water amount. most of this water is found in the dough with xanthan and dew – approximately 4.0 – 2.0% of the total amount of `water respectively as compared to wheat dough sample. water in cmc samples is almost the same as in the control sample, whereas in the gelatin sample it is lower than in the control sample, by 3.64% of the total amount of water. the adsorbically bound moisture in samples of dough made from corn flour amounts to 18.49 – 23.13% of the total amount of water, a higher amount being determined in dough with additives of carbohydrate origin. the amount of adsorbically bound moisture in corn dough with additives is lower in all the samples approximately by 6.0 – 11.0% than in wheat dough – 29.23%. during heating over 145oc (v interval) water loss is caused by the start of the oxidation of organic elements and removal of chemically bound water, and further on by the destruction of material. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 173 the amount of removed water in samples with structure forming additives of carbohydrate origin increases in this interval up to 8.21 – 9.07% of the overall water amount, as compared to 5.80% of the wheat dough, and the amount of removed moisture in the sample with protein structure forming additive decreases approximately by 1.0%. so, the amount of moisture of macroand micro-cappillars increases in dough samples with protein structure-forming additives, while osmotically bound moisture in sample with dew increases by 2%. the amount of osmotically bound moisture (in the case of xanthan use) increases in corn dough with carbohydrate structure-forming additives as well as the chemically bound moisture. energy of activation energy of activation is higher – by 0.68 – 1.47kj/mole in the samples with xanthan and cmc and by 3.13 – 3.87 kj/mole in the samples with protein structure forming additives as compared to wheat dough. obviously, a high content of moisture of macroand micro-cappillars (samples with protein structure forming additives) and amount of tighly bound water (samples with xanthan and cmc) has the most effect on the energy of activation for these systems. the results of description of derivatograms of samples made from corn dough without structure forming additives are shown in table 3. the data of table 3 show that the amount of unbound water in the dough sample which contains part of scalded corn flour is higher than in control sample – by 1.45% of the total water amount. the sample with extruded flour is 2 times lower than this previous amount. the amount of moisture of macroand micro-cappillars in the sample with extruded corn flour is 53.48 % of the total water amount. in dough with part of scalded flour this amount is not by much higher than in control sample – 41.60% as compared to 39.81% of the total water amount in wheat dough. the amount of osmotically bound water in the dough with 20% of extruded flour is less by 5.25% of the total water amount than in the control sample; and in the dough with part (10%) of scalded flour is insignificantly higher (by 0.83%). the amount of adsorbically bound water in the samples with use of extruded and scaled corn flour is lower by 6.32% and 8.77% of the total water amount than in the control sample. obviously, during scalding of fine meal corn flour centers of micells get broken and van der waals’ forces reduce [3]. evidently, the same things take place during starch gelatinization of corn flour in the extrusion process. the energy of activation in corn macaroni dough samples without addition of structure forming ingredients is higher than in the dough samples made from wheat flour which has an energy of activation of 4.06 kj/mol. table 3 results of description of derivatograms of samples made from corn dough without use of structure forming additives dough samples made from flour temperature interval, °c moisture loss, mg moisture loss, w, % energy of activation, kj/mole % of sample mass % of overall moisture mass wheat (control) і. 20-34 4.15 0.41 1.15 4.06 іі. 34-110 143.33 14.33 39.81 ііі. 110-120 86.42 8.64 24.00 iv. 120-145 105.21 10.52 29.23 v. 145-179 20.90 2.09 5.80 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 174 corn with extruded corn flour (20%) і. 20-36 2.53 0.25 0.70 6.61 іі. 36-106 192.54 19.25 53.48 ііі. 106-116 67.51 6.75 18.75 iv. 116-143 82.47 8.25 22.91 v. 143-160 14.95 1.50 4.15 corn with part (10 %) of scalded corn flour і. 20-35 5.20 0.52 1.45 8.48 іі. 35-103 149.76 14.98 41.60 ііі. 103-119 89.39 8.94 24.83 iv. 119-139 73.67 7.37 20.46 v. 139-159 41.98 4.20 11.66 the moisture sorption-desorption isotherms of macaroni samples are shown in the fig. 2. data of moisture of monomolecular layer, polimolecular layer and hygroscopic moisture are given in the table 4 for the corn macaroni samples with addition of structure forming ingredients and in the table 6 for the macaroni samples without addition of structure forming ingredients. it was found that macaroni products made with use of structure forming ingredients of carbohydrate origin exert sorbtion capability that is marginally higher (table 5). the amount of adsorbed moisture is 101 – 102% for macaroni products with addition of cmc and xanthan gum as compared to the amount of adsorbed moisture of samples made from wheat flour. nevertheless, the amount of moisture of monomolecular layer for the corn macaroni samples with xanthan gum and cmc is significantly higher (aproximately by 1.5 times) – 18.6% and 17.4% respectively of the overall water amount as compared to 12.4 % in wheat macaroni samples. thus, the amount of moisture of polymolecular layer by 0.5 – 1.5% and moisture of hygroscopic state approximately by 4.5% get reduced in the experimental samples when compared to the control sample. it stands for the increase of energy of moisture bond in the samples with structure forming additives. macaroni samples with structure forming additives of protein origin have slightly reduced sorbtion capability when compared to wheat samples and corn with additives of carbohydrates origin. the corn macaroni samples with use of dry egg white contain the lowest amount of adsorbed moisture – 88.0% when compared to its amount in wheat flour. it suggests less evident sorbing effect of these macaroni samples during storage. the macaroni samples with gelatin and dry egg white absorb higher amount of moisture of monomolecular layer by 1.4 and 1.2 times respectively when compared to the control sample. thus, the amount of cappillar moisture reduces up to 83% when compared to wheat samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 175 fig. 2 – isotherms of sorbtion-desorbtion of water in macaroni products made from flour a) wheat; b) corn with addition of xanthan gum; c) corn with addition of cmc; d) corn with gelatin; e) corn with dry egg white; f) corn with addition of extruded flour; g) corn with part of scalded fine meal corn flour. by means of the adsorbtion-desorbtion curves analysis, the structural characteristics of the macaroni samples with structure forming additives were calculated by the equation bet [15] (see table 5). the results suggest that in the case of equal micropore volumes corn macaroni samples with xanthan gum and cmc have less diameter of pores, and, respectively, bigger square of pores by approximately 1.4 times when compared to wheat samples. it explains the increase in the amount of adsorbed moisture of monomolecular layer energy of moisture bond by 1.4 times. macaroni samples with gelatin and dry egg white have slightly reduced volume of pore, while their diameter is also less, but their square is bigger. due to this, the energy of moisture sorption is higher – 4.26 and 4.76 kj/mole when compared to 3.41% kj/mole in wheat samples. the overall amount of adsorbed moisture in macaroni sample with extruded flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 176 without addition of structure forming ingredients is evidently higher when compared to other samples – 113% of control sample (table 6). table 4 effect of different structure forming additives on the adsorbed moisture content in the gluten-free macaroni products macaroni samples made from amount of adsorbed moisture monomolecular layer (p/ps =0 0,36 g/g) polymolecular layer (p/ps = 0,36 0,74 g/g) hygroscopic state (p/ps = 0,74-1,0 g/g) total а , g /g d s % t o f th e c o n tr o l % of the total amount of water а , g /g d s % t o f th e c o n tr o l % of the total amount of water а , g /g d s % o f th e c o n tr o l % of the total amount of water а , g /g d s % o f th e c o n tr o l wheat flour (control sample) 0.036 100 12.4 0.071 100 24.1 0.184 100 63.5 0.290 100 corn with xanthan (0.7%) 0.055 153 18.6 0.067 97 22.6 0.174 94 58.8 0.296 102 corn with cmc (0.3%) 0.051 142 17.4 0.069 99 23.6 0.173 94 59.0 0.293 101 corn with gelatine (1.0%) 0.051 141 18.0 0.070 99 24.7 0.163 89 57.4 0.284 98 corn with dew (5.0%) 0.044 122 17.0 0.060 84 23.5 0.151 83 59.2 0.255 88 table 5 structural characteristics of the macaroni samples with use of structure forming additives macaroni samples square of pores, s, m2/g volume of pores, vs, cm3/g diameter of pores, d, 10-10 m sorbing energy, kj/mole wheat (control, no additives) 110 0.29 105 3.41 corn with xanthan (0.7%) 149 0.29 77 4.92 corn with cmc (0.3 %) 151 0.29 78 4.59 corn with gelatine (1.0 %) 139 0.28 81 4.26 corn with dew (5.0 %) 139 0.26 75 4.76 macaroni products made from the part of scalded corn fine meal flour contain an overall amount of adsorbed moisture that is slightly higher when compared to wheat sample. the amount of monomolecular layer’s moisture of these samples is higher – 13.5 and 16.7 % of the total amount of adsorbed water in macaroni samples with extruded flour and part of scalded flour respectively. amount of moisture of polymolecular adsorption in these samples is slightly reduced when compared to the control sample. macaroni products with extruded flour also contain higher amount of hygroscopic state moisture – 66.4 % of overall water amount. evidently, such results of amount of adsorbed water in the macaroni with extruded flour are associated with their more porous structure. as can be seen from the table 6 corn macaroni samples with extruded flour provide the maximal volume and diameter of pores – 0.33cm3/g and 115·10-10m respectively. nevertheless, square of pores is slightly increased as compared to the control sample, which results in lowering the amount of moisture of monomolecular layer than in the other corn samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 177 table 6 amount of adsorbed water in macaroni samples made without use of structure forming additives macaroni samples made by amount of adsorbed water monomolecular layer (p/ps =0 0,36 g/g) polymolecular layer (p/ps = 0,36 0,74 g/g) hygroscopic state (p/ps = 0,74-1,0 g/g) total а , g /g d s % o f th e c o n tr o l % of the total quanti ty of water а , g /g d s % o f th e c o n tr o l % of the total quantit y of water а , g /g d s % o f th e c o n tr o l % of the total quantit y of water а , g /g d s % o f th e c o n tr o l wheat flour (control, no additives) 0.036 100 12.4 0.070 100 24.1 0.184 100 63.5 0.290 100 corn with extruded flour (20%) 0.044 121 13.5 0.066 94 20.2 0.217 118 66.4 0.327 113 corn with part of scalded flour (10%) 0.050 137 16.7 0.069 98 23.1 0.180 98 60.2 0.299 103 table 7 structural characteristic of macaroni samples made without use of structure forming additives macaroni samples made from square of pores, s, m2/g volume of pores, vs, cm3/g diameter of pores, d, 10-10 m sorbing energy, kj/moll wheat flour (control, no additives) 110 0.29 105 3.41 corn with extruded flour (20 %) 115 0.33 115 4.62 corn with part of scalded flour (10 %) 156 0.30 77 4.59 the energy of moisture sorption of these macaroni samples is higher – 4.62 kj/mole. the samples that were obtained with addition of part scalded flour provide more fine-pored structure – contain pores of smaller diameter, while their square is maximum. energy of moisture sorption for these macaroni samples is also high – 4.59 kj/mole. so, to summarize, the energy of moisture sorption and amount of the adsorbed water are both associated with the porous structure and square of pores. the results of the previous experimental researches [8] show correlations between structural characteristics of the macaroni samples and their quality. as shown by our study, in the case of using structure forming additives in samples with smaller diameter pores and high energy of sorption, the best quality of macaroni is provided, in particular, both in terms of strength and passing of dry substances into the cooking water. for example, the macaroni samples with xanthan gum (0.7% of the weight of flour) and dry egg white (5.0% of the weight of flour) provide greater strength, while the passing of dry matters into the cooking water remains lower when compared to other samples – 9.8% and 11.8% of the dry matters respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 178 4. conclusion the results obtained show correlations between moisture of different bond forms in macaroni dough made from corn flour with and without use of different structure forming additives. it may determine both the rheology of dough and mechanism of drying process of macaroni products. on the ground of analysis of sorptiondesorption curves, the sorbtion capability of gluten-free macaroni products made from corn flour with and without addition of structure forming ingredients was determined. the characteristics of microporous structure were shown, as well as their impact both on the energy of sorption and forms of moisture bond. it was proven that the moisture of macro and micro-capillaries is prevalent in corn dough – 39.40 – 54.69% of overall water amount. this amount of moisture bond is bigger in the dough samples with gelatin and dry egg white. the osmotically bound moisture amounts to 18.75-28.04% of the total water amount, being mostly contained by the dough samples with xanthan and gelatin. content of adsorbed bound moisture in dough samples made from corn flour amounts to 18.49 – 23.13% of total water amount, thus its bigger amount being determined in the dough samples with additives of carbohydrates origin. the increase in the moisture amount of macro and microcapillaries and lower content of adsorbed bound water in dough made from corn flour obviously will provide fastening of drying of corn macaroni products. total amount of adsorbed moisture in macaroni products made from corn flour is slightly changed from this index in wheat samples. macaroni samples with extruded corn flour are an exeption, where sorption capability is higher. thus, corn gluten-free macaroni samples contain significantly higher amount of moisture of monomolecular layer – by 1.2 – 1.5 times, and, the energy of sorption is also higher. it may be explained by the microporous structure of gluten-free macaroni products which is characterized by smaller diameter of pores (excepting the samples with extruded flour), since the square of pores is higher. this research might be considered a basis of developing regulatory documentation in view of producing gluten-free macaroni products from corn flour. 5. references [1]. mukoid r. m., yemelyanova n. o., chumakova e. v. gluten. why it can not all? // modern directions of theoretical and applied research: materials of the international scientificpractical conference. – 2013 р. – о. : 2013 – issue 1. – vol. 4. – p. 81-84. (in ukrainian). [2]. skurikhin n. m., volgareva m. n. chemical composition of food products: reference tables for the content of amino acids, fatty acids, vitamins, macroand microelements, organic acids and carbohydrates, // m.: agropromizdat. – book 1. – 1987. – 224 p. (in russian). [3]. marti a., pagani m. a. what can play the role of gluten in gluten free pasta? // trends in food science and technology. – 2013. – vol. 31. – p. 63–71. [4]. patent ep 0792109b1, ipc a23l 1/16 alimentary paste containing indian corn flour / luigi marconato; applicant molino di ferro s. r. l. – no. 95938590.7; stated. 22.11.1995; has published july 29, 1998 [5]. padalino l, mastromatteo m. effects of hydrocolloids on chemical properties and cooking quality of gluten‐free spaghetti // international journal of food science and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 olexandr rozhno, vira yurchak, tatjana golikova, water bond forms in the dough and sorbtion properties of gluten-free macaroni products made from corn flour, food and environment safety, volume xvi, issue 3 –2017, pag. 167179 179 technology. – 2013. – access mode: http://www.researchgate.net/publication/263057900 [6]. rozhno o. v., podobiy o. v., yurchak v. g. the influence of methods of making structurants on the quality of gluten-free pasta // 8th central european congress on food 2016 – food science for well-being (cefood 2016): book of abstracts. – 23-26 may 2016. – k.: nuft, 2016. – p. 143. [7]. rozhno o. v., podobiy o. v., yurchak v. g. research of the rheological properties of gelatine solutions for production of gluten-free pasta // ukrainian food journal, internatinal scientific journal. vol.5. – issue 2. – national university of food technologies. – к.: nuft. – 2016. – p. 265-273. [8]. rozhno o. v., yurchak v. g. creation of gluten-free pasta products will help ensure the population of ukraine with dietary products // bread and confectionery business. – k.: llc «bioprom company» – 2016. – № 10. – p. 27-28. (in ukrainian). [9]. patent 113395 ua, ipc a23l 7/109 (2016.01) method of making pasta products / yurchak v. g., rozhno o. v. – no. u201607670; stated. 07/12/2016; has published jan 25, 2017, bul. no. 2 (in ukrainian). [10]. patent 113396 ua, ipc a23l 7/109 (2016.01) method of making pasta products / yurchak v. g., rozhno o.v. – № u201607672; stated. 07/12/2016; has published jan 25, 2017, bul. no. 2 (in ukrainian). [11]. nazarov n. i. technology of pasta products. // m.: food industry. – 1978 – 287 p. (in russian). [12]. voloshchuk g. i. development of technology of pasta products with pectin and pectin-containing raw materials: author's abstract. dis ... cand. tech sciences: 05.18.01 // ukr. state university of food technologies. – k. – 2000. – 20 p. (in ukrainian). [13]. yurchak v. g. scientific substantiation and development of technology of pasta products of improved quality and preventive purpose through the use of non-traditional raw materials and nutritional additives // dis ... dr. techs. sciences: 05. 18. 01. – k. – 2003. – 336 p. (in ukrainian). [14]. pimenova l. n. thermography: methodological instructions for the discipline «physico-chemical methods of research» // tomsk: publishing house of tomsk. architect-build university. – 2005. – 19 p. (in russian). [15]. greg s., singh k. adsorption, specific surface and porosity // m.: svit. – 1980. – p. 300. (in russian). http://www.researchgate.net/publication/263057900 1. introduction issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvii, issue 4 31 december 2018 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 contents: 1. new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana roxana puscaselu, sonia amariei 512 2. (imidazole-5-yl) ylidenthiazolidones: a new class of antimicrobial agents for food industry vitaliy chornous, alina grozav, victoria antoniychuk, nina yakovychuk, mykhaylo vovk 13-17 3. study of heavy metals effects on in vitro cultures of sedum telephium ssp. maximum l. mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți 18 26 4. calcium requirement in relation to milk fever for high yielding dairy cows: a review angassa tesfaye 27 35 5. quality estimation of frozen desserts with polyfunctional composition olena grek, tatyana osmak, larisa chubenko, artur mykhalevych 36 43 6. the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits sorina ropciuc, cristina damian, ancuța elena prisacaru 44 51 7. research on the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips alina kovtun, vladimir kovbasa, vitaliy pichkur 52 59 8. the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo 60 66 9. author instructions i v 10. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 microsoft word 13 u._kuzmyk_corectat 12 mai.doc 224 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 224 232 development of mathematic model of spiced sour-milk pastas quality viktor goots1, nataliia yushchenko2, *ulyana kuzmyk2 1national research institute of industrial safety and labor protection, kiev, ukraine 2national university of food technologies, kiev, ukraine ukuzmik@gmail.com *corresponding author received 8th march 2018, accepted 27th june 2018 abstract: comprehensive assessment effectiveness and food products quality forecast was substantiated using organoleptic estimation, profiles construction and usage of differential equations. the pastas with spices based on soft diet cottage cheese produced of variable recipes quality was selected as an object of the study.in the pastas composition such spices as allspice, black pepper, ginger, cinnamon, anise, cloves, cardamom, fenugreek and nutmeg were used in amount of 0.27…1.1%. profiles of square area quality and differential equation for solving and analyzing by a computer program with usage of «maple» pack were developed based on mathematical model in order to assess the quality of these dairy products.the analytical research results on product quality were obtained from organoleptic parameters and content of biologically active substances (tannin, catechin, and rutin) with usage of symbolic computer mathematics and graphic images methods. keywords: nutritious system condition, comprehensive evaluation of quality, mathematic model, differential equation, sour-milk pasta, spices 1. introduction nowadays consumer’s demands for food products quality have changed dramatically. food products have to be not only tasty and of appearance attractive, but also safe for consumption and serve as a source of nutritious and biologically active substances. thus, the assessment of food products quality, including dairy products, comes to solving of complex criterial tasks. the question of complex criterial tasks of food products is considered in the works of such scientists as bazarnova, burova, ishevsky, & zyukano, 2004 [1]. the most important product characteristics, which are of interest for consumers, are the following criteria: organoleptic properties, content of the main nutrients (proteins, fats and carbohydrates), biologically active substances, etc. the comparison of the quality indicators of the studied product between the basic sample – the closest to the analogue properties or "ideal" ones gives an opportunity to evaluate effectiveness of the solutions proposed in the scientific research: improvement of recipe composition, introduction of new processing methods, technological parameters refinement, etc. these products have sticky pasty consistency and are intended for direct consumption [2]. the main feature that determines the high nutritional value of the base for pastas – cottage cheese, is the high content of proteins – 10…16 % compared to whole milk (3.2±0.5) % [3]. sour-milk pastas have better protein and fat digestibility compared to similar dairy products produced without fermentation [4]. introduction of natural components to products composition allows additional food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 225 enrichment by a complex of nutrients and biologically active substances [5]. it is known [3], that spices are characterized by high content of biologically active substances; they are used in products’ composition in small amounts, faster food’s digestibility by increasing body’s metabolism, have bactericidal and antioxidant properties, so that they are able to stabilize nutrition products’ quality during storage. combining of natural spices in the new types of sour-milk pastas’ technologies will allow enriching them by a complex of biologically active substances and provide products with original taste and flavoring properties. one of the most important consumer characteristics of sour-milk pastas, as they are products of multiple raw material components is the organoleptic estimation, since organoleptic products characteristics such as appearance, taste, smell, color and consistency provide consumers with general idea of product quality [6]. thus through compilation of recipes of such products complex approach based on sensory analysis methods with the elements of mathematical modeling and analysis of material system state should be applied. 2. materials and methods the study subject is the quality of sour-milk pastas with addition of spices obtained by using different recipes. the subject of research is food products quality profiles, computer program and mathematic models, based on differential equations. the research objective was to develop a method of computer simulation for complex evaluation and forecast quality of sour-milk pastas with spices obtained using different recipes. as sour milk base soft diet fat-extracted soft curd (moisture content 80 %, acidity – 150ºт) was used. spices are to be dispensed in dry ground form with particles size no bigger than 0,4 mm; common salt “extra” (extra class) – in amount 0.4 %. the paste with dry garlic concentrate was used as control one. the compositions include spices as allspice, black pepper, ginger, cinnamon, anise, cloves, cardamom, fenugreek and nutmeg. the desired content of spice compositions was determined – from 0.27 to 1.1 %. to make harmonious products of taste characteristics, an insertion of common salt of 0.4 % is provided. in scientific studies for assessing quality of dairy products identification methods are commonly used. a numerical score is usually included in. it allows setting the level of partial (by individual indicators) or general (by a complex of indicators) quality of the evaluated products and express its numerical value [7]. such approach to products quality evaluation does not always give an objective assessment, therefore a new recipe usage of profiles square area quality and differential equations is proposed to forecast and determine the quality of food products. development and further usage of mathematic models, different in their complexity and functional purpose, allows not only an objective evaluation of the product, but also forecasting the dynamics of a complex indicator of its quality under varying conditions [8]. analytical studies of analysis methods of material system state in determining quality of food products indicate that objective mathematic models shall be considered the ones built on differential equations. they give opportunity to describe a state of a product by main predefined quality indicators, when chemical, biological, structural changes and other various by their nature transformations happen [9]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 226 therefore, the development of computer programs with usage of mathematic modeling for objective evaluation and forecast behavior of nutrition systems under varying conditions is a relevant objective scientific study. the sour-milk pastas recipe composition with spices was developed by authors through previous studies (table 1). sourmilk pastas are produced from soft curd and/or sour cream with addition of flavoring fillers and additives; the pastas are a source of highly digestible complete dairy proteins and they are characterized by a certain content of fats, vitamins, minerals etс. [10]. table 1 the recipes of sour-milk pastas with spices norm, kg for 1000 kg without losses recipe, № raw material basic recipe (control) 1 2 3 4 5 soft diet cottage cheese (fatextracted), moisture content no higher than 80 % 487.0 993.0 993.3 992.25 992.0 992.0 sour cream, fat content no lower than 20 % 500.0 common salt, dry solids weight ratio 99 % 5.0 4.0 4.0 4.0 4.0 4.0 spices: allspice:ginger:cinnamon=1:1:1 3.0 cloves:allspice:ginger=0.8:1:1 2.8 anise:cloves:ginger:black pepper =1:0.8:1:1 3.75 allspice:ginger:cardamom: fenugreek=1:1:0.8:1.2 4.0 anise:ginger:nutmeg:black pepper =1:1:1:1 4.0 dry garlic concentrate 3.0 xanthum gum 5.0 total 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 into a mixing container a calculated amount of soft diet fat-extracted curd cottage cheese, spices and common salt were added. the mixture was stirred for (5±1) min until homogenous consistency was obtained. after that, the paste was packed in 100 g glass jars, which were covered by lids and placed in a refrigerator, where the paste cooled down to a temperature (4±2)ºс. for analysis of quality of sour-milk pastas with spices compositions, the authors have proposed to apply a mathematic model in the form of a differential equation [1, 9]: 0)( )(  ncky dc cdy , (1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 227 where у – general index of product quality (numerical score); с – estimated variable characteristic functionally linked to a product recipe; n – ratio of area of product quality profile polygon, taken for the ideal area of product quality profile of each of the studied products; k – area of quality profile of ideal product at the selected coding level for individual metrics. equation (1) contains main indicators of sour-milk pastas quality. in order to obtain an adequate result usage of the equation (1) for sour-milk pastas quality forecast by determining the value of the general index was proposed. comprehensive evaluation was made by 6 indicators (descriptors), using a 30-point rating scale. nomenclature of quality indicators and coefficients of significance of each indicator were defined by an expert commission taking into account their importance for consumer’s assessment of sour-milk pastas with spices. determination of tannin, rutin, catechin content in spices was made by water suspension titration method chopped to particle size no bigger than 0.4 mm of spices by solution кмno4 with concentration0.1 mole /dm3. the content of the relevant biologically active substances in sour-milk pastas with spices was defined by computational method taking into account their recipe composition. to determine general index of sour-milk pastas with spices quality and forecast quality of the same pastas, but prepared by using different recipes, a computer program was developed. it allows determining the general index of quality under organoleptic (assessment in points) or natural estimation of individual indicators, when their numerical values are brought to dimensionless view. the computer program allows: calculating areas of individual of so called quality profiles, which may be obtained both for specific sour-milk pastas recipes (recipe-1, recipe-2, recipe-3, recipe-4, recipe-5), and for individual quality indicators (consistence, taste, scent, color, appearance etc.). using a system of computer mathematics “maple”, for differential equations solving we calculated the value of general index of product quality under selected mathematic model in a form of first order differential equation, perform its analytical research and we presented the results graphically [11]. 3. results and discussion the analysis was made and sequence of mathematic model (1) usage in pack with computer program “maple”, of sour-milk pastas quality analysis was considered. the equation (1) solving in a generic form will be: (2) the constant of integration с1 we shall find having accepted the initial condition, which corresponds to the maximum point value of the general index of quality of conditionally ideal product. the initial condition needed for solving of differential equation in an analytical form will be: y(1)=y1, for different dairy products у1=5; 15; 30 is accepted. then we shall write down the final equation (2) solving: (3) the equation (2) is multipurpose. when it is necessary to make a comparative analysis of product quality not only to ideal, but also to, for example, control sample, or between samples, different values of coefficient k are selected, which equals area of quality profile of selected sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 228 we shall consider the example sour-milk pastas obtained on the bases of cottage cheese (table 1) sequence of determination and analysis of general index of quality as compared with conditionally ideal product. by using single point coding of quality organoleptic estimation results the following indicators were obtained: consistence, color, scent, taste, appearance, content of biologically active substances; the results are shown in table 2. table 2 the results of organoleptic estimation of sour-milk pastas with spices indicator ideal control recipe-1 recipe-2 recipe-3 recipe-4 recipe-5 у1consistence 1 1 1 0.95 0.92 1 0.9 у2-color 1 0.8 0.9 0.95 0.91 0.9 0.9 у3-scent 1 0.9 0.95 0.85 0.89 0.92 0.82 у4-taste 1 0.85 0.9 0.92 0.9 0.85 0.9 у5appearance 1 0.84 0.91 0.9 0.91 0.92 0.92 у6cbas 1 0.6 0.92 1 0.94 0.91 0.89 the results of organoleptic estimation of products in coded form transfer into interface pattern of the computer program (table 3). it is obligatory to fill in boxes “maximum score” and “amount of indicators”.then, one should pass the mouse to the box “calculate areas” and give a command “calculate”. table 3 an interface pattern of the computer program with inserted experimental data indicator ideal control recipe-1 recipe-2 recipe-3 recipe-4 recipe-5 consistence 1 1 1 0.95 0.92 1 0.9 color 1 0.8 0.9 0.95 0.91 0.9 0.9 scent 1 0.9 0.95 0.85 0.89 0.92 0.82 taste 1 0.85 0.9 0.92 0.9 0.85 0.9 appearance 1 0.84 0.91 0.9 0.91 0.92 0.92 cbas 1 0.6 0.92 1 0.94 0.91 0.89 s1 s2 s3 s4 s5 s6 s7 s k 2.60 1.78 2.25 2.24 2.16 2.18 2.05 2.60 ideal control recipe -1 recipe -2 recipe -3 recipe -4 recipe -5 si/s 1.00 0.68 0.86 0.86 0.83 0.84 0.79 c s/si 1 1.46 1.16 1.16 1.2 1.19 1.27 n maximum score 1 amount of indicators 6 at the end we shall receive k, n, c of equation. they are needed to complete the calculations for differential equation (2). the program calculates the polygons area. s1=2.60; s2=1.78; s3=2.25; s4=2.24; s5=2.16; s6=2.18; s7=2.05 and values of characteristics k=2.60; c=1; c0=0.68; c1=0.86; c2=0.86; c3=0.83; c4=0.84; c5=0.79 та n=1; n0=1.46; n1=1.16; n2=1.16; n3=1.2; n4=1.19; n5=1.27. the automatic mode displays graphic images on the monitor. the products’ quality profiles by organoleptic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 229 characteristics: consistence, color, taste, scent, appearance, content of biologically active substances are shown in the figure 1, and the quality profiles of products conditionally ideal, controled and obtained by using different recipes are shown in the figure 2. fig. 1. products quality profiles by organoleptic characteristics and content of biologically active substances fig. 2. quality profiles of products conditionally ideal, control and obtained using different recipes the developed computer program also allows, besides quality profiles, which characterize the quality of the product according to area of polygon, comparative assessment card of quality of conditionally ideal, control and obtained using different recipes products (figure 3). the results of calculations allow visual performing of a comparative analysis of product quality obtained by using different recipes as compared to conditionally ideal, control and between themselves by the values of polygon areas (s1, s2…s7) and through the card analysis (figure 3). with the help of this program it is possible to calculate areas of quality profiles of sour-milk pastas obtained using different recipes at different point scores, at 1, which is used in technology of dairy products. when comparing sour-milk pastas obtained using different recipes to conditionally ideal product we adopt: k=2.6, to compare to control k=1.78. where k – area of polygon with single coding, у1=5 – indicator of ideal product quality with selected fivepoint score. these values are automatically entered as fixed into the corresponding program line of the “maple” system, which was used for solving and analyzing differential equation (1). we shall consider an example of practical use of the mathematical model analysis program for analysis of sour-milk pastas with spices quality using a 30-point evaluation system, when k=2.60; y1=30; с=0.86; n=1.16. having used an equation, we shall find a value of general index of quality. it equals у=16.5. the result was obtained after substitution of data into the equation, derivatives k; y1; c; n. (4) the calculations by using a mathematic model (1) values of general index of quality with different point scores (у1=30; 15; 5) are listed in the table 4. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 230 0 0,2 0,4 0,6 0,8 1 ideal control recipe -1 recipe -2 recipe -3 recipe -4 recipe -5 si/s fig. 3. comparative assessment card of sour-milk pastas quality obtained using different recipes and conditionally ideal product table 4 values of general index of quality with different point scores (5; 15; 30) of sour-milk pastas quality (c; n; y1 – characteristic of the mathematic model of the product quality calculation) characteristics m.m.* ideal control recipe-1 recipe-2 recipe-3 recipe-4 recipe-5 c 1 0.68 0.86 0.86 0.83 0.84 0.79 n 1 1.46 1.16 1.16 1.20 1.19 1.27 у1=30 3.13 16.50 16.50 13.42 14.32 9.36 у1=15 2.38 8.80 8.80 7.40 7.80 5.58 у (с) у1=5 1.39 3.18 3.18 2.80 2.9 2.34 *m.m. – mathematic model the positive side of the food products quality modeling program is that if one needs to use other levels of point evaluation, the program gives an opportunity by changing the y1 parameter, get value of general index у (c) product quality and build 3d cards. visually the results of calculations in the range of change n=1…1.5; c=1…0,5 using a 30point evaluation system of product quality are presented graphically in the figure 4. the use of various levels of point evaluation of dairy products quality is pictorially presented in figures 5-6. fig. 4. 3d card of dependence of general index of quality y(c) using a 30-point evaluation system of product quality food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 231 when using a 15-point evaluation system of product quality in figure 5. fig. 5. 3d card of dependence of general index of quality y(c) using a 15-point evaluation system when using a 5-point evaluation system of product quality on figure 6. fig. 6. 3d card of dependence of general index of quality y(c) using a 5-point evaluation system analysis of graphic dependencies shown in figure 4-6 testifies that the smaller the area of quality profile of sour-milk pastas is, the lower point score on general indexes of quality the product has. with a decrease of value of the characteristic n of the mathematic model and accordingly an increase in the characteristic с , the product quality improves. 4. conclusion the proposed computer program allows determining and forecasting quality of sour-milk pastas with spices obtained by using different recipes based on their organoleptic estimation, quality profiles and usage of differential equations. by using it we can build 3d cards and visually conduct analytical researches of product quality, forecast its quality for different content of biologically active substances, develop new products by improving both their recipes and technological modes of production. the suitability of the computer program and its adequacy is confirmed by the development of new types of sour-milk pastas with spices. 5. references [1]. bazarnova y., burova t., ishevsky a., zyukanov v. (2004). possibilities of applying the principles of chemical kinetics to assessment of quality of nutrition products. storage and processing of agricultural raw materials, 11, 33-36. [2]. gulich, m. (2011). rational nutrition and a healthy lifestyle – the main factors of preserving health of the population. the problems of aging and longevity, 2, 128-132. [3]. young w. park. (2009). bioactive components in milk and dairy products. singapore, 439. [4]. perecichniy, m., palamarek, k. (2013). mineral and vitamin composition of cheesevegetable pastes with high iodine content. goods and markets, 2, 125-131. [5] du, j., cullen, j., buettner g. (2012). ascorbic acid: chemistry, biology and the treatment of cancer. biochim biophys acta, 20, 443–457. [6]. mardar m. (2012). sensory analysis of extruded products enriched with meat components. food science and technology, 12, 57-60. [7]. kanther v., matison v., fomenko m. (2002). consumer esstimation of products – the most important component of marketing research. meat industry, 8, 11-13. [8]. chugunova o., zavorokhina n. (2010). the usage of tasting analysis methods in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 viktor goots, nataliia yushchenko, ulyana kuzmyk, development of mathematic model of spiced sour-milk pastas quality, food and environment safety, volume xvii, issue 2 – 2018, pag. 224 – 232 232 modeling of food recipes with specified consumer properties, 148. [9]. lipatov n., gevorgyan a. (2003). organismic approaches to the assessment of food production facilities. magazine dairy industry, № 3(6), 24-27. [10] yushchenko, n., mуkoliv, i., kuzmyk, u. (2016). development of natural spices compositions for dairy products, 8th central european congress on food «food science for well-being». kyiv: nuft. [11]. dyakonov v. (2006). maple 9.5/10 in mathematics, physics and education, 720. – m.: solon-press. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 105 near infrared spectroscopy – an alternative to determine the crude fiber content of forages monica hărmănescu1, alexandru moisuc1, iosif gergen2 1 banat’s university of agricultural sciences and veterinary medicine, faculty of agriculture, timisoara, calea aradului nr. 119, ro-300645, romania, monicaharmanescu@yahoo.com 2 banat’s university of agricultural sciences and veterinary medicine, faculty of food technology, timisoara, calea aradului nr. 119, ro-300645, romania. abstract: in our days nir spectroscopy represent a promising alternative to the chemical methods for crude fiber contents of forages. the main objective of this study was to obtain a nir calibration model for prediction this parameter of forages harvested in june 2009 from hill permanent grassland (grădinari, caraş-severin district). the experimental field was organized in ten experimental trials fertilized organic, mineral, and organo-mineral. the floristic composition of forages from this period was determined gravimetrically. from poaceae were present festuca rupicola and calamagrostis epigejos. fabaceae family was represented by trifolium repens and lathyrus pratensis. from other botanical family: rosa canina, filipendula vulgaris, galium verum and inula britanica. like input data for nir calibration were used the results for this qualitative parameter by chemical method and the reflectance values from 150 nir spectra for all analysed samples. partial last square (pls) regression was used to obtain the “nir total fiber” model, implemented in panorama program (version 3, labcognition, 2009). the statistical parameters (r2=0.80; rmsec=2.73) and the differences between references and predicted values situated in range 0.03 and 9.24% suggest a medium quality of calibration model, but it is promising to use it to predict the crude fiber contents of forages from grassland in this period of year using higher number of samples for calibration. key words: forages quality, complex fertilizers, plsnir model, grassland. introduction since romania becomes part of european union the national authorities must adjust the legislation to those european also regarding the quality and safety of the food. the 150/2004 low from our country transpose partially the ce 178/2002 rule, which establish the most important recommendations on the qualities of row matter, from vegetal and animal origin, destined to obtain the food [8]. the security and quality of food with animal’s origin must be discussed in direct correlation with the requests for forages quality used like animal’s feed. the forages from permanent grassland represent the cheaper source of feed for ruminants. fiber content of these forages, alongside of protein, lipids, minerals and vitamins, represents one of the most important parameter which characterize the forages quality [3]. ruminants have the capacity to digest and use crude fiber like source of nutrients [2]. for example the cellulose content of forages must be in their ratio between 2325% [1], representing the key in intestinal transit, in stimulation of satiety sensation [2], like energetic source in animal’s metabolism [6]. the chemically determination of this parameter request a high reagents consumption, qualified human resources to perform the operation, a long time to obtain the results [7]. an important food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 106 alternative for these chemical models is represented in our days by nir spectrometry, a non-destructive method which allows obtaining very fast the final results (appreciatively 3 minutes), without reagents consumption, medium pollution and samples destroying [4, 5]. experimental the forages were harvested in june 2009 from hill permanent grassland (grădinari, caraş-severin district). the soil of permanent grassland was calcic luvisol and the annual average temperature around 10.4oc. the experimental field was organized in ten fertilized trials in randomized plots, in multiple stage blocks with five replications. it was used for mineral fertilization: 15:15:15 npk complex, ammonium nitrate, potassium salt, superphosphat). like organic fertilizer was chose fermented sheep manure. the fermented sheep manure was applied at each two years, even the mineral fertilizers yearly. the fertilization process was made during the period 2003-2008. the ten trials were: v1-unfertilized trial, v2-20 t/ha sheep manure, v3-40 t/ha sheep manure, v4-60t/ha sheep manure, v5-20 t/ha sheep manure + 50p2o5(kg/ha), v6-20 t/ha sheep manure + 50p2o5 (kg/ha) + 50 k2o (kg/ha), v720 t/ha sheep manure + 50 p2o5 (kg/ha) + 50 k2o (kg/ha) + 50n(kg/ha), v8-100 n (kg/ha) + 50 p2o5 (kg/ha) + 50 k2o (kg/ha), v9-150 n (kg/ha) + 50p2o5(kg/ha) + 50 k2o (kg/ha), v10 (100+100)n (kg/ha) + 50 p2o5 (kg/ha) + 50k2o(kg/ha). the floristic composition of forages from the ten trials for this period of year was determined gravimetrically. from poaceae familly dominant was festuca rupicola (varied between 16.00 – 52.00%), followed by calamagrostis epigejos (5.00-13.00%). fabaceae family was represented mainly by trifolium repens (dominant) and lathyrus pratensis. from other botanical family were present rosa canina (7.0018.00%), filipendula vulgaris (3.009.00%), galium verum (3.00-7.00%) and inula britanica (5.00%). nir calibration model was obtained by pls (partial last square) regression, implemented in panorama software (variant 3, labcognition, 2009). like input data were selected the chemical data for crude fiber content, determined by jaoac 962.09/1990 [7] method (samples are sequentially refluxed in dilute base followed by dilute acid), and the reflectance values from 150 nir spectra. the v670 spectrophotometer by abblejasco was the instrument used to scan the spectra in the range 800-2500 nm, and than was selected with panorama software three spectral ranges favourable to perform the calibration model for crude fiber determination. these spectral ranges were specific for the overtones of fundamental frequencies of oh bound, characteristic for fiber compounds (table 1). table 1. calibration data for the „nir-cf” model with 3 spectral ranges no. selected spectral ranges number of wavelengths 1 [1282.5 .. 1433.5] 303 2 [1542.0 .. 1949.0] 815 3 [2263.0 .. 2356.0] 187 „nir-cf” nir-crude fiber model food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 107 for all the grounded dried samples the chemical results and nir spectra were obtained in triplicate. results and discussion statistical parameters for “nir-cf” model with the three selected spectral ranges are presented in table 2: table 2. statistical parameters for „nir-cf” model with three selected spectral ranges r2 0.80 rmsec 2.73 sd 4.75 these parameters suggest a medium quality of “nir-cf” model, but were better than the case when were used the entire spectral domain (r2 = 0.7355, rmsec = 3.28, sd = 4.39). the quality of nir calibration model is underlined also in the graphical presentation of prediction for crude fiber by „nir-cf” model with three spectral ranges (figure 1) and also by the differences between the chemical results and those predicted for control samples. the control samples were harvested in the same period of year and grassland and conditioned in the same manner with those used to perform the „nir-cf” calibration model (table 3). table 3. the results of crude fiber (%) prediction for the control samples forages (june2009) by „nircf” calibration model with 3 spectral ranges crude fiber (%) control sample’s name real (chemical method) predicted (nir model) differences between real predicted 101a 32.10 34.11 -2.01 101b 43.99 34.75 9.24 101c 18.85 24.24 -5.39 101d 31.06 26.92 4.14 102a 34.57 34.38 0.19 102b 24.89 27.08 -2.19 102c 25.46 23.27 2.19 102d 28.25 26.31 1.94 103a 36.45 36.35 0.10 103b 32.08 32.28 -0.20 103c 21.07 23.42 -2.35 103d 28.22 28.68 -0.46 104a 38.85 34.97 3.88 104b 32.98 30.26 2.72 104c 20.72 23.36 -2.64 104d 30.82 26.35 4.47 105a 35.17 34.70 0.47 105b 20.73 29.55 -8.82 105c 24.33 25.05 -0.72 105d 32.08 30.91 1.17 106a 33.01 33.91 -0.90 106b 22.60 27.64 -5.04 106c 24.74 23.06 1.68 106d 31.02 26.77 4.25 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 108 107a 36.41 36.30 0.11 107b 29.78 31.26 -1.48 107c 20.03 24.18 -4.15 107d 31.62 29.87 1.75 108a 36.44 37.69 -1.25 108c 22.42 24.18 -1.76 108d 29.55 30.21 -0.66 109a 33.75 38.03 -4.28 109b 28.22 30.83 -2.61 109c 24.37 24.34 0.03 109d 36.07 35.46 0.61 figure 1. prediction of crude fiber by the „nir-cf” model with 3 selected spectral ranges the differences between values obtained by chemical method and those predicted by „nir-cf” model were situated between 0.03 and 9.24%. almost 31.43% from these values were under 1.00%; 40.00% in range 1.01-3.00%; 17.14% between 3.015.00%; and 11.43% in range 5.01-9.24%. these results indicate a medium quality of performed calibration model, but encourage us to continue these researches using a high number of samples to characterize better the concentrations of this qualitative parameter of forages from the permanent grassland conclusion the pls regression model “nir-cf” with three selected spectral ranges for the determination of crude fiber of forages harvested in june 2009 had a medium quality. but this model promised to be used with success to determine routinely this parameter for the samples harvested in this period of year from the permanent grassland after the enrichment with a higher number of forages samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 109 acknowledgement the authors are grateful to cncsis uefiscsu (romania) for financial support (pd project / 28 sept.2010: on the applications of spectroscopic and chromatographic methods to establish the effects pf fertilisation on the quality of forages from grasslands). references 1. drinceanu, d., 1994, alimentatia animalelor, ed. eurobit, p: 16-17. 2. georgescu, gh., mărginean, gh., petcu, m., 2007, cartea producătorului şi procesatorului de lapte, vol. 2, editura ceres, bucureşti, isbn: 978-973-40-0773-8. 3. linskens h.f, jackson j.f., 1989, plant fibers. modern methods of plant analysis, vol. 10, springer-verlag berlin heidelberg, germania. 4. mcclure w.f., 1992, making light work: advances in near-infrared spectroscopy, eds. murray i., and cowe i.a., vch publishers, new york, p.4-7. 5. sugiyama j., mcclure w.f., hana m., 1992, in advances in near-infrared spectroscopy, eds. murray i., and cowe i.a., vch publishers, new york, p.61-66. 6. van soest, p. j., 1994, nutritional ecology of the ruminant, 2nd ed. cornell university press, ithaca, ny. 7. *** jaoac official methods of analysis, 1990, 962.09 – fiber (crude) in animal feed. ceramic fiber filter method, edited by herlich kenneth, 15 edition, publshed y association of official analytical chemists, arlington, virginia, sua. 8. www.immromania.ro 20 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue xvii2018, pag. 20 31 pork supply chain, consumption and risk factors for infections of consumers in the abidjan district (côte d'ivoire) * koua atobla 1 , edwige essoma akoa 1 , carole aya bonny 1 , adjehi dadié 2 , germain tago karou 1 , sebastien niamke 1 1laboratory of biotechnologies, department of research and training unit of biosciences, félix houphouët-boigny university, cocody, abidjan, côte d'ivoire, 22 bp 582 abidjan 22, atobla@yahoo.fr 2laboratory of biotechnology and microbiology of foods, department of foods sciences and technologies, nangui abrogoua university, abobo, abidjan, 02 bp 802 abidjan 02, côte d'ivoire *corresponding author received 9th october 2017, 23th march 2018 abstract: the ban on the consumption of game throughout côte d'ivoire by the emergence of ebola haemorrhagic fever prompted consumers to turn to other sources of animal protein, such as pork. there is more frequent consumption of pork in more urban areas. it is in this context that a transversal and retrospective survey was performed in 12 communes of the abidjan district with 3,200 people in order to assess pork supply chain, the level of consumption and environmental infection risks of consumers. pork meat and pork products supply chain for marketing were revealed. of the 3,200 people surveyed, 2,400 (75%) reported being consumers of pork at home and out-ofhome. pork was consumed smoked, fried and fresh cooked in sauce at home. thus, male (63.6%) and female (44.9%) have a preference for smoked pork at home. out-of-home, baked pork was the most consumed by women (41.3%). consumers reported some infections of pork after consumption. symptoms such as diarrhea was more common in men (47.9%) followed by abdominal pain (22.8%), fever (21.1%) and vomiting (2.5%). thus, the conditions of transport, hygiene and the mode of consumption and handling of pork observed during the investigation could constitute risk factors for infections in the abidjan district. therefore, good hygiene practices in the processing of pork from farm to slaughter, transport, marketing and consumption can decrease the risk of infection. so, the best means of preventing the risk for pork consumers remains the compliance with good hygiene practices. keywords: pork consumption, supply, environmental infection risks, good hygiene, public health. 1. introduction pork is the most widely eaten meat in the world, accounting for over 36% of the world meat intake, followed by poultry and beef, with about 35% and 22%, respectively [1]. pork imports and exports in each country are now part of international trade rather than simply domestic pork supply and demand [2]. pork, despite religious considerations and taboos, is one of the most widely consumed meats in the world in terms of its cost and on the other hand, to its nutritive, taste and organoleptic qualities. unfortunately, pork can be the source of thirty-five biological hazards to humans [3]. this obligation especially concerns foodborne zoonotic hazards transmitted to humans through pork consumption, such as thermophilic campylobacter spp., listeria monocytogenes, salmonella enterica and yersinia enterocolitica [4, 5]. although pork is still only about 10% of http://www.fia.usv.ro/fiajournal mailto:atobla@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 21 the meat consumed in africa, the accelerated urbanization of the past two decades has given new impetus to its production. indeed in 2005, 800000 tons of pork were consumed, compared with 500000 tons in 1990 [6]. in côte d'ivoire, pork consumption is increasing in the face of the high cost of other protein sources (beef, sheep meat, chicken meat, fish, etc.) on the market. moreover, with the emergence of haemorrhagic fever in ebola leading to a ban on the consumption of bushmeat throughout côte d'ivoire, the need for consumers to look for other sources of animal protein is necessary [7], thus increasing pork consumption. a risk model is built that covers the food pathway from farm to fork comprising six modules as the model developed by [8]. the six modules are: (1) primary production, (2) transport and lairage, (3) slaughterhouse, (4) post-processing, (5) distribution and storage and (6) preparation and consumption [8]. slaughtering of pigs remains an open process with many possibilities for contamination of the carcass [9]. among foodborne infectious diseases, foodborne zoonoses are a public health problem. these biological hazards present in the food, which may have an adverse effect on health, are most often associated with inapparent carriage in animals [10]. insufficient or non-existent information on the supply of pork sales outlets does not allow traceability of the production chain to consumers. in addition, understanding the supply chain for pork will identify the areas to be promoted in order to increase the availability and accessibility of this meat. in this context, the objective of this study is to assess pork supply chain, practices surrounding pork consumption and to estimate the environmental infection factors associated with pork consumption in côte d'ivoire. 2. materials and methods 2.1. study area the study took place from june 2015 to november 2015 in abidjan district. a survey was carried out to describe the attitudes, practices surrounding consumption of pork, the supply and marketing system for pork. within this transversal study and retrospective survey took place in 12 communes in abidjan district, namely yopougon, cocody, treichville, adjamé, port-bouët, abobo, marcory, attecoubé, plateau, koumassi, bingerville and anyama (figure 1). the areas have been chosen because abidjan district cities are an important hub with regard to pork marketing and its supply with large quantities of pig carcasses from surrounding cities and also, the existence of public markets for pork and the presence of many restaurants specialized in pork consumption. 2.2. data collection 2.2.1. estimation of sample size the sample size was determined by the following formula [11]:   2 96,1 1*         e pp n with n, sample size required; proportion of people surveyed in abidjan district p = 50%; and e margin of error at 5%; 1,96 at a confidence level of 95%. in this study, n represents the proportion of people surveyed consuming pork or not. given the lack of data on the level of pork consumption in côte d'ivoire, the prevalence of 50% was retained. the minimum size of surveyed required, estimated from this formula is 384. however, in order to have a representative food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 22 sample size and maximize the finding of more consumers, the sample size within the framework of this study was finally set at 3,200 for all the twelve (12) communes. fig. 1. map of abidjan district 2.2.2. conducting the survey methods of collecting data were face-toface interviews using standardized questionnaires (written surveys) for both to consumers or non-consumers. written surveys allowed asking questions to consumers or not. the same questionnaire was used for both consumers and nonconsumers. the survey was conducted at home (in households) and out-of-home (in streets and market) in 12 communes in abidjan district. in the selected sites, the people predisposed whether they were consumers or not, were randomly questioned. at home, interviews were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 23 carried out with the people in charge of cooking the family meal.the questionnaire focused on themes, namely the place of supply, eating habits of pork consumption, frequency of consumption, and diseases related to pork consumption. sociodemographic information such as age, gender and level of education were also collected. in addition, sales and consumption practices were observed during the survey. in order to determine pork supply, marketing and to investigate the factors that may lead to contamination of pork, a survey was carried out among breeders, at the slaughterhouse and meat sellers of pigs in the markets of each selected commune. the questionnaire survey was coupled with direct observation on the selected sites. the main objective of this survey was to determine the places of supply, distribution, means of transport, storage of pork and environmental infection factors. 2.3. data analysis data collected was analyzed using the statistical package for social sciences (spss) version 20.0 (ibm corporation) software to constitute the database. the descriptive statistical methods (frequency, mean) were used for the quantitative variables. the chi-square test was used to test the relationships between the variables. the difference between the variables was considered significant at p < 0,05. 3. results and discussion 3.1. pork marketing and supply chain pork supply and marketing are partly informal. the breeding pigs are slaughtered either directly in the farming areas (farms) or transported to be slaughtered at the slaughterhouse of sivac (ivorian society of slaughter and pork butchery). pigs slaughtered in slaughterhouses by specialized agents are distributed by refrigerated trucks in swine processing factories and distributed to supermarkets in respect of the cold chain. pork is also distributed on the wholesale and semi-wholesale market or in areas for the sale of pork (figure 2). pigs from traditional farms are slaughtered at the slaughterhouse or on the farm by breeders (sales slaughterers) who ensure their distribution on markets or in areas equipped for sale, without respect the cold chain. pork is carried by means of load carriers ("rickshaws", wheelbarrows) or vehicles (motorcycle, taxi, tarpaulin, truck, etc.). pork is subject to many sources of contamination (environmental contamination, cross-contamination, etc.) related to the types of transport. pork products are then stored and transported, frozen or chilled to wholesale, retail and catering facilities for ultimate sale to consumers. in addition, some breeders sell pork directly to sellers or consumers. pork is consumed smoked, fresh or fried at home or out of home in the form of baked pork, braised, minced pork (figure 2). the inadequacy or lack of information on the supply of pork sales outlets does not allow traceability of the production chain to consumers. in addition, understanding the supply chain for pork will identify the areas to be promoted in order to increase the availability and accessibility of pork. while the food choices reflect the preferences and eating habits of the population, the composition of a meal also relies on the availability and accessibility of food sources. the ivorian pig sector is supported by a dense marketing network, with a large number of farms concentrated mainly on abidjan district areas but also inside the country. with the advent of ebola and the ban on game consumption throughout the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 24 country, consumers would be looking for new sources of protein [8], leading to an increase in pork consumption. as a result, the quantitative and qualitative improvement in production could increase pork supply. the intensification of production through the creation of farms appears as an option to improve pork supply. the production-to-marketing route is partly informal. as a result, some breeders sell pork directly to consumers and sellers. also in abidjan district, the cold chain is not always respected when transporting pork meat, especially after traditional slaughter. pork is transported either in refrigerated trucks for industrial slaughter for supermarkets or in taxis, sheeted boards or even in load-carriers called "rickshaws" for traditional slaughter for local markets. similary reference [12] reported in a study conducted in chad that most purchases of live pigs are made directly from producers due to the absence of a physical market. pork is transported by vehicle (taxi, tarpaulin, truck etc.). it has been found that transport of pigs from production areas to consumption places is carried out by means of load-carriers, bicycles and mopeds when distances are low and in vehicles when they are far away [13, 14]. moreover, the reference [14] showed that production in côte d'ivoire is dominated by modern livestock farming, which is aimed at profitability, compared to the traditional breeding farms in the country whose production is generally intended for self-consumption, saving. 3.2. pork consumption of the 3200 people surveyed, 2400 said they were consumers of pork, i.e. pork consumption rate of 75% in abidjandistrict and 800 (25%) were nonconsumers. the global size of people survey to be investigated was distributed evenly communes selected, resulting in 200 consumers per commune. according to the data collected, 1554 (48,6%) consume pork at home. on the other hand, 846 (26,4%) respondents consume or prefer to consume this meat out-of-home (table 1). table 1. distribution of consumers and non-consumers of pork surveyed in abidjan district consumption locations effective (n) rate (%) consumers home 1554 48,6 out-of-home 846 26,4 nonconsumers 800 25 total 3200 100 for the rest of the results, only those who consumed pork were taken into account (n= 2400). in côte d'ivoire, studies on the consumption of foodstuffs are generally rare. however, they allow us to better understand the uses of these food products and the environment of consumption whether at home or in collective catering. emergence of ebola virus disease (evd) in west africa in 2014 has led to a ban and stigmatization of this essential food source in rural zones. the prohibition of game consumption throughout the extent of ivorian territory due to the occurrence of ebola hemorrhagic fever has incited consumers to turn toward other sources of animal protein [8], favoring an increase in pork consumption. the survey conducted in this study revealed that three quarters (75%) of the population surveyed in abidjan district consume pork. this consumption in abidjan district is higher than that of pork in africa which was 8.9% in 1997 reported by [15]. indeed, following the ban on game consumption, people were increasingly interested in pork consumption because of its organoleptic quality. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 25 fig.2. pigs and pork supply chain, marketing in côte d'ivoire and indication of potential contamination sites factory (cutting, packing, processing) pig breeding (standing pigs), farm modern breeding of pigs traditional pig breeding (farm or walking pig) pig slaughterhouses modern slaughter traditional or clandestine slaughter modern transport (refrigerated truck) of pork traditional transport (motorcycle, taxi, truck, etc.) of pork market (fresh meat, smoked pork) respect of the cold chain: freezing refrigeration (storage, conservation) space for the sale of pork, butchery (wholesale, semiwholesale and retail or distributor) supermarket (sausage, ham, pâté, etc.) consumption out-of-home (maquis, restaurant, street): baked pork, braised, minced pork consumption at home (households): smoked pork, fresh, fried in sauce non-compliance with the cold chain: ambient temperature, under ice, refrigeration breeding consumers storage, butchers, market slaughter and transport environmental contamination carcass contamination by faeces and offal or by instruments or environmental environmental contamination or man-made contanination environmental or crosscontamination food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 26 3.3. pork consumption frequency housewives typically consume pork twice (55,1%) a day, with the majority of staff consuming once (79,9%) in the day after purchasing prepared or cooked fresh pork (figure 3). the social classes that consume the most pork are in particular housewives, traders, artisans, school pupils and students. the statistical analysis shows that there is a significant difference between daily consumption frequencies of socioprofessional categories (p = 0,02<0,05). the preferential choice of pork among other types of meat by modest socioeconomic categories was previously mentioned by [16]. indeed, this author has shown that households with the lowest incomes and the rural ones buy and consume more pork than other meats. in addition, he noted that pork would have strengths making it popular with consumers and giving it a place of choice in daily food [17]. other authors have pointed out that pork enjoys a good reputation because of its affordable price, its ease of preparation, its many possibilities of accommodation due to its not too pronounced taste and its role as a component of traditional eating habits in rural areas [18, 19]. also, pork formed part of the staple diet in many households and was held in high regard in terms of its versatility, cost effectiveness and taste. 3.4. pork consumption at home 3.4.1. consumption forms of pork at home, pork was preferentially consumed in sauce in three forms (fresh, smoked and fried). males (63,6%) and females (44,9%) have a preference for smoked pork. females with a frequency of 29,1% consume more fresh pork compared to males (17,3%) (figure 3). fig. 3. pork consumption frequency by socioprofessional categories the chi-square test revealed a significant difference between the forms of pork consumption by both genders (chi-square = 137858a, p= 0,000) (figure 4). consequently, the gender of the respondents influences the forms of pork consumption. fig. 4. different forms of pork consumption at home the study also reveals that at home, pork is consumed in sauce especially in three forms (fresh, smoked and fried). males (63,6%) and females (44,9%) have a preference for smoked pork at home. previous studies have justified this preference. according to references [20] and [21], smoking reduces the high fat content, partially masks the often unpleasant smell of fresh pork meat; which contributes to improving the nutritional food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 27 and organoleptic characteristics of the cooked product. also, pork was consumed for several reasons, including for breakfast, lunch, dinner, snack and for emotional reasons. it was perceived to be a comfort food, also consumed to cure a hangover [22]. 3.5. pork consumption out-of-home in general, females (31,1%) and males (23,6%) prefer to consume baked pork outside their home. in addition, other forms of pork consumption have been reported. for males, the consumption of skewer is 7,5% and consumption of offal soup by females is 7,4% (figure 5). fig. 5. distribution of pork products consumed out-of-home on the other hand, out-of-home, pork consumption in a baked form is most appreciated by both women and men. this consumer preference out-of-home can be explained by the greater availability and convenience of consuming meat in this form. indeed, cooking preparation in sauce is relatively difficult, requires a variety of logistics and ingredients that are not always easy to bring together in an out-ofhome environment. in addition, there is a convenience of service, consumption of the baked pork and its association with the attiéké (grated cassava) or alloco, menus entering the eating habits in urban environment outside the homes and sometimes at home. in addition, the presentation of the baked meat is similar to the barbecue, which leads to a product locally called choukouya often consumed during the ceremonies of rejoicing (marriage, baptism, etc.) in côte d'ivoire. the forms of consumption encountered in n'djamena in chad are similar to those in abidjan district. indeed, grills and soups are most often consumed outside the home while sauces are consumed in the home. meat is also used for ceremonies and friendly and family meetings [12]. survey revealed that pork was consumed at each major eating occasion, for breakfast, lunch and dinner. additionally, main consumption of pork takes place during feast times [22]. growth of pathogenic bacteria can occur if the cold chain is not maintained during transport to the home. raw meat should be packed in separate bags or containers away from other foods, particularly ready-to-eat foods, to avoid potential crosscontamination. the use of insulated bags or freezer bags is recommended during transportation. food should be refrigerated, cooked or frozen as soon as possible following purchase. 3.6. mean time of pork cooking pork is cooked mostly in the range of 31 minutes to 1 hour in both females (56%) and males (45,1%). meanwhile, 44,7% of males cooked meat less than 30 minutes, compared to 43,8% of females (figure 6). data received from housewives and out-ofhome salesmen revealed that they generally cook pork in sauce to avoid exposing their family and client to possible infections related to insufficient cooking. however, cross-contamination after cooking would be the main risk factor for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 28 the transmission of disease to the consumer as reported previously by [23] and [24]. fig. 6. mean cooking time of pork by gender of consumers compliance with hygiene rules of good practice would be a prerequisite for reducing the risk of contamination of zoonosis and parasitic infestations by pork consumption. thus, the need to inform and train the actors of the pig farms, vendors and industry on methods of packaging and preparations in breaking the chain of contamination is necessary [22]. nevertheless, it should be recalled that in côte d'ivoire, food in general and especially meat are consumed frequently cooked, which a priori limits the transmission of the micro-organisms that they contain. on the other hand, these practices do not exclude a transfer of pathogenic microorganisms in the kitchen, in particular via cross contamination related to the handling of contaminated raw foods. moreover, the evolution of the lifestyle that allows the consumption of new products cooked or raw suggests the transmission of pathogenic microorganisms. côte d'ivoire has been involved for decades in sanitary inspection of meat in slaughterhouses. but this inspection does not allow absolute control of the dangers for the meat consumer. in addition, small-scale breeders set up clandestine slaughter systems for animals intended for direct sale to consumers. all of which is a public health concern today. meat inspection could contribute effectively to the eradication of zoonoses when associated with the presence of macroscopic clinical or lesional signs in animals and their carcasses. it does not make it possible to combat the zoonoses associated with an inapparent (asymptomatic) carriage as the case of animal yersiniosis and campylobacteriosis [4]. 3.7. symptoms after pork consumption and levels of risk to consumers the most common symptoms were diarrhea (47,9%), abdominal pain (22,8%), fever (21,1%) and vomiting (2,5%) for males. vomiting was more reported for females (6,8%) than males (2,5%) (table 2). foodborne illness is caused as a result of the consumption of pork, or contact with, food that has been contaminated with some type of microbiological, biological, chemical or physical hazard. pork production is affected by a variety of risk factors among which are quality of pig feed, number of subclinical carriers within a farm, conditions during transportation and lairage premises before slaughtering, slaughter line contamination by carrier animals, conditions during processing and retailing of pork products, conditions of handling pork products during catering and home-food preparation [25-28]. thus, risk of pork contamination increases when supply chains become longer (i.e. due to poor slaughter and other post-harvest handling practices) as practiced in abidjan district. consumers reported experiencing digestive problems such as diarrhea, abdominal pain, nausea, vomiting consecutive to pork consumption. these discomforts revealed consumers face in pork consumption. in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 29 table 5. symptoms after pork consumption by gender gender symptoms male n (%) female n (%) abdominal pain 144 (22.8) 214 (20.4) vomiting 16 (2.5%) 71 (6.8%) nausea 9 (1.4%) 13 (1.2%) diarrhea 302 (47.9) 471 (44.8) fever 133 (21.1) 161 (15.3) abdominal pain + diarrhea 23 (3.7%) 80 (7.6%) vomiting + nausea 0 19 (1.8%) abdominal pain + vomiting + diarrhea 0 21 (2%) total 631 1051 statistical analysis khi-square of pearson 61.076a 61.076a p value 0.000 0.000 addition. studies have highlighted the failure of the street food vendor's hygiene system [29] and the risk factors for infection in humans [30]. it is found. as revealed by the investigation. that pork is also sold and consumed in the street; which exposes the consumer to a contamination whose origin may not always be an agent intrinsically transported by pork. the domestic cycle refers to the transmission pattern occurring in swine herds. pigs can acquire infection by eating the infected flesh of other pigs or rats [31]. consumption of undercooked pork and cross-contamination of consumer products during processing of pork products are high-risk factors [32]. thus. the protection of public health depends on reliable rules of monitoring and reporting animal salmonella infections and retailing facilities contamination. but also the undertaking of educational programs for increasing the awareness of food workers and consumers on food safety during food preparation [33-35]. the consumption of raw or undercooked pork poses a risk of infection as does the cross-contamination of ready to eat foods with bacteria from raw pork or its juices. we can use separate cooking utensils and plates for raw meat and cooked foods and always store separately to avoid cross-contamination. therefore. sensitize and educate farmers and other value chain actors on the control of pathogenic bacteria. parasitic and virus are needed to achieve efficient and sustainable control. thus. good animal husbandry practices should be adhered to and pigs sourced from microbiologically reliable sources. the implementation of good biosecurity measures and good quality feed and water will ensure a healthy herd. 4. conclusion pork supply and marketing circuit were revealed during this investigation. three quarters of the people surveyed in abidjan district consume pork. so. pork is widely consumed at home and out-of-home. the type of processing undergone by the meat affects the choice of consumers according to the place of consumption. at home. smoked pork in a sauce is more prized. out-of-home. baked pork is appreciated by consumers. this investigation also revealed that conditions of transport. handling of pork are a risk factor. particular attention should be paid to some people who consume pork in the form of boiled. smoked. fried and / or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 30 undercooked. today. in africa. and particularly in côte d'ivoire. a change in eating habits and the development of fastfood restaurants are most evident in the urban population. it is therefore advisable to apply good hygiene practices in the processing. sale and pork consumption. our results refer to insufficient hygiene management in all stages of the food chain. educating consumers about the found risk factors presents an important action to decrease the incidence of bacterial infections. information and sensitization of population on bacteria risk factors and preventive measures must be observed. 5. acknowledgments the researchers are grateful to the staff of laboratory of biotechnologies and to the slaughterhouse of sivac (ivorian society of slaughter and pork butchery). 6. references [1]. fao. sources of meat. food and agricultural organization. animal production and health. (2014). [online] available: http://www.fao.org/ag/againfo/themes/en/meat/bac kgr_sources.html. accesed in 24 august 2017. [2]. oh s.-h.. see m. t.. pork preference for consumers in china. japan and south korea. asian-australasian journal of animal sciences. 25 (1): 143 – 150. (2012). [3]. fosse j.. magras c.. h. seegers. evaluation quantitative des risques biologiques pour le consommateur de viande de porc. journées recherche porcine. 39: 207-214. (2007). [4]. fosse j.. valeur informative d'indicateurs ante et post mortem pour la détection des dangers biologiques pour le consommateur de viande porcine. thèse de vie-agro-santé. sciences de la vie et de l'environnement. rennes. p. 408. (2008). [5]. fosse j.. seegers h.. magras c.. prevalence and risk factors for bacterial foodborne zoonotic hazards in slaughter pigs: a review. zoonoses public health. 56 (8): 429-454. (2009). [6]. holness d. h.. porcs. des atouts sous la menace. élevage de porcs dans les zones tropicales. cta/macmillan. (éd. révisée). isbn 0-333-791487. cta n° 1235. 10 unités de crédit. p.160. (2005). [7]. dindé a.o.. mobio a. j.. konan a. g.. fokou g.. yao k.. esso e. l. j. c.. fantodji a.. koussemon m.. bonfoh b.. response to the ebola-related bushmeat consumption ban in rural côte d’ivoire. agriculture and food security. 6 (28) : 1-9. (2017). doi 10.1186/s40066-017-0105-9. [8]. hill a. a.. england t. j.. snary e. l.. kelly l. a.. cook a. j. c.. wooldridge m.. a' farm-to-consumption' risk assessment for the adverse effects to human health of salmonella typhimurium in pigs. centre for epidemiology and risk analysis. veterinary laboratories agency. new haw. surrey. united kingdom. kt15 3nb. proceedings of the 10th international symposium on veterinary epidemiology and economics. (2003). [online] available : www.sciquest.org.nz. [9]. amos m. a.. le danger yersinia enterocolitica. thèse pour le doctorat vétérinaire. alfort. faculté de médecine de créteil. p. 116. (2003). [10]. singer r. s.. cox jr l. a.. dickson j. s.. hurd h. s.. phillips i.. miller g. y.. modeling the relationship between food animal health and human foodborne illness. preventive veterinary medicine. 79 (2): 186-203. (2007). [11]. oms. manuel d’épidémiologie pour la gestion de la santé au niveau du district. jouve (eds.). p.186. (1991). [12]. mopaté l. y. kaboré-zoungrana c. y.. production. commerce et consommation de viande porcine dans quelques villes d’afrique de l’ouest et du centre. revue scientifique du tchad – décembre 2013. pp. 43-44. (2013). [13]. mopaté l. y.. kaboré-zoungrana c. y.. gouro a.. commerce et consommation de la viande porcine dans la zone de n’djaména (tchad). revue sénégalaise de la recherche agricoles et agroalimentaires (rsraa). 1 (2) : 39-48. (2006). [14]. mopaté l. y.. matna m. m.. approvisionnement. transformation et consommation hors-foyer de porcs dans la ville de moundou (tchad). revue scientifique du tchad (rst). 11 (1spécial) : 53 – 62. (2012). [15]. d’orgeval r.. le développement de la production porcine en afrique: l’analyse des systèmes d’élevage du porc local africain au sudbénin. thèse ina-pg. paris. france. p. 272. (1997). [16]. legendre v.. les consommateurs de viande de porc frais et de charcuterie : qui sont-ils ? eclairage sociologique. techniporc. la revue technique de l’ifip. vol. 31. no4. (2008a). [17]. legendre v.. les déterminants de la consommation de porc en france : effets économiques. attentes et perceptions des http://www.sciquest.org.nz/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 koua atobla, edwige essoma akoa, carole aya bonny, adjehi dadié, germain tago karou, sebastien niamke, pork supply chain, consumption and risk factors for infections to consumers in abidjan district (côte d'ivoire), food and environment safety, volume xvii, issue 1 – 2018, pag. 20 – 31 31 consommateurs. journées de la recherche porcine. 40 : 69-78. (2008b). [18]. rainelli p.. l’image de la viande de porc en france. attitude des consommateurs. le courrier de l’environnement. vol. 42. p. 10. (2001). [19]. cazes-valette g.. le rapport à la viande chez le mangeur français contemporain“. rapport d’étude. groupe esc toulouse/ccit. ministère de l'agriculture. de l'alimentation. de la pêche et des affaires rurales. (2004). [20]. stolzenbach s.. lindahl g.. lundström k.. chen g.. byrne d. v.. perceptual masking of boar taint in swedish fermented sausages. meat science. 81 (4): 580-588. (2009). [21]. chevillon p.. bonneau m.. le strat p.. guingand n.. courboulay v.. quiniou n.. gault e.. lhommeau t.. acceptabilité par les consommateurs des viandes de porc mâle entier transformées en saucisses. lardons. saucissons secs et jambons cuits. techni-porc. 32 (5): 15-20. (2009). [22]. anonymous. consumer focused review of the pork supply chain. (2008). [online] available : http://www.safefood.eu/safefood/media/safefood library/documents/publications/research%20rep orts/safefood_pork_cfr_fullreport_2.pdf. accesed in 30 august 2017. [23]. friedman c. r.. hoekstra r. m.. samuel m.. marcus r.. bender j.. shiferaw b.. reddy s.. ahuja s. d.. helfrick d. l.. hardnett f.. carter m.. anderson b.. tauxe r. v.. emerging infections program foodnet working group. risk factors for sporadic campylobacter infection in the united states: a case-control study in foodnet sites” clinical infectious diseases. 38 (suppl. 3): s285–296. (2004). [24]. wilson d. j.. gabriel e.. leatherbarrow a. h.. cheesbrough j.. gee s.. bolton e.. fox a.. fearnhead p.. hart a. c.. diggle p. j.. tracing the source of campylobacteriosis. plos genetics. 4 (9). e1000203. (2008). doi:10.1371/journal.pgen.1000203 [25]. boyen f.. haesebrouck f.. maes d.. van immerseel f.. ducatelle r.. pasmans f.. non-typhoidal salmonella infections in pigs: a closer look at epidemiology. pathogenesis and control. veterinary microbiology. 130: 1-19. (2008). [26]. kranker s.. dahl j.. wingstrand a.. bacteriological and serological examination and risk factor analysis of salmonella occurrence in sow herds. including risk factors for high salmonella seroprevalence in receiver finishing herds. berliner und munchener tierarztliche wochenschrift. 114 ( 9-10): 350-352. (2001). [27]. van der wolf p. j.. wolbers w. b.. elbers a. r.. van der heijden h.m.. koppen j.m.. hunneman w.a.. van schie f.w.. tielen m. j.. herd level husbandry factors associated with the serological salmonella prevalence in finishing pig herds in the netherlands. veterinary microbiology. 78 (3): 205-219. (2001). [28]. lo fo wong d. m .a.. dahl j.. stege h.. van der wolf p. j.. leontides l.. von altrock a.. thorberg b.m.. herd-level risk factors for subclinical salmonella infection in european finishing-pig herds. preventive veterinary medicine. 62 (4): 253-266. (2004). [29]. dawson p.. han i.. cox m.. black c.. simmons l.. resident time and food contact time effects on transfer of salmonella typhimurium from tile. wood and carpet: testing the five second rule. journal of applied microbiology. 102 (4) : 1364–5072. (2006). [30]. secke c.s.. contribution à l'étude de la qualité bactériologique des aliments vendus sur la voie publique de dakar. thèse pour obtenir le grade de docteur vétérinaire (diplôme d'état) de l’université cheikh anta diop de dakar. (2007). [31]. schad g. a.. dyffy c. h.. leiby d. a.. murrell k. d.. zirkle e. w.. trichinella spiralis in an agricultural ecosystem: transmission under natural and experimental modified on-farm conditions. the journal of parasitology. 73 (1): 95102. (1987). [32]. prendergast d. m.. duggan s. j.. gonzalesbarron u.. fanning s.. butler f.. cormican m.. duffy g.. prevalence. numbers and characterizations of salmonella spp. on irish retail pork. international journal of food microbiology. 131 (2-3): 233-239. (2009 [33]. su l.-h.. wu t.-l.. chiu c.-h.. decline of salmonella enterica serotype choleraesuis infections. taiwan. emerging infectious diseases.. 20 (4): 715-716. (2014). [34]. efsa. the european union summary report on trends and sources of zoonoses. zoonotic agents and food-borne outbreaks in 2010. efsa journal. 10 (3). 2597. pp. 442. (2012). doi:10.2903/j.efsa.2012.2597. [online] available: www.efsa.europa.eu/efsajournal. [35]. king r.p.. backus g. b.c.. van der gaag m.a.. incentive systems for food quality control with repeated deliveries: salmonella control in pork production.” european review of agricultural economics. 34 (1): 81-104. (2007). http://www.safefood.eu/safefood/media/safefoodlibrary/documents/publications/research%20reports/safefood_pork_cfr_fullreport_2.pdf http://www.safefood.eu/safefood/media/safefoodlibrary/documents/publications/research%20reports/safefood_pork_cfr_fullreport_2.pdf http://www.safefood.eu/safefood/media/safefoodlibrary/documents/publications/research%20reports/safefood_pork_cfr_fullreport_2.pdf http://www.efsa.europa.eu/efsajournal 1. introduction therefore. sensitize and educate farmers and other value chain actors on the control of pathogenic bacteria. parasitic and virus are needed to achieve efficient and sustainable control. thus. good animal husbandry practices should be adhered to and pi... 4. conclusion microsoft word 17 tatjana kalevska_pe format.doc 252 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 252 260 slaughter characteristics and weight losses (shrinkage) of lambs from an organic and conventional system * tatjana kalevska 1 , ljupce kocoski 2, elena joshevska2, zora uzunoska1, viktorija stamatovska1, 1faculty of technology and technical sciences,university st.kliment ohridski, veles, r.macedonia, ttfv@ uklo.edu. mk 2faculty of biotechnical sciences, university st.kliment ohridski, bitola, r.macedonia, fbn@uklo.edu.mk. *corresponding author tkalevska@gmail.com received 3rd march 2018, accepted 27th june 2018 abstract: the aim of the research was to determine the differences in slaughter characteristics, weight loss in transport and cooling, and the ph value of lamb meat grown in an organic and conventional system. the study included 120 lambs, 60 of which were bred in a conventional and 60 in an organic system for a period of 75 days. the established differences in the slaughter results (carcass weight, dressing percentage) and the proportion of internal organs relative to the weight of lamb before slaughter) among the lambs bred in an organic and conventional system are statistically not significant (p>0.05). significant difference (p<0.05) is determined for total shrinkage (transport and shrinkagein lairage facility) between lambs of group i from the organic and conventional system and a significant difference from (p<0.01) between lambs of group ii of the organic and conventional system. the discrepancies found in the cooling shrinkage of lamb carcases between groups from the organic and conventional system are significant (p<0.01). in the ph value measured 1 and 24 hours post-mortema statistically significant difference was found (p<0.05) between lamb meat from the organic and conventional system. keywords: quality, carcass, meat, breeding systems 1. introduction with the increasing needs and interest in the domestic and foreign market for lamb, there are demands for increasing its quality, covered by the new eu regulations concerning the quality of the animals for slaughter [1]. in accordance with [2] there is no general model for breeding, which could be applied in all farms and in all conditions. there are different breeding systems in the world, which are conditioned by natural and economic factors, as well as from the tradition of countries or regions.in the conventional breeding system, the traditional way is popular, with a technology of early rejection of the lambs from 25 to 30 days, and intensive feeding with concentrated fodder mixtures and hay at will, up to a certain slaughter weight or age. the main objective of this method of breeding is to intensify sheep farming, by achieving solid weight gaining performance in earlier rejected lambs and getting bigger amount of milk per sheep [3]. compared to conventional, organic sheep production is based on compliance with standards and legal regulations for controlling each stage of the production cycle, which contribute to respect for animal welfare and the production of safe products of animal origin. the cultivation of lambs in an organic system is carried out in accordance with the organic production rules, equivalent to the european union regulation 834/2007, according to which, in order to respect animal welfare, the lambs should be rejected at the 45 days old. the birth weight represents the basis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 253 for the growth and development of lambs, [4]. lambs grown in a conventional and organic system have similar production and slaughter yields, [5]. long transport of animals, adverse conditions and improper handling of animals during transport negatively affect animals and the quality and sustainability of the meat. transport shrinkage (loss of body mass), when transporting lambs to a slaughterhouse depends on the influence of several factors such as: breed, sex, health status of lambs, degree of nutrition, the treatment of the lambs before and after transport, mode of transport, length of transport, type of transport vehicle, and the season. the shorter relation when transporting lambs from farm to slaughterhouse leads to a reduction in stress [6], [7]. the loss of body weight and the stress experienced during the transportation of lambs negatively affects the quality and sensory properties of the lamb, which means that the lambs should be placed in the slaughterhouse before the slaughter for the normalization of the physiological condition, and therefore be properly prepped for slaughter. with the rest in the slaughter shed many negative consequences caused by transport are eliminated or mitigated, i.e., the irritated vasomotor centres in the brain, ejection of harmful products from the metabolism and withdrawal of the bacteria from the blood. during the stay in the shed in the lambs, a loss of body mass occurs, primarily as a result of excretion of bodily fluids by evaporation, sweating, urination, etc. [8]. the weight of the lambs before slaughter affects the carcass weight of the lamb carcass, [9]. slaughter characteristicsof the lamb carcass, i.e. the quantity and quality of the resulting meat vary depending on farming systems, [10]. when slaughtering the lambs, the dressing percentageis an important economic indicator that represents the relationship between the slaughter and the body weight of lambs before slaughter, expressed in percent. dressing percentageof slaughter varies greatly and depends on the influence of several factors such as: breed, sex, age, health, manner of nutrition and degree of nutrition and weight of the lambs. the mass of the internal organs and parts of the lamb carcases is a parameter that is often used to evaluate the conformation of lamb carcasses. the loss in the mass of carcasses so called. cooler shrink occurs because of the draining and evaporation of the lamb carcasses in the cooling chambers. shrinkage of cooling depends on the influence of several factors, primarily on the time of the first measurement, which represents the basis for determining the shrinkage, the quality of the meat and the coverage of the body with fatty tissue that depends on the race of lambs and the diet, the speed of cooling of the carcasses, air circulation in the chambers and the relative importance of the air, [11]. ph value is one of the factors that determine the quality of the meat associated with biochemical processes during the transformation of the muscles in the meat. measuring the ph value of the meat allows for monitoring of the intensity of changes occurring in the muscles in the post-mortem period which also affect the sensory properties of the meat, [12]. lowering of the ph value of the meat must be gradual, [13]. if the animal is under stress immediately before slaughter, the glycogen reserves in the muscles are reduced and a sufficient amount of lactic acid is not generated, and the ph value of the meat remains high and causes the occurrence of dfd dark, firm and dry meat [14], while too rapid a decrease in ph value can lead to pse pale, soft, exudative meat, [15]. the ph value of the meat is influenced by many factors such as breed, animal fatigue before slaughter, muscle glycogen content, carbohydrate-rich diet, acute starvation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 254 before slaughter, and meat storage temperature, [16]. 2. matherials and methods the study covered 120 lambs, 60 of which were grown conventionally and 60 in an organic system, over a period of 75 days. in the conventional system, two groups of 30 lambs were formed. the lambs of group imade up of crossbreeds of domestic breed refined with virtemberg, and in group ii, the lambs were made up of crossbreeds of domestic breed refined with east-friesian breed. the lambs of both groups in the conventional system were raised the traditional way, weaned on the 30th day and until the end of the breeding were fed with a commercial conventional fodder mixture. two groups of 30 lambs were formed in the organic system, out of which the lambs in group i made up of crossbreeds of domestic breed refined with virtemberg, and in group ii, the lambs were made up of crossbreeds of domestic breed refined with east-friesian breed, certified for organic production. lambs from the organic system were breed according to organic production rules, according to which lambs were weaned on the 45th day and by the end of the breeding were fed with a concentrated fodder mixture obtained from an organic produced and certified food from own production cycle. body mass control of the lambs from both systems was done every fifteen days starting from the first day (birth weight), to the 75th day when they were slaughtered. the transport of lambs from both systems to the slaughterhouse, which is 75 km away, was carried out in the same way, with special means of transport for animals. the last measuring of the weight of the lambs was done on the farm; just before loading them into the vehicle.after arriving and unloading them at the slaughterhouse, the lambs were measured on an electronic livestock scale and placed in a lairage facility. by calculating the difference in body mass before and after transport, the transport shrinkage of the lambs is determined. after the 7 hour stay at the lairage facility, the lamb shrinkage is determined in the same manner. the killing of the lambs was done with stunning, to humanize the procedure. after the slaughter of the lambs and the processing of the carcases, individual measurements of the internal organs, taken from the lamb carcass, were performed. after 24 hours in the cooling chambers at a temperature of 0°c, the carcases were individually weighted and on the basis of the difference in the weight of the warm and cold carcasses the loss in the mass during cooling was determined. in determining the slaughter value, the following parameters for the dressing percentage of the lambs were determined: warm carcass with head and internal organs (r i), cold carcass with head and internal organs (r ii), carcass with head without internal organs (r iii), cold carcass without head and internal organs (r iv). the first measurement of the ph value of the meat was conducted one-hour post mortem, and the second measurement of the ph value was 24 hours after the stay in the cooling chambers at a temperature of 0°c. 3. results and discussion the established average values for the birth and body weight of the lambs, on the 75th day of the breeding, before the transport of the lambs to the slaughterhouse are presented in table 1. the determined birth weight of the lamb’s fromgroup iof the organic system is 3.991kg, i.e. 4,189kg of group ii from the organic system. a similar birth weight of 4.226 kgand 4.338 kg, it was found in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 255 lambs of group i and ii from the conventional system. the difference in the birth weight between lambs of group i from the organic and conventional system, as well as the difference between group ii lambs from the organic and conventional system, is statistically not significant (p>0.05) [17]. table 1 birth and body weight of lambs from breeding systems birth weight x sd cv s x о (i) 3.991 0.522 13.08 0.095 о (ii) 4.189 0.566 13.52 0.103 c (i) 4.226 0.615 14.55 0.112 c (ii) 4.338 0.561 12.95 0.102 body weight (75th day) x sd cv s x o (i) 17.995 1.812 10.07 0.331 o (ii) 18.363 2.161 11.77 0.395 c (i) 18.480 2.447 13.24 0.452 c (ii) 19.149 2.339 12.22 0.428 similar value results for the birth weight of the lambs were stated [18]. on the 75th day, i.e. at the end of the breeding the highest body weight of 19.149 kg was established in lambs from group iifrom the conventional system, and the lowest body mass of 17.995 kg of group i lambs from the organic system. the difference in body weight of lambs found at the end of breeding (75 days), between groups in the same system, as well as the established differences between the same groups of different systems (organic and conventional system) is statistically not significant (p>0.05), [17]. results for the body weight of lambs from the organic system at the end of the breeding can be compared with the results obtained by [19]. figure 1 presents the total shrinkage of lambs (transport and shrinkage in a lairage facility) of both groups of organic and conventional system breeding. from the data presented in figure 1, it can be stated that the total shrinkage (transport and lairage facility shrinkage) in lambs in the organic system is significantly lower, compared to the total shrinkage in lambs of the conventional system. fig.1 total shrinkage (loss) ofthe weight of the lambs the total shrinkage in the organic system for lambs of group i is 5.61% and 5.97% for lambs of group ii. in the conventional system, total shrinkage in group i lambs is 6.02% and 7.09%. differences in the total shrinkage of lambs between group i from the organic and group i from the conventional system, as well as the difference between group ii in the organic and group ii in the conventional system, are statistically significant at the level of (p<0.05), that is, (p<0.01), respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 256 from the above it can be concluded that apart from the breeding system, the breed of the lambs, as well as the stress during transport has a certain influence on the total shrinkage. in table 2 are presented relative participation of internal organs and parts of the lamb carcass table 2 relative participation of internal organs and parts of the lamb carcass system/group o(i) o(ii) c(i) c(ii) indicators kg % kg % kg % kg % live weight 16.987 100 17.267 100 17.368 100 17.79 100 head 728.0 4.28 760.0 4.40 755.0 4.35 790.0 4.44 liver 380.0 2.24 400.0 2.32 405.0 2.33 425.0 2.39 lung 320.0 1.88 350.0 2.08 340.0 1.96 370.0 2.08 heart 90.0 0.52 100.0 0.57 95.0 0.54 105.0 0.59 tallow 103.9 0.61 106.2 0.62 111.5 0.61 119.6 0.67 tonsils 158.0 0.93 166.6 0.96 169.0 0.97 176.0 0.99 stomach with intestines 3.890 22.89 3.905 23.25 3.995 23.31 4.230 23.77 skin 1.950 11.48 2.038 11.80 2.020 11.70 2.115 11.89 feet 530.0 3.12 557.0 3.23 546.0 3.14 586.0 3.29 with lambs of group i from the organic system, the body weight before slaughter is 16.987kg, that is, 17.267 kg for lambs of group ii, while the weight before slaughter in lambs of group i in the conventional system is 17.368 kg, or 17.792 kg in lambs of group ii. the observed differences in body mass before slaughter of lambs from the groups in the systems are statistically not significant (p>0.05). the percentage share of skin in lambs of group i from the organic system is11.48% i.e. 1.80% in lamb’s fromgroup ii, i.e 11.70% in group i and 11.89 in lamb’s from group ii from the conventional system. similar results were obtained by [11]. the percentage share of the liver in lambs from group i and ii of the organic system is 2.24%, i.e. 2.32% and in the conventional system 2.33%, i.e. 2.39%. the percentage share of the the lungs in the lambs from group i and ii of the organic system is 1.88%, i.e. 2.08, while and in the conventional system in group i and ii with 1.96%, i.e. 2.08%. the heart accounts for 0.52% in lambs from group i and 0.57% in lambs from group ii in the organic system, while and in the conventional system the heart accounts fоr 0.54% in group i and 0.59% in group ii. similar results to ours were obtained by [20], for the percentage of liver 2.20±0.05, for the lungs 2.06±0.09, for the heart 0.46% and for the head 4.97, [21],obtained almost the same values as ours, for the percentage of the liver weight in male lambs from 0.23 ± 0.01 kg, i.e. 0.22 ± 0.01 kgin female lambs, i.e. 1.91% in unweaned lambs and 1.60% in weaned lambs and percentage shareof the heart of 0,52% in unweaned and 0.48% in weaned lambs.the differences in relative share of the internal organs of lambs from group i of the organic and conventional system and group ii of the organic and conventional systems are not significant (p>0.05), which is understandable because the weight of the lambs from the organic and conventional breeding system before the slaughter is approximate in lambs from both breeding systems. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 257 fig 2 mass losesof lamb carcasses in cooling chambers the differences in the average values for cooling shrinkage in lamb carcases between groups of the organic and conventional system are significant (p<0.01). the discrepancy found is understandable and stems from the influence of several factors such as: the time of the first weighing, which represents the basis for determining the shrinkage, the quality of the meat and the coverage of the body with fatty tissue that depends on the breed of lambs and the diet, the speed of cooling of carcasses, circulation and the relative importance of air in the chambers [11]. fig 3 slaughter values of the lambs of i and group ii from the organic and conventional system differences in carcass weight among groups in the systems are not statistically significant (p>0.05), which is understandable because the live weight before slaughter is approximate in lambs between both breeding systems. fig 4 dressing percentage of lambs from an organic and conventional system food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 258 the dressing percentage of slaughter is very variable and dependent of the influence of several factors such as: breed, sex, age, diet, fat, etc. during the research a difference is determined between lambs from group i of the organic and conventional system, in the dressing percentagefrom: (ri-0.89%; rii-0.84%; r iii-0.65% and r iv-0.59%), as well as a difference in the dressing percentagebetween lambs from group ii from the organic and conventional system of (ri-0.74%; rii-0.40%; riii-0.24% and riv-0.23%). the observed differences in the average value of dressing percentage in group i lambs from the organic and conventional system and group ii of the organic and conventional system in all cases are statistically not significant (p>0.05) which is understandable, because the live weight of the lambs and the slaughter weight of the lamb carcasses from the organic and conventional system is similar [17]. similar results for dressing percentage of a warm carcass in lambs grown in an organic system of 51.66% i.e. 48.90% in lambs of the control group were found [19]. the smaller dressing percentage of the organic system, is probably due to quicker weaning, changes in diet of the lambs as well as stress during transport [22], determined values fordressing percentage of a warm carcass that deviate from our results for the lambs from the conventional system from 49.27±0.607, i.e. 49.36±0.525 in lambs from the organic system. in the research ofslaughter characteristicsof creska lambs [21] stated similar results of ours in the carcass weight of the lambs 19.04 ± 0.22 and dressing percentage of slaughter 50.50 ± 0.60. the average ph1 and ph2 values of meat from lambs of both groups in the organic and conventional system are shown in figure 5, where by it can be stated that the ph value of meat from lambs from the organic system has characteristic values for the normal course of post-mortem glycolysis. the established difference in the ph value of the lamb between the groups of the organic and conventional system is significant (p<0.05). identical results were obtained by [23], [24], whereby the average ph value after 45 minutes from the slaughter was 6.15, i.e. 5.55 after 24 hours. similar results to ours for the ph value, 1 and 24 hours after the slaughter were obtained by [25]. results from our research coincide with the data obtained for the ph value of lamb, 1 and 24 hours after the slaughter of the lambs from [10], [11], [26]. fig 5 ph value of lamb from organic and conventional system food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 259 4. conclusion the study did not determine statistically significant (p>0.05) influence of the system on the breeding of lambs (organic and conventional one) on production and slaughtering results. the statistically significant influence of the breeding system, but also partly on the racial characteristics, was established in some of the investigated parameters: totalshrinkage (transport andlairage facility shrinkage) between group i of the organic and group i of the conventional system at the level of (p<0.05) and between group ii of organic and group ii of the conventional system at the level of (p<0.01), the cooling shrinkagebetween lamb carcasses from the groups in the organic and conventional system (p<0.01). significant difference (p<0.05) was determined for the ph value of lamb meat between groups of the organic and conventional system. 5. references [1] žujović m., tomić z., pavlovski z., lukić m., pavlović m., ivanović s., randman mesa jagnjadi meleza pirotske pramenke i merino landšaf rase kao pokazatelj proizvodnje mesa. biotechnology in animal husbandry 20 (12), p 75-80, (2004) [2] osamu s., kazuo i., yoshitaka n., breeds and breeding systems of dairy sheep in the mediterranean countries. experimental herbivore, 29, 39-48, (2005) [3] pacinovski n., eftimova e., gorgovska n., dubrova mateva n., naletoski z., palasevska a., production results of early weaned lambs from domestic merinized sheep in macedonia. journal of mountain agriculture on the balkans, vol. 14. (1), 76–89, troyan, bulgaria, (2011) [4] petrović m., ružić-muslić d., maksimović n., evaluation of genetic potential of sheep in different production systems. biotechnology in animal husbandry 25(5-6), p 421-429, (2009) [5] fisher av, enser m, richardson ri, wood jd, nute gr, kurt e, sinclair la and wilkinson rg., fatty acid composition and eating quality of lamb types derived from four diversebreed x production system. meat science 55, 141–147, (2000) [6] blake, f., the handbook of organic husbandry. crowood press, ramsbury,(1981) [7] gade, p.b. 2002. welfare of animal production in intensive and organic systems with special reference to danish organic pig production. meat science, 62: 353-358. [8] petrovski i.,higiena i tehnologija na mleko i meso. rabotni listovi za ucenici vo sredno zemjodelsko uciliste. bitola, (2007). [9] čaušević z., milanović a., glogovac ž., lelek m., prilog poznavanju proizvodnjesudžuka. radovi poljoprivrednog fakulteta. sarajevo, (1987). [10] diaz m.t., velasco, caneque v., lauzurica s.,physico-chemical characteristics of caracass and meat manchengo-breed suckling slaughtered at different weights. meat science, 65:1247-1255,(2002). [11] danev m., ispitivanje uticaja načina tova jagnjadi na randman klanja i neka kvalitetna svojstva mesa. doktorska disertacija.veterinarski fakultet.beograd. (1984). [12] beriain m.j., bas p., purroy a.,treacher t., effect of animal and nutritional factors and nutrition on lamb meat quality. zaragoza: ciheam, cahiers options. mediterraneenes; n.52 pag.75-86. (2000). [13] lanza a., biondi l., miglioramento e valutazione della qualità della carne negli ovicaprini. in: atti del ii simposio internazionale: "nuove prospettive della ricerca sugli ovi-caprini", varese-ville ponti, 23 novembre: 129-170, (1990). [14] sarti d.m., qualità delle carni ovine. in: "ovinicoltura" unapoc, roma: 311-314. (1992) [15] renieri c., silvestrelli m., stradaioli g., cavallucci c., trabalza marinucci m., la qualità della carne nella razza ovina fabrianese. l'allevatore di ovini e caprini, 5: 7-11, (1993). [16] dzinleski b., meso i prerabotka na meso. i и ii del, skopje, (1990). [17] kalevska t., kocoski lj, joshevska e., uzunoska z., stamatovska v., slaughter characteristics of lamb’s meat according to breeding systems. maced. j. anim. sci., 7 (1–2) 67–73 (2017) . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska, slaughter characteristic and weight losses (shrinkage) of lambs from an organic and conventional system, food and environment safety, volume xvii, issue 2 – 2018, pag. 252 260 260 [18] pacinovski n., dzabirski v., kozarovski n., belicovski s., andonov c., i gordana dimitrovska. prirast i klanicni osobini na jagninjata krstoski megu ovnite od rasata hios i domasnata merinizirana ovca odgledni na pasa. jubileen zbornik. simpozium za stocarstvo so megunarodno ucestvo (77-82), struga, (2002). [19] morbidini l., pollidori p., sarti d.m., valigi a., carcass, meat and fat quality in italian merino derived lambs obtained with"organic"farming systems. dept. of animal science, faculty of agraria, borgo xx giugno 74, 06121 perugia, italy, (2001). [20] źgur s., cividini a., kompan d., birtićd., the effect of live weight at slaughter and sex on lambs carcass traits and meat, characteristics agriculturae conspectus scientificus, vol. 68. no. 3 (155-159), (2003). [21] mioč b., paviš v., vnuţec i., prpiš z., suńiš v., baraš z., klaoniĉki pokazatelji i odlike trupa creske janjadi .44th croatian & 4th international symposium on agriculture,(2009). [22] hanoğlu hülya, i̇brahim ak, deniz soysal: determination of organic lamb fattening performanceand slaughter caracteristics in the sout marmara conditions in turkey. macedonian journal of animal science, vol. 3, no. 2, pp. 135– 142, (2013). [23] vergara h., gallego l., effect of type suckling and lenghtof lactacion period of carcass and meat quality and intensive lamb production systems. meat sci. 53.211-215,(1999). [24] sanudo c., sierra i., olleta j.l., martin l., campo m.m., santolaria p., wood j.d., nute g.r., influence of weaningon carcass quality, fatty acid compositionand meat quality in intensive lamb production systems. animal. sci., 66 175-187, (1998). [25] esterhuizen j., groenewald i.b., strydomp.e. and hugo a., the performance and meat quality of bonsmara steers raised in a feedlot, on conventional pastures or on organic pastures. south african journal of animal science.38 (4), (2008). [26] dzabirski v., kozrovski n., plasevski b., andonov s., pacinovski n, naletovski z., dinamika na prirastot na nekoi klanicni parametric kaj jagninjata melezi od f1 genercija pomegu domasna merinizirana ovca i rasata hios. macedonian veterinary rewiew 29 2/3 p 37-42, (2000). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 contents: 1. comparative analysis of the proximate, fatty acids and mineral composition of selected fish species alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola 136 149 2. determination of color and anthocyanins in three romanian red wines cristina ghinea, ana leahu, florin ursache 150 155 3. activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire. jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje 156 166 4. distribution of polycyclic aromatic hydrocarbons (pahs) in some charcoal roasted foods commonly consumed in port harcourt, nigeria virginia i. p. nitonye, michael horsfall jnr., mark o. onyema 167 175 5. nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan dina anwar, ghadir el-chaghaby 176 190 6. effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp. jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio 191 200 7. existing regulations on different markets on the obtaining and marketing of foods with benefits for health micşunica rusu 201 207 8. statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov 208 213 9. influence of ph and acidity on the fermentation of finger millet spiced ogi abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf 214 222 10. effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters elena todosi sănduleac, gheorghe gutt 223 234 11. effect of different packaging methods on consumers eating quality of beef ahmedin abdurehman musa 235 245 12. state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo 246 257 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 13. author instructions i v 14. subscription information vi title …………………… 276 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 276 281 assessment of potential contamination and health risk associated with metals in drinking waters from copsa mica region *maria-alexandra hoaghia1,2, oana cadar1, erika levei1, cecilia roman1, lacrimioara senila1, dumitru ristoiu2 1incdo-inoe 2000, research institute for analytical instrumentation, 67 donath, ro-400293 cluj-napoca, romania, 2babeş-bolyai university, faculty of environmental science and engineering, 30 fantanele, ro-400294 cluj-napoca, romania, alexandra.hoaghia@icia.ro *corresponding author: received september 5th 2015, accepted september 29th 2015 abstract: metal contamination of drinking water sources from copsa mica region (copsa mica village, tarnava villages and medias town, sibiu county, romania) was assessed using the metal index, while health risk associated with metals from the drinking water sources was assessed using the chronic daily intake and the hazard quotient. the mi values increase in following order s1>s3>s2>s4>s5, indicating a possible metal contamination of three drinking water sources from the studied area. however, according to the hazard quotient and the total hazard quotient, the studied drinking waters are safe for human consumption. the calculated hazard quotients for studied metals decrease in the order: as>cd>mn>zn≥ cu>ni ≥ pb>cr for sample s1, as>cu ≥ mn ≥ pb>cd ≥ ni>zn>cr> for sample s2, cd ≥mn ≥ pb>cu ≥ ni>as>cr ≥zn for sample s3, zn>cd ≥ cu>mn ≥ ni>cr>pb for sample s4 and cd ≥ni>cu>mn>cr ≥pb ≥zn>as for sample s5. keywords: drinking water, metal index, chronical daily intake, hazard quotient 1. introduction drinking water contamination with metals represents a significant issue for the environment and human health [1-3]. metals content in water sources have anthropogenic (industrial, agricultural activities) and natural origins (weathering, rock erosion) [1-4]. the use of unnapropriate or poorly monitorized water sources (well water, surface water) as drinking water exposes the population to potential health risks. the raise of population awarenes related to the risk of metal contaminated water consumption could avoid noumerous health problems (cancer, kidney damages, heart disease) [4-6]. drinking water quality monitoring with respect to chemical compounds (metals, pesticides, nitrogen compounds) is important due to the pollution exposure issues which implies potential negative effects on health [7, 8]. for that purpose increasing numbers of studies all over the world were performed [9, 10]. studies on metal pollution are important due to the metals toxicity, ubiquity and persistence in the environment [11, 12]. thus, different indices (metal index, chronic daily intake, hazard quotients) for metal pollution, risk assessment and drinking water quality were elaborated and applied in order to http://www.fia.usv.ro/fiajournal mailto:alexandra.hoaghia@icia.ro food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu, assessment of potential contamination and health risk associated with metals in drinking waters from copsamica region, food and environment safety, volume xiv, issue 3 – 2015, pag. 276 – 281 277 assess the chemical status of drinking water soursec [3, 4, 13]. the metal index (mi) is an arithmetical tool used to assess the metal pollution status and represents the sum of the ratio between the monitored concentrations and the maximum admissible concentrations (macs) of the studied metal [4, 13]. to assess the non-carcinogenic risk posed by metal contaminated drinking waters, healh risk indices such as the chronic daily intake (cdi), the hazard quotient (hq) and the total hazard quotient (thq) are used [3]. copsa mica is known as a “hot spot” of pollution, due to the metal industry and its negative effects on human and animal health and on plants, soil, air and water qulity [14]. presently, the industrial activities ceased, but the chemical compounds still persist in the environment [14]. the aim of the present study is to evaluate the metal contamination status of drinking water sources using the mi and to assess the potential human health risk of metal contaminated waters using the cdi, the hq, and the thq indices. 2. materials and methods study area and sampling well water samples from tarnava village (s2, s3), copsa village (s4, s5) and a public spring water sample (s1) from the vicinity of medias town, sibiu county, romania used by inhabitants as drinking water sources were studied (figure 1). fig. 1. study area and drinking water sampling points these villages are close to copsa mica town, known for the environment pollution with cu, fe, zn, cd, cu [14]. all water samples were colected in august of 2014 in polyethylene bottles (500 ml) and kept at 4 ºc in a refrigerator until analysis. chemical analysis drinking water samples were filtered using 0.45µm filter membrane and acidified with hno3 before analysis of the as, cd, cr, cu, fe, mn, ni, pb and zn content using drc ii perkin-elmer icp-ms. metal index (mi) the metal index (mi) is based on total trend assessment of present status [15, 16]. a high metal concentration associated with its mac indicats a low water quality [15]. the formula (eq 1) for the mi rating is [16, 17]:      n i i i mac c mi 1 (1) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu, assessment of potential contamination and health risk associated with metals in drinking waters from copsamica region, food and environment safety, volume xiv, issue 3 – 2015, pag. 276 – 281 278 where ci represents the metal concentration and mac is the maximum allowable concentration according to moghaddam [18]. according to the mi results, waters can be classified as potable (mi< 1), non-potable (mi> 1) and water on the threshold of danger (mi= 1) [17, 18]. if mi> 1 it represents a threshold of warning, even if the ci does not exceed the macs for all metals [17]. human health risk assessment indices chronic daily intake (cdi) the cdi was calculated in order to estimate the health risk for adults associated with the consumption of contaminated drinking water. the cdi raiting was calculated using eq 2 [3, 19, 20]: bw dic cdi   (2) where, c represents the metal concentration in water samples (µg/l), di is the average daily intake rate (2l/day) and the body weight is represented by bw (72 kg) [2, 3]. hazard quotient (hq) the hq is a human health risk assessment index, which quantifies the noncarcinogenic risk (eq 3) [2, 21]. rfd cdi hq  (3) where, cdi is the chronic daily intake and rfd is the oral toxicity reference dose. for the hq computation the used rfd values were 1.5mg/kg-day for cr, 5.0 104mg/kg-day for cd, 3.7 10-2mg/kg-day for cu, 1.4 10-1mg/kg-day for mn, 2.0 102mg/kg-day for ni. 3.6 10-2mg/kg-day for pb, 3.0 10-1mg/kg-day for zn and 3.0 10-4mg/kg-day for as [2, 3, 22]. if hq< 1, the safety of the exposed population is assumed [2, 3, 23, 24]. in case of hq>1, there might be noncarcinogenic risk and potential for toxic effects for the population [24]. 3. results and discussion for the mi rating, as, cd, cr, cu, fe, mn, ni, pb and zn were selected in order to evaluate the possible metal contamination of drinking water sources in copsa mica region. the mi values revealed possible metal pollution of drinking water samples s1, s2 and s3 with mi values higher than the critical value of 1 (table 1). table 1. metal index (mi) of the drinking water samples s1 s2 s3 s4 s5 mac* cr 5.0 1.5 4.6 3.8 3.4 50 cd 0.3 0.03 0.1 0.04 0.04 3 cu 5.8 6.9 6.2 2.8 1.5 1000 fe 151 94 94.7 50.4 27.2 200 pb 4.2 0.7 0.8 0.4 0.6 1.5 zn 38.6 40.9 58 176 21.7 5000 ni 2.5 1.6 3.9 0.9 1.4 20 as 1.0 0.3 0.9 <lq <lq 50 mn 7.3 6.6 7.7 4.9 0.7 50 mi 4.1 1.2 1.5 0.79 0.71 *according tomoghaddam et al., 2014 [18] <lq – values are bellow the limit of quantification food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu, assessment of potential contamination and health risk associated with metals in drinking waters from copsamica region, food and environment safety, volume xiv, issue 3 – 2015, pag. 276 – 281 279 thought, the measured concentrations for studied metals do not exceed the macs used for the mi count. the drinking water samples with mi<1 are suitable for drinking. the cdi values (table 2) for the studied metals in s1 water source situated near medias decrease in the order of cd<as<ni<pb<cr<cu<mn<zn. for samples collected from tarnava village (s2 and s3) the calculated cdi increase in the order cd<as<pb<ni≤cr<mn<cu<zn and cd<pb<as<ni<cr<cu<mn<zn, respectively. table 2. chronic daily intake (cdi), hazard quotient (hq) and total hazard quotient (thq) for drinking water metal s1 s2 s3 s4 s5 cdi hq cdi hq cdi hq cdi hq cdi hq cr 1.4 x10-4 0.1x10-3 0.4x10-4 0.3x10-4 1.2x10-4 0.1x10-3 0.1x10-3 0.1x10-3 0.9x10-4 0.1x10-3 cd 0.1x10-4 0.2x10-1 0.1x10-5 0.2x10-2 0.3x10-5 0.6x10-2 0.1x10-5 0.2x10-2 0.1x10-5 0.2x10-2 cu 1.6x10-4 0.4x10-2 1.9x10-4 0.5x10-2 1.7x10-4 0.5x10-2 0.8x10-4 0.2x10-2 0.4x10-4 0.1x10-2 mn 2.0x10-4 1.0x10-2 1.8x10-4 0.5x10-2 2.1x10-4 0.6x10-2 1.4x10-4 0.1x10-2 0.4x10-4 0.3x10-3 ni 0.7x10-4 0.3x10-2 0.4x10-4 0.2x10-2 1.1x10-4 0.5x10-2 0.3x10-4 0.1x10-2 0.6x10-3 0.2x10-2 pb 1.2x10-4 0.3x10-2 0.2x10-4 0.5x10-2 0.2x10-4 0.6x10-2 0.1x10-4 0.3x10-3 0.2x10-4 0.1x10-3 zn 10.7x10-4 0.4x10-2 0.1x10-2 0.1x10-3 1.6x10-3 0.1x10-3 4.9x10-3 0.2x10-1 0.2x10-4 0.1x10-3 as 0.3x10-4 0.9x10-1 0.1x10-4 0.3x10-1 0.3x10-4 0.4x10-3 thq 0.13 0.05 0.03 0.02 0.01 for samples from copsa mica village (s4 and s5) the calculated cdi increase in the order as<cd<zn<cr<pb<ni<cu<mn for sample s4 and cd<pb≤zn<cu≤mn<cr<ni for sample s5. the highest cdi values were obtained for zn in samples s1, s2, s3 and s4, followed by the cdi for ni in sample s5 and cr in sample s4. samples s2, s3, s4 and s5 present the lowest cdi for cd. 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 s1 s2 s3 s4 s5 h q drinking water samples as ni zn pb mn cu cd cr fig. 2. the hazard quotient (hq) variation for drinking water according to the hq values, the sample s1 and s2 present the highest risk exposure to metals, mainly as and cd. sample s4 is characterized with the highest zn concentration. the lowest hq values were obtained for sample s5, as shown in figure 2. the thq ranged from 0.01 (s5) to 0.13 (s1). according to the hq and the thq results, the inhabitants using the studied water samples as drinking water sources do not present potential non-carginogenic risk. 4. conclusion the mi indicates a possible metal contamination of drinking water samples from medias town and tarnava village, respectively water samples s1, s2 and s3. however the metal concentrations do not exceed the macs used for the mi calculation. the health risk assessment (cdi, hq and thq indices) indicates that the studied drinking water samples do not food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu, assessment of potential contamination and health risk associated with metals in drinking waters from copsamica region, food and environment safety, volume xiv, issue 3 – 2015, pag. 276 – 281 280 present potential non-carcinogenic risk, adverse or toxic effects for the inhabitants. 5. acknowledgments the research is financed by romanian financial authority cncs-uefiscdi, partnership project vulmin, contract no. 52/2012 and the sectorial operational programefor human resources development 2007-2013, co-financed by the european social fund, under the project posdru/159/1.5/s/133391 “doctoral and postdoctoral excellence programs for training highly qualified human resources for research in the fields of life sciences, environment and earth”. 6. references [1] ahmet d., fevzi y., tuna a.l., nedim o., heavy metals in water, sediment and tissues of leuciscuscephalus from a stream in southwestern turkey, chemosphere, 63: 1451– 1458, (2006). [2] muhammad s., shah m.t., khan s., arsenic health risk assessment in drinking, water and source apportionment using multivariate statistical techniques in kohistan region, northern pakistan, food and chemical toxicology, 48: 2855–2864, (2010). [3] muhammad s., shah m.t., khan s., health risk of heavy metals and their source apportment in drinking water of kohistan region, northern pakistan, microchemical journal, 98: 334343, (2011). [4] prasanna m.v., praveena s.m., chidambaram s., nagarajan r., elayaraja a., evaluation of water quality pollution indices for heavy metal contamination monitoring: a case study from curtin lake, miri city, east malaysia, environmental earth science, 67: 1978-2001, (2012). [5] arain m.b., kazi t. g., baig j.a., afridi h.i., sarajuddin, brehman k.d., panhwar h., arain s.s., co-exposure of arsenic and cadmium through drinking water and tobacco smoking: risk assessment on kidney dysfunction, environmental science and pollution research, 22 (1): 350-357, (2015). [6] sofuoglu s.c., lebowitz, m.d., o’rourke m.k., robertson g.l., dellarco m., moschandreas d.j., exposure and risk estimates for arizona drinking water, journal american water works association, 95: 67–79, (2003). [7] delpla i., benmarhnia t., lebel a., levallios p., rodrigueq m. j., investigating social inequalities in exposure to drinking water contaminants in rural areas, environmental pollution, 207: 88–96, (2015). [8] battaleb-looie, s., moore, f., malde, m.k., jacks, g., fluoride in groundwater, dates and wheat: estimated exposure dose in the population of bushehr, iran. journal of food and composition analysis, 29: 94–99, (2013). [9] hoaghia m.a., roman c., levei e.a., ristoiu d., footprint and direct impact of anthropic activities on groundwaters from medias area, studiauniversitatis babes-bolyai, chemia, lx (1): 109-118, issn 1224-7154, (2015). [10] xu, p., huang, s., wang, z., lagos, g., daily intakes of copper, zinc and arsenic in drinking water by population of shanghai, china, science of the total environment 362: 50–55, (2006). [11] zhang l., mo z., qin j., li q., wei y., ma s., xiong y., liang g., qing l., chen z., yang x., zhang z., zou y., change of water sources reduces health risks from heavy metals via ingestion of water, soil, and rice in a riverine area, south china, science of the total environment, 530–531: 163–170, (2015). [12] razak n.h.a, praveena s.m., aris a.z, hashim z., drinking water studies: a review on heavy metal, application of biomarker and health risk assessment (a special focus in malaysia), journal of epidemiology and global health, (2015). doi: 10.1016/j.jegh.2015.04.003. [13] edet a.e., offiong o.e., evaluation of water quality pollution indices for heavy metal contamination monitoring. a study case from akpabuyo-odukpani area, lower cross river basin (southeastern nigeria), geojournal, 57: 295304, (2002). [14] muntean e., muntean n., duda m., heavy metal contamination of soil in copsa mica area, proenvironment, 6: 469-473, (2013). [15] bakan g., boke o.h., tulek s., cuce h., integrated environmental quality assessment of kızılırmak river and its coastal environment, turkish journal of fisheries and aquatic sciences, 10: 453–462, (2010). [16] goher m.e., hassan a.m., abdelmoniem i.a., fahmy a.h., el-sayed s.m., evaluation of surface water quality and heavy metal indices of ismailia canal, nile river, egypt, egyptian journal of aquatic research, 40: 225-233, (2014). [17] tamasi g., cini r., heavy metals in drinking waters from mount amiata. possible risks from arsenic for public health in the province of http://www.sciencedirect.com/science/journal/02697491 http://www.sciencedirect.com/science/journal/02697491/207/supp/c food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 maria-alexandra hoaghia, oana cadar, erika levei, cecilia roman, lacrimioara senila, dumitru ristoiu, assessment of potential contamination and health risk associated with metals in drinking waters from copsamica region, food and environment safety, volume xiv, issue 3 – 2015, pag. 276 – 281 281 siena, science of the total environment, 327: 41– 51, (2004). [18] moghaddam m.h., lashkaripour g.r., dehghan p., assessing the effect of heavy metal concentrations (fe, pb, zn, ni, cd, as, cu, cr) on the quality of adjacent groundwater resources of khorasan steel complex, international journal of plant, animal and environmental sciences, 2 (1): 511-518, (2014.) [19] u.s. epa, “risk assessment guidance for superfund. vol. i. human health evaluation manual. part a. interim final,” office of emergency and remedial response, u. s. environmental protection agency, washington, dc, (1989). online source, accessed in 18.08.2015: http://www.epa.gov/oswer/riskassessment/ragsa/pdf /rags_a.pdf. [20] qaiyum m.s., shaharudi m.s., syazwan a.i., muhaimin a., health risk assessment after exposure to aluminum in drinking water between two different villages, journal of water resource and protection, 3: 268274, (2011). [21] u.s. epa, guidance for performing aggregate exposure and risk assessments, office of pesticide programs, washington, dc, (1999). online source, accessed in 18.08.2015: http://www.epa.gov/scipoly/sap/meetings/1999/febr uary/guidance.pdf. [22] u.s. epa integrated risk information system, a-z list of substances. online source accessed at 28.08.2015: http://cfpub.epa.gov/ncea/iris/index.cfm?fuseaction =iris.showsubstancelist. [23] u.s. epa, guidelines for carcinogen risk assessment, epa/630/p-03/001f, risk assessment forum, washington, dc, (2005). online source, accessed in 18.08.2015 http://www.epa.gov/reg3hwmd/risk/human/rbconcentration_table/documents/cancer_guide lines_final_3-25-05%5b1%5d.pdf. [24] kavcar p., sofuoglub a., sofuoglub s.c., a health risk assessment for exposure to heavy metals via drinking water ingestion pathway, international journal of hygiene and environmental health, 212: 216–227, (2009). microsoft word 3 journal nr 4 2010 din 8 noiembrie.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 78 fruit-based concentrated products, iron fortified, intended for prevention and diet therapy of iron deficiencies of vulnerable population groups luminiţa catană1, monica catană1, mioara negoiţă1, enuţa iorga1, gabriela lilios2 1national institute of research&development for food bioresources – iba bucharest, 021102, bucharest 2, 6 dinu vintila street, romania, e-mail: lumi_catana@yahoo.co.uk 2ovidius university, 900527, constanţa, 124 mamaia street, romania, e-mail: liliosgabriela@yahoo.com abstract. enrichment of food products by micro-nutrients is an essential element of strategies against nutritional deficiencies, such as iron deficiency, especially, in populations within developing countries. the addition of a micro-nutrient has to be done on the basis of scientific researches, so that its concentration in product is optimal for correction of nutritional deficiency, but, at the same time keeping the sensorial properties of product (appearance, colour, taste and smell). this paper presents some results of researches made to obtain two fruit-based concentrated products (apricots, plums) fortified with iron. as fortification agents, ferrous sulphate, ferrous lactate and ferrous gluconate were used, and the fortification levels were of 4 mg/100 g in the end product and 6.5 mg/100 g, respectively. the increase of iron bio-availability in the human body and, at the same time, assurance of an optimal acidity of fruit-based concentrated products should be done by addition of ascorbic acid into their composition. the fruit-based concentrated products fortified with iron were analysed from sensorial, biochemical and microbiological point of views. the used fortification agents have not modified the product sensorial characteristics (appearance, colour, taste and smell), in comparison with the control sample (jams non-fortified with iron). the iron content of the fortified products with iron, achieved within the national institute of research&development for food bioresources – iba bucharest, was determined through atomic absorption spectrometry (λ = 248.3 nm) from samples mineralised on dry way. the iron content of fruit-based concentrated products fortified with iron varied in the range 4.27 – 7.05 mg fe/100g. keywords: iron, fortified, food, content introduction enrichment of food products with micronutrients is an essential element of strategies against nutritional deficiencies, such as iron deficiency, especially, in populations within developing countries. the adding of one micro-nutrient has to be done on the basis of scientific researches, so that its concentration in product is optimal for correction of nutritional deficiency, but, at the same time keeping the sensorial properties of product [1]. at the international level, important researches on the development of iron fortification technologies of food products were made [2]. although, in comparison with meat products, the processed fruits have lower iron content, nutritionists recommend them in diet-therapy of iron deficiencies. researches achieved by nutritionists underline that, the value of a food product as iron source, is influenced much more by the chemical state of this element than by its total iron content. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 79 high solubility, easy ionization and ferrous valence are properties which increase iron assimilation grade. in the case of fruits, due to ascorbic acid content, the trivalent iron is reduced to bivalent iron, which favours its bio-availability in human body [3]. this paper presents the results of some researches made to achieve two fruit-based concentrated products (apricots, plums) fortified with iron: "apricots jam with nut, fortified with iron", "plums jam fortified with iron". experimental the experiments aimed at in order to obtain iron fortified food products, were made within the micro-production pilot plant of the national institute of research&development for food bioresources – iba bucharest. to obtain fruit-based concentrated products fortified with iron, we used the following raw materials and materials: apricots (the best hungary variety), plums (stanley variety), sugar, nut, ascorbic acid, ferrous sulphate, ferrous gluconate, ferrous lactate, glass jars of 370 ml and 220 ml capacity (twist – off closing system). in order to obtain food products fortified with iron, more experimental variants were performed, the variable factors being the following: iron fortification agent iron fortification level ascorbic acid fortification level to achieve fruit-based concentrated products, fortified with iron, the following technological flow was used: raw materials, materials and packaging reception, sorting, washing, cleaning division, product preparation, packaging preparation, dosing, closing, pasteurisation, cooling, conditioning of full jars, storage. the iron content of the fortified products with iron, achieved within the national institute of research&development for food bioresources – iba bucharest, was determined through atomic absorption spectrometry (λ = 248.3 nm), from samples mineralised on dry way [4]. results and discussion fortification of food products is legislated through regulation (ec) no. 1925/2006 of european parliament and council, on 20 december 2006. this document specifies: requirements concerning the addition of vitamins and minerals, restrictions concerning the addition of vitamins and minerals, and vitamins and minerals sources which can be added in food products. since iron deficiency and feripive anaemia have an increased incidence among vulnerable population groups (children, teen-agers, pregnant women, etc.), the achievement of some fruit-based processed products, fortified with iron, represents a necessity. taking into consideration all these aspects, our researches were performed at laboratory level and we achieved two fruitbased processed products, fortified with iron: "apricots jam with nut, fortified with iron" and "plums jam fortified with iron" at the same time, we achieved control samples of fruit-based processed products (products unfortified with iron). the fruit varieties used in experiments have superior sensorial characteristics (appearance, taste, flavour) and have a complex biochemical composition, excelling in glucids, vitamin c, minerals and ß – carotene content (only apricots). food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 80 figure 1. apricots – the best hungary variety figure 2. plums stanley variety table 1 biochemical analysis of apricots – the best hungary variety biochemical characteristics results dry soluble solids (brix) 12.6 titratable acidity (g malic acid/100 g) 1.15 soluble glucids (%) 9.85 proteins (%) 0.82 lipids (%) 0.1 ß–carotene (mg/100 g) 2.25 ascorbic acid (mg/100 g) 12.45 ash (%) 0.67 iron (mg/100 g) 0.60 table 2 biochemical analysis of plums – stanley variety biochemical characteristics results dry soluble solids (brix) 17.2 titratable acidity (g malic acid/100 g) 0.85 soluble glucids (%) 14.10 proteins (%) 0.72 lipids (%) 0.20 pectic substances (%) 0.92 ascorbic acid (mg/100 g) 14.15 ash (%) 0.54 iron (mg/100 g) 0.42 directive 2006/125/ec of european community commission on 5 december 2006, concerning cereal-based preparations and food products for children, intended for suckers and infants, imposes a maximum limit of iron addition in order to fortify: 3 mg fe/100 kcal. taking into consideration this recommendation, in the case of the two products "apricots jam with nut, fortified with iron" and "plums jam fortified with iron" we achieved the following fortification levels: 4 mg fe/100 g product and 6.5 fe mg/100 g product. to increase iron bio-availability in the human body and, concomitantly, to ensure optimal acidity of fruit-based concentrated products, fortified with iron, ascorbic acid was added in their composition, in the following concentrations:  70 mg ascorbic acid/100 g product, 95 mg ascorbic acid/100 g product (in the case of product "apricots jam with nut, fortified with iron")  90 mg ascorbic acid/100 g product, 125 mg ascorbic acid/100 g product (in the case of product "plums jam fortified with iron") for each fruit-based concentrated product fortified with iron we achieved, besides control samples (concentrated products, unfortified with iron), 12 experimental variants (3 iron fortification agents, 2 iron fortification levels and 2 ascorbic acid fortification levels). all of them were analysed from the sensorial, biochemical and microbiological point of views. the sensorial analysis of products "apricots jam with nut, fortified with iron" and "plums jam fortified with iron" proved that in the case of all experimental variants, the used fortification agents (ferrous sulphate, ferrous lactate, ferrous gluconate) have not determined modification of sensorial characteristics (appearance, colour, taste and smell), in comparison with the control samples (jams unfortified with iron). thus, the two fruitbased concentrated products, fortified with iron (fortification agents: ferrous sulphate, ferrous lactate, ferrous gluconate) comply food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 81 with the provisions of sr 3183:1990 „jams” from the sensorial point of view. according to the biochemical analysis, the product "apricots jam with nut, fortified with iron" achieved in 12 experimental variants has high nutritional value, excelling through its content in soluble glucids, minerals, ß – carotene and ascorbic acid. the iron content of the product "apricots jam with nut, fortified with iron" is in the range 4.48–7.05 mg/100 g, and that of ascorbic acid in the range 21.58–32.75 mg/100 g. after assessing the sensorial and nutritional characteristics of this product, we selected, as optimal variants, for each fortification agent, the following: v4 (fortification agent ferrous sulphate) v8 (fortification agent ferrous gluconate) v12 (fortification agent ferrous lactate) table 3 biochemical analysis of product "apricots jam with nut, fortified with iron" biochemical characteristics v4 v8 v12 dry soluble solids (brix) 68.0 68.0 68.1 titratable acidity (g malic acid/100 g) 1.44 1.46 1.42 soluble glucids (%) 64.14 64.00 64.27 proteins (%) 1.35 1.29 1.30 lipids (%) 2.24 2.14 1.94 pectic substances (%) 0.30 0.28 0.25 ß–carotene (mg/100 g) 1.60 1.51 1.42 ash (%) 0.608 0.622 0.603 iron (mg/100 g) 6.98 7.01 7.05 potassium (mg/100 g) 271.9 250.1 267.1 5 calcium (mg/100 g) 12.60 12.15 13.03 magnesium (mg/100 g) 10.03 9.42 10.21 ascorbic acid (mg/100 g) 30.42 31.67 32.75 according to the biochemical analysis, the product "plums jam fortified with iron", achieved in 12 experimental variants has high nutritional value, excelling through its content in soluble glucids, minerals and ascorbic acid. the iron content of the product "plums jam fortified with iron" is in the range 4.27 – 6.80 mg/100 g, and that of ascorbic acid in the range 28.95 – 40.15 mg/100 g. after assessing the sensorial and nutritional characteristics of this product we selected, as optimal variants, for each fortification agent, the following: v4 (fortification agent ferrous sulphate) v8 (fortification agent ferrous gluconate) v12 (fortification agent ferrous lactate) table 4 biochemical analysis of product "plums jam fortified with iron" biochemical characteristics v4 v8 v12 dry soluble solids (brix) 68.15 68.00 68.10 titratable acidity (g malic acid/100 g) 1.57 1.50 1.55 soluble glucids (%) 64.92 64.65 64.83 proteins (%) 0.51 0.55 0.44 pectic substances (%) 0.57 0.61 0.53 ash (%) 0.405 0.416 0.400 iron (mg/100 g) 6.80 6.75 6.70 potassium (mg/100 g) 153.15 152.28 154.4 5 calcium (mg/100 g) 11.10 10.85 11.42 magnesium (mg/100 g) 8.42 8.31 8.18 ascorbic acid (mg/100 g) 39.55 40.15 39.27 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 82 0 1 2 3 4 5 6 7 8 ir o n ( m g /1 00 g ) c v 1 v2 v3 v 4 v5 v6 v7 v8 v9 v10 v 11 v 12 variation of iron content in "apricots jam with nut, fortified with iron" product iron (mg/100g) v1v4 fe rrou s su lph ate v5v8 fe rrou s gl u con ate v9v12 ferrou s lactate figure 3. variation of iron content in "apricots jam with nut, fortified with iron" product 0 1 2 3 4 5 6 7 ir on (m g/ 10 0 g) c v 1 v 2 v 3 v 4 v 5 v 6 v 7 v 8 v 9 v 10 v 11 v 12 variation of iron content in "plums jam, fortified with iron" product iron (mg/100g) v1 v4 fe rrou s su l ph ate v5 v8 fe rrou s glu con a te v9 v1 2 fe rrou s lacta te figure 4. variation of iron content in "plums jam fortified with iron" product figure 5. "apricots jam with nut, fortified with iron" figure 6. "apricots jam with nut, fortified with iron" – experimental variant v1 (4 mg fe/100 g, fortification agent – ferrous sulfate) figure 7. "plums jam fortified with iron" figure 8."plums jam fortified with iron" – experimental variant v2 (4 mg fe/100 g, fortification agent – ferrous sulphate) the microbiological analysis has shown that fruit-based concentrated products, fortified with iron, achieved in 12 experimental variants and control samples, are comply with the legislation in force from the microbiological point of view. conclusions food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava year ix, no3 2010 83 1. at the national institute of research&development for food bioresources – iba bucharest we achieved two fruit-based processed products, fortified with iron ("apricots jam with nut, fortified with iron" and "plums jam fortified with iron"), we used ferrous sulphate, ferrous lactate and ferrous gluconate as fortification agents of the achieved products. 2. the iron content of the achieved fruitbased concentrated products varied in the range 4.27 – 7.05 mg fe/100g. 3. the used fortification agents have not determined modifications of sensorial characteristics (appearance, colour, taste and smell), of the fruit-based concentrated products, fortified with iron, in comparison with those of control products. acknowledgments the experiments were performed within the contract no. 51-092/18.09.2007, financed through programme 4 “partnerships in priority s&t domains” 2007 – 2013 – national centre for projects management. references 1. berger, j., 2003, enrichissement des aliments en micronutriments: élément d’une stratégie intégrée de lutte contre les carences en micronutriments, en particulier en fer, dans les pays en developpement. iième atelier international, voies alimentaires d’amelioration des situations nutritionnelles, vol. 11, p. 23-28. 2. mehansho, h., 2006, iron fortification technology development: new approaches, j. nutr. 136, p. 1059-1063. 3. mogoş, v.t., 1997, alimentaţia în bolile de nutriţie şi metabolism, vol. 1, editura didactică şi pedagogică, bucureşti 4. bordei d., 2007, controlul calităţii în industria panificaţiei – metode de analiză, editura academica, galaţi microsoft word 3 journal fia nr 1 2010 final_36-41.doc journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 40 study on identifying butter faking by substitution with pork fat or with margarine rodica rotar ştefan cel mare university of suceava, faculty of food engineering, 13 universitaţii st., 720229, suceava, romania, e-mail: rodicas@usv.ro abstract: experimental study’s objective is to set the relevant parameters that allow the identification of the presence of animal or nonanimal fat substitutes in butter as a fake by substitution, but also the indication of butter subtituient dose in faked sample. evaluated parameters for comparative study of the characteristics of the original butter butter blank and the ones of the faked by substitution with pork fat at different rates were: changes in the melting point of the glycerides (after bomer limprick), refractive index, iodine index, reichert meissl and polenske index. in the second part of the experimental study, for the category of falsified butter by adding margarine,the parameters evaluated for comparative study of the characteristics of original butter blank, falsified by the addition of margarine, in various percentages, and margarine were: reichert meissl index and iodine index. of all parameters, iodine index varies not relevanlyt for the purpose of identification or dosing of mix-ups pork fat and butter rates. iodine index varies relevanlyt for identification or dosing the margarine sustitute in butter. iodine index varies relevanlyt for the purpose of identification or dosing mix-ups with margarine. since that can not be considered a single parameter determining the dose of substitutes, the subsequent research will be able to establish a mathematical model, with the variables comprising the parameters settled to be relevant and sufficient to indicate the dose of substitution, applicable to both categories of fakes by substitution used in the experimental study. key words: melting point, refractive index, iodine index, reichert meissl index introduction of animal fat, butter is the most exposed to fraudulent practices by partial substitution with various types of low-value fat, animal (pork or beef fat) or vegetable fat as margarine or seeds oil [1]. value given by cow's milk fat parameters are conditioned by the presence of lower saturated fatty acids with 4 to 10 carbon atoms, absent from other fat, and by the low share of fatty acids with a large number of carbon atoms [2]. identifying butter faking by substitution with pork fat is based on changes in the melting point of glycerides and fatty acids in falsified samples. the constant value of the melting point of the sample fat is due to the relatively constant of the chemical composition (nature and proportion of acylglycerols and fatty acids) of an animal fat [3]. the melting point can be considered as a criterion for checking the purity of fat. margarines are fatty food products, containing min 80% fat, obtained by mixing fluid oil, hydrogenated and solid or semisolid (vegetable or animal) and the aqueous phase (dispersed) up to 16.6%. margarine also contains a number of additives: emulsifiers, vitamins, flavoring and preservatives [4]. with a similar composition to that of butter, margarine is a potential alternative, frequently used in faked butter. since natural fatty acids are normally found in the cis form, by hydrogenation are partly converted as trans form, their presence in butter is a proof of faked butter by substitution with margarine or other hydrogenated fats [5]. sensory analysis is not always relevant; whereas faked butter may contain a number of compounds apparently natural (β-carotene, diacetyl, acetic acid) giving journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 41 the sensory qualities similar to original butter, so only the physical-chemical analysis is capable to certify and identify faked butter [6]. both determining the nature and structure of acylglycerols and fatty acids as well as the indices characterizing the fat phase, particularly saponification indices, reichert meissl and polenske indices, provides the most secure information for deceleration faking, other indices (melting point, refractive index, index iodine) gives only helpful information, because margarines value depends mainly on the degree of hydrogenation of fats [7]. experimental for the experimental study on the identification of butter faking by substitution with pork fat were made determinations on 6 samples of butter (unfaked and faked at different rates of pork fat substitution). evaluated parameters for comparative study of the characteristics of the original butter butter blank and the samples of falsified butter by substitution with pork fat at different rates were: changes in the melting point of the glycerides (after bomer limprick), refractive index, iodine index, reichert meissl and polenske indeces. in the second part of the experimental study, for the category of falsified butter by adding margarine, parameters evaluated for comparative study of the characteristics of original butter blank, falsified by the addition of margarine, in various percentages, and margarine were: reichert meissl index, and iodine index. falsifications were applied by dosing and mixing in bagmixer. to identify the presence of pork fat in butter bomer limprick method was used based on differentiation of melting points; it involves sample crystallization with anhydrous acetone and ether, to determine the melting point of the glycerides and to separate and determine the melting point of fatty acids. butter is the only animal fat that contains volatile lower saturated fatty acids with 410 carbon atoms (butyrate, caproate, caprylic, caprynic). the first two may be charged with water vapor (butyrate, caproate), while the other two (caprylic, caprynic) may be charged with water vapor, but are insoluble in it. these qualities are prerequisites to determine reichert-meissl index and polenske index. according to the data presented in the literature, the index correlated with butyric acid content and caproate reichert meissl index, at cow fat (21 36) indicates a higher content of these fatty acids comparatively with caprylic and caprynic, insoluble in water, represented by polenske index (1.5 3.5). in pork fat four volatile lower saturated fatty acids with 4-10 carbon atoms (butyrate, caproate, caprylic, caprynic) are found in reduced content, as revealed by reichert meissl index (0.3 to 0.9) and polenske index (max 0.5). therefore, for detection of butter faking by substituting with pork fat, in the experimental study we considered it relevant to determine reichert meissl index. reichert meissl index (index of soluble volatile fatty acids) is the number of mg of potassium hydroxide required to neutralize the volatile fatty acids, soluble in water, from 1g fat and polenske index (index of insoluble volatile fatty acids) is the number of mg of potassium hydroxide required to neutralize the volatile fatty acids, insoluble in water, from 1g fat. validation of these two indices is the basic criterion for assessing the authenticity of butter or milk fat, so a reliable means to detect substitution of foreign fat. if partial substitution, the values of these two indices will fall below the lower limits of normal, and if the total substitution these values tend towards zero. melting point of fat, determined by journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 42 capillary method, depends on the length of the fatty acids components carbohydrate chain and on the degree of unsaturation. thus, the values of the melting point of saturated fatty acids increase as the carbohydrate chain length increases. the presence of one or more double bonds leads to decreasing melting point (introduction of a double bond to c 18:0 leads decreasing melting point from 69.6 º c to 13.4 ° c). steric configuration is also extremely important; cis type double bonds have a weaker crystal setting density and melt at a lower temperature than the trans forms. refractive index determination is a reliable method that could indicate the origin of fat, but is rather uncertain in case of fat mixture. refractive index is changed when a higher water content than the maximum allowed as for grease alteration. determination was made with abbe-zeiss refractometer. iodine index, determined by hanus method, is the chemical principle of fatty acids to react with halogens, whom addition to double links. depending on the degree of unsaturation, unsaturated fatty acids are additional 2, 4 or 6 halogen atoms, while saturated fatty acids behave differently. the iodine index means the quantity of iodine, in grams, which may be additioned to fatty acids from 100 g fat. saponification index identifies possible fraud, but is also used to determine molecular weight fatty acids. the amount of koh needed for saponification is variable, depending on the molecular weight of fatty acids components of fat. the higher molecular weight will be, the less will be needed koh to neutralize and saponificate, as the number of acid molecules contained in one gram of fat will be lower. results and discussion for the experimental study on the identification of butter faking by substitution with pork fat were made determinations on 6 samples of butter, according to table 1. falsifications were applied by dosing and mixing in bagmixer (for samples with substitution). evaluated parameters for comparative study of the characteristics of unfaked butter blank and faked by the substitution with pork fat at different rates, were: changes in the melting point of the glycerides (after bomer limprick), iodine index, reichert meissl index and polenske index (represented in figure 1) and refractive index (represented in figure 2). table 1 evaluated samples for identification of butter faking by substitution with pork fat evaluated samples percentage of pork fat substitution no pork fat content butter sample (unfaked butter) (blank) faked by substitution sample (p1) 4% faked by substitution sample (p2) 8% faked by substitution sample (p3) 12% faked by substitution sample (p4) 16% faked by substitution sample (p5) 20% to determine the melting point variation of glycerides and fatty acids, 50g fat from each sample were subjected to crystallization(5successive crystallization). melting points of glycerides increase with increasing rate of substitution of butter with pork fat. chemical composition of animal fat is relatively constant (nature and proportion acylglycerols and fatty acids), so the melting point of fat has constant values, which is a criterion for checking the purity of fat. an 8% substitution percentage sample has a variation of the melting point of glycerides and fatty acids significantly (66.6) compared with blank sample (61.5) and even with the sample with 4% rate of substitution (63.6) indicating that any substitution of a higher percentage than 4% is identifiable with the relevant parameter variation of the melting point of glycerides journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 43 and fatty acids. iodine index values are not relevant for dosing or identification purpose of butter substitution with pork fat. 34 32 30 28 27 2628 30 31 32 33 34 61,5 63,6 66,6 67,1 68,7 69,8 0 10 20 30 40 50 60 70 tg+2d (oc), iodine index, reichert-meissl index blank (0%) 4% 8% 12% 16% 20% evaluated samples (percentage of pork fat substitution) tg+2d iodine index reichert meissl index figure 1: changes in parameters of evaluation and identification of falsifications by substituting butter with pork fat data represented in figure 1, indicate the correlation with butyric acid content and caproate reichert meissl index; if unfaked butter, indicates higher content of these acids (34) than samples of faked butter (26), corresponding to the maximum percentage of substitution (20%). expressed by the refractive index, refraction can be the basis for assessing the origin of fat, is a reliable method if relates to one fat and relatively uncertain if fat mixture. refractive index is a relevant parameter identifying butter faking by substitution with pork fat. refractive indices of samples submitted evaluations to identify faked butter by substitution with pork fat in different percentages are represented in figure 2: 1,4524 1,453 1,4531 1,4534 1,4538 1,454 1,4515 1,452 1,4525 1,453 1,4535 1,454 refractive index (20/40oc) blank (0%) 4% 8% 12% 16% 20% evaluated samples (percentage of pork fat substitution) figure 2: refractive index of samples submitted evaluations journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 44 refractive index of butter the blank is 1.4524. faked butter with 8% substitution with pork fat, with refractive index 1.4530, is slightly detectable by determining this parameter. maximum rate of substitution of sample 6 shows a refractive value index 1.4540. for the experimental study on the identification of faked butter by substitution with nonanimale fat (margarine) were assessed 4 samples of butter and margarine, according to table 2: tabelul 2 evaluated samples for identification of butter faking by substitution with margarine evaluated samples percentage of substitution with margarine no margarine content butter sample (unfaked butter) (blank) faked by substitution sample (p1) 30% faked by substitution sample (p2) 60% margarine (p3) 100% evaluated parameters for comparative study of the characteristics of unfaked butter blank, faked butter by substitution with margarine and margarine were iodine index and reichert meissl index. iodine index of butter blank is 34 and easy detectable by determining the iodine index is faking with 30% margarine subsitute, sample in which iodine index is 68. maximum rate of substitution of sample 3 shows an iodine value index 128. according to the data represented in figure 3, reichert meissl index if unfaked by substitution butter with margarine, indicates higher content of these acids (34) compared with samples of faked butter (13), according to the percentage of substitution (60%) and minimum value, 0.6, corresponding to maximum percentage of substitution (100%). 34 22 13 0,6 34 68 93 128 0 20 40 60 80 100 120 140 iodine index, reichert-meissl index blank (0%) 30% 60% margarine (100%) percentage of substitution with margarine iodine index changes depending on the percentage of substitution reichert meissl index changes depending on the percentage of substitution figure 3: changes in parameters of evaluation and identification of falsifications by substituting butter with margarine journal food and environment safety of the suceava university – food engineering, year ix, no1 2010 45 conclusion sample with 8% percentage of substitution of butter with pork fat indicates a significant variation of the melting point of glycerides and fatty acids, compared with the blank sample and even with the sample with 4% percentage of subtitution indicating that any subtitution with a higher than 4% percentage is identifiable with the relevant variation of the point of melting of glycerides and fatty acids. refractive index of evaluated blank butter is 1.4524. faked butter with 8% percentage of substitution with pork fat is slightly detectable by determining the refractive index (1.4530). maximum rate of substitution of sample 6 shows a maximum value for refractive index. reichert meissl index, if unfaked butter indicates higher content of butyric acid content and caproate compared with index values at samples of faked butter by substitution with pork fat, corresponding to the maximum percentage of substitution (20%). of all parameters, iodine index varies not relevantly for the purpose of identification or dosing of mix-ups pork fat and butter rates. since this is the same type of animal fat, this parameter can not be very closely related to faking by substitution of butter with pork fat. iodine index varies relevant for identification or dosing the margarine sustitute in butter. iodine index varies relevant for the purpose of identification or dosing mix-ups with margarine. iodine index of butter blank is 34 and easy detectable by determining the iodine index is faking with 30% margarine subsitute, sample in which iodine index is 68. maximum rate of substitution of sample 3 shows an index of iodine 128. the index which is correlated with butyric acid content and caproate – reichert meissl index if unfaked by substituting butter with margarine, indicates higher content of these acids compared to samples of faked butter, according to the percentage of substitution (60%,) and minimum value, corresponding to maximum percentage of substitution (100%). since that can not be considered a single parameter determining the dose of substitutes, the subsequent research will be able to establish a mathematical model, with the variables comprising the parameters settled to be relevant and sufficient to indicate the dose of substitution, applicable to both categories of fakes by substitution used in the experimental study. references 1. m. bulancea – autentificarea, expertizarea şi identificarea falsificărilor produselor alimentare, ed. academica, galaţi, 2002 2. i. bondoc, e.v. sindilar – controlul sanitar veterinar al calitaţii şi salubritatii alimentelor, vol. 1, ed. ion ionescu de la brad, iaşi, 2002 3. n. popescu, s. meica – bazele controlului sanitar veterinar al produselor de origine animală, ed. diacon coresi, bucureşti, 1995 4. e. sindilar – controlul igienic al produselor şi subproduselor de origine animală. vol. ii, ed. i.n.s.c.r., iaşi, 2000 5. g. georgescu – laptele şi produsele lactate, ed. ceres, bucureşti, 2000 6. c. ciotau – controlul şi expertiza alimentelor şi depistarea falsurilor, editura universităţii din suceava, 2009 7. c. savu – igiena şi controlul produselor de origine animală, ed. semne, bucuresti, 2008 title …………………… 340 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 42015, pag. 340 344 impact of food waste on climate change * cristina ghinea1,2 , maria gavrilescu2,3 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania, cristina.ghinea@fia.usv.ro 2“gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof. dr. doc. dimitrie mangeron street, 700050 iasi, romania 3academy of romanian scientists, 54 splaiul independentei, ro-050094 bucharest, romania *corresponding author received november 7th 2015, accepted december 18th 2015 abstract: food waste represents an issue that needs to be solved in order to achieve sustainable development and reduce its environmental impacts. intensive utilization of resources sustained many decades of growth in wealth and wellbeing in europe. today, efforts are being made to get efficient use and development of resources, thus complying with the eu vision for 2050: all resources will be sustainably managed, waste will be managed as a resource and climate change milestones will be reached etc. in this work we focus on the actions of sustainable food production and consumption necessary to diminish the impacts of food waste on the environment. it is considered that food wastage arises from an inefficient use of ecosystem services but also can be considered as an important contributor to climate change. the greenhouse gas (ghg) calculations in the early stages of decision making process, for different waste management strategies, could provide us the quantification and comparison of ghg emissions for different categories of food waste and allow the understanding of the efficiency of proper waste management on ghg emissions. in this paper we have applied a specific tool to calculate ghg emissions generated by food waste considering three waste management scenarios developed for iasi city, romania, based on the targets established by legislation. keywords: environmental impacts, food, ghg, waste. 1. introduction food waste is considered one of the most sensitive issues at global level. a significant amount of food waste is produced by consumers in the higher income countries, while the population from other countries are suffering from the lack of food (sub-saharan africa) [1, 2]. approximately 1.3 billion tonnes per year of food are lost or wasted globally [3-5], while 89 million tonnes of food are wasted each year in the european union. about 222 million tonnes of food are wasted every year by the consumers from the industrialized countries [1]. according to [4], fruits and vegetables loss has the highest percentage (44%), followed by roots and tubers (20%) and cereals (19%), while the lowest percentage is registered for meat (4%), oilseeds and fish and seafood (2%) (fig.1). it is well known that, from initial agricultural production to final household consumption, food is wasted or lost [5]. the activities responsible for food loss in the supply chain are: farming and husbandry (agriculture), drying, sieving, http://www.fia.usv.ro/fiajournal mailto:cristina.ghinea@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 cristina ghinea, maria gavrilescu, food waste impact on climate change, food and environment safety, volume xiv, issue 4 – 2015, pag. 340 – 344 341 milling, grinding, mixing, cooking, moulding etc. (food processing and manufacturing), markets, grocers, bakers, supermarkets (retail), household, restaurants, cafes, institutions (consumption) [6]. the food waste quantities from manufacturing, households, retail and food services produced in eu27 are illustrated in fig. 2. fig. 3 presents the food waste discarded from manufacturing, household, retail and food services in eu27 and romania in 2010. cereals 19% roots and tubers 20% fruits and vegetables 44% oilseeds and pulses 3% meat 4% milk 8% fish and seafood 2% loss and waste by weight, % fig. 1. food losses for different commodities (100%=1.3 billion tonnes) [4] 70 76 8 25 food waste amounts in eu27, kg/capita manufacturing households retail food service fig. 2. food waste quantities produced in eu27 in 2006 [7] 0 5 10 15 20 25 30 35 40 45 manufacturing household retail food service % food waste, 2010 romania eu27 fig. 3. food discarded in eu and romania (according to [8]) at the household level, the food waste generated: avoidable (slices of bread, apples), possibly avoidable (bread crusts, potato skins) and unavoidable food waste (egg shells, banana skins) [7]. according to [9] the quantities of food discarded from households differed between 5% -30%. the negative environmental impacts, but also social and economic impacts of food waste are becoming increasingly apparent as the food waste amount is growing, while global food security is becoming more pressing [6]. considering the food waste importance and the impacts on the environment, in this paper we have developed three scenarios considering two food waste treatments (composting and anaerobic digestion) that have been evaluated in terms of climate change. 2. policy and regulations according to [10] there are 52 eu legislative acts impacting on food waste including: 29 regulations, 10 directives, 3 decisions and 10 communications. the complexity of the food waste challenge is reflected by eu legislative framework. legislation is potentially implying: food waste generation, food waste management, food waste reduction, food use optimization etc. [10]. the european commission sets out a 50% food waste reduction target for 2020 [11]. european parliament proposed in 2012 a 50% prevention target on avoidable food waste by 2025 [12]. at european level there is a number of directives on waste management, the first one was established in 1975 waste framework directive (75/442/eec) which aims: to harmonize national measures concerning waste, to encourage the preparation of waste management plans, to prevent generation of waste and encourage recycling. other general guidelines have been established as well: hazardous waste food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 cristina ghinea, maria gavrilescu, food waste impact on climate change, food and environment safety, volume xiv, issue 4 – 2015, pag. 340 – 344 342 (91/686/eec), waste transport (259/93eec), incineration of hazardous waste (94/67/eec), waste incineration and existing facilities (84/369/eec and 89/429/eec), waste disposal (99/31/ec) etc. [13]. these represent the most important directives for waste management planning at the municipal level [14]. the european legislation on waste has been adopted by the romanian legislation, and finally the national waste management strategy (nwms) was founded to set waste management objectives. they aim at creating the framework for developing and implementing an integrated waste management and the national waste management plan (nwmp) representing nwms implementation plan [15]. to ensure sustainable development in terms of solid waste management it is necessary that society wastes to be managed in an efficient manner, environmentally effective, economically affordable, and socially acceptable [16]. 3. current situation in 2011, the composition of household waste in romania was as follows: paper and cardboard 9.3%, glass 3.62%, metals 2%, plastics 8.6%, organic waste (biodegradable) 57.82%, wood 1.7% and others 16.9% [17]. in iasi, the household waste composition included paper and cardboard (7.68%), glass (4.35%), plastic (6.17%), metals (1.78%), organic waste (biodegradable, food waste) (47.15%) and others (32.87%) [18]. it can be observed that organic waste is the fraction with the highest percentage followed by paper and plastic. in iasi a total of 120 containers were placed for the collection of organic waste or bio-waste [18]. in recent years, municipal biodegradable waste percentage dropped from 64% in 1998 to approx. 48% in 2012. according to the regulation 1999/31/ec on the landfill of waste, transposed at national level by gd 349/2005 on waste disposal, with subsequent amendments, the targets for the reduction of dumped biodegradable waste, relative to the total amount (expressed in weight) produced in 1995, are: july 16, 2010 to 75%; july 16, 2013 to 50%; july 16, 2020 to 35% [18]. the amount of biodegradable waste generated in the year 1995 in iasi county was of 115,659 tons and, therefore, according to the corresponding targets the waste quantity dumped should be diminished by: 16 july 2010 28915 tons; july 16, 2013 57830 tons; july 16, 2020 75178 tons [18]. there is only one composting station in iasi county (10000 t/year capacity) and no other facilities for the treatment of biodegradable waste, which implies that currently, biodegradable waste and similar are eliminated shared with all waste by land filling [19]. the project “integrated waste management system in iasi county” also plans to build a mechanical-biological treatment plant [19]. the capacity will be of 95000 t per year and the plant will consist of a mechanical treatment step and biological treatment: aerobic decomposition, maturing platform for further biodegradation [19]. 4. food waste and (ghg) greenhouse gas emissions an amount of 180 kg per person food is wasted in the eu according to [11]. it is considered that 17% of direct greenhouse gases emissions are caused by food and drink value chains. a significant amount of food is still suitable for human consumption [11], meaning that over 60% of food waste is avoidable [20]. it is estimated that, over the whole life cycle of food wasted, almost 1.9 t co2 eq./t are emitted in europe [20]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 cristina ghinea, maria gavrilescu, food waste impact on climate change, food and environment safety, volume xiv, issue 4 – 2015, pag. 340 – 344 343 three scenarios for waste management were developed considering the targets imposed by the legislation mentioned above: in the first scenario (s1), it was considered a recycling rate of food waste of 25%; in s2 the recycling rate was of 50% and in the last scenario (s3) the recycling rate of food waste was set to 65%. an annual waste quantity of 694 kg solid waste/cap/yr with 40% food waste was considered. to calculate ghg emissions of scenarios we used swm-ghg calculator, a tool developed by ifeu institute germany [21]. this tool calculates the total ghg emissions in co2 equivalents. the calculation method follows the life cycle assessment (lca) method, but it is not a lca study fully fledged [21]. ghg emissions for the recycled waste fractions are calculated based on the mass of waste recycled and a ghg emission factor. the average electricity demand for composting is calculated as 30 kwh/t organic wastes and for diesel demand 1.5 l/t organic waste [21]. the country specific electricity mix was chosen. co2 emissions from electricity generation was of 337 g/co2 eq./kwh, which only refers to direct co2 emissions from fuel combustion [21]. the results are illustrated in figs. 4-5. the ghg emissions caused by food waste composting are indicated by the first bar from the figure (emissions – debits), the emission savings are represented by the second bar (avoided emissions or credits, negative values) and the net results signify the difference between debits and credits. it can be observed that emission savings increase with the waste recycling rate. the results obtained provide additional information for the decision making process. -8.00e+03 -6.00e+03 -4.00e+03 -2.00e+03 0.00e+00 2.00e+03 4.00e+03 6.00e+03 8.00e+03 t c o 2 e q ./ y r s1 s2 s3 ghg emissions of food waste composting emissions avoided emissions net results fig. 4. ghg emissions from food waste composting -5.00e+03 -4.00e+03 -3.00e+03 -2.00e+03 -1.00e+03 0.00e+00 1.00e+03 2.00e+03 3.00e+03 4.00e+03 5.00e+03 t c o 2 e q ./ y r s1 s2 s3 ghg emissions of food waste digestion emissions avoided emissions net results fig. 5. ghg emissions from food waste digestion 5. conclusions in this paper, three scenarios were developed considering different food waste recycling rates and evaluated with swmghg calculator from climate change perspective. the results obtained with the swm-ghg calculator provide an accurate quantitative approximation of the ghg impacts of different strategies. the results show that waste recycling rate has a significant influence on emission savings. also the values of emissions from composting are higher than those from anaerobic digestion. in further studies the lca methodology will be applied to evaluate food waste not only in terms of climate change but also considering other impacts categories such as: acidification, eutrophication, abiotic depletion, photochemical ozone formation etc. the lca provides detailed information about food impact on climate change and not food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 cristina ghinea, maria gavrilescu, food waste impact on climate change, food and environment safety, volume xiv, issue 4 – 2015, pag. 340 – 344 344 only. also, the whole food waste cycle will be taken into consideration and the results will be compared. different scenarios will be developed considering other treatment methods such as mechanical-biological treatment, aerobic/anaerobic biological treatment for waste valorization as raw materials etc. 6. references [1]. ec, counting the cost of food waste: eu food waste prevention, european union committee, 10th report of session 2013–14, (2014) [2]. ilea m., chiciudean g., chiciudean d., analysis regarding food waste and consumption in romania, annals of oradea, ecotoxicology, animal husbandry and food industry technologies, xiii/a: 293-296, (2014) [3]. fao, food wastage footprint impacts on natural resources, online at: http://www.fao.org/docrep/018/i3347e/i3347e.pdf, (2013) [4]. lipinski b., hanson c., lomax j., kitinoja l., waite r., searchinger t., reducing food loss and waste, working paper, instalment 2 of creating a sustainable food future, washington, dc: world resources institute, online at: http://www.worldresourcesreport.org, (2013) [5]. vanham d., bouraoui f., leip a., grizzetti b., bidoglio g., lost water and nitrogen resources due to eu consumer food waste, environmental research letters, 10, doi:10.1088/1748-9326/10/8/084008, (2015) [6]. papargyropoulou e., lozano r., steinberger j.k., wright n., bin ujang z., the food waste hierarchy as a framework for the management of food surplus and food waste, journal of cleaner production, 76: 106–115, (2014) [7]. wrap, household food and drink waste in the uk, banbury, uk, isbn: 1-84405-430-6, (2009) [8]. kretschmer b., smith c., watkins e., allen b., buckwell a., desbarats j., kieve d., technology options for recycling agricultural, forestry and food wastes and residues for sustainable bioenergy and biomaterials, report for the european parliament, stoa, as part of the study ‘technology options for feeding 10 billion people’, ieep: london, (2013) [9]. stefan v., romanian consumers’ perceptions and practices regarding food waste, master thesis, aarhus school of business, aarhus university, (2011) [10]. fusions, review of eu legislation and policies with implications on food waste, final report, bologna, (2015) [11]. ec, roadmap to a resource efficient europe, communication from the commission to the european parliament, the council, the european economic and social committee and the committee of the regions, brussels, (2011a) [12]. ec, analysis associated with the roadmap to a resource efficient europe, part i. commission staff working paper, brussels, (2011b) [13]. schiopu a.m., apostol i., hodoreanu m., gavrilescu m., solid waste in romania: management, treatment and pollution prevention practices, environmental engineering and management journal, 5: 451-465, (2007) [14]. den boer e., den boer j., jager j., waste management planning and optimization, handbook for municipal waste prognosis and sustainability assessment of waste management systems, ibidemveralg, stuttgart, (2005) [15]. nwmp, national waste management plan, ministry of environment and water management, bucharest, romania, (2004) [16]. ghinea c., waste management models and their application to sustainable management of recyclable waste, phd thesis. gheorghe asachi technical university of iasi, romania, (2012) [17]. anpm, national state of the environment report – 2012, ministry of environment and climate change, national environmental protection agency, bucuresti, romania, (2012) [18]. anpm, report regarding environment in iasi county for the year 2014, environmental protection agency iasi, romania, (2014) [19]. iasi county council, integrated waste management system in iasi county, report on environmental evaluation study, (2011) [20]. ec, assessment of resource efficiency in the food cycle, final report, european commission, (2012) [21]. ifeu, tool for calculating greenhouse gases (ghg) in solid waste management (swm), manual swm-ghg calculator, (2009) microsoft word 0a first pages 2_2017.docx stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvi, issue 2 30 june 2017 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania title …………………… 239 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 239 244 the influence of regular and genetically modified soybeans on postnatal development of rats n. n. omelchenko1, g. v. dronik2, *і. а.winkler3, м. s. rogozynskyi1, v. а. kucheriava1 1 chernivtsi faculty, national technical university “kharkiv polytechnic institute”, chernivtsi, ukraine 2 bucovina state agricultural research station, naas of ukraine, chernivtsi, ukraine 3 bucovina state medical university, chernivtsi, ukraine *corresponding author: winkler@bsmu.edu.ua received 19th october 2017, accepted 21th december 2017 abstract: ukraine is one of the european and world leaders in soybean cultivation. according to some estimation, 30-90 % of the crops area is used currently for cultivation of genetically modified varieties. as modified soybean areas are expanding, the potential nutritional threats related to this product should be thoroughly assessed. the influence of nutrition ration consisting of 20 % of the thermo-treated genetically modified soybeans on postnatal development of rats has been investigated over two generations in comparison with nutrition by regular thermo-treated beans. the number of alive and dead newborns, average number of offspring and the survival rate has been calculated as well as general estimation of physical development of the newborn rats was made. the experimental results prove that the reproductive function of rats and the offspring development are not seriously influenced by nutrition with genetically modified soybeans within the first and second generations. no statistically reliable difference was found between the characteristic parameters of the experimental and control groups, which remained within their regular physiological limits. however, some decrease in the suckling age rats’ number was registered in the experimental group. keywords: regular soybean, genetically modified soybean, rats, postnatal development, survival rate. 1. introduction as molecular biology, biotechnology and gene engineering reach new advance, construction of new plants varieties and cattle breeds becomes easier and more available for agricultural and industrial users avoiding traditional selection methods and procedures. moreover, it is quite feasible now to construct even the genotypes that never existed before by introducing the alien genes into dna of a given variety. this way new functions or characteristics of the variety can be obtained. such genetically modified organisms (gmo) are becoming more and more popular in many fields of medicine, agriculture, cattle breeding, food production and processing. the widest introduction of gmo in crop production is known for soybean, maze, cotton, rape, sugar beet, papaya, pumpkin, paprika, tomato, rice, potato, plum, haricot bean, alfalfa, wheat, groundnuts, mustard, cauliflower and chili pepper [1, 2]. soya is extensively used in many fields of food production and agriculture because of its useful merits. soybean consists of 36-48 % of proteins, 13-27 % of oils, 17-34 % of carbohydrates and brings other useful components like vitamins (thiamin, riboflavin, pyridoxine, folic acid, choline, vitamin e and c), macroand micro elements (k, р, ca, mg, fe, zn, mn and se) and bioactive compounds (phospholipids, tocopherols, phytoestrogens and some others) [3-5]. high nutritional values and optimal composition ensure wide distribution of soybeans as a component of various http://www.fia.usv.ro/fiajournal mailto:winkler@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 n.n. omelchenko, g.v. dronik, і.а.winkler, м.s. rogozynskyi, v.а. kucheriava, an influence of regular and genetically modified soybeans on postnatal development of rats, food and environment safety, volume xvi, issue 4 – 2017, pag. 239 – 244 240 foods and food additives. soya is used in human nutrition directly as well as soybean oil, milk, tofu cheese and as an additive for a wide variety of food such as chocolate and sausage. it is reported that about 60 % of regular food consist of some soybean products [4]. some gene modifications (gm) are being made in order to achieve higher crop productivity and better resistance of soya against diseases, pests, herbicides, abiotic stresses and other negative influences. first commercial gm soya varieties have been introduced in 1996 while according to the estimation of international service for the acquisition of agri-biotech applications [6], current crop area occupied by various gm plants has reached 185.1 mln ha in 2016. more than 50 % of this area is occupied by four cultures: soybean, cotton, maze and rape). more than 30 countries are involved in the gm crops cultivation while more than 60 countries – in the consumption. ukraine is one of world leaders and the european leader in gm soya cultivation. current estimation shows that 30-90 % of all soya crops in ukraine is occupied by the gm varieties [7] still remaining beyond strict government control of the seed grain quality and amount. this estimation has also been proven by independent analysis of the cattle forage and additives performed in 2013-2015 [8]. further uncontrolled spreading of gmo can provoke more or less deeper ingression of the modified soya in human food. there are still vivid discussions on possible threats and food safety of gmo. as seen from analysis of hundreds scientific papers dealing with this problem reviewed in [9], there is still no doubtful evidence of distinct negative effects of gmo or gmocontaining food on human health. there is still no correlation found between consumption of the gmo-containing food and digestion, kidney diseases, diabetes, cancer, autism, obesity and various allergy symptoms. on the other hand, the idea of potential threat of genetically engineered products is also popular and has many supporters stating that these components should be considered as dangerous as long as no indisputable safety evidences are found. moreover, some primary deviations were found in the viscera morphogenesis as a result of nutrition with gmo components. chronic intoxications and some diseases have also been reported for next generations of the lab animals [10-18]. that is why the discussion of possible safety/unsafety of gmo is still open and requires more data and evidences. this work deals with investigation of possible influence of nutrition with traditional and genetically modified soybeans on postnatal development in the first and second generations of rats. 2. experimental all experiments were carried out with three generations of the wistar line rats: parents (f0), first (f1) and second (f2). the source population consisted of males and females aged 3-3.5 months. the experiment lasted for 9 months. the animals were kept in a vivarium, in the standard plastic boxes with thermo-treated wood shavings substrate at 20-22 0c, relative humidity 50-60 % and under the standard day/night regime. all rats were divided into three groups: one control and two experimental. all the groups were formed by random selection of the animals with the only account of the body weight. each group consisted of 12 rats: 8 females and 4 males. the control group animals were fed according to the standard vivarium ration. the second group animals were fed with the food containing 20 % of thermo-treated soybeans of variety chernivetska 9. unlikely to the traditional procedure, the soybeans were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 n.n. omelchenko, g.v. dronik, і.а.winkler, м.s. rogozynskyi, v.а. kucheriava, an influence of regular and genetically modified soybeans on postnatal development of rats, food and environment safety, volume xvi, issue 4 – 2017, pag. 239 – 244 241 steeped in water during 12 hours then boiled during 60 minutes and finally dried at 115-125 0c. the third group animals were fed using the above procedure but with the genetically engineered soya roundup ready, line gts 40-3-2: 20 % of their ration consisted of this gm material. all rations were optimized according to the standard norms and requirements and provided to the respective groups throughout the entire experimental period. the reproduction capacity characteristics and some postnatal development data during first two months of life were investigated in all the groups. following postnatal development characteristics were controlled: ratio between alive and dead youngs, average offspring value, visual estimation of general physical development, body weight and survival rate. further development of the rats was assessed by the auricle opening time, body hair eruption time, teething and eyes opening time. male and female rats were combined for the fertilization with ratio 2:1 for a single estrous period (5 days). the baby rats were put apart from their mothers on the 30th day and provided with the same adult ration. further experiments were carried out with the descendants obtained from different mothers in order to randomize the investigation and avoid incest. 3. results and discussion investigation of reproductive capacity/fertility is one of integral indicators for the hygienic evaluation of various potentially unsafe or threatening processes or phenomena. this capacity is very sensitive function that can be disordered under influence of many unfavorable impacts and effects. on the other hand, evaluation of reproductive capacity is very labor and time demanding procedure, which puts obstacles on wide introduction of this method to the food and food additives safety analysis. this approach is recommended only for an optional usage in case its necessity is obvious. even after more than 20 years long efforts in investigation of possible adverse effects of gmo on the human and cattle health, there is still not enough evidence to support or reject this assumption. that is why results of investigation of reproductive capacity should shed more light on this problem. it has been found that the pregnancy time was unchanged in both experimental groups comparing to the value of the control group. all baby rats were delivered after 21-23 days of pregnancy. an average offspring number was 9.4 ± 2.4 individuals in the control group f1 generation; 8.0 ± 2.1 individuals – in the second group and 8.5 ± 1.5 individuals in the third group. no deviations were found in physical development of young rats from all the groups [19]: the auricle opening time was 3-4 days; body hair erupted after 5-6 days; teething time was 9-10 days and eyes opening time was 15-16 days. no visible mutilations were registered. survival rate of the f1 rats was quite high in all the groups (see table 1). it is easy to see that the corresponding values for different groups are rather close. death rate for the first five days was 5.3 % (control group), 6.2 % (second group) and 7.3 % (third group). same rates for the next 25 days period were correspondingly 4.2 %, 8.3 % and 12.7 %. gender ratio in the control group was balanced while insignificant prevalence of the young males was registered in both experimental groups. it has been shown [20] that physical condition of the female rats is the key factor governing reproductive capacity of the rodent. fat deposits and body weight are growing during the pregnancy time to store enough metabolic resource to be used for lactation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 n.n. omelchenko, g.v. dronik, і.а.winkler, м.s. rogozynskyi, v.а. kucheriava, an influence of regular and genetically modified soybeans on postnatal development of rats, food and environment safety, volume xvi, issue 4 – 2017, pag. 239 – 244 242 table 1. survival rate indexes for the f1 offspring group number of baby rats, individuals first 5 days survival rate next 25 days survival rate individuals % individuals % і 75 71 94.7 68 95.8 іі 64 60 93.8 55 91.7 ііі 68 63 92.7 55 87.3 gaining body weight during pregnancy is the key factor for the successful pregnancy ratio, fertility, offspring gender balance, baby rats’ survival ratio, the rate of their development and overall reproductive capacity in the population. insufficiently fed females bring more male babies. this gender regulation occurs due to the embryonic mortality and results in lowering of the female number in the population, which decreases its reproductive potential. it has been found that the body weight increase rate stayed within normal values for all three groups over early postnatal time and mixed feeding period. no difference has been found between the first generation of rats raised under traditional and genetically modified soybean feeding. all indexes of their development didn’t exceed corresponding normal values. survival rate parameters remained high for the f2 generation animals as well (see table 2). for instance, the initial five days death percentage was 6.7 % (control group); 6.1 % (second group) and 8.9 % (third group) while the next 25 days death percentages were correspondingly 7.1 %, 8.1 % and 16. 4 %. table 2. survival rate indexes for the f2 offspring group number of baby rats, individuals first 5 days survival rate next 25 days survival rate individuals % individuals % і 60 56 93.3 52 92.9 іі 66 62 93.9 57 91.9 ііі 67 61 91.1 51 83.6 it is seen that postnatal death rate for the third group f2 animals was somewhat higher than that in the second and control group. this effect can be caused by a long term influence of phytoestrogens and other bioactive compounds coming to the organism from gm soybeans. normal embryonal development can be disturbed by these compounds resulting in the birth of debilitated or unviable individuals. this effect is especially dangerous for the reproductive hormones balance both for males and females [21] and, according, to some recent data affects primarily normal functioning of hypothalamus and hypophysis through the regime of growth hormone generation [22]. all adverse effects of phytoestrogens disappear after stopping of feeding with gm materials. an average f2 offspring number in the control group was 7.5 ± 1.7 individuals; in the second group – 8.3 ± 1.8 and in the third group – 8.4 ± 1.9. as seen from these data, the f2 offspring number in the control group was somewhat lower than that in the other groups but these values remained comparable and within the physiological norms. no relevant difference has been found between the groups in the f1 baby rats’ weight. this parameters also stayed within physiological norms for the corresponding age (m (group i) = 5.7 ± 0.54 g; m (ii) = 5.2 ± 0.49 g; m (iii) = 5.3 ± 0.52 g). same food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 n.n. omelchenko, g.v. dronik, і.а.winkler, м.s. rogozynskyi, v.а. kucheriava, an influence of regular and genetically modified soybeans on postnatal development of rats, food and environment safety, volume xvi, issue 4 – 2017, pag. 239 – 244 243 pattern was registered also for the f2 animals’ weight after 28 days (see fig. 1). no statistically relevant disturbances in the offspring gender balance have been registered. besides, overall physical development of all f2 baby rats was found satisfactory: the auricle opening time was 3-4 days; body hair eruption time was 4-6 days; teething time was 9-10 days and eyes opening time was 15-17 days. all these values also lay within corresponding physiological norms [19]. no difference was found between animals’ development for the control and experimental groups. 0 10 20 30 40 50 0 5 10 15 20 25 30 age, day m a s s , g control ii group iii group fig. 1. body weight dynamics for the f2 baby rats 4. conclusions no relevant evidence of negative or positive effect of feeding the rats with a food containing 20 % of thermo-treated genetically modified soya on reproductive capacity and physical conditions of the rats were found. all the animals showed similar rates and indexes of physical shape and postnatal development parameters despite different feeding regimes over the experimental and control groups. the parameters remained within physiological norms with the exception of some decrease in the suckling age rats number in the population fed with the genetically engineered soybean ration. 5. references [1]. grodzynsky d., et al. gene engineering investigation in nas of ukraine, bulletin of nas of ukraine, 8: 3-12, (2006) (in ukrainian). [2]. eldyshev yu., konov a., modern biology: myths and reality, “tidex”, moscow, 196 p (2004) (in russian). [3]. anderson j.w., smith b.m., washnock c. s., cardiovascular and renal benefits of dry bean and soybean intake, amer. j. clin. nutrit., 70(3): 464-474, (1999). [4]. soares l.l., lucas a.m.m., boaventura g.t., can organic and transgenic soy be used as a substitute for animal protein by rats? brazil, j. med. bio. res., 38: 583-586, (2005). [5]. petibskaya v.s., soya: chemical composition and usage, “poligraf-yug”, maykop, 432 p (2012) (in russian). [6]. isaaa brief 52-2016. global status of commercialized biotech. gm crops: 2016. https://www.isaaa.org/resources/publications/briefs/ 52/download/isaaa-brief-52-2016.pdf (accessed oct. 11, 2017). [7]. chekhov a.s., demand and supply on the ukrainian soybean market, economy field, 106: 127-134, (2016) (in ukrainian). [8]. kushnir g.v., control of the genetically modified plants as a food safety guarantee, sci. bulletin of lviv nat. uni. veterinary med and biotech., 18(3(71)): 167-169 (2016), (in ukrainian). [9]. genetically engineered crops: experiences and prospects, https://www.nap.edu/catalog/23395/geneticallyfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 n.n. omelchenko, g.v. dronik, і.а.winkler, м.s. rogozynskyi, v.а. kucheriava, an influence of regular and genetically modified soybeans on postnatal development of rats, food and environment safety, volume xvi, issue 4 – 2017, pag. 239 – 244 244 engineered-crops-experiences-and-prospects (accessed oct. 11, 2017). [10]. yermakova i.v., an influence of the soya with epsps cp4 gene on reproductive and physiological functions of rats over two generations, modern problem. sci. and edu., 5: 15-21, (2009) (in russian). [11]. seralini g.e. et al, retracted: long term toxicity of a roundup herbicide and a rounduptolerant genetically modified maize, food and chem. toxicol., 50(11): 4221-4231, (2012). [12]. malatesta m. et.al, a long-term study on female mice fed on a genetically modified soybean: effects on liver ageing, histochem. cell. biol., 130: 967–977, (2008). [13]. brasil f.b. et al, the impact of dietary organic and transgenic soy on the reproductive system of female adult rat, anat rec. (hoboken), 292(4): 587-594, (2009). [14]. konovalova m.a, blinov v.a., morphology indexes and peculiarities of the mice blood ferments spectra in case of feeding with genetically engineered soya, proceedings of 2 russian symposium on transgene plants physyology and biosafety problems., 48 (2007) (in russian). [15]. lukashenko t.m., an influence of the soybean additives on the reproductive capacity of rats, bulletin of grodno state medical uni., 2(26): 189-191, (2009) (in russian). [16]. dolaychuk o.p., fedoruk r.s., kovalchuk i.i., an influence of natural and genetically modified soybean components on some immune and reproductive capacity indexes of female rats, physiol. j., 59(2): 65-70, (2013) (in ukrainian). [17]. kotsyumbas g.i., samsonuyk i.m., shkil m.i., reproductive capacity and morphological condition of the rat uterus after feeding with the mixtures containing 20 % of genetically engineered soya, sci bulletin lviv nat. uni. veter. med. and biotech.: veterinary sci., 19(73): 126-130, (2017) (in ukrainian). [18]. samsonyuk i.m., morphological conditions of gastrointestinal tract of the first generation of rats fed with genetically engineered soya, sci. bulletin inst. animal. biology and state inst. veterinary medicine and nutrit. additives., 14(3-4): 238243, (2013) (in ukrainian). [19]. zapadnyuk i.p et al. experimental animals: breeding, raising and experimental usage, kiev, high school, 243-271 (1983) (in russian). [20]. nazarova g.g., maternal influence on the descendant’s adaptability and population size. author’s abstract of d. sc. thesis in zoology, novosibirsk, 51 (2007) (in russian). [21]. hooper l., et al. effects of soy protein and isoflavones on circulating hormone concentrations in preand post-menopausal women: asystematic review and metaanalysis, hum. reprod. update, 15(4): 423-440, (2009). [22]. mesiano s., et al. phytoestrogens alter adrenocortical function: genistein and daidzein suppres glucocorticoid and stimulate androgen production buy cultured adrenal cortical cells, j. clin. endocrinol. metab., 84(7): 2443-2448, (1999). 1. introduction it is seen that postnatal death rate for the third group f2 animals was somewhat higher than that in the second and control group. this effect can be caused by a long term influence of phytoestrogens and other bioactive compounds coming to the organis... an average f2 offspring number in the control group was 7.5 ± 1.7 individuals; in the second group – 8.3 ± 1.8 and in the third group – 8.4 ± 1.9. as seen from these data, the f2 offspring number in the control group was somewhat lower than that in th... no relevant difference has been found between the groups in the f1 baby rats’ weight. this parameters also stayed within physiological norms for the corresponding age (m (group i) = 5.7 ± 0.54 g; m (ii) = 5.2 ± 0.49 g; m (iii) = 5.3 ± 0.52 g). same pa... no statistically relevant disturbances in the offspring gender balance have been registered. besides, overall physical development of all f2 baby rats was found satisfactory: the auricle opening time was 3-4 days; body hair eruption time was 4-6 days;... fig. 1. body weight dynamics for the f2 baby rats 4. conclusions 363 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 -2018 , pag. 363 371 the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties *ana leahu 1 , cristina ghinea 1 , cristina damian 1 1food engineering faculty, stefan cel mare university of suceava, 13 university street, 720229 suceava, romania, *analeahu@gmail.com *corresponding author received 15th october 2018, accepted 28th december 2018 abstract: the objective of this research was to investigate the effect of substituting fructose by different levels of inulin (3 and 6%) and the addition of psyllium fibers (3 and 6%) on the quality attributes of lingonberry ice cream. five formulations comprised of the control sample using fructose and the other four samples applying inulin and psyllium fibers, respectively, were examined to determine: ph, total acidity, sugar content, viscosity, water activity, color analysis, and sensory properties in order to find their optimum ratios. a positive linear correlation between psyllium fibers addition and apparent viscosity of the lingonberry ice cream was observed. supplementation of inulin fibers revealed that lingonberry ice cream with 3 % inulin had the most appealing sensory characteristics. the results suggest that the addition of inulin and psyllium fibers as fiber-enriching agents can be used in the ice cream industry in order to fortify the diet. keywords: lingonberry, inulin, psyllium fibers, color analysis, sensory attributes ice cream. 1. introduction ice cream, popular throughout the world, is one of our favorite desserts of high sugar content. this product is consumed especially during the warmer seasons, because of its cooling properties and refreshing sensation produced. ice cream, a complex dairy product, is produced in a wide variety of flavors and colorscustard/french or reduced fat and no-fat versions [1]. fruit ice cream may be made by mixing different ingredients including milk, cream, fresh/frozen fruit, fruit juices, dietary fibers, stabilizers, emulsifiers, artificial or natural flavoring and colorants. the lingonberry (vacciniumvitis-idaea l.), a small shrub and perennial, belongs to the ericaceae family, is most widely spread around the high regions of the eastern carpathian mountains. lingonberries (vacciniumvitis-idaea l.) are a valuable natural resource of bioactive compounds such as antioxidants, vitamins and minerals. many studies have shown the pro-health effects (reduced risks of diseases such as cancers, diabetes, kidney and urinary disorders, cardiovascular diseases) of antioxidants from diets with lingonberry (vacciniumvitis-idaea l.) [2.3]. according to ho et al. [4], tannins isolated from v. vitis‐idaea l with antimicrobial activity could potentially be used for the treatment of periodontal disease [4]. lingonberries are consumed regularly in the season, fresh (sometimes as juice and tea) or preserved (dried fruits, jam, marmalade, sorbets), and also used as dietary supplements in food industry, cosmetics and medicine. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 364 in the last several decades, the research has focused on identifying the natural sources of dietary fibers, defined as portions of plant foods that were resistant to digestion by human digestive enzyme, so their applicability in the food industry have increased. additionally, dietary fibers play an important role in the treatment of various diseases such as obesity, coronary heart disease [5], type 2 diabetes [6] and colorectal cancer [7]. the recommended daily dietary fiber intake depends on age and gender: 87 g per day for women younger than 50, 21 g per day for women older than 50; 38 g per day for men younger than 50, and 30 g per day for men older than 50 [8]. inulin belongs to the carbohydrates called fructans and is distributed in various plants such as chicory roots, jerusalem artichoke, dahlia tubers, asparagus, leek, onion, bananas, wheat and garlic [9]. psyllium is a water-soluble fiber, from the plantago ovata plant, which has the satiety-inducing effect, similar to other fiber sources. supplementation with soluble fiber, such as psyllium fibers in daily diets and lifestyle modifications may provide cardiovascular benefits via its effect on blood lipids, improving glycemic control, and has other health benefits, such as obesity and metabolic syndrome (mets) risk factors and body weight management [10]. supplementation with soluble and moderately fermentable dietary fiber such as psyllium improves the global symptoms of irritable bowel syndrome (ibs) [11]. other studies have shown that total cholesterol was significantly lower in the psyllium (psy) groups compared to control at 3 and 6 months, for the participants who have consumed 5 g waterinsoluble fiber of their supplement before meals [12]. thus, the general objective of this study was to produce and evaluate the characteristics of ice cream with inulin and psyllium fibers and contribute to the manufacturing of a new nutritional and functional ice cream, respectively. 2. materials and methods 2.1. samples and storage conditions the lingonberry fruit is collected from vacciniumvitis-idaea l., in the spontaneous flora in suceava county, romania. the fruits were harvested at full ripeness from the shrubs, they were cleaned manually to remove all foreign material and broken seeds, and stored at 5 0c. ice cream was prepared according to the traditional recipe, mixing gently the ingredients: raw milk (with 3.5 % fat), fat content of ice cream was standardized to 9% using milk cream (with 40% fat), fructose syrup (purchased from a local store) and lingonberry fruit (16 % of the total component content). the mixture was held at 85 0c not less than 30 s for pasteurization and cooled to 43 0c. in this time, pasteurized mixture was divided to five lots in 200 ml plastic cups. five ice cream formulas were produced the control using fructose and the other four samples containing inulin (3 and 6%), psyllium fibers (3 and 6%), respectively. all the samples were gently stirred for 1 min and aged overnight at 4 0c. each ice cream samples was frozen at -24 0c in a freezer and stored at -18 0c. three complete replications of the experiment were performed. 2.2. physico-chemical determinations the ph values of ice cream samples were measured using a digital ph meter at 25 0c calibrated with ph 4 and 7 buffers (hanna instruments, italy). titratable acidity was determined by titrating samples with 0.1n naoh solution up to ph 8.3, and it was expressed as a percentage of lactic acid (1 ml of 0.1 n naoh = 0.009 g of lactic acid). https://www.sciencedirect.com/topics/medicine-and-dentistry/plantago-ovata https://www.sciencedirect.com/topics/medicine-and-dentistry/plantago-ovata https://www.sciencedirect.com/topics/medicine-and-dentistry/cardiovascular-system food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 365 the sugar content was determined using an abbe refractometer. viscosity measurements were carried out on the ice cream samples at 4°c, with a brookfield viscometer (brookfield engineering inc, model rvdv ii pro+) at 20 rpm with rv5 spindle. measurement of water activity (aw) at 20 °c was carried out using an aw meter analyzer (aqua lab model) was used with a 0.5 g sample. three replications were conducted for each sample. 2.3. color measurement l* (whiteness/darkness), a* (redness/greenness), and b* (yellowness/blueness), color parameters of the samples was conducted using a minolta chroma meter (model cr-400/410, minolta camera co. ltd., japan), [13]. 2.4. sensory analysis of the ice cream samples was carried by thirty-three (33) panelists comprising of students of the faculty of food engineering in the “stefan cel mare” university of suceava using a 9point hedonic scale (1 disliked extremely; 9 liked extremely), [14]. color, taste, smell, texture and consistence, flavor and odor, and overall acceptability of ice cream samples were evaluated. 2.5. statistical analysis the results were shown as an arithmetic mean (± standard deviation). all statistical calculations were performed using minitab software and one-way anova was applied. p-values<0.05 were considered statistically significant. 3. results and discussion 3.1. chemical and rheological analysis the results of proximate analysis for the five lingonberry ice creams are shown in table 1. as expected, there were differences between the levels of apparent viscosity in the four different types of ice creams, where the apparent viscosity actual of control, 600 mpa*s, and lingonberry ice cream fortified with psyllium fibers 7560, and 10560 mpa*s, respectively. the results indicate that the addition of dietary fiber increases the size and weight of casein micelles by the formation of aggregates caused by dissolution of colloidal calcium phosphate [15]. however, no differences were found among the five types of lingonberry ice creams between ph values. table 1. physico-chemical characterization of ice cream (mean ± sd, n = 3) characterization s1(control) s2 s3 s4 s5 ph 6.31±0.01 6.21±0.01 6±0.003 6.17±0.03 6.3±0.003 acidity (% lactic acid) 0.031±0.0003 0.033±0.0005 0.037±0.0005 0.02±0.005 0.017±0.0005 viscosity (mpa*s) 600±0.58 600.66±0.88 800±0.58 7560±1.15 10559.66±0.33 sugar 0brix 97.18±0.72 117.04±0.58 122.94±0.53 133.24±0.62 153.32±0.66 aw 0.978±0.0005 0.978±0.001 0.985±0.0005 0.984±0.0005 0.986±0.0003 results are the means of three analyses. s1 lingonberry ice-cream control; s2lingonberry ice cream fortified with 3% inulin; s3lingonberry ice cream fortified with 6% inulin; s4lingonberry ice cream fortified with 3% psyllium fibers; s5 lingonberry ice cream fortified with 6% psyllium fibers; aw water activity. the titratable acidity varied significantly from 0.037% to 0.017% of lactic acid, and the tendency of this value to decrease was observed in the samples with high contents of psyllium fibers. the sugar content differed among samples, and it was observed that an increased content of psyllium fibers content resulted in higher values of the lingonberry ice creams (table 1), which ranged from 97.18 to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 366 153.32. the water activity ranged significantly from 0.978 to 0.986, and the increase in the content of dietary fiber protein resulted in a lower water activity. 3.2. instrumental color analysis color measurements for the control samples were compared to samples containing 3 or 6 % of sugar substituted by either inulin, and to samples containing 3 or 6 % psyllium fibers, color parameters of the lingonberry ice creams are presented in the tables 3 and 4. with regard to lightness (l∗), the sample with 6 % psyllium fibers presented high values (l∗>75), indicating that the ice cream could be considered bright. psyllium fibers added positive values for parameter b*, indicating the presence of the yellow pigment inside and at the sample surface. table 2. color parameters for surface lingonberry ice cream samples sample l * a * b * h * c * s1 62.5 0.5 0.1 11.31 0.51 s2 57.6 2.6 -4.3 -58.78 5.02 s3 70.7 9.6 -19.2 -63.43 21.47 s4 66.9 -6 27.9 -77.86 28.54 s5 89.6 -1.6 5.7 -74.31 5.92 chroma (c∗), considered the quantitative attribute of colorfulness, the higher the chroma value, the higher the color intensity perceived by human vision [16]. lingonberry ice creams with % psyllium fibers were significantly darker as compared to all treatment samples including the control. inulin fiber was responsible for increasing the values of a*coordinate (redness) and hue angle, while psyllium fibers showed a tendency to decrease the redness of assays. tabel 3. color parameters for lingonberry ice cream samples inside sample l * a * b * h * c * s1 71.9 8.7 -20.5 -67.04 22.27 s2 73.4 4.2 -10.3 -67.8 11.12 s3 72.4 3.1 -8.1 -69.04 8.67 s4 71.2 -7.7 24.1 -72.28 25.3 s5 89.1 -5.5 27.7 -78.78 28.24 3.3. sensory analysis a comparison of the sensory rating test scores for the evaluated ice cream samples is shown in fig. 1. assay number 3 (6% inulin) and 5 (6% psyllium fibers), got the highest mean scores for acceptability (8.7 and 8.8, respectively), suggesting that even the ice cream with higher proportions of fibers were well accepted. https://www.sciencedirect.com/science/article/pii/s0308814610000944#tbl3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 367 fig. 1. sensory quality of ice cream samples; s1 lingonberry ice-cream control; s2lingonberry ice cream fortified with 3% inulin; s3lingonberry ice cream fortified with 6% inulin; s4lingonberry ice cream fortified with 3% psyllium fibers; s5lingonberry ice cream fortified with 6% psyllium fibers similar results were reported for fat-free dairy desserts, the addition of inulin to these desserts increased sweetness, thickness and creaminess [17]. ice cream containing 15 % cape gooseberry (physalis peruviana l.), known as golden berry in english speaking countries, was the highest-scored of color and appearance, flavor, body and texture, and general acceptability characteristics by the panelists [18]. 3.4. statistical evaluation one-way analysis of variance (anova) was used to establish if there are statistically significant differences between ice cream samples based on the physicochemical parameters analyzed. in order to examine (using interval plotting) and to compare (using the tukey and 95% confidence method) the group of means and to determine how well the model matched the data (calculation of s, r2 and r2 pred.) and whether the model meets the assumptions of the analysis, anova was applied. the following null hypothesis was established: all means are equal, and as an alternative hypothesis: at least one average is different. the significance level was set as follows: α = 0.05. the confidence level was 95, and the type of confidence interval was two-sided. equal variants for analysis were assumed. the comparison procedure used is tukey. results showed that p-values for all indicators are lower than  value which means that the factors are statistically significant, the null hypothesis is rejected and not all group means are equal. fig. 2a illustrates the confidence intervals for the mean: ph vs types of samples and it can be observed that the highest average has the control sample, and the less average has the sample s3. the bars are overlapped for samples s and s5 meaning that the means are not different for these samples and can be grouped in one category (a). the same result was observed also in the case of sample s2 and s4 which can be grouped in one category b, different from sample s3. from fig. 2b it can be seen that the highest average value had s3, followed by sample s2, while the lowest average value had s5. the difference between the means is likely to be significant in s1-s3 samples because the intervals are interleaved, they can be grouped into a single category (a), and samples s4 and s5 can be grouped into another category c. all samples belong to different categories when sb vs types of sample are investigated (fig. 2c). it can be 7.5 8 8.5 9 aspect color texture (firminess) flavouraroma taste acceptability s1 s2 s3 s4 s5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 368 seen that the highest average value had s5, followed by sample s4, while the lowest average value had s1. the lowest average for aw has the control sample (s1), instead the highest average value has s5, followed by sample s3 (fig. 2d). the last three samples can be grouped into one category a, while the other two are grouped into another category b. the confidence intervals for the difference between s5-s1 samples range from -0.0854 to 0.0587, and for s4-s2 samples from -0.1154 to 0.0287 (fig. 3a). these ranges contain zero, indicating that the difference between these means is not statistically significant. the differences between the means for the other samples do not include the zero value, indicating that the difference between these means is statistically significant. the intervals that contain zero, when acidity is evaluated (fig. 3b), are: s2–s1 (-0.01054; 0.01387), s3-s1 (0.00654; 0.01787), s4-s1 (-0.02354; 0.00087), s3-s2 (-0.00820; 0.01620) and s5-s4 (-0.01520; 0.00920). for sb no interval does not contain zero resulting that means are significantly different, while for aw only s2-s1, s4-s3, d5-s3, s5-s4 included zero value. the individual confidence level was 99.18%. a) b) c) d) fig. 2. intervals (95% confidence interval for mean): a) ph, b) aciditiy (a), c) sugar 0brix (sb), d) water activity (aw) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 369 fig. 3. tukey simultaneous 95% cis, differences of means for a) ph, b) acidity in order to find out how well the model matches, r2 data were calculated, the s values were also determined to show how well the model describes the response so that the following values were obtained, for: ph: s = 0.02, r2 = 96.38%, r2 (pred) = 91.86%. the r 2 value shows that the factor explains 96.38% of the response variation, while the standard deviation between the data points and the fixed s values is 0.02 units; acidity: s = 0.004, r2 = 81.40%, r2 (pred) = 58.16%; sugar 0brix (sb): s = 1.08, r2 = 99.77%, r 2 (pred) = 99.48%; water activity (aw): s = 0.0012, r 2 = 92.23%, r 2 (pred) = 82.52%. a) b) c) d) fig. 4. normal probability plot: a) ph, b) v, c) sb, d) aw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 370 it can be considered that the model fits the data when high values were obtained for r2 close to 100. from the normal probability graph, it can be observed if the data set is roughly distributed normally. when the points in this graph form an almost linear pattern, it shows that the normal distribution is a good model for the evaluated data as in the case of fig. 4 b, d. 4. conclusions thus, the aim of the present research was to investigate the effects of inulin and psyllium fibers levels on physical and sensory characteristics of lingonberry icecream. the increase of dietary fiber concentration led to products with better physical and sensory properties. the addition of 6 % inulin improved viscosity, and had effect on sensory properties of the samples. the obtained results showed that the ph value of the lingonberry ice cream samples registered minor changes, depending on the added fiber content. also, the results showed that the investigated factors are statistically significant. lingonberry ice-cream fortified with dietary fibers could be used as a good source of fibers for consumers. 5. references [1]. kilara a., chandan r.c., hui y.h., ice cream and frozen desserts, handbook of food products manufacturing, 593-633, (2007) [2]. grace m.h., esposito d., dunlap k.l., lila m.a., comparative analysis of phenolic content and profile, antioxidant capacity, and antiinflammatory bioactivity in wild alaskan and commercial vaccinium berries, journal of agricultural and food chemistry, 62:4007-4017, (2013) [3]. bujor o.c., extraction, identification and antioxidant activity of the phenolic secondary metabolites isolated from the leaves, stems and fruits of two shrubs of the ericaceae family, doctoral dissertation, universitéd' avignon, (2016) [4]. ho k.y., tsai c.c., huang j.s., chen c.p., lin t.c., lin c.c., antimicrobial activity of tannin components from vacciniumvitis‐idaea l, journal of pharmacy and pharmacology, 53:187191, (2001) [5]. wolk a., manson j.e., stampfer m.j., colditz g.a., hu f.b., speizer f.e., ... & willett w.c., long-term intake of dietary fiber and decreased risk of coronary heart disease among women, jama, 281:1998-2004, (1999) [6]. schulze m. b., liu s., rimm e.b., manson j.e., willett w.c., hu f.b., glycemic index, glycemic load, and dietary fiber intake and incidence of type 2 diabetes in younger and middle-aged women, the american journal of clinical nutrition, 80:348-356, (2004) [7]. bingham s.a., day n.e., luben r., ferrari p., slimani n., norat t., ... & tjφnneland a., dietary fibre in food and protection against colorectal cancer in the european prospective investigation into cancer and nutrition (epic): an observational study, the lancet, 361:1496-1501, (2003) [8]. elleuch m., bedigian d., roiseux o., besbes s., blecker c., attia h., dietary fibre and fibre-rich by-products of food processing: characterisation, technological functionality and commercial applications: a review, food chemistry, 124:411-421, (2011) [9]. shoaib m., shehzad a., omar m., rakha a., raza h., sharif h. r., ... & niazi s., inulin: properties, health benefits and food applications, carbohydrate polymers, 147:444-454, (2016) [10]. jane m., mckay j., pal s., effects of daily consumption of psyllium, oat bran and polyglycoplex® on obesity-related disease risk factors: a critical review, nutrition, 57:84-91 (2018) [11]. el-salhy m., ystad s.o., mazzawi t., gundersen d., dietary fiber in irritable bowel syndrome, international journal of molecular medicine, 40:607-613, (2017) [12]. pal s., ho s., gahler r.j., wood s., effect on insulin, glucose and lipids in overweight/obese australian adults of 12 months consumption of two different fibre supplements in a randomised trial, nutrients, 9:91, (2017) [13]. leahu a., damian c., oroian m., ropciuc s., rotaru r., influence of processing on vitamin c content of rosehip fruits, scientific papers animal science and biotechnologies, 47: 116-120, (2014) [14]. sagdic o., ozturk i., cankurt h., tornuk f., interaction between some phenolic compounds and probiotic bacterium in functional food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 ana leahu, cristina ghinea, cristina damian, the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties, food and environment safety, volume xvii, issue 4 – 2018, pag. 363 371 371 ice cream production, food and bioprocess technology, 5: 2964-2971, (2012) [15]. alvarez v.b., wolters c.l., vodovotz y., ji t., physical properties of ice cream containing milk protein concentrates, journal of dairy science, 88:862-871, (2005) [16]. granato d., ribeiro j.c.b., castro i.a., masson m.l., sensory evaluation and physicochemical optimisation of soy-based desserts using response surface methodology, food chemistry, 121:899-906, (2010) [17]. tárrega a., costell e., effect of inulin addition on rheological and sensory properties of fat-free starch-based dairy desserts, international dairy journal, 16:1104-1112, (2006) [18]. erkaya t., dağdemir e., şengül m., influence of cape gooseberry (physalisperuviana l.) addition on the chemical and sensory characteristics and mineral concentrations of ice cream, food research international, 45:331-335, (2012) microsoft word 6 olena krasulya_corectat.doc 165 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 22018 pag. 165 175 the investigation of the potentional complex from plantago major to coagulate milk proteins olena grek1, *olena krasulya1, larisa chubenko1, alla tymchuk1 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str. 68, kyiv, ukraine, olena_krasulya@ukr.net *corresponding author received 5th january 2018, accepted accepted 26th june 2018 abstract: the possibility of coagulating milk proteins by wild plants was confirmed. the prospects of this direction in the technology of milk – protein concentrates obtaining was provided. the potential ability of plantago major leaves to coagulate milk proteins is considered in the article. the mass fraction of dry matters in the juice was within (4.5±0.23). it was found, that the addition of plant coagulant in the amount of 7...9 % from the general amount of milk allows obtaining a clot with such indicators: active acidity – 6.3…6.4 units, moisture mass fraction – 66.00…69.00 %, water – retaining capacity – 60.61...64.26 %. organoleptic indicators of samples are suitable for consumption. the yield of the protein clot was 184.0...232.7 g from 1000 ml of normalized milk mixture. the state of moisture in herbal – protein clots was established by the method of infrared spectroscopy. the intensive zone of с=о – groups valence oscillations in the area of 1638 cm-1 was observed. this fact indicates the presence of tightly applied moisture. microstructural study of clot samples, which were obtained by the use of plantago major juice showed more developed spatial configuration of gel frame as compared with the control sample. high structural and mechanical characteristics and therefore water-retaining capacity of the clot were studied. the obtained results are the base for snack cheese products, semi-finished and processed cheeses technology decisions. keywords: plantago major leaves’ juice, coagulation of milk proteins, protein –herbal clot, clot’s quality indicators. 1. introduction the inventions of milk – protein products of general purpose with use of local plant raw materials are actual. these raw materials serve as an enrichers and technological ingredients. the production of different types of cheeses is accompanied by the milk proteins coagulation. destabilization of the colloid state of the casein micelle occurs under the action of proteolytic enzymes and the change of the ph value. milk gel is a structure that consist of a gel frame filled with serum. a characteristic feature of the gel is the synthesis – reducing of the water-retaining ability under the influence of temperature, ph and mechanical action [1-3]. the process of milk coagulation occurs as follows: χ-casein binds free calcium, what causes casein deposition. as a result, the milk coagulates and is divided into two fractions – protein clot (casein in insoluble form) and whey (containing soluble proteins) [4]. the different methods of coagulation are used for the cheese making: acid, acidrennet (the most widespread), thermo-acid and thermocalcium. enzyme milk coagulation – is modification of casein micelles with a help of imited hydrolysis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 166 of casein by the enzyme. this process is accompanied with micelles’ aggregating [5]. various factors have contributed to the search for alternative sources of milk collection, which include higher cheese prices, aspects of restrictions on use related to religion, diet (vegetarianism), or the prohibition of the use of veal sticks in some countries (france, germany, and the netherlands) [6]. a series of studies, that were aimed at milk-clotting enzyme determination for provision of the classical rennet enzyme replacement were conducted [7]. milk coagulation can be carried out by proteases capable of providing χ-casein’ hydrolytic decomposition. the conditions of such coagulants’ industrial introduction were formulated. also the specific physico-chemical and technological properties which should provide the appropriate characteristics of protein concentrates were determined. it is necessary that the proteolytic activity of the enzyme was not very high, otherwise, the splitting of excessive amounts of peptide bonds and the formation of a significant amount of soluble proteins may occur, and as a result, formation of weak clot [8]. as a result, the loss of dry matters and the acceleration of the serum secretion from the clot will occur. microbial enzymes (proteases) are used as a rennet enzymes substitutes. they synthesize proteinases that are localized in cells (endo-enzymes), or are extracted into the culture medium (exoenzymes). there are proteinases of bacteria, mold, fungi and yeasts. inside these groups they are divided into acid, neutral and alkaline. proteinases of mold fungi are used for a long time in the food industry for cooking food and seasonings, soy sauces and in the technology of certain types of soft cheeses [9]. herbal milk-clotting enzymes are the subject of increasing scientific and practical interest of specialists in the field of dairy – protein production. the interest in using increases due to the availability of sources and the relative simplicity of their obtaining. according to the literature [8, 10] almost all types of plant tissues have proteolytic enzymes. they have the ability to coagulate milk proteins in the appropriate conditions [8]. enzymes that are used as the above-mentioned coagulants belong to the aspartic proteases. cysteine and serine proteases are also fit for milk coagulation. according to the data [6] there is production of cheeses with a use of artichoke cynara spp. as a herbal coagulant in spain and portugal. cheeses from the sheep's milk: serra and serpa [11], los pedroches, la serena [12], torta del casar. los ibores cheese – from the goat’s milk. flor de guía from the mix of sheep’s and cow milk [13-15]. extracts of cynara spp. were used for the production of both spanish and portuguese cheeses. an extract of calotropis procera is used for cheese production in nigeria and benin republic [6]. except of the mentioned above, there are a lot of other plants that can be used as the source for milk coagulation: nettle (urticadioica), fig (ficuscarica), burdock (arctium minus), black currant (centureaspp.), ambrosia artemisiifolia, and others. nevertheless, the excessive proteolytic activity of most of plant coagulants limited their use in the milk-protein products manufacturing. as a result – law cheese yield and flavor and texture defects [16]. hemlock (coniummaculatum) and ricinus seeds (ricinuscommunis) cause the combined coagulation of milk with a help of acids and enzymes. mainly they are used in the production of cheeses with soft dough. therefore, the search for new potential enzymes from traditional herbal plants is relevant. proteases are divided into groups on the ground of the catalytic mechanism, which food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 167 is used during the hydrolytic process. the main catalytic types are aspartate, serine and cysteine [17]. types and sources of milk – clotting proteases are presented in table 1. table 1. the characteristic of milk-clotting plant proteases [8] type of protease the name of the protease source link cardosins and cyprosins cynara cardunculus (spanish artichoke) [12, 15, 18-26] protein extract silybum marianum (saint-mary-thistle) [27-28] oryzasin oryza sativa (rice) [29] protein extract centaurea calcitrapa (maize thistle) [30-32] procirsin cirsium vulgare (sow thistle) [33] aspartic acid (aspartic) protein extract solanum elaeagnifolium (silver-leaved nightshade) [34] cystein protein extract helianthus annuus (common sunflower) [35] neriifolin, neriifolin s euphorbia neriifolia (euphorbia) [36-37] dubiumin solanum dubium fresen (solanum) [38-40] serin lettucine lactuca sativa (cutting lettuce) [41] serine and cysteine proteases catalytically differ from asparagine and metalloproteases. inasmuch as the nucleophile of the catalytic node is a part of the amino acid, while in the other two groups, it is a molecule with the activated moisture [42]. the wild plantain (plantago major) – is the source of alternative kinds of plant milk-clotting enzymes. it belongs to the genus of singleand perennial grasses, less often half shrubs of the plantain family (plantaginaceae). plantago media is widespread on ukrainian territory. the coagulating ability of milk proteins depends not only from the presence of proteases, but also from organic acids.thus, the acid complex of plantain leaves is represented by the following components: fumaric, oxalic (31...103 mg%) [43], wine (1.60...1.87 %), citric (1.22...1.53 %), apple (0.20...0.51 %), malonic (0.11...0.35 %) and succinic (0.25...0.55 %) acids [44]. the total content of organic acids is 10...12%, of which up to 60 % are bound acids. the dominant components of the complex are wine and citric acid. the plananthosine enzyme from the plantago maior’ leaf extract was identified by affinity chromatography on bacitracin sepharose and ion exchange chromatography on mono q in fplc at 70 °c and ph 11 [45]. plant milk-clotting enzymes have a number of technological and economic benefits: low cost, ease of allocation, a wide range of producer plants, etc. this allows to consider them as promising technological components for the cheeses production. it is necessary to take into account the special characteristics of plant milk coagulants. the conditions of their use for the production of certain types of cheeses need additional research. the specific chemical composition makes possible to use plantago major juice for the milk proteins’ coagulation in the technology of soft cheese. the aim of study was to investigate the potential capacity of the plantago major juice from the aerial part of plant to coagulate milk proteins. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 168 2. matherials and methods 2.1. obtaining of plantago major juice the plantago major leaves were collected on the territory of the kyiv-svyatoshinsky district of the kyiv region. the juice of herbaceous plant was obtained according to the scheme shown in figure 1. fig. 1. technological scheme of herbaceous plant’ juice obtaining in the plantago major juice, the amount of dry matter was (4.5±0.23) %, the index of active acidity at the level (5.85±0.18). 2.2. the obtaining and study of herbal – protein clots to prepare model samples was used normalized milk with the following indicators: dry matters mass fraction – (12.3±0.62) %, fat – (2.6±0.13) %, protein– (2.8±0.14) %, with active acidity – (6.9±0.35) рн units, density– 1027 kg/m3. the preliminary preparation of normalized milk according to classical technology was carried out for the milk proteins’ coagulation. the plantago major juice was introduced into the milk heated to 96…98 °с. the mixture was slightly stirred and held for 1 to 2 minutes till the clott was formed. the mass of the wet protein clot for each sample was determined. it was fixed by weighing after self-pressing for 15 minutes. the yield of herbal – protein mass was 184.0…232.7 g from 1000 ml of normalized mixture. the samples, obtained by the coagulation by plant coagulant in the amount 5…12 % from the normalized milk volume were prepared for the experiment. the indicators of active acidity were determined by the potentiometric method (sartorius ph meter). the moisture mass fraction was determined by drying the sample to a constant mass. the moisture-retaining capacity was determined by grau-hamm method in modification of a.a. alekseyev. this technique consists in determination of water mass that allocates from the product in mild compression and is absorbed by the filter paper [46]. solvent cake dregs grinding receipt of raw materials, sorting, rejection of the ground part, roots rough filtering lightning and decantation of juice juice decantation storage at the temperature (6±2) ºс solvent cake for processing drying washing, drying, storage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 169 2.3. infrared (ir) spectroscopy to determine and compare the moisture condition, ir spectra of milk – protein and herbal–protein clots were shooted by the method of a drop, inflated between the rrs-5 windows on the ir-fourier nexus spectrometer of thermo nicolet company, usa. nujol, which has no additional strips in the field of water absorption, was used as an internal standard. the conditions of spectrum shooting: scan range – 400...4000 cm-1, number of scans per second – 7, scan interval – 1 cm-1, resolution – 1 cm-1. 2.4. microphotographs of herbal – protein clots microphotographs of herbal – protein clots were obtained with a help of a luminescent and phase-contrast microscope xsp-139atp with a digital camera canon-66 (nngoa compani ltd). organoleptic indicators were determined according to the generally accepted methods for soft cheeses. 3. results and discussion the amount of plantago major juice that was used in the milk proteins coagulation and its influence on the active acidity and the moisture mass fraction of the protein – herbal clots were studied in the first stage of research. the corresponding results are presented in figure 2. 40 45 50 55 60 65 70 75 80 85 90 6 6,1 6,2 6,3 6,4 6,5 5 6 7 8 9 10 11 12 c lo ts ' m oi st ur e m as s fra ct io n, % a ct iv e ac id ity o f he rb al pr ot ei n cl ot s, un its рн the amount of plant coagulant, % active acidity of herbal – protein clot with a us e of plantago magor juice, units clot’ mois ture mas s fraction with a us e of plantago major juice,% fig. 2. the dependence of active acidity and moisture mass fraction of herbal –protein clot from the amount of plantago juice experimental studies (fig. 2) showed a tendency to decrease the index of active decrease when the amount of plant coagulant increases. the addition of 12 % plantago major juice to milk promotes the setting of active acidity at the level 6.25 units. with decreasing of coagulant amount, the level of clot’s active acidity, which was 6.45 ph units at the adding of 5 % of juice, increases. perhaps it is connected with a presence of organic acids, namely oxalic, citric, apple, wine, and others in the plantago major juice. they affect the process of milk proteins’ coagulation. the obtained results of experimental samples correspond to the ph of the adyghey soft cheese – (5.9±0.3). it is produced by thermo-acid coagulation with a help of milk whey with increased acidity. the indicator of moisture mass fraction of the clots, obtained by the adding of plant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 170 coagulant from 7 to 9 %, changed significantly and varied from 66.00 to 69.00 %. the moisture-retaining ability of the herbal – protein clots depending from the plant coagulant amount was investigated. this rate affects on such soft cheese indicators as structure and quality of the finished product (figure 3). fig. 3. the dependence of moisture – retaining capacity of herbal – protein clot from the amount of plant coagulant the results of research (fig. 3) indicate a strengthening of the clot with the increasing of plantago major juice amount. moisture -retaining capacity of clots’ model samples increased with a compaction of their structure. thus, with the maximum amount of plantago major juice, the moisture-retaining capacity index is 80.68 %, which indicates about active removal of the surface charge from the micelle, that is a factor of the milk protein phase’ colloid destabilization. the organoleptic evaluation of herbal – protein clots’ experimental samples is presented in table 2. table 2. the organoleptic evaluation of herbal – protein clots the amount of plantago major juice introduction into the milk 5±0.25 8±0.4 11±0.55 indicator taste and smell dairy with a weakly herbal flavor dairy with herbal flavor excessive herbal taste and smell color white with a light green tint light green saturated green consistence the clot is mild, weak, and there is a slight whey selection the clot is soft, smeared moderately tight the clot is strong, too tight due to the change of color and its intensity, it is necessary to take into account the colority of the samples. a similar situation occurs with taste and smell. the samples, obtained by proteins coagulation with a help of 10…12 % of plantago major juice have the excessive taste, color and smell. it is necessary for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 171 them to apply organoleptic assessment as a limiting factor for wide use in the form of the basis for milk-protein products. as a result of the investigation of the moisture’ state by the ir spectroscopy, in both samples – control and herbal – protein clot , the assignment of the bandwidths of the ir spectra of the experimental samples and the identification of the functional groups were obtained. the examples of experimental clots samples graphic image are shown in fig. 4 (a-b). a b fig. 4. infrared transmittance spectra of samples: a) milk – protein (control); b) herbal – protein the similarity of the main zones was observed at the comparison of the studied samples’ spectra (fig. 4). the spectra of protein clot, obtained by acid – rennet coagulation differed from the herbal – protein clot in the presence of absorption zones 3269 сm-1. the wide zone in the area 3400…3200 сm-1 was caused by valence fluctuations of h-bound oh groups. the appearance in the infrared spectrum of an intensive zone of c=o-groups valence fluctuations at 1638 cm-1 showed a deformation oscillation of adsorptionbound water. absorption zones at 1550…1542 cm-1 are caused by deformation oscillations of amino acids’ nh groups as the part of protein portion of the protein clot. as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 172 shown in fig. 4 (a-b), this zone was present in all spectra. the 1549 cm-1 zone is characteristic for a carboxylate ion: с о о( ) . the wide zone in the 2133…2127 cm-1 area refers to a mobile proton, which is capable to form h-ligaments of the "bridge" type. the formed in this area zone indicates on the presence of weakly bound moisture. in the herbal – protein clot’ spectra there are small changes in the area of the c-h and o-h bonds fluctuations in comparison with the spectrum of the milk-protein clot. the appearance of absorption zones of the oh –group’s valence oscillations to 2962…2865 cm-1 indicates about the formation of carboxyl groups. the 1449, 1453 cm-1 zones, that indicates on deformation vibrations of methyl and methylene groups of the matrix were present in all samples. a number of zones in the 1243-1041 cm-1 region relates predominantly to c-o, c-c bonds of matrices. in order to learn the mechanism of various types of h-bound moisture adsorption in the herbal–protein clot, the relative transmission intensity of the zones was calculated, where r1 – indicator of tightly bonded moisture; r2 – is an indicator of weakly bound moisture. the square calculation is made by the triangle method. as the baselines, the corresponding tangents are taken – ab, cd and dc. the internal standard of calculation was the 1539 cm-1 zone, which objectively characterized the change in the state of moisture in the experimental mixtures. for example, below is a calculation of r1 and r2 for milk-protein clot (control) (figure 4a): 1640 1 1539 s r s  ; 2127 2 1539 s r s  . s – area (or integral intensity) of the corresponding zone. relative intensities of various h-bound water types (x = 0.95; n = 3; sch = 10 %) of the experimental control and herbal – protein clots are presented in table 3 table 3. the relative intensities of various h-bound water types (x = 0.95; n = 3; sch = 10 %) experimental samples indicators milk-protein clot herbal-protein clot r1 9.8 7.4 r2 14.8 10.7 in order to determine the peculiarities of the herbal – protein clot’ structure, which was obtained with a use of plantago major juice, the comparative studies of the control and experimental samples microstructure were carried out (fig. 4). the results of the microstructural analysis were compared with the data obtained at the evaluating of the qualitative parameters of soft cheese samples. the cheese was obtained by thermo-acid coagulation with a help of milk whey with an titrated acidity index (150±2) °t. it is clear from the presented material (figure 4), that the microstructure of the clot, formed in the presence of plantago major juice had distinct features from control. thus, in the experimental samples of clots made with plant coagulant, it is possible to see more structural units in protein clot. they have expressed homogeneous consistency and their food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 173 microstructure is more homogeneous and includes uniformly distributed micropores of minimal size. in the microphotographs of clot obtained with the plantago major juice, the protein structure consists of particles that are more closely interconnected than in the control sample. obviously, the clot structure has a more advanced spatial configuration, representing a "carcass". it is typical to connected disperse systems, which obviously prevents the free reciprocal movement of its links. more structure rigidity provides the best structural – mechanical properties and moistureretaining ability. а) b) fig. 4. microphotographs of clots: а) control (protein clot); b) herbal–protein clot obtained with a use of plantago major juice in the amount of 8% 4. conclusion the possibility of plantago major juice using for milk proteins complex coagulation at the temperature of thermoacid coagulation by a classical technology was proven. a rational amount of plantago major juice for milk proteins coagulation at the level of 7…9 % was established. the active acidity of the herbal-protein clot was 6.3...6.4 ph units, the moisture mass fraction was 66.00...69.00 %. at this case, the yield of herbal-protein clot varied from 184.0...232.7 g from the 1000 ml of normalized mixture. the results of moisture-retaining capacity of herbal-protein clots with a meaning 60.61 to 64.26 % at addition of 7...9 % of the plantago major juice, were obtained. the limiting factor for this type of clots is colority. the efficiency of the ir spectroscopy method for determining the state of moisture in herbal–protein clots was proved. it was confirmed, that the coagulation of milk proteins by plantago major proteases and organic acids promotes active synergetic occurrence. this was detected by the appearance of an intensive zone of c=o-groups valence oscillations in the infrared spectrum at 1638 cm-1,that indicated about deformation oscillation of adsorption-bound water. the microstructure of the clot’s samples with a use of plantago major juice was observed. it was determined that clots had a more developed spatial configuration than the control. the "skeleton" of samples obtained with a maximum content of plant coagulant, results in greater rigidity of the structure. therefore, it provides the best structural and mechanical characteristics and, accordingly, moisture-retaining ability. the obtained results confirm the ability of the juice of the herbaceous part of plantago major of coagulating milk proteins. the mechanism of the process requires an additional investigation. the obtained data are the basis for technology developing of the snack cheese pastes, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 174 semi-finished products and processed cheese. 5. references [1]. ostroumov, l.v., husnullina, n.v., the investigation of acid-rennet milk coagulation with the dls admixture, technique and technology of food production, 1(16): 7–10, (2010). [2]. bobylin, v.v., physico–chemical and biochemical basis of soft acid-rennet cheeses production, kemerovo, 208, (1998). [3]. ramanauskas, r., the regularities of milk rennet coagulation kinetics, dairy industry, 8: 24– 26, (1994). [4]. shlyapnikova, s.v., bogatyriova, e.r., the particular qualities of milk coagulation rennet enzyme preparation and its analogs, biomic, 9 (1): 33–41, (2014). [5]. fox, p.f., mcsweeney, p.l.h., dairy chemistry and biochemistry, springer – verlag: 145–239, (1998). [6]. roseiro, l.b., barbosa, m., ames, j.m., wilbey, r.a., cheesemaking with vegetable coagulants – the use of cynara l. for the production of ovine cheeses, int j dairy technol, 56: 76–85, (2003). [7]. jacob, m., jaros, d., rohm, h., recent advances in milk clotting enzymes, int j dairy technol, 64: 14–33, (2011). [8]. shah, m. a., mir, s.a., paray, m.a., plant proteases as milk-clotting enzymes in cheesemaking: a review, dairy sci. & technol, 94: 5–16, (2014). [9]. raghunath, t. mahajan and shamkant, b., badgujar biological aspects of proteolytic enzymes: a review, journal of pharmacy research, 3(9): 2048–2068, (2010). [10]. tamer, m.i., mavituna, f., protease from freely suspended and immobilized mirabilis jalapa, process biochem, 32: 195–200, (1997). [11]. macedo, i.q., faro, c.j., pires, e.m., specificity and kinetics of the milk-clotting enzyme from cardoon (cynara cardunculus l.) toward bovine κ-casein, j agric food chem, 41: 1537– 1540, (1993). [12]. roa, i., lόpez, m.b., mendiola, f.j., residual clotting activity and ripening properties of vegetable rennet from cynara cardunculus in la serena cheese, food res int, 32: 413–419, (1999). [13]. fernández-salguero, j., sanjuán, e., montero, e., a preliminary study of the chemical composition of guía cheese, j food compos anal, 4: 262–269, (1991). [14]. fernández-salguero, j., sanjuán, e., influence of vegetable and animal rennet on proteolysis during ripening in ewes’ milk cheese, food chem, 64: 177–183, (1999). [15]. sanjuán, e., millán, r., saavedra, p., carmona, m.a., gόmez, r., fernández-salguero, j., influence of animal and vegetable rennet on the physiochemical characteristics of los pedroches cheese during ripening, food chem, 78: 281–289, (2002). [16]. lo piero, a.r., puglisi, i., petrone, g., characterization of “lettucine”, a serine-like protease from lactuca sativa leaves, as a novel enzyme for milk clotting, j agric food chem, 50: 2439–2443, (2002). [17]. bah, s., paulsen, b.s., diallo, d., johansen, h.t., characterization of cysteine proteases in malian medicinal plants, j ethnopharmacol, 107: 189–198, (2006). [18]. silva, s.v., malcata, f.x., studies pertaining to coagulant and proteolytic activities of plant proteases from cynara cardunculus, food chem, 89: 19–26, (2005). [19]. silva, s.v., allmere, t., malcata, f.x., andrén, a., comparative studies on the gelling properties of cardosins extracted from cynara cardunculus and chymosin on cow’s skim milk, int dairy j, 13: 558–564, (2003). [20]. agboola, s.o., chan, h.h., zhao, j., rehman, a., can the use of australian cardoon (cynara cardunculus l.) coagulant overcome the quality problems associated with cheese made from ultrafiltrated milk? lwt food sci technol, 42: 1352–1359, (2009). [21]. barros, r.m., ferreira, c.a., silva, s.v., malcata, f.x., quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate, enzyme microb technol, 29: 541–547, (2001). [22]. esteves, c.l.c., lucey, j.a., pires, e.m.v., rheological properties of milk gels made using coagulants of plant origin and chymosin, int dairy j, 12: 427–434, (2002). [23]. esteves, c.l.c., lucey, j.a., hyslop, d.b., pires, e.m.v., effect of gelation temperature on the properties of skim milk gels made from plant coagulants and chymosin, int dairy j, 13: 877–885, (2003). [24]. low, y.h., agboola, s., zhao, j., lim, m.y., clotting and proteolytic properties of plant coagulants in regular and ultrafiltered bovine skim milk, int dairy j, 16: 335–343, (2006). [25]. ordiales, e., martín, a., benito, m.j., hernández, a., ruiz-moyano, s., cόrdoba, m.g., technological characterisation by free zone capillary electrophoresis (fzce) of the vegetable rennet (cynara cardunculus) used in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, olena krasulya, larisa chubenko, alla tymchuk, the investigation of the potentional comlex from plantago major to coagulate milk proteins, food and environment safety, volume xvii, issue 2 – 2018, pag. 165 – 175 175 “torta del casar” cheese-making, food chem, 133: 227–235, (2012). [26]. pino, a., prados, f., galán, e., mcsweeney, p.l.h., fernándezsalguero, j., proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet, food res int, 42: 324–330, (2009). [27]. vairo-cavalli, s., claver, s., priolo, n., natalucci, c., extraction and partial characterization of a coagulant preparation from silybum marianum flowers. its action on bovine caseinate, j dairy res, 72: 271–275, (2005). [28]. vairo-cavalli, s., silva, s.v., cimino, c., malcata, f.x., priolo, n., hydrolysis of caprine and ovine milk proteins, brought about by aspartic peptidases from silybum marianum flowers, food chem, 106: 997–1003, (2008). [29]. asakura, t., watanabe, h., keiko, a., soichi, a., oryzasin as an aspartic proteinase occurring in rice seeds: purification, characterization, and application to milk-clotting, j agric food chem, 45: 1070–1075, (1997). [30]. domingos, a., cardos, p.c., xue, z.t., clemente, a., brodelius, p.e., pais, m.s., purification, cloning and autoproteolytic processing of an aspartic proteinase from centaurea calcitrapa, eur j biochem, 267: 6824–6831, (2000). [31]. salvador, s.m., novo, c., domingos, a., evaluation of the presence of aspartic proteases from centaurea calcitrapa during seed germination, enzyme microb technol, 38: 893–898, (2006). [32]. reis, p.m., lourenço, p.l., domingos, a., clemente, a.f., pais, m.s., malcata, f.x., applicability of extracts from centaurea calcitrapa in ripening of bovine cheese, int dairy j, 10: 775–780, (2000). [33]. lufrano, d., faro, r., castanheira, p., parisi, g., veríssimo, p., vairocavalli, s., simões, i., faro, c., molecular cloning and characterization of procirsin, an active aspartic protease precursor from cirsium vulgare (asteraceae), phytochem, 81: 7–18, (2012). [34]. néstor, g.m., rubí, c.g.d., héctor, j.c., exploring the milk-clotting properties of a plant coagulant from the berries of s. elaeagnifoliun var. cavanillies, j food sci, 71: 89– 94, (2012). [35]. egito, a.s., girardet, j.m., laguna, l.e., poirson, c., mollé, d., miclo, l., humbert, g., gaillard, j.l., milkclotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κcasein, int dairy j, 17: 816–825, (2007). [36]. yadav, r.p., patel, a.k., jagannadham, m.v., purification and biochemical characterization of a chymotrypsin-like serine protease from euphorbia neriifolia linn, process biochem, 46: 1654–1662, (2011). [37]. yadav, r.p., patel, a.k., jagannadham, m.v., neriifolin s, a dimeric serine protease from euphorbia neriifolia linn.: purification and biochemical characterisation, food chem, 132: 1296–1304, (2012). [38]. ahmed, i.a.m., morishima, i., babiker, e.e., mori, n., characterisation of partially purified milk-clotting enzyme from solanum dubium fresen seeds, food chem, 116: 395–400, (2009a). [39]. ahmed, i.a.m., morishima, i., babiker, e.e., mori, n., dubiumin, a chymotrypsin-like serine protease from the seeds of solanum dubium fresen, phytochem, 70: 483–491, (2009b). [40]. ahmed, i.a.m., babiker, e.e., mori, n., ph stability and influence of salts on activity of a milk-clotting enzyme from solanum dubium seeds and its enzymatic action on bovine caseins, lwt food sci technol, 43: 759–764, (2010). [41]. lo piero, a.r., puglisi, i., petrone, g., characterization of “lettucine”, a serine-like protease from lactuca sativa leaves, as a novel enzyme for milk clotting, j agric food chem, 50: 2439–2443, (2002). [42]. bruno, m.a., trejo, s.a., aviles, x.f., caffini, n.o., lopez, l.m.i., isolation and characterization of hyeronymain ii, another peptidase isolated from fruits of bromelia hieronymi mez (bromeliaceae), protein j, 25: 224– 231, (2006). [43]. guil, j.l., torija, m.e., gimenez, j.j., rodríguez, i., identification of fatty acids in edible wild plants by gas chromatography, journal of chromatography, 719: 229–235, (1996). [44]. olennikov, d.n., tanhaieva, l.m., mykhailova, t.m. samuelsen, a.b., organic acids of herbal plants 1. plantago major l., chemistry of natural connections, 4: 354–355, (2005). [45]. bogacheva, a.m., rudenskaya, g.n., dunaevsky, y.e., chestuhina, g.g., golovkin, b.n., new subtilisin like collagenase from leaves of common plantain, biochimie, 83 (6): 481–486, (2001). [46]. grek, o.v., yushchenko, n.m., osmak, t.g., et al., labaratory cource of milk and milk products technology, k.: nuft: 431, (2015). 262 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 262 268 research on the quality indicators of curd products based on protein-herbal clots olena grek 1 , *alla tymchuk 1 , larisa chubenko 1 , kira ovsiienko 1 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str.68, kyiv, ukraine *corresponding author: 589112@ukr.net received 29th october 2017, accepted 19th december 2017 abstract: the article presents the research of qualitative indicators of curd products with different nutritional ingredients based on protein-herbal clots. the effect of the number of rumex juice and the duration of thermoacid processing on the process of precipitation of milk proteins was determined. it was established that the introduction of vegetative coagulant in the amount (9 ± 0.5)% at a temperature (93 ... 95) °c and endurance (3 ... 5) min provides the optimal yield of protein-herbal clot taking into account restrictions according to organoleptic parameters. the effect of white sugar and apple pectin in fiber on the organoleptic, physico-chemical and rheological indicators curd products was investigated. the dietary fibers increase moisture-proof ability and effective viscosity of samples, and white sugar reduces these indexes due to dehydrating properties. the optimal option is to add to the protein-herbal bunch at the same time two components when mixing white sugar and apple pectin in fiber in quantities of 15% and 2% respectively. taking into account the influence of individual non-dairy ingredients rumex juice of white sugar and apple pectin in fiber on curd products, the performance of the finished product can purposefully be affected. keywords: protein-herbal clones, sorrel, cheese products, white sugar, apple pectin in cellulose. 1. introduction extension of assortment and improvement of quality of curd products is carried out as follows main directions:  improvement of milk-protein concentrates, which are produced without significant changes of technology on existing equipment;  creation of new types of curd products based on modified dairy raw materials from modern nutritional ingredients;  use of innovative constructive ideas and technological processes. ingredients are generally administered in small quantities, taking into account to their efficacy. ago special attention should be paid to the issue of preparation for their uniform distribution in raw materials, including milk-protein mixtures. to do this, you need to evaluate the structural state of raw material or semi-finished. choose the stage in the process and the modes for making. take into account the commercial form of the preparation (in the form of a solution, emulsion, dry powder, and granule), concentration (immobilized preparation on the carrier, composition, concentrate) and technological properties (water, fat solubility, salt stability, swelling, degree dispersion) and others [12]. vegetable ingredients are used as a technological component in many production processes. so, the thermoacid coagulation of milk proteins occurs under action, as serum with acidity (150 ... 200) °t, and coagulants of vegetable origin with relevant characteristics. the latest trend is aimed at expanding the production of milkprotein http://www.fia.usv.ro/fiajournal mailto:589112@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 263 products and also modernization of the assortment. scientists have developed technologies for coagulation of milk proteins as berry raw materials [3-6], and other receptions. they provide a separation process сonstituents of milk with juice or extracts of plants including wild plants. consequently, the aim is to study the qualitative indicators of curd products on the basis of protein-herbal clots with different nutritional ingredients. the object of the study is the technology of cheese products based on protein-herbal clots and also study of their quality indicators depending on the content of various foods ingredients. the subject of research protein-herbal clots obtained by thermoacid coagulation of skimmed milk with sorrel juice, food fibers (apple pectin in fiber), white sugar, indicators of quality of model samples of curd products. the task is:  to get protein-herbal clots by thermal acid coagulation of fat-free milk in the presence of sorrel juice as a coagulant;  research on the effect of white sugar and food fibers on organoleptic, physico-chemical and rheological indicators of curd products on the basis of protein-herbal clots. 2. materials and methods sorrel (rumex) was used as a coagulant for protein-herbal clots. it belongs to the genus of one-, twoand perennial herbaceous plants of the family buckwheat, the order of buckwheat flowers [7]. the water content of rumex leaves is (90 ... 92)% protein 1,5%, total carbohydrates (3.0 ... 5.3)%, fiber 1%, minerals, in (mg%): na (15), k (500), ca (47), mg (85), p (90), fe (2,0). vitamins, (mg%): c (43 ... 81), b1 (0.19), b2 (0.1), pp (0.3), βcarotene (2.5). the amount of oxalic acid is determined at the level of 360 mg% [811]. according to the literature [12, 13] oxalic acid is divided into organic and inorganic. in the first case, the acid contained in the fresh sorrel is useful for human health substance. it saturates the body with such necessary for the hemorrhage microelements as iron and magnesium. in the inorganic form it is a substance that is formed after heat treatment plants at temperatures above 100 ° с. the acid is coupled with free calcium, forming salts oxalic acid. oxalates contribute to the formation of stones in the kidneys, the development of articular rheumatism and arthritis, destruction of teeth and bones. with moderate amounts intake of salt of oxalic acid, it is easily derived from the human body. the harmful amount is 50 mg per 100 g of product [14]. for thermal oxidation deposition from the leaves of rumex received juice in the following sequences. the ground part was sorted, inspected from dirt and mechanical impurities, washed, dried. chopped to a homogeneous state for (2 ... 3) minutes on the device of the brand dex dhb-572 power of 750 w. the raw material where a coagulation process was held is skim milk, which was pasteurized at a temperature (76 ± 2) ° с with a holding (15 ... 20) s. mass fraction of dry matter is (11.2 ± 0.7)%, protein (3.7 ± 0.2)%, titrated acidity (17 ± 1.0) ° t, density 1032 kg / m3. previous studies that took into account organoleptic constraints and protein release mass, the optimum amount of vegetative coagulant with ph (3.1 ... 3.5) is determined at the level (9 ± 0.5)% by weight of milk [3, 4]. this amount changes the active acidity of the mixture for providing a balanced isoelectric state of protein in the entire volume. it leads to their active coagulation for classical modes. in heated to a temperature (93 ... 95) ° c, skim milk it was added rumex juice. during the application was moderately stirred and kept (3 ... 5) minutes until a clot was formed. complex food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 264 the effect on high-temperature milk protein and acid reagent leads to the maximum complete their deposition. the process of coagulation was set visually for intense formation protein clot and serum release. the resulting protein-herbal clot had the following qualitative indicators: the mass fraction of moisture at the level (64 ± 2)%, titrated acidity (80 ± 1) ° t. the organoleptic characteristics of the clot were as follows. color was light pistachio, not uniform; consistency of the clot was soft, plastic and tight; taste was milkprotein, cheese, without foreign smells, with a slight herbal flavor. at the next stage of the research, model samples of cheese products were made of proteinherbal clots with white sugar and food fibers (ff). the protein base was added into the mixer at a temperature of (12 ± 3) ° c and mixed with pre-sieved white sugar after partial mixing; prepared ffs were added to the mixture. if necessary, flavoring substances are introduced to disguise the color in the quantities recommended by producer (apple, kiwi e600 e699). the average duration of mixing was (5 ... 10) min. as an ff, apple pectin in fiber was used (apf), which is produced in ukraine according to tu u 30335750.001-2000. apf contains extruded wheat bran 60%, apple powder 38%, pectin 2%. chemical composition of 100 g above mentioned the food fibers the following, in%: the mass fraction of moisture – 6.0; sugar 48.7, including кeducing – 40.9; sucrose 7.8, organic acids (by apple acid) 5.0; cellulose13.4, pectin substances – 12.4, including water-soluble – 5.1, waterinsoluble – 7.3; proteins 6.7; fats 6.8; potassium 0.08; magnesium 0.73; calcium – 0.32; phosphorus 0.240. the vitamin complex of apf is represented by carotenoids (0.8 mg) and ascorbic acid (76.4 mg) [15]. during the experiment, the physical and chemical parameters of the model samples curd products were studied. mass fraction of moisture was determined by drying the samples to a constant mass on the laboratory electronic scales-moisture series of ads manufactured by the company "ahis". titrated acidity a titration method based on neutralization of acidic substances contained in 100 g of product with a solution of sodium hydroxide or potassium concentration of 0.1 mol / dm3. moisture-retaining ability (hu) by grauhamm's method of modifications a.a. alekseev, based on the determination of the amount (mass) of water released from the product is lightly compressed and absorbed by a filter paper. effective viscosity on a rotating viscometer "reotest ii". the viscometer uses a twocylinder a measuring system that allows determining the structural mechanical characteristics of the syrup products. the measurement of the shear stress τ (pa) was carried out with a change in the gradients of the shear voltage γ in the range of 0.33 ... 145.8 s-1 for direct traffic. the indicators of viscometer data were specified maximum angle of deviation of the arrow on the instrument scale. displacement voltage (pa) was calculated by the formula (eq. 1): τ = z ∙ α, (1) where z is the cylinder constant, p ∙ unit. scale; α is the measure of measurement. the effective viscosity (pа⋅с) was determined by the formula (eq. 2): , (2) where γ is the gradient of shear force, c-1 [16-18]. all analytical definitions were performed at least three times. indicator is average the standard deviation value. the statistical analysis was conducted with the help of microsoft excel version 2010. γ τ ef η  food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 265 3. results and discussion the influence of the amount of rumex juice on the process of coagulation of proteins in low-fat milk and output of protein-herbal clot has been confirmed. the output of the latter under the same conditions process and adjusted according to the amount of dry matter prepared rumex is presented on fig. 1. * mass fraction of moisture of protein-grassy clot (64 ± 2) % fig. 1. the yield of a protein-grassy bunch, depending on the amount of rumex juice organoleptic parameters are a limiting factor due to color variations of clots from barely green to intensely green (for 11% juice). not formed clot that complicates the process of self-pressing (for 5% juice) is also a limiting factor. number juice at the level (9 ± 0.5)%, taking into account the above restrictions, is optimal. entrance protein-herbal clot is (203 ± 5) g of 1000 g of skimmed milk. as for the course process, the result of coagulation is a partial dehydration, a decrease in the charge hydrated protein molecules and rapid precipitation in the precipitate. curd products with a polycomponent composition should be elastic and plastic. this will allow the formation and packaging of products commonly accepted in the industry way on existing equipment [19]. as a result of the research, it was discovered the effect of the individual prescription components of sugar and food fiber with the above water absorption capacity on quality indicators of cheese products on the basis of protein-herbal clots. so, the amount of white sugar in the recipes of the classic soured items the range varies from 10% to 20% (creams, syrup masses, glazed curds, semi-finished products, etc.). to stabilize the moisture and enrichment of protein clots was used apf. the basis of the distribution of ingredients is the need to clarify the effects of individual components of non-milk origin and their compositions on physicochemical and rheological indicators of curd products. results of determining the change in effective viscosity, moisture retention capacity and titrated acidity of the samples under study are presented in table. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 266 table 1 dependency of titratable acidity, moisture-retaining ability and effective viscosity on the composition of curd products based on protein-grassy clots № s a m p le ratio of prescription components in curd products,% indicators of curd products t it ra te d a c id it y , о т m o is tu re re ta in in g a b il it y , % e ff e c ti v e v is c o si ty , p a · s protein-herbal clots white sugar apf 1 100 80 ± 1 57.5 ± 1.3 353.3 ± 3.3 2 90 10 78 ± 1 49.7 ± 1.0 293.5 ± 2.8 3 85 15 77 ± 1 46.4 ± 1.1 264.2 ± 2.7 4 80 20 76 ± 1 43.2 ± 1.3 234.8 ± 3.0 6 99 1 80 ± 1 60.3 ± 1.2 372.2 ± 3.0 7 98 2 79 ± 1 66.5 ± 1.1 411.3 ± 1.9 8 97 3 79 ± 1 71.8 ± 1.4 469.6 ± 2.5 10 89 10 1 79 ± 1 57.3 ± 2.1 345.99 ± 2.2 11 83 15 2 78 ± 1 61.1 ± 1.7 381.60 ± 2.5 12 77 20 3 77 ± 1 67.2 ± 1.6 401.03 ± 2.8 according to experimental data (table 1), non-dairy ingredients that are added to the protein-herbal clot (namely, white sugar and / or apf), do not amplify the course of processes and do not activate the increase of acidity. this indicator basically depends from the constituent and qualitative parameters of the protein concentrate obtained by the way thermal coagulation of milk proteins with rumex juice. white sugar slightly more reduces the acidity of the mixture than the apf. samples containing 20% sugar and 80% proteinherbal clots have acidity of 3 ° t less than samples with the same amount protein component and 3% apf. the apf lowers the moisture content of the model samples, since they consist of hollow ones cells whose walls are cellulose. other components are hemicellulose and pectin, which are in mostly located between empty cells. hydrophilic substances of food fibers interact with moisture of dairy products due to the formation of h-bound polyasocyanates with the participation of water molecules and h-bound functional groups of hydrophilic substances. molecules hydrophobic groups become more ordered, as evidenced by a decrease in entropy. due to dispersion forces hydrophobic groups are aggregated. when combined with moisture, they quickly get it adsorb, increase in size, which increases the viscosity of the system [20]. one of the most important physicochemical indicators of model samples of curd products is moisture-retaining ability, which is associated with effective viscosity. previous studies [19] found that for curd products, the index of moisture retaining ability (mra) on levels above (82.0 ± 2)% are sufficient. growth of the mra leads to too dense consistency. it is found that white sugar lowers the mra and the effective viscosity of the model specimens. with adding a sugar to the protein-herbal clots in the amount of 20% of the mra is reduced by 14.3%, and effective viscosity almost 118.5 pa · s. intensively increase from (60.3 ± 1.2)% to (71.8 ± 1.4)% moisture-retaining ability curd products apf with high water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 267 absorption capacity and hydrophilicity. at added to the protein-herbaceous clot above the above non-dairy ingredient in an amount from 1 to 3%, mra is increased by 11.5%. at the same time, the effective viscosity increases from (372.2 ± 3.0) pa · s to (469.6 ± 2.5) pa · s. the ability of the ff to absorb water is related to the degree hydrophilicity and the number of biopolymers present in them, the nature of the surface and porosity particles, their sizes. they combine in their composition biopolymers with different kinship to water. thus, pectin substances and some hemicelluloses are classified as hydrophilic colloid. this causes their ability to keep water. however, a significant increase in moisture retaining ability and effective viscosity of sour cream products are also undesirable because of this complication is transportation and mechanical the formation of products. concerning the conditions of the introduction, the optimal option is to add at the same time two components white sugar and apf. so, when added to the protein-herbal clot. 15%of white sugar and 2% of food fibers (sample number 11), moisture-retaining ability rises from (57.3 ± 2.1)% to (61.1 ± 1.7)% (table 1), and the effective viscosity increases from (345.99 ± 2.2) pa · s to (381.60 ± 2.5) pa · s. the samples under study have organoleptic characteristics, which satisfy the requirements for such products. 4. conclusions the possibility of using rumex juice in the amount (9 ± 0.5)% as a coagulant of milk proteins has been confirmed. this makes it possible to obtain organoleptically suitable protein-herbal clots of thermoacid coagulation of skimmed milk for regimes: temperature(93 ... 95) ° c, duration (3 ... 5) min. adding prescription components of sugar and apf to protein-herbal clots in different ways affects the physico-chemical and rheological indices of model samples of curd products. it has been confirmed that the moisture-retaining ability and effective viscosity of the samples increase food fibers. the process is due to the hydrophilicity of biopolymers. white sugar has dehydrating properties and reduces the moisture-retaining ability and effective viscosity of the samples. the acidity of curd products based on protein-herbal clots is determined by acidity the protein base is from (76 ± 1) ° t and up to (80 ± 1) ° t. taking into account the influence of individual components non-dairy origin of the quantity of juice rumex, white sugar and apf for curd products, you can purposefully influence the performance of the finished product. its addiction will make it possible ensure compliance with regulatory documents and production conditions. 5. references [1]. bal-prilipko l.v., venglyuk yu.p., zaletok s.p., moiseenko a.m., improving the technology of combined food products using high quality ingredients and dietary supplements, food industry, 2: 23–26, (2013). [2]. bal-prilipko l.v., innovative technologies of quality and safe meat products, 207 p., (2012). [3]. grek o., ouopriichuk o., pshenychna t., the rationalization ofthe parameters ofmilk proteins' thermo acid coagidation by berry coagulants, food and environment safety, 1: 1–7, (2017). [4]. schetinin m.p, koltyugina o.v., kosynkina a.a., cheese product with berry components, dairy industry, 10: 58, (2011). [5]. ostroumov l.a., bobylin v.v., smirnova i.a., rafalovich s.r., investigation of the process of thermo-acidic coagulation of milk using various coagulants, storage and processing of agricultural raw materials, 7: 26–27, (1998). [6]. bychkova m.v., technology development of production of thermo-acid cheeses with various coagulants, polzunovsky almanac, 4: 92–94, (2009). [7]. chernykh v.p., pharmaceutical encyclopaedia, 1632 р., (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 olena grek, alla tymchuk, larisa chubenko, kira ovsiienko, research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, food and environment safety, volume xvi, issue 4 – 2017, pag. 262 – 268 268 [8]. evtefeev yu. v., zykovich s.n., investigation of the chemical composition and nutritional status of rumex k-1 sorrel, bulletin of the altai state agrarian university, 76–80, (2011). [9]. vysochina g.v., phenoliс compounds in the taxonomy and phylogeny of the buckwheat family: diss. doc. of biological sciences: 03.00.05, novosibirsk: 410 p., (2002). [10]. kim j.m., lee y.m., jang d.s., kim j., constituents of stems of rumex japonicus with advanced glycation end products (ages) and rat lens aldose reductase (rlar) inhibitory activity, agric. chem. biotechnol, 49(1): 24–27, (2006). [11]. rao k.v., sunitha c., banji d., a study on the nutraceuticals from the genus rumex, hygeia journal for drugs and medicines, 2: 76–88, (2011). [12]. pereira c., barros l., carvalho a., ferreira i., use of uflc-pda for the analysis of organic acids in thirty-five species of food and medicinal plants, food anal. methods, 6: 1337–1344, (2013). [13]. rao k., ch s., banjii d., sandhya s., saikumar p., pharmacognostic studies on rumex vesicarius, asian journal of plant science and research, 1: 102–115, (2011). [14]. viligorskaya k.v., kuhlina o.s., dismetabolic disorders of the exchange of oxalic acid as a provocative factor for the development of chronic obstructive pulmonary disease, achievements of clinical and experimental medicine, 4: 26–28, (2014). [15]. perfilova o.v., mitrofin m.a., use of powders from fruit and vegetable extracts for the purpose of expansion of assortment of flour confectionery products, achievements of science and technology of agrarian and industrial complex, 8: 48–50, (2008). [16]. onopriichuk o.o., improving the technology of cheese products with grain ingredients: diss. сand. tech sciences: 05.18.16. «food technology», kiev: 270 p., (2008). [17]. kosoy v. d., control of quality of dairy products by methods of physico-chemical mechanics, 208 р., (2005). [18]. grek o.v., yushchenko n.m., osmak t.g., onopriichuk o.o., rybak o.m., tуmchuk a.v., krasulya o.o., workshop on milk and dairy technology: teaching. manual, 431 p., (2012). [19]. tуmchuk a.v., onopriichuk o.o., grek o.v., puhlyak a.g., pasichny v.m., modern approach to the development of cheese products, food industry of agroindustrial complex, 1: 25–29, (2015). [20]. grek, o.v., krasulya o.o., determination of forms of bound moisture in serum drinks with food fibers, food industry, 14: 58-62, (2013). 1. introduction 4. conclusions 317 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue3 2018, pag. 317 326 consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban-zambia *maureen s. banda 1 , rosemary n. likwa 2 , phoebe bwembya 2 , jeremiah banda 3 and allan mbewe 4 1maureen s. banda, c/o amos banda, department of mathematics and statistics. university of zambia, box 32379, lusaka, zambia, maureens.banda@gmail.com 2rosemary n. likwa school of public health, department of community and family medicine. university of zambia, box 50110, lusaka, zambia likwandonyodr@gmail.com 2phoebe bwembya school of public health, department of community and family medicine. university of zambia, box 50110, lusaka, zambia pabwembya@gmail.com 3jeremiah banda school of public health, department of epidemiology and biostatistics. university of zambia, box 50110, lusaka, zambia jbanda6053@gmail.com 4allan mbewe school of public health, department of environmental health. university of zambia, box 50110, lusaka, zambia rabmbewe@gmail.com *corresponding author e-mail:maureens.banda@gmail.com received 7th june 2018, accepted 27th september 2018 abstract: aflatoxin contamination is a major global public health problem especially in developing countries. consumption of contaminated peanut butter poses a serious public health challenge. aflatoxin is implicated in cancer, low immunity, and stunting among children and increases morbidity and mortality. to investigate aflatoxin contamination levels in peanut butter from local and international suppliers in selected lusaka urban district outlets, a cross sectional quantitative study was carried out. a total of 109 peanut butter samples collected from local and international sources were tested for aflatoxin contamination levels using accuscan reveal q+ test. the findings showed that nine (8.3%) of the 109 samples satisfied the 0 to 4ppb european standards, and were deemed safe for public consumption, while the majority (91.7%) were not. however, using the codex alimentarius commission (cac) standard, of up to 15ppb, 83 (76.1%) samples were found to be safe for consumption. there was a marked difference in aflatoxin contamination of products with p-value of less than 0.00001. the study revealed high aflatoxin contamination in local compared to international products at 15ppb standard where 25 local products compared to 1 product of the international origin were contaminated above 15ppb. to protect the safety of consumers, it is recommended that the regulatory agency, the zambia bureau of standards (zabs) provides guidance on standards and monitor compliance. to control the movement of contaminated peanut products across borders and their consumption, collaborative research and consumer awareness will be critical. keywords: contamination, international, local, products 1. introduction aflatoxin contamination is a major global public health threat accounting for 28% of all global liver cancer cases particularly in developing countries [1]. several epidemiological studies in africa and asia have demonstrated an association between dietary aflatoxin and liver cancer. according to the international agency for research on cancer (iarc) in 1988, aflatoxin b1 was on the list of human carcinogens [2]. furthermore, the occurrence and extent of aflatoxin contamination varies and has been influenced by many environmental factors which include geographical location, suboptimal agronomic practices, susceptibility to pre-harvest fungal invasions, http://www.fia.usv.ro/fiajournal mailto:maureens.banda@gmail.com mailto:likwandonyodr@gmail.com mailto:pabwembya@gmail.com mailto:jbanda6053@gmail.com mailto:rabmbewe@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 318 transportation, storage type and processing [3,4]. historically, aflatoxins were first discovered as a result of the deaths of over 100, 000 young turkeys in england in 1960 from a new disease termed “turkey x disease” [5]. this disease outbreak also occurred in more than 150 villages in western india in 1974 where 397 persons were affected and 108 individuals died [6]. investigations on the poultry and ducklings outbreak of “turkey x disease” revealed that there was an association between the feed given to birds, a brazilian peanut meal and the high toxins [7]. since then, aflatoxin has been of interest for study among scientists throughout the world [8]. in rural kenya, aflatoxin outbreak happened in april 2004, where 317 cases and 125 deaths of mostly children were reported because of eating aflatoxin contaminated maize [9]. when highly contaminated products with aflatoxin are consumed or just chronically eaten in low doses, health problems such as, liver cancers, aflatoxicosis, liver cirrhosis, stunted growth, low immunity and eventually death may occur [10]. furthermore, literature reveals that highly contaminated peanut products contribute to economic loss for traders [11]. if peanut is not properly sorted by separating quality peanut from immature and discolored type before storage, there is an increased chance of moisture creation and insect manifestation [12] occurrence that increases aflatoxin levels in peanut and in turn reduces its quality. when such low quality peanut is used for peanut butter processing, aflatoxin contamination levels are likely to be raised. the maximum tolerable limit of aflatoxin contamination for developing countries according to the european union (eu) is between 4ppb and 30ppb [13]. furthermore, legislation in the eu sets maximum aflatoxin levels for groundnuts destined for processing at 10ppb and those for direct human consumption at 4ppb [13]. in some developing countries including zambia where limits are not yet in place, 15ppb has been prescribed as the maximum acceptable level of aflatoxin for safe consumable peanut products [14]. the presence of aflatoxin contaminated peanut butter in lusaka’s outlets may affect the nutrition status of consumers, loss of income for traders, ill health and eventually deaths of consumers. in zambia, there is limited documentation of aflatoxin contamination that compares local with international peanut butter aflatoxin levels. furthermore, aflatoxin contamination is not well known by many people in zambia. however, very high aflatoxin contamination of upto 10,740ppb, way above the recommended 15ppb, have been recorded [15]. similarly, this result may be true for both local and international peanut butter products that are sold in the outlets, a situation that predisposes consumers to dangers of aflatoxin contamination [11]. this concern led to the need for the study to investigate aflatoxin contamination in peanut butter from local and international suppliers. this is a unique study to be conducted in lusaka, zambia which included samples drawn from processing plants. peanut butter is used widely in zambia. results of the study may influence policy makers to enact a regulation or develop clear guidelines on how aflatoxin contamination in peanut butter can be reduced or controlled. to curb the consumption of contaminated peanut butter, the need to explore suitable interventions cannot be overmphasised. by ensuring government and zambia bureau of standards strengthen regulations to adhere to the set standards, and creating public awareness will support and safeguard the health of consumers. the objective of this paper was to investigate aflatoxin contamination of peanut butter from local and international origin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 319 2. material and methods this study was conducted in lusaka urban district, with a total population of 1, 747 152 [16]. there were three strata that provided cans of peanut butter for testing, one market (a), with 14 stand points, three processing plants (b, c and d), where local samples were purchased. the three commercial stores (e, f and g) represented the points from where international samples were purchased among the 20 distribution points. cans were proportionally allocated to the stores based on the number of outlets each store had. soweto market was chosen because of its popularity and being the cheaper supplying source for peanut butter products to customers. similarly, soweto was also known to have been serving as a retail market for lusaka and beyond. the processing plants were included in order to assess aflatoxin contamination at plant level while commercial stores were included as a source for international peanut butter supply to consumers in lusaka urban city. sample selection the suppliers (commercial store, processing plants and soweto market) were purposively selected because they served and supplied peanut butter to the majority of consumers in lusaka. processing plants were identified as one of the sources for peanut butter sampling sites with the help of staff from zabs. the managers or supervisors at the processing plants were asked to provide referrals for other processing plants where peanut butter samples could be purchased. most of these plants were located outside of lusaka which made them ineligible, as eligibility criteria included only those within lusaka urban district. actual stores were randomly selected from the list frame. from commercial store e, one of the nine sub-outlets was selected, from commercial store f, one of the six suboutlets and from commercial store g, one of the five sub-outlets was selected. in these selected sub-outlets, a list of peanut butter cans were developed to assist in selecting desired number. the number of cans purchased from each outlet was arrived at by the use of systematic sampling of two in one until the required number was reached. this selection was irrespective of texture or brand. however, the three processing plant owners could not allow the investigator to enter their warehouses to randomly sample units. peanut butter can samples table 1 main outlets source of origin site or id sample size local (1) soweto 1 a 14 processing plant 1 b 12 processing plant 2 c 15 processing plant 3 d 14 international (2) store 1 e 24 store 2 f 16 store 3 g 14 managers instead were instructed to systematically sample the number of cans that were needed which they did by picking the first and leaving the next until they had food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 320 the number required. at soweto market, samples of peanut butter were collected from processors who were available at the time. the processors were selected systematically. purchased peanut butter samples were kept at room temperature for two weeks before taking them to the laboratory for testing. samples were kept close to the way they were at the time of purchasing. a total of 109 peanut butter cans were purchased and of these, 55 products were local while 54 were international origin. use of total population “n” and total sample “n” (n/n = z) interval was used to get actual cans from sub-outlets as shown in table 1. data collection methods a check list was used in the outlets to collect peanut butter samples from the origin and site. each sample was uniquely coded to avoid mix up of products drawn from different collection sites and for easy identification. a can with an identification number 2, 3 and 1 would therefore be indicating that a sample was drawn from the main outlet 2, source of origin 3, store g and sample number 1 of the 14 required. the same approach was repeated throughout the sample collection. peanut butter was tested using reveal q+ test which is a competitive direct enzyme-linked immunosobernt assay (cd-elisa) in order to obtain aflatoxin concentration in parts per billion (ppb). the following materials were used: 65% ethanol solution, sample collection cups with lids, a scale, a timer, sample cup rack, aflatoxin stat tablet reader, graduated cylinder, filter paper, sample collection tubes with cups, 100 µl pipettes with tips, 500µl pipette, tips, paper towels and gloves. in addition, neogen test strips, sample diluents and clear sample cups were also used. clear sample dilution cups were labeled and placed into a rack, 20 grams of peanut butter in cups were weighed and mixed with 60ml of 65% ethanol which was then shaken vigorously for 3 minutes in order to extract aflatoxin from the mixture. the sample was then allowed to settle for (one) 1 minute before filtration. thereafter, 500µl of sample diluents was put in a clear cup using a 500 µl pipette to which 100µl of sample extract was added and then mixed by pipetting up and down for 5 times. thus from this mixture, a 100 µl was later taken and transferred into a new clear sample cup where a test strip end was inserted. a timer was then set for 5 minutes to allow the strip to develop after which the strip was removed from the sample cup. finally, the result was read using the stat reader, which automatically analyzed the strip. the results were ready within 6-minutes of incubation. data entry and analysis the quantitative data for aflatoxin concentration levels was entered and analysed using spss version 20 software package. the comparisons between local and international aflatoxin contamination was carried out using two sample t-test which was performed. in determining aflatoxin contamination, samples met specific set categories. the categories considered were 0 4ppb, 4 to 15 and above 15ppb. all tests were performed at 5% level of significance and 95 percent confidence intervals. 3. results and discussion aflatoxin contamination the results in table 2 below show the descriptive summary of findings that have been presented while infusing the discussion. the results revealed aflatoxin contamination levels to be between 1.75ppb and 147.2ppb. generally, high level values were observed in samples drawn from local outlets while low values were from international outlets. samples from site f had the lowest (5.3625ppb) average amount of aflatoxin while samples from site c had the highest 68.38ppb average amount of aflatoxin contamination. there were wide variations in local samples than in international samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 321 summary of aflatoxin contamination table 2 for local samples, standard deviation ranged from 1.69ppb to 46.85ppb while for the international samples, the range was from 1.19ppb at site f to 5.86ppb at site e and these were from 55 (50.5%) and 54 (49.5%) samples from local and international sources respectively. the international peanut butter products yielded lowest values of 1.75ppb to 32.95ppb while for the local products the range was between 2.275ppb and 147.2ppb. median values of aflatoxin concentration table 3 origin no. minimum median maximum mean std. deviation international local total 54 55 109 1.75 3.30 1.75 4.92 13.10 6.95 32.95 147.20 147.2 5.87 31.62 18.86 4.085 36.066 28.738 table 3 highlights the median for both local and international samples. the findings show that the median and the mean are reasonably close for international samples as opposed to local samples. the standard deviation of aflatoxin contamination for local sample is about nine times greater than that of the international. however, the median for samples was 6.95ppb and maximum was 32.95ppb for international products while for local product maximum was 147ppb. of these, 25 (22.9%) samples were unsafe for human consumption. these findings were different from those cited in malawi [17] where aflatoxin contamination for imported peanut butter had a median of 2.7μg/kg but all the local products were in the range of 34.2μg/kg to 115.6 μg/kg and these were cited as unsafe globally for human consumption. aflatoxin contamination is a problem in many parts of the world although the values may be a little lower in zambia where this study has revealed aflatoxin contamination levels to be in a range of 1.75ppb to 147.2ppb regardless of source of origin. of these, 0.9% and 22.9% represented unsafe products for international and local samples respectively with over 15ppb. the findings further indicate that, regardless of the source of origin, majority (91.7%) of peanut butter samples from both local and international were contaminated as they were above the european set standard of up to 4ppb. thus if codex alimentarius commission of 0 to 15ppb is used, the results show that 83 (76.1%) of the sample as outlet of origin sample aflatoxin contamination in ppb site no. mean min max std. deviation local a 14 32.50 3.30 147.20 46.848 b 12 6.32 3.70 9.15 1.690 c 15 68.38 42.90 122.50 25.104 d 14 13.06 8.85 16.30 2.275 international e 24 6.40 3.25 32.95 5.859 f 16 5.36 3.95 8.10 1.194 g 14 5.54 1.75 10.35 2.166 total 109 18.86 1.75 147.20 28.738 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 322 safe for consumption. the results were found to be lower than those found in haiti [3] where aflatoxin contamination were in the range of 137µg/kg and 2720µg/kg. this study also shows that aflatoxin contamination is a real problem in many parts of the world, hence the need for more research to seek for interventions. this study has revealed that, peanut butter from soweto market and processing plants in zambia had higher aflatoxin contamination levels than products from commercial stores in contrast to the study done in zimbabwe [18] where high aflatoxin contamination levels were detected from commercial peanut butter than from markets. in addition, another study in india [19] found aflatoxin contamination of above 30ppb with the highest values having been detected from peanut butter samples (42.5%). moreover, the findings in this study where 26 (23.9%) samples exceeded 15mg/kg limit contrasted with the study that was conducted in taiwan [20] where aflatoxin contamination was even higher (52.8%). inferential results a parametric approach was used to carry out the test of proportions for sample units between local and international products. data here was found to be skewed to the right in both local and international sources (figure 1). fig 1: aflatoxin contamination by source of origin furthermore, variation was higher in the local as compared to the international data set. for the international peanut butter, most of the values were in the range of 0 to 10ppb while for local, they were between 0 and 20ppb with values going as higher as above 140ppb. thus to achieve normality, the dependent variable was transformed by taking a natural log (ln). ln (aflatoxin in ppb) satisfied the normality assumption and a common variance could then be assumed (figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 323 fig 2: log of aflatoxin contamination a comparison of aflatoxin contamination levels between local and international samples was carried out using the transformed variable which showed that the hypothesis that the mean values between local and international peanut butter are the same was rejected because the observed t value was -8.019 (-1.22426/0.15266) which is outside the interval (-1.98, 1.98) suggesting that the result is highly statistically significant with a p-value of less than 0.00001. similarly, the 95% confidence interval of -1.22426 was estimated (1.52690, -0.92163) and this also confirmed it to be significant since 0 is not contained in the interval. further tests were carried out to compare differences in aflatoxin contamination between local and international peanut butter products. european standard of 0 to 4ppb the findings in this category was that 7 out of 54 (12.96%) and 2 out of 55 (3.64%) international and local samples respectively, fell within 0 – 4ppb. thus, the difference in proportions was -0.094 (2/55 – 7/54) with an estimated standard error of 0.053 and a zscore value of -1.77 at 95% confidence interval of -0.011 to 0.197. this result shows that there was no significant difference (pvalue = 0.0768) in aflatoxin contamination levels between local and international products according to the european set standard of 0 – 4ppb since both origins only yielded less than 10 samples as safe for consumption (table 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 324 table 4 proportion of contamination in specific standards codex alimentarius commission 0-15ppb this study has revealed that aflatoxin contamination at 0 to 15ppb was significantly different for local and international products at p-value of less than 0.00001. the difference in proportions was 0.43603 with an estimated standard error of 0.07081, these values yeild a z-score of 6.158 with an estimated 95% confidence interval of 0.29566 to 0.57640. the findings in this study (76.1%) are comparable to the study conducted in kenya [4] where 71.8% of the peanut butter products satisfied both european and kenyan bureau of standard as safe for consumption. the study results are contrasted with the findings reported in the survey undertaken in zambia [15] where aflatoxin contamination in eight brands tested were in the range 73% (130μg/kg), 80% (10,740μ/kg) and 53% (1000μ/kg) respectively. using 15ppb exposure limit to aflatoxin contamination in peanut butter, this study has revealed that local products had higher aflatoxin contamination compared to international products. this is in contrast with report that stated that south african peanut butter had the highest aflatoxin contamination than the zambian local peanut butter products [15]. the high aflatoxin contamination in peanut butter could be attributed to the observed lack of cleanliness among traders from which peanut butter was purchased in the market. some looked unkempt and had no protective dress code. these findings imply that aflatoxin contamination is a problem which needs to be addressed in order to protect the population and minimise health concerns among the people. peanut butter is one of the products that is consumed by many people in zambia which motivate business people to buy it from soweto market and/or other sources; and later repack it for sale after labeling the cans. the results from this study are similar to a review of nut–pasticcio trade [21] that stated that nations tend to trade with other nations that have identical aflatoxin standards. this led countries without legal standards to import contaminated products and supplying such to consumers. such activities expose people to risks of aflatoxin contamination and in turn affect their health which would culminate into deaths as reported in other nations. therefore, it is important to search for interventions that will contribute to reducing aflatoxin contamination in peanut butter for safe human consumption. 4. conclusion this study has revealed that aflatoxin contamination is high in locally compared to internationally processed peanut butter products based on 15ppb set standard. using the european standard of up to 4ppb, both local and international peanut butter products origin range of aflatoxin contamination in (%) total 0 4ppb > 4 15ppb >15ppb international 7 (6.4) 46 (42.2) 1 (0.9) 54 (49.5) local 2 (1.8) 28 (25.7) 25 (22.9) 55 (50.5) total 9 (8.3) 74 (67.9) 26 (23.8) 109 (100) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 325 were found to be contaminated and less than ten (10) products were safe for human consumption. this implies that the population in zambia is consuming highly aflatoxin contaminated peanut butter products. aflatoxin contamination seems not to be well known and as a result it is recommended that awareness campaigns be amplified to enlighten the public on the dangers of consuming aflatoxin contaminated products. further, a recommendation to zabs as a regulatory agency to ensure regulatory limits are set, followed and complied with by processors. government must endorse collaborative local and international research to keep abreast on current trends of aflatoxin contamination reduction measures among stakeholders. 5. acknowledgments this study received monetary help from the faculty in the school of public health. a great recognition is extended to them in a special way otherwise this study could not have been done in the specified period. much gratitude goes to my husband amos who assisted in reading through the statistical section of the paper. munsanda and hendrix helped much in the laboratory works at the school of agriculture, microbiology laboratory, i wish to appreciate them greatly. 6. references [1]. liu, y. and wu, f. “global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment”. environmental health perspectives”. 2010: 118 (1): 818-824. [2]. iarc. “some traditional herbal medicines, some mycotoxin”, naphthalene and styrene: iarc monogr eval carcinog risks hum. 2002:82(2):171-300. [3]. schwartzbord, j. r. & brown d. l. “aflatoxin contamination in haitian peanut products and maize and the safety of oil processed from contaminated peanuts.” 2015 food control. 56(4): 114-118. [4]. mutegi, c., wagacha, c. j., kimani, j. and karanja, l., “effect of storage on conditions on quality and aflatoxin contamination of peanuts (arachis hypogaea l.).” international journal of agri science. 2013:3(5):746-758. [5]. guo, b., yu, j., holbrook, c., cleveland, t., nierman, w. and scully, b. 2009. “strategies in prevention of pre-harvest aflatoxin contamination in peanuts: aflatoxin biosynthesis, genetics and genomics.” peanut science. 36, 11 – 20. [6]. krishnamachari, k. a, r. v. bhat, v. nagarajan and t. b. tilak. 1975. hepatitis due to aflatoxicosis: an outbreak of hepatitis in parts of western india. lancet 1:1061-1063. [7]. blount, w. 1961. turkey “x” disease. turkeys. 9, 52 – 55 [8]. shephard, g.s. 2008. “risk assessment of aflatoxins in food in africa. food additives and contaminants.” 25, 1246 – 1256. [9]. lewis, l., m. onsongo, h. njapau, h. schurz-rogers, g. luber, s. kieszak, et al. 2005. “aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern kenya.” environ. health perspect 113:1763-1767. [10]. nyikal, j., misore, a., nzioka, c., njuguna, c., muchiri, e., njau, j., maingi, s., njoronge, j., mutiso, j., onteri, j., langat, a., kilei, i., nyamongo, j. & ogana, g. 2004. “outbreak of aflatoxin poisoning -eastern and central provinces, kenya.” centers for disease control and prevention. morb mort. january july 2004. ed. [11]. n’dede, c.b., jolly, c., vodouhe, s.d., and jolly, p. 2012. “economic risks of aflatoxin contamination in marketing of peanut in benin.” economics research international. 2012, 1 – 12. [12]. uçkun o & işil v. “monitoring of aflatoxins in peanuts.” türk tarım ve doğa bilimleri., 2014: 6, 1310-1314. [13]. fao, 2003. “mycotoxin regulations in and current developments. factors affecting the constitution of mycotoxin regulations in food and feed.” food control. 26, 580-586. [14]. zambia bureau of standards, zs 723:2008. “zambian peanut butter specification.” zambia: government printers. ics: 67.040., 2-3. [15]. njoronge, s.m., matumba, l., kanenga, k., siambi, m., wariya, f. maruwo, j. and monyo, e. 2016. “a case for regular aflatoxin monitoring in peanut butter in sub-saharan africa: lessons from 3-year survey food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 maureen s. banda, rosemary n. likwa, phoebe bwembya, jeremiah banda, allan mbewe, consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban – zambia, food and environment safety, volume xvii, issue 3 – 2018, pag. 317 – 326 326 in zambia.” journal of food protection. 79, 795 – 800. [16]. central statistical office (cso), 2010. “census of population and housing” zambia population and demographic projections 2011 2035. lusaka, zambia. [17]. matumba, l., poucke c. v., monjerezi, m., ediage, e.n., de seager, s. “concentrating aflatoxins on the domestic market through groundnut export: a focus on malawian groundnut value and supply chain.” 2015. food control. 51(10):236-239. [18]. mupunga, i., lebelo, s.l., mngqawa, p., rheeder, j.p. and katerere, d.r. 2014. “natural occurrence of aflatoxins in peanuts and peanut butter from bulawayo, zimbabwe.” journal of food protection. 77, 1814 – 1818. [19]. koirala, p. kumar, s., yadav, b. k., premarajan, k.c. 2005. “occurrence of aflatoxin in some of the food and feed in nepal.” indian journal of medical sciences. 59, 331 – 336. (20). chen ying-chun, liao chia-ding, lin hsu-yang, chiueh lih-ching, yangchih shih, daniel, 2013. “survey of aflatoxin contamination in peanut products in taiwan from 1997 to 2011.” food and drug administration, department of health, taipei 115, taiwan. (21). bui-klimke, t.r., gulch, h., kensler, t.w, yuan, j.m, wu, f. 2014. “aflatoxin regulations and global pistachio trade: insights from social network analysis plos one” 9(3): e92149. doi:10.1371/journal.pone.0092149. aflatoxin contamination 4. conclusion 300 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 2018, pag. 300 308 the potential role of chicken meat in transmission of campylobacter jejuni to consumers *nagwa thabet elshraway 1 , ahmed ibrahim youssef 2 mohamed shaker abdel hafez 3 1food hygiene department, 3poultry diseases department, 1,3faculty of veterinary medicine, assuit university, new valley branch, egypt, 1dr.nagwa2004@yahoo.com 2division of zoonoses, department of animal hygiene and zoonoses, faculty of veterinary medicine, suez canal university, ismailia, egypt. *corresponding author received 6th january 2018, accepted 24th september 2018 abstract: food-borne zoonotic diseases are a significant and widespread global public health threat. campylobacter jejuni is a most common contaminant of chicken meat. this study was conducted to investigate the presence of c. jejuni and some of its virulence genes in chicken meat in new valley, egypt and determine their zoonotic impacts. a total of 300 chicken meat specimens were collected from 100 freshly slaughtered from breast (pectoral), thigh, and liver (100 of each) from (healthy and diarrheic) live bird markets and analyzed for c. jejuni load using specific and selective nutrient media and molecular diagnosis. the identified strains were screened for virulence factors (virb11, fla a and iam) genes. the results revealed that throughout 300 meat samples, c. jejuni was detected in 74 (24.67%). the highest microbial load was in liver samples (26%) followed by pectoral (25%) then thigh muscle samples (23%). the virulence gene markers of c. jejuni was detected in chicken meat and liver samples as virob11 (2.27%), fla a gene (3.34%) and iam gene (26.66%). out of 50 diarrheic patients with food-poising signs 18 (36%) was positive for c. jejuni. the virulence markers genes in the human isolates showed that the prevalence of virb11 gene, fla a, and iam genes was 14.29 %, 71.43%, and 35.71%, respectively. in conclusion, this survey revealed that raw poultry meat available for consumers in elkharga, egypt was contaminated with zoonotic c. jejuni that might represent a threat to public health. keywords: chicken, campylobacter jejuni, enterotoxine, food poisoning 1. introduction foodborne diseases especially food poisoning is going up worldwide and becomes an important issue in public health impact by its fast contagiousness high morbidity and lethality. food-borne zoonotic pathogens caused by consuming undercooked food or drinking water contaminated by pathogenic microorganisms. many of food-borne zoonotic diseases are commonly found in the intestines of healthy food-producing animals and birds. chicken is one of the major food-borne pathogens source by contaminated raw or undercooked chicken meat [1]. live bird markets are places where customers can buy domestic bird where either slaughter birds in the retail shops or at homes where minimal, if any, food safety standards or veterinary inspection are applied [2]. these markets related to traditional preference for consumption of freshly slaughtered poultry. live poultry markets are an important part of the poultry supply chains in different parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonosis, in egypt, the poultry meat trade depends mainly on that types markets that characterized by unhygienic slaughtering process, lack of marketing infrastructures. http://www.fia.usv.ro/fiajournal mailto:dr.nagwa2004@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 301 c. jejuni is the major pathogen causing foodborne diseases worldwide [3]. the incident of c. jejuni is one of the most common causes of bacterial enteritis in human. poultry products play an important role in transmission of campylobacter bacteria to humans. c. jejuni contamination of poultry meat during processing has been reported [4]. campylobacter is most often detected in fresh broiler meat and in the eu the prevalence of these bacteria in broiler carcasses identified at the retail level varied from 3.1% to 58.8%, depending on the member state [5]. several studies showed that certain bacterial factors are essential for the pathogenesis of c. jejuni including the motility and adherence to intestinal mucosa, capability of the bacteria to invade enterocytes as well as toxin production [6]. recently, some genes have been recognized as responsible for the expression of pathogenicity such as flagellin gene fla a, virb11 and invasion associated markers (iam) genes which are genetic markers for c. jejuni [7]. it is necessary to know the extent to which the public is exposed to zoonotic c. jejuni infections via poultry meat as observed in slaughtered markets. the main objectives of this study were to assess the role of fresh chicken meat sold on the egyptian live bird markets in the transmission of c. jejuni entero-pathogene, detection of these genes have been recognized as responsible for the expression of pathogenicity and identifying its zoonotic implications. 2. material and methods ethical approval: this study has been approved by the animal rights and ethical use committee of suez canal and assiut universities. the study area: this study was conducted in elkharga which, is the capital of new valley governorate which is a part of the oasis which is located to the west of the nile valley, 232 kilometers to the south of assuit governorate. the new valley governorate represents about 45% from the total egypt area. samples collection: from farm birds, a total of 300 meat specimens were collected from 100 freshly slaughtered from 3 sites which were breast (pectoral) meat, thigh meat, and liver (100 of each) from new valley public from (healthy and diarrheic) live bird markets. all data was recorded and samples were transported with minimal delay in an ice-box to the laboratory for microbiological and molecular examinations. samples preparation: samples were prepared following the protocols of apha, [8] as following: ten grams from each sample were weighted under complete aseptic conditions, and transferred into sterile polyethylene bag containing 90 ml of sterile 0.1% peptone water (oxoid). samples were blended in a stomacher (lab-blender, 400) for one minute to provide 10-1 dilution. human samples: to identify the occurrence of c. jejuni food poisoning bacteria among human cases, 50 stool swabs were collected from the outpatients suffering from diarrhea and fever of elkharga general hospital. stool swab samples were examined for c. jejuni infections. patients were interviewed and filled out a standardized questionnaire addressing the family’s chicken consumption and purchasing and preparation conditions for chicken and their contacts with people having presented with an episode of diarrhea within 2-5 days after ingestion. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 302 microbiological examination: total bacterial isolation was performed by adding 0.1ml of each dilution to the following media in duplicate: standard plate count agar (oxoid) using drop technique. the plates were incubated at 35oc/24hr. plates with distinct colonies counted between 30-300 colony were enumerated as colony forming units (cfu) and in preston enrichment broth were incubated at 37ºc for 24 hours. after enrichment, 0.1 ml of the broth was streaked onto modified campylobacter selective agar base cefoperazone charcoal desoxycolate agar (oxoid) containing antibiotic supplement (oxoid). the plates were then incubated at 42ºc for 48 hours under microaerophilic conditions (5% o2, 10% co2, and 85% n2) using campylobacter gas generating kits (oxoid) [9]. all the isolates were picked up and preserved on nutrient agar (oxoid) and preston broth (oxoid) examined microscopically by gram's stain to observe the morphological arrangement and staining reaction and pure cultures of the isolates were biochemically identified using catalase test, oxidase test, urea hydrolysis test, hydrogen sulphide (h2s) production, citrate utilization test and rapid hippurate hydrolysis test according to quinn et al. [10]. ten pure positive isolates for c. jejuni further enterotoxins and pathogenicity gene identification by pcr. pcr detection: genomic dna was extracted from the selected c. jejuni isolates from chicken and human samples using the qia amp dna mini kit (qiagen, germany, gmbh) with modifications from the manufacturer’s recommendations. briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase k and 200 µl of lysis at 56○c for 10 min. after incubation, 200 µl of 100% ethanol was added to the lysate. the sample was then washed and centrifuged following the manufacturer’s recommendations. nucleic acid was eluted with 100 µl of elution buffer provided in the kit. primers used were supplied from (biobasic, canada) as listed in table (1). primers were utilized in a 25µl pcr reaction containing 12.5 µl of 2x dreamtaq green mastermix kit (fermentas, germany), 1 µl of each primer of 10 pmol concentration, 4.5 µl of water, and 6 µl of dna template. the reactions were performed in applied biosystem 2720 thermal cycler. the thermal cycle condition was used as initial denaturation at 94○c for 4 min, followed by 35 cycles of 94○c denaturation for 30 sec, annealing (temperature differed as mentioned in table 1), for 30 sec, and extension at 72○c for 45 sec. followed by on cycle of final extension at 72○c for 5 min. table 1 primers sequences, target genes, amplicon sizes and annealing temperature of pcr reactions. target gene primers sequences (5` to 3`) amplified segment (bp) annealing reference flaa aataaaaatgctgataaaacaggtg 855 53˚c datta et al., 2003 taccgaaccaatgtctgctctgatt virb11 tcttgtgagttgccttacccctttt 494 53˚c cctgcgtgtcctgtgttatttaccc iam gcgcaaatattatcaccc ttcacgactactactatgcgg 518 46˚c wieczorek et al., 2012 the pcr products were separated by electrophoresis on 1.2% agarose gel (applichem, germany, gmbh) in 1x tbe buffer at room temperature using gradients of 5v/cm. for gel analysis, 10 µl of pcr products were loaded in each gel slot and 100 bp plus dna ladders (qiagen, germany, gmbh) were used to determine the fragment sizes. control positive and control negative were included in each reaction. the gel was photographed by a gel documentation system (alpha innotech, biometra). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 303 table 2 total prevalence of c. jejuni among chicken meat and stool samples from diarrheic human samples examined number of the examined samples no (%) chicken pectoral meat 100 25 (25%) chicken thigh meat 100 23 (23%) chicken liver 100 26 (26%) total 300 74 (24.67%) human samples 50 14 (28%) 3. results 1total prevalence of c. jejuni in chicken meat and stool samples from diarrheic human as listed in the table (2) and figure (1), the results revealed that the total prevalence of c. jejuni in chicken samples was of 24.67% whereas the total prevalence in diarrheic human stool samples was 28%. the higher c. jejuni prevalence was in liver samples 26 (26%) followed by pectoral muscles 25 (25%) and thigh muscle 23 (23%). all these percentage of chicken meat and liver exceeded the egyptian organization specification (eos) levels of c. jejuni: considered unfit for human consumption because of high microbial load of c. jejuni exceeding the permissible limits. fig. 1 . the percentage of chicken meat and liver samples exceeding the eos standards of c. jejuni. 2prevalence of virb11, fla a, and iam virulence genes in c. jejuni isolates from chicken: the prevalence of three main virulence genes in ten c. jejuni from chicken meat and liver samples was illustrated in table (3) and figure (2) as following: the total prevalence of virb11, fla a, and iam genes was 6.67%, 3.34%, and 26.66%, respectively. virb11 gene was detected in 2 (20%) in pectoral samples. fla a gene was detected in 1(10%) only in thigh meat. the highest rate was detected in iam gene by a percentage of 3 (30%) in pectoral and liver samples and 2 (20%) of the thigh muscles. 25% 23% 26% 21% 22% 23% 24% 25% 26% 27% pectoral meat thigh meat liver food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 304 pcr amplification of the enterotoxins (virb11) 494 bp products of dna extracted from c. jejuni, enterotoxins (iam): 518 bp molecular size ladder. enterotoxins (flaa): 855bp of c. jejuni isolates from chicken samples respectively. fig. 2 agarose gel electrophoresis of specific dose-dependent amplification of c. jejuni pathogenic genes (virb11,iam, flaa). table 3 prevalence of three virulence genes of in c. jejuni isolates from chicken meat and human stool samples samples virulence c. jejuni genes virb11 no (%) fla a no (%) iam no (%) chicken pectoral meat 2 (20%) *nd 3 (30%) chicken thigh meat *nd 1 (10%) 2 (20%) chicken liver *nd *nd 3 (30%) total no=30 2 (6.67%) 1 (3.34%) 8 (26.66%) human samples 2/50 (14.29%) 10 (71.43%) 5 (35.71%) *nd: not detected 3prevalence of virb11, fla a, and iam virulence genes in human samples as illustrated in the table (3) and figure (2), out of 50 diarrheic patients with food poising signs, 18 (36%) for c. jejuni, among c. jejuni isolates, the virulence gene markers were detected as following virb11, 2 (14.29%), fla a 10 (71.43%), iam 5 (35.71%). all human subjects were living in rural areas and all were buying chicken from public live bird markers. 4. discussion chicken are susceptible to many bacterial diseases which may be caused by a lack of hygiene, extensive exposure to the microbial contaminants and being carriers of these diseases due to their genetic immunity. many chicken contaminating pathogens have potential risk for consumers directly from eating contaminated food by the microorganisms or through their enterotoxins. c. jejuni are associated in a wide range of food sources such as poultry. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 305 symptoms of food poisoning are ranged from mild to severe include stomach pain, abdominal cramps, nausea and vomiting, diarrhea, fever, and dehydration [11]. egyptians have been costumed to buy live chicken from public live bird markets and slaughter immediately after selection in the markets using the same primitive manual equipment in slaughtering that considered excellent sources to spreading microbial contamination to the slaughtered bird. that ‘on-the-spot’ slaughter, plucking and evisceration in the live bird markets lead to carcass contamination and poses hazards of food borne diseases to the consumers [1, 2]. meat handlers could be sources of c. jejuni contamination particularly in case of following improper personal hygienic precautions. moreover, shutting down live poultry markets is extremely effective in preventing human cases. egyptian organization specification, [12] and european regulations, (european commission, [13]) prohibit consumption of any food containing any amount of c. jejuni. in the present study, the detection of c. jejuni with zoonotic potentials that exceed the standards indicate public health hazards to the consumers and raise the needs for proper implementation of preventative programs and regular surveillance. c. jejuni infect intestinal tract of several domestic poultry types frequently which be more sever in chicks which known as enter-invasive transient diarrhea resulting as watery droppings, hepatic focal necrosis, focal hemorrhage, jejunum distention or the absence of clinical signs. infected bird shed c. jejuni after 2-3 weeks of infection [14]. however, it was illustrated that 100% of chickens infected asymptomatically by c. jejuni in their intestinal tracts and shading the organism through feces [15]. in this study, the total prevalence of c. jejuni among chicken meat and liver was 24.67% which was consistent with other studies that recorded 22.6% in egypt [2] and 25% in england [16]. moreover, a higher, this higher percentage could be attributed to the sampling of stool samples from human in contact with food animals [17]. however, lower c. jejuni isolation rate of 16.7% was reported in chicken meat of giza, egypt by hassanien et, al., [17], 21.5% in bin-suef, egypt chickens meat by el fadaly, et. al., [18]. other studies reported a very high prevalence of c. jejuni reached 40.4 % and 37.5 % in chicken liver and meat in egypt [19], 51.06% in malaysian [20], 60% in portugal [21]and even over 90% [22]. in 2008, an extensive survey on the prevalence of campylobacter spp. in broiler carcasses from slaughterhouses in the european union showed that 75.8% of samples were contaminated with these bacteria with 100% records in some countries [23]. the differences in the prevalence of c. jejuni could be related to differences in geographical area and population. this study was conducted in a desert area and very hot and dry weather might be a determinant of the prevalence rate. regarding the site of contamination of the examined bacteria, pectoral muscle the total contamination rate of c. jejuni was highest in liver samples 26 (26%), followed by breast muscles 25 (25%) and thigh muscles 23 (23%). ilida and faridah, [20] reported that poultry samples contaminated by c. jejuni were (66.67%) of breast & liver and (75%) of thigh. these results concluded that all poultry meat are subjected to contamination by c. jejuni from intestinal contents that might be occur during improper slaughtering, buckling and evisceration processes. compared with the numbers found on poultry meat and liver surfaces, bacterial numbers inside tissues are low but nonetheless may be significant when undercooking occurs [20]. regarding human samples, the prevalence rate of c. jejuni among diarrheic human samples was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 306 28%. c. jejuni was detected in 35% children stool in 3 governorates of egypt [19]. campylobacter organism is one of the most common causes of human bacterial gastroenteritis. about 15 of every 100,000 people are diagnosed with campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor campylobacter in their gut. all this emphasizes the importance of chickens as a potential reservoir and source of c. jejuni infection in human. some potential genetic markers of bacterial virulence have been identified. fla a gene involved in adhesion and colonization, virb11gene as pathogenic genes responsible for the expression of invasion and iam gene is associated with invasiveness and play a role in the transmission of campylobacter and/or its adaptation to different hosts [6, 7, 24, 25]. in this study, the prevalence of virb11 gene, fla a, and iam genes was 2.27%, 3.34%, and 26.66%, respectively in chicken meat and liver. results of virb11 gene in chicken was lower than that reported by many authors; 6.1% [26], 7.3% [27] and 9.5% [5]. the fla a gene was detected in 3.34% of chicken meat, which was much lower than previous reports, which reached even 100% wiseczorek et al., [26]. the iam gene was detected in 26.66% of chicken meat which was higher than the previous study by gonzalez-hein et al, [27] (6.2%), whereas it was much lower than that detected by wiseczorek et al., [26] who recorded 53.8%. the virulence markers genes in the human isolates showed that the prevalence of virb11 gene, fla a, and iam genes was 14.29 %, 71.43%, and 35.71%, respectively. results of virb11 gene was lower than that reported by datta et al., [5] who recorded 10·7%. another study showed that the virb11 gene was detected in 3.6% isolates from human [27]. the fla a was detected in a high percentage of the isolates tested (71.34%). these results were much lower than the data previously reported by other authors [5, 28]. the iam gene of campylobacter was another virulence marker detected in this study. differences in the prevalence of the iam factor were found by other authors [24, 28, 29]. the iam gene has been detected in the majority of invasive c. jejuni retrieved from humans. furthermore, the detection of iam in c. jejuni isolated from two important hosts, humans and chickens, suggested a role for this marker in c. jejuni’s colonization of multiple hosts. moreover, it was proposed that the iam gene is not only essential for the colonization of the chicken gut but is also responsible for the induction of diarrhea in humans [28, 29]. the detection of high rates of virulence genes among chicken and human samples referred to the potential of zoonotic infection of human by eating undercooked or improperly cooked chicken meat and liver. 5. conclusion in conclusion, this survey revealed that raw poultry meat available for consumers in egypt was often contaminated with zoonotic c. jejuni that might represent a threat to public health. furthermore, several strains were positive for the several putative virulence marker genes. the consumption of undercooked meat crosscontaminated with c. jejuni may pose a serious threat to consumer health. therefore, adequate cooking of chicken meat, the staff’s and equipment’s cleanliness and chemical disinfectants, hygienic handling, storage and effective processing of chicken meat are warranted. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 307 6. acknowledgments this study was funded by the corresponding author. 7. references [1] gwida m, el-gohary a., prevalence and characterization of antibiotic resistance food borne pathogens isolated from locally produced chicken raw meat and their handlers. journal of dairy, veterinary & animal research. 3(1): 00062, (2015). [2] khalafalla fa, abdel-atty ns, soad a abdel-wanis, adel s hanafy, food poisoning microorganisms in chicken broiler meat. global veterinaria 14(2): 211-218, (2015). [3] scallan e., hoekstra r.m., angulo f.j., tauxe r.v., widdowson m.a., roy s.l., jones j.l., griffin p.m., foodborne illness acquired in the united states--major pathogens. emerg infect dis. 17(1):7-15, (2011). [4] berrang, m. e., buhr, r. j., cason, j. a. & dickens, j. a. broiler carcass contamination with campylobacter from feces during defeathering. j food prot 64, 2063–2066, (2001). [5] datta s, niwa h., itoh k., prevalence of 11 pathogenic genes of campylobacter jejuni by pcr in strains isolated from humans, poultry meat and broiler and bovine faeces. journal of medical microbiology 52, 345–348, (2003). [6] dasti ji, tareen am, lugert r, zautner ae., gross u., campylobacter jejuni: a brief overview on pathogenecityassociated factors and disease-mediating mechanism. international journal of medical microbiology 300: 205–211, (2003). [7] nuijten, j., van den berg, j., formentini, i., van der zeijst, a., jacobs, a., dna rearrangements in the flagellin locus of an flaa mutant of campylobacter jejuni during colonization of chicken ceca. infect immun 68: 7137–7140, (2000). [8] american public health association (apha), compendium of methods for the microbiological examination of food 4th ed. apha technical committee on microbiological method for foods, washington d.c.usa, (2002). [9] skirrow, m. b., campylobacter enteritis: a “new” disease. british medical journal 2: 9, (1977). [10] quinn, p.j., m.e. carter, b.k. markey, g. carter, clinical veterinary microbiology, mosby yearbook europe ltd, (1994). [11] manal, f. abdelmalek, ayako, s., cynthia, g., aynur, u., ryan, c., richard, j., anna, m. d., increased fructose consumption is associated with fibrosis severity in patients with nafld. hepatology. 51(6):1961-71, (2010). [12] egyptian organization for standardization and quality, egyptian standards poultry meat. e.s.: 4177-2005, ics: 67.120.10, (2005). [13] european commission (ec), commission regulation no. 466/2005. setting of maximum limits for certain contaminants in foodstuffs. off. j. l77, (2005). [14] shane, m., campylobacter infection of commercial poultry. rev. sci. tech. off. int. epiz., 19(2): 376-395, (2000). [15] wagenaar, a, french p., havelaar h., preventing campylobacter at the source: why is it so difficult? clin infect dis. 2013; 57(11):1600-6, (2013). [16] jørgensen f, bailey r, williams s, henderson p., wareing r., prevalence and numbers of salmonella and campylobacter spp. on raw, whole chickens in relation to sampling methods. int j food microbiol 76(1-2): 151-164, (2002). [17] hassanain, n. a., antimicrobial resistant campylobacter jejuni isolated from humans and animals in egypt. global veterinaria 6 (2): 195-200 (2011). [18] el fadaly1, h. a., barakat, a. a., abd el-razik, k. a., abuelnaga, a. s. m., elgabry, e. a., incidence of zoonotic campylobacter jejuni in fast meal meat, grill chickens and symptomatic egyptians. african journal of microbiology research. 10(18): 608615, (2016). [19] barakat a., mona m. sobhy, elfadaly h., nagwa s. rabie, nashwa o. khalifa, eman r. hassan, kotb, m., zeinab m., girh, a., dalia m. sedeek, mona s. zaki., zoonotic hazards of campylobacteriosis in some areas in egypt. life science journal. 12(7): 9-16, (2015). [20] ilida n., faridah s., prevalence of campylobacter jejuni in chicken meat and chickenbased products. j. trop. agric. and fd. sc. 40 (1): 63–69, (2012). [21] antunes p, reu c, sousa c, peixe l., pestana n., incidence of salmonella from poultry products and their susceptibility to antimicrobial agents. int j food microbiol 82(2): 97-103, (2003). https://www.ncbi.nlm.nih.gov/pubmed/?term=scallan%20e%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=hoekstra%20rm%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=angulo%20fj%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=angulo%20fj%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=tauxe%20rv%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=widdowson%20ma%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=roy%20sl%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=roy%20sl%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=jones%20jl%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/?term=griffin%20pm%5bauthor%5d&cauthor=true&cauthor_uid=21192848 https://www.ncbi.nlm.nih.gov/pubmed/21192848 https://www.ncbi.nlm.nih.gov/pubmed/20301112 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez, the potential role of chicken meat in transmission of campylobacter jejuni to consumers, food and environment safety, volume xvii issue 3 – 2018, pag. 300 – 308 308 [22] rozynek e, dzierzanowskafangrat k, korsak d, konieczny p, wardak s, szych j, jarosz m., dzierzanowska d., comparison of antimicrobial resistance of campylobacter jejuni and campylobacter coli isolated from humans and chicken carcasses in poland. journal of food protection 71, 602–607, (2008). [23] tang h, mohamad ghazali f, saleha aa., nishibuchi m., son r., comparison of thermophilic campylobacter spp. occurrence in two types of retail chicken samples. international food research journal 16, 277–288, (2009). [24] sanad m, kassem i, liu z, lin j, lejeune t., rajashekara g., occurrence of the invasion associated marker (iam) in campylobacter jejuni isolated from cattle. bmc res notes. life sci j;12(7):9-14, (2011). [25] young kt, davis lm., dirita vj., campylobacter jejuni: molecular biology and pathogenesis. nature reviews microbiology 5, 665–679, (2007). [26] wieczorek k, szewczyk r., osek j., prevalence, antimicrobial resistance, and molecular characterization of campylobacter jejuni and campylobacter coli isolated from retail raw meat in poland. veterinarni medicina. 57(6):293-299, (2012). [27] gonzález-hein g, huaracán b, garcía p, figueroa g., prevalence of virulence genes in strains of campylobacter jejuni isolated from human, bovine and broiler. braz j microbiol. 10: 44 (4):1223-9, (2013). [28] rozynek e, dzierzanowskafangrat k, jozwiak p, popowski j, korsak d., dzierzanowska d., prevalence of potential virulence markers in polish campylobacter jejuni and campylobacter coli isolates obtained from hospitalized children and from chicken carcasses. j med microbiol, 54:615619, (2005). [29] korsak d, dzierzanowskafangrat k, popowskip j., rozynek e., incidence of the virulence markers iam in campylobacter jejuni and campylobacter coli strains isolated from poultry carcases. rocz panstw zakl hig, 55:307-312, (2004). 2. material and methods 3. results 4. discussion rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl2 nephropathy 261 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 2018, pag. 261 271 rat kidney tissues: oxidative modification of proteins under water and salt loads associated with hgcl2 nephropathy * alla velyka 1 1department of medicinal and pharmaceutical chemistry, bucovinian state medical university, chernivtsi, ukraine, velyka.alla@bsmu.edu.ua *corresponding author received 10th april 2018, accepted 14th september 2018 abstract: some changes in the main indexes of the pro-oxidation system of rat’s kidney tissues exposed to water and salt load on background of the nephropathy caused by mercury chloride were investigated. conditions of the free-radical oxidation of kidney tissues’ proteins under water and salt load alone or complicated by the nephropathy have been analyzed. it is found that water and salt load leads to a higher content of the thiobarbiturate reaction products (tba-rp) in kidney tissues in comparison with the control experiments while the content of the protein oxidative modification products remains unchanged. thus, water and salt load causes a higher activation level of free radical oxidation of lipids. a histochemical investigation has been performed to evaluate a degree of the oxidative modification of proteins in rat kidney tissues under water and salt load complicated by the mercuric nephropathy and a higher level of the oxidative modification of proteins has been determined by the red/blue coefficient in rat kidneys irrespectively of the load type in case of intoxication with mercury chloride. keywords: water and salt load, oxidative protein modification, kidney, mercury chloride, tbareaction products, environmental contamination 1. introduction heavy metals are known as one of the most dangerous environmental pollutants while soluble salts of mercury are among those, which cause very tough and longlasting consequences. mercury (ii) chloride is a well soluble salt, which is still in use in some branches of chemical industry, pharmaceutical and agricultural applications such as disinfection, seeds sterilization and so on. due to its extreme toxicity, ability to cumulate in tissues and aggravate along the food chains, this compound can cause devastating effect on the human and other organisms and misbalance the whole ecosystem for years. mercury chloride is a xenobiotic that exhibits rather wide toxicity effects affecting various organs and system but kidney is among those, which suffer the most of this kind of intoxication. that is why it is important to carry out wide investigations of its toxicity mechanism, effect on different organs and tissues, and changes caused by this toxic agent alone or in combination with other aggravating or mitigating conditions. kidney is a leading efferent organ of human and animals, which is responsible for maintenance of the water-electrolyte balance, acid-basic and osmotic homeostasis [1]. this organ is capable to provide highly adjustable and selective excretion of water and various ions to maintain stability of the inner biological liquids [2]. there is a dynamic equilibrium between proand antioxidants in the http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 262 tissues and organs of living organism, which can be disturbed by oxidative stresses and shifted towards more active formation of the free radicals. they facilitate oxidation of the macromolecules resulting in some disorders in the cell membranes structure and functioning [3]. a wide spectrum of issues related to disorders in the kidney function is being extensively investigated in various directions in the context of environmental protection, effect of various toxicants on biosystems and their mitigation, etc. that is why many efforts are targeted on experimental researches dealing with the oxidative, salt/water and intoxication stresses alone or on the background of other intoxications. morphological and functional characteristics of the nephropathy caused by mercury chloride facilitate its using as an adequate model for the kidney pathology in comparison to both clinical and experimental puromicin or adriamicin nephrites [4, 5]. on the other hand, it is important to understand what kind of changes can be caused to the kidney tissues suffering of the mercurytype intoxication together with salt and/or water load. glomerotubular disorders have been reported for pathogenesis of the kidney functionality affected by the experimental nephrites. these disorders are related to injuries to the proximal tubules epithelium in comparison to more resistive epithelium of the distal tubules [69]. the disorder in normal rats’ kidney functioning is still noticeable, even one to three weeks after mercury chloride intoxication. it is interesting that the comparison between the results obtained after simultaneous water or salt load and the intoxication proves that the one-weekdisorder is more significant than the threeweek one in the former combination while total intoxication effects become worse in three weeks at the latter combination [10]. proteinuria and reduced excretion of creatinine are the main signs of this disorder. an increasing proteinuria on the background of acceleration of nitrates and deceleration of nitrites excretion has been reported for the combined intoxication by hgcl2 and ccl4 [11]. an interaction between hgcl2 intoxication and oxidative stress induced by angiotensin ii proved that even specific anti-angiotensin treatment is capable to diminish the content of peroxide radicals in the rat kidney tissues and reduce tubular damage caused by the intoxication [12]. in general, any heavy metal intoxication (including mercury chloride) leads to significant changes in antioxidant activities and lipid peroxidation values [13]. therefore, it is obvious that various issues related to the heavy metals nephropathy alone or in combination with water or salt load remain interesting in the context of nephrology, toxicology and/or kidney tissues histology. our investigation has been aimed onto biochemical evaluation of influence of the water and salt load on content of the oxidate modified proteins and lipids in the rat kidney under experimental nephropathy followed by additional histological analysis of the kidney tissues. 2. materials and methods this investigation has been carried out with the white nonlinear male adult rats with weight 180±10 g. the animals were kept in the vivarium at stable temperature and illumination and subdivided into eight groups: group 1 (6 individuals) – control, no load. group 2 (6 individuals) – water load induced (5 ml per 100 g of live weight). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 263 group 3 (6 individuals) – the 0.75 % salt load induced (0.75 % solution of nacl was given as 0.65 mmol (14.8 mg of na) per 100 g of live weight). group 4 (6 individuals) – the 3 % salt load induced (3 % solution of nacl was given as 2.56 mmol (59 mg of na) per 100 g of live weight). group 5 (6 individuals) – the 0.1 % solution of mercury chloride was injected subcutaneously 5 mg per 1 kg of live weight. group 6 (6 individuals) – the 0.1 % solution of mercury chloride was injected subcutaneously 5 mg per 1 kg of live weight followed by the water load (same as for the group 2) induced in 72 hours. group 7 (6 individuals) – the 0.1 % solution of mercury chloride was injected subcutaneously 5 mg per 1 kg of live weight followed by the salt load (same as for the group 3) induced in 72 hours. group 8 (6 individuals) – the 0.1 % solution of mercury chloride was injected subcutaneously 5 mg per 1 kg of live weight followed by the salt load (same as for the group 4) induced in 72 hours. the water and salt loads were injected by the metal endogastric probe 2 hours before euthanasia. the euthanasia was realized by decapitation under the light ether narcosis according to the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (etc 123) [14]. the kidneys were taken out of the decapitated rats as soon as possible, dried by the filter paper and separated into three parts: cortex, medullar and papilla. then the 5 % supernatant of the rat kidneys was prepared using the 50 mm tris-hcl buffer solution (ph=7.4) containing 0.1 % solution of trilon b by centrifugation at 900 g for 10 min. all these operations were performed at the temperature below 4 0c. afterwards, the free radical oxidation conditions for lipids and proteins were determined in the post-nuclear supernatants by the content of tba-rp and the oxide-modified proteins products (omp-p) [15, 16]. a mikel calvo bromophenol blue staining method [17] was used for histochemical evaluation of the omp-p samples and the “colorpic” software was employed for the computer spectrometry of the histological micro sections. the r/b coefficient representing a ratio between the red (r, acidic proteins) and blue (b, basic proteins) staining of the cytoplasm was used to characterize a degree of the oxidative modification of proteins. all the pathological processes were recorded by taking digital photos of the micro samples parts by the camera olympus c740uz and the microscope lumam-p8 equipped with various interchangeable lenses. 3. results and discussion it has been found that both salt and water load can cause changes in characteristics of the free radical oxidation of macromolecules within various kidney layers. a rise for 35.7 % in comparison to the control samples value 44.3±4.26 mole/l has been registered for the tba-rp content in the kidney cortex tissue (see table 1). a rise for 48.1% has been found for the same content in the medullar tissue while the rise in papilla tissue was only for 17.3 %. same values for the 3 % salt load case were 66.4 % for the cortex samples, 42.5 % for the papilla and 24 % for the medullar tissues. finally, the 0.75 % salt load gave rise for 58.4 % in the papilla, 55 % cortex and 45.7 % medullar layers in comparison to the corresponding control samples. the photometry investigation at 370 and 430 nm showed no reliable changes in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 264 oxide proteins modification products contents after 5 % water, 3 % or 0.75 % salt loads (table 1). on the other hand, both 3 % and 0.75 % salt loads result in approx. 25 % rise in the tba-rp content in various kidney tissues comparing to those after the water load. this may be caused by a deeper stress imposed by the salt load followed by more intense activation of the free-radical oxidation of lipids. table 1 normalized optical densities (nod) for the “blue” and “red” spectral zones (370 and 430 nm respectively) parameters groups tba-rp, mole/g (tissues) omp-p (370), nod (tissues) omp-p (430), nod (tissues) control group cortex kidney tissues 43.3±4.26 10.9±0.46 20.2±1.55 medullar kidney tissues 60.8±4.51 13.3±1.95 20.1±0.60 papilla kidney tissues 57.0±1.15 10.3±0.45 18.5±1.25 5 % water load cortex kidney tissues 60.1±7.56* 11.4±0.41 18.5±1.10 medullar kidney tissues 90.1±5.88* 11.2±1.36 18.0±1.55 papilla kidney tissues 66.9±3.98* 10.1±0.87 18.2±0.26 3% salt load cortex kidney tissues 73.7±2.86* 11.8±0.30 19.9±0.56 medullar kidney tissues 75.4±2.09* 10.9±0.10 18.7±0.45 papilla kidney tissues 81.1±3.59* 10.1±0.36 17.7±1.20 0.75% salt load cortex kidney tissues 68.8±4.00* 11.9±0.85 18.5±0.21 medullar kidney tissues 88.6±9.88* 11.7±0.55 16.9±0.85 papilla kidney tissues 90.3±3.99* 10.5±0.7 17.9±1.58 note: р≤ 0.05 comparing to the corresponding kidney tissues in the control series a measuring method for the ratio between the basic and acidic protein groups is based on comparison between optical densities measured at 370 nm (“blue” spectral zone, related to the neutral aldehydoand ketone-derivatives of the proteins) and 430 nm (“red” zone, the basic derivatives) [17]. all optical densities obtained in the experiments were normalized per 1 g of the tissue. then the r/b coefficient was calculated as a quotient of division the normalized “red” zone optical density by the “blue” zone optical density [17]. it is known [3, 18] that the free radical oxidation of proteins causes formation of new carboxyl groups and other acidic functions that results in shifting the r/b coefficient towards more acidic values [19]. that is why a value of r/b can be used as a characteristic of the oxidative modification of proteins. when its value is equal to 1, the content of the acidic and basic product is the same and it is greater than 1, the content of the acidic products is greater than the basic ones [17]. data of table 2 prove various depth of the oxide proteins modification in the epithelium of tissues of the kidney cortex sinuous tubules. as seen from the data, no clear changes were registered in the cytoplasm of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 265 epitheliocytes in the kidney sinuous tubules after 0.75 % salt load. however, both 5 % water and 3 % salt load caused shifts in the amino vs carboxyl groups in the proteins towards the latter. this can be considered as a sign of more intense oxidative modification of the proteins. it is interesting that for the 3 % salt load the above changes are more distinct for the samples taken at 8 pm than that for the 8 am samples. the value of r/b becomes three times greater after injection of mercury chloride and again the 8 pm coefficients are some greater than the 8 am ones. on the other hand, the water load cause some decrease in the r/b values but they remain far above the control group. no significant changes in the r/b values were registered at the simultaneous mercury chloride intoxication and 0.75 % salt load while these parameters showed the highest level after same intoxication combined with 3 % salt load. it should also be stressed that r/b for the hyaline and granular cylinders of the sinuous tubules lumen was higher than that in the control group, which evidences deeper oxidative modification in the cell remainders. table 2 r/b coefficients for the epithelium of sinuous tubules of the rat kidney cortex tissue samples taken under various experimental conditions group sampling time deviation probability 8.00 20.00 control 1.02±0.004 1.03±0.005 р>0.05 water load 1.12±0.006 1.14±0.008 р>0.05 0.75 % salt load 1.03±0.006 1.04±0.007 р>0.05 3 % salt load 1.14±0.008 1.17±0.009 р=0.032 hgcl2 2.94±0.014 3.21±0.017 р<0.001 hgcl2 and water load 2.46±0.015 2.71±0.017 р<0.001 hgcl2 and 0.75 % salt load 2.90±0.019 3.23±0.018 р<0.001 hgcl2 and 3 % salt load 3.26±0.018 3.43±0.020 р<0.001 note: р≤ 0,05 comparing to the corresponding kidney tissues in the control series the above mentioned changes can also be proved by the series of microphotographs of the kidney core layer tissues. all the samples were stained with bromphenol blue by mikel-calvo method (see figs. 1-8). as seen from the comparison between the intact animals (fig 1) and the animals exposed to 5 % water load (fig. 2), an average percentage of the cells affected by granular degeneration has increased in the latter case. this situation can be caused by a higher functional load on epitheliocytes leading to more intense running of physiological processes, which knocks out the weakest cells and results in the changes represented in fig. 2. it should also be noted that some signs of plethora can be seen in the venules of interstitium for the animals exposed to water load both for the 8 am and 8 pm euthanasia. however, no statistically relevant increase in the total interstitium volume was determined. application of mercury chloride causes some deep morphological changes of the kidney tissues. the severest affection can be noted for epithelium of the proximal tubules of core kidney tissues. coagulation necrosis has stricken 40 % of the proximal tubules for the 8 am euthanasia (fig. 5a) and 70 % for the 8 pm euthanasia (fig 5b). besides, significant destruction of the cell nuclei (karyolysis) can be noted for both series of euthanasia (fig 5 a and b). therefore, the total affection of epitheliocytes with the alteration process can be stated. this process implies first reversible changes in the tissues structure followed by irreversible effects after a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 266 longer exposition to the pathological processes. the tubules lumen is almost completely clogged with fragments of necrotized or desquamated cells. it can be concluded that the mercury chloride intoxication (caused, for example, by environmental contamination or some other reasons) results in deep histological and morphological changes in the kidney tissues. that is why further experiments were aimed onto investigation of combined actions of the mercury chloride intoxication on the background of the salt and/or water load. a b fig. 1. core tissues of the intact animals. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . a b fig. 2. core tissues of the rats exposed to 5 % water load. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 267 a b fig. 3. core tissues of the rats exposed to 0.75 % salt load. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . a b fig. 4. core tissues of the rats exposed to 3 % salt load. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 268 a b fig. 5. core tissues of the rats exposed to mercury chloride. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10х. ocular magnification 10х. as seen from the fig. 6, water load causes some relief in the kidney core tissues affected by the mercury chloride nephropathy. although almost all epitheliocytes were involved in the process of alteration, its depth was lower and the process manifested itself mostly through the granular and hydropic degeneration. for example, comparison between fig. 6a and b proves that only 9 % of epitheliocytes were necrotized for the 8 am euthanasia while for 8 pm that parameter was 16 %. no interstitium edema can be notice for any of the above cases. a b fig. 6. core tissues of the rats exposed to 5 % water load combined with the mercury chloride intoxication. а) euthanasia at 8 am; b) euthanasia at 8 pm. necrosis spots (1) and granular or hydropic degeneration spots (2) are indicated. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10х. ocular magnification 10х. 1 1 2 2 2 1 1 1 1 1 2 2 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 269 obvious positive changes can be seen from comparison between fig 5 (mercury chloride nephropathy alone) and fig 6 (same as in fig. 5 but on background of the extra 5% water load): the kidney core tissue status is improving and the necrosis affects a lesser area of the tissue. a b fig. 7. core tissues of the rats exposed to 0.75 % salt load combined with the mercury chloride intoxication. а) euthanasia at 8 am; b) euthanasia at 8 pm. bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . a b fig. 8. core tissues of the rats exposed to 3 % salt load combined with the mercury chloride intoxication. а) euthanasia at 8 am; b) euthanasia at 8 pm. necrosis spots are indicated (1). bromophenol blue staining by mikel-calvo method for the acidic and basic proteins. lens magnification 10 х . ocular magnification 10 х . 1 1 1 1 1 1 1 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 270 in contrary to the water load, serious deterioration in the kidney core tissue status was caused by the 3 % salt load exposed together with the mercury chloride intoxication. massive necrosis has covered more than 90 % of the tubules in the juxtamedullary area of kidney. the rest of epitheliocytes were affected by degeneration, mostly of the hydropic type, which is more threatening than the granular one. the comparison between fig. 8 a and b proves that about 70 % of the tubules were affected by deep necrosis for the 8 am euthanasia while this parameter reached 73 % for the 8 pm experiment. all these processes took place on the background of total involvement of the tubules epithelium in the process of alteration. 4. conclusion a content of tba-rp in kidney tissues samples changes under both water and salt loads while contents of omp-p remain almost unchanged. regardless of the sampling time, both types of the loads cause moderate changes in the depth of oxidative modification of proteins. the injection of mercury chloride followed by water and/or salt load results in the activation of free-radical oxidation of proteins because of the damage of cell membranes. a value of the oxide proteins modification index can bring important information related to pathogenesis and histology of the kidney tissues. in general, it can be concluded that only moderate and reversible morphological changes were found in the kidney tissues underwent 5 % water and 3 % salt load while no morphological changes were found in the tissues after 0.75 % salt load. these morphological changes are in good agreement with histochemical data of the oxidative modification of proteins. a classical necrotic nephrosis has been found in the animals after the mercury chloride intoxication. the nephrosis symptoms were more severe at 8 pm comparing to those at 8 am. besides, the nephrosis symptoms were relieved partially by the water load while 3 % salt load caused worsening of the kidney tissues injury especially in case of the 8 pm results. no significant changes in the nephrosis symptoms were found after additional 0.75 % salt load. these results are also in good agreement with the histochemical data related to oxidative modification of proteins. therefore, it can be concluded that water load can provide some relieving effect on the mercury chloride nephrosis while salt load results in further aggravation of its symptoms. 5. references [1]. gozhenko, a., sluchenko, a., functional conditions of the rat’s kidney under water and salt load at pregnancy and the mercury chloride nephropathy. nephrology, 10(1): 72-76 (2006) (in ukrainian). [2]. vladimirov, yu., archakov, a., peroxide lipids oxidation in biological membranes. moscow, nauka, 252 p (1972) (in russian). [3]. matsyopa, i., grygorieva, n., freeradical lipids oxidation products, proteins contents and antioxidation system activity in the rat’s kidney under changed photoperiod and exposition to melatonin and extraction of echinacea purpurea. bulletin of biol. and med. problems, (2009), 3: 7881 (2009) (in ukrainian). [4]. jeansson, m., bjorck, k., tenstad, o., haraldsson, b., adriamycin alters in glomerular endothelium to induce proteinuria. j. am. soc. nephrology, 20(1): 114–122 (2009). [5]. besse eschman, v., et al., regulation of the proximal tubular sodium/proton exchanger nhe3 in rats with puromycin aminonucleoside (pan) – induced nephrotic syndrome. j. am. soc. nephrology, 13(9): 2199–2206 (2002). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 alla velyka, rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl 2 nephropathy, food and environment safety, volume xvii, issue 3 – 2018, pag. 261 – 271 271 [6]. tompson, s., blantz, r., glomerulotubular balance, tubuloglomerulo feedback, and salt homeostasis. j. am. soc. nephrology, 19(12): 2272–2275 (2008). [7]. heyman, s., et al. proximal tubular injury attenuates outer medullary hypoxic damage: studies in perfused rat kidneys. exp. nephrol., 10(4): 259– 266 (2002). [8]. gobe, g., jonson, d., distal tubular epithelial cells of the kidney: potential support for proximal tubular cell survival after renal injury. int. j. biochem., 39(9): 1551–1561 (2007). [9]. gadau, j., et al. mechanisms of tubular volume retention in immune mediated glomerulonephritis volume retention in acute gn. kidney int., 75(7): 699–710 (2009). [10]. gozhenko, a., et al, a functional kidney reserve at chronic toxic nephropathies. actual problems of the transport medicine, 1(7): 131-134 (2007) (in russian). [11]. nasibulin, b., et al, structural effects caused by some mercury compounds alone or in combination with carbon tetrachloride. morphology bulletin, 11(1): 18-21 (2005) (in russian). [12]. pena, c., et al. renal oxidative stress and renal cd8+ t-cell infiltration in mercuric chlorideinduced nephropathy in rats: role of angiotensin ii. j. immunotoxicology, 13(3): 324-334 (2016). [13]. apayudin, p., acute effects of low dose lead nitrate and mercury chloride exposure on kidney of rats. environ. toxicol. and pharmacol., 41: 219-224 (2016). [14]. http://www.coe.int/en/web/conventions/fulllist/-/conventions/treaty/123 (european convention for the protection of vertebrate animals used for experimental and other scientific purposes). accessed: april 03, 2018. [15]. frankel, e. n., lipid oxidation. prog. lipid. res., 19: 1-22 (1980). [16]. drachuk. v., zasmorskiy, i., horoshko, o., morphological changes of kidney tissues when using glutathione at rhabdomyolytic acute kidney injury. german science herald, 4: 34-37 (2016). [17]. patent of ukraine u200509673. a method of measuring of the oxidative modification of proteins in the placenta tissues. authors: o. shenderyuk, i. davydenko. industrial property, 4. published 17.04.2006. [18]. pishak, v., velyka, a., matsyopa, a., changes in the lipids and proteins peroxide oxidation indexes under the water and salt load. ukr. j. clinic. and lab. medicine, 6(4): 38-40 (2011) (in ukrainian). [19]. shenderyuk, o., davydenko, i., a method of measuring of the oxide modification of proteins in the placenta tissues. clinical anatomy and operational surgery, 5(2): 101 (2006) (in ukrainian). http://www.coe.int/en/web/conventions/full-list/-/conventions/treaty/123 http://www.coe.int/en/web/conventions/full-list/-/conventions/treaty/123 1. introduction title …………………… 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1 2018, pag. 5 8 elaboration of a method to determine the dry white wine falsification with sucrose * igor winkler 1,2 , antonia vitvitska 2 1 department of medicinal and pharmaceutical chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine winkler@bsmu.edu.ua 2department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine *corresponding author received 17th october 2017, accepted 19th march 2018 abstract: a standard method of reducing sugars determination using fehling-muller reagent has been modified and applied to control the presence of sucrose in white dry wine that can be considered as an indication of its falsification. natural dry wine should not contain any significant amounts of sucrose since its content in regular grape is below 0.9 wt % and it would be fermented almost completely in the course of wine maturing and manufacturing. however, some sucrose can be added by fraudulent producers to the source wine materials in order to accelerate its fermentation. this results in a higher content of residual sucrose in dry wine that can exceed its maximum permissible level of 4 g/l. the modified method of the reducing sugars determination has shown good durability and reproducibility and can be used to determine the residual sucrose concentration above 1.33 g/l. however, excessive sulfites and other reducing preservatives present in some wines (especially in the low-grade samples) can distort the analysis results and should be removed in advance. potentially, this method can also be applied to analyze wine blending samples and to control their affinity by the ratio between reducing and non-reducing sugars contents. keywords: white wine; wine falsification; sucrose; modified fehling-muller method; reducing sugars 1. introduction residual sugars content in dry white wine is limited by the value 4 g/l, which should consist of almost no sucrose [1]. this requirement is based on a low content of sucrose in the raw grapes, where glucose and fructose represent almost all the sugars in the berries. those insignificant amounts of sucrose coming with grape should be completely fermented during maturing of wine. therefore, any considerable amount of sucrose found in the dry wine can be considered as an evidence of its possible falsification by fraudulent producers through adding of the ‘extra’ sucrose to accelerate the fermentation [2, 3]. it is known that an estimated amount of falsified wines coming to the world markets is very large. this fraudulent practice started as long as wine has been made in antiquity and it is still popular [4]. there are numerous methods of wine manipulation and adding of disaccharides is one of them. technically, there are several approaches to determination total sugars content in wine, which involve photometry, hplc or traditional chemical methods using oxidation of the monosugars by fehlingmuller reagent [2]. http://www.fia.usv.ro/fiajournal mailto:winkler@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor winkler, antonia vitvitska, elaboration of a method to determine the white dry wine falsification with sucrose, food and environment safety, volume xvii, issue 1 – 2018, pag. 5 – 8 6 however, only the latter one is recognized as the official method of determination the sugar content in wines [1, 5]. both reagents are in fact same complex compound – ditartratocopper (ii) hydroxide with the only difference in their preparation methods. following both methods, any sugars present in wine should be previously converted into the monosugars (glucose, fructose) and then the concentration of the latter can be determined according to the reagent amount used for their oxidation [1]. an idea beyond our experimental method is following. theoretically, disaccharides are not subject of oxidation by fehlingmuller reagent since they do not undergo any chemical transformation under the conditions set for the monosaccharides transformation. in reality, it is not true and disaccharides can be partially oxidized as well, bringing some experimental errors in the results. on the other hand, the value of this error is comparatively low and can be neglected in preliminary investigations. therefore, we can establish two parallel determinations with two samples of the same wine. one sample would remain untreated preserving all sugars in their original form (monosaccharides remain monosaccharides while disaccharides also remain unconverted). another sample would be treated according to the standard sugar inversion method that converts all present sugars into monosaccharides. then both samples should be analyzed according to the fehling-muller method [1, 2] followed by cross-comparison between the results obtained. any difference between them would indicate presence of some disaccharides in the source material. more exact results can be obtained from the advanced method including parallel idle experiments taking into account unavoidable natural decomposition of the reagents and other factors beyond our control. it is important to check if this method can work in reality for the samples of white wines available in regular stores and understand if its sensitivity would be sufficient to find disaccharides concentrations expected for the fraudulent wines. due to massive amounts of reducing agents present naturally in the red wines and their intense color, our investigations were restricted by the white brands only. 2. experimental all experiments were carried out using three randomly selected ukrainian-made wine samples: a cheap unbranded table wine in the tetrapack-type carton box (1); and two other bottled wines of chardonnay (2) and sauvignon (3) types. all beverages were declared by their producers as the table dry wines and were purchased in local grocery stores. each wine was opened right before the investigation and then two equal samples were taken from each bottle/pack. one of them was left untreated while the other sample underwent the acidic sugars inversion according to [1]. all dilution ratios were kept similar for the both samples. fehlingmuller reagent and other working solutions were prepared not longer than 6 hors prior usage according to detailed description [1,6]. then the samples were analyzed and the sugars contents were determined in such a way that the first (untreated) sample result corresponded to the monosaccharides content only while the second sample result corresponded to the total sugars content. a difference between the second and the first values gave us a possible content of disaccharides in the wine. in order to understand if the method’s sensitivity is sufficient for determination of the disaccharides content that can be expected in case of wine falsification, we repeated all the above procedures with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor winkler, antonia vitvitska, elaboration of a method to determine the white dry wine falsification with sucrose, food and environment safety, volume xvii, issue 1 – 2018, pag. 5 – 8 7 another set of samples after intentional adding of 1,33 g/l (4 mmole/l) of sucrose before investigation. since this concentration is three times lower than the maximum permissible sugar content for the dry wine, it was considered as an indicator of wine falsification that can be expected in real conditions. 3. results and discussion all results of the sugars contents determination in the source and intentionally falsified wine samples are shown in table 1. table 1. determined sugars contents (x, mmole/l) in different wine samples sample 1 х (source wine) 2 х (intentional falsification) 3 (1), inverted 1.8 19.7 (1), untreated 1.7 12.9 (2), inverted 2.2 4.8 (2), untreated 1.4 4.2 (3), inverted 2.3 5.0 (3), untreated 1.2 4.5 as seen from the data of table 1, column 2, almost no sucrose was found in the sample 1 (its inverted and untreated x values were 1,8 and 1,7 or almost identical) while in two other samples the concentration of sucrose was about 1 mmole/l (0,34 g/l). indeed, comparing the x values for the inverted and untreated samples of the wines (2) and (3), one can see that the difference between them was 0.8 for the wine (2) and 1,1 for the wine (3). this content is rather insignificant and it can be considered as a residue of the initial natural sucrose remained in the source blend after its fermentation. absence of sucrose in the sample (1) can be caused by a comparatively lower content of the compound in the source blend or by a deeper fermentation of the source material. therefore, it can be initially concluded that there is no any signs of forgery with sucrose in the wine samples taken for this investigation. on the other hand, the suitability of any analytical method proposed for identification of the food falsification should be tested for the falsified samples in order to assess general qualification and sensitivity of the method. in this context, the data of the column 3, table 1, which is related to the intentionally falsified samples should be analyzed and discussed. as seen from the table, the results for the samples (2) and (3) are quite predictable: sugar contents rise approximately by 4 mmole/l after adding extra 4 mmole of sugar. for instance, the total content of all sugars in the sample (2) was 2,2 mmole/l initially and then it increased to 4,8 mmole/l after intentional adding of sucrose. same data for the sample (3) are 2,3 and 5,0 mmole/l simultaneously. one can notice some deficiency in the sugar contents since 2,2 initial mmole/l plus 4 mmole/l (added) had to result in the content 6,2 mmole/l while in reality it was only 4,8 mmole/l (sample(2)). this deficiency in the total sugar content can be caused by partial sucrose decomposition and presence of some impurities in the sucrose used for ‘falsification’. as a result we added lesser amount of sucrose and some more products of its hydrolysis during the process of intentional forgery. the result of falsification of the sample (1) is paradoxical as the sugar content in the inverted sample has risen for approximately 18 mmole/l and for ~11 mmole/l in case of untreated wine. in our opinion, this result may be caused by considerable amounts of reducing preservatives (for instance, sulfites) present in this sort of the cheap wine. these compounds may distort results of the sugars content determination by fehling-muller method since they also consume copper tartratic complex actively food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor winkler, antonia vitvitska, elaboration of a method to determine the white dry wine falsification with sucrose, food and environment safety, volume xvii, issue 1 – 2018, pag. 5 – 8 8 for the reactions of own reduction. this assumption seems real taking into account lower price category of this wine. that is why potential applicability of the proposed method for the dry wines falsification with sucrose should be limited by the lowpreservatives containing wines. 4. conclusion the proposed modification of the well known and reliable fehling-muller method makes feasible its utilization in the wine genuineness/falsification control. this method ensures determination of disaccharides presence in wine even with contents below their maximum permissible level. on the other hand, the presence of wine preservatives, sulfites or other reducing agents may cause serious distortion of the results. the method can not be applied directly to red and some other wines with intense color and/or containing considerable amounts of natural reducing agents and tannin-like compounds. extra attention should be given to application of this method in the case of analysis of lowgrade wines. 5. references [1]. dstu gost13192:2009. wines, wine materials and cognacs: a method of sugars content determination. http://document.ua/vinavinomaterialy-i-konjaki_-metod-opredelenijasaharov-std4532.html (accessed october 10, 2017) (in ukrainian). [2]. wine sugar analysis: it’s just a matter of… fehling. https://www.cdrfoodlab.com/newstopics/sugar-analysis-wine-methods/ (accessed october 02, 2017). [3]. steve kupina, mark roman, determination of total carbohydrates in wine and wine-like beverages by hplc with a refractive index detector, j. aoac intl., 97(2): 498-505 (2014). [4]. jancis robinson, julia harding, the oxford companion to wine, 4th edition, oxford university press, oxford, uk (2015). [5]. law of ukraine “grape and grape wines” http://zakon4.rada.gov.ua/laws/show/2662-15 (accessed september 22, 2017) (in ukrainian). [6]. an iodometric method of the reducing compounds determination using the muller’s reagent. http://mydocx.ru/10-84362.html (accessed september 22, 2017) (in russian). http://document.ua/vina-vinomaterialy-i-konjaki_-metod-opredelenija-saharov-std4532.html http://document.ua/vina-vinomaterialy-i-konjaki_-metod-opredelenija-saharov-std4532.html http://document.ua/vina-vinomaterialy-i-konjaki_-metod-opredelenija-saharov-std4532.html https://www.cdrfoodlab.com/news-topics/sugar-analysis-wine-methods/ https://www.cdrfoodlab.com/news-topics/sugar-analysis-wine-methods/ http://zakon4.rada.gov.ua/laws/show/2662-15 http://mydocx.ru/10-84362.html 1. introduction on the other hand, the suitability of any analytical method proposed for identification of the food falsification should be tested for the falsified samples in order to assess general qualification and sensitivity of the method. in this context, the d... as seen from the table, the results for the samples (2) and (3) are quite predictable: sugar contents rise approximately by 4 mmole/l after adding extra 4 mmole of sugar. for instance, the total content of all sugars in the sample (2) was 2,2 mmole/l ... this deficiency in the total sugar content can be caused by partial sucrose decomposition and presence of some impurities in the sucrose used for ‘falsification’. as a result we added lesser amount of sucrose and some more products of its hydrolysis d... the result of falsification of the sample (1) is paradoxical as the sugar content in the inverted sample has risen for approximately 18 mmole/l and for ~11 mmole/l in case of untreated wine. in our opinion, this result may be caused by considerable am... 4. conclusion microbial characteristics of egg-white creams with reduced sugar 147 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 147 152 microbial characteristics of egg-white creams with reduced sugar nataliia hryhorchak 1 , olena ukrainets 1 , alina bilko 2 *iryna sokolovska 2 , julya kambulova 2 1department of biotechnology and microbiology, national university of food technologies 2department of bakery and confectionary goods technologies, national university of food technologies, volodymyrska str.,68, kyiv, ukraine, 01033, *corresponding author: sokolovska.ir@gmail.com received 21th june 2017, accepted 25th september 2017 abstract: one of the most relevant problems in developed countries is the reduction of free sugars in confectionary goods. the article focuses on the microbial analysis of egg-white creams for cakes and pastries decoration with reduced sugar. it was determined the possibility of replacing the traditional sucrose by fructose or glucose in the technology of egg-white creams production. the results of research showed the possibility of reducing sugar in creams up to 40%. sugar gives the products high dry matter content. sugar reduction causes the growth of free humidity content and microbial activity in confectionary goods. the free humidity binding is provided by complex of polysaccharides – pectin and sodium alginate. the binding ability of structurants provides minor changes in humidity and ph of egg-white creams during storage. creams with reduced sugar correspond to all the state standard requirements by microbial indicators. new recipes could be used in the production of cakes and pastries decorated by egg-white creams. keywords: glucose, fructose, sucrose, microbial activity, polysachharide complex. 1. introduction in the new "guideline: sugar intake for adults and children" of the world health organization recommendations of reducing consumption level of free sugars are given [1-2]. consumption of free sugars by adults and children should be reduced to less than 10% of the total caloric content of consumed food. a decrease to less than 5% of the total caloric content of consumed food will provide further health benefits. based on these recommendations, developed countries have identified priority ways to improve the technology of food products, especially confectionery products with reduced sugar [3-5]. previous studies determine that it is possible to reduce sucrose about 25% in the recipe of egg-white creams that are common semi-finished goods for cakes and pastries decorating [6]. along with this, we have proposed to improve technological scheme of egg-white creams production and special diet food, which replaces sucrose by glucose or fructose. in new recipes the normative quantity of glucose is decreased about 30% in comparison to traditional recipes. the quantity of fructose is decreased about 40% without compromising the structuralmechanical, physico-chemical and organoleptic properties. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova, microbial characteristics of egg-white creams with reduced sugar, food and environment safety, volume xvi, issue 3 – 2017, pag. 147 – 152 148 but, accordance to such parameters as high stability and sweetness of creams with reduced sugars is not enough for such product as egg-white cream. the determining factor to reduce sugar complies with the requirements of regulatory documents by microbial parameters of creams. egg-white creams refer to products with high water activity in which (aw) 0.88-0.98. in such systems, a variety of bacteria, mold, and yeast can develop [7-9]. sugar is a natural preservative [10-13], and the concentrations of sugar used in creams as a rule prevent the rapid growth of microorganisms. therefore, the indicators of microbial safety were studied in creams with reduced sugars. 2. matherials and methods three samples were analyzed: egg-white cream with sucrose (-25%), egg-white cream with glucose (-30%), egg-white cream with fructose (-40%). to stabilize the structure of egg-white foam, a complex of natural stabilizers – pectin and sodium alginate was introduced in cream recipe. cream with sucrose and fructose is produced with technological scheme for raw protein cream. this scheme includes the preparation of prescripted components, whipped egg-white with sugar and addition of citric acid. to make cream with glucose, the technological scheme of custard eggwhite cream was used. the syrup of glucose and maltose treacle was brewed and introduced (t ~ 90˚c) in egg-white foam during whipping. according to the recommended storage rules of creams by the ukrainian standard [14] the shelf life of products decorated with raw egg-white creams is 12 hours, with custard cream –is 5 days at a temperature of 6 ± 2 oc. the following microbial indicators were monitored [14]: the quantity of mesophilic aerobic and facultative anaerobic microorganisms (qmafanm), yeasts and molds, the quantity of spore-forming bacteria (sfb), and pathogens, including salmonella and s. aureus in number of conventional units (ncu) per 1 g. the number of spore-forming bacteria is not provided by standards for cakes and pastries in ukraine. but, from the literature data, it is known that the high number of sfb in raw materials and finished goods may contain enough pathogenic bacteria bacillius cereus, which is dangerous for human health [15-17]. the analysis of samples was carried out within 5 days of storage at temperatures of 6 ± 2 oc. the microbial determination was made in compliance with lvs iso 21257-2:2008. ph was measured by az-8690 ph-meter, precision 0.01 (standard method lvs iso 5542:2010). moisture content was determined by verified balance with precision ±0.001g; mass loss was determined by weighing samples on scales (lvs iso 1442: 1997). 3. results and discussion the results of microbial analysis are given in the table 1. the analysis of tabular data allows determining the new egg-white creams recipes with reduced sugars as safe for human health during their use (within 5 days). the microbial content of all the tested parameters does not exceed required limits. pathogenic bacteria such as s. aureus and salmonella were not found. this indicates that the complex of polysaccharides introduced to the system of egg-white creams has the ability of water binding. this ability allows reducing water activity, thereby preventing growth of microbial activity. the analysis of colony morphotypes isolated from the egg-white creams shows that all the samples have several common types of microorganisms’ colonies. during storage the ratios of colonies changed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova, microbial characteristics of egg-white creams with reduced sugar, food and environment safety, volume xvi, issue 3 – 2017, pag. 147 – 152 149 common for all the egg-white cream samples were bacterial colonies such as white with irregular edges, round glow yellow, round white and round colorless. the main part of all colonies was coccoid. cells of these colonies were placed singly or in clusters. they were represented by aerobic and facultative anaerobic bacteria. the correlation and changes of colonies during storage are given in figure 1. table 1. microbial indicators of egg-white creams indicator normative by ukrainian standard (dstu) time of storage, days cream with sucrose with fructose with glucose qmafanm, ncu in 1 g, not more than 1.0х104 – 2×102 1×102 3×102 1 2×102 2×102 4×102 2 3×102 2×102 5×102 3 3×102 2×102 5×102 4 2×103 1×103 3×103 5 3×103 2×103 5×103 s. aureus in 1 g not allowed 1-6 not found salmonella in 25 g not allowed 1-6 not found mold funghi, ncu in 1 g, not more than 100 1 not found 2 <5 <5 <5 3 <10 <10 <10 4 <10 <10 <10 5 <15 <15 <15 yeasts, ncu in 1 g, not more than 50 1-2 not found 3 <5 <5 <5 4 <10 <10 <10 5 <10 <10 <10 sfb, ncu in 1g not standardized – 1×102 1×102 1×102 1 2×102 1×102 2×102 2 3×102 3×102 4×102 3 4×102 3×102 4×102 4 5×102 4×102 5×102 5 5×102 4×102 6×102 fig. 1. correlation of morphotypes of egg-white creams during storage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova, microbial characteristics of egg-white creams with reduced sugar, food and environment safety, volume xvi, issue 3 – 2017, pag. 147 – 152 150 in samples of freshly made egg-white creams predominate clear round colonies of medium size (32%). during storage of the samples, the number of white round colonies of medium size (45%) increases. this is due to the ability of microorganisms of absorbing nutrients from the product and interspecific competition for nutrients. the development of microorganisms and the rate of their growth depend on different factors such as product’s composition, its properties and production environment. first of all, the growth rate determines the presence of moisture available to microorganisms. with decreasing of humidity, the intensity of the reproduction of microorganisms decreases. after reaching sthe pecified moisture, the growth of humidity content stops. the research results on how the humidity content changes in samples are given in figure 2. the analysis of moisture changes during storage (figure 2) shows that the samples with sucrose and glucose behave identically. on the 4th night they retain the moisture content at the initial data. after 4 days of storage the samples begin to lose humidity due to desorption, so this indicator begins to decrease. on the contrary, the cream with fructose shows the ability of raising humidity. it is connected with high hydrophilic abilities of fructose. the sample starts to absorb water even when φ is about 45 ... 50%. the ph indicator is also one of the most important factors that affect the activity of microorganisms. all the microorganisms grow well at ph 6.0-8.0. however, with a ph below 4.5 only certain types of bacteria are able to develop, including fungi and yeasts. pathogenic bacteria at a ph below 4.5 do not reproduce. also at increased ph, such as above 9.0, many bacteria stop their growth. the diagram (figure 3) shows that the ph values of cream samples are optimal for the growth of microorganisms. this is another factor that reduces the activity of microorganisms. during storage the ph remains virtually unchanged, but the samples of sucrose and glucose have the trend of increasing the values. the ph value of cream with fructose decreases. the reduction of the ph values can be explained by higher moisture content in sample of fructose. it is a consequence of some higher activity of complex enzymatic of egg-white and amino acids accumulation. but even after 8 days ph does not reach the optimal values for microorganisms. however, total acidity changes for all the samples with the same regularity (table 2). fig. 2. changes of humidity of egg-white creams with reduced sugar food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova, microbial characteristics of egg-white creams with reduced sugar, food and environment safety, volume xvi, issue 3 – 2017, pag. 147 – 152 151 fig. 3. changes of ph of egg-white creams with reduced sugar table 2. changes of the total acidity of egg-white creams with reduced sugar egg-white cream total acidity freshly prepared on 4 th day on 8 th day degrees % solids degrees % solids degrees % solids with sucrose 4.2 6.36 4.9 7.42 4.7 6.96 with fructose 4.5 7.26 4.5 7.5 4.3 7.17 with glucose 4.3 6.83 4.7 7.46 3.6 5.54 in the first period of storage up to 4 days there is an increase of total acidity. it is explained by the accumulation of active acid-reacting compounds as a result of enzymatic hydrolysis of proteins. in the second period of storage the process slows down. this happens due to a decrease of moisture content. some substances are able of breaking down into simpler compounds and as a result the total acidity rate decreases. the most significant decrease in terms of the tendency of accumulating acid-reacting compounds is observed for the sample with glucose. it is connected with the start of its crystallization and therefore with the negative impact on the growth of microorganisms. 4. conclusions regardless of the type of sugar in the recipe of egg-white creams all the microbial indicators correspond to the state standard. the microflora activity does not increase during storage. this is achieved as a result of the polysaccharide complex introduction and presence of sugar as preserving agents. binding water and reducing its activity in the product inhibit the growth of microorganisms. at the same time, the ph of samples changes slightly during storage time. the active development of microflora prevents the changes of ph of creams that are out of bounds for optimal activity of bacteria, fungi and yeasts. creams produced by new recipes are safe and can be used in technology of cakes and pastries production. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 nataliia hryhorchak, olena ukrainets, alina bilko, iryna sokolovska, julya kambulova, microbial characteristics of egg-white creams with reduced sugar, food and environment safety, volume xvi, issue 3 – 2017, pag. 147 – 152 152 5. references [1]. diet, nutrition and the prevention of chronic diseases: report of a joint who/fao expert consultation, who technical report series, no. 916. geneva: world health organization, (2003). [2]. guideline: sugars intake for adults and children. geneva: world health organization, (2015). [3]. stieger m., van de velde f., microstructure, texture and oral processing: new ways to reduce sugar and salt in foods, current opinion in colloid & interface science, 18(4): 334348, (2013). [4]. panel, new zealand beverage guidance, policy brief: options to reduce sugar sweetened beverage (ssb) consumption in new zealand, pac health dialog, 20(1): 98-102, (2014). [5]. gallagher e., o’brien c.m., mscannell a.g., arendt e.k., evaluation of sugar replacers in short dough biscuit production, journal of food engineering, 56(2): 261-263, (2003). [6]. sokolovska i., rational using of pectin and sodium alginate in sugar reduced egg-white creams technology, 23, (2015). [7]. soares n.f.f., rutishauser, d.m., melo, n., cruz, r.s., andrade n.j., inhibition of microbial growth in bread through active packaging, packaging technology and science, 15(3): 129-132, (2002). [8]. wijtzes t., rombouts f.m., kantmuermans m.l., van riet k., zwietering m.h., development and validation of a combined temperature, water activity, ph model for bacterial growth rate of lactobacillus curvatus, international journal of food microbiology, 63(1): 57-64, (2001). [9]. pandey a., solid-state fermentation, biochemical engineering journal, 13(2): 81-84, (2003). [10]. jackson p., ward n., russell p., moral economies of food and geographies of responsibility, transactions of the institute of british geographers, 34(1):12-24, (2009). [11]. rowe r.c., sheskey p. j., weller p.j., handbook of pharmaceutical excipients, (washington, dc: american pharmacists association, 2006). [12]. siddique n.a., islam n., habib r., ur-rashid h., islam a., afrin s., evaluation of the quality of sweetened condensed milk of different brands available in local markets of bangladesh, international journal of natural and social sciences, 4(1): 64-70, (2017). [13]. smith f.x., ophthalmic and contact lens solutions containing simple saccharides as preservative enhancers, u.s. patent application no. 15/278, 660. [14]. dstu 4803:2007, cakes and pastries. general specifications, 22, (2007). [15]. horswill a.r., dudding a.r., escalante-semerena j.c., studies of propionate toxicity in salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth, journal of biological chemistry, 276(22): 1909419101, (2001). [16]. le loir y., baron f., gautier m., staphylococcus aureus and food poisoning, genetics and molecular research, 2(1): 63-76, (2003). [17]. lapidus a., goltsman e., auger s., galleron n., ségurens b., dossat c., land m.l., broussolle v., brillard j., guinebretiere m.h., sanchis v., nguenthe c., lereclus d., richardson p., wincker p., weissenbach j., ehrlich s.d., sorokin a., extending the bacillus cereus group genomics to putative food-borne pathogens of different toxicity, chemico-biological interactions, 171(2): 236-249, (2008). 1. introduction 4. conclusions issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xviii, issue 4 31 december 2019 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania звіт з ндр 29-81 за 2007 – 2009 р 67 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2 – 2017, pag. 67 74 the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus liliana luca 1 , *mircea oroian 1 , andrei lobiuc 1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 15th february 2019, accepted 29th june 2019 abstract: probiotic intake in human nutrition is particularly important for maintaining an optimal physiological state a de man-rogosa-sharpe (mrs) broth medium free of fermentable carbon source and a basic mrs broth was used to investigate the ability of the lactobacillus plantarum and lactobacillus rhamnosus strains to grow on selected prebiotic. criteria for the evaluation of the use of prebiotics were the growth of lactobacillus and acidification rate as compared against the results obtained in the control media. lactobacillus plantarum and lactobacillus rhamnosus were monitored for 10 hours in culture media with starch, inulin, glucose and media without glucose to assess the opportunity of sugar substrates as a support for the development of probiotics for micro-encapsulation. the largest number of final cfu was obtained for inulin, followed by oligo-fructose and starch. the oligo-fructose and inulin did not reach the stationary phase after 10 hours, while the stationary phase for starch, glucose and medium without glucose was recorded at 8 hours, 6 hours and 6 hours, respectively. the development of lactic microorganisms was also observed by decreasing the ph in the culture medium during monitoring. keywords: probiotics, prebiotics, lactobacillus rhamnosus, lactobacillus plantarum introduction in the last years, the interest in better living and improved health led to a significant increase in demand for new healthpromoting foods, including probiotic functional foods [1]. the word "probiotic" comes from the greek words "pro" and "bios" which together means "for life" and are therefore widely considered to be beneficial microorganisms. according to the world health organization, probiotics must be administered in adequate amounts (10⁷ cfu gˉ¹ or cfu/ml) to confer health benefits to the host (fao/ who, 2001) at the time of consumption [1], [2]. in order to be able to offer these benefits, microorganisms must survive during harsh conditions throughout the food production cycle (such as ph, temperature, fat and humidity), storage as well as stress in the gastrointestinal tract [3]. therefore, new approaches which could protect and deliver probiotics are required, for instance, microencapsulation [4] (wu, y., & zhang, g. 2018). research trends are now increasing with regard to the combined use of prebiotics and microencapsulation to enhance and maintain the viability of probiotics 1 ningtyas et al., 2019. some authors such as [5], [6], emphasize the fact that many of food oligosaccharides and polysaccharides have been claimed to have prebiotic activity, but not all dietetic carbohydrates are prebiotic. the majority of probiotic candidates are non-digestible oligosaccharides such as lactulose, refinose, fructooligosaccharides, lactose, saccharides, isomaltooligosaccharides, etc., but also include highly polymerized saccharides such as inulin, resistant starch, pectin, etc. it was also shown that this combination of probiotics and prebiotics, known as symbiotic, improves probiotic proliferation in the intestine and helps to modify the intestinal community [5], [6]. http://www.fia.usv.ro/fiajournal mailto:m.oroian@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 68 probiotics. regarding the known probiotic microorganisms, [7], known species of lactic acid bacteria (lab) (e.g., lactococcus, lactobacillus, streptococcus, and enterococcus), bifidobacterium, bacillus, and yeast, are considered to play important roles in maintaining the intestinal ecosystem and stimulating their host's immune system [8]. according to figure 1, lactobacillus, under the lab group, was the earliest discovered probiotic. in their paper, [9] könig and fröhlich, 2009 stated that this genus of lactobacillus, gram-positive bacteria, comprises 183 species and they are used in many industrial processes such as the production of preservatives, the production of food flavors, pharmaceutical industry, the cosmetics industry, etc. regarding the selection of probiotic microorganisms, it usually follows a strategy similar to that shown in figure 2 [7]. prebiotics. in 2016, a panel of experts summoned by the international scientific association for probiotics and prebiotics (isapp) defined prebiotics as "a substrate selectively used by microorganisms that provide health benefits" [10]. prebiotics such as inulin, oligofructose, starch, lactulose etc. are indigestible carbohydrates (not digested by human enzymes but act as a fermentable substrate for probiotics in the colon) [1] that selectively promote the growth of probiotics and demonstrate that prebiotics have the potential to alter in a positive manner the composition of intestinal microbiotherapy [4]. fig.1. the phylogenetic relationship of the different probiotic bacterial groups through16s rrna gene sequences retrieved from genbank database. [7]pereira et.al 2018 studies conducted on the growth of some lactobacillus strains (l. paracasei ktn-5, l. delbruckii tu-1, l. plantarum 22a-3), inulin and fructose showed that the strain of l. plantarum 22a3 could use fructose but not inulin. furthermore, in co-culture with the other 2 strains from the study grown on inulin, the growth of the l. delbrurckii strain was not as affected as the l. paracasei strain [5]. [11] lopes et al., conducted a study in 2017 on the prebiotic activity of inulin and oligofructose extracted from stevia rebaudiana on seven strains of bifidobacteria and lactobacilli. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 69 fig. 2. screening approaches used to characterize probiotic strains according to who / fao and studies quoted by [7] pereira et al. 2018 they found that in vitro, oligofructose molecules with a low degree of polymerization are preferred by microorganisms in comparison with inulin that has a higher degree of polymerization. this study investigated the influence of prebiotics used as a carbon source on the increase in the biomass of lactobacillus rhamnosus and lactobacillus plantarum. 2. materials and methods media and substrates the prebiotics used were: inulin in chichory (inu), potato starch soluble (sth) and oligofructose (oli). the manrogosa-sharpe (mrs) agar (sigma aldrich, germany), mrs broth from sigma-aldrich (saint louis, usa) was used for cultivation in 4 variants. for control variant, basic mrs recipe, including glucose (20 g/l), was followed, while, for testing carbohydrate substrates, the glucose in basic mrs recipe was replaced by different amounts of the respective substrates (20 g/l of each). chicory inulin, potato starch and oligofructose have been used as a carbon source by replacing glucose in the original mrs recipe. as a negative control, a carbohydrate-free medium and an uninoculated medium were used. for comparison of bacterial growth, the simple mrs broth was used. all the chemicals were purchased from sigma-aldrich (saint louis, usa). before being used, the modified culture media were adjusted at the ph of 6.2 and the mrs agar medium at 5.7, and then sterilized for 20 minutes at 121c. microorganisms and cultivations the bacterial strains used in this study were lactobacillus plantarum and lactobacillus rhamnosus (bioprox rp 80, france, lactobacillus cells count = 7±2x10e10) the lyophilized bacterial strains were cultivated in the mrs broth at 37c for 24h and then they were inoculated on the 4 modified media (medium mrs broth without carbohydrate source: mrs wg, mrs – inu, mrs – sth, mrs – oli) and on a mrs broth. the last culture medium was used as a standard medium. the growth of the bacteria was quantified through plate counting. all assays were performed in triplicate yielding a total of 109 experiments. bacterial strains and preparation of bacterial suspension food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 70 the bacterial strains used in this study were lactobacillus plantarum and lactobacillus rhamnosus, these strains were obtained from lyophilised culture bioprox rp80 (noyant, france) distributed by sc enzimes & derivates sa (romania). lactobacillus cells count = 7 ± 2x10e10 cfu /dose. bacterial strains were reactivated in mrs broth medium at 37 ° c for 24 h. from the reactivated culture (24 h) bacterial suspensions (1 ml) were used for inoculation in each test medium. later, the media were incubated at 37 ° c for 12 hours and tested every 2 hours. all determinations were made in triplicates. prebiotic activity testing prebiotic activity, reported in this study, reflects the ability of some substrates (inu, sth, oli) to support the growth of probiotic organisms using as a standard non-prebiotic substrate – glucose (mrs) and a carbon-free medium (mrs wg). assuming that these carbohydrates (inu; sth; oli) will be metabolized by probiotic strains such as glucose. criteria for the evaluation of the use of prebiotics were also the acidification rate as compared against the results obtained in the control medium. rate of acidification by these strains was determined as changes in ph. measurement of biomass growth optical density of culture media was measured (jenway 6400, jenway ltd uk) after inoculation (zero time) every 2 hours, for 12 h. bacterial growth was evaluated as the change in absorbance (at 600 nm) of the medium during 12 hours of incubation at 37 ° c, interpolated in a macfarland standard curve, (figure 3 with the regression coefficient of r²=0,978. the change of the acidity of the medium was determined once every 2 hours for 12 h by measuring the ph using a ph meter (hach lange hq11d digital ph meter). viable cell counts the growth of bacteria was quantified through plate counting. mrs agar medium was used as a control for bacterial growth and bacterial viability. mrs agar was inoculated from subculture starting at point 0 then once at 2 hours for 12h using serial dilution method. mrs agar plates were incubated at 37°c for 48 hours. all the inoculations were made in triplicates. after 48h colonies on plaques were counted and expressed as colony forming units per ml. statistical analysis for the statistical evaluation of data, the anova test followed by the post-hoc tukey test was used (p <0.05). statistical replication was provided by using 3 sets of probiotic culture tubes for each type of culture medium, inoculated and analyzed independently and the same for mrs agar plates. fig. 3. the calibration curve of the mcfarland standard y = 8e-10x r² = 0.978 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 2e+09 4e+09 a b s 6 0 0 n m conc. cel. /ml food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 71 3. results and discussion biomass growth the number of bacterial cells after 0, 2, 4, 6, 8, 10 h of incubation is given in figure 4 and figure 5. in the first 4 hours of incubation, the growth of probiotic cells was observed in all 5 culture media. fig. 4. biomass growth in comparison with the control cultures, in the case of mrs inu and mrs oli followed by mrs sth cultures it was observed that lactobacilli had a significant increase (p > 0.05). after 6 h the culture of l. rhamnosus and l. plantarum reached the stationary phase on the mrs sth medium and on the culture media with inu and oli it was still growing. on the other hand, it was observed that on culture media used as control media (standard), probiotics entered in a regression phase. growth rate to analyze the growth rate of culture, the following formula was used: rₓ₋y = (vy₋vₓ) / (ty-tₓ) => rₓ₋y = (vy₋vₓ) / 2 cfu / h, where r = growth rate, v = 2 hours, t = reading interval = 2h, x and y time interval where y = x+2h . analyzing the growth rate of the probiotic culture (figure 6) on each substrate, there was a sharp increase in mrs sth at the 3rd measurement (after 6 h of incubation). the culture entered the logarithmic phase with a growth rate of 133% as compared to the second measurement. by accumulating in this phase a maximum amount of biomass, the substrate was quickly exhausted and the culture went into the stationary stage of growth starch is composed of amylose and amylopectin. amylose is a linear chain of glucose α(1 → 4) with a plant-specific degree of polymerization of 200-6000. amylopectin consists of linear chains α(1 → 4) with linked side chains α(1 → 6). amylolytic degradation of amyloses by α and β-amylase and amyloglucosidase leads to linked maltodextrins respectively, maltodegas and glucose. hydrolysis of amylopectin requires amylopululanase or pullulanase to cleave the α-(1→6) branch points; the hydrolysis of amylopectin produces in addition α d-glu-α(1-6) -dglu (isomaltose) and oligosaccharides with bonds α-(1-→4) and α-(1-→6) mixed [12] gänzle and follador (2012). according to a study conducted by [13]-humblot et al. 2014, which cultivated l. plantarum a6 on mrs sth, simple carbohydrate consumption takes place over the first 3 hours, followed by maltodextrins with 3 to 7 glucose units. on the other hand, inulin and its partially hydrolyzed derivative oligofructose are made up of linear β-(2→1) glycosidic bonds of d-fructose (linkages that differ in a high (10–60) (inulin) and low (3–7) 0 50 100 150 200 250 300 350 400 450 2 4 6 8 10 c f u time (h) mrs mrs fg mrs i mrs o mrs s food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 72 (oligofructose) number of fructose monomers), often with a terminal glucose moiety that is linked by an α-(1→2) glycosidic bond, as in sucrose. oligofructose can contain both chains of fructose (fm type) and fructose chains with a terminal glucose unit (gfn type) [14, 15]. in a 2005 study, it was shown that the consumption of free fructose occurs within 4 hours of fermentation, oligofructose was preferentially metabolized during the exponential growth phase, and rapid degradation of long-chain inulin resulted in an increased concentration of fructose and sucrose after 8 hours of growth [16]. as for inu and oli, the other substrates analyzed in this study, there were significant growth rates (p> 0.5) as compared to the second measurement (6h). because probiotic culture had a growth rate of 82% on mrs oli versus second reading and a growth rate of 7.5% versus the third reading, probiotic culture started the logarithmic growth phase at 6 h and entered the 10h stationary growth phase. regarding the growth rate on mrs inu, the logarithmic growth phase began at 4h but without the initiation of stationary growth phase. some lactobacilli species can use inulin in two steps, first to degrade it to fructose and then to use the fructose molecules inside the cell [5]. regarding the evolution of ph, after 10h of incubation, the results showed that with the increase in cellular concentration, the ph decreases at each interval reading (table 1). thus the ph from 6.2 decreased to 4.32 for inulin, to 4.27 for oli, and 4.36 for sth as a result of consumption of the carbon source and, probable, its transformation into lactic acid [17]. fig. 5 the growth of the bacterial strains in the 4 types of culture media: a)-mrs; b) carbon free mrs;d) mrs chicory inulin;c)mrs oligofructose 0.000e+00 3.000e+09 6.000e+09 9.000e+09 1.200e+10 1.500e+10 0 2 4 6 8 10 12 c f u / m l time (h)a) mrs 0.000e+00 3.000e+09 6.000e+09 9.000e+09 1.200e+10 0 2 4 6 8 10 12 c f u /m l time(h)b) mrs wg 0.000e+00 3.000e+09 6.000e+09 9.000e+09 1.200e+10 1.500e+10 0 2 4 6 8 10 12 c f u /m l time (h) c) mrs oligofructose 0.000e+00 3.000e+09 6.000e+09 9.000e+09 1.200e+10 1.500e+10 0 2 4 6 8 10 12 c f u /m l time h food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 73 4. conclusions this study compared the effect of three carbon substrates in order to increase the biomass of a probiotic culture of l. plantarum and l. rhamnosus. after 10h incubation, the culture had a significant increase on all three substrates, which led to an improvement in the cultivation of this culture. further studies will test the viability and degree of encapsulation of probiotics in the most favorable substrates. since the best encapsulation substrates are carbohydrate polymers with a low polymerization degree [18]-huang et al., 2017), studies will be conducted on inulin, oligo-fructose and starch by determining the most effective concentrations, degree of encapsulation and viability of probiotics in simulated conditions. 5. acknowledgement this work was supported by a grant of the romania national council for higher education funding, cnfis project number cnfis-fdi-2019-0600. figure 6 the growth rate of the probiotic culture table.1 the ph variation during 10h of monitoring media type time (h) 0 2 4 6 8 10 avg sd avg sd avg sd avg sd avg sd avg sd mrs sth 5.90 0.012 4.97 0.032 4.65 0.078 4.41 0.006 4.40 0.142 4.36 0.111 mrs oli 5.95 0.006 5.01 0.012 4.66 0.035 4.49 0.023 4.36 0.006 4.27 0.017 mrs inu 5.90 0.006 5.08 0.017 4.71 0.023 4.51 0.010 4.35 0.021 4.32 0.023 mrs 5.90 0.006 5.16 0.006 5.06 0.006 4.99 0.015 4.81 0.012 4.50 0.006 mrs wg 6.00 0.006 5.12 0.005 4.94 0.006 4.55 0.007 4.43 0.010 4.38 0.006 6. reference. [1] de araújo etchepare, m., raddatz, g. c., de moraes flores, é. m., zepka, l. q., jacoblopes, e., barin, j. s., ... & de menezes, c. r. (2016). effect of resistant starch and chitosan on survival of lactobacillus acidophilus microencapsulated with sodium alginate. lwt-food science and technology, 65, 511517. [2]. ningtyas, d. w., bhandari, b., bansal, n., & prakash, s. (2019). the viability of probiotic -100 -50 0 50 100 150 r₁₋₂ r₂₋₃ r₃₋₄ r₄₋₅ c f u mrs wg mrs sth oli inu food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 liliana luca, mircea oroian, andrei lobiuc, the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus, food and environment safety, volume xviii, issue 2 – 2019, pag. 67 – 74 74 lactobacillus rhamnosus (non-encapsulated and encapsulated) in functional reduced-fat cream cheese and its textural properties during storage. food control, 100, 8-16. [3]. castro, j. m., tornadijo, m. e., fresno, j. m., & sandoval, h. (2015). biocheese: a food probiotic carrier. biomed research international, 2015 [4]. wu, y., & zhang, g. (2018). synbiotic encapsulation of probiotic latobacillus plantarum by alginate-arabinoxylan composite microspheres. lwt, 93, 135-141 [5]. takagi, r., tsujikawa, y., nomoto, r., & osawa, r. (2014). comparison of the growth of lactobacillus delbrueckii, l. paracasei and l. plantarum on inulin in co-culture systems. bioscience of microbiota, food and health. [6]. sathyabama, s., & vijayabharathi, r. (2014). co-encapsulation of probiotics with prebiotics on alginate matrix and its effect on viability in simulated gastric environment. lwt-food science and technology, 57(1), 419-425. [7]. de melo pereira, g. v., de oliveira coelho, b., júnior, a. i. m., thomaz-soccol, v., & soccol, c. r. (2018). how to select a probiotic? a review and update of methods and criteria. biotechnology advances sánchez, [8]. sánchez, m. t., ruiz, m. a., lasserrot, a., hormigo, m., & morales, m. e. (2017). an improved ionic gelation method to encapsulate lactobacillus spp. bacteria: protection, survival and stability study. food hydrocolloids, 69, 67-75. [9]. könig, h., unden, g., & fröhlich, j. (2009). biology of microorganisms on grapes, in must and in wine. springer. [10]. quigley, e. m. (2018). prebiotics and probiotics in digestive health. clinical gastroenterology and hepatology. [11]. lopes, s. m. s., krausová, g., carneiro, j. w. p., gonçalves, j. e., gonçalves, r. a. c., & de oliveira, a. j. b. (2017). a new natural source for obtainment of inulin and fructooligosaccharides from industrial waste of stevia rebaudiana bertoni. food chemistry, 225, 154-161 [12]. gänzle, m., & follador, r. (2012). metabolism of oligosaccharides and starch in lactobacilli: a review. frontiers in microbiology, 3, 340. [13]. humblot, c., turpin, w., chevalier, f., picq, c., rochette, i., & guyot, j. p. (2014). determination of expression and activity of genes involved in starch metabolism in lactobacillus plantarum a6 during fermentation of a cereal-based gruel. international journal of food microbiology, 185, 103-111 [14]. roberfroid, m. b., van loo, j. a., & gibson, g. r. (1998). the bifidogenic nature of chicory inulin and its hydrolysis products. the journal of nutrition, 128(1), 11-19. [15]. leenen, c. h., & dieleman, l. a. (2007). inulin and oligofructose in chronic inflammatory bowel disease. the journal of nutrition, 137(11), 2572s-2575s. [16] makras, l., van acker, g., & de vuyst, l. (2005). lactobacillus paracasei subsp. paracasei 8700: 2 degrades inulin-type fructans exhibiting different degrees of polymerization. appl. environ. microbiol., 71(11), 6531-6537. [17]. pimentel-gonzález, d. j., campos-montiel, r. g., lobato-calleros, c., pedroza-islas, r., & vernon-carter, e. j. (2009). encapsulation of lactobacillus rhamnosusin double emulsions formulated with sweet whey as emulsifier and survival in simulated gastrointestinal conditions. food research international, 42(2), 292-297 [18]. huang, s., vignolles, m. l., chen, x. d., le loir, y., jan, g., schuck, p., & jeantet, r. (2017). spray drying of probiotics and other food-grade bacteria: a review. trends in food science & technology, 63,1 113 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 113 130 screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast *odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke laboratory of biotechnology, faculty of biosciences, felix houphouet-boigny university, abidjan, 22 bp 582 abidjan, ivory coast *corresponding author: odykoffi@gmail.com received 28th december 2017, accepted 25th june 2018 abstract: cocoa beans (theobroma cacao l.) are the raw material for chocolate production. the variability of cocoa beans quality, due to the activity of microbiota involved in cocoa fermentation causes huge economic losses in cocoa-producing countries. to resolve this issue, 743 yeast strains isolated in ivorian cocoa fermentation were investigated in order to identify potential starters by their ability to resist to physico-chemical stress conditions and to produce enzyme necessary to cocoa fermentation. thus, out of the 743 investigated yeast strains, 113 showed high fermentative capacity, 19 being able to produce high ethanol contents up to 4,18 % w/v. out of 19 yeast strains high ethanol producers, two strains saccharomyces cerevisiae yb14 and pichia kudriavzevii yp13 were able on one hand to resist to most parameters such as temperature, ph, ethanol, organics acid, and on the other hand they were able to produce specific enzymes like pectinase, β-glucosidase, protease necessary to have a good cocoa fermentation process, and finally to produce acetoin which is desirable for flavor development in the fermentation process. these two strains could therefore be used as starter cultures which may contribute to the control of cocoa fermentation in ivory coast. keywords: yeasts, ethanol, cocoa fermentation, starters 1. introduction cocoa fermentation plays an important role in the chocolate production chain with major impacts on product quality and value [1]. it’s a spontaneous process which involves the action of complex microbial interactions, led mainly by yeasts, lactic acid bacteria (lab), acetic acid bacteria (aab) and aerobic spore forming bacilli (bacillus sp.). the microbial activity generates metabolites and conditions that kill the embryo of beans, triggering an array of biochemical reactions, essentials for cocoa quality development [2, 3]. among microflora of cocoa, yeasts are frequently isolated from fermenting cocoa beans. they perform mainly an alcoholic fermentation of pulp sugars to produce ethanol and carbon dioxide, and secondary metabolites, such as higher alcohols, organic acids, esters, aldehydes, ketones, sulfur, and nitrogen volatiles, which will diffuse into the bean to affect the final quality of chocolate [4, 5]. moreover, others functions such as acceleration of carbohydrate consumption, pectinolytic activity and inhibition of growth of pathogenic microorganisms, essential to cocoa fermentation are also attributed to yeast metabolism [6, 7]. regarding all these aspects, the use of yeast starter cultures has been proposed as one the key to conduct controled cocoa fermentations. in this context, some species including saccharomyces cerevisiae, kluyveromyces http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 114 marxianus, torulaspora delbrueckii, pichia kluyveri, hanseniaspora uvarum and candida sp. were used as starter, and the results obtained contributed to successful changes in cocoa aroma profile with respect to spontaneous cocoa fermentation [7-11]. although, these different species improved cocoa fermentation process and the chocolate quality, the results obtained are still insufficient for cocoa fermentation process standardization. therefore, studies to select appropriate yeast starter have to be pursue in order to solve the great problem of the variability of cocoa beans quality linked to the spontaneous nature of fermentative cocoa process [12]. in this context, the use of yeast starters with high ethanol production capacity was suggested. in fact, ethanol plays an important role in cocoa fermentation. it contributes to the brown color of good fermented cocoa beans; because it facilitates glycosidase and polyphenoloxidase action, responsible to color change [13-15]. through its oxidation in acetic acid, ethanol contributes highly to characteristics aromas formation of chocolate [16, 17]. indeed, during cocoa fermentation, ethanol produced mainly by yeasts, is oxidized into acetic acid by acetic acid bacteria. the ethanol oxidation in acetic acid increases cocoa mass temperature which kill the cocoa-beans embryo. the death of cocoa beans allows the permeability of cellularwall, which facilitate the diffusion of metabolites (ethanol, acetic acid) within cotyledons. this diffusion activates reserve-cellules enzymes, which produce cocoa aromas precursors [18-20]. more to the point, the studies of [11] showed that the addition of yeasts starter sole at the beginning of fermentation contributes to have higher aabs concentration in mass fermenting and a better cocoa beans quality comparatively to spontaneous fermentation. in addition, according to [21], a high acetic acid amount would contribute to obtain a good cocoa-beans quality. therefore, this high acetic acid amount during the fermentative process could come from high ethanol amount from yeasts activity. thus, yeasts with high ethanol production capacity may be useful for cocoa fermentation. this would allow aabs to have the early ethanol for conversion to flavor precursors, but also could contribute to the reduction of fermentation time. so, the aim of this study is to select potential yeast starters with high ethanol production capacity for the control of cocoa fermentation. this study, considered as a promising approach, is the first which pointed exceptionally the effects of stress conditions from cocoa fermentation on ethanol production for the selection of yeast starters. 2. materials and methods 2.1. yeast strains isolation a total of 743 yeast strains were previously isolated from six cocoa fermentations carried out in ivory coast. theses strains were stored in mygp broth supplemented with 20 % glycerol for further tests [12]. 2.2. screening of potentials yeast starters 2.2.1. screening of high fermentativecapacity yeasts the fermentative capacity of 743 yeast strains was studied according to the method of [22] with a slight modification. from pre-culture of 24 hours, pure yeasts culture were suspended in saline tryptone to get an optical density of 0.7 at 600 nm and 100 µl of this suspension were used to inoculate 10 ml of ypg medium containing a durham tube into essay tube. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 115 then, the culture was incubated at 30 °c for 6 days, without agitation. fermentative capacity was also determined by measuring the gas production in durham test. yeasts usually oxidize sugars into ethanol with production of gas [23]. this volume of gas and ethanol produced is related to fermentative strength of strain [24]. 2.2.2. screening of high ethanolproducer yeasts the screening was carried out by the method described by [25] with a slight modification. from pre-culture of 24 h, pure yeasts culture was suspended in saline solution buffer to have an optical density of 0.7 at 600 nm. 100 µl of this suspension were used to inoculate 10 ml of ypg medium. then, the culture was incubated at 30 °c for 3 days, without agitation. after 72 h, the culture was centrifuged at 15000 rpm for 10 min at 4 °c and the supernatant was retained. the supernatant containing ethanol produced by yeasts is quantified by uv method using alcohol dehydrogenase (adh) and nad as cofactor. thus, the produced ethanol is measured by following the formation of nadh to 340 nm at spectrophotometer (eppendorf, germany) according to the reaction: rch2oh+nad + rcho+ nadh+h+ the reactional medium is composed of 0.75 ml of h2o; 0.25 ml of buffer 60 mm tris-hcl (ph 9); 0.05 ml of 1.5 mm nad+ (sigma); 1µl of adh (500u/ml, sigma) and 50 µl of the supernatant to be analyzed. the ethanol production was determined using a standard curve carried out under the same conditions as the test with 99 % pure ethanol. 2.3. effects of cocoa fermentation environmental conditions on ethanol production by yeasts the yeasts with high ethanol production capacity were further investigated to assess the individual impact of various environmental conditions (notably ph and temperature) and organic compounds (ethanol, citric acid, lactic acid and acetic acid) on ethanol production according to the method of [25] with slight modifications. 2.3.1. ethanol production under temperature and ph stress strains were grown in a standard liquid medium containing 0.05 % yeast extract; 0.3 % casein peptone; 1% glucose at ph 5.6. to assess the influence of temperature on ethanol production by yeasts, 10 ml of standard liquid medium contained in a 50 ml test tube, were inoculated with 100 μl of yeasts pre-culture, od600 = 0.7. then, cultures were incubated for 72 h at variable temperatures ranging from 30 to 45 °c. influence of ph variations on ethanol production were analyzed in the same medium at different ph values (3; 4 and 5), and incubated at 30 °c.the ethanol production was quantified by the uv method using alcohol dehydrogenase (adh) and nad. 2.3.2. ethanol production under carbohydrates stress the influence of sugars (glucose, fructose and sucrose) on ethanol production by yeasts was also individually assessed. the liquid medium contained 0.05 % yeast extract; 0.3 % casein peptone with sugar at different concentrations. the different sugar concentrations studied were 2; 4 and 8 % for glucose and fructose, and for sucrose 2; 4 and 6 %. after inoculation, each medium was incubated at 30 °c during 3 days.the ethanol production was adh food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 116 quantified by the uv method using alcohol dehydrogenase (adh) and nad. 2.3.3. ethanol production under ethanol and organic acidsstress the influence of each organic compound (ethanol, citric acid, lactic acid and acetic acid) on ethanol production by yeasts was individually assessed. for this purpose, the standard liquid medium free of these compounds was used as negative control. for example, to assess the influence of ethanol on ethanol production, yeasts were grown in the standard medium containing variable ethanol concentrations (4; 8 and 12 %), incubated at 30 °c and then ethanol production in these variable ethanol concentrations was compared with the negative control. the same type of experiment was performed individually for each compound at variable concentrations of acetic acid (0.5; 1 and 2 %), lactic acid (0.5 and 1 %) and citric acid (1; 2 and 3 %). the ethanol production was quantified by the uv method using alcohol dehydrogenase (adh) and nad. 2.4. enzymatic activities and acetoin production of high ethanol-producer yeasts 2.4.1. enzymatic activities the yeast strains were analyzed for enzymatic activities, and in particular for the esterase, β-glucosidase, glycosidase, protease and pectinase activities, according to the methods reported in [26]. screening of enzymatic activity was performed in triplicate on agar plates by spot inoculation method. the positive yeast controls, used as the reference in order to verify the reliability of the tests, were part of the collection of the laboratory of biotechnology of felix houphouet-boigny university. 2.4.2. acetoin production acetoin production was demonstrated according to the method described by [27]. the clark and lubs medium is inoculated with yeast strains and incubated at 30 °c for 48 h. after incubation, 1 ml of the medium is taken and introduced in a test tube. to this medium are added two drops of an alcoholic solution of 6 % alphanaphthol in 90 ° alcohol and two drops of a 16 % sodium hydroxide solution in distilled water. the tube tests are stirred and heated on the flame of a bunsen beginning of boiling. after stirring for 30 seconds on a vibrating stirrer of vortex type, a cherry red color indicates a positive reaction. 2.5. phenotypic and molecular characterization of potential starters 2.5.1. phenotypic characterization of potential starters the best high ethanol-producer yeasts were investigated for growth at different culture conditions according to the method described by [28]. from pre-culture of 24 h, pure yeasts culture was suspended in saline solution buffer to have an optical density of 0.7 at 600 nm. 100 µl of this suspension were used to inoculate 10 ml of ypg medium in 50 ml test tube. the strains were tested for growth in ypg at different ph (3; 4 and 5) and incubated 30 °c with 150 rpm orbital shaking during 3 days. the growth was also assessed in ypg (natural ph 5.6) at different temperatures 30; 35; 40; 45 and 50 °c with 150 rpm orbital shaking during 3 days. after incubation time, the growth was determined by measuring the optical density at 600 nm at spectrophotometer (pioway medical labs, singapore). different sugars were tested using a base of yeast extract (0.05 %) and peptone (0.3 %) additioned of glucose (2; 4 and 8 %), fructose (2; 4 and 8 %) and sucrose (2; 4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 117 and 6 %). furthermore, growth in presence of ethanol, lactic acid, citric acid and acetic acid was tested. the different concentrations were tested for ethanol (4; 8; 12 and 16 %), citric acid (1; 2; 3 %), acetic acid (0.5; 1; 2 %), lactic acid (0.5; 1%) and supplemented by ypg. the high osmotic tolerance test was carried out according to [29] in two steps. firstly, a basal medium containing 0.5 % yeast extract and 0.5 % peptone was supplemented with 50 % of glucose to determine the ability to growth yeast at high level sugar [30]. secondly, a medium containing 10 % sodium chloride and 5% glucose [31] was realized to determine the ability to resist at high osmotic pressure. for both experiments, the growth was assessed by measuring the optical density at 600 nm after 3 days of incubation at 30 °c with shaking at 150 rpm. 2.5.2. molecular characterization of potential starters yeasts were grown at 30 °c to the mid-log phase in ypd medium before harvesting. yeast genomic dna was extracted using the classic phenol / chloroform method described by [32]. the d1/d2 region of 26s rdna was pcr amplified as described by [33]using the eukaryotic universal primer gc-nl1 (5’ cgcccgccgcgcgcggcgggcgggc gggggccatatcaatagcggagga aaag 3’) and the reverse primer ls2 (5’ attcccaaacaactcgactc 3’). the d1/d2 pcr program consisted to one cycle at 94 °c for 3 min, followed by 30 cycles (95 °c for 1 min, 52 °c for 1 min, 72 °c for 1 min), and a final extension at 72 °c for 10 min. pcr-amplified d1/d2 regions of 26s rdna were sequenced by biofidal (lyon, france) using the pseq d1/d2 primer (5’gggccatatcaataagc3’). sequences were then compared to the ncbi data genbank using the blast program (http://blast.ncbi.nlm.nih.gov/blast.cgi). sequences showing a high percentage of identity (≥ 97 %) were considered as belonging to the same species. 3. results and discussion 3.1. fermentative capacity and ethanol production of yeast strains among the 743 analyzed yeast strains for the fermentative capacity, only 113 showed a high fermentative capacity with gas amount above to 4 cm3 (table 1). table 1. co2 production of yeast strains involved into cocoa fermentation in ivory coast. these yeast strains are susceptible to product high level ethanol because during ethanol fermentation, gas produced is correlated to ethanol produced according to [24]. indeed, fermentations that develop a stronger presence of highly fermentative yeasts are more likely to have a greater concentration of ethanol [34-36] than those which develop less-fermentative yeasts [37]. hence, the 113 yeast strains with a high fermentative capacity strains were investigated to select among them, the high ethanol-producer yeasts. the results indicated that 62 (54.87 %) yeast strains have ethanol production inferior to 1 % (w/v) while 32 (28.32 %) yeast strains have ethanol production within 1-2 % (w/v) and 19 (16.81 %) yeasts strains produce ethanol above 2 %. the table 2 fermentative capacity volume of co2 number of isolates low level [0 ; 2 cm3 [ 506 middle level [2 ; 4 cm3 [ 124 high level [4 ; 6 cm3] 113 http://blast.ncbi.nlm.nih.gov/blast.cgi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 118 shows the distribution of ethanol production level for the 19 yeasts strains high ethanol-producer. table 2. ethanol production by yeast strains with high fermentative capacity each data is the average of triplicate tests analysis. data following with the same letter were similar statistically at 95 % according to tukey test. during most cocoa heap fermentation, the ethanol concentration into pulp doesn’t exceed 20 mg/g cocoa beans whether 2 % (w/v) during the first 24-72 h [6, 9, 35, 3841]. however, according to [42] and [43], ethanol concentrations in excess of 40 mg/g (4 %) are needed to kill the bean, important step into cocoa fermentation. indeed, the death of bean due to ethanol and acetic acid diffusion will trigger endogenous biochemical reactions that produce the chocolate flavor precursors [2, 44, 45]. these lasted will be transformed into distinct chocolate notes during the subsequent industrial processes [46]. ethanol produced by yeasts, could contribute also into brown color obtain of cocoa beans fermented, as will facilitate the bean glycosidase activity necessary to convert the bean anthocyanins (purple-red) to the colorless form [13, 14] and the bean polyphenoloxidase activity needed to transform phenolic compounds to brown polymers [15]. thus, the nineteen (19) yeast strains able to produce ethanol more than 2 % (w/w) will contribute to have ethanol concentrations in excess of 40 mg/g (4 %), which, are needed to kill the bean in order to reduce the fermentation time and obtain a good cocoa beans fermented. however, during cocoa fermentation, the environmental stresses impact the microflora activity, which influence the efficiency of cocoa fermentation. the impact of stress conditions prevailing during cocoa fermentation process on the ability of these 19 yeast strains to produce ethanol must be study for an efficiency selection of appropriate starters. 3.2. effect of ph and temperature on the ethanol production the influence of temperature on yeasts for ethanol production indicates that all the 19 yeast strains are able to produce ethanol at 30; 35 and 40 °c. in addition, the maximal productions were recorded to 30 – 35 °c for most strains. nevertheless beyond 40 °c, an inhibition of fermentative yeasts activity was observed (figure 1). these results showed that the increasing temperature influences negatively the fermentative activity of yeasts. indeed, temperature is the main environmental parameter which has an important impact on physiology and activity of microorganisms [47]. it affects growth rate, speed production, co2 production, ethanol production and cellular viability [48]. also, according to [49] and [48], beyond 35 °c it appears to most yeasts a real decreasing of ethanol rate production. however, in our study, twelve (12) yeast yeast strains ethanol production (% w/v) yp20 4.19±0.16a yt13 3.23±0.03b yt8 3.09±0.05bc yp11 2.77±0.07cd yg14 2.69±0.09de yp9 2.58±0.10def yh7 2.49±0.20def yt11 2.37±0.29efg yp3 2.31±0.00efg yg28 2.28±0.00fg yp23 2.27±0.20fg yb11 2.19±0.01fgh yb10 2.08±0.03ghi yp13 2.07±0.00ghi yp12 2.05±0.01ghi yb14 1.79±0.16i yc4 1.78±0.10i yg12 1.78±0.24hi yp1 1.72±0.22i food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 119 strains showed a good ethanol production at 35 °c and these strains kept 50 % of their ethanol production at 40 °c comparatively to 30 °c. fig. 1. effect of temperature on ethanol production of yeasts the influence of ph on ethanol production indicates that these twelve (12) yeast strains, able to produce ethanol with ethanol production capacity under high temperature, are also able to produce ethanol at ph 3, ph 4 and ph 5 (figure 2). fig. 2. effect of ph on ethanol production of yeasts however, the high levels of ethanol production were recorded for ph 3 and ph 4. among these twelve yeast strains, seven (7) strains (yp3, yp13, yp20, yb14, yt8, yt13 and yg12) showed high production levels at ph 3 and ph 4 comparatively to the other yeasts; and the ethanol production for these strains ranged to 1.70 – 3.84 % (w/v) for ph 3 and 1.69 – 3.07 % (w/v) for ph 4. at the beginning of cocoa fermentation, the cocoa pulp has acid ph (3-4) which constitute favorable conditions to yeasts growth [20]. thus, these seven (7) yeast strains, which are able to produce high ethanol rate at 35 – 40 °c and at ph 3 and ph 4, could be able to start the cocoa fermentative process but will also be able to produce ethanol during the first 48 – 72 h when temperature reach 35 – 40 °c [20]. however, ph and temperature are not sole stress conditions which prevailing into cocoa fermentation. variations of sugar, ethanol and organic acid concentrations influence strongly the yeasts activity. it arranges to study the effect of these stresses on ethanol production by yeasts for selecting appropriate starter cultures. 3.3. effect of carbohydrates and organic compounds on the ethanol production the figure 3 (a, b, c) showed that all the seven (7) yeast strains realized an alcohol fermentation with glucose and fructose until 8 %, and sucrose until 6 %. however, the ethanol production differs to one yeast strain to another for each sugar. the results indicate also that all yeast strains excepting yt8 showed ethanol production more than 2 % with different sugars studied. the ethanol production by these yeast strains confirms the known role of yeasts, to degrade sugars into ethanol as reported by literature data [29, 50]. thus, the mucilaginous cocoa bean rich-pulp in fermentable sugars as glucose, 2.4–6.6 % (w/w), fructose 4.2–7.4 % (w/w), and sucrose, 2.1–3.2 % (w/w) [16, 34, 37, 51, 52] would constitute favorable conditions for fermentative activity of these yeast food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 120 strains during fermentation process of beans. fig. 3. effect of glucose (a), fructose (b) and sucrose (c) on ethanol production of yeasts so, the seven yeast strains tested excepted yt8 would be susceptible to realize easily alcohol fermentation during the cocoa fermentative process to produce ethanol necessary at good fermentation. the figure 4a showed that ethanol production decreases when ethanol concentration increases until to have an inhibition in presence of ethanol 8 % in the medium. fig. 4a. effect of ethanol concentration on ethanol production of yeasts this indicates that ethanol is able to limit the ethanol production itself as reported by some authors [53-55]. indeed, according to these authors, when ethanol concentration increases into the culture medium, a decrease of rate growth, cellular viability, metabolic activity and production ability is observed. however, some yeast strains as yp13, yb14 and yt13 showed a resistance to ethanol indicated by their ethanol production at presence of ethanol 4 % in the medium. among these yeast strains, yp13 showed good ethanol production in presence of 4 % ethanol in the medium compared toyb14 and yt13. a lower production in presence of ethanol 8 % in the medium is recorded by yp3 while ethanol production by yb14 and yt13 were inhibited. these results corroborated those of [56] and [57] which reported a threshold and maximum tolerance level 4 % (w/v) and 8 % (w/v) ethanol for yeasts, respectively. the maximum concentrations of ethanol reported in the pulp and nib fractions vary from as little as 0.5–2 % [6, 35, 37, 58] to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 121 values as high as 3–8 % [28, 34, 36, 59]. although, our yeast strains can’t produce ethanol above ethanol 8%, they may be relevant to cocoa fermentation because the high ethanol concentrations produced by yeasts during the first 48-72 h to cocoa fermentation time, could rapidly convert by acid acetic bacteria (aab) which are dominating in this time. indeed, ethanol produced by yeasts would be oxidized into acetic acid by aab, which will diffuse into cotyledons, for formation of aroma precursors future chocolate [20, 46, 60]. citric, acetic and lactic acids have key roles in the development of chocolate characterics [44, 59, 61]. apart from their direct impacts on sensory quality, their diffusion in and out of the nib modulates bean ph which indirectly affects the activity of bean enzymes such as proteases and invertase involved in the production of free amino acids, peptides and reducing sugars [17, 44, 62]. they have an inhibitor effect on the growth and activity of fermentative microbiota notably yeasts; due to their un-dissociated forms [63]. indeed, in acid medium, the un-dissociated acids can diffuse through plasmic membrane, in intracellular environment more alkaline (cytoplasm) where the dissociation happens [64-66]. these dissociated acids disarrange cellular activity employing for example an uncoupling effect on respiration or inhibition on some enzymes activities notably the obstruction of some transport mechanisms, which can cause the loss of cellular activity then cellular death. the results of this study indicate that citric acid, main acid in unfermented cocoa pulp, influences fermentative activity of yeasts (figure 4b). out of the 7 yeast strains tested, yp3, yp13, yb14 and yt8 showed ethanol production inferior to 1 % (w/v) in presence of citric acid 1 % (w/v) in the medium. however, beyond 1 % citric acid, it was observed an inhibition of fermentative activity for all the 7 yeast strains. this inhibition activity of yeast strains would confirm that the citric acid use is more often connected with the growth of certain species of lactic acid bacteria, which are present at the first hours of the fermentation time with yeasts, such as l. plantarum as reported by [67] and [68] or lactococcus lactis and l. fermentum as reported by [16]. fig. 4b. effect of citric acid concentration on ethanol production of yeasts during cocoa fermentation, the citric acid in pulp is variable depending on cocoa pods source, probably due to different varieties of cocoa pods which exist in the world. in indonesia, cocoa pulp contains 2.1-2.4 % (w/w) citric acid while in west africa notably in ivory coast, cocoa pulp is around 1 % citric acid [13, 34, 38, 69]. thus, yp3, yp13, yb14 and yt8 could be able to start cocoa fermentative in ivory coast with a high ethanol production under conditions where acid citric concentration don’t exceed 1% citric acid. concerning acetic acid, the figure 4c indicates that acetic acid decreases fermentative activity of yeast strains. indeed, rapid fall on fermentative activity was observed between 0-0.5 % acetic acid (figure 4c). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 122 these results corroborate those of [70] who reported that no ethanol was produced when 0.5 % (w/v) acetic acid was present in brewing mash. moreover, according to [71], acetic acid in excess of 0.1 % (w/v) decreased the rate of ethanol production. however, yp13, yt8, yt13 and yg12 showed ethanol production counting to 0.11 – 0.53 % (w/v) in presence of acetic acid 0.5 %. fig. 4c. effect of acetic acid concentration on ethanol production of yeasts among these yeast strains, yp13 and yt8 produce lesser ethanol values around 0.1 % (w/v) with 1 % acetic acid in medium. during the first hours of ivorian, malaysian and ghanaian cocoa heap fermentation, the acetic acid concentrations counting 0.6 – 1 % [38, 67, 69]. moreover, the production of acetic acid at levels of 1–2 % during fermentation is necessary to kill the bean [43] and it is generally considered that this production mainly comes from the oxidation of ethanol by acetic acid bacteria [2, 17, 44]. despite their low production recorded at 1 % acetic acid, yp13 and yt8 would be able to resist to acetic acid stress conditions encountered during cocoa fermentative process. however, the high level of ethanol production by these yeasts at the beginning of cocoa fermentation, when acetic acid content is 0.04 % [34, 36], could allow aab to produce the acetic acid necessary to kill the bean and trigger endogenous biochemical reactions for the formation of chocolate flavor precursors [2, 16, 45]. as for lactic acid, the figure 4d indicates that lactic acid inhibits highly fermentative yeasts activity. indeed, the yeast strains cannot produce ethanol to 0.5 and 1 % lactic acid. these results corroborate those of [72] which reported that lesser concentrations of 0.2-0.6 % w/v greatly affected glucose utilization and the rate of ethanol production, until totally shut down glucose utilization and ethanol synthesis in presence of lactic acid 0.6 % (w/v) in glucose-mineral salts medium. fig. 4d. effect of lactic acid concentration on ethanol production of yeasts however, lactic acid content often attained 0.6 0.8 % (w/w) even 1.1 % (w/w) during cocoa fermentation carried out around the world [6, 39]. this indicates that, although yeasts initiate cocoa fermentation process, they can’t realize totally fermentative process single in order to obtain cocoa beans good fermented, moreover the lactic acid contribution to obtain fermented dry cocoa beans that give a typical chocolate flavor is more often controversial and considered as not essential to cocoa fermentative process because of its nonvolatility. indeed, the high migration of lactic acid into the beans may contribute to the high acidity of cocoa beans from some geographical regions and poorer chocolate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 123 quality [60]. thus, cocoa beans with lactic acid exceeding 5 mg/g (even 0.5 % w/w) dry weight tend to have a ph value less than 5.0 and have lesser quality [60, 61, 73]. this issue could be resolved by aab notably acetobacter species which oxidize a part of lactic acid produced by the lactic acid bacteria (lab) into acetic acid and acetoin desirable components for flavor development in fermentation process [69, 74]. 3.4. enzymatic profile and selecting of potential starters the results of the screening for the presence of specific enzymatic activities of the 7 yeast strains are reported in table 3. table 3. enzymatic profile and acetoin production of yeast strains +: positive reaction, – : negative reaction β-gluc: β-glucosidase, prot: protease; este: esterase; pecti: pectinase; acetoin prod: acetoin production β-glucosidase activity was found by yp20, yb14 and yg12, while protease and pectinase activities were from yb14 only. no yeast strain gave positive result for ester-hydrolase activity. protease activity could be beneficial for the microorganisms during the fermentation process, by liberating assimilable nutrient sources, such as amino acids and peptides necessary to microorganisms’growth. indeed, proteins content in cocoa pulp range of 0.4 to 0.6 % w/w [45]. thus, the protease activity by yb14 would contribute to rapid microbiota growth in mass fermenting. moreover, endo and exo protease activities influence the concentration of free amino acids within the beans [16], contributing to chocolate flavor due to the formation of pyrazines during roasting. β-glucosidase has a great potential to be used in various biotechnological processes like liberation of aromas, flavours, and isoflavone aglycones to oligosaccharides and alkylglycosides [75-77]. indeed, βglucosidase in yeasts is responsible for the release of the flavoring compounds like nerol, geraniol, linalool, benzyl and phenylethyl alcohols [78]. thus, βglucosidase activity produced by yb14 is essential to cocoa fermentation as it will contribute to develop aromas molecules of chocolate. moreover, this enzyme would contribute to degrade the minimal quantity of cellulosic biomass founded into cocoa pulp. pectinases are responsible for the degradation of pectic substances that occur as structural polysaccharides in the middle lamella and the primary cell walls of plant tissues [79]. some yeasts, as kluveromyces marxianus, produce pectinolytic enzymes which are believed to play a central role in the degradation of the viscous pectin-rich pulp [80, 81]. the action of yeast pectinolytic enzyme is essential for the growth of other microorganisms involving in cocoa fermentation [80]. indeed, by their depectinization activity, yeasts are responsible for liquefying the cocoa pulp, which causes pulp drainage (release of sweatings) and reduces pulp viscosity that in turn allows air ingress into the fermenting cocoa pulp-bean mass. this aeration of fermenting cocoa pulp-bean mass favorize the growth of aab and others bacteria necessary to cocoa fermentative process [20]. thus, pectinase activity from yb14 constitutes a potential yeast strains βgluc prot este pecti acetoin prod yp3 + yp13 + yp20 + + yb14 + + + + yt8 yt13 + yg12 + + food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 124 from characteristic for this strain in the starter selecting to control cocoa fermentation. besides, all 7 strains excepted yt8 are able to produce acetoin (table 3). thus, the ability of these yeast strains to produce acetoin is a potential way in the aromas precursors’ production of chocolate. indeed, citric acid metabolism lead to produce some molecules aroma such as acetoin (3-hydroxy-2-butanone) which are desirable flavor in fermentation process [82]. taken together, our results indicate that yb14 and yp13 constitute the potential yeast starters to control cocoa fermentation in ivory coast. yb14 showed the best resistance profile through their ethanol production for most parameters used for screening such as temperature, ph, as well as organic compounds stress. moreover, this cocoa-specific strain showed positive results for almost all the enzymatic activities, particularly β-glucosidase, protease and pectinase. as for yp13, it showed an excellent profile of screening with ethanol and organic acids stress only. 3.5. phenotypic characterization of yb14 and yp13 growth was assessed under stress conditions in order to determine the functional properties of selected yeast strains (figures 5 a, b, c, d, e, f, g h, i). the results showed that yb14 and yp13 were able to grow up to 45°c, with the higher tolerance at 40 °c. the data indicate also that yb14 and yp13 showed acid ph tolerance up to ph 2. the both strains showed a good tolerance at ph 3 and ph 4 while at ph 5, yp13 showed a higher growth comparatively to yb14. the ability of these two strains to resist under temperature and stress conditions, as found in cocoa fermentation, constitute the potential properties of these strains to grow and dominate the cocoa fermentative process in ivory coast as reported by [83]. furthermore, growth assessment was also done to investigate the use of different carbon sources, growth in ethanol or organic acids and to test the high osmotic stress tolerance. the two strains grew well on carbon source as glucose, fructose and sucrose. however, high growth is observed for glucose 2 %, fructose 2 % and sucrose 4 % with the two strains. concerning growth under ethanol stress, the results indicate that the growth was successively impaired with ethanol raise. though, yb14 showed alcohol-resistance up to ethanol 12 %, while yp13 showed alcohol-resistance up to ethanol 8 %. also, these two yeasts were able to grow in a medium containing citric acid up to 4 %, acetic acid up to 1 % and lactic acid at 1.5 %, respectively. the figure 5j shows that yb14 and yp3 well grew under high osmotic stress due by glucose 50 % or mixed glucose 5 % with nacl 10 %, respectively. the growth of these yeast strains under stress conditions confirm the ability of these yeast strains to be used as starters into many food process notably cocoa fermentation process. 3.6. molecular characterization the molecular characterization indicates that yb14 was identified as saccharomyces cerevisiae and yp13 as pichia kudriavzevii. saccharomyces cerevisiae already showed some performance as starter in some biotechnological applications of fermentation [8, 84]. even, pichia kudriavzevii also showed performance asstarter recently in a study leaded by [41]. however, these two-species taken together food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 125 fig.5. effects of temperature (a), ph (b), glucose (c), fructose (d), sucrose (e), ethanol (f), citric acid (g), acetic acid (h), lactic acid (i) and high osmotic pressure (j) on yeasts growth. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume …., issue .. – … 126 have never been used in a microbial cocktail. thus, considering the important potentialities that they release, it should be carried out cocoa fermentation with these two-strains for a control of the ivorian fermentation process. 4. conclusion this study showed that two yeast strains, notably yb14 (saccharomyces cerevisiae) and yp13 (pichia kudriavzevii), with high ethanol producer under stress conditions were able to produce specific enzymes necessary to cocoa fermentation and produce acetoin, which is desirable flavor in fermentation process. thus, these two strains can be used as starter cultures in cocoa fermentation which may contribute to the control of the cocoa fermentative process in ivory coast. 5. acknowledgments contribution of waves laboratories team, to bingerville (ivory coast), was highly appreciated during the quantification of ethanol by uv method. also, the contribution of laboratory to adaptation microbiology and pathogeny, at lyon (france), was appreciated for the molecular identification of yeast strains. 6. references [1]. pereira g. v. m., soccol.v.t., soccol c. r., current state of research oncocoa and coffee fermentations. current opinion in food science, 7, 50-57, (2016). [2]. schwan r. f., wheals .a.e., the microbiology of cocoa fermentation and its role in chocolate quality. crit rev food sci nutr, 44(4), 205–221, (2004). [3]. ouattara h. g., ban-koffi .l., karou g. t., sangare a., niamke s. l., diopoh j. k., implication of bacillus sp. in the production of pectinolytic enzymes during cocoa fermentation. world journal of microbiology and biotechnology, 24, 1753-1760, (2008). [4]. rodriguez-campos j., escalonabuendiah. b., orozco-avila i., lugocervantes e., jaramillo-flores m. e., dynamics of volatile and non-volatile compounds in cocoa (theobroma cacao l.) during fermentation and drying processes using principal components analysis. food research international, 44, 250-258, (2011). [5]. rodriguez-campos j., escalonabuendiah b., contreras-ramos s. m., orozcoavila i., jaramillo-flores e., lugo-cervantes e., effect of fermentation time and drying temperature on volatile compounds in cocoa. food chemistry, 132, 277-288, (2012). [6]. lefeber t., papalexandratou z., gobert w., camu n., de vuyst l., on-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof, food microbiol, 30, 379–392, (2012). [7]. mahazar n. h., sufian n.f., meorhussin a. s., norhayati h., mathawan m., rukayadi y., candida sp. as a starter culture for cocoa (theobroma cacao l.) beans fermentation. international food research journal, 22, 1783-1787, (2015). [8]. meersman e., steensels j., struyf n., paulus t., saels v., mathawan m., allegaert l., vrancken g., verstrepen k. j., tuning chocolate flavour through development of thermotolerant saccharomyces cerevisiae starter cultures with increased acetate ester production. appl. environ. microbiol., 82(2), 732-746, (2016). [9]. crafack m., mikkelsen m., saerens s., knudsen m., blennow a., lowor s., takrama j., swiegers j. h., petersen g. b., heimdal h., nielsen d., influencing cocoa flavor using pichia kluyveri and kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation, international journal of food microbiology, 167, 103-116, (2013). [10]. leal g. a., gomes l.h., efraim p., tavares f. c., figueira a., fermentation of cacao (theobroma cacao l.) seeds with a hybrid kluyveromyces marxianus strain improved product quality attributes, fems yeast research, 8, 788798, (2008). [11]. visintin s., ramos l., batista n., dolci p., schwan f., cocolin l., impact of saccharomyces cerevisiae and torulaspora delbrueckii starter cultures on cocoa beans fermentation, international journal of food microbiology, 257, 31–40, (2017). [12]. koffi o., samagaci l., goualie b., niamke s., diversity of yeasts involved in cocoa fermentation of six major cocoa-producing regions in ivory coast. european scientific journal, 13(30), 496-516, (2017). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 127 [13]. pettipher g. l., an improved method for the extraction and quantitation of anthocyanins in cocoa beans and its use as an index of the degree of fermentation. j. sci. food agric, 37, 289–296, (1986). [14]. wollgast j., anklam e., review on polyphenols in theobroma cacao: changes in composition during the manufacture of chocolate and methodology for identification and quantification. food res. int., 33, 423–447, (2000). [15]. kyi t.m., daud w.r.w., mohammad a.b., wahid samsudin m., kadhum a.a.h., talib m.z.m., the kinetics of polyphenol degradation during the drying of malaysian cocoa beans. int. j. food sci. technol, 40, 323–331, (2005). [16]. ho v. t., zhao .j., fleet g., yeasts are essential for cocoa fermentation. international journal of food microbiology, 174, 72-87, (2014). [17]. thompson s., miller k., lopez a., camu n., cocoa and coffee, in food microbiology: fundamentals and frontiers, m.p. doyle, buchanan, r.l. (eds.), 881–889, (2013). [18]. cros e., relation précurseursdéveloppement de l’arôme cacao, in 11 ème conférence internationale sur la recherche cacaoyère, a.l.o.c.w.d.u. editor., 632-638., yamoussoukro (côte d’ivoire), (1993). [19]. barel m., la première transformation du cacao, in cahiers des charges ingénieurs agronomes, 14-15, (1998). [20]. de vuyst l., weckx s., the cocoa bean fermentation process: from ecosystem analysis to starter culture development. journal of applied microbiology,1-13, (2016). [21]. soumahoro s., typage biochimique et moleculaire de bacteries acetiques a forte capacite de production d’acide acetique isolees de la fermentation du cacao de six regions de côte d’ivoire : recherche de potentiels starters et application biotechnologique, in biosciences, université félix houphouet boigny, côte , p.190, (2016). [22]. dung n. t. p, phong .x.h., screening thermoand ethanol tolerant bacteria for ethanol fermentation. american journal of microbiological research, 1(2), 25-31, (2013). [23]. hesclot h., vladescu b., la levure dans les industries alimentaires,(1994). [24]. dung n. t. p., pornthap t., huynh x. p., screening useful isolated yeasts for ethanol fermentation at high temperature. international journal of applied science & technology, 4, 65-71, (2012). [25]. racker e., alcohol dehydrogenase from baker’s yeast methods in enzymology, 1, 500-503, (1955). [26]. englezos v., rantsiou k., torchio f., rolle l., gerbi v., cocolin l., exploitation of the non-saccharomyces yeast starmerella bacillaris (synonym candida zemplinina) in wine fermentation: physiological and molecular characterizations. int. j. food microbiol., 199, 33 – 40, (2015). [27]. king n., modification of voges-proskauer test for rapid colorimetric determination of acetyl methyl carbimol plus diacetyl in butter. dairy inductries, 13, 860-866, (1948). [28]. pereira g., miguel m., ramos c., schwan r., microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture. appl. environ. microbiol., 78(15), 5395-5405, (2012). [29]. kurtzman c.p.,pichia. e.c. hansen (1904), in the yeasts: a taxonomic study c.p. kurtzman, fell, j.w., boekhout, t. (eds.), 685–707, (2011). [30]. yarrow d.,saccharomyces meyen ex reess., in the yeasts, a taxonomic study. n.j.w. kregervan rij (ed.), 379-395, (1984a). [31]. wickerham l. j., taxonomy of yeast. united states. washington dc, department of agriculture, . 1029, 1-55, (1951). [32]. hoffman c. s., preparation of yeast dna. current protocols in molecular biology. 39, 3p, (2001). [33]. hamdouche y., guehi t., durand n., kedjebo k. b., montet d., meile j. c., dynamics of microbial ecology during cocoa fermentation and drying: towards the identification of molecular markers. food control, 48, 117-122, (2015). [34]. ardhana m. m., fleet g.h., the microbial ecology of cocoa bean fermentations in indonesia. international journal food microbiology 86, 87-99, (2003). [35]. papalexandratou z., camu n., falony g., de vuyst l., comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in ivory coast and brazil. food microbiol.,. 28, 964– 973, (2011a). [36]. schwan r.f., cocoa fermentations conducted with a defined microbial cocktail inoculum. appl environ microbiol, 64, 1477–1483, (1998). [37]. lagunes-galvez s., loiseau g., paredes j., barel m., guiraud j., study on the microflora and biochemistry of cocoa fermentation in the dominican republic. int j. food microbiol., 114(1), 124–130, (2007). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 128 [38]. papalexandratou z., de vuyst l., assessment of the yeast species composition of cocoa bean fermentations in different cocoaproducing regions using denaturing gradient gel electrophoresis. fems yeast res, 11(7), 564–574, (2011). [39]. papalexandratou z., lefeber t., bahrim b., lee o.s., daniel h.-m., de vuyst l.,hanseniaspora opuntiae, saccharomyces cerevisiae, lactobacillus fermentum, and acetobacter pasteurianus predominate during well performed malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process. food microbiol, 35, 73–85, (2013). [40]. lefeber t., janssens m., moens f., gobert w., de vuyst l., interesting starter culture strains for controlled cocoa bean fermentation revealed by simulated cocoa pulp fermentations of cocoa-specific lactic acid bacteria, appl. environ. microbiol. , 77, 6694–6698, (2011b). [41]. pereira g., alvarez j.p., neto d. p., soccol v. t., tanobe v. o., rogez h., goes-neto r., soccol c., great intraspecies diversity of pichia kudriavzevii in cocoa fermentation highlights the importance of yeast strain selection for flavor modulation of cocoa beans. lwt-food science and technology, 84, 290 – 297, (2017). [42]. roelofsen p.a., giesberger g., onderzoekingen over cacao bereiding. arch. koffiecult. ned.-indie. 16, 119–159, (1947). [43]. quesnel v.c., agents inducing the death of cacao seeds during fermentation. j. sci. food agric, 16, 441–447, (1965). [44]. de vuyst l., lefeber t., papalexandratou z., camu n., the functional role of lactic acid bacteria in cocoa bean fermentation. in: mozzi, f., raya, r.r., vignolo, g.m. (eds.), biotechnology of lactic acid bacteria novel applications. wiley-bl., (2010). [45]. lima l.j.r., almeida m.h., nout m.j.r., zwietering m.h., theobroma cacao l., “the food of the gods”: quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation. crit. rev. food sci. nutr., 51, 731–761, (2011). [46]. afoakwa e.o., paterson a., fowler m., ryan a., flavour formation and character in cocoa and chocolate: a critical review, crit rev food sc nutr, 48, 840–857, (2008). [47]. watson j. d., hopkins n.h., roberts j. w., stteitz j. a., wainer a. m., yeast as the e. coli of eucaryotic cell. molecular biology of the gene. benjamin-cummings, 550-593, (1987). [48]. aldiguier s. a., alfenore s., cameleyre x., goma g., uribelarrea j. l., guillouet s. e., molina-jouve c., synergistic temperature and ethanol effet on saccharomyces cerevisiae dynamic behaviour in ethanol bio-fuel production. bioprocess biosyst. eng, 26, 217-222, (2004). [49]. torija m.j., b.g., novo m., poblet m., guillamón j.m., mas a., rozès n., effects of fermentation temperature and saccharomyces species on the cell fatty acid composition and presence of volatile compounds in wine. int j food microbiol, 85(1-2), 127-136, (2003b). [50]. barnett j. a., entian k.-d., a history of research on yeasts 9 : regulation of sugar metabolism1. yeast, 22(11), 835–894, (2005). [51]. jespersen l., nielsen d.s., hønholt s., jakobsen m., occurrence and diversity of yeasts involved in fermentation of west african cocoa beans, fems yeast research, 5, 441– 453, (2005). [52]. nielsen d., teniola o., ban-koffi l., owusu m., andersson t., holzapfel w., the microbiology of ghanaian cocoa fermentations analyzed using culture-dependent and culture independent methods. int. j. food micro., 114, 168–186, (2007). [53]. gryta m., morawskia., tomaszewska m., ethanol production in membrane distillation bioreactor, catalysis today,159-165, (2000). [54]. aguilera a., peinado r.a., millan c., ortega j. m., mauricio j. c., relationship between ethanol tolerance, h+-atpase activity and the lipid composition of the plasma membrane in different wine yeast strains, int. j. food microbiol., 110, 34-42, (2006). [55]. hirasawa t., yoshikawak., nakakura y., nagahisa k., furusawa c., katakura y., shioya s., identification of target genes conferring ethanol stress tolerance to saccharomyces cerevisiae based on dna microarray data analysis. j. biotechnol., 131, 34-44, (2007). [56]. piper w.p., talreja k., panaretou b., moradasferreira p., byrne k., praekelt u.m., meacock p., recnacq m., boucherie h., induction of major heat-shock proteins of saccharomyces cerevisiae, including plasma membrane hsp30, by ethanol levels above critical threshold, microbiology-uk, 140, 30313038, (1994). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 129 [57]. banat i.m., marchant r., characterization and potential industrial applications of ®ve novel, thermotolerant, fermentative yeasts strains, world journal of microbiology and biotechnology, 11, 304-306, (1995). [58]. camu n., gonzalez a., de winter t., van schoor a., de bruyne k., vandamme p., takrama j.s., de vuyst l., influence of turning and environmental contamination on the dynamics of lactic acid bacteria and acetic acid bacteria populations involved in spontaneous cocoa bean heap fermentation in ghana. appl environ microbiol, 74, 86–98, (2008b). [59]. roelofsen p., fermentation, drying and storage of cacao beans. adv. food res. int., 8, 225– 296, (1958). [60]. jinap s., organic acids in cocoa beans a review. asean food j.,. 9, 3–12, (1994). [61]. holm c., aston j., douglas k., the effects of the organic-acids in cocoa on the flavor of chocolate, j. sci. food agric., 61, 65–71, (1993). [62]. hansen c., del olmo m., burri c., enzyme activities in cocoa beans during fermentation. journal of the science of food and agriculture, 77, 273-281, (1998). [63]. levine a.s, fellers c.r., action of acetic acid on food spoilage microorganisms. j. bacteriol., 39, 499–515, (1940). [64]. imai t., ohno t., the relationship between viability and intracellular ph in the yeast saccharomyces cerevisiae, appl. environm. microbiol, 61(10), 3604-3608, (1995). [65]. ferreira m. m., loureiro-dias m.c., loureiro v., weak acid inhibition of fermentation by zygosaccharomyces bailii and saccharomyces cerevisiae, int j food microbiol, 36(3), 145-153, (1997). [66]. pampulha m.e., loureiro-dias m.c., energetics of the effect of acetic acid on growth of saccharomyces cerevisiae, fems microbiol. lett., 184, 69–72, (2000). [67]. lefeber t., janssens m., camu n., de vuyst l., kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation, appl. environ. microbiol., 76, 7708–7716, (2010). [68]. ouattara h. d., ouattara h.g., droux m., reverchon s., nasser w., niamke s. l., lactic acid bacteria involved in cocoa beans fermentation from ivory coast: species diversity and citrate lyase production. international journal of food microbiology, 256, 11–19, (2017). [69]. camu n., de winter t., verbrugghe k., cleenwerck i., takrama j.s., vancanneyt m., de vuyst l., dynamics and biodiversity of lactic acid bacteria and acetic acid bacteria populations involved in spontaneous heap fermentations of cocoa beans in ghana. appl environ microbiol., 73, 1809–1824, (2007). [70]. graves t., narendranath v.n., dawson k., power r., interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by saccharomyces cerevisiae in corn mash. appl microbiol biotechnol., 73, 1190–1196, (2007). [71]. abbott da, ingledew w., buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of saccharomyces cerevisiae and ethanol production, biotech lett, 26, 1313–1316, (2004). [72]. narendranath n.v., thomas k.c., ingledew w.m., effects of acetic acid and lactic acid on the growth of saccharomyces cerevisiae in a minimal medium, j ind microbiol biotechnol, 26, 171–177, (2001). [73]. jinap s., dimick p.s., hollender r., flavor evaluation of chocolate formulated from cocoa beans from different countries. food control, 6, 105–110, (1995). [74]. adler p., frey l.j., berger a., bolten c.j., hansen c.e., wittmann c., the key to acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp fermentation simulating conditions. appl environ microbiol, 80, 4702– 4716, (2014). [75]. pyo y.h., lee t.c., lee y.c., enrichment of bioactive isoflavones in soy milk fermented with beta-glucosidase-producing lactic acid bacteria. food res int, 38, 551-559, (2005). [76]. yin l.j., li l.t., liu h., saito m., tatsumi e., effects of fermentation temperature on the content and composition of isoflavones and beta-glucosidase activity in sufu. biosci biotechnol biochem, 69, 267-272, (2005). [77]. otieno d.o., ashton j.f., shah n.p., evaluation of enzymic potential for biotransformation of isoflavone phytoestrogen in soymilk by bifidobacterium animalis, lactobacillus acidophilus and lactobacillus casei, food res int, 39, 394-407, (2006). [78]. rosi i., vinella m., domizio p., characterization of betaglucosidase activity in yeasts of oenological origin. j appl bacteriol, 77, 519-527, (1994). [79]. martos m. a., zubreskie.r., garro a. o., hours r. a., production of pectinolytic enzymes by the yeast wickerhanomyces anomalus isolated from citrus fruits peels. biotechnology, research international, 7p, (2013). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke, screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast, food and environment safety, volume xvii, issue 2 – 2018, pag. 113 – 130 130 [80]. schwan r., rose a., board r., microbial fermentation of cocoa beans, with emphasis on enzymatic degradation of the pulp, j. appl. bacteriol. symp. sup., 79, 96–107, (1995). [81]. lopez a. s., dimick p.s., cocoa fermentation. in: reed, g., nagodawithana, t. w. (eds.), enzymes, biomass, food and feed. vch, weinheim., 561-577, (1995). [82]. illeghems k., weckx s., de vuyst l., applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a singlespontaneous cocoa bean fermentation process sample, food microbiology, 50, 54-63, (2015). [83]. visintin s., alessandria v., valente a., paola dolci p., cocolin l., molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentation in west africa, international journal of food microbiology, 216, 69-78, (2016). [84]. ramos c.l., dias d.r., miguel m.g.c.p., schwan r.f., impact of different cocoa hybrids (theobroma cacao l.) and s. cerevisiae ufla ca11 inoculation on microbial communities and volatile compounds of cocoa fermentation, food res int, 64, 908–918, (2014). 1. introduction effect of essential oil of mentha spicata l 123 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 123 134 effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c *euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue laboratory of research and study in applied chemistry, polytechnic school of abomey-calavi, university of abomey-calavi, 01 p.o.b: 2009, cotonou, benin *corresponding author: eulogesenan@yahoo.fr; euloge.adjou@epac.uac.bj received 27th june 2017, accepted 24 th september 2017 abstract: the present study aims to evaluate the effect of essential oil from fresh leaves of mentha spicata l. on the quality of mango puree in storage model food systems at 4°c and 25°c. the results of physico-chemical characterization of mango puree underlined its high nutritional potential, with carbohydrates, carotenoids and vitamin c contents of 9.5±0.4%, 20.05±0.03 mg/100g and 21.03±0.05 mg/100g respectively. the microbiological analyses using taxonomic schemes primarily based on morphological characters of mycelium and conidia revealed that aspergillus parasiticus, fusarium versicolor and mucor spp. were the most common fungi identified in mango puree in the southern benin. antifungal assay, performed by the agar diffusion assay, indicated that essential oil exhibited high antifungal activity against the growth of fungi. the minimal inhibitory concentration (mic) of the essential oil was found to be 2.0 μl.ml-1for aspergillus parasiticus and fusarium versicolor; and 1.0 μl.ml-1 for mucor spp. the chemical analysis of the oil made by gc/ms led to the identification of 35 components, characterized by carvone (67.5%), and limonene (12.0%) as major components. the results obtained during the evaluation of the microbiological, physico-chemical and sensorial characteristics of the mango puree stored by adding essential oil, revealed the high potential of the essential oil of mentha spicata l. in food quality preservation. this essential oil offers a novel approach to the management of fruit derivate products during storage. keywords: mentha spicata l., plant extract, chemical composition, conservation, benin. 1. introduction in africa, several studies reported that the problems encountered by producers during the post-harvest period of agricultural products have been neglected, because they were combined with those related to production. meanwhile, post-harvest losses are increasing, due to the fact that traditional storage technologies are generally inadequate [1]. thus, to contribute to the reduction of food insecurity problems, agricultural production should be increased and local products should be valued through the judicious use of technical knowledge and biotechnological tools. for example, the valorization of tropical fruits through their biotransformation into better stabilized and value-added products should be promoted in low income countries. african flora has a lot of tropical species including mango tree (mangifera indica), whose fruit is highly appreciated for its high content of carotenoids, flavonoids, vitamins and fibers [2]. in benin, the low valorization of this fruit causes a significant post-harvest loss due to the harvesting or storage conditions that attract many microorganisms and parasites. thus, http://www.fia.usv.ro/fiajournal mailto:euloge.adjou@epac.uac.bj food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 124 one of the solutions envisaged is its transformation into value-added products, such as mango puree. however, the conservation of fruit products is a serious problem because of the rapid growth of micro-organisms. to overcome this problem, unspecified heat treatments or antimicrobials products from chemical synthesis are often used. unfortunately, the nutritional value of fruit derivate products are often modified by heat treatments, and the application in high concentrations of synthetic chemicals products in food preservation increases the risk of toxic residues in food products [3]. due to the increasing sensitivity of consumers to this residual pollution and the toxic effects of many antimicrobials from chemical synthesis, the importance of using natural products becomes necessary [4]. similarly, the restriction imposed by the food industry and regulatory agencies on the use of some synthetic food additives has led to a renewed interest in the search for alternatives, such as natural compounds [3]. plant extracts have many properties, the antimicrobial activity being one of the most important [5]. many researches have investigated the systematical study of the essential oils extracted from the aromatic plants commonly used in traditional pharmacopoeias in benin [6, 7], and the importance of the use of essential oils in the food preservation are also reported by konfo et al. [8], soumanou and adjou [9], and adjou et al. [10]. the use of essential oils as antimicrobial agents has two main advantages: the first refers to their natural origin which means more safety for the population and environment, and the second is that they are considered to have a low risk of development of resistance by pathogenic microorganisms [11]. thus, the objective of our study was to evaluate the efficacy of the essential oil extracted from fresh leaves of mentha spicata l. against the spoilage flora of mango puree in benin by physico-chemical characterization and microbiological analysis. 2. matherials and methods collection of plant leaves plant materials used for essential oil (eo) extraction were fresh leaves of mentha spicata l. plants were collected at abomey-calavi (6°26′54″ north /2°21′20″ east) (south benin) and identified at the benin national herbarium, where voucher specimens are deposited. essential oil extraction the eo tested was extracted by the hydrodistillation method using clevenger-type apparatus. the oil recovered was dried over anhydrous sodium sulfate and stored at 4 °c until it was used [12]. gas chromatography–mass spectrometry analysis the eo were analyzed by gas chromatograph (perkin elmer auto xl gc; waltham, ma, usa) equipped with a flame ionisation detector, and the gc conditions were equity-5 column (60 m x 0.32 mm x 0.25 µm); h2 as the carrier gas; column head pressure 10 psi; oven temperature program isotherm 2 min at 70 °c, 3 °c/min gradient 250 °c, isotherm 10 min; injection temperature, 250 °c; detector temperature 280 °c. gas chromatography–mass spectrometry (gcms) analysis was performed using a perkin elmer turbomass gc-ms. the gc column was equity-5 (60 m x 0.32 mm x 0.25 µm); fused silica capillary column. the gc conditions were injection temperature, 250 °c; column temperature, isothermal at 70 °c for 2 min, then programmed to 250 °c at 37 °c/min and held at this temperature for 10 min; ion source temperature, 250 °c. helium was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 125 the carrier gas. the effluent of the gc column was introduced directly into the source of ms and spectra obtained in the ei mode with 70 ev ionisation energy. the sector mass analyzer was set to scan from 40 to 500 amu for 22 s. the identification of individual compounds is based on their retention times, retention indices relative to c5 – c18 n-alkanes, and matching spectral peaks available in the published data [13]. collection of mango and puree production the mango samples used in this study were purchased at the large mango selling area of dantokpa market in cotonou (6° 21' 45'' north/2° 25' 32'' east) (south benin). these samples consist of ripe mangoes of eldon variety, characterized by a yellow-orange color and a strong aroma of turpentine. mango samples were washed and pulped. the pulp obtained is chopped and then ground to obtain the mango puree. the figure 1 shows the technological diagram used for the production of mango puree. fig. 1. technological diagram for mango puree production microbiological analysis for the microbiological analysis, 25 g of sample and 225 ml of peptone water was added and homogenized. from the initial concentration, appropriate decimal dilutions were prepared and aliquots were plated in duplicates on various media. plate count agar was used for the total bacterial count according to iso 4833-1 (1) methods. plates were incubated at 30°c for 72 h. desoxycholate was used for the total coliforms count according to nf v08050, and plates were incubated at 30°c for 24 h. desoxycholate was also used for the faecal coliforms count. in this case, plates were incubated at 44°c and the identification was made using eosine methylene blue (emb) medium. bair parker medium was used for staphylococcus spp. count according to nf en iso 6888-1/a1, and plates were incubated at 37°c for 24h. tryptone sulfite neomycin agar was used for anaerobic sulfito-reducer (asr) count, according to iso 7937 methods and tubes were incubated at 37°c for 24 h. after incubation, the number of colonies was tracked, using a colony counter. the number of bacteria expressed as colony forming units per gram (cfu/g) was then determined by calculation, considering dilution factor. all the media used for microbiological analysis were prepared as indicated by the manufacturer. after their isolation, bacteria were also controlled with api system (biomérieux france). fungal isolation the isolation of fungi from samples was performed using dilution plating method. ten grams of each juice sample were added separately to 90 ml of sterile water containing, 0.1% peptone water. this was thoroughly mixed to obtain the 10-1 dilution. further, 10fold serial dilutions up to 10-4 were made. 1 ml volume of each food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 126 dilution was separately placed in petri dishes, over which, 10 to 15 ml of potato dextrose agar amended with 60 µg/ml chloramphenicol (pdac) was poured. the plates were incubated at 28 ± 2°c for 7 days. fungal isolates from pdac were sub-cultured on malt extract agar (mea) and identified by using a taxonomic schemes primarily based on morphological characters of mycelium and conidia [10]. antifungal assay antifungal assay was performed by the agar medium assay [5]. yeast extract sucrose medium (yes) with different concentrations of essential oil (0.15, 0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 2.50 μl/ml-1) and tween 20 (0.01%) were prepared by adding appropriate quantity of eo to melted medium, followed by manual rotation of flask to disperse the oil in the medium. about 20 ml of the medium were poured into glass petri dishes (9 cm). fungal isolates from mango puree on malt extract agar (mea) are transplanted (subcultured), using a disc of 6 mm in diameter which carries spores from the anamorph mold, on the surface of a petri dish containing the former medium yeast extract sucrose and eo at different concentrations. positive control plates (without eo and inoculated following the same procedure) and negative control plates were also used. plates were incubated at 25 °c for 5 days. determination of the fungistatic or fungicidal activity with the experimental concentrations where neither growth nor germination was observed, the fungistatic or fungicidal activity was tested. this assay consisted by taking the mycelial disc not germinated at the end of the incubation of the petri dish and reintroducing it in a new culture medium (former one) without eo. if the mycelial growth is always inhibited, the plant extract is fungicidal at this concentration and allows the determination of the minimum fungicidal concentration (mfc). in the contrary case, it does fungistatic activity which is related to the minimum inhibitory concentration (mic) [5]. conservation of mango puree with essential oil to evaluate the conservation potential of the eo of mentha spicata l., on mango puree, five eo concentrations were tested. these are 0.50, 1.00, 1.50, 2.00 and 2.50 μl/ml-1. these concentrations were chosen taking into account the high fragrant nature of the eo. a negative control (mango puree without eo) was also produced. samples were placed at 4°c at 25 °c. after thirty (30) days of conservation at these temperatures, microbiological and physico-chemical qualities of canned mango purees were then evaluated. the ph of the samples was determined in 10ml of mango puree using a digital ph-meter. vitamin c (l-ascorbic acid) and carotenoids concentrations were determined using method described by adjou et al. [14]. organoleptic tests organoleptic tests were performed using a panel comprising 30 panelists selected on the basis of good health conditions, time availability, no allergy to plant products and their ability to appreciate taste, flavor, color, texture, appearance and after taste. the overall acceptability was determined on a scale by using the 9 point hedonic test as described by bisla et al. [15]. the standard mango puree purchased from retail shops was used as control sample. statistical analysis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 127 the experiments were performed in triplicates, and data analyzed are means ± sd subjected to one-way anova. means are separated by the tukey’s multiple range test where anova was significant (p<0.05) (spss 10.0; chicago, il, usa). 3. results and discussion by hydrodistillation, fresh leaves of mentha spicata l. yielded 1.85 % of eo. the chemical analysis by gc and gc-ms analysis of eo enabled the identification of 35 components, (table 1) representing 98.5 % of the eo. in the volatile extract, different groups of terpenes and terpenoids, such as hydrogenated monoterpenes (15.2%) and oxygenated monoterpenes (78.0%) were detected. the eo has chemical composition characterized by carvone (67.5%), and limonene (12.0%) as major components. the result of microbial analysis and isolation of fungi in pure culture revealed that the most contaminated microflora of mango puree were fungi (table 2). fungal isolates include aspergillus parasiticus, fusarium versicolor and mucor spp. the results of physico-chemical characterization of mango puree (table 3) indicated that the ph was 6.8±0.1, with carbohydrates, carotenoids and vitamin c contents of 9.5±0.4 %, 20.05±0.03 mg/100g and 21.03±0.05 mg/100g respectively. eo exhibited pronounced antifungal activity against the growth of aspergillus spp., fusarium spp and mucor spp. the mic of the eo was found to be 2.0 µl/ml for aspergillus parasiticus and aspergillus versicolor; and 1.0 µl/ml for mucor spp. the mfc was recorded to be 2.5 µl/ml for aspergillus parasiticus and fusarium versicolor and 1.5 µl/ml for mucor spp. the results obtained during the storage tests of mango puree with the eo of mentha spicata l. at different concentrations (tables 4 and 5) indicated strong antimicrobial activity of the eo against the spoilage flora, depending on the storage model food system used. indeed, with the essential oil concentration of 1.50 μl.ml-1 in a storage model food system of 4°c, there was an important antifungal activity against aspergillus parasiticus, fusarium versicolor and mucor spp. (table 4). however, the high antifungal activity of this eo is obtained only with eo concentration of 2.0 μl.ml-1 in a storage model food system of 25°c (table 5). tables 6 and 7 present the results of the evaluation of the physicochemical characteristics of the mango puree conserved by adding eo and stored respectively at 4°c and 25°c. these results indicated a relative stability of physicochemical parameters such as ph, carotenoids and vitamin c contents, in a storage model food system of 4°c and eo concentration of 1.5 μl.ml-1 after 30 days of storage (table 6). however, in mango puree stored with eo at concentrations of 0.5 – 1.5 μl.ml-1, in a storage model food system of 25°c, there was a significant difference in ph, carotenoids and vitamin c contents, after 30 days of storage (table 7). the results of organoleptic evaluation are shown in table 8. they indicated that mango puree samples conserved with eo of mentha spicata were found to be organoleptically satisfactory at different concentrations in the tested and stored model food systems used. the comparison of organoleptic quality of mango puree conserved with eo at different concentrations and the standard revealed that mean scores of mango puree conserved with eo were more acceptable than those of the standard. however, it was also observed that the concentration of eo of mentha spicata in samples was inversely proportional to the acceptability food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 128 scores. the best overall acceptability scores were identified for eo concentration of 1.5 μl.ml-1 at 4°c. edible fruits become an alternative source of food with high potential of vitamins, minerals and other interesting elements, particularly during seasonal food shortage [16]. they are also known to have nutritional and medicinal properties that can be attributed to their antioxidant effects and they can be used to fortify staple foods particularly for malnourished children [17]. the results of the proximate analyses revealed that mango puree is a good source of carbohydrates, carotenoids and vitamin c. the carbohydrate content was similar to that reported in sour soup fruit [18] and higher than that reported in palmyra juice [14] and in cashew apple juice [19]. the carotenoid content was similar to that reported in palmyra juice [14]. vitamin c content is lower than those reported in oranges juice [10] and higher than those reported in palmyra juice [14]. the high nutritional potential of mango puree, justified its uses as supplement in infant feeding in benin. the results obtained from microbial analysis revealed that mango puree was contaminated with microorganisms. the most dominant flora was fungi aspergillus parasiticus, fusarium versicolor and mucor spp. these fungi species are known as spore formers and their growth can result in the production and accumulation of mycotoxins. the moisture content of mango puree would also encourage microbial growth and so deterioration. the mango puree contamination by fungi does not only reduce its quality, but it may also lead to mycotoxin production [20]. the present study also explores the bioefficacy of eo of mentha spicata l. as the promising plant-based antimicrobial against mango puree-infecting fungal growth. this eo was found to be effective against all aspergillus, and mucor strains tested, and also in the preservation of mango puree quality. this bioefficacy may be due to the presence of some highly fungitoxic components in the oil such as terpenoids. indeed, mentha spicata oil has a chemical composition characterized by terpenes and terpenoids as the main chemical groups. several studies have indicated that terpenes do not represent a group of constituents with a high inherent antimicrobial activity. for example, koutsoudaki et al. [21] compared the effect of -pinene, ß-pinene, p-cymene, ßmyrcene, limonene, and γ-terpinene against escherichia coli, staphylococcus aureus, and bacillus cereus and reported that their antimicrobial activity was low or absent. rao et al. [22] also reported that pcymene and γ-terpinene were ineffective as fungicides against saccharomyces cerevisiae. in contrast, terpenoids are a large group of antimicrobial compounds that are active against a broad spectrum of microorganisms [23]. their antimicrobial activities are linked to their functional groups and it has also been reported that the hydroxyl group of phenolic terpenoids and the presence of delocalized electrons are important for the antimicrobial activity [23]. the antimicrobial activity of menthol, thymol and linalool, against listeria monocytogenes, enterobacter aerogenes, eschericha coli, and pseudomonas aeruginosa were reported by bassole et al. [24]. these results confirm the high antimicrobial activity of a broad collection of terpenoids, which are the major components of the eo of mentha spicata l. a range of eo components (menthol, thymol, eugenol, carvone, cinnamaldehyde, vanillin, carvacrol, citral, and limonene) has been accepted by the european commission for their intended use as flavorings in food products [25]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 129 table 1. chemical composition of mentha spicata l. essential oil investigated components kovats index percentage (%) α-pinene 932 0.5 sabinene 969 0.6 β-pinene 974 0.8 myrcene 988 0.8 limonene 1024 12.0 1,8-cineole 1026 3.6 (z)-β-ocimene 1032 0.2 ȣ-terpinene 1054 0.2 linalol 1095 0.5 oct-1-en-3-yl acetate 1110 0.1 neo-alloocimene 1140 0.1 trans-limonene oxide 1137 0.1 cis-chrysanthenol 1160 0.2 δ-terpineol 1166 0.1 cis-dihydrocarvone 1191 0.2 dihydrocarveol 1192 0.6 neo-dihydrocarveol 1212 0.3 isodihydrocarveol 1212 3.4 neoiso-dihydrocarveol 1226 0.1 carvone 1239 67.5 piperitone 1249 0.9 piperitenone 1343 0.5 eugenol 1356 0.2 α-copaene 1374 0.4 methyl eugenol 1403 0.1 (z)-caryophyllene 1408 0.5 β-gurjunene 1431 0.6 α-humulene 1454 0.1 germacrene-d 1484 0 .9 δ-cadinène 1522 0.1 cis-calamenene 1528 0.9 (e)-nerolidol 1561 0.3 caryophyllene oxide 1582 0.4 1,10-di-epi-cubenol 1618 0.4 α-cadinol 1652 0.5 total 98.5 table 2. microbiological quality of investigated mango puree (cfu/ml) microbiological parameters total bacterial count total coliforms count e.coli s. aureus a.s.r. count yeast count fungi count mango puree sample 4.7 x 102 00 00 00 00 00 8.1x101 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 130 table 3. physicochemical quality of investigated mango puree physico-chemical parameters ph carbohydrates (%) vitamin c (mg/100g) carotenoids (mg/100g) mango puree sample 6.8±0.1 9.5±0.4 21.03±0.05 20.05±0.03 table 4. microbiological quality (ufc/ml) of investigated mango puree after 30 days of conservation at 4°c parameters concentrations of essential oil (μl/ml) 0 0.50 1.00 1.50 2.00 2.50 total bacterial count 2.0x103a* 1.0 x 103b* 101c* 00d* 00e* 00e* fungi count 1.7.0x103a* 2.0 x 102a* 101b* 00c* 00d* 00d* *values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. table 5. microbiological quality (ufc/ml) of investigated mango puree after 30 days of conservation at 25°c parameters concentrations of essential oil (μl/ml) 0 0.50 1.00 1.50 2.00 2.50 total bacterial count 3.0x107a* 2.0 x 105b* 102c* 10d* 00e* 00e* fungi count 1.0x105a* 1.0 x 102a* 1.2 x101b* 10c* 00d* 00d* *values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. table 6. physico-chemical quality of investigated mango puree after 30 days of conservation at 4°c physico chemical parameters mango puree ‘s characteristic s at the beginning of the conservation tests characteristics of the mango puree after 30 days of conservation concentrations of essential oil (μl/ml) 0 0.50 1.00 1.50 2.00 2.50 ph 6.8±0.1a* 4.5±0.1b* 4.97±0.30b * 5.7±0.2b* 6.2±0.3a* 6.21±0.10a* 6.22±0.80a* carbohydrate s (%) 9.5±0.4a* 2.97±0.10b * 5.85±0.20c* 6.5±0.1c* 9.4±0.1a* 9.43±0.10a* 9.42±0.20a* carotenoids (mg/100g) 20.05±0.03a* 5.91±0.07b * 9.09±0.03c* 11.73±0.01d * 18.03±0.05a * 18.05±0.03a * 19.07±0.03a * vitamin c ( mg/100g ) 21.03±0.05a* 3.09±0.02b * 4.08±0.09c* 11.01±0.03d * 20.04±0.07a * 20.09±0.04a * 21.01±0.05a * *values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 131 table 7. physico-chemical quality of investigated mango puree after 30 days of conservation at 25°c physicochemical parameters mango puree ‘s characteristics at the beginning of the conservation tests characteristics of the mango puree after 30 days of conservation concentrations of essential oil (μl/ml) 0 0.50 1.00 1.50 2.00 2.50 ph 6.80±0.10a 2.40±0.10b 4.90±0.40b 5.40±0.60b 5.70±0.20c 6.20±0.30a 6.23±0.70a carbohydrates (%) 9.50±0.40a 1.80±0.20b 5.70±0.30c 6.20±0.10c 8.10±0.60d 9.40±0.10a 9.39±0.30a carotenoids (mg/100g) 20.05±0.03a 4.51±0.07b 9.06±0.04c 11.53±0.02d 14.03±0.06e 18.03±0.05a 19.02±0.07a vitamin c ( mg/100g ) 21.03±0.05a 1.06±0.03b 4.07±0.09c 10.01±0.04d 12.08±0.07d 20.04±0.07a 21.01±0.03a values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. table 8. results of organoleptic evaluation of mango puree preserved with eo of mentha spicata type of mango purees appearance color texture after taste overall acceptability standard 8.12 ± 0.57 7.04 ± 0.29 7.57 ± 0.42 8.29 ± 0.15 8.38 ± 0.14 storage at 4°c samples a (conserved with 1.5 μl.ml-1 of eo) 8.27 ± 0.24 8.32 ± 0.57 8.4 ± 0.23 8.40 ± 0.21 8.74 ± 0.26 samples b (conserved with 2.0 μl.ml-1 of eo) 8.20 ± 0.13 8.27 ± 0.62 8.02 ± 0.74 8.25 ± 0.32 8.60 ± 0.19 samples c (conserved with 2.5 μl.ml-1 of eo) 8.10 ± 0.82 8.04 ± 0.52 7.82 ± 0.54 8.16 ± 0.11 8.45 ± 0.65 storage at 25°c samples d (conserved with 2.0 μl.ml-1 of eo) 8.17 ± 0.51 8.32 ± 0.72 8.3 ± 0.71 8.37 ± 0.54 8.69 ± 0.37 samples e (conserved with 2.5 μl.ml-1 of eo) 7.63 ± 0.25 8.29 ± 0.43 8.07 ± 0.56 8.28 ± 0.62 8.73 ± 0.39 all values are found to be nonsignificant. the united states food and drug administration (fda) also classify these substances as generally recognized as safe (gras). in our study, gc–ms data depicted remarkable variation in the earlier reports on the oils [24, 26]. the chemical profile of eo is reported to be influenced by the harvest period, and by climatic, seasonal, and geographical conditions, which can significantly affect the amount and composition of the active constituents [27]. thus, the biologically activity of eos should be qualitatively standardized before their recommendation for practical exploitation as has been done in the present investigation. the results of the organoleptic evaluation revealed that the use of eo of mentha spicata in other to preserve the quality of mango puree does not have a negative impact on the sensorial characteristics, such as appearance, color, texture, after taste of the canned mango food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 132 purees and its overall acceptability was determined by panelists during the degustation tests. in the available literature, many areas of food science are concerned with the use of essential oils. in food products, essential oils have been used in bakery [28], cheese [29] and fruit [30], among others. however, many of individual oil components used as approved food flavorings can also impart a certain flavor to foods. then, the organoleptic impact on treated food must also be evaluated, as it has been done in the present investigation. 4. conclusions this work underlined the bioactivity of eo of fresh leaves of mentha spicata l. from benin as a fungal growth suppressor in mango puree. based on its antifungal potential, this natural plant product may successfully replace synthetic chemicals and provide an alternative method to the preservation of mango puree as well as of other fruit derivate products with high nutritional significance against the contamination and growth of bacteria and fungi. 5. acknowledgments the authors are grateful to the department of food engineering of polytechnic school of abomey-calavi university, for the financial support. they also like to thanks mr gad fayomi and mrs adidjath mounirou for their continuous help and assistance during the experiment. conflicts of interest: none. 6. references [1]. fandohan p., hell k., marasas w.f.o., infection of mayse by fusarium specifies and contamination with fumonisin in africa, african journal of biotechnology, 2: 510-579, (2003). [2]. djioua t., charles f., lopez-lauri f., filgueiras h., coudret a., freire m., ducamp-collin m.n., sallanon h., improving the storage of minimally processed mangoes (mangifera indica l.) by hot water treatments, postharvest biology and technology, 52: 221–226, (2009). [3]. hammer k.a., carson c.f., ridley c.v., antimicrobial activity of essential oils and other plants extract, journal of applied microbiology, 86: 985-990, (1999). [4]. bankole s.a., effect of essential oil from two nigerian medicinal plants (azadirachta indica and morinda lucida) on growth and aflatoxin b production in maize grain by a toxigenic aspergillus flavus, letters in applied microbiology, 24: 190–192, (1997). [5]. yehouenou b., noudogbessi j.-p., sessou p., avlessi f., sohounhloue d., etude chimique et activités antimicrobiennes d’extraits volatils des feuilles et fruits de xylopia aethiopica (dunal) a. rich. contre les pathogènes des denrées alimentaires, journal de la société ouest-africaine de chimie, 029 : 19 – 27, (2010). [6]. sohounhloue d.k., dangou j., gnonhossou b., garneau f.x., gagnon h., jean f.i., leaf oils of three eucalyptus species from benin: e. torellina f. muel, e. citriodora hook and e. tereticornis smith., journal of essential oil research, 8: 111-113 (1996). [7]. alitonou g.a., huiles essentielles extraites de plantes aromatiques acclimatées au bénin : étude chimique, évaluation biologique et applications potentielles. thèse de doctorat en cotutelle, université d’abomey-calavi et université de montpellier ii., (2006), 282p. [8]. konfo c.t.r., chabi n.w., dahouenon-ahoussi e., cakpo-chichi m., soumanou m.m., sohounloue ckd., improvement of african traditional sorghum beers quality and potential applications of plants extracts for their stabilization: a review, journal of microbiology, biotechnology and food sciences, 5(2):190-196, (2015). [9]. soumanou m.m. adjou e.s., sweet fennel (ocimum gratissimum) oils in: preedy, v.r. (ed.), essential oils in food preservation, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 133 flavor and safety, academic press, 765–773, (2016). [10]. adjou e.s., dahouenon-ahoussi e., dègnon g.r., mongazi c., soumanou m.m., sohounhloue d.c.k., chemical composition and biological activity of essential oil from cymbopogon citratus leaves on the quality of fresh orange juice during storage, international journal of health, animal science and food safety, 4: 01 – 12, (2017). [11]. tatsadjieu n., jazet m., ngassoum m.b., etoa x., mbofung c.m.f., investigations on the essential oil of lippia rugosa from cameroon for its potential use as antifungal agent against aspergillus flavus link ex. fries, food control, 2: 161–166, (2009). [12]. de billerbeck v.g., roques c.g., bessière j.m., fonvieille j.l., dargent r., effect of cymbopogon nardus (l) w. watson essential oil on the growth and morphogenesis of aspergillus niger, canadian journal of microbiology, 47:9–17, (2001). [13]. adams r.p., identification of essential oil components by gas chromatography ⁄ mass spectrometry. allured publishing corporation (no. ed. 4) (2007), 804p. [14]. adjou e.s., amamion h., tchobo f.p., aissi v.m., soumanou m.m., extraction assistée par enzyme du jus de la pulpe fraîche du rônier (borassus aethiopum mart.) acclimaté au benin: caractérisation physico-chimique et microbiologique, international journal of biological and chemical sciences, 7(3): 11351146, (2013). [15]. bisla g., choudhary s., chaudhary v., evaluation of the nutritive and organoleptic values of food products developed by incorporated catharanthus roseus (sadabahar) fresh leaves explore their hypoglycemic potential, the scientific world journal id304120: 1-5, (2014). [16]. umaru h.a., adamu r., dahiru d., nadro m.s., levels of antinutritional factors in some wild edible fruits of northern nigeria, african journal of biotechnology, 6(16):19351938, (2007). [17]. barminas j.t., charles m., emmanuel d., mineral composition of non conventional leafy vegetables, plant foods for human nutrition, 53 (1): 29-36, (1998). [18]. degnon g.r., adjou e.s., noudogbessi j.-p., metome g., boko f., dahouenon-ahoussi e., soumanou m.m., sohounhloue d.c.k., investigation on nutritional potential of soursop (annona muricata l.) from benin for its use as food supplement against protein-energy deficiency, international journal of biosciences, 3(6): 1-10, (2013). [19]. gbohaïda v., mossi i., adjou e.s., agbangnan p., yehouenou b., sohounhloué d.c.k., morphological and physicochemical characterizations of cashew apples from benin for their use as raw material in bioethanol production, international journal of pharmaceutical research review and research, 35(2): 7-11, (2015). [20]. sultan y., magan n., mycotoxigenic fungi in peanuts from different geographic regions of egypt, mycotoxin research, 26:133–140, (2010). [21]. koutsoudaki c., krsek m., rodger a., chemical composition and antibacterial activity of the essential oil and the gum of pistacia lentiscus var. chia., journal of agricultural and food chemistry, 53: 7681– 7685, (2005). [22]. rao a., zhang y., muend s., rao r., mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the tor pathway. antimicrob, agents chemother, 54: 5062–5069, (2010). [23]. dorman h.j.d., deans s.g., antimicrobial agents from plants: antibacterial activity of plant volatile oils, journal of applied microbiology, 88: 308–316, (2000). [24]. bassolé i.h.n., lamien-meda a., bayala b., tirogo s., franz c., novak j., nebié r.c., dicko m.h., composition and antimicrobial activities of lippia multiflora moldenke, mentha spicata l. and ocimum basilicum l. essential oils and their major monoterpene alcohols alone and in combination, molecules, 15:7825–7839, (2010), [25]. hyldgaard m., mygind t., meyer r.l., essential oils in food preservation: mode of action, synergies and interactions with food matrix components, front microbiol, 3(12):1–24, (2012). [26]. sokovic m., marin p.d., brkc d., van griensven l.j.l.d., chemical composition and antibacterial activity of essential oils of ten aromatic plants against human pathogenic bacteria, food, 1: 220-226, (2007). [27]. kpadonou-kpoviessi b.g.h., yayiladekan e., kpoviessi d.s., gbaguidi f., yehouenou b., quetin-leclercq j., figueredo g., moudachirou m., accrombessi g.c., chemical variation of essential oil constituents of ocimum gratissimum l. from benin, and impact on antimicrobial properties and toxicity against artemia salina leach, chemistry & biodiversity, 9: 139–150, (2012). [28]. nielsen p.v., rios r., inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 euloge s. adjou, rené g. degnon, bertin a. gbaguidi, edwige dahouenon-ahoussi, mohamed soumanou, dominique c.k. sohounhloue, effect of essential oil of mentha spicata l. from benin on the quality of mango puree in storage model food systems at 4° and 25°c, food and environment safety, volume xvi, issue 3 – 2017, pag. 123-134 134 active packaging with special emphasis on mustard essential oil, international journal of food microbiology, 60: 219–229, (2000). [29]. vazquez b.i., fente c., franco c.m., vazquez m.j., cepeda a., inhibitory effects of eugenol and thymol on penicillium citrinum strains in culture media and cheese, international journal of food microbiology, 67: 157–163, (2001). [30]. lanciotti r., gianotti a., patrignani f., belleti n., guerzoni m.e., gardini f., use of natural aroma compounds to improve shelf life and safety of minimally processed fruits, trends in food science & technology, 15: 201–208, (2004). 1. introduction fig. 1. technological diagram for mango puree production [3]. hammer k.a., carson c.f., ridley c.v., antimicrobial activity of essential oils and other plants extract, journal of applied microbiology, 86: 985-990, (1999). [6]. sohounhloue d.k., dangou j., gnonhossou b., garneau f.x., gagnon h., jean f.i., leaf oils of three eucalyptus species from benin: e. torellina f. muel, e. citriodora hook and e. tereticornis smith., journal of essential oil research, 8: 111-113 ... [8]. konfo c.t.r., chabi n.w., dahouenon-ahoussi e., cakpo-chichi m., soumanou m.m., sohounloue ckd., improvement of african traditional sorghum beers quality and potential applications of plants extracts for their stabilization: a review, journal of ... 167 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 167 175 distribution of polycyclic aromatic hydrocarbons (pahs) in some charcoal roasted foods commonly consumed in port harcourt, nigeria virginia i. p. nitonye 1 , michael horsfall jnr. 1 , *mark o. onyema 1 1department of pure and industrial chemistry, faculty of science, university of port harcourt, port harcourt, nigeria, onyemark@yahoo.com *corresponding author received 21th june 2019, accepted 29th september 2019 abstract: sixteen (16) priority polycyclic aromatic hydrocarbons (pahs) were investigated in commonly consumed charcoal roasted foods prepared by roadside vendors in port harcourt, nigeria. charcoal roasted fish (cr-fh), meat (cr-mt), plantain (cr-pn) and yam (cr-ym) were homogenized, extracted, fractionated and for pah analysis, gas chromatography flame ionization detector (gc-fid) system was used. eleven (11) pahs were identified in cr-fh, 14 in cr-mt, 11 in cr-pn and 13 in cr-ym with total concentrations of 166.48 g/kg, 91.46 g/kg, 68.39 g/kg and 70.36 g/kg respectively. fluoranthene was observed to be the most prominent pah in all the charcoal roasted food samples, being the most abundant in cr-fh, cr-mt and cr-pn with the concentrations of 113.6 µg/kg (68.23%), 37.4 µg/kg (40.87%) and 39.8 µg/kg (58.21%) respectively and the next most abundant in cr-ym with a concentration of 17.8 µg/kg (25.36%). the compositions of pahs in the charcoal roasted foods revealed the 4-6 ring high molecular weight (hmw), from 97.21 98.75%, were considerably dominant over the 2-3 ring low molecular weight (lmw), from 1.25 2.79%, which were only minor. ratio values of pah isomers for phe/ant 0.05; fth/pyr, from 2.00 4.89 and baa/chr, from 0.60 17.63, indicate pahs in the charcoal roasted foods were pyrogenic and derived from the combustion of foods. from the results, charcoal roasting of fish, meat, plantain and yam prepared by roadside vendors and commonly consumed in port harcourt, nigeria contaminated the foods with pahs. keywords: charcoal roasted foods, pahs, fluoranthene, abundance, 4-6 ring hmw, port harcourt. 1. introduction the knowledge of the harmful effects of polycyclic aromatic hydrocarbons (pahs) has increased the concern about their presence in the environment [1], [2]. two major sources of pahs in the environment are petrogenic and pyrogenic. petrogenic sources include raw oil, while pyrogenic are from the incomplete combustion organic materials such as petroleum fuels (gasoline, kerosene, diesel and lubricating oil), coal, wood, tires, plastics, asphalt, incinerators and electronic waste [3-5]. more than 100 different pahs are known, but the united states environmental protection agency (usepa) has classified sixteen (16) as priority pollutants because they are toxic, mutagenic and carcinogenic in humans [6], [7]. this group of pahs consist of two to six aromatic rings fused together e.g. naphthalene, anthracene, benzo[a]anthracene, and benzo(a)pyrene. pahs are chemically stable, highly lipophilic and persist in the environment [8]. http://www.fia.usv.ro/fiajournal mailto:onyemark@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 168 food is one of the major routes by which humans are exposed to pahs. pahs in foods originate from environmental deposits or pyrolysis of organic substances like fats, carbohydrates and proteins which are at temperatures above 200oc. cooking techniques such as frying, drying, grilling, smoking and roasting generate and increase the level of pahs in foods [9-12]. investigation of various grilled and smoked foods (meat, chicken and vegetables) showed the presence of 16 priority pahs with phenanthrene having the highest mean concentration of 54.9 μg·kg−1, which accounts for 37.1% of the total pahs [13]. it was observed that conditions such as the type of heat source, duration of grilling and fat content influenced pahs formation in the foods. in milk and meat/fish based baby foods available on the italian market, 14 pahs were found to frequently occur and their average concentrations were higher than the permissible eu limits [14]. however, in polystyrene food contact materials lowring pahs were detected while the highring pahs (>4 rings) were not found. eight low-ring pahs were detected with phenanthrene and naphthalene having the highest average concentrations which collectively accounted for over 80% of the σ8pah total concentrations in all the samples [15]. the concentrations of 16 pahs in baked ready-to-eat cake, sausage roll, meat pie, burger and bread hawked in nigeria, showed that baked foods were mainly contaminated with 2-, 3-, and 4ring pahs [16]. this study is aimed at identifying pahs and their distributions in some commonly consumed foods (fish, meat, plantain and yam) in port harcourt, nigeria, prepared by charcoal roasting method. 2. matherials and methods 2.1. description of sampling area the location of sampling is port harcourt, situated in rivers state, southern nigeria. rivers state has tropical rain forest vegetation greatly blessed with aquatic and agricultural resources thus, the natives are predominantly fishers and farmers. the riverine communities are well-known for fishing, while the upland communities are noted for farming of livestock, and crops such as palm oil, yam, cassava, plantain, melon, banana and vegetables. port harcourt is a city in rivers state and a commercial nerve center. notable markets in the city include mile 1, mile 3, rumuokoro and oil mill. 2.2. sample collection and preparation foods used for this study are considered as ready-to-eat delicacies and are commonly consumed in port harcourt, rivers state. they include fish, meat, plantain and yam. the foods were obtained from the popular mile 3 market in obio/akpor local government area (l.g.a) of port harcourt. the food samples were each divided into two sets. the first set of samples were sent to roadside vendors within the campus of the rivers state university of science and technology (rsust), port harcourt, who prepared the foods in their usual way for consumption by charcoal roasting method. the second set was not prepared (raw) to serve as the control. the surface and near surface layers of the charcoal roasted fish (crfh), plantain (cr-pn) and yam (cr-ym) were cut off from the bulk, leaving the inner most parts, while the charcoal roasted meat (cr-mt) was not cut. all the food samples (charcoal roasted and raw) were air-dried for 4 days, homogenized, labelled appropriately and taken to laboratory for analysis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 169 2.3. sample extraction and clean up five (5) grams of each homogenized food sample was weighed out into a conical flask. 20 ml of hexane/dichloromethane (1:3 v/v) was measured out, poured into the conical flask, stirred, and agitated with a mechanical vibrator for 30 minutes for efficient extraction. the mixture was filtered and the filtrate concentrated by evaporation under a gentle stream of dry nitrogen in a fume cupboard. clean up of the concentrated extracts was achieved on a glass chromatographic column (25cm x 1cm) packed with activated silica gel (mesh 100-200) and 0.5g of sodium sulphate to absorb any moisture. hexane was poured onto the top of the column to elute the saturate fraction and dichloromethane poured to elute the aromatic hydrocarbons. the aromatic hydrocarbon fraction, which contains the pahs, was concentrated in a fume cupboard under a gentle stream of nitrogen. 2.4. polycyclic aromatic hydrocarbon (pah) analysis analysis of the polycyclic aromatic hydrocarbons in the food samples was achieved with an agilent 7890b gas chromatography (gc) system fitted with a hp-5 silica capillary column (30 m x 320 µm id and 0.25 µm film thickness) and coupled to a flame ionization detector (fid). one microlitre (1µl) of each cleaned up extract was introduced into the gc capillary column with the aid of a g4513a automatic liquid sampler (als) using the splitless injection mode. helium was used as the carrier gas and oven temperature for the analysis was programmed from 35 oc to 325oc at 10oc/min with 2 mins hold at 35oc and 10 mins hold at 325oc. pahs were identified by comparing their retention times with internal standard. quantification of each identified peak was acquired by area integration and processed by chemstation open lab cds edition. 3. results and discussion 3.1. distribution of pahs in the charcoal roasted foods gas chromatographic (gc) analysis of the charcoal roasted food samples revealed the presence of pahs, which were absent in the raw samples. the distributions of the pahs identified in the charcoal roasted food samples; fish (cr-fh), meat (crmt), plantain (cr-pn) and yam (crym), are shown in figures 1-4 respectively. total pahs identified in the food samples were 11 in cr-fh, 14 in cr-mt, 11 in cr-pn and 13 in cr-ym. the pahs identified in the food samples are: cr-fh, from anthracene to benzo[g,h,i]perylene; cr-mt, acenaphthylene, acenaphthene and from phenanthrene to benzo[g,h,i]perylene; cr-pn, anthracene to benzo[g,h,i]perylene and cr-ym, acenaphthene, fluorene and from anthracene to benzo[g,h,i]perylene (figures 1-4). this result indicate the charcoal roasted food with the most pahs identified is meat (cr-mt), followed by yam (crym), with fish (cr-fh) and plantain (crpn) having same number. profile of pahs fluoranthene was observed to be the most prominent pah in all the charcoal roasted food samples (figures 1-4). it is the most abundant pah in cr-fh, cr-mt and cr-pn with concentrations of 113.6 µg/kg, 37.4 µg/kg and 39.8 µg/kg, which constitute 68.23%, 40.87% and 58.21% of total pahs in the charcoal roasted food samples respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 170 fig. 1. gas chromatogram showing the distribution of polycyclic aromatic hydrocarbons (pahs) in charcoal roasted fish (cr-fh) sample. fig. 2. gas chromatogram showing the distribution of polycyclic aromatic hydrocarbons (pahs) in charcoal roasted meat (cr-mt) sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 171 fig. 3. gas chromatogram showing the distribution of polycyclic aromatic hydrocarbons (pahs) in charcoal roasted plantain (cr-pn) sample. fig. 4. gas chromatogram showing the distribution of polycyclic aromatic hydrocarbons (pahs) in charcoal roasted yam (cr-ym) sample. in cr-ym, dibenzo[a,h]anthrancene was the most abundant pah with a concentration of 19.4 µg/kg (27.52%) followed by fluoranthene with 17.8 µg/kg (25.36%). the total concentrations of pahs in the charcoal roasted food samples are shown in the figure 5. the total concentration of the 11 pahs identified in cr-fh was 166.48 g/kg, 14 pahs identified in cr-mt was 91.46 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 172 g/kg, 11 pahs identified in cr-pn was 68.39 g/kg and 13 pahs identified in cr-ym was 70.36 g/kg. the total pah concentrations decreased in the order crfh > cr-mt > cr-ym > cr-pn. when organic substances in foods are in contact with flame, pyrolysis generates pahs. but the highest concentrations of pahs are linked to the pyrolysis of fats, which melts, drips onto the heat source and the pahs generated deposits on the food being roasted as the smoke goes up [17]. consequently, the total pah concentration was highest in the charcoal roasted fish sample which is consistent with the pyrolysis of its fats and oil content. fig. 5. profiles of total pah concentrations of in the charcoal roasted food samples. cr-fh, charcoal roasted fish; cr-mt, charcoal roasted meat, cr-pn, charcoal roasted plantain; and cr-ym, charcoal roasted yam. source of pahs in the charcoal roasted foods pahs derived from different sources have characteristic composition which is used for source identification and differentiation in environment forensic [18]. compositions of pahs dominated by the low molecular weight (lmw) 2-3 ring are usually of raw oil source with the high molecular weight (hmw) 4-6 ring being minor components, but dominant in products from combustion (pyrolysis) of organic materials [19],[20]. in cr-fh, the lmw 2-3 ring pahs identified was anthracene; in cr-mt, acenaphthylene, acenaphthene, phenanthrene and anthracene; in cr-pn, anthracene and in cr-ym acenaphthene, fluorene and anthracene. however, ten (10) hmw 4-6 ring pahs from fluoranthene to benzo[g,h,i]perylene were identified in cr-fh, cr-mt and cr-pn, with only benzo[a]pyrene absent in cr-ym. total lmw pah concentrations were 2.98 g/kg, 2.08 g/kg, 1.91 g/kg and 0.88 g/kg in cr-fh, cr-mt, cr-pn and crym respectively. these lmw pah concentrations were significantly lower than the total hmw concentrations of 163.50 g/kg, 89.38 g/kg, 66.48 g/kg, and 69.48 g/kg respectively. abundance of lmw over hmw (>1) signifies a probable petrogenic (petroleum) origin for pahs, while the abundance of hmw are generally considered to indicate combustion source [20]. the pah concentrations in the charcoal roasted foods revealed the hmw 4-6 ring pahs, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 173 with compositions of 98.21%, 97.72%, 97.21%, and 98.75% were considerably dominant over the lmw 2-3 ring pahs, which were only minor components with compositions of 1.79%, 2.28%, 2.79% and 1.25% respectively. this indicates that pahs in all the charcoal roasted foods were derived from combustion source. ratios of pah isomers, phenanthrene (phe) and anthracene (ant), 3 rings; fluoranthene (fth), pyrene (pyr), benzo[a]anthracene (baa) and chrysene (chr), 4 rings, are employed as source type diagnostic parameters for environmental samples. calculated diagnostic ratios of the pah isomers are shown in the table 1. table 1. diagnostic ratios of polycyclic aromatic hydrocarbons employed for oil spill study lmw hmw phe/ant fth/pyr baa/chr cr-fh 2.98 163.50 n.d. 4.89 2.59 cr-mt 2.08 89.38 0.05 3.11 1.38 cr-pn 1.91 66.48 n.d. 3.23 0.60 cr-ym 0.88 69.48 n.d. 2.00 17.63 n.d. not determined phe/ant ratios for cr-fh, cr-pn and cr-ym were not determined because of the absence of phenanthrene in these samples (figs. 1, 3 and 4), while the ratio for cr-mt was 0.05. fth/pyr ratio for the charcoal roasted food samples ranged from 2.00 4.89 and baa/chr ratio ranged from 0.60 – 17.63 (table 2). these results reveal ant, fth and baa were dominant over their isomers, phe, pyr and chr. phe, pyr and chr are petroleum characteristic compounds derived from crude oil in which they are the dominant isomers, while ant, fth and baa are preferentially generated during combustion of organic materials [21]. phe/ant ratio >10 suggest crude oil (petrogenic) source for pahs, with degraded crude oils having ratios <10 and combustion (pyrogenic) products having ratios <1. fth/pyr ratios <1 suggest petrogenic origin and ratios >1 indicate pyrogenic processes, while baa/chr ratios for crude oils are < 0.02 with higher values indicating pyrogenic input from combustion of organic material [22]. from the ratios, the high abundance of the pyrogenic pahs (ant, fth and baa) in all the food samples indicated combustion process via charcoal roasting contaminated the foods with pahs. 4. conclusion gas chromatographic (gc) analysis of charcoal roasted fish (cr-fh), meat (crmt), plantain (cr-pn) and yam (cr-ym) commonly consumed in port harcourt, rivers state, nigeria, showed the presence of polycyclic aromatic hydrocarbons (pahs) in foods. the united states environmental protection agency (us epa) priority analyzed pahs in the charcoal roasted foods and 11 were identified in cr-fh, 14 in cr-mt, 11 in cr-pn and 13 in cr-ym. the total pah concentrations decreased in the following order: cr-fh > cr-mt > cr-ym > crpn. fluoranthene was observed to be the most prominent pah in all the charcoal roasted food samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 174 the compositions of the hmw 4-6 ring pahs (≥ 97.21%) were considerably dominant over the lmw 2-3 rings pahs (≤ 2.79%), which were only minor components. this indicates that pahs in all the charcoal roasted food samples were derived from combustion source. pah isomer ratios of phe/ant, fth/pyr and baa/chr, indicated pyrogenic source for pahs from the combustion of foods. 5. references [1].agency for toxic substances and disease registry (atsdr), toxicology profile for polycyclic aromatic hydrocarbons. public health service, u. s. department of health and human services, atlanta, georgia, (1995) assessed from https//www.atsdr.cdc.gov [november 7, 2016.] [2].bansal, v., kim, k., review of pah contamination in food products and their health hazards environment international 84: 26-38, (2015). [3].khalili, n.r., scheff, p.a., holsen, t.m., pah source fingerprints for coke ovens, diesel and gasoline engines, highway tunnels, and wood combustion emissions. atmospheric and environment, 29: 533-542, (1995). [4].hsu, w.t., liu, m.c., hung, p.c., chang, s.h., chang, m.b., pah emissions from coal combustion and waste incineration. journal of hazardous materials, 318: 32-40, (2016). [5].onyema, m.o., obasi, c.m., okoye, n.h. and osuji, l.c., distribution of polycyclic aromatic hydrocarbons in soils within vicinity of an electronic waste open burning workshop in aba, south east nigeria. international journal of sciences: basic and applied research (ijsbar), 35(1): 286-293, (2017). [6].united state environmental protection agency (us epa), polycyclic aromatic hydrocarbons (pahs) fact sheet. national center for environmental assessment, office of research and development, united state environmental protection agency, washington dc, (2008) assessed from http://www.epa.gov/osw/hazard/wastemin/minimiz e/factshts/pahs.pdf [august 12, 2015]. [7].silva, b.o., adetunde, o.t., oluseyi, t.o., olayinka, k.o., alo, b.i., effects of the methods of smoking on the levels of polycyclic aromatic hydrocarbons (pahs) in some locally consumed fishes in nigeria. african journal of foods science, 5(7): 384-391, (2011). [8].abdel-shafy, h.i., mansour, m.s.m., a review on polycyclic aromatic hydrocarbons: source, environmental impact, effect on human health and remediation. egyptian journal of petroleum, 25: 107-123, (2016). [9].knize m.g., salmon c.p., pais p., felton j.s., impact of processing on food safety. advances in experimental medicine and biology, 459: 179-193, (1999). [10]. ogbuagu, d.h., ayoade a.a., presence and levels of common polynuclear aromatic hydrocarbons (pahs) in staple foods of nigerians. food and public health, 2(1): 50 – 54, (2012). [11]. manda p., dano d.s., ehile e.s., koffi m., aman n., ass y.a., evaluation of polycyclic aromatic hydrocarbons (pahs) content in foods sold in abobo market, abidjan, côted' ivoire. journal of toxicology and enviromental health sciences, 4(6): 99 105, (2012). [12]. singh, l., varshney j.g., agarwal t., polycyclic aromatic hydrocarbons’ formation and occurrence in processed food. food chemistry, 199: 768-781, (2016). [13]. alomirah, h., al-zenki, s., alhooti, s., zaghloul, s., sawaya, w., ahmed, n., kannan, k., concentrations and dietary exposure to polycyclic aromatic hydrocarbons (pahs) from grilled and smoked foods. food control, 22(12): 2028-2035, (2011). [14]. santonicola, s., albrizio, s., murru, n., ferrante, m.c., mercogliano, r., study on the occurrence of polycyclic aromatic hydrocarbons in milk and meat/fish based baby food available in italy. chemosphere, 184: 467-472, (2017). [15]. li, s., ni, h., zeng h., pahs in polystyrene food contact materials: an unintended consequence. science of the total environment, 609: 1126-1131, (2017). [16]. iwegbue, c.m.a. concentrations and hazards of polycyclic aromatic hydrocarbons in hawked baked ready-to-eat foods in nigeria. acta alimentaria, 45(2): 175-181, (2015). [17]. center for food and safety (cfs), chemical hazard evaluation. food and environmental hygiene. 2004. assessed from: https://www.cfs.gov.hk [january 31, 2017]. [18]. zemo, d.a. 2009. use of parent polycyclic aromatic hydrocarbon (pah) proportions to attribute pah sources in sediments: a case study from the pacific northwest environmental forensics, 10(3): 229-239. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 virginia nitonye, michael horsfall jnr., mark onyema, distribution of pahs in charcoal roasted foods consumed in port harcourt, nigeria, food and environment safety, volume xviii, issue 3 – 2019, pag. 167 175 175 [19]. chen, b., xuan, x., zhu, l., wang, j., gao, y., yang, k., shen, x., lou, b. 2004. distributions of polycyclic aromatic hydrocarbons in surface waters, sediments and soils of hangzhou city, china. water resource. 38: 3558–3568. [20]. wang, z. 2009. oil weathering and chemical fingerprinting of spilled or discharged petroleum in the environment. ioc/westpac workshop, qingdao, china. [21]. peters, k., walters, c., moldowan, j., the biomarker guide: biomarkers and isotopes in petroleum systems and earth history. vol. 2, 2nd edition, cambridge university press, new york, (2005). [22]. wang, z., fingas, m., page, d.s., oil spill identification a review. journal of chromatography. a, 843: 369 411, (1999). 1. introduction microsoft word 6 ana leahu_fens 2_2017_final 31 iulie.docx 104 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2 2017, pag. 104 110 study on the acceptability of yoghurt with carrot juice 1ana leahu*, 1cristina e. hretcanu 1faculty of food engineering, stefan cel mare university of suceava, romania analeahu@fia.usv.ro *corresponding author received 15th april 2017, accepted 27th june 2017 abstract: in this paper we focus on the results of a study regarding the evaluation of the sensory characteristics and consumers’ acceptance of unsweetened yoghurt with carrot juice. the yoghurts were obtained considering three carrot juice levels (10%, 20% and 30%, respectively) and two starter cultures. carrot yoghurt was prepared in the laboratory by adding carrot juice to milk and inoculated with a 3% yoghurt culture. the effects of the carrot juice on the sensory quality and consumers’ acceptance of the yoghurt samples were determined. the data obtained on various parameters were statistically analyzed. keywords: yogurt, carrot juice, sensory quality, consumer acceptability, preference map. 1. introduction. yoghurt is a popular healthful dairy product obtained by fermentation of milk with lactic culture producing fermentation of lactose; the ph decreases due to lactic acid accumulation and coagulation of casein. yoghurt is a traditional product across the country and for all income segments of population and therefore, its production and consumption has increased in recent years in romania. the data delivered by the national institute of statistics for the years 2003 and 2015 show some evolution in terms of production of yoghurts which stands for a general upward trend in the analyzed interval [1]. moreover, yoghurt is a product with multiple health benefits since it contains viable bacteria that are considered probiotics and may help adjusting the microflora in the intestines. the addition of fruit or fruit mixtures, fruit flavors and sugar in yoghurt and yoghurt drink improves the nutritional and sensory properties [2]. several investigations into different additions of fruit, fibers and protein fortifying agents in yoghurt have been conducted. boycheva, s., et al., 2011, evaluate the effect of goat’s milk yoghurt supplemented by aronia and blueberry juice. the results obtained show that the addition of aronia and blueberry juices increased the amount of unsaturated fatty acids in yoghurt by 6.9% and 8.5%, respectively. polyunsaturated fatty acids increased by 11.2% in yoghurt with aronia juice in comparison with natural yoghurt [3]. hashim, i. b., et al., 2009 reported that an increase in the acceptance ratings of yoghurt fortified by up to 3% date fiber (df) was registered as compared with the control yoghurt [4]. a recent study has proposed the direct use of scalding water from quince fruits scalded in a set style yoghurt on yoghurt quality for 28 days of refrigerated storage [5]. the addition of banana marmalade [6], mulberry pekmez [7], ethanol extracts of four different grape varieties (cabernet sauvignon, chardonnay, shyrah, and merlot) and grape callus/cell culture [8], sour cherry pulp [9] has also been tested on dairy products. the addition of carrot juice and yoghurt produces a nutritionally balanced food because carrot is rich in beta carotene, ascorbic acid and tocopherol [10]. carrots (daucus carota) are important vegetables food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 105 in diets, being associated with their carotenoids, dietary fibers, vitamins, minerals and bioactive compounds content. thus, carrots are recommended for improving eyesight, to lower cholesterol and for skin care, in case of nervous disorders or indigestion [11]. in a study on the content of total polyphenols, ß-carotene and antioxidant activity in five varieties of carrots (jitka, kardila, katlen, rubína and koloseum), bystrická, j., 2015 and others have shown that total polyphenols were recorded in the variety of koloseum (113.69 ± 11.57 mg/kg), emphasizing that carrot is a rich source of carotenes [12]. daneshi, m., 2013 investigated the changes of ph, acidity, sedimentation and sensory quality of the milk/carrot juice drink. the scores allocated for colour, flavour, taste and consistency showed that during the first 5 days of the storage period all samples had the highest sensory acceptability [13]. the most important five characteristics of the products to be analyzed in this paper are: taste, color, appearance and consistency of the clove, fat content and whey. intensity of color, in the case of acidic dairy products, stands for the characteristic color of juice (in the case hereby for that of carrot). yellow may be interpreted as follows: the product is either too ripened and matured or it got altered, whereas a too intense color in the case of fruit products would mean a higher presence of artificial colorants or chemical chemical additives. flavor should vary according to the recipe and the sales package data; appearance and consistency of the coagulum: fine, compact or with a fluid consistency like fresh cream, visible coagulum particles are admitted. taste should be as natural as possible if it is simple, milk, not too pronounced (sour, acidified, pungent, ugly) but pleasant, characteristic, sour, refreshing, odorless or foreign taste. as regards whey, the less they contain, the fresher and better the acidic airy products are. thus, the aims of this study was to analyze new fruit yoghurts by adding carrot juice to milk and inoculated with a 3% freeze-dried starter yoghurt culture and to determine the effect of different concentrations of carrot juice on their sensory characteristics. 2. materials and methods. materials raw cows’ milk was obtained from a local milk dispenser in suceava (romania). fat content and ph of this fresh milk were 2.8 % and 6.56, respectively. the freezedried starter yoghurt culture (chr. hansen co. (denmark), fd-dvs chn-22, containing lactococcus lactis subsp. cremoris and flora danica, containing lactococcus lactis subsp. diacetylactis ware purchased from sc enzymes@derivates sa romania (costisa neamt). preparation of carrot juice mature carrot samples (cultivars suceava) were freshly harvested from the field in october 2016.the carrots were washed and manually peeled using a stainless steel knife prior to juicing in a robot type fruit squeezer. manufacture of yoghurt samples yoghurt was prepared in such a way to contain various concentrations of carrot juice of 10%, 20 % and 30% and it was inoculated by two types of culture. milk used for the yoghurt production was heated at 85°c for 30 min and then rapidly cooled to 43°c in order to inoculate the starter. after being cooled, the samples were inoculated with fd-dvs chn-22 type culture (noted as c1), the others were inoculated with flora danica type culture. the inoculation rate was of 3 % for all samples. once the starter was completely mixed, it was then incubated at 41-43°c for 3-4 hours to complete the preparation. the fermentation was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 106 considered completed when a ph of 4.64.7 was reached. for sensory descriptive testing, yoghurt samples were kept under normal refrigeration conditions (4°c) for 24 hours before assessments. table 1 depicts the different yoghurt samples by percentage of carrot juice and type culture included in the formulation. table 1. experimental design of yoghurt sensory study for eigh total treatments type of culture included composition of yoghurt cod control (0% carrot juice) p0 – c1 10% carrot juice p1 – c1 20% carrot juice p2 – c1 fd-dvs chn-22 (c1) 30% carrot juice p3 – c1 control (0% carrot juice) p0 – c2 10% carrot juice p1 – c2 20% carrot juice p2 – c2 flora danica (c2) 30% carrot juice p3 – c2 methods: sensory evaluation twenty semi-trained panelists regularly yoghurt consumers (60% female; 22 – 56 year old) were voluntarily recruited and instructed on how to perform the sensory evaluation by the staff and students of the faculty of food engineering, stefan cel mare university of suceava, romania. informed consent was obtained from all the participants. yoghurt samples were presented in opaque white plastic cups at room temperature (20 ± 1°c). all samples were marked with 3-digit codes, and the order of presentation of samples was randomized for each panelist. the panelists rated general appearance, taste, color, odor, flavor and texture (firmness) using a point hedonic scale (1 point for “dislike very much” and 7 points for „like very much”) [14]. the results of evaluation were written down by all panelists for every sample. statistical analysis data sets were evaluated using descriptive statistics for average ± standard deviation. means of sensorial characteristics were compared by using two-way analysis of variance (anova), in order to assess significant differences among samples. a two-way anova was used to assess the effects of carrot juice level (10%, 20%, 30%) and bacterial cultures (c1, c2). the threshold for significance was set at p<0.05. if significant differences between samples were found, then tukey’s hsd was used to determine which samples differed significantly. a biplot and an external preference mapping (prefmap) were represented for sensory and hedonic analysis of samples to compare and to evaluate the interrelationships between attributes for all 6 types of yoghurts (3 carrot juice levels: 10%, 20%, 30% and two cultures c1 and c2). a biplot from the principle component analysis (pca) is studied to interpret the position of the objects in prefmap as a function of the objective criteria. all the analyses were performed by using microsoft excel with xlstat™ (trial version, addinsoft, u.s.a.). 3. results and discussion for the sensory examination, we chose semi-trined panelists regular yoghurt consumers who, on the basis of the tasting sheet and the tasting of the samples presented in table 1, responded to the questions asked. during the sessions of analysis, the assessors analyze the sensory properties of each sample by objective evaluations. the results of evaluation were written down by every panelist for all samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 107 a. b. c. fig. 1. yoghurt samples: (a) control (0% carrot juice); (b) yoghurt with c1 (fd-dvs chn-22); (c) yoghurt with c2 (flora danica); where 10% (p1), 20% (p2) and 30% (p3) carrot juice, respectively the following table shows the sensory qualities for consumers’ acceptance of yoghurt with carrot juice. mean hedonic scores for general appearance, color, taste, odor, flavor and texture (mouth-feel) and overall liking can be found in the table 2. table 2. scores for sensory attributes for yoghurt samples with juice carrot (0%, 10%, 20%, 30%) and c1, c2 cultures yoghurt samples taste odor flavor texture (firmness) color appearance p0 – c1 5.20a±1.44 5.20a±1.44 5.15a±1.39 5.80a±1.11 5.80a±1.01 6.20a±0.81 p1 – c1 5.50b±1.36 5.40b±1.47 5.40b±1.47 5.40b±1.54 5.80a±1.06 5.80b±1.03 p2 – c1 5.50b±1.36 6.00c±1.03 6.00c±1.03 5.05c±1.73 5.70a±1.34 6.20a±1.03 p3 – c1 5.75c±1.37 5.80d±1.11 5.85d±1.09 4.55d±1.91 5.90a±1.17 6.10a±0.94 p0 – c2 4.95d±1.43 5.20a±1.44 5.15a±1.39 5.80a±1.11 5.95a±0.95 6.10a±0.83 p1 – c2 5.50b±1.36 5.40b±1.47 5.40b±1.47 5.50e±1.43 5.80a±0.95 5.80c±0.92 p2 – c2 5.50b±1.36 5.90c±1.02 6.00c±1.03 5.05c±1.73 5.40b±1.47 6.10a±1.09 p3 – c2 5.70c±1.42 5.75e±1.16 5.85d±1.09 4.60f±1.93 5.65c±1.42 6.10a±0.99 mean values ± standard deviation of scores (n = 20 assesors). different superscripts letters (a-f) within indicate statistically significant differences (*p <0.05) according to tukey’s test applied after anova. samples were evaluated on 7-point hedonic scale (1=dislike extremely and 7=like extremely). a two-way anova, with factors factor 1 (% of juice carrot added in yoghurt) and factor 2 (c1 and c2 culture) was performed on the sensory data and statistically significant differences (*p <0.05) was analyzed, using fcrit = 9.27. this revealed that the most apreciated taste is obtained by the sample p3 c1 and p3 – c2, which display significant differences (f = 20.82 , p = 0.016) as compared to the other samples. factor 2 does not have any influence on the taste of yoghurt samples (p = 0.29). regarding the scores for samples’ odor, the most appreciated samples were p2 – c1 and p2 – c2, which display significant differences (f = 204.6, p = 0.0006). factor 2 does not have any influence on the odor of yoghurt samples (p = 0.215). regarding the scores for samples’ flavor, the most appreciated samples were p2 – c1 and p2 – c2, which display significant differences (f = 83767·e11, p = 0.0000). factor 2 does not have any influence on the flavor of yoghurt samples (p = 0.215). the most apreciated texture (firmness) is obtained by sample p0 c1 and p0 – c2, which display significant differences (f = 485.36, p = 0.00015) as compared to the other samples. factor 2 does not have any influence on the taste of yoghurt samples (p = 0.29). the most apreciated color is obtained by sample p0 c1, which does not display significant differences (f = 1.740, p = 0.33) as compared to the other samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 108 factor 2 does not have any influence on the taste of yoghurt samples (p = 0.42). the overall acceptability (figure 2) of yoghurt samples with different percentages (10%, 20%, 30%) of carrot juice and two types of bacterial cultures (c1, c2) was determined on the basis of the average of the total score obtained for different sensory attributes (taste, odor, flavor, texture or firmness, color and appearance). figure 2 shows that the highest overall acceptability score was obtained by yoghurt with 20% carrot juce and culture c1 (p2-c1) which obtained a main scores of 5.74 points, followed by p3-c1 (yoghurt with 30% carrot juce and culture c1) and p2-c2 (yoghurt with 20% carrot juce and culture c2) which obtained a main scores of 5,66 on a scales of 7-point hedonic points (1 point for dislike extremely and 7 points for like extremely). fig. 2. overall acceptability of yogurt samples fig. 3. the mean scores for sensorial atributes of all yogurt samples mean values ± standard deviation of scores (n = 8 yoghurt samples). different superscripts letters (a-d) within indicate statistically significant differences (*p <0.05) according to tukey’s test applied after anova. samples were evaluated on 7point hedonic scale (1=dislike extremely and 7=like extremely). the control sample and carrot juice yoghurts (c1, c2) did not differ significantly one from another in terms of organoleptic characteristics mentioned above (f = 0.40, fcrit = 2.01) but the sensorial attributes (taste, odor, flavor, texture or firmness, color and appearance) have significant differences (f = 7.91, fcrit=2.23, p=0.02e-5). no interaction between samples and sensorial characteristics were found (f= 1.36, fcrit=1.43, p=0.07). figure 3 shows that the most appreciated sensorial attributes are: appearance, followed by color, flavor and odor, taste and texture. also, we remark that the type of culture (c1 or c2) did not influence the sensorial caracteristics of samples. the biplot for scores of yoghurt samples (mentioned in table 1) are shown in figure 4 (a), where one can evaluate the interrelationship between the sensorial attributes (taste, odor, flavor, texture or firmness, color and appearance). it was conducted using the mean sensory scores for the sensory attributes on the correlation matrix (table 3). we can remark that the texture is negatively correlate by taste (r = -0.880), the flavor (r = -0.859) and the odor (r = 0.828), respectively. moreover, the flavor is positively correlate by odor (r=0.994) and the taste (r=0.745). the total variance is given by the first two principal components (f1 and f2) for samples of yoghurt with different percentages (10%, 20%, 30%) of carrot juice and two types of bacterial cultures (c1,c2), respectively (fig. 4 a.). the first principal component (f1) provides the greatest amount of information, approximately 65.57% and the second (f2) contributes by 23.72% of the data distribution. also, the preference map for yoghurt samples is shown in figure 4 (b). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 109 table 3 correlation matrix (pearson (n)) for sensorial atributes of yoghurt samples variables appearance color taste odor flavor texture appearance 1 color -0.567 1 taste -0.133 -0.230 1 odor 0.411 -0.598 0.710 1 flavor 0.392 -0.630 0.745 0.994 1 texture -0.205 0.320 -0.880 -0.828 -0.859 1 values in bold are different from 0 with a significance level α=0.05 a. b. fig. 4 a. biplot b. preference map for yoghurt samples the main source of variance between the samples, as shown by the first principal component (f1), is given by the odor and flavor. the second principal component shows that the second most important sources of variance between the samples are apparence, taste and color. as shown by other authors [16], the changes in composition of yoghurt are strongly influenced by the addition of carrot juice in different percentages, this fact influenced the flavor, odor, apparence and color. the external preference mapping (prefmap) of yoghurt samples and sensorial properties provides a multidimensional representation ([16]) of yoghurt samples with carrot juice based on their sensory profile given by taste, flavor, odor, color, texture or apparencer, obtained through principle component analysis (pca) of a data matrix with products as rows and external data as variables or columns [17]. the analysis given by the preference mapping of yoghurt samples is to fit the consumers’ data in the sensory space, using a polynomial model to regress the hedonic scores obtained by samples on the coordinates of the products in the sensory space. a circular model, described by carroll [18], was used in this representation. the preference score for each object, whose value is between 0% and 100%, is calculated from the prediction of the model used. thus, we can remark that the most apreciated samples were p3-c2 and p2-c1, which have the position in the red area (which corespods to a rate of 80% 100%) and the most important factors are flavor and odor. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 ana leahu, cristina hretcanu, study on the acceptability of yoghurt with carrot juice, food and environment safety, volume xvi, issue 2 – 2017, pag. 104 – 110 110 4. conclusions yoghurt is a product with multiple health benefits given by viable bacteria. the addition of carrot juice in yoghurt can improve its nutritional and sensory properties. the mean hedonic scores for general appearance, color, taste, odor, flavor and texture (mouth-feel) and overall liking revealed that the most apreciated taste is obtained by sample p3 c1 and p3 – c2. regarding the scores for samples’ odor, the most appreciated samples were p2 – c1 and p2 – c2. using the scores for samples’ flavor, we have remarked that the most appreciated samples were p2 – c1 and p2 – c2 and the most apreciated texture (firmness) is obtained by sample p0 c1 and p0 – c2. flavor and odor are important factors for the acceptability of carrot juice yoghurt and the samples p3-c2 (yoghurt with 30% carrot juice and culture c2) and p2-c1 (yoghurt with 20% carrot juice and culture c2) satisfy the tasters' acceptability requirements. 5. references [1]. dobra i. b., sandru f. l., analytical overview on the romanian dairy market in the context of economic changes, academic journal of economic studies, 2(4), 172-186, (2016). [2]. cakmakci s., tahmas kahyaoglu d., erkaya t., cebi, k., hayaloglu a.a., β‐carotene contents and quality properties of set type yoghurt supplemented with carrot juice and sugar, journal of food processing and preservation, 38(3), 1155-1163, (2014). [3]. boycheva s., dimitrov t., naydenova n., mihaylova g., quality characteristics of yogurt from goat’s milk, supplemented with fruit juice, czech j. food sci, 29(1), 24-30, (2011). [4]. hashim i. b., khalil a. h., afifi h. s., quality characteristics and consumer acceptance of yogurt fortified with date fiber, journal of dairy science, 92(11), 5403-5407, (2009). [5]. trigueros l., pérez-alvarez j. a., viuda-martos m., sendra e., production of lowfat yogurt with quince (cydonia oblonga mill.) scalding water, lwt-food science and technology, 44(6), 13881395, (2011). [6]. çakmakçi s., çetin b., turgut t., gürses m., erdoğan a., probiotic properties, sensory qualities, and storage stability of probiotic banana yogurts, turkish journal of veterinary and animal sciences, 36(3), 231-237, (2012).. [7]. celik s., bakirci i., some properties of yoghurt produced by adding mulberry pekmez (concentrated juice), international journal of dairy technology, 56(1), 26-29, (2003). [8]. karaaslan m., ozden m., vardin h., turkoglu h., phenolic fortification of yogurt using grape and callus extracts, lwt-food science and technology, 44(4), 1065-1072, (2011). [9]. şengül m., erkaya t., şengül m., yildiz h., the effect of adding sour cherry pulp into yoghurt on the physicochemical properties, phenolic content and antioxidant activity during storage, international journal of dairy technology, 65(3), 429-436, (2012). [10]. salwa a.a., galal e.a., neimat a. e., carrot yoghurt: sensory, chemical, microbiological properties and consumer acceptance, pakistan journal of nutrition, 3(6), 322-330, (2004). [11]. leahu a., damian c., carpiuc n., oroian m., avramiuc m., change in colour and physicochemical quality of carrot juice mixed with other fruits, journal of agroalimentary processes and technologies, 19, 241-246, (2013). [12]. bystrická j., kavalcová p., musilová j., vollmannová a., tomáš t. ó. t. h., lenková m., carrot (daucus carota l. ssp. sativus (hoffm.) arcang.) as source of antioxidants, acta agriculturae slovenica, 105(2), 303-311, (2015). [13]. daneshi m., ehsani m. r., razavi s. h., labbafi m., effect of refrigerated storage on the probiotic survival and sensory properties of milk/carrot juice mix drink, electronic journal of biotechnology, 16(5), 5-5, (2013). [14]. hashim i. b., khalil a. h., afifi h. s., quality characteristics and consumer acceptance of yogurt fortified with date fiber, journal of dairy science, 92(11), 5403-5407, (2009). [15]. cliff m. a., fan l., sanford k., stanich k., doucette c., raymond n., descriptive analysis and early-stage consumer acceptance of yogurts fermented with carrot juice, j. dairy sci. 96:4160–4172, (2013). [16]. http://www.sensorysociety.org/knowledge/sspwiki/ pages/external%20preference%20mapping.aspx, accesed in march 2017. [17]. meullenet j-f., xiong. r., findlay, c., multivariate and probabilistic analyses of sensory science problems. ift press. blackwell publishing, ames, ia, (2007). [18]. carroll j.d., individual differences and multidimensional scaling. in r.n. shepard, a.k. romney, and s.b. nerlove (eds) multidimensional scaling: theory and applications in the behavioral sciences, vol 1 (pp. 105-155). new york: seminar press (1972). title …………………… 196 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 196 201 quinoa as a substitute for semolina: some aspects and problems of introduction sergiy boruk 1 , *igor winkler 2 , olga romanovska 3 , olga gerych 1 1yu. fedkovych national university of chernivtsi, ukraine 2bucovina state medical university, chernivtsi, ukraine 3chernivtsi institute of trade and economics, chernivtsi, ukraine *corresponding author: winkler@bsmu.edu.ua received 7th october 2017, accepted 19th december 2017 abstract: semolina is one of the most widely used kinds of finely ground cereals, which is easy to digest and assimilate. however, it consists of significant amount of heavy gluten which is not suited for people suffering from celiac diseases. besides, the content of proteins, vitamins and microelements in semolina is comparatively poor. quinoa is a pseudo-grain crop imported mostly from south america. quinoa can be considered as a substitute for semolina since it is rich in vitamins, proteins, amino acids and microelements, and targeted especially to the customers suffering from celiac disease, allergic to gluten or having special dietary requirements. it is important to investigate and compare some rheological properties of quinoa and semolina in order to ensure safe and effective interchanges in the technology of cereals production. it has been found that swelling of the quinoa mixtures is higher than that of the semolina mixtures. a rise in temperature causes initial increase in volume of the dispersed phase particles followed by fast destruction of the cereal disperse systems. viscosity of the quinoa mixtures shows a sharp rise when mass percent of the cereal exceeds 13 % while in case of semolina mixtures, the same phenomenon occurs after 16 %. a rise in viscosity is unwanted since it lowers the quality of the material used. it can be avoided to some extent by partial substitution of semolina with quinoa as 1:0.8. keywords: quinoa; semolina; mixtures viscosity; viscosimetry. 1. introduction semolina is a widely used kind of finely ground wheat cereals, which is popular due to its easy digestion and assimilation, low price and availability for any group of consumers. on the other hand, the proteins, vitamins and microelements contents in semolina are comparatively low [1]. moreover, it is not recommended for people under 2 and over 45 because of phytin, which causes decalcification and loss of magnesium. besides, its mixture with milk is highly caloric and may cause obesity or gaining extra weight. quinoa is a wheat-type pseudo-grain culture imported from south america. it is very valuable cereal with high content of vitamins, microelements, amino acids and proteins. the year 2013 was proclaimed in uno as “quinoa year” [2-5]. it is even called as “vegetable breast milk”. it is especially important because there is no gluten in quinoa [6-8]. nutritive values of both cereals are close (360 and 368 kcal/100 g) while the protein content in quinoa is somewhat higher as compared to that in semolina (14-23 g/100g vs 12.7 g/100 g respectively) [1, 6]. many food processing operations involve mechanical manipulation with materials http://www.fia.usv.ro/fiajournal mailto:winkler@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 sergiy boruk, igor winkler, olga romanovska, olga gerych, quinoa as a substitute for semolina: some aspects and problems of introduction, food and environment safety, volume xvi, issue 4 – 2017, pag. 196 – 201 197 (mixing, plasticizing and others). in this context, a nature of the cereal, its mechanical characteristics are the key factors governing quality of various confectionary items and half-finished products. for instance, pudding production requires only extra or first quality grade cereal like semolina. other substitutes can only be used in case they do not depreciate the final product’s consumer qualities. substitution raw materials can ensure some decrease in the product price and/or widen a circle of potential consumers by lifting some dietary restrictions. complete or partial substitution of the wheat flour is one of many ways to reach the above aims. however, some physicochemical parameters of the wheat and quinoa flours are significantly different (first of all, water absorbing capacities), fact which puts serious obstacles on the possibility of direct substitution of semolina with quinoa. different water absorbing capacities cause changes in the flour particles swelling, influence the distance between the particles, inter-particle gap width and, as a result, cause changes in the system’s internal friction (viscosity) value. therefore, a comparative analysis of water absorbing capacity values should be carried out for semolina and quinoa cereals in order to evaluate and elaborate changes in the corresponding food processing flowcharts. our paper addresses this problem as well as it investigates the influence of cereals’ concentration on the rheological conditions of their water suspensions. 2. experimental all investigations were carried out with different aqueous suspensions of quinoa and semolina. source cereals characteristics were the following. table 1. granulometric distribution of the source semolina particles size, μm content (%) 0 – 50 15,5 50 – 100 65,5 100 – 250 15,0 semolina grains: water content – 15 %, packing density 1.1 cm3/g, granulometric particles distribution – see table 1. quinoa cereals: water content 6.5 %, packing density 0.9 cm3/g, granulometric particles distribution – see table 2. table 2. granulometric distribution of the source quinoa particles particle size, μm content (%) 0 – 50 27.5 50 – 100 58.0 100 – 250 14.5 sedimentation cylinders were used to determine water absorbing capacity of the samples following the procedure: a sample was placed in a beaker and then 5-6 ml of water was added. the mixture was stirred with a glass stick until a uniform viscous mass was formed. if needed, 2-3 ml of water were added and then the mixtures were placed in the sedimentation cylinders, filled with water to the upper mark and left under fixed temperature. the sedimentation values were recorded in 0.5, 1, 2 and 12 hours. all these experiments were carried out at initial temperature 20, 50 and 70 0c. all systems were heated up to the corresponding temperatures and then they were naturally cooled down to room temperature. shearing stress value and viscosity were measured using the rotational viscosimeter rheotest-2, s and s3 cylinders sets and the following calculation formulas (1), (2) were used: τr = z × a, (1) where z – cylinder constant, din/cm2; a – reading of the shearing scale, τr – shearing stress value; food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 sergiy boruk, igor winkler, olga romanovska, olga gerych, quinoa as a substitute for semolina: some aspects and problems of introduction, food and environment safety, volume xvi, issue 4 – 2017, pag. 196 – 201 198 η = τr ÷ dr × 100, (2) where η – effective viscosity, pa·s, τr – shearing stress value, din/cm2, dr – shearing rate, s-1. 3. results and discussion as seen from the experimental data, the swelling degree of semolina (fig. 1) and quinoa (fig. 2) are significantly different. the swelling degree of quinoa is higher than that of semolina and this fact should be accounted by the substitution of the latter with the former. this difference is caused by the higher content of the smaller particles in quinoa cereals and lower water content in this grain. on the other hand, quinoa can be easily hydrolyzed causing the formation of white jellylike hydrolysis products shell on each grain. 0 2 4 6 8 10 12 14 16 3.3 6.6 10 13.3 16.6 20 disperse phase concentration, % d is p e rs e p h as e s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 20 ºс (293 к) 0 2 4 6 8 10 12 14 16 3.3 6.6 10 13.3 16.6 20 disperse phase concentration, % d is p e rs e p h as e s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 50 ºс (323 к) 0 5 10 15 20 25 3.3 6.6 10 13.3 16.6 20 disperse phase concentration, % d is p e rs e p h a se s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 70 ºс (343 к) fig. 1. swelling degree for semolina samples as seen from fig. 1, the maximum disperse phase sediment height is reached at 20 0c in 1 hour followed by gradual compacting of the sediment. in the beginning, the compacting runs slowly, then its rate accelerates. it can be seen visually that the sediment becomes non-uniform, the particles agglutinate and thin layers of the liquid appear in its bulk. the same tendencies were registered for the systems at 50 0c. when the temperature was of 70 0c, the maximum sediment height was reached food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 sergiy boruk, igor winkler, olga romanovska, olga gerych, quinoa as a substitute for semolina: some aspects and problems of introduction, food and environment safety, volume xvi, issue 4 – 2017, pag. 196 – 201 199 even in 30 min and then it compacted at comparatively high rate. some rise in the initial sediment volume with temperature is also evident. we believe that this effect is caused by faster formation of the particle shells at higher temperature. however, higher temperatures push up the rate of various microbes’ proliferation, which manifests itself through intense gas release conjugated with raise in the solution turbidity after 24 hours at the initial temperature 20 0c and after 12 hours – at 50 and 70 0c. 0 2 4 6 8 10 12 14 16 18 20 3. 3 6. 6 10 13 .3 16 .6 20 disperse phase concentration, % d is p e rs e p h a se s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 20 ºс (293 к) 0 2 4 6 8 10 12 14 16 18 20 3. 3 6. 6 10 13 .3 16 .6 20 disperse phase concentration, % d is p e rs e p h a se s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 50 ºс (323 к) 0 5 10 15 20 25 30 3. 3 6. 6 10 13 .3 16 .6 20 disperse phase concentration, % d is p e rs e p h a se s e d im e n t h e ig h t, c m 30 mi n 1 hour 2 hours 12 hours 70 ºс (343 к) fig. 2. swelling degree for quinoa samples the temperature swelling coefficient was calculated as a ratio between the system’s volume at the temperature t+10 divided by the ratio at the temperature t. these values for semolina were ~1.05 at the temperatures 20-50 0c and ~1.07 for the range 5070 0c. the same coefficients for the quinoa mixtures were 1.06 and 1.1 correspondingly, which is quite close to the semolina values. all these values are subject to change with time because of decomposition of the structures formed initially in the mixtures. that is why all the coefficients were determined after 30 min of keeping of the mixtures. it is unlikely that any mixfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 sergiy boruk, igor winkler, olga romanovska, olga gerych, quinoa as a substitute for semolina: some aspects and problems of introduction, food and environment safety, volume xvi, issue 4 – 2017, pag. 196 – 201 200 tures would be stored longer during real manufacturing. the concentration changes in viscosity of semolina and quinoa water suspensions are shown in the fig. 3. it can be seen that both values rise with concentration, which is a proof of dependence of the system’s viscosity on volumetric concentration of the disperse phase. 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 disperse phase concentration, % v is co si ty , p a * s fig 3. dependence of the semolina and quinoa water suspensions viscosity on their concentration at 293 k and shifting rate 27 s -1 . the dependences for other shifting rates were similar to that in fig. 3. the semolina suspension viscosity shows a sharp increase after its concentration exceeds 16 % (see fig. 3, bottom line) while the same jump occurs for the quinoa suspensions after 13 % (upper line in fig. 3). further increase in the disperse phase concentration causes rise in the dough viscosity of 0.8÷1.1 pa*s for each 10 % of concentration. this is an evidence of the stable coagulation structure based on the cereal particles or aggregates that appear in the system. therefore, both suspensions are stable against sedimentation and can self-recover after destruction. however, effective homogenization and stirring of such systems at viscosity over 2÷2.5 pa*s may require too much energy. therefore, it can be recommended to keep quinoa and semolina contents in the mixtures below 13 % and 16 % correspondingly. in the case of substitution of semolina with quinoa, this should be done according to the ratio 1 g of semolina for 0.8 g of quinoa. further investigation of the highly concentrated mixtures’ viscosity showed that it rose with concentration more slowly than it should in the case of complete structure that involves all disperse phase particles. in this case, some particles in the structure become its nodes while the others can get located in the interstices structural space. this distribution is dynamic and some interstice particles can become nodes and vice versa: disperse particle (structure node) ↔ disperse particles (interstice space) the interstice particles also participate in the inter-particle interaction that keeps distance fixed between the nodes even if their influence is less important than that of the node particles. low concentrated suspensions exhibit low viscosity but they are unstable and decompose easily and irreversibly. if the concentration is higher than critical, the system’s viscosity rises sharply with concentration while the system’s stability against external influences decreases. this can be caused by complete filling of all node locations at the critical concentration followed by filling the interstices at further increase in the particles content. interstice particles cause extra load on the formed structure and lower its stability. semolina quinoa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 sergiy boruk, igor winkler, olga romanovska, olga gerych, quinoa as a substitute for semolina: some aspects and problems of introduction, food and environment safety, volume xvi, issue 4 – 2017, pag. 196 – 201 201 4. conclusion it has been shown that swelling of the quinoa mixtures is more intense because of the higher content of fine-dispersed particles and lowers its water content. hydrolysis of the quinoa mixtures is also more intense than for the semolina compositions. an initial volume of the disperse phase particles increases with temperature while stability of the formed structures decreases. microbes’ functioning is the most influential factor of the systems’ decomposition. the critical concentration of semolina in water suspensions is of 16 % and for quinoa of 13 %. higher contents cause sharp increase in the systems’ viscosity. interchange between semolina and quinoa should be done according to the ratio 1 g of semolina for 0.8 g of quinoa. 5. references [1]. gost 7022.97. semolina: specifications and manufacturing conditions. http://rshn32.ru/files/n_gost-7022-97.pdf accessed october 02, 2017 (in russian). [2]. abugoch j., quinoa (chenopodium quinoa willd.): composition, chemistry, nutritional, and functional properties, elsevier inc., 31 (2009). [3]. cauda c., quinoa in the kitchen, borgaro. turin. 14-16 (2013) [4]. zong g., et al. whole grain intake and mortality from all causes, cardiovascular disease, and cancer: a meta-analysis of prospective cohort studies, circulation, 133(24): 2370-2380 (2016). doi: 10.1161/circulationaha.115.021101. [5]. garcia c. et al, use of quinoa extract as cosmetic and pharmaceutical slimming agent and/or as an agent preventing the formation of new fats in the human body. us paten applic. 20100061945. wo2008034989, publ. 27.03.2008 (2008). [6]. the 21-day weight loss breakthrough diet quinoa. http://www.doctoroz.com/recipe/21-dayweight-loss-breakthrough-diet-quinoa accessed oct. 01, 2017. [7]. gallagher e., gluten-free food science and technology, wiley-blackwell (2009). [8]. valencia-chamorro s. a., in: encyclopedia of food science and nutrition. ed.: caballero b., 4895-4902, academic press, amsterdam. (2003). http://rshn32.ru/files/n_gost-7022-97.pdf http://www.doctoroz.com/recipe/21-day-weight-loss-breakthrough-diet-quinoa http://www.doctoroz.com/recipe/21-day-weight-loss-breakthrough-diet-quinoa 1. introduction the temperature swelling coefficient was calculated as a ratio between the system’s volume at the temperature t+10 divided by the ratio at the temperature t. these values for semolina were ~1.05 at the temperatures 20-50 0c and ~1.07 for the range 50-... the concentration changes in viscosity of semolina and quinoa water suspensions are shown in the fig. 3. it can be seen that both values rise with concentration, which is a proof of dependence of the system’s viscosity on volumetric concentration of t... fig 3. dependence of the semolina and quinoa water suspensions viscosity on their concentration at 293 k and shifting rate 27 s-1. the dependences for other shifting rates were similar to that in fig. 3. the semolina suspension viscosity shows a sharp increase after its concentration exceeds 16 % (see fig. 3, bottom line) while the same jump occurs for the quinoa suspensions afte... therefore, it can be recommended to keep quinoa and semolina contents in the mixtures below 13 % and 16 % correspondingly. in the case of substitution of semolina with quinoa, this should be done according to the ratio 1 g of semolina for 0.8 g of qui... further investigation of the highly concentrated mixtures’ viscosity showed that it rose with concentration more slowly than it should in the case of complete structure that involves all disperse phase particles. in this case, some particles in the st... disperse particle (structure node) ↔ disperse particles (interstice space) the interstice particles also participate in the inter-particle interaction that keeps distance fixed between the nodes even if their influence is less important than that of the node particles. low concentrated suspensions exhibit low viscosity but they are unstable and decompose easily and irreversibly. if the concentration is higher than critical, the system’s viscosity rises sharply with concentration while the system’s stability against ... 4. conclusion 375 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 4 2015, pag. 375 384 studies regarding the photocatalytic transformation of erythrosine b *laura carmen apostol1,2, anca florentina caliman2, maria gavrilescu2,3 1”stefan cel mare” university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania; laura.apostol@fia.usv.ro 2“gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof.ph d.docent d. mangeron street, 700050 iasi, romania 3academy of romanian scientists, 54 splaiulindependentei, ro-050094 bucharest, romania *corresponding author received november 26th 2015, accepted december 16th 2015 abstract: heterogeneous photochemical processes using semiconductors as a photocatalyst have gained wide interest in the treatment of dyes effluents from industries, where biological methods did not succeed, i.e. especially because of dyes toxicity on sludge. experiments concerning the photocatalytic degradation of erythrosine b xanthene dye were conducted in the presence of different semiconductor catalysts (tio2 p-25, tio2-rutile, zro and sb2o3). the experiments showed that tio2 p-25 exhibited the best removal efficiency. photocatalytic degradation rate decreased from 0.9 to 0.01 mg l -1 min -1 when erythrosine b concentration increased from 30 mg l -1 to 300 mg l -1 . the addition of hydrogen peroxide leads to increase of the reaction rate from 0.06mgl -1 min -1 to 0.14mgl -1 min -1 . photocatalytic degradation of erythrosine b led to a complete detoxification of compounds, but the complete mineralization was not achieved. keywords: food dye degradation, reaction rate, slurry reactor 1. introduction synthetic organic pollutants in wastewater can generate serious environmental problems in the aquatic system when these pollutants are improperly disposed and treated. the wastewater containing dyes can induce eco-risk, beside the dyes themselves, by toxic by-products which can be formed naturally trough oxidation or reduction pathways. to overcome the problems that dye effluents can induce when they are released into receivers, a wide range of biological and chemical treatments have been applied in order to decrease the environmental effects that can appear in these conditions. advanced oxidation processes (aop) represent a chemical procedure designed to remove particularly the toxic and nonbiodegradable organic pollutants from wastewater. the objective of the wastewater treatment using aop is the minimization of the pollutants concentration and toxicity so that the cleaned wastewater may, nevertheless, be dumped into a conventional sewage treatment [1]. heterogeneous photochemical processes using semiconductor as a photocatalyst have gained wide interest in the treatment of dyes effluents from industries, where biological methods did not succeed, especially due to dyes toxicity on sludge [2, 3]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 376 the method can result in the mineralization of the hazardous pollutants rather than transferring them to another phase, such as in the case of adsorption on different materials [4, 5]. biodegradation of recalcitrant dyes is an inefficient method due to the dyes resistance to reductive processes [6, 7]. the majority of the researchers reported that the process can generate the break of the dye, but rarely complete mineralization can be achieved in an economically and environmental friendly ways [8]. from the semiconductors tested by different researchers, the statistics showed that tio2 has received the biggest interest for wastewater purification [9]. the conversion of organic pollutants by tio2 p-25 represents an effective and economical method due to the rapidity of its electron transfer to molecular oxygen for decomposition of pollutants, chemical stability and nontoxicity [10]. the biggest challenge of tio2 as a photocatalyst is its wide band gap, which limits its photoactivity under visible light and the work in slurry system. erythrosine b is authorised as a food additive in the eu and is a water-soluble dye of xanthene class used in different industries such as a colorant in foodstuffs (baked goods, fruits cocktails), cosmetics (lipsticks, bubble baths) or drugs (tablet, capsule). the large majority of the dyes from this class are toxic, carcinogenic or mutagenic for humans and animals [11]. the amount of erythrosine b used in food products is between 150 and 200 mg kg-1 [12, 13]. hasnat et al. published in 2007 two papers containing preliminary studies regarding erythrosine b photodegradation [14, 15]. in their papers it is presented the influence of two catalysts: tio2 and zno on erythrosine b photodegradation for a low dye concentration comparing with the concentration used in the industries starting with aspects regarding the adsorption of the dye on the catalysts. erythrosine b (ery b) has a complex chemical structure which showed to be toxic for the mixed culture of microorganisms (i.e. anaerobic granular sludge) [2]. the purpose of this study was to to evaluate further the photocatalytic degradation of erythrosine b, the influence of uv in the presence of the catalyst degusa tio2 aeroxide p25, on erythrosine b removal from aqueous solution was tested. experiments were conducted to evaluate the influence of different parameters (catalyst type, ph, catalyst loading, initial dye concentration, chargetrapping species h2o2 and reactor type) on the removal efficiency of the dye. 2. materials and methods 2.1. materials the compound investigated in this study was chosen because of its use in different industries despite the toxic and carcinogenic effects. the erythrosine b is a red odourless powder or granules with a calculated log p (octanol-water) of 4.95 at 25°c [16], soluble in water (≤ 9% w/w) and ethanol. the chemical was purchased from sigma–aldrich (c.i. acid red 51, 45430). a stock solution of 1000 mg l-1 was prepared by dissolving an accurately weighed amount of erythrosine b in 500 ml distilled water. for the experimental assays, dilution of the stock was done to achieve the desired concentration. the semiconductor used in this study degusa tio2aeroxide p25 was purchase from aerosil; the physico chemical properties are presented in table 1. 2.2. experimental procedures and analysis the experiments were conducted in a close pyrex cell, with a capacity of 500 ml, (9 cm diameter, 15 cm height) provided with hole for bubbling air (fig. 1). the reaction mixture was maintained in suspension using a magnetic stirrer and the pumped air. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 377 table 1. physico-chemical data for degusa tio2 aeroxide p25 (cas no. 13463-67-7) properties unit typical value behavior towards water hydrophilic specific surface area (bet) m2/g 50 ± 15 average primary particle size nm 21 tapped density g/l 130 ph 3.5 – 4.5 in order to achieve the sorption equilibrium of the dye onto the semiconductor catalyst surface the mixture was kept in dark for 15 minutes. the irradiation was performed with a 9w central lamp. the spectral response of the irradiation source (osramdulux s 9w/78 uv-a) according to the producer is ranged between 350 nm and 400nm with a maximum at 366nm and two additional weak lines in the visible region [17, 18, 19]. the photon flow per unit volume of the incident light was determined by chemical actinometry using potassium ferrioxalate [20]. the initial light flux, under exactly the same conditions as in the photocatalytic experiments, was evaluated to be 6.46×10−4einstein l−1 min−1[17, 21]. fig. 1.photocatalytic reactor used in the study: 1. reactor (v=500 ml); 2. pump for air bubbling; 3. magnetic stirrer; 4. osram dulux s 9w/78 uv-a type lamp specific quantities of samples were withdrawn at periodic intervals and filtered through a 0.2 μm filter in order to remove the catalyst particles. in order to assess the extent of color removal, changes in the concentration of the dye were observed from its characteristic absorption band using a t60 uv–vis spectrophotometer. solutions of h2so4 0.1 n and naoh 0.1 n, respectively were used to adjust the solution ph. the ph values of the solution were monitored with a hanna ph-meter; the reactions were conducted at room temperature (~27ºc). the degree of erythrosine b removal as function of time is gived by eq. (1):  removal b eerythrosin % 100 c c -1 0         (1) where c0 and c are the dye concentration at initial and any subsequent reaction time (mg l-1). the photocatalytic degradation rate of most organic compounds is described by first order kinetics. direct photocatalyitc pathway can be described using langmuir hinshelwood (l – h) equation, which can be applied for the heterogeneous photocatalys is based on the formation of electrons and holes by catalyst light activation. the hole is entrapped by the adsorbed dye molecule on the catalyst surface to form a reactive radical state, which can decay as a result of recombination with an electron [1]. langmuir-hinshelwood expression is given by eq. (2): (2) ckkkr arr 111  food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 378 where r is the reaction rate of the oxidation of reactant (mg l-1 min-1), kr is the specific reaction rate constant for the oxidation of the reactant (mg l-1 min-1), ka is the equilibrium constant of the reactant l mg1, c is the dye concentration. when the pollutant concentration c0 is a millimolar solution, the equation can be simplified to an apparent first-order equation (3): ln(c0/c)=kkt=kappt or c=c0exp(kappt) (3) a plot of lnc0/c versus time represents a straight line, the slope of which upon linear regression equals the apparent first – order rate kapp. the toxicity tests were conducted in triplicate using 20 seeds for germination on each petri dish. 3 ml solution of 25 mg l1ery b, 50 mg l-1ery b, and the corresponded degradation products, respectively were used to wet the supported paper of the seeds. the petri dishes were kept at room temperature (~25ºc) for 72 hours. the number of seeds germinated, formed biomass, stem length and root length were measured to determine the germination degree. 3. study of factors influencing the heterogeneous photocatalytic process 3.1. effect of photocatalyst type on dye degradation experiments concerning the photocatalytic oxidation of erythrosine b were conducted in the presence of different semiconductor catalysts. results of the photolysis of 50 mg l−1 dye solution containing 0.5 g l−1 tio2 p-25, tio2-rutile, zro and sb2o3, respectively are presented in fig. 2, where the amount of the organic molecule present in the supernatant is plotted as a function of irradiation time. tio2 p-25 exhibited the best results. under the experimental conditions and in the presence of 0.5 g l−1 tio2 p-25, 50 mg l-1 erythrosine b was complete removed after 60 min. in the presence of other catalysts, such us tio2-rutile, zro and sb2o3, the degradation was a very slower process, considering the fact that after 120 min of irradiation, around 70% of dye initial concentration remained in solution when tio2-rutile, zro and sb2o3 catalysts were used. hasnat et al. (2007) [14] and uddin et al.(2007) [15] obtained also low decolorization efficiency for ery b with zno as catalyst. 0 20 40 60 80 100 120 140 0 10 20 30 40 50 e ry th ro s in e b ( m g l -1 ) time (min) tio 2 p25 tio2 rutile zro sb 2 o 3 fig. 2. influence of catalyst type on erythrosine b photodegradation (natural ph = 5.6) 3.2. effect of catalyst concentration on dye degradation the effect of tio2 p-25 on the degradation kinetics of ery b was investigated employing different concentration of catalyst: 0.25, 0.35, 0.5, 0.75 and 1 g l-1. photodegradation of dye increased rapidly with increasing the amount of tio2. hasnat et al. (2007) [14] specified that the optimum tio2 loading was 2.0 g/l taking into account just the degradation rate of 22 μm ery b and ignoring the degradation efficiency; the irradiation time is not specified. in this study the optimum catalyst concentration was established at 0.5 g l1for 30 min of irradiation. for higher values, the removal percentage remained food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 379 constant (100%) and the degradation rate value increased (figs. 3 and 4). the increase in the amount of catalyst rises the number of active sites on the tio2 surface, which widens the number of oh and o2 radicals that conducted at a growing of the degradation rate. -20 0 20 40 60 80 100 120 140 0 10 20 30 40 50 e ry th ro s in e b ( m g l -1 ) time (min) 0.25 g l -1 0.35 g l -1 0.5 g l -1 0.75 g l -1 1 g l -1 fig. 3. influence of catalyst concentration on erythrosine b (ci= 40 mg l-1) photodegradation on time 0.0 0.3 0.6 0.9 1.2 0.00 0.05 0.10 0.15 0.20 0.25 r e m o v a l e ff ic ie n c y ( % ) [tio 2 p25] (mg l -1 ) k ( m g l 1 m in 1 ) 0 20 40 60 80 100 fig. 4. effect of different tio2 p25 concentration (30 min of irradiation) on erythrosine b (ci=40 mg l-1) degradation 3.3. effect of pollutant concentration on dye degradation the photocatalytic degradation of different concentration of ery b has been investigated in aqueous suspension of tio2 as a function of irradiation time (fig. 5). 0 30 60 90 120 150 180 210 240 270 300 330 0 30 60 90 120 150 180 210 240 270 300 [e ry th ro s in e b ] (m g l -1 ) irradiation time (min) 30 mg l -1 ery b 40 mg l -1 ery b 50 mg l -1 ery b 80 mg l -1 ery b 100 mg l -1 ery b 150 mg l -1 ery b 200 mg l -1 ery b 300 mg l -1 ery b fig. 5. influence of irradiation time on initial erythrosine b concentration on dye photodegradation by 0.5 g l-1 tio2 p25 the rate of dye photodegradation was investigated by varying the pollutant concentration from 30 mg l-1 to 300 mg l1. it was observed that the photocatalytic degradation rate decreases from 0.9 to 0.01 mg l-1 min-1 because the concentration of active site remained the same for a fixed catalyst, the number of dye molecules adsorbed on tio2 surface increased and free radicals reactivity decreases (fig. 6). 0 50 100 150 200 250 300 0.00 0.02 0.04 0.06 0.08 0.10 k ( m g l -1 m in -1 ) [erythrosine b] (mg l-1) fig. 6. rate of photocatalytic degradation of erythrosine b by tio2 p25 (0.5 g l-1) hasnat et al. (2007) [14] found that the decolorization mechanism of ery b under their reaction condition followed zeroorder kinetics over tio2/zno surface. in our case, the reaction followed first order food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 380 kineticsfor a higher dye concentration and a higher amount of oxygen (provided by the bubbled air beside the magnetic steered). 3.4. effect of initial ph on dye degradation the ph value is an important operational variable in wastewater treatment. in photocatalysis systems, ph value is also one of the factors influencing the rate of degradation. different phs were tested in order to evaluate the effect of this parameter on dye degradation. the role of ph on the rate of photocatalytic degradation of ery b was studied in the rage of 5 – 9 for 50 mg l-1ery b (fig. 7). -20 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 [e ry th ro s in e b ] (m g l -1 ) ph i =5.27 ph i =6.34 ph i =7.25 ph i =8.56 irradiation time (min) fig. 7. influence of initial ph of erythrosine b (50 mg l-1) on dye photodegradation the solution ph was adjusted initially and no buffered system was used to maintain constant ph during the reaction. the ph variation in time was also monitored (fig. 8). it can be observed that after the sorption process the ph tends to stabilize in the range of neutral field. when the photodegradation process started the ph values decreased with around two ph unit due to the products released after dye degradation. it was observed that the degradation rate decreased with increasing in ph (fig. 10). since adsorption became strongly around zero charge point of tio2 the maximum reaction rate was obtained for the ph values closely to the point of zero charge (pzc). also, it can be estimated that because the increasing of color intensity the ph increases, the solution opacity increases and the light penetration become more difficult. hasnat et al. (2007) [14] and uddin et al. (2007) [15] found that, with increasing of the medium ph from 5.0 to 7.0 the degradation rate, determined for the zero order reaction, increased and remained steady up to 9.0. -30 0 30 60 90 120 150 180 210 4 5 6 7 8 9 ph i =5.27 ph i =6.34 ph i =7.25 ph i =8.56 p h irradiation time (min) fig. 8. variation of the ph of the solution in photocatalytic degradation of erythrosine b 0 10 20 30 40 50 0 1 2 3 ph 5 ph 6 ph 7 ph 8 ln ( c 0 /c ) irradiation time (min) fig. 9. firstorder kinetics of the photocatalytic degradation of ery b at different phs 3.5. influence of charge-trapping species – h2o2 on dye degradation the addition of strong powerful oxidizing agents, such as hydrogen peroxide, to tio2 suspensions leads in many cases to an increase in the photooxidation rate through food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 381 generation of more radicals in the presence of light. the role of h2o2 in the process of photocatalytic degradation is dual [22]. it accepts a photogenerated electron from the conduction band and thus promotes the charge separation and it also forms oh radicals via superoxide. 4 5 6 7 8 9 0.00 0.01 0.02 0.03 0.04 0.05 k ( m g l 1 m in 1 ) ph fig. 10. rate of photocatalytic degradation of erythrosine b different initial phs by tio2 p25 a possible reaction of h2o2 with the photogenerated intermediates also cannot be excluded. when h2o2 is in excess, it may act as holeor oh radical dot scavenger or react with tio2 and form peroxo compounds that are detrimental to the photocatalytic action. this explains the need for an optimal concentration of hydrogen peroxide for the maximum effect [23 25]. the photocatalytic activity increases with increasing the amount of hydrogen peroxide until it reaches a maximum at the concentration of 200 mg l−1 oxidant (fig. 11). table 2. erythrosine b degradation rate obtained for different h2o2 concentration h2o2 concentration (mg l-1) reaction rate (mg l-1 min-1) correlation coefficient (r2) 0 0.05417 0.9978 20 0.06132 0.9932 50 0.0672 0.9824 100 0.09359 0.9977 200 0.1479 0.9963 the process removal efficiency increases from 50%, for the assay without h2o2, to 98 % for the analysis conducted in the presence of 200 mg l-1 oxidizing agent. the effect of the amount of h2o2 on the initial reaction rates, calculated for the first 30 min of reaction, is presented in table 2. under the studied conditions, the addition of hydrogen peroxide accelerates the degradation by 3 times comparative with the assay without h2o2. a relative high positive effect was obtained in the presence of the of charge-trapping species – h2o2 considering that the degradation rate increased from 0.06 mg l-1 min-1 to 0.14 mg l-1 min-1 for an increasing of h2o2 concentration from 25 mg l -1 to 200 mg l-1. 0 50 100 150 200 40 60 80 100 r e m o v a l e ff ic ie n c y ( % ) [h2o2] (mg l -1 ) fig. 12. removal efficiency of dye obtained for different h2o2 concentration 0 10 20 30 40 50 60 0 1 2 3 4 5 20 mg l -1 h 2 o 2 50 mg l -1 h 2 o 2 100 mg l -1 h 2 o 2 200 mg l -1 h 2 o 2 ln ( c 0 /c ) irradiation time (min) fig. 13. firstorder kinetics of the photocatalytic degradation of ery b in the presence of different h2o2 concentration food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 382 the results are in concordance with those of hasnat et al. (2007) [14] that explained the phenomena based on the activity of •oh radicals that are the major oxidant for dye decolorization. therefore, the excess •oh radicals added to the system can accelerate the decolorization rate. fig. 14. time-dependent uv-vis absorption spectra for decolorization of erythrosine b 4. cod and toc data during erythrosine b degradation by heterogeneous photocatalysis. products analysis the reduction of chemical oxygen demand (cod) reflects the extent of degradation or mineralization of an organic species (eq. 4). therefore the percentage change in cod was studied for 50 mg l-1 erythrosine b under optimized conditions (catalysts dose 0.5g l-1, ph = 5) as a function of irradiation time using uv (table 3). the percentage of cod removal was 22 %, after 2 h concentrated uv irradiation treatment. c20h6i4na2o5 + 18 o2 20co2 + 4h + + 4i+ 2na+ + h2o (4) table 3. cod and toc value for aqueous solution photodegradation containing erythrosine b in the presence of tio2 irradiation time (min) cod (mg l-1) 0 55.6 5 54.3 10 52 20 49.6 30 46.1 60 43.3 120 39.2 4. conclusions the experiments on the photocatalytic degradation of erythrosine b xanthene dye conducted in the presence of different semiconductor catalysts (tio2 p-25, tio2rutile, zro and sb2o3) showed that tio2 p-25 exhibited the best removal efficiency. photocatalytic degradation rate decreased from 0.9 to 0.01 mg l-1 min -1 when erythrosine b concentration increased from 30 mg l-1 to 300 mg l-1, because of new picks after 0.5 1h of irradiation initial erythrosine b picks (0 -30 min) (*20 dillution) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 383 the given catalyst concentration (1 g l-1), active site number being constant, the number of dye molecules adsorbed on tio2 surface increased and free radicals reactivity decreased. the addition of 20 and 200 mg l-1 hydrogen peroxide led to an increase of reaction rate from 0.06 mgl-1min-1 to 0.14 mgl-1min-1. the presence of chargetrapping h2o2 decreased the irradiation time with 30 min required to achieve a removal efficiency of 99% to 69% in the absence of added h2o2. photocatalytic degradation of erythrosine b led to a complete detoxification of the compounds but the complete mineralization was not achieved. 5. acknowledgment the authors would like to thank brain project id 6681, financed by the european social found and romanian government. 6. references [1].rauf m.a., ashraf s.s., fundamental principles and application of heterogeneous photocatalytic degradation of dyes in solution, chemical engineering journal, 151, 10–18, (2009). [2].apostol l.c., pereira l., pereira r., gavrilescu m., alves m.m., biological decolorization of xanthene dyes by anaerobic granular biomass, biodegradation, 23, 725–737, (2012). [3].pereira l., pereira r., oliveira c.s., apostol l.c., gavrilescu m., pons m.n., zahraa o., alves m.m., uv/tio2 photocatalytic degradation of xanthene dyes, photochemphotobiol., 89, 33–39, (2013). [4].satuf m.l., pierrestegui m.j., rossini l., brandi r.j., alfano o.m., kinetic modeling of azo dye photocatalytic degradation in aqueous tio2 suspention. toxicity and biodegradability evaluation, catalysis today, 161, 121-126, (2011). [5].apostol l.c., gavrilescu m., bean husk used for erythrosine b removal, the 14th international symposium on cellulose chemistry and technology, iasi, romania, (2010). [6].pereira l., coelho a.v., viegas c.a., dos santos m.m.c., robalo m.p., martinsa l.o., enzymatic biotransformation of the azo dye sudan orange g with bacterial cota-laccase, journal of biotechnology, 139, 68–77, (2009). [7].van der zee f.p., cervantes f.j., impact and application of electron shuttles on the redox (bio)transformation of contaminants: a review, biotechnol. adv., 27, 256–277, (2009). [8].kumar p., photocatalytic degradation of procion yellow dye, master of technology thesis, thaparuniversity, india, (2010). [9].akpan u.g., hameed b.h., parameters affecting the photocatalytic degradation of dyes, using tio2-based photocatalysts: a review, journal of hazardous materials, 170, 520–529, (2009). [10]. luenloi t., chalermsinsuwan b., sreethawong t., hinchiranan n., photodegradation of phenol catalyzed by tio2 coated on acrylic sheets: kinetics and factorial design analysis, desalination, 274(1-3), 192199, (2011). [11]. dees c., foster j.s., ahamed s., wimalasena j., dietary estrogens stimulate human breast cells to enter the cell cycle, environ health perspect, 105, 633–636, (1997). [12]. food standards, initial assessment report, application a603, red 3 erythrosine in food colouring preparations, on line at: http://www.foodstandards.gov.au/standardsdeve lopment/, (2008). [13]. european parliament and council directive 94/36/ec of 30 june 1994 on colours for use in foodstuffs. 10.09.1994, 13. [14]. hasnat m.a., uddin m.m., sameda a.j.f., alama s.s., hossain s., adsorption and photocatalyticdecolorization of a synthetic dye erythrosine on anatase tio2 and zno surfaces, journal of hazardous materials, 147, 471–477, (2007). [15]. uddin m.m., hasnat m.a., samed a.j.f., majumdar r.k., influence of tio2 and znophotocatalysts on adsorption and degradation behaviour of erythrosine, dyes and pigments 75, 207 – 212, (2007). [16]. european food safety authority, scientific opinion on the re-evaluation of erythrosine (e 127) as a food additive. reevaluation of erythrosine (e 127) as a food additive, efsa journal, 9(1), 1854, (2011). [17]. caliman f.a., antoniadis a., poulious i., macoveanu m., kinetic study on heterogeneous photocatalytic degradation of reactive orange 16 into a sllury reactor, environmental engineering and management journal, 5, 649 – 659, (2006). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 laura carmen apostol, anca florentina caliman, maria gavrilescu, studies regarding the photocatalytic transformation of erythrosine b, food and environment safety, volume xiv, issue 4 – 2015, pag. 375 – 384 384 [18]. betianu c., caliman f.a., gavrilescu m., cretescu i., cojocaru c., poulios i., response surface methodology applied for orange ii photocatalytic degradation in tio2 aqueous suspensions, journal of chemical technology and biotechnology, 83, 1454–1465, (2008). [19]. poulios i., kositzi m., pitarakis k., beltsios s., oikonomou i., photocatalytic oxidation of methomyl in the presence of semiconducting oxides, international journal of environment and pollution, 28(1-2), 33 – 44, (2006). [20]. kuhn h.j., braslavsky s.e., schmidt r., international union of pure and applied chemistry organic and biomolecular chemistry division, subcommittee on photochemistry, chemical actinometry, iupac technical report, pure appl. chem., 76, 2105– 2146, (2004). [21]. caliman a.f., cojocaru c., antoniadis a., poulios i., optimized photocatalytic degradation of alcian blue 8 gx in the presence of tio2 suspensions, journal of hazardous materials, 144, 265 – 273, (2007). [22]. bizani e., fytianos k., poulios i., photocatalytic degradation of an azo – dye and textile wastewater in tio2 suspension under uv/vis irradiation, proceedings of the 9th international conference on environmental science and technology, rhodes island, greece, september, a190 – a196, (2005). [23]. caliman a.f., betianu c., robu b.m., gavrilescu m., poulious i., study concerning the influence of oxidizing agents on heterogeneous photocatalytic degradation of organic pollutants, environmental engineering and management journal, 6, 483 – 489, (2007). [24]. fujishima a., zhang x., tryk d.a., tio2photocatalysis and related surface phenomena, surface science reports, 63, 515 – 582, (2008). [25]. roselin l.s., selvin r., photocatalytic degradation of reactive orange 16 dye in a zno coated thin film flow photoreactor, science of advanced materials, 3, 251–258, (2011). 209 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 209 215 enrichment of pasta products using beetroot *péter sipos1, márta horváth2, cintia adácsi2, brigitta horváth 2 , beáta babka 2 , zoltán győri 1 1institute of nutrition of university of debrecen, debrecen, hungary, h-4032 138 böszörményi street, 2institute of food technology, faculty of agricultural and food sciences and environmental management of university of debrecen, debrecen, hungary, h-4032 138 böszörményi street, *corresponding author, siposp@agr.unideb.hu received 19th october 2017, accepted 19th december 2017 abstract: beetroot has numerous advantageous health effects due its chemical composition, such as fibres, mineral elements, vitamins and phenolic compounds. it has not only anti-tumour effects but its consumption is favourable for the cardiovascular system, too. it is used as natural colouring agent in several products. in this research wheat pasta products were made with 8 and 16% beetroot juice and pulp addition to evaluate that the advantageous nutritional effects of beetroot could be utilized in pasta production as well and the consumer’s opinion on these products. besides the general physical parameters (dry matter content, drying water loss, cooking water absorption) total phenol content and flavonoid concentrations were measured and a sensory analysis was performed. it was found that the beetroot addition did not influence the most the physical properties of pasta as compared to the control, but the use of beetroot pulp resulted in a significant increase in the water absorption during cooking. unfortunately the high polyphenol content of beetroot could not be utilized in pasta consumption. although the dry pasta had significant total phenol content, the cooking led to serious leaching and the same value were experienced as for the control pasta. despite these facts, this kind of product can be beneficial for the producers as the sensory analysis showed that the overall acceptance and the taste and texture were found to be advantageous for potential consumers. keywords: beetroot, pasta products, fortification, antioxidants. 1. introduction red beetroot (beta vulgaris l.) is grown all over the world and it can be consumed in many ways, such as canned vegetable and juice, but it is used more and more in the food industry in dried and frozen form as natural colouring agent in different products [1]. besides, its health promoting effect it is more and more widely known, therefore it became more and more popular. its health-benefiting compounds are both inorganic compounds (like minerals such as calcium, iron, zinc, sodium, potassium, phosphorus) and organic ones like vitamins (biotin, folic acid, niacin, vitamin b6) and it contains high amounts of high soluble fibres. beetroot is one of those vegetables, which contains large concentrations of bioactive substances, like betalain, nitrate, antioxidants and phenol [2]. betalains are water soluble nitrogen containing pigments and the food industry uses it as a natural colouring agent. betalains are claasified in two main groups: red-violet betacyanins and yelloworange betaxanthins, these two main pigments determine the colour of the beetroot variety. the antioxidant activity of betalains is well known, it is referred as being higher than that of the ascorbic acid [3]. betacyanins have strong antitumor effect, being able to slow tumour growth and help treat prostate, ovarian and lung cancer [4, 5]. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 210 beetroot effects on the vasculature is attributed to its high nitrate content, and several reports indicate its potential for significantly reducing blood pressure in humans [6] and improves the results of athletes by the increase of o2 supply of muscles, as it was found in an experiment that the beetroot juice consumption made the results of swimmers better [7]. the red beetroot is a good source of fibres. pectin is its water soluble polysaccharide, fibre that bypasses enzymatic digestion of the small intestine, but it is easily degraded by the microflora of the colon. due to this aspect, it improves cholesterol and lipid metabolism and diabetes control and prevention [8]. due to its antioxidant, antiinflammatory, anticarcinogenic and vascular effect, beetroot is very popular in nutrition. pasta products are one of the most popular staple foods nowadays because they are easy to use, quickly ready to consume, can be combined with a wide range of foods, have a diversified appearance both by shape and size and can be stored for a long time. on the other hand, there are many criticisms against it because of its low nutritional value. pasta products are rich in carbohydrates and therefore energy and the modern trends in dietetics suggest other side dishes instead of it. a trend in the development of the pasta products is the improvement of its nutritional value by addition of plant and animal originated health promoting components to increase its protein, fatty acid or mineral composition or fibre content [9-12]. this study aimed to answer the question whether the positive effects of beetroot consumption could be utilized in the case of pasta products. beetroot is used as natural colourant in these products but the question is open that its advantageous components can be useful in pasta products too. to answer this question we made wheat pasta products with different concentrations of beetroot juice and pulp and their phenol and flavonoid contents were evaluated by the main physical and sensory parameters. 2. matherials and methods preparation of experimental samples flour samples used for the evaluation were of bl 55 type commercial flour (white wheat fine flour with 0.55% ash content). the beetroot was also purchased from a local market. the beetroots were peeled, washed and shredded by a commercial juicer. the beetroot was added to the flour as pulp containing all the juiced parts of the beetroot and as juice when the solid particles were removed from the liquid part. in the case of pulp the ratio of solid and liquid parts were measured by laboratory scale and applied in their original ratios avoiding the differences resulted by the sedimentation of fibre parts. the control pasta was made using 70 g wheat flour, 30 g tap water and 1 g nacl. the other samples contained 8 and 16% beetroot pulp or juice in the amount of dough, i.e. 8 or 16 g beetroot was added to 100 g pasta, while the amount of water was decreased to 22 or 14 g, respectively. the preparation and molding of dough were done by hand. the short-cut pasta was dried at room temperature for 3 days avoiding the nutrient losses caused by the high drying temperature. the dried pasta samples were boiled in hot (100°c) water until they reached the readyto-eat texture and the time required for boiling was measured. evaluation of pasta samples for the determination of drying weight loss 100 g of pasta was separately dried and their weights were measured after drying by a laboratory scale. the drying weight food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 211 loss (dwl) was determined using the following eq. (1): dwl (m/m%)= (1) where y is the weight of the dried pasta. dry matter content was determined by the measurement of weight loss during drying to constant weight by the msz 20500/2-85 hungarian standard. for the cooking water absorption capacity (cwa) 10 g of pasta was cooked in boiling 400 ml tap water. after the pasta was cooked, it was rinsed by cold tap water, let to rest for cooling down and weighted by a laboratory scale. the water absorption during cooking was determined using eq. (2): cwa (m/m%)= (2) where x is the weigh after cooking and cooling and y is the weight before cooking. the acidity degree of pasta was determined by the msz 20500/2-85 hungarian standard. pasta samples were grinded under 315 µm particle size by laboratory grinder and 10 g was measured and added to 100 ml distilled water. after 15 minutes phenolphthalein indicator was added and this mixture was titrated by 0.1 mol/l naoh until reaching pink colour. the used naoh in cm3 is the acidity degree of the pasta. total phenol content was determined by the method of kim et al. [13]. 10 g of grinded samples were extracted by 100 ml of 80% aqueous methanol and it was filtered and 0.5 ml was measured and 2.5 ml folin-ciocalteu’s reagent was added. after 5 minutes 2 ml 75 g/l naco3 was added and this mixture was rested for two hours in dark at room temperature. after resting, the absorbance of mixture was measured using a spectrophotometer on 760 nm and the total phenol content was determined by a calibration line which was made from gallic acid. total phenol content is presented as mg gallic acid equivalent (gae)/100 g sample. the total flavonoid content was determined by the method of meda et al. [14]. the same filtered 80% aqueous methanol extract was used as in the case of total phenol content. in a test tube the following was added in the following order: 4 ml distilled water, 1 ml extract, 0.3 ml nano3 and five minutes later 2 ml 1 mol/l alcl3. the test tubes were completed to 10 ml by distilled water and the absorbance was measured at 510 nm. the total flavonoid content was determined by a calibration line what was made from catechin and the total phenol content is presented as mg catechin equivalent (ce)/100 g sample. sensory analysis of pasta samples was performed by the msz 20500/3-1986 hungarian standard. the pasta samples were boiled and a five-member committee performed the sensory analysis. four parameters: appearance, odour, taste and texture had to be evaluated and scored from 0 to 5. the values were corrected by weighting factors: 1.1 for the appearance, 0.7 for the odour, 0.9 for the taste and 1.3 for the texture. the final score is the sum of the corrected scores and the result of test report that the pasta is first class when this score is higher than 18, the product is classified into the second class when it is between 16 and 18. a preference order and opinion were also asked from the testers. 3. results and discussion the different kinds of beetroot added to the pasta did not influence significantly the dry matter content of the dried pasta products; the values ranged from 87.2 to 87.7%. the lowest value was found in the case of control product while the beetroot addition increased this value in all the cases. the requirement on the moisture food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 212 content of pasta products is 13% at most; therefore all the products meet this demand (table 1). the other legal requirement on the quality parameters of these products is the acidity value: it has to be less than 5 cm3. it can be seen in fig. 1 that all the pasta products met this requirement too, but the beetroot addition increased the acid value. the 8% pulp and juice addition resulted in one and a half to two times higher values in comparison to the control product, but the 16% beetroot addition resulted in three times higher values. table 1. dry matter content, drying loss, cooking water absorption and cooking time of pastas made with beetroot juice and pulp addition dry matter content, % drying loss, % water absorption during cooking, % cooking time, min control 87.3 27 226.60 11 8% beetroot juice 87.7 27 223.38 14 16% beetroot juice 87.6 30 222.92 17 8% beetroot pulp 87.7 28 261.32 20 16% beetroot pulp 87.7 25 259.52 22 0,00 0,50 1,00 1,50 2,00 2,50 control 8% beetroot juice 16% beetroot juice 8% beetroot pulp 16% beetroot pulp a c id v a lu e , m l fig. 1. acid values of pasta made with beetroot juice and pulp addition the drying losses were also influenced by the composition. as table 1 shows 8% beetroot addition did not lead to changes in water loss, 27 and 28% values were experienced and therefore almost all the added water was removed during the natural drying. in contrast, the 16% beetroot juice addition increased the water loss by 3% and the same amount of beetroot pulp addition decreased it by 2% in comparison to the control pasta. the composition also influenced the amount of absorbed water during cooking; the control and the 8 and 16% beetroot juice enriched pasta had 222 to 226% water absorption, but the absorption of beetroot pulp enriched the resulted values around 260%, probably due to the higher fibre content. the presence of beetroot had also effect on the required cooking time. the beetroot food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 213 juice addition resulted in a moderate increase in the cooking time, while the addition of the beetroot pulp resulted in two time higher cooking times. the concentrations of total phenolic components (tpc) were also measured from the raw materials and the pasta products. the beetroot juice had 185 mg gae/100 g tpc and the beetroot juice and the beetroot pulp had 128 mg gae/100 g. the tpc of the control dry pasta was 28.4 mg gae/100 g and both the pulp and juice addition doubled it. much less difference can be seen amongst the concentrations but we can state that the higher additions resulted in slightly higher concentrations and the use of pulp resulted in a little bit higher tpc. these advantages in nutrient value had been lost by cooking: the tpc varied from 7 to 17 mg gae/100 g, therefore we can state that the tpc content of pasta products cannot be increased by beetroot addition, although the colour of the additive results significant change in the colour of the dry and cooked pasta (fig. 2). the total flavonoid content (tfc) was also measured for the raw materials and the products, but while the beetroot juice and pulp had significant concentrations (185 and 128 mg ce/100 g, respectively), the tfc were under the detection limit for both the dry and cooked pasta products. fig. 2. total phenol contents of pastas made with beetroot juice and pulp addition the sensory analyses were made to determine how the consumers accept the products (table 2). on average the products got 4.8 points from the maximum 5.5 for the appearance, 2.94 from 3.5 for the odour, 3.82 from 4.5 for the taste and 5.62 from 6.5 for the texture, what shows that the overall acceptance is good for the products. the average score for the sensory analysis is 17.2; this means second class products by classification. one product got higher values that 18 what is required for first class quality; the one made with 8% beetroot pulp. this was due its high values for appearance, odour and taste, but its texture score registered also food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 214 the highest values. the pasta made with 16% beetroot pulp addition scored 15.84% and was classified under the second class, because of its low scores for appearance. it is interesting to see that the control pasta got the lowest scores for taste and odour, therefore we concluded that the addition has advantageous effects on the main sensory values. when the testers were asked to make a preference order for the products, the highest position was achieved by the pasta made with beetroot pulp addition independently from the concentration (18 scores for both the 8 and 16% addition), the third was the one made with 16% juice addition (17 score) and the forth is the one made with 8% addition (15 score). the control pasta got only 7 score that meant that almost all the testers would prefer the least. the tasters found the pasta products made with beetroot addition have intensive taste what makes them enjoyable. table 2. results of sensory evaluation of pastas made with beetroot juice and pulp addition appearance odour taste texture sensory value control 4.84 2.8 3.42 5.72 16.78 8% beetroot juice 4.84 2.8 3.78 5.98 17.4 16% beetroot juice 4.18 2.94 3.78 4.94 15.84 8% beetroot pulp 5.28 3.08 4.14 5.98 18.49 16% beetroot pulp 4.84 3.08 3.96 5.46 17.34 average 4.8 2.94 3.82 5.62 17.17 4. conclusions the results show that while the beetroot pulp and juice addition in 8 and 16% concentrations significantly influences the colour of products the other physical properties are only slightly modified. the addition has only moderate effect or no effect on the moisture content and the drying loss (and not even on the acidity value) while the water absorption during cooking is increased by the fibre parts of the beetroot pulp. the expected nutritional effect that this antioxidant rich component would increase the nutritional value of pasta was not proved; although the dry enriched pasta had significant total phenol concentration thanks to the gentle natural drying, cooking resulted in decrease probably due to leaching. in the case of the total flavonoid content the enrichment had no effect on the flavonoid content of dry or cooked products. in addition to all of these apsects we concluded that the production of this kind of enriched products can be advantageous, because the consumers found them attractive and the sensory analysis proved that they can be competitors of traditional pasta products both in texture, taste and odour. they could consume it willingly and it can be an opportunity for the expansion of assortment. 5. references [1]. kanner j., harel s., granit r., betalains: a new class of dietary cationized antioxidant, journal of agriculture and food chemistry, 49(11): 5178-5185, (2001). [2]. vanajakshi v., vijayendra s.v.n, varadaraj m.c., venkateswaran g., agrawal r., optimization of a probiotic beverage based on moringa leaves and beetroot, food science and technology, 63: 1268-1273, (2015). [3]. ravichandran k., saw t. m. m. n., mohdaly a.a.a., gabr m.m.a., kastell a., riedel h., cai z., knorr d., smetanska i., impact of processing of red beet on betalain content and antioxidant activity, food research international, 50: 670-675, (2013). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 péter sipos, márta horváth, cintia adácsi, brigitta horváth, beáta babka, zoltán győri, enrichment of pasta products using beetroot, food and environment safety, volume xvi, issue 4 – 2017, pag. 209 – 215 215 [4]. kapadia g. j., azuine m. a., rao g. s., arai t., lida a., tokuda h., cytotoxic effect of the red beetroot (beta vulgaris l.) extract compared to doxorubicin (adriamycin) in the human prostate (pc-3) and breast (mcf-7) cancer cell lines, anti-cancer agents in medicinal chemistry, 11(3): 280-284, (2011). [5]. lechner j. f., wang l. s., rocha c.m., larue b., henry c., mclntyre c. m., riedl k. m., schwartz s. j., stoner g. d., drinking water with red beetroot food color antagonizes esophageal carcinogenesis in nnitrosomethylbenzylamine-treated rats, journal of medicinal food, 13(3): 733-739, (2010) [6]. clifford t., howatson g., west j. d., stevenson j. e., the potential benefits of red beetroot supplementation in health and disease, nutrients, 7(4): 2801-2822, (2015). [7]. pinna m., roberto s., milia r., marongiu e., olla s., loi, a., migliaccio g. m., padulo j., orlandi c., tocco f., concu a., crisafulli a., effect of beetroot juice supplementation on aerobic response during swimming, nutrients, 6(2): 605– 615, (2014). [8]. lattimer m.j., haub d.m., effects of dietary fiber and its components on metabolic health, nutrients, 2(12): 1266-1289, (2010). [9]. kaur g., sharma s., nagi h. p. s., dar b. n., functional properties of pasta enriched with variable cereal brans, journal of food science and technology, 49(4): 467–474, (2012). [10]. mogra r., midha s. value addition of traditional wheat flour vermicelli, journal of food science and technology, 50(4): 815–820, 2013. [11]. mridula d., gupta r. k., bhadwal s., khaira h., tyagi s.k., optimization of food materials for development of nutritious pasta utilizing groundnut meal and beetroot, journal of food science and technology, 53(4): 1834–1844, (2016). [12]. anbudhasan p., asvini g., surendraraj a., ramasamy d., sivakumar t., development of functional pasta enriched with omega 3 fatty acids, fishery technology, 5i: 1-6, (2014) [13]. kim d.o., jeong s.w., lee, c.y., antioxidant capacity of phenolic phytochemicals from various cultivars of plums, food chemistry, 81(3): 321-326, (2003). [14]. meda a., lamien c.e., romito m., millogo j., nacoulma g.o., determination of the total phenolic, flavonoid and proline contents in burkina fasan honey, as well as their radical scavenging activity, food chemistry, 91(3): 571577, (2005). 1. introduction 4. conclusions применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 contents: 1. effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan" caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa 513 2. effect of season on the major physicochemical parameters of honey product destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun 14 24 3. antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains hayet messaoui, salima roudj, nour-eddine karam 25 39 4. healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets marianna barus, vita antsupova, igor chifurko, yana ushko 40 47 5. review: the main raw materials used for production of 1 st and 2 nd generation bioethanol vasile-florin ursachi, gheorghe gutt 48 68 6. physicochemical and sensory properties of barley bread enriched by freeze dried date pomace powder amal messaoudi , djamel fahloul 69 75 7. research of functional-technological properties of the protein complex and natural color in the composition of restructured ham products iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak 76 83 8. practices of biosecurity measures and their consequences on poultry farms in abidjan district gblossi bernadette goualie , souleymane bakayoko, kalpy julien coulibaly 84 91 9. effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacter xylinum kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun 92 97 10. author instructions i v 11. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 microsoft word 2 krasulya_corrected_l2.docx 71 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2 2017, pag. 71 76 investigation on lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant olena grek1, *olena krasulya1, alla petryna2 1educational and research institute of food technology, national university of food technologies, 2branch office of the national university of food technologies in lviv 01601, volodymyrska str. 68, kyiv, ukraine, olena_krasulya@ukr.net *corresponding author received 8th may 2017, accepted 27th june 2017 abstract: the results of biochemical activity of lactose fermenting yeasts in the wort based on whey, obtained by thermo acid coagulation of milk by berry raw material (sterilized black currant paste) are shown. the extraction of black currant’ valuable components occur in the protein foundation and colored whey, which can be used in the production of fermented beverages with high biological and nutrition value. it was found from the analysis of lactose fermenting yeasts’ biomass accommodation that the biggest growth of yeasts in the wort based on colored whey was in the samples which are fermented by zygosaccharomyces lactis 868-k – the general amount of cells was (78.1...79.9)∙106 cfu/ml for 48 hours. the optimal fermentation temperature (30…32 °c) was established by the parameters of fermentation activity: accumulation of ethyl alcohol and carbon dioxide in the wort, the total amount of yeasts cells.the obtained results were used in the technology development of nonalcocholic beverages based on colored whey. keywords: colored whey, fermentation activity, lactose fermenting yeasts, wort. 1. introduction milk whey is the valuable proteincarbohydrate side raw material. the yield of whey is 70...90 %, at the rennet cheeses production, 70...80 % at the cottage cheese and 75 % at the casein [1, 2]. in ukraine, about 50 % of whey is being processed, the rest of it being recycled, herewith the valuable raw material is lost and the environmental problems are getting worse. the last one is connected with high values of chemical oxygen consumption, which consist of 75 g/l for acid whey and 57 g/l for deprotonated. besides this aspect, the content of organic nitrogen in acid whey is about 1.03 g/l [3-5]. the additional costs for effluent treatment plants or whey transportation to specialized plants reduce economic indicators of plant generally. there are a lot of industrial methods of whey processing. one of the most effective and the least expensive is the manufacturing of fermented non-alcoholic beverages which already exists in the dairy plants equipment. the use of milk whey in the capacity of water phase in the beverages manufacturing promotes enrichment with biological components such as γ-casein, β-lactoglobulin, whey albumin, immunoglobulin and proteosepeptone, lactose, glucose, galactose, lactulose, arabinose, microelements and ultramicroelements, vitamins (retinol, tocopherol, thiamine, riboflavin, pirodoxin), milk fat, etc. the energy value of milk whey is somewhat lower than in skimmed milk, and the biological value is approximately the same, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 olena grek, olena krasulya, yuliia pakhomova, investigation of lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant, food and environment safety, volume xvi, issue 2 – 2017, pag. 71 – 76 72 this makes possible to be used in the manufacturing of dietary foods [2, 6 8]. there are several methods to get milk whey: hard and soft rennet cheeses production; casein making process; cottage cheese production by acid, acid-rennet and thermoacid methods and etc. also the obtaining technology of the proteinberry clots by thermo acid coagulation of milk proteins by berry raw material (black currant paste), with receiving of milk whey was developed [9]. black currant is one of the most widespread berry crops that grow in ukraine. medicinaland-prophylactic properties are determined by the fact that berries contain vitamins, macro and microelements, polysaccharides (pectin), polyphenols and others which are necessary for humans. berries contain a high amount of iron, phosphorus and calcium salts in the form of organic compounds, which are easily digested by the human body.[10, 11]. the extraction of black currant’ valuable components occur in the same way as in the protein foundation in whey. the addition of black currant puree with high acidity permits to increase the yield of clot. these berries contain pigments that give light crimson hue to the cheese clot and intensive crimson red to the whey obtained. colored whey is a valuable raw material for the production of fermented non-alcoholic beverages due to its intensive coloration, which excludes the necessity of adding artificial colourants or flavorants. also, most of the water-soluble constituents of berry puree – vitamin c, carbohydrates, polyphenols, macroand microelements, pass into the whey, thus increasing its biological value. carbohydrates of puree at 95 % are represented by reducing sugars (glucose and fructose), which, probably can be the growing medium for yeasts and consequently they can accelerate the process of colored whey fermentation. the aim of the work was to investigate the biochemical activity of lactose fermenting yeasts in the wort based on whey, obtained by thermo acid coagulation of milk by berry raw materials. 2. materials and methods microorganisms to carry out the experimental research, zygosaccharomyces lactis 868-k, kluyveromyces lactis 2452, saccharomyces lactis 95 yeasts from the «сollection of microorganism strains and plant lines for food and agricultural biotechnology» of the research institution «institute of food biotechnology and genomics’ of the national academy of science of ukraine» were used . the production of colored whey having in view a fermented drink base, the whey was obtained from the milk-protein clots production by the method of thermo acid coagulation and the classic technology from the whole milk was used. the sterilized blackcurrant paste (тм «liqberry» manufacturer, ukraine technical conditions 15.3-24110704-003:2011), was used as a coagulant. the composition of paste is shown in the table 1 [12]. the paste was prepared in industrial conditions, by improved technology with the use of hydrodynamical (cavitational) processing of raw material at the tek-cm installation. the berries were processed in accordance with the developed regime in order to get the necessary degree of homogenization and industrial sterility [12]. the (10±0.5) % of berry coagulant from the general mass was added to the prepared normalized mixture. the whey obtained had the following indicators: the amount of dry matters – (7.47±0.37) %; reducing sugars – (5.57±0.58) %; protein – (1.3±0.07) %; the value of active acidity – (4.8±0.27); color – dark pink, bright, saturated; taste and smell – sour with a taste of berry coagulant. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 olena grek, olena krasulya, yuliia pakhomova, investigation of lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant, food and environment safety, volume xvi, issue 2 – 2017, pag. 71 – 76 73 table 1. the composition of homogenized black currant paste indicator amount fats, g/100 g 0.2 proteins, g/100 g 0.8…1.0 soluble dry matters, g/100 g 18.9 cellulose, g/100 g 1.5 value of рн 3.5 polyphenols, mg/100 g 350…400 organic acids, g/100 g 2.0…2.5 vitamin с, mg/100 g 20…40 pectin, g/100 g 0.9…1.1 carbohydrates, g/100 g total amount 14.92 reducing sugars 14.32 fructose 1.96 glucose 12.36 minerals, mg/100 g к 300…350 na 25…32 ca 30…36 mg 26…31 p 28…33 fс 1.0…3.0 preparation of experimental samples for the wort preparation, the colored milk whey was separated from the residual proteins and other factored particles, pasteurized at the temperature of (78±1) °с without exposure and cooled down to the temperature of (30±2) °с. the lactose fermenting yeasts in amount of at least 40 mln per 1 cm3 of wort were introduced into the obtained mixture. the fermentation was carried out at the temperature of 30 °с during 48 hours in accordance with the theoretical data [2]. the control sample – milk whey, obtained from the manufacturing of milk-protein clots by thermo acid coagulation of classical technology without using berry raw materials. the physiological condition of yeast was estimated by the total number of yeast cells (106 cfu/ml) and it was directly counted in the goryaev chamber. the amount of carbon dioxide exhaled during the fermentation process was controlled by gravimetric method [13]. the fermented wort was distilled in order to determine the mass fraction of alcohol in the distillate by conventional methods [14]. the content of reducing matters was investigated by iodometric method [15]. the statistical analysis all the analytical determinations were performed at least three times and the value reported for determined characteristics was the average value ± of the standard deviation (s.d.). the statistical analysis was performed by the microsoft excel statistical software version 2010. 3. results and discussion the determination of the lactose-fermenting yeast productivity the results of lactose fermenting yeasts’ biomass accumulation in the colored whey in 48 hours are shown in the fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 olena grek, olena krasulya, yuliia pakhomova, investigation of lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant, food and environment safety, volume xvi, issue 2 – 2017, pag. 71 – 76 74 fig. 1. accumulation of yeasts cells in the colored milk whey the research results confirm that yeast cells develop more actively in the medium with colored whey. obviously, this effect was associated with a high amount of reducing sugars, due to the transfer of glucose and fructose from the berry coagulant. herewith the amount of growth of the yeast cells varied from 55.9∙106 to 77.26∙106 cfu/ml of wort depending on the type of yeasts. the biggest amount of lactose fermenting yeast growth in the medium with colored whey was registered in the samples fermented by zygosaccharomyces lactis 868-k microorganisms – the general amount of yeasts cells was of 77.26∙106 cfu/ml of wort. thus, the fermentation of whey wort with colored whey by lactose fermenting yeasts accelerates the process of fermentation. the obtained data confirm the possibility of using such whey in the process of fermented colored whey beverages manufacturing. the determination of the fermentation rational temperature range the temperature range of whey wort fermentation was specified according to the zygosaccharomyces lactis 868-k yeasts biomass accumulation, amount of exhaled carbon dioxide and ethyl alcohol. the dependence of the change of these indicators from the temperature of fermentation in wort is shown in the table 2. table 2. the dependence of zygosaccharomyces lactis 868-k yeasts biomass accumulation, the amount of exhaled carbon dioxide and formation of ethyl alcohol in the wort from the fermentation temperature yeast cells concentration, (106 cfu/ml) co2 content, g/100 ml ethanol content, vol. % fermentation temperature, °с whey (control) colored whey whey (control) colored whey whey (control) colored whey 24 61.2±1.84 65.3±1.96 0.69±0.02 0.73±0.02 0.20±0.01 0.26±0.01 26 65.9±1.98 69.8±2.09 0.73±0.02 0.79±0.02 0.22±0.01 0.28±0.01 28 69.2±2.08 75.7±2.27 0.83±0.02 0.85±0.03 0.25±0.01 0.29±0.01 30 69.3±2.08 78.1±2.34 0.93±0.02 0.96±0.03 0.26±0.01 0.35±0.01 32 71.2±2.14 79.9±2.40 0.95±0.03 0.99±0.03 0.30±0.01 0.38±0.01 34 70.5±2.12 76.4±2.92 0.94±0.03 0.96±0.03 0.25±0.01 0.33±0.01 36 70.1±2.10 78.5±2.36 0.86±0.03 0.88±0.03 0.20±0.01 0.31±0.01 according to the research results, shown in table 2, the optimal temperature of medium growth for the interested yeast species (control and model samples) is 30…32 °с, at which the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 olena grek, olena krasulya, yuliia pakhomova, investigation of lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant, food and environment safety, volume xvi, issue 2 – 2017, pag. 71 – 76 75 maximal accumulation of microorganisms – (61,2…79,9)∙106 cfu/ml can be observed. with the further temperature increase or decrease, the amount of yeast cells reduce, that can be explained by the lowering of their ferments’ activity. wort substrate from the colored whey with the amount of yeasts biomass 79,9∙106 cfu/ml of wort with the temperature 32 с showed high quality of culture medium for lactose fermenting yeasts. the worst result was registered by the wort that was fermented at the temperature of 24 °с – 65,3∙106 cfu/ml. in all the samples, a similar dynamics of the biomass accumulation of yeast species zygosaccharomyces lactis 868-k was observed. the amount of accumulated carbon dioxide varied from 0,69 to 0,99 g per 100 ml of wort. the highest rate was observed at the temperature of 30...32 °c and was 0.73...0.99 g per 100 ml of medium. with the temperature increase or decrease the yeasts fundamentally reduced their activity and consequently the amount of exhaled carbon dioxide decreased. the content of ethanol in all the samples was in the range from 0.2 to 0.38 vol. %. the maximal amount of ethyl alcohol 0.38 vol. % was accumulated at the temperature of 32 °с in the wort with colored whey. in the control sample, this indicator is lower by 16…30 %, that testifies the positive impact of carbohydrate composition of colored milk whey on lactose fermenting yeasts’ biological activity. according to the requirements of regulatory documents on non-alcoholic beverages [16], the permissible amount of ethanol in the product is of most 1.2 vol. %, thus the obtained worts can be the foundation for fermented beverages. the determination of the reducing matters amount the metabolism of lactose-fermenting yeasts is due to the course of chemical reactions, catalyzed by enzymes and connected with hydrolysis of lactose and monosaccarides of berry puree as an additional source of carbohydrates. therefore, the amount of reducing matters, left over after the completion of the fermentation of wort from the colored whey was determined. according to the research results, the initial content of reducing matters in colored whey differs essentially from the control sample and was of 5.57 % and 4.6 % correspondingly. after the wort fermentation during 48 hours at the temperature 30...32 °с the amount of reducing sugars decreased significantly to 0.07 % – for the wort based on the colored whey and 1.1 % for the control sample, respectively. this accounts for an almost complete process of carbohydrates fermentation and utilization, as well as for high activity of enzymes that catalyze the hydrolysis of lactose. the obtained data prove that the zygosaccharomyces lactis 868-k yeasts have a high fermentation activity not only in relation to carbohydrates of whey, but probably to monosaccarides of black currant paste as well. 4. conclusions in the process of research, the possibility of lactose–fermenting yeasts usage for the fermentation of wort based on whey, obtained by thermo acid coagulation of milk by berry raw materials – sterilized black currant paste was proved. the basic regularities of multiplication of the diferrent yeast species on the base of colored whey were studied. it was determined that zygosaccharomyces lactis 868-k are the most effective ones for the fermentation at the temperature of 30…32 °с and maximately accumulate the yeast biomass (the amount of cells – (78,1...79,9)∙106 cfu/ml) for 48 hours.the obtained results can be used in the processing technology of nonalcoholic beverages based on colored whey. 5. references [1]. ramos o.l., pereira r.n., rodrigues r.m., teixeira j.a., vicente a.a., malcata f.x., whey and whey powders: production and uses / reference module in food science, from encyclopedia of food and health: 498–505, (2016). [2]. hramthsov a.g., the phenomenon of milk whey, monograph, spb: profession, 802 p., (2011). [3]. klasnja m.t., sciban m.b., osnovi procesa anaerobnog prečišćavanja otpadnih voda food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 olena grek, olena krasulya, yuliia pakhomova, investigation of lactose fermenting yeasts activity in the whey obtained by coagulation of milk proteins by berry coagulant, food and environment safety, volume xvi, issue 2 – 2017, pag. 71 – 76 76 prehrambene industrije i industrije pića, acta periodica technologica, 31: 740–748, (2000). [4]. jelen p., whey processing. utilization and products, reference module in food science, from encyclopedia of dairy sciences (second edition), 731–737, (2011). [5]. shershenkov b., suchkova e., the direct microbial synthesis of complex bioactive compounds as perspective way of milk whey utilization, energy procedia, 72: 317–321, (2015). [6]. cui s.w. nie s., roberts k.t., functional properties of dietary fiber, comprehensive biotechnology (second edition), 4: 517–525, (2011). [7]. jeličić i., božanić r., tratnik l.j., napitci na bazi sirutke − nova generacija mliječnih proizvoda, mljekarstvo, 58: 257–274, (2008). [8]. smithers g.w., whey and whey proteins – from «gutter-to-gold», international dairy journal, 18: 695–704, (2008). [9]. grek o., pshenychna t., krasulya o., pakhomova y., ivashchenko k., the influence of berry puree on microbiological indicators of cheese product during storage, food and environment safet, xv, is. 4: 334–340, (2016). [10]. makarkina m.a., yanchuk t.v., the characteristics of the black currant grades by sugars and organic acids content, modern gardening, 2: 9– 12, (2010). [11]. shevchuk l.m., the quality of black currant in the carpathian region, ukrainian academy of agricultural sciences, 10: 24–26, (2008). [12]. ivashchenko k.y., improving of homogenized fruit products technology using hydrodynamic processing: synopsis of a thesis..phd,speciality 05.18.13. «technology of canned and chilled products» nuft: 20 p., (2015). [13]. gost (all union state standard) 6687.3-87 carbonated soft drinks and drinks from bread raw materials. the methods of carbon dioxide determination, (1987). [14]. gost (all union state standard) 6687.7-88 non-alcoholic beverages and kvasses. a method of alcohol determination, (1988). [15]. gost (all union state standard) 3628-78 milk products. the methods of sugar determination, (1978). [16]. dstu 4069:2002 «non-alcoholil drinks. general technical conditions», (2002). 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017, pag.5 12 study of the evolution of microand macroelements during the winemaking stages: the importance of copper and iron quantification *liliana norocel1, *gheorghe gutt1 1faculty of food engineering, ștefan cel mare university of suceava, universitatii 13, ro 720229 suceava, romania liliana.norocel@fia.usv.ro, g.gutt@fia.usv.ro *corresponding author received february 15th 2017, accepted march 27th 2017 abstract: knowledge of the concentration of mineral elements from winemaking products, particularly from the final product is important because of their influence on wine quality. some metal ions such as iron and copper can induce haze formation and changes in the sensory proprieties of wine. the presence of heavy metals in wine is due to different factors including vineyard soil, agricultural practices (the use of fertilizers and pesticides), and can be at the same time a result of environmental pollution. in addition, the acidity of wine and grape must (freshly pressed grape juice) can dissolve cr, cu, ni, and zn from winemaking equipment like pumps and taps. as wine is the most widely consumed alcoholic beverage, analytical control of mineral elements content is required during the whole process of wine production, from the grapes used to the final product. in this study the content of microand macroelements in grape pomace, yeast sediment, grape must and wine was determined by inductively coupled plasma-mass spectrometry (icp-ms). samples of winemaking products originating from five grape varieties were analyzed in four forms in order to determine to what measure the values varied the pca (principal component analysis). the obtained results using pca highlighted major differences in the content in trace elements between samples. keywords: grape pomace, grape must, icp-ms, wine, yeast 1. introduction the content in mineral elements of food products has been extensively studied, in recent papers [1-4]. the analysis of mineral elements in alcoholic beverages [5] is particularly centered on the heavy metal content. among alcoholic beverages, wine has been the most popular and widely-consumed worldwide since early civilization [6]. according to the international organization of vine and wine, the worldwide production of wine was estimated at 259 mhl in 2016. wine is a product obtained exclusively through the alcoholic fermentation of fresh grapes. the final beverage is a complex matrix containing water, alcohol, sugar, and a great variety of components, organic as well as inorganic ones [7]. wine quality depends on the chemical composition, which is influenced by both geographic factors (climate, soil, grape variety and culture) and factors related to the production process (winemaking, transport and storage). inorganic ions concentration in wine is of great interest [8], as in some cases it can have a major impact on the quality of the final product. mineral elements are absorbed from the http://www.fia.usv.ro/fiajournal mailto:liliana.norocel@fia.usv.ro, mailto:g.gutt@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 6 soil through the vine roots and are accumulated in the cellular walls, skin and seeds of the grape. during winemaking, the minerals from grapes pass into wine. therefore, the mineral composition of wine reflects its origin and development, making it unique and identifiable. oroian (2015) achieved an authentication of romanian white wines varieties in terms of the content in 27 mineral elements [9]. it contributes significantly to wine’s sensory characteristics, affecting color, clearness, flavor and aroma [10]. most of the minerals are important to the alcoholic fermentation: calcium, potassium, magnesium, and sodium maintain an adequate ph and ionic balance stabilizing the cellular metabolism of yeasts [11]. minerals in lower concentration such as copper, iron, manganese, vanadium, zinc are also favorable for yeasts, particularly saccharomyces cerevisiae. copper alongside iron and manganese can cause sensory quality changes after bottling and influence the stability in old wine. the presence of high levels of both fe and cu leads to the formation of hazes in wine [12]. this undesirable oxidation process has an impact on the commercial acceptance of wine and can induce potentially toxic effects [13]. in this context, measurement of metal content is essential to provide the final product with high quality. the mineral constituents of wine can be assessed by several analytical methods. atomic spectrometry techniques such as inductively coupled plasma-mass spectrometry (icp-ms) [14], icp atomic emission spectrometry (icp-oes) [15], and flame atomic absorption spectrometry (faas) have been extensively used for the elemental analysis of wine [16, 17]. the aim of this paper was to determine the metal ions in the winemaking process, namely grape pomace, yeast, grape must, and wine, and to study the changes in the concentration of these elements during winemaking. 2. materials and methods samples. samples of winemaking products (grape pomace, yeast, grape must, and wine) obtained from five different grape varieties were analyzed by icp-ms. the grape varieties were feteasca neagra, merlot, chasselas, riesling, ottonel. all grapes used in winemaking originated from the region of focsani, romania. sample preparation. 5 grams of grape pomace and yeast were mineralized in an electric furnace at a temperature of 600°c, for 6 hours. the resulted ash was transferred into a 50 ml volumetric flask, where it was dissolved by adding a mixture of nitric acid and deionized water until the concentration of nitric acid in the solution was of 1% [18]. the elemental analysis of grape must was conducted according to toaldo et al. (2013). an aliquot of 500 µl of sample was diluted to 10 ml with 0.14 mol/l nitric acid, then directly analyzed by icp-ms [19].wine samples were prepared according to oroian (2015). reagents. the reagent used was of high purity grade: double deionized water (18 mω cm resistivity) produced by a water purification system (thermofisher, germany) was used in all solutions. samples were digested with concentrated nitric acid (65% hno3, sigma aldrich, germany) and hydrogen peroxide (30% h2o2 pure, sigma aldrich, germany). apparatus. the determination of 12 elements was performed in a mass spectrometer with inductively coupled plasma, (icp-ms) agilent technologies 7500 series (agilent, usa). statistical analysis. statistical analysis, pca and anova, was performed using xlstat 2016. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 7 3. results and discussion in this study the content of grape pomace, yeast sediment, grape must and wine in the following mineral elements: v, mn, fe, ni, au, hg, mg, al, ca, cu, zn, and cr were determined. to estimate the concentration of these elements in the sample following equation was used: m tm v xvc c  where: c = concentration of the element in the sample; cm = concentration of the elements in the diluted sample; vt = final volume of the measurement solution, in ml; vm = aliquot volume of wine, in ml [20]. the determined concentrations of micro and macroelements are presented in table 1. as it can be observed, magnesium was the most abundant mineral element, with concentrations that varied from 43.25 to 298.69 mg/l. high levels of calcium were also determined in all the samples analyzed. for the merlot grape variety, grape pomace, must, yeast and the final sample of wine had a significantly higher content of magnesium and calcium. the grape variety merlot, alongside feteasca neagra had a remarkable content of copper and zinc. for feteasca neagra variety, the copper content ranged between 1.7 mg/l in wine and 41.1 mg/l in pomace. compared to this, the copper content of grape pomace from merlot variety was slightly higher (44.3 mg/l). zinc concentration was comparable in these two grape varieties, especially in pomace and wine. aluminum and chromium are found in an increased concentration in all the samples analyzed. among microelements, nickel and vanadium displayed significant variation of concentration during different stages of winemaking. the main microelement in winemaking products, nickel was determined in higher concentration in the merlot variety. in contrast, the other red grape variety analyzed in this paper had a notable content of iron. finally, the winemaking products obtained by processing varieties of white grapes (chasselas, riesling and ottonel) had greater levels of au. fig. 1. major mineral elements in winemaking products food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 8 fig. 2. minor mineral elements in winemaking products multifactorial anova was performed to evaluate the influence of interactions between factors (mineral element, analyzed sample and grape variety) on the concentration in minerals. five grape varieties were analyzed in the mentioned forms, and the pca (principal component analysis) was used to determine to what measure the values varied. of the major mineral elements in winemaking products, magnesium and calcium had the highest concentrations in all the samples analyzed, irrespective of the grape variety (fig. 1). during the mechanical processes of crushing and pressing a proportion of minerals contained by grapes passes into must, while a large quantity of these elements remains in the pomace. with the removal of yeast sediment, the mineral elements concentration decreases and therefore the final product (wine) has the lowest mineral content. a similar variation in the concentration determined in winemaking products was also observed for minor mineral elements (fig. 2). compared to other minerals, au and hg were found in lower concentration in yeast sediment samples. multifactorial anova showed a significant interaction between the mineral element and grape variety (p < 0.001). fig. 3. pca for fetească neagră fig. 4. pca for merlot food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 9 fig. 5. pca for chasselas fig. 6. pca for riesling fig. 7. pca for ottonel principal component analysis (fig. 3-7) indicated positive correlations between the main mineral elements ca, mg and al and the four samples analyzed. the other minerals determined are displayed in opposed quadrant, and are found in lower concentrations. of particular interest were the concentrations of copper and iron. it is known that the presence of copper and iron ions can induce haze formation, which consequently leads to changes in the organoleptic properties of wine. the iron and copper content of the samples is presented in fig. 8-9. concentrations of around 0.5 mg/l copper and 10 mg/l iron or higher can cause a metallic taste, haze, browning and other undesirable effects on wine. its presence in higher concentrations in the must can even have an impact on the alcoholic fermentation, as it has been reported on several occasions, being capable of inhibiting the growth and development of several groups of certain naturallyoccurring microorganisms. according to the data presented in table 1, the copper content of wine varied from 0.35 mg/l (riesling) to 1.70 mg/l (feteasca neagra). the maximum level of copper specified in the european wine regulations and the compositional guidelines set by the international organization of vine and wine is of 1 mg/l. on the other side, it is generally accepted that levels above 0.5 mg cu/l are very likely to induce copper casse. based upon this consideration, all the samples of wine analyzed excepting that of riesling variety are susceptible to this type of oxidation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 10 m g 16 1. 09 10 6. 71 10 1. 19 10 5. 57 14 2. 33 14 0. 05 11 4. 36 10 2. 86 29 6. 48 17 0. 14 10 5. 81 92 .8 1 25 2. 30 17 8. 35 15 1. 86 43 .2 5 29 8. 69 92 .7 8 48 .3 1 59 .6 8 a l 20 .5 9 20 .5 0 20 .8 2 17 .2 6 35 .4 1 30 .2 1 12 .7 8 16 .1 5 10 1. 71 72 .4 1 31 .6 8 12 .5 1 10 9. 08 17 .7 8 14 .3 3 12 .7 0 37 .1 4 19 .3 9 11 .7 5 11 .0 5 c a 27 .2 0 27 .1 9 20 .8 4 20 .9 3 21 2. 03 21 1. 73 98 .5 4 38 .1 1 19 7. 46 37 .1 1 23 .0 1 36 .9 8 13 1. 97 10 7. 47 60 .9 8 52 .6 3 34 3. 29 63 .1 1 33 .3 4 13 .0 0 c u 41 .1 0 6. 69 1. 74 1. 70 44 .3 4 31 .3 7 2. 82 1. 35 6. 96 5. 30 1. 24 0. 68 5. 88 2. 22 1. 50 0. 35 7. 19 1. 78 1. 22 1. 13 z n 10 .9 2 0. 39 0. 24 0. 32 11 .3 4 7. 74 0. 75 0. 21 1. 80 0. 23 0. 08 0. 09 1. 32 0. 62 0. 27 0. 14 1. 84 0. 34 0. 28 0. 28 c r 5. 53 4. 83 1. 89 1. 68 6. 37 3. 22 5. 14 1. 60 20 .8 4 6. 87 3. 60 3. 52 14 .1 5 16 .7 3 2. 21 1. 67 7. 10 4. 44 1. 79 0. 70 v 0. 56 0. 54 0. 12 0. 50 0. 80 0. 55 0. 55 0. 09 1. 86 0. 78 0. 43 0. 38 1. 45 1. 09 0. 13 0. 75 0. 55 0. 43 0. 14 0. 03 m n 2. 04 1. 97 1. 86 1. 12 8. 44 4. 79 3. 92 1. 47 26 .2 5 10 .5 8 5. 23 3. 83 20 .0 0 12 .7 6 11 .6 9 9. 07 7. 73 6. 07 2. 37 0. 54 f e 0. 97 0. 70 0. 07 0. 50 0. 48 0. 33 0. 28 0. 05 0. 98 0. 36 0. 17 0. 13 0. 79 0. 64 0. 08 0. 37 0. 54 0. 25 0. 06 0. 02 n i 1. 73 1. 04 1. 30 1. 07 4. 55 4. 04 1. 48 1. 48 1. 13 1. 16 1. 32 0. 20 3. 00 0. 81 1. 07 0. 07 0. 87 1. 00 0. 21 0. 28 a u 0. 18 0. 19 0. 19 0. 04 0. 07 0. 36 0. 10 0. 14 1. 07 0. 36 0. 30 0. 44 2. 92 0. 08 1. 89 0. 04 3. 07 2. 80 1. 56 0. 04 h g 0. 05 0. 04 0. 01 0. 01 0. 02 0. 11 0. 07 0. 04 0. 03 0. 02 0. 02 0. 03 2. 10 0. 02 0. 02 0. 01 0. 01 0. 02 0. 07 0. 00 a na ly ze d sa m pl es g ra pe p om ac e y ea st g ra pe m us t w in e g ra pe p om ac e y ea st g ra pe m us t w in e g ra pe p om ac e y ea st g ra pe m us t w in e g ra pe p om ac e y ea st g ra pe m us t w in e g ra pe p om ac e y ea st g ra pe m us t w in e g ra pe v ar ie ti es fe te as ca n ea gr a m er lo t c ha ss el as r ie sl in g o tto ne l t ab le 1. c on ce nt ra tio ns o f m ic ro a nd m ac ro el em en ts in w in em ak in g pr od uc ts [ m g/ l ] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 11 unlike the concentration of copper. iron did not exceed the maximum level allowed and therefore a haze formation caused by this metal is not posible. in the case of copper the increased content could be attributed to prolonged use coppercontainig fungicide and particularly bordeaux mixture (copper sulphate).this scenario is highly likely due to the fact that all grapes used in this study originated from a vineyard which is known to require this type of treatment in order to reach the desired quality and production. moreover, the traditional winemaking process used in this case din not imply a removal of copper and iron through the addition of stabilizing agents (e.g. potassium ferricyanide). fig. 8. copper content in analyzed samples [mg/l], s1-s5grape varieties (s1-feteasca neagra, s2merlot, s3chasselas, s4-riesling, s5-ottonel) fig. 9. iron content in analyzed samples [mg/l],s1-s5 grape varieties (s1-feteasca neagra, s2merlot, s3chasselas, s4-riesling, s5-ottonel) 4. conclusions the results of this study show a decrease in the concentration of mineral elements in the winemaking process from grape pomace, must, yeast sediment to wine. multifactorial anova indicated a significant interaction of the mineral element with the grape variety (p < 0.001). pca analysis highlighted a strong correlation between the elements with higher concentration and the corresponding winemaking products. 5. references [1]. ma g., zhang y., zhang j., wang g., chen l., zhang m., lu c., determining the geographical origin of chinese green tea by linear discriminant analysis of trace metals and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 liliana norocel, gheorghe gutt, study of the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification, food and environment safety, volume xvi, issue 1 – 2017, pag. 5 – 12 12 rare earth elements: taking dongting biluochun as an example, food control, 59: 714-720, (2016). [2]. barone g., giacominellistuffler r., storelli m. m., evaluation of trace metal and polychlorinated biphenyl levels in tea brands of different origin commercialized in italy, food and chemical toxicology, 87: 113-119, (2016). [3]. griboff j., wunderlin d. a., monferran m. v., metals, as and se determination by inductively coupled plasmamass spectrometry (icp-ms) in edible fish collected from three eutrophic reservoirs. their consumption represents a risk for human health, microchemical journal, 130: 236-244, (2017). [4]. mleczek m., niedzielski p., kalač p., budka a., siwulski m., gąsecka m., sobieralski k., multielemental analysis of 20 mushroom species growing near a heavily trafficked road in poland, environmental science and pollution research, 23(16): 16280-16295, (2016). [5]. alkiş i̇. m., öz s., atakol a., yilmaz n., anli r. e., atakol o. investigation of heavy metal concentrations in some turkish wines, journal of food composition and analysis, 33(1): 105-110, (2014). [6]. tariba b., metals in wine impact on wine quality and health outcomes, biological trace element research, 144(1-3): 143-156, (2011). [7]. dehelean a., voica c., determination of lead and strontium isotope ratios in wines by inductively coupled plasma mass spectrometry, romanian journal of physics, 57: 1194-1203, (2012). [8]. sadak o., sundramoorthy a. k., gunasekaran s., highly selective colorimetric and electrochemical sensing of iron (iii) using nile red functionalized graphene film, biosensors and bioelectronics, 89: 430436, (2017). [9]. oroian m., romanian white wine authentication based on mineral content, journal of agroalimentary processes and technologies, 21: 9-13, (2015). [10]. coli m.s., rangel a. g. p., souza e. s., oliveira m. f., chiaradia na. c. n, chloride concentration in red wines: influence of terroir and grape type, food science and technology (campinas), 35(1): 9599, (2015). [11]. pohl p., what do metals tell us about wine?, trac trends in analytical chemistry, 26(9): 941-949, (2007). [12]. rousseva m., kontoudakis n., schmidtke l. m., scollary g. r., clark a. c., impact of wine production on the fractionation of copper and iron in chardonnay wine: implications for oxygen consumption, food chemistry, 203: 440-447, (2016). [13]. trujillo j. p. p., conde j. e., pont m. l. p., câmara j., marques j. c., content in metallic ions of wines from the madeira and azores archipelagos, food chemistry, 124(2): 533-537, (2011). [14]. taylor v. f., longerich h. p., greenough j.d., multielement analysis of canadian wines by inductively coupled plasma mass spectrometry (icp-ms) and multivariate statistics, journal of agricultural and food chemistry, 51(4): 856-860, (2003). [15]. álvarez m., moreno i. m., jos á. m., cameán a. m., gonzález a. g., study of mineral profile of montilla-moriles “fino” wines using inductively coupled plasma atomic emission spectrometry methods, journal of food composition and analysis, 20(5): 391-395, (2007). [16]. stafilov t., karadjova i., atomic absorption spectrometry in wine analysis, macedonian journal of chemistry and chemical engineering, 28(1): 17-31, (2009). [17]. bora f. d., donici a., voica c., rusu t., cimpoiu c., nicula c., anca p., bunea i. c., pop n., mihăiescu d. e., inductively coupled plasma-mass spectrometry (icp-ms) characterization of some white wines from dealubujorului vineyard by their mineral content, aab bioflux, 8 (3):156-174, (2016). [18]. sidor a.m., the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophaerhamnoides l.) berries and juice, journal of faculty of food engineering, ştefan cel mare university of suceava, romania, xiv(3): 327 – 330, (2015). [19]. toaldo i. m., fogolari o., pimentel g. c., de gois j. s., borges d. l., caliari v., bordignon-luiz m., effect of grape seeds on the polyphenol bioactive content and elemental composition by icp-ms of grape juices from vitislabrusca l. lwt-food science and technology, 53(1):1-8, (2013). [20]. compendium of international analysis of methods – oiv multielemental analysis using icp-ms, oiv-maas323-07. food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xvi, issue 1 – 2017 contents: 1 study on the evolution of microand macroelements during the winemaking stages: importance of copper and iron quantification liliana norocel, gheorghe gutt 5 12 2 sensory characteristics of peach and plum jams with differents weeteners viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski 13-20 3 assessment of environmental impact of food loss: case study apple fruits cristina ghinea 21-28 4 oxidative stability of refined sunflower oil at room temperature and during conventional frying gorica pavlovska, natasha shurkova, vezirka jankuloska 29-33 5 determination of chlorpyrifos in apple from the resen region vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski 34-39 6 chemical composition and energy value in the meat of macedonian and ohrid trout aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski 40-46 7 rationalization of the parameters of milk proteins’ thermo acid coagulation by berry coagulants olena grek, olena onopriichuk, tetiana pshenychna 47-53 8 impact of adulteration with glucose, fructose and hydrolysed inulin syrup on honey physicochemical properties sorina ropciuc, mircea oroian, vlad olariu 54 60 author instructions i v subscription information vi 89 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2-2019, pag.89 96 photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica oriolowo oloruntoba, *abdulhamid hamza department of chemical engineering, ahmadu bello university zaria, nigeria, ahamza@abu.edu.ng *corresponding author received 29th april 2019, accepted 29th june 2019 abstract: production of biodiesel from waste cooking oils via alkali catalyzed transesterification reaction is hampered by the unacceptably high amount of free fatty acids (ffa) present in waste cooking oils. therefore, catalytic esterification of waste cooking oil with small alcohols is performed before the transesterification reaction. herein, zno/silica composite was synthesized via mechanochemical route and characterized using x-ray diffraction, x-ray fluorescence spectroscopy and scanning electron microscopy. photochemical and photocatalytic esterification of free fatty acids in a sample of waste cooking oil with methanol was performed under visible light irradiation. the efficiency of photochemical esterification was found to be strongly dependent on the power of the light used. significant increase in the efficiency of the photochemical esterification was observed when synthesized zno/silica composite was used as the photocatalyst. the photocatalytic activity of the zno/silica composite under visible light irradiation is attributed to the impurities present in the silica support. photocatalytic esterification using zno/silica composite is a promising method to reduce the ffa content of waste cooking oil under ambient conditions. keywords: biodiesel, waste cooking oil, esterification, photocatalyst, zno/silica composite. 1. introduction biodiesel is a clean biofuel produced from renewable feedstock, mainly vegetable oils, green algae and animal fats. biodiesel is a renewable alternative to petroleum based diesel that can be used in compression-ignition engines with little or no modification [1]. biodiesel is mostly produced via catalytic transesterification of triglycerides contained in fats and oils with methanol [2]. utilization of waste cooking oil for biodiesel production will add value to it, and reduce environmental pollution. production of biodiesel from waste cooking oil via alkali catalyzed transesterification reaction is hampered by the unacceptably high amount of free fatty acids present in waste cooking oil with may cause soap formation [3,4]. soap formation reduces the yield of biodiesel, and also leads to increase in catalyst consumption [5]. this results in high production cost of biodiesel. therefore, fats and oils containing high level of free fatty acids must undergo esterification of their free fatty acids with small alcohols before the actual transesterification process is performed. esterification of free fatty acids to yield alky esters in the presence of an acidic catalyst is the conventional route to reduce free fatty acids of oils used in biodiesel production. esterification is commonly http://www.fia.usv.ro/fiajournal mailto:ahamza@abu.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 90 carried out in a homogeneous phase in the presence of acidic catalysts because of its high conversion efficiency and lower cost. however, the use of the homogenous acidic catalysts has some drawbacks such as effluent disposal problem, corrosion of process equipment, loss of catalysts, high amount of alkali required for neutralization of the effluent, etc. in view of these problems of homogeneous catalysis, heterogeneous catalysis has been considered in order to minimize environmental pollution and reduce production cost of biodiesel. several heterogeneous catalysts have been developed for esterification of biodiesel feedstock with high free fatty acids [6, 7]. photocatalysis is a light induced catalytic process that involves oxidization and/or reduction of organic molecules via redox reactions activated through the electronhole pair generated on the surface of semiconductors upon light irradiation [810]. heterogeneous photocatalysis has many advantages over thermal catalytic processes because photocatalysis can be performed at room temperature and atmospheric pressure using direct solar energy or artificial light sources, thereby reducing operating costs. it is environmentally friendly process and enables reusability of the catalyst which minimizes its consumption [11]. most research works on photocatalysis focus on treatment of polluted water and air [10, 12, 13] very few works have been reported on free fatty acids esterification by a heterogeneous photocatalytic process [14, 15]. zno is one of the most extensively used heterogeneous photocatalysts for various processes under solar and/or ultraviolent irradiation [16, 17]. although the free fatty acids could be photocatalytically esterified using zno alone, separation of zno particles after photocatalytic esterification is a difficult problem. this problem can be solved by anchoring zno particles on suitable support materials. thus, corro et al [15] synthesized zno/silica and applied it for photocatalytic esterification of jatropha curcas oil under uv irradiation. 96 % conversion of free fatty acids was achieved at a fixed methanol to oil ratio of 12:1, photocatalyst dosage of 15 wt% and uv irradiation time of 4 hours [4]. to the best of our knowledge, there is no any published literature report on the photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation. thus, this paper focuses on photochemical and photocatalytic esterification of free fatty acids in waste cooking oil using zno/silica composite under visible light irradiation. 2. materials and methods materials the waste cooking oil was obtained from a restaurant in zaria, kaduna state, nigeria. the waste cooking oil was filtered to remove impurities. the oil was analyzed following standard methods [18]. the saponification value of the waste cooking oil was determined according to titremetric method described by pearson [18]. the acid value (a) of the raw oil before and after the photoesterification reaction was determined according to equation (1): m cv a   1.56 (1) where v is the volume (ml) of standard volumetric potassium hydroxide (koh) solution used, c is the concentration (mol/dm3) of the standard volumetric koh solution used, m is the mass (g) of the oil sample and 56.1 is the molecular mass of koh. the molecular weight (m) of the oil was determined from saponification value (sv) and acid value (a) according to equation (2): asv m    310001.56 (2) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 91 the amorphous silica used was obtained via dealumination of kankara kaolin [19]. the procedure for mechanochemical synthesis of zno has been described elsewhere in details [20]. briefly, a mixture of zncl2, na2co3 and nacl were mixed and milled using a high energy ball milling machine (kera bv) for 8 hours (the molar ratio of nacl to zncl2 was 8:1). nacl was used as inert diluent to prevent agglomeration of the nanoparticles being formed. after the milling step, the products obtained were then calcined in an electric furnace (nabertherm gmbh) at 800 oc for 4 hours to yield zno. the product was thoroughly washed with deionized water and dried. silica supported zno was prepared by solid state dispersion method. thus, the required amount of zno was thoroughly mixed with the required amount of silica in absolute ethanol at 40 0c. ethanol was then evaporated. the zno/silica powder was heated in an electric furnace at 300 0c for 4 hours. the phase composition of the synthesized zno/silica composite was examined using a powder x-ray diffractometer (panalytical) employing cu kα radiation (λ=0.154nm). the morphology of the zno/silica composite was investigated using phenom pro-x scanning electron microscope (sem). the chemical composition of the zno/silica composite was obtained via x-ray fluorescence (xrf) spectroscopy using minipal-4 x-ray fluorescence spectrometer. photocatalytic and photochemical esterification experiments a 500-w halogen lamp was used in the experiments because it simulates solar radiation [21, 22]. all experiments were carried out at room temperature (30 ± 5 °c) and atmospheric pressure. photoesterification of the waste cooking oil was performed in a 500 ml conical flask. a mixture of waste cooking oil, zno/silica composite and methanol was poured into the flask. the waste cooking oil:methanol ratio was 12:1. the mixture was stirred with a magnetic stirrer under visible light irradiation provided by the 500w halogen lamp for 4 hours. samples were taken at 1 hour interval. the analytical samples were filtered to remove the particles of the zno/silica composite photocatalyst. the clear esterified waste cooking oils were then analyzed for free fatty acids. photochemical experiments were performed under similar conditions without the use of zno/silica composite. to study the effect of the power of the incident visible light on the photochemical esterification process, three lamps with different power ratings (100w, 200w and 500w) were used. the conversion of free fatty acids (% cfree fatty acids) was obtained using equation (3): %100%    i ti y acidsfree fatt a aa c (3) where ai is the acid value of the raw waste cooking oil and at is the acid value of the waste cooking oil after photocatalytic or photochemical esterification over a given irradiation time. 3. results and discussion properties of the waste cooking oil the acid value of the waste cooking oil used in this study is 11.22 mgkoh/g which corresponds to a free fatty acid value of 5.64 %. the waste cooking oil has saponification value and molecular weight of 180.29 mgkoh/g oil and 995.45 g/mol, respectively. it has been reported that the content of free fatty acids in oils and fats for base catalysed transesterification reactions shall not exceed 1% [23]. therefore, it is necessary to perform a pretreatment process (esterification) on such lipids prior to biodiesel production via base catalysed transesterification. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 92 characterization of the synthesized zno/silica catalysts table 1 shows the chemical composition of the synthesized zno/silica. the main constituents are sio2 (49.6 wt%) and zno (45.22 wt%). apart from zno and sio2, that were predominant, the catalyst also contained lower amounts of other metal oxides (k2o, cao, tio2, mno, fe2o3 etc). these impurities may alter the bandgap of zno thereby extending its activity into the visible region of the electromagnetic spectrum [24, 25]. table 1. chemical composition of the synthesized zno/silica composite component concentration, wt% component concentration, wt % sio2 49.600 zno 45.220 so3 0.400 cr2o3 0.019 cao 0.404 ti2o 0.870 tio2 0.071 er2o3 0.038 k2o 0.601 nd2o3 0.087 mno 0.023 bao 0.100 fe2o3 0.293 ag2o 1.980 seo2 0.220 cuo 0.017 the xrd pattern depicted in fig. 1 was employed to determine phase composition and crystallite size of the synthesized zno/silica composite. the peaks at 2θ = 31.770, 34.420, 36.240, 47.520, 56.570, 62.840, 66.350, 67.910, 69.080, and 74.480 are in good agreement with the powder diffraction standard data (jcpds no. 361451), corresponding to the hexagonal wurtzite structure of zno. the strong and sharp diffraction peaks indicate that the synthesized zno via mechanochemical method is of high crystallinity. a crystalline peak with low intensity appeared at bragg’s angle of 26.650 due to crystalline silica, which also known as quartz [19]. the other peaks observed were rough background peaks due to the presence of amorphous silica. thus, the xrd pattern of the zno/silica composite is dominated by the characteristics peaks of the wurtzite phase of zno; this shows that the amouphous silica does not alter the crystalline structure of zno. the same observation was also reported by other researchers [26]. the highest intensity peaks in the xrd pattern (fig. 1) were used for computing the crystallite size (d) of the prepared zno/silica composite using the debye-scherrer formula given by equation (4):   cos d 9.0 (4) where λ is the wavelength of cu kα radiations (0.1542nm), β is the width at half maximum in radians and θ the angle obtained from 2θ value of the reference peak in the xrd pattern. the computed crystallite size of the synthesized zno/silica composite was found to be 20.92 nm. the scanning electron microscopic (sem) image presented in fig. 2 reveals deposition of zno particles on the silica. zno forms small crystallites on the external surface of the silica. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 93 fig. 1. xrd patterns of the synthesized zno/silica composite fig. 2. scanning electron micrographs of the synthesized zno/silica composite photochemical esterification of the waste cooking oil the results of photochemical esterification of waste cooking oil are depicted in fig. 3, from where it can be seen that the conversion of free fatty acids increases with increase in irradiation time in the absence of a photocatalyst. it has been reported that esterification of carboxylic acids with various alcohols in carbon tetrachloride can be accomplished efficiently by exposure of the solution to uv light [27]. in this work, light power for the photochemical esterification process was varied between 100w and 500w. as apparent in fig. 3, conversion of free fatty acids increases with increase in lamp power. these results indicate that reduction of free fatty acids in waste cooking oil could be achieved without a photocatalyst. however, photochemical esterification of waste cooking oil strongly depends on the light power. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 94 fig. 3. effect of irradiation time and lamp power on photochemical esterification of the waste cooking oil in the absence of a photocatalyst photocatalytic esterification of the waste cooking oil using the synthesized zno/silica composite fig. 4 shows the results of photocatalytic esterification of waste cooking oil using zno/silica composite. compared to the results presented in fig. 3, there is a significant enhancement of conversion of the free fatty acids by the zno/silica photocatalyst. for example, after one hour of irradiation with the 500 w halogen lamp, the conversion of free fatty acids achieved via photochemical esterification and photocatalytic esterification are 35 % and 87 %, respectively. therefore, photocatalytic esterification using zno/silica is by far more efficient than photochemical esterification. the conversion of free fatty acids increases with increase in irradiation time. the reaction was very fast during the first one hour due to the high driving force. at irradiation time of 4 hours, 94 %conversion of free fatty acids of the waste cooking oil was achieved. this illustrates the positive role of zno/silica in photocatalysing the esterification reaction. therefore, the use of zno/silica will reduce the cost of photocatalytic esterification process. fig.4. effect of irradiation time on photocatalytic esterification of waste cooking oil using zno/silica composite and 500 w lamp food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 95 4. conclusion the reduction of free fatty acids in the waste cooking oil via photochemical esterification is possible, but the photochemical esterification process is less efficient than the photocatalytic esterification. the efficiency of photochemical esterification is strongly dependent on the intensity of light. photocatalytic esterification is a promising process to reduce the ffa content of waste cooking oil under visible light irradiation at room temperature and atmospheric pressure. 5. references [1].guo m, song w, buhain j. bioenergy and biofuels: history, status, and perspective. renewable and sustainable energy reviews. 2015;42:712-25. [2].baskar g, aiswarya r. trends in catalytic production of biodiesel from various feedstocks. renewable and sustainable energy reviews. 2016;57:496-504. [3].urrutia c, sangaletti-gerhard n, cea m, suazo a, aliberti a, navia r. two step esterification–transesterification process of wet greasy sewage sludge for biodiesel production. bioresource technology. 2016;200:1044-9. [4].boz n, degirmenbasi n, kalyon dm. esterification and transesterification of waste cooking oil over amberlyst 15 and modified amberlyst 15 catalysts. applied catalysis b: environmental. 2015;165:723-30. [5].sirisomboonchai s, abuduwayiti m, guan g, samart c, abliz s, hao x, et al. biodiesel production from waste cooking oil using calcined scallop shell as catalyst. energy conversion and management. 2015;95:242-7. [6].veillette m, giroir-fendler a, faucheux n, heitz m. esterification of free fatty acids with methanol to biodiesel using heterogeneous catalysts: from model acid oil to microalgae lipids. chemical engineering journal. 2017;308:101-9. [7].zhou y, niu s, li j. activity of the carbonbased heterogeneous acid catalyst derived from bamboo in esterification of oleic acid with ethanol. energy conversion and management. 2016;114:188-96. [8].belver c, bedia j, gómez-avilés a, peñas-garzón m, rodriguez jj. semiconductor photocatalysis for water purification. nanoscale materials in water purification. 2019. p. 581-651. [9].sharma mp, kumari vd, subrahmanyam m. photocatalytic degradation of isoproturon herbicide over tio2/al-mcm-41 composite systems using solar light. chemosphere. 2008;72(4):644-51. [10]. vasilache v, catalin p, vasilache t. new materials for photocatalytic purification of air–a review. food and environment safety journal. 2017;10(4). [11]. apostol lc, caliman af, gavrilescu m. studies regarding the photocatalytic transformation of erythrosine b. food and environment safety journal. 2016;14(4). [12]. ahmed sn, haider w. heterogeneous photocatalysis and its potential applications in water and wastewater treatment: a review. nanotechnology. 2018;29(34):342001. [13]. kobasa i. photocatalytic degradation of methylene blue on nanostructured composites based on tio2-bi2o3. food and environment safety journal. 2017;10(4). [14]. manique mc, silva ap, alves ak, bergmann cp. application of hydrothermally produced tio2 nanotubes in photocatalytic esterification of oleic acid. materials science and engineering: b. 2016;206:17-21. [15]. corro g, pal u, tellez n. biodiesel production from jatropha curcas crude oil using zno/sio2 photocatalyst for free fatty acids esterification. applied catalysis b: environmental. 2013;129:39-47. [16]. ong cb, ng ly, mohammad aw. a review of zno nanoparticles as solar photocatalysts: synthesis, mechanisms and applications. renewable and sustainable energy reviews. 2018;81:536-51. [17]. lee km, lai cw, ngai ks, juan jc. recent developments of zinc oxide based photocatalyst in water treatment technology: a review. water research. 2016;88:428-48. [18]. anyasor g, ogunwenmo k, oyelana o, ajayi d, dangana j. chemical analyses of groundnut (arachis hypogaea) oil. pakistan journal of nutrition. 2009;8(3):269-72. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 oriolowo oloruntoba, abdulhamid hamza, photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica, food and environment safety, volume xviii, issue 2 – 2019, pag. 89 – 96 96 [19]. salahudeen n, ahmed a, almuhtaseb a, dauda m, waziri s, jibril b. synthesis and characterization of micro-sized silica from kankara kaolin. journal of engineering research. 2014;19:27-32. [20]. ao w, li j, yang h, zeng x, ma x. mechanochemical synthesis of zinc oxide nanocrystalline. powder technology. 2006;168(3):148-51. [21]. yoong l, chong fk, dutta bk. development of copper-doped tio2 photocatalyst for hydrogen production under visible light. energy. 2009;34(10):1652-61. [22]. lai y-k, huang j-y, zhang h-f, subramaniam v-p, tang y-x, gong d-g, et al. nitrogen-doped tio2 nanotube array films with enhanced photocatalytic activity under various light sources. journal of hazardous materials. 2010;184(1-3):855-63. [23]. berrios m, siles j, martin m, martin a. a kinetic study of the esterification of free fatty acids (ffa) in sunflower oil. fuel. 2007;86(15):2383-8. [24]. bora lv, mewada rk. visible/solar light active photocatalysts for organic effluent treatment:fundamentals, mechanisms and parametric reviews. renewable and sustainable energy reviews. 2017;76(1):1393-1421. [25]. hamza a, john ij, mukhtar b. enhanced photocatalytic activity of calcined natural sphalerite under visible light irradiation. journal of materials research and technology. 2017;6(1):1-6. [26]. siddiquey ia, furusawa t, sato m, bahadur nm, alam mm, suzuki n. sonochemical synthesis, photocatalytic activity and optical properties of silica coated zno nanoparticles. ultrasonics sonochemistry. 2012;19(4):750-5. [27]. hwu jr, hsu c-y, jain ml. efficient photolytic esterification of carboxylic acids with alcohols in perhalogenated methane. tetrahedron letters. 2004;45(26):5151-4. 1. introduction 27 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 12019, pag. 27 35 calcium requirement in relation to milk fever for high yielding dairy cows: a review *angassa tesfaye 1 1faculty of agricultural science, bule hora university, p.o.box: 144, bule hore, ethiopia, angassatesfaye@yahoo.com *corresponding author received 16 th december, 2018, accepted 28 th march 2019 abstract: the purpose of this paper is to review the predisposing factors and advances in controlling principles of milk fever in high yielding dairy cows. dietary imbalances in dairy cows can be one of fundamental cause of numerous and complicated types of health problems that are usually categorized as metabolic disorders. high producing dairy cows are among highly susceptible animal to such metabolic disorders during their transitional periods; three weeks before and after calving. in these periods, the animal struggles to maintain their calcium homeostasis; which is the most common metabolic disorder of high yielding dairy cows, due to their underlying physiological and pathological factors. thus, those factors predisposing dairy cows to milk fever are age, stage of lactation and parity, body condition and diet. usually, the basic principle of controlling milk fever is feeding the cows from diet constituting lower calcium and potassium contents in dry period. so that calcium mobilization from bone would be promoted, which ensures the increased metabolic processes of calcium mobilization during the transition period? furthermore, supplementations of various anionic salts which includes magnesium sulfate, calcium sulfate, ammonium sulfate, calcium chloride, ammonium chloride and magnesium chloride are commonly used to raise blood calcium level, thus, minimizes risks of milk fever. also, body condition management of cows during dry period is used to prevent milk fever. finally, a recent research indicates that serotonin intravenous injections have significantly reduced the incidence of hypocalcemia in high yielding dairy cows. keywords: calcium, dairy cows, hypocalcemia 1. introduction mineral nutrients are inorganic elements required for normal physiological and anatomical functioning and health of the animals. based on their abundance in the body, the minerals can be macro-mineral or micro-mineral; were the later one is referred as trace elements, due to its relatively smaller amounts. but macrominerals are those minerals that are relatively, required with higher quantities than micro-minerals. therefore, these macro-minerals are calcium, phosphorus, magnesium, sodium, potassium, chloride and sulphur. calcium is the most abundant mineral in the body of the animals, where its significant proportion (99 % of calcium) is dominantly found in the bone. in the bones, together with phosphorus, it forms hydroxyapatite, which is an important component of the skeletal structure of vertebrates. while, the remaining amount of calcium is extracellular, it plays a crucial role of muscle contractions and normal functioning of nervous system. however, the calcium in the bones is not a static mass, but it is in continuous exchange with the extracellular fluids. the intracellular calcium fraction is involved in signaling within the cell. in plasma, calcium is found as protein-bound calcium, http://www.fia.usv.ro/fiajournal mailto:angassatesfaye@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 28 ionized calcium and calcium bound in complexes with the anions. the fraction of ionized calcium must be maintained at a constant level to ensure the normal functioning of muscles and nerves [1]. milk fever (mf) is one of the most common mineral-related metabolic conditions affecting dairy cows during transition, a disorder that occur immediately after or shortly before calving as a result of a low level of calcium in the blood (hypocalcaemia) [2]. milk fever and subclinical hypocalcemia (total blood calcium ≤ 2.0 mmol/l) are the most important macro-mineral disorders affecting transitioned high yielding dairy cows [3]. however, a mild degree of hypocalcemia in the majority of cows during the peripartum period and usually associated calving problems, retained placenta, uterine prolapse, mastitis, ruminal stasis, depression of the immune system and generally reduced reproductive performance of the animals [4]. in a small proportion of animals, hypocalcemia becomes severe and results in paresis, lying down and occasionally death [5]. about 50% of dairy cows in their second lactation and greater have blood ca concentrations that fall below the threshold for subclinical hypocalcemia after calving [6]. this insufficient blood calcium concentration can make a cow to lose its ability to rise up to her feet, as calcium is responsible for normal nerve and muscle functioning. this can cause a metabolic disorder termed as milk fever, also known as post-parturient hypocalcemia or parturient paresis. it is most common metabolic disorder of older and high yielding dairy cows [7]. most of the cows experience subclinical hypocalcemia after calving, but some experience severe blood calcium drops that leads to milk fever. the normal blood calcium level is ranges between 8 and 12 mg/dl. however, when blood calcium is reduced to 7.5 – 5mg/dl, the stomach motility is reduced by 30 and 70 % respectively [8]. the reduced abomasum motility due to lowered blood calcium leads to displaced abomasum. the low calcium requirements of cows during the dry period become suddenly elevated after parturition. this sudden calcium drain from blood must be counter balanced by increased calcium absorption from gut or calcium mobilization from bone. thus, during this time the parathyroid hormone is released to enhance calcium resorption from bone. but, hypocalcemia occurs when the mechanism of calcium absorption and resorption are insufficient to meet calcium demands. therefore, if not treated soon, most of the wows will die within a few days [9]. therefore, this paper is a review on predisposing factors and the preventive strategies of milk fever and subclinical hypocalcemia in high yielding dairy cows. 2. what is milk fever? milk fever and subclinical hypocalcemia are the most important macro-mineral disorders that affect transition. milk fever and subclinical hypocalcemia (total blood calcium ≤2.0 mmol/l) are the most important macro-mineral disorders that affect transition dairy cows [10]. adequate circulating ca concentrations throughout the transition period are necessary for productive lactation, but large quantities of ca are lost from maternal ca pools into milk and colostrum. a rapid, large drop in maternal blood ca causes 5 – 10 % of cows to be afflicted with clinical hypocalcemia and additional 50 % to suffer from subclinical hypocalcemia. subclinical hypocalcemia and clinical hypocalcemia were the major reason for early lactation removal of cows from the herd [11] [6] [12]. the transition period is an extremely critical period of milk fever in high yielding dairy cows, were jersey cows are being more susceptible than holstein food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 29 friesian [13] [14]. at this time to insufficient blood calcium levels at the onset lactation, depending on the extent of dropped blood calcium, the animals exhibit a range of clinical symptoms [15]. in clinical hypocalcemia the blood calcium level drops below 1.4 mmol/l, while in subclinical hypocalcemia the total blood calcium level drops into 1.4 – 2.00 mmol/l [11]. the cows that are once affected with parturient paresis are more susceptible to several metabolic disorders such as ketosis, retained placenta, displaced abomasum and muscle weakness [11] [13] [16] [17]. overall, the study conducted in usa, indicated that the prevalence of milk fever and subclinical hypocalcemia are more common in jersey cows; due to their higher milk production per unit body weight. in general, dairy cattle exhibit a delay in calcium resorption from bone resulted in manifestation of milk fever [13]. 3. prevention strategies of milk fever 3.1. feeding of low-calcium diets conventionally, it was known that feeding with low calcium diets during dry period is one of milk fever prevention strategy. this was achieved through feeding the cows with less than 50 g per day. therefore, to do so, high calcium forages such as alfalfa, have to be eliminated from animal’s diet. forages such as corn silage and grass hay have to be routinely used in dry from to reduce calcium composition within it [9]. calcium requirements for the non-lactating cow are as minimal as 41 grams/day for a 1400lb cow [18], being necessary only for maintenance and fetal skeletal development. hence, when calcium requirements are met through dietary feed, the metabolic processes of calcium mobilization from bone will be inactivated. however, upon parturition and onset of lactation, there is an immediate demand for large amounts of calcium for colostrum production. at this time, the dietary calcium could not be met the sudden demand of calcium, normally, due to dry matter intake decreases a few weeks before parturition. therefore, calcium must be mobilized from bone in order to meet this sudden demand of calcium. however, since calcium requirement during dry period being met from diet, the mobilization of calcium from bone is very minimal, so that the cows experience milk fever [9]. in contrary, underfeeding of calcium during dry period promotes calcium mobilization from bone and ensures calcium homeostasis during parturition. in addition, calcium absorption from small intestine is proportionally higher when the cow fed lower calcium during dry period. thus, a low-calcium diet will promote efficient resorption of calcium from bone, as well as calcium absorption from small intestine [9]. 3.2. feeding of anionic salts the anionic salts are simply minerals that possess high proportion of anions. anions are ions that are negatively charged and cations are ions that are positively charged. the tissues of living being maintain a balance of cations and anions, so that they remain neutral. so that, in order to not interfere with anionic and cationic balance, their net sum in the feeds of the animals should be near neutral. however, certain cations and anions, such as sodium, potassium, chloride and sulfur have significant effects on acid-base balance of the body. therefore, quantifying these major cations and anions in animal diet are very important. the quantification is done by calculating the dietary cation-anion difference (dcad); which is achieved by a subtracting a sum of dietary cations (i.e. sodium and potassium) from anions (i.e. chloride and sulfur) [9]. a negative dcad, diet contains more equivalents of anions than cations, a zero food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 30 dcad diet contains equal equivalents, and a positive dcad diet contains more cation equivalents. this was used to estimate the influence of cation and anions in the diet on acid-base status of animals. hence, the anionic diet (negative dcad) will induce metabolic acidosis and lower blood ph [7]. the induced metabolic acidosis, was therefore, suggested to increases tissue responsiveness to parathyroid hormone, in resorption of calcium from bone [19] [20]. the desired effect of feeding anionic salts is to decrease blood ph, which is highly correlated with reduced urine ph and increased blood calcium. thus, urine ph is used as indicator for blood ph. thus, an anionic diet, a negative dcad diet, induces a mild metabolic acidosis, normal blood calcium and a urine ph between 6.5 and 5.5 in close up cows [7]. urine ph can be monitored on the farm using ph paper or a ph meter, thus when urine ph is greater than 7.0, it should be considered to supplement anionic salts. but, when urine ph is less than 5.5, the anionic salts salt intake is very high, so that consider anionic salts intake reduction [19]. in contrary, excessive anionic salts in the diet are usually, associated with reduced feed intake, displaced abomasum and kidney overload. however, the addition of anionic salts such as magnesium sulfate, calcium sulfate, ammonium sulfate, calcium chloride and magnesium chloride are the only means of achieving a negative dcad [21]. although supplementing close-up dry cow with the diets of anionic salts can reduce the incidence of milk fever, but excessive intake of anionic salts can pause potential problems to dairy cows. for instance, it can cause severe reduction in dry matter intake. further, the severe reduction in dry matter intake near parturition can predispose animals to metabolic disorders such as displaced abomasum, milk fever and ketosis, it is necessary to closely monitor in close-up cow, not to feed excessive level of anionic salts [9]. 3.3. management of body condition score (bcs) management of body condition score during dry period and calving is another important means of achieving milk fever. the research finding reported that, the dairy cows that are over-conditioned at calving are up to four times more likely to develop milk fever [22]. it is uncertain that why this case is happening, but several hypotheses have been suggested that firstly, dairy cows with higher bcs at calving have higher ca output in milk, which makes them more prone to milk fever. secondly, it has been widely appreciated that over-conditioned dairy cattle have a reduced feed intake relative to thinner cows, in the last 7 – 10 days of precalving. this may result in reduction of ca and mg intake and predisposing the animals to the development of hypocalcemia [3]. 3.4. the intravenous infusion of serotonin obviously, colostrum and milk synthesis rapidly deplete ca from the maternal circulation and therefore, ca must be mobilized from maternal bone to maintain adequate circulating ca concentrations become dip below the animal’s minimal threshold for ca, via a classic negative feedback loop [23]. circulating ca concentrations are tightly regulated and controlled by one of the parathyroid hormones related-protein (pthrp) hormones. recently, the research experiments demonstrated that mammary serotonin (5-hydroxytryptamine) regulates induction of pthrp. thus, the manipulation (injection) of serotonin, induces pthrp synthesis near the end of the pregnancy period that could be critical in preventing the onset of hypocalcemia during the early lactation period [24]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 31 however, though the overall circulating serotonin concentration are not constant within the course of given lactation period, its average is approximated at 1700ng/ml. thus, it decreases around the time of calving period (from 0 to 2 days) and bounce back by ten days into lactation [25]. therefore, according to hernandez [26], the intravenous infusion of 5-htp in lactating, non-pregnant, multiparous holstein dairy cows have shown, a significant increase in circulating serotonin concentrations and altered ca dynamics and it can be used to prevent hypercalcemia in high yielding dairy cows. 4. predisposing factors for hypocalcemia according to reports of several authors [27] [28] [29] [30] [31] [32], the factors contributing to milk fever and which influence its incidence and degree of severity are quite numerous. therefore, these factors include: age parity of cows, breeds differences, body condition score, dietary factors, potassium supplementation and pathophysiology. 4.1. age and parity of cows the incidence of developing milk fever is increased with age cow [14][33]. obviously, milk yield in dairy cows, from third lactation onwards, start to increase, which lead to high calcium demand for milk production. in addition to increase in milk production, increase in age also results in diminishing of the capability of mobilizing calcium from bone and declining of active transportation of calcium from intestine, which was resulted in increased risks milk fever incidence [34]. thus, hypocalcemia is usually highly frequent from third to seventh calving due to reduced adaptation capacity of cows during calving [35]. the hypocalcemia at calving is age related and most marked in cows from third to seventh parturition; it is infrequent at the first parturition. this is because while some degree of hypocalcemia occurs during the first few days of lactation, they are able to adapt rapidly to the high demands of calcium for lactation [36]. 4.2. breed certain breeds of dairy cows have shown more susceptibility to milk fever than other breeds. specifically, cross breed or temperate breeds are more susceptible to milk fever than tropical zebu breeds. this could be attributed to their high milk yield and low ability to maintain their calcium homeostasis of cross breed and temperate breed [38]. the other important reason of higher susceptibility to milk fever is lower number of intestinal receptors for 1, 25(oh) d, which responsible intestinal calcium absorption and bone calcium resorption [37]. 4.3. body condition score according to ostergard et al., [22], a high body condition score of dairy cows can raise the risk of milk fever. the dairy cows that are over conditioned at calving are four times greater in developing milk fever than skinny cows. this is due to, the dairy cows with higher bcs at calving have higher calcium output in milk and reduced feed intake due to loss of appetite. hence, a combined effect of calcium loss through milk and reduced feed intake predispose high yielding dairy cows to parturient paresis [39]. furthermore, bewley and schultz [39], suggested that the cows with over conditioned during calving and/or excessive weight loss just after calving demonstrated increased risks of dystocia, retained placenta, metritis, milk fever, cystic ovaries, lameness, mastitis, ketosis and metabolic disorders. 4.4. dietary factors boda and cole [39], indicated that providing a diet with high daily calcium food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 32 intake to non-lactating cows are associated with increased incidence of parturient paresis. primarily, during the dry period, the maintenance requirement of calcium can be predominantly, met by passive absorption since active absorption of dietary calcium and bone resorption are then suppressed. the cows in this condition are not able to quickly place blood calcium lost in milk and become severely hypocalcemia. excessive dietary phosphorus intake (>80g/day) during late gestation can also induce milk fever and the severity of hypocalcemia by raising blood phosphorus concentrations to the point that can inhibits renal synthesis of 1, 25 – (oh), which is then reduces intestinal calcium absorption [41]. generally, the pre-partum diets high in cations like sodium and potassium are associated with increasing incidence of milk fever, while the diets with high in anion such as chlorides and sulfides results in decreased milk fever incidence. the addition of anions to dairy cow’s diet prior to parturition are claimed effective in reducing the incidences of milk fever by inducing metabolic acidosis, calcium bone resorption [42]. 4.5. potassium supplementation high potassium forages have been highly associated with milk fever in high yielding dairy cows. usually, alfalfa has long been considered as high potassium contents and leads to milk fever. hence, in perspective high potassium levels may have been the cause of milk fever, in association with alfalfa-based dry cow diets. grass hay can have lower level of potassium, but overfertilizing with potassium on hay fields increases, both soil and hay potassium levels. high soil potassium often occur in the fields were manures have been applied for many years. but, harvesting of forages from such fields and separately feeding dry cows are recommended, since potassium level drops as plants mature and get dried [9]. 4.6. pathophysiology during the dry period, calcium demand is relatively low. thus, the activity of intestinal and/or bone resorption of calcium is relatively sluggish during this time. the onset of lactation at the time of parturition results in sudden loss of calcium into milk. however, at this time when the calcium homeostasis is unable to meet the demand of calcium for milk production, the cow develops milk fever [43].this fall in blood calcium level stimulates the calcium homeostatic mechanism to improve intestinal calcium absorption and increase bone resorption. approximately 24 hours of elevated 1, 25 dihydroxy vitamin d3 is required to improve intestinal transport of calcium and an increased rate of bone resorption requires 48-hours of pth stimulation. but, when these compensatory mechanisms are prolonged for more than 48-hours, then the cows develop clinical hypocalcemia. consequently, most of the cows with clinical hypocalcemia have shown higher level of pht and 1, 25 dihydroxy vitamin d3 [44] [45]. 5. conclusion milk fever and subclinical hypocalcemia are the most important macro mineral disorders of the transitioning cows. since, it is highly corelated with several production diseases and reproductive complication milk fever was one of economically important disease among most of highly yielding dairy herds. milk fever and subclinical hypocalcemia to large extent can be prevented through good dietary management of cows during dry period. the dietary calcium levels must be low in the weeks leading up to calving in order minimizes the risk of milk fever. anionic salt feeding, body condition food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 33 management, feeding low calcium and the recent outcome of serotonin infusion to high yielding dairy cows are among the most important mechanisms of controlling milk fever. 6. references [1]. k. cecilia, minerals to dairy cows with focus on calcium and magnesium balance. doctoral thesis swedish university of agricultural sciences uppsala 2011. [2]. a. khan, m. hassan,a. mushtaq, m. wali khan,chaudhry, and a. hussain, descriptive epidemiology and seasonal variation in prevalence of milk fever in kpk (pakistan). global vet.,2015, vol.14, pp.472-477. [3]. m. finbar, o.g. luke,r. desmond, and d. michael, production diseases of the transition cow: milk fever and subclinical hypocalcemia. irish veterinary journal,2006. vol.59 (12), pp.697-702. [4]. f. wubishet, t. dechassa, a. nejash, and m.a. wahid, milk fever and its economic consequences in dairy cows: a review. global veterinaria,2016, vol.16(5), pp.441-452. [5]. m.s. bhanugopan, and j. lievaart, survey on the occurrence of milk fever in dairy cows and the current preventive strategies adopted by farmers in new south wales, australia. aust vet j.,2014, vol.92, pp.200-205 [6]. t.a. reinhardt, j.d. lippolis, b.j. mccluskey, j.p. goff, and r.l. horst, prevalence of subclinical hypocalcemia in dairy herds. vet. j.2011, vol.188, pp.122124. [7]. j.p. goff, the monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. vet. j.2008,vol.176, pp.50-57. [8]. r.c.w. daniel, motility of the rumen and abomasum during hypocalcemia. can. j. comp. med. 1983, vol.47, pp.276. [9]. b. greg, v. rick, and f.s. john, controlling milk fever and hypocalcemia in dairy cattle: use of dietary cationanion difference (dcad)in formulating dry cow rations. technical report of extension dairy specialist, 1998, kansas state university. [10]. f.j. mulligan, l. o’grady, d.a. rice and m.l. doherty, a herd health approach to dairy cow nutrition and production diseases of the transition cow. animal reproduction science, 2006,96: 331–353. [11]. p.j. degaris, and i.j. lean, milk fever in dairy cows: a review of pathophysiology and control principles. vet. j.2008, vol.176, pp.58-69. [12]. t. roberts, n. chapinal, s.j. leblanc, d.f. kelton, j. dubuc, and t.f. duffield, metabolic parameters in transition cows as indicators for earlylactation culling risk. j. dairy sci.2012, vol.95, pp.3057-3063. [13]. g.r. oetzel, parturient paresis and hypocalcemia in ruminant livestock. vet. clin. north am. food anim. pract.1988, vol.4(2), pp.351-364. [14]. national research council (nrc). subcommittee on dairy cattle nutrition. in nutrient requirements of dairy cattle, 7th eds. national academy press, 2001, washington dc. [15]. r. adams, v. ishler, and d. moore. trouble-shooting milk fever and downer cow problems. das. ivef, 1996, vol.216, pp.1-7. [16]. n. chapinal, m. carson, t.f. duffield, m. capel, s. godden, m. overton, j. e. santos, and s. j. leblanc, the association with serum metabolites with clinical disease during the transition period. j. dairy sci.2011, vol.94, pp.48974903. [17]. n. chapinal, s.j. leblanc, m.e. carson, k.e. leslie, s. godden, m. capel, j.e.p. santos, m.w. overton, t.f. duffield. herd-level association of serum metabolites in the transition period with disease, milk production, and early food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 34 lactation reproductive performance. j. dairy sci.2012, vol.95, pp.5676-5682. [18]. national research council (nrc), nutrient requirements of dairy cattle, sixth revised edition. national academy press, 1989, washington, d.c. [19]. r.l. horst, j.p. goff, t.a. reinhardt, and d.r. buxton. strategies for preventing milk fever in dairy cattle. j. dairy sci.,1997, vol.80, pp.1269. [20]. j. davidson, l. rodriguez, t. pilbeam, and d. beede, urine ph check helps avoid milk fever. hoard’sdairyman,1995, vol.140, pp.16:634. [21]. s.j. moore, m.j. vandehaar, b.k. sharma, t.e. pilbeam, d.k. beede, h.f. bucholtz, j.s. liesman, r.l. horst, and j.p. goff. varying dietary cation anion difference (dcad) for dairy cattle before calving. j. dairy sci.1997, vol.1, pp.170. [22]. s. ostergaard, j. sorensen and h. houe, a stochastic model simulating milk fever in a dairy herd. prev vet. med.,2003, vol.58, pp.125-143. [23]. j.p. goff, macro mineral disorders of thetransition cow. vet clin of north america.,2004, vol.20,pp.471-494. [24]. l.l. hernandez, k.a. gregerson, and n.d. horseman, mammary gland serotonin regulates parathyroid hormonerelated protein and other bone-related signals. am. j. physiol. endocrinol. metab,2012, vol.302(8), pp.1009-1015. [25]. s.a.e. moore, j. laporta, t.d. crenshaw, and l.l. hernandez, patterns of circulating serotonin (5-ht) and related metabolites in multiparous dairy cows in the periparturm period. j. dairy sci.,2015, vol. 98(6), pp.3754-3765. [26]. l.l. hernandez, s.w. limesand, j.l. collier, n.d. horseman, and r.j. collier, the bovine mammary gland expressed multiple functional isoforms of serotonin receptors. j. endocrinol, 2009, vol.203, pp.123-131. [27]. j.r. roche, and d.p. berry, periparturient climatic, animal and management factors influencing the incidence of milk fever in grazing system. j. dairy sci.,2006, vol.89, pp.2775-2783. [28]. j. rehage, and m. kaske, interaction between milk yield and productive diseases in dairy cows. proc. soc. nutr.physiol.,2004, vol.13, pp.177-182. [29]. l.e. mcdowel, recent advances in mineral and vitamins on nutrition of lactating cows. pak j. nutr.,2002, vol.1, pp.8-19. [30]. p.m. fleischer, metzner, m., beyersbach, m., hoedemaker, m. and klee, w. the relationship between milk yield and the incidence of some diseases in dairy cows. j. dairy sci.,2001, vol.84,pp.2025-2035. [31]. v.j. taylor, hattan, a.j., bleach, e.c., beever d.e., and wathes, d.c. reproductive function in average and high yielding cows. brit. soc. anim. sci.,2001, vol.26, pp.495-498. [32]. h. houe, s. ostergaard, t. thilsinghansen, r.j. jorgensen, t. larsen, j.t. sorensen, j.f. agger, and j.y blom, milk fever and subclinical hypocalcemia. an evaluation of parameters on incidence risk, diagnosis, risk factors and biological effects as input for a decision support system for disease control. acta vet. scan.,2001, vol.42, pp.1-29. [33]. d.j. rezac, dietary cation anion difference and acidified co-products: effects on peripartum dairy cows. msc. thesis, manhattan, kansas: b.s., kansas state university, 2010. [34]. p.e. jawor, j.m. huzzey, s.j. leblanc, and m.a.g. von keyserlingk, associations of subclinical hypocalcemia at calving with milk yield and feeding, drinking and standing behaviors around parturition in holstein cows. journal of dairy science,2012, vol.95(3), pp.12401248. [35]. e. tadesse, and l. belete, an overview on milk fever in dairy cattle in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 angassa tesfaye, calcium requirement in relation to milk fever for high yielding dairy cows: a review, food and environment safety, volume xviii, issue 1 – 2019, pag. 27 – 35 35 and around west shoa. world j. biol and medical sci.,2015, vol.2, pp.115-125. [36]. s. anteneh, t. guadu, t. fentahun, and m. chanie, incidence of milk fever on dairy cows and its risk factors in gondar town, northwest ethiopia. global vet.,2012, vol.9, pp.659-662. [37]. w.p. weiss, e. azem, w. steinberg, and t.a. reinhardt, effect of feeding 25 hydroxy vitamin d3 with a negative cation-anion difference diet on calcium and vitamin d status of periparturient cows and their calves. j. dairy sci.,2015. vol.98, pp.5588-5600. [38]. d. harris, t. rukkwamsuk and t. wensing, relationship between over feeding and over conditioning in the dry period and the problems of high producing dairy cows during the post parturient period. vet. quarterly,1999, vol.21, pp.7177. [39]. j.m. bewley, and m.m. schutz, review: an interdisciplinary review of body condition scoring for dairy cattle. prof. anim. sci.,2008, vol.24, pp.507-529. [40]. j.m. boda, and h.h. cole, calcium metabolism with special references to parturient paresis in dairy cattle. j. dairy sci.,2003, vol.39, pp.1027. [41]. g.r. oetzel, non-infectious diseases: 2: 239-245 milk fever. encyclopedia of dairy sciences,2011, vol.2, pp.239-245. [42]. p. bradford, disorders of calcium metabolism. large animal internal medicine, 2nd edition, donlading press, usa, 1996, pp. 1464-1470. [43]. c.f. ramberg, e.d. johnson, and r.d. fargo, calcium homeostasis in cows with special reference to parturient hypocalcemia. american journal ofphysiology,1994, vol.246, pp.698. [44]. d.j. harris,factors predisposing to parturient paresis. australian veterinary journal,2002, vol.57, pp.357-361. [45]. a. iggo, activity of peripheral nerves and junctional regions. physiology of domestic animals, 10 editions, california university press, usa. 1994, vol.96, pp.331-353. 4.5. potassium supplementation title …………………… 398 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 42015, pag. 398 406 establishing the antioxidant activity based on chemical composition of wild edible mushrooms * ana leahu 1 , cristina damian 1 , mircea oroian 1 , sorina ropciuc 1 1 faculty of food engineering, stefan cel mare university of suceava, universitatiistr. 13,suceava, romania, analeahu@usv.ro *corresponding author receivednovember10 th 2015, accepted december 15 th 2015 abstract: the aim of this study is to determine the chemical compositions of fruiting bodies of widely used wild edible mushroom species (agaricus albolutescens, armillaria mellea, russula virescens, cantharellus cibarius and boletus edulis), commonly consumed in the bukovina region, from the northeast of romania. the content in water, crude protein, lipids, carbohydrates and ash, in fruiting bodies of mushroom samples was assessed. the antioxidant activities were performed on the 1,1´diphenyl-2-picrylhydrazyl (dpph) test. in addition to this, phenolic compounds were also analyzed. the carbohydrate contents were in the following order: agaricus albolutescens>boletus edulis>armillaria mellea>russula virescens and cantharellus cibarius. the protein also varied and ranged from 10.12 to 15.15 % dry weight. the polyphenol content was the same in the two mushrooms, whereas the antiradical activity was higher in russula virescens. the results suggested that consumption of agaricus albolutescens, armillaria mellea, russula virescens,cantharellus cibarius and boletus edulis might be beneficial to the antioxidant protection system of the human body against oxidative damage and others complications. keywords: mushrooms, carbohydrate, antioxidant, total phenolic content. 1. introduction mushrooms are an important dietary component for the modern consumer across the world, used for nutritional and medicinal purposes, due to the presence of metabolites with pharmacological potential. wild mushrooms are becoming more and more important in our diet due to their nutritional characteristics [1-3]. vegetables and mushrooms occupy an important position among functional food products due to their contents of many bioactive components that have a beneficial effect on human health and sense of wellbeing [4]. dietary fibers are the compounds resistant to hydrolysis, which the human body cannot use, but which play a considerable role in the prevention of diseases. they are: cellulose, chitin, and glucans. the impact of fiber consumption is positive because it can influence the digestion rate and it was also associated with a reduced risk of type 2 diabetes. recent clinical studies recommended mushrooms for inclusion in "functional foods" [5]. functional foods are those foods enriched/modified and consumed as normal diet to provide health benefits [6]. manzi et al., [7] evaluated the chemical composition of mushrooms, commercial samples, widely consumed in italy (boletus group, agrocybe aegerita and pleurotus eryngii). the results obtained in this study consider that the dried and rehydrated boletus samples showed higher levels of soluble and insoluble dietary fiber mailto:analeahu@usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 399 than the other samples (agrocybe aegerita and pleurotus eryngii) [7]. mushrooms have low energy content but remarkable protein content, from this point of view their consumption being advantageous, as compared to other vegetable products. moreover, as compared to meat products or milk, fat content is negligible. these considerations justify the recommendation and preference to include mushrooms in a consumer’s usual diet. wild mushrooms are rich sources of proteins and have low amounts of fat, making it an ideal snack material [1]. dried pleurotus ostreatus and lentinula edodes mushrooms exhibited high contents of protein, total dietary fiber, k, mg, zn, fe, cu and low contents of fat, na, ca [8]. carbohydrates constitute the most important component in terms of weight, the dry matter of mushrooms being the largest source of energy. di anibal et al. [9] tested the chemical composition and nutritional value of four edible mushroom varieties (champignons, portobellos, girgolas and shiitakes) commonly cultivated and consumed in argentina. the study reveals that edible mushrooms are rich sources of carbohydrates and have low amounts of fats; also they are rich in potassium and phosphorus whereas they have a low content of sodium [9]. mushrooms contain water-soluble compounds including reducing sugars and soluble polysaccharides. the dominant compound is mannitol, which in a. bisporus has the concentration of 330g/100g dry matter [10]. other simple sugars, such as: glucose, galactose, mannose and fructose have been also identified. edible mushrooms have a discreet aroma, but individualized depending on the species, which contributes to their attractiveness to the consumer. previous scientific reports have demonstrated the contribution of about 150 volatile compounds that outlines their aromatic profile[10]. according to ashmore l. et al. [11], the characteristic volatile compounds found in fresh “raw” agaricus bisporus mushroom sample are 4-methyl, 3-penten 2-one, 1-octen-3-ol, 3-octanone, benzaldehyde, 3-octanol, and 2-octen-1-ol, and the dried sample had a mild aroma but upon rehydration, a stronger cooked aroma developed [11]. among the sensory qualities, the most valued by the consumer are the visual ones: color, shape and size. studies on the components which determine the color were focused on the carotenoids. the analysis of the extracts obtained from the seventeen fresh mushrooms, collected from different places in north-eastern portugal, allowed the identification of carotene only in c. cibarius [12]. cultivated mushrooms (agaricus bisporus/white, agaricus bisporus/brown, lentinus edodes, and pleurotus ostreatus) were found to be good sources of vitamin b2, niacin, and folates, with contents varying in the ranges 1.8-5.1, 31-65, and 0.30-0.64 mg/100 g dry weight (dw), respectively [13]. it is considered that mushrooms are the only products that contain whole complex b. mushrooms contain high levels of pro-vitamin d2 (ergosterol) [14]. edible mushrooms also provide a nutritionally significant content of minerals, may constitute a good source of iron, zinc and copper. various studies have been carried out on the minerals composition of the european edible wild mushrooms from several countries like romania [15-16]; greece [17]; turkey [18]; croatia [2]; portugal [1]; finland [13]. mushrooms are known for their ability to accumulate heavy metals and radioactive elements, due to the texture of very good conducting properties in comparison to plants. that is why food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 400 mushrooms could be used as potential bio sorbents for heavy metals from aqueous solutions [19]. mushrooms are considered bio indicators of environmental pollution. as mentioned previously, the antioxidant activity of mushrooms is positively correlated firstly to the amount of phenolic compounds, followed by the flavonoids, tocopherols, ascorbic acid and carotenoids [20]. many studies have confirmed some large parts of traditional knowledge regarding the medicinal effects of mushrooms due to their antifungal, antibacterial, antioxidant and antiviral properties, besides being used as functional foods [21]. the main focus of this work was to determine the nutritional value and chemical composition, including the antioxidant activity in five types of wild mushrooms, agaricus albolutescens, armillaria mellea, russula virescens, cantharellus cibarius and boletus edulis. 2. materials and methods 2.1. samples and storage conditions mushrooms grow abundantly in the wild during the rainy season in every part of dragomirna forrest, suceava county. the fruits of five mushrooms (agaricus albolutescens, armillaria mellea, russula virescens, cantharellus cibarius and boletus edulis) species were harvested during early mature fruiting stage, in dragomirna forrest, suceava county (northeast of romania) in autumn 2014. sample preparation: the mushroom sample (600 g each) was cleaned, sliced and dried on paper towel prior to weighing. mushrooms were air-dried in the oven at 40 0 c before making the analysis. 2.2. chemical analysis the determination of moisture (according sr. iso 1026:2008) in mushrooms samples was made by the drying process in a drying chamber at the temperature of 105 °c, until it reached a constant weight. total ash composition was obtained by calcination of 5g of sample at 600 °c for 240 min (according sr iso 763: 2008). the protein content (nx4.38)of the samples was analyzed by the kjeldahl method. the oil content was determined in a soxhlet apparatus, extracting the fats from 10 g of mushrooms sample with petroleum ether, followed by extract evaporation to dryness and gravimetric determination. the carbohydrate content was estimated by difference of the other components using the following formula [8]: carbohydrate content = 100% (% moisture + % protein + % fat + % ash) (1) (1). energy was expressed as kilocalories/100g, using the factors mentioned in the romanian legislation: energy (kcal) = 4.1x (g protein + g carbohydrate) + 9.3x (g lipid) (2) (2) the content of total polyphenolic compounds in dried mushroom methanol extracts diluted 1/10 was determined by folin-ciocalteu method. for the preparation of the calibration curve 0.5 ml aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 μm/ml aqueous gallic acid solution were mixed with 10.0 ml folin-ciocalteu reagent (diluted ten-fold) and 1.0 ml sodium carbonate (20.0%) and the volume made up to 10.0 ml with h2o. the absorbance was read after 2 h at 25°c, at 760 nm. all determinations were performed in triplicate. total phenols were determined as gallic acid equivalents on a dry weight (mg gae g -1 d.w.). 2,2-di (4-tert-octylphenyl)-1pycrilhydrazyl (dpph) scavenging capacity assay the method used to determine the antioxidant activity of mushrooms is based food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 401 on scavenging 2,2-di (4-tert-octylphenyl)1-picrylhydrazyl (dpph) radicals. the mushroom samples aliquot (0.5 ml) was added to freshly prepared dpph reagent. after incubating for 5 min, the absorbance of the resulting solutions was measured at 517 nm using a spectrophotometer t70 uv-vis pg instruments ltd. the control was conducted in the same manner, except that the distilled water was used instead of sample. lower absorbance of the reaction mixture indicated higher free radical scavenging activity. the capability to scavenge the dpph radical was calculated using the following equation: ic50 (%) = [(a0-a1/a0) x 100] (3) where a0 was the absorbance of the control reaction and a1 the absorbance in the presence of the sample of mushroom species extract [22]. 2.3. statistical analysis three replications were used to obtain average values and standard deviations for proximate biochemical properties. multiple linear regression (pls), a method based on the criterion of covariance has been used, to analyze the relationship between the explanatory variables (chemical characteristics of mushrooms) or independent variables (different species of mushrooms) and between explanatory variables and independent. 3. results and discussion figure 1 presents the contents of macro components in five types of wild edible mushrooms: agaricus albolutescens, armillari amellea, russula virescens, cantharellus cibarius and boletus edulis. fig. 1. the contents of macrocomponents, g/100 g dry matter of dried agaricus albolutescens, armillaria mellea, russula virescens, cantharellus cibarius and boletus edulis mushrooms varied in their content of raw protein more than the other macroelements determined. all dried mushrooms were found to be good sources of proteins, with moisture % crude protein % fat % ash % 0 2 4 6 8 10 12 14 16 b. edulis c. cibarius r. virescens a. mellea a. albolutescens food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 402 contents varying in the ranges of 10.12– 15.15 g/100 g dry weight (dw), while the fat content was very low (0.93 –1.48 g/100 g dw). minimum and maximum ash levels in the present study were 1.75 mg/100 g d.w. and 2.47 mg/100 g d.w. for russula virescens and boletus edulis, respectively. the distribution of minerals in the fruiting bodies of the mushrooms is not uniform, caps concentrating, generally, the largest amount. barros et al. [1] reported similar ash content (0.81 ± 0.03 g/100 g), but higher moisture (93.05 ± 0.51g) and protein (2.12 ± 0.08 g/100 g of weight matter) levels in samples from northeast of portugal, while ouzouni et al. [17] described similar moisture (87.6 g/100 g) and ash (6.25 ± 0.02 g/100 g) contents, and higher protein levels (27.17 ± 0.15 g/100 g weight matter) in boletus edulis samples from forests in the west macedonia and epirus. carbohydrates, calculated by difference, were also an abundant macronutrient and ranged from 70.85 g/100 g in boletus edulis and 74.94 g/100 g in agaricus albolutescens. the relative homogeneity in wild mushrooms suggests a uniform composition of the soil, which is the main reservoir of nutrients. the energy value differed significantly (p < 0.001) in samples tested dried products, while the higher energy value was found 367.64 kcal/100g for dried russula virescens (figure 2). fig. 2. the contents of energy value, kcal/100 g dry matter, of dried agaricus albolutescens, armillaria mellea, russula virescens, cantharellus cibarius and boletus edulis generally talking, mushrooms are characterized by low energy value. the content of total phenolic compounds was determined in methanol extract of mushrooms samples and characterized using gallic acid equivalents (gae) per 1 g of mushroom dry matter. total polyphenol content reveals a significant difference between species, boletus edulis proving to be the richest in such compounds and was found to be 18.38 mg gae/g weight matter (figure 3). jeena et al. [23] determined total poliphenol content of three mushrooms types pleurotus (p. sajorcaju, p. ostreatus et p.sapidus) and the study revealed that p. 352 354 356 358 360 362 364 366 368 370 a. albolutescens a. mellea r. virescens c. cibarius b. edulis energy value kcal/100g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 403 sajorcaju 1.53 mg/g>p. ostreatus 1.32 mg/g> p. sapidus 1.10 mg/g [23]. the results obtained by gençcelep and hüseyin [24] indicated that total phenolics in the methanolic extracts were the highest in boletus edulis. reis et al. [25] analyzed the total polyphenol content of different species of mushrooms, thus, in agaricus bisporus (brown) was 37.33 mg gae/g extract, while in pleurotus eryngiiit has a lower value: 7.14 mg gae/g extract. fig. 3.total phenolicsmg gae/ g of analyzed samples concerning mushroom sample extracts, the russula virescens extract was the one that had the strongest antiradical activity (ic50 of 15.154), followed by armillaria mellea and cantharellus cibarius extracts, with very similar activities (ic50 of 20.12 and 21.55, respectively). alam n. et al. [26] reported that the antioxidant activities of the fruiting bodies of pleurotus ferulae was lower than the synthetic antioxidant, bht and toc, respectively at 0.5 mg/ml and at 0.5–20.0 mg/ml concentration. their antioxidant activities of the acetonic, methanolic, and hot water extracts of p. ferulae ranged from 52.71% to 95.63%, 69.51% to 96.52%, and 34.88% to 93.22%, respectively [26]. fig. 4. free radical scavenging activities % of analyzed samples 0 2 4 6 8 10 12 14 16 18 20 a. albolutescens a. mellea r. virescens c. cibarius b. edulis t o ta l p h e n o li cs ( m g g a e /g ) 0 5 10 15 20 25 30 35 a. albolutescens a. mellea r. virescens c. cibarius b. edulis ic 5 0 ( % ) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 404 in a research conducted by ferreira, i. c., et al.[27] it was reported that the extracts obtained from the mushroom cap proved to be a better source of antioxidants than extracts from mushroom stem [27]. similar studies reported that the antioxidant activity of ethanolic extracts from mushrooms is due to phenolic compounds content [28]. statistical analysis using the pls (partial least squares regression) application dependent variables (content in carbohydrate, fat content and the quantity of minerals-ash %) for mushroom samples analyzed indicates that the energy is determined by lipid content (fig. 5). in b. edulis samples it can be observed the high amount of protein and minerals content. the analyses conducted on armillaria mellea and russula virescens revealed a high energetic value and fats concentration, respectively. samples with high humidity belong to the species of fungus cantharellus cibarius. the high carbohydrate content occurs in agaricus albolutescens mushrooms. fig. 5.correlation between carbohydrates % and the energy value kcal/100g for analyzed samples table 1. pearson correlative bond of chemicals characteristics of mushroom samples variables energy, kcal/100g carbohydrate, % moisture % protein, % fat % ash % energy value, kcal/100g 1.000 -0.140 -0.958 0.663 0.830 -0.176 carb. content (cho) % -0.140 1.000 0.397 -0.933 -0.064 -0.687 moisture % -0.958 0.397 1.000 0.472 -0.798 -0.073 protein, % 0.663 -0.933 0.472 1.000 0.265 0.469 fat % 0.830 -0.064 -0.798 0.265 1.000 0.148 ash % -0.176 -0.687 -0.073 0.469 0.148 1.000 energy value, kcal/100g carbohydrate content (cho) % moisture % crude protein, % fat % ash % a. albolutescens a. mellea r. virescens c. cibarius b. edulis -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 t2 t1 correlations on axes t1 and t2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 405 the correlation coefficients (table 1) between chemicals’ characteristics analysis reveal that the protein gives to fungus a medium energy values (r=0.663) and once the humidity increases, the energy values decrease significantly.the protein content representstheonlysource of minerals in mushrooms. pearson coefficient shows a significant correlation between the percentage of ash and protein content. 4. conclusion nutritional content represents the rational motivation for mushroom consumption. due to their low fat content, the risk of cholesterol and triglyceride accumulation is eliminated. the main nutritional characteristics are presented in the current study (high water and protein content, but low fat content) along with the content of macroand micro-nutrients. the fruiting bodies of widely used wild edible mushroom have all attracted considerable attention as sources of phenolic compounds. all of these recommend them as food with health benefits. the results of the research suggest that dried wild mushrooms can be used as additives in food products. it was also noted a high degree of biodiversity resulting from the specificities of the species, substrate composition, and harvesting conditions. 5. references [1]. barros, l., baptista, p., correia, d. m., casal, s., oliveira, b., & ferreira, i. c., fatty acid and sugar compositions, and nutritional value of five wild edible mushrooms from northeast portugal. food chemistry, 105(1), 140-145, (2007). [2]. beluhan, s., & ranogajec, a., chemical composition and non-volatile components of croatian wild edible mushrooms. food chemistry, 124(3), 1076-1082, (2011). [3]. muszyńska, b., & sułkowska-ziaja, k., analysis of indole compounds in edible basidiomycota species after thermal processing. food chemistry, 132(1), 455-459, (2012). [4]. sobieralski, krzysztof, marek siwulski, and iwona sas-golak., potential utilisation of vegetables and mushrooms as components of functional food. folia horticulturae 25.1: 79-88, (2013). [5]. elefterios,e., vassilis,m.g. & cleanthes i., the potential use of mushrooms ß-glucans in the food industries, international journal of biotechnology for wellness industries,vol 3,15-18, (2014), [6]. patel, y., naraian, r., & singh, v. k. medicinal properties of pleurotus species (oyster mushroom): a review. world journal of fungal and plant biology, 3(1), 1-12, (2012). [7]. manzi, p., marconi, s., aguzzi, a., & pizzoferrato, l., commercial mushrooms: nutritional quality and effect of cooking. food chemistry, 84(2), 201-206, (2004). [8]. r., julita, and m. siwulski., dried shiitake (lentinulla edodes) and oyster (pleurotus ostreatus) mushrooms as a good source of nutrient." acta sci. pol., technol. aliment 6.4 135142, (2007). [9]. di anibal, c., farenzena, s., rodríguez, m. s., & albertengo, l., chemical composition and nutritional value of argentine commercial edible mushrooms. journal für verbraucherschutz und lebensmittelsicherheit, 1-10:155-164, (2015). [10]. bernaś, e., jaworska, g., & kmiecik, w., storage and processing of edible mushrooms. acta scientiarum polonorum technologia alimentaria, 5, 5-23, (2006). [11]. ashmore, l., j. d. craske, and g. srzednicki., volatile compounds in fresh, cooked fresh, dried and cooked dried agaricus bisporus using ambient temperature vacuum distillation." international food research journal 21(1): 263-268, (2014). [12]. ribeiro, b. et al., do bioactive carotenoids contribute to the colour of edible mushrooms, the open chemical and biomedical methods journal, vol 4, pp 14-18, (2011). [13]. mattila, p., könkö, k., eurola, m., pihlava, j. m., astola, j., vahteristo, l.,& piironen, v., contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. journal of agricultural and food chemistry, 49(5), 2343-2348, (2001). [14]. chen, s. y., yu, h. t., kao, j. p., yang, c. c., chiang, s. s., mishchuk, d. o., & slupsky, c. m. consumption of vitamin d 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 ana leahu, cristina damian, mircea oroian, sorina ropciuc, establishing the antioxidant activity based on chemical composition of wild edible mushrooms, food and environment safety, volume xiv, issue 4 – 2015, pag.398 – 406 406 enhanced mushrooms is associated with improved bone health. the journal of nutritional biochemistry, (2015). [15]. radulescu, c., stihi, c., popescu, i. v., busuioc, g., gheboianu, a. i., cimpoca, v. g., & diaconescu, m, determination of heavy metals content in wild mushrooms and soil by edxrf and faas techniques. ovidus uni. ann. chem, 21(1), 9-14, (2010). [16]. stihi, c., radulescu, c., busuioc, g., popescu, i. v., gheboianu, a., &ene, a., studies on accumulation of heavy metals from substrate to edible wild mushrooms. romanian journal of physics, 56(1-2), 257-264, (2011). [17]. ouzouni, p. k., petridis, d., koller, w. d., & riganakos, k. a. nutritional value and metal content of wild edible mushrooms collected from west macedonia and epirus, greece. food chemistry, 115(4), 1575-1580, (2009). [18]. sarikurkcu, c., tepe, b., solak, m. h., & cetinkaya, s. metal concentrations of wild edible mushrooms from turkey. ecology of food and nutrition, 51(4), 346-363, (2012). [19]. vimala, r., and nilanjana das., biosorption of cadmium (ii) and lead (ii) from aqueous solutions using mushrooms: a comparative study. journal of hazardous materials 168.1: 376382, (2009). [20]. naknaen, phisut, teerarat itthisoponkul, and phantipha charoenthaikij. proximate compositions, nonvolatile taste components and antioxidant capacities of some dried edible mushrooms collected from thailand. journal of food measurement and characterization: 9: 259-268, (2015). [21]. tan, y. s., baskaran, a., nallathamby, n., chua, k. h., kuppusamy, u. r., & sabaratnam, v., influence of customized cooking methods on the phenolic contents and antioxidant activities of selected species of oyster mushrooms (pleurotus spp.). journal of food science and technology, 52(5), 3058-3064, (2015). [22]. elmastas, m., isildak, o., turkekul, i., & temur, n. determination of antioxidant activity and antioxidant compounds in wild edible mushrooms. journal of food composition and analysis, 20(3), 337-345, (2007). [23]. jeena, g. s., punetha, h., prakash, o., chandra, m., & kushwaha, k. p. s., study on in vitro antioxidant potential of some cultivated pleurotus species (oyster mushroom). indian journal of natural products and resources, 5(1), 56-61, (2014). [24]. gençcelep, hüseyin. antioxidant properties of wild edible mushrooms." journal of food processing & technology (2011). [25]. reis, f. s., barros, l., martins, a., & ferreira, i. c., chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study. food and chemical toxicology, 50(2), 191197, (2012). [26]. alam, n., yoon, k. n., lee, j. s., cho, h. j., & lee, t. s. consequence of the antioxidant activities and tyrosinase inhibitory effects of various extracts from the fruiting bodies of pleurotus ferulae. saudi journal of biological sciences, 19(1), 111-118, (2012). [27]. ferreira, i. c., baptista, p., vilasboas, m., & barros, l. free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast portugal: individual cap and stipe activity. food chemistry, 100(4), 1511-1516, (2007). [28]. fidler, g., a., butu, rodino, s., m., butu, & p., cornea. antioxidant activity, bioactive compounds and antimicrobial effect of mushrooms extracts. bulletin of university of agricultural sciences and veterinary medicine cluj-napoca. animal science and biotechnologies, 72(1), (2015). 404 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 2018, pag. 404 412. determination of oxytetracycline residue levels in edible tissues of slaughtered cattle *engelbert bilashoboka 1 , boyd mudenda 1 , nosiku munyinda 1 , fabiola vincent moshi 2 , dominic m. kambarage 3 1department of public health, school of medicine, university of zambia, , 2 school of nursing and public health, the university of dodoma p.o. box 259 dodoma, tanzania, 3mwalimu julius k.nyerere university of agriculture and technology, musoma, tanzania, *corresponding author, bilashoboka.engelbert@gmail.com received 11th november 2018, accepted 28th december 2018 abstract: worldwide, antimicrobials are widely used in food producing animals for treatment and/or prophylaxis of diseases. in tanzania, irrational administration of antimicrobials, notably oxytetracycline (otc) is common because of the under-developed animal health service delivery system. the administration of otc and other drugs is often done by animal keepers, thereby leading to drug residues in meat and milk. the study established the level of otc residues in edible tissues of cattle slaughtered within dodoma city tanzania. this was cross-sectional analytical study design which quantified drug residues in muscle, liver, kidney from licenced and unlicenced premises. samples from licenced as well as unlicenced premises were collected and used in determining otc levels using high performance liquid chromatography (hplc) method. the hplc method was validated to establish the reliability of the method in quantifying otc residues. the results indicated that otc residues were 0.6 mg/kg, 0.25mg/kg and 1.28 mg/kg for muscle, liver and kidney tissues, respectively against corresponding maximum residue limit (mrl) 0.2mg/kg, 0.6mg/kg and 1.2mg/kg. regardless of the licensure status and source of the sample, 53% of muscle, 65% of liver and 7.1% of kidney tissues were above mrl. the quantities of otc residues in samples from licensed and unlicensed food vending settings were not significantly different (p = 0.3676) but above the mrl. the above results show unacceptably high levels of otc residues in meat with most beef value chain actors being ignorant of the impending public health threat/risks of consuming beef with high drug residues. keywords: toxic/anaphylactic reactions, public health threat, antibiotic resistant strain of bacteria, beef value chain actors 1. introduction antibiotics are among the most widely used veterinary drugs in food-producing animals for therapeutic purposes and as dietary supplements. they may be administered orally as food additives like oxytetracycline (otc) and sulfadimidine or directly by injection like otc injection, peni-streptomycin, gentamycin and sulfadimidine [1]. among the veterinary antibiotics, oxytetracycline is one of the most widely used antibiotics due to its broad spectrum activity and easy access by practitioners [2] and animal keepers as pointed out, oxytetracycline works by interfering with the ability of bacteria to add new amino acids to the peptide chain to produce essential proteins. without these proteins, the bacteria cannot grow, multiply and increase in numbers [3]. otc therefore stops the spread of the infection and the remaining bacteria are thereby killed by the immune system or eventually http://www.fia.usv.ro/fiajournal mailto:bilashoboka.engelbert@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 405 die. resistance against this antibiotic in humans is often attribute to drug residues in animal products [4]. in developing countries, due to scarcity of veterinary professionals and lack of enforcement of veterinary regulations, the otc and other veterinary drugs are often administered into sick animals by lay persons. according to bedada and zewde [5] the control of drugs from the government authorities and information on the actual rational drug use pertaining to veterinary drug in developing countries is very limited. some antibiotics are sold casually by street vendors like any other commodity with no regard for their use. consequently there is a widespread and indiscriminate use of tetracyclines, the once effective broad spectrum antibiotic against both gram negative and positive organisms. the codex alimentarius commission(cac)/ maximum residue limit stipulates that once the animal is injected with oxytetracycline, the antibiotic residues remain in the body of an animal for 28 days and during this period, the animal should not be slaughtered for human consumption [6]. this however does not happen as many of these animals are sent for slaughter before the drug washout period has elapsed. such a malpractice can have a disastrous impact to the health of meat consumers. the beef consumed with high levels of drug residue has cumulative effect of causing toxic and anaphylactic reactions to consumers. this study therefore aims at determining the levels of oxytetracycline residues in beef consumed in dodoma as part of the desire to formulate appropriate public health strategies. the oxytetracycline drug residues pose a real public health hazard to consumers. such hazards include anaphylactic reactions and development of drugresistant pathogens. according to codex alimentarius commission of fao and who, the current standard maximum residue level (mrl) for otc in beef muscles is 0.2 microgram per gram of beef [6]. however, due to profit motives and resource poor related challenges, animal keepers and business men do not follow the fao and who guidelines. even at the ranch setting where hazard analysis critical control points (haccp) is not applicable, the animals are likely to be sent for slaughter before the recommended withdrawal period. thus, the meat is likely to enter the market with high otc residues. in most of developing countries, this is further compounded by the fact that slaughter houses/slaughter slabs do not have facilities for detecting drug residues. consumption of meat with high drug residues can lead to toxic reactions, anaphylactic reactions and development of antibiotic resistant strains of bacteria. as stipulated in the tanzania food, drugs and cosmetic act [7], the washout periods of drugs are supposed to be observed before animals are sent for slaughter. but due to absence of serious enforcement systems, animals are often slaughtered without taking into account the drug withdrawal requirements; thereby posing a real threat to public health. despite this threat,to date, little is known on the oxytetracycline residue levels in edible cattle tissues in central tanzania and the animal keepers’ awareness on the withdraw period. 2. matherials and methods study site and setting dodoma city is in dodoma region which is in the central part of tanzania. following declaration of dodoma as as the capital city of the country there has been a lot of influx of people. this has created a high demand of beef. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 406 despite the high demand of beef, dodoma city has only one ultra modern abattoir. the abattoir slaughters an average of 20 animals a day and ante mortem and post mortem inspections are done by municipal meat inspectors. study design this was an analytical cross-sectional study that was done october and november 2015. sample size and sampling method sample size the sample size at confidence interval of 95% with prevalence of positive samples estimated at being 70% [5] was determined using the following formula where z=1.96, p=0.7, =0.08 (1.96/ .08 )2 x0.3x0.7 +35% = 171 therefore the sample size was 171 specimens sampling method this study focused on the laboratory quantification of the levels of otc residues in 171 beef samples from five wards namely majengo, viwandani, makole, makulu and ntyuka which were randomly selected out of 14 wards of dodoma city from 1st october to 30th november 2015. of these samples, 128 were collected from licensed vending outlets. this involved samples from 30 butchery shops, five supermarkets and one abattoir) and 43 specimens were from unlicensed vending outlets, especially the pubs and bars where often animals were slaughtered under a backyard system. each sample collected weighed approximately 250g. the samples as shown in table 1 were collected using the properly marked sampling bags, stored in a cool box and transported to the laboratory. data collection growth inhibition method this method which detects antibiotic residues through inhibition of growth of bacillus subtilis on nutrient agar at 37ºc when incubated over night was planned to be used to screen positive samples that were then subjected to hplc analysis. however, due to lack of conclusive answers with this method, in the study all 171 were subjected to hplc analysis. high performance liquid chromatography (hplc) frozen samples were initially thawed and cut with scissors to remove fat and fascia before being weighed. the samples were then homogenised using the ultra-turrex t25 tissue homogenizer. the homogenized muscle, liver or kidney samples which weighed approximately 5 grams were then mixed with 30ml of extraction solvent (5% trichloroacetic acid with 0.5% disodium ethylenediaminetetraacetate, edta) and acetonitrile-water (3:7) and then centrifuged for 25 min at 5300 rpm. the supernatant was then filtered through a 0.45 µm filter paper into hplc vial. 20µl of filtered solution of each sample was then injected into hplc for analysis. hypersil bdc c18 column was used and otc was separated at 24 0 c using a mobile phase of methanol-acetonitrile-0.2m oxalic acid (1:1:3.5) at ph=2.0 (with 28% aqueous ammonia) at a flow rate of 0.6 ml/min and wavelength detector set at 360 nm. otc residues were determined by using hplc (model shimadzu class-vp series, kyoto, japan) equipped with sil10autoinjector with sample cooler and lc10 on-line vacuum degassing solvent delivery unit. chromatographic control, data collection and processing were carried out by using shimadzu class vp data software by aid of computer connected to the detector. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 407 the quantities established were compared to the mrl set by cac and categorized as being within, below or above the mrl. data analysis the laboratory data were analyzed as follows: continuous variables (quantities of otc residues in mg/kg) slightly failed to be symmetrical, but right skewed. the assumption of normal distribution was made based on the smallness of sample size (171 samples). the test for normality by the shapiro-wilk w test (quantilequantile plot) was done. since there was no evidence of gross deviation from normal distribution of the continuous variables, the descriptive statistics for continuous variables was hinged on pairwise comparison of the means between the licensed and unlicensed premises. the pairwise comparison of the means was done using benferron’s and studentnewman keuls’ methods. the generalized linear model was used to select the best predictors of otc residues in our samples after subjecting the predictor variable to univariate and multivariate analyses. all analyses were done by using stata software version 13. 3. results and discussion in this study, one hundred seventy one (171) beef samples were collected from five (5) wards within dodoma municipality catchment area. the concerned wards were majengo, viwandani, makole, makulu and ntyuka. table 1 shows the results of the samples which were subjected to growth inhibition technique using nutrient agar seeded with bacillus subtilis. for muscles from licensed premise, there were 60 samples, 21 samples from unlicensed premise, 18 samples of liver and 14 samples of kidneys which inhibited growth but at this stage it was not possible to know the type of antibiotic and quantities till the next method of high performance liquid chromatography (hplc). the antibiotic residues pose a real public health threat. this is consequent to the lack of adequate control on the rational use of these antibiotics and lack of observation of drug withdrawal periods. such a scenario has made the antibiotic residues find their way into the food value chain. this study established 53% of muscle tissues regardless of the licensure status and source of the sample to have levels of otc residues above the maximum residue limit (mlr) of 0.2mg/kg. only 34.1% (47/137) of the muscle tissue had residues below the maximum residue limit whereas 12.4% (17/137) had otc residues within the limit. these results were similar to the results of the study which was conducted in ethiopia where out of the total 384 table 1 types of beef samples sample type number of sample per ward samples from licensed 1 premises from 5 wards samples from unlicensed 2 premises from 5 wards total number of samples muscles 27 94 43 137 liver 4 20 0 20 kidney 3 13 1 14 1officially recognized beef vending outlets like butchery shops and supermarkets; 2unofficially recognized beef vending outlets like bars and under the tree. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 408 samples analyzed for tetracycline residues, 71.3% had detectable oxytetracycline levels [5]. these results are comparatively higher in relation to canada where there is zero tolerance of drug residues in food products [8]. table 2 growth inhibition method results (bacillus subtilis) sample type sample source no growth growth total samples a. muscles licensed premise 60 34 94 unlicensed premise 21 22 43 b. livers licensed premise 18 2 20 unlicensed premise 0 0 0 c. kidneys licensed premise 14 0 14 unlicensed premise 0 0 0 total 171 table 2 shows the otc residue results for muscle, liver and kidney samples by hplc whereby 73 muscle samples, 13 liver samples and 1 kidney sample were above the set residue limit. table 3 otc residue quantification in beef by hplc source of samples type mrl1 range of results licensed unlicensed total n % n % n % a. muscle 0.2mg/kg i. below limit (<0.2mg/kg) 27 19.71 20 14.6 47 34.31 ii. within limit(0.2mg/kg) 12 8.76 5 3.65 17 12.41 iii.above limit (>0.2mg/kg) 55 40.15 18 13.14 73 53.28 b. liver 0.6mg/kg i. below limit (<0.6mg/kg) 7 35 0 0 7 35 ii.above limit (>0.6mg/kg) 13 65 0 0 13 65 c. kidneys 1.2mg/kg i. below limit (<1.2mg/kg) 13 92.86 0 0 13 92.86 ii.above limit (>1.2mg/kg) 1 7.14 0 0 1 7.14 1maximum residue limit as set by codex alimentarius commission according to different tissue types. table 3 shows to what level the mean otc residues are above the maximum residue limit (mrl) in muscles and liver while below the limit in the kidney. the anomaly of the kidney to have residues below the limit is explained in the subsequent discussion. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 409 table 5 otc residue quantification by tissue, licensure and sample source variable mean (sd) 1 mrl(mar) 2 tissue type muscle 0.60(1.19) 0.20(10.72) liver 0.25(0.43) 0.60(3.00) kidney 1.28(1.03) 1.20(1.67) licensure status licensed 0.63(0.89) (15.94) unlicensed 0.73(1.70) (10.72) sample source butchery 0.64(0.83) (5.34) supermarket 0.77(1.12) (5.94) slaughterhouse 0.39(0.80) (2.95) bars 0.37(0.38) (1.35) emergency slaughters 2.29(3.64) (10.72) 1mean residues in mg/kg; 2standard deviation; 3maximum residue in mg/kg table 6 shows the mean residues of the samples from the two groups based on licensure status whereby the samples from unlicensed premise had slightly higher otc residues (0.73mg/kg) compared to those from licensed premises(0.63). but table 4 mean levels of otc residues according to tissue type tissue type number of samples mean (sd) 1 mrl 2 muscle 137 0.60(1.19) 0.20 kidney 14 0.25(0.43) 0.60 liver 20 1.28(1.03) 1.20 1 mean residues levels (standard deviation) in mg/kg; 2 maximum residue limit as set by cac in mg/kg 1 mean residues levels (standard deviation) in mg/kg; 2maximum residue limit (mrl) as set by cac in mg/kg against the maximum actual residues (mar) as found in the samples in mg/kg based on tissue, licensure and sample source. table 6 comparison of the mean otc residues by licensure licensure status mean residues 1 sd 2 maximum 3 95% confidence interval (ci) licensed 0.629 0.8962 5.944 -.302388 .498497 unlicensed 0.727 1.7030 10.716 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 410 the comparison of the mean residues from the two groups by benferron’s and student-newman keuls’ methods revealed no significant difference between the two groups at 95% confidence interval (.302388, .498497). as evidenced from the results, most of the muscle tissues and the liver tissues had mean residues above the mrls. but most of the kidney tissues had low mean residues (table 6). there was one muscle sample with unexpectedly high levels of otc residues up to 10.72mg/kg. the sample was taken from unlicensed premise (emergency slaughter) at makulu ward. this is explained by confession of the interviewed farmers who treated the animals but when death was eminent they resorted to slaughtering of the animal. given the pharmacokinetics of otc, the sick animal has high concentration of otc due to alteration of haemodynamics especially drop in blood pressure, cardiac output, glomerular filtration, renal blood flow and decreased hepatic drug metabolism all of which bottleneck to lower clearance rate and longer half-life. the kidney tissues which were expected to have comparatively higher levels of otc residues were found to have low levels of otc due to the similar reason of pharmacokinetics of the drug. another reason behind high levels of residues in that muscle sample is the injection sites through which the drugs are administered into the animals which are neck and buttocks muscles. the sample from the injection site of an animal which has been injected recently is expected to have high levels of residues compared to muscle away from the injection site. given the current animal marketing system even at the slaughterhouse, the traceability of the slaughtered animal was difficult to underscore with the aim of feedback to the culprits for corrective action. based on the licensure status of the premise, the mean residues of the samples from the two sources, the samples from unlicensed premise had slightly higher otc residues (0.73mg/kg) compared to those from licensed premises (0.63) as shown in table 8. but the comparison of the residues among the two groups by benferron’s and student-newman keuls’ methods revealed no significant difference between the two groups at 95% confidence interval (-.302388, .498497). from the licensed premises the residues ranged from 0 to 5.94mg/kg whereas from unlicensed premises the residues ranged from 0 to 10.72mg/kg. the results are indicating how people who are sourcing beef from licensed and unlicensed premises are all exposed to consumption of drug residues. adjusting for the licensure status and source of the samples, 65% (13/20) of the liver tissues were found to have otc residue levels above the mrl of 0.6mg/kg while 35% (7/20) had residues below the limit. these results are in line with the results of the the similar study done in akure, nigeria by olatoye and ehinimowo [9] where most of the tissues with residues above mrl were the liver tissues. this is due to the fact that the liver is the organ for metabolism and excretion of these drugs. these tissues are preferred by many, hence a lot of consumers at high public health risk. the kidney tissues were found to have 7.1% (1/14) of the samples above the mrl of 1.2mg/kg whereas 92.9% (13/14) of the samples were below the mrl. but these results were not like the results of the study done by olatoye and ehinimowo [9] where more kidney tissues had residues above the limit. viewed from the source of the sample without taking into account of tissue types and licensure status, the butcheries, supermarkets and slaughterhouses had mean residues of 0.64mg/kg, 0.77mg/kg and 0.39mg/kg respectively. the samples from the bars and emergency slaughters food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 411 (slaughter under the tree) had otc residues of 0.37mg/kg and 2.30mg/kg respectively. from the above findings, the entire source of beef is not safe in terms of drug residues because the samples from the entire source are all above the mrls. the samples from the emergency slaughter had the highest mean of otc residues. remarkably, the emergency slaughter source was the one with the sample with highest levels of otc residues of 10.72mg/kg which is unacceptably high. with such high levels of residues, it was quite evident that in the bars most of the beef from unlicensed premised managed to penetrate into the food chain. this presents a threat to people who go to the bar to enjoy bites with liquor or soft drinks. the beef products with high levels of drug residues were likely to have been sourced from unlicensed premises more precisely the slaughter under the tree and emergency slaughters. alarmingly, the public health is at risk. this needs joint efforts from regulators and stakeholders to address the problem. the fight against the problem demands all the key players along the value chain addition to work together to fight against this public health threat. table 7 determinants of otc residues predictors univariate analysis multivariate analysis p value confidence interval p value confidence interval tissue type 0.051 -.0009856 .507935 0.028 0.03118 0.56067 licensure 0.629 -.2995204 .495629 0.850 -0.8853 0.72952 source 0.424 -.0757791 .180373 0.399 -0.1475 0.37022 table 7 shows the predictor variables for otc residues after subjection to univariate and multivariate analysis whereby the tissue type was found to be a significant predictor variable (p value = 0.028). the generalized linear model was run through the variables to detect the best otc residue predictor variable between tissue type, licensure and source. the univariate analysis of the variables revealed the tissue type to be a significant predictor at p value 0.05 while licensure and source were not significant at p values 0.63 and 0.42 respectively. when considering the predictor variables of otc residues holistically (tissue type, licensure and source) by using the generalized linear model (table 7), it was established that the tissue type was a significant predictor at p value = 0.028 at 95% ci (0.03118 0.56067) at multivariate analysis. this study has managed to unveil the truth about residues and knowledge of stakeholders and other beef value chain actors. however, the levels of residues to which the consumers are constantly exposed to have been brought to light. it is also undeniable fact that dodoma capital city is centrally located at the heart of tanzania. it is receiving animals from different parts of tanzania. hence the samples taken represent the true picture of the other parts of tanzania. 4. conclusion this study established the beef food chain in dodoma to be contaminated by otc residues at unacceptably high levels. this in turn poses a real public health threat to the consumers who are likely to succumb to toxic/anaphylactic reactions, drug resistance eratogenic risks to the foetus and can cause discolouration of primary and secondary teeth as well as hypoplasic development of teeth when consumed by infants or pregnant mothers during the last 2 terms. it is highly recommended for the ministry of livestock development and fisheries to intervene in screening the animals at the market and enhance traceability of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage , determination of oxytetracycline residue levels in edible tissues of cattle slaughtered, food and environment safety, volume xvii, issue 4 – 2018, pag. 404 – 412 412 animals by improving the animal marketing system in tanzania. the law enforcers should also actively seek find and take action against the practice of slaughter under the tree. it is also recommended to have effective control of veterinary drugs so that some of them like antibiotics should be bought from licensed shops with prescription. 5. acknowledgements this paper is a part of dissertation thesis of the correspondent author ([10]). 6. references [1]. mesgari abasi m, rashidi mr, javadi a, bannazadeh amirkhiz m, mirmahdavi s, zabihi m., levels of tetracycline residues in cattle meat, liver, and kidney from a slaughterhouse in tabriz, iran. turkish j vet anim sci. 2009;33(4):345–9. [2]. kaneene jb, miller r., problems associated with drug residues in beef from feeds and therapy. rev sci tech [internet]. 1997;16(2):694–708. available from: http://www.scopus.com/scopus/inward/record.u rl?eid=2-s2.00031196635&partnerid=7tdmeqzl&rel=3.0.0 &md5=7a507fd45103361884aba16a3e3afc9d [3]. chopra i, roberts m. , tetracycline antibiotics : mode of action , applications , molecular biology , and epidemiology of bacterial resistance tetracycline antibiotics : mode of action , applications , molecular biology , and epidemiology of bacterial resistance. microbiol mol biol rev [internet]. 2001;65(2):232–260. available from: http://www.facm.ucl.ac.be/full-textsfacm/vanbambeke-2006-4.pdf [4]. mitchell jm, griffiths mw, mcewen sa, mcnab wb, yee aj., antimicrobial drug residues in milk and meat: causes, concerns, prevalence, regulations, tests, and test performance. j food prot [internet]. 1998;61(6):742–56. available from: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink .fcgi?dbfrom=pubmed&id=9709262&retmode =ref&cmd=prlinks%5cnpapers3://publication/ uuid/a8513747-f2d9-45ab-b22cf2b2023fd74e [5]. bedada ah, zewde bm, zewde bm., tetracycline residue levels in slaughtered beef cattle from three slaughterhouses in central ethiopia. glob vet. 2012;8(6):546–54. [6]. shahbazi y, ahmadi f, karami n. screening, determination and confirmation of tetracycline residues in chicken tissues using four-plate test, elisa and hplcuv methods: comparison between correlation results. food agric immunol [internet]. 2015;26(6):821–34. available from: http://dx.doi.org/10.1080/09540105.2015.1036 357 [7]. tfda., the tanzania food, drugs and cosmetics act, 2003. 2003;1–89. [8]. gosting dc, doyle me. food safety 1990: an annotated bibliography of the literature [internet]. 1991. p. 400–11. available from: https://books.google.com/books?id=qqdlba aaqbaj&pgis=1 [9]. olatoye i, ehinmowo a. oxytetracycline residues in edible tissues of cattle slaughtered in akure, nigeria. niger vet j [internet]. 2010;31(2):93–102. available from: http://www.ajol.info/index.php/nvj/article/view /68952/57010 [10]. http://dspace.unza.zm:8080/xmlui/bitstr eam/handle/123456789/4675/otc%20disse rtation%20final.pdf?sequence=1&isall owed=y http://dspace.unza.zm:8080/xmlui/bitstream/handle/123456789/4675/otc%20dissertation%20final.pdf?sequence=1&isallowed=y http://dspace.unza.zm:8080/xmlui/bitstream/handle/123456789/4675/otc%20dissertation%20final.pdf?sequence=1&isallowed=y http://dspace.unza.zm:8080/xmlui/bitstream/handle/123456789/4675/otc%20dissertation%20final.pdf?sequence=1&isallowed=y http://dspace.unza.zm:8080/xmlui/bitstream/handle/123456789/4675/otc%20dissertation%20final.pdf?sequence=1&isallowed=y 1. introduction study site and setting despite the high demand of beef, dodoma city has only one ultra modern abattoir. the abattoir slaughters an average of 20 animals a day and ante mortem and post mortem inspections are done by municipal meat inspectors. study design data collection growth inhibition method data analysis growth inhibition method results (bacillus subtilis) 4. conclusion this study established the beef food chain in dodoma to be contaminated by otc residues at unacceptably high levels. this in turn poses a real public health threat to the consumers who are likely to succumb to toxic/anaphylactic reactions, drug resist... 123 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2 2019, pag. 123 129 human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria *ijeoma oboh 1 , benjamin okpara 1 , peace wilfred ekprikpo 2 1faculty of life sciences, department of animal and environmental biology, university of benin, pmb 1154, benin city, nigeria, obohij@yahoo.com 2 nigerian institute of oceanography and marine research, lagos. nigeria *corresponding author received 15th february 2019, accepted 28th june 2019 abstract: the purpose of this paper is to evaluate the accumulation of heavy metals (ni, zn, pb, fe and cr) in two commercially available fishes clarias gariepinus and parachanna obscura, and estimate the health risks they pose to humans through consumption. fish samples were collected between july and october, 2017. heavy metals were determined using the atomic absorption spectrophotometer while health risk to consumers was evaluated using estimated daily intake (edi), target hazard quotient (thq) and hazard index (hi). heavy metal accumulation followed the order fe>cr>zn>pb>ni and cr>fe>zn>pb>ni in the liver and muscle of clarias gariepinus while concentrations in the liver and muscle of parachanna obscura followed the order fe>pb>cr>zn>ni and fe>cr>zn>pb>ni respectively. chromium and lead exceeded the permissible limits for fish food. target hazard quotient was cr >pb> ni with values of 0.040, 0.014 and 0.002 for c. gariepinus and 0.013, 0.010 and 0.002 for p. obscura. the hazard index was 0.056 and 0.025 for c. gariepinus and p. obscura respectively. the hazard indexes for the two fishes were below one and thus indicate minimal risk of health concerns to consumers of these fishes. regular monitoring of heavy metal levels in fishes is recommended. keywords: heavy metals, consumption, health risk, hazard index, c. gariepinus, p. obscura, ikpoba river 1. introduction worldwide, there has a global concern over the contamination of rivers, water bodies and aquatic animals by heavy metals. as a result of the increasing rate of industrialization in nigeria, a lot of harmful substances, including heavy metals, are now being discharged into the environment particularly water bodies. this has translated to an increase in the concentration of various heavy metals in the aqueous environment beyond their natural levels [1] however, the severity of these effects depends on the type, properties, dosage and exposure duration [2]. in the aquatic environment, heavy metals may affect organisms directly by accumulating in their body or indirectly by transferring to the next trophic level of the food chain. fish are often at the top of aquatic food chain and may concentrate large amounts of these metals from the water [3]. in humans, heavy metal accumulation has hazardous effects on the brain, liver, kidneys, lungs, and muscles [4,5]. reported damages include enhanced lipid peroxidation, dna damage, enzyme inactivity and the oxidation of protein sulfydryl groups [6,7]. excessive levels of heavy metals in food is associated with the etiology of a number of diseases, especially cardiovascular, renal, neurological, http://www.fia.usv.ro/fiajournal mailto:obohij@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 124 cancer and bone diseases [6,8]. for these reasons, evaluation of heavy metal levels in commercially important fish species is necessary in verifying whether there is a significant health risk arising from the consumption of these fishes. human health risk assessment is considered as the characterization of the potential adverse health effects of humans as a result of exposures to environmental hazards [9]. the target hazard quotients (thq) has been recognized as a reasonable index for the evaluation of heavy metals intake by consumption of contaminated food [10]. thq is a ratio of consumed dose of a toxic metal via an oral reference dose (rfd). a thq value above 1 means that contaminated food intake has likely some noticeable harmful effects on the exposed population. higher thq value indicates a higher probability of hazard risk. the hazard index (hi) is the sum of thqs of all the metals in the fish and it is calculated in view of the fact that more than one metal can often be bioaccumulated in fish tissue and could have interactive or synergistic effects. this study is aimed at assessing heavy metal levels in clarias gariepinus and parachanna obscura and to estimate the human health risk of consumption of these two fishes which are of great economic importance to nigerians. 2. materials and methods 2.1. study area the ikpoba river a fourth order stream is located in benin city, edo state, nigeria. it lies between longitudes 5° 25’ e and 50 40’ e and latitudes 6° 20’ n and 60 30’ n (fig 1) water samples were collected from two sampling points between the hours of 9:00 am and 12:00 noon from july to october 2017. standard methods and procedures were followed during sample collection. physico-chemical parameters were determined according to procedures outlined in the standard methods for the examination of water and wastewater. the surface water temperature was taken in-situ with the use of mercury-in-glass thermometer [11].the extracted liver and muscle portions were wrapped in foil paper, labeled and oven dried at a temperature of 105°c for 1 hour. the dried samples were ground to powdered form with plastic mortar and pestle, sieved to obtain a uniform particle size and preserved in well-labelled containers. heavy metals – nickel (ni), zinc (zn2+), lead (pb2+), iron (fe2+) and chromium (cr2+) in both water and fish were determined using atomic absorption spectrophotometer (aas). data analysis the levels of selected heavy metals in the liver and muscle of c. gariepinus and p. obscura were characterized using basic statistical measurement of central tendency and dispersion. paired sample t-test was used in comparing levels of heavy metals in the liver and muscle of the two fishes. human health risk assessment for fish consumption the concentration of heavy metals in the muscles of the two fishes was used in calculating the estimated daily intake (edi), target hazard quotient (thq) and hazard index (hi). estimated daily intake of metals (edi) edi (mg/kg − bw/day/week) = where m if = mass of fish ingested per day; c mf = concentration of metal in fish and bw = body weight (60kg for adult). the per capital consumption of fish and shellfish in nigeria for human food is 9.0kg, which is equivalent to 24.7kg per day [12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 125 target hazard quotient the thq is an estimate of the noncarcinogenic risk level due to pollutant exposure and calculated by the following equation: thq = × 10-3 where; thq is the target hazard quotient, ef = exposure frequency (365 days/year); ed is the exposure duration (52.62 years) which corresponded to average life expectancy of a nigerian (male and female); at = average exposure time for non-carcinogens (365 days/year x ed). the oral reference dose (ord) is an estimate of daily exposure to human population (including sensitive subgroup) that is likely to be without an appreciable risk of deleterious effect during a life time. the oral reference dose (ord) (mg/kg/day) used were ni 2.0 × 10-2, zn 3.0 × 10-1, pb 3.5 × 10-3 fe 7.0 × 10-1 and cr 1.5 × 10-3 [10]. hazard index the hazard index (hi) from the consumption of clarias gariepinus and parachanna obscura obtained from ikpoba and owan rivers, edo state was calculated as the sum of thqs of all the metals in the fish samples and was expressed as follows; hi = thqpb + thqcr + thqzn + thqfe+thqni where hi is the hazard index;thqpb = the target hazard quotient for pb intake; thqcu = the target hazard quotient for cu intake thqzn = the target hazard quotient for zn intake; thqfe = the target hazard quotient for fe intake and thqni = the target hazard quotient for ni intake fig. 1: map of the ikpoba river showing sampling point; insert (a) nigeria (b) edo state food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 126 3. results and discussion a human health risk assessment process uses the tools of science, engineering, and statistics to identify and measure a hazard, determine possible routes of exposure, and finally use that information to calculate a numerical value to represent the potential risk [13]. heavy metals concentrations varied in the two organs (liver and muscle) and also in the two fishes species studied. significant factors that influence differential accumulation of heavy metals in fishes include age of fish, lipid content in the tissue and mode of feeding [14]. in clarias gariepinus, mean levels of ni, zn, pb, fe and cr in the liver were 0.16 mg/kg, 0.93 mg/kg, 0.21 mg/kg, 2.81 mg/kg and 1.91 mg/kg while mean levels in the muscle were 0.18 mg/kg, 0.48 mg/kg, 0.26 mg/kg, 1.24 mg/kg and 1.63 mg/kg respectively. parachanna obscura recorded mean levels of 0.18 mg/kg, 0.39 mg/kg, 0.99 mg/kg, 3.74 mg/kg and 0.63 mg/kg in the liver and 0.18 mg/kg, 0.28 mg/kg, 0.19 mg/kg, 0.77 mg/kg, 0.52 mg/kg in the muscle. significant differences (p < 0.05) were observed between the zinc concentrations in the liver and muscle of both c.gariepinus and p. obscura. the order of accumulation of heavy metals in the liver and muscle of clarias gariepinus were fe > cr > zn >pb> ni and cr > fe > zn >pb> ni respectively, while the order observed in the liver and muscle of p. obscura was fe >pb> cr > zn > ni and fe > cr > zn >pb> ni (tables 1 and 2). the levels of chromium and iron in both fishes exceeded the who permissible limits for fish foods. observation from this study correlates with results from other studies on bioaccumulation of heavy metals in tissues and organs, which revealed that fish muscle has a lower concentration in comparison to other tissues. this could be attributed to the low metabolic activity of the muscles. in addition, the liver is the principal organ responsible for the detoxification, transportation, and storage of toxic substances and it is an active site of pathological effects induced by contamination [15]. health risk assessment for consumption of c. gariepinus and p. obscura are shown in tables 3 and 4. estimated daily intakes (edi) for clarias gariepinus were in the order cr > fe > zn > pb > ni while the thq was cr >pb> ni > fe = zn with risk values of 0.040, 0.014 and 0.002 respectively. an edi order of fe > cr > zn >pb> ni was observed for p. obscura while the thq was in the order cr >pb> ni with risk values of 0.013, 0.010, and 0.002. the hazard index was 0.025. for both fishes with cr being the highest contributor. e\stimated daily intake (edi) values in this study were higher than values reported for scomber scrombus [16]. although the concentrations of chromium and lead were above recommended limits for food fish [17] they may not pose human health risk in view of the fact that their edi values were less than their respective rfd values. target hazard quotient (thq) for the metals were in the order cr >pb> ni > fe > zn with all values < 1, suggested minimal risk of non-carcinogenic consequence. target hazard quotient > 1 is known to indicate potentials of noncarcinogenic risk to human exposures [1820] particularly with increase in metal exposure. the thq of metals in this study were all < 1 and this is consistent with the findings of previous researchers [21 and 22]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 127 table 1 summary of heavy metals in tissues of clarias gariepinus from the ikpoba river n=4 organs pvalue standards liver muscle heavy metals (mg/kg) mean±sd min max mean±sd min max ni 0.16±0.13 0.01 0.32 0.18±0.18 0.02 0.42 p>0.05 0.5 [17] zn 0.93±0.11 0.82 1.04 0.48±0.26 0.13 0.70 p<0.05 30 [23] pb 0.21±0.08 0.12 0.31 0.26±0.24 0.04 0.60 p>0.05 0.5 [17] fe 2.83±1.89 1.34 5.60 1.24±0.35 0.95 1.73 p>0.05 0.5 [17] cr 1.91±0.79 0.88 2.80 1.63±0.65 0.87 2.46 p>0.05 0.15[17] table 2 summary of heavy metals in tissues of parachanna obscura from the ikpoba river n=4 organs pvalue standards liver muscle heavy metals (mg/kg) mean±sd min max mean±sd min max ni 0.18±0.05 0.11 0.22 0.18±0.06 0.10 0.23 p>0.05 0.5 [17] zn 0.39±0.17 0.16 0.53 0.28±0.17 0.05 0.46 p<0.05 30 [23] pb 0.99±0.46 0.40 1.50 0.19±0.08 0.10 0.29 p>0.05 0.5 [17] fe 3.74±3.07 1.34 8.20 0.77±0.27 0.40 1.01 p>0.05 0.5 [17] cr 0.63±0.40 0.20 1.05 0.52±0.57 0.07 1.36 p>0.05 0.15[17] table 3 health risk assessment for the consumption of clarias gariepinus and parachanna obscura from ikpoba river clarias gariepinus parachanna obscura heavy metals risk model % contribution of metal to hi risk model % contribution of metal to hi edi thq edi thq ni 0.08 0.002 0.40 0.07 0.002 8.0 zn 0.20 0.000 0.00 0.12 0.000 0.0 pb 0.11 0.014 25.00 0.08 0.010 40.0 fe 0.51 0.000 0.00 0.32 0.000 0.0 cr 0.67 0.040 71.43 0.22 0.013 52.0 hi 0.056 no risk 0.025 no risk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 128 hazard index (hi) values for c. gariepinus and p. obscura were < 1, an indication of minimal risk exposure of no significant health risk to consumers of these fishes. this agrees with the findings of [24] for hemichromis fasciatus and [25]. several studies have reported that hi should not be > 1 in order to ease public health concern [26, 27]. chromium contributed the highest value to the hi and this corroborates with the reports of [28] for sarotherodon melanotheron. 4. conclusion assessment of heavy metals in fish from contaminated areas can be extremely important in evaluating the potential health risks to humans associated with consumption of fish from these contaminated catchments to safeguard human health. it also improves our knowledge on the biological status of the aquatic ecosystems and how it adapts or changes according to the change in the surrounding environmental conditions. the hazard index showed that the fishes do not pose any health risk to humans through consumption. however, it is recommended that routine assessment of heavy metal bioaccumulation in fishes of the ikpoba river be carried out to provide baseline data for accurate estimation of health risks to humans and to protect the health of the aquatic ecosystem. 5. references [1] oboh, i. p and edema, c. u., levels of heavy metals in water and fishes from the river niger.journal of chemical society of nigeria 32 (2): 29 -34, (2007) [2] omoigberale m. o. and ikponmwosa-eweka o., evaluation of heavy metals of the palaemonid shrimp macrobrachiumvollenhoveniiherklots, 1851 in ovia river, nigeria. bioscience research communications, 22 (5): 247 – 254, (2010). [3] mansour, s. a. and sidky, m. m., “ecotoxicological studies: heavy metals contaminating water and fish from fayum governorate, egypt,” food chemistry, 78 (1): 1522, (2002) [4] petera j. and viraraghavanb, t., a review of public health and environmental concerns.environ. int. 31: 493 – 501, (2005) [5] youn-joo, a. total, dissolved and bioaccumualation in freshwater fish with emphasis on the dietary influence, water res.34: 4234 – 4242, (2003) [6] taiz, l. and zeiger, e., plant defences: surface protectants and secondary metabolites. in taiz l and zeiger e (ed.s).plant physiology, sinauer associates, massachusetts.pp 347 – 377, (1998) [7] obasohan, e. e., oronsaye, j. a. o., eguavoen, o. i., a comparative assessment of the heavy metal loads in the tissues of a common catfish (clarias gariepinus) from ikpoba and ogba rivers in benin city, nigeria. african scientist9 (1): 13 – 23, (2008) [8] chailapakul, o., korsrisakul s., siangproh w. and grudpan k., fast and simultaneous detection of heavy metals using a simple and reliable microchip-electrochemistry route: an alternative approach to food analysis. talanta, 74:83–689, (2008) [9] united states environmental protection agency (usepa) (2012) risk assessment guidance for superfund: vol iii part a. process for conducting probalistic risk assessment epa, 540r-02-002 oswer 9285745pb2002 963302. usepa, human health risk assessment. (available at http://www.epa.gov/riskk/healthrisk.html retrieved. 6th june, 2017) [10] li j, huang z., hu y., yang h., potential risk assessment of heavy metals by consuming shellfish collected from xiamen, china. environ sci. pollut. res. int. 20 (5): 2937-47, (2013) [11] american public health association (1998).standard methods for the examination of water and waste water.20th edn.american public health association, washington. pp 1134. [12] douye p. n. and michael, h., assessment of non-carcinogenic human health risk of some heavy metals in land snails commonly consumed in bayelsa state, nigeria. research journal of chemical sciences, 5 (10): 6166, (2015) [13] lushenko m. a., a risk assessment for ingestion of toxic chemicals in fish from imperial beach, california: san diego state university, (2010) http://www.epa.gov/riskk/health-risk.html http://www.epa.gov/riskk/health-risk.html food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ijeoma oboh , benjamin ok para , peace wilfred ekprikpo, human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria, food and environment safety, volume xviii, issue 2 – 2019, pag. 123 – 129 129 [14] eneji, s. i., ato, r. s. and annune, p. a., bioaccumulation of heavy metals in fish (tilapia zilliand clarias gariepinus) organs from river benue, north central nigeria, pak. j. anal. environ. chem. 12 (1-2):25-31, (2011) [15] saeed m.s., and shaker m.i., assessment of heavy metals pollution in water and sediment and its effect on oreochromisniloticusin the northern delta lakes egypt, 8th international symposium on tilapia in aquaculture, egypt 1: 475-490, (2008) [16] abubakar, a., uzairu, a., ekwumemgbo, p. a. and okunola, o. j., risk assessment of heavy metals in imported frozen fish scomberscombrus species sold in nigeria: a case study in zaria metropolis.journal of aquatic sciences, 21 (1): 23 – 28, (2015). [17] fao/who (2003) code of practice for fish and fishery products. joint fao/who foods standards programme. report cac/rcp 52-2003. fao, rome. (rev. 2004, 2005, 2007.) [18] harmanescu, m., alda, l. m., bordean, d. m., gogoasa, i. and gergen, i., heavy metals health risk assessment for population via consumption of vegetables grown in old mining area; a case study: banat county, romania. chem central j., 5: 64 – 70, (2011) [19] abdou, h. m. and hassan, m. a., protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver and kidney of female rats.biomed res in,10: 1155 – 1162, (2014) [20] jovic, m. and stankovic, s., human exposure to trace metals and possible public health risks via consumption of mussels mytilusgalloprovincialis from the adriatic coastal area, food chemtoxicol70: 241–251, (2014) [21] tao, y., yuan, z., xiaona, h. and wei, m., distribution and bioaccumulation of heavy metals in aquatic organisms of different trophic levels and potential health risk assessment from taihulake, china. ecotoxicol environ saf, 81: 55-64, (2012) [22] taweel, a., shuhaimi-othman, m. and ahmad, a. k., assessment of heavy metals in tilapia fish (oreochromisniloticus) from the langat river and engineering lake in bangi, malaysia, and evaluation of the health risk from tilapia consumption, ecotoxicol environ saf.93: 45-51, (2013) [23] fao (food and agriculture organization), “compilation of legal limits for hazardous substances in fish and fishery products,” fao fisheries circular. 464: 5-100, (1983) [24] enuneku, a. a. and adelusi, m. a., heavy metal levels in hemichromisfasciatus from ubeji creek, warri, delta state nigeria: implications on human health through consumption european international journal of science and technology, 4(7):24 – 31, (2015) [25] malakootian, m., mortazavi, m. s. and ahmadi, a. (2016).heavy metals bioaccumulation in fish of southern iran and risk assessment of fish consumption. environmental health engineering and management journal 3 (2): 61–68. [26] islam, m. s., ahmed, m. k., almamun, m. h., islam, k. n., ibrahim, m. and masunaga, s., arsenic and lead in foods: a potential threat to human health in bangladesh. food add contam part a. 10.1080/19440049, (2014) [27] zodape, g. v., metal contamination in commercially important prawns and shrimps species collected from kolaba market of mumbai (west coast) india. int. j. agri. sci.4: 160–169, (2014). [28] moslen, m. and miebaka, c. a., concentration of heavy metals and health risk assessment of consumption of fish (sarotherodonmelanotheron) from an estuarine creek in the niger delta, nigeria.iosr journal of environmental science, toxicology and food technology (iosr-jestft) 11 (3): 68-73, (2017) 1. introduction 180 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 180 189 biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks * tsvetko prokopov 1 , nayden delchev 2 , bozhidar bozadzhiev 3 , donka taneva 1 , milena nikolova 1 , danislav spasov 2 1faculty of economic, department of engineering ecology, university of food technologies, plovdiv, bulgaria, tsvetko_prokopov@ abv.bg, 2faculty of technology, department of analytical chemistry , university of food technologies, plovdiv, bulgaria, naiden555@ abv.bg, 3faculty of technology, department of technology of cereal, bread and confectionary products, university of food technologies, plovdiv, bulgaria, bbozadjiev@ yahoo.de, *corresponding author received 11th june 2017, accepted 27 th september 2017 abstract: the purpose of this paper is to tested jerusalem artichoke stalks as a cheap biosorbent for its ability to remove mn (ii) ions from aqueous solution. batch experiments were carried out to evaluate the effects of ph, biosorbent particle size, dosage, initial metal concentration and contact time. the maximum removal efficiency of about 97.0 % was reached at ph 8.0 by using of biosorbent particle size 530-850 m, adsorbent dosage 30 g/l, initial metal concentration 10 mg/l, temperature 20 oc, agitation speed 120 rpm and contact time 90 min. pseudo-first order and pseudo-second order models were applied to describe the obtained kinetic data. the pseudo-second order model provided the best fit for experimental data with coefficient of determination r2 > 0.99. freundlich and langmuir isotherm models were used to describe metal adsorption. equilibrium data agreed well with langmuir isotherm with r2 = 0.993. keywords: removal, manganese, water, biosorbent. 1. introduction environmental pollution with industrial wastewaters contaminated with various metals has become one of the major ecological problems nowadays. the raw and wastewaters that consist of heavy metals ought to be treated, because it will be harm the ecosystems and public health. heavy metals are non-degradable and causing various diseases and disorders [1]. the existing methods used for metal removal from the water could be classified in three main groups such as physical, chemical and biological. adsorption is one of the successful methods of physicochemical treatment process to remove heavy metals from aqueous solution [2]. recently, there has been a tendency to shift conventional adsorbents with natural sorbents, especially waste materials, because of its low price, large availability, high efficiency, biodegradability and safety. the ability of the large number of microbial and plant waste materials to bind heavy metals have been reported in the literature. most of the adsorption studies focused on using plant waste materials, such as leaf powder, rice husks, sunflower stalks, peanut hull pellets, marine algae, etc. [3]. the jerusalem artichoke stalks have been applied as cheap biosorbent for removal of http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro mailto:mariap@fia.usv.ro mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 181 cu (ii) and fe (iii) ions from aqueous solutions [4, 5]. bio-sorption may be defined as a process of removing the metal or metalloid components from the aqueous solution by biological materials. the mechanisms involved in the process of bio-sorption include chemical sorption, complex formation, surface adsorption, inner porous adsorption, ion exchange, etc. it has been found that in the cellular and tissue materials exist a large number of groups (cooh, -oh, -sh, =nh, -nh2, etc.) which have ability to bind metal and therefore they are ionic functional groups of the biomass [6]. numerous empirical models for single solute systems have been employed to describe the bio-sorption equilibrium, namely langmuir, freundlich, brunaueremmet-teller, sips, dubininredishkevich, temkin and toth models. in kinetic modelling the pseudo-first and pseudo-second order equations are considered as the most celebrated models [7]. manganese is common constituent in waters. it is known that mn (ii) exposure causes neurotoxicity, low hemoglobin levels and gastrointestinal accumulation [8-10]. the who set a maximum acceptable drinking water concentration for manganese of 0.05 mg/l. therefore, it is necessary to treat the manganese contaminated water in order to reduce the environmental and human health risks. many studies have been conducted recently on the bio-sorption of mn (ii) from water. as adsorbents usually are used bacteria, corncob biomass, green tomato husks, sewage activated sludge, white rice husk ash, yields and spent mushroom compost [11-17]. to the best of our knowledge, absorbent prepared from jerusalem artichoke had not been used for the removal of mn (ii) ions from aqueous solution. therefore, this study examines the performance of jeruslalem artichoke stalks, as cheap biosorbent, for its ability to remove mn (ii) ions from aqueous solution. the effects of different process parameters, such as ph, adsorbent dosage, adsorbent particle size, contact time and initial metal concentration on manganese removal efficiency were determined. the mn (ii) ions biosorption equilibrium and kinetic modeling were carried out. 2. matherials and methods matherials. in the present study the powder obtained from stalks of jerusalem artichoke by cutting, drying (40oc), milling and sieving was used. for the removal of certain interfering components (pigments, etc.) the plant material was extracted twice with distillated water (1:7) for 45 min at 25oc under continuously stirring. after that the material was dried at 40oc. the particle size distribution was determined by mechanical sifting of driеd fractions of powder. sieves with standard light openings: 1 (2000-3000 m), 2 (1250-2000 m), 3 (850-1250 m), 4 (530850 m), 5 (400-530 m), 6 (250-400 m), 7 (150-250 m) and 8 (0-150 m) were used. the sifting was performed on a laboratory sifting machine. the function of the density distribution was calculated [18,19]. all reagents used in the experiments were of analytical grade. stock solution of mn (ii) was prepared by dissolving of mnso4.5h2o (merk) in distillated water. this solution was diluted with distillated water to obtain desired concentrations of working solutions for the batch experiments study. the ph value of the samples was adjusted by adding 0.1 m naoh or hcl solutions. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 182 biosorption batch experiments. biosorption experiments were carried out in 250 ml erlenmeyer glass flasks with 100 ml volume of manganese solution. batch experiments were conducted by varying the ph value (3, 4, 5, 6, 7, 8 and 9), initial mn (ii) concentration (5, 10, 30 and 40 mg/l) and biosorbent dosage (from 1.0 to 35.0 g/l). experiments were carried out at contact time of 24 h in order to reach equilibrium, agitation speed 120 rpm and ambient temperature 20.0±0.5oc. all experiments were performed in duplicate. analytical methods. for determination of mn(ii) concentration in the solutions before and after biosorption, 10 ml of samples were withdrawn, filtered (wathman no. 42) and filtrate was analyzed spectrophotometrically according to standard formaldoxim spectrometric method [20-21]. the metal uptake q (mg/g) was determined by employing the mass balance. if c0 and ce are the initial and final metal concentration (mg/l), respectively; v (l) is the initial volume of mn (ii) solution and m (g) is the mass of biosorbent material, the equilibrium metal uptake qe (mg/g) can be calculated as: m v).cc( q e0e   (1) the performance of biosorption was evaluated in terms of its removal efficiency as re (%), estimated by the following equation: 100. c )cc( re 0 t0  (2) where ct is the mn (ii) concentration at time t. kinetic experiments. batch kinetic experiments were carried out. for this purpose, 3.0 g of biosorbent were contacted with 100 ml of mn (ii) aqueous solution with initial metal concentrations 5, 10, 30 and 40 mg/l in 250 ml erlenmeyer glass flasks on a magnetic stirrer at 120 rpm, ph 8.0 and temperature 20.0±0.5oc. at different time intervals ranging from 5 to 180 min the concentrations of mn (ii) in the treated solutions were determined as described in analytical methods. kinetic modeling. the lagergren model was employed due to its simplicity and good fit. two different kinetic models were used to model experimental data. the pseudo-first-order model is expressed as: )qq(k dt dq eads,1  (3) where qe (mg/g) and q are amounts of adsorbed metal ions on the biosorbent at the equilibrium and any time t, respectively; and k1,ads is the lagergren rate constant of the first-order biosorption (min-1). the model is based on the assumption that the rate is proportional to the number of free site [7, 22]. integrating eq. (3) between the limits, t=0 to t=t and q=0 to q=q yields the linearized version of this model: 303.2 t.k qlog)qqlog( ads,1 ee  (4) linear plots of log(qe-q) versus t were plotted to evaluate this kinetic model and to determine rate constant and qe from the slope and intercept, respectively. the pseudo-second-order model is based on the assumption that biosorption follows a second-order mechanism, whereby the rate of sorption is proportional to the squire of the number of unoccupied sites [7, 22]: 2 eads,2 )qq(k dt dq  (5) where k2,ads is the rate constant of secondorder biosorption (g/mg.min). integrating eq. (5) from t=0 to t=t and q=0 to q=q and linearization yields: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 183 e 2 eads,2 q t q.k 1 q t  (6) the parameters qe and k2,ads are calculated from the slope and the intercept of the plot t/q versus t. it is not necessary to independently determine qe to apply this model. isotherm experiments. equilibrium sorption experiments were performed as follow. biosorbent (3 g) were exposed to 100 ml mn (ii) solution with an initial concentrations 5, 10, 30 and 40 mg/l at constant ph 8.0, agitation speed 120 rpm and ambient temperature 20.0±0.5oc for 24 h in order to rich equilibrium. sorption isotherm is plotted of the sorbate uptake (qe) versus the equilibrium concentration of the residual sorbate remaining in the solution (ce). sorption isotherm modeling. the freundlich isotherm which has been widely used in correlating equilibrium data can be expressed by the following linearized logarithmic form: efe clg n 1 klgqlg  (7) where qe (mg/g) is the amount of mn (ii) removed per unit mass of the adsorbent, ce (mg/l) is the residual mn(ii) concentration of the aqueous solution, kf and n are freundlich constants and measures of adsorption capacity and adsorption intensity, respectively. a higher n value (lower value of 1/n) implies stronger sorbent-pollutant interaction whereas 1/n equal to 1 indicates linear adsorption leading to identical adsorption energies for all sites [7]. the langmuir isotherm is based on three assumptions: namely adsorption is limited to monolayer coverage, all surface sites are alike and only can accommodate one adsorbed atom and the ability of a molecule to be adsorbed on a given site is independent of its neighboring sites occupancy [7]. this isotherm can be described by the following linearized form: maxemaxle q 1 c 1 qk 1 q 1          (8) where qe (mg/g) is the equilibrium amount of mn (ii) adsorbed, ce (mg/l) is the equilibrium concentration of mn (ii) in the solution, qmax (mg/g) and kl (l/mg) are langmuir constants representing the maximum monolayer adsorption capacity for the solid phase loading and the energy constant related to the heat of adsorption, respectively. for the langmuir isotherm analysis, the separation factor (rl) value is of special importance: 0l l ck1 1 r   (9) where c0 (mg/l) is the initial mn (ii) concentration in the solution. four possibilities for the separation factor values which determine the isotherm type: rl = 0 (irreversible isotherm), rl = 1 (linear isotherm), rl>1 (unfavorable isotherm) and rl<1 (favorable isotherm) have been reported [23]. 3. results and discussion effect of ph. hydrogen ion concentration (ph) of the aqueous solutions is an important parameter since it affects the surface charge of sorbent and the degree of speciation and ionization of sorbent during adsorption [1]. from the practical point of view, the most important was to be determined the optimal ph at which the investigated system could reach its maximum removal efficiency [15]. the effect of ph on the mn (ii) removal efficiency from the aqueous solution by studied biosorbent is illustrated in figure 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 184 it was found that the maximum removal efficiency (81.3 %) was reached at ph 8.0, than it sharply decreased probably due to formation of mn(oh)2. fig. 1. effect of ph on the removal efficiency of mn (ii) from aqueous solution by powder of jerusalem artichoke stalks (initial metal concentration 10 mg/l, biosorbent dosage 1 g/l, 20 o c, 24 h, agitation speed 120 rpm) effect of adsorbent particle size and dosage. the biosorbent particle size and dosage are other two major factors affect the biosorption efficiency. for the evaluation of particle size effect on mn (ii) biosorption, batch experiments were carried out with eight size ranges of bioserbent particle and dosage of 1 g/l. to investigate the effect of biosorbent dosage on biosorption, the experiments were conducted with constant manganese concentration of 10 mg/l and samples with different biosorbent dosage ranging from 1.0 to 35.0 g/l were used under the constant temperature 20oc and ph 8.0. data are given in figures 2 and 3. regarding to the influence of the biosorbent particle size on mn (ii) removal efficiency, the results in figure 2 indicate that the optimal particle size yielded the maximum metal removal of 81.3% was from the range 530-850 m. the mn (ii) removal efficiency was found to increase from 81.3% to 97.0% with the increase of the biosorbent dosage from 1.0 to 30.0 g/l (figure 3), which can be explained by an increase of the contact surface available for adsorption and the presence of a larger number of groups, binding the mn (ii) ions. application of higher biosorbent dosage above 30.0 g/l did not lead to significant increase (p < 0.05) in metal removal efficiency due to possible aggregation of the biomass particles and disturbing of the mass transfer of the metal ions from the liquid to solid phase of the system. similar results have been reported by other researchers [12, 15, 24, 25]. effect of initial metal concentration. the initial metal concentration is essential for the duration of biosorption and the rate of metal removal from aqueous solutions. results for the influence of the initial mn (ii) concentration in the solution on metal removal efficiency are presented in figure 4. fig. 2. effect of biosorbent particle size on the removal efficiency of mn (ii) from aqueous solution by powder of jerusalem artichoke stalks (initial metal concentration 10 mg/l, ph 8.0, 20 o c, biosorbent dosage 1 g/l, 24 h, agitation speed 120 rpm) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 185 fig. 3. effect of biosorbent dosage on the removal efficiency of mn (ii) from aqueous solution by powder of jerusalem artichoke stalks (initial metal concentration 10 mg/l, ph 8.0, 20 o c, 24 h, agitation speed 120 rpm) fig. 4. effect of initial metal concentration (c0, mg/l) on the removal efficiency of mn (ii) from aqueous solution by powder of jerusalem artichoke stalks (biosorbent dosage 30 mg/l, ph 8.0, 20 o c, 24 h, agitation speed 120 rpm) as shown in figure 4, the removal efficiency of mn (ii) ions from aqueous solutions by used biosorbent was found to decrease with the increase of the initial metal concentration, probably due to the restriction or saturation of the biosorbent active groups. the highest level of removal efficiency (97.0%) was established at an initial metal concentration of 10 mg/l. similar results have been reported for biosorption of mn (ii) ions from aqueous solutions by corncob biomass [12]. adsorption kinetics data. the contact time of the biosorbent and sorbate also has a significant impact on the biosorption efficiency. biosorption kinetics of mn (ii) ions on powder of jerusalem artichoke stalks at four different initial metal concentrations in the aqueous solution are presented in figure 5. the results indicated that the biosorption process can be divided into two main stages. the initial rapid stage where biosorption was fast and second slow stage which refers to gradual biosorption before manganese uptake reached equilibrium. a similar phenomenon was observed by others also [12, 26, 27]. the established times taken to reached equilibrium for mn(ii) concentrations of 5, 10, 30 and 40 mg/l were 30, 90, 150 and 120 min, respectively. kinetic data were fitted onto two kinetic models of pseudo-first and pseudo-second order with acquired parameters listed in table 1. by comparing the fitting results the pseudo-second model seems to give better representation of the experimental data. moreover, calculated values of equilibrium metal uptake (qe cal, mg/g) from pseudosecond model agree quite well with experimentally obtained values (qe exp, mg/g). equilibrium isotherms data. equilibrium adsorption isotherms study is of fundamental importance in the design of adsorption systems, since it provides the basic physicochemical data to evaluate the suitability of the sorption process for removal of different pollutants, incl. heavy metals, from aqueous solutions. adsorption equilibrium is usually described by an isothermal equation whose parameters express the surface properties and the affinity of the adsorbent at a given temperature and ph of the solution. therefore, an adequate mathematical description of the experimentally obtained equilibrium isotherm is essential for the efficient design of the system. in this work, two of the most frequently used model were tested [7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 186 fig. 5. biosorption kinetics of mn (ii) ions on powder of jerusalem artichoke stalks (biosorbent dosage 30 mg/l, ph 8.0, 20 o c, agitation speed 120 rpm) table 1. kinetic parameters for the biosorption of mn (ii) ions onto powder of jerusalem artichoke stalks initial mn(ii) ions concentration (c0), mg/l experimental metal uptake (qe exp), mg/g pseudo-first order model pseudo-second order model qe cal, mg/g k1ads, min-1 r2 qe cal, mg/g k2ads, g/mg.min r2 5 0.1600 0.0122 0.126 0.999 0.1614 21.58 1.0 10 0.3233 0.0227 0.044 0.615 0.3253 4.98 1.0 30 0.8270 0.2003 0.021 0.976 0.8378 0.336 0.999 40 0.9983 0.3525 0.035 0.928 1.0188 0.275 0.999 the results of the application of langmuir and freundlich linearized models to the experimental data for biosorption of mn (ii) ions from aqueous solutions by the investigated biosorbent under equilibrium conditions are presented in figures 6 and 7. model parameters were determined using linear regression toolbox in microsoft excel software and are summarized in table 2. fig. 6. linearized langmuir isotherm for biosorption of mn (ii) ions on powder of jerusalem artichoke stalks (biosorbent dosage 30 mg/l, ph 8.0, 20 o c, 24 h, agitation speed 120 rpm) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 187 table 2. isotherm parameters of langmuir and freundlich models for biosorption of mn (ii) ions onto powder of jerusalem artichoke stalks langmuir isotherm model freundlich isotherm model qmax, mg/g kl, l/mg r2 1/n kf, mg/g r2 1.08 0.95 0.993 0.4163 0.404 0.933 fig. 7. linearized freundlich isotherm for biosorption of mn (ii) ions on powder of jerusalem artichoke stalks (biosorbent dosage 30 mg/l, ph 8.0, 20 o c, 24 h, agitation speed 120 rpm) the regression parameters and coefficients of determination (r2) presented in table 2 indicate that the langmuir and freundlich models fitted very well the experimental data with the calculated coefficients of determination (r2) of 0.993 and 0.933, respectively. similar results were reported for mn (ii) biosorption by corncob biomass [12]. the value of the r2 for langmuir model is slightly higher than that for freundlich model and the langmuir adsorption capacity (qmax) is larger than that obtained by application of freundlich model (kf). the langmuir rl values were found to be between 0 and 1, which confirmed that the biosorbent prepared from the jerusalem artichoke stalks is favourable for biosorption of mn (ii) ion under conditions used in this study. 4. conclusion present research showed that powder of jerusalem artichoke stalks is a potential biosorbent for removal of mn (ii) ions from aqueous solution. the batch study parameters, such as ph of solution, biosorbent particle size, biosorbent dosage, contact time and initial manganese concentration were found to be important on the biosorption process. finally, the experimental results of present study indicated that maximum removal efficiency of mn (ii) ions of 97.0% was obtained at ph 8.0, biosorbent particle size 530-850 m, adsorbent dosage 30 g/l, initial metal concentration 10 mg/l, temperature 20 oc, agitation speed 120 rpm and contact time 90 min. the pseudosecond order kinetic model was found to fit well experimental data with r2 > 0.99 and the adsorption isotherm followed langmuir model (r2 = 0.993). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 188 5. references [1]. dahlan i., hassan s., hakim m., removal of iron (fe2+) from aqueous solution using siliceous waste sorbent, sustain. environ. res., 23(1): 41-48, (2013) [2]. volesky b., detoxification of metalbearing effluents: biosorption for the next century, hydrometalurgy, 59: 203-206, (2001) [3]. wang j., chen c., biosorbents for heavy metals removal and their future, biotech. adv., 27: 195-226, (2009) [4]. prokopov t., removol of copper (ii) from aqueous solution by biosorption out powder of jerusalem artichoke, ecologicol engineering and environmental protection, 1: 24-32, (2015) [5]. prokopov t., delchev n., taneva d, biosorption of fe (ііі) from aqueous solutions by jerusalem artichoke (helianthus tuberosus l.) powder, journal of food and packaging science, technique and technologies, 3: 64-68, (2014) [6]. gardea-torresdey j., rosa g., peralta-videa j., use of phytofiltration technologies in the removal of heavy metals: a review, pure appl. chem., 76(4): 801813, (2004) [7]. febrianto j., kosasih n., sunarso j., ju y., indraswati n., ismadji s., equilibrium and kinetic studies in adsorption of heavy metals using biosorbent: a summary of recent studies, journal of hazardous materials, 162: 616-645, (2009) [8]. vêliz g., mora s., gómez p., dossi m., montie i., arriagada c., aboitiz f., aguilar s., behavioral effects of manganese injected in the rat sub stantiaciagra are potentiated by dicumarol, dt-diaphoras inhibitor, pharmacology biochemistry and behavior, 77(2): 245-251, (2004) [9]. burgoa c., rios c., mercado l., serrano s., valle f., wynter r., sangrador j., barragan j., agudelo y., montes s., exposure to manganese health effects on the general population, a pilot study in central mexico, environmental research, 85(2): 90-104, (2001) [10]. henrik s., thomsen m., svendsen o., klastrup s., increased manganese concentration in the liver after oral intake, academic radiology, 11(1): 38-44, (2004) [11]. gialamonidis d., mitrakas m., liakopoulow-kyriakides m., equlibrium, thermodynamic and kinetic studies on biosorption of mn (ii) from aqueous solution by psudomonas sp., staphylococcus xylosus and blakeslea trispora cells, journal of hazardous materials, 182: 672-680, (2010) [12]. adeogum a., ofudje a., idowu m., kareem s., equlibrium, kinetic and thermodynamic studies of the biosorption of mn (ii) ions from aqueous solution by raw and acid treated concorb biomass, bioresources, 6(4): 4117-4134, (2011) [13]. garcĺa-mendieta a., olguin m., solache-rios m., biosorption properties of green tomato husk (physalis philadelphica lam) for iron, manganese and iron-manganese from aqueous systems, desalination, 284: 167-174, (2012) [14]. hasan h., abdullah s., kofli n., kamarudin s., isotherm equilibria of mn2+ biosorption in drinking water treatment by locally isolated bacullus species and sewage activated sludge, journal of environmental management, 111: 34-43, (2012) [15]. tavlieva m., genieva s., georgieva v., vlaev l., thermodynamics and kinetics of the removal of manganese (ii) ions from aqueous solutions by white rice husk ash, journal of molecular liquids, 211: 938-947, (2015) [16]. fadel m., hassanein n., elshafei m., mostafa a., ahmed m., khater h., biosorption of namganese from groundwater by biomass of saccharomyces cerevisiae, hbrc journal, 13(1): 106-113, (2017) [17]. kamarudzaman a., chay t., amir a., talib s., biosorption of mn (ii) ions from aqueous solution by pleurotus spent mushroom compost in a fixed-bed column, procedia-social and behavioral sciences, 195: 2709-2716, (2015) [18]. stieß m., mechanische verfahrenstechnik 2, springer-verlag, (2001) [19]. schubert h., handbuch der mechanischen verfahrenstechnik, wileyvch, (2002) [20]. marczenco z., dosage spectrophotometrique de differents metaux (mn, ce, v, ni, fe) au moyen de la formaldoxime, analitica chimica acta, 31: 224-232, (1964) https://www.amazon.de/matthias-stie%c3%9f/e/b0045azjpw/ref=dp_byline_cont_book_1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 tsvetko prokopov, nayden delchev, bozhidar bozadzhiev, donka taneva, milena nikolova, danislav spasov, biosorption of mn (ii) ions from aqueous solution by jerusalem artichoke (helianthus tuberosus l.) stalks, food and environment safety, volume xvi, issue 3 – 2017, pag. 180-189 189 [21]. din 38406-2, german standard methods for the examination of water, waste water and sludge, cations (group e), determination of manganese (e2), deutsches institut fur normung e.v., (1983) [22]. el-naas m., al-zuhar s., m. alhaija m., reduction of cod in refinery wastewater through adsorption on date-pit activated carbon, journal of hazardous materials, 173: 750-757, (2010) [23]. foo k., hameed b., insights into the modeling of adsorption isotherm system, chemical engineering journal, 152: 2-10, (2010) [24]. suguna m., reddy a., kumar n., krishnaiah a., biosorption of manganese ions from aqueous solution by glutaraldehyde cross-linked chitoson beads. equilibriaum and kinetic studies, adsorption science and technology, 28 (3). p. 213-228, (2010) [25]. zhang y., zhao j., jiang z., shan d., lu y., biosorption of fe (ii) and mn (ii) ions from aqueous solution by rice husk ash, hindawi biomed research international, article id 973095, 10 pages http://dx.doi.org/10.1155/2014/973095 [26]. el-sayed g., dessouki h., ibrahiem s., removal of zn, cd, mn from aqueous solution by adsorption on maize stalks, the malaysian journal of analytical sciences, 15(1): 8-21, (2011) [27]. vijayaraghavan k., winnie h., balasubramanian r., sorption characteristics of crab shell particles for the removal of manganese (ii) and zinc (ii) from aqueous solutions, desalination, 266: 195200, (2011) http://dx.doi.org/10.1155/2014/973095 1. introduction 4. conclusion 306 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 306 309 the nitrogen compound content of some natural mineral waters from bukovina, romania,versus their bottled form *anca-mihaela sidor1 1 food engineering faculty, stefan cel mare university of suceava, romania, ancagatlan@gmail.com *corresponding author received september 4th 2015, accepted september 28th 2015 abstract: the purpose of this paper is to study the problem of water contamination by nitrogen compounds such as nitrates, nitrites and ammonium in three zones of interest: one drill river in păltiniș reservoir and „rău” and „chiril” rivers flowing through crucea village in bukovina area, romania. the work direction was to investigate the contamination level in the rivers and to compare the obtained results with the levels found in the bottled form of the water originating from these rivers, given the obligatory drinking water standards. this issue occured during debates with the local society. after developing the study we can remark that the water contamination levels by nitrogen compounds in the water sources rivers in the three chosen zones of interest and in the bottled still spring water as well, are lower than the maximum allowable value, but not 0. mention should be made that there are no efforts made during the industrial water conditioning process to reduce the levels of nitrate, nitrite and ammonium in the water to be consumed by the population. keyword: water, contamination level, nitrogen compounds, zones of interest, bottled still water 1. introduction under the conditions of life existence, in general, and the conditions of ongoing human activities, especially, the water in different hydrological formations presents a double importance. first of all, it is an environmental factor, respectively,a habitat of a variety of living organisms. on the other hand, for people water composes a medium widely used in different types of their economical activity, but the chief use of water is for consumption [1]. the quality is the main dimension of water and the subject of mineral water quality is particularly interesting due to concerns about the concept of natural mineral water, consumption benefits and the original purity of bottled mineral water, a subject about which there have been many speculations lately [2].for this purpose, water is drawn from various sources. in many areas,the surface water is only used after the removal of the pollutants through treatment processes.from among water contaminants particularly burdensome are inorganic nitrogen compounds.in aerobic waters nitrogen is mainly present as n2and no3 -, and depending on environmental conditions it may also occur as n2o, nh3, nh4 +, hno2, no2 -or hno3.ammonium,nitrate and nitriteplay the most important role in biochemical processes [3]. nitrogen is essential for all living things as it is a component of protein. however, http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro http://www.lenntech.com/hazardous-substances/nitrate.htm http://www.lenntech.com/hazardous-substances/nitrite.htm food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 anca-mihaelasidor, the nitrogen compounds content of some natural mineral waters from bucovina, romania versus their bottled form,food and environment safety, volume xiv, issue 3 – 2015, pag. 306 – 309 307 excessive concentrations of nitratenitrogen or nitrite-nitrogen in drinking water can be hazardous to health, especially for infants and pregnant women [4].the primary health hazard from drinking water with nitrate-nitrogen occurs when nitrate is transformed to nitrite in the digestive system [5]. the nitrite oxidizes the iron in the hemoglobin of the red blood cells to form methemoglobin, which lacks the oxygen-carrying ability of hemoglobin. this creates the condition known as methemoglobinemia (sometimes referred to as „blue baby syndrome”), when blood iron in hemoglobin (fe2+) is reduced to its oxidized form,fe3+, the blood lacks the ability to carry sufficient oxygen to the individual body cells causing the veins and skin to appear blue [6]. a possibility exists that nitrate can react with amines or amides in the body to form nitrosamine which is known to cause cancer. nitrate must be converted to nitrite before nitrosamine can be formed. the magnitude of the cancer risk from nitrate in drinking water is not known [7]. the ammonium does not directly harm the human body in typical ph values (6,5 to 9,5) applied in drinking water treatment. however, it may form nitrite ions under oxidative conditions.beside the possible nitrite formation, the other issue related to the presence of ammonium in drinking water is the decrease of the chlorination disinfection efficiency. the ammonium reacts with chlorine forming chloramines, and thus reducing the amount of the disinfectant available for microorganism inactivation. the less efficient disinfection may cause secondary water pollution in the distribution system. moreover, the resulting chloramines cause the unpleasant smell, which may lead to customer complaints [8]. in the presented research, it is discussed the problem of water contamination with nitrogen compounds such as nitrates, nitrites and ammonium in three zones of interest: one drill river in păltiniș reservoir and „rău” and „chiril” rivers flowing through crucea village inbucovina area, romania.the aim of the work was to investigate the contamination level in the rivers and to compare the obtained results with the levels found in the bottled form of the water originating from these rivers, given the obligatory drinking water standards. this issue occured during talks with local society. 2. materials and methods 2.1. location of the research for this study, there were selected a number of three rivers from two localities in bucovina region, romania. the selection criteria of these localities, crucea and paltinis, were linked to their location, geographically speaking, and to the specific anthropogenic activities conducted in the area. subsequently, the three rivers wherefrom the water samples were drawn were chosen because they are the source of water for a big romanian company with the domain of activity linked to water treatment and bottling, and whose main marketing strategy is the spread of information that nitrogen compounds are absent in the spring still water they commercialize. crucea is a village located in the bistrita valley, in suceavacounty. the specific activities of the population incrucea village is the ore exploitation, farming and logging. the paltinis natural mineral water deposit is situated on the eastern rim of the calimani mountains, at the southern limit of the dorna depression. the deposit is hosted in the magnesium limestones in the cristalline-mesozoic area of the eastern carpathians which sank westward under the thick stack of the calimani mountain volcanic rocks. 2.2. materials food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 anca-mihaelasidor, the nitrogen compounds content of some natural mineral waters from bucovina, romania versus their bottled form,food and environment safety, volume xiv, issue 3 – 2015, pag. 306 – 309 308 the analysis of the nitrogen compounds content in the samples drawn from the three rivers and in the still spring water sample marketed by the company in question was performed using the hach lange dr 3800 spectrophotometer and the lck 353 kits. 2.3. methods to determine the nitrite content of the samples it was analysed a water volume of 2 ml. this volume was inserted into the kit vial, after the aluminum foil of the kit was unbent, followed by the stirring of the vial and an idle time of 10 minutes. after the specified time, the vial was placed in the spectrophotometer, wherein it was determined the amount of no2 contained in the test samples, at a λ =515 nm wavelength. to determine the nitrate content of the samples it was analysed a water volume of 1 ml and 0,2 ml of a reagent. this volume was inserted into the kit vial, after the aluminum foil of the kit was unbent, followed by the strong stirring of the vial and an idle time of 15 minutes. after the specified time, the vial was placed in the spectrophotometer, wherein it was determined the amount of no3 contained in the test samples, at a λ = 345 nm wavelength. to determine the ammonium content of the samples it was analysed a water volume of 0,2 ml. this volume was inserted into the kit vial, after the aluminum foil of the kit was unbent, followed by the stirring of the vial and an idle time of 15 minutes. after the specified time, the vial was placed in the spectrophotometer, wherein it was determined the amount of nh4 contained in the test samples, at a λ = 690 nm wavelength. 3. results and discussion from the correlation of the results it can be easily observed that in all the four studied samples the nitrate amount was lower than the maximum allowable value. however, a fraction of the nitrate contained in the samples was transformed to nitrite, their amount in the analysed samples being half of the maximum allowable value. in this context, it should be noted, however, that it were found certain quantities of nitrates and nitrites in the bottled still spring water sample marketed by the company in question, contrary to the commercials and the statements they broadcast in the media environment. fig. 1. the nitrate content (mg/l) in the water samples 0 10 20 30 40 50 maximum allowable value rău spring chiril spring păltiniș spring bottled still spring water 50 3,27 3,7 2,39 2,44 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 anca-mihaelasidor, the nitrogen compounds content of some natural mineral waters from bucovina, romania versus their bottled form,food and environment safety, volume xiv, issue 3 – 2015, pag. 306 – 309 309 fig. 2. the nitrite content (mg/l) in the water samples the amount of nitrates and nitrites found in the bottled still spring water sample is almost equal to the amount of nitrate and nitrite contained in the samples taken from the source river of the company which commercialises it. table 1 the ammonium content (mg/l) in the water samples nh4 (mg/l) rău spring 0 chiril spring 0,014 păltiniș spring 0,011 bottled still spring water 0,009 maximum allowable value 0,5 given that the maximum allowable value for ammonium ions in drinking water is 0,5 mg/l, the values obtained for the studied samples small, nonexistent in some cases (rău spring). this proves the purity of those sources in terms of domestic and industrial waste, which is not uncommon for mountain waters. 4. conclusion in conclusion we can remark that the water contamination levels with nitrogen compounds such as nitrates, nitrites and ammonium in the water sources rivers in the three chosen zones of interest and in the bottled still spring water as well,are lower than the maximum allowable value, but not 0. it can be noted that there are no efforts made during the industrial water conditioning process to reduce the levels of nitrate, nitrite and ammonium in the water to be consumed by the population. 5. references [1] pawełczyk a., assessment of health hazard associated with nitrogen compounds in water, water sci technol., 66(3): 666-672, 2012. [2] diţoiu v., modificari antropice ale mediului, editura orizonturi universitare, timisoara, 2005. [3] mason c., biology of freshwater pollution. fourth edition, essex, pearson education limited, 2002. [4] skipton s., dvorak b., woldt w., cassada c., drinking water: nitrate-nitrogen, university of nebraska-lincoln extension, institute of agriculture and natural resources, 2013. [5] robillard p.d., sharpe w.e., swistock, b.r.., nitrates in drinking water, college of agricultural sciences, u.s. department of agriculture, and pennsylvania counties cooperating, 2006. [6] beatson, c.g., methaemoglobinaemia nitrates in drinking water, environ. health, 86: 3133, 1978. [7] rousta m.j., lotfi e., shamsalam n., mousavi f., heshmati l.s., ghasemyfard s., nitrate situation in some vegetables and the necessity of crop production via organic farming, 19th world congress of soil science, soil solutions for a changing world 1–6 august, brisbane, australia, 2010. [8] takó s., ammonium removal from drinking water comparison of the breakpoint chlorination and the biological technology, conference of junior researchers in civil engineering, p. 248-254, 2012. 0 0,1 0,2 0,3 0,4 0,5 rău spring chiril spring păltiniș spring bottled still spring water maximum allowable value 0,232 0,205 0,254 0,251 0,5 272 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 272 278 impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria *henry uzoma anuforo 1 , campbell onyeka akujobi 2 , ethelbert uchechukwu ezeji 3 , confidence chiamaka okehi 1 1department of biology, school of biological sciences, federal university of technology, owerri, nigeria, henry.anuforo@futo.edu.ng 2department of microbiology, school of biological sciences, federal university of technology, owerri, nigeria 3department of biotechnology, school of biological sciences, federal university of technology, owerri, nigeria *correspondence author received 17th november 2019, accepted 27th december 2019 abstract: cassava tubers and soil samples on which they were cultivated were collected at 10m, 15m and 20m from two sections of roadsides of owerri-aba expressway. samples from section 1 were analyzed for the presence of lead (pb), cadmium (cd), copper (cu) and manganese (mn) while those from section 2 were analyzed for lead, zinc (zn), chromium (cr), cadmium and nickel (ni) using atomic absorption spectrophotometer (aas). results obtained showed that at section 1, the concentrations of heavy metals in cassava samples were in the range (0.0-13.5mg/kg) pb, (0.00.5mg/kg) mn, (1.5-16mg/kg) cu and (0.05-0.4mg/kg) cd. at section 2, they ranged from 3.225.48mg/kg (pb), 8.70-14.18mg/kg (zn), 0.0-0.16mg/kg (cr), 1.70-2.12mg/kg (ni) and 0.03-0.22mg/kg (cd). similarly, in soil samples from section 1, the concentrations were (0.0-25mg/kg) pb, (0.54.5mg/kg) mn, (0.0-10mg/kg) cu and (0.1-0.55mg/kg) cd, while those from section 2 were (7.49.6mg/kg) pb, (9.5-15.5mg/kg) zn, (0.2-0.7mg/kg) cr, (3.1-4.8mg/kg) ni and (0.3-0.5mg/kg) cd. in both samples from section 1, as well as only soil samples from section 2, the average concentrations of heavy metals studied decreased from 10 to 20m from the roadsides, except for pb. moreover, at section 1, the order of concentration of heavy metals is pd>cu>mn>cd for both soil and cassava tuber samples but zn>pb>ni>cr>cd in samples from section 2. only pb in cassava tubers harvested at most of the locations in the two sections far exceeded the 2.0 mg/kg maximum permissible limit (mpl) set by who/fao. furthermore, result of bioaccumulation factor showed that the cassava samples moderately bioaccumulated the heavy metals. keywords: vehicular traffic, heavy metals, cassava tubers, food safety, roadsides 1. introduction vehicular traffic constitutes a major nonpoint source of environmental pollution with its emissions polluting nearby air, land and water bodies [1]. studies have shown that fumes from exhausts of automobile account for about 80% of air pollution due to heavy metals in nigeria [2], [3]. in addition, mechanical abrasions of vehicular components including alloys, wires, tyres, oil and brake pads release some metals like cadmium, zinc, lead, iron and copper into roadside soil and plants [4]. thus, there is an increasing concentration of heavy metals in urban roadside soils and plant samples due mostly to higher traffic [5]. soil is http://www.fia.usv.ro/fiajournal mailto:henry.anuforo@futo.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 273 considered major reservoir or sink for metal contaminants in automobile emissions [1]. some heavy metals like cu, co, mn, ni, v, mo, fe and zn are essentially required in minute quantities by organisms for normal metabolic activities. for instance, cu and zn are known cofactors or activators of some enzymes [6]. however, when in excess, these elements may become harmful to organisms with associated neurological and cardiovascular impairments. other heavy metals like cd, pb, hg are toxic with no beneficial effects to organisms. heavy metal pollution is of very serious public health concern due to their persistence, toxicity and nonbiodegradability in the environment [7], [8]. these heavy metals may be found in the atmosphere in particulate form and can be transferred to land or water surfaces by wind, precipitation and occult deposition [9]. plants exposed to these heavy metals may absorb them through the leaves and roots [10]. cassava (manihot esculenta crantz) is a major staple food and feedstock in the world [11]. over 2 billion people worldwide, with about 700 million in tropical and sub-tropical regions depend on cassava tubers for the provision of carbohydrates and energy [12]. in fact, cassava is the fourth staple crops, following rice, sugar, and corn. the global output of cassava is about 183 million tons a year [13]. studies have indicated increasing presence of harmful levels of heavy metals such as cadmium, nickel, manganese, chromium, zinc, mercury, lead and iron in cassava and its associated products [14], [15]. this calls for an increasing public health concern and monitoring to avert the harmful effects on plants, animals and humans which share the same food chain [8]. 2. materials and methods study area the samples used in this study were collected from randomly selected farms on aba-owerri expressway, imo state, nigeria. the gps locations are 159428n, 512590e; 159333n, 513089e; 158814n, 514161e. collection of samples the cassava (manihot esculenta) tubers were harvested and samples of soil on which they were growing collected from three cassava farms cultivated over a year ago along aba -owerri expressway in imo state, nigeria. at each of the six locations studied, both cassava tubers and soil samples were collected at three distances of 10m, 15m and 20m from the roadside respectively. soil samples were collected using well cleaned soil auger at depths of 0.30m. the samples were properly labeled and sent to the laboratory for further processing. both soil and cassava samples from section 1 were analyzed for cadmium (cd), copper (cu), manganese (mn) and lead (pb) while those from section 2 were analyzed for cadmium (cd), nickel (ni), chromium (cr) zinc (zn) and lead (pb). sample preparation and analysis for heavy metals the collected cassava tubers were carefully peeled, pelletized, sun dried, pounded and then packaged in clean containers fitted with lids. as described by akinyele and shokunbi [16] with modifications, “wet digestion of samples was performed using mixtures of acids; hno3:hcl (3:1). twenty millilitres of the acid mixture was used for each 1 g sample digested. each mixture was heated up to 150 °c for 2½ h on the heating digestion block. then the acid digest was allowed to cool and filtered into a 25 ml volumetric flask, using whatman no 1 filter paper and made up to mark with de-ionized water. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 274 this way, organic matter was destroyed in the sample and a high concentration of the sample was achieved. the blank digests and spiked samples were similarly processed. the concentrations of pb, cd, mn, cr, zn and cu, depending on the section involved, were obtained using the atomic absorption spectroscopy (aas).” also, the modified method described by akinyele and shokunbi [16] was adopted in processing and analyzing soil samples. “one gram of each dried sample was weighed into a porcelain crucible and dryashed in a muffle furnace by stepwise increase of temperature up to 500 °c within 1 h and then leaving to ash at this temperature for additional 12 h. the residue was dissolved in 1 m nitric acid, filtered into a 25 ml volumetric flask using whatman no 1 filter paper and made up to mark with the nitric acid (1 m). the blank digests was similarly processed. the concentrations of pb, cd, mn, cr, zn and cu, depending on the section involved, were obtained using the atomic absorption spectroscopy (aas).” 3. results and discussion the concentrations of all the selected heavy metals studied were expectedly higher in soil samples than in cassava samples. the results further revealed that at section 1, the heavy metals concentrations ranged from 0.0 to 13.5 mg/kg, 0.0 to 0.5 mg/kg, 1.5 to 16mg/kg and 0.05 to 0.4mg/kg for lead, manganese, copper and cadmium in cassava samples respectively. similarly, at section 2, the concentrations ranged from 3.22 to 5.48mg/kg, 8.70 to 14.18mg/kg, 0.0 to 0.16mg/kg, 1.70 to 2.12mg/kg and 0.03 to 0.22mg/kg for pb, zn, cr, ni and cd in cassava samples respectively. these findings are similar to the results reported by kalagbor et al [17], but lower than the reports of osakwe and okolie [1], ogundele et al [18]. in soil samples from section 1, the concentrations were however, 0.0 to 25 mg/kg, 0.5 to 4.5 mg/kg, 0.0 to 10mg/kg and 0.1 to 0.55mg/kg for lead, manganese, copper and cadmium in soils samples respectively. the concentrations were 7.4 to 9.6mg/kg, 9.5 to 15.5mg/kg, 0.2 to 0.7mg/kg, 3.1 to 4.8mg/kg and 0.3 to 0.5mg/kg for pb, zn, cr, ni and cd respectively for soil samples in section 2. the results of this study lend credence to those reported by rahman and zaim [19], but lower than the concentrations reported by ogundele et al [18], asdeo [20], mbong et al [21], yahya et al [22]. it was also found that the concentrations of pb in the cassava tubers harvested at most of the locations in the two sections far exceeded the 2.0 mg/kg maximum permissible limit (mpl) set by who [23], who/fao [24], ahmad et al [25]. however, this corresponds to the results reported by kalagbor et al [17], ogundele et al [18]. such excessive concentration of pb in cassava tuber samples studied poses great risk to vulnerable populations including children [26]. conversely, the concentrations of chromium in all the cassava tuber samples were well below the 20mg/kg by who [23], who/fao [24] at all the locations studied. similarly, the concentrations of cadmium in all the tubers from the two sections studied were below the 0.5mg/kg limit by who [23], who/fao [24]. also, the concentrations of copper in the cassava tuber samples were far below 30mg/kg mpl by who [23], sauerbeck [27]. the concentrations of manganese in the tubers studied were far below the 55.5mg/kg mpl of who. moreover, the concentrations of zinc were found to fall below 60mg/kg mpl of who [23]. the concentrations of nickel were well below the 10mg/kg mpl set by who [23]. kalagbor et al [17] have earlier reported that the concentrations of most heavy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 275 metals in cassava samples studied were below the mpl. for soil samples from both sections studied, the concentrations of the heavy metals were quite below the mpl of 2300mg/kg, 20-100mg/kg, 140mg/kg, 3.0 mg/kg set by fepa [28], eu [29], 35mg/kg by ogundele et al [18], 30.8219.23mg/kg and 2-100mg/kg by mbong et al [21] for pd, mn, cu, cd, ni, zn and cr. the low concentrations of most of the heavy metals studied are clear evidence of low industrialization and limited anthropogenic activities in imo state, nigeria [30]. distribution of heavy metals in cassava tubers on roadside the average concentrations of heavy metals studied decreased from 10 to 20m distance away from the roadside at both sections, except for pb. this is corroborated by the results reported by yan et al [31]. these results are shown in figures 1 and 2 respectively. at section 1, the order of concentration of the heavy metals is pd>cu>mn>cd for both soil and cassava tuber samples. at section 2, it was zn>pb>ni>cr>cd for both soil and cassava tuber samples. these orders are related to the findings of osakwe and okolie [1] except for pb, but differ from the reports of mbong et al [21] and yan et al [31]. the higher concentrations of zn and pb in the soil samples, compared to other heavy metals studied could be due to the use of zn in manufacture of brake linings owing to its heat conducting properties. it may be released onto the road due to mechanical abrasion of vehicular parts like tyres, cables as well as from engine oil combustion [32-34]. similarly, the high concentration of lead could be attributable to anthropogenic activities which include fuel combustion, vehicular emissions, use in electronic devices, and a constituent of lead-acid batteries in car and tyres which release it to the soil through corrosion [18]. fig. 1: average concentrations of heavy metals in cassava tubers harvested at various distances away from the roadside at section 1. fig. 2: average concentrations of heavy metals in cassava tubers harvested at various distances away from the roadside at section 2. distribution of heavy metals in soil on roadside at section 1, the concentrations of the heavy metals in the soil increased from 10 m to 20m away from the roadside, except for lead. conversely, at section 2, the concentrations for all the heavy metals decreased with distance away from the roadside, except for lead. these are shown in figure 3 and 4 respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 276 fig.3: average concentrations of heavy metals in soil samples collected at various distances away from the roadside, at section 1. fig. 4: average concentrations of heavy metals in soil samples collected at various distances away from the roadside, at section 2. heavy metal bioaccumulation factor (baf) heavy metal bioaccumulation factor is a measure of the rate of uptake of each jeavy metal by plants. values above 1 indicates that the plant is a hyperaccumulator of heavy metals. when it is below 1, then the plant moderately bioaccumulated the heavy metal. this is of great importance because heavy metals at excessive concentrations have harmful effects to both plants and animals when ingested. also, plants can serve as indicators to accessible fraction of metals [22]. this was computed using the equation 1; baf = (1) table 1 bioaccumulation factors for cassava samples in section 1. distance (m) cd cu mn pb 10 1.22 1.89 0.11 0.06 15 1.00 0.76 0.09 0.67 20 0.38 0.30 0.08 1.89 table 2 bioaccumulation factors for cassava samples in section 2 distance (m) cd ni cr zn pb 10 0.24 0.40 0.04 1.2 0.60 15 0.31 0.43 0.20 1.50 0.57 20 0.52 0.34 0.42 1.0 0.62 the results shown in tables 1 and 2 indicate that cassava tubers did not hyperbioaccumulate heavy metals at most of the locations studied except for cd at secton 1 and zinc at section 2. these are related to the results reported by yahya et al [22], yan et al [31], opaluwa et al [35]. thus, the metals studied were moderately bioaccumulated in the cassava tuber samples (0.01-1.0) [1]. 4. conclusion the results obtained revealed the presence of lead, cadmium, nickel, manganese, zinc, chromium and copper in most of the soil and cassava samples studied. the trands exhibited by the concentrations of heavy metals are pd>cu>mn>cd and zn>pb>ni>cr>cd for both soil and cassava tuber samples from sections 1 and 2 respectively. moreover, the concentrations of all the heavy metals studied were below maximum permissible limits set by fao/who, except for the concentration of lead in cassava tuber in both sections. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 277 similar to the results obtained from soil samples of section 2, the concentrations of heavy metals in cassava tuber of both sections also decreased with increasing distance 10 to 20m away from the roadside. however, it increased with increasing the distance from roadside. the results showed that cassava cultivated on roadsides of owerri-aba expressway were moderately bioaccumulating heavy metals. 5. references [1]. osakwe s. a., okolie l. p., physicochemical characteristics and heavy metals contents in soils and cassava plants from farmlands along a major highway in delta state, nigeria, journal of applied science and environmental management,19(4): 695 – 704, (2015) [2] onianwa p. c., fakayode s. o., lead contamination of topsoil and vegetation in the vicinity of a battery factory in ibadan, environmental geochemistry and health, 22: 211218, (2000) [3] adriano d. c., trace elements in terrestrial environment, 2nd edition, springerverlay company, new york. pp. 12 -34, (2001) [4] quasem m. j., kamal a. m. contamination of roadside soil, plants and air with heavy metals in jordan: a comparative study, turkish journal of chemistry, 23:209-220, (1999) [5] onder s., dursun s., gezgin s., demirbas a., determination of heavy metal pollution in grass and soil of city centre green areas, konya, turkey, polish journal of environmental study, 16(1):145-154, (2007). [6] nematshahi n., lahouti m., ganjeali a., accumulation of chromium and its effect on growth of (allium cepa cv. hybrid), european journal of experimental biology, 2(4): 969-974, (2012) [7] adeleken b., abegunde k., heavy metal contamination of soil and ground water at automobile mechanic village in ibadan, nigeria, international journal of the physical sciences, 6: 1045-1058, (2011) [8] mtunzi f. m., dikio e. d., moja s. j., evaluation of heavy metal pollution on soil in vaderbijl park, south africa, international journal of environmental monitoring and analysis, 3(2): 44-49, (2015) [9] shrivastav r., atmospheric heavy metal pollution, development of chronological records and geological monitoring, (2001). accessed from www.ias.ac.in [10] ambika a., mohnish p., umar n. effects of heavy metals on plants: an overview, international journal of application and innovation in engineering & management, 5(3): 56-66, (2016) [11] vincenza f., clara p., keith t., manuela e. p., cassava (manihot esculenta crantz) and yam (dioscorea spp.) crops and their derived foodstuffs: safety, security and nutritional value. critical reviews in food science and nutrition, 56(16): 2714-2727, (2016) [12] montagnac j. a., davis c. r., tanumihardjo s. a., processing techniques to reduce toxicity and antinutrients of cassava for use as a staple food. comprehensive review of food science, 8:7–27, (2009) [13] faostat, cassava. food and agricultural organization of the united nations: commodity balances, (2012). available from http://faostat3.fao.org/home/index.html#downlo ad. accessed in may 2019 [14] idodo-umeh g., ogbeibu a. e., bioaccumulation of the heavy metal in cassava tubers and plantains fruits grown in the soils impacted with petroleum and non-petroleum activity. resource journal of environmental science, 4(1): 33–41 [15] emmanuel o., otutu o. j., otuya o. b. concentration of heavy metal in soils, tubers and leaves of cassava plants grown around some oils spills and gas fairing zones in delta state, nigeria, applied science resource journal, 1(1): 1–6, (2013) [16] akinyele i. o., shokunbi o. s., comparative analysis of dry-ashing and wet digestion methods for the determination of trace and heavy metals in food samples, food chemistry, 173: 682-684, (2015) [17] kalagbor i. a., dighi n. k., james r. levels of some heavy metals in cassava and plantain from farmlands in kaani and kpean in khana local government area of rivers state, journal of applied science and environmental management, 19 (2): 219 – 222, (2015) [18] ogundele d. t., adio a. a., oludele o. e., heavy metal concentrations in plants and soil along heavy traffic roads in north central nigeria, journal of environmental analysis and toxicology, 5(6): 1-5, (2015) [19] rahman h. a., zaim f. a. concentration level of heavy metals in soil at vegetables areas in kota bharu, kelantan, malaysia, international journal of environmental science and development, 6(11): 843-848, (2015) http://www.ias.ac.in/ http://faostat3.fao.org/home/index.html#download http://faostat3.fao.org/home/index.html#download food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi , impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 272 – 278 278 [20] asdeo a. toxic metal contamination of staple crops (wheat and millet) in periurban area of western rajasthan, international refereed journal of engineering and science, 3(4): 08-18, (2014) [21] mbong e. o., akpan e. e., osu s. r., soil-plant heavy metal relations and transfer factor index of habitats densely distributed with citrus reticulata (tangerine), journal of research in environmental science and toxicology, 3(4), 6165, (2014) [22] yahya a., naiem n. e., mona m., giesy j. p. seif a. metals in agricultural soils and plants in egypt, toxicological & environmental chemistry, 96(5): 730-742, (2014) [23] who/fao, permissible limits of heavy metals in soil and plants (geneva: world health organization), switzerland, (1996) [24] who/fao, joint fao/who food standard programme codex alimentarius commission 13th session. in: report of the thirty eight session of the codex committee on food hygiene, houston, united states of america, 103p, (2007) [25] ahmad i., zaman a., samad n., ayaz m. m., rukh s., atomic absorption spectrophotometry detection of heavy metals in milk of camel, cattle, buffalo and goat from various areas of khyberpakhtunkhwa (kpk), pakistan. journal of analytical and bioanalytical techniques, 8(3): 1-6, (2017) [26] wang s., zhang j., blood lead levels in children. china environmental research, 101:412418, (2006) [27] sauerbeck d., funktonen, güte und belastbarkeit aus agrikulturchemischer sicht. materialien zur umweltforschung 10, kohlhammer, stuttgart, (1985) [28] fepa, guidelines and standard for environmental impact assessment in nigeria, 87– 95, (1997) [29] european union, heavy metals in wastes, european commission on environment (2002). http://ec.europa.eu/environment/waste/studies/pdf/h eavy_metalsreport.pdf. retrieved on 06 october, 2019 [30] aksoy a., ozturk, m. a., nerium oleander l. as a biomonitor of lead and other heavy metal pollution in mediterranean environments, science of the total environment, 205:145-150, (1997) [31] yan x., zhang f., zeng c., zhang m., devkota l. p., yao t. relationship between heavy metal concentrations in soils and grasses of roadside farmland in nepal, international journal of environmental research and public health, 9: 3209-3226, (2012) [32] hjortenkrans d., diffuse metal emission to air from road traffic: a case study of kalmar, sweden, environmental science sector bulletin, 1-55, (2003) [33] dolan m. s., clapp c. e., allmaras r. r., baker j. m., molina j. a. e. soil organic carbon and nitrogen in a minnesota soil as related to tillage, residue and nitrogen management, soil tillage & research 89: 221-231, (2006) [34] saeedi m., hosseinzadeh m., jamshidi a., pajooheshfar s. p., assessment of heavy metals contamination and leaching characteristics in high way side soils, iran. environmental monitoring assessment, 151(1/4): 231-241, (2009) [35] opaluwa o. d., aremu m. o., ogbo l. o., abiola k. a., odiba i. e., abubkar m. m., nweze n. o. heavy metal concentrations in soils, plant leaves and crops ground around dumpsites in lafia metropolis, nassarawa state, nigeria, advances in applied science research, 3(2): 780 – 784, (2012) http://ec.europa.eu/environment/waste/studies/pdf/heavy_metalsreport.pdf http://ec.europa.eu/environment/waste/studies/pdf/heavy_metalsreport.pdf 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 5 12 new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana * roxana puscaselu1, sonia amariei1 1faculty of food engineering, stefan cel mare university, suceava, romania, roxana.puscaselu@usm.com, *corresponding author received 15th february 2019, accepted 28th march 2019 abstract: the purpose of this paper is the development of fully biodegradable and edible biopolymer materials used for the packaging of powdered food products. stevia is a natural sweetener used in the food and beverage industry. the films were obtained through cast method, from agar and sodium alginate, plasticized with glycerol. stevia was added in proportion of 1,25% of the total ingredients used. the films made with stevia added were fine, soft, transparent, odorless, but sweet, resilient and elastic. their surface was free of cracks and, where there were pores, they did not pierce the material. microbiological determinations and the absence of tested microorganisms indicate the safety of consumption of these membranes. the results indicate the possibility of using these materials to the detriment of the synthetic ones, as well as their transposition on an industrial scale. keywords: agar, sodium alginate, biofilm, powdered product 1. introduction nowadays, population growth and increased food consumption have led to the need to pack products in order to keep them for a longer time. but with this necessity, the industry has created and developed plastic packaging that is extremely damaging to the environment. [1-3] studies have shown that even the first piece of plastic ever thrown can be found in nature. besides the environmental problems and the impossibility of selective waste recycling, plastics can interact with the constituent compounds of the products they contain. a viable alternative to plastic packaging is the use of bio-packages entirely made of biopolymers. [4-7] they have advantages in use; are completely biodegradable and compostable, obtained from renewable resources, at low cost, are edible and can be enriched with various additives (essential oils [8], antioxidant substances, medicinal herbs, spices, natural dyes or flavorings, etc.). edible films and coatings made from biopolymers have become a real alternative to conventional packaging materials. the importance of the use of films and coatings derives, in particular, from the advantages of their use: they extend the shelf life, improve the quality of fresh products, prevent the loss of nutrients, volatile compounds, maintain or even improve the appearance of the products they protect [9]; there are a selective barrier to moisture transfer, prevents access to oxygen and, implicitly, to lipid oxidation, but also to harmful microorganisms. in addition, the replacement of conventional packaging with edible or completely biodegradable http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 6 packaging has the effect of reducing environmental pollution. [10] polysaccharides are most commonly used to develop edible films because of their outstanding film forming capability. polyols, such as glycerol or sorbitol, are especially used to plastify the films. glycerol is often used as it improves physical properties and solubility, but modifies the mechanical properties of hydrophilic films; prevents the destruction of the films during handling or storage. it is a non-volatile substance that reduces the hydrogen bonding between polymeric chains, increases molecular volume, and improving film flexibility. [11] stevia (stevia rebaudiana) is a natural sweetener used in the food and beverage industry. due to its chemical composition, it is considered to be the best substitute for sugar, especially for patients with diabetes [12-14], due to the chemical and nutritional composition; this is a good source of carbohydrates, proteins, fibers, minerals, amino acids, sterols, chlorophyll, organic acids and inorganic salts. [15], [16] the most important bioactive compounds are tannins and phenolic compounds alkaloids, flavonoids, that are able to improve and prevent many diseases. [17-20] because of these benefits, stevia is the ideal substitute for sugar. the purpose of the research was to obtain edible films from bioplymers, enriched with stevia. they can be used for packing instant drinks such as soluble coffee, capuccino (when replacing sugar in their composition), or for coating powdered mixtures of the type of dehydrated vegetables. they can also be eaten by adults, children, elderly people or specialpurpose individuals. in this way, they successfully replace conventional, multicomponent, composite packaging made from plastic and metal, difficult to select and recycle when they become waste. 2. materials and methods the biomaterials were obtained from agar, sodium alginate, and glycerol in variable proportions; the mass of the stevie remained constant (1,25% of total ingredients). water was used to solubilize the constituents and to plasticize the biofilms. except for agar (which was made available by b & v the agar company, italy), the sodium alginate and glycerol were purchased from sigma aldrich company. stevia was obtained from local certified manufacturers. in this way, 30 samples were obtained by the cast method. the main objectove of this survey is to obtain an intelligent material tested for use as edible packaging for dehydrated food. 2.1. determination of microstructure, physical and optical properties; texture profile analysis evaluation. in order to determinate the physical characteristics and microstructre of each membrane, a number of determinations were made, such taste or smell, adhesion to the drying surface, thickness, retraction ratio, and transmitance. film transmission was identified with the ocean optics hr 4000 cg-uv-nir spectrometer at 660 nm wavelength. an important property of the material intended for the packaging of products that may be affected by light is transmitance. to establish the roughness and observe the microstructure, the films were tested with the mar surf cwm 100 microscope (mahrsurf company) and the images obtained were processed with the mountain map software. the texture profile analysis (tpa) the adesivness, elasticity and rupure point were made with the perten tvt 6700 textrometer (perten instruments of australia). specific test accessories, probe, perforated device, and 10 kg loading cell were used. the determinations were performed according to the standard method for gelatin sheets described in the user manual. [21] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 7 2.2. the evaluation of mechanical properties. the samples were tested for tensile strength and elongation at break by using esm 301 mark 10 texturometer and the grips for thin films and films as attachments. for determination purposes, stas astm d882 (standard test method for tensile properties of thin plastic sheeting) was used. [22] tests were performed at ambient temperature of 24.4°c. 2.3. solubility assesment. to establish the suitable film for the development of a completely soluble and edible material, determinations were made in order to characterize the material from this point of view: moisture, water solubility, and water activity index. for moisture determination, film samples (3x3 cm) were weighed and maintained for 24 hours at 110°c. they were then reweighed, and the results were noted in the moisture calculation formula. solubility in water implied the use of the same type of sample; thus, 3x3 cm pieces were cut, weighed, immersed in water for 8 hours (22°c temperature), dried in an oven for 24 hours at 110°c and reweighed. [23] the water activity index (aw) was determined using the aqualab equipment at 22.8°c ±1.5°c. the value recorded represents the sum of five determinations. 2.4. determining antimicrobial safety. in order to be safe for consumption, the material must also be tested from a microbiological point of view. thus, both the obtained films and the ingredients used were tested for the identification of coliforms, enterobacteria, e. coli, staphylococcus aureus, and yeasts and molds. for this purpose, specific culture media compact dry type were used. all determinations were made in triplicate. 3. results and discussion 3.1. characterization of the materials obtained. the films obtained were soft, fine, thin, flexible, without pores or cracks into structure, with intense gloss, no odor, but sweet taste; exhibited low adhesion to the silicone support used for drying, had regular edges, and were pleasing to touch. from the point of view of physical characteristics, they can successfully compete with conventional packaging. fig.1. values obtained for thickness determination fig.2. values obtained for retraction ratio thickness was reduced, sample s30, with high glycerol content (1,50 g) into composition showed a value of 37,80 μm (fig. 1). the thickest film was s24 (0.75 g glycerol), with a value of 52,00 μm. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 8 retraction ratio (fig. 2) is an important parameter when the production recipe is used at industrial level so the results indicate the possibility of controlling the final thickness of the membrane (e.g., if a film with the ingredients and sample thickness s1 is desired and the value of the withdrawal ratio is known 35,78 μm in this case then the manufacturer must pour the film-forming solution and level it so that it has an initial thickness of approximately 73,15 μm). sample s2 (without agar into composition) was a very thin film, smooth, glossy and transparent, with no pores or cracks, no non-solubilized particles, regular and well defined edges, no odor, low sweet taste, low solubility, medium mechanical properties. it can be successfully used for packing whole or sliced meat products, cheeses, cut fruits and vegetables or other high moisture products. s9 (with 1,25 g agar and 1,70 g sodium alginate) is a film with low transparency and gloss, but uniform, homogeneous, with regular edges, without pores or cracks in the structure. with good mechanical properties and high solubilitycan be used for packaging products with medium (reduced) moisture content or powdered products. s12 (without agar into composition and 0,50 g glycerol) showed intense gloss, was transparent, soft, without pores or cracks, with regular edges, allows multiple bending, very flexible, odorless, slightly perceptible sweet taste, rapid total solubility. in terms of solubility, is best suited for packaging pulverulent products. although the s20 (high amount of agar) film is thick, unlike the other films, the film is flexible but with low gloss and transparency. high solubility promotes it to pack instant drinks, but requires improvements (increasing alginate content could be a solution). medium mechanical properties. the s24 sample (0,75 g glycerol) was a touchy, lightweight film with medium gloss, flexible, moderate sweet taste, odorless, very low solubility as opposed to other samples, reduced mechanical resistance but elasticity above average; can be used for packaging highmoisture or high-fat products or lightsensitive compounds (dyes, antioxidants, etc.). the images and microstructre of these films can be observed in fig. 5. fig.3. values obtained for transmittance determination fig. 4. water activity index values for biofilms film transmittance is an important parameter when the packaged product can be degraded due to light radiation. sample s10, s20, s24, and even s28, due to their low transmittance values, can be used as packaging materials for food products with high lipid concentration into composition (fig. 4). for the other biofilms, s2, s12, s30, with high vales of transmittance, the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 9 films can be improved by the addition of substances that can lower the values obtained, such as natural food dyes, for example. the table 1 shows the correlations that can be established between the thickness and the mechanical properties and texture profile of the membranes with stevia. considerable positive correlations between thickness, breaking strength and rupture point are noted. elasticity correlates negatively with roughness and adhesion. from the data obtained, it is not possible to establish clear connections between thickness and elasticity. roughness correlates negatively with the break point, which is absolutely normal. table 1. pearson correlation of stevia-added biofilms t ts e r rpt pa atp a etp a t 1 0.90 0.10 0.35 0.70 0.04 0.09 ts 1 0.34 0.24 0.20 0.12 0.11 e 1 0.76 0.04 0.19 0.13 r 1 -0.15 0.14 0.20 rp tpa 1 0.13 0.18 at pa 1 0.24 et pa 1 t – thickness, (µm), rt – tensile strength, (mpa), e-elongation, (%), r-roughness, (nm), rptparupture point (tpa determination), atpa – adhesiveness (tpa determination), etpa – elasticity (tpa determination). the complete solubility of samples s2, s12, s26 and s27 highlights the ability of sodium alginate to produce biofilm that is completely dissolved. thus, rewinding was impossible, hence the lack of values in table 2. the other samples with agar in the composition retained their integrity even after 20 minutes immersion in water at ambient temperature (21 ± 2°c). according to the results obtained, we can conclude that sodium alginate has the ability to form films with higher solubility than agar (which prevents water absorption), although biofilms obtained from equal or relatively equal amounts of biopolymers (s17, s18, s29, s30) showed high solubility. the moisture content increased directly in proportion to the increase in the amount of sodium alginate in the composition s2, s3, s19 (table 2). however, the maximum moisture content of sample s10, obtained from approximately equal amounts of agar, sodium alginate and glycerol, can be observed. table 2. solubility determinations of membranes with stevia into composition sample moisture (%) water solubility, (%) s1 11.09 ± 0.08 40.24 ± 0.48 s2 13.70 ± 0.65 solubilization s3 12.72 ± 0.57 62.85 ± 0.66 s4 8.00 ± 0.64 43.73 ± 0.41 s5 8.36 ± 0.81 41.24 ± 0.23 s6 11.53 ± 0.56 69.07 ± 0.33 s7 10.32 ± 0.70 48.70 ± 0.85 s8 10.89 ± 0.25 38.30 ± 0.50 s9 7.01 ± 0.30 61.13 ± 0.21 s10 15.96 ± 0.16 58.03 ± 0.18 s11 8.46 ± 0.36 54.61 ± 0.74 s12 11.12 ± 0.50 solubilization s13 9.31 ± 0.34 51.60 ± 0.59 s14 7.56 ± 0.12 59.50 ± 0.75 s15 8.76 ± 0.36 65.01 ± 0.31 s16 8.51 ± 0.54 72.26 ± 0.96 s17 6.89 ± 0.46 86.60 ± 0.51 s18 8.33 ± 0.95 76.63 ± 0.48 s19 12.85 ± 0.70 63.12 ± 0.94 s20 9.71 ± 0.17 87.03 ± 0.64 s21 10.87 ± 0.91 91.00 ± 0.17 s22 7.22 ± 1.09 13.12 ± 0.80 s23 8.16 ± 0.38 12.83 ± 0.38 s24 8.47 ± 0.58 12.19 ± 0.92 s25 9.03 ± 0.81 11.93 ± 0.10 s26 11.67 ± 0.70 solubilization s27 11.13 ± 0.43 solubilization s28 8.94 ± 0.50 71.00 ± 0.41 s29 9.52 ± 0.91 73.81 ± 0.33 s30 7.11 ± 0.33 96.00 ± 0.92 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 10 fig. 5. images and microstructures of stevia-added films food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 11 after microbiological testing, no microorganisms have been developed on the culture media. thus, the consumption of this material is safe from the point of view of the incidence of coliforms, staphylococcus aureus, escherichia coli, yeasts and molds. reduced values of the water activity index (fig. 5) indicate the probability that on the surface of the membranes will not develop microorganisms on the entire shelf life. 4. conclusion stevia is considered the best substitute for sugar, the benefits of consumption referring to the nutritional and biochemical composition. the addition of stevie has improved the physical characteristics of the material, which was transparent, fine, very glossy, with no odor, but sweet taste, with homogeneous microstructure, without pores or cracks. the presence of pores was identified on the higher agar-containing membrane in the composition; but it should be noted that the pores are at the surface of the material that has not been pierced. at the same time, solubility has increased, the desired aspect in this case. though added in small quantities, stevia has moved from the relatively sweetener threshold to the ingredient ingredient of materials that can be used successfully in the food industry and other adjacent industries. in conclusion we can remark the possibility of using stevia as an ingredient in biofilm production. 5. references [1] moore, c.j., et al., a comparasion of plastic and plankton in the north pacific central gyre. marine pollution bulletin 42(12): 1927-1300, (2001); [2] thakur, s., et al., sustainability of bioplastics: opportunities and changes. current option in green and sustainability chemistry, 13:68-75; (2018), https://doi.org/10.1016/j.cogsc.2018.04.013; [3] wroblewska-krepsztul, j., et al., recent progress in biodegradable polymers and nanocomposite-based packaging materials for sustenaible environment. international journal of polymer analysis and characterization, 23(4):383395; (2018), doi: 10.1080/1023666x.2018.1455382; [4] maizura, m, et al., antibacterial activity and mechanical properties of partially hydrolized sago starch – alginate edible film containing lemongrass oil. journal of food science, 72(6): c324-c330, (2007), doi: 10.1111/j.1750-3841.2007.00427.x; [5] cagri, a, ustunol, z, ryser, e., antimicrobial films and coatings. review. journal of food protection, 67(4):833-848, (2004); [6] yadav, a., et al., biopolymers as packaging material in food and allied industry. international journal of chemical studies, 6(2):2411-2418, (2018); [7] elsabee, mz, abdou, e.s., chitosan based edible films and coatings: a review. material science engineering, c 33: 1819–1841, (2013); doi: 10.1016/j.msec.2013.01.010; [8] puscaselu, r., amariei, s., the antibacterial properties of seaweed biopolymer based films incorporated with essential oils, journal of agroalimentary processes and technologies, 23(3):157-163, (2017); [9] rojas-grau, m.a., et al., effects of plant essential oils and oil compound on mechanical, barrier and antimicrobial properties of alginateapple puree edible films. journal of food engineering, 81: 634-641, (2007); doi: 10.1021/jf061717u; [10] ruiz-navajas, y., et al., in vitro antibacterial and antioxidant properties of chitosan edible films incorporated with thymus moroderi and thymus piperella essential oils. food control, 30:386-392, (2013); [11] borges, j.a. et al., influence of different starch sources and plasticizers on properties of biodegradable films. international food research journal 22(6): 2346-2351, (2015); [12] hossain, m.f. et al., cultivation and uses of stevia: a review. afr.j.food agr. nutr dev, 17(4): 12745-12757, (2017). [13] parker, m., et al., consumer acceptance of natural sweeteners in protein beverages. journal of dairy science, 101(10): 8875-8889, 2018; https://doi.org/10.3168/jds.2018-14707; [14] suresh, v., et al., uses of stevia. journal of medicinal plant studies. 6(2):247-248, 2018; [15] samuel, p., et al., stevia leaf to stevia sweetener: exploring its science, benefits, and https://doi.org/10.1016/j.cogsc.2018.04.013 https://doi.org/10.1080/1023666x.2018.1455382 https://doi.org/10.3168/jds.2018-14707 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 roxana puscaselu, sonia amariei, new trends in food packaging technology: biopolymers based materials enriched with stevia rebaudiana, food and environment safety, volume xviii, issue 1 – 2019, pag. 5 – 12 12 future potential. the journal of nutrition, 148(7):1186s-1205s, (2018), https://doi.org/10.1093/jn/nxy102; [16] ciriminna, r., et al., a bioeconomy perspective for natural sweetener stevia. biofuels, bioproducts & biorefining, (2018); https://doi.org/10.1002/bbb.1968; [17] amchra, f. et al., effect of stevia rebaudiana, sucrose and aspartame on human health: a comprehensive review. journal of medical plants studies, 6(1): 102-108, (2018); [18] ghandi, s., et al., natural sweeteners: health benefits of stevia. foods and raw materials. 6(2):392-402, (2018), http://doi.org/10.21603/2308-4057-2018-2-392402; [19] rizwan, f., et al., preliminary analysis of the effect of stevia (stevia rebaudiana) in patients with chronic kidney disease (stage i to stage iii). contemporary clinical trials communications, 12:17-25, (2018); https://doi.org/10.1016/j.conctc.2018.08.007; [20] ucar, a., et al., a research on the genotoxicity of stevia in human lymphocytes. drug and chemical toxicology, 41(2): 221-224, (2018); https://doi.org/10.1080/01480545.2017.1349135 [21] gelatin leaf burst strength by puncture, perten instruments method description tvt method 71-04.02; [22] stas astm d882 -standard test method for tensile properties of thin plastic sheeting [23] puscaselu, r., amariei, s., the application of the peleg model in order to obtain completely soluble materials for food product packaging. journal of applied packaging research, 10(1):98-106, (2018). https://doi.org/10.1093/jn/nxy102 https://doi.org/10.1002/bbb.1968 http://doi.org/10.21603/2308-4057-2018-2-392-402 http://doi.org/10.21603/2308-4057-2018-2-392-402 https://doi.org/10.1016/j.conctc.2018.08.007 https://doi.org/10.1080/01480545.2017.1349135 1. introduction 53 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 12018, pag. 53 58 comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water muhammad haroon 1 , *muhammad vaqas 2 1 department of chemistry, minhajuniversity lahore, lahore, pakistan 2health, safety, enviornment and quality department, attock refinery limited, rawalpindi, pakistan, muhammad_vaqas@yahoo.com *corresponding author muhammad vaqas received november 22th 2017, 23th march 2018 abstract: the purpose of this study was to compare the concentrations of micronutrients (maganese, zinc, copper, iron and chromium) and toxic metals (lead and cadmium) in edible part of ten different species of vegetables, irrigated by sewage/fresh (tube well) water. it was found that the irrigation source changes the concentration of micronutrients and toxic metals in the edible part of vegetables leading to risks for humans’ health. keywords: micronutrients, edible part of vegetables, humans’ health risk, effect of irrigation source 1. introduction irrigation is an artificial application of water to soil. it is used to assist the growth of agricultural crops, maintenance of landscapes, and revegetation of disturbed soils in dry areas, during periods of inadequate rainfall; irrigation also includes protecting plants against frost [1]. vegetables are important protective food, highly beneficial for the maintenance of health and prevention of disease. increasing fruit and vegetable consumption up to 600g per day (baseline) could reduce the total worldwide burden of disease by 1.8% and reduce the burden of ischaemic heart disease by 31% and ischaemic stroke by 19%. for stomach, oesophageal, lung and colorectal cancer, the potential reductions were 19%, 20%, 12% and 2% respectively [2]. manganese is essential for development, metabolism and the antioxidant system. nevertheless, excessive exposure or intake may lead to neurodegenerative disorder cause dopaminergic neuronal death [3]. zinc is typically the second most abundant transition metal in organisms after iron and it is the only metal which appears in all enzyme classes [4]. excess zinc can be harmful. excessive absorption of zinc suppresses copper and iron absorption. copper deficiency leads to tissue injury while high intake may increase the chances of cancer especial liver cancer. the patients with hemochromatosis have high risk of liver cancer and malignancies [5]. iron is involved in numerous biological processes and excess iron typically damages cells in the heart, liver and elsewhere, causing adverse effects that include coma, metabolic acidosis, shock, liver failure, coagulopathy, adult respiratory distress syndrome, long-term organ damage, and even death [6]. in the form trivalent chromium cr3+, chromium is identified as an essential http://www.fia.usv.ro/fiajournal mailto:muhammad_vaqas@yahoo.com http://en.wikipedia.org/wiki/water http://en.wikipedia.org/wiki/soil http://en.wikipedia.org/wiki/agriculture http://en.wikipedia.org/wiki/landscape http://en.wikipedia.org/wiki/revegetation https://en.wikipedia.org/wiki/neurodegeneration https://en.wikipedia.org/wiki/manganese#cite_note-emsley2001-23 https://en.wikipedia.org/wiki/enzyme#naming_conventions https://en.wikipedia.org/wiki/zinc#cite_note-broadley2007-97 https://en.wikipedia.org/wiki/heart https://en.wikipedia.org/wiki/liver https://en.wikipedia.org/wiki/coma https://en.wikipedia.org/wiki/metabolic_acidosis https://en.wikipedia.org/wiki/shock_(circulatory) https://en.wikipedia.org/wiki/liver_failure https://en.wikipedia.org/wiki/coagulopathy https://en.wikipedia.org/wiki/adult_respiratory_distress_syndrome https://en.wikipedia.org/wiki/adult_respiratory_distress_syndrome https://en.wikipedia.org/wiki/iron#cite_note-cheney-152 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 muhammad haroon, muhammad vaqas, comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water, food and environment safety, volume xvii, issue 1 – 2018, pag. 53 – 58 54 nutrient. its role is important in the action of insulin [7]. in contrast, hexavalent chromium cr6+ is highly toxic and mutagenic when inhaled [8]. lead is a toxic metal and primary cause of lead's toxicity is its interference with a variety of enzymes because it binds to sulfhydryl groups found on many enzymes [9]. cadmium intake through diet associates to higher risk of endometrial, breast and prostate cancer as well as to osteoporosis in humans [10-12]. the main objective of this survey was to determine the concentration of micronutrients and toxic metals in the edible part of vegetables so all the samples were collected from gujrawala, punjab pakistan. 2. matherials and methods the collected samples were the edible part of the vegetables irrigated either by fresh water (tube well) or sewage water. all the samples were collected in sunny days, temperature range 25-30oc in paper bags with complete labeling of name, date and location. the vegetables with some soil and dust were washed with tap water first and then with distilled water. all the samples were primarily dried in open air covered with filter papers and further dried in an oven at temperature 70 80oc. all the dried samples were grinded in wooden mortar in such a way that mortar was cleaned thoroughly each time after use to avoid the intermixing of the samples. sieving of the grinded samples was done in a stainless steel sieve of 5 mm mesh. the grinded samples were kept in air tight polythene bags labeled with sample number, in a dark and cool place [13, 14]. the samples were digested by taking one gram of the dried sample in a 100 ml beaker and added 20 ml nitric acid (conc.). the beaker was covered with a watch glass and was allowed to stand for two hours then placed on a hot plate inside a fuming chamber until the solid particles nearly disappeared then removed from hot plate and allowed to cool. after that 10 ml of 72% perchloric acid was added and again placed on the hot plate. heated gently first and then vigorously until solution in beaker became clear and volume reduced to about 5-6 ml then cooled and added 3ml of 50% hydrochloric acid, again heated on hot plate until volume of the solution is reduced to 5 ml at the end it was allowed to cool and added some distilled water. the solution was transferred carefully to 100 ml measuring flask and made the volume up to mark with distilled water, shacked well and allowed to stand overnight, filtered, and collected in labeled plastic bottles. all the samples prepared likewise and stored in dark. a blank test solution was also prepared by adopting the same procedure without taking vegetable sample [15]. all the vegetable samples were analyzed by atomic absorption spectrometer [perkin elemer pinaacletm 900t] and calculations were performed by following formula [16]. metal in vegetable sample: (mg/kg) = (s-b)*dt*v / w s = sample reading; b = blank reading; dt = dilution factor; v = volume of first dilution; w = weight of sample. 3. results and discussion manganese (mn): the highest concentration of manganese was found 94,53mg/kg in the spinach (spinacia oleracea) grew near drain nala in the village of kot mand gujranwala and irrigated with sewage water which comes out from industries and houses. the concentration of manganese was observed in the range of 10,34 – 94,53 mg/kg in the https://en.wikipedia.org/wiki/chromium#cite_note-53 https://en.wikipedia.org/wiki/hexavalent_chromium https://en.wikipedia.org/wiki/hexavalent_chromium https://en.wikipedia.org/wiki/mutagen https://en.wikipedia.org/wiki/chromium#cite_note-58 https://en.wikipedia.org/wiki/enzyme https://en.wikipedia.org/wiki/sulfhydryl_group https://en.wikipedia.org/wiki/lead#cite_note-pearson03-181 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 muhammad haroon, muhammad vaqas, comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water, food and environment safety, volume xvii, issue 1 – 2018, pag. 53 – 58 55 vegetable samples irrigated with sewage water. the level of manganese was found to be 8,75 – 78,78 mg/kg in the vegetables irrigated with fresh water. in the nine vegetable species, the manganese found higher in the vegetables irrigated with sewage water and lower in the vegetables irrigated with fresh water (fig. 1). fig. 1. manganese (mg/kg) zinc (zn): the level of zinc in the vegetables irrigated with sewage water was ranged from 8,82 – 23,26 mg/kg. in the nine vegetable species, the level of zinc was found higher in the vegetables irrigated with sewage water and lower in the vegetables irrigated with fresh water. the level of zinc in the vegetables irrigated with fresh water was found in the range of 4,28 – 14,28 mg/kg. similar concentration of zinc was observed in the vegetable specie radish (raphanus sativus) irrigated with both type of irrigation waters (fig. 2). fig. 2. zinc (mg/kg) copper (cu): the level of copper in the vegetables irrigated with sewage water was found in the range of 8,86 – 26,57 mg/kg. the concentration of copper in the vegetables irrigated with fresh water was determined to be 16,84 – 5,80 mg/kg. the highest amount of copper was found 26,57mg/kg in the fenu-greek (trigonella foenum graecum) grew near drain nala in the village of qila deso singh gujranwala and irrigated with sewage water (fig. 3). fig. 3. copper (mg/kg) iron (fe): the concentration of iron in the vegetables irrigated with sewage water was found to be 48 584 mg/kg. in the eight vegetable species, the amount of iron was found higher in the vegetables irrigated with sewage water and lower in the vegetables irrigated with fresh water. the concentration of iron in the vegetables irrigated with fresh water was determined to be 30 – 412 mg/kg. the highest amount of iron was found 584 mg/kg in the spinach (spinacia oleracea) grew near drain nala in the village of kot mand gujranwala and irrigated with sewage water which comes out from industries and houses (fig. 4). fig. 4. iron (mg/kg) chromium (cr): it was examined that most of the vegetables irrigated with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 muhammad haroon, muhammad vaqas, comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water, food and environment safety, volume xvii, issue 1 – 2018, pag. 53 – 58 56 sewage water contained the higher concentrations of chromium as compared to the vegetables irrigated with fresh water. the concentration of chromium in the vegetables irrigated with fresh water was evaluated in the range of 0.17 1.34mg/kg. the chromium concentration in the vegetables irrigated with sewage water was determined to be 0.09 2.30 mg/kg. the concentrations of chromium were not found in the vegetables lady’s finger (hibiscus rsculentis) and radish (raphanus sativus) irrigated with fresh water (fig. 5). fig. 5. chromium (mg/kg) lead (pb): the level of pb concentrations in the vegetables irrigated with sewage water was found in the range of 0.48 4.23mg/kg. the concentration of lead was observed in the range of 0.27 2.87 mg/kg in the vegetable samples irrigated with fresh water. the concentration of lead was determined higher in the fenu-greek (trigonella foenum graecum) irrigated with fresh water as compared to that irrigated with sewage water because it was grown near the road side (fig. 6). fig. 6. lead (mg/kg) cadmium (cd): it was noted that most of the vegetables contained higher concentrations of cadmium in the vegetables irrigated with sewage water than those irrigated with fresh water. the level of cadmium in the vegetables irrigated with sewage water was found in the range of 0.04 1.86 mg/kg. the highest concentration of cadmium was determined 1.86 mg/kg in the couliflower (brassica oleracea) irrigated sewage water grew in the field of francisabad, gujranwala near the drain stream containing industrial and domestic effluents. the concentrations of cadmium were determined to be 0.01 0.34 mg/kg in the vegetables irrigated with fresh water (fig. 7). fig. 7. cadmium (mg/kg) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 muhammad haroon, muhammad vaqas, comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water, food and environment safety, volume xvii, issue 1 – 2018, pag. 53 – 58 57 table1. concentration of metals in edible part of vegetables irrigated by sewage/ fresh (tube well) water s. no . sampl e no. vegetable name botanical name mn zn cu fe cr pb cd mg/k g mg/k g mg/k g mg/k g mg/k g mg/k g mg/k g 1 1sw lady’s finger hibiscus esculentis 34.46 8.34 14.53 120 0.09 2.34 0.36 2 1fw lady’s finger hibiscus esculentis 40.24 5.86 9.8 84 nd* 1.78 0.08 3 2sw mint mentha spicata 58.26 23.26 16.25 247 0.92 4.23 0.06 4 2fw mint mentha spicata 42.48 10.67 12.61 188 0.46 2.87 nd* 5 3sw fenugreek trigonella foenumgraecum 26.73 8.92 26.57 435 1.64 0.8 0.62 6 3fw fenugreek trigonella foenumgraecum 23.98 6.44 14.73 421 0.52 1.26 0.03 7 4sw spinach spinacia oleracea 94.53 16.23 20.44 584 2.3 2.48 0.68 8 4fw spinach spinacia oleracea 78.78 9.45 16.84 408 1.06 1.24 0.06 9 5sw bell pepper capsicum annuum 38.51 20.53 13.52 133 0.26 3.21 0.04 10 5fw bell pepper capsicum annuum 29.76 14.28 12.78 141 0.35 2.08 nd* 11 6sw cauliflowe r brassica oleracea 32.13 12.79 19.63 98 1.8 0.94 1.86 12 6fw cauliflowe r brassica oleracea 30.42 10.33 15.32 98 0.98 0.57 0.34 13 7sw radish raphanus sativus 24.55 8.82 10.27 161 0.36 2.86 0.05 14 7fw radish raphanus sativus 18.98 8.82 11.62 30 nd* 2.04 0.02 15 8sw bitter gourd momordica charantia 75.84 22.84 22.34 61 2.16 0.48 0.22 16 8fw bitter gourd momordica charantia 57.47 16.45 14 54 1.34 0.27 0.22 17 9sw vegetable marrow cucurbita pepo 35.65 9.82 8.86 48 0.42 2.24 0.04 18 9fw vegetable marrow cucurbita pepo 27.94 4.28 8.22 34 0.17 1.91 0.01 19 10sw apple gourd praecitrullu s fistulosus 10.34 10.44 9.58 72 1.42 2.53 0.94 20 10fw apple gourd praecitrullu s fistulosus 8.75 5.33 5.8 55 0.87 1.94 0.28 nd*= not dectected 4. conclusion sewage water irrigation is practicing in various developing countries like pakistan and normally considers a source of good production. this study shows that concentrations of micronutrients (mn, zn, cu, fe and cr) and toxic metals (pb and cd) in edible parts of the vegetables were higher in vegetables irrigated by sewage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 muhammad haroon, muhammad vaqas, comparative study of toxic metals and micronutrients in vegetables irrigated by sewage/fresh water, food and environment safety, volume xvii, issue 1 – 2018, pag. 53 – 58 58 water so using the sewage water as irrigation source is hazardous to humans health. 5. acknowledgments we are thankful to dr. izhar ul haq (minhaj university lahore) and dr. muhammad adnan iqbal (university of agriculture faisalabad) for their support and guidance. 6. references [1]. snyder r. l. and melo-abreu j. p., frost protection fundamentals practice and economics, vol. 1, food and agriculture organization of the united nations, (2005). [2]. siebert s., jippe h., petra d., jeanmarc f., sebastian f., and karen f., the digital global map of irrigation areas– development and validation of map version 4, tropentag, conference on international agricultural research for development. university of bonn, (2006). [3]. emsley j., manganese, nature's building blocks: an a-z guide to the elements. oxford, uk: oxford university press, 249-253 (2001). [4]. broadley m. r., white p. j., hammond j. p., zelko i., lux a., zinc in plants. new phytologist, 173(4): 677-702, (2007). [5]. fabris n., mocchegiani e., zinchuman diseases and aging. aging clinical and experimental research, 7: 77-93, (1995). [6]. cheney k., gumbiner c., benson b., tenenbein m., survival after a severe iron poisoning treated with intermittent infusions of deferoxamine. journal of toxicology: clinical toxicology. 33(1): 61-6, (1995). [7]. anderson r. a., chromium as an essential nutrient for humans. regulatory toxicology and pharmacology, 26 (1 pt 2): s35s41, (1997). [8]. wise s. s., wise jp. sr., chromium and genomic stability. mutation research /fundamental and molecular mechanisms of mutagenesis, 733(1-2): 78-82, (2012). [9]. abraham m. r., colin d. r., margaret k. h., george e. l., norman j. s., lead. rudolph's pediatrics (21st ed.) mcgrawhill professional, 369 (2003). [10]. julin b., wolk a., johansson j. e., andersson s. o., andren o., akesson a., dietary cadmium exposure and prostate cancer incidence: a population-based prospective cohort study. british journal of cancer, 107(5): 895-900, (2012). [11]. engstrom a., michaelsson k., vanter m., julin b., wolk a., akesson a., associations between dietary cadmium exposure and bone mineral density and risk of osteoporosis and fractures among women. bone. 50(6): 1372-1378, (2012). [12]. julin b., wolk a., bergkvist l., bottai m., akesson a., dietary cadmium exposure and risk of postmenopausal breast cancer: a population-based prospective cohort study. cancer research. 72(6): 1459-1466, (2012). [13]. agency for toxic substances and disease registry. toxicological profile for chromium. u.s. public health service, department of health and human services, atlanta, us, (1998). [14]. amma m. k., plant and soil analysis. rubber research institute, rubber board, kottayam-686009, keral, (1990). [15]. jones j., benton, plant tissue analysis for micronutrients, in j. j. mortvedt et al., (ed) micronutrients in agriculture. soil science society america, madison, wisconsin, u.s.a., 319-348, (1972). [16]. rechards l. a., diagnosis and improvement of saline and alkali soils. (ed) 1954, oxford and ibh publishing company, new delhi, (1968). https://books.google.com/?id=j-xu07p3ckwc https://books.google.com/?id=j-xu07p3ckwc https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4138963 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4138963 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3425979 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3425979 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3425979 1. introduction 4. conclusion 74 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 12018, pag. 74 80 use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets *valentina bantea-zagareanu faculty of food technology, technical university of moldova, town, country, 168 stefan cel mare blvd., md-2004 chisinau, republic of moldova *corresponding author: v_bantea@yahoo.com received 16th october 2017, accepted 23th march 2018 abstract: the purpose of the research consists in enhancing the value of the secondary products of the seed oil industry, especially of the defatted walnut flour (juglans regia l.). by adding it in the composition of sweets, the aim is to obtain a new product, with a balanced ensemble of nutritional substances – fats and carbohydrates. also, it will be possible to enlarge the assortment of the confectionery products, which due to the low amount of carbohydrates, could be consumed by persons of all age, without the risk of sickening of diabetes.physico chemical methods were used to determine humidity, amount of fats, acid value, peroxide value and amount of total ash as well as the defatted walnut flour’s quality, used in the production of sweets. the product was subjected to physico – chemical, microbiological and sensory analysis. due to the amount of fats contained in defatted walnut flour, the peroxide value in the finite product was determined. the aim is to notice wether fats’ oxidation occurs during storage which results in the worsening of organoleptic characteristics of the product – taste and flavor. the results show microbiological stability of the product, absence of oxidation of fats contained in defatted walnut flour, confirmed by sensory analysis. no taste or flavor drawbacks of the product that could be caused by fats’ oxidation were identified. keywords: dried fruits, fats, oxidation, peroxide value, sweets, defatted walnut flour, inulin. 1. introduction confectionery products are food products with a high amount of sugar, with sweet taste and different forms, composition, consistency, structure, flavor and are characterized by a high energetic value, good digestibility, having a pleasant taste, flavor and look. confectionery products are divided into two categories bakers' confections and sugar confections. the main ingredient of confectionery products is sugar or sugar substitutes (sweetener). sweets are products manufactured form sugar, glucose syrup, cocoa products, peanuts, walnuts’ kernels, dairy products, food coloring and flavors, and other ingredients [1-3]. production volume of sugar confections in republic of moldova shows a quantitative evolution from 12.0 thousand tons in 2005 up to 14.0 thousand tons in 2015, but it doesn’t exceed the production volume of bakers’ confections, which is 2.5 times higher in 2015 (33.6 14.0 thousand tons) [4]. results are given in figure 1. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 75 fig. 1. quantitative evolution of production volume of sugar confections (2005-2015), thousand tons [4] nuts are one of the main raw materials used at sugar confections’ manufacturing. the basic characteristic of nuts is high amount of fats and proteins, which determines high energetic value of confectionery products. nuts are used at obtaining of marzipan and praline masses, at the production of caramel fillings, candied roasted nuts, dragees, halvah or are added in chocolate tablets, because of high tasteful qualities, which it gives to the finite product [5, 6]. confectionary products were used to investigate walnut waste value determination. the choice of confectionary products for the investigation was determined due to their heterogenic structure, which often contains low amount of essential nutrients. supplementing confectionary products with walnut waste would improve their structure and nutrition value as well as reduce their cost. products from halva and sponge cake as well as cookies „macarons” were chosen for this investigation. the quality of elaborated confectionary products depends majorly on the grinding size and antioxidant properties of walnut waste, which also has a big impact on the organoleptic and physiochemical qualities of the final products [7]. in this paperwork we studied use of defatted walnut flour in amaretti cookies production, which are normally produced on the base of almond flour according to their classic recipe. it was detected that adding walnut waste to the cookies recipe positively influences on their organoleptic and physiochemical properties as well as the microbiological stability reducing their costs [8]. one of walnut waste use in food industry is its use in milk industry to produce a certain type of cheese. this type of cheese contains 1.5-2 % of walnut waste from the whole mass of the product. the walnut waste use is more preferable than one of the walnuts due to the fact that walnut waste contains lower amount of oil as well as it is oxidized slower, extending the final product shelf life. in such a way, walnut waste use improves functional and organoleptic properties of the product, increases its nutrient value and shelf life [9]. defatted walnut flour (juglans regia l.) is a rich source of lecithin, vitamins (a, b, c, e, p), microand macro elements (sodium, potassium, iron, iodine, phosphorus, magnesium, calcium), biologically active substances which helps at improvement of heart’s activity, purification of blood vessels, brain’s stimulation in period of intensive intellectual activity, helps at muscles’ strengthening and at removing of tiredness caused by physical effort. another important component of defatted walnut flour is cellulose. it has to remove toxins from the organism, this process improving the state of skin and speeding the loss of weight. the amount of vitamin c in defatted walnut flour is 5 times higher than in red currant. iodine which is contained in defatted walnut flour helps at maintaining of thyroid’s health [10]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 76 because of high amount of proteins and their partial solubility, defatted walnut flour (juglans regia l.) has good functional properties and may be a good source of functional and protein ingredient in heterogeneous food systems like emulsions, foams, suspensions [11]. also, antioxidant properties of walnuts are well outlined from peanuts and almonds’ properties, a fact which increases the use of walnuts not only for the pleasant taste and flavor of confectionery products, but also for extending their term of validity [12]. 2. materials and methods for the research were used raw materials such as by-product from the production of cold-pressed walnut oil (juglans regia l.), harvest of 2014 year, kogalniceanu kind [13], dried plums [14], raisins [14], powdered sugar [15], the dietary fiber – inulin, cocoa powder [16] and ascorbic acid (e300) [17]. fig. 2. defatted walnut flour (juglans regia l.), harvest of 2014 year (produced under laboratory conditions the technique for making dried plum or raisins sweets with addition of walnut waste implies raw materials preparation for production process – inspecting and washing dried fruit, separating walnuts waste from ferromagnetic impurities, sorting according to the grind size, auxiliary materials screening (cocoa and sugar powder, inulin). after being washed, dried plums are steamed and boiled in sugar syrup. plums and raisins are dried until their humidity level is decreased to 20-25 %, but walnut waste is heated at 60-65 ºc during 30-35 minutes to reduce its humidity level. to obtain the recipe mixture, plums and raisins are grinded up, dried walnut waste and auxiliary materials (cocoa and sugar powder, inulin and ascorbic acid) are added. then, bases of sweets are formed, being further covered with chocolate glaze, which was filtered and warmed up to 29-32 ºc preventively. glazed sweets are cooled to +4 +6 ºc during 5-7 minutes. for determination of defatted walnut flour’s quality, which was used for production of sweets, were used physic chemical methods, for determination of humidity [18], amount of fats [19], acid value [20], peroxide value [21] and amount of total ash [22]. sensory analysis of defatted walnut flour was performed according to gost 13979.4-68 [23]. for sensory analysis of sweets with addition of walnut waste an analytical method of appreciation was applied 5 point scale. there is a 5 point scale to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 77 appreciate each feature (taste, flavor, composition, aspect) [24]. taster team consisted of 7 members (students and teaching stuff). each taster had an individual file for sensory analysis. based on the results, a general file as well as a sensory profile of defatted walnut flour was created. in table 1 presented scale of appreciation grades. table 1. 5 point scale of appreciation grades [24, p.91]. grade of appreciation grade points general description of appreciation grades very good 5 exceptional qualities good 4 adequate qualities satisfactory 3 with some defects unsatisfactory 2 with evident defects poor 1 with strong defects very poor 0 unacceptable for determination of microbiological characteristics of sweets were determined the quantity of mesophilic aerobes and facultative anaerobes [25], the number of yeasts and molds [26], presence of coliform bacteria (escherichia coli) [27]. because of amount of fats contained in defatted walnut flour, was determined peroxide value [21] in the finite product. the goal is to notice if during the storage take place the fats’ oxidation which has as result worsening of organoleptic characteristics of the product – taste and flavor. 3. results and discussion the new product contains vegetal proteins from walnuts’ kernel, with a high biological value. fats are represented by the oil remained in the cake after the nuts’ pressing and the carbohydrates are represented by the powdered sugar and dried fruits. even if the carbohydrates’ quantity is large, the major are fibers, which are contained in the dried fruits. in table 2 are presented physic-chemical characteristics of the defatted walnut flour, determined in the research. to get valid results, 5 analyses were carried out identifying experiment data precision table 2. quality characteristics of defatted walnut flour. physic-chemical characteristics experimental values technical conditions amount of fats, % 35.26 ± 0.2 41.0-45.0 humidity, % 7.62 ± 0.2 5.8 amount of total ash, % 2.53 ± 0.03 4.0 peroxide value, millimoles per kilogram 8.82 ± 0.1 10.0 acid value, mg koh/g oil 1.79±0.01 4.0 it shows that experimental values are according to technical acts. it means that this defatted walnut flour can be used for manufacturing of sweets. in the production recipe was used inulin (the dietary fiber), obtained under laboratory conditions. inulin is also a sugar replacement. the new product received a good appreciation from the tasters’ team. equally was appreciated the consistence, taste and flavor of the sweets (4,85), while the structure is considered to be a critical parameter [2]. sensory profile of the product with the defatted walnut flour is shown in figure 3. https://en.wikipedia.org/wiki/millimole https://en.wikipedia.org/wiki/kilogram https://en.wikipedia.org/wiki/potassium_hydroxide food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 78 fig. 3. sensory profile of the product with the defatted walnut flour this thing is owed to an insufficient mixing of defatted walnut flour with dried fruits’ mass. from tasters’ observations was noticed that they would prefer a product, which will have a higher content of defatted walnut flour, which will contribute at increasing of energetic value of the product. because of actual tendency to release on market products which have a long term of validity, is important to study and check the parameters, which can have a negative influence above the product at the storage. the oil content that remains in walnut waste after cold pressing (approximately 40 %) is perishable. oils which walnut waste contains can be affected by the process of rancidity, which leads to product taste change. rancidity is a process of oil oxidization caused by oxygen from the air and it can be developed by microorganisms. this process causes products taste and flavor change. to stop or avoid rancidity, various inhibitors of freeradicals reactions or inhibitors which create heavy-metal complexes are used (сitric acid, ascorbic acid). oxidization reactions are exothermic thus, if storage or transportation conditions are unsatisfactory, walnut waste can be self-ignited [12]. for the sweets with addition of defatted walnut flour is important to know if the fats are oxidized, because this process can cause depreciation of sensory characteristics – taste and flavor. after determination of peroxide value in sweets with defatted walnut flour, using a method for confectionery products, can be said that after a month peroxide value grew up, but the value (4,56 millimoles per kilogram) is according to the peroxide value for walnuts’ oil, which maximum is 10,0 millimoles per kilogram [28]. so, for establishing the term of validity for this product is necessary to continue the research and check monthly physicchemical parameters. also, is necessary to establish suited conditions for the storage, taking into account all the ingredients from the product and processes which can take place at different temperatures. microbiological stability is also an important element, which should be taken in account before releasing a product on market. according to [29], the quantity of mesophilic aerobes and facultative anaerobes are 5,0·10³ colonies per gram of product. evolution of quantity of mesophilic aerobes and facultative anaerobes at the storage of sweets with dried fruits is shown in figure 4. https://en.wikipedia.org/wiki/millimole https://en.wikipedia.org/wiki/kilogram https://en.wikipedia.org/wiki/millimole https://en.wikipedia.org/wiki/kilogram food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 79 as we can see, even after 4 months of storage at temperature 18±3 ºc, the number of aerobic and optionally anaerobic microorganisms doesn’t exceed the maximum value. microbiological load of the sweets with defatted walnut flour is poor, because molds and yeasts were not identified after examination. the main cause is the lack of conditions, which can cause molds and yeasts’ growth. humidity of sweets, determined by the method [30], is 21 %, value which is corresponding with technical conditions [29]. fig. 4. evolution of quantity of mesophilic aerobes and facultative anaerobes at the storage of sweets with dried fruits and defatted walnut flour at temperature 18±3 ºc escherichia coli wasn’t identified in the sweets. this means that during the manufacturing and storing them were respected hygiene conditions of equipment used during the process, of storage space and the staff which participated in the manufacturing process respected good hygiene practices. 4. conclusions sweets with body made from dried fruits can be a good alternative instead of confectionery products, which contain large quantities of sugar – caramels or fondant sweets. the substitution of chocolates or caramels with sweets made from dried fruits and defatted walnut flour will stop gaining weight, but receiving the same pleasure from eating sweets. such sweets contain a small quantity of sugar, vitamins of all groups, necessary for intellectual and physical growth of children. application of defatted walnut flour (juglans regia l.) at manufacturing of sweets with dried fruits has an influence about energetic value of the product, enriching them with a balanced quantity of fats. in comparison with other confectionery products, where whole nuts are used, the presented product contains a smaller quantity of fats, but the same of proteins, which has the same benefits for human’s organism. also, the small quantity of fats from defatted walnut flour has a positive influence above microbiological stability of sweets, and using of ascorbic acid protects fats from defatted walnut flour from oxidation, which can affect organoleptic characteristics of finite product. 5. acknowledgments this work was benefited from support through the 11.817.04.40a project, “elaboration of methods to protect walnut lipids (juglans regia l.) from oxidative degradation”, funded by the academy of science of moldova and moldavian government. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentina bantea-zagareanu, use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets, food and environment safety, volume xvii, issue 1 – 2018, pag. 74 – 80 80 6. references [1]. government decision of the republic of moldova, no. 204 from 11.03.2009. regarding the approval of the technical regulation "confectionery". [2]. kuznetsova, l., s., sidanova, m., yu. technology and organization of confectionery production, moscow, "academy" publishing center, 285 p., (2006). [3]. lurye, i., s. confectionery production technology, moscow, agropromizdat, 399 p., (1992). [4]. statistical yearbook of the republic of moldova (2015) http://www.statistica.md/. [5]. banu, c., ianiţchi, d., jantea, c., bărăscu, e. confectionery technology, bucharest, agir publishing house, 200 pp., (2013). [6]. skobelskaya, z., g., goryacheva, g., n. technology of sugar confectionery production, moscow, profobrizdat 416 p., (2002). [7]. grosu, c. validation of nuts and confectionery: phd thesis, chisinau, 2016, 173 pp., (2016). [8]. ghendov-moşanu, a., banteazagareanu, v., tatarov, p. the use of nutmeg meal (juglans regia l.) in the manufacture of amaretti biscuits. in: meridian engineering, no. 3, p. 62-65, (2016). [9]. onopryko a., v., masliy e., a., onopryko v., a. cheese product with nut component "souvenir" and the way of its production. patent for invention ru 2473228, published: 27.01.2013. [10]. http://liveprodukt.zakupka.com/p/9615454shrot-greckogo-oreha-istochnik-lecitinavitaminov-i-makroelementov/. [11]. grosu, c., gorbuleac, v., scripcari, i., ciumac, j., tatarov, p. functional properties of nutmeg (juglans regia l.), in: international conference mtfi-2014, modern technologies in the food industry, chisinau, pp. 198-202, (2014). [12]. makarova n. v., dmitrieva, a. н. antioxidant properties of mineral water, materials international scientific and practical conferences, voronezh, 25-26 september 2014., pp. 305-308, (2014). [13]. government decision of the republic of moldova, no.174, dated 02.03.2009, regarding the approval of the technical regulation "fruit of nutraceuticals. quality and marketing requirements". [14]. government decision of the republic of moldova, no.1523, dated 29.12.2007, regarding the approval of the technical regulation "dry and dehydrated fruits and vegetables". [15]. government decision of the republic of moldova, no.774, dated 03.07.2007, regarding the approval of the technical regulation "sugar. production and marketing ". [16]. gost 108-2014. cocoa-powder. specifications. [17]. government decision of the republic of moldova, no. 229 of march 29, 2013, regarding the approval of the technical regulation "health regulations on food additives". [18]. gost-13979.1-68 oilcakes, oil meals and powdered mustard seed cake. methods for determination of water and volatile substance content. [19]. gost 13979.2-94 oilcakes, oil meals and powdered mustard seed cake. method for determination of oil and extractive substances mass fraction. [20]. official methods and recommended practicles of the american oil chemists’ society. method cd 3d-63. acid value. champaign: aocs press, (1999). [21]. sr 13531. food products. method for determination of peroxide value. [22]. gost 13979.6-69 oilcakes, oil meals and powdered mustard seed cake. determination of ash content. [23]. gost-13979.4-68 oilcakes, oil meals and powdered mustard seed cake. determination of color, odour, dark impurities and small particles of oilcake. [24]. banu, c., and colab. quality and sensory analysis of food, bucharest, agir publishing house, 574 p., (2007). [25]. gost 10444.15-94 food products. methods for determination quantity of mesophilic aerobes and facultative anaerobes. [26]. gost 10444.12-88 food products. method for determination of yeasts and mould. [27]. sm iso 4832:2013 microbiology of food products and fodder. horizontally method for counting of coliform bacteria. method for colonies’ counting. [28]. government decision of the republic of moldova no. 434 of 27.05.2010 regarding the approval of the technical regulation "edible vegetable oils". [29]. gost 4570-93. sweets. general specifications. [30]. gost 5900-73 confectionery. methods for determination of moisture and dry substances. 1. introduction 358 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 42015, pag. 358 367 fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content raphael amon akpossan1, *edmond ahipo due1, djary michel koffi2 , patrice kouame1 1laboratory of biochemistry and food technology, nangui abrogoua university, 02 bp 801, abidjan 02, cote d’ivoire. assieloub@yahoo.fr , ahipoedmond@yahoo.fr , kouame_patrice@yahoo.fr 2laboratory of biotechnology, felix houphouet boigny university, 02 bp 801 abidjan 02, cote d’ivoire, djaryss@yahoo.fr *corresponding author received november 18th 2015, accepted december 18th 2015 abstract: imbrasia oyemensis larva is a widely prized and consumed caterpillar in côte d’ivoire. the oil extracted from this insect was analyzed for physicochemical properties, mineral composition and fatty acid constituents using standard methods. the analysis revealed a refractive index of 1.4675 ± 0.0002, specific gravity of 0.942 ± 0.02, moisture and volatile matter of 7.51 ± 0.1, acid value of 2.81 ± 0.02, iodine value of 105.19 ± 1.62, saponification value of 184.20 ± 1.62, unsaponifiable matter of 45.0 ± 1 and free fatty acid of 1.49 ± 0.37 (% oleic acid). this oil was found to be a very rich source of mineral elements with a 100 g dry sample containing phosphorus (26.82 g), potassium (14.97 g), magnesium (5.63 g), calcium (5.39 g), zinc (0.63 g) and iron (0.31 g). as regards fatty acids profile, the major unsaturated fatty acids of imbrasia oyemensis oil were linolenic (26.22 ± 0.03 %), linoleic (11.33 ± 0.02 %) and arachidonic (10.26 ± 0.04 %), while the major saturated fatty acids were stearic (20.92 ± 0.03 %) and palmitic (18.65 ± 0.05 %). the unsaturated fatty acids accounted for 55.7 % of the total fatty acids, whereas the saturated fatty acids constituted 44.3 % of the fatty acids. these values when compared with those observed in oils which have been considered to be of high quality, suggest that imbrasia oyemensis oil has potentials that could be exploited by the nutritional and pharmaceutical companies. keywords: fatty acids profile, imbrasia oyemensis, mineral composition, oil, physico-chemical parameters. 1. introduction insects are often considered a nuisance to human beings and mere pests for crops and animals. yet this is far from the truth. they provide food at low environmental cost, contribute positively to livelihoods, and play a fundamental role in nature [1, 2, 3]. however, these benefits are largely unknown to the public. contrary to popular belief, insects are not merely “famine foods”, eaten in times of food scarcity or when purchasing and harvesting “conventional foods” becomes difficult; many people around the world eat insects out of choice, largely because of the palatability of the insects and their established place in local food cultures [4]. people in the world eat insects as regular parts of their diets [2]. they may do so not only because conventional meats such as beef, fish and chicken are unavailable and http://www.fia.usv.ro/fiajournal mailto:assieloub@yahoo.fr mailto:ahipoedmond@yahoo.fr mailto:kouame_patrice@yahoo.fr mailto:djaryss@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 359 insects therefore are vital sources of protein, but also because insects are considered important food items, often delicacies [5, 6, 7]. in countries where edible insects constitute regular elements in traditional diets, the shift towards western foods constitutes a real threat to entomophagy. to counter this, efforts are being made to merge the traditional practice of insect eating with more popular foods [1]. caterpillars are among the world’s most diverse groups of edible insects. they are not only valuable sources of protein and other micronutrients; they also make valuable contributions to livelihoods in many parts of the world. the consumption of caterpillars is especially pervasive in sub-saharan africa, where 30 percent of all edible insect species are caterpillars [4]. in the democratic republic of the congo, caterpillars make up 40 percent of the total animal protein consumed [8]. the most popular and profitable caterpillar on the african continent is undoubtedly the mopane caterpillar, imbrasia sp. although the caterpillars are important sources of nutrition in lean times, they also form a regular part of the diet [9]. the protein content of the mopane caterpillar is 48–61 percent and fat content is 16–20 percent, of which 40 percent is essential fatty acids. mopane caterpillars are also a good source of calcium, zinc and iron [10, 11]. in cote d'ivoire, imbrasia oyemensis is one of the most widely eaten insects. this caterpillar, sold at high prices on abidjan markets, is consumed by an important population fringe of the country in replacement of meat and fish [12]. the present paper investigates the content and composition of oil extracted from imbrasia oyemensis larvae. indeed, edible insects are a considerable source of fat [13]. womeni et al. [14] investigated the content and composition of oils extracted from several insects. these authors showed that insect oils are rich in polyunsaturated fatty acids and frequently contain the essential linoleic and α-linolenic acids. the nutritional importance of these two essential fatty acids is well recognized, mainly for the healthy development of children and infants [15]. furthermore, the fatty acid composition of insects appears to be influenced by the plants on which they feed [16, 17]. 2. materials and methods larvae collection and sample preparation dried caterpillars i. oyemensis were obtained from the “gouro” market of adjamé (abidjan, côte d’ivoire). after collection, they were sorted and free from any kind of waste. then, dried caterpillars (2 kg) were ground using a porcelain motar to obtain the full-fat flour. oil extraction oil was extracted from 3 g of caterpillar flour (full-fat flour) with 70 ml of nhexane in a soxhlet extractor [18]. then, the solvent was gently evaporated with a rotary evaporatorty (heidolph, hei-vap, germany). the extracted lipid was weighted to determine the oil content of caterpillar. crude oil was stored at 4 °c in airtight brown sterile glass bottle until further use for physicochemical analysis. physicochemical analysis moisture content 10 g of oil was weighed into a petri-dish of known weight. the weighed sample was put into an oven pre-set at 110 °c for 3 h. the sample was removed and cooled in a dessicator at room temperature and the weight was determined after which it was returned into the oven at 110 °c for 30 min until constant weight was obtained [1 8]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 360 ash content ash content was determined according aoac [18] method. 5 g of oil was weighed in a silica crucible. the crucible was heated first over a low flame till all the material was completely charred, followed by heating in a muffle furnace for about 3– 5 h at 600 °c. it was cooled in a desiccator and weighed to ensure completion of ashing. to ensure completion of ashing, it was heated again in the furnace for half an hour, cooled and weighed. this was repeated consequently till the weight became constant (ash became white or greyish white). weight of ash gave the ash content. specific gravity and refractive index specific gravity and refractive index of i. oyemensis oil was determined at 25 °c following the iupac [19] method by using a pycnometer and a refractometer (abbe, optic ivymen, spain), respectively. uv-vis spectra uv-vis spectra of oil sample was determined by measuring absorbance of hexanic oil solution (1%) by using a uvvis spectrophotometer (t80+, pg instruments, england) in the range of 200 to 600 nm [20]. acid, peroxide, iodine and saponification values acid, peroxide, iodine and saponification values were determined following the aoac [18] methods. acid index of i. oyemensis oil was determined according to aoac official method with the morn iso-9001. a volume of 100 ml ethanol was neutralised with a solution of naoh (n/10). the titration was performed using a solution of koh (1n) in the presence of phenolphthalein. iodine value of i. oyemensis oil was determined according to aoac [18] official method. a volume of 30 ml carbon tetrachloride was used to dilute 0.4 g oil in the presence of 25 ml of wijs reagent and 10 ml of acetate mercury. the titration was carried out using a solution of sodium thiosulfate (0.1 n). the peroxide value was determined according to aoac [18] method. a solution made out of potassium iodine added to a mixture of acetic acid – chloroform: 3/2 (v/v) has been used. the titration was carried out using a solution of sodium thiosulfate (n/100). saponification value was determined according to aoac [18] official method. an amount of 2 g oil has been treated using alcoholic potash (0.5 n) and titrated hot with hydrochloric acid (0.5 n) in the presence of phenolphthalein. unsaponifiable matter unsaponifiable matter content of oil sample was determined following the iupac [19] method. for this, 100 mg of unsaponifiable fraction was dissolved in 2 ml of chloroform. to 1 ml of aliquot, 4 ml of a trifluoroacetic-chloroform (1:3, v/v) solution was added. the absorbance was measured at 620 nm using a spectrophotometer (t80+, pg instruments, england). mineral composition mineral composition was determined by scanning electron microscopy / energy dispersion spectrometric (sem/eds) methods, from 2 g of ash. fatty acid composition the fatty acids were converted to their methyl esters (fames) as described by the european communities [21]. about 0.1 g of oil sample was mixed with 2 ml of nheptane and 0.2 ml of a methanolic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 361 solution of potassium hydroxide (2n). the whole mixture was shaken up for 30 s and allowed to settle for 5 min. the top layer containing the fames was used for gas chromatography (gc) analysis. fames solution (1 μl) containing the internal standard (erucic acid) was injected into a gas chromatograph (shimadzu, gc 14 a, japan) equipped with a flame ionization detector (fid) and a capillary column trd1 (60 m x 0.25 mm i.d. x 0.25 μm film thickness). the carrier gas was nitrogen and the flow rate was adjusted to 23 ml/min. temperatures of detector and injector were 250°c. the initial column temperature was fixed to 100°c and programmed to increase by 5°c per min intervals until 220°c and, kept for 10 min at this temperature. the fatty acid methyl esters peaks were identified by comparing their retention times with those of standards. after adjusting areas with the internal standard (erucic acid), the yield of each fatty acid was calculated as follow: area of the fatty acid/areas of total fatty acids in the oil sample × 100 (%). statistical analysis each sample was analyzed in triplicates and data are reported as mean ± standard deviation (sd). analysis of variance (anova) was performed using spss version 11.0 software. statistical significance was set at p ≤ 0.05. 3. results and discussion extraction yield and physicochemical composition edible insects are well known as fat and oil sources [14, 22]. as far as concerned i. oyemensis, its oil content was amounted to 23.96 ± 0.82 % (dry matter) (table 1). with regard to this value, i. oyemensis larvae are lipid-rich than most of edible insects such as cirina forda [23], zonocerus variegates [14], oryctes rhinoceros and rhynchophorus pheonicis [23]. physicochemical characteristics of i. oyemensis oil are shown in table 1. values of specific gravity and refractive index were found to be 0.942 ± 0.02 and 1.4675 ± 0.0002 respectively (table 1). the refractive index value obtained for i. oyemensis (1.4675) was in close agreement with values reported for conventional edible oils [24]. the high refractive index of this oil seems to confirm the high number of carbon atoms in its fatty acids [25]. refractive index also increases as the double bond increases [26]. furthermore, i. oyemensis oil could be classified as nondrying fats in view to its refractive index value [27]. this property would underline its quality of edible fat and also disqualify him for varnish manufacturing in chemical industry. as regards specific gravity (0.942), it is reported for denser than specific gravity (0.89) reported for rhynchophorus phoenicis [22]. the specific gravity and refractive index for i oyemensis oil is similar to those for arachis oil, linseed oil and olive oil [28]. this implies that the oil from this insect is lighter as these oils that have been considered to be of high quality and as such find much use in the pharmaceutical industries. in addition the oil is also unsaturated as these oils, which suggests that it might be fluid at room temperature [22]. the acid value is low, acid value of 0.00 to 3.00 mg koh/g oil is recommended for oil to find application in cooking [29]. thus the oil from i. oyemensis larvae could be suitable for cooking. the free fatty acid value of 1.49 ± 0.37 falls within the maximum limit of 5% for free fatty acids in high grade palm oil in nigeria [30]. the data relevant to acid value and free fatty acid further indicate that i. oyemensis oil is not much susceptible for fat degradation process during oil extraction too. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 362 saponification value is an index of average molecular mass of fatty acids in oil sample. the higher saponification value (184.20 ± 1.62) in the i. oyemensis oil suggest that the mean molecular weight of fatty acids is higher as that of rhynchophorus palmarum larvae (189.22 ± 0.92) oil or that the number of ester bonds is similar to that of rhynchophorus palmarum larvae [31]. this might imply that the fat molecules were intact. high saponification value indicated the presence of greater number of ester bonds, suggesting that the fat molecules were intact. these properties make it useful in soap making industry [32]. table 1 extraction yield and physicochemical properties of i. oyemensis oil parameters values extraction yield (%) 23.96 ± 0.82 moisture and volatile matter at 105 °c (%) 7.51 ± 0.1 acid value (mg koh/g mg) 2.81 ± 0.02 free fatty acid (% oleic acid) 1.49 ± 0.37 saponification value (mg koh/g mg) 184.20 ± 1.62 iodine value (g i2/100 g) 105.19 ± 1.62 refraction index 26°c 1.4675 ± 0.0002 specific gravity 0.942 ± 0.02 unsaponifiables (g/kg) 45.0 ± 1 total sterols content (mg / kg) 1589.0 ± 2 values are mean ± standard deviation of three measurements (n = 3). mineral composition it is noteworthy that minerals play an important part in biological processes. the recommended dietary allowance (rda) and adequate intake are generally used to quantify suggested daily intake of minerals. the mineral composition of oil from i. oyemensis larva is shown in table 2. this oil was found to be a very rich source of mineral elements with a 100 g dry sample containing phosphorus (26.82 g), potassium (14.97 g), magnesium (5.63 g), calcium (5.39 g), zinc (0.63 g) and iron (0.31 g). the consumption of 100 g dry oil would provide 1407 %, 3831 %, 318 %, 539 %, 3875 %, and 5727 % of the recommended dietary allowances of magnesium, phosphorus, potassium, calcium, iron and zinc respectively [33]. therefore, this oil can be used in diet to prevent against some mineral deficiencies. for example, zinc deficiency is a core public health problem, especially for child and maternal health. zinc deficiencies can lead to growth retardation, delayed sexual and bone maturation, skin lesions, diarrhea, alopecia, impaired appetite and increased susceptibility to infections mediated via defects in the immune system [34]. otherwise, the sodium and potassium levels in the larva oil are 0.67 g/100 g and 14.97 g/100 g, respectively, resulting in potassium to sodium ratio of approximately 22:1. this favourable potassium/sodium ratio renders the oil from i. oyemensis larva a potential component of diets for the management of hypertension. potassium intake has been found to lower blood pressure by antagonizing the biological effect of sodium [35]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 363 table 2 mineral composition of i. oyemensis oil minerals values (g/100 g) intake recommendation for 25-year-old males (g per day)* na 0.67 ± 0,08 1.50 mg 5.63 ± 0,05 0.40 al 0.00 si 0.00 p 26.82 ± 0.24 0.70 s 0.00 cl 0.00 k 14.97 ± 0.15 4.70 ca 5.39 ± 0.14 1.00 mn 0.85 ± 0.04 0.0023 fe 0.31 ± 0.03 0.008 cu 0.36 ± 0.16 0.0009 zn 0.63 ± 0.15 0.011 values are mean ± standard deviation of three measurements (n = 3). *dietary reference intakes (dris): recommended dietary allowances and adequate intakes, minerals, food and nutrition board, institute of medicine, national academies. uv-vis spectrum the uv-vis spectrum of i. oyemensis oil is depicted in figure 1. fig. 1. uv-vis spectrum of i. oyemensis oil maximum absorbencies were obtained at 220 and 300 nm corresponding respectively to absorbance values of 1.9 and 0.5. absorbance of the studied oil decreases quickly from 0.5 to 0 in the range of 250 – 400 nm. this wavelength interval includes that of the uv-b and uva range varying from 290 to 400 nm. given that in the uv-b and uv-a wavelengths range, absorption of uv light is responsible for most of cellular damages, i. oyemensis oil may be used in cosmetic formulations of uv protectors that provide protection against both uv-b and uv-a [20]. fatty acids composition figure 2 depict the fatty acids profile of i. oyemensis. the data given in the table 3 showed that unsaturated fatty acids (55.7 %) are higher than saturated (44.3 %). the high percentage of polyunsaturated fatty acids in the oil from i. oyemensis is supported by the high iodine value obtained for the oil in this study. the abundances of unsaturated fatty acids in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 364 the oils were desirable from the nutritional and health view points as unsaturated fatty acids consumption will not lead to heart related diseases while the consumption of foods rich in saturated fatty acids is implicated with certain cardiovascular disorders like atherosclerosis, cancer and aging [36, 37]. the major unsaturated fatty acids in i. oyemensis oil were linolenic (26.22 ± 0.03 %), linoleic (11.33 ± 0.02) and arachidonic (10.26 ± 0.04), while the major saturated fatty acids were stearic (20.92 ± 0.03) and palmitic (18.65 ± 0.05). the unsaturated fatty acids accounted for 55.7 % while the saturated acids accounted for 44.3% of total fatty acids (table 3). the presence of both saturated and unsaturated fatty acids in this insect could be an advantage since they may complement the functions of one another. linoleic, linolenic and arachidonic acids are important essential fatty acids required for growth, physiological functions and body maintenance [38]. as far as concerned palmitic and stearic acids, which are the main saturated fatty acids of the studied oil, previous studies have shown that they are free from deleterious effect on plasma cholesterol [39]. in addition, they are often used in food industries to provide texture and softness to products [40]. ratio of polyunsaturated to saturated fatty acids (pufa/sfa) has been used widely to indicate the cholesterol lowering potential of a food. a pufa/sfa ratio of 0.2 has been associated with high cholesterol level with high risk of coronary heart disorders while a ratio as high as 0.8 is associated with desirable levels of cholesterol and reduced coronary heart diseases [41]. the pufa/sfa ratio of 1.07 in i. oyemensis larva (table 3) tends to suggest that the larva oil has the potential of being used in the dietetic management of certain coronary heart diseases. ratios of linoleic and arachidonic acids to the linolenic one (n-6/n-3), calculated for i. oyemensis (0.82), are extremely lower than the critical ratio (5.00) recommended by nutritionists to reflect the need for a nutritional equilibrium [42]. therefore, i. oyemensis oil would be nutritionally balanced than most of the conventional oils such as soybean, walnut, olive, corn and sunflower oils with respective n-6/n-3 ratios of 8, 8.1, 34, 72.5 and 670 [24]. fig. 2. gas chromatograms of fatty acids methyl esters of fatty acids methyl esters of i. oyemensis oil analysed by flame ionization detector food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 365 table 3 fatty acids composition of i. oyemensis oil fatty acid saturation values (%) essential linoleic acid omega 6 polyunsaturated 11.33 ± 0.02 linolenic acid omega 3 polyunsaturated 26.22 ± 0.03 arachidonic acid omega 6 polyunsaturated 10.26 ± 0.04 non-essential caprylic acid saturated 0.56 ± 0.045 capric acid saturated 1.47 ± 0.02 lauric acid saturated 1.38 ± 0.01 myristic acid saturated 1.30 ± 0.03 palmitic acid saturated 18.65 ± 0.05 stearic acid saturated 20.92 ± 0.03 oleic acid omega 9 monounsaturated 7.91 ± 0.01 ratios sfa 44.3 mufa 7.91 pufa 47.79 pufa / sfa 1.08 n6 / n3 0.82 values are mean ± standard deviation of three measurements (n = 3). 4. conclusion based on the present study, it appears that i. oyemensis larva is a good source of edible oil, contains high percentage of polyunsaturated fatty acids (55 %) mainly consisting of arachidonic, linolenic and linoleic acids which were desirable from the nutritional and health view points. furthermore, the pufa/sfa and n-6/n-3 ratios suggest that i. oyemensis oil would be nutritionally more balanced than most of the conventional oils such as soybean, walnut, olive, corn and sunflower. this oil was also found to be a very rich source of minerals especially phosphorus, potassium, magnesium, calcium, zinc and iron. therefore, it can be used in diet to prevent against some mineral deficiencies. the oil extract exhibited good physicochemical properties and could be useful for nutritional, pharmaceutical and industrial applications. 5. references [1]. defoliart g.r., the human use of insects as food and as animal feed. bulletin entom. soc. am. 35: 22–35, (1989). [2]. defoliart g.r., edible insects as minilivestock. biodiversity conservation, 4(3): 306–321, (1995). [3]. van huis a., van itterbeeck j., klunder h., mertens e., halloran a., muir g. and vantomme p., edible insects: future prospects for food and feed security, fao forestry paper 171. fao, rome, italy, (2013). [4]. van huis a., potential of insects as food and feed in assuring food security. annual review of entomology, 58: 563-583, (2013). [5]. defoliart g.r., an overview of the role of edible insects in preserving biodiversity, ecology and food nutrition, 36: 109–132, (1997). [6]. fao., biodiversity and sustainable diets: united against hunger, report presented at world food day/world feed week, 2–5 november, 2010, rome, (2010). [7]. ramandey e. and van mastrigt h., edible insects in papua, indonesia: from delicious snack to basic need. in p.b. durst, d.v. johnson, r.l. leslie. & k. shono, eds. forest insects as food: humans bite back, proceedings of a workshop on asia-pacific resources and their potential for development. pp. 105–114. bangkok, fao regional office for asia and the pacific, (2010). [8]. latham p., edible caterpillars and their food plants in bas-congo. canterbury, mystole publications, (2003). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 366 [9]. stack j., dorward a., gondo t., frost p., taylor f. and kurebgaseka n., mopane worm utilisation and rural livelihoods in southern africa. paper presented at the international conference on rural livelihoods, forests and biodiversity, bonn, germany, 19–23 may 2003, (2003). [10]. glew r.h., jackson d., sena l., vanderjagt d.j., pastuszyn a. and millson m., gonimbrasia belina (lepidoptera: saturniidae), a nutritional food source rich in protein, fatty acids and minerals. american entomology, 45, 250–253, (1999). [11]. headings m.e. and rahnema s., the nutritional value of mopane worms, gonimbrasia belina (lepidoptera: saturniidae) for human consumption. presentation at the ten-minute papers: section b. physiology, biochemistry, toxicology and molecular biology series, 20 november 2002. ohio, usa, ohio state university, (2002). [12]. akpossan r.a., digbeu y.d., koffi d.m., kouadio j.p.e.n., due e.a., kouame p.l., protein fractions and functional properties of dried imbrasia oyemensis larvae full-fat and defatted flours. international journal of biochemistry research and review, 5(2): 116-126, (2015). [13]. defoliart g., insect fatty acids: similar to those of poultry and fish in their degree of unsaturation, but higher in polyunsaturates. the food institut newsletter, 4(1): 1-4, (1991). [14]. womeni h.m., linder m., tiencheu b., mbiapo f.t., villeneuve p., fanni j. and parmentier m., oils of insects and larvae consumed in africa: potential sources of polyunsaturated fatty acids. ocl – oléagineux, corps gras et lipides, 16(4): 230–235, (2009). [15]. michaelsen k.f., hoppe c., roos n., kaestel p., stougaard m., lauritzen l. and mølgaard c., choice of foods and ingredients for moderately malnourished children 6 months to 5 years of age. food nutrition bulletin, 30(3): 343–404, (2009). [16]. bukkens s.g.f., the nutritional value of edible insects. ecolology food nutrition, 36: 287–319, (1997). [17]. bukkens s.g.f., insects in the human diet: nutritional aspects. in m.g. paoletti, ed. ecological implications of minilivestock; role of rodents, frogs, snails, and insects for sustainable development, pp. 545–577. new hampshire, science publishers, (2005). [18]. aoac, 2004. official methods of analysis. association of official analytical chemists international washington, dc, usa. [19]. iupac., standard methods for the analysis of oils, fats and derivatives. pergamon: oxford, (1979). [20]. besbes s., blecker c., deroanne c., lognay g., drira n.e. and attia h., quality characteristics and oxidative stability of date seed oil. food sciences and technology international, 10: 333-338, (2004). [21]. european communities, determination of free fatty a cid methyl esters. in: oj, (ed). characteristics of olive oil and the relevant methods of analysis. geneva, pp. 64 -67, (1991). [22]. ekpo k.e., onigbinde a.o. and asia i.o., pharmaceutical potentials of the oils of some popular insects consumed in southern nigeria. african journal of pharmacy and pharmacology, 3(2): 051-057, (2009). [23]. akinnawo o. and ketiku a.o., chemical composition and fatty acid profile of edible larva of cirina forda (westwood). african journal of biomediation research, 3: 93 –96, (2000). [24]. codex alimentarius, fats and vegetable oils. fao/who, geneva, p. 160, (1993). [25]. falade o.s., adekunle s.a., aderogba m.a., atanda o.s., harwood c. and adewusi s.r.a., “physicochemical properties, total phenol and tocopherol of some acacia seed oils”. journal of sciences and food agriculture, 88: 263268, (2008). [26]. eromosele c.o. and pascal n.h., “characterization and viscosity parameters of seed oils from wild plants”. journal of bioresources and technology, 86: 203-205, (2003). [27]. rossell j.b., vegetable oils and fats. in: rossell jb (ed). analysis of oilseeds, fats and fatty foods. london. pp. 261-325, (1991). [28]. pearson d. the chemical analysis of foods. 7th edition. churchill living stone (publisher). pp. 491-516, (1976). [29]. oderinde r.a., ajayi i.a. and adewuyi a., “characterization of seed and seed oil of hura crepitans and the kinetics of degradation of the oil during heating”. elect. j. env. agr. food chem, 8(3): 201-208, (2009). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame, fatty acids, mineral composition and physico-chemical parameters of imbrasia oyemensis larvae oil with unusual arachidonic acid content, food and environment safety, volume xiv, issue 4 – 2015, pag. 358 367 367 [30]. nifor., nigeria institute for oil research. history, activities and achievement. 2nd ed. benin city, nigeria, 26-28, (1989). [31]. due e.a., zabri h.c.b.l., kouadio e.nj.p., kouame l.p., fatty acid composition and properties of skin and digestive fat content oils from rhynchophorus palmarum l. larva. african journal of biochemistry research, 3(4): 089-094, (2009). [32]. denniston k.j., topping j.j., caret r.l., general, organic and biochemistry, 4th ed. mcgraw hill companies, new york, pp: 432-433, (2004). [33]. food and nutrition board. recommended dietary allowances 9th ed. national academy of sciences/national research council, national academy press, washington d.c, (1980). [34]. fao/who., human vitamin and mineral requirements, rome, (2001). [35]. einhorn d, landsberg l., nutrition and diet in hypertension: in shils me, young vr eds. modern nutrition in health and disease ed. 7, philadelphia. lea and febiger, (1988). [36]. law m., dietary fat and adult diseases and the implications for childhood nutrition: an epidemiologic approach. american journal of clinical nutrition, 72: 1291-1296, (2000). [37]. anita b.s., akpan e.j., okon p.a., umoren i.u., nutritive and anti-nutritive evaluation of sweet potatoes leaf. pakistan journal of nutrition, 5: 166-168, (2006). [38]. fao/who., fats and oils in human nutrition. report of a joint expert consultation organised by the food and agriculture organization of the united nations and the world health organization rome, 19-26 october. 10, 19-26, (1993). [39]. hunter k., crosbie l.c., weir a., miller g.j., dutta-roy a.k., a residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, haemostatic variables and platelets in young healthy men. journal of nutrition and biochemistry, 11: 408-416, (2000). [40]. dubois v., breton s., linder m., fanni j., parmentier m., fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. european journal of lipid sciences and technology, 109: 710732, (2007). [41]. mann j., diseases of the heart and circulation: the role of dietary factors in aetiology and management: in human nutrition and dietetics eds j.s. garrow and w.p.t. james 619-650. churchill, livingstone london, (1993). [42]. legrand p., bourre j.m., descomps b., durand g., renaud s., lipids. in: martin a (ed). the advisable nutritional benefits for french population. paris. pp. 6382, (2001). 32 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue xvii2018, pag. 32 36 antioxidant capacity of local berries in complex food products * violina popovici 1 , rodica sturza 2 1faculty of food technology. technical university of moldova, 168 stefan cel mare blvd., md-2004, chisinau, republic of moldova, *violina.popovici@toap.utm.md *corresponding author received 15th october 2017, 21th march 2018 abstract: complex food degradation is caused by lipid oxidation process and as a result there may occur a rancid odor, colour change and texture may be modified, thus negatively influencing the sensory qualities of food. the results obtained through analysis of different methods of research has found that the oxidation process can be prevented or slowed down by using horticultural oily extracts fortified with natural antioxidants. the study determined whether the oily extracts enriched with antioxidants are characterized by a higher antioxidant capacity as compared to control samples and the highest value is characteristic for rosehip extract with 73,2%. in terms of sensory aspect, the products with high lipid content was appreciated positively, so we can conclude that products based on vegetable fat and enriched with natural antioxidants are an appropriate alternative to produce harmless and safe food products for consumers’ health. it was determined that oily extracts of sea buckthorn , hawthorn , rosehip berries show increased antioxidant capacity as compared to the control samples of sunflower oil. the highest value of antioxidant capacity is characteristic for rosehip extract with 73,2%. we can conclude that lipid products enriched with natural antioxidants are an appropriate alternative to produce natural, harmless and safe food for consumers’ health. keywords: oxidation, rosehip, sea buckthorn, hawthorn, extracts, lipids, antioxidants. 1. introduction a permanent pursuit of the modern food industry is to ensure an optimal storage term for food products. one of the main causes of the degradation of complex food products such as pastries (biscuits, waffles, creams) is the oxidation of lipids. lipids are a slightly perishable fraction of food, so the storage term and conditions depend largely on their nature and concentration[1]. as a consequence of this phenomenon caused by the degradation of some fragile constituents of the lipid fraction in food, there may be the appearance of a scent, the colour change and, in some cases, the food texture change, which negatively influences the sensory qualities of food products. the nutritional value of foods may also be affected to a considerable extent because of oxidative degradation of the lipid fraction [2]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 violina popovici, rodica sturza, antioxidant capacity of local berries in complex food products, food and environment safety, volume xvii, issue 1 – 2018, pag. 32 – 36 33 the most important risk is the ingestion of lipid oxidation products, as they present enormous toxicological risks and, if will be consumed for a longer term, can cause degenerative pathologies such as arteriosclerosis, cancer, and so on. in biological materials, the lipids are protected from oxidation by the presence of antioxidants and cell membranes, which diminish the access of oxidants to fragile fractions [3]. in complex foods, the impact reduction of lipid oxidation can only be ensured by appropriate packaging and antioxidants that block the propagation or decomposition of the hydroperoxides and is manifested by the inhibition of the oxidation process. the industrially manufactured complex food products usually contain antioxidants of synthetic origin (propyl gallate e-311 or octyl-e312, butylhydroxyanisole (bha) e-320, etc.) and their effect on health human is not very beneficial. so the purpose of the research is to obtain lipid rich food products enriched with natural antioxidants that will be safe and harmless for human health [4-7]. 2. materials and methods plant material of moldavian origin was used. the vegetal material (fresh berries) was harvested from a local forest. as extracting solution was used refined sunflower oil which was purchased from a local market [8, 9]. all chemicals used were of analytical reagent grade. reagents used for analytical methods of analysis include: hydrogen peroxide (0,1 m), ammonium molybdate (3%), sulfuric acid (2m), potassium iodide (1,8m), sodium thiosulfate (5,09 mm), concentrated nitric acid, hexane, ethyl alcohol (70%), phenolphthalein, glacial acetic acid, starch solution (50%). for extraction procedure all berries were dried and finely grounded. refined sunflower oil was used as extracting solution. the oily extracts of sea buckthorn, hawthorn and rosehip were obtained under laboratory conditions. the finely grounded plant material (sea buckthorn, hawthorn, rosehip) was exactly weighed in portions of 1g for each 20 ml of extracting solution. the extraction process took place at controlled temperature of 45°c. the decanted extracts were kept in dark glass vials and stored under reduced light and temperature (+24°c). 2.1. the antioxidant capacity of plant extracts in order to determine the antioxidant capacity of plant extracts the hydrogen peroxide scavenging activity method were used [10]. for the determination of hpsa, in the titration flasks, was weighed with precision 1 ml of plant sample and was mixed with 1 ml of hydrogen peroxide solution (0,1 mm). then 2 drops of ammonium molybdate, 10 ml of sulfuric acid (2m) and 7 ml of potassium iodide (1,8m) were added. the obtained solution was titrated with sodium thiosulfate (5,09 mm) until the yellow colour disappeared. the volume of sodium thiosulfate (v1) used for titration was recorded. parallel to the basic determination, control titration is performed, without plant sample. the volume of sodium thiosulfate (v0) used for titration was recorded. to calculate the antioxidant capacity of plant extracts the following formula (1) was used: (1) [%]%,100% 0 10 22    v vv oh food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 violina popovici, rodica sturza, antioxidant capacity of local berries in complex food products, food and environment safety, volume xvii, issue 1 – 2018, pag. 32 – 36 34 2.2. peroxide value in order to determine the peroxide value for plant extracts, the following method were used [11, 12]. in the titration flask was weighed 3 g of the investigated vegetable plant extract, then added 10 cm3 of hexane, the sample was quickly dissolved, then poured 15 cm3 of glacial acetic acid and 1 cm3 of potassium iodide, then the flask was closed and stirred for 1 minute and left for 5 minutes in a dark place. then added 75 cm3 of distilled water, stirred and added 1 ml of starch solution until a pale blue tint appeared and the released iodine was titrated with sodium thiosulfate solution until a white colour appeared and it was stable for 5 seconds. the volume of sodium thiosulfate (vsample) used for titration was recorded. parallel to the basic determination, control titration was performed, and the sodium thiosulfate volume (vref) used for titration was recorded. to calculate the peroxide value for plant extracts the following formula (2) were used:   ]/[, 1000 fatperoxide sample thiosrefsample kgmol m nvv pv   (2) 2.3. acid value in order to determine the acid value for plant extracts, the following method were used [13]. in a 50 ml conical flask, was weighed 1 g of the plant extract with a precision of 0,01 g. then 5 ml of hexane and 5 ml of ethyl alcohol was added to the sample. the flask content was stirred and after added a few drops of phenolphthalein. the analyzed sample of plant extract was titrated by stirring continuously with potassium hydroxide solution with a molar concentration of 0,1 mol/dm3 until a pale pink colour appeared and was stable for 30s. the volume of potassium hydroxide (vkoh) used for titration were recorded. to calculate the acid value of plant extracts the following formula (3) were used: ]1/[, 611,5 samplekoh sample kohkoh gmg m nv av   , (3) 3. results and discussion series of experiments have shown that antioxidant capacity is considerably higher in the case of rosehip extract, and for sea buckthorn and hawthorn extracts it exceeds nonessential the antioxidant capacity value for the control sample, being within the range of certainty. it has therefore been established according to the results obtained (figure 1) that the rosehip extract is characterized by the highest capacity to resist to oxidation process which can take place in the food products with an antioxidant capacity of 73,2%. sea buckthorn and hawthorn extracts are also characterized by a higher antioxidant capacity compared to the control sample. thus, plant extracts present great interest for the food industry in order to replace synthetic antioxidants with natural antioxidants obtained from local horticultural resources [14, 15]. fig. 1. content of inhibited hydrogen peroxide (%): hpsa confidence interval ± 1,2% the acidity value (mg koh / g) for the control sample is within the permitted limits (0,6 mg koh / g) according to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 violina popovici, rodica sturza, antioxidant capacity of local berries in complex food products, food and environment safety, volume xvii, issue 1 – 2018, pag. 32 – 36 35 normative documents for the refined oil [8]. in the case of rosehip and sea buckthorn extracts, the acidity value exceeds the value of the control sample and the hawthorn extract shows a nonessential drop. the obtained values are within the permitted limits according to the normative documents (figure 2). fig. 2. acidity value (mg ko /g): the confidence interval av ± 0,04 (mg koh/g) the acidity value for the analyzed samples (figure 2) exceeds the acidity value of the control sample, which is explained by the increase of the amount of free fatty acids in the plant extracts. the acidity value of the hawthorn extract is lower than the acidity value of control sample, because of the active substances from the horticultural sources with which the extract has been enriched, the process of formation of fatty acids occurs slower and the oxidation process is slowed down. the peroxide value for control sample is within the permitted limits (max 10 mechiv o2/kg) according to normative documents [8]. in the examined extracts the peroxide value is considerably lower in the case of rosehip extract by 0,5 mechiv o2/kg, and in the case of sea buckthorn and hawthorn extracts by about 1,0 units less (figure 3). it has been found that samples enriched with natural antioxidants are characterized by a lower peroxide value compared to the peroxide value of the control sample, which means that the active substances in the horticultural sources considerably slow down the formation of the peroxides, respectively slowing down the oxidation process of the investigated product. the sea buckthorn extract shows the lowest value of the peroxide index (3,66 mechiv o2/kg), the hawthorn and rosehip extracts also have a low value compared to the control sample, demonstrating that they are slowing down the oxidation process. fig. 3. peroxide value (active o2 mechiv/kg): the confidence interval pv ± 0,13 (mechiv o2/kg) 4. conclusions according to the experiments made and the results obtained can be motivated the possibilities of using the horticultural oily extracts as components for the production of food products with high lipid content and enriched with natural antioxidants. it has been established that sea buckthorn, hawthorn and rosehip oily extracts present an increased antioxidant capacity compared to the control sample, and the highest antioxidant capacity value is characteristic for the rosehip oily extract with 73,2%. there was highlighted that the effect of the active substances in horticultural extracts are slowing down the formation of primary and secondary oxidation products, the free fatty acids, so therefore the oxidation process of lipid rich food is slowing down too. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 violina popovici, rodica sturza, antioxidant capacity of local berries in complex food products, food and environment safety, volume xvii, issue 1 – 2018, pag. 32 – 36 36 5. references [1]. wasowicz e., gramza a., hes m, oxidation of lipids in food, faculty of food science, the august africultural universitz of poznan, poland, 2004, vol. 13/54, si 1, pp.87-100. [2]. f.m. maries, research on sensory and physicochemical changes during storage of animal fat, phd thesis resume, cluj-napoca, 2010. [3]. m. repetto, j. semprine, a. boveris; lipid peroxidation: chemical mechanism, biological implications and analytical determination; chapter 1; 2012. [4]. m. gomez, syntetic antioxidants: role, function and uses in foods; fstc 605: chemistry of foods, april 21, 2016. [5]. s. cartofeanu, g. musteata, p. tatarov, antioxidants in vegetal raw material and evaluation of stabilization methods in food products, chisinau; tehnica, utm, 1996. [6]. c. popovici, p. alexe, e. dmitrieva, o. deseatnicova, the influence of natural and synthetic antioxidants on the oxidation stability of heated treat walnut oil, proceedings of international conference, modern technologies in the food industry, volume i, chisinau, 2014. [7]. s. m. djilas, m. jasna, antioxidants in food; faculty of technology; university of novi sad; review paper; 2002. [8]. government decision nr. 434 from 27.05.2010 on technical regulation „edible vegetable oil“; [9]. a.m câmpău; c.r cristian.; production technology of vegetable oil; north university, baia mare, 2012. [10]. z. sroka, w. cisowski, hydrogen peroxide scavenging antioxidant and antiradical activity of some phenolic acids, food and chemical toxicology, volume 41, issue 6, jume 2003. [11]. peroxide value, acetic acid – chloroform method, aocs official method 8-53, sampling and analysis of commercial fats and oils, 2003. [12]. sr 13531, food products, peroxide value determination, july 2008. [13]. acidvalue, aocs official method cd 3d-63, sampling and analysis of commercial fats and oils, 1999 [14]. d. ciobanu, r.c. ciobanu, food products chemistry, part i, tehnica-info, chisinau, 2001. [15]. d. ciobanu, r.c. ciobanu, food products chemistry, part ii, tehnica-info, chisinau, 2001. 331 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 42015, pag. 331 339. the role of functional food in the improvement of consumer’s health status *katerina bojkovska1 , elena joshevska1, nikolche jankulovski1, gordana dimitrovska1, vangelica jovanovska1 1faculty of biotechnical sciences bitola.university „st. kliment ohridski” bitola partizanska bb, bitola, republic of macedonia,katerina.bojkovska@uklo.edu.mk, *corresponding author received september 5th 2015, accepted december 15th 2015 abstract: the main objective of this study is to present advances in understanding the relationship between nutrition and health that lead to the development of the concept of functional foods, which means a practical and new approach to achieve optimal health status by promoting the state of well-being and possibly reducing the risk of desease. for this purpose, the paper will look into the factors that influence consumers’ awareness of functional foods and their purchasing decisions of functional food products in the republic of macedonia. the challenge of understanding the connection between diet and health has resulted in the creation of a new concept of functional products, which means a new practical approach to balance the diet with the aim to achieve optimal health and possibility t o reduce the risk of deseases. functional foods are foods that provide health benefits beyond basic nutrition ones. biologically active compounds in functional foods may impart health benefits or desirable physiological effects. increasing awareness of healthier food choices, its possibilities and advantages, provides higher quality of life. however, for a functional food product to be good and widely accepted, it is necessary to link research, marketing and relevant feedback information obtained by monitoring consumers’ attitudes towards functional product, i n o r d e r t o meet consumers’ expectations. one important fact in the whole concept of functional foods is informing consumers about the possibilities of functional foods, but any information relating to health must necessarily be true, attractive, simple and intelligible to the consumer s o that the concept of functional foods b e i n g a b l e i n continuing to get developped. keywords: functional food, consumer health, consumer buying behavior, marketing. 1. introduction food preference is determined by both nutrition and pleasure derived from food consumption. with the development, the "society of abundance" faces new challenges such as increasing costs for health care, extension of the life span of individuals, and the new scientific knowledge and new technologies lead to significant changes in the lifestyle. due to all these, functional food plays a major role in improving the standard of living, balancing and maintaining maximum physiological functions, in preserving health and reducing the risk of diseases [1, 2]. changes of consumer’s attitudes about diet and the connection between diet and health can be achieved by placing an emphasis on targeted and balanced diet, maximizing physiological functions of the body in order to reduce the risk of disease or formulating functional food as an important source of specific nutrients from which are of great importance [2]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 332 although there is no existing unique and regulatory definition for functional foods, the scientific community agrees that those foods include a variety of foods and food components which are believed to improve overall health and well-being, reduce the risk of specific diseases, or minimize the effects of other health concerns. one of the most comprehensive definitions states that food can be regarded as functional “if it is satisfactorily demonstrated to affect beneficially one or more target functions of the body” [3]. besides the usual foods seen as functional, like fruits and vegetables with their healthful components, breads and cereals made of whole grains, milk with its high intake of calcium, fortified foods and beverages, functional foods can also include dietary supplements. concept of functional food the concept of balanced diet is a result of long research in the field of science of food and nutrition. a balanced diet is the most important support for the recommendations of dietary intake of certain nutrients. the beginning of the 21st century, however, puts the science of food in front of new challenges [4]. food is no longer viewed only from the point of view of a need for adequate intake for proper growth, development and regeneration of the body. food today has got a leading role in the quality of human life. for these reasons, balancing a diet grows at a level for optimal diet balancing, which focuses on optimizing the daily intake of both nutrients and nonnutritive components of food, in order to promote health and to reduce the risk of chronic, non-contagious diseases. functional food plays an important role in the concept of optimally balanced diet. in 1998, the japanese government set up a project in which they conducted research on the potential positive function of food in order to reduce the costs of medical treatment. the category of food with potential positive health effects, which occurs as a result of this research, is known as "food for specific non-medical purposes" (foshu food). this food category appeared in 1991 and is expected to exert certain, beneficial health effects as a result of the presence of certain components. foshu (food for specified health uses) includes the set of rules for specific health statements for functional food [5, 6, 7, 8]. according to the ministry of health of japan, foshu is [8]: • food that is expected to have specific health impacts due to its composition or food from which allergens are removed; • food in which the influence of additives or removed substances is scientifically validated and permits the claims about specific positive effect on health is obtained. in most countries there are no legal definitions of the term "functional food" and the boundaries between conventional and functional food are challenge even for nutritionists and food technologists [9, 10]. also, there are several different definitions for functional food (ff). one of the definitions that explain the concept of ff in a simple way is the definition which stipulates that some food can be called "functional" if in addition to its basic nutritional values, it affects one or more target functions of the body in a positive and satisfying way by reducing the risks of developing certain diseases [8]. functional food can improve the overall health of the body, reduce the risk of various diseases, it can even be used during the treatment of certain disease states [9, 7]. ific (international food information council) has given a definition which states that: functional food is that food which provides greater health benefits than basic nutrition. fufose (the european commission concerted action on functional food science in europe) has food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 333 given the unique features of functional food: • should be conventional and daily food; • the possibility of consumption as a part of normal diet; • natural (as opposed to synthetic) with components that can be naturally found in the food or added to the food to a greater amount of the concentration specific to the food; • has a positive effect on physiological functions; • can improve the overall health and reduce the risk of disease; • has confirmed and substantiated health claims. [8, 3] functional food may not necessarily be functional for the entire population and the combination of individual biochemical needs with specific food components can affect the progress in understanding the interaction between genes and diet. however, it is very important to understand the distinction between food and medicine. if indications for the treatment or prevention of certain diseases are recognized in a product, then the product is a medicine and a certain degree of toxicity is tolerant, but if indications for treatment or prevention of certain diseases are not recognized in the product, then it is a food and in this case, in normal amounts it must not cause any toxic effects. economic aspects of the development of functional food the market of functional food functional food market is constantly changing and the competition in winning consumers is growing relentlessly and bluntly. key factors affecting the purchase of products represent a recurring cycle that shows the relationship between quality, price, convenience and ease of preparation and of course, a positive impact on health, which means that functional food or functional food manufacturers must find a response to consumers’ demands for convenient and easy preparation, positive health impact and of course, the good taste. also, it should not be forgotten that consumers are not willing to compromise at the expense of taste, in favour of a positive impact on health [11]. to be an excellent functional food product, it must offer special health properties, and at the same time when compared with conventional food, it must develop its own strategy by linking research and marketing [9]. it is necessary to monitor consumers’ attitude towards a product so that it is possible, to a measurable way, to establish the idea of products and that the new product meets consumer’s expectations and that all information relating to health are truthful and attractive. by tracking consumer’s attitudes, the development of new methods of market research and consumer’s preferences large losses in investments can be reduced [12]. development and marketing of functional food functional food is a sustainable food category on the market due to long-term trends in the society, such as sociodemographic trends [13,14,15] which imply a higher life standard, longer life expectancy, better health care, higher level of education in a large part of the population, and more. scientists and, most importantly, consumers are beginning to accept the fact about the connection between health and nutrition [7]. consumers are more and more informed about health aspects of proper nutrition and willingly accept the changes in their eating habits [16, 17]. the aspect in which functional food helps preserving the overall health status and/or reduces and prevents the risk of developing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 334 certain diseases is not negligible. it is also beyond doubt that increasing awareness of health food choices, its possibilities and advantages indirectly provides increased quality of life, and affects the reduction of the mortality rate, which in the end is of economic and public interest [14, 18]. it should be taken into account that the prices of functional food are higher than those of conventional food which seems to be of interest for all the participants in the supply chain [19]. the development of new functional components and technological solutions can be expensive and it requires a large research effort.this involves identification of the functional components and determining their physiological impact; development of suitable matrix products, determination of the bioavailability of functional components and their potential changes during processing and preparation of the products, consumer’s education and clinical studies on the safety and efficacy of the product to obtain approval for use of claim about the positive health impact [19, 17]. all these represent a multi-stage process in which commercial, academic and legal inputs or legal interests are required, all with the aim of achieving recognition and acceptance by consumers [14]. the acceptance of the concept of functional food and better understanding of its determinants by the consumers has been identified as a key factor for market orientation towards functional food, product development driven by the feedback from customers and market opportunities [4, 20]. the acceptance of functional food or such product by consumers depends largely on the sociodemographic factors such as level of education, place of residence, presence of children in the household, gender and age. on the basis of the socio-demographic factors it can be stated that typical consumers of functional food are persons with higher levels of education, older people and people with health problems who must adhere to a specific diet regime. from the point of view of gender, women are more frequent consumers and they are more interested in functional foods than men [17]. 2. matherial and methods considering the fact that the subject of this research is consumer’s behavior towards functional food in the republic of macedonia, the main group consists of respondents aged over 18 years. the research was conducted on a sample case. the research involved participants from the northeast and southwest of the republic of macedonia. 500 questionnaires that were defined as a target group were made for the survey, but relatively low turnout of participants always occurs with the use of written surveys. thus, from the total number of 500 distributed surveys (450 electronic and 50 physical) only 313 surveys were answered and only those involved in the further analysis, which is of 62.6%. from all the respondents, only 198 respondents answered that they were familiar with the concept of functional food, which represents 63.26% of their total number. the respondents who were not familiar with the term were excluded from further analysis. statistical methods were used in the analysis of data collected by polling. when determining the demographic and socio-economic features, distribution of respondents by place of residence, gender, age, qualification and monthly income was determined. within the analysis of ordinal variables, the selected indicators of descriptive statistics were calculated, while the nominal variables were determined by the respective distributions. hi-square test was applied to test reliability of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 335 selected features. statistically significant differences are considered the differences confirmed at the level of p<0.05. 3. results and discussion part of the questionnaire contained questions related to the consumer’s familiarity with the concept of functional food and their buying habits associated with such products. as previously pointed out, 198 of the 313 subjects included in the study (63.26%) have heard of the term "functional food". table 1 contains the distribution of respondents considering their position on what the term "functional food" means for them. table 1 the distribution of respondents due to the perception of the term "functional food" the perception of the term “functional foods” number of respondents percentage healthy food food with specific benefits to the body expensive food all above 17 144 1 36 8.59 72.73 0.51 18.18 total 198 100.00 for most of them the term “functional food” means food with certain benefits for the system. only one respondent thinks that “functional food” is expensive food. table 2 presents the distribution of respondents according to the defined characteristics (residence, gender, age, education and monthly income) and the perception of the term "functional food". the table also lists the results of hi-square test for independence of the features that is used to examine the relationship between the defined features and the term "functional food". since only one person had a position that functional food is expensive food, in order to satisfy the assumptions of the tests, that answer was neglected in the analysis. in all the groups analyzed there are persons who perceive functional food as food with specific benefits to the body. among the respondents from the northeast part of macedonia, as well as those from the southeast, the number of persons who perceive the term "functional food" as all together (healthy food, food with specific benefits to the body and expensive food) is higher, while the number of those who perceive the functional food only as a healthy food is lower. the same characteristic was registered by the distribution of responses by gender. according to the hi-square test there is no statistically significant relationship between the place of residence and the perception of the term "functional food". also, there is no statistically significant dependence between the gender and the perception of the term "functional food". the existence of statistically significant relationship between age and perception of the term "functional food" is confirmed by hi-square test. in all age groups, as already noted, most respondents answered that functional food is food with certain benefits to the body. these respondents were most prevalent in the older age group, and least prevalent in the young age group. regarding the other respondents, the youngest respondents in greater number have perceived the term "functional food" as being all previously stated (healthy foods, foods with specific benefits to the body and expensive food). in the older group, the number of respondents who food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 336 perceived functional foods as healthy food and as all stated was equal. table 2 distribution of respondents according to the defined characteristics and the perception of the term "functional food" with the results by the chi-square test characteristics perception of the term "functional food" chi-square test healthy food food with particular benefits for the organism as all together residence northeast part of macedonia southwest part of macedonia 14 (12.39%) 3 (3.57%) 80 (70.80%) 64 (76.19%) 19 (16.81%) 17 (20.24%) x2=4.842 p=0.089 gender male female 4 (4.44%) 13 (12.15%) 70 (77.78%) 74 (69.16%) 16 (17.78% 20 (18.69%) x2=3.882 p=0.144 age under 24 25-38 39 or more 8 (10.53%) 7 (8.75%) 2 (4.88%) 46 (60.53%) 61 (76.25%) 37 (90.24%) 22 (28.95%) 12 (15.00%) 2 (4.88%) x2=13.621 p=0.009 education university education secondary education 11 (13.10%) 6 (5.36%) 63 (75%) 80 (71.43%) 22 (11.90%) 26 (23.21%) x2=6.74 p=0.035 monthtly income (denars) less than 10.000,00 10.000,00-30.000,00 to 30.000,00 or more 7 (11.67%) 2 (2.47%) 8 (14.29%) 30 (50.00%) 73 (90.12%) 41 (73.21%) 23 (38.33%) 6 (7.41%) 7 (12.50%) x2=33.338 p<0.001 according to the hi-square test, there is statistically significant relationship between the professional qualification and the perception of the term "functional food". the percentage of persons who associate functional food with healthy food with specific benefits for the body was equal in both groups. but, compared with those who had secondary education, among those with university education, there were people who perceived the functional food as healthy food. the percentage of people with secondary education who experienced functional food as all stated was greater than the group of respondents with university education. the hi-square test confirmed the existence of a statistically significant relationship between the monthly income and the perception of the term "functional food". the largest percent is that of respondents food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 337 who perceive functional food as food with specific benefits to the body, determined among those who earn between 10 000,00 and 30000,00 per month. such persons are less recorded in the lowest profitable group. in the group with the least income, the number of persons who stated that they associate the term "functional food" with all stated (healthy food, food with specific benefits to the body and expensive food) is significant. the percentage of respondents who perceive functional food as a healthy food was the highest among respondents whose monthly income amounted to 30.000,00 denars or more. the questionnaire contained two questions related to the participation in the buying of functional food and functional beverages. respondents' answers to these questions were measured on likert scale which has 4 degrees (1 – never, 2 occasionally, 3 – often, 4 regularly). table 3 contains selected indicators from the descriptive statistics (arithmetic mean, median, mode, standard deviation and coefficient of variation) calculated for the two mentioned variables. table 3 selected descriptive statistical indicators relating to the frequency of purchase frequency of purchase arithmetic mean median mode standard deviation coefficients of variation functional food 2.35 2.00 2.00 0.71 30.18 functional beverages 2.05 2.00 2.00 0.69 33.52 although there are conflicting opinions on the use of mean and its key indicators in the case of measured variables on the likert scale, we applied them in the analysis in order to facilitate decision making. based on the calculated arithmetic mean it can be concluded that respondents buy more functional food than functional beverages. in both cases, the median had the same value. according to the median, half of the respondents expressed purchase frequency of functional food and functional beverages by 2 or lower, and the other half by 2 or higher. and, in both cases the mode has a value of 2. thus, most respondents answered that they bought functional food and functional beverages. the standard deviation and coefficient of variation suggest that the degree of variability in the response to both questions can be considered small. table 4 contains the distribution of respondents according to the category of functional food that they usually buy. table 4 distribution of respondents considering the category of functional foods usually bought categories of functional food number of respondents percentage dairy products products enriched with omega-3 fatty acids fruit juices other products all above 70 27 46 15 35 36.27 13.99 23.83 7.77 18.13 total 193 100.00 the results showed that five respondents who had heard of the term "functional food" never bought such products. most respondents answered that they usually bought dairy products as functional food, which make up more than a third of the respondents. the next respondents are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 338 those who answered that they buy fruit juices. all stated (dairy products, products enriched with omega-3 fatty acids, fruit juices and other products) are usually bought by less than the fifth of the respondents. the number of persons who answered that they usually buy other products was the least. 4. conclusion functional food is a promising and dynamic part of the food industry that is rapidly evolving thanks to a better understanding of the close connection between diet and health.therefore, it is necessary to develop a new approach on the development of new products for consumers. the presentation of functional food is not successful unless it is not carried out simultaneously with successful informing of consumers. the information and the increased awareness of the opportunities of healthy eating allow easier choice in buying the products and their inclusion in the daily diet. with an increasing demand for a healthy lifestyle and general well-being, food and drink manufacturers are provided with opportunities to reformulate their products to address the needs on this emerging market. among these ones, fortified/functional food and drinks has been the most dynamic and fast moving category of new product development on the health and wellness market. although the enhanced health benefits attracted consumers’ attention, concerns on the artificial ingredients and effectiveness of the health benefits have formed negative attitudes towards functional products. thus, the understanding of factors which influence consumers’ perceptions and acceptances of functional food is essential for food and drink manufacturers in their new product development (npd) strategies making. factors that affect the increasing development of functional products are aging population, increasing costs for health care, autonomy in health care and awareness and desire to improve personal health, new research and scientific evidence that diet can change the frequency and progression of a disease. in the conducted research, for most of the respondents the term "functional food" is food with certain benefits to the body. a frequency of respondents is made according to the defined characteristics (residence, gender, age, education and monthly income) and the perception of the term "functional food". most respondents answered that they buy occasionally functional food and functional beverages. the standard deviation and the coefficient of variation suggest that the degree of variability in the response of both questions may be considered small, suggesting that the trend of functional food is accepted. most of the respondents stated that they mostly buy dairy products as functional food. this was the case of more than a third of the respondents. then, there come the respondents who answered that they buy fruit juices. it can be concluded that functional food as a product among the consumers of the republic of macedonia was accepted, but there are significant differences depending on the place of residence, monthly income, age and education of the consumers who have a major influence on the purchase decision. however, functional food for the macedonian food industry is a promising category primarily due to the greater expressiveness and close connection between food and health, the innovation in the food industry, and the consumer’s awareness. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska, vangelica jovanovska, the role of functional food in the improvement of consumer’s health status, food and environment safety, volume xiv, issue 4 – 2015, pag. 331 – 339 339 the information and the increase of the awareness for healthy diet options influence the decision making of purchasing products and of more frequent and regular usage. for this purpose, the macedonian food industry should take maximum advantage of their potential and adapt to the european and world trends to produce functional food and thus, to increase the financial results, but also to influence the change of macedonia customers’ food habits. 5. references [1]. milner j., functional foods: the us perspective, american journal of clinical nutrition, 71 (1654s– 1659s), (2000) [2]. roberfroid, m., global view on functional foods: european perspectives, british journal of nutrition, 88(suppl. 2) (s133–s138), (2002) [3]. diplock at., aggett p., ashwell m., bornet f., fern e., roberfroid m., scientific concepts of functional foods in europe, consensus document. british journal for nutrition, 81 (suppl 1): 1-27, (1999) [4]. ares, g., gámbaro, a., influence of gender, age and motives underlying food choice on perceived healthiness and willingness to try functional foods, appetite, 49, 2007: 148–158. [5]. burdock, g. a., carabin, i. g., & griffiths, j. c., the importance of gras to the functional food and nutraceutical industries, toxicology, 221: 17–27, (2006) [6]. kwak, n. s., jukes, d. j., functional foods. part 1, the development of a regulatory concept, food control, 12: 99–107, (2001a). [7]. menrad, k., market and marketing of functional food in europe, journal of food engineering, 56: 181– 188, (2003). [8]. roberfroid, m. b., an european consensus of scientific concepts of functional foods, nutrition, 16: 689–691, (2000b). [9]. mark-herbert, c., innovation of a new product category—functional foods, technovation, 24: 713– 719, (2004). [10]. niva, m., all foods affect health: understandings of functional foods and healthy eating among health-oriented finns, appetite, 48: 384–393, (2007). [11]. urala, n., lähteenmäki, l., consumers changing attitudes towards functional foods, food quality and preference, 18: 1–12, (2007). [12]. shepherd, r., sparks, p., bellier, s., & raats, m., the effects of information on sensory ratings and preferences: the importance of attitudes, food quality and preference, 3: 147– 155, (1991). [13]. bech-larsen, t., & scholderer, j., functional foods in europe: consumer research, market experiences and regulatory aspects, trends in food science & technology, 18: 231–234, (2007) [14]. jones, p. j., jew, s., functional food development: concept to reality, trends in food science & technology, 18: 387–390, (2007) [15]. van kleef, e., van trijp, h. c. m., luning, p., jongen, w. m. f., consumeroriented functional food development: how well do functional disciplines reflect the voice of the consumer?, trends in food science & technology, 13: 93–101, (2002). [16]. frewer, l. et al., consumer acceptance of functional foods: issues for the future. british food journal, 10: 714-730, (2003). [17]. siró i., kápolna e., kápolna b., andrea lugasi, functional food. product development, marketing and consumer acceptance a review, appetite 51: 456–467, (2008). [18]. van kleef, e., van trijp, h. c. m., luning, p., functional foods: health claimfood product compatibility and the impact of health claim framing on consumer evaluation, appetite, 44: 299–308, (2005). [19]. kotilainen, l., rajalahti, r., ragasa, c., & pehu, e.: health enhancing foods: opportunities for strengthening the sector in developing countries, agriculture and rural development discussion paper, 30: 11-38, (2006). [20]. gilbert, l., the functional food trend: what’s next and what americans think about eggs, journal of the american college of nutrition, 19: 507–512, (2000). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 contents: 1. selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products taisa honcharenko, oksana topchyi 258 265 2. optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology anca-mihaela gâtlan , gheorghe gutt 266 271 3. impact of vehicular traffic on concentrations of selected heavy metals in cassava tubers harvested from roadside in owerri, nigeria henry uzoma anuforo, campbell onyeka akujobi, ethelbert uchechukwu ezeji, confidence chiamaka okehi 272 278 4. traditional uses of wild berries in the bukovina region (romania) ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea 279 286 5. some quality attributes of sausage roll produced from wheat tigernut composite flour emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni,omobolanle omowunmi olorode 287 297 6. physicochemical parameters of romanian raspberry honey daniela pauliuc, mircea oroian 298 307 7. application of water quality index in the drinking water quality assessment of a southeastern nigeria river emeka donald anyanwu, chinonyerem sylva emeka 308 314 8. consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria saheed adewale omoniyi, phidelia ramatu waziri-ugwu 315 320 9. the potential of electronic noses in quality and freshness prediction of fresh and stored meat products. review leonard fweja 321 336 10. prospects of using secondary energy resources anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko 337 346 11. author instructions i v 12. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 256 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 32015, pag. 256 268 effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire *constantacho1, lessoyzoue1, niamkey adom1, sébastien niamke1 1biotechnology laboratory, biosciences faculty, félix houphouet-boigny university, po box 582, abidjan 22, côte d’ivoire, conxty977@gmail.com, *corresponding author received august24th 2015, accepted september 25th 2015 abstract: this study aimed to evaluate the effect of sun drying on nutrient and antioxidant properties of five leafy vegetables (basella alba, colocasia esculenta, solanum melongena, talinum triangulare and corchorius olitorus) commonly used in southern côte d’ivoire. the result of this study revealed that sun drying increased some nutrient contents by concentration phenomenon after 1, 2 and 3 days at 30-32 °c. ash, fibres, proteins, lipids and carbohydrates contents varied after 3 days of sun drying as follow: 10.12 ± 0.00 to 25.78 ± 0.00 %, 16.50 ± 0.00 to 29.81 ± 0.01 %, 16.67 ± 0.01 to 23.68 ± 0.00 %, 6.28 ± 0.00 to 14.24 ± 0.00 % and 10.21 ± 0.00 to 37.68 ± 0.00 %. the mineral contents increased with respective values after 3 days of sun drying: calcium (82.86-481.65 mg/100 g), magnesium (81.98-298.46 mg/100 g), phosphorus (63.41-297.69 mg/100 g), potassium (419.81-993.41 mg/100 g), iron (20.05-90.37 mg/100 g), sodium (19.43-150.51 mg/100 g) and zinc (15.76-64.39 mg/100 g). however, anti-nutritional factors such as oxalates varied from 123.01 to 815.97 mg/ 100 g for the same period of drying. losses of vitamin c and carotenoids were estimated to 85.12-96.42% and 98-100% respectively. contrary to these losses, the antioxidant activity increased and ranged from 75.92 to 82.30% after 3 days of sun drying. all these results suggest that sun drying technique could contribute efficiently to the nutritional requirements and to the food security of ivorian population. keywords:sun drying, nutritive value, antioxidant properties, leafy vegetables. 1. introduction hunger and malnutrition threaten millions of people in sub-saharan andthe increase in consumption of african leafy vegetables (alvs) can have a positive effect on nutrition, health and economic well-being of both rural and urban populations [1]. traditional african leafy vegetables are eaten by many african families because they are rich in micro nutrients needed by humans for good health, growth and development [2]. these plants occupy an important place among the food crops as they provide adequate amounts of many vitamins and minerals for humans[3]. the ethno-botanical reports offers information on medicinal properties of alvs like antidiabetic, anti-histaminic, anti-carcinogenic, hypolipidemic and antibacterial activity [4,5]. however ways of leafy vegetables preparation and preservation may affect significantly the concentration and availability of minerals, vitamins and other essential compounds. indeed, losses of nutrients from vegetables during drying and cooking have been noted in previous studies[6, 7].drying is the process of removal of moisture due to simultaneous heat and mass transfer actions. it is the http://www.fia.usv.ro/fiajournal mailto:conxty977@gmail.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 257 classical method for food preservation which serves lighter weight for transportation and small space for storage[8].apart from moisture losses, the changes in organoleptic quality of dried vegetables are: optical properties (colour, appearance), sensory properties (odour, taste, flavour), and structural properties (density, porosity, specific volume, textural properties). dried vegetables are generally tasty, nutritious, lightweight, easy-to prepare, and easy-to-store and use[9]. in rural areas of southern côte d’ivoire (ivory coast) were population are not provided by refrigerator, sun drying is the method used for the preservation of leafy vegetables before their consumption through recipes made of sauces and starchy staples foods[10]. ethno-botanical studies have stated that most people in southern côte d’ivoire consume indigenous green leafy vegetables such as basella alba “epinard”, colocasia esculenta “taro”, corchorus olitorius “kplala”, solanum melongena “aubergine” and talinum triangulare “mamichou” [11, 12, 13]. earlier reports have highlighted the nutritive potential of these fresh leafy vegetables [14] but there is a lack of scientific data with regards to the effect of sun drying methods on their physicochemical and nutritive characteristics. the aim of this study was to determine the effect of sun drying method on chemical composition of five leafy vegetables consumed in southern côte d’ivoire. 2. matherials and methods 2.1materials 2.1.1 samples collection leafy vegetables(basella alba, colocasia esculenta, corchorus olitorius, solanum melongena and talinum triangulare) were collected fresh and at maturity from cultivated farmlands located at dabou (latitude: 5°19′14″ north; longitude: 4°22′59″west) (abidjan district). the samples were harvested at the early stage (between one and two weeks of the appearance of the leaves). these plants were previously authenticated by the national floristic center (university felix houphouët-boigny, abidjan-côte d’ivoire). 2.1.2 samples processing the fresh leafy vegetables were destalked, washed with deionized water and edible portions were separated from the inedible portion. the edible portions were allowed to drain at ambient temperature and separated into two portions of 250 g each. the first portion was spread on black polythene sheet and dried under the sun (35-38°c) for 1, 2 and 3 days during 8 hours per day [15]. the leaves were constantly turned to avert fungal growth. the second 250 g portion of leafy vegetables was not subjected to any form of drying and used as the control (raw). after drying period, the dried leaves were ground with a laboratory crusher (culatti,france) equipped with a 10 μm mesh sieve and stored in air-tight containers for further analysis. 2.2 methods 2.2.1 nutritive properties 2.2.1.1 proximate analysis proximate analysis was performed using official methods [16]. the moisture content was determined by the difference of weight before and after drying the sample (10 g) in an oven (memmert, germany) at 105°c until constant weight. ash fraction was determined by the incineration of dried sample (5 g) in a muffle furnace (pyrolabo, france) at 550°c for 12 h. the percentage residue food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 258 weight was expressed as ash content. for crude fibres, 2 g of sample were weighed into separate 500 ml round bottom flasks and 100 ml of 0.25 m sulphuric acid solution was added. the mixture obtained was boiled under reflux for 30 min. thereafter, 100 ml of 0.3 m sodium hydroxide solution was added and the mixture were boiled again under reflux for 30 min and filtered through whatman paper. the insoluble residue was then incinerated, and weighed for the determination of crude fibres content. proteins were determined through the kjeldhal method and the lipid content was determined by soxhlet extraction using hexane as solvent. carbohydrates and calorific value were calculated using the following formulas [17]: carbohydrates: 100 – (% moisture + % proteins + % lipids + % ash + % fibres). calorific value: (% proteins x 2.44) + (% carbohydrates x 3.57) + (% lipids x 8.37). 2.2.1.2 mineral analysis minerals contents were determined by the icp-ms (inductively coupled argon plasma mass spectrometer) method [18].the dried powdered samples (5 g) were burned to ashes in a muffle furnace (pyrolabo, france). the ashes obtained were dissolved in 10 ml of hcl/hno3 and transferred into 100 ml flasks and the volume was made up using deionized water. the mineral composition of each sample was determined using an agilent 7500c argon plasma mass spectrometer. calibrations were performed using external standards prepared from a 1000 ppm single stock solution made up with 2% nitric acid. 2.2.1.3 anti-nutritional factors determination oxalates content was performed by using a titration method [19]. one (1) g of dried powdered sample was weighed into 100 ml conical flask. a quantity of 75 ml of sulphuric acid (3 m) was added and stirred for 1 h with a magnetic stirrer. the mixture was filtered and 25 ml of the filtrate was titrated while hot against kmno4 solution (0.05 m) to the end point.phytates contents were determined using the wade’s reagent colorimetric method [20]. a quantity (1 g) of dried powdered sample was mixed with 20 ml of hydrochloric acid (0.65 n) and stirred for 12 h with a magnetic. the mixture was centrifuged at 12000 rpm for 40 min. an aliquot (0.5 ml) of supernatant was added with 3 ml of wade’s reagent. the reaction mixture was incubated for 15 min and absorbance was measured at 490 nm by using a spectrophotometer (pg instruments, england). phytates content was estimated using a calibration curve of sodium phytate (10 mg/ml) as standard. 2.2.2 antioxidant properties 2.2.2.1 vitamin c and carotenoids determination vitamin c contained in analyzed samples was determined by titration [21]. about 10 g of ground fresh leaves were soaked for 10 min in 40 ml metaphosphoric acidacetic acid (2%, w/v). the mixture was centrifuged at 3000 rpm for 20 min and the supernatant obtained was diluted and adjusted with 50 ml of bi-distilled water. ten (10) ml of this mixture was titrated to the end point with dichlorophenolindophenol (dcpip) 0.5 g/l. carotenoids were extracted and quantified following a spectrophotometric method [22]. two (2) g of ground fresh leaves were mixed three times with 50 ml of acetone until loss of pigmentation. the mixture obtained was filtered and total carotenoids were extracted with 100 ml of petroleum ether. absorbance of extracted food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 259 fraction was then read at 450 nm by using a spectrophotometer (pg instruments, england). total carotenoids content was subsequently estimated using a calibration curve of β-carotene (1 mg/ml) as standard. 2.2.2.2 polyphenols determination polyphenols were extracted and determined using folin–ciocalteu’s reagent [23]. a quantity (1 g) of dried powdered sample was soaked in 10 ml of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min. an aliquot (1 ml) of supernatant was oxidized with 1 ml of folin–ciocalteu’s reagent and neutralized by 1 ml of 20% (w/v) sodium carbonate. the reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm by using a spectrophotometer (pg instruments, england). the polyphenols content was obtained using a calibration curve of gallic acid (1 mg/ml) as standard. 2.2.2.3 antioxidant activity antioxidant activity assay was carried out using the 2, 2-diphenyl-1-pycrilhydrazyl (dpph) spectrophotometric method [24]. about 1 ml of 0.3 mm dpph solution in ethanol was added to 2.5 ml of sample solution (1 g of dried powdered sample mixed in 10 ml of methanol), filtered through whatman no. 4 filter paper and was allowed to react for 30 min at room temperature. absorbance values were measured with a spectrophotometer (pg instruments, england) set at 415 nm. the average absorbance values were converted to percentage antioxidant activity using the following formula: antioxidant activity (%) = 100 – [(abs of sample – abs of blank) x 100/abs positive control] 2.2.3 statistical analysis all the analyses were performed in triplicate and data were expressed as mean ± standard deviation (sd). data were analyzed using excell and statistica 7.1 (statsoft). differences between means were evaluated by duncan’s test. statistical significant difference was stated at p < 0.05. 3. results and discussion nutritive and anti-nutritive properties:the results of moisture, ash, fibre, protein, lipid, carbohydratecontents are presented in table 1. table 1: proximate composition of sun dried leafy vegetables consumed in southern côte d’ivoire moisture (%) ash (%) fibres (%) proteins (%) lipids (%) carbohyd. (%) energy (kcal /100g) c. esculenta raw 82.35 ± 2.83a 2.65 ± 0.00d 4.23 ± 0.01d 1.72 ± 0.00d 1.47 ± 0.00d 7.56 ± 0.70d 44.53 ± 0.04d 1 day 58.43 ± 1.12b 6.58 ± 0.00c 10.46 ± 0.02c 5.72 ± 0.02c 3.69 ± 0.00c 15.09 ± 0.08c 98.81 ± 0.03c 2 days 19.42 ± 0.70c 13.05 ± 0.00b 23.07 ± 0.01b 13.73 ± 0.02b 7.51 ± 0.00b 23.18 ± 0.05a 179.18 ± 0.04b 3 days 9.79 ± 1.23d 16.13 ± 0.00a 29.81 ± 0.01a 16.67 ± 0.01a 9.26 ± 0.00a 18.28 ± 0.07b 183.60 ± 0.08a b. alba raw 89.82 ± 1.24a 2.01 ± 0.00d 1.67 ± 0.00d 1.00 ± 0.00d 0.69 ± 0.00d 4.78 ± 0.10d 25.36 ± 0.02d food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 260 data are represented as mean ± sd (n = 3). means in the column with no common letter differ significantly (p<0.05) for each leafy vegetable. the moisture contents ranged between 5.55-12.07 % after 3 days. moisture generally refers to the presence of water, often in trace amounts [25]. high moisture content in vegetables is indicative of freshness as well as easy perishability [26]. higher moisture content of vegetables also suggests that the vegetable could not be stored for long time without microbial spoilage [27,28]. during drying, warm temperatures cause the moisture to move quickly from the food to the air [29].the low moisture content of the dried vegetables makes them suitable for longer storage period [25]. ash is defined as the inorganic residue remaining after the water and organic matter [30,31]. in this study, ash content was found to be in the range of 1.34-5.20 % (fresh leaves) and 10.12-25.78 % (dried leaves) after 3 days. these results suggest that the dehydration could retain more minerals which may be benefit for consumers. indeed, it has been reported that leaves should contain 3 % ash are considered as beneficial human foods [32].the values of 1 day 76.08 ± 2.72b 5.09 ± 0.02c 4.46 ± 0.00c 3.26 ± 0.06c 2.03 ± 0.00c 9.05 ± 0.70c 57.36 ± 0.13c 2 days 31.52 ± 1.37c 15.58 ± 0.01b 16.07 ± 0.00b 12.84 ± 0.03b 8.43 ± 0.00b 15.52 ± 0.50a 157.44 ± 0.06b 3 days 12.07 ± 0.29d 22.36 ± 0.00a 23.83 ± 0.00a 17.24 ± 0.00a 14.24 ± 0.00a 10.21 ± 0.10b 197.79 ± 0.00a s. melongena raw 74.38 ± 0.72a 5.20 ± 0.01d 3.50 ± 0.00d 3.16 ± 0.00d 0.69 ± 0.00d 13.04 ± 0.02d 71.11 ± 0.09c 1 day 17.71 ± 0.65b 17.69 ± 0.00c 12.07 ± 0.00c 14.06 ± 0.01c 2.44 ± 0.00c 36.59 ± 0.05a 185.45 ± 0.02b 2 days 13.01 ± 1.93c 20.29 ± 0.01b 13.59 ± 0.02b 17.27 ± 0.04b 6.38 ± 0.00b 29.42 ± 0.08b 200.64 ± 0.03a 3 days 8.43 ± 0.90d 24.56 ± 0.00a 17.01 ± 0.00a 19.90 ± 0.01a 9.17 ± 0.00a 20.88 ± 0.50c 199.98 ± 0.03a t. triangulare raw 90.20 ± 0.21a 2.17 ± 0.00d 1.37 ± 0.00d 1.68 ± 0.00d 0.48 ± 0.00d 5.17 ± 0.01c 26.58 ± 0.01d 1 day 65.77 ± 0.57b 8.47 ± 0.00c 4.98 ± 0.00c 6.01 ± 0.00c 1.75 ± 0.00c 12.98 ± 0.05b 75.79 ± 0.00c 2 days 28.43 ± 1.27c 19.28 ± 0.00b 11.00 ± 0.00b 13.21 ± 0.00b 5.51 ± 0.00b 22.53 ± 0.70a 158.9 ± 0.02b 3 days 9.96 ± 0.30d 25.78 ± 0.00a 16.50 ± 0.00a 18.17 ± 0.00a 7.33 ± 0.00a 22.22 ± 0.02a 185.12 ± 0.00a c. olitorius raw 84.28 ± 0.34a 1.34 ± 0.00d 1.80 ± 0.00d 3.32 ± 0.00d 0.51 ± 0.00d 8.73 ± 0.01c 43.60 ± 0.00d 1 day 30.40 ± 4.03b 6.22 ± 0.00c 8.84 ± 0.00c 15.70 ± 0.00c 2.30 ± 0.00c 36.50 ± 0.50b 187.94 ± 0.00c 2 days 14.93 ± 4.82c 8.26 ± 0.00b 13.16 ± 0.00b 20.15 ± 0.00b 4.57 ± 0.00b 38.89 ± 0.50a 226.33 ± 0.00b 3 days 5.55 ± 0.30d 10.12 ± 0.00a 16.64 ± 0.00a 23.68 ± 0.00a 6.28 ± 0.00a 37.68 ± 0.01a 244.95 ± 0.00a food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 261 fibre contents ranged from 4.46 ± 0.00 % to 12.07 ± 0.00 after 1 day and from 16.50 ± 0.00 % to 29.81 ± 0.01 % after 3 days of sun-drying. the higher fibre contents may be advantageous since their consumption could enhance digestion and prevent constipation. high crude fibre in the vegetable according could also help in blood cholesterol attenuation, as well as blood glucose attenuation when consumed [33,34,35]. the protein contents in the five dehydrated samples were in the ranged of 3.26-5.72 %, 12.84-20.15 % and 16.6723.68 % after 1, 2 and 3 days respectively. the sun drying method increased protein content of the studied vegetables compared to their controls. the increase in protein was due to loss of moisture during drying processing. many workers had reported similar phenomenon [36]. it is known that loss of moisture increases nutrient content and extends keeping quality of the food [37]. protein helps in building and maintaining all tissues in the body, forms an important part of enzymes, fluids and hormones of the body [38]. plant proteins may be less digestible because of intrinsic differences in the nature and the presence of other factors such as fibre, which may reduce protein digestibility.nevertheless, dietary studies show the adequacy of plant foods, as sole sources of protein [39]. the lipids contents after 3 days of drying were in the range 6.28-14.24 %. the lipid content of dried leaf samples were also higher than their fresh counter parts but leafy vegetables could not be considered as rich source of fat [40, 41]. the relatively low lipid content of the dried vegetables makes them suitable for people who suffer from heart related diseases [25]. vegetables in their fresh state have been noted to be poor sources of carbohydrate [40,42,43]. however, after drying, carbohydrate content increased and varied from 10.21 to 37.68 % after 3 days of sun drying. carbohydrates are the most important food energy provider among the macronutrients, accounting for between 40 and 80 percent of total energy intake [44,45]. the low caloric values obtained in this study could be explained to low proteins, lipids and total carbohydrate contents. the result of anti-nutritional factors (oxalates and phytates) contents of the sun dried leafy vegetables were presented in fig.1. the values ranged within 123.01-815.97 mg/ 100 g and 0.65-3.60 mg/100 g after 3 days of sun drying for oxalates and phytates, respectively. contrary to the phytate contents, oxalates content increased with sun drying time compared to their controls. oxalates and phytates are considered as anti-nutritionnal factors because of their ability to chelate minerals such as calcium, iron, magnesium and zinc [46,47]. mineral composition: sun drying method had concentration effect on mineral composition of leafy vegetables consumed in southern côte d’ivoire (table 2). levels minerals contents were as follow: calcium (82.86-481.65 mg/100 g), magnesium (81.98-298.46 mg/100 g), phosphorus (63.41-297.69 mg/100 g), potassium (419.81-993.41 mg/100 g), iron (20.0590.37 mg/100 g), sodium (19.43-150.51 mg/100 g) and zinc (15.76-64.39 mg/100 g) after 3 days. with regards to the recommended dietary allowances (rda) as mg/day/person for minerals, the level of iron and zinc in the samples could cover rda and contribute substantially for improving human diet [48,49,50]. iron is known to be an essential part of red blood cells (haemoglobin) and enzymes (cytochromes) and consumption of these leafy vegetablescould reduce considerably the risk of anaemia [51]. to predict the effect of phytates and oxalates on food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 262 nutrients, anti-nutritional factors/minerals ratios were calculated (table 3). fig.1. effect of sun drying on oxalate (a) and phytate (b) contents of leafy vegetables consumed in southern côte d’ivoire the calculated phytates/ calcium, phytates/iron and oxalates/calcium ratios of the studied leafy vegetables were below the critical level of 0.5, 0.4 and 2.5, respectively [44]. this implies that phytates and oxalates contents of the dried leaves would have deleterious effects on human nutrition. table 2. mineral composition of sun dried leafy vegetables consumed in southern côte d’ivoire ca mg p k fe na zn c. esculenta raw 103.64 ± 0.01c 61.29 ± 0.00d 139.08 ± 0.02d 402.70 ± 0.06d 25.30 ± 0.00d 6.96 ± 0.00d 6.58 ± 0.00d 1 day 247.21 ± 0.02b 150.70 ± 0.02c 238.91 ± 0.02c 562.68± 0.03c 63.43 ± 0.01c 10.84 ± 0.02c 10.43 ± 0.01c 2 days 302.53 ± 0.02a 156.58 ± 0.01b 274.64 ± 0.01b 577.03 ± 0.02b 67.71 ± 0.01b 18.16 ± 0.01b 10.92 ± 0.00b 3 days 302.13 ± 0.05a 171.21 ± 0.04a 297.69 ± 0.04a 628.14 ± 0.04a 73.98 ± 0.00a 19.43 ± 0.02a 15.76 ± 0.01a b. alba raw 76.38 ± 0.00d 76.74 ± 0.00d 39.71 ± 0.00d 275.31 ± 0.02d 7.88 ± 0.00d 56.50 ± 0.07d 6.84 ± 0.00d food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 263 1 day 102.47 ± 0.07c 81.83 ± 0.09c 95.74 ± 0.05c 349.64 ± 0.06c 10.26 ± 0.00c 84.95 ± 0.08c 17.46 ± 0.03c 2 days 135.49 ± 0.03b 105.44 ± 0.05b 104.77 ± 0.02b 387.98 ± 0.04b 19.64 ± 0.00b 101.08 ± 0.05b 19.07 ± 0.02b 3 days 175.16 ± 0.00a 107.95 ± 0.00a 136.63 ± 0.40a 419.81 ± 0.00a 20.05 ± 0.00a 135.90 ± 0.01a 27.59 ± 0.00a s. melongena raw 204.07 ± 0.00d 123.46 ± 0.00d 95.93 ± 0.00d 578.01 ± 0.01d 35.73 ± 0.00d 82.78 ± 0.02d 16.58 ± 0.00d 1 day 380.48 ± 0.01c 208.71 ± 0.00c 132.28 ± 0.01c 883.38 ± 0.01c 60.63 ± 0.01c 92.47 ± 0.01c 31.65 ± 0.00c 2 days 423.80 ± 0.07b 269.47 ± 0.02b 168.66 ± 0.04b 893.15 ± 0.04b 66.66 ± 0.00b 103.57 ± 0.05b 45.71 ± 0.00b 3 days 481.65 ± 0.02a 298.46 ± 0.01a 171.56 ± 0.02a 993.41 ± 0.02a 90.37 ± 0.01a 150.51 ± 0.01a 64.39 ± 0.00a t. triangulare raw 58.93 ± 0.00d 74.08 ± 0.00d 23.47 ± 0.00d 495.21 ± 0.00d 10.02 ± 0.00d 25.50 ± 0.00d 3.53 ± 0.00d 1 day 69.81 ± 0.02c 80.76 ± 0.00c 33.41 ± 0.00c 637.37 ± 0.02c 37.10 ± 0.00c 32.75 ± 0.01c 16.74 ± 0.00c 2 days 70.27 ± 0.03b 84.90 ± 0.03b 48.38 ± 0.00b 656.79 ± 0.04b 42.94 ± 0.01b 38.13 ± 0.02b 23.27 ± 0.01b 3 days 82.86 ± 0.00a 92.22 ± 0.00a 63.41 ± 0.00a 672.83 ± 0.01a 46.93 ± 0.00a 47.39 ± 0.00a 29.51 ± 0.00a c. olitorius raw 58.00 ± 0.00d 36.86 ± 0.00d 49.80 ± 0.00d 412.26 ± 0.05d 15.34 ± 0.00d 4.36 ± 0.00d 3.88 ± 0.00d 1 day 78.96 ± 0.00c 54.45 ± 0.00c 58.38 ± 0.00c 447.80 ± 0.00c 26.03 ± 0.00c 19.07 ± 0.00c 5.59 ± 0.00c 2 days 83.37 ± 0.02b 63.69 ± 0.00b 63.02 ± 1.35b 473.83 ± 0.03b 33.44 ± 0.00b 25.86 ± 0.01b 15.77 ± 0.00b 3 days 86.83 ± 0.01a 81.98 ± 0.00a 72.20 ± 0.00a 549.90 ± 0.01a 42.61 ± 0.00a 37.82 ± 0.00a 30.24 ± 0.00a data are represented as mean ± sd (n=3). means in the column with no common letter differ significantly (p<0.05) for each leafy vegetable table 3. anti-nutritional factors/mineral ratios of sun dried leafy vegetables consumed in southern côte d’ivoire phytates/ca phytates/fe oxalates/ca c. esculenta raw 0.04 0.18 0.99 1 day 0.03 0.11 0.99 2 days 0.01 0.05 1.04 3 days 0.01 0.01 1.04 b. alba raw 0.03 0.26 0.87 1 day 0.02 0.16 0.91 2 days 0.01 0.06 0.91 3 days 0.01 0.02 0.94 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 264 s. melongena raw 0.05 0.30 0.12 1 day 0.02 0.13 0.13 2 days 0.01 0.04 0.14 3 days 0.01 0.01 0.16 t. triangulare raw 0.05 0.29 0.86 1 day 0.02 0.13 0.87 2 days 0.01 0.04 0.91 3 days 0.01 0.03 0.94 c. olitorius raw 0.11 0.40 2.11 1 day 0.04 0.15 1.79 2 days 0.01 0.05 1.75 3 days 0.01 0.03 1.76 antioxidant properties: antioxidantcomponentsare substances that may protect cells from the damage caused by molecules known as free radicals. most common antioxidants in vegetables and spices are vitamin c, e, phenolic compounds and carotenoids [52]. vitamin c and carotenoids contents of the studied leafy vegetables are shown in fig. 2. fig.2. effect of sun drying on vitamin c (a) and carotenoids (b) contents of leafy vegetables consumed in southern côte d’ivoire food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 265 vitamin c losses were estimated to 85.1296.42 %, after 1 day of sun drying. this decrease could be explained by the fact that vitamin c is subjected to oxidation by exposure to sunlight [41]. carotenoids are important precursors of retinol (vitamin a) and they have also been studied for their potential protection against numerous cancers [52,53]. after sun drying processing, carotenoids contentsdecreased in all the analyzed samples. carotenoid losses were estimated to 97.4898 % and 99.07-100 % after 1 day and 3 days of sun drying respectively. the drying techniqueused in this work involved subjecting the vegetables to heat, light and oxygen and all of these factors will accelerate the rate of oxidation of carotenoids[54]. phenolic coupounds and antioxidant activity of the studied leafy vegetables are shown in fig.3. fig.3. effect of sun drying on polyphenols c (a) and antioxidant activity (b) contents of leafy vegetables consumed in southern côte d’ivoire phenolic agents are major class of antioxidants that are found in plant foodswith relatively high concentration[55, 56]. the phenolic contents of the samples increased during sun drying. the values were in the range of 35.44-191.01 mg/100 g and 198.53-388.69 mg/100 g at 1 day and 3 days, respectively. phenolic compounds are secondary metabolites synthesized by plants, both during normal development and in response to stress conditions (infection, wounding, uv radiation and others) [57]. they also have antioxidant properties that enable them to food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 266 quench free radicals in the body [58]. phenolic compounds have potentially beneficial effect on human health by reducing the occurrence of coronary heart disease, age-related-eye diseases and atherogenic processes [59, 60]. moreover, this increase in phenolic contents caused the increase of antioxidant activity because there is a direct correlation between the concentration of antioxidant compounds and the antioxidant activity [61]. antioxidant activity ranged from 77.36 ± 0.00 % to 82.30 ± 0.00 % after 3 days of sun drying. this increase could to be advantageous from the consumers because antioxidants prevent many diseases [62]. 4. conclusion the results of the present study showed that basella alba, colocasia esculenta, solanum melongena, talinum triangulare and corchorius olitorus consumed as leafy vegetbles in southern côte d’ivoire are good sources of nutrients. it was also observed that sun-drying was the method used to process leafy vegetables for long term preservation by decreasing moisture. moreover, this technique resulted in concentration of nutrients content of the vegetables (protein, ash, fat, crude fiber and carbohydrates). however, factors such as heat, light and oxygen caused the decreaseof vitamin c and carotenoids. a comparaison with the major methods as solar drying, shadow drying and oven drying must be studied to determine the best preservation method of leavy vegetables. 5. references [1]. obel-lawson e. the contribution of the awareness campaign of the african leafy vegetables project to nutrition behaviour change among the kenyan urban population: the case of nairobi. thesis, university of nairobi, kenya. 161 p. (2005). [2]. abugre c. assessment of some traditional leafy vegetables of upper east region and influence of stage of harvest and drying method on nutrients content of spider flower (cleome gynandral.). thesis, kwamenkrumah university of science and technology, kumasi, ghana, pp. 136. (2011). [3]. subhash b. k., neeha v.s. dehydration of green leafy vegetable: review. (2014). [4]. kubo, i., fijita, k., kubo, a., nehi, k., gura, t. antibacterial activity of coriander volatile compounds against salmonella choleraesuits. journal of agricultural and food chemistry. vol. 52, 33293332. (2004). [5]. raju, m., varakumar, s., lakshminarayany. r., krishnakantha, t.p., baskaran, v. carotenoid composition and vitamin a activity of medicinally important green leafy vegetables. food chemistry. vol. 101, 15981605. (2007) [6]. kachik, f., mudlagiri, b.g., gary, r.b., joanne, h., lusby, w.r., maria, d.t., barrera, m.r. effects of food preparation on qualitative and quantitative distribution of major carotenoids constituents of tomatoes and several green vegetables. journal of agricultural and food chemistry. vol. 40, 390-398. (1992) [7]. yadav, s.k., sehgal, a. effect of home processing on ascorbic acid and beta carotene content of bathua (chenopodium album) and fenugreek (trigonellafoenungraecum) leaves. plant food for human nutrrition. vol. 50, 239-247. (1997) [8]. bukola, o. bolaji, tajudeen, m.a., olayanju, taiwo, o. falade. performance evolution of a solar wind ventilated cabinet dryer. west indian journal of engineering. vol. 33, 12-18 (2011). [9]. satwase, a. n., pandhre, g. r., sirsat, p.g., wade, y.r. studies on drying characteristic and nutritional composition of drumstick leaves by using sun, shadow, cabinet and oven drying methods. vol. 2, 584 doi:10.4172/scientificreports.584. (2013) [10]. cnra. socio-economical importance of leafy vegetables for the urban populations of côte d’ivoire, cnra ed., 2011 (2011). [11]. kouame n. m. contribution à l’étude des plantes spontanées alimentaires du department d’oumé (côte d’ivoire). mémoire de d.e.a d’ecologie tropicale. université de cocody-abidjan, côte d’ivoire, pp. 122 (2000). [12]. fondio l., kouamé c., n’zi j. c., mahyao a., agbo e., djidji a. h. survey of indigenous leafy vegetables in the urban and periurban areas of côte d’ivoire. in: m. l. chadha et al. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 267 (eds). indigenous vegetables and legumes: prospects for fighting poverty, hunger and malnutrition. acta horticulturae. vol. 752, 287-289 (2007). [13]. n’dri m. t., kouame g. m., konan e., traore d. plantes alimentaires spontanées de la région du fromager (centre-ouest de la côte d’ivoire) : flore, habitats et organes consommés. science of natural. vol.1, 61-70 (2008). [14]. acho f.c., zoué l.t., akpa e.e., yapo v.g., niamkél.s. 2014. leafy vegetables consumed in southern côte d’ivoire: a source of high value nutrients. journal animal and plant sciences. vol. 20, 3159-3170 (2008). [15]. mepba h.d., eboh l., banigo d. e.b. effects of processing treatments on the nutritive composition and consumer acceptance of some nigerian edible leafy vegetables. african journal of food, agricultural, nutrition and development. vol. 7, 1 (2007). [16]. aoac. official methods of analysis. association of official analytical chemists ed., washington dc. 684 p (1990). [17]. fao. food energy-methods of analysis and conversion factors. fao ed, rome. 97 p (2002). [18]. ceaeq, détermination des métaux. méthode par spectrométrie de masse à source ionisante au plasma d’argon. ma 200 – met 1.2, rev 4. quebec, 2013, 24 p. (2013). [19]. day r.a., underwood, a.l. quantitative analysis. 5th ed. prentice hall. 701 p. (1986). [20]. latta m., eskin m. a simple method for phytate determination. journal of agricultural and food chemistry. vol. 28, 1313-1315.(1980) [21]. pongracz g., weiser h., matzinger, d. tocopherolsantioxydant. fat science and technology. vol. 97, 90-104 (1971). [22]. rodriguez-amaya d.b. a guide to carotenoids analysis in foods. ilsi press, washington dc. 64 p (2001). [23]. singleton v.l., orthofer r., lamuela-raventos r.m. analysis of total phenols and other oxydant substrates and antioxydants by means of folin-ciocalteu reagent. methods enzymol.,vol. 299, 152-178 (1999). [24]. choi c.w., kim s.c., hwang s.s., choi b.k., ahn h.j., lee m.z., park s.h., kim s.k. antioxidant activity and free radical scavenging capacity between korean medicinal plant and flavonoids by assay guided comparison. plant sciences. vol.163, 1161-1168 (2002). [25]. seidu j. m., bobobee e.y. h., kwenin w. k. j., frimpong r., kubge s. d., tevor w. j., mahama a. a. preservation of indigenous vegetables by solar drying. journal of agriculture and biology sciences. vol. 7, 407-415 (2012) [26]. adepoju o. t., oyewole o. e. nutritional importance and micronutrient potential of two non-conventional indigenous green leafy vegetables from nigeria. agricultural journal. vol. 5, 362-365 (2008). [27]. ejoh r. a., nkonga d. v., inocent g., moses m. c. nutritional components of some nonconventional leafy vegetables consumed in cameroon. pakistan journal of nutrition. vol. 6, 712717 (2007) [28]. ladan, m.j., abubakar m.g., lawal m. effect of solar drying on the nutrient composition of tomatoes. nigeria journal renew energy. vol. 5, 67-69 (1997). [29]. harrison j. a., andress e. l. preserving food: drying fruits and vegetables. university of georgia cooperative service. page 2. (2000). [30]. mcclements d. j. analysis of food products. chenoweth lab, room 238. www.unix.oit.umass.edu/~mcclemen/581rheology. html. (accessed on 20 may 2015) (2003). [31]. ekpe o. o., umoh i. b., eka o. u. effect of a typical rural processing method on the proximate composition and amino acid profile of bush mango seeds (irvingiagabonensis )african journal of food, agriculture, nutrition and development. vol. 7, 12 p. (2007). [32]. pivie, n.w., butler j.b. a simple unit leaves. proceedings of the nutrition society. vol. 36, 136-139. (1977). [33]. oguche g.h.e. effect of drying methods on chemical composition of spinach “aieifo” (amaranthusaquatica) and pumpkin leaf (telfairiaoccidentalis) and their soup meals. pakistan journal of nutrition. vol. 10, 1061-1065 (2011). [34]. cfw. all dietary fiber is fundamentally functional. c. f. w. aacc report. publication no. w2003-0407-01o. 128 / may-june, 2003, vol. 48, 3. www.aaccnet.org/news/pdfs/dfreport.pdf. (accessed in may, 2015) (2003). [35]. komal m., kaur a. reviews: dietary fibre. dieticians, adipostat clinic, 103-104, lady ratan tata medical centre, bombay 400 021. int. j. diab. dev. countries, vol. 12, 12-18. (1992) [36]. elegbede j.a. legumes. nutritional quality of plant foods. in: post harvest research unit. osagie au, eka, qu (eds). fakeye io (2009). nigerian leafy vegetable, nigerian journal of food sciiences. vol. 1, 55 (1998) [37]. osagie, a.u., onigbide a.o. effect of growth, maturation and storage on the composition of http://www.unix.oit.umass.edu/~mcclemen/581rheology.html http://www.unix.oit.umass.edu/~mcclemen/581rheology.html food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 3 – 2015 constant acho , lessoy zoue, niamkey adom , sébastien niamke, effect of sun drying on nutritive and antioxidant properties of five leafy vegetables consumed in southern côte d’ivoire, food and environment safety, volume xiv, issue 3 – 2015, pag. 256 – 268 268 plant foods. nutrition quality of plant foods. pp: 214-216. (1992) [38]. johnson, r.s. ‘key nutrients’, cooperative extension service, iowa state university of science and technology, ames, iowa available at http://www.scribd.com/doc/6646054/keynutrients (accessed on 23 may 2015) (1996). [39]. afpa. human protein requirements. www.afpafitness.com (accessed in july, 2015). (2010). [40]. uwaegbute a. c.. vegetables nutrition and utilization. in: b.n. mba and d.o. nnanyelugo (eds). food crops production, utilization and nutrition. dotam publ. ltd. ibadan, pp: 145-153 (1989). [41]. joshi p., mehta d. effect of dehydration on the nutritive value of drumstick leaves. journal of metabolomics systems biologica. vol.1 : 5-9 (2010) [42]. kolawole o.m., ayiboye a.e., aturu e.e., anibijuwun i.i. effect of solar drying on the proximate and microbial composition of abelmoschusesculentus. journal of microbial and biotechnology research. vol. 1, 71 – 81 (2011). [43]. rossello, c., berna, a., mulet, a. solar drying of fruits in a mediterranean climate. drying technology, vol.8, 305 – 321 (2000). [44]. hassan, s.w., umar, r.a., maishanu, h.m., matazu, i.k., faruk, u.z., sani, a.a. the effects of drying method on the nutrients and nonnutrients composition of leaves of gynandropsisgynandra (capparaceae). asian journal of biochemistry. vol. 5, 349-353 (2007). [45]. dga. dietary guidelines for americans. www.healthierus.gov/dietaryguidelines. (accessed in 19 may, 2015). (2005) [46]. oguche, g.h.e. the effect of sun and shade drying on chemical composition of vitexdoniana, ipomoea aquaticaand cohcorusand their soups. international journal of nutrition and metabolism. vol 4, 121-129. (2012). [47]. vainio, h., bianchini, f. irrc hand book of cancer-prevention fruit and vegetables vc/8lyon, france, pp. 27-30 (2003). [48]. fao. human vitamin and mineral requirements. fao. ed. pp: 361 (2004). [49]. nas. national academy of sciences, dietary references intake (dris): recommended intakes for individuals, elements. food and nutrition board, institute of medicine, national academies. available online at: http//www.nap.edu (accessed in may 2015) (2004). [50]. fnd. dietary reference intake for energy, carbohydrate, fibre, fat, fatty acids, cholesterol, protein and amino acid (micro-nutrients). food and nutrition board, www.nap.edu. (accessed in may 2015) (2005). [51]. soetan, k. o., olaiya c. o., oyewole o. e. the importance of mineral elements for humans, domestic animals and plants: a review. african journal of food sciences.vol. 4, 200-222 (2010). [52]. priecina, l., karklina, d. natural antioxidant changes in fresh and dried spices and vegetables. international journal of biology veterinary, agricultural and food engineering. vol. 8, 480-484 (2014). [53]. rickman, c.j., barrett, m.d., bruhn, m.c. nutritional comparison of fresh, frozen and canned fruits and vegetables. part 1. vitamins c and b and phenolic compounds. journal of the sciences of food and agriculture. pp: 15. (2007). [54]. mcdowell, r.l. vitamin in animal nutrition, comparative press inc; new york. nutrition journal. pp. 365 – 427 (1989). [55]. larrosa, m., liorach, r., espin, j.c., tomás-baraberán, f.a. increase of antioxidant activity of tomato juice upon functionalisation with vegetable by product extracts. lebensm-wiss. utechnology.vol. 35, 532-542 (2002). [56]. robards, k. strategies for the determination of bioactive phenols in plants, fruit and vegetables. journal of chromatography a. pp. 657-691 (2003). [57]. naczk, m., shahidi e. “phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis”. journal of pharmaceutical and biomedical analysis. vol. 41, 1523-1542 (2006). [58]. norman, j.t., kerri, k.g. fruit, vegetables and the prevention of cancer: research challenges. nutrition. vol.19, 467-470 (2003). [59]. morton, l.w. caccetta, r.a.a. puddey, i.b., croft, k.d. chemistry and biological effects of dietary phenolic compounds: relevance to cardiovascular disease. clinical and experimental pharmagology and physiology. vol. 27, 152-159 (2000). [60]. john, s., haseeb, n., joseph, p., gauri, m., yukio, k., yueming, j. extraction polyphenolics from plant material for function foodsengineering and technology. food review international. vol. 21, 139-166 (2005). [61]. lan, w. effect of chlorogenic acid on antioxidant activity of floslanicerae extracts. journal of zhejiang university of sciences biological. vol 8, 673-679 (2007). [62]. islam, s. m., yonshinioto, m., yahara, s., okuno, s., ishiguro, k., yamakawa, o. identififcation and characterization of folialpolypheolic composition in sweet potato (ipomeabatatas l.) genotypes. journal of agriculture and food chemistry. vol. 50, 3718-3722 (2002). http://www.scribd.com/doc/6646054/keyhttp://www.nap.edu/ 34 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017, pag. 34 39 determination of chlorpyrifos in apple from the resen region * vezirka jankuloska1, ilija karov2, gorica pavlovska1, ilija buzlevski3 1faculty of technology and technical sciences, veles, university “st. kliment ohridski”, bitola, dimitar vlahov 57, 1400 veles, republic of macedonia, gorica.pavlovska@gmail.com, 2faculty of agriculture,"goce delcev" university , krste misirkov 10-a posh. box 201, 2000 shtip, republic of macedonia, ilija.karov@ugd.edu.mk, 3center for healthy food“ in-10” mite bogoevski 37, 7310 resen, republic of macedonia, ilij_buzle@hotmail.com *corresponding author: vezirka.jankuloska@gmail.com received february 15th 2017, accepted march 27th 2017 abstract: the aim of this research is to determine the presence of chlorpyrifos in two varieties of apples: golden delicious and idared in two different locations (evla and krveni) from the resen region in the country. chlorpyrifos is organophosphate pesticide (insecticide) used to protect apples from insects which can cause significant damage in apple production. the apples are analyzed in four development phases and in each phase the presence of chlorpyrifos is determined. chlorpiryfos analysis of the apples is performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) followed by an extraction/separation cleansing with acetonitrile and the dispersive spe quechers method. the results are compared with the maximum residue levels (mrl) prescribed by the legislation of the republic of macedonia. the conclusion is that chlorpyrifos is variously represented in most phases, regardless of the phase and location where the apple is grown. at certain phase the concentration exceedes mrl, however during the harvest, apples are safe to be consumed. higher presence of chlorpyrifos is found in idared apples at kriveni location as compared to evla. тhe presence of chlorpyrifos in golden delicious is almost equal in both locations. from the statistical analysis of the data it is noticeable that the use of chlorpyrifos does not depend on the (type) variety of apple but rather on the location where the apple is grown. keywords: chlorpyrifos, pesticides, apples, golden delicious, idaret 1. introduction the apple is the most important deciduous fruit consumed during a year. its fruits can be frozen or used for various types of products (juice concentrate, apple acid and etc.) [1]. in the republic of macedonia, the most common fruits are: apples with about 62%, 13% plums, cherries 7%, peaches 7%, table grapes 7% and other types of fruit (pears, apricots, almonds, walnuts, etc.) with about 4% [2]. domestic consumption of apples is 12 kg per citizen [3]. it is estimated that about 25% of the total production of apples is delivered to domestic market while the remaining 75% is processed or sold on foreign markets [2]. fruit production occupies about 2.75% of the arable agricultural land, about 14 000 ha with 9 000 000 trees concentrated in regions at an altitude of 300-800 m (including the resen region). in macedonia, the total number of trees apple is 4 082 328, fruitful trees 3 811 548 with total production of 136 931 t or 36 kg per tree. in resen, the number of fruit trees is 2 458 300, out of which fruitful trees are 2 256 800 while the apple production is of 90 450 tons of apples or 40 kg of apples per tree [3]. this shows that apple is mostly cultivated in resen. it is said that apple is a measure for the development of fruit growing in every country, since increasing the number of apple trees it increases the intensity of fruit growing production, globally and conversely [4]. in http://www.fia.usv.ro/fiajournal mailto:gorica.pavlovska@gmail.com, mailto:ilija.karov@ugd.edu.mk, mailto:ilij_buzle@hotmail.com mailto:vezirka.jankuloska@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski, determination of chlorpyrifos in apple from resen region, food and environment safety, volume xvi, issue 1 – 2017, pag. 34 – 39 35 resen the following varieties of apples are being grown: idared, golden delicious, mutcu, red delicious, jonagold, granny smith, fuji, gala and rubistar [5]. idared is a variety of apple grown in moscow, idaho, by lejf verner. it is a diploid and secondary lush variety, blooming early. the fruit is coarse to very coarse, with a round shape. the epidermis is firm, elastic and smooth. the main color is green to red. the inner part of the fruit is white, mixed with chippings of green color, firmly, crispy, with good structure, juicy and sweet. it is harvested from late september to early november [6]. golden delicious is diploid, secondary lush variety of apples. its blooming lasts long and late, being harvested very late. the fruits are medium large, round, have greenish yellow surface mulch, which turns to golden yellow color at stage of maturity. it is harvested in the second half of september and can be stored until the end of may [6]. producers of apples in resen are facing with different kinds of pests in their fruit tree plantations that cause major economic damage. the most important apples pests are: codling moth (cydiapomonella), apple green aphid (aphis pomi) and phytophagous spiders, european red mite (panonychus ulmi) and red spider mite (tetranychus urticae) [4]. to protect apples from various diseases pesticides are used. pesticides are chemical compounds with active substance affecting the suppression of the pest and cause of the disease. poulsen et. al., in 2009 pointed out that pesticides are used in the production of fruit, vegetables and cereals [7]. residuals of pesticides (residues) must be monitored and controlled. the european commission has adopted a list of maximum residue levels (mrl) of pesticides used in food production and animal feed regulated by regulation ec 396/2005 [8]. maximum residues levels of certain substances (pesticides, mycotoxins, heavy metals and other contaminants) in the republic of macedonia are given in the regulation on general requirements for food safety [9]. chlorpyrifos (o, o diethyl o 3, 5, 6trichloro – 2 pyridyl phosphorothioate) is organophosphate pesticide and is used as an insecticide in order to increase the yield of agricultural products. chlorpyrifos is mostly used in the following crops: cotton, corn, almonds, vegetables and fruits, especially oranges and apples [10]. chlorpyrifos is moderately toxic to humans. poisoning by chlorpyrifos can have a major impact on the central nervous system, cardiovascular system and respiratory system [11]. it can cause irritation to skin and eyes. symptoms of acute and chronic exposure include: numbness, tingling, incoordination, headache, dizziness, nausea, abdominal cramps, sweating, blurred vision, difficulty in breathing or respiratory depression, and slow heartbeat. very high doses may result in unconsciousness, incontinence, and convulsion fatality. integral protection of apples in recent years has gained in importance because there are major advantages. it involves using natural enemies to eliminate use of selective insecticides, which preserve the environment and the production of food safety [4, 12]. student t-test is the most often used parametric test of significance testing а statistical significant difference between two arithmetic averages. the interpretation of the obtained values of the t-test is based on student t-distribution with a number of degrees of freedom and table of critical values of t-distribution. if statistical t-value < tcritical value (ss and 0.05), the null hypothesis (ho) is accepted and rejecting the alternative hypothesis (ha), of p = 0.05; p = 0.95 (95%). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski, determination of chlorpyrifos in apple from resen region, food and environment safety, volume xvi, issue 1 – 2017, pag. 34 – 39 36 the samples are statistically significantly different if the obtained p (probability) value is less than the p critical value. if the obtained p-value is greater than or equal to the limit p-value, it says that the samples do not differ significantly. in the first case, the null hypothesis is rejected, while the second is accepted [21, 22]. we made student t-test for the following hypothesis: does the presence of chlorpyrifos in both varieties of apples taken from the kriveni and evla are significantly different? the aim of our research is to analyze this pesticide in two varieties of apples, in two different locations. 2. matherials and methods for the analysis two varieties of apples were taken: golden delicious and idared. samples were collected from two different locations (evla and kriveni) in four developmental phases of apples: i phase, apple at the size of hazelnuts; ii phase apple at the size of a walnut; iii phase, early ripening of apple; iv phase during the apple harvest. fresh apples with peel and mesocarp were analyzed. the sampling dates for analysis are the following: 31. 05. 2016, 09. 07. 2016, 21. 08. 2016, 25. 09. 2016 for each phase. lc-ms/ms has become the most frequently used analytical method for quantification of polar pesticides in food [13-17]. chlorpyrifos analysis was performed with modern methods lcms/ms following acetonitrile extraction/partitioning and clean-up by dispersive spe quechers-method with mks en 15662: 2011 method. this method is accredited in a flexible range of the institute for accreditation of the republic of macedonia (iram) for mks en iso/iec 17025 : 2006 under number lt – 036 [18]. the quechers method uses acetonitrile, with the application of adequate combination of salts, dinatrium hidrogen citrata sesquihydrate, water-free magnesium sulfate and sodium chloride with the purification procedure by primary-secondary amine (psa) with the addition of water-free magnesium sulfate which results in a better separation of phases without dilution [19, 20]. the homogenized sample of apple with weight of 10 g is transferred to a 50 ml centrifuge tube. 10 ml of acetonitrile is added and e.g. 100 μl of the internal standard solution, shaking vigorously for 1 min. this is the first extraction step. after extraction, in a vortex mixer for 1 minute, 4 g of magnesium sulfate anhydrous (mgso4), 1 g of sodium chloride (nacl), 1 g of trisodium citrate dehydrate and 0.5 g of disodium hydrogencitrate sesquihydrate were added and the mixture was shaken for 1 min and centrifuged for 5 minutes at 3000 rpm. this is the second extraction with phase separation. after centrifugation, 1 ml of the extract was transferred into a clean-up tube containing 900 mg of mgso4 and 150 mg of primary-secondary amine (psa). shake for 30 sec. centrifuge for 5 minutes at 4500 rpm. the cleaned and acidified extracts are transferred into autosampler vials to be used for the multiresidue determination. the analyses were carried out with a liquid chromatograph (lc) (agilent 6420) with triple quadrupole mass spectrometry. 3. results and discussion the results are presented in graphs and diagrams. in order to see if the insecticide chlorpyrifos in the analyzed fruit is within the limits, the obtained values are compared with the maximum residue levels (mrl) according to the legislation of the republic of macedonia [9]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski, determination of chlorpyrifos in apple from resen region, food and environment safety, volume xvi, issue 1 – 2017, pag. 34 – 39 37 fig. 1. the presence of chlorpyrifos in idared from evla and kriveni and compared with the mrl fig. 1 represents the content of chlorpyrifos in idared from two different locations. it is noticeable that chlorpyrifos at evla location is present only in the first phase. in this phase, its concentration is 0.52 mg / kg and it is slightly above the allowed values. in the other phases chlorpyrifos is not present or is below the limit of detection. at the location kriveni chlorpyrifos is more present than evla and it is present in all three phases at a concentration that is acceptable according to the legislation of the republic of macedonia. it is seen that during the harvest time, apples do not contain residues of chlorpyrifos. the presence of chlorpyrifos in golden delicious in two different locations is presented in fig. 2. fig. 2. the presence of chlorpyrifos in golden delicious from evla and kriveni and compared with the mrl in golden delicious of evla the chlorpyrifos is present in all four phases with the highest concentration of 0.19 mg / kg in the first phase and the lowest concentration of 0.03 mg / kg in the fourth phase, which is approximately 17 times less than the maximum allowed. at the location kriveni it is present in the first two phases. the concentration of chlorpyrifos is reduced from 0.28 mg / kg in the first phase and finds with 0.05 mg / kg in the second phase which is 10 times smaller than the maximum residue levels. in the third and fourth phase the chlorpyrifos is not present in the analyzed samples. we made a comparison of the presence of chlorpyrifos in both varieties of apples at both locations (figs. 3 and 4). from fig. 3 it may be noticed that in the location evla this pesticide is present in golden delicious in all phases and in idared only in the first phase. fig. 3. the presence of chlorpyrifos in idared and golden delicious from location evla fig. 4 shows that the chlorpyrifos is more present in golden deliciuous than in idared. from the diagrams can be noticed that chlorpyrifos in idared is more present at kriveni. the presence of chlorpyrifos in golden delicious is almost equal in both locations because it is present in all the phases. 0 0,5 1 1 phase 2 phase 3 phase 4 phase 0,52 0,13 0,04 0,11 0,5 0,5 0,5 0,5 apple idared evla kriveni mrl (mg/kg) mg/kg 0 0,2 0,4 0,6 1 phase 2 phase 3 phase 4 phase 0,19 0,05 0,13 0,03 0,28 0,05 0,5 0,5 0,5 0,5 apple golden delicious evla kriveni mrl (mg/kg) mg/kg 0 0,2 0,4 0,6 1 phase 2 phase 3 phase 4 phase m g/ kg location "evla" idaret golden delicious food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski, determination of chlorpyrifos in apple from resen region, food and environment safety, volume xvi, issue 1 – 2017, pag. 34 – 39 38 fig. 4. the presence of chlorpyrifos in idaret and golden delicious from location kriveni the obtained statistical values are presented in table 1. table1. representation of the parameters from student’s ttest statistical parameters idared golden delicious evla kriveni evla kriveni x 0.13 0.07 0.1 0.0825 t-test 0.534169 0.385922 t-critical value 3.182446 p-value 0.630237 0.725295 p critical value 0.025 table 1 presents the mean of value (x) on the concentration of chlorpyrifos shown in mg / kg in the two varieties of apples from both locations. the value of t-test for idared in both locations is 0.534169 and it is lower than the critical t-value 3.182446. the value of t-test for golden delicious from both locations is 0.385922 and is less than the critical value of t-test. that means that null hypotheses can be accepted and that there is a significant difference in the presence of chlorpyrifos in apples from both locations. in both varieties of apples p-values are greater than the critical p-value. that means that null hypothesis can be accepted but the difference between the samples is not statistically significant. 4. conclusions analyses were made on two varieties of apples: golden delicious and idared from two different locations kriveni and evla in the resen region. chlorpyrifos is detected by lc ms / ms and quechers extraction is applied with the standard method mks en 15662: 2011. analyses show that at evla chlorpyrifos is represented in the idared variety only in the first phase while in the other phases it is absent. in golden delicious at location evla chlorpyrifos is represented in all four phases but with approximately 17 times lower concentration than the maximum allowed in the fourth phase. at the location kriveni chlorpyrifos in golden delicious is present in both phases with allowed concentration, while in idared is present in all three phases. although chlorpyrifos is available in two varieties of apples they are safe for consumption. statistical analysis of data on apple from kriveni and evla showed that the use of chlorpyrifos depends on the variety of apple, but not on the location where it is grown. further investigation will be made on other varieties of apples as well as in other locations. 5. references [1]. nikolić d., fortić m., apple breeding in the world, proceedings of the ii conference "innovations in fruit growing", faculty of agriculture, department of pomology beograd, 523, (2009). [2]. facts and figures, ministry of agriculture, forestry and water management, 13-14, (2010). [3]. agriculture, orchards and vineyards, state statistical office, republic of macedonia, 53, (2015). [4]. miletić n., tamaš n., integrated apple pest and disease management, proceedings of the ii conference "innovations in fruit growing", 0 0,1 0,2 0,3 0,4 0,5 m g/ kg location "kriveni" idaret golden delicious food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 vezirka jankuloska, ilija karov, gorica pavlovska, ilija buzlevski, determination of chlorpyrifos in apple from resen region, food and environment safety, volume xvi, issue 1 – 2017, pag. 34 – 39 39 faculty of agriculture, department of pomology beograd, 95-106, (2009). [5].http://www.resen.gov.mk/default.aspx?lcid=2 62, accessed on 18.10.2016 [6]. jackson j., biology of apples and pears, cambridge university press, 22-42, (2003). [7]. poulsen e. m., naef a., christen d., rasmussen p., influence of different disease control pesticide strategies on multiple pesticide residue levels in apple, journal of horticultural science & biotechnology, 58–61, (2009). [8]. european commission., regulation (ec) no. 396/2005 of the european parliament and the council of 23 february 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending council directive 91/414/eec, (2005). [9]. list of permitted active substances, official gazette of the republic macedonia, no. 105, (2013). [10]. knezević z., serdar m., screening of fresh fruits and vegetables for pesticide residues on croatian market, food control, 20(4): 419-422, (2009). [11]. kumar p., singh s.p., shrikant k., madhukar d., analysis of buffalo liver samples for the presence of chloropyrifos residues by using hight performance liquid chromatography, turk. j.vet.anim.sci., (35)4: 219-226, (2011). [12]. fenik j., tankiewicz m., biziuk m., properties and determination of pesticides in fruits and vegetables, trends in analytical chemistry, (30 6: 814-826, (2011). [13]. ferrer j., garcia reyes j., mezcua m., thurman m. e., alba a., multi residue pesticide analysis in fruits and vegetables by chromatography – time – of – light mass spectrometry, journal of chromatography, 1-10 (2005). [14]. dasika r., tangirala s.,padmaja naishadham p., pesticide residue analysis of fruits and vegetables, journal of environmental chemistry and ecotoxicology, 4(2): 19-28, (2012). [15].chamkasem n., harmon t., ollis l., lee s., mercer g., analysis of 136 pesticides in avocado using a modified quechers method with lc-ms/ms and gc-ms/ms, j. agric. food chem., 61: 2315−2329, (2013). [16]. desmarchelier a., mulihid c., racault l., perring l., lancova k., analysis of patulin in pearand apple-based foodstuffs by liquid chromatography electrospray ionization tandem mass spectrometry, j. agric. food chem., 59: 7659–7665, (2011). [17]. sinhaa s., rao m., vasudev k., odetokunc m., a liquid chromatography mass spectrometry-based method to measure organophosphorous insecticide, herbicide and nonorganophosphorous pesticide in grape and apple sample, food control, 25: 636646, (2012). [18]. institute for accreditation of the republic of macedonia, mks en/iec 17025:2006 number lt – 036. [19]. vukovic, g., stereva d., bursic, v., mladenova, r., lazic, s. application of gcmsd and lc-ms/ms for the determination of priority pesticides in baby foods in serbian market, lwt-food science and technology, 49(2): 312319, (2012). [20]. anastassiades m.s., fast and easy multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce, j aoac int, 86(2): 412-431, (2003). [21]. newbold p., carlson w., thorne b. (2007).statistic for business and economist.pearson education, inc., new jersey, usa (2007). [22]. levine d., stephan d., krehbiel t., berenso m., statistics for managers using microsoft excel, pearson prentice-hall, new jersey, usa, (2008). http://www.resen.gov.mk/default.aspx?lcid=2 308 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 308 314 application of water quality index in the drinking water quality assessment of a southeastern nigeria river *emeka donald anyanwu 1 , chinonyerem sylva emeka 1 1college of natural sciences, department of zoology and environmental biology, michael okpara university of agriculture, umudike, nigeria, ekadon@yahoo.com. *corresponding author received 18th october 2019, accepted 27th december 2019 abstract: the physico-chemical parameters of ikwu river were studied between january and june 2016 in 3 stations to assess its drinking water quality using water quality index. weighted arithmetic water quality index (wawqi) method was used. thirteen parameters were evaluated which included temperature, ph, electrical conductivity (ec), dissolved oxygen, total dissolved solid (tds), chemical oxygen demand (cod), nitrate, alkalinity, phosphate, sulphate, chloride and calcium. the physicochemical parameters recorded were within acceptable limits except dissolved oxygen and ph. oneway analysis of variance (anova) showed that ph, ec, tds, cod and calcium were significantly different and station 2 was observed to be the source. the water quality index (wqi) for stations 1 and 3 were 56.49 and 57.21 respectively; categorised as poor drinking water quality while station 2 was 79.70; categorised as very poor drinking water quality. this study also showed that ph and dissolved oxygen were the contributing parameters that rendered ikwu river unfit for drinking. it was concluded that water of ikwu river is unsuitable for human consumption. keywords: physico-chemical, anthropogenic, limits, potable water, ikwu river. 1. introduction rivers have always been the most important freshwater resources, ancient civilizations have flourished along their banks while most developmental activities are still depends on them [1]. man’s utmost concern since the beginning of civilization has been the search for good quality domestic water. one of the world most challenging environmental problems resulting in the increasing demand for domestic water is the scarcity and search for clean freshwater [2]. rivers and streams are sources of water for the domestic and other uses for rural community dwellers along the water fronts [3]. water is essential in the sustenance of life, and a satisfactory (adequate, safe and accessible) supply must be made available to all [4]. since it is directly linked with human welfare, the quality of water is of great concern to mankind [5]. surface water (lakes, rivers, streams, springs, etc.) and ground water (borehole water and well water) are the two principal natural sources of drinking water [6, 7]. water pollutants that exceed certain standards are a threat to public health [8]. consequently, monitoring of surface water quality is an important issue for evaluating spatiotemporal variations of the surface water resources [9]. water resources challenges have long existed in sub-sahara africa, but their impacts have been exacerbated by recent trend such as increasing urbanization, industrialisation, agriculture and climate change. these challenges highlight the fact that the quantity and quality of water available is rapidly diminishing [10]. water quality indices are tools to evaluate the conditions http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 309 of water quality and require basic knowledge of water quality assessment like any other tool [11]. it is an acceptable way of expressing water quality with a reliable standard of measurement which reflects changes in the critical components of water [12]. water quality index (wqi) is a method of expressing an aggregated numerical value showing level of water quality [13]. it simplifies large amounts of water quality data in a reliable way that can be appreciated by the decision-makers and general public [14].the overall water quality of a certain location and time derived from a number of water quality parameters can be presented in a single number using water quality index (wqi) [15]. the aim of this study is to assess the drinking water quality of ikwu river using water quality index. 2. matherials and methods study area and sampling stations the portion of ikwu river studied is located in ohuhu community, along umuahia – uzoakoli road, umuahia, abia state, nigeria. it is located within 5º34’11.988˝ 5º34’48.000˝n and 7º28’44.400˝ 7º28’52.764e (figure 1). the river is near the popular oban international market in umuahia. the river flowed through umuire and umuegwu okpula villages before discharging into the imo river basin [16]. three sampling stations were selected along the river for the purpose of this study. station 1 on the right side along umuahia-uzoakoli road; is the upstream and control station. there were limited human activities such as extraction of water for horticultural, agricultural and drinking purposes. raffia palm (rafia africana) was the dominant plant while reissantia indica was the dominant aquatic macrophyte; station 2 which is about 150 metres downstream of station 1 was located on the left side of umuahia – uzoakoli road. in the upper part of this station, bathing, washing of cars, tricycles and motorcycles as well as abstraction of water for drinking were observed. cattle are also watered in the station by fulani herdsmen; resulting in defecation into the water. napoleona vogelli, aneilema aequinoctiale and diplazium sammatii were the common aquatic macrophytes encountered and station 3, about 530m downstream of station 2 was located within umuire village and serve as a major source of water for domestic purposes. aquatic macrophytes, hyptis suaveoleus and solenostemon monostachyus were encountered. abstraction of water for drinking, washing of clothes, fermentation of cassava and bread fruit, bathing and swimming are some of the human activities observed in this station. fig. 1: map of umuahia, southeast nigeria showing the sampling stations of ikwu river. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 310 sample collection and analysis water samples were collected from ikwu river monthly from january to june 2016. the samples were stored in sterilized 1litre plastic bottles and then taken to the laboratory for analysis. the physicochemical parameters were analyzed using standards methods described by [17]; as follows: water temperatures and ph (digital ph meter/ thermometer (hach ec 20), electrical conductivity (hach conductivity meter (hach co. 150), tds (gravimetric method), do (winkler method with azide modification), cod (open reflux method), nitrate, sulphate, and chloride (hach dr 1900 uv spec), phosphate (stannous chloride method), sodium (technicon auto analyzer flame photometer iv), calcium and alkalinity (titrimetic method). all the results were statistically analysed using single factor anova to test for significant difference among the stations and tukey pairwise test was performed to determine the location of any significant difference. the past software package (version 3.24) was used in the analysis [18]. the weighted arithmetic index method as described by [19] was used for the calculation of wqi in this study. thirteen (13) parameters were evaluated and world health organisation (who) drinking water quality guideline [20] was used as the recommended standard. the weighted arithmetic water quality index (wawqi) method is used in the classification of the water quality based on the level of purity; using the most commonly measured water quality parameters (table 1). the method has been widely used by the various scientists [15, 21 – 26] and the calculation of wqi was made as described by [12] using the equation: wqi = (1) the quality rating scale (qi) for each parameter is calculated by using this expression: qi = (2) where, vi is estimated concentration of ith parameter in the analysed water, vo is the ideal value of this parameter in pure water. vo = 0 (except ph = 7.0 and dissolved oxygen = 14.6 mg/l), si is recommended standard value of ith parameter. the unit weight (wi) for each water quality parameter is calculated by using the following formula: wi = k/si (3) where, k = proportionality constant. table 1: water quality rating as per weight arithmetic water quality index method wqi value rating of water quality grading 0 – 25 excellent water quality a 26 – 50 good water quality b 51 – 75 poor water quality c 76 – 100 very poor water quality d > 100 unsuitable for drinking purpose e source: [19]. 3. results and discussion the summarised results of the physicochemical parameters analysed in the ikwu river water samples and the calculated water quality index (wqi) are presented in table 2. most of the parameters (electrical conductivity, total dissolved solids, nitrate, sulphate, chloride, chemical oxygen food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 311 demand, phosphate, sodium and calcium) evaluated were generally within acceptable limits and lower in stations 1 and 3, except dissolved oxygen. some of the parameters were significantly different and point to station 2 as the source of the variation. the more serious problem observed in this study was ph and dissolved oxygen, which had values outside acceptable limits and point to pollution attributed to increased human activities. the temperature values ranged between 22.0 and 28.0 ºc. the lowest temperature values were recorded in stations 2 and 3 in january 2016 while the highest temperature value was recorded in station 1 in february 2016. the water temperatures were influenced by seasons. there was no significant difference in temperature (p > 0.05) in all the stations. though the temperature values recorded in this study were within acceptable limits, some were relatively high; [27] observed that temperature has a significant effect on the flavour of tap water, and a temperature that is 20 to 25 oc cooler than body temperature is generally considered to provide optimum taste. cool water is more palatable than warm water and high water temperature enhances the growth of microorganisms and may increase taste, odour, colour and corrosion problems [28]. table 2 summary of physico-chemical parameters and wqi recorded at ikwu river, umuahia, nigeria parameters station 1 x±s.e.m station 2 x±s.e.m station 3 x±s.e.m p-value who standard[20] water temperature (ºc) 25.85 ± 0.78 (22.0 – 27.5) 25.92 ± 0.81 (22.0 – 27.5) 26.45 ± 0.92 (22.0 – 28.0) p > 0.05 f = 3.02 40 ph 6.58 ± 0.27a (5.46 – 7.34) 8.46 ± 0.48b (7.22 – 10.32) 6.39 ± 0.38a (5.14 – 7.45) p < 0.05 f = 8.74 6.5 8.5 electrical conductivity (µs/cm) 22.85 ± 1.56a (18.5 – 27.8) 36.97 ± 2.88b (28.4 – 45.5) 26.38 ± 1.96a (22.1 – 33.6) p < 0.05 f = 11.1 100 total dissolved solids (mg/l) 15.3 ± 1.03a (12.4 – 18.6) 24.78 ± 1.93b (19.0 – 30.5) 17.68 ± 1.32a (14.8 – 22.5) p < 0.05 f = 11.2 500 dissolved oxygen (mg/l) 5.42 ± 0.57 (4.2 – 7.7) 4.72 ± 0.39 (3.9 – 6.3) 5.9 ± 0.35 (4.5 – 6.8) p > 0.05 f = 2.91 > 5 chemical oxygen demand (mg/l) 25.93 ± 1.57a (18.6 – 29.5) 49.08 ± 9.31b (35.5 – 94.0) 39.93 ± 2.68a (33.4 – 49.4) p < 0.05 f = 4.23 100 nitrate (mg/l) 2.8 ± 0.13 (2.5 – 3.3) 3.0 ± 0.13 (2.5 – 3.5) 2.68 ± 0.11 (2.3 – 3.0) p > 0.05 f = 2.53 50 alkalinity (mg/l) 0.63 ± 0.18 (0.28 – 1.19) 0.69 ± 0.17 (0.38 – 1.25) 0.73 ± 0.15 (0.16 – 1.2) p > 0.05 f = 1.93 50 phosphate (mg/l) 1.24 ± 0.15 (0.86 – 1.88) 1.46 ± 0.16 (0.98 – 2.14) 1.36 ± 0.30 (0.75 – 2.44) p > 0.05 f = 2.00 100 chloride (mg/l) 6.23 ± 0.22 (5.3 – 6.8) 6.78 ± 0.41 (5.3 – 8.4) 6.73 ± 0.25 (5.9 – 7.7) p > 0.05 f = 2.99 250 sulphate (mg/l) 0.42 ± 0.02 (0.37 – 0.48) 0.66 ± 0.15 (0.3 – 1.27) 0.75 ± 0.14 (0.28 – 1.13) p > 0.05 f = 2.82 200 sodium (mg/l) 4.81 ± 1.16 (2.55 – 9.8) 5.7 ± 1.04 (2.8 – 9.2) 3.86 ± 0.47 (2.24 – 5.7) p > 0.05 f = 2.71 250 calcium (mg/l) 13.01 ± 1.86a (8.0– 20.0) 22.77 ± 1.10b (17.6 – 25.3) 13.65 ± 0.38a (12.6 – 15.0) p < 0.05 f = 18.4 75 wqi 56.49 79.70 57.21 a, b = means with different superscripts across the rows are significantly different at p<0.05; sem= standard error of mean. the ph values ranged between 5.14 and 10.32. some of the ph values were not within the acceptable limits set by who. the lowest ph value was recorded in station 3 in january 2016 while the highest ph value was recorded in may 2016 in station 2. station 2 was significantly difference (p < 0.05) from the other food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 312 stations. some of the ph values were outside the acceptable limits and according to [29], extremes of ph as recorded in this study are attributable to pollution. prolonged intake of water with low ph may predispose one to the dangers of acidosis, which health experts believe could lead to cancer or cardiovascular damage including blood vessels constriction and reduction in oxygen availability even at low levels [30]. abnormal values of ph causes bitter taste to water, affect mucous membrane, cause corrosion and also affect aquatic life [31]. the dissolved oxygen values ranged between 3.9 and 7.7 mg/l. some of the dissolved oxygen values recorded in this study was below acceptable limits (> 5 mg/l). the lowest dissolved oxygen value was recorded in february 2016 in station 2 while the highest dissolved oxygen value was recorded in june 2016 in station 1. there was no significant difference (p > 0.05) in all the stations. some of the dissolved oxygen values are lower than acceptable limits. a high do level in a community water supply is good because it makes drinking water taste better [32]. organic wastes can enter a body of water in the of form leaves, grass clippings, dead plants and animals, animal droppings and sewage [33] and these organic materials eventually are broken down by bacteria, which require oxygen for decomposition process [34]. [35] reported that a low do indicates a high cod. the highest cod value was recorded in station 2 and could be attributed to anthropogenic and seasonal influences. [33] and [28] reported that depletion of dissolved oxygen in water supplies can encourage the microbial reduction of nitrate to nitrite and sulphate to sulphide as well as mobilization of trace metals. the results showed that the wqi varied from 56.49 to 79.70. the results indicated that stations 1 and 3 with wqi of 56.49 and 57.21 respectively, are of poor water quality (grade c) while station 2 with wqi of 79.70 was considered very poor water quality (grade d) when compared to the wqi standards for the weighted arithmetic index method by [19] in table 1. the ph of the samples contributed to the high wqi values recorded in this study especially in station 2, where ph contributed as high as 11.49 to the sum of the product of their quality rating scale and unit weight (qiwi). in [36], ph contributed as low as 2.63 4.17 to qiwi, which resulted in lower wqi values while in [25], ph contributed as high as 22.03 to qiwi. dissolved oxygen also contributed to high qiwi especially in station 2, where anthropogenic impacts (including watering of cattle) were intense. the water quality index values recorded was a reflection of the nature and intensity of the anthropogenic activities going on in the river. 4. conclusion based on the water quality index values recorded in this study, it can be concluded that waters of ikwu river are unsuitable for human consumption especially in station 2; though it is the only source of water for drinking and other domestic uses in the area. traditional system of water conservation includes certain activities that are restricted, while some sections of the river are demarcated for special purpose. water can also be boiled, cooled and filtered before drinking. 5. acknowledgments we sincerely appreciate mr. mike ndiuche of department of plant science and biotechnology, michael okpara university of agriculture, umudike, nigeria, who identified all plants and aquatic macrophytes. we also appreciate mr. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 313 chinedu ogbodo of department of geography, university of nigeria, nsukka, nigeria, who produced the study map. 6. references [1] al obaidy, a.m.j., talib, a.h. and zaki, s.r., application of water pollution index for assessment of tigris river ecosystem. international journal of advanced research, 3(2):219-223, (2015). [2] bustanmante., r, butterworth, j., flierman, m., herbas, d., hollander, m., meer, s.v., ravenstijn, p., reynanga, m. and zurita, g., livelihoods in conflict: dispute over water for household level productivity uses in tarata bolivia [online], (2004). available at www.irc.nl. accessed 23rd september 2016. [3] ani, c, okogwu, o.i., nwonumara, g.n., nwani, c.d. and nwiniyimagu, a.j., evaluation of physicochemical parameters of selected rivers in ebonyi state, southeast, nigeria. greener journal of biological sciences, 6(2):34-41. doi: http://doi.org/10.15580/gjbs.2016.2.020216030, (2016). [4] world health organization (who), guidelines for drinking-water quality. first addendum to third edition. volume 1 recommendations. world health organisation publication, geneva, switzerland. 595 pp., (2006). [5] singh, s., negi, r.s. and dhanai, r., a study of physico-chemical parameters of springs around srinagar garhwal valley, uttarakhand. international journal of engineering development and research, 2(4):3885–3887, (2014). [6] mcmurry, j. and fay, r.c., hydrogen, oxygen and water. in: mcmurry fay chemistry. hamann, k.p. (ed.). 4th edn. pearson education, new jersey, 575-599, (2004). [7] mendine, u., the nature of water. in: the theory and practice of clean water production for domestic and industrial use. lacto-medals publishers, lagos, nigeria, 1-21, (2005). [8] celiker, m., yildiz, o. and sonmezer, y.b., assessing the water quality parameters of the munzur spring, tunceli, turkey. ekoloji, 23(93):43-49. doi: 10.5053/ekoloji.2014.936, (2014). [9] armagan, b., gok, n., and ucar, d., assessment of seasonal variations in surface water quality of the balikligol lakes, sanliurfa, turkey. fresenius environmental bulletin, 17(1):79-85, (2008). [10] any, f., water as a stress factor in subsaharan africa. european union inst. security studies. brief issue 12. 4 pp., (2013). available on www.iss.europa.eu/publications/detail/article/water -as-a-stress-factor-in-sub-saharan-africa/. accessed 29th august 2016. [11] nikbakht, m., the effect assessment of ahvaz no.1, 2 water treatment plant on karoon water quality. m.sc thesis, ahvaz: islamic azad university of najafabad, iran, (2004). [12] brown, r.m., mccleiland, n.j., deiniger, r.a. and o’connor, m.f.a., water quality index – crossing the physical barrier. (jenkis, s.h. ed.) proceedings in international conference on water pollution research jerusalem, (6)787-797, (1972). [13] bordalo, a.a., teixeira, r. and wiebe, w.j., a water quality index applied to an international shared river basin: the case of the douro river. environmental management, 38:910– 920. doi: 10.1007/s00267-004-0037-6, (2006). [14] bharti, n. and katyal, d., water quality indices used for surface water vulnerability assessment. international journal of environmental sciences, (2)1:154-173, (2011). [15] etim, e.e., odoh, r., itodo, a.u., umoh, s.d. and lawal, u., water quality index for the assessment of water quality from different sources in the niger delta region of nigeria. frontiers in science, 3(3):89-95 doi: 10.5923/j.fs.20130303.02, (2013). [16] wikipedia, umukabia, (2016). http://en.m.wikipedia.org/wiki/umukabia. accessed 20th february 2016. [17] american public health association (apha), standard methods for exanimation of water and wastewater. 20th ed. american public health association, washington, d.c. 1193 pp., (1998). [18] hammer, o., harper, d.a.t. and ryan, p.d., past: paleontological statistics software package for education and data analysis, paleontologia electronica, 4:9, (2001). [19] tyagi, s., sharma, b., singh, p. and dobhal, r., water quality assessment in terms of water quality index. american journal of water resources, 1(3):34-38. doi:10.12691/ajwr-1-3-3, (2013). [20] world health organization (who), guidelines for drinking-water quality. world health organisation publication, geneva, switzerland. 595pp., (2010). [21] chauhan, a. and singh, s., evaluation of ganga water for drinking purpose by water quality index at rishikesh, uttarakhand, india. report and opinion, 2(9):53-61, (2010). http://www.irc.nl/ http://doi.org/10.15580/gjbs.2016.2.020216030 http://www.iss.europa.eu/publications/detail/article/water-as-a-stress-factor-in-sub-saharan-africa/ http://www.iss.europa.eu/publications/detail/article/water-as-a-stress-factor-in-sub-saharan-africa/ https://doi.org/10.1007/s00267-004-0037-6 http://en.m.wikipedia.org/wiki/umukabia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emeka donald anyanwu, chinonyerem sylva emeka, application of water quality index in the drinking water quality assessment of a southeastern nigeria river, food and environment safety, volume xviii, issue 4 – 2019, pag. 308 – 314 314 [22] rao, c.s., rao, b.s., hariharan, a.v.l.n.s.h. and bharathi, n.m., determination of water quality index of some areas in guntur district andhra pradesh. international journal of applied biology and pharmaceutical technology, 1(1):79-86, (2010). [23] balan, i.n., shivakumar, m. and kumar, p.d.m., an assessment of ground water quality using water quality index in chennai, tamil nadu, india. chronicles of young scientists, 3(2):146-150, (2012). [24] chowdhury, r.m., muntasir, s.y. and hossain, m.m., water quality index of water bodies along faridpur-barisal road in bangladesh. global engineers and technologists review, 2(3):1-8, (2012). [25] anyanwu, e.d. and nwigwe, n.c., assessment of bottled water quality using physicochemical indicators. applied science research journal, 3(1):1-12, (2015). [26] anyanwu, e.d, and ukaegbu, a.b., index approach to water quality assessment of a south eastern nigerian river. international journal of fisheries and aquatic studies 7(1): 153-159, (2019). [27] osaka municipal waterworks bureau (omwb) water quality, (2011). www.city.osaka.lg.jp/contents/wdu030/english/user /qa/f_1.html. accessed 29th august 2016. [28] world health organization (who), guidelines for drinking-water quality. 4th ed. world health organization publication, geneva, switzerland. 564pp., (2011). [29] radojevic, m. and bashkin, v.n., practical environmental analysis. royal society of chemistry. cambridge, uk. 466pp, (1999). [30] ogundipe, s. and obinna, c., safety of table water goes beyond the bottle in: good health weekly, vanguard newspapers tuesday, may 20, 2008 p.42, (2008). [31] narasimha, r.c., dorairaju, s.v., bujagendra, r.m. and chalapathi, p.v., statistical analysis of drinking water quality and its impact on human health in chandragiri, near tirupati, india [online]. eco services international, (2011). available on www.ecoweb.com/edi/111219.html. accessed 29th august 2016. [32] apec water, how exactly does dissolved oxygen affect water quality? (nd). available on www.freedrinkingwater.com/water_quality/quality1/1-how-dissolved-oxygen-affectswater-quality.html. accessed 27th august 2016. [33] murphy, s., basin: general information on dissolved oxygen. city of boulder/usgs water quality monitoring, (2007). available on bcn.boulder.co.us/basin/data/new/info/do.html. accessed 27th august 2016. [34] mahre, m.y., akan, j.c., moses, e.a. and ogugbuaja, v.o., pollution indicators in river kaduna, kaduna state, nigeria. trends in applied sciences research, 2:304 – 311. doi: 10.3923/tasr.2007.304.311, (2007). [35] hunt, m.s.q., loughlin, m.d. and quintanilla, a.t., instrumentation for environmental monitoring. 2nd edn., john wiley and sons, new york, pp: 155 – 184, (1986). [36] ushurhe, o., origho, t., ohwohere-asuma, o. and ewhuwheezo, j., a comparative assessment of water quality index (wqi) and suitability of river ase for domestic water supply in urban and rural communities in southern nigeria. international journal of humanities and social science 4(1):234 – 245, (2014). http://www.city.osaka.lg.jp/contents/wdu030/english/user/qa/f_1.html.%20accessed%2029th%20august%202016 http://www.city.osaka.lg.jp/contents/wdu030/english/user/qa/f_1.html.%20accessed%2029th%20august%202016 http://www.eco-web.com/edi/111219.html.%20accessed%2029th%20august%202016 http://www.eco-web.com/edi/111219.html.%20accessed%2029th%20august%202016 http://www.eco-web.com/edi/111219.html.%20accessed%2029th%20august%202016 http://www.freedrinkingwater.com/water_quality/quality1/1-how-dissolved-oxygen-affects-water-quality.html.%20accessed%2027th%20august%202016 http://www.freedrinkingwater.com/water_quality/quality1/1-how-dissolved-oxygen-affects-water-quality.html.%20accessed%2027th%20august%202016 http://www.freedrinkingwater.com/water_quality/quality1/1-how-dissolved-oxygen-affects-water-quality.html.%20accessed%2027th%20august%202016 1. introduction 4. conclusion original research paper 282 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 282 286 recyclable porous materials for the uptake of bisphenol a rodica sturza 1 , mihai melenciuc 1 , denisa nistor 2 , farida boudissa 3 , thiziri terkani 3 , nou-houda hadj abdelkader 3 , azzouz abdelkrim 3* 1technical university of moldova, stefan cel mare bd. 168, chishinau, md-2004, republic of moldova 2university „vasile alecsandri” of bacău, calea mărăşeşti str. 157, bacău, 600115, romania 3université du québec à montréal c.p. 8888, succ. centre-ville, montréal (québec), h3c 3p8, canada *corresponding author: sturzar@yandex.ru received 14th october 2017, accepted 29th november 2017 abstract: the presence of pollutants in the environment, food, drinking water and other compounds in direct or indirect contact with humans is a major environmental issue. in this regard, a privileged place reverts to the capture or decomposition of pollutants without producing traces of residual toxins. an interesting approach for depollution is the use of natural, non-toxic, recyclable materials as adsorbents and catalysts. the aim of this work was to study the ozonation of bisphenol-a in water in the presence of bentonite and its acid-activated counterparts aab1, aab4, aab8, aab15 and aab24 obtained after 1, 4, 15 and 24 h of acid treatment. for comparison, one also used two deriving montmorillonite-rich materials ion-exchanged with na+ and fe2+ cation, namely, namt, fe(ii)mt. the kinetic and thermodynamic parameters of the process were assessed through uv-vis spectrophotometric measurements, and were discussed in correlation with thermo-programmed desorption technique (tpd) data. calculations of the retention capacities of co2 and h2o (crc and wrc) revealed high basic character (high crc) for namt but lower hydrophilic character (low wrc) as compared to fe(ii)mt. this explains the higher catalytic activity of fe(ii)mt by enhanced interaction with basic bisphenol (pka = 9.2 at 25oc), which favors adsorption, and higher dispersion in water, which offer higher contact surface as compared to namt. this provides valuable date for designing effective oxidative water treatments using clay materials and other natural’s counterpart. keywords: bisphenol; ozonation; bentonite; montmorillonite; iron cation, thermal programmed desorption. 1. introduction bisphenol a is used in polycarbonates production as well as a precursor for epoxy resins [1]. in 2011, about 5.5 million tons of bisphenol a were produced, which were subsequently used for the manufacture of polycarbonate bottles and tableware, bottles for infant formula, and so on [[1], [2], [3]]. by multiple molecular mechanisms, bisphenol a has a strong disrupting effect on estrogenic activity, as well as other endocrine activities [[3], [5], [6], [7]]. several studies and researches have shown that all age categories are affected by the action of bisphenol a because it is detected in the urine of most adults is children tested [[2], [8]]. however, small children and babies are more sensitive when exposed to the action of bisphenol a. this compound has already been detected in the serum of pregnant women, breast milk, placental tissues and fetal liver, which shows that the child is exposed to the action of bisphenol a at the stage of fetal development [[2], [7], [9], [10]]. http://www.fia.usv.ro/fiajournal mailto:sturzar@yandex.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 rodica sturza, mihai melenciuc, denisa nistor, farida boudissa, thiziri terkani, nou-houda hadj abdelkader, azzouz abdelkrim , recyclable porous materials for the uptake of bisphenol a, volume xvi, issue 4 – 2017, pag. 282 – 286 283 the heat and contact with any of the components, whether basic or acidic, will accelerate the hydrolysis of the ester bonds that bind bisphenol a in the polycarbonates and resins. it is the same hydrolysis and the same thermal action that takes place during the processes of pasteurization or sterilization or by microwave heating. preheating food or even washing food containers allows bisphenol a to enter the food [[7]]. because of increasing environmental pollution, bisphenol a has become one of the most important pollutants and the need to eliminate it is of great importance [11]. an interesting approach for pollution abatement and prevention is the use of natural, non-toxic, recyclable materials as adsorbents and catalysts [12], [12]. this approach also involves more environmentally friendly methods of synthesizing and modifying such materials that do not use or produce potential pollutants. this will certainly open up promising prospects for a more "green" chemistry for the decontamination of water used in the food, pharmaceutical and cosmetic industries as well as for environmental sanitation in general. to achieve this objective, an experimental attempt made it possible to highlight the beneficial role of the combination of ozone with a porous aluminosilic surface such as natural bentonite and montmorillonite, its purified derivative. 2. materials and methods to perform the experiments we used bisphenol a (2,2-bis(4-hydroxyphenyl)propane) with the following parameters: assay – 97%, bp – 220°c/4 mmhg(lit.), mp – 158-159°c(lit.) [14]. bisphenol a has been subjected to the ozonation process using a 20 ml aqueous solution (10-4 m) at different ozone exposure duration (1, 2, 3, 4, 5, 7, 10, 15, 20 and 25 minutes), using the respective ozonation: 10 mg / 1 min, 20 mg / 2 min, 30 mg / 3 min, 40 mg / 4 min, 50 mg / 5 min, 70 mg / 7 min, 100 mg / 10 min, 150 mg / 15 min, 200 mg / 20 min, 250 mg / 25 min. to intensify the effect of ozone, one used two types of bentonite (benta and bentb), ion exchange with sodium (namt) and iron (femt) montmorillonite, as well as bentonites having undergone treatment with sulfuric acid for 1, 4, 8, 15 and 24 hours (aab1, aab4, aab8, aab15 and aab24). for the ozonation tests, 40 mg/l of each catalyst was previously inserted in 20 ml of bisphenol a solution, before triggering the bubbling of ozone at a rate of 6 mg / h. the basic and hydrophilic characteristics were evaluated by thermo-programmed desorption analysis (dtp or tpd) of 40 mg of each catalyst, previously saturated with carbon dioxide and water vapor. 3. results and discussion the uv-vis spectra made it possible to trace the evolution of the absorbance of each of the two spectral bands of bisphenol a (fig. 1, fig. 2, fig. 3, fig. 4,fig. 5). fig. 1. bisphenol a uv-vis spectra at different ozonation time without catalyst: concentration – 10-4 m; volume: 20 ml; ozonation regimes: 10 mg/1min, 20 mg/2 min, 30 mg/3 min, 40 mg/4 min, 50 mg/5 min, 70 mg/7 min, 100 mg/10 min, 150 mg/15 min, 200 mg/20 min, 250 mg/25 min. -0.2 0.3 0.8 1.3 1.8 2.3 2.8 3.3 190 240 290 340 390 440 490 540 590 640 690 740 790 a b s o rb e n c e w ave lenght, [nm] o3, 0 min o3, 1 min o3, 2 min o3, 3, min o3, 4 min o3, 5 min o3, 7 min o3, 10 min o3, 15 min o3, 20 min o3, 25 min food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 rodica sturza, mihai melenciuc, denisa nistor, farida boudissa, thiziri terkani, nou-houda hadj abdelkader, azzouz abdelkrim , recyclable porous materials for the uptake of bisphenol a, volume xvi, issue 4 – 2017, pag. 282 – 286 284 fig. 2. bisphenol a uv-vis spectra at different ozonation time with bentonite as catalyst: concentration – 10-4 m; volume: 20 ml bisphenol + 40 mg benta; ozonation regimes: 10 mg/1 min, 20 mg/2 min, 30 mg/3 min, 40 mg/4 min, 50 mg/5 min, 70 mg/7 min, 100 mg/10 min, 150 mg/15 min, 200 mg/20 min, 250 mg/25 min. fig. 3. bisphenol a uv-vis spectra at different ozonation time with femt as catalyst: concentration – 10-4 m; volume: 20 ml bisphenol + 40 mg femt; ozonation regimes: 10 mg/1 min, 20 mg/2 min, 30 mg/3 min, 40 mg/4 min, 50 mg/5 min, 70 mg/7 min, 100 mg/10 min, 150 mg/15 min, 200 mg/20 min, 250 mg/25 min. fig. 4. bisphenol a uv-vis spectra at different ozonation time with namt as catalyst: concentration – 10-4 m; volume: 20 ml bisphenol + 40 mg namt; ozonation regimes: 10 mg/1 min, 20 mg/2 min, 30 mg/3 min, 40 mg/4 min, 50 mg/ 5 min, 70 mg/7 min, 100 mg/10 min, 150 mg/15 min, 200 mg/20 min, 250 mg/25 min. fig. 5. bisphenol a uv-vis spectra at different ozonation time with bent b, aab1, aab4, aab8, aab15 and aab24 as catalyst: concentration – 10-4 m; volume: 20 ml bisphenol + 40 mg catalyst; ozonation regimes: 50 mg/5 min. the analysis of the uv-vis spectra of bisphenol a subjected to the action of the ozonation process has shown that fe (ii) mt is the most effective than namt. this is probably due to its acidic nature, which makes it possible to retain the most basic molecule of bisphenol a (pka = 9.2 at 25°c). this hypothesis is confirmed by the analysis of thermo-programmed desorption (tpd). fig. 6fig. 7 and others relating to fe (ii) mt have been used to calculate the values of the co2 and water retention capacities (crc and wrc). these are summarized in table 1. calculations of co2 and water retention capacities (crc and wrc) revealed a high basic character (high crc) for namt but a lower hydrophilic character (wrc low) compared to mt fe (ii). this explains the higher catalytic activity of fe (ii) mt by a stronger interaction with basic bisphenol (pka = 9.2 at 25°c), which promotes its adsorption, and a greater dispersion in water, which offers a contact area greater than that of namt in water. this provides valuable data for designing effective treatments for oxidizing water using clay materials and other natural counterparts. -0.2 0.3 0.8 1.3 1.8 2.3 2.8 3.3 190 240 290 340 390 440 490 540 590 640 690 740 790 a b s o rb e n c e w ave lenght, [nm] o3, 0 min o3, 1 min o3, 2 min o3, 3, min o3, 4 min o3, 5 min o3, 7 min o3, 10 min o3, 15 min o3, 20 min o3, 25 min -0.2 0.3 0.8 1.3 1.8 2.3 2.8 190 240 290 340 390 440 490 540 590 640 690 740 790 a b s o rb e n c e w ave lenght, [nm] o3, 0 min o3, 1 min o3, 2 min o3, 3, min o3, 4 min o3, 5 min o3, 7 min o3, 10 min o3, 15 min o3, 20 min o3, 25 min -0.2 0.3 0.8 1.3 1.8 2.3 2.8 190 240 290 340 390 440 490 540 590 640 690 740 790 a b s o rb e n c e w ave lenght, [nm] o3, 0 min o3, 1 min o3, 2 min o3, 3, min o3, 4 min o3, 5 min o3, 7 min o3, 10 min o3, 15 min o3, 20 min o3, 25 min -0.5 0 0.5 1 1.5 2 2.5 3 3.5 190 240 290 340 390 440 490 540 590 640 690 740 790 a b s o rb e n c e w ave lenght, [nm] bp bp+bentb bp+aab1 bp+aab4 bp+aab8 bp+aab15 bp+aab24 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 rodica sturza, mihai melenciuc, denisa nistor, farida boudissa, thiziri terkani, nou-houda hadj abdelkader, azzouz abdelkrim , recyclable porous materials for the uptake of bisphenol a, volume xvi, issue 4 – 2017, pag. 282 – 286 285 fig. 6. tpd diagrams of co2 – temperature relation for the three samples of femt. co2: purge debit: 14 ml/min; tpd debit: 6 ml/min; temperature domain: 23 ÷ 450°c. fig. 7. tpd diagrams of h2o – temperature relation for the three samples of femt. co2: purge debit: 14 ml/min; tpd debit: 6 ml/min; temperature domain: 23 ÷ 450°c. table 1. crc and wrc values for the studied catalysts nr. of sample crc, [μmol/g] wrc, [μmol/g] namt tpd1 3821±5 111±5 femt tpd1 1109±5 116±5 benta tpd1 2697±5 161±5 bentb tpd1 2081±5 119±5 aab1 tpd1 2554±5 102±5 aab4 tpd1 1976±5 97±5 aab8 tpd1 2422±5 84±5 aab15 tpd1 2080±5 70±5 aab24 tpd1 2415±5 73±5 4. conclusions the results of this research are of great importance as they provide compelling evidence that natural materials such as clays can be used to develop inexpensive water decontamination technologies. this is all the more interesting taking into account the wide availability of such materials, their ease of handling and modification, the non-use of solvents and other polluting chemicals, and especially the high chemical stability of bisphenol a, opens promising prospects for the treatment of water, especially that related to human health such as those used or discharged by the food and pharmaceutical industries and in domestic waste. 5. acknowledgments the presented research was supported by francophone university agency as well as the subventions from mdeie-fqrnt and fodar-uq (qc, canada). 6. references [1]. eddo j. hoekstra & catherine simoneau, “release of bisphenol a from polycarbonate – a review”, critical reviews in “food science and nutrition” 53:4, 2013, 386-402 [2]. johanna r. rochester, “bisphenol a and human health: a review of the literature”, reproductive toxicology (2013), 96 p. [3]. ruthann a. rudel, janet m. gray, connie l. engel, teresa w. rawsthorne, robin e. dodson, janet m. ackerman et al., “food packaging and bisphenol a and bis(2-ethyhexyl) phthalate exposure: findings from a dietary intervention”, environmental health perspectives, 119 : 7, 2011, 914-920 [4]. hoa h. le, emily m. carlson, jason p. chua, scott m. belcher, “bisphenol a is released from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in developing cerebellar neurons”, toxicol letters, jan 30, 208 176(2), 2008, 149-156 [5]. sarah j oppeneer, kim robien, “bisphenol a exposure and associations with obesity among adults: a critical review”, public health nutrition: 18(10), 2014, 1847-1863 [6]. beverly s. rubin, “bisphenol a: an endocrine disruptor with widespread exposure and multiple effects”, journal of steroid biochemistry & molecular biology 127, 2011, 27-34 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 2 2 .0 0 0 3 8 .5 0 0 5 5 .0 0 0 7 1 .5 0 0 8 8 .0 0 0 1 0 4 .5 0 0 1 2 1 .0 0 0 1 3 7 .5 0 0 1 5 4 .0 0 0 1 7 0 .5 0 0 1 8 7 .0 0 0 2 0 3 .5 0 0 2 2 0 .0 0 0 2 3 6 .5 0 0 2 5 3 .0 0 0 2 6 9 .5 0 0 2 8 6 .0 0 0 3 0 2 .5 0 0 3 1 9 .0 0 0 3 3 5 .5 0 0 3 5 2 .0 0 0 3 6 8 .5 0 0 3 8 5 .0 0 0 4 0 1 .5 0 0 4 1 8 .0 0 0 4 3 4 .5 0 0 c o 2 , μ m o l temperature, °c femt tpd1 femt tpd2 femt tpd3 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 2 2 .0 0 0 4 0 .6 6 7 5 9 .3 3 3 7 8 .0 0 0 9 6 .6 6 7 1 1 5 .3 3 3 1 3 4 .0 0 0 1 5 2 .6 6 7 1 7 1 .3 3 3 1 9 0 .0 0 0 2 0 8 .6 6 7 2 2 7 .3 3 3 2 4 6 .0 0 0 2 6 4 .6 6 7 2 8 3 .3 3 3 3 0 2 .0 0 0 3 2 0 .6 6 7 3 3 9 .3 3 3 3 5 8 .0 0 0 3 7 6 .6 6 7 3 9 5 .3 3 3 4 1 4 .0 0 0 4 3 2 .6 6 7 h 2 o , μ m o l temperature, °c femt tpd1 femt tpd2 femt tpd3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 rodica sturza, mihai melenciuc, denisa nistor, farida boudissa, thiziri terkani, nou-houda hadj abdelkader, azzouz abdelkrim , recyclable porous materials for the uptake of bisphenol a, volume xvi, issue 4 – 2017, pag. 282 – 286 286 [7]. cheryl erler, julie novak, “bisphenol a exposure: human risk and health policy”, journal of pediatric nursing, 25, 2010, 400-407 [8]. jenny l. carwile, henry t. luu, laura s. bassett, daniel a. driscoll, caterina yuan, jennifer y. chang et al., “polycarbonate bottle use and urinary bisphenol a concentrations”, environmental health perspectives, 117 : 9, 2009, 1368-1372 [9]. joe m. braun, amy e. kalkbrenner, antonia m. calafat, kimberly yolton, xiaoyun ye, kim n. dietrich, bruce p. lanphear, “impact of early-life bisphenol a exposure on behavior and executive function in children”, pediatrics, 128 : 5, 2011, 873-882 [10]. thit j. mørck, giuseppina sorda, nicoletta bechi, brian s. rasmussen, jesper b. nielsen, francesca ietta et al., “placental transport and in vitro effects of bisphenol a”, reproductive toxicology 30, 2010, 131-137 [11]. henryk metcer, gary klecka, “treatment of wastewaters containing bisphenoi a: state of the science review”, water environment research, 83: 7, 2011, 650-666 [12]. alisa v. arus, m. nazir tahir, radia sennour, tze c. shiao, lamyaa m. sallam, ileana d. nistor, rene roy, abdelkrim azzouz, “cu0 and pd0 loaded organo-bentonites as sponge–like matrices for hydrogen reversible capture at ambient conditions”, chemistry select 2016, 1, 1452-1461 [13]. d. nistor, d.n. miron, a. azzouz, “ cinétique de rétention des éléments radioactifs sur des argiles pontées d’origine roumaine, studies and scientific research, vol. iii, abstract, issue: scscc6, volume vii, no. 3 (2006), (issn 1582-540x). [14]. http://www.sigmaaldrich.com/catalog/pr oduct/aldrich/133027?lang=en&region=ca http://www.sigmaaldrich.com/catalog/product/aldrich/133027?lang=en&region=ca http://www.sigmaaldrich.com/catalog/product/aldrich/133027?lang=en&region=ca title …………………… 345 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 4 2015, pag. 345 351 investigation of the effect of vegetable powders on aqueous and oil phase in butter paste oksana vasheka1, aleksandra niemirich1, *oksana petrusha1, natalya drozd1 1faculty of health improving products technology and food expertise, national university of food technologies, 68 volodymyrska str., kyiv, ukraine, petrushaoo@ukr.net, *corresponding author received october 4th 2015, accepted december 15th 2015 abstract: the paper presents a method of enriching oil salt complex natural mixture of micronutrients by a slurry of white sesame seeds and powders from carrot and tomatoes obtained by cold spray drying, in amounts of 2%, 8% and 4% respectively. the control sample used to regulate the skimmed milk solids content moisture content in the test samples was 48%. the feasibility of introducing selected herbal ingredients is confirmed by numerous positive results reported in the literature: medical valuable micronutrients selected plant raw materials have a positive effect on the immune, nervous, cardiovascular and respiratory systems, improve liver, promote regeneration of skin cells and the like. the slurry of white sesame seeds produced immediately before introducing it into the oil mixture by grinding seeds in special conditions. on the basis of microstructure analysis, we studied the structural elements that formed plant components with the fat and aqueous phases salt butter paste, as well as we determined the size and distribution of droplets characterized by plasma product. the paper studied the effect of carrot and tomato powders and especially of designed suspension of sesame-rich plasma in the dispersion of oil mixture. it is found that the introduction of plant ingredients in the specified amounts increases the number of droplets with sizes of up to 5 microns by 1.8 times. the method of microstructure analysis of the interacting components of vegetable raw materials with fatty and aqueous phases and oil mixture revealed the formation of sinter mesh structure. the investigation of crystallization of fat phase was determined by differential scanning calorimetry. these results indicate that there are components of plant raw materials which alter crystallization of the fat phase of the product. when storing the product of the fat phase, greatest changes occur up to three days. it is found that the fat phase recrystallization is fusible and medium melting triglyceride. keywords: glycerides, agglomerate, cellular structure, crystalline linear structure, dispersion, plasma, differentiation, crystalline phase, recrystallization 1. introduction analysis of the results of recent statistical research indicate a direct connection to reduce the content of natural micronutrients in the diet of the population and distribution, and a significant the rejuvenation of chronic no communicable diseases (ncd) [1-3]. according to who, 80 % of the adult working age population mortality in european countries due to the ncd, among them about 50 % of cases due to cardiovascular diseases and 20 % of cancers. leading experts from the who identified six groups of major risk factors that have the greatest impact on the emergence and development of ncd, and the two of them are directly related to the daily food intake of the population [1]. for the purpose of a positive decision, "hidden hunger" and harmonization of nutrition in most european countries and in ukraine http://www.fia.usv.ro/fiajournal mailto:petrushaoo@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 346 they implemented a range of activities related to the enrichment of traditional foods with micronutrients and natural setting strict state control over the quality and safety of feedstock, semi-finished and finished products [2, 3]. the daily diet of the population consists of dairy products which are perceived as traditional products with pleasant taste properties. however, in recent years there has been a general trend of increasing consumption of dairy products with reduced fat content. this is often due to the existence of views on the harmful effects of one of the components in milk fat cholesterol on the cardiovascular system of the human body. it must be said that the results of long-term research conducted in academic centres leading countries, indicate that cholesterol is involved in the synthesis of bile acids and some hormones used in the construction of the myelin sheaths of nerve cells and cell membranes. dutch doctors and scientists from boston university, based on long medical research found that people with low cholesterol diet have a reduced ability to remember and logical thinking, often in bad moods, tendency to depression and mental disorders [4, 5]. also, leading scientific journals reported the presence in milk fat and fat globules, a number of medical components capable of, even in small concentrations, acting as inhibitors in carcinogenesis of some cardiovascular and gastrointestinal diseases [4, 5]. given the above, there is a need to create new types of products based on butter with a high content of natural micronutrients. these include oil mixture. in ukraine such products are not produced in industrial conditions. at the same time in many european countries a mixture of oil and oil paste are popular enough foodstuff, as evidenced by the presence of existing documentation for these products and the significant volumes of industrial production. taking into account modern trends in the development of food science, a method of enriching complex salt-oil mixture of natural micronutrients was developed. according to the proposed technology of making specially slurry of white sesame seeds and powders from carrot and tomatoes obtained by cold spray drying consists in machining the basic oily mixture ingredients. it is pointed out that the enrichment of white sesame seeds is used in the form of slurry which is produced directly before making it into an oil mixture by grinding seeds under special conditions. the feasibility of introducing selected herbal ingredients is confirmed by numerous positive results presented in the literature: medical valuable micronutrients, selected plant raw materials have a positive effect on the immune, nervous, cardiovascular and respiratory systems, improve liver function, contribute to the regeneration of skin cells, etc. [6-8]. according to the results of experimental research and based on the high sensory properties of the finished product, rational concentration of herbal supplements was established. during the production of carrot powder an amount of 2% tomato powder 8% and sesame paste 4% respectively were used. the paste is white sesame seed produced on own technology. the setting of the number of herbal supplements was performed at the best structure and taste of the product. it was revealed that the introduction of selected additives in specific amounts reduces the hardness of the finished product and reduces the liquid fat excretion of the product. also, butter paste has a high ability to retain its shape at high temperatures (30°c). the experimental results indicate the internal interaction and the formation of additional bonds between the components of vegetable raw materials and water and fat phase rich oil mixture. to confirm this hypothesis and for better understanding of the effect of additives on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 347 the formation of plant properties of the oilenriched mixture, we studied its microstructure and its changes occurring in the aqueous and fatty phases of the finished product. 2. materials and methods we investigated the butter paste made with carrot powder in an amount of 2% tomato powder 8% and sesame paste 4% respectively. salt was added to enhance the taste of the product. a sample control was used to monitor the changes occurring under the influence of herbal supplements. it was manufactured using the same technology as the research oily paste, but instead of herbal supplements, milk powder was used. moisture content in the test samples was of 48%. micro-structural methods of studying the structural elements are formed by plant components with fatty and aqueous phases of the product. the microstructure characterizing droplet sizes of plasma products and their placement in the structure of the product were analyzed. these microstructures were prepared by „crushed drops." view preparations were carried out at a temperature of 18 ± 1°c; using optical polarizing microscope mbi15 with lighting "the passage" and in polarized light. individual fields were photographed with the scale of ocular micrometer. the research on phase glycoside changes of milk fat in samples was performed by thermodynamic differential scanning calorimeter in the temperature range between -50°c and 50°c. the analysis of crystallization was carried out by obtaining curves, focusing on the values of their highs and substrate temperature melting peak groups of triglyceride fat phase in finished products. 3. results and discussion pictures of the microstructure of salt butter paste in the light "on transmission" and polarized light are shown in the figure 1. plasma drops of control sample (fig. 1, d) are distributed in non-uniform structure and the product is of different magnitudes. the average size is in the range 2.5...7.5 m (fig. 2). it is pointed out that 30% of the plasma droplets of the control structure have dimensions of 5 ... 6 mm. when microscopically viewed, individual plasma drops of a diameter bigger than 10 microns were identified in the oil mixture without additives. in the literature there is a study which indicates a direct correlation between plasma dispersion of droplets of butter and microbial spoilage [9]. the authors found that with increasing the number of plasma drops with dimensions greater than 5 microns, the microbial spoilage rate increases. the total number of diameters drops in the control sample is about 48 %. the results of research make it possible to predict a low microbiological stability of the oil mixture without herbal supplements during storage. the microstructure of the sample butter pasta with herbal supplements has many fine droplets of plasma. the structure of the product is placed evenly. the vast majority (70.5%) of plasma droplets have a size of up to 2,5 mm (fig. 2). enriched butter paste with droplets greater than 5,0 mm is not more than 5%. this indicates high resistance to microbial spoilage of the finished products during storage. we should mention that while watching the preparations of enriched butter paste we also found particles of tissue made from herbal ingredients, and the structural elements that are not specific to a control example. figure 1 a and b show the picture of the microstructure of butter paste enriched with vegetable powder particles. one can see in the picture traces of the leading c and d in stored tissues. tissue particles have a size of about 5...10 mm and 60...80 mm respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 348 when viewing the preparations in polarized light (fig. 1, b and d) at 18°c the surface of powder particles has a bright pale yellow and light orange color. that indicates the interaction of the components of powders with a fat phase in butter paste. as a result of the interaction between particles the surface of tissue is easily crystallized (hmt) and medium melting (mmt) triglycerides. fig. 1. microstructure of oil mixtures: a, b – an enriched butter paste microstructure when viewed in light of "the passage"; с, d microstructure enriched oil mixture when viewing the preparations in polarized light; a, f – interparticle cellular structure; b – oil droplet plasma mixture; c – store and particle d – conducting tissues vegetable powders food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 349 the fig. 1 shows agglomerate f which has a honeycomb structure. on the sides of the agglomerate linear crystals are placed. they are well visible in polarized light. also, agglomerates can be seen (fig. 1a), which consist of polygons of irregular shapes. the size of the faces of its cells varies between 5...15 mm. a characteristic feature of agglomerate is that they have strongly bound moisture on the surface of its faces, as indicated by viewing the respective portions in the light microscope slide "on transmission", and in polarized light. these results confirmed the previous research and well-known results obtained by leading scientists in the study of such structures [10, 11]. fig. 2. the fineness of plasma drops so, microstructural investigations of salt enriched butter paste indicate the interaction between the components of vegetable raw materials with water and fat phase of the final product, creating new structural elements a honeycomb structure of the agglomerates. at the edges of cells sinter layers, firmly bound aqueous phase and crystalline linear structure are formed from groups lmt and hmt. changes in the fatty phase of butter paste which were kept 3 days at 6±1°c were studied by differential scanning calorimetry. the determination of group composition of the crystalline quality of the fat phase of prototypes was carried out according to the method proposed by professor tamara rashevsky. it was obtained by endoderm melting of the crystalline phase of the product laid out on a simple (gaussian) by least squares method using the program “peak.fi”. the laws of normal distribution curves gauss were used. the peaks of outlined characterized the change of crystallization processes in the butter paste. melting endothermic curve prototypes mathematically processed of oil mixtures are presented in figure 3. the nature of specific heat control (fig. 3.a) indicates the gradual melting of the crystalline phase of the product. when carrying out the mathematical processing of the melting curve, low gaussian temperatures with maximum of –21,3°c and –12,1°c were registered. according to the literature it is known that these peaks characterize the transition from the amorphous phase to the liquid triglyceride fat amorphous phase control. the highest maximum peak was registered at -2,6°c which was formed by melting water and the partial melting of low-melting triglycerides (lph) fat phase of the product. the presence of diffuse peak in the total heat capacity curve and gaussian one at 7,8°c indicates the completion of melting triglyceride fraction in the crystalfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 350 melting fat phase of the product. the peak with a maximum at 20°c is the result of co-melting fuse light and medium-melting triglycerides (mmt). the diffuse peak at 31.2°c characterizes the melting of high melting triglycerides (hmt) crystalline phase of milk fat. we observed that the groups of fungal microflora present initially were also identified at the end of the drying process but with different level of contamination depending on the surface drying and the origin of beans (figures 2 and 3). fig. 3. mathematically treated curves melting endothermic crystalline phases of oil mixtures: a – control; b – enriched butter paste. from the beginning to the end of drying, several factors in combination may contribute to impact differently the microbial growth in contaminated cherries. this is strongly supported by the fact that the anova was performed before and at the end of drying, showing a distribution of contamination levels of cherries initially uniform in samples from different areas, but this distribution is no longer uniform at the end of drying. moreover, an interaction of different factors has been evidenced. by comparing control endothermic and enriched butter paste it is evident that the final melting of the crystalline phase occurs over a wide range and is described by a large gaussian number and peaks melting triglycerides individual groups are more pronounced. this nature of the curve indicates discrete crystallizing of triglyceride fat in a crystalline phase, an enriched butter paste during its storage. the components in butter paste with vegetable should also be mentioned: structural transformations in amorphous milk fat 14°c and 9°c; the maximum comelting of water and fat phases lph product ≈ 5°c. clearly, such processes are due to the content in sesame oil linoleic acid with a low melting point. the gaussian number also increased in the finished butter paste, changing the temperature of highs, having in view that the peak melting lph in enriched butter paste was 4.4°c. the maximum co-melting lph and lng (fig. 3.b.) compared to the control sample is reduced to 13°c. this explains the processes of recrystallization of triglycerides. the melting group mmt occurs at higher temperatures than in the control sample, with a peak at 23.5°c. melting processes described two gaussian fractions hmt with peaks at 34.2°c and 48.8°c. it should be noticed that the first peak melting temperature as compared to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd, investigation of the effect of vegetable powders into aqueous and oil phase in butter paste, food and environment safety, volume xiv, issue 4 – 2015, pag. 345 – 351 351 control was registered at above 3°c. an additional melting peak hsv was obtained at 48.6°c, its area being negligible, whereas for the control sample it was not specific. it was obviously created by melting stearic acid and palmitic sesame oil. by analyzing the data obtained, it was found that the introduction of the mixture of powders in the oil from carrots and tomatoes, and specially prepared sesame seeds leads to discrete crystallization and recrystallization of individual triglyceride groups in products during storage. these data confirm the results of microstructural investigations. 4. conclusion 1. the effect of powders of carrot and tomato paste and sesame on the dispersion of the enriched butter paste plasma mixture was analyzed. it is found that the introduction of plant ingredients in the specified amounts increases the number of droplets with sizes of up to 5 microns by 1,8 times. 2. the method of microstructural analysis revealed the interaction between the components of vegetable raw materials with fatty and aqueous phases and butter paste revealed the formation of sinter mesh structure. 3. it was found that the plant components accelerate the process of recrystallization group small lmt and mmt in a crystalline fat phase rich in oil mixture. these processes take place the most intensively during the first 3 days of storage. 5. references [1]. course on safely. report on health in europe 2012, 196, (2013) [2]. the concept of the national program «health 2020: ukrainian dimension», approved by cabinet of ministers of ukraine, № 1164-r., (2011) [3]. s. linnik, the directions of realization in ukraine who european strategy for prevention and control of non-communicable diseases, scientific work. governance, 196, 208: 106-111, (2012) [4]. m. ancelin, i. carriеre, j. boulenger, gender and genotype modulation of the association between lipid levels and depressive symptomatology in communitydwelling elderly (the esprit study), biological psychiatry. 68, 2: 125-132, (2010) http://www.biologicalpsychiatryjournal.com/article/ s0006-3223(10)00393-8 [5]. v. spitsberg, invited review: bovine milk fat globule membrane as a potential nutraceutical, journal of dairy science, 88, 7: 2289-2294, (2005) [6]. m. simonova, carotenoids: structure, properties and biological biologichni studiyi. studia biologica. 4, 2: 159-170, (2010) [7]. а. rajoria, j. kumar, а. chauhan, anti-oxidative and anti-carcinogenic role of lycopene in human health a review, journal of dairying, foods and home sciences, 29, 3: 157165, (2010) [8]. v. isaev, polyunsaturated fatty acids and their role in cerebral blood flow, http://www.trinita.ru/articles/pnzhk_mozg.html [9]. e. kaneva, f. vyshemirsky, the fineness of the plasma and ability to persist for butter, сontribution of science to the development of butter and cheese: scientific and engineering. conf., uglich, 116, 3-4 october, (1994) [10]. a. karpenko, j. vashek, o. nemіrіch, microstructural investigation of an aqueous suspension sesame seeds, scientific papers of young scientists and students, odesa: 167-169, (2014) [11]. m. mahonina, t. rashekska, the fatty phase microstructure slurry additives flax seed, food science and technology, 2(15): 50-53, (2011). file:///e:/ktn_останне/холестерин/abstract_2.htm file:///e:/ktn_останне/холестерин/abstract_2.htm http://www.biologicalpsychiatryjournal.com/article/s0006-3223(10)00393-8 http://www.biologicalpsychiatryjournal.com/article/s0006-3223(10)00393-8 http://www.cabdirect.org/search.html?q=au%3a%22avneet+rajoria%22 http://www.cabdirect.org/search.html?q=au%3a%22jitendra+kumar%22 http://www.cabdirect.org/search.html?q=au%3a%22chauhan%2c+a.+k.%22 http://www.trinita.ru/articles/pnzhk_mozg.htm 245 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 245 249 sensory evaluation of goat milk cheese under different freezing conditions *eugenia mihaela pricop faculty of food science and engineering, “dunarea de jos” university of galati,111 domneasca str., 800201 galati, romania, *corresponding author: mihaela.pricop@ugal.ro received 13th october 2017, accepted 19th december 2017 abstract: the purpose of this paper is to investigate the effects of different freezing treatments on textural and sensorial characteristics of goat cheese in order to explore the possibility of storing for a longer period of time. goat cheese samples were prepared from raw goat milk and treated under four different freezing methods:1) natural convection of air (200c); 2) deep freezing (-700c);3) immersion in eutectic solution of nacl 23% and 4) cryogenic freezing using liquid nitrogen and compared with a control sample kept at 4-60c. after freezing, the samples were stored for 3 month at -18…-200c then they were thawed and stored for 14 days at 4…60c. the textural and sensory aspects were undertaken using 12 panelists. at the end, the samples were classified, depending on the score obtained from good to unsatisfactory. keywords: goat cheese, freezing methods, long term storage, sensorial aspects. 1. introduction the goat milk and cheeses are considered ideal foods for their high nutritional value due to its higher digestibility, its dietary characteristics with smaller diameters fat globules, density, variety, availability, good taste and affordable price [1, 2]. many of cheese characteristics such as flavor and texture are determined by the spatial arrangement of the main compounds including: casein particles which form the proteic matrix, fat globules, the dispersed water and minerals [3]. changes that occur under different freezing conditions can alter the curd protein's micro-structure and thus can degrade the functional properties of the final product. the sensorial and textural perception ( sensory perception of cheese structure) [4], represents a very important analysis for dairy products especially for cheeses. sensory quality of food refers to a set of characteristics such as appearance, colour, taste, smell, aroma, texture and auditory properties [5]. sensory analysis techniques have developed into a powerful tool for understanding how the appearance, flavor and texture attributes of dairy products drive the consumer preferences [6]. the aim of this research is to evaluate the potential changes of the texture and sensorial characteristics of frozen goat cheese samples through human perception. 2. matherials and methods 2.1. preparation of goat milk curd-style cheese the raw whole milk was acidified by its native microbiota. the coagulation http://www.fia.usv.ro/fiajournal mailto:mihaela.pricop@ugal.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 eugenia mihaela pricop, sensory analysis of goat milk cheese characteristics under different freezing conditions, food and environment safety, volume xvi, issue 4 – 2017, pag. 245 – 249 246 process of milk took place at 320c by adding about 1, 5 % of commercial calf rennet (strength 1/10000). the coagulum was cut into pieces (8 -10 mm cubes). the coagulum was left to stand for 45 minutes to remove the whey, then transferred into the cotton bags for drainage. the draining was done after 5 hours at 200c. 2.1.1. sampling the blocks of curd – style cheese weighing 2 -2.5 kg were divided into samples with dimensions of 50 × 40 × 25 mm (approximately 50 g). the samples thus prepared were subjected to vacuum prepackaging or directly freezing according to freezing method. 2.2. set up of the freezing methods the freezing procedures used are presented in table 1. table 1. characteristics of freezing processes freezing procedures code parameters of freezing processes troom, [ 0 c] t i sample, [ 0 c] t f , * [ 0 c] w c , [cm/h] τ c, [h] freezing type equipment freezing by natural convection of air -20 0 c mcl -20...-22 6...8 -20 0.45...0.5 5.5 slow home freezer deep freezing -70 0 c qac -70 6...8 -25 1...1.5 2.5 fast superfreezer angelantoni -industries nacl 23% solution (eutectic solution) rqa -15 6...8 -15 3.3...4 0.5-0.6 very fast immersion freezing (direct contact with samples) cryogenic freezing sbt -196 6...8 -25 15...20 0.150.18 ultra fast cryogenic freezer (cryogen sprayed on samples) control sample prs 4...6 refrigeration tf, freezing temperature, 0c;ti sample sample initial temperature, 0c tf, sample final temperaturei, 0c;wc average freezing rate, cm/h; τcaverage freezing time, h. 2.3. sensory and texture analysis the sensory evaluation was performed by 12 panelists [7, 8]. the textural profile included the following properties: springiness (ability to recover its initial thickness rapidly after compression and deformation), firmness (resistance that the sample presents with small displacements of the jaws), friability (ability to generate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 eugenia mihaela pricop, sensory analysis of goat milk cheese characteristics under different freezing conditions, food and environment safety, volume xvi, issue 4 – 2017, pag. 245 – 249 247 several pieces from the beginning of chewing), deformability (ability of the sample to deform successively or stretch easily before breaking the sample in the oral cavity), adhesiveness (moving the tongue to detach the sample stuck in the palate or teeth), and cohesiveness (firmness of the internal joints in the cheese sample) [9, 10]. to determine the fully sensorial profile the following attributes has been used: external appearance, section appearance, odor, taste, colour and consistency. sensory analysis was organized according to stas 12656-88: sensory analysis. method of scoring scales [11]. a continuous, one (minimum) to five (maximum), scale was used to estimate the felt intensities for each attribute. global sensorial qualities of frozen goat milk cheese samples were evaluated by comparation between samples using a 20 points scale. table 2. correlation of average score with total qualifying average score total qualifying 18.1.....20 very good 15.1...18 good 11.1......15 satisfactory 7.1....11 unsatisfactory 0...7 inadequate 3. results and discussion sensory analysis the sbt (freezing at -700c) and qac (cryogenic freezing) samples had greater values for elasticity than the control sample (prs) (fig. 1). this allows us to appreciate that the rapid and ultra-rapid methods of freezing which generate small crystals of ice into [10, 11], extraand intracellular space, do not adversely affect the textural characteristics but it could improve some of these (such as elasticity). significant changes of hardness have been observed for the rqa sample (frozen in salt solution), which was the least appreciated by the evaluators in the terms of all textural parameters. the lowest score awarded by evaluators to rqa sample, may have been due to very high concentrations of salt, about 3.8%. the cheese texture could be affected by the high salt concentration by changing the moisture content, the casein structure and interactions of calcium-p-caseinate phosphate complex [12]. the high salt content from rqa samples had unfordable consequences on all atributes. the panelists consider that sample to be undesirable for consumption (table 3). as we expected, control sample recorded the highest score. however, the score obtained demonstrate that goat cheese sample treated under cryogenic method (sbt) and cheese sample treated under 700c (qac) doesn’t change their quality atributtes especially external appearance, section appearance. fast freezing methods have little or no effect on the external appearance of the samples because the freezing process took place quickly and the ice crystals formed were small and uniform. cheese sample frozen under conventional procedures (-200c) obtained a medium score (~3.5). after correlations of the obtained scores by panelists the analyzed samples were classified as follow in table 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 eugenia mihaela pricop, sensory analysis of goat milk cheese characteristics under different freezing conditions, food and environment safety, volume xvi, issue 4 – 2017, pag. 245 – 249 248 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 springiness hardness friability deformability adhesiveness cohesiveness prs mcl sbt rqa qac fig. 1. graphical representation of scores obtained for textural characteristics of goat milk cheese samples after freezing procedures 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 external appearance section appearance consistency color taste odor prs mcl sbt rqa qac fig. 2. score of sensory attributes of goat milk cheese samples after freezing procedures table 3. correlation of the average score with the total qualifying for the analyzed samples code sample total average score total qualifying prs control 17.518 good sbt frozen at -700c 16.816 good qac liquid nitrogen 14.730 satisfactory mcl frozen at -20°c 13.094 satisfactory rqa frozen in nacl 23% 10.238 unsatisfactory food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 eugenia mihaela pricop, sensory analysis of goat milk cheese characteristics under different freezing conditions, food and environment safety, volume xvi, issue 4 – 2017, pag. 245 – 249 249 4. conclusions conventional freezing by natural convection of air at -200c determined several changes which affect sensorial aspects. the high score obtained by deep freeze sample demonstrates that the frozen rate and temperature have a major impact on sensorial aspects. after the analysis of textural aspects, it was found that the samples have not suffered significant textural changes except the goat milk cheese sample frozen in eutectic salt solution which was not agreed by the evaluators. freezing can be used as a method for longterm preservation of goat milk cheese, but the freezing operation should be performed only after the establishment of optimal parameters to ensure minimal structural changes of sensory atributtes. 5. references [1]. bogue j.d., market-oriented methodologies to optimize consumer acceptability of cheddar-type cheeses, british food journal, 101: 301-316, (1999). [2]. nascimento t.v.c., gonçalves de almeida jr. w.l., lopes e.s. jr. et.al, physical and chemical characteristics of milk from goats supplemented with different levels of total digestible nutrients in the dry period, acta scientiarum. animal sciences, maringá, 39(4): 429-435, (2017). [3]. everett d.w., microstructure of natural cheeses, in: structure of dairy products, ed a. tamime, oxford, uk, blackwell publishing ltd, (2007). [4]. foegeding e.a., drake m.a., invited review: sensory and mechanical properties of cheese texture, j dairy sci., 90 (4):1611-24, (2007). [5]. kalit m.t., markovic k., et.al. application of electronic nose and electronic tongue in the dairy industry, mljekarstvo, 64(4): 228 244, (2014). [6]. inovations in dairy, www.mintinnovation.com. (2005) [7]. banu c., vizireanu c., calitatea si analiza senzoriala a produselor alimentare, bucuresti: ed. agir, (2007). [8]. lawless h., heymann h., sensory evaluation of food, principles and practices, u.s.a: aspen publication, (1999). [9]. caro i., soto s., fuentes l., méndez –gutiérrez n., et.al. compositional, functional and sensory characteristics of selected mexican cheeses, food and nutrition sciences, 5: 366-375, (2014). [10]. rohm h., jaros d, properties, analytical methods. principles and significances in assesing rheological and textural, in: encyclopedia of dairy science, elsevier, (2011). [11]. stas 12656-88., sensory analysis. method of points scale. [12]. akalin a.s., et.al. variation in organic acids content during ripening of pickled white cheese, journal of dairy science, 85:1670-1676, (2002). 1. introduction 4. conclusions удк 664 337 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 337 346 prospects of using secondary energy resources anatoly sokolenko 1 , olexandr shevchenko 1 ,* konstantin vasylkivsky 1 , oleg stepanets 1 , inna vinnichenko 1 1national university of food technologies, volodymyrska str. 68, kyiv, ukraine tmipt@ukr.net *corresponding author received 27th october 2019, accepted 27th december 2019 abstract: the paper deals with the peculiarities of thermodynamic analysis of technological systems of food production with generation of secondary steam. system analysis is based on information about production, equipment operation, modern scientific achievements and modeling methods in chemical, physical, mathematical and other sciences. the information about applied energy and thermodynamic criteria, criteria of optimization of systems, list of sequence of actions on improvement of systems is given. secondary energy resources of food industries are often represented by thermal energy of liquid media or secondary steam. regeneration of the secondary steam allows, during its subsequent condensation, to return the heat potential of the steam to the media. this means that in the systems where the increase in the thermodynamic parameters of steam due to its compression, there is the use of carno's return cycle. such use occurs both in closed and open cycles. the description of the physical processes accompanying the work of the thermocompressors is subject to mathematical modeling based on the laws of conservation of energy, mass conservation and conservation of impulses. the assessment of the suitability for using secondary energy resources is based on exergy methods in order to determine the directions for their further improvement. for identical pressures in the mixing chamber, the efficiency of the actual compressor is higher than the ratio of the ejection coefficients of the real and ideal compressors, since internal irreversible losses along with the reduction of the ejection rate result in the increase of the specific exergy of the compressed flow. key words: thermodynamics, recuperation, secondary energy resources, secondary steam, enthalpy, exergy, energy transformation. 1. introduction functioning of technological systems of food production is provided in the presence of chemical, energy, mechanical and microbiological processes at different stages of existence. their cumulative flow, structural and parametric interconnection require the involvement of system analysis to assess the effectiveness of technologies objectively and identify areas and prospects for improvement and optimization. information on the construction and operation of productions, modern scientific advances and modeling methods in chemical, physical, mathematical and biological sciences is the basis for system analysis. the project of the existing system at the stage of its creation and implementation has its reflection in the form of the target function, and its optimization is the ultimate goal for system analysis [1, 2]. in the general definition, optimization tasks are divided into parametric and structural. technological development of individual subsystems leads to the conclusion that the determining role belongs to the choice of optimal structure of the system, the economic effect of which is an order of magnitude higher than the effect of optimal process control [3]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 338 determination of the extremum of the target function without specifying conditions for other values leads to the case of unconditional optimization, and in cases when it is necessary to take into account certain conditions that are superimposed on other values, there is a conditional optimization. the type of criterion for an optimal or target function is determined by a specific optimization task. the list of criteria used in system evaluations includes economic, energy, technical, environmental, reliability, flexibility, etc. in connection with the further interests of the study, we will give information on energy and thermodynamic criteria. to energy criteria include: criteria for the maintenance of heat ltlgpgm ηηqqη  , (1) where g q , pq , lgη and lpη are heat fluxes and coefficients of heat loss from the medium giving (g) and perceiving (p) heat; heat utilization factor maxqqη maxuh  , (2) where q and max q are real and maximum possible heat fluxes in the heat exchanger, j; kirpichov’s criterion maxаlqе к  , (3) where q – real heat flux; al – the work of the superchargers; glaser's criterion maxnqη т  , (4) where n – power of the supercharger, w. the thermodynamic criteria include: criterion of thermodynamic reversibility (grassman) maxδаδаη gptr  , (5) where gδа and pδа – increase in the robustness of given and perceived warmth, j; thermodynamic coefficient of gui-stodol max аа δаδа 1η pg pg t     , (6) where g а and p а – robustness of given and perceived warmth, j. the choice of the optimization criterion for the investigated energy technology system should correspond to the transparent scientifically substantiated physical content and reflect the most important aspects of the process. sama (1995) notes that notes that classical thermodynamics with its second law is the fundamental basis for the synthesis of both new systems and for optimization of the parameters and structure of the existing ones, and since the processes proceed on the macroscopic level, this allows to establish direct interconnections between the parameters of heat, work and efficiency criteria. the comprehensive goal of the thermodynamic analysis of the system is formulated by the following sequence: assessment of the degree of thermodynamic perfection of the system; definition of the cause and source of imperfection; finding ways to eliminate imperfections; formulation of measures to improve the efficiency of the system, which correspond to the chosen criterion of effectiveness. the list of possible areas for optimization of thermodynamic systems is related to energy, entropy, exergy, thermoeconomic and pinch analysis. these methods lead to thermodynamic relations, which relate to the possibilities of intensive energyabsorption, however, the in-depth improvement of technologies of energy transformations requires more extensive use of heat engineering, technological and biochemical variations. an assessment of the suitability of using secondary energy resources requires recourse to the second law of thermodynamics, in which the correspondence to all types of heat flows is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 339 characterized by the quality of indicators, that is, the ability to perform work. work is possible only in a system that is not in equilibrium with the environment, when the pressure and temperature in the system are greater than those of the environment with which the system interacts. as the work is done, the isolated system approaches the equilibrium state with the environment where the work is stopped. the quality indicator of various types of heat (exergy) is the maximal ability of matter to work in such a process, the final state of which is determined by equilibrium with the environment. the exergy method is used for the purpose of energy optimization of technological systems and determination of directions of their further thermodynamic perfection [5]. real energy processes are irreducible and this irreversibility is the cause of their imperfection. however, the physics of the essence of the energy balance does not reflect the loss of the system from nonfailure [6]. the general representation of the components of exergy is associated with the concept of entropy. the types of energy that can be completely transformed into others include mechanical, electrical and nuclear (for them entropy s' = 0). in the cases of the chemical energy of intermolecular bonds, the chemical potential and the heat s ≠ 0, their transformations occur with certain losses. in the general list of technological operations in the food industry, cases of heat treatment of media with intensive generation of secondary steam are presented, the recuperative return of the potential of which in the system greatly limits the costs of primary energy resources. among the classical cases, it is possible to include processes of brewing beer wort, evaporation of beet juice, syrups, bards, milk, crystallization of sugar, etc. the hardware design of processes at the same time depends on the ultimate goal, which can take into account the technological features of their flow or is only a matter of concentration of solutions. the classic theory of evaporation has been reflected in the processes of condensation of solutions during boiling, which occurs in volume, when the steam pressure of the solvent is equal to the total pressure in the ingenious volume. the feature of the process is the stability of the boiling temperature at this pressure and a certain composition of the solution. in this case, water evaporates and is removed in the form of secondary steam, and dissolved substances remain in solution in an unchanged amount. these processes are carried out in evaporators of technological purpose, and if their implementation takes place under vacuum, then such devices are called vacuum machines. for one-time evaporation in a separate evaporator, the cost of a heated steam is about 1 kg per 1 kg of evaporated water, which is economically unprofitable. the use of secondary steam is carried out in multibody evaporation units or in evaporators with heat pumps. in the latter case, the technological apparatus becomes a component of the system, which is the realization of the heat pump, since it simultaneously serves as an evaporator and a condenser [7-11]. 2. matherials and methods the tasks of static optimization are solved for processes in tranquil modes with the decision of questions of creating optimal models, and in cases of dynamic optimization, the creation and implementation of systems with optimal control over non-sustained operating modes is carried out. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 340 3. results and discussion fig. 1 shows the scheme of the system with mechanical compression of the secondary steam and fig. 2 is the corresponding diagram in coordinates t-s. fig. 1. scheme of system with mechanical compression of secondary steam the primary steam supplied in the heating shirt provides an increase in the energy potential of the solution to the boiling point, and the prescribed pressure level is stabilized, which results in the inclusion of a portion of the secondary steam with the compressor. coupling of steam is accompanied by an increase in its temperature. fig. 2. diagram of the system with mechanical compression of the secondary steam in coordinates t-s this means that the energy potential of the secondary steam, determined at the temperature 1 t , is supplemented by the compression energy. taking into account the speed of compression in the first approximation, we will consider this process as adiabatic, and the theoretical work of the compressor is equal to the difference between enthalpies '' 2 i and '' 1 i , that is, '' 1 i '' 2 i theor  (7) from this assumption comes the dependence k 1k 1 р 2 р 1 т 2 т           for an adiabatic process with gas it is possible to be transferred to the second steam. however, for the compilation of energy balances and, taking into account the possible need to increase the motive factor in the heat transfer process, it is possible to get into the zone of correlation between parameters 2 і and the heat of vaporization r, in which the indicated balances are not achieved. an increase in the temperature of the secondary steam, which corresponds to a certain value '' 2 i , means simultaneous reduction of the heat of vaporization r and hence the increase of the difference should be deduced from it. r '' 2 iδ  (8) if the thermodynamic parameters of the wort melting remain constant during a flow of time, this should correspond to a stabilized input flow of secondary steam. in this case, the condition must be fulfilled (fig. 3). it can be seen from the figure 3 that such condition can be achieved, so that the flow of the secondary steam will be stabilized. кm primary steam condensate of secondary steam 1 2 2' 3' 1' 4 т s ev q 1 5 6 4 3 2 0 т2 т1 condensate of primary steam food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 341 '' 1 ir '' 2 iδ  (9) at the same time, technological requirements, for example, of wort boiling may apply to situations in which the flow of secondary steam can increase or decrease. obviously, the achievement of such changes will be related to the parameters δ and pressure c p , and obtaining the given stabilized parameters in the compressor with the use of modern methods of regulating does not cause the complexity. fig. 3. diagram to determine the parameter δ in figure 1 the system is shown with two separate heating surfaces. obviously, such an option is possible in its existence, but the prospect should have a variant, in which the transformed flow of the secondary steam returns to the heating shirt. this is due to the fact that the superficial heating shirt of the wortboiling apparatus, in its absolute value, is approaching the surfaces of the apparatus itself, which are in direct contact with the wort. hence the conclusion is that the creation of an additional cooking surface with equivalent parameters looks problematic, which is amplified by the existing limitations of the temperature of the heating surface. however, the system with an additional heating surface can be used in devices with significant dimensions, for which designers are required to provide internal additional heating surfaces or external circulating contours with such surfaces. although the root cause of such problems is known and has the form of a correlation of the heating surface to the volume of the apparatus, however, in such cases the use of heat pumps based on the mechanical compression of the secondary steam is perspective. of course, the presence of the internal surface of the heating should affect the hydrodynamics of the steam-liquid medium in the apparatus. by the traditional implementation of cylindrical apparatus with semi-spherical bottoms, a stable circulation circle with a rising peripheral flow and a downstream center is achieved. the introduction of an additional heating surface does not displace the ascending circular peripheral flow, but it is accompanied by a central upstream flow, and the downstream flow will be formed between the two ascending. however, the features of the heating surfaces and their arrangement do not neglecte the main conclusion concerning the possibility and necessity of full use of the potential of the secondary steam. moreover, such use must be carried out within the closed circuit circuits. the practical implementation of this idea continues to interfere with the possible presence of air in the secondary steam. such air in the system of heat transfer forms air "coat" and requires special intervention. the limitations of this disadvantage are achieved by blowing out or discharging part of the steam-water mixture into the environment or providing the whole system of airtight nature so that the air does not enter the circulation circuit along with the secondary steam. recent features of the arrangement of systems with closed circulating power i r р, мpа 0,1 рk δ r  2 i 0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 342 supply circuits refer to cases in which the output of the formed secondary steam is compulsory. this leads to the use of a heatexchanging surface. an example with a wortboiling device is not typical, but it was considered in connection with the need to achieve three results of heat treatment, namely, the achievement of an aseptic state, a deep extraction of substances from the hops, and output from the process of 10% liquid phase in the form of secondary steam. the choice of the thermodynamic parameters of the system with the mechanical compression of the secondary steam is carried out taking into account the desired temperature difference on the surface of the heat transfer. received characteristics of saturated water steam in the range of possible use are given below (table 1). тable 1 parameters of saturated water steam (by pressure) р, mpа t, °c 'i , kj/kg '' i , kj/kg r, kj/kg ' s , kj/(kg∙к) '' s , kj/(kg∙к) 0.100 0.120 0.140 0.160 0.180 0.200 0.220 0.240 99.640 104.81 109.33 113.32 116.94 120.23 123.27 126.09 417.40 439.40 458.50 475.40 490.70 504.80 517.80 529.80 2675 2683 2690 2696 2702 2707 2711 2715 2258 2244 2232 2221 2211 2202 2193 2185 1.3026 1.3606 1.4109 1.4550 1.4943 1.5302 1.5630 1.5929 7.360 7.298 7.246 7.202 7.163 7.127 7.096 7.067 numerical energy relations in this case are determined by the equations (7) (9), and the coefficient of recuperative return of the energy of the secondary steam is reduced to the form: 1 і 2 і (c) r theor (c) r β    (10) where the parameter of the compressed steam corresponds to the heat of condensation )(сr . during the transition to the use of a thermocompressor in the form of an ejector (fig. 4), the necessary pressure in the system is formed by a combination of the primary and secondary steam to form a mixture that corresponds to the equation of thermal and material balance:       , sec m prim m mix m ; sec і sec m prim і prim m mix і mix m (11) where mix m , prim m and sec m – respectively the mass of the mixture, primary and secondary steam. fig. 4. scheme of the ejection thermocompressor: 1 working nozzle; 2 receiving camera; 3 mixing chamber; 4 – diffuser 1 secondary steam primary steam 1 2 2 1 2 3 4 steam mixture food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 343 hence the relation between masses or mass flows:   mix і prim і sec і mix і sec m prim m    (12) in the absence of a heat pump, the primary steam consumption to generate the secondary one will be: prim і sec і sec m prim m    (13) the perfection of jet apparatustransformers is determined by the magnitude of the efficiency, which is the ratio of the exergy received by the secondary steam flow to the exergy spent by the flow of the primary steam:   mix е prim е sec е mix eu η    (14) where mix e , prim e and sec e – specific exergy of mixture, primary and secondary streams respectively; prim m sec mu  – ejection coefficient; sec m and і prim m – mass flows of the secondary and primary steam respectively. specific exergy is determined by the dependence:   env s 0 s env т env і 0 іе  , kj/kg (15) where 0 і and 0 s – respectively specific enthalpy and specific entropy of the primary steam in the isoentropically inhibited state; env і and env s –specific enthalpy and the specific entropy respectively of the primary steam in a state of equilibrium with the environment. let’s compare the efficiency of the ideal and real jet transformers at identical initial parameters of the primary and secondary steam: idem prim р  ; idem prim е  ; idem sec р  ; idem sec і  ; idem mix р  . in fig. 5 we have a diagram in the coordinates i-s with the specified parameters of flow interactions. the state of the primary flow of steam in front of the apparatus is determined by the point p: entropy prim s ; enthalpy prim i ; pressure prim p . the state of the secondary steam flow in front of the transformer is determined by the point n: entropy sec s ; entalpiy sec i ; pressure sec p . fig. 5. parameters of the interaction of the flow of ejection thermal transformer in terms of the coefficient of ejection ' u in an ideal apparatus, enthalpy ' mix i is determined on the basis of the law of conservation of energy: ' u1 sec s ' u prim i ' mix i    (16) in an ideal jet device without loss, the entropy of the system does not change. therefore, the entropy of the compressed stream is: ' u1 sec s ' u prim s ' mix s    (17) the point ' с in the section p-n, which і p s c' n с pmix psec іprim. іmix. іsec. іmix. sprim s'mix ssec. smix δsc. pprim food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 344 connects in the diagram points p and n corresponding to the initial thermodynamic parameters of the system satisfies equations (16) and (17). obviously, in a nonideal ejection machine, the process takes place with losses, so the entropy of the system increases. then some point c may correspond to a state of compressed stream, which entropy is ' mix s mix s  . under the same pressure ( idem 0 p  ), the enthalpy of the compressed stream in the real apparatus is ' mix i mix i  . herewith: u1 sec us prim i mix i    (18) as sec i prim i  , then ' uu  corresponds to the condition ' mix i mix i  . this means that for given pressures prim p , sec p and mix p the ejection coefficient of actual jet engine is less than ideal. taking into account the condition (14) for the ideal compressor, we will write:   mix е prim е sec е mix eu η    (19) where ' u – the ideal compressor ejection coefficient; ' mix e – specific exergy of the compressed stream in the ideal compressor. dividing the condition (14) into (19), we obtain: mix eprime mix ' e prim e sec emix ' e sec e mix e ' u u η       (20) it is obvious that ' mix e mix e  . condition (15) follows: δs env t mix δi ) ' mix s mix (s env t ' mix i mix i ' mix e prim e    (21) where mix i and mix s – enthalpy and entropy of the medium in a state that corresponds to a point с; mix ' i і mix ' s – the same in the state of the point ' c . but mix δs med t mix δi  ; approximately 2 ' c t c t med t   , where ct and ' c t – appropriate temperatures to the points с and с'. then s) env t med (t ' mix e mix e  (22) at env t med t  ' mix e mix e  . as c e ' c e  , then 1 sec e ' mix e sec e mix e    and 1 mix e prim e ' mix e prim e    hence, ' u u η  . (23) condition (23) indicates that for the same pressures mix p , the efficiency of the real compressor is greater than the ratio of the ejection coefficients of the real and ideal compressors, since internal irreversible losses along with the reduction of the ejection coefficient result in the increase of the specific exergy of the compressed flow. 4. conclusion secondary energy resources of food industries are often represented by thermal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 345 energy of fluid media or secondary steam. the use of energy potentials of liquids or their approximate flow structures is more often achieved by heat exchange with countercurrent flows. the result of this interaction is the energy recovery. it is shown that for the use of secondary energy resources of steam or gas phases, the regenerative heat exchange by physical strength can be supplemented by regeneration and the proving of thermodynamic parameters to values that allow to remove from systems or to limit the action of energy primary sources. regeneration of the secondary steam allows the process of its subsequent condensation to return to the environment the potential of heat of vaporization. this means that in the systems in which the increase of thermodynamic parameters of steam due to its compression is restored, there is a use of carnot's reverse cycle. such application takes place both in closed and open loops. the work of compressing the secondary steam is much less than the heat of its condensation. that is what determines the high efficiency of regenerative regimes. however, the direct use of secondary steam in condensation modes eliminates the expediency of the presence of thermal pumps in such systems. the basis of the calculations of secondary steam recovery systems is material and thermal balances. the description of the physical processes that accompany the work of the thermocompressors is subject to mathematical modeling based on the laws of conservation of energy, mass conservation and conservation of impulses. the assessment of the suitability for using secondary energy resources is based on exergy methods in order to determine the directions for their further improvement. for identical pressures in the mixing chamber, the efficiency of the real compressor is greater than the ratio of the ejection coefficients of the real and ideal compressors, since internal irreversible losses along with the reduction of the ejection coefficient result in the increase of the specific exergy of the compressed flow. mathematical models of transformation of energy-material flows of secondary steam are developed. it is shown that the secondary steam with respect to the medium in which it is generated has an exergy that is zero, since without the transformation of its thermodynamic parameters, it is impossible to return the heat of condensation. 5. references [1]. samilyenko s. methodological bases of optimization of heat-technological complex of sugar production, k.: nuft, 23, (2014). [2]. samilyenko s., vasilenko o., bulyandra a. methodological principles of thermodynamic analysis of heat exchange systems of sugar production, scientific works of national university of food technologies, 23 (1): 61-70, (2012). [3]. klemesh j., panchyk r. synthesis of the optimal structure of heat transfer systems, theoretical foundations of chemical technology, 11 (4): 488-499, (1987). [4]. sama d.a. the use of the second law of thermodynamics in process design, journal of energy resources technology, 117: 179-184, (1995). [5]. brodyansky v., verhiver g., karchev y. exergy calculations of technical systems. kyiv: scientific thought, 360, (1991). [6]. brodyansky v. prospects for the use of low ambient temperatures in heat and refrigeration engineering, refrigeration equipment, 8: 15-17, (2003). [7]. sokolenko a. intensification of heat and mass exchange processes in food technologies. kyiv: phoenix, 536, (2011). [8]. sokolenko a. energy transformations and energy saving in food technologies. kyiv: phoenix, 484, (2012). [9]. sokolenko a., poddubny v., chagayda a. energy transformations in food technologies based on closed circuits. kyiv: kondor,300, (2015). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anatoly sokolenko, olexandr shevchenko, konstantin asylkivsky, oleg stepanets, inna vinnichenko, prospects of using secondary energy resources, food and environment safety, volume xviii, issue 4 – 2019, pag. 337 346 346 [10]. shevchenko a., vinnichenko o., stepanets o., boyko o. features of transformations of material and energy flows in fermentation media, scientific works of national university of food technologies, 23 (3): 107-115, (2017). [11]. sokolenko a., shevchenko a., bilyk o., mironenko s. energy potentials of gas-liquid media, scientific works of national university of food technologies, 24 (1): 107-118, (2018). 327 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3 2015, pag. 327 330 the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice *anca-mihaela sidor1 1stefan cel mare university of suceava, food engineering faculty, romania, anksidor@yahoo.com * corresponding author received august 26th 2015, accepted september 29th 2015 abstract: the main goal of this study is to quantify the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) in different forms: berries and juice. the relevance of the subject approached in this paper lies in the importance of the sea buckthorn as a plant with immense medicinal and therapeutic potential. the analyzed sea buckthorn fruits were collected in botoșani county, romania and the sea buckthorn juice was obtained trough the cold pressing of the sea buckthorn berries, followed by pasteurization. the analysis of samples was done using atomic emission in plasma coupled with mass spectrometry technique with icp-ms agilent technologies 7500 series. five of the essential minerals – calcium, iron, magnesium, manganese, sodium, and four of the trace elements chromium, cobalt, selenium, vanadium were determined, all of them necessary for the properly functioning of the human organism. considering the presence of all these valuable essential minerals and oligoelements in sea buckthorn berries and juice, and from the scientific knowledge of their importance, it can be stated that sea buckthorn should be more and more used as one of the main nutritional sources in the diet. keywords: sea buckthorn, essential minerals, oligoelements, nutritional source. 1. introduction adequate intakes of the mineral nutrients are only obtained by following a pattern of food selection, especially the inclusion of plant foods [1]. in our “plastic” world the role of the herbs and the medical products is rising. one of the important herbs is sea buckthorn [2]. sea buckthorn is a medicinal and aromatic plant that is both cultivated and naturally grown in various parts of the world. the distribution of sea buckthorn ranges from the himalayan regions, including india, nepal, bhutan, pakistan and afghanistan, to china, mongolia, russia, kazakhstan, hungary, romania, switzerland, germany, france, and britain, and northwards to finland, sweden, and norway [3]. sea buckthorn (hippophae rhamnoides l.) is widely grown all over the world for its valuable berries [4]. sea buckthorn, an ancient crop with modern virtues has recently gained worldwide attention, mainly for its nutritional and medicinal value [5]. medicinal applications of sea buckthorn have been well-documented since ancient times, and are still in use in europe and asia [6]. the sea buckthorn berries contain different kinds of nutrients and bioactive compounds including vitamins, fatty acids, free amino acids and elemental components [5]. the main goal of this study is to quantify the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) in different http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 anca-mihaela sidor, the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice, food and environment safety, volume xiv, issue 3 – 2015, pag. 327 – 330 328 forms: berries and juice. the relevance of the subject approached in this paper lies in the importance of the sea buckthorn as a plant with immense medicinal and therapeutic potential [7]. 2. materials and methods 2.1. sample material fruits of sea buckthorn (h. rhamnoides l.) were collected in botoșani county, romania, gps location: n: 47°39'44.9", e: 26°48'15.2" (fig. 1), from ten randomly selected 5-year old male and female trees, grown at a 2×3 m spacing without fertilization and irrigation. the fruits were preserved by freezing at -30°c until the analyses could be carried on. fig. 1. the sampling area the analyzed sea buckthorn juice was obtained trough the cold pressing of the sea buckthorn berries, followed by filtration and preservation through htst (high temperature short time) pasteurization at 82-85°c for 15 seconds. then, the syrup was bottled in glass containers which were cooled down to 45°c temperature. 2.2. sample preparation 5 grams of sea buckthorn fruits, respectively 5 grams of sea buckthorn juice, were weighed with an accurate of 0,01 g. the organic matter in the samples was destroyed by calcination in an electric furnace at a 600°c temperature for 4 hours. the resulted ash was transferred into a 50 ml volumetric flask, wherein it was dissolved using a nitric acid and deionized water solution, so that the concentration of the nitric acid in the resulting solution into the volumetric flask to be 1%. 2.3. apparatus research on trace-level concentrations requires analytical techniques that are versatile, robust, of the highest sensitivity and capable of providing accurate and reliable information on concentrations and species identity. with respect to most of these criteria, determination of trace elements by icp ms is performing extremely well and is unchallenged by other ms techniques [8]. therefore, the analysis of samples was done using atomic emission in plasma coupled with mass spectrometry technique with icp-ms agilent technologies 7500 series device with a detection limit of 10-12 [9]. 3. results and discussion within the study, there was determined the mineral content of the sea buckthorn berries and juice: five of the essential minerals – calcium, iron, magnesium, manganese, sodium, and four of the trace elements chromium, cobalt, selenium, vanadium (tab. 1), all of them necessary for the properly functioning of the human organism. b o t o ș a n i c o u n t y n: 47°39'44.9" e: 26°48'15.2" food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 anca-mihaela sidor, the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice, food and environment safety, volume xiv, issue 3 – 2015, pag. 327 – 330 329 tab. 1. the mineral content (ppb) in the samples content (ppb) in sea buckthorn fruits content (ppb) in sea buckthorn juice calcium 120.000 60.000 chromium 60.000 32.000 cobalt 150 88 iron 8.500 4.900 magnesium 4.900 4.500 manganese 7.100 3.500 selenium 120.000 56.000 sodium 9.900 11.000 vanadium 270 160 analyzing the data set obtained from the analysis of samples through the mentioned procedure, it can easily bee seen that all targeted mineral elements are present in significant proportions in both studied samples. the data presented in the table above illustrates the importance of sea buckthorn as a source of minerals for the human body. it is noteworthy the high selenium content, known for its antioxidant properties and role in boosting immunity and fertility [10]. it is also remarkable the semnificative content in calcium of the sea buckthorn, a mineral known for its role in maintaining solidity of bones in the body, but also for the functioning of nerve and muscle cells [11]. an important issue resulted after a closer examination of the data is the difference between the mineral intake brought by the seabuckthorn juice consumption compared to sea buckthorn berries consumption. it can be remarked a halving of the mineral content when processing the fruits for obtaining the sea buckthorn juice. besides determining the essential minerals and trace elements content, another goal of the study was to detect traces of heavy metals in the analyzed sea buckthorn samples. therefore, it was concluded that the intake of heavy metals in the body brought by the consumption of sea buckthorn berries and juice is negligible, significantly below the maximum amount allowed by regulations (fig. 1). fig. 1. the heavy metal content of the samples vs. the maximum allowable amount 4. conclusion in conclusion, it can be noted that the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice is considerable, especially by the presence of high amounts of calcium and selenium. at the same time, there are noteworthy the low heavy metals' levels in the analyzed samples. therefore, considering the presence of valuable essential minerals and oligoelements in sea buckthorn berries and juice, and from the scientific knowledge of their importance, it can be stated that sea buckthorn should be more and more used as one of the main nutritional sources in the diet. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 sea buckthorn fruits sea buckthorn juice maximum allowable amount sample the mineral element amount (mg/kg) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 anca-mihaela sidor, the intake of minerals in the diet brought by the consumption of sea buckthorn (hippophae rhamnoides l.) berries and juice, food and environment safety, volume xiv, issue 3 – 2015, pag. 327 – 330 330 5. references [1] anderson j.j.b., nutrition of minerals in relation to human function, reference module in biomedical sciences, (2014). [2] vincze i, banyai-stefanovits e, vatai g., concentration of sea buckthorn (hippophae rhamnoides l.) juice with membrane separation, separation and purification technology,57(3): 455-460, (2007). [3] zeb a., chemical and nutritional constituents of sea buckthorn juice, pakistan journal of nutrition, 3: 99-106, (2004). [4] górnaś p, šnē e, siger a, segliņa d., sea buckthorn (hippophae rhamnoides l.) vegetative parts as an unconventional source of lipophilic antioxidants,saudi journal of biological sciences, in press, accepted manuscript, (2015). [5] bal l. m., meda v., naik s.,satya s., sea buckthorn berries: a potential source of valuable nutrients for nutraceuticals and cosmoceuticals, food research international, 44(7): 1718-1727, (2011). [6] zeb a., important therapeutic uses of sea buckthorn (hippophae); a review,journal of biological sciences, 4: 687-693, (2004). [7] suryakumar g., gupta a., medicinal and therapeutic potential of sea buckthorn (hippophae rhamnoides l.), journal of ethnopharmacology, 138(2): 268–278, (2011). [8] ammann a.a., inductively coupled plasma mass spectrometry (icp ms): a versatile tool, j. mass spectrom., 42: 419–427, (2007). [9] amariei s., gutt g., oroian m., bodnar a., study on toxic metal levels in commercial marine organisms from romanian market, ovidius university annals of chemistry, 25 (2): 59-64, (2014). [10] burk r. f., selenium, an antioxidant nutrient, nutrition in clinical care, 5: 75–79, (2002). [11] ilich j.z., kerstetter j.e., nutrition in bone health revisited: a story beyond calcium, journal of the american college of nutrition, 19(6): 715-737, (2000). http://www.sciencedirect.com/science/article/pii/b9780128012383002348 http://www.sciencedirect.com/science/journal/aip/01620134 study of chemical composition influence on the rheological and textural properties of various types 282 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 32015, pag. 282 292 study of chemical composition influence on the rheological and textural properties of various types of animal and vegetal pâté *elena todosi sănduleac1, gheorghe gutt1, andreea ianovici iordache1, silviu gabriel stroe1 1faculty of food engineering, stefan cel mare university of suceava, romania sanduleacelena@yahoo.com, g.gutt@fia.usv.ro, andreea_iordache88@yahoo.com, silvius@fia.usv.ro *corresponding author received september 5th 2015 , accepted september 28th 2015 abstract: the aim of the study was to establish the influence of chemical composition of animal and vegetal pâtés on the rheological and textural properties.the analysis and methods utilized in the present study were the following: determination of dry substance content, moisture content, starch, protein and fat using chemical methods and rheological and textural properties utilizing tpa method. the main chemical parameter to be determined was starch content from wheat flour or starch from corn used as raw materials. the results obtained confirm the fact that starch added in animal and vegetal pâtés could have a strong influence on their rheological and textural properties. for tpa determination the samples were cut as cubes with 20x20 mm sides, displacement of 10 mm/min. and the result interpretation was made using the mesur gauge software of mark 10 texturometer. the main functions of the starch in food products are the following: thickness agent, coloidal stabilizer, agent for moisture retention, gelling agent, binding agent and covering agent. the main role of starch added in pâtés is of thickness agent or agent for moisture retention. keywords: animal and vegetal pâtés, tpa, texturometer, viscosity, hardness, adhesivity, cohesivity, elasticity, gumminess, chewingness, starch, moisture, protein, fat. 1. introduction pâté is one of the dishes with creamy consistency, homemade or industrial obtained, of various ingredients such as: pork meat, beef, fish (tuna), ham, liver, butter, cream, spices, salt, food additives, flavours. the most appreciated is foie gras pâté, made from goose or duck liver, grown especially for this purpose. the industrial manufactured pates are very different from foie gras made from goose or duck liver. the typical composition of an industrial pâté includes: 20% liver, water, meat, non-hydrogenated vegetable oil, soybean protein, starch from corn, iodized salt, condiments, starch from wheat, condiment extract, mustard, flavours, stabilizers, glucose syrup, emulsifiers, thickness agents, antioxidants, dye, preservatives. on the market there are also vegetal pâtés which reproduces the composition of pâtés made from liver-meat, containing the following: water, non or hydrogenated vegetable oil (margarine), soybean protein, starch from corn, iodized salt, condiments, starch from wheat, condiment extract, mustard, flavours, stabilizers, glucose syrup, emulsifiers, thickness agents, antioxidants, dye, sugars, dehydrated vegetables, acidifier, carob flour, paprika extract. several studies regarding the composition of different types of pates and its influence on the textural properties of pates were conducted worldwide. the http://www.fia.usv.ro/fiajournal mailto:@yahoo.com mailto:g.gutt@fia.usv.ro mailto:andreea_iordache88@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 283 effect of fat content (30%, 35% and 40%) on physical, microbial, lipid and protein changes, during chill storage, of foal liver pâté [2], represents one of the studies regarding the composition of pates. the antioxidant effect of two plant essential oils (sage and rosemary) and one synthetic antioxidant (bht) on refrigerated stored porcine liver pâté (4 °c/90 days) was also evaluated [1]. another study is regarding the effect of fat content on physicochemical properties and lipid and protein stability of foal liver pâté. for this purpose, two batches (10 units per batch) of foal liver pâté with different pork back fat content [30% (30f) and 40% (40f)] were manufactured [3]. all physicochemical parameters were affected by the storage time, especially instrumental texture, which indicated emulsion instability during storage due to increased hardness, elasticity and gumminess [4]. other researchers have studied the physicochemical characteristics and oxidative stability of liver pâtés with different fat content. pâtés with high-fat contents presented a smaller cooking yield than pâtés with medium and low fat contents, mainly due to a higher loss of lipids. fat content was closely related to the calorific value of pâtés, these being more calorific in those with higher fat contents [5]. 2. matherials and methods the aim of the study was to establish the correlation between chemical composition (starch content and moisture, dry substance, proteins and fat of nine pâtés samples: three pork pâtés, three chicken pâtés and three vegetal pâtés having different chemical compositions. physicochemical determinations were made such as: moisture, starch, heavy metals and they were correlated with the textural parameters by using tpa (texture profile analysis) as determination method. for tpa determination the samples were cut as cubes with 20x20 mm sides, displacement of 10 mm/min., and data interpretation using mesur gauge software of mark 10 texturometer. 2.1. determination of physico-chemical properties. physico-chemical analysis. quality control includes physico-chemical analysis of canned vegetal and animal origin patees and has the following physico-chemical parameters:  moisture content determination(dry substance) in pâtés or water content sr iso 1442: 2010, [6]  method of drying in the oven. the principle of the method is water evaporation from the sample by heating in the oven at 120 ± 2° c until a constant mass is reached [6].  determination of protein content (kjeldahl method) sr iso 937: 2007, [10] represents the nitrogen content of meat and other meat products and consists in determination of nitrogen quantity corresponding to nitrogen produced from the decomposition of proteins. principle of method: a sample digestion of concentrated sulphuric acid which transforms organic nitrogen in ammonium ions under the catalytic action of copper sulphate (ii); alkalinization, distillation of ammonia released in excess of boric acid solution, titration with hydrochloric acid of ammonia combined with the boric acid and calculation of nitrogen content from the samples, starting from the produced ammonia. where: v0volume of hydrochloric acid solution 0,1 n used as blank, expressed in ml; v1-volume of hydrochloric acid solution 0,1 n used for determination, expressed in ml; m-mass of the sample, expressed in g. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 284  determination of fat content (soxhlet method) sr iso 1444: 2008, [11] principle of method: fat extraction using an adequate solvent in a closed system through repeated jets, solvent removal by drying in the oven, dosing the fat extracted by weighting. the extraction can be made directly from the food or product or dry residue. the fat content of the sample it follows from the formula: fat % 100 1  m m where: m-quantity of fat extracted expressed in g (difference between the weight of the balloon with fat extracted after the balloon has dried); m1-quantity of product results obtained: qualitative identification of starch from meat products is realized when it comes to products without starch addition and quantitative identification reffers to products made from recipes in which auxiliary materials based on starch are included. the identification could be made on a sample extracted from product or directly on the section of the product.  determination of starch content (elser method) sr iso 9297: 2001 [7] principle of method: the starch is converted by acid hydrolysis into direct reducing sugar (glucose), using fehling solution as oxidizing solution and iodometric titration. results are expressed in % starch. expression of results: starch content is calculated with the following formula: (1) where: mıthe inverted sugar mass, expressed in mg, corresponding to the volume of the iodine used for titration (cm3); mmass of product for determination, in g; vvolume of solution for determination from the flask, in cm3 ; 1000flask volume; 0.9starch conversion factor from invert sugar. 2.2 texture parameters determination for tpa determination the samples were cut as cubes with 20x20 mm sides, displacement of 10 mm/min. and the results interpretation was made using the mesur gauge software of mark 10 texturometer. fig. 1 load diagram in two cycles used for texture profile analysis (tpa). mechanical textural properties: hardness, cohesivity, viscosity, elasticity, adhesivity, fracturability, chewingness, gumminess [8], [9]. table 1 primary and secondary texture parameters defined according to iso 11036/2007 [9] the primary parameters of texture parameter texture popular adjectives notation (figure1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 285 hardness soft, firm, hard h cohesivity brittle, crisp, friable, chewable, hard, soft, short and firanaceous, pasta gummy a2/a1 viscosity fluid, thin, viscous c elasticity plastic, malleable, elastic d2/d1 adhesivene ss sticky, gummy, greasy a3 secondary parameters of texture fracturabilit y (fragility) it is related to the primary parameters of hardness and cohesion b gumminess it is related to the primary parameters of hardness and cohesion of semisolid foods when cohesion is reduced hxa2/a1 chewiness it is related to the primary parameters of hardness, cohesion and elasticity hxa2/a1xd 2/d1 3. results and discussion to study the influence of the chemical composition of pâtés samples on their texture parameters it was used the statistical analysis of experimental data. in order to identify and quantify the relations between the variables studied, the calculation of pearson correlation coefficient (r) was used, as well as the principal component analysis (pca). these methods are widely utilized in statistic analysis for measuring linear relationships, positive or negative between variables. the experimental data were processed using the software component analyze-it for microsoft office excel (v. 4.10 – the evaluation version) pearson correlation matrix only between the dependent variables (texture parameters) is shown in table 3. fig. 2 dry substance and moisture content of animal and vegetal pâtés fig. 3 starch content of animal and vegetal pâtés 2.3. stereomicroscope images of animal and vegetal pâté samples 66.610 60,837 57,977 62,125 66,224 72.270 63,055 61,105 62.660 33.390 39,163 42,023 37,875 33,776 27.730 36,945 38,89537.340 0 10 20 30 40 50 60 70 80 p1 p2 p3 p4 p5 p6 p7 p8 p9 moisture dry substance dry substance and moisture content of animal and vegetal pâtés % 15,95 12,33 14,85 10,06 12.10 7,28 5,25 6,25 11,39 0 2 4 6 8 10 12 14 16 18 p1 p2 p3 p4 p5 p6 p7 p8 p9 starch content of animal and vegetal pâtés pâté samples % food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 286 fig. 4 pork pâté p1 fig. 5 pork pâté p2 fig. 6 pork pâté p3 fig. 7 chicken pâté p4 fig. 8 chicken pâté p5 fig. 9 chicken pâté p6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 287 fig. 10 vegetal pâté p7 fig. 11 vegetal pâté p9 fig. 12 vegetal pâté p8 fig. 13 tpa diagram pork pâté p1 fig. 14 tpa diagram pork pâté p2 fig. 15 tpa diagram pork pâté p3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 288 fig. 16 tpa diagram chicken pâté p4 fig. 17 tpa diagram chicken pâté p5 fig. 18 tpa diagram chicken pâté ( p6) fig. 19 tpa diagram vegetal pâté p7 fig. 20 tpa diagram vegetal pâté p8 fig. 21 tpa diagram vegetal pâté p9 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 289 table 2 texture properties and chemical composition of animal and vegetal pâtés table 3 pearson correlation matrix between the dependent variables pearson's r viscosity hardness adhesivity cohesivity elasticity gumminess chewingness viscosity 1 hardness -0.177 1 adhesivity 0.385 -0.502 1 cohesivity 0.241 0.682 -0.288 1 elasticity -0.480 -0.338 0.281 -0.802 1 gumminess -0.085 0.982 -0.445 10.801 -0.447 1 chewingness -0.377 0.929 -0.421 0.482 0.002 0. 0.889 1 by studying the values of correlation coefficient from the table above it is found very strong positive associations between the texture parameters hardness and gumminess (r = +0.982) and hardness and chewingness (r = +0.929), as well as very strong negative associations between cohesivity and elasticity (r = -0.802). n r . c r t. s a m p le t y p e v is c o si ty [n ] h a r d n e ss [n ] a d h e si v it y [n ·m m ] c o h e si v it y e la st ic it y g u m m in e ss [n ] c h e w in g n e ss [n ·m m ] s ta r c h [ % ] d r y s u b st a n c e [% ] m o is tu r e [% ] p r o te in [% ] f a t [% ] 1. pork pâté (p1) 0.520 2.440 7.337 0.299 0.472 0.731 0.345 15.950 42.650 57.350 9.000 16.000 2. pork pâté (p2) 0.300 3.440 3.786 0.286 0.671 0.986 0.662 12.330 39.163 60.837 11.000 13.900 3. pork pâté (p3) 0.340 2.640 4.494 0.246 0.678 0.651 0.441 14.850 29.023 57.977 10.600 14.700 4. chicke n pâté (p4) 0.260 2.660 3.531 0.247 0.688 0.659 0.453 10.060 37.875 62.125 9.000 17.000 5. chicke n pâté (p5) 0.240 3.500 3.048 0.275 0.747 0.965 0.721 12.100 33.776 66.224 9.100 10.800 6. chicke n pâté (p6) 0.280 4.640 3.891 0.304 0.767 1.413 1.084 7.280 28.730 71.270 9.100 10.400 7. vegeta l pâté (p7) 0.260 2.040 3.649 0.264 0.793 0.539 0.427 5.250 36.945 63.055 2.000 26.000 8. vegeta l pâté (p8) 0.240 1.880 8.650 0.209 1.153 0.394 0.454 6.250 38.895 61.105 2.100 27.300 9. vegeta l pâté (p9) 0.360 2.220 4.572 0.202 1.072 0.450 0.482 11.390 37.340 62.660 3.100 21.000 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 290 low negative associations is found between viscosity and gumminess (r = 0.085), as well as low positive associations between elasticity and chewingness (r = +0.002). also, in order to obtain a complete characterization of the texture parameters variance it must be studied the association between the independent variables (composition of pâtés samples) and dependent variables (texture parameters). thus, the pearson correlation matrix between the independent and dependent variables is shown in table 4. table 4 pearson correlation matrix between independent and dependent variables pearson's r viscosity hardness adhesivity cohesivity elasticity gumminess chewingness starch 0.703 0.043 0.014 0.213 -0.589 0.052 -0.265 dry substance 0.376 -0.564 0.470 -0.117 -0.026 -0.483 -0.606 moisture -0.541 0.707 -0.465 0.267 0.220 0.658 0.876 protein 0.218 0.645 -0.360 0.635 -0.763 0.651 0.334 fat -0.136 -0.847 0.499 -0.664 0.613 --0.828 -0.61717 from studying the values of the correlation coefficient in table 2 results very strong negative associations between the independent variable fat and the dependent variable hardness (r = -0.847), which indicates the fact that an increased concentration of fat in pâtés samples leads to a lower value of hardness and vice versa. also, it shows a very strong negative association between the independent variable fat and dependent variable gumminess (r = -0.828), which indicates the fact that an increased concentration of fat in pâtés samples leads to a lower value of gumminess and vice varsa. a high positive value of pearson correlation coefficient it is noted between independent variable moisture and dependent variable chewingness (r = +0.876). values of pearson correlation coefficient close to 0 is observed between the independent variable dry substance and dependent variables adhesivity and hardness (r = +0.014, respectively r = +0.043). principal component analysis (pca) by utilizing statistic method principal component analysis (pca) it is shown the possible correlations between variables. the relative positions between the dependent variables are found in figure 22 fig. 22 principal component analysis (pca) relationship between studied independent and dependent variables food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 291 it is shown that the first two principal components explain 82.8 % of data variance (pc1 = 55.9%; pc2 = 26.9%). vectors. position of variables hardness and gumminess towards the first principal component (pc1) from figure 2 shows that between them is a strong, direct connection. the correlation between them is positive, which can be observed also due to the small angle between the corresponding the position of variables hardness and chewingness also shows a very strong positive connection. between cohesivity and gumminess, as well as between hardness and chewingness it can be observed a very strong positive association, which results from the very small angle between the vectors corresponding to the two pairs of variables. vectors positions corresponding to variables cohesivity and elasticity shows a very strong negative association. 4. conclusion this experimental study was conducted to highligh the influence of starch addition on the rheological, textural and technological properties of the animal and vegetal pâtés. the main functions of starch in food products are the following: thickening agent; colloidal stabilizer; agent for retaining moisture; gelling agent; binding agent; covering agent. the main role of starch addition in animal and vegetal pâtés is of thickening agent and agent for retaining moisture. new studies have highlighted the fact that not all types of starch have the same effect on the body. one of them are digested very quickly and produces a sharp increase of blood glucose, meanwhile other types of starch are slowly digested, having a minor effect on the glycemic index. moreover, the resistant starch it is not digested in the small intestine and does not increase blood glucose level almost at all. from the experimental results obtained from the three types of pâtés: pork, chicken and vegetal the influence of the starch content is very obvious as well as the influence of chemical composition on the rheological and textural properties. using statistic methods of pearson correlations and principal component analysis we can remark semnificative correlations between dependent variables – texture parameters of pâtés samples, at different values of independent variables (starch, dry substance, moisture, protein and fat and certain formed groups of these variables. 4. references [1]. estévez m., ventanas s., cava r., effect of natural and synthetic antioxidants on protein oxidation and colour and texture changes in refrigerated stored porcine liver pâté, meat science, 74 (2): 396–403, (2006) [2]. lorenzo j.m., pateiro m., garcía fontán m.c., carballo j., effect of fat content on physical, microbial, lipid and protein changes during chill storage of foal liver pâté, food chemistry, 155: 57–63, (2014) [3]. lorenzo j.m., pateiro m., influence of fat content on physico-chemical and oxidative stability of foal liver pâté, meat science , 95(2): 330–335, (2013) [4]. amaral d.s., silva f.a.p., bezerra t.k.a., arcanjo n.m.o., guerra i.c.d., dalmás p.s., madruga m.s., effect of storage time and packaging on the quality of lamb pâté prepared with ‘variety meat, food packaging and shelf life, 3:39-46 (2015)[5]. estévez m., ventanas s., cava r., physicochemical properties and oxidative stability of liver pâté as affected by fat content, food chemistry, 92(3): 449–457, (2005) [6]. sr iso 1442/2010 moisture content determination [7]. sr iso 9297/2001 starch content determination http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/journal/03091740 http://www.sciencedirect.com/science/journal/03091740/74/2 http://www.sciencedirect.com/science/article/pii/s0308814614000594 http://www.sciencedirect.com/science/journal/03088146 http://www.sciencedirect.com/science/journal/03088146 http://www.sciencedirect.com/science/journal/03088146/155/supp/c http://www.sciencedirect.com/science/article/pii/s0308814614000594 http://www.sciencedirect.com/science/journal/03091740 http://www.sciencedirect.com/science/journal/03091740/95/2 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/article/pii/s2214289414000738 http://www.sciencedirect.com/science/journal/22142894 http://www.sciencedirect.com/science/journal/22142894 http://www.sciencedirect.com/science/journal/22142894/3/supp/c http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/article/pii/s0309174006001124 http://www.sciencedirect.com/science/journal/03088146 http://www.sciencedirect.com/science/journal/03088146/92/3 http://www.sciencedirect.com/science/journal/03088146/92/3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 elena todosi sănduleac, gheorghe gutt, andreea ianovici iordache, silviu gabriel stroe, study of chemical composition influence on the rheological and textural properties of various typesof animal and vegetal pâté, food and environment safety, volume xiv, issue 3 – 2015, pag. 282 – 292 292 [8]. amariei s., gutt g. oroian m. a., sănduleac e., pădureţ s., automated process for achieving food texture profile, proposal of invention, osim file a00673 / 2013. [9]. sr iso 11036/2007senzory analysis methods-texture profile. [10]. sr iso 937: 2007 determination of protein content (kjeldahl method) [11]. sr iso 1444: 2008 determination of fat content (soxhlet method) 235 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 235 245 effect of different packaging methods on consumers eating quality of beef review * ahmedin abdurehman musa 1 1college of agriculcure, oda bultum. university, p.o.box 226, chiro, ethiopia, ahmedin133@ gmail.com, *corresponding author received 28th march 2019, accepted 27th september 2019 abstract: protecting meat and meat products during the distribution process, including storage and transport, from contamination by dirt, bacteria, moulds, yeasts, parasites, toxins and weight loss are very prominent. the aim of this review was to summarize the effect of packaging on consumers eating quality of beef. if meat is packaged in different packaging methods there may be a change in some beef quality parameters. three types of packagesl are in general used today i.e. vacuum, atmospheric and active one. if meat is packaged in modified atmosphere with high oxygen content the colour stability of beef increases, but other quality parameters are negatively affected. vacuum packaging has considerable advantage on all meat eating quality except of colour and purge problem. it has been observed that active packaging systems maintain as well as extend meat eating quality and shelf-life beef. keywords: packaging, beef; eating quality 1. introduction raising cattle to produce safe, high quality protein for the human diet is an important element in the nutritional well-being of the world. therefore, production practices that optimize the wholesomeness, nutritional quality and palatability of beef are all critical to consumer satisfaction [1]. beef is defined as the flesh of cattle used as food. fresh meat includes meat from recently processed animals as well as vacuum-packed meat or meat packed in controlled-atmospheric gases, which has not undergone any treatment other than chilling to ensure preservation. the diverse nutrient composition of meat makes it an ideal environment for the growth and propagation of meat spoilage microorganisms and common food-borne pathogens. it is therefore essential that adequate preservation technologies are applied to maintain its safety and quality [2]. the processes used in meat preservation are principally concerned with inhibiting microbial spoilage, although other methods of preservation are sought to minimize other deteriorative changes such as colour and oxidative changes. beef quality can be affected by many factors. the following three are the main factor determine beef quality: palatability, nutritional quality and safety (wholesomeness). there are numerous pre and post-harvest management practices and technologies that positively affect all three of these factors. palatability refers to the overall beef-eating experience which is determined by the tenderness, juiciness and flavor of the beef. while tenderness, juiciness and flavor are all important in http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 236 creating the best overall eating experience, there is some controversy among meat scientists as to which factor is the most important and detectable by the consumer [1]. the purpose of packaging is primarily to protect foodstuffs during the distribution process, including storage and transport, from contamination by dirt, bacterias, moulds, yeasts, parasites, toxic substances or those influences affecting smell and taste or causing loss of moisture. packaging should help to prevent spoilage, weight losses and enhance customer acceptability. simple packaging without further treatments is less effective in prolonging the shelf-life of meat and meat products. frequently full advantage of packaging can only be achieved in combination with preservation methods. though meat handling, storage and consumption may differ from one place to another, the factors limiting the shelf-life of these products are the same. there are endogenous factors, such as: ph-value or the degree of acidity of the product, the amount of moisture available in the product and exogenous factors, such as: oxygen (from the air), micro-organisms, temperature, light; and evaporation and desiccation [3]. primary functions of packaging are: protect against physical change, protect against chemical change, protect against microbes and present the product to the consumer in an attractive manner. advantages of packaging to industry and the consumer are: economy of scale (cutting and packaging carried out at a limited number of centralized locations), transportation costs reduced, better sanitation is achieved (increased shelf life), better inventory and product control for retailer, trim losses minimized: weight losses due to evaporation also minimized and enhances palatability due to controlled aging [4]. consumers base their purchasing choices on perceived quality and a bright red colour of beef is to many a sign of freshness and good meat quality, making colour the most important quality attribute for retailers. meat colour depends on both the degree of pigment and the muscle structure. retailers have reported an increasing problem with colour stability in beef [5]. if meat is packaged in different packaging methods there may be a change for some beef quality parameters. therefore, the aim of this review was to summarize the effect of different packaging methods on consumers eating quality of beef. 2. effect of different packaging methods on consumer eating quality for all meats the two main costs for production is animal husbandry and storage [6]. the high investment in production and storage makes it even more important to have the right type of packaging, also considering environmental damage and recycling of packaging material [5]. the role of meat packages is to protect the meat and increase the shelf life but it should also help to sell the product. many interrelated factors influence the shelf life and freshness of meat such as temperature, oxygen, endogenous enzymes, moisture, light and most important microorganisms [7]. consumers have become increasingly concerned about food-borne risks and personal health [8]. packaging protects products against deteriorative effects, which may include discolouration, off-flavour and off-odour development, nutrient loss, texture changes, pathogenicity and other measurable factors. variables that influence shelf life properties of packaged fresh meat are product type, gas mixture, package and headspace, packaging food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 237 equipment, storage temperature and additives [7]. three types of packaging in general are used today i.e. vacuum, atmospheric and active packaging [4]. modified atmosphere packaging (map) modified atmosphere packaging can be defined as; “the removal and/or replacement of the atmosphere surrounding [meat] before sealing in with vapourbarrier materials” [9], [10]. the most common consumer packaging method of beef in sweden and most of the western world is high-oxygen modified atmosphere packaging (map), with the gas composition 80% o2 and 20% co2 [5]. packaging of fresh beef in high-oxygen map is predominantly used for retail display of steaks and minced meat. by using modified atmosphere packaging the shelf life of fresh red meat can be extended [11], compared with meat wrapped in airpermeable overwrap. the amount of meat that is packaged in high-oxygen map is increasing due to the increased colour stability and better hygienic quality of the meat compared with meat wrapped in airpermeable plastic [12]. the high-oxygen content gives the beef a stable bright red oxymyoglobin colour that is desirable to consumers at the moment of purchase [13]. the report of [14] showed high oxygen map systems resulted in less palatable juiciness, flavour and overall acceptability of beef. the first research to show a detrimental effect of oxygen in map on sensory tenderness was [15], who showed that storage of beef steaks in 10 – 20% gas flushed map packs reduced the sensory tenderness scores, relative to vacuum packaged beef steaks. successively, [16] demonstrated that high oxygen map packaging systems increased toughness. more recently, [17] investigated various packaging treatments on sensory scores for lamb longissimus thoracic lumborum and semimembranosus muscles. using a similar consumer testing protocol to that used in the current experiment, packaging longissimus thoracics lumborum and semimembranosus muscles in high oxygen map decreased tenderness scores by 10 to 14 sensory units compared with vsp packs when tested after 5 or 10 days in the packaging treatments. however not all experiments have reported a toughening effect of high oxygen map. similarly, [18] showed a trend for higher shear force in the map treatment, although it failed to achieve significance. previous studies have proposed several mechanisms by which the high oxygen map penalty could occur. [19] reported that high oxygen map increased crosslinking of myosin heavy chain through disulfide bonding, and the content of protein thiols decreased indicating increased protein oxidation. their study also suggested that the high oxygen map pack promoted oxidation and inhibition of calpains. [20] reported a lower myofibrillar fragmentation index suggesting lower proteolysis in high oxygen map. [21] concluded that packaging in modified atmosphere containing a high level of oxygen resulted in protein cross-linking and reduced tenderness and juiciness of the meat. more recently [17] and [22] failed to show any effect of high oxygen map packaging on desmin degradation, suggesting that the toughening effect of high oxygen map was not due to inhibition of post-mortem proteolysis. the high oxygen map effect was independent of aging rate and from the literature would most likely have been due to myosin cross linking, but further verification would be needed [14]. the magnitude of the map effect tended to vary according to muscle [23]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 238 their study showed a higher response in the semimembransous muscle compared to a low response in the rectus femoris and vastus lateralis muscles. also a recent report by [24] showed a much higher map effect in shear force for the biceps femoris compared with the longissimus lumborum muscles. they concluded that the difference was likely due to differences between muscles in proteolysis rates. the addition of 20-30% co2 prolongs the shelf life by inhibiting bacterial growth [5]. however, high-oxygen map will allow growth of aerobic bacteria and therefore has a reduced shelf life compared to vacuum packaging [25]. most of the shelf life properties of meat are extended by use of map, but anoxic forms of map without carbon monoxide (co) do not provide bloomed red meat color and map with oxygen (o2) may promote oxidation of lipids and pigments [5]. the use of co for meat packaging is not allowed in most countries due to the potential toxic effect, and its use is controversial in some countries [26]. [27] concluded that the major disadvantages of co-map of red meat were the negative image of co held by consumers because of its potential toxicity. however, [28] and [29] showed that addition of 0.4% co to modified atmospheres for chilled beef did not mask spoilage, even when the color stability was increased. currently, consumers are not informed by the package itself regarding use of co or elevated o2 levels in the headspace of map meats. due to the lack of consumer understanding of the science and being misinformed about this technology, consequently, to improve consumer attitudes about co packaging of fresh meat, communications should be designed to not only inform consumers about the use of co, but also familiarize consumers with the science of this technology. an increase in personal knowledge and media exposure influenced acceptance of co-map negatively [26]. the disadvantage of high-oxygen content when packaging beef in modified atmosphere (map) is the increased level of lipid oxidation [12]. it has also been shown that high-oxygen content in map can negatively affect the tenderness in beef [30-32] and increase cross-linking of proteins in pork [19] and beef [33]. highoxygen map has been shown to increase the breaking strength of individual beef muscle fibers [33]. moreover, sensory attributes such as juiciness and meat flavour are negatively affected and the amount of off-flavour increases in highoxygen map [30], [32]. in other studies storage of beef in high oxygen map generated high off-flavour or warmed over flavour (wof) or both [21], [30] and [31]. beef packaged in high-oxygen map resulted in a large increase in wof and tbars as well as a decrease in juiciness and tenderness compared with packages without oxygen [31]. the correlation between sensory analysis and tbars (2thiobarbituric acid reactive substances) has been found to be high; consequently tbars is a good predictor of the perception of rancidity [34]. after storage in high-oxygen map, several volatile compounds, mainly carbonyls such as ketones and aldehydes from the lipid oxidation, were identified to be responsible for the rancid flavour in beef, using gas chromatography-olfactometry [35]. the lower tenderness and juiciness scores found in beef steaks packed in highoxygen map may be due to protein oxidation leading to crosslinking/aggregation of myosin, and hence a deterioration in sensory quality [21]. the shelf life of high-oxygen map is not as long as that in vacuum packages but it is still about twice that obtained in air [36]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 239 nonetheless, the high-oxygen content may promote oxidation of lipids, proteins and pigments in the meat, which leads to inferior beef quality. generally, if meat is packaged in modified atmosphere with high oxygen content the colour stability of beef increases, but other quality parameters such as shear force, tenderness, juiciness and meat flavour are negatively affected due to oxidation of proteins and fat [5]. low o2 map may be used as a barrier package with an anoxic atmosphere of n2 and co2. n2 is an inert gas that is not reactive with meat pigments or absorbed by the meat; therefore, it maintains integrity of the package by its presence in the headspace. however, co2 reacts with meat, changing the properties [37]. vacuum packaging (vp) vacuum packages are commonly used for packaging whole muscles for the initial ageing period before they are cut and packaged in consumer packages. the considerable advantage of meat packaged in vacuum is that the tenderization process continues in the package leading to more tender meat. the vacuum packages are easy to handle and store, and the long shelf life may prevent the need for short-time frozen storage [37]. vacuum packaging materials for primal cuts are usually three layered co-extrusions of ethyl vinyl acetate/polyvinylidene chloride/ethyl vinyl acetate, which generally have an o2 permeability of less than 15.5 ml/m2 /24 h at 1 atmosphere as a result of the polyvinylidene chloride layer [37]. vacuum packaging extends the shelf life of beef even further than high-oxygen map and the tenderization continues throughout the storage time. vacuum packing eliminates the air surrounding the meat and consequently the meat colour changes from a red oxymyoglobin colour to a purple deoxymyoglobin colour [36]. vacuum packed steaks were perceived as more tender than high oxygen map steaks after both 5 and 15 days of storage [5], but the two main problems with selling meat to consumers in vacuum packages are i) the colour, since the meat has a purple deoxymyoglobin colour and ii) the amount of purge in the vacuum packages, which does not look appealing to consumers. the lack of the bright red colour of skin packed beef was regarded as a possible disadvantage in marketing [39]. vacuum and skin packed steaks had higher scores for meat flavour and juiciness compared with map steaks. the decreased juiciness for map steaks was also combined with a higher total water loss. in contrast to this, reduced juiciness scores for steaks in high-oxygen map could not be explained by weight loss in the study by [31]. the lower whc might be influenced by an increased level of protein oxidation causing limited degradation of cytoskeletal proteins and hence increased shrinkage of the overall muscle cell [40], even though this was not verified by the result of [5] protein oxidation analysis. the lack of o2 in packages may minimise the oxidative deteriorative reactions, and reduce aerobic bacteria growth, which usually causes pigments to be in the deoxymyoglobin state. low o2 vacuum packages for retail meat cuts are usually vacuum skin packaging (vsp) systems for placing the retail cut in a barrier styrene or polypropylene tray and vacuum sealing barrier films that are heat shrunk to conform to the shape of the product [41]. vsp packaging equipment removes atmospheric air or flushes the air from the package with gaseous mixtures such as n2, co2 or mixtures of n2 and co2 before heat sealing the film layers [37]. n2 is inert, i.e. it does not react with meat product components such as fat or myoglobin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 240 its function is to replace the atmospheric oxygen (o2) and thus prevents o2 induced negative impacts. co2, has a protective function, as it inhibits to some extend the growth of bacteria and moulds [42]. the common construction for the top and bottom package webs is nylon barrier polymer of polyvinylidene chloride or ethylene vinyl alcohol, tie layer and ionomer. nylon provides bulk, toughness and low melting point, while the barrier layer prevents vapour permeation and the ionomer gives necessary seal characteristics [37]. a variation of vsp is for the lidding film to have outer barrier and inner air-permeable layers so that before retail display, the outer barrier film layer is peeled away from the permeable layer so that air can then contact the meat product and result in a bloomed colour [41], [43]. in [13], it investigated whether consumer preferences for beef colors (red, purple, and brown) or for beef packaging systems (modified atmosphere, map; vacuum skin pack, vsp; or overwrap with polyvinyl chloride, pvc) influenced taste scores of beef steaks and patties. to test beef color effects, boneless beef top loin steaks (choice) and ground beef patties (20% fat) were packaged in different atmospheres to promote development of red, purple, and brown color. to test effects of package type, steaks and patties were pretreated with carbon monoxide in map to promote development of red color, and some meat was repackaged using vsp or pvc overwrap. finally they conclude that, appearance scores and likelihood to purchase were correlated (r=0.9). however, color or packaging did not affect (p>0.5) taste scores. thus, consumer preferences for beef color and packaging influenced likelihood to purchase, but did not bias eating satisfaction. vacuum packaged meats have been marketed successfully for years in many countries. however, the darkpurplish color of deoxymyoglobin in vacuum packaged retail beef has not been accepted by consumers [5], [44]. active packaging active packaging systems are developed with the goal of extending shelf life for foods and increasing the period of time that the food is high quality [45]. is the incorporation of specific compounds into packaging systems that interact with the contents or environment to maintain or extend product quality and shelf life, while intelligent or smart packaging provides for sensing of the food properties or package environment to inform the processor, retailer and/or consumer of the status of the environment or food [46]. active packaging technologies include some physical, chemical, or biological action which changes interactions between a package, product, and/or headspace of the package in order to get a desired outcome. active packaging is typically found in two types of systems; sachets and pads which are placed inside of packages, and active ingredients that are incorporated directly into packaging materials [45]. active packaging functions and technologies include moisture control, o2permeable films, o2 scavengers or absorbers, o2 generators, co2 controllers, odour controllers, flavour enhancement, ethylene removal, antimicrobial agents and microwave susceptors [47] in addition to indicators of specific compounds [48] and temperature control packaging. the most important active packaging systems applied to meat and meat products are oxygen scavengers, antimicrobial, antioxidant, and carbon dioxide emitting/generating packaging [47], [49]. i. oxygen scavengers an oxygen scavenger is a substance that scavenges oxygen chemically or enzymatically and therefore, protects the packaged food completely against food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 241 deterioration and quality changes due to oxygen [50]. often the oxygen absorber or scavenger is enclosed in a porous sachet or packet but it can also be part of packaging films and structures. steaks packaged without oxygen scavengers had more discoloration and significantly higher proportions of metmyoglobin when compared to steaks packaged with oxygen scavengers. prevention of metmyoglobin formation was influenced by the number but not the type of oxygen scavenger employed [42]. oxygen scavengers effectively prevent oxidative damage in a wide range of food constituents such as (i) oils and fats to prevent rancidity, (ii) muscle pigments and flavours to prevent discolouration of meat and loss of taste and (iii) nutritive elements, e.g., vitamins to prevent loss of the nutritional value [50]. ii. antimicrobial active packaging the aims of using antimicrobial active packaging are to extend shelf life and to ensure food safety of meat and meat products. there are four basic categories of antimicrobial packaging [51]: 1) incorporation of antimicrobial substances into a sachet/pad inside the package. the antimicrobial sachet/pad can be produced by generating antimicrobial compounds in situ with subsequent release, or by using sachets to carry and then release the antimicrobials [52]. 2) direct incorporation of the antimicrobial agents into the packaging film. this can be achieved by the conventional heat treatment method such as co-extrusion of packaging films with the antimicrobials although high loss of bioactive compounds will occur. alternatively, non-heating methods such as solvent compounding, electrospinning, and casting can be used to maintain the maximum antimicrobial activity of the packaging films [53]. 3) coating of packaging with a matrix that acts as a carrier for antimicrobial agents so that the agents can be released onto the surface of food through evaporation into the headspace (volatile substances) or migration into the food (non-volatile substances) through diffusion. this matrix can be a plastic film or any food safe materials such as wax or polysaccharides incorporating antimicrobials and directly coated on the food. 4) use of polymers that are inherently antimicrobial. two examples of these polymers used in food packaging and coatings are chitosan and poly-l-lysine. the charged amines of the polymers interact with negative charges on the microorganism cell membrane which cause leakage of intracellular constituents and then cell death [54]. iii. antioxidant active packaging high levels of oxygen in meat packaging can facilitate microbial growth, lipid oxidation, development of off-flavours and off-odors, colour changes and nutritional losses. lipid oxidation not only results in the development of rancidity, but also the potential formation of toxic aldehydes and the loss of nutritional quality because of polyunsaturated fatty acid (pufa) degradation [55]. therefore, control of oxygen levels in meat packaging is important to limit the rate of such deteriorative and spoilage reactions [49]. the effects of an alginate-based edible coating containing natural antioxidants (rosemary and oregano essential oils) on lipid oxidation, color preservation, water losses, texture and ph of beef steaks during 14 days of display were studied. the coatings significantly decreased color losses, water losses and shear force compared to the control. the coatings had a significant effect on consumer perception of odor, flavor and overall acceptance of the beef. generally, they conclude that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 242 coated meat was redder, had a more intense chroma and was more tender [56]. the addition of essential oils (eo) from oregano (or, oreganum heracleoticum l.) or thyme (th, thymus vulgaris l.) into soy-based edible films (ef) as an antioxidant activepackaging is effective in retarding oxidative changes in meats [57]. iv. carbon dioxide emitting/scavenger carbon dioxide emitters can be used either alone or with oxygen scavengers to extend the shelf life of meat. co2 acts as an antimicrobial agent and it also prevent the collapse of the pack, which is caused by the development of a partial vacuum as the residual oxygen is utilized. use of co2 emitter in conjunction with an oxygen scavengers to replace the oxygen removed with an equivalent volume of co2, has proven to be successful in the prevention of pack collapse. carbone dioxide scavengers do not have application in the packaging of meat [42]. the action of co2 has differential effects on microorganisms; for instance aerobic bacteria such as pseudomonas can be inhibited by moderate to high levels of co2 (10-20%) whereas lactic acid bacterial proliferation can be stimulated by co2. furthermore, proliferation of pathogens such as c. perfringens, c. botulinum and l. monocytogenes is only minimally inhibited by co2 levels lower than 50%. there is concern that by inhibiting spoilage microorganisms, a food product may appear edible while in fact containing a high quantity of pathogens that have multiplied due to a lack of indigenous competition [58]. 3. conclusion the high-oxygen content of map may promote oxidation of lipids, proteins and pigments in the meat, which leads to inferior beef quality. generally, if meat is packaged in modified atmosphere with high oxygen content the colour stability of beef increases, but other quality parameters such as shear force, tenderness, juiciness and meat flavour are negatively affected due to oxidation of proteins and fat. vacuum packaging extends the shelf life of beef even further than high-oxygen map and the tenderization continues throughout the storage time. vacuum packing eliminates the air surrounding the meat and consequently the meat colour changes from a red oxymyoglobin colour to a purple deoxymyoglobin colour., but the two main problems with selling meat to consumers in vacuum packages are i) the colour, since the meat has a purple deoxymyoglobin colour and ii) the amount of purge in the vacuum packages, which does not look appealing to consumers. the lack of the bright red colour of skin packed beef was regarded as a possible disadvantage in marketing. active packaging refers to the incorporation of additives into packaging systems with the aim of maintaining or extending meat product quality and shelflife. active packaging systems include 1) oxygen scavengers, 2) carbon dioxide scavengers and emitters, 3) antioxidant and 4) antimicrobial packaging technologies. 4. references [1]. montgomery, ted, and jennifer leheska. effects of various management practices on beef eating quality, (2008) [2]. aymerich, t., picouet, p. a., & monfort, j. m. decontamination technologies for meat products, meat science, 78, 114 – 129, (2008) [3]. fao. manual on simple methods of meat preservation, (1990) [4]. gazalli, humaira, altaf hussain malik, heena jalal, saima afshan, ambreen mir, and humaira ashraf. packaging of meat. international journal of food nutrition and safety international journal of food nutrition food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 243 and safety journal homepage, int. j. food nutr. saf ,4 (42):70–80, (2013) [5]. åsa lagerstedt norström. packaging methods and storage time, jounal of meat science, 88 (3):391-396, ( 2011) [6]. smulders, f.j.m., hiesberger, j., hofbauer, p., dogl, b. & dransfield, e. modifiedatmosphere storage under subatmospheric pressure and beef quality: ii. color, drip, cooking loss, sarcomere length, and tenderness, journal of animal science, 84(9), 2456-2462,( 2006) [7]. zhou, g. h, x. l xu, and y. liu. preservation technologies for fresh meat – a review, 86(1):119–128, (2010) [8]. wezemael, lynn van, wim verbeke, marcia d de barcellos, joachim scholderer, and federico perez-cueto. consumer perceptions of beef healthiness : results from a qualitative study in four european countries.( 2010) [9]. mcmillin, kw, huang, ny, ho, cp, smith, bs. quality and shelf-life of meat in dynamic modified atmosphere packaging. abstracts of papers of the american chemical society 215, u16-u16. (1998) [10]. mcmillin, k.w. where is map going? a review and future potential of modified atmosphere packaging for meat, meat science 80(1), 43-65,(2008) [11]. zhao, y., wells, j.h. & mcmillin, k.w. applications of dynamic modified atmosphere packaging systems for fresh red meats: review, journal of muscle foods 5(3), 299-328, (1994) [12]. jakobsen, m. & bertelsen, g. x. colour stability and lipid oxidation of fresh beef. development of a response surface model for predicting the effects of temperature, storage time, and modifed atmosphere composition, meat science 54(1), 49-57, (2010) [13]. carpenter, c., cornforth, d.p. & whittier, d. consumer preferences for beef colour and packaging did not affect eating satisfaction, meat science 57(4), 359-363, (2001) [14]. polkinghorne, r j, j philpott, jessira perovic, j lau, l davies, and w mudannayake. the effect of packaging on consumer eating quality of beef, meat science elsevier:59–64, (2018) [15]. fu, a.h., molins, r.a., & sebranek, j.g. storage characteristics of beef rib eye steaks packaged in modified atmosphere, journal of food science, 5:283-287, (1992). [16]. tørngren, m.a. effect of packing method on colour and eating quality of beef loin steaks. pages 495-496 in proceedings of the 49th international congress of meat science and technology, campinas, brazil. (2003) [17]. frank, d.c., geesink, g., alvarenga, t.i.r.c., polkinghorne, r., stark, j., lee, m., & warner, r.. impact of high oxygen and vacuum retail ready packaging formats on lamb loin and topside eating quality, meat science, 123:126-133, (2017) [18]. lopacka, j., poltorak, a., & wierzbicka, a. effect of map, vacuum skinpack and combined packaging methods on physicochemical properties of beef steaks stored from up to 12 days. meat science, 119: 147-153, (2016). [19]. lund, m. n., christensen, m., fregil, l., hviid, m. s. & skibsted l. h.. effect of modi fi ed atmosphere packaging on strength of single muscle fi bres from beef and pork. in proceedings of the international congress of meat science and technology (icomst), beijing, china. (2007) [20]. estevez, m. protein carbonyls in meat systems: a review. meat science, 89:259-279, (2011). [21]. kim, yuan h, elisabeth hufflonergan, joseph g sebranek, and steven m lonergan.. high-oxygen modified atmosphere packaging system induces lipid and myoglobin oxidation and protein polymerization, meat science, 85:759–67, (2010) [22]. geesink, g., robertson, j., & ball, a. the effect of retail packaging method on objective and consumer assessment of beef quality traits, meat science, 104: 85-89, (2015). [23]. seyfert, m., hunt, m.c., mancini, r.a., hachmeister, k.a., kropf, d.h. & unruh, j.a. beef quadriceps hot boning and modified-atmosphere packaging influence properties of injection enhanced beef round food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 244 muscles, journal of animal science, 83: 686693, (2005). [24]. moczkowska, m., poltorak, a., montowska, m., pospiech, e., & wierzbicka, a. the effect of the packaging system and storage time on myofibrillar protein degradation and oxidation process in relation to beef tenderness, meat science, 130: 7-15, (2017) [25]. jeremiah, l. e. packaging alternatives to deliver fresh meat using short or long-term distribution, food research international, 34(9), 749-772, (2001) [26]. djenane, djamel and roncaler pedro. carbon monoxide in meat and fish packaging, 1–34, (2018) [27]. cornforth, d.p.; hunt, m.c. lowoxygen packaging of fresh meat with carbon monoxide: meat quality, microbiology, and safety. in the american meat science association (amsa) white paper series number 2; american meat science association: savoy, il, usa, 8, (2000). [28]. hunt, m.c.; mancini, r.a.; hachmeister, k.a.; kropf, d.h.; merriman, m.; delduca, g.; milliken, g. carbon monoxide in modified atmosphere packaging affects color, shelf life, and microorganisms of beef steaks and ground beef, j. food sci., 69:45–52, (2004) [29]. brooks, j.c.; alvarado, m.; stephens, t.p.; kellermeier, j.d.; tittor, a.w.; miller, m.f.; brashears, m.m. spoilage and safety characteristics of ground beef packaged in traditional and modified atmosphere packages, j. food prot., 71:293–301, (2008) [30]. clausen, i. sensory evaluation of beef loin steaks stored in different atmospheres. in: proceedings of 50th international congress of meat science and technology, helsinki, finland, 8-13 p. 77, (2004) [31]. clausen, i., jakobsen, m., ertbjerg, p. & madsen, n.t. modified atmosphere packaging affects lipid oxidation, myofibrillar fragmentation index and eating quality of beef, packaging technology science, 22(2): 85-96, (2009) [32]. sørheim, o., wahlgren, m., nilsen, b.n. & lea, p. effects of high oxygen packaging on tenderness and quality characteristics of beef longissimus muscles. in: proceedings of 50th international congress of meat science and technology, helsinki, finland. pp. 120, 4 pages on cd. (2004) [33]. lund, m. n., lametsch, r., miklos, r., hviid, m. s. & skibsted l. h. protein oxidation in meat during chill storage in highoxygen atmospheres. in proceedings of the 55th international congress of meat science and technology, (icomst), copenhagen, denmark. (2009). [34]. m.m. campo, g.r. nute, s.i. hughes, m. enser, j.d. wood, r.i. richardson. flavour perception of oxidati on in beef, elsevier meat science, 72: 303–311, (2006) [35]. resconi, c. v., escudero, a., beltrán, j. a., sañudo, c., ferreira, v. & campo, m. m. effect of modi fi ed atmosphere in aroma development of beef. in proceedings of the 55th international congress of meat science and technology (icomst). copenhagen, denmark. (2009) [36]. shay, b.j. & egan, a.f. the packaging of chilled red meats, food technology in australia, 36: 283-285, (1987) [37]. jenkins, w. a., & harrington, j. p. packaging foods with plastics. technomic pub. co. http://agris.fao.org/agrissearch/search.do?recordid=us9169521 (1991) [38]. resurreccion, a.v.a. sensory aspects of consumer choices for meat and meat products, meat science, 66(1): 11-20, (2003). [39]. taylor, a.a., down, n.f. & shaw, b.g. a comparison of modified atmosphere and vacuum skin packing for the storage of red meats, international journal of food science and technology, 25(1): 98-109, (1990) [40]. huff-lonergan, e. & lonergan, s.m. mechanisms of water-holding capacity of meat: the role of postmortem biochemical and structural changes, meat science, 71(1): 194204,(2005) [41]. belcher, j n. industrial packaging developments for the global meat market. (2006) [42]. shaikh, anas ejaz. processed meat packaging, food, (2015) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 ahmedin abdurehman musa, effect of different packaging methods on consumers eating quality of beef, food and environment safety, volume xviii, issue 3 – 2019, pag. 235 – 245 245 [43]. jeyamkondan, s., jayas, d.s. & holley, r.a. review of centralized packaging systems for distribution of retail-ready meat. journal of food protection, 63(6):796-804, (2000). [44]. gazalli, humaira, altaf hussain malik, heena jalal, saima afshan, ambreen mir, and humaira ashraf. packaging of meat. international journal of food nutrition and safety international journal of food nutrition and safety, int. j. food nutr. saf, 4 (42):70– 80, (2013) [45]. pavelková, adriana, and erika flimelová. active packaging system for meat and meat products, potravinarstvo, 6 (3):21– 27, (2012). [46]. kerry, j p, m n o ’grady, and s a hogan. past, current and potential utilisation of active and intelligent packaging systems for meat and muscle-based products: a review. (2006) [47]. brody, aaron l., betty bugusu, jung h. han, claire koelsch sand, and tara h. mchugh. innovative food packaging solutions. journal of food science 73 (8). (2008) [48]. vermeiren, l, f devlieghere, m van beest, n de kruijf, and j debevere.. developments in the active packaging of foods, 10(3):77-86, (1999) [49]. fang, zhongxiang, yanyun zhao, robyn d. warner, and stuart k. johnson. active and intelligent packaging in meat industry, 61 (january), elsevier ltd:60–71, (2017) [50]. vermeiren l, heirlings l, devlieghere f, and debevere joxygen, ethylene and other scavengers. novel food packaging techniques: an introduction, 24–49, (2003) [51]. coma, véronique. bioactive packaging technologies for extended shelf life of meat-based products, (2008) [52]. otoni, caio g, paula j p espitia, roberto j avena-bustillos, and tara h mchugh. trends in antimicrobial food packaging systems: emitting sachets and absorbent pads. frin 83:60–73, (2016) [53]. sung, suet-yen, lee tin sin, tiamting tee, soo-tueen bee, a r rahmat, w a w a rahman, ann-chen tan, and m vikhraman. antimicrobial agents for food packaging applications. (2013) [54]. maurice and e rosenberg. mechanism of enhancement of microbial cell. strain 172 (10):5650–54, (1990) [55]. gomez-estaca, j., lopez-dedicastillo, c., hernandez-mu~noz, p., catala, r., & gavara, r. advances in antioxidant active food packaging. trends in food science & technology, 35:42-51, (2014) [56]. pelaes vital, ana carolina, ana guerrero, jessica de oliveira monteschio, maribel velandia valero, camila barbosa carvalho, benício alves de, abreu filho, grasiele scaramal madrona, ivanor nunes, and do prado. effect of edible and active coating (with rosemary and oregano essential oils) on beef characteristics and consumer acceptability, (2016) [57]. kodal, betül, co kun, eda çalikog, ˘ lu, zehra karagöz, emirog ˘ lu, kezban candog, and ˘ an. antioxidant active packaging with soy edible films and oregano or thyme essential oils for oxidative stability of ground beef patties, 37(3):203–312, (2014) [58]. yingyuad, songchai, sompoch ruamsin, dhaweerat reekprkhon, supaporn douglas, suwassa pongamphai, and ubonrat siripatrawan. effect of chitosan coating and vacuum packaging on the quality of refrigerated grilled pork, packaging technology and science, 19 (3):149–57, (2006) 1. introduction modified atmosphere packaging (map) vacuum packaging (vp) active packaging i. oxygen scavengers ii. antimicrobial active packaging iii. antioxidant active packaging iv. carbon dioxide emitting/scavenger 327 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 -2018 , pag. 327 331 validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes *veronica tanasa1, 2, madalina doltu2, dorin sora2, radu i. tanasa3, narcisa babeanu1 1university of agronomical sciences and veterinary medicine in bucharest, 59 marasti blvd, district 1, bucharest, 011464, romania 2institute of research and development for industrialization and marketing of horticultural products horting, 5n drumul gilaului, district 4, bucharest, romania 3national institute of research “cantacuzino”, 103 splaiul independentei, district 5, 050096, bucharest, romania e-mail: vero.tanasa@yahoo.co.uk *corresponding author received 12th july 2018, accepted 19th september 2018 abstract: a high performance liquid chromatography method for carbendazim residues determination was adapted to the condition of our laboratory and validated for tomatoes samples. carbendazim eluted at 3.13 minutes. the signal was linear over the concentration range 1 to 15 µg/ml with correlation coefficient 0.999092. the detection limit and the quantitation limit values were 0.002 mg/kg and 0.02 mg/kg respectively. relative standard deviation of repeatability was 3.47% and 4.78% and recovery was 99.67% and 113,11% for two levels of concentration. the adapted method allowed a simple and rapid separation and quantification of carbendazim in tomatoes by high performance liquid chromatography. keywords: carbendazim, tomatoes, validation parameters, hplc. 1. introduction it is well known the human health impact of pesticide exposure: asthma, diabetes, parkinson's disease, leukaemia, cancer [1]. carbendazim (metil n-benzimidazol-2carbamat) belongs to carbamate pesticide class and is a fungicide used in fruit and vegetable growing and viticulture. carbendazim is not approved for use in the european union [2], but is used in brazil, china and some other non-eu/eea countries to preserve agricultural crops. for this reason carbendazim residues in food are monitored in the european union. carbendazim residues are determined by high performance liquid chromatography (hplc) coupled with uv, diode array, fluorescence or ms detectors, or by gas chromatography [3-10]. the aim of this study was to adapt a hplc method for carbendazim residues determination described by phansawan et al. [10] for tomatoes samples using our equipments, and to develop an internal validation study focused on the following performance characteristics: linearity, accuracy, precision (repeatability), sensitivity (detection limit, quantitation limit). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 3, 2018 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes, food and environment safety, volume xvii, issue 3 – 218 , pag. 327 331 328 2. materials and method 2.1. samples tomatoes (hybrid f1 primadona) free of carbendazim were obtained from the department of horticultural cultures in protected areas, horting institute, romania, and were kept at -200 c before analysis. 2.2. reagents and standards carbendazim (97 %) was purchased from aldrich. a stock solution (250 µg/ml) was prepared in methanol and used for the preparation of working standard solutions necessary for calibration curve (1, 2.5, 5, 7.5, 10, 12.5 and 15 µg/ml). all other reagents used were p.a. grade and solvents were hplc grade. 2.3. the analytical procedure has been adapted in the chemistry and biochemistry laboratory, horting institute, following a previously published protocol [10]. tomatoes samples were well blended, and 5 g sample were extracted in 25 ml methanol. the extracts were filtered through whatman no. 1 paper; the filtrates were cleanup on oasis mcx cartridges (waters, ireland) following the manufacturer instructions, then were concentrated using a turbovap equipment (caliper lifesciences), so that the injected volume contained an amount of carbendazim within the linear range of the diode array detector. finally the samples were filtered through 45 μm filters prior to injection. in our case, the chromatographic separation was performed using a lichrocart purospher rp-18 column (250 * 4 mm), with 5μ particle size (merck kgaa, germany) and, the mobile phase consisted of water and methanol (25:75,v/v) under isocratic chromatographic conditions, with a flow rate of 1 ml/min. the column temperature was set at 200c. carbendazim was detected at 286 nm by the diode array detector. the data acquisition and processing used software chromquest 4.2. (thermofinnigan). the results were statistically processed using graphpad prism (version 5.00, graphpad software inc., san diego, 2007). 3. results and discussion carbendazim peak eluted at 3.13 minute in standard solutions (fig. 1), and that is important if analysis time is of a higher priority. increasing of organic solvent in mobile phase, we obtained a retention time (rt) shorter than phansawan et al. [10], with a value around 12.5 minutes. a good separation of carbendazim peak for tomatoes spiked samples was also obtained (fig. 2). the signal was linear over the concentration range 1.0 15.0 µg/ml, with correlation coefficient of 0.999092 (fig.3); this range covers the eu maximum residue level tolerance for carbendazim in tomatoes (0.3 mg/kg) [2]. the accuracy of the method was examined through the results of the recovery by means of spiking procedure. for the recovery assays known amounts of carbendazim were added to the tomatoes samples to achieve 1 mg/kg (level 1) and 0.5 mg/kg (level 2) respectively. good recovery was obtained: 99.67% and 113.11% respectively (table 1). han et al. [9] reported recovery values between 72.0%-86.5% for tomatoes samples spiked with carbendazim at level of 5-50 ng/g. phansawan et al. [10] reported also good recoveries of carbendazim from spiked pooled vegetable samples (including tomatoes) ranged from 92.5% to 96.0 % at spiked levels of 0.050.30 mg kg-1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 3, 2018 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes, food and environment safety, volume xvii, issue 3 – 218 , pag. 327 331 329 the precision was evaluated by relative standard deviation (rsd) at two levels of concentration previously presented. rsd was 3.47% and 4.78% respectively (table 1). rsds that ranged from 2.1% to 7.5 % were obtained by phansawan et al. [10] for different carbendazim amounts. limit of detection (lod) is given as the concentration of the analyte that gives an absorbance signal three times higher than background noise, while limit of quantitation (loq) is given as the lowest concentration of analyte that can be determined with an acceptable accuracy in terms of methods of analysis. using the signal to noise ratio registered for standard carbendazim solutions by high performance liquid chromatograph, we calculated lod and loq values (table 1). lod and loq (0.002 mg/kg and 0.02 mg/kg respectively) were comparable with those obtained by phansawan et al. [10] (0.003 mg kg-1 and 0.03 mg/kg respectively). lod value was greater than that reported by han et al. [9] (0.55 ng/g). minutes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 m a u 0 50 100 150 200 250 300 350 400 m a u 0 50 100 150 200 250 300 350 400 42 05 1 .3 03 37 61 1 .5 25 16 75 5 1 .8 17 35 02 7 1 .9 50 41 18 2 2 .3 17 c ar be nd az im 3 93 25 23 3 .1 35 21 61 4 .1 77 57 13 4 .3 70 87 5 4 .5 87 20 6 4 .7 03 pda-286nm name area retention time rt (min) fig. 1. chromatogram of carbendazim standard solution (10 μg/ml) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 3, 2018 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes, food and environment safety, volume xvii, issue 3 – 218 , pag. 327 331 330 minutes 0.0 0.5 1.0 1.5 2.0 2. 5 3. 0 3.5 4.0 4.5 5.0 m a u 0 100 200 300 400 500 m a u 0 100 200 300 400 500 21 41 0 .3 35 51 36 0 .6 00 19 13 61 1 .3 82 46 25 6 1 .6 17 17 02 43 1 1 .9 58 66 16 39 2 .4 48 c ar be nd az im 4 02 40 23 3 .1 90 11 40 45 4 .1 05 30 67 7 4 .5 77 pda-286nm name area retention time rt (min) fig. 2. chromatogram of a tomato sample spiked with carbendazim (1 mg/kg) concentration (μg/ml) fig. 3. carbendazim calibration curve linear fit ax + b a = 394806. b = 0.000000 goodness of fit (r^2): 0.999092 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 3, 2018 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes, food and environment safety, volume xvii, issue 3 – 218 , pag. 327 331 331 table 1. validation parameters for carbendazim determination by hplc method precision (rsd(%)) 1mg/kg (level 1) 0.5 mg/kg (level 2) (n=5) (n=5) recovery(%)±rsd 1mg/kg (level 1) 0.5 mg/kg (level 2) (n=5) (n=5) lod (mg/kg) loq (mg/kg) 3.47 4.78 99.67±3.35 113.11±4.86 0.002 0.02 4. conclusion the adapted method is simple, fast, accurate, precise and sensitive in order to detect carbendazim residues in tomatoes samples below the maximum residue level of 0.3 mg/kg, according to the eu tolerance. 5. acknowledgments this work was supported by the romanian ministry of education and scientific research executive agency for higher education, research, development and innovation funding (uefiscdi), under the national r&d&i plan ii partnering program, grant pn ii-pt-pcca-20134-0128, contract no. 147/2014 to m.d. and r.i.t. some expenditure was supported by the doctoral school in engineering and plant and animal resources management of the university of agronomical sciences and veterinary medicine in bucharest, for v.t. and n.b. all the authors declare no conflict of interest. 6. references [1]. kim k.h., kabir e., jahan, s.a., exposure to pesticides and the associated human health effects, science of the total environment, 575: 525-535, (2017) [2]. commission regulation (eu) no 559/2011 of 7 june 2011 amending annexes ii and iii to regulation (ec) no 396/2005 of the european parliament and of the council as regards maximum residue levels for captan, carbendazim, cyromazine, ethephon, fenamiphos, thiophanatemethyl, triasulfuron and triticonazole in or on certain products. [3]. fernandez-alba a.r., tejedor a., aguera a., determination of imidacloprid and benzimidazole residues in fruits and vegetables by liquid chromatography-mass spectrometry after ethyl acetate multiresidue extraction, journal of aoac international, 83(3): 748-755, (2000) [4]. blasco c., fernandez m., pico y., font g., manes j., simultaneous determination of imidacloprid, carbendazim, methiocarb and hexythiazox in peaches and nectarines by liquid chromatography–mass spectrometry, analytica chimica acta, 461 (1): 109-116, (2002) [5]. singh b.s., foster d.g., khan u.s., determination of thiophanate methyl and carbendazim residues in vegetable samples using microwaveassisted extraction, journal of chromatography a, 1148: 152–157, (2007) [6]. hu y., yang x., wang c., zhao j., li w., wang, z., a sensitive determination method for carbendazim and thiabendazole in apples by solidphase microextraction−high performance liquid chromatography with fluorescence detection, food additives & contaminants: part a, 25(3): 314-319, (2008) [7]. liu s.x., tong f.z., zhen l., huang x. d., gao x., xu y.c., simultaneous analysis of thiabendazole, carbendazim and 2aminobenzimidazole in concentrated fruit juices by liquid chromatography after a single mix-mode solidphase extraction cleanup, journal of environmental science and health, part b> pesticides, food contaminants, and agricultural wastes, 44 (6): 591597, (2009) [8]. paranthaman r., sudha a., kumaravel s., determination of pesticide residues in banana by using high performance liquid chromatography gas chromatography – mass spectrometry, american journal of biochemistry and biotechnology, 8(1): 1-6, (2012) [9]. han d., tang b., thian m., row k.h., solid-phase extraction combined with dispersive liquid-liquid microextraction for the determination of three benzimidazole pesticides (carbendazim, thiabendazole, and thiophanate-methyl) in tomatoes, analytical letters, 46 (4): 557-568, (2013) [10]. phansawan b., prapamontol t., thavornyutikarn p., chantara s., mangklabruks a., santasup c., a sensitive method for determination of carbendazim residue in vegetable samples using hplc-uv and its application in health risk, chiang mai journal of science, 42 (3): 681-690, (2015) microsoft word 14 ogunremi_corectat pe 27 june.doc 233 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 233 240 phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms *omotade ogunremi 1, oladotun ishola 2, hannah ogunedina 3 1department of microbiology, michael okpara university of agriculture, umudike, nigeria, tadeogunremi@yahoo.com, 2department of chemical sciences, samuel adegboyega university, ogwa, nigeria 3department of biological sciences, samuel adegboyega university, ogwa, nigeria *corresponding author received 27th january 2018, accepted 27th june 2018 abstract: recently, plant by-products are being explored as safe and sustainable sources of bioactive compounds. this is the first study to screen phytochemicals and evaluate the antimicrobial activity of extracts from mucuna pruriens husk for potential application as food preservative. aqueous and ethanol extract from mucuna pruriens seed husk were examined for presence of phytochemicals. the antimicrobial activities of the extracts against the predominant food spoilage bacterial and fungal isolates were evaluated by agar well diffusion method. flavonoids, phenol, glycosides, steroids, saponins and anthraquinones were present in both aqueous and ethanol extracts while tannins and phlobatannins were absent. the test isolates except candida sp. fy22 were sensitive to at least one extract. aqueous extracts showed stronger and wider spectrum of antimicrobial activity with 100 mg/ml of the extract demonstrating highest activity index of 1.15 compared to gentamycin (10 µg) against pseudomonas sp. bb22. this study confirms the potential of extracts from mucuna pruriens husk as a natural source food preservative. keywords: mucuna pruriens, phytochemicals, antimicrobial, husk 1. introduction food spoilage and safety remain among the most serious economic and public health concerns globally [1]. it is estimated that more than half of the world’s harvested and traditionally processed crops are lost to microbial deteriorations [2]. up to 1.5 billion cases of morbidity and more than three million deaths occur in children every year as a result of food and water contamination. conventional methods and synthetic additives employed to promote shelf-life stability and eliminate microbial hazards in foods are not affordable and with safety concerns to consumers. in addition, most of the target microorganisms have developed resistance to these strategies [3]. this rationalizes the recent exploration of natural sources, including plants, animals and microorganisms for novel antimicrobials that meet the criteria of safety, availability, affordability, eco-friendliness and efficacy for food applications. compounds derived from different plant parts have been used since ancient times for flavoring, coloring and preserving foods without reports of harm to consumers [3, 4]. preparations from plants have been used orally for centuries in traditional medicine to treat infectious diseases. antimicrobial activity of plant materials is due to the presence of some chemical compounds, including; alkaloids, flavonoids, phenolics, quinones, saponins and tannins [3]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 234 mucuna pruriens, also known as yerepe in yoruba is a twinning annual plant that belongs to the family fabaceae [5]. it is widespread in tropical and subtropical regions of the world, thriving under warm and moist conditions [6]. its leguminous fruit is a longitudinal pod that contains about 4 to 6 shiny black or brown seeds [7]. the husk of the pod is densely covered with pale-brown or grey trichomes that cause irritating blisters if they come in contact with the skin. mucuna pruriens has gained prominence as a valuable plant in the pharmaceutical and food industries. the seed was reported to have hypocholesterolemic, hyploglycemic, antiparkinson, antioxidant, laxative, aphrodisiac, immunostimulatory, anthelmintic and antimicrobial effects [8]. in addition, it is rich in polyunsaturated fatty acids, dietary minerals and highly digestible proteins [8]. hence, the high demand on the seed of mucuna pruriens. mucuna prurience seed husk is a byproduct that is largely underutilized and typically considered as wastes. some studies have shown that plant husks can have similar or even higher proportions of bioactive compounds than the regularly used parts [3]. they could be abundant and readily available sources of phytochemicals that have several functionalities such as antimicrobial activity. high concentration of phenol and antimicrobial activity against staphylococcus aureus was reported in aqueous extract of walnut green husk [9]. coconut husk extract inhibited the growth of some biofilm-forming bacteria such as pseudomonas sp., alteromonas sp. and gallionella sp. [10]. however, there is no report on the value of mucuna pruriens seed husk as a potential source of bioactive compounds. therefore the objectives of this study are to screen for the phytochemicals and determine the antimicrobial properties of different extracts from mucuna pruriens seed husk against isolated food pathogenic and spoilage microorganisms. 2. materials and methods 2.1 plant materials dry pods of mucuna pruriens were handpicked from the matured plants in eguare quarters, ebele, igueben local government area, edo state, nigeria in october, 2015. the plant was identified in biological sciences department, samuel adegboyega university, ogwa, edo state. husk was obtained by breaking the pod and separating the seed. 2.2 preparation of aqueous and ethanol extracts of husk the husk of mucuna pruriens was sundried, crushed and milled into fine powder. ten grams each of mucuna pruriens husk powder was weighed with an analytical balance (setra model bl200s) and added to 200 ml of distilled water and 98% ethanol in separate conical flasks at ambient temperature (28-30 oc) for 48 hours. the solutions were decanted and the supernatant were filtered using clean muslin cloths. the filtrates were concentrated to dryness by evaporating the solvents in the water bath at 45 0c [11]. 2.3 isolation and characterization of test microbial strains enumeration and isolation of microorganisms from deteriorating bread, fish and meat samples were carried out using the following media: nutrient agar for bacteria, brilliant green bile agar for coliforms and yeast peptone dextrose (ypd) agar (supplemented with streptomycin) for yeasts. distinct isolates were selected after incubation and characterization using morphological and biochemical methods [12, 13]. probable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 235 identification of isolates was done by reference to standard descriptions [14, 15]. 2.4 phytochemical screening of extracts aqueous and ethanol extracts of mucuna pruriens seed husks were tested for the presence of alkaloids, anthraquinones, flavonoids, glycosides, phenols, phlobatannins, saponins, steroids and tannins using standard methods [16]. 2.5 antimicrobial activity assay of extracts antimicrobial properties of the aqueous and ethanol extracts were determined by agar well diffusion assay. test bacteria and yeasts were inoculated in sterile nutrient and ypd broth respectively and incubated for 18 hours to obtain fresh cultures. cell pellets were obtained from the broth cultures after centrifugation (12000 g for 15 min) and suspended in sterile distilled water to a turbidity that is equivalent to 0.5 mcfarland standards. a total of 100 µl of suspensions of bacteria and yeasts were spread on already solidified muellerhinton and ypd agar respectively. for both extracts 5 mg/ml and 10 mg/ml were prepared in sterile distilled water. wells (6 mm diameter) were bored in the agar and 200 µl of extracts were dispensed into them. the plates were kept in the refrigerator for 2 hours for diffusion of the extracts into the agar and incubated at 37 0c for 24 hours. the plates were examined for zones of inhibition around the wells and the zones were measured in diameters [4]. 2.6 antibiotic sensitivity assay the antibiotic sensitivity patterns of bacterial isolates were determined using the agar overlay disc diffusion method of national committee for clinical laboratory standards [17]. mueller-hinton agar plates were seeded by spread plate method with 100 µl of 18 hours old standardized bacterial suspension and left to dry at 30 0c for 15 mins. disc containing different concentration of antibiotics were placed on seeded agar with the aid of sterile forceps under aseptic conditions. the antibiotics disc were; ceftazidime (30 µg), efuroxime (30 µg), gentamicin (10 µg), cefixime (5 µg), ofloxacin (5 µg), augmentin (30 µg), nitrofurantin (30 µg) and ciprofloxacin (5 µg). the plates were incubated at 37℃ for 24 hours. the resultant visible zones of inhibition were measured in millimeters (mm). zones lesser than 14 mm were regarded as resistant (r), those ranging from 14 to 17 mm were indicated as intermediate (i) and zones greater than 17 mm were indicated as susceptible (s) [18]. 2.7 determination of activity index of selected extract the activity index (ai) of the extract that showed the widest spectrum of antibacterial activity with reference to selected antibiotics towards which all test isolates showed zones of inhibition were calculated as described by erhabor et al. [19]. 3. results and discussion 3.1 microbial analysis enumeration of the microbial load revealed that the deteriorating food samples were heavily contaminated with bacteria and fungi (table 1). fish sample had the highest total bacteria count (8.2175 log10 cfu/g) while bread sample had the lowest total bacteria count (7.2765 log10 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 236 cfu/g). the total coliform counts range from 6.0000 to 8.8325 log10 cfu/g. yeast was not cultured from meat sample but bread and fish samples had yeast counts of 8.0000 and 7.1139 log10 cfu/g respectively. in developing countries, food handling practices, especially the processing of fresh fish and meat are carried out with poor implementation of good manufacturing and hygienic practices, thereby exposing them to several sources of microbial contamination. these foods have high water activity and supply complete range of nutrients for microbial growth; hence, they are prone to spoilage [20]. table 1 microbial counts in deteriorating food samples food sample total bacteria (log10 cfu/g) coliform (log10 cfu/g) yeast (log10 cfu/g) bread 7.2765 8.1367 8.0000 fish 8.2175 8.8325 7.1139 meat 7.2625 6.0000 ng key: ng: no growth, cfu/g: colony forming units per gram table 2 probable identity of selected bacteria and yeast isolates from deteriorating food samples probable identity food source pseudomonas sp. bb21 bread pseudomonas sp. bb22 bread lactobacillus sp. bm21 bread listeria sp. fm21 fish aeromonas sp.fn21 fish pseudomonas sp. mm1 meat bacillus subtilis mn1 meat saccharomyces cerevisiae by21 bread kluyveromyces africanus by22 bread candida sp. fy22 fish based on morphological examination and biochemical tests, the bacteria were probably identified as species of aeromonas, bacillus, lactobacillus, listeria and pseudomonas. the yeasts are candida sp., kluyveromyces africanus and saccharomyces cerevisiae (table 2). the bacteria identified in this study have been implicated in the spoilage of fresh meat and fish and their respective products [2]. yeasts are important food spoilage microorganisms, including foods with low water activity such as bread [22]. 3.2 phytochemical analysis table 3 shows the phytochemical constituents of the aqueous and ethanol extracts from husk of mucuna pruriens. four and five chemicals were identified in the aqueous and ethanol extracts respectively. in both extracts, phenols, glycosides and saponins were present while tannins and phlobatannins were absent. flavonoids and anthraquinones were detected in ethanol extract. husks of seeds which constitute about 45% of plant biomass have been noted to be more concentrated with phytochemicals such as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 237 flavonoids, phenols and tannins compared to their respective seeds [3, 23]. as far as we know, this is the first report on the phytochemicals in mucuna pruriens seed husk extracts. however, studies on the aqueous and ethanol extracts of mucuna pruriens seeds and leaves also revealed the presence of similar phytochemicals [24, 25]. in agreement with ours, studies that explored phytochemicals in husks from other plants reported high concentration of phenol in juglans regia (walnut) and cocos nucifera (coconut) husk extracts [9, 10]. table 3 phytochemical constituents of mucuna pruriens husk extracts phytochemical constituent aqueous extract ethanol extract tannins -ve -ve flavonoids -ve +ve phenols +ve +ve glycosides +ve +ve steroids +ve -ve phlobatannins -ve -ve saponins +ve +ve anthraquinones -ve +ve key: +ve: present, -ve: absent 3.3 antimicrobial activity table 4 shows the antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens against the food spoilage bacteria and yeast isolates. the spectra of antimicrobial activity of both extracts increased with concentration, however, aqueous extract demonstrated wider spectrum than the ethanol extract. all test isolates were resistant to 50 mg/ml of ethanol extract while 4 test isolates were susceptible to the 100 mg/ml of the same extract. 50 mg/ml of aqueous extracts inhibited 6 isolates with zones of inhibition ranging from 5-10 mm while 100 mg/ml of the extract demonstrated stronger and wider spectrum of activity, inhibiting 9 test isolates with zones of inhibition of 5-15 mm. table 4 antimicrobial activity of mucuna pruriens husk extracts diameter of inhibition zones (mm) test microorganisms aqueous extract (50 mg/ml) aqueous extract (100 mg/ml) ethanol extract (50mg/ml) ethanol extract (100mg/ml) pseudomonas sp. bb21 -ve 10 -ve 5 pseudomonas sp. bb22 -ve 15 -ve 10 lactobacillus sp. bm21 10 15 -ve 5 listeria sp. fm21 5 5 -ve 1 aeromonas sp.fn21 5 10 -ve -ve pseudomonas sp. mm1 5 5 -ve 1 bacillus subtilis mn1 -ve 5 -ve -ve s. cerevisiae by21 10 15 -ve -ve k. africanus by22 5 15 -ve -ve candida sp. fy22 -ve -ve -ve -ve food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 238 key: -ve: no inhibition lactobacillus sp. bm21 and saccharomyces cerevisiae by21 were more sensitive to the aqueous extract while pseudomonas sp. bb22 was the most sensitive test isolate to ethanol extract. candida sp. fy22 was resistant to all the extracts. the results obtained in this study present almost similar antimicrobial capacity with juglans regia husk aqueous extract, inhibiting gram positive and negative bacteria but unable to inhibit candida albicans and cryptococcus neoformans [9]. table 5 antibiotic sensitivity pattern of test microorganisms diameter of zone of inhibition (mm) test isolate g ra m r ea ct io n p e f c n a p x z a m r c p x s sx t e o f x a u c h sp pseudomonas sp. bb21 -ve 17 (s) 19 (s) na na -ve (r) na 21 (s) -ve (r) -ve (r) na 25 (s) -ve (r) 17 (i) 24 (s) pseudomonas sp. bb22 -ve 23 (s) 13 (i) na na 15 (i) na 27 (s) -ve (r) -ve (r) na 15 (i) -ve (r) 17 (i) 23 (s) lactobacillus sp. bm21 +ve 19 (s) 17 (s) -ve (r) -ve (r) -ve (r) 8 (r) 25 (s) -ve (r) -ve (r) -ve (r) na na na na listeria sp. fm21 +ve 23 (s) 23 (s) -ve (r) -ve (r) -ve (r) -ve (r) 26 (s) 17 (s) 13 (r) -ve (r) na na na na aeromonas sp.fn21 -ve 21 (s) 13 (i) na na -ve (r) na 23 (s) -ve (r) -ve (r) na 27 (s) -ve (r) 15 (i) 17 (s) bacillus subtilis mn1 +ve 15 (i) 15 (s) -ve (r) -ve (r) -ve (r) -ve (r) 23 (s) -ve (r) -ve (r) -ve (r) na na na na key: am:30 µg amoxacillin, apx:30 µg ampiclox, au:25 µg augmentin, ch:30 µg chloramphenicol, cn:10 µg gentamycin, cpx:10 µg ciprofloxacin, ofx:30 µg tarivid, pef:10 µg pefloxacin, r:25 µg rocephin, s:30 µg streptomycin, sp:10 µg sparfloxacin, sxt:30 µg septrin, z:20 µg zinnacef, -ve: no inhibition, na: not applicable, resistant (r), intermediate (i), susceptible (s) table 6 antimicrobial activity index of 100 mg/ml aqueous extract from mucuna pruriens husk against selected antibiotics test isolate pef cn cpx pseudomonas sp. bb21 0.59 0.53 0.48 pseudomonas sp. bb22 0.65 1.15 0.56 lactobacillus sp. bm21 0.79 0.88 0.60 listeria sp. fm21 0.22 0.22 0.19 aeromonas sp.fn21 0.48 0.77 0.44 bacillus subtilis mn1 0.33 0.33 0.22 cn: 10 µg gentamycin, cpx: 10 µg ciprofloxacin, pef: 10 µg pefloxacin most reports support stronger antimicrobial activity when extraction is done using organic solvents. however, considering the extraction conditions and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 239 plant material, there are few reports of comparatively better extraction and higher antimicrobial activity with aqueous extracts. gomaa et al. [26] reported the release of more phytochemicals and wider spectrum of antimicrobial activity in aqueous extracts from seeds of jatropha curcas, simmondsia chinensis and datura metel compared to their respective ethanol extracts. similarly, ali et al. [27] reported that aqueous extract of vitex doniana stem back demonstrated higher antibacterial activity than the methanolic extract. in a similar manner, aqueous extracts from stem bark, stem wood and root of ternimalia brownii exhibited the strongest activity against both bacteria and fungi compared to petroleum ether, dicloromethane and methanol extracts [28]. the antibiotic sensitivity and resistance pattern of the test isolates to different types of antibiotics is presented in table 5. all test isolates were sensitive to pefloxacin, gentamycin and ciprofloxacin. the gram positive test isolates were resistant to ampiclox, erythromycin and zinnacef while the gram negative isolates were resistant to augmentin. the activity indexes of 100 mg/ml aqueous extract from mucuna pruriens husk compared to gentamycin (10 µg), ciprofloxacin (10 µg) and pefloxacin (10 µg) are presented in table 6. the highest activity index value (1.15) was shown against gentamycin for pseudomonas sp. bb22 while the least activity index value (0.19) was recorded against ciprofloxacin for listeria sp. fm21. relative to standard antibiotics, the activity index of the 100 mg/ml aqueous extract is considered to be significant against the test isolates. the ranges of activity index reported in this study have also been reported for extracts from other plant materials [19, 30]. 4. conclusion the present study has revealed the presence of valuable bioactive compounds in mucuna pruriens husk extracts. in addition, the extracts exhibited strong antimicrobial activities against food spoilage and pathogenic bacteria and fungi. this by-product that was hitherto discarded as wastes could be an affordable, eco-friendly and sustainable sources of food additives for preservation purposes. 5. references [1]. antolovich, m., prenzler, p. d., patsalides, e., mcdonald, s., robards, k., methods for testing antioxidant activity, analyst, 127: 183–198, (2002) [2]. sanni, a. i., onilude, a. a., fadahunsi, i. f., afolabi, r. o., microbial deterioration of traditional alcoholic beverages in nigeria, food research international, 32: 163-167, (1999) [3]. gyawali, r., ibrahim, s. a., natural products as antimicrobial agents, food control, 46: 412-429, (2014) [4]. dhiman, r., aggarwal, n., aneja, k. r., kaur, m., in vitro antimicrobial activity of spices and medicinal herbs against selected microbes associated with juices, international journal of microbiology, article id 9015802, 1-9, (2016) [5]. salau, a. o., odeleye, o. m., antimicrobial activity of mucuna pruriens on selected bacteria, african journal of biotechnology, 6 (18): 2091-2092, (2007) [6]. raina, a. p., tomar, j. b., dutta, m., variability in mucuna pruriens l. germplasm for l-dopa, an anti parkinsonian agent. genetic resources and crop evolution, doi 10.1007/s10722-012-9836-4, (2012) [7]. agharkar, s.p., medicinal plants of bombay presidency. scientific publication, jodhpur, india: 1–2, (1991). [8]. pugalenthi et al., 2005 pugalenthi, m., vadivel, v., siddhuraju, p., alternative food/ feed perspectives of an underutilized legume mucuna pruriens var. utilis a review, plant foods for human nutrition, 60:201-18, (2005) [9]. oliveira, i., sousa, a., ferreira, i. c. f. r., bento, a. and pereira, j. a., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 omotade ogunremi, oladotun ishola, hannah ogunedina, phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms, food and environment safety, volume xvii, issue 2 – 2018, pag. 233 – 240 240 total phenols, antioxidant potential and antimicrobial activity of walnut (juglans regia l.) green husks, food and chemical toxicology, 46: 2326–2331, (2008). [10]. viju, n., satheesh, n. s., vincent, s. g. p., antibiofilm activity of coconut (cocos nucifera linn.) husk fibre extract, saudi journal of biological sciences, 20: 85–91, (2013). [11]. jarriyawattanachaikul, w., chaveerach, p. and chokesajjawatee, n., antimicrobial activity of thai-herbal plants against food-borne pathogens e. coli, s. aureus and c. jejuni, agriculture and agricultural science procedia, 11: 20 – 24, (2016) [12]. cheesbrough, m., discrete laboratory practice in tropical countries. part 1. cambridge second editions, published by press syndicate of the university of cambridge, chapter 5. 258pp, (2005) [13]. kurtzman, c. p., jack, w., teun, b., vincent, r. methods for isolation, phenotypic characterization and maintenance of yeasts. in: the yeasts, a taxonomic study, c.p. kurtzman, and j.w. fell eds. elsevier, b.v, amsterdam: 87–110, (2011) [14]. buchanan, r. e., gibbons, n. e., bergey’s manual of determinative bacteriology, 8th edition. williams and wilkins, baltimore. 1268 pp, (1974) [15]. sanni, a. i., lonner, c., identification of yeasts isolated from nigerian traditional alcoholic beverages. food microbiology, 10:517–523, (1993) [16]. amadi, b. a., agomuo, e. n., ibegbulem, c. o. research methods in biochemistry, supreme publishers, owerri, nigeria, (2004) [17]. clsi, clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing; approved standardeleventh edition, (2014) [18]. harley, j. p., prescot, l. m., laboratory exercises in microbiology. 5th edition. mac graw hill, new york. 449pp, (2002) [19]. erhabor, j.o., oshomoh, e.o., enerijiofi, k., erhabor, c. r., ogbonnaya, c.s., idu, m., antibacterial activity and phytochemical screening of the acetone extract of the root of aloe vera (l.) burm. f. against microorganisms associated with male infertility. sau science-tech journal, 2(1): 7-16, (2017). [20]. gill, c.o. microbiological contamination of meat during slaughter and butchering of cattle, sheep and pigs. in: davies, a. and board, r. (eds), the microbiology of meat and poultry. london: blackie academic and professional: 118-157, (1998). [21]. jay, j. m. modern food microbiology. 6th ed. gaithersburg (md): aspen. p 679, (2000) [22]. deák, t., handbook of food spoilage yeasts, second ed. crc press, usa, (2008) [23]. viswanath, v., urooj, a., malleshi, n. g., evaluation of antioxidant and antimicrobial properties of fingermillet polyphenols (eleusine coracana). food chemistry, 114:340– 346, (2009). [24]. kumar, a., rajput, g., dhatwalia, v. k., srivastav, g., phytocontent screening of mucuna seeds and exploit in opposition to pathogenic microbes. journal of biological and environmental sciences, 3(9), 71-76, (2009). [25]. pandey, j., pandey, r., study of phytochemical and antimicrobial activity of alcohlic extract of mucuna pruriens (l.) leaves. international journal of applied research, 2(2): 219-222, (2016) [26]. gomaa, e. e. d. g., esmaiel, n. m., salem, m. z. m., gomaa, s. e., in vitro screening for antimicrobial activity of some medicinal plant seed extracts. international journal of biotechnology for wellness industries, 5: 142152, (2016) [27]. ali, m., aminu, f., ibrahim, i. s., invitro assessment of antibacterial activity and phytochemical screening of vitex doniana on clinical isolate of salmonella typhi. international journal of advanced academic research science, technology and engineering, 3 (1): 9-16, (2017). [28]. mbwambo, z. h., moshi, m. j., masimba, p. j., kapingu, m. c., nondo, r. s. o., antimicrobial activity and brine shrimp toxicity of extracts of terminalia brownii roots and stem, bmc complementary and alternative medicine, 7:9 doi:10.1186/1472-6882-7-9, (2007). microsoft word 10 larisa chubenko_corectat 2mai.doc 197 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2-2018, pag. 197 204 technological aspects of production of frozen dessert with protein-herbal component olena grek1, tatyana osmak1, *larisa chubenko1, artur mykhalevych1 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str.68, kyiv, ukraine, lorkachub@gmail.com *corresponding author received 12th march 2018, accepted 27th june 2018 abstract. the aim of scientific work is to improve the technology of frozen desserts enriched with protein-herbal component by the obtained thermo & heat coagulation of skimmed milk with rumex juice. the emergency of the development of new frozen desserts with protein-milk concentrates, obtained by different coagulation methods, is substantiated. the advantages of using the thermo & heat coagulation of milk proteins are taken into consideration. having in view the complex influence on the proteins of high temperature milk and the rumex acid reagent, the parameters of the process are specified. as raw material for coagulation, skimmed milk with a mass fraction of dry matter of 11.2 ± 0.7%, protein, 3.7 ± 0.2%, titrated acidity, 17 ± 1.0°т, density, 1032 kg/m³ was selected. the amount of coagulant with a ph of 3.1 at a level of 7 ± 0.5% of the weight of skimmed milk satisfies the requirements for protein-herbal clots according to quality indices. the technological process of production of frozen dessert with protein-herbal component was provided, which envisaged the receipt of raw materials and preparation of the milk basis, production of protein-herbal component, mixing, milling and packing. the refined technological parameters of production and the biological value of dessert are determined. thus, it was established that the introduction of the protein-herbal component into the frozen dessert composition at the ratio of the milk basis to protein-herbal 80:20 will allows getting the product with the corresponding organoleptic and physico-chemical indicators of quality with increased nutritional and biological value. keywords: sorrel (rumex), thermo & heat coagulation of proteins, biological value, ice cream 1. introduction the nutrition structure of the population of ukraine to a large extent does not correspond to modern principles rational nutrition and practical diet. in the food ration of the population is an excess of bakery products and potatoes. there are few main sources of complete food protein (meat, fish and dairy products), food fibers, micronutrients (vegetables, fruits, nuts, etc.). the results of studies on the actual state of nutrition in different regions of ukraine testify to the fact that the food status of both children and adults is characterized by serious violations: the lack of valuable (animal) proteins; polyunsaturated fatty acids; vitamins c, b, e, folic acid, retinol, β-carotene); macroand trace elements: ca, fe, zn, f, se, i; food fibers. on the contrary, there is an over-consumption of animal fats and easily digestible carbohydrates. fat consumption is higher than the recommended norm more than 32% of the caloric intake of the diet. the deficiency of protein intake is on average 20%, most of the vitamins and trace elements are 55%, of food fibers 30% [1]. when forming a modern assortment of food products should take into account the main stages selection of prescription food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 198 components indicators of economic efficiency from possible introduction into production. according to the scheme, it is necessary to highlight the most important directions in this area. they should take into account the need for further research work. priority directions of the complex the problem is to provide healthy nutrition to the population. the structure of nutrition requires considerable correction in the direction of greater balance. food products should increase the body's resistance to conditions of unfavourable environment. providing mass release of such products is possible improve the quality of life of the sick person. a healthy person helps to reduce the risk of the most widespread diseases. ensure adaptation of the body to adverse living and working conditions. above this information is fully applicable to dairy products, especially those widely used [2]. ice cream is a stuffed, frozen dessert, a popular product from the population of our country. this is due not only to its pleasant flavoring properties, but also to its high nutritional and biological value. fig.1. stage of components selection for modern food products on average, the energy value of milk and fruit ice cream is 560.7 616.2 kj / kg, creamy up to 836.0 kj / kg, pencil up to 1010 kj / kg. chemical composition of ice cream (in %): carbohydrates, 14 25; fat, 0.5 20; proteins, 3.5 4.5, minerals, up to 0.7. ice cream is digested by the body on 95 98% [ 3 ]. protein in ice cream on a milk basis is represented mainly by casein. whey proteins albumin and globulin partly coagulate when pasteurizing mixtures of ice cream. scientists have already developed a technology of ice cream with the addition of cheese sour milk low-fat obtained by acid coagulation of milk proteins [4] and a frozen dessert enriched with soy protein component [5]. at present, vegetable ingredients are used as technological components in the production processes. so, the thermo & heat coagulation of milk proteins occurs under the action of whey with acidity 150 200° t, and coagulants of vegetable origin with corresponding characteristics [6, 7]. sour milk cheese is obtained according to the classical technology with the thermo food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 199 acid deposition of proteins allows you to get the finished product enriched with serum proteins. the aim of the work is to improve the technology of enriched frozen desserts protein-herbal component. the object of the study is the technology of frozen desserts on the basis of phc. subject of research – skimmed milk; rumex juice; phc; quality indicators and biological value of a frozen dessert with proteinherbal component. the task is: establishment of a rational ratio of milk base and protein-herbal component in mixtures for frozen dessert; development of technology of frozen dessert with protein-herbal component; research of qualitative indicators and biological value of model samples of frozen desserts. 2. materials and methods method of obtaining protein herbal component. raw material for coagulation skimmed milk with a mass fraction of dry matter of 11.2 ± 0.7%, protein, 3.7 ± 0.2%, titrated acidity, 17 ± 1.0°т, density, 1032 kg/m³. earlier studies, which took into account organoleptic constraints and protein yield, the optimal amount of coagulant with ph 3.1 was determined at a level of 7 ± 0.5% of the milk mass. that is the number that changes active acidity in the mixture to ensure a balanced is electric state of proteins in all volume and leads to their active coagulation in classical modes. in a heated temperature of 93 ... 95 ° с, skim milk was introduced by rumex juice with a mass fraction of dry substances 3.2%, moderately stirred and kept at 3 ... 5 minutes until a clot was formed. comprehensive impact on high temperature milk proteins and acid reagent leads to their full deposition. the coagulation process was set visually for intense formation of strong protein clot and release whey [8]. the paper uses both standard and adapted methods for determining the quality indices. the mass fraction of dry substances in ice cream was determined by the arbitration method by drying for temperature 102 ÷ 105°с (gost 3626). the titratable acidity of an ice cream is titer-metric (gost 3624). the loss of ice cream was determined according to dstu 4733: 2007. resistance to melting of ice cream expressed through the accumulation time of 10 cm³ mixture (in minutes), which it is formed due to the melting of ice cream in a thermostat at a temperature of 25 °c by ion-exchange liquid-column chromatography on the automatic analyzer of amino acids t339 was determined which based on the acidbase properties of amino acids. to register the amino acids in elutes use the detection method with ninhydrin. it interacts with the amino acid by amino group and forms a hydrindantin compound, which causes a coloration of 560 nanometers. qualitative and quantitative composition of the amino acid mixture is determined by comparing the standard chromatogram and the studied mixtures of amino acids and calculating the peak area of each amino acid. amino acid fast the ratio of the content of the essential amino acid product to the corresponding content an indispensable amino acid "ideal protein" on the fao / who scale: af = eaapr / eaae (2.1) where af amino acid rate,%; eaapr the content of the essential amino acid in the product, g/1 g protein; eaaep the content of the "essential protein" of the "essential protein" on the fao / who scale, g / l protein. the coefficient of difference of amino acid fast (kdaf) and biological value of protein (bv) was determined by the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 200 fig. 2. parametric scheme of production of frozen dessert with protein-herbal component method m.p. chernikov [9]. it is based on the postulate that the assimilation of eaa is limited to limiting content amino acids that is, the kdaf is the average arithmetic excess of the eaa amino acid accelerator compared to the smallest limit of limiting amino acids. kdaf are found by the formula: kdaf= (σdaf) / n, (2.2) where kdaf coefficient of difference of an amino acid,%; daf is the difference in the amino acid rate for each nac compared with the amino acid soon limiting amino acids, %; n the number of amino acids. the smaller the value of kdaf, the more is fully used by eaa for the needs of biosynthesis. so the biological value of the protein is calculated by the formula: bv = 100 kdaf, (2.3) where bv biological value of protein,%. acceptance and preparation of raw materials drafting of milk mixtures t=40±5°c 20…30 min filtering homogenization pasteurization t=80-85°c, 50 s t=92-95°c without endurance cooling t=0…6°c maturation t=0…6°c not less than 4 hours reception and preparation of skimmed milk pasteurization t=93-95°c acid precipitation of proteins whey pressing the bunch reception and preparation of herbal raw materials washing drying grinding pressing 1-2 min juice e shrot for drying freeze packing tempering storage mixing of milk mix with protein-herbal component preparation of normalized milk mixture preparation of protein-herbal component t= -4 6°c t= -25-42°c for recycling t=75-85°c р=9 -15 мpа food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 201 3. results and discussion the basis of the technology of a new kind of frozen dessert is the classical technological scheme ice cream production. technology of production of frozen dessert with protein-herbal component was carried out in three stages. technological scheme of production is shown in fig. 2. preparation of a normalized mixture for ice cream (scheduled figures: mass the share of fat 3.5%, the skimmed milk powder balance 12%, sugar 14%). preparation of phc with a mass fraction of moisture 64% was made as follows: mixing of normalized milk base and phc in ratio 80:20. the limitations of adding protein herbal component are as specific organoleptic characteristics and color, and ph at the level of 5.4 ... 5.7. the ground part of rumex was used to get the protein-herbal clot as a coagulant with a thermo & heat coagulation [10, 11]. the obtained protein-herbal component had the following qualitative indices: the mass fraction of moisture at the level (64 ± 2)%; titrated acidity (80 ± 1) °t; color light pistachio, not uniform; consistency – soft slightly broken on the cut, as tight as it is; taste, milk-protein, cheesy, without foreign smells, with light herbal flavor. after maturation, the milk base was mixed with protein-herbal component in the ratio of 80:20%. it is expedient to carry out this operation immediately before the freeze of the mixture. it provides admission to the free homogeneous mixture, without increasing the titrated acidity. organoleptic and physico-chemical parameters of freshly prepared samples of frozen dessert from protein-herbal component are given in tables 1 and 2. it is known that the biological value of a product depends on the quality of the proteins that are part of the raw materials ingredients and are characterized by the content of essential amino acids. to study the biological value of the developed dessert, its amino acid composition was determined. samples were studied for protein-herbal component, milk ice cream (control) and frozen dessert with phc. the results of research on the amino acid composition of these samples are showed in table 3. table 1 organoleptic characteristics of frozen dessert with protein-herbal component indicator characteristics of a frozen dessert taste and smell pure, delicious sour-milk taste with a light herbal flavor, no foreign smells structure and consistency homogeneous throughout the mass, delicate creamy color light pistachio table 2 physico-chemical parameters of frozen dessert with protein-herbal component characteristic indicator rate mass fraction,%, not less: fat / sugar / dry matter 3.5/14.0/29.5 acidity, ° t 45 active acidity, ph 6.15 damage, % 52 resistance to melting, min 43 it was revealed (table 3) that the total content of amino acids of frozen dessert in comparison with protein-herbal component has decreased, as frozen desserts contain more carbohydrates and less amount of protein. the calculation of the amino acid rate of the essential amino acids was carried out on the basis of the obtained data it was found that the limiting amino acid in milk ice cream is methionine + cysteine, and for groups of experimental samples of protein-herbal component and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 202 table 3 amino acid composition of protein-herbal component and frozen desserts phc milk ice cream frozen dessert with phc the name of the amino acid mg/100g % to the total mg/100g % to the total mg/100g % to the total lysine 752.393 9.07 161.707 7.36 301.4922 8.82 histidine 327.7321 3.95 57.105 2.60 119.9816 3.51 arginine 377.5142 4.55 59.318 2.70 109.0431 3.19 aspartic acid 365.8984 4.41 157.169 7.15 219.1117 6.41 threonine 315.2866 3.8 105.477 4.80 135.3638 3.96 serine 270.4827 3.26 129.391 5.89 131.9456 3.86 glutamic acid 1749,011 21.08 471.154 21.43 726.7261 21.26 proline 513,5852 6.19 287.500 13.08 310.7216 9.09 glycine 92,09687 1.11 48.320 2.20 45,80494 1.34 alanine 233.1461 2.81 82.540 3.75 125.7927 3.68 сysteine+ 274,6312 3.31 11.667 0.53 63.92182 1.87 valine 588,2584 7.09 117.000 5.32 216.7189 6.34 methionine 269.653 3.25 49.434 2.25 83.74784 2.45 isoleucine 301.1816 3.63 95.469 4.34 141.8586 4.15 leucine 1011.406 12.19 219.715 9.99 389.342 11.39 tyrosine++ 314.4569 3.79 42.843 1.95 86.14063 2.52 phenylalanine 540.1357 6.51 102.785 4.68 210.566 6.16 total amino acid 8297.015 100.00 2198.595 100.00 3418.279 100.00 frozen dessert threonine (table 4).the amino acid rate of each amino acid does not give a general idea of the biological value of the product. to evaluate the degree of use of the protein was calculated by the coefficient of difference of the amino acid af (kdfa) and biological protein value (bv) by the method of m.p. chernikov [9]. all the excess amino acids are used by the body for energy needs, not by protein biosynthesis. kdfa is an arithmetic mean of af essential amino acids relative to the af of the limiting amino acid. differences of amino acid af essential amino acids of the experimental samples are presented in fig.3. table 4 amino acid rate of protein-herbal component and frozen desserts amino acid af, % the name of the amino acid standard fao / who, g / 1 g "essential protein" phc milk ice cream frozen dessert with phc lysine 5.5 165 134 160 threonine 4 93 120 99 methionine + cysteine 3.5 187 79 123 valine 5 142 106 127 isoleucine 4 95 108 104 leucine 7 174 143 163 phenylalanine+ tyrosine 6 172 111 145 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 203 fig. 3. difference of amino acid af essential amino acids of the experimental samples according to fig. 3, the maximum excess, characteristic of protein-herbal component, provides methionine + cysteine, and samples of dessert leucine. the smaller the value of kdfa, the more fully is used by the eaa for the needs of biosynthesis. the determination of biological value (bv) was carried out through the coefficient of difference of the amino acid af: bv = 100 kdfa, % the relevant indicators are presented in fig. 4. it was revealed (fig. 4) that the introduction of the frozen dessert phc in the amount of 20% will not allow only to enrich the product with whey proteins, but also to increase its biological value to 62%. 0 20 40 60 80 in di ca to r va lu e, % kdfa bv name of indicator protein-herbal component milk ice cream frozen des sert with protein-herbal component fig. 4. indicators of biological value of experimental samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, technological aspects of production of frozen dessert with protein-herbal component,food and environment safety, volume xvii, issue 2 – 2018, pag. 197 204 204 the technology of the frozen dessert with the natural biologically complete proteinherbal component is used to bring the composition of the product closer to the optimal ratio of the main nutrients. the development can be implemented in dairy enterprises, restaurants, shops of low capacity with periodic action freezers. 4. conclusions 1. the authors established a rational ratio of the normalized mixture for milk ice cream and protein-herbal component at the level of 80:20, which ensures the receipt of the product with the corresponding organoleptic and physico-chemical indicators with high nutritional and biological value. 2. the technology of frozen dessert with phc is developed, which involves the introduction of protein enrichment in cooled, normalized basis after ripening at a temperature of 0 ... 6 °с for at least 4 hours, directly before freezing. 3. the chemical composition of the developed frozen dessert determines the high digestibility level of all nutrients, which is a particularly valuable characteristic of food. we found that the introduction into the composition of icecream protein enrichment will increase the biological value of ice cream by 3.33%. consequently, the inclusion of a frozen dessert with protein-herbal component to a diet for adults and children is promising. its consumption will contribute to the daily physiological needs for basic nutrients and energy. 5. references [1]http://medved.kiev.ua/web_journals/arhiv/nutriti on/2008/1-2_08/str32.pdf [2] bal-prilipko l.v., venglyuk yu.p., zaletok s.p., moiseenko a.m., improving the technology of combined food products using high quality ingredients and dietary supplements, food industry, 2: 23–26, (2013) [3] oleney y.,tyorogova n., kazakova l., soloviev l.,handbook for the production of ice creamv. 800 p ( 2004 ) [4] osmak t., ryabokon n., soy-protein protein component promising ingredient of frozen dessert, food industry of agroindustrial complex, 1: 38–41, (2014) [5] skorchenko t., osmak t., ice cream technology of diabetic "syrok" , dairy business,3: 12-14, (2006) [6] ostroumov l.a., bobylin v.v., smirnova i.a., rafalovich s.r., investigation of the process of thermo-acidic coagulation of milk using various coagulants, storage and processing of agricultural raw materials, 7: 26–27, (1998) [7] grek, o., ouopriichuk o., pshenychna t., the rationalization of the parameters ofmilk proteins' thermo acid coagidation by berry coagulants, food and environment safety, 1: 1–7, (2017) [8] grek, o.,tymchuk a., chubehko l., ovsienko k., research of quality indicators of curd products on basis of protein-herbal clots, food and environment safety, 4: 262-268, (2017) [9] chernikov n., on the chemical methods for determining the quality of food proteins, questions nourishment, 1: 42-45, (1986) [10] chernykh v. p., pharmaceutical encyclopaedia, 1632 р., (2010). [11] evtefeev yu. v., zykovich s.n., investigation of the chemical composition and nutritional status of rumex k-1 sorrel, bulletin of the altai state agrarian university, 76–80, (2011). 272 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 2018, pag. 272 277 advantages of using proteins in the production of truncated semi-finished products *iryna shevchenko 1 , alexey skochko 1 1educational and research institute of food technology, national university of food technologies,kyiv, ukraine, irinanuht@ukr.net *corresponding author received 12th may 2018, accepted 19th september 2018 abstract: in world practice, recently, the question of using physico-chemical methods of treating biotechnological objects by means of substances of cryoprotective action is increasingly being discussed. the principle of these substances is to create an amorphous structure throughout the volume of the product and to reduce the number of crystallization centers and water activity. on the basis of proteins in blood plasma vepro 75 psc, wheat sodium caseinate, flax fiber and cellulose plantain were used to get protein-polysaccharide compositions with high functionaltechnological and cryoprotective properties and to produce chopped semi-products of deep freezing and extended storage at a temperature of 18 ºс. the conducted studies confirmed that developed composite mixtures have high functional and technological properties, which increase the moistureretaining, fat-retaining ability and form stable properties of meat systems. the mechanism of cryoprotective action of the developed compositions is associated with the formation of an amorphous structure in the middle of meat system, as well as with the decrease in the number of crystallization centers and a decrease in water activity. keywords: freezing, meat systems, food fibers, crystallization, cryoprotectants. 1. introduction. a necessary condition for the preservation of quality indices of chopped semi-finished products after defrosting is compliance with the optimal conditions of refrigeration (freezing temperature, humidity, air flow velocity). however, this does not always allow maintaining high consumer properties of frozen semi-finished products, as the degree of destructive impact of low temperatures on muscle fibers of raw meat also depends on the functional and technological properties of raw meat [1-3]. recently, there has been a steady trend in food development with minimal content of chemical (synthetic) origin, with the simultaneous increase in the popularity of dietary supplements and natural ingredients. this allows obtaining products that best meet the requirements of a healthy diet [2-4]. recently, according to world's practice, the issue of the use of physico-chemical processing methods of biotechnological objects through the use of substances of cryoprotective action is being discussed more frequently. these substances' principle of operation is to create an amorphous structure throughout the volume of the product, to reduce the number of crystallization centers and to reduce the activity of water. in order to develop food products that meet the requirements of healthy diet and reduce the negative effects of freezing on the functional and technological properties of raw meat, as well as to prevent significant crystallization and slow down the freezing process, cryoprotective properties of protein-polysaccharide mixtures of animal proteins and plant fiber were studied. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 iryna shevchenko, alexey skochko, advantages of using proteins in the production of truncated semi-finished products, food and environment safety, volume xvii, issue 3 – 2018, pag. 272 – 277 273 2. materials and methods protein-polysaccharide composition mixtures as a functional, technological and cryostating component in meat stuffing, four composite mixtures were studied, with protein and polysaccharide the ratio 1 to 1. their structure includes: blood plasma protein, sodium caseinate and wheat cellulose mixture no. 1; blood plasma protein, sodium caseinate and flax fiber mixture no. 2; blood plasma protein, sodium caseinate and plantain fiber mixture no. 3; blood plasma protein, sodium caseinate, plantain fiber and flaxseed mixture no. 4. during the determination of organoleptic, physical and chemical indices of modulated minced meat systems and chopped semi-finished products, standard methods of research were used. structural-mechanical indices were determined on a penetrometer ulab331m. the penetration properties of chopped products were determined by the depth of the immersion of the indenter in a test sample at a temperature of 20 ° c. three measurements were made on the exposed surface of the sample at a distance of not less than 10 mm from the edge of the product and at the maximum distance from the points of other measurements, so that the deformed part of the surface did not interfere with the measuring zone, after which the penetration value was recalculated in the penetration tension value. the water activity index aw of the model fermented systems and meat brooded semifinished products was determined using the rotronic hygro palm-23 analyzer. measurement of the cryoscopic temperature of the model fermented systems and chopped half-finished products was carried out by the method of thermal analysis, which is based on the construction of curves of temperature changes in time. preparation of experimental samples protein-polysaccharide mixtures were added to the modulated minced systems made on the basis of single-grade beef (50%) and semi-bulk pork (50%) in the amount of 2% to replace a single-breed beef. for control, a meat-stuffed meat system without animal proteins and food fibers was used. the minced samples were subjected to stirring at a temperature of 12 °c for 15 minutes and frozen at a temperature of minus 18 °c. the duration of storage at the indicated temperature was of 30 days. in all samples, the organoleptic, physico-chemical, functional-technological, structural and mechanical parameters, as well as water activity aw and cryoscopic temperature values were determined before freezing, after defrosting and thermal treatment. the statistical analysis all the analytical determinations were performed at least three times and the value reported for the determined characteristics was the average value ± of the standard deviation (s.d.). the statistical analysis was performed at the microsoft excel statistical software version 2010. 3. results and discussion the development of a rational composition of chopped semi-finished products was carried out by computer optimization method based on the chemical composition of the recommended ingredients and the results of research on their functional and technological properties (ftp). as a target function, moisture retainer (hu) and fatretaining ability (luw) were selected, which are criteria of maintaining the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 iryna shevchenko, alexey skochko, advantages of using proteins in the production of truncated semi-finished products, food and environment safety, volume xvii, issue 3 – 2018, pag. 272 – 277 274 stability of moisture and fat content in meat systems. knowledge of ftw is necessary for the rational processing of raw meat. it provides an opportunity to predict and direct the quality characteristics of finished products [5-7]. selected as cryoprotectants of plasma proteins vepro 75 psc and sodium caseinate are thermostable functional proteins used both at minus temperatures and in pasteurization regimes. they are high molecular weight substances that can reduce the growth rate of crystals and protect the muscle cells from osmotic changes. in addition, they have high functional properties and the ability to stabilize meat systems. wheat fiber, flax fiber and plantain fiber are multifunctional components that can improve consistency, adsorb water, reduce mass loss, and enrich the final product with ballast substances, but as for most fibers containing a mixture of soluble and insoluble fractions, have a low the ability to adsorb fat [1-3]. in the organoleptic study of modulated samples of meat chopped meat systems, it has been established that the use of developed functional compositions in the amount of 2% does not lead to significant changes in the organoleptic properties of meat systems. all samples before freezing had a pinkish-red color, a smell characteristic for fresh meat and a gentle consistency. the experimental specimens were characterized by a high degree of adhesion compared to the control sample. after defrosting, the prototype had a darker color. the greatest changes in the organoleptic parameters after freezing, storage for 30 days and defrosting were characteristic of control samples characterized by a loose structure of muscle tissue and a sour smell. in order to develop recommendations for the use of investigated proteinpolysaccharide mixtures as functional and technological ingredients with cryostating properties in the production of chopped semi-finished products, the chemical composition and functional and technological properties (ftw) of the model meat-stuffed meat systems with their use were studied. the results of studies are shown in the table.1. the conducted studies suggest that the addition of protein-polysaccharide compositions as cryoprotectants positively influence the increase in moisture-retaining capacity by 9.7 17.3% and the fatretaining ability by 9.4 9.7% in the modulated meat systems, which certainly contributes to the improvement of the structure of the modulated chopped semifinished products (table 2). an important indicator of the quality of semi-finished products is the stability of the forage systems of chopped semifinished products. this indicator characterizes the amount of water and fat bound in the meat system [2, 3]. the results of the studies indicate an increase in the stability of the meat systems of the experimental samples by 15.7 16.5% more, as compared with the control sample. the consistent use of animal proteins and fibers contributes to the improvement of structural and mechanical performance of modulated meat systems. the results of studies are shown in the table. 2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 iryna shevchenko, alexey skochko, advantages of using proteins in the production of truncated semi-finished products, food and environment safety, volume xvii, issue 3 – 2018, pag. 272 – 277 275 table. 1 chemical composition and functional and technological properties of model meat stuffing systems (n = 3; p ≥ 95) indexes control sample №1 sample №2 sample №3 sample №4 mass fraction of moisture,% 69.95 71.47 71.50 72.67 72.56 bulk protein,% 9.89 10.61 10.63 10.60 10.65 mass fraction of fat,% 19.46 17.19 17.14 16.00 16.06 mass fraction of ash,% 0.70 0.73 0.73 0.73 0.73 ph 6.0 6.02 6.07 6.08 6.07 moisture-binding ability,% 78.20 79.09 79.85 80.10 81.14 moisture capacity,% 67.90 74.50 74.70 75.1 75.79 fat-retaining ability,% 68.00 74.50 74.52 74.63 74.57 emulsifying capacity,% 69.80 79.90 79.86 79.91 79.88 emulsion stability,% 77.30 89.40 90.40 89.95 90.02 entrance 74.58 80.76 81.30 81.78 81.83 table2. structural mechanical characteristics of experimental samples of truncated semi-finished products after thermal treatment sample no penetration (depth of immersion of the needle indenter, mm) penetration voltage, pa control 28.4±0,15 17.25±0,09 sample № 1 19.7±0,1 24.87±0,12 sample № 2 17.9±0,09 27.37±0,14 sample № 3 17.6±0,1 27.54±0,12 sample № 4 17.09±0,09 27.63±0,14 the results of the study of these indicators give a complete picture of the meat systems, their structure, their ability to absorb and retain moisture during heat treatment [2-9]. the addition of the above mentioned substances as cryoprotectants in the modulated meat system positively affects the rate decrease in water activity aw 0.027-0.034 as compared with a control sample. the results of the study are presented in fig. 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 iryna shevchenko, alexey skochko, advantages of using proteins in the production of truncated semi-finished products, food and environment safety, volume xvii, issue 3 – 2018, pag. 272 – 277 276 fig. 1 dynamics of changes in the activity of water of heat-treated samples of truncated semi-finished products after 30 days of storage the decrease of aw value in experimental samples is associated with an increase in the concentration of soluble substances in meat juice, which in its turn involves the lowering of temperature at the beginning of the crystallization of moisture in meat systems and, accordingly and changes the nature of ice crystals formation in the cellular structure of meat systems [7-11]. the results of the study of the effect of cryoprotectants on the change in the water activity criterion of the chopped semifinished products indicate an increase in the value of this indicator in comparison with the values for freezing in the experimental samples by 0.002, and in the control sample by 0.005, which is explained by the partial destruction of cell walls and the separation of meat juice , which is more significant for a control sample which does not contain proteinpolysaccharide ingredients [7-9]. the value of the cryoscopic temperature for the prototype samples was: for the control 1.75 ° с, for the sample number 1 -3.84 ° с, for the sample number 2 -4.16 ° с, for the sample number 3 -4.58 ° с, for sample number 2 -4.56 ⁰c (fig. 2). during freezing, storage, defrosting and heat treatment in meat systems for the production of chopped semi-finished products, a slight decrease in the ph value is observed at 0.02 ... 0.04 units relative to the initial level due to the decomposition of the glycogen remaining in the meat until freezing and the formation of lactic acid [10 ,11, 12]. fig.2.cryoscopic points of model samples of truncated semi finished products when introducing different types of proteinpolysaccharide mixtures of all the test samples of heat-treated split model meat products, the biggest changes in organoleptic quality (after freezing, storing for 30 days and thawing) were observed in control samples, characterized by a lack of succulence, fragility structure and lower mass losses and higher than 6.18 7.25 % losses due to heat treatment. the best qualitative characteristics were registered by the prototypes using a protein-polysaccharide mixture of plasma proteins, sodium caseinate, fiber of plantain and flax. however, they all had increased juiciness and a more dense consistency. 0.85 0.9 0.95 1 сontrol sample 1 sample 2 sample 3 sample 4 аw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 iryna shevchenko, alexey skochko, advantages of using proteins in the production of truncated semi-finished products, food and environment safety, volume xvii, issue 3 – 2018, pag. 272 – 277 277 4. conclusion thus, two promising directions of use of protein polysaccharide mixtures in the production of chopped semi-finished products can be singled out: this is the stabilization of the functional and technological properties of raw meat and the cryostabilization of meat systems in the manufacture of frozen semi-finished products. the results of complex studies on the quality of the modulated chopped semifinished products show the stabilizing properties of protein-polysaccharide mixtures, which manifest themselves in improving their qualitative and structuralmechanical parameters, reducing losses in heat treatment and increasing the yield of finished products. 5. references [1]. rogov i.a., chemistry of food. book. 1 (proteins: structure, functions, role in nutrition), rogov i.a. m .: koloss, 2007. 853 p. [2]. salavatulina r.m., rational use of raw materials in sausage production, salavatulin r.m. spb .: zao trading house geord, 2005. 236 p. [3]. feiner g., meat products handbook. practical science and technology, feiner g. boston, ny, washington: crc press, woodhead publ, 2006 356 p. [4]. feiner g., meat products. scientific bases, technologies, practical recommendations, feiner g.; translation from english n.v. magda spb .: professional, 2010. 720 p. [5]. syazin ie, kasynov g.i. the phenomenon of cryoprocessing products. monograph. saarbrücken, germany: palmarium academic publishing, 2012. 296 p. [6]. sharpe a. a., n. g. azarova, e. d. yankovaya, aa bliznyuk, the effect of freezing on the functional and technological properties of meat systems, food science and technology. 2009. no. 2 (7). p. 12-14. [7]. pak a.о., m.o. yancheva, yu.v.yakovleva, influence of the composition of cryoprotective action on the amount of frozen moisture in meat-stuffed semifinished products, thematic collection of scientific papers donetsk. nats university of economics and trade them. m. tuganbaranovsky. 2011. vip. 27. p. 281-286. [8]. nechaev ap, shchub is, anoshina o.m. technology of food production. moscow: koloss, 2005. 768 p. [9]. kutsakova v.e., av baranenko, t.e. burov, mi kremenovskaya, refrigeration technology of food products. part iii biochemical and physico-chemical bases, spb .: giord, 2011. -272 p. [10]. kasynov g.i., o.i kvasenkov, ie syazin., av poker. – krasnodar, innovative technologies of cryopreparation o,f agricultural raw materials: monograph, publishing house of the federal state administration of education and science of the higher educational institution "kubshtu", 2013 147 pp. [11]. semenova a.a, trifonov mp, kholodov fv, a new look at the production of frozen semi-finished products, all about meat. no12008. [12]. vinnikova l.g., o. a. glushkov, e. d. yankovaya, estimation of quality of frozen meat semifinished products with cryoprotective additives, food science and technology. no. 2 (11). p. 47-48, 2010. [13]. kyshenko i.i., oi skachko, estimation of influence of substances of cryoprotective effect on quality indices of truncated semi-finished products, food industry, – no. 21. -s.89-94 2017 . 40 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 40 47 healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets marianna barus 1 , *vita antsupova 2 , igor chifurko 1 , yana ushko 2 1hsee of ukraine “bukovinian state medical university”, chernivtsi, ukraine barus.m@bsmu.edu.ua 2bohomolets national medical university, kyiv, ukraine vitaantsupova@gmail.com *corresponding author received 29thdecember 2019, accepted 30th march 2020 abstract meat is an important source of protein and a popular food product in ukraine. a survey of teenagers in a secondary school in bukovina showed that meat makes up almost half of the daily ration of schoolchildren. the most consumed types of meat: pork and chicken (79% of the total meat ration of the respondents), the most popular places for buying meat: organized urban markets (44% of the families of the respondents). given the widespread use of antibiotics in animal husbandry, the problem of antibiotic content in meat as a food product is very important and relevant. quality food is one of the components of shaping the health of the future generation. in ukraine, the analysis of meat regarding the presence of antibiotic residues in laboratories in organized markets is not mandatory. theoretically, the likelihood of the presence of antibiotic residues in meat and their ingress into the body of a teenager when eating meat is not excluded. to look into this problem, a study was conducted on popular meat varieties from organized markets in chernivtsi (2017-2018) in terms of the presence of antibacterial residues. the identification of antibiotic residues in meat was carried out according to standard methods. the following results were obtained: 91% of meat samples contained residues of antibacterial drugs. of these, 100% pork and 86% chicken (the most consumed meat) contained antibiotic residues. the most commonly detected antibiotics are the aminoglycoside group and the highly toxic streptomycin. 91% of the studied samples in 2017 contained two or more antibiotics, in 2018 – 46%. in 2018, compared with 2017, there was a slight decrease in the number of meat samples containing antibiotics (f = 4.3; p = 0.05). studies have shown that more meat samples contain antibiotics. given the small number of samples, the data obtained are preliminary and require further study. key words: antibiotics; meat; teenagers’ food safety; quality reaction; sensitivity threshold. 1.introduction nutrition is one of the most important factors in human health and its proper physical development. food, entering the body, performs many important functions. healthy lifestyle priorities are food safety and quality issues. more than 90% of human exposure to harmful substances comes from the consumption of contaminated food (1). due to the fact that the consumption of animal products has http://www.fia.usv.ro/fiajournal mailto:barus.m@bsmu.edu.ua mailto:vitaantsupova@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 41 increased significantly, there is a risk of co-occurrence of antibiotics with meat, milk, fish and eggs (2). the developing organism has the greatest risk of exposure to various biologically active substances. frequent use of such products can cause diseases of the gastrointestinal tract, decreased immunity, allergic reactions and a number of dangerous diseases (3). eating quality products will help preserve the health of the nation (4). one of the main foods is animal products. almost half of the need for proteins, which are the basic structural elements of all tissues of the human body, we get with meat. according to the norms of a balanced diet a person needs an average of 44-45 g of animal protein per day (5). the most protein is found in beef and chicken (up to 20 grams), the lowest protein content in pork – up to 12 grams. in modern animal husbandry for various purposes, antibiotics are widely used (6). the widespread use of antibiotics as medicines in medicine, veterinary medicine, various fields of agriculture, food and canning industry contributed to the creation of a special industry – the antibiotic industry (7). it also caused the emergence of semi-synthetic and synthetic analogues. in animal husbandry, antibiotics are used not only for the treatment and prevention of diseases, but also to stimulate growth, fattening, improve the productive characteristics of animals (8). as a result, the meat supplied to the consumer contains substances that, when they are received continuously, pose a serious risk to human health (9). the use of antibacterial promotes the formation of antibiotic-resistant bacteria in the body of farm animals that enter the food further and pose a threat to the health of the consumer (10). passive use of antibiotics can cause intestinal dysbiosis, decreased immunity. a very dangerous and undesirable effect of antibiotics is the sensitization of the human body with subsequent allergic reactions (8). penicillin, streptomycin and aminoglycosides are considered to be the strongest allergens (4; 11). streptomycin and tetracycline act on pregnant women as teratogens, causing anomalies in embryonic development. widely used in veterinary medicine, levomycetin, extremely dangerous for antibiotic sensitive people, can cause toxicosis, aplastic anemia, which turns into leukemia (12). the problem of residues of antibiotics in food, including their impact (contamination) on human health and the environment, is receiving great attention in almost all countries in europe, canada and the united states. the urgency of the problem is determined by three aspects: general biological, medical and socioeconomic, which are closely related (10). the general biological aspect of the problem is that microorganisms resistant to disease are trapped in the digestive canal of diseased humans and animals. under the influence of antibiotics, sensitive cells die, and resistant, multiplying, become the predominant part of the micro flora. this leads to an increase in the duration of diarrheal and other diseases, the need to increase doses of drugs, which is undesirable both in terms of toxicity and in terms of the possibility of super infections. the problem is acute in pediatrics and in the clinic of immunodeficiency states (13). for the prevention of the adverse effects of the use of bioactive substances, there are mandatory conditions for keeping animals before slaughter until the antibiotics are completely removed from their body, but these conditions are not always met. according to literature, in ukraine about 30-40% of all meat contains antibiotics food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 42 (14). in europe, this figure is less than 1% (10). meat is one of the most sought after products. for healthy eating, the information on the presence of antibiotics in the meat we consume is very important. to study this issue, the following tasks and goals were set: to determine the meat diet of adolescents in bukovina, students of chernivtsi secondary school i-iii degrees №4, to study the content of antibiotic residues in meat from the markets of chernivtsi for 2017-2018. to substantiate the control of antibiotic content in meat in organized urban markets as a guarantee of a safe and healthy diet. 2. materials and methods in order to determine the meat diet of adolescents within one family, we conducted a survey of students of chernivtsi secondary school №4 grades 11 to 17 years. respondents were asked, with the permission of their parents, to voluntarily and honestly answer questions regarding their nutrition. including: 1. what meat is in your diet? 2. where do you usually buy meat from? 3. in what markets do you buy meat? 4. do you know about the physiological protein intake recommended for adolescents? 5. do you know about the importance of animal protein for the healthy development of a teenager? 6. do you know about the content of antibiotics in the meat you consume? 7. do you know about the harm of antibiotics to the human body? closed questions (№2, №4-7) were used because they are easy to handle, in addition, the respondent answers the question more willingly and quickly. as well as open-ended questions (№1, №3) were used in order not to influence the respondents by the researcher’s opinion, to direct further work and to narrow the circle of purchase of products. during 2017-2018, 23 meat samples from the chernivtsi market were surveyed. for qualitative reactions, we used a concentrate extract from the test sample of meat, which was prepared for each sample separately (13). weighing the prepared minced meat according to state standard 20235.0-74, weighing 5 g, was transferred into a conical flask of 20 ml of twice boiled distilled water; with shaking three times, withstood for 15 min. the resulting extract was filtered. the filtrate was used to perform qualitative reactions. for the study of each sample, 13 standard qualitative reactions were performed (15). antibiotics are chemical compounds that have certain chemical functional groups, each of which can be determined by an appropriate qualitative reaction. due to the fact that amino acids, which are a component of animal protein, may have identical functional groups with antibiotics, they were denatured by ten minutes of boiling the filtrate (14). it should be noted that in the samples tested, the initial concentration is much lower than the concentration of these substances in the animal body. for each of the qualitative reactions, there is a concept known as a "threshold of sensitivity" that is directly dependent on the concentration of the substance being determined. that is why when studying the filtrate of one sample in the experimental series of qualitative reactions in some cases, we observed the formation of sediment, gas release, turbidity were interpreted as a positive analytical effect of "+" (positive reaction); in others, these signs were absent, which was interpreted as a negative food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 43 analytical effect of "-" (negative reaction) (14). qualitative analysis of the presence of antibiotics in the samples was carried out taking into account the chemical classification of antibiotics by standard methods. tetracyclines reaction i. the reaction is carried out with concentrated sulfuric acid. as a result, tetracycline derivatives are formed that have a specific color. reaction ii. formation of colored complex salts in an alcohol medium from ferric (iii) chloride is brown or reddish brown. reaction iii. formation of colored complexes with cuprous salts (ii), zinc. antibiotics of aromatic series reaction iv. hydrolytic cleavage reactions are used in acidic or alkaline media, with subsequent identification of the products formed. thus, when heated with levomycetin with sodium hydroxide solution, a yellow color first appears, which turns to red-orange (due to the formation of acinitroform), and further heating produces a brick-red precipitate and an ammonia odor. reaction v. levomycetin stearate when heated with hydrochloric acid concentrated hydrolyzes – stearic acid is formed, which floats to the surface in the form of oily droplets that solidify upon cooling. reaction vi. in the rapid analysis, the reaction of levomycetin with cuprum (ii) sulfate in an alkaline medium is used in the presence of n-butanol – the alcohol layer is colored in blue-violet due to the formation of a complex salt. penicillins reaction vii. non-pharmacopoeial reactions: reaction of formation of cuprum (ii) (green) or ferum (iii) (red) of penicillinohydroxamates after hydroxylaminolysis of the β-lactam cycle of penicillins. cephalosporins reaction viii. under the action of a mixture of sulfuric and nitric acids, cephalexin turns yellow, cephalothin turns olive green, then it turns red-brown. streptomycin reaction ix. when interacted with ferric ions (iii) in an acidic environment, maltol forms compounds that have a purple color. express methods of identification of streptomycin: reaction x. isolation of ammonia by heating the substance with sodium hydroxide solution. reaction xi. formation of brown color with potassium tetraiodomercurate alkaline (nessler reagent). reaction xii. formation of red precipitate when heated with copper-tartrate reagent (aldehyde group). aminoglycosides reaction xiii. reaction to the aliphatic amino group of aminoglycosides – violet color is formed when heated with ninhydrin. the results of qualitative reactions to the content of antibacterial drugs in the test samples are shown in tables 1, 2. 3. results and discussion. a survey of schoolchildren (286 respondents) of the chernivtsi secondary school №4 showed the following results: 48% – most often use pork; 44% buy meat in organized city markets; 62% – recognize the importance of animal protein for the healthy development of adolescents; 93% – do not reliably know about the presence of antibiotics in the purchased meat; 61% of respondents are aware of the dangers of these substances for the human body. 79% of the meat from the diet of chernivtsi teenagers is pork and chicken. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 44 this result of the survey prompted us to investigate the content of antibiotics primarily in the most common types of meat from organized markets in chernivtsi. for the study of antibiotic content in meat samples, 156 qualitative reactions were performed in 2017, 28 of which gave a positive analytical effect, in 18% of specific reactions had a sufficient threshold for sensitivity in antibiotic meat samples tested (table 1). it should be emphasized that 100% of the tested samples showed the presence of antibiotics. most often, the positive reactions were for the following groups: aminoglycosides – 100% (reaction xiii); tetracycline – 50% (reaction ii); aromatic antibiotics and streptomycin – 42% respectively (reactions iv; xi). table 1 the results of the study of the presence of antibacterial preparations in meat products from the markets of chernivtsi for 2017 № meatsample kindofmeat samplingmarket reactionnumber іі іv xi xiii і,ііі,v,vі,vіі,vііі,іх,х,хіі 1. rabbit lower + + all samples had a negative analytical effect 2. chicken main + + + + 3. pork main + + + 4. chicken main + + + 5. veal central + 6. pork central + + 7. chicken central + + 8. chicken lower + 9. veal main + + 10. veal central + + + 11. pork central + + + 12. pork lower + + i; ii; iii; iv; v; vi; vii; viii; ix; x; xi; xii; xiii – see materials and methods division; "+" – positive reaction; "-" – is a negative reaction. more than one type of antibiotic was detected in 91% of the samples. such data are cause for concern, since they indicate the likelihood of concomitant uncontrolled penetration of antibiotic residues into the body of a teenager during meat consumption, which can have adverse consequences. in the theoretical part of our work we have described the negative impact of these substances on the body. to study the content of antibiotics in meat samples in 2018, we performed 143 qualitative reactions, 21 of which gave a positive analytical effect, that is, 15% of specific reactions had a sufficient threshold for the detection of antibiotics in the tested meat samples (table 2). this is a 3% decrease compared to 2017, which may indicate a decrease in the level of antibiotics in consumed meat. it should be emphasized that in 18% (two samples: №1 chicken; №2 rabbit), the qualitative reactions gave a negative analytical effect, that is, we can assume that the tested meat contains no antibiotics, but 82% (9 samples) – contains antibacterial residues. more than one type of antibiotic was detected in 46% (5 samples), which means that while consuming meat, although in residual quantity, a complex of dangerous groups of antibacterial drugs falls (see table 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 45 most often in 2018, meat samples tested positive for the following antibiotic groups: streptomycin – 64% (reaction xi); aminoglycosides – 46% (reaction xiii); tetracyclines – 46% (reaction i); aromatic antibiotics – 36% (reaction iv) (see table 2). table 2 the results of the study on the presence of antibacterial preparations in meat products from the markets of chernivtsi for 2018 № meatsample kindofmeat sampling market reaction number і іv xi xiii іі,ііі,v,vі,vіі,vііі,іх,х,хіі 1. chicken central all samples had a negative analytical effect 2. rabbit main 3. chicken lower + 4. veal central + + + + 5. pork central + + + 6. veal central + + + 7. chicken main + 8. pork lower + 9. pork main + + + 10. veal central + 11. pork central + + + + notes:i; ii; iii; iv; v; vi; vii; viii; ix; x; xi; xii; xiii – see materials and methods division; "+" – positive reaction; "-" – is a negative reaction. in 2018, the content of two or more antibiotics in the tested samples was 1.8 times lower than in 2017, which may indicate a decrease in the amount of incoming residues of various groups of antibacterial drugs in the consumer's body. the validity of the difference (f=4.3; p˂0.05) was confirmed by the fisher test. this result is encouraging. however, the number of meat samples containing highly toxic streptomycin has increased. interestingly, chicken sample №1, 2018 did not contain antibiotic residues, and sample №2, 2017 revealed 4 different groups (see tables 1 and 2). this case indicates that the content of harmful substances in the meat does not depend on its type. for safe nutrition, you must focus on the authority of the manufacturer and the protocols of laboratory testing of meat products. during the experiment, it was found that only 9% of meat (2 of 23 samples: chicken and rabbit) were not contaminated with antibacterial agents. the most common type of meat pork in 100% (8 samples) contained antibiotics, chicken – 86% (6 out of 7). all veal samples contained antibiotics. half of the rabbit samples were contaminated with antibacterial agents. available materials encourage the dissemination of information on the basics of safe and balanced nutrition among adolescents and indicate the need for educational work among the population. based on an experimental study of the antibiotic content in meat consumed by the population of chernivtsi, we give some recommendations on how to eliminate the passive use of antibiotics from our menu: 1. meat must be soaked in salted water for several hours (the minimum procedure should be 15 minutes). 2. boil meat for 20-30 minutes and drain the broth. statistics show that boiling for 3 hours reduces the content of antibiotics by 90%. in this case, 70% of the harmful food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 46 substances go into the broth, so it must be drained. 3. to minimize the consumption of byproducts (kidneys, liver), since they have the highest accumulation of harmful substances (4). 4. buy meat from certified sellers and private producers who meet the health standards of their products. 5. require lab meat testing protocols. if consumers pay more attention to the quality of what they buy, then entrepreneurs will also have to improve production technologies and, eventually, we will be able to eliminate antibiotics from the menu. 6. if you do not like cooked meat, you can use the oven. antibiotics are starting to split already at 130°c. 7. when dividing the meat, be sure to remove the skin. in it often accumulate harmful substances. 8. it is also important to store the meat properly. to do this, select a separate shelf in the freezer, preferably the bottom. freezing meat reduces antibiotic content by 20-25%. 9. never buy foods with a very long shelf life: they contain many harmful substances and toxins. eating quality products will help keep the younger generation healthy. 4. conclusion 79% of the entire meat diet of adolescents is pork 48% and chicken 31%; 91% of the test meat is contaminated with antibacterial residues. the most commonly used types of meat, such as pork and chicken, contained antibiotic residues in 100% and 86% of the samples, respectively. residues of antibiotics from the group of aminoglycosides and streptomycin were most often found in the studied meat samples. 9% of meat (one sample of chicken and rabbit meat) was not contaminated with antibacterial agents. the content of harmful substances in meat does not depend on its type. for safe nutrition, it is necessary to focus on the authority of the manufacturer and the protocols for laboratory studies of meat products. laboratory monitoring of the content in antibiotics in meat in organized urban markets, as a guarantor of a safe and healthy diet, is advisable. in order to disseminate information about safe balanced nutrition, educational conversations are necessary. given the small number of samples, the data obtained are experimental in nature and require further study. 5. acknowledgements the authors are grateful to tatyana golubchik, biology teacher at general education school of і-iii degrees №4, chernivtsi, ukraine, for their help in conducting a survey of schoolchildren. 6. references [1] ashraf n., rashid a., naz u., ashraf m.m., khaliq s., khan i.a., nazir a., mohsin n.u., sarwar a., asif r. & malik a. detection of antibiotics residues in protein containing diets (meat and eggs) of human through different methods. j univ med dent coll 9:111. (2018). [2] codex alimentarius commission (cac). maximum residue limits (mrls) and risk management recommendations (rmrs) for residues of veterinary drugs in foods. cac/mrl 2-2017. updated as at the 40th session of the codex alimentarius commission. (2017). [3] darko g., borquaye l.s., acheampong a., oppong k. veterinary antibiotics in dairy products from kumasi, ghana. cogent chem 1;3:1343636. (2017). [4] al awar m.s., al shaibani e.a., salih e.m. & al eryanim m.a. (the protective effect of nabk honey against pathological effects of penicillin and streptomycin on histological food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2019 marianna barus, vita antsupova, igor chifurko, yana ushko, healthy food of teenagers: monitoring the content of antibiotics in meat from organized city markets, food and environment safety, volume xix, issue 1 – 2020, pag. 40 – 47 47 structure and functions of guinea pigs liver. j. appl. pharm. 3:s1–s6. (2013). [5] baynes r.e, dedonder k., kissell l., mzyk d., marmulak t., smith g., tell l., gehring r., davis j. & riviere j. e. health concerns and management of select veterinary drug residues. food chem toxicol 29:112-22. (2016). [6] al-mashhadany d.a., nahla a.a., zaki a.m. & mohammad v.s. detection of antibiotic residues among poultry meat in erbil city and impact of thermal processing on remnants. res j life sci bioinform pharm chem, 3:237-47. (2018). [7] elnasri h.a., salman a.m. & el rade s.a. screening of antibiotic residues in poultry liver, kidney and muscle in khartoum state, sudan. j. appl. ind., 2:116–122. (2014). [8] el-wehedy s.e., darwish w.s., tharwat a.e. & hafez a.e. estimation and health risk assessment of toxic metals and antibiotic residues in meats served at hospitals in egypt. j vet technol 9: 2. (2018). [9] ahmedin abdurehman musa. effect of different packaging methods on consumers eating quality of beef. journal of faculty of food engineering, ştefan cel mare university of suceava, romania v. xviii, issue 3, p. 235-245. 3:s1–s6.(2019). [10] pacheco de carvalho b.r. economic efficiency, markets, institutions and food security. int. j. food system dynamics, 4(2): 88-105.(2013). [11] pyun c.w., abd el-aty a.m., hashim m.m.m., shim j.h., lee s.k., choi k.d., park k.h., shin h.c. & lee c. monitoring of streptomycin and dihydrostreptomycin residual levels in porcine meat press juice and muscle via solid-phase fluorescence immunoassay and confirmatory analysis by liquid chromatography after post-column derivatization. biomed. chromatogr, 22: 254–259. (2008). [12] pegah a., ingela l., barbro s. & karin ö. green-lean synergy – root-cause analysis in food waste prevention. int. j. food system dynamics, 6 (2): 99-109. (2015). [13] shalaby a., salama n.a., abouraya s.h., emam w.h. & mehaya f.m. validation of hplc method for determination of tetracycline residues in chicken meat and liver. food chem. 124:1660–1666. (2011). [14] bezugliy p.a. pharmaceutical chemistry. vinnytsia: the new book, 560 p. (2006) [15] mohamed g.g., khalil s.m. & mahmoud s.h. spectrophotometric determination of some fluoroquinolone drugs in tablets. world j. pharm. pharm. 3:844–857. (2013). microsoft word 7 doaa mohamed abd_corected 29 june.doc 176 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 22018 pag. 176 182 effects of natural feed additives and probiotics on productive performance and meat quality of pigeon *doaa abd el-aziz1 , ghada abdel-raheem2 1faculty of veterinary medicine, assiut university, department of food hygiene, assiut, egypt doaassiut@yahoo.com 2 faculty of veterinary medicine, assiut university, department of animal and clinical nutrition, assiut, egypt *corresponding author received 31th january 2018, accepted 25th june 2018 abstract: the purpose of this paper is to evaluate the effects of moringa oleifera, fenugreek (trigonella foenum-graecum) seeds and yeast (saccharomyces cerevisiae) on pigeon performance, carcass traits, pigeon meat quality and composition. twenty four pairs of parent local egyptian baladi pigeons (20-22 month-old) were used in the experiment. live body weight (lbw), body weight gain (bwg), feed intake (fi), feed conversion ratio (fcr) and feed efficiency were reported. the birds were slaughtered at the end of experiment (90days) and carcass meat attributes and composition were recorded. the best lbw, bwg, fcr and feed efficiency were noticed when feeding yeast (saccharomyces cerevisiae), while the best carcass weights, carcass yields, head and liver weights resulted with moringa oleifera feeding. also, the best result for cooking loss percent reduction was occurred when feeding with moringa oleifera. all treatments improved the microbiological quality of pigeon meat, in particular fenugreek when compared with control. all treatments had no significant effects on color, odor or ph. the highest protein content in pigeon meat was estimated with moringa oleifera followed by fenugreek, while the best reduction effect on fat contents occurred with yeast feeding.thus, natural feed additives and probiotics could improve pigeon productive performance and meat quality. keywords: safe additives, yeast (saccharomyces cerevisiae), pigeon performance, pigeon meat 1. introduction pigeon meat is tasty and nourishing, but little is known about pigeon rearing and its meat quality. pigeon meat is very rich in proteins and other nutrients and pigeons can be simply reared. addition of probiotics and feed additives as herbs can be used instead of antibiotics to enhance the health, performance, carcass characteristics and meat quality of birds [1]. moringa oleifera has many uses; it is used to prevent underfeeding in infants, as a good source of iron, applied in culinary uses, and also applied for medical uses as an alternative to antibiotic [2]. fenugreek (trigonella foenum-graecum); a famous plant which is rich in calcium and can be used as vegetable, herbs, or as spice and applied in medical uses to reduce blood sugar and to improve digestion [3]. so, moringa oleifera and fenugreek can be used as feed additives in pigeon feed as good source of protein and improve protein utilization. probiotics are bacteria or yeasts that birds or animals can be fed with to improve their growth performance parameters traits. yeast (saccharomyces cerevisiae) has been known as feed additive for its contents of high quality protein as it includes lysine and vitamin-b complex. also, yeast enhances immune response, intestinal development, feed conversion ratio and decreases the toxic effect of aflatoxins. consequently, yeast food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 177 (saccharomyces cerevisiae) has good application in medicinal purposes [4]. the aim of this study is to evaluate the effects of addition of moringa oleifera, fenugreek (trigonella foenum-graecum) and yeast (saccharomyces cerevisiae) on pigeon performance, carcass characteristics and pigeon meat quality and composition when added to pigeon diet. 2. materials and method a total of 24 pairs of parent local egyptian baladi pigeons (20-22 month-old), with an original average body weight of 330 ± 9 g were employed in this experiment (sex ratio of pigeons 1:1). pigeon divided into four treatments of 6 pairs each (3 replicates of 2 pairs each). birds were reared in the laboratory animal house in the faculty of veterinary medicine, assiut university, assiut, egypt. the birds housed in identical environmental conditions and rooms (4×4 m2 floor and 6 m height). feed was inhibited 8 hours before slaughter, but birds were watered till slaughtered. 2.1. treatments the four treatments were: 1) basal diet (control) (table 1), 2) basal diet + 2% moringa oleifera, 3) basal diet +2% fenugreek (trigonella foenum-graecum) seeds, and 4) basal diet + 2% yeast (saccharomyces cerevisiae). the basal diet was prepared according to abou khashaba et al [5]. live body weight (lbw), body weight gain (bwg), feed intake (fi), feed conversion ratio (fcr) and feed efficiency (inverse of feed conversion ratio) were recorded weekly till the end of experiment (90days). the birds were slaughtered at the end of experiment, scalding in hot water at 85°c, directly after slaughter. carcass weight was calculated after head removal and evisceration. also, weights of head, heart, liver and gizzard were recorded. in addition, the carcasses were kept frozen till ph, cooking loss, sensory and microbiological analyses were carried out. table 1 composition and chemical analysis of the basal diet ingredients% yellow corn 68.50 soybean meal,44 % 22.00 oil 5.20 limestone 1.40 bon meal 2.30 common salt 0.30 vit. & min. mix.* 0.30 total 100 calculated values raw protein, % 15.51 me kcal/kg 3201.36 raw fiber,% 3.183 ether extract,% 2.813 calcium, % 1.346 available phosphorus, ap % 0.403 lysine, % 0.806 methionine,% 0.280 methionine + cysteine % 0.533 c/p ratio 1/212 *vit. & min. mix: each 1kg contains: 10,000 iu vit. a; 2,000 iu vit d3 10 mg vit. e; 1mg vit. k; 1mg vit. b1; 5mg vit. b2; 1,5mg vit b6; 0.1mg vit. b12; 0.3mg; niacin, 10 mg ; panatothenic acid, 0.5 mg, biotin; i mg folic acid;250 mg choline chloride; 60 mg manganese; 30 mg iron; 50 mg zinc; 4 mg copper;0.3 mg iodine; 0.1 mg seleinium and 0.1mg cobalt.** calculated according to nrc [6]. 2.2. carcass yield the birds slaughtered and the carcasses were weighed without head, neck, feet or viscera to give carcass weight. carcass yield (dressing %) is the relation of the carcass weight to the live body weight (lbw) [7]. carcass yield= carcass weight/ lbw × 100 2.3. sensory analysis sensory analysis was carried out by using nine-point-hedonic scale for odor and color [8]. 2.4. ph measurement food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 178 ten grams of meat sample added to 100 ml of distilled water after slaughter for one hour. (adwa two points ph calibration, romania) [9]. 2.5. cooking loss the sample was weighed at room temperature and placed at 80°c for 45 minutes then cooled at room temperature and it was weighed once again to calculate cooking loss [8]. cooking loss= initial weight of sample weight of sample after cooking/ initial weight of sample × 100 2.6. microbiological analysis carcasses were rinsed by 100 ml of distilled water and ten-fold serial dilutions were prepared and plating on standard plate count agar for aerobic bacterial count, violet red bile agar (vrb) for coliforms count, and eosin methylene blue agar(emb) for detection of e.coli count. the plates were incubated at 35±2 °c for 24-48 hours [10]. 2.7. chemical analysis the pigeon samples were analyzed for moisture, protein, fat, and ash contents according to the aoac methods [11]. 2.8. statistical analysis data were analyzed using one way anova with spss 13.00 software (spss inc., chicago, il, usa). 3. results and discussion 3.1. performance as shown in table 2, performance effect of moringa and fenugreek improved feed intake as compared to yeast, but with less weight gain. this result is in accordance with sanchez et al. [12] who found that an increased level of moringa oleifera in the diet enhanced the feed intake and digestibility of the diet. the authors owed the improvement in the feed conversion ratio and weight gain when feeding moringa due to its high nutrient contents.[2] in contrast to gakuya et al. [13] who found that feeding broiler with moringa oleifera above 7.5% caused reduction in feed intake. also, feeding fenugreek recorded high feed intake which in agreement with elagib et al. [14] as a result of its high contents of galactomannan that improve appetite and digestion. there is a significant increase in body weight in control and treatments till the second month, then the weight continued to increase only with yeast feeding in the third month compared with control. the best overall mean body gain was observed when feeding with yeast followed by moringa. the best feed conversion ratio was recorded when feeding with yeast (saccharomyces cerevisiae); which indicates less feed consumed to gain one kilogram per body weight, by comparing with other treatments and control. this result is in accordance with paryad and mahmoudi [15], who found that the addition of 1.5% yeast to the diet of broiler caused body weight gain and feed conversion ratio getting better. also, hosseini [16] and onifade [17] observed that feeding broiler with yeast advanced body weight and feed conversion. in addition, rutz et al. [18] reported that feeding with yeast extract enhanced the growth performance of broiler and accredited this to the effects of the nucleotide contents in yeast extract and to the occurrence of glucans/mannan/ fructooligosaccharides in yeast. on the other hand, duru et al. [3] established that feeding of broiler chicks with higher levels of fenugreek (20 and 40g/kg) negatively affect the feed efficiency ratio and the weight gain. 3.2. carcass parameters moringa showed an improving effect on the carcass yield (dressing %), while yeast and fenugreek showed lower carcass yield food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 179 than the control (table 3). qwele et al. [19] reported that addition of moringa oleifera to broiler finisher ratio with or without corn maize increased carcass weight. also, hosseini [16] found that addition of yeast caused reduction in the carcass weight, and somewhat similar result was recorded by onwurah and okejim [20] who found that levels of yeast at 1.5g/l and 2 g/l in water for broiler chicken gave dressing percentage of 64.51 and 69.13, respectively. on the other hand, elagib et al. [14] found that feeding of broiler chicks with fenugreek gave dressing percentage of 69, while cinnamon and ginger gave dressing percentage of 72.05 and 73.65, respectively. also moringa had significantly improved head and liver weights than other treatments. these results showed similarity to sarker et al. [2] who revealed that addition of moringa oleifera to nourish commercial chicken broiler significantly improved heart and liver weights and dressing percentages. table 2 effect of moringa oleifera, fenugreeks seed, and yeast (saccharomyces cerevisiae) on pigeon performance parameters control moringa oleifera fenugreeks seed yeast feed intake (kg) 1st month 0.65 ± 0.03b 0.97 ± 0.03 a 0.85± 0.01 a 0.55 ± 0.02 b 2nd month 0.50 ± 0.05 b 0.85 ± 0.11 a 0.75 ± 0.01a,b 0.50 ± 0.03 b 3rd month 0.49± 0.02 b 0.85± 0.06 a 0.74 ± 0.06 a 0.65± 0.08 b monthly fi of a replicate 0.53± 0.02 b 0.89 ± 0.05 a 0.78 ± 0.03 a 0.55 ± 0.03 b total fi 1.64 b 2.67 a 2.34 a 1.7 b body weight (kg) 1st month 1.36 ± 0.03 1.37 ± 0.008 1.37 ± 0.005 1.37± 0.01 2nd month 1.38± 0.023 b 1.44± 0.01 a,b 1.55± 0.02 a 1.44± 0.012 a,b 3rd month 1.40 ± 0.03 a,b 1.38 ± 0.02 b 1.37 ± 0.02 b 1.50 ± 0.01 a over all (mean) 1.38 ± 0.01 b 1.39 ± 0.02 b 1.43 ± 0.02 a 1.44 ± 0.01 a body gain (kg) 1st month 0.03± 0.0002 c 0.08± 0.002 a 0.07 ± 0.002 a,b 0.06± 0.0007 b 2nd month 0.02± 0.013 c 0.07±0.005 b 0.18± 0.003 a 0.07±0.006b 3rd month 0.02± 0.02 b -0.06± 0.001 c -0.18± 0.013 c 0.06±0.009 a monthly bwg of a replicate (mean) 0.02± 0.012 c 0.03± 0.003 b 0.02± 0.006 c 0.06± 0.003 a total bwg for a replicate 0.07 c 0.09 b 0.07 c 0.19 a fcr 23.4 29.7 33.4 8.9 feed efficiency 4.3 b 3.4 b 3 b 11.2 a a, b, c differences between means in the same row with different letters are significantly different (p<0.05). table 3 effect of moringa oleifera, fenugreeks seed, and yeast on carcass and organ characteristics of pigeon parameters control moringea oleifera fenugreeks seed yeast l.b.w(g) 343.5 ± 32.6 340 ± 23.5 330± 3.41 335.5± 10.61 carcass weight(g) 245 ± 39.5 a 255 ± 28.26 a 212.5 ± 1.02 b 220 ± 10.31 b carcass yield % 71.3 b 75 a 64.4 c 65.6 c head(g) 16.5 ± 0.28b 17 ± 1.09 a 16.2 ± 0.21b 15 ± 0.92c heart(g) 3.06 ± 1.49 3.83± 0.13 3.12 ± 0.19 3.24 ± 0.04 liver(g) 6.45 ± 0.69 a,b 7.01 ± 0.89 a 6.81 ± 0.62 a,b 5.81 ± 0.22 b gizzard(g) 6.73 ± 1.54 6.43 ± 0.52 7.45 ± 0.55 7.21 ± 0.52 a, b, c differences between means in the same row with different letters are significantly different (p<0.05) values are means of replicate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 180 3.3. meat quality the effects of treatments on ph, color or odor were non-significant. this is in agreement with loddi et al. [21] when reported that addition of any of probiotics or antibiotics did not affect the sensory characters of broiler meat. higher cooking loss observed in fenugreek and yeast, but non-significant in the case of moringa as compared with the control. on the contrary qwele et al. [19] recorded that the addition of moringa lowered the cooking loss of broiler breast meat. alternatively, milewski and zaleska [22] found that the addition of saccharomyces cerevisiae dried yeast to lamb feed had no effect on the cooking loss percent. also, pelicia et al. [4] observed that feeding yeast with prebiotics to free-range broiler chicken increased the cooking loss percent, but decreased ph of broiler meat. feeding with fenugreek gave the best reduction effect on apc, coliforms and e.coli counts, followed by moringa and yeast as compared with control. the result is in agreement with sharma et al. [23] who found that fenugreek had a reduction effect on e.coli and ascribed this to its flavonoids and phenol contents which have antibacterial and antioxidants effects (table 4). table 4 effect of moringa oleifera, fenugreeks seed, and yeast on sensory, physicochemical and microbiological quality of pigeon meat treatments color odor ph cooking loss (%) apc (cfu/ml) coliforms (cfu/ml) e.coli (cfu/ml) control 7.4 7.2 6.7 22 b 1.7x106a 1x103 1x10 moringa oleifera 7.6 7.2 6.7 26 b 2.3x104b <10 <10 fenugreeks seeds 7.4 7 6.6 37a 1.6x104b <10 <10 yeast 7.6 7.2 6.7 32 a 5.8x104b 1x102 1x10 a, b, c differences between means in the same column with different letters are significantly different (p<0.05) 3.4. chemical analysis as shown in table 5, yeast has the best effect on decreasing fat contents which is good for heart diseases prevention, but had no significant effect on protein contents. these findings are in contrast to milewski and zaleska [22] who reported that the addition of saccharomyces cerevisiae dried yeast in lamb ration increased fat contents, but in agreement that no effect on protein contents as compared to control. moringa oleifera has a good effect on the increase in protein content and decrease in fat contents as compared with the control. also, fenugreek caused increase in the protein contents. as well, the authors reported that moringa and fenugreek are considered good sources of protein [2, 3].these results are in compliance with khaksefidi and rahimi [24] who recognized that the addition of probiotics to chicken broiler diet improved the protein and moisture content in meat. table 5 effect of moringa oleifera, fenugreeks seed, and yeast on proximate composition of pigeon meat treatments moisture% protein% fat% ash% control 71.90 ± 2.34 a,b 18.54±1.51 b 7.30±1.73 a 2.12±0.31a moringa oleifera 72.22±2.57 a,b 19.71 ± 1.91a 6.43 ± 1.46 b 1.52± 0.57 b fenugreeks seeds 71.19 ±2.24 b 19.52 ± 1.64 a 7.42 ± 1.57 a 1.77± 0.58 a,b yeast 73.15 ± 2.78a 18.82 ± 1.74 b 5.95 ± 1.84b 1.96 ± 0.54 a,b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 181 a, b, c differences between means in the same column with different letters are significantly different (p<0.05) 4. conclusion addition of yeast (saccharomyces cerevisiae) to pigeon diet could improve pigeon performance (lbw, bwg, fcr and feed efficiency), while moringa oleifera enhanced some carcass traits (carcass weights, carcass yields, head and liver weights). also, moringa oleifera reduced the cooking loss percent. all treatments, in particular fenugreek improved the microbiological quality of pigeon meat when compared with control, but had no significant effects on color, odor or ph. the highest protein contents in pigeon meat were estimated with moringa oleifera followed by fenugreek, while the best reduction effect on fat contents occurred with yeast feeding. so, these natural feed additives and probiotics could improve pigeon performance, carcass traits and meat quality and composition when added to pigeon diet. 5. references [1]. roberts t., wilson j., guthrie a., cookson k., vancraeynest d., schaeffer j., moody r., , clark s. new issues and science in broiler chicken intestinal health: emerging technology and alternative. j. appl. poult. res. 24:257–266, (2015) [2]. sarker md. sazedul karim, rana md. masud, khatun halima, faruque shakila, sarker nathu ram, sharmin farhana , islam md. nazrul moringa leaf meal as natural feed additives on the growth performance and meat quality of commercial broiler chicken, asian j. med. biol. res., 3 (2): 240-244, (2017) [3]. duru m., erdoğan z., duru a.,küçükgül a., düzgüner v.,kaya d. a. , şahin a. effect of seed powder of a herbal legume fenugreek (trigonella foenum-graceum l.) on growth performance, body components, digestive parts, and blood parameters of broiler chicks. pakistan j. zool. 45(4), 1007-1014, (2013) [4]. pelicia k., mendes a.a., saldanha e.s.p.b., pizzolante c.c., takahashi s.e., moreira j., garcia r.g., quinteiro r.r., paz i.c.l.a., komiyama c.m., use of prebiotics and probiotics of bacterial and yeast origin for free-range broiler chickens. brazilian journal of poultry science, 6(3), 163169, (2004) [5]. abou khashaba h.a., sayed m. a. m., mariey y.a., ibrahem m. a., nutritional and management studies on the pigeons: estimate of metabolizabol energy rquirements. egypt poult. sci., 29(ii), 481-501. (2009) [6]. nrc, national research council : nutrient requirements of poultry. 9th rev. ed. national academy press, washington, dc. , (1994) [7]. wang k. h., shi s. r., dou t. c., sun h. j., effect of a free-range raising system on growth performance, carcass yield, and meat quality of slow-growing chicken. poultry science, 88, 2219–2223, (2009) [8]. apata e. s., koleoso i. m., tijani l. a., obi o. o., okere i. a., effect of sex on meat quality attributes of pigeon birds (columbia livia) in abeokuta metropolis. international journal of agricultural sciences and natural resources, 2(2), 19-23, (2015) [9]. okuskhanova e., rebezov m., yessimbekov z., suychinov a., semenova n., rebezov y., gorelik o., zinina o., study of water binding capacity, ph, chemical composition and microstructure of livestock meat and poultry. annual research & review in biology, 14(3), 1-7, (2017) [10]. dickens j. a., berrang, m. e.; cox, n. a. efficacy of an herbal extract on the microbiological quality of broiler carcasses during a simulated chill. poultry science, 79, 1200–1203, (2000) [11]. a.o.a.c., official methods of analysis, 19th edition aoac international inc. washington dc., (2000) [12]. sanchez n.r., sporndly e., ledin i., effect of feeding different levels of foliage of moringa oleifera to creole dairy cows on intake, digestibility, milk production and composition. livest. sci., 101, 24-31, (2005) [13]. gakuya d.w., mbugua p.n., kavoi b., kiama s.g., effect of supplementation of moringa oleifera leaf meal in broiler chicken feed. international journal of poultry science, 13 (4), 208-213, (2014) [14]. elagib h.a. a., abbas s. a., elamin k. m., effect of different natural feed additives compared to antibiotic on performance of broiler chicks under high temperature. bull. env. pharmacol. life sci., 2 (11), 139-144, (2013) [15]. paryard a., mahmoudi m., effect of different levels of supplemental yeast (saccharomyces cerevisiae) on performance, blood constituents and carcass characteristics of broiler food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 doaa abd el-aziz, ghada abdel-raheem, effects of natural feed additives and probiotics on productive performance and meat quality of pigeon, food and environment safety, volume xvii, issue 2 – 2018, pag. 176 – 182 182 chicks. j. african agric. res., 3(12), 835-842, (2008) [16]. hosseini s.c., the effect of utilization of different levels of saccharomysec cerevisiae on broiler chicken’s performance. global veterinaria, 6(3), 233–236, (2011) [17]. onifade a.a., growth performance, carcass characteristics, organs measurements and haematology of broiler chickens fed a high fiber diet supplemented with antibiotics or dried yeast. nahrung., 41(6), 370–374,( 1997). [18]. rutz f., anciuti m.a., rech j.l., gonclaves f.m, delgado a.d., rosa e.r., zauk n., ribeiro c.l.g., sila r.r., dallmann p.r., desempenho e., caracteristica de carcacas de frangos de corte recebonde extracto de levedura na dieta. j. ciencia animal brasileira, 7, 349-355,( 2006) [19]. qwele k., muchenje v., oyedemi s. o., moyo b. , masika p. j., effect of dietary mixtures of moringa (moringa oleifera) leaves, broiler finisher and crushed maize on anti oxidative potential and physico-chemical characteristics of breast meat from broilers. african journal of biotechnology, 12(3), 290-298, (2013) [20]. onwurah f. b. , okejim j. c., effect of graded levels of baker’s yeast (saccharomyces cerevisiae) in water on carcass and organ characteristics of broiler chickens. academic research international, 5(5), 128-133, (2014) [21]. loddi m.m., gonzalez e., takita t.s., mendes a.a., roca r.o., roca r., effect of the use of probiotic and antibiotic on the performance, yield and carcass quality of broilers. revista brasileirade-zootecnia., 29(4), 1124-1131, (2000) [22]. milewski s., zaleska b., the effect of dietary supplementation with saccharomyces cerevisiae dried yeast on lambs' meat quality. journal of animal and feed sciences, 20, 537–545, (2011) [23]. sharma v., singh p., rani a., antimicrobial activity of trigonella foenumgraecum l. (fenugreek) european, journal of experimental biology, 7, 1-4,( 2017) [24]. khaksefidi a., rahimi sh., effect of probiotic inclusion in the diet of broiler chickens on performance, feed efficiency and carcass quality. asian-aust. j. anim. sci., 18 (8), 11531156, (2005) применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 contents: 1. in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer 98 115 2. study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county sonia amariei , alina mihalcea 116 121 3. aerated confectionery: physicochemical and textural evaluation raluca olimpia zimbru, sergiu pădureţ, sonia amariei 122 130 4. impact of corn and rice syrup adulteration on physico-chemical properties of tilia honey paula ciursa, mircea oroian 131 138 5. the effect of microencapsulation and potato starch on the survival of lactobacillus strains liliana luca, mircea oroian 139 147 6. physicochemical parameters of romanian sunflower honey daniela pauliuc, mircea oroian 148 155 7. feedstocks used for production of 2 nd and 3 rd generation bioethanol – review vasile-florin ursachi, gheorghe gutt 156 169 8. impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk 170 175 9. total phenolic contents of some natural fruit juices and nectars from romanian market veronica tanasa 176 179 10. author instructions i v 11. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xiv, issue 4– 2015 contents: 1 the role of functional food in the improvement of consumer’s health status katerina bojkovska, elena joshevska, nikolche jankulovski, gordana dimitrovska 331 339 2 impact of food waste on climate change cristina ghinea, maria gavrilescu 340 344 3 investigation of the effect of vegetable powders into aqueous and oil phase in butter paste oksana vasheka, aleksandra niemirich, oksana petrusha, natalya drozd 345 351 4 investigation of structural-mechanicalproperties of the recipe composition for special purpose fondants olena dudkina, olexandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk 352 357 5 fatty acids, mineral composition and physico-chemical parameters of imbrasiaoyemensis larvae oil with unusual arachidonic acid content raphael amon akpossan, edmond ahipo due, djary michel koffi, patrice kouame 358 367 6 the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda 368 374 7 studies regarding the photocatalytic transformation of erythrosine b laura carmen apostol, anca florentina caliman, maria gavrilescu 375 384 8 water phase condition in the butter paste with red beet powder oksana podkovko, tamara rashevskaya 385 390 9 wheat fibre impact on flour quality of confectionery products olena kobets, viktor dotsenko, oksana arpul, iryna dovgun 391 397 10 establishing the antioxidant activity based on chemical composition of wild edible mushrooms ana leahu, cristina damian, mircea oroian, sorina ropciuc 398 406 author instructions i v subscription information vi food and environment safety journal of faculty of food engineering, ştefancelmareuniversity suceava volume xiv, issue 4– 2015 153 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 32017, pag. 153 159 study on the influence of heat treatments on the antioxidant properties of ginger amalia cosmovici1, sonia amariei*1 1faculty of food engineering, ștefan cel mare university of suceava, universitatii 13, ro 720229 suceava, romania *corresponding author received 7th september 2017, accepted 28th september 2017 abstract: the purpose of this paper is to study the influence of thermal treatments such as refrigeration, freezing, boiling, and drying on the antioxidant properties of ginger. the determination of antioxidant activity was performed by the spectrophotometric method with the dpph reagent (2,2diphenyl-1-picrylhydrazyl) and the photometric dosing of the polyphenols by the folin-ciocalteu method. the vitamin c concentrations were also determined by hplc as well as the spectrophotometric β-carotene content. the analyses performed showed that thermal processes have a significant influence on the antioxidant capacity of ginger. in the drying process there occurs an increase in antioxidant capacity, and in the case of frozen ginger the polyphenol content is higher than in the fresh form or aqueous extract. also, the content of β-carotene and vitamin c was higher in the case of dried ginger than in the refrigerated one. key words: ginger, antioxidant capacity, β-caroten, c vitamin 1. introduction a quarter of the antioxidants discovered to date are found in ginger. the major source of antioxidants in ginger is represented by: flavonoids, polyphenols (gingerols, shogaols, and catechins), tannins, vitamin c, and beta-carotene. the chemical composition of fresh ginger varies depending on the cultivation mode, the location where it is cultivated, the extraction methods, and the processing methods [1]. a proximate composition of fresh ginger rhizome is: moisture 80.9%, crude protein 2.3%, fat 0.9%, mineral 1.2%, crude fibre 2.4%, and carbohydrates 12.3%. with such a composition, ginger root is an excellent source of nutrients the body needs and active principles with remarkable therapeutic properties for health [2], [3]. ginger active compounds are volatile oils, 1% -3% (bisabolen, zingiberene, zingiberol), non-volatile oils represented by spicy oleoresin components [4], and phenolic compounds (gingerol and (6)-shogaol), and curcuma, geraniol, gingersulfonic acid, [5] etc. the compounds that give the specific taste are gingerol, (6)-shogaol and zingerone. gingerol is the active ingredient of fresh ginger, with many therapeutic effects. gingerol has got anti-inflammatory, antiplatelet, antibacterial and anti-tumor properties which are remarkable [6]. thus, by administering an amount of about 500mg / kg body of ginger, the level of prostaglandins was significantly lower, these results suggesting that ginger could be used as an anti-thrombotic and antiinflammatory agent [7], [8] and as a reducer for the platelets deposition on the blood vessels. ginger exerts many direct http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 154 and indirect effects on blood pressure and heart rate [9]. among the beneficial effects on body health, the most appreciated and highlighted ones are: it is a powerful vasodilator antioxidant-by attenuating or preventing free radical formation, immuno-stimulating by increasing thymus and spleen activity, and production of phagocytes, stimulating the digestive system by increasing the secretion of protective mucus of the gastric mucosa that prevents the occurrence of gastric or duodenal ulcers. it has also antiviral and antifungal properties, while being considered one of the most rewarding cancer enemies [10], even considered to have anti-tumoral effect in leukemia [11], [12].in vitro studies have shown that gingerol present in the root of ginger inhibits the development of helicobacter pylori, the major cause of ulcer, gastric and colon cancer [13]. the ginger root has been shown to have antiemetic effect, reduce nausea and post-operative vomiting or chemotherapy effects due to the presence of gingerol [14]. it has effects of calming and reducing migraines [15]. the mechanism of ginger action in the control of diabetes is due to the inhibition of carbohydrate-metabolizing enzymes, alpha-amylase and alpha-glucosidase, inhibition produced by gingerol, with an effect in reducing glucose absorption [16] [17]. an important study highlighted the antimicrobial action of ginger against e. coli, salmonella typhi and bacillus subtilis [18], [19], [20] and the ethanolic ginger extract showed the largest inhibition zone against salmonella typhi, it showed antibacterial activity against periodontal bacteria such as candida albicans [21], [22], [23], [24] and inhibition of bacteria of the genus mycobacterium avium and mycobacterium tuberculosis in vitro. ginger”s root has a strong antibacterial activity against gram-negative and grampositive bacteria such as klebsiella pneumoniae, proteus vulgaris, streptococcus pyogenes and staphylococcus aureus that can be compared with antibiotics such as trimethoprim, chloromphenicol, gentamicin and erythromycin [25]. like other antioxidants and β-carotene shows sanogenic characteristics for the body in the prevention of certain forms of cancer, heart disease, cataracts, alzheimer's disease, depression, epilepsy, migraines, stomach burns, hypertension, infertility, rheumatoid arthritis, schizophrenia and skin disorders[26]. the antioxidant properties of ascorbic acid are explained by the fact that vitamin c acts as an electron donor. thus, it donates electrons to a series of enzymes that act in the body, part of them participate in the hydrolysis of collagen, others interact in the metabolism of tyrosine, and another category is needed in norepinephrine biosynthesis [27]. stability studies of the active compounds with antioxidant properties in the various stages of raw material processing are required to ensure the effectiveness of the finished product. processing techniques involving extraction solvent, a certain ph, light and heat, can significantly influence the levels and efficacy of bioactive compounds in dietary supplements [28]. 2. materials and methods ginger was purchased from local market. all reagents used were of analytical grade from the sigma aldrich and merck. all analysis were done in triplicate for each of the samples, fresh ginger, oven dried, refrigerated ginger and frozen ginger. determination of antioxidant activity of ginger by dpph method dpph (2,2-diphenyl-1-picrylhydrazyl) is one of the most stable organic nitrogen radicals with a maximum absorption in uv-vis at 517 nm. 2.5 g of each sample food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 155 to be tested was brought into a 50 ml volumetric flask, which was added over a concentration of 99.8% methanol. samples were stirred for 15 minutes, after which they were filtered. the absorbance of the 0.004% dpph standard solution in methanol was read and then the absorbance values of the samples. percent inhibition of free radicals (i%) is calculated by comparing with the dpph solution, with the relation(1): (1) i% inhibition percentage of radicals; aetalon absorbance of the standard sample; aprobă absorbance of the analyzed sample. total polyphenol content. the total phenolic content of the extract was determined by the folin–ciocalteu method. the principle of this method is to reduce the mixture of phosphotungstic acid (h3pw12o40) and phosphomolybdic acid(h3pmo12o40), in the presence of phenols in a basic medium, to yield the blue oxides of tungsten (w8o23) and molybdenum(mo12o23). polyphenols, due to their redox properties, can be oxidized by the folin-ciocalteu reagent, resulting in a blue color whose maximum absorption is 750nm. 1 ml of the liquid sample (obtained by ethanol extraction of 2.5 g sample), diluted 1/5, 50 ml distilled water, is mixed with 5 ml of folin-cobalt reagent with stirring and 20 ml of 20% na2co3 20%solution. after standing for 30 minutes at room temperature, in the dark, the absorbance of the mixture was measured at 750 nm using a double beam uv-vis-nir spectrometer 3600 shimadzu. in order to obtain the calibration curve, gallic acid solutions were used in the following concentrations: 0; 0.2; 0.4; 0.6; 0.8; 1 g / l. the total phenolic content was calculated from the calibration curve, and the results were expressed as mg of gallic acid equivalent per g dry weight. the folin-ciocâlteu (fc) index is calculated by the relation (2): (2) were: fc –folin-ciocâlteu index; a750 – the absorbance at 750 nm; d – sample dilution; determination of vitamin c by high performance liquid chromatography hplc. 4 grams of ginger were milled with 12 ml of acid solution (10% perchloric acid + 1%, a o-phosphoric acid). the preparation obtained was transferred into a volumetric flask of 50 ml, the mixture is stirred vigorously for 2 minutes and subsequently filled to the mark with mobile phase, phosphate buffer solution (tfa) to a ph of 3.5. the standard solution of ascorbic acid has the concentration of 1000 mg / l, and for calibration curve plotting standard solutions were prepared at concentrations of 300, 200, 100, 75, 50 and 25 mg / l ascorbic acid respectively hplc-lc10advp– shimadzu. is provided with automatic injector and uv-vis detection system (dad) and an analytical column with a reverse phase c18 type, length of 250 mm and diameter of 4.6 mm filled with particles of 5 μm diameter. determination of carotenoids content 7.5 g of ginger were milled, over which cyclohexane is added to 25 ml. after extraction the resulting liquid is used to determine the carotenoids, by measuring the absorbance at uv-vis spectrophotometer at a wavelength of 470 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 156 nm. the carotene concentration is calculated using the relationship (3): (3) were : a470 – sample absorbance measured at 470 nm. 3. results and discussion quantities of 210, 150, 90, 30, 10 g of frozen, refrigerated, boiled, tea and dried ginger were analyzed. the analyzed samples have the composition shown in table 1. table 1. the component of samples to be analyzed composition (µl) 1 2 3 4 5 analyzed sample 210 150 90 30 10 methanol 2790 2850 2910 2970 2990 dpph 0,004 % 500 500 500 500 500 from the point of view of the heat treatment of the ginger rhizome, it is observed that the ginger subjected to the drying has the greatest inhibition capacity of radicals, followed by the refrigerated, boiled, frozen one and ginger in the form of the tea with the smallest percent of inhibition rates, as shown in the table 2. table 2. percent inhibition of ginger samples sample/% inhibition i210 i150 i90 i30 i10 refrigerate d 93.76 91.72 91.18 89.42 88.33 frozen 91.59 91.11 90.91 90.64 89.83 boiled 93.01 92.61 90.50 89.55 89.15 tea 89.01 88.61 88.61 88.60 88.54 dried 98.77 98.57 98.30 97.96 96.61 the high antioxidant activity in the case of dried ginger is due to the concentration process by which water is removed, which determines the concentration of the active substance. this high activity for the dried ginger was shown in the study conducted by r. offei okyne according to which, ginger dehydrated by oven had the highest antioxidant power to reduce iron. also, some of the ginger antioxidants are heatresistant, which is also explained by the rather high antioxidant capacity of boiled ginger. according to the results, it can be noticed that thermal treatments influence the phenolic compounds content as shown in figure 1. fig. 1. polyphenol content in ginger it is noticed that the content of phenols is higher in frozen ginger than in the fresh one. this has been demonstrated by r. offe-okyne in his study, according to which frozen ginger presented the highest content of phenols and flavonoids as compared to dehydration in the oven ginger, dried ginger, ginger under the influence of solar heat and fresh ginger. also, in the liquid in which the ginger has been boiled, the phenolic compounds pass in a very small amount, due to their reduced solubility in water. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 157 fig. 2. β-caroten content in ginger the largest share of β-carotene, as shown in the figure 2, shows dried ginger, while the difference between refrigerated and frozen ginger is insignificant. the high content of β-carotene for dried ginger, as compared to the refrigerated and fresh one is due to the dehydration process of ginger, whereby water is removed from the product, this one being concentrated in the active substances. the variation in the amount of ascorbic acid for the three types of ginger: refrigerated, frozen and dried ginger is shown in figure 3. fig. 3 . variation of ascorbic acid concentration in the analyzed samples as can be seen from figure 3, the ginger dried up has the highest amount of ascorbic acid, while the difference between the refrigerated and the frozen one is very small. the destruction of vitamin c during the freezing depends very much on the freezing time and freezing temperature, freezing in this case was carried out in a short period of time of about 29 days. 4. conclusions according to the performed analyses, it can be appreciated that the thermal processes have a significant influence on the antioxidant capacity of ginger. thus, the drying process by dehydration, produces an increase in antioxidant capacity, and the frozen ginger has a higher polyphenols content than the fresh or aqueous extract. also, the content of βcarotene and vitamin c was much superior in the case of dried ginger than the refrigerated one. 5. references [1]. shyur lf. et al., antioxidant properties of extracts from medicinal plants popularly used in taiwan, international journal of applied science and engineering, 3, 3: 195-202, (2005) [2]. samira shahsiah, eskandar moghimipour, nooshin shamsizadeh, azardokht khosravi, evaluation of antibacterial effect of eucalyptus and ginger on dentinal tubules enterococcus faecalis in the presence of smear layer, biosci. biotech. res. comm. 10(2): 225232, (2017) [3]. yiming li, van h. tran, colin c. duke, basil d. roufogalis, preventive and protective properties of zingiber officinale (ginger) in diabetes mellitus, diabetic complications, and associated lipid and other metabolic disorders: a brief review, evidence-based complementary and alternative medicine volume article id 516870, 10 pages doi:10.1155/2012/516870, (2012) [4]. shirin adel p. r and jamuna prakash, chemical composition and antioxidant properties of ginger root (zingiber officinale), journal of medicinal plants research vol. 4(24), pp. 2674-2679, (2010); [5]. n. azu, r onyeagba, antimicrobial properties of extracts of allium cepa (onions) and zingiber officinale (ginger) on escherichia coli, salmonella typhi and food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 158 bacillus subtilis, journal of tropical medicine volume 3 number 2, 1-7; [6]. brett white, md, ginger: an overview, american family physician, volume 75, number 11, 1689-1691, (2007); [7]. sandeep shah, commentary on therapeutic role of ginger (zingiber officinale) as medicine for the whole world, international journal of pharmacognosy and chinese medicine, volume 1 issue 1, 1-3, (2017); [8]. yiming li, van h. tran, colin c. duke, and basil d. roufogalis, preventive and protective properties of zingiber officinale (ginger) in diabetes mellitus, diabetic complications, and associated lipid and other metabolic disorders: a brief review, evidence-based complementary and alternative medicine, article id 516870, 10 pages doi:10.1155/2012/516870, (2012); [9]. nafiseh shokri mashhadi, reza ghiasvand, gholamreza askari, mitra hariri, leila darvishi, mohammad reza mofid, anti-oxidative and anti-inflammatory effects of ginger in health and physical activity: review of current evidence, international journal of preventive medicine, apr; 4(suppl 1): s36–s42, (2013) [10]. e.ernst, r.h.pittler, efficacy of ginger for nausea and vomiting: a systematic review of randomized clinical trials, british journal of anaesthesia 84(3): 367-71, (2000); [11]. badreldin h. gerald blunden b , musbah o. tanira, abderrahim nemmar, some phytochemical, pharmacological and toxicological properties of ginger (zingiber officinale roscoe): a review of recent research2008, food and chemical toxicology 46 409–420, (2008); [12]. leila abar, ana rita vieira, dagfinn aune, christophe stevens, snieguole vingeliene, deborah a. navarro rosenblatt, doris chan, darren c. greenwood, teresa norat, blood concentrations of carotenoids and retinol and lung cancer risk: an update of the wcrf–aicr systematic review of published prospective studies, cancer medicine 5(8):2069–2083, (2016); [13]. gail b. mahady, susan l. pendland, gina s. yun, zhi-zhen lu, adina stoia, ginger (zingiber officinale roscoe) and the gingerols inhibit the growth of cag a+ strains of helicobacter pylori, anticancer res., 23(0): 3699–3702, (2003); [14]. elin yulinah sukandar, neng fisheri kurniati, pratiwi wikaningtyas, deani agprikani, antibacterial interaction of combination of ethanolic extract of zingiber officinale var rubrum rhizome, boesenbergia pandurata rhizome, and stevia rebaudiana leaves with certain antibiotics against infectious mouth microbial, asian j pharm clin res, vol 9, issue 1, 332-335, (2016); [15]. mehdi maghboali et. al., comparision between the efficacy of ginger and sumatripan in the ablative treatment of the common migraine, phytother. res. 28: 412–415 , (2014); [16]. variyar, p.s., gholap, a.s, sharma, a. changes, flavor components inirradiated fresh ginger (zingiber officinale) rhizomes during storage. journal of herbs, spices and medicinal plants, 12, 25-35, (2006); [17]. arshad h rahmani, fahad m al shabrmi, salah m aly, active ingredients of ginger as potential candidates in the prevention and treatment of diseases via modulation of biological activities, int j physiol pathophysiol pharmacol.; 6(2): 125– 136, (2014); [18]. n. azu, r onyeagba, antimicrobial properties of extracts of allium cepa (onions) and zingiber officinale (ginger) on escherichia coli, salmonella typhi and bacillus subtilis, journal of tropical medicine, volume 3 number 2, 1-7. [19]. zahra atai, manijeh atapour, maryam mohseni, inhibitory effect of ginger extract on candida albicans, american journal of applied sciences 6 (6): 1067-1069, (2009); [20]. sandeep shah, commentary on therapeutic role of ginger (zingiber officinale) as medicine for the whole world, international journal of pharmacognosy and chinese medicine, volume 1 issue 1, 1-3, (2017) https://www.ncbi.nlm.nih.gov/pubmed/?term=mashhadi%20ns%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=ghiasvand%20r%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=ghiasvand%20r%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=askari%20g%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=hariri%20m%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=darvishi%20l%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=darvishi%20l%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=mofid%20mr%5bauthor%5d&cauthor=true&cauthor_uid=23717767 https://www.ncbi.nlm.nih.gov/pubmed/?term=rahmani%20ah%5bauthor%5d&cauthor=true&cauthor_uid=25057339 https://www.ncbi.nlm.nih.gov/pubmed/?term=shabrmi%20fm%5bauthor%5d&cauthor=true&cauthor_uid=25057339 https://www.ncbi.nlm.nih.gov/pubmed/?term=shabrmi%20fm%5bauthor%5d&cauthor=true&cauthor_uid=25057339 https://www.ncbi.nlm.nih.gov/pubmed/?term=aly%20sm%5bauthor%5d&cauthor=true&cauthor_uid=25057339 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4106649/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4106649/ food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xvi, issue 3 – 2017 amalia cosmovici, sonia amariei, study on the influence of heat treatments on the antioxidant properties of ginger, food and environment safety, volume xvi, issue 3 – 2017, pag. 153 159 159 [21]. arash azizi, shabnam aghayan, saeed zaker, mahdieh shakeri, navid entezari, shirin lawaf, in vitro effect of zingiber officinale extract on growth of streptococcus mutans and streptococcus sanguinis, international journal of dentistry, volume 1-5, (2015); [22]. ruth t. s. ofongo-abule, elijah i. ohimain, antimicrobial effect induced by fresh ginger root extracts in broilers, british biotechnology journal 9(1): 1-6, (2015); [23]. giriraju a, yunus gy. assessment of antimicrobial potential of 10% ginger extract against streptococcus mutans, candida albicans, and enterococcus faecalis: an in vitro study. indian j dent res.24:397-400, (2013); [24]. suhad a. ahmed, iman i. jabbar and hamssah e. abdul wahed, study the antibacterial activity of zingiber officinale roots against some of pathogenic bacteria, al mustansiriya j. sci, vol. 23, no 3, 2012 [25]. shirin adel p. r , jamuna prakash, chemical composition and antioxidant properties of ginger root (zingiber officinale), journal of medicinal plants research vol. 4(24), pp. 2674-2679, (2010); [26]. leila abar, ana rita vieira, dagfinn aune, christophe stevens, snieguole vingeliene, deborah a. navarro rosenblatt, doris chan, darren c. greenwood, teresa norat, blood concentrations of carotenoids and retinol and lung cancer risk: an update of the wcrf–aicr systematic review of published prospective studies, cancer medicine 5(8):2069–2083, (2016); [27]. r. offei-okyne, j. patterson, l. t. walker, martha verghese, processing effects on phytochemical content and antioxidative potential of ginger zingiber officale, food and nutrition sciences, 6, 445-451, (2015); [28]. badreldin h. gerald blunden b , musbah o. tanira a , abderrahim nemmar, some phytochemical, pharmacological and toxicological properties of ginger (zingiber officinale roscoe): a review of recent research2008; food and chemical toxicology 46 409–420, (2008). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 contents: 1. the use of sunflower lecithin in the technology on gluten-free bread with enzymatic modification of flour starch iryna medvid, olena shydlovska, viktor dotsenko 352 362 2. the influence of inulin and psyllium addition to ice-cream and its effects on the sensorial properties ana leahu, cristina ghinea, cristina damian 363 371 3. characterization of the effect of modulated dry heat processing conditions on essential and non essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds azubuike cumezuruike, joel ndife, chinwe nwachukwu 372 384 4. the contribution of hellenic food authority to the training field of food businesses staff from the authorityestablishment to its modern reorganization elias chaidoutis, dimitrios kostakis 385 394 5. determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka 395 403 6. determination of oxytetracycline residue levels in edible tissues of cattle slaughtered engelbert bilashoboka, boyd mudenda, nosiku munyinda, fabiola vincent moshi, dominic m. kambarage 404 412 7. effect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough galyna voloshchuk, natalia pashova, tatjana golikova 413 – 421 8. author instructions i v 9. subscription information vi title …………………… 135 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 135 139 comparative study of adsorption activity of activated charcoal and silica gel in case of water/toluene mixtures *igor winkler 1,2 , kateryna bodnaryuk 2 1department of medicinal and pharmaceutical chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine 2department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine *corresponding author winkler@bsmu.edu.ua received 17th july 2017, accepted 24th september 2017 abstract: efficiency of the liquid phase adsorption of toluene has been investigated for two common and widely used adsorbents: activated charcoal and silica gel. the adsorption values were determined using the uv-photometry method, where the solution’s absorbance has been measured at the wavelength, which corresponds to the characteristic “benzene” peak of the adsorptive. it has been found that charcoal is a highly effective adsorbent for toluene, while silica gel reveals almost no adsorption activity towards this substance no matter if it is used raw or after desiccation. this conclusion can be used in the development of various adsorbents designed to decontaminate water or wastewaters from toluene and its homologues. such adsorbents should be based mostly on activated charcoal as the most active substrate for the benzene-series hydrocarbons. keywords: adsorption, liquid phase, toluene, activated charcoal, silica gel. 1. introduction toluene and other benzene-series compounds are present, among other contamination agents, in many kinds of municipal, industrial wastewaters and, consequently, can be found in contaminated natural water bodies. aromatic compounds are known as highly toxic and persistent pollutants [1-3]. therefore, a problem of effective extraction and decontamination from these pollutants is important and topical for many fields of technology, industry and environment protection. basically, various solutions can be employed in this context: biological cleaning, chemical destruction, physical destruction and adsorption. however, efficiency of many of them remains insufficient, which complicates fast, effective and comparatively inexpensive decontamination of liquids from the benzene homologies. indeed, even nonnuclear aromatic hydrocarbons are typical xenobiotics and the rate of their aerobic or anaerobic biodegradation is comparatively low [4, 5]. remediation and biodegradation of many aliphatic pollutants usually occur much faster [6-8]. it should be noticed that phenol and its homologues are especially persistent and their biodegradation takes the longest time [8]. on the other hand, this method remains the only option for remediation of contaminated soils, water bodies or other similar objects. low reactivity of aromatic compounds decelerates their chemical decomposition and requires quite strong oxidizing agents. apart from low efficiency and apparent high cost of this option, its applicability is seriously restricted by potential danger of formation of the secondary pollution agents, which may be more toxic and dangerous than the primary pollutants. for inhttp://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 igor winkler, kateryna bodnaryuk, comparative study of adsorption activity of activated charcoal and silica gel in case of water/toluene mixtures, food and environment safety, volume xvi, issue 3 – 2017, pag. 135 – 139 136 stance, oxidative decomposition of phenols, halogen phenols and some other aromatic compounds may result in formation of highly toxic dioxins [9, 10]. physical destruction of aromatic pollutants can be performed, for instance, by uv irradiation solely or in combination with some chemical oxidizing agents (such as h2o2) [11]. this method is comparatively safe, effective and competitive. various substrates can be used for adsorption removal of aromatic compounds from water. in this context it is important to pay attention to adsorption efficiency (1) and post-adsorption utilization of adsorbent (2). second issue assumes both remediation and recycling of adsorbent (in this case the pollutant is collected and reused, buried or utilized otherwise) and direct utilization of the adsorbent without any remediation (by burning the activated carbon together with captured contamination). both adsorption efficiency and adsorbent usability should be considered for the first issue. in this context, silica gel seems interesting since this material is very cheap, stable, and easy to handle with and can be separated from the liquid systems by very simple technological operations. on the other hand, it is highly hydrophilic and expectable adsorption efficiency for aromatic compounds to be captured from aqueous solution is arguable. the main aim of this work was to investigate adsorption efficiency of silica gel in respect to toluene to be captured from its aqueous solution. 2. experimental all experimental investigations have been performed using toluene\distilled water mixtures with the solute concentration 0.2 ml/l. changes in concentration of toluene were measured by determination of absorbance at the wavelength 258 nm corresponding to the ‘benzene’ peak for this compound [12, 13]. since absorbance is in the direct dependence on the optically active component molarity, this parameter can be used in further analysis directly instead of the concentration. further details of the experimental procedures can be found in [13-15]. two kinds of adsorbents were used in our work: the medicinal activated charcoal in the form of regular detoxification pills (0.5 g each) available at drugstores and regular commercial silica gel balls. the charcoal was used directly while silica gel was divided into two experimental series. in one series we used raw material while in another one we used desiccated silica gel after preliminary thermo treatment at 185 0c during 48 hours. the weight of the latter silica gel samples was controlled repeatedly after the treatment and cooling in the desiccators box and, in case the constant weight value was reached, the balls were considered desiccated and ready for adsorption. no grinding was applied to both adsorbents because the adsorbent dispersion can only influence the rate of adsorption, not its value [13, 15, 16]. the dummy experiments were carried out preliminary with a system containing water and silica gel or charcoal without toluene. no changes in absorbance were registered in that series, which indicates no optical activity in the dummy systems without toluene. therefore, this experimental method can be applied to the system under investigation. then, 1 liter of distilled water was mixed with 0.2 ml of toluene, stirred thoroughly and placed in an inert and closed plastic container for 24 hours to reach stabilization of the solute’s concentration. afterwards, the absorbance was measured using lomo sf-46 spectrophotometer (5 times for each sample) and an averaged value was taken as the initial point. then some amounts of different adsorbents (silica gel and activated charcoal) were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 igor winkler, kateryna bodnaryuk, comparative study of adsorption activity of activated charcoal and silica gel in case of water/toluene mixtures, food and environment safety, volume xvi, issue 3 – 2017, pag. 135 – 139 137 added to the liquid and temporal changes in the absorbance were measured during 23 hours. at last, the final absorbance values were measured after next 24 hours for each system. 3. results and discussion a dynamics of temporal changes in absorbance for the system with the thermotreated silica gel is represented in fig. 1. it can be seen that adsorption activity of silica gel towards toluene remains very insignificant. it changes within 5-8 % from its initial value that does not even go beyond experimental error range. no adsorption activity has been registered for the raw silica gel and temporal dependencies in this case remained almost horizontal with some very insignificant scattering of the data because of unavoidable experimental errors. fig. 1. temporal changes in absorbance (conventional units) for the systems toluene/water with different samples of silica gel. an approximate experimental error range (5%) is shown for one of the series therefore, these changes in absorbance cannot be considered as a reliable mark of active adsorption capturing of toluene by silica gel within the indicated time frame. in contrary to the above, the activated charcoal shows quite active adsorption of toluene (see fig. 2). as seen in this case, the changes in absorbance go far beyond the experimental errors range ensuring reliability of the data that proves toluene adsorption on activated charcoal within time frame of the experiment. another proof can be obtained from comparison between the initial and final adsorption values for two adsorbents (table 1). the data of table 1 prove that the thermo treated silica gel does not show appropriate adsorption efficiency towards toluene to be adsorbed from aqueous solutions. amount of the captured adsorptive is very low and it stays beyond accuracy of our experimental investigation. in contrary, adsorption efficiency of untreated medicinal activated charcoal is much higher and it is capable to retain up to 50-60 % of toluene extracted from the 0.2 ml/l aqueous solution. 0.15 0.17 0.19 0.21 0.23 0.25 0.27 0.29 0 20 40 60 80 100 120 140 time, min a b s o rb a n c e , c o n v e n ti o n a l u n it s 5g 7 g 10 g 15 g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 igor winkler, kateryna bodnaryuk, comparative study of adsorption activity of activated charcoal and silica gel in case of water/toluene mixtures, food and environment safety, volume xvi, issue 3 – 2017, pag. 135 – 139 138 fig. 2. temporal changes in absorbance for the systems toluene/water with different samples of activated charcoal. an approximate experimental error range (5%) is shown for one of the series table 1. relative amounts of the solute retained on the adsorbent’s surface for the silica gel and activated charcoal samples adsorbent mass, g initial absorbance i0, c. u. final absorbance if, c.u. amount of adsorbed solute (*), % silica gel (thermally treated) 5 0.218 0.206 5.5 7 0.255 0.234 8.2 10 0.242 0.229 5.4 15 0.261 0.246 5.7 activated charcoal 2 0.391 0.168 57.0 5 0.369 0.127 65.6 (*) any amount of the solute retained by the substrate was calculated as a ratio %100* 0 0 i ii f  , which can be taken as a relative amount of the solute (in percent) captured by the adsorbent 4. conclusions lower cost, higher durability and handling characteristics of silica gel do not compensate its low adsorption efficiency in toluene/water systems. this substrate cannot be any alternative for usage of activated charcoal in various water/wastewater treatment solutions and other environmental protection technologies. 5. references [1]. kim k. h., jahan s.a., kabi e., brown r.j.c., a review of airborne polycyclic aromatic hydrocarbons (pahs) and their human health effects, environment international, 60: 71-80, (2013). [2]. scwarzenbach r.p., et all, environmental organic chemistry, chapter 7, john wiley & sons inc., 213-386, (2013). [3]. wang l., asimakopoulos a.g., kannan k., accumulation of 19 environmental phenolic and xenobiotic heterocyclic aromatic compounds in human adipose tissues, environment international, 78: 45-50, (2015). [4]. diaz e., jiménez j.i., nogales j., aerobic degradation of aromatic compounds, current opinion in biotechnology, 24(3): 431-442, (2012). [5]. boll m., löffler c., morris b.e., kung j.w., anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme a esters: organisms, strategies and key enzymes, environmental microbiology, 16(3): 612-627, (2014). [6]. lasiecki p., biodegradation of diesel/biodiesel blends in saturated sand microcosms. fuel, 116(1): 321-327, (2014). 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 20 40 60 80 100 120 140 time , min a b s o rb a n c e , c o n v e n ti o n a l u n it s 2 g 5 g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 igor winkler, kateryna bodnaryuk, comparative study of adsorption activity of activated charcoal and silica gel in case of water/toluene mixtures, food and environment safety, volume xvi, issue 3 – 2017, pag. 135 – 139 139 [7]. varjani s.j., microbial degradation of petroleum hydrocarbons, bioresource technology, 223(1): 277-286, (2017). [8]. krastanov a., microbial degradation of phenol and phenolic derivatives, engineering in life sciences, 13(1): 76-87, (2013). [9]. shi x., zhang r., zhang h., xu f., zhang q., wang w., influence of water on the homogeneous gas-phase formation mechanism of polyhalogenated dioxins/furans from chlorinated/brominated phenols as precursors, chemosphere, 137: 142-148, (2015). [10]. fernandez-castro p., román m.f.s., ortiz i., theoretical and experimental formation of low chlorinated dibenzo-p-dioxins and dibenzofurans in the fenton oxidation of chlorophenol solutions, chemosphere, 161: 136-144, (2016). [11]. bahnmuller s., loi c.h., linge k.l., von guntena u., canoni s., degradation rates of benzotriazoles and benzothiazoles under uv-c irradiation and the advanced oxidation process uv/h2o2, water research, 74: 143-154, (2015). [12]. ardyukova t., et all, aromatic and hheterocyclic spectra atlas. vol. 7, nauka, novosibirsk, (1973) (in russian). [13]. winkler i., agapova n., determination of water pollution by the oil products through uv photometry, environmental monitoring and assessment, 168: 115-119, (2010). [14]. winkler i., diychuk v., adsorption of toluene from the mixed water−toluene system on medicinal activated carbon, canadian j. chem., 92(5): 392-396, (2014). [15]. winkler i., saviuc a., adsorption and electroadsorption of toluene on some graphite/clay materials, revue roumaine de chimie, 61(10): 773778, (2016). [16]. winkler i., kuvila i., decontamination of toluene pollution in water using raw walnuts shells, food and environment safety, 14(3): 241244, (2015). 1. introduction 4. conclusions microsoft word 4 avramiuc_2_2017.docx 92 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2 2017, pag. 92 97 the influence of slow thawing on evolution of some biochemical compounds in frozen fishes marcel avramiuc* 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received may 17rd 2017, accepted 24th june 2017 abstract: the aim of this work was to study the evolution of ph, amino nitrogen and nitrogen from aminoacids in four fish species, during 48 hours of slow thawing, in order to assess the fish spoilage speed in these keeping conditions. the biological material was represented by frozen fishes (carp, catfish, mackerel and hake) which were subjected to slow thawing at room temperature (+20..+22°c), by analysing, at certain time intervals, ph, amino nitrogen an (mg %), and nitrogen from aminoacids naa (g %). the ph was determined with a digital ph-meter type hanna, and the nitrogen from aminoacids according to sörensen method. the amino nitrogen was determined by the difference between the nitrogen content of volatile bases and the nitrogen content of the ammonia and primary amines. as compared to frozen samples, both ph and the amino nitrogen values of all fish samples showed constant and significant increases up to the end of the analyzed period, while the nitrogen from aminoacids only in the first 30 hours of thawing. the amino nitrogen and the nitrogen from amino acids values have indicated the highest spoilage speed in catfish and hake, and the least speed in mackerel. keywords: ph, freshness, amino nitrogen, nitrogen from aminoacids. 1. introduction the conservation methods using low temperatures, such as freezing, can prevent or limit the modification of nutritional and sensory qualities of raw materials and foodstuffs. as a consequence of biochemical changes taking place in the proteins and lipid fractions during chilling storage of fishes, the deterioration in sensory quality, loss of nutritional value and changes in physicochemical properties occur [1, 2, 3]. according to matsumoto [4], some deterioration occurs, including changes in flavour, colour, odour, and texture, during the freezing, thawing, and frozen storage of fish muscle. the freeze-thaw process caused fibre distortion and an increased gap between fibres in whole tiger shrimp [5]. also, freeze-thaw accelerated protein and lipid oxidation, changed the structure of the myofibrillar protein, caused muscle discoloration, and led to the loss of myofibrillar protein function [6, 7]. by some authors [8, 9, 10], the rate of fish outage varies from one species to another; the deterioration of quality of both wild and farmed fish species is mainly due to action of intrinsic enzymes and microbes. the protein and lipid oxidation occur and have an important influence on product acceptability, during the frozen storage of meat [11]. the fish products are very susceptible to oxidation due to their high levels of long-chain polyunsaturated fatty acids, and this oxidation leads to the formation of lipid hydroperoxides and free radicals [12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 marcel avramiuc, the influence of slow thawing on evolution of some biochemical compounds in frozen fishes, food and environment safety, volume xiv, issue 2 – 2017, pag. 91 – 97 93 however, there are many compounds, including certain low-molecular-weight sugars and polyols, as well as many amino acids, carboxylic acids and polyphosphates that have cryoprotective properties [13]. in this work, the evolution of ph and two nitrogen compounds was investigated, during slow thawing (+20..+22°c) of four frozen fish species, to see what species have a higher spoilage speed on these conditions. 2. experimental 2.1. research material and samples preparation the biological material was represented by four fish species: carp (cyprinus carpio l.), catfish (silurus glanis l.), mackerel (scomber japonicus houttuyn), and hake (merluccius merluccius l.), whose weight ranged from 0.250 kg to 0.900 kg. carp and catfish were caught in romanian streams and brought fresh (in containers of water) at laboratory, where they were slaughtered. after evisceration, meat samples were immediately frozen (at – 29°c), and kept two months up to the experiment. mackerel and hake were bought eviscerated and frozen from retail providers. 2.2. procedure and research methods the fish samples was subjected to slow thawing at room temperature (+20...+22°c), analyzing, at certain intervals, ph values, amino nitrogen, and nitrogen from aminoacids. the amino nitrogen, an (mg %) was evaluated by the difference between the nitrogen content of volatile bases and the nitrogen content of the ammonia and primary amines [14]. the nitrogen from aminoacids, naa (g %) was determined titrimetrically, according to sörensen method [14]. the ph values were determined with a digital ph-meter type hanna. 2.3. statistical analysis four replicates of each determination have represented the data of experiments, which were statistically processed, using sas version 8.02 [15]. to analyze the significance of differences among samples, generalized linear model analysis was carried out. for multiple comparisons duncan’s multiple range test was used (p<0.05). 3. results and discussion the values of ph, amino nitrogen, an (mg %), and nitrogen from aminoacids, naa (g %) in frozen fish samples are shown in the table 1. table 1 biochemical indices values in frozen fish samples fish species biochemical indices carp catfish mackerel hake ph 6.35±0.78cc* 6.40±0.91cc* 6.38±1.04cc 6.41±0.37cc an (mg %) 0.37±0.02hi* 0.50±0.09hi* 0.73±0.07hi 0.52±0.03hi naa (g %) 0.06±0.008d* 0.07±0.005d* 0.05±0.006d 0.04±0.007d *means with the same letters within a row are not statistically diferent (p<0.05) as can be seen from the table, the values of these indices show small and non significant differences between fish species. in the table 2 the biochemical indices determined at certain time intervals of the thawing process are reproduced. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 marcel avramiuc, the influence of slow thawing on evolution of some biochemical compounds in frozen fishes, food and environment safety, volume xiv, issue 2 – 2017, pag. 91 – 97 94 table 2 biochemical indices values during fish thawing test intervals spl. test 3 h 9 h 15 h 21 h 30 h 36 h 42 h 48 h ph 6.38± 0.25 cc* 6.65± 0.78 bc* 6.83± 1.07 bb 7.05± 0.69 bb 7.35± 0.93 ab 7.40± 1.32 ab 7.58± 0.49 ab 7.66± 0.92 aa an (mg %) 0.56± 0.09 hi* 1.53± 0.11 h* 4.68± 1.05 fg 6.03± 1.57 f 8.53± 0.64 e 9.44± 0.09 e 10.33± 1.19 de 13.75± 0.72 c fs1 naa (g %) 0.08± 0.007 cd* 0.09± 0.005 cd 0.18± 0.03 c* 0.23± 0.05 c 0.31± 0.04 bc 0.25± 0.08 c 0.23± 0.03 c 0.17± 0.01 c ph 6.40± 0.97 cc 6.74± 0.63 bc 6.90± 0.48 bb 7.12± 0.88 bb 7.46± 0.59 ab 7.58± 0.45 ab 7.64± 1.23 aa 7.85± 0.38 aa an (mg %) 1.03± 0.04 h 2.36± 0.07 gh 7.58± 1.37 ef 9.91± 0.89 de 12.98± 1.88 cd 13.75± 1.75 c 15.08± 1.08 bc 17.13± 1.92 a fs2 naa (g %) 0.1± 0.07 cd 0.15± 0.04 cd 0.21± 0.09 c 0.34± 0.07 bc 0.38± 0.09 bc 0.32± 0.05 bc 0.25± 0.06 c 0.19± 0.09 c ph 6.42± 1.04 cc 6.65± 0.39 bc 6.73± 0.65 bc 6.94± 0.32 bb 7.35± 0.98 ab 7.37± 0.21 ab 7.49± 0.55 ab 7.58± 0.63 ab an (mg %) 0.88± 0.09 h 0.98± 0.05 h 3.95± 0.74 g 5.27± 0.56 fg 8.03± 1.08 e 9.14± 1.23 e 9.96±1 .08 de 11.81± 0.98 d fs3 naa (g %) 0.07± 0.007 d 0.10± 0.08 cd 0.17± 0.03 c 0.23± 0.09 c 0.29± 0.07 bc 0.25± 0.09 c 0.21± 0.08 c 0.19± 0.06 c ph 6.48± 1.02 cc 6.70± 1.12 bc 6.85± 0.74 bb 7.02± 0.88 bb 7.41± 0.56 ab 7.50± 0.93 ab 7.61± 0.38 aa 7.73± 1.12 aa an (mg %) 0.95± 0.08 h 1.19± 0.04 h 4.87± 1.12 fg 6.53± 1.09 f 9.52± 0.89 de 10.89± 1.34 de 11.35± 0.88 d 13.30± 1.56 cd fs4 naa (g %) 0.10± 0.09 cd 0.14± 0.07 cd 0.27± 0.05 c 0.36± 0.03 bc 0.42± 0.08 b 0.35± 0.05 bc 0.31± 0.04 bc 0.21± 0.07 c spl.= samples; fs1=carp; fs2=catfish; fs3=mackerel; fs4=hake; an=amino nitrogen; naa=nitrogen from aminoacids; *means with different letters within a row are statistically diferent (p<0.05) as compared to the blank sample (frozen fish table 1), at 48 hours of carp slow thawing, the ph of this fish sample recorded an increase by 1.31 ph units. significant increases of ph values were registered at 9, 15, 30 and 48 hours of thawing (p<0.05). as seen in the table 2, at 48 hours of slow thawing, the amino nitrogen, an (mg %), of carp has increased by 37.2 times compared with frozen fish (table 1), the largest and significant increase being recorded in the range 42-48 hours (p<0.05). during thawing of carp, the nitrogen from aminoacids, naa (g %), constantly increased recording a maximum at 30 hours, that is 5 times higher than the blank sample (frozen fish – table 1), then it has food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 marcel avramiuc, the influence of slow thawing on evolution of some biochemical compounds in frozen fishes, food and environment safety, volume xiv, issue 2 – 2017, pag. 91 – 97 95 decreased until 48 hours, reaching 2.8 times higher than blank. at 48 hours of thawing, the ph of the catfish samples recorded an increase by 1.45 ph units, compared to the blank (frozen fish – table 1). significant increases of ph values have been recorded at 9, 15, 30 and 42 hours of thawing (p<0.05). during thawing of catfish, an (mg %) increased by 34.3 times compared with the blank (frozen fish), the largest increase being recorded in the range 9-15 hours (p<0.05). during catfish thawing, naa (g %) increased steadily, recording a maximum at 30 hours (5.4 times higher than blank), then decreased until 48 hours, reaching 2.7 times higher than blank. compared to the blank (frozen fish table 1), at 48 hours of mackerel thawing, its ph recorded an increase by 1.20 ph units. significant increases of ph values were recorded at 9, 21, and 30 hours of thawing (p<0.05). after 48 hours from the start of thawing, an (mg %) of mackerel increased 16.2 times compared to the blank (frozen fish table 1), the largest increase being recorded in the range 9-15 hours (p<0.05). during mackerel thawing, naa (g %) increased constant, recording a maximum at 30 hours (5.8 times higher than blank), then decreased, being at 48 hours 3.8 times higher than blank. at 48 hours of thawing, the ph of the hake samples recorded an increase by 1.32 ph units, compared with the blank. significant increases of ph values were recorded at 9, 15, 30, and 42 hours of thawing (p<0.05). during hake thawing, an (mg %) increased by 25 times compared with the blank (frozen fish), the largest increase being recorded in the range 9-15 hours (p<0.05). during thawing of hake, naa (g %) increased steadily, reaching a maximum at 30 hours (10.5 times higher than blank), then decreased until 48 hours, being, finally, 6 times higher than blank. in figure 1 is shown the comparative evolution of amino nitrogen (an) in fish samples during thawing. 1.03 0.88 2.36 0.98 7.58 3.95 9.91 5.27 12.98 8.03 13.75 9.14 15.08 9.96 17.13 11.81 0 2 4 6 8 10 12 14 16 18 3h 9h 15h 21h 30h 36h 42h 48h thawing time intervals a n ( m g% ) carp catfish mackerel hake fig. 1. the comparative evolution of an (mg %) in fish samples during thawing as seen in fig 1, the amino nitrogen (an) increased, during slow thawing, in all analysed fish samples, the lowest values being registered in mackerel (0.88 – 11.81 mg %), and the greatest ones in catfish (1.03-17.13 mg %). the amino nitrogen (trimethylamine) values can indicate the freshness degree of the fish food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 marcel avramiuc, the influence of slow thawing on evolution of some biochemical compounds in frozen fishes, food and environment safety, volume xiv, issue 2 – 2017, pag. 91 – 97 96 meat. according to castell and triggs, cited by [14], 0-1 mg % amino nitrogen (trimethylamine) indicates fresh fish, 1-5 mg % relative fresh fish, and 5 mg % altered fish. from fig. 1 it can be seen that, at 3 hours of thawing, an values show, in all cases, fresh fish, but at 9 hours only mackerel was still fresh, while the others were already fishes with relative freshness, with smaller values and close in carp and hake, and much greater in catfish (p<0.05). starting with 15 hours of thawing, in all analyzed fishes the amino nitrogen values have shown an altered state. the fig. 2 reproduces the comparative evolution of nitrogen from aminoacids (naa) in fish samples during slow thawing. 0.070.1 0.15 0.1 0.18 0.27 0.23 0.36 0.29 0.42 0.25 0.35 0.21 0.31 0.17 0.21 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 3h 9h 15h 21h 30h 36h 42h 48h thawing time intervals n a a (g % ) carp catfish mackerel hake fig. 2. the comparative evolution of naa (g %) in fish samples during thawing as seen in the graph, for all fish samples the nitrogen from aminoacids (naa) values had a similar evolution, that is a significant increase up to 30 hours of thawing, compared to frozen fishes (p<0.05). at 30 hours, it followed a constant decrease of naa up to the end of analyzed period (48 hours). according to beschea and toma [14], naa content more than 0,1 g per 100 g of product is usually associated with the beginning of fish alteration. the evolution of naa during thawing (table 2 and fig. 2), highlights, at 9 hours, a beginning of alteration only in catfish and hake, and at 15 hours in all the fish samples analyzed. at 30 hours of thawing, hake registered the highest naa value, compared to frozen sample, and to carp, catfish and mackerel with close values (p<0.05). although naa values decreased, steadily, in all fish samples between 30 and 48 hours of slow thawing, in the end that values were significant higher than those of frozen fishes. in order to extend the keeping quality of fish it should lower their body temperature [16], but the freeze-thaw accelerated protein and lipid oxidation, changed the structure of the myofibrillar protein, caused muscle discoloration, and led to the loss of myofibrillar protein function [6, 7]. the deterioration of quality of both wild and farmed fish species is mainly due to action of intrinsic enzymes and microbes [8, 9, 10]. the decrease of naa values after 30 hours of thawing can be attributed to the conversion of aminoacids (released by proteolysis) in subcomponents, by means food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 marcel avramiuc, the influence of slow thawing on evolution of some biochemical compounds in frozen fishes, food and environment safety, volume xiv, issue 2 – 2017, pag. 91 – 97 97 of biochemical processes like: decarboxylation, deamination and desulfuration. 4. conclusions the evolution of amino nitrogen, nitrogen from aminoacids and ph values in frozen fishes (carp, catfish, mackerel, and hake), during 48 hours of slow thawing at room temperature (20-22°c), showed differences between these species. the amino nitrogen values indicated a fresh fish at 3 hours of thawing, in all cases, a fish with relative freshness at 9 hours of thawing (except mackerel which was still fresh), and an altered state of all fishes after 15 hours of thawing. during fish slow thawing, the evolution of nitrogen from aminoacids has emphasized the beginning of alteration, at 9 hours, only in catfish and hake, and at 15 hours in all analyzed fishes. slow thawing at room temperature have proved that, from the four frozen fish species analyzed, catfish and hake had the highest spoilage speed, and mackerel the lowest one. 5. references [1]. bennour m., el marrakchi a., el ouadaa m., chemical and microbiological assessments of mackerel (scomber scombrus) stored in ice, j food prot, 54: 789–792, (1991). [2]. nunes m., batista i., campos r.m. physical, chemical and sensory analysis of sardine (sardine pilchardus) stored in ice, j food sci agric, 59: 37-43, (1992). [3]. olafsdottir g., martinsdottir e., oe,hlenschlage j., dalgaard p., jenson b., undeland i., mackie i.m., henehan g., nielsen j., nilson h., methods to evaluate fish freshness in research and industry, trends in food sci technol, 8: 258-265, (1997). [4]. matsumoto j.j., (1980) in chemical deterioration of muscle proteins during frozen storage, ed. by whitaker j.r., fujimaki m. (american chemical society, washington, 1980), pp. 95-124. [5]. boonsumrej s., chaiwanichsiri s., tantratian s., suzuki t., takai r., effects of freezing and thawing on the quality changes of tiger shrimp (penaeus monodon) frozen by air-blast and cryogenic freezing, j. food eng, 80: 292-299, (2007). [6]. xia x.f., kong b.h., liu q., liu j., physicochemical change and proteinoxidation in porcine longissimus dorsi as influenced by different freeze-thaw cycles, meat sci, 83: 239-245, (2009). [7]. xia x.f., kong b.h., xiong y.l., ren y.m. meat sci. 85 (3), pp. 481-487, (2010). [8]. mehta naresh kumar, elavarasan k., reddy manjunatha a. and shamasundar b.a. (2011) effect of ice storage on the functional properties of proteins from a few species of fresh water fish (indian major carps) with special emphasis on gel forming ability. journal of food science and technology© association of food scientists & technologists (india) 201110.1007/s13197-0110558-y, published online: 12 october 2011. [9]. hsieh r., kinsella j.e., oxidation of polyunsaturated fatty acids: mechanisms, products and inhibition with emphasis on fish, adv food nutr res, 33:233–341, (1989) [10]. pigott g.m., tucker b.w., science opens new horizons for marine lipids in human nutrition, food rev int, 3: 105-138, (1987). [11]. eymard s., baron c.p., jacobsen c., oxidation of lipid and protein in horse mackerel (trachurus trachurus) mince and wash mince during processing and storage, food chem, 114: 57-66, (2009). [12]. kong b., guo y., xia x., liu q., li y., chen h. (2013) cryoprotectants reduce protein oxidation and structure deterioration induced by freeze-thaw cycles in common carp (cyprinus carpio) surimi. food biophysics© springer science+business media new york 201310.1007/s11483-012-9281-0, published online: 9 january 2013. [13]. campo-deaño l., tovar c.a., pombo m.j., solas m.t., borderías a., j. food eng, 94: 26-34, (2009). [14]. beschea magda, toma gabriela copybook of organic chemistry and special biochemistry practical works (fasc. 1 and 2), galaţi, pp. 131-133, (1984). [15]. sas institute, sas user’s guide. statistical analysis system institute, cary, nc, (2005). [16]. pigott g.m., tucker b.w., sea food: effect of technology on nutrition, marcel dekker, new york and basel inc. p 362, (1990). microsoft word 7 myrohn_rogozynskyi_2_2017_31 iulie.doc 111 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 2, pag. 111 116 low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina natalia omel’chenko1, valeriy sinchenko2, *myron rogozynskyi1, oksana mykytyuk3 1 chernivtsi faculty, national technical university “kharkiv polytechnic institute”, chernivtsi, ukraine, rmyron@ukr.net 2 l.i. medved’s research center of preventive toxicology, food and chemical safety, ministry of health, chernivtsi, ukraine 3 dept. of propedeutics of internal medicine, bukovinian state medical university, ukraine *corresponding author received 27th march 2017, accepted 27th june 2017 abstract: investigation on food products contamination by cesium-137 and strontium-90, detection of content of natural radionuclides (radium-226, thorium-232, potassium-40) in raw materials and finished products of building industry, monitoring of some radionuclides content in soils of mountainous area of northern bukovina was carried out. all the results were analyzed and discussed in view of the life safety position. data on radionuclides content in surrounding natural environment as well as in building and food industry production confirm northern bukovina territory’s attractiveness and safety for recreation areas development. keywords: radionuclides, safety, northern bukovina, recreation. 1. introduction increase of the recreational potential is an actual development strategy of chernivtsi region (northern bukovina). the air and water quality, presence of great quantity of mineral water sources with widely known useful properties are topical criteria of attractiveness of the region for tourists. in parallel, we have to keep in mind the existence of some areas with higher radionuclides pollution level in bukovina that is controlled by the authorities of committee on hydrometeorology of the ukraine [1-4]. current level of territory pollution by different agents is relevant and needs to be taken into consideration when developing and implementing programs and strategy for improvement of regional perspectives in recreational tourism. it is well known that the technogenic disaster at the chernobyl atomic electric station in 1986 was a reason for several “spot places” polluted by radionuclides on the territory of bukovina [2]. the presence of the above mentioned polluted areas on the map of the region is the reason of necessity for permanent radiological monitoring of radionuclides spreading and transformation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk, low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina, food and environment safety, volume xvi, issue 2 – 2017, pag. 111 116 112 in natural environment. in parallel, raw materials for industrial sections and manufacturing companies located in the northern bukovina should be checked for radiation activity level as well. significance of food and building industry for tourism is determined by the fact that their production manufactured with the use of local raw materials should be essential when establishing and building recreational areas. hence, reliable information on radiological control results and presence of both artificial and natural radionuclides in the environment of chernivtsi region, especially in places meant for future location of recreational areas should be especially assessed. such data should be taken into consideration when implementing safety improvement and health restoring strategy in the region as well [5, 6]. 2. materials and methods contamination level of food products by cesium-137 and strontium-90, detection of content of natural radionuclides (radium-226, thorium-232, potassium40) in raw materials and finished products of building industry, monitoring of content of natural radionuclides in mountainous area soils of some districts of northern bukovina were the aim of the present investigation. all the mentioned data are important to estimate attractiveness of northern bukovina region in view of investments into recreational areas, resorts and tourism, considering life safety aspect. radiation control of environment was performed by means of estimation of natural radionuclides effective specific activity according to the actual legislation [7]. the total specific activity аς was defined by аς = ara + 1,31ath + 0,085aк ratio, which takes into account specific activity of radium-226 ara, thorium-232 ath and potassium-40 ak. radiation doses were standardized as well. soil samples were collected according to standard operative procedures of testing methods [8] in three mountainous districts of chernivtsi region closely to transport communication areas. points of samples collection were located at the suburbs or beyond settlements in order to avoid misinterpretation caused by human activities’ influence. the irradiation dose was measured in sample collection points as well. samples of food products and raw materials used for their manufacturing were obtained from enterprises and companies acting on the territory of northern bukovina. by analogy, samples of building materials were got from enterprises producing building materials. all data on collected samples were stored in special databases according to local regulations of standard-technical documents management. the detection of content of cesium-137, radium-226, thorium-232 and potassium40 was done by gammasprectrophotometric analytic method employing scintillating detector and multichannel fmf-04а analyzer of spectrum with a modernized software [9,10]. sample volume was equal to 1 liter, different concentration methods were used in case of necessity. the determination of strontium-90 was conducted using method of radiochemical analysis according to standard technique [8]. measurements of intensity of β-irradiation were done using ump-1500 equipment. the irradiation dose was established by using dosimeter drg-01т as an average value for a set including 8 measurements. 3. results and discussion investigation data on radionuclides content in soils of different districts in the northern bukovina are provided in table 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk, low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina, food and environment safety, volume xvi, issue 2 – 2017, pag. 111 116 113 the average values of specific activity for two measurements are presented there. the provided values for the content of natural radionuclides in soils prove that it is the same for all mountain districts of chernivtsi region. such similarity exists probably due to the affinity of rock mediums and similar type of climatic and hydrologic conditions for geological material destruction within bukovinian carpathians. the absolute values of specific activity of investigated radionuclides in crystalline rocks from bukovina were lower as compared with the activity values of samples obtained from the northern regions of the ukraine (zhytomyr region) [11]. the specific activity values similar in magnitude caused also insignificant fluctuations of radiation doses in places of soils sampling. furthermore, the radiation dose value of tested soils was close to the generally accepted normal range for natural background 14 µr/hour. the specific activity of cesium-137 аcs varied within 18-33 bq / kg for different sampling points within a specific district (table 1). such values of аcs pointed on low level of contamination of the territory. a conclusion about radiological safety of soils in highland districts of northern bukovina may be drawn on the basis of detection results of radionuclide content in soils and rocks. soils radiational safety is a factor which stands for the region’s attractiveness in view of establishing and developing recreation areas and resorts. table 1 average values of radionuclides specific activity a (bq/kg) in soils of settlements and radiation doses d (µr/hour) no settlement аcs-137 аra-226 аth-232 аk-40 а d 1 vyzhnytsia 18.7 16.1 29.9 381 87.6 15 2 putyla 24.6 14.9 31.3 462 95.2 16 3 seliatyn 19.5 15.6 23.6 390 79.7 17 4 dolishniy shepit 21.2 17.4 33.2 435 97.9 15 5 beregomet 23.3 19.8 42.1 418 110.5 14 6 krasloil’sk 33.2 25.4 32.1 372 99.1 16 7 quarry run stone < 3 39.6 68.3 748 192.6 – the content of natural radionuclides in raw materials for building industry production was taken into consideration. values of specific activity of some natural radionuclides in different types of raw materials extracted in the chernivtsi region are shown in table 2. on the contrary to quarry run stone (table 1), crushed stone (table 2) is obtained from local rocks. lower normal range of total specific activity is of 370 bq/kg for building materials. table 2 specific activity a (bq/kg) of natural radionuclides in building materials no material аk-40 аra-226 аth-232 а 1 clay 440 12 52,5 118 2 sand 348 38 9 62 3 gravel 81 17 5 31 4 slag 370 92 40 176 5 brick 351 36 53 135 6 wood 8 3 – – 7 adobe 2200 60 262 590 8 crushed stone 495 67 85 220 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk, low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina, food and environment safety, volume xvi, issue 2 – 2017, pag. 111 116 114 according to our results, all the investigated samples of raw materials except adobe had specific activity value even below the lower limit of normal range. hence, raw materials and manufactured products of building companies of the chernivtsi region are safe and they may be used without limitations for building of recreational areas and resorts where investigations had been made. it should be noticed that wood as a building material does not require increased attention from the point of view of radiological control as far as measurements of its content in radionuclides demonstrate that results comply with the level of sensitivity of the devices used (table 2). such fact is confirmed by a number of observations regarding the transfer of radionuclides by root system of herbal plants and trees [4]. additional risk of accumulation of radionuclides by tree leaves should be considered as minimal due to significant precipitation and seasonal change of crown. therefore, radionuclides contamination of food produced and soil in the northern bukovina from life safety aspects was taken into consideration. table 3 displays the results of measurements of cesium-137 and strontium-90 content in different food products. we paid greater attention to the previously mentioned radionuclides as far as their presence in food should be standardized [7]. the product samples were provided by manufacturers in view of compliance with the hygienic standards according to the requirements of actual legislation. the normal ranges of values are provided in table 3 as well. the average values of specific activity were determined for different quantity of samples (n). standard deviation  for each of the investigated radionuclides was provided on the basis of the normal probability law. table 3 average specific activity a of cesium-137 and strontium-90 (bq/kg), standard error of the mean , and acceptable levels (bq/kg) for food products acceptable levels no title of product / raw material samples quantity аcs cs аsr sr аcs аsr 1 beef meat 13 4.77 1.38 1.96 0.82 200 20 2 pork meat 4 < 3 – < 1 – 200 20 3 mutton meat 3 3.27 0.38 2.11 0.97 200 20 4 milk 7 < 3 – < 1 – 100 20 5 flour 5 < 3 – < 1 – 20 5 6 dried fruits 6 8.83 3.28 4.83 1.27 40 20 7 fresh mushrooms 6 8.63 3.12 13.42 4.63 500 50 8 dried mushrooms 2 14.31 4.78 11.09 3.78 2500 250 special attention should be given to the detection of radionuclides’ presence in fish products. such need is maintained by the fact, that major part of fish stocks is located in closed ponds with low water drainage in kitsman’ district of bukovina (a portion of kitsman’district area has status of radionuclide contamination of fourth grade). hence, a part of our research was conducted as an attempt to detect and estimate the content of some radionuclides (cesium-137, strontium-90, radium-226 and potassium-40) in fresh fish, in pond water and in benthal deposits. all the investigated samples were obtained from stavchany pond in the kitsman’ district. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk, low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina, food and environment safety, volume xvi, issue 2 – 2017, pag. 111 116 115 measurements results of radionuclides content in two types of industry fish are provided in table 4. the content of cesium-137 and strontium-90 in fish and fish products from northern bukovina was normal (135 and 35 bq/kg correspondently). it is obvious that radionuclides content is almost the same in live fish, pond water and benthal deposits. such balance is typical for equilibrium in fish-pond biosystems. we observed a higher potassium-40 content in live fish by comparing levels in water and benthal deposits. the external intake of potassium-40 and accumulation in fish for a long time period is probably the reason for such findings. this hypothesis was revealed by the investigation on radionuclides level determined in feed put into pond to feed the fish [12, 13]. values of specific activity of potassium-40 in feed for fish are also provided in table 4. table 4 specific activity a (bq/kg) of radionuclides in carp and crucian fish, benthal deposits, fish feed, pond water of stavchany village radionuclide carp crucian pond water benthal deposits feed cesium-137 0.3 0.5 0.7 18.5 – strontium-90 0.9 0.7 0.3 22.1 – potassium-40 239.2 196.9 1.9 342.4 822.3 radium-226 23.4 21.3 5.2 27.4 – 4. conclusions a conclusion may be drawn that fresh fish meets the criteria of safety for consumption. at the same time, great attention should be paid to a fact of possible presence of nondetected radionuclide potassium-40 in feed for fish. the investigation results of radionuclides content in the surrounding natural environment and production of building and food industry confirm fact that the bukovina territory is safe and attractive for investments and development of recreation areas [7, 14-16]. thus, the possibility of unlimited use of local industry production for building is substantiated. the results of the present investigation confirm the objectivity of advertising messages on attractivity and safety of recreation areas within northern bukovina. it should be marked that our study was a pilot one, and our deductions are generalized and prefatorial, as far as samples quantity for radionuclides content detection varied from a few ones to dozens that might influence dispersion of results. radionuclides’ propagation over the territory, their content in food products and building materials should be investigated more fundamentally in case of necessity by means of collecting and analyzing unified data corresponding to control agencies of chernivtsi region. the field research may be further continued by means of increase of both quantity of samples and points of their collection. the list of controlled isotopes in natural environment and industry products should be widened in future as well. 5. references [1]. kalugin v.o., pishak v.p., mineral waters of bukovina and perspectives of their application, recreating sourses of the carpathians and surrounding territories: materials of the conference. – chernivtsi: p.3-6, (1999). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 natalia omel’chenko, valeriy sinchenko, myron rogozynskyi, oksana mykytyuk, low content of radionuclides in natural environment and production as a rationale for development of recreational potential of northern bukovina, food and environment safety, volume xvi, issue 2 – 2017, pag. 111 116 116 [2]. lapshyn f.v., mineral waters and resorts of bukovina, uzhgorod: carpaty, 128 p, (1965). [3]. vlasyk l.i., horbunov v.v., volyns’ka y.m., omel’chenko n.m., medical-ecological aspects of use of mineral sources in foothill deistricts of chernivtsi region, hygiene of settlements, 42: 88-92. (2003). [4]. the list of settlements designated to zones of radioactive pollution due to chernobyl disaster by statement of cabinet of ministers of the ukraine №106 from 1.07.91 year and order of the cabinet of ministers №17 from 12.01.93, (1993). [5]. gorbunov v.v., omel’chenko n.m., sinchenko v.g., about radionuclides content in natural environment and production as a background for development of recreational areas of bukovina, materials of 3rd international scientific conference “youth’ role in solving out regional and trans-border problems of ecological safety. perspectives of formation of pan-european ecological network”, green bukovina, chernivtsi, 162-169, (2004). [6]. program of tourism development in chernivtsi region for 2016-2020 years, approved by the resolution №20/4-16 of іv session, vii convocation of chernivtsi regional board [7]. standards of radiational safety. state hygieic standards, department of polygraphy of ukrainian center of state sanitaric-epidemiologic control of the ministry of health protection of the ukraine, kiev, 121 p, (1997). [8]. methodological recommendations on sanitary control of radioactive substances content in environmental objects (ed. by marei a.n, zykova a.s.) medicine, moscow, 121 p., (1980). [9]. levchuk с., reference book on principal methods of determination of radionuclides activity, kiev, 119 p., (2016). [10]. acceptable levels of radionuclides cs-137 and sr-90 content in food products and potable water, kiev., 47 p., (1997) [11]. krasnov v.p., radioecology of the woods of polessie, ukraine, volyn, zhytomyr, 112 p., (1998) [12]. konstantinov m.p., zhurbenko o.a., radiational safety, “university book”, sumy, 68-72, (2003). [13]. sinchenko v.g., omel’chenko n.m. indexes of radiational safety of mineral and water resources of northern bukovina in context of it’s recreational development // actual aspects of hygiene and ecological safety of the ukraine. materials of scientific and practical conference (sixth marzeev’s readings, 20-21 may 2010), 10:128-130, (2010). [14]. yatsiv m.y. estimation of ecological situation in chernivtsi region, ecology of environment and life safety, 4: 43-51, (2008). [15]. fomenko n.v. recreational sources and balneology, centre of educational literature, кyiv, 312 p., (2007). [16]. omel’chenko n.m., rogozynskyi m.s., slyvka n.b. functional nutrition as a component of touristic-recreational complex of chernivtsi region // evaluation of ecological state of the territory and perspectives of development of tourism and recreation in chernivtsi region. horbunov’s readings (19 apr 2012), chernivtsi: cf ntu «kpi»: p.64-66, (2012). 309 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3, pag. 309 316 modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia *katerina bojkovska 1 , fanche joshevska 2 , andrijana velevska 1 , sasho bojkovski 3 1faculty of biotechnical sciences, partizanska bb, university st. kliment ohridski-bitola, republic of macedonia, katerina.bojkovska@uklo.edu.mk 2faculty of technical sciences, university st. kliment ohridski-bitola, republic of macedonia 3faculty of economics, university st. kliment ohridski-bitola, marksova 133, 7500, prilep, republic of macedonia *corresponding author received 16th may 2018, accepted 24th september 2018 abstract: the basis of marketing concept refers to the fact that companies exist in order to satisfy consumers’ needs. in order to achieve its objectives and be successful, a company needs to be aware of its customers’ wishes and preferences, so that it should gather information that could be obtained through market research. regardless of the business they are included and performing in, the marketing managers are faced with the need to make decisions constantly, i.e. solve certain problems every day. the quality of the decision mainly depends on the quality of information they have in the moment of decision. the main objective of marketing research is to provide relevant information, increase of success and decrease of risk in company’s working and making right marketing decisions on the basis of gathered marketing information. in order to achieve certain sales level, each company should choose promotional activities by means of which it will achieve the desired effect in the most efficient way. the research study is focused on manager’s attitude towards the utilization of marketing comunication. the objective of the research is to show the significance of marketing research in modern working conditions not only of companies in general, but of companies in milk processing industry in the republic of macedonia as well, because it provides significant information for the market, consumers’ needs, wishes and requests, the demand for certain type of products, influence of prices on the consumers’ decision for buying milk and dairy products, etc. also, the objective of the research is to study the way in which promotion as part of marketing mix affects consumers’ decision making for buying certain product and significance of promotion as a factor of sales volume increase; it aims at increasing the company’s profit, as well as it takes into consideration the effects of application of the promotional activities and improvement of competitive advantage of companies in milk industry. keywords: marketing research, integrated marketing communications, milk processing industry, marketing campaigns, promotion. 1. introduction the concept of marketing communication is used to estimate the strategic role of different instruments of promotion and to combine them, in order to provide clear and consistent messages, that is, maximum communication influence of consumers, is concept of marketing communication [1].the research results from different authors indicate that marketing communication increases the communication influence, makes creative ideas effective and provides bigger communication consistence regarding the traditional marketing communication [2]. also, one of the main reasons that the companies accept the concept of http://www.fia.usv.ro/fiajournal mailto:katerina.bojkovska@uklo.edu.mk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 310 promotional mix understands the values (significance) of strategic integration of different communication instruments regarding their individual application, as reflected in avoidance of duplication and achievement of synergy of the effects of multiple instruments of marketing communication [3]. scholars long have recognized that the organizational context of integrated marketing communications (imc) implementation is an important area for empirical research [4]. no promotional tool should be used in isolation or without regard to the others; hence there is a trend toward integrated marketing communications [5]. some authors point out that promotion campaigns may result in increased sales volume during these activities, which in turn leads to piling up stocks and subsequent decreased sales, but on the other hand, it may lead to consumers reducing the purchases of given product before expected sales promotion activities [6]. to develop customer-orientated messages, the manufacturing company should realize the importance of the information held by retailers and other intermediaries about customer wants and needs [7]. well-coordinated campaign of promotional marketing communication contributes to completion of synergistic effects that are reflected in achievement of communication objectives, such as increase of consciousness, knowledge and interest for certain product, positive attitude of consumers regarding the product, thereby increasing the positive image of the company and its products, as well as increased intention for buying products. prior to making decision to buy certain product, consumers are going through cognitive and affective phase in the buying process. affect and cognition represent the consumers’ psychological answer to the promotional stimulants. the affect is manifested in various kinds, such as: emotions, specific feelings, mood and estimations; while cognition is comprised of processes, such as: attention, perception, thinking, believing, learning and reminding [8]. going through the mentioned phases, the consumers possess different level of awareness, knowledge, beliefs, preferences and actions [8], i.e. readiness to buy, and therefore they need information on the basis of which they shall form their beliefs and attitudes toward the product and the company, which are being provided through various promotional campaigns conducted by company’s via media. the objective of the research in this article is to determine the effects from usage of multiple instruments of promotional mix by the companies in the milk processing industry. in the literature, not much attention is paid to exactly that synergistic effect [3]. the consideration of the effects from application of the instruments of promotional mix through respective marketing research brings multiple potential advantages [9]: it enables to determine how much the investment in promotional activities contributes to increase of financial assets through bigger sales volume; the base is evaluation of communication options (potential messages, instruments and media), and the selection of their most effective combinations enables to determine the effects from different promotional strategies; it represents basis for planning; it increases the effectiveness of promotional instruments by means of elimination of nonproductive alternatives and application of productive elements of the promotional program, considered by appropriate marketing research. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 311 reasons for the significance of the measurement of effect from optimal combination of the instruments of promotional mix through application of appropriate marketing research as its determinant, could be found in valuation of different promotional strategies which are based on estimation of the extent in which each media could be used; in estimation when one message is more effective that the others; right distribution of the promotional budget; determination are the objectives achieved etc. if the instruments of the promotional mix are successfully integrated, which means that each instruments “cooperates” with the others regarding the design of promotional messages, time, exhibition, target audience to whom the message has been sent etc., their synergistic effects are realized, i.e. overall results from the application of certain promotional instruments are bigger than the sum of the effects of separate instruments [10]. in order to measure the success of marketing communications by the company, it is necessary to perceive its effects. "given the fact that some companies are investing significant resources in marketing communications and especially in advertising, measuring and analyzing the efficiency of marketing communications has great practical significance" [11]. only then can the field move toward the successful implementation of this rather old innovation in marketing thinking [12]. organising and manipulating the numerous components of the promotional mix help to generate a unified consumer-focused message, thereby contributing towards achieving various organisational objectives [2]. imc scholars suggest that the concept can be implemented effectively only if all elements of the marketing mix are coordinated, to produce a consistent and integrated brand message [13]. in order to heighten consumer understanding about the source of their food and to market agriculture products, organizations representing food producers use integrated marketing communication strategies and tactics to build trust and brand awareness amongst target markets [14]. strategic task of management of companies – producers of milk and dairy products is continuous monitoring of the modern world trends and finding new ways for marketing research as determinant of promotional mix (advertising, promotion of sells, public relations and personal selling) which will enable coordinating with the flexible goals, available resurces and their possibilities for changing conditions in the target markets that they will decide to take part in a certain time period [15]. 2. materials and methods several methods of research are applied in the construction of this article in order to ensure bigger systematization and scientific basis of the thesis. scientific method –extensive scientific and professional literature by outstanding experts, scientific associations and relevant institutions from the country and abroad was used. survey method – resides in the examination of business entities from the milk industry through previously structured questionnaire in order to assess the attitudes of marketing managers towards their products’ sufficient promotion and whether the promotion has impact on the consumers’ buying decision of certain dairy product. this empirical research was conducted in order to determine the extent of performed promotional activities and their effects on the operation of 40 business entities in milk processing industry which are 60% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 312 from the whole dairy industry companies in the republic of macedonia. taking into consideration the design of this study and after assessing various research questions and hypotheses, the use of well structured questionnaire was adopted for respondent at company level. the survey research method was used by the distribution of copies of questionnaire to solicit information from respondents. thus, the work enhanced adequate uniformity of responses. the survey of the agricultural marketing firms was implemented from january to may 2018. in that period researchers contacted all targeted firms and their marketing managers and sent them questionare by e-mail. after one week, all managers sent us back the answered questionaires. a questionnaire comprised of general questions about the entity and questions about promotional activities and the impact of those activities on the work in milk processing capacities was used for that purpose. the questionnaire for the dairy industry company’s employees was divided into three major sections. the first part of the questionare contained questions on the demographic profile of the respondents and their organisation in terms of age, gender, civil status, educational attainment and position in the company. in the second part of the questionare are listed questions about the company, its activity, number of employees, production capacity and possibilities for its expansion, working problems of the dairy industry companies and using of marketing research by the companies. in the third part of the questionare are listed questions about the promotion and its effects on succesifuly work of the dairy industry companies, combining of the promotional mix elements for promotion of the company and their products, using of promotional channels (internet, social media etc.), conducting market research to perceiving the effects of promotional campaign and interest in using the services from the specialized marketing agencies which will promote dairy industry companies and their products. the research includes 40 business entities from milk industry in the republic of macedonia. statistical method the gathered data is statistically processed, interpreted and afterwards analyzed, and appropriate conclusions were made on the basis of those analyses. 3. results and discussion promotion is one of the marketing mix elements, which is very significant for sales increase and attaining higher profit from each product, including milk and dairy products. as proof, a lot of companies in the republic of macedonia have the leading place in their area by using numerous promotional activities and performing big promotional campaigns. this empirical survey is conducted in order to determine the extent of performed promotional activities and their effects on the operation of business entities in milk processing industry in the republic of macedonia. data in table 1 indicate that most of the respondents that were included in the survey are women at the age of 30 to 40, employed in marketing and sales department, marketing managers, technologists, human resources managers, directors etc. results and discussion should be presented with clarity and precision and should be explained, but largely without referring to the literature and should interpret the findings in view of the results obtained. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 313 table 1. survey sample (n=40) characteristics n % characteristics n % age production capacity of the enterprise (tons/daily) 30-40 27 66.7 <50 27 67.5 41-50 7 17.5 50-100 3 7.5 > 51 6 15.0 >101 10 25.0 male 7 16.7 number of employees <50 20 50.0 51-250 13 32.5 >251 7 17.5 enterprise activity primary production 7 17.5 processing 20 50.0 combined (production and processing) 13 32.5 resource: own research results this research includes small enterprises (up to 50 employees), middle (up to 250 employees) and big enterprises, above 251 employees, which occupation is production and processing of milk and dairy products. most of the companies are small, up to 50 employees – even 50 % of the companies covered with this research are small ones, middle companies, with up to 250 employees comprise 32.5% of the sample, and finally, only 17.5% of the companies are big, with more than 250 employees (table 1). regarding the activity of the company, i.e. whether it is engaged in primary production, processing, or combination, the following results are obtained: most of the companies (50% of total number of companies) are engaged in milk and dairy products processing, 32.5% are engaged both in production and processing of milk and dairy products, and only 17.5% are engaged in primary production (table 1). milk processing capacities which are included in this research are with different production capacities. most of the milk processing capacities that were subject of examination have production capacity of up to 50 tons of milk – 67.5% in percentage, i.e. that is the amount of milk which they process daily, 25.0% of the milk processing capacities regarding the activity of the company, i.e. whether it is engaged in primary production, processing, or combination, the following results are obtained: most of the companies (50% of total number of companies) are engaged in milk and dairy products processing, 32.5% are engaged both in production and processing of milk and dairy products, and only 17.5% are engaged in primary production (table 1). milk processing capacities which are included in this research are with different production capacities. most of the milk processing capacities that were subject of examination have production capacity of up to 50 tons of milk – 67.5% in percentage, i.e. that is the amount of milk which they process daily, 25.0% of the milk processing capacities included in the research process above 101 tons of milk per day, while none of the companies responded that daily processed amount of milk is in the range between 50 and 100 tons of milk. this condition indicates small volume of production, but considering the fact that on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 314 the question “are you thinking of expanding your capacities”, even 100% of the companies included in the research answered that they are thinking of expanding their capacities, and an increase in the volume of production of milk and dairy products in the republic of macedonia could be expected in the future, which will have positive impact on import decrease of such product from abroad, and consequently, decrease of the country’s trade deficit. in modern conditions, many enterprises invest larger part of their assets in promotion of their products. promotion delivers information by means of which the buyer is acquainted with the products and services he needs. it reminds him, encourages him and motivates him to buy what he needs. awareness for the buying process is built with the promotion, and it has impact on the buyers to satisfy their needs in efficient way. the promotion, that is, the marketing activities as a whole, creates values in the buyers’ consciousness by indicating what those values mean to the buyers. promotion means influence on the consumers by developing and sending informative and persuading messages to the buyers, i.e. the target audience. from the conducted research and the obtained results it could be perceived that all promotional assets are used in big percentage, i.e. all companies included in the research make combination of all assets for promotion, since each of them has its own advantages, as well as disadvantages, and their combination solves that issue, more or less (83,3% of the companies included in servey apply advertising, 66,7% use direct marketing and 50% of the companies apply publicity and public relations and sales promotion in their combination of promotion assets) (fig. 1). fig. 1. using of promotional assets from the dairy industry companies resource: own research results to the question “can you perceive the effects from the promotion campaign you are using”, asked in the questionnaire, 66.7% of the companies included in the research answered that they can perceive the effects from the performed promotion campaign, and 33.3% cannot perceive the effects, which means that their promotion campaign is not good enough, or maybe they invest too small amount of money in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 315 their promotion campaign and therefore certain marketing activities that would contribute to increase the sales of their products, and thereby making bigger profit, should be performed (figure 2). fig. 2. perceiving the effects from used promotion campaign resource: own research results the market research is a process of gathering data in order to determine whether certain product of service is going to satisfy the potential consumers’ needs. information about its competitors, economic changes, demography, current market trends and consumers’ habits could be obtained by efficient research. the market research could help the companies to find out all of their competitors, to follow their progress, as well as the competitors’ growth, and in that way, they can develop business strategies which will take him a step in front of his competitors. also, the loss of a business could be decreased by market research, it bring success to marketing campaigns and guarantees successful sales. it means that the market research helps in the design of marketing campaigns which are directly concentrated on the interest of the potential consumers and helps to increase the sales. besides that, market research could also be conducted in order to perceive the effects from performed promotional activities. so, to the question “have you conducted market research in order to perceive the effects from performed promotional activities”, 77% of the companies from milk processing industry included in the research answered that they have done a market research; while 23% answered that they have not done a market research, and and these are actually small companies which did not conduct a market research to mesure the effect of the marketing campaign. because of lack of financial assets, are not able to invest a lot in promotion of their products, which results with smaller sales volume, there by smaller profit, too (figure 3). fig. 3. perceiving the effects from used promotion campaign resource: own research results 4. conclusion buyers make buying decisions in different ways and with different reasons. in order to be able to attract the buyers, the reason for which they acquire a certain product, they should be familiar with. since he knows that, the producer is able to direct the promotional activities towards buyers, in order to inform, intrigue and persuade them to buy his products. business entities from milk processing industry in the republic of macedonia pay great attention to the way of promoting their products, especially bigger companies from this area, and therefore they can assess the effects of performed promotions. they perform their promotional campaigns in very creative way, using different combinations of assets and media, following the latest trends of promotion. however, smaller companies, because of lack of financial assets, are not food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski, modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia, food and environment safety, volume xvii, issue 3 – 2018, pag. 309 316 316 able to invest a lot in promotion of their products, which results in smaller sales volume, thereby a smaller profit, too. in recent years, the internet is experiencing big expansion, so it would be good for the companies from the milk processing industry to use it in order to promote their products, as much as possible. any information could be obtained very quickly via internet, so consumers can be informed about certain companies and their products in really easy way, and thereby an increase in sales volume could be expected. from all above, it could be concluded that the main function of promotion is to provide information on products and services, to transmit suggestions and ideas and to encourage the acquisition process, having in view that the objective of marketing research is to obtain information about the market, that is, the existing and potential buyers, in order to determine current and future needs and priorities, attitudes and behavior on the market, and to determine the value and changes in the business environment that could affect the size and nature of the market in future. if all of that is familiar to the marketing managers in the companies from any industry, as well as the marketing managers in the companies from milk processing industry, it will be much easier to make decision about the type of action that need to be taken in order to achieve previously defined marketing objectives. 5. references [1]. vantamay s., performances and measurement of integrated marketing communications (imc) of advertisers in thailand, journal of global management: 1-12, (2011) [2]. kitchen p. j., burgmann i., integrated marketing communication/sheth j., malhotra n. k. (eds.). wiley international encyclopedia of marketing: 1-21 (2010) [3]. belch g. e., belch m. a., advertising and promotion: an integrated marketing communications perspective. new york: mcgrawhill education., 10th ed: 647-653, (2012) [4]. moriarty s., schultz d., four theories of how imc works. advertising theory, s. rodgers and e. thorson, (eds.) new york: rutledge: 491-505, (2012) [5]. czinkota m. r.. ronkainen i. a., international marketing. 8th edition. mason, ohio: thomson/south-western: 646, (2007) [6]. mesaroš i., đokić n., penić m, measuring the communication effects of sales promotion in a food company, economics of agriculture, 60(1): 49-64, (2013) [7]. shet n. j.m., malhotra n., wiley international encyclopedia of marketing: 100-103, (2011) [8]. salai, s., grubor, a., marketing communications. subotica: faculty of economics: 173-105, (2011) [9]. shah k., d‘souza a., advertising and promotions: an imc perspective. noida: tata mc graw hill: 580-590, (2009) [10]. keller k. l., unlocking the power of integrated marketing communications: how integrated is your imc program?, journal of advertising, 45(3): 286-301, (2016) [11]. hanić h., simeunović i., domazet i., market research – determinant of successful strategic marketing in financial organizations, journal of marketing, 44(4): 310-320, (2013) [12]. ots m., nyilasy g., integrated marketing communications (imc): why does it fail?, journal of advertising research, 55(2): 132-145, (2015) [13]. kotler ph., keller k.l., marketing management, 14th ed. prentice hall/pearson, new jercey: 474-500, (2016) [14]. de pelsmacker p., geuens m., van den bergh j., marketing communications: a european perspective, forth edition, harlow: prentice-hall/financial times: 610, (2007) [15]. bojkovska k., petkovska t., possibilities and perspectives for export of milk and dairy products from republic of macedonia., journal of the institute of economics skopje: economic development, 18(1-2): 59-78, (2016) 1. introduction 4. conclusion 320 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 32015, pag. 320 326 physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil bernard assielou 1 , *edmond ahipo due1, djary michel koffi2 and patrice kouame1 1laboratory of biochemistry and food technology, nangui abrogoua university, 02 bp 801, abidjan 02, cote d’ivoire. 2laboratory of biotechnology, felix houphouet boigny university, 02 bp 801 abidjan 02, cote d’ivoire. * corresponding author: ahipoedmond@yahoo.fr received august 16th 2015, accepted september 28th 2015 abstract: oryctes owariensis is one of the most widely prized edible insects consumed in côte d’ivoire. oil extracted from this insect was analyzed for physicochemical properties and fatty acid constituents using standard methods. results revealed that with oil content of 18.88 ± 1.3 %, oryctes owariensis larvae are lipid-rich than most of edible insects. this oil shows a refractive index of 1.344 ± 0.002, specific gravity of 0.71 ± 0.10, acid value of 3.73 ± 0.02, iodine value of 105.27 ± 0.15, saponification value of 120.14 ± 1.5, unsaponifiables of 2.99 ± 0.31 %, peroxide value of 0.96 ± 0.02 and free fatty acid of 1.12 ± 0.03 (% oleic acid). as regards fatty acids profile, the unsaturated fatty acids in oryctes owariensis oil were oleic (46.09 ± 0.07%), linoleic (3.25 ± 0.02 %) and palmitoleic (1.06 ± 0.01 %), while the major saturated fatty acids were palmitic (39.55 ± 0.03 %) and stearic (10.05 ± 0.03 %). the unsaturated fatty acids accounted for 50.4 % of the total fatty acids whereas the saturated fatty acids constituted 49.6 % of the fatty acids. these values when compared with that observed in oils which have been considered to be of high quality, suggest that oryctes owariensis oil has potentials that could be exploited by the industrial and pharmaceutical companies. keywords: physicochemical characterization, fatty acid composition, oryctes owariensis larvae, oil, edible insect. 1. introduction insects are the most common multicellular organisms on planet earth and are thought to account for >70% of all species. insects are also among the most diverse groups of organisms in the history of life [1]. although mainly recognised as pests or nuisances affecting human, plant and animal health, insects play an essential role in minimising of food safety in addition to providing ecosystem services (such as pollination, waste degradation and biological control). in a recent review, van huis [2] outlined the important role of insects in assuring food and feed security. globally, it is believed that 1,900 species of insects are consumed by about 2 billion people, mainly in the developing world [2]. this has become especially important as the need for alternative protein sources increases due to rapid urbanisation in developing countries and the shifts in the composition of global food demand. people in africa, asia and latin america eat insects as regular parts of their diets. they may do so not only because conventional meats such as beef, fish and http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. 320 – 326 321 chicken are unavailable and insects therefore are vital sources of protein, but also because insects are considered important food items, often delicacies [3]. insects are a highly nutritious and healthy food source with high content of nutrients (fats, protein, vitamins, fibre and minerals) required by humans and animals. however, the nutritional composition of edible insects between and within species is highly variable, depending upon metamorphic stage, habitat and diet of the insect [4]. edible insects are a considerable source of fat. many authors investigated the content and composition of oils extracted from several insects [5, 6, 7, 8]. their oils are rich in polyunsaturated fatty acids and frequently contain the essential linoleic and α-linolenic acids. the nutritional importance of these two essential fatty acids is well recognized, mainly for the healthy development of children and infants [9]. greater attention has been paid to the potential deficient intake of these omega-3 and omega-6 fatty acids in recent times, and insects could play an important role, in particular in landlocked developing countries with lower access to fish food sources, by supplying these essential fatty acids to local diets [10]. in cote d'ivoire, oryctes owariensis larva is one of the most widely prized and eaten insects. this larva is commonly hunted in dead trunks of palms by the villagers. thus, it is either eaten raw, boiled, smoked or fried [11]. the present study was aimed at evaluating the physicochemical properties and fatty acid composition of dried o. owariensis larvae oil, with a view to revealing any possibility of its use as conventional oils for human and animal nutrition and industrial purposes. 2. matherials and methods larvae collection and sample preparation fresh o. owariensis larvae were collected from dead trunks of raffia palms at saïoua (6°29'31" n and 6°15'32" w) in côte d’ivoire. after collection, the larvae were placed in a cooler with ice to keep their freshness and then transported to the laboratory for flour preparation. fresh larvae (1 kg) were cleaned to distilled water then, drained and dried at 65°c in an oven for 72 h. dried larvae were ground using a porcelain motar to obtain the crude flour. oil extraction oil was extracted from 3 g of crude flour with 70 ml of n-hexane in a soxhlet extractor [12]. then, the solvent was gently evaporated with a rotary evaporatorty (heidolph, hei-vap, germany). the extracted lipid was weighted to determine the oil content of catepillar. crude oil was stored at 4 °c in airtight brown sterile glass bottle until further use for physicochemical analysis. physicochemical analysis refractive index refractive index of o. owariensis oil was determined at 25 °c following the iupac [13] method by using a refractometer (abbe, optic ivymen, spain), respectively. acid, peroxide, iodine and saponification values acid index of o. owariensis oil was determined according to aoac [12] official method with the morn iso-9001. a volume of 100 ml ethanol was neutralised with a solution of naoh (n/10). the titration was performed using a solution of koh (1n) in the presence of phenolphthalein. iodine value of o. owariensis oil was determined according to aoac [12] official method. a volume of 30 ml carbon tetrachloride was used to dilute 0.4 g oil in the presence of 25 ml of wijs food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. 320 – 326 322 reagent and 10 ml of acetate mercury. the titration was carried out using a solution of sodium thiosulfate (0.1 n). the peroxide value was determined according to aoac [12] method. a solution made out of potassium iodine added to a mixture of acetic acid – chloroform: 3/2 (v/v) has been used. the titration was carried out using a solution of sodium thiosulfate (n/100). saponification value was determined according to aoac [12] official method. an amount of 2 g oil has been treated using alcoholic potash (0.5 n) and titrated hot with hydrochloric acid (0.5 n) in the presence of phenolphthalein. unsaponifiable matter unsaponifiable matter content of oil sample was determined following the iupac [13] method. for this, 100 mg of unsaponifiable fraction was dissolved in 2 ml of chloroform. to 1 ml of aliquot, 4 ml of a trifluoroacetic-chloroform (1:3, v/v) solution was added. the absorbance was measured at 620 nm using a spectrophotometer (t80+, pg instruments, england). fatty acid composition the fatty acids were converted to their methyl esters (fames) as described by the european communities [14]. about 0.1 g of oil sample was mixed with 2 ml of nheptane and 0.2 ml of a methanolic solution of potassium hydroxide (2n). the whole mixture was shaken up for 30 s and allowed to settle for 5 min. the top layer containing the fames was used for gas chromatography (gc) analysis. fames solution (1 μl) containing the internal standard (erucic acid) was injected into a gas chromatograph (shimadzu, gc 14 a, japan) equipped with a flame ionization detector (fid) and a capillary column trd1 (60 m x 0.25 mm i.d. x 0.25 μm film thickness). the carrier gas was nitrogen and the flow rate was adjusted to 23 ml/min. temperature of detector was 325°c and that of injector was 275°c. the initial column temperature was fixed to 60°c and programmed to increase by 1°c per min intervals until 325°c and, kept for 10 min at this temperature. the fatty acid methyl esters peaks were identified by comparing their retention times with those of standards. after adjusting areas with the internal standard (erucic acid), the yield of each fatty acid was calculated as follow: area of the fatty acid/areas of total fatty acids in the oil sample × 100 (%). statistical analysis each sample was analyzed in triplicate and data are reported as mean ± standard deviation (sd). analysis of variance (anova) was performed using spss version 11.0 software. statistical significance was set at p ≤ 0.05. 3. results and discussion physicochemical properties fat is the chief form in which energy is stored in insect larvae [15]. it is usually present in greatest amounts in the mature larvae before metamorphosis [16]. as regards o. owariensis, its oil content was amounted to 18.88 ± 1.3 % (dry matter) (table 1). this value shows that o. owariensis larvae are lipid-rich than most of edible insects such as cirina forda [17], zonocerus variegatus [6], oryctes rhinoceros and imbrasia belina [18]. physicochemical characteristics of o. owariensis oil are shown in table 1. the oil from the studied larvae is a yellow brown and a colourless liquid (data not shown). values of specific gravity and refractive index were found to be 0.71 ± 0.10 and 1.344 ± 0.002 respectively (table 1). these values are lower than those of arachis oil, linseed oil and olive oil [19]. this implies that the oil from this insect is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. 320 – 326 323 lighter than these seed oils that have been considered to be of high quality and as such would find much use in pharmaceutical industries. the oil of o. owariensis is more unsaturated than the seed oil which suggests that it might be more fluid at room temperature and less viscous at low temperatures. the low acid value (3.73 ± 0.02 mg koh/g mg) is an indication of its lower susceptibility to rancidity which depicts a higher shelf life. table 1 extraction yield and physicochemical properties of o. owariensis oil parameters values extraction yield (%) 18.88 ± 1.3 refractive index 1.344 ± 0.002 specific gravity 0.71 ± 0.10 acid value (mg koh/g mg) 3.73 ± 0.02 free fatty acid (% oleic acid) 1.12 ± 0.03 saponification value (mg koh/g mg) 120.14 ± 1.5 iodine value (g i2/100 g) 105.27 ± 0.15 unsaponifiables (%) 2.99 ± 0.31 peroxyde value (meq o2/kg) 0.96 ± 0.02 values are mean ± standard deviation of three measurements (n = 3) the acid value is low, acid value of 0.00 to 4.00 mg koh/g oil is recommended for oil to find application in cooking [20]. thus the oil from o. owariensis larvae could be suitable for cooking. the free fatty acid value of 1.12 ± 0.03 falls within the maximum limit of 5% for free fatty acids in high grade palm oil in nigeria [21]. the data relevant to acid value and free fatty acid further indicate that o. owariensis oil is not much susceptible for fat degradation process during oil extraction too. the peroxide value is used as an indicator of deterioration of oils, thus low peroxide value indicates resistance of the oil to peroxidation during storage. fresh oils have peroxide values lower than 10 meq o2/kg and before oil becomes rancid, its peroxide value must be between 20 and 40 meq o2/kg [22]. the peroxide value of oil from o. owariensis larvae is very low (0.96 ± 0.02 meq/kg) compared to the maximum acceptable value of 10 meq koh/g set by the codex alimentarius commission for groundnut seed oils [23]. the oil is thus stable and would not easily go rancid. peroxide formation is an indication that lipid oxidation is on-going, these compound react with low molecular weight metals to produce free radicals that are capable of further lipid oxidation [24]. the iodine value is a measure of the degree of unsaturation in oil and could be used to quantify the amount of double bonds present in the oil which reflects the susceptibility of oil to oxidation. the iodine value of o. owariensis oil was 105.27 ± 0.15 g i2/100 g. this was close to the 108.00 ± 0.15 reported for macrotermes bellicosus oil by ekpo and onigbinde [25]. this value was however, higher than the 92.62 ± 0.33 g i2/100 g and 48.35±0.55 g i2/100 g reported for coptotermes gestroi and rhynchophorus palmarum l. larva respectively [5, 8]. saponification value is an index of average molecular mass of fatty acids in oil sample. the lower value of saponification value in o. owariensis oil (120.14 ± 1.5 mg koh/g) suggest that the mean molecular weight of fatty acids is lower than that of r. palmarum l. larva oil (198.26±0.99 mg koh/ g) or that the number of ester bonds is less when compared to that of r. palmarum l. larva oil [5]. nevertheless, this saponification value is consistent to make the studied oil useful in soap industry. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. 320 – 326 324 fatty acid profile figure 1 depicts the fatty acids profile of o. owariensis. the data given in the table 2 showed that the unsaturated fatty acids in o. owariensis oil were oleic (46.09 ± 0.07%), linoleic (3.25 ± 0.02 %) and palmitoleic (1.06 ± 0.01 %), while the major saturated fatty acids were palmitic (39.55 ± 0.03 %) and stearic (10.05 ± 0.03 %). this insect oil could be classified in oleic acid group. fig. 1. gas chromatograms of fatty acids methyl esters of o. owariensis oil analysed table 2 fatty acids composition of o. owariensis oil fatty acid saturation values (%) essential linoleic acid omega 6 polyunsaturated 3.25 ± 0.02 non-essential palmitoleic acid omega 7 monounsaturated 1.06 ± 0.01 oleic acid omega 9 monounsaturated 46.09 ± 0.07 palmitic acid saturated 39.55 ± 0.03 stearic acid saturated 10.05 ± 0.03 ratios sfa 49.6 tufa 50.4 mufa 47.15 pufa 3.25 pufa / sfa 0.07 values are mean ± standard deviation of three measurements (n = 3). abbreviations: sfa = saturated fatty acids tufa =total unsaturated fatty acids; mufamonounsaturated fatty acids; pufa: polyunsaturated fatty acid. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. … – 325 the unsaturated fatty acids accounted for 50.4 % while the saturated acids accounted for 49.6 % of total fatty acids (table 2). the presence of both saturated and unsaturated fatty acids in this insect could be an advantage since they may complement the functions of one another. oleic acid, a monounsaturated fatty acid, has been shown to be hypocholesterolemic [26]. linoleic acid is important essential fatty acid required for growth, physiological functions and body maintenance [27]. furthermore, linoleic acid is one of the most important polyunsaturated fatty acids in human food because of its prevention of distinct heart and vascular diseases. apart from preventing cardiovascular disorders such as coronary heart diseases and atherosclerosis; linoleic acid prevents high blood pressure. also linoleic derivatives serve as structural components of the plasma membrane and as precursors of some metabolic regulatory compounds [28]. as far as concerned palmitic and stearic acids, which are the main saturated fatty acids of the studied oil, previous studies have shown that they are free from deleterious effect on plasma cholesterol [29]. in addition, they are often used in food industries to provide texture and softness to products [30]. 4. conclusion based on the present study, it appears that o. owariensis larva is a good source of oil, contains high amount of unsaturated fatty acids (50.4 %) mainly consist to oleic acid (46.09 %) which was found to be hypocholesterolemic and thus, desirable from the nutritional and health view points. so, this insect oil could be classified in oleic acid group. also, the studied oil exhibited good physicochemical properties and could be useful for nutritional, pharmaceutical and industrial applications. 5. references [1]. van huis a., van itterbeeck j., klunder h., mertens e., halloran a., muir g. and vantomme p., edible insects: future prospects for food and feed security, fao forestry paper 171. fao, rome, italy, (2013). [2]. van huis a., potential of insects as food and feed in assuring food security, annual review of entomology, 58: 563-583, (2013). [3]. aguilar-miranda e.d., lopez m.g., escamilla-santana c., barba de la rosa a.p., characteristics of maize flour tortilla supplemented with ground tenebrio molitor larvae, journal of agricultural and food chemistry, 50(1): 192–195, (2002). [4]. rumpold b.a., schlüter o.k., potential and challenges of insects as an innovative source for food and feed production, innovative food science and emerging technologies, 17: 1-11, (2013). [5]. due e.a., zabri h.c.b.l., kouadio e.nj.p., kouame l.p., fatty acid composition and properties of skin and digestive fat content oils from rhynchophorus palmarum l. larva, african journal of biochemistry research, 3(4): 089-094, (2009). [6]. womeni h.m., linder m., tiencheu b., mbiapo f.t., villeneuve p., fanni j., parmentier m., oils of insects and larvae consumed in africa: potential sources of polyunsaturated fatty acids, ocl – oléagineux, corps gras, lipides, 16(4): 230– 235, (2009). [7]. agomuo e.n., proximate and fatty acid compositions of termites (macrotermes bellicosus), international sciences research journal, 3: 93–96, (2011). [8]. mathew t.j., ndamitso, m. m., shaba e.y., mustapha s., muhammed s.s., adamu a., phytochemical, physicochemical, antinutritional and fatty acids composition of soldier termites (coptotermes gestroi) from paikoro local governvernt, niger state, nigeria, journal of environmental science, toxicology and food technology, 7(1): 7175, (2013). [9]. michaelsen k.f., hoppe c., roos n., kaestel p., stougaard m., lauritzen l., mølgaard c., choice of foods and ingredients for moderately malnourished children 6 months to 5 years of age, food and nutrition bulletin, 30(3): 343– 404, (2009). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 bernard assielou, edmond ahipo due, djary michel koffi, patrice kouame, physicochemical characterization and fatty acid composition of oryctes owariensis larvae oil, food and environment safety, volume xiv, issue 3 – 2015, pag. 320 – 326 326 [10]. roos n., nurhasan m., thang b., skau j., wieringa f., khov k., friis h., michaelsen k.f., chamnan c. winfood cambodia: improving child nutrition through improved utilization of local food. poster for the winfood project, denmark, department of human nutrition, university of copenhagen, (2010). [11]. kelemu s., niassy s., torto b., fiaboe k., affognon h., tonnang h., maniania n.k., ekesi s., african edible insects for food and feed: inventory, diversity, commonalities and contribution to food security, journal of insects as food and feed, 1(2): 103-119, (2015). [12]. aoac, official methods of analysis of the association of official analytical chemists, 16th ed. virginia. u.s.a., 1995. [13]. iupac. standard methods for the analysis of oils, fats and derivatives. pergamon: oxford, (1979). [14]. european communities, determination of free fatty a cid methyl esters. in: oj, (ed). characteristics of olive oil and the relevant methods of analysis. geneva , pp. 64 -67, (1991). [15]. chapman r.f., the insects: structure and function. the english language book society. stoughton and hodder. printed in great britain for hodder and stoughton educational. pp. 83-106, (1980). [16]. fast p.g., insect lipids in: progress in the chemistry of fats and other lipids. 11(2): 181242, (1970). [17]. akinnawo o., ketiku a.o., chemical composition and fatty acid profile of edible larva of cirina forda (westwood). african journal of biomedical research, 3: 93–96, (2000). [18]. ekpo k.e., onigbinde o., nutritional potentials of orycetes rhinoceros larva, nigerian journal of nutrition sciences, 26: 54-59, (2007). [19]. pearson d., the chemical analysis of foods. 7th edition. churchill living stone (publisher). pp. 491-516, (1976) [20]. oderinde r.a., ajayi i.a, adewuyi a., characterization of seed and seed oil of hura crepitans and the kinetics of degradation of the oil during heating. electronic journal of environment, agricultural and food chemistry, 8(3): 201-208, (2009). [21]. nifor, nigeria institute for oil research. history, activities and achievement. 2nd ed. benin city, nigeria, 26-28, (1989). [22]. akubugwo i.e., ugbogu a.e., physicochemical and studies on oils from five selected nigerian plant seeds. pakistan journal of nutrition, 6: 75-78, (2007). [23]. abayeh o.j., aina e.a., okounghae c.o., oil content and oil quality characteristics of some nigerian oils seeds, journal of pure and applied sciences, 1(1): 17-23, (1998). [24]. kilic b., richards m.p., lipid oxidation in poultry döner kebab: pro-oxidative antioxidative factors. journal of food sciences, 68: 686–689, (2003). [25]. ekpo k.e., onigbinde a.o., asia i.o. pharmaceutical potentials of the oils of some popular insects consumed in southern nigeria, african journal of pharmacy and pharmacology, 3(2): 051-057, (2009). [26]. mensink r.p., katan m.b., effect of a diet enriched with monounsaturated or polyunsaturated fatty acids on the levels of low-density and high density lipoprotein cholesterol in healthy women and men. the new england journal of medecine, 321: 43641 (1989). [27]. fao/who, fats and oils in human nutrition. report of a joint expert consultation organised by the food and agriculture organization of the united nations and the world health organization rome, 10, 19-26, (1993). [28]. matos l., nzikou j.m., kimbonguila a, ndangui c.b., pambou-tobi n.p.g., abena a.a., silou t.h., scher j., desobry s., composition and nutritional properties of seeds and oil from terminalia catappa l., advance journal of food sciences and technology, 1(1): 72-77 (2009). [29]. hunter k., crosbie l.c., weir a., miller g.j., dutta-roy a.k., a residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, haemostatic variables and platelets in young healthy men, journal of nutrition and biochemistry, 11: 408-416, (2000). [30]. dubois v., breton s., linder m., fanni j., parmentier m., fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. european journal of lipid sciences and technology, 109: 710732, (2007). 60 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 60 66 the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae *ijeoma p. oboh 1 , o. a. sanni 1 , n. k. egun 1 and c. p. wilfred-ekprikpo 2 1faculty of life sciences, department of animal and environmental biology, university of benin, pmb 1154, benin city, nigeria 2 nigerian institute of oceanography and marine research, lagos. nigeria *corresponding author: obohij@yahoo.com received 6th march 2019, accepted 29th march 2019 abstract: the high susceptibility of harvested fresh fish to immediate deterioration has resulted in the fundamental need for fish preservation to maintain its nutritional quality, minimize changes in its physical appearance, taste and lengthen shelf life. the insecticide, pirimiphos methyl (actellic dust) has been utilized in the storage of dried products against insect infestation, thereby increasing their shelf life. this study investigated the effect of pirimiphos methyl on the proximate and mineral composition of synodontis nigrita and tilapia mariae in jamieson river which were purchased from the fish landing site in sapele delta state, nigeria. the fish samples were divided into three batches of fresh samples (batch i), oven – dried samples (batch ii) and actellic dust treated samples (batch iii). the proximate composition (moisture, protein, fat, ash, crude fibre, and carbohydrate) and mineral content (sodium, potassium, calcium, and magnesium) of the samples were determined using the aoac standard methods. results showed that actellic dust treatment greatly increased the protein (19.35 to 23.31%), fat (8.12% to 22.31%), and raw fibre content (0.69 to 8.03%) in s. nigrita and protein (7.09 to 25.46%), fat (5.13 to 11.70%), and raw fibre content (3.40 to 14.23%) in t. mariae. the mineral elements analyzed showed no well-defined order across the treatment levels with potassium being the most abundant mineral element in all fish samples. actellic dust has been shown to improve the proximate composition and shelf life of both fish species studied, and its use as a fish preservative is therefore recommended. keywords: pirimiphos methyl, proximate composition, mineral content, synodontis nigrita, tilapia mariae, fish preservation, jamieson river. 1. introduction fish production plays an important role in food security and nutrition especially in many developing countries. fish is one of the healthiest and inexpensive sources of animal protein and other important nutrients required in human diets in africa [1]. the importance of fish in human nutrition as attributed to its nutrient profile which is superior to all terrestrial meats such as beef, pork, chicken, etc. [2]. it is an excellent source of high quality animal protein and highly digestible energy, with a good source of sulphur and essential amino acids such as lysine, leucine, valine and arginine, and therefore suitable for supplementing diets of high carbohydrates contents. beyond its approval as a balanced source of animal protein and vitamins, it also provides polyunsaturated fatty acids (pufas) and minerals required for optimal health [3], [4]. the fishery sector is http://www.fia.usv.ro/fiajournal mailto:obohij@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 61 estimated to supply 3.5% of nigeria's gross domestic product (gdp) and provides direct and indirect employment to over six million people [5]. fishes are highly perishable and they spoil rapidly if not properly handled. fish deterioration is a complex process brought about by action of enzymes, bacteria and chemical constituents. the main principle involved in fish preservation is the prevention of microbial spoilage by reduction in moisture content or simply creating unfavourable environment for microbial growth [6], [7]. various food preservation techniques such as freezing, chemical preservation, salting/brining, sundrying and smoking have been used to improve the microbial safety, extend the shelf life of fish and increase fish availability to the consumers [6], [8], [9]. despite these preservation methods, processed fish products are still susceptible to bio-deterioration due to microbial attack and insect infestation during storage or distribution. control measures against insect infestation of dried and smoked fish include the use of chemical insecticides such as dichlorvos, actellic dust, synergized pyrethrins ddt and heptachlor to keep away insects and other pests [10], [11]. pirimiphos-methyl (actellic dust) is a broad-spectrum organophosphorus insecticide with both contact and fumigant action. when applied to stored agricultural commodities (such as grain and nuts) it provides longer-lasting pest control. it is also effective in controlling various mites on vegetables and fruits. at high doses, pirimiphos-methyl show a mechanism of toxicity similar other organophosphorus insecticides and produce acute symptoms such as nausea, vomiting and cholinergic effects in humans [12]. insecticides have been used extensively on dried fish samples to prevent insect infestation and improve the organoleptic properties of fish samples but very little information on its effects on the proximate and mineral composition of fish is available. therefore, this study aims to examine and compare the effect of actellic dust (pirimiphos methyl) on the nutrient and mineral composition of synodontis nigrita and tilapia mariae from jamieson river, sapele, delta state. 2. materials and methods 2.1 study area this study was carried out in jamieson river which is located in the niger-delta region of nigeria. the region has two prevailing climatic seasons: wet season (may-october) and dry season (novemberapril). jamieson river a tributary of the benin river lies between longitudes 5041' to 5058"e and latitudes 5054' to 6008"n. the total length of the river is estimated to be 70km. the depth and width of the river is estimated to be between 1.7-2.1m and between 17m and 25m respectively [13]. 2.2 fish collection and identification the fish species chosen for this study are synodontis nigrita and tilapia mariae. the fish samples were procured fresh from landing sites at jamieson river. a total of 54 fish specimens were collected over a period of three months, february to april, 2017. the samples were preserved in ice blocks and transported to the laboratory, where they were properly identified using taxonomic guides [14], [15]. routine measurements of standard length, total length and body weight of all fish samples were taken to the nearest 0.1cm and 0.1g using a metre rule and digital electronic weighing balance [mettler toledo (pl203 model)] respectively. the fish samples were thoroughly washed with distilled water to remove any adhering contaminants and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 62 drained under folds of filter paper. the head was also discarded and the samples were stored in a deep freezer prior to analysis. 2.3 experimental set-up the fish samples were divided into three batches. this is important for valid conclusions to be drawn. each batch contained 9 fish specimens of synodontis nigrita and tilapia mariae.  batch i were made up of fresh fish samples  batch ii samples were oven-dried at a temperature of 105oc for 6 hours using an electric oven [bran drying oven (dhg-9023a model)].  batch iii fish samples were treated with pirimiphos methyl (actellic dust) 2.4 actellic dust treatment fresh fish samples were washed thoroughly with distilled water and submerged in 1 litre of actellic v solution for 15 seconds after which they were oven-dried for 6 hours in an electric oven at 105oc. after drying, they were sprayed with 5 ml of actellic solution, sun-dried for 3 hours and stored in a desiccator for 3weeks at 28oc . at the end of 3 weeks, they were analysed for their proximate and mineral composition. the insecticide solution was prepared by adding 12ml of actellic dust to 10 litres of distilled water. 2.4 proximate analysis the proximate composition (moisture, fat, ash, protein, crude fibre, and carbohydrate) and minerals content of synodontis nigrita and tilapia mariae were determined using the standard methods [16]. 2.5 data analysis all statistical analysis was computed using microsoft excel and statistical package for social sciences (spss) version 21. the proximate values arising from fish samples in batches i – iii were analysed for their range values, mean and standard errors. one-way analysis of variance (anova) was used to test for significant difference (p<0.01) between means and the source of significant differences identified using duncan’s multiple range (dmr) test. 3. results and discussion the total length ranged from 17.50 to 27.90cm, standard length (12.7 to 19.50cm) and body weight from (31.30 to 209.08g) for synodontis nigrita. the total length for tilapia mariae ranged from 12.50 to 21.00cm, standard length (10.20 to 17.80cm) and body weight (41.29 to 233.12g) for tilapia mariae. the results of the proximate and mineral composition of fresh, oven-dried and actellic treated samples of synodontis nigrita and tilapia mariae are presented in table 1 and 2. the variations observed in all the values recorded in all parameters examined showed high significant different between the means of the different treatments. in tables 1 and 2, the highest mean moisture content of (79% and 78.87%) were observed in fresh samples while the lowest (0.18 and 2.19%) were observed in oven-dried samples of synodontis nigrita and tilapia mariae. the moisture content of the muscles of fresh samples for synodontis nigrita and tilapia mariae in this study were within acceptable levels of 60% to 80% [17]. the observed high significant (p<0.01) reduction in moisture content in oven dried samples of synodontis nigrita and tilapia mariae is consistent with reported observed reduction in moisture content from 67.33 to 4.11%, 58.50 to 15.60% and 66.33 to 4.97% in oven-dried samples of tilapia zilli and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 63 synodontis clarias [18], [19], [20]. actellic treated fish samples recorded higher moisture content of 22.00% and 19.75% in s. nigrita and t. mariae after storage. in contrast, low moisture content of 8.89%, 10.11% and 16.62% in actellic treated samples were recorded for heterobranchus longifilis, heterotis niloticus and chrysichthys nigrodigitatus, respectively [21]. increase in moisture content after storage can be attributed to variation in relative humidity and optimum temperature during the period of storage which led to oxidation of carbohydrate and yield carbon dioxide, water and energy. fish lipids are rich in polyunsaturated fatty acid particularly omega-3-fatty acids which have important role in disease prevention and health promotion [20] [22], [23]. the fat content was higher in oven-dried and actellic treated samples of s. nigrita and t. mariae. table 1. summary of the proximate composition of fresh, oven-dried and actellic treated synodontis nigrita from jamieson river proximate composition fresh oven-dried actellic p value + sd min max + sd min max + sd min max moisture 79.00±2.35c 76.65 81.35 2.19±0.86a 1.33 3.05 22.00±1.46b 20.54 23.46 <0.01 fat 3.43±0.46a 2.97 3.89 8.12±0.36b 7.71 8.34 22.31±1.10c 21.20 23.40 <0.01 ash 6.22±1.05b 5.17 7.27 30.10±0.93c 29.17 31.03 1.89±0.16a 1.70 2.00 <0.01 protein 7.09±0.16a 6.93 7.25 19.35±0.39b 18.96 19.74 23.31±0.70c 22.83 24.12 <0.01 raw fibre 0.40±0.02a 0.38 0.42 0.69±0.03a 0.66 0.72 8.03±0.45b 7.58 8.48 <0.01 carbohydrate 3.86±0.70a 3.16 4.56 39.55±0.76c 38.93 40.39 22.46±3.46b 18.84 25.72 <0.01 note: similar superscripts – a, b, c indicate values that are not significantly different from each other (p < 0.01), while dissimilar superscripts – a, b, c indicate values that are significantly different from each other (p < 0.01) table 2. summary of the proximate composition of fresh, oven-dried and actellic treated tilapia mariae from jamieson river proximate composition fresh oven-dried actellic p valu e + sd min max + sd min max + sd min max moisture 78.87±0.66 c 78.2 1 79.5 3 0.18±0.04a 0.14 0.22 19.75±1.15 b 18.6 0 20.9 0 <0.0 1 fat 0.67±0.04a 0.63 0.71 5.13±0.58b 4.55 5.71 11.70±0.70 c 11.0 0 12.4 0 <0.0 1 ash 6.68±0.56b 6.12 7.24 22.10±0.70 c 21.4 6 22.8 5 4.90±0.30a 4.60 5.20 <0.0 1 protein 8.84±0.40a 8.44 9.24 7.09±0.30a 6.79 7.39 25.46±2.34 b 23.1 2 27.8 0 <0.0 1 raw fibre 1.80±0.20a 1.60 2.00 3.40±0.42a 2.98 3.82 14.23±2.02 b 12.2 1 16.2 5 <0.0 1 carbohydrat e 3.14±1.06a 2.08 4.20 62.10±0.29 c 61.7 7 62.3 2 23.96±4.51 b 19.4 5 28.4 7 <0.0 1 note: similar superscripts – a, b, c indicate values that are not significantly different from each other (p < 0.01), while dissimilar superscripts – a, b, c indicate values that are significantly different from each other (p < 0.01) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 64 the observed increase in fat content of oven-dried samples of s. nigrita and t. mariae supports the reported increase in fat content from 12.85 to 20.25% (oreochromis niloticus) and 2.73 to 13.34% (synodontis clarias) in oven-dried samples [23], [20]. actellic dust treatment greatly increased fat content from 8.12% to 22.31% and 5.13% to 11.70% in s. nigrita and t. mariae. in contrast, low fat content of 3.60%, 3.03% and 3.00% were recorded in actellic treated samples of heterobranchus longifilis, heterotis niloticus and chrysichthys nigrodigitatus respectively [21]. in comparison, the mean fat content of fresh and oven-dried samples of s. nigrita was higher than t. mariae. fresh samples of s. nigrita and t. mariae in this study belong to the low and lean fat categories respectively. in this study, high ash content (30.10% and 22.10%) was recorded in oven-dried samples of s. nigrita and t. mariae, due to moisture loss and concentration of chemical components in the fish samples. similar trend of high ash content in ovendried samples were reported in t. zilli [18], c. gariepinus [19] and s. clarias [20]. high ash content has been reported to lower bacterial and fungal activities leading to better shelf life in fishes. the mean percentage ash content of actellic treated samples of s. nigrita (1.89%) and t. mariae (4.90%) were significantly low. similar trend was observed in actellic treated samples of heterobranchus longifilis (5.99%), heterotis niloticus (4.00%) and chrysichthys nigrodigitatus (3.03%) [21]. protein is one of the major nutrients in fish and their levels define the nutritional status of a particular animal. the proximate composition of s. nigrita and t. mariae in this study revealed low protein content in fresh samples of s. nigrita (7.09%) and t. mariae (8.84%). the differences in protein content may be attributed to variation in fish size, age, seasonal variations i.e spawning, migration and starvation, level of assimilation of essential nutrient from their diets and the conversion rates of these nutrients for various metabolic activities. actellic dust treatment increased protein content from 19.35% to 23.31% and 7.09% to 25.46% in s. nigrita and t. mariae respectively. this agrees with the reported high protein content in actellic treated fish samples of heterobranchus longifilis (50.42%), heterotis niloticus (58.64%) and chrysichthys nigrodigitatus (67.32%) [21]. actellic treated samples had the highest crude fibre content in s. nigrita (14.23%) and t. mariae (8.03%). while in a similar experimental setup, highest crude fibre content was reported in fresh samples of heterobranchus longifilis, heterotis niloticus and chrysichthys [21]. increase in crude fibre content could be attributed to the oxidation of poly-unsaturated fatty acids (pufa) components, contained in their tissues to products such as peroxides, aldehydes, ketones and free fatty acids [24]. the highest carbohydrate content values (39.55 and 62.10%) in this study were recorded in oven-dried samples and lowest (3.86 and 3.14%) in fresh samples of s. nigrita and t. mariae. similar trend of high carbohydrate content in oven-dried samples of tilapia zilli and synodontis clarias has been reported [20], [18]. the low carbohydrate values observed in fresh samples could be attributed to the fact that glycogen does not contribute significantly to the reserves in the fish body tissue [25]. the study of mineral elements present in living organisms is of biological importance; since many of such elements take part in some metabolic processes and are known to be indispensable to all living things [26]. although fish is very unlikely food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 65 to be the only source of an essential mineral in the diet, fish provide a wellbalanced supply of minerals in a readily usable form [27]. the mineral elements analyzed showed no well-defined order across the treatment levels, as the most abundant mineral element present in s. nigrita and t. mariae across the treatment levels was potassium. previous investigation observed a similar trend [28]. sodium and potassium were greatly reduced in actellic treated samples of s. nigrita and t. mariae. similar trend of low sodium and potassium concentration was observed in actellic treated samples of c. nigrodigitatus [21]. oven-dried samples of s. nigrita and t. mariae had higher sodium (47.70 mg/kg and 15.80 mg/kg) and calcium (42.90 mg/kg and 13.20 mg/kg) content than values recorded in fresh samples. the concentration of sodium in fresh samples of s. nigrita and t. mariae could be attributed to the concentration of sodium in the water body. calcium content in actellic treated samples was significantly high (p < 0.01) in t. mariae (32.70 mg/kg) but significantly low (p < 0.01) in s. nigrita (3.10 mg/kg). the observed higher calcium content in fresh samples of synodontis nigrita over tilapia mariae, can be attributed to its benthic nature and its relative preference for consumption of fish scales, crustaceans, insect parts and larvae. magnesium function as a cofactor of many enzymes involved in energy metabolism, (proteins, rna and dna) synthesis and maintenance of the electrical potential of nervous tissues and cell membranes [29]. in both fish species, magnesium was almost completely eroded in actellic treated samples. this corroborates with the reported findings that magnesium content in actellic treated samples of chrysichthys nigrodigitatus was almost completely eroded [21]. the differences observed in the content of magnesium could be due to variation in individual species, season and temperature. 4. conclusion the use of actellic dust insecticide in fish preservation and its effects on the proximate and mineral composition of fish has been studied. in general, there was noticed a significant influence of actellic dust on improving the proximate and mineral composition, and shelf life of s. nigrita and t. mariae. therefore, the use of the insecticide as a fish preservative is recommended. 5. references [1]. sadiku, s.o.e., oladimeji, a.a., relationships of proximate composition of lates niloticus, synodontis schall and sarotherodon galilaeus from zaria dam, nigeria. bioscience biotechnology research communications, 3(1): 2940 (1991). [2]. amiengheme, p., the importance of fish in human nutrition: a paper delivered at a fish culture forum, federal department of fish farmers, abuja, nigeria. 21pp (2005). [3]. clandinin, m.t., et al. omega-3 fatty acid intake results in a relationship between the fatty acid composition of ldl cholesterol ester and ldl cholesterol content in humans, biochimica.biophysica acta, 1346(3):247–252 (1997). [4]. raatz, s.k., et al. issues of fish consumption for cardiovascular disease risk reduction, nutrients 5(4): 1081–1097 (2013). [5]. olayemi, f.f., et al. proximate composition of catfish (clarias gariepinus) smoked in nigerian stored products research institute (nspri): developed kiln. international journal of fisheries and aquaculture 3(5): 96-98 (2011). [6]. ghazala, s., new packaging technology for seafood preservation shelf life extension and pathogen control. in: fisheries processing biotechnological applications (ed. a. m. martin). chapman and hall, london. pp. 83-110 (1994). [7]. ayolabi, c.i., fagade, e.o., mycological evaluation of smoked fish (ethmalosa fimbriata) from retail outlets in ago-iwoye, ogun state, nigeria. journal of life and physical sciences, 3(2):64-68 (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 ijeoma p. oboh, o. a. sanni, n. k. egun, c. p. wilfred-ekprikpo, the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae, food and environment safety, volume xviii, issue 1 – 2019, pag. 60 – 66 66 [8]. nickelson, r.i., et al. fish, crustaceans and pre-cooked sea foods in: compendium of methods for the microbiological examination of foods, 4th edition, downes, f. p., ito, k. (eds.). american public health association, washington, dc, pp. 497-505 (2001). [9]. abolagba, o.j., et al. salting of fish as a means of reducing damage by dermestes maculates (coleoptera). nigerian journal of entomology, 13: 81-86 (1996). [10]. eyo, a.a., fish processing technology in the tropics. national institute for freshwater fisheries research. university of ilorin press. pp. 1 – 20 (2001). [11]. bhuiyan, m.n.h., et al. organochlorine insecticides (ddt and heptachlor) in dry fish: traditional washing and cooking effect on dietary intake. bangladesh journal of pharmacology, 4: 46-50 (2009). [12]. food and agriculture organization (fao), monitoring progress towards the world food summit and millennium development goals. state of food insecurity in the world, rome, italy. 120pp. (2004). [13]. niger delta development commission (nddc), environment and hydrology study report. 1: 60-136 (2003). [14]. babatunde, d.o., raji, a., field guide to nigerian freshwater fishes. federal college of freshwater fisheries technology, new bussa, niger state, nigeria. pp 106 (1998). [15]. idodo-umeh g., freshwater fisheries of northern nigeria taxonomy, ecological notes, diet and utilization. isbn 978-8052-01-0. (2003). [16]. association of official analytical chemists (a.o.a.c.), official methods of analysis of the association of official analytical chemists. 18th edition, washington d.c. usa (2010). [17]. gallagher, m. l., et al, variation in lipid and fatty acid contents of atlantic croakers, striped mullet and summer flounder. transactions of the american fisheries society, 120(5):614-619 (1991). [18]. fapohunda, o.o., ogunkoya, m., effect of smoke-drying on the proximate composition of tilapia zilli, parachanna obscura and clarias gariepinus obtained from akure, ondo-state, nigeria. animal research international, 3(2): 478 – 480 (2006). [19]. oparaku, n.f., nwaka, f.c., effect of processing on the nutritional qualities of three fish species: synodontis clarias, trachurus trecae and clarias gariepinus. international journal of biology and biological sciences, 2(10): 143-149 (2013). [20]. omoruyi, k., et al. effect of oven–drying and smoke–drying on the nutritional quality of snakehead (parachanna obscura) and upsidedown-catfish (synodontis clarias) in delta state. fuw trends in science & technology journal, 2(1): 239-243 (2017). [21]. nwuba, l.a., et al. the effect of actellic 25 ec on mineral composition on cured freshwater fishes: heterobranchus longifilis, heterotis niloticus and chrysichthys nigrodigitatus. animal research international, 1(2): 86 – 89 (2004). [22]. omotosho, o. e., et al. comparative effects of local coagulants on the nutritive value, in-vitro multi enzyme protein digestibility and sensory properties of wara. international journal of dairy science, 6(1): 58-65 (2011). [23]. ogbonnaya, c., influences of drying methods on nutritional properties of tilapia fish (oreochromis niloticus). world journal of agricultural sciences, 5(2): 256-258 (2009). [24]. daramola, j.a., et al. changes in physicochemical and sensory characteristics of smoke-dried fish species stored at ambient temperature. african journal food, agriculture nutrition and development, 7(6): 1684-5358 (2007). [25]. das, s., sahu, b.k., biochemical composition and calorific content of fishes and shellfishes from rushikulya estuary, south orissa coast of india. indian journal of fisheries, 48: 297-302 (2001). [26] shul’man, g. e., life cycle of fish: physiology and biochemistry, halsted press: division of john wiley and son inc. n.y. (1st ed.). pp. 101-104. (1974). [27]. ako, p.a., salihu, s.o., studies on some major and trace metals in smoked and oven-dried fish. journal of applied sciences and environmental management. 8(2): 5-9 (2004). [28]. adeniyi, s.a., et al. nutritional composition of three different fishes (clarias gariepinus, malapterurus electricus and tilapia guineensis). pakistan journal of nutrition, 11(9): 891-895 (2012). [29]. food and agriculture organization/world health organization, vitamin and mineral requirements in human nutrition: report of a joint fao/who expert consultation. bangkok, thailand. turkish journal of fisheries and aquatic sciences, 12: 761-770 (1998). 216 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 216 221 perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining *anastasiia sachko, igor kobasa, olesya moysyura yu. fedkovych chernivtsi national university, ukraine *corresponding author: an.sachko@chnu.edu.ua received 9th october 2017, accepted 27th november 2017 abstract: the present paper analyzes the efficiency of various mineral fining agents (nano dispersion composites sio2, tio2, tio2-sio2, bentonite and saponite) in the process of white and red wines fining. the influence of both the mass of the adsorbent and the time of the storage in wine on the optical density of wine was investigated.the best results were obtained with tio2, amd-2 and sio2 for white wines, and bentonite and amd-2 – for red ones. it was shown that the optimal time of contact for wine with the fining agent is: four days for white wines and two days for red ones. keywords: fining agent, adsorbent, wine, tio2, sio2, bentonite. 1. introduction stable transparency of beverages and wines is an indispensable feature of their quality. at the end of fermentation, young wine materials self-illuminate, that is, they become transparent. to accelerate the process of juices and wine materials’ clarification, special techniques are used: fining, filtering and combining of them. the substances used to fine wines must meet a number of requirements, among which: to have a developed surface, to cause coagulation of organic suspensions (proteins, polysaccharides), do notexhibit the toxic effects, easily stand out from wines, do not change the organoleptic properties, etc. in addition to the above, their utilization should be economically feasible. there are a lot of substances that might be used as wine fining materials. among them: minerals, proteins, woodcharcoal, synthetic polymers, enzymes and many others. in the wine industry coal [1] and natural mineral sorbents, in particular "bentonites" and "saponites" [2], are quite popular because of their ability to bind tannins and other organic substances [3]. recently, the effectiveness of the wine fining with casein, potassium caseinate products, isinglass and egg albumin was confirmed [4]. other works describe the development of conditions for utilization of chitin and chitosan [2], as well as the use of mineral adsorbents in a mixture with proteins or other organic substances [5]. however, it has been established that the applying of organic fining agents, especially proteins, may cause appearance of food allergies to consumers [6]. hence, the search for new effective, health-safe, economically viable wine fining materials and the establishment of the mechanism and conditions of their applying remain current. the action of bentonites, carbon, ferrocyanides and other mineral agents has been studied in detail. much less attention is paid to the use of silicon and titanium oxides [7]. for example, previously it was believed that the applying of silicon dioxide is ineffective, but its combination with gelatin gives a better result [8]. although the utilization of nanodispersed oxide http://www.fia.usv.ro/fiajournal mailto:an.sachko@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 anastasiia sachko, igor kobasa, olesya moysyura, perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining, volume xvi, issue 4 – 2017, pag. 216 – 221 217 materials is not very widely covered in the literature, they can be successfully applied as fining agents. therefore, our aim was to consider the posibility of using nanosized sio2, tio2, and tio2-sio2 compositions for white and red wines’ fining. the conclusion about the possibility of oxide materials utilization as fining agents was based on the efficiency of clarification of wines. the content of dry substances and the ethanol content of the beverage were monitored before and after the fining procedure. 2. materials and methods as fining agents, the following materials were selected: bentonite al2[alsi3o9×(oh)](oh)2×nh2o; saponite (ca,na)0.3(mg,fe 2+)3(si,al)4o10(oh)2×4h 2o. nanodispersed sio2 – product which was obtained by flaming hydrolysis of sicl4 vapors in an air-aqueous mixture at a temperature of 700 to 1100 °c. the size of the particles is of 10-20 nm, the specific surface area sbet = 300 m 2/g. amd-2 – high-disperse silicon dioxide modified by depolymerizate d-4, specific surface area sbet = 280 m 2/g.tio2 – nanosized titanium dioxide. tіo2-sіo2 –a composite containing 6.0% wt of sio2. the fining agents were placed in wine in the powder form, without any pretreatment. some of them (especially amd2) exhibit hydrophobic properties and the preparation of aqueous suspensions is not possible. for standardization of start conditions of this research, it was decided to put powders in wine without additional previous preparation. powders stayed in wine from one to nine days. for each sample of the fining agent, three parallel experiments were performed. the final values presented in the work the result of averaging the values obtained for each sample. at the end of the fining process, the powder-wine mixtures were filtered. the content of dry substances in white wine before fining was 8%, in red – 17% (at 20° c).the ethanol content did not change after fining: in white wine it was 13%, in red – 15%. we researched samples of red and white wine made from isabella and flamingo grapes, respectively, in laboratory conditions using the traditional technology.these varieties of grapes are grown in the territory of western ukraine. they are actively used by locals for the production of home-made wines. although, it should be noticed that these grape varieties are not used in industrial winemaking.the efficiency of fining was calculated based on the results of the optical density measurement (eq. 1): , % (1) where а0 is the optical density of wine sample before fining (0.555 for white, 1.957 for red), a is the optical density of the wine sample after fining procedure. the optical density was measured with agilent cary 60 spectrophotometr. white wine was studied at the wavelength λ = 340 nm, absorbing layer thickness 5.075. the red wine was studied at the wavelength λ=540 nm, and the thickness of the absorbing layer was of 3.070. the comparing solution was water. the content of dry substances was evaluated refractometrically (atago pal-1 blt digital brix refractometer). 3. results and discussion it is thought [8] that fining agents can act in two mechanisms: electrostatic interaction and adsorption. in the case of electrostatic interaction, the particles of the fining powder bind to the oppositely charged particles contained in wine. the result: large particles’ formation and their deposition. for adsorption interactions – the process takes place on the surface of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 anastasiia sachko, igor kobasa, olesya moysyura, perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining, volume xvi, issue 4 – 2017, pag. 216 – 221 218 the adsorbents and in such a way that it is accompanied by the resulting composites separation from wine. in general, the effectiveness of the fining agent depends on many factors, such as: the nature of the substances, the method of its manufacturing and addition, concentration, external conditions, preliminary preparation of wine materials, and others. it is known from the literature that the optimal concentrations of bentonite range from 0.2–1.5 g/l [9, 10] or 10–13 g/l of wine [11]. therefore, for our studies we have chosen a row of concentrations 1–3– 10 g/l of wine (or 0.05–0.15–0.5 g/ 50 ml). this work consisted of two blocks. the first one is to evaluate the fining efficiency within 7 days after mixing the adsorbent with wine. the second is to observe the changes in the optical density of wines over time in order to determine the optimal time of wine – fining agent contact. white wine. the research results show that all of the test substances show good efficiency as white wine clarifiers. fig. 1 is a diagram illustrating the fining efficiency of wine with the studied agents at their various contents per 50 ml of wine. fig 1. efficiency of fining with different agents for a white wine for varies mass on 50 ml of wine from the obtained results we concluded that the most effective is nano-dispersed tio2, regardless of its content. amd-2 and sio2 are demonstrating really good results. their effectiveness significantly decreases with the increase of their content in wine. it should be noticed that the highest fining efficiency and сlarity were achieved with the use of 0.05 g amd-2on 50 ml of wine (fig. 2). this is an important observation, because the applying of minimum quantities of fining agents influences minimally the taste and physical and chemical properties of wine. this result is somewhat unexpected. on the one hand, amd-2 has a large specific surface area. on the other hand, it is a highly hydrophobic powder. unlike other substances, it formed a film on the wine-air surface and did not fall into the volume of the liquid. fig. 2. the result of amd-2 fining of white wine bentonite and saponite are the worst of the studied fining agents for white wines, which may be due to the fact that we did not pre-prepare the suspension, and all the substances were used as powders. the preliminary preparation is a factor that has a significant effect on the final result of fining. on the other hand, it is known that bentonite possesses a low ability to color reduction and weakly binds wine tannins [8]. from the obtained results, it can be argued that the fining efficiency decreases with the weight increase for all powders except sio2. refractometric studies conducted after removing of adsorbents showed that the content of dry matter in the wine practically did not change as compared with the original wine. based on the results of previous investigations on the effect of luminaries, several samples were selected for detailed re-studies to determine the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 anastasiia sachko, igor kobasa, olesya moysyura, perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining, volume xvi, issue 4 – 2017, pag. 216 – 221 219 optimal time for the wrapping of wines on the illuminator. red wine. similar studies have been conducted for red wines. however, in the case of red wines, the substances showed a significantly worse fining ability. fig. 3 shows the results of the study on the fining efficiency for red wine samples. in general, bentonite has shown the best action as fining agent. it should be noticed that the most effective at low content (0.05 g / 50 ml of wine) were tio2 and amd-2. in the case of tio2, the fining effect varied insignificantly with an increase in powder weight. a similar situation was observed in the case of white wines. for sio2, the fining effectiveness became negative with an increase of its weight (0.5 g / 50 ml of wine), which means that the optical density of wine after clarification became larger than before. fig 3. efficiency of fining with different agents for a red wine for varies mass on 50 ml of wine it has been shown that for bentonite and saponite there takes place a significant improvement in the fining effect with an increase of powders’ weight. at the same time sio2 has proved to be ineffective for the treatment of red wines. a comparison of silica gel and bentonite showed that bentonite is not the most effective fining agent [12]. silica gel used to provide a better binding of proteins contained in the wine and had less effect on the product’s organoleptic properties. selection of optimal fining time. based on the results of previous investigations, several samples were selected for detailed re-studies. the subject matter of re-studies was the determination of the optimal time for the delaying of the wines with powder. the information described in the literature is somewhat controversial. for example for bentonite, on the one hand, it is recommended to provide fining "in flow" in order to minimize the contact time of the mineral with wine. it is believed that when the "bentonite-wine" mixture is standing, some of the adsorbed proteins may return to the solution [9]. on the other hand [8, 11], it is best to delay the wine with the fining agent for 2 days. white wine. of all the previously selected fining agents, the best for the white wine were: amd-2, sio2, tio2. to establish the time dependence "optical density – delay time", the wine was kept in contact with the powders from one to nine days (fig. 4). a). b). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 anastasiia sachko, igor kobasa, olesya moysyura, perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining, volume xvi, issue 4 – 2017, pag. 216 – 221 220 c). fig. 4. dependence of the fining efficiency of white wine in time: a – sio2, b – amd-2, c – tio2 (0.05-0.15-0.5 g / 50 ml of wine) from the submitted data it is possible to draw the following conclusion: the maximum fining efficiency is reached after 4 days. further exposure of wine with the powders is unreasonable and worsens its properties. however, our results contradict the described data [8, 11]: in the case of white wines, the contact for two days is insufficient. these three fining agents can be placed in a row in a descending order of efficiency: tio2> amd-2 > sio2. the highest effective for white wines was tio2. moreover, the lowest values of the optical density were obtained using the least amounts of both sio2 and tio2 (0.05 g per 50 ml of wine). the minimization of the quantities of agents that are introduced in wine to improve its characteristics is important for wine industry. the refractometric studies before and after fining showed that the content of dry matter in the investigated wine practically did not change and was of 7.5-7.8%. red wine. bentonite and amd-2 were selected for studying the optimal contact time fining agents with red wine. bentonite was chosen for comparison, since it is one of the most studied fining agents. and amd-2 was chosen because it showed the highest efficiency with contents of 0.05 g per 50 ml of wine. the obtained dependences are presented in fig. 5. the analysis of fig. 5 a shows that the maximum amd-2 fining efficiency was achieved on the second day, therefore the further exposure of suspension was unwarranted. all the studied powders have a highly developed surface that is why with the increase of contact time, adsorption-readsorption of the components in wine on the surface of adsorbents could take place. such processes may affect adversely wine quality. a). b). fig. 5. dependence of the fining efficiency of red wine in time: a – amd-2, b – bentonite (0.050.15-0.5 g/50 ml of wine) similar to amd-2, bentonite exhibits the best fining result on the second day (fig. 5 b). this result is fully consistent with the regularities described in the literature [8, 11, 13-14]. an interesting fact is that at low concentrations (0.05 g / 50 ml of wine), bentonite exhibited lower efficacy than amd-2. this makes amd-2 a promising agent for fining procedures. it is well known that bentonite affects significantly the red wines’ color due to its ability to bind positively charged anthocyanin monomers [8]. as a result of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 anastasiia sachko, igor kobasa, olesya moysyura, perspectives of utilization of nanodispersive materials based on sio2, tio2 and sio2–tio2 for wine fining, volume xvi, issue 4 – 2017, pag. 216 – 221 221 this binding, the intensity of red color decreases. so, since in our studies we observed a change in the color intensity, it is natural that bentonite showed the best result. at the same time, bentonites can be successfully used for fining wines’ particles of which are positively charged. the mechanism of removing protein particles from wine under bentonite influence was induced by the interaction of negatively charged colloidal particles of bentonite with positively charged particles of protein substances, as well as their adsorption and subsequent sedimentation [15]. refractometric control of dry matter in the samples of red wine before and after fining did not show significant changes. the content of dry matter in the samples of red wine ranged from 16.5 to 16.9%. 4. conclusions on the basis of the obtained results it can be argued that bentonite, nano-dispersed oxides of silicon, titanium and their composition can be proposed as an alternative to classical mineral adsorbents. in particular, for white wines, these are samples: tio2 > amd-2 > sio2 (in order of efficiency decrease). a significant advantage of these substances is the high fining efficiency at small contents (0.05 g / 50 ml of wine). the utilization of amd-2 can be recommended for red wine. similar to previous studies it exhibits high efficiency (comparable with bentonite) at low content (0.05 g per 50 ml of wine). the optimal contact time of the wine with fining agents is of 4 days for white wine, and 2 days – for red wine, respectively. 5. references [1]. corcho-corral b., olivaresmarín m.,valdes-sánchez e., fernándezgonzález c., macías-garcía a., gómezserrano v., development of activated carbon using vine shoots (vitis vinifera) and its use for wine treatment, j. agric. food chem., 53(3): 644–650, (2005). [2]. quintela s., villarán m.c., lópez de armentia i., elejalde e., ochratoxin a removal from red wine by several oenological fining agents: bentonite, egg albumin, allergen-free adsorbents, chitin and chitosan, j. agric. food chem., 55(8): 3127–3133, (2007). [3]. moreno-arribas m.v., polo c., wine chemistry and biochemistry, springer science & business media, 735, (2009). [4]. cosme f., ricardo-da-silva j.m., laureano o., protein fining agents characterization and red wine fining assays, ital. j. food sci., 1(19): 39– 56, (2007). [5]. cosme f., ricardo-da-silva j.m., laureano o., interactions between protein fining agents and proanthocyanidins in white wine, food chemistry, 106(2): 536–544, (2008). [6]. weber p., steinhart h., paschke a., investigation of the allergenic potential of wines fined with various proteinogenic fining agents by elisa, j. agric. food chem., 56(1): 158–165, (2008). [7]. pachova v., ferrando m., güell c., lóapez f., protein adsorption onto metal oxide materials in white wine model systems, journal of food sci., 67(6): 2118–2121, (2002). [8]. zoecklein b.w., fugelsang k.s., gump b.h., nury f.s., wine analysis and production, springer science & business media new york, 638, (1999). [9]. jacobson l., introduction to wine laboratory practices and procedures, springer science & business media, 375, (2006). [10]. salazar f.n., ahaerandio i., labb m.a., gell c., lpez f., comparative study of protein stabilization in white wine using zirconia and bentonite:  physicochemical and wine sensory analysis, j. agric. foodchem., 54(26): 9955–9958, (2006). [11]. valuiko g.g., technology of grape wines. simferopol: tavrida, 624, (2001), (in russian). [12]. armada l., falque e., repercussion of the clarification treatment agents before the alcoholic fermentation on volatile composition of white wines, eur food res technol, 225: 553–558, (2007). [13]. pocock k.f., salazar f.n., wate e.j., the effect of bentonite fining at different stages of white winemaking on protein stability, australian journal of grape and wine research, 17:280–284, (2011). [14]. catarino s., madeira m., conteiro f., rocha f., curvelo-garcia a.s., bruno de sousa r., effect of bentonite characteristics on the elemental composition of wine, j. agric. food chem, 56(1): 158–165, (2008). [15]. aleksanyan k.a., tkachuk l.a., technology of production of fruit and berry natural wines, minsk: belarus. nova, 246, (2012), (in russian). 1. introduction 9 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1-2018, pag. 9 19 heat transter at evaporative concentration of down flowing in vertical pipes solutions in annular regimes valentin petrenko 1 , *yaroslav zasiadko1 1department of termal engineering and industrial refrigeration, national university of food technologies, 68, volodymyrska st.; 01601 kyiv; ukraine *corresponding author iaroslav@nuft.edu.ua received 20th november 2017, accepted 19th march 2018 abstract: in many branches of food processing, particularly in sugar production, the solution concentration takes place within industrial multi effect evaporator; here, the solution moves down as a liquid film forming annular descending flow in vertical heated pipes. it should be noticed that within the evaporation station the liquid which is being evaporated significantly changes its physical properties due to the fact that the concentration of the solution increases by the range of 14…65% of dry matter. the operational process parameters (pressure, temperature, etc.) also change to a noticeable degree. these factors determine a wide range of liquid films flow patterns, namely film thickness, surface wavy structures, velocity distribution across the films, interphase shear stress and, as a result of these, the making of a certain heat transfer mechanism including bubble boiling or its suppression. unfortunately, the reliable methods of prediction and calculation of heat transfer coefficients are unavailable, which seriously hampers the designing and calculation of efficient evaporators and other heat transfer equipment. the work presents the results of modeling heat transfer to saturated liquid films of solutions flowing down inside the vertical pipes as in the regime of evaporation from the interphase surface and in that of surface boiling. the correlations given allow calculating the heat transfer coefficients for liquid films being concentrated in the vertical pipes of industrial evaporators keywords: heat flux, heat transfer coefficient (htc), depression, interphase surface, nucleate boiling 1. introduction an extensive analysis of the mechanisms of heat transfer to the saturated turbulent and laminar liquid films of sugar solutions in the regime of evaporation from the developed wavy structures in given in [1,2]. the analyzed pattern of film movement is considered to be dominant in the long vertical pipes of industrial evaporators [3]. the data presented in [1,2] and respective correlations for calculation heat transfer coefficients (htc) for laminar and turbulent films adequately reflect the mechanisms of heat transfer in long vertical pipes of industrial evaporators within the regime of evaporation from film free surface. at the same time in [4-7] it has been shown that the regime of heat transfer which has been identified as that of evaporation from the interphase surface can be realized only to the certain limiting value of heated wall superheat related to the local saturation temperature, above which the mechanism of heat transfer undergoes a drastic change as being affected by pulses resulted by appearing and move of steam bubbles. http://www.fia.usv.ro/fiajournal mailto:iaroslav@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 10 nomenclature  – film thickness; v г – volumetric liquid flux; a –temperature conductivity;  –cinematic viscosity coefficient; sat t –saturation temperature; w t – wall temperature; i t – interphase temperature  –heat conduction of liquid; q – heat flow;  –liquid density; 2  –steam density;  – surface tension; c – mass concentration u – average liquid film velocity 2 u − steam velocity; g –acceleration of gravity; a u a г pe v   44 – the peclet number;   v г re 4 – the reynolds number. in [6], the intensifying effect of the nucleate boiling upon the heat transfer at a free down flowing water films on the outside of a cylinder and a constant along the channel heat flux has been determined as a multiplier:                   36.1 05.01 incip incipw q qq (1) to the basic equation for htc determination at the regime of evaporation from the film free surface. in eq.(1) the subscripts denote: w-related to wall, incip-at bubble boiling inception. the minimum value of the heat flux at which the bubble boiling starts effect the heat transfer mechanism has also been found. according to [6], the heat flux at which the bubble boiling starts in some inception points is recommended to determine as 2 2.14 m kw q incip  . at the same time, it has been noted that the onset of boiling is influenced by liquid convection, therefore a much more adequate factor which reflects the beginning of boiling as well as its effect upon the intensity of heat transfer would be a complex ur q presented in [4] in which the onset of boiling is identified as: 3.0 24.0 4 pr105.1             a u ur q . (2) it should be marked that the complex in eq. (2) ur q does not account for the effect of pressure or vacuum upon the critical heat flux at which the surface nucleate boiling starts. on top of this, neither incip q in eq. (1), nor ur q account for the local roughness of heat transfer surface, which presents a decisive factor in the determination of a critical value of the wall superheat leading to the onset of bubbles generation. it is necessary to stress specifically, that the majority of experimental works deal with the heat transfer processes in the liquid films heated to the saturation temperatures in experimental stands which model the conditions that occur in the industrial evaporators. in these conditions of simultaneous effect of mutually regulating factors, it is practically impossible to separate and distinguish effect of any individual factor. therefore the recommended expressions which are used for calculations of heat transfer intensity in the saturated liquid films have a limited applicability due to the narrow range of the regime parameters, in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 11 which they are valid, as well as due to their applicability only to some geometrical configurations. due to the above mentioned, they hardly may be applied to determine the conditions of the bubble boiling onset in the pipes of real industrial evaporators. 2. materials and methods a direct experimentation of heat transfer in down flowing liquid films of sugar solutions heated to the saturation temperatures has been carried out in the experimental unit with the independent formation of phases’ mass flow rates and heat flux. the main core of the experimental unit is formed by a stainless still pipe with the inside diameter of 20 mm and 1.8 m long. the experimental tube was separated into the initial 1.5 m stabilization section and 0.3 m measurement section. the down flow of water (sugar solutions) film has been formed by means of overflowing over the tube’s upper rim. in the event of steam-liquid flow modelling, dry saturated steam has been supplied in co-current regime. the liquid falling film has been heated by dry saturated steam which was supplied into outside heating sections attached to the experimental tube. the heating chambers were designed in a way as to provide an individual heating of the stabilization section and the experimental one. the said sections were hooked up to the individual vacuumcondensation sections which allowed for the keeping of different pressures in each chamber. such arrangements allowed also maintaining vacuum down to 0.85 bars and thus, vary the temperature head between the heating steam temperature and evaporation temperature. special probes for taking samples of liquid to determine its concentration and measurements of temperatures were positioned directly after the measurement section. a detailed description of the experimental unit is given in [11]. the experimental unit closely reflects the actual operational conditions of sugar industrial film evaporators. the installation consist of heat transfer pipe from stainless steel, 9 m long with internal diameter of 30 mm partitioned in 20 section each of 40 mm long. each section was equipped with the special chamber which allowed collecting condensate which was generated on the respected section. the experimental pipe and condensate chambers were positioned into the heating jacket. then this condensate was transferred to the adiabatic measurement glasses which allowed measurements of the condensate collected from each test section and thus to determine the heat flux acting on the respective section. the heating of the experimental pipe has been done by means of supplying dry saturated steam into the pipe’s heating jacket. between each of 20 test section a special probe for taking samples of liquid and static pressure were installed. the wall temperature of each section and the temperature at the access of the pipe were taken by means of copperconstantan thermo-couple (type t). a detailed description of the experimental unit is given in [4]. 3. results and discussion a generally accepted parameter for the identification of the onset of nucleate boiling in films has been suggested by chun and seban [5]. the value of the minimal superheat of a rough wall over which a liquid film flows down looks a reliable factor which may be used for identification of the onset of steam bubbles appearing. the value can be calculated based upon the combining the laplace and clapeyron-clausius expressions, which correlate the value of a wall critical superheat min t with a certain radius of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 12 indents and micro cracks c r at a certain pressure (or vacuum): c sat min rr t t 2 2    , (3) were sat t absolute saturation temperature. in the event of solution boiling, the wall superheat will be bigger by the value of the physical-chemical depression fc  , and thus eq.(3) is to be rearranged as: fc c sat min rr t t     2 2 . (4) it is clear that the intensification of heat transfer at the surface boiling is caused by a gradual increase in the new centers of steam bubbles generation along with the growth of the temperature superheat, as it is evidently follows from (3,4). equally this parameter adequately reflects the effect of pressure upon the inception of bubble appearing on the heated surface. therefore the parameter which may be used to characterize the heat transfer intensification might be developed by use of the temperature head min tt  which later will be presented as a multiplier to the basic equation for calculation of htc in liquid films at evaporating from the interphase surface as: n boil t tt ck            min min1 , (5) where c and nempirical coefficients derived from the experimental data. the basic equation for calculation the intensity of teat transfer at the regime of evaporation from the free interphase of liquid films of water and sugar solutions derived in [2] is extremely complex, especially in case of co-current steam flow, therefore its simplified version for engineering calculations is given as eq. (6): , (6) where boil k correction factor accounting for the nucleate boiling, l k correction factor accounting for the channel length and diameter. a comparison of experimental and calculated according to eq.(6) data on htc to water and sugar solution free flowing films in 20 mm diameter pipes at the regime of evaporation from the free intrerphase surface is given in figure 1 and in case of co-current steam flow – on figures 2, 3 and 4. a w кm2, 4000 1 2 3 4 5 3000 2000 10 4 s m2 ,гv 1 3 4 5 2 fig.1. comparison of date calculated as per (6) and experimental data [11] 1 – water, t = 100 о с; 2, 3, 4, 5 – sugar solution; 2 –dm = 30%; 3 – 40; 4 – 50; 5 – 60 1 4 3 2 4000 6 12 18 24 30 4500 6000 5000 5500 2 1 3 4 a w кm 2, u 2 , /m s fig.2. correlation   2 uf . comparison of data calculated by eq.(6) with experimental data [11] water, t = 100 o c. 1 – гv = 0.1 10 -3 2 – 0.2; 3 – 0.3; 4 – 0.5   1/3 2 1/3 0.2 4 0.86 0.2 2 0.2 6 2 2 1.12 re 0.85 0.01 4.5 10 pr 1 7.5 10 re boil l pe pe g k k                                 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 13 a w km2, 2000 2400 2800 3200 3600 5 1 23 10 15 20 25 30 35 1 2 3 u2, /m s fig.3. correlation   2 uf . comparison of data calculated by eq.(6) with experimental data [11]sugar solution, dm = 40%, t = 100 o c ; 1 – гv = 0.1 10 -3 2 – 0.2; 3 – 0.35 a w кm2, 800 5 10 15 20 25 30 1 2 3 900 1000 1100 u , / 2 m s 1 2 3 fig.4. correlation   2 uf . comparison of data calculated by eq.(6) with experimental data [11] sugar solution dm = 70 %, t = 100 o c, 1 – гv = 0.1 10 -3 2 – 0.3; 3 – 0.55 analysis of the obtained results shows that a close concurrence may be achieved by substituting into (3) the values of micro cracks dimensions of 5105.0   c r m, which corresponds to the values of new heat transfer pipes’ roughness. respective values of 4.0c and 1.2n  found from experimental data are to be used in eq. (5). an additional factor affecting the accuracy and singularity of the experimental data on heat transfer to the solutions processing would be a unevenness of the concentration distribution across the film. as a result of the gradual evaporation of solvent (water in case of sugar solutions or fruit juices) from the liquid film, there appears a thin layer adjacent to the interphase in which the concentration of solution is somewhat bigger than that in the bulk of the film. if only the molecular diffusion had caused the equalizing of the concentration field, the process would have taken an extremely long time. in fact, the surface of the film is covered by a complex wavy structure, which even at low reynolds numbers exerts turbulence penetrating the bulk of the film, which in turn results in an uncertain form of the concentration distribution across the film. the temperature of liquid film of solution being concentrated at a regime of evaporation from the free surface i t should be equal to the saturation temperature of the solvent at a given pressure and physical-chemical depression at a concentration on the interphase surface i c , thus   ifcsati сtt  . keeping in mind stated above concerning the uncertainties in the concentration field across the film, it would be grounded enough to suggest that the waves equalize the concentrations in the bulk of the film, so that the mean film temperature will be  сtt fcsati  . using this value as a reference temperature and determining htc as )/( iw ttq  , one may obtain:   ctt q fcsatw   , (7) where c mean across the film solution concentration. since the film temperature profile is nonlinear (usually in the laminar stationary film it is parabolic), the mean film temperature mav t . in general may be derived as eq.(8):     2 0 . iw mav tt dy u yu ytt     , (8) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 14 where    yuyt , temperature and velocity profiles, respectively. at the same time, the results of direct measurements have proven that the experimental mean mass film temperature of solution exp m.av t , which had been measured in the adiabatic calorimeter immediately at the exit of the experimental stand correlates with eq.(8) only at conditions of free down flowing at the absence of interphase shear stress. the schematic of measurements is given in figure 5. ex p .mav t sat t w t u2 q fig. 5. schematics of calorimetric temperature measurements in flowing down liquid films in case of a co-current flow of steam above the film, the experimental mean mass solution temperature exp m.av t turned out lower than that determined by eq.(8) exp .. mavmav tt  . the deviation observed was the bigger the bigger was the steam flow velocity and liquid mass flow rate. since the experimental temperature exp m.av t in the contrast to i t adequately reflects any change in the regime parameters and being the value which is directly measured in the experiments, then the htc to solutions are to be correctly defined as eq.(9): exp .mavw m tt q   , (9) which will adequately reflect the effect of phases mass flow rates change. this immediately yields m  .since htc determined as per eq.(6) led to the generalized correlation eq.(9), the calculation of heat flux (or as a result evaporation capacity) should take into consideration the value of the mean concentration of the solution in the liquid film and thus appearing effect of physicalchemical temperature depression. it is necessary to stress specifically that since the correlation presents data of the discreet-local character, which means that they were obtained within comparatively short sections of the channel, in which it was acceptable to assume a constancy of all regime parameters, phase flow rates and their properties. this determines a successive-iterative method of channel evaporative capacity calculations. a channel being under the calculation is to be split into a number of sections, and the calculation process starts from the upper inlet of the channel. insofar as it can be seen from eqs.(6) and (11), the local values of heat flux do not affect htc directly, there is no need to initial guess of q to start iterative calculations to reach a convergence of guessed and obtained q values. rather, it would be necessary to first calculate the local value of heat flux on the first section as a first approach, then to calculate the amount of steam generated in this section, and after this – determine all parameters dependent on the steam velocity. the next iteration would be calculation of a new value of htc by eq.(6) and (11) with the respective new values of heat flux and obtained in the previous calculation q and eventually steam velocity, phase flow rates, concentrations, etc. the values of the local heat flux are to be determined with taking account of local value of physical-chemical temperature depression as well as its suppression by the concurrent steam flow food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 15   tcttq fcsatwm  , (10) where     iwmavw ttttt  exp . -a function which accounts for the suppression effect of interphase shear stress. the suppression effect may be explained by the fact that the steam being generated from the film has the temperature of the saturated solvent (water), whereas the upper layer of the film has local temperature sufficiently higher due to the concentration growth of the soluble substance. being in the contact with the steam flow at a lower temperature, the surface of the film cools down; this leads to the decrease in physical-chemical temperature depression. analysis of the experimental data on the heat transfer to liquid films of sugar solutions with the concentrations up to 70% dm (dry matter) in the regimes of evaporation from the interphase surface as well as at bubble boiling with the concurrent steam flow allowed deriving a temperature function as eq.(11):    fx pewet   32103.2exp1 .(11) the comparison of htc calculations by eq.(6) with the experimental data on the heat transfer to the free down flowinh liquid films and films flowing co-currently with the steam at a velocity of 25 m/s of water and sugar solutions with the concentartion up to 75% within the regimes of evaporation from the free surface and nucleatete boiling are shown in figures 6 and 7. the transition to the regime of nucleate boiling is identified by the beginning of upward line  qf on the graphs. it is clearly seen from the data given above that the transition to regime of nucleate boiling with the steam flow growth shifts towards to greater heat fluxes, which satisfactorily is identified by eqs. (4) and (5). for rough pipes ( mr c 5 105.0   ), for example, after cleanning with the hard metal brushing or superimposin an artificial roughness according to eq. (3) the transition to the nucleate boiling regime accompanied with the respective heat transfer intensification at lower values of heat flux. apart from the mentioned above factors, the heat transfer intensity is greatly affected by geometry of the channel, since as the height as well as the diameter of the channel determines the character of wavy structure on the film surface. namely, in the 9 m boiling tubes of industrial evapolators along with the distance from the film distributor down to 3…4 m takes plase an increase in heat transfer intensity comared to that in the short pipes at the same regime parameters and mass flow rates. on top of this, it has been found in [8,9] that an increase in pipe diameter entails the increase in length and amplitude of big waves that significantly intensifies the mixing in films. unfortunately, it is impossible to separate the effect of certain factors on the intensity of heat transfer within the system under consideration. it is necessary to mark specifically that within the experimental stands, which simulate the conditions of actual film evaporators, all working and regime parameters manifest themselves in the unseparable mutually regulating unity. for example, at a certain set of regime parameters and initial solution flow rate, down the therefore, modelling of heat transfer to free falling films in such conditions becomes possible only within the upper sections of experimental pipe. here the velocity of cocurrent steam is negligibly low but, at the same time, the wavy structure will be undeveloped.the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 16 data for films with the developed waves might be taken at the middle or exit sections of pipes, but here the film flow will be affected by the strong shear stress fron the steam flow. fig. 6. correlation  qf for free falling films of water and sugar solutions, ( m.r,ct c o 5 1050100   ). data[11]. а. water, 1 – гv = 1∙10 -4 m2/s; 2 – 2∙10 -4 ; 3 – 3∙10 -4 ; 4 – 5.5∙10 -4 ; b. sugar solution, dm = 30 %, 1 – гv = 1∙10 -4 m2/s; 2 – 1.6∙10 -4 ; 3 – 2.2∙10 -4 ; 4 – 4.5∙10 -4 ; c. sugar solution, dm = 50 %, 1 – гv = 0.7∙10 -4 m2/s; 2 – 1.5∙10 4 ; 3 – 2.2∙10 -4 ; 4 – 4∙10 -4 ; d. sugar solution, dm = 70 %, 1 – гv = 0.5∙10 -4 m2/s; 2 – 2∙10 -4 ; 3 – 3∙10 -4 ; 4 – 5.5∙10 -4 . lines – calculated to eq.(6) at the same mass flow rates. 1 1 10 3.5 4.5 4.0 5.0 5.5 20 30 40 50 60 2 2 33 4 4 km kw , 2  2 m kw ,q 1 2 3 4 1 10 2.5 3.0 4.0 3.5 20 30 40 50 60 2 3 4 km kw , 2  2 m kw ,q 1 2 3 4 10 1.5 1.8 2.1 1 2 4 3 20 km kw , 2  2 m kw ,q 10 15 0.6 0.8 1.0 5 2 1 3 4 4 2 3 1 km kw , 2  2m kw ,q a b c d food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 17 km kw , 2  2 m kw ,q 1 1 2 2 3 3 4 4 2010 4.5 5.5 6.0 6.5 5.0 30 40 50 km kw , 2  2 m kw ,q 1 2.6 10 20 30 3.0 3.4 2 3 1 2 3 4 4 km kw , 2  2m kw ,q 1 1 2 2 3 3 4 4 2.0 2.2 2.4 2.6 2.8 10 20 30 km kw , 2  2 m kw ,q 1.4 1.0 5 1 2 3 4 1510 1.2 1 2 3 4 fig.7. correlation  qf at steam velocity s m u 25 2  , ( m.r,ct c o 5 1050100   ). data [11] а. water, 1 – гv = 1∙10 -4 m2/s; 2 – 2∙10 -4 ; 3 – 3∙10 -4 ; 4 – 5.5∙10 -4 ; b. sugar solution, dm = 40 %, 1 – гv = 1∙10 -4 m2/s; 2 – 1.5∙10 -4 ; 3 – 2∙10 -4 ; 4 – 3.5∙10 -4 ; c. sugar solution, dm = 50 %, 1 – гv = 1∙10 -4 m2/s; 2 – 2∙10 -4 ; 3 – 3∙10 -4 ; 4 – 6∙10 -4 ; d. sugar solution, dm = 70 %, 1 – гv = 1∙10 -4 m2/s; 2 – 2∙10 -4 ; 3 – 3∙10 -4 ; 4 – 5.5∙10 -4 lines – calculated to eq. (6) at the same mass flow rates. therefore, the comparison and generalization of the experimental data obtained in long evaporating channels at the local level (within a comparatively short sections to suggest the constancy of parameters) appear grounded only in the event of equality all respective regime parameters, flow rates and physical properties of phases. in such case the data can be compared and generalized with those obtained in the stands allowing individual formation of pfase flows and thus providing for the comparable working conditions. data in figure 8 illustrate the given above. it shows the fields of scatter for data obtained in the experiments in short and long pipes of different diameters in the comparable conditions and their comparison with the results of calculation by eq.(6). d c a b d food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 18 the correction factor accounting for the effect of pipe length and diameter on the heat transfer was found by examining data obtained in pipes of different geometries but exclusively in the comparable conditions as eq.(12):  3 0.35 0.06 1 0, 06 1 exp 0, 05 o l o d d o k l d d                                 ,(12) where md o 02.0 , s m о 2 6 103.0   , valid for l >1.0 m. km kw 2 , m,l 1 3 2 2 4 6 8 4.5 5.5 6.0 5.0 km kw 2 , m,l 1.5 2.0 1 1 2 2 1.0 2 4 6 8 fig. 8. correlation  lf . а. water, s m u 15 2  ; b. sugar solution, dm = 70 %, s m u 30 2  . 1 –data scatter field, d = 20 mm, l = 1.8 m[11]; 2 – data scatter field [4] , d = 30 mm, l = 9 m; 3 – data scatter field [10] d = 32 mm, l = 4.9 m. lines – calculated by eq.(6), 1 – d = 20 mm, 2 – 30 mm. the comparison of calculated by eq. (6) values of heat flux along the height of a 9 m pipe with the experimental data [4] at various temperature differences is given in figure 9. as it is clearly seen from figure 9 the proposed correlations and methodology allows quire closely predict the evaporative capacity of boiling channels of induslrial film evaporators with pipes up to 9 m height within a vide range of working parameters. even at a big temperature difference (wall – saturation) of 50c, figure 9(c) when the local values of heat flux reach 35 kw/m2 at the lower sections and, thus, one may expect rather significant values of the cocurrent steam flow velocity with affecting film surfacial wave structure, its supressing effects, named above, which manifest itself inseparably with the local heat flux – the recommeded method gives remarcably good coincidece of measured and predicted data. 27 29 31 33 35 3718 20 22 24 1 2 3 4 5 6 7 8 9 4 6 8 10 q, kw m/ 2 l,m a b c fig.9. heat flux distribution at a height of the evaporative pipe lines – calculation by (6) (6 ); dots experimental data [4]. a – δt = 2.2 o c, tw – tsat = 1.2; b – 7.5, tw – tsat = 3.7; c – 11, tw – tsat = 5.1; гv = 0.3 10 -3 m 2 /s, water, tsat = 100 o с. a b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 valentin petrenko, yaroslav zasiadko, heat transfer at evaporative concentration of down flowing in vertical pipes solutions in annular regimes, food and environment safety, volume xvii, issue 1 – 2018, pag. 9 – 19 19 4. conclusion equations (3) and (4) which take into account the value of local wall superheat and heating surface roughness are recommended to be used as a factor determining the onset of nucleate boiling in films. the temperature of liquid solution film at a regime of evaporation from free surface with the co-current steam flow is to be determined with taking into account the value of physical-chemical temperature depression eq. (11). the eq. (6) along with the methodology of its application may be used for the calculations of evaporative capacities of boiling channels of industrial film evaporators of sugar industry. the methodology is valid in application to water and sugar solutions with the concentrations up to 70% dm, at regimes of evaporation from free surface and nucleate boiling, within the range of vacuum up to 0.85 bar, heat fluxes up to 60 kw/m2, liquid mass flow rate density 0.05…600 m2/s, co-current steam flow velocities up to 40 m/s, for evaporative pipes 20…32 mm diameter. 5. references [1]. petrenko v., zasyadko y. heat transfer modeling in down-flowing laminar films with the developed wavy structure with co-current steam flow. food and environment safety, vol 15, issue 3, рр. 203 – 215, (2016) [2]. petrenko v., zasyadko y. heat transfer in down flowing turbulent evaporating liquid film with developed wavy structure and co-current steam flow. food and environment safety (fes), vol 15, issue 4, 2016. рр. 284 – 298, (2016) [3]. ganchev b. g. cooling of nuclear reactor of elements by film flow. energoatomizdat, moscow, рр. 192, (1987) [4]. ardashev v.a. investigation of heat transfer at evaporation gravitationally falling liquid film in a vertical pipe. – g.: phd dissertation , 188 p.(1982) [5]. heat transfer in evaporating liquid films. heat transfer, no. 4, pp. 71 – 77, (1971) [6]. cerza m., sernas v. nucleate boiling in thermally developing and fully developed laminar falling water films. heat transfer, no.4, pp. 185 – 174. (1988) [7]. fujita t., ueda t. heat transfer to falling liquids films and film breakdown – part іі. saturated liquid films with nucleate boiling int. j.heat mass transfer, vol. 21, no. 2, pp. 109 – 118.(1978) [8]. kulov n. n. hydrodynamics and a mass exchange in the film and disperse streams descending, dissertation, moscow,409 p.(1984) [9]. chu k.j., dukler a.e. statistical characteristics of thin, wavy films. pt 111, structure of the large waves and their resistance to gas flow. aiche.j., vol.21, n 3, pp. 583 – 593.(1975) [10]. leverash v.i. experimental studies into the heat transfer to liquid in the industrial evaporators with falling films in application to the conditions of distillation units. phd dissertation.moscow. mosdow power institute.1964 [11].riabchuk о. м., thermal and hydrodynamic processes evaporation in downstream film flows of sugar solutions, dissertation, kyiv , 167 р.(2013) 1. introduction 4. conclusion microsoft word 0b cuprins 2_2018_20 iunie.doc food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 contents: 1. antifungal effect of spice extracts possible solutions for biological preservation of food monica marian, oana mare roşca, lucia mihalescu, zorica voşgan, anamaria lazăr 103 112 2. screening of potential yeast starters with high ethanol production for a small-scale cocoa fermentation in ivory coast odilon koffi, lamine samagaci, bernadette goualie, sébastien niamke 113 130 3. cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo 131 140 4. complex quality indicator of sugars and sugar substitutes and their use in the production of confectionery products antonella dorohovych, victoria dorohovych, liubov mazur 141 153 5. perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia rehrahie mesfin, getnet assefa, fassil assefa 154164 6. the investigation of the potentional complex from plantago major to coagulate milk proteins olena grek, olena krasulya, larisa chubenko, alla tymchuk 165 175 7. effects of natural feed additives and probiotics on productive performance and meat quality of pigeon doaa abd el-aziz, ghada abdel-raheem 176 182 8. assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry 183 191 9. desaturation of cultural media in processes of anaerobic fermentation olexandr shevchenko, anatoliy sokolenko, konstantin vasylkivsky, oleg stepanets 192 196 10. technological aspects of production of frozen dessert with protein-herbal component olena grek, tatyana osmak, larisa chubenko, artur mykhalevych 197204 11. environmental evaluation of pork meat chain: a romanian case study cristina ghinea, ana leahu 205 212 12. positive impact of frequent milk and dairy products consumption on bone mineral density of over-50 aged macedonian women zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska 213 223 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 13. development of mathematic model of spiced sour-milk pastas quality viktor goots, nataliia yushchenko, ulyana kuzmyk 224 232 14. phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from mucuna pruriens husks against some foodborne microorganisms omotade ogunremi, oladotun ishola, hannah ogunedina 233 240 15. electrochemical biosensor based on the use of spe for the detection of iron content in wine liliana norocel, gheorghe gutt 241 245 16. effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin florina dranca, mircea oroian 246 251 17. slaughter characteristics and weight losses (shrinkage) of lambs from an organic and conventional system tatjana kalevska, ljupce kocoski, elena joshevska, zora uzunoska, viktorija stamatovska 252 260 18. author instructions i v 19. subscription information vi 75 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2 2019, pag. 75 81 hand hygiene practices among street food vendors *alexander fiifi ghartey 1 , barima kwabena antwi 2 1school of medicine and health sciences, central university, p.o. box 2305, tema,miotso, ghana, xff_ghartey@yahoo.com, 2faculty of social sciences, university of cape coast, ghana, kantwi@ucc.edu.gh. *corresponding author received 26th march 2019, accepted 29th june 2019 abstract: the purpose of this paper is to assess the nature and extent of hand hygiene practices in the street food business in selected districts in southern ghana. though street-vended foods have many health and socio-economic benefits, they often pose significant public health risk to consumers. codex alimentarius standards identify the hand of the vendor (or the consumer) as a ‘high-risk’ factor or conduit in street food contamination. the study applied cross-sectional descriptive survey with observation of street food vendor practices using a structured questionnaire in a face to face interview of 413 vendors randomly selected. while almost half of vendors (47.9%) were observed to handle food with their bare hands in the selling process, nearly one-fifth of vendors (18.4%) did not wash their hands with water and soap after visiting the toilet. vendor fingernail hygiene was however not a major risk factor. it is also revealed that there is a significant positive relationship between food vendor’s educational status and proper hand washing practices. it is recommended that respective local authorities in collaboration with other stakeholders be more proactive in the enforcement of food safety regulations regarding hand hygiene and food handling. keywords: street-vended food, codex alimentarius, hand, contamination, food-borne diseases. 1. introduction street-vended food (svf) refers to a wide range of ready-to-eat-food, prepared and sold along streets and several other public places such as lorry stations, parks, schools, construction sites and all locations where there is a high number of potential customers. about 2.5 billion people worldwide patronize svf on daily basis [1]. in most developing countries including ghana, svfs are commonly and widely patronized by public/civil servants, school children, travelers/tourists, market women, petty traders, artisans, and the general public [2,3]. the street-vended food trade thus contributes significantly to food security and nutrition by providing comparatively inexpensive, and easily accessible tasty foods to millions of consumers. additionally, it provides vast employment opportunities in the private sector, thus guaranteeing a decent livelihood for millions of street-food vendors (sfvs).however, svf often pose significant public health risk to consumers. concerns about svf safety stem from the risk of faecal contamination of street food with the human hand as a conduit, potentially culminating in infection and outbreak of food borne diseases (fbds) and outcomes of death or personal injury to the consumer. as an endogenous risk factor, contaminated hand thus plays a major role in faeco-oral transmission through food [4,5]. the purpose of this study was to assess the nature and extent of hand hygiene practices in the street food business with the aim of adding on to existing knowledge in food hygiene and safety. benchmarks of the who codex http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 76 alimentarius for street-vended foods constitute the international standards for assessing food safety. who five keys to safer food relate to specific practices including washing the hands before handling food and during food preparation. others include washing the hands thoroughly with water and soap after visiting the toilet to prevent food contamination from faecal matter; washing the hands after handling raw food such as meat, changing baby’s diaper, blowing the nose and after handling rubbish, maintaining trimmed and clean fingernails and washing the hands thoroughly with water and soap before eating [6]. the individual street-food vendor, the consumer, and respective local authorities are all stakeholders in striving to achieve street-vended food safety [7]. surveys in africa, asia, and latin america suggest that in developing countries, ready-to-eat food sold along the streets and other public places, pose a significant public health burden to consumers. poor personal hygiene of street food vendors, especially with respect to hand washing and food handling practices is considered a major preventable ‘high-risk’ factor [8, 9, 10, 11, 12]. even in advanced countries, streetfood vendors contact-serve foods with bare hands [13]. in ghana, foods mainly prone to contamination are foods “handled excessively after cooking” and that the risk of “contamination was reduced where vendors sold food from the cooking pots” [14]. in ghana between 70% and 95% of food vendors wash their hands regularly in the process of vending [15,16,17]. this assertion is however contradicted by some recent studies in northern ghana [18] hence the justification for this study. a principal contamination risk factor of public health concern linked to food handling is the concurrent handling of food and money because of the presence of certain pathogens such as e. coli, s. aureus and salmonella spp. on banknotes or currencies [19-21].similarly, food handler nasal carriage of staphylococcus aureus is a potential food contaminant through nose picking[22]. on the other hand, fingernail hygiene practices differ from one geographical area to the other [23-25]. the tendency of food vendors to properly wash their hands also depends on access to improved water sources [26-28]. ultimately deficiencies in food safety and hygiene practices may be attributable to inadequate food regulation enforcement by the local authorities [29]. 2. materials and methods 2.1 study area the study was carried out in two selected districts of the central region of ghana, namely komenda edina eguafo abrem (keea) municipal district (figure 1) and the ajumako enyan essiam (aee) (figure 2) district between january and may 2015. the keea municipality is located along the coast of southern ghana. the keea, whose capital elmina, houses the st george castle, a unesco designated world heritage site, is a major tourist destination. notable socio-economic activities in the municipality are petty trading including food vending. on the other hand, the aee district with ajumako as its capital is located inland away from the coast. this district, predominantly rural, covers an area of about 521.3sq.km. dominating the district economy is agriculture and the sales sectors consisting of a large number of street-food vendors operating at various market centers and public places. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 77 figure 1: map of the keea municipal district. methods & sample size the main objective of this survey was to study the role of the hand of food vendor as a risk factor to food safety. a crosssectional survey of food vendors selling along streets and other public places was used. a study sample size of 413 food vendors was performed. in all a number of 266 food vendors were interviewed in the keea, and 147 in the aee district using a structured questionnaire. the selected food vendors were obtained from one-third of localities in each district randomly sampled from a sampling frame of localities for each district. the sampling frame for the keea municipality had 36 localities while that of the aee district had 45 localities. food vendors were mainly interviewed at low peak hours (inbetween breakfast, lunch and dinner hours) to minimize interruptions of interviews by customers. alongside the interview process was the observation of vendor food safety practices and behaviors. data analysis data entry and analysis were conducted using spss version 21 software. the validity and reliability of the data were enhanced by pre-testing the instruments and the constructs revised appropriately before data collection. averagely seven minutes were used in keying each questionnaire data into the spss data editor. 3. results and discussion 3.1 vendor socio-demographic profile overwhelming majority (97.3%) of street food vendors in the study area were females (table 1). the study thus confirms street-food vending as a major source of income and livelihood for middle-aged women in the private sector. additionally, table 2 reveals that majority of food vendors were illiterates. overall, 84.5 per cent of vendors had education below the senior high school level. whereas only 1.2 percent had tertiary education, 15.3 percent of vendors had had no formal schooling. majority (39.2%) of vendors also fell within the age bracket of 30 to 39 years with an arithmetic mean age of 34.98 years (table 2). it was also found that majority of food vendors (83.3%) plied their trade at a stationary public place whiles 16.7 per cent were mobile vendors usually called hawkers. this finding has a major implication for town and urban planning. in the main it was found that designated food vending areas lacked adequate sanitary facilities. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 78 table 1 socio-demographic profile of vendors keea aee total freq (266) % freq (147) % freq (413) % types of vendors stationary 198 74.4 146 99.3 344 83.3 mobile 68 25.6 1 0.7 69 16.7 sex male 9 3.4 2 1.4 11 2.7 female 257 96.6 145 98.6 402 97.3 education no schooling 42 15.8 21 14.3 63 15.3 primary 24 9 35 23.8 59 14.3 jhs middle 128 48.1 75 51 203 49.2 voc tech 11 4.1 5 3.4 16 3.9 shs 49 18.4 10 6.8 59 14.3 tertiary 4 1.5 1 0.7 5 1.2 arabic 8 3 0 0 8 1.9 table 2 age (years) distribution of street-food vendors n range minimum maximum mean std dev variance ages in years 413 50 15 65 34.98 8.958 80.138 as shown in table 3, majority of vendors in the study area (61.3%) said they washed their hands with water and soap before preparing food and during vending (even though this could not be verified). overall, over one-third of vendors (38.7 percent) conceded to not washing their hands with water and soap during the vending process while nearly half of vendors (47.9%) handled food with their bare hands in the vending process. also 81.6 percent of vendors intimated that they washed their hands with water and soap after visiting the toilet. this implies that overall nearly onefifth of vendors (18.4%) did not wash their hands with water and soap after visiting the toilet. table 3 also reveals that over onethird of food vendors (35.1%) were caring for children at the time of the study. and of all food vendors studied, 16.7 percent who were carriers of children said they handled food with their bare hands. these findings suggest that nearly one-fifth of vendors who changed used children dippers, handled vended food with bare hands thus making ‘child care’ an indirect risk factor to food handling and food safety. this makes food handling a key risk factor in food vending in the study area and brings to question the effectiveness of regulation enforcement practices. the length and cleanliness of vendor fingernails is also an important consideration in hand hygiene practices because safe hand washing practices includes keeping clean and trimmed fingernails. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 79 figure 2: map of ajumako enyan essiam district table 3 suggests that vendor fingernail hygiene was not a major risk factor in the study area. only 9 (2.2%) of food vendors were observed to wear long fingernails. the tendency for food vendors to properly wash their hands may depend on access to improved water sources. table 3 food vendor hand hygiene practices keea aee total freq (266) % freq (147) % freq (413) % fingernail hygiene long nails 2 0.8 7 4.8 9 2.2 short nails 264 99.2 140 95.2 404 97.8 handling of food bare hands 125 47.0 73 49.7 198 47.9 protected hand 141 53.0 74 50.3 215 52.1 vendor handwashing no hand washing 40 15.0 4 2.7 44 10.7 only water 35 13.2 79 53.7 114 27.6 muddy water 0 0 1 0.7 1 0.2 water and soap 191 71.8 62 42.2 253 61.3 wiping with cloth/napkin 0 0 1 0.7 1 0.2 handwashing after toilet don’t wash hands 3 1.1 0 0 3 0.7 wash hand with water 33 12.4 39 26.5 72 17.4 wash hands/ water & soap 230 86.5 107 72.8 337 81.6 bath before vending 0 0 1 0.7 1 0.2 child care yes 83 31.2 62 42.2 145 35.1 no 183 68.8 85 57.8 268 64.9 a chi-square hypothesis test (table 4) conducted shows that there is a significant positive relationship between food vendor educational status and hand washing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 80 practices in the study area. chi-square pearson’s correlation coefficient (r= 69.349, df= 20, p-value= 0.000. pvalue of 0.000 < statistical significance of 0.05, i.e. 5%). this finding signals the importance of education and schooling to food safety. significantly, access to potable water sources was not a major challenge. table 4 chi-square hypothesis testseducation status and hand washing practices value df asymp. sig. (2-sided) pearson chi-square 69.349 a 20 .000 likelihood ratio 52.210 20 .000 linear-by-linear association 12.972 1 .000 n of valid cases 413 4. conclusion hand hygiene is one of the most important strategies for infection control. proper hand washing is necessary because hands frequently transport microorganisms and could be a major source of food contamination. scrupulous hand-washing by food handlers results in the prevention of the spread of pathogenic microorganisms. the study reveals an unsatisfactory handwashing practices among food vendors. the findings therefore bring to question the effectiveness of food regulation enforcement practices. strengthening food safety programmes help to ensure protection of consumer health, reduce food-borne disease and mortality burden, and thereby increase human wellness and decrease poverty. 5. acknowledgements the researchers are very grateful to the management of central university, ghana, for sponsoring this research. the ghana health directorates and the environmental health offices in both study districts are equally acknowledged for their cooperation and support during data collection. 6. references [1]. food and agricultural organization of the united nations, west african cities in street food vending. potential and challenges. fao regional office for africa, brazzaville (2012). [online accessed on 10 june 2014 from http://www.fao.org/africa] [2]. haleegoah, j., ruivenkamp, g., essegbey, g., frempong, g., & jongerden, j., street-vended local food systems actors perceptions on safety in urban ghana: the case of hausa koko, waakye and ga kenkey. advances in applied sociology, 5: 134145, (2015). [3]. tortoe, c., johnson, p.n.t., ottah-atikpo, m. & tomlins, k. i. systematic approach for the management and control of food safety for the street/informal food sector in ghana. food and public health, 3(1):5967, (2012). [4]. who regional office for africa, initiative to estimate the global burden of foodborne diseases. who africa region office, brazzaville, (2014). [5]. nurudeen, a. a., lawal, a. o. and ajayi, s. a., a survey of hygiene and sanitary practices of street food vendors in the central state of northern nigeria. journal of public health and epidemiology, 6(5):174-181, (2014). [6]. who, five keys to safer foods manual. world health organization, department of food safety, zoonoses and foodborne diseases, (2006). http://www.fao.org/africa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 alexander fiifi ghartey & barima antwi, hand hygiene practices among street food vendors, food and environment safety, volume xviii, issue 2 – 2019, pag. 75 81 81 [7]. ahwoi, k. local government & decentralisation in ghana: unimax macmillan ltd, accra, ghana, (2010). [8]. who & unicef, progress on sanitation and drinking-water: 2010 update. geneva & london, (2010). [9]. muhonja, f. & kimathi, g. k., assessment of hygienic and food handling practices among street food vendors in nakuru town in kenya. science journal of public health, 2(6): 554-559, (2014). [10].faruque, q., haque, q.f., shekhar, h.u., & begum, s., institutionalization of healthy street food system in bangladesh: a pilot study with three wards of dhaka city corporation as a model, final report (2010). [11] muyanja, c., nayiga, l., brenda, n. & nasinyama, g., practices, knowledge and risk factors of street food vendors in uganda. food control journal, 22 (10):1551–1558, (2011). [12]. gawande h.a., mishra a.a., shukla r.n. & jyoti j., socio-economic profile of street food vendors and quality evaluation of samosa and panipuri in allahabad city, (up) india. international journal of agriculture and food science technology, 4(3): 275-280, (2013). [13]. burt, b. m., volel, c., & finkel, m., safety of vendor-prepared foods: evaluation of 10 processing mobile food vendors in manhattan. public health reports (2016). [14]. mensah,p., yeboah-manu, d, owusu-darko, k & ablordey, a., street foods in accra, ghana: how safe are they? geneva: bulletin of the world health organization, 80(7): 546-554, (2002). [15]. food and agricultural organization of the united nations, street food in urban ghana. a desktop review and analysis of findings and recommendations from existing literature, (2016). [16]. apanga, s., addah, j., & sey, d.r., food safety knowledge and practice of street food vendors in rural northern ghana, food and public health, 4(3):99-103, (2014). [17]. monney, i., agyei, d., & owusu, w., hygienic practices among food vendors in educational institutions in ghana: the case of konongo. food and public health, scientific and academic publishing, 4(6):306-315, (2013). [18]. dun-dery, e. j. & addo, h.o., food hygiene awareness, processing and practice among street food vendors in ghana. food and public health journal, 6(3): 65-74, (2016). [19]. angelakis, e., azhar, e. i., bibi f., yasir, m., al-ghamdi a. k., ashshi, a. m., et. a.l., paper money and coins as potential vectors of transmissible disease. future microbiology, 9(2): 249-261, (2014). [20]. luure, p., asare, w., cobbina, s. j., duwiejuah, a. b., & nkoom, m., microbial contamination of ghanaian cedi notes from traders of the tamale central market, ghana. british microbiology research journal, 5(2): 139, (2015). [21]. vriesekoop, f., russell, c., alvarez-mayorga, b., aidoo, k., yuan, q., scannell, a., et al., dirty money: an investigation into the hygiene status of some of the world's currencies as obtained from food outlets. foodborne pathogens and disease, 7(12): 14971502, (2010). [22]. el-shenawy, m.,el-hosseiny, l., tawfeek, m., el-shenawy, m., baghdadi, h., saleh, o., et. al., nasal carriage of enterotoxigenic staphylococcus aureus and risk factors among food handlers-egypt. food and public health, 3(6): 284-288, (2013). [23]. odonkor, s.t., adom t., boatin, r., bansa d., & odonkor, c.j. evaluation of hygiene practices among street food vendors in accra metropolis, ghana. elixir food science international journal 41: 5807-5811, (2011). [24]. iwu, a.c., uwakwe, k.a., duru, c.b., diwe, k.c., chineke, h.n., merenu, i.a., oluoha, u.r., madubueze, u.c., ndukwu, e. & ohale, i., knowledge, attitude and practices of food hygiene among food vendors in owerri, imo state, nigeria. occupational diseases and environmental medicine, 5:11-25, (2017). [25]. aluko, o.o, ojeremi, t.t., olaleke, d.a., ajidagba, e.b., evaluation of food safety and sanitary practices among food vendors at car parks in ile ife, southwestern nigeria. food control, (40): 165–171, (2014). [26]. ghana statistical service 2010 population & housing census. national analytical report, (2013). [27]. ghana statistical service 2010 population & housing census. summary report of final results (2012), p. 96. [28]. donkor, e.s., kayang, b.b., quaye, j. & akyeh, m.l., application of the who keys of safer food to improve food handling practices of food vendors in a poor resource community (chorkor) in ghana. international journal of environmental research and public health, 6(11): 2833–2842, (2009). [29]. annan-prah a, amewowor dh, osei-kofi j, amoono se, akorli sy, saka e, ndadi ha. street foods: handling, hygiene and client expectations in a world heritage site town, cape coast, ghana. african journal of microbiology research, 5(13):1629-1634, (2011). http://www.sciencedirect.com/science/article/pii/s095671351100034x http://www.sciencedirect.com/science/article/pii/s095671351100034x http://www.sciencedirect.com/science/article/pii/s095671351100034x http://www.sciencedirect.com/science/article/pii/s095671351100034x http://www.sciencedirect.com/science/journal/09567135 http://www.sciencedirect.com/science/journal/09567135 http://www.sciencedirect.com/science/journal/09567135/22/10 http://www.sciencedirect.com/science/article/pii/s0956713513006312 http://www.sciencedirect.com/science/article/pii/s0956713513006312 http://www.sciencedirect.com/science/article/pii/s0956713513006312 http://www.sciencedirect.com/science/journal/09567135 http://www.sciencedirect.com/science/journal/09567135 http://www.sciencedirect.com/science/journal/09567135/40/supp/c http://www.ncbi.nlm.nih.gov/pubmed/?term=kayang%20bb%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=quaye%20j%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=akyeh%20ml%5bauth%5d 1. introduction microsoft word 3 elutade_corectat 27 june.doc 131 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 131 140 cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria *olayinka o. elutade1, olubukola m. oyawoye2, ediga b. agbo3 1faculty of sciences, bowen university, iwo, nigeria, olayinkaelutade@yahoo.com 2faculty of science, adeleke university, ede, nigeria, oyawoyeom@yahoo.com 3school of science, abubakar tafawa balewa university, bauchi, nigeria, ediga_agbo@yahoo.com *corresponding author received 18th december 2017, accepted 27th june 2018 abstract: this work reports the comparison in the physical properties of tiger nut tubers (cyperus esculentus), a crop extensively cultivated in northern nigeria as well as in the biochemical changes which occur during the spontaneous fermentation of the soaked tubers in water for tiger nut beverage production. both the brown and yellow tuber varieties of c. esculentus were used in this study. for each variety, the length and width of 100 randomly selected tubers alongside the weight of 1000 tubers were determined. furthermore, 10g of each variety was steeped in sterile distilled water for periods of time that ranged from 0-96 hours in order to highlight the bacterial and fungal diversities occuring among the tubers. results of the physical properties revealed that the yellow variety tubers were highly significantly longer in length, bigger in size, have higher length/width ratio, and heavier in weight than the brown variety tubers. when steeped in water, the mean range count of log 2.763 – 10.20 cfu/g for lactic acid bacteria and log 2.073 – 7.73 for yeast/mold count in the brown tubers were significantly higher than the range of log 2.51 -6.14 and 1.56 – 8.17 obtained for the same parameters, respectively, in the yellow tubers. the dominant microorganisms during the steeping process, regardless of the variety, included bacillus species, gram-negative group enterobacteriaceae, as well as yeasts, moulds, and acetic acid bacteria. these array of microorganisms are for assessment and proper selection of desirable strains, as potential starter cultures, towards tiger nut beverage production. keywords: tigernuts, brown variety, yellow variety, tuber length/width, lactic acid bacteria, microbial profile 1. introduction cyperus esculentus is an erect perennial weed plant which occurs in the tropics, subtropics, mediterranean and warm temperate regions [1,2] and produces edible tubers. in nigeria, the tubers are commonly known as “ayaya” in hausa, “ofio” in yoruba, and “akiausa” in igbo and are cultivated extensively in the northern part of the country; three varieties (black, brown and yellow) are cultivated and of these, only two varieties, yellow and brown, are readily available in the local market [3]. the tubers are widely consumed raw or unprocessed. they could be dried, mixed with groundnut or soaked in water for varied time-lengths of about 3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 132 days [4]. they are particularly savoured for the production of non-alcoholic dairylike beverage with a worldwide acceptance and which is non-indigenous to nigeria. in spain, the beverage is known as “horchata de chufa” particularly in the valencia region [5]. in recent times, the beverage has increasingly become popular in many local social functions as substitute to industrially-produced conventional carbonated drinks, perhaps probably due to its low price, and wide acceptance by the people. traditionally, in nigeria, production of the consumed beverage extracts is by spontaneous fermentation. the extracts are prepared by steeping the tubers in water for different periods of time. this may range from a few hours to a full day; or for about three to four days, after which the harvested tubers could be consumed directly or sold in the public or hawked as delicacies. information is not available on the effects of periods of steeping in water in terms of the microbial diversity and biochemical properties of the resultant extracts. such information is therefore necessary in the quest to transform the local process into a conventional technological process. this research aims to determine and compare the physical characteristics of the two varieties of tiger nut tubers, locally available in the market, and the biochemical changes produced during the natural fermentation of the steeped tubers 2. materials and methods 2.1. collection of samples: the tiger nuts used in the study were the yellow and brown varieties, identified locally as the ‘big’ and ‘small’ varieties. these were obtained directly from local markets in biliri (gombe state) and labeled “bbf” and “vsf,” respectively. a weight of five kilogrammes (5kg) of each variety was purchased. these were brought into the laboratory, and were separately sorted as described by [6], in order to remove foreign materials, broken and damaged tubers. the selected tubers were then used in every experimental procedure in the course of the study. 2.2. physical properties of tubers 2.2.1. estimation of the length and width of tubers a vernier caliper was used to measure the length and width of 100 randomly selected tubers from each variety [7]. the obtained values were then appropriately noted and recorded. 2.2.2. weight of 1000 tubers the weights of randomly selected 1000 tubers were measured collectively in grammes using a pre-weighed large beaker and the obtained values recorded [7]. this procedure was repeated in six replicates for each variety to measure the quality of the tubers in terms of filling. 2.3. isolation of microbial species associated with soaked tiger nut tubers the microbial analytical methods described by [8] and [9] were utilized for the study. a weight of 10g of each variety of tiger nut tubers was thoroughly washed in three changes of sterile distilled water. these were then steeped in 90ml of sterile distilled water, contained in plastic bowls with covers, to make 10-1 dilution, and the bowls covered. at regular intervals, that is, 0 hour, 24 hours, 48 hours, 72 hours, and 96 hours, 1ml of the steeping water was aseptically serially diluted and plated using the pour-plate technique on solid medium. based on the results of some preliminary trial experiments, diluents values 10-1, 10-3, 10-5, 10-7, and 10-9 of the steeping water were pour-plated on solid medium at 0 hour, 24 hours, 48 hours, 72 hours, and 96 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 133 hours, respectively. the experiments were triplicated. the solid media used for enumeration were nutrient agar, for aerobic colony count; eosin methylene blue agar, for enterobacteriaceace count; mann rogosa sharpe agar for lactic acid bacterial count; and potato dextrose agar, for yeast and mould count. inoculation into each media plate was done in three replicates, except for the lactic bacterial count where dual replicates were used. the plates for bacterial isolation were incubated at 37°c for 24 hours while plates for fungi were incubated at 25°c for 48 hours. the viable cells obtained on the inoculated media plates after incubation were counted using digital colony counter (gallenkemp, england). the average colony counts obtained were expressed as colony forming units per gram (cfu/g). pure bacterial and yeasts cultures obtained from the study were identified to conventional biochemical identification keys. moulds were identified by [10] methods. 2.4. statistical analyses the various experimental data obtained were subjected to statistical analysis using the genstat release 7.22 de, version 2008 (vsn, international limited). the unpaired students’ t-test was used to analyze the physical properties of tiger nut tubers. all other analyses were by two-way analysis of variance (anova). probability values (p ≤ 0.05) were considered as statistically significant. 3. results and discussion 3.1. physical properties the yellow tubers were highly significantly longer in length, bigger in size, have higher length/width ratio, and heavier in weight than the brown coloured tubers (figure 1). several authors, [11-13] have reported the yellow variety is preferred to all the other varieties in nigeria, because of its inherent properties like its bigger size, attractive colour and fleshier body. these attributes may have been responsible for this variety’s preference by the consumers. the length, length/width ratio, and unit weight of tiger nut tuber are as high heritable traits in tiger nut plants and soil texture has significant effect on the average tuber size [14]. tubers grown in sandy soil have been significantly observed to be larger in diameter than those grown in clay [15, 14]. it is worth noting that the ratio values of length and length/width recorded for both the brown and yellow tuber were 0.68±0.10cm, 0.96±0.15cm and 1.17±0.13, 1.03±0.11, respectively. these values are different from the tubers of ‘gegant africana,’ ‘liagueta alboraia’ and ‘ametilla bonrepos’ cultivars, selectively cultivated in spain, and described by [14] to have a length and length/width ratio values of 1.73cm, 1.62; 1.52cm, 1.68; and 1.24cm, 1.16, respectively. 3.2 microbial species and ph changes associated with soaked tiger nut tubers the aerobic colony count on nutrient agar of tiger nut tubers soaked in water at ambient temperature is shown in figure 2. generally, aerobic plate count in the yellow and brown tubers increased with time and varied between 2.647 to 9.54 log cfu/g, and 2.53 to 8.933 log cfu/g, respectively (figure 2a). statistically, the mean values obtained at each of the sampled periods were significantly different in the yellow tubers; whereas in the brown tubers, the mean values were significantly different at 0 hour, 24 hours, 48 hours and 72 hours. the enterobacteriaceace count on eosin methylene blue (emb) agar of tubers soaked in water at ambient temperature is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 134 key: vsf-brown variety bbfyellow variety fig. 1: physical properties of tiger nut tubers* (a) and weight of 1000th tubers÷ *each parameter is a mean of 100 replicates ÷mean of six replicates shown in figure 2b. generally, the enterobacteriaceace counts in the yellow and brown tubers increased with steeping time and varied between 2.357 to 9.40 log cfu/g, and 2.257 to 8.673 log cfu/g, respectively. the mean values obtained at each of the sampled periods were significantly different in the yellow tubers; whereas in the brown tubers, the mean values were highly significantly different at 0 hour, 24 hours, 48 hours and 72 hours. the fungal and yeasts, on potato dextrose agar (pda), from tubers soaked in water at ambient temperature is shown in figure 2c. generally, the yeast and mold counts increased with steeping time, and the mean counts in the yellow and brown tubers ranged from 1.5667 to 8.1733 log cfu/g, and 2.0733 to 7.7367 log cfu/g, respectively. at the onset of the soaking process (0 hour), the yeast and mold counts from the brown tubers (2.0733 log cfu/g) were higher than the value (1.5667 log cfu/g) obtained in the yellow. statistically, the mean fungal counts recorded at each of the sampling points, in the different varieties, were significant key: vsf-brown variety; bbfyellow variety fig. 2: aerobic colony counts (a), enterobacteriaceace counts (b), yeasts and mould count (c) and lactic acid bacterial counts (d) from varieties of tiger nut tubers steeped in water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 135 the mean lactic acid bacterial (lab) count in the soaked yellow and brown tubers is expressed in figure 2d. mean counts ranging from 2.510 to 6.143 log cfu/g and 2.763 to 10.205 log cfu/g in the yellow and brown tubers, respectively, were obtained. the counts obtained increased concomitantly with time. statistically, the mean lab counts obtained at each of the sampling periods in the different tubers were significant. the change in ph, during the soaking of tiger nut tubes in water at ambient temperature, is shown in figure 3. the ph fell from 6.86 4.73 and 7.13 – 4.82 within 72 hours of soaking in the yellow and brown tubers, respectively. within 24 hours of soaking, there was a steep fall from 7.14 – 5.40 in the brown tubers, and a gradual fall from 6.86 – 5.69 in the yellow tubers. between 72 hours and 96 hours of soaking, the two types of tubers experienced a further fall in ph values, i.e. 4.13 and 4.30 in yellow and brown tubers, respectively. cyperus esculentus tubers are underground stems which are harvested when ploughed from the soil. bacteria have been reported to be the most dominant group of microorganisms in soils, while fungi are more numerous in the surface areas of well-aerated, cultivated soils [16]. it is also envisaged that the field-soil or storage conditions, or the combination of both, may have been responsible for the higher yeast and fungal count in the brown than yellow tuber, observed at the onset (0 hour) of soaking in water. key: vsf-brown variety bbfyellow variety fig. 3: changes in ph during soaking of tiger nut tubers in water at ambient temperature the identities and distribution of microorganisms obtained from tiger nut tubers soaked in water is shown in table 1. bacillus species, lactobacillus plantarum, non-lactose fermenters such as serratia marscens and providencia rettgeri and the lactose-fermentors, like. klebsiella pneumonia, were the dormant bacteria isolated from the tubers at the onset of the soaking process. these mentioned organisms are all bacilli (rod-shaped). [16] have reported that bacilli are very common in soils, and some of them like bacillus species can persist in unfavourable conditions via formation of resistance structures called endospores which can withstand prolonged desiccation and high temperature. similarly, aerobic epiphytic microflora found present at the start of fermentation on raw plant material, have been known to include bacillus species, pseudomonadaceae, fungi, enterobacteriaceae, lactic acid bacteria, and yeasts [17]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 136 furthermore, all microorganisms of the bacillus species are known to generate fermentable reducing sugars from raw plant materials via their ability to synthesize extracellular alpha-amylase enzymes that degrade starch molecules [18, 19] into products which are likely to stimulate the growth of other organisms such as lactic acid bacteria. [20] and [17] have explained that most lactic acid bacteria lack the ability to hydrolyse starch or are deficient in the use of amylolytic enzymes; consequently, the generation of fermentable carbohydrates by the bacillus species helps to ensure the initiation of lactic acid fermentation. bacillus species are also associated with the fermentation of cereals such as for ‘burukutu’ and ‘ogi’, and during the soaking of cassava tubers in water for the production of ‘lafun’ [21, 22]. the lactic acid bacterium, lactobacillus plantarum, was persistently commonly isolated from the two varieties of tubers throughout the period of soaking. since, it was isolated from the onset of the soaking process; it may belong to the epiphytic microflora group of these tubers. l. plantarum has also been isolated as part of the microflora in ‘kocho’, a fermented, acidic, starchy, and staple food in ethiopia; prepared from the plant, ensette (ensette ventricosum) [23]. similarly, [24] have isolated l. plantarum during the retting of cassava tubers in water, in the traditional production of the fermented food ‘fufu’ (cassava flour), popularly consumed in the west african sub-region. l. plantarum, l. acidophilus and l. fermentum come from the environment, and are associated with plant material [25]. l. plantarum and l. fermentum are heterofermenters which produce equimolar amounts of lactic acid, ethanol/acetic acid, and carbon dioxide from glucose [26]. heterofermentative lactic acid bacteria contribute to flavor development [17]. l. acidophilus is a homofermenter which produces only lactic acid from fermentable carbohydrates [17]. lactic acid bacteria are gram-positive organisms which grow readily in most food substrates and can lower the ph rapidly to a point where competing organisms are no longer able to grow [27]. hence, the rapid fall in ph from 7.10 – 5.50 in the brown tubers and 6.90 – 6.20 in the yellow tubers, within 24 hours of soaking in water, may be attributable to the metabolic activities of lactic acid bacteria. lactic acid bacteria are important microorganisms for biotechnological applications the spectrum of enterobacteriaceae lactose-fermentor organisms obtained from yellow and brown tubers during the soaking process were the same, and these organisms were klebsiella pneumonia, k. oxytoca, enterobacter aerogenes, e. cloacae and citrobacter diversus, (table 1). the increased dominance in the isolation of these organisms, particularly observed from 24 hours of sampling, could have stemmed from the conducive nearneutral ph of 6.20 and 5.50 observed in the yellow and brown tubers, respectively, during the soaking process. in [17] it have reported that members of enterobacteriaceae, particularly enterobacter and klebsiella species dominate the early stages of fermentation because of their high specific growth rates at neutral ph. klebsiella species are known to contribute to the decay of cassava roots during the traditional submerged fermentation of cassava to ‘fufu’ and ‘lafun’ production [22]. it is envisaged that the klebsiella species, along with the other lactose-fermenting organisms obtained in this study would have performed similar role that is of decaying of tubers. table 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 137 identity and distribution of microorganisms isolated from tiger nut tubers steeped in water fermentation hours number of _________________________________ species identified isolates 0 24 48 72 96 yellow variety bacillus substilis 2 1 1 0 0 0 b. megaterium 1 1 0 0 0 0 lactobacillus plantarum 9 3 2 2 1 1 l. acidophilus 3 0 1 0 0 2 l. fermentum 3 0 0 1 2 0 klebsiella pneumoniae 7 2 2 2 0 1 serratia marscens 1 1 0 0 0 0 pseudomonas aeruginosa 1 1 0 0 0 0 enterobacter aerogenes 1 0 1 0 0 0 e. cloacae 1 0 1 0 0 0 acetobacter species 5 0 0 2 2 1 citrobacter diversus 2 0 0 1 1 0 klebsiella oxytoca 2 0 0 0 1 1 acinetobacter species 3 0 0 0 1 2 aspergillus flavus 5 1 1 1 1 1 aspergillus niger 5 1 1 1 1 1 aspergillus species 5 1 1 1 1 1 fusarium species 5 1 1 1 1 1 candida krusei 5 1 1 1 1 1 candida glabrata 1 0 0 0 0 1 candida zeylanoides 2 1 1 0 0 0 saccharomyces cerevisae 4 0 1 1 1 1 rhodotorula mucilaginosa 2 0 0 1 1 0 cryptococcus laurenti 1 1 0 0 0 0 total 76 16 15 15 15 15 brown variety bacillus substilis 1 1 0 0 0 0 lactobacillus plantarum 12 3 3 2 2 2 l. acidophilus 3 0 0 1 1 1 klebsiella pneumoniae 10 2 3 2 2 1 providencia rettgeri 1 1 0 0 0 0 serratia marscens 1 1 0 0 0 0 enterobacter aerogenes 2 0 1 1 0 0 citrobacter diversus 3 0 1 1 1 0 acetobacter species 2 0 0 1 0 1 enterobacter cloacae 1 0 0 1 0 0 klebsiella oxytoca 1 0 0 0 1 0 acinetobacter species 3 0 0 0 1 2 aspergillus flavus 5 1 1 1 1 1 aspergillus niger 5 1 1 1 1 1 aspergillus species 5 1 1 1 1 1 candida krusei 5 1 1 1 1 1 candida glabrata 1 0 0 0 1 0 candida zeylanoides 3 2 1 0 0 0 saccharomyces cerevisae 4 0 1 1 1 1 rhodotorula mucilaginosa 2 0 0 1 0 1 total 70 14 14 15 14 13 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 138 enterobacteriaceae count obtained from the two varieties of tubers, as soaking time progressed, may be attributable to favourable conditions, such as the nearneutral ph and availability of suitable substrates, for microbial activity. the acetic acid bacteria, acetinobacter and acetobacter species were the dominant microorganisms that were increasingly isolated from 48 hours of soaking the yellow and brown tubers in water. acetic acid bacteria, as reported by [28] and [29], belong to the family acetobacteriaceae, and are obligate aerobic gram-negative organisms. they are widely distributed in the natural habitat and have been isolated from alcoholic beverages, vinegar, fruits, flowers, honey, bees, sugar cane, juices, soil and water [30]. the observation that the isolated acetic acid bacterium became increasingly obvious only after 48 hours of soaking, suggest that this bacterium could have resulted from the accumulates of metabolites in the steeping water. [31]) and [32] reported that acetic acid bacteria can use a variety of substrates such as glucose, ethanol, lactic acid or glycerol as energy sources. acetic acid bacteria are known to play a fundamental role in the development of chocolate flavor precursors in the spontaneous fermentation of cocoa beans [9]. the dominance of acetic acid bacteria population in cocoa fermented in lohdjibouna, cote d’ivoire, has been reported by [33]. the yeast species candida krusei, c. zeylanoides, cryptococcus laurenti and c. krusei, c. zeylanoides isolated from yellow and brown tubers, respectively, at the onset (0 hour) of the soaking process probably belong to the epiphytic flora of the tubers (table 1). in the spontaneous fermentation of cocoa beans, in the dominican republic, [9] reported the presence of candida zeylanoides among the yeast species isolated, and which produced pectinolytic enzymes that resulted in mucilage breakdown in the cocoa beans, and hence, release of fermentable carbohydrate substrates. it may probably be that the yeast species forming the epiphytic flora on the tiger nut tubers in this study could have probably produced similar pectinolytic activity. nevertheless, majority of epiphytic yeasts in plant fermented foods are known not to ferment sugar, but rather use pectin as a carbon source [34, 17]. yeast species of sacchromyces cerevisae was continuously isolated from 24 hours until the end of the fermentation study (96 hours) in both varieties of tubers. in pickled vegetable, s. cerevisae is regarded as fermentable yeast because it is able to remove fermentable substrates, and consequently, preserve the pickled vegetable [17]. similarly, candida glabrata and rhodotorula mucilaginosa were among the dominant yeast isolates obtained from the varieties of tubers between 24 hours and 96 hours of fermentation in water. rhodotorula species have also been associated with fermenting ‘kocho’, a staple acidic, starchy food in ethiopia, though the role of these yeast species in the fermentation process, was not be specified [23]. the study [17] reported the existence of two major groups of yeast, based on their roles in naturally fermented products of plant origin; fermentative yeasts and oxidative yeasts. the authors explained that fermentative yeasts metabolize fermentable substrates, and are important in preservation, while oxidative yeasts are associated with deterioration via breakdown of organic acids, which lead to increase in ph and growth of deteriogens, acid-sensitive organisms. the role of the yeast species obtained in the present study was not assessed. generally, yeast species ferment food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 139 simple sugars to ethanol and carbon dioxide [9]. 4. conclusion in nigeria, the yellow tubers of c. esculentus significantly differ from the brown variety in length, width, length/width ratio and weight. during steeping, changes in apc, enterobacteriaceae count and yeast and mould count were significantly higher in the yellow tuber variety than in the brown one. nevertheless, the dominant microbial species associated with the natural fermentation of these tubers are similar, and present potentials of being used as starter cultures for not only the commercial production of the beverage, but perhaps also in the formulation and development of derived fermented beverages. 5. acknowledgements: we are grateful to the laboratory staff in bowen university, iwo and abubakar tafawa balewa university, bauchi, where this research work was carried out. 6. references [1] kay d., crop and product digest number 2 root crops, 2nd edition tropical development and research institute, new zealand. pp 308, (1987) [2] djomdi r., ndjouenkeu r., soaking behavior and milky extraction performance of tigernut (cyperus esculentus) tubers journal of food engineering 78: 546-550, (2007) [3] oladele a., aina j., chemical composition and functional properties of flour produced from two varieties of tigernut (cyperus esculentus) african journal of biotechnology 6 (21): 2473-2476, (2007) [4] temple v., ojobe t., kapu m., chemical analysis of tigernut (cyperus esculentus) journal of the science of food and agriculture 50 (2): 261-263, (1990) [5] pascual b., muroto v., lopezgulama s., sanbautista a., alagarda j., lopez g,. morphological and productive characteristics of nine “chufa” (cyperus esculentus l-var sartivus boeck) clones. acta-horticulturae 1 (614): 85-88, [2003] [6] turhan m., sayar s., gunasekaran s., application of peleg model to study water absorption in chickpea during soaking. journal of food engineering 53: pp 153-159, [2002] [7] maziya-dixon b., king j., okooruwa a., physical, chemical and water absorption characteristics of tropical maize hybrids african crop science journal 8 (4): 419-428, [2000] [8] badau m., microorganisms associated with pearl millet cultivars at various malting stages. inter journal of food safety 8: 66-72, [2006] [9] lagunes g., loiseau g., paredes j., barel m., guiraud j., study of the microflora and biochemistry of cocoa fermentation in the dominican republic. international journal of food microbiology 114: 124-130, [2007] [10] brown e., benson’s microbiology applications, 9th edition. mc graw-hill, new york. pp 57-62, 249263, (2005) [11] okafor j., mordi j., ozumba a., solomon h., olatunji o., preliminary studies on the characterization of contaminants in tiger nuts (yellow variety). in: proceedings of 27th annual nigerian institute of food science and technology (nifst) conference. 13-17 october, 2003. pp 210-211, [2003] [12] belewu m., abodunrin o., preparation of kunu from unexploited rich food; tigernut (cyperus esculentus) world journal of diary and food sciences 1: 19-21, [2006] [13] adejuyitan j., otunola e., akande e., bolarinwa i., oladokun f., some physiochemical properties of flour obtained from the fermentation of tigernut (cyperus esculentus) sourced from a market in ogbomosho, nigeria african journal of food science 3 (2): 51-55, [2009] [14] pascual b., maroto v., lopezgulama s, sanbautista a., alagarda j., chufa (cyperus esculentus l-var sartivus boeck): an unconventional crop, studies related to applications and cultivation economic botany 54 (4): 439-448, [2000] [15] pascual b., maroto j., influencia de la textura en la cuantia y calidad de los tuberculos de las chufas (cyperus esculentus l.). agricultura 592:873-875, [1981] [16] rangaswami g., bagyaraj d., agricultural microbiology 2nd edition prentice-hall of india private limited, new dehli, india. pp 205208, [2004] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olayinka o. elutade, olubukola m. oyawoye, ediga b. agbo, cyperus esculentus: physical properties of tubers and microbiological changes during natural fermentation in nigeria, food and environment safety, volume xvii, issue 2 – 2018, pag. 131 – 140 140 [17] nout m., rombouts f., fermentative preservation of plant foods journal of applied bacteriology symposium supplement 73: 136s147s, [1992] [18] njoku h., okemadu c., biochemical changes during the natural fermentation of the african oil bean (pentaclethra macrophylla) for the production of ugba. journal of the science of food and agriculture 49: 457-465, [1989] [19] eliana de oliveira s., merie-leslis l.,. effect of medium composition on formation of amylase by bacillus species brazilian archives of biology and technology 46: 129-134, [2003] [20] santos e., martins m., effect of medium composition on formation of amylase by bacillus species brazilian archives of biology and technology 46 (1) : 129-134, [2003] [21] achi o., microbiology of ‘obiolor’ a nigerian fermented non-alcoholic beverage journal of applied bacteriology 69: 321-325, [1990] [22] oyewole b., cassava processing in africa. in national research council applications of biotechnology to traditional fermented foods, national academy press, washington d.c., usa, pp 8992, [1992] [23] gashe b., kocho fermentation journal of applied bacteriology 62: 473-477, [1987] [24] brauman a., keleke s., malonga m., miambi e., ampe f., microbiological and biochemical characterization of cassava retting, a traditional lactic acid fermentation for foo-foo (cassava flour) production applied and environmental microbiology 62 (8): 2854-2858, [1996] [25] nout m., sakar p., lactic acid fermentation in tropical climates antonie van leeuwenhoek 76: 395-401, [1999] [26] adams m., moss m., food microbiology the royal society of chemistry, cambridge, uk. pp 316-317 [2008] [27] steinkraus k., lactic acid fermentations in: applications of biotechnology to traditional fermented foods, national research council, national academy press, washington d.c., usa, pp 4351, [1992] [28} matsushita k., toyama h., adachi o., respiratory chains and biogenetics of acetic acid bacteria; in rose and ah (ed.), advances in microbial physiology academic press, pp 247-301, [1994] [29] adachi o., moonmangmee d., toyama h., yamada m., shinagawa e., matsushita k., new developments in oxidative fermentation applied microbiol. biotechnol. 60: 643-653, [2003] [30] yamada y., hosono r., lisdyanti p., widyastuti y., saono s., uchimura t. and komagata k., identification of acetic acid bacteria isolated from indonesian sources, especially of isolates classified in the genus glucobacter, journal gen. appl. microbiology 45: 23-28, [1999] [31] de ley j., gillis m., swings j., bergey’s manual of systematic bacteriology, williams and wilkins baltimore, baltimore pp 267-278, [1984] [32] rasooli i., sharafi s., beheshtimaal k., isolation, characterization and optimization of indigenous acetic acid bacteria and evaluation of their preservation methods iranian journal of microbiology 2 (1): 41-48, [2010] [33] kousame l., doue g., adom n., ouattara h., niamke s., biochemical characterization of microbial populations involved in loh-djiboua cocoa’s fermentation in cote d’ivoire food fermentation and safety 14 (2): 196-205, [2015] [34] dobolyi c., kecskes m., ecophysiological characteristics of the epiphytic yeast populations of some agricultural plants zentralblatt fur mikrobiologie 145: 334, (1990) issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvii, issue 3 30 september 2018 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 130 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2 2019, pag. 130 135 study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant ofelia arvinte 1 , *liliana norocel 1 , sonia amariei 1 1faculty of food engineering, stefan cel mareuniversity, suceava, romania, liliana.norocel@fia.usv.ro, *corresponding author received 15 may 2019, accepted 28 june 2019 abstract: the aim of this study is to establish the antioxidant capacity and phenolic content of one of the most popular medicinal plants in bucovina area of romania, named volovatic or mountain volovatic (carduus glaucinus), a plant of the asteraceae family, carduus genus, defloratus carduus species, carduus glaucinus (glaucus) subspecies, less studied but widely used in the area of origin. it was determined volovatic antioxidant potential, respectively polyphenol content and antioxidant activity, contents of metals and metalloids from the plant in order to assess a possible toxic effect. keywords: volovatic, polyphenol content, dpph, heavy metals 1. introduction medicinal plants constitute one of the main sources of dietary supplements, phytochemicals and pro-vitamins very important in maintaining good health. most medicinal plants are used in the form of teas. the role of herbal tea in disease prevention and cure is due mostly to antioxidant properties of their constituents [1]. although knowledge and literature about herbs are very vast, resources are not exhausted. in the mountainous area of suceava county there are still plants used for therapeutic purposes by the native population, plants that are not at all or sporadically occurring references related to their therapeutic properties, respectively the content in active principles. in the dorna area, volovatic (carduus glaucinus) flowers are used from ancestral herds to the preparation of liver tea [2, 3]. tea is bitter, which confirms the existence of bitter substances that might be bitter or not. carduus glaucinus is a perennial herbaceous plant in which renewal buds appear under the layer of dead leaves of basal rosette [4]. belongs to the genus thistle, although no spikes. taxonomic classification and botanical characterization of the volovatic is the following: kingdom – plantae, phylum – spermatophyta, class dicotoledonatae, order -asteridae/ compositae, familyasteraceae, gender-carduus, species carduus glucinus/carduus defloratus. the stem is simple, high 30-50 cm, up to 80 cm, without leaves and covered with bristles. the leaves are lanceolate to top undivided. they form a basal rosette, and if they are arranged on the stem, they alternate. the upper part of the leaves is milky; the bottom is covered with hairs [5]. the tubular flowers, purple, hermaphrodite form a chapel surrounded by sculptors. it blooms from june (july in the mountain area) until september. flowers are pollinated by insects. the fruit is an achene clear ovoid with a white doll made of hairs [5]. finally, in place of the flowers, the crook is made up of a tusk of white hairs like silver. under each puff there is a nacelle, and inside a chicken of the plant. the wind http://www.fia.usv.ro/fiajournal mailto:liliana.norocel@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ofelia arvinte, liliana norocel, sonia amariei, study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant, food and environment safety, volume xviii, issue 2 – 2019, pag. 130 – 135 131 is blowing, the puffs are loosening, the sun lounges are stretched, and the baby is wrapped in the diapers [6]. these are an alpine species, which appear from the boreal to the subalpine. these love light, are not pretentious with regard to temperature, hears well the dryness and acidity of the mountain coast [7]. although the sub-mountain hills and the coasts of the dornelor depression are full of blooming shrimps in the summer, volovatic is a rare plant. it is mentioned in "the flora and vegetation of moldova" as appearing in suceava county and rarely in neamt county [2]. it is not mentioned in the flora of other mountain areas (retezat, ciucaş, bucegi) [8-10]. other species of carduus (thistles) appear, such as: c. candicans, c. kerneri, c. viridis, c. achantoides, c. personata, în bucegi mountains [9]. perhaps because volovatic is a little widespread plant on the territory of romania, and also quite rarely encountered in the world, there are few studies that remind it. besides the fact that the inflorescences of this plant are used by the local population to prepare the tea for the regeneration and proper functioning of the liver, in the carduus genus of which it is part, we find a number of representatives recognized for their sanogenic effects but which do not grow in the mountainous area of suceava county: carduus marianus, carduus benedicta, carduus nutans, silybum marianum, armurariu [11]. in view of the likely local ancestral use of volovace in combating indigestion/liver problems and the fact that many of its close relatives have demonstrated various beneficial actions on the liver, it is very necessary and challenging to research and find the active principles it contains as well their applicability to phytotherapy. taking into account these premises, the antioxidant potential of the volovate, namely the content of polyphenols and the antioxidant activity, as well as the mineral content, was determined. particularly the presence of heavy metals and their content was compared with the maximum admissible limits. 2. materials and methods materials harvested plants were used from the commune of coşna, suceava county, from two neighboring areas, located at 47,378558 latitude and 25,171808 long, stereo gps coordinates 70 x = 513111,239m, y = 653287,138 m respectively 47,375816 and 25,175646 long, stereo coordinates 70: x = 513349,851 m, y = 6256978,629m and at about 860m altitude. for the determinations, aerial parts of plants were harvested during the flowering and naturally dried, shade, and fresh roots, autumn harvested at the end of the vegetation period. reagents. all reagents were of analytical grade and were purchased from sigma aldrich (germany). methods the moisture (w) was determined by weighing at the analytical balance of samples from all parts of the plant, dried in the oven until the mass remained constant using zrd-a5055 oven (zhicheng, china). w% = (msample – mdried sample)*100/msample where: msample sample mass before drying mdried samplesample mass after drying the ash was obtained by calcining at 8000c the samples from all parts of the plant using a nabertherm le 2/11/r6 muffle furnace. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ofelia arvinte, liliana norocel, sonia amariei, study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant, food and environment safety, volume xviii, issue 2 – 2019, pag. 130 – 135 132 determination of antioxidant capacity was achieved by the dpph (2.2 -diphenyl1-picrylhydrazyl) method. the methanolic solution of dpph has a maximum absorption at the wavelength of 517 nm due to the unpaired electron. in the presence of an antioxidant, the electron is mated to form the discoloured dpph-h form, the discoloration being directly proportional to the number of electrons captured. the determinations were carried out for the aqueous extract: 5 g sample to 200 ml water, 30 min boiled extract and alcohol extract: 1 g sample to 50 ml ethanol obtained in an ultrasonic bath at 40°c and 25 khz frequency from the aerial parts of the plant: leaves, flowers, stem. the samples of 0.5 ml were mixed with 2.5 ml of dpph solution, 6 x 10-5 m. the absorbance was measured with 3600 schimadzu uv-vis-nir spectrophotometer. the percent inhibition of free radicals, calculated by comparison with dpph, was calculated using the formula: i% = a0 – ap/a0 x100 (1) where: a0 = the absorbance of the reference substance, ap = absorbance of the analysed sample. determination of the polyphenol content was achieved by the folin-ciocalteu method. as aromatic chemical compounds as several hydroxyl groups inserted on the aromatic ring, the polyphenols can be oxidized by the folin ciocalteu (fc) reagent, with a blue colour formation, with a maximum absorption of 750 nm. the folin ciocalteu index is specific only for phenolic compounds with reducing properties. total phenolic content was determined with folin ciocalteu method, using gallic acid as a standard. the results were expressed as g of gallic acid equivalents (gae)/l on a dry weight basis [16]. for analysis 2 ml of extract, 1 ml of folin ciocalteu reagent and 8 ml of 7.5% na2co3 were mixed for 5 min and stored in the dark for 30 min then it was measured the absorbance at 750 nm [17]. determination of the mineral content in different samples was performed using a mass spectrometer with inductively coupled plasma (icp-ms agilent technologies 7500 series) digestion of the samples was performed with concentrated nitric acid and hydrochloric acid, using double deionized water obtained with a water purification system thermofisher. concentration (c) of heavy metals in samples obtained is expressed in µg/g sample and is calculated from the formula [2]: c m v a  (2) where: a concentration value measured, [ppb]; v volume of acid that dissolved sample [ml] m mass of mineralized sample [g]. 3. results and discussion the ash content shows (table 1) the mineral richness of the plant, and its determination was used for their elemental analysis. table 1. water and ash content the various anatomical parts of the medicinal plant carduus glaucinus samples moisture % ash % dried leaves 6.25 8.54 dried flowers 6.05 3.85 dried strain 6.38 2.24 fresh root 30.58 5.24 it is noticeable that the highest mineral content was found in the leaves, followed by root, flowers and plant stem. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ofelia arvinte, liliana norocel, sonia amariei, study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant, food and environment safety, volume xviii, issue 2 – 2019, pag. 130 – 135 133 table 2. metals and metalloids content of the various anatomical parts of the medicinal plant carduus glaucinus, in μg/g. tea 1 (leaves) tea 2 (flowers) li 0.366 9.129 na 30.853 656.689 mg 32.361 675.819 al 0 13.035 ca 305.613 2789.826 ti 59.375 553.567 v 0.065 3.958 cr 1.649 39.300 mn 1.928 55.729 fe ii 0.116 3.545 fe iii 0 0.907 co 0 1.219 ni 0.142 10.130 cu 0.100 9.102 zn 0.014 1.687 ga 0 0.104 as 0.072 0.350 se 0.083 1.101 sr 0 136.398 mo 57.546 519.011 pd 0 0.013 ag 0 0.014 cd 0.630 0.735 in 4.019 0.527 sn 0 6.117 sb 0.105 0.193 te 0 2.065 cs 0.278 4.432 ba 0.270 4.360 pt 0 0 au 0 0.657 hg 0.059 0.056 tl 0.010 0.144 pb 0 0 u 0 0 table 2 shows the high mg and ca content of the plant flowers, followed by those of the root and then of the stem, the smallest values being in the leaves. fe2+, se and zn are high in flowers and roots. in terms of heavy metals, pb is absent in the entire plant, and hg and cd to be well below the maximum limit of 10mg/kg for children. [18]. tea 3( strain) tea 4 (root) li 5.097 2.982 na 290.811 302.707 mg 185.585 247.688 al 294.393 1019.286 ca 2021.654 2749.519 ti 360.831 822.616 v 1.831 13.717 cr 19.751 2.744 mn 25.737 200.609 fe ii 1.979 8.144 fe iii 0.417 1.782 co 0.571 1.697 ni 4.769 8.384 cu 4.572 6.818 zn 1.095 1.493 ga 0.081 0.248 as 0.387 0.492 se 1.015 4.179 sr 157.211 4674.264 mo 597.403 4542.413 pd 0.010 0.039 ag 7.393 0.019 cd 0.763 0.010 in 0.786 0.276 sn 7.394 3.884 sb 0.178 0.143 te 0.428 1.338 cs 9.218 10.729 ba 8.839 13.805 pt 0 0 au 0 0 hg 0.032 0.046 tl 0.083 0.121 pb 0 0 u 37.234 0 from the analysis of the results it can be concluded that, from the point of view of the elemental composition, the flowers are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ofelia arvinte, liliana norocel, sonia amariei, study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant, food and environment safety, volume xviii, issue 2 – 2019, pag. 130 – 135 134 most suitable for use in the form of tea. cadmium, arsenic, mercury and antimony were within the safety limits and lead was below the detection limits. table 3. antioxidant capacity of the various anatomical parts of the medicinal plant carduus glaucinus (i %) samples aqueous extract alcoholic extract leaves 61.348 2.033 flowers 89.947 20.444 strain 78.652 30.497 it can be seen that for all parts of the plant, the aqueous extract is much richer in antioxidants, the biggest difference being observed in the case of the leaves. table 4. polyphenol content of the various anatomical parts of the medicinal plant carduus glaucinus samples aqueous extract alcoholic extract dried leaves 8.526 g/l 0.865 g/l dried flowers 9.107 g/l 1.009 g/l dried strain 11.116 g/l 0.755 g/l in according with the previous result, the concentration of polyphenols is much higher in the aqueous extract than in the alcohol, being the highest in the strain. in the alcoholic extract the highest amount of polyphenols is found in the flowers. 4. conclusion volovatic (carduus glaucinus) is a rich source of antioxidants and phenolic compounds and can protect the human body from the action of free radicals. the determined metals and metalloids show that the traditional medicinal plant is safe to use. along with the other plants of the cardus species, tea from carduus glaucinus can be used for the regeneration and proper functioning of the liver especially in the treatment and control of liver disease. 5. references [1]. d. ivanaova, d. gerova, t. chervenkov, t.yankova, polyphenols and antioxidant capacity of bulgarian medicinal plants, journal of ethnopharmacology, volume 96, issues 1–2, 4 january, pages 145-150 (2005). [2]. chifu toader, mânzu ciprian, zamfirescu oana, flora and vegetation of moldova (romania), alexandru ioan cuza university publishing house iaşi, (2006). [3]. https://it.wikipedia.org/carduus_defloratus; [4]. https://dexonline.ro/definiţie/hemicriptofita [5]. mititelu d., viţalariu gh., chifu t., ştefan n., dăscălescu d., horeanu cl., – flora of calimani mountains, (in romanian) an. şt. univ. „al. i. cuza” iaşi, 32, s. ii a., biol.: 28-30 (1986); [6]. simionescu ion, flora of romania, editura albatros, (in romanian) (1973) [7]. aeschimann d., lauber k., moser d. m., theurillat j.-p., – alpin flora. atlas des 4500 plantes vasculaires des alpes, vol. i-iii, belin, paris, p. 622-623 vol. i, 560-561, 576-577 vol.ii (2004); [8]. nyaradi e. i. flora and vegetation of retezat mountain, (in romanian), publishing house of the romanian people's republic (1958). [9]. beldie alexandru, flora and vegetation of bucegi mountain, publishing house of the academy of the socialist republic of romania, (in romanian) (1967). [10]. ciucă maria, flora and vegetation of grassland from ciucaş mountain, publishing house of the academy of the socialist republic of romania, (in romanian), (1967). [11]. buhner, stephen harrod, plants for liver disease publishing house of guides, bucureşti (2006). [12]. rabea parveen, sanjula baboota, javed ali, alka ahuja, suruchi s. vasudev, and sayeed ahmad, effects of silymarin nanoemulsion against carbon tetrachlorideinduced hepatic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 ofelia arvinte, liliana norocel, sonia amariei, study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant, food and environment safety, volume xviii, issue 2 – 2019, pag. 130 – 135 135 damage, arch pharm res vol 34, no 5, 767-774, (2011); [13]. goina, t., constantinescu e, ciulei i et al., pharmacognosy, didactic and pedagogical publishing house, bucharest, (in romanian), (1967). [14]. istudor viorica, pharmacognosy, phytochemistry of phytotherapy, vol i, medical publishing house, bucharest, (in romanian), 20 (1998). [15]. pârvu c. the universe of plants little enciclopedia, enciclopedia publishing house bucharest, (in romanian), (1991). [16]. su, x., duan, j., jiang, y., duan, x., & chen, f., polyphenolic profile and antioxidant activities of oolong tea infusion under various steeping conditions. international journal of molecular sciences, 8(12), 1196-1205. (2007). [17]. sripakdee, t., sriwicha, a., jansam, n., mahachai, r., & chanthai, s. determination of total phenolics and ascorbic acid related to an antioxidant activity and thermal stability of the mao fruit juice. international food research journal, 22(2), (2015). [18]. amariei, s., gutt, g., oroian, m., study on toxic metal levels in food supplements. revista de chimie, 68(6), 1298-1301 (2017). [19]. aleksandra stanojkovi -sebic, radmila pivic, dragana josic, zoran dinic, aleksandar stanojkovic, heavy metals content in selected medicinal plants commonly used as components for herbal formulations, journal of agricultural sciences, 21 317-325, (2015). 1. introduction 4. conclusion 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 5 13 effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan" *caroline yaya abbe 1 , ghislaine chépo dan 1 , pascal amédée ahi 1 and nestor aboa 1 1laboratory of biochemistry and food technology, nangui abrogoua university, 02 bp 801 abidjan 02, côte d’ivoire, akpouichia@gmail.com *corresponding author received 18th november 2019, accepted 20th march 2020 abstract: the purpose of this paper is to evaluate the effect of ripening at different stages on nutrient properties in solanum anguivi lam berries at different stages. fresh berries were collected at agboville (100 km from abidjan). nutritional values were investigated using standard methods, while minerals profiles were performed by using an atomic absorption spectrophotometer. results showed that green berries had the highest content in ash (06.90 ± 0.01 %) and in fiber (21.67 ± 0.02 %) while red berries had the highest content in protein (14.02 ± 0.03 %). ash and protein content decreased during boiling, but the fiber content increased. the losses registered after 15 min of boiling were the following: protein (10.35 – 10.41 %) and ash (1.62 – 3.04 %). during ripening, calcium, potassium, phosphorus and zinc amount increased whereas magnesium, manganese and iron quantity decreased. generally, all minerals decreased during boiling times. in this view, these berries constitute good source of nutrients and could contribute efficiently to the nutritional requirement and food security of ivorian populations. keywords: solanum anguivi lam, boiling, proximate composition, minerals. 1. introduction native fruit vegetable, solanum anguivi lam is part of the family solanaceae, [1]. it is a spontaneous food plant widespread in the tropical and temperate zones [2]. commonly called "gnagnan", these berries of s. anguivi lam are highly appreciated by the ivorian population specifically that of central côte d'ivoire. they are characterized by a bitter aftertaste due to the presence of various phenolic compounds that give them antioxidant properties and are able to fight against stress and cell aging [3]. at physiological maturity, the berries of s. anguivi lam change color successively over time. they pass from the green state to the red state (optimum ripening) by passing through the yellow state, then orange. the green berries contain a lot of vitamin c, phenolic compounds, iron and magnesium. however, red berries are rich in protein, cellulose, total sugars, lipids, potassium [4]. in addition, fruits, leaves, roots and even seeds are used in traditional medicine to treat various pathologies including malaria, high blood pressure, prostate, abdominal pain, diabetes etc [5]. traditionaly, in côte d’ivoire, berries are eaten raw, boiled or are preserved by drying at sun preceded or not by cooking with water or steam of water [3]. however, various studies have shown that cooking caused negative impact by reducing nutritive value but positive impact by increasing some nutrients. thus, cooking with water causes a drop in minerals (ca, mg, p, k, na, zn, fe) in vitamin c, in ash, in protein yet raises fiber content [6-7]. earlier reports have high lighted the nutritive potential of these fresh http://www.fia.usv.ro/fiajournal mailto:akpouichia@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 6 berries but there is a lack of scientific data with regards to the effect of cooking. 2. matherials and methods samples collection s. anguivi lam berries at different stage of ripening were collected from cultivated farmlands located at agboville (5°55'40" n and 4°12'47" w), and authenticated at the departement of national center of floristic research (felix houphouët boigny university, cocody-abidjan. they were analysed in the laboratory at nangui abrogoua university. berries (green, yellow, orange and red) were directly dried into an oven at 45ºc for 72 hours. samples preparation berries were harvested as follows [8]: green berries at 90 days after growth, yellow berries about 6 days after green stage, orange berries about 3 days after yellow stage, and red berries about 2 days after orange stage. the fruits were stored at a temperature of 28°c ± 3 °c. color analysis was done using a chroma meter (konica minolta, inc. color reader cr_ 10 (japon). after harvesting at different stages of ripening, fresh berries were washed with deionised water and allowed to drain at ambient temperature. each sample was divided into two lots. the first lot (raw) was dried in an oven (memmert, germany) at 45ºc for 72 h according to chinma and igyor [9]. then, the dried berries were ground with a moulinex-type mixer. the powder obtained was sieved (100 μm) and samples were stored in clean dry air-tight bottles at 4ºc until required for analyses. the second lot was cooked by using the method of randrianatoandro, [10] modified as follow: 250 g of berries were immersed in 1.5 l of boiled water in stainless steel container for 10 and 15 min. the boiling solution was discarded and the boiled samples were cooled, drained at ambient temperature and subjected to the same treatment using for raw samples. proximate analysis the dry matter contents were determined by aoac [11]. ash, proteins, lipids and minerals were determined using standard methods aoac [12]. for total fibers, 2 g of dried powdered sample were digested with a solution of 0.25 m sulphuric acid and 0.3 m sodium hydroxide. the insoluble residue obtained was washed with hot water and dried in an oven (memmert, germany) at 100 °c until constant weight. the dried residue was then incinerated, and weighed for the determination of fiber content. glucids and calorific value were calculated using the following formulas fao [13]: glucids: 100 – (% moisture + % proteins + % lipids + % ash). calorific value: (4 x % proteins) + (4 x % glucids) + (9 x % lipids). the results of ash, fibres, proteins, lipids and glucids contents were expressed on dry matter basis. minerals including calcium, magnesium, iron, zinc, manganese, phosphorus, sodium and potassium were determined using an atomic absorption spectrophotometer, aas (model 372, perkin-elmer, beaconsfield, uk) by wet digestion while phosphorous level was determined using the phosphovanado molybdenate method [11]. statistical analysis all experiments were carried out in triplicate and data were expressed as mean ± standard deviation (sd) or standard error of mean (sem).two ways analysis of variance (anova) and the duncan test at significant level p = 0.05 was conducted to compare treatment means using statistica 7.1 software. principal component analysis (pca) was also used food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 7 to distribuate samples in terms of nutritional parameters. 3. results and discussion nutritional values mineral content is an essential component of the nutritive value of vegetable. table 1 showed the effect of cooking on mineral composition of s. anguivi lam during ripening. green berries of s. anguivi lam had the highest levels of magnesium, manganese and iron while red berries are higher in calcium, potassium and zinc. after boiling, all mineral content decreased significantly (p <0.05). the registered losses were as follow: calcium (66.81 – 75.34 %), magnesium (56.99 – 61.9 %), phosphorus (51.51 – 60.53 %), potassium (28.53 – 57 %), iron (63.90 – 72.07), zinc (66.95 –77.47 %) and manganese (50 – 70.58 %). these observed reductions may be due to leaching of the mineral compounds into the boiling water [14]. this finding is in line with the report of davidson and monulu [15] who observed significant reduction in the mineral concentration of boiled and steamed eggplant leaves (solanum macrocarpon). it is known that calcium and phosphorus play a major rôle in ossification and dentition and has a preventive effect on artériel hypertension in the elderly [16]. concerning magnesium, this mineral prevents cardiomyopathy, muscle degeneration, growth retardation, congenital malformations and bleeding disorders [17]. potassium is an essential mineral and a major electrolyte found in the human body. potassium and sodium are involved in membrane and cellular exchange, thus contributing to the regulation of plasma volume, acid-base balance and muscles contraction [18]. iron plays important role in prevention of anemia which affects more than one million people worldwide [19]. considering the recommended dietary allowance (rda) for iron (8 mg/day), consumption of 15 min boiling, berries could cover rda [19, 20]. zinc is metal present in all cells. it is co-factor for morethan 300 enzymes, and it is necessary for wide variety of biological functions. it is also required for synthesis of dna, normal growth, gene expression, gene regulation, cell division and immunity [21]. nutritional content during the ripening and cooking berries of s. anguivi lam were shown in table 2. during boiling time, dry matter, ash, protein, fat and calorific energy contents decreased significantly (p <0.05) while fiber and glucids quantity increased. the decline of dry matter content is provoked by the weakness of the berries tissues and movement of water in the cell walls [22]. a similar reduction in dry matter content was reported by lo scalzo and al. [23] in three boiled eggplants (7.8 5.6 %; 9.18 6.2 % and 8 – 5.2 %) respectively in tunisia, buia and l 305 after 10 min. in green berries, after 15 min of boiling, dry matter (24.97 ± 0.03 %) were higher than red berries (22.23 ± 0.01%). quantity of ash varied from 6.90 ± 0.01 to 6.69 ± 0.02 % in green berries and from 6.17 ± 0.02 to 6.07 ± 0.03 % in red berries. the lowering of ash content in these studied berries may be a result of minerals leaching into the boiling water [24]. in spite of ash losses, s. anguivi the studied berries may be considered as good sources of minerals after cooking at 15 min when compared values of boiled leaves of solanum nigrum (1.24 %) [14] and boiled yam (1.53 %) [25]. fibers revealed a significant slight increase with 15.37 ± 0.02 % and 27.12 ± 0.02 % after 15 min. this finding is in agreement with some reports that cooking caused increase in soluble fiber content [26]. with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 8 regard to their fibers content at 15 min, fiber intake has a number of health benefits, including maintenance of healthy laxation and the reduced risk of cardiovascular diseuse and cancer [27, 28]. these results are in agreement with those of oulai and al. [6] who observed an increase in the fiber content when leafy vegetables were cooked with water (h. sabdariffa (14.27 14.55 %), a. digitata (12.56 – 13.10 %)). table 1 mineral composition of raw and cooked of solanum anguivi lam at differents stages of ripening minerals (mg/100g ms) berries cooking time (min) 0 10 15 green 517.05±0.04i 289.50±0.05g 171.57±0.02d calcium yellow 531.38±0.02j 290.17±0.02h 142.14±0.02a orange 551.53±0.03k 248.29±0.02f 144.44±0.02b red 647.42±0.02l 235.39±0.02e 159.64±0.05c green 421.36±0.01l 364.57±0.02k 163.53±0.01g magnesium yellow 339.11±0.01j 152.03±0.01f 130.08±0.01c orange 269.78±0.02i 141.70±0.02e 136.22±0.02d red 257.59±0.01h 125.05±0.02b 110.79±0.01a green 120.44±0.04i 94.29±0.04g 47.53±0.03a phosphorus yellow 123.22±0.02k 97.87±0.03h 91.42±0.02f orange 125.96±0.03l 84.34±0.01e 78.51±0.01d red 122.06±0.03j 70.56±0.03c 59.18±0.02b green 1925.79±0.02i 1780.43±0.03f 1540.06±0.03d potassium yellow 2046.26±0.06j 1869.97±0.02h 1647.01±0.01e orange 2296.05±0.01k 1781.31±0.01g 1484.99±0.02b red 2304.58±0.08l 1516.56±0.04c 827.95±0.04a sodium green 256.70±0.02e 140.58±0.02b 100.28±0.02a yellow 310.20±0.01g 189.18±0.03c 151.10±0.03b orange 356.12±0.02h 300.07±0.02f 223.03±0.03d red 460.17±0.02j 371.13±0.03 235.06±0.03d green 454.45±0.02l 204.15±0.05f 116.02±0.02c iron yellow 443.96±0.01k 213.60±0.10h 100.29±0.02b orange 356.46±0.03j 206.21±0.01g 164.06±0.03e red 338.12±0.02i 133.22±0.02d 94.43±0.03a green 20.70±0.01l 12,88±0,02k 4,68 ± 0,20d zinc yellow 8.08±0.02h 5,28 ± 0,02e 3,94 ± 0,01c orange 12.64±0.02j 9,40 ± 0,02i 6,84 ± 0,04g red 6.17±0.02f 2,45 ± 0,04b 1.82 ± 0.02a manganese green 0.17±0.02e 0.08±0.00bcd 0.05±0.02ab yellow 0.11±0.01d 0.08±0.02bcd 0.04±0.01a orange 0.09±0.03cd 0.05±0.02ab 0.04±0.02a red 0.10±0.03cd 0.07±0.02bc 0.05±0.01ab values are expressed as mean ± sd. (n =3). means assigned to the same letter for the same parameter are not significantly differents (p ≤ 0.05) as concern proteins content, cooking processing used in this study caused 10.35 – 10.41 % reduction after 15 min. these losses of protein during heat treatments of food could be due to heat destruction of protein peptide bonds [29]. according to oulai and al. [6] cooked leafy vegetable are poor in crude protein compared to raw leafy. however, after 15 min of cooking, s. anguivi lam berries could be considered food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 9 as a significant source of protein because the minimum value recommended for protein-rich foods is 12% [30]. it could play a significant role in providing cheap and available proteins for rural communities. in fact, proteins were also essential for the growth of children and adolescents, as well as for the formation of the fetus in pregnant women [6]. during boiling times, lipid content decreased significantly (p <0.05) in green berries (1.37 ± 0.03 to 0.94 ± 0.04 %) and in red berries (1.95 ± 0.05 to 1.54 ± 0.02 %). with boiling the fat must have melted into the boiling water thus causing a reduction in the fat content [14]. lipids of s. anguivi lam’ s berries was in agreement with the results of many authors which showed that vegetables are poor sources of fats [31], [6], [32]. therefore, the consumption of these berries may be recommended to individuals suffering from obesity [6]. the glucids content in green berries of s. anguivi lam (4.66 ± 0.01 5.31 ± 0.02 %) increased significantly (p <0.05) during boiling time. this result is similar to the most cooked leafy vegetables which are generally not good sources of carbohydrates [32]. in addition, other authors obtained an increase in carbohydrate content in boiled (83.62 84.41 %) and steamed bananas (83.62 83.76 %) compared to fresh fruits [33]. energy value generally decreased significantly (p <0.05) during boiling time from 84.65 ± 0.01 to 77.82 ± 0.03 kcal/100g in green berries and from 76.07 ± 0.04 to 72.34 ± 0.02 kcal/100g in red berries. the relatively low energy values of s. anguivi lam berries were consistent with the observations made on vegetables. according to the general observation, vegetables have low energy values because of their low crude fat content and relatively high moisture content [34]. table 2: proximate composition of raw and cooked berries of solanum anguivi lam at differents stages of ripening parameters berries cooking time (min) 0 10 15 green 26.35 ± 0.05i 25.36 ± 0.04h 24.97 ± 0.03 g dry matter (%) yellow 24.18 ± 0.03f 24.02 ± 0.02ef 23.85 ± 0.02de orange 23.67 ± 0.05d 23.88 ± 0.02de 23.35 ± 0.01c red 22.75 ± 0.03b 22.55 ± 0.02b 22.23 ± 0.01a green 6.90 ± 0.01k 6.78 ± 0.02j 6.69 ± 0.02h ash (%) yellow 6.74 ± 0.04i 6.61 ± 0.01g 6.58 ± 0.02g orange 6.32 ±0.02f 6.27 ± 0.02e 6.22 ± 0.02d red 6.17 ± 0.02c 6.13 ± 0.03b 6.07 ± 0.03a green 21.67 ± 0.02g 25.13 ± 0.02h 27.12 ± 0.02i fiber (%) yellow 18.42 ± 0.02e 20.33 ± 0.02f 21.33 ± 0.03g orange 14.00 ± 1.00b 15.10 ± 0.01c 16.17 ± 0.03d red 12.17 ± 0.03a 14.33 ± 0.03b 15.37 ± 0.02c green 13.42 ± 0.02f 12.18 ± 0.03e 12.03 ± 0.01a proteins (%) yellow 13.76 ± 0.02g 12.33 ± 0.02b 12.22 ± 0.01ab orange 13.94 ± 0.20gh 12.55 ± 0.02c 12.30 ± 0.02b red 14.02 ± 0.03i 12.96 ± 0.04d 12.56 ± 0.02c green 1.37 ± 0.03cd 1.12 ± 0.05ab 0.94 ± 0.04a lipids (%) yellow 1.49 ± 0.02de 1.15 ± 0.02ab 1.01 ± 0.01ab orange 1.80 ± 0.04fg 1.45 ± 0.05de 1.17 ± 0.01bc red 1.95 ± 0.05g 1.65 ± 0.02ef 1.54 ± 0.02de food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 10 glucids (%) green 4.66 ± 0.01i 5.13 ± 0.03k 5.31 ± 0.02k yellow 2.19 ± 0.01e 3.71 ± 0.03g 4.04 ± 0.01h orange 1.14 ± 0.02b 3.26 ± 0.01f 3.86 ± 0.02f red 0.61 ± 0.02a 1.59 ± 0.02c 2.06 ± 0.03d calorific energy (kcal/100g) green 84.65 ± 0.01j 79.92 ± 0.02i 77.82 ± 0.03h yellow 77.21 ±0.01g 75.39 ± 0.04d 74.94 ± 0.02c orange 76.52 ± 0.05f 77.93 ± 0.02h 75.17 ± 0.02d red 76.07 ± 0.04e 73.93 ± 0.02b 72.34 ± 0.02a values are expressed as mean ± sd. (n =3). means in the same parameter followed by different letters differed significantly (p ≤ 0.05) classification of solanum anguivi berries samples the principal component analysis (pca) based on the nutritional values obtained for the berries at different stages of ripening are presented in figure 1. the fresh green, yellow and orange berries (gb0, yb0, ob0), green (gb10)) cooked for 10 min and green berries (gb15) cooked for 15 min obtained a high positive score while fresh red berries (rb0), yellow (yb10), oranges (ob10) and red berries (rb10) cooked for 10 min and yellow (yb15), oranges (ob15) and red berries (rb15) cooked for 15 min. had a negative score in pc 2 (fig 1). fig. 1 circle of correlation of biochemical composition of raw and cooked solanum anguivi lam berries during ripening on axis 1 and 2. fib : fiber, zn : zinc, lip : lipids, na : sodium, p : phosphorus, k : potassium, prot : protein, ca : calcium, fe : fer, mn : manganese, mg : magnesium, cev : calorific energy value, dm : dry matters, glu : glucid ob0, rb0, ob0, yb10, and gb15 were near zero in pc2. rb0 are near zero in pc1. the correlation circle provides information about correlations between the measured properties (fig. 2). the observation of the correlation circle shows that the dry food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 11 matter, ash, fibers, glucide and energy content levels are positively correlated with each other. this group is negatively correlated with the group consisting of proteins, lipids, iron, magnesium, potassium, manganese, sodium, calcium, potassium and zinc. gb0 gb10 gb15 yb0 yb10 yb15 ob0 ob10 ob15 rb0 rb10 rb15 -10 -8 -6 -4 -2 0 2 4 6 8 fact. 1 : 51,62% -8 -6 -4 -2 0 2 4 6 8 10 f a c t. 2 : 3 6 ,7 6 % fig. 2. . sample plot of principal components 1 and 2 raw and cooked berries of solanum anguivi lam gb0: raw green berries, yb0: raw yellow berries, ob0: raw orange berries, rb0: raw red berries gb10: green berries boiled during 10 min, yb10: yellow berries boiled during 10 min, ob10: orange berries boiled during 10 min, rb10: red berries boiled during 10 min gb15: green berries boiled during 15 min, yb15: yellow berries boiled during 15 min, ob15: orange berries boiled during 15 min, rb15: red berries boiled during 15 min 4. conclusion in conclusion, green berries contain appreciable amounts of fiber, ash, magnesium, iron and zinc while red berries are rich in protein, calcium, phosphorus, potassium and sodium. whatever the stages of ripening, this content decrease during boiling times.yet, the result of this study revealed that cooked solanum anguivi lam berries at 10 min preserved more nutrients. 5. acknowledgments the authors thank the reviewers for their constructive comments, which helped to improve the manuscript. 6. references [1]. n'dri m.t.k., gnahoua m.g., konan e.k., traore d., plantes alimentaires spontanées de la région du fromager (centre ouest de la côte d'ivoire) flore, habitats et organes consommés. sciences & nature, 5, 61-70, (2008). [2]. kouadio a. i., chatigre k. o. ; dosso b. m., phytochemical screening of the antimicrobial fraction of solanum indicum l. berries extract and evaluation of its effect against the survival of bacteria pathogens of plants. international journal of biotechnology and food science, 2 (1): 21-30, (2014). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 12 [3]. cnra, bien conserver les fruits frais de gnagnan en côte d’ivoire. fiche technique conservation, transformation des produits nº1, (2017). [4]. dan c.g., kouassi k.n., ban k.l., nemlin g.j., kouame p. l., influence of maturity stage on nutritional and therapeutic potentialities of solanum anguivi lam berries (gnagnan) cultivated in côte d’ivoire. international journal of nutrition and food sciences, 3(2): 1-5 (2014). [5]. dan c. g., evolution des paramètres biochimiques et physico-fonctionnels des baies de solanum anguivi lam récoltées en côte d’ivoire au cours du mûrissement. these de doctorat de l’université nangui abrogoua. ufr des sciences et technologie des aliments spécialité: biochimie et technologie des aliments, 169 p, (2015). [6]. oulai d. p., zoue t. l., bedikou e. m., megnanou r., niamke l.s., impact of cooking on nutritive and antioxidant characteristics of leafy vegetables consumed in northern côte d’ivoire. international journal of plant, animal and environnemental sciences, 4 (3), issn 22314490.9 (2014). [7]. acho f. c., zoue l. t., niamke s. l., nutritional and antioxidant characterization of blanched leafy vegetables consumed in southern côte d’ivoire (ivory coast). biotechnology journal international, 6 (4), 154-164. (2014) https://doi.org/10.9734/bbj/2015/ 14509. [8]. n’dri d., calani l., mazzeo t., scazzina f., rinaldi m., del rio d., and al., effects of different maturity stages on antioxidant content of ivorian gnagnan (solanum indicum l.) berries. molecules, 15 (10), 7125-7138 (2010). [9]. chinma c.e., igyor m.a., micronutriments and anti-nutritional content of select tropical vegetables grown in south-east, nigeria. nigerian food journal, 25(1): 111– 116, (2007). [10]. randrianatoandro v.a., identification et caractérisation des plats sources en micronutriments consommés en milieu urbain (manjakaray, madagascar): étude des plats à base de légumes-feuilles, thèse, université d’antananarivo, madagascar, (2010). [11]. aoac. (1995). official methods of analysis of the association of official analytical chemists, 16th ed. virginia. u.s.a; 1995 [12]. aoac. (1990). official methods of analysis. association of official analytical chemists ed., washington dc, 684 p. [13]. fao (2002). food energy-methods of analysis and conversion factors. fao ed, rome, 97 p. [14]. ajala l., the effect of boiling on the nutrients and anti-nutrients in two non conventional vegetables. pakistan journal of nutrition, 8 (9): 1430-1433 (2009). [15]. davidson g. and monulu a., vitamins and minerals composition of eggplant (solanum macrocarpon) and ‘ukazi’ (gnetum africanum) leaves as affected by boiling and steaming. journal of scientific research & reports 21(4): 1-8 (2018). [16]. turan m., kordali s., zengin h., dursun a. and sezen y., macro and micromineral content of some wild edible leaves consumed in eastern anatolia. plant soil science, 53: 129-137 (2003). [17]. chaturvedi v.c., shrivastava r., upreti r.k., viral infections and trace elements: a complex trace element. curr. sci., 87: 15361554, (2004). [18]. akpanyung e.o., proximate and mineral élement composition of bouillon cubes produced in nigeria. pakistan journal of nutrition 4 (5) : 327 329, (2005). [19]. trowbridge f., martorell m. forging effective strategies to combat iron deficiency. journal of nutrition, 85: 875880, (2002). [20]. fao (2004). human vitamin and mineral requirements. fao ed, 361 p. [21]. vidyavati sd, sneha a, katti sm (2016). zinc: the importance in human life. international journal of healthcare and biomedical research, 04, (04), 18-20 (2016). [22].vodouhe s., dovoedo a., anihouvi v.b., tossou r.c., soumanou m.m., influence du mode de cuisson sur la valeur nutritionnelle de solanum macrocarpum, amaranthus hybridus et ocimum gratissimum, trois légumes-feuilles traditionnels acclimatés au bénin. international journal of biological chemistry sciences, 6(5): 1926-1937 (2012). [23]. lo scalzo r., fibiani m., francese g., d’alessandro a., rotino g. l., conte p., and al., cooking influence on physico-chemical fruit characteristics of eggplant (solanum melongena l.). food chemistry, 194: 835–42 (2016). https://doi.org/10.1016/j. foodchem.2015.08.063 [24]. oboh g., effect of blanching on the antioxidant properties of some tropical green leafy vegetables. food science and technology, 38:513517 (2005). [25]. kone d., kone f., dje k.m., , dabonne s, kouame p., effect of cooking time on biochemical and functional properties of flours from yam “kponan” (dioscorea cayenensisrotundata) tubers. current journal of applied science and technology, 4(23), 3402-3418, (2014). https://doi.org/10.9734 /bjast/2014/10414. [26]. lintas c., cappelloni m., dietary fiber, resistant starch and in vitro starch digestibility of https://doi.org/10.9734/bbj/2015/%2014509 https://doi.org/10.1016/j.%20foodchem.2015.08.063 https://doi.org/10.1016/j.%20foodchem.2015.08.063 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 caroline yaya abbe, ghislaine chépo dan, pascal amédée ahi, nestor aboa, effect of ripening on nutritional values of solanum anguivi lam berries "gnagnan", food and environment safety, volume xix, issue 1 – 2020, pag. 5 – 13 13 cereal meals. food science of nutrition, 42: 117– 124, (1998). [27]. murphy n., nora t., ferrari p., jenab m., bueno-de-mesquita b., skeie g. and al., dietary fibres intake and risks of cancers of the colon and rectum in the european prospective investigation into cancer and nutrition (epic). plos one, 7(6): e39361 (2012). https://doi.org/10.1371/journal. [28]. madhu c., krishna k. m., reddy k. r., laksm j., kumar kelari e., estimation of crude fibre content from natural food staffs and its laxative activité induced in rats. international journal of pharma research and health science, 5(3):1703-06 (2017). [29]. abeke f.o., ogundipe s.o., oladele s., sekoni a.a., dafwang le.l., adeyinka le.a., and al., effect of duration of cooking of lablab purpureus bans on the performance organ weight and haematological paramètres of shika brown pellet clicks. journal of biological sciences, 7 (3): 562-565 (2007). [30]. ali a., proximate and mineral composition of the marchubeh (asparagus officinalis). world dairy food sciences, 4: 142-149, (2009). [31]. ejoh, a.r., tchouanguep m.f., fokou e., nutrient composition of the leaves and flowers of colocasia esculenta and the fruits of solanum melongena. plant food for human nutrition, 49 (2): 107-112, (1996). [32]. zoro a. f., zoue l. t., bedikou m. e., kra s. a. niamke s. l., effect of cooking on nutritive and antioxidant characteristics of leafy vegetables consumed in western côte d’ivoire. scholars research library 6(4): 114 – 123 (2014). [33]. baiyeri, k. p., aba, s. c., otitoju, g. t., mbah, o. b., the effects of ripening and cooking method on mineral and proximate composition of plantain (musa sp. aab cv. ‘agbagba’) fruit pulp. african, journal of biotechnology 10(36) 6979-6984, (2011). [34]. sobowale s.s., olatidoye o.p., olorode o.o., akinlotan j.v. nutritional potentials and chemical value of some tropical leafy vegetables consumed in south west nigeria. journal science multidisciplinary ressource, 3: 55-65 (2011). https://doi.org/10.1371/journal 1. introduction [19]. trowbridge f., martorell m. forging effective strategies to combat iron deficiency. journal of nutrition, 85: 875880, (2002). title …………………… 13 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 13 17 (imidazole-5-yl)ylidenthiazolidones: a new class of antimicrobial agents for food industry *vitaliy chornous 1 , alina grozav 1 , victoria antoniychuk 1 , nina yakovychuk 2 , mykhaylo vovk 3 1 department of medicinal and pharmaceutical chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine chornous.vitalij@bsmu.edu.ua 2 department of microbiology and virology, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine 3department of mechanics of organic reactions, institute of organic chemistry national academy of science of ukraine, 2 murmanska st., kyiv, 02660, ukraine *corresponding author received 23th january, 2019 accepted 28th march 2019 abstract: an antimicrobial and antifungal activity of new derivatives of (imidazole-5yl)ylidenthiazolidinones and its dependence on the substance structure and the type of substitutes in the imidazole and thiazole nucleus have been investigated. it was found that the sulphur of thiazolidone section is the key factor that governs antimicrobial activity of the compounds. high antibacterial and antifungal activity against pathogenic microbes s. aureus atcc25923, e. coli atcc25922 and fungi c. albicans atcc885-653 was determined for some representatives of the compounds series. since the evaluated acute toxicity of the compounds is quite low, they can be recommended for further systematic investigation as possible prospective disinfection agents for food industry. keywords: organic synthesis; imidazoles; thiazolidinones; antimicrobial and antifungal activity; foodstuff treatment and production 1. introduction a risk of microbial and/or fungal contamination is one of important problems endangering foodstuff processing, production and storage at every stage of manufacturing. that is why, extensive efforts are made in various directions to safeguard the foodstuff and raw materials from primary and/or secondary bacterial/fungi contamination or to ensure that the pathogenic objects would not be able to grow and develop. strict observation of all sanitary and hygienic requirements is considered as one of the most effective actions protecting the food materials from pathogenic germs. disinfection chemicals are known as inexpensive and very effective solution to reduce or exterminate unwanted microorganisms or yeast-like fungi such as e. coli, s. aureus and c. albicans at every stage of food processing technologies [1]. a number of chemical agents of different nature are used for this purpose: phenolic compounds, chloroorganic compounds, alcohols, aldehydes, oxygen-containing and oxidizing substances, various mixtures of quaternary ammonium salts, guanidine derivatives etc. [2]. however, many of them are either low efficient or toxic, corrosive to the equipment parts or destructive for the foodstuff components. moreover, it usually takes shorter and shorter time now for the pathogenic germs to develop strong resistance against regular antibiotics or disinfectants [3], which pushes new investigahttp://www.fia.usv.ro/fiajournal mailto:chornous.vitalij@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 vitaliy chornous, alina grozav, victoria antoniychuk, nina yakovychuk, mykhaylo vovk (imidazole-5yl)ylidenthiazolidones: a nex class of antimicrobial agents for food industry, food and environment safety, volume xviii, issue 1 – 2019, pag. 13 – 17 14 tions directed onto finding of more effective chemical disinfectants. that is why it is strongly topical problem to investigate and develop new stable disinfection compounds with prolonged activity, low toxicity and other adhere effects. it is known that 1,2,4-substituted 5formylimidazoles can be considered as interesting candidates for this category. they have already been used widely to synthesize various biologically active components [4-6] and it has been reported [7] that some derivatives of thiazolidone exhibit distinct antimicrobial and antifungal activity. that is why a deeper investigation of antimicrobial potential of imidazolylmethylenthiazolidones seems topical and promising. 2. experimental all target 5-[(1-aryl-1n-imidazole-5yl)methylen]-1,3-thiazolidine-2-thio-4ones (fig. 1, structure 1), 5-[(1-aryl-1nimidazole-5-yl)methylen]-1,3-thiazolidine2-thio-4-ones (fig.1, structure 2) and 5-[(1aryl-1n-imidazole-5-yl)methyl]-1,3thiazolidine-2-thio-4-ones (fig.1, structure 3) were synthesized by the condensation of available 1-arylimidazole-5-carbaldehydes with rhodanine or thiazolidine-2,4-diones under the basic catalysis conditions [8]. general structures of the synthesized compounds are shown in fig. 1. n n n s o s r r r h 1 2 3 n n n s o o r r r h 1 2 3 n n n s o o r r r h 1 2 3 1 (a-f) 2 (a-f) 3 (a-f) fig. 1. structural composition of three classes of antimicrobial/antifungal agents synthesized and investigated in this work. substitutes r 1 , r 2 and r 3 were combined as shown in table 1. the degree of bioactivity of all the substances has been tested on the museum strains of s. aureus atcc25923, e. coli atcc25922 and pathogenic fungi c. albicans atcc885-653 by the double serial dilution method [9]. according to this method, the disposable 96-hole pads and the 8-channel titrator were used to evaluate an antimicrobial activity of the compounds libraries. the clinical strains mentioned above are known to cause usual invasions in humans and were chosen as the testcultures to evaluate disinfection potential of the substances synthesized in this project. the clear bacterial colonies were cultivated in the meat-peptone broth for 24 hours at 37±1 0c until the number of the cells reached 105 cfu/ml. the clear fungi colonies of c. albicans were cultivated in saburo nutritional agar for 7 days at 30±1 0c until the suspension concentration reached 10 cfu/ml. then concentration of all solutions was standardized to 0.5 mcfarland units by visual comparison. all compounds under investigation were used to prepare a series of solutions with concentrations ranged from 500 μg/ml to 7.8 μg/ml and then the minimum inhibition concentration was found after 24 hours of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 vitaliy chornous, alina grozav, victoria antoniychuk, nina yakovychuk, mykhaylo vovk (imidazole-5yl)ylidenthiazolidones: a nex class of antimicrobial agents for food industry, food and environment safety, volume xviii, issue 1 – 2019, pag. 13 – 17 15 cultivation in case of bacteria or after 4872 hours in case of fungi. the minimum bacteriostatic (mbsc), minimum bactericide (mbcc), minimum fungi static (mfsc) and minimum fungicide concentrations (mfcc) were determined as the lowest solution concentration of a given compound that results in the distinct oppression of the test-cultures growth (static concentrations) or extermination of the colonies (mbcc and mfcc). 3. results and discussion it was found that antimicrobial activity of imidazolylmethylenthiazolidones against various types of pathogenic germs depends significantly on the compound structure and composition (see table 1). table 1. antimicrobial activity of the 1-3 compound series (μg/ml) № r1 r2 r3 s. aureus atcc25923 e. coli атсс25922 c. albicans атсс885-653 mbsc mbcc mbsc mbcc mfsc mfcc 1 a ch3 h cl 250 250 250 250 31.3 31.3 1 b c6h5 h cl 3.9 3.9 31.3 62.5 7.81 7.81 1 c 4-clc6h4 h cl 15.6 31.3 7.81 15.6 31.3 31.3 1 d ch3 h sch2co2 h 31.3 31.3 31.3 125 15.6 31.3 1 e 4-clc6h4 h sch2co2 h 250 250 250 1000 15.6 125 1 f 4-ch3c6h4 cl cl 7.8 7.8 250 250 31.3 62.5 averaged activity amongst 1 а-f 95.7 283.9 48.2 2 а c6h5 h cl 62.5 125 250 500 125 250 2 b 2-ch3c6h4 h cl 125 250 250 500 62.5 125 2 c 3-ch3c6h4 h cl 125 250 250 500 125 250 2 d 4-ch3c6h4 h cl 125 250 250 500 62.5 125 2 e 4ch3oc6h4 h cl 500 1000 500 1000 250 500 2 f 1-c10h7 h cl 62.5 125 125 500 125 250 averaged activity amongst 2 а-f 333.3 500 250 3 а c6h5 h cl 62.5 125 250 500 125 250 3 b 2-ch3c6h4 h cl 125 250 250 500 62.5 125 3 c 3-ch3c6h4 h cl 125 250 250 500 125 250 3 d 4-ch3c6h4 h cl 125 250 250 500 62.5 125 averaged activity amongst 3 а-d 218.8 500 187.5 total averaged activity amongst 1-3 series 215.6 450.2 158.7 *control *7.8 *15.6 *7.8 *15.6 **3.9 **1.9 *furacilinum (furacin or nitrofurazone) and **clotrimazole were used in the control experiments as the reference antimicr obial and antifungal compounds correspondingly. it should be noted that comparatively high antimicrobial and antifungal activities have been registered for all the compounds involved in this work. the total averaged mbcc for the entire series in case of s. aureus was 215.6 μg/ml, in case of e. coli – 450.2 μg/ml while mfcc was even lower – 158. 7 μg/ml. in our opinion, such a high antifungal efficiency can be caused by imidazole nucleus present in all compounds of the series 1-3. since bactericide and fungicide indexes are more important than food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 vitaliy chornous, alina grozav, victoria antoniychuk, nina yakovychuk, mykhaylo vovk (imidazole-5yl)ylidenthiazolidones: a nex class of antimicrobial agents for food industry, food and environment safety, volume xviii, issue 1 – 2019, pag. 13 – 17 16 corresponding ‘static’ concentrations, we skipped the former parameters in the further analysis. a dependence of antimicrobial activity on the structure of thiazolidone section can easily be understood from comparison between subseries 1 (containing the exocyclic atom of sulphur) and subseries 2 (containing the exocyclic atom of oxygen). an influence of reduction of the methylene fragment to methyl on antimicrobial activity can also be understood from comparison between subseries 2 and 3. for instance, it can be noted that the series 1 and 2 have same structural composition while antimicrobial and antifungal activities of the sulphur-containing series 1 are higher than those of the oxygen-containing series 2. an average mbcc and mfcc for the set 1 are (μg/ml): 95.7 (s. aureus), 283.9 (e. coli) and 48.2 (c. albicans) correspondingly. similar values for the set 2 are sufficiently higher that proves lower activity of these compounds: 333.3 (s. aureus), 500 (e. coli) and 250 μg/ml (c. albicans). transformation of the methylene structural bridge in the set 2 to the methyl one (set 3) results in some increase of averaged antimicrobial and antifungal activity of the compounds and the corresponding values of mbcc and mfcc decrease down to 218.8, 500 and 187.5 μg/ml (see table 1). therefore, it is obvious that the compounds 1a-f reveal the highest germ-suppressing activity and should be analyzed further to find the most effective representative. as seen from the data of table 1, the structure of the imidazole part of a compound is the key factor influencing germsuppressing activity. for instance, both antimicrobial and antifungal activities are increasing after exchange of a substitute in the first position from methyl (1a) to phenolic (1b) or 5-chlorophenolic (1c). on the other hand, a substitute in the second position of imidazole cycle also seems influential. when the polar atom of chlorine is inserted in the r2 position, the value of mbcc improves to 7.8 μg/ml in case of s. aureus while mfcs reaches 62.5 μg/ml. in contrary, this substitute makes no effect on the value of mbcc in case of e. coli (see comparison between 1a and 1f). fig. 2. comparison of minimal bactericide (mbcc) and fungicide (mfcc) concentrations (mg/ml) for some representatives of the subseries 1 and the control compounds. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 vitaliy chornous, alina grozav, victoria antoniychuk, nina yakovychuk, mykhaylo vovk (imidazole-5yl)ylidenthiazolidones: a nex class of antimicrobial agents for food industry, food and environment safety, volume xviii, issue 1 – 2019, pag. 13 – 17 17 it is also interesting to analyze an influence of a substitute to the fourth position of imidazole cycle (r3). as seen from the comparison between the compounds 1a and 1d, both mbcc and mfsc values get improved for all three types of the germs when chlorine is substituted with the hydrophilic fragment of thioglycolic acid. when there is the aryl fragment in the first position of imidazole fragment (1c and 1e), substitution of chlorine in the fourth position with the thioglycolic acid fragment results in a serious decrease of antigerm activity especially against s. aureus and c. albicans (compare corresponding values of mbsc, mbcc and mfcc for 1c and 1e). it is interesting to note that the change of mfsc in this case is opposite and it is improving because of the above-mentioned substitution. the above discussion is visualized graphically in fig. 2 for better comprehension of comparative bactericide and fungicide activities of some representatives of the subseries 1 and the corresponding control compounds. finally, toxicity of all the synthesized compounds has been evaluated using the computer simulation software gusar online [10]. the toxicity of the entire class of the synthesized compounds has been found low: rat oral ld50 = 1027 mg/kg; rat sc ld50 = 1366 mg/kg. 4. conclusion a wide spectrum of bactericide and fungicide or static activities has been found for some representatives of the investigated compounds. mbcc and mfcc values were close or sometimes better than those of the control agents. since predicted toxicity was appropriately low, (imidazole-5-yl) ylidenthiazolidones can be used in further search for the low toxic bacterioand fungicide agents for food industry. 5. references [1]. g. wirtanen, s. salo, disinfection in food processing – efficiency testing of disinfectants., rev. environ. sci. and biotech., 2(2–4): 293– 306 (2003). [2]. g. mcdonnell, a. d. russell, antiseptics and disinfectants: activity, action, and resistance. clin. microbiol., rev., 12(1): 147–179 (1999). [3]. a. j. mcbain, p. gilbert, d. g. allison, biofilms and biocides: are there implications for antibiotic resistance? rev. environ. sci. and biotech., 2(2–4): 141–146 (2003). [4]. v. a. chornous, m. k. bratenko, m. v. vovk, polyfunctionalimidazoles: i. synthesis of 1-substituted 4-chloro-1h-imidazole-5carbaldehydes by wilsmeier-haack reaction. rus. j. org. chem., 45(8): 1210–1213 (2009). [5]. v. a. chornous, a. n. grozav, l. d. todoriko, m. v. vovk, synthesis and biological activity of 4-chloro-1h-imidazole-5carbaldehyde thiosemicarbazones. pharm. chem. j., 47(10): 524–526 (2014). [6]. v. a. chornous, a. a. palamar, i. n. yaremii, m. v. vovk, synthesis and antioxidant activity of [(1-aryl-5-formylimidazol-4yl)thio]acetic acids. pharm. chem. j., 47(2): 96–98 (2013). [7]. n. c. desai, a. m. dodiya, p. n. shihora, a clubbed quinazolinone and 4thiazolidinone as potential antimicrobial agents. med. chem. res., 21(8): 1577–1586 (2012). [8]. v. o. chornous, o. ya. melnyk, o. m. hliebov, m. v. tykhonenko, l. m. sheremeta, o. k. yarosh, et al., the synthesis, hypoglycemic activity and acute toxicity of new imidazole-thiazolidines hybrid structures. j. org. pharm. chem., 14(1): 46-52 (2016). [9]. s. n. kulikov, r. z. hayrullin, s. a. lisovskaya, antimycotic activity of chitozan with various molecular weights and its influence on the cell morphology in some yeast-like fungi. problem. medic. mycol., 12(2): 32-36 (2010) (in russian). [10]. http://pharmaexpert.ru/gusar/acutoxpredict. html (accessed jan. 05, 2019). http://pharmaexpert.ru/gusar/acutoxpredict.html http://pharmaexpert.ru/gusar/acutoxpredict.html 1. introduction it should be noted that comparatively high antimicrobial and antifungal activities have been registered for all the compounds involved in this work. the total averaged mbcc for the entire series in case of s. aureus was 215.6 μg/ml, in case of e. coli... a dependence of antimicrobial activity on the structure of thiazolidone section can easily be understood from comparison between subseries 1 (containing the exocyclic atom of sulphur) and subseries 2 (containing the exocyclic atom of oxygen). an influ... for instance, it can be noted that the series 1 and 2 have same structural composition while antimicrobial and antifungal activities of the sulphur-containing series 1 are higher than those of the oxygen-containing series 2. an average mbcc and mfcc f... transformation of the methylene structural bridge in the set 2 to the methyl one (set 3) results in some increase of averaged antimicrobial and antifungal activity of the compounds and the corresponding values of mbcc and mfcc decrease down to 218.8, ... therefore, it is obvious that the compounds 1a-f reveal the highest germ-suppressing activity and should be analyzed further to find the most effective representative. as seen from the data of table 1, the structure of the imidazole part of a compound is the key factor influencing germ-suppressing activity. for instance, both antimicrobial and antifungal activities are increasing after exchange of a substitute in th... on the other hand, a substitute in the second position of imidazole cycle also seems influential. when the polar atom of chlorine is inserted in the r2 position, the value of mbcc improves to 7.8 μg/ml in case of s. aureus while mfcs reaches 62.5 μg/m... the above discussion is visualized graphically in fig. 2 for better comprehension of comparative bactericide and fungicide activities of some representatives of the subseries 1 and the corresponding control compounds. finally, toxicity of all the synthesized compounds has been evaluated using the computer simulation software gusar online [10]. the toxicity of the entire class of the synthesized compounds has been found low: rat oral ld50 = 1027 mg/kg; rat sc ld50 =... 4. conclusion estimation of quality of frozen desserts with polyfunctional composition 36 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 36 43 quality estimation of frozen desserts with polyfunctional composition olena grek 1 , tatyana osmak 1 , *larisa chubenko 1 ,artur mykhalevych 1 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str.68,kyiv,ukraine, lorkachub@gmail.com *correspondingauthor received22ndjanuary 2019, accepted 29 march 2019 abstract:the article analyzes the structure of consumption of animal and plant origin food products. the characteristic of milk-protein concentrates is being looked at for the purpose of their use in the technology of frozen desserts. the relevance of the development of new frozen desserts with polyfunctional composition is substantiated. the technological process of production of poly-functional ingredients soy-containing protein component and protein-herbal semi-finished product is being dealt with. formulations of frozen desserts with proteins of various origins were developed on the basis of a complex analysis of organoleptic indicators and physico-chemical characteristics. the aim of the work is to evaluate the quality of frozen desserts with poly-functional composition. the object of research is the technology of frozen desserts.it is proved that by using the graph-mathematical method it is possible to predict the quality of frozen desserts with soy-containing protein component in the range of 40...50% and protein-herbal semi-finished products at the level of 20...25%. an analysis of the amino acid composition of frozen desserts has confirmed that the introduction of protein components of different origins in formulation will lead to high biological value products. keywords: low-fat cottage cheese, soy-containing protein component, protein-herbal semi-finished product, frozen dessert, graph-mathematical method. 1. introduction. in recent years, the structure of food consumption has changed dramatically. the results of dynamic observations of actual nutrition of the adult and infant population, as well as data from the state statistics committee of ukraine, show a decrease in the consumption of animal products, as well as vegetable oils, fruits and vegetables. this tendency is caused by a decrease in social accessibility and an appropriate quality assortment. most people consume cheap products with a low biological value, but a high energy content of the diet [1]. as a result of the reduction in the consumption of dairy products, the population is poorly provided with full protein, easily digestible calcium and phosphorus. in the diet of the people, the consumption of animal proteins decreased from 110 g / day (in 1990) to 78 g / day (2015) [1]. actual questions arise in terms of adequate nutrition, with reduced content of fats, sugars and high content of complete proteins. scientific and practical interest lay on the development and implementation of technologies of frozen desserts using milk and protein concentrates, which will reduce the cost of raw materials and lead to the production of high nutritional value food. the raw materials for the aforementioned products are cottage cheese, soy-containing protein component and protein-herbal http://www.fia.usv.ro/fiajournal mailto:lorkachub@gmail.com foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 37 semi-finished product and other traditional ingredients. cottage cheese is characterized by high digestibility. the main feature of cottage cheese is the high content in proteins (10... 18%), most of which is casein. the protein composition of cottage cheese contains all the essential amino acids, which determine the biological value of the product. the object of research is the technology of frozen desserts [2]. soy protein the main component of soycontaining protein concentrate, 88...95% is represented by a water-soluble fraction, which includes soluble globulins (60...81%), albumins (8...25%) and difficult soluble globulins (3...7%). soybean proteins contain the necessary set of essential amino acids, which are almost identical in quality to animal protein [3,4]. the introduction of protein-herbal semifinished products into a frozen dessert will allow the finished product being enriched by whey proteins, thus providing the daily intake of main nutrients. an objective indicator for assessing the quality of frozen desserts is a complex analysis of organoleptic and physicochemical characteristics. thus, the development of a complex assessment combining the above mentioned indicators of frozen desserts with poly-functional composition, will allow the obtainingof a product with a balanced composition and of guaranteed quality. the main tasks of this scientific work are: to use the graph-mathematical method in order to establish a ratio between the milk base and protein components in the formulation of frozen desserts with polyfunctional composition; to study the biological values of model samples of frozen desserts with polyfunctional composition. the purpose of the articleis to evaluate the quality of frozen desserts with polyfunctional composition, using the graphmathematical method. 2. materials and methods for the soy-containing protein component the following were used soy protein concentrate, dried skim milk, drinking water, dvs starter (lactococcuslactis subsp. lactis, lactococcuslactis subsp. cremoris, lactococcuslactis subsp. lactisbiovar. diacetylactis). the technological process was carried out in the following sequence: the recipe components (soy protein concentrate and skimmed milk powder) are taken into consideration in terms of quantity and quality, weighed, sifted and restored at the temperatures of 35...40 °c. for better dissolution, the resulting mixture can be maintained for 30... 40 minutes. in order to isolate the insoluble particles of dry components, the mixture is subjected to filtration. after that, the resulting mixture is pasteurized at the temperature of 76...78 °c and cooled to a temperature of inoculation of the mixture, which occurs by the starter culture at a temperature of 30...34 °c. the incubation of the mixture takes place during 6...8 hours. after that, there occurs the process of whey extraction and self-pressing and pressing of the resulted protein base (soy-containing protein component) [3,5]. the indicators of the soy-containing protein component are shown in table 1 [3,6]. to obtain protein-herbal semi-finished products as a coagulant, the ground part of sorrel rumex [7] was used during the thermos-acid precipitation of milk proteins at a temperature of 96...98 ° с. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 38 table 1 indicators of soy-containing protein component indicator characteristic taste and smell sour milk, with a slight vegetable flavor structure and consistency homogeneous, tender, creamy color white with a grayish tinge titrated acidity, ºt 174...180 active acidity, ph 4.2...4.3 mass fraction of moisture,% 74...76 according to the literature data, the water content in the leaves of rumex is (90...92)%, protein 1.5%, total carbohydrates (3.0... 5.3)%, fiber 1%. minerals are fixed at (mg%): na (15), k (500), ca (47), mg (85), p (90), fe (2,0), vitamins, in (mg% ): c (43...81), b1 (0.19), b2 (0.1), pp (0.3), β-carotene (2.5). the amount of oxalic acid is determined 360 mg%, which will allow the use of this type of plant material as a coagulant [8,9]. the obtained protein-herbal semi-finished product had the following qualitative parameters: the mass fraction of moisture (64 ± 2)%, titrated acidity (80 ± 1) ° t, color light pistachio, not uniform, consistency mild, slightly broken on slices, to a degree tight, taste milkprotein, cheesy, without foreign smells, with a slight herbal flavor. the parametric scheme of production of protein-herbal semi-finished products is shown in figure 1. fig. 1. parameter scheme of production of protein-herbal semi-finished products reception and preparation of skimmed milk pasteurization t= 93...95c thermo-acid precipitation of milk proteins whey self-pressing of bunch acceptance and sorting of herbal raw materials washing drying grinding pressing 1…2 min juice meal for drying for recycling foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 39 the determination of physico-chemical and organoleptic indicators of frozen desserts was carried out according to generally accepted methods [10]. amino acid composition of frozen desserts – by ion-exchange liquid-column chromatography on automatic analyzer of amino acids t339. the coefficient of difference of amino acid fast (kdaf) and biological value of protein (bv) was determined by the method m.p. chernikov [4]. it is based on the postulate that the assimilation of eaa is limited to limiting content amino acids that is, the kdaf is the average arithmetic excess of the eaa amino acid accelerator as compared to the smallest limit of limiting amino acids. kdaf are found by the formula: kdaf= (σdaf) / n, (2.2) where kdaf coefficient of difference of an amino acid,%; daf is the difference in the amino acid rate for each nac compared with the amino acid soon limiting amino acids, %; n the number of amino acids. the smaller the value of kdaf is, the more fully used by eaa for the needs of biosynthesis. so, the biological value of the protein is calculated by the formula: bv = 100 kdaf, (2.3) where bv biological value of protein,%. the graph-mathematical method is the determination of the optimal value (point of intersection) of complex indicators by graphic representation of the simultaneous influence of two variables, which are reflected on parallel axes of the ordinate on a function, which is reflected on the axis ofabscise. the complex indicator of organoleptic evaluation was calculated using the equations below: where мt=0.4;mcon,=0.3; мcol=0.3 – coefficients of weight of the corresponding group of indicators: taste and smell, structure and consistency, colour and appearance; са, conа, colа – absolute values of the corresponding group of indicators: taste and smell, structure and consistency, colour and appearance; сb, conb, colb – basic values of the corresponding group of indicators: taste and smell, structure and consistency, colour and appearance. the complex index of physical and chemical characteristics was calculated by the equations given below: whereмa=0.4;мdef,=0.3; мr=0.3 – coefficients of weighting of the corresponding group of indicators: acidity, defeat, resistance to melting; defа, rа, aа – defb, rb, ab absolute values of the corresponding group of indicators: loss, resistance to melting, acidity; defb, rb, ab – basic values of the corresponding group of indicators: loss, resistance to melting, acidity. 3. results and discussion at the department of milk and dairy products technology of the national university of food technologies, there were developed formulations of frozen desserts with the use of low-fat cottage cheese, soy-containing protein component and protein-herbal semi-finished product as a protein enrichment agent [3, 5, 6]. at the first stage, organoleptic and physicochemical parameters of freshly prepared samples of frozen desserts with polyfunctional components were determined. the results are shown in the tables 2 and 3. for an objective assessment of the quality of frozen desserts in a laboratory, a 30point system was developed that included foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 40 the following groups of indicators: taste and smell – 15, structure and consistency – 10, colour – 5. when defects of quality of frozen desserts were detected, a decrease in the maximum predicted score for a certain number of points was made. frozen dessert with an estimate below 24 points is discarded. table 2 organoleptic indicators of frozen desserts with poly-functional components indicator characteristics of a frozen dessert with low-fat cottage cheese with soy-containing protein component with protein-herbal semi-finished product taste andsmell pure, delicious sourmilk taste, without foreign flavors and odors a pleasant sour-milk taste with a slight flavor of soy, without foreign smells pure, delicious sourmilk taste with a light herbal flavor, without foreign smells structureandconsistency homogeneous throughout the mass, delicate creamy color white white, with a grayish tinge lightpistachio table 3 physico-chemical indicators of frozen desserts with poly-functional components indicator indicator rate for frozen dessert with low-fat cottage cheese with soy-containing protein component with protein-herbal semi-finished product mass fraction,%, not less: fat / sugar / dry matter 3.5/14.0/29.5 3.5/14.0/29.5 3.5/14.0/30.5 acidity, ° t 84 80 85 active acidity, ph 6.1 6.2 6.15 defeat,% 54 51 52 resistancetomelting, min 52 55 43 for the objective assessment of the influence of organoleptic and physicochemical indicators on the consumer properties of the product, a general integrated quality index is used. the assessment of the quality of products by the complex indicator was conducted on the following scale: 0.9...1 rating "excellent", 0.8...0.89 score "good", 0.7...0.79 the score "satisfactory". the value of a complex indicator of organoleptic evaluation and physical and chemical indices of experimental samples of frozen desserts are shown in the table 4. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 41 table 4 complex index of organoleptic and physico-chemical evaluation of frozen desserts indicator sample of frozen dessert with low-fat cottage cheese (control) with soy-containing protein component with proteinherbal semifinished product organoleptic evaluation 1.0 0.935 0.957 physico-chemical indicators 1.0 0.942 0.95 the rational mass contribution of the protein component in the formulations of frozen dessert was determined by the graph-mathematical method [11], by assessing the optimal ratio the milk basis: the protein component, taking into account the complex interaction of the indicators of organoleptic evaluationko and physicochemical characteristicskp-c (figure 2). fig. 2. graph-mathematical method of determining the mass fraction of the introduction of protein components as can be seen from the chart below, the optimum mass fraction of making soycontaining protein component was of 40...50%, protein-herbal semi-finished product 20... 25%. the technology of production of frozen desserts with poly-functional composition was carried out in three stages: the preparation of a normalized mixture (scheduled parameters: a mass fraction of fat – 3.5%, a skimmed milk residue 12%, sugar 14%) and protein component, mixing of the normalized milk base and protein component. the mixing of milk and protein basis is assumed with the following ratio of components: with use of low-fat cottage cheese milk basis: cottage cheese, low-fat cheese, as (50... 60): (40...50); with use of a soy-containing ingredient-milk basis: soy-containing component, as (50...60): (40...50); with use of protein-herbal semifinished products milk basis: proteinherbal semi-finished product, as (75... 80): (20...25). foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 42 the restriction of the amount of addition of protein components is due to an increase in acidity up to 100 ° t and specific organoleptic characteristics due to the taste of the incorporated protein ingredients. it is advisable to mix the milk base with the protein component directly before the freezing of the mixture, which ensures the addition of a homogeneous mixture to the freezer, without increasing the titratable acidity. to study the biological value of the developed frozen dairy dessert, its amino acid composition was determined. the following samples were studied: frozen dessert with cottage cheese (control) and desserts with soy-containing component and protein-herbal semi-finished products. to assess the degree of using protein, the coefficient of difference between amino acid rate (cdaf) and biological value of protein (bv) was calculated. the difference between the amino acid rates of the essential amino acids of the experimental samples is shown in figure 3. fig. 3. difference between the amino acid af of the essential amino acids of the experimental samples as can be seen from figure 3 the maximum surplus, characteristic for frozen desserts with cottage cheese and proteinherbal semi-finished product, provides leucine, and a sample of frozen dessert with soy-containing protein component threonine. the smaller the value of cdaf is, the more fully used by eaa for the needs of biosynthesis. the indicators of biological value (cdaf and bv) are shown in figure 4. fig.4. indicators of biological value of experimental samples the given biological values indicate that the introduction into the formulation of a frozen dessert of protein-herbal semifinished products will result in a product whose biological value is 62%, which is practically close to the control sample 71%. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 olena grek, tatyana osmak, larisa chubenko, artur mykhalevych, quality estimation of frozen desserts with polyfunctional composition,foodandenvironmentsafety , volume xviii, issue 1 – 2019, pag. 36 – 43 43 4. conclusions 1. by using the graph-mathematical method, it was established that the mass fraction of protein component in formulations of frozen milk desserts varies in the range of 40...50% (with soycontaining protein component), 20…25% ( with protein-herbal semi-finished product), providing the optimal ratio of organoleptic and physico-chemical parameters of experimental samples. 2. an analysis of the amino acid composition of frozen desserts has shown that the incorporation of protein components into the formulation will provide the product with a biological value of 37% (for a protein containing protein soya) and 62% (for protein-herbal semifinished products), respectively. 5. references [1] smolyar v.the state of actual nutrition of the population of independent ukraine, problems of nutrition,5-9, (2012). [2] savchenko o., grek o., krasulya o.topical issues of milk and protein concentrate technology: theory and practice, 293 p., (2015). [3] osmak t., ryabokon n. soy-protein protein component a promising ingredient of frozen dessert, food industry of agroindustrial complex, 1:38-41,(2014). [4] chernikov n., chemical methods for determining the quality of food proteins, questions of nutrition, 1:42-45, (1986). [5] osmak t. development of technology of frozen dessert with sugar substitutes: author's abstract. diss.cand.tech sciences: 05.18.04. «food technology», kyiv: 24 p., (2013). [6] grek o., osmak t., chubenko l., mykhalevych a. technological aspects of production of a frozen dessert with protein-herbal component, food and environment safety, 2: 197204, (2018). [7] grek o., tymchuk a., chubenko l., ovsienko k. research of quality indicators of curd products on the basis of protein-herbal clots, food and environment safety, 4: 262-268, (2017). [8] evtefeev yu., zykovich s. investigation of chemical composition and nutritional status of rumex k-1 sorrel, bulletin of the altai state agrarian university, 76-80, (2011). [9] bagry a. polyphenolic compounds of some sorrel species (rumex): author's abstract. dis for the sciences. degree candidate farm sciences, kyiv, 17 p., (1965). [10] grek o., yushchenko n., osmak t. workshop on milk and dairy products technology: teaching manual, 431 p., (2013). [11] dorokhovich v.scientific substantiation and development of technologies of flour confectionery products of special dietary consumption author's abstract diss.cand. tech sciences: 05.18.16 «food technology», kyiv: 39 p., (2010). statistical analysis of the results of trans-resveratrol investigation in grapes of storgozia, kaylashki rubin, trapezitsa, rubin, bouquet and pinot noir varieties 208 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag.208 213 statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties vanyo haygarov 1 , tatyana yoncheva 1 , *dimitar dimitrov 1 , emil tsvetanov 2 1department of selection, enology and chemistry, institute of viticulture and enology, pleven, bulgaria, ilv@el-soft.com 1department of technologies and plant protection, institute of viticulture and enology, pleven, bulgaria, ilv@el-soft.com *corresponding author: dimitar_robertov@abv.bg received 4th may 2019, accepted 27th september 2019 abstract: a study for statistical analysis of the results of the trans-resveratrol content in grapes of hybrid bulgarian vine varieties was carried out. the chemical composition of the grapes was analyzed. excellent technological grapes maturity (194.33 ± 19.13 g/dm3 244.00 ± 20.80 g/dm3 of accumulated sugars) was achieved. the detected ranges of titratable acids (5.66 ± 0.25 g/dm3 7.05 ± 0.33 g/dm3), tartaric acid (3.04 ± 0.15 g/dm3 4.66 ± 0.25 g/dm3), malic acid (4.42 ± 0.27 g/dm3 6.33 ± 0.15 g/dm3) and ph (3.24 ± 0.03 3.60 ± 0.32) were typical for grapes obtained from vines grown in the region of the danube plain, central northern bulgaria. trans-resveratrol was found in the highest amount (3.52 ± 0.59 mg/dm3) in the kaylashki rubin grapes. its content was close to this (3.46 ± 0.40 mg/dm3) of the control variety pinot noir. the lowest (1.78 ± 0.17 mg/dm3) was the amount of trans-resveratrol in the grapes of storgozia variety. the statistical processing divided the varities into two groups (by trans-resveratrol content): the first group had higher trans-resveratrol content and included the varieties kaylashki rubin (3.52 ± 0.59 mg/dm3), bouquet (3.18 ± 0.15mg/dm3) and pinot noir (3.46±0.40 mg/dm3); the second group consisting of the varieties rubin (2.40±0.09 mg/dm3), trapezitsa (2.30±0.80 mg/dm3) and storgozia (1.78±0.17 mg/dm3) with lower trans-resveratrol content. keywords: grapes, wine, chemical composition, resveratrol, intraspecific and interspecific hybridization. 1. introduction the trans-resveratrol as a chemical compound in grapes has been given attention by the scientific community since 1990 when the compound has been shown to have cardioprotective properties. transresveratrol, as a gift of nature, has a wide range of health-promoting effects, including antibacterial, antifungal, antioxidant, anti-inflammatory, cardioprotective and anti-tumor activities. it protects vine from ultraviolet rays, viruses, bacteria, fungi and acts as a natural antibiotic [1]. the trans-resveratrol is an extremely rare substance. in addition to grapes and wine, certain amounts of it are contained only peanuts and some rare species of forest fruits such as blueberries, blackberries, strawberries, mulberries and cherries. chemically, it is a type of natural polyphenol (stilben) [2]. it is produced naturally by some plants (including vine) when they are attacked by pathogens such as bacteria or fungi. the other factors for its accumulation include climate conditions (the big differences between daily and nightly temperatures), weather anomalies resulting in stress for the plant. its http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro mailto:mariap@fia.usv.ro mailto:dimitar_robertov@abv.bg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov, statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties, food and environment safety, volume xviii, issue 3 – 2019, pag. 208 213 209 accumulation is the plant response to overcoming the unfavorable environmental conditions. the trans-resveratrol is a powerful antioxidant. it has a high ability to capture and block the free oxidative radicals and prevents cells from malignant alterations. the substance has a number of benefits: anticancer and antiviral effect, it increases the energy and improves heart function, it normalizes blood glucose levels and stimulates the nervous system and significantly improves the sport achievements. the trans-resveratrol is an antidepressant that reduces “bad” ldlchlesterol. it is also thought to block the formation of cox-2 enzyme that promotes the development of colon cancer [1]. 2. materials and methods the study was carried out at the institute of viticulture and enology – pleven (ive), during the period 2015-2017. red grape varieties storgozia, kaylashki rubin, trapezitsa, rubin and bouquet, selected at ive – pleven, r. bulgaria by the intraspecific and interspecific hybridization [3-4] were the object of the study. they were cultivated in certain micro-regions throughout the country (pleven, sadovets, barkach, suhindol, pavlikeni, brestovitsa, karnobat, burgas, blagoevgrad and sandanski). the vine plantation was located at the experimental base of the ive, on an area of 0.2 ha for each studied variety. the vineyards were fruit-bearing. the vines were grown on a medium stem training system. the planting distance was 3.00 m / 1.20 m. berlandieri x riparia so4 was the used rootstock. during the vegetation period standard agricultural practices and plant protection measures were carried out. pleven is situated in central northern bulgaria. this region is characterized by cold winters and hot summers. the sum of the average air temperatures during the vegetation period (from 16th april until 31st october) was approximately 4000 °с. the average air temperature of the warmest month was always above 20°с. temperatures below 20°с and over 30°с were not recorded. the conditions were fovarable for obtaining of quality red grapes [5]. the brief climatic characterization of the years of the study was presented on table 1. table 1. brief climatic characterization of study years probability (р %) of precipitation and average air temperature (т°) for the years of the study years 2015 2016 2017 n (v –ix) % 17 (very wet) 35 (wet) 6 (very wet) тᵒ (v –ix) 29 (average hot) 56 (average cool) 33 (average hot) р – probability, n – (precipitations, тᵒ average air temperature with regard to the average air temperatures, the years 2015 and 2017, could be defined as average hot and 2016 as an average cool. as for the precipitation 2015 and 2017 were very wet and 2016 – wet. the soil in the region was leached chernozem on loess foundation. it was suitable for vine cultivation. the intraspecific varieties storgozia, kaylashki rubin and trapezitsa and the interspecific variety bouquet were actually resistant to stress factors (lower winter temperatures and mildew) [3-4]. the parental forms of the studied varieties were as follows: storgozia – bouquet х villard blanc12375 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov, statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties, food and environment safety, volume xviii, issue 3 – 2019, pag. 208 213 210 kaylashki rubin – pamid x hybrid vі-2-15 х gamay noir х vitis amurensis trapezitsa – dunavska gamza х marseilles early dunavska gamza – bouquet х villard blanc 12375 rubin – nebbiolo x syrah bouquet – mavrud х pinot noir the control variety was pinot noir of vitis vinifera, which according to the available literature data had the highest content of trans-resveratrol [6]. upon reaching of the technological maturity (20-23% sugars), the grapes were harvested and analyzed. the main chemical indicators of the grape pulp from the studied varieties were determined according to the methods generally accepted [7,2,8]. for determining of the trans-resveratrol content in the grapes for each sample, twofold extraction of the grape pulp with methanol was carried out. high pressure liquid chromatography (hplc), according to the modified method of anli et al. [9], was used for determining the transresveratrol content in the wine samples. the results were statistically processed by one-factor analysis of variance (anova) [10]. the aim of the present study was to analyze statistically the results of transresveratrol content in grapes of hybrid bulgarian grape varities. 3. results and discussion table 2 presents the grapes chemical composition and the date of harvest for each variety studied. table 2. grapes chemical composition of the studied varieties (average with ±sd) for the period 2015 2017 from the indicators presented in table 2, it was evident that grapes have accumulated sugars from 194.33±19.13 g/dm3 (trapezitsa) to 244.00±20.80 g/dm3 (rubin). the sugars in must of ripe and healthy grapes are in the range of 150.00 250.00 g/dm3 [2]. the results of this study correlated with this finding. the grapes were reached good technological maturity. the titratable acids were within the range from 5.66±0.25 g/dm3 (trapezitsa) to 7.05±0.33 g/dm3 (kaylashki rubin). the variety storgozia kaylashki rubin trapezitsa rubin bouquet pinot noir harvest date 06.10.2015 10.09.2016 18.09.2017 16.09.2015 11.09.2016 12.09.2017 02.09.2015 30.08.2016 28.08.2017 03.09.2015 31.08.2016 12.09.2017 20.09.2015 12.09.2016 26.09.2017 03.09.2015 30.08.2016 30.08.2017 reducing sugars g/dm 3 232.33±9.45 212.33±10.50 194.33±19.13 244.00±20.80 221.00±11.53 234.00±13.52 titratable acids g/dm 3 5.97±0.36 7.05±0.33 5.66±0.25 6.81±0.55 6.67±0.59 6.80±0.33 tartaric acid g/dm 3 3.30±0.42 4.50±0.32 4.66±0.25 3.04±0.15 3.32±0.12 3.56±0.17 malic acid g/dm 3 5.52±0.21 5.84±0.19 4.95±0.10 6.33±0.15 5.49±0.25 4.42±0.27 рн 3.60±0.32 3.40±0.28 3.45±0.22 3.58±0.26 3.39±0.31 3.24±0.03 transresveratrol mg/dm 3 1.78±0.17 3.52±0.59 2.30±0.80 2.40±0.09 3.18±0.15 3.46±0.40 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov, statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties, food and environment safety, volume xviii, issue 3 – 2019, pag. 208 213 211 acid content of the grape juice is dependent of the grape variety, geographic location and climatic conditions of the region in which it is grown [11]. at the conditions of bulgaria, the grapes acids are in the range of 4.00 12.00 g/dm3 [11]. the results for the “titratable acidity” indicator in the present study fully correlated with the range of acidity variations in mature grapes. the tartaric acid is essential for grapes. its quantitative accumulations are influenced exclusively by the location of the area to the south (2.00 3.00 g/dm3); in the northern regions (6.00 g/dm3 and above) [11]. the highest quantitative presence (4.66 ± 0.25 g/dm3) of tartaric acid was found in the grapes of the kaylashki rubin variety. the lowest (3.04 ± 0.15 g/dm3) was its amount in the rubin grapes. the region where the grapes were grown was the danube plain, central northern bulgaria. all established tartaric acid concentrations in the grapes of the varieties studied exceed 3.00 g/dm3, which was typical for this northern region. the malic acid is the second by quantitaty. in the grapes of the northern region it is present in quantities of 4.00 7.00 g/dm3 and in the southern 1.00 3.00 g/dm3 [11]. in the present study, it was found in the highest amount (6.33 ± 0.15 g/dm3) in rubin grapes and in the lowest (4.42 ± 0.27 g/dm3) in pinot noir. its variations were typical for grapes grown in the northern region of bulgaria. the ph of grapes must be within 2.8 3.8 (chobanova, 2012). in the present study, the ph was recorded in the range of 3.24 ± 0.03 (pinot noir) 3.60 ± 0.32 (storgozia). the data was in agreement with the presented by [2]. the data showed that the amount of transresveratrol in the grapes from the studied varieties in the region of pleven was almost equal and comparable to that of pinot noir control of vitis vinifera. the results for trans-resveratrol (average for 2015, 2016 and 2017 harvests) and its statistical analysis are presented in table 3. the quantitative characterization distinguished the hybrid kaylaski rubin variety as the quntitavely dominated of trans-resveratrol (3.52 ± 0.59 mg/dm3). the difference between it and the pinot noir control (3.46 ± 0.40 mg/dm3) was negligible. in all other studied varieties, lower amounts of trans-resveratrol were observed in comparison with pinot noir. the control variety showed its genetic superiority for increased synthesis of resveratrol. the lowest amount of the stilbene (1.78 ± 0.17 mg/dm3) was found in the must of storgozia variety. table 3 presents the statistical processing of the obtained results. on the average, for the three years of the research, the studied varieties could be divided into two groups. the first group had higher trans-resveratrol content and included the varieties kaylashki rubin (3.52±0.59 mg/dm3), bouquet (3.18±0.15 mg/dm3) with pinot noir control (3.46±0.40 mg/dm3). the difference between them was insignificant and statistically unproven. the second group consisting of the varieties rubin (2.40±0.09), trapezitsa (2.30±0.80) and storgozia (1.78±0.17) had proven lower trans-resveratrol content compared to the first one and the difference between the varieties in the group was also statistically unproven. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov, statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties, food and environment safety, volume xviii, issue 3 – 2019, pag. 208 213 212 table 3. statistical analysis of the results for the content of trans-resveratrol in grapes of studied grape varieties variants average value, mg/dm 3 trapezitsa pinot noir kaylashki rubin bouquet storgozia differ. sign. differ. sign. differ. sign. differ. sign. differ sign. trapezitsa 2.30±0.80 x x pinot noir 3.46±0.40 1.16 ++ x x kaylashki rubin 3.52±0.59 1.22 ++ 0.07 n.s x x bouquet 3.18±0.15 0.88 + -0.28 n.s -0.34 n.s x x storgozia 1.78±0.17 -0.52 n.s -1.68 ---1.75 ---1.40 -x x rubin 2.40±0.09 0.10 n.s -1.06 --1.13 --0.78 0.62 n.s 5% (+) (-); 1% (++) (--); 0.1% (+++) (---); < 5% (n.s) 4. conclusion from the results obtained, the following conclusions can be made: • the studied grapes reached an excellent technological maturity with accumulated sugars of 194.33 g/dm3 (trapezitsa) to 244.00 ± 20.80 g/dm3 (rubin). • the presence of titratable acids (5.66 ± 0.25 g/dm3 7.05 ± 0.33 g/dm3) was typical for grapes grown in the region of central northern bulgaria. • the tartaric and malic acids were found in concentrations typical for grapes of northern bulgaria. • the quantitative characteristic for the presence of the stilbene trans-resveratrol distinguished the grapes of the kaylashki rubin variety. the grapes from this variety contained 3.52 ± 0.59 mg/dm3 transresveratrol. the established quantity was close to the amount found in the control (3.46 ± 0.40 g/dm3) pinot noir. • the chemical indicators of the studied varieties selected through the intraspecific and interspecific hybridization were not different from those of vitis vinifera control. these results indicate that the grapes of the varieties studied are suitable for the production of quality red wines. • the statistical processing divided the varieties into two groups: first witn the highest content of resveratrol kaylashki rubin (3.52±0.59), bouquet (3.18±0.15) and rubin (2.40±0.09); the second with the lowest resveratrol content storgozia (1.78±0.17), trapezitsa (2.30±0.80) and pinot noir control (3.46±0.40).the statistical difference between the two groups of varieties was insignificant and statistically unproven. the amount of resveratrol in the grapes from the studied varieties cultivated in the region of pleven, central northern bulgaria was almost equal and comparable to those of pinot noir control from vitis vinifera. 5. references [1]. fartsov k., alyakov m., onisiforu d., pargov m., videnova r., toshev, d., trans-resveratrol on red wines and its physiological effect, at print, lymasol, cyprus, (2013). (bg) [2] chobanova d., manual for enology exercises, academic press of university of food technologies, plovdiv, bulgaria, (2007). (bg) [3]. roychev v., selection evaluation of hybrid vine forms for the quality of wine, viticulture and enology magazine, 2: 6-14, (2014). (bg). [4]. ivanov m., hybridization in the vine selection. monography, academic publishing of agricultural university, plovdiv, (2016). (bg). [5]. haygarov v., study the possibilities for production of quality white wines in the region of pleven. dissertation thesis, plovdiv, bulgaria: university of food technologies, 246 pp., (2012). (bg) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 vanyo haygarov , tatyana yoncheva , dimitar dimitrov , emil tsvetanov, statistical analysis of the results of trans-resveratrol content in grapes of hybrid bulgarian grape varieties, food and environment safety, volume xviii, issue 3 – 2019, pag. 208 213 213 [6]. videnova r., investigation of changes in resveratrol content in the processing of berries. dissertation thesis, plovdiv, bulgaria: university of food technologies, (2017). (bg) [7]. ivanov t., gerov s., yankov a., bambalov g., tonchev t., nachkov d., marinov m., practicum in wine technology, plovdiv, bulgaria: publ. house hristo g. danov, 530 pр., (1979). (bg) [8]. pandeliev s., harizanov a., botyanski p., roychev v., kemilev s., practical advices of vine and wine, dionysus, sofia, bulgaria, (2010). (bg) [9]. anli e., demirey n., fzkan m., trans–resveratrol and other phenolic compounds in turkish red wines with hplc, journal of wine research, 17(2): 117–125, (2006). [10]. dimova d., marinkov e., experimental work and biometry. academic publishing house of higher institute of agriculture, plovdiv, pp. 263.,(1999). (bg) [11] abrasheva p., bambalov k., georgiev a., viticulture and enology, ed. matkom, sofia, bulgaria, pp. 344, (2008). (bg) 1. introduction 4. conclusion 84 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 12020, pag. 84 91 practices of biosecurity measures and their consequences on poultry farms in abidjan district *gblossi bernadette goualie 1 , souleymane bakayoko 2 , kalpy julien coulibaly 2 1laboratory of biotechnology, faculty of biosciences, felix houphouet-boigny university, abidjan, 22 bp 582 abidjan, ivory coast, bettygoualie@yahoo.fr 2institut pasteur of côte d’ivoire, abidjan, côte d’ivoire *corresponding author received 18th february 2020, accepted 25th march 2020 abstract: in côte d'ivoire, poultry farming is an important source of income for many people. however, the industry faces many health constraints that cause enormous economic losses. since implementation of biosecurity measures is known to reduce the risk of disease transmission, the aim of this study is to analyze the biosecurity measures applied in poultry farms in abidjan district. therefore, 91 farms were investigated using a face‐to‐face interview‐based questionnaire. the questionnaire consisted of question sets related to production characteristics and the biosecurity components. the results revealed that the majority of farms were broilers chicken (69.65 %) and laying hens (28.35%). regarding isolation of farms, 45% had no physical barriers around the farms and in 41% of the farms visited, the presence of other farm animals was observed. concerning sanitation, the biosecurity level was low for 98.5% of farms with only one cleaning process of poultry houses between two flocks. however, using disinfectants is implemented in all visited farms. the most frequently symptoms observed are loss of appetite, weakness, weight loss, respiratory problems, coughing, fever, and diarrhea. but in 79 % of cases many animal deaths were due to diarrheal diseases. the majority of farmers (89 %) use as soon as the first symptoms appear, various antibiotics like macrolides, betalactamines, tetracyclines, fluoroquinolones, aminosides and polymycines. the low implementation level of biosecurity measures and overuse of antibiotics in poultry farms suggests that government officials should monitor the issues of biosecurity and the use of antibiotics in this sector very seriously. keywords: poultry sector, hygien practices, biosecurity, drugs overuse 1. introduction poultry farming is a key factor in the development of many nations, both for nutritional and economic reasons [1]. in côte d'ivoire, the poultry production sector accounts for nearly 5% of agricultural gross domestic product (gpd) and 2% of total gpd [2]. in 2017, for example, total revenue was estimated at around us$48 million [3], corresponding to 44,000 tons of poultry meat and 36,000 tons of eggs. this covered 90% of the population's poultry meat needs and 100% of its egg needs. in addition, the ivorian government intends to increase this performance by reaching 60,000 tons of poultry meat and more than 1.678 billion eggs/year in 2020 to fully cover the population's animal protein needs [3]. the poultry industry is made up of two sectors, traditional poultry farming and modern poultry farming. in the modern production system, breeders generally use imported breeds that have a very high productivity compared to local breeds, making the poultry sector very dynamic compared to the traditional production system in côte d'ivoire [3]. however, this sector faces many problems including various pathologies that lead to a significant drop in meat and egg production. the most common pathologies are gumboro and newcastle diseases, http://www.fia.usv.ro/fiajournal mailto:bettygoualie@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 85 salmonellosis, collibacillosis and avian coccidiosis [4]. the production loss caused by these diseases is estimated at 39.45%, corresponding to more than us$ 5.2 million [5]. the occurrence of these diseases is generally due to poor control of biosecurity measures [6]. during poultry production indeed, biosecurity measures help to prevent the introduction of pathogens into the farm (external biosecurity or bio-exclusion) and to prevent the spread of the disease to uninfected animals on the farm or to other farms where the pathogen is already present (internal biosecurity or biocontainment) [7]. these biosecurity measures include the application of hygiene rules in poultry farms and the implementation of preventive means such as vaccination [8]. thus, the aim of this study is to analyze the biosecurity measures applied in poultry farms in abidjan district. these data could contribute to a better understanding of the economic losses due to many diseases affecting animals during the rearing process, to improve sanitary conditions in poultry farms and reduce the use of antibiotics in this sector in our country. 2. matherials and methods study area and farm selection criteria the study was carried out from january to june 2018 on poultry farms located in district of abidjan (in the south of côte d'ivoire). the subdivisions bingerville, port-bouet, ayama and yopougon were chosen due to the importance of poultry farming in these locations (figure 1). the district of abidjan lies between longitude 4° 1' 59.999" w of the greenwish meridian and latitude 5° 19' 0.001" n of the equator. this region is characterized by a typical humid tropical climate with temperature ranges between 22 ° c and 32 ° c and a precipitation evaluated to 787 mm and 1500 mm throughout the year [9]. fig 1. map of the district of abidjan (in the south of côte d'ivoire) indicating the study area food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 86 questionnaire design and selection of farms the questionnaire consisted of question sets related to production characteristics (farm size, breed and production system, using of antibiotics among others) and biosecurity components (isolation and sanitation items). questions were constructed based on the epidemiology of common chicken's diseases in côte d'ivoire. the survey based on the formulated questionnaire was carried out through a face‐to‐face interview between the researcher and the farmers and personal observations of the researcher. only farm’s with at least 100 chicken heads were selected for the survey. determination of the adoption level of evaluated parameters by farmers the adoption level of a given parameter was obtained by dividing the total number of farms applying that measure by the total number of visited farms. the ratio was expressed as a percentage. 3. results and discussion general characteristics of poultry husbandry from visited farms a total of 91 poultry farms were visited during this survey. of these farms, 27 were in bingerville, 33 in port-bouet, 21 in ayama and 10 were located in yopougon. generally, in all visited farms, the number of animals ranged from 150 to 2,000 heads. moreover, broilers chicken and laying hens farming is the most developed with respectively in 69.65% and 28.35% of visited farms. these results are similar with finding of bitty [10] that has showed that poultry production in abidjan area is dominated by the breeding of broilers chicken. according to this author, increase of chicken meat consumption in this area would lead to the increase of broilers chicken breeding. among the visited farms, only 35% are headed by farmers with basic certificate in poultry production system. despite, only 5.4 % regularly use a veterinary service for animal health monitoring. this fact could lead to bad poultry breeding conditions, misdiagnosis in cases of diseases and therefore inadequate therapeutic treatment [11, 12]. application of biosecurity measures in general, the application of the biosecurity procedures is essential for the success of any type of animal production. indeed, biosecurity measures reduce the risk of introducing pathogens during the poultry production processes [13] and then reduce financial losses [14]. in this study, we focused on two aspects of biosecurity during the poultry production process, namely farm isolation and on-farm hygiene practices. the isolation of the farm on 45% of the farms visited, no physical barriers were observed around the farms. however, 39.5% of the farms had a metal or plastic sheeting fence, while 15.5% had a real brick fence around the farm. the absence of a physical barrier such as real brick in most of the poultry farms visited could promote the free movement of rodents and wild birds, which are potential sources of contamination of chickens by various pathogenic microbes [15]. in addition, proximity between the different farms visited (within 100 meters) was observed in more than 98% of cases. dosso [16] made the same observations in poultry farms in the agnibilékro area. in 41% of the farms visited, the presence of other farm animals (pigs, rabbits, other poultry, etc.) and pets (dogs, cats) was observed. these animals could be microbial sources of contamination of chickens. indeed, pigs are generally carriers of many pathogens such as salmonella, e. coli, coccidia. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 87 on the other hand, the proximity observed between farms could allow the spread of pathogenic microorganisms from one farm to another. indeed, studies conducted in denmark, netherlands and australia have shown a relationship between the risk of infection of chickens and the distance between farms respectively for gumboro disease [17], h7n7 avian influenza [18] and newcastle disease [19]. these authors indicated that the probability of occurrence of these infections decreased with increasing distance between farms. vandekerchove et al. [20] also showed that the probability of colibacillosis decreased six-fold with a 1 km increase in distance between herds. therefore, the proximity observed between the farms visited could explain the different pathologies affecting many farms and the huge economic losses estimated at more than us$ 5.2 millions per year in the poultry sector in côte d'ivoire [2123]. hygiene practices on farms in general, poultry are housed in a poultry house built with a concrete floor (100 %). this could be a major advantage that could facilitate the disinfection process [24]. poultry farmers reported that cleaning of poultry houses is carried out once (98.5 %) or twice (1.5 %) between two flocks. the majority (64.71 %) of owners use a sodium hypochlorite solution for this disinfection. some owners (31.51 %) also reported that they use other disinfectants such as lime, methylene blue and formaldehyde in addition to sodium hypochlorite solution. in all farms visited, farmers also indicated that they cleaned feeders at the same frequency. however, the use of detergents (such as sodium hypochlorite) during cleaning could help to maximize the removal of pathogenic flora that contaminates poultry houses. in addition, on most of the farms visited, farmers cleaned the water troughs daily, using simple water without detergent. this daily operation avoids the proliferation of pathogenic flora in the drinkers but is insufficient due to the absence of disinfectant. symptoms of poultry diseases and antibiotic treatment more than 98% of farmers in the study area reported that the symptoms frequently encountered in cases of disease include loss of appetite, weakness, weight loss, respiratory problems, coughing, fever, diarrhea (figure 2). moreover, according to these farmers, the pathologies actually identified are fowl plague (7.69 %), gumboro disease (9.89 %), cocidiosis (12.08 %) and infectious bronchitis (5.49 %). thus, in the majority of cases (more than 64 %), the causes of these symptoms remain unknown or are not investigated. it should also be noted that 79 % of farmers indicated that many animal deaths were due to diarrheal diseases. according to the farmers surveyed, losses due to these diseases can range between 5 and 100 % of the animals. since the late 1970s, the poultry sector has witnessed the emergence or re-emergence of at least one infectious disease every year on average [25]. according to barnes et al. [26], these diseases can be divided into two classes, namely production diseases affecting animal performance and classical pathologies. production diseases can be responsible for growth retardation and, in severe cases, can lead to the death of the majority of the flock within a few days. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 88 fig 2. symptoms reported by abidjan disctrict poultry farmers the occurrence of production diseases is generally due to a lack of biosecurity measures on farms [27]. indeed, the problem seems to have become more alarming in recent decades; the prevalence of these diseases is increasing, particularly on modern farms. to overcome these problems, farmers are turning to the overuse of antibiotics. in our survey, the majority of farmers surveyed (89 %) indicated that, as soon as the first symptoms appear, various antibiotics are used (table 1) in order to better cover many types of pathologies. these drugs are most often used in combination (71.42 %) in order to increase their effectiveness. these combinations generally include tylosin combined with tetracyclines and/or amoxicilineclavulanic acid, colistin and streptomycin. these antibiotics would be used prophylactically (in 100 % of the farms visited), although antibiotics such as tylosin and enrofloxacin are well known to be growth promoters.the use of several families of antibiotics is probably responsible for the emergence of bacterial strains resistant to these and other antibiotics of these families in the poultry industry in côte d'ivoire [28, 29]. indeed, several studies have shown that the increase in antibiotic-resistant bacterial strains could be linked to selection pressure for these strains resulting from the uncontrolled use of antibiotics in the livestock sector [30 32]. in addition, some families of identified antibiotics are also used in human medicine, suggesting a risk of therapeutic failure in the treatment of human bacterial infections [33]. it is therefore necessary to seek new approaches to limit the use of antibiotics in poultry farming. furthermore, according to the survey data, 85.41% of the farmers surveyed generally purchase these drugs without a prescription from a veterinarian, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 89 while only 14.59 % of poultry farmers always seek the services of a veterinarian before using antibiotics. these data confirm the absence of a system for monitoring the supply and use of antibiotics in semi-industrial poultry farms in côte d'ivoire, as previously observed by ouattara et al. [34]. table 1: antibiotics used in the farms visited drugs use in visited farms antibiotic family frequency (%) tylosin macrolide 26.37 amoxicillin-clavulanic acid beta-lactamine 2.11 association of amoxicillin-colistin beta-lactam and polymycin 3.29 tetracyclin tetracyclines 20.87 flumequin quinolones 5.49 enrofloxacin fluoroquinolones 8.79 streptomycin aminosides 12.08 oxytetracyclin tetracyclines 18.68 association of oxytetracyclin-doxycline tetracyclines 6.59 gentamycin aminosides 3.29 association of gentamycin-doxycline aminosides and tetracyclines 6.59 colistin polymycin 8.79 4. conclusion in conclusion, the low implementation level of bm suggests that farmers still have a long way to go and that government officials should handle the issue of biosecurity in poultry farming sectors very seriously. moreover, the overuse of antibiotics could lead to the increase in antibiotic-resistant bacterial flora. therefore, it is necessary to seek new approaches to limit the use of antibiotics in poultry farming. 5. acknowledgments the authors would like to thank the poultry producers of the district of abidjan for their kind cooperation for the success of the study. 6. references [1]. kouam m.k., jacouba m., moussala j.o., management and biosecurity practices on pig farms in the western highlands of cameroon (central africa), veterinary medicine and science published 00:1–10, 2019. [2]. boka e.e.j., pratique des mesures de biosecurité dans les marchés de volailles vivantes en côte d’ivoire : cas du district d’abidjan. doctorat en médecine vétérinaire de l’université cheikh anta diop de dakar. 133 pages, 2009. [3]. ipravi, la lettre avicole, bulletin d’information de la filière avicole de côte d’ivoire. hors-série n°4, 20 pages, 2017. [4]. m’bari k.b., contribution à l’identification des contraintes au développement de l’aviculture moderne en côte d’ivoire. thèse : méd. vét. : dakar ; 4, 2000. [5]. kone y., contribution a l’évaluation de l’incidence socio-économique de la grippe aviaire en cote d’ivoire au cours de l’année 2006, thèse, faculté de médecine, de pharmacie et d’odonto stomatologie, ecole inter états des sciences et médecine vétérinaires, université cheikh anta diop de dakar. 151, 2007. [6]. itavi, cirad, ofival, le marché mondial des viandes de volailles . in : la production de poulets de chair en climat chaud. itavi, rennes (france).pp 6-20, 2012. [7]. fao (food and agriculture organization), l’élevage dans le monde en 2011 : contribution de l’élevage à la sécurité alimentaire. rome. 48 pages. available on: http://www.fao.org/3/i2373f/i2373f00.htm. accessed on: 30/01/2020., 2012. http://www.fao.org/3/i2373f/i2373f00.htm food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 90 [8]. anonyme, répertoire des localités: district autonome d'abidjan. available on: http://www.ins.ci/n/documents/rgph/abidjan.pdf; accessed on: 16/02/2020, 2015. [9]. bitty z.a.b., contribution à l’amélioration de la gestion sanitaire et des pratiques médicales en élevage avicole moderne dans la zone péri-urbaine d’abidjan (côte d’ivoire), thèse, université cheikh anta diop de dakar, états des sciences et médecine vétérinaires. 120, 2013. [10]. bessin r., belem a.m.g., boussini h., compaore z., kaboret y., dembele m.a., causes of young guinea fowl mortality in burkina faso. revue élev. méd. vét. pays trop., 51 (1): 87-93, 1998. [11]. n’guessan y.t.n.c., pratiques de biosécurité et risques biologiques potentiels dans les élevages avicoles à agnibilékrou et en zones périurbaines d’abidjan, thèse: médecine.véterinaire, dakar ; 21, 2009. [12]. shane s.m., preventing erosive diseases in broiler parents, zootecnica international, 16 (5): 58–60, 1993. [13]. gifford d. h., shane s. m., hughjones m., weigler b.j., evaluation of biosecurity in broiler breeders. avian diseases, 31 (2): 339–344, 1987. [14]. selma o. and alloui n., biosecurity in poultry production. available on: https://www.researchgate.net/publication/28056632 9_traite_de_biosecurite_en_aviculture. accessed on: 30/01/2020, 2015. [15]. dosso s., analyse des pratiques avicoles et de l’usage des antibiotiques en aviculture moderne dans le département d’agnibilékrou (cote d’ivoire). doctorat en médecine vétérinaire de l’université cheikh anta diop de dakar. 152 pages, 2014. [16]. sanchez j. stryhn h., flensburg m., ersboll a.k., and dohoo i., analysis of the 1999 outbreak of acute clinical infectious bursal disease in broiler flocks in denmark, preventive veterinary medicine, 71(3-4): 209-23, 2005. [17]. boender g.j., hagenaars t.j., bouma a., nodelijk g., elbers a.r.w., de jong m.c.m., van boven m., risk maps for the spread of highly pathogenic avian influenza in poultry. plos computational biology, 3(4), e71.doi:10.1371/journal.pcbi.0030071, 2007. [18]. east i., kite v., daniels p., garner g., a cross-sectional survey of australian chicken farms to identify risk factors associated with seropositivity to newcastle disease virus, preventive veterinary medicine, 77: 199–214, 2006. [19]. vandekerchove d., de herdt p., laevens h., pasmans f., colibacillosis in caged 741 layer hens: characteristics of the disease and the aetiological agent, avian pathology, 33, 117742 125. doi: 10.1080/ 03079450310001642149, 2004. [20]. kone y., contribution a l’évaluation de l’incidence socio-économique de la grippe aviaire en cote d’ivoire au cours de l’année 2006, thèse, faculté de médecine, de pharmacie et d’odonto stomatologie, ecole inter états des sciences et médecine véterinaires, université cheikh anta diop de dakar,151, 2007. [21]. knöbl t., moreno m.a., paixao r., gomes t.a.t., vieira m.a.m., leite d.s.d., blanco e.j., ferreira p.j.a., prévalence d'un gène sfa de clonage d'escherichia coli pathogène aviaire (apec) au brésil, scientific world journal , doi: 10.1100 / 2012/437342, 2012. [22]. ipravi, lettre avicole n° 2 hors-serie. 26 pages. accessed on: 30/01/2020. available: http://www.ipravi.ci/lettre-avicole/, 2013. [23]. drouin p., les principes de l’hygiène en productions avicoles, sciences et technologies avicoles. (hors série): 10-14, 2000. [24]. racicot m., vaillancourt j.p., evaluation of biosecurity measures based on video surveillance in poultry farms in quebec and main failures. bulletin de l'académie vétérinaire france, tome 162, n°3, http://www.academie-veterinairedefrance.org, 2009. [25]. barnes h.j., guy j., vaillancourt j.-p. poult enteritis complex. maladies aviaires : conséquences pour le commerce international et la santé publique, revue scientifique et technique (international office of epizootics), 19 (2) : 565– 588, 2000. [26]. barnes h. j., vaillancourt j-p., presentations at the 100th necad anniversary– poultry diseases in the year 2003 congrès annuel de la northeastern conference on avian diseases ; orono, maine. pp 13–21, 2003. [27]. goualie g.b., akpa e.e., kakoun’gazoa e.s., guessennd n., bakayoko s., niamké l.s., dosso m., prevalence and antimicrobial resistance of thermophilic campylobacter isolated from chicken in côte d’ivoire. international journal of microbiology 2012: 5 pages, 2012. [28]. bonny c., karou t. g., ake m. d. f., dadie a. t., bohoua l. g., niamké s.l., distribution of serovars and antibiotic resistance genes of salmonella isolated from chicken gizzards in abidjan, côte d’ivoire. international journal of innovation and applied studies. vol. 11 no. 3, pp. 567-578, 2015. http://www.ins.ci/n/documents/rgph/abidjan.pdf https://www.researchgate.net/publication/280566329_traite_de_biosecurite_en_aviculture https://www.researchgate.net/publication/280566329_traite_de_biosecurite_en_aviculture http://www.ipravi.ci/lettre-avicole/ http://www.academie-veterinaire-defrance.org/ http://www.academie-veterinaire-defrance.org/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 gblossi bernadette goualie, souleymane bakayoko, kalpy julien coulibaly, practices of biosecurity measures and their consequences on poultry farms in abidjan district, food and environment safety, volume xix, issue 1 – 2020, pag. 84 – 91 91 [29]. mcewen s.a., fedorka‐cray p.j. antimicrobial use and resistance in animals. clinical infectious diseases, 34(s3), s93– s106. doi:10.1086/340246, 2002. [30]. alfredson d.a., korolik v., antibiotic resistance and resistance mechanisms in campylobacter jejuni and campylobacter coli”, fems microbiology letters. 277: 123–132, 2007. [31]. ewnetu d. and mihret a., prevalence and antimicrobial resistance of campylobacter isolates from humans and chickens in bahir dar, ethiopia‖. foodborne pathogens. dis.7: 667-670, 2010. [32]. rahimi e., momtaz h., ameri m., ghasemian-safaei h., ali-kasemi m., prevalence and antimicrobial resistance of campylobacter species isolated from chicken carcasses during processing in iran. poultry sciences, 89: 1015–1020, 2010. [33]. goualie g.b., akpa e.e.,. kakoun’gazoa s.e ., ouattara h.g., niamke s.l., dosso m., antimicrobial resistance and virulence associated genes in campylobacter jejuni isolated from chicken in côte d’ivoire. journal of infections in developing contries, 13(8):671-677. doi:10.3855/jidc.11355, 2019. [34]. ouattara n.d., guessennd n., gbonon v., toe e., dadié t., tiécoura b., consommation des antibiotiques dans la filiére aviaire à abidjan: cas de quelques fermes semiindustrielles. europeen journal of scientifics reseach, 94: 80-85, 2013. 1. introduction 4. conclusion [16]. sanchez j. stryhn h., flensburg m., ersboll a.k., and dohoo i., analysis of the 1999 outbreak of acute clinical infectious bursal disease in broiler flocks in denmark, preventive veterinary medicine, 71(3-4): 209-23, 2005. [18]. east i., kite v., daniels p., garner g., a cross-sectional survey of australian chicken farms to identify risk factors associated with seropositivity to newcastle disease virus, preventive veterinary medicine, 77: 199–214, 2006. [24]. racicot m., vaillancourt j.p., evaluation of biosecurity measures based on video surveillance in poultry farms in quebec and main failures. bulletin de l'académie vétérinaire france, tome 162, n 3, http://www.academie-veterinaire-defrance.org, ... [25]. barnes h.j., guy j., vaillancourt j.-p. poult enteritis complex. maladies aviaires : conséquences pour le commerce international et la santé publique, revue scientifique et technique (international office of epizootics), 19 (2) : 565–588, 2000. issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xix, issue 1 31 march 2020 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 97 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2 2019, pag. 97 104 biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje *gordana šebek1, valentina pavlova2, tatjana popović3 1biotechnical faculty, university of montenegro, podgorica, montenegro, sebek@t-com.me, 2faculty of technology and technical sciences, university st. kliment ohridski, bitola, republic of macedonia, valentina.pavlova2@gmail.com, 3biotechnical faculty, university of montenegro, podgorica, montenegro, tatjanapopovic@t-com.me *corresponding author received 8th may 2019, accepted 28th june 2019 abstract: this study described some biochemical and pomological parameters of fruits in 4 commercial medlar cultivars (‘domestic medlar’, ‘plovdivska’; ‘royal medlar’; ‘rasna’) grown in ecological conditions of bijelo polje (montenegro) in the period from 2010 to 2012. recording of biochemical parameters such as dry matter, total soluble solids, total acidity and ph was the most important segment of this research. the study also focused on comprised pomological traits such as fruit weight (g), fruit size (mm) and length (mm) and petiole length (mm). the values for fruit dry mater ranged from 26.2% to 28.8%, total soluble solid contents ranged from 20.45% to 22.25%, titrable acid contents ranged from 1.9% to 2.28%. the values for fruit weights ranged from 21.4g to 25.5g, fruit length ranged from 34.5mm to 38.4mm, fruit widths ranged from 31.5mm to 36.2mm, and petiole length ranged from 19.8mm to 23.2mm. over the years of study, all researched cultivars had yields in the agroecological conditions of bijelo polje. the ‘royal medlar’ cultivar is the cultivar we would recommend to producers, due to its fruit dry matter content of 28.2%, the total soluble solid contents of 22.25%, and the total titrable acid content of 2.28%. moreover, the fruit mass was the highest for the ‘royal medlar’ cultivar (25.5g). the length and width of the fruit in the same cultivar had also maximum values (38.4mm and 36.2mm, respectively), as compared to other researched cultivars. keywords: ‘royal medlar’, dry mater, soluble solid contents, titrable acid contents, fruit weights. 1. introduction the medlar (mespilus germanica l.) is a member of the rosaceae family and is native to the eastern mediterranean area. it was already cultivated about three thousand years ago in the caspian sea region of northern iran [1]. medlar (mespilus germanica l.) belongs to rosaceae family, and it is called ‘döngel’ or ‘beşbıyık’ in turkey, ‘ezgil’ in azerbaijan, ‘bushmala’ in georgia and ‘german’ or ‘germanic medlar’ in the most of european countries. until the seventeenth century, the medlar was the most important fruit crop. however, interest in it gradually faded away, and later it was replaced by other, more productive and undemanding crops. currently, medlar is grown quite rarely and, mainly, in botanical gardens or in small farms. mespilus germanica is indigenous to southwest asia and possibly also southeastern europe – from http://www.fia.usv.ro/fiajournal mailto:tatjanapopovic@t-com.me food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 98 northern turkey (some occurrence in greece and on the crimea) to the caucasus and transcaucasus and the north-eastern part of iran. its tree is generally 3-5 m tall, but it may reach nearly 8 meters. it is self-fertile and long-lived tree. it lives approximately 3050 years. there are 100 year old trees in uk as well. the flowers are white-pink and hermaphrodite. flower buds are formed in may-june, and each bud has one flower. fruit shape may be variable. fruits are inedible during tree maturity stage because of tannin content. it becomes edible when the skin color becomes chocolate brown. medlar contains organic acids, sugars, pectin, vitamin c, and small amounts of vitamin a [2]. medlar is a climacteric fruit, and as such, important biochemical changes occur during the respiratory climacteric, just before the full maturation stage [1] .the medlar shows better pest and climate resistance than most other fruit species of landscaping importance. very large number of researchers in europe studied the biochemical and pomological characteristics of medlar cultivars [1-5]. the data for medlar trees in the territory of bijelo polje (montenegro) are not statistically processed, which refers to the fact that the cultivation of this fruit cultivar is very low. we certainly wanted to make the existing situation less severe by presenting data for the quality of the fruits of certain medlar cultivars, which are grown in smaller family orchards in the territory of bijelo polje municipality. 2. matherials and methods bijelo polje is situated between 43° and 43°5′ north latitude and 19°40′ and 19°50′ east longitude. the municipality of bijelo polje is situated at the altitude from 520m (estuary of the river kanjska to river lim) to the altitude of 2017m (komovi mountain). the terrain of bijelo polje municipality is following the direction of river lim flow, i.e. the north and northwest direction. larger and betterquality agricultural land is located on the near proximity of the river lim, and its tributaries [6]. the municipality of bijelo polje has average annual temperature of 8.9°c. the warmest period is during the months of june, july and august, with an average temperature from 16.3°c to 18.1°c. the coldest period is during the months of december and january, with an average temperature from 0.1°c to -1.6°c. the lowest daily temperatures on annual level are around -3°c. the winter period is characterized with intense negative temperatures, which can reach up to 27.6°c. the spring period is characterized with low temperatures, which can be more than critical when it comes to fruit production (late spring frosts). namely, the month of april was recorded in some years with the lowest temperature of up to -8°c. the average minimal temperature during april is -2.8°c for the bijelo polje territory. the annual precipitation level in bijelo polje municipality is 893.7 mm/m2. the maximum precipitation is occurring during the period of october december, while the period with the lowest precipitation is from june august. the dominance of the cold and rainy period over the warm and dry one is expressed in relation of 54% vs. 46%. the materials of this study were commercial cultivars of medlar: ʽdomestic medlar’, ʽplovdivska’, ʽroyal medlar’ (fig. 1.) and ʽrasna’ grown in ecological conditions of bijelo polje (montenegro) in the period from 2010 to 2012. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 99 fig. 1. royal medlar tree fig. 2. royal medlar fruit these researches were conducted on a larger number of private production orchards, which are in near proximity one from another and are all situated within the territory of bijelo polje. all cultivars were grafted on the vegetative rootstocks quince ba-29.the orchards were of mixed type, and the trees of researched cultivars were with average age of 7-10 years, and were in the fruiting period. when it comes to agro-technical measures, winter cutting and winter spraying were applied. the study focused on few segments. the first one included recording of biochemical traits dry matter, total soluble solids, ph, and total acidity. the second segment comprised pomological traits [fruit weight (g), fruit diameter (mm), fruit length (mm), petiole length (mm)]. the dry mater was determined by drying at 105°c. the total soluble solid content was determined by refractometer. the acidity was measured by titration with 0.1 n naoh. the fruit weight was determined by measuring by the electric scale metler 1200. the result was shown in grams with the accuracy of 0.01g. fruit dimensions length and width were measured by vernier scale. 3. results and discussion the results of biochemical and morphological characteristics of fruits of researched medlar cultivars are shown in table 1 and table 2. fruit dry mater, total soluble solids, ph and titrable acid content of four medlar cultivars are shown in table 1. table 1. biochemical characteristics of fruit of some commercial medlar cultivars (average 2010-2012) medlar cultivar fruit dry mater (%) total soluble solids(%)(tss) ph (0 -14) total acidity(%)(ta) domestic medlar 26.20 20.45 3.59 2.20 plovdivska 27.80 21.82 3.40 2.26 royal medlar 28.80 22.25 3.86 2.28 rasna 27.25 21.60 3.57 1.90 lsd 0.05 lsd 0.01 1.09 1.70 1.01 1.28 0.28 0.37 0.18 0.29 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 100 the values for fruit dry mater ranged from 26.2% (cv.‘domestic medlar’) to 28.8% (cv.‘royal medlar’); total soluble solid contents ranged from 20.45% (cv.‘domestic medlar’) to 22.25% (cv.‘royal medlar’); ph ranged from 3.40 (cv.‘plovdivska’) to 3.86 (cv.‘royal medlar’); titrable acid contents ranged from 1.90% (cv.‘plovdivska’) to 2.28% (cv.’royal medlar’). the results of our research, related to the parameter of dry matter for the cultivars ‘domestic medlar’, ʽplovdivska’, ʽroyal medlar’, and ‘rasna’, showed significantly lower values in comparison to data of 11 medlar genotypes, selected from tokat province. in the research of genotypes from tokat province, total dry matter was determined between 27.34% 44.11% [2]. the fruit soluble solid contents increased in the ripening period for all of the researched medlar cultivars (table 1). the fruit soluble solid contents were measured between 20.45% and 22.25% at the time of harvest. the highest fruit ph value was found in cv. ‘royal medlar’(3.86). previous similar researches show that the total soluble solids (tss) and ph values of medlar fruits grown in different agro climatic regions of turkey (kocaeli province) are between 16.4% 22.2% and 3.68% 4.02%, respectively [7]).the soluble solid contents varied between 17.0% and 24.0% in selected types of wild medlars in earlier studies in turkey (tonya district of trabzon province) [8]. our findings regarding total soluble solids (tss), and ph values showed results that were similar to these studies. the recorded variations of total soluble solids and ph in medlar fruits could be the result of different genetically based characteristics and the effect of the different agro ecological conditions, where the cultivars are grown. significant variation in chemical and morphological characteristics of fruit were recorded among cultivars of different fruit species, such as apple [9],plum [10], pear [11-12] and sour cherry [13], which were all grown in conditions of bijelo polje (montenegro) as well. the above listed biochemical characteristics of fruit dry mater and total soluble solids for all the researched medlar cultivars can be compared with the data for the same biochemical characteristics for commercial cultivars of pear from the same territory (bijelo polje, montenegro). in the commercial pear cultivars, the values for fruit dry mater ranged from 16.6% ± 0.39 to 18.08% ± 0.32, total soluble solid contents ranged from 11.99% ± 0.25 to 15.66% ± 0.42. [11]. our comparative analysis between the fruits of researched medlar cultivars and the fruits of researched pear cultivars showed that the level of the aforementioned biochemical parameters for medlar cultivars is significantly higher compared to parallel data in commercial pears from the same area. having this in mind, we believe that the medlar is an unjustifiably neglected fruit species for cultivation in this area. also, the experience has showed us that there is a large demand for all types of products from the fruits of medlar, whereas the supply is on an extremely low level. in order to confirm abovementioned, we can emphasize the exceptional quality and taste of medlar fruit liqueur (fig. 3.), produced by the original recipe in the monastery of gračanica, kosovo and metohija (serbia). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 101 fig. 3. medlar fruit liqueur fruit weight, length, width and petiole length are given in table 2. the results of our research, as show in table 2, indicated that the values for fruit weights ranged from 21.4 g (cv.‘domestic medlar’) to 25.5g (cv.‘royal medlar’), fruit length ranged from 34.5mm (cv.‘domestic medlar’) to 38.4 mm (cv.‘royal medlar’), fruit widths ranged from 31.5 mm (cv.‘domestic medlar’) to 36.2 mm (cv.‘royal medlar’), and petiole length ranged from 19.8mm (cv.‘domestic medlar’) to 23.2 mm (cv.‘plovdivska’). the scientific works that were also concerned with the study of the morphological properties of medlar genotypes indicate a wide divergence in terms of fruit weight, fruit width, fruit length and petiole length. we compared our data with researches conducted in serbia and turkey. table 2. morphological characteristics of fruit of some commercial pear cultivars (average 2010-2012) cultivar fruit weight (g) fruit length (mm) fruit widths (mm) petiole length (mm) domestic medlar 21.4 34.5 31.5 19.8 plovdivska 23.3 35.9 34.2 23.2 royal medlar 25.5 38.4 36.2 22.1 rasna 22.8 35.5 35.1 22.0 lsd 0.05 lsd 0.01 0.28 0.37 0.08 0.11 0.08 0.10 0.25 0.33 seedless medlar genotype ‘pomoravka’ was found in pomoravlje, in the close vicinity of svilajnac (serbia), in 1994. it was grafted and transferred at three locations so as to be protected from deterioration. apart from being used fresh, its fruit is very interesting for processing industry for making pastes, jelly, mash, liqueur etc. this genotype can be beneficial in the breeding aimed at development of seedless medlar cultivars of satisfactory fruit size. this genotype has a relatively small fruit (8.2g) but high stone flesh ratio (96.5%) and it is highly qualitative especially when it is overripe. due to its high using values this genotype should be more propagated especially in view of the fact that the presence of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 102 pathogen erwinia amylovora has not been detected on any young tree of this medlar genotype [14]. namely, the medlar genotype ‘pomoravka’ has very small fruits (8.2g) and it is inferior in regards to fruit weight in comparison to all medlar cultivars we researched in our work, especially compared to ‘royal medlar’(fig. 2.) cultivar, which average size was 25.5g. the fruit weight of the ‘royal medlar’ cultivar is three times larger than the fruit weight of medlar genotype ‘pomoravka’. the serbian research team must have researched the medlar genotype ‘pomoravka’ for its resistance to the pathogen of erwinia amylovora, and also because of the fact that there are no seeds within the fruit. the randman is 96.5% [14]. in tokat province (turkey), the average fruit weight of medlar's genotipes ranged between 17.71g 32.46g in 2011, and 15.99g 37.54g in 2012. fruit diameter was determined around 21.07mm 41.05mm in 2011, and 17.49 mm 43.63mm in 2012. fruit length was 18.25 mm-38.27 mm in 2011, and 14.96mm 35.68 mm in 2012 [2]. the interesting fact is that some genotypes from tokat province were with larger fruit weight even from cultivars from our research. this suggests that the work on selection of new cultivars can be continued, and that natural populations can produce cultivars of large fruits. morphological characterization continues to be the first step for germplasm description and classification [1516]. the existence of a large variability in fruits has been demonstrated in different species such as prunus persica (l.) batsch [17], prunus spp. [18 20], malus sp. [20], diospyros spp. [22], castanea sativa mill. [2324], pseudocydonia sinensis schneid. [25], vitis vinifera l. [26], ziziphusjujuba mill. [27], for the mespilus germanica breeders or plant biologists, the description of the fruit morphology is of significant importance for phenomics studies. 4. conclusion based on the obtained results in terms of morphological and biochemical characteristics of researched medlar cultivars (ʽdomestic medlar’, ʽplovdivska’, ʽrojal medlar’, and ʽrasna’), the following conclusions can be drawn: 1. the values for fruit dry mater ranged from 26.2% (cv.'domestic medlar') to 28.8% (cv.' rojal medlar '); total soluble solid contents ranged from 20.45% (cv. 'domestic medlar') to 22.25% ( cv.'rojal medlar'); ph ranged from 3.40 (cv.'plovdivska') to 3.86 (cv.'rojal medlar ' ); titrable acid contents ranged from 1.90 % (cv.'plovdivska') to 2.28% (cv.'rojal medlar '). 2. the results of our research indicate that the values for fruit weights ranged from 21.4 g (cv.'domestic medlar') to 25.5g (cv.'rojal medlar'), fruit length ranged from 34.5mm (cv. 'domestic medlar') to 38.4 mm (cv. 'rojal medlar'), fruit widths ranged from 31.5 mm (cv. 'domestic medlar') to 36.2 mm (cv. 'rojal medlar'), and petiole length ranged from 19.8 mm (cv. 'domestic medlar' ) to 23.2 mm (cv.'plovdivska'). 3.the ‘royal medlar’(fig. 1. and fig. 2.) cultivar is the cultivar we would recommend to producers, due to its fruit dry matter content of 28.2%, the total soluble solid contents of 22.25%, and the total titrable acid content of 2.28%. moreover, the fruit mass was the highest for the ‘royal medlar ‘cultivar (25.5g). the length and width of the fruit in the same cultivar had also maximum values (38.4mm and 36.2mm), compared to other researched cultivars. 4. at the end of these studies, one general conclusion can be made, as an answer to set goals and the very task of our research: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 103 agro-ecological conditions of bijelo polje and its surrounding environment fully correlate to the intensive medlar production; hence, the agro-biological characteristics of researched cultivars can be demonstrated economically. 5. references [1]. veličković m, radivojević d, oparica č, nikićević n,živković m, đorđević, vajs v, tešević v. volatile compounds in medlar fruit (mespilus germanica l.) at two ripening stages, hemijska industrija. 67 (3): 437–441 (2013) [2] yilmaz a, gercekcioglu r, atasever o. determination of pomological and chemical properties of some medlar (mespilus germanica l.) genotypes, journal of new results in science. 11:118-124 (2016) [3] gruz j., ayaz a., torun h., strand m. phenolic acid content and radical scavenging activity of extracts from medlar (mespilus germanica l.) fruit at different stages of ripening, food chemistry. 124 271–277. (2011) [4] ercisli s., sengul m., yildiz h., sener d., duralija b., voca s., dujmovic d. phytochemical and antioxidant characteristics of medlar fruits (mespilus germanica l.), journal of applied botany and food quality. 85:85-90. (2011) [5] glew r., ayaz c., sanz d., vanderjaght h., huang l., chuang l., strand m. changes in sugars, organic acids and amino acids in medlar (mespilus germanica l.) during fruit development, food chemistry, 83363– 369. (2003) [6] šebek g. (2011) organic fruit production. austrian development agency, austria (lmuc project). [7] sulusoglu durul m., unver h. morphological and chemical properties of medlar (mespilus germanica l.) fruits and changes in quality during ripening. agrofor international journal, vol.1. issue no.2 133-140. (2016). [8] yilmaz p. selection of wild types medlars in tonya district of trabzon province, msc thesis, ordu university, orduturkey (2015). [9] šebek g. autochthonous cultivars of apple from the area of the upper polimlje, agriculture & forestry, 59. (3).pp. 67-74. (2013). [10] šebek g. the phenological and pomological traits of autochthonous plum cultivars in the area of north montenegro, acta sci. pol. hortorum cultus, 15(4), 45–63. (2016) [11] šebek g. chemical and pomological characteristics of fruit of some commercial pear cultivars grown in conditions of bijelo polje, journal of hygienic engineering and design. vol.25, pp.56-61(2018) [12] šebek g. the phenological and pomological traits of selected genotypes of wild pear important for the production of generative rootstocks, acta scientarium polonorum hortorum cultus, 18 (2), 133-145, doi: 10.24326/asphc.2019.2.13 (2019) [13] šebek g. pomological and chemical characteristics of fruit of some sour cherry cultivars grown in the conditions of bjelo polje, journal of hygienic engineering and design, vol.26, pp.100104. (2019) [14] nikolić m. pomoravka – seedless medlar genotype, journal of pomology, vol.39.br.150, 155-160 (2005). [15] badenes l., martinez-calvo j., liacer g. analysis of a germplasm collection of loquat (eriobotrya japonica lindl.). euphytica, vol. 114, no. 3, p. 187-194. (2000). [16] nazari a., zamani z., fatahi r., sofla h. s., morphological characterization of prunus incana pall by multivariate analysis. plant syst. evol., vol. 298, p. 1805-1814. (2012) [17] scorza r. characterization of four distinct peach tree growth types. j. am. soc. hort. sci., vol. 109, p. 455-457. (1984) [18] zhang q., yan g., dai h., zhang x., li c., zhang, z. characterization of tomentosa cherry (prunus tomentosa thunb.) genotypes using ssr markers and morphological traits. sci hortic., vol. 118, no. 1, p. 39-47.(2008) [19] perez-sanches r., gomez-sanches m. a., morales-corts r. agromorphological characterization of traditional spanish sweet cherry (prunus avium l.), sour cherry (prunus cerasus l.) and duke cherry (prunus gondouinii rehd.) cultivars. span. j. agric. res., vol. 6, no. 1, p. 4255. (2008). [20] nazari a., zamani z., fatahi r., sofla h. s. morphological characterization of prunus incana pall by multivariate analysis. plant syst. evol., vol. 298, p. 18051814. (2012). [21] mratinić e., akšić, m. phenotypic diversity of apple (malus sp.) germplasm in south serbia. braz. arch. biol. technol., vol. 55, no. 3, p. 49-358.(2012) [22] grygorijeva o., abrahamova v., karnatovska m., bleha r., brindza j. morphological characteristic of fruit, drupes and seeds genotypes of ziziphus jujuba mill. potravinarstvo slovak journal of food sciences, vol. 8, no. 1, p. 306-314.( 2014). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 gordana šebek, valentina pavlova, tatjana popović, biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje, food and environment safety, volume xviii, issue 2 – 2019, pag. 97 – 104 104 [23] poljak i., vahčić n., gačić, m., idžojić, m. morphological characterization and chemical composition of fruits of the traditional croatian chestnut variety 'lovran marron', food technol biotechnol., vol. 54, no. 2, p. 189-199. (2016) [24] grygorijeva o., klymenko s., vergun o., hundy n., nikolieva n., schubertova z., palamarchuk o., brindza j., morphological characteristics and determination of volatile organic compounds of diospyros virginiana l. genotypes fruits. potravinarstvo slovak journal of food sciences, vol. 11, no. 1, p. 612-622. (2017) [25] monka a., grygorijeva o., chlebo p., brindza j. morphological and antioxidant characteristics of quince (cydonia oblonga mill.) and chinese quince fruit (pseudocydonia sinensis schneid). potravinarstvo slovak journal of food sciences, vol. 8, no. 1, p. 333-340. (2014). [26] lamine m., zemni h., ziadi s., chabane a., melki i., mejri s., zoghlami n.. multivariate analysis and clustering reveal high morphological diversity in tunisian autochthonous grapes (vitis vinifera) : insights into characterization, conservation and commercialization. journal international des sciences de la vigne et du vin .vol 48, no. 2, p. 111122. (2014) [27] ivaniševa e., grygorijeva o., abrahamova v., schubertova z., terentijeva m., brindza, j. characterization of morphological parameters and biological activity of jujube fruit (ziziphus jujuba mill.). journal of berry research, vol. 7, no. 4, p. 249-260. (2017). 1. introduction 4. conclusion 372 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 -2018 , pag. 372 384 characterization of the effect of modulated dry heat processing conditions on essential and non essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds *azubuike c umezuruike 1 , joel ndife 2 , chinwe nwachukwu 3 1scientific directorate, ministry of science and technology/absiec, abia state government service, umuahia, nigeria, realmira4ac@gmail.com 2 department of food science and technology, college of applied food sciences and tourism, michael okpara university of agriculture, umudike nigeria. 3 department of food science and technology, imo state university, owerri *corresponding author received 16th august 2018, accepted 27th december 2018 abstract: the objective of this study was to determine the functional relationship between roasting temperature (rt), roasting time (rm) and feed quantity (fq) and the yield of all essential and non-essential amino-acids obtainable through modulated roasting of breadfruit seeds harvested from undomesticated breadfruit tree. roasted seeds of breadfruit (v. decne) consumed as snacks represent an important source of protein/amino acids needed for good health in humans. however, these amino acids are needed in certain levels in order to achieve their metabolic usefulness. processing methods influence their useful concentrations. central composite design at 3 process variables (rt, rm, fq) and 5 levels (-1.682, -1, 0, 1, 1.682) was used for the experimental runs. amino acid assay was conducted and the results obtained from experimentation were statistically analyzed. similar aminoacids but in different contents were present both in raw and roasted seeds. roasting temperature, time and feed quantity were significant (p < 0.05) terms in the models which influenced the quantum of amino acid, either in linear, squared or interactive terms. cysteine and methionine showed some heat stability. other non-essential amino acids were observed at concentration range of 0.29 g to about 7.0 g. tryptophan was not detected at temperatures higher than a hundred and forty degrees celsius. the predicted optimum total of amino acids of roasted seed samples was 66.02 % of the total amino acids of the control. the predicted values for optimum process condition were in good agreement with experimental data. hence, roasting of breadfruit seeds for snacking at the identified optimum variable combination will supply safe and recommended daily levels of amino acids in humans. keywords: breadfruit seeds, roasting, essential, non -essential amino-acids. 1. introduction in nigeria, breadfruit ( v.decne) seeds are consumed in roasted forms as snacks. the roasted snack seeds are popular convenient snacks among travellers and commuters or when packaged in bottles shared as social gifts. roasted breadfruit (v. decne) seeds is an important source of protein/amino acids for consumers. the reported amino acids of breadfruit and other legume seeds include alanine, arginine, aspartic acid, glutamic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine phenylalanine, proline, serine, threonine, tyrosine,tryptophan and valine [1,2]. http://www.fia.usv.ro/fiajournal mailto:realmira4ac@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 373 histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine are essential amino acids that must be provided in the human diets for metabolic development, immune boosting, neurotransmission, fertility, inhibition of reactive oxygen species, anti-inflammation etc. [3.4.5.6.7]. only methionine, cysteine and tryptophan are limited in most legumes including breadfruit seeds [8] other studies that explored the extraction and composition of breadfruit seeds [8,9] reported important health nourishing nutrients of breadfruit seeds. the protein content of breadfruit (v. decne) is comparable to other legumes [10]. breadfruit seeds maybe seasoned with salt or spices before roasting. usually the seeds are roasted without being hulled. after roasting the breadfruit seeds are consumed as snacks. traditional roasting of legume seeds and nuts for snacking takes about 3040 min at very high temperatures with obvious deleterious effect on the nutrients [11] the outcome of processing treatments are influenced by the processing parameters of temperature, time, heat transfer rates, moisture content, seed chemistry, seed physics, processors skill etc. [12,13]. previous studies focused more on parboiled seeds with little attention on roasted seeds. studied of iwe and ngoddy [12] and nwabueze [13] successfully optimized the process conditions nutritive properties of parboiled breadfruit (v.decne) seeds but no such magnitude of research efforts on dry heat processing are recorded for roasted seeds. this suggests that consumers of roasted seeds may be accessing low nutrients. as the global food trend tilts towards convenience food, emphasis is put now on roasted high energy density convenient snacks such as roasted breadfruit seeds, groundnuts, almonds etc. more studies are needed to determine optimum process variable combination for enhanced yield of amino-acids and other health promoting factors of roasted breadfruit seeds consumed as snacks. heat treatment improves protein digestibility, amino acid availability in diets [14,15, 16], reduces the amino acids content of our diet to levels that are nontoxic in humans [17,18]. as most safe levels of amino acids fall within 5g-10g/ day [19], it is important to determine the processing points of convergence as processing variable optima between safety and critical needs of amino acids from roasted breadfruit (v. decne) seeds consumed as snacks. for instance, essential amino acids such as methionine, histidine and cysteine which are very toxic to human in higher concentrations and they could be modified to tolerable and useful levels by roasting. roasting though leads to denaturation and decomposition of amino acids, still it remains an indispensable food processing operation for the production of roasted breadfruit snack seeds. this study was designed as an evolving study carried out to determine and characterize the process variable combinations for optimum dry heat processing spectrum and yield of essential and non-essential amino acids in the roasting of breadfruit seeds by using overall desirability approach. desirability function approach is used to transform multiresponses problem into a single response objective function (overall desirability) by means of mathematical transformation. overall desirability approach has been successfully applied in many optimization studies [20]. it involves the optimization of the desired product quality within conflicting operational process variables in the processing region. ([21]. the results of this study are envisaged as a guide to breadfruit seed processors for the production of safe and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 374 nutritionally adequate levels of amino acids in roasted breadfruit snack seeds. 2. materials and methods collection of materials freshly pulped seeds of breadfruit (v. decne) were provided from undomesticated breadfruit tree growing in umudike, nigeria. the seeds were screened for stones, sands, debris etc, washed with portable water and dried under shade at 28±40c for 34 hours. seeds were not seasoned with salt or spices for flavour. experimental design the central composite rotable design was used for the study. three (3) variables 5 levels experimental layout at 8 factorial, 6 axial and 6 replications at the centre (table 1) were employed. effects of roasting temperature, roasting time and feed quantity on amino acids content of roasted seeds were examined. definition of operational variables were carried out by using minitab statistical software version 15. the input variable values were chosen based on the information in literature and reasonable enough to accommodate statistically valid deductions [22]. the range and values at star, axial and center points were calculated using the equation (1) as outlined in table 2 x1= xi – x10 (1) where x1= independent variable code value xi= independent variable actual value xio= independent variable actual value of centre point = step change value experimental roasting of samples the unseasoned seeds ( samples) were roasted in electric oven (fishers scientific co. uk) according to the experimental layout. the roasted seeds are cooled, hulled using a locally fabricated huller. the roasted hulled seeds are edible snacks. for amino acid assays, the hulled seeds were milled and sieved into flour using a 200mm mesh. the flour samples were placed in sterilized plastic bowls, appropriately labeled and stored at ambient 28 ± 20c) temperature before use. table 1 central composite rotable design code variables combination replications experiments x1 x2 x3 ±1 ±1 ±1 8 1 8 ±1.682 0 0 2 1 2 0 ±1.682 0 2 1 2 0 0 ±1.682 2 1 2 0 0 0 1 6 6 table 2 range and levels of experimental runs codes -1.682 -1 0 1 1.682 roasting temperature rt (0c) x1 123.36 140 160 180 193.64 roasting time rm (min) x2 31.59 35 40 45 48.4 feed quantity fq (g) x3 331.80 400 500 600 668.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 375 determination of aminoacid profile: the amino-acids profile of raw and roasted breadfruit flour were determined using the method described by [23, 24]. the samples were dried to constant weights in oven, defatted, hydrolyzed, and then evaporated using rotary evaporator. thirty milligrams of each sample were mixed with 7ml of hcl in a glass ampoule. oxygen was expelled from the mixture by passing nitrogen into the ampoule. the glass ampoule was heat sealed using a bunsen burner flame and placed in an oven (1050c) for 22 hours. after heating, the glass ampoules were let to cool, the tip was opened and the content was filtered. the filtrate was evaporated to dryness using rotary evaporator at 400c. the residue was dissolved in 5ml acetate buffer (ph. 2.0) and stored in refrigerator using plastic bottle. 5-10 microliters of each sample were placed in amino-acid cartridge and loaded into aminoacid analyzer. the tsm analyzer separated and analyzed the amino-acids in samples. the data generated from the technicon sequential multi-sample analyzer were quantitatively determined against the standard technicon auto analyzer (no 011648-0) chart. statistical analysis of results data of the study were analyzed and presented as tables. for statistical evaluation, data of study were analyzed using minitab statistical software (version 15 of minitab inc.pen. usa) which described the optimized global setting for input variables and predicted optimum yield of amino acids at 0.0 1.0 desirability bar. the effect of roasting condition on amino acids of breadfruits was described in regression terms by + (2) where y= response, β0 = intercept, xiji= independent variables, e=error maximization of the fitted models was performed using unified goals with defined (0.1-1.0) limits. adequacy of models was defined by co-efficient of determination r2 and adjusted r2. the significant (p<0.05) variables were identified and their effects described in linear, squared or cross product terms. the coefficients of determination were complemented with their adjusted values and standard error of estimate s in judging the adequacy of fitted models. 3. results and discussion adequacy of regression models of amino acids the co-efficient of determination r, adjusted r2 and standard error of estimate s were adequate to predict the variability in amino acid content of the processed breadfruit snack seeds. the co-efficient of determination of r2 ranged from70% to 95% and all adjusted r2 values were above 65%. all s values indicated that data points were 0.23 to 1.16% of the fitted line and less than 7% which statistically satisfied the prediction intervals. significance (p=0.5) of input process variables x are described in linear, squared and interaction terms. the amino acid profile of the processed breadfruit seeds flour is shown in table 3. all amino acids in the control were contained in the processed flour samples in varying proportions. the amino acids identified were: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, cysteine, threonine, aspartic acid, glutamic acid, glycine, proline, serine, threonine, tyrosine, tryptophan, arginine and valine. the values of all the amino acids analyzed progressively reduced as the roasting temperature, roasting duration and mass were extended simultaneously from 1400c, 1600c and 1800c to 31.59 min, 53 min, 40 min , 45 min, 48 min and 331.80g. 400g, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 376 500g, 600g 668.20g for roasting temperature, roasting time and feed quantity, respectively. table 3 amino acid profile of processed samples (g/16 gn) essential amino acids s/n experimental runs h is iu ly me ph th tp va 1 1400c/35m/400g 2.23 2.90 5.70 4.18 0.73 3.99 2.70 trace 3.18 2 1400c/35m/600g 2.25 3.01 6.00 4.27 0.75 4.05 2.73 trace 3.25 3 1400c/45m/400g 2.00 2.89 5.10 3.11 0.70 3.80 2.57 nd 3.04 4 1400c/45m/600g 2.03 2.77 5.15 4.00 0.71 3.89 2.57 nd 3.10 5 1800c/35m/400g 1.80 1.70 4.03 3.30 0.60 3.20 2.21 trace 3.05 6 1800c/35m/600g 1.82 1.77 4.33 3.21 0.61 3.50 2.33 nd 3.90 7 1800c/45m/400g 1.10 2.30 3.46 2.88 0.55 3.86 2.10 nd 2.96 8 1800c/45m/500g 1.50 2.38 3.59 2.91 0.57 3.39 2.01 nd 2.95 9 1260c/40m/500g 2.29 3.11 4.16 3.87 0.81 3.90 1.81 trace 2.90 10 193640c/40m/500g 1.00 1.30 2.79 2.16 0.43 2.64 1.14 nd 2.03 11 1600c/31.59m/500g 2.30 3.11 7.53 3.64 0.69 3.47 2.71 nd 2.97 12 1600c/48.41m/500g 1.19 2.05 3.60 2.51 0.61 3.10 2.55 nd 2.90 13 1600c/40m/331.80g 2.13 2.11 3.37 3.00 0.59 3.05 1.95 nd 2.47 14 1600c/40m/668.20g 2.16 2.49 3.66 3.81 0.65 3.59 2.69 nd 2.95 15 1600c/40m/500g 2.20 2.28 3.49 3.49 0.63 3.35 2.31 nd 2.59 control 3.16 3.60 6.8 7.10 1.53 6.10 3.22 0.4 4.5 non-essential s/n experimental runs al ar as cy gl gy pr sr ty 1 1400c/35m/400g 2.66 5.58 6.00 1.50 8.10 3.97 2.00 3.63 2.35 2 1400c/35m/600g 3.01 5.62 6.10 0.95 8.20 4.05 2.53 3.30 2.41 3 1400c/45m/400g 2.35 5.15 5.58 0.90 7.71 3.93 2.30 3.56 2.25 4 1400c/45m/600g 2.33 5.10 5.91 0.95 7.60 4.00 2.41 2.30 2.17 5 1800c/35m/400g 2.07 4.60 5.40 0.90 6.70 3.41 2.37 2.41 2.07 6 1800c/35m/600g 2.17 4.71 5.70 0.91 6.48 3.53 2.39 2.37 1.99 7 1800c/45m/400g 1.85 4.14 5.30 0.80 6.00 2.91 2.30 2.30 2.10 8 1800c/45m/500g 1.88 4.25 5.35 0.88 6.16 2.94 2.29 2.28 1.71 9 1260c/40m/500g 2.05 4.77 5.99 0.90 7.60 3.99 3.01 3.01 2.51 10 193640c/40m/500g 1.80 3.25 3.01 0.45 5.13 2.21 1.21 1.20 1.07 11 1600c/31.59m/500g 2.04 4.90 5.90 0.80 7.61 3.19 2.60 2.60 2.41 12 1600c/48.41m/500g 1.19 3.90 3.89 0.65 5.88 3.00 2.67 2.66 2.07 13 1600c/40m/331.80g 1.85 3.17 3.96 0.71 6.40 3.01 2.21 2.23 1.97 14 1600c/40m/668.20g 1.90 3.53 4.01 0.75 6.72 3.10 2.33 2.33 2.13 15 1600c/40m/500g 2.01 3.28 3.99 0.73 6.88 3.20 2.27 2.27 2.01 control 4.5 9.86 11.01 2.00 19.50 5.15 4.00 5.10 3.5 key h = histidine, is= isoleucine, lu = leucine, ly = lysine , me = methionine, ph = phenylalaine, th = threonine, tp = tryptophan, va = valine; al= alanine; gy = glycine; tyrozine, ag =arginine; as= aspartic acid; cy = cysteine, gl = glutamic acid, pr = proline, sr = serine; ty = tyrosine food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 377 effect of processing variable of amino acid of roasted breadfruit seeds basic amino acids (basic side chains at neutral ph) histidine content of processed sample ranged from 1.0 to 2.29g/16n. roasting temperature had significant effect on the contents of histidine and accounted for 95% variations in histidine content. increases in temperature resulted in loses of histidine. the maximum (68.35%) loss occurred at 193.640c with less than 50% of such losses occurring below 1600c. the author [25] reported a lesser reduction of 16.5% for roasted groundnuts at similar temperature. the relationship between histidine content and process variables is described by equation 3. his = 16.8033 + 0.153 x1 0.005x1 2 r2 (0.90) (3) lysine content in processed samples ranged from 2.16 to 4.18g. lysine content of control was 7.10g/16g n. roasting condition was responsible for 90% per unit change of lysine of processed seeds. simultaneous increases in roasting temperature, time and feed quantity resulted in losses of lysine with a maximum (60.42%) loss observed at 193.64°c at 45min mark. the presence of the highly reactive 6-hn2 group of lysine may be responsible for observed reductions in lysine value. drawing from the significant terms the relationship between processed variables and lysine content of samples can be written as: lys = 5.650 + 0.0418x1 – 0.1142x2 – 0.030x3 – 0.023x1x2r 2 (0.90) (4) the content of arginine in the processed seed samples, varied between 3.17 to 5.62g/16g n which represents about 32 – 57.35% of the value of control. about 70.94% variation in arginine content was attributable to their roasting condition. however, no significant (p>0.05) effect of roasting variables on arginine was observed. acidic amino-acids (acid side chains at neutral ph) aspartic acid loss of processed seeds was 50% of its original content in control. the maximum loss in value of aspartic acid occurred at the extreme (193.64%) temperature. all roasting variables showed no significant effect on aspartic acid content of roasted breadfruit seeds. glutamic acid was observed to be heat susceptible with losses as much as 66.0% of its initial value in raw samples (19.50g/16g n) at 1200c -1400c. however, as it was observed with aspartic acid, roasting condition did not significantly reduce glutamine acids. the most probable reason could be an inherent ability of certain amino acids to inhibit the maillard reactions. hydroxylcontaining amino acids threonine content of roasted seeds was influenced by the processing conditions. though relatively heat stable at low temperature, threonine showed significant reductions at 1600c. threonine content in roasted seed sample ranged from 1.14g to 2.81g compared with 3.22g/16g n of control. the significant effect of roasting temperature and time on threonine can be described using the equation. thre = 6.4176 – 0.0642x1 – 0.3954x2 (r2) (0.85) (5) increasing roasting temperature and time significantly influenced threonine content by about 50%. serine content in roasted breadfruit seeds showed losses attributable to roasting variable conditions. computed loss variations showed a spread of between 1.47g to 3.9g/16g n, from 1230c to 193.640c, respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 378 branched chain amino acids isoleucine content of roasted breadfruit seed samples was between 1.30 and 3.11g/16gn. after processing isoleucine content varied from 13. 61% to 63.88% of control value (2.60g/16g n). the range of losses of isoleucine in roasted breadfruit seeds was comparable with the reports on groundnuts (25) and luffa seeds [26]. the roasting variables showed linear and interactive effects on isoleucine as: iso = 26.8837 – 1.0131x1 – 0.6994x2 – 0.0580x3 + 0.0185x1x2.r 2 (0.83) (6) leucine was relatively more heat liable than isoleucine. maximum loss (4.01g) occurred at 193.640c which showed the rate of change as each unit change of process variable reflecting 72% variability in leucine content of roasted samples. the significant relationship between leucine and process variables is expressed using equation 7, as: leu = 62.3574 – 0.2441x1 – 1.5157x2 – 0.0188x1x2r 2 (0.72) (7) neutral non polar amino-acids valine losses in roasted seeds were between 35.55% and 54.89%. it was observed that increases in roasting temperature and time simultaneously resulted in reduction of valine content of samples. roasting temperature and time significantly influenced valine content in a manner described by the equation: val = 24.9529 – 0.0489x1 – 0.7546x 2 + 0.0550x1x2r 2 (0.79) (8) alanine and glycine contents of roasted breadfruit seeds responded similarly to the effect of processing variables. alanine in samples was reduced to about 43.5% at temperature range of 1600c and 1800c, respectively. roasting variables of temperature, time and feed quantity did not significantly influence alanine contents of seed samples. aromatic amino acids phenylalanine content in roasted seeds was influenced by roasting variables. the relationship between contents of phenylalanine with roasting temperature, time and feed quantity using significant terms can be described by equation 9, as: phy = 19.3587 – 0.0919x1 0.2623x2 – 0.850x3 + 0.290x1x2r 2 (0.78) (9) increases in roasting temperature and time resulted in linear, quadratic and interactive effects. the linear and quadratic effect occurred below 1600c. above 1600c the model described an interactive effect between the roasting variables. a maximum loss of 56.72% occurred at 193.64%. tyrosine content of control was 3.5g/16g n. after roasting tyrosine values were between 1.0 and 2.5g with a maximum loss at 193.640c. tyrosine values of roasted seeds were related to processing conditions without observing any significant influence of roasting temperature, time and mass on the tyrosine content. the range of losses of tyrosine in breadfruit as observed by this study is in agreement with the findings of passmore and eastwood [27] in legumes. heterocyclic amino acids tryptophan content of raw unprocessed seeds was 0.4g/16g n, but it was undetected at processing temperatures above 123.640c. though breadfruit seeds are low in tryptophan, the rapid reduction observed for roasted breadfruit seeds could be due to the highly reactive nh-group of the inidole ring of tryptophan [28]. proline content of processed samples ranged from 1.21g to 4.9g/1.6gn (control). proline values were related to treatment conditions but they were not significantly (p>0.05) influenced by any processing variable (temperature, time and feed quantity). maximum loss (75.30%) of proline occurred at 193.640c. proline was more heat sensitive than histidine. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 379 sulphur-containing amino acid methionine contents of processed seed flour samples were influenced by roasting temperature and time. the loss variations of methionine depicted heat stability with content of processed seeds ranging from 0.81 to 1.53g/16g n of control. the summarization of the effects of roasting temperature and time using significant terms results in the equation 10 as: meth = 2.3326 – 0.0534x1 – 0.4203x2 – 0.0330x1x2r 2 (0.93) (10) increase in roasting temperature and time sequence resulted in initial linear effect up to 1400c. the observed square effect above that temperature was probably due to the sustained oxidation of sulphur fraction of methionine [28]. the result contrasted with the reported ones for methionine during roasting of legumes [29].the cysteine content of control was 2.0g/16g n. after roasting the cysteine content ranged between 0.45 and 1.50g which showed a loss range of 33.04% to 67.12% of cysteine at extreme conditions of 140°c to193.64°c. the results comply with the heat stability of cysteine up to 1600c and plausible for the fact that cysteine is limited in legumes. although the roasting variables had no significant effect on cysteine values of processed seeds, they significantly influenced similar sulphur containing methionine content of samples. the high toxicity risk factor of methionine in muscular damage should be a nutritional concern over high consumption of roasted breadfruit snack seeds. the global optimum for process variables and amino acids are summarized in table 4. optimum process variables resided at -1, -1, 1 at the composite desirability of 0.96. the optimum variable combination was 142.450c (temperature) 40.12mm (time) and 509.27g (feed quantity). table 4 experimental and predicted optimum process variables for amino acids process variables optimum values non-coded coded roasting temperature (0c) 142.45 -1 roasting time (min) 40.12 -1 feed quantity (g) 509.29 1 amino acids experimental (g/16gn) predicted (g/16gn) rda (mg/kg adult) alanine 3.10 2.99 arginine 5.62 4.87 aspartic acid 6.10 7.03 glutamic acid 8.21 9.11 phenyalanine 4.05 3.98 3.0 histidine 2.29 2.00 10.0 gylcine 4.05 4.16 lysine 4.27 4.19 30.0 threonine 2.81 2.30 15.0 methionine 0.75 0.89 4.1 proline 3.01 3.2 cysteine 0.95 1.02 0.4 serine 3.63 4.10 leucine 6.00 5.99 39.0 isoleucine 3.10 4.03 20.0 tyrozine 2.51 1.99 25.0 valine 3.25 4.16 26.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 380 the predicted optimum total of amino acids was 66.02% of control. for technical convenience, the processed variables could be adjusted to 140c, 40min and 500g. the experimental and predicted values are comparable (fig.1). hence the content of amino-acids in roasted breadfruit as revealed by the study satisfied about twothirds of the needed safe aggregates of amino-acids for humans [30] fig 1: total variation in different classes of amino acid of processed samples nutritional implication of amino-acid profile of roasted snack seeds the observed variations in amino acids are indicative of the influence of processing conditions on breadfruit seeds. oxidation, maillard reaction and formation of cross linkages involving amino acid side chains are some of the reported reasons for observed reductions of amino acid content during heat processing [28]. the high protein, carbohydrate [31] and anti nutrients [32] of breadfruit seeds infer the concomitance of oxidation, maillard reaction and cross linking of the active sites of amino acid side chains during dry heat processing of breadfruit snack seeds. the losses in amino-acid content are comparable with values reported for roasted groundnuts [25] bambara groundnuts [33], cashew [34] benniseeds [35] and sphenostylic stenocarpa [36]. under optimum roasting conditions (142.450c, 40.12mm500g) high contents of arginine (5.62g), aspartic acid (6.102g), glutamic acid (8.21g), leucine (6.0g) were in agreement with the reported information on those amino acids in the literature for roasted leguminous seeds and nuts [37, 38,39,40]. reports of studies under similar roasting conditions showed that roasted breadfruit seeds have higher content of lysine, arginine, threonine, glycine and serine than roasted groundnuts, almonds, cashew and sphenostylis stenocarpa. this study confirms the positive correlation between heat susceptibility and essential amino acids of roasted legumes but disagreed with the changes in methionine and threonine content of roasted groundnuts as reported by anatharaman and capenter [29]. the high susceptibility of the sulphur fraction of methionine and cysteine to oxidation contests their finding on methionine. the prominence of breadfruit roasted seeds snack as an important source of aminoacids needed for good health cannot be contested. the safe concentration levels of amino acids observed at optimum roasting conditions are able to satisfy the rda of amino acids for humans. the presence of histidine essential for infants and children [41] phenylalanine an important constituent of thyroid hormones, methionine an antioxidant needed for thiamine synthesis and mineral absorption, lysine essential for tryptophan and niacin metabolism, arginine an antioxidant immune booster and vasodilator and tyrosine need for neurotransmission and hormonal synthesis point to the nutritional importance of roasted breadfruit snack seeds. the leucine-isoleucine ratio as 14.31 12.18 39.34 30.5 20.22 43.02 experment control food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 381 observed promotes the sustenance of the complimentary role of leucine and isoleucine as a protection against high leucine induced adverse mental and physical disorder in population whose diets contain more of breadfruit seeds [41,42] the possibility of a nitrogen imbalance in the amino acid pool resulting from low lysine initiated pressure between methionine and lysine is inhibited by the high contents of methionine and lysine in roasted breadfruit seeds. roasted breadfruit snack seeds would be unable to satisfy the rda of cysteine for humans. the low glutamic acid content is also a minus for the proper development of skin and immune system in human [43,44] however, the observed concentrations of cysteine and glutamic acid of roasted breadfruit seeds pose no adverse toxicity risks of breadfruit to humans. it could be inferred through observed amino acid values that roasted snack seeds of breadfruit are nutritionally more wholesome in amino acid density, safety, digestibility and toxicity as compared with commonly consumed snack seeds of roasted groundnuts, almonds, cashews, sphenostylis stenocarpa and walnuts. the important nutritional contributions of roasted groundnuts, bambara groundnuts, almonds, cashews, sphenostylic stenocarpa and walnuts are limited by their low content of histidine, lysine, threonine, glycine and leucine. many physiological diseases have been traced to the deficiency of these important amino-acids in humans and experimental animals. moreover infants and growing children would need to consume large amounts of roasted almonds, cashews, groundnuts or other commonly consumed roasted snack seeds in order to achieve their rda for histidine. while roasted breadfruit seeds are able to deliver about 66% of total amino-acids, almonds with their high content of 23g glutamic acid, 0.26g tryptophan [37] can only deliver 37.25% total amino acids for humans. walnuts, cashews, sphenostylis sternocarpa and bambara groundnuts can only deliver between 11-24%, total aminoacids on consumption. the high content of glutamic acid and aspartic acid in almonds, cashews and walnuts are physiological risk factor for the development of mental and physical stressful conditions in humans. however the ability of the human body to convert glutamic acid to glutamine through the glutamate ammonium ligase reaction during trauma underscores the need for high content of glutamic acids in diets. glutamine is rapidly depleted during trauma [44] which emphasizes the need for high glutamic acid in the body for the maintenance of proper neurotransmission and brain functions. the usefulness of high glutamic acid in diet for the maintenance of glutamine homeostasis during trauma does not however challenge the findings which associate high glutamic acid with adverse neuro-physiological functions. under traumatic conditions roasted almonds are snacks of choice. digestibility of amino acids in animal gut is reduced by the presence of high lipid and dietary fibers in the fed diets [45 ,46] the reported lipid values in the literature are 14-54% almonds [37], 34% groundnuts [25],13% cashews [34] ,16% bambara groundnuts [33] as compared with 9 – 11% of roasted breadfruit seeds [9,47,]. the nutritional implication is that other roasted snack seeds could potentially impair amino acid digestibility in humans. groundnuts and almonds have higher soluble dietary fibers (about 50% of their 3.5 -5% raw fiber values) than breadfruit seeds. soluble fibers have higher impeding actions on nutrient metabolism than other forms of fibers. the inhibitory actions of lipid and dietary fibers on nutrient metabolism occur at the enzyme-nutrient interface, blocking the active side chains and the promotion of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 382 physical barriers at nutrient absorption sites in humans. another nutritional risk factor for other roasted snack seeds is atherogenesis [48].the atherogenic implications of roasted nuts with high lipid contents could probably be ameliorated by the presence of high dietary fiber (9-13%) in nuts. the presence of high fibers though important in preventing atherogenic reactions does not prevent the initiation of the maillard reaction during roasting and the development of some undesirable metabolites. during roasting of seeds, the maillard reaction occurs between sugars and aminoacids resulting in production of metabolites and color development through different biochemical reaction pathways. acrylamide, a carcinogenic toxic [49] has been shown to get formed in food during roasting [50]. acrylamide accumulation in roasted seeds is defined by roasting temperature, time, seed architecture and chemical composition of seeds [51]. high fat content is a risk factor for accumulation of acrylamide during the roasting process [52]. seed architecture on the other hand influences heat transfer rates and substrate interactions inside the seed. thick seed hull inhibits accumulation of acrylamide in seeds transiting the acrolein metabolic pathway [53,54]. high lipid contents of roasted almonds, groundnuts, cashews and walnuts are suggestive of high accumulation of acrylamide in them. also the roasting of almonds or of walnuts without shells offers no protection against acrylamide accumulation. in addition to these nutritional drawbacks of other snack seeds and nuts, some studies have reported walnuts, almonds, groundnuts and cashew as major sources of food allergens [55]. no such implicating reports for roasted breadfruit seeds as allergenic to humans have been documented. 4. conclusion the process parameters used in this study demonstrated good performance. identified optimum process variables combination occurred at about 1400c and 40min. above 140° 40 min. point, roasting of breadfruit seeds results in significant reductions in amino acid concentration to levels below the values needed to satisfy the rda of amino acids for humans. the results of this study are an important template for proper dry heat processing of unseasoned breadfruit seeds for human consumption as snacks. 5. references [1]. nkatamiya, uu.,madebo, aj., manji, d., haggai, o.. nutrient contents of seeds of some weld plants. j. african of biotechnology: 6 (4) 1665 – 69. 2007 [2]. nwosu, jn., ubaonu, cn.,banigo,eoi and uzouah, a.. the effects of processing on amino acid profile of ‘oze’ seed flour. life science journal; 5 (4): 69 – 74. 2008 [3]. makinde, ma.,.arukwebo and p. pettet.ukwa seed (treculiaafricana). protein i. chemical evaluation of the protein quality. j. agric. food. chem. 33 – 72 – 74. 1985. [4]. health and welfare canada.report of the expert advisory committee on amino acids. ministry of supply and services. canada catelog no h42-2/40. 3-60. 1990. [5]. mariod, aa., ahmed,sy,abdelwahi si , cheng sf,eltam am, yacoub ss. s. w. gouke. effects of roasting and boiling on the chemical composition, amino-acid and oil stability of safflower seeds. intl. j. of food sci. and technology. 47 (8): 1737 – 1743. 2012. [6]. okaka, jc. anc. okaka.foods composition, spoilage and shelf-life extension. ocjano academic pub. enugu. 2005 [7]. sfgate.recommended essential amino acids. in healthy eating. m. appleby (ed). available www.safeguard.com/accessed 13/31/2015. 2014 [8]. nwabueze, tu.,iwemo. e.n.t. akobundu. unit operations and analyses for african breadfruit based spaghetti type product at extreme process combination. j. food. tech. 5 (1): 42 – 45. 2007. http://www.safeguard.com/accessed%2013/31/2015 http://www.safeguard.com/accessed%2013/31/2015 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 383 [9]. nwokochalm.,ugbomoiko. effect of parboiling on the composition and physiochemical properties of treculia africans seeds. pakistan j. nutrient. 7(2):317-320. 2008. [10]. arawande, jo. oluwasani, ai. adewumi bl. .nutritional significance of husked and dehusked seeds of african breadfruit and characteristics of its oil.j. res. int. nat. dev. (7) 1 – 5. 2007. [11]. fassasi, os.,eleyimmi, af. fasasiar. o. r. karim. chemical properties of raw and processed breadfruits (treculiaafrican) seed flour. j. food. agric. env. 1459 – 1465. 2004. [12]. iwe, m.o., p.o. ngoddy. .development of mechanical process for the african, breadfruit (treculiaafricana). nig. food. j. (19): 8 – 16. 2001. [13]. nwabueze, tu. kernel extraction and mechanical efficiency in dehulling parboiled african breadfruit (treculiaafricana) seeds. j. food quality. 32: 669-683. 2009. [14]. anyalogba, e, eoyeiken.,monanu mo. effects of heal treatment on the amino acid profile of plukenetiaconophora seeds kernel flours. international j. of biochem. research of review: (3); 121 – 131. 2015. [15]. enwere, nj. . food of plant origin. afro orbis pub. nsukka. 194 – 199. 1998. [16]. lehningers, al.,nelson dl., mm. cox.principles of biochemistry, w. h. freeman and co. pp 301-416. 2000. [17]. fao/who/unn protein and amino-acid requirements in human. who press – 150. 2007. [18]. enujiugh, v. n. chemical and functional characteristics of canephor nut. pakist. j. nutr. 2 (6) 335-338.2003 [19]. francis, d. nutrition for children. blackwell. oxford.1986 [20]. dziesk, jd.. taking the gamble out of product development. food technology. 125: 110 – 117. 1990. [21]. ruguo, s. food product design-computer aid statistical approach. crc. press fl. usa.1999. [22]. nwabueze, t.u. basic steps in adapting response surface methodology as mathematical modeling for bioprocess optimizations in the systems. international journal of food science technology. doi.io.iiii/j.1362-2621.2010.02256x. accessed 11/12/2012. 2010. [23]. spackman, de,, stein h, and s. mooxe. .automatic recording apparatus for use in the chromatography of amino acids. analytical chemistry. 30: 1190 – 1101. 1958 [24]. aoac international..official methods of analysis. association of analytical chemists maryland. 2000. [25]. adeyeye, et. effect of cooking and roasting on the amino acid composition of raw groundnut (arachushypogaea) seeds. acta, scient, polonoium tech. alimentara (2) 201 – 216. .2010. [26]. olaofe, o., okoiribiti, by.,aremu, o..chemical evaluation of the nutrition value of smooth luffa seed kernel. elect. j. environ. agric. food chem. 7 (10): 3444 – 3752. 2008 [27]. passmore, r., ma. eastwood..human nutrition and dietetics. 8 edn. churchill livingstone. london. 1981 [28]. dworschak, e., non enzyme browning and the effect on protein nutrition critical rev. food. sci. nutri. 13 (1): 1-40. 1980. [29]. amantharaman, k.., capenter, kj. effects of heat processing on the nutritional value of groundnut products ii. individual amino-acids. j. sci. food. agric. 22 (3) dio.org/10.10021/jsfa.2740220807. 1971. [30]. american society for nutritional sciences asns. the nature of human hazard associated with excessive intake of amino acids. journal of nutrition. available www.mjn.nutriiton.org/content/134/1633. accessed 03/31/2012. 2004. [31]. umezuruikeac ,nwabuezet.u , akobundu ent. anti nutrient and hemagglutinin activity o food grade flour of roasted african breadfruit seeds produced under extreme conditions. fuuast j. biol. 6(2):135-139. 2016. [32]. gilani, g. s., xiao, c. w., cockell, k. a. impact of anti-nutritional factors in food protein on the digestibility of protein and bioavailability of amino acids and on protein quality. brit. j. nutrition. 108 (2): 5315-5332. doi.org/10.1017/50007114512002371. 2012. [33]. abdulsalami, ms., sheriff, hb. effects of processing on the peroxide composition and mineral content of bambara groundnuts (v. suberanean). j. plant. appl. sci. 3 (1): 123-190. 2010. [34]. fagbemi, t n. effect of processing on chemical composition of cashew nut (anacardiumoccidentale). j. food. sci. tech. 46 (1) 36-40. 2009 [35]. adegunwa, mu.,adebowale, aa., solano, eo. effect of the thermal processing on the biochemical composition, anti-nutritional factors and functional properties of beniseed (s. indicum) flour. am. j. biochem. mol. biol. 2 (3): 175-182. doi.10.3923/9jbmb.2012. 175-182. 2012 [36]. uche, sn., ndidi, c., olagunju, a., muhammadu, a, billy, fg., okpe o. proximate, ant nutrients and mineral composition of raw and processed (boiled and roasted http://www.mjn.nutriiton.org/content/134/1633 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 azubuike c umezuruike, joel ndife,chinwe nwachukwu, characterization of effect of modulated dry heat processing conditions on essential and non-essential amino acid profile of unseasoned breadfruit (v. decne) snack seeds, food and environment safety, volume xvii, issue 4 – 2018, pag. 372 – 384 384 sphenostylisstenocarpa seeds from southern kaduna, north-west, nigeria. issrn nutrition. doi.dio.org/10.1155/2014/280837.1-8.2014 [37]. adu, ob., ogundeko, to.,ogunrinola, oo et. al.. the effect of thermal processing on protein quality and freeze amino acid profile of terminaliacatappa (indian almond). j. food sci. technol. doi:10.1007/s/3197.0141490-8. 2005. [38]. odoemelams. chemical composition and functional properties of canephor nut flour. intl. j. food sci. technol. 3 (6) 729-734. 2003. [39]. kita, a. figiel, a.. effect of roasting on properties of walnuts. pol. j. food. nutr. sci. 57 (2): 89-94. 2007 [40]. ozdemir, m., ackurt, f., yildiz, biringena,m,curcan t., loderm. effect of roasting on some nutrient of hazalnut food. chem. 13 (2) 185-190. 2001 [41]. imura, k. and okada, a. (1998).amino-acid metabolism in pediatric patients. nutrition 14 (1): 143 – 8. doi.10.1016/50899-9007(97)00230 – x. accessed 03/31/2015. [42]. munro, hm. nutritional consequences of excessive amino-acid intake adv. exp. med. biol. 105: 119-129. 1978 [43]. balch, p., balch, j. .prescriptions for nutritional healing. avery books. 2000. [44]. labow, b. i.,soubaw. w.. glutamine. world j. surrg. 24 (12): 1503-1513. 2000 [45]. li, s. ,saver, wc. hardini, rt.. effect of dietary fibre level on amino acid digestibility in young pigs. canadian j. anim. sci. 74: 327-333. 1994. [46]. akande, ke. and fabiyi, ef..effects of processing methods on some nutritional factors in legumes seeds for poultry feeding. international journal of poultry science. 9 (10) 996-1001. 2010 umezuruike ac, nwabuezet.u.nutritional and health potentials of the seasonal changes in some nutrients, anti nutrients and minerals of treculia africans food crop. am j. foodsci. technology.6(1):12-18. doi:10.12691/ajfst-61-3. 2018. [47]. anderson, jw ,deakin, da,floor, tc., smith. bm.,whitis, se. dietary fibre and coronary heart. disease. food sci. nutr. 29:95147. 1990. [48]. duxbury, d. acrylamide in food. cancer risk or mystery. j. food technol. 58 (12) 91-93. 2004 [49]. xu, v., cui, b., ran, r., liu, y., chen h., kai, g., shi, j. risk assessment formation and mitigation of dietary acrylamide: current status and future prospects. food and chem. toxixol. 69: 1-12. 2014. [50]. friedman, m. chemistry biochemistry and safety of acrylamide. a rev. j. agric. food chem. 51: 4505-4526. 2003 [51]. muesser, s. goskel, ts., omer, o. effect of different roasting temperature on acrylamide formation of some different nuts. j. env. sci. toxicol. food technol. 11 (4) 38-43. 2017 [52]. lukac, h. ,amrein,t.m., perren, r., conde-petit b, amado, r., escher, f. influence of roasting condition on the acrylamide content and colour of roasted almonds. j. food. sci. 77 (1) 33-39. 2007. [53]. zhang y., zhang, y. formation and reduction of acrylamide in mailard reaction. a review basedfon the current state of knowledge. crit. rev. food. sci. nutr. 47 (5): 521-542. 2009. [54]. stein, n.. are roasted cashews healthy. livestrong.com.www.livestrong.com/article44104, 2017 effect of plant protein isolates to the structural – mechanical properties of wheat dough 413 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 2018, pag. 413 421 effect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough galyna voloshchuk 1 , natalia pashova 2 , * tatjana golikova 3 , 1institute of postgraduate education of national university of food technologies, estonska str. 8a, kyiv, ukraine, volo_g@ukr.net 2 educational-scientific institute of food techmologies of national university of food technologies, volodymyrska str. 68, kyiv, ukraine, pashova.n@gmail.com, 3technology of nutrition and restaurant business dept., national university of food technologies, volodymyrska str. 68, kyiv, ukraine, tatjana.golikova12@gmail.com *corresponding author received 25th november 2018, accepted 28th december 2018 abstract: this paper offers investigation of fractionally defatted flour from seeds of walnut, pumpkin, sesame, topinambur flour and their mixes influence on the rheological properties of rye dough. the investigated raw materials provide increase of water absortion and decrease of softening of dough due to the farinograph values. the use of fractionally defatted flour and topinambur flour reduces gelatinization temperature and viscosity of suspension by amylograph indexes. nonetheless, dough for malt rye bread with addition of certain types of fractionally defatted flour (up to 4 %) has lower viscosity that causes increase in strength of structural links in dough after kneading and its decrease after one hour of fermentation. the developed mix of fractionally defatted flour from seeds with topinambur flour led to increased rheological properties of dough and provided proper structuralmechanical properties of dough till the end of fermentation that were identical to the control samples. keywords: fractionally defatted flour, topinambur flour, rye dough, rheological properties. 1. introduction malt rye bread is quite popular bread and is highly profitable for bakeries. but because of its high content in starch and its degrading products with high gi, of reduced biological value, dieticians do not recommend this bread for people suffering from carbohydrate-lipid metabolism disorder [1, 2]. in order to fortify bread with physiologicallyfunctional components – inulin, mineral matters, high nutritional proteins and lipids, dietic fibers, it is necessary to produce malt rye bread with addition of topinambur flour (tf) and fractionally defatted flour (fdf) from seeds of walnut, pumpkin, sesame [3-5]. fractionally defatted flour is a product of processing of meal pellets from oil seeds that after partial oil extraction contains most part of nutritional bioactive matters [6, 7, 8]. tf and fdf include specific protein matters, hydrophilic polysaccharides, lipids and lipoids, ascarides, mineral matters, enzyme complexes, acids. producers regularize quality of fdf by mass fat content (till 15 % to the dry matters), mass water content (till 8.0 %), and particles size. due to the grain size this flour has similar particle sizes as rye flour t130 [9, 13]. optimal dosage of topinambur flour for providing of 30...70 % daily intake of http://www.fia.usv.ro/fiajournal mailto:volo_g@ukr.net mailto:pashova.n@gmail.com mailto:tatjana.golikova12@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 414 inulin in case of consuming 130…277 g of bread and minimum content of sugar in rye bread is 3 % [8, 9, 10]. authors believe that increase in dosage causes worsening of taste and flavor properties of bread, decreases elasticity of crumb. scientists proved that the addition of fractionally defatted flour from walnut fortifies products with protein matters, polyunsaturated fatty acids, lecitin, mineral matters, and selenium [12]. but even small dosage of walnut additive decreases gas production and gas retention of dough and reduces porosity of bread and elasticity of crumb [13]. fdf from pumpkin contains more than 50 % of protein that is reached with tryptophan, valuable mineral composition. dough with pumpkin seed products increased intensity of fermentation (gas production), shape stability, prolongation of shelf life, elastic crumb [13, 14]. but a bigger dosage leads to stickiness of crumb and flavor of pumpkin oil. fdf in sesame fortifies bread with mineral complexes ca, mg and other insufficient mineral matters, monounsaturated fatty acids, dietic fibers [15]. bread with fdf in sesame had highest quality factors of crumb, higher specific volume, but big amount of sesame product reduces gas production and gas retention of dough. mix of fdf made from sesame, pumpkin and walnut was determined by chemical composition of rye flour t130 and fdf by means of the program “optima” proposed by professor larysa arseniyeva [16]. it has been demonstrated that combined use of fdf with tf in amount of 10 % in relation to flour leads to the obtaining of bread with increased nutritional value and good degustation quality indexes [17]. mix composition of tf and fdf had positive influence on the technological process and intensification of fermentation process. so, the most important criteria of dosage of new products were their chemical composition and influence on the quality of dough. the rheological properties of dough provide quality indexes of bread; they determine choice of equipment for transporting and processing of dough [18]. the aim of the work was to investigate effect of tf and fdf made from sesame seeds, pumpkin and walnut on the structural-mechanical and rheological properties of dough from rye flour t130. 2. matherials and methods ingredients rye flour t130 with falling number 220 c and 165 c was used. rye malt flour, salt, water, ready to use rye sourdough (lactic acid bacteria: l. plantarum 30, l.casei 26, l.fermenti 34, l.brevis and yeast s. minor, s.cerevisiae) with flour content 15 % to the general weight of flour in dough and moisture content 71,0±1,0 %, acidity 10,0±1,0 grade. brewing (pregelatinized flour – 5.0 % rye flour, 5.0 % of rye malt flour with saccharification period (time of brewing) 180 min, moisture content 64.0±1.0 %. dough preparation included 3 steps: liquid sourdough – saccharified brewing – dough. topinambur flour was added to dough. moisture content of dough – 51.0±0.2 %, temperature of dough 29 °с, last fermentation time 60 min (3600 с) fractionally defatted four from walnut, pumpkin seeds and sesame (company richoil) had fat content 10.0…12.0 % of dry matters and scientific-production company “elitphito” with fat content 10.0…15.0 % to the dry matters. topinambur flour “dar”® with total fructan content 44 %, average molecular mass – 15 points of linearity, acidity 15…22 grades was used. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 415 structural and mechanical properties the effect of the raw materials studied in terms of structural and mechanical properties and water absorption of rye dough have been estimated by means of farinograph (brabender, germany). the trial samples with general weight 300 g were prepared according to the instructions. the quantity of water for mixing the dough has been chosen experimentally in order to obtain dough with standard consistency 500 units. rye flour t130 and flour (fractionally defatted flour from seeds and topinambur flour) were used for test samples. registration of the changes in rheological characteristics of test samples was determined during 25 min from the start moment of mixing. this method is widely accepted to estimate the water binding as well as structural and mechanical properties of dough [ 19]. viscoelastic properties the viscoelastic properties of dough made from fractionally defatted flour were estimated by means of amylograph. (brabender® gmbh & co. kg, germany) due to the icc-standard no. 126/1 iso 7973 aacc method no. 22-10 for measurement of gelatinization properties and enzyme activity of flour and whole meal [20]. dough samples were prepared from pregelatinized flour (brewing) and rye liquid sourdough. fdf have been added in a dosage that favorizes the obtaining of bread with proper sensorial characteristics. the viscoelastic properties of dough have been estimated after the mixing of dough and after 60 min of fermentation accordingly to the technological instruction. rheological characteristics rheological characteristics have been estimated by means of rotational viscosemeter “rheotest-2”. test samples of dough were prepared using pregelatinized flour and liquid rye sourdough. rheological characteristics were investigated after mixing and after 1 hour of fermentation. 3. results and discussion structural and mechanical properties farinograms of dough with fdf and tf had a curve that is common to rye flour, without expressed section of stability dough. the results of farinograms’ description of rye flour as well as its mixes with fdf and tf are shown in the table 1. the addition of tf decreases water absorption of flour probably due to the high content of sugar [21]. degree of dough softening also decreases due to the presence of inulin and pectin substances [22]. it was proven that the addition of tf leads to minor increase of water absorption of dough (of 0.3 – 1.7 %) and to significant decrease of dough softening (of 11 – 57 %). the dough development time with additives is in frame ± 30 с. fdf of sesame and walnut express the highest effect on reduction of degree of softening – between 40…80 points. fdf of walnut increases elasticity of mixed dough of 5…11 %. the addition of fdf of sesame and pumpkin seeds decreases elasticity of dough, but in the case of a bigger dosage it increases elasticity of dough. obviously, such influence is determined by increased content of dietary fibers as well as high acidity of fdf especially fdf of walnuts. the applying of a mix of fdf and tf causes some increase in water absorption and dough development time, but it significantly increases elasticity of dough and degree of softening. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 416 table 1 effect of fdf on the farinogram parameters of dough made from rye flour t130 viscoelastic properties due to the results of amylograms analyses of rye flour with additional raw materials (see table 2) tf decreases by 1 °с temperature of initial gelatinization of starch and 20 points (or 4 %) maximum viscosity of suspension. the addition of fdf reduces the gelatinization temperature to 2…3 °с. herewith amylograph’ curves had prolonged up to 5 min section of maximum viscosity of suspension. the addition of fdf significantly reduces the maximum viscosity of water-flour suspension of rye flour – from 520 to 320…360 points (of 31….39 %). table 2 effect of ftf to the carbohydrates-amylase complex of rye flour sample dosage, % to the mass of flour initial gelatinization temperature, °с maximum viscosity, pа∙с control sample (without additives) 55 520 tf 3 54 500 fdf of walnut 2 53 345 4 53 340 fdf of pumpkin seeds 2 53 360 4 53 355 fdf of sesame seeds 2 52 360 4 53 345 mix of fdf 7 53 320 mix of tf and fdf 10 52 330 sample dosage, % to the weight of flour quality indexes of dough water sorbing capacity, cm3/100 g dough development time, min elasticity, points degree of softening, points control sample (without additives) 60.6…59.8 2.5 74 140 tf 3 59.8…59.0 2.5 74 100 fdf of walnut 1 60.8…60.0 3.0 77 100 2 61.0…60.2 2.5 77 65 4 61.0…60.2 3.5 80 60 fdf of pumpkin seeds 1 61.0…60.8 2.0 73 120 2 61.4…60.6 2.0 73 110 4 61.0…60.2 2.5 75 100 fdf of sesame seeds 1 60.8…60.0 2.0 73 125 2 61.2…60.4 2.5 74 100 4 60.8…60.0 2.0 74 95 mix of fdf 7 60.9…60.2 2.5 80 95 mix of fdf and tf 10 60.5…60.0 2.5 78 100 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 417 the use of fdf and tf challenges minimum viscosity of starch paste and lower initial gelatinization temperature – 51 °с. we believe that such effect may be caused by big amount of water-soluble substances in fdf that have an influence on accessibility of starch in rye flour gelatinization. rheological characteristics in order to investigate the rheological characteristics of dough we installed that dynamic viscosity of un-destroyed system grows during fermentation of dough on 1.6 – 2.0 %, and dynamic viscosity of destroyed system remains unchangeable (see table 3). border of capability of dough to the dynamics of destroying decreases while strength of structural links in fermented dough grows due to the capability of rye pentosans to make a sticky water solution that after oxidation turns into a strong gel. thus, rye dough during fermentation gets more sticky, elastic and slows stretching. the addition of tf has minor effect on the viscosity parameters of dough after mixing but during the fermentation process, the dynamic viscosity together with the strength of structural network decrease (see fig. 1, 2). the results of table 3 and fig. 1, 2 show that fdf effects change the rheological parameters of dough during fermentation. table 3 effect of fdf on the rheological characteristics of dough from rye flour t130 sample d y n am ic v is c o si ty o f u n c ru sh e d st ru ct u re , η 0 , p a. с * 1 0 ³ d y n am ic v is c o si ty o f c ru sh e d st ru ct u re , η 0 , p a. с * 1 0 ³ a n o m a ly o f v is c o si ty , η 0 – η m , p a . с * 1 0 ³ c h a ra c te r o f c re a te d s y sy te m р к 1 , p а * 1 0 ³ d y n am ic l im it o f fl o w in g s y te m c a p a ci ty , р к 2 , p а * 1 0 ³ s tr e n g th o f st ru c tu ra l n et w o rk , р m , p а * 1 0 ³ s tr e n g th o f st ru c tu ra l li n k s р к 1 /р к 2 , p а s tr e ss r a n g e, р m /р к 1 , p а dough after mixing control sample 29.55 0.34 29.21 9.84 13.00 14.90 0.76 1.51 3 % tf 29.55 0.32 29.23 9.85 12.50 14.00 0.78 1.42 1% fdf walnut 28.07 0.29 27.78 9.35 11.00 12.50 0.85 1.34 2% fdf pumpkin seeds 29.06 0.33 28.73 9.68 11.50 13.00 084 1.34 4% fdf sesame 29.05 0.32 28.73 9.72 11.20 12.70 087 1.30 10 % mix of tf and fdf 31.52 0.35 31.16 10.50 12.30 15.00 085 1.43 dough after fermentation control sample 30.04 0.34 29.70 10.00 12.50 14.80 080 1.48 3 % tf 28.07 0.29 27.78 9.35 11.00 11.80 085 1.26 1% fdf walnut 27.58 0.29 27.29 9.18 11.10 11.75 0.83 1.28 2% fdf pumpkin seeds 28.58 0.32 28.24 9.51 11.70 12.70 0.81 1.33 4% fdf sesame 28.56 0.32 28.24 9.61 11.60 12.40 0.82 1.29 10 % mix of tf and fdf 30.53 0.34 30.19 10.17 12.20 14.26 0.83 1.40 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 418 a – dough after preparation b – dough after 60 min of fermentation fig. 1. influence of tf and fdf on viscosity curves a – dough after preparation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 419 b – dough after 60 min of fermentation fig. 2. influence of tf and fdf on flow curves dosage of some types of fdf in amount from 1 % to 4 % decreases the dynamic characteristics of dough at the beginning and at the end of fermentation process as well when compared with the control sample. viscosity curves of rye dough samples prove that the addition of fdf decreases values of maximum and minimum viscosity (η0, ηm,) that refers to un-destroyed and practically destroyed systems of dough after mixing (table 3, fig. 1). but in fermented dough the effect of fdf is less evident. the lowest viscosity values have been obtained for dough samples with addition of 1 % of fdf of walnut (28072,1 pа∙с – after mixing of dough and 27579,6 pа∙с – after fermentation). the strength of structural network of control rye dough sample almost has no changes but the strength of structural links increases (table 3, fig. 3). the addition of tf in dough provides an increase of structural links at the beginning of the fermentation process and at the end as well. but topinambur ingredients significantly decrease strength of structural network of dough. fdf of seeds and mixes undergo an increase of dough strength of structural links after mixing but further its decrease during fermentation. herewith, the addition of certain types of fdf decreases the strength of structural carcass of dough just after mixing and during fermentation. the addition of mix of fdf with tf provides hybrid effect and increases the values of viscosity and structure strength and decreases diapasons of intentions of dough when compared to control sample. the samples of structural network and structural strength of dough with mix decrease during fermentation up to the parameters of control samples after fermentation. such effect may be explained by the addition of high amount of dietic fibers, proteins and bioactive substances of fdf. it caused strengthening of structural links of dough after mixing, at the beginning of fermentation and decreasing of rheological parameters of dough at the end of fermentation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 420 fig. 3. influence of tf and fdf on strength of structural links thus, the quality of dough with developed mix is identical to the control sample at the end of fermentation. it allows recommending the production of rye bread with addition of mix of fdf and tf by means of production line without technological adjustments of dough shaping and transporting equipment. 4. conclusion investigation of structural and mechanical properties of dough showed increase in water absorption and decrease in the degree of softening of 15…80 pharinograph points due to the addition of fractionally defatted flour from walnut, pumpkin seeds and sesame. fdf and tf provide decrease in gelatinization temperature and suspension viscosity of rye flour by amilograph values. the maximum viscosity of rye flour suspension decreases by 160…200 points and gelatinization temperature is of – 3…4 °с. the addition of certain type fdf up to 4 % causes increase in strength links after mixing of dough in brewing and sourdough both with decrease in the structural dough network after one hour of fermentation. the addition of mix fdf of walnut, pumpkin seeds, sesame and topinambur flour has positive influence on dough preparation – it allows to fortify dough with functional ingredients and increases rheological properties of dough for rye malt bread. this mix of fdf and tf increases viscosity of dough and strength of structural links relative to the reference dough sample and lowers these rheological properties after the fermentation process, close to the control sample. such influence of mix is explained by the formation of carbohydate complexes between fdf and tp that borrow water in rye flour and determine changes of rheological properties. 5. references [1]. kuznetsova l. (2003). production of rye malt bread / l. kuznetsova, o. afanasieva, n. sinyavskaya, e. flenova. snt. peterburg. – 298 p. in russian). [2]. lebedenko t. (2016) aspects of using natural sweetener in breadmaking technology / t. lebedenko, n. sokolova// materials of international scientific conference “food technologies, bread and compound feed”. – food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 natalia pasova, galyna voloshchuk, tatjana golikova, eeffect of fractionally defatted flour of seeds on the structural and mechanical properties of rye dough, food and environment safety, volume xvii, issue 4 – 2018, pag 413–421 421 odessa, september 13-17, 2016 – p. 24 – 25. (in ukrainian). [3]. patent 120600 ua, ipc a21d 2/36(2006.01) a21d8/02(2006.1) fortified rye malt bread / pashova n., voloshchuk g. – № u201705030; stated. 25/05/2017; has published nov. 10, 2017, bul. no. 21 (in ukrainian). [4]. patent 120601, ipc а21d 2/36(2006.1) a21d8/04(2006.1) fortified rye malt bread / pashova n., voloshchuk g. – № u201705031; stated. 25/05/2017; has published nov. 10, 2017, bul. no. 21 (in ukrainian). [5]. patent 120603, ipc а21d 2/36(2006.1) a21d8/04(2006.1) fortified rye malt bread / pashova n., voloshchuk g. – № u201705031; stated. 25/05/2017; has published nov. 10, 2017, bul. no. 21 (in ukrainian). [6]. nutritional value, chemical composition and calority. data sourse usda sr-23 [electronic resourse]. – access http: // intelmeal.ru/nutrition/foodinfo-wheat-sprouted.php [7]. raw material compendium (1996): a compilation of worldwide data sources. novus europe s.a.brussels, p. 541. [8] codină g. g., mironeasa s (2016). application of d-optimal mixture design to optimize the wheatpumpkin composite flour for the bread production // food and environment safety, vol. xv, issue 1 – 2016, pag. 10 – 20 [9]. dotsenko v. f. (1991). scientific grounding and development of bread technology with using of new carbohydrates containing raw materials and sweeteners / ext. abstract of doctor sci. (eng.) dissertation. – 05.18.01 “technology of bread, macaroni and confectionery products”– кyiv.: – 1994. – 49 p.(in ukrainian). [10]. bantea-zagareanu v. (2018) use of walnuts (juglans regia l.) waste from physical extraction of oil to produce flour and sweets // food and environment safety, vol. xvii, issue 1 – 2018, pag. 74 – 80. [11]. pashova n. (2017). optimization of bread production for rye malt bread with topinambur flour / n. pashova, g. voloshchuk, v. khrut’ba, h. waihang // storage and processing of grain. – 2017. – № 7. – p. 60 65. (in ukrainian). [12]. mao x. (2014). amino acid composition, molecular weight distribution and gel electrophoresis amino of walnut (juglans regia l.) proteins and protein fractionations / mao xiaoying, yufei hua, guogang chen / x. mao, h. yufei, c. guogang. // international journal of molecular sciences open access journal. – 2014. – №15 (2). – p. 2003–2014. [13]. chornyi v. (2017) influence of oil cake from seeds and walnut on rye bread production / v. chornyi, g. voloshchuk, n. pashova // materials of x ukrainian student scientific practical conference “natural and humanitarian sciences. actual questons”. ternopil – 2017 –p. 225 – 226 (in russian). [14]. makarenko ye. (2017). microbiota of rye dough with topinambur and oil cakes / ye. makarenko, n. pashova, n. gregirchak, g. voloshchuk // modern achievements of pharmaceutical technology and biotechnology. – kharkiv. – 2017. – p. 177 180 (in ukrainian). [15]. drobot v. (2018). use sesame flour in the production of whet bread / v. drobot, yu. bondarenko, o. bilyk, a.grishenko // collection of scientific works "food resources". 11: 101-110 (in ukrainian). [16]. arsenieva l. yu. (2007). scientific grounding and development of technology of functional bread with vegetable origin and micronutrients / doctor sci. (eng.) dissertation. – 05.18.01– кyiv.: – 2007. – 402 p. (in ukrainian). [17]. patent 120604, ipc а21d 2/36(2006.1) a21d8/02(2006.1) fortified rye malt bread / pashova n., voloshchuk g. – № u201705064; stated. 25/05/2017; has published nov. 10, 2017, bul. no. 21 (in ukrainian). [18]. cherevko o.i. (2005). calculation of machines and apparatus of food production / o. cherevko, v. majiak, o. majiak kharkiv – 2005. – 223 p. (in ukrainian). [19]. iso 5530 – 1:2015. “wheat flour. physical characteristics of dough. part 1: determination of water absorption and rheological properties using a farinograph”. [20]. icc-standard no. 126/1 iso 7973 aacc method no. 22-10. brabender® amylograph®-e for measurement of gelatinization properties and enzyme activity of fl our and whole meal. [21]. dotsenko v. f. (1991). influence of topinambur products on structural and mechanical properties of wheat dough / dotsenko v., horbatiuk l. –кyiv.: – 1991. – 49 p.(in ukrainian). [22]. dorokhovich v. (2003). investigation of influence of inulin on structural-mechanical properties of flour confectionary products / v. dorokhovich // scientific works of donetsk state uniersity of economy and trade 2003. – – 1(17). p. 95-100 (in ukrainian). http://www.intelmeal.ru/nutrition/foodinfo-wheat-sprouted.php 1. introduction 136 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 3 2019, pag. 136 149 comparative analysis of the proximate, fatty acids and mineral composition of selected fish species *alexander ikechukwu ajai 1 , mohammed mohammed ndamitso 1 , lucky ekwoba 1 and abidemi adedayo koleola 1 1department of chemistry, federal university of technology minna, niger state, nigeria ajai.ike@futminna.edu.ng; talk2alexajai@gmail.com *corresponding author received 30th may 2019, accepted 20th september 2019 abstract: proximate, minerals, fatty acids and cholesterol compositions were determined in five different freshwater fish species (oreochromis leucostictus, clarias garipinus, gynarchus niloticus, hyperopisus bebe occidentallis and synodotus clarias) obtained from shiroro lake, niger state, nigeria using standard analytical methods. the proximate compositions were found to be 39.00 – 51.18% protein, 19.43 – 48.47% fat, 2.13 – 14.74% carbohydrate, 4.91 – 19.59% ash and 4.57 – 5.63% moisture content, with significant difference at p < 0.05 among fish species, whereas the fatty acids were found to be composed of 38.93 – 42.81% saturated fatty acids (sfa), 33.63 – 45.37% monounsaturated fatty acid (mufa) and 7.79 – 26.10% polyunsaturated fatty acids (pufas) with significant difference at p < 0.05 among fish species. the cholesterol content was significantly higher in tilapia (oreochromis leucostictus) with 8.38 ± 0.02 mg/100g and the lowest in catfish (clarias garipinus) with 2.74 ± 0.04mg/100g respectively. the mineral composition (na, ca, k, mg, fe and zn) in the five species of fish ranged from 31.63 – 49.59mg na/100g, 8.56 – 33.60mg ca/100g, 8.68 – 20.69mg k/100g, 6.33 – 11.77mg mg/100g, 0.07 – 2.05mg fe/100g and 0.13 – 0.33mg zn/100g respectively with significant difference at p < 0.05 among the fish species. in this study, gynarchus niloticus and synodotus clarias have registered the highest amount of poly-unsaturated and the lowest amount of saturated fatty acids and therefore they are recommended for consumption. notwithstanding other species should also be harnessed for their outstanding mineral contents in order to improve the essential nutrients derivable from them. keywords: fish species, cholesterol, proximate composition, minerals, polyunsaturated fatty acids 1. introduction fish is one of the most diverse groups of animals known to man and there are more species of fish than all other vertebrates. it has remained as one of the most important sources of animal protein due to its availability, being relatively cheap as compared with other sources of proteins such as meat, excellent taste, easy digestibility, lack of any cultural or religious taboos associated to its consumption by any particular ethnic group, high content of essential nutrients and unsaturated fatty acids which are very relevant for functionality of protein in the body [1-4]. fish lipids act as important sources of energy for the biochemical activities of cell membranes. it is composed of mono and poly unsaturated fatty acids such as omega three (ω-3) and omega six (ω-6) [5, 6] that can help reduce the cholesterol content of the body and the risk of cardiovascular diseases [7-10]. from the nutritive point of view, various studies have been carried out to determine http://www.fia.usv.ro/fiajournal mailto:ajai.ike@futminna.edu.ng mailto:talk2alexajai@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 137 the proximate composition of fish. the results obtained from these studies, show that fish is composed of between 30 to 90% water [11], 60-75 % protein [12], 30 to 50% lipids [13], 0.1 – 1% carbohydrates [4], essential minerals such as sodium, magnesium, calcium, phosphorus, potassium, iodine and appreciable quantity of vitamins such as a, d, e, k [14]. these proximate compositions according to the report may vary depending on the geographical location, season of the year, the feed intake, sex, species, age and maturity or size of the fish [14]. one of the most common fish species in nigeria is clarias garipinus (african sharp-tooth catfish) which belongs to the family of clarida and is found in freshwaters, lakes, rivers and swamps. other species are tilapia, which belong to the tilapine cichlid family [15], hyperopisus bebe occidentallis (elephant fish) to the family of mormyridae [16], synodontis clarias (fresh water catfish), to the genus mochokidae [17-18] and gynarchus niloticus (trunk fish) [16]. this study was aimed at determining the proximate, essential minerals, cholesterol and fatty acids composition of selected fish species obtained from shiroro river, nigeria and their oil extracts. these species were selected to be studied due to their economic importance and consumers' demand. therefore, detailed information about their proximate, essential minerals and fatty acids composition is important from nutritional point of view. 2. materials and methods the fish species, oreochromis leucostictus, clarias garipinus, gynarchus niloticus, hyperopisus bebe occidentallis and synodotus clarias, used for the study were purchased from fishermen at shiroro river of niger state, nigeria. the fish species were taken to the laboratory and were properly washed with water and lacerated. the non-edible parts were removed and then properly washed again with tap water and rinsed with distilled water. the samples were oven dried at 90°c until a constant weight was obtained. the dried samples were ground and homogenized. the moisture, ash, raw protein, raw fiber and raw fat contents of the samples were determined in triplicate according to the method describe by the association of official analytical chemist methods [19]. carbohydrate contents of the samples were determined by difference as reported by other workers [20]. the concentration of calcium, magnesium, zinc and iron were determined using atomic absorption spectrophotometry after wet digestion using the mixture of concentrated hno3 and hclo4. the digestion was done in a fume hood using a hotplate at 70°c. fatty acid profile was determined using gas chromatography/mass spectrophotometer (gc-ms) after extraction of the sample with a soxhlet extractor, purification and cleanup using a packed column. free fatty acid (ffa), saponification value, peroxide value, iodine value, acid value and level of cholesterol of extracted oil in the fish species were determined as described in the association of official analytical chemist methods [19]. 3. results and discussion the result of proximate compositions of the studied fish species per dried weight are shown in the table 1. from the results, there were significant differences (p < 0.05) in the mean concentrations of biomolecules (carbohydrate, fats, protein) and other parameters such as (ash fiber, moisture) between the species of the fish studied. the concentration of raw protein in the fish species was in the following order: gynarchus niloticus (51.1±80.36%) https://en.wikipedia.org/wiki/palmitoleic_acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 138 > clarias garipinus (48.15±0.02%) > synodotus clarias (46.13±0.03%) > oreochromis leucostictus (45.17±0.04%) > hyperopisus bebe occidentallis (39.00±0.02%) respectively. the relatively high raw protein content in the fish species could be attributed to the fact that fish is good source of protein and it is likely to meet the daily protein intake needed by human consumption. higher protein compositions were observed by several works carried out on nigeria freshwater fish. alfa et al reported a range of 49.4761.90 % in studies carried out on fish sold within the bida market in niger state, nigeria [21]. also, adesola reported protein compositions between 38 % 50 % in some important fish species obtained from lagos, nigeria [22]. the differences in the protein concentration between fish may be due to their feeding life style or absorption capability and the conversion potentials of the essential minerals in their diets and environment into various biochemical molecules in their body system [23]. . table 1 proximate compositions of selected fish species from shiroro river fish specie moisture (%) raw fat (%) raw protein (%) raw fiber (%) ash (%) carbohydrate (%) gynarchus niloticus 5.99 ± 0.47b 23.34 ± .20d 51.18 ±0.36a 0.90 ± 0.01c 9.01 ± 0.03c 9.58 ± 0.03b clarias garipinus 4.57 ± 0.02c 31.79 ±0.05b 48.15 ±0.02b 1.17 ± 0.02b 9.89 ± 0.03a 6.53 ± 0.02d oreochromis leucostictus 6.01 ± 0.02b 22.43 ±0.01e 45.17 ±0.04d 2.00 ± 0.21a 9.65 ± 0.28b 14.74 ± 0.03a synodotus clarias 7.87 ± 0.02a 26.71 ±0.02c 46.13 ±0.03c 1.20 ± 0.10b 8.59 ± 0.01d 9.51 ± 0.01c hyperopisus bebe occidentallis 4.60 ± 0.20c 48.57 ±0.02a 39.00 ±0.02e 0.85 ± 0.03c 4.90 ± 0.02e 2.13 ± 0.02e means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) the order in which the concentration of raw fat in the fish species were significantly higher than the other one was the following: hyperopisus bebe occidentallis (48.57±0.20 %) > clarias garipinus (31.79±0.05 %) > synodotus clarias (26.71±0.02 %) > gynarchus niloticus (23.34±0.2%) > oreochromis leucostictus (22.43±0.0 1 %). alfa et al explained that raw fat in fish varies from each other because of seasonal variation, species, geographical location and variation in their age and maturity [21]. based on the classification made by bennion and scheule, fish species can be categorized as fatty fish if they all have fat content greater than 10 % by weight [24]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 139 the raw fiber content of oreochromis leucostictus (2.00±0.21 %) was significantly higher than those of the other fish species. the fiber contents of synodotus clarias (1.20±0.10%) and clarias garipinus (1.17±0.02 %) were not significantly different from each other but were significantly higher than those of gynarchus niloticus (0.90±0.90 %) and hyperopisus bebe occidentallis (0.85±0.03 %), which also were not significantly different from each other. the raw fiber content of gynarchus niloticus and hyperopisus bebe occindentallis were within the range of the values (0.54 0.95 %) obtained by alfa et al for clarias garipinus, synodotus clarias and oreochromis leucostictus fish species in bida region of niger state [21]. also taiwo et al, who studied cultured and wild clarias garipinus and synodotus clarias fish species reported values that ranged between 0.78 1.23% [25]. the order in which the ash contents (index of mineral content of biota) of the fish species were higher than in each other was the following:clarias garipinus (9.89±0.03%), oreochromis leucostictus (9.65+0.28%), gynarchus niloticus (9.01±0.03%), synodotus clarias (8:59±0.01%), and hyperopisus bebe occidentallis (4.90±0.02%). low ash content in hyperopisus bebe occidentallis as compared to other species may be due to the lesser amount of skeleton it possesses. the results obtained in this study fall within the ones obtained by mazumder et al for different fish species which ranged between 4.91% and 19.59% [26] and also similar to the work carried out by alfa et al in freshwater fish species from bida which ranged from (5.32 – 42.44%) in the same species of fish [21]. however, it is higher when compared with the work carried out by taiwo et al and osibona which ranged between 1.16-1.26 % and 1.22-1.40 % in clarias garipinusi and oreochromis leucostictus respectively [15, 27]. the order in which the carbohydrate composition of the fish species studied were significantly different (p < 0.05) from each other was oreochromis leucostictus (14.74±0.03%), gynarchus niloticus, (9.58+0.03%), synodotus clarias (9.51±0.01%), clarias garipinus (6.53±0.02%) and hyperopisus bebe occidentallis (2.13±0.02 %). the results were high, except that of hyperopisus bebe occidentallis which was comparable to that obtained by alfa et al of between 2.695.28 % for five freshwater fish species [21]. also ayeloja et al obtained the range of 2.10-12.57 % for freshwater fish species from the western part of nigeria [28]. instead, the range of 1.9511.95 % was obtained by effiong and fakunle, in common five fish species from the kainji lake [29]. the moisture content which has effects on spoilage ranged from 4.57-7.87 % with maximum value in synodotus clarias (7.87 %) which was significantly higher than in other species. the moisture content of oreochromis leucostictus (6.01%) and gynarchus niloticus (5.99%) was not significantly different from each other but significantly higher than that of hyperopisus bebe occidentallis (4.60%) and clarias garipinus (4.57%). hyperopisus bebe occidentallis and clarias garipinus were not significantly different from each other. the values obtained on the basis of dried matter were similar to those obtained by effiong and fakunle and ande et al in fish species from the kainji lake and river lafia respectively which gave the range of 5.10 10.50% and 5.67 9.50% respectively [29, 30]. the values obtained in this study are within the range of 5-8 % for moisture content for fish products on dry basis [31].the results of the proximate parameters obtained in this present study are higher than those reported in s. nigrita of 7.09 to 25.46% protein, 5.13 to 11.70% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 140 fat, and 3.40 to 14.23% raw fiber content in t. mariae [32]. the variation can be attributed to environmental factors, such as the size of fish and its feeding habits. other researchers also reported various proximate parameters in fish that were dependent on environmental factors and feeding habits [33]. the mineral and cholesterol concentration in the fish species studied is shown in the table 2. there were significant differences (p < 0.05) in the mean mineral and cholesterol concentration between fish species. the concentration of calcium in fish species ranged from 4.28±0.02 to 16.80±0.01 mg/100g. the concentration of calcium in synodotus clarias (16.8mg/100g) and oreochromis leucostictus (16.79mg/100g) was not significantly different from each other but was significantly higher than that of the other species. the concentration of calcium in clarias garipinus (8.57mg/100g) was significantly higher than that of gynarchus niloticus (7.73mg/100g) and hyperopisus bebe occindentallis (4.28mg/100g). gynarchus niloticus had its calcium content significantly higher than that of hyperopisus bebe occindentallis. alfa et al reported a lower concentration of calcium (0.003 -0.014 mg/100g) in freshwater fish sold in bida markets [21] than that obtained in this study. the calcium concentration of fish species was higher than the recommended standard [31] except for hyperopisus bebe occindentallis that fell below. the magnesium concentration in the fish species ranged from 3.17±0.02 to 5.88±0.04 mg/100g. this falls within the recommended range of 4.5-452 mg/100g [31]. the order of magnesium content in different fish species were synodotus clarias (5.84mg/100g) > clarias garipinus (5.16mg/100g) > orechromis leucustictus (4.03mg/100g) > hyperopisus bebe occindentallis (3.45±0.01 mg/100g) > gynarchus niloticus (3. l7±0.02mg/l00g). these results were lower than those obtained by other researchers [34], who reported a range of mg/l00g to 20 mg/l00g which is also higher than the result obtained by alfa et al of 0.06 mg/l00g to 1.19 mg/l00g [21]. table 2 mineral and cholesterol composition of selected fish species from shiroro river fish specie ca (mg/100g) mg (mg/100g) fe (mg/100g) zn (mg/100g) cholesterol (mg l-1) gynarchus niloticus 7.73 ± 0.03c 3.17 ± 0.02e 0.50 ± 0.02a 0.08 ± 0.02b 548 ± 0.02b clarias garipinus 8.57 ± 0.03b 5.16 ± 0.03b 0.48 ± 0.01a 0.06 ± 0.02b 274 ± 0.04d oreochromis leucostictus 16.79 ± 0.03a 4.03 ± 0.03c 0.51 ± 0.04a 0.17 ± 0.00a 838 ± 0.02a synodotus clarias 16.80 ± 0.01a 5.84 ± 0.04a 0.03 ± 0.02c 0.14 ± 0.01a 503 ± 0.02c hyperopisus bebe occidentallis 4.28 ± 0.02d 3.47 ± 0.01d 0.21 ± 0.02b 0.08 ± 0.02b 509 ± 0.04c who/fao, 2011 5.00 – 502.00 50.00 – 451.00 1.00 – 5.60 0.23 – 2.10 ≤ 30000 means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) the iron concentration in the fish species ranged from 0.03±0.02 to 0.51±0.04 mg/100g, which falls below the standard concentration range of 1.0 – 5.6mg/100g [31]. the concentration of magnesium in oreochromis leucostictus (0.51mg/100g), gynarchus niloticus (0.50mg/100g), clarias garipinus (0.48mg/100g) was not significantly different from each other but it was significantly higher than that of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 141 hyperopisus bebe occidentallis (0.2 1mg/l00g) and synodotus clarias (0.03mg/100g). hyperopisus bebe occidentallis was significantly higher than synodotus clarias. the results obtained were within the range of those obtained by guerin et al in fish from french market which gave a range of 0.13-1.9 mg/100g [35] except that of synodotus clarias which fell below the lowest concentration. from the analysis carried out by alfa et al on fish samples from bida markets of niger state, nigeria which gave the range of 1.42-2.00 mg/100g [21] resulted higher values than those obtained in this study. the concentration of iron in oreochromis leucostictus was the highest and that of synodotus clarias was the lowest one. the concentration of zinc in this study ranged from 0.07±0.02 to 0.17±0.00 mg/100g which was below the standard range of 0.23 – 2.1mg/100g [31]. the concentration of zinc in oreochromis leucostictus (0.17mg/100g) and synodotus clarias (0.14mg/100g) was not significantly different from each other but it was significantly higher than that of gynarchus niloticus (0.08mg/100g), hyperopisus bebe occidentallis (0.08mg/100g) and clarias garipinus (0.06mg/100g). the results obtained were lower than those of tao et al who obtained values of 0.64 – 0.81mg/100g in farmed fish in china [36] as well as those obtained by alas et al in fish sampled from beysehir lake in turkey [37] but they fell within the range reported by guerin et al in fish from french market with value ranging from 0.13 – 2.5mg/100g [35].the cholesterol contents in oil extract of the studied fish species in the table 2 ranged between 274±0.04 838±0.02 mg/l. in oreochromis leucostictus registered higher content of cholesterol (838mg/l) while clarias garipinus (274.0mg/l) had the lowest one. oreochromis leucostictus oil had its cholesterol content significantly higher than other fish species. the cholesterol content in gynarchus niloticus (548mg/l) was significantly higher than that of hyperopisus bebe occindentalis (509mg/l), synodotus clarias (503mg/l) and clarias garipinus (274mg/l). hyperopisus bebe occindentallis and synodotus clarias oils were not significantly different in their cholesterol content but they were significantly higher than that of clarias garipinus. the values obtained were all lower than the recommended standard of 30000mg/l [31] which implies that fish species can be recommended as suitable for consumption by humans without any negative implication on their health with respect to total cholesterol. the chromatograms showed that oil in the fish species was made up by long chains of fatty acids, with a minimum carbon chain length of 10 22 carbons (table 3), which is a typical characteristic of fish oil [38]. each of the fish species was found to be composed of different fatty acids. still, some fatty acids were not detected in some of the fish species studied. fatty and non-fatty acid compositions of oils in the selected fish species are shown in the table 4. the poly-unsaturated fatty acids (pufa) content in the fish gave a range of 7.79 26.10% which was less than the saturated fatty acids (sfa) of 38.93 41.58% and mono-unsaturated fatty acids (mufa) of 33.63 45.37% respectively. the differences in fatty acids composition can be attributed to the influence of environmental factors and nutritional habits of fish [39]. other researchers have also reported that fresh water fish has lower content of polyunsaturated fatty acids (pufa), because freshwater fish feeds largely on vegetation and plant materials [22]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 142 table 3 percentage (%) fatty acid composition of selected fish species from shiroro river fame systemic name fish species gynarchus niloticus clarias garipinus oreochromis leucostictus synodotus clarias hyperopisus bebe occidentallis 10:0 decanoic acid 2.01 ± 0.06a nd nd nd 1.82 ± 0.03b 13:0 tridecanoic acid nd 0.55 ± 0.02a 0.47 ± 0.02b 0.41 ± 0.04c nd 14:0 tetradecanoic acid 2.76 ± 0.02c 1.71 ± 0.01d 3.30 ± 0.04b 1.40 ± 0.01e 4.70 ± 0.01a 15:0 pentadecanoic acid 1.54 ± 0.03c 0.83 ± 0.06d 2.98 ± 0.01b 0.47 ± 0.02e 3.35 ± 0.02a 16:0 hexadecanoic acid 14.95 ± 0.02c 17.72 ± 0.02c 18.84 ± 0.05b 22.91 ± 0.02a 17.07 ± 0.02d 17:0 heptadecanoic acid 4.52 ±0.02a 2.03 ± 0.02b 1.27 ± 0.01c nd 0.70 ± 0.02c 18:0 octadecanoic acid 11.07 ± 0.03d 14.18 ± 0.01b 11.61 ± 0.02c 16.39 ± 0.02a 8.93 ± 0.02e 20:0 eicosanoic acid nd 2.36 ± 0.01b nd nd 2.93 ± 0.03a 21:0 heneicosanoic acid nd nd nd nd 0.54 ± 0.02a 22:0 docosanoic acid 2.02 ± 0.00b 1.28 ± 0.02c nd nd 2.80 ± 0.03a 18:1ω-12 6octadecenoic acid nd nd 1.09 ± 0.03a nd nd 18:1ω-9 9-octadecenoic acid 1.90 ± 0.01d 27.89 ± 0.01a 23.63 ± 0.02b nd 21.98 ± 0.01c 18:1ω-7 11octadecenoic acid 23.49 ± 0.04b 4.76 ± 0.03e 8.24 ± 0.05d 35.76 ± 0.03a 9.57 ± 0.02c 18:1ω-9 12octadecenoic acid 8.25 ± 0.04a nd nd nd nd 20:1ω-9 11-eicosanoic acid nd nd nd nd 4.86 ± 0.00a 22:1ω-9 13-docosenoic acid nd 4.10 ± 0.02c 4.75 ± 0.02b 8.15 ± 0.02a nd 24:1ω-9 15tetracosenoic acid nd nd nd 1.46 ± 0.03a 1.43 ± 0.02a 16:3ω-3 7,10,13hexadecatrienoic acid nd nd 2.44 ± 0.02b nd 4.51 ± 0.03a 18:2ω-6 9,12,15octadecadienoic acid nd 1.49 ± 0.01b 2.82 ± 0.03a 1.17 ± 0.02c nd 18:2ω-6 9,12octadecadienoic acid 9.43 ± 0.03c 16.77 ± 0.02a nd 6.16 ± 0.02d 11.82 ± 0.02b 20:4ω-6 5,8,11,14eicosatetraenoic acid 1.58 ± 0.01c 1.44 ± 0.04d 3.44 ± 0.04a nd 2.36 ±0.03b 20:5ω-3 5,8,11,14,17eicosapentaenoic acid, epa 8.70 ± 0.02b nd 8.75 ± 0.03a nd nd 22:6ω-3 4,7,10,13,16,19docosahaenoic acid, dha 6.39 ± 0.06a 2.14 ± 0.01b 1.94 ± 0.04c nd nd means (± standard deviation) on the same row with different superscripts are significantly different (p < 0.05) the highest saturated fatty acid (sfa) was found in the oil oreochromis leucostictus (42.81%) (table 4) followed by those of hyperopisus bebe occidentallis (41.58%), synodotus clarias (41-28%), clarias garipinus (40-66%) and gynarchus niloticus (38-93%). of these fatty acids identified from the study, palmitic acid (16:0) was the predominant one. the maximum palmitic acid (hexadecanoic acid) content was 22.91 % in hyperopisus bebe occindentallis and the minimum was 14.95 % in gynarchus niloticus. the amount of acid in the oil of hyperopisus bebe occindentallis was significantly higher than that of the other fish species food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 143 which was significantly higher than in each other in the following order synodotus clarias (18.84 %) > clarias garipinus (17.72%) > oreochromis leucostictus.( 17.07%) > gynarchus niloticus. the amount of palmitic acid in fish was within the range of the findings made by adesola and rahman et al that obtained the concentration range of 10.11 21.15 % and 12.7 26.6 % in freshwater fish species in lagos and malaysia respectively [22, 40]. table 4 fatty and non-fatty acid (%) composition of selected fish species from shiroro river fatty acids type gynarchus niloticus clarias garipinus oreochromis leucostictus synodotus clarias hyperopisus bebe occidentallis who/fao, 2011 sfa 38.93 40.66 42.81 41.28 41.58 ≤10 mufa 33.63 36.75 37.84 37.71 45.37 ≥12 pufa 26.10 21.83 19.69 19.39 7.79 ≥6 non-fatty acid 1.34 0.76 0.66 1.62 5.28 pufa/sfa 0.67 0.54 0.46 0.47 0.19 ≥0.1 ω-6 11.01 18.21 14.18 6.26 6.62 ≥8 ω-3 15.09 3.62 4.51 13.13 1.17 ≥2 ω-3/ω-6 1.37 0.20 0.31 2.10 0.18 ≤4 sfa: saturated fatty acid, mufa: mono-unsaturated fatty acid, pufa: poly-unsaturated fatty acid the mufa content was the highest in hyperopisus bebe occidentallis (45.37%) followed by oreochromis leucostictus (37.84%), synodotus clarias (37.71%), clarias garipinus (36.75%) and gynarchus niloticus (33.63%), with the smallest one. the major mono-unsaturated fatty acids were 18:1 .a-9 and 18: l-7 (oleic and vaccenic acid). similarly, osibona reported 18:1 a -9 and 18:1 ω-7 as major mufa in freshwater fish species from lagos, nigeria [27]. this is contrary to that reported by kaneiwaa et al which were of 18:1 and 16:1 (oleic and palmitic oleic) found in freshwater fish species from china [38]. the variation can be attributed to environmental difference. guler et al and osman et al reported that high oleic acid is a major fatty acid component of freshwater fish species [41, 42]. the maximum oleic acid (18:1 ω -9, 9-octadecenoic acid) content was found in clarias garipinus (27.89%) and the minimum was 1.90 % in gynarchus niloticus. the highest vaccenic acid (18:17, 11-octadecenoic acid) was 35.75 % found in hyperopisus bebe occindentallis and the minimum content was 4.76 % in clarias garipinus. the monounsaturated fatty acids are responsible for physiological activities and also in the reduction of serum cholesterol [25]. majority of polyunsaturated fatty acids (pufas) are essential in carrying out physiological activities in the body. pufas of the oil of the samples ranged between c16 to c22 in chain length and are ω-3 and ω-6 type. freshwater fishes usually contained higher amounts of ω-6 fatty than ω-3 when compared with marine fish [43]. similar pattern was observed in the fish species studied in this work except in gynarchus niloticus and synodotus clarias, which corresponds to the findings of aclcman and mcleod who reported higher omega-3 pufas in fish from cold northern freshwaters fish species [44]. it could therefore be deduced that environmental factor plays vital roles in the pufas content of fish. fish oil goes through oxidative deterioration and hydrolytic spoilage due to the presence of unsaturated fatty acids (mono-unsaturated and poly-unsaturated). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 144 in this present study the oxidative stability of oil in the five (5) species of freshwater fish was determined using different analytical parameters, such as peroxide value, free fatty acid, and saponification, iodine and acid values to assess the quality of fish oil under room conditions. the results are shown in the tables 5 – 9. table 5 change in oxidative stability of synodotus clarias oil during storage under room temperature storage days peroxide value (meqo2/kg) free fatty acid (%) saponification value (mgkoh/g) iodine value (mgkoh/g) acid value (mgkoh/g) 0 1.32 ± 0.05d 2.75 ± 0.20e 160 ± 0.01e 170 ± 0.02a 5.50 ± 0.20e 15 1.40 ± 0.10d 2.98 ± 0.01d 165 ± 0.11d 165 ± 0.01b 5.96 ± 0.01d 30 2.71 ± 0.01c 3.26 ± 0.05c 168 ± 0.12c 163 ± 0.01c 6.52 ± 0.05c 45 4.50 ± 0.11b 3.53 ± 0.01b 172 ± 0.01b 162 ± 0.04d 7.05 ± 0.01b 60 4.93 ± 0.12a 6.21 ± 0.11a 199 ± 1.15a 155 ± 1.00e 12.42 ± 0.11a means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) freshness of lipid is determined by its ability to withstand oxidation reaction during storage, measured by its peroxide value [45]. in this study the peroxide values in meqo2/kg within storage period of 0 to 60 days were 1.32±0.05 4.93±0.12 for synodotus clarias (table 5), 1.89±0.01 5.12±110 for clarias garipinus (table 6), 1.13±0.10 3.71±0.01 for oreochromjs leucostictus (table 7), 1.00±0.10 3.80±1.00 for hyperopisus bebe occidentallis (table 8) and 0.98±0.01 3.98±0.03 for gynarchus niloticus (table 9). in the entire sample an increase in peroxide value with increased storage time was observed except in hyperopisus bebe occidentallis where a decrease of 3.87 meqo2/kg to3.80 meqo2/kg occurred between 45 60 days of storage, which were statistically comparable. the increase after 15days from the initial day was not significant for all the species except gynarchus niloticus, with significant increase. alicia et al and gokhan et al reported peroxide values of raw fish oil as 3 – 20 meqo2/kg [46, 47] but the values obtained by the five fish species did not exceed 10 meqo2/kg. free fatty acid is the measure of the extent to which by hydrolysis reaction fatty acids are released from their ester linkages with the parent triglyceride molecules [48]. willy et al stated that free fatty acid formation due to the lipid hydrolysis provides suitable means of assessment of oil spoilage during storage [49]. table 6 change in oxidative stability of clarias garipinus oil during storage under room temperature storage days peroxide value (meqo2/kg) free fatty acid (%) saponification value (mgkoh/g) iodine value (mgkoh/g) acid value (mgkoh/g) 0 1.89 ± 0.01c 1.58 ± 0.01e 162 ± 0.12e 175 ± 0.10a 3.15 ± 0.01e 15 2.25 ± 0.12c 2.70 ± 0.11d 164 ± 0.01d 168 ± 0.12b 5.40 ± 0.11d 30 2.51 ± 0.10c 3.25 ± 0.10c 168 ± 0.12c 166 ± 0.11c 6.50 ± 0.10c 45 3.90 ± 0.10b 3.49 ± 0.13b 170 ± 0.10b 162 ± 0.10d 6.98 ± 0.13b 60 5.12 ± 1.10a 5.89 ± 0.15a 190 ± 1.12a 150 ± 0.01e 11.78 ± 0.15a means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05 the percentage free fatty acids content of the five fish species during the periods of storage from 0 to 60 days ranged from 2.75±0.20 6.2l±0.11 for synodotus clarias (table 5), 1.58±0.01 5.89±015 for clarias garipinus (table 6), 1.00±0.01 – 4.57+002 for oreochromis leucostictus (table 7), l.58±0.0l -2.5l±0.l2 for hyperopisus bebe occidentallis (table 8) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 145 and 1.07 4.42 % for gynarchus niloticus (table 9). free fatty acid significantly increased (p < 0.05) as the time of storage increased except for hyperopisus bebe occidentallis where the increase between 30 and 60 days was not significant (p > 0.05). bimbo suggested the maximum acceptable values of 5%, [50] however in this study synodotus clarias and clarias garipinus had values above 5% after 45days and 60 days of storage. table 7 change in oxidative stability of oreochromis leucostictus oil during storage under room temperature storage days peroxide value (meqo2/kg) free fatty acid (%) saponification value (mgkoh/g) iodine value (mgkoh/g) acid value (mgkoh/g) 0 1.13 ± 0.10c 1.00 ± 0.01e 175 ± 0.01e 165 ± 0.06a 2.00 ± 0.01e 15 1.22 ± 0.02c 1.95 ±0.05d 180 ± 0.05d 163 ± 0.03b 3.89 ± 0.05d 30 1.95 ± 0.11b 2.75 ± 0.03c 181 ± 0.01c 159 ± 0.01c 5.50 ± 0.03c 45 3.69 ± 0.10a 3.91 ± 0.01b 183 ± 0.20b 152 ± 0.01d 7.81 ± 0.01b 60 3.71 ± 0.01a 4.57 ± 0.02a 230 ± 0.01a 139 ± 1.00e 9.14 ± 0.13a means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) saponification value (sv) is an indication of the molecular weights of triglycerides of oils. high saponification value indicates high proportion of low fatty acids since saponification value is inversely proportional to the average molecular weight or length of fatty acids [51]. therefore the shorter the average chain length (c4 c12), the higher the saponification value [52]. the saponification values in mgkoh/g of analyzed fish oil significantly increase (p < 0.05) with increase in storage period. the saponification values of the fish species within the storage periods of 0 to 60 days ranged from 160±0.01 199±1.15 for synodotus clarias (table 5), 162±0.12 – 190±1.12 for clarias garipinus (table 6), 175±0.01 230±0.01 for oreochromis leucostictus (table 7) and 172±0.01201±0.12 for hyperopisus bebe occindentallis (table 8). it is very possible that the end product of oxidation, such as aldehydes and ketones may have contributed to the increase in saponification value, which may be responsible for high value observed in the last three species of fish. the values obtained were between l60.00 203.00 mg koh/g. these values are within the recommended range of 195 -205 mg koh/g for oil [53]. the values show that the oils are well suited for soap making. table 8 change in oxidative stability of hyperopisus bebe ocidentallis oil during storage under room temperature storage days peroxide value (meqo2/kg) free fatty acid (%) saponification value (mgkoh/g) iodine value (mgkoh/g) acid value (mgkoh/g) 0 1.00 ± 0.10c 1.58 ± 0.01c 172 ± 0.01e 185 ± 0.04a 3.15 ± 0.01b 15 1.37 ± 0.12c 2.37 ± 0.10b 175 ± 0.10d 183 ± 0.01b 4.74 ± 0.10a 30 2.82 ± 0.03b 2.40 ± 0.02ab 178 ± 0.02c 180 ± 0.01c 4.81 ± 0.02a 45 3.87 ± 0.11a 2.44 ± 0.10ab 179 ± 0.01b 173 ± 0.12d 4.89 ± 0.10a 60 3.80 ± 1.00a 2.51 ± 0.01a 201 ± 0.12a 169 ± 0.10e 5.02 ± 1.00a means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) the iodine value, defined as the measure of unsaturation in an oil sample, is a useful tool in detecting the level of spoilage in oil. the iodine values of the oils in each of the fish species significantly decreased as the storage time increased from 0 to 60 days. the iodine value of oils as the storage time increased to 60 days decreased from 170.00±0.02 to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 146 155.00±1.00 for synodotus clarias (table 5), 175.00±0.10 to 150.00±0.01 for clarias garipinus (table 6), 165.00±0.06 to 139.00±1.00 for oreochromis niloticus (table 7), 185.00±0.04 to 169.00±0.10 for hyperopisus bebe occidentallis (table 8) and 162.00±0.10 to 143.00±0.10 for gynarchus niloticus (table 9). the percentage decrease was calculated to be 8.65 % for hyperopisus bebe occidentallis, 8.82 % for synodotus clarias, 11.73 % for gynarchus niloticus, 14.29 % for clarias garipinus and 15.75 % for oreochromis niloticus. the decrease in iodine value shows the decrease in the degree of unsaturation during the storage of oils [49]. table 9 change in oxidative stability of gynarchus niloticus oil during storage under room temperature storage days peroxide value (meqo2/kg) free fatty acid (%) saponification value (mgkoh/g) iodine value (mgkoh/g) acid value (mgkoh/g) 0 0.98 ± 0.01e 1.07 ± 0.01e 175 ± 0.01e 162 ± 0.10a 2.14 ± 0.01e 15 1.21 ± 0.01d 1.99 ± 0.20d 176 ± 0.10d 160 ± 0.02b 3.98 ± 0.20d 30 1.90 ± 0.11c 2.57 ± 0.09c 179 ± 0.05c 159 ± 0.10c 5.14 ± 0.09c 45 3.02 ± 0.10b 3.48 ± 0.12b 180 ± 0.20b 154 ± 0.05d 6.95 ± 0.12b 60 3.98 ± 0.03a 4.42 ± 0.02a 216 ± 0.10a 143 ± 0.10e 8.83 ± 0.02a means (± standard deviation) on the same column with different superscripts are significantly different (p < 0.05) the acid value is the measure of free fatty acid in oils. the acid values in mgkoh/g of oils in the five fish species significantly increased (p < 0.05) with increase in the storage time from 0 to 60days. the acid values for each of the oils from day 0 to the 60th day ranged from 5.50±0.20 to 12.42±0.11 for synodotus clarias (table 5), 3.15±0.01 to 11 78±0.15 for clarias garipinus (table 6), 2.00±0.01 to 9.14±0.13 for oreochromis leucostictus (table 7), 3.15±0.01 to 5.02±1.00 for hyperopisus bebe occidentallis (table 8) and 2.14±0.01 to 8.83±0.02 for gynarchus niloticus (table 9). the suitable limit for acid value is 7 8mgkoh/g as reported by de-koning [48]. the study revealed that the acid values of the oils of synodotus clarias, clarias garipinus and oreochromis leucostictus exceeded the maximum limit after 60 days of storage. the acid value of oils within the storage period of 45days in all the fish species fell within the maximum limit reported by dekoning [54], while after 60 days of storage, the oil of hyperopisus. bebe occidentallis was below this limit. 4. conclusion there were some levels of significant differences in the proximate minerals, fatty acids and cholesterol compositions between the different freshwater fish species analyzed in this study. the protein content of the fish species, per dried weight, was generally high, with gynarchus niloticus having the highest concentration of above 50% and hyperopisus bebe occidentallis the lowest one, below 40%. this implies that all the fish species can be used as protein supplements. the fish species were also found to be the oily fish with low saturated fatty acids and high polyunsaturated fatty acid. they are also rich in omega-6 and 3 fatty acids and therefore they can be recommended for human consumption especially gynarchus niloticus and synodotus clarias species. also, oreochromis leucostictus has the highest cholesterol content while clarias garipinus species has the lowest one. the values are general lower than those recommended by the fao /who as regards the toxicity limit of cholesterol. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 147 5. acknowledgements the authors appreciate the assistance of the technical staff of central and soil science laboratory, federal university of technology, minna, nigeria and ahmadu bello university, research laboratory, zaria, nigeria. 6. references [1] jitender k. j., pal a. k.., devivaraprasad r., sahu n. p., venkateshwarlu g. & vardia h. k.., fatty acids composition of some selected indian fishes. africa journal of basic & applied sciences, 4(5), 155-160, (2012). [2] stone n. j., fish consumption, fish oil, lipids, & coronary heart disease. circulation, (2010). [3]. deslypere j. p., effect of fish consumption compares to intake of fish oil, bibliotheca nutrition et dieta, 46, 53 – 69, (1990) [4]. eyo a. a., fish processing in the tropics. university of ilorin press, nigeria, 153-189, (2001) [5] knuth b., connelly n. a., sheeshica j. & patterson j., weighing health benefits and health risk information when consuming sport caught fish. risk analysis, 23, 11851197, (2003) [6] darren j. h. & bruce j. h., omega-3 fatty acids from fish oils and cardiovascular disease. in: molecular and cellular biochemistry. kluwer academic publishers, netherlands, 2004, 263, 217 225. [7] wang c., harris w.s., chung m., lichtenstein a.h., balk e.m., kupelnick b., jordan h.s & lau j., omega-6 fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid benefit cardiovascular disease outcomes in primary and secondary prevention studies: a systematic review. america journal of clinical nutrition, 84(1):5-17 (2006) [8] dhanapal k.., reddy a.d., & reddy g.v.s., beneficial effects of fish oil and its applications in human health. int. j. med. bio., 17(12): 137-156, (2011) [9] bhaskar n., miyashita k. & hosokawa m., physiological effects of eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) – a review. food rev int, , 22: 291307, (2006) . [10] daviglus m., sheeshka j. & murkin e., health benefits from eating fish. comments toxicology, 8, 345-348, (2002) [11]. wheaton f. w. & lawson t. b., processing aquatic food products. new york; john wiley, (1985) [12]. suzuki t., krill and krill protein. processing technology. applied science publishers, london, (1981) [13]. drevon c. a. ω-3 fatty acids in health disease, fish fats and your health. proceedings of the international conference on fish lipids and their influence on human health, suanøy foundation, 6965 svanybukt, norway, 19-25, (1989). [14]. syvaoja e. l., salminem k.., piironen v., varo p., kerojoki o & koivistoinen p., tocepherols and tocetrienols in finnish foods: fish and fish products. journal of american oil chemical society, 62(8), 1245 – 1248, (1985) [15] idodo-umeh g., freshwater fisheries of northern nigeria. (taxonomy, ecological notes, diet and utilization), isbn 978-8052-01-0 (2003) [16] froese r. & pauly d., tilapia jallae (boulenger 1896). fish base, (2015). [17] ayoola s. a. & abotti c. e., morphology of abba knife fish (gymnarchus niloticus). world journal of fish and marine science, 2(5), 354-356, (2011) [18] stephen k.., stumbauer c., verheyen e., meyer a., & salzburger w., mitochondrial phylogeny and phylogeography of east african squeaker catfishes (siluriformes: synodontis). bmc evolutionary biology, 6, 49, (2006) [19] aoac, official methods of analysis, 18th edition. method 960.52a. association of official analytical chemists, washington dc, (2007). [20] enyisi 1., umoh v. j., whong c. m. z., abdullahi 1.o. & alabi o., chemical and nutritional value of maize and maize products obtained from selected markets in kaduna state, nigeria. african journal of food science and technology, 5, 100-110, (2014) [21] alfa y. m., nda-umar u. i., salihu a. b. & nma n. y., proximate composition and minerals components of some species of fish sold in bida fish market. international journal of current research in chemistry and pharmaceutical sciences, 1(8), 1924, (2014) [22] adesola o. o., comparative study of proximate composition, amino acid and fatty acid of some economically important fish species in lagos, nigeria. african journal of food science, 5, 581 – 588 (2011) [23] adewole o. s., fawole o. o. & food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 148 omotosho j. s., concentration of selected elements in some freshwater fishes in nigeria. science focus, 4, 106 – 108, (2003) [24] bennion m & scheule b., introductory foods; prentice-hall, new jersey, usa, 575, (2000) [25] taiwo o. e., usman k., ogono t. h. & osoniyi r. d., proximate and lipid profile analysis of cultured wild african catfish, clarias gariepinus (burchell), ife journal of science, 16(1), 50-52, (2014) [26] mazumder m. s. a., rahman m. m., ahmed a. t. a., begum t. & hossain m. a., proximate composition of some small indigenous fish species (sis) in bangladesh. international journal of sustainable crop production, 3(3), 18-23, (2008) [27] osibona a. o., comparative study of proximate composition, amino and fatty acids of some economically important fish species in lagos, nigeria. african journal of food science, 5(10), 581-588, (2011) [28] ayeloja a. a., george f. o. a., dauda t. o., jimoh w. a. & popoola m. a., nutritional composition of captured clarias garipinus and oreochromis niloticus. international research journal of natural science, 1(1), 9 – 13, (2013) [29] effiong b. n. & fakunle j. o., proximate and mineral content of traditional smoked fish species from lake kainjj, nigeria. bulletin of environmental pharmacology and life sciences, 1, 43-45, (2012) [30] ande s., leke i., eneji i. & yakubu s., proximate analysis of smoked and unsmoked fish (catfish and tilapia) in ombi river lafia, nassarawa state, nigeria. elixir food science, 53, 11801 – 11803, (2012) [31] fao/who, human vitamin and mineral requirements rome: fao, (2011) [32].ijeoma p. o., sanni o. a., egun n. k. & wilfred-ekprikpo c. p., the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae , food and environmental safety, 18(1), , 60 – 66, (2019) [33] sivakova b. & blazhekovikj – dimovska d., the impact of different diet and environmental conditions on chemical composition of rainbow trout (oncorhynchus mykiss walbaum, 1792) from macedonian aquaculture facilities, journal of faculty of food engineering, 15(3), 227 – 233, (2016) [34] adeniyi s. a., orjieke c. l., ehiagbonare j. e. & josia s. j., nutritional composition of three different fishes (claria gariepinus, malapteru, electricus’ and tilapia guineensis) pakistan journal of nutrition, 11, 793-797, (2012) [35] guerin t., chekri r., vastel c., sirot v., volatier j. l, leblanc j. c. & noel l., determination of 20 trace elements in fish and other seafood from the french market. food chemistry, 127,934 -942, (2011) [36] tao n. p., wang l. y., gong x. & liu y., comparison of nutritional composition of farmed puffer fish muscles among fugu obsurus, fugu fladivus and fugus rubripes. journal of food composition analysis, 28, 40-45, (2012) [37] alas a., ozcan m. m. & hannakaya m., mineral contents of head, caudal, central fleshy part and spinal colum of some fishes. environmental monitoring assessment, 186, 889894, (2014) [38] kaneniwa m., miaob s., yuanb c., jidac h. & fukudad y., lipid components and enzymatic hydrolysis of lipids in muscle of chinese freshwater fish. journal of the american oil chemical society, 77: 825-830, (2008) [39] keriko j. m., chege c.w., magu m.m., mwachiro e.c., murigi a. n., githua m. n., kareru p.g., fish lipid contents and classes of selected fish species found in lake naivasha (kenya) and the fish feeding habits of the lakes inhabitants. africa journal of pharmacy and pharmacology, 4(10): 745-753, (2010) [40]. rahman s. a., huah t. s., hassan o. & daud n. m., fatty acid composition of some malaysian freshwater fish. food chemistry, 54(1), 45—49, (2015) [41]. guler g. o., kiztanir b., aktumsek a., citil o. b. & ozparlak h., determination of the seasonal changes on total fatty acid composition and co-3/ w-6 ratios of carp (cyprinus carpio l.) muscle lipids in beysehir lake (turkey). food chemistry, 108, (2008) [42] osman h., suriah a. r. & law e. c., fatty acid composition and cholesterol content of selected marine fish in malaysian waters. food chemistry, 73, 55-60, (2001) [43] muhamad n. a. & muhamad s., fatty acids composition of selected malaysia fishes (komposis asis le’rnakllcan terpilh malaysis). sains malaysiana, 41(1), 81-94, (2012) [44] ackman r. g. & mcleod c., lipids and fatty acids of five freshwater food fishes of india. journal of food lipids, 9, 127 – 145, (2002) [45] malheiro r., rodrigues n., manzke g., bento a., pereir j. a. & casal s., the use of olive leaves and tea extracts as effective antioxidants against the oxidation of soybean oil food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 alexander ikechukwu ajai, mohammed mohammed ndamitso, lucky ekwoba and abidemi adedayo koleola, comparative analysis of the proximate, fatty acids and mineral composition of selected fish species, food and environment safety, volume xviii, issue 3 – 2019, pag. 136 – 149 149 under microwave heating, industrial crops and products, 44, 37– 43, (2013) [46] alicia r., vanesa l., angelica l., vilma q., julia v. & santiago p.a., development of lipid changes related to quality loss during the frozen storage of farmed coho salmon (oncorhynchus kisutch). journal of the american oil chemists society, 84, 727-734, (2007) [47] gokhan b., muhammet b. & hikmet k., seasonal changes in proximate composition of anchovy and storage stability of anchovy oil. journal of food quality, 31, 503-513, (2006) [48]. salhin m. a. & abdurahman m. a., determination of free fatty acids in palm oil samples by non aqueous flow injection using salicyaldehyde-2,4-dinitrophenylhydrazone as colorimetric reagent. chemical and materials engineering, 1(3), 96-103, (2013) [49] willy p. w., abdulan a. s., fatima b., abubakar n. b. s., zamari b. i. & hamid a. a., lipid quality deterioration of bagriclac catfish (mystus nemurus) during storage research journal of biological sciences, 4, 525530, (2009) [50]. bimbo a. p., guidelines for characterizing food-grade fish oils. hertfordshire: uk, , 9(5). 1998 [51]. muhammad e., al-maqbaly r. & mansour m.h., proximate composition, amino acid and mineral contents of five commercial nile fishes in sudan. africa journal food sciences, 10, 650-654, (2011) [52]. tamzid h. m., alam m. t. & islam m. a. u., physico-chemical and nutritional studies of terminalia belerica roxb seed oil and seed kernel. journal of bio-science, 15, 117 – 126, (2007) [53]. aocs, official methods and recommended practices of the american oil chemist society (5th ed.) champaign, 2009. [54]. de-koning a .j., the free fatty acid content of fish oil, part v. the effect of microbial contamination on the increase in free fatty acid content of fish oils during storage at 25°c. fat/lipid, 101(5), 184-186, (1999) звіт з ндр 29-81 за 2007 – 2009 р 37 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 12018, pag. 37 40 influence of thermal treatment of the basalt tufa on its phase compos ition and sorption capacity volodymyr diichuk1, iryna diichuk2, *igor kobasa1 1yurii fedkovych chernivtsi national university, ukraine, ikobasa@chnu.edu.ua 2bukovinian state medical university i.kobasa@chnu.edu.ua * corresponding author received 5th december 2017, 23th march 2018 abstract: the results reflecting influence of thermal treatment of basalt tufa (bt) on its phase composition and capacity of some heavy metal ions (cu 2+ , mn 2+ , ni 2+ , pb 2+ , zn 2+ ) sorption under the static conditions are presented. it has been found that thermal modification of tufa at 400–1000 0 с results in weight loss of the material from 5,79 % (at 400 0 c) to 10,18 % (1000 0 c) caused by changes in the mineral phase composition such as loss of the chemically bound water, decomposition of carbonates and other compounds. it has been shown that the sorption value for the heavy metal ions, being captured on surface of the thermally modified bt samples, is governed by the ion’s nature and temperature of its thermal treatment. the negatively charged surface-active adsorption centers were identified for all bt samples since they exhibited the positive suspension effect. therefore, a competition between adsorption of h + and heavy metal ions can take place. keywords: thermal modification, basalt tufa, phase composition, sorption capacity, suspension effect 1. introduction the sorption technologies for natural water and wastewater cleaning using various mineral adsorbents play an important role for environment protection and contamination prevention. since natural sorbents such as montmorillonites, diatomites, bentonites are easily available, inexpensive and widely functional [1, 2], they are used extensively in many wastewater treatment and water cleaning solutions. zeolites are minerals with some unique properties [3, 4] (ability to ensure high ion exchange capacity, well-ordered lattice with uniform molecule-sized pores, protonic and aprotonic acidity), which are also used widely as effective dryers, gas and liquids cleaning substrates and so on. basalt tufa is a volcanogenic mineral having chemical and structural compositions close to zeolites [5]. there are vast deposits of tufa available for mining, but the detailed properties of the mineral are still insufficiently investigated. it should be mentioned that high chemical and thermal durability of tufa facilitates its usability as a raw mineral for various environment protection, agrochemical, technical and biological solutions. sorption capacity is one of important characteristics controlling sorption efficiency of any substrate. it was found previously that bt exhibits polyfunctional adsorption, which depends substantially on the conditions of preliminary thermal, chemical and chemo-thermal treatment [6]. in this context, the main aim of this work was to investigate an influence of the thermal treatment temperature on phase composition and sorption capacity of basalt tufa. http://www.fia.usv.ro/fiajournal https://fia.usv.ro/webmail/src/compose.php?send_to=i.kobasa@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 volodymyr diichuk, iryna diichuk, igor kobasa, influence of thermal treatment the basalt tufa on its phase composition and sorption capacity, food and environment safety, volume xvii, issue 1 – 2018, pag. 37 – 40 38 2. experimental all experiments were done using the basalt tufa obtained from polytske-2 deposit (ukraine). the samples were dried in air at 105 0c and then modified thermally at 400, 600, 800 and 1000 0c. sintering was carried out until stable weight of the sample (approx. 3 hours). then xrd analysis was employed to determine the tufa phase composition. ion sorption was investigated using the powdered samples (d=80–120 m) of bt. the well-known low temperature argon adsorption method was employed to determine the specific surface area. sorption of the heavy metal ions cu2+, mn2+, ni2+, pb2+ and zn2+ was investigated at the room temperature under static conditions with aqueous solutions of corresponding nitrates (c0=210 -4 mole/l) keeping 1:100 phase ratio between the solid and liquid components. it took 18–24 hours for the sorbent/sorbate equilibrium to establish. all concentrations of the metal ions were determined by aas using kas-120 m1 spectrophotometer operating acetylene/air flame [7]. suspension effect value (δрн), which used to identify negatively or positively charged surface centers [6], was measured by ph-meter and was calculated according to δрн=рн1рн0; рн0 – initial ph value of solution without basalt tufa; рн1 – ph value of suspension with basalt tufa. 3. results and discussion it is known that both phase composition and structure of bt undergo serious influence after thermal processing of the material [8, 9]. many processes can occur during this treatment: decomposition of some mineral compounds, sintering and pore formation, which cause various changes in the tufa properties. an influence of the thermal treatment regime on the samples weight change is shown in figure 1. as expected, the higher temperatures correspond to greater weight loss. for instance, 5,79 wt % were lost after treatment at 400 0c while same processing at 1000 0c results in loss of 10,18 wt %. these changes can be resulted by evaporation of the crystal water and decomposition of carbonates and some other thermally unstable compounds. 400 600 800 1000 0 2 4 6 8 10 % c 0 fig. 1. influence of thermal treatment temperature on the basalt tufa weight loss phase composition of the thermally modified bt samples is represented in table 1. table 1. phase composition of the thermally modified bt temperature, [0с] phase composition 105 al2o3·54sio2 sio2 mgsio3 fe2o3 400 al2si4o10 sio2 mgsio3 fe2o3 600 283sio2·al2o3 sio2 fe2o3 800 fe2o3 al2o3·54sio2 sio2 1000 mgsio3 fe2o3 sio2 data of table 1 prove that phase composition of the samples is substantially dependent on the temperature of their treatment. a 400 0c treatment results in changes in the aluminosilicate composition: the structure al2o3·54sio2 transforms into al2si4o10 then the latter compound undergoes transformation into food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 volodymyr diichuk, iryna diichuk, igor kobasa, influence of thermal treatment the basalt tufa on its phase composition and sorption capacity, food and environment safety, volume xvii, issue 1 – 2018, pag. 37 – 40 39 283sio2·al2o3 at 600 0c. a 1000 0с treatment results in formation of mgsio3, fe2o3, sio2. this means that a ratio si/al in aluminosilicates is being changed with temperature of thermal treatment, which leads to some changes in adsorption capacity of the tufa. adsorption of the heavy metal ions was found depending on the ion nature and temperature of the tufa treatment (see figure 2). 0 200 400 600 800 1000 0,008 0,012 0,016 0,020 0,024 s o rp ti o n c a p a c it y , m m o l/ g 0 c 1 2 3 4 5 fig. 2. influence of the tufa thermal treatment temperature on its adsorption capacity in regard to some heavy metal ions: 1 – ni2+, 2 – pb2+, 3 – zn2+, 4 – cu2+, 5 – mn2+ as seen from fig. 2, the ions adsorption value grows in the row: ni2+ < pb2+ < zn2+ < cu2+ < mn2+. a 400 0c treatment of the samples causes some rise in the adsorption value followed by further decrease at the higher temperature treatment. a comparison between adsorption value and specific surface area proves that both parameters change similarly (see table 2) as it is typical for the fine-dispersed adsorbents. table 2. area of specific surface for the thermally modified bt т,[°с] 105 400 600 800 1000 sbet, [m 2/g] 7.25 7.81 7.65 4.54 1.82 according to [8], following groups: ≡si–он and ≡al–он play role of the aluminosilicate surface active centers. thermal treatment causes their partial dissociation according to the mechanism that depends on the treatment temperature. a mechanism of dissociation has been identified using ir-spectroscopy, which proved [10] that the dissociation runs through removal of h+ ion at the temperature under 400 0c and formation of of the negatively charged surface centers (≡s–оand =al–о-). alternatively, this process may run through removal of oh groups resulting in formation of the positively charged surface centers (≡si+ and =al+). this explanation is based on the results of ph-measuring carried out with aqueous aluminosilicate suspensions (table 3). as seen from table 3, all the systems under investigation has revealed the positive suspension effect that means that the charge of the surface active centers is negative and a competing adsorption of h+ and mn2+ is possible. this is also proved by another experiments were ∆рн for the suspensions containing cu2+ is decreasing for all modified bt samples. another experiment has been carried out for the grains (d=1-2 mm) of bt similarly to the powdered samples. results of this investigation are shown in figure 3. it can be seen that the sorption value of the heave metal ions is growing within the sequence pb2+ < zn2+ < mn2+, which is similar to the one obtained for the powdered samples. again, a thermal treatment at 400 0c results in some increase in the adsorption values while any table 3. influence of the basalt tufa thermal processing temperature on the suspension effect value suspension medium suspension effect value for the samples modified at the temperature, [0с] 105 400 600 800 1000 distilled water, рн 2.005 2.743 4.108 3.382 2.748 solution of cu(nо3)2, рн 1.916 1.947 3.843 3.228 2.582 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 volodymyr diichuk, iryna diichuk, igor kobasa, influence of thermal treatment the basalt tufa on its phase composition and sorption capacity, food and environment safety, volume xvii, issue 1 – 2018, pag. 37 – 40 40 higher temperature of the treatment causes temperature of the treatment causes decrease in the heavy metal ions adsorption. a comparison between the granule and powder samples adsorption values shows that the former is somewhat higher than the latter because of a higher specific surface area of the powdered samples. 0 200 400 600 800 1000 0,005 0,010 0,015 0,020 0,025 s o rp ti o n c a p a c it y , m m o l/ g 1 2 3 0 c fig, 3. an influence of the bt thermal treatment temperature on adsorption of some heavy metal ions: 1 – pb2+, 2 – zn2+, 3 – mn2+ 4. conclusion it has been found that thermal treatment of basalt tufa results in significant changes in its phase composition, which depends greatly on the temperature regime of sintering. weight loss during thermal treatment is ranged from 5,79 % (at 400 0c) to 10,18 % (1000 0c). adsorption of the heavy metal ions on the powdered tufa samples decreases for the sequence: ni2+ < pb2+ < zn2+ < cu2+ < mn2+. when the sample undergoes thermal treatment, its adsorption capacity increases while further increase in the thermal treatment temperature results in decrease in this value because of drop in the specific surface area of the samples. 5. references [1]. tarasevych, yu. i., klimova, g. m., obtaining of the modified sorbents and its usage in the field of heavy metals removal from water. water chem. and tech. 28(2): 107–116 (2006) (in ukrainian). [2]. lichareva, o. b., ivanov, m. g., matern, a. i., increase of the opoca’s sorption activity through its thermal and chemical modification. chem. for sustain. develop. 16: 415– 420 (2008) (in russian). [3]. basaraba, yu. b., zasadnyi, т. м., prospects of using zeolites from sokyrnytsya deposit in the water cleaning technologies. sci. and tech. j. 11(1): 46–51 (2015) (in ukrainian). [4]. vatin, n. i., chechevichkin, a. v., chechevichkin, v. n., shilova, e. s., usage of the clinoptilolitovite type zeolites for cleaning of the natural water. j. eng. and construct., 2: 81–88 (2013) (in ukrainian). [5]. biletsky v. s. volcanic tufa. short mining encyclopedia. ed. donetsk: donbass, vol.1: а–к. 200 р. (2004) (in ukrainain). [6]. kobasa, i.m., tsymbalyuk, v.v., basalt tufa: composition, properties and ways of utilization. chernivtsi, publishing house of chernivtsi national university. 200 p. (2015) (in ukrainian). [7]. tsymbalyuk, v.v., kobasa, i.m., woloshuk, a.g., sorption of ammonium cations and heavy metal ions from the natural artesian waters with the basalt tufa samples. advanced water supply and wastewater treatment : a road to safer society and environment. eds.: hlavinek, p., winkler, i., marsalek, j., mahrikova, i. springer. 325–328 (2011). [8]. tsymbalyuk v.v., woloshuk a.g, kobasa i.m., an influence of thermotreatment on sorption characteristics of basalt tufa. ukr. j. chem., 75(12): 85–90 (2009) (in ukrainian). [9]. woloschuk, a.g., tsymbalyuk, v.v., kobasa, i.m., influence of chemical modification of the basalt tufa on its physicochemical properties, sci. bullet. chernivtsi univ. series chem., 453: 63–68 (2009) (in ukrainian). [10]. tsymbalyuk, v.v., woloshuk, a.g., kobasa, i.m., heavy metals ions sorption on the acid-modified basalt tufa samples, sci. bullet. chernivtsi univ. series chem., 555: 84–88 (2011) (in ukrainian). in this context, the main aim of this work was to investigate an influence of the thermal treatment temperature on phase composition and sorption capacity of basalt tufa. sensory characteristics of peach and plum jams with different sweeteners 13 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017 pag. .1320 sensory characteristics of peach and plum jams with different sweeteners * viktorija stamatovska1, ljubica karakasova2, zora uzunoska1, tatjana kalevska1, valentina pavlova1,* gjore nakov3, aleksandar saveski1 1faculty of technology and technical sciences, university st. kliment ohridski bitola, dimitar vlahov bb, 1400 veles, republic of macedonia 2faculty for agricultural sciences and food, st. cyril and methodius university, edvard kardelj, bb, 1000, skopje, republic of macedonia 3department of biotechnology and food technologies, ruse “angel kanchev” university, branch razgrad, 47aprilsko vastanie blvd., razgrad 7200, bulgaria vikistam2@gmail.com, gore_nakov@hotmail.com *corresponding author received february 10th 2017, accepted march 23th 2017 abstract: sensory characteristics are of great significance for all food products. these are features which every consumer evaluates on a daily basis and based on that assessment decides whether to buy or not a product.the aim of this paper is to examine the sensory characteristics of peach and plum jams, prepared with different sweeteners (sucrose, fructose, sorbitol and agave syrup) and determine which of them is the most acceptable one for consumers. sensory analysis was conducted by applying the scoring system, which assessed individual quality criteria (smell, taste, color and consistency). the results of analysis showed that the color, smell, taste and consistency of tested jams are acceptable. jams with sorbitol, assessed with the highest average total grade have better sensory characteristics as compared with jams prepared with other sweeteners. keywords: sensory analysis, jams, different sweeteners, peach, plum 1. introduction sensory charactarestics of the food products (appearance, taste, color, consistency, smell) play a significant role in consumers’ afinity to use certain food product [1, 2]. for these reasons, the sensory properties of the products are regulary tested either by potential consumers (the food is estimated as delicious, tasteful, aromatic etc.) or by a highlyqualified team [3]. some disadvantages of aesthetic character, can reduce product quality in the eyes of the consumer despite the fact that its nutritional value is at a satisfactory level [1]. the perception of sensory characteristics is rather subjective and preconditioned by nutritional education, current trends, age, health, religion etc. in order to minimize these factors and obtain relevant statistical information, various procedures were developed [3]. the jam belongs to the group of products based on pectin gel. it is made by boiling fresh, frozen or semi processed fruit or fruit pieces, with a corresponding amount of sugar, pectin and acid. sugar and pectin form a network which gives certain texture to each product [4, 5]. consumers who want to control the level of blood sugar are not allowed to use traditional jams because of the extra amount of sucrose (sucr). according to parsayee et al. [6], the high content of sucrose in jams, contributes to excessive weight and health problems such as diabetes and hyperglycemia. http://www.fia.usv.ro/fiajournal mailto:vikistam2@%20gmail.com mailto:gore_nakov@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 14 there is a great interest for jam production with reduced energy value i.e with a lower amount of sucrose or with another type of sweetener replacement. recently, there is a high tendency of various sweeteners usage as a replacement for sucrose [7]. it was found [8-10] that sweeteners like fructose (fru), sorbitol (sorb), honey, maple syrup, agave syrup (ag), stevia and etc. did not affect the nutritional and sensory quality of the products. therefore, it was suggested that these sweeteners can be a suitable replacement for sucrose [8-10]. fructose is a natural low-calorie sweetener which has a positive impact on human health initiating a lower concentration of glucose in the blood which protects the blood vessels and the myocard from atherosclerosis preventing hypertension, diabetes, obesity, teeth disease and higher appetite [11]. sorbitol is a sugar alcohol (polyol), being applied in a low-calorie and diabetic products, and like other polyols it does not stimulate an increase in blood glucose on ingestion. however, frequent sorbitol usage can induce diarrhea [10]. agave syrup is a very popular and frequently used sugar substituent because of its low glycemic index compared to other sweeteners or to honey [12, 13], with antioxidative [14] and probiotic capacity [13]. low-energy jams and marmalades from: raspberry, blackberry, strawberry and redcurrant obtained by tepić et al. [15] using fructose, sorbitol, cyclamate and saccharin as sweeteners are an adequate example of high quality jams with good sensory characteristics. the aim of this study was to determine which of three sweeteners would be the best substitute of sucrose in plum and peach jam in terms of the best evaluated sensory characteristics. 2. materials and methods peach and plum dietetic jams were used as a material for sensory testing. dietetic jams were manufactured according to the recopies and long experience of the factory "vitalia nikola" llc skopje, which comply with the regulations of the republic of macedonia [16]. the regulations recommend that the standard jam should contain 60% of dry materia. however, dietetic jam contained 30% lower amount of sugar compared to standard jam, so the final dry materia content was 42% (+/2%). during the preparation of each type of jam, the ratio between dry materia of the sweetener and the fruit was applied according to the recommendation of the raw material producers and the experience of the factory. during the production: sucrose (a low amount), fructose, sorbitol and agave syrup were used as sweeteners. technological process of jams production covered the following operations: reception and storage of fruits, preparation of fruits (washing, cutting, inspection), measurement, heat treatment in open stainless steel vats on direct fire, at temperature of ≈ 100 °c, for 15 minutes (boiling with adding suitable sweetener with constant stirring, adding pectin, calcium citrate and acid, boiling with stirring) packaging in jars, closing, cooling and storage. the sensory analysis of jams was performed by scoring method for jelly products assesment [1]. the sensory characteristics (smell, taste, color and consistency) were assessed by highly experienced 10 testers, using a different number of points: for color 0-4, for smell 0-2, for taste 0-8, and consistency 0-6 and their maximum sum was 20. evaluations were performed in the laboratory for fruit and vegetables processing at the faculty of agricultural sciences and food in skopje (fig. 1). the tests were repeated three times over a three-year period (2011, 2012, 2013). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 15 fig. 1. preparation jam samples for sensory evaluation the results of the research were presented, analyzed and statistically processed by using microsoft excel and statistical package spss statistics version 19. 3. results and discussion the obtained data by sensory analysis of jams produced in three different years (2011, 2012, 2013) are shown in tables 1 and 2. the differences between the jams with different sweeteners in each of the productive years were statistically processed and their significance was determined. table 1. the results obtained by sensory analysis of the peach jams with different sweeteners year peach jam color (0-4) ±sd smell (0-2) ± sd taste (0-8) ± sd consistency /texture (0-6) ± sd total points (0-20) ± sd 2011 sucr 3.93±0.16b 1.93±0.16b 7.90±0.21b 5.95±0.16b 19.71±0.23b fru 3.93±0.16b 1.88±0.19 b 7.72±0.40b 5.93±0.16b 19.46±0.45b sorb 3.95±0.16b 1.98±0.06 b 7.79±0.38b 5.89±0.17b 19.61±0.43b ag 3.17±0.53a 1.44±0.42 a 6.12±1.03a 5.52±0.46a 16.25±1.66a 2012 sucr 3.53±0.82ab 1.85±0.34 7.10±1.10 5.65±0.41ab b 18.13±2.32b ab fru 3.70±0.42ab 1.90±0.32 7.25±0.82 5.65±0.47ab b 18.50±1.65b ab sorb 3.95±0.16b 1.95±0.16 7.20±1.01 5.60±0.57b ab 18.70±1.60ab b ag 3.39±0.50a 1.80±0.42 6.30±1.25 4.80±1.03a a 16.29±2.25aa 2013 sucr 3.80±0.42b 2,00±0,00b 7.60±0.52b 6.00±0.00b 19.40±0.70ab b fru 3.70±0.48b 1.90±0.32ab 7.50±0.71b 5.50±0.71ab 18.60±0.84b b sorb 3.90±0.32b 2.00±0.00b 7.80±0.42b 5.90±0.32b 19.60±0.52a b ag 2.90±0.74a 1.70±0.48a 6.40±0.84a 5.10±0.57a 16.10±1.79ab a 2011-2013 sucr 3.75± 0.20 1,93± 0,08 7.53± 0.40 5.87± 0.19 19.08± 0.84 fru 3.78±0.13 1.89±0.01 7.49±0.24 5.69±0.22 18.85±0.53 sorb 3.93± 0.03 1.98± 0.03 7.50± 0.30 5.80± 0.17 19.30± 0.52 ag 3.15±0.25 1.65±0.19 6.27±0.14 5.14±0.36 16.21±0.10 * average value; sd standard deviation; a, b – values in the same column with no common superscript differ significantly (p<0.05); a,b – values in the same column with no common superscript differ significantly (p<0.01). sucr-sucrose, fru-fructose, sorb-sorbitol, ag-agave syrup color evaluation. the term color implies the presence of colored substances in the products. the intensity of color depends on pigment content in the product. it is very important for color to be the same in all parts of the product. color quality depends mostly on raw material and the loss of color intensity depends on the conducting of the technological process of production. basic standard for sensory assessment product's color is a comparison with the natural color of the raw material, so it is required to make the deviation from raw material's natural color as small as possible, according to hue and intensity [1]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 16 table 2. results obtained by sensory analysis of the plum jamswith different sweeteners year plum jam color (0-4) ±sd smell (0-2) ± sd taste (0-8) ± sd consistency /texture (0-6) ± sd total points (0-20) ± sd 2011 sucr 3.86±0.21ab b 1.86±0.21ab 7.66±0.45b 5.77±0.28a ab 19.15±0.83b fru 3.91±0.19ab b 1.98±0.06b 7.90±0.18b 5.93±0.16ab b 19.72±0.28b sorb 3.74±0.35b ab 1.89±0.22b 7.50±0.55b 5,80±0.27ab b 18.93±1.06b ag 3.03±1.13a a 1.57±0.69a 5.55±2.23a 4.66±1.80a a 14.81±5.50a 2012 sucr 3.85±0.34b 1.95±0.16ab 7.80±0.35b 5.75±0.42b 19.35±0.78b fru 3.68±0.78b 2.00±0.00b 7.55±0.69b 5.95±0.16b 19.18±1.02b sorb 4.00±0.00b 2.00±0.00b 7.95±0.16b 5.85±0.34b 19.80±0.35b ag 2.89±0.72a 1.74±0.54a 5.60±1.26a 5.00±0.82a 15.23±2.51a 2013 sucr 3.70±0.48b ab 1,90±0.32 7.20±0.63b 5.50±0.53ab 18.30±1.06b fru 3.50±0.53ab ab 1.80±0.42 7.10±0.88b 5.50±0.53ab 17.90±1.10b sorb 3.80±0.42 ab b 1.80±0.42 7.50±0.53b 5.80±0.42b 18.90±1.10b ag 3.10±0.74a a 1.70±0.48 5.50±0.97a 5.20±0.63a 15.50±1.43a 2011-2013 sucr 3.80± 0,09 1.90± 0.05 7.55± 0.31 5.65±0.13 18.93± 0.56 fru 3.70±0.21 1.93±0.11 7.52±0.40 5.79±0.25 18.93±0.93 sorb 3.85± 0.14 1.90± 0.10 7.65± 0.26 5.82± 0.03 19.21± 0.51 ag 2.98±0.16 1.67±0.09 5.55±0.05 4.95±0.27 15.18±0.35 * average value; sd standard deviation; a, b – values in the same column with no common superscript differ significantly (p<0.05); a,b – values in the same column with no common superscript differ significantly (p<0.01). sucr-sucrose, fru-fructose, sorb-sorbitol, ag-agave syrup the results shown in table 1 (peach jams with different sweeteners) suggested that in three different years of testing (2011, 2012, 2013), the highest average color value (3.95, 3.95 and 3.90 points respectively) were registered by the sorb jams, and the lowest average color value (3.17, 3.39 and 2.90 points respectively) by the ag jams. the determined differences in 2011 and 2013, between ag and sucr, fru and sorb jams were significant in terms of color (p<0.01). according to the same parameter, in the year 2012, a significant difference (p<0.05) between ag and sorb jams only was detected. the highest average grade for color quality during 2011-2013(3.93), was notified in sorb jams, compared to the lowest grade for color quality (3.15) in ag jams. different results were obtained when plum jams made with the above mentioned sweeteners were analyzed (table 2). in the year 2011, the fru jams had the highest color quality (3.91) compared to ag jams with the lowest number of points (3.03). in the same year, a significant difference in terms of color quality between ag jams and sucr, fru (p<0.01) and sorb jams ((p<0.05) was detected. in the year 2012 and 2013, the highest number of points (4.00 v. 3.80 respectively) was assessed in sorb jams, while the lowest number of points (2.89 v. 3.10) was detected in ag jams. in the year 2012, there was a significant difference in color quality value between ag and sucr, fru and sorb jams (p<0.01). in 2013, the difference between ag and sorb jams was significant (p<0.01) as well as between the ag and sucr jams (p<0.05). the same results in terms of the highest average grade for color quality during 2011-2013 as in the case of sorb peach jams were obtained when plum sco jams were analyzed (3.85). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 17 the results obtained suggest strongly that the sorb jams characterized with the highest average points in terms of color, due to the great stability of the sorbitol during the heat treatment and the low possibility of creating new compounds that would cause discoloration [17]. kerdsup and naknean [18] examined the impact of sorbitol on physical, chemical and sensory characteristics of low-energy mango jams and concluded that the jams produced had a color that did not deviate from the natural color of mango fruit, the results that were compatible with our results (sorb jams had the same color with peach and plum fruit in our study). in our study, ag jams had the lowest average points in terms of color, due to the coloration of the agave syrup and the changes that occur during heat treatment. we also should underline the fact that the agave syrup contains mineral substances [19] that can react with anthocyanins present in fruits and thus create compounds that cause tanning [20]. smell evaluation. the smell of the product is a multidimensional phenomenon, due to the presence of a great variety of aromatic volatile compounds (alcohols, esters, ethers, organic acids, aldehydes, ketones, etc.). the smell of the product will meet the required standards, if the smell is similar to the raw material from which it is derived, without foreign uncharacteristic smells [1]. as regards the smell analysis (table 1), it was concluded that in three different years of testing, sorb jams registered the highest number of points (1.98, 1.95 and 2.00 respectively) compared to ag jams which had the lowest number of points (1.44, 1.80 and 1.70 respectively). in 2011, a significant difference (p<0.01) between ag jams and sucr, fru and sorb jams in context of smell was determined. in the year 2012, in terms of smell there were not any significant differences between the jams (p>0.05) but in 2013 there was a significant difference between ag jams and sucr and sorb jams (p<0.05), but not with fru jams. sorb jams were assessed with the highest average grade for the whole three-year period (1.98), while ag jams had the lowest average grade for smell (1.65). the results from the analysis of plum jams (table 2) suggested that fru jams had the highest number of points (1.98) while the lowest number was detected in ag jams (1.57) in 2011 (the difference was significant p<0.05). in 2012, the fru and sorb jams were assessed with maximum points (2.00). a significant difference between ag and fru and sorb jams was detected (p< 0.05). in 2013, the sucr jams had the highest number of points (1.90) and significant differences between different types of jams were not detected. three-year average grade for smell shows the highest value in fru jams (1.93), while ag jams had the lowest average grade (1.67). our results suggest that sorbitol had the least effect on the smell of jams. however, the ag jams registered the lowest average points in terms of smell, probably due to the possibility of creating new compounds during the heat treatment which can induce a change of smell. taste evaluation. in terms of food taste, it should be completely satisfactory, the consumer will disregard smaller deficiencies in terms of color, consistency etc. [1]. according to the results obtained from the sensory analysis of the peach jams (table 1) in 2011 harvest sucr jams had the highest number of points (7.90), whereas the ag jams the lowest number of points (6.12). in 2012 vintage, fru jams had the highest number of points (7.25), and the ag jams the lowest number (6.30). in 2013 harvest, ag jams had also the lowest number of points (6.40) in relation to the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 18 same parameter while the highest number was detected in sorb jams (7.80). from the data statistical analysis obtained in terms of taste of peach jams, the years 2011 and 2013 showed significant differences (ag jam compared to sucr, fru and sorb jams, p<0.01) while no significat differences were found in 2012, p>0.05). the peach sucr jams were assessed with the highest for 3 year harvest follow up average grade for taste (7.53), while the ag jams with the lowest (6.27). from the results obtained for each year and 3 years average taste value of investigation (plum jams, table 2) it can be concluded that ag jam showed the lowest taste values compared to 3 other types of jam. the highest taste values in 2011 harvest was observed in fru jams (7.90) while in the next two years sorb jams had the highest value (7.95 v.7.50). during the three-year period the ag jams taste values differ significantly from the other jams (p<0.01). the above results obtained, suggest that in general, sorb jams were characterized with the highest average points in terms of taste, confirming thus the fact that sorbitol with its mild sweetness provides products with taste typical to the fruit which it originates from [7]. the results are compatible with the findings of other authors [18]. the jams with agave syrup have been assessed with the lowest average points in terms of taste, which is probably due to the properties of the agave-syrup and the changes that occur during heat treatment. namely, the creation of new compounds during heat treatment can affect the taste of jams. consistency evaluation. consistency is specific to each type of product. jams require particular degree of jelling and presence of whole fruits or parts of the fruits preserved [1]. according to the results obtained from the sensory analysis of peach jams (table 1), it was concluded that in all 3 years investigation, sucr jams had the highest consistency values, but in 2012 these values were equal with fru jams values. the interesting observation was that the values in context of previously interpreted results for color, smell and taste, showed similar results. namely, ag jams showed the lowest values compared to other types of jams (significant difference was determined). from the data presented in table 2 (plum jams), it was concluded that in the years 2011 and 2012, the frujams had the highest number of points in terms of consistency (5.93 v. 5.95), whereas during 2013 it was found sorb jams had the highest value as well (5.80). however, ag jams had the lowest consistency values during the whole investigation. (4.66 v. 5.00 v. 5.20). significant difference was noticed between the values of ag jams compared to fr, sorb and sucr jams during 2011 and 2012, and to sorbjams only during 2013. our results indicated that sorb jams had the highest average points in terms of consistency, which showed that sorbitol provided the product with the texture required, without making it too sweet [17, 21]. the ag jams registered the lowest average points in terms of consistency, probably due to the changes occuring during heat treatment since it contains large amounts of fructose which can induce certain chemical reactions during heat treatment of the fruit, affecting the consistency of jams [19, 22, 23]. total sensory evaluation. the total assessment of sensory characteristics of jams was obtained by adding the individual points for color, smell, taste and consistency with a possibility of maximum total point score of 20 points. according to the total sum of points for peach jams (table 1), it was found that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 19 sorb jams had the highest total average grade compared to ag jams with the lowest total sum (19.30 v 16,21). from the data shown in table 2 (plum jams) it can be concluded that during the three-year period, sorb jams had the highest total average grade compared to ag jams with the lowest value (19.21 v. 15.18). our results strongly suggest that sorbitol used as a sweetener in both types of jams was the best option for sucrose replacement, resulting in best sensory characteristics which are compatible with other researchers’ findings [24, 25]. 4. conclusions based upon the results obtained by sensory characteristics analysis of peach and plum jams prepared with different sweeteners (fructose, sorbitol, agave syrup and a low amountof sucrose) it can be concluded that plum and peach jams with sorbitol had the highest total average score and better sensory characteristics as compared to jams where other sweeteners were used. the agave sweetener when added to peach and plum jams did have the weakest sensory characteristics as compared to other sweeteners used. the results provided can help fruit producers in choosing a sweetener which can replace sucrose in jam production. the production of such fruit products is highly recommended for people who need a limited amount of sugar intake. 5. references [1]. vračar o.lj., manual for quality control of fresh and processed fruits, vegetables and mushrooms and refreshing soft drinks, faculty of technology, novi sad, (2001). [2]. koprivnjak о., the quality, safety and preservation of food, textbook „introduction to food technology" for students of sanitary engineering, rijeka, (2014). [3]. gulaboski r., chemical control of the quality of the products, agricultural faculty -štip, university goce delčev štip, (2010). [4]. figuerola e.f., chapter 13: berry jams and jellies, part iii processing technologies for developing value-added berry fruit products, in: berry fruit: value added products for health promotion (ed. zhao y.), crc press, boca raton, fl, 367-387, (2007). [5]. levaj b., technology of fruit and vegetables, ii part, faculty of food technology and biotechnology, zagreb, (2013). [6]. parsayee a.s., zonuz a.a., zadeh g.b., ayase a., low calorie sour cherry jam: optimization of ingredient levels using response surface methodology, international journal of applied sciences and engineering research, 2(1): 35–43, (2013). [7]. malčev e., mollov p., technology canning of fruits and vegetables concentrates and fruit sugar products, second edition, “kontrast”, stara zagora, (2008). [8]. chetana r., studies on the use of sucrose alternatives in traditional sweetmeats, thesis, university of musore, india, (2004). [9]. asif m., low caloric sweeteners for diabetes and obesity care and their clinical inferences for tackle the prevalence, journal pharm care, 1(3): 104-113, (2013). [10]. gwinn r., technology and ingredients to assist with the reduction of sugar in food and drink. fhis, campden bri, (2013). [11]. gałkowska d., fortuna t., prochwicz-zagórska w., physicochemical quality of selected strawberry jams with fructose, potravinárstvo, ročnik, 4(2): 22-24, (2010). [12]. foster-powell k., holt h.a.s., brand-miller c., international table of glycemic index and glycemic load values: 2002, the american journal of clinical nutrition, 76: 5– 56, (2002). [13]. mellado-mojica e., lópez-pérez g.m., comparative analysis between blue agave syrup (agave tequilana weber var. azul) and other natural syrups, agrocoencia, 47(3): 233-244, (2013). [14]. phillips m.k., carlsen h.m., blomhoff r., total antioxidant content of alternatives to refined sugar, journal of the american dietetic association, 109 (1): 64–71, (2009). [15]. tepić n.a., vujičić l.b., dimić p.n., popov-ralić v.j., the quality of dietetic jelly products, acta periodica technologica, 32: 33-38, (2001). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 2017 viktorija stamatovska, ljubica karakasova, zora uzunoska, tatjana kalevska, valentina pavlova, gjore nakov, aleksandar saveski, sensory characteristics of peach and plum jams with different sweeteners, food and environment safety, volume xvi, issue 1 – 2017, pag. 13 – 20 20 [16]. regulation for special security requirements of fruitjams, jellies, marmalades and sweetened chestnutpurée (republic ofmacedonia official gazette no. 3/2007). [17]. bemiller n.j., carbohydrate chemistry for food scientists. aacc international, prevod na deloto: ars lamina, 2011, skopje, (2007). [18]. kerdsup p., naknean p., effect of sorbitol substitution on physical, chemical and sensory properties of low-sugar mango jam, proceeding science and engineering, 12-16, (2013). [19]. conis e., agave syrup's benefits are in debate, los angeles times, (2009), online source, accessed in 04.02.2017: http://articles.latimes.com/2009/mar/30/health/henutrition30. [20]. tepić a., colored matter fruits and vegetables, university of novi sad, faculty of technology, novi sad, serbia, (2012). [21]. walters d. e., all about sweeteners, (2008), online source, accessed in 04.02.2017: http://www.sweetenerbook.com/neodhc.html. [22]. barclay t., ginic-markovica m., cooper d. p., petrovsky n., the chemistry and sources of fructose and their effect on its utility and health implications, j. excipients and food chem., 3(2): 67-82, (2012). [23]. quintas a. c. m., fundo f. j., silva l. m. c., sucrose in the concentrated solution or the supercooled ‘‘state’’: a review of caramelisation reactions and physical behaviour, food engineering reviews, 2 (3): 204-215, (2010). [24]. karakashova lj., stamatovska v., babanovska-milenkovska f., the quality properties of raspberry jams with different sweeteners, symposium proceeding, international symposium for agriculture and food, skopje, 12-14 december 2012, volume ii, 848-855, (2013). [25]. saveski а., stamatovska v., pavlova v., kalevska t., vaskoska s. r., sensory analysis of raspberry jam with different sweeteners, scientific works of university of food technologies, lxii(lxii): 294-297, (2015). http://www.sweetenerbook.com/neodhc.html 1. introduction sensory charactarestics of the food products (appearance, taste, color, consistency, smell) play a significant role in consumers’ afinity to use certain food product [1, 2]. for these reasons, the sensory properties of the products are regulary tested... 4. conclusions 5. references sensory evaluation 310 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 3-2015, pag. 310 319 sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies for optimum formulation by the mixture experimental design silvia mironeasa1, *georgiana gabriela codină1 1faculty of food engineering, stefan cel mare university, suceava, romania, silviam@fia.usv.ro; codina@fia.usv.ro *corresponding author received september 5th 2015, accepted september 28th 2015 abstract: the aim of the present investigation was to prepare and optimize the formulation of millet yellow flaxseedhemp composite flour gluten-free cookies by applying experimental design technique. simplex-lattice design and desirability function were used to evaluate and then to establish the optimal recipe for gluten-free cookies. maximum sensorial quality and general acceptability were selected as dependent variables. regression equations and contour plots were used to relate dependent variables, i.e. color, taste, smell, aroma, texture and general acceptability of cookies, with independent variables.the results of the experimental design revealed that the independent factors millet flour, yellow flax seed flour and hemp flour are significantly influenced by the general acceptability of the cookies. based on the computed value of desirability function, it was concluded that the composite flour containing 21.50% millet flour, 20.00% yellow flaxseed flour and 58.50% hemp flour was in the optimum mixture proportions based on the maximum sensory characteristics of cookies samples. key words: composite flour, gluten-free cookies, sensory evaluation, simplex-lattice design, desirability function 1. introduction in general, cookies are products favored by consumers being an important part of their daily diet. the main ingredient of cookies is wheat flour, but the health problems associated with celiac disease has limited the use of this type of flour in such products. therefore, nowadays the requirement for gluten-free products is increasing worldwide. considering this demand for obtaining cookies only from composite flours we used hemp, yellow flaxseed and millet flours which allowed us to improve the quality of cookies from the nutritional point of view due to the fact that these flours are nutrient-rich and also gluten-free. in the food industry it is using only hemp (cannabis sativa l., cannabaceae) with a low level of δ-9-tetrahydrocannabinol [1]. it presents a great nutritional value generally composed of 25-35% lipids, 2025% protein, 20-30% carbohydrates, and 10-15% fibers [2]. in addition to these components, hemp seeds also contain minerals and vitamins like phosphrous, potassium, magnesium, sulphur, calcium, iron, zinc, carotene and e, c, b1, b2, b3, b6 vitamins [3]. the lipid portion of hemp http://www.fia.usv.ro/fiajournal mailto:silviam@fia.usv.ro mailto:codina@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 311 seeds is a healthy one due to it content up to 80% polyunsaturated fatty acids [4] notably essential linoleic acid (omega-3) and α-linolenic acid (omega-6) in the favorable ratio of 3:1 [2, 3, 5]. moreover, hempseed oil contains γ-linolenic and stearidonic acid which can only be found in a few plant families [4]. hemp seeds are also a good source of easily digestible proteins which contains all essential amino acids [2]. in the seeds contents substances such as methionine and cystine were also identified [6]. the hemp seed protein contains 65% globulin called edestin and 33% albumin [7]. the flaxseed (linum usitatissimum l.) is rich in lipids, proteins, lignans and dietary fibers. the golden flaxseed contains a lower amount of fiber and a higher amount of soluble carbohydrates and fatty acids omega-3 and omega-6 than the brown variety [8]. in general, regarding the lipid content, flax seeds are rich in polyunsaturated fatty acid in especially omega-3 fatty acid with the highest amount of any seed oil. omega-6 fatty acid is only about 16% from the polyunsaturated fatty acid content [9]. from the protein content point of view the flaxseed presents an amino acid composition similar to that of soybean protein, which is viewed as one of the most nutritious plant proteins [10]. the major proteins in flaxseed are albumins and globulins. flax seeds contain also large amounts of minerals and vitamins such as vitamin e present as gammatocopherol, which function as an antioxidant [11], vitamin b (thiamine (b1), riboflavin (b2), niacin (b3), pantothenic acid (b5), pyridoxine (b6), folate and cyanocobalamin (b12)) [12], potassium and phosphorus like major mineral elements and significant quantities of iron, zinc and manganese [13]. the term “millet” is used for several small seeded annual grases and can be cultivated in a wide range of soils and climates with a special importance in semiarid regions [14]. this food cereal is the forth tropical crop in the world.[15]. millet presents high levels of calcium, iron, zinc, lipids and high quality proteins [16]. compared to other cereals millet has a better amino acid profile than maize and sorghum and a similar one to barley, rice and wheat [17]. the lysine and methionine content of pearl millet is with 40% and respectively 30% higher than the corn [18]. however, millet does not contain high level of lysine comparatively with other essential amino acids like methionine and tryptophan [19]. regarding the lipid content, millet is rich in oil which contains linoleic acid and αlinolenic acid [20]. linoleic acid is about 4% of the total fatty acids from this oil in a more high content than in maize [17]. sensory properties of food have the highest influence on food choice in most situations. these sensory properties are perceived through the senses of smell, sight, touch, taste and hearing and are evaluated by different scientific methods. the evaluations of food preference from the sensorial point of view are based on foods accepted or rejected by using different preference sensorial tests. mixture experiment design is a statistical technique that can be used for evaluating the preference and acceptance of the final product based on the relative proportions of the components in the mixture. this technique describes through an equation how the response variables are affected by the independent variables in a mixture experiment [21]. simplex-lattice design can be applied as standard tool in order to find the optimal response for any mixture of the ingredients, and to obtain the influence on the response of each ingredient singly and in combination with other ingredients. the main objective of this study was to optimize the mixture of the three ingredients, hemp, yellow flaxseed and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 312 millet flours from the gluten-free cookies recipe, with the purpose of achieving the maximum sensorial quality and overall acceptability of produced cookies. simplex-lattice design was applied as standard mixture designs in order to determine the optimal blend for high acceptance gluten-free cookies. 2. materials and methods 2.1. materials the millet seed, yellow flaxseed and hemp seed were purchased from the romanian supermarket and used as raw materials for composite flour formulation. the glutenfree flours from these seeds were obtained through mechanical milling of the seeds, and then the mill was passed through a 150 m mesh sieve in order to obtain fine flour. 2.2. methods 2.2.1. experimental design in this study, the simplex-lattice design was used with a three components mixture, with lower and upper limit restrictions, for the determination of the optimum combinations of the gluten-free flours (components) in the recipe of cookies to produce acceptable product and to estimate the most acceptable product with the highest sensory score. the lower and upper limit restrictions of each component (gluten-free flour) were based on preliminary work. in this mixture design the following three factors: millet flour (x1), yellow flaxseed flour (x2) and hemp flour (x3) were evaluated by changing their levels (proportions) simultaneously and keeping total level (proportions) constant (x1 + x2 + x3 = 1) [22]. blends of the formulations were the pure (x1, x2, x3), two-component mixtures (x1x2, x1x3, x2x3) and the three-component mixtures (x1x2x3) according to montgomery (2000) [23]. the experimental design required 13 combinations of gluten-free flours, levels and experimental design in term of pseudo components and real levels which are presented in table 1. the sensory characteristics such as: colour, taste, smell, aroma, texture and general acceptability were selected as the response variables. the value of response variable is a function of the level of ingredients present in a cookie recipe [24]. the goal of this experimental design is to model the mixture surface with a form of polynomial equation so that the responses (sensory characteristics) for any blend of ingredients from recipe of cookies can be predicted. 2.2.3. preparation of cookies the flour mixtures composed of hemp, yellow flaxseed and millet flours at different proportions (table 1) were used for the cookie production. the other ingredients used were sodium chloride 1% and water 20% (on 100% weight basis of mixed flour). dry flour mixture were homogenized using dough mixture for 3 min and sodium chloride dissolved in about 5 ml water were added into the mix. further mixing was done with the rest of the water for 10 minutes. the dough was sheeted to thickness of 0.5 cm. the sheeted dough was made into circular shape samples by circular cookie cutter of 4 cm diameter. these circular samples were transferred in a baking oven and baked at 180-2000c. the baking time varied between 10 and 13 minutes. after baking, cookies samples were cooled by resting on a paper napkin at room conditions for 30 minutes. cookies were stored in hdpe bags in a refrigerator at 40c before analysis. 2.2.4. sensory evaluation consumer affective testing was carried out using a nine point structured hedonic scale food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 313 (1 = disliked and 9 = liked extremely) for the evaluation of the color, taste, smell, aroma, texture and general acceptability. the test was conducted in the sensorial analysis laboratory at the ştefan cel mare university, faculty of food engineering. samples (approximately 5 g) of each formulation coded with three-digit numbers were presented to 63 students on plastic plates. unflavored, dry crackers and taste free water were provided for palate cleansing between samples. for minimize sensory fatigue there was a 3 minute break after each sample and a 15 minute break after the seventh sample. 2.2.5. statistical analysis and optimization statistical analysis and modeling of response variables were carried out using stat ease design expert 7.0.0 software package (trial version). the multiple regression analysis with the intercept set to zero was used to generate polynomial models based on scheffé’s canonical model for each of the response variables. the adequacy of the regression model was verified by the sequential model sum of squares, lack of fit, multiple correlation coefficients (r2) and other adequacy measures. in order to determine the significance of each term an analysis of variance (anova) with a level of significance of 95% was performed, whereby the resulting model equations describe the sensory characteristics on the three components (millet flour, yellow flaxseed flour, and hemp flour) and their interactions. the optimum levels of the factors (components used in recipe) were obtained by using the numerical optimization technique. this technique searches the design space, using the models created during analysis to find factor settings that meet the defined goals. to find out an effective solution, a multiple response method called desirability was applied [25]. the desired goal for each factor and response was selected so: the proportion of millet flour, yellow flaxseed flour and hemp flour were permitted to be at any level within range of the design, while all of the response variables were desired maximal. after applying these constraints, the optimal levels for ingredients used in cookies recipe and the predicted values for sensory quality and overall acceptability were obtained. table 1 experimental design for ternary mixture of millet, yellow flaxseed and hemp flour in pseudo components and in real levels formulation component proportion in the composite flour pseudo components real levels (%) millet flour (x1) yellow flaxseed flour (x2) hemp flour (x3) millet flour yellow flaxseed flour hemp flour 1 1.000 0.000 0.000 50.0 20.0 30.0 2 0.500 0.000 0.500 35.0 20.0 45.0 3 0.167 0.167 0.667 25.0 25.0 50.0 4 0.000 0.500 0.500 20.0 35.0 45.0 5 0.333 0.333 0.333 30.0 30.0 40.0 6 0.000 0.000 1.000 20.0 20.0 60.0 7 1.000 0.000 0.000 50.0 20.0 30.0 8 0.000 1.000 0.000 20.0 50.0 30.0 9 0.000 1.000 0.000 20.0 50.0 30.0 10 0.000 0.000 1.000 20.0 20.0 60.0 11 0.500 0.500 0.000 35.0 35.0 30.0 12 0.167 0.667 0.167 25.0 40.0 35.0 13 0.667 0.167 0.167 40.0 25.0 30.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 314 3. results and discussion 3.1. fitting for the best model a simplex-lattice design was applied in this study to perform the experiments by changing the concentrations of component simultaneously and keeping total concentrations constant (table 1). the quadratic model (eq.1) was found the best fitted for all sensory characteristics of gluten-free cookies because has low standard deviation, low predicted sum of squares, and high predicted r-squared [21]. y = β1x1+ β2x2+ β3x3+ β12x1x2+ β13x1x3 + + β23x2x3 (1) where y is the dependent variable; β1, β2, β3, β12, β13 and β23 are the constant coefficients for each linear and interaction term in the predicted models, and x1, x2, x3 the proportions (levels) of each pseudocomponents. analysis of variance (anova) showed that the sensory characteristics were significantly (p < 0.01) influenced from the linear terms of the predicted models despite the fact that the sensory characteristics were not significantly influenced from two component interactions (table 2). significance of model fitting was also assessed using anova and p value was always less than 0.01. the r2 value obtained through the analysis of predictive models was found to be always greater than 0.88, indicating a great fitting model. table 2 regression coefficients and analysis of variance of the models for sensory characteristics coefficientsa sensory characteristics colour taste smell aroma texture general acceptability β1 6.83 ** 7.29** 7.37** 7.54** 7.21** 6.84** β2 6.79 ** 5.96** 6.01** 6.00** 5.77** 6.26** β3 8.04 ** 8.19** 8.45** 8.41** 8.25** 8.29** β12 2.11 * 4.20* 3.82* 5.29* 5.48* 2.97* β13 1.62 * 0.40 1.68 -0.25 1.56 1.94 β23 -0.58 1.20 -2.86 -0.65 -1.92 -0.74 significance of the model (p) 0.0004 0.0036 0.0019 0.0006 0.0018 0.0007 lack of fit of the model 0.2920ns 0.1922 ns 0.0502 ns 0.2773 ns 0.2570 ns 0.0610 ns r2 0.9408 0.8835 0.9033 0.9300 0.9057 0.9271 a β1, β2, β3 correspond to the effects of millet flour, yellow flaxseed flour and hemp flour, respectively, in the model; ns no significant effect at level  0.05; * significant at the level of p  0.05; ** significant at the level of p  0.001. 3.2. sensory evaluation figure 1 shows the regression model of sensory characteristics for the milletyellow flaxseed-hemp composite flour gluten-free cookies in a triangular plot of the statistically analyzed experimental design. each corner of the equilateral triangle shown in figure 1 represents a concentration of 50% (w/w) of millet flour and yellow flaxseed flour, and 60% of hemp flour, respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 315 3.2.1. color according to table 2, the color scores response was well fitted to the quadratic model with high determination coefficient (r2 = 0.9408). two-component interaction, millet flour-yellow flaxseed flour (x1x2) and millet flour-hemp flour (x1x3) had significant influence (p < 0.05) on the color score response of cookies samples (table 2). in the contour plot of the color score response, the highest values were given in the x3 vertex and to edge of x3 (figure 1a). 3.2.2. taste the predicted regression coefficients for taste showed that the quadratic model is appropriate for this sensory characteristic (table 2). for taste response, the interaction between millet flour and yellow flaxseed flour (x1x2) had a more notable influence on taste scores as shown by the higher coefficient value (table 2). higher taste scores were observed in the x3 edge, in the vertex of the x3 and to some extend in x3x2 edge. increased yellow flaxseed (x2) concentration decreased sample taste while, increased millet flour (x1) also increased sample taste (figure 1b). high millet flour levels in cookies gave products with more taste scores compared to products with low levels added millet flour. similar results were reported in the literature in which the composition on gluten-free flour cookies was optimized [26]. 3.2.3. smell as can be observed in table 2, r2 value of the predicted model for smell response were higher than 0.90, indicating that this were relatively adequate for the prediction purpose. figure 1c) illustrates ternary contour plot that indicates the effects of processing components on the smell sensory characteristic of cookies samples. the smell contour values increased toward the hemp flour (x3) edge where maximum smell contour could be seed; however, the lowest smell scores were at yellow flaxseed flour (x2) vertex and to some extend in x2 edge. from this point of view, it could be said that the use of hemp flour in recipe gluten-free cookies increased the smell of the final product, while use of the others decreased. 3.2.4. aroma the aroma response was well fitted to the quadratic model with high determination coefficient (r2 = 0.9300). generally, the linear terms had a significant effect on aroma scores of cookie samples; however, interaction between millet flour and yellow flaxseed flour had lower coefficient compared to the linear terms (table 2). contour plot (figure 1d) shows that aroma score was higher in the vertex of the hemp flour (x3) and edge of millet flour (x1). the lowest aroma scores were at yellow flaxseed flour (x2) vertex. therefore, high yellow flaxseed flour levels gave glutenfree cookie products with no acceptable aroma compared to products with low levels of this flour. 3.2.5. texture the quadratic regression model for texture response was highly significant (p < 0.01). two-component interaction, millet flour and yellow flaxseed flour (x1x2) had significant influence (p < 0.05) on the texture response of cookie samples (table 2). contour plot showed that texture score seemed to behave symmetrical when yellow flaxseed flour concentration was equal to the millet flour (figure 1e). also, the highest texture score to be at millet flour and hemp flour edges and hemp flour vertex. however, the lowest texture scores were at yellow flaxseed flour vertex. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 316 consequently, it could be said that the use of hemp flour and millet flour in recipe increased the texture of the cookie samples. a) b) c) d) e) f) fig. 1. ternary contour plots of the effects of processing components on sensory characteristics of cookies: a) color, b) taste, c) smell, d) aroma, e) texture and f) general acceptability food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 317 3.2.6. general acceptability general acceptability of millet-yellow flaxseed-hemp composite flour gluten-free cookies were significantly (p < 0.01) influenced from the linear terms of the predicted model as indicated by the adjusted regression model developed for general acceptability were not significantly (p  0.05) influenced from two component interactions, millet flour-hemp flour (x1x3) and yellow flaxseed flour-hemp flour (x2x3). general acceptability score was higher at the points closest to hemp flour vertex and millet flour-hemp flour edge, indicating that the cookies samples were more preferred by the panelist when the millet flour and hemp flour was added in recipe at the higher levels comparatively with yellow flax seed flour (figure 1f), probably because of the synergistic effects of the flours upon the sensory characteristic of the gluten-free cookie samples. 3.3. mixture proportion optimization mixture proportion optimizations were performed to determine the optimum levels of gluten-free flours (millet flour, yellow flaxseed flour and hemp flour) used in the recipe of cookies to achieve the desired response goals. finding maximum scores of sensory characteristics is the desired goal to obtain an acceptable product in terms of sensory characteristics. the scores of following sensory characteristics: colour, taste, smell, aroma, texture and general acceptability were desired maximal. the best combinations between the proportions of these glutenfree flours from recipe of cookies in order to obtain maximum scores of sensory characteristics were extracted by design expert software that performs from the thousands of iterations and calculation the maximum desirability score and the conditions on which it was arrived. using this approach, a set of combination of millet flour, yellow flaxseed flour and hemp flour was found but the best formulation (table 3) to obtain an acceptable product in terms of sensory characteristics, at a total desirability value (d) of 0.955. maximum colour (8.060), taste (8.169), smell (8.477), aroma (8.358), texture (8.268) and general acceptability score (8.305) could be obtained from this optimum mixture proportion (formulation of samples). table 3 optimum mixture proportion components actual proportions (%) millet flour 21.50 yellow flaxseed flour 20.00 hemp flour 58.50 overlay plots can also be produced by superimposing the contour for the sensory characteristics. by defining the limits of the color, taste, smell, aroma, texture and general acceptability score desired, the zone at the bottom right of the plot, as shown in figure 2, defines the permissible scores of the response variables. fig. 2. overlaid contour plot of sensory characteristics for millet-yellow flaxseed-hemp composite flour gluten-free cookies food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 318 according to these results, high proportion of hemp flour comparatively with millet flour and yellow flaxseed flour, are needed to obtain maximum sensorial quality and overall acceptability of produced cookies from millet-yellow flaxseed-hemp composite flour gluten-free. 4. conclusion the results of this study showed that simplex-lattice mixture design approach is a fitting method for optimization of millet-yellow flaxseed-hemp composite flour gluten-free cookies formulation. according to experimental results and contour plots were observed that hemp flour and millet flour significantly influenced sensory characteristics of cookies and increase levels of yellow flaxseed flour in recipe of cookies decreased sensory characteristics, especially smell characteristic. therefore, millet-yellow flaxseed-hemp flour can be use as a mixture to produce gluten-free cookies when optimum combination proportions of these flours are taken account. 5. references [1]. wang x.s., tang c.h, yang x.q., gao w.r., characterization, amino acid composition and in vitro digestibility of hemp (cannabis sativa l.) proteins, food chemistry, 107: 11-18, (2008) [2]. leizer c., ribnicki d., poulev a., dushenkov s., raskin i., the composition of hemp seed oil and its potential as an important source of nutrition, journal of nutraceuticals, functional & medical foods, 2: 36-53, (2000) [3]. borhade s.s., chemical composition and characterization of hemp (cannabis sativa) seed oil and essential fatty acids by hplc method, archives of applied science research, 5: 5-8, (2013) [4]. petrovic´ m., debeljak z., kezic´ n., dzidara p., relationship between cannabinoids content and composition of fatty acids in hempseed oils, food chemistry, 170: 218-225, (2015) [5]. vonapartis e., aubin m.p., seguin p., mustafa a.f., charron j.b., seed composition of ten industrial hemp cultivars approved for production in canada, journal of food composition and analysis, 39: 8-12, (2015) [6]. odani s., isolation and primary structure of a methionine and cystine-rich seed protein of cannabis sativa l. , bioscience biotechnology and biochemistry, 62: 650-654, (1998) [7]. malomo s.a., aluko r.e., a comparative study of the structural and functional properties of isolated hemp seed (cannabis sativa l.) albumin and globulin fractions, food hydrocolloids, 43: 743-752, (2015) [8]. machado m.sc. a.m, paula d.sc. h., cardoso d.sc. l.d., costa n. m.b., effects of brown and golden flaxseed on the lipid profile, glycemia, inflammatory biomarkers, blood pressure and body composition in overweight adolescents, nutrition, 31: 90-95, (2015) [9]. ganorkar p. m., jain r. k., effect of flaxseed incorporation on physical, sensorial, textural and chemical attributes of cookies, international food research journal, 21: 15151525, (2014) [10]. youle r.j., haung a.h.c., occurrences of low molecular weight and high cysteine containing albumin storage proteins in oilseeds of diverse species, american journal of botany, 68:44-48, (1981) [11]. mantzioris e., james m.j., gibson r.a., cleland l.g., dietary substitution with a alpha-linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentration in tissues, american journal of clinical nutrition, 59:13041309, (1994). [12]. bhatty r.s., cherdkiatgumchai p., compositional analysis of laboratory-prepared and commercial samples of linseed meal and of hull isolated from flax, journal of american oil chemist society, 67:79–84, (1990) [13]. carter j.f., potential of flaxseed and flaxseed oil in baked goods and other products in human nutrition, cereal foods world, 38: 753-758, (1993). [14]. lorenz k., dilsaver w., proso millets. milling characteristics, proximare compositions, nutritive value of flours, cereal chemistry, 57: 1620, (1980) [15]. osman n.a., effect of traditional fermentation process on the nutrient and antinutrient contents of pearl millet during preparation of lohoh, journal of the saudi society of agricultural sciences, 10: 1-6, (2011) [16]. klopfenstein cf, hoseney rc, nutritional properties of sorghum and the millets, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 silvia mironeasa, georgiana gabriela codină, sensory evaluation of millet-yellow flaxseed-hemp composite flour gluten-free cookies by the mixture experiment design, volume xiv, issue 3 – 2015, pag. 310 – 319 319 st. paul: american association of cereal chemists inc 2: 125-168, (1995) [17]. ranasalva n., visvanathan r., development of bread from fermented pearl millet flour, journal food process technology, 5: 1-5, (2014) [18]. burton g.w., wallace a.t., rachile k.o., chemical composition and nutritive value of pearl millet, crop science, 12: 187–188, (1972) [19]. glew r.s., chuang l.t., roberts j.l., glew r.h., amino acid, fatty acid and mineral content of black finger millet (eleusine coracana) cultivated on the jos plateu of nigeria, food – global science books, 2: 115-118, (2008) [20]. fernandez d.r., vander jagt d.j., milsson m., huang y.s., chuang l.t., pastuszyn a., glew r.h., fatty acid, amino acid and trace mineral composition of eleusine corocana (pwana) seeds from northen nigeria, plant foods for human nutrition, 58: 1-10, (2008) [21]. cornell j.a, experiment with mixtures, designs, models and the analysis of mixture data, 3rd edition, john wiley & sons , inc, usa., (2002) [22]. derringer g, suich r, simultaneous optimization of several responses variables, journal of quality technology 12(4):214–219, (1980) [23]. montgomery d. c., design and analysis of experiments, fifth edition, john wiley & sons, inc., (2000) [24]. okpala l. c. , okoli e. c, optimization of composite fl our biscuits by mixture response surface methodology, food science and technology international, 19: 343–350, (2013) [25]. myers rh, montgomery dc, response surface methodology: process and product optimization using designed experiments, john wiley & sons, inc., new york, (1995) [26]. hathan b.s., prassana b. l., optimization of fiber rich gluten-free cookie formulation by response surface methodology, world academy of science, engineering and technology, 5: 12-24, (2011) 1 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 1 7 amino-acid content in grain protein of tetraploid opaque-2 maize andrei palii, *grigorii batîru department of plant biology, faculty of agronomy, state agrarian university of moldova, chishinau, 2049, republic of moldova, grigore.batiru@mail.ru *corresponding author received october 27th 2013, accepted december 7th 2013 abstract: amino acid composition in grain protein of a diploid (2х=20) single-cross maize hybrid containing opaque-2 (o2) gene and tetraploid (4х=40) derivatives of this hybrid was assayed. this mutant gene is widely used in maize breeding to improve quality of grain protein. protein content was determined by the kjeldahl method and analysis of protein amino acids by ion exchange chromatography. tetraploids, depending on genotype, showed higher protein content in grains than diploid forms and high variability in amino acid composition. some derivatives contained higher levels of such amino acids like, lysine, arginine, aspartic acid and lower levels of leucine in protein in comparison with the diploid grains indicating possibilities to select valuable tetraploid forms. grains with o2 endosperm from duplex-heterozygote self-pollinated ears showed higher content of lysine, arginine, valine, aspartic acid and glycine and reduced content of leucine, glutamic acid, proline and tyrosine than vitreous grains from the same ears, which highlights the specificity of gene effect. the analysis of dosage effect of the o2 gene revealed that lysine content increases proportionally to the allele number of o2 in the endosperm of diploid and, partly, in tetraploid grains. a possible maternal influence is indicated to explain the result. keywords: maize – diploid – tetraploid – gene – opaque-2 – amino acid – lysine. 1. introduction maize has spread in culture as a diploid species (2x=20). the first tetraploids (4x = 40) were obtained in 1932 by l. f randolph using high temperature [1]. since the first research on tetraploid forms a number of valuable morphological, physiological and biochemical features were highlighted. the grains of tetraploid maize are characterized by high content of protein, carotenoids and low fat [2-7]. increasing the amount of grain protein is an important objective in maize breeding, as compared to other cereals, maize is low in protein content with an average of 10%. studies have shown, however, that with increasing protein in grain, respectively, increases the rate of alcohol soluble fraction zein, very low in some essential amino acids such as lysine and tryptophan [8]. the research on improving the quality of maize protein opened up new perspectives after the discovery of the biochemical effect of the recessive mutation opaque-2 (o2), which determines a floury texture of the endosperm and a dramatic shift in protein metabolism resulting in considerable increase of lysine and tryptophan [9]. even in the early years of investigations of new forms created on the basis of the o2 gene in various institutions valuable practical results have been obtained. after using mutant maize grains in animal feed it was revealed a significant increase in the weight of different species of monogastric animals (mice, rabbits, pigs, poultry) and an food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 2 essential reduction of feed consumption [10-12]. currently the biochemical effect of the o2 mutation is quite well studied in diploid maize, but, virtually, no information is known about the action of this gene in tetraploid maize. in just one paper of m. i. hadjinov and v. s. sherbak [7] is shortly stated that studying the biochemical effect of o2 gene in tetraploid maize, no significant changes in the content of lysine in grain protein was found. given the hexaploid nature of the endosperm in tetraploid grains remains still unclear the dosage effect of the o2 recessive gene on the content of lysine and other amino acids from protein. in this paper we present the results of studying the action of o2 mutation on amino acid composition of proteins in tetraploid maize grain compared to the original diploid form. also, the influence of the gene dosage in the endosperm of diploid and tetraploid grains on aminoacid composition is shown. 2. material and methods as biological material were used: diploid single-cross maize hybrids chisiniovschi 307pl with o2 endosperm and porumbeni 331mrf with regular texture of the endosperm, tetraploid synthetic population b (usa) with normal consistency (regular) of endosperm (kindly offered by dr. e. hatefov from kabardino balkarian institute of agriculture, rusia) and tetraploid o2 maize forms obtained by colchicine treatment from hybrid chisiniovschi 307 pl at the state agrarian university of moldova in 2010 [13]. as these o2 tetraploids derived from a hybrid that segregated in the following generations of reproduction, they were grouped in several segregating families (p38-3, p38-5, p39-3, u3-2, u4-12 and u7-11) in order to assess the possibility to select lines for protein quality. field experiments were performed at the moldavian institute of plant breeding "porumbeni" in 2010-2012. study of gene dosage effect was accomplished on material obtained from reciprocal crosses between genotypes with vitreous and o2 endosperms at both ploidy levels. grains for biochemical analysis were obtained by controlled pollination. protein content was determined by the kjeldahl method for the quantitative determination of nitrogen and subsequent conversion to protein by the coefficient 6.25, specific for maize. analysis of protein amino acid content was performed by ion exchange chromatography on an automatic amino acid analyser t339m. 3. results and discussion the product of the o2 gene is a transcriptional activator of zein gene expression. this gene is active in normal grains and inactive in mutant grains, which causes a sudden reduction of zein synthesis and increase content of albumins, globulins and glutelins. as zeins lack lysine, and other fractions are rich in this amino acid, redistribution of protein fractions conditions increased lysine content in the mutant endosperm [14, 15]. at the amino acid level of the protein it was found that o2 mutation also causes a higher content of histidine, arginine, aspartic acid, glycine, cysteine, tryptophan, reduces the content of glutamic acid, alanine, methionine, leucine, tyrosine, phenylalanine, as well as, increases the content of free amino acids [9, 14, 16-18]. in our research, in the study of protein amino acid composition of diploid and tetraploid grains containing o2 gene, it was found that with increasing ploidy level, with some exceptions, there is a tendency to increase the content of lysine, threonine, leucine, glutamic acid, aspartic acid and serine (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 3 table 1 content of amino acids in protein of diploid and tetraploid grains of maize, % amino acids genotype/family/ploidy mean for o2 tetrapl oids porumbeni 331 mrf 2x (+) chisinio vschi 307 pl 2x (o2) sin. b 4x (+) p38-3 4x (o2) p38-5 4x (o2) p39-3 4x (o2) u3-2 4x (o2) u4-12 4x (o2) u7-11 4x (o2) essential amino acids lys 3.02 4.78 3.17 6.08 4.30 5.08 5.18 3.90 4.36 4.82 his 2.71 3.53 3.37 3.82 2.97 3.28 3.63 3.22 3.09 3.34 arg 4.39 6.32 4.85 3.69 3.78 6.73 7.31 4.15 3.33 4.84 thr 2.66 2.76 2.23 3.16 2.85 3.21 3.06 3.09 2.95 3.05 phe 4.22 3.60 3.98 3.39 3.76 3.41 3.44 4.05 2.94 3.50 ile 3.03 2.87 2.51 2.36 2.10 2.16 2.43 2.22 2.09 2.23 leu 12.78 9.66 13.54 10.45 11.91 7.24 10.18 10.10 10.77 10.11 met 0.53 0.47 0.63 0.43 0.56 0.46 0.63 0.23 0.56 0.48 val 4.42 5.03 4.14 4.29 3.54 4.24 4.32 3.59 3.91 3.98 σ 37.75 39.04 38.41 37.67 35.77 35.83 40.19 34.55 34.01 36.34 non-essential amino acids glu 26.92 20.55 22.86 23.56 22.23 24.59 22.71 25.03 24.16 23.72 pro 9.12 8.56 10.95 6.98 7.15 5.83 6.82 9.19 7.81 7.30 ala 6.84 5.32 6.14 5.63 6.02 5.21 5.31 5.73 4.90 5.47 asp 5.58 7.51 5.01 9.17 8.71 10.51 10.36 8.62 9.58 9.49 tyr 2.69 2.69 3.35 3.15 3.19 2.94 3.10 1.94 2.75 2.85 cys 0.89 1.18 1.02 0.84 0.84 1.03 1.10 1.08 1.14 1.01 ser 4.60 4.34 4.54 5.97 5.30 5.16 5.11 5.37 4.73 5.27 gly 3.30 4.69 3.60 4.34 3.45 3.58 3.60 4.08 3.10 3.69 σ 59.95 54.82 57.47 59.65 56.88 58.85 58.11 61.06 58.17 58.79 σ total 97.69 93.86 95.88 97.32 92.65 94.68 98.29 95.60 92.19 95.12 protein, % dry matter 12.90 11.91 13.60 13.81 14.06 13.50 13.74 14.30 14.13 13.92 the content of arginine, isoleucine, valine, proline, cysteine, glycine decreased, at the same time no clear trend was found in the contents of histidine, phenylalanine, methionine, alanine and tyrosine. a relatively high quantity of lysine was revealed in family p38-3 (6.08%) and u3-2 (5.18%). the sum of the essential amino acids of o2 tetraploids tends to be lower than that of diploids and the amount of nonessential higher. however, the results can be influenced both by ploidy level and gene action. in order to broader assess the biochemical effect of the o2 mutation on grain quality in our experiments we analyzed the amino acid composition of the protein from vitreous and o2 grains developed on the same tetraploid ears after self-pollination of heterozygous plants for o2 gene (o2o2/o2o2) which segregated in a phenotypic ratio of 35:1, grains with vitreous and floury endosperm, respectively (table 2). from the data obtained it can be observed that on the average, in tetraploid genome of maize the o2 gene increases the content of lysine, arginine, valine, aspartic acid and glycine in grain protein, while the content of leucine, glutamic acid, proline and tyrosine tends to lower. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 4 table 2 content of amino acids in protein of tetraploid grains with vitreous and o2 endosperm selected from self-pollinated duplex heterozygotes (o2o2o2o2), % amino acid number of segregating ear mean 1 2 3 4 5 + o2 + o2 + o2 + o2 + o2 + o2 essential amino acids lys 3.24 4.63 3.11 5.06 3.98 5.30 3.10 4.45 3.02 5.78 3.29 5.05 his 3.23 3.14 2.91 3.94 3.80 3.65 3.76 3.46 3.35 2.85 3.41 3.41 arg 4.98 5.21 2.98 3.20 3.89 6.85 3.83 5.22 1.45 5.36 3.43 5.17 thr 2.94 3.42 2.90 3.16 3.40 3.55 3.05 3.11 2.97 2.89 3.05 3.22 phe 3.71 3.46 3.50 3.95 3.83 3.20 3.44 3.89 3.78 2.96 3.65 3.49 ile 2.17 2.41 2.19 2.31 2.44 2.21 2.41 2.20 2.12 2.56 2.27 2.34 leu 12.38 9.31 12.68 10.75 12.94 9.11 11.17 9.91 13.26 7.99 12.49 9.42 met 0.30 0.38 0.14 0.47 0.45 0.66 0.44 0.47 0.22 0.45 0.31 0.49 val 3.62 4.70 3.48 3.82 3.58 4.12 3.44 4.20 3.47 4.79 3.52 4.33 σ 36.57 36.66 33.89 36.66 38.31 38.66 34.64 36.90 33.65 35.63 35.41 36.90 non-essential amino acids glu 25.06 26.14 26.11 24.96 26.95 23.82 25.99 24.72 27.75 23.63 26.37 24.66 pro 7.50 4.97 8.30 5.82 3.76 5.45 10.59 6.73 7.63 7.32 7.56 6.06 ala 5.58 5.91 5.42 5.71 4.29 4.92 6.59 5.73 6.11 5.10 5.60 5.48 asp 5.71 10.88 6.17 9.79 7.10 9.98 5.91 8.71 6.40 9.55 6.26 9.78 tyr 3.20 2.61 3.32 3.34 3.52 3.07 2.79 2.84 3.03 3.10 3.17 2.99 cys 0.85 1.24 0.95 0.78 1.31 1.03 1.20 1.27 0.97 1.40 1.06 1.14 ser 5.13 5.65 5.20 5.48 5.51 5.45 5.42 5.58 5.43 4.77 5.34 5.39 gly 2.84 3.52 2.99 4.09 2.33 3.90 3.26 3.91 2.76 3.41 2.83 3.77 σ 55.87 60.93 58.46 59.97 54.78 57.63 61.74 59.48 60.07 58.29 58.18 59.26 σ total 92.44 97.59 92.34 96.63 93.09 96.29 96.38 96.38 93.72 93.92 93.59 96.16 protein, % dry matter 13.85 12.97 15.16 14.44 13.25 13.19 12.97 13.75 14.19 13.93 13.88 13.66 regarding the content of the amino groups (essential and nonessential) one can see that the essential group is higher in o2 grains. the results can be easily influenced by the fact that grains on the segregated ears contain different doses of the o2 gene. it is known that maize endosperm is an important biological tissue resulting from double fertilization and is triploid by nature i.e. made up of two maternal and one paternal genomes. this allows, by directing pollination, to obtain and study grains with different endosperm genotypes and number of doses (1, 2 and 3) of endosperm genes (dosage effects). dosage effect in maize endosperm was first reported by p. c. mangelsdorf and g. s. fraps in 1931 [19], who found a direct relationship between the number of alleles y1 (yellow) and content of provitamin a in grains. in research conducted by l. s. bates [17], related to dosage effect of the o2 gene in diploid maize it was established that lysine content in grain increases proportionally with allele number. the endosperm of tetraploid grains of maize is hexaploid, contains 60 chromosomes (40 maternal and 20 paternal) that is four maternal and two paternal genomes. this allows by reciprocal crosses, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 5 to obtain tetraploid grains with a different number (2, 4 and 6) of alleles of a gene (dominant and recessive). in special literature, at the moment, there is no information about obtaining and biochemical study of different doses of endosperm genes (gene dosage) in tetraploid maize grains. based on the mentioned, one of the objectives of our research was to obtain o2 maize tetraploid forms containing high (almost double) lysine content in protein grain. it was also considered the possibility to obtain tetraploid forms of maize with 4 doses of the o2 gene in endosperm, which would have a normal endosperm (glassy), but with a higher content of lysine in protein than diploid grains homozygous for o2, which could facilitate the implementation of o2 maize in production. in our experiments possible variants of endosperm genotype were obtained, which allowed us to study the influence of gene dose on protein quality at both diploid and tetraploid levels (table 3). table 3 dosage effect of o2 gene in endosperm on protein amino acids of diploid and tetraploid maize grains, % amino acid number of recessive alleles in endosperm 2x 4x ++/+ ++/o2 o2o2/+ o2o2/o2 ++++/ ++ ++++/ o2o2 o2o2o2o2/ ++ o2o2o2o2/ o2o2 essential amino acids lys 3.02 3.63 3.76 4.78 3.17 4.07 3.03 5.18 his 2.71 3.49 3.60 3.53 3.37 3.28 3.39 3.63 arg 4.39 5.68 5.53 6.32 4.85 4.22 5.53 7.31 thr 2.66 2.60 2.54 2.76 2.23 3.14 2.99 3.06 phe 4.22 3.58 3.46 3.60 3.98 4.12 3.90 3.44 ile 3.03 2.79 2.77 2.87 2.51 2.25 2.42 2.43 leu 12.78 12.91 11.81 9.66 13.54 10.27 14.45 10.18 met 0.53 0.44 0.47 0.47 0.63 0.23 0.28 0.63 val 4.42 3.66 4.77 5.03 4.14 3.65 3.70 4.32 σ 37.75 38.78 39.17 39.04 38.41 35.24 39.69 40.19 non-essential amino acids glu 26.92 22.31 23.56 20.55 22.86 25.46 25.80 22.71 pro 9.12 11.83 8.92 8.56 10.95 9.35 7.58 6.82 ala 6.84 6.18 6.28 5.32 6.14 5.83 6.13 5.31 asp 5.58 5.66 5.31 7.51 5.01 8.77 5.76 10.36 tyr 2.69 3.05 3.13 2.69 3.35 1.98 3.50 3.10 cys 0.89 1.24 1.53 1.18 1.02 1.10 0.93 1.10 ser 4.60 4.74 4.93 4.34 4.54 5.46 5.38 5.11 gly 3.30 3.94 4.19 4.69 3.60 4.15 2.69 3.60 σ 59.95 58.96 57.85 54.82 57.47 62.10 57.76 58.11 σ total 97.69 97.73 97.02 93.86 95.88 97.34 97.46 98.29 protein. % dry matter 12.90 12.25 13.19 11.91 13.60 14.06 12.75 13.74 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 6 as a result of the study we have found that the content of lysine increased proportionately with dose number in endosperm of diploid grains, which confirmed the research carried out by l. s. bates [17], but in the case of tetraploid grains data obtained are inconsistent. the presence of two recessive alleles of o2 gene in hexaploid endosperm determined an increase in the content of lysine in protein (4.07%) compared to the dominant homozygote genotype (3.17%). for reasons unclear it is difficult to explain lysine content reduction in protein in the presence of 4 doses in endosperm (3.03%). at the same time, in the case of maximum number of doses (six o2 recessive alleles), the content of lysine exceeded all other variants of gene dosage of diploid and tetraploid levels (5.18%). therefore, with the exception of the variant with four doses, the phenomenon of gene dosage effect of the o2 gene has been found, at both diploid and tetraploid levels. it is, also, to be mentioned the pleiotropic action of the mutation on the content of other amino acids. thus, a similar pattern as lysine was found for arginine at both diploid and tetraploid levels, due, perhaps, to their biochemical interdependence, but leucine showed a negative trend. other amino acids manifested an unclear tendency. uncertainties related to these data can easily be influenced by differences between genotypes involved in crosses, especially the effects of reciprocal crosses (maternal influence). in order to exclude this, similar genotypes and isogenic lines are needed. preventive information with respect to the positive influence of the number of doses of o2 gene on lysine content in tetraploid maize grains proves the need to extend the research in this field. the results allow us to admit the possibility to select tetraploid genotypes with a higher content of lysine in grain protein than diploid grains and obtaining of forms heterozygote for o2, with vitreous endosperm and high protein quality. 4. conclusions tetraploid maize forms, that contain the o2 gene, exhibit considerable variability on proteinogenic amino acid content in grains. lysine content in protein varied from 3.90 to 6.08 %. this allows performing selection for desirable quality characteristics of grain protein at tetraploid level. the amino acid analysis of the protein composition of tetraploid grains with vitreous and o2 endosperm obtained from the same heterozygote self-pollinated ears showed that the o2 gene in tetraploid genome increases the content of lysine, arginine, valine, aspartic acid and glycine and reduces the content of leucine, glutamic acid, proline and tyrosine, discriminating gene effect from ploidy level. in a dosage effect study of the o2 gene it was found that the content of lysine increases proportionally with dose number in the diploid genome and, partly, in tetraploid. as we used different genotypes in the investigation, there could be differences in results due to maternal cytoplasm involved in crosses, so to further understand and confirm this expression pattern of o2 gene in tetraploid maize, a different series of genotypes and more sophisticated biochemical analyses are required. 5. references [1] randolph l. f.. some effects of high temperature on polyploidy and other variations in maize. proc natl acad sci. 18. p.222-229. (1932). [2] ellis g. h.. randolph l. f.. matrone g.. a comparison of the chemical composition of diploid and tetraploid corn. j agr res. 72 (3). p.123130. (1946). [3] freeman j. e.. yahl k. r.. watson s. a.. a chemical and structural analysis of the caryopsis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 andrei palii, grigorii batîru, amino-acid content in grain protein of tetraploid opaque-2 maize, food and environment safety, volume xiii, issue 1 2014, pag. 1 7 7 of an autotetraploid corn (zea mays l.). crop sci. 7. p.655-658. (1967). [4] randolph l. f.. hand d. b.. relation between carotinoid content and number of genes per cell in diploid and tetraploid corn. j agr res. 60. p.51-64. (1940). [5] kovarskii a. e.. obersht v. m.. chalyk t. s.. experience in maize breeding at tetraploid level in moldova. izv akad nauk mold ssr ser biol khim nauk. 4. 1970. p.84-85. (1970) (in russian). [6] rotar a. i.. obersht v. m.. chalyk t. s.. pashkar s. i.. biochemical and morphological characteristics of pollen and seeds in tetraploid maize developed experimentally. cytol genet. acad sci ussr. 1 (4). p.15-23. (1970) (in russian). [7] hadzinov m. i.. shcherbak v. s.. polyploidy in maize. theoretical and practical problems of polyploidy. moscow: nauka. p. 27-42. (1974) (in russian). [8] palii a.. genetic aspects of improving the quality of maize grain. chishinau: shtiinta. 175p. (1989) (in russian). [9] mertz e. t.. bates l. s.. nelson o. e.. mutant gene that changes protein composition and increases lysine content of maize endosperm. science. 145. p.279-280. (1964). [10] mertz e. t.. veron o. a.. bates l. s.. nelson o. e.. growth of rats fed on opaque-2 maze. science. 148. p.1741-1742. (1965). [11] pickett r. a.. opaque-2 corn in swine nutrition. proc. high lysine corn. conf.. purdue univ.. washington. 1966. p.19-22. (1966). [12] hadzinov m. i.. budnaja m. v.. potehin s. a.. zima k. i.. corn opaque-2 in the diets of weanlings pigs. dokl vses akad s-kh nauk im v. i. lenina. 4. p.27-31. (1970) (in russian). [13] palii a.. batiru gr.. experimental production of tetraploid opaque-2 maize. in: improving maize and using cytoplasmic male sterility in seed production. internat. conf. dedicated to tikhon chalyk. 90 years from birth. chishinau. p.88-97. (2011) (in romanian). [14] plotnikov v. k.. on the 40th anniversary of the discovery of the biochemical actions of opaque-2 mutations in the high-lysine corn. the amino acid nutrition of animals and the problem of protein resources. cuban. state agr. univ. krasnodar. p.257-312. (2005) (in russian). [15] plotnikov v. k.. retrospective and contemporary look at the molecular basics of creating the high-lysine corn. genetics. breeding and cultivation technology of maize. p.134-138. (2009) (in russian). [16] wang x.. larkins b. a.. genetic analysis of aminoacid accumulation in opaque-2 maize endosperm. plant physiol. 145. p.1766-1777. (2001). [17] bates l. s.. amino acid analysis. proc. high lysine corn. conf.. purdue univ.. washington. p.55-65. (1966). [18] vynnychenko a. n.. shtemenko n. i.. features of amino acid exchange of the highlysine corn. biol. science. 2. p.98-112. (1990) (in russian). [19] egesel c. o.. wong j. c.. lambert r. j.. rocheford t. r.. gene dosage effects on carotenoid concentration in maize grain. maydica. 48. p.183-190. (2003). 279 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 279 286 traditional uses of wild berries in the bukovina region (romania) *ana leahu 1 , cristina elena hretcanu 1 , alice iuliana roșu 1 , cristina ghinea 1 1faculty of food engineering, stefan cel mare university of suceava, 13 university str., 720229 suceava, romania, analeahu@gmail.com *corresponding author received 5th october 2019, accepted 17th december 2019 abstract: in recent years there has been given increased attention to the health-promoting dietary recommendations as regards the use of wild berries in human nutrition. this study analyzes the differences in the consumption of wild berries in the bukovina area (in the northern part of romania). bilberries (vaccinium myrtillus l.), sea-buckthorn (hippophae rhamnoides l), rosehip (rosa canina l.) and hawthorn (crataegus monogyna l.) are the plants harvested from spontaneous flora, with diverse uses, such as preparation of juices, tinctures and decoctions or tea beverages due to their medicinal and nutritional potential. wild berries are also processed for production of beverages, jams, as functional food products, and, to some extent, they are consumed fresh as well. the consumers’ preferences were established by the analysis of questionnaires of 101 respondents interviewed concerning different botanicals used in a particular geographical and cultural context. the results show that the selections of wild berries containing many biochemical compounds are mostly used as teas or decoctions due to their nutritional role and health benefits. the preference for sea-buckthorn (hippophae rhamnoides l) was strongly associated with its dietary benefits for the immune system. the consumption of wild berries is considered important by the romanians living in bukovina in fresh form, as beverages or in the dried form. key words: consumers’ preferences, wild berries, bukovina, romania. 1. introduction bukovina has a population of over 630 thousand people, the majority living in the 97 villages spread throughout the mountainous area. the use of traditional medicines made from wild berries fruit to prevent diseases has been present in bukovina since immemorial times and the villagers have made a craft of such practices. thus, over the last decades, the interest in harvesting wild berries has increased considerably in many mountain regions of romania. sea-buckthorn (hippophae rhamnoides l), hawthorn (crataegus monogyna l.) and rosehip (rosa canina l.), collected in the wild constitute a good source of natural antioxidant substances and the consumption of fruits plays an important role in the maintenance of health and in disease prevention [1]. wild berries represent a rich natural resource in bukovina, but their collection and capitalization have not been developed enough in the region. consumers’ preferences (color, taste, flavor, aroma, texture) and information on health benefits may also be important in increasing the interest in wild berry picking as an activity. the fruits of sea-buckthorn are the most favored fruits of edible wild plants, and probably the most frequently used ones http://www.fia.usv.ro/fiajournal mailto:analeahu@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 280 today. in romania, local people used sea buckthorn berry to treat urticaria, burns and other skin wounds, gastric and duodenal ulcers, liver diseases, anemia, atherosclerosis and cardiovascular disorders, fatigue and even stress. sea buckthorn pulp oil treatment prevents atherosclerosis by lowering triglyceridemia, cholesterolemia and blood pressure (strong effects) and by reducing oxidative stress, inflammation and insulin resistance (weak effects) [2]. the fruit of sea buckthorn (hippophae rhamnoides l) contains proteins, lipids, carbohydrates, vitamins (c and e), phenols, carotenoids, flavonoids, minerals, and volatile compounds and has various health benefits. the author of the study also reported that sea buckthorn berry cultivars (ssp. carpatica) exhibited higher oil content than other european or asiatic sea buckthorn subspecies [3]. moreover, the pulp/peel oils of ssp. carpatica were found to contain high levels of oleic acid and slightly lower amounts of linoleic and α-linolenic acids [3]. in another study, the authors evaluated the content of berries and leaves of six varieties of carpathian sea buckthorn (hippophae rhamnoides l., ssp. carpatica) in terms of their carotenoid composition (free and esterified, (pop et al. 2014). total carotenoid content varied between 53 and 97 mg/100g dry weight in berries [4]. volatile compounds of sea buckthorn are responsible for specific flavors and bacteriostatic action. in a study made by socaci et al. 2013, the volatile compounds, in the headspace of sea buckthorn samples originating from the carpathians, romania, were separated. the most abundant derivatives were ethyl esters of 2-methylbutanoic acid, 3methylbutanoic acid, hexanoic acid, octanoic acid and butanoic acid, as well as 3-methylbutyl 3-methylbutanoate, 3methylbutyl 2-methylbutanoate and benzoic acid ethyl ester [5]. the fruit of blueberries (vaccinium myrtillus l.) are eaten fresh, used in the preparation of tea, juice, as a remedy for gastric and duodenal ulcers, some types of cancer, being also recommended in the prevention of coronary diseases. blueberry peels may be used in pharmaceutical products, due to their potential beneficial effects on human health; they have been reported to improve neuronal and cognitive brain functions and ocular health. on the other hand, recent studies indicated that the high contents of polyphenols and acid ascorbic in blueberries can play a remarkable role in the northern european diet, especially when redox homeostasis is imbalanced [6]. blueberry low-sugar jams and juices are recommended for diabetics and people with specific health problems [7]. the fleshy part of the rosehip fruit is used in the pharmaceutical industry, as health supplements and for the preparation of herbal tea, due to its therapeutically properties: choleretic, diuretic, vitamin provider, antioxidant, astringent, antiinflammatory and antidiabetic. it is also used in food industry in juices, as ingredients in pro-biotic drinks, yoghurt, jellies, jams and alcoholic beverages etc. various studies over the last decades have shown that the consumption of rose hip fruits may reduce the risk of many diseases, including, gastrointestinal, osteoarthritis, rheumatoid arthritis, inflammation, renal, gynecological, lung and nerve diseases [8, 9 and 10]. clinical and experimental investigations show that hawthorn preparations can be safely used for the treatment of hypertension, it prevents ischemia and generally slows the heart rate, it also prevents chronic degenerative diseases especially atherosclerosis and cancer [11]. nowadays, however, the consumption of wild berries is determined by the pleasure of gathering wild resources, recreating traditional practices and enjoying the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 281 characteristic flavors [12]. some authors studied the consumer perceptions of 3 formulations of frozen dessert bars made from both soy and wild blueberries [13]. the formulations tested in this study have got low acceptability, (chocolate and vanilla) flavor being mentioned as an essential reason when choosing frozen soy products [13]. most studies on wild edible berries focus on nutrient contents in terms of macronutrients, minerals, and vitamins [1, 8, 9 and 10]. size and texture are the ones of the most important quality parameters of wild berries strictly related to consumer perception [14]. other studies have attempted to optimize pomegranate juice with blueberry, raspberry, or blackberry added in juices, to increase acceptance and consumption. pomegranate/blueberry juice with 20% and 50% of blueberry juices have shown a higher acceptance for consumers, being the best option for the consumers interested in health benefits [15]. documentation on the use of wild berries in treating some diseases is important for traditional medicinal practices and has contributed to the development of new dietary supplements. therefore, the objective of the present work is to provide information on the consumption and to study the use of traditional medicines made from the wild berries fruit registered in bukovina (romania). these data would be useful in rendering the wild berries’ picking more important and providing nutritional, economic and cultural benefits. 2. materials and methods methods 101 students were recruited from the faculty of food engineering of the suceava university, situated in bukovina region (fig. 1), located in the northeastern part of romania. students were interviewed based on questionnaires about their consumption way and health benefits regarding wild berries used in traditional contexts. the respondents were explained the purpose of the questionnaire and they were asked to give their consent for collected data dissemination. fig. 1 geographical location of the study area food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 282 wild berries preference checklists four wild berry fruits were taken into consideration in order to establish a preference checklist. statistical analysis data sets were evaluated using descriptive statistics for average ± standard deviation. a principal component analysis (pca) was performed with xlstat™ (version 2017, by addinsoft, u.s.a.). consumers test research was conducted in the faculty of food engineering, stefan cel mare university of suceava, romania. a total of 101 of wild berries consumers recruited from the students who participated in the test (15th – 25th june 2018). the gender of participants being 78% female and 22% male, and their ages ranged from 19 to 24 years old. the mean age, sex and bmi (body mass index) of respondents who answered the questionnaire are given in the table 1. table 1. the structure of respondents who answered the questionnaire category sex percent male female mean ± sd of age 20.32±1.52 21.15±3.86 mean ± sd of bmi 23.71±2.67 21.82±3.23 underweight (bmi<18.5) 9 8.91% normal range (18.5 bmi<25) 18 58 75.25% overweight (25 bmi<30) 4 12 15.84% 3. results and discussion the comparison between the variations of cultures and factors involved in the wild berries choice may have important implications. the answers to the 1st question (are you consumer of any specific berries (cranberries, sea buckthorn, rosehip, hawthorn)? if yes, how often do you eat these wild berries?) are shown in fig. 2 . from the analysis of fig. 2, we can observe that cranberries are eaten by 47.53% of respondents in season (june august) and occasionally (less than weekly) by 34.65% of respondents; sea buckthorn is not eaten by 52.47% of respondents and it is eaten occasionally by 31.68% of respondents; rosehip is not eaten by 63.36% of respondents and it is eaten occasionally by 29.7% of respondents; hawthorn is not eaten by 90.1% of respondents. the fig. 3 displays the answers to the 2nd question (what makes you choose a certain kind of wild berries? (with the variants: (a) because they are healthy; (b) i like the taste; (c) as treatment for various conditions; (d) something else, please specify). the respondents can mark more than one of the variants (a) to (d) for every wild berries. we can remark that the majority of respondents (50.49%) eat wild berries because they like taste and 37.62% eat them because they are healthy. fig. 2 how often do you eat these specified berries (cranberries, sea-buckthorn, rosehip, hawthorn)? a = i do not eat; b= occasionally (less than weekly); c = eat only in season (june august); d = eat several times a week food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 283 fig. 3 motivation to eat a certain kind of wild berries (cranberries, sea buckthorn, rosehip and hawthorn) a = because they are healthy; b = i like the taste; c=as treatment for various conditions; d = other else, please specify fig. 4 the appropriate quality of wild berries which is appreciated by respondents (a) healthy, (b) tasty, (c) sweet, (d) bitter, (e) sour, (f) sophisticated, (g) intense color the answers to the 3rd question (please specify the quality which makes you eat this kind of wild berries) can be viewed in the fig. 4. the respondents can mark more than one of the variants (a) to (h) for every wild berry. we can remark that wild berries would be eaten due to their healthy properties: 73.26% of respondents indicate cranberries, 66.33% sea buckthorn, 48.51% rosehip and 32.67% hawthorn. also, cranberries would be eaten for taste (71.28%) and intense color (64.35%). the answers to the 4th question indicate the acceptability of wild berries on a hedonic scale ranged from 1 to 5 points, where 1 corresponds to the response i dislike it very much and 5 corresponds to i like it very much. the pca (principle component analysis) for scores of sensorial attributes (taste, odor, color and flavor) are shown in the fig. 5. the total variances are given by the first two principal components (f1 and f2) for wild berries (such as cranberries, sea buckthorn, rosehip and hawthorn, respectively). regarding the taste of wild berries (fig. 5 (a)), the first principal component (f1) provides the greatest amount of information, about 49.7%, and the second one (f2) contributes by 22.55% of the data distribution. for the odor of wild berries (fig. 5 (b)), the first principal component (f1) provides the greatest amount of information, about 53.23%, and the second one (f2) contributes by 24.08% of the data distribution. for the color of wild berries (fig. 5 (c)), the first principal component (f1) provides the greatest amount of information, about 53.66%, and the second one (f2) contributes by 22.28% of the data distribution. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 284 regarding the flavor of wild berries (fig. 5 (d)), the first principal component (f1) provides the greatest amount of information, about 50.52%, and the second (f2) contributes by 23.46% of the data distribution. moreover, we can remark that sea buckthorn and cranberries scores given for their color are very similar (having 0.525 correlation coefficient from the correlation matrix (pearson (n)), different from 0, with a significance level α=0.05) and also, the taste of hawthorn and rosehip have similar scores (having 0.513 correlation coefficient from the correlation matrix (pearson (n)), different from 0, with a significance level α=0.05). fig. 5 the pca for scores of sensorial attributes: (a) taste, (b) odor, (c) color and (d) flavor the answers to the 5th question (please fill in the table below, by ticking x in the cell corresponding to the quality of wild berries from your own point of view) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 285 indicate the way of consumption of wild berries (fig. 6). from fig. 6 we remark that cranberries are consumed as fresh fruit by most respondents (73.26%) whereas sea buckthorn (40.59%), rosehip (40.59%) and hawthorn (27.72%) are consumed, in general, as tea. recently, the results of research data on the use of wild food plants used in manyas region (turkey) showed that the plants used are eaten raw [16]. traditional wild fruit consumption can be enhanced by incorporating them into contemporary diets, being rich in soluble and insoluble fiber, ash, na, ca, and total vitamin c [17]. fig. 6 the way of consumption of wild berries: a = consumed as fresh fruit, b = consumed as jam, c = consumed as tea, d = added to yoghurt, e = added to ice cream, f = consumed as fruit juice, g = in other foods 4. conclusion in this paper we have studied some aspects concerning the use of wild berries (cranberries, seabuckthorn, rosehip, and hawthorn) on the territory of bukovina, emphasizing the significance of wild berries in the lives of local people in this region. we suggest that wild berries should be used as ingredients in new healthier diets because they are highly available and accessible. these data are important in demonstrating differing reasons when it comes to berries’ choices, still wild berries are not being consumed so frequently, this fact being explained by the low interest in or knowledge about their health benefits. this documentation makes bukovina become an important area of exploitation of wild berries fruits and creates perspectives for further research on the use of wild berries in food and non-food applications (such as the pharmaceutical ones). 5. references [1]. leahu, a., hretcanu, c. e., oroian, m.,ropciuc, s.: evaluation of ascorbic acid and phenolic content of four traditional romanian medicinal berry species, food and environment safety, 15: 123-130, (2016). [2]. virgolici b, lixandru d, casariu e d, stancu m, greabu m et al,: sea buckthorn pulp oil treatment prevents atherosclerosis in obese children, isrn oxidative medicine, 1-9, (2013). [3]. dulf, f. v.: fatty acids in berry lipids of six sea buckthorn (hippophae rhamnoides l., subspecies carpatica) cultivars grown in romania, chemistry central journal, 6: 1-12, (2012). [4]. pop, r. m., weesepoel, y., socaciu, c., pintea, a., vincken, j. p. et al,: carotenoid composition of berries and leaves from six romanian sea buckthorn (hippophae rhamnoides l.) varieties, food chemistry, 147 1-9, (2014). [5]. socaci, s. a., socaciu, c., tofană, m., raţi, i. v., pintea, a.: in‐tube extraction and gc–ms analysis of volatile components from wild and cultivated sea buckthorn (hippophae rhamnoides l. ssp. carpatica) berry varieties and juice, phytochemical analysis, 24: 319-328, (2013). [6]. kleiner, d., kurucz, d., bersényi, a., szentmihályi, k., skesters, a., et al.: berries, the antioxidant sources of the boreal cold and arid regions, acta alimentaria, 1-6, (2015). [7]. poiana, m. a., alexa, e., mateescu, c.: tracking antioxidant properties and color changes in low-sugar bilberry jam as effect of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 ana leahu, cristina elena hretcanu, alice iuliana roșu, cristina ghinea, traditional uses of wild berries in the bukovina region (romania), food and environment safety, volume xviii, issue 4 – 2019, pag. 279 – 286 286 processing, storage and pectin concentration, chemistry central journal, 6, (2012). [8]. andersson, s. c., rumpunen, k., johansson, e., olsson, m. e.: carotenoid content and composition in rose hips (rosa spp.) during ripening, determination of suitable maturity marker and implications for health promoting food products, food chemistry 128: 689-696 (2011). [9]. patel, s.: rose hips as complementary and alternative medicine: overview of the present status and prospects, mediterranean journal of nutrition and metabolism, 6: 89-97, (2013). [10]. ropciuc, s., cretescu, i., leahu, a., velicevici, v.: biometric and chemical characteristics of the species rosa canina l. used as a natural and functional food, 48. hrvatskii 8. međunarodni simpozij agronoma, dubrovnik, hrvatska, 425-429, (2013). [11]. froehlicher, t., hennebelle, t., martin-nizard, f., cleenewerck, p., hilbert, j. l., et al,: phenolic profiles and antioxidative effects of hawthorn cell suspensions, fresh fruits, and medicinal dried parts, food chemistry 115: 897-903, (2009). [12]. pardo-de-santayana, m., tardío, j., blanco, e., carvalho, a. m., lastra, j. j. et al,: traditional knowledge of wild edible plants used in the northwest of the iberian peninsula (spain and portugal): a comparative study, journal ethnobiology and ethnomedicine, 3, 1, (2007). [13]. teh, t., dougherty, m. p., camire, m. e.: how do consumer attitudes influence acceptance of a novel wild blueberry–soy product, journal of food science, 72: s516-s521, (2007). [14]. donahue, d. w., work, t. m.: sensory and textural evaluation of maine wild blueberries for the fresh pack market, journal of texture studies, 29: 305-312, (1998).[15]. vázquez araújo, l., chambers, iv., adhikari, e. k., carbonell barrachina, á. a.: sensory and physicochemical characterization of juices made with pomegranate and blueberries, blackberries, or raspberries, journal of food science, 75: s398-s404, (2010). [16]. kayabaş, n. p., tümen, g., polat, r.: wild edible plants and their traditional use in the human nutrition in manyas (turkey), indian j traditional knowledge, 17: 299-306, (2018). [17]. ruiz-rodríguez, b. m., morales, p., fernández-ruiz, v., sánchez-mata, m. c., camara, m. et al,: valorization of wild strawberry-tree fruits (arbutus unedo l.) through nutritional assessment and natural production data, food research international 44: 1244-1253 (2011). research was conducted in the faculty of food engineering, stefan cel mare university of suceava, romania. a total of 101 of wild berries consumers recruited from the students who participated in the test (15th – 25th june 2018). the gender of partici... table 1. the structure of respondents who answered the questionnaire 3. results and discussion [9]. patel, s.: rose hips as complementary and alternative medicine: overview of the present status and prospects, mediterranean journal of nutrition and metabolism, 6: 89-97, (2013). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 contents: 1. modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction linda loucif seiad, soraya demim 180 191 2. effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage kitisart kraboun, putkrong phanumong, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, ithinath tantivitittapong, pornrak chowvanayotin 192 198 3. impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio 199 209 4. analysis for the determining factors of the evolution of hepatitis a viral in suceava county, romania, in the period 2018 2019 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan 210 218 5. occurrence and assessment of physical contaminants based on food recalls in canada mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid 219 227 6. physicochemical and color evaluation of confectionery mousses raluca olimpia zimbru, sergiu pădureţ, sonia amariei 228 236 7. physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review vasile-florin ursachi, gheorghe gutt 237 259 8. author instructions i v 9. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 звіт з ндр 29-81 за 2007 – 2009 р 81 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1 2018, pag. 81 86 basalt tufa as a bactericide filler for some packaging materials *igor kobasa 1 , mariya vorobets 1 , larysa аrsenieva2 1yu. fedkovych national university of chernivtsy, ukraine, i.kobasa@chnu.edu.ua 2national university of food technologies, kyiv, ukraine * corresponding author received 27th november 2017, accepted 19th march 2018 abstract: a dependence of the catalytic and antibacterial activity of some basalt tufa samples on their qualitative and quantitative composition, preliminary thermal and/or chemical treatment conditions were investigated. as seen from the chemical x-ray phase and atom adsorption analyses, the tufa is a highly siliceous (si/al mass ratio is about 4.7÷5.9) zeolite-like mineral with comparatively high content of iron (68÷74 g/kg), some bioelements (мg, ca, к, na) and microelements (ti, mn, zn, сu). the positive (≡si+)s and negative (≡siо -)s surface active centers were determined by ir spectroscopy. these points ensure high catalytic and bactericide activity of the material. the influence of preliminary thermal and chemical treatment of basalt tufa on its bactericide activity against pathogenic staphylococcus aureus and escherichia coli was also investigated. possible utilization of natural basalt tufa as an antibacterial filler for the packaging materials is discussed. keywords: basalt tufa, catalytic and antibacterial activity, packaging materials, staphylococcus aureus, escherichia coli. 1. introduction quality of packaging materials is an important factor influencing consumer acceptance, competitive potential, storage time and safe transportation of various goods. as a result, the modern packaging materials industry shows strong extension, new technologies are developing and new materials are coming to the market. construction of the packaging materials consisting of some bioactive, bactericide agents, enzymes and genuine films is one of promising directions in this branch. such materials can effectively preserve biologically and nutritionally valuable components and keep high overall quality of the foodstuff. the potential usability of the composites based on the natural alumosilicate basalt tufa (bt) in construction of such bactericide materials is comparatively high. bt can be obtained in the form of magmatic rock (volcanic glass, basalt, slag) or as a mineral (plagioclase, pyroxene) [1]. bt tufa is one of the waste materials formed massively at modern industrial basalt production. many efforts are being made to elaborate effective solutions for its utilization. depending on the chemical composition, bt can be used as highly effective pigments [2], sorbents for drinking water and wastewaters treatment [1], as a substrate for the toxic waste and exhaust gas decontamination [3] and so on. since this mineral is easily available and ecologically safe, it can potentially be proposed as a http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor kobasa, mariya vorobets, larysa аrsenieva, basalt tufa as a bactericide filler for some packaging materials, volume xvi, issue 4 – 2017, pag. 287 – 292 82 filler for the composite antibacterial materials. it is known that the adsorption, antibacterial and catalytic parameters of bt can vary depending on its chemical composition, structure, porosity, dispersability and preliminary thermal and/or chemical treatment [1, 4]. therefore, it is possible to govern the catalytic and antibacterial activity of bt by changes in the above characteristics that can be through thermal or chemical modification of the material. this way, either highly active catalysts and bactericide compositions or low active passive materials can be obtained. both solutions seem promising in the context of construction and development the mineral fillers for modern packaging. this paper deals with the investigation of dependence of catalytic and bactericide characteristics of bt on its qualitative and quantitative composition and conditions of its thermal and chemical modification. a possibility of developing bactericide packaging filler using various modified and treated bt samples is also discussed. 2. experimental the tufa samples obtained from polytske-2 (ukraine) deposit (see chemical composition data in table 1) and products of their thermotreatment at 250–1000 0c or chemical treatment with h2so4, hcl, hno3, h3po4 were used in this experimental series. chemical composition of the samples was determined by classical chemical methods (for si, al, fe, mg, ca, р and s) and aas (for na, к, zn, ni, cu, co) at kac-120 m1 spectrometer in the acetylene/air flame [5]. the powder surface was analyzed with xray photoelectron spectroscopy (xps) also referred to as electron spectroscopy for chemical analysis (esca) using escala™ xi+ x-ray photoelectron spectrometer microprobe. xps is an elemental analysis technique, which is capable of detecting all elements except for h and he and has a nominal detection limit of ~0.1 atom %. spectral interferences may prohibit the detection of some elements in relatively low concentrations. samples were measured at a 90° take-off-angle yielding a sampling depth of ~10 nm. the analysis area was ~500 µm in diameter. analyses were performed with a monochromatic al kα x-ray source. the powder particle size analysis was performed with a field emission scanning electron microscopy (fesem) using hitachi su70 electron microscope. fesem images depict topographic features of the sample surface. fesem imaging was performed at 2 kev. one hundred particles of each powder were measured to provide an average particle size. both powders were coated with ~100 å of gold to facilitate analysis [6]. surface area of the samples has been determined using bet method by the lowtemperature argon adsorption while all ir spectra were recorded with avatar 320 ft1r spectrophotometer. antibacterial activity of the samples was investigated using the diffusion method (also known as the disks method) [7]. the bacterial colonies were cultivated from the atcc standard strains escherichia coli and staphylococcus aureus, while a paper disk impregnated with antibiotic novobiocin was used as a control experiment. 3. results and discussion following components were identified in natural tufa samples: zeolites (35–40 %), montmorillonite (30–40 %), feldspars (10– 15 %), silica materials (4–5 %) and hematites (3–5 %) [1]. averaged data of the tufa composition are shown in table 1 as mass percents of the corresponding oxides. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor kobasa, mariya vorobets, larysa аrsenieva, basalt tufa as a bactericide filler for some packaging materials, volume xvi, issue 4 – 2017, pag. 287 – 292 83 table 1. chemical composition of the basalt tufa from polytske-2 deposit sio2 тіо2 al2o3 fe2o3 мnо mgo cao na2o k2o p2o5 so3 67.44 1.75 12.82 10.14 0.09 5.02 0.46 0.94 1.06 0.12 0.11 the data in table 1 prove that bt is in fact an alumosilicate having the mass ratio si/al=4.7÷5.9, which also contains 68÷74 g/kg of fe. besides that, the mineral also consists of detectable amounts of microelements mn, zn, cu, ni, co ranged from 0.71 to 0.08 g/kg [1]. it was also found that many physicochemical parameters of the tufa, which are responsible for its adsorption, catalytic and antibacterial properties, can be sufficiently altered after its thermal treatment. in this context, an influence of the treatment temperature of the tufa grains porosity and specific surface area was carried out for the four hours long modification at 105, 250, 400, 500, 750 and 1000 °с in air. figure 1. influence of the modification temperature on the bt’s specific surface area and porosity as seen from fig. 1, the area of specific surface and porosity of the grains sized from 1 to 2 mm depend on the thermotreatment conditions. both dependencies look similarly – reach their maximum values for 200–400 0c and then decrease for the higher modification temperatures. such a pattern may be caused by losses of the hygroscopic and zeolite’s water that occurs at this temperature [8]. adsorption capacity of the tufa also increases for the temperatures 105–400 0с followed by the decrease for the higher temperature values. pores, surface holes and channels can be seen in the electron microscope images taken with zoom x10000 (see fig. 2). porosity of the grains is rising for temperatures 250–400 0c and then drops at further increase of temperature. this effect is caused by sintering of the powdery particles ended by melting of the mineral at 1050 0c. mechanic strength of a material is one of important parameters outlining its possible utilization as a catalyst or bactericide agent. this parameter is linearly growing with the thermal modification temperature. а в с d fig. 2. electronic microscope images of the bt disperse particles during the thermomodification process: а – natural source material; b–d – after thermomodification at 250 0с, 400 0с and 1000 0с respectively. no evidence of the new phases formation was found in the x-ray structure analysis data embracing the range 105–1000 0c. therefore, a rise in the mechanical strength can be caused by a deeper structuring of the granules because of the constitution water losses. an acidic modification of the tufa samples was performed using the 3 mole/l solutions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor kobasa, mariya vorobets, larysa аrsenieva, basalt tufa as a bactericide filler for some packaging materials, volume xvi, issue 4 – 2017, pag. 287 – 292 84 of h2so4, hcl, hno3 and h3po4 during 3 hours at the room temperature for the ratio solid phase/solution 1:1.5. following conclusions can be drawn from analysis of the acidic modification results shown in table 2.  the acids can be placed in the sequence нсl > h2so4 > н3ро4 > hno3 according to their activity in elution of calcium;  similar sequence based on the magnesium elution efficiency would be h2so4 > нс1 > н3ро4 > hno3;  total amount of eluted aluminum and iron in the form of their oxides m2o3 decreases in the row h2so4 > нс1 > н3ро4 > hno3 [9]. as seen from table 2, h2so4 and hcl are the most effective acidic modifiers of the bt materials. table 2. concentrations of са 2+ , mg 2+ and (al 3+ + fe 3+ ) found in the acidic extracts (c) and their elution degrees (α) modifying acid са 2+ mg 2+ al 3+ + fe 3+ as me2о3 с (g/l) α, % с (g/l) α, % с (g/l) α, % h2so4 0.3360 68.08 0.1345 0.97 4.9280 14.30 hcl 0.5605 93.57 0.0895 1.97 4.3900 12.74 н3ро4 0.4485 90.88 0.0560 1.23 4.5280 13.14 hno3 0.0745 15.09 0.0220 0.49 2.4680 7.16 the acidic-soluble phases are being eluted from the surface of bt in course of its acidic modification, which results changes in the chemical composition, increase of the tufa’s defect ratio, specific surface area and concentration of the active centers. irspectroscopy data prove that the bronstedtype (≡si–oh) and lewis-type (≡si....o(h+)н)surface singular points act like catalytic and antibacterial centers of bt [1]. these conclusions were alternatively verified by chemical analysis, which evidenced rise in sio2 content after acidic modification of the tufa samples. a comparative study of catalytic activity (ca) of the source and modified tufa was realized using a model reaction of hydrogen peroxide decomposition. an experimental mixture contained н2о2 – naoh – н2о (рн=10) and bt powder (weight ratio was 1:20) at 20 0с. permanganate titration was used to determine concentration of peroxide. as a result, ca of the thermally modified bt was found 1.6–3 times higher than that for the natural untreated material. catalytic activity of tufa depends on the thermotreatment temperature and increases for the sequence: untreated tufa < modification at 250 0c < 400 0с < 1000 0с. this result proves that chemical composition of the tufa surface that changes in course of the modification is the crucial factor governing its catalytic activity. as mentioned above, adsorption and catalytic activity of natural alumosilicates can sufficiently influence their adsorption and catalytic properties. this kind of treatment causes changes in qualitative and quantitative composition of the material and generates new surface active centres [10]. detailed results of the catalytic activity values are shown in fig. 3 for various modification conditions and concentrations of h2o2. it has been found that ca of the chemically modified samples increases for 1.2–4.5 times if the ph of the medium is alkaline comparing to the activity values of untreated tufa. besides, the type of the modifying agent also influences the tufa activity and the following sequence can be built according to increase of the agents’ efficienfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor kobasa, mariya vorobets, larysa аrsenieva, basalt tufa as a bactericide filler for some packaging materials, volume xvi, issue 4 – 2017, pag. 287 – 292 85 cy: untreated tufe < modified by naoh < hno3 < h3po4 < h2so4 < hcl. next stage of the investigation was aimed onto determination of antibacterial activity of various tufa samples. it was found that antibacterial activity of both thermally and chemically modified examples against escherichia coli is higher than that against staphylococcus aureus (see table 3). further elaboration of practical aspects related to possible utilization of these materials as antibacterial components leads to the concept of the tufa-containing packaging materials that should be developed, constructed and then compared with other packaging materials exhibiting or not exhibiting some antibacterial properties. figure 3. catalytic activity of the chemically modified tufa samples in the system н2о2 – naoh – н2о с0(н2о2) = 0.05 (a); 0.1 (b); 0.2 (c); 0.4 (d); 0.8 (e); 1.6 (f) м/l: 1 – untreated bt; 2–6 – after modification with hno3, hcl, h2so4, h3po4, naoh correspondingly. table 3. antibacterial activity of thermally and chemically modified tufa samples against escherichia coli and staphylococcus aureus in this context, the tufa-containing samples of packaging paper with 10 wt % of the basalt tufa modified at 250 0c were made and then used for the iso 27447:2009(e) antibacterial activity tests [6]. rather high antibacterial activity of both samples has been detected since even 4 hours long test ensured elimination of 95.7 and 92.3 % of escherichia coli and staphylococcus aureus respectively while complete disinfection was registered after 8 hours long treatment no antibacterial activity has been detected in the similar experiments carried out with no tufa containing paper packaging. this gives us ground to envisage good prospects for construction and further development sample number of escherichia coli (staphylococcus aureus) after treatment with tufa and % of remaining microbes initial after 4 h % after 8 h % bt after thermotreatment at 250 ºс 7.8·104 (6.7·104) 2.8·103 (1.6·103) 96 (98) 0 0 100 (100) bt after chemical treatment with solution of hcl 7.3·105 (9.7·105) 2.2·104 (1.4·104) 97 (99) 0 0 100 (100) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 igor kobasa, mariya vorobets, larysa аrsenieva, basalt tufa as a bactericide filler for some packaging materials, volume xvi, issue 4 – 2017, pag. 287 – 292 86 of the non-toxic tufa-containing food packaging materials. 4. conclusion high antibacterial activity against the standard strains of escherichia coli and staphylococcus aureus has been established for the composite materials containing some thermal or chemically modified basalt tufa. this activity remains at sufficient level for the paper packaging materials containing such composites. therefore, they can be considered as antibacterial fillers for the foodstuff packaging manufacturing. 5. references [1]. kobasa, i.m., tsymbalyuk, v.v., basalt tufa: composition, properties and ways of utilization. chernivtsi, publishing house of chernivtsi national university, 200 p. (2015) (in ukrainian). [2]. mikula, j., hebdowska-krupa, m., kobasa, i., glanowski, p., tuf jako wypelniacz mineralny, zwalaszcza antykorozyjny, sposob jego otrzymywania i sposob wytwarzania z nim powlok lakierniczych. poland patent application pl 218359 b1. 28.11.2014 wup 11/14. [3]. rakitskaya, t.l., kiose, t.a., volkova v.ya., sorption parameters of the basalt tufa and catalytic activity of the acidic complexes of pd(ii) and cu(ii) fixed on its surface in the reaction of carbon monoxide oxidation, ukr. j. chem., 74(4): 80–85 (2008) (in ukrainian). [4]. tsymbalyuk, v.v., woloshuk, a.g., kobasa, i.m., an influence of thermotreatment on sorption characteristics of basalt tufa. ukr. j. chem., 75(12): 85–90 (2009) (in ukrainian). [5]. tsymbalyuk, v.v., kobasa, i.m., woloshuk a.g., sorption of ammonium cations and heavy metal ions from the natural artesian waters with the basalt tufa samples. advanced water supply and wastewater treatment: a road to safer society and environment [eds.: hlavinek, p., winkler, i., marsalek, j., mahrikova, i.] 325–328 (2011). [6]. kobasa, i., vorobets, m., arsenieva, l., nanosized titanium dioxide as an antibacterial admixture for the food packaging materials. food and env. safety j., 14(4): 306–311 (2016). [7]. kobasa, i., bilokopyta, g., arsenieva, l., nanodispersed composite antimicrobial materials based on calcium hydroxylapatite. food and env. safety j., 14 (1), 89–92 (2015). [8]. malyovanyi, v.s., odnorig z.s., guzova i.s., natural zeolites modification and utilization prospects, chem. indust. of ukraine, 5: 10–12 (1999) i(in ukrainian). [9]. woloschuk, a.g., tsymbalyuk, v.v., kobasa, i.m., influence of chemical modification of the basalt tufa on its physico-chemical properties, sci. bullet. chernivtsi univ. series chem., 453: 63–68 (2009) (in ukrainian). [10]. tsymbalyuk, v.v., woloshuk, a.g., kobasa, i.m., heavy metals ions sorption on the acid-modified basalt tufa samples, sci. bullet. chernivtsi univ. series chem., 555: 84–88 (2011) (in ukrainian). sensory evaluation of goat milk cheese under different freezing conditions 250 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 250 255 supplementation of wheat flour with soy flour, sensory and physico-chemical evaluation of fortified biscuits flavia pop 1*, zorica voșgan1, lucia mihalescu1 1department of chemistry and biology, technical university of cluj-napoca, north university center of baia mare, 76a victoriei str., 430122, baia mare, romania, *corresponding author: flavia_maries@yahoo.com received 23th october 2017, accepted 18th december 2017 abstract: the research was conducted in order to evaluate the physico-chemical and quality characteristics of biscuits fortified with soy flour that could be used as a protein supplemented food. wheat flour has been replaced at different levels with soy flour that were 30% (r4), 20% (r3), 10% (r2) and without soy flour, control (r1). the fat content of control was 16.36% and increased to 19.4% in the case of 30% soy flour formulation that could be due to the increase in the proportion of soy flour in the flour blend. the highest mineral content was obtained in r4 (1.98%) and the lowest in r1 (0.552%), soy flour brought a significant contribution to mineral substances, and the ash content increased gradually with the increase in the percentage of soy flour. from the sensory evaluation, the biscuits with 10 and 20% soy flour addition presented similar characteristics, biscuits supplemented with up to 20% soy flour have shown superior nutritional characteristics as compared to wheat flour biscuits. keywords: soy flour, biscuits, fat content, saturation capacity, sensory evaluation. 1. introduction soy is particularly rich in lecithin, a very valuable emulsifier, in proteins and polyunsaturated fatty acids. the water absorption capacity of soy flour is much higher than that of wheat flour. in combination with wheat flour, soy flour is used for the preparation of bread or various pastry products. soy flour does not contain cholesterol, is ideal for people who are lactose intolerant, does not contain gluten, is rich in fat but poor in starch. soy flour contains vitamins b, vitamin e, magnesium, phosphorus and calcium [1, 2]. cracked biscuits are prepared from flour with medium or low gluten, because a high gluten content leads to hard biscuits.to reduce the high gluten protein content of a flour, it can be used wheat, potato or corn starch, which reduces the elasticity and increases the plasticity of the dough. due to its complex composition, the biscuits dough is more difficult to prepare compared to the dough intended for bread preparation, because additional additives reduce the moisture capacity of the flour and gluten elasticity [3-6]. the dough for sugary biscuits involves an initial mixing of powdered sugar with fats, until it reaches an aliphous consistency without any roughness. the cream is mixed with sugars, eggs and essences homogenised in advance, and finally the flour is added. after homogenization the leavening agents are added and the kneading continues for 20-30 minutes. the increasing of the mixture temperature to an optimal value, favors the fluidization of certain components, the ability to hydrate the flour and reduce kneading time. although in dough with higher humidity the development of the gluten network is http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 flavia pop, zorica voșgan, lucia mihalescu, supplementation of wheat flour with soy flour, sensory and physicochemical evaluation of fortified biscuits food and environment safety, volume xvi, issue 4 – 2017, pag. 250 – 255 251 faster, it is not recommended to use a very wet dough, because it occurs a negative influences on the texture and shape of biscuits. after kneading the dough is left at rest, when the dough is matured, by repairing gluten threads damaged at kneading, that leads to the improvement of the rheological characteristics of the dough [7-9]. during the rest, the decomposition of the leavening agents begins, under the action of component acidity; the resulting gases loosen the dough and diminish its consistency. inappropriate storage or low humidity, leads to defective biscuits such as absence of volume and porosity, ruptures, surface blisters. also, if the thickness of the dough layers is too high, the dough layers in the lower part are heated, chemical decomposition is very fast and they break apart completely during the resting phase. the formed gases will be largely eliminated at rolling and the final products will have low porosity and volume. the cooling of biscuits should be done as soon as possible, to strength the structure of the product, making it more resistant [10-12]. the objective of the study was to fortify wheat flour with soy flour at different levels that were 30% (r4), 20% (r3), 10% (r2) and without soy flour as control (r1) and to study their effects on the biscuits quality. to assess the quality of biscuits the following parameters were determined: fat content, alkalinity, determination of total ash, saturation capacity, acidity and moisture content during storage, as well as sensory analysis. 2. materials and methods materials wheat flour was replace with soy flour in three proportions 30% (r4), 20% (r3) and 10% (r2) respectively, whereas r1 was kept as control (without soy flour). table 1. formulation of the biscuits the fineness or size of the flour particles are of particular importance because influences the rate of colloidal and biochemical processes and the baking characteristics of the dough. wheat flour contains 12% proteins, 70% carbohydrates, 2% lipids, 14% water, as well as mineral substances, vitamins and pigments [4]. physicochemical examination acidity is based on the extraction of the biscuits acids and their titration with sodium hydroxide 0.1 n, using phenolphthaleine, as an indicator. acidity was expressed in ml of naoh 0.1 mol/l solution by 100 g of biscuits [13]. fat content was determined by soxhlet method witch consisted of extracting the fat from the analyzed sample with ethyl ether. determination of moisture content is based on drying of a sample with known initial mass to a constant mass. alkalinity determination is based on the titration of the alkaline substances extracted from the product with hcl solution in the presence of blue-bromo-thymol. the alkalinity of pastries was expressed in degrees. degrees of alkalinity mean the number of milliliters of acid, normal solution, used to neutralize the alkalinity from 100 g of a product and based on dry matter. determination of total ash is based on the calcination of a known mass of product up to constant weight [13]. saturation capacity consists in weighing of one or two biscuits that are placed in a pot sam ples wheat flour (%) soy flour (%) sugar (g) butter (g) sodium bicarbon ate (g) r1 100 0 100 100 1.0 r2 90 10 100 100 1.0 r3 80 20 100 100 1.0 r4 70 30 100 100 1.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 flavia pop, zorica voșgan, lucia mihalescu, supplementation of wheat flour with soy flour, sensory and physicochemical evaluation of fortified biscuits food and environment safety, volume xvi, issue 4 – 2017, pag. 250 – 255 252 of water and allowed to soak for 15-20 minutes, and then are weighed again. determination of elasticity is based on the application of an known external force on the dough of known initial length and tracking its response. to assess the normal taste of biscuits we used the descriptive sensory analysis, which is an ideal technique to identify flavors in a product and to distinguish the products between them, using tasters familiar with methods of scoring and sensory language. sensory analysis was performed on a sample of 10 trained panelist, aged between 20 and 55 years, according to the method described by farzana and mohajan, [14]. values of different parameters were expressed as the mean ± standard deviation. significant differences between samples were tested by student’s t-test. 3. results and discussion dough acidity increased directly proportional with the increase of soy flour content in the biscuits formulatios. according to farzana and mohajan, soy flour has a 24.9% fat content in the nutritional composition, and brings a significant contribution of fatty acids to biscuit dough, and it represents a source of linoleic and linolenic acids that are not produced by the body [14]. the dough lost from elasticity directly proportional with the increase of soy flour and the reduction in gluten content. the fat content of control biscuits was 16.36% and increased to 19.4% in the case of 30% soy flour formulation (table 2). the increase in fat content is in agreement with several studies [7, 9, 10]. the increase in the present study could be explained as soy flour is considered as an edible oil source, containing a higher percentage of fat than wheat flour. farzana and mohajan reported that the fat content of soy flour is between 20-24%, the white wheat flour has a 0.9-1.1% content and most of them are unsaturated fatty acids. the trend of increase in fat content could be due to the increase in the proportion of soy flour in the flour blend [14]. the highest mineral content was determined for r4 (1.98%) and the lowest for r1 (0.552), soy flour brought a significant contribution to mineral substances. the data showed that the ash content gradually increased with the increase in the percentage of soy flour, results which are in agreement with the findings of ayo et al., and siddiqui et al., on the supplementation of soy flour for the preparation of biscuits [15, 16]. acidity presented an increasing trend during four weeks storage, due to the hydrolysis processes occurring in the fat formulation. biscuits presented a higher acidity than the dough, due to the amount of carbon dioxide resulted from thermal decomposition of chemical agents. a more pronounced increase in acidity was recorded for r3 and r4 (fig. 1). in the present study, the highest moisture content was determined for wheat flour biscuits (3.6%), and the content gradually decreased to 2.07% for biscuits supllemented with 30% soy flour. the data showed that moisture decreased with the increase in soy flour witch is in agreement with the study of banureka and mahendran [17]. this may be explained by the fact that soy flour contains a higher amount of solid dry substance with emulsifying properties compared to wheat flour. during biscuits storage the moisture content increased, but r3 and r4 showed greater storage stability (fig. 2). the saturation capacity decreased during four weeks of storage, between the moisture content and saturation capacity there was an inverse correlation (fig. 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 flavia pop, zorica voșgan, lucia mihalescu, supplementation of wheat flour with soy flour, sensory and physicochemical evaluation of fortified biscuits food and environment safety, volume xvi, issue 4 – 2017, pag. 250 – 255 253 table 2. proximate analysis of biscuits parameters r1 r2 r3 r4 fat (%) 16.34 ± 0.03 17.31a ± 0.02 19.07b ± 0.05 19.84bc ± 0.06 moisture (%) 3.62 ± 0.08 2.95a ± 0.04 2.63ab ± 0.02 2.07b ± 0.03 ash (%) 0.75 ± 0.01 1.45ab ± 0.05 1.59b ± 0.06 1.86bc ± 0.01 alkalinity (degrees of alkalinity) 2.73 ± 0.02 2.38a ± 0.01 1.82ab ± 0.01 1.25b ± 0.04 saturation capacity (g) 14.31 ± 0.05 18.82ab ± 0.03 21.76b ± 0.04 23.54bc ± 0.02 dough elasticity (%) 40.63 ± 0.04 29.35bc ± 0.04 26.12c ± 0.03 23.78cd ± 0.05 values are means of triplicates ± standard deviation. values with the same superscript in a column are not significantly different (p > 0.05). fig. 1. acidity variation during storage of biscuits fig. 2. moisture variation during storage of biscuits fig. 3. saturation capacity variation during storage of biscuits food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 flavia pop, zorica voșgan, lucia mihalescu, supplementation of wheat flour with soy flour, sensory and physicochemical evaluation of fortified biscuits food and environment safety, volume xvi, issue 4 – 2017, pag. 250 – 255 254 farzana and mohajan studied the incorporation of soy flour to wheat flour and the quality of biscuits fortified with mushroom. the researchers reported that protein content of soy flour-supplemented biscuits increased from 11.07% to 17.86% as compared to control and a significant increased in fat (17.36-20.89%), fiber (0.48-0.92%), iron (1.56-1.99 mg/100 g), and energy value. the results from chemical analyses indicated that good quality biscuits can be prepared by replacing wheat flour with 15% soy flour and addition of mushroom powders may affect the backing quality [14]. mildnerszkudlarz et al., studied the incorporation of white grape pomace in wheat flour at levels of 10, 20 and 30% and its influence on rheological, nutraceutical, physical and sensory properties of dought and biscuits. the researchers concluded that dough with different levels of white grape pomace showed a decrease in water absorption and the addition of white grape pomace reduced hardness and caused deterioration in brightness and yellowness of all the enriched samples. the results showed that the smallest addition of white grape pomace caused an approximately 88% increase in total dietary fibre content as compared to control. the content of phenolic compounds increased with the addition of white grape pomace and enhanced the antioxidant properties of biscuits, while acceptable biscuits were obtained when incorporating 10% white grape pomace [18]. to assess the state of freshness we used the descriptive sensory analysis, which is an ideal technique to identify flavors in a product and to distinguish the products between them, using tasters familiar with methods of scoring and sensory language. the organoleptic test of the products was done by the 5-point hedonic scale and was performed on a sample of 10 trained panelist, aged between 20 and 55 years, according to the method described by farzana and mohajan, [14]. selected attributes were: smell, taste, color, appearance and texture (fig. 4-6). 0 1 2 3 4 5 smell taste colorappearance consistency fig. 4. sensory analysis of biscuits with 10% soy flour incorporation fig. 5. sensory analysis of biscuits with 20% soy flour incorporation 0 1 2 3 4 5 smell taste colorappearance consistency fig. 6. sensory analysis of biscuits with 30% soy flour incorporation for sensory analysis the descriptive scale was the following: 1 = very little normal, 2 = less than normal, 3 = moderately normal, 4 = almost normal, 5 = normal. the taste is the primary factor which food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 flavia pop, zorica voșgan, lucia mihalescu, supplementation of wheat flour with soy flour, sensory and physicochemical evaluation of fortified biscuits food and environment safety, volume xvi, issue 4 – 2017, pag. 250 – 255 255 determines the acceptability of any product, which has the highest impact as far as market success of product, is concerned. in the present study, sensory scores of biscuit enriched with 10% (r2) and 20% (r3) soy flour, showed that with regard to taste, color, smell, appearance, and consistency, the sensory characteristics of r2 were found to be the best. 4. conclusions during four weeks of storage at ambient temperature, acidity and moisture content showed an upward trend and were within the maximum admissible limits. biscuits fortified with soy flour had a higher fat and mineral content as compared to wheat flour biscuits. from the sensory evaluation, the biscuits with 10 and 20% soy flour addition presented similar characteristics. the biscuits supplemented with up to 20% soy flour have shown superior nutritional characteristics as compared to wheat flour biscuits. soy flour can be used efficiently in biscuit production technology. 5. references [1]. hegstad h.g., nutritional and health benefits of soybean. soy protein quality evaluation report, food and agriculture organization of the united nations, food and nutrition paper no. 71, rome, italy, (2008). [2]. slavin j.l., dietary fiber and body weight, nutrition, 21: 411-418, (2005). [3]. sutharshan s., sriwardana t.d., mahendran t., development of maize-soy based supplementary food and evaluation of quality parameters, proceedings of the 57th annual sessions, sri lanka association for the advancement of science, 26th november 1st december, (2001). [4]. pop f., milling technology, mega publishing house, cluj-napoca, pp. 70-90, (2017). [5]. mihaly cozmuta a., pop f., bakery technology, risoprint publishing house, clujnapoca, pp. 120-140, (2008). [6]. wong s.y., colorimetric determination of iron and hemoglobin in blood. ii, journal of biology and chemistry, 77: 409-412, (1999). [7]. akubor p.i., ukwuru m.u, functional properties and biscuit making potential of soybean and cassava flour blends, plant foods for human nutrition, 58: 1-12, (2005). [8]. gopalan c., shastri b.u.r., balasubramaniam s.c., nutritive value of indian foods, national institute of nutrition, india, pp. 1-156, (2000). [9]. vernaza m.g., gularte m.a., chang y.k., addition of green banana flour to instant noodles: rheological and technological properties, ciencia agrotecnologia lavras, 35: 1157-1165, (2011). [10]. sobczyk m., haber t., witkowska k., the influence of an addition of oat flakes on the quality of dough and wheat bread, acta agrophysica, 16: 423-433, (2012). [11]. baltsavias a., jurgens a., van vliet t., fracture properties of shortdough biscuits: effect of composition, journal of cereal science, 29: 235-244, (2001). [12]. chevallier s., della valle g., colonna p., broyart b., trystram g., structural and chemical modifications of short dough during baking, journal of cereal science, 35: 1-10, (2003). [13]. mihaly cozmuta a., pop f., codre a., practical guidance on milling, bakery and pasta products technology, risoprint publishing house, cluj-napoca, pp. 21-37, (2007). [14]. farzana t., mohajan s., effect of incorporation of soy flour to wheat flour on nutritional and sensory quality of biscuits fortified with mushroom, food science & nutrition, 3(5): 363-369, (2015). [15]. ayo j.a., ayo v.a., popoola c., omosebi m., joseph l., production and evaluation of malted soybean-acha composite flour bread and biscuit, african journal of food science and technology, 5: 21-28, (2014). [16]. siddiqui n.r., hassan m., raza s., hameed t., khalil s., sensory and physical evaluation of biscuits supplemented with soy flour, pakistan journal of food sciences, 12: 45-48, (2003). [17]. banureka v.d., mahendran t., formulation of wheat-soybean biscuits and their quality characteristics, tropical agricultural research and extension, 12: 2, (2010). [18]. mildner-szkudlarz s., bajerska j., zawirska-wojtasiak r., gorecka d., white grape pomace as a source of dietary fibre and polyphenols and its effect on physical and nutraceutical characteristics of wheat biscuits, journal of the science of food and agriculture, 93: 389-395, (2013). *corresponding author: flavia_maries@yahoo.com 1. introduction 40 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 12017, pag. 40 46 chemical composition and energy value in the meat of the macedonian and ohrid trout * aleksandar saveski1, tatjana kalevska1, viktorija stamatovska1, dragan damjanovski 1 1faculty of technology and technical science veles, university st. kliment ohridski, bitola, add. dimitarvlahovnn, veles, r. macedonia saveskia@ yahoo.com *corresponding author received february 12th 2017, accepted march 27th 2017 abstract: the aim of this research is to conduct a comparative analysis and to verify the quality properties of meat of the macedonian trout (salmo macedonicus) and the ohrid trout (salmo letnica), from aquaculture production, bred on a fish farm under controlled conditions. in order to determine the quality of meat of the macedonian and ohrid trout, analyses of the chemical composition have been conducted: the amount of proteins, fat, water, minerals, as well as a determination of the energy value of the meat. the analyses have been made on samples from macedonian and ohrid trout, with size of consumption between 200g and 300g. by these analyses, it has been confirmed that in the meat of the macedonian trout, the amount of water is 77.122%, 17.800% proteins, 3.333% fat and 1.667% minerals, whereas in the meat of the ohrid trout 75.923% water, 16.783% proteins, 5.403% fat and 1.080% minerals. though the established difference in the amount of water and fat is not significant, the differences determined in the contents of proteins and minerals are significant on a level p>0,05. the energy value of macedonian trout meat is 435.29 kj/100gm, i.e. and 498.49 kj/100gm in the ohrid trout meat. keywords: salmo macedonicus, salmo letnica, quality, aqua cultural production 1. introduction the republic of macedonia has excellent conditions for the development of fishery such as the climatic conditions, the tradition of fishing and the abundance of water areas. at the beginning the macedonia fishery was present in freshwater lakes, but after the second world war, there was a fast development of the aquaculture [1]. in the waters of macedonia representatives of different types of fish can be found such as: salmonidae (trout), esocidae, cyprinidae, cobitidinae, siluridae, anguillidae (eels), percidae, blennidae, poeciliidae. the family salmonidae includes nine different genera, with forty types of fish. in the lakes in our state, there are four genera with a large number of types and subtypes. the macedonian trout – salmomacedonicus (karaman, 1924) is an indigenous and endemic type of fish, characteristic of the macedonian lakes, whereas the ohrid trout – salmoletnica (karaman, 1924) is an endemic type that lives only in the ohrid lake and at the same time it is being bred in aquaculture, under controlled conditions on fish farms. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 41 the protein composition of fish from aquaculture production has a very high quality and large biological and nutritive value, and speaking from an ecological point of view their breeding ranks among the cleanest and most natural breeding of animals [2]. the annual consumption of fish in macedonia is very small, around 3.5 kg per person (together with the imported and processed fish), which is far from the annual world`s average of 15-20 kg fish per person. the main reason for small consumption and processing of fish is thought to be the insufficient domestic production, the small assortment of fish, little knowledge of the nutritive value and quality of the meat, a lack of habit for regular consumption of fish meat by the population and most often the consumption is connected with fasting religious holidays. therefore, there is a need of informing people about the benefits for their health by consuming fish, as well as the advantages that fish meat has when compared with different types of meat: easy digestion, small amount of fat, high percent of omega-3 unsaturated fatty acids that reduce the cholesterol levels in the blood and so on [1]. the nutritive and health significance of fish and fish products in the human diet is the reason for continued higher demand on the market, especially for fish from aquaculture conditions [14, 22]. fish meat is the most valuable and useful food product of animal origin used in human nutrition [27]. the high nutritious value of fish is manifested through the favorable content and proportion of proteins, fat, carbohydrates and minerals [3, 4]. from the components that are present in fish meat, the human organism on average uses 95-96% of proteins and up to 91% of fat. fish that are bred in aquaculture show slight, predictable variations in the chemical content. the controlled conditions of breeding, the content of the food such as the amount of proteins and fat, the environmental conditions, the size of the fish and the genetic potential are all factors that have influence on the content as well as on the quality of the fish bred in aquaculture [5]. the amount of nutrients in fish meat varies in accordance with the type of fish, diet, age, external conditions, season, etc. [7]. the nutritional characteristics, quality and biological value of fish meat, apart from the other factors, also depend on its chemical composition and is characterized by easy digestion, nutritiousphysiologically nutritionally-physiological favorable relationship of amino acids and great amount of vitamins and minerals. speaking from a nutritious point of view, fish is an important source of substances such as proteins which are a necessary component for human life and development [6]. proteins are the most important content in fish meat, which combined with fats and carbohydrates form the base of healthy diet. the proteins from fish meat are equally as valuable as proteins from other kinds of meat. the amount of proteins in fish meat is 12% to 24%, which is reduced during spawning because the roe is rich in proteins, while the fish loses about 25% of its weight [8, 9]. the main characteristic of proteins is easy digestion (on average between 2 and 3 hours), better usage, favorable amino acid relation that is connected with the presence of the essential amino acids such as: methionine, lysine, tryptophan, arginine, histidine. the amount of fat in fish varies between 0.7% and 20% and depends on the diet, type, gender, age of the fish and season [10]. according to the amount of fat, fish is classified in: lean fish that has less than 0.5% fat, medium-fat fish which has 510% fat and fatty fish with more than 10% fat [12]. the fat in fish usually consists of triglycerides, i.e. complex substance which food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 42 is a mix of glycerol and higher fatty acids. the amount of fat in fish is not constant and varies during the year, usually being inversely proportional with the content of water [13]. the percent of fat in fish, which can be reabsorbed in the gastrointestinal tract, is known as the coefficient of digestibility of fat in fresh, frozen and smoked [5]. the average chemical content in low fat fish is: 77-82% water, 18-19% proteins, 0.1% -1.0% fat, 1.0% 2.0% minerals whereas in high fat fish the content is as follows: 55-79% water, 14.5-21.5% proteins, 1.1 -2.9% fat and 1.0-2.0% minerals. [16] depending on the type of fish differences of water and fat content in the meat occur [17, 18]. analyses have been made in order to determine the influence of the season on the chemical content of meat from different types of carp. in the examined samples the amount of proteins varied between 13.0 – 21.9%, fat 0.3 – 23.9%, water 59.8 – 84.2% and minerals from 0 to 16% [20]. the results show that the season has no big influence on the chemical content of the carp (proteins, water, fat and minerals) [21]. fish is very important for the human diet, due to the presence of minerals which range from 1.0% to 1.5%. mineral matters in fish meat have been identified such as salts of potassium, sodium, calcium, magnesium and phosphorus, but also small amounts of iron, copper, iodine, chromium, zinc and fluorine. the large amount of fluorine and iodine in fish meat increases its biological value, whereas the small amount of sodium makes it preferable as a dietary food [23, 24]. the presence of carbohydrates in fish meat changes between 0.5 and 0.8%. however, the small amount of carbohydrates in fish does not make them less significant [26]. the amount of carbohydrates in fish meat is relatively low as compared to other types of meat [13, 25]. the most common carbohydrate is glycogen [10]. the amount of carbohydrates in fish meat depends on: fatigue, stress and diet. if the fish is well fed, rested and has not been subjected to stress, it contains a great amount of glycogen [23]. in the meat of the california trout the average value of its contents is 20% proteins, 2% fat and 1.2% minerals. due to its ideal content this fish has been recommended to children, old people and people with certain health issues [19]. with the conducted analyses on the chemical composition of the meat of california trout, it has been determined that its average content is: proteins 18.8 – 19.3%, water 73.8 – 78.0% and minerals 1.2% [5]. with the examinations and analyses of the meat of california trout it has been determined that it contains: 77.2% water, 13.7% proteins, 5.5% fat and 2.0% minerals [15]. with different examinations in the meat of the california trout, the following chemical content has been determined: 75% water, 20% fat, 3.8% fat, 1.2% minerals [12]. 2. materials and methods in this research as a material for work, we used samples of the macedonian trout (salmo macedonicus) and the ohrid trout (salmoletnica) with size of consumption between 200g and 300g. the trout has been bred in a fish farm with spring water from the spring of the river beleshnica, which is situated near the village belica in the region poreche in macedonia, and at the same time the fishpond is the reproduction center for ohrid trout. the average temperature of the water in the fishpond depends on the weather conditions and season, and usually varies between 7-12oc. both types of fish are fed with the same complete food for trout. in order to determine the quality of the meat of the trout, the following analyses have been made: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 43 analyses of the chemical composition of the fish meat the amount of water is determined by drying the material at a temperature of 1050c until a constant weight is reached. the amount of proteins has been determined according to the kjeldahl method, whereas the amount of fat has been determined with the method of extraction according to soxhlet. the presence of minerals has been confirmed by using the method of burning the material at a temperature of 500oc. the appearance of phosphorus (p) in the meat was determined by using atomic emission spectrometry with inductively coupled plasma (icp – aec). the quantity of potassium (k) was verified by burning the material with concentrated h2so4 and its determination with flame photometer. the energy value of the meat of both the macedonian and ohrid trout was determined through the already confirmed quantity of proteins and fat. by multiplying the amount of proteins (%) by the factor 17.16 and multiplying the amount of fat (%) by the factor 38.96 and then calculating the sum of the two already calculated values, we reckon the energy value of the fish meat expressed in kj/100g [11]. 3. results and discussion the results from our examination of the chemical content in the meat of both the macedonian and ohrid trout are shown in table 1. table 1. comparison of the chemical content in the meat of the two types of trout type of fish water % ̅ ± s ̅ proteins % ̅ ± s ̅ fat % ̅ ± s ̅ minerals % ̅ ± s ̅ macedonian trout 77.122 ± 0.61 17.800 ± 0.17 3.333 ± 0.44 1.667 ± 0.06 ohrid trout 75.923 ± 0.60 16.783 ± 0.22 5.403 ± 0.93 1.080 ± 0.09 with the results shown in table 1, we conclude that the average content of water in the macedonian trout is 77.122 ± 0.61% i.e. 75.923 ±0.60% in the ohrid trout. the amount of proteins in the macedonian trout is 17.800 ± 0.17%, whereas in the ohrid trout, proteins are present with 16.783 ±0.22%. the amount of fat in the macedonian trout is 3.333 ± 0.44% and is lower than in the ohrid trout where fat is present with 5.403 ± 0.93%. there are 1.667 ± 0.06% minerals in the meat of the macedonian trout, whereas in the ohrid trout the amount of minerals is smaller and is determined to be 1.080 ± 0.09% (fig. 1). the verified differences in the amount of water and fat in the two types of trout have no statistical significance. however, a significant difference is determined in the content of proteins and minerals in the two types of trout (p>0.05). the amount of potassium (k) and phosphorus (p) in the meat of the macedonian and ohrid trout is shown in table 2. table 2. content of k and p in the meat of the macedonian and ohrid trout type of fish parameters (%) potassium (к) ̅ ± s ̅ phosphorus (р) ̅ ± s ̅ macedonian trout 1.413 ± 0.02 1.888 ± 0.03 ohrid trout 1.012 ± 0.06 1.540 ± 0.02 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 44 fig. 1. comparison of the chemical content in the meat of the macedonian and ohrid trout the amount of potassium in the meat of the macedonian trout is 1.413 ±0.02%, whereas in the ohrid trout potassium is 1.012 ±0.06%. on the other hand, there is 1.888 ± 0.03% phosphorus in the meat of the macedonian trout, whereas there is 1.540 ±0.02% phosphorus in the ohrid trout (fig. 2). the determined differences in the amount of phosphorus and potassium in the meat of the macedonian and ohrid trout are significant (p>0.5). fig. 2. comparison of the amount of phosphorus and potassium in the meat of the macedonian and ohrid trout the energy value of the meat of the macedonian and ohrid trout is shown in fig. 3. the energy value of the meat of the macedonian trout is 435.29 kj/100g, i.e. 498.49kj/100g of the meat of the ohrid trout. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 45 higher energy value is determined in the meat of the ohrid trout as a result of the higher amount of fat. the energy value of the meat of the ohrid trout is significantly higher (p>0.05) in comparison with the meat of the macedonian trout. fig. 3. energy value in the meat of macedonian and ohrid trout 4. conclusions no significant differences were determined about the amount of water in the meat of the trout (in the macedonian trout 77.122 ±0.61% and 75.923 ± 0.60% in the ohrid trout), as well as in the amount of fat in the trout (in the macedonian trout 3.333 ±0.44% and 5.403 ± 0.93% in the ohrid trout). a significant difference (p>0.05) was determined in the amount of proteins (17.800 ±0,17% in the macedonian trout and 16.783 ±0.22% in the ohrid trout). a significant difference (p>0.5), as well, was determined in the amount of minerals (1.667 ± 0.06% in the macedonian trout and 1.080 ± 0.09% in the ohrid trout). in the amount of phosphorus and potassium a significant difference was determined in the two types of trout (p>0.05). the energy value of the meat of the ohrid trout is 498.49 kj/100g and it is significantly higher (p>0.05) when compared with the energy value of the meat of the macedonian trout 435.29 kj/100g, explained by the higher amount of fat in the ohrid trout. due to the favorable chemical content, i.e. the significant content of proteins and the small amount of fat, both the macedonian and the ohrid trout are one of the most nutritionally valuable products of animal origin, which should often be a part of the human diet, because of the nutritive and health benefits for its consumers. 5. references [1]. hristovski m., stojanovski s., biology and fish diseases, national forum for animal protection in macedonia,7-16; 39-46; 126134; 156-163, skopje, (2005). [2]. stevanovski v., hristova k. v., technology for processing and canning fish, faculty of biotechnological sciences, 43-70; 263271, bitola, (2010). [3]. conor w. e., importance of n-3 fatty acids in health and desease. american journal of clinical nutrition 71s, 171–175, (2000) [4]. sidhu k. s., health benefits and potential risks related to consumption of fish or fish oil, regulatory toxicology and pharmacology, 38 (3): 336–344, (2003). [5]. vranić d., baltić z. m., the californian trout oncorhynchus mykiss): chemical composition, cholesterol content and fatty acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 1 – 2017 aleksandar saveski, tatjana kalevska, viktorija stamatovska, dragan damjanovski, chemical composition and energy value in the meat of macedonian and ohrid trout, food and environment safety, volume xvi, issue 1 – 2017, pag. 40 – 46 46 composition of the fillets, meat technology, 53(1): 26–35, (2012). [6]. marosević d., freshwater fisheries, fish as a foodstuff, 553, (1982). [7]. hamić a., aquaculture in bih, coron's, sarajevo, (2003). [8]. šoša b., hygiene and technology of processing sea fish, zagreb, schoolbook, (1989). [9]. kiessling a., pickova j., changes in fatty acid composition in muscle and adipose tissue of farmed rainbow trout (oncorhynchus mykiss) in relation to ration and age, food chem., 73: 271– 284, (2001). [10]. ingemansson t., lipids in light and dark muscle of farmer rainbow trout (oncorhynchus mikiss). j. sci. food. agric., 57: 443-447, (1991). [11]. vitchenko a., kopilov â., fisihing, 175, moskva, (1981). [12]. grujić r., the science of nutrition, banja luka, faculty of technology, atlantik, (2000) [13] zhlender, b., sea fish vs freshwater fish, 42-43, (2000) [14]. burger j., gochfeld m., perceptions of the risks and benefits of fish consumption: individual choices to reduce risk and increase health benefits, environmental research, 109: 343– 349, (2009). [15]. philips a.m., brockway d.r., the nutrition of trout ii, protein and carbohydrate, progr, fish-cult, 19(4), (1956). [16]. tadejević v., merchandising with the basics of technology and science of nutrition, zagreb, schoolbook, 310–316, (1971). [17]. rasoaraho j. r. e., barnathan g., influence of season on the lipid content and fatty acid profiles of three tilapia species (oreochromisni loticus, o. macrochir and tilapia rendalli) madagascar: food chemistry, 91(4): 683–694, (2005). [18]. ćelik m., diler a., kucukgulmez a., a comparison of the proximate compositions and fatty acid profi les ofzander (sander lucioperca) from two different regionsand climatic conditions, food chemistry, 92(4): 637–641, (2005). [19]. ćirković m., jovanović, b., maletin s., fisheries, faculty of agriculture, novi sad, (2002). [20]. milinković r., the effect of steroids and nutrition on reproductive and productive traits of rainbow trout at different growing conditions. the business community in the production, processing and marketing of livestock, livestock products and animal feed, beograd, (1988). [21]. stolywho a., kolodzijeska i., sikoroski y. e., long chain polyunsaturated fatty acid in smoked atlantic mackerel and baltic sprats, 94: 585–595, food chemistry, (2006). [22]. sveinsdottir k., martindottir e., sensory characteristics of different cod products related to consumer preferences and attitudes, food quality and preference, 20: 120–132, (2009). [23]. anon., nutritional aspects of fish, bord iascaigh mhara, co. dublin: www.bim.ie, (2003). [24]. bogut i., opaćak a., stević s., nutritional and protective value of fish with emphasis on omega-3 fatty acids, fisheries, 54(1): 21-38, (1996). [25]. konosu s., yamagući k., martineta l r. e., the flavor components in fishandshellfish in: chemistryandbiochemistry of marine food products, 367-404, connecticut: avi publishing co, (1982). [26]. stripković f., changes properties of sardines (hunting areas korcula pool) during the year and their impact on the production of "sardines in oil", master's thesis: faculty of food technology, university of zagreb, (1982). [27]. nistor e. c., pagu i. b., study of meat physical-chemical composition of three trout breeds farmed in salmonid exploitations from moldova. animal science and biotechnologies, 47(2): 190–195, (2014). . http://www.bim.ie, 258 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 258 265 selection of components of the compositional proteincarbohydrate mixture to improve the quality of the chopped semi-finished products *taisa honcharenko 1 , oksana topchyi 1 1 national university of food technologies, technology of meat and meat products department e-mail: yataya@ukr.net* *corresponding author received 18th november 2019, accepted 27th december 2019 abstract: meat is one of the most significant and nutritious foods available for the masses, which helps to satisfy most of the needs of the body. it has is a good source of proteins, zinc, iron, selenium, and phosphorus followed by vitamin a and b-complex vitamins. the modern production of meat products develops in the direction of expanding assortment of production, combining and optimizing the component composition in order to increase the nutritional value, substances by including in the recipe of the functional ingredients. the article presents the results of the study of qualitative indicators of animal origin protein preparations and protein-carbohydrate composition based on beef collagen protein and mixture of oilseed cultures flour for use in the recipe of chopped semi-finished products to improve their biological value. the aim of the study was to determine and compare the qualitative indicators of animal origin protein preparations, to create a compositional proteincarbohydrate mixture based on the selected animal protein and flour of oilseed cultures, to study its functional-technological properties in view of being used in the recipe of the chopped semi-finished products. based on the study and comparative analysis of qualitative indicators of animal origin protein preparations, the use of helios-11 beef collagen protein in the composition of a proteincarbohydrate composition was selected and substantiated. ratios of protein and mixture of oilseed cultures flour (1: 4) have been determined, which create the most favorable conditions for the correction of meat systems by their functional-technological properties, providing an acceptable and stable level of emulsifying and water-binding properties. keywords: meat products, animal proteins, oilseed cultures flour 1. introduction. meat is one of the most important and nutritious products available to the masses, helping to satisfy most of the body's needs. it is an important component of a wellbalanced diet, as it is a good source of protein, zinc, iron, selenium and phosphorus, as well as vitamins a and b vitamins. the range of meat products available on the ukrainian market today has a number of characteristics that reflect the key trends in the modern world. thus, given the active rhythm of life of modern man, fast food products are especially popular semi-finished products, due to the quality of which accurate demands are made, since today a culture of healthy nutrition is becoming a vital necessity. the active rhythm of life provokes irregular and monotonous nutrition, which leads to a decrease in the consumption of irreplaceable components. irrational nutrition and environmental degradation make relevant the search for natural substances containing vitamins and http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 259 minerals necessary to increase the body's resistance to the negative effects of environmental damage. domestic and foreign experience shows that the most effective way to combat nutritional deficiency is to enrich the vitamins and minerals of mass-produced products, including meat semi-finished products. at the same time, one of the most important challenges facing the meat industry is to reduce the cost of the finished product. this requires the revision and improvement of the traditional methods of production of meat products, which is possible through the use of food additives and protein preparations of plant and animal origin, which allow to purposefully change the functionaltechnological and structural-mechanical properties of meat systems and to obtain the desired technological effect. today, the problem of product quality is of concern for both manufacturers and consumers. the popularity of healthy lifestyles encourages people to carefully choose the components of their diets, preferring natural ingredients. at the same time, the tense modern lifestyle determines a clear tendency to increase the frequency of consumption of semi-finished products, including meat. the analysis of the market of meat semi-finished products in recent years shows that chopped semi-finished products in cooled and frozen form are characterized by high demand among modern consumers, being a common family food [1]. meat, which is a valuable protein raw material, in many cases still needs to be enriched with vitamins, minerals and other substances that are lacking for a balanced diet. this issue is of scientific and practical interest and does not lose its relevance, as shown by studies of domestic and foreign scientists [2]. a detailed analysis of scientific sources containing the results of experimental studies has shown that one of the most promising ways of enriching traditional meat products is the use of vegetable raw materials, in particular, oils and flour in their recipe. in the studies of simakhina g., pasichny v., slobodyanyuk n. and etc. the scientific bases and practical aspects of functional additives based on vegetable raw materials in production technologies of chopped semi-finished products and minced meat have been highlighted [3]. the study of oilseeds cultures as promising sources for the production of biologically active components of food is described in the works of stetsenko n., as well as in the works of foreign scientists [4-7]. recently, in the meat industry, special attention is given to the ingredients of the protein nature and animal origin due to their compatibility with the main raw materials. animal proteins significantly improve the structural and mechanical properties of foods, and above all their consistency, while playing the role of stabilizers, jelly-forming agents, improving the appearance of finished products. protein preparations based on connective tissue proteins, mainly collagen, are used to increase nutritional value, functionaltechnological and rheological parameters of finished products. such proteins in physiological effect equate to dietary fibers having cytoprotective activity and normalize gut microbiocenosis. scientists claim that collagen proteins improve the intestinal motility, and the breakdown products of these proteins, which are formed during heat treatment, stimulate the secretory function of the body [8]. experimental and clinical trials by foreign scientists have shown that collagen-based dietary supplements can have a beneficial effect on skin health [9]. works of yancheva m., polumbrik m. and others are devoted to the study of animal proteins qualitative indicators for the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 260 purpose of use in the recipes of meat products [10]. 2. materials and methods. determination of the protein mass fraction was performed using the keldal method, which is based on complete ashing (mineralization) of the sample in the process of heating with concentrated sulfuric acid in the presence of a catalyst (cuo), with futher determination of ammonia, bounded by acid. the amount of bound ammonia is determined by adding alkali (naoh), whereby the ammonium sulfate is decomposed to ammonia, which is subsequently converted into a titrated acid solution taken in a certain amount, where it binds to the formation of ammonium salt. then the acid residue is titrated and the amount of ammonia, which recalculated to the protein, determined [11]. the ash content was determined by ashing the dried sample at 500… 700 ° c in a muffle furnace to constant weight [11]. determination of the mass fraction of moisture was performed by method of drying to constant weight. the sample of the product is dried in the drying cabinet at a temperature of 100 105 ° c for 3 5 hours, cooled in a desiccator from 20 to 120 minutes and weighed on analytical balance. then again dried from 0.5 to 1.5 hours, cooled and weighed. if the difference between the last two weighings is greater than 0.0004 g, it is dried again and again until the mass is constant [11]. mass fraction of fat was determined by a method based on repeated extraction of fat from a dried sample by volatile solvents followed by removal of the solvent and drying of the extracted sleeve to constant weight. the extraction was carried out in a soxhlet apparatus, hexane was used as solvents [11]. determination of moisture-retaining and fat-retaining capacity was performed by gravimetric methods as the difference between the mass fraction of water (fat) in the mince and the amount of water (fat) released during the heat treatment. the maximum amount of added water (oil), at which does not separate the aqueous (fat) phase during the test, in terms of 1 g of the preparation, was taken as the value of moisture-retaining (fat-retaining) ability [11]. determination of the emulsifying ability of protein preparations was carried out by the method developed by inklaar r. and fourtuin j., by preparation of emulsions based on 1% aqueous dispersions and vegetable oil . homogenization is carried out at the speed of rotation of the motor shaft 3000 rpm. for each sample, a series of emulsions with a fat phase content from 10% to 90% with an interval of 10% is prepared. then, using a syringe, the emulsion is poured into tubes with a diameter of 5 mm, thermostated at 85 ° c for 20 minutes, then cooled with running water and centrifuged for 30 minutes at 6000 rpm. the criterion of the emulsions stability at the initial ratio of fat and water phases is the average for test tubes of the phase ratio in the system [11]. determination of moisture-binding capacity was carried out by the "pressmethod", which was developed by grau r. and hamm r. in modification of v. volovinskaya and b. kelman. the method is based on the separation of water by prototype with light pressing, adsorption of water released by filter paper and determining the amount of separated moisture by the size of the area of the stain left on the filter paper [11]. the determination of protein preparations ph was determined by a potentiometric method. the method is based on the measurement of the electromotive force of an element consisting of a comparison food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 261 electrode with a known potential value and an indicator (glass) electrode, the potential of which is due to the concentration of hydrogen ions in the test solution [11]. the degree of swelling was determined by the weight method, which is to determine the difference between the weight of the sample before and after swelling: where m0 the mass of the original mixture; m the mass of the swollen mixture. the viscosity was determined using a rototest 2 viscometer [11]. the required speed of the rotor was setby means of a switching actuator by setting the lever in the position of the stages from 1a to 12 a. the rheological characteristics of the product were determined by the magnitude of rotation of the rotor (velocity gradient) and the resistance force of its rotation. 3. results and discussion. previous studies have found that sesame, linseed, sunflower and pumpkin flour with unique chemical composition and pharmacological properties are promising raw materials for the enrichment of traditional meat products, in particular, chopped semi-finished products. it has been found that the ability of meat systems to bind moisture is increased with an increase in the degree of crushing of seeds, which can be explained by the microcrystallization of cellulose, which occurs in the process of fine grinding of grains. this is known to increase the ability of cellulose to absorb moisture and form a stable colloidal gel. it is also determined that the introduction into the meat systems of seeds, crushed to a particle size of 350 μm, increases fatholding capacity, which is important technological value, since this indicator provides a gentle and homogeneous texture of the product, eliminates the separation of fat and reduces weight loss during heat treatment. analytically, on the basis of the analyzed literature sources, it was established that all types of oilseeds we study have high nutritional value and functional and technological characteristics. and since, one of the most important indicators that determine the possibility of using any additive is its organoleptic properties, due to the peculiarities of the composition, the content of the individual components was determined based primarily on the results of sensory evaluation. for this purpose, the seeds of sesame, flax, sunflower and pumpkin, crushed to a particle size of 350 µm, were mixed in different proportions. after analyzing the results of organoleptic studies, it was decided to have an equal ratio of seeds four types in the mixture composition [12]. thus, for further research, a mixture of seeds, which was finely ground to flour. in order to simplify the technological process, finished partially skimmed flour of tm “sto pudov” (ukraine) oil cultures was used. animal proteins play an important role in the production of meat products. their concentration in the finished product determines the protein and energy value of the finished products. animal proteins have a high moisture retaining, emulsifying ability and thermal stability, which determines their multi-purpose. therefore, in meat systems, there must be a sufficient amount of protein to realize these properties. equally important advantages of animal proteins are the ease of use and preservation of their properties during long-term storage. the use of animal protein in the formulations of meat products can increase the yield of finished products and, thus, ensure the high quality food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 262 of the finished product while reducing its cost. full-fledged animal proteins are significantly better than plant proteins in terms of biological value. it has been proved that, compared with plant, animal proteins are more balanced in amino acid composition and to a greater extent meet the body's needs for essential amino acids. special attention should be paid to beef proteins, which are almost identical to natural meat in amino acid composition. the degree of beef collagen protein hydrolysis allows the enzymes of the body to break down and convert it into energy necessary for human activity. neutral ph and no odor make it possible to mix beef protein with different ingredients at different stages of meat production. on the basis of the market analysis of animal protein preparations for the purpose of qualitative indicators research and comparison, protein preparations were selected: "scanprotm t95", "collagen plus", "helios-11". scanpro t95 is an animal protein based on collagen-containing raw materials (pork). it is used for the production of various types of meat products, including the production of chopped semi-finished products. it is applied in dry form or in the form of gels with a degree of hydration up to 1:15. in terms of organoleptic characteristics it is a fine powder (less than 106 µm) of light gray color with a neutral odor. collagen plus is a beef collagen protein used in the meat industry. in the preparation of minced meat protein is added in dry form or in gel form. capable of binding up to 20 units of water. the protein powder has a light color and a neutral odor. helios-11 is a long-fiber beef collagen protein (fiber sizes greater than 400 µm) of light gray color, with a neutral odor. used in the technology of meat products in the form of gels, granules and in the dry form. one unit of protein is able to bind up to 20 units of water. the results of the study of the physicochemical parameters of the experimental protein preparations are shown in table 1. differences in the amount of protein, ash and fat can be explained by the different chemical composition of the raw materials entering the production and the different parameters of its technological processing. the moisture content of proteins is determined by modes, drying methods and the subsequent holding time before feeding the raw material for grinding (the material may collect moisture from the air). table 1. physico-chemical parameters of experimental protein preparations parameters « s c a n p ro t m t 9 5 » « c o ll a g e n p lu s» « h e li o s1 1 » protein content,% 90.10 95.2 94.90 ash content,% 2.61 1.14 0.98 fat content,% 3.20 1.45 1.37 moisture content,% 4.09 2.21 2.75 рн 8.10 8.10 7.85 «collagen plus» (3.82%) and «helios-11» (2.21%) have the lowest moisture content. the low fat content in the protein preparation «helios-11» (0.72) is explained by the use of higher quality raw materials and better alkaline treatment. the high content of ash in the proteins «scanprotm t95» (2.81%) is a consequence of the inefficient technology of washing and removal of calcium salts. the high protein content of manufacturers such as «collagen plus» and «helios-11» indicates a high level of raw material preparation technology for protein food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 263 production. the ph in the range of 5-9 does not directly affect the qualitative characteristics of the proteins. to assess the level of functional and technological properties (moistureretaining capacity mrc and fat-retaining capacity frc) of the experimental protein preparations, appropriate studies were conducted, the results of which are shown in figure 1. the results of moisture-retaining and fatretaining capacity analysis of the experimental protein preparations indicate that all protein preparations have high functional and technological properties, however, the «helios-11» protein provides the highest performance. thus, the investigated additives flour mixture from oil cultures and beef collagen protein «helios-11» are promising and scientifically valid components for further use in the composition of complex mixtures for regulating the functionaltechnological properties of meat systems. fig. 1 comparative moisture-retaining and fatretaining capacity of animal proteins in the next stage, a compositional proteincarbohydrate mixture obtained from flour of oils cultures (sesame, flax, sunflower and pumpkin) and collagen beef protein was investigated. the mixture was prepared by mixing the components in a dry form. to further justify the use of composite mixtures, their functional properties were studied under different component ratios and different degrees of hydration. the study of the swelling of the proteincarbohydrate composition was performed using the ratio of protein : flour mixture from oils cultures equal to 1:4, 1:2, 1:1, 2:1, 4:1. the results obtained are presented in figure 2. fig. 2 changes in the degree of swelling of the protein-carbohydrate composition depending on the ratio of protein : flour mixture from oils cultures the results of the studies show that the swelling of the test mixture increases in the samples where the content of oil cultures flour is higher. this can be explained by the fact that the chemical component of the flour mixture is fiber, which binds to water chemically and capillary-osmotic, while the protein is only chemical, and depends on the content in its structure of free amino and carboxyl groups. in order to establish the optimum value of the hydromodule for the proteincarbohydrate composition, a series of experiments was conducted, based on the addition to the mixture of different mass fraction of moisture at a temperature of 2025 ºc. the results of the experiments showed that one part of the mixture can firmly bind 3-3.5 parts of water, so the recommended hydromodule is 1:3. the investigated composite mixtures are intended to be used in the composition of emulsions, therefore, in the next stage, the stability and emulsifying ability were studied without heating the samples and with heating to 70 ° c for 20 min. (fig. 3 and fig. 4). thus, the results of the studies show that the highest rates have samples with a ratio food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 264 of protein : flour mixture from oils cultures 1:4 with a degree of hydration 1:3. fig. 3 changes in the emulsifying ability of the protein-carbohydrate composition depending on the ratio of protein: flour mixture from oils cultures fig. 4 changes in the emulsifying ability of the protein-carbohydrate composition depending on the ratio of protein: flour mixture from oils cultures next, the optimal duration of gel retention and the effect of temperature on its viscosity were determined experimentally. the results are presented in the form of a graph (fig. 5). fig. 5 effect of temperature and exposure time on gel viscosity studies have shown that the optimum duration of the gel edurance is 30 minutes, during this time the gel stabilizes and with further aging its strengthening is not observed. as can be seen from figure 5, heating to a temperature of 30-40 ° c increases the viscosity of the gel, and with a further increase in temperature this figure decreases slightly, which can be explained by the denaturation of proteins. 4. conclusions. the use of beef collagen protein «helios11» as a part of a protein-carbohydrate composition was substantiated on the basis qualitative indicators of studies and comparison of animal origin protein preparations in order to use them in the formulation of chopped semi-finished products and to improve their quality. ratios of protein and flour mixture from oils cultures (1:4) were selected, which create the most favorable conditions for the correction of meat systems by functional-technological properties, which contribute to the regulation of quality and are able to counteract the deficiencies of meat raw materials. the revealed features of functionaltechnological properties of composite protein-carbohydrate mixtures depending on the ratio of components and the level of hydration, what are the basis for further research. 5. references [1]. oliynyk l., modern directions of technologies improvement of meat semi-finished products, scientific bulletin of the poltava university of economics and trade, 1(78): 22–28, (2016) [2]. strashynskiy i., fursik o., pasichniy v., marynin a., goncharov g., influence of functional food composition on the properties of meat mince systems, eastern european journal of advanced technology, 6(11): 53-58, (2016) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 taisa honcharenko, oksana topchyi, selection of components of the compositional protein-carbohydrate mixture to improve the quality of the chopped semi-finished products, food and environment safety, volume xviii, issue 4 – 2019, pag. 258 – 265 265 [3]. simakhina g., korikhalova o., increasing the bioavailability of grain nutrients by mechanical activation, progressive techniques and technologies of food production in the restaurant industry and trade: coll. of scientific works, 2(10): 431–435, (2009) [4]. kraevskaya s., stetsenko n., analysis of the chemical composition of pumpkin, sesame and flax seeds as promising sources for the production of biologically active food additives, quality strategy in industry and education: ix international. conf, may 31 june 7, varna,: 95-97, (2013) [5]. ganorkar p., jain r., flaxseed a nutritional punch, international food research journal, 20(2): 519–525, (2013) [6]. e onsaard., pomsamud p., audtum p., functional properties of sesame protein concentrates from sesame meal, asian journal of food and agro-industry, 3(4): 420-431, (2010) [7]. adeel a., sohail a., masud t., characterization and antibicterial study of pumpkin seed oil (cucurbita pepo), life sciences leaflets: 53-64 (2014) [8]. zhongkai z., guoying l., bi s., physicochemical properties of collagen, gelatin and collagen hydrolysate derived from bovine limed split wastes, journal of the society of leather technologists and chemists, 90: 23-28, (2006) [9]. choi sy, ko e, lee yh, kim bg, shin hj, seo db, lee sj, kim bj, kim mn, effects of collagen tripeptide supplement on skin properties: a prospective, randomized, controlled study, j cosmet laser ther, 16(3): 132-139, (2014) [10]. ukrainits a., pasichny v., zheludenko yu., polumbrik m., the influence of collagen-based protein-containing compositions on the quality of sausage products, scientific and production journal "food science and technology", 10(3): 50-55, (2016) [11]. kyshenko i., starchova v., goncharov g., technology of meat and meat products, workshop, kyiv, nuft, (2010) [12]. topchii o., kotliar ye., honcharenko t., petryna а., tarasiuk о., use of oilseed polyfunctional supplements in the manufacture of meat products, food science and technology, 13(2): 78-86, (2019) «collagen plus» (3.82%) and «helios-11» (2.21%) have the lowest moisture content. the low fat content in the protein preparation «helios-11» (0.72) is explained by the use of higher quality raw materials and better alkaline treatment. the high conten... to assess the level of functional and technological properties (moisture-retaining capacity mrc and fat-retaining capacity frc) of the experimental protein preparations, appropriate studies were conducted, the results of which are shown in figure 1. 131 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 22020, pag. 131 138 impact of corn and rice syrup adulteration on physicochemical properties of tilia honey *paula ciursa 1 , mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, ciursa.paula@yahoo.com, *corresponding author received 25th april 2020, accepted 28th june 2020 abstract: the aim of this study was to highlight the impact of corn and rice syrups adulteration on physico-chemical parameters of tilia honey. for this purpose, tilia honey was adulterated by addition of concentrated corn and rice syrups in different percentages: 5%, 10%, 20% and 50%. the parameters chosen to study the influence of adulteration agents in honey were the following: ph, free acidity, electrical conductivity, color, moisture content, hydroxymethyl furfural content and the sugar content (fructose, glucose, sucrose, maltose, melesitose, turanose, trehalose, raffinose and f/g ratio). the result indicated that the adulteration with corn and rice syrup caused significant chances on most of physico-chemical parameters values. the moisture content of adulterated honey with both types of syrup increased depending on the degree adulteration from 16.75% in authentic honey to 19.39% for 50% adulterated honey with corn syrup and to 17.03% for adulterated honey with 50% rice syrup. the hydroxymethyl furfural content of honey samples did not exceed the maximum allowed limit (40 mg/kg). the free acidity content decreased from 14.56 meq/kg in authentic honey to 8.96 meq/kg in adulterated honey with 50% corn syrup and increased from 14.56 meq/kg to 18.17 meq/kg in adulterated honey with 50% rice syrup. electrical conductivity decreased 1.91 times in adulterated honey with 50% corn syrup and increased 1.41 times in adulterated honey with 50% rice syrup. keywords: tilia honey, adulteration, syrups, physico-chemical parameters 1. introduction honey is a natural produce with a characteristic taste and known for its therapeutic properties (antioxidant, antimicrobial, anti-inflammatory, antitumor) [1]. the composition of honey include about 200 substances, mainly sugars and water but also other substances such as proteins, organic acids, enzymes, vitamins, minerals, phenolic compounds, pigments and solid particles (results from honey harvesting) [2]. honey composition depends on various factors (floral source, soil, and climate) [3]. due to a high worldwide demand for bee products, the adulteration of honey has become an increasingly common problem. illegal honey producers add various types of syrups at a lower cost price, seeking to obtain greater economic benefits. thus, consumer confidence in the honey quality and safety is undermined, having also a negative effect on the competitiveness and profitability of honest producers [4]. in recent years, honeybee has been adulterated with different concentrated carbohydrate syrups such as: sugar cane, inverted sugar, rice syrup, agave syrup, date syrup, corn syrup, hfcs (highfructose corn syrup), fructose, glucose, zaharose and maltose syrups[5]. at present, guaranteeing the quality and authenticity of honey represents a very http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 132 important issue for both the national and international market. therefore, it is necessary to establish an analytical procedure for the detection of adulterated honey [4]. rice syrup is obtained from rice in three stages: hydrolysis (acid or enzymatic), refining and concentration. the main chemical components of rice syrup are glucose, maltose, trisaccharides, tetrasaccharides, etc. [6]. corn syrup is obtained from corn starch by enzymatic reaction using the enzymes α-amylase and glucoamylase.if the reaction is continued with the enzyme glucose isomerase, high fructose corn syrup (hfcs) of different degrees of isomerization (hfcs-90, hfcs-55, hfcs-42) can be obtained[7]. the chemical composition of these syrups is similar to that of authentichoney, but thenutritionalvalueismuchlower. thus, the detection of adulterated honey with corn and rice syrups has become an important topic for the production and quality of honey, but also for the nutrition and health of consumers. adulterated honey by addition of sugars may present changes in some physico-chemical parameters (color, free acidity, electrical conductivity etc.) and/or enzymatic activity, the content of various compounds such as fructose, glucose, sucrose, fructose/glucose ratio, maltose, proline and hydroxymethyl furural[8]. in this study, the influence of corn and rice syrups adulteration on the physicochemical parameters of romanian tilia honey is presented.to highlight the differences between authentic and adulterated honey samples, the analysis of variance (anova) was used. 2. materials and methods tilia sp. honey was purchased from a local beekeeper located in suceava county, romania. the syrups produced in south korea, were purchased from importing companies: daesang europe b.v. for corn syrup and from panaisia de handels gmbh for rice syrup. authentic honeywas adulterated with these types of syrups by addition in different percentages (5%, 10%, 20% and 50%) (w/w). the physico-chemical characteristics analyzed were: color, electrical conductivity, moisture content, ph, free acidity, hydroxymethyl furfural content and sugar content. before performing the analysis, all the samples were liquefied at 50° and homogenized. to measure the color of honey samples two instruments were used: a portable chromameter cr-400 (konica, minolta, japan) and a photometer pfund hi 96785 (hanna instruments, usa). the electrical conductivity was measured with a portable conductometer hq14d (hach, usa). for this purpose, 20 g of honey were dissolved in 100 ml of distilled water. the results were expressed in micro siemens per centimeter (µs·cm-1). the moisture content was analyzed using an abbé refractometer (leica mark ii plus), based on refractometry. this is a method that determines the refractive index of honey and the determination of the water content (%) is done using the chataway table [9]. a 10% honey aqueous solution was prepared to measure the ph with a mettler toledo fivego ph-meter (mettler toledo, usa). the free acidity was determined by titrimetric method with a titroline easy device (schott instruments, germany). the results were expressed in milliequivalents/kg of honey. the hydroxymethyl furfural (hmf) presence in honey was determined using the method proposed by white [10].the absorbance of the reference samples (containing 0.2% sodium bisulphite solution) and samples (containing distilled water) was read at 284 nm and 336 nm using a uv-vis-nir 3600 spectrophotometer (schimadzu food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 133 corporation, japan). the results were expressed in mg/kg honey. high performance liquid chromatography (hplc) was used to determine the sugar content according to the method published by bogdanov& baumann [11]. the mobile phase was the mixture acetonitrile: water (80:20) and standard substances used were: fructose, glucose, sucrose, maltose, melesitose, turanose, trehalose and raffinose. one-factor analysis of variance (anova) was the statistical analysis used to present the differences between samples. the anova is based on the law of total variance. 3. results and discussion color honey color is an important factor that depends largely on the polen content and the source of nectar. the various pigments that derive from these such as phenolic acids, flavonoids, anthocyanins along with the mineral content constitute the basic color of honey[12]. depending on the degree of adulteration, l*, a*, b*, δe* color parameters showed significant (p<0.001) or partially significant (p<0.01) changes for both types of syrups. the values of l* ranged between 37.76 for authentic honey and 40.81 for adulterated honey with 50% corn syrup and between 37.76 for authentic honey and 27.10 for adulterated honey with 50% rice syrup.the value of l* for corn syrup was 38.22 and for rice syrup was 34.13. ribeiro et al. [13] observed an increase of l* parameter for adulterated honey with high fructose corn syrup. a* parameter decrease for adulterated honey with corn syrup from 3.01 in authentic honey to 0.48 in adulterated honey with 50% syrup and increase for adulterated honey with rice syrup from 3.01 to 9.02 in adulterated honey with 50% syrup. the variation of b* parameter was between 28.88 and 31.17 for adulterated honey with corn syrup and between 28.88 and 13.41 for adulterated honey with rice syrup. δe* parameter ranged from 0.66 to 4.47 for adulterated honey with corn syrup and from 2.82 to 19.73 for adulterated honey with rice syrup. positive values for both coordinates (a* and b*) indicate that all the samples had nuance of color between yellow and red (first quadrant of cie l*a*b* color space). on the pfund scale adulterated with corn syrup decreased significantly from 35.64 in autentic honey to 27.72 in adulterated honey with 50% syrup (acording to the pfund scale, the color changed from extra light amber to white), and for adulterated honey with rice syrup increased significantly from 36.64 to 52.47 in adulterated honey with 50% syrup (the color changed from extra light amber to light amber acording to the pfund scale). the value for corn syrup (100%) was 33.46 and for rice syrup (100%) was 41.98. depending on the adulteration agent all the parameters from cie l*a*b* space showed significant changes (p<0.001). all these results are shown in table 1 and table 2.a. and table 2.b. ph a low ph prevents the development of microorganisms. the acidic content of honey bee is relatively low, influencing its texture, stability and term of validity [14]. depending on the degree of adulteration, the ph results for adulterated honey with corn syrup were not significant (p>0.05), the values being between 4.05 for authentic honey and 4.27 for adulterated honey with 50% syrup (table 2.a.). for adulterated honey with rice syrup the results were less significant (p<0.05), existing as in the case of adulterated honey with corn syrup an increase, with values between 4.05 for authentic honey and 4.36 for adulterated honey with 50% syrup (table 2.b.). ribeiro et al. [13] observed an increase in the case of adulterated honey with high fructose corn syrup. food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 134 depending on the adulteration agent, the changes were not significant, the ph values being 4.13 for corn syrup and 4.15 for rice syrup (table 1). free acidity free acidity is given by the presence of organic acids and organic ions. a high degree of acidity may indicate the fermentation of sugars into organic acids [15]. adulterated honey with corn syrup presented a significant decrease of free acidity, against to adulterated honey with rice syrup that presented a significant increase (p<0.001). the values ranged from 14.46 meq/kg for authentic honey to 8.96 meq/kg and 18.17 meq/kg, for adulterated honey with 50% corn syrup and 50% rice syrup, respectively, as shown in table 2.a. and table 2.b. depending on the adulteration agent, all the changes were significant, the values being 12.65 meq/kg for corn syrup and15.78 meq/kg for rice syrup (table 1). none of the analyzed samples showed free acidity values superior to 50 meq/kg [16]. electrical conductivity electrical conductivity depends on various factors such as the content of ash, proteins, organic acids, some sugars and polyols[14]. the electrical conductivity values decreases significantly (p<0.001) for adulterated honey with corn syrup from 333.63µs·cm-1 in authentic honey to 270 µs·cm-1in adulterated honey with 20% syrup and to 175.02 µs·cm-1for adulterated honey with 50% syrup (table 2.a.).the values for adulterated honey with rice syrup has shown a significant increase from 333.63 µs·cm-1in authentic honey to 397.12 µs·cm-1 (for adulterated honey with 20% syrup) and to 492.36 µs·cm-1 (for adulterated honey with 50% syrup) (table 2.b.). regardless of the adulteration agent, the changes were significant, too. the value for corn syrup was 279.70 µs·cm-1 and for rice syrup was 387.60 µs·cm-1 (table 1). all the analyzed honey samples showed values of electrical conductivity lower than the maximum allowed limit of 800µs·cm1[16]. moisture the moisture content of honey varies from year to year depending on the environmental conditions and the manipulation of beekeepers during the harvest period. high moisture content could increase the activity of water with negative effects on the development of yeasts and accelerate the crystallization of certain types of honey [15]. depending on the degree of adulteration, the moisture content presented a significant increase for adulterated honey with corn syrup, the values ranged from 16.75% (authentic honey) to 19.39% (adulterated with 50% syrup) (table 2.a.).for adulterated honey with rice syrup the increase was not significant (p>0.05), values ranged from 16.75% to 17.03% (adulterated honey with 50% syrup), as it can be observed in table 2.b. ribeiro et al. [13] observed also an increase for the adulterated honey by high fructose corn syrup (hfcs). depending on the adulteration agent, the changes were significant. the values for corn and rice syrups were 17.65% and 16.85% (table 1). the moisture content did not exceed the maximum allowed limit (20%) established by codex alimentarius and eu honey directive for any sample. hydroxymethyl furfural content (hmf) the hmf content is a parameter that indicates the degree of freshness of honey. this is a product of the maillard reaction, the maximum allowed value being 40 mg/kg of honey [12]. in this study, the hmf content did not exceed the established limit. depending on the degree of adulteration and on the adulteration agent the changes for hmf food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 135 content were significant (p<0.001). for adulterated honey with corn syrup the hmf content decreased from 13.34 mg/kg in authentic honey to 8.07 mg/kg in adulterated honey with 50% syrup and for adulterated honey with rice syrup the hmf content presented a significant increase from 13.34 mg/kg to 20.03 mg/kg adulterated honey with 50% syrup (table 2.a. and table 2.b). the values for corn and rice syrups (100%) were 11.55 mg/kg and 15.70% mg/kg (table 1). table 1. physico-chemical parameters of adulteration agent ns – not significant (p>0.05), * p<0.05, ** p<0.01, *** p<0.001, a-e –significant differences between sample sugar content for adulterated honey with corn syrup the significant changes (p<0.001) were in the case of fructose, glucose, turanose, maltose, trehalose and melesitose, the partially significant were in the case of raffinose (p<0.01) and for sucrose and f/g ratio the changes were not significant (p>0.05). in the case of fructose, glucose, turanose, maltose, trehalose, raffinose and f/g ratio for adulterated honeywith corn syrup was a decrease from 33.65% to 16.85% (fructose), 30.61% to 16.08% (glucose), 0.36% to 0.18% (turanose), 1.15% to 0.58% (maltose), 1.46% to 0.73% (trehalose), 0.19% to 0.15% (raffinose) and 1.09% to 1.04% (f/g ratio). an increase was in the case of melesitose from 0.75% to 17.89%. sucrose was not present in either authentic honey or corn syrup. for adulterated honey with rice syrup the changes were significant (p<0.001) for all sugar content analyzed in this study. a decrease was in the case of fructose (from 33.65% to 16.95%), glucose (from 30.61% to 22.57%), turanose (from 0.36% to 0.18%), maltose (from 1.15% to 0.57%), trehalose (from 1.46% 0.72%), raffinose (from 0.19% to 0.09%), f/g ratio (from parameter adulteration agent f – ratio interaction corn syrup rice syrup agent – degree of adulteration l* 38.22(3.93)a 34.13(1.50)b 311.07*** 118.24*** a* 2.78(1.27)b 6.01(2.12)a 10248.90*** 2186.62*** b* 30.12(1.04)a 24.65(6.19)b 947.05*** 327.26*** δe* 1.91(1.63)b 6.70(7.27)a 11377.98*** 3755.05*** color (mm pfund) 33.46(3.14)b 41.98(6.16)a 1214.70*** 317.96*** ph 4.13(0.09)a 4.15(0.12)a 1.16ns 0.32ns free acidity (meq/kg) 12.65(2.1)b 15.78(1.36)a 1157.91*** 316.39*** electrical conductivity (μs/cm) 279.70(59.52)b 387.60(59.63)a 2429.31*** 664*** moisture (%) 17.65(1.01)a 16.85(0.21)b 52.21*** 14.36*** hmf (mg/kg) 11.55(1.98)b 15.70(2.62)a 2176.06*** 595.20*** fructose (%) 27.93(6.30)a 27.97(6.26)a 0.04ns 0.01ns glucose (%) 25.67(5.45)a 27.88(3.02)a 164.13*** 44.83*** sucrose (%) 0b 0.02(0.03)a 1874.70*** 506.89*** turanose (%) 0.29(0.07)a 0.29(0.07)a 0.03ns 0.03ns maltose (%) 0.95(0.21)a 0.95(0.21)a 0ns 0ns trehalose (%) 1.21(0.27)a 1.20(0.27)a 0ns 0ns melesitose (%) 6.58(6.43)b 6.77(6.64)a 11.56** 3.17ns raffinose (%) 0.17(0.01)a 0.15(0.03)a 96.94*** 26.25*** f/g ratio 1.07(0.02)a 0.98(0.13)b 173.69*** 56.41*** food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 136 1.09 to 0.74). the content of sucrose and melesitose increased from 0% in authentic honey to 0.07% in adulterated honey with 50% syrup (sucrose) and from 0.75% to 18.47% (melesitose). regardless of the adulteration agent, the changes were not significant for fructose, turanose, maltose, trehalose (p>0.05). the values of fructose, glucose, trehalose, melesitose, raffinose and f/g ratio were 27.93% for corn syrup and 27.97% for rice syrup (fructose), 25.67% for corn syrup and 27.88% for rice syrup (glucose), 1.21% for corn syrup and 1.20% for rice syrup (trehalose), 6.58% for corn syrup and 6.77% for rice syrup (melesitose), 0.17% for corn syrup and 0.15% for rice syrup (raffinose) and 1.07 for corn syrup and 0.98 (f/g ratio) (table 1). all these results are shown in table 1 and table 2.a. and table 2.b. table 2.a. physico-chemical parameters of tilia honey adulterated with corn syrup tilia honey analysis results parameter degree of adulteration for corn syrup f ratio 0.00% 5.00% 10.00% 20.00% 50.00% l* 37.76(0.54)bc 36.8(0.52)c 37.48(0.54)bc 38.24(0.54)b 40.81(0.58)a 15.98** a* 3.01(0.04)c 3.93(0.06)a 3.55(0.05)b 2.95(0.04)c 0.48(0.007)d 1963.05*** b* 28.88(0.41)b 30.48(0.44)a 29.14(0.42)b 31.17(0.45)a 30.93(0.44)a 11.85** δe* 0e 2.08(0.03)c 0.66(0.008)d 2.33(0.03)b 4.47(0.06)a 4756.80*** color (mm pfund) 35.64(0.51)a 35.64(0.51)a 34.65(0.49)ab 33.66(0.48)b 27.72(0.40)c 95.64*** ph 4.05(0.06)b 4.07(0.06)b 4.09(0.06)b 4.14(0.06)ab 4.27(0.06)a 4.63ns free acidity (meq/kg) 14.56(0.21)a 13.99(0.20)b 13.44(0.19)c 12.32(0.18)d 8.96(0.13)e 296.82*** electrical conductivity (μs/cm) 333.63(4.77)a 317.77(4.54)bc 301.91(4.31)c 270.19(3.86)d 175.02(2.50)e 478.56*** moisture (%) 16.75(0.24)c 17.01(0.24)c 17.28(0.25)bc 17.80(0.26)b 19.39(0.28)a 34.08*** hmf (mg/kg) 13.34(0.19)a 12.81(0.18)b 12.28(0.17)c 11.23(0.16)d 8.07(0.12)e 314.67*** fructose (%) 33.65(0.48)a 31.96(0.46)b 30.29(0.43)c 26.92(0.39)d 16.85(0.24)e 533.57*** glucose (%) 30.61(0.43)a 29.15(0.42)b 27.70(0.40)c 24.80(0.36)d 16.08(0.23)e 480.28*** sucrose (%) 0 0 0 0 0 0ns turanose (%) 0.36(0.007)a 0.34(0.008)b 0.32(0.002)c 0.28(0.005)d 0.18(0.0005)e 354.91*** maltose (%) 1.15(0.02)a 1.09(0.01)b 1.03(0.01)c 0.92(0.01)d 0.58(0.007)e 467.11*** trehalose (%) 1.46(0.02)a 1.38(0.01)b 1.31(0.02)c 1.16(0.01)d 0.73(0.007)e 621.39*** melesitose (%) 0.75(0.007)e 2.46(0.03)d 4.17(0.06)c 7.60(0.11)b 17.89(0.25)a 5749.03*** raffinose (%) 0.19(0.007)a 0.18(0.002)ab 0.17(0.006)ab 0.17(0.0007)b 0.15(0.001)c 21.53** f/g ratio 1.09(0.01)a 1.08(0.02)a 1.08(0.02)a 1.07(0.02)ab 1.04(0.01)b 3.09ns ns – not significant (p>0.05), * p<0.05, ** p<0.01, *** p<0.001, a-e –significant differences between sample food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 137 table 2.b. physico-chemical parameters of tilia honey adulterated with rice syrup tilia honey analysis results parameter degree of adulteration for rice syrup f ratio 0.00% 5.00% 10.00% 20.00% 50.00% l* 37.76(0.54)a 36.39(0.52)b 35.36(0.50)b 34.08(0.49)c 27.10(0.39)d 144.83*** a* 3.01(0.042)d 5.43(0.078)c 5.41(0.078)c 7.23(0.11)b 9.02(0.13)a 1211.89*** b* 28.88(0.41)a 29.38(0.42)a 27.05(0.39)b 24.52(0.35)c 13.41(0.19)d 657.06*** δe* 0e 2.82(0.04)d 3.86(0.05)c 7.09(0.10)b 19.73(0.28)a 6285.09*** color (mm pfund) 35.64(0.51)d 39.6(0.57)c 38.61(0.55)c 43.56(0.62)b 52.47(0.75)a 231.60*** ph 4.05(0.06)b 4.08(0.06)b 4.11(0.06)b 4.17(0.06)b 4.36(0.06)a 8.89* free acidity (meq/kg) 14.56(0.21)d 14.92(0.22)cd 15.28(0.22)c 16.00(0.23)b 18.17(0.26)a 80.33*** electrical conductivity (μs/cm) 333.63(4.77)e 349.50(4.99)d 365.38(5.22)c 397.12(5.67)b 492.36(7.04)a 254.31*** moisture (%) 16.75(0.24)a 16.78(0.24)a 16.81(0.24)a 16.86(0.24)a 17.03(0.24)a 0.41ns hmf (mg/kg) 13.34(0.19)e 14.03(0.20)d 14.73(0.21)c 16.12(0.23)b 20.30(0.29)a 297.30*** fructose (%) 33.65(0.48)a 31.98(0.46)b 30.31(0.43)c 26.97(0.38)d 16.95(0.24)e 526.97*** glucose (%) 30.61(0.43)a 29.80(0.43)ab 29.00(0.41)b 27.39(0.39)c 22.57(0.32)d 128.54*** sucrose (%) 0d 0.009(0.002)c 0.01(0.003)c 0.03(0.001)b 0.07(0.001)a 506.89*** turanose (%) 0.36(0.007)a 0.35(0.008)b 0.32(0.002)c 0.28(0.005)d 0.18(0.003)e 348.06*** maltose (%) 1.15(0.02)a 1.09(0.01)b 1.03(0.01)c 0.92(0.01)d 0.57(0.009)e 454.88*** trehalose (%) 1.46(0.02)a 1.38(0.01)b 1.31(0.02)c 1.16(0.01)d 0.72(0.008)e 615.74*** melesitose (%) 0.75(0.007)e 2.52(0.04)d 4.29(0.06) 7.84(0.11)bc 18.47(0.26)a 5746.84*** raffinose (%) 0.19(0.007)a 0.17(0.005)b 0.16(0.005)b 0.15(0.0008)c 0.09(0.001)d 129.01*** f/g ratio 1.09(0.01)a 1.06(0.02)ab 1.03(0.02)b 0.97(0.02)c 0.74(0.01)d 162.51*** ns – not significant (p>0.05), * p<0.05, ** p<0.01, *** p<0.001, a-e – significant differences between sample 4. conclusion in this study the impact of honey adulteration with corn and rice syrups on the physico-chemical properties was analyzed. statistical analysis indicated that most of the physico-chemical parameters presented significant differences (p<0.001) between authentic and adulterated honeys. free acidity, electrical conductivity, moisture and hmf contents did not exceed the maximum allowed limit for any analyzed sample. ph values had a low increase from 4.05 to 4.27 for adulterated honey with corn syrup and from 4.05 to 4.36 for adulterated honey with rice syrup. the color parameters on both color cie l*a*b* and pfund scales showed significant changes (p<0.001 in the case of adulterated honey with rice syrup and significant and partially significant (p<0.01) for adulterated honey with corn syrup, due to the intensity degree of color of the adulteration agent. food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xix, issue 2 – 2020 paula ciursa, mircea oroian, impact of corn and rice syrups adulteration on physicochemical properties of tilia honey, food and environment safety, volume xix, issue 2 – 2020, pag. 131 138 138 the content of fructose, glucose, turanose, maltose, trehalose and melesitose decreased significantly for adulterated honey with corn and rice syrups. for turanose, maltose and trehalose the changes were similar in the adulterated honey with both types of syrups. depending on the adulteration agent the changes for the following parameters were not significant (p>0.05): ph and sugar content (fructose, turanose, maltose and trehalose). 5. acknowledgment this work was supported by “decide development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu/380/6/13/125031, supported by project co-financed from the european social fund through the 2014 – 2020 operational program human capital”. 6. references [1]. fernandes, l., ribeiro, h., oliveira, a., silva, a. s., freitas, a., henriques, m., & rodrigues, m. e. portuguese honeys as antimicrobial agents against candida species. journal of traditional and complementary medicine, (2020). [2]. da silva, p. m., gauche, c., gonzaga, l. v., costa, a. c. o., & fett, r. honey: chemical composition, stability and authenticity. food chemistry, 196, 309-323, (2016). [3]. pereira, j. r., campos, a. n. d. r., de oliveira, f. c., silva, v. r., david, g. f., da silva j. g., ... & denedai, â. m. physicalchemical characterization of commercial honeys from minas gerais, brazil. food bioscience, 100644, (2020). [4]. geană, e. i., ciucure, c. t., costinel, d., & ionete, r. e. evaluation of honey in terms of quality and authenticity based on the general physicochemical pattern, major sugar composition and δ13c signature. food control, 109, 106919, (2020). [5]. oroian, m., paduret, s., & ropciuc, s. honey adulteration detection: voltammetric e‐ tongue versus official methods for physicochemical parameter determination. journal of the science of food and agriculture, 98(11), 43044311, (2018). [6]. cai, j., wu, x., yuan, l., han, e., zhou, l., & zhou, a. determination of chinese angelica honey adulterated with rice syrup by an electrochemical sensor and chemometrics. analytical methods, 5(9), 23242328, (2013). [7]. parker, k., salas, m., & nwosu, v. c. high fructose corn syrup: production, uses and public health concerns. biotechnol mol biol rev, 5(5), 71-78, (2010). [8]. geană, e. i., & ciucure, c. t. establishing authenticity of honey via comprehensive romanian honey analysis. food chemistry, 306, 125595, (2020). [9].bogdanov, s., lüllmann, c., martin, p., von der ohe, w., russmann, h., vorwohl, g., ... & flamini, c. honey quality and international regulatory standards: review by the international honey commission. bee world, 80(2), 61-69, (1999). [10]. white jr, j. w. spectrophotometric method for hydroxymethylfurfural in honey. journal of theassociation of official analytical chemists, 62(3), 509-514, (1979). [11]. bogdanov, s., & baumann, e. bestimmung von honigzuckermit hplc. mitt geblebensmitteluntershyg, 79, 198-206 (1988). [12]. can, z., yildiz, o., sahin, h., turumtay, e. a., silici, s., & kolayli, s. an investigation of turkish honeys: their physicochemical properties, antioxidant capacities and phenolic profiles. food chemistry, 180, 133-141, (2015). [13]. ribeiro, r. d. o. r., mársico, e. t., da silva carneiro, c., monteiro, m. l. g., júnior, c. c., & de jesus, e. f. o. detection of honey adulteration of high fructose corn syrup by low field nuclear magnetic resonance (lf 1h nmr). journal of food engineering, 135, 39-43, (2014). [14]. oroian, m., olariu, v., & ropciuc, s. influence of adulteration agents on physicochemical and spectral profile of different honey types. international journal of food engineering, 4(1), (2018). [15]. gomes, s., dias, l. g., moreira, l. l., rodrigues, p., & estevinho, l. physicochemical, microbiological and antimicrobial properties of commercial honeys from portugal. food and chemical toxicology, 48(2), 544-548, (2010). [16]. eu, 2001. council directive 2001/110 relating to honey. official journal of the european communities. 1. introduction 269 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 269 275 sustainable packaging solutions for organic fresh berries *elisabeta elena tănase1, adina alexandra baicu1, vlad ioan popa1, amalia carmen miteluț1, mihaela draghici 1 , andreea stan 1 , paul alexandru popescu 1 , mona elena popa 1 university of agronomic sciences and veterinary medicine of bucharest, 59 marasti blvd, district 1, bucharest, romania, elena.eli.tanase@gmail.com * corresponding author received 30th october 2017, accepted 8th december 2017 abstract: climate changes and particularly global warming are topics carefully treated by specialists already since decades. the most pregnant factor that influences climate change is pollution, namely the high level carbon dioxide emissions. besides other substances used by the most of the industries (oil, charcoal, fertilizers, etc.), plastics are not to be ignored when talking about pollution. plastic waste affects animals and humans, as well as their habitat. in this respect, food industry engages in preserving the good functioning of the environment by developing and using biodegradable and bio-based resources for food packaging. the aim of this literature review was to identify the optimal sustainable packaging solution used for berries. the results of the study pointed out that the most used environmentally friendly packaging technique is the one that involves modified atmosphere. in terms of packaging materials, the literature is limited when it comes to biodegradable/bio-based solutions. however, active packaging gains popularity among researchers, considering the endless possibilities to include sustainable compounds in a biopolymer based matrix, in order to prolong the shelf-life of berries or fruits in general. keywords: organic fresh berries, sustainable packaging, biodegradable/bio-based materials 1. introduction over the last few years, consumers started to appreciate the advantages of convenience food in general, and of fresh fruits and vegetables in particular. this trend comes together with a series of challenges for the food business operators that try to stay on the market by offering the desired attributes of fresh produce and seek for better post-harvest solutions to avoid operating at a loss [1]. it became common knowledge that fruits and vegetables are rich in components that sustain human health and wellbeing, and, as a results, they are promoted to be consumed in a daily basis. the benefits of consuming fresh fruits and vegetables are highlighted by the correlations between specific compounds of the produce and their favorable effects in the fight against cancer, neurodegenerative diseases, obesity, inactivity, poor diet or even erectile dysfunctions. the recent scientific evidence in that respect acts as an engine for promoting consumption of fresh fruits and vegetables and it leads to a market growth of such products [2], [3], [4]. the perks of having a larger part of the population interested in this kind of goods come with the reality of producing enough marketable harvest. the most coming to the forefront challenge of fresh fruits and vegetables producers is the fast decline of the quality characteristics, as well as the short shelf-life. an important role in the changes that occur on the quality of fresh fruits and vegetables is played by the metabolism of the products, as well as by the mechanical http://www.fia.usv.ro/fiajournal mailto:elena.eli.tanase@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 270 swelling or physiological defects. these undesired changes can lead to depreciation of the nutritional value, taste, color, texture, and make the products less attractive to the consumers. in addition, they can lead to the deterioration of the quality attributes and in financial loss for the food business operator, [1], [5], [6], [7]. food is a dynamic system [8] and it is subject of both chemical and biological spoilage. chemical spoilage can occur as a result of the oxidation process, but the most widely spread type of spoilage is the biological one. it can be caused by enzymatic degradation, contamination (with protozoa, parasites, or viruses), or microbial contamination. in the case of microbial spoilage, the nature of the food (ph, texture, water activity, amount/type of nutrients and oxygen contained), plays a vital aspect with regards to the microorganism that can cause spoilage – be it bacteria, yeast or moulds [9]. in the case of fruits, as they are mostly acid products, their resistance to bacteria is higher than to fungi [10], [11]. in order to reduce post-harvest waste and loss, as well as to prolong the timeframe in which the fruits meet the consumer demands and expectations, post-harvest technologies are being developed and optimized accordingly. in this respect, optimal methods and techniques are still being investigated, having the purpose of maintaining high quality of fruits and to extend their shelf-life. amongst the most recognized and successfully applied chemical or physical techniques are: the use of ozonized water, sanitizers, heating, uv light treatments and irradiating [1], [12], [13]. food packaging in general and also of fruits in particular, has the primary function of protecting them from mechanical injuries to maintain quality and to delay microbial growth [9], [14]. the most widely spread material used in packaging is plastic, which is known to have negative effects on the environment. plastic waste affects animal and humans, as well as their habitat. the united nations draws attention that before 2050, plastics will exceed the fish in the oceans if people keep using plastic at the same rate [15]. this research proposes to point out possible suitable methods for sustainable packaging for berries. 2. materials and methods this study consists of a literature review. it aims to identify optimal solutions for sustainable packaging for organic fresh berries. in order to deliver our statements and arguments, 53 articles in english have been taken into our attention. after being analyzed, 38 out of 53 articles proved to be relevant for our investigation. 3. overview of available, promising packaging options antimicrobial packaging the concept of active packaging is fairly new, but the research in this field knows a rapid development and the directions taken within the subject are multiplying [15], [16]. the main purpose of the active packaging technology consists of creating a mutual action between food and the package itself, at the same time as assisting the microenvironment generated within the unit. active packaging should provide microbial safety, maintain the quality of the product, its nutritional value and prolong its shelf-life [17]. antimicrobial packaging can drag out the time frame of realistic usability by deterring the development of target food spoilage microorganisms either by broadening their lag-phase or by reducing their growth rate [9], [16]. the mechanism used for designing active packages roots in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 271 introducing antimicrobial agents (in this case, chemical) into the packaging structure. later on, however, edible coatings are available, as well as emitting sachets, sheets, films or gases. in addition, it is also possible to induce vacuum, which does not only inhibit the oxidation process of foods, but it also has antimicrobial and antifungal characteristics, targeting aerobic bacteria [9]. having in mind the actual need of environmentally friendly options, to avoid plastic and food waste, a dynamic film based on methylcellulose and maqui berry (aristotelia chilensis) extract was challenged against microorganisms and oxidative reactions. because methylcellulose’s availability to bind with water, it was cross-linked with glutaraldehyde at various concentrations. the glutaraldehyde concentration added proved to be proportional with the amount of the antioxidant agent released [18]. similarly, extruded recycled pet was enriched with potassium sorbate in two concentrations (2,5 and 4,0 wt%). the rate of the botrytis cinerea growth was proportional to the quantity of potassium sorbate added as antifungal agent. after the migration tests were performed for potassium sorbate, it was found that the results were within the legal limits [19]. another successful experiment done on wild berries was performed by adding a naturally occurring volatile compound, 2nonanone to an equilibrium modified atmosphere package [20]. outstanding results were also obtained by using a combination of modified atmosphere packaging and eugenol or thymol. grapes were stored in said conditions for 56 days. after the observations, it was concluded that losses caused by sensorial, functional, nutritional and fungal decay were significantly lower on the samples where eugenol or thymol was used [21]. biomaterials biomaterials, besides being environmentally friendly and highly demanded by consumers, can be cheap to procure, as they can be by-products from other industries or the materials can be easily found in nature [22], [23]. pectin, a resource that is available in apples and citrus fruit, is biodegradable, non-toxic and renewable. it is already used in biomedicine and films made with pectin proved to extend shelf-life of foodstuffs and can be used in drug delivery systems [23]. polylactic acid (pla) is synthetized from corn and it is also a widely used material as an alternative to conventional plastics [24]. almenar et al. (2008) recommend pla to be used in packaging for fresh berries – it prolonged the postharvest life of blueberries at different temperatures and suggests sensory tests to be performed concerning flavour discrepancies [25]. a combination of rice starch, fish protein and oregano essential oil proved to be worth considering in the development of active, biodegradable packaging [26]. aloe vera gel was also used for an edible coating on grapes, proving to have the capacity of inhibiting microbial (yeast and moulds) growth [10]. chitosan is another remarkable biomaterial used for extending the post-harvest life of fresh fruits and vegetables. it was effectively used to extend the shelf-life of apples, litchi fruits and peaches [22]. in an interesting experiment carried out on strawberries stored at 7oc, chitosan beads supplemented with lavender essential oil extended the fruits’ shelf-life to 8 days, comparing to the control sample that after 2 days of storage was already mouldy [27]. sodium alginate was also used in edible coatings for apples and for sweet cherries delaying their ripening time outstandingly [28]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 272 essential oils (eos) with respect to natural antimicrobials, scientific literature suggests that the most common methods to apply the treatments are by spraying, rinsing, washing, vaporing or edible coatings [29]. essential oils continue to prove efficiency in fighting and inhibiting spoilage microorganisms in both fresh fruits and fresh vegetables. thyme, oregano and lemongrass eos in combination with map were successfully used to extend the shelf-life of cabbage [30]. in strawberries, botrytis cinerea was inhibited or delayed using thyme or lavender eo [30], [31], [32]. in order to avoid the disadvantage of eos, they can be introduced into a polymer matrix, so the release is done gradually. a study of jurmkuwan et al. (2016) showed that by encapsulating thyme and lavender essential oils into a chitosan matrix, inhibited the growth of b. cinerea and maintained the quality of strawberries, without interfering with their taste [32]. the concentrations of eos used in food packaging are being optimized, as the taste resulted after the treatments can be unacceptable by the consumers. consequently, studies with panels are organized and it can be remarked that the right concentration of eo can be both effective in prolonging the post-harvest life of food and adequate for the consumers [20]. modified atmosphere packaging modified atmosphere packaging (map) has been used already since the first half of the previous century, when new zealand shipped lamb and beef to the uk stored under co2 conditions [33]. ever since then, methods and gas concentrations are being optimized and perfected. as a result of its relatively long history, the technology of map has been largely approached in literature too. however, we will bring to bear on recent research done on the use of map to extend the shelf-life of fruits and vegetables and particularly, of berries. the effect of map was tested on pomegranate seed pods. after being flushed with low o2 atmosphere, the seeds were suitable for consumption for up to 6 and 9 days (depending on the tray they were placed in). differently, when the pomegranate arils were flushed with high o2 atmosphere, their accepted quality was prolonged to over 9 days. the authors of this experiment (banda et al., 2015) recommend optimization of gas levels, and to avoid introducing low levels of o2 in trays sealed with high barrier polymeric films, since this combination leads to offodours [34]. thus, it can be observed that failing to create appropriate environment may and in shortening the shelf-life of the foodstuffs [35]. table grapes have also been taken into research, results showing that map was effective in extending the period in which the rachises kept their green color. this attribute is directly correlated with the quality of the grapes after harvest. this study suggests, however, that are still elements to be optimized for assuring a stable potential technology [36]. when storing blueberry under map, it was found to be more effective when the sample kept in perforated bag was fumigated with so2. differently, the blueberries placed under map only, were subject of weight loss, decay, softening and dehydration [37]. another experiment performed on table grapes tested whether proper map can have beneficial effects on the sugar, anthocyanin and organic acid content, as well as on the antioxidant activity. the fruits were stored for 3 months at 0oc either in perforated polyethylene bags or in zoepac map bags, with or without sachets emitting ethanol vapours (antimold®30, antimold®60 or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 273 antimold®80) or pads emitting so2. it was observed that so2 was effective in maintaining the sugar and organic acid levels, as well as preserving the anthocyanin content, but it transferred an off-taste to the grapes [38]. 4. conclusions after reviewing the relevant scientific literature, it can be concluded that many options are potentially effective in extending the post-harvest life of berries. however, not much information is available on suitable biodegradable materials. from what recent literature provides, we can observe that science knows various directions with regards to shelf-life prolongation of fresh fruits and vegetables, but are still many details to be polished before we can benefit from technologies that can represent the knowledge transfer from laboratory level to industry scale. in addition, aside the need adjust the said details, it should be considered researching for further solution regarding convenient biodegradable materials to be combined with technologies intended to assist shelflife and quality of fresh fruits and vegetables. 5. acknowledgments financial support for this project is provided by funding bodies within the core organic plus fp7-era-net618107 ensuring quality and safety of organic food along the processing chain and with cofounds from the european commission uefiscdi era net for romanian partner usamvb “innovative and eco-sustainable processing and packaging for safe and high quality organic berry products with enhanced nutritional value” ecoberries. 6. references [1] hussein, z., caleb, o. j., opara, u.l. perforation-mediated modified atmosphere packaging of fresh and minimally processed produce a review, in food packg and shelf life, v6, pages 7-20, (2015). [2] allende, a., tomas-barberan, f.a., & gil, m.i. minimalprocessingfor healthy traditional foods.trendsinfoodsci&tech, 17, 513–519, (2006). [3] ramos, b., miller, f. a., brandão, t. r. s., teixeira, p., & silva, c. l. m. fresh fruits and vegetables—an overview on applied methodologies to improve its quality and safety. innovative food science and emerging technologies, 20, 1–15. (2013). [4] cassidy, a., franz, m., & rimm, e. b. dietary flavonoid intake and incidence of erectile dysfunction. the american journal of clinical nutrition, 103(2), 534–541, (2016). [5] mahajan, p. v., caleb, o. j., singh, z., watkins, c. b., & geyer, m. postharvest treatments of fresh produce. philosophical transactions of the royal society a, 372, 1–19, (2014). [6] opara, u. l., al-ani, m. r., & al-rahbi, n. m. effect of fruit ripening stage on physicochemical properties, nutritional composition and antioxidant components of tomato (lycopersicum esculentum) cultivars. food and bioprocess technology, 5(8), 3236–3243, (2012). [7] sandhya. map of fresh produce: current status and future needs. a review. lwt—food sci and techn, 43, 381–392, (2010). [8] luning, p.a., marcelis, w.j. food quality management. technological and managerial principles and practices. wageningen academic publishers, wageningen, netherlands. p. 48 (2009). [9] jung h. han antimicrobial packaging systems, in food sci and techn, academic press, london, pages 80-107, innovations in food packaging (2005). [10] long n., joly, c., dantigny, p. active packaging with antifungal activities, int j of food micr 220, 73–90 (2016). [11] pitt, j., hocking, a.d.fungi and food spoilage. aspen publishers, inc., gaithersburg, maryland 593 pp, (1999). [12] hong, p., hao, w., luo, j., chen, s., hu, m., & zhong, g. combination of hot water, bacillus amyloliquefaciens hf-01 and sodium food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 274 bicarbonate treatments to control postharvest decay of mandarin fruit. postharvest biology and technology, 88, 96–102, (2014). [13] maghoumi, m., gomez, p. a., artes hernandez, f., mostofi, y., zamania, z., & artes, f. hot water, uv-c and superatmospheric oxygen packaging as hurdle techniques for maintaining overall quality of freshcut pomegranate arils. journal of science of food and agriculture, 93, 1162–1168, (2012). [14] kelly, m., steele, r., scully, a. rooney, m. enhancing food security through packaging. intlrev. food sci. technol. 11,115-117, (2003). [15] world economic forum (2016) – the new plastics economyrethinking the future of plastics. available:http://www3.weforum.org/docs/wef_th e_new_plastics_economy.pdf[accessed: 27/10/17] [16] gutiérrez, l., sánchez, c., batlle, r., & nerín, c. new antimicrobial active package for bakery products. trends in food science and technology, 20, (2009). [17] king, k. packaging and storage of herbs and spices.handbook of herbs and spices.3.86-102, (2006). [18] de dicastillo, c.l., rodríguez, f., guarda, a., galotto, m.j. antioxidant films based on cross-linked methyl cellulose and native chilean berry for food packaging applications, carbohydrate polymers 136, 1052–1060 (2016). [19] junqueira-gonçalves, m.p., keshavan niranjan, e.a. development of antifungal packaging for berries extruded from recycled pet, in food control, volume 33, issue 2, pages 455-460 (2013). [20] almenar, e., catala, r., hernandez-munoz, p., gavara, r. optimization of an active package for wild strawberries based on the release of 2-nonanone, lwt food sci and techn 42, 587–593, (2009). [21] valero, d., valverde, j.m., mart´inez-romero, d., guillen, f., castillo, s., serrano m., the combination of modified atmosphere packaging with eugenol or thymol to maintain quality, safety and functional properties of table grapes, postharvest biology and techn 41, 317–327 (2006). [22] cazon, p., velazquez, g., ramírez j.a., vazquez m. polysaccharide-based films and coatings for food packaging: a review, food hydrocolloids 68, 136-148, (2017). [23] nesic, a., ruzic, j., gordic, m., ostojic, s., micic, d., onjia, a. pectinpolyvinylpyrrolidone films: a sustainable approach to the development of biobased packaging materials, composites part b 110, 56-61, (2017). [24] mistriotis, a., briassoulis, d., giannoulis, a., d’aquino, s. design of biodegradable bio-based equilibrium modified atmosphere packaging (emap) for fresh fruits and vegetables by using micro-perforated poly-lactic acid (pla) films, in postharvest biology and technology, 111, 380-389, (2016). [25] almenar, e., samsudin, h., auras, r., harte, b., rubino, m. postharvest shelf life extension of blueberries using a biodegradable package, food chemistry 110, 120–127, (2008). [26] romani, v.p., prentice-hernández, c., guimarães martins, v. active and sustainable materials from rice starch, fish protein andoregano essential oil for food packaging, industrial crops and products 97, 268–274, (2017). [27]sangsuwan,j.,pongsapakworawat, t., bangmo, p., sutthasupa, s. effect of chitosan beads incorporated with lavender or red thyme essential oils in inhibiting botrytis cinerea and their application in strawberry packaging system, lwt food science and technology 74, 14-20, (2016). [28] davidson, p.m., bozkurt cekmer, h., monu, e.a., techathuvanan, c. the use of natural antimicrobials in food: an overview, handbook of natural antimicrobials for food safety and quality, edited by t.m. taylor,, woodhead publishing, oxford, pages 1-27, (2015). [29] hyun, j.-e., bae, y.-m., song, h., yoon, j.-h. and lee, s.-y. antibacterial effect of various essential oils against pathogens and spoilage microorganisms in fresh produce. j food saf, 35: 206–219, (2015). [30] reddy, m. v. v., angers, p., gosselin, a., & arul, j. characterization and use of essential oil from thymus vulgaris against botrytis cinerea and rhizopus stolonifera in strawberry fruits. phytochemistry, 47, 1515-1520, (1998). [31] rattanapitigorn, p. arakawa, m., tsuro, m. vanillin enhances the antifungal effect of plant essential oils against botrytis cinerea. internat jr of aromatherapy, 16, 193-198. (2006). [32] jurmkwan, s., titimon, p., peeraya., b., sutthira, s. effect of chitosan beads incorporated with lavender or red thyme essential oils in inhibiting botrytis cinerea and their application in strawberry packaging system. lwt food sci and techn. 74, (2016). [33] farber, j.m., dodds, k. principles of modified-atmosphere and sous vide product packaging. crc press, canada, (1995). [34] banda, k., caleb, o.j., jacobs, k., opara, u.l. effect of active-modified atmosphere packaging on the respiration rate and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 elisabeta elena tănase, adina alexandra baicu, vlad ioan popa, amalia carmen miteluț, mihaela draghici, andreea stan, paul alexandru popescu, mona elena popa, sustainable packaging solutions for organic fresh berries, food and environment safety, volume xvi, issue 4 – 2017, pag. 269 – 275 275 quality of pomegranate arils (cv. wonderful), postharvest biology and techn 109,97–105 (2015). [35] mangaraj, s., goswami, t.k., mahajan, p.v. applications of plastic films for modified atmosphere packaging offruits and vegetables: a review.food eng.rev.1, 133–158, (2009). [36] silva-sanzana, c., balic, i., sepúlveda, p., olmedo, p., león, defilippi b.g., blanco-herrera, f., campos-vargas, r. effect of modified atmosphere packaging (map) on rachis quality of ‘red globe’ table grape variety, postharvest biology and technology 119, 33–40, (2016). [37] rodriguez, j., zoffoli, j.p. effect of sulfur dioxide and modified atmosphere packaging on blueberry postharvest quality, postharvest biology and techn 117, 230–238, (2016). [38] ustun, d., candir, e., ozdemir, a.e., kamiloglu, o., soylu, e.m., dilbaz, r. effects of map packaging and ethanol vapor treatment on the chemical composition of ‘red globe’ table grapes during storage, postharvest biology and techn68, 8–15, (2012) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 contents: 1. rat kidney tissues: oxidative modification of proteins under water and salt loads associated with the hgcl2 nephropathy alla velyka 261 271 2. advantages of using proteins in the production of truncated semi-finished products iryna shevchenko, alexey skochko 272 277 3. improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables lessoy zoue , sébastien niamke 278 285 4. determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia rehrahie mesfin, getnet assefa, fassil assefa 286 299 5. the potoential role of chicken meat in transmission of campylobacter jejuni to consumers nagwa thabet elshraway, ahmed ibrahim youssef, mohamed shaker abdel hafez 300 308 6. modern aspects of marketing research application as determinant of promotional mix in business entities from milk processing industry in the republic of macedonia katerina bojkovska, fanche joshevska, andrijana velevska, sasho bojkovski 309 316 7. consumption of aflatoxin contaminated peanut butter: a health threat to the population in lusaka urban zambia maureen s. banda, rosemary. n. likwa, phoebe bwembya, jeremiah banda, allan. mbewe 317 326 8. validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu 327 331 9. chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda) aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke 332 340 10. pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon jean sonchieu, john frunsoh, caroline nain waingeh 341 351 11. author instructions i v 12. subscription information vi 176 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag.176 190 nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan dina anwar 1 , *ghadir el-chaghaby 1 1 regional center for food and feed, agricultural research center, giza, egypt ghadiraly@yahoo.com *corresponding author received 23th july 2019, accepted 27th september 2019 abstract: as the world population is increasing, tofu and seitan may play an important role in human nutrition as inexpensive protein-rich food sources. in order to increase the consumption of tofu and seitan, modified processes are needed to meet the needs of different consumers. in this study, the aim of the work is to set up fried tofu and seitan that couple high nutritional value and good sensory features and to evaluate their nutritional profile and protein quality as well as the consumer’s acceptance. the average values of tested organoleptic parameters of tofu and seitan resulted in a positive influence after frying process. cooking loss due to frying was lower in case of fried seitan over fried tofu. the protein content of both fried tofu and seitan samples was higher than 20%, fat and energy of the control were increased after frying process. minerals and antioxidant capacity were higher in the modified products as compared to the traditional ones. most of essential amino acid values were enhanced after frying seitans especially with wheat flour coated seitan, however, raw tofu was proven to be a rich source of essential amino acids over fried ones and different seitan samples. tofus were found to be higher in essential amino acid index (eaai) and biological value (bv) over seitan samples. tofu samples with higher amino acids content had higher values of digestibility and pdcaas. overall, the new formulations of tofu and seitan could be used to enhance their nutritional quality and taste. keywords: nutritional profile, protein quality, sensory evaluation, mineral content 1. introduction proteins are the main constituents of agricultural raw materials with two main functions: bioand techno-function. biofunctionality of proteins is related to their nutritional and physiological properties, while techno-functionality is related to their physico-chemical ones [1]. one of the earliest indications of the importance of the increment of food protein resources was to overcome the world malnutrition. it is expected that the world demand for proteins of animal origin would be doubled by 2050 [2]. the replacement of animal proteins by novel plant proteins has been driven by sustainability assurance in the protein food sources and considered the simplest way to solve the shortage in animal protein sources. protein rich plantbased foods are legumes, grains, nuts and dairy replacers. the current ready-made meat and dairy replacers are based on wheat, soy and lupine, more than other plant based protein sources from the fact that they have an amino acid composition of quite high quality [3]. a growing awareness in the population about healthy and sustainable foods has lead to a rising interest in plant protein based meat alternatives in many european countries and worldwide [4]. soybean foods are rich in protein and have been shown to offer specific health benefits. tofu (soy bean curd) is one of the most important food products made from soy bean protein [5]. it is an important food for eastern asians due to its good http://www.fia.usv.ro/fiajournal mailto:ghadiraly@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 177 nutrition and digestibility [6]. western countries have recently increased interest in eating tofu due to its benefits to human health and the united states has increased tofu consumption [7]. it has been increasingly used in numerous asian dishes, replacing dairy products due to comparatively low cost and high protein bioavailability [8]. tofu is usually considered as salt – or acid – coagulated water based gel, with soy lipids and proteins trapped in its gel networks [5]. it is a cheap, nutritious and versatile meat or cheese substitute with bland taste and porous texture and hence called “tofu is meat without bone”. the main health benefits of tofu will show up in significantly lower total cholesterol, triglycerides and low-density lipoprotein (bad cholesterol) if one eats tofu regularly instead of meat [9]. on the other hand, cereals (e.g wheat, oats, etc) can be considered the other side of plant protein sources. wheat gluten is becoming a very large player in the industry, with new production facilities for gluten and textural wheat products expanding all over the world [10]. gluten can be readily prepared by gently washing dough under a stream of running water. this removes the bulk of the soluble and particulate matter to leave a protein aceous mass that retains its cohesiveness on stretching [11]. soy protein and wheat gluten have been the dominant raw materials for meat surrogates for a long time. these proteins have unique techno-functional taste and nutritional properties depending on their origin and how they are processed [4]. historically, wheat protein has been used for thousands of years as a meat substitute called seitan in china, japan, korea, and russia [10]. gluten can be flavored in a variety of ways; when simmered in a traditional broth of soy sauce, ginger, garlic, it is called seitan. seitan contains small amount of sodium and extremely low fat protein. in addition, gluten (setian) has a very similar texture to meat, making it ideal for vegetarian dishes meant to mimic meat-based ones. seitan is a great option for vegans who cannot eat soy. recently, western countries have increased the interest in eating tofu and seitan which are considered as high protein source especially for vegetarians. despite their high functional properties, they are not typically consumed in the basic arab countries diet because of their unpleasant flavor. the acceptability level of foods depends mostly on cultural habits, therefore, when a food is created or modified, it is extremely important to evaluate it with consumers or potential consumers. thus, the objective of this study is to prepare tofu and seitan by two different methods and to evaluate the products in terms of sensory characteristics, nutritional quality, amino acid scores and antioxidant capacity. 2. matherials and methods 2.1. raw material dried soy beans (glycine max), giza 111 cultivar were obtained from the field crops research institute, agricultural research center. wheat flour was purchased from the local market. the chemicals were obtained from elgomhoria company for chemicals (elmataria, egypt). 2.2. preparation of tofu tofu was prepared by the following method [12]: one kilogram of soy bean seeds were washed, soaked in 4l of water for 16 hrs at room temperature, drained and rinsed with water. soaked seeds were then processed by hand dehulling and the cotyledons were ground in low speed blender with addition of water (8 l) for 2 minutes followed by high speed for 5 minutes. the resultant bean puree was heated to 80-85 ͦ c with constant stirring. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 178 the slurry was filtered through cheese cloth to separate soy milk from residue (okara) and the liquid was heated to boiling. the soy milk was allowed to cool to 70 ͦ c and while stirring, a saturated solution of calcium sulphate (2% of dry soy bean weight) was added to soy milk until it began to coagulate. the suspension was allowed to form a curd without further stirring. after one hour, the curd was transferred into cheese cloth lined on plastic strainer. the curd was pressed to separate the whey. at the end of pressing, the curd had attained the required firmness and consistency of tofu. the cloth was removed and the tofu was stored refrigerated until used. 2.3. fried tofu preparation fried tofu was prepared by mixing optimum quantities of onion paste (12%), corn starch (4.2%), bicarbonate (0.7%), salt (1.5%), spices (0.6%) and chili sauce together with fresh tofu (74%). the tofu mix was then formed into discs of 14g. the produced tofu patties were coated with wheat flour (7.0%) and fried using sunflower oil at 170 ͦc for 20 sec. the fried tofus were cooled till examination. 2.4. seitan preparation to produce seitan, wheat flour was mixed with tap water as needed to make dough. the dough was washed repeatedly under running water to remove starch and some bran until a glutenprotein was observed. seitan was stored in a refrigerator until used. 2.5. fried seitan preparation seitan (wheat gluten) was soaked in mixed ingredients of onion juice, yoghurt, lemon juice, soy sauce, salt and spices and allowed to rest for one hour. seitan was crosswise cut into four pieces then simmered in water for 15 min. cooked seitan was cut into small pieces then coated with wheat flour (1) or chickpea flour (2). coated seitans were fried using sunflower oil at 170 ͦ c for 20 sec and the fried seitan was cooled till examination. 2.6. investigations for sensory evaluation: samples from each of tofu and seitan patties were fried in a preheated pan for 5 min (2.5 min. on each side) before coded and evaluated for test, color, odor, texture, bite and overall acceptability using 10-point descriptive scales, where a score of 1 denotes for extremely poor and a score of 10 denotes for excellent (as described before by [13]. the weight of tofu and seitan patties were measured before and after frying to determine cooking loss and calculated according to the equation below: cooking loss% = cooked weight – raw weight x 100 (1) raw weight 2.7. chemical determinations 2.7.1. proximate analysis moisture, protein, fat (ether extractable), fiber and ash contents were determined according to methods described in [14]. while, total carbohydrates were estimated by difference according to [15]. 2.7.2. minerals content potassium, magnesium, phosphorous and iron were analyzed by atomic absorption spectrophotometry 3300 perken elmer, while, calcium was analyzed by icp optima 2000 dv perken elmer according to the method described in the [14]. 2.7.3. soluble, in soluble and total dietary fiber soluble dietary fiber (sdf), in soluble dietary fiber (idf) and total dietary fiber (tdf) were determined according to the methods in [16]. 2.7.4. antioxidant assay total antioxidant capacity of the samples was determined using the phosphomolebdenum method [17] using ascorbic acid as standard. the results were expressed as milligram ascorbic acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 179 equivalent per 100 milliliters (mg aae/100 ml). 2.7.5. amino acids content amino acids determination was performed according to [14]. the system used for the analysis was eppemdorf lc 3000 ez chrom. aessential amino acid index (eaai) the eaai was calculated using eq. (1) [18]. eaai = (2) where n is the number of the essential amino acids; a, b,….h are the concentrations of the essential amino acids (lysine, isoleucine, valine, theronine, leucine, phenylalanine, histidine and methionine) in test sample, and av, bv, …..hv are the concentrations of the essential amino acids in standard protein (%) (casein) bnutritional index (ni) the ni was calculated using eq. (2). ni (%) = eaai x protein (%) (3) 100 cbiological value (bv) the bv was calculated using eq. (3) [1819]: bv = 1.09 x eaai – 11.7 (4) dpredicted protein efficiency ratio (p-per) the p-per was calculated using the regression equation, eq. (4) [20-21]: p-per = 0.468 + 0.454 (leu) – 0.105 (tyr) (5) 2.7.6. determination of amino acid scores determination of the amino acid scores was first based on casein. in this method, essential amino acids were scored. secondly, amino acid score was calculating using the following formula by [22]: uncorrected amino acid score% = mg of essential amino acids 1g of test protein x100 (6) mg of amino acid 1g of reference protein 2.7.7. in-vitro protein digestibility determinations the in vitro protein digestibility (ivpd) was measured using a multienzyme system (pepsin, chloramphenicol and pancreatin) according to the method of [23]. crude protein (cp) was determined by the macro kjeldahi technique (%n x 6.25) [14]. protein digestability was calculated with the formula: % protein digestibilty = (cp sample – cp undigested) / (cp sample) x 100 (7) 2.7.8. protein digestibility corrected amino acid score (pdcaas) the indicator of protein quality was calculated based on the total amino acid contents of the sample, considering for its calculation t he limiting aa (g/16gn) of the sample in relation to the same aa of a reference protein and multiplied by the samples % digestibility according to [24]. the pdcaas was calculated according to the following equation: pdcaas (protein digestibility corrected amino acid score) = lowest uncorrected amino acid score x protein digestibility (%) (8) 3. results and discussion 3.1. sensory quality of fried tofu and seitan the sensory evaluation was done on a ten-point scale by a ten panelists and the results are shown in table (1). fried tofu was scored higher than 8.0 for the parameters considered (taste, odor, color, texture, bite and overall acceptability). in addition, 95% of consumers expressed the will of buying it. fried tofu’s taste had a level of acceptance near ‘extremely like’, therefore, incorporation of the ingredients in fried patties formulation increased the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 180 pleasant taste of tofu. tofu has a mild flavor and a porous texture and, due to its neutral sensory characteristics, the tofu texture has an important role in the quality of the product, as well as in the consumer’s acceptance [25]. table 1 sensory evaluation of fried tofu and seitan products parameters tofu seitans fried tofu fried seitan (1) fried seitan (2) taste 9.55 8.70 8.80 color 9.45 8.60 8.45 odor 8.70 8.15 9.00 texture 8.25 7.80 7.80 bite 9.05 6.80 7.15 over all acceptability 9.00 8.01 8.24 cooking loss% 26.76% 2.15% 1.56% (1) fried seitan coated with wheat flour (2) fried seitan coated with chickpea flour the frying process increased the pleasant taste of the products; it also maintained the high average of texture. tofu had high cooking loss (26.76%) which is probably due to its lower ability to hold the moisture during the frying process. table (1) also shows the mean scores of the consumer’s acceptance for fried seitan coated with wheat or chickpea flour. both of them were scored higher than 8.0 for taste, color, odor and overall acceptability. although, the tested seitans were characterized by similar ingredient contents, the use of chickpea flour instead of wheat flour involved an increase of the taste, odor and overall acceptability, probably due to a greater acceptance of chickpea by consumers. taste and aroma are mostly the important attributes that influence the sensory properties of products. cooking seitan (deep fat frying/70 ͦ c) resulted in low cooking loss percentage. 3.2. proximate composition the proximate composition of tofus and seitans are presented in table (2). the results showed that moisture percentage was higher in raw tofu compared to fried tofu. while, changes in moisture content of different seitan samples were not noticeable. the protein content of raw tofu was 11.29% of fresh matter which was lower than that of fried tofu (21.21%), probably due to the high moisture content of raw tofu. table 2 proximate composition (g/100g) and the energy value of tofus and seitans products macronutrients tofus seitans raw tofu fried tofu raw seitan fried seitan (1) fried seitan (2) moisture 74.92 44.61 49.30 48.30 51.59 protein 11.29 21.21 34.27 21.71 24.52 fiber 0.36 0.37 0.16 0.02 0.36 carbohydrate 4.15 14.20 15.24 22.17 16.82 lipid 7.84 15.08 0.78 6.40 5.09 ash 1.44 4.53 0.25 1.40 1.62 energy (kcal/100g) 132.3 277.36 205.06 233.12 211.17 (1) fried seitan coated with wheat flour (2) fried seitan coated with chickpea flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 181 tofu manufacture is in many respects similar to cheese-making, the essential difference being that a lactic acidproducing bacterial starter culture is not employed in its preparation [26]. solids of tofu are composed chiefly of protein of high qualitythe net protein utilization (npu) of tofu is reported to be 65% making it equivalent to chicken meat in terms of assimilability and digestibility. soybean protein showed hypochloesterolemic and soy isoflavones have antiatherogenic effect [27]. it was also reported that the soy protein decreased effectively serum concentration of total cholesterol and triglycerides as compared to animal proteins [28]. on the other hand, seitan contained 34.27% of protein while it reached 21.71% and 24.52% of fresh matter for fried seitan coated with wheat or chickpea flour, respectively. it was clear that use of chickpea flour caused an increase in protein content of fried seitan over wheat flour. the fiber content of tofus and seitans was comprised between 0.02% and 0.37% of fresh matter. frying tofu result an increase in carbohydrates (10%), lipid (7.24%) and ash (3.1%). deep-fat frying of tofu with subsequent fat absorption increase the energy value (from 132.3 to 277.36 kcal/100g for tofus before and after frying, respectively). similar trends were observed for seitan samples, the fried seitan either coated with wheat of chickpea flour contained high carbohydrates, lipids and ash in compare with raw seitan. the highest energy value was recorded for fried seitan with wheat flour (233.12 kca/100g) over other seitans. the high energy value in cooked burger patties was attributed to the reduction in moisture content during cooking [29]. the predicted dietary protein intake of 100g of raw and fried tofu and seitan for males and females was shown in fig (1). raw tofu can supply about 21.2 and 24.5% of the protein requirement for males and females, respectively and this percentage would increase with fried tofu. fig. (1): % dietary protein intake of raw and fried tofu and seitan on the other hand, raw seitan can supply about 61.2 and 74.5% of recommended daily protein for males and females, respectively while these percentages were decrease with fried seitans but it still provide more than 38% of the protein requirement for both males and females. 3.4. mineral content mineral content of raw and fried tofu is shown in the table (3). the results showed that k and mg were the most abundant minerals in raw tofu; however, ca and p recorded higher mean values over other minerals in fried tofu. the ratio of ca/p of raw and fried tofu was 0.707 and 1.383, respectively. according to many authors a good food has a ca/p ratio higher than one, while a poor food has a ratio lower than 0.5[30]. the ratio of ca/p in the food is related to teeth and bone formation and therefore children and youth food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 182 need higher intakes of calcium and phosphorus. since the ratio of ca/p in tofus either raw or fried is above 0.5, tofu can be considered a good food. as regards the seitan samples, the lowest mean value of fe, k and p was recorded by raw seitan. fried seitan coated with wheat flour contained big amounts of k and p which recorded the highest value among other seitan samples. on the other hand, fried seitan with chickpea flour yielded the highest mg level followed by raw seitan and fried seitan with wheat flour. calcium was not detected in raw seitan but it was recorded as 204.0 and 177.3 mg/kg for fried seitan with wheat and chickpea flour, respectively. these results may be related to the yoghurt addition in the seitan’s broth. a slight increase was also shown in ca/p ratio of fried seitan with chickpea flour over wheat flour. finally, the different ingredients added to tofu and seitan recipes before the frying process enhanced their mineral content and thus, fried tofu and seitan can provide high amount of minerals which are associated with good human health. table 3 mineral composition (mg/kg) of tofus and seitans products minerals tofus seitans raw tofu fried tofu raw seitan fried seitan (1) fried seitan (2) fe 24.00 13.24 22.46 76.88 71.02 k 239.6 411.1 225.8 2688 506.4 mg 3010 137.3 184.4 69.23 309.0 ca 12.19 4597 nd 204.0 177.3 p 17.23 3323 3.51 1603 918.3 ca/p 0.707 1.383 0.127 0.193 (1) fried seitan coated with wheat flour (2) fried seitan coated with chickpea flour 3.5. dietary fiber content the results of soluble, insoluble and total dietary fiber shown in table (4) indicate that total dietary fiber (tdf) levels in the tofu and seitan ranged from 2.12 to 5.95%. on the other hand, the content of soluble diatery fiber (sdf) and insoluble dietary fiber (idf) in raw tofu was lower than that in fried tofu which may be due to the lost soluble carbohydrates in the whey during tofu making. these results were in agreement with previous results [31] .in the case of seitans, fried seitan with chickpea flour had highest content of idf followed by raw seitan and seitan with wheat flour. table (4): dietary fiber (%) of tofus and seitans products fiber tofus seitans raw tofu fried tofu raw seitan fried seitan (1) fried seitan (2) idf 1.88 2.82 1.57 1.09 4.45 sdf 1.61 1.66 1.77 1.03 1.50 tdf 3.49 4.48 3.34 2.12 5.95 (1) fried seitan coated with wheat flour (2) fried seitan coated with chickpea flour dietary fibre is regarded as one of the most important ingredients in human diet [32]. the characteristics of fibre such as particle size, bulk volume, surface area and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 183 adsorption as well as organic molecules are more effective in human digestive system [32-33]. it was observed that the addition of dietary fibre in foods can improve the overall qualities such as cooking prosperities and textural characteristics as well as taste [34]. 3.6. antioxidant capacity the antioxidant capacity of raw and fried tofu and seitan is shown in the fig (2). fried tofu showed high antioxidant level (861.6 mgaae/100g), up to 1.8 fold of antioxidant capacity of raw tofu (487.9 mgaae/100g). in this context, it was reported that isoflavones, aglycones and proteins present in tofu have antioxidant properties protecting from lipid oxidation [35]. fried seitan coated with wheat flour contained higher levels, up to 2.4 fold of antioxidant than raw seitan. although it is believed that that deep-fat frying changes the quality of fried foods and nutritional quality of foods [36], but in the present study, the use of onion paste in fried tofu recipe and lemon juice for seitan may be the reason for improvement of their antioxidant capacity which can reduce the harmful effects of frying process. fig. (2): antioxidant activity of raw and fried tofu and seitan 3.7. amino acids profile and protein quality of tofus and seitans amino acid composition is critical to evaluate the quality of a dietary protein source through quality also relates to other food properties (e.g presence of metabolic interfering substances digestibility and chemical integrity) with potential to manipulate the proportion of utilizable amino acids. particularly, the essential amino acids content in protein source primarily influences the quality of that protein [37]. table (5) shows the amino acids composition of the amino acids composition of tofu and seitan before and after frying in g/16gn. the sum of essential amino acids (teaa) ranged from 25.77g in fried seitan with chickpea flour to 37.72g in raw tofu. raw tofu was higher than 36% considered for an ideal protein [38] but it is lower than that in casein standard of 39.18%. leucine and lysine content were higher in tofus and these values were higher than the recommended dietary requirements of fao for adult foods while, the methionine content was the lowest amino acid in tofus. seitan samples contained low amounts of lysine. however, fried seitan coated with chickpea flour caused an increase in lysine content, up to 1.18 fold than wheat flour and 1.23 fold than raw seitan.nevertheless, seitans contained appreciable amount of leucine and phenylalanine, but it is still lower than that in tofu samples. fried seitan coated with wheat flour contained 1.76g/100g of methionine which recorded the highest amount in seitan samples followed by raw seitan (1.66g/100g) and fried seitan with chickpea flour (1.65g/100g). these relative food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 184 deficiencies in lysine in seitan samples and that of methionine in fried tofu can be largely attributed to their disparate composition of major storage proteins. raw and fried tofus were found to be rich in valine and phenylalanine. the histidine content in the studied samples followed a similar pattern, being the highest in tofus (table 5). histidine has multiple roles including that in protein interactions, histamine synthesis, and repair and tissue growth [39]. table 5 amino acid profile of tofus and seitans treatments t1 t2 t3 t4 t5 (1) rda%* amino acids essential amino acids (g/100g cp.) theronine 3.86 3.78 2.44 2.54 2.51 1.50 valine 5.06 4.90 3.44 4.00 3.30 2.60 isoleucine 4.68 4.62 3.30 3.69 3.30 2.00 leucine 7.91 7.38 6.51 7.02 6.52 3.90 phenylalanine 5.17 5.03 4.66 4.64 4.28 2.50 histidine 2.71 2.50 2.03 2.14 1.89 1.00 lysine 6.40 5.22 1.89 1.97 2.32 3.00 methionine 1.93 1.40 1.66 1.76 1.65 1.50 conditionally essential amino acids (g/100g cp.) tyrosine 3.71 4.09 3.20 3.11 2.84 arginine 6.97 6.55 3.33 3.40 3.57 cysteine 2.13 1.98 2.29 2.59 2.72 proline 4.84 5.37 11.71 11.85 11.86 glycine 4.48 4.15 3.45 3.95 3.44 non-essential amino acids (g/100g cp.) aspartic acid 10.77 10.41 3.08 3.66 4.38 serine 4.88 4.64 4.20 4.45 4.01 glutamic acid 18.20 18.45 39.30 33.80 28.94 alanine 4.73 4.30 2.77 3.50 2.78 teaa 37.72 34.83 25.93 27.76 25.77 tcea 22.13 22.14 23.98 24.90 24.43 tneaa 60.71 59.94 73.33 70.31 64.54 (1)t1= tofu, t2 = fried tofu, t3 = seitan, t4 = fried seitan with wheat flour and t5 = fried seitan with chickpea flour (2) teaa=total essential amino acids, tcea=conditionally amino acids, tneaa=total non essential amino acids, tsaa=total sulphur amino acids, taraa=total aromatic amino acids, taaa=total acidic amino acids, tbaa= total basic amino acids. *rda%=recommended dietary allowanced fao/who, 2007 [38]. recent evidences have indicated the dietary essentiality of traditionally considered non essential (dispensable) amino acids as there is lack of substantive research on the assumption that nutritionally non-essential amino acids are adequately synthesized in human beings to meet the basal requirements [40]. these amino acids, termed functional amino acids include aspartic acid, serine, glutamic acid, alanine, arginine, proline and glycine and also play important roles among others in intestinal integrity [41], immune responses [42], cell growth and differentiation [43] and antioxidant defense [44]. as shown in the table (5), glutamic acid was the predominant amino acid amongst the other amino acids ranging from 39.3g/100g in raw seitan to 18.20g/100g in raw tofu. aspartic acid content was the highest in tofu samples while proline was found to be the highest in seitan samples. both asparatate and serine play as precursors of other amino acids. over all, the amino acids content of proteins in different samples can vary due food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 185 to the kind of seed’s protein and the procedures carried out to get the final products. variation was found in the content of some of the amino acids including lysine, aspartic acid and glutamic acid. the nutritional quality of tofus and seitans is presented in the table (6). tofu samples contained higher ratio of essential amino acids than total amino acids (teaa/taa %) as compared with seitans. however, the values of tofus and seitans were above 26% for ideal protein food for children and 11% for adult [45]. the sum of the acidic amino acids was higher in seitan samples than that in tofus. conversely, tofu samples were found to have higher total basic amino acids than seitans. an observation on asp/glu showed that the level of asp appeared to affect the glutamic value and vice versa as shown in the table (6). table 6 protein quality of tofu and seitan treatments t1 t2 t3 t4 t5 (1) parameters nutritional quality (2) total amino acids (taa) 98.43 94.77 99.26 98.07 90.31 teaa/taa % 38.32 36.75 26.12 28.30 28.53 tneaa/taa% 61.67 63.24 73.87 71.69 71.46 teaa/tneaa 0.62 0.58 0.35 0.39 0.40 tsaa ( meth. + cys ) 4.06 3.38 3.95 4.35 4.37 cys / tsaa % 52.46 58.57 57.97 59.54 62.24 taraa ( pheny + tyr) 8.88 9.12 7.86 7.75 7.12 leu / ileu ratio 1.69 1.60 1.97 1.90 1.98 leu-ileu (difference) 3.23 2.76 3.21 3.33 3.22 leu / ileu % 25.66 23.00 32.72 31.09 32.79 taaa ( asp + glu) 28.97 28.86 42.38 37.46 33.32 asp/ glu 0.59 0.56 0.08 0.10 0.15 arg/lys 1.09 1.25 1.76 1.73 1.54 tbaa ( arg + lys) 13.37 11.77 5.22 5.37 5.89 teaa+arg+his/taa % 48.15 46.30 31.52 33.95 34.58 p-per 2.734 2.453 2.152 2.393 1.705 eaai % 95.90 87.12 64.67 69.07 64.92 bv % 92.83 83.26 58.79 63.58 59.06 nutritional index % 43.15 33.26 43.72 29.00 32.88 (1)t1= tofu, t2 = fried tofu, t3 = seitan, t4 = fried seitan with wheat flour and t5 = fried seitan with chickpea flour (2) teaa=total essential amino acids, tcea=conditionally amino acids, taa=total amino acids, tneaa=total non essential amino acids, tsaa=total sulphur amino acids, taraa=total aromatic amino acids, taaa=total acidic amino acids, tbaa= total basic amino acids, p-per= predicted protein efficiency ratio, eaai =essential amino acid index, bv= biological value. thus, the lowest asp/glu was recorded in raw seitan (0.08) while, it was of 0.59 in raw tofu and 0.56 in fried tofu. the arg/lys ratios obtained in this work ranged from 1.09 to 1.76. it was previously reported that high ratio of arg/lys in the diet can produce beneficial hypocholesterolemic effects, improving the cardiovascular health, in addition to helps in hypertension regulation [46-47]. the values of predicted protein efficiency ratio (p-per) of seitan samples were between 1.705 in fried seitan coated with chickpea flour to 2.393 in fried seitan with wheat flour whilst, it reached 2.453 for fried tofu and 2.734 for raw tofu (table 6). the p-per in all seitan samples were lower than 2.88 in that was recorded in whole hen’s egg [48] and 2.5 that found in reference casein [49]. nevertheless, the p-per in raw tofu was higher than the value for reference casein and lower than whole hen’s egg. the eaai of tofu samples (raw and fried) were 95.90 and 87.12%, respectively, therefore, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 186 tofus are considered a good nutritional quality since the eaai is ranged 80 – 90% as previously reported [19]. however, seitan samples were lower in the present study as compared with the values previously reported [19] showing that protein based food is in adequate when it’s eaai below 70%. the predicted bv exhibited the highest percentage in raw tofu followed by fried tofu and then fried seitan coated with wheat flour, while, raw seitan was the lowest. scientifically, it is well known that a protein-based food nutritional is of good quality when its biological values (bv) are as high as 70 and up to 100% [19].the increase in bv of the tofu samples either raw or fried is an indication of improved digestibility potential and effective utilization. table (6) also showed the nutritional index (ni) of tofu and seitan samples. the results showed that the raw tofu and seitan had higher ni followed by fried tofu and fried seitan with chickpea flour while, fried seitan with wheat flour was the lowest one. 3.7.1. amino acid scores amino acid content in foods can be used to calculate the amino acid score, which provides a way to predict how efficiently protein will meet a person’s amino acid needs [38]. table (7) shows the amino acid scores for tofu and seitan samples analyzed in this study based on the essential amino acid content and the pattern for casein standard. as expected, lysine was the first limiting amino acid in cereal products. seitans expressed the wheat protein (gluten) and therefore, lysine amino acid was the lowest score among other amino acids. table 7 essential amino acid score % of tofu and seitan relative to casein standard t1= tofu, t2 = fried tofu, t3 = seitan, t4 = fried seitan with wheat flour and t5 = fried seitan with chickpea flour however, the highest score of seitan samples was recorded for phenylalanine. the results of the amino acid scores in tofu samples indicated that methionine was the limiting amino acid. it was also reported that legumes have low values of sulfurcontaining amino acids such as methionine [50]. generally, raw tofu had the highest score in all essential amino acid as compared with fried tofu and different seitan samples. 3.8. in-vitro protein digestibility and protein digestibility corrected amino acid scores (pdcaas) the in vitro protein digestibilities of tofu and seitan samples are presented in table (8). the lowest values were observed for fried seitan with chickpea flour and fried seitan with wheat flour. fried seitan with wheat flour had a relatively high amino acid score as compared to other seitans. treatments t1 t2 t3 t4 t5 casein standard essential amino acids theronine 102.1 100.0 64.55 67.19 66.40 3.78 valine 86.94 84.19 59.11 68.72 56.70 5.82 isoleucine 103.0 101.7 72.69 81.27 72.69 4.54 leucine 95.53 89.13 78.62 84.78 78.74 8.28 phenylalanine 113.6 110.5 102.4 101.9 94.07 4.55 histidine 106.2 98.03 79.61 83.92 74.12 2.55 lysine 90.26 73.62 26.66 27.78 32.72 7.09 methionine 75.09 54.47 64.59 68.48 64.20 2.57 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 187 however, protein quality is not on the amino acid profile, but also on the protein digestibility. on the other hand, fried seitan with chickpea flour had low digestibility but had higher protein content than wheat flour and these differences in protein digestibility indicated that the preparation method may have an effect on the availability of amino acids. the main determinant of food protein quality is the content and availability of essential amino acids. these nutrients have been shown to play an important role in the growth, reproduction and maintenance of the human body [38]. table 8 % protein digestibility and % pdcaas of tofu and seitan samples foods % protein digestibility % pdcaas tofu 93.00 70 fried tofu 89.64 49 seitan 84.88 23 fried seitan (1) 77.37 22 fried seitan (2) 63.62 21 (1) fried seitan coated with wheat flour (2) fried seitan coated with chickpea flour bongnar [51] reported that frying without any additional ingredients, as it is normally the case, does not change the digestibility of protein. when reducing substances are added to the food that is fried, for instance, carbohydrates (flour), protein digestibility is lowered slightly, albeit significantly. on the other hand, the previous data showed that kind of flour can also affect the protein digestibility and use of wheat flour as a coating substance of seitan before frying is better than the use of chickpea flour to enhance the protein digestibility. as regards tofu samples, raw tofu sample was the highest in % digestibility (93%) as compared to other samples. these results are in accordance with previously reported results [52] showing that the digestibility for tempeh was 91.41 and this value was higher than that found in meat (90.79) or pure beef burger (90.04), even though temph is a form of soya and it was processed via fermentation without thermal processing. this study provided information on the amino acid content and protein digestibility of fried tofu and seitan and this is new information that was not available for consumers. the protein digestibilitycorrected amino acid score (pdcaas) method has been considered to be a simple and scientifically sound approach for routine assessment of dietary protein quality of humans [21]. higher pdcaas for raw were recorded for tofu samples as compared with seitan samples (table8). the difference between the pdcaas of raw and fried seitan was small but the pdcaas of raw tofu were higher than that of fried tofu. it was earlier reported that a high protein quality of diets when pdcaas values above 70 – 80%. the vegetable source foods with the lowest pdcaas like wheat, maize and cassava, it is only when 50% of the protein is exchanged with an animal food source (i.e milk) that the increases are up to a level of 80% or above[53]. the addition of 25% milk powder brings pdcaas values to a reasonable level, above 70 %, while the level, when adding meat, is only of 60 % or slightly above, for wheat and maize. the data on the composition of fried tofu and seitan are important to determine the nutritional value of these diets and can be useful for improving their protein quality to make a significant impact on growth. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 188 4. conclusions plant-based food preparations produced from pulses or grains have always been a major source of protein in human diet. tofu and seitan have a very protein contents and can be considered as meat alternatives. this recent trend is being accompanied and promoted by a growing number of new food products from various raw materials. frying method had a good effect on the pleasant and acceptability of tofu and seitan. fried tofu and seitan contained a moderate amount of protein, which was above of 20% and high content of carbohydrates, lipids and energy in compare with traditional ones. however, the new recipes followed by frying process of tofu and seitan caused an increment in desired minerals and antioxidant activity. fried tofu proved to be a good source of essential amino acids with respect to protein quality. a correspondence was found between essential amino acids of fried products and % digestibility and pdcaas, since tofu samples with higher amino acids content had higher values of digestibility and pdcaas. 5. acknowledgments the authors are thanksfull to the regional center for food and feed at the agricultural research center, giza, egypt. 6. references [1] anna i., minkiewicz pi., protein as the source of physiologically and functionally active peptides. acta scientiarum polonorum. technologia alimentaria. 6(3): 5-15, (2007) [2] fao. dietary protein quality evaluation in human nutrition. in report of an fao expert consultation. fao food and nutrition paper 92. rome, italy: food and agriculture organization of the united nations, (2013) [3] tijhuis m.j., ezendam j., westenbrink s., van rossum c. & temme l., replacement of meat and dairy by more sustainable protein sources in the netherlands. in: quality of the diet. rivm. letter report 350123001/2011. pp. 163, (2011) [4] wild, f., czerny, m., janssen, a. m., kole, a. p. w., zunabovic, m., & domig, k. j. the evolution of a plant-based alternative to meat. from niche markets to widely accepted meat alternatives. agro food industry hi-tech, 25(1), 45-49., (2014) [5] kohyma k., sano y. & doi e., rheological characteristics and gelation mechanism of tofu (soybean curd). journal of agricultural and food chemistry 43, 1808 – 1812, (1995) [6] tsai s.j., lan, c.y. kao c.s. & chen s.c., studies on the yield and quality characteristics of tofu. journal of food science46, 1734 – 1740, (1981) [7] wang h.l., tofu and tempeh as potential protein sources in the western diet. j. am. oil chem soc. 61(3), 528 – 535, (1984) [8] dey a., tofu: technological and nutritional potential. indian food industry magazine. 36, 3, (2017) [9] jubayer m.f., uddin m.b. & faruque. m.o., standardization parameters for production of tofu using wsd-y-1 machine. journal of bangladesh agricultural university, 11 (2): 307 – 312, (2013) [10] cooper n., veggie burgers and meat analogs. diabetes self-management. 18:53 – 58, (2001) [11] shewry, p.r.; n.g. halford; p.s. belton and a.s. tatham. the structure and properties of gluten: an elastic protein from wheat grain. philosophical transactions of the royal society b: biological sciences,357, 133 – 142, (2002) [12] wang h.l., cavins j.f., yield and amino acid composition of fractions obtained during tofu production. cereal chemistry, 66(5), 359 – 361, (1989) [13] kassem m. a. g., emara, m. m. t., quality and acceptability of value-added beef burger. world journal of dairy and food sciences. 5(1): 14 – 20, (2010) [14] aoac. official methods of analysis of aoac international.20th ed. arlington: aoac international, (2016) [15] egan h.i., kirk r.s. & sawyar r., pearson’s chemical analysis of food (8th ed.). churchill livingistogen, edintugh. uk, (1981) [16] aoac. official methods of analysis of aoac international.17th ed. arlington: aoac international, (2000); [17] prieto p., pineda m. & aguilar m., spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 189 to the determination of vitamin e. analytical biochemistry, 269, 337 – 341, (1999) [18] ijarotimi s.o., keshinro o.o., determination of nutrient composition and protein quality of potential popcorn. african locust and bambara groundnut seed flour. polish journal of food and nutrition sciences63: 155 – 166, (2013) [19] oser b. l., an integrated essential amino acid index for predicting the biological value of proteins. in: albanese, a.a. (ed.), protein and amino acid nutrition. academic press, new york, pp. 295 – 311, (1959) [20] nieman d.c.d., butterworth e. & nieman c.n., nutrition. wm. c. brown publishers, dubuque, ia., usa, p. 540, (1992) [21] aremu m.o., osinfade b., basu s., & ablaku b., development and nutritional quality evaluation of kersting’s groundnut-ogi for african weaning diet. american journal of food technology. 6: 1021 – 1033, (2011) [22] fao/who., protein quality evaluation. report of the joint fao/who expert consultation. fao food and nutrition paper 51. fao/who. rome, italy, 1 – 66, (1991) [23] caprita, r., adrian c., iuliana c. & valeria n., in vitro determination of wheat dry matter solubility and protein digestability. animal science and biotechnologies. 45(2): 113 – 116, (2012) [24] schaafsma, g., the protein digestibilitycorrected amino acid score. journal of nutrition, 130(7): 1865s – 1867s, (2000) [25] rekha, c.r., vijayalakshmi, g., influence of processing parameters on the quality of soycurd (tofu). journal of food science technology. 50, 176 – 180, (2013) [26] shurtleff, w., aoyagi, a., the book of tofu. ballantine books, new york, usa, (1979) [27] carrol, k. review of clinical studies on cholesterol-lowering response to soy protein. journal of the american dietetic association. 91: 820 – 827, (1991). [28] potter, s., soy protein and cardiovascular disease: the impact of bioactive components in soy. nutrition reviews, 56(8): 231 – 235, (1998) [29] bilek, a.e., turhan. s., enhancement of the nutritional status of beef patties by adding flaxseed flour. meat science, 82: 472 – 477, (2009) [30] aremu, m.o., olaofe o. & akintayo t.e., a comparative study on the chemical and amino acid composition of some nigerian underutilized legume flours. pakistan journal of nutrition, 5: 34 – 38, (2006) [31] yang y., modification and application of dietary fiber in foods. review article, 9340427, 8, (2017) [32] dhingra, d., m. michael, h. rajput and r.t. patil. dietary fibre in foods: a review. journal of food science and technology. 49(3); 255 – 266, (2012) [33] guillon f., auffret a., robertson j.a., thibault j.f. & barry j.l., relationships between physical characteristics of sugar beet fibre and its frementability by human fecal flora. carbohydrate polymers, 37 (2): 185 – 197, (1998) [34] nassar, a.g., abd elhamied a.a. & elnaggar e.a., effect of citrus by-products flour incorporation on chemical, rheological and organoleptic characteristics of biscuits. world journal of agricultural sciences, 4(5): 612 – 616, (2008) [35] jackson, c.j.c., dini j.p., lavandier c., rupasinghe h.p.v., faulkner h., poysa v., buzzell d., degrandis, s., effects of processing on the content and composition of isoflavones during manufacturing of soy beverage and tofu. process biochemistry, 37, 1117 – 1123, (2002) [36] choe, e., min, d. b., chemistry of deep-fat frying oils. journal of food science. 72 (5), r77 r86, (2007); [37] shaheen n., islam s., munmun s., mohiduzzaman m. & longvah t. amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key food in bangladesh. food chemistry, 213, 83 – 89, (2016) [38] fao/who., protein quality evaluation. report of the joint fao/who expert consultation. food and nutrition. no.69. rome: food and agriculture organizations and the world health organization, (2007) [39] liao, s.m., q.s. du, j.z. meng, z.w. pang and r.b. huang. the multiple roles of histidine in protein interactions. chemistry central journal, 7(1): 44, (2013) [40] hou, y., y. yin and g. wu. dietary essentiality of “nutritionally non – essential amino acids “for animals and humans. experimental biology and medicine. 240 (8), 997 – 1007, (2015) [41] dai, z.i., g. wu and w.y. zhu. amino acid metabolism in intestinal bacteria: links between gut ecology and host health. front bioscience, 16 (1), 1768 – 1786, (2011) [42] ren, w., l. zou, z. ruan, n. li, y. wang, y. peng and g. liu. dietary l-proline supplementation confers immunostimulatory effects on inactivated pasteurella multocida vaccine immunized mice. amino acids, 45 (3): 555 – 561, (2013) [43] kim, j.y., r.c. burghardt, g. wu, g.a. johnson, t.e. spencer and f.w. bazer. select nutrients in the ovine uterine lumen. vii. https://www.hindawi.com/18936576/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 dina anwar, ghadir el-chaghaby, nutritional quality, amino acid profiles, protein digestibility corrected amino acid scores and antioxidant properties of fried tofu and seitan, food and environment safety, volume xviii, issue 3 – 2019, pag. 176 190 190 effect of arginine, leucine, glutamine, and glucose on trophectoderm cell signaling. proliferation and migration. biology of reproduction, 84(1): 62 – 69, (2011) [44] hou, y., l. wang, d. yi, b. ding, z. yang and j. li. nacetylcysteine reduces inflammation in the small intestine by regulating redox, egf and tlr4 signaling. amino acids, 45(3): 513 – 522, (2013) [45] fao/who/unu., energy and protein requirements report of a joint expert consultation. who technical report series, no 724. geneva: world health organization, (1985) [46] malomo, s.a., r. he and r.e. aluko.structural and functional properties of hemp seed protein products. journal of food science, 79 (8): c 1512 – c 1521, (2014) [47] vallobha, v.s., a. tapal, s.v. sukhdeo, k. govindaroju and p.k. tiku. effect of arginine: lysine ratio in free amino acid and protein on l-name induced hypertension in hypercholesterolemic wistar rats. rsc advances, 6, 73388 – 73398, (2016) [48] paul, a.a and d.a.t. southgate. the composition of foods (4th ed.) london, hmso, (1976) [49] oyarekua, m.a., eleyinmi, a. f., comparative evaluation of the nutritional quality of corn, sorghum and millet ogi prepared by modified traditional technique. journal of food, agriculture and environment, 2, 94 – 99, (2004) [50] caire-juvera, g., f.a. vázquezortiz and m.i. grijalva-haro.amino acid composition, score and in vitro protein digestibility of foods commonly consumed in norhwest mexico. nutricion hospitalaria, 28(2): 365 – 371, (2013) [51] bongnar a., comparative study of frying to other cooking techniques influence on the nutritive value. grasas aceites. 49 (3,4): 250 – 260, (1998) [52] babji a.s. s., fatimah y. abolhassani and m. ghassem. nutritional quality and properities of protein and lipid in processes meat-products a perspective. international food research journal, 17: 35 – 44(2010) [53] golden m., nutrient requirements of moderately malnourished populations of children. food and nutrition bulletin,30(3):s267-s342 (2009) 1. introduction 4. conclusions microsoft word 15 norocel liliana_corectat.doc 241 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 241 245 electrochemical biosensor based on the use of spe for the detection of iron content in wine *liliana norocel1, gheorghe gutt1 1faculty of food engineering, ștefan cel mare university of suceava, romania liliana.norocel@fia.usv.ro *corresponding author received 25th january 2018, accepted 27th june 2018 abstract: the purpose of this paper is to develop a new method for iron analysis in wine. the determination of iron in winemaking products is particularly important because a content higher than 10 mg/l can lead to turbidity or a change in color, which causes ferric casse. the new method is based on the use of screen printed electrodes (spe), previously immobilized with protein a-agarose and connected to a potentiostat which displays a cyclic voltammogram. to verify the method, the samples of wine were also analysed by a reference method, namely aas. in order to visualize the deposition of the iron ions on the electrode, surface analyses were perfomed by a microscope which combines confocal microscopy with white light interferometry. the main features of this sensor are simplicity of operation, good sensitivity and low limit of detection. keywords: aas, cyclic voltammetry, iron casse, surface analysis 1. introduction the analysis of trace metals in wine is of great importance for the quality of a product, and also for the authenticity control of wine. the content of mineral elements in wine can be attributed to natural sources or to contamination during the wine-making process [1, 2]. it is known that there are certain species that significantly contribute to the destabilization of wines and their oxidative evolution. these species include the oxygen molecules, which is a process initiator, polyphenols as oxidative matter, and certain metal ions, such as fe, cu and mn as process activators, which are also present [3]. at low concentration, iron ions have an important role in metabolism and fermentation processes as stabilizer, enzyme activator and functional component of proteins. at a higher concentration, it plays other roles such as altering redox systems of the wine in favour of oxidation, affecting sensory characteristics and participates with chemical compounds (tannins and phosphates) to instabilities. at concentrations above 10 mg/l iron forms insoluble suspensions which are known as ferric casse [4]. the analysis of iron in wine before bottling, can prevent the casse formation through the addition of potassium hexacyanoferrate(ii), which eliminates part of iron content and significantly decreases its concentration [5]. the most commonly used technique for determining iron in wine is atomic absorption spectrometry (aas) due to its precision [6]. also, many papers have been published reporting the use of icp-ms methods for the determination of mineral elements in wine fingerprints [7, 8]. the proposed methods for the analysis of metals in wine by these techniques usually involves sample preparation [9], thus it is necessary to develop a simple and easy to prepare method with effective costs. the use of quick and cheap biosensors for the detection of mineral elements represents an important requirement in the wine sector food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 liliana norocel, gheorghe gutt, electrochemical biosensor based on the use of spe for the detection of iron content in wine paper, food and environment safety, volume xvii, issue 2 – 2018, pag. 241 245 242 [10]. several studies have been reported on the use of miniaturized electrodes coupled with various detection techniques in the study of trace metals. electrochemical biosensors bring a number of significant advantages such as miniaturization, sensitivity, portability, selectivity, low sample volumes and effective costs. in the last decades, advances have been made in electro-analytical chemistry through the development of ultra-microelectrodes, molecular devices and intelligent sensors [11]. cyclic voltammetry (cv) is very popular electrochemical technique commonly used to investigate the reduction and oxidation processes of molecular species [12]. in many papers are studied the interactions between iron and proteins in wine. a simple, cost-effective method of iron is accomplished by using lactoferrin as part of a biosensor to detect iron in samples [13]. normally, iron entraps a variety of protein transports such as transferrin and lactoferrin and is stored in proteins such as ferritin and hemosiderin [14]. the purpose of this study was to develop a simple biosensor for iron determination in wine, through the use of protein-a as biological element which was immobilized on screen printed electrodes. 2. materials and methods protein a-agarose and iron sulphate were purchased from sigma aldrich (germany) and the 15 wine samples used for iron analysis were bought from wine cellars in suceava (romania). preparation of screen printed electrodes: 1µl of protein a-agarose was immobilized on spe and used in the iron analysis with a potentiostat which displays a cyclic voltammogram. voltammetric measurements were carried out with a pgstat204 autolab at a minimum potential of -0.6 v and a maximum of 0.6v. preparation of wine samples for aas: the wine samples for aas analyses were prepared according to [15]. thus the alcohol was evaporated from the wine by reducing the sample volume to half the original quantity using a rotary evaporator (50 to 60°c), and then it was made up with distilled water to the initial volume. the absorbance was read at 248.3 nm. spectrometric measurements were carried out with an atomic absorption spectrometer 6300 shimadzu. the microscopic analysis of the working electrode surface after immobilization and use in the wine was performed with the mahrsurf cwm 100 microscope, which combines confocal microscopy with white light interferometry. 3. results and discussion in this study, immobilized protein (protein a agarose) was used as biological element. in fig. 1 is given the calibration curve with maximum and minimum intensities of the artificial samples and fig. 2 illustrates the cyclic voltammogram of the five iron concentrations, respectively 1, 2.5, 5, 7.5, 10 mg/l, analyzed at 120 seconds after immersion. from both the voltammogram and the calibration curve it can be easily observed that the values extracted can provide good results and can be used to calculate the iron concentration of the solution. the performance of the developed biosensor was determined through the sensitivity and limit of detection. the results of these characteristics are presented in table 1. table 1 parameters of performance characteristics results time of immersion (s) 120 sensitivity 1.48e-06 limit of detection (ppm) 0.900 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 liliana norocel, gheorghe gutt, electrochemical biosensor based on the use of spe for the detection of iron content in wine paper, food and environment safety, volume xvii, issue 2 – 2018, pag. 241 245 243 fig. 1 calibration curve fig. 2 cyclic voltammogram of the five iron concentration sensitivity was calculated as slope of calibration curve on the surface electrode area. regarding the limit of detection, this biosensor does not have low enough values to detect the smallest iron concentrations in wine; however, it can analyze wine samples and provide values up to the limit that can stop the ferric casse phenomenon. for testing the developed method, the results obtained with the biosensor were compared to those obtained with the aas method, which was used as reference method. for this purpose, 15 samples of wine were analized and the values obtained are presented in fig. 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 liliana norocel, gheorghe gutt, electrochemical biosensor based on the use of spe for the detection of iron content in wine paper, food and environment safety, volume xvii, issue 2 – 2018, pag. 241 245 244 fig. 3 iron concentration from this chart it can be observed that there is no general trend, some values being higher than those obtained with the reference method and others being lower than the reference values. in order to visualize the sample surface analysis, microscopic images of the unused electrode were captured and analyzed by software in terms of profile. fig. 4 3d images of the spe and skewness and kurtosis histograms for electrodes a) unused, b) immobilized, c) used in wine it was also analyzed the surface of the immobilized electrodes and the spe after the analysis of the iron content in wine. the results are shown in the 3d figures presented below (fig. 4). as can be observed, the electrode used in the analysis of the iron content of wine has deposits, which leads to a higher average height than the immobilized electrode, analyzed from the profile. a) b) c) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 liliana norocel, gheorghe gutt, electrochemical biosensor based on the use of spe for the detection of iron content in wine paper, food and environment safety, volume xvii, issue 2 – 2018, pag. 241 245 245 the coefficient of skewness and the coefficient of kurtosis have lower values for the electrode used in wine compared to the immobilized electrode, obtained by profile analysis, and the immobilized electrode has lower values of these two coefficients than the unsed electrode. 4. conclusion the biosensor proposed in this study provided excellent results in artificial iron solutions, both in terms of sensitivity and detection limit and also for r2 of the calibration curve. in the case of the wine samples the results don’t have a general trend, some values being higher than those obtained with the reference method and other values being lower than the ones obtained through aas method. in the future, the selectivity of the biosensor for the determination of iron ions will be studied on the mineral elements present in wine which can interfere. 5. references [1] pyrzyńska, k. (2004). analytical methods for the determination of trace metals in wine. critical reviews in analytical chemistry, 34(2), 69-83. [2] norocel, l., & gutt g. (2017). study on the evolution of micro-and macroelements during the winemaking stages: the importance of copper and iron quantification. food and environment safety journal, 16(1), 5 – 12. [3] benitez, p., castro, r., & barroso, c. g. (2002). removal of iron, copper and manganese from white wines through ion exchange techniques: effects on their organoleptic characteristics and susceptibility to browning. analytica chimica acta, 458(1), 197-202. [4] pyrzynska, k. (2007). chemical speciation and fractionation of metals in wine. chemical speciation & bioavailability, 19(1), 1-8. [5] ketney, o., lengyel, e., tita, o., & țifrea, a. (2013). content variation of iron and copper in wine obtained from wine vineyards recas. acta universitatis cibiniensis. series e: food technology, 17(1), 39-45. [6] olalla, m., gonzález, m. c., cabrera, c., & lópez, m. c. (2000). optimized determination of iron in grape juice, wines, and other alcoholic beverages by atomic absorption spectrometry. journal of aoac international, 83(1), 189-195. [7] avram, v., voica, c., hosu, a., cimpoiu, c., & măruţoiu, c. (2014). icp-ms characterization of some romanian white wines by their mineral content. revue roumaine de chimie, 59(11-12), 1009-1019. [8] oroian, m. (2015). romanian white wine authentication based on mineral content. journal of agroalimentary processes and technologies, 21, 913. [9] ferreira, s. l., souza, a. s., brandao, g. c., ferreira, h. s., dos santos, w. n., pimentel, m. f., & vale, m. g. r. (2008). direct determination of iron and manganese in wine using the reference element technique and fast sequential multi-element flame atomic absorption spectrometry. talanta, 74(4), 699-702. [10] rodriguez-mendez, m. l., medinaplaza, c., garcia-hernandez, c., de saja, j. a., fernández-escudero, j. a., barajas-tola, e., & medrano, g. (2014, november). analysis of grapes and wines using a voltammetric bioelectronic tongue: correlation with the phenolic and sugar content. in sensors, 2014 ieee (pp. 2139-2142). [11] norocel, l., & gutt, g. (2017) electrochemical biosensor for detection of copper ions in wine. journal of agroalimentary processes and technologies, 23 (3), 120-124. [12] elgrishi, n., rountree, k. j., mccarthy, b. d., rountree, e. s., eisenhart, t. t., & dempsey, j. l. (2017). a practical beginner’s guide to cyclic voltammetry. journal of chemical education, 95, 2, 197-206. [13] kruzel, m. l. (1996). u.s. patent no. 5,516,697. washington, dc: u.s. patent and trademark office. [14] paffetti, p., perrone, s., longini, m., ferrari, a., tanganelli, d., marzocchi, b., & buonocore, g. (2006). non-proteinbound iron detection in small samples of biological fluids and tissues. biological trace element research, 112(3), 221-232. [15] compendium of international methods of analysis, iron, oiv-ma-as322-05a: r2009. issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xviii, issue 2 30 june 2019 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania title …………………… 287 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 287 – 295 study on the kinetics of biomass blends dewatering as a stage of thermal degradation 1yaroslav zasiadko*, 1mykola pryadko, 1pavlo zasiadko, 1olexiy buliandra 1 national university of food technologies, 68, volodymyrska st.; 01601 kyiv; ukraine *corresponding author iaroslav@nuft.edu.ua received 26th october 2017, accepted 21th december 2017 abstract: solid biomass co-combustion with coal in industrial furnaces, including those of power plants, looks promising in terms of fuel base diversification and also presents a way to reduce significantly the emission of air harmful pollutants, particularly carbon dioxide. co2 resulting from biomass burning is environmentally neutral, since the same amount of carbon dioxide will be consumed at the next cycle of biomass photosynthesis. in case of the ukrainian low reactive anthracite schtib used in such technology, the radical differences in the kinetic characteristics of co-fired fuels become critical issues. a significant difference in the attainable degree of fuel milling fineness due to the prohibitively high expense for solid biomass milling makes the problem more complicated. the values of biomass particle fineness are approximately 5…15 mm; whereas the fineness of pulverized coal reaches as low as 30...50 mkm. the moisture and volatiles content for the fuel mentioned also differ noticeably. therefore, the stages of these fuels burn out will be extremely complex in terms of various time scales of subsequent stages and their positioning in the furnace. the case becomes even more aggravated insofar the kinetic characteristic of the stages of biomass combustion are not known yet in details. the present work is dedicated to the research into the kinetics of biomass blends demoisturization and aimed at the deriving of the arrhenius type equations, allowing to develop submodels of the demoisturization as an individual stage of biomass burn out for 3-d models. keywords: biomass, gravimetry, activation energy, moisture, 1. introduction the release of moisture from the heated solid fuel which undergoes combustion usually happens quickly, if compared to the total time of fuel burn out. this generally holds when the moisture content of the fuel is negligibly low, approx. 3-5%, which is typical for coal. in case of burning solid biomass, one deals with the moisture content of 30…40% and in some cases as high as 70% for sugar pulp, for example. keeping in mind that a typical particle size of solid biomass prepared for pulverized combustion falls between 5…25mm, one should expect an extended duration of the demoisturization stage along with the significant heat consumption for moisture evaporation. extensive emission of vapor will also contribute to the formation of the concentration field of flue gases components. to correctly simulate the process of pulverized co-firing of coal and solid biomass it is necessary to have precise kinetic characteristics of biomass demoisturisation as an individual fuel, and as blends of various types of biomass. the kinetic characteristics of individual stages of biomass thermal degradation are being determined using thermogravimetric methods [1-7]. http://www.fia.usv.ro/fiajournal mailto:iaroslav@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 288 the essence of the method of dynamic thermal gravimetrical analysis (tga) [1-7] consists in the continuous measurement during the experiment of temperature and weight of a sample and comparison them with those of the standard, which does not participate in the reaction. the sample and the standard are both heated with a certain constant speed. thus, registering the time of each measurement, weights and temperatures continuously, it becomes possible to determine the time derivative (dm/dt) for the sample along with the thermal effect of the process. the time change in the degree of conversion in a process can mathematically be described using the respective physical constants and corresponding model functions by the equation [4-12]: )(   fk d d  (1) where α the degree of conversion; τ time; k constant of the reaction rate; f(α) a function that describes a particular reaction model. the degree of conversion within a demoisturization process is determined using the continuous data of the sample weight measurements as follows: )/()( 00   mmmm  (2) where mo initial mass of the sample; m∞ sample mass at the end of the process. moisture release; mτ -is the sample mass at a time τ. thus, the degree of conversion characterizes the current proportion of moisture which evaporates to the instant τ ),/exp( rteak  where a frequency factor, 1/s; e activation energy, j/mole; r universal gas constant – 8.314 j/(mole k); t temperature, k. obviously, it is critical to determine the exact instants of the beginning and end of the process and register respective values mo, m∞. it is also important to determine with the maximum accuracy the time instants and corresponding values of mτ and sample temperatures. in the practical conditions determining of the certain process” start and finish does not look an easy problem, since there might be a superimposition of various processes at one temperature range, which leads to the change in the sample mass. for example, in case of demoisturization process, the moisture removal ending coincides with the beginning of devolatilization, which significantly hampers the precise determination of the dewatering process limits. therefore, for dewatering, the point where the second derivative of the mass change function passes through a zero value in the range of 170...200°c and the local reaction rate acquires minimum value that corresponds to the practically complete removal of moisture usualy is considered the process ending. given the arrhenius equation, the eq. (1) acquires the form: )()/exp(    frtea d d  (3) if the sample is heated at a constant rate, the time – temperature function will be [410]: to the initial mass of moisture in the sample. the constant of the reaction rate is given in the form of the arrhenius equation [1-10] t = βτ + c (4) where βthe rate of heating. combining eq. (3) and (4), we obtain: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 289 exp ( ) d a e f dt rt             (5) typically, f (α) is used extensively for the description of heterogeneous reactions of devolatilization in the form of power or special functions [5,7]. common in the processing of tga data is the assumption of the first order of reaction. by grouping variables and taking logarithms of eq.(5) we obtain: 1 1 d e ln lna dt rt             (6) equation (6) forms the methodological basis of the differential approach to the determination of kinetic constants on the basis of tg studies. a detailed analysis of data processing specifics and interpretation based on the application of eq. (6) is given in [5,7,12,13]. despite its visibility and simplicity, this approach has significant drawbacks, related to the need to calculate the left-hand side of eq. (6). it contains the conversion rates and their time derivative which is a varying mass change function, and has to be taken at the precisely same time instants. in this case, the value of the temperature in the right side of eq. (6) must correspond to the same moment. the critical point of this methodology is also the need to scale the value of the derivative, based on the actual curve mτ [3,7,12,13] . in accordance with the integral approach, which has become widely used [2-7,11-13] in recent years, equation (6) is integrated to the form: 0 0 1 exp (1 ) t n a d dt rt              (7) if the integration of the expression on the left side does not cause complications at any choice of the reaction model, then the expression on the right side could be represented as an approximating asymptotic series, and, upon the integration, yields [7,12,13,14-21]: 0 exp t e dt rt         2 1 2 e rt art rt e e e       (8) this method, called the integral one is transformed depending on the accepted model of the reaction. for the first-order reaction at n=1 we obtain from eq. (8):   2 ln 1 1 2 exp art rt e e e rt                  (9) which after taking logarithms yields: 2 ln(1 ) 1 ln ln (1 2 ) e ar rt r t e t e                    (10) 2. matherials and methods the kinetic characteristics of the samples of ground solid biomass blends were determined based on the differential thermal gravimetry curves obtained derivatigraph q-1500 d (paulik-erdey). all measured data were recorded in analogue format with a six channel recorder with measurement limits for each channel from 50 μv to 5 mv / 250 mm. the parameters of the derivatograph operation corresponded to the following values: medium oxidizing (air), weight of weight gain 50±4 mg (error±1%), heating rate 20oc/min, inert material al2o3, crucible platinum, size particles polyfractional. the sample was heated from room temperature (25±3ºс) to 900ºс. samples of the biomass from which mixtures were formed, are shown in figure food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 290 1 and the results of the biomass technical analysis are presented in table 1. fig. 1 biomass samples a-beet sugar pulp, b-wheat straw, cpinewood sawdust, d-sunflower husk table 1. biomass technical analysis data biomass volatiles, v d % ash, a d % moisture, w r t % beet sugar pulp 80.5 2.0 10 wheat straw 76.0 16.7 10 pinewood sawdust 85.0 1.75 10 sunflower husk 77.1 15.8 7 special samples of biomass blends were prepared with the weight of 40...50 mg of individual components in a 1: 1 proportion to be used in the experimental studies of the stages of thermal destruction kinetics. the blends sawdust-straw, pulp-straw, pulp-husk, pulp-sawdust were studied. experimental samples of individual biomass were pre-dried in a muffle furnace snol 24/200 at a temperature of 105...110°c, ground and sifted through sieves to have a nearly mono-dispersed structure with particle size of 1...3 mm and mixed in a given proportion. the calculated moisture was added to get the initial moisture content of the sample mixture at 10%. the bulk weight of a sample was 50±4 mg and was selected from the condition that in the process of thermal decomposition the fuel would covere the bead of the thermocouple at the bottom of the platinum crucible and also to reduce the temperature gradient between the center and the sample surface. the samples were heated from room temperature (25 ± 3°c) to 900°c at a constant speed of 6.6...7.2°c/min. (0.11...0.12 k/s) to ensure maximum proximity to isothermal conditions in the oxidizing medium (air). as an inert material, al2o3 was used. experiments were carried out under the same conditions in order to obtain comparable results for various mixtures of biofuels. 3. results and discussion the processing of the obtained data and their interpretation were performed on the basis of the non-isothermal integral method based on the coates-retfern model [6,7,11-14,16-22]. the data were presented as a dependence of y = f(1/t), where:                e rt tlnln 21/1 2  (11) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 291 as can be seen from the structure of the equation for y, its application is possible by the method of successive approximations, that is, the complex y is calculated preliminarily, on the basis of processing the graphs y = f(1/t).   2 1 /1 tlnln  (12) values e1 and a1 are determined as the first approximation, for which the correction factor is calculated. obtained thus initial values e1 and a1 allow calculating the correction multiplier:                1 21 e rt , which, in turn, allows calculating y2, followed by the definition of a new pair of values e2 and a2. as a rule, a one-time iteration provides a deviation of e2 and a2 from the pair of e1 and a1 to values of less than 2...3%. the obtained data in the form y = f(1/t) with the serial iterations of e and a are shown in figure 2. fig. 2. dependence y=f (1/t) for the mixture of pulp-husk in the zone of dehydration with correction multiplier, no correction the analysis of the data obtained in conjunction with the data [12,13] for pure biomass shows that at small values of α, less than 0.05 and at values α, greater than 0.95, there is a significant deviation from the approximating lines. this particular circumstance is noticeable in the process of moisture release. the results of studies into the drying process indicate that the initial free moisture release (constant speed drying) and the subsequent period of the release of chemically bound moisture [12] are essentially different. therefore, the representation of the demoisturization process by the first order reaction looks somewhat arbitrary. from the data shown in figure 3 (a) and (b) follows that it is expedient to approximate them by two lines on the abscissa sections 0.0032...0.0026 and 0.0026...0.0022 separately. y = -4196.5x 3.3282 y = -4 276.80x 3.28 -18 -17 -16 -15 -14 -13 -12 2.00e-03 2.20e-03 2.40e-03 2.60e-03 2.80e-03 1/t food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 292 a. b fig. 3. dependence y = f(1/t) for the mixture of pulp-wheat straw in the zone of dehydration. a approximated within a whole range of reverse temperatures; b approximated in sections of 1/t range it should be noted that in [12,13] data were generalized, starting with α>0,05. when involving the generalization of the lower values of α, the need to generalize the process of moisture release on the two segments of 1/t axis becomes apparent. in figure 2 (a) shows the obtained data for a mixture of wheat strawpulp. for the comparison, here are given the lines calculated as recommended in [12,13] for the pure components of the mixture, as well as the approximation line for the whole range of the process. the data clearly prove that the kinetics of the blend demoisturization corresponds with the kinetics of the individual components of the mixture, since the data for the mixture lay closely to and within the lines for the components. it should be marked that the equation of the approximating line is strongly dependent upon the 1/t range of approximation. the lower (and equally the higher) the values of α are to be included into the approximation the bigger the deviation of the experimental points from the approximating line should be expected. this proves distinctly the necessity to introduce the two section approximation of the experimental data aimed at the increasing accuracy of the calculations, which is shown in figure 2. kinetic constants thus obtained are shown in table 2 and 3. y = -4022.6x 3.3202 -18 -17 -16 -15 -14 -13 -12 -11 -10 lnln corrected data pulp only [20] data straw only [20] 2.00 e-03 2.20e-03 2.40e-03 2.60e-03 2.80e-03 1/t y = -7795x + 7.3092 r² = 0.833 y = -2012x 8.001 r² = 0.9281 -18 -17 -16 -15 -14 -13 -12 -11 -10 lnln cor start lnln finish linear trend start linear trend finish 2.00 e-03 2.20e-03 2.40e-03 2.60e-03 2.80e-03 1/t food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 293 table 2 kinetic constants for the whole range and sections blend constants average 1 section 2 section а, 1/s е, кj/mole а, 1/с е, кj/mole а, 1/s е, кj/mole pulp-straw 145.4 33.44 1.16е07 64.8 0.675 16.73 pulp-husk 154.9 34.92 8.9е08 82.72 0.70 18.26 pulp-sawdust 1603 39.96 10.4е12 106.37 12.325 24.85 straw-sawdust 522 39.2 9.4е09 87.65 3.28 23.25 table 3 approximating equations blend line equation average 1 section 2 section pulp-straw -4023х-3.2 -7798х+7.3 -2012х-8.0 pulp-husk -4200х-3.3 -9552х+11.4 -2196х-8.1 pulp-sawdust -4806х-1.1 -12794х+20.5 -2989х-5.5 straw sawdust -4712х-2.2 -10542х+13.7 -2797х-6.75 data on the degree of conversion calculated by the basic equation (3) and by using the data given in table 2 and tabl3 in mathcad are presented in figures 4 and 5 fig. 4 . degree of moisture conversion at heating within 300…500 k of wheat straw-pulp blend approximated within a whole region 300 350 400 450 500 1 0.8 0.6 0.4 0.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 294 fig. 5 . degree of moisture conversion at heating within 300…500 k of wheat straw-pulp blend approximated in two sections as it can be clearly seen, the approximation within the two sections of the temperature range of the demoisturization section allows obtaining much more accurate correspondence of the experimental data to those calculated, which, in turn, allows precise determination of the target product yield. 4. conclusion the kinetics of the dewatering stage within a whole process of thermal degradation of the set of ground solid biomass blends was determined by means of the dynamic thermogravimetry analysis. data processing and determination of process kinetic constants were done within the modified coats-redfern method. it has been shown that the process of demoisturization can hardly be distinguished on the tga curves, since the first derivative of mass never reaches zero because the process of dewatering at its end coincides with the next process of devolatilization. the accuracy of data generalization which leads to the determination of kinetic constants depends strongly on the marked limits of the process. the method of the approximation data on two sections within the process temperature range has been proposed. the method allows much closer match of the experimental data to the results of calculations. proposed kinetic constants may be used at calculations of the demoisturisatrion stage of biomass blends during their thermal degradation and at deriving 3-d sub models of biomass combustion. 5. references [1]. thermogravimetry (tg) or thermogravimetric analysis (tga) or thermal gravimetric analysis. anderson materials evaluation, inc., http://www.andersonmaterials.com/tga.html [2]. standard test method for decomposition kinetics by thermogravimetry, bulletin of american society for testing and materials: astme 1641-98 [3]. gaisford s., kett v., haines p., principles of thermal analysis and calorimetry, royal society of chemistry, 2-nd edition, p. 268, (2016) [4]. naian l., weicheng f., dobashi r., qizhao l., new mass loss kinetic model for 300 350 400 450 500 1.0 0.8 0.6 0.4 0.2 http://www.andersonmaterials.com/tga.html food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 yaroslav zasiadko, mykola pryadko, pavlo zasiadko, olexiy buliandra, study on the kinetics of biomass blends dewatering as a stage of thermal degradation , food and environment safety, volume xvi, issue 4 – 2017, pag. 287 – 295 295 thermal decomposition of biomass, chinese science bulletin, vol. 46, no. 16 – p. 1398-1402, (2001) [5]. yao f., qinglin w., yong l., weihong g., yanjun x., thermal decomposition kinetics of natural fibers: activation energy with dynamic thermogravimetric analysis, polymer degradation and stability, № 93, p. 9098, (2008) [6]. johannes i., kruusement k., veski r., bojesen-koefoed j. a., characterization of pyrolysis kinetics by rock-eval basic data, oil shale, vol. 23, no. 3, p. 249–257, (2006) [7]. han y., theoretical studies of thermal analysis kinetics, theses and dissertations— mechanical engineering, 35, p.91, (2014), http://uknowledge.uky.edu/me_etds/35 [8]. gašparovič l. koreňová z., jelemenský ľ., kinetic study of wood chips decomposition by tga, 36th international conference of ssche, tatransk´e matliare, slovakia, le-we-4, .p 178, (2009) [9]. ledakowicz s., stolarek p., kinetics of biomass thermal decomposition, presented at the 29th international conference of the slovak society of chemical engineering, tatranské matliare, chem. p. 56(6)37-381, (2002) [10]. yu f., ruan r., steele p., consecutive reaction model for the pyrolysis of corn cob, transactions of the american society of agricultural and biological engineers, issn 00012351, vol. 51, no. 3, p 1023-1028, (2008) [11]. kořistková m., vallová s., kolat p., co-combustion coal and waste alternative fuel: thermogravimetric analysis. transactions of the všb, technical university of ostrava, mechanical series no. 1, vol. lvii article no. 1848, p.133-139, (2011) [12]. zasyadko i., myroshnik m., dunaevska n., zasyadko p., tga biomass demoisturization modelling, renewable energetics, no.1, p. 85-95, (2012) [13]. zasyadko i., .myroshnik m., zasyadko p., thermogravimetric studies of beet pulp thermal decomposition, sugar of ukraine. no.2 (74), p. 29-32, (2011) [14]. yao f., wu q., zhou d., thermal decomposition of natural fibers: global kinetic modeling with nonisothermal thermogravimetric analysis, journal of applied polymer science, vol. 114, p 834–842, (2009) [15]. gao-jiin u., wu s., lou r., kinetic study of the thermal decomposition of hemicellulose isolated from corn stalk, bioresources, vol.5, no 2, p. 1281-1291, (2010) [16]. weerachanchai p., tangsathitkulchai c., tangsathitkulchai m., comparison of pyrolysis kinetic models for thermogravimetric analysis of biomass, suranaree j. sci. technol, vol. 17, no. 4, p 387-400, (2010) [17]. yang q., shubin w., wheat straw pyrolysis analysis by thermogravimetry and gas chromatography-mass spectrometry, cellulose chemistry and technology, vol. 43, no. 4-6, p. 123131, (2009) [18]. gomes р., claudia j., velo e., puigjaner l., comparative thermogravimetriy/mass spectrometry study of woody residuals and herbaceous biomass crop using тga techniques, etseib, e-08028, barcelona, spain [19]. chen h., liu n., new procedure for derivation of approximation for temperature integral, aiche 52,12, p 4181-4185 [20]. noorfidza y. h., afaz m. t. thermal decomposition kinetics of forest residue, journal [21]. of applied scienses, vol. 10, no. 12, p. 1122-1127, (2010) [22]. kumar sachin, agrawalla ankit, and singh r. k., thermpgravimetric analysis of groundnut cake. international journal of chemical engineering and applications, vol. 2 , no. 4 , p.267-271, (2011) http://uknowledge.uky.edu/me_etds/35 1. introduction 4. conclusion 293 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 32015, pag. 293 299 sensory evaluation of lamb’s meat according to conventional and organic breeding systems * tatjana kalevska 1 , ljupce kocoski 2, elena joshevska2, viktorija stamatovska1, aleksandar savevski1 1faculty of technology and technical sciences,university st.kliment ohridski petre prlicko 42, veles, r.macedonia, 2faculty of biotechnical sciences, university st.kliment ohridski, partizanska b.b, bitola, r.macedonia tkalevska@gmail.com received september 5th 2015, accepted september 29th 2015 abstract: the purpose of this paper is to determine the sensory properties of lamb meat according to conventional and organic breeding systems. the research was carried out on 120 lambs, out of which 60 were reared in a conventional and 60 in an organic system. during the research done by the heat treatment of lamb meat at a temperature of 175°, a significantly greater (p<0.05) weight loss of the meat from the conventional system as compared to organic system was found. the shrinkage during thermal processing of meat from organic system is of 31.45±0.97%, being of 2.59% smaller than the meat shrinkage from conventional system 34.04±0.78%. the overall acceptability score for sensory attributes (smell, juiciness, taste, aroma, texture and softness) was significantly (p < 0.01) higher for meat from group i and ii from the organic system, as compared to meat from group i and ii from conventional system. the results showed that the breeding system had a significant effect in lamb’s sensory profile. keywords: sensory properties, lambs meat, oragnic and conventional systems, weight loss, thermal processing 1. introduction lately, because of concern for their own health, saving the environment and respecting animal welfare, the consumers’ habits and requests are changed, and they prefer meat produced in organic system, which has better nutritive and sensory quality is safe for consuming, and which shall be widely accepted from domestic and foreign consumers [1]. the quality of the lamb must be acceptable for the consumers, who are on the top of the lamb production chain. the factors that affect the acceptability of the meat are divided in sensory (look, taste, smell, aroma, tenderness, juiciness, texture), health correctness of meat, stability of the product, nutritional value, suitability for use and awareness about the product. during thermal processing of the meat, loss of the weight of meat is occurring as a result of loss of certain amount of water, fat and soluble substances. the loss of weight which occurs during thermal processing of the meat is very significant, because it affects the look, the taste and the nutritional value of the meat [2]. the loss of weight during thermal processing show specific variations, depending on several factors, especially http://www.fia.usv.ro/fiajournal mailto:tkalevska@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 294 the method and time of thermal processing, temperature, size, i.e. surface of the piece and the nutritional composition of the meat. according to [3], the meat freezingpoint, storing and thawing causes specific biochemical and histological changes that manifest in bigger are smaller loss of tissue liquids (juices) while thawing. the inability of meat to bind the juice released while thawing the ice crystals is a result of protein denaturation. the average weight loss of meat after completed dry thermal processing (roasting) at 1750с temperature is 20-23%, while bigger weight loss occurs at higher roasting temperature, up to 2220с [3]. in their research, [4] determine weight loss of meat during thermal processing, wherein he stated weight loss of meat from 24.01% when it’s boiled, that is 31.46% when it’s roasted. during the thermal processing of the meat [5] stated bigger weight loss of the meat from conventional group, from 24.54%, that is 13.41% of the meat from organic system, as a result of meat chemical composition. the sensory attributes of meat depend on the breed, the type, the sex, and the age of animals, as well as the meat storing and thermal processing conditions [6]. the most important factors which impact the acceptability of the lamb are: taste, smell, juiciness and tenderness of the meat [7]. the consumers request tender, soft meat, with specific taste and smell [8]. the smell of the meat is one of the most important factors that determine the acceptability of meat by the consumers. the smell of the meat is less pronounced in younger animals compared to older ones [9]. the meat aroma is one of the most important factors in determination of acceptability of the meat by the consumers. the basic meat aroma comes from meat components which are soluble and insoluble in water, like: sugars, amino acids and nucleotides. the specific meat aroma in different species is conditioned by the proportion of different fatty acids [10]. the textural attributes of the meat, like softness, tenderness and juiciness of the meat, which depend on the breed, the specie, the sex, the age, etc, are important sensory characteristics [6]. the juiciness of the lamb is more pronounced and depends on the ability to retain the natural water. this ability decreases with aging of the lamb and directly affects the decrease of meat juiciness [11]. 2. materials and methods 2.1 source of lamb’s meat the experimental part of the research is performed on a sheep farm in property of zk kicevo-ad kicevo located in cer, 30 km (18.6 miles) from kicevo, republic of macedonia, certificated for organic production. the lambs bred on that farm are traditionally exported to italy for easter, and belong to the first category, with average slaughter weight from 7.0 to 12.0 kg. the experimental part of the research began in the middle of january, and ended on the 75th day of lamb breeding. the research includes 120 lambs, 60 of them are bred in organic system, and the other 60 are bred in conventional system. in the conventional system of breeding two groups are formed, each one with 30 lambs. lambs in group i are crossbreeds from domestic breed refined with virtemberg, and the lambs in group ii are crossbreeds from domestic breed refined with east friesian. the lambs from both groups in the conventional system are bred on traditional way, the breast-feeding was interrupted at the 30th day and they were fed with conventional commercial fodder, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 295 alfalfa hay and ad libitum water until the end of breeding. in the organic system of breeding two groups are formed, each one with 30 lambs. the lambs in group i are crossbreeds from domestic breed refined with virtemberg, and the lambs in group ii are crossbreeds from domestic breed refined with east friesian, certificated for organic production and bred according to the rules for equivalence with eu/reg. 834/2007, according to which with the respect of animal’s welfare, they were weaned at the 45th day and fed with concentrated fodder from organic and certificated food, produced in own mill. the lambs from the conventional and the organic system are bred in special, physically separated and marked parts of the farm, with lot of straw on the floor. 2.2 measuring, marking and lambs feeding the formation of the groups and the selection of the lambs is done shortly after the partus. right after the measuring, each lamb is marked with numbers inflicted with paint, and on the second control weight measuring on the 15thday the lambs are marked with earmarks provided from the authorized veterinary station. in the first ten days of breeding, the lambs from the conventional and the organic system of breeding were constantly with their mothers, just breast fed, while after the tenth day the lambs were separated from their mother for few hours during the day in order to adjust to concentrated food. the chemical composition of organic and conventional fodder for lambs, are presented in (table 1). table 1 chemical composition of fodder lambs in conventional and organic system index conventional fodder organic fodder moisture (%) 15.44 14.98 dry matters (%) 84.56 85.02 crude proteins (%) 14.86 14.76 crude fats (%) 1.21 1.20 crude fiber (%) 3.63 3.96 total ash (%) 4.92 3.81 nitrogen free extracts (%) 59.94 61.29 2.3 statistical data processing for the calculation of basic statistical indicators the software package tools (data analysis) was used. results were presented as the mean value % of total peak areas of three repeated analyses. the statistical significance of the effect considered was evaluated by means of the variance analysis at the level of 0.05 and 0.01. the variations between each mean value were also tested by applying the ttest. 2.4 samlpes of lamb meat and panels samples of lamb shoulder and thigh from group i and ii from organic system and from group i and ii from conventional system, which after 24 hours cooling are wrapped into polyethylene foil and frozen to -180с, are taken for examining the weight loss that is determining the shrinkage during thermal processing of the meat. meat defrostration is performed at room temperature from 18 do 200с. the shrinkage from the meat thawing is determined on the basis of difference between meat weight before freezing and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 296 after thawing. all the samples are measured and registered before roasting. the thermal processing of the meat (roasting) is performed in professional oven at 1750с to reaching temperature from 800с ±1 in depth of the samples. after roasting, the meat is left to cool at room temperature, and measurements of roasted meat weight are performed afterwards. the weight loss of the meat that is the shrinkage during thermal processing is determined on the basis of the difference between meat weight before and after roasting. the evaluation of the sensory attributes of lamb from both systems is conducted by 38 different evaluators. the meat samples are encrypted with letters: a,b,c and d. the evaluation of the sensory attributes of lamb is conducted with the scoring method, [12]. coefficient of relevance is determined for each attribute of the meat: smell, juiciness, taste, softness, texture and aroma. the sensory attributes are evaluated with grades 1-5, which multiple with the coefficient of relevance, and their sum is expressed in (%) and indicates the percentage of maximum possible quality. mean value, or weighted overall evaluation of meat quality is obtained by dividing the maximum possible quality with the sum of coefficient of relevance (σ=20). 3. results and discussion examination of evaluation results during thermal processing of lambs meat from bought breeding systems are shown in table 2. table 2 weight loss during thermal processing of lambs meat according to breeding system *o(i)organic system i group, o(ii)-organic system ii group, c(i)-conventional system i group, c(ii) conventional system ii group, a,b, means in a column not having a common superscript letter are different (p<0.05) biggest meat thawing shrinkage is determined in meat from c (ii) in amount of 2.99 ±0.74 %, and 2.78% in meat from c (i). the meat thawing shrinkage in the meat from o (i) is 2.49±0.71%, or 2.34% in meat from o (ii). the bigger meat thawing shrinkage in lamb from conventional system is due to different chemical composition of meat, or bigger amount of water compared to the meat from organic system which is released during meat thawing, i.e. during thawing the ice crystals. from the research results it can be concluded that weight loss during thermal processing, that is the shrinkage during thermal processing of frozen and thawed meat from o (i) is 30.20±1.09%, and it is 2.51% smaller than the weight loss from c (i) (32.71±0.84%). the shrinkage during thermal processing of meat from o (ii) is 31.45±0.97%, and it is 2.59% smaller than the meat shrinkage from c (ii) system weight before frozen (kg) thawing shrinkage (%) weight before roasting (kg) weight after roasting (kg) roasting shrinkage (kg) roasting shrinkage (%) o (i) 4.754±0.74 2.34±0.63a 4.645±0.83 3.242±0.79 1.403±0.81 30.20±1.09a o(ii) 4.709±0.76 2.49±0.71b 4.595±0.80 3.150±0.93 1.445±0.66 31.45±0.97b c(i) 4.728±0.81 2.78±0.71a 4.600±0.69 3.095±0.55 1.505±0.66 32.71±0.84a c(ii) 4.773±0.89 2.99±0.74b 4.635±0.74 3.057±0.67 1.578±0.75 34.04±0.78b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 297 (34.04±0.78%). the ascertained difference in thermal processing shrinkage between the meat from o (i) and c (i) is significant (p<0.05). the weight loss during roasting determined in lamb from conventional system compared to the ones from the organic system are bigger, and derive from breeding system and way of feeding the lamb, which is the reason for different chemical composition of the lamb from organic and conventional system. it is obvious that the bigger weight loss of meat from conventional system during thermal processing is due to bigger content of water and fat in the meat, which are released during thermal processing of the meat. our results are in accordance to ascertainment that the weight loss of meat during thermal processing indicates specific variations depending on the impact of various factors, especially the method and time of thermal processing, the temperature, the size, i.e. the area of piece and the meat composition, the freezingpoint, the meat storing and thawing, which cause bigger or smaller loss of tissue liquids (juices) [3]. table 3 sensory evaluation of lamb’s meat sensory attributes organic system conventional system i ii i i intensity 4.24±0.16a 4.13±0.17a 3.97±0.17b 3.05±0.21b juiciness 4.34 ±0.11 a 3.91±0.13b 3.87±0.16b 3.55±0.17c taste 4.26±0.15 4.05±0.16 4.10±0.15 3.03±0.18 aroma 3.92±0.19a 3.86±0.18a 3.76±0.17b 2.74±0.18c texture 4.13± 0.12 a 4.03±0.16 a 3.74±0.16 b 3.32±0.19c softness 4.34± 0.12 4.50± 0.13 3.81±0.15 3.47±0.20 overall acceptability score 4.29 4.08 3.92 3.31 *o(i)organic system i group, o(ii)-organic system ii group, c(i)-conventional system i group, c(ii) conventional system ii group, a,b,c means in a row not having a common superscript letter are different (p<0.01) the results from the evaluation of sensory attributes of lamb from bought breeding group are represented in table 3. there are no significant differences in intensity and taste of lamb from organic system and from conventional system [13]. the meat smells from o (i) is normal and pleasant, evaluated with highest evaluation 4.24±0.16. the smell of the lamb from c (ii) is more pronounced and unpleasant, evaluated with lowest evaluation 3.05±0.21. the determined differences between the smell of the meat from o (i) and c (ii) are statistically significant (p<0.01). there are no determined significant differences in the smell of the meat between groups in the same breeding system. the lamb from organic breeding system, compared to the lamb from conventional breeding system is more acceptable and desirable among the consumers, mostly because of meat juiciness [14]. meat which has bigger ability to retain water is juicier. meat juiciness of lamb from o (i) is evaluated with highest evaluation 4.34 ±0.11, and the juiciness of lamb from c (ii) is evaluated with lowest evaluation 3.55±0.17. the determined difference in juiciness is significant (p<0.01). the meat aroma is one of the most important factors of the acceptability of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 298 meat among consumers. from the shown results, a large deviation in evaluation of meat aroma from both systems is ascertained, i.e. the aroma of the lamb from o (i) is evaluated with 3.92±0.19, and the meat aroma from c (ii) is evaluated with 2.74±0.18. the determined difference in meat aroma is statistically significant (p<0.01). ascertainment that meat from younger animals has less pronounced aroma, which is more and more pronounced with aging because of increase of intramuscular tissue does not match with the results from this research, because the meat from both systems if from lambs of same age, [15]. the specific meat aroma from different breeds is a result of the different relation of fatty acids, especially unsaturated fatty acids which are more susceptible to oxidation. the way of feeding the lambs also impacts meat aroma [15]. the differences which occur in the evaluations of texture and softness of highest and lowest evaluated meat are statistically significant, and a consequence of breed characteristics. our ascertainments match to [16] ascertainments. from the results from the sensory evaluation it is ascertained that lamb from group i and ii from the organic system has much better acceptability of sensory qualities that the meat from group i an ii from the conventional system. the lamb from o (i) is evaluated with highest average evaluation 4.29, the meat from o (ii) is evaluated with 4.08, the meat from c (i) is evaluated with 3.92, and the meat from c (ii) is evaluated with lowest average evaluation 3.31. the general acceptability of lamb is highest evaluated in meat from o (i), and lowest evaluated in meat from c (ii), and thereby it is ascertained that different sensory attributes of the meat derive from different breeding system and way of feeding, the composition of animal feed mixes, the chemical composition of the meat, as well as the breed characteristics of the lambs. 4. conclusion on the basis of the results from the evaluation of the sensory quality of thermal proceeded lamb from group i and ii from the organic and the conventional system it is ascertained that the general acceptability of lamb, wherein all the sensory attributes (smell, juiciness, taste, aroma, texture and softness) are summed up, is highest evaluated in meat from group i and ii from the organic system, compared to meat from group i and ii from conventional system. thereby, it can be ascertained that the determined differences in sensory attributes of the meat derive from the different breeding system that is the way of feeding which is practiced in the organic system, and the meat chemical composition. also, there are differences in the sensory attributes due to breed characteristics of the lambs. after performed thermal processing a significantly bigger (p<0.05) weight loss of lambs from conventional system compared to the weight loss of lamb from the organic system is determined. 5. references [1] hermansen j e., organic livestock production systems and appropriate development in relation to public expectations, livestock production science vol. 80, issues 1–2, 3–15, (2003); [2] benoit, m., veysset p, conversion of cattle and sheep suckler farming to organic farming: adaptation of the farming system and its economic consequences. livestock production science, 80, 141-152, (2003); [3] stojanovski m, technology of meat and meat products, faculty of biotechnical sciences, 91104, (2011); [4] petrović m., ružić-muslić d., maksimović n., novi tehnološki postupak za proizvodnju kvalitetnog jagnjećeg mesa, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 3 – 2015 tatijana kalevska, ljupce kocoski, elena joshevska, viktorija stamatovska, aleksandar savevski, sensory evaluation of lambs meat according to conventional and organic breeding systems, food and environment safety, volume xiv, issue 3 – 2015, pag. 293 – 299 299 biotechnology in animal husbandru 26, 31-40, (2010); [5] morbidini, l., d.m sarti, p. pollidori, a. valigi, carcass, meat and fat quality in italian merino derived lambs obtained with ‘‘organic’’ farming system. options méditerraneénnes série a, 46, 29– 31, (2001); [6] schreurs f.j.g., postmortem changes in chicken muscle: some key biochemical processes involved in the conversion of muscle to meat. ph d thesis, university of wageningen, wageningen, the netherlands, (1999); [7] cividini a., kompan d., potočnik k., žgur s., the senzor quality of lamb meet produced in different rearing systems of autochthonous jezersko-solcava sheep breed. biotechnology in animal husbandru 25(5-6), 935944, (2009); [8] boutonnet, j. p., perspectives of the sheep meat world market on future production systems and trends. small ruminant research, 34, 189–195, (1999); [9] young, o.a., berdague, j.l., viallon, c., rousset-akrim, s.a., theriez m, fatborne volatiles and sheepmeatodour. meat sci., 45: 183-200, (1997); [10] ivanovć s., savić s., baletić ž., uticaj starosti jagnjadi na senzorne osobine mesa jagnjadi. veterinarni glasnik 61(1-2), 65-73, (2007); [11] teixeira, a., s. batista, r. delfa,v. cadavez: lamb meat quality of two breeds with protected origin designation. influence of breed, sex and liveweight. meat science 71(3), 530-536, (2005) [12] radovanovic r., popov-raljic j, senzorna analiza prehrambenih proizvoda. beograd, 242-245, (2000); [13] revilla i., vivar-quintana a.m., luruena-martinezm.a., palacios c., severiano-perez p, organic vs conventional suckling lamb production: product quality and consumer acceptance.in: cultivating the future based on science. vol. 2. sofar, bonn: 514–517, (2008); [14] fisher, a.v., enser, m., richardson, r.i., wood, j.d., nute, g.r., kurt, e., sinclair, l.a., wilkinson, r.g, fatty acid composition and eating quality of lamb types derived from four diverse breed x production system. meat sci. 55, 141–147, (2000); [15] ružić-muslić d., grubić g., petrović m., žujović m., muslić h., efekat izvora proteina u obroku na proizvodne perfomanse jagnjadi u tovu. biotechnology in animal husbandru 23(1-2), 175-184, (2007); [16] revilla i., vivar-quantina a.m., palacios c., severiano p, organic vs conventional suckling lamb production quality and consumer acceptance. in: cultivating the future based on science. vol.2 isofar, bonn: 514-517, (2009). 278 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3, pag. 278 285 improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables * lessoy zoue1 , sébastien niamke1 1faculty of biosciences, biotechnology laboratory, felix houphouet boigny university, abidjan, côte d’ivoire, y.lessoy@yahoo.fr *corresponding author received 11th july 2018, accepted 14th september 2018 abstract: the purpose of this study was to assess in vitro bioaccessibility of provitamin a carotenoids in fermented leafy vegetables. these fermented leafy vegetables are often used to prepare dishes in some parts of tropical africa. carotenoids were extracted from fermented leafy vegetables and analyzed by using high performance liquid chromatography (hplc). in order to simulate the human digestion tract, the in vitro bioaccessibility method known as simple, inexpensive and reproductible tool was performed. all leafy vegetables contained considerable amount of provitamin a carotenoids (26.61 ± 1.98 – 305.85 ± 14.38 μg/g dw) with the concentration varying from one source to another. fermentation of leafy vegetables resulted in losses (24.5 – 78.22%) of provitamin a carotenoid contents. the in vitro bioaccessibility of provitamin a carotenoids of non fermented leafy vegetables ranged from 3.58 to 5.21% while that of fermented leafy vegetables was siginificantly different (p ˂ 0.05) and ranged from 11.51 to 15.32%. the results also showed that fermented leafy vegetables may contribute between 9.48 and 111.31% of the vitamin a daily requirement (rda) estimated to 550 μg for individuals in developing countries. this study highlighted that fermentation appears as a suitable non thermal processing to improve the bioaccessibility of provitamin a carotenoids of leafy vegetables. thus, an increased consumption of fermented leafy vegetables could help combat vitamin a deficiency (vad) which remains a major public health problem in developing countries. keywords: bioavailability, carotenoids, fermentation processing, leafy vegetables, vitamin a 1. introduction dietary carotenoids are fat-soluble pigments found in high concentrations in fruit and vegetables [1]. these bioactive components include carotenes (β-carotene, α-carotene and lycopene) and xanthophylls (zeaxanthin, lutein, α and βcryptoxanthin) and they may prevent several diseases like cancer, cardiovascular disease and mainly blindness due to vitamin a deficiency (vad) [2,3]. vad remains a major public health problem in developing countries where children and pregnant women are usually affected. for these population groups, the largest contribution of vitamin a intake is provided by provitamin a carotenoids from plant foods, which may contribute up to 82% of the total vitamin a [4]. therefore, food-based strategies for combating vitamin a deficiency in africa include promotion and consumption of tropical leafy vegetables. indeed, these plants are available, cheaper and provitamin a carotenoids-rich [5]. commonly, tropical leafy vegetables are prepared at home by using different cooking methods (boiling, stewing, frying, and blanching) that have been reported to degrade 5 to 78% of β-carotene [6,7]. considering that important amount of carotenoids needed by individuals may be lost during household cooking, the question of bioaccessibility and http://www.fia.usv.ro/fiajournal mailto:y.lessoy@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 279 bioavailability of provitamin a carotenoids from tropical leafy vegetables remains important. the term bioavailability refers to the fraction of an ingested nutrient available for utilization in normal physiological functions and storage. however, bioaccessibility is defined as the amount of an ingested nutrient that is available for absorption after digestion [8]. many factors may affect the bioavailability of carotenoids and the term slamenghi used to describe these factors was formulated as follow [9]: species of carotenoid, molecular linkage, amount of carotenoids in a meal, matrix in which the carotenoid is incorporated, effectors of absorption and bioconversion, nutrient status of the host, genetic factors, hostrelated factors, and mathematical interactions. absorption of carotenoids involves the release from the food matrix, dispersion and solubilization into mixed bile salt micelles, uptake by intestinal mucosal cells and circulation through the lymphatic system into the bloodstream [10]. for green leafy vegetables, the main factor that affects the bioaccessibility of carotenoids is the leaf matrix which may hinder the release of the carotenoids during digestion [11]. in order to reduce this matrix effect, traditional processing methods such as boiling, blanching and pounding are commonly used in households [12]. some reports also indicated that boiling or blanching leafy vegetables resulted in 2 to 12% bioaccessibility of β-carotene [11]. another scarce processing method used to reduce the leaf matrix effect is fermentation. indeed, some populations of west and central africa ferment leafy vegetables for 3 to 4 days to prepare dishes with a distinct flavor. in addition, fermentation can enhance the nutritional quality of leafy vegetables by producing beneficial by-products. to the best of our knowledge, the bioaccessibility of provitamin a carotenoids in such relishes is not documented and little information were reported about the ability of such fermented tropical leafy vegetables to supply daily vitamin a requirement. in vitro model method is known as simple, inexpensive and reproductible tool to stimulate the human digestion tract [13]. therefore, the aim of this study was to assess the impact of fermentation on the bioaccessibility of provitamin a carotenoids from tropical leafy vegetables by using in vitro model. 2. materials and methods 2.1 chemicals the solvents (methanol, dichloroethane, methyltertiary-butyl diethyl ether and formic acid) were all hplc grade and purchased from fisher scientific. the purified carotenoids standards (lutein, alltrans-β-carotene, 13-cis-β-carotene, 9-cisβ-carotene, α-carotene, β-apo-8-carotenal) and enzymes (porcine pepsin, pancreatin, bile extract) were from sigma-aldrich. 2.2 fermentation processing of leafy vegetables twelve (12) leafy vegetables widely consumed in côte d’ivoire were selected (table 1). all the leaves were collected at maturity from a periurban farmland (latitude: 5°19′14″ north; longitude: 4°22′59″west) located in abidjan district (côte d’ivoire). the collected leaves were authenticated by national floristic center (university felix houphouët-boigny, abidjan). the leaves were washed several times with distilled water, drained at ambient temperature, cut into small pieces and separated into two portions of 250 g each. the first portion was wrapped in clean papaya leaves for 4 days to induce natural fermentation in a covered plastic box. afterwards, the fermented (f) leaves were oven-dried (50°c/3 days) and ground food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 280 into a powder with a laboratory crusher. the second portion was not subjected to the fermentation step but was used as control (non-fermented nf leaves). all the dried and powdered samples were stored at -18°c in airtight containers for further experimentation. table 1. common and local names of selected tropical leafy vegetables leafy vegetables common name local name abelmoschus esculentus okra gombo amaranthus hybridus green amaranth boronbrou basella alba indian spinach epinard celosia argentea common cockscomb soko colocasia esculenta taro taro corchorus olitorius jew’s mallow kplala hibiscus sabdariffa roselle dah ipomea batatas sweet potato patate manihot esculenta cassava manioc myrianthus arboreus bush pineapple tikliti solanum melongena eggplant aubergine talinum triangulare ceylon spinach mamichou 2.3 carotenoid analysis 2.3.1 carotenoid extraction total carotenoids of f and nf leaves were extracted using a previously reported method [14] with slight modifications. powdered samples (0.05 g) were mixed with 5 ml ethanol containing butylated hydroxytoluene (0.1%, w/v) and heated in a water bath at 85°c for 5 min. then, 400 μl koh in water (80% w/v) was added for saponification and the suspension was mixed using a vortex for 20 s and heated in a water bath at 85°c for 5 min. the tubes containing the reaction mixture were placed in ice after introducing 3 ml deionized water and carotenoids were extracted three times with 4 ml hexanes. β-apo-8’-carotenal was used as an internal standard and was added after saponification to account for mechanical losses. the combined extracts were dried under nitrogen and reconstituted in 1 ml 50:50 methanol-dichloroethane. 2.3.2 hplc analysis for the carotenoid identification and quantification, 25 μl extract was injected into an hplc system (waters corporation; milford, ma, usa) consisting of a 717 autosampler, 1525 binary pump, and a 2996 photo-diode array detector (pda). the column used was a c30 ymc carotenoid column (4·6 x 250mm, 3mm). the hplc solvent gradient included methanol-water (92:8, v/v) with 10 mm ammonium acetate (solvent a) and 100 % methyltertiary-butyl ether (solvent b). samples were analyzed at 1 ml/min with a 30-min linear gradient from 70 to 40% solvent a. lutein, β-carotene (including all-trans, 13-cis, and 9-cis), and α-carotene were identified and quantified using hplc-purified standards. chromatograms were generated at 450 nm. 2.4 in vitro bioaccessibility of provitamin a carotenoids determination of in vitro bioaccessibility of provitamin a carotenoids was carried out according to an in vitro digestion method [15] with slight modification. about 0.5 g of powdered vegetable sample was mixed with 10 ml ascorbic acid (1%, w/v). for the digestion, 5 ml of a 0.5% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 281 (w/v) porcine pepsin solution in hcl 0.1m with physiological amounts of calcium (3.6 mmol), magnesium (1.5 mmol), sodium (49 mmol), potassium (12 mmol) and phosphate (6.4 mmol) were added. ph was adjusted to 2.0 with hcl 2 m and nitrogen gas was blown into the flask and the scraw capped test tube was incubated at 37°c in a shaking water bath at 250 rpm for 1 h. after the gastric digestion phase, the ph value was adjusted to 5 with naoh 1m and 3 ml of pancreatin-bile solution (4 g/l bile extract and 25 g/l pancreatin dissolved in 0.1m nahco3) was added. the ph value was further increased to 7.5 and the mixture was incubated again for 30 min. after intestinal digestion phase, the sample was centrifuged at 5000 g for 20 min. the supernatant was collected and mixed with 5 ml of 25% (w/v) nacl and 5 ml of ethanol stabilised with 0.1% (w/v) bht. afterwards, carotenoids were extracted two times by centrifugation (5000 g; 20 min) with 5 ml hexane and the combined extracts were dried under nitrogen before reconstitution in 1 ml 50:50 methanol-dichloroethane. the released provitamin a carotenoids were analyzed and quantified by hplc as described in 2.3.2. 2.5 retinol activity equivalent determination the calculation of retinol activity equivalent (rae) of fermented leafy vegetables after in vitro bioaccessibility was based on the bioconversion factor defined as 2 μg accessible β-carotene to 1 μg of retinol (2:1)[16] . 2.6 statistical analysis values were reported as means ± sd. data were analyzed using xlstat 2016.2 (addinsoft, ny, usa). carotenoid composition and bioaccessibility values were compared using one-way anova. differences among processed leaves and treatment groups were determined using least significant differences (lsd) at p ˂ 0.05. 3. results and discussion 3.1 carotenoid profile carotenoid contents and their chromatographic profile are given in table 2 and fig. 1, respectively. the identified carotenoids were lutein and provitamin a carotenoids (13-cis-β-carotene, α-carotene, all-trans-β-carotene, and 9-cis-β-carotene). all leafy vegetables contained considerable amount of provitamin a carotenoids (26.61 ± 1.98 – 305.85 ± 14.38 μg/g dw) with concentration varying from one source to another. many factors affect carotenoid content of leafy vegetables, such as variety, location, cultivation, and post-harvest handling practices [17]. considering non fermented (nf) leafy vegetables, the best sources of provitamin a carotenoids were a. esculentus (305.85 ± 14.38 μg/g dw), c. olitorius (200.56 ± 16.21 μg/g dw), m. esculenta (233.20 ± 13.13 μg/g dw) and s. melongena (220.71 ± 2.23 μg/g dw). fermentation of leafy vegetables resulted in losses (24.5 – 78.22%) of provitamin a carotenoids contents. it’s important indicating that the traditional fermentation of leafy vegetables involves lactic acid bacteria as main fermenting microorganisms [18]. the losses of carotenoids in this study is not surprising since carotenoids degradation occurs during lactic acid fermentation of vegetables as indicated by some authors [19]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 282 fig. 1. chromatrogram of carotenoids from selected tropical leafy vegetables (a): lutein; (b): 13-cis-β-carotene; (c): α-carotene; (d): all-trans-β-carotene; and (e): 9-cis-β-carotene table 2. total provitamin a contents of fermented (f) and non fermented (nf) leafy vegetables leafy vegetables total provitamin a carotenoids (μg/g dw) non fermented (nf) fermented (f) a. esculentus 305.85±14.38a 193.90±3.07 b a. hybridus 129.41±2.41a 33.49±0.27 b b. alba 83.07±3.01a 62.71±6.55 b c. argentea 148.46±5.55a 32.33±1.48 b c. esculenta 189.78±3.23a 80.40±3.71 b c. olitorius 200.56±16.21a 122.88±3.21 b h. sabdariffa 26.61±1.98a 17.16±4.23 b i. batatas 156.97±1.90a 48.85±8.92 b m. esculenta 233.20±13.13a 140.78±1.88 b m. arboreus 73.02±8.98a 38.11±0.64 b s. melongena 220.71±2.23a 128.86±3.10 b t. triangulare 178.25±1.62a 45.98±0.43 b data are presented as means of triplicate analyses ± sd. means with the same superscript letter in the same line for a single vegetable are not different at p > 0.05 3.2 in vitro bioaccessibility bioaccessibility of food components such as vitamins is an important feature to assess their role in human health. the in vitro bioaccessibility model has been developed as simple, inexpensive, and reproducible tools to study the human digestion of different food components (ascorbic acid, carotenoids, chlorophylls, polyphenols) [20]. the in vitro bioaccessibility of provitamin a carotenoids of non fermented leafy vegetables ranged from 3.58 to 5.21% while that of fermented leafy vegetables was siginificantly different (p ˂ 0.05) and ranged from 11.51 to 15.32% (table 3). the results for provitamin a carotenoids bioaccessibility of non fermented leafy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 283 vegetables were consistent since less than 5% provitamin a carotenoids is generally released from unprocessed food matrix as indicated by some authors [21]. indeed, the first step in digestion of carotenoid comprises disintegration of the food matrix in order to release carotenoids [22]. in green leaves, carotenoids components are organised in pigment–protein complexes located in cell chloroplasts, which have to be accessed before making carotenes available [23]. contrary to non-fermented leaves, fermentation processing increased bioaccessibility of provitamin a carotenoids. the values of bioaccessibility of fermented leaves were similar to those (6-29%) of cooked or blanched leafy vegetables without oil [11,15]. moreover, accessible percentage of provitamin a carotenoids from fermented leafy vegetables were slightly lower than the factor 1/6 (16,7%) proposed by the us institute of medicine (iom) for the bioaccessibility of all-trans-β-carotene from vegetable food items [16]. thus, fermentation of leafy vegetables appears as a suitable non thermal technique to improve the bioaccessibility of provitamin a carotenoids. table 3. in vitro bioaccessibility of fermented (f) and non fermented (nf) leafy vegetables leafy vegetables bioaccessibility (%) non fermented (nf) fermented (f) a. esculentus 4.55±0.01a 12.63±0.90 b a. hybridus 5.21±0.02a 14.33±0.01 b b. alba 5.05±0.01a 11.51±0.10 b c. argentea 4.05±0.03a 13.38±0.01 b c. esculenta 4.16±0.78a 12.11±0.69 b c. olitorius 4.34±0.78a 15.32±0.49 b h. sabdariffa 4.31±0.05a 12.16±0.01 b i. batatas 4.69±0.01a 14.58±0.02 b m. esculenta 5.03±0.17a 13.89±0.56b m. arboreus 5.02±0.15a 14.21±0.05 b s. melongena 3.58±0.20a 12.76±0.35b t. triangulare 4.85±0.02a 13.72±0.05 b data are presented as means of triplicate analyses ± sd. means with the same superscript letter in the same line for a single vegetable are not different at p > 0.05 3.3 contribution to vitamin a requirements assuming that the minimum intake of fermented leafy vegetable relish in a single meal is 50 g dry matter, the corresponding amount of accessible provitamin a carotenoids from the results of in vitro digestion was between 104.33 and 1224.47 μg (table 4). the conversion factor adopted by iom [16] assumes that onesixth (1/6) of the total carotene content is absorbed into the mucosa, and that onehalf (1/2) of absorbed β-carotene is converted to retinol. these conversion factors were used to estimate how much fermented leafy vegetables might contribute to the daily requirement (rda) of vitamin a. in this study, the in vitro digestion data were used instead of the one-sixth (1/6) factor with assumption that 100% of the carotenes released after in vitro digestion is absorbed into the mucosa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 284 [23]. considering the one-half (1/2) factor of accessible provitamin a carotenoids, the retinol activity equivalent (rae) varied from 52.16 μg (fermented h. sabadariffa leaves) to 612.23 μg for fermented a. esculentus leaves. the contribution to the rda of retinol was calculated and the results showed that fermented leafy vegetables may contribute between 9.48 and 111.31% the rda estimated to 550 μg vitamin a for individuals in developing countries [24]. therefore, increasing consumption of fermented leafy vegetables could help alleviate vitamin a deficiency (vad) which remains a major public health problem in developing countries. table 4. contribution of fermented leafy vegetables to daily vitamin a requirement fermented leafy vegetables accessible provitamin a carotenoids (μg) retinol activity equivalent (μg) contribution to daily vitamin a requirement (%) a. esculentus 1224.47 612.23 111.31 a. hybridus 239.95 119.97 21.81 b. alba 360.89 180.44 32.80 c. argentea 216.28 108.14 19.66 c. esculenta 486.82 243.41 44.25 c. olitorius 941.26 470.63 85.56 h. sabdariffa 104.33 52.16 9.48 i. batatas 356.11 178.05 32.37 m. esculenta 977.71 488.85 88.88 m. arboreus 270.77 135.38 24.61 s. melongena 822.12 411.06 74.73 t. triangulare 315.42 157.71 28.67 4. conclusion in this study, the fermented leafy vegetables showed relatively important in vitro bioaccessibility of provitamin a carotenoids. thus, they may contribute to the daily requirement of vitamin a. these findings could be useful in dietary intervention programmes to alleviate vitamin a deficiency (vad) in developing countries. however, several foodand host-related factors may influence vitamin a bioavailability at different points. thus, in vitro methodology for provitamin a bioaccessibility should be validated in different in vivo conditions. 5. acknowledgments the authors thank the us government for the fulbright fellowship granted to the first author lz at wisconsin-madison university, usa. 6. references [1]. liu r.h., health-promoting components of fruits and vegetables in the diet, advances in nutrition, 4:384–392, (2013). [2]. krinsky n.i., johnson e.j., carotenoid actions and their relation to health and disease, molecular aspects of medicine, 26:459–516, (2005). [3]. zampatti s., ricci f., cusumano a., marsella l.t., novelli g., giardina e., review of nutrient actions on age-related macular degeneration, nutrition research, 34:95–105, (2014). [4]. van den berg h., faulks r., granado h.f., hirschberg j., olmedilla b., sandmann g., et al., the potential for the improvement of carotenoid levels in foods and the likely systemic effects. journal of the science of food and agriculture, 80:880–912, (2000). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 lessoy zoue , sébastien niamke, improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, food and environment safety, volume xvii, issue 3 – 2018, pag.278 285 285 [5]. bloem m.w., de pee s., darnton-hill i., new issues in developing effective approaches for the prevention and control of vitamin a deficiency, food and nutrition bulletin, 19:137– 48, (1998). [6]. rahman m.m., wahed m.a., ali m.a., β-carotene losses during different methods of cooking green leafy vegetables in bangladesh., journal of food composition and analysis., 3:47– 53, (1990). [7]. vimala b., thushara r., nambisan b., sreekumar j., effect of processing on the retention of carotenoids in yellow-fleshed cassava (manihot esculenta crantz) roots, international journal of food science and technology, 46:166– 169, (2011). [8]. eriksen j.n., luu a.y., dragsted l.o., arrigoni e., adaption of an in vitro digestion method to screen carotenoid liberation and in vitro accessibility from differently processed spinach preparations, food chemistry, 224:407–413, (2017). [9]. tanumihardjo s.a., factors influencing the conversion of carotenoids to retinol: bioavailability to bioconversion to bioefficacy, international journal for vitamin and nutrition research, 72:40–45 (2002). [10]. tanumihardjo s.a., palacios n., pixley k.v., provitamin a carotenoid bioavailability:what really matters?, international journal for vitamin and nutrition research, 80:336–350, (2010). [11]. mulokozi g., hedrén e., svanberg u., in vitro accessibility and intake of β-carotene from cooked green leafy vegetables and their estimated contribution to vitamin a requirements, plant foods for human nutrition, 59:1–9, (2004). [12]. van het hof k.h., gärtner c., west c.e., tijburg l.b., potential of vegetable processing to increase the delivery of carotenoids to man, international journal for vitamin and nutrition research, 68:366–370, (1998). [13]. failla m.l., huo t., thakkar s.k., in vitro screening of relative bioaccessibility of carotenoids from foods, asia pacific journal of clinical nutrition, 17:200–203, (2008). [14]. howe j.a., tanumihardjo s.a., carotenoid-biofortified maize maintains adequate vitamin a status in mongolian gerbils, journal of nutrition, 136:2562–2567, (2006). [15]. hedrén e., diaz v., svanberg u., estimation of carotenoid accessibility from carrots determined by an in vitro digestion method, european journal of clinical nutrition, 56:425– 430, (2002). [16]. institute of medicine (us) panel on micronutrients. dietary reference intakes for vitamin a, vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. washington (dc): national academy press (us); 2001. available from: https://www.ncbi.nlm.nih.gov/books/nbk222310/ doi: 10.17226/10026. [17]. schmaelzle s., gannon b., crawford s., arscott s.a., goltz s., palacios-rojas n., et al., maize genotype and food matrix affect the provitamin a carotenoid bioefficacy from staple and carrot-fortified feeds in mongolian gerbils (meriones unguiculatus), journal of agricultural and food chemistry, 62:136–143, (2013). [18]. swain m.r., anandharaj m., ray r.c., parveen rani r., fermented fruits and vegetables of asia: a potential source of probiotics, biotechnology research international, article id:250424, (2014). [19]. bartkiene e., vidmantiene d., juodeikiene g., viskelis p., urbonaviciene d., lactic acid fermentation of tomato : effects on cis / trans lycopene isomer ratio , β-carotene mass fraction and formation of l (+) and d (–) -lactic acid, food technology and biotechnology, 51:471–478, (2013). [20] granado f., olmedilla b., herrero c., perez-sacristan b., blanco i. and blazquez s., bioavailability of carotenoids and tocopherols from broccoli: in vivo and in vitro assessment. experimental biology and medicine, 231(11):1733-1738, (2006). [21]. gomes s., torres a.g., godoy r., pacheco s., carvalho j., effects of boiling and frying on the bioaccessibility of β-carotene in yellow-fleshed cassava roots (manihot esculenta crantz cv. brs jari), food and nutrition bulletin, 34:65–74, (2013). [22]. bohn t., mcdougall g.j., alegría a., alminger m., arrigoni e., aura a.m., et al., mind the gap-deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites-a position paper focusing on carotenoids and polyphenols, molecular nutrition and food research, 59:1307– 1323, (2015). [23]. hedrén e., mulokozi g., svanberg u., in vitro accessibility of carotenes from green leafy vegetables cooked with sunflower oil or red palm oil. international journal of food science and nutrition, 53:445–53, (2002). [24]. world health organization (who), global prevalence of vitamin a deficiency, mdis working paper #2, p. 116, (1995) 1. introduction 4. conclusion microsoft word 11 ghinea and leahu_corectat 15 mai.doc 205 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 22018, pag. 205 212 environmental evaluation of pork meat chain: a romanian case study *cristina ghinea1, ana leahu1 1faculty of food engineering, stefan cel mare university of suceava, romania, cristina.ghinea@fia.usv.ro *corresponding author received 5th february 2018, accepted 25th june 2018 abstract. the aim of this paper was to establish the environmental impacts of pork meat chain using life cycle assessment (lca) methodology. for this study the system boundaries included: pig farm, slaughterhouse, meat processing, transport and waste treatment, which represent the main and secondary activities of a romanian pork meat producer. all inputs and outputs data necessary for the inventory analysis were collected from this producer, gabi database and other sources. the impact assessment phase was performed with gabi software which includes lca methods like cml2001 jan. 2016, recipe 1.08, ubp 2013 and edip 2003. the results showed that pork meat chain has negative impact on the environment mainly contributing to the acidification potential (ap), photochemical ozone creation potential (pocp), eutrophication potential (ep) and global warming potential (gwp 100 years). according to the results obtained with cml2001 jan. 2016 method, the main activities that contribute to global warming potential are manure storage (67.10%), central heating system (13.56%) and intensive pigs growth (9.59%). similar results were obtained by applying of ubp 2013 method which indicated also that the manure storage is the main contributor to gwp (66%) followed by central heating system (14.33%) and intensive pigs’ growth (9.43%). wastewater treatment is obvious the main contributor to ‘water pollutants’ category, while water consumption has a significant impact on ‘water resources’ according to ubp 2013 method. resources like water and energy are necessary in very large quantities in meat production from which solid waste and wastewater result, thus increasing the environmental impacts of this process. keywords: food industry, global warming potential, life cycle assessment, waste 1. introduction food commodities such as meat and dairy products have significant impacts on the environment [1, 2]. from all activities included in the life cycle of meat products, starting from farming stage until final waste disposal, there result environmental emissions [1]. pork meat is the most popular meat in the world according to [3], the larger producer is china followed by europe [1]. approximately 22.2 million tonnes of this meat was produced in europe, in 2014 (fig. 1). in the same year 13 million tonnes of poultry meat, 7.3 million tonnes of bovine meat and 0.8 million tonnes meat from sheep and goats were produced and processed [4]. most slaughtered pigs in the eu in 2014 were in poland, over 1000 tonnes, followed by romania with 800 tonnes and hungary with about 500 tonnes (fig. 2). at global level the average pork meat consumption is of 15 kg/capita/year, while in europe is of approximately 40 kg/capita/year [5]. in romania, there was a continuing decline in the production of meat for consumption [6]. between 2001 and 2013 the largest meat production in romania was in 2003, namely 710 thousand tonnes, and the smallest production of 553 thousand tonnes in 2010. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 206 fig. 1. meat production by species, eu-28, 2009-2014 (million tonnes) (according to [4]) fig. 2. pigs slaughtering in 2014, in europe (adapted from [4]) life cycle assessment (lca) is a useful tool which can be applied in the determination of environmental impacts resulted from complex food systems. lca was applied in various studies to evaluate agricultural and food processing activities (among them: production of rice, fruits, bread, milk, beef, pork meat etc.): all life cycle from paddy field to the supermarket of rice production system were evaluated by [7]. they showed that the production of 1 kg of rice produces 2.9 kg of co2; ghinea [10] evaluated the production, consumption and loss of apple fruits from the environmental point of view; vinyes et al. [11] investigated the production, distribution and consumption of fruits like apple and peach in the mediterranean area, while longo et al. [12] compared the environmental impacts of organic and conventional apple production from italy; 0.97 to 1.24 kg co2 eq. per loaf of bread (800 g) are the results obtained by [8] after evaluation of bread produced and consumed in the uk; the environmental impacts of 1 kg of packaged ultra-high temperature (uht) milk were determined by [9] and for the global warming potential, these authors obtained a value of 0.73 kg co2eq; beauchemin et al. [13] investigated greenhouse gas emissions (ghg) from beef production and estimated the intensity of ghg at 22 kg co2 equivalent (kg carcass)-1; gonzalez-garcía et al. [1] calculated the environmental impacts of portuguese pork meat production and reported a value of 3.3 kg co2 eq kg-1 pig carcass weight; food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 207 mcauliffe et al. [14] compared the environmental performances of intensive pig production units and estimated a value of 3.5 kg co2-eq per kg carcass weight. in this study lca was applied in order to establish the environmental impacts of pork meat chain considering the main activities of a romanian pork meat producer: pig farm, slaughterhouse, meat processing, transport and waste treatment. 2. materials and methods 2.1. goal, scope and functional unit the main aim of this study was to determine the environmental impacts resulting from a romanian pork meat producer by applying lca methodology. one tool namely gabi software, which includes lca methodology [15-17], was used for the modelling of the considered system. the system boundaries established for this evaluation are illustrated in fig 3. the functional unit considered was the amount of meat products obtained in one year by one romanian pork meat producer. table 1 presents the potential emissions which may result from the investigated activities. 2.2. inventory analysis activities included in the evaluated system were: growing and fattening of pigs, preparation of hot water, fresh water supply, meat products manufacturing, primary treatment of wastewater and manure management, organic waste incineration and other related activities. it is considered that for pigs growing from 25 kg up to 110 kg of live weight, about 260 kg of feed is consumed [18]. also, from pig breeding and fattening are resulting 14000 kg/day of manure. the main raw materials used in the production of meat products are: pork, beef and poultry, organs and edible by-products of slaughterhouse, bacon and other animal fats. the auxiliary materials are composed of: spices and food additives, membranes and coating materials, packaging materials. it was considered a production level of 14,000 heads/year and 7700 maximum accommodation capacities (for pig breeding and fattening); 350 pigs (weighing between 95 and 105 kg each) slaughtered daily and 10,000 t/year meat products processed under normal operating conditions of the target. the annual quantities of raw materials, auxiliaries and fuels used to obtain pork meat products are presented in table 2. within the outputs of the process besides the meat products can be mentioned: organic waste 475 tonnes, plastic waste 25 tonnes, wood waste 100 tonnes, bones 30 tonnes, paper and cardboard waste 7 tonnes, ash from the incinerator – 16 tonnes and others. the utilities necessary for the technological process are: groundwater captured from the underground, electric energy taken from the national system, thermal energy obtained in its own plant, sawdust for the production of smoke in its own generators, ammonia and gas butane. annual energy consumption is approximately 3600 mwh (2013-2014) and specific consumption is within the limits recommended by [18]. this consumption is represented by: slaughterhouse approx. 1875 mwh; the food factory approx. 1492 mwh; zootechnical complex approx. 250 mwh. chemical substances used (annual consumption) or owned: naoh 8500 kg (for wastewater treatment), h2so4 8 l; ch3cooh 2 l; hcl 1 l; (c2h5)2o 2l; petroleum ether 15 l; naphthylamine 25 g; k2cro4 1 kg; acetone (c3h6o) 1l; nano2 1 kg. other chemicals used for different purposes: detergents (280 l/month), descaler (230 l/month), disinfectants (115 l/month), degreasers (218 l/month), liquid soap (210 l/month), fe2(so4)3 (4 tonnes/month for wastewater treatment) polyacrylamide (125 kg/month for wastewater treatment). emissions of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 208 gaseous pollutants from the process and the heat and power plant are: co2, co, no, no2, nox, so2 and particulate matter. ammonia (nh3) is emitted in air from animal housing, storage of manure and land spreading of manure. methane (ch4) comes from animal housing, storage of manure and manure treatment. from animal housing, manure storage and land spreading is also emitted nitrous oxide (n2o) and odour (e.g. h2s) [18, 19]. the mass emissions of pollutants from the thermal plant registered in one year are: 1242 t of co2, 46.2 kg of co, 2712 kg of nox, 1627 kg of so2 and 172 kg of particulate matter. the determined and admitted values by the legislation are presented in the table 3 [20]. from smoking cells are emitted approximately 76.25 mg/nm3 co. in the case of the incinerator the air emissions established were: no2 (56 mg/nm3), so2 (47 mg/nm3), co (0.12 mg/nm3). these values fall within the permissible limits [20]. the total average water requirement is 681.5 m3 and the maximum total water demand is 723 m3. the daily water consumption was 380 m3 and the specific consumption of water for pigs was calculated at 31 l/head/ day. the values of the emissions in the water are below the limits allowed by the romanian legislation (table 4, [21]). also, the concentration of nitrate (n no3), ammonium (n-nh4), total nitrogen (n), total phosphorus (p), chlorides and synthetic detergents falls within the limits set by ntpa 001/2005 [21]. from land spreading and manure storage are emitted in soil in groundwater: nitrogenous compounds, phosphorus, k and na, heavy metals and antibiotics [18]. fig. 3. system boundaries table 1. potential emissions of activities included in the evaluated system [18, 19] activities potential emission housing of animals air emissions, odour, manure, dust, noise, wastewater storage of feed and feed additives dust storage of manure air and soil emissions, odour storage of other residues soil and groundwater emissions, odour storage of carcases odour manure landspreading emissions to air, soil, water and groundwater, odour, noise incineration of residues air emissions, odour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 209 table 2. raw materials, auxiliaries and fuels used for producing of meat products raw materials, auxiliaries and fuels unit annual quantity pork, beef and poultry meat t 7000 spices and additives t 500 membrane m 7500 flexible plastic packaging t 25 wood sawdust t 20 fuels t 450 detergents and hygienic substances t 10 table 3. emissions from the thermal plant admitted and determined values emissions concentration, mg/nm3 co no2 so2 particulate matter determined values 8.8 311 189 32.8 admitted values, alert threshold according to the order mappm 462/93 [20] 119 315 1190 35 admitted values, intervention threshold according to the order mappm 462/93 [20] 170 450 1700 50 table 4. wastewater emissions admitted and determined values wastewater cu2+ (mg/l) ni2+ (mg/l) zn2+ (mg/l) cd2+ (mg/l) pb2+ (mg/l) hg 352/2005 ntpa 001 [21] 0.1 0.5 0.5 0.2 0.2 determined value 0.02 0.021 0.32 0.007 0.015 soil analysis has shown that emissions fall within the limits imposed by current legislation (order 756/1997): cd (0.13 mg/kg determined – 3 mg/kg admitted), total cr (7.52 mg/kg determined – 100 mg/kg admitted), cu (13 mg/kg determined – 100 mg/kg admitted), ni (26 mg/kg determined – 75 mg/kg admitted) [22]. for transportation stage of meat products were calculated the air emissions (co2, co, nox, n2o, pm10, ch4, so2 and hydrocarbons) considering the emissions resulted from burning 1 kg of diesel. based on the values presented above (and others) were calculated, determined and estimated all the necessary inputs and outputs for each activity included in the system (fig. 3). 2.3. life cycle impact assessment, results and discussion in the impact assessment stage the data obtained in the inventory phase were modelled with gabi software. the values for impact categories such as: acidification potential (ap), eutrophication potential (ep), global warming potential (gwp), human toxicity potential (htp), photochemical ozone creation potential (pocp), main air pollutants (map), aquatic eutrophication (ae), terrestrial eutrophication (te), stratospheric ozone depletion (sod), climate change ecosystems (cche), climate change human health (cchh) were determined by applying lca methods like cml2001 jan. 2016, recipe 1.08, ubp 2013, edip food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 210 2003. the emissions which contribute to these impact categories are presented in [15-17]. in fig. 4 are illustrated the main contributors to the environmental impacts. it can be observed that the main activities that contribute to global warming potential are manure storage (67.10%), central heating system (13.56%), transport (%) and intensive pigs’ growth (9.59%). the environmental impacts of pork products obtained by applying cml 2001 jan. 2016 and edip 2003 lca methods are presented in fig. 5. the results obtained with cml method indicate that the manufacturing of pork products contribute mainly to ap (61%), followed by pocp (30%) and to a lesser extent to the other impact categories (fig. 5a). the results obtained with the second method (edip 2003) indicate that production of pork products mainly influences photochemical ozone formation potential (impact on human health (pocp – hh) and impact on vegetation (pocp-v)) and terrestrial eutrophication (fig. 5b). with recipe method were obtained the normalised values (in pe = person equivalents) for climate change impact category (cche, cchh) (fig. 6). three cultural perspectives: egalitarian (e), hierarchist (h), individualist (i) were considered for the evaluation. it can be observed that the pork meat chain has negative impacts on the environment since all the values are positive. for the climate change impact on human health were registered the highest values. also, fig. 6 shows that the individualist perspective provides the higher values compared with the other two. a) b) fig. 4. contribution to the environmental impacts of central heating, manure storage, intensive pigs growth and transport a) cml 2001 jan. 2016 and b) ubp 2013 methods a) b) fig. 5. environmental impacts of manufacturing of pork products obtained by applying: a) cml 2001 jan. 2016 and b) edip 2003 lca methods food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2018 cristina ghinea, assessment of environmental impact of food loss: case study apple fruits, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 211 fig. 6. impact on climate change of pork meat supply chain (ccheclimate change ecosystems; cchhclimate change human health) according to [19] the greenhouse emissions for pig production in eu27 there were estimated at 7.55 kg co2-eq./kg of pork meat produced (including ch4 – 0.74 kg co2-eq./kg of pork meat produced, n2o – 1.71 kg co2-eq./kg of pork meat produced, co2 related to energy consumption – 2 kg co2-eq./kg of pork meat produced, co2 related to land use – 3.1 kg co2-eq./kg of pork meat produced). 3. conclusions the environmental impacts of the main and secondary activities of a romanian pork meat producer (pig farm, slaughterhouse and meat processing, transport and wastewater treatment, incineration of organic waste) were investigated in this paper. determination of these impacts was performed by applying lca methodology. the results showed that the central heating is the main emitter of pollutants contributing to all impact categories. manure storage, transport and intensive pig growth are the activities which contribute significantly to gwp. also, intensive pig growth has a major contribution to ep. the main air pollutants come from central heating and manufacturing of pork products contributes mainly to ap and pocp. 4. acknowledgments this paper was performed with the support of gabi life cycle assessment software. 5. references [1]. gonalez-garcía s., belo s., dias ac., rodrigues j.v., da costa r.r., ferreira a., de andrade l.p., arroja l., life cycle assessment of pigmeat production: portuguese case study and proposal of improvement options, journal of cleaner production, 100:126139, (2015) [2]. notarnicola b., tassielli g., renzulli p.a., castellani v., sala s., environmental impacts of food consumption in europe, journal of cleaner production, 140: 753765, (2017) [3]. fao, fao's animal production and health division: meat & meat products, food and agricultural organization of the united nations, rome, (2014), on line at: www.fao.org/ag/againfo/themes/en/meat/backgroun d.html [4]. eurostat, meat production statistics, (2015), on line at: http://ec.europa.eu/eurostat/statisticsexplained/index.php/meat_production_statistics [5]. ahdb, eu per capita consumption, ahdb/ahdb pork calculations using data from eurostat, (2017), on line at: https://pork.ahdb.org.uk/pricesstats/consumption/eu-per-capita-consumption [6]. luca l., evolution of the localization of pig and poultry meat production in romania after eu accession, impact of socio-economic and technological transformations at national, european and global level, 7:152-159, (2015) [7]. blengini g.a., busto m., the life cycle of rice: lca of alternative agri-food chainmanagement systems in vercelli (italy), journal of environmental management, 90: 1512– 1522 (2009) [8]. espinoza-orias n., stichnothe h., azapagic a., the carbon footprint of bread, the international journal of life cycle assessment, 16:351–365, (2011) [9]. gonzalez-garcía s., castanheira e.g., dias a.c., arroja l., using life cycle assessment methodology to assess uht milk production in portugal, science of the total environment, 442: 225-234 (2013) [10]. ghinea c., assessment of environmental impact of food waste: a case study apple fruits, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 cristina ghinea, ana leahu, environmental evaluation of pigmeat chain: a romanian case study, food and environment safety, volume xvii, issue 2 – 2018, pag. 205 – 212 212 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava, xvi, (1):21 – 28, (2017) [11]. vinyes e., asin l., alegre s., muñoz p., boschmonart j., gasol c.m., life cycle assessment of apple and peach production, distribution and consumption in mediterranean fruit sector, journal of cleaner production, 149: 313-320, (2017) [12]. longo s., mistretta m., guarino f., cellura m., life cycle assessment of organic and conventional apple supply chains in the north of italy, journal of cleaner production, 140: 654-663, (2017) [13]. beauchemin k.a., janzen h.h., little s.m., mcallister t.a., mcginn s.m., life cycle assessment of greenhouse gas emissions from beef production in western canada: a case study, agricultural systems, 103:371-379, (2010) [14]. mcauliffe g.a., takahashi t., mogensen l., hermansen j.e., sage c.l., chapman d.v., lee m.r.f., environmental trade-offs of pig production systems under varied operational efficiencies, journal of cleaner production, 165:1163-1173, (2017) [15]. ghinea c., gavrilescu m., impact of food waste on climate change, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava, xiv, (4): 340 – 344, (2015) [16]. ghinea c., petraru m., bressers h.th. a., gavrilescu m., environmental evaluation of waste management scenarios – significance of the boundaries, journal of environmental engineering and landscape management, 20(1): 76-85, (2012) [17]. pe international, handbook for life cycle assessment (lca) using the gabi education software package, germany, (2009) [18]. ec, integrated pollution prevention and control (ippc), reference document on best available techniques for intensive rearing of poultry and pigs, european commission, (2003) [19]. santonja g.g., georgitzikis k., scalet b.m., montobbio p., roudier s., sancho l.d., best available techniques (bat) reference document for the intensive rearing of poultry or pigs; eur 28674 en; doi:10.2760/020485 [20]. mappm, order no. 462 of 1 july 1993 for the approval of the technical conditions for atmospheric protection and the methodological norms for determination of the pollutants emissions from stationary sources, ministry of waters, forests and environmental protection, (1993) [21]. decision no. 352/2005 regarding the modification and completion of the government decision no. 188/2002 approving some norms regarding the discharge conditions in the aquatic environment of the waste waters, normative on the establishment of limits for pollutant loading of industrial and urban waste water to evacuation to natural receptors, ntpa-001/2005, (2005) [22]. order 756/1997 for the approval of the regulation on environmental pollution assessment, ministry of waters, forests and environmental protection, (1997) 28 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2014, pag. 28 33 research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt *mihaela adriana tiţa1, ovidiu tiţa1, adriana dabija 2, alexandra majdik 1 1lucian blaga university of sibiu, faculty of agricultural sciences, food industry and environmental protection, 7-9 dr.i. ratiu street, 550012 sibiu, romania, mihaela.tita@ulbsibiu.ro 2 stefan cel mare university of suceava, faculty of food engineering, 13 university street, 720229 suceava, adriana.dabija@fia.usv.ro *corresponding author received february 19th 2014, accepted march 17th 2014 abstract: yogurt is a dairy product with high calcium content thatshould be consumed each day. yogurt,a dairy product of fermentation of milk, has a gel-like texture and a sour taste because of fermentation of milk sugar (lactose) into lactic acid. it has many nutritional and health benefits. with or without fruit, diet or not, yogurt remains one of the essential food for our health. quinoa is one of the most nutritious plants in the world. its beans are rich in proteins both quantitatively and qualitatively, contain the eight essential amino acids and are a rich source of minerals (ca, k, zn, fe, mg), b vitamins and fiber, which makes quinoa a complete and modern food. oat bran decreases the absorption of fat and sugar. but on the same principle, they allow the absorption of vitamins and other valuable nutrients for the body. oat bran is a good source of dietary fiber, thiamine, magnesium, phosphorus and iron proteins, in addition, they have a low sodium content. keywords: dairy product, ekomilk, health benefits, dietary fiber 1. introduction yogurt is the most specific and prevalent assortment of dairy acids products. initially, yogurt is obtained only from sheep's or buffalo milk; currently the most widespread assortment of yogurt is made from cow's milk and is produced under different types [1, 2]. yogurt is a dairy product from fermentation of milk, has a gel like texture and a sour taste because of fermentation of milk sugar (lactose) into lactic acid. it has many nutritional and health benefits. it is an important ally for the silhouette, is low calorie but high in nutrients. regular yogurt contains only 49 calories per 100 g, the fat 38 calories while yogurt made from whole milk, considered the most caloric, has only 64 calories per 100 g. it contains vitamins of group b, essential to the proper functioning of the nervous system and restore tissue affected by aging or disease, vitamin pp, which helps to increase vitamin a, which gives skin the glow and suppleness, and many minerals (calcium, potassium, sodium, zinc, and so on). the calcium in yogurt is fully assimilated better than from milk. yogurt with cereal for breakfast gives us energy for the day. the main advantage of the yogurt is calcium, which assimilate more easily and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik, research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt , volume xiii, issue 1 2014, pag. 28 – 33 29 better than from milk. this is because the milk components converted into yogurt become more affordable to the human body [3, 4, 5]. yogurt is a good source of phosphorus, b complex vitamins, monounsaturated fats. because of its contents, yogurt reduces the risk of cardiovascular disease, prevents high cholesterol, gastrointestinal disorders, and, at the same time holds off constipation. we can enjoy the benefits of yogurt if we eat the whole grains-wheat or just the oat flakes [6, 7]. yogurt with cereal strengthens the immune system and supports the normal functioning of the muscular system. cereals are an ideal ingredient of a healthy breakfast. containing fiber, minerals, vitamins and carbohydrates, grains are very important to maintain balance and therefore health food. you will be in top form and you'll be able to accomplish all your goals. fatigue and hunger will be removed. it was found that the grain also prevent heart diseases, obesity, diabetes, intestinal problems, and even different types of cancer [8, 9, 10]. quinoa has a similar composition and use of cereals (wheat, oats, barley and rye), it is generally assimilated, but is actually a pseudo cereal as it stands at the crossroads of cereals and grasses, being related to spinach and beets. it is a very strong and less demanding plant able to survive at high altitudes and cold thin air, the scorching sun or sandy terrain. there are many varieties of quinoa, but the most appreciated of all this is quinoa real (royal quinoa) having large grain and increased nutritional load. an extensive list of anti-inflammatory nutrients appears in quinoa. but regarded controversial aspect is the presence of saponin. this is a bitter nutrient soluble in water, which is the outer layer of the seed. quinoa saponins has been shown to have anti-inflammatory properties and antioxidant properties, however through the thermal preparation, it is removed. the current research is trying to establish the link between inflammatory effects of quinoa and the saponin. even with this limitation, the number of antiinflammatory nutrients to the quinoa remains impressive. the content of fiber and protein helps to regulate the blood sugar. because chronic inflammation is a key factor in developing type 2 diabetes, inflammatory nutrients found in quinoa reduces the risk of developing this disease. inflammatory nutrients help protect the blood vessels, which reduces the risk of cardiovascular disease, including atherosclerosis. the high fiber content manages to lower the blood cholesterol, control blood sugar levels and helps maintain weight [11, 12, 13]. evaluations show that it is a complete nutritional source of protein (14% by weight), comparable to milk as a source of fiber and phosphorus, as well as being rich in magnesium and iron. quinoa is a great source of calcium, very appreciated by vegetarians, but also by people who are lactose intolerant. quinoa is rich in riboflavin substance known to reduce the frequency of migraines, having a beneficial effect on the brain and muscle cells. oat bran is rich in insoluble fiber and pectin, contributing to the absorption of significant amounts of water. thus, if we take oat to each of main meal fiber mass contained facilitates digestion. they help to stabilize blood sugar levels, helps to reduce blood cholesterol levels. fibers are not absorbed by our body. by allowing the body to rapidly eliminate all or part fats and carbohydrates in foods eaten in a day, oat bran manages to reduce the number of calories ingested. that is why they have won over the years a leading position in weight loss diets. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik, research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt , volume xiii, issue 1 2014, pag. 28 – 33 30 2. experimental raw material for yogurt is mainly represented by cow's milk, by the content of varied nutrients especially proteins, milk is a particularly valuable food for nutrition especially children, sick people or those working in toxic environments. physico-chemical analysis of the milk samples are performed using ekomilk total analyzer. milk analyzer provides rapid test results of: fat, protein, non-fat substance, lactose, density, freezing point, added water, ph, temperature and conductivity in fresh milk [14, 15]. determination of milk acidity is carried out by titration with sodium hydroxide, in the presence of phenolphthalein as an indicator. determination of dry matter of yogurt with ml-50 thermobalance, rapid and uniform heating with halogen lamp and technology sra (secondary radiation assist). determining the total number of germs of yogurt samples. the method allows assessment of the degree of contamination of the product by seeding on nutrient solid media, and colony count results [14]. insemination is made in two petri dishes for each dilution: place 1 ml dilution first and then sterilized solid medium in a test tube. thermostating at 37°c for 48 hours after which it makes the counting of the colonies [14]. determination of the number of coliform bacteria of milk samples. the principle is based on the property of coliform bacteria to ferment lactose, producing gas release. 3. results and discussion the main objective of this work is to obtain a yogurt with added quinoa. in addition to that, another object is to obtain yoghurt with different nutrients such as milk powder, and the mixture of oat bran and oat bran quinoa (table 1). table 1 yogurt samples analyzed samples yogurt with quinoa yogurt with milk pouder yogurt with oat bran yogurt with oat bran and quinoa lp1 5g lp2 10g lp3 15g lq1 5g lq2 10g lq3 15g lt1 5g lt2 10g lt3 15g lq1 + lt1 2.5g 2.5g lq2 + lt2 5g 5g lq3 + lt3 7.5g 7.5g symbols lp1, lp2, lp3: yogurt with milk pouder; lq1, lq2, lq3: yogurt with quinoa; lt1, lt2, lt3: yogurt with oat bran; lq1+lt1, lq2+lt2, lq3+lt3: yogurt with oat bran and quinoa. were made 3 determinations for each sample at three different concentrations (table 1). 3.1. physico-chemical analysis analysis of milk, raw material was performed by ekomilk obtaining the following results: fat content 3.28 % non-fat substances content 9.94% protein content 3.74 % lactose content 5.45% density 1.0343 ph 6.43 due to the increasing amount of milk powder (lp3 = 15g milk powder), increases dry matter. low in fat content of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik, research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt , volume xiii, issue 1 2014, pag. 28 – 33 31 grain, quinoa and oat bran causes a lower dry matter. after storage of yogurts, cereals absorb water and dry matter content increases, as shown in lq3 + lto3. he results obtained for the dry matter of the samples of yogurt is shown in figure 2. figure 2. evolution of the dry matter determination of acidity of yoghurt samples after the first day. the results of this determination are shown in figure 3. figure 3. determination of acidity of yoghurt samples after the first day the acidity of the yogurt milk powder samples is higher due to the viscosity of yogurt, curd format that is more dense. the acidity of the yogurt samples with quinoa, oat bran and mixtures thereof having a constant acidity as curd formed is not so dense. determination of acidity of yoghurt samples after the seventh day. the results of this determination are shown in figure 4. figure 4. determination of acidity of yoghurt samples after the seventh day after storage, the acidity of the yogurt samples and quinoa milk powder increases, and the curd formed is more dense. the acidity of the samples with a mixture of oat bran and oat bran and quinoa decreases because the format curd and consistency of the yogurt is not compact and homogeneous. determination of acidity of yoghurt samples after the fourteenth day. the results of this determination are shown in figure 5. after fourteen days storage, the acidity of the samples increases due to the formation of compact and homogenous curd without whey disposal. figure 5. determination of acidity of yoghurt samples after the fourteen day. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik, research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt , volume xiii, issue 1 2014, pag. 28 – 33 32 3.2. microbiological analysis determining the total number of germs of yogurt samples. the number of the colony and theirs evolutions are shown in figure 6. figure 6. the number of the colony and theirs evolutions after long periods of storage of test plates, the culture medium develops several colonies. from figure 6 we can conclude that during the study period the yogurt with quinoa has formed the most colony in all the situations. determination of the number of coliform bacteria. after thermostating at 37°c for 48 hours, we obtained the following results: for the first 5-positive, for the second 5-positive, for the third series 2positive, representing 5-5-2 combination obtained is 540×103 colliforms 100 cm3/milk. 4. conclusions the final products correspond to the sensory characteristics: appearance and consistency, color, taste and smell [15]. appearance and consistency of yogurt with: milk powder: compact curd, homogeneous, without gas bubbles without whey removed; yogurt with quinoa: compact curd, homogeneous, without gas bubbles without whey removed; yogurt with oat bran: compact curd, homogeneous, without gas bubbles without whey removed; yogurts with the mixture of oat bran and quinoa: compact curd, homogeneous, without gas bubbles without whey removed. taste and smell: pleasant sour flavor, specific formula, quinoa and oat bran. after determining the acidity of the milk used to make yogurt, the milk corresponds in terms of standards. results obtained using the apparatus ekomilk, corresponding to a fresh milk with a high fat content. evolution acidity of yogurt samples, indicating an increase in value. the highest values recorded are those of the quinoa milk, milk powder and oat bran due to lactic acid of fermentation of lactose. the lowest results are obtained from a mixture of quinoa and oat bran that inactivate fermenting lactose. evolution of the dry matter depends on the cereal quantities added to yoghurts, as the amount of oat bran and quinoa is higher the dry matter increase due to water absorption power of cereals. from a microbiological point of view, yogurts obtained correspond to the standards. 5. references [1]. georgescu g., milk and dairy products, ceres, bucharest, (2000) [2]. georgescu g., book of producer and milk processor, ceres, bucharest, (2005) [3]. serafeimidou, a., et al. chemical characteristics faty acid composition and conjugated linloeic acid (cla) content of traditional greek yogurts, food chenistry, vol.134, issue 4, 1839-1846, (2012) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik, research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt , volume xiii, issue 1 2014, pag. 28 – 33 33 [4]. grygorczyk, a., el al., extraction of consumer texture preferences for yogurt: comparison of the preferred attribute elicitation method to conventional profiling, food quality and preference, vol.27, issue 2, 215-222, (2013) [5]. seo, m.h., et al., physicochemical, microbial, and sensory properties of yogurt supplemented with nanopowdered chitosan during storage, journal of dairy science, vol.92, issue 12, 5907-5916, (2009) [6]. soukoulis, c., et al., industrial yogurt manufacture: monitoring of fermentation process and improvement of final product quality, journal of dairy science, vol. 90, issue 6, 2641-2654, (2007) [7]. lureati, m., sustainability and organic production: how information influences consumer’s expectation and preference for yogurt, food quality and preference, vol.30, issue 1, 1-8, (2013) [8]. desai, l., et al., sensory properties and drivers of liking for greek yogurts, journal of dairy science, volume 96, issue 12, 74547466, (2013) [9]. van loo, e., et al., consumer attitudes, knowledge and consumption of organic yogurt, journal of dairy science, volume 96, issue 4 , 2118-2129, (2013) [10]. trigueros, l., et al., use of date blanching water for reconstituting milk powder: yogurt manufacture, food and bioproducts processing, volume 90, issue 3, 506-514, (2012) [11]. isik, u., et al., frozen yogurt with added inulin and isomalt, journal of dairy science, volume 94, issue 4, april 2011, 16471656, (2011) [12]. isleten, m., karagul-yuceer, y., effects of dried dairy ingredients on physical and sensory properties of nonfat yogurt, journal of dairy science, volume 89, issue 8, 2865-2872, (2006) [13]. hashim, i.b., khalil, a.h., afifi, h.s., quality characteristics and consumer acceptance of yogurt fortified with data fiber, journal of dairy science, volume 92, issue 11, 5403-5407, (2009) [14]. tiţa, m.a., handbook of analysis and quality control in the dairy industry " publishing lucian blaga university of sibiu, (2002) [15]. *** state collection of dairy industry standards, ministry of food and agriculture, cocpcia s.a., bucuresti, (1990) title …………………… 352 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 42015, pag. 352 357 investigation of structural-mechanical properties of the recipe composition for special purpose fondants * olena dudkina1 , aleksandra nemirich1, andriy gavrysh1, tatiana ischenko1, yuriy tkachuk1 1 faculty of hotel, restaurant and tourism business, national university of food technologies, ukraine busetko@gmail.com *corresponding author received november 4th 2015, accepted december 18th 2015 abstract: in ukraine, scientists and experts have paid great attention to the development of culinary wellness products and products of special purpose.these products include sweet dishes, in particular fondants, which are popular among population. therefore, researches have been done to justify the composition and structural-mechanical properties of dough and fondants made from gluten-free rice flour, using innovative ingredients cocoa butter, condensed milk, chicken eggs, white sugar and citron powder.the main technology characteristics of fondants have been presented. surfactants were used in order to obtain the desired structure. the necessity of using surfactants in fondants has been proved. this article analyses the advantages and disadvantages of the new recipe composition, in search for solutions to optimize formulations, taking into account the introduction of surfactants. on the basis of theoretical analysis and analyzing the classic ingredients of hot dessert, the authors have chosen the formulation of special purpose fondants. the article also presents the results of experimental studies on parameters such as porosity, adhesion, general deformation of dense part of the fondant. therefore, it is reasonable to conduct the experimental research and develop the technical documentation for new types of fondant desserts using alternative raw materials. as a result of research on dough viscosity, the porosity of the dense part of fondant optimized the ratio between flour and surfactant. it is shown that the adhesion values of dough and total deformation of dense part of fondants are 1.3 times greater, and by 11% greater respectively in comparison with the control sample. keywords: hot sweet dishes, surfactant, porosity, adhesion, optimization. 1. introduction in ukraine, there is a group of diseases, their socially dangerous relevance and importance being determined not only by their high prevalence, but also by lack of a range of special purpose products. one of such diseases is the celiac disease – metabolic intolerance to cereal protein by the body. as a result of the united european gastroenterology week in 2002 in geneva at a plenary meeting it was determined that every hundredth citizen of the country has food intolerance to proteins of wheat, rye, barley and cerealmade products. it is known that the majority of patients suffering from celiac disease are children of different ages. in connection with the absence of a wide range of gluten-free products on the territory of ukraine, and considering the widespread popularity of hot sweet dishes among different age groups, recipes of special purpose fondants have been developed on the basis of gluten-free rice flour in order to implement them in public restaurant business units. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 olena dudkina, aleksandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk investigation of structural-mechanical properties of the recipe composition for fondans special purpose, food and environment safety, volume xiv, issue 4 – 2015, pag. 352 – 357 353 currently, the implementation of traditional technologies of fondants (chocolate fondants fig.1) is at an early stage, though this dessert is becoming more and more popular in the restaurant business establishments of ukraine. but, unfortunately, there are still no profound researches on diversification and adaptation of assortment for different populations. these factors lead to further improvement of the technological production process of recipe compositions of special purpose fondants using mathematical modeling. the classic technology of cooking fondant consists of the following steps: introduction of the stuffing – chocolate cream ganache which is frozen, then addition to form inside the biscuit dough and baking for five minutes. for the preparation of the traditional product the following ingredients are used: dark chocolate (28...30%), cream butter (13...14%), wheat flour (20%), eggs (22...25%), white sugar (13...14%). however, the use of wheat flour in the production of gluten-free products is impossible. given the characteristics of the developed product, the recipe of special purpose fondants is hereby given: rice flour (gost 27168-86), cocoa butter (dstu 5004:2008), white sugar (dstu 4623:2006), and citron powders (gost 6829-89), eggs (dstu 5028:2008), condensed milk (dstu 4274:2003). during the technological production process, one of the key steps is preparing the recipe mixture for semifinished special purpose fondants and formation of a complex colloidal system. however, given the selection of these components in terms of their physicochemical properties, research goals and objectives were established. the purpose of the article was to study the structural-mechanical properties of a recipe mixture for special purpose fondants to further optimize the recipe. tasks: 1. to justify the nomenclature of the list of indicators which form the structuralmechanical properties; 2. to investigate viscosity, shear stress and porosity recipe mixture for special purpose fondants; 3. to establish correlations between porosity and viscosity of the finished product. 4. to investigate adhesion strength and overall deformation of the thick part of fondants. 2. matherials and methods the main objective of this survey is to analyze fondants made from wheat flour as control sample and those made from rice flour.traditional methods of determining viscosity, shear stress were used: viscometer reotest-2, porosity – by means of the computerized method, adhesion – by means of adhesion tester, total deformation – by penetrometer, optimization – by the method of least squares using software packages. 3. results and discussion despite the exceptional variety of foods that are used in the food industry and establishments of restaurant business, in most cases at different technology stages fig. 1. chocolate fondant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 olena dudkina, aleksandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk investigation of structural-mechanical properties of the recipe composition for fondans special purpose, food and environment safety, volume xiv, issue 4 – 2015, pag. 352 – 357 354 of their processing, they are nonhomogeneous systems. by homogeneous system we understand that system which consists of two or more phases, each one having its own phase contact area and being mechanically separated from the other phase. any non homogeneous binary system consists of an internal or disperse system and the external or dispersion medium in which dispersed phase particles are located. a system in which the external phase is a liquid is called the liquid non homogeneous system. depending on the dispersive and the dispersed phase the following liquid heterogeneous systems are distinguished: suspension – a liquid which contains therein solid suspended particles; emulsion – a system in which the liquid dispersion medium contains therein particles of one or more other liquids, wherein all the components of the system must be mutually insoluble; foam – systems which consist of a liquid dispersion medium and therein particles gas. in food industry and restaurant business units, homogeneous solutions, suspensions, emulsions were prepared by mixing the dispersion process inseparably. dispersing is a grinding process of solid and gaseous substances in the liquid. among these processes, there are three main ones: emulsification, homogenization and atomization of liquids. the above-mentioned systems are used to obtain dough for special purpose fondants formed during heat treatment (baking) homogeneous internal component, similar to cream ganache in traditional fondants. as main type of gluten-free raw materials for special purpose fondants we selected rice flour, which is taken in parts by weight of 20, 25 and 30%. the resulting system is a polidispersoid which is a combination of emulsion and dispersion of particular matter in the fatty phase, and does not provide the organoleptic quality of the finished product at control level. to improve the data quality and functional properties in food processing industries surfactants are used to stabilize the emulsion and foam structure, modifying the crystalline form of fat compositions, regulating the structural-mechanical properties of semi-finished and hot sweet dishes. over the last years, esters of various fatty acids (including citric acid ester) have been increasingly used as emulsifiers both in our country and abroad during the production of food products. hydrolysed and standardised lecithin is the most common one in multicomponent food systems. standardised lecithin has a more pronounced effect on the process and quality of finished products due to the differences of its hydrophilic-lipophilic balance. the recommended dosage of lecithin is 0.7% in the mass of the composition recipe. hydrolysed lecithin has also a number of advantages. it is the most hydrophilic, as determined by the degree of hydrolysis, thus allowing the use of such emulsions in cream lecithins. its structure is similar to that of monoglycerides, having only one fatty acid which is more effective in baking and provides a delicate product, stable to storage. the best result is achieved by a lecithin content of 0.7% in the weight of the composition recipe. among the wide range of emulsifiers, citric acid ester of the company «grindsted®citremsp 70» was chosen, which complies with the regulation № 1829/2003 and 1830/2003 and meets the requirements of the european union (e 472 c) and is completely safe for use in the food industry. citric acid ester of mono-, diglycerides made from edible refined sunflower oil and palm oil. it reduces the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 olena dudkina, aleksandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk investigation of structural-mechanical properties of the recipe composition for fondans special purpose, food and environment safety, volume xiv, issue 4 – 2015, pag. 352 – 357 355 tension between lipid and aqueous phase to stabilize the liquid emulsion, ensures the purity and stability of the aqueous dispersion medium with a high fat content, also has a high water binding capacity, and prevents the cracking of the product during baking and sticking to the surface of the molds. the recommended dosage is of 1.0% by weight of the composition recipe. based on the experimental data defining the viscosity of the product of the number of rice flour x1 (v%) and surfactant x2 (y%), the quadratic regression equation found, is approximately given this dependence. y = 172.39 + 1.43 x1 + 2085.83 x2 + 0.31 x1 2 – 2683.33 x2 2 + 2.50 x1 x2. (1) multiple determination coefficient r2 = 0.9877, that is accuracy is quite high. the corresponding surface is as follows (figure 2). fig. 2. response surface of dosing rice flour and surfactant on viscosity of dough for special purpose fondants obviously (figure 2), that in the area of changes factors x1 and x2, the viscosity of the product has no extrema. from a practical point of view, acceptable value of viscosity of the product is the value of y = 800 pa · s, similar to the viscosity of the test of control. therefore, we can write the equation of the corresponding line level: 172.39 + 1.43 x1 + 2085.83 x2 + 0.31 x1 2 – 2683.33 x2 2 + 2.50 x1 x2 = 800, (2) or 0.31 x1 2 – 2683.33 x2 2 + 2.50 x1 x2 + 1.43 x1 + 2085.83 x2 = 627.61, (3) which defines hyperbole, the schematic graph being shown below: fig. 3. the line of viscosity of fondant dough depending on rice flour and surfactant dosage in addition to viscosity of the product, its porosity was investigated as well. based on the experimental data, the following equation of the quadratic regression was obtained: y = – 88.50 + 8.93 x1+ 129.17 x2 – 0.14 x1 2 – 50.00 x2 2 – 3.50 x1 x2. (4) multiple determination coefficient r2 = 0.8132, that is accuracy is quite high. the corresponding surface looks as follows: fig. 4. response surface of dosing rice flour and surfactant on the porosity and viscosity of dough 800 pa.s. for special purpose fondants food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 olena dudkina, aleksandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk investigation of structural-mechanical properties of the recipe composition for fondans special purpose, food and environment safety, volume xiv, issue 4 – 2015, pag. 352 – 357 356 the investigation of the porosity function of the extremum was done on the condition that the viscosity of the product is 800 pa·s. the task was done on conditional extremum. the analysis was carried out, giving the point of maximum x1 = 24,583% rice flour, x2 = 0.495% ester of citric acid, ymax = 55.52%. an important factor is to ensure appropriate adhesive properties of glutenfree dough. accordingly, we investigated the adhesion strength of the recipe composition according to the optimized structure by comparing to the control sample– figure 5. fig. 5. adhesion strength of test samples as seen in figure 5, the strength of experience is 1.3 times greater than the strength of the control. thus, it is explained the action of gluten-free raw materials and surfactants due to the lack of gluten proteins. in addition to the positive physiological effects, rice flour provides higher emulsifying (es) (stability of the emulsion from 42 to 65%) and fat binding (fbc) (from 28 to 68%) capacities as compared with the control sample. based on these investigations, the general deformation of the dense part of fondants was determined using penetrometer (figure 6). as seen in figure 6, we can conclude that the experiment sample formed a more tender structure that is 11% less than the control one, due to the chemical composition of innovative recipe ingredients. fig. 6. general deformation of dense part of fondants 4. conclusion thus, in conclusion we can remark based on the multifaceted complex researches of structural-mechanical properties that the formation of the structure by optimizing recipe fondants’ composition of gluten-free rice flour is similar to traditional fondants made from wheat flour. 1. based on the features of special purpose fondants, namely the presence of the liquid component during cutting, similar filling (cream ganache in traditional fondant) and dense part (porous shell) of sweet dish, indicators of structural-mechanical properties – dough viscosity and adhesion, porosity and overall deformation of dense part were selected. 0 50 100 150 200 250 control test adhesion , pa 0 20 40 60 80 100 control test general deformation of dense part of fondants food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 4 – 2015 olena dudkina, aleksandra nemirich, andriy gavrysh, tatiana ischenko, yuriy tkachuk investigation of structural-mechanical properties of the recipe composition for fondans special purpose, food and environment safety, volume xiv, issue 4 – 2015, pag. 352 – 357 357 2. as a result of research on viscosity of fondants’ dough made from wheat flour and those made from rice flour for special purpose, the equation of quadratic regression was found, which roughly defines the dependence of dough viscosity on dosing of rice flour and surfactants, and of the constructed surface as response of this dependence. 3. from a practical point of view, an unacceptable viscosity value of the product is the value of y = 800 pa·s, similar to the viscosity of control sample dough, on the basis of experimental data and their mathematical processing the line of dough viscosity level is built for special purpose fondants (800 pa·s), depending on the dosage of rice flour and surfactant. 4. the response surface of porosity of the dense part of special purpose fondants depends on the dosage of rice flour and a surfactant with a dough viscosity of 800 pa·s. 5. it is found that the adhesion strength of dough in the test sample of fondants has by 23% greater importanc than that in the control sample, which is due to the technological properties of gluten-free raw materials and surfactant and the lack of gluten proteins. 6. in the special purpose fondants a more tender structure of the dense part than in control one was formed, fact that may be explained in terms of the overall deformation of the dense part of this sweet dish. 5. references [1]. dudkina o.o., gubenko s.o., gavrysh a.v., nemirich a.v. justification recipe composition fondans special purpose, progressive technique and technology of food production restaurant economy and trade: collection of scientific papers / kharkiv state university of food technology and trade, vol. 1 (21), 331-343 (2015). [2]. gavrysh a.v., ischenko t.i., nemirich a.v., dudkina o.o., gubenko s.o. fondan special purpose, patent for utility model, № 100479 (2015). [3]. dotsenko v.f., gavrysh a.v., kulikova kh.v. composition of chocolate fondan, patent for utility model, № 82998 (2013). [4]. vyshnyak m.n. pastries for gluten-free food, polzunovsky almanac. vol. 2, 95-96 (2009). [5]. kozubaeva l.a. gluten-free flour products for prophylactic and therapeutic feeding. modern problems of technique and technology of food production: mater. eleventh intern. scientific and practical. conf., 73-74 (2008). [6]. obolkina v.i., zalevskaya n.a., gureeva v.s., kishko s.a. study of the effectiveness of mixtures of emulsifiers in confectionery, bakery and confectionery industry ukraine vol. 5, 11-12 (2011). 59 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1 2018, pag. 59 65 the use of dog-rose hips (rosa canina) fruits in the production of marshmallow-type candy *aliona ghendov-moșanu 1 1faculty of of food technology, technical university of moldova, chisinau, republic of moldova *corresponding author: aliona.mosanu@tpa.utm.md; a_mosanu@mail.ru received 19th october 2017, accepted 19th march 2018 abstract: the marshmallow is a high-energy sugar-based product with a pleasant taste and aroma, chewable consistency, foamy structure with fine porosity, consumed by children. artificial dyes are added during the manufacture of these candies to give them an appealing look. studies show that the ingestion of tartrazine combined with a diet rich in trans fats, nitrites, nitrates and low fiber intake are linked to malignant tumors, especially in the esophagus, breast, rectum, stomach and ovaries. hence, the substitution of the synthetic dye tartrazine with a natural food dye obtained from dog-rose (rosa canina) fruits as a source of biologically active substances (tannins, polyphenolic compounds) is of particular interest. marshmallow candy with the addition of dog-rose fruit i.e. 1.0% and 2.0% hydroalcoholic extract, and 1.0%, 2.0% and 2.5% dog-rose hip powder to the product mass were produced. the control sample was prepared in a similar way, by replacing the plant addition with 0.02% synthetic dye tartrazine. the physico-chemical and organoleptic indices of the samples corresponded to the acceptable values. it was found that the mass fraction of dry matter, the reducing substances and the acidity do not change significantly during storage. following the determination of the (dpph) antiradical activity of the candy by in vitro method, it was found that the percentage of dpph inhibition in the control sample was negative, while in the samples with the addition of dogrose fruit, the antiradical capacity had significant positive values. keywords: sugar-based products, vegetable matter, antiradical activity, quality. 1. introduction the sugar-based products are characterized by high nutritional value, pleasant taste and aroma, attractive appearance, so that the demand for this product group is continuously increasing. according to fao who cifocoss food consumption database, sugar-based products are highly demanded by different age groups, especially children [1]. on average 13100 tons of confectionery products were produced annually in the republic of moldova between 2007 and 2014, according to statistical data [2]. marshmallow is a sugar-based product which is not traditional for the consumers of the republic of moldova. it is however similar to small-sized zephyr with a chewable consistency and a foamy structure with fine porosity. initially, marshmallow was manufactured by using the common marh mallow (althaeae radix) root, which when boiled produces a gluey and gelatinous mass, that can then be used to manufacture foamy products. over time, the marshmallow root has been replaced with gelatin and starch [3]. currently, marshmallow is becoming more and more popular, especially among children, due to http://www.fia.usv.ro/fiajournal mailto:aliona.mosanu@tpa.utm.md mailto:a_mosanu@mail.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 60 the variety of its forms, appearances and flavours. during the manufacture of these candies, artificial dyes are added to give them an appealing look. studies show that ingestion of artificial dyes, such as tartrazine, combined with a diet rich in trans fats, nitrites, nitrates and low fibre intake is related to malignant tumours, especially in the oesophagus, breast, rectum, stomach and ovaries [4]. other harmful observed effects are the allergic reactions, which can cause either simple allergies or even severe asthma and bronchitis [5]. moreover, the literature shows that around 13% 22% of people with aspirin allergies also have the same reactions after eating foods with tartrazine [6]. at present, a particular attention is paid to technologies based on natural sources of biologically active compounds, rich in antioxidants substances with beneficial health effects [7-10]. the dog-rose (rosa canina) hips are a part of the forest fruits, which are an important source of bioactive components (vitamin c, polyphenolic compounds, proanthocyanidins, flavonoids, carotenoids, etc.) [11 14]. rose hips are known to be efficacious in strengthening the body defence, especially against colds and for decreasing slightly the total cholesterol in humans [15-17]. the extract of hips significantly reduced the level of glucose in the blood, substantially inhibiting the weight gain and/or the accumulation of visceral fat, without affecting food intake in mice [18]. thus, the substitution of the synthetic tartrazine with natural colourant made from dog-rose hips for the production of marshmallow candy, especially for children, is appropriate.the objective of this research was to develop the production technology of the marshmallow with the use of hydroalcoholic extract and powder of the dog-rose fruit in order to diversify the assortment of functional products. the studies were focused on the following directions: the analysis of the chemical composition of the hydroalcoholic extract; the determination of the quality indicators, the microbiological stability and the antiradical activity of candy. 2. matherials and methods the hydroalcoholic extract of the autochthonous dog-rose hips (rosa canina) harvested in 2014 was used for this study. the hips were washed and dried at 65°c to a humidity of 8,0±0,25%. then, the dried hips were ground to the powder and sieved. the granulation of the powder after sieving was 140µ. the obtained meal was subjected to hydroalcoholic extraction using 50% ethanol. the extraction was carried out in a solid-liquid ratio of 1:15 in the water bath under stirring. the obtained extract was filtered, distilled in a rotary evaporator at 65°c. an extract with a dry mass fraction 73,0±0,1% was obtained and stored in dark-coloured containers at 5,0±1,0°c. the marshmallow-type candy were prepared using gelatin, sugar, molasses and water, with the addition of dog-rose fruit i.e. 1.0% and 2.0% hydroalcoholic extract and 1.0%, 2.0% and 2.5% rose hip powder of the mass of the product as a natural dye, but also as a source of biologically active compounds. the mixture of corn starch and powdered sugar was used to sprinkle candy. the control sample was prepared in a similar way, by replacing the plant addition with 0.02% tartrazine. the vacuum packed samples were stored in the refrigerator at 5±1°c. the following analytical methods were used to determine the content of biologically active compounds in the dogrose extract: the content of tannins by the folin-ciocalteu method [19], the phenolic compounds content by the folin-ciocalteu method [19] and antiradical activity by the dpph method [20]. the physicochemical characteristics of the products were determined according to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 61 [21]. the mass fraction of the dry substance by the refractometric method; the acidity; the mass fraction of reducing substances were analyzed. the sensory analysis of the products was carried out according to bs iso 6658-2005 sensory analysis methodology general guidance [22] and the technical regulation “confectionery food products” of the republic of moldova [23]. the microbiological analysis was carried out in accordance with the rules on microbiological criteria for foodstuffs [24]. the total viable count was estimated. the antiradical activity of the candy was measured using the spectrophotometric method with the free dpph (2.2-diphenyl1-picrylhydrazyl) radical [20]. the antiradical dpph• activity of the products has been determined in vitro in order to simulate gastric digestion in the presence of pepsin (150 mg/100g of product), at ph=2,0±0,1 (1.5 m hcl), temperature 37.0±0.1ºc in water bath, under agitation at 60 min-1 for 2 hours [25]. the samples have previously been centrifuged at 6000 min-1 for 10 min, at 20±1ºc temperature, they have been filtered and tested as well [20]. according to bibliographical estimating, the correlation degree between in vitro scientific results, made by monsen’s model and the results obtained in vivo varie between 60 70% [26]. the variance analysis of the results was carried out by least square method with application of student coefficient and microsoft office excel program version 2007. the differences were considered statistically significant if probability was greater than 95% (p-value <0,05). all assays were performed in triplicate at room temperature 20±10c. the experimental results are expressed as average±sd (standard deviation). 3. results and discussion table 1 shows the content of tannins, phenolic compounds and the antiradical activity of the dog-rose fruit extract used in the manufacture of the candy [27]. the analysis of the results showed that the obtained extract is rich in biologically active compounds. table 1. the content of tannins, phenolic compounds and the antiradical activity of rose fruit extract tannins, mg tae/g d.w. total phenols, mg gae/g d.w. antiradical activity,% 106.41±1.34 26.98±0.36 85.11±0.02 the mass fraction of dry matter and the acidity were determined on the 6th and 12th days from the production date to observe the changes during product storage. table 2 presents quality physicochemical indicators of the marshmallows with 1,0% and 2,0% rose hip extract and with 1,0%, 2,0% and 2,5% rose hip powder to mass product as compared to the control sample with 0,02% tartrazine, during storage. the results obtained in table 2 showed that the dry mass fraction increased with the concentration of rose hip extract and powder compared to the control sample with synthetic dye, on the 6th day after production. thus, the addition of 1% rose hip extract enhanced the mass fraction of dry substances by 0.41% and the addition of 2% by 1.08%. the rose hip powder addition at the concentration of 1%, 2%, and 2.5% increased the dry mass fraction by 0.7%, 0.8% and 2.1%. respectively. during the 12-day storage period, the dry mass fraction of the candy obtained increased insignificantly by an average of 1.2%. at the same time, it was observed that during the initial preservation period, the marshmallow samples absorbed the moisture and a layer of sugar-saturated solution was formed on the surface. due to the diffusion of moisture from the upper food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 62 layers to the inner layers of the sample, the partial crystallization of the sugar occurred and the samples turned to the crystalline state [3]. this state was formed when the moisture balance of samples was reached. thus, reducing the storage moisture content leads to increased content of dry matter, which demonstrates that the developed product is stable over time [21]. it was found that acidity augmented with increasing concentration of rose hip extract and powder and varied within the range of 1.3 – 2.5 degrees of acidity on the sixth day and within 1.4 – 2.6 degrees of acidity, after 12 days of storage. the rise in acidity can be explained by the chemical composition of the dog-rose fruit (lascorbic acid, fruit acids, organic acids, amino acids) [11]. table 2. the change of the quality physicochemical indicators of the marshmallow candy manufactured using rose hip extract, rose hip powder, and a synthetic dye during storage marshmallow -type candy quality indicators the mass fraction of the dry matter, % acidity, degrees the mass fraction of reducing substances, % after 6 days after 12 days after 6 days after 12 days after 6 days after 12 days control sample 70.25±0.35 71.25±0.25 0.7±0.20 0.8±0.05 8.01±0.23 n.d. with 1.0% extract 70.66±0.29 71.83±0.15 1.3±0.10 1.4±0.10 8.51±0.22 n.d. with 2.0% extract 71.33±0.44 72.60±0.24 1.6±0.20 1.7±0.20 8.68±0.18 n.d. with 1.0% powder 70.95±0.12 71.75±0.15 1.1±0.10 1.2±0.05 8.21±0.26 n.d. with 2.0% powder 71.05±0.30 72.50±0.10 2.1±0.10 2.2±0.10 8.36±0.32 n.d. with 2.5% powder 72.35±0.15 73.80±0.16 2.5±0.05 2.6±0.10 8.53±0.31 n.d. n.d. – not determined at the same time, the mass fraction of the reducing substances augmented insignificantly with the increase of the plant matter concentration. in the case of the extract, this indicator varied between 8.51 – 8.68%, while in the case of rose hip powder between 8.21 – 8.36%, but it did not exceed the maximum value of 10% [23]. the analysis of the obtained results showed that the physicochemical parameters of the candy corresponded to the regulated admissible values according to the government decision no. 204 [23] after 6 days and 12 days from the production date. the organoleptic analysis of the marshmallow was also carried out in order to place the product on the market. the organoleptic evaluation of the candy was carried out using the 5 point system. obviously, the smell and taste of fortified candies with extracts and powder from rose hips differed from candy with synthetic dye, fig. 1. the appearance of the candy containing rose hip powder varied from the control sample by the presence of fruit particles in their structure, which did not influence food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 63 essentially this index. at the same time, the powder of rose hips contained soluble and insoluble fibres that influenced the structure and consistency of marshmallow candy. fortified candies with 2% powder had a more elastic consistency than the control sample, but with increasing admixture, other organoleptic markers such as appearance and taste worsened. the best results have been obtained for marshmallow fortified with 2% fruit powder to the weight of the product. fig.1. profiles of marshmallow candy with: a) rose hip extract; b) rose hip powder compared to the control sample with tartrazine. the influence of the rose hip extract and powder on the microbiological stability of candy against the synthetic dye was also investigated. table 3 shows the evolution of total viable count (tvc)* during the storage of marshmallows obtained with the addition of rose hip extract, rose hip powder and synthetic dye tartrazine. the study has shown that the total viable count decreased with increasing concentration of the plant matter, which confirms its antimicrobial activity [28]. table 3. the evolution of the total viable count (tvc)* during the storage of marshmallows containing rose hip extract and rose hip powder parameter marshmallow-type candy tvc (total viable count), % of the maximum admissible number after 6 days after 12 days control sample 6.81 21.32 with 1.0% extract 5.27 16.81 with 2.0% extract 4.65 16.32 with 1.0% powder 2.27 15.45 with 2.0% powder 2.29 13.74 with 2.5% powder 1.81 13.07 * nutrient agar all tested products had the total viable count corresponding to the acceptable value, after 6 and 12 days of storage [24]. the presence of biologically active compounds essentially influences the stability and the antioxidant ability of food products [29]. the antiradical activity (% inhibited dpph free radicals) of the candy containing rose hip extract and rose hip powder in the conditions of the stomach digestion was investigated in this study. it was found that the control sample containing tartrazine had dpph-negative antiradical activity i.e. (-29.33±1.21%), exhibiting an oxidizing action. in the samples with the optimal concentration of dog-rose fruit extract and powder, the antiradical activity was positive (12.35±0.56% and 21.29±1.11% respectively), which presents an extremely important argument in favour of these products. 4. conclusion the analysis of the organoleptic indices, physicochemical indicators, the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 64 microbiological stability and the antiradical activity of marshmallow has shown that this product is competitive as it exhibits functional properties due to the presence of the biologically active compounds of the dog-rose hips, and can thus be recommended for consumption. 5. acknowledgments this work was benefited from support through the 16.80013.5107.22/ro project, “the substitution of synthetic food additives with bioactive components extracted from natural renewable resources”, funded by the academy of science of moldova and moldavian government. 6. references [1]. www.fao.org/nutrition/en/ (accessed 18 september 2016). [2]. statistical yearbook of the republic of moldova (in romanian), chisinau: 566, (2015). [3]. magomedov g., plotnikova i., zhuravlev a., shevjakova t., popova a., perfection of gelatin mini-zephyr (marshmallow) technology (in russian), konditerskoe i hlebopekarnoe proizvodstvo, 11– 12: 6-9, (2014). [4]. prado m., godoy h., artificial food dyes (in portuguese), alimentos nutrição, 14 (2): 23750, (2003). [5]. cosentino h., effects of ionizing radiation on natural food dyes (in portuguese), thesis doctorate in science, instituto de pesquisas energéticas e nucleares, autarquia associada à universidade de sã o paulo, são paulo: 149, (2005). [6]. durinezio jose de almeida, bennemann g., bianchi c., freitas g., colorful, cute, attractive and carcinogenic: the dangers of dyes, cancer research journal, 2 (6-1): 42, (2014). [7]. block g., patterson b., subar a., fruit, vegetables, and cancer prevention: a review of the epidemiological evidence, nutrition cancer, 18: 1-29, (1992). [8]. bonina f., leotta c., scalia g., puglia c., trombetta d., tringali g., et al., evaluation of oxidative stress in diabetic patients after supplementation with a standardised red orange extract. diabetes, nutrition & metabolism, 15: 1-19, (2002). [9]. felgines c., talavera s., texier o., gil-izquierdo a., lamaison j. l., remesy c., blackberry anthocyanins are mainly recovered from urine as methylated and glucuronidated conjugates in humans, journal of agricultural and food chemistry, 53: 7721-7727, (2005). [10]. ferruzzi m., blakeslee j., digestion, absorption, and cancer preventative activity of dietary chlorophyll derivatives, nutrition research, 27: 1-12, (2007). [11]. sezai ercisli, chemical composition of fruits in some rose (rosa spp.) species, food chemistry, 104: 1379-1384, (2007). [12]. chai j., ding z., nutrients composition of rosa laevigata fruits, science technology in food industry, 3: 26-29, (1995). [13]. demir f., ozcan m., chemical and technological properties of rose (rosa canina l.) fruits grown wild in turkey. journal food engineering, 47: 333-336, (2001). [14]. türkben c., uylaser v., incedayi b., celikkol i., effects of different maturity periods and processes on nutritional components of rosehip (rosa canina l.), journal of food, agriculture and environment, 8: 26-30, (2010). [15]. duru n., feryal k., erge h., changes in bioactive compounds, antioxidant activity and hmf formation in rosehip nectars during storage, food and bioprocess technology, 5 (7): 2899-2907, (2012). [16]. szentmihályi k., vinkler p., lakatos b., illés v., then m., rose hip (rosa canina l.) oil obtained from waste hip seeds by different extraction methods, bioresource technology, 82 (2): 195-201, (2002). [17]. rein e., kharazmi a., winther k., a herbal remedy, hyben vital (stand. powder of a subspecies of rosa canina fruits), reduces pain and improves general wellbeing in patients with osteoarthritis: a double-blind, placebo-controlled, randomised trial, phytomedicine, 11: 383-391, (2004). [18]. ninomiya k., matsuda h., kubo m., morikawa t., nishida n., yoshikawa m., potent antiobese principle from rosa canina: structural requirements and mode of action of transtiliroside, bioorganic & medicinal chemistry letters, 17: 3059-3064, (2007). [19]. waterman p.g., mole s., analysis of phenolic plant metabolites, ecological methods and concepts, wiley: 248, (1994). [20]. brand-williams w., cuvelier m., berset c., use of free radical method to evaluate http://www.fao.org/nutrition/en/ http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahukewinlpzmx7jxahvqoqqkhxgxcfqqfggumae&url=http%3a%2f%2fwww.statistica.md%2fpageview.php%3fl%3den%26idc%3d263%26id%3d2193&usg=aovvaw2qetuhngva9r3vt0fttgfr http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahukewinlpzmx7jxahvqoqqkhxgxcfqqfggumae&url=http%3a%2f%2fwww.statistica.md%2fpageview.php%3fl%3den%26idc%3d263%26id%3d2193&usg=aovvaw2qetuhngva9r3vt0fttgfr https://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjzmcochpdwahueepokhybdd9kqfggnmaa&url=https%3a%2f%2fwww.ncbi.nlm.nih.gov%2flabs%2fjournals%2fdiabetes-nutr-metab%2f&usg=aovvaw3_neklawcorfe67rnuwcrq https://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjzmcochpdwahueepokhybdd9kqfggnmaa&url=https%3a%2f%2fwww.ncbi.nlm.nih.gov%2flabs%2fjournals%2fdiabetes-nutr-metab%2f&usg=aovvaw3_neklawcorfe67rnuwcrq https://www.ncbi.nlm.nih.gov/pubmed/?term=gil-izquierdo%20a%5bauthor%5d&cauthor=true&cauthor_uid=16190623 https://www.ncbi.nlm.nih.gov/pubmed/?term=lamaison%20jl%5bauthor%5d&cauthor=true&cauthor_uid=16190623 https://www.ncbi.nlm.nih.gov/pubmed/?term=remesy%20c%5bauthor%5d&cauthor=true&cauthor_uid=16190623 http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewihv4x9hpdwahwnkjokhui1bk4qfggtmaa&url=http%3a%2f%2fpubs.acs.org%2fjournal%2fjafcau&usg=aovvaw0degyj7j9k4krwux_ppc6q http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewihv4x9hpdwahwnkjokhui1bk4qfggtmaa&url=http%3a%2f%2fpubs.acs.org%2fjournal%2fjafcau&usg=aovvaw0degyj7j9k4krwux_ppc6q https://www.ncbi.nlm.nih.gov/pubmed/?term=ill%c3%a9s%20v%5bauthor%5d&cauthor=true&cauthor_uid=12003323 https://www.ncbi.nlm.nih.gov/pubmed/?term=then%20m%5bauthor%5d&cauthor=true&cauthor_uid=12003323 https://www.ncbi.nlm.nih.gov/pubmed/?term=morikawa%20t%5bauthor%5d&cauthor=true&cauthor_uid=17400451 https://www.ncbi.nlm.nih.gov/pubmed/?term=nishida%20n%5bauthor%5d&cauthor=true&cauthor_uid=17400451 https://www.ncbi.nlm.nih.gov/pubmed/?term=yoshikawa%20m%5bauthor%5d&cauthor=true&cauthor_uid=17400451 https://www.ncbi.nlm.nih.gov/pubmed/?term=yoshikawa%20m%5bauthor%5d&cauthor=true&cauthor_uid=17400451 http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewieiihyh_dwahwdcjokhui9dv8qfggnmaa&url=http%3a%2f%2fwww.sciencedirect.com%2fscience%2fjournal%2f0960894x&usg=aovvaw0tpjkm8hratodx7fiwqnkm http://www.google.md/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewieiihyh_dwahwdcjokhui9dv8qfggnmaa&url=http%3a%2f%2fwww.sciencedirect.com%2fscience%2fjournal%2f0960894x&usg=aovvaw0tpjkm8hratodx7fiwqnkm https://www.google.md/search?hl=ru&tbo=p&tbm=bks&q=bibliogroup:%22ecological+methods+and+concepts%22&source=gbs_metadata_r&cad=2 https://www.google.md/search?hl=ru&tbo=p&tbm=bks&q=bibliogroup:%22ecological+methods+and+concepts%22&source=gbs_metadata_r&cad=2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 aliona ghendov-moșanu, the use of dog-rose hips (rosa canina) fruits in the production of marshmallow type candy, food and environment safety, volume xvii, issue 1 – 2018, pag. 59 – 65 65 antioxidant activity, lebensm wiss technology, 28: 25-30, (1995). [21]. official methods of analysis of aoac international 20th edition, (2016). [22]. bs iso 6658-2005 sensory analysis methodology general guidance, (2006). [23]. decision of the government of the republic of moldova no. 204 from march 11, 2009 regarding the approval of technical regulation on “confectionery food products” (in romanian), official monitor of the republic of moldova, 20.03.2009, no. 57-58, art. 254. [24]. decision of the government of the republic of moldova no. 221 from march 16, 2009 regarding the approval of rules on microbiological criteria for foodstuffs (in romanian), official monitor of the republic of moldova, 24.03.2009, no. 59-61, art. 272. [25]. monsen e.r. iron nutrition and absorption: dietary factors which impact iron bioavailability, journal of the american dietetic association, 88:786-790, (1988). [26]. miller d., schricker b., rasmussen r., van campen d., an in vitro method for estimation of iron availability from meals, the american journal of clinical nutrition, 34 (10): 2248-2256, (1981). [27]. railean a., ghendov-moşanu a., sturza r., the study of the addition of dog-rose hips fruits extract on the quality of candies „gummy bears” (in romanian), technical scientific conference of collaborators, doctoral students and students tum, chisinau, 26th-28th november, 2: 51-58, (2015). [28]. yilmaz s., ercisli s., antibacterial and antioxidant activity of fruits of some rose species from turkey, romanian biotechnological letters, 16 (4): 6407-6411, (2011). [29]. savvin p., bolotov v., the study of antioxidant properties of fruit jelly (in russian), himija rastitel'nogo syr'ja, 4: 177-179, (2008) 1. introduction microsoft word 8 golikova_corrected.doc 117 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 22017, pag. 117 122 effect of plant protein isolates on the structural – mechanical properties of wheat dough valeriy makhynko1, vira drobot1, *tatjana golikova2, 1educational-scientific institute of food technologies, national university of food technologies, volodymyrska str. 68, kyiv, ukraine, makhynkovaleri@gmail.com, 2technology of nutrition and restaurant business dept., national university of food technologies, volodymyrska str. 68, kyiv, ukraine, tatjana.golikova12@gmail.com *corresponding author received 29th may 2017, accepted 29th june 2017 abstract: the results of using isolates of soya, pea and rice flour as well as of dry wheat gluten in the making of bread dough have been presented. taking into account the high water absorption capacity of these products, effect of the protein isolates on the structural-mechanical properties of the dough has been investigated. on the basis of farinogram curves the additional quantity of water needed to obtain proper structure of dough made from all types of raw materials has been determined. a formula of calculation the additional quantity of water has been proposed. it proves that most quantity of water is needed for dough with isolate of soya protein – 2.3 g per 1 g of added isolate. isolate of pea protein needs additionally 1.5 g of water, dry wheat gluten – 1.3 g, and isolate of rice protein – 0.9 g of water. the proposed calculation has been checked for mixes with different proportion of raw materials and its effectiveness has been proven. the calculation method was used to determine the additional quantity of water required to obtain wheat dough with necessary structural and mechanical properties. keywords: soya, pea, rice, gluten, farinograph, protein isolate, water absorption. 1. introduction bread products (especially made from wheat flour of a high extraction rate) have non-balanced chemical composition: poor protein content and over-saturated with carbohydrates [1, 2]. it seems topical to increase nutritional value of bread products as daily-used products. the main part of carbohydrates is brought about in bread products by flour, so it is hard to decrease significantly its content without losses of technological properties of dough and quality of ready products [2, 3]. the use of additional high protein raw materials is a perspective way to approximate chemical content of bread to the physiological norms [1, 2, 3, 4, 5]. in order to choose protein fortifiers, it is necessary to take into account safety, price, and protein content and balance by the main essential amino acids [2, 3]. the additional advantage of such fortifiers is a certain surplus of the lysine amino acid due to fact that protein of most bread products has poor lysine content. protein of vegetable crops (soya, pea, nut etc.) is mostly met to the mentioned demands [2, 6, 7]. but legumes contain certain amount of anti-nutritional substances. so, it is advisable to use highly purified forms of this protein – isolates. technology of isolates’ obtaining provides almost full purifying of all anti-nutritional substances, and high protein content – near 90 % encourages minimizing the dosage of the isolates [2, 7]. it does not exert food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 valeriy makhynko, vira drobot, tatjana golikova, effect of adding of plant protein isolates to the structural – mechanical properties of the wheat dough, food and environment safety, volume xvi, issue 2 – 2017, pag. 117 – 122 118 significant impact on the technological properties of dough, and, at the same time, it provides ready products with increased protein content. due to their high nutritional and biological value, isolates are being more applied in different food productions [5, 6, 7, 8]. it is known that extraneous protein has negative effect on the forming process of dough from the wheat flour and on its structural and mechanical properties. this problem has been investigated by different researchers [1, 3, 6]. 2. materials and methods isolates isolates of soya protein (isp) іsopro 900 em-upi shandong sinoglory group co., ltd (prc), isolate of pea protein (ipp) cosucra groupe warcoing s. a. (belgium) and isolate of rice protein (irp) growing naturals, llc (usa) have been investigated. flour the wheat flour of a high extraction rate with gluten content 26 % that had quality estimation as acceptable strong (extensibility – 12 cm, elasticity – 75 units of device ddk), type of flour is 550 units was used in the making of control sample [9]. taking into account the fact that bread with no more than 20 % of additional protein raw materials is present on the market of high-protein bread products we have decided to replace 20 % of wheat flour in samples by a certain type of isolate. the dry wheat gluten (dwg) (cargill, kazakhstan) was used as control protein additive in the same quantity. structural and mechanical properties the effect of the researched high-protein plant raw materials (hprm) on the structural and mechanical properties of the dough have been estimated by means of farinograph® – ts (brabender, germany). the instrument consists of a drive unit with continuous speed control and an attached measuring mixer for mixing the dough to be tested. the farinogram shows the quality characteristics of the analyzed flour. water absorption: the more water a flour can absorb at a definite consistency of a dough, the greater the dough yield per sack of flour; dough development time optimum mixing time for optimum doughs stability the longer the stability, the greater the fermentation and the higher the forces required for mixing. degree of softening the sooner the weakening, the shorter the fermentation and the less the abuse the flour can withstand. the trial samples with general weight 300 g were prepared according to the instruction. quantity of water for kneading the dough has been chosen experimentally in order to obtain dough with standard texture 500 units. registration of the changes of rheological characteristics of test samples was determined during 25 min from the moment of start kneading. this method is widely accepted for estimating the water bindingas well as structural and mechanical properties of the dough [10]. 3. results and discussion farinograms of the researched samples are shown in figures 1-5. the farinogram analysis of dough with addition of 20 % high-protein plant raw materials shows that all samples absorb higher amount of water than the control sample: the least of all with addition of rice isolate – 5 % more than control sample, the most – with addition of soya isolate – 32 % more than control. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 valeriy makhynko, vira drobot, tatjana golikova, effect of adding of plant protein isolates to the structural – mechanical properties of the wheat dough, food and environment safety, volume xvi, issue 2 – 2017, pag. 117 – 122 119 dough development time changes too: the sample with rice isolate has less duration than the control and with soya isolate – 2 times longer, with pea isolate – in 4 times longer. the most deviation takes place in sample with dry wheat gluten – 8 times longer than control sample. we believe that this difference is caused by fraction protein content of different types of high-protein plant raw materials. in particular, it is well known that more than 60 % of protein nitrogen of rice is a glutelin fraction. it is the largest of all the types of raw materials used. the structuralmechanical properties of hydrated glutelin promote high dough development. isolate of soya protein contains till 10 % of glutelin, and isolate of pea protein contains no more than 5 %, so it exerts some impact on the process of dough kneading. dry wheat gluten as well as flour contains prolamin and glutelin protein fractions, but its proportion differs significantly, it also has an impact on the behavior of dough during forming [11]. fig. 1. farinograms of control sample fig. 2. farinograms with replacing of 20 % wheat flour of premium class to the dwg fig. 3. farinograms with replacing of 20 % wheat flour of premium class to the isp to rq ue [ fe ] te m pe ra tu re [ °c ] time [mm:ss] to rq ue [ fe ] te m pe ra tu re [ °c ] time [mm:ss] to rq ue [ fe ] te m pe ra tu re [ °c ] time [mm:ss] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 valeriy makhynko, vira drobot, tatjana golikova, effect of adding of plant protein isolates to the structural – mechanical properties of the wheat dough, food and environment safety, volume xvi, issue 2 – 2017, pag. 117 – 122 120 fig. 4. farinograms with replacing of 20 % wheat flour of premium class to the ipp fig. 5. farinograms with replacing of 20 % wheat flour of premium class to irp numerical data of farinogram decryption is presented in the table 1. table 1 data of farinograms with addition of high-protein plant raw materials with replacement of 20 % wheat flour in: index control sample (without additives) dwg іsp іpp іrp water absorption, % 61.8 73.3 94.2 78.1 66.5 dough development time, min. 2.1 16.4 4.5 8.1 1.3 dough stability, min. 12.6 25 7.4 7.4 13.5 degree of softening 18 6 29 6 21 the addition of hprm has effects on dough stability. the sample with rice protein isolate is similar to the control sample, whereas that with addition of 20 % of spi – is 1.5 times less. the degree of softening increases in samples with addition of irp and isp by 3 and 11 % in accordance with the control sample too, though sample the with ipp has this index 3 times lower than that in the control sample. such behavior of different samples may be explained firstly due to the redistribution of water between high-protein plant raw material and components of wheat flour that has negative effect on the swelling of colloidal substances of flour and decreases the forming of dough structure. the development of protein-protein complexes between wheat gluten and protein of added raw materials may take place. on the ground of obtained data it is possible to make further recommendations in order to correct the duration of dough kneading. all the types of added highprotein raw materials, excepting rice protein isolate, require prolonged duration of kneading, especially those made from dry wheat gluten. and it is necessary to increase the quantity of water to be added in the dough. it depends on the type and quantity of added high-protein plant raw materials. since the industrial production of bread requires to rq ue [ fe ] te m pe ra tu re [° c ] time [mm:ss] to rq ue [ fe ] te m pe ra tu re [° c ] time [mm:ss] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 valeriy makhynko, vira drobot, tatjana golikova, effect of adding of plant protein isolates to the structural – mechanical properties of the wheat dough, food and environment safety, volume xvi, issue 2 – 2017, pag. 117 – 122 121 different dosages of hprm, it may take place as well by using compositions with different proportions. thus, the method of calculating the water absorption coefficient of dough with hprm was proposed. due to farinogram data we obtained that 1 g of flour absorbs approximately 0.6 g of water. the calculating formula of the conversion coefficient required by the quantity of water for dough kneading is proposed: where wa – water absorption capacity of high-protein plant raw materials; gf – weight of flour, g; кf – coefficient that takes into account water absorption of control sample (кf = 0.6); gad – quantity of added hprm, g. according to this formula, the conversion coefficient required by the quantity of water for dough kneading with dwg is: it is possible to calculate coefficients for isp (2.3), ipp (1.5) and irp (0.9) in the similar way. so, replacement of 1 g of flour to the dwg will need adding of 1.3 g of water; 1g of isp – 2.3 g of water, 1g of ipp – 1.5 g and 1 g of irp – 0.9 g of water. this method is used in calculations of different dosages and proportions of dwg and hprm. for example, in the case of proportion 10 dwg:10 spi additional quantity of water will be 36 g: addition of 10 g of dwg requires additionally 13 g of water (10×1.3), and addition of 10 g of isp – 23 g of water (10×2.3). to verify the theoretical calculation, the analysis of the calculated dough by means of farinograph was implemented (fig. 6). 10 % of dwg and 10 % of isp have been replaced by 20 % of wheat flour. fig. 6. farinograms wuth replacing of 20 % of wheat flour to 10 % dwg and 10 % isp. the experimental results show that the proposed calculation method may be applied in the obtaining of dough with necessary structural and mechanical properties. to rq ue [ fe ] te m pe ra tu re [° c ] time [mm:ss] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 2 – 2017 valeriy makhynko, vira drobot, tatjana golikova, effect of adding of plant protein isolates to the structural – mechanical properties of the wheat dough, food and environment safety, volume xvi, issue 2 – 2017, pag. 117 – 122 122 4. conclusion the changes of structural and mechanical properties of wheat dough with addition of different kinds of high-protein plant raw materials have been investigated. the necessity of prolonging kneading period has been carried out by the farinogram data (excepting the addition of rice protein isolate) and addition of extra quantity of water. the coefficients that take into account the type of raw material and its water absorption capacity have been calculated: for isolate of soya protein – 2.3, for isolate of pea protein – 1.5, for dry wheat white – 1.3, for isolate of rice protein – 0.9. the use of these coefficients in calculating the additional quantity of water in case of adding of high-protein raw materials will provide dough with necessary structural and mechanical properties. 5. references [1]. buresova i, tokar m, marecek j., hrivna l, famera o., sottnikova v. the comparison of the effect of added amaranth, buckwheat, chickpea, corn, millet and quinoa flour on rice dough, rheological characteristics, textural and sensory quality of bread // journal of cereal science, vol. 75, may 2017, p. 158 – 164, (2017). [2]. chernysh l., makhynko v., berezna o. influence of vegetable protein isolates on structural-mechanical properties of wheat dough. – 8th central european congress on food 2016 – food science for well-being. – p. 167, (2016). [3]. marti a., bick j., pagani m.a., ismail b., k. seetharamon. structural characterization of protein of wheat flour doughs enriched with intermediate wheatgrass flour // food chemistry, vol. 194, 1 march 2016, p. 994 – 1002. [4]. peng b. yougian li, shiyong ding, jun yang. transgenic sorghum with suppressed synthesis of kafirin subclasses: effect on flour and dough rheological characteristics // journal of cereal science, vol. 75, may 2017, p. 69 – 76. [5]. stone a., karalash a., tyler r., warkentin t., nickerson m. functional attributes of pea protein isolates prepared using different extraction methods and cultivars // food research international, volume 76, part 1, october 2015, pages 31-38. [6]. amagliani l., o'regan j., kelly a., o'mahony j. the composition, extraction, functionality and applications of rice proteins: a review //trends in food science & technology, volume 64, june 2017, pages 1-12. [7]. mccarthy n., kennedy d., hogan s., kelly p., thapa k., murphy k., fenelon m. emulsification properties of pea protein isolate using homogenization, microfluidization and ultrasonication // food research international, volume 89, part 1, november 2016, pages 415-421. [8]. girgih a, chao d., lin l., rong he, jung s., aluko r. enzymatic protein hydrolysates from high pressure-pretreated isolated pea proteins have better antioxidant properties than similar hydrolysates produced from heat pretreatment. // food chemistry, volume 188, 1 december 2015, pages 510-516. [9] . iso 21415-1:2006 wheat and wheat flour gluten content part 1: determination of wet gluten by a manual method [10]. iso 5530-1:2013 «wheat flour. physical characteristics of doughs. part 1: determination of water absorption and rheological properties using a farinograph». [11]. drobot v., sylchuk t., bilyk o. effect of dry wheat gluten to the technological process and bread quality // storage and processing of grain. 2005. – no 4(70), 53-55 (in ukrainian). 385 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 4 2015, pag. 385 390 water phase condition in the butter paste with red beet powder *oksana podkovko1, tamara rashevskaya1 1the national university of food technologies, kyiv, ukraine oa_podkovko@mail.ru received december 6th 2015, accepted december 29th 2015 abstract. the technology of butter paste with red beet powder based on butter with addition of dry skimmed milk, flaxseeds and inulin is developed. the quantity of the plant food additives in the ready product is the following: red beet powder – 0.80 %, inulin – 1.50 %, flaxseeds – 2.90 %.the condition of water phase in the fresh product was investigated for the indicators: dispersion of plasma and forms of moisture connection. dispersion was determined by computing the drops of moisture under the micromed xs-2610 microscope. the article concludes that the addition of red beet crio-powder, flaxseeds, inulin with simultaneously mechanical processing generates dispersion of moisture in the butter paste with the increasing of droplets with the diameter of 1...5 microns. the main forms of moisture connection research were conducted by the thermogravimetry method using a pauli-erdene q-1500 d derivatograph system. these results suggest that the selected complex of plant food additives leads to the increasing of the amount of monomolecular and polymolecular firmly bound moisture. key words: butter paste, plant food additives, dispersion of plasma, forms of moisture connection. 1. introduction. nutrition plays an important role in everyday life. the components of food provide the body with vitamins, minerals, dietary fibers, polyunsaturated fatty acids, which in their turn determine the state of health, support the immunity, physical and mental efficiency. considering this, it is important and appropriate to develop and improve food technologies that will correspond to the status of health, prophylactic and functional purpose. an assortment of butter with functional properties containing pectin and inulin polysaccharides, red beet, black currant and carrot plant criopowders, artichoke and flaxseeds was developed in the national university of food technologies (nuft) led by prof. rashevskaya t. tests on butter with pectin, inulin, red beet powder and black currant buds were conducted in the clinics within the institute of ecological hygiene and toxicology m.i. medvedya and in the institute of microbiology and virology national academy of sciences biomedical and accordingly, the ukraine health ministry drew some conclusions on the use of this assortment for health, prophylactic and dietary nutrition [1]. it is important nowadays to develop and produce moderate calorie foods with simultaneously increase of the biological value and dietary properties. the reducing of energy value of butter can be achieved by correcting its composition and correlating its components. the continuation of the work in this direction with these requirements has limitations, since the content of milk fat in butter should be at least 51.00 %; it is not recommended to use non-dairy ingredients, http://www.fia.usv.ro/fiajournal mailto:oa_podkovko@mail.ru food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 oksana podkovko, tamara rashevskaya, water phase condition in the butter paste with red beet powder, food and environment safety, volume xiv, issue 4 – 2015, pag. 385 – 390 386 since the traditional color of the product is preferred. therefore, it is appropriate to create a new group of moderate calorie products with typical consumer characteristics for the traditional composition of butter. in such groups butter paste could be included – dairy product with fat emulsion base of fat mass fraction of 39...49 % that is produced from cow's milk, dairy products and (or) milk processing byproducts using stabilizers, but not substitutes of milk component parts by nondairy components or without them. the composition and technologies of butter paste with honey, cocoa, chicory, fruit and berry, vegetable and fungal additives, spices, herbs were science-based in the all-russian research institute of dairy and cheese manufacturing by vishemirskii f. [2,3]. chocolate butter paste "for tea" is produced in belarus with fat mass fraction of 40.00 % and consists of butter, drinking water, sugar, dry skimmed milk, cocoa powder, stabilizers, iodized salt, potassium sorbate and citric acid. the disadvantage of the above assortment is that these additives do not have functional properties. preference is given to the use of food additives made from plant raw materials, which are a source of biologically active substances. butter paste assortment with the complex of biologically active substances, containing honey, carrot powder, micronutrients, viburnum syrup, on antidiabetic purpose, was developed by the young scientists in nuft led by prof. rashevsky t.[4]. the use of inulin and flaxseed into the composition was the common basis for these types of products, which are technological and have functional properties [5-7]. recently, red beet has attracted the attention of medical and food industries. it is rich in anthocyanin coloring agents (betaine and betanin), catechins, flavonoid glycosides, vitamins and minerals that help strengthen capillaries and blood vessels, increase the hemoglobin content and the amount of red blood cells, reduce blood pressure, prevent cancer, reduce cholesterol blood, enhance detoxification, toxins, salts of heavy metals and radionuclides from the body[8]. in this direction, we have developed the technology of red beet powder butter paste with fat mass fraction of 42.00 % based on butter with addition of dry skimmed milk. into the milk-based plant product, red beet crio-powder, flaxseeds and inulin were added. plant additives were used as suspension in buttermilk. the method of producing red beet powder butter paste was awarded the ukraine patent for invention [9]. in previous studies it was found out that red beet crio-powder, flaxseeds and inulin influenced the quality indicators of butter paste. the selected complex of plant additives improves heat resistance of the ready product, the structure ability to keep the liquid phase of fat, promotes its plasticizing and forms the structure with optimal ratio of coagulation and crystallization connections [10,11]. the butter paste is characterized by high moisture content (47.00 %), which is why the water phase state, dispersion of plasma, forms of moisture connection with the components plays an important role in providing the quality of the ready product. it is acknowledged that the addition of crio-powders and polysaccharides changes the ratio of forms of moisture connection, the amount of strongly bound absorption moisture and dispersion of moisture on the microand nanoscale increases, the number of drops with the diameter higher than 5 microns is reduced, coalescence of droplets during storage is broken [6, 12, 13]. according to the above information, the aim of this work is to analyze the impact of red beet crio-powder, flaxseeds food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 oksana podkovko, tamara rashevskaya, water phase condition in the butter paste with red beet powder, food and environment safety, volume xiv, issue 4 – 2015, pag. 385 – 390 387 and inulin on the state of water phase in butter paste. 2. materials and methods. the objects of the study were samples of fresh butter paste with red beet powder and butter with fat mass fraction of 63.00 % as a control. the determination of dispersion of plasma product was performed by counting the drops of water under a microscope. by applying a piece of the sample with help of a metal needle with size less than 1 mm microscopic pattern were prepared, carefully covered by cover slip, set a 100 g weight and left for 5 min at 20 0c. the prepared patterns were studied under a microscope micromed xs-2610 with lighting "on the passage". at least five most typical fields and the scale line of the object-micrometer were photographed to calculate dispersion. according to the obtained images size of droplets of plasma, fractions were determined and shared: (1...2) mm, (3...4) mm, (5...6) mm, (7...8) mm, (9…10) mm. the content of each droplets fraction and the average diameter in the samples were determined as well. the analysis of bond moisture forms was made by the method of thermogravimetry, which allows the determining of temperature of the sample, its mass change, rate of mass change and enthalpy change simultaneously. curves were reflected on the derivatograph paulierdene q-1500 d system with speed increasing temperature 50c/min. the weight of the samples was of (150...200) mg. tape speed was 2mm/min. simultaneous getting of derivatograph curves – differential thermal analysis (dta), thermogravimetric (tg), differential-thermogravimetric (dtg) and temperature (t) of moisture removal allows the determination of the chemical and physical transformations in the studied samples and conduct of qualitative and quantitative assessment of these changes. according to the obtained derivatogram curves, the temperature peaks and intervals of the removal with different types of connection were determined. according to (tg) curve the number of removed moisture in percent was determined. classification of bond forms was moisture held by rehbinder p., who identified three groups: chemical, physico-chemical and physico-mechanical [14]. 3. results and discussion. the dispersion of plasma product affects the stability of the ready product by microbial and oxidative deterioration. the research results are presented in fig. 1. fig. 1. dispersion of butter paste with red beet powder plasma and butter-control 0 20 40 60 1…2 3…4 5…6 7…8 9…10a m o u n t o f d ro p le ts , % size of droplets, мicrons butter paste with red beet powder butter-control food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 oksana podkovko, tamara rashevskaya, water phase condition in the butter paste with red beet powder, food and environment safety, volume xiv, issue 4 – 2015, pag. 385 – 390 388 according to the obtained results the amount of drops of plasma with diameter (1...2) microns is of 48 % in fresh butter paste and of 41 % in butter-control, respectively. moisture droplets with diameter (9...10) microns in the paste are not available, however in the control there are 7 %. in our opinion, the dispersion of plasma droplets in the butter paste occurs due to the addition of red beet criopowder, flaxseeds and inulin together with simultaneous homogenization process during the addition of supplements into the product composition. in previous studies it has been established that red beet criopowder, flaxseeds and inulin are characterized by the ability to connect free moisture and to swell [6,15,16]. due to such properties of plant additives and simultaneous use of mechanical processing of mixing, the presence of a higher number of plasma droplets with diameter (1...5) microns was caused, increasing the stability of the ready product to oxidation and microbial deterioration. food products are systems in which moisture has different forms of connection with the hard skeleton. we can identify them, using the method of thermogravimetry. the deryvatohramies of the butter paste with red beet powder and butter-control are shown in fig.2. fig. 2.deryvatohramms of the fresh butter paste with red beet powder and (a) and butter-control (b) analyzing dta curves of the butter paste with red beet criopowder and buttercontrol, four main forms of connection of moisture were highlighted by temperature peaks. by tg curves their number in percent was defined (table 1). butter-control is characterized by a weakly bound in an amount of 11.91 % and 35.09 % strongly bound moisture. removal of free moisture is held in several stages: first, mechanically connected one at temperatures of 33 0c and 42 0c in an amount of 3.76 % is removed, then – 8.15 a) b) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 oksana podkovko, tamara rashevskaya, water phase condition in the butter paste with red beet powder, food and environment safety, volume xiv, issue 4 – 2015, pag. 385 – 390 389 % osmotic at 98 0c. firmly connected moisture is presented by polymolecular form that is removed at temperatures of 107 0c and 109 0c in an amount of 33.21 % and by monomolecular with the fixed peak at 131 0c in an amount of 1.88 %. table 1 forms of connection of moisture in the butter paste with red beet powder and butter-control forms of connection of moisture name of fresh sample butter paste with red beet powder butter-cjntrol moisture, % temperature, 0с moisture, % temperature, 0с absolute relative absolute relative weakly bound: 8.02 17.05 11.91 25.33 mechanical 0 0 36 0.63 3.13 1.33 6.67 33 42 osmotic 8.02 17.05 94 8.15 17.33 98 firmly bound: 38.98 82.95 35.09 74.67 polymolecular 32.10 68.30 108 27.57 5.64 58.67 12.00 107 109 monomolecular 6.88 14.65 127 1.88 4.00 131 unlike butter-control, in the butter paste with red beet powder mechanically bound moisture was not found. weakly bound moisture is presented by only osmotic connections in an amount of 8.02 %. the temperature peak of removal is 94 0c. the number of firmly bound monomolecular moisture increases by 5.00 % in comparison with the butter-control. in our opinion, such redistribution of moisture connection forms is due to the microstructure of plant supplements. in previous studies it has been established that red beet criopowder has indestructible mechanical and leading texture which connects free moisture [15]. so, when adding criopowder, the joining of adsorption of moisture of the butter paste by additive particles is done. it has been proved that inulin has the ability to form strong hydrogen bonds in the structure of butter, increasing the content of tightly bound monomolecular moisture [13]. it is known that the addition of flaxseeds into the composition of butter helps thin dispersion of water phase droplets due to the formation of additional intermolecular bonds [16]. we assume that inulin and flaxseed have similar properties in the structure of the butter paste as well. due the complex effects of plant food supplements, the mechanical weakly bound moisture in the ready product is not available, thus increasing the number of monomolecular tightly connected ones. 4. conclusion. the addition of the complex of plant food additives promotes the dispersion of plasma in the ready product, increasing the amount of droplets with diameter of (1...5) microns. red beet criopowder, flaxseeds and inulin lead to the increase in the amount of firmly bound, especially monomolecular and absence of mechanical weakly bound moisture in the butter paste. 5. references [1]. rashevskaya t., gulyi i., ukrainets a. biochemical studies of the butter with plant food additives during storage, food industry, 2: 15–18, (2003) [2]. vishemirskii f., topnikova e., lobacheva t. assortment of the butter with flavor filler, cheese and butter manufacture, 6: 37– 39, (2005) food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 oksana podkovko, tamara rashevskaya, water phase condition in the butter paste with red beet powder, food and environment safety, volume xiv, issue 4 – 2015, pag. 385 – 390 390 [3]. topnikova e. butter with low fat mass and his analogues, cheese and butter manufacture, 3: 10–12, (2006) [4]. ivanov s., rashevskaya t. the butter paste with complex of biologically active plant micronutrients of antidiabetic purpose, the scientific works of nuft, 43: 85–94. [5]. barclay t. inulin – a versatile polysaccharide with multiple pharmaceutical and food chemical uses, j. excipients and food chem., 1(3): 27–50, (2010) [6]. ivanov s. flaxseed additive application in dairy products production, procedia food science, 1: 275–280, (2011) [7]. muir a. flax seed constituentsand human health, flax: the genus linum, ed. by a.d muir, n.d westcott, taylor & francis inc.: 243–257, (2003) [8]. upur l., kovalev v. the research of biologically active substances in beetroot, physiologically active substances, 2(32): 82–86, (2001) [9]. rashevskaya t., goncharov g., podkovko o. the method of the producing of the butter paste.patent ua, no. 108443, 2015. [10]. rashevskaya t., goncharov g., podkovko o. butter paste with red beet powder, the scientific works of nuft, 53: 7–14, (2013) [11]. podkovko o., rashevskaya t. indicators of structure and consistency of butter paste research, scientific works of uft (plovdiv), lxi, i: 163–166, (2014) [12]. rashevskaya t. analysis of water connection in the butter with red beet criopowder, news of higher education. food technology, 1: 36–38, (1999) [13]. rashevskaya t. influenceofpectinonwaterandfattyphasesstatesinbu tter, journaloffoodphysics, xi–xii: 39–45, (1999) [14]. rebinder p.surfactants in science and technology. moscow, 1961.46 p. [15]. podkovko o. rashevskaya t. investigation of technological characteristics of red beet powders for use in the composition of the butter paste, collection scientific works of vnau. technical science, 2(90): 62–66, (2015) [16]. machonina m., rashevskaya t., ukrainets a. dispersion of the plasma in butter with flaxseeds, the scientific works of nuft, 32: 94–96, (2010) microsoft word 8 ama lethicia manizan_corectat 19 aprilie 2018.doc 183 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 22018 pag. 183 191 assessment of the risk factors associated with aflatoxin contamination during the marketing of peanuts in côte d'ivoire *ama léthicia manizan1, david akaki2, isabelle piro-metayer3, didier montet3, catherine brabet3, rose koffi-nevry1 1 faculty of food science and technologies, biotechnology and food microbiology laboratory, manizanlethicia@yahoo.fr university of nangui abrogoua, 02 bp 801 abidjan, côte d’ivoire. rosenevry2002@gmail.com, 2 department of chemical and agri-food engineering, industrial process synthesis, environment and new energies laboratory, national polytechnic institute houphouet-boigny, bp 1313 yamoussoukro, côte d’ivoire. davidakaki@yahoo.fr, 3 umr 95 qualisud / cirad/ université of montpellier, ta b-95 /16, 73 rue j-f breton, 34398 montpellier cedex 5, france. isabelle.piro-metayer@cirad.fr, didier.montet@cirad.fr , catherine.brabet@cirad.fr *corresponding author received 31th january 2018, accepted 26th june 2018 abstract: this study was conducted to assess the different risk factors related to practices of sale of peanuts for better control of aflatoxin contamination and also to improve the safety of peanuts during marketing. the sales practices are similar regardless of the area surveyed. of all the respondents in this survey, 61.7% were men as against 38.3% women and 70% of the respondents do not have any formal education. the marketing of peanut seeds is generally an activity done by men (100%) while that of peanut paste is assigned to women (100%). the unsold peanut paste is kept from 1 day to 3 months at room temperature until sale, mainly in market stalls used to sell it (42.4%) or in stores (39.4%). the losses are mainly due to fungi (37.9%) and insects (13.6%) followed by humidity and high temperature (4.5%). during sale of peanut paste, the site, the surroundings of sales points and the storage containers are potential fungi growth factors and eventual risk points for mycotoxin contamination. proper handling and hygiene might reduce the aflatoxin contamination of peanut to ensure better sanitary quality of peanuts and protect consumer’s heath. keywords: mycotoxins, sale, market, peanut, risk. 1. introduction native to the caribbean area, peanut (arachis hypogaea l.) is a major food source [1]. in africa, peanuts represent a major cash crop that contributes significantly to food security and poverty reduction [2, 3]. this oilseed is very rich in protein (25%) and lipid (50%) and it is thus an important nutrient source for populations [4]. in côte d'ivoire, peanuts are mainly produced in the center and north regions [5]. however, their production is not selfsufficient. the totality of ivorian production is entirely meant to selfconsumption [6]. in côte d'ivoire, peanut is a staple food used for oil production but also for paste produced from roasted seeds [6]. peanut paste is widely used in the preparation of soup but also as a spread [7]. in côte d'ivoire, peanut paste is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 184 traditionally produced and marketed under unsanitary conditions [8]. like most crops, peanut production and marketing face losses caused by insects and rodents, but also contamination by mycotoxins produced by toxinogenic fungi. contamination of food by toxinogenic fungi and their mycotoxins has been recognized by the world health organization as a major source of foodborne diseases [9]. mainly produced by aspergillus genus, including aspergillus flavus, aspergillus parasiticus and aspergillus nomius, aflatoxins are the main mycotoxins commonly associated with peanuts. aflatoxin contamination of peanuts may occur before harvest, during storage and during marketing [10]. numerous studies have reported that contamination of peanuts sold on markets by aspergillus and aflatoxins, in côte d'ivoire [11], congo [12] and ethiopia [10]. aflatoxins are highly toxic, mutagenic, immunosuppressive, teratogenic and carcinogenic which target primarily the liver [13, 14]. aflatoxins are thermostable and it is thus difficult to eliminate them from food. however, studies have shown that foods contaminated with aflatoxins can be detoxified by using inorganic salts, organic acids, ammonia, and aflatoxin b1 binding agents [14]. according to elmore et al. [15], african cooking courses such as heat treatment and fermentation cannot reduce aflatoxin contamination to an acceptable level. in contrast, some processing methods such as roasting could significantly reduce aflatoxin levels by 89% in the final product [14]. aflatoxin contamination does not only limit exports to the european countries due to the application of standards, but it also reduces the revenue of peanut producers and represents a risk for consumer’s health. the establishment and control of good hygiene practices and production could help to limit aflatoxin contamination. this study aims to contribute to control aflatoxin contamination during the sale of peanuts and derivate products on the ivorian markets by the determination of sales practices. 2. matherials and methods 2.1. material study sites. the study was carried out in four major cities of côte d'ivoire, namely, abidjan, bouaké, korhogo and daloa which represent the main food supply areas respectively in the southern, central, northern and western areas of the côte d'ivoire. abidjan, the economic capital of côte d'ivoire is the largest consumer market. bouaké, the second city of côte d’ivoire has a leading role in marketing since it is a food collect and exporting centre. daloa is the centre for food products collection in the western region and korhogo a food collect for the north. figure 1 shows the location of these study areas. fig. 1. map of côte d'ivoire showing sample collection areas for rice, maize and peanut food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 185 2.2. methods survey. a socio-demographic survey was conducted in the main markets belonging to the cities of abidjan (26), bouaké (10), korhogo (13) and daloa (11). the study population of 60 sellers consisted of 3 types of sellers mainly unshelled peanut sellers (8), grain wholesalers (38) and peanut paste sellers (20) which means wholesalers, retailers and peanut paste sellers. the sales conditions were investigated and direct interview was conducted to identify the characteristics of the sellers (sex, age, educational status, methods of supply, duration and storage conditions, conditions of the peanut paste production, loss or deterioration, management of the unsold peanut paste etc.). statistical analyses. the data obtained on the characteristic of the sellers and the handling of peanut during selling were submitted to an analysis of spss statistics version 21, using khi-deux pearson test with respect to the conditions of peanut sale investigated during aflatoxin risk factors assessment. 3. results and discussion socio-demographic profile of traders is shown in table 1. 61.7% of all in this survey were men. the result of this study is different from that of mutegi et al. [16] who, in a similar study in kenya, found that only 25% of peanut sellers were men. the age of traders ranged between 15 and 60 years old or more, with a majority (85%) between 30 and 60 years old (table 1). while peanut seed sale is exclusive to men between ages of 30 and 60, women only make the peanut butter sale. according to the respondents, seed trade requires a lot of money, which explains why this activity is done by men. in addition, 70% of traders surveyed have no level of study. in addition, only 1.7% has a high level of education (table 1). these results corroborate those of boli et al. [17] who, after a study of the main markets of the city of abidjan, reported that 73.4% of peanut paste sellers are illiterate. the peanut trade therefore does not require a specific level of study. however, this lack of education could affect knowledge and the need to use good production practices. table 1. socio-demographic profile of peanut traders interviewed socio-demographic profile number of individuals (n=60) percentage (%) locality bouaké korhogo daloa abidjan 10 13 11 26 16.7 21.7 18.3 43.3 sex male female 37 23 61.7 38.3 age 15-30 years 30-60 years > 60 years 4 51 5 6.7 85.0 8.3 level of education primary secondary superior none 13 4 1 42 21.7 6.7 1.7 70.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 186 practices and forms of peanut marketing. table 2 showed that the majority of traders are independent (91.7%), most of them (70%) having more than 20 years of experience in the field and with peanut sale as their main source of revenue (63.3%). this could be explained by the fact that most traders inherit a family business. they have learned for most part, the trade since childhood, as evidenced by the number of experience years. similar observations were made by broutin and rouyat [18] who indicated that in urban areas, the majority of food processors and sellers benefit from the support of family members who replace them during their periods of inactivity. table 2 also showed that peanuts are marketed in several forms. the main forms are seeds (63.3%), followed by paste (30%) and dried pods (11.7%). fresh pods are the least marketed at 6.7%. however, it is important to mention that peanuts are sometimes marketed at the same time with cereals such as maize (46.7%), rice (38.3%), millet (16.7%) and cashew (8.3%). peanuts are mainly marketed as shelled seeds or paste. they are often marketed on the same display as other cereals such as maize and millet, which could lead to cross contamination between peanuts and other marketed products. table 2. practices and forms of peanut marketing in côte d'ivoire number of individuals (n=60) percentage (%) trader type independent in association 55 5 91.7 8.3 years of experience in peanut trade 1-10 years 10-20 years > 20 years 10 8 42 16.7 13.3 70.0 main source of revenue yes no 38 22 63.3 36.7 forms marketed* fresh pods dried pods shelled seeds peanut paste 4 7 38 18 6.7 11.7 63.3 30.0 other commercialized products* rice corn cashew mil bean sorghum bambara peanut pistachio 23 28 5 10 4 2 1 1 38.3 46.7 8.3 16.7 6.7 3.3 1.7 1.7 (n) = number of individuals surveyed *proportions sum is greater than 100% because only one person can have more than one choice for this question food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 187 sales circuit and storage mode. table 3 highlights the sale circuit and storage methods for pods, seeds and peanut paste. peanut trade in côte d'ivoire uses the same circuit regardless of the zone and the type of interviewed traders. the sales circuit takes into account several actors including, farmers, trackers, wholesalers and semiwholesalers. in the sale circuits, it appeared that 39.4% of traders obtained peanuts from wholesalers, 27.3% from farmers, while 27.3% were self-supplied. it should be noted that the trackers (1.5%) intervened very little in this circuit (table 3). trackers, wholesalers and semiwholesalers systematically scour villages for field buying of cereals and other food products. this trade circuit differs very little from that established by demont [19] on marketing systems for food products (maize and peanuts) in côte d'ivoire. from tracker to wholesaler and retailer, peanut undergoes transport and storage times that can promote fungi proliferation. in 68% of cases, peanuts sold in ivorian markets can be kept for a period varied from one day to three months regardless of storage location at room temperature, mainly in markets (42.4%) and in warehouses (39.4%). for reasons of profitability and according to the market demand, traders keep the same stock until it is completely exhausted before buying a new one. similar results obtained by boli et al. [17], in abidjan markets, showed that peanut paste could be marketed over a period of 3 months. peanuts pods and/or seeds are either stored in polythene bags (45.5%) or in aluminium bowls for peanut seeds (24.2%) or paste (35%) and in plastic pots with lids only for peanut paste (85%). these results are consistent with those of xu et al. [20]. according to them in a similar study in gambia, most women reported using sealed plastic containers (40%) or bags (44%), or a combination of both to store their peanuts. in addition, the risk to peanut paste contamination with aflatoxins increases with the duration of marketing due to bad practices and many manipulations [21]. the conditions and duration of peanut storage in our markets represent a risk of increased contamination of peanuts by aflatoxins. causes of losses and quality control of peanuts. the majority of traders (48.5%) said they did not have any losses during peanut sale. about 36.4% of traders suffer losses of 2 to 50% during sale. these losses could be explained mainly by the action of fungi (37.9%) and insects (13.6%). about 50% of the respondents have already observed peanut contamination by fungi. the results of this study confirm those of other authors where losses were also recorded after long periods of peanut storage. as examples, in benin losses of 2 to 5% have been recorded [22] and 5 to 15% in ghana [23] after a sorting in view to eliminate mouldy seeds. however, the majority, 74.2% of interviewed traders, did not use any ways of control. the mains means of control to reduce losses was drying and sorting. the choice of these methods could be justified by the lack of education of majority of traders and absence of training in good hygienic practices. these results corroborate with those obtained by mutegi et al. [16] who indicated that the main control methods are sorting, drying and sieving. some traders (wholesalers) recommended reducing shipping and storage time by making only purchases on order. for peanut paste sellers, the only way to reduce contamination would be to avoid any contact with water and to put oil on the surface of paste. indeed, most fungi involved in peanut deterioration are aerophilic. as a result, the presence of oil on the surface creates an anaerobic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 188 environment unfavourable to fungi development. the quality of peanut seeds is usually verified by visual, taste or touch control. however, 28.8% of traders did not apply any quality criteria. visual inspection permits to detect presence of mouldy seeds or foreign bodies. taste control is used to determine water content of seeds and paste quality. the water content is also estimated by touch. this expertise could be explained by the number of experience years acquired by the majority of traders. in a comparative study in ghana, most participants reported using visual appreciation to identify poor quality peanuts [23]. table 3. supply and storage conditions of peanuts in the markets according to the forms marketed in côte d'ivoire percentage (%) (n=66) selling practices peanuts pods (n=8) peanuts seeds (n=38) peanuts paste (n=20) % total method of obtaining (a)* myself on field wholesalers semi-wholesalers trackers 0 37.5 (3) 37.5 (3) 37.5 (3) 0 2.6 (1) 39.5 (15) 52.6 (20) 13.2 (5) 2.6 (1) 85.0 (17) 0 15.0 (3) 0 0 27.3 27.3 39.4 12.1 1.5 storage (b) yes no 62.5 (5) 37.5 (3) 84.2 (32) 15.8 (6) 100 (20) 0 86.4 13.6 storage container (a)* bag plastic bag aluminium bowl bucket with lid 62.5 (5) 0 12.5 (1) 0 65.8 (25) 10.5 (4) 21.0 (8) 0 0 0 35.0 (7) 85.0 (17) 45.5 6 24.2 25.8 storage place (b) house market mill warehouse warehouse or truck 12.5 (1) 12.5 (1) 0 0 37.5 (3) 0 28.9 (11) 0 52.6 (20) 2.6 (1) 0 80.0 (16) 5.0 (1) 15.0 (3) 0 1.5 42.4 1.5 39.4 1.5 storage time (b)** 1-5 days 1-2 weeks 2-3 weeks 1-2 months 3 months unknown 0 12.5 (1) 0 12.5 (1) 0 37.5 (3) 2.6 (1) 15.8 (6) 2.6 (1) 15.8 (6) 2.6 (1) 44.7 (17) 15.0 (3) 55.0 (11) 5.0 (1) 20.0 (4) 0 5.0 (1) 6.1 27.3 3 16.7 1.5 31.8 (n) = number of individuals surveyed; (a) = strong correlation between variable and trader type (degree of significance of khi2 <0.05 and phi> 70%); (b) = no correlation between variable and trader type (degree of significance of chi2> 0.05); (c) = low correlation between variable and trader type (degree of significance of chi2 <0.05 and phi <70%). *proportions sum is greater than 100% because only one individual can have multiple answers to this question. **proportions sum is less than 100% because this question concern only the 86.4% of traders witch conserving the unsold. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 189 table 4. loss factors, means of control and quality control of peanuts according to the marketed form in côte d'ivoire losses during selling percentage (%) (n=66) peanuts pods (n=8) peanuts seeds (n=38) peanuts paste (n=20) % total losses (b) 0 % 2-10 % 10-50 % unknown 12.5 (1) 62.5 (5) 25.0 (2) 0 39.5 (15) 26.3 (10) 7.9 (3) 26.3 (10) 80.0 (16) 15.0 (3) 5.0 (1) 0 48.5 27.3 9.1 15.2 loss factors (b)* insects fungi humidity temperature long conservation grinding-tri others (pebbles, sand, transport) 25.0 (2) 62.5 (5) 12.5 (1) 0 0 0 12.5 (1) 8.4 (7) 50.0 (19) 5.3 (2) 7.9 (3) 2.6 (1) 0 10.5 (4) 0 5.0 (1) 0 0 0 10.0 (2) 0 13.6 37.9 4.5 4.5 1.5 3.0 7.6 contamination by fungi (c) yes no 87.5 (7) 12.5 (1) 68.4 (26) 31.6 (12) 0 100 (20) 50.0 50.0 means of control (a) drying-tri sieving-tri reduced transport time purchase on order avoid contact with water cover the surface with oil none 12.5 (1) 25.0 (2) 12.5 (1) 0 0 0 50.0 (4) 23.7 (9) 0 0 2.6 (1) 0 0 73.7 (28) 0 0 0 0 10.0 (2) 5.0 (1) 85.0 (17) 15.2 3.0 1.5 1.5 3.0 1.5 74.2 quality control of peanuts (c) visual-touch (check if dry) visual-taste taste none 37.5 (3) 0 0 62.5 (5) 89.5 (34) 0 0 10.5 (4) 25.0 (5) 10.0 (2) 15.0 (3) 50.0 (10) 63.6 3.0 4.5 28.8 (n) = number of individuals surveyed; (a) = strong correlation between variable and trader type (degree of significance of khi2 <0.05 and phi> 70%); (b) = no correlation between variable and trader type (degree of significance of chi2> 0.05); (c) = low correlation between variable and trader type (degree of significance of chi2 <0.05 and phi <70%). *proportions sum is less than 100%, because we only counted those who experienced losses. 4. conclusion peanut trade practices in côte d'ivoire are the same regardless of the surveyed area. along the circuit sale, duration, location and storage containers of peanuts constitute risk factors for fungi contamination and growth. training in good hygiene and manufacturing practices of peanut traders would be an alternative to the reduction of aflatoxin contamination, in order to ensure a better sanitary quality of peanuts in the ivorian markets. 5. acknowledgments food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 190 this work is resulting from the eu project 3civoire (europeaid/129596/l/act/ci dci-nsapvd/2010/64). this research was also supported by a grant for ivorian phd students awarded by the service of co-operation and cultural action of the french embassy in côte d’ivoire and the ministry of higher education and scientific research of côte d'ivoire. 6. references [1]. yussif i., kwoseh c., osman m., kwabena a., yirzagla j., farmers’ perception and farming practices on the effect of early and late leaf spots on groundnut production in northern ghana. journal of biology, agriculture and healthcare, 4(19): 22 28, (2014). [2]. ntare b.r., arachis hypogaea l. dans van der vossen, h.a.m & mkamilo, g.s (editeurs). resources végétales de l’afrique tropicale 14. oléagineaux, 261pp, (2007). [3]. smart m.g., shotwell o.l., caldwell r.w., pathogenesis in aspergillus ear rot of maize: aflatoxin b1 levels in grains around wound inoculation sites. phytopatology, 80: 1283-1286, (1990). [4]. thiaw c., coly e., djiba s., diop m., ndoye o., cisse n., sembene m., senna occidentalis l., une plante prometteuse dans la lutte contre caryedon serratus ol. (coleoptera, bruchidae), insecte ravageur des stocks d’arachide au sénégal. international journal of biological and chemical sciences, 9 (3): 1399, (2015). [5]. anader (agence nationale de developpement rural)., la production vivrière : un enjeu national. bulletin de liaison de l’agence nationale de développement rural. bulletin de liaison de l’agence nationale de développement rural, no 14: 12p., (2009). [6]. sangare a., edmond k., fataye a., cheikh a.f., rapport national sur l’état des ressources phytogénétiques pour l’alimentation et l’agriculture. ministère de l’agriculture, republique de côte d’ivoire, (2009). [7]. tsigbey f.k., brandenburg r.l., clottey v.a., peanuts production methods in northern ghana and some disease perspectives, 9p, (2004). [8]. boli z.a., zoue l.t., alloueboraud w.a.m., kakou c.a. koffinevry r., proximate composition and mycological characterization of peanut butter sold in retail markets of abidjan (côte d’ivoire). journal of applied biosciences 72: 5822 29, (2003). [9]. who., stratégie mondiale de l’oms pour la salubrité des aliments: une alimentation à moindre risque pour une meilleure santé. programme pour la sécurité sanitaire des aliments 2002. organisation mondiale de la santé (oms), genève, suisse, (2002). [10]. guchi e., ayalew a., dejene m., ketema m., asalf b., fininsa, c., occurrence of aspergillus species in groundnut (arachis hypogaea l.) along the value chain in different agro-ecological zones of eastern ethiopia. journal of applied & environmental microbiology 2 (6): 309 3017, (2014). [11]. boli z.a., zoue l.t., alloueboraud w.a.m., kakou c.a. koffinevry r., proximate composition and mycological characterization of peanut butter sold in retail markets of abidjan (côte d’ivoire). journal of applied biosciences, 72 (1): 5822 29, (2014). [12]. kamika i., mngqawa p., rheeder j.p., teffo s.l., katerere d.r., mycological and aflatoxin contamination of peanuts sold at markets in kinshasa, democratic republic of congo, and pretoria, south africa. food additives & contaminants: part b, 7 (2): 120 26, (2015). [13]. efsa (european food safety authority), opinion of the scientific panel on contaminants in the food chain on a request from the commission related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and derived products. question n efsaq-2006-174. efsa journal, no. 446: 1 127, (2007). [14]. usaid (united states agency for international development), aflatoxine: une synthèse de la recherche en santé, agriculture et commerce. rapport final. 82p, (2012). [15]. elmore s.e., mitchell n., mays t., brown k., marroquin-cardona a., romoser a., phillips t.d., common african cooking processes do not affect the aflatoxin binding efficacy of refined calcium montmorillonite clay. food control, 37: 27 32, (2014). [16]. mutegi c., wagacha m., kimani j., otieno g., wanyama r., hell k., christie m.e., incidence of aflatoxin in peanuts (arachis hypogaea linnaeus) from markets in western, nyanza and nairobi provinces of kenya and related market traits. journal of stored products research, 52: 118 27, (2013). [17]. boli z.a., kakou c.a., toka d.m., koffi-nevry r., factors of medical risks related to the production and the sale of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 ama léthicia manizan, david akaki, isabelle piro-metayer, didier montet, catherine brabet, rose koffi-nevry, assessment of the risk factors associated to aflatoxin contamination during the marketing of peanuts in côte d'ivoire, food and environment safety, volume xvii, issue 2 – 2018, pag. 183 – 191 191 groundnut paste in the markets of the town of abidjan (côte d’ivoire). international journal of science and research, 5 (5): 2504 2508, (2016). [18]. broutin c., rouyat j., education qualifiante des jeunes et des adultes (eqja) etude sur les formations des femmes dans le domaine de [19]. l’agroalimentaire, groupe de recherche et d’échanges technologiques. contrat unesco – no. 803.065.4., 94p, (2004). [20]. demont m., système de commercialisation des produits vivriers en côte d’ivoire: étude pour le maïs (zea mays) et l’arachide (arachis hypogaea). document de travail no. 10 projet ‘renforcement des études agro-économiques à l’idessa’ idessa k.u.leuven, 79p, (1997). [21]. xu y., doel a., watson s., routledge m.n., elliott c.t., moore s.e., gong y.y., study of an educational hand sorting intervention for reducing aflatoxin b1 in groundnuts in rural gambia. journal of food protection, 80 (1): 44 49, (2017). [22]. kapseu c., production, analyse et applications des huiles végétales en afrique. oléagineux, corps gras, lipides, 16 (4-5-6): 215 29, (2009). [23]. n’dede c.b., jolly c.m., vodouhe s.d., jolly p. e., economic risks of aflatoxin contamination in marketing of peanut in benin. aflatoxins recent advances and future prospects. economics research international, 377395, (2013). awuah r.t., fialor s.c., binns a.d., kagochi j., jolly c.m., factors influencing market participant’s decision to sort groundnuts along the marketing chain in ghana. peanut science, 36 (1): 68 76, (2009). 160 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 3 2017, pag. 160 166 the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people antonella dorohovych 1 , *mykola petrenko 1 1 department of bakery and confectionery goods technology, national university of food technology volodymyrska str. 68, kyiv, ukraine, 01601, captainartyaderain@gmail.com *corresponding author received 22th august 2017, accepted 27th september 2017 abstract: the article reviews the potential rationale of the addition of inulin and soy proteins to harddough biscuits recommended for senior people’s diet. hard-dough biscuits were considered as the objects of this nutritional enrichment since they have an optimal balance of proteins, fats and carbohydrates compared to the rest of flour-baked foods. our study aimed to examine the role that inulin and protein play in the technological processes associated with producing hard-dough biscuits; in particular, special consideration was given to the selection of the optimal proportion of the new raw components, as well as the effect they cause on the quality of the hard-dough biscuits, the emulsion and the dough. the study identifies the role that inulin and soy protein isolate play in the heat treatment processes and storage of hard-dough biscuits intended for senior people. recommendations have been given as to the healthy diet for senior people.the authors have proved a significant effect of the new raw materials on the structural and mechanical characteristics of dough that assumes additional elasticity due to the increase in the volume of bound moisture in the hydrated flour. it has been established that the addition of inulin and soy isolate prolongs the duration of the heat treatment of hard-dough biscuits. by consuming 100 g of special-purpose hard-dough biscuits, it is possible to fill up to 30% of the daily need for fiber in humans. the study has proved the advisability of using soy protein isolate and inulin when producing special-purpose hard-dough biscuits for senior people because of its enrichment with complete proteins, irreplaceable amino acids and food fibers. keywords: functional foods, hard-dough biscuits, geriatric dietetics, soy protein, food fibers 1. introduction one of the most promising directions in the development of confectionery industry is the design of new functional food items that are capable of providing the necessary quantity of biologically active substances in the daily ration of a modern person. in the setting of the rising trend of diminished caloric value of food, the need for macroand micronutrients remains the same, since their lack can lead to weaker immunity, health deterioration and a poorer quality of life. among the products that are considered wholesome are functional foods that contain a high volume of biologically active components (antioxidants, vitamins, amino acids, minerals, food fibers), with a lower caloric value and low in sugar and fat. consumption of such products improves physiological bodily processes, strengthens immunity and the person’s overall wellbeing, as well as helps prevent chronic diseases [1]. when designing new functional products, a special focus should be placed on the requirements for the diets of certain http://www.fia.usv.ro/fiajournal mailto:captainartyaderain@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 161 categories of population. the nutrition of senior people is studied and advised by geriatric dietetics. below is some of such advice: 1. an energetically balanced diet aligned with the body’s actual energy demands. 2. nutrition aimed at treatment and prevention of diseases. 3. a ration well balanced in irreplaceable dietary factors. 4. intake of products that normalize intestinal microflora. 5. consumption of foods that are easily processed by food enzymes. 6. the diet protein/fat/carbohydrates ratio should be 1/0.9/3.8 [1]. one of the promising ways of making functional products is the enrichment of existing products in biologically active substances through the use of innovative raw materials [2]. among flour-baked foods, hard-dough biscuits make a good object for this enrichment, as of all other kinds of biscuits this one is characterized by the most balanced chemical composition of proteins, fats and carbohydrates, which makes it an appropriate foundation for designing healthy food recipes. the list of scientists who conducted research into the design of special-purpose bakery includes such prominent names as a. tumanova, a. dorokhovich, o. yaremenko, m. levachova, g. sevryukova, and others [3, 4, 5, 6, 7]. in particular, a vast range of research on baking hard-dough biscuits low in caloric value and sugar was conducted in 2008 by o. yaremenko [3]. these studies prove the practical sense of using food fibers and sugar substitutes in hard-dough biscuit recipes in order to impart them functional properties [4]. the studies also offer a deep analysis of the impact the new raw materials have on the processes of cooking, heat treatment and storage of biscuits. to improve the chemical formula of biscuits and align it with the prescriptions of geriatric dietetics, it was decided to enrich it in fiber and protein components, since these components are among the scarcest ones in today’s ration of senior people. the inulin has high degree of polymerization, allows it to stay undecomposed in the small intestine and carry out all the functions that are typical for decomposable food fibers the isolated soy protein was chosen as the source of complete protein. inulin is a straight chain of fructofuranose residues connected with β-glycoside links; it belongs to the group of polysaccharides. it has a neutral sweet taste without unpleasant aftertaste, poorly soluble in water. a regular intake of inulin can help lower sugar in blood, which is of great importance for people with diabetes [8]. consumption of inulin facilitates a whole range of functions in the human body, specifically lowers cholesterol in blood, stimulates intestines, reduces the risk of formation of malignant tumors in it, etc [9]. inulin is characterized by a low caloric value in the range of 1.0-1.5 kcal/g, which allows using it in functional recipes. soy isolate is a source of complete protein, high in irreplaceable amino acids [10]. soy isolates contain over 92% of protein, 2.5% of carbohydrates, 0.5% of fat, 0.5% of raw fiber and 4.5% of ash, they have a low water content. they are produced through the chemical extraction of protein from defatted protein meal. food cooked from soy isolates is especially rich in protein and low in fat, calories, cholesterol. additionally, isolated soy proteins are much better digested than the proteins contained in soy flour. the protein isolate as a finished product is a white amphoteric powder without distinct taste or smell, which allows adding it to biscuit recipes without changing their organoleptic parameters and makes it a cheap and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 162 practicable protein-enrichment substance for functional food products [11]. the study aims at designing a recipe of hard-dough biscuits according to the current recommendations of geriatric dietetics as to the diet of senior people and identifying the way the new raw materials (inulin and soy isolate) affect the quality of hard-dough biscuits, emulsion and the dough used for their production, as well as the heat treatment and storage of those biscuits. 2. matherials and methods the study examined the following objects: hard-dough biscuits, semi-finished products (dough, emulsion) and the raw ingredients for their recipes. first-grade wheat flour was used as a baseline raw ingredient, and other ingredients included powdered inulin and soy protein isolate and other components present in the harddough biscuits recipe. the water content of the biscuits, semi-finished products and raw materials was being controlled using a sesh-3 drying oven. the gluten strength of the dough was measured with a gluten deformation measuring device idk-2. the structural and mechanical performance of the dough was measured using the strukturometer st-1 device and a brabender farinograph [12]. the waterbinding modes in the examined samples were identified through a thermogravimetric analysis using the derivatograph q-1500d device, which involved measuring the speed of water removal and the values of heat effects [13]. the sorption and desorption properties of hard dough and biscuits made of it were examined using a macben sorption pump [14]. 3. results and discussion an important stage of designing a new variety of hard-dough biscuits consisted in determining an optimal quality of novel raw components in the recipe that would ensure that the resulting biscuits would have organoleptic properties no worse than those of customary biscuits. three biscuit samples were studied: the first sample was a reference one, with the second sample inulin was added in the dough, and the third sample was cooked with soy protein isolate. the quality of the biscuits was evaluated based on their organoleptic properties in compliance with dstu 3781–14 [15].the biscuits were cooked according to a traditional technology in laboratory environment [16, 17]. the examined samples of biscuits cooked of the new ingredients are of comparatively bigger volume, a homogeneous structure when broken in parts, a higher density and a better taste. the optimal quality of the biscuits can be observed when the proportion of inulin is 7% and of protein isolate – 12% of the dough weight. the effect that the raw materials cause on the structural and mechanical properties of hard dough was established through measuring its boundary shear and adhesion value. research has shown that the changes in the adhesion of hard dough are insignificant, whereas both inulin and soy isolate notably reinforce the dough structure, due to which the boundary shear of hard dough increases by 9% and 14% correspondingly. this can be explained by a greater content of bound liquid in the dough attributed to the active processes of hydration initiated by inulin and soy isolate whose water absorption capabilities are much higher than those of flour. the quality of the finished hard-dough biscuits is mostly determined by the structural and mechanical properties of the dough they are made of. dough for the hard-dough variety of biscuits should have an elastic, springy structure and retain it during the kneading, rolling and forming of dough balls. that elastic and springy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 163 structure is attained thanks to the gluten frame of the dough that forms as a result of the hydration of gluten-forming proteins. to establish how inulin and soy isolate affect the properties of the gluten frame of hard dough, the authors tested (see table 1) the dough samples prepared according to recipes no. 1, 2 and 3 respectively, with inulin and isolated soy protein added to the dough.having evaluated the obtained data, it is possible to argue that adding inulin and soy protein isolate to the dough slackens the gluten complex of hard dough; specifically, it reduces gluten stretchability and strength. the introduction of the new ingredients also results in a reduced content of raw gluten and a significant decrease in its hydration capacity (by 8% and 22% respectively), which indicates the reduced volume of free water in the dough and, obviously, can be attributed to the binding of free water by inulin and proteins added to the dough. table 1. characteristics of the gluten complex of hard-dough biscuits characteristics sample no. #1 reference #2 with inulin #3 with inulin and protein isolate gluten content, % of flour weight 25.3 21.3 19.0 hydration capacity, % 181.0 173.0 159.0 stretchability, cm 15 13 12 strength idk-2, device units 72.0 81.0 75.0 the dough test conducted using a farinograph (table 2) verifies the assumptions put forward above as to the effect of the new raw materials on the dough structure. the dough containing inulin and soy isolate is 18.6% higher in its water absorption capacity and has lower stretchability, and that is a sign of a diminished volume of free water in the system and slower structure building processes, which is indicated by the double increase in the time needed for the dough to develop. the water content in dough was measured using thermogravimetric analysis. the analysis was carried out in the temperature range of 20-200°с using the derivatograph q-1500d device. table 2. results of farinogram analysis characteristics sample no. #1 reference #2 with inulin #3 with inulin and protein isolate water absorption capacity, cm3/100 g 58.6-55.6 44.8-43.3 71.0-69.5 dough development time, min 1.5 1.5 3.0 stability, min 0.0 0.0 0.0 dough softening, device units 120 140 120 elasticity, device units 120 130 105 the test was applied to dough samples with water content of 27.0%. derivatograms were analyzed according to the commonly accepted food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 164 methodology, with results given in table 3. table 3. results of derivatographic analysis characteristic sample no. #1 reference #2 with inulin #3 with inulin and protein isolate total water content, % 27.0 27.0 27.0 free water content, % 59.6 57.1 53.2 bound water, % 40.4 42.9 46.8 activation energy, kj/mole 5.32 6.76 8.93 the derivatographic analysis shows that the addition of inulin and soy isolate significantly affects the free to bound water ratio in hard dough. the addition of inulin to hard dough increases the volume of bound water by 2.5%, while the addition of soy protein isolate results in an increase of 6.4%. this is accompanied by the increase in the value of activation energy in the samples different from the reference one, which also indicates that when added to the recipe, the new raw ingredients generate stronger forms of bound water in the dough. such notable increase in the volume of bound liquid can be explained by the presence of stronger forms of water binding in the samples different from the reference one. this is accompanied by the increase in the value of activation energy in the samples different from the reference one, which also indicates the emergence of stronger forms of bound water in the dough and that they require more energy to be broken down. the increase in the amount of water bound in the dough will affect the duration of the heat treatment of hard-dough biscuits and will require correcting the baking time and temperature for the new hard-dough biscuits. besides, the reduced content of free water will slow down the hardening of the hard-dough biscuits and positively affect its shelf life during storage. however, the shelf life and storage conditions of the biscuits are determined not only by the amount of bound water, but also by the intensity of sorption and desorption processes that take place in the course of storage, so the authors examined the aforementioned sorption and desorption processes using a macben sorption pump for the dough (fig. 1 and fig. 2) and for the finished hard-dough biscuits (fig. 3). judging from the moisture adsorption graph, it is possible to say that the samples were adsorbing liquid quite slowly up to the pressure of p/ps=0.35, because the pores inside them were filled before the start of the adsorption and did not clear in time. this is also testified by the practically missing surface, as closed pores do not take in liquid; however later on they got more active, since the sample was being diluted under the pressure of liquid and evaporated water was being absorbed, for the pores in the samples were quite large. sample #2 demonstrated the lowest moisture sorption capacity, while sample #1 – the highest one. the hysteretic loop was also wide in the case of sample #2, which indicates that having sucked in the water, the sample took a while to dry up to its initial state at the same temperature, and did not release all its water because its adsorption capacity at zero pressure is higher than the initial one. the greatest number of the smallest pores was registered in the sample #1, which is evident when looking at the pore radius distribution graph (fig. 2); the peak sizes of the pores of the samples #2 and #3 are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 165 almost identical — 16.8 and 16.2 å (1.68 and 1.62 nm), whereas the sample #1 has slightly narrower pores of 13.2 å (1.32 nm). fig. 1. sorption/desorption isotherms for the hard dough used to cook the biscuits: 1 – reference sample, 2 – sample with inulin, 3 – sample with inulin and soy protein isolate fig. 2. pore radius distribution graphs for the hard-dough biscuit sample: 1 – reference sample, 2 – sample with inulin, 3 – sample with inulin and soy protein isolate fig. 3. sorption/desorption isotherms for the hard dough used to cook the biscuits: reference sample fig. 4. sorption/desorption isotherms for the hard dough used to cook the biscuits: sample with inulin and soy protein isolate the calculation of the adsorption energy based on the obtained data shows that for the sample #1 it is equal to 4.818 kj/mole, for the sample #2 – 4.869 kj/mole, and for the sample #3 – 4.419 kj/mole. following the analysis of the hard-dough biscuits sorption isotherms (fig. 3), it was established that the equilibrium moisture values for the reference sample and the sample with inulin and soy protein isolate in the range of aw = 0.75…0.85 are equal to 12.0–12.5%, which in turns implies that the effect of the new raw ingredients on the sorption capacity of the biscuits is insignificant. yet for hard-dough biscuits that are capable of actively absorbing moisture during storage, high sorption capacity would negatively affect the quality of the product and its shelf life, so we recommend packing the finished biscuits in vacuum package. 4. conclusion the study has shown that, provided they are used in optimal proportions, inulin and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 3 – 2017 antonella dorohovych, mykola petrenko, the use of inulin and soy protein isolate in producing hard-dough biscuits for senior people, food and environment safety, volume xvi, issue 3 – 2017, pag. 160 166 166 soy protein isolate do not cause any negative effect on the technological characteristics of the dough and the finished biscuits, which means they can be produced using the existing machinery and equipment. the novel hard-dough biscuits have the closest to optimal balance of proteins, fats and carbohydrates compared with ordinary hard-dough biscuits chosen as a reference product. since neither inulin nor soy protein isolate have distinct taste or flavor, the organoleptic properties of the finished biscuits are identical to the ordinary hard-dough biscuits. the caloric value of the hard-dough biscuits with soy protein isolate and inulin is 389 kcal/100 g; in addition, it has a higher nutritional and biological value and satisfies 30% of human daily requirement for fiber and 15% of daily requirement for proteins (the content of essential amino acids in 100 g: isoleucine – 0,75 g, leicine – 1,16 g, lizin – 0,65 g, methіonіne+cystine – 0,36 g, phenylalanіne+tyrosine – 0,97 g, treonine – 0,55 g, tryptophan – 0,21 g, valin – 0,75 g), which makes it reasonable to label these biscuits as a functional product. the chemical formula of these biscuits also takes into account the prescriptions of geriatric dietetics, so these biscuits are recommended for consumption by senior people. also, due to the absence of white granulated sugar in the recipe of the biscuits, the finished product will also be suitable for consumption by people suffering from diabetes. 5. references [1].kharchenko n. dietology: textbook, kyiv, meridian: 526, (2012) [2].ostrik a., dorohovich a. use of nontraditional raw materials in the confectionery industry: handbook, kiev, urozhaj: 112, (1989) [3].yaremenko o., dorohovich a. development of innovative technologies of sugar,bold and long-lasting low-calorie biscuits, strategic directions of development of enterprises of food production, restaurant industry and trade: international. sci. pract. conf., kharkiv: 56-57, (2008) [4].yaremenko o., dorohovich a. development of sugar, bacon, prolonged biscuits with low glycemic index, proceedings of the national university of food technologies, ukraine, №25: 84-86, (2008) [5].tumanova a. scientific bases of creation of confectionery products of functional purpose, proceedings of the universities. food technology, kuban state technological university, №5-6: 4850, (2003) [6].levachova m., shubina o., kochetkova a. use of polydextrose and lactitum in sugar cookies, bread products, moskow, №11: 30-33 (2006) [7]. sevryukova g., katayeva a. new in the technology of production of long-lasting cookie varieties, baking and confectionery industry, moskow, no. 2: 24-25 (1992) [8]. polumbrik m. carbohydrates in food products and human health, kyiv, academiperiodica: 487, (2011) [9]. roberfroid m. inulin-type fructans: functional food ingredients, crc press: boka ration: 392, (2005) [10]. mahinko v., chernich l. high-protein additions in bakery, storage and processing of grain, ukraine, №6: 57-60, (2014) [11]. kaprelyants l. functional foods, odessa, print: 312, (2003) [12]. drobot v., yurchak v., bilyk a. chemical control of raw bakery and pasta: teach, nat. university of food. techn, kyiv, condor: 172, (2015) [13]. pimenova l. thermography. methodical instructions for laboratory work, tomsk, tgsau: 19, (2005) [14]. al muhtaseb a., magee t. moisture sorption isotherm characteristics of food products: a review, transaction of the institytion of chemical engineers, №80: 118-128, (2002) [15]. dstu 3781–2014, biscuit. general technical conditions, kyiv, state committee of ukraine, 16, (2014) [16]. sokolovsky a. confectionery production technology, moscow, pyshcheprom, 710, (1959) [17]. manley d. technology of biscuits, crackers and cookies, elsevier, 632, (2011). 1. introduction 332 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 -2018 , pag. 332 340 chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda) aderonke o. lawal-are 1 ,*olatunji r. moruf 1 , damilola a. junaid 1 , moruf o. oke 2 1shellfish research laboratory, department of marine sciences, university of lagos, akoka, nigeria 2department of food science and engineering, ladoke akintola university of technology, ogbomoso, nigeria tunjimoruf@gmail.com *corresponding author received 5th june 2018, accepted 19th september 2018 abstract cuttlefish is one of the most economically important cephalopods with rich taste and few inedible parts. the study was aimed at evaluating the nutrient, non-nutrients, free fatty acid and cholesterol compositions and functional properties of raw and processed sepia officinalis using standard methods. moisture and raw protein in raw sample of the cuttlefish were significantly higher (𝑃< 0.05) than in processed samples. raw fat showed significant differences with fried sample having highest value (22.39±0.54 %) to raw (16.24±0.10 %) and boiled samples (4.09±1.42 %). raw sample contained more ash with highest mg/100g levels of calcium, phosphorus, magnesium, iron and manganese. processed cuttlefish registered significant reduction in levels of trypsin inhibitor, phytate and oxalate compared to the raw. raw protein of fried cuttlefish exhibited positive relationships with all the non-nutritional factors while total ash only showed positive relationships with phytate (𝑟 = 0.998) and trypsin inhibitor (𝑟 = 0.999). cholesterol content of raw cuttlefish was of 0.923±0.02 mg/100g being reduced by boiling (0.711±0.02 mg/100g) and increased by frying (1.037±0.02 mg/100g). a significantly high percentage of oleic was observed in fried cuttlefish (4.47±0.10) while boiled sample had the lowest value (1.4±0.05). the results showed significantly high percentage of water and oil absorbing capacities for raw cuttlefish while foam and emulsion stabilities were higher in the boiled sample. by frying, the highest foam and emulsion capacities of 8.8 and 4.4% respectively were obtained. the study suggests that sepia officinalis will be highly desirable for preparing comminuted sausage products due to its good functional and nutritional properties. keywords: nutritional, mollusc, shellfish, nigeria 1. introduction sepia officinalis (cephalopoda) is one of the most important demersal marine invertebrates inhabiting the continental shelf. global annual catch of cuttlefish ranged from 11,000 to 15,000 tons [1] where mediterranean countries are the main exporters of frozen cuttlefish in the world. the connective tissue of cephalopods is highly developed compared to fish in general [2-3]. cuttlefish is rich in taste and has few inedible parts. as fat content of fishes varies with species, age, size and season, cuttlefish contains low level of lipid; however, omega-3 polyunsaturated fatty acid presented the majority of the total lipid [4, 2]. the fat content of raw fishes can also influence fat exchanges and interactions between the http://www.fia.usv.ro/fiajournal mailto:tunjimoruf@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 333 culinary fat and that of the fish during processing [5]. according to okpanachi et al.[5], various processing methods (boiling, frying, smoking, roasting etc) used in the preparation of raw fish for consumption could have varying effects on the fish nutrient, texture and flavor. nutrient chemical bio-compounds are traditionally used as nutritional indicators of fish. the proximate composition and mineral profiles of fish are determined largely by several factors e.g species, sex, size, maturity, feeding regimes and season [6-7]. information on daily dietary intake of nutrients, especially cholesterol, is quite important for especially those with cardiovascular problems [8]. the nutritional composition of cuttlefish has been reported by some researchers [9, 10, 2]. however, this composition could vary among species due to geographical differences of fishing grounds [11]. in addition, compositions can also vary with processing methods. data on the macronutrient content of cuttlefish is only available for raw sepia spp and there seems to be a scarcity of information on the processed ones. cuttlefish was chosen because they are readily available, cheap, affordable and within the reach of an average nigerians. this work is thus a preliminary investigation on the proximate composition, mineral, anti-nutrient, cholesterol contents and functional properties (absorbing, foaming and emulsion capacities) of raw, boiled and fried cuttlefish, sepia officinalis commonly retailed in nigeria with a view to showcase the nutritive significance for human health benefit. 2. materials and methods 2.1 sample preparation the cuttlefish samples (fig. 1) used in this study were obtained from ajeloro fish market in apapa, lagos state-nigeria. the fishes were thoroughly washed, cut into pieces (50 g) and washed again with distilled water. the head region was discarded. the samples were then separated into three parts, one part was analyzed raw; a second part was boiled in water while the third part was deep-fried with vegetable oil in a frying pan. boiling was done in distilled water, kept boiling for about 20 minutes until the pieces were cooked and tender. frying was achieved within 15 minutes and the temperature was about 240°c. all processing methods followed the usual procedures used to prepare fish for table consumption in nigeria but without the addition of any ingredient. all samples were homogenized prior to analysis. fig. 1: dorsal view of the cuttlefish, sepia officinalis (mollusca: cephalopoda) 2.2 analytical procedures proximate analysis was carried out according to standard procedure [12]. for the moisture content, fish muscle duplicate samples were kept in an oven, at 102105°c for 24 h. the ash content was determined by incineration of 5 g of the sample at 600°c for 8 hours. the determination of crude fat content was conducted by soxhlet extraction method using n-hexane as solvent. the n-content was multiplied by 6.25 to estimate the protein of the samples. the content of total carbohydrates was determined by difference according to the formula below: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 334 100 – [% water + % proteins + % lipids + % ashes]. the proximate compositions were measured in dry matter basis. mineral components were evaluated from solution obtained by first wet-ashing the samples and dissolving the ash with deionized water and concentrated hydrochloride acid in standard flask. the solution was analyzed for minerals content using atomic absorption spectrophotometer. phosphorus was analyzed for by employing the method reported by vanado molybate and read on colorimeter [12]. the estimation of anti-nutritional contents was by the colorimetric procedure as modified by [13]. all determinations were done in triplicate. cholesterol content was estimated using liebermann-burchard reagent. standard cholesterol solution used was 0.4 mg/ml. liebermann-burchard reagent was prepared with 0.2 ml concentrated sulfuric acid and 2 ml glacial acetic acid and was covered with aluminum foil. fatty acid of fat extracted from samples was determined by gas chromatography (gc) of methyl esters. methyl esters were prepared by transmethylation using 2 m koh in methanol and n-heptane according to the method described by aoac with minor modifications [12]. the modified methods reported by souissi et al. [14] were used to determine the oil absorbing, water absorbing, emulsion and emulsion stability. the foam formation and the foam stability were determined by optical measurements. the foams were produced with a homogenizer for 2 min at 17 500 rpm, in 3 ml of solution (50 mmtris–hcl – 0.5 m nacl, ph 7.5), which contained 1.5% protein. the initial height of the solution and the foam height were recorded at intervals of 0, 2, 10, 20 and 30 min, using a caliper. the foaming capacity17 was expressed as the proportion of foam height at 0 min to solution height. the foaming stability (fs) was conveyed by the percentage of foam height at some time to 0 min. the measurement of the height was rapid and accurate to three digits after the decimal point. 2.3 statistical analysis data obtained was subjected to analysis of variance (anova), pearson correlation coefficient and where there was significant difference at p≤0.05, duncan multiple range test (dmrt) was used to sort out the differences in the means. 3. results 3.1 proximate composition the moisture and crude protein contents for raw sample of cuttlefish were significantly higher (𝑃< 0.05) than the processed samples (table 1). also, the raw fat showed significant differences between the samples with the fried sample (22.39±0.54%) having significantly higher values than the raw (16.24±0.10%) and boiled samples (4.09±1.42%). raw sample contained more ash compared to the processed form. raw fiber was not detected in the cuttlefish; raw or processed. 3.2 mineral content raw sample of cuttlefish has the highest mg/100g levels of calcium, phosphorus, magnesium, iron and manganese as follows: 342.16±3.99, 5.75±0.04, 368.81±4.21, 1.22±0.03 and 4.07±0.02 respectively. with the exception of calcium, frying had a significant increase in mineral level as compared to boiling (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 335 table 1 proximate and mineral compositions of raw and processed cuttlefish (sepia officinalis) composition raw boiled fried % moisture 73.33±0.68 a 68.00±0.42 b 60.19±0.67 c % raw protein 78.55±0.76 a 75.96±0.62 b 73.7±0.94 c % raw fat 16.24±0.10 b 4.09±1.42 c 22.39±0.54 a % raw fibre nd nd nd % total ash 3.35±0.09 a 3.18±0.04 b 2.12±0.01 c % carbohydrate 1.86±0.03 b 16.77±0.04 a 1.79±0.20 c calcium (mg/100g) 342.16±3.99 a 321.05±2.85 b 319.76±1.09 phosphorus (mg/100g) 5.75±0.04 a 5.11±0.01 c 5.35±0.08 b magnesium (mg/100g) 368.81±4.21 a 306.74±1.92 c 325.67±3.31 b iron (mg/100g) 1.22±0.03 a 0.94±0.02 c 0.97±0.02 b manganese (mg/100g) 4.07±0.02 a 3.12±0.21 c 3.95±0.02 b 3.3 non-nutrients the non-nutrient content (oxalate, phytate and trypsin inhibitor) was significantly (𝑃< 0.05) higher in raw sample compared to the processed sample (fig.2). in this work, frying was found to have greater efficiency in the elimination of the levels of oxylate and phytate available in the cuttlefish than boiling which seemed to eliminate trypsin inhibitor more efficiently when compared with frying. fig.2: non-nutrient levels in raw and processed cuttlefish (sepia officinalis) samples 3.4 correlation analyses the correlation matrix showed some important relationships between the chemical bio-components analyzed. table 2 showed the correlation matrix for raw cuttlefish in which case there were approximately perfect positive relationships between crude protein and phytate (𝑟 = 0.967) and between crude protein and trypsin inhibitor (𝑟 = 0.992). similar relationship was exhibited by total ash with phytate (𝑟 = 0.995) and trypsin inhibitor (𝑟 = 0.974) with significance at the 0.05 level. oxalate showed negative relationship with all the proximate composition except with carbohydrate (𝑟 = 0 10 20 30 raw boiled fried 25.02 21.35 20.27 5.25 3.56 3.4 10.25 7.62 9.71 oxalate phytate trypsin inhibitor food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 336 0.942). for boiled cuttlefish sample, only crude fat showed negative relationship with the non-nutritional factors; oxalate (𝑟 = -0.607), phytate (𝑟 = -0.006) and trypsin inhibitor (𝑟 = -0.356) (table 3). the crude protein of fried cuttlefish exhibited positive relationships with all the nonnutritional factors while total ash only showed positive relationships with phytate (𝑟 = 0.998) and trypsin inhibitor (𝑟 = 0.999) at significance of 0.05 levels (table 4). table 2 correlations for raw cuttlefish (sepia officinalis) sample moisture raw protein raw fat total ash carbohydrate oxalate phytate trypsin inhibitor moisture 1 raw protein 0.999 1 raw fat 0.919 0.923 1 total ash 0.937 0.939 0.999 1 carbohydrate -0.064 -0.058 0.335 0.289 1 oxalate -0.395 -0.389 -0.001 -0.048 0.943 1 phytate 0.966 0.967 0.989 0.995 0.197 -0.148 1 trypsin inhibitor 0.991 0.992 0.963 0.975 0.067 -0.272 0.992 1 table 3 correlations for boiled cuttlefish (sepia officinalis) sample moisture raw protein raw fat total ash carbohydrate oxalate phytate trypsin inhibitor moisture 1 raw protein 0.196 1 raw fat -0.975 -0.409 1 total ash 0.521 0.939 -0.697 1 carbohydrate 0.583 0.911 -0.749 0.997 1 oxalate 0.415 0.974 -0.607 0.993 0.982 1 phytate 0.217 0.914 -0.006 0.721 0.667 0.799 1 trypsin inhibitor 0.140 0.998 -0.356 0.918 0.886 0.959 0.936 1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 337 table 4 correlations for fried cuttlefish (sepia officinalis) sample moisture raw protein raw fat total ash carbohydrate oxalate phytate trypsin inhibitor moisture 1 raw protein 0.867 1 raw fat 0.801 0.396 1 total ash 0.124 0.602 -0.495 1 carbohydrate 0.806 0.403 0.999 -0.488 1 oxalate 0.500 0.001 0.919 -0.798 0.916 1 phytate 0.182 0.649 -0.443 0.998 -0.435 -0.760 1 trypsin inhibitor 0.169 0.638 -0.455 0.999 -0.447 -0.770 0.999 1 3.5 cholesterol and oleic contents the cholesterol content of raw cuttlefish was 0.923±0.02mg/100g, being reduced by boiling (0.711±0.02 mg/100g), but increased by frying (1.037±0.02 mg/100g). a significantly high percentage of oleic was observed in fried cuttlefish sample (4.47±0.10 %), while the boiled sample had the lowest value (1.4±0.05 %)(table 5). table 5 cholesterol and free fatty acid contents of raw and processed cuttlefish (sepia officinalis) raw boiled fried p-value cholesterol (mg/100g) 0.923±0.02 0.711±0.02 1.037±0.02 0.00* ffa as oleic (% ) 3.95±0.10 1.4±0.05 4.47±0.10 0.00* 3.5 functional properties the result as shown in the fig.3 revealed significantly high percentage of water and oil absorbing capacities of raw cuttlefish sample, while foam and emulsion stabilities were the highest in the boiled sample. by frying, the highest foam and emulsion capacities with 8.8 and 4.4% respectively were obtained. fig. 3: functional properties of raw and processed cuttlefish (sepia officinalis) 0 100 200 300 water absorbing capacity oil absorbing capacity foam capacity foam stability emulsion stability @3hrs emulsion capacity (g/ml) p e rc e n ta g e l e v e l functional property raw boiled fried food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 338 4. discussion 4.1 proximate and mineral contents the nutritional elements showed variable values in the cuttlefish analyzed, raw protein recording the highest values both in raw and processed forms. the significant decrease in raw protein levels (p < 0.05) in boiled and fried, when compared with the raw sample, is in agreement with the report of okpanachi et al.[5] and this suggested that the protein was leached and denaturized in the boiling and frying respectively. as observed from the results, frying decreases the moisture content that result in desirable non-enzymatic browning reactions, but increases the raw fat content of the cuttlefish. fat contents are vital in the structural and biological functioning of cells and they help in the transportation of nutritionally essential fat soluble vitamin [15]. ash, a measure of the mineral content of food item indicates that the cuttlefish is a good source of minerals. the amount of carbohydrate obtained in this study was higher than the amount observed in the report of [2] on sepia recurvirostra which might be due to genetic factors as well as extrinsic factors such as feeding regimes and/or exercise significant change in some structural and flesh quality parameters of the two cuttlefish species. according to [16], cephalopods living in hypo-osmotic environment absorb minerals using digestive gland as they swallow massive quantities of sea water during and after feeding. raw sample of the cuttlefish recorded the highest values for all the measured minerals while boiled sample recorded the lowest with the exception of calcium which lowest value was observed in fried sample. in general, there were significant influences of boiling and frying on the proximate and mineral compositions of the cuttlefish. 4.2 non-nutritional content non-nutritional factors are generally toxic and may negatively affect the nutrient value of fish by impairing protein digestibility and mineral availability. however, they are heat labile and hence may be inactivated by processing methods involving heat generation [17]. the lower values of phytate and oxalate suggest that the nutritive value of cuttlefish would be impaired to a comparatively lesser extent. nwosu[18]indicated a significant reduction in phytate and trypsin inhibitor contents following cooking which is in agreement with this research. generally, processing reduced the level of all the non-nutrients analyzed to their permissible levels. 4.3 correlation the correlation analyses for raw and processed samples suggest that phytate and trypsin inhibitor will negatively affect the raw protein and mineral level (ash), making these nutrients unavailable. in the fried cuttlefish sample, oxalate has no significant effect on mineral level (ash) which is contrary to the report of [19] that suggests that oxalate forms complexes with minerals. according to ndidi et al. [17], although processing reduces nonnutrient level to permissible limits, however some quantity of the nutrients could still be affected. this may offer some explanations why processing does not increase, in some cases, the level of nutrients. 4.4 oleic acid and cholesterol contents significant differences in oleic acid and cholesterol content were observed among raw and processed samples (p < 0.05). similar observation was reported by ozogul et al.[20] on frying process significantly reducing the sterol content of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 339 some selected mollusc except for common cuttlefish. several studies have shown that cephalopod lipid profiles display high concentration of phospholipids, cholesterol and polyunsaturated fatty acids. [21] reported a content of cholesterol in several cephalopods such as 123 mg/100 g in cuttlefish, 180 mg/100 g in squid and 139 mg/100g in octopus. 4.5 functional properties the cuttlefish (sepia officinalis) has a low value of water absorbing capacity in both the raw and processed samples as compared to that reported by [22] on clarias lazera while oil absorbing capacity is similar to the report of [23] on gymnarcus niloticus. water absorbing capacity reflects the extent of denaturation of the protein while oil absorbing capacity acts as flavor retainer and improves the mouth feel of food. foam formation and stability are a function of the type of protein, ph, processing method, viscosity and surface tension [15]. [24] reported that foams are used to improve the texture, consistency and appearance of foods. fried sample of the cuttlefish has the highest values of foam and emulsion capacities while boiling received the highest values for foam and emulsion stabilities. the cuttlefish sample in the present study has low values of emulsion capacity and emulsion stability in comparison with clarias lazera[22] but higher than the values reported for some selected sea foods [25]. this suggests that cuttlefish would be highly desirable for preparing comminuted sausage products. 5. conclusion different nutritional components of cuttlefish undergo different changes at elevated temperatures. processing drastically reduced the level of nonnutrients in cuttlefish with minimal effect on the nutritional quality. therefore, in view of the nutrient availability, low nonnutritional content, and the quality of functional properties after processing, the consumption of sepia officinalis could help to combat malnutrition in developing countries. further work should be geared toward essential and non-essential amino acid analyses and organoleptic property of this species of cuttlefish for knowledge of its complete nutritional benefit. 6. acknowledgement the second author is grateful to mrs. oluboba t.f of the nigerian institute for oceanography and marine research, lagos, nigeria for the cuttlefish image used in this paper. 7. references [1]. sreeja v., bijukumar a., cephalopod resources of india: diversity, status and utilization. science chronicle, 5(10):4492-4497, (2013). [2]. nurjanah a.m., jacoeb r.n., suhana s.n., siti k., proximate, nutrient and mineral composition of cuttlefish (sepia recurvirostra). advance journal of food science and technology, 4(4): 220-224, (2012). [3]. moruf r.o., lawal-are a.o., growth pattern, whorl and girth relationship of the periwinkle, tympanotonus fuscatus var radula (linnaeus, 1758) from a tropical estuarine lagoon, lagos, nigeria. international journal of fisheries and aquatic studies, 3(1): 111-115, (2015). [4]. thanonkaew a., benjakul s., visessanguan w., chemical composition and thermal property of cuttlefish (sepia pharaonis) muscle. j. food compos. anal, 19(2-3): 127-133, (2006). [5]. okpanachi m.a., yaro c.a., bello, o.z., assessment of the effect of processing methods on the proximate composition of trachurus trachurus (mackerel) sold in anyigba food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 aderonke o. lawal-are, olatunji r. moruf, damilola a. junaid, moruf o. oke, chemical bio-compounds and functional properties of raw and processed cuttlefish, sepia officinalis (mollusca: cephalopoda), food and environment safety, volume xvii, issue 3 – 218 , pag. 332 – 340 340 market, kogi state. american journal of food science and technology, 6 (1): 26-32, (2018). [6]. richard k., prithiviraj n., andrews a., ayisi c.l., xiaojie d., biochemical composition of predatory carp (chanodichthys erythropterus) from lake dianshan, shanghai, china. egyptian journal of basic and applied sciences, 4: 297–302, (2017). [7]. akinjogunla v.f., lawal-are a.o., soyinka o.o., proximate composition and mineral contents of mangrove oyster (crassostrea gasar) from lagos lagoon, lagos, nigeria. nigerian journal of fisheries and aquaculture, 5 (2): 36 – 49, (2017). [8]. periyasamy n., srinivasan m., devanathan k., balakrishnan s., nutritional value of gastropod babylonia spirata (linnaeus, 1758) from thazhanguda, southeast coast of india. asian pac j trop biomed, 2: 49–52, (2011). [9]. villanueva r., riba j., capillas c.r., gonzalez a.v., baeta m., amino acid composition of early stages of cephalopods and effect of amino acid dietary treatments on octopus vulgaris paralarvae. aquaculture, 242(14): 455478, (2004). [10]. ozyurt g., duysak o., akama e., tureli c., seasonal change of fatty acids of cuttlefish sepia officinalis l. (mollusca: cephalopoda) in the northeastern mediterranean sea. food chem, 95(3): 382-385, (2006). [11]. moruf r.o., adekoya k.o., molluscan and crustacean genetic and biotechnology interventions: a review. animal research international, 15(1): 2906 – 2917, (2018). [12]. aoac (official methods of analysis of the association of official analytical chemists) international 17 th ed. published by the association of official analytical chemists international, suite 4002200 wilson boulevard, arlington, virginia usa .pp. 22201-3301, (2000). [13]. enujiugha v.n., olagundoye t.v., comparative nutritional characteristics of raw fermented and roasted african oil bean (pentaclethra macrophylla benth) seed. revista italiana delle sostanze grasse, 78: 247250, (2001). [14]. souissi n., bougatef a., trikiellouz y., nasri m., biochemical and functional properties of sardinella (sardinella aurita) by-product hydrolysates. food technol. biotechnol., 45 (2): 187–194, (2007). [15]. omotosho o.t., chemical composition and nutritive significance of the land crab, cardiosoma armatum (decapoda). african journal of applied zoology and environmental biology, 7: 68-72, (2005). [16]. lourenco h.m., anacleto p., afonso c., ferraria v., martins m.f. carvalho m.l. lino a.r., nunes m.l. elemental composition of cephalopods from portuguese continental waters. j. food chem., 113(4): 1146-1153, (2009). [17]. ndidi u.s., ndidi c.u., olagunju a., muhammad a., billy f.g., okpe o., proximate, antinutrients and mineral composition of raw and processed (boiled and roasted) sphenostylis stenocarpa seeds from southern kaduna, northwest nigeria. isrn nutrition, 10: 19, (2014). [18]. nwosu j. n., effects of soaking, blanching and cooking on the antinutritional properties of asparagus bean (vigna sesquipedalis) flour. nature and science, 8(8): 163– 167, (2010). [19]. adegunwa m. o., adebowale a. a., solano e. o., effect of thermal processing on the biochemical composition, antinutritional factors and functional properties of beniseed (sesamum indicum) flour. american journal of biochemistry and molecular biology, 2(3): 175–182, (2012). [20]. ozogul f., kuley e., ozogul y., sterol content of fish, crustacea and mollusc: effects of cooking methods. international journal of food properties,18: 2026–2041, (2015). [21]. okozumi m., fujii t., nutritional and functional properties of squid and cuttle fish. japan: national cooperate association of squid processors, 11(2000). [22]. aremu m.o., ekenude o.e., nutritional evaluation and fuctional properties of clarias lazera (african catfish) from river tamma in nasarawa state, nigeria. american journal of food technology, 3(4): 264274, (2008). [23]. adeyeye e.i., adamu a.s., chemical composition and food properties of gymnarchus niloticus (trunk fish). biosci.biotech. res. asia,3: 265-272, (2005). [24]. akubor p.i., chukwu j.k., proximate and selected functional properties of fermented and unfermented african oil bean (pentaclethra macrophylla) seed flour.plant food for human nutrition, 54: 227238, (1999). [25]. ogundele i.o., olaofe o., fadare i., chemical composition, amino acids and nutritional properties of selected sea foods. journal of food, agriculture and environment,3(2): 130-133, (2005). 391 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 4 2015, pag. 391 398 wheat fibre impact on flour quality of confectionery products *olena kobets1, viktor dotsenko1, oksana arpul1, iryna dovgun1 hotel-restaurant and tourism business faculty, national university of food technologies, ukraine elenka-021991@mail.ru *corresponding author received december 7th 2015, accepted december 29th 2015 abstract: the article reviews the current state and prospects of the development of products enriched by dietary fibres, including wheat fibre, and their importance to the public. the importance of the dietary fibres for people suffering from diabetes and cardiovascular disease is emphasized. the features of the chemical and fractional composition of wheat fibre are described. the impact of wheat fibre and emulsifier mixture on wheat flour gluten complex is determined. the influence of wheat fibre and emulsifier mixture on physical and chemical indicators of the quality of the biscuit semi-finished product is studied. keywords: dietary fibres, biscuit semi-finished products, emulsifier, gluten. 1. introduction flour confectionery products are among the most popular and affordable food products in the world, their consumption possesses the first place in all population groups. however, they have high calorie content and contain many easily digestible carbohydrates, which are starch and sucrose, and unbalanced nutrient composition, as this product has a small amount of biologically active substances. an issue of the enrichment of flour confectionery products with physiologically functional food ingredients is important as it reduces the risk of the most common “diseases of civilization” (cardiovascular, cancer, diabetes, and obesity). a solution to this problem is to create functional food products, because their segment is almost empty in the market of ukraine today. it will provide an opportunity to the enterprises of confectionery industry, including the units of restaurant business, to use a wide range of the flour confectionery products enriched by biologically active substances in their manufacture [1]. biscuit semi-finished products are primary or component part of many flour confectionery products. biscuit dough is a thermodynamically unstable foam-like food system; a stability of this system has technological importance in its production. the introduction of the flour confectionery products of new generation is due to their enrichment with vital and biologically active substances: dietary fibres, unsaturated fatty acids, macro-and micronutrients. dietary fibres are among the most important physiologically functional ingredients that can ensure a real correction of the food products towards improving their health benefits. there is credible evidence of the link between dietary fibres consumption and cholesterol decrease that is a risk factor for cardiovascular disease. a sufficient http://www.fia.usv.ro/fiajournal mailto:elenka-021991@mail.ru food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 392 amount of the soluble dietary fibres in the diet reduces the risk of atherosclerosis and coronary heart disease. low consumption of the dietary fibres is related to the fact that the products enriched with them are often less attractive to taste than refined [2]. choosing an appropriate type of the fibres with different properties, according to specific tasks, enables to develop the products with their high content without compromising organoleptic properties of the latter. proper selection of the fibres provides certain technological and economic advantages. in accordance with the recommendations of the world health organization, the product, 100 grams of which contain 3 grams of the dietary fibres, is a source of this functional ingredient, and with the content of 6 grams of the dietary fibres per 100 grams of the product – enriched with the dietary fibres [3]. however, with the introduction of the dietary fibres in the food system, including flour confectionery products, many technical problems appear. the introduction of the dietary fibres influences on taste, smell, colour, texture, and volume indicators. the level of these indicators change largely depends on the source of the dietary fibres and their technological properties. the analysis of the literature shows that the use of the dietary fibres in the manufacture of the flour confectionery products is poorly studied [4, 5]. however, conducted research concerning the use of different types of the dietary fibres in the manufacture of the flour confectionery products, including biscuit semi-finished products, does not reflect a full picture of their impact on physical and chemical processes occurred in the production; as well as the change of the structural and mechanical properties of the dough and baked semi-finished products. we use a secondary plant material such as wheat fibre produced by vitatsel (germany) as enriching supplements [6]. it contains a significant amount of the dietary fibres, thus, it is important to study its chemical composition and functionaltechnological properties. 2. materials and methods the aim is to study the prospects of using the product of wheat processing – wheat fibre (wf) in the technology of the biscuit semi-finished product. the objective of the work is to determine the general chemical and fractional composition of the wheat fibre and its polysaccharide complexes as well as the impact on the quantity and quality of gluten wheat flour and indicators of the quality of the biscuit semi-finished product. the protein content is determined by the modified kjeldahl’s method, ash – by wet ashing method with the use of the accelerator, mass fraction of fat – by the refractometric method. the quantitative determination of cellulose is determined by the nitrogen-alcoholic method (kiurshner’s method), pectin substances – by the calcium-pectin method, lignin – by the method of vilshtetter tsehmeister, the total hemicellulose content – according to the technique [7], the content of their separate fractions – using the technique [7], the mass fraction of starch – by the volumetric method. quality indicators of the finished product – porosity, specific volume, ratio of lifting and baking loss are described by the generally accepted methods. 3. results and discussion using experimental and literature data, a comparative evaluation of the content of food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 393 the nutrient and bioactive substances of wheat fibre, wheat bran and extra wheat flour is performed, which is presented in table 1. table 1 the content of nutrients and bioactive substances in the raw materials studied, g/100g main components, g/100 g extra wheat flour wheat bran wheat fibre proteins 10.3±0.3 14.6±0.2 0.1±0.1 fats 1.1±0.1 5.5±0.2 0.1±0.1 carbohydrates, including 73.6±2.0 63.1±2.0 95.4±2.5 mono and disaccharides 1.6±0.1 4.6±0.1 0.1±0.1 starch 68.5±2.0 20.6±1.0 0.1±0.1 dietary fibres 3.5±0.2 37.9±1.0 95.2±2.0 ash 0.5±0.1 1.8±0.1 1.0±0.1 energy value, kcal 342.0 165.0 4.0 the research results in table 1 show that wf yields to extra wheat flour and wheat bran with protein and fat content because its composition contains only traces of them. at the same time the content of df in wf is 95.4g/100 g that is 28 times larger than their content in extra wheat flour and 2.5 times than in wheat bran. a significant number of df can reduce the percentage dosage of wf to flour weight in the fcp technology to ensure physiologically desired concentration of df. another significant advantage of wf is its low energy value – only 4.0 kcal / 100 g – that can also reduce the caloric content of the finished product as a result. the direction of physiological effect of df, their technological features depend on the content and value of the individual components of df. thus, df content in the raw materials is investigated by fractions (table 2). table 2 the content of the dietary fibres and their individual components in the studied raw materials. g/100 g the name of the raw materials polysaccharides cellulose hemicellulose pectin substances lignin together extra wheat flour 0.6±0.1 2.5±0.1 0.3±0.1 0.1±0.1 3.5±0.1 wheat bran 17.3±0.3 13.7±0.2 3.1±0.1 3.8±0.1 37.9±0.3 wheat fibre 53.6±1.0 24.1±0.5 10.1±0.3 7.4±0.3 95.2±1.0 cellulose is the main component of the cell walls of the plants and has high water absorbing ability, which as a result has a positive effect on the human gastrointestinal tract. the experimental data, presented in table 2, show that this polysaccharide is a basis of the dietary fibres of wf – 53.6 g / 100 g of the raw materials. its content compared to wheat flour and wheat bran is 90 and 3 times larger respectively, due to the peculiarities of the wf technology. hemicelluloses are characterized by a high hydrophilic nature. the binding process of water with xylan, which is a chemical component of hemicelluloses, is similar to the processes of starch swelling, but they do not undergo retrogradation. hemicelluloses in the composition with fcp are able to slow down the process of hardening but do not have a significant impact on increasing their capacity and output. table 2 shows that wf contains a significant amount of food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 394 hemicellulose, namely 24.1 g / 100 g that is 9.6 and 1.8 times larger than their content in wheat flour and wheat bran, apparently due to the differences in the chemical composition of the studied raw materials [8]. the study of the fraction composition of wf hemicelluloses shows (tab. 3) that hemicelluloses of the group “b” (waterinsoluble) constitute their basis, the content of which is higher compared to hemicelluloses of the group “a” (watersoluble fraction) in average 1.2 ... 2.0 times respectively. table 3 hemicellulose fractional composition of wheat fibre, g / 100 g hemicellulose fraction product containing df extra wheat flour wheat bran wheat fibre group „a” 1.1±0.1 7.3±0.2 7.8±0.2 group „b” 1.4±0.1 6.4±0.2 16.3±0.3 pectin substances have complex forming properties that determine their participation in the metabolic processes and the ability to rid from the human body of heavy metals and radionuclides. there is evidence that pectin substances influence on the course of biochemical and microbiological processes in the dough; change the rheological properties of the semi-finished products and structural and mechanical properties of the finished products. the degree of this impact depends on the ratio of watersoluble and insoluble pectin components. therefore, the study of the fractional composition of the pectin substances in the raw materials is conducted (tab. 4). table 4 pectin substances fractional composition in the studied raw materials, g / 100 g fraction extra wheat flour wheat bran wheat fibre water-soluble pectin 0.08±0.01 1.1±0.1 3.8±0.1 protopectin 0.22±0.01 2.0±0.2 6.3±0.2 the amount of pectin substances 0.30±0.01 3.1±0.2 10.1±0.3 research results of the fractional composition of the pectin substances in the raw materials (tab. 4) indicate that their main proportion is protopectin. the content of this component in wheat fibre is 1,7 times larger compared to the content of water-soluble fraction of these substances. thus, the results that determine the chemical composition of wf show that it has a high content of bioactive substances, especially dietary fibres, which are mainly represented by cellulose and hemicellulose complex [9]. to investigate the wf impact on structural and physical properties of the dough, modelling experiments are conducted to determine the quantity and quality of gluten, namely elasticity, extensibility, ability, and hydration. the indicators of the quality of gluten depend not only on the parameters of technological process of the dough, but also on the composition and properties of its prescription components. the technology of the biscuit products provides the use of wheat flour with low gluten; otherwise, semi-finished products food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 395 have a small volume and low porosity due to a significant resistance of the strong gluten to the expansion of the air bubbles with temperature increasing. the use of the wheat fibre enables to prevent it, as this raw material does not form a fibre. in this regard, the impact of the wheat fibre on the quality of gluten dough is determined (tab. 5). the necessity to investigate the impact of the studied products on the content and quality indicators of gluten is due to the peculiarities of their chemical composition, high degree of their dispersion. it is possible that fine grinding of the product leads not only to the changes in the quantitative and qualitative content of its individual components, enzyme activity, degree of digestibility, changes of the mechanism of its influence on the dough biopolymers. wheat fibre is added to the dough in quantities of 25, 30 and 35% instead of wheat flour. the amount of wheat fibre adding is established at the rate of 25% of the daily need of the dietary fibres. table 5 wheat fibre impact on the quality indicators of gluten flour mixture the content of wet gluten,% the content of dry gluten,% quality indicators of gluten extensibility, cm elasticity, units idg the hydration ability, % extra wheat flour 28.5±1.1 9.5±0.5 19.0±1.1 81.6±2.2 188.5±3.1 extra wheat flour, wheat fibre, 25% 26.7±1.0 9.0±0.5 17.0±0.9 66.9±2.1 184.1±3.3 extra wheat flour, wheat fibre, 30% 25.5±0.8 8.4±0.4 16.0±0.8 64.6±2.1 181.3±2.8 extra wheat flour, wheat fibre, 35% 24.2±0.7 7.8±0.3 13.0±0.5 62.8±2.0 176.8±2.5 the research results show (tab. 5) that wf introduction causes the decrease of the amount of wet and dry gluten. thus, the use of 25 ... 35% wf the content of wet gluten decreases by 22.3 ... 53.4% and dry gluten – 5.2 ... 12.6% respectively. increasing the dosage of all supplements in the studied range, gluten strengthens. it becomes more resilient and has a lower extensibility. strengthening performance of the additives is associated with high hydrophilic ability of the constituents of their polysaccharide complexes that have a significant dehydrating impact on the dough biopolymers. it is evidenced by lowering hydration ability of dough gluten with this raw material, namely 2.3 ... 6.2% by adding wf compared to hydration of gluten washed from the dough without it. it should be noted that the formation of strong gluten frame might cause an excessive compaction of the dough structure due to a significant resistance of the elastic gluten to the expansion of the air bubbles with temperature increase during baking and getting not enough aerated crumb of the baked biscuit semifinished products and reduce their volume and porosity [10]. table 4 shows that the introduction of the wheat fibre strengthens gluten, which is a negative factor in relation to the biscuit dough, so it is appropriate to provide the introduction of the surface-active agents (surfactants) that enable to weaken it and thus contribute to obtaining finished products with greater porosity and specific volume. surfactants are chemical compounds that concentrating on the interface cause the food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 396 reduction of the surface tension. the most common surfactants that gained their recognition in the fcp technology are nonionic. they are generally compatible with other classes of surfactants, added to the raw materials in small quantities and are relatively inexpensive. non-ionic surfactants enable to form an even thinwalled structure of the fcp crumb able to be kept fresh for a long time and to create a weakening effect on dough gluten. non-ionic surfactants are offered to use as surfactants for the biscuit semi-finished product that are a mixture of the emulsifiers «grindsted cake» produced by danisco (denmark) [11]. it consists of propylene glycol ester and fatty acids (e 477), monoand diglycerides of fatty acids (e 471) , lactic acid stearate sodium (e 481). the addition of «grindsted cake» improves the overrun of the liquid biscuit dough and increased volume of the finished product, and provides an even structure. the next stage of the research is to determine the impact of wf with adding the mixture of the emulsifiers «grindsted cake» on the quality of gluten of extra wheat flour (tab. 6). table 6 impact of the emulsifier «grindsted cake» on the quality indicators of gluten flour mixture extensibility, cm elasticity, units idg extra wheat flour 19±1.0 81.6±3.0 extra wheat flour, wheat fibre, 30% 16±0.6 64.6±2.0 extra wheat flour, wheat fibre, 30%+ «grindsted cake» 19.5±0.8 83.7±1.4 table 6 shows that the included emulsifier enables to weaken gluten, improve its elasticity and extensibility. therefore, we can predict that the finished products with added «grindsted cake» will have larger volume, better porosity and structure. to determine the wf impact on the indicators of the quality of the biscuit semi-finished product, baking tests are carried out with the replacement of the wf flour in the amount of 25, 30 and 35% and their quality indicators are determined, in particular – their specific volume, porosity, lifting and baking loss coefficient (tab. 7). table 7 quality indicators of the biscuit semi-finished product with adding wheat fibre indicators control with the replacement of 25% with the replacement of 30% with the replacement of 35% specific volume, cm3 / g 2.46±0.2 2.43±0.2 2.36±0.2 2.31±0.3 porosity, % 68.80±1.4 67.30±1.2 65.6±1.3 63.20±1.1 lifting coefficient, units 1.83±0.2 1.81±0.1 1.76±0.1 1.71±0.2 baking loss, % 9.90±0.4 9.10±0.3 8.40±0.2 7.90±0.1 table 7 shows that the addition of wf degrades the quality of the baked semifinished products for all indicators except baking loss whose value increases with increasing the dosage of wf, apparently due to its high water absorbing and water retaining ability. based on the data obtained above on food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 397 weakening the gluten of wheat flour when adding «grindsted cake» emulsifier (tab. 6) it is decided to determine its impact on the quality of the biscuit semi-finished product with wheat fibre addition (tab. 8). table 8 quality indicators of the biscuit semi-finished product with wheat fibre addition and mixture of «grindsted cake» emulsifiers indicators control with the replacement of 25% with the replacement of 30% with the replacement of 35% specific volume, cm3 / g 2.46 ±0.2 2.58 ±0.2 2.51 ±0.2 2.43 ±0.1 porosity, % 68.80 ±1.2 73.20 ±1.4 71.40 ±1.1 67.30 ±1.0 lifting coefficient, units 1.83 ±0.1 1.92 ±0.2 1.88 ±0.1 1.79 ±0.1 baking loss, % 9.90±0.2 9.10 ±0.3 8.40 ±0.2 7.90 ±0.2 table 8 shows that the addition of «grindsted cake» leads to the increase of porosity, specific volume and lifting coefficient of the finished product with the addition of wheat fibre, thus, enabling to approach the quality indicators of the biscuit semi-finished product to the quality indicators of control. specifically, the specific volume of the finished product increases by 2%, porosity by 3.7% and lifting coefficient by 2.7% respectively for the sample with the replacement of wheat flour into wheat fibre in the amount of 30%. thus, it can be concluded that the addition of the «grindsted cake» emulsifier is appropriate to improve the quality of the biscuit semi-finished products with wheat fibre addition. 4. conclusion thus, wheat fibre addition is a promising source for the enrichment of the flour used in confectionery products with dietary fibres, because its composition contains large amounts of cellulose, hemicellulose, lignin and pectin substances. the determined results of the fractional composition of wf indicate that dietary fibres are largely represented by cellulose and hemicellulose complex. quite low energy value of the raw materials reduces the energy value of the final product. it is investigated that wf has a strengthening impact on flour gluten. it becomes more resilient and has lower extensibility. reinforcing effect of wf can be associated with high hydrophilic ability of its polysaccharide component systems. it is determined that the introduction of wf together with a mixture of the emulsifiers «grindsted cake» in the dough increases flexibility and extensibility of flour gluten that affects positively the quality of the finished products. during baking tests of the biscuit semifinished products with the replacement of wheat flour into wf in the amount of 25 ... 35% and addition of a mixture of the emulsifiers «grindsted cake», it is determined that the quality indicators, including porosity, specific volume and lifting coefficient, are improved in comparison with the indicators of control. thus, wf can be recommended to use in the flour confectionery products technology as a source of functional ingredients. 5. references [1]. dorohovich v. v., the use of carrot juice with biscuits developed functionality, products and ingredients, vol. 8, 22-23 (2013). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xiv, issue 4 – 2015 olena kobets, viktor dotsenko, oksana arpul, iryna dovgun, wheat fibre impact on flour confectionery products quality, food and environment safety, volume xiv, issue 4 – 2015, pag. 391 – 398 398 [2]. shenderov b. a., current state and prospects of development of the concept of "functional food", food industry, vol. 5, 4-7 (2003). [3]. marina n. v., novoselova g. n., shavnin s. a. products with increased biological value out of non-traditional plant raw materials, news of the samara scientific center of the russian academy of sciences, vol. 12, 1(8), 20792082 (2010). [4]. brennan c.s. evaluation of starch degradation and textural characteristics of dietary fiber enriched biscuits, international journal of food properties, vol. 7, 3, 647-657 (2004). [5]. arsenev l.y., dotsenko v. f.,concentrates nutritive fibers, bakery and confectionery industry ukraine, vol. 12, 109, 49-51 (2013). [6]. conclusion sanitary and epidemiological expertise № 05.03.02-03/11418 from 18.02.2014. [7]. constandache m., the influence of fortification of bread with exogenes proteins on the protein digestibility, journal of agroalimentary processes and technologies, vol. 8, 2, 461-466 (2007). [8]. ocheme o., chinma c. effects of soaking and germination on some physicochemical properties of millet flour for porridge production, journal of food technology. vol. 6. № 5. p. 185188 (2008). [9]. hidvegi m., lasztity r. phytic acid content of cereals and legumes and interaction with proteins, periodica polytechnica. ser.chem.eng.,vol. 46. № 1–2. p. 59–64 (2002). [10]. drobot v.i., arsenyeva l.yu., bilyk o.a. etc. laboratory workshop on technology of bakery and macaroni production. kyiv: center of educational literature, 341 (2006). [11]. conclusion sanitary and epidemiological expertise № 05.03.02-03/84115 from 17.09.2013. http://www.pp.bme.hu/ch/2002_1/ch2002_1_08.html http://www.pp.bme.hu/ch/2002_1/ch2002_1_08.html http://www.pp.bme.hu/ch/2002_1/ch2002_1_08.html 352 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 -2018 , pag. 352 362 the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch * iryna medvid1, olena shydlovska1, viktor dotsenko1 1department of hotel and restaurant business, national university of food technologies, kyiv, ukraine, *medvidrina@gmail.com, *corresponding author received 5th october 2018, accepted 28th december 2018 abstract: the necessity of search of new ways to improve the quality of gluten-free bread for people suffering from celiac disease is considered in the article. as one of the directions of this problem solution the use of surface-active substances has been suggested. the efficiency of the use of sunflower skimmed lecithin for production of rice bread with the use of enzymatic modification of flour starch has been proved. its influence on the intensity of fermentation in the rice dough, the activity of fermentative microflora and the change of specific volume in the process of maturation has been investigated. the positive influence of emulsifier in the technology of rice bread on the indicators as ready-made products, in particular specific volume, porosity and elastic properties of the crumb have been studied out. it was established that the addition of sunflower skimmed lecithin to vegetable oil in the process of production of rice bread using flour starch hydrolysis promotes the extension of its shelf life and freshness. keywords: celiac disease, rice bread, gluten, surface-active substances, lecithin, starch, enzymatic modification. 1. introduction one of the most complicated tasks of food science is the development of baked goods for special use, which are recommended to be consumed by people suffering from certain diseases. the most emerging problem nowadays is the considerable spread of gastroenterological diseases. these ones include the celiac disease (gluten-sensitive enteropathy), caused by the violation of nutrients absorption in the small intestine and the damage of its mucous membrane due to consumption of gliadin and wheat glutenin, as well as prolamynes of rye barley and triticale [13]. gliadin and glutenin are the main fractions of gluten, which play a role in structure-forming in the bread dough and take part in the formation of desirable volume and texture of dough system [4]. during baking, the protein substances are denatured; as a result they create a dense framework of the goods and provide them with their high quality. since going on a gluten-free diet during the whole life is the only method of treatment and prevention of celiac disease, the use of gluten-free cereals flour (corn, rice, buckwheat, amaranth etc) which due to their chemical composition do not contain proteins is important in the production of bread for special use. that is why baked goods “without gluten” have low, if compared with traditional wheat bread, organoleptic and structural-mechanical properties. the point of search for new ways of quality improvement of gluten-free bread is rather acute and relevant for scientists and and specialists in the bakery industry. rice flour is an alternative raw material for production of gluten-free bread, which http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 353 contains rather little amount of prolamine (1.5-3.5%) and does not take part in the formation of gluten complex. only gliadin fractions are toxic, and as rice proteins do not contain them, it is not dangerous for the people suffering from celiac disease. moreover, rice flour is a source of vegetable protein, complete for amino acid composition, it contains sodium, potassium, phosphorus, magnesium, vitamins в1, в2 and рр, high amount of easily digestible carbohydrates, has soft taste, white colour and hypoallergenic properties [5, 6]. its use in bread baking is limited to form the dough with the necessary structural-mechanical properties and to ensure high quality of the goods [7]. the study of literature shows that in order to achieve desirable bread quality from rice flour, scientists suggested the use of hydrocolloids (xanthan gum and guar gum, carboxymethylcellulose, hydroxymethyl propyl cellulose) and emulsifiers (glycerol and diacetyl vinyl and fatty acid esters (datem), sodium stearyl alkylate, distilled monoglycerides) [6, 8, 9]. one of the directions of this problem is the modification of the main chemical components of rice flour with the help of enzymes. the use of transglutaminase as a formulation agent in the rice dough is suggested in the research [10], the addition of which leads to crosslinking of proteins that allows creating a gluten-like network. in the national university of food technologies a study was conducted, the results of which substinated the feasibility of using the amylolytic enzymes, particularly α-amylase and glucoamylase in the rice bread technology [11]. it was established, that the use of rice flour starch fermentative modification in dough preparation promotes more intensive microbiological processes, which is manifested in activation of gas and acid accumulation. it is made up by increase in the amount of nutrients for fermentative microflora of sugars, which appear as a result of flour starch hydrolysis under the influence of α-amylase and glucoamylase. though, the use of amylolytic enzymes does not allow getting the bread with high specific volume and porosity, which is related to low gas-holding capacity of this type, resulting in the formation of carbon dioxide during fermentation and does not lead to the loosening of dough preparation. one of the ways of solving out this problem is the use of surface-active substances (sas), which, when added to the dough come into contact with the starch fraction of flour, forming the complex compound, which play an important role in forming the structure of semi finished products and quality of finished products [12]. however, the use of sas promotes gas-holding in the dough that, in turn, depends on its structural mechanical properties and the intensity of fermentation. therefore, in order to improve the quality of gluten-free bread by using of fermentative modification of rice flour starch we suggested the addition of sas to dough. as the tendency of eating healthily is growing, the use of lecithins is of perspectives, which do not only play the role of sas, but also they are a valuable source of phospholipids from the physiological point of view. one of the most important functions of phospholipids is to provide and maintain protein-lipid metabolism in the organism. the main technological functions of lecithins in food systems are emulsification, interaction with proteins, change of viscosity and modification of crystals due to chemical structure of these compounds. lecithin contains polar hydrophilic (negatively charged phosphoric acid residue and a cationic core group of choline) and nonpolar hydrophobic (higher food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 354 fatty acid radicals) groups, that are located in the opposite sides of molecula. hydrophilic group provides with solubility in water, hydrophobic in fat. this explains that in heterogeneous systems lecithin molecules are oriented at the boundary between two phases, reducing the surface tension and playing an important role as emulsifiers in foods. taking into the consideration lecithin’s technological functions and its beneficial properties, the use of the sas is relevant to improve the quality of gluten-free bread [13, 14]. the degree of lecithin improvement in the production of bread is determined by the way of sas production, on which the value value of the hydrophilic-lipophilic balance (hlb) and the content of phospholipids depend. as known, the emulsifiers with the index of hlb 6...14 have positive effect on the quality of bakery products [13]. the industrial production of lecithin is mainly due to the processing of soy phosphate concentrate, which is a byproduct of oil purification from concomitant substances. taking into the consideration the fact that practically all soy is genetically modified and can lead to allergic reactions [14], the search for an alternative sas of natural origin, which has the status of being completely harmless, is relevant. sunflower lecithin is widely used in modern food industry, which unlike soy processing products, is environmentally friendly product, does not contain genetically modified organisms, phytoestrogens and substances that cause allergic reactions. considering the range of hlb emulsifiers values, which are recommended to improve quality of the bread, it is advisable to use low-fat lecithin with an hlb index of 7...8. the choice of this sas is explained by high content of phospholipids, and as a result, high emulsifying capacity, higher index of hlb, as compared with standard lecithin and phosphatidic concentrate, powdered consistency, which greatly facilitates the process of food additive dosing, as well as the absence of inhibitory effects on the activity of microorganisms of phospholipid lysosomes, which are contained in hydrolysed lecithin. the purpose of this article is to research the possibility of using sunflower skimmed lecithin in order to improve the structural mechanical properties of rice dough with fermentative modification of flour starch and the quality of ready-made product. 2. materials and methods 2.1. materials as the main raw material for bread preparation, rice flour was labeled as “crossed spike” with the following characteristics: the mass fraction of moisture was 11.5%, titrated acidity 2.2 deg., ash content 0.5%, protein content 8%, mono and disaccharides 0.7%, starch 80.3%. for the hydrolysis of rye flour starch amylolytic enzymes were used, particularly α-amylase with an amylolytic activity of 5000 skb/g, which has an optimal ph of 4.7, the temperature of 37…40ºс (mühlenchemie, germany), and glucoamylase with the activity of 500 amg/g, an optimal ph 3.0-5.5 and the temperature of 40-64ºс (germany). sunflower fat-free lecithin was used in the research (scientific and production center "dniprotehnologii", ukraine), containing 96.5% of phospholipids insoluble in acetone and has the following characteristics: the mass fraction of moisture 1.2%, the mass fraction of oil 1.4%, acid number 8.9 mg koh/g, ph 1% aqueous solution 6.7. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 355 in the production of bread, other raw materials were used as well: pressed baking yeast, salt, citric acid, drinking water and sunflower oil. 2.2. methods conducting enzymatic fermentation of rice flour starch in order to provide with the optimal conditions for simultaneous action of αamylase and glucoamylase, the ambient temperature of 40°c and ph 4.7 were set. to maintain the appropriate ph conditions, citric acid in the amount of 0.065% by flour weight was used. the enzymes were pre-dissolved in water at a temperature of 25-30ºc at a ratio of 1:10. for enzymatic modification, the mixture of rice flour, citric acid, enzymes and water was prepared, which had been hydrolyzed in the thermostat at a temperature of 40ºс for 2 hours before the accumulation of sugars 5.5-6%. the mass fraction of the moisture in the mixture was 65% [11]. dough preparation and bread baking pressed baking yeast (3%) was dissolved in water at a temperature of 26-32ºс. aqueous saline solution was prepared at a concentration of 1.2% at 30ºс and filtered. on the basis of pre-prepared hydrolyzed semi-finished product with a humidity of 53% the dough was kneaded with the addition of a yeast suspension, a salt solution and a second part of the flour according to the formulation. then dough preparations were formed and fermented them in forms in the cabinet for 45 minutes at a temperature of 32ºс and relative humidity of 85%. it was baked for 25...30 minutes at a temperature of 180ºс. the ready-made bread was cooled for 180 minutes at a temperature of 23-27°с. lecithin was added in the stage of dough kneading in the amount of 0.5%, 1.0% and 1.5% by flour weight. the control sample was prepared by using enzymatic modification of flour starch without sas addition. investigation on the change in the specific volume of dough, intensity of its fermentation and activity of fermentation microflora gas holding capacity of the dough was evaluated by the change of the specific volume of the samples in measuring cylinder by 250 сm3 in the thermostat at a temperature of 30ºс from the beginning of fermentation till the moment of dough dropping and showed in сm3/g. the intensity of alcoholic fermentation in the dough was determined by volumetric method on ag-1 device on the indicator of its gas-forming ability, i.e. the volume of со2 allocated, сm 3/100g, at a temperature of 30ºс during the dough fermentation. determination of yeast lifting power was carried out by the standard method, which is the determination of raising speed of dough, prepared by the full formulation in the conditions of thermostat and characterized by the time from the moment of putting the dough into the form until it touched the bar, i.e. the raise for the height of 70 mm. the activity of lactic acid bacteria in the dough was determined by the time needed for the blue color of samples to disappear in the presence of methylene blue as an indicator. evaluation of bread quality the determination was carried out not earlier than 3 hours after baking for all the samples. specific volume was determined as the ratio of volume they occupy, to their mass (сm3/g). bread porosity was determined by zhuravlyov device. from a piece of crumb at a distance not less than 1 cm three slots were made by the cylinder of the device, after which they were weighed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 356 the porosity (%), was calculated by the formula 1: , v m v п    (1) v – the general volume of the slots, сm3; m – mass of the slots, g; ρ – density of a porous crumb, g/сm3. the density of a porouse crumb of glutenfree bread was determined in the following way. the slot was made by zhuravlyov device, carefully squeezed it with the wooden piston in the cylinder of the device in order to remove the pores. then, the slot in the form of pressed cylinder was weighed and the height was measured. the density of a porous crumb, g/сm3, was calculated by the formula 2: hr m   2   , (2) m – mass of the slot, g; r – radius of the slot, cm; h – height of the slot, cm. the determination of bread crusting was carried out using the following methods: measuring the degree of deformation of the bread crumb on the automated penetrometer of the company ar-4/1 and determining the fragmentation of the bread by the amount of crumbs resulting from the shaking in the vibration mixer. 3. results and discussion the dosing of skimmed lecithins in dough was done accordingly to the recommendations of literary sources and manufacturers, namely 0.1…0.5% to the mass of flour. however, such amount of sas is intended to improve the quality of bread from wheat flour. taking into consideration the complete lack of gluten, in order to improve structural mechanical properties of rice dough and quality of ready-made products, an increased dosing of lecithin 0.5%, 1.0%, 1.5% to mass of flour was chosen. dry and skimmed lecithin was added in the form of colloidal solution (suspension). due to the results of research the influence of lecithin on the dough properties and the quality of rice bread were determined (table 1). the data obtained (table 1) makes it possible to state that the positive effect of lecithin studied is to increase the specific volume of bread, to improve the structure of the crumb; its porosity becomes more even and thin-walled. it is known that the volume increase of bread as a result of adding sas is explained only by the increase of gas-holding capacity of the dough in the stage of fermentation. however, lecithins of natural origin have also functional properties, namely their phospholipid content improves permeability of the membranes of yeast cells, which promotes more effective access to food sugars, amino acids, and vitamins. thus, the formation of gas in the dough during the period of its maturation increases to 21.2…22.4% when added sunflower skimmed lecithin as compared with the control. the use of lecithin positively influences the accumulation of acids in dough. after 45 minutes of maturation of dough preparations by adding sas, titrated acidity increases by 0.7...0.8 deg., while a control sample of dough made from enzymatic modification of rice flour starch by 6 deg. the change of active acidity correlates with the increase of titrated one, which naturally decreases. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 357 table 1 the influence of sunflower skimmed lecithin on dough properties and quality of bread with enzymatic modification of flour starch indicators control sample (with enzymes, without sas) samples with lecithin, % to mass of the flour 0.5 1.0 1.5 dough gas formation for 45 mins off fermentation, сm3/100 g of the dough 330 419 425 425 titrated acidity, deg. the initial one 2.6 2.6 2.6 2.7 the final one 3.2 3.3 3.4 3.4 active acidity, un. of the device the initial one 4.75 4.74 4.73 4.71 the final one 4.20 4.21 4.05 4.04 specific volume (at the end of fermentation), сm3/g 1.32 1.45 1.55 1.57 bread specific volume, cm3/g 1.51 1.67 1.80 1.82 acidity, degr. 2.8 2.8 2.9 2.9 porosity, % 41.4 44.3 47.4 47.6 lecithin in dozing of 1.0% to mass of flour causes the best improving action on the quality indicators of ready-made bread. in this case specific volume of the products is 16.2% as compared to the control sample. a similar pattern is observed in the study of the effect of sas on porosity of rice bread, which increases by 12.7% as compared to the control. the addition of lecithin in the amount of 1.5% to mass of flour does not lead to significant increase in quality of readymade bread, which, obviously, is related to the increase of viscosity of dough due to the formation of protein-phospholipid complexes, which are decisive in the formation of its rheological properties [15]. it is known, that the use of skimmed lecithins contributes to the creation and stabilization of emulsions such as "oil in water", which is conditioned by the presence in their hydrophilic and hydrophobic groups of their molecules. considering this, it was expedient to study the effect of sunflower skimmed lecithin on the bread quality in the presence of vegetable oil. in this regard, the determination of the effect of lecithin dose chosen on dough quality and bread if sunflower oil addition is compatible was performed. sas and vegetable oil were added after enzymatic modification of rice flour starch; the mixture was dispersed for 2-3 minutes, and the dough was kneaded after. with the help of trial laboratory baking the dosage of sunflower oil in a quantity of 3% to the mass of the flour was predetermined. further research on the effect of lecithin while adding it to change the specific volume of dough, using enzymatic modification of flour starch in the process of maturation, the properties of pressed baking yeast and lactic acid bacteria, quality indicators of ready-made products and on the process of getting stale is required. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 358 the results of the change of specific volume of dough in the process of fermentation are shown in the figure 1. fig. 1. the change of specific volume of the dough in the process of fermentation: a control (with enzymes); b with enzymes and lecithin the increase in specific volume of dough with enzymatic modification of flour starch by adding the lecithin is more intensive as compared to the control sample without sas; this is explained by the technological functions of the improver in food systems. thus, phospholipids of lecithin, due to their differentiated structure, are oriented to the boundary between two phases, reducing the surface tension, which contributes to the good formation of drops and protects them from coalescence. emulsion becomes more stable, as coalescence is hampered by a rather strong energy barrier, which is created by emulsion pellicle on the surface of a drop of water. the emulsifier effects on water and fat molecules so that they repel from each other, or creates stable, often very viscous or even solid system of protective layers around the droplets. as a result of this process, phospholipids create specific barriers on the surface of the water droplets, due to which gas-holding ability of the dough is increasing, which leads to preservation of со2 oozed in the process of alcoholic fermentation of dough semi-finished products. the data in the figure 1 also show that the maximum amount of dough with lecithin addition in the presence of vegetable oil is reached in 45…50 mins of fermentation and is held for longer period in comparison to the control sample, which indicates the improvement of gas-holding capacity of the dough with sas. the effect of sas on the fermentation activity of yeast was characterized by its lifting power (figure 2) and it depends on the initial biological activity of saccharomycetes and adaptats to the anaerobic conditions of life in the dough system, as well as on its recipe components. the analysis of obtained data has shown that the addition of lecithin in dough using enzymatic modification rice flour starch has a positive effect on yeast fermentation activity. this fact, evidently, is explained by the improvement of availability of sugars formed during the hydrolysis process, to the yeast cell due to the action of phospholipid component of sas on its shell and the cytoplasmic membrane. it also substantiates the more intense release of carbon dioxide in the test with 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 10 20 30 40 50 60 1.18 1.24 1.28 1.31 1.32 1.321.27 1.4 1.51 1.59 1.63 1.63 s p e c if ic v o lu m e o f th e d o u g h , с m 3 /g the duration of fermentation, min a b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 359 the addition of lecithin (table 1). thus, in the presence of sas yeast lifting power increases by 15.6%, in comparison to the dough sample with enzymes without lecithin addition. fig. 2. the change of yeast lifting power a control (with enzymes), b with enzymes and lecithin while maturation of yeast dough, in addition to alcohol fermentation, there is also lactic acid one, which is due to the activity of homoand heterofermentative lactic acid bacteria which convert sugars mainly into lactic acid, as well as to a small amount of volatile acids. for a clearer picture of the effect of sas on the dough microflora, the activity of lactic acid bacteria in the samples was investigated in terms of the intensity of their discoloration in the presence of methylene blue immediately after mixing and in 45 minutes of fermentation (figure 3). fig. 3. the activity of lactic acid bacteria in the rice dough: a control (with enzymes), b with enzymes and lecithin as a result of the analysis of the time difference for discoloration of methylene blue by lactic acid bacteria for the investigated samples, it has been established that the vital activity of these microorganisms has a similar pattern with a change in the yeast lifting power. it was determined that the duration of discoloration of the indicator for dough sample with the use of enzymatic modification rice flour starch with the addition of lecithin is lower by 6.7% after the fermentation in comparison to the control, indicating an increase in the activity of lactic acid bacteria with the use of enzymes and sas. the obtained research results are explained by the fact, that the presence of lecithin in dough and the hydrolysis of rice flour starch before kneading the dough promotes the activation of lactic acid bacteria due to the accumulation of simple sugars that are easily digested by microorganisms, resulting in more intensive allocation of 0 10 20 30 40 50 60 a b 32 27 t h e d u r a ti o n o f li ft in g , m in 150 160 170 180 190 200 a b 195 187 180 168 t h e d u r a ti o n o f d is c o lo r a ti o n o f m e th y le n e b lu e , m in s after mixing at the end of fermentation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 360 life products organic acids, carbon dioxide, which provide with orientation and completeness of colloidal, physical and biochemical processes in dough semi finished products. so, the addition of sas and vegetable oil in the rice dough with the use of enzymatic modification of flour starch promotes the activation of fermentation microflora, that leads to the intensification of processes of alcohol and lactic acid fermentation, as well as gas retention capacity, which is expected to improve the porosity structure, and accordingly, the volume of ready-made bread. in the course of the further studies on the effect of lecithin added in the amount of 1.0% to the mass of flour in presence of vegetable oil on the indicators of bread quality using enzymatic modification of starch and the process process of getting stale was determined (table 2). table 2 the effect of lecithin and vegetable oil on the rice bread quality with enzymatic modification of flour starch indicators control sample (with enzymes, without sas) sample with enzymes, lecithin (1.0%) and vegetable oil (3%) specific volume, cm3/g 1.51 1.92 acidity, degr. 2.8 3.2 porosity, % 41.4 51.5 fragility, % in 3 hrs 1.30 1.02 in 24 hrs 2.60 2.01 deformation of bread crumb, un. of penetrometer in 3 hrs 59 72 in 24 hrs 46 61 it was established that the use of lecithin, when adding it in sunflower oil, leads to an increase in specific volume of the bread, improvement of crumb structure and porosity and its elasticity as compared with the control. thus, porosity increases by 19.7%, specific volume of the bread by 21.4%, the general deformation of the crumb increases by 18.1%. it can be explained by the capacity of oil to create thin fat pellicles between starch granules that make the dough more plastic, as well as increases its capacity to be stretched without rupture under the pressure of carbon dioxide, formed during fermentation. the obtained data correlate with the results of the research on the change of specific volume of dough in the process of fermentation indicating an improvement in its gas-holding capacity (figure 1). it is known that during bread storage there occur changes in the structure of its protein-carbohydrate matrix due to the retrogradation of amylose and starch syneresis and the interaction of protein molecules with amylose and amylopectin with the formation of new bonds, as well as the redistribution of moisture between polymers, which leads to consolidation of the structure of the crumb and the change of its rheological properties. considering this, it was determined the effect of raw material on the quality indicators of bread, that characterize its suitability for storage, namely fragility and elastic properties of the crumb. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 361 it was found that the addition of lecithin and sunflower oil affects the process of bread staling, contributing to the preservation of its freshness. it can be seen from the table 2 that the use of this raw material leads to less intensive increase of fragility and the reduction of general deformation of crumb of the rice bread during storage. it can be explained by the formation of complexes of starch polysaccharides with lipids, which contribute to the inhibition of the process of retrogradation of starch. evidently, the lipids create thin pellicles that wrap the particles of the polyesterized starch grains, which hinder the release of water in the process of bread storage. besides, lecithin can create complexes with starch amylose; as a result the process of crystallization is slowing down. thus, the most positive effect on the quality of bread with the use of enzymatic modification of rice flour starch is observed when using sunflower skimmed lecithin when added to vegetable oil. 4. conclusion as a result of the theoretical and experimental studies, the feasibility of using sas in the technology of rice bread with the use of enzymatic modification of starch flour has been substantiated. as an emulsifier, sunflower oil has been chosen as skimmed lecithin, which besides the technological function of sas in the food systems, due to the high content of phospholipids has an important physiological significance. it was determined that the dosage of lecithin in the amount of 1.0% to the mass of flour contributes to the improvement of gas accumulation in the rice dough with the premade enzymatic hydrolysis has a positive effect on the porosity and specific volume of the ready-made bread. considering the presence of a hydrophobic group in the molecule of lecithin, it is substantiated that the addition of sas to the dough is compatible with sunflower oil in the amount of 3% by mass of flour. the use of phospholipid in the technology of gluten-free bread with the use of enzymatic modification of rice flour starch contributes to the improvement of vitality of dough fermentable microflora, increasing its gas-retaining capacity; as a result ready-made products are characterized by improved quality indicators. in addition, the use of lecithin and vegetable oil in the production of rice bread allows prolonging shelf-life and freshness. 5. references [1]. houben a., hochstotter a., becker t., possibilities to increase the quality in glutenfree bread production: an overview, european food research and technology, 235: 195-208, (2012) [2]. remesy ch., zeenhardt f., fordet a., donner un nouvel avenir au pain dans le cadre d`une alimentation durable et preventive, cahiers de nutrition et de dietetique, 50: 39-46, (2015) [3]. elif turabi, gulum sumnu, serpil sahin, rheological properties and quality of rice cakes formulated with different gums and an emulsifier blend, food hydrocolloids, 22: 305-312, (2008) [4]. demirkesen i., mert b., sumnu g., sahin s., rheological properties of gluten-free bread formulations, journal of food engineering, 96: 295-303, (2010) [5]. hatta e., matsumoto k., honda y., bacillolysin, papain, and subtilisin improve the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue – 4, 2018 iryna medvid, olena shydlovska, viktor dotsenko, the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, volume xvii, issue 4 – 2018, pag. 352 362 362 quality of gluten-free rice bread, journal of cereal science, 61: 41-47, (2015) [6]. nunes m.h.b., moore m.m., ryan liam a.m., arendt e.k., impact of emulsiwers on the quality and rheological properties of gluten-free breads and batters, eur food res technol, 228: 633-642, (2009) [7]. hera e., martinez m., gómez m., influence of flour particle size on quality of glutenfree rice bread, food science and technology, 54(1): 199-206, (2013) [8]. hryshchenko a.m., udvorheli l.i., mykhonik l.a., kovalevska ye.i., research of structural and mechanical properties of exalbuminous dough with guar gum and xanthan gum, food science and technology, 1(10): 63-65, (2010) [9]. drobot v.і., mykhonik l.a., hryshchenko a.m., the influence of structure forming food additives on the quality of gluten free bread made from the mixture of rice and corn flour, scientific works of nuft, 23(6): 169175, (2017) [10]. snanina o.m., lobacheva n.l., zverev v.o., the effect of transglutaminase enzyme on the properties of flour proteins, easterneuropean journal of enterprise technologies, 5/11(71): 28-33, (2014) [11]. medvid і., shydlovska о., dotsenko v., influence of fermentative modification of rice flour starch on bread quality for patients with celiac disease, ukrainian food journal, volume 6. issue 4: 632-647, (2017) [12]. tkachuk y., shydlovska о., dotsenko v., maltseva о., characteristics and use of surface-active substances (sas), bakery and confectionery industries in ukraine, 6 (55): 1822, (2009) [13]. polodiuk v.s., arsenieva l.y., dotsenko v.f., lecithin efficiency in bread baking, scientific works of nuft, 15: 35-38, (2004) [14]. shulga s.m., glukh a.i., glukh i.s., drozdov o.l., sckola o.i., the use of dry sunflower lecithin in the production of bakery and margarines, thematic collection of scientific works of donetsk national university of economics and trade, 28: 169-174, (2012) [15]. bortnichuk о.v., gavrish а.v, niemirich о.v., dotsenko v.f., innovate approaches in the technology of bakery products with dry whey, food science and technology, 2(31): 97-102, (2015) 1. introduction 4. conclusion issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xvi, issue 4 30 december 2017 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, sapporo, japan alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 92 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 12020, pag. 92 97 effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacter xylinum kamonwan rojsuntornkitti 1 , nitipong jittrepotch 1 , teeraporn kongbangkerd 1 , putkrong phanumong 2 , mayura thongchuang 3 , *kitisart kraboun 2 1department of agro-industry, faculty of agriculture, natural resources and environment, naresuan university, phitsanulok 65000, thailand. 2division of food safety management and technology, department of science, faculty of science and technology, rajamangala university of technology krungthep, bangkok 10120, thailand. 3department of chemistry, faculty of science, ramkhamhaeng university, bangkok 10240, thailand. kitisart.k@mail.rmutk.ac.th *corresponding author received 10th march 2020, accepted 30th march 2020 abstract: pineapple juice (ananascomosus l. cv. pattavia) has high amounts of sugar, making it potential to be produced into nata.the study will enable efficient utilization of pineapple juice and will also provide a new product dimension to the pineapple farmers who are not getting the right price for their product. the objectives of this research were to study the various contents of (nh4)2so4on total soluble solids, ph, % ethanol, thickness and yield of nata de pina from pineapple juice (ananascomosus l. cv. pattavia) fermented by acetobacter xylinum. (nh4)2so4 was used as a nitrogen source and its contents for nata de pina production at 30oc for 15 days were 0.3, 0.4 and 0.5 % w/v. increasing 0.3 0.5 %w/v (nh4)2so4 contents improved to utilize total soluble solids and % ethanol of nata de pina and the lowest ph of nata de pinawas obtained from using 0.5 % w/v of (nh4)2so4. using 0.5 %w/v of (nh4)2so4led to the highest values of thickness and yield of nata de pina after 15 days which were 12.14 mm and 100.02 g, respectively. keywords: (nh4)2so4, nata, acetobacter xylinum 1. introduction nata de coco, a kind of bacterial cellulose is an indigenous dessert well known in thailand, which is produced from coconut water fermented by acetobacter xylinum. after a period of the fermentation, a layer of gelatinous sheet is formed on the surface of the fermented coconut water. the sheet is cut into cubes. as a dessert, the washed cubes are usually served with flavoured syrup, jelly or other fruit cocktails. presently, nata de coco is manufactured at an industrial scale not only in thailand but also in indonesia and some are exported to countries like japan [1]. a. xylinum is widely found in nature and is a common contaminant in the industrial production of vinegar by a. aceti. a. xylinum has been isolated from rotting fruits, vegetables and fermenting coconut water. many strains of a. xylinum are able to produce cellulose in varying amounts and growing on a wide variety of substrates, such as glucose, sucrose, fructose, invert sugar, ethanol and glycerol [2]. cellulose production by a. xylinum has been noted both in static as well as agitated cultures [3] and is known to be affected by the type and concentration of sugar, nitrogen source and ph [4]. commonly, nata de coco is produced by coconut water fermentation with a. xylinum, the substrate http://www.fia.usv.ro/fiajournal mailto:kitisart.k@mail.rmutk.ac.th http://www.foodnetworksolution.com/wiki/word/1507/acetobacter-xylinum food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue1 – 2020 kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun, effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacterxylinum, food and environment safety, volume xix, issue 1 – 2020, pag. 92 – 97 93 is changed to pineapple juice which itis interesting since changing the substrate might affect specific characteristics of nata such as texture, flavor, taste, color and others. in addition, nitrogen source is an important factor for microorganism growth, which can influence on cellulose production [5].ramana et al.[6]reported that the effect of nitrogen sources on cellulose membrane production by acetobacter xylinumwas evaluated. the strain was able to utilize a wide range of protein and nitrogen sources such as (nh4)2so4, peptone, soybean meal, glycine, casein hydrolysate, and glutamic acid for cellulose synthesis. it was found that (nh4)2so4 could produce higher yields of bacterial cellulose compared with other nitrogen sources. moreover,jagannath[7]studied the effect of (nh4)2so4concentrations of 0.25 0.50 %w/w on the production of nata de coco, a form of bacterial cellulose, by acetobacter xylinum. maximum thickness of nata was obtained 0.5 %w/w of (nh4)2so4. this (nh4)2so4 concentration also produced the good qualities of nata de coco with a smooth surface and soft chewy texture. however, there are no studies of the various contents of (nh4)2so4 on total soluble solids, ph, % ethanol, thickness and yield of nata obtained from pineapple juice (ananascomosusl. cv. pattavia) fermented by acetobacter xylinum. therefore, nata de pineapple production by pineapple juice supplemented with (nh4)2so4 is expected to be a new product, which might be specific characteristics unlike nata de coco product. the present work used pure cultures of a. xylinum to obtain a consistent product and will benefit the pineapple based farmers and communities to produce good quality nata de pinawith superior physical properties. 2. matherials and methods 2.1 maintenance of a. xylinum acetobacter xylinum (tistr 975) culture obtained from thailand institute of scientific and technological research (tistr), thailand was maintained on tomato agar slants. two hundred grams of fresh tomatoes and 500 ml of distilled water were boiled for 30 min. this tomato infusion was filtered and mixed with 100 g yeast extract, 50 g sucrose, 2.5 g peptone and 20 g agar. the volume was made up to 1,000 ml with distilled water and sterilized at 121oc for 15 min. a. xylinum was streaked on these slants and incubated at 30oc for 4 days. 2.2 preparation of nata de pina starter a. xylinum grown on tomato agar slants was inoculated into sterilized media containing 20 g-l glucose, 5 g-l yeast extract, 5 g-l peptone and 2.7 g-l k2hpo4. the original glucose medium bywatanabe&yamanaka[2]was modified to exclude citrate and the ph adjusted to 4.2 with glacial acetic acid. the inoculated media was incubated statically at 30oc for 7 days. 2.3 preparation of pineapple juice medium pineapple juice (ananascomosusl. cv. pattavia) was obtained from pineapple fruits purchased locally in phitsanulok, thailand. the juice was steamed for 30 min in a laboratory autoclave, then supplemented with varying amounts of (nh4)2so4( 0.3, 0.4 and 0.5 % w/v) , 6 %v/v of ethanol 95 % and adjusting the ph 5 with glacial acetic acid. the surface pellicle from the agar slants were removed and inoculated (10%w/v) into 2,000 ml the medium taken in trays (l x b x h:30 x 25 x 6 cm). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue1 – 2020 kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun, effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacterxylinum, food and environment safety, volume xix, issue 1 – 2020, pag. 92 – 97 94 the trays were covered with muslin cloth and allowed to stand for 15 days. 2.4 harvesting and processing of nata de pina the sheet of nata de pinaformed after 15 days was harvested when it was about 0.8 1.0 cm thick, washed repeatedly with water to remove glacial acetic acid and cut into cubes of equal dimensions. the cut cubes of nata were immersed in water for 24 h with repeated changing of water to remove the sour odour. 2.5 thickness and yield the thickness of the nata layer was measured with a micrometer. this was done in 5 different points of the surface of the nata. the average data obtained were used. the yield of nata de pina was measured in weight (g) [8]. 2.6 ph and ethanol concentration the ph of the samples was measured by using a ph meter (thermo orion model 420, usa). the measurement was carried out in triplicate [9]. ethanol concentration was measured using an ebulliometer (alla model 99002-ca, france)[8]. total soluble solids (tss) the nata was used for extracting juice with a commercial juice extractor. filtered supernatant juice was used for determination of tss. tss was measured by a digital refractometer (atago pr101, tokyo, japan). 2.7 statistical analysis all determinations were performed in triplicate and results were expressed as the mean + standard deviation calculated using spreadsheet software microsoft excel. this was carried out in a completely randomized design (crd) which the data were analyzed by an analysis of variance (p<0.05) and means were compared with duncan’s multiple range test. the results were processed by spss 16.0 (spss inc., chicago, il, usa) for windows. 3. results and discussion 3.1 tss, ph and % ethanol of nata de pina tss and ethanol valuesof nata de pina produced by all percentages of (nh4)2so4decreased throughout the fermentation, affecting ph decreased as well ( fig. 1) . this might be a cause of using reducing sugars or carbon sources, appeared in pineapple juice, by a. xylinum in order to be substrates for the production of gluconic and ketogluconic acid[10]. increasing in the range of 0.3 0.5 %w/v of (nh4)2so4 promoted carbon and nitrogen sources utilization by a. xylinum. furthermore, among different (nh4)2so4percentages, the organism preferred 0.5 %w/v of (nh4)2so4in the presence of pineapple juice during the fermentation. on day 15, tss value of nata de pinaproduced by 0.5 %w/v of (nh4)2so4 was used 2.52 times; however, tss value of the control ( no addition of (nh4)2so4)was used 1.40 times.ramana et al.[6]confirmed that among various nitrogen sources evaluated, (nh4)2so4was found to be a suitable substrate of a. xylinum along with one of the carbon sources such as sucrose/glucose or mannitol. 3.2 thickness and yield of nata de pina increasing 0.3 – 0.5 %w/v of (nh4)2so4 affected higher values of thickness and yield of nata de pina, but thickness and yield of nata de pina decreased when 0.7 %w/v of (nh4)2so4 added to pineapple juice ( fig. 2) . in the present study, 0.5 %w/v of (nh4)2so4 gave the highest food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue1 – 2020 kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun, effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacterxylinum, food and environment safety, volume xix, issue 1 – 2020, pag. 92 – 97 95 values of thickness and yield of nata de pina produced in the end of experiment which were 12.14 mm and 100.02 g, respectively, indicating higher than those of nata de coco ( 8 . 6 mm of thicknessand 61.3 g of yield) [7]. this was in agreement with yamanaka et al. [11] who studied 0.5 %w/v of (nh4)2so4affecting to maximize the thickness of nata produced. this result indicated thatpineapple juice as a medium was adjusted to ph 5 with acetic acidwhich was suitable for nata de pina. this isin disagreement withjagannath et al.[7]whomodified to exclude citrate and adjusted to ph 4.2 with acetic acid was suitable for nata de coco produced by a. xylinum. the use of acetic acid to bring down the ph of the juice has a better effect as compared to other acids on the growth of a. xylinum and nata de pina formation [11].vandamme et al. [12] opined that the role of acetic acid was that of an in situ control of ph. acetic acid breaks down to co2 and water generating extra atp and thereby leading to more efficient utilization of sugars for cellulose synthesis. fig. 1 tss (a), ph (b)and % ethanol (c) of nata de pina supplemented with various amounts of (nh4)2so4, α = 0.05. further the bacteria were able to grow equally well in media containing an appropriate concentration of (nh4)2so4as the nitrogen source. the bacteria were able to make this transition effectively as evidenced by the good yield of nata de pina obtained. however, the precursor in cellulose synthesis is uridine diphosphoglucose and hence bacterial cellulose production involves use of sucrose or glucose as a carbon source for growth and polysaccharide formation [12]. these conditions also positively influenced on the quality of nata de pina in terms of water holding capacity and hardness. thickness has a direct effect on the amount of water that nata can hold which in turn affects the softness of the final product. hence, nata has high hydrophilicity which shows significant water holding capacity as well [7]. moreover, in this experiment, ethanol was added in pineapple juice, which is a significant factor in the production of cellulose by a. xylinum [13]. as well, pineapple juice adjusted to ph 5 played an important role in the production of nata cellulose ( fig. 1,2) .budhiono et al.[14] reported that ph 5 of the pineapple juice medium had a significant effect on the thickness of nata produced. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue1 – 2020 kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun, effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacterxylinum, food and environment safety, volume xix, issue 1 – 2020, pag. 92 – 97 96 verschuren et al. [10] confirmed that oxygen concentration also influenced on nata formation. therefore, the growth of a. xylinum in trays should be adequate aeration even under static conditions. the work ofverschuren et al. [10] has established that the first stage of cellulose fermentation isentirely oxygen controlled. when the dissolved oxygen in the medium is used up, bacteria existing only in the vicinity of the surface can maintain their activity and produce cellulose in the form of gel. fig. 2 thickness (a)and yield (b) of nata de pina supplemented with various amounts of (nh4)2so4, α = 0.05. 4. conclusion addition of 0.5% w/v of (nh4)2so4 led to benefits in production of nata from pineapple juice by giving better yield, thickness since a. xylinum could use more ethanol and tss. bacterial cellulose in the form of nata de pina is virtually indigestible because of its high dietary fiber content. many health benefits like prevention of colon cancer, heart attack, and increase in blood pressure or hypertension have been attributed to nata [15]. this work will be database for nata production from other kinds of fruits to give a new product dimension to the general farmers who are not getting proper price for their produce. 5. references [1] iguchi m., yamanaka, s.&budhiono, a. review bacterial cellulose a masterpiece of nature’s arts. journal of materials science,35:261270, (2000). [2] watanabe k.& yamanaka s. effects of oxygen-tension in the gaseous-phase on production and physical-properties of bacterial cellulose formed under static culture conditions. bioscience, biotechnology, and biochemistry, 59: 65-68, (1995). [3] chao y., ishida t., sugano y. & shoda m. bacterial cellulose production by aacetobacter xylinum in a 50-l internal-loop airlift reactor. biotechnology and bioengineering, 68: 345-352, (2000). [4]embuscado me., marks js.&bemiller jn.bacterial cellulose. i. factors affecting the production of cellulose by acetobacter xylinum. food hydrocolloids, 8: 407-418, (1994). [5] sanchez pc. philipines fermented foods: principles and technology. quezon city: the university of the philippines press, (2008). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue1 – 2020 kamonwan rojsuntornkitti, nitipong jittrepotch, teeraporn kongbangkerd, putkrong phanumong, mayura thongchuang, kitisart kraboun, effect of (nh4)2so4 on some qualities of nata de pina produced by acetobacterxylinum, food and environment safety, volume xix, issue 1 – 2020, pag. 92 – 97 97 [6] ramana kv., tomar a.& singh l. effect of various carbon and nitrogen sources on cellulose synthesis by acetobacter xylinum.world journal of microbiology and biotechnology, 16: 245-248, (2000). [7] jagannath a., kalaiselvan a., manjunatha ss., raju ps. & bawa as. the effect of ph, sucrose and ammonium sulphate concentrations on the production of bacterial cellulose (nata-de-coco) by acetobacter xylinum.world journal of microbiology and biotechnology, 24: 2593-2599, (2008). [8] krystynowicza., czaja w., wiktorowska-jezierska a., gonçalves-miśkiewicz m., turkiewicz m. & bielecki, s. factors affecting the yield and properties of bacterial cellulose. journal of industrial microbiology and biotechnology. 29: 189-195, (2002). [9] zhang x., kong b.&xiong yl. production of cured meat color in nitrite-free harbin red sausage by lactobacillus fermentum fermentation.meat science, 77: 593-598, (2007). [10] verschuren pg., carodona td., nout mjr., gooijer kd. & van den heuvel jc. location and limitation of cellulose production by acetobacter xylinum established from oxygen profiles. journal of bioscience and bioengineering, 89: 414-419, (2000). [11] yamanaka s., watanabe k.&kitamura n. the structure and mechanical properties of sheets prepared from bacterial cellulose. journal of materials science, 24: 31413145, (1989). [12] vandamme ej., baets s., vanbaelen a., joris k. & wulf p. improved production of bacterial cellulose and its application potential. polymer degradation and stability, 59: 93–99, (1998). [13] hutchens sa., leon rv., o’neill hm. & evans br. statistical analysis of optimal culture conditions for gluconacetobacterhansenii cellulose production. letters in applied microbiology, 44: 175-180, (2007). [14] budhiono a., rosidi b., taher h. & iguchi m. kinetic aspects of bacterial cellulose formation in nata-de-coco culture system. carbohydrate polymers, 40: 137-143, (1999). [15] keshk s.&sameshima k. the utilization of sugar cane molasses with/without the presence of lignosulfonate for the production of bacterial cellulose.applied microbiology and biotechnology, 72: 291-296, (2006). 1. introduction 4. conclusion food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 contents: 1. the prebiotic potential of some carbohydrate substrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus liliana luca, mircea oroian, andrei lobiuc 67 74 2. hand hygiene practices among street food vendors alexander fiifi ghartey, barima kwabena antwi 75 81 3. optimization of sequential ultrasound-assisted extraction – heating treatment to obtain pectin from malus domestica ‘fălticeni’ pomace florina dranca, mircea oroian 82 88 4. photochemical and photocatalytic esterification of waste cooking oil under visible light irradiation using mechanochemically synthesized zno/silica oriolowo oloruntoba, abdulhamid hamza 89 96 5. biochemical and pomological characteristics of fruit of some commercial medlar cultivars (mespilus germanica l) grown in bijelo polje gordana šebek, valentina pavlova, tatjana popović 97 104 6. the soils examination and recommendations for creating better conditions of raspberries planting jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska 105 110 7. polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction methods abel inobeme, alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka 111 122 8. human health risk assessment of heavy metal levels in clarias gariepinus and parachanna obscura from the ikpoba river, edo state, nigeria ijeoma oboh, benjamin okpara, peace wilfred ekprikpo 123 129 9. study of volovatic (carduus glaucinus) active principles for potential use as a medicinal plant ofelia arvinte, liliana norocel, sonia amariei 130 135 10. author instructions i v 11. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 103 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 103 112 antifungal effect of spice extracts possible solutions for biological preservation of food * monica marian 1, oana mare roşca 1, lucia mihalescu 1, zorica voşgan 1, anamaria lazăr 2 1 faculty of sciences, technical university of cluj-napoca, north university center of baia mare, baia mare, romania, monica.marian@cunbm.utcluj.ro, 2transilvania university of braşov, romania *corresponding author received 29th january 2018, accepted 25th june 2018 abstract: aromatic plants used as spices are effective sources for food additives, used both as taste correctors and for the purpose of preserving food. the paper aims at identifying aromatic herbal extracts that summarize several qualities: to help improve the taste and olfactory qualities of food, to stimulate digestive secretions by facilitating digestion and, last but not least, to biologically preserve food, contributing to the reduction in the number and amount of synthetic additives. for this purpose, aqueous extracts of cinnamomum zeylanicum ritidom, laurus nobilis leaves and eugenia caryophyllata floral buds, from commercial sources, were prepared. the extracts were tested on saprophytic fungi cultures, which usually infest food by causing alteration and they were obtained using the ser 148 extractor. the aspergillus, mucor and penicillium cultures were made by selection from environment. the extracts of the three spice species have demonstrated significant fungal activity, inhibiting mold growth. the most powerful effect is recorded by the cloves extract, eugenia caryophyllata, followed by the cinnamon extract. having in view the results of these experimental studies we consider that spice extracts can be used in the medium term food storage, as they reduce the amount of synthetic preservatives and replace them by natural products. keywords: biological methods, mold, vegetal extracts. 1. introduction fungal colonies are spread throughout the environment. they have an extensive capacity to colonize organic materials, soils, leaves and wood, but also food, textiles, paper, archives, museums and libraries, generating their degradation. it generates significant damage. their presence in food is particularly harmful due to aflatoxins, with carcinogenic potential. long-term storage of food in safe conditions, using as few as possible preservatives, is an objective of the current food industry. the aromatic plant species used as spices owe their digestive effect to essential compounds such as monoterpenoids and sesquiterpenoids. these substances are also known to have inhibitory effects on the development of both microbial and tumor cells. more recently, a novel antimicrobial peptide namely “plantaricin cs” with a wide antibacterial activity was isolated from coriander leaf extract and the greatest antimicrobial effect of it was shown on s. aureus strain but also against fusarium and aspergillus species [113]. in recent time, a new antimicrobial peptide namely “plantaricin cs” was isolated from coriander leaf extract and has shown a highly effective antifungal activity against penicillium lilacinum (mic = 2.5 mg/ml) and a. niger (mic = 2.3 mg/ml) [13]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 104 exotic spices, as well as aromatic plants in the european spontaneous flora, are tested for their popular properties, including the ability to inhibit mold growth [14]. components of these plants, such as essential oils and other substances are responsible for germicidal effects. the allelopathic effect can be extended to yeast strains and bacteria, highlighting these plants as potential alternatives to synthetic antibiotics. plants from the local flora might present a new alternative source for possible bioactive substances. the culinary herbs and spices have major advantages being inexpensive, safe (used since generations), and easily accessible. nevertheless, fractionation, purification, and isolation processes are underway with the aim to isolate and chemically modify bioactive natural compounds [15-17]. the antifungal effect of spice extracts is not limited to preserving food. it can be used in the preservation of any organic material likely to be degraded by molds, such as textiles, art objects, and construction materials. the purpose of using plant extracts can be also the reduction of synthetic fungicides, polluting to the natural environment [4, 18-20]. most of the prevalent synthetic preservatives have multiple side effects to the health and environment. in this context, plant essential oils which have been used in traditional medicine and pharmaceutical preparations are gaining interest by the food industries for the development of ecofriendly food preservatives with functional properties [21]. 2. matherials and methods isolation of fungi from local soil was performed on dichloran 18% glycerol (dg18) agar (merck, darmstadt) and dichloran rose bengal chloramphenicol (drbc) agar (merck, darmstadt). under aseptic conditions, 20 g of soil was homogenized in 180 ml of sterile peptone water (0.1 g of peptone/100 ml of distilled water). after this, samples were shaken for 10 min at 200 rpm [22]. one milliliter of the obtained stock solution was transferred into a petri plate (∅ 9 cm), in which the medium was poured and samples were incubated for 7 days at 25 ± 2 c. in order to obtain pure cultures and perform the identification, colonies that were assumed to belong to the aspergillus, mucor and penicillium genus (according to the macromorphological characteristics) were re-inoculated to the czapek yeast autolysate agar, cya (nano3 3 g; k2hpo4 1 g; kcl 0.5 g; mgso4 ּ 7h2o 0.5 g; feso4 ּ7h2o 0.01 g; yeast extract 5 g; sucrose 30 g; agar 20 g; distilled water 1000 ml). after this, samples were incubated for 7-14 days at 25 ± 2 c. the isolates assumed to belong to the genus aspergillus and mucor were inoculated to the csapek-dox agar, in order to obtain monosporic cultures. monosporic cultures were incubated for 10 and 14 days under a cyclic regime-mode with 12 h of combined light (fluorescent light and nuv e near ultraviolet) and 12 h of darkness at 25 c in order to stimulate the formation of conidiogenous structures. obtained pure cultures of fungi were identified according to the keys for determination (colony diameter, color and texture; microscopic characteristics e hyphae and conidiophore appearance, size and shape of vesicles, metulae, phialides and conidia). seven-day fungal cultures grown on czapek-dox agar were used for preparation of the fungal spore suspension tests. the spores were harvested with sterile loop in 10 ml of medium which contained 0.5 ml/100 ml tween 80 and 0.2 g/100 ml agar in sterile distilled water and aseptically transferred into sterile test tubes. the spore suspensions were adjusted with the same solution to give a final spore concentration of 106 spores / ml by using the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 105 hemocytometer. csapek-dox was the medium used for antifungal investigations. csapek-dox was utilised in the following variants: v1csapek-dox was poured in sterile petri plate (∅ 9 cm), 12 ml into each plate. plates were centrally inoculated by spotting the 1 ml of spore suspension (106 spores/ml) in the middle of the plate using an inoculation needle to give a circular inoculum of approximately 2 mm in diameter (one inoculum per plate). after inoculation, the petri plates were closed with a parafilm and incubated at 25 ± 2 c for 5 days. the filter paper rings, 6 mm in diameter, were soaked in the dilute extracts as follows: v1.1: 1:2; v1.2: 1:5, v1.3: 1:10. paper loops have been applied to fungal crops. after 24 and 48 hours of incubation, the inhibition area around the rounds is measured. v2 csapek-dox was divided into equal volumes (10 ml), poured into erlenmeyer (50 ml) flasks and autoclaved at 121 c for 15 min and then cooled to 45 c. each of the extracts were added in the liquid medium at following concentration: v2.1 – 1 ml extract in 10 ml medium, v2.2. 2 ml extract in 10 ml medium, v2.3 3 ml of extract in 10 ml medium, v2.4 without vegetal extract. after inoculation, the erlenmeyer were closed with a parafilm, incubated and shaken at 200 rpm, at 25 ± 2 c for 3 days. v3 csapek-dox medium was poured in sterile petri plate (∅ 9 cm), 10 ml into each plate. in each plate was mixed one extract in following variants: v3.1 1 ml extract mixed with 10 ml medium, v3.2 – 2 ml extract in 10 ml mediu, v3.3 3 ml extract in 10 ml medium and v3.4. without vegetal extract. v4 – csapek-dox medium was poured in sterile petri plate (∅ 9 cm), 10 ml into each plate. in all tested (for each extract concentration), plates were inoculated 1 ml of spore suspension (103 spores/ml) in zig –zag modality of entire surface of the plate, using an inoculation needle. before spores inoculated, undiluted extracts were applicated on the half of developed colonies, and the other half was preserved as witness. the effect of the each extracts on the growth of fungi and fungal count were carried out in 3 replications. extracts. ripen and dried fruits of carum carvi, coriandrum sativum and anethum graveolens were obtained by commercial sources, washed with tap water followed by distilled water to remove the dust particles. the fruits were allowed to dry at room temperature (37 0c). hundered grams of fruits were crushed and soaked in 100 ml of double distilled water. the mixtures of water and seeds were submitted for 1h to water distillation using a ser 148-type apparatus to produce an hidroextraction. also for utilised spices: twenty grams of: cinnamomum zeylanicum barks, laurus nobilis leaves, eugenia caryophyllata flower buds from local market were soaked in 100 ml of double distilled water. the mixtures of water and spices were submitted for 1h to water distillation using a ser 148-type apparatus to produce an hidroextraction. the final volume of the each extract obtained was 30 ml and were stored at refrigerator in sterile bottles for further use. 3. results and discussion in experimental variant v1, 18 petri dishes were tested. on average 7-8 colonies of aspergillus and penicillium/petri dish were grown. of the six extracts used in three proportions each, only two samples have been shown to have a visible macroscopic effect on the inhibition of fungi culture. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 106 this is the 1: 2 or 1: 3 cloves extract with the culture medium. the inhibition is reduced in the fungal colonies, the area being no more than 0.5 mm around the filter paper disc imbedded with the cloves extract. we believe that the method applicable to bacteria is not relevant for antifungal testing. in the disks used, the concentration of the extracts is much lower than an effective dose for the inhibition of mold species (table1). table 1. the observed results on the inhibition of plant hydroextracts on fungal cultures tested by the antibiotic method experimental variants /plant species v1 – 24 h v1 – 48 h v1.1 v1.2 v1.3 v1.1 v1.2 v.1.3 results ø of the inhibition zone mm ø of the inhibition zone mm cinnamomum zeylanicum 0.5 0.5 0 0.5 0.5 0 laurus nobilis 0 0 0 0 0 0 eugenia caryophyllata 0.5 0.5 0 0.5 0.5 0 carum carvi 0 0 0 0 0 0 coriandrum sativum 0 0 0 0 0 0 anethum graveolens 0 0 0 0 0 0 table 2. inhibition of fungal cultures caused by the inclusion of the hydro-extracts in the culture medium experimental variants /plant species v2 v3 v4 v2.1 v2.2 v2.3 v2.4 v3.1 v3.2 v3.3 v3.4 96h results percentage development of colonies, relative to the volume of culture medium percentage development of colonies, relative to the surface of the plate the degree of colony development % cinnamomum zeylanicum 70 50 20 100 0 0 0 100 20 laurus nobilis 75 30 50 100 70 50 30 100 15 eugenia caryophyllata 60 40 10 100 0 0 0 100 5 carum carvi 80 80 50 100 100 90 90 100 30 coriandrum sativum 80 60 50 100 100 100 90 100 50 anethum graveolens 90 70 50 100 100 100 100 100 30 fig. 1. inhibition of caraway, coriander and dill extracts at 96 hours after inoculation by including plant extracts in both the liquid culture medium and the agarized medium, the fungicidal effect of all plant extracts is evident at all concentrations tested. the inhibition of colony development relative to the control and relative to the concentration used is so evident that it can be estimated in the percentage of colony development either in the volume of the liquid medium or on the surface of the agar medium. each of the petri plates was treated on the right half with plant extracts, observing an obvious inhibitory effect thereof. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 107 the order of extracts used is the following: carum carvi, coriandrum sativum, anethum graveolens (figure 1). c in n . l a u . e u g . c a r. c o r. a n e th . 12 24 36 48 60 72 84 96 108 % c o lo n ie s d e v e lo p e d v2.1 v2.2 v2.3 v2.4 v 2 .1 v 2 .2 v 2 .3 v 2 .4 0 20 40 60 80 100 120 140 160 180 % fig. 2. inhibition of plant extracts included in the liquid medium fig. 3. variation of the inhibitory effect, depending on the concentration of the extracts in the culture medium the six species analyzed are grouped into two categories, both in terms of their climatic zones and the fungicide effect. on the one hand there are exotic spices from tropical and subtropical climates, on the other hand the species belonging to the apiaceae family, coming from the spontaneous flora of the temperate zone (figure 4, figure 7). 50 45 40 35 30 25 20 15 10 5 d is ta n c e c a r. a n e th . c o r. e u g . c in n . l a u . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 108 fig. 4. cluster analysis of the experimental variants for the six tested plant species c in n . l a u . e u g . c a r. c o r. a n e th 0 50 100 150 % o f d e v e lo p e d c o lo n ie s v3.1 v3.2 v3.3 v3.4 v 3 .1 v 3 .2 v 3 .3 v 3 .4 0 20 40 60 80 100 120 140 160 180 % o f d e v e lo p e d c o lo n ie s fig. 5. inhibition of plant extracts included in the solid medium fig. 6. variation of the inhibitory effect, depending on the concentration of the extracts in the culture medium 160 140 120 100 80 60 40 20 d is ta n c e c a r. c o r. a n e th l a u . e u g . c in n . fig. 7. cluster analysis of the experimental variants for the six tested plant species an analysis of the intensity of the inhibitory effect reveals that the most powerful effect is exotic spice extracts. the cinnamon extract introduced into the liquid medium inhibits the development of colonies in varying percentages, depending on concentration, and introduced into the agar medium, completely prevents the growth of spores and colonies (table 2, figure 2, figure 5). the previous studies reveal that also cinnamon oil in vapour phase is a strong inhibitor for fungal and bacterial growth [18, 23-26]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 109 laurel leaves have a pronounced inhibitory effect, although among the three exotic species, it is in third place (figure 7). laurus nobilis essential oils presented a good source of bioactive compounds such as 1,8-cineole, methyl eugenol, a-terpinyl acetate and linalool, which were considered as powerful bactericides [2728]. laurus nobilis etheroleum has antifungal activity probably due to the presence of the major monoterpenes and sesquiterpenes identified, likely acts interfering with the cell wall biosynthesis and ionic permeability of the membrane, and has deleterious effects on c. albicans biofilm adhesion and formation [29-31]. in the experiments carried out, the most potent inhibitor of mold species is the extract of cloves (figure 2, figure 5). in particular, the extract in all tested proportions included in the agar medium, completely inhibits spore development. clove (syzigium aromaticum l., family myrtaceae) is considered to have an enormous potential as a food preservative against spoilage and pathogenic bacteria [11, 32]. the phytochemical constituents of this plant includes eugenol, transcaryophyllene, a-humulene, eugenol acetate, syzygin a, syzygin b, caffeic acid, ferulic acid and ellagic acid [2, 7, 33]. the extract of cloves used as such in various concentrations, as well as in nanocomposites, has certain therapeutic effects [34-35]. the second group is that of the apiaceae family. this family is rich in phytochemicals and secondary metabolites which are potential source of drugs such as terpenoids, triterpenoid saponins, flavonoids, coumarins, polyacetylenes and steroids [36]. carum carvi is known to be one of the species with potential for aflatoxigenic reduction aspergilli, which are cosmopolitan fungi with air-borne conidia as infective propagules, they routinely contaminate foods, feeds and agricultural commodities such as peanuts, corn, pistachio nuts and oil seeds all over the world [37]. the results clearly show that the eos of carum carvi may have potential for use as natural preservatives in controlling aflatoxins contamination of foods, feeds and agricultural commodities in practice [38]. the antibacterial activity of carum carvi could be attributed to the high polyphenolic compounds present in the extract [9, 31]. in studies conducted, 3:10 caraway extract with culture medium, inhibits the growth of mold colonies by 50%. although the bibliographic studies show coriander extract as effective against candida, fusarium, aspergillus and penicillium strains [5-6, 8, 39-40], in our experiments, umbellifer species inhibit the growth of fungal cultures in proportions of 10-50%. at the level of use in preserving food, the effect is beneficial and consistent with similar studies. coriander essential oil could inhibit the growth of fungal in the cake and could be thus, used as a potential antifungal agent in foodstuffs especially those containing lipids [41-43]. the coriandrum sativum etheroleum showed excellent antifungal activity against seed borne pathogens of paddy: pyricularia oryzae, bipolaris oryzae, alternaria alternata, tricoconis padwickii, drechslera tetramera, drechslera halodes, curvularia lunata, fusarium moniliforme, fusarium oxysprorum [10, 20, 30, 44]. in the case of fungal strains grown in symbiotic relation with ant species, the coriander has shown an inhibitory capacity between 23, 3, and 100% of the fungal biomass [24, 45]. anethum graveolens is a widely grown species with certain aromatic qualities.various pharmacological actions of a. graveolens such as antimicrobial, antispasmodic, antidiabetic, antifood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 110 hypercholesterolaemic, and antiinflammatory have been reported [12, 4647]. in relation to saprophytic fungi, it diminishes the ability of colony development in liquid nutrient medium and also has a retarding effect on colonies (table 2). 4. conclusion all plant species, through their hydrodextracted compounds, demonstrate significant abilities in inhibiting the development of saprophytic molds. the effect is stronger on solid culture media, especially at the level of colonies developed on surfaces. the inhibitory effect is persistent over time, being visible even at 96 hours after contact with fungal colony (table 2). efficiency is better as the plant extract is more concentrated (figure 3, figure 6). the plant extracts tested can be used to impregnate solid food packaging, being able to prevent or slow the growth of mold colonies. 5. references [1]. ahmada, a., taloua, t., saadb, z., hijazib, a., meraha, o., the apiaceae: ethnomedicinal family as source for industrial uses, industrial crops & products, 109: 661–67, (2017) [2]. ajiboye, t.o., mohammed, a.o., bello, s.a., yusuf, i.i., ibitoye, o.b., muritala, h.f., onajobi, i.b., antibacterial activity of syzygium aromaticum seed: studies on oxidative stress biomarkers and membrane permeability, microbial pathogenesis, 95: 208-215, (2016) [3]. bakkali, f., averbeck, s., averbeck, d., biological effects of essential oils – a review, food chem. toxicol., 46: 446–475, (2008) [4]. bakkali, f., averbeck, s., averbeck, d., zhiri, a., baudoux, d., idaomar, m., antigenotoxic effects of three essential oils in diploid yeast (saccharomyces cerevisiae) after treatments with uvc radiation, 8mop plus uva and mms, mutat. res., 606: 27– 38, (2006) [5]. cantore, p.l., iacobellis n.s., demarco, a., capasso, f., senatore, f., antibacterial activity of coriandrum sativum l. and foeniculum vulgare miller var.vulgare (miller) essential oils, j. agric. food chem., 52: 7862– 7866, (2004) [6]. casetti, f., bartelke, s., biehler, k., augustin, m., schempp, c.m., frank, u., antimicrobial activity against bacteria with dermatological relevance and skin tolerance of the essential oil from coriandrum sativum l. fruits, phytother res., 26: 420–424, (2012) [7]. cortes-rojas, d.f., fernandes de souza, c.r., oliveira w.p., clove (syzygium aromaticum): a precious spice, asian pac. j. trop. biomed., 4: 90-96, (2014) [8]. darughe, f., barzegar, m., sahari, m.a., antioxidant and antifungal activity of coriander (coriandrum sativum l.) essential oil in cake, int food res j.,19: 1253–1260, (2012) [9]. deb roy, s., thaku, s., negi, a., kumari, m., sutar, n., jana, k.g., in vitro antibiotic activity of volatile oils of carum carvi & coriandrum sativum, int. j. chem anal sci, 1: 149– 50, (2010) [10]. duman, a.d., telci, i., dayisoylu, k.s., digrak, m., demirtas, i., alma, m.h., evaluation of bioactivity of linalool-rich essential oils from ocimum basilucum and coriandrum sativum varieties, nat prod commun., 5: 969–974, (2010) [11]. devi, k.p., nisha, s.a., sakthivel, r., pandian, s.k., eugenol (an essential oil of clove ) acts as an antibacterial agent against salmonella typhi by disrupting the cellular membrane, j. ethnopharmacol., 130: 107-115, (2010) [12]. fatope, m.o., marwah, r.g., onifade, a.k., ochei, j.e., al mahroqi, y.k.s., c13 nmr analysis and antifungal and insecticidal activities of oman dill herb oil, pharm. biol. 44: 44–49, (2006) [13]. freires, i.a., murata, r.m., furletti, v.f., sartoratto, a., de alencar, s.m., figueira, g.m., oliveira rodrigues, j.a., teixeira duarte, m.c., rosalen, p.l., coriandrum sativum l. (coriander) essential oil: antifungal activity and mode of action on candida spp., and molecular targets affected in human whole-genome expression, plos one, 9: 99086, (2014) [14]. silva, f., ferreira, s., queiroz, j.a., domingues, f.c., coriander (coriandrum sativum l.) essential oil: its antibacterial activity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 111 and mode of action evaluated by flow cytometry, j med microbiol., 60: 1479–86, (2011) [15]. jelmar, z., al-kalaldeh, f., abudahab, r., afifi, f.u., volatile oil composition and antiproliferative activity of laurus nobilis, origanum syriacum, origanum vulgare and salvia triloba against human breast adenocarcinoma cells, nutrition research, 30: 271–278, (2010) [16]. unlu, m., ergene, e., unlu, g.v., zeytinoglu, h.s., vural, n., composition, antimicrobial activity and in vitro cytotoxicity of essential oil from cinnamomum zeylanicum blume (lauraceae), food and chemical toxicology, 48: 3274–3280, (2010) [17]. lixandru, b.e., drăcea, n.o., dragomirescu, c.c., drăgulescu, e.c., coldea, i.l., anton, l., dobre, e., rovinaru, c., codiţă, i., antimicrobial activity of plant essential oils against bacterial and fungal species involved in food poisoning and/or food decay, roum arch microbiol immunol., 69: 24–230, (2010) [18]. lopez, p., sanchez, c., batlle, r., nerin, c., solid and vaporphase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains, j. agric. food chem., 53: 6939–6946, (2005) [19]. el-maatia, m.f.a.s.a., labiba, s.m., al-gabya, a.m.a., ramadan, m.f., phenolic extracts of clove (syzygium aromaticum) with novel antioxidant and antibacterial activities, european journal of integrative medicine, 8: 494– 504, (2016) [20]. mandal, s., mandal, m., coriander (coriandrum sativum l.) essential oil: chemistry and biological activity, asian pac j trop biomed., 5: 421–428, (2015) [21]. kiran, s., kujur, a., prakash, b., assessment of preservative potential of cinnamomum zeylanicum blume essential oil against food borne molds, aflatoxin b1 synthesis, its functional properties and mode of action, innovative food science and emerging technologies, 37: 184–191, (2016) [22]. kocic-tanackov, s.d., dimic, g.r., mojovi, l.v., pejin, j.d., tanackov, i.j., effect of caraway, basil, and oregano extracts and their binary mixtures on fungi in growth medium and on shredded cabbage, lwt food science and technology, 59: 426-432, (2014) [23]. friedman, m., henika, p.r., mandrell, r.e., bactericidal activities of plant essential oils and some of their isolated constituents against campylobacter jejuni, escherichia coli, listeria monocytogenes and salmonella enteric, j. food protect., 65: 1545–156, (2002) [24]. msaada, k., jemia, m.b., salem, n., bachrouch, o., sriti, j., tammar, s., bettaieb, i., jabri, i., kefi, s., limam, f., marzouk, b., antioxidant activity of methanolic extracts from three coriander (coriandrum sativum l.) fruit varieties, arabian journal of chemistry, 10: 3176–3183, (2017) [25]. ooi, l.s.m., li, y.l., kam, s.l., wang, h., wong, e.y.l., ooi, v.e.c., antimicrobial activities of cinnamon oil and cinnamaldehyde from the chinese medicinal herb cinnamomum cassia blume, am. j. chin. med., 34: 51–522, (2006) [26]. santiago-adame, r., medinatorres, l., gallegos-infante, j.a., calderas, f., gonzalez-laredo, r.f., rocha-guzman, n.e., ochoa-martinez, l.a., bernard, m.j., spray dryingmicroencapsulation of cinnamon infusions (cinnamomum zeylanicum) with maltodextrin, lwt food science and technology, 64: 571-577, (2015) [27]. merghni, a., marzouki, h., hentati, h., aouni, m., mastouri, m., antibacterial and antibiofilm activities of laurus nobilis l. essential oil against staphylococcus aureus strains associated with oral infections, pathologie biologie, 64: 29–34, (2016) [28]. peixotoa, l.r., rosalenb, p.l., ferreirra, g.l.s., freiresb, i.a., galbiatti de carvalhoa, f., castellanoa, l.r., dias de castroa, r., antifungal activity, mode of action and anti-biofilm effects of laurus nobilis linnaeus essential oil against candida spp. archives of oral biology, 73: 179–185, (2017) [29]. hassiotis, c.n., dina, e.i., the effects of laurel (laurus nobilis l.) on development of two mycorrhizal fungi, international biodeterioration & biodegradation, 65: 628-634, (2011) [30]. pawar, v.a., bhagat, t.b., toshniwal, m.r., mokashi, n.d., khandelwal, k.r., formulation and evaluation of dental gel containing essential oil of coriander against oral pathogens, int res j pharm., 4: 48–54, (2013) [31]. soares, b.v., morais, s.m., dos santos fontenelle, r.o., queiroz, v.a., vila-nova, n.v., preira, c.m.c., brito, e.s., neto, m.a.s., brito, h.s., cavalcante, c.s.p., castelo-branco, d.s.c.m., rocha, m.f.g., antifungal activity, toxicity and chemical composition of the essential oil of coriandrum sativuml. fruits, molecules, 17: 8439–8448, (2012) [32]. thippeswamya, n.b., akhilender naidu, k., achur, r.n., antioxidant and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 monica marian, oana mare roșca, lucia mihalescu, zorica voşgan, anamaria lazăr, antifungal effect of spice extracts – possible solutions for biological preservation of food, food and environment safety, volume xvii, issue 2 – 2018, pag. 103 – 112 112 antibacterial properties of phenolic extract from carum carvi l., journal of pharmacy research, 7: 352-357, (2013) [33]. santoro, g.f., cardoso, m.g., gustavo, l., guimaraes, l., mendonca, l.z., soares, m.j., trypanosoma cruzi: activity of essential oils from achillea millefolium l., syzygium aromaticum l. and ocimum basilicum l. on epimastigotes and trypomastigotes, exp. parasitol., 116: 283-290, (2007) [34]. shukri, r., mohamed, s., mohamed, n., cloves protect the heart, liver and lens of diabetic rats, food chem., 122: 1116-1121, (2010) [35]. thompson, a., meah, d., ahmed, n., conniff-jenkins, r., chileshe, e., phillips, c.o., claypole, t.c., forman, d.w., row, p.e., comparison of the antibacterial activity of essential oils and extracts of medicinal and culinary herbs to investigate potential new treatments for irritable bowel syndrome, bmc complement altern med, 13:338-342, (2013) [36]. kouki, b.l.k., m`hamdi, m., bettaieb, t., coriander (coriandrum sativum l.) and its bioactive constituents, fitoterapia, 103: 9–26, (2015) [37]. razzaghi-abyaneh, m., shamsghahfarokhi, m., razaee, m.b., jaimand, k., alinezhad, s., saberi, r., yoshinari, t., chemical composition and antiaflatoxigenic activity of carum carvi l., thymus vulgaris and citrus aurantifolia essential oils, food control, 20: 1018–1024, (2009) [38]. simic, a., socovic, m.d., ristic, m., grujic-jovanovic, s., vukojevic, j., marian, p.d., the chemical composition of some lauraceae essential oils and their antifungal activities, phytother. res. 18: 713–717, (2004) [39]. singh, g., maurya, s., de lampasona, m.p., catalan, c.a.n., studies on essential oils, part 41. chemical composition, antifungal, antioxidant and sprout suppressant activities of coriander (coriandrum sativum) essential oil and its oleoresin, flavour fragance j, 21: 472–9, (2006) [40]. morais, w.c.c., lima, m.a.p., zanuncio, j.c., oliveira, m.a., braganc, m.a.l., serrao, j.e., della lucia, t.m.c., extracts of ageratum conyzoides, coriandrum sativum and mentha piperita inhibit the growth of the symbiotic fungus of leaf-cutting ants, industrial crops and products, 65: 463–466, (2015) [41]. kubo, i., fujita, k.i., kubo., a., nihei, k.i., ogura, t., antibacterial activity of coriander volatile compounds against salmonella choleraesuis, j agric. food chem., 52: 3329–3332, (2004) [42]. shan, b., cai, y.z., brooks, j.d., corke, h., antibacterial properties and major bioactive components of cinnamon stick (cinnamomum burmannii): activityagainst foodborne pathogenic bacteria, j. agr. food chem., 55: 5484–5490, (2007) [43]. vejdani, r., shalmani, h.r.m., mirfattahi, m., sajed-nia, f., abdollahi, m., zali, m.r., alizadeh, a.h.m., bahari, a., amin, g., the efficacy of an herbal medicine, carmint, on the relief of abdominal pain and bloating in patients with irritable bowel syndrome: a pilot study, dig dis sci., 51: 1501–1507, (2006) [44]. rattanachaikunsopon, r., phumkhachorn, p., potential of coriander (coriandrum sativum) oil as a natural antimicrobial compound in controlling campylobacter jejuni in raw meat, biosci biotechnol biochem., 74: 31–5, (2010) [45]. shahverdi, a.r., monsefesfahani, h.r., tavasoli, f., zaheri, a., mirjani, r., trans-cinnamaldehyde from cinnamomum zeylanicum bark essential oil reducesthe clindamycin resistance of clostridium difficile in vitro, j. food sci., 72: 55-58, (2007) [46]. kazemi, m., chemical composition and antimicrobial, antioxidant activities and antiinflammatory potential of achillea millefolium l., anethum graveolens l., and carum copticum l. essential oils, journal of herbal medicine, 5: 217– 222, (2015) [47]. matusiak, k., machnowski, w., wrzosek, h., polak, j., rajkowska, k., smigielski, k., kunicka-styczynska, a., gutarowska, b., application of cinnamomum zeylanicum essential oil in vapour phase for heritage textiles disinfection, international biodeterioration & biodegradation 30: 1-9, (2017) 1. introduction 4. conclusion 44 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 -2019 , pag. 44 51 the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits *sorina ropciuc 1 , cristina damian 1 , ancuța elena prisacaru 1 1food engineering faculty, stefan cel mare university of suceava, romania sorina.ropciuc@fia.usv.ro *corresponding author received 17th december 2018, accepted 27th march 2019 abstract: soil analysis was determined in order to identify the type of soil through nutrient analysis and granulometric analysis, features that may occur in the fructification of the species rosa canina l. the studied area includes the plateau of suceava and obcina mare, where the lowest elevation is in the floodplain area of suceava (292 m) and in dărmănești station and the highest research point is palma with an altitude of 1080 m. the field gradient is between 0.1 ° and 13.4 °, the dominant slopes have values of 0.8 ° and 2.6 ° and they are characteristic to the plateau of suceava. the assessment of geodeclivity was obtained based on grid information using gis software. the obtained results led to the classification of the studied soils based on fertility; statistical interpretation has been made in terms of influence of soil fertility on the fructification of rosa canina l. by using statistical pca and mca programs we were able to classify soils by chemical constituents. the correlation index determined by pearson matrix showed a significant correlation level between the chemical characteristics of soil and the productivity of rosehip bushes. keywords: chemical constituents, biometric characters, granulometry, productivity. 1. introduction as environmental factor, soil represents a major importance because it influences directly or indirectly the production potential of the station. taking into account the soil, rosehip is one of the least demanding species that vegetate even on poor and dry grounds [1, 2]. it achieves good growth on compact, clayey and clayloamy soils, slightly acidic, neutral to stony, skeletal, eroded or subject to erosion, and even on great slopes exposed to insolation rosa canina l. harnesses well any type of soil from soils which are usually medium textured, with skeletal, reduced content to the alluvial soils from plains, stuffy and humid [3, 4]. in our country rosa canina l. is the most popular of the many species of rosa. it is common in all parts of the country, from the black sea up to an altitude of 1100 – 1200 m. the studied area is represented by the route suceava–palma and it includes the following stations: suceava, pătrăuţi, dărmănești, costîna, părhăuţi, todireşti, cajvana, arbore, solca, clit, marginea, rădăuţi, suceviţa, and palma. from a geomorphological point of view, the studied territory includes obcina mare and suceava plateau, the lowest elevation stands in the meadow of suceava 292 m and in dărmăneşti station and the highest research point is palma, with an altitude of 1080 m. the field gradient is between 0.1° and 13.4° and the dominant slopes have values of 0.8° and 2.6° and they are characteristic to the suceava plateau. stations with higher gradients are found in suceava and in the following stations: părhăuţi, arbore and clit which are located on sloping grounds. http://www.fia.usv.ro/fiajournal mailto:sorina.ropciuc@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 45 2. material and methods for the analysis of the chemical constituents and for the granulometric analysis, soil samples were taken from each studied station and were collected during the vegetative rest period (early march). thus, average samples were formed from the surface layer (0-20cm). for the analysis of the biometric characteristics, the rosehip beerries were harvested by hand randomly, from 42 biotypes marked on the field, in rosehip bushes respectively. of each biotype, there were harvested 10 berries; only the whole, healthy, fully riped berries were harvested. the harvested samples were labeled and analyzed in a laboratory. 2.1. soil analysis methods soil samples were gathered with a probe with a prior dismission of the vegetation layer. from each station there were collected average samples of 1 kg. the soil samples have been processed in the laboratory for pedological analyses. thus, the samples were dried in the air for 24 hours and then the foreign parts have been removed (roots, other plant remanents, stones). then the following operations grinding, mixing and screening took place. chemical constituents foodstuffs in the soil samples were determined by standard analysis methods of soil. 2.2. fruit analysis methods the biometric measurements of rosehip fruit were made on freshly picked fruit during the years. thus, from 3 bushes per station there were harvested randomly 10 berries each, i.e. a total of 30 berries per station. the measurements were made with callipers and the length of each berry was determined and the value was expressed in mm. the weight of the fruits was determined by weighing 100 g of randomly harvested berries from three different bushes. 2.3. statistical analysis using statistical analysis computer packages excel and xlstat (version 2017, addinsoft, u.s.a.), the data were statistically processed via analysis methods of main elements (pca) and the method of multiple variables analysis (mca). 3. results and discussion the analysis of soil collected from the studied stations was made for the chemical and granulometric analysis of soil, two values that can interfere in the productivity of bushes and in the biometric changes of the pseudofruit of the species rosa canina l. the results of the soil analysis from the point of view of nutrients and of granulometry are shown in table 1 (a, b) and table 2. from the point of view of the reaction of soil in aqueous solution, cajvana presents a strongly acid reaction, the soils in todireşti and pătrăuţi are strongly acidic, the soils in părhăuţi and clit are moderately acidic, the soils in costîna, solca, arbore, palma and rădăuţi are slightly acidic, the soils in suceava, dărmăneşti, marginea and suceviţa present a neutral to a weak alkaline reaction (figure 3). the humus content (%) in the 0-20 cm layer ranges between 0.4% (clit) and 5,94% (palma) and indicates extremely low values of humus content. the total nitrogen indicates limits between low and high content (0,019% clit and 0,313% palma). the granulometric composition was statistically processed by analyzing the main components in order to obtain accurate information on the division of the stations according to this variable. the analysis of the main components divides the analyzed stations into two planes, on the left and on the right of the f1 axis, figure 4. on the right of the f1 axis there are grouped the stations that significantly correlated with coarse sand, fine sand and ph. the best correlation with coarse sand takes place at dărmăneşti station and in the same plane can be found pătrăuţi, sucevița, rădăuți and marginea. rădăuți station correlates with fine sand. on the left of the f1 vertical axis, arbore station significantly correlates with clay whereas palma and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 46 costîna significantly correlate with physical clay. dărmăneşti station is distinguished by its high content in coarse sand (43.13%) while marginea presents the highest values of fine sand content (76.47%). soil classification according to the fertility. table 1. the analysis results of nutrients in soil (a,b) and granulometric analysis stations carbonates% humus% nt(s.u.) % p-al ppm k-al ppm sb me/100g soil acidity/alcalinity suceava 3.41 3.49 0.166 45.3 250 0 7.48 patrauti 0 3.87 0.193 11.7 80 6.83 4.99 darmanesti 0 4.77 0.227 17.8 96 0 7.12 costina 0 4.16 0.218 64.9 134 29.98 6.15 parhauti 0 1.96 0.109 13.2 105 17.1 5.4 todiresti 0 2.6 0.140 30.1 74 11.47 5.38 cajvana 0 2.78 0.138 39.7 53 5.63 4.34 arbore 0 3.26 0.163 10.7 161 21.33 6.38 solca 0 3.11 0.153 6.7 117 19.32 6.04 clit 0 0.4 0.019 10 51 7.44 5.17 marginea 0.21 0.87 0.039 32.6 46 0 7.29 radauti 0 2.91 0.132 7.8 166 17.1 6.56 sucevita 2.98 3.84 0.175 37 265 0 7.31 palma 0 5.94 0.313 17.3 238 31.99 6.33 nt (total nitrogen); p-al (mobile phosphorus); k-al (mobile potassium); sb (sum of basic cations); sh (total exchange acidity); ahme (hydrolytic acidity); sh (total exchange acidity); ah (hidrolitic acidity); al (changeable aluminium according); ctss (total mineral content); ce (electrical conductivity); u (umidity); i.n. (nitrogen index). the infield bonitation is a complex action of research and quantitative estimation of the main conditions that determine growth and fruit-bearing, of establishing the favourability of these conditions for each use and culture (as a field may be unfavourable to certain uses and cultures but favourable to other ones). since soil bonitation and the determination of their suitability for the studied species is more laborious and more difficult, soil assessment was carried out in the 14 stations based on the results obtained from analyses. the optimal level of the analysed results received the mark 3, the average level 2, the satisfactory level 1 and the unsatisfactory level 0, according to the data in table 3. stations sh me/100g ah me/100g v-ah % al me/100g c.e simens/cm ctss mg/100g u % i.n suceava 0 0 0 0 174.71 111.81 3.11 0 patrauti 8.57 7.31 48.3 3.11 102.3 65.47 3.96 1.87 darmanesti 0 0 0 0 108.32 69.32 3.07 0 costina 7.73 6.01 83.3 0 90.85 58.14 3.08 3.46 parhauti 16.97 15.21 52.92 0.21 37.56 24.04 3.11 1.04 todiresti 11.03 9.98 53.47 0.17 55.91 35.78 3.21 1.39 cajvana 10.98 8.97 38.56 8.2 40.18 25.72 3.17 1.07 arbore 6.16 5.2 77.59 0 78.62 50.32 3.08 2.62 solca 12.41 9.5 67.04 0 61.15 39.13 3.02 2.08 clit 8.56 7.69 49.17 0.63 25.32 16.21 3.17 1.02 marginea 0 0 0 0 141.52 90.57 3.2 0 radauti 5.01 3.99 77.34 0 45.42 29.07 3.12 2.36 sucevita 0 0 0 0 203.07 129.96 3.1 0 palma 8.9 6.88 78.23 0 115.54 73.95 3.14 4.89 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 47 the analysis results may highlight the following: the soil in palma has a very good fertility. a satisfactory fertility can be found in suceava, dărmăneşti, costîna, todireşti, arbore, solca, marginea, rădăuţi and suceviţa. table 2. the results of the granulometric analysis ng (coarse sand); nf (fine sand); p (dust), a (clay), af (phisical clay) table 3. the relative fertility potential of soils specification s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 s14 ph 3 0 3 2 1 1 0 3 2 1 3 3 3 3 humus 1 1 2 1 0 1 1 1 1 0 0 1 1 3 nitrogen in 0 0 0 1 0 0 0 1 1 0 0 1 0 2 phosphorus 2 1 1 3 1 2 2 1 0 1 2 0 2 1 potassium 3 1 1 2 1 1 0 2 1 0 0 2 3 3 average 1.8 0.6 1.4 1.8 0.6 1.0 0.6 1.6 1.0 0.4 1,0 1,4 1,8 2,4 s1suceava; s2patrauti ; s3darmanesti; s4costina; s5parhauti; s6todiresti; s7cajvana; s8arbore; s9 solca; s10clit; s11marginea; s12radauti; s13sucevita; s14palma given that the studied species are not part of cultivated plants, and they are rather spontaneous species that can be located on different categories of land, we must emphasize the fact that we are dealing with a natural fertility of soils with a satisfactory level and less demanding species in terms of soil fertility of soils with a satisfactory level and less demanding species in terms of soil fertility. as an environmental factor, soil has a major importance directly or indirectly influencing the productive potential of the station. regarding the soil, rosehip is one of the least demanding species that vegetate even on poor, dry soil. stations granulometric analysis ng nf p a af suceava 4.26 44.1 20.67 30.97 39.12 patrauti 10.08 42.62 25.57 21.73 33.65 darmanesti/ lunca sucevei 43.13 39.55 8.51 8.81 12.96 costina 7.43 44.12 28.76 19.69 32.09 parhauti 2.28 46.12 27.99 23.61 36.21 todiresti 3.28 50.53 27.27 18.92 30.9 cajvana 5.11 48.96 25.57 20.36 32.71 arbore 4.57 37.75 30.93 26.75 42.07 solca 2.57 41.55 29.33 26.55 40.03 clit 1.76 41.74 33.77 22.73 37.21 marginea 10.61 76.47 6.15 6.77 8.96 radauti 8.28 49.86 20.47 21.39 30.41 sucevita 7.63 48.11 19.84 24.42 33.2 palma 3.9 31.3 32.53 32.27 49.71 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 48 according to the studies in the field, rosehip achieved good growth in compact, loamy or clay-loamy soils, slightly acidic to neutral and even in stony, skeletal, eroded or subject to erosion soils. on the other hand, rosehip does not resist in wetlands, alluvial soils in meadows and mires [5-7]. the granulometric analysis offers the possibility of determining soil texture. for example, this analysis shows the fact that in suceava station, the soil texture comprises: coarse sand 4.26%, fine sand 44.10%, dust 20.67%, clay 30.97%. comparing these values to the chart for textural classes, it results the fact that the soil belongs to the structural clay class with a medium clay type. soil acidity is emphasized by the results of the analysis of exchangeable aluminium in soil. from this point of view, the situation is critical in cajvana (8.2 me/100g) and pătrăuţi (3.11 me/100g). in this situation it is recommended immediate action in order to correct soil reaction by applying amendments and avoiding administration of nitrogenous fertilizers, which increase the acidity of soil [8, 12]. overall, from the point of view of soil reaction (ph), the studied soils can be regarded as good ones. determining humus content is of great importance because it helps to identify the presence of nitrogen in soil. fig. 1. the relationship between soil granulometric features within the stations suceava-palma this is explained by the organic matter which contains more than 90% of the soil nitrogen. moreover, based on the content of humus and the degree of base saturation, the value of the nitrogen index (in) was calculated. the results of the biometric data show that the length of beans on the suceava palma route records a higher ph-ul ng nf dust clay suceava pătrăuţi dărmăneşti costâna părhăuţi todireşti cajvana arbore solcaclit marginea rădăuţi suceviţa palma -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 f 2 ( 2 1 ,1 0 % ) f1 (75,85 %) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 49 amplitude of variation within the same station and a narrower amplitude at the inter population level. thus, the average length of the bush is between 17.1 mm (minimum) in the resort of clit and 31.3 mm (maximum) in the todiresti resort (measured in 2010, the results of the measurements are not shown). biotopes from arbore, clit, marginea and palma resorts have shorter grain lengths, averaging between 17.1 mm (minimum) and 2.0 mm (maximum) [10,11]. within the analysed populations, a mass of 100 grains was determined in terms of quantity, which contributes to the appreciation of fruit quality and the productivity of bushes. biotopes do not differ significantly in the length of the grain. we noted the small value of grain weights in stations 4 and 8 (fig. 2). fig. 2. the weight of 100 grains in terms of the influence of soil composition on biometric features, it is estimated that phosphorus is a stimulating factor for grain growth and pulp ratio in the fruit [12, 13, 15]. acidic soils and those with high nitrogen index do not positively influence the length of the fruit and, implicitly, low weight. table 3. the correlation matrix between the soil features and the biometric features at rosehip fruits variabile p-al ppm nf sb me/100g sh me/100g ah me/100g v-ah u % i.n nf 0.264 sb -0.082 -0.472* sh -0.291 -0.288 0.545* ah -0.297 -0.260 0.493* 0.994*** v-ah -0.258 -0.445 0.907*** 0.662** 0.620* u -0.175 0.028 -0.195 0.092 0.109 -0.008 i.n -0.113 -0.515* 0.936 0.432 0.372 0.877*** 0.022 avg l, 2009 0.344 0.156 -0.295 0.138 0.140 -0.297 0.218 -0.332 avg l, 2010 0.323 0.219 -0.248 0.388 0.410 -0.170 0.152 -0.360 avg, g 2009 0.111 0.125 -0.620** 0.033 0.057 -0.543 0.412 -0.540* avg, g 2010 0.340 0.106 -0.223 0.403 0.435 -0.167 0.212 -0.327 studies on rosa canina l. harvested at altitudes of 1800 m in new zeeland present length values ranging between 17, 29 and 19.68 mm [14]. the results obtained by weighing 100 freshly harvested beans range in the interval confirmed by other studies as well. the indicated weight was of 1.884.95 g/fruit [12]. the pearson correlation matrix between the soil composition and the medium length of the fruit, the weight of 100 berries and the percentage of pulp in fruit, indicates positive correlations 50 100 150 200 250 300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 w e ig h t , g station food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 50 between mobile phosphorus in soil and fruit length in 2007, r = 0,558* according to the detailed data in table 4. phosphorus correlates significantly positively with the fruit weight, r = 0.698** and correlates significantly with the percentage of pulp in fruit r = 0.489*. the soil fine sand content correlates with the content of pulp in fruit in 2007 and 2008. the basic cations correlate significantly negatively with the berries weight r = -0.620* and with the percentage of pulp in fruit r = -0.443*. hydrolytic soil acidity correlates significantly negatively with the average weight of 100 berries r = -0.543*. the nitrogen index correlates significantly negatively with the fruit weight r = -0.540* in 2008. the physiology research is confirmed by the fact that nitrogen and its components increase vegetative organs, while phosphorus affects the growth of the reproductive organs. 4. conclusions under the influence of soil composition on biometric features, it is estimated that phosphorus is a stimulating factor for grain growth and pulp ratio in the fruit. the acidic and high nitrogen index does not positively influence the development of fruit and, implicitly, low weight and poor fruit pulp. rosehip is ruderal species, ecological role, being included in restoring forest ecosystems, wildlife conservation, the development of ecotourism, planning and the gaps in hardwood stands. depending on the resistance to anthropogenic factors, species rosa canina l. urbanofobic moderate in nature with a tendency towards non-urban areas was recovered well irrespective of soil type oil, with sandy loam soil preferences. under the aspect of soil composition, the influence on biometric features it is estimated that phosphorus is a grain growth stimulating factor. acid soils and high nitrogen index positively influence hawthorn fruit length and thus lead to lower values and relative weight of the fruit pulp decrease. hilly area stations provide fruits with higher values of biometric features in the sub-mountainous. 5. references [1]. celik, f., kazankaya, a. and ercisli s., 2009. fruit characteristics of some selected promising rose hip (rosa spp.) genotypes from van region of turkey. african journal of agricultural research 4 (3), 236-240 [2] celik f, kazankaya a, ercisli s., 2009. fruit characteristics of some selected promising rose hip (rosa spp.) genotypes from van region of turkey. afr j agric res;4(3):236–240 [3]. ahmed, j., gȕvenc, a., 2009. morfological and anatomical investigation on the rose hips sold in some herbalists in ankara. turkish journal of pharmaceutical sciences, vol 6/2: 135 146. [4]. cvetkovic, b., filipčev, b.v., bodroza-solarov, m., bardic, ž.m., sakač, m.b, 2009. chemical composition of dried fruits as a value added ingredient in bakery product. food &feed research, 36 (1-2):15-20. [5]. güner, ş. t. , özkan, k., çömez, a., 2011. key factors in the site selection of rosa canina (l.)applying generalized additive model. polish journal of ecology 599(3), 475-482 [6]. damian c., 2013. a study on biodiversity in agriculture, international scientific symposium „modern agriculture – achievements and prospects”, 80th anniversary of state agrarian university of moldova,chișinău, october 09-11 [7]. ercişli s, güleryüz m (2006) fruit properties of promising rose hips (rosa spp.) from the northeastern anatolia region of turkey. asian j chem 18(1):239–242 [8]. güner, ş. t. , özkan, k., çömez, a. , 2011. key factors in the site selection of rosa canina (l.)applying generalized additive model. polish journal of ecology 599(3), 475-482. [9]. šindrak, z., jemrić, t., baričević, l., han dovedan, i, fruk, g., 2012. fruit quality of dog rose seedlings (rosa canina l). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue – 1, 2019 sorina ropciuc, cristina damian, ancuța elena prisacaru, the relationship between physico-chemical characteristics of ground and biometrics rosehip fruits, food and environment safety , volume xviii, issue 1 – 2019, pag. 44 – 51 51 journal of central european agriculture 13(2), 321330 [10]. soare r., bonea d., iancu p., niculescu m., 2015. biochemical and technological properties of rosa canina l. fruits from spontaneous flora of oltenia, romania. bulletinuasvm horticulture 72(1) / 2015 [11]. soare r, iancu p, soare m, bonea d, manila g, panita o, 2014. morfological and biochemical issues to some rosa canina l. populations from the spontaneous flora from soutwest romania. micro and nano tehnologies advances in biotechnology 1:432-442 [12]. dincu, i., lăcătușu, gh., 2002. technological bases of agricultural crops (in romanian), editura ceres [13]. ercişli s., eşitken a., 2010. fruit characteristics of native rose hip (rosa spp.) selections from the erzurum province of turkey. new zealand journal of crop and horticultural science. [14]. mabellini a & al., 2011. chemical and physical characteristics of several wild rose species used as food or food ingredient, international journal of industrial chemistry, 2(3), 158-171 [15]. leahu, a., gutt, s., gutt, g., hretcanu, c., 2011. the impact of fertiliser types on soil's macronutrients and microlements content, environmental engineering, vols 13,pages: 182-187, 8th international conference environmental engineering, vilnius, lithuania, may 19.5-20.5/ 2011. http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&excludeeventconfig=excludeiffromfullrecpage&sid=r2cis9jyijjukjma5gv&field=au&value=leahu,%20a&cacheurlfromrightclick=no http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&excludeeventconfig=excludeiffromfullrecpage&sid=r2cis9jyijjukjma5gv&field=au&value=gutt,%20s http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&excludeeventconfig=excludeiffromfullrecpage&sid=r2cis9jyijjukjma5gv&field=au&value=gutt,%20g http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&excludeeventconfig=excludeiffromfullrecpage&sid=r2cis9jyijjukjma5gv&field=au&value=gutt,%20g http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&excludeeventconfig=excludeiffromfullrecpage&sid=r2cis9jyijjukjma5gv&field=au&value=hretcanu,%20c 260 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 260 266 tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression daniela pauliuc 1 , paula ciursa1, florina dranca 1 , sorina ropciuc 1 , *mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, m.oroian@fia.usv.ro, *corresponding author received 5th october 2020, accepted 29th december 2020 abstract: the aim of this study was to assess the usefulness of fourier transform infrared (ftir) spectroscopy coupled with partial least squares regression (pls-r) to predict the fructose, glucose and sucrose content of tilia honeys. in order to achieve the aim of this study, 22 samples of tilia honey were purchased from suceava, neamt and iasi county in the year of 2020. the fructose, glucose and sucrose content was determined prior the pls-r prediction using highperformance liquid chromatography coupled with refractive index detector (hplc-rid). the fructose content of tilia honeys ranged from 31.94 to 35.22%, the glucose content ranged between 26.76 and 33.15%, while sucrose content ranged between 0 and 2.20%. for the prediction of fructose, glucose and sucrose it was used the 3000 2800 + 1700 1600 +1540 700 cm-1 spectral range. the spectral data was submitted to different mathematical pretreatments in order to reduce the noise and to improve the prediction of results. for the prediction of fructose content the suitable pretreatment was multiplicative scatter correction – msc, for glucose prediction the suitable pretreatment was standard normal variate – snv, while for the prediction of sucrose the suitable pretreatment was 1st derivate. keywords: tilia honey, fructose, glucose, sucrose, ft-ir, prediction 1. introduction honey is the oldest sweetener used by humankind and is worldwide known by its taste, color, aroma, and medicinal and nutritional properties as antioxidant, antiinflamatory, antitumor, anticancer and antimicrobial [1]. it is considered that honey has more than 200 substances as: sugars, organic acids, proteins, aminoacids, enzymes, vitamins, minerals, phenolic compounds and some solid particles (e.g. wax, pollen or other particles originating from its harvesting) [2,3]. the sugar composition of honey is a complex mixture of mono-, di-, tri-, oligo and polysaccharides, but the main sugars present are fructose and glucose [4]. the main method used to determine the sugar composition of honey is high-performance liquid chromatography coupled with refractive index detector (hplc-rid) [5]. fourier transform infrared (ft-ir) spectroscopy presents information from frequencies of fundamental molecular vibrations and mostly reveals sharp peaks and different spectral characteristics [6,7]. fourier transform infrared spectroscopy was used in many non-destructive methods for the characterization of honey and to determine different parameters, as follows: moisture content, sugar composition, free acidity, authentication of the botanical origin of honeys or to estimate the phenolic compounds and antioxidant capacity [7-10]. partial least squares regression (pls-r) is a multivariate regression method used for the complex data (e.g. ft-ir spectra, raman http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 261 spectra, large number of data, uv-vis spectra) and physicochemical data [11-13]. the pls-r models are able to provide reduced models constructed on the original data in order to reduce data complexity [13, 14]. in this study is presented the prediction of fructose, glucose and sucrose from tilia honeys using ft-ir data with partial least squares regression (pls-r). 2. materials and methods materials 22 samples of tilia honeys were collected from suceava, neamt and iasi county from 2020 production. methods the fructose, glucose and sucrose content were determined using a high-performance liquid chromatography (hplc) method based on the protocol of bogdanov & baumann [4]. ft-ir analysis an atr-nicolet is-20 spectrometer (thermo scientific, karlsruhe, dieselstraße, germany) was used for honey spectra acquisition in the mid-infrared region of 4000-650 cm-1 with a resolution of 4 cm-1. the sample was placed on the atr crystal and the spectra were collected using omnic software (version 32, thermo scientific). statistical analysis partial least squares regression (pls-r) was realized using the unscrambler x 10.1 software (camo, norway). 3. results and discussion sugar content in table 1 are presented the mean, minimum and maximum concentrations of fructose, glucose and sucrose from the analyzed tilia honey samples. as it can be observed, the fructose concentration was higher in all samples, while sucrose content ranged between 0 and 2.20 %. the concentrations of fructose + glucose were higher than 60% in all the honey samples therefore meeting the threshold established by the eu legislation [15]. the fructose/glucose ratio was higher than 1 which confirmed the liquid state of the analyzed samples. table 1. fructose, glucose and sucrose content in tilia honeys parameter mean min max fructose (%) 33.14 31.94 35.22 glucose (%) 28.79 26.76 33.15 sucrose (%) 0.36 0 2.20 f/g 1.15 fig. 1. ft-ir spectra of tilia honeys food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 262 table 2. regression parameters of the calibration and validation procedure calculated for the ft-ir spectral data submitted to partial least square regression (pls-r) analysis in order to predict glucose, fructose and sucrose in tilia honey food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 263 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 264 fig. 2. reference vs predicted fructose, glucose and sucrose content using ft-ir spectra and pls-r ft-ir spectra the ft-ir spectra collected for the analyzed honey samples are shown in figure 1. according to the scientific literature, the spectral range typical for sugars in honeys are 3000-2800 + 1700-1600 +1540-700cm-1 as it can be observed in figure 1, the signal for the spectral range presented above is high in the tilia honey samples that were analyzed in this study. pls-r prediction the pls-r model was used for the prediction of fructose, glucose and sucrose based on ft-ir spectral data. as it can be seen in figure 1, the noise recorded for the honey samples was high and to reduce it the spectra must be submitted to different mathematical procedures, as follows: multiplicative scatter correction – msc, standard normal variate – snv, baseline, normalization, 1st derivate, 2nd derivate, smoothing (table 2). as it can be observed in table 2, each parameter predicted has a different pretreatment: fructose – multiplicative scatter correction – msc, glucose – standard normal variate – snv and sucrose – 1st derivate. the suitable model for each parameter is highlighted in table 2 with different colours. the calibration and validation coefficients for the fructose and sucrose were higher than 0.861 proving the suitability of the proposed models, and in the case of glucose, the calibration parameters have a good magnitude, however, the validation parameters cannot be considered satisfactory. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 265 for improving the correlation coefficients of the glucose prediction it would be necessary a higher number of samples. in figure 2 is presented the evolution of reference and predicted values for fructose, glucose and sucrose. 4. conclusion in this study it was determined the concentration of fructose, glucose and sucrose in 22 samples of tilia honeys from suceava, neamt and iasi county. as it was observed, the concentration of fructose was higher than that of glucose in all the samples, while the concentrations of fructose + glucose met the threshold established by the ue legislation. the ftir spectra of the tilia honey samples were used for the prediction of glucose, fructose and sucrose content. the pretreatment of the ft-ir spectra increased the values of the prediction parameters. the fructose and sucrose prediction and validation parameters were satisfactory (r2 > 0.860), while the glucose parameters where not satisfactory in the validation step. for improving the correlation coefficients of the glucose prediction, a higher number of samples would be necessary. 5. acknowledgment this work was supported by a grant of romanian ministry of education and research, cncs-uefiscdi, project number pn-iii-p1-1.1-te-2019-0583, and by the romania national council for higher education funding, cnfis, project number cnfis-fdi-2020-0615. 6. references [1]. fernandes, l., ribeiro, h., oliveira, a., silva, a. s., freitas, a., henriques, m., & rodrigues, m. e. portuguese honeys as antimicrobial agents against candida species. journal of traditional and complementary medicine, (2020). [2]. da silva, p. m., gauche, c., gonzaga, l. v., costa, a. c. o., & fett, r. honey: chemical composition, stability and authenticity. food chemistry, 196, 309-323, (2016). [3]. pereira, j. r., campos, a. n. d. r., de oliveira, f. c., silva, v. r., david, g. f., da silva j. g., ... & denedai, â. m. physicalchemical characterization of commercial honeys from minas gerais, brazil. food bioscience, 100644, (2020). [4] pascual-maté, a., osés, s. m., marcazzan, g. l., gardini, s., muiño, m. a. f., & sancho, m. t. sugar composition and sugar-related parameters of honeys from the northern iberian plateau. journal of food composition and analysis, 74, 34-43, (2018). [5] bogdanov, s., martin, p., & lullmann, c. harmonised methods of the international honey commission. swiss bee research centre, fam, liebefeld. (2002). [6] schönbichler, s. a., falser, g. f. j., hussain, s., bittner, l. k., abel, g., popp, m., ... & huck, c. w. comparison of nir and atr-ir spectroscopy for the determination of the antioxidant capacity of primulae flos cum calycibus. analytical methods, 6(16), 6343-6351, (2014). [7] tahir, h. e., xiaobo, z., zhihua, l., jiyong, s., zhai, x., wang, s., & mariod, a. a. rapid prediction of phenolic compounds and antioxidant activity of sudanese honey using raman and fourier transform infrared (ft-ir) spectroscopy. food chemistry, 226, 202-211, (2017) [8] anjos, o., campos, m. g., ruiz, p. c., & antunes, p., application of ftir-atr spectroscopy to the quantification of sugar in honey. food chemistry, 169, 218-223, (2015) [9] zhu, x., li, s., shan, y., zhang, z., li, g., su, d., & liu, f., detection of adulterants such as sweeteners materials in honey using near-infrared spectroscopy and chemometrics. journal of food engineering, 101(1), 92-97, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian, tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, food and environment safety, volume xix, issue 4 – 2020, pag. 260 266 266 [10] jandrić, z., haughey, s. a., frew, r. d., mccomb, k., galvin-king, p., elliott, c. t., & cannavan, a., discrimination of honey of different floral origins by a combination of various chemical parameters. food chemistry, 189, 52-59, (2015). [11] jović, o., smolić, t., primožič, i., & hrenar, t., spectroscopic and chemometric analysis of binary and ternary edible oil mixtures: qualitative and quantitative study. analytical chemistry, 88(8), 4516-4524, (2016). [12] monfreda, m., gobbi, l., & grippa, a. blends of olive oil and seeds oils: characterisation and olive oil quantification using fatty acids composition and chemometric tools. part ii. food chemistry, 145, 584-592., (2014). [13] li, s., ng, t. t., & yao, z. p., quantitative analysis of blended oils by matrix-assisted laser desorption/ionization mass spectrometry and partial least squares regression. food chemistry, 334, 127601, (2020). [14] wold, s., sjöström, m., & eriksson, l., pls-regression: a basic tool of chemometrics. chemometrics and intelligent laboratory systems, 58(2), 109-130, (2001). [15] council directive 2001/110/ec of 20 december 2001 relating to honey 1. introduction art 97 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 12018, pag. 97 102 study on total phenolic content in some romanian forest mushroom species, before and after heat treatment marcel avramiuc 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received 17th november 2017, accepted 23th march 2018 abstract: the purpose of this work was to study the phenolic compounds in ten forest mushroom species to see if and to what extent the thermal processing can modify the content of these compounds. the biological material consisted of ten forest mushroom species, colected from suceava county area: agaricus campestris, morchella esculenta, cantharellus cibarius, armillaria mellea, lactarius deliciosus, boletus edulis, pleurotus cornucopiae, russula vesca, sparassis crispa, ramaria aurea. the experiment consisted in mushrooms thermal processing (boiling for 30 and 60 minutes, and roasting for 10 minutes at 160°c), followed by the analysis of total phenolic content (tpc) which values were compared to the ones obtained from raw samples. as compared to raw material, the boiling process led to a significant increase of percent of tpc between 13.71% and 31.91% (after 30 min. of boiling) and between 12.33% and 61.36% (after 60 min. of boiling). as compared to raw material, the roasting of mushrooms for 10 minutes (at 160°c) led to significant increases of the phenolic compounds between 29.31% and 95.23% in all samples. high tpc values recorded by some species may be explained by the specific tissue structure of these species, which promotes a penetration and a more intense heat action on bound phenols. keywords: phenolic content, mushroom, boiling, roasting 1. introduction widespread in plants and present in many species, phenolic compounds highlight various properties, from antioxidant, antimicrobial, anti-inflammatory, antiallergenic to cardio-protective and vasodilatory ones [16]. there is a relationship between the consumption of phenolic-rich food products and a low incidence of coronary heart disease, atherosclerosis, certain forms of cancer and stroke [7-10]. according to huang et al., 1992 [11], due to their antioxidant properties, plant phenolic compounds have potential benefits for health. plant species and varieties influence the content of phenols and of other metabolites [1216]. lately, a number of scientific papers have revealed the presence of some mineral elements (ca, mg, p, cd, se), vitamins (b1, b2, b12, niacin, folates, c, d), flavonoids, lignans and phenolic acids in different species of cultivated mushrooms [17]. also, have been reported some mushroom species used to prevent hypertension, hypercholesterolemia and even cancer [18, 19, cited by 20), due to the presence of chitin [21], and of beta glucans with β (1-3), β (1-4) and β (1-6) glycosidic linkages [22, 23 cited by 20]. the heat processing influences the concentration and quality of food nutrients http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 marcel avramiuc, study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, food and environment safety, volume xvii, issue 1 – 2018, pag. 97 102 98 (carbohydrates, lipids, proteins), but there are less information, in this regard, on natural antioxidants and their activity. cooking induces changes in physiological and chemical composition, influencing the concentration and bioavailability of bioactive compounds in food, the thermal treatments decreasing the total phenolics in squash, peas and leek [24]. dewanto et al., 2002a [25] showed that in sweet corn, cooking led to an increase in the level of phenolic compounds. this paper studies the phenolic compounds in ten forest mushroom species in order to see if and to what extent the thermal processing can modify the content of these compounds. 2. experimental 2.1. research material and samples preparation the biological material was represented by ten forest mushroom species, colected from suceava county area: agaricus campestris, morchella esculenta, cantharellus cibarius, armillaria mellea, lactarius deliciosus, boletus edulis, pleurotus cornucopiae, russula vesca, sparassis crispa, ramaria aurea. all these species are edible and tasteful, being consumed with pleasure by people. from each species they have prepared control (raw material) and thermal processed samples. 2.2. procedure and research methods the experiment consisted in thermal processing (boiling and roasting), whose results were compared to those ones obtained from raw samples. for boiling 30 minutes and 60 minutes, they have taken (for each procedure) 50 g mushrooms of each sample, which have been placed in a stainless steel vessel of two liters capacity. the boiling has done in one liter of tap water in the pot covered, for 30, respectively 60 minutes (timed from the moment when the water began to boil). for roasting, 50 g of each sample were heated for 10 minutes at 160°c, in an electric oven. in order to determine total phenolic content (tpc), first an extract for each sample (raw or thermal processed) was obtained, weighing 10 g mushrooms, which were ground and subjected to extraction with a mixture methanol and water (80/20), by stirring, centrifuging and recovering the supernatant. the estimation of total phenolic contents in extract was carried out through a colorimetric assay, by measuring its reducing capacity with folinciocalteu reagent [26, 27]. tpc was expressed as mg gallic acid equivalent/100 g matter (mg gae/100g). for this purpose, a standard curve was generated, representing the absorbance values of gallic acid standard solutions in relation to their concentrations [28]. because during boiling, some of the cellular compounds can pass into the boiling water, tpc was dosed from both mushroom samples and boiling water, calculating the total amount for each sample. 2.3. statistical analysis the data of experiments, coming from four replicates of each determination, were statistically processed, using sas version 8.02 [29]. to analyze the significance of differences among samples, generalized linear model analysis was carried out. for multiple comparisons duncan’s multiple range test was used (p<0.05). 3. results and discussion in the table 1 are rendered the values of total phenolic content (tpc), in ten forest mushroom species. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 marcel avramiuc, study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, food and environment safety, volume xvii, issue 1 – 2018, pag. 97 102 99 table 1 comparative tpc mean values (±sd) in raw and thermal processed forest mushrooms test tpc (mg gae/100 g) samples (species) raw material boiled (30 min.)* boiled (60 min.)* roasted (10 min.) agaricus campestris 23.25±1.82 c 27.70±0.78 bc 29.67±1.23 b 35.82±0.72 ab morchella esculenta 18.43±1.07 cd 22.06±1.25 c** 26.03±1.84 bc 29.56±0.88 b cantharellus cibarius 16.55±0.96 d 20.12±1.47 c** 22.85±0.39 c 27.43±1.09 bc armillaria mellea 28.74±1.35 b 34.34±0.93 ab 37.23±0.96 ab 40.27±1.94 a lactarius deliciosus 19.88±0.78 cd 23.53±0.74 c 23.66±1.02 c 28.09±0.48 b boletus edulis 22.07±0.54 c 27.30±1.43 bc** 27.49±0.45 bc** 30.05±2.01 b pleurotus cornucopiae 22.38±1.36 c 24.89±2.01 c 25.14±1.58 bc 28.94±1.65 b russula vesca 24.66±0.97 c 28.04±1.44 bc 29.27±0.91b 33.67±1.03 ab sparassis crispa 17.39±1.04 cd 22.94±1.09 c 28.06±1.35 bc 33.95±0.57 ab ramaria aurea 20.12±0.89 c 26.15±0.53 bc 31.43±0.48 b 38.44±1.22 a sd=standard deviation; *sum of boiled sample and its boiling water; **means with the same letters within a row or a column are not statistically diferent (p<0.05) as seen from the table, in the raw material the values of tpc ranged between 16.55±0.96 (cantharellus cibarius) and 28.74±1.35 mg gae/100 g (armillaria mellea). beetween these extreme values, there are some close ones (without significant differences among them) in five species (agaricus campestris, boletus edulis, pleurotus cornucopiae, russula vesca and ramaria aurea) on one hand, and in other three species (morchella esculenta, lactarius deliciosus and sparassis crispa), on the other hand. as as result of boiling process, the tpc values showed modifications depending on boiling duration and species. thus, after 30 minutes the highest tpc value was registered by armillaria mellea, followed by agaricus campestris, boletus edulis, russula vesca and ramaria aurea, with close values (p<0.05). the other species registered the least values and close between them. after 60 minutes the highest tpc value was registered by armillaria mellea, followed by agaricus campestris, russula vesca and ramaria aurea, with close values, and by morchella esculenta, boletus edulis, pleurotus cornucopiae and sparassis crispa. cantharellus cibarius and lactarius deliciosus had the least values (p<0.05). the roasting process caused the highest modifications of tpc values in all mushroom species. the greatest tpc values was registered by armillaria mellea and ramaria aurea, followed by agaricus campestris, russula vesca and sparassis crispa (with close values). the other four species registered the least values. comparing the tpc of mushrooms thermal processed with raw materials, one can observe a raising of tpc values after heating in all analyzed samples (p<0.05). the table 2 highlights the mushrooms tpc increase percents after boiling and roasting. thus, compared to raw materials, boiling for 30 minutes led to a significant raising percent of tpc (except pleurotus cornucopiae) beetween 13.71% (russula vesca) and 31.91% (sparassis crispa). compared to raw materials, 60 minutes of boiling determined significant raising percent of tpc in all samples, the great values being registered in sparassis crispa and ramaria aurea, and the least one in pleurotus cornucopiae. from 30 to 60 minutes of boiling were four species whose tpc values have not singnificantly modified (cantharellus cibarius, armillaria mellea, lactarius deliciosus and boletus edulis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 marcel avramiuc, study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, food and environment safety, volume xvii, issue 1 – 2018, pag. 97 102 100 table 2 tpc mean values raising percent in mushroom heated samples, compared to raw material samples (species) tpc raising percents boiled (30 min.)* boiled (60 min.)* roasted (10 min.) agaricus campestris 19.14% 27.60% 54.00% morchella esculenta 19.70% 41.24% 60.40% cantharellus cibarius 21.57% 38.10% 65.74% armillaria mellea 19.48% 29.54% 40.12% lactarius deliciosus 18.36% 19.00% 41.30% boletus edulis 23.70% 24.56% 36.16% pleurotus cornucopiae 11.22% 12.33% 29.31% russula vesca 13.71% 18.69% 36.54% sparassis crispa 31.91% 61.36% 95.23% ramaria aurea 29.97% 56.21% 91.05% *sum of boiled sample and its boiling water investigating the effect of thermal treatment on corn, xu and chang, 2009 [30], found changes of phenols caused by liberating of free forms from bound phenols. the results of the thermal treatment (boiling) applied to samples of the ten mushroom species, analysed in this work, are consistent with data reported by choi et al, 2006 [31], which searching the effect of heat treatment (at 100°c and for 15 or 30 min.) on shitake (lentinus edodes) mushroom extracts, found the polyphenolic contents and antioxidant activities increased as heating temperature and time increased. although the increase in tpc mean values on heating could be attributed to the release of free phenolics from combinations, the differences between the studied species could be explained by the difference in structure between these species. for example, some species have their body composed by thin and wavy formations (sparassis crispa) or by a number of vertical and cylindrical branches (ramaria aurea), which increase the contact surface of the internal tissues with the heat agent, favoring a more intense heat action on cellular compounds, including bound phenols. as to mushrooms roasting, the highest raising percent was in sparassis crispa, followed by ramaria aurea, and the least one in pleurotus cornucopiae. the thermal treatment causes phytochemical degradation, oxidation, and maillard reactions resulting in changes in antioxidant property [32]. by lin et all [33], maillard reaction products may protect phytochemicals from oxidation, and can maintain or even enhances the overall antioxidant properties of food products [34]. heat treatment at 150°c for 40 min. liberated bound phenolics in citrus peels having as result a significant increasing of tpc after treatment [35]. heating at 121°c for 30 min shitake mushroom extracts, choi et al, 2006 [31] found the free polyphenolic content increased by 1.9-fold compared to that in the extract from the raw sample. 4. conclusions the thermal processing of mushrooms, belonging to ten species colected from suceava county area, significantly influenced their total phenolic content (tpc). thus, as compared to raw material, the boiling process led to a significant increase in percent of tpc between 13.71% and 31.91% (after 30 min. of boiling) and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 marcel avramiuc, study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, food and environment safety, volume xvii, issue 1 – 2018, pag. 97 102 101 between 12.33% and 61.36% (after 60 min. of boiling). compared to raw material, the roasting of mushrooms for 10 minutes (at 160°c) evidenced in all samples, significant increases of the phenolic compounds between 29.31% and 95.23%. it seems that heating at this temperature, for 10 minutes, led to an increase in the free phenols content by releasing bound phenols. high tpc values recorded by some species may be explained by the specific tissue structure of these species, which promotes a penetration and a more intense heat action on bound phenols. 5. references [1]. benavente-garcia o., castillo j.,marin f.r., ortuno a., del rio j.a. uses and properties of citrus flavonoids. journal of agricultural and food chemistry, 45, 4505-4515 (1997) [2]. samman s., lyons wall p.m., cook n.c. flavonoids and coronary heart disease: dietary perspectives. in c.a. rice-evans & l. packer (eds.), flavonoids in health and disease (pp. 469-482), new york: marcel dekker (1998) [3]. middleton e., kandaswami c., theoharides t.c. the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. pharmacological reviews, 52, 673-751 (2000) [4]. puupponen-pimiä r., nohynek l., meier c., kähkönen m., heinonen m., hopia a., et al. -antimicrobial properties of phenolic compounds fom berries. journal of applied microbiology, 90, 494-507 (2001) [5]. manach c., williamson g., morand c., scalbert a., rémésy c. bioavailability and bioefficacy of polyphenols in humans. i. review of 97 bioavailability studies. american journal of clinical nutrition, 81 (suppl), 230s – 242s (2005) [6]. balasundram n., sundram k., samman s. phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. food chemistry 99, 191-199 (2006) [7]. hertog m.g.l., fesrens e.j.m., hollmann p.c.h., katan m.b., krombout ddietary antioxidant flavonoids and risk of coronary heart disease: the zutphen elderly study. lancet 342, 1007-1011. (1993) [8]. diaz m.n., frei b., vita j.a, keaney j.f. antioxidants and atherosclerotic heart disease. new engl. j. med., 337, 408-416 (1997) [9]. ito n., hirose m. antioxidantscarcinogenic and chemo-preventive properties. adv. cancer res. 53, 247-302 (1989 [10]. ness a.r., powles j.w. fruit and vegetables and cardiovascular disease: a review. int. j. epidemiol. 26, 1-13 (1997) [11]. huang m.t., ho c.t., lee c.y. phenolic compounds in food and their effects on helth ii: antioxidants and cancer prevention. american chemical society symposium series 507. washington, dc: american chemical society, pp 2-7 (1992) [12]. abdel-aal e.-s.m., hucl p. a rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats. cereal chem., 76, 350–354 (1999) [13]. zhao z.h., egashira y., sanada h. phenolic antioxidants richly contained in corn bran are slightly bioavailable in rats. j.agric.food chem., 53, 5030–5035 (2005) [14]. pedreschi r., luis c.-z antimutagenic and antioxidant properties of phenolic fractions from andean purple corn (zea mays l.). j. agric. food chem., 54, 4557–4567 (2006) [15]. li w., wei c.v., white p.j., beta t. high-amylose corn exhibits better antioxidant activity than typical and waxy genotypes. j. agric. food chem., 55, 291–298 (2007) [16]. lopez-martinez l.x., oliart-ros r.m., valerio-alfaro g., lee c.-h., parkin k.l., garcia h.s. antioxidant activity, phenolic compounds and anthocyanins content of eighteen strains of mexican maize. lwtfood sci. technol. 42, 1187–1192 (2009) [17]. mattilla p., könkö k., eurola m., pihlava j.m., astola j., vahteristo l., hietaniemi v., kumpulainen j., valtonen m., piironen v. contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. agric food chem. 2001, may, 49(5): 2343-8 (2001) [18]. bobek p., galbavy s. hypocholesterolemic and antiatherogenic effect of oyster mushroom (pleurotus ostreatus) in rabbit. nahrung, 43(5), 339-342 (1999) [19]. bobek p., ozdyn l., kuniak l. the effect of oyster mushroom (pleurotus ostreatus), its ethanolic extract and extraction residues on cholesterol levels in serum lipoproteins and liver of rat. nahrung, 39, 98-99 (1995) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 marcel avramiuc, study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, food and environment safety, volume xvii, issue 1 – 2018, pag. 97 102 102 [20]. manzi p., aguzzi a., pizzoferrato l. nutritional value of mushrooms widely consumed in italy. food chemistry 73, 321-325 (2001) [21]. manzi p., aguzzi a., vivanti v., paci m., pizzoferrato l. mushrooms as a source of functional ingredients. euro food chem x european conference on: functional foods. a new challenge for the food chemist. 22-24 september, budapest, hungary, vol. i, 86-93 (1999) [22]. manzi p., pizzoferrato l beta glucans in edible mushrooms. food chemistry, 68, 315-318 (2000) [23]. mullins j.t. regulatory mechanism of βglucan synthetases in bacteria, fungi and plants. physiological plantaurm, 78, 309-314 (1990) [24]. turkmen n., sari f., velioglu y.s. the effect of cooking methods on the total phenolics and antioxidant activity of selected green vegetables. food chem., 93, 713-718 (2005) [25]. dewanto v., wu x., liu r.h. processed sweet corn has higher antioxidant activity. journal of agricultural and food chemistry, 50, 4959-4964 . (2002a) [26]. adom, k.k., liu r.hantioxidant activity of grains. j. agric. food chem. 50, 6182–6187 (2002) [27]. dewanto v., wu x., adom k.k., liu r.h. thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. j. agric. food chem. 50, 3010-3014 (2002b) [28]. moore jeffrey and yu liangli methods for antioxidant capacity estimation of wheat and wheat-based food products in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 147-150 (2008) [29]. sas institute sas user’s guide. statistical analysis system institute, cary, nc (2005) [30]. xu b. and chang s.k.c. total phenolic, phenolic acid, anthocyanin, flavan-3-ol, and flavonol profiles and antioxidant properties of pinto and black beans (phaseolus vulgaris l.) as affected by thermal processing. journal of agricultural and food chemistry, 57, 4754–4764 (2009) [31]. choi, y., lee, s.m., chun, j., lee h.b., lee j. influence of heat treatment on the antioxidant activities and polyphenolic compounds of shiitake (lentinus edodes) mushroom. food chemistry, vol. 99, issue 2, 381-387 (2006) [32]. cheng z., su l., moore j., zhou k., luther m., yin j.j., yu l. j. agric. food chem., 53, 2433–2440 (2006) [33]. lin c.j., guo g., mennel d.l. effects of postharvest treatments, food formulation, and processing conditions on wheat antioxidant properties in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 78-79 (2008) [34]. slavin j.l., jacobs d., marquart l. crit. rev. food sci. nutr., 40 (4), 309-327 (2000) [35]. jeong s.-m., kim s.-y., kim d.-r., jo s.c., nam k.c., ahn d.u., lee s.-c. effect of heat treatment on the antioxidant activity of extracts from citrus peels. journal of agricultural and food chemistry, 52, 3389-3393 (2004) 156 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 22020, pag. 156 169 feedstocks used for production of 2nd and 3rd generation bioethanol review *vasile-florin ursachi 1, gheorghe gutt1 1faculty of food engineering, ”ștefan cel mare” university, suceava, romania, florin.ursachi@fia.usv.ro *corresponding author received 23th aprilie 2020, accepted 27th june 2020 abstract: global biofuel production has increased significantly over the last decade, but firstgeneration biofuels have been identified as a major concern, especially their sustainability as they are produced from food crops (such as cereals, sugar cane and vegetable oils). depending on the feedstocks and cultivation technique, the production of second and third generation biofuels has the potential to provide benefits, such as the recovery of residues and unusable land. therefore, second and third generation biofuels are indicated to meet the increasing demand for energy and contribute considerably to the development of rural areas and the increasing of bioeconomy. this short review shows that there may be different types of feedstocks (agricultural residues, forest residues, energy crops and algae) which can be used for the production of 2nd and 3rd generation bioethanol without affecting food security. keywords: celullose, hemicelullose, starch, lignin, bioethanol 1. introduction the feedstocks used to obtain biofuels are plants and cereals that are intended for human consumption. lignocellulosic biomass (lcb) is easily accessible worldwide and is found in the form of residues and agricultural biomass such as corn straw, wheat straw and rice straw. the production of biofuels aims to protect the environment, to meet new energy requirements, to reduce the import and production of conventional fuels, thus stimulating the development of agriculture [1]. second-generation biofuels are largely derived from lcb, which includes most plant-based, non-food materials that are inexpensive and found in huge quantities. currently, the production of secondgeneration biofuels is not cost-effective because it requires overcoming technical barriers to obtaining them. in terms of bioethanol production, lcb is one of the most abundant and least used resources. lcb is usually burned for the production of heat and electricity, although it could be used to produce liquid biofuels. however, the production of biofuels from agricultural by-products could only meet a certain proportion of the growing demand for liquid biofuels, crops dedicated to the production of lcb are an important solution for the production of biofuels [2]. compared to first generation biofuels which are mostly obtained from corn or sugar cane, biofuels obtaining from lcb is more expensive because lignocellulosic materials have a complex structure and require a specific technological process [3, 4]. microalgae production is the key to the development of the third generation of bioethanol, as they could provide an alternative in terms of biomass production [5, 6]. algae are also the fastest growing plants on earth. depending on how they are recovered, algae can be used to produce biofuels such as biodiesel, http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 157 bioethanol, but also other valuable substances [7]. 2. feedstocks used in the production of second generation bioethanol (2ndg) in the food industry, significant quantities of non-food lignocellulosic biomass can be used to produce 2ndg bioethanol, thus making it a promising alternative for fossil fuels. lignocellulosic biomass is one of the most abundant renewable resources on earth and has a relatively low price [8]. lignocellulosic feedstocks are renewable and cheap sources. these can come from the forestry, agricultural fields (grains, wheat straw, rice straw and sugar cane), agro-industrial as well as significant quantities of food residues [9]. lcms are made of cellulose, hemicellulose and lignin which form a complex structure and are resistant to physico-chemical and biological treatments. one of the best strategies is to convert sugars from lcb by enzymatic hydrolysis because it does not require high energy consumption and is a clean process. however, it should be noted that the enzymatic step in the technological process of obtaining bioethanol also has a disadvantage, namely that related to the rigid structure between cellulose and lignin. therefore, in order to facilitate enzymatic hydrolysis and implicitly to obtain high concentrations of cellulose, a pretreatment step is required, being considered a key step in obtaining an increased bioethanol yield from lcms [10]. in literature several methods of pretreatment are described and are known as [10]: physical pretreatment (milling and milling, microwave oven and extrusion); chemical pretreatment (alkaline, acid, organosolvent, ozonolysis and ionic liquid); physico-chemical pretreatment (steam explosion, hot water, afex ammonia fiber explosion, wet oxidation and co2 explosion); biological pretreatment. cellulose cellulose (c6h10o5)n is a carbohydrate found in agricultural and woody biomass [10]. it is a linear polymer composed of glucose (d-glucose) molecules that have β (1,4) -glycoside bonds [11, 12]. cellulose is insoluble in water therefore, a hydrolysis process must be applied to convert this polysaccharide into glucose molecules [13]. general hydrolysis of cellulose produces only glucose, which can be transformed into different forms of biochemical and chemical substances. various biochemical and chemical substances such as bioethanol, organic acids, glycerol, sorbitol, mannitol, fructose, enzymes and biopolymers can be obtained through biological processes [14, 15]. figure 1 shows the enzymatic hydrolysis of cellulose and the enzymes involved in this process. hemicellulose hemicellulose (c5h8o4)n is a short polymer that has a branched structure, comprising sugars such as pentoses (dxylose and l-arabinose) and hexoses (dglucose, d-mannose and d-galactose) [16]. hemicelluloses are found in plants in the form of xyloglucans or xylans hemicelluloses are present in woody biomass, softwood and hardwood [17]. due to its branched structure, hemicellulose is easier to hydrolyzate as opposed to cellulose. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 158 fig 1. enzymatic hydrolysis of cellulose [18] xylose can be used to obtain xylitol. xylitol is a non-carcinogenic sweetener, with the same sweetening power of sucrose. xylose can be transformed with the help of microorganisms into proteins, fuels and solvents. there are certain yeast strains that can ferment xylose and transform it into bioethanol (pichia stipitis, candida sheratae) [15]. lignin lignin [c9h10o3 (och3)0,9–1.7]n is an organic compound and has a branched structure consisting of 3 different monomers (coniferyl alcohol, synapyl alcohol and pcoumaryl alcohol) [19]. lignin is a barrier in the fermentation process of lcb and is resistant to chemical and biological degradation. also, its presence affects the yield of bioethanol [20]. by utilizing lignin, carbon fibers, emulsifiers, dispersants, sequestrants, surfactants, binders and other chemicals can be obtained [21]. the chemical composition (%) and the main constituents of lcms are shown in figure 2. 2.1. agricultural residues, municipal solid residues and different types of grass agricultural residues (corn cobs, corn stover, sugarcane bagasse, rice straw, and wheat straw) are important sources for 2ndg bioethanol production. the grain harvesting period is relatively short and so these residues are available throughout the year. each year, between 350 and 450 million tonnes of crops are harvested, resulting in huge quantities of agricultural residues [18]. for example, up to 1 3 tons of straw can grow from 1 acre area grown with wheat. from the cost point of view, the price of sugar cane and maize rises to 60.9 usd / tonne respectively, 185.9 usd / tonne, while those for the sugarcane bagasse and corn stover the price is much lower, of only 36.4 usd / tonne and 58,5 usd / tonne respectively [22]. one should know that almost 70% of the cost of bioethanol production is represented by the cost of obtaining the feedstocks [23]. therefore, for half the costs it would be preferable to use agricultural residues and not to use energy crops. by capitalizing these residues, forestry and arable land held by herbaceous plants (switchgrass, miscanthus) would be reduced. municipal solid residues and residues from food industry have been studied for ethanol production [23, 25], because it has an important carbohydrate content, and the protein and mineral content can support the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 159 fermentation process. the study by matsakas et al. (2014) showed that food residues can be transformed successively into bioethanol after a double fermentation. after the enzymatic hydrolysis and also the fermentation phase completed, an ethanol content of 43 g / l was obtained. then, a microwave-assisted hydrothermal pretreatment was applied to the remaining solid residue and again subjected to fermentation.after the second fermentation was completed, an alcohol content of 59 g / l of ethanol was obtained [26]. fig.2. chemical composition (%) and main constituents of lcm [24] switchgrass is a feedstock that has high glucose content, is highly resistant to disease and has high biomass productivity. miscanthus giganteus is another type of grass that can be used for biofuel production, especially as it has a fast growth rate. it is native to asia, but it is also cultivated in europe. this grass represents 50 70% of the total biomass feedstocks (including forest wood biomass and agricultural residues) that are used for the production of cellulosic biofuels [18]. it was estimated that approximately 133 × 109 l of ethanol could be produced if 9.3% of us agricultural land were cultivated with this plant, thus 1/5 of the country's gasoline consumption could be replaced [27]. scagline-mellor et al. (2018) argue that bioethanol yield is higher for miscantus compared to switchgrass [28]. in the mediterranean area, lignocellulosic materials are found that can be used for the production of 2ndg bioethanol. these raw materials include: cereal crops, olives, tomatoes, grapes and residues resulting from the processing of grapes, solid residues of olives, "date" palm trunks, perennial lignocellulosic herbs (arundodonax, saccharum spont. aegyptiacum and miscanthusus giganteus) or the cactus species luffa cylindrica and luffa prickly pear. stipa tenacissima or esparto grass belongs to the family poaceae; it is a perennial plant that has a fast growth rate. the leaves of this plant have high fiber content [29] and can reach up to 1 m in height. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 160 stipa tenacissima bushes have a circular and homogeneous shape when they are young, but as they age they dry out and in the center empty spots form. the leaves are thin, ribbon-shaped, smooth, shiny, and solid and at the base they are covered by a hairy sheath. esparto leaves reach maturity between the fourth and eleventh months after flowering, depending on the geographical area and climatic conditions [30]. stipa tenacissima is spread over an area of about 3 million hectares in algeria [31] and over 400 thousand hectares in tunisia, located mainly in the ksserine, sidi-bouzid, gafsa and kairouan regions. for about four decades, the alfa plant has been considered of great importance for the production of fibers intended for the manufacture of paper. for example, every year tunisia produces an amount of alpha pulp in excess of 30,000 tonnes [30, 32]. given that stipa tenacissima is a plant that has adapted to the semi-arid climate and does not require large quantities of water to grow, it is an important source for bioethanol production. the central-western part of tunisia faces water shortages, and by cultivating energy plants that require significant quantities of water would put huge pressure on food crops. specifically, various authors argue that in terms of adaptation, but also environmental sustainability, it would be advisable to grow energy-tolerant drought plants, such as sweet sorghum [33]. table 1 shows the quantities of some lcms and their potential for bioethanol production. table 2 presents a series of lcms with their main constituents. table 1 the worldwide available quantity of the main agricultural residues and their potential for bioethanol production [34] feedstock for 2 nd g bioethanol worldwide quantities of agricultural residues (tg) potential bioethanol production (gallons) total bioethanol (gallons) gasoline equivalent (gallons) corn grain residue 20.7 14.38 72.98 52.4 corn stover 203.62 58.6 barley grain residue 3.66 2.46 20.56 14.8 barley straw 58.45 17.1 oat grain residue 0.55 0.39 3.17 2.27 oat straw 10.62 2.78 rice grain residue 25.44 16.8 221.4 159 rice straw 731.34 204.6 wheat grain residue 17.2 11.33 115.13 82.71 wheat straw 354.35 103.8 sorghum grain residue 3.12 2.14 4.93 3.54 sorghum straw 10.32 2.79 sugarcane residue 3.2 1.59 52.89 38 sugarcane bagasse 180.73 51.3 2.2. forest wood biomass forest wood biomass is known as one of the most promising renewable feedstocks for the production of 2ndg bioethanol. wood biomass can be obtained from maintenance or forestry exploitation. this has a high energy value and the acquisition costs are low, therefore it could be used for bioethanol production [41]. in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 161 the us, woody biomass accounts for about 30% of total biomass used annually to generate bioenergy [42]. the wood forest materials used in the usa generally come from 3 species of resinous pinus contorta, pseudotsuga menziesii and pinus ponderosa. these conifer species have a high content of hemicellulose (18 33%) and cellulose (39 55%) [42]. nearly 90% of the dry weight of forest wood biomass is composed of lignin, hemicellulose, cellulose and pectin [43]. specifically, woody biomass comprises 30 60% cellulose, 15 40% hemicellulose and 10 25% lignin [17, 22, 37]. table 2 chemical composition for different lcms [35, 36] feedstock carbohydrate compositions (%) references cellulose hemicellulose lignin sugarcane tops 35 32 14 [37] sugarcane bagasse 32 48 19 25 23 32 [36, 38] corn stover 38 40 26 28 7 21 corn cob 45 35 15 [38] sorghum stalks 27 25 11 [39] sorghum straw 32 24 13 sweet sorghum bagasse 34 45 18 28 14 22 [36, 39] barley straw 31 45 27 38 14 19 [39] rice straw 28 38 23 32 12 14 [36, 39] rice husk 37 29 24 [40] wheat straw 33 41 20 32 13-20 [36, 40] cotton, flax, etc. 80 95 5 20 [36, 39] coir 36 43 0.15 0.25 41 45 [39] switchgrass 40 45 30 35 12 [17] leaves 15 20 80 85 0 [38] grasses 2543 8 50 8 30 [17, 23] agriculture residues 37 50 25 50 5 15 [17] industrial residue from chemical pulp 50 80 20 30 2 10 [17, 36] newspaper 40-55 25 40 18 30 [39] paper residues 65 13 1 [40] there are 35 species of the genus populus that have a fast growth rate and can produce large quantities of woody material that can be used to obtain bioethanol 2ndg. the harvest time of forest wood biomass is more flexible as compared to agricultural residues. forest residues, such as dry trees, wood chips and sawdust, could be an important feedstock that can be converted into bioethanol [17]. the carbohydrate content as well as of other wood extractable substances and the bark of the various trees are presented in the tables 3, 4, 5 and 6. 3. feedstocks used in the production of third generation bioethanol (3rdg) currently, the use of algae biomass for 3rdg biofuel production is of high interest, as well as investments in the biofuel, petroleum and agri-food industries. it has been proven that major biofuel producing countries, such as the us, europe and asia, cannot produce sufficient quantities of corn, soy or rapeseed for their biofuel targets. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 162 table 3 proportion of major main constituents of wood [47] sample of wood holocellulose (%) cellulose (%) hemicellulose (%) lignin (%) beech 82.5 46.7 35.8 20.7 birch 84.2 45.4 38.8 17.7 alder 77.2 44.1 33.1 22.0 maple 80.1 44.6 35.5 24.9 spruce 77.8 50.0 27.8 26.5 pine 73.1 47.3 25.8 25.6 oak 69.4 39.1 30.3 22.8 table 4 content of carbohydrates and other extractable substances (%) of some trees in north america [44, 45] scientific name common name x a g ga m ua ac lg ash hardwoods acer rubrum red maple 19 0.5 46 0.6 2.4 3.5 3.8 24 0.2 acer saccharum sugar maple 15 0.8 52 <0.1 2.3 4.4 2.9 23 0.3 betula alleghaniensis yellow birch 20 0.6 47 0.9 3.6 4.2 3.3 21 0.3 betula papyrifera white birch 26 0.5 43 0.6 1.8 4.6 4.4 19 0.2 fagus grandifolia beech 19 0.5 46 1.2 2.1 4.8 3.9 22 0.4 platanus occidentalis sycamore 15 0.6 43 2.2 2.0 5.1 5.5 23 0.7 populus deltoides eastern cottonwood 15 0.6 47 1.4 2.9 4.8 3.1 24 0.8 populus tremuloides quaking aspen 17 0.5 49 2.0 2.1 4.3 3.7 21 0.4 quercus falcata southern red oak 19 0.4 41 1.2 2.0 4.5 3.3 24 0.8 softwoods abies balsamea balsam fir 6.4 0.5 46 1.0 12 3.4 1.5 29 0.2 gingo biloba ginko 4.9 1.6 40 3.5 10 4.6 1.3 33 1.1 larix laricina tamarack 4.3 1.0 46 2.3 13 2.9 1.5 29 0.2 picea abies norway spruce 7.4 1.4 43 2.3 9.5 5.3 1.2 29 0.5 picea glauca white spruce 9.1 1.5 45 1.2 11 3.6 1.3 27 0.3 picea mariana black spruce 6.0 1.5 44 2.0 9.4 5.1 1.3 30 0.3 picea rubens red spruce 6.2 1.4 44 2.2 12 4.7 1.4 28 0.3 pinus resinosa red pine 9.3 2.4 42 1.8 7.4 6.0 1.2 29 0.4 pinus rigida pitch pine 6.6 1.3 47 1.4 9.8 4.0 1.2 28 0.4 pinus sylvestris scots pine 7.6 1.6 44 3.1 10 5.6 1.3 27 0.4 pinus taeda loblolly pine 6.8 1.7 45 2.3 11 3.8 1.1 28 0.3 pseudotsuga menziesii douglas-fir 2.8 2.7 44 4.7 11 2.8 0.8 32 0.4 thuja occidentalis northern white cedar 10.0 1.2 43 1.4 8.0 4.2 1.1 31 0.2 tsuga canadensis eastern hemlock 5.3 0.6 44 1.2 11 3.3 1.7 33 0.2 xxylose, aarabinose, gglucose, gagalactose, mmannose, uauronic acids, acacetyl, lglignin 3.1. macroalgae and microalgae carbohydrate percentages for seaweed depend on the species and hydrolytic treatment used. these sugars can be fermented by microorganisms and converted into bioethanol and / or biobutanol [49]. researches on brown algae have shown that from 50 g / l sugar the ethanol yield is 7.0 9.8 g / l, and the fermentation process lasted for 40 hours (acidic medium) [50]. in the case of acid hydrolysis of green macroalgae (ulva), a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 163 content of 15.2 g sugars/l was obtained and the average yield of butanol was 4 g/l [50]. table 5 content of carbohydrates and other extractable substances in the bark of some trees [44, 46] species x a g ga m rh ua ac abies amabilis 3.2 3.2 37.4 1.6 8.0 5.6 0.8 picea abies 4.8 1.8 36.6 1.3 6.5 0.3 picea engelmannii 3.8 3.3 35.7 2.4 2.9 8.0 0.5 pinus contoria inner bark 3.7 10.6 40.9 4.3 2.5 9.9 0.2 outer bark 3.4 5.5 26.8 4.2 2.5 7.7 0.8 pinus sylvestris 5.8 2.1 30.2 2.4 5.4 0.3 pinus taeda inner bark 2.1 5.6 21.3 3.1 2.5 0.3 4.6 outer bark 3.8 1.8 15.8 2.5 2.6 0.1 2.1 betula papyrifera inner bark 21.0 2.7 28.0 1.0 0.2 2.2 fagus sylvatica 20.1 3.1 29.7 3.1 0.2 1.2 quercus robur 16.4 2.0 32.3 1.3 0.5 0.5 xxylose, aarabinose, gglucose, gagalactose, mmannose, rhrhamnose, uauronic acids, acacetyl table 6 the content of fermentable sugars from hydrolyzed biomass and the ethanol content resulting from fermentation [48] mixed sugar content ethanol content sample amount (2/1) percentage (%) amount (g/l) percentage (%) sawdust 18.20 36.40 8.51 17.02 corn residues 19.24 38.48 8.99 17.98 in recent years it has been found that microalgae are a promising starting material for bioenergy production, because they have a high content of carbohydrates that can be used for the purpose of obtaining bioethanol and biobutanol, respectively a lipid content that could be used to obtain biodiesel. also, a series of gaseous biofuels, such as biomethane and biohydrogen, can be produced from microalgae or their residues (after obtaining bioethanol and biodiesel) [51]. unlike plants that do not grow in the aquatic environment, microalgae do not have biopolymers like lignin and hemicelluloses in the chemical structure [52 54]. under specific conditions, the biomass formed from the microalgae can undergo a hydrolysis step and the carbohydrates can be fermented by the yeasts in bioethanol [55]. in the case of fermentation of microalgae biomass for the release of fermentable sugars it is possible not to use chemical and enzymatic pretreatments. it is known that in the case of cellulose feedstocks by applying these pretreatments, significant amounts of energy are consumed. however, mechanical pretreatments are still needed to disintegrate algal cells by various techniques [56]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 164 different microalgae species, such as chlamydomonas sp., chlorella sp., spirulina sp., spirogyra sp. also dunaliella sp. can be used to obtain 3rdg bioethanol because they have a starch content of about 64% [57]. another important aspect is that they have a fast development rate, high photosynthesis activity and high co2 absorption capacity [58]. liyamen and ricke (2012) concluded that microalgae can produce about 10 times more bioethanol than maize on the cultivation surface. in recent years, with the help of genetic engineering, species of microalgae have been created that have a higher carbohydrate content, resulting in higher yields of 3rdg bioethanol. for example, chlamydomonas reinhardtii and chlorella vulgaris jsc-6 cultivated under controlled conditions had a carbohydrate content of 71% and 54%, respectively [59 60]. table 7 carbohydrate content of different algae and microalgae species [61] algal species carbohydrate content (%) reference c. vulgaris 20.99 55.0 [62, 63] chlorella sorokiniana 35.67 [64] chlorella minutissima 61 [65] chlorella homosphaera 54 [66] chlamydomonas reinhardtii utex 90 60.0 [67] spirulina platensis 30.21 30.21 [64] spirulina platensis leb 52 65 [66] scenedesmus dimorphus 21 52 [69, 70] scenedesmus obliquus 46 [71] scenedesmus ecosystem 42 53 [72] nannochloropsis oceanica 22.70 [73] spirogyra sp. 33 64 [74] porphyridium cruentum 40 57 ulva lactuca 55-60 [75] dunaliella salina 32 [70] dunaliella tertiolecta 21.69 [76] tetraselmis sp. 24 [77] porphyra 40-76 [78] palmaria 38-74 [79] also, several researchers claim that from microalgae a bioethanol yield can be obtained with values between 0.240 and 0.888 g ethanol / g substrate, at 25 30 ° c [56, 80, 81]. laboratory research has shown that bioethanol yield from biomass formed from microalgae under optimal conditions is about 65% [56]. chlorella vulgaris biomass was enzymatically hydrolyzed, and the resulting carbohydrates were fermented by saccharomyces cerevisiae and converted to ethanol. the yields obtained for sugars following hydrolysis and ethanol were 0.55 and 0.17 g / g biomass respectively [82]. after extracting from schizochytrium sp. lipids and proteins, the remaining carbohydrates (dglucose and l galactose), were transformed by escherichia coli ko11 into bioethanol. following the fermentation process of the concentration of 25.7 g / l glucose, a yield of 11.8 g ethanol / l was obtained [83]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 165 it is estimated that in a year, between 5,000 15,000 gallons of ethanol / acre (46,760 140,290 l / ha) can be produced from microalgae [84]. table 7 shows the carbohydrate content of algae by species. table 8 shows the bioethanol yield for different algae and microalgae. table 8 bioethanol yield for different species of algae and microalgae [84] feedstock bioethanol reference chlorococcum infusionum 260 g ethanol/kg algae [85] spirogyra 80 g ethanol/kg algae [86] chlorococcum humicola 520g ethanol/kg microalgae [87] chlamydomonas reinhardtii utex 90 11.73 ethanol g/1 [88] chlamydomonas reinhardtii 29.2 % [89] chlamydomonas fasciata 19.4 [90] chlorella vulgaris 11.66 % ethanol g/1 [91] arthrospira platensis 16% g etoh per g of dry biomass. [91] 4. conclusion by reviewing the current state of research regarding biofuel production, the following conclusions can be drawn: every year, huge quantities of lignocellulosic materials (lcm) are generated from agriculture, which instead of being wasted can be converted into second-generation 2ndg bioethanol. the forestry sector generates a huge amount of wood biomass which is relatively cheap and can be used for bioethanol production. the efficiency of lignocellulosic feedstocks depends mainly on their availability and composition (cellulose, hemicellulose, lignin, ash). in order to obtain high yields of carbohydrates, respectively bioethanol 2ndg, it is indicated that different pretreatments described in the literature should be applied on lignocellulosic biomass (lcb). recent research has shown that algae / microalgae are a source of biomass from which significant quantities of thirdgeneration bioethanol (3rdg) and biodiesel could be produced. 5. references [1]. eckerta c.t., frigob e.p., albrechtc l.p., albrechtc a.j.p., christa d., santosc w.g., berkembrockc e., egewarth v.a., maize ethanol production in brazil: characteristics and perspectives, renewable and sustainable energy reviews, 82 3907–3912, (2018) [2]. gomez l.d., clare g.s., mcqueenmason j., sustainable liquid biofuels from biomass: the writing’s on the walls, new phytol;178:473–85, (2008). [3]. lee d.s., wi s.g., lee s.j., lee y.g., kim y.s., bae h.j., rapid saccharification for production of cellulosic biofuels, bioresour technol 2014;158:239–47, (2014) [4]. zhu s., luo f., huang w., huang w., wu y., comparison of three fermentation strategies for alleviating the negative effect of the ionic liquid 1-ethyl-3-methylimidazolium acetate on lignocellulosic ethanol production, appl energy;197:124–31, (2017) [5]. lam m.k., and lee k.t., “microalgae biofuels: a critical review of issues, problems and the way forward,” biotechnology advances 30(3), 673-690. doi: 10.1016/j.biotechadv.2011.11.008, (2012) [6]. canadell j. g., and schulze e. d., “global potential of biospheric carbon management for climate mitigation,” nature communications, 5. doi: 10.1038/ncomms6282, (2014) [7]. schubert c., “can biofuels finally take center stage? ” nature biotechnology 24(7), 777784. doi: 10.1038/nbt0706-777, (2006) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 166 [8]. stevens c.v., verhe r., renewable bioresources scope and modification for nonfood application, john wiley and sons ltd, england, (2004) [9]. maitan-alfenas, g.p., visser, e.m., guimaraes, v.m., enzymatic hydrolysis of lignocellulosic biomass: converting food waste in valuable products, curr. opin. food. sci., 1 44e49, (2015) [10]. balat, m., production of bioethanol from lignocellulosic materials via the biochemical pathway: a review, energy convers. manag., 52 (2) 858e875, (2011) [11]. ebringerová, a., thomas, h., hemicellulose, adv polym sci, 186(8), 1–67, (2005) [12]. haghighi mood s., hossein golfeshan a., tabatabaei m., salehi jouzani g., najafi g.h., gholami m., ardjmand m., lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. renewable and sustainable energy reviews, 27, 77–93, (2013) [13]. hamelinck c.n., van hooijdonk g., faaij a.p.c., ethanol from lignocellulosic biomass: techno-economic performance in short-, middleand long-term. biomass and bioenergy, (2005) [14]. mäki-arvela p., salmi t., holmbom b., willför s., murzin d. y., synthesis of sugars by hydrolysis of hemicellulosesa review. chemical reviews, 111(9), 5638–5666, (2011a) [15]. pereira n., couto m., anna l. s., biomass of lignocellulosic composition for fuel ethanol production and the context of biorefinery, brazilian national library (vol. 2), (2008) [16]. kuhad r.c., gupta r., khasa y.p., singh a., zhang y.h.p., bioethanol production from pentose sugars: current status and future prospects, renewable and sustainable energy reviews, (2011) [17]. limayem a., ricke s.c., lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects, prog. energy combust. sci. 38 (4) 449e467, (2012) [18]. saini j.k., saini r., tewari l., lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments, 3 biotech 5:337–353, (2015) [19]. sánchez c., lignocellulosic residues: biodegradation and bioconversion by fungi. biotechnology advances, (2009) [20]. taherzadeh m.j., karimi k., pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. international journal of molecular sciences (vol. 9), (2008) [21]. rosas j.m., berenguer r., valeroromero m.j., rodrãguez-mirasol j., cordero t., preparation of different carbon materials by thermochemical conversion of lignin, frontiers in materials, 1, 1–17. (2014) [22]. rosales-calderon o., arantes v., a review on commercial-scale high-value products that can be produced alongside cellulosic ethanol. biotechnol. biofuels. 12(1), 240, (2019) [23]. smuga-kogut m., piskier t., walendzik b., szymanowskapowałowska d., assessment of wasteland derived biomass for bioethanol production, electron. j. biotechn,. 41, 1-8, (2019) [24]. amin f.r., khalid k., zhang h., rahman s., zhang r., liu g., chen c., mini-review pretreatment methods of lignocellulosic biomass for anaerobic digestion, amb expr 7:72, (2017) [25]. branco r.h., serafim l.s., xavier a.m., second generation bioethanol production: on the use of pulp and paper industry wastes as feedstock, ferment., 5(1), 4, (2019) [26]. matsakas l., kekos d., loizidou m., christakopoulos p., utilization of household food waste for the production of ethanol at high dry material content. biotechnol. biofuels. 7(1), 4, (2014) [27]. heaton e.a., dohleman f.g., long s.p., meeting us biofuel goals with less land: the potential of miscanthus, global change biol., 14(9), 2000-2014, (2008) [28]. scagline-mellor s., griggs t., skousen j., wolfrum e., holásková i., switchgrass and giant miscanthus biomass and theoretical ethanol production from reclaimed mine lands, bioenergy res., 11(3), 562-573, (2018) [29]. mabrouk b.a., kaddami h., boufi s., erchiqui f., dufresne a., cellulosic nanoparticles from alfa fibers (stipa tenacissima): extraction procedures and reinforcement potential in polymer nanocomposites, cellulose 19:843–853, (2012) [30]. belkhir s., koubaa a., khadhri a., ksontim h., smit. s., vanations in the morphological characteristics of stipa tenactssima fiber: the case of tunisia, ind. crops prod., 37. 200-206, (2012) [31]. akchiche o., messaoud boureghda k., esparto grass (stipa tenacissima l.), raw material of papermaking, first part. j. chem. raw plant 4 (c), 25–30, altai university, (2007) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 167 [32]. marrakchi z., khiaria r., oueslatic h., maureta e., mhenni f., pulping and paper making properties of tunisian alfa stems (stipa tenacissima) effects of refining process, industrial crops and products 34 1572– 1582, (2011) [33]. dalla marta a., mancini m., orlando f., natali f., capecchi l., oriandini s., sweet sorghum for bioethanol production: crop responses to different water stress levels, biomass bioenerg., 64, 211-219, (2014) [34]. seungdo k., bruce e.d., global potential bioethanol production from wasted crops and crop residues, biomass and bioenergy, 26, 361 – 375, (2004) [35]. cheah w.y., sankaran r. , show p.l., ibrahim n.b., chew k.w. , culaba a., chang j-s., pretreatment methods for lignocellulosic biofuels production: current advances, challenges and future prospects, biofuel research journal, 25 1115-1127, (2020) [36]. jitendra k.s., reetu s., lakshmi t., lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments, 3 biotech, 5:337–353, (2015) [37]. menon v., rao m., trends in bioconversion of lignocellulosc: biofuels, platform chemicals & bioretinery concept, prog. energy combust, 38(4), 522-550, (2012) [38]. kumar a.k., sharma s., recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review, bioresour. bioprocess, 4(1), 7, (2017) [39]. saini j.k., saini r., tewari l., lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments, 3 biotech, 5:337–353, (2015) [40]. baruah j., nath b.k., sharma r., kumar s., dcka r.c., baruah d.c., kalita e., recent trends in the pretreatment of lignocellulosic biomass for value-added products. front. energy res. 6, 141 (2018) [41]. zheng, j., choo k., bradt c., lehoux r., rehmann l.,. enzymatic hydrolysis of steam exploded corncob residues after pretreatment in a twin-screw extruder, biotechnol. rep., 3, 99-107, (2014) [42]. liang s., gu h., bergman r.d., life cycle assessment of cellulosic ethanol and biomethane production from forest residues, bioresources, 12(4), 7873-7883, (2017) [43]. prasad a., sotenko, m., blenkinsopp, t., coles, s.r., life cycle assessment of lignocellulosic biomass pretreatment methods in biofuel production, int. j. life cycle assess, 21(1), 44-50, (2016) [44]. geffert a., geffertova j., dudiak m., direct method of measuring the ph value of wood, forests,10, 852; doi:10.3390/f10100852, (2019) [45]. pettersen r.c., the chemistry of solid wood, advances in chemistry series 20, chapter 2, pp. 57-126, washington, dc, (1984) [46]. rowell r.m., pettersen r., tshabalala m.a., cell wall chemistry from: handbook of wood chemistry and wood composites, crc press, (2012) [47]. fengel d., wegener g., wood: chemistry, ultrastructure and reactions, w. de. gruyter, berlin, (1984) [48]. ahmed s.a., islam s., hoque a., doraisingam y., production of bioethanol from tropical woody biomass, open journal of renewable energy and sustainable development, volume 1, number 2 (2014) [49]. chen, h., zhou, d., luo, g., zhang, s., chen, j., "macroalgae for biofuels production: progress and perspectives," renewable and sustainable energy reviews, vol. 47, pp. 427-437, (2015) [50]. kim n.-j., li h., jung k., chang h.n., lee p.c., "ethanol production from marine algal hydrolysates using escherichia coli ko11," bioresource technology, vol. 102, pp. 7466-7469, (2011) [51]. cheah w.y., ling t.c., show p.l., juan j.c., chang j.s., lee d.j., cultivation in wastewaters for energy: a microalgae platform, appl. energy, 179, 609-625, (2016a) [52]. fao, food and agriculture organization of the united nations "algae-based biofuels: a review of challenges and opportunities for developing countries", (2010) [53]. u.s.d.o.e., "national algal biofuels technology roadmap. u.s. department of energy, office of energy efficiency and renewable energy, biomass program.," workshop and roadmap sponsored by the u.s. department of energy office of energy efficiency and renewable energy office of the biomass program (2010) [54]. chisti y., "fuels from microalgae," biofuels, vol. 1, pp. 233-235, (2010) [55]. enzing c., ploeg m., barbosa m., sijtsma l., vigani m., parisi c., cerezo e. r., microalgae-based products for the food and feed sector: an outlook for europe, publications office, (2014) [56]. zhu l., hiltunen e., antila e., zhong j., yuan z., wang z., "microalgal biofuels: flexible bioenergies for sustainable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 168 development," renewable and sustainable energy reviews, vol. 30, pp. 1035-1046, (2014) [57]. cheah, w.y., ling, t.c., juan, j.c., lee, d.j., chang, j.s., show, p.l., biorefineries of carbon dioxide: from carbon capture and storage (ccs) to bioenergies production, bioresour. technol, 215, 346-356, (2016) [58]. cheah w.y., show p.l., chang j.s., ling t.c., juan j.c., biosequestration of atmospheric co2 and flue gas-containing co2 by microalgae. bioresour. technol, 184, 190-201, (2015) [59]. wang x., ruan z., sheridan p., boileau d., liu y., liao w., two-stage photoautotrophic cultivation to improve carbohydrate production in chlamydomonas reinhardtii, biomass bioenergy, 74, 280-287, (2015) [60]. wang y., guo w., cheng c.l., ho s.h., chang j.s., ren n., enhancing biobutanol production from biomass of chlorella vulgaris jsc-6 with sequential alkali pretreatment and acid hydrolysis, bioresour. technol., 200, 557564, (2016) [61]. özçimen, d., i̇nan, b., an overview of bioethanol production from algae, biofuels status and perspective, (2015) [62]. brennan, l., owende, p., biofuels from microalgae – a review of technologies for production, processing, and extractions of biofuels and co-products, renew. sust. energ. rev. 14,557– 577, (2010) [63]. lardon l., hélias a., sialve b., steyer j.-p., bernard o., "life-cycle assessment of biodiesel production from microalgae," environmental science & technology, vol. 43, pp. 6475-6481, (2009) [64]. chen w.h., wu z.y., chang j.s., isothermal and non-isothermal torrefaction characteristics and kinetics of microalga scenedesmus obliquus cnw-n., bioresour. technol., 155, 245-251, (2014a) [65]. margaritas a.c.f., da costa j.a.v., increment of carbohydrate concentration of chlorella mintaissinza microalgae for bioethanol production, int j. eng. res. appl., 4 (3) 80-86, (2014) [66]. margarites a.c., spirulina platensis is more efficient than chlorella homosphaera in carbohydrate productivity, environ. technol., 38 (17) 2209-2216, (2017) [67]. a. hirano, r. ueda, s. hirayama, y. ogushi, co2 fixation and ethanol production with microalgal photosynthesis and intracellular anaerobic fermentation, energy, 22 (2) 137-142, (1997) [68]. jena u., das k.c., kastner j.r., effect of operating conditions of thermochemical liquefaction on biocrude production from spirulina platensis, bioresour technol., 102(10), 6221-6229, (2011) [69]. yuan j., kendall a., zhang y., "mass balance and life cycle assessment of biodiesel from microalgae incorporated with nutrient recycling options and technology uncertainties," gcb bioenergy, (2014) [70]. castrillón l.j.r., carmona m.e.r., salazar y.v.,. microalgas para la industria alimenticia. editorial universidad pontificia bolivariana, colombia, (2013) [71]. ho s.-h., chen c.-y., chang j.-s., effect of light intensity and nitrogen starvation on co, fixation and lipid/carbohydrate production of an indigenous microalga scenedesmus obliquus cnw-n, bioresour. technol., 113 244-252, (2012) [72]. gonzalez-fernandez c., molinuevo-salces b., garciagonzalez m.c., open and en-closed photobioreactors comparison in terms of organic matter utilization, biomass chemical profile and photosynthetic efficiency, ecol. eng., 36 (10) 14971501, (2010) [73]. chen w.h., huang m.y., chang j.s., chen c.y., thermal decomposition dynamics and severity of microalgae residues in torrefaction. bioresour. technol. 169, 258-264, (2014) [74]. milano j., chyuan h., masjuki h.h., chong w.t., kee m., microalgae biofuels as an alternative to fossil fuel for power generation, renew. sust. energy. rev., 58, 180-197, (2016) [75]. i̇nan b., utilization of algae for bioethanol production. msc. thesis. yıldız technical university, (2014) [76]. shuping z., yulong w., mingde y., chun l., junmao t., pyrolysis characteristics and kinetics of the marine microalgae dunaliella tertiolecta using thermogravimetric analyzer, bioresour. technol., 101(1), 359-365, (2010) [77]. schwenzfeier a., wierenga p.a., gruppen h., isolation and characterization of soluble protein from the green microalgae teraselmis sp, bioresour. technol., 102 (19) 91219127, (2011) [78]. jensen a., present and future needs for algae and algal products, hydrobiologia, 260–261, 15–23, (1993) [79]. ross a.b, jones j.m, kubacki m.l, bridgeman t., classification of macroalgae as fuel and its thermochemical behaviour, bioresour. technol., 99, 6494–6504, (2008) [80]. john r.p., anisha g., nampoothiri k.m., pandey a., "micro and macroalgal biomass: a renewable source for bioethanol," food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 vasile-florin ursachi, gheorghe gutt, feedstocks used for production of 2nd and 3rd generation bioethanol review, food and environment safety, volume xix, issue 2 – 2020, pag. 156 – 169 169 bioresource technology, vol. 102, pp. 186-193, (2011) [81]. dębowski m., zieliński m., grala a., dudek m., "algae biomass as an alternative substrate in biogas production technologies review," renewable and sustainable energy reviews, vol. 27, pp. 596-604, (2013) [82]. moncada j., jaramillo j.j., higuita j.c., younes c., cardona c.a., production of bioethanol using chlorella vulgaris cake: a technoeconomic and environmental assessment in the colombian context, industrial & engineering chemistry research, 52(47):16786-94, (2013) [83]. kim j.k., um b.h., kim t.h., bioethanol production from micro-algae, schizocytrium sp., using hydrothermal treatment and biological conversion, korean journal of chemical engineering, 29(2):209-14, (2012) [84]. patidar, j., sharma, r., yadav, m., tiwari, a., microalgae as sustainable renewable energy feedstock for bioethanol production, sch. acad. j. biosci.,; 5(7):536-542, (2017) [85]. harun r., danquah m.k., forde g.m., microalgal biomass as a fermentation feedstock for bioethanol production, journal of chemical technology and biotechnology, 2010 feb 1; 85(2):199-203, (2010) [86]. eshaq f.s., ali m.n., mohd m.k., production of bioethanol from next generation feed-stock alga spirogyra species, int. j. eng. sci. technol., 2011 feb; 3(2):1749-55, (2011) [87]. harun r., danquah m.k., enzymatic hydrolysis of microalgal biomass for bioethanol production, chem. eng. j., 168, 1079-1084, (2011) [88]. choi w.y., han j.g., lee c.g., song c.h., kim j.s., seo y.c., lee s.e., jung k.h., kang d.h., heo s.j., cho j.s., bioethanol production from ulva pertusa kjellman by hightemperature liquefaction, chemical and biochemical engineering quarterly, 2012 apr 1; 26(1):15-21, (2012) [89]. nguyen t.h., bioethanol production from marine algae biomass: prospect and troubles, journal of vietnamese environment, 2012 nov 6; 3(1):25-9, (2012) [90]. asada c., doi k., sasaki c., nakamura y., efficient extraction of starch from microalgae using ultrasonic homogenizer and its conversion into ethanol by simultaneous saccharification and fermentation, nat. resour., 3, 175–179, (2012) [91]. ho s.h., huang s.w., chen c.y., hasunuma t., kondo a., chang j.s., bioethanol production using carbohydrate-rich microalgae biomass as feedstock, bioresour. technol., 135, 191–198, (2013) [92]. markou g., angelidaki i., eorgakakis d., microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels, applied microbiology and biotechnology, 2012 nov 1; 96(3):631-45, (2012) 1. introduction 246 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 3 – 2019, pag. 246 257 state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré *adou akpa guy blanchard gnagne 1 , ahou irène kouadio 1 , kouamé affourmou 2 , brou lazare yao 3 , moussa sanogo 2 1laboratory of biochemistry and food sciences, training and research unit of biosciences. félix houphouëtboigny university of abidjan (côte d’ivoire). 22 bp 582 abidjan 22. 2national laboratory to agricultural development support – veterinary central laboratory of bingerville (côte d’ivoire). bp 206 bingerville. 3national laboratory to agricultural development support – central laboratory of agro-chemistry and ecotoxicology of abidjan (côte d’ivoire). 04 bp 612 abidjan 04. adoublanc@gmail.com *corresponding author received 24 july 2019, accepted 27th september 2019 abstract: a survey was carried out among 129 pesticide applicators of the departments of agboville and soubré, in order to identify the phytosanitary practices in field’s treatment adjacent to a fish farm. it turns out that pesticide application is a male task. among these pesticide applicators, 59.7% of those from soubré are illiterate as compared to 12% of those from agboville. most applicators in both localities (64% in agboville and 95% in soubré) have no qualifications. only 1.5% of them are wearing appropriate protective equipment when spraying. 50.7% and 9.7% of applicators of agboville and soubré respectively are dressed in partial equipment. poor practices in the dosing and preparation of porridges, as well as the management of empty packs could expose fish farms to pesticide residues contamination; fields being treated virtually every month in the year. among the pesticides listed, insecticides accounted for 47% followed by 44% herbicides. growth regulators and fungicides represent 5% and 4% respectively. at the end of this survey, it appears that pesticides with toxic effects on human health and environment have been listed. thiamethoxam and glyphosate are respectively the predominant insecticide and herbicide contaminants. keywords: survey, fish farms, thiamethoxam, glyphosate, health risks. 1. introduction at the dawn of its independence, côte d’ivoire concentrated its economic development on agricultural sector [1]. thanks to its important natural potentialities characterized by a favorable climate, fertile lands and important hydrological resources, a varied range of vegetal productions is available on ivorian markets [2]. in response to the increase in consumption imposed by its growing population, adoption of the master plan for agricultural development 1992 – 2015 has promoted the use of plant protection products (fertilizers and pesticides) in agriculture [3]. the use of pesticides in ivorian agriculture is only recent, but has developed rapidly. already in 1996, the amount of pesticides used was estimated at more than 128,000 tons [4]. of course, the use of pesticides has significantly increased agricultural productions. but at the same time, they http://www.fia.usv.ro/fiajournal mailto:adoublanc@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 247 have a negative influence on biodiversity and lead to health risks related to their exposure [5, 6]. some of these pesticides are indeed very remnant products; their half-life extending over several years in the order of ten years and beyond. in tropical countries as in the case of côte d’ivoire, they can degrade rapidly under action of ultraviolet radiation into derived products which are generally more toxic and more stable than the initial products [7]. following a spread, the fraction of pesticides that reaches the ground, joins the groundwater by infiltration or surface water by runoff. and yet, the dangerousness of these products is well proven. they would be responsible for decreasing immunity [8], of appearance of cognitive and neurodegenerative disorders [9]. thus, use of agricultural pesticides is not without consequences on environment, health of farmers themselves and that of consumers. in benin, in the department of borgou, 37 deaths of people and 36 cases of severe intoxication were recorded following endosulfan poisoning between may and september 1999 [10]. in côte d’ivoire, according to the occupational health service of the hospital and university center of yopougon (abidjan), high clinical signs suggestive of organophosphorus and carbamate in many patients would have been detected [11, 12]. to this end, in the interest of public health, it is therefore imperative to identify the phytosanitary practices made on crops by ivorian farmers. the purpose of this study was to identify agricultural pesticides used in fields near fish farms, and the phytosanitary practices of applicators in agboville and soubré departments with the aim of making a prognosis of the state of environmental pollution and that of fishes from these farms. 2. materials and methods 2-1. presentation of the study areas this study was carried out in the departments of agboville and soubré. agboville department covers an area of 11,300 km2 and is located 80 km from the ivorian economic capital, abidjan. characterized by a dense and humid forest, this department is part of the southern forest of côte d’ivoire [13]. it has a very rugged terrain characterized by presence of many valleys and hills. the climate is subequatorial with two dry seasons and two rainy seasons all of unequal length. the average annual rainfall is 1400 mm of rain. agboville is mainly irrigated from east to west by agneby’s river and has a ferralitic type soil. however, there are nonpermanent rivers that can drought in dry season including rivers abé, bebasso, gorké, kavi, mafou, mé, m’brou and séguié. the average annual temperature is around 25 °c, and economic activity is based on primary sector dominated by cocoa farming. soubré’s department is located in the south-west of côte d’ivoire, 380 km from abidjan and extends on 4779 km2. the relief consists of vast plateaus surmounted by hills in places and a subequatorial climate type, characterized by a dry season and two rainy seasons of unequal length. the hydrographic network consists of permanent watercourses, the most important of which are sassandra’s river and its main tributaries, the davo, gôh, lobo and n’zo. soubré has a ferralitic type soil and has vegetation with enormous agricultural and forestry potential making the department one of the most important economic centers in the country. economic activity is based on primary sector dominated by cocoa cultivation, and 70 % of the population is rural. figure 1 present the map of the study areas. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 248 2-2. choice of investigations sites survey was held in five villages or camps in each department. it was interested in pesticides applicators whose treated plantations are within one kilometer of a fish farm. in agboville department, applicators concerned come from the villages of kamabroux, mucho, offoriguié, offoumpo and oress-krobou. in soubré, they come from camps of amarakro, carrière, djoutoubou, kouakoudankro and sayo. the choice of these sites was motivated by the fact that sales of fish from these fish farms was made at least once. the farming system in these two departments varies from semiintensive to intensive. 2-3. progress of the investigation the survey was conducted with 129 planters and / or pesticide applicators from the two departments, of which 67 are from agboville and 62 are from soubré. it was carried out using a questionnaire giving information on the type and qualifications of applicator, age range, periods of application, commercial names of the pesticides used, places of purchase, preparation of porridge, wearing of personal protective equipment (ppe), any food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 249 discomfort experienced by spraying suites and the management of empty packaging. the survey was conducted according to snowball method [14]. it consisted of interviewing owners and / or pesticide applicator of fields adjoining fish farm on a radius of about one kilometer, then to interview another following recommendation of the previous one. it is completed in a village when people recommended by the previous ones have already been interviewed in the radius considered. farms observations were made during pesticide application. the survey lasted 27 days in agboville from april 7 to june 11, 2017, and then 18 days in soubré over the period from september 7 to september 26, 2017. 2-4. data analysis the survey data was entered using cs pro version 6.3 software. data were later taken to excel 2010 for clearance, regulation and graphing. stata version 14.2 software has been used for statistical processing. the chi2 statistical binding test between variables was established at 5% threshold (p < 0.05). 3. results 3-1. gender and age range of applicators the survey revealed that pesticides application in fields is a task almost exclusively male. the male gender represents 97% and 93.5% respectively in agboville and soubré. they are adults, most of whom are aged 31 to 40, followed by those aged 41 to 50. however, applicators over 50 are more abundant in agboville than in soubré (figure 2). 3-2. schooling level and qualification of applicators while rate of applicators enrolled in agboville is high and reaches 88% dominated by secondary level, that of soubré is low with 40.3% whose primary level is dominant (figure 3). the rate of qualified applicators is relatively low in both localities. this survey shows that 36% of applicators are qualified to apply phytosanitary products in agboville, while 5% are concerned in soubré. for more cases in both localities, application is made by the plots owners or entrusted to maneuvers, whose generally aren’t qualified. these owners both applicators represent 59.4% and 90.3% in agboville and soubré respectively. exception is made in offoumpo’s village where application is mainly entrusted to specialists trained to that task. 3-3. spraying periods and wearing of ppe contrary to soubré, pesticides are almost sprayed every month in the year in agboville. however, insecticides and fungicides are preferentially sprayed in the months of january, august and december. as for herbicides, they are preferentially sprayed in february, march and april depending on the locality (figures 4 & 5). applicators interviewed don’t have adequate ppe against pesticides. in soubré, protection is almost non-existent and comes down to wearing ordinary boots and clothes, often torn. however, in camps of amarakro, kouakoudankro and sayo some of them protect airways using a nose shield. in agboville, however, the majority of applicators protect themselves from pesticides inhalation using a nose shield or gas mask, and/or contact with eyes using glasses. only 1.5% protect themselves from inhalation, contact with eyes and body using a suit (figure 6). at 5% threshold, a statistical link is established food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 250 between ppe’s wearing and schooling level (p<0.05) (table 1), and between ppe’s wearing and the lack of qualification (p < 0.05) (table 2). 3-4. discomfort felt 79% and 77% of applicators of agboville and soubré respectively feel discomfort, of which itching, sneezing and skin irritation are the most common; sometimes, eyes irritation and cold. on the other hand, some admit not to feel effects (figure 7). the null hypothesis of independence between the variables ppe’s wearing and existence of discomfort is accepted at 5% threshold. there is no statistical link between these two variables (p > 0.05) (table 3). 3-5. places of purchase pesticides and management of empty packaging in both localities, 53.2% and 78.1% of agboville and soubré applicators respectively buy pesticides at local markets (figure 8), where they are exposed without any protection from sun. a statistical link is established between choice of the places of purchase of pesticides and their cost and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 251 proximity at the threshold of 5% (p < 0.05) (table 4). empty packs generally are mismanaged. there are left intact in fields after use (67% of cases in agboville and 89% in soubré). in other cases, there are reused for domestic use as a container for drinking water or palm wine called "bandji", but also for preservation of cooking ingredients such as salt (figure 9). there is a statistical link between the management of empty packaging and qualification of applicators at the 5% threshold (p < 0.05) (table 5). 3-6. listed pesticides and their active ingredients at end of the survey, 72 different pesticides were counted, 15 of which were unregistered, ie 20.8%. there are 47% of insecticides, 44% of herbicides, 5% of growth regulators and 4% of fungicides. twelve insecticidal active ingredients have been identified, of which thiamethoxam is the most important, belonging to nicotinoids family (figure 10). in herbicides, eleven active ingredients have been identified of which glyphosate is the most represented molecule (figure 11). in fungicides, six active ingredients have been identified among which metalaxyl is predominant. in growth regulators ethephon is predominant. table 1 cross table and independent test of chi2 between the variables ppe’s wearing and applicator schooling levels. ppe’s wearing schooling level total unschooled primary secondary higher no 40 26 22 0 88 yes 5 10 25 1 41 total 45 36 47 1 129 pearson chi2 (1) = 21.2173 pr = 0.000 table 2 cross table and independent test of chi2 between the variables ppe’s wearing and applicator qualifications. ppe’s wearing applicator qualification total none qualified no 81 7 88 yes 21 20 41 total 102 27 129 pearson chi2 (2) = 28.1686 pr = 0.000 table 3 cross table and independent test of chi2 between the variables protection equipment and discomfort felt. protection equipment discomfort total none presence full equipment 0 1 1 none protection 21 67 88 partial protection 7 33 40 total 28 101 129 pearson chi2 (3) = 0.9347 pr = 0.627 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 252 3-7. preparation of porridge during observations made when porridge is prepared, the dosage prescribed on packaging is not followed. it is usually done under the order of owner of plantation treated when applicator is a specialist. when this one is lacking, the dosage is arbitrary and depends on its judgement or a third person. applicators sometimes make mixture of different pesticides. it concerns 21.7% and 17.7% of applicators of agboville and soubré respectively. while 65% of mixture consists of mixing several insecticides in agboville, 70% of mixture made in soubré concern several herbicides. table 4 cross table and independent test of chi2 between the variables places of purchase pesticides and the reasons of that choice. places of purchase pesticides reasons for choosing this place total effectiveness lower cost no expense payment condition facilitated proximity quality authorized seller 1 1 0 1 0 20 23 cooperative 0 0 0 12 1 0 13 gift of state 1 1 22 0 2 0 26 local market 21 61 4 6 41 9 142 street vendor 1 1 0 3 1 1 7 tracker 0 0 0 7 2 2 11 total 24 64 26 29 47 32 222 pearson chi2 (4) = 378.9245 pr = 0.000 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 253 table 5 cross table and independent test of chi2 between the variables management of empty packs and applicator's qualification. management of empty packs applicator qualification? total none qualified demolished and buried 4 9 13 garbage dump 4 0 4 reused 11 1 12 thrown into fields 83 17 100 total 102 27 129 pearson chi2 (5) = 21.4686 pr = 0.000 4. discussion the survey showed that use of phytosanitary in both study localities for harvests optimization, is almost systematic as meant by several authors of other countries [15, 16, 17, 18]. according to some interviewees, this situation is due to lack of manpower. application is an almost masculine task, as was also reported by [19] in yamoussoukro market gardeners. these applicators are all adults aged 21 to 60 ; with the age group 31 to 40 being the majority. in soubré, the majority of pesticides applicators are illiterate (60%), as are those in morocco for the health protection of spearmint [20]. in agboville, most of them are literate (87%), and 54% of them have reached secondary level. the few women pesticides applicators surveyed are all market gardeners and illiterate. according to those women, they would have learned phytosanitary application by initiation with their husbands as muliele et al [21] pointed out, but also to ensure the survival and that of their family, because they would be either divorced or widowed. despite the high proportion of literate applicators in agboville, rate of qualified in phytosanitary application is low; it’s even more so in soubré. this observation is consistent with that made by muliele et al [21]. this could explain bad practices observed in pesticides use as underlined by several authors [22, 23, 24]. indeed, certainly literate applicators could be ignorant of good phytosanitary practices. according to some respondents, solicitation of specialist applicators is expensive, while pesticides and machines are sold over-the-counter. these bad practices refer in particular to the wearing of ppe, misuse of pesticides, their origin, the various mixtures made, management of empty packaging and precautions to be taken for any surrounding watercourse. in agboville, 48% of applicators give little interest to wearing of ppe ; as for soubré, 90% are concerned. similar observation is made by doumbia and kwadjo [25] among market gardeners in abidjan and two of its suburbs. some applicators with no education level or qualification interviewed, not wearing any protection during spraying, claim to be immune to pesticides that would then be ineffective on them. this same remark was made by toe et al [26] in burkina faso among the cotton producers of the hauts-bassins, the cascades and the boucle of the mohoun. logically, this attitude of ignoring the wearing of protective equipment should be at the origin of listed discomforts, as well as numerous cases of intoxication and deaths that would have occured in camps as some respondents have indicated. these discomforts have also been observed by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 254 sonchieu et al [27] among bread sellers from bamenda in cameroon. and yet, results of our study show that being dressed in ppe does not induce absence of discomfort. this situation could be explained by the fact that applicators considered to be equipped with protection during spraying, are dressed only in partial and inadequate equipment such as use of nose shield instead of the gas mask and / or wearing ordinary clothes instead of the suit. in addition, only 1.5% of applicators interviewed confessed to detaining and wearing full protective equipment when spraying. the source of pesticides is also a source of concern. pesticides are usually bought in local markets exposed to sun, as it is the case with tomato producers in burkina faso [22]. this could explain use of unapproved and unauthorized products in côte d’ivoire, as indicated by doumbia and kwadjo [25]. also, according to some interviewees aware of using unauthorized products, there would cost less compared to those authorized. phytosanitary practices in our two study localities concerning dosage of porridges, mixtures of different pesticides and management of empty containers could have a negative impact on environment. in fact, the dosages carried out are generally beyond the requirements in the case of insecticides and herbicides. concerning the case of insecticides, it is thought that overdose is due to resistance of insect pests to cocoa, which is dominant crop, and also to gardeners. this result is supported by the work of gnankine et al [28]. according to these authors, overdose of pesticides by tomato producers in burkina faso is related to its pest resistance (h. armigera and b. tabaci) to insecticides. this situation could also explain the mixtures of pesticides carried out, with intention of producing a more effective porridge on these pests. observations made in farm environment have revealed mismanagement of empty packaging. there are either reused for drinking or abandoned in fields as shown by eddaya et al [20]. however, good phytosanitary practices would advise that there be demolished and buried. these practices could prove damaging for the water environment. from results of work of keddal and n’dri [29] and mawussi et al [16], water resources represent the catchment area for a surge of pesticides residues. given that investigation concerned plantations located in the vicinity of fishponds, it would therefore be likely to assume that by runoff or infiltration, pesticides residues could reach these ponds and then accumulate in fish; spraying being carried out almost every month. studies have shown such contamination in surface water [30], groundwater [31] and fish [32, 33, 34]. for this purpose, since lambdacyhalothrin, thiamethoxam and glyphosate are the predominantly identified pesticide active ingredients; consumption of fish from fish farms could constitute a risk for the health of populations. this is also the case of endosulfan of which the toxic effects are well known. indeed, lambdacyhalothrin dissipates rapidly in water due to its adsorption on particles and aquatic organisms [35]. it is extremely toxic to many aquatic organisms; hence its ban in china [36]. works have shown the involvement of pyrethroids in coronary artery desease in chinese population [37]. similarly, tests in rats, mice and rabbits revealed neurotoxic effects of lambdacyhalothrin [38] and decrease of body weight [39]. other tests in mice fed thiamethoxam for 18 months at concentration of 500 to 2,500 ppm, have shown that one of its metabolites cga330050, would be responsible for liver tumors [40]. as for glyphosate, it has food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 255 been classified as a probably carcinogenic substance for human by the international agency for research on cancer [41]. the results of de roos et al [42], have shown that glyphosate would be involved in the prevalence of myeloma or kahler’s disease, a type of cancer that affects plasma cells. several authors have also shown other effects of pesticides including decline in intellectual abilities and intelligence level in children [43], the onset of type 2 diabetes [44] or disorders of reproduction [45]. 5. conclusion faced with the challenge of food security, the use of pesticides has won over ivorian agriculture. however, poor pesticides use could expose population to risk related to consumption of food, especially fish from fish farms adjacent to plantations undergoing pesticides treatments. in view of continuing this work, it is planned to carry out qualitative analyses for the detection of pesticides residues in fish from fish farms, in order to assess their level of contamination. 6. acknowledgement the authors of this article would like to express their gratitude to the national association of aquaculturists of côte d’ivoire (anaquaci) for facilitating access to fish farmers, the ekleyo’s cooperative of soubré for full adherence to this work and the administrative and village authorities of agboville and soubré departments for their hospitality. 7. references [1]. kouadio ka, monsan v, gbongue m. investissement et dynamique de la pauvreté en milieu agricole ivoirien. politique economique et pauvreté (pep), rapport final pep-pmma; 39 p., (2007) [2]. sangaré a, koffi e, akamou f, fall ca. etat des ressources phytogénétiques pour l’alimentation et l’agriculture. ministère de l’agriculture, second rapport national; 65 p., (2009) [3]. fleischer g, andoli v, coulibaly m, randolf t. analyse socio-économique de la filière des pesticides en côte d’ivoire. publication du projet de politique des pesticides en collaboration avec la direction de la protection des végétaux et de la qualité du ministère de l’agriculture et des ressources animales de côte d’ivoire; n°6, 112 p., (1998) [4]. zadi dr. profil national actualisé sur la gestion des produits chimiques en côte d’ivoire. ministère de l’environnement, des eaux et forêts; 80 p., (2008) [5]. gnago ja, danho m, agneroh ta, fofana ik, kohou ag. efficacité des extraits de neem (azadirachta indica) et de papayer (carica papaya) dans la lutte contre les insectes ravageurs du gombo (abelmoschus esculentus) et du chou (brassica oleracae) en côte d’ivoire. int j biol chem sci; 4(4): 953-966, (2010) [6]. monkiedje a, njine t, tamatcho b, demanou j. assessment of the toxic effects of the fungicide ridomil plus 72 on aquatic organisms and soil micro-organisms. environmental toxicology; 15: 65-70, (2000) [7]. pnue. evaluations régionales des substances toxiques persistantes. rapport de la région subsaharienne. (2002); available from : http://www.chem.unep.ch/pts/regreports/translated %20reports/sub%20saharan%20africa%20fr.pdf [8]. dewailly e, ayotte p, bruneau s, gingras s, belles-isles m, roy r. susceptibility to infections and immune status in inuit infants exposed to organochlorines. environ. health perspect mar; 108(3): 205-211, (2000) [9]. costa lg, ashner m, vitalone a, syversen t, soldin op. developmental neuropathology of environment agents. annu. rev. pharmacol toxicol; 44: 87-110, (2004) [10]. ton p, tovignan s, davo s. endosulfan deaths and poisonings in benin. pesticides news; 47: 12-14, (2000) [11]. manda p, dano ds, kouassi ym, oga as, dembélé a, wognin sb et al. evaluation de l’exposition aux organophosphorés et aux carbamates des applicateurs de produits phytosanitaires. edition universitaires de côte d’ivoire (educi), j sci pharm biol; 6(1): 53-60, (2005) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 256 [12]. kouassi ym, wognin sb, manda p, yeboue-kouame by, tchicaya af, bonny js et al. intoxications chroniques professionnelles diagnostiquées au chu de yopougon-abidjan de 1990 à 2002. cahier de santé publique, editions universitaires de côte d’ivoire (educi), université de cocody-abidjan; 3(1) : 61-66, (2004) [13]. n’guessan k, zirihi gn, boraud nkm. etude ethnopharmacologique des plantes utilisées pour faciliter l’accouchement, en pays abbey et krobou, au sud de la côte d’ivoire. int j biol chem sci; 4(4) : 1004-1016, (2010) [14]. bahouayila mcb. cours de pratiques des enquêtes. institut africain de la statistique; 18 p., (2016) [15]. belhadi a, mehenni reguieg l, yakhlef h. pratiques phytosanitaires de serristes maraichers de trois localités de l’est des ziban et leur impact potentiel sur la santé humaine et l’environnement. revue agriculture, numéro special; 1: 9-16, (2016) [16]. mawussi g, kolani l, devault da, alate kka, sanda k. utilisation de pesticides chimiques dans les systèmes de productions maraîchères en afrique de l’ouest et conséquences sur les sols et la ressource en eau : cas du togo. 44ème congrès du groupe français des pesticides, 26-29 mai, schoelcher (2015); pp : 45-53, (2014) [17]. akan jc, jafiya l, mohammed z, abdulrahman fi. organaphosphorus pesticide residues in vegetable and soil samples from alau dam and gongulong agricultural areas, bomo states, nigeria. int. j. environ. monit anal; 1(2): 58-64, (2013) [18]. cissé i, tandian aa, fall st, diop es. usage incontrôlé des pesticides en agriculture péri-urbaine : cas de la zone des niayes au sénégal. cah agric; 12(3): 181-186, (2003) [19]. tano bf, abo k, dembele a, fondio l. système de production et pratiques à risque en agriculture urbaine : cas du maraîchage dans la ville de yamoussoukro en côte d’ivoire. int. j biol chem sci; 5(6): 2317-2329, (2011) [20]. eddaya t, boughdad a, becker l, chaimbault p, zaïd a. utilisation et risques des pesticides en protection sanitaire de la menthe verte dans le centre-sud du maroc. j mater environ sci; 6(3): 656-665, (2015) [21]. muliele mt, manzenza mc, ekuke wl, diaka pc, ndikubwayo md, kapalay mo et al. utilisation et gestion des pesticides en cultures maraîchères : cas de la zone de nkolo dans la province du kongo central, république démocratique du congo. j appl biosci; 119: 11954-11972, (2017) [22]. son d, somda i, legreve a, schiffers b. pratiques phytosanitaires des producteurs de tomates du burkina faso et risques pour la santé et l’environnement. cah agric; 26, 25005, (2017) [23]. nare nwa, savadogo pw, gnankambary z, nacro hb, sedogo pm. analysing risks related to the use of pesticides in vegetable gardens in burkina faso. agriculture, forestry and fisheries; 4(4): 165-172, (2015) [24]. assogba-komlan f, anihouvi p, achigan e, sikirou r, boko a, adje c, et al. pratiques culturales et teneurs en éléments antinutritionnels (nitrates et pesticides) du solanum macrocarpum au sud du benin. african journal of food, agriculture, nutrition and development; 7(4): 1-21, (2007) [25]. doumbia m, kwadjo ke. pratiques d’utilisation et de gestion des pesticides par les maraichers en côte d’ivoire : cas de la ville d’abidjan et de deux de ses banlieues (dabou et anyama). journal of applied biosciences; 18: 9921002, (2009) [26]. toe am, ouedraogo m, ouedraogo r, ilboudo s, guissou pi. pilot study on agricultural pesticides poisoning in burkina faso. interdiscip toxicol; 6(4): 185-191, (2013) [27]. sonchieu j, nsoh fj, waingeh nc. pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon. food and environment safety, 17(3): 341-351, (2018) [28]. gnankiné o, mouton l, savadogo a, martin t, sanon a, dabire rk, vavre f, fleury f. biotype status and resistance to neonicotinoids and carbosulfan in bemisia tabaci (hemiptera : aleyrodidae) in burkina faso, west africa. international journal of pest management; 59(2): 95-102, (2013) [29]. keddal h, n’dri jy. impact de l’intensification agricole sur la qualité des eaux de surface et des eaux souterraines. révue hte; 138: 13-29, (2008) [30]. bao g, wang mh, william lc, dao jc, zheng js. risk assessment of cyhalothrin on aquatic organism in paddy weld in china. regulatory toxicology and pharmacology; 48: 6974, (2007) [31]. ngom s, seydou t, thiam bm, manga a. contamination des produits agricoles et de la nappe phréatique par les pesticides dans la zone des niayes au sénégal. rev sci technol., synthèse; 25: 119-130, (2012) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 adou akpa guy blanchard gnagne, ahou irène kouadio, kouamé affourmou, brou lazare yao, moussa sanogo, state of play in the use and management of pesticides and risk of contamination of fish ponds in agricultural production areas in côte d’ivoire: case of the departments of agboville and soubré, volume xviii, issue 3 – 2019, pag. 246 257 257 [32]. agbohessi pt, toko ii, ouedraogo a, jauniaux t, mandiki snm, kestemont p. assessment of healthstatus of wild fish inhabiting a coton basin heavily impacted by pesticides in benin (west africa). sciences of the total environment; 506-507; 567-584, (2015) [33]. toko ii, attakpa ey, tobada pc, ble cm, guedegba ln, elegbe h. impact des pesticides agricoles sur les performances physiologiques des poissons : cas du tihan 175 o-teq sur la reproduction des femelles de clarias gariepinus exposées à des doses chroniques. agronomie africaine; 26(3): 247-259, (2014) [34]. adetola jo, ndimele pe, onuoha s. acute toxic effects of endosulfan (organochlorine pesticides) to fingerlings of african catfish (clarias gariepinus, burchell, 1822). american-eurasian j agric & environ sci; 10(5): 884-892, (2011) [35]. li-ming h, john t, albert w, kean g. environmental chemistry, ecotoxicity and fate of lambda-cyhalothrin. review of environmental contamination and toxicology; 100: 71-91, (2008) [36]. çakmak mn, gorgon a. toxic effect of a synthetic pyrethroid insecticide (cypermethrin) on blood cells of rainbow trout (onchorhychus mykiss, walbaum). journal of biological sciences; 3: 694698, (2003) [37]. han j, liqin z, luo m, liang y, zhao w, wang p, zhou z, liu d. non occupational exposure to pyrethroid and risk of coronary diseases in the chinese population. environmental sciences and technology; 51(1): 664-670, (2017) [38]. el-demerdash fm. lambda-cyhalothrininduced changes in oxydative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxydants. toxicol. in vitro.; 21(3): 392-397, (2007) [39]. ali a, khan ja, khaliq t, javed i, muhammad f, aslam b, khan mz. hematobiochemical disruptions by lambdacyhalothrin in rats. pak vet j; 34(1): 54-57, (2014) [40]. green t, toghill a, lee r, waechter f, weber e, noakes j. thiamethoxam induced mouse liver tumors and their relevance to humans ; part 1 : mode of action studies in the mouse, toxicological sciences; 86(1): 36-47, (2005) [41]. iarc. some organophosphate insecticides and herbicides: tetrachlorvinphos, parathion, malathion, diazinon and glyphosate. iarc working group, monographs volume 112 on the evaluation of carcinogenic risks to humans; (in press) (2015) [42]. de roos aj, blair a, rusiecki ja, hoppin ja, svec m, dosemeci m, sandler dp, alanvanja mc. cancer incidence among glyphosate exposed pesticide applicators in the agricultural health study. environmental health perspective; 113(1): 49-54, (2005) [43]. lanphear bp, horung r, khoury j, yolton k, baghurst p, bellinger dc. low-level environmental lead exposure and children intellectual function: an international pooled analysis. environ. health perspect; 113: 894-899, (2005) [44]. azandjeme cs. exposition aux pesticides et risques de diabète de type 2 : une étude au nord du benin (afrique de l’ouest). mémoire de thèse de philosophiae doctor (phd) en nutrition, au département de nutrition, faculté de médecine, université de montréal; 256 p., (2014) [45]. anger jp, kintz p. difficultés analytiques de la caractérisation des pesticides dans le sang. ann toxicol anal; 21(3): 131-141, (2009) from results of work of keddal and n’dri [29] and mawussi et al [16], water resources represent the catchment area for a surge of pesticides residues. given that investigation concerned plantations located in the vicinity of fishponds, it would theref... indeed, lambdacyhalothrin dissipates rapidly in water due to its adsorption on particles and aquatic organisms [35]. it is extremely toxic to many aquatic organisms; hence its ban in china [36]. works have shown the involvement of pyrethroids in coron... 5. conclusion faced with the challenge of food security, the use of pesticides has won over ivorian agriculture. however, poor pesticides use could expose population to risk related to consumption of food, especially fish from fish farms adjacent to plantations und... 6. acknowledgement the authors of this article would like to express their gratitude to the national association of aquaculturists of côte d’ivoire (anaquaci) for facilitating access to fish farmers, the ekleyo’s cooperative of soubré for full adherence to this work a... 7. references [1]. kouadio ka, monsan v, gbongue m. investissement et dynamique de la pauvreté en milieu agricole ivoirien. politique economique et pauvreté (pep), rapport final pep-pmma; 39 p., (2007) [2]. sangaré a, koffi e, akamou f, fall ca. etat des ressources phytogénétiques pour l’alimentation et l’agriculture. ministère de l’agriculture, second rapport national; 65 p., (2009) [3]. fleischer g, andoli v, coulibaly m, randolf t. analyse socio-économique de la filière des pesticides en côte d’ivoire. publication du projet de politique des pesticides en collaboration avec la direction de la protection des végétaux et de la qua... [4]. zadi dr. profil national actualisé sur la gestion des produits chimiques en côte d’ivoire. ministère de l’environnement, des eaux et forêts; 80 p., (2008) [5]. gnago ja, danho m, agneroh ta, fofana ik, kohou ag. efficacité des extraits de neem (azadirachta indica) et de papayer (carica papaya) dans la lutte contre les insectes ravageurs du gombo (abelmoschus esculentus) et du chou (brassica oleracae) en... [6]. monkiedje a, njine t, tamatcho b, demanou j. assessment of the toxic effects of the fungicide ridomil plus 72 on aquatic organisms and soil micro-organisms. environmental toxicology; 15: 65-70, (2000) [7]. pnue. evaluations régionales des substances toxiques persistantes. rapport de la région sub-saharienne. (2002); available from : http://www.chem.unep.ch/pts/regreports/translated%20reports/sub%20saharan%20africa%20fr.pdf [8]. dewailly e, ayotte p, bruneau s, gingras s, belles-isles m, roy r. susceptibility to infections and immune status in inuit infants exposed to organochlorines. environ. health perspect mar; 108(3): 205-211, (2000) [9]. costa lg, ashner m, vitalone a, syversen t, soldin op. developmental neuropathology of environment agents. annu. rev. pharmacol toxicol; 44: 87-110, (2004) [10]. ton p, tovignan s, davo s. endosulfan deaths and poisonings in benin. pesticides news; 47: 12-14, (2000) [11]. manda p, dano ds, kouassi ym, oga as, dembélé a, wognin sb et al. evaluation de l’exposition aux organophosphorés et aux carbamates des applicateurs de produits phytosanitaires. edition universitaires de côte d’ivoire (educi), j sci pharm biol;... [12]. kouassi ym, wognin sb, manda p, yeboue-kouame by, tchicaya af, bonny js et al. intoxications chroniques professionnelles diagnostiquées au chu de yopougon-abidjan de 1990 à 2002. cahier de santé publique, editions universitaires de côte d’ivoir... [13]. n’guessan k, zirihi gn, boraud nkm. etude ethnopharmacologique des plantes utilisées pour faciliter l’accouchement, en pays abbey et krobou, au sud de la côte d’ivoire. int j biol chem sci; 4(4) : 1004-1016, (2010) [14]. bahouayila mcb. cours de pratiques des enquêtes. institut africain de la statistique; 18 p., (2016) [15]. belhadi a, mehenni reguieg l, yakhlef h. pratiques phytosanitaires de serristes maraichers de trois localités de l’est des ziban et leur impact potentiel sur la santé humaine et l’environnement. revue agriculture, numéro special; 1: 9-16, (2016) [16]. mawussi g, kolani l, devault da, alate kka, sanda k. utilisation de pesticides chimiques dans les systèmes de productions maraîchères en afrique de l’ouest et conséquences sur les sols et la ressource en eau : cas du togo. 44ème congrès du group... [17]. akan jc, jafiya l, mohammed z, abdulrahman fi. organaphosphorus pesticide residues in vegetable and soil samples from alau dam and gongulong agricultural areas, bomo states, nigeria. int. j. environ. monit anal; 1(2): 58-64, (2013) [18]. cissé i, tandian aa, fall st, diop es. usage incontrôlé des pesticides en agriculture péri-urbaine : cas de la zone des niayes au sénégal. cah agric; 12(3): 181-186, (2003) [19]. tano bf, abo k, dembele a, fondio l. système de production et pratiques à risque en agriculture urbaine : cas du maraîchage dans la ville de yamoussoukro en côte d’ivoire. int. j biol chem sci; 5(6): 2317-2329, (2011) [20]. eddaya t, boughdad a, becker l, chaimbault p, zaïd a. utilisation et risques des pesticides en protection sanitaire de la menthe verte dans le centre-sud du maroc. j mater environ sci; 6(3): 656-665, (2015) [21]. muliele mt, manzenza mc, ekuke wl, diaka pc, ndikubwayo md, kapalay mo et al. utilisation et gestion des pesticides en cultures maraîchères : cas de la zone de nkolo dans la province du kongo central, république démocratique du congo. j appl bio... [22]. son d, somda i, legreve a, schiffers b. pratiques phytosanitaires des producteurs de tomates du burkina faso et risques pour la santé et l’environnement. cah agric; 26, 25005, (2017) [23]. nare nwa, savadogo pw, gnankambary z, nacro hb, sedogo pm. analysing risks related to the use of pesticides in vegetable gardens in burkina faso. agriculture, forestry and fisheries; 4(4): 165-172, (2015) [24]. assogba-komlan f, anihouvi p, achigan e, sikirou r, boko a, adje c, et al. pratiques culturales et teneurs en éléments antinutritionnels (nitrates et pesticides) du solanum macrocarpum au sud du benin. african journal of food, agriculture, nutr... [25]. doumbia m, kwadjo ke. pratiques d’utilisation et de gestion des pesticides par les maraichers en côte d’ivoire : cas de la ville d’abidjan et de deux de ses banlieues (dabou et anyama). journal of applied biosciences; 18: 992-1002, (2009) [26]. toe am, ouedraogo m, ouedraogo r, ilboudo s, guissou pi. pilot study on agricultural pesticides poisoning in burkina faso. interdiscip toxicol; 6(4): 185-191, (2013) [27]. sonchieu j, nsoh fj, waingeh nc. pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon. food and environment safety, 17(3): 341-351, (2018) [28]. gnankiné o, mouton l, savadogo a, martin t, sanon a, dabire rk, vavre f, fleury f. biotype status and resistance to neonicotinoids and carbosulfan in bemisia tabaci (hemiptera : aleyrodidae) in burkina faso, west africa. international journal of... [29]. keddal h, n’dri jy. impact de l’intensification agricole sur la qualité des eaux de surface et des eaux souterraines. révue hte; 138: 13-29, (2008) [30]. bao g, wang mh, william lc, dao jc, zheng js. risk assessment of cyhalothrin on aquatic organism in paddy weld in china. regulatory toxicology and pharmacology; 48: 69-74, (2007) [31]. ngom s, seydou t, thiam bm, manga a. contamination des produits agricoles et de la nappe phréatique par les pesticides dans la zone des niayes au sénégal. rev sci technol., synthèse; 25: 119-130, (2012) [32]. agbohessi pt, toko ii, ouedraogo a, jauniaux t, mandiki snm, kestemont p. assessment of healthstatus of wild fish inhabiting a coton basin heavily impacted by pesticides in benin (west africa). sciences of the total environment; 506-507; 567-584... [33]. toko ii, attakpa ey, tobada pc, ble cm, guedegba ln, elegbe h. impact des pesticides agricoles sur les performances physiologiques des poissons : cas du tihan 175 o-teq sur la reproduction des femelles de clarias gariepinus exposées à des doses ... [34]. adetola jo, ndimele pe, onuoha s. acute toxic effects of endosulfan (organochlorine pesticides) to fingerlings of african catfish (clarias gariepinus, burchell, 1822). american-eurasian j agric & environ sci; 10(5): 884-892, (2011) [35]. li-ming h, john t, albert w, kean g. environmental chemistry, ecotoxicity and fate of lambda-cyhalothrin. review of environmental contamination and toxicology; 100: 71-91, (2008) [36]. çakmak mn, gorgon a. toxic effect of a synthetic pyrethroid insecticide (cypermethrin) on blood cells of rainbow trout (onchorhychus mykiss, walbaum). journal of biological sciences; 3: 694-698, (2003) [37]. han j, liqin z, luo m, liang y, zhao w, wang p, zhou z, liu d. non occupational exposure to pyrethroid and risk of coronary diseases in the chinese population. environmental sciences and technology; 51(1): 664-670, (2017) [38]. el-demerdash fm. lambda-cyhalothrin-induced changes in oxydative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxydants. toxicol. in vitro.; 21(3): 392-397, (2007) [39]. ali a, khan ja, khaliq t, javed i, muhammad f, aslam b, khan mz. hematobiochemical disruptions by lambdacyhalothrin in rats. pak vet j; 34(1): 54-57, (2014) [40]. green t, toghill a, lee r, waechter f, weber e, noakes j. thiamethoxam induced mouse liver tumors and their relevance to humans ; part 1 : mode of action studies in the mouse, toxicological sciences; 86(1): 36-47, (2005) [41]. iarc. some organophosphate insecticides and herbicides: tetrachlorvinphos, parathion, malathion, diazinon and glyphosate. iarc working group, monographs volume 112 on the evaluation of carcinogenic risks to humans; (in press) (2015) [42]. de roos aj, blair a, rusiecki ja, hoppin ja, svec m, dosemeci m, sandler dp, alanvanja mc. cancer incidence among glyphosate exposed pesticide applicators in the agricultural health study. environmental health perspective; 113(1): 49-54, (2005) [43]. lanphear bp, horung r, khoury j, yolton k, baghurst p, bellinger dc. low-level environmental lead exposure and children intellectual function: an international pooled analysis. environ. health perspect; 113: 894-899, (2005) [44]. azandjeme cs. exposition aux pesticides et risques de diabète de type 2 : une étude au nord du benin (afrique de l’ouest). mémoire de thèse de philosophiae doctor (phd) en nutrition, au département de nutrition, faculté de médecine, université d... [45]. anger jp, kintz p. difficultés analytiques de la caractérisation des pesticides dans le sang. ann toxicol anal; 21(3): 131-141, (2009) enrichment of pasta products using beetroot 222 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvi, issue 4 2017, pag. 222 233 determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and the first survey of the bulgarian market *valentina christova-bagdassarian 1 , julieta tishkova 1 , anton tachev1 1department of chemical substances and mixes & food contact materials, national center of public health and analyses; 15, acad. iv.geshov bul. 1431 sofia, bulgaria *corresponding author: v.hristova@ncpha.government.bg received 9th october 2017, accepted 19th december 2017 abstract: phthalates are a large group of compounds with similar chemical structure widely used as plasticizers for plastics. they can easily be released from the polymer during usage and in this way enter the human body. their adverse effect on health is known as "phthalate syndrome" and can impact children and adults alike. for children in particular, there is an additional potential risk of exposure to phthalates via toys intended for oral use. the regulation (ec) no 1907/2006 (reach) prohibits the use of bis (2-ethylhexyl) phthalate (dehp), di butyl phthalate (dbp) and benzyl butyl phthalate (bbp) as a substance or constituent of preparations at concentrations higher than 0.1 % in the plasticized material meant to toys and childcare items. the aim of this article is to quantify the content of dehp, dbp and bbp in soft toys intended for children up to 3 years of age, including toys designed to be placed in the mouth. a method consisting of an extraction procedure of phthalates from polymers, purification of the extract, followed by gc/ms identification and quantification was validated. three different techniques for phthalates’ extraction (soxhlet, ultrasonic and vibrator) were compared and the most effective one was chosen.this method was applied to the analysis of dehp, dbp and bbp in soft toys from the bulgarian market to establish their compliance with reach requirements. all the tested toys contained at least one phthalate. exceedence of the permitted content for dehp, dbp and bbp (as sume), mainly due to the high levels of dehp, was observed in several toys from retail. keywords: phthalates; dbp; bbp; dehp; plastic toys; gc/ms. 1. introduction phthalates (phthalate esters) are a large group of compounds which have a similar chemical structure and are widely used as plasticizers for plastics, in solvents, in cosmetics and others. they are not covalently bound to the molecules of the plastic and they can easily be released directly or through migration, evaporation, leaching and abrasion into food or into the environment. this is a prerequisite for their penetration in the human organism through various pathways ingestion, inhalation, dermal absorption, and even intravenously from medical devices. phthalates have a low acute toxicity (ld50 1-30 g/kg), but their toxic effects at low exposure levels affect the liver and reproductive system, especially in males. in animal studies, phthalates have been identified as endocrine disrupters. phthalate exposure has an impact on humans of all ages. "phthalate syndrome" which refers to the wide range of effects of phthalates on males includes infertility, decreased sperm count, cryptorchidism, hypospadias and other malformations of the reproductive organs. studies on young girls also showed possible correlation http://www.fia.usv.ro/fiajournal mailto:v.hristova@ncpha.government.bg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 223 between exposure to phthalates and shortening of the pregnancy duration or breast cancer. for the general population the main sources of exposure to phthalates are foods, followed by inhalation of stuffy and/or polluted air. in animal studies it has been established that phthalates can pass to the offspring through the placenta and through the stern. to the sources of exposure for children and adolescents there should be added the risk of exposure through toys, breastmilk, children's cosmetics and personal care products. in infants, human milk and toys intended for chewing and scratching the teeth, can additionally contribute to phthalate exposure [1, 2]. children and adolescents consume more calories per kilogram body weight, they eat more frequently and more fatty foods and they have higher minute ventilation than the adults. therefore, their phthalate exposure is expected to be higher [3]. the european union, united states and a number of other countries have regulated certain phthalates in children’s products. the regulation (ec) no 1907/2006 of the european parliament and of the council on 18th december 2006 concerning the registration, evaluation, authorisation and restriction of chemicals (reach), which established an european chemicals agency, by entry no. 51 to annex xvii “restrictions on the manufacture, placing on the market and use of certain dangerous substances, preparations and articles” prohibits the use of a substance or constituent in preparations at concentrations higher than 0.1 % by mass of the plasticised material, in toys and childcare articles of the following phthalates: bis (2-ethylhexyl) phthalate (dehp) cas № 117-81-7; dibutyl phthalate (dbp) cas № 84-74-2; benzyl butyl phthalate (bbp) cas № 85-68-7[4]. restrictions on other phthalates were introduced by reach, too. despite the restrictions, phthalates have been found in 69% of plasticisers in the usa, 92% in western europe and 81% in japan [5]. furthermore, it was found that the overall exposure for children and adolescents of the most common plasticizer dehp is higher than that for adults in all ways of administration, excluding food intake. in children aged 5 months to four years of age, the risk is the highest. studies on the relationship between exposure to phthalates and the development of the nervous system of infants show an impact on mental development and psychomotor indices in infants, especially on post six month development [6]. some effects include deformation of the reproductive organs (hypospadias, cryptorchidism), decreased anogenital distance, impact on future fertility and pregnancy, weight reduction of the reproductive organs, disturbance in testicular functioning, impact on the organization and functioning of the germ and spermatogonial stem cells [7-10]. apart from the information on the main polymer from which an article has been made, labels should describe data of the other substances involved in the composition plasticizers, fillers, colorants, etc. the control of the composition of polymers is associated with the availability of analytical methods for determining ingredients in different matrices. the purpose of this work is to develop a combination of extraction procedure and gas chromatography mass spectrometry analysis (gc/ms) to quantify the content of three phthalates (dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp)) in polymeric toys, intended for children up to 3 years of age or to be placed in the mouth. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 224 2. materials and methods experimental design ten different articles of soft plastic toys with different composition, country of origin and place of distribution (large chains and small retailers) were purchased from the bulgarian market. all toys were designed for children up to 3 years of age or for oral use. the information on the toys was collected from labels. descriptions of the plastic toys are presented in the table 1 and in fig 1. table 1. description of the plastic toys for analysis of phthalates № description suitable age distributor for bulgaria country manufacturer type of plastic note 1. yellow bath duck „duck 5,5 сm”, yellow „happy people” ом+ not specified on the label a large retail chain peoples republic of china unknown, not stated on the label distributor germany art. no.40189 2. white dog honey baby for children over 6 months little shop peoples republic of china unknown, not stated on the label art. no.tl.306 lot. no. bt130134 3. pink teddy bear honey baby for children over 6 months little shop peoples republic of china unknown, not stated on the label art. no. tl.306 lot. no. bt130134 4. teether "foot" for children between 0 and 18 months a large retail chain hong kong special administrative region of the people's republic of china polyester item no.08/8434/в lot. no. rhsc-141004 5. teeth "dino" for children over 3 years a large retail chain peoples republic of china unknown, not stated on the label import from turkey chocolate egg with a toy surprise barcode no:8699462607173 barcode box:8699462607 6. vampire teeth carnival accessories not specified on the label a large retail chain peoples republic of china unknown, not stated on the label item no.04017 7. doll "baby", 12 сm for children over 12 months a large retail chain peoples republic of china unknown, not stated on the label lot. lm140112wj item no.w10786j (8082) 8. stand"color rings"for order in chimney stack „rainbow colored stacker for children over 10 months a large retail chain peoples republic of china unknown, not stated on the label imported from romania nd.710v 9. teether "three fruits"ed, green and yellow for children over 6 months a large retail chain peoples republic of china unknown, not stated on the label item no.34471-1 batch: 20130530 10. teether-rattle „teetherfeeding bottle" baby rattles series not specified on the label a large retail chain peoples republic of china unknown, not stated on the label item no.fs34691-2 batch no. 20141220 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 225 fig. 1 representative photos of the analyzed toys the type of polymer was mentioned on one item only. a lack of marking and proper labeling of the toys was discovered, which violates consumers’ rights to be informed about the quality of the toys. all the items were in quantity of 5 or more, allowing an average sample to be formed from the batch. the samples were cut at retail and average samples and analytical samples were formed. the choice of extraction method of the phthalates from the polymeric material was made as a result of comparative experimental tests as described below. reagents and chemicals all the chemicals used were of analytical grade purity for gc: dichloromethane for gc, pestanal®, (fluka); hexane for gc (merck); glass microfiber filters whatman®, grade gf/a circles, ø 47 mm; syringe filters “chromatography” ptfe, 0.45 µm, ø 25 mm; medical syringes of 5 ml and 10ml, agilent certified caps with integrated septa (ptfe/red rubber) on 2 ml vials wide opening screw and 2 ml vials for automatic injector, wide opening screw, colorless; cellulose extraction thimble for soxhlet, 30x80 mm; acetone and methanol for gc (merck). certified reference materials/reference materials crm/rm di-n-butyl phthalate (dbp) cas # 84-74-2, crm, 96.8 % purity (accustandard®,inc.); benzyl butyl phthalate (bbp) cas #85-68-7, 96.9 % purity (acros organics); bis-(2ethylhexyl)-phthalate pestanal®, cas #117-81-7, rm, 99.7 % purity (sigma aldrich). equipment and gas chromatographic conditions agilent technology gc-7890 gc/ms with split/splitless capillary injector, autoinjection system and quadrupole mass spectrometric detector (msd-5975 inert) with electronic ionization (ei) working in sim and scan mode. the gas chromatograph was equipped with a fused silica capillary column slb ™ -5ms 30 m x 0.25 mm i.d. x 0.25 μm film thickness (sigma-aldrich, supelco). the temperature of column was programmed as follows: the initial temperature 50ºс (2 min), 30˚c/min to 280˚c (10 min); injector temperature was 280˚c; the transfer line temperature was 280˚c, helium carrier gas with a flow rate 1.3 ml/min in splitless mode; sample size (injection volume)1μl; solvent delay 5.80 min. the duration of a single analysis is of 20 min. additional equipment soxhlet extraction system with adjustable heating consists of soxhlet extractor, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 226 cartridge, reflux condenser, 250 ml roundbottom flask with heating mantle; glass tubes with screw caps of 40 ml volume, shaker with temperature-controlled water bath; ultrasonic bath with temperature control; temperature-controlled rotary evaporator; analytical balance; buchner filtration system. identification of dbp, bbp and dehp in msd scan and msd sim-modes the identification of the phthalates is carried out by the retention times of standard solutions of crm/rm of dbp, bbp and dehp in msd scan mode. using the chromatographic conditions described above, the retention times of dbp, bbp and dehp were 9.15 min, 10.48 min and 11.17 min, respectively. identification was confirmed by comparing their mass spectra with the nist specialized library. to improve the parameters of the method, after specifying the chromatographic conditions, a msd sim mode was used with the introduced characteristic ions for each phthalate. some important physical and chemical properties and the monitored ions for mass spectrometric identification of dbp, bbp and dehp are presented in the table 2. phthalates are common laboratory contaminants, so special attention was paid to eliminating the external contamination. an important factor is the cleanliness of the glassware. the laboratory dishes were washed with distilled water and rinsed with solvent. the solvents used must be of high grade of purity and not in contact with plastic parts, elements or gaskets, with the exception of polytetrafluoroethylene ptfe, which is resistant to chemicals. all the samples are performed in 5 replicates, spikes are also used. calibration stock standard solutions a of each phthalate at a concentration of 1000 μg/ml were prepared individually by weighing of crm/rm of dbp, bbp or dehp and suitable diluted with hexane. the storage period at 4 ° c in the dark is 1 month. intermiate standard solutions b for each phthalate at a concentration 100 μg/ml were prepared from the stock solutions by appropriate dilutions with hexane. mixed standard calibration solutions of dbp, bbp and dehp in concentration range from 0.5 to 10 μg/ml for dbp and 1.0 to 50 μg/ml for bbp and dehp were prepared by appropriate dilutions with dichloromethane from the standard solutions b. the calibration curves were plotted separately for each phthalate concentration against peak areas. calibration solutions were analyzed in scan mode and the characteristic ions for each phthalate were monitored in sim mode. the regression equations of dependencies of peak areas (in absolute indexes) of the concentrations (µg/ml) for each phthalate in sim mode are presented in table 3. limit of detections (lod) and limit of quantifications (loq) values of all the standards were calculated by signal to noise s/n ratio multiplied by three and ten, respectively. all values are recorded in five replicates for each experiments. table 4 summarizes the parameters of gc/ms method in scan and sim modes. the relative standard deviation rsd is calculated at the concentration level of 5 μg/ml. sample preparation before analyzing the samples (at least 3 items) from each kind of articles, they were cleaned by a microfiber cloth to remove dust and impurities from their surfaces. then they were crushed / milled to obtain particles of about 1-2 mm2 which increase the effective extraction surface (fig 2). about 1 g, with an accuracy of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 227 0.0001 g, was weighed for analysis by analytical balance for each item type. the sample with a standard addition of a mixed standard solution (spike-sample) was analyzed every time for each sample. dichloromethane is a suitable solvent which can either dissolve or soften the polymeric material. table 2. physico-chemical properties and the monitored ions for mass spectrometric identification of dbp, bbp and dehp substance cas no molecular formula molar mass, boiling point, basic ion characteristic monitored ions g.mol-1 0c q1 q2 q3 di-n-butyl phthalate(dbp) 84-74-2 c16h22o4 278.34 340 149 205 223 278 (m +) butyl benzyl phthalate(bbp) 85-68-7 c19h20o4 312.36 370-380 149 206 91 238 di-(2-ethylhexyl) phthalate(dehp) 117-81-7 c24h38o4 390.56 385 149 167 279 table3. mean values in absolute indexes of the peak areas (n = 5) of the calibration solutions (sim mode) peak area, abs.ind. phthalate concentration of the phthalates in the calibration solutions, µg/ml calibration equation 0.5 1.0 5.0 10 50 dbp 210329 473781 5941599 18477433 bbp 55740 542674 1891438 26693875 dehp 66228 468432 1561130 16541333 table 4. parameters of the gc/ms method for the determination of dbp, bbp and dehp in scan and in sim modes of the msd phthalate scan-mode sim-mode linearity range, (r2) rsd,a % lod, µg/ml loq, µg/ml rsd,a % lod, µg/ml loq, µg/ml μg/ml, dbp 18.4 0.27 0.49 8.6 0.03 0.09 0.5–10 (r2=0.9738) bbp 14.9 0.21 0.50 5.2 0.01 0.04 1.050 (r2=0.9890) dehp 14.6 0.14 0.41 4.7 0.02 0.07 1.0 50 (r2=0.9925) a rsd is calculated at a concentration level of 5 μg / ml for each phthalate http://www.commonchemistry.org/chemicaldetail.aspx?ref=85-68-7 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 228 fig.2 average samples of toys prepared for extraction 3. results and discussion 3.1. selection of an extraction procedure three different extraction techniques were compared: soxhlet extraction [11, 12] ultrasound extraction [13, 14, 15] shaker extraction [16, 17] the samples were extracted by one of the extraction techniques mentioned above; the resulting extracts were purified to remove co-extracted solid particles either by centrifugation or by filtration through suitable filters. soxhlet extraction samples of 1 g ± 0.0001 g were weighed into cellulose extraction thimbles and were extracted by soxhlet using about 180-200 ml dichloromethane at the temperature of the round-bottomed flask, which guarantees no vigorous solvent boiling. the duration of the extraction procedure was 120 min, 240 min or 360 min, respectively. the cooled extracts were filtered through a glass microfiber filters whatman®, gf/a, via buchner-system and were concentrated using a rotary evaporator at a bath temperature of about 32-34 °c to received exact volumes of 10 ml. in parallel, samples with spike 10 μg/ml for dbp and bbp and 50 μg/ml for dehp, calculated to the final volume of 10 ml, were prepared, too. ultrasound extraction the samples (1 g) and the spike samples (see above) were wetted with 20 ml of dichloromethane in the glass tubes, closed tightly and placed in an ultrasonic bath for 15 min, 30 min or 60 min at a bath temperature of 25 °c, 35 °c or 38 °c. extracts were filtered through a glass microfiber filter whatman®, gf/a via buchner-system, transferred quantitatively into graduated tubes and evaporated in a stream of nitrogen to the exact volume of 10 ml. shaker extraction the samples (1 g) and the spike samples are filled with 10 ml of dichloromethane in glass tubes, sealed tightly and stirred on a shaker at 25 ºc of the water bath. the durations of the treatments were of 30 min 60 min or 120 min. the extracts were filtered and concentrated, as it was described in „ultrasound extraction” to the exact volume of 10 ml. approximately 2 ml of the concentrated extracts were filtered through a filter-ptfe using a medical syringe directly into vials for gc-ms analysis. the analyses were performed in the above-described chromatographic conditions; the concentrations of the phthalates were calculated by regression equations. recovery (r) was calculated using parallel analysis of samples and spike-samples. spikes were added at concentrations 10 μg/ml for dbp and bbp, and 50 μg/ml for dehp. recovery (r) was calculated as follows (eq. 1): food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 229 , % (1) where ccalc. is the spike-concentrations of the each individual phthalate, calculated from the corresponding regression equation after the analysis, сsp. is the concentration of the spike, added to the sample. the results obtained are presented in table 5. they indicate that the use of a soxhlet extraction for retrieving the phthalates from the polymer showed the highest recovery, varying in the range of 91.7% 101.1% for the analyzed phthalates. the highest rates are for bbp (98.5%) and dehp (101.1%) and were observed at the duration of the extraction 120 min. for dbp through soxhlet extractions close values of recoveries at two hours (97.8%) and six hours (98.7%) were observed. following these results, the sample preparation was selected to be held by soxhlet extraction with dichloromethane over 120 min. table 5. comparison of recoveries of phthalates aby the three methods of sample preparation № sample preparation recovery r, % dbp bbp dehp i. soxhlet extraction i.1. soxhlet extraction, 120 min 97.8 98.5 101.1 i.2. soxhlet extraction, 240 min 96.9 96.9 91.7 i.3. soxhlet extraction, 360 min 98.7 96.0 92.8 ii. ultrasound extraction: ii.1. ultrasound extraction at 25 0с, 15min 78.5 76.3 77.1 ii.2. ultrasound extraction at 25 0с, 30 min 69.5 78.4 80.8 ii.3. ultrasound extraction at 25 0с, 60 min 70.8 77.1 79.4 ii.4. ultrasound extraction at 350с, 15 min 46.6 42.3 38.4 ii.5. ultrasound extraction at 350с, 30 min 41.2 47.0 51.2 ii.6. ultrasound extraction at 350с, 60 min 40.7 45.3 47.9 ii.7. ultrasound extraction at 380с, 15min 54.7 55.5 59.1 ii.8. ultrasound extraction at 380с, 30 min 50.4 47.8 55.6 ii.9. ultrasound extraction at 38 0с, 60 min 51.2 49.7 55.2 iii. shaker extraction iii.1. shaker extraction at 25 0с, 30 min. 72.9 68.4 71.2 iii.2. shaker extraction at 25 0с, 60 min. 70.9 71.1 75.2 iii.3. shaker extraction at 25 0с, 120 min. 70.3 70.6 74.7 3.2. analysis of soft toys the soft plastic toys were pre-cut and 1 g ± 0.0001 g of anaverage samples were extracted by soxhlet using dichloromethane, as described above. the extracts were filtered, concentrated and analyzed by gc/ms in sim mode. the iidentification was carried out by the retention times and by comparison with a nist library. the chromatograms of sample 1 and sample-1spike (yellow bath duck „duck 5.5 сm” in sim-mode after pretreatment by soxhlet extraction for 2 hours are presented in the fig. 3 and 4, respectively. the concentrations were calculated using the calibration equations (table 3). the results were recalculated in %, taking into account the exact weighed quantity for each sample (table 6). the content of each food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 230 phthalate in the sample, x, in %, was calculated as follows (eq. 2): (2) where cx is the calculated concentration in the sample of each phthalate, in μg/ml; w is the weighted amount of the sample in g. fig. 3. chromatogram of sample 1 (yellow bath duck „duck 5.5 сm” in sim-mode after pretreatment by soxhlet extraction for 2 h fig. 4. chromatogram of sample-1spike (yellow bath duck „duck 5.5 сm” in sim-mode after pretreatment by soxhlet extraction for 2 h risk assessment for dbp, bbp and dehp takes into account their additive effect all three phthalates impact on the reproduction and development of young organisms. for this purpose, the results for individual phthalates should be summed and compared, as a percentage, to the acceptable limit of 0.1 %, according to the entry no.51 of annex xvii to regulation reach. the results for the dbp, bbp and dehp contents in analyzed samples are presented in the table 6. all the tested toys contain at least one phthalate. dehp was identified and quantified in 9 samples; dbp in 8 samples. bbp was found rarely and was detected in one sample only. the total content of phthalates in one of the samples sample no.1 was 0.09%, which was within the acceptable value of 0.1%, but very close to it. in three samples (samples no.no. 2, 3 and 7), the total content of phthalates exceeds the acceptable value of 0.1% under regulation reach, as the excess is due to the content of dehp. the permissible total content of phthalates was exceeded significantly in two samples (no. no. 2 and 3), 13 and 26 times, respectively, which were exactly the samples purchased from small shops. dehp, most commonly present in the results, is defined as endocrine disruptor in humans that causes decreased sperm motility and damage to the chromatin, germ cell developmental disturbances and it has the potential to alter androgenresponsive brain development in humans. fetuses and newborn babies are more susceptible to the testicular toxicity of dehp than mature individuals and certain stages of fetal and neonatal development are critical.the toxicity of dbp is focused on the male reproductive system [7, 9]. a similar toxicity in humans can lead to impaired fertility and may not manifest itself for many years until adulthood. the highest risk is present in children from 5 months to 3 years of age, because phthalates can be taken in the body (except through food) by mouth, by licking or chewing toys or other objects containing phthalates. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 231 the potential risk of toxicity of toys threatens child health and depending on the dose and timing of exposure of the child to phthalates could lead to health problems in children or unlock the hidden diseases in later years. despite the limited scope of this study, some trends have been identified. the required labels on most toys did not provide the necessary information for the customer.the quality of toys purchased from large chains meets the requirements of the european legislation regarding phthalates, but caution should be exercised by regulatory authorities regarding toys from retail shops (so-called "shopping for one euro"). a clear declaration of the composition of the items on the labels will allow users to choose alternatives. table 6. the results for the phthalates dbp, bbp, dehp in soft toys № article identified phthalate concentration, µg/ml in the extract, µg/ml weight of the sample, g phthalate content in the sample, g/kg phthalate content in the sample, % total content of phthalates, % 1 yellow bath duck dbp dehp 14.612 79.209 1.0369 0.1409 0.7639 0.0141 0.0764 0.09 2 white doc dehp 1322.473 1.0130 13.0550 1.3055 1.31 3 pink teddy bear dehp 2650.781 1.0159 26.0929 2.6093 2.61 4 teether„foot“ dbp dehp 2.872 9.082 1.0056 0.0286 0.0903 0.0029 0.0090 0.01 5 teeth “dino” dbp dehp 10.123 13.285 1.0076 0.1005 0.1319 0.0100 0.0132 0.02 6 vampire teeth dbp bbp dehp 3.572 7.933 8.264 1.0899 0.0328 0.0728 0.0758 0.0033 0.0073 0.0076 0.02 7 doll “baby” dbp dehp 5.045 121.111 1.1029 0.0457 1.0981 0.0046 0.1098 0.11 8 rainbow colored stacker dbp dehp 6.376 4.873 1.0852 0.0588 0.0449 0.0059 0.0045 0.01 9 teether “three fruits” dbp 8.014 1.0313 0.0777 0.0078 0.01 10 teether„feeding bottle“ dbp dehp 5.641 64.789 1.0197 0.0553 0.6354 0.0055 0.0635 0.07 4. conclusions a method for determining the three phthalates dbp, bbp and dehp in polymeric materials was developed. the method includes a choice of an extraction procedure and gc/ms identification and quantification. three different extraction techniques for extracting the phthalates from the polymeric material were compared: soxhlet extraction, ultrasound extraction and shaker extraction. experimentally, it was found that soxhlet extraction provides the most efficient recovery. gc/ms analysis was performed on system agilent technologies gc 7890 system with quadrupole mass selective detector model msd 5975 inert with electronic ionization (ei); fused silica capillary column slb ™ -5ms 30 m x 0.25 mm i.d. x 0.25 μm film thickness at programmed mode chromatographic column. the identification of phthalates was performed using method of external standards and retention times of dbp, bbp and dehp in msd scan-mode and was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 232 confirmed by the specialized nist mass spectra library in msd sim-mode. the method was applied to the analysis of dbp, bbp and dehp in soft toys intended for children up to 3 years of age, some of which designed to be placed in the mouth and chewed. this study was done in order to establish the compliance with the requirements of reach regulation. it was proved that at least one phthalate is contained in each sample. in 30% of the samples there was found an exceeding of the permissible total content of phthalates under reach was exceeded, mainly due to high content of dehp in the polymer. excess of around 10-30 times was found in 20% of the analyzed samples purchased from retailers in small shops. a lack of marking and labeling of the toys was found, which violates the consumers’ rights to be informed about the quality of toys. conflict of interest: none. 5. references [1]. mortensen g. k., main k. m., andersson a.-m., leffers h., skakkebaek n.e., determination of phthalate monoesters in human milk, consumer milk, and infant formula by tandem mass spectrometry (lc– ms–ms), analytical and bioanalytical chemistry, 382(4): 1084–1092, (2005). [2]. silva m.j., barr d.b., reidy j.a., malek n.a., hodge c.c., caudill s.p., calafat a.m., urinary levels of seven phthalate metabolites in the u.s. population from the national health and nutrition examination survey (nhanes) 1999-2000, environmental health perspectives, 112(3):331–338, (2004). [3]. etzel r.a., balk s.j. (ed.), pediatric environmental health, 3rd edition, mdamerican academy of pediatrics, 907, (2011). [4]. regulation (ec) no 1907/2006 of the european parliament and of the council of 18 december 2006 concerning the registration, evaluation, authorisation and restriction of chemicals (reach), establishing a european chemicals agency, amending directive 1999/45/ec and repealing council regulation (eec) no 793/93 and commission regulation (ec) no 1488/94 as well as council directive 76/769/eec and commission directives 91/155/eec, 93/67/eec, 93/105/ec and 2000/21/ec (text with eea relevance) (2006). [5]. johnson s., saikia n., sahu r., phthalates in toys available in indian market, bulletin of environmental contamination and toxicology, 86(6): 621, (2011). [6]. kim y., ha e., kim e., park h., ha m., kim j., kim b., prenatal exposure to phthalates and infant development at 6 months: prospective mothers and children’s environmental health (moceh) study, environmental health perspectives, 119(10):1495–1500, (2011). [7]. kavlock r., boekelheide k., chapin r., cunningham m., faustman e., foster p., zacharewski t., ntp center for the evaluation of risks to humanreproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate, phthalate reports, 16(5): 489–527, (2002). [8]. foster p.m., cattley r., mylchreest e., effects of di-n-butyl phthalate (dbp) on male reproductive development in the rat: implications for human risk assessment, food and chemical toxicology, 38(suppl. 1):s97–s99, (2000). [9]. swan s., main k., liu f., stewart s., kruse r., calafa a. , sullivan s., decrease in anogenital distance among male infants with prenatal phthalate exposure, environ health perspect, 113:1056–1061, (2005). [10]. doyle t.j., bowman j.l., windell v.l., mclean d.j., kim k.h., transgenerational effects of di-(2-ethylhexyl) phthalate on testicular germ cell associations and spermatogonial stem cells in mice, biology of reproduction, 88(5):112, (2013). [11]. rastogi s.c., jensen g.h., worsøe i.m., compliance testing of phthalates in toys. analytical chemical control of chemical substances and products.neri research notes no. 185, national environmental research institute ministry of the environment. denmark, 26 p., (2003). [12]. huang l., liu z., yi l., liu c., yang d., determination of the banned phthalates in pvc plastic of toys by the soxhlet extraction-gas chromatography/mass spectrometry method, international journal of chemistry, 3(2):169, (2011). [13]. schreiber a., fu f., yang o., wane., gu l., leblanc y., increasing selectivity and confidence in detection when food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvi, issue 4 – 2017 valentina christova-bagdassarian, julieta tishkova, anton tachev, determination of dibutyl phthalate (dbp), benzyl butyl phthalate (bbp) and bis (2-ethylhexyl) phthalate (dehp) in soft plastic toys and first survey of bulgarian market, food and environment safety, volume xvi, issue 4 – 2017, pag. 222 – 233 233 analyzing phthalates by lc-ms/ms., absciex, publication number: 3690411-01, 6 p., (2011). [14]. dimple s., burgess j., one-minute method for the screening of phthalates in toys at regulatory limits using uplc/ms and empower software [pdf]. waters corporation, milford, ma usa (2010). [15]. goodman w., gc/ms determination of phthalate concentration in plastic toys [pdf]. perkinelmer inc, 62, the application notebook, (2008). [16]. tihg k.-c., gill m., garbin o. ecl report 2008-03 mobile laboratory screening method for phthalate esters in children’s toys by gas chromatography/mass spectrometry. california, dept. of toxic substances control (2008). [17]. ting k.-c., gill m., garbin o., gc/ms screening method for phthalate esters in children’s toys, journal of aoac international, 92(3): 951–958, (2009). 1. introduction 4. conclusions title …………………… 41 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 1 2018, pag. 41 47 edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies * oksana shulga 1 , natalya pritulska 2 , oksana petrusha 3 , jelyzaveta smirnova 4 1educational and research institute of food technologies, national university of food technologies, kyiv, ukraine, shulgaos@nuft.edu.ua 2kyiv economics and trade university, kyiv, ukraine, pritulska@knteu.kiev.ua 3educational and research institute of food technologies, national university of food technologies, kyiv, ukraine, petrushaoo@ukr.net faculty of hotel and restaurant and tourism business, national university of food technologies, kyiv, ukraine, smirnovaliza777@ukr.net *corresponding author shulgaos@nuft.edu.ua received 5th november 2017, accepted 20th march 2018 abstract: the article presents a study on the changes occurring in the qualitative and quantitative characteristics of “korivka” fondant candies when replacing traditional paper wrapper with edible coating. paper industry refers to sources of intensive pollution. it is expedient to use edible films and coatings for confectionery products in order to comply with the requirements of the directive 94/62 / eu of the european parliament and union on packaging and packaging waste, aimed at minimizing packaging materials waste. edible coating is made of natural polymers – starch, gelatin together with plasticizer – urea or glycerol and hydrophobic component – linseed oil and solvent. the proposed composition of edible coating is completely utilized due to its use along with candies. candies have been studied according to organoleptic parameters, humidity and mass change, as well as substances in view of reducing 3 month-shelf life. in addition, the change in sucrose crystals size during the storage period through a microscope with further image rendering by computer program has been determined. keywords: edible coating fondant candy, paper wrapper, storage. 1. introduction the first mentioning about edible coatings can be found in the works of the 40's of the last century. chocolate or confectionery glaze can also be attributed to edible coatings, but some foreign experts call them chocolate films [1]. however, edible coating is an independent term, which for the time being is not fixed yet in normative documentation of ukraine. distinguishing edible coating as a separate term is necessary and valid, since the composition of glazes obligatory includes 12% or more fat (cocoa butter, confectionery fat, etc.). however, the composition of edible coating can not contain fat at all; it is added in the amount of 10% or less in order to improve their barrier properties [2]. for edible coatings, the term of edible films [3] is also used as they are made from natural carbohydrate polymers (starch, pectin, cellulose, etc.), proteins (soy, whey, etc.), but, as a rule, obtained films have small elongation index values, therefore the term “edible coating” occurs. http://www.fia.usv.ro/fiajournal mailto:shulgaos@nuft.edu.ua mailto:pritulska@knteu.kiev.ua mailto:petrushaoo@ukr.net mailto:smirnovaliza777@ukr.net mailto:shulgaos@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 42 most researchers do not see the difference between the notion “edible film” and “coating”. these notions are used interchangeably to вуашту a thin layer of edible material that can be applied directly to a food product or placed as a barrier between the product and the environment and consumed together with this product. others believe that edible coating is a thin layer applied directly to the product, and the previously obtained film, serving as a wrapper then, is the edible film itself. in this aspect, based on classifying edible packaging into films and coatings, there are only different methods for their obtaining. films are pre-obtained materials, for example, by extrusion. coatings are applied to food directly by casting film-forming solutions, spraying, immersion or using a brush, and are formed when dried on the product. it is expedient to use edible films and coatings for confectionery products in order to comply with the requirements of the directive 94/62/eu of the european parliament and union on «packaging and packaging waste», aimed at minimizing waste of packaging materials. today, the literature provides data on edible film use for biscuit only [4]. edible coatings do not only help to preserve the freshness of products, but also can have functional properties [5]: antimicrobial [6], probiotic [7] and others. ukrainian confectionery industry does not use edible films and coatings yet, although the problem of storage extension term of some confectionery, including fondant candy remains unsolved. the main obstacle for long-term storage of these products is presented by desorption processes indicated by a. kokhan and a. dorokhovich [8]. at present, to extend fondant candies shelf life one uses invertase, glazing [9], packaging into a sealed polymer container [11], as well as adding to their recipes substances that could hamper the removal of moisture from bodies (increase of hygroscopic – retaining agents wet) [11]. today «korivka» candies are packed in paper materials. it is known [12] that paper industry belongs to sources of intense enviromental pollution. a plant with a capacity of 15000 tons of packaging paper per year consumes annually 500000 m3 of wood, 95000 tons of fuel oil or 82000 m3 of natural gas, 1070 mw of electricity per year, and 24800000 m3 of fresh water; it also requires the removal of almost 150 hectares of land from agricultural property for the construction of a concrete reservoir for collecting river water, reservoirs with a capacity of 150 m3 each providing treatment facilities with technical water. wastewater treatment involves the construction of water treatment plants with capacity of 100,000 m3/day, including incoming equipment block, septic tanks and water treatment reagent filters. consequently, reducing the consumption of paper materials is necessary regarding the complicated environmental situation in the world. "korivka" are candies produced with liquid stuffing. traditional "korivka" includes sugar, whole condensed milk (with sugar), butter, syrup, flavoring. the name was obtained due to cow picture on the wrapper. candy viscosity lasts for the first 10-14 days, and then it is slightly saccharified. taste is not affected by the presence of viscosity, in fact, as well as its quality. the сandy caloric content is 351 kcal per 100 g of product. the objective of the study is to determine the effect of edible coating on candies "korivka" quality during their storage and raising their biological value. 2. materials and methods the coatings applied to the surface of “korivka” candies were made in the following way: starch (corn or potato), gelatin (e 441) as film formers, urea (e food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 43 927b) or glycerol (e 422) as plasticizers, linseed oil as hydrophobic component and the source of vitamin f were added. film formers were mixed in dry form, water was added and solution was heated to dissolve gelatin and starch gelling, after that plasticizer was added. the solution was cooled to 40 °c and linseed oil was added whisking until homogeneous emulsion. emulsion obtained was applied to candies surface and held up until the film became dry completely (6-8 h). control sample was stored in traditional paper wrapper. the samples were stored in a dry, clean, well-ventilated room without a foreign smell, without pest infection of grain stocks at the temperature of (18 ± 3) °с and relative air humidity not more than 75% for 3 months. candies were not exposed to direct sunlight. organoleptic parameters of candies have been determined by tasting in accordance with the state standard of ukraine (ssu) 4683:2006. the content of reducing substances has been determined in accordance with ssu 5059:2008. humidity hss been defined by the refractometric method according to ssu 4910:2008. the crystals size change of “korivka” candy solid phase has been estimated by microscopy with subsequent processing using imagej – the open source software for analysis and image processing, written in the java language by the national institute of health and distributed without license restrictions as public property. this program was also used to determine edible coating thickness on the product surface. 3. results and discussion edible coating after drying on the surface of candies is transparent and smooth, so it does not change the appearance, as seen in figure 1. according to ssu 4135:2014, on the basis of organoleptic parameters taste, smell, appearance, shape are determined. fig. 1. “korivka” candies in edible coating taste of proposed edible coating is neutral, therefore, the eating of candies with coating will not change the usual taste of the classic product, which will not lose traditional consumers and involve new groups to consume this product. in order to create contrast while determining coating thickness on candies, red dye was added to coating, as shown in figure 2. after image processing using the imagej computer program, thickness of coating was determined as 0,504 ± 0,05 mm at the top of the product, 0,382 ± 0,05 mm on the lateral surfaces and 0,111 ± 0,05 mm at the bottom. different coating thickness on the surface of candies is explained by the lack of adhesion between the forming solution in the cut to determine coating thickness of edible coating on the surface of product and therefore the bottom and lateral fig. 2. picture of«korivka» candies food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 44 0 2 4 6 8 10 12 14 16 0 7 14 28 42 56 70 90 duration of storage, days h u m id it y o f c a n d ie s , % paper w rapper edible coating fig. 3. humidity changes in of “korivka” candies during storage according to the results shown in figure 3, the reduction in mass fraction of moisture content in products with edible coating is at the same level as in products with classical paper packaging. the given regularity is grounded, since edible coatings possess barrier properties, in particular, vapor permeability [13]. it should be noted that vapor permeability is the value that numerically equals to the amount of water vapor in milligrams passing for 1 hour through a layer of material with 1 m2 area and 1 m thick, provided that the temperature of opposite sides is the same, and the difference in partial pressure of water vapor equals to 1 pa [14]. changing the mass fraction of moisture will cause change of products’ mass, therefore it is also advisable to determine the reduction of products mass during the storage period. results of study are presented in table 1. according to the results obtained, mass reduction of products in edible coating is at the same level as of products in paper packaging. consequently, the barrier properties of coating allow moisture to be kept inside the product, which in turn allows storing a lot of products during the shelf life. change of reducing substances content characterizes stability of sugar confectionery products during the term of storage. accumulation of reducing substances gives them stickiness due to their hydrophilic properties. however, higher content of reducing substances will allow delaying fondant sweets aging and increase the coarse-sugar crystal phase, which will be considered further. results of study of quantitative change “korivka” candies reducing substances are given in figure 4. results of study (figure 4) show that reducing substances content in edible coatings is slightly higher due to slightly higher amount of moisture in products (see figure 1). higher reducing substances content allows to keep fresh fondant candy longer, and edible coating protects from stickness, which can be caused by higher content of reducing agents. one of the factors characterizing freshfondant candy is also the size of sucrose crystals, which is known to increase throughout the storage period [9]. according to the results of study, the increase of fraction with larger size of sugar crystals in products with edible coating occurs more slowly compared with products in paper packaging. thus, for example, in 2 week storage in candy with paper wrapper, sugar fraction with crystal size of 46-55 μm appears. table 1. “korivka” candies mass reduction during storage period type of packaging shelf life (days),% 14 28 42 56 70 90 paper wrapper 0.52 1.60 3.18 4.02 4.89 5.78 edible coating 0.61 2.11 3.63 4.67 5.01 6.24 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 45 13.913.3 12.9 12.211.9 11.3 10.9 10.3 14.3 12.7 13.913.3 11.911.4 10.9 10.3 0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 0 7 14 28 42 56 70 90 storage term , days r e d u c in g s u b s ta n c e s , % paper w rapper edible coating fig. 4. content change of reducing agents depending on packaging material type table 2. size and number of sugar fractions in candies during storage period, μm duration of storage, days type of packaging number of sugar crystal fractions (μm),% 15 25 26 35 36 45 46 55 56 and more 0 paper wrapper, edible coating 21 49 30 14 paper wrapper 16 37 25 22 edible coating 25 50 25 28 paper wrapper 33 50 17 edible coating 50 39 11 42 paper wrapper 38 25 37 edible coating 62 28 10 56 paper wrapper 50 33 17 edible coating 22 33 34 11 70 paper wrapper 50 50 edible coating 66.7 16.7 16.6 90 paper wrapper 29 43 28 edible coating 11 78 11 on the 28th day of storage in both samples sugar crystals fraction of 55 μm or more in size appears, but this fraction disappears in both samples during further storage. this can be explained by two factors: change in climatic conditions in the room where candies were stored (increasing humidity due to dissolution of large crystals) or place where sample was taken for microscopy is closer to candy’s edges). at the end of candy’s shelf-life in paper wrapper, 28% of the fraction had the crystals size of 46-55 μm, while of that in edible coating such a fraction was absent. this can be explained by barrier properties of edible coating: the ability to pass moisture, which can dissolve crystals of sugar, and then it evaporates. in order to ensure demand for proposed change in “korivka” candy packaging, the price of edible coating is also important. the cost of paper packaging for “korivka” candies today amounts to $ 349,79 per 1 ton of sweets on the average, according to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 46 technological design norms of confectionery industry, paper wrapper is spent in the amount of 5,8 kg/ton, the label paper – 45,3 kg/ton. the cost of proposed edible coating in accordance with calculated and approved recipe is $ 341,02 per 1 ton of sweets. consequently, proposed edible coating will not increase the cost of candies, even a little decrease, which will positively affect of products retail price. 4. conclusion it has been proposed to replace paper packaging (wrapper) of “korivka” candy with edible coating. the feasibility has been confirmed by organoleptic parameters. moisture mass fraction change and products mass reduction occur at the same level. at the end of the shelf life, reducing substances content of candy in paper wrapper is 13.87%, and that of candies in edible coating is 14.33%, which is 0.5% within the error margin. fraction increase of larger size sugar crystals in products with edible coating occur more slowly compared with those in paper packaging. the cost of proposed edible coating is lower by $ 8.77 per 1 ton of candies compared with traditional paper material. the results obtained will help producers, as well as consumers, to take an active part in setting the environmental situation in case of support of proposed environmental packaging. 5. acknowledgments anastasia chorna – the assistant of foodstuff expertise department, national university of food technologies, kyiv, ukraine, who helped in the experimental work. 6. references [1]. biquet b., & labuza t. p., evaluation of the moisture permeability characteristics of chocolate films as an edible moisture barrier, journal of food science, 53(4): 989-998, (1988). [2]. garcia m. a., martino m. n., & zaritzky n. e., lipid addition to improve barrier properties of edible starch‐based films and coatings. journal of food science, 65(6): 941-944, (2000). [3]. torres j. a. edible films and coatings from proteins. protein functionality in food systems: 467-507, (1994). [4]. panchev i. n., baeva m. r., & lambov s. i., influence of edible films upon the moisture loss and microstructure of dietetic sucrose-free sponge cakes during storage. drying technology, 23(4): 925-940, (2005). [5]. falguera v., quintero j. p., jiménez a., muñoz j. a., & ibarz a., edible films and coatings: structures, active functions and trends in their use. trends in food science & technology, 22(6): 292-303, (2011). [6]. seydim a. c., & sarikus g., antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. food research international, 39(5): 639-644, (2006). [7]. kanmani p., & lim s. t., development and characterization of novel probiotic-residing pullulan/starch edible films. food chemistry, 141(2): 1041-1049, (2013). [8]. kohan o. o., dorohovich a. m., innovative technologies of long-term storage of confectionery products. resource and energy-saving technologies of production and packaging of food products the basic principles of its competitiveness: materials iii international specialized scientific-practical conference, 9th of september, kyiv, ukraine: 41-47, (2014). [9]. kohan o. o., dorohovich a. m. patent 70679 а, a23g 3/20, a23g 3/34 (2006.01). the method of manufacturing fondant chocolates. applicant national university of food technology. № food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 1 – 2018 oksana shulga, natalya pritulska, oksana petrusha, jelyzaveta smirnova, edible coatings – environmental replacing of traditional candy paper wrapper of “korivka” candies, food and environment safety, volume xvii, issue 1 – 2018, pag. 41 – 47 47 20031212096; stated 23.12.2003; posted 15.10.2004, bulletin № 10. [10]. the state standard of ukraine 4135:2014. candies general specifications, 24, (2015). [11]. gavva o.o. improving the technology of non-glazed chocolates in order to extend the shelf life. candidate's dissertation, (2006). [12]. ecology: chemical and pulp and paper industry. access mode http://childflora.org.ua/?page_id=28 [13]. al-hassan a. a., & norziah m. h., starch–gelatin edible films: water vapor permeability and mechanical properties as affected by plasticizers. food hydrocolloids, 26(1): 108-117, (2012). [14]. standard astm e96 / e96m – 16. standard test methods for water vapor transmission of materials. annual book of astm standards. designation e96-e80:7 0739, (1989). 1. introduction 4. conclusion 105 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2-2019, pag. 105 110 soil examination and recommendations for creating better conditions for raspberry planting *jovica momirceski 1 , julijana tomovska 1 , dijana blazhekovikj-dimovska 1 1university "st. kliment ohridski" – bitola, faculty of biotechnical sciences – bitola, macedonia zlatce_moe@yahoo.com *corresponding author received 7th may 2019, accepted 29th june 2019 abstract: when choosing land for raising a new planting of vegetables or fruit crops, the composition of the soil plays an important role; therefore, the composition of soil is examined, and chemical analysis of soil is made. the culture that we examine is a raspberry planting. raspberry is a multi-annual plant in the form of a bush or semi-shrub, with more annual roots and with one-year or two-year-old offshoots belonging to the rosaceae family. raspberries are irrigated by a drop-by-drop system, because through that system, remediation of soil can be carried out by adding appropriate crystalline fertilizers. nitrogen (n) is the element with the greatest movement in the soil itself and its content is from 0.05 0.14 %. the ph value affects the calcium content in soil. calcium plays an important role in the hardness of fruit and the sensitivity of physiological disturbance amounts to 12.1 %. phosphorus is important for the fruit properties during storage and amounts to 14.7 40 kg / da. potassium is a major element and plays a major role in producing good yields in fruit trees and amounts to about 14-50 kg / da. when mentioning magnesium, its role is not the most important and amounts to 74.5-221 ppm. the main role of magnesium lies within the ratio of potassium (k / mg), which should range from 2: 1 to 3: 1. with the obtained soil analysis data, in addition to the climatic conditions, type and structure, which are equally important for raising a raspberry plant, we have a full understanding of the soil processes and guidelines for suitable conditions for growing raspberries. key words: soil, raspberry, planting, chemical elements, soil acidity. 1. introduction raspberry is a multi-annual plant in the form of a bush or semi-bush with perennial roots and with one-year or two-year-old growths from the roots belonging to the rosaceae family. raspberries enter the order of the most important varieties of berries in the world, even in the balkans. the biological properties, the technological and nutritional value of the fruit make this culture quite significant and fairly cost-effective [1]. raspberry is well adapted to various climatic and production conditions; it can be grown in hillymountainous areas at an altitude of up to 800 meters. proper selection and preparation of soil will greatly influence the development of the bushes raspberries, the growth of swords (growths on the surface), the yield and the quality of the product itself. therefore, it is necessary to select the area for raising raspberry plantations, and to begin with timely and proper preparation of the soil [2]. when choosing a soil for raising a new planting of vegetable or fruit crops, the composition of the soil is important. depending on the needs of each plant, appropriate soil is selected, which will meet the requirements and needs of the plant. for this to be done http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska, the soils examination and recommendations for creating better conditions of raspberries planting, food and environment safety, volume xviii, issue 2 – 2019, pag. 105 – 110 106 correctly, it is best to carry out field analysis and therefore chemical analysis of the soil. soil analysis should be carried out a few months before the planting begins, by taking samples from several places in that area from a depth of 30-60 cm. in larger plots, sampling from only a few parts can lead to incorrect results, and thus to improper solutions when intervention on the same surface is involved [3]. the best is to take 4-5 specimens of a 1 hectare area to get the best and most relevant results. the application and accumulation of different elements are always different in the various phases of plant development. values that the soil should contain for raising a raspberry plant are: ph from 5,5 – 6,5; k2o (light soils) 20 – 25 mg / 100g; k2o (heavy soils) 25 – 30 mg / 100g; p2o5 > 12 – 15 mg; mgo > 10 mg (best ratio k/mg from 2:1 to 3:1). it is necessary to be careful about the content of mg, the lack of which can prevent photosynthesis and lead to colorful leaves. soil in nature appears as a product of pedogenic factors (creation factors of soil), which should be especially emphasized: soil, climate, relief, vegetation and human influence [4]. when water in the soil reacts with the elements, a solution (soil solution) is obtained, which represents the ratio of the hydrogen (h+) and hydroxyl (oh-) ions. soil response influences: the availability of nutrients and the efficiency of fertilization, nutrient mobility and soil pollution, stability of structural aggregates of the soil, soil, water mobility and its aeration, plant growth, pesticide and environmental performance [5]. when analyzing soil, soil samples are extracted with certain solvents. in addition, the content of the nutrients determined in the extract of soils shows the level (content) of the nutrients in the soil. today there are many soil solvents used to extract nutrients. for example, today the double lactate method is used, which uses a lactic acid content as a solvent for the extract, as well as the calcium acetate method, used to determine the content of the available k and p in the soils [6]. 2. materials and methods the culture being examined is a raspberry plant. for that purpose, 4 soil samples were taken to make initial examination of the soil composition of that land. samples were taken at a depth of 30 to 60 cm at different places in that plot in order to get a better image of that land. the plant, in the case of raspberries, is irrigated by a dropby-drop system. the samples were taken, packed in black plastic bags and with individually transported to an accredited laboratory. first, the laboratory gave directions about the proper way of taking samples, and how far the distance between the sampling points should be. thus, over a period of 2 hours, the samples were transferred to the appropriate laboratory and left for further examination. the samples were taken at a length of about 100m in a diagonal direction in order to cover as much as possible from the target surface and to obtain more relevant results. there were 4 soil samples from the entire area of their own choice of location, but certainly the determined distance between all samples was taken into account. it was also pointed out that the samples were not taken from a water surface or a covered mud. no earlier processing was performed on the soil in order to prevent layering. examination of the samples is done on a sample of 1 kg of soil in a period of 15 days from the end of november and the beginning of december. appropriate accredited methods were used for each examined element by the respective laboratory. for ph analysis, the ts iso 10390 method was used with a ph-meter with potentiometer, sature food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska, the soils examination and recommendations for creating better conditions of raspberries planting, food and environment safety, volume xviii, issue 2 – 2019, pag. 105 – 110 107 ortamda (h2o). to obtain the percentage of caco3, the method ts 8335 iso 10693 was used with an instrument called kalsimetrik. in order to obtain the electrical conductivity, the ts iso 11265 method was used with the same instrument as when determining the ph. to apply the saturation, the texture in percent was used with the aid of ts 8333, by the use of the saturasyon instrument. for the study percent of organic matter in the soil, the method ts 8336 the walkley black instrument was used. the ts 8337 iso 11261 method, i.e. the kjeldahl method, was applied to obtain the percentage representation of total nitrogen in the soil. the absorbed phosphorus (p) was tested by the use of the ts 834 method and the spectrophotometer type spektrofotometrik olsen. the method ts 8341 / n amo was used for the examination content of the potassium (k), calcium (ca) and magnesium (mg) at ase / icp. the presence of other elements in the soil such as iron (fe), manganese (mn), zinc (zn), and copper (cu) is examined with the aid of the ts iso 14870 dtpa / icp method. all methods used for examining soil samples for raspberry plantations are accredited methods by the institute for accreditation of the republic of north macedonia (iarsm). 3. results and discussion тhe chemical analysis of soil was made with regard to each sample and repeated three times, then the results obtained were statistically calculated and their average value is shown in table 1. table 1. basic parameters of soil for a raspberry plant basic analysis of soil for raspberries planting no. of samples рн limecalcium carbonate (%caco3) electroconductivity ec / (ds/m) t ( 0 c) salty nacl / % saturation – texture / % organic matter / % 1 7.7 1.0 0.287 / (21.70c) 0.01 71.51 2.05 2 6.28 1.3 0.237 / (20.70с) 0.07 51.04 2.8 3 6.35 1.6 0.215 / (21.160с) 0.06 53.09 1.64 4 6.11 2.1 0.225 / (21.10с) 0.009 55.44 1.24 acidification of the soil: according to the obtained soil acidity results, it is necessary to implement measures only to the surface from which the sample number 1 is taken, where the ph is 7.7 (weak alkaline), because when the ph is above 7.0 it can come to the lack of some elements such as fe and zn. in this case, acidification of the soil can be done with sawdust or with a comminute (which is obtained after digestion of brandy). other samples produce ph (weakly acidic) results provided by the planting, raspberry requirements. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska, the soils examination and recommendations for creating better conditions of raspberries planting, food and environment safety, volume xviii, issue 2 – 2019, pag. 105 – 110 108 chemical elements in soil: the results of the analysis show presence of several chemical elements in soil that are presented in table 2. table 2. analysis of chemical elements in soil for planting a raspberry plant chemical analysis of soil for raspberries planting chemical elements simples method of analyzes / instrument 1 2 3 4 total nitrogen (n) / % 0.10 0.14 0.08 0.06 ts 8337 iso 11261/ kjeldahl absorbed phosphorus (p) p2o5 kg/da 16.03 40.36 24.7 14.77 ts 834 olsen /spektrofotometrik absorbed kalium (k) k2o kg/da 17.1 49.7 23.6 14.0 ts 8341 / 1n amo. ase / 1cp – 0es absorbed calcium (ca) / ppm 3745.0 1235.0 2170.0 1113.0 ts 8341 / n amo. ase / icp absorbed magnesium (mg) / ppm 74.58 138.7 220.9 184.7 ts 8341 / n amo. ase / icp absorbed iron (fe) /ppm 1.163 81.92 157.8 76.20 ts iso 14870 dtpa / icp – 0es absorbed manganese (mn) /ppm 1.283 21.00 24.25 12.12 ts iso 14870 dtpa / icp absorbed zink (zn) /ppm 1.031 5.473 2.570 1.848 ts iso 14870 dtpa / icp absorbed copper (cu) /ppm 3.309 4.655 4.322 2.822 ts iso 14870 dtpa / icp fertility: raspberries work hard all summer to produce large leaves and berries, so they need increased fertility than other shrubs and perennial plants. spread commercial fertilizer -such as a 20-20-20 formula at a rate of 6 pounds per 100 feet of raspberry plants -in the spring as new growth emerges [7]. give fallbearing raspberries an additional 1 to 2 pounds of fertilizer as the canes start to bloom. if you prefer, spread manure or compost in the fall after you harvest the raspberries [8]. calcification: the ph value also shows the calcium content in the soil. in soil with a ph higher than 7.0, blocking certain microelements like fe and zn may occur [9]. calcium, important for the soil, is also important for the availability of other elements in the soil. calcium has an important role for the hardness of the fruit and for the sensitivity of physiological disorders [10]. the adequate presence of calcium in the soil, as can be seen from the obtained results (lime soils), facilitates absorption of other elements and increases the activity in the soil itself. in this case, it is recommended to minimize the addition of calcium on this surface in order to bring calcium into the ideal content required by the raspberry planting. further calcification can be performed every 3 years to replenish the evaporated or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska, the soils examination and recommendations for creating better conditions of raspberries planting, food and environment safety, volume xviii, issue 2 – 2019, pag. 105 – 110 109 extracted carbonate from the soil by natural processes [11]. electroconductivity of the soil: the conductivity results / (ds / m) of the ions from the present sample elements, which have an appropriate temperature of 20.70c 21.70c and no salt (nacl), are in the normal range, which is the result of the absence or minimum presence of salt in the soil. the percentage of nacl is from 0.009 to 0.07% and corresponds to the requirements for the raspberry planting. plants require nutrients to grow, and, if we fail to supply the proper nutrients in the proper concentrations, plant function is affected [12]. saturation texture of the soil: from the results it can be seen that only in the first sample a higher value of 71.51% was obtained, compared to other samples whose value ranges from 51.04% to 55.44%. from the values we conclude that the first sample is a surface with sand deposits or sandy soil, while as far as the other three samples are concerned, we are dealing with clayey rocky soil. 4. conclusion тhe results show that this soil is good for growing the desired crop plantations of raspberries. with small interventions and addition of certain fertilizers in soil the optimal soil quality is achieved. due to the fact that the elements which are most necessary for the quality production of raspberries are in good proportion and well contained in soil, it can be freely stated that this country is good for raising a new raspberry plantation. from the results one can also get recommendations that actually represent the conclusions of this research: the land that is chosen for raising longterm plantations of raspberries is mostly used, i.e. deep land, because the native raspberries system reaches up to about 50 cm, has good permeability, the humus content is 3-5%, the ph value is to move within the limits of 5.5 6.5 (weakly acidic) and medium heavy soil (about 50% clay). the irrigation method in this case is important, because through that system the land can be renovated by adding appropriate crystalline enzymes, in order to achieve a better quality of the composition of the soil and, if possible, to reach the requirements of the raspberries for the composition of the soil for raising an appropriate plantation [13]. nitrogen (n) is the element with the greatest mobility in the soil, which from an ecological point of view can be a significant problem [14].uy the content and the representation of nitrogen in the soil can most easily be determined by the percentage of plant growth and the development of the flower itself. uncontrolled application of fertilizers can lead to a series of unwanted consequences that are manifested through various forms of disadvantages, accumulation of varied foods, unevenness of the elements in the soil, but also in the plant itself. the most important elements that should be included in soil for raspberry cultivation are: phosphorus (p2o5) in amounts of 10mg and potassium (k2o) in the amount of 40mg per 100g soil [15]. in addition to climatic conditions, the type and structure of the soil, the results of the chemical soil structure tests will give a complete image of the best conditions for raising the plant sediment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 jovica momirceski, julijana tomovska, dijana blazhekovikj-dimovska, the soils examination and recommendations for creating better conditions of raspberries planting, food and environment safety, volume xviii, issue 2 – 2019, pag. 105 – 110 110 5. references [1]. aleksandar soskic, malina (raspberry); belgrade, 1997, page 164, [2]. vocar (fruit), published 28 february 2015 [3]. asad jeleskovic, dipl. ing. agr tehnologija uzgoja maline (technology of raspberry growing), may, 2014 [4]. dushko mukaetov, soil fertility, ukim, agricultural institute – skopje (2013) [5]. rubin gulabovski, agrochemistry and plant nutrition, ugd, faculty of agriculture stip, 2013. [6]. fidanka trajkova msc vasko zlatkovski, guide for soil samples from agricultural landguidelines for proper soil soil sampling for agrochemical analysis of soil; publisher: university "goce delcev" shtip, faculty of agriculture, march, 2017. [7]. r. r. maxwell, synthetic nitrogen products: a practical guide to the products and processes, kluwer academic new york, 2004. [8]. julie christensen, ideal growing conditions for raspberry plants, december 14, 2018 [9]. natasha kljajic, eficiency of invest in milk production, belgrade, 2014, p.717 [10]. e. plaster, soil science and management (5th edition), delmar cengage learning, 2008, new york. j. filipovski, pedology, 1984. [11]. http://www.caritas.ba/dok/1418119408.p df [12]. richards, l. a, diagnosis and improvement of saline and alkali soils. usda,agriculture handbook 60, washington d. c.(ed.) 1954. [13]. rulebook on analysis of inorganic fertilizers, government of the republic of macedonia, 2009. [14]. http://vocarstvo.rs/literatura [15]. https://www.smartfertilizer.com/articles/phosphorus http://www.caritas.ba/dok/1418119408.pdf http://www.caritas.ba/dok/1418119408.pdf http://vocarstvo.rs/literatura https://www.smart-fertilizer.com/articles/phosphorus https://www.smart-fertilizer.com/articles/phosphorus звіт з ндр 29-81 за 2007 – 2009 р 368 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 4 – 2014, pag. 368 374 the application of basaltic tuffs in the technology of cultivation the live f eed for fish – preliminary study oleksii khudyi1, *igor kobasa1, olga kushniryk1, lidiia khuda1 1 yuriy fedkovych chernivtsi national university, ukraine, imk-11@hotmail.com * corresponding author received november 24th 2015, accepted december 18th 2015 abstract: the research work is meant to investigate the possibility of using the zeolite-containing tuffs in the technology of live fish feed cultivation by example of cladocera. it is shown that the use of natural and treated at the temperature of 150 °c basaltic tuffs from „polytske-2” basalt deposit during moina macrocopa cultivation leads to an increase of the culture density by 2 and 2.3 times, respectively. in the experiment with simocephalus vetulus the increase of culture density has not been found, however basaltic tuffs allow to stabilize the culture growth and slow its extinction rate. besides, it is observed the effective purification of cultural medium of dissolved nitrogen forms probably by adsorption. the use of basaltic tuffs, calcined at a temperature of 1000 °c, leads to the death of m. macrocopa and s. vetulus cultures in 6 days. this circumstance makes the use of calcined tuffs impractical. keywords: zeolites, cladocera, cultivation medium, ammonium, nitrites, nitrates 1. introduction basaltic tuffs are natural aluminosilicates from zeolite group of minerals. zeolites possess several unique features, including adsorption, and therefore, they are used in the technological process of fluids purification, including wastewaters [1-3]. this is especially true for systems with closed cycle of water use. in this regard, tuffs are widely used in the recirculation of aquaculture systems as components of water treatment [4, 5]. live feed is widely used to obtain quality planting material for commercial fish farming and for decorative aquaculture objects [6]. among all the diversities of forage organisms, an important place is taken by cladocera crustaceans. one of the problems that occur with periodic method of zooplankton cultivation is exhaustion of culture medium caused by the accumulation of metabolic products in it [7, 8]. the use of substances with high adsorption properties, such as zeolites, may have effects on prolonging use of culture media without their update that reduces the costs of live feed. in addition, zeolites possess the ion-exchange properties which can positively affect the availability of micro and macro-elements for cultivating organisms. given the above, the purpose of the research was to find the applicability of zeolites from «polytske-2» basalt deposit during cladocera cultivation. 2. materials and methods the mineral samples received from „polytske-2” basalt deposit, situated in the rivne region of ukraine, were used to study the possibility of basaltic tuffs applied in the http://www.fia.usv.ro/fiajournal mailto:imk-11@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 369 technology of cultivation of live food for fish. the determination of basaltic tuffs composition was performed using classical methods of chemical analysis [9], the content of si, al, fe, mg, ca, p, s – by flame photometry method, na and k – by atomic absorption spectroscopy. the studies were conducted on laboratory cultures of cladocera crustaceans – simocephalus vetulus (muller) and moina macrocopa (straus). cultivation of zooplankton was carried out in 0.5 dm³ jars using standardized by chemical composition synthetic medium adam [10], at a temperature of 21±2 °c, cold light fluorescent glow 2,5–4 klux and 16 hour photoperiod. to determine the effective amounts of minerals during formation of experimental groups, the tuff was introduced into jars in amounts of 30, 40 and 50 g per 1 liter. according to the results of previous studies we found that the porosity and specific surface area of basaltic tuff granules depends on the temperature of heat treatment. these parameters reach the maximum values in the temperature range of 150–400 °c. there is a steady trend towards a decrease in the specific surface area and porosity after tuff calcination in the temperature range of 500–1000 °c. it may be caused by the removal of hygroscopic and zeolite water [11]. taking into account above stated in the present study it was used both natural and thermally activated at temperatures of 150 °c and 1000 °c tuffs, which differ in their sorption properties. group of organisms, in cultivation medium in which the tuff has not been introduced, served as a control. the feeding of cultures was performed every 48 hours, yeast saccharomyces cerevisiae at a dose of 0,05 g/l were used as feed. measurements of ph level were carried out using i-160мi ion meter, determination of total mineralization level in culture medium was performed indirectly through conductivity using conductivity meter sx-650 ulab. the content of ammonium, nitrites and nitrates was determined in the culture medium. measurings the concentration of nh4 + and no3 ions were performed by potentiometric method using i-160мi ion meter with ion-selective electrodes elis-121 nh4 + and elis-121-121no3 -, concentration of no2 was determined by photocolorimetric methods [12]. 3. results and discussion the mineralogical composition of basaltic tuffs, obtained from „polytske-2” basalt deposit which was used in the study, includes: zeolites (35–40%), montmorillonites (30–40%), feldspars (10–15%), silicas (4–5%), hematites (3–5%) [13]. the statistically average results of the chemical composition analysis of the tuff are expressed in mass percents of oxides. the summary of the results shows that the basaltic tuff is an aluminum silicate with a mass ratio si/al = 4,7÷5,9, containing iron in amounts of 68–74 g/kg. except elements, whose compounds in terms of oxides were presented in table 1, the investigated tuffs also contain trace elements (mn, zn, cu, co, ni) in amounts of 0,71– 0,08 g/kg. toxic elements (as, pb, hg, cr) were not found. table 1 the chemical composition of basaltic tuff from basalt deposit „polytske-2” in terms of oxides, the mass fraction expressed in % oxides mass fraction, % oxides mass fraction, % sio2 67.44 cao 0.46 тіо2 1.75 na2o 0.94 al2o3 12.82 k2o 1.06 fe2o3 10.14 p2o5 0.12 мnо 0.09 so3 0.11 mgo 5.02 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 370 the results of the studies showed that the use of basaltic tuffs from „polytske-2” basalt deposit enhances the growth intensity of both zooplankton cultures. a positive effect from tuffs using was manifested at 7–8 days of cultivation in experiment with m. macrocopa. the maximal development of moina culture was achieved both in experimental and control groups on the 14th day, but the density of cultures that were grown using tuffs was 2 times higher (figure 1 a). the culture of s. vetulus is characterized by much lower growth rates with achieving of maximum density on the 4th day, the introduction of natural basaltic tuff in the experimental concentrations (30, 40 and 50 g/l) does not affect the density of s. vetulus culture (figure 1 b). a b c d figure 1. the dynamics of m. macrocopa (a, c) and s. vetulus (b, d) density using different concentrations of natural unmodified (a, b) and thermally (150 °с) calcined (c, d) basaltic tuffs however, it was found that the use of basaltic tuff allows stabilizing the development of culture and slowing its extinction rate. it is known that the temperature of basaltic tuffs calcination affects their elemental composition and adsorption properties. respectively, the heat treatment can influence the biological objects that are grown in the presence of basaltic tuffs [14]. in accordance with the research results, it was established that thermally activated tuffs differ0 400 800 1200 1600 2000 2400 2800 3200 3600 0 2 4 6 8 10 12 14 16 18 in d . / l days 30g/l 40g/l 50g/l к 0 20 40 60 80 100 120 140 160 180 0 2 4 6 8 10 12 14 16 in d . / l days 30g/l 40g/l 50g/l к 0 400 800 1200 1600 2000 2400 2800 3200 3600 0 2 4 6 8 10 12 14 16 18 in d . / l days 30g/l 40g/l 50g/l к 0 20 40 60 80 100 120 140 160 180 0 2 4 6 8 10 12 14 16 in d . / l days 30g/l 40g/l 50g/l к food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 371 ently affect density and dynamics of both zooplankton species. the introduction of tuffs, thermally treated at 150 °c, into jars for m. macrocopa cultivation is accompanied by increase of culture density by 2.3 times as compared to the control in contrast to the experiment with natural tuffs, where the difference was 2 times (figure 1 c). the use of tuffs treated at high temperature (150 °c) was accompanied by no changes in s. vetulus culture density, while the stabilization of the culture and slowdown of its extinction have been seen like in the case where natural tuffs were used (figure 1d). the negative impact on the culture in both cases was not observed. due to this fact, the use of zeolite-containing tuffs to increase the mentioned species density is not appropriate. a negative effect of tuffs, calcined at the temperature of 1000 °c, on the growth of both studied cultures (m. macrocopa and s. vetulus) was observed in the experiment (figure 2). a b figure 2. the density of m. macrocopa (a) and s. vetulus (b) cultures using thermally calcined (1000 °с) basaltic tuffs during the first 6 days of the experiment the complete extinction of two zooplankton species has been seen, that is associated with significant changes in the structure and functions of basaltic tuffs. changes of ph level during m. macrocopa cultivating were observed in the cultivation medium due to the accumulation of metabolic products of living organisms (figure 3). figure 3. the ph dynamics during m. macrocopa cultivation with use of different concentrations of natural unmodified basaltic tuffs 0 10 20 30 40 50 60 0 2 4 6 in d . / l days 30g/l 40g/l 50g/l 0 10 20 30 40 50 60 0 2 4 6 in d . / l days 30g/l 40g/l 50g/l 7.1 7.6 8.1 8.6 0 2 4 6 8 10 12 14 16 18 days p h 30g/l 40g/l 50g/l к food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 372 instead, significant increase in ph level in the experiment with s. vetulus was not observed, being associated with minor zooplankton density. the influence of changes on ph, using different concentrations of natural form of zeolite-containing tuff, has not been observed, and does not differ greatly from the control experiment. as mentioned above, tuff calcined at a temperature of 1000 °c inhibited the development of both investigated cultures. however, despite the degradation of culture, the trend of ph level raise is observed, which is obviously associated with the accumulation of food residues, decomposition of dead organisms and ionexchange processes between solution and tuffs. the dynamics of total mineralization values during the period of zooplankton cultivation is caused by the accumulation of waste products in the medium, food residues and dead organisms. the marked influence of changing on total mineralization when using thermally activated (150 °c) and natural form of basaltic tuffs at investigated concentrations was not found. indicators of total mineralization in experimental groups did not differ from the control (figure 4). a b c d figure 4. the change of total mineralization values of medium using natural form and thermally calcined tuffs: m. macrocopa (a) and s. vetulus (b) with natural tuffs; m. macrocopa (c) and s. vetulus (d) with thermally calcined at 150 °c tuffs the impact of basaltic tuffs on the dynamics of metabolites accumulation in the cultivation medium, including ammonium ion and the products of its oxidation, nitrates and nitrites, attracts a particular attention. the increased density of the culture is accompanied by increased concentrations of soluble forms of nitrogen. the research results show that the use of the basaltic tuffs provides the slowdown accumulation of nh4 + ions and products of 750 790 830 870 910 950 990 1030 2 4 6 8 10 12 14 16 18 m g /l days 30g/l 40g/l 50g/l 750 790 830 870 910 950 990 1030 2 4 6 8 10 12 m g /l days 30g/l 40g/l 50g/l 750 790 830 870 910 950 990 1030 2 4 6 8 10 12 14 16 18 m g /l days 30g/l 40g/l 50g/l к 750 790 830 870 910 950 990 1030 2 4 6 8 10 12 m g /l days 30g/l 40g/l 50g/l к food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 373 its oxidation no2 and no3 -, apparently due to adsorption processes (figure 5). accordingly, the increase of concentration of the zeolite-containing tuff from 30 to 50 g/l improves the efficiency of cleaning the liquid phase of cultivation medium due to increasing of adsorption area. a b c d e f figure 5. the dynamics in content of nitrogen soluble forms in the medium for m. macrocopa (a, c, e) and s. vetulus (b, d, f) cultivation using different concentrations of basaltic tuffs 4. conclusion 0 0,05 0,1 0,15 0,2 0,25 0 2 4 6 8 10 n h 4 + , m m o l / l days 30g/l 40g/l 50g/l к 0 0,05 0,1 0,15 0,2 0,25 0 2 4 6 8 10 n h 4 + , m m o l / l days 30g/l 40g/l 50g/l к 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0 2 4 6 8 10 n o 2 , m m o l / l days 30g/l 40g/l 50g/l к 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0 2 4 6 8 10 n o 2 , m m o l / l days 30g/l 40g/l 50g/l к 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 0 2 4 6 8 10 n o 3 , m m o l / l days 30g/l 40g/l 50g/l к 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 0 2 4 6 8 10 n o 3 , m m o l / l days 30g/l 40g/l 50g/l к food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 4 – 2014 oleksii khudyi, igor kobasa, olga kushniryk, lidiia khuda, the application of basaltic tuffs in the technology of cultivation the live feed for fish – preliminary study, food and environment safety, volume xiv, issue 4 – 2015, pag. 368 – 374 374 we can remark that the use of natural and thermally activated at a temperature of 150 °c basaltic tuffs has a positive impact on the development dynamics of zooplankton cultures. maximal density of m. macrocopa cultures in the experiments with natural and activated tuffs was higher than in control group by 2 and 2.3 times respectively. the application of tuffs during s. vetulus cultivation did not cause an increase in the density of culture, but slowed the rate of extinction. the use of basaltic tuffs, calcined at a temperature of 1000 °c, leads to the death of m. macrocopa and s. vetulus cultures in 6 days. basalt tuffs provide the effective cleaning of cultivation medium from nitrogen soluble forms. the maximal effect is observed when using 50 g of tuffs for 1 liter of cultivation medium. 5. references [1]. kallo d., applications of natural zeolites in water and wastewater treatment, rewiews in mineralogy and geochemistry, 45 (1): 519-550, (2001). [2]. tarasevych yu.i., klimova g.m., obtaining of modified sorbents and their application for water purification from heavy metals, journal of water chemistry and technology, 28 (2): 107-116, (2006). (in russian). [3]. petrus r., malyovanyy m., varchol j., odnorig z., petrushka i., leskiv g., technologies of wastewater treatment using natural dispersed sorbents, chemical industry of ukraine, 2: 20-22, (2003). (in ukrainian). [4]. vatin n.i., chechevichkin a.v., chechevichkin v.n., sorption treatment of industrial highly concentrated waters from the ammonium ion using natural zeolites. st. petersburg: 36, (2007) (in russian). [5]. sirakov i., velichkova k., stoyanova s., dinev d., staykov y., application of natural zeolites and macrophytes for water treatment in recirculation aquaculture systems, bulgarian journal of agricultural science, 21 (1): 147-153, (2015). [6]. das p., mandal s.c., bhagabati s.k., akhtarm.s., singhs.k. important live food organisms and their role in aquaculture. in: frontiers in aquaculture. (ed.) m. sukham, narendra publishing house: 69-86, (2012). [7]. hedstrom a., ion exchange of ammonium in zeolites: a literature review, j. environ. eng., 127: 673-681, (2001). [8]. pabalan r.t., bertetti f.p., cationexchange properties of natural zeolites, rewiews in mineralogy and geochemistry, 45 (1): 453518, (2001). [9]. tsimbalyuk v.v., voloshchuk a.g., kobasa i.m., effect of heat treatment on sorption properties of basaltic tuff, ukrainian chemistry journal, 75 (12): 85-90, (2009) (in ukrainian). [10]. kluttgen b., dulmer u., engels m., ratre h.t., adam, an artificial freshwater for the culture of zooplankton, water res., 28 (3): 743-746, (1994). [11]. kobasa i., mikula j., woloschuk a, tsymbaliuk w., influence of thermal treatment on the physical, mechanical and sorption characteritics of the basalt tufa, technical transactions. mechanics, 5-m (15): 3-9, (2011). [12]. linnik p. determination of the nitrite nitrogen concentration in water. in: methods of hydroecological researches of surface water. logos. kyiv: 242-244, (2004). (in ukrainian). [13]. goliar n., tuffs: the use in economic sectors. rivne: 30, (in ukrainian). [14]. melnychuk v. zeolite-smectite tuff of rivne region: the biological aspects of application. volynski oberegy. rivne: 184, (2005). (in ukrainian). 266 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 266 271 optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology * anca-mihaela gâtlan 1 , gheorghe gutt 1 1faculty of food engineering, “ștefan cel mare” university, suceava, romania, anca.gatlan@fia.usv.ro *corresponding author received 28th november 2019, accepted 28th december 2019 abstract: sea buckthorn (hippophae rhamnoides l.) is a unique and valuable shrub cultivated all over the world. food industry is the most important sector of use of the sea buckthorn, where it is used as a raw material to obtain functional food or food supplements. sea buckthorn fruits are among the most complex nutritionally of all berries. the aim of the present study was to offer an optimum solution for the valorization by alcoholic fermentation of the press residues obtained after processing the sea buckthorn fruit to obtain juice. the present research was conducted with the application of response surface methodology (rsm) for optimization, using a box-behnken design. the determined optimum conditions for a 3 days-fermentation were: a temperature of 25.38 °c, a ph of 4.62 and an inoculum size of 0.18 % to obtain a 3% vol. alc. beverage. this study provides useful information that can help beverage processing industries achieve optimum yield in low alcoholic beverage production using sea buckthorn press residues as sole substrate. keywords: hippophae rhamnoides, capitalization, fermentation, optimization, box-behnken design 1. introduction fermentation is a cheap technology and plays an important role as the main biotechnological application of food processing in many developing countries. benefits associated with fermentation include improving the sensory properties and nutritional value of foods, reducing toxic and anti-nutritional factors, and extending the shelf life of foods [1]. the process of alcoholic fermentation, through its form of manifestation, has been known and used in the technique of preparing beverages, since ancient times [2]. alcoholic fermentation is a process extensively used in beverage industry, generating value-added, less perishable products, in which ethanol is a major constituent and other minor secondary metabolites are also formed [3]. yeast plays pivotal role in winemaking and its activity is mainly influenced by factors such as temperature and ph, which play crucial role in controlling the growth and metabolism rate of yeast and in synthesizing or transforming bioactive compounds in wine [4]. sea buckthorn (hippophae rhamnoides l.) is a unique and valuable species, being cultivated all over the world [5]. in addition to the different areas of importance of all the component parts of the plant, but also of the shrub, as a whole [6], the most important sector of use of the sea buckthorn remains the food industry, where it is used as a raw material to obtain functional food or food supplements [7]. research carried out in the country and abroad has shown that the leaves, fruits and shoots of sea buckthorn contain a number of biologically active substances http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anca-mihaela gâtlan, gheorghe gutt, optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology, food and environment safety, volume xviii, issue 4 – 2019, pag. 266 – 271 267 with an essential role in regulating metabolism [8]. sea buckthorn fruits are among the most complex nutritionally of all berries [9]. in romania, sea buckthorn has an exceptional chemical content, perhaps the richest in the world. nowhere in the world, where it grows naturally and even the improved varieties do not have a composition at the level of the sea buckthorn grown on the territory of romania, especially in the sub-carpathian region [10]. the aim of the present study was to offer an optimum solution for the valorization by alcoholic fermentation of the press residues obtained after processing the sea buckthorn fruit to obtain juice. by identifying the optimal fermentation conditions, it was obtained a novel low alcohol refreshing, energizing and healthy sea buckthorn beverage. the obtained product will have superior nutritional and sensory properties in comparison to other similar fruit beverages on the market due to the special raw material used in its production. several statistical approaches have been used to optimize the process factors maximizing yield of the product. response surface methodology (rsm) is one such approach that has been extensively studied and applied in bioprocess technology. it minimizes the number of observations and thus provides more precise and accurate results [11]. the present research was conducted with the application of response surface methodology (rsm) for optimization, using a box-behnken design. 2. matherials and methods 2.1. yeast inoculum preparation the yeast used for the experimental trials was a strain of saccharomyces cerevisiae killer (former bayanus), (ioc 18 2007), purchased from enzymes & derivates sa company, piatra neamț, romania. the yeast was rehydrated in water at 37°c (1: 10, w: v), in a clean container. the content was slowly stirred and then left to rest for 20 minutes. then, must was added progressively so that the yeast starter can acclimatize to the specific must conditions (ph, sugars, temperature, etc.). in order to obtain an active starter culture sufficiently concentrated to achieve desired fermentation, hydra pc was incorporated (1: 1, w: w) in the yeast stock solution. hydra pc is an adjuvant naturally rich in magnesium, also bought from enzymes & derivates sa. it optimizes yeast rehydration by reinforcing its plasma membrane, which is essential to yeast growth and metabolism. 2.2. fermentation of hippophae rhamnoides press residues fruits of sea buckthorn (h. rhamnoides l.) were collected in botoșani county, romania. fruits were cleaned and the juice was extracted by cold pressing. the press residues were collected and submitted to a maceration phase in water in 1:2 proportion (w:v), at 25˚c for 5 hours. maceration step will contribute to all the specific characteristics of smell, appearance and taste of the final product. since the fermentation medium obtained thereby lacked in the requisite sugar content (1.3˚brix), sugar level was adjusted to 7˚brix. also, 100 ppm of so2 was added in the form of potassium bisulfite (sigma aldrich) to inhibit the growth of unfavorable microorganisms. further, a mix of ammonium salts, inactivated yeast, thiamin and diammonium phosphate was added to ‘help’ the fermentation process. this ameliorated sea buckthorn marc juice was further inoculated with target concentration of yeast and the ph was set using sodium carbonate of absolute purity, from sigma aldrich. the fermentation processes were conducted for three days, according to the respective runs as suggested in a boxbehnken design (bbd) matrix (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anca-mihaela gâtlan, gheorghe gutt, optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology, food and environment safety, volume xviii, issue 4 – 2019, pag. 266 – 271 268 2.3. experimental design and optimization the fermentation process for the sea buckthorn marc was established by response surface methodology (rsm), using design-expert 12 software. rsm is an empirical statistical modeling technique employed for multiple regression analysis using quantitative data obtained from properly designed experiments to solve multivariable equations simultaneously [12]. this system was employed to determine the best combination of variables for desired ethanol concentration (3%). box–behnken design with three factors at three levels (-1, 0 and +1) was undertaken with six center points in a set of 18 experiments. for statistical calculations, the independent variables were coded as x1 (coded temperature, range 18 ÷ 30 °c), x2 (coded ph, range 2.85 ÷ 5) and x3 (coded inoculum size, range 0.02 ÷ 2 %), according to table 2. this design is an experimental design of response surface methodology that studies the effect of the chosen variables and their interactions. table 1. experimental design (box-behnken design matrix) run order temperature (ºc) ph inoculum size (% w/v) ethanol (% v/v) 1 24 2.85 0.2 2.08 2 24 3.925 0.11 2.21 3 24 3.925 0.11 2.21 4 24 3.925 0.11 2.11 5 24 3.925 0.11 2.15 6 18 3.925 0.02 1.32 7 30 3.925 0.02 2.96 8 24 5 0.2 3.29 9 24 3.925 0.11 2.32 10 18 5 0.11 1.84 11 30 5 0.11 3.19 12 30 3.925 0.2 3.5 13 18 2.85 0.11 0.91 14 24 2.85 0.02 2.05 15 24 3.925 0.11 2.33 16 18 3.925 0.2 1.52 17 24 5 0.02 2.3 18 30 2.85 0.11 3.53 table 2. factors involved in box-behnken design in terms of actual values factor name low value high value x1 temperature 18 30 x2 ph 2.85 5 x3 inoculum size 0.02 0.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anca-mihaela gâtlan, gheorghe gutt, optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology, food and environment safety, volume xviii, issue 4 – 2019, pag. 266 – 271 269 2.4. fermentation monitoring and ethanol determination bluesens sensors were used to monitor the fermentation trials. the collected data were logged with bacvis software. to this extent, spectral sensors in infrared domain were mounted on the fermentation recipients. this modern gas analysis allowed the control of fermentation processes in real-time and allowed the overseeing of the ethanol content during the fermentation processes of the sea buckthorn press residues. 3. results and discussion the data of ethanol yield according to box-behnken design shown in table 1 indicated that there was a variation in each ethanol production in response to the eighteen trials employed (ethanol concentration: 0.91 – 3.53%). these variations reflected the importance of medium optimization in obtaining the desired ethanol concentration. also, the experiment successfully indicated that all the three independent variables, i.e., temperature, ph and inoculum size had a profound effect on ethanol yield, 3d response surface plots at intermediate level of temperature, ph and inoculum size and interaction between different variables as depicted in fig. 1a-c. fig. 1. 3d surface plots of temperature vs. ph (a), temperature vs. inoculum size (b) and ph vs. inoculum size (c) on ethanol content food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anca-mihaela gâtlan, gheorghe gutt, optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology, food and environment safety, volume xviii, issue 4 – 2019, pag. 266 – 271 270 on the entire domain of ph, high temperatures lead to high concentrations of ethanol (fig. 1a), while the gradual increase of both temperature and inoculum size (fig. 1b) and ph and inoculum size (fig. 1c) resulted in higher values of ethanol (fig. 1b). these trends of variations of ethanol yield considering those three independent variables were also observed in earlier reports of kumar et al. (2009) for mango fermentation [13] and of sevda & rodrigues (2011), too, for guava fermentation [14]. several previous studies suggested that these three primary factors have an important influence on the fermentation kinetics [15, 16]. thus, the present study indicated that hippophae rhamnoides press residues could be successfully capitalized in the production of a refreshing drink with target ethanol content of 3% vol. in 3 days, with the following optimized conditions: temperature of 25.38 °c, ph of 4.62 and inoculum size of 0.18 %. analysis of the variance (anova) showed that models are significant with respect to ethanol concentration (table 3). table 3. analysis of variance (anova) for the selected quadratic model of box-behnken design source sum of squares df mean square f-value p-value model 8.9 9 0.1 49.7 < 0.0001 significant x1 -temperature 7.2 1 7.2 362.3 < 0.0001 x2 ph 0.5 1 0.5 26.4 0.0009 x3 inoculum size 0.4 1 0.4 19.5 0.0022 x1x2 0.4 1 0.4 20.3 0.0020 x1x3 0.03 1 0.03 1.4 0.2623 x2x3 0.2 1 0.2 11.6 0.0093 x1 2 0.002 1 0.002 0.09 0.7700 x2 2 0.07 1 0.07 3.4 0.1002 x3 2 0.03 1 0.03 1.5 0.2541 residual 0.2 8 0.02 lack of fit 0.1 3 0.04 5.0 0.0561 not significant pure error 0.04 5 0.008 cor total 9.0 17 the quadratic model thus generated could successfully explain that all three variables have a significant effect on the ethanol content, especially temperature, with p < 0.0001. in addition, the interactions between temperature and ph (p < 0.01) and ph and inoculum size (p < 0.01) were also having a significant effect on the ethanol content. the model f-value of 49.70 implies the model is significant. coefficient of determination r-square value was 0.9824 indicated model’s goodness of fit. the predicted r-square value of 0.7821 was in reasonable agreement with the adjusted r-squared value of 0.9627. thus, the final equation of process variables in terms of coded values on developed model for the production of hippopae rhamnoides low alcoholic refreshing drink is as follows (equation 1): ethanol concentration = 2.22 + 0.9487 x1 + 0.2562 x2 + 0.22 x3 0.3175 x1x2 + food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 anca-mihaela gâtlan, gheorghe gutt, optimization for alcohol fermentation conditions of sea buckthorn press residues using response surface methodology, food and environment safety, volume xviii, issue 4 – 2019, pag. 266 – 271 271 0.0850 x1 x3 + 0.24 x2x3 + 0.0204 x1 2 + 0.1254 x2 2 + 0.0829 x3 2 (1) to validate the predicted response, another fermentation process as a confirmatory run with the optimized conditions was conducted, in triplicate. the result found was in accordance with the predicted response and the ethanol content observed was 3 ± 0.2%. 4. conclusion in conclusion, the fermentation conditions were successfully optimized using response surface methodology and boxbehnken design by analyzing the individual and interactive effects of fermentation temperature, inoculum size and ph on fermentation kinetics. the determined optimum conditions for a 3 days fermentation were: a temperature of 25.38 °c, a ph of 4.62 and an inoculum size of 0.18 % for obtaining a 3% vol. alc. beverage. hence, sea buckthorn press residues can be capitalized by means of alcoholic fermentation in order to obtain a novel refreshing drink. therefore, it can be stated that this study provides useful information that can help beverage processing industries achieve optimum yield in low alcoholic beverage production using sea buckthorn press residues as sole substrate. 6. references [1]. misihairabgwi j., cheikhyoussef a., traditional fermented foods and beverages of namibia, journal of ethnic foods, 4(3): 145-153, (2017). [2]. cotea v. d., pomohaci n., gheorghiţă m. șt., oenologie, editura tipo moldova, iași, 315 p., (2010). [3]. room r., babor t., rehm j., alcohol and public health, lancet, 365(9458): 519-530, (2005). [4]. arroyo-lópez f. n., orlić s., querol a., barrio e., effects of temperature, ph and sugar concentration on the growth parameters of saccharomyces cerevisiae, s. kudriavzevii and their interspecific hybrid, international journal of food microbiology, 131(2-3): 120-127, (2009). [5]. li t., mcloughlin c., sea buckthorn production guide, pacific agri-food research centre, agriculture & agri-food canada summerland, 21 p., (1997). [6]. zeb a., chemical and nutritional constituents of sea buckthorn juice, pakistan journal of nutrition, 3(2): 99-106, (2004). [7]. beveridge t., li t.s., oomah b.d., smith a., sea buckthorn products: manufacture and composition, journal of agricultural and food chemistry, 47(9): 3480-3488, (1999). [8]. rați i.v., rați l., cătina albă în exploatații agricole, ministerul agriculturii, pădurilor, apelor și mediului, agenția națională de consultanță agricolă, 127 p., (2003). [9]. small e., catling p. m., li t., blossoming treasures of biodiversity: sea buckthorn (hippophae rhamnoides) an ancient crop with modern virtues, biodiversity, 3(2): 25-27, (2002). [10]. manea ș., cătina și uleiul de cătină. aliment, miracol, sanătate, echilibru, hofigal, 113 p., (2004). [11]. mundaragi a., thangadurai d., process optimization, physicochemical characterization and antioxidant potential of novel wine from an underutilized fruit carissa spinarum l. (apocynaceae), ciência e tecnologia de alimentos, 38(3): 1-6, (2017). [12]. sevda s., rodrigues l., extraction and optimization of guava juice by using response surface methodology, american journal of food technology, 7: 326-339, (2012). [13]. kumar y. s., prakasam r. s., reddy o. v. s., optimisation of fermentation conditions for mango (mangifera indica l.) wine production by employing response surface methodology, international journal of food science & technology, 44(11): 2320-2327, (2009). [14]. sevda s., rodrigues l., fermentative behavior of saccharomyces strains during guava (psidium guajava l.) must fermentation and optimization of guava wine production, journal of food processing & technology, 2: 2-9, (2011). [15]. reddy l. v. a., reddy o. v. s., effect of fermentation conditions on yeast growth and volatile composition of wine produced from mango (mangifera indica l.) fruit juice, food and bioproducts processing, 89(4): 487-491, (2011). [16]. bleoanca i., bahrim g., overview on brewing yeast stress factors, romanian biotechnological letters, 18(5): 8559-8572, (2013). 1. introduction 4. conclusion microsoft word 4 dorohovych_mazur_corrected.doc 141 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 141 153 complex quality indicator of sugars and sugar substitutes and their use in the production of confectionery products antonella dorohovych1, victoria dorohovych1 *liubov mazur1 1 department of bakery and confectionery goods technology, national university of food technology volodymyrska str. 68, kyiv, ukraine, 01601, confkiev2017@gmail.com *corresponding author received 4th december 2017, accepted 25th june 2018 abstract: the paper presents the results of calculation on the basis of the basic principles of theoretical qualimetry of the complex index of quality of sugars and sugar substitutes. when evaluating sugars according to the complex indicator as the differential indicators, there were selected sweetness, glycemic index, solubility and melting point, caloric content, which have the greatest influence on the organoleptic, technological and physiological parameters of sugar quality. when calculating the complex index of sugar substitutes, it was suggested to introduce three indicators: the heat dissolution, water activity and the degree of tolerance. according to the selected indicators, which values have been transformed into a 10-point scale, profilograms have been constructed. formulas of calculating the complex quality index were developed taking into account the validating factors of each indicator determined by the delphi method. the calculation of the complex index of sugars’ quality showed a significant advantage of tagatose and the expediency of its use in the manufacture of confectionery products. the calculation of the complex index of quality of sugar substitutes showed that the highest score was registered by erythritol, fact that determines the expediency of its use in the manufacture of confectionery products with dietary function. the calculation showed the following values of the complex index of sugars’ quality – sucrose, glucose, fructose, maltose, lactose, tagatose, trehalose: 0.53, 0.46, 0.72, 0.44, 0.26, 0.81, 0.44, 0.47 respectively, and sugar substitutes – sorbitol, xylitol, isomaltotol, maltitol, erythritol, lactitol: 0.34, 0.42, 0.37, 0.47, 0.86, 0. 37 respectively. the article describes the sorption-desorption properties of various groups of confectionery products using sugars and sugar substitutes. keywords: theoretical qualimetry, complex index, delphi method, differential indicators 1. introduction today food products for special purposes are in great demand in the world. according to the codex alimentarius food products for special purpose are divided into functional items that have health properties and dietary products intended for nutrition for people with different diseases who are unable to consume original products [1]. carbohydrates are of great importance for human life – they are a source of energy and perform very important physiological functions: regulatory, toning, protective, adsorption. according to iupac nomenclature carbohydrates are divided into simple (monosaccharides) and complex (oligo and polysaccharides) [2]. mono and disaccharides form a group of carbohydrates called sugars. it should be noted that the experts of the food and agriculture organization of the un (fao) and the world health organization (who) use the term "sugars" referring to mono and disaccharides [1]. the suffix "ose" is present in the title of sugars like: sucrose, glucose, fructose, lactulose, etc. according food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 142 to the international standard codex stan 212-1999 the group of sugars include: white sugar, powdered sugar, yellow sugar, brown sugar, glucose anhydrous, glucose crystalline hydrate, the powder of glucose, glucose syrup, dry glucose syrup, lactose, fructose, raw sugar from sugar cane [3]. all sugars have a sweet taste and in food production have structure functions which mean that they participate in the formation of disperse structures with coagulation, crystal and coagulating-crystalline properties. in the manufacture of confectionery sugars participate in the formation of amorphous, crystalline, gellike, pasty, cream structure [4]. in the manufacture of confectionery for dietetic purpose sugar substitutes are used instead of sugar. substances with a sweet taste that by organoleptic, physicochemical and structural-mechanical indicators can replace sugar in food products belong to the group of sweeteners. according to the rules of nomenclature of iupac (international union of pure and applied chemistry), formulated in 1996, polyols, acrospires, hydrogenated carbohydrates which are synonyms of one class of derivatives of carbohydrates belong to sugar substitutes. all sweeteners have the suffix "-tol": sorbitol, xylitol, lactitol and other [2]. the "sugar substitutes", "acrospires", "hydrogenated carbohydrates", "polyols" are the synonyms of one subclass of derived of carbohydrates. the alcohol group is presented in the compounds (сн2он), which replaces the carbonyl group (-c=o) in aldoses and ketoses of different sugars. as polyols do not have a carbonyl group, they can’t form a cyclic structure which is characteristic for monosaccharides and disaccharides, so they are usually depicted in fischer projections. sugar substitutes do not participate in the reaction of melanoyodin formation, because they have no reductive properties [5]. all sugar substitutes-polyols are obtained by catalytic hydrogenation of the corresponding carbohydrates. sorbitol is produced from glucose, xylitol – from dxylose, isomalt from sucrose, lactitol from lactose, maltitol from maltose. erythritol is the only sugar substitutes, which is obtained not by catalytic hydrogenation of carbohydrates but by microbial synthesis. as microorganisms, osmophilic yeast and fungi pollinis, trigonopsis, or torulopsis are used [4]. this work is dedicated to the assessment of the quality of sugars and sugar substitutes by the integrated indicator of quality. while determination by a integrated indicator, the sweetness, glycemic index, solubility, melting point, calorific value were selected as the quality differential indicators, which have the greatest influence on organoleptic, technological and physiological indicators of the quality of the product. the sweetness has an important functional role in the manufacturing of confectionery products. sweetness is estimated relatively to sucrose, which sweetness is taken as 1 unit of sweets [6]. the glycemic index in recent years has become the main indicator of the quality of food, especially for dietetic purpose. the concept of glycemic index was first introduced in 1981 by dr. david jenkins, a professor at the university of toronto in canada in order to determine what meals are the most beneficial for people with diabetes. the impact of food on glucose levels in the blood is determined by the glycemic index (gi). the lower the ability of foods to raise glucose level in blood, the more favorable their effect on insuline and the lower risk of developing diabetes, cardiovascular disease, obesity [7]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 143 solubility has a great importance in assessing the quality of sugars by an integrated measure. most sugars are well soluble in water, with increasing temperature the solubility increases. solubility is accompanied by the destruction of the crystal structure, hydrogen bonds with water molecules in aqueous solutions as well as the internal and intermolecular hydrogen bonds, which strength will depend on the concentration of carbohydrate solutions and temperature. the ability of carbohydrates to dissolve determines their effect on structure formation processes of various food products of amorphous, crystalline, prescriptive, spumy structure [8, 9]. the melting point is the temperature at which a crystalline solid turns into liquid state, which affects energy costs, that is, with the increase of the melting temperature the amount of energy increases that is required to transfer the food product into the liquid state [8]. calorific value is an important indicator of the assessment. it is the amount of energy (kcal, kj) that is released in the human organism from food products to ensure physiological functions [10]. when calculating the integrated indicator of the quality of the sugar substitutes, it was suggested to introduce three indicators: the heat of dissolution, water activity and degree of tolerance in addition to differential metrics which were used in assessing the quality of sugars (solubility, melting point, calorie, sweetness, glycemic index). the heat of solution is of great importance as teplophisic characteristics, affects the organoleptic characteristics, so the consumption of sorbitol, xylitol and especially erythritol there is a strong cooling effect [4]. water activity is of great importance when storing products and it describes the hygroscopic properties and microbiological stability of the product [8]. the degree of tolerance is important in assessing the quality of polyols. polyols can cause undesirable gastro-intestinal disorders (laxative effect) when used by sensitive persons, so excessive consumption can cause gastrointestinal complications. there are limitations to an amount of consumption of polyols. so it is shown in the codex alimentarius that if in the consumption of the food product it is assimilated more than 20 grams of polyols, the product must contain a marking that it has laxative effect [11] 2. matherials and methods the solubility of sugars was determined by the mass of the substance that can dissolve in 100 g of solvent at a certain temperature to make the solution saturated [8]. the melting point was determined using a capillary method using a device which consists of a heat-resistant round-bottom flask of 100-150 ml with a long neck; heatresistant glass test tube which is inserted in a flask so that it does not reach its bottom by 1 cm; capillary tubing made of nondurable glass with 0.9 – 1.1 mm in diameter; mercury thermometer with a price division of 0.5 ° с; heating sources gas burner, electric heater. the flask is charged at ¾ with a suitable liquid, for example, with distilled water (for substances with tmel to 80 ° c), vaseline or silicone oil, concentrated sulfuric acid (for substances with tmel from 80 to 260 ° c), a solution of three parts of potassium sulfate in seven parts of concentrated sulfuric acid (for substances with tmel> 260 ° c). the difference between definitions should not exceed 1°с. the beginning of melting is the appearance of the first droplet of liquid and the end the complete transition of matter into a liquid state. this temperature food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 144 range (range tmel) must not exceed 2°c [9]. energy value was determined taking into account the amount of energy released from 1g of the product, taking into account the content of proteins, fats and carbohydrates [10]. sweetness was determined by the organoleptic method using sensory analysis. in the evaluation of organoleptic parameters from 12 to 15 experts participated [12]. determination of the glycemic index involves testing by people with normal weight index and tolerance to glucose and food consumption on an empty stomach (two hour’s break after the last meal) with the absence of physical activity. it is desirable to conduct testing at 10 o'clock in the morning after a 10-14 hour rest break. it is also recommended when taking a certain amount of food or white bread, if it is chosen as the standard, drink them with a standard amount of water, which is 250 ml. when using glucose as a standard, firstly it must be dissolved in 250 ml of water. duration of consumption of liquid food products should be 5-10 minutes, and solid and semi-hard 10 20 minutes. the first measurement of blood glucose should be done exactly 15 minutes after consuming the first portion of food or the first drink of the beverage. sweetness of glucose and high osmotic pressure of its aqueous solutions can lead to a decrease in stomach activity, and therefore, as a standard, instead of glucose, sometimes white (wheat) bread is used. another advantage of white bread is that its consumption results in an increased insulin response. the value of glycemic indexes for white bread (gib) is higher than the value of gig (gib = 100/73 gig). it is also appropriate to obtain at least two values of gi for each person [13]. the heat of solution is determined using calorimetry [14]. water activity and sorption-desorption properties are investigated on a mac-ben device, according to sorption-desorption curves. the method is used to obtain information about the kinetics of the determination of sorption equilibrium and sorption capacities at different media humidity (steam vapor activity). measurements are carried out on the vacuum balances of mac-ben with a quartz spiral, sensitivity of it is at least 1 milligram / mm. deformation of the spiral is recorded using a catheter km.-9 with an accuracy of ~ 0.01 mm. the method of measurement is as follows: a sample of an adhesive in the form of an individual film or film on a substrate weighing at least 100 mg is reinforced on a quartz spiral, which is placed in a sorption column. after thermostating at a given temperature for 30-40 minutes, the system is vacuumed to a residual pressure of 10-5 mm.m.c. with the continuous registration of the desorption process of volatile components from the sample to the determination of the constant weight of the sorbent [8]. integrated indicator of quality was calculated according to the basic principles of qualimetry [15]. 3. results and discussion in this work, a comparison of the quality of sugars: sucrose, glucose, maltose, lactose, tagatose, trehalose, lactulose is conducted by an integrated measure (table 1), which takes into account physic-chemical properties. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 145 table 1 basic physical and chemical parameters of sugars caloric content melting point solubility at 293 k (20 ° c),% glycemic index (gi),% kj / g kcal/g к °с sweetness, unit name of sugar р1 р2 р3 р4 р5 saccharose 69.00 68.00 16.75 4.00 453.00 180.00 1.00 glucose 47.00 100.00 16.75 4.00 419.00 146.00 0.80 fructose 78.00 20.00 16.75 4.00 377.00 104.00 1.50 maltose 47.00 105.00 16.75 4.00 381.00 108.00 0.40 lactose 16.00 45.00 16.75 4.00 525.00 252.00 0.35 tagatose 58.00 3.00 6.28 1.50 407.00 134.00 0.92 trehalose 68.90 72.00 14.49 3.46 483.50 210.50 0.45 lactulose 75.20 46.00 16.75 4.00 442.00 169.00 0.50 analysis of the data indicates that all indicators have different units of measure. to calculate the integrated indicator of quality according to the basic principles of qualimetry differential indicators it should be transformed to the same scale of measurement. it was decided to transform the indicators into ten-point scale. profile grapgics were built due to the obtained data (fig. 1), which area of characterizes the overall quality of sugars. fig. 1. the profile graphics of indicators of quality of sugars analysis of profile graphics shows that the largest area has tagatose, and the lowest – lactose. each individual indicator has different influence on the overall assessment of the quality of the product. the validityt of influence of indicators on overall quality is not taken into account in profile graphics. according to the basic principles of quality control the influence of individual indicators on the quality was determined taking into account the validity coefficients. there are many methods of determining the validity coefficients. we decided to determine the validity factors by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 146 the method of delphi. the condition for determining the validitycoefficients is: 1 m i i m const   (1) in our case, the validity factors were determined according to the following formula: м1 + м2 + м3 + м4 + м5 = 1 (2) table 3 shows the definition of the validity coefficients by the method of delphi (round ii) table 2 definition of validity factors the value of the validity factor expert number м1 м2 м3 м4 м5 sum of the validity coefficients the first expert 0.20 0.15 0.25 0.20 0.20 1.00 the second expert 0.20 0.15 0.25 0.15 0.25 1.00 the third expert 0.15 0.20 0.20 0.15 0.30 1.00 the fourth expert 0.25 0.15 0.20 0.15 0.25 1.00 the fifth expert 0.20 0.10 0.20 0.20 0.25 1.00 the sixth expert 0.10 0.20 0.25 0.20 0.25 1.00 the seventh expert 0.30 0.10 0.25 0.10 0.25 1.00 the eighth expert 0.25 0.15 0.25 0.10 0.25 1.00 the ninth expert 0.20 0.15 0.25 0.15 0.25 1.00 a tenth expert 0.20 0.15 0.24 0.16 0.25 1.00 the average value 0.20 0.15 0.24 0.16 0.25 1.00 the average point was taken into account in the calculation of the integrated indicator, which was carried out according to the following formula: 1 31 2 0 1 2 3 1 2 3 54 4 5 4 5 б б б б б рр р к м м м р р р рр м м р р       (2) where м1, м2, м3, м4, м5 – the value of the validity factors of the indicator р1б, р2б, р3б, р4б , р5б – the values of the corresponding indicators in the baseline sample, which equals 10 points р1, р2, р3, р4 , р5 the corresponding value from the profile graphics. using the data in profile graphics and table 2 integrated indicators of the quality of sugars were calculated according to the formula (2) whiche values are given in the table 3. table 3 the value of the integrated indicator of quality of sugars name of sugar the value of the integrated indicator of quality saccharose 0.53 glucose 0.46 fructose 0.72 maltose 0.44 lactose 0.26 tagatose 0.81 trehalose 0.44 lactulose 0.47 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 147 quality assessment by an integrated indicator showed significant benefits of tagatose relatively to other sugars. a comparison of the quality of sugar substitutes: sorbitol, xylitol, maltitol, isomaltitol, lactitol, erythritol by an integrated indicator is made. main physico-chemical parameters are given in the table 4. profile graphics are built due to the obtained data (fig. 2). table 4 main physical-chemical properties of sugar substitutes caloric content melting point solubility at 293 k (20 °c),% glycemic index (gi),% kj/g kcal/g к °с sweetness, unit the heat of solution kj/kg water activity tolerance, g/day name of sugar substitute р1 р2 р3 р4 р5 р6 р7 р8 sorbitol 75.0 9.0 8.62 2.6 373.0 100.0 0.6 -110 0.72 24 xylitol 58.0 8.0 17.01 4.06 367.0 94.0 0.6 -153 0.82 60 isomaltitol 24.5 9.0 10.05 2.4 419.0 146.0 0.55 -39 0.88 66 maltitol 65.0 36.0 10.05 2.4 421.0 148.0 0.9 -23 0.85 87 erythritol 37.0 0.2 2.09 0.5 399.0 126.0 0.65 -192 0.91 132 lactitol 55.0 2.0 10.05 2.4 423.0 150.0 0.35 -65 0.90 54 fig. 2. the profile graphics of quality of sugar substitutes analysis of profile graphics shows that the most square takes erythritol, and the less – sorbitol. table 5 shows the validity factors that were determined by the method of delphi. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 148 table 5 definition of validity factors the value of the validity factor sum of the validity coefficients expert number м1 м2 м3 м4 м5 м6 м7 м8 the first expert 0.1 0.35 0.2 0.03 0.15 0.01 0.05 0.11 1.00 the second expert 0.1 0.25 0.1 0.05 0.2 0.05 0.15 0.1 1.00 the third expert 0.1 0.2 0.2 0.05 0.1 0.05 0.1 0.2 1.00 the fourth expert 0.2 0.15 0.1 0.08 0.05 0.05 0.05 0.32 1.00 the fifth expert 0.1 0.3 0.2 0.02 0.05 0.08 0.15 0.1 1.00 the sixth expert 0.1 0.25 0.3 0.04 0.1 0.08 0.1 0.03 1.00 the seventh expert 0.05 0.15 0.2 0.1 0.15 0.03 0.08 0.24 1.00 the eighth expert 0.1 0.4 0.1 0.05 0.05 0.02 0.1 0.18 1.00 the ninth expert 0.05 0.3 0.2 0.03 0.1 0.03 0.14 0.15 1.00 a tenth expert 0.1 0.15 0.4 0.05 0.05 0.1 0.08 0.07 1.00 the average value 0.1 0.25 0.2 0.05 0.1 0.05 0.1 0.15 1.00 taking into account the validity coefficients an integrated indicator of the quality of sugar substitute is counted according to the formula (4) (table. 8). 1 31 2 0 1 2 3 1 2 3 5 64 4 5 6 4 5 6 7 8 7 8 7 8 б б б б б б б б рр р к м м м р р р р рр м м м р р р р р м м р р           (4) where м1, м2, м3, м4, м5, м6, м7, м8 – the values of the validity factors of the indicator; р1б, р2б, р3б, р4б , р5б , р6б, р7б , р8б – the values of the corresponding indicators in the baseline sample, which equals 10 points р1, р2, р3, р4 , р5, р6, р7, р8 – the corresponding values from the profile graphics. table 6 the value of the integrated indicator of the quality of sugar substitutes name of sugar substitute the value of the integrated indicator of the quality sorbitol 0.34 xylitol 0.42 isomaltitol 0.37 maltitol 0.47 erythritol 0.86 lactitol 0.37 analysis of the data showed that the highest mark was registered by erythritol. studies have shown a significant outgrowth of tagatose among sugars and erythritol among sugar substitutes. d-tagatose (tagatose) is a monosaccharide identified in chemical nomenclature (cas) by formula c6h12o6. tagatose is a white crystalline powder similar to sucrose. tagatose is found in nature in various fruit, milk and other products. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 149 in the united states tagatose received gras status ("recognized as safe") in october, 2001. in december, 2005 tagatose was approved for use in the eu as an innovative food ingredient. tagatose promotes the growth of lactobacillus and lactic acid bacteria in the human body, and also has prebiotics action. tagatose is characterized by a low glycemic index, which is equal to 3 %. long-term consumption of tagatose lowers the level of glucose and blood cholesterol, reducing the risk of obesity, diabetes. in addition, it is considered to be potentially useful in the treatment of anemia, hemophilia, infertility has crioprotector and antioxidant properties [16, 17]. erythritol is one of the most promising substitutes of a new generation. in the environment erythritol is in plant foods: pear, melon, grape. in the human body erythritol also contains, in particular in blood serum, tissues of the eye. erythritol is an inhibitor of the breakdown of red blood cells, induced by diazole compounds, reduces vascular damage caused by hyperglycemia. a positive feature of erythritol is also that it belongs to the prebiotics. this poliol does not cause any gastrointestinal complications and loose stools. erythritol is absorbed in the small intestine by passive diffusion. erythritol is get by the microbiological synthesis, using such microorganisms trigonopsis, torulopsis, pollinis. cultivation of microorganisms is carried out in an aqueous medium at a ph from 3 to 6 and a temperature of 35...38°c for 3 to 8 days. carbohydrates (e.g., glucose, sucrose), nitrogenous substances (malt extract, urea), inorganic salts of potassium, calcium, iron are used for the cultivation of microorganisms [18, 19]. however, in the manufacture of confectionery products, their technological properties and the possibility of their use in the manufacture of confectionery products of various structures are of great importance. an important characteristic of the quality of confectionery products during storage is their sorption-desorption properties. at the national university of food technologies, kyiv, the innovative technology of chewing caramel of dieticfunctional purpose on the basis of the monosaccharide tagatose is developed, that deserves the label "product with reduced calorie" and reduced glicemicity. studies have shown that chewing caramel with tagatose quickly lose chewing properties during storage, therefore, in order to maintain organoleptic characteristics, that is, chewing effect, and the structure, polyols maltitol and glycerol were proposed to enter in the formulation of the product [20]. studies have shown that despite the high value of the complex quality index of tagatose, chewing caramel that is made on its basis, while stored quickly loses chewing properties, therefore, in order to preserve the necessary chewing effect and the desired structure, it was proposed to introduce polyol maltitol to the formulation of the product, the complex quality index of which is rather low, but the technological properties of maltitol will improve the quality of chewing caramel on tagatose. the study of sorption-desorption samples of chewing caramel, made according to the traditional recipe on sucrose and using the proposed composition on tagatose and maltitol (table 7) was carried out. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 150 table 7 the value of the equilibrium humidity content in chewing caramel by zones of sorption isotherms the value of equilibrium humidity, % first zone φ = 0 … 25% аw = 0.0 … 0.25 second zone φ = 25 … 75 % аw = 0.25 … 0.75 third zone φ = 75 … 100 % аw = 0.75 … 1.0 kind of chewing caramel on sorption desorption sorption desorption sorption desorption sucrose 0.0…0.0 11.0…13.0 0.0…13.0 13.0…24.1 8.0…75.0 24.1…75.0 tagatosemaltitol 0.0…0.0 11.0…13.0 0.0…8.0 13.0…35.6 8.0…85.4 35.6…85.4 experiments have shown that the equilibrium humidity content in caramel produced on a mixture of tagatose-maltitol with aw = 0.75 equals 8%, as well as for caramel produced on sucrose, but the initial humidity content of the finished caramel using tagatose is 8.1%. this suggests that during storage of caramel there was a slight humidity separation, which affects the reduction of the chewing effect by increasing the degree of crystallinity of the structure. the calculation of the complex quality index has shown good indicators for fructose, but from a technological point of view, fructose is a hygroscopic substance, so its use in confectionery products greatly increases sorption properties. for example, in the production of candy caramel, using only fructose and molasses as anticrystallizer, moisture absorption during storage is significantly increased (table 8) and when developing new confectionery products, it is necessary to introduce substances with low hygroscopicity into the prescription composition. among a number of polyols, isomalt and maltitol during storage do not absorb moisture, therefore, recipe mixes of isomalt-fructose and maltitol-fructose were developed in the production of candy caramel. the use of sugar substitutes and fructose in the manufacture of confectionery products can reduce their caloric content and glycemicity, which will allow consuming them by patients with diabetes, as well as providing products with prebiotic properties. in new samples of caramel, sorption-desorption properties were investigated (table 8). table 8 the value of the equilibrium humidity content of candy caramel by zones of sorption isotherms the value of equilibrium humidity, % first zone φ = 0 … 25% аw = 0.0 … 0.25 second zone φ = 25 … 75 % аw = 0.25 … 0.75 third zone φ = 75 … 100 % аw = 0.75 … 1.0 kind of caramel on sorption desorption sorption desorption sorption desorption fructose 0.0 – 0.0 9.0-17.0 0.0-11.0 17.0-30.0 11.0-125.0 3.0 -125.0 isomalt-fructose 0.0 – 0.0 8.0-16.0 0.0-2.5 16.0-18.0 2.5-133.0 18.0 – 133.0 maltitol-fructose 0.0 – 0.0 7.5-14.0 0.0-3.5 10.0-14.0 3.5-154.0 10.0 – 154.0 analysis of data shows that caramel on fructose will greatly absorb moisture, because the humidity according to the recipe is 2 4%, and equilibrium humidity is 11%. the use of polyols can reduce equilibrium humidity to moisture according to recipes, so such caramel will food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 151 retain the necessary properties throughout the expiration date. we conducted a complex of studies about the use of sugar substitutes and sugars in the production of marshmallows, among which the use of mixtures of isomalt-fructose and maltitol-fructose allowed to obtain a good quality product. the determination of the behavior of new samples of marshmallows during storage was very important, so their sorptiondesorption properties were investigated (table 9). table 9 the value of the equilibrium humidity content of marshmallow by zones of sorption isotherms the value of equilibrium humidity, % first zone φ = 0 … 25% аw = 0.0 … 0.25 second zone φ = 25 … 75 % аw = 0.25 … 0.75 third zone φ = 75 … 100 % аw = 0.75 … 1.0 kind of marshmallow on sorption desorption sorption desorption sorption desorption sucrose 0.0 – 0.0 0.0 – 1.0 1.0 10.0 12.0-40.0 10.0-130.0 40.0-130.0 isomalt-fructose 0.0 – 0.0 0.0 – 1.0 1.0 33.0 9.0-40.0 33.0-163.0 40.0-163.0 maltitol-fructose 0.0 – 0.0 0.0 1.0 1.0 31.0 11.0-37.0 31.0-158.0 37.0-158.0 analysis of the data shows that new samples of marshmallow will largely absorb moisture, since the humidity according to the recipe is 18 20%, and the equilibrium moisture content of the new samples is 31 33%, therefore, such marshmallow in the process of storage will absorb moisture, which leads to the need to pack marshmallow in a waterproof container.the complex of researches on the rational use of erythritol in the production of cakes is conducted in nuft. the aim was to get a cake based on erythritol that deserves the label "product with reduced calories" and "reduced glycemicity". however, erythritol has a high heat of dissolution (-192 kj/kg), which influences on the organoleptic properties, i.e. provides unwanted cooling effect. experiments have shown that the elimination of the cooling effect may be when changing the temperature and duration of baking cake. traditional temperature of baking for the cake with sucrose is 453 k (180 °c), cooking duration is 30 min. experiments showed that the temperature of baking cake with erythritol must be 413 k (140 °c), and the duration of baking must be increased by 50 % to reach 45-50 min [21]. the study of sorption-desorption properties of samples of cakes made on the basis of erythritol was conducted. a sample on sucrose was choosen as a control sample (table 10). table 10 the value of the equilibrium humidity content of cakes by zones of sorption isotherms the value of equilibrium humidity, % first zone φ = 0 … 25% аw = 0.0 … 0.25 second zone φ = 25 … 75 % аw = 0.25 … 0.75 third zone φ = 75 … 100 % аw = 0.75 … 1.0 kind of cake on sorption desorption sorption desorption sorption desorption sucrose 0.0 – 0.0 3.1 – 1.8 0.0-11.6 3.1 – 26.98 11.6-52.0 29.98-52.0 erythritol 0.0 – 0.0 2.8 – 3.95 0.0-9.0 3.95 – 79.3 9.0 – 102.18 79.3 – 102.18 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 152 according to the obtained data (table 12), it is determined that at p / p0 = 0.75 the equilibrium moisture content of cake based on the sucrose is 11.6%, on the basis of the erythritol 9.0, which is significantly less than the moisture content of the finished cakes (w = 25 ... 27%), this indicates that when storing all samples will lose moisture, that is, will undergo the process of drawing, therefore, to preserve the necessary properties of products, they must be packed. 4. conclusion research showed the feasibility of determining the assessment of quality of sugars and sugar substitutes by the integrated indicator taking into account the basic principles of quality control. the expediency of calculation of the integrated indicator of quality of sugars by such differential parameters such as solubility, caloric value, glycemic index, sweetness, melting point was justified. the calculation of the integrated indicator of sugar substitutes was carried out by solubility, caloric value, glycemic index, sweetness, melting point, water activity, degree of tolerance and heat of solution. when assessing quality for the integrated indicator the validity factors were taken into account and they were determined by the method of the expert survey after delphi. it was established that sugar tagatose has the highest value of the integrated indicator of quality (k=0.81). it is established that the sugar substitute erythritol has the highest value of the integrated indicator of quality (k=0.86). the obtained data for the calculation the complex quality index increase the interest to the use of tagatose and erythritol in the manufacture of confectionery, but it is necessary to take into account the technological properties of sugars and sugar substitutes in the manufacture of each individual product. so, tagatose may be used in the production of chewing caramel in a mixture with maltitol. studies of sorption-desorption properties have shown that such caramel will insignificantly lose moisture and get stale. the widespread distribution of confectionery products was made because of isomalt and maltitol in a mixture with fructose, for example, lollipop caramel and marshmallow were produced using various recipe compositions. designed samples of candy caramel, according to the data of sorption isotherms, will have high quality during the guaranteed expiration date, and marshmallow absorb moisture, which requires considerable attention when packaging products and the approach to the type of packaging in general. the use of erythritol in the production of cakes significantly affects the quality of the product, so when stored, they will lose moisture and get stale, but it significantly reduces the caloric content of the product and improves its healing properties, therefore, such products require careful packaging to maintain the required quality. 5. references [1]. report of a joint fao/who (food and agriculture organization of the united nations) expert consultation, rome:140, (1998) [2]. mcnaught d. nomenclature of carbohydrates, pure & appl. chem., vol. 68, no. 10, uk: 1919-2008s, (1996) [3]. codex standard for formula foods for use in weight control diets codex stan 181: 6, (1991) [4]. polumbrik m. carbohydrates in food products and human health, academiperiodica, kyiv: 487, (2011) [5]. levin g. sugar substitutes: their energy values, bulk characteristics and potential health benefit, am. j. clin. nutr.. v. 62 (suppl.), usa: 1161-1168s (1995) [6]. kier l. a molecular theory of sweet taste, j. pharm. sci. v. 61: 1394-1397s (1972) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 antonella dorohovych, victoria dorohovych, liubov mazur, complex indicator of quality of sugars and sugar substitutes and their use in the production of confectionery products, food and environment safety, volume xvii, issue 2 – 2018, pag. 141 – 153 153 [7]. jenkins d., wolever t., taylor r. glycemic index of foods: a physiological basis for carbohydrate exchange, am. j. clin. nutr. v. 34, usa: 362-366s, (1981) [8].francis f. wiley encyclopedia of food science and technology. 2th edition, j. wiley&sons, new york: 2816 p (1999) [9].belitz h.-d., grosch w., schieberle p. food chemistry. 4th revised and expanded edition, springer-verlag, berlin: 1070 p, (2009) [10]. faseb the evaluation of the energy of certain sugar alcoohols used as food ingredients. bathesda, md: life sciences research office, federation of american societies for experimental biology, (1994) [11]. korpachev v. sugars and sweetener, кnyga plus, kyiv :320 (2004) [12]. zhuk v. sensory analysis, litopys, kyiv: 231, (1999) [13]. brouns f., bjorck i., frayn k. glycaemic index methodology, nutr. res. rev. v. 18: 145 – 171s, (2005) [14]. skoog d., west d., holler f. fundamentals of analytical chemistry. 7th. ed., saunders college publishing, fort worth: 293, (1996) [15]. azgaldov g., glichev a., rayhman e. qualimetry is a science about measuring the product’s quality, standards and quality, moskow: 34-35s, (1968) [16]. levin g. tagatose, the new gras sweetener and health product, j. med. food. v. 65: 243-249s, (2002) [17]. lu y., levin g., donner t. tagatose, a new antidiabetic and obesity control drug, diab. obes. metab. v. 10: 109-134s, (2008) [18]. o’brien-nabors l. alternative sweeteners. third edition, crc press, n. y.: 572 p., (2001) [19]. mitchell h. sweeteners and sugar alternatives in food technology, wiley-blackwell publishing, oxford: 432 p., (2006) [20]. dorokhovych a., bogok o., mazur l. the tagatose and maltitol are innovation raw materials for production of masticatory caramel, food science and technology, odessa: 43-48s, (2016) [21]. abramova a. dorohovich v. innovation technologies of semi-finished cake products with the use of erythritol – a sweetener of the new generation, almata: 22-231s, (2013) 8 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2014, pag. 8 13 chemical mutagenesis in the curranт (ribes l.) and gooseberry (grossularia mill.) selective breeding igor e. buchenkov1 1international sakharov environmental university, belarus, butchenkow@mail.ru *corresponding author received january 15th 2014, accepted march 5th 2014 abstract: the optimum solution concentration for the treatment of mutagen varieties of black currants, red currants and gooseberries is defined within 12 hour exposure as: 0.005% nitrosoethylurea, 0.01% diethyl sulfate, 0.1% ethyleneimine, 0.05% dimethyl sulfate. when using higher concentration of mutagens (0.5 to 1%) along with an increase in the total number of mutants, the percentage of plants with economic traits is reduced. if the mutagen solution concentration is 1% the growth of browses from apical buds does not occur due to their dieback. the great number of somatic mutations is induced by nitrosoethylurea and diethyl but less by ethylenimine and dimethyl sulfate. mutant forms of black currants, red currants and gooseberries with morphological lamina change and leaf chlorophyll distortion revealed a clear relation to bush yield and mien. keywords: currant, gooseberry, chemical mutagenesis 1. introduction the food program doesn’t lose its actuality as the years go by. the root of this matter is in the discrepancy between an increased product demand of the population and performance capabilities of the agricultural industry, limited by land resources and its own development level. due to this fact, there is a need of change-over to agricultural plant selection techniques providing their maximal compliance with the culture biological requirements to a new integrated utilization strategy of genetic, natural and man-induced factors providing stable growth of productivity, resource conservation and an environmental safety. the variety role will be increasingly rising in the integrated system of agricultural plant production as it is the most reliable and ecologically beneficial factor of productivity improvement and stability. in perspective the increase in food products and other agricultural goods in the world will be primarily determined by the new technology development and implementation [1]. working process in selecting initially various genotypes based on the hybridization technique precedes a new plant variety establishment. however, this fruit and berry shrub method of selective breeding requires 15-20 years to be implemented. one of the methods to obtain assorted in many features primary sources in the shortest possible time is the induced mutagenesis. as compared with traditional methods of selection the induced mutagenesis main advantage is fast primary sources improvement for further selective purposes (by one or several agriculturally important characters) [2, 3]. however, early determination of dominant mutations and comparatively frequent varfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 igor e. buchenkov, chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding, volume xiii, issue 1 2014, pag. 8 13 9 ious chromosomal aberrations caused by physical mutagens (high and cold temperatures, x-rays, α, β and γ-rays, uv radiation) create significant difficulties to more productive operation with the induced mutagenesis in selection process. to a great degree that can be limited by using chemical mutagens which can reduce drastically the amount of chromosomal aberrations and increase the percentage of dominant mutations [4, 5]. among small fruits, cultivated in belarus, the very important cultures are currants (ribes l.) and gooseberries (grossularia mill.). their berries enriched in complex of vitamins, minerals and enzymes play significant role in balanced nutrition, maintenance and effective treatment of many human diseases. in this regard, selective breeding of these cultures is of prime importance [6]. large scale variability of such an important practical features as resistance to american powdery mildew, high productivity, berry size, taste, chemical composition, color etc. is typical to currants and gooseberries [6]. at this stage an extensive factual material has been accumulated about currant and gooseberry mutational variability. special sorts of black currant with high practical value (high growth, shortened internodes, elongated racemes, king-size berries, better taste, late ripening, anthracnose resistant) have been selected in germany by γ-rays exposure. according to r. bauer opinion, selection of new black currant variety by the method of radiation mutagenesis is more effective then intervarietal hybridization [7]. mutant forms with different ripening periods, leaf form, growth rate have been selected in switzerland [8]. the most effective mutagenic factors, doses, various sorts of black currant mutability and morphological characteristics variability have been studied in latvia. it has been established that black currant sprigs γ-ray exposure with the test doses cause dramatic rise in variability, whereas 0.7%6.7% of all changes fall beyond the species features [9]. grober k. from germany has selected mutant species with high berry ascorbic acid content [10]. yurtsev v.k. and gorlanov n.a. have observed a positive effect of black currant sprigs γ-ray exposure on roots regeneration process, planted sprigs growth and development [11]. in experiments with black currant chronic γ-ray exposure during its vegetation period ravkin a. s. has established the optimal dose to obtain radiomorphosis and highly resistant mutable varieties [12]. experimental studies of a group of mutagenic effect chemical compounds on plants have been conducted concurrently with the effect of ionizing radiation. however, the first experiments on somatic mutations detection were ineffective. that was due to specific plants reaction on treatment with chemical compounds [13]. further experimental study on black currant induced chemical mutagenesis was based on the culture specific features development, new particular characteristics, the obtained morphosis studies and detailed consideration of all hereditary changes frequency and range. at the same time black currant ability to vegetative reproduction provides stability of inherited somatic and bud mutations in the next vegetative generations [14, 15, 16]. chemical mutagen implementation on black currant and gooseberry selection is at an initial stage of studying process in belarus at present (including effective mutagens, doses, exposure time, variety mutability and features type of variation study). starting from 1976 – 1980 mutant forms of black currant with changes in leaves and browse morphology, berry size, ripening period, yield, immunity, winter resistance, growth rate and self-fertility have been obtained by bavtuto g.a. [6]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 igor e. buchenkov, chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding, volume xiii, issue 1 2014, pag. 8 13 10 in spite of visible success many aspects of black currant, red currant and gooseberry chemical mutagenesis remain unclear today. 2. materials and methods the buds treatment of black currants, red currants and gooseberries was conducted by supermutagens during the period 19982003. the analysis on selection and study of mutant species was conducted during 19992009 at an agrobiological station of bspu named after maxim tank, and 2009-2012 at a substation of poles su. the objects of the study were such varieties as black currant (pamyati vavilova, minay shmiryov, kantata 50, tserera, katyusha), red currant (nenaglyadnaya, golandskaya krasnaya, prygazhunya), gooseberry (yarovoy, belarussky krasny, masheka). apical buds of varieties named above were treated by nitrosoethylurea (nel), nitrosomethylurea (nml), ethyleneimin (ei), dimethyl sulfate (dms) and diethyl sulfate (des) in concentration 0.001; 0.005; 0.01; 0.05; 0.1; 0.5; 1% within 6, 12, 24 hours exposure. being treated the apical buds of named above varieties were placed into gelatin capsule filled with mutagen aquatic solution of appropriate concentration. in each capsule with different concentration were placed 160-180 buds. after certain exposure effect the buds were waterwashed. next year the grown browses from treated buds were cut and rooted. the quantity indicator of transformed plants, grown from treated by chemical mutagen buds as the criteria of different currant and gooseberry variety test was used. the sensitivity of sprigs, grown from treated by mutagen buds was tested in a 2year. the field experiments and observations were made in line with program and techniques of fruit, berry, and nut-fruited crop variety research [17]. 3. results and discussion during the research period 4808 buds were treated and 404 plants were grown. there were 81 selected with more than 20 various types of morphosis and mutation. it is stated that the frequency of mutative change depends on primary variety, mutagen and its concentration and exposure effect. on average, black currant – 2.07±0.28%; red currant – 1.94±0.15%; gooseberry – 1.07±0.08% (see figure 1). figure 1 generalized average data of currant and gooseberry variety apical buds treatment crop treated buds, in pieces evolved apical buds rooted plants transformed plants selected varieties with economic traits pieces % pieces % pieces % pieces % black currant 1783 1041 58.38 154 8.64 37 2.07 4 0.22 red currant 1341 921 68.68 152 11.33 26 1.94 2 0.15 gooseberry 1684 1002 59.50 98 5.82 18 1.07 3 0.18 the research of chemical mutagen influence on black and red currant and gooseberry varieties showed the linear dependence of mutant plants development degree on concentration and exposure of mutagen influence. the analysis of the obtained results showed: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 igor e. buchenkov, chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding, volume xiii, issue 1 2014, pag. 8 13 11 1) to get varieties with economic traits the optimum concentration of the solutions for the treatment of mutagens varieties of black currants, red currants and gooseberries is 0.005% nml, 0.01% des, 0.1% ei, 0.05% dms within 12 hours exposure. 2) with the use of higher concentration of mutagens (0.5 to 1%) along with an increase in the total number of mutants, the percentage of plants with economic traits is reduced. 3) if the mutagen solution concentration is 1% the growth of browses from apical buds does not occur due to their dieback. it is stated that the most mutable sorts are pamyati vavilova (4.38%), minay shmiryov (4.26%), kantata 50 (3.87%), yarovoy (2.63%), belarussky krasny (2.79%), nenaglyadnaya (4.32%); less mutable tserera (0.84%), katyusha (0.78%), masheka (0.63%), golandskaya krasnaya (0.72%), prygazhunya (0.71%). the study shows that chemical mutagens induce a great number of currant and gooseberry hereditary changes the most part of which is not relevant to economic traits (figure 1). nearly all characteristics and properties change under mutagen influence: increase or reduction in berry size and shape; fruit flavor reversion; reduction in number of seeds; early or late ripening; improvement or reduction of disease resistance; deformation of the ability to self-pollination, dwarfness, mien change, size and form variation, leaf coloration and its parameter, change of phenophase occurance period. the study of the derived and selected forms according to their main economic traits made it possible to distinguish among revealed deviations macroand micromutations. mutant forms of the first group stand in marked contrast with parental forms in leaf structure, mien and branch growth status. mutant forms of the second group keep predominant of mother variety but there are some leaf morphology deviations. the most valuable for selective breeding currant and gooseberry neoplasms are their much strong growth, shortened internodes, long racemes, tree-shaped shrub mien, fruit enlargement, fruit taste control, improvement of disease resistance. the frequency of desirable mutations for practical selective breeding, however, is rather small. the desirable features of derived forms combine with the impairment of fertility that phenotypically is presented by small in size berries, their quantity reduction, and fruit and germ drop. in whole, there are only 9 forms selected that exceed primary varieties on the strength of all traits. somatic mutations of currant and gooseberry were also studied. apparent phenomic changes reasoned by chlorophyll deficiency and leaf morphology (pattern of limbus lamina laceration, its surface, pinnation, size and form) were used. the record was maintained at the end of browses growth. the great number of somatic mutations is induced by nitrosoethylurea and diethyl but less by ethylenimine and dimethyl sulfate. in most cases nel and des facilitated chlorophyll mutation emergence, but ei and dms caused accompanying each other chlorophyll and morphological mutations. the study of currant and gooseberry somatic mutations was conducted to ascertain mutant trait correlation evident at the early stage of growth (chlorophyll deficiency, leaf morphology) with economic traits (tree-shaped mien, large fruit and et cetera) evident at the late stage. the studied mutant forms of morphological type were divided into three groups according to leaf change rate with: 1 – marked pinnation 2 – severe lamina surface strain 3 – lamina size change. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 igor e. buchenkov, chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding, volume xiii, issue 1 2014, pag. 8 13 12 originally selected modified forms underwent vegetative breeding. correlation of lamina size change rate with other traits is shown in the results of maintained records in the second and third vegetative voltine. the first group of shrubs is characterized by slow growth up to dwarfing and late phenophase stage. the majority of shrubs in this group are characterized by chlorophyll deficiency. the zone of transformed by colour tissue can be found lengthwise big ribs. the second group is characterized by impairment of fertility and chlorophyll deficiency in the form of light green leaf colour. the shrubs of this group are marked with low yield due to the reduction of inflorescence number, florets and berry size. the third group does not differ from the original parental varieties (the power of growth, leaf colour), but is characterized by high pollen sterility, reduction of inflorescence number and small fruit size. it is revealed that there is a clear connection of leaf lamina change to a complex of other traits such as shrub mien, yield and et cetera. the chlorophyll mutant forms presented by shrubs with leaf colour change, having been observed during a year are investigated. as opposed to morphological mutant forms with a slight amount of chlorophyll deficiency the named above forms are characterized by the colour change of the whole leaf lamina. in whole, all educed forms with chlorophyll change can be unified into three groups: 1 – homochromous – yellow, light green, greenish-yellow 2 – bicolorous – a part of shrub leaves is light green or yellow-green, the rest one is habitual 3 – colour interchange – from green to inconspicuous yellowy-green during vegetation period. the eduction of correlation between groups of chlorophyll mutants with other economic traits during three vegetative voltines showed that the first group is characterized by slow growth or treeshaped shrub mien, the second group – tight shrub mien with elevated branch, the third – sprawling shrub and lagged phenophase development. the educed regularity is inherent to black currants, red currants and gooseberries. 4. conclusion the possibility of currant and gooseberry selection induced by chemical mutagenesis has been shown in the following study results: 1. optimum concentrations of mutagen solutions for agriculturally important species of black currant, red currant and gooseberry selection are: 0.005% neu (nitroso-n-ethylurea); 0.01% diethyl sulfate (des); 0.1% ethyleneimin (ei) and 0.05% dimethyl sulfate (dms) within 12 hours exposure. when using higher concentration of mutagens (0.5 to 1%) along with an increase in the total number of mutants, the percentage of plants with economic traits is reduced. if the mutagen solution concentration is 1% the growth of browses from apical buds does not occur due to their dieback. 2. the most mutable varieties are pamyati vavilova (4.38%), minay shmiryov (4.26%), kantata 50 (3.87%), yarovoy (2.63%), belarussky krasny (2.79%), nenaglyadnaya (4.32%); less mutable tserera (0.84%), katyusha (0.78%), masheka (0.63%), golandskaya krasnaya (0.72%), prygazhunya (0.71%). 3. the great number of somatic mutations is induced by nitrosoethylurea and diethyl but less by ethylenimine and dimethyl sulfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 igor e. buchenkov, chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding, volume xiii, issue 1 2014, pag. 8 13 13 fate. nel and dms in most cases enforce onset of chlorophyll mutations but ei and dms usually cause concurrent chlorophyll and morphological mutations. 4. mutant forms of black currants, red currants and gooseberries with morphological lamina change and leaf chlorophyll distortion revealed a clear relation to shrub yield and mien. 5. four varieties of black currant, 2 – red currant and 3 – gooseberry exceeded the original parental variety in a complex of characteristics have been selected. the selected variety represents the original breeding material for further improvement. 5. references [1]. rossikhin v. p. evolutionary trend in agricultural plants selection, zernograd, 48 p, (2006). [2]. zoz n. n. survey technique of chemical mutagens used for agricultural plants selection, moscow: science, p. 217-230, (1968). [3]. strelchuk s. i. the first principles of experimental mutagenesis, kiev: vizha shkola, p. 216, (1981). [4]. auerbach s. mutagenesis problems, m: mir. 458 p, (1978). [5]. rapoport i. a. dominance genetic resources in chemical mutagenesis and their selection value, moscow: nauka, p. 3-33, (1978). [6]. bavtuto g. a. gene pool enrichment and fruit and berry crops parent material selection based on experimental polyploidy and mutagenesis: thesis abstract. dr. of biol. science: 03.00.05, tartu, 49 p. (1980). [7]. bauer, r.: the induction of vegetative mutations in ribes nigrum: heredidas., no 2., p. 323-337, (1957). [8]. nybom, n. induced mutations and breeding methods in vegetatively propagated plants: rad. bot., no 4, p. 661-678, (1965). [9]. melekhina a. a. blackcurrant variability induced by ionizing radiation, latvian academy of science proceedings, no10 (231), p. 83-88. (1966). [10]. grober, k. some results of mutation experiments an apples and black currants, bonn, p. 377-382, (1967). [11]. yurtsev v. k. gamma rays effect on currants and gooseberries root formation and quickset segments development, moscow: kolos, p. 67-79, (1969). [12]. ravkin a. s. types of black currant induced chimeras and some aspects of their formation, m., p. 322-331, (1972). [13]. ravkin a. s. ionizing radiation and chemical mutagens effects on plants with vegetative reproduction, moscow: nauka, 192 p. (1981). [14]. potapov s. p. chemical mutagenesis in black currant selection process, moscow: mgu, 131-141, (1977). [15]. salnikova t. v.: factors affecting mutants range and types induced by chemical mutagenesis, moscow: nauka, p. 38-51, (1983). [16]. eglite m. a. the effect of nnitroso-nethylurea on blackcurrant in a year of treatment, moscow: nauka, p. 379-388, (1971). [17]. program and methodology of fruit, berry and nut crops investigation, vniispk; edited by sedov e. n., ogoltsova t. p., oryol, 608 p., (1999). issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiv, issue 4 31 december 2015 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladislav reșitca technical university of moldova, chisinau republic of moldova alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldova yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:fiajournal@fia.usv.ro mailto:gutts@fia.usv.ro mailto:cristina.hretcanu@fia.usv.ro mailto:gabriela.constantinescu@fia.usv.ro 214 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 214 222 influence of ph and acidity on the fermentation of finger millet spiced ogi *abosede m. adisa 1 , adefisola b. adepeju 1 , ayowunmi k. yusuf 1 1department of food science and technology, joseph ayo babalola university, ikeji arakeji, osun state, nigeria. abosedeadis@gmail.com *corresponding author received 12 july 2019, accepted 27th september 2019 abstract: the purpose of this research work was to evaluate the effect of ginger and garlic on the fermentation dynamics of finger millet made ogi. it is important to allow cereals achieve acidification during fermentation in order to improve the organoleptic properties and safety of the product. spiced finger millet (fm) ogi was produced in the ratios of 100% fm, 99% fm: 1% ginger, 95% fm: 5% ginger, 90% fm: 10%, 99% fm: 1% garlic, 95% fm: 5% garlic, 90% fm: 10% garlic. the data generated were subjected to statistical analysis and means were separated using analysis of variance. the results of microbial associations of spiced finger millet ogi samples investigated at 48 h during the secondary fermentation stages ranged from (5.0×103 – 43.0×103) cfu/ml for total bacteria count, (1.0×103 – 3.0×103) cfu/ml for total fungi count, (2.0× 103 – 10.5× 103) cfu/ml for total coliform count and (7.5×103 – 70×103) cfu/ml for total lab count. all the isolates that were catalase negative, gram positive, non-spore forming were identified as lactic acid bacteria and sugar fermentation pattern revealed that they belong to the specie of lactobacillus. during steeping the acidity increased with consequent drop in ph, the trend which was sustained during the souring stage, the ph decreased gradually (p<0.05) from 5.98 to 4.01 at 10% garlic inclusion, while it decreased significantly (p<0.05) from 5.54 to 3.42 at 10% inclusion of ginger. keywords: traditional, lactic acid bacteria, safety, dominate, acidification 1. introduction. finger millet (eleusine coracana) locally known as ‘tamba’ in nigeria, ‘ragi’ and ‘mandua’ in india belongs to the family poaceae and genus eleusine. it is consumed as one of the main staple food that supplies calories and protein to a relatively large population of rural dwellers and low income earners in these countries [1]. it is quite interesting that despite its excellent nutritional profile, finger millet is still being under-utilized as only a few people are aware of its health benefits and nutritional value [2]. the nutritional properties of the grains is outstandingly comparable with other cereals such as rye, barley and oats, being a rich source of riboflavin, thiamine, iron, methionine, leucine, isoleucine, phenylalanine and other essential amino acids [3]. finger millet’s chemical composition consist of high content of protein (6%– 13%), dietary fiber (18%), minerals (2.5%–3.5%), calcium (0.38%), carbohydrate (65%–75%), phytates (0.48%), tannins (0.61%), phenolic compounds (0.3–3%) and trypsin inhibitory factors [3]. it is recognized for its health benefits such as anti-diabetic, anti-diarrheal, anti-inflammatory, antiulcer, atherosclerogenic effects, antioxidant and antimicrobial properties [4, 5]. http://www.fia.usv.ro/fiajournal mailto:abosedeadis@gmail.com http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/eleusine http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/riboflavin http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/thiamine http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/methionine http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/leucine http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/isoleucine http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/phenylalanine http://www.sciencedirect.com/topics/nursing-and-health-professions/amino-acid http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/carbohydrate http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/phytic-acid http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/tannin http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/trypsin http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/antioxidant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 215 ogi is a traditionally fermented food prepared by steeping cereal grains such as millet, maize, sorghum in water for 72 h, followed by wet milling and sieving using muslin clothe. the filtrate obtained is allowed to settle to give the slurry referred to as ogi and the supernatant discarded. the traditional processing of ogi is characterized with a lot of nutrient loss which include proteins, water soluble vitamins and minerals [6]. as a result several researchers have made attempts at improving the nutritional status of this unique food by enriching it with both plant and animal protein substrate [711]. however, this nutritional fortification has been reported to lower the pasting viscosity and also adversely affect the sensory attribute for which the product is desired [12]. the predominant organisms involved in the fermentation are mainly lactic acid bacteria and yeast which are also responsible for microbial stability, characteristic sour taste, flavour and aroma development of ogi. the health benefit of some herbs and spices on humans has led to the gradual drifting towards natural, antimicrobial and traditional preservative techniques. currently there is increased awareness in the use of spices such as ginger, garlic, conophor nuts etc in the production of ogi to improve its organoleptic properties, enhance shelf stability and provide other therapeutic benefits [13, 14]. in order to attain its desired sensorial quality and safety it is important to study and establish the fermentation kinetics of the fermenting grain and slurry as the degree of sourness which reflects its level of acidity varies with consumers and region. this study is therefore under-taken to evaluate the effect of ginger and garlic on the microbial load and fermentation dynamics of ogi produced from finger millet. 2. materials and methods collection of samples for this study, finger millet (eleusine coracana), cloves of fresh garlic (allium sativum) and ginger roots (zingiber officinale) were all purchased from owena market in oriade local government area of osun state, nigeria. preparation of finger millet into ogi this was done according to the methods described by 15 and [16] with slight modifications. spiced finger millet ogi was produced in the ratios of (0:100, 1:99, 5:95, and 10:90 w/w) for both ginger and garlic. prior to fermentation the dried finger millet grains were cleaned to remove pebbles and dirt, one thousand grams of the grain was weighed and washed thoroughly in clean water. fresh ginger rhizomes and garlic cloves were washed peeled with a sharp knife and steeped together with the cleaned grains in 2000 ml of sterile water in a plastic container for 48 h. the steeped grains were wet-milled together with the steeping water using a sterile laboratory blender. the slurry was sieved using sterile muslin clothe and the filtrate allowed to settle for 48 h at 28±2 oc to undergo souring (secondary fermentation) and give a smooth starchy. samples were aseptically taken 12 hourly for analyses during the primary and secondary fermentation period. microbiological analysis of spiced finger millet ogi samples the samples were analyzed for total bacterial, fungal, coliform and lactic acid bacteria [lab] counts according to the methods stated in [17]. about 10 g of each composite mixture was homogenized with90 ml of sterile distilled water to give a ten-fold serial dilution of 10-1 level. from this, subsequent dilution levels were made food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 216 and cultured on appropriate agar medium using the pour plate technique. pure cultures of isolated colonies of fermenting organisms (lab) on de man rogosa and sharpe (mrs) medium were obtained by incubating inoculated plates at 37 oc for 48 h under anaerobic conditions with successive streaking on plates and slants. the pure isolates were subjected to further characterization based on appropriate, cultural, morphological and biochemical tests to establish their identity. determination of ph and titratable acid the ph of the finger millet ogi samples during the primary and the secondary fermentation stage were determined at 12 h intervals for 48 h as described by [18] using a digital ph meter (hanna instruments, model hi 8314). the titratable acid (ta) of the fermenting finger millet grain during the primary and the secondary fermentation stage was determined by taking 10-ml sample and titrating against 0.1m naoh, using 2 to 3 drops of phenolphthalein as indicator until a faint pink color was observed. this was carried out at 37 oc for 48 h, with a 12 hourly monitoring. the titratable acidity was calculated as follows: % acid (w/w) = 𝑁×𝐸 ×𝐸𝑄 𝑊 × 100 where n= normality of titrant e= volume of titrant eq= equivalent weight w= mass of sample statistical analysis data obtained from the study were subjected to analysis of variance (anova) using a statistical package for the social sciences (spss). differences among means were separated using duncan’s multiple range test and significances accepted at 5% level (p≤0.05). total viable counts were expressed as means of three replicates. 3. results and discussion microbial association of spiced finger millet ogi samples table 1. microbial counts (cfu/ml) of spiced finger millet ogi samples sample tbc tfc tcc tlc fm 5.0 x 103 1.0 x 103 2.0 x 103 7.5 x 103 fgn1 8.0 x 103 18.0 x 103 12.5 x 103 t.n fgn5 8.0 x 103 3.4 x 104 6.0 x 103 7.0 x 104 fgn10 6.0 x 104 5.6 x 104 4.1 x 104 t.n fgr1 1.0 x 103 10.0 x 103 9.0 x 103 t.n fgr5 2.0 x 104 3.0 x 103 10.5 x 103 t.n fgr10 4.3 x 104 n.g n.g t.n values are means of triplicate measurement tbc =total bacterial count, tfc=total fungal count tcc=total coliform count, tlc=total lab count t.n = too numerous, n.g=no growth, fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% 10% ginger, fgr1 = 99% 1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 217 the growth kinetics of the microbial population investigated at 48 h of secondary fermentation stage as shown in table 1 ranged from 5.0 x 103 to 4.3 x 104 cfu/ml for total bacteria counts and 1.0 x103 3.0 x 103 for total fungi counts, while the values obtained for total coliform and lactic acid bacteria (lab) counts ranged from 2.0 x103 10.5 x 103 and 7.5 x103 70 x 103 respectively. this study revealed that the population of coliform and fungal organisms at this stage were relatively low compared to total bacteria and lactic acid bacteria count due to low ph of the slurry which could be partly responsible for the inhibition of the latter because most coliform organism cannot survive under low ph [19]. furthermore, the presence of gingerol and allicin in both ginger and garlic respectively could also contribute to the low coliform and total bacteria counts as also reported by [20]. total bacterial count was lower than total lab count possibly because lab are anaerobic organisms requiring a more fastidious medium for growth and may not be able to grow on a general medium such as plate count agar used for total bacteria counts. previous studies have reported the co-existence and symbiotic association of yeast and lab in the fermentation of ogi where they contribute to the flavor and aroma development [15, 21 – 22]. the morphological and biochemical characteristic of the purified representative isolates on mrs agar is presented in table 2. all the isolates were revealed to be catalase negative, gram positive, non-spore forming and rod shaped which characterizes lactic acid-producing bacteria. sugar fermentation pattern revealed that the isolates belong to the species of facultative heterofermentative lactobacillus. these were present throughout the fermentation process with their growth followed by simultaneous acidification of the product. table 2. morphological and biochemical characteristics of lactic acid bacteria isolates characteristics isolate 101 isolate 102 isolate 103 colony/cell morphology large, white spreading colonies, non-sporing rods small whitish distinct colonies, non sporing rods small creamy shiny distinct colonies, short medium rods gram’s reaction + + + motility non-motile non-motile non-motile catalase test _ _ _ oxidase test _ _ _ vogue proskauer _ _ _ methyl red test _ _ _ urease production _ _ _ nitrate reduction _ _ _ citrate utilization + + + growth at 30 o c + + + growth at 37 o c + + + growth at 45 o c growth in ftm f/a f/a f/a co2 production + + + mannitol utilization + + + galactose utilization + + + glucose utilization + + + lactose utilization + + + food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 218 fructose utilization + + + sucrose utilization + + + maltose utilization + + + probable identity lactobacillus spp lactobacillus spp lactobacillus spp notes: ftm, fluid thioglycolate medium, +positive, negative, f/a facultative anaerobic this relates well with the work of [23] which confirmed the presence of heterofermentative lab during ogi fermentation. the results obtained were also in accordance with the report of other authors [24 – 26] and were also referenced to bergey’s manual of modern bacteriology. changes in ph during fermentation of spiced finger millet ogi tables 3 to 6 show the changes in ph and tta during fermentation of spiced finger millet ogi. there was a general steady reduction in ph and simultaneous significant increase in tta during the 48 h steeping period. the trend was also sustained during the souring stage, the ph decreased gradually (p<0.05) from 5.98 to 4.01 at 10% garlic inclusion, while it decreased significantly (p<0.05) from 5.54 to 3.42 at 10% inclusion of ginger this might be as a result of the utilization of sugars and subsequent production of lactic acid by the fermenting organisms responsible for the fermentation of ogi [18], [27]. this observation also agrees with the reports of [5, 15, 28, 29]. low ph inhibits the growth and activities of many microorganisms and at the same time is a determinant of the type of microorganisms that grows and dominates the fermentation process for which lactic acid bacteria have been implicated to dominate [18. this activity which is also due to the high counts of lactic acid bacteria, accumulation of lactic acid and organic acids produced during the fermentation can contribute to the safety of finger millet ogi in addition to the activity of the added spices [18]. the degree of sourness attained is relative to individual consumer and determines the time of termination of the fermentation process. table 3. changes in ph during fermentation of spiced ogi at the steeping stage period of fermentation (hours) sample 0 12 24 36 48 fm 6.38a ±0.00 5.49ab ± 0.00 5.39a ± 0.00 5.19a ± 0.00 4.83ab ± 0.00 fgn1 6.23a ±0.10 5.50ab ± 0.10 5.50a ± 0.10 5.22a ± 0.10 4.79abc± 0.10 fgn5 5.86b ±0.10 5.40a ± 0.58 5.45a ± 0.10 4.96ab± 0.10 4.86a ± 0.10 fgn10 5.86b ±0.10 5.33a ± 0.10 5.00a ± 1.00 4.89ab ± 0.10 4.62c ± 0.10 fgr1 5.55 cd ±0.10 5.56a ± 0.10 5.28a ± 0.10 4.63b ± 0.44 4.66bc ± 0.10 fgr5 5.49 d ±0.10 5.51ab ± 0.10 5.43a ± 0.10 5.02a ± 0.10 4.77abc ± 0.10 fgr10 5.70bc ±0.10 5.49ab ± 0.10 5.41a ± 0.10 5.12a ± 0.10 4.79abc ± 0.10 notes: means with different superscripts on the same column are significantly different (p<0.05) values are means ±sd of triplicate measurement fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% 10% ginger, fgr1 = 99% 1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 219 table 4. changes in tta of spiced finger millet ogi at the steeping stage period of fermentation (hours) sample 0 12 24 36 48 fm 0.20a ± 0.00 0.20a ± 0.00 0.30c ± 0.00 0.80b ± 0.00 0.40b ± 0.00 fgn1 0.20a± 0.10 0.20a ± 0.10 0.30c ± 0.10 0.60c ± 0.10 0.50ab ± 0.10 fgn5 0.20a ± 0.10 0.20a ± 0.58 0.60a ± 0.10 1.00a ± 0.10 0.60a ± 0.10 fgn10 0.20a ± 0.10 0.20a ± 0.10 0.40bc ± 1.00 0.80b ± 0.10 0.60a ± 0.10 fgr1 0.20a ± 0.10 0.40± 0.10 0.50ab ± 0.10 0.60c ± 0.46 0.60a ± 0.10 fgr5 0.20a ± 0.10 1.50a± 0.10 0.40bc ± 0.10 0.50c ± 0.10 0.60a ± 0.10 fgr10 0.20a ± 0.10 0.30a ± 0.10 0.40bc ± 0.10 0.80b ± 0.10 0.60a ± 0.10 notes: means with different superscripts on the same column are significantly different (p<0.05) values are means ±sd of triplicate measurement fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% -10% ginger, fgr1 = 99% -1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic table 5. changes in ph during fermentation of spiced finger millet ogi at the souring stage period of fermentation (hours) sample 0 12 24 36 48 fm 5.36d ± 0.10 4.82bc ± 0.10 4.31bcd± 0.10 3.79c ± 0.10 3.40d ± 0.15 fgn1 5.70abc ± 0.10 4.66c ± 0.42 4.39abc ± 0.10 3.86c± 0.10 3.64c± 0.10 fgn5 5.60bcd ± 0.40 5.14a ± 0.01 4.26cd ± 0.10 3.82c± 0.12 3.59c ± 0.11 fgn10 5.54cd ± 0.12 5.19a ± 0.01 4.17d ± 0.01 4.00c± 0.05 3.42d ± 0.01 fgr1 5.89 ab ± 0.00 4.58c ± 0.00 4.25cd ± 0.10 4.09b± 0.10 3.89b ± 0.10 fgr5 5.92 ab ± 0.01 4.78bc ± 0.02 4.42ab ± 0.01 4.10b± 0.00 3.91b ± 0.12 fgr10 5.98 ± 0.01 5.07ab ± 0.01 4.50a ± 0.01 4.28c ± 0.10 4.09a ± 0.10 means with different superscripts on the same column are significantly different (p<0.05) values are means ± sd of triplicate measurement fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% 10% ginger, fgr1 = 99% 1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic table 6. changes in tta of spiced finger millet ogi at the souring stage period of fermentation (hours) sample 0 12 24 36 48 fm 1.90a ± 0.10 1.80cd±0.10 3.77e ± 0.15 6.20a ± 0.00 7.90b ± 0.10 fgn1 2.03a ± 0.06 1.73d± 0.53 3.90de ± 0.10 4.90c ± 0.10 8.40a± 0.10 fgn5 1.07b ± 0.15 1.70d± 0.10 4.30c ± 0.10 6.20a± 0.10 7.90b ± 0.10 fgn10 2.00a ± 0.10 1.90cd±0.10 4.70a ± 0.10 4.60d ± 0.10 8.40a ± 0.10 fgr1 1.20b ± 0.00 2.00c± 0.10 4.10cd ± 0.10 5.00c ± 0.10 6.70c ± 0.10 fgr5 1.20b ± 0.10 3.10b± 0.10 4.10cd ± 0.10 5.20b ± 0.10 5.60d ± 0.10 fgr10 1.10b ± 0.00 1.10b± 0.00 4.50b ± 0.10 5.30b ± 0.10 5.60d ± 0.10 means with different superscripts on the same column are significantly different (p<0.05) values are means ± sd of triplicate measurement fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% 10% ginger, fgr1 = 99% 1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 220 antimicrobial effect of ogi samples against pathogens. traditionally fermented foods are known to exhibit antimicrobial and health promoting properties which has been demonstrated indigenously by rural dwellers who administer raw ogi slurry to diarrhea patient [26, 29]. the ability of lab to produce organic acids, hydrogen peroxide and bacteriocins and other metabolites confer on them the potential to inhibit the growth of food-borne pathogens [29]. all the ogi samples showed clear zones of inhibition against the tested pathogenic organisms. the highest inhibition zone of 20.0 mm against e. coli was observed by 10% garlic spiced ogi (fgr10), while the control fm which is the un-spiced ogi gave the lowest inhibition zone of 5 mm against e.coli. incorporation of some spices to groundnut products significantly reduced the microbial load [30]. this result also corresponds with the findings of several authors who have reported antibacterial activities of garlic, ginger and lab isolates against pathogenic organisms [5, 25, 31]. the large clear zones of inhibitions observed for the spiced ogi is due to the result of the synergistic effect of the antimicrobial activity of the isolates and antibacterial properties present in the spices as spices have also been reported to inhibit microbial growth [32]. table 7. inhibitory effect of samples against selected pathogenic organisms diameter of inhibition zones in mm isolate code escherichia coli klebsiella pneumonia proteus mirabilis fm 5.0 5.0 10, fgn1 16.0 9.0 13.0 fgn5 18.0 9.0 12.5 fgn10 14.0 15.0 7.0 fgr1 18.0 11.0 6.0 fgr5 14.5 6.0 9.0 fgr10 20.5 9.0 13.0 fm = 100% finger millet, fgn1 = 99% -1% ginger, fgn5 = 95%-5% ginger, fgn10 = 90% 10% ginger, fgr1 = 99% 1% garlic, fgr5 = 95%-5% garlic, fgr10 = 90%-10% garlic 4. conclusion this study revealed that the inclusion of ginger and garlic contributed to the improvement of the organoleptic quality of finger millet ogi and also able to inhibit pathogens and spoilage microorganisms, thus ensuring safety of the product. the addition of spice in ogi and probably other food items would reduce the risk of food contamination, protect the consumer from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 221 different foodborne diseases and improve health status by using small quantities as low as 1%. the study of the fermentation dynamics of ogi will further facilitate the use of starter culture for a controlled fermentation and more so, enhance its process optimization for improved ogi. it is therefore important to allow cereals to achieve the right fermentation by attaining a considerable low ph in order to enhance the organoleptic qualities and also improve the safety of the fermented food. 5. conflict of interests the author(s) did not declare any conflict of interest. 6. references [1]. kennedy, m.m.o., grootboom, a., and shewry, p.r (2006). harnessing sorghum and millet biotechnology for food and health. journal cereal science. 44 (3), 224–235 [2]. upadhyaya, h.d., gowda, c.l.l., reddy, v.g (2007). morphological diversity in finger millet germplasm introduced from southern and eastern. africa journal agricultural resources, 3 (1), 1–3 [3]. dida, m.m., wanyera, n., dunn, m.l.h., bennetzen, j.l., and devos, k.m (2008). population structure and diversity in finger millet (eleusine coracana) germplasm. tropical plant biology. 1(2), 131–141. [4]. devi p. b., vijaya bharathi, r., sathyabama s., malleshi n.g., and priyadarisini v. b (2014). health benefits of finger millet (eleusine coracana l.) polyphenols and dietary fiber. a review journal food science technology. 51, 1021–1040 [5]. adisa, a. m., and ifesan, b. o (2016). probiotic potential of lactic acid bacteria (lab) isolated from wholegrain millet sourdoughs, annals. food science and technology. 17(2), 459463. [6]. adeleke, m.a., olaitan, j.o., abiona, o., canice, j, and olajide, s (2014). molecular characterization and antibiotic susceptibility patterns of bacteria isolated from wara (west african cheese) sold in osun state, nigeria. innov. romanian food biotechnol. 15, 2330 [7]. akinrele, i.a (1970). fermentation studies on maize during the preparation of a traditional african starch-cake food. journal of the science of agriculture. 21(12), 619-625. [8]. fasasi, o.s., adeyemi, i.a and fagbenro o.a (2007) functional and pasting characteristics of fermented maize and nile tilapia (oreochromis niloticus) flour diet. pakistan journal of nutrition. 6(4), 304309 [9]. ajanaku, k.o., ajani,o., siyanbola, t.o, akinsiku a.a, ajanaku, c.o and oluwole o (2013) dietary fortification of sorghum ogi using crayfish (paranephrops planifrons) as supplement in infancy. food science and quality management. 15, 1-9 [10]. odunlade, t.j., taiwo, k.a and adeniran h.a (2016) functional and antioxidative properties of sorghum ogi flour enriched with cocoa. annals. food science and technology. 17(2), 497-506 [11]. ojo, d.o and enujiugha, v.n (2016) physico-chemical properties, chemical composition and acceptability of instant ‘ogi’ from blends of fermented maize, conophor nut and melon seeds. journal food process technology 7:640.doi:4172/2157-7110.1000640 [12]. osungbaro, t.o (2009) physical and nutritive properties of fermented cereal foods. african journal of food science, 3(2), 23-27. [13]. soomro, a.h., masud, t., and awaar, k (2002). role of lactic acid bacteria (lab) in food preservation and human health, a review pakistan nutrition journal. 1, 20-24. [14]. eidi a, eidi m, and esmaeili e (2006). antidiabetic effect of garlic (allium sativum l.) in normal and streptozotocin-induced diabetic rats. phytomedicine. 13(9-10), 624-9. [15]. odunfa, s.a., and adeyele, s (1985). microbiological changes during the traditional fermentation of ogi-baba, west african fermented sorghum gruel. journal of cereal science. 3, 173-180. [16]. sanni ai, asiedu m, ayernor g.s (2001). influence of processing conditions on the nutritive value of ogi-baba, a nigeria fermented sorghum gruel. plant food hum. nutr. 56(3), 217223 [17]. omemu a.m and fanman o. w (2011) assessment of the antimicrobial activity of lactic acid bacteria isolated from two fermented maize productsogi and kunun-zaki. malaysian journal of microbiology, 7(3), 124-128 [18]. adesokan, i.a., avanrenren, e.r., salami, r.t., akinlosotu, i.o. and olayiwola, d.t (2008). management of spoilage and pathogenic organisms during food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 abosede m. adisa, adefisola b. adepeju, ayowunmi k. yusuf, influence of ph and acidity on the fermentation of finger millet spiced ogi, food and environment safety, volume xviii, issue 3 – 2019, pag. 214 – 222 222 fermentation of nonoan indigenous fermented milk product in nigeria. journal of applied bioscience. 11, 564-569. [19]. savadogo, a., ouattara, c.a., bassole, i.h., and traore, s.a. (2006). bacteriocins and lactic acid bacteriaa mini review. african journal biotechnology, 5(9), 678-683. [20]. kolapo, a.l., popoola, t.o.s., sanni, m.o., and afolabi, r.o. (2007). preservation of soybean daddawa condiment with dichloromethane extract of ginger. research journal of microbiology. 3, 254-259. [21]. oyewole, o.b (1997). lactic acid fermented foods in africa and health benefits. food control. 8, 5-6 [22]. omemu, a.m., oyewole, o.b., and bankole, m.o., (2007). significance of yeasts in the fermentation of maize for ogi production. food microbiology. 24, 571-576. [23]. akinleye, o.m., fajolu,i.o., fasure, a.k., osanyinpeju, o.s., aboderin, a.o., and salami.o.o (2014) evaluation of microorganisms at different stages of production of ogi in alimosho community, area southwest, lagos, nigeria. american journal of research communication. 2(10), 215-230 [24]. thamaraj, n. and n.p. shah, 2003. selective enumeration of lactobacillus delbrueckii ssp. bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, bifidobacteria, lactobacillus casei, lactobacillus rhamnosus and propionibacteria. journal dairy research, 86, 2288-2296. [25]. daka, d. (2009). antibacterial effect of garlic (allium satium) on staphylococcus aureus. an vitro study. african journal of biotechnology. 10 (4), 666-669 [26]. estifanos hawaz, (2014). isolation and identification of probiotic lactic acid bacteria from curd and in vitro evaluation of its growth inhibition activities against pathogenic bacteria. african journal of microbiology [27]. omemu, a.m. (2011) fermentation dynamics during production of ogi, a nigerian fermented cereal porridge. report and opinion. 3(4), 8-17. [28]. adebolu, t.t., olodun, a.o. and ihunweze, b.c (2007) evaluation of ogi liquor from different grains for antibacterial activities against some common diarrhoeal bacteria in southwest nigeria. african journal of biotechnology. 6 (9), 1140-1143 research. 8(13), 1419-1425 [29]. abdus-salaam, r.b., adepoju, p.a., olaleye o.n., and adeoye, i.a., (2013). the antimicrobial effect of corn steep liquor on some diarrhea causing organisms. african journal of biotechnology. 13(2), 332-335 [30]. adegunwa, m.o, alamu, e.o. bakare, h.a and godwin, p.o (2011). effect of fermentation length and varieties on the qualities of corn starch (ogi) production. american journal of food nutrition, 1(4) 166-170 [31]. olayiwola, j.o., inyang, v. and bello, m.a. (2017). bacteriological and proximate evaluation of ginger-fortified fermented maize (ogi). american journal of food technology, 12: 374-378 [32]. amusa, n.a., ashaye, o.a and oladapo, m.o (2005). microbiological quality of ogi and soyogi (a nigerian fermented cereal porridge) widely consumed in southern nigeria. journal of food, agriculture & environment, 3(2), 81-83 385 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 4 2018, pag. 385 394 the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization *elias chaidoutis1, dimitrios kostakis1 hellenic food authority directorate of training, communication & it. athens, greece, echaidoutis@gmail.com, *corresponding author received 8th november 2018, accepted 28th december 2018 abstract: the greek government among other reformative measures in the field of public administration promotes the public sector rationalization in order to be managed more effectively and more efficiently in view of the strict fiscal policy which is currently being applied in greece. in the context of the state transformation, the ministry of rural development and food proceeds to the organizational reorganization of the supervising bodies. hellenic food authority (efet) since its establishment up to nowadays has significantly contributed to the achievement of its mission through the training of food businesses staff. this article reports with conciseness the efet work that concerns training subjects of food businesses staff before the modern revision of the authority organization. the efet administration proceeded to the approval of the revised authority organization with purpose of its more effective function, continuing to offer its training services to the new configuration aiming to the protection of greek consumers in the food sector. keywords: training, food business staff, food safety, trainer registry, reorganization, hellenic food authority. 1. introduction the european policy evolution for food safety european citizens must have access to safe and healthy foods which meet the highest strong specifications [1,2]. food legislation has been a major issue of public interest within the european union. effective food control as well as achievement of a high safety level constituted an aim which can be achieved through assurance of food hygiene and safety as well as the effective protection of the interests of european consumers [3]. nowadays, the european union (eu) constitutes a global example for defining high specifications for food safety [1]. most of 100.000 adequately trained inspectors who have specialized inspection capabilities control with impartiality and independence 25 million food businesses annually throughout length of the agro-food chain on the basis of farm-to-table policy to guarantee the safety approximately 500 million eu consumers. the european union (eu) tried to capture the conversation about the foundations of food legislation. thus, in 1997, eu published the green paper which goes after the completeness of legislation over the expectations of european consumers and food businesses, the principles of impartiality, independence and effectiveness of official food control in order to provide healthy and safe food on the market [3]. also, the aim of the green paper was the submission of proposals on behalf of the european commission to receive suitable measures for the development of the eu legislation for food. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 386 table 1. key objectives of the green paper on eu food legislation. source: european commission, 1997 2. the scientific approach in the food sector and the establishment of competent authorities in order to put foundations of scientific approach in the food sector in eu level, european parliament issued on 25th february 1993 the directive 93/5/eec with aim to provide assistance towards member states and their cooperation with european commission over scientific evolution and examination of food-related issues [4]. concerning the enactment of competent authorities which can cooperate and assist commission for food sector, receiving of necessary measures on behalf of member states is predicted by the directive. specifically, particular importance is given to the public health protection through cooperation of scientific disciplines that associated with food sector. the scientific disciplines which are involved in the eu directive are branches of medicine, nutrition, health, biology, toxicology, food technology, chemistry etc. [4]. as regards the content of cognitive object, the distribution of experts for the application of haccp principles is identical at any stage of food chain is required [5,6]. each member state should define a specific responsible authority for the effective function of the cooperation procedure. in greece, the responsibilities that related to the food sector were being distributed to different bodies and exerted separately depending on scientific character of body. particularly, the ministry of agriculture (today, it is called ministry of agricultural development and food) was responsible for the primary production and the animal and vegetable foods, the ministry of health was responsible for public health and hygiene issues, the ministry of development was competent for industry and the ministry of finance (general chemical state laboratory) that was responsible for the 1. ensuring a high level of public health and consumer safety 2. ensure free movement of goods in the internal market 3. ensure legislation based on risk assessment and scientific data 4. ensuring the competitiveness of the food industry and enhancing exports 5. set primary responsibility for food business operators and use haccp principles 6. ensure coherent and rational legislation “from the stall to the table” food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 387 conduct of laboratory tests of chemical factors [7]. different facts in the late 1990s which concerned food have shown that, there is a need for enactment of general principles and rules for food and feed at eu level. in this context, the european commission designed a completed approach in the food safety “from farm to table” which is illustrated in the white paper for food safety. this approach covers all the food chain sectors such as feed production, food processing and storage as well as transport and retail sale [1]. as continuation of the green paper of european commission for food legislation, the current legislative framework is proposed through the white paper (general food safety legislation, food hygiene package, official controls) [2]. 3. efet establishment and its enactment as competent authority to the food sector in greece the greek government with application of eu common perception in the food sector established hellenic food authority (efet) under act no. 2741/1999 [8]. the basic purpose of efet is the consumer protection and the translucence reinforcement [7,8]. all responsibilities of ministries and organizations that is associated with food sector and separately exerted are accumulated in efet [9] by aiming the hygiene assurance and concurrently the protection of consumers. similarly, the distribution of efet staff is come from the whole range of scientific disciplines that related with food sector [10]. the bodies (public services, ministries etc.) from whom efet responsibilities come from become co-competent, their executive authorities take part in configuration of policy for food safety [7,10,11] and the control authorities remain in their responsibilities area by participating in the single framework of official controls on the basis of the planning of the competent authority [10,12]. efet has exclusive responsibility for determining of food quality specifications, the accomplishment of official control and the enforcement of sanctions. the above fact doesn’t affect the mission of ministries and services that already possessed the relevant responsibilities because their executive authorities become co-competent bodies to the coconfiguration of policy for the food controls through national control policy council for food. the authority was developed at central executive level and regional control level [7] specific aims of efet are the prosecution of businesses to food businesses, the provision of technical assistance such as the training of businesses staff as well as the communication with consumer with purpose its training about food safety issues as well as its information [12]. 4. executive role of training, communication and information technology directorate. efet in addition to fulfillment of inspections is responsible for assisting in food businesses at technical level such as training of businesses staff and inspectors and communication with consumers by aiming to their training on food safety issues and their information on efet responsibility issues [7,9]. considering the factors which contribute to the achievement of the efet goals, directorates with executive character are created in its central service (figure 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 388 fig.1 efet development according to its initial organization (pd 223/2000). source: hellenic food authority, 2000 the creation of training, communication and ιτ directorate had the following targets: i. the fulfillment of responsibilities that concern the staff training of executive authorities in order to achieve a uniform accomplishment of inspections and the cultivation of interdisciplinary cooperation. ii. the training provision to the food businesses staff by reinforcing the effectiveness of market operation and the contribution to the health protection of consumers. iii. the information provision to the consumers with aim its education about nutrition, safety and other issues that is relevant with efet responsibilities. the training, communication and information technology directorate were the competent service for continuous training of the efet staff and the representatives of professional branches, the communication with consumers, food businesses and professional branches. the directorate included the training, communication and information technology and computerization departments [9]. the training department has been responsible for devising of the necessary training programs for the representatives of food professional food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 389 branches, the efet staff and the keeping of relevant records. the communication department was competent for food businesses information and any other interested body about the current legislation on food control issues and information of consumers on efet responsibility issues through newsletters, multimedia and any other appropriate media. finally, information technology department had responsibilities which are related to informatics (software and hardware). in order to fulfill its mission and by amending its founding law under no. 2741/1999 (law 3438/2006, issue a, official government gazette, 33/14.02.2006), efet extends its competence with the possibility to develop compulsory training programs for food business staff and the granting of a certificate of successful follow-up of these programs after the evaluation of the trainees by a three-member committee of efet employees, who are defined by decision of its board of directors [13]. following the authorization given by law, the responsible development minister issued the 14708/2007 ministerial decision (official government gazette, issue b, 1616/17.08.2007) for the determination of the terms and conditions for the preparation and approval of the compulsory training programs of food businesses staff which belong to the efet responsibility [14]. the same ministerial decision arrange the terms and conditions of training of co-responsible controls authorities and the bodies that exert official control to food such communal legislation defines [15,16]. efet proceeded to the constitution of trainers’ registry from which the trainers should be selected by interested implementation body of training, has developed a training package with a basic training manual, slides and audiovisual material that is exclusively used by the registered trainers who take part in efet programs in order to ensure homogeneous overall and integrated training of food businesses staff [17]. 5. efet training actions for the food businesses staff training which concerns the food hygiene is an objective with fundamental importance for the operation of food businesses [18]. unsuitable practices of handling can provoke food contamination resulting in appearance of food-borne diseases and harm to the health of consumers [5,19]. staff must know its role about the food protection from contaminations. food handlers must have necessary knowledge and skills so that they can handle food with a sanitary acceptable manner [18,20]. the staff training is legal requirement of regulation (ec) 852/2004 [14,21]. according to codex alimentarius, food handlers are considered persons who ‘’handle directly packaged or unpacked food, food equipment and utensils or surfaces in contact with food’’ [5]. the directive 93/43/1993 is issued by european communities council in order to be regulated issues about the food hygiene [22], this directive predicted the training of food handlers depending on executed works. greek government has complied with directive by adopting joint ministerial decision 487/2000 for the food hygiene. by 2004, efet takes care of the training of food businesses staff through training actions (e.g. training of food handlers which are volunteers in the limits of the olympic games, training of catering facilities staff of hoosegow etc.) [12] by using of training material (training manual and video) which includes 62 knowledge food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 390 assessment questions with 5 thematic areas about food hygiene (table 2) according to joint ministerial decision 487/2000 [23]. table 2. knowledge assessment questions per thematic area of basic training manual on the food hygiene. source: hellenic food authority, 2004 the assessment writing examinations took place at implementation place by the program executor after the end of the procedure, by using questions from the basic training manual and the relevant documents are sent to the efet after the program completion. by 2006, the number of trained food handlers was estimated at 56,100 [11]. after the adoption of ministerial decision with number 14708/2007 which regulates the terms and conditions for development and approval of compulsory training programs for food businesses staff the procedure takes different direction [14]. the aim of training programs is the quality assurance and the production of safe products for the consumer protection. the certification is given to food handlers who will successfully complete their assessment to the examinations which are organized and conducted by efet [14]. training helps to the accomplishment of regulation (ec) 852/2004 requirement for the food hygiene. this regulation predict that, food businesses managers ensure training concerning food hygiene for those who handle food within their limits businesses as well as those who are responsible for training and maintenance of procedures based on the haccp principles [21]. the procedure is applied to the businesses staff who processes, stores, transports, distributes food or deals with retail, food sale, mass catering, confectionery or is a food production, processing lab with small capacity. excluded from the compulsory character of participation in training programs of efet are the businesses that apply the haccp principles to full development due to reasons of hazard control in the limits of their responsibility [14]. also, after modification of ministerial decision ordinances, excluded from the procedure is the staff that has a certificate which clearly shows that the food handler is adequately trained in food safety and hygiene matters [24]. the least content which is approved by efet as well as the terms and conditions that are put in the context of adults’ training are followed for the questions thematic areas measurements number microbes world 14 food contamination and food poisoning 16 food safety-supplies/receiving 10 keeping food safe 11 safe preparation before serving 11 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 391 accomplishment of training programs. the programs can last for 8 to 12 hours and after the completion of program, the participators food handlers take part in examinations for their knowledge assessment [14]. from the beginning of new training framework for the food businesses staff to 2012, the total of participators food handlers in the efet writing examinations is come up to 2148. specifically, for the year 2012, became 87 training programs in which 1180 food handlers participated from 360 food businesses throughout greece [25]. the last numerical data which are cited by efet show that the number of trainees exceeds 60000 food handlers [26]. 6. registry of the food business trainers taking into account the legislation for hygiene and safety of food, the national policy and european strategy for the training of involved in food chain, efet structured a registry of trainers according to the article 11 of the law 3438/2006 with purpose the improvement of the training system, the quality assurance of provided training services and the enhancement of training force through programs of food businesses staff, authorities and control bodies [17]. the meaning of training for staff that is involved in the production and control of food as well as the trainer who should have typical and substantive qualifications which are required for carrying out of the specific work or profession as well as the necessary knowledge and skills for the approach of trainees are determined by adopting of the ministerial decision 14707/2007 which is authorized by the above law. also, administrative matters about the provision of training work by civil servants who are enrolled in the registry as well as matters concerning conflict of interests are defined in ministerial decision ordinances [17]. the registry of the food businesses trainers includes information about the typical qualifications and the professional and teaching experience of the trainers and their additional information (pedagogical training, participation in other training programs etc.). the registry is accepted registrations by persons who possess diploma that is relevant with the food sector (figure 2). fig.2 registered in the register of trainers of food businesses staff per professional field. source: hellenic food authority, 2018 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 392 therefore, the conditions for interdisciplinary approach in the staff training food sector are created. currently, about 900 registered trainers from various specialties are found in the registry of trainers of food businesses staff [27]. 7. modern data on the structure of efet training services greek government, among others reformative measures promoted in the field of public administration, promotes the rationalization of the public sector so that it can be managed more effectively and efficiently in view of strict fiscal policy implemented in conjunction with the accomplishment of the stability program [28]. in the context of the transformation of reformative actions that have been decided by the greek government for each ministry, the organizational reorganization of the state services is included as a basic purpose. following the above government policy, the efet boards of directors proceeded in the approval of the revised efet organization and its mission under the supervision from ministry of agricultural development and food. subsequently, with the publication of presidential decree number 71/2018, efet acquired a new organizational chart [29,30]. according to the revised plan of the efet organization, the body services have changed with the creation and abolition of organic structures as well as the scientific disciplines of the food sector that assist the body works. from the beginning of the efet new organization force, the training, communication and information technology directorate is abolished and its services are reshaped. the reformative training department is under the new directorate for risk assessment and nutrition [30]. as regards, the training of food businesses staff, at the body mission, among others is included the care of continuous training of food businesses staff according to the requirements of food legislation. at the new training department remains the issue of circulars and guidelines for the definition of the training programs content and the responsibilities about the approval process of the training programs for food businesses staff, the maintenance of implementation terms and conditions as well as the good performance control of the training programs of the food businesses staff, the conduct of the evaluation procedures for the trained food operators and the issue of the relevant certificate in the administrative and financial services directorate (human resources department) [30]. the accomplishment of the training programs for business staff falls under the general directorate, since it has not been included in any particular organizational unit. 8. conclusions food in the european union should be safe and healthy. community legislation includes a total of rules which guarantee the achievement of this aim. member states must apply the food legislation, attend and verify if relevant requirements are observed by responsible of businesses at all production, processing and distribution stages of food through the official controls [15, 16]. concerning food safety, the people behavior is an important aspect of food businesses management and bodies which configure the public policy due to epidemic outbreaks of food-borne explanation occurrences which are due to inadequate food handling [5,31]. hellenic food authority due to its responsibilities food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 393 plays a significant role in the configuration of the policy for food safety with determination of terms and conditions for the effective training of the food businesses staff [7]. following the publication of the 71/2018 presidential degree in the government gazette, the new organization of efet has been put in application. the efet reorganization is expected to offer more possibilities to the provision of its training services on the sector of food businesses staff as well as correct inexpediencies which are created and detected since its establishment up to nowadays with the aim to protect the greek consumer’s health in the food sector [29]. however, time is required for the application of new organization in order to demonstrate its adequacy in comparison with objective needs of the food businesses, to cover gaps and clarify unclear ordinances about its services responsibilities in the sector of the food businesses staff training [30]. 9. conflicts of interest this article presents part of the work of the authors in the training, communication & technology directorate of the hellenic food authority and also their critical view. publishing of this article does not imply acceptance of the author's opinions on behalf of the organization. the authors declare no conflict of interest. 10. references [1]. european commission. food safety: overview food safety (2018). https://ec.europa.eu/food/overview_en (accessed may 21, 2018). [2]. european commission. white paper on food safety. brussels, (1999). [3]. european commission. the general principles of food law in the european union: commission green paper. luxembourg: office for official publications of the european communities, (1997). [4]. european commission. council directive 93/5/eec of 25 february 1993 on assistance to the commission and cooperation by the member states in the scientific examination of questions relating to food; l 052:0018–21, (1993). [5]. codex alimentarius j. general principles of food hygiene cac/rcp 1-1969, rev. 4-2003:23, (2003). [6]. european commission. sanco/1955/2005 guidance document on the implementation of procedures based on the haccp principles, and on the facilitation of the implementation of the haccp principles in certain food businesses, (2005). [7]. hellenic parliament. “hellenic food authority and other matters of competence of the ministry of development”, (1999). [8]. hellenic republic. law no. 2741/1999. “hellenic food authority, other affair of competence of the ministry of development and other provisions”, (1999). [9]. hellenic republic. presidential decree no. 223/2000. “hellenic food authority’s organization” 2000. [10]. hellenic republic. joint ministerial decision no 052/2004 “efet co-operation with public authorities and agencies”, (2004). [11]. hellenic food authority. hellenic food authority, ten years of efet leaflet, (2009). [12]. hellenic food authority. hellenic food authority. annual report, (2006). [13]. hellenic republic. law no. 3438/2006 «regulation of ministry of development issues», (2006). [14]. hellenic republic. ministerial decision no.14708/2007. “terms, conditions, and procedure for the implementation of compulsory education and training of food business personnel and official control authorities”, (2007). [15]. eu. regulation (ec) no 882/2004 of the european parliament and of the council of 29 april 2004. core eu legislation, london: macmillan education uk; p. 288–318. doi:10.1007/978-1-13754482-7_27, (2004). [16]. eu. regulation (eu) 2017/625 of the european parliament and of the council of 15 march 2017 on official controls and other official activities performed to ensure the application of https://ec.europa.eu/food/overview_en food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 elias chaidoutis and dimitrios kostakis, the contribution of hellenic food authority to the training field of food businesses staff from the authority establishment to its modern reorganization, food and environment safety, volume xvii, issue 4 – 2018, pag. 385 394 394 food and feed law, rules on animal health and welfare, plant health and plant protection products, amending regulations (ec) no 999/2001, (ec) no 396/2005, (ec) no 1069/2009, (ec) no 1107/2009, (eu) no 1151/2012, (eu) no 652/2014, (eu) 2016/429 and (eu) 2016/2031 of the european parliament and of the council, council regulations (ec) no 1/2005 and (ec) no 1099/2009 and council directives 98/58/ec, 1999/74/ec, 2007/43/ec, 2008/119/ec and 2008/120/ec, and repealing regulations (ec) no 854/2004 and (ec) no 882/2004 of the european parliament and of the council, council directives 89/608/eec, 89/662/eec, 90/425/eec, 91/496/eec, 96/23/ec, 96/93/ec and 97/78/ec and council decision 92/438/eec (official controls regulation)text with eea relevance. 2017:142, (2017). [17]. hellenic republic. ministerial decision no.14707/2007. “hellenic food authority’s register of trainers", (2007). [18]. chaidoutis elias. the new guide to good practice for confectioners. an instrument to aid food business operators with compliance with european food law. e-journal of science & technology (e-jst), 12 (3):83–7, (2017). [19]. zanin lm, da cunha dt, de rosso vv, capriles vd, stedefeldt e. knowledge, attitudes and practices of food handlers in food safety: an integrative review. food research international, 100:53–62, (2017). doi,: 10.1016/j.foodres.2017.07.042. [20]. hellenic food authority. good practice guide for confectioners, (2016). [21]. regulation (ec) no 852/2004 of the european parliament and of the council. core eu legislation, london: macmillan education uk; p. 183–6. doi:10.1007/978-1-137-54482-7_19, (2004). [22]. the council of the european communities. council directive 93/43/eec of 14 june 1993 on the hygiene of foodstuffs, (1993). [23]. hellenic republic. joint ministerial decision no.487/2000 “food hygiene in compliance with council directive 93/43 / eec”, (2000). [24]. hellenic republic. ministerial decision no 439 /2017 “modification of ministerial decision no.14708/2007, terms, conditions, and procedure for the implementation of compulsory education and training of food business personnel and official control authorities”, (2017). [25]. hellenic food authority. annual report 2012. athens, greece: hellenic food authority, (2012). [26]. hellenic food authority. annual report 2016. athens, greece: hellenic food authority, (2016). [27]. hellenic food authority. food business staff training register of trainers (2018). http://www.efet.gr/portal/page/portal/efetnew/autho rities_control/control_auth_training/registers. (accessed may 21, 2018). [28]. hellenic republic ministry of administrative reconstruction. organization of public services. athens, greece, (2018). [29]. hellenic food authority. approval of the new hellenic food authority agency, (2018). [30]. hellenic republic. presidential decree no. 71/2018, “hellenic food authority’s organization”, (2018). [31]. who. five keys to safer food manual, geneva, switzerland, (2006). www.who.int/foodsafety/consumer/5keys/en/index. html (accessed july 21, 2018). http://www.efet.gr/portal/page/portal/efetnew/authorities_control/control_auth_training/registers http://www.efet.gr/portal/page/portal/efetnew/authorities_control/control_auth_training/registers http://www.who.int/foodsafety/consumer/5keys/en/index.html http://www.who.int/foodsafety/consumer/5keys/en/index.html 180 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 32020, pag. 180 191 modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction *linda loucif seiad, soraya demim département de chimie, faculté des sciences, universitém'hamed bougara, avenue de l'indépendance, 35000, boumerdès, algérie. *loucifsiad_linda@univ-boumerdes.dz received18th may 2020, accepted 28thjuly 2020 abstract: the aim of this study is to improve extraction of biologically active compounds (bacs) found in rosmarinus officinalis l. from algeria country using green extraction process. the effect of the main process variables (time, ethanol concentration, solid-to-liquid ratio) on maceration efficiency has been studied using factorial design (fd) to verify the single factors effects. total polyphenol content (tpc) and total flavonoids content (tfc) were measured to control the maceration efficiency in different experimental conditions. the iso-response curves and the response contours have been exploited. the best experimental results were 0.15 g/ml, 50 %, 1hour and 0.15 g/ml, 50 %, 72hours for tpc and tfc respectively. the values of tpc and tfc were 2.5 mg gae/g dw and 1 mg qe/g dw respectively. these results were in perfect agreement with the expected theoretical model. the relationship between the tpc and tfc provides a high correlation coefficient (0.82). keywords: rosmarinus officinalis l., bacs, green extraction, modelling. 1. introduction biologically active compounds (bacs) are known to have large properties. they can be used in food industry as antioxidants, coloring agents, preservatives, fortifiers, etc.[1-3]. bacs sources are essentially from mediterranean aromatic plants [4], [5]. the targeted plant on this study, which is rosemary represent, one of them. it was reported that bacs products in rosemary have antifungal activity as well as antithrombotic, anti-inflammatory, antiulcerogenic and antidepressant effects [6]. according to the european medicines agency recommendations, rosemary essential oil (reo) can be used for many medical purposes. it can be used for peripheral circulatory disorders, alleviate muscular pain help, and dyspepsia [7]. it was also reported the use of rosemary extracts (re) and reo for commercial purpose as a natural food biopreservative. it is important to know that the antioxidant property is closely linked to phenolic acids and flavonoids [7-9]. they are the major classes of compounds with antioxidant activity among other vitamins (c and e). these natural antioxidants can be extracted from plants and substitute synthetic antioxidants, source of toxicity [5]. many extraction techniques from plants are used: soxhlet extraction, maceration, ultrasoundassisted extraction, heated reflux extraction, accelerated supercritical fluid extraction and microwave-assisted extraction. maceration technique is chosen because frequently it gives good results taken into account the principles of green chemistry. http://www.fia.usv.ro/fiajournal mailto:loucifsiad_linda@univ-boumerdes.dz foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 181 some parameters must be taken into consideration such as time, solid-to-liquid ratio, solvent, etc. and even the plant itself [10]. optimizing the extraction technique in the bacs is the most important step. this is due principally to the plant matrices complexity and different physico-chemical properties of bacs. another more rigorous aspect is the use of green chemistry. after having published the twelve principles of green chemistry and green engineering [11], [12], six new principles of green extraction were established [13]. among these principles, alternative solvents were used (water or agro-solvents). the present study investigates and discusses the valorization of algerian plants rosmarinus officinalis l. by improving the bacs extractions using an agro-solvent, taking into consideration the green extraction process. the lack of papers available about the plant used in this work has highly motivated this study. factorial design (fd) was used to verify the single factors effect’s (time, solvent concentration, and solid-to-liquid ratio) on extraction efficiency. tpc and tfc were measured to control the efficiency of maceration at different experimental conditions conducting to determine the interactions between those factors. the isoresponse curves and the response contours have been explored to collect the maximum amount of data. 2. matherials and methods 2.1. characterization of plant matrix rosmarinus officinalis l. was collected in algiers (algeria) during the month of march. the leaves were removed from the stems and have been dried at room temperature for three weeks in order to preserve the maximum integrity of their molecules. the dried leaves were crushed into fine particles and ground to powder using a mortar then passed through a 1.12 mm sieve. the powder was stored in a cool and dark place, protected from light and moisture for subsequent use. 2.2. reagents and chemicals folin–ciocalteu reagent, gallic acid and quercetine were purchased from sigma– aldrich, steinheim, germany). etoh of 96% purity (rieldel-de haen, germany), sodium carbonate (bhd chemicals ltd, england) and aluminum chloride (merck, germany) were used. 2.3. analysis 2.3.1. total polyphenols content tpc was determined by folin-ciocalteu according to singleton et al. [14] expressed as gallic acid equivalents (mg gae/g dw). absorbance was measured at 760 nm on a spectrophotometer (uv-vis secomam s250, france). the results were calculated using a standard gallic acid curve of (1–30 μg/ml). 2.3.2. total flavonoids content tfc estimated was determined by colorimetry according to the miliauskas method [15], expressed as quercetin equivalents (mg qe/g dw). absorbance was measured on a spectrophotometer (uv-vis secomam s250, france) at 430 nm. the results were calculated using a standard curve of quercetin (1 – 40 μg/ml). 2.4. experimental design and statistical analysis fd was used to study the effects of three variables: extraction time (x1), ethanol concentration(x2) and solid-to-liquid ratio (x3). the low and high levels for each factor in the experimental design are given. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 182 the responses (tpc and tfc) were expressed individually as a function of independent variables. the experimental design presented nine (9) combinations, including three replicates of the central point in order to estimate pure error and to assess the lack of fit for the proposed models. all experiments were performed randomly. a first-order polynomial equation (eq.1) was used to express tpc (y1) and tfc (y2) of rosemary as a function of independent variables asfollow: yi = β0+ β1 x1 + β2 x2 + β3 x3 + β12 x1x2+ β13 x1x3+ β23 x2x3 (1) whereyi represents the variables response, β0 is the model constant, β1, β2 and β3 are the linear coefficients.β12, β13 and β23 are the interaction coefficients respectively. x1, x2 and x3 are the levels of the independent variables. the model appropriateness was also evaluated by the coefficient model (r2) and adjusted coefficient model (r2adj) and also by the statically significant model (p-value model) properly tested by anova method. 3. results and discussion 3.1. choice of solvent in order to use environmentally friendly methods, the use of water ethanol and agro-solvents has been encouraged [16], [17]. however, using pure ethanol could dehydrate the vegetable cells, making the bacs diffusion process difficult from the plant material to the extracting liquid. a combination of ethanol/water is then used in the actual study in order to adjust the polarity [18]. 3.2. fitting model three factors that may affect the experimental responses were selected as independent variables at three levels. table 1 summarizes the minimum, central and maximum values for each factor and the different independent variables of the extracts. the experiments were performed according to the design of experiments shown in tables 2. observed and predicted responses on two levels are shown on the same table. statistical analysis of experimental data was performed by using "modde 6" software [19]. table 1. coded/ real levels used in factorial design of algerian rosmarinus officinalis l. maceration. coded levels -1 0 +1 time (hours) (x1) 1 36.5 72 ethanol concentration (%) (x2) 50 65 80 solid-to-liquid ratio (g/ml) (x3) 0.06 0.105 0 .15 the results obtained indicate that the tpc levels ranged from 1.35 mg gae/g dw to 2.5 mg gae/g dw confirmed the influence of the studied parameters (time, ethanol concentration, solid-to-liquid ratio). the best results were observed in run 5 at 2.5 mg gae/g dw followed by 2.4 mg gae/g dw in run 6. the tfc levels ranged from 0.45 mg qe/g dw to 1 mg qe/g dw. the best results related to the lower levels were observed in run 8 at 1 mg qe/g dw followed by 0.83 mg qe/g dw in run 1. these results indicate the presence of low content of polyphenols. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 183 table 2. factorial design of three variables with their experimental and predicted responses of algerian rosmarinus officinalis l. maceration. run coded variable tpc (mggae/g dw) tfc (mgqe/g dw) x1 x2 x3 experimental predicted experimental predicted 1 -1 -1 -1 1.80 1.82 0.83 0.77 2 +1 -1 -1 1.65 1.66 0.49 0.49 3 -1 +1 -1 1.35 1.34 0.45 0.47 4 +1 +1 -1 1.60 1.62 0.59 0.59 5 -1 -1 +1 2.50 2.5 0.80 0.81 6 +1 -1 +1 2.40 2.3 0.75 0.73 7 -1 +1 +1 1.76 1.78 0.63 0.63 8 +1 +1 +1 2.15 2.14 1.00 0.99 9 0 0 0 1.93 1.91 0.70 0.69 10 0 0 0 1.90 1.91 0.69 0.69 11 0 0 0 1.92 1.91 0.68 0.69 it should be noted that some research have shown lower phenolic contents [20-22]. further researches have given higher values [23]. they pointed out that the extract composition changes according to many factors (type of sample, location, and time... etc.). other work has shown the influence of plant matrix and solvent [24]. oliveira et al. have found that optimum yield reaches 89.8% using a hydroalcoholic solution of ethanol-water with 70% v/v, liquid-to-solid ratio of 5 ml/g and 55 min [25]. tpc levels range from 4.58 mg gae/g dw to 28.06 mg gae/g dw for 0% etoh at ph 2. bucickojić et al. have found similar results with 50% etoh [26]. table 2 shows that experimental conditions, which give an extract with the best tpc (run 5); do not give the highest tfc. this is the reason to studied both tpc and tfc at the same time. the results also show that experimental data are closely to the predicted values. the estimate goodness of fit results for all responses analyzed by anova variance analysis, have been summarized in table 3. the data indicated that all the final models resulting from anova analysis in terms of coded variables were significant at 95 % confidence level, with all p-values of regression ≤ 0.05 (from 0.0397 to 9.38 10-10 and 0.03011 to 5.957 10-9, respectively for tpc and tfc). lack of fit (lof) were higher than 0.05. fig. 1(a) for tpc effects indicates that solid-to-liquid ratio has a higher effect. it is the most important factor (positive effect) followed by an important effect of ethanol concentration (negative effect) and by the interaction of ethanol concentration and time (positive effect). interaction of solid-to-liquid ratio and time shows weak effect. regarding the tfc shown in fig. 1 (b), the interaction of ethanol concentration and time has a high effect. it is the most important factor. on the other side, ethanol concentration and time show weak effect. the reproducibility and models validity foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 184 values of tpc and tfc show a good agreement for all the responses. solid-toliquid ratio effect is positive for tpc and tfc because decreasing the solvent proportion increases the concentration gradient, which consequently leads to an increase in the diffusion of solid compounds in the solvent. the effect of ethanol concentration is negative in this case. there has to be a compromise between the amount of ethanol and water. therefore, increasing ethanol concentration induces consequently a decrease in maceration time. other research has shown that tpc yield increases with the concentration of ethanol, up to 50% the tpc yield decrease [27], [28]. on the other hand, time showed a weak effect because maceration process is carried at room temperature. a longer process causes phenolic compounds degradation [29] and this is not the case here. the fitted mathematical models (y1 andy2) for tpc and tfc with 95% coefficient level were given in eq. 2 and eq. 3 respectively. the values of r2 were 99.8% and 99.7% respectively. y1 = 1.91 + 0.05 x1 0.19x2 + 0.30 x3 + 0.11 x1x2 + 0.02 x1x3 0.06 x3x2 (2) y2 = 0.69 + 0.01 x1 0.02 x2 + 0.10 x3 + 0.11 x1x2 + 0.06 x1x3 + 0.04 x3x2 (3) from theses equations, the preponderant effect for the actual maceration process was solid-to-liquid ratio for y1 and y2. the high r2adj values indicated that there is a good correlation and relationship between the experimental data and the obtained model data. anova data (table 3) shows a p-value of lof around 0.234 and 0.301 for tpc and tfc respectively. it confirms the high efficiency of fitting model suitability and explanation of experimental data, while coefficients of determination for its polynomial model equation are r2 = 0.998, r2adj of 0.996 and r 2 = 0.997, r2adj of 0.993. the experimental versus predicted response are shown in fig. 2. table 3. analysis of variance (anova) for the fitted model of algerian rosmarinus officinalis l. maceration tpc tfc df model 6 6 lack of fit 2 2 pure error 2 2 sum of squares model 1.1568 0.2421 lack of fit 0.00153105 0.0463636 pure error 0.0466664 0.02 f value model 385.799 243.206 lack of fit 3.28084 2.31818 p value model 0.000 0.000 lack of fit 0.234 0.301 r2 0.998 0.997 r2adj 0.996 0.993 in the present study, the values of r2 were 99.8% and 99.7% respectively for tpc and tfc. the values of r2adj were 0.996 % and 0.993 % for tpc and tfc respectively. these results showed a close agreement between the experimental and the predicted values. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 185 histograms below (fig. 1) present the factors effect and their interactions. fig. 1. histogram of tpc and tfc effects from algerian rosmarinus officinalis l. maceration: (a) histogram of tpc effects, (b) histogram of tfc effects. fig. 2a and fig. 2b reveal the presence of linear relationship between them with high correlation coefficient. it indicates normal distribution of error around the mean and a good applicability of model for interpreting the experimental data. these plots are required to check the normality assumption in fitting model. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 186 fig. 2. predicted values of tpc and tfc versus experimental values from algerian rosmarinus officinalis l. maceration: (a) predicted values of tpc versus experimental values, (b) predicted values of tfc versus experimental values. 3.3. response surface and iso-response plots 3d surface plots serve to predict the response variation as a function of two chosen factors. in order to investigate the interactive effects of operational parameters on responses, threedimensional response surface plots were generated by plotting the response on the z-axis against two independent variables keeping the other independent variables at zero level. the advantage of this illustration is the possibility to visualize the evolution of responses according to two factors at the same time over the entire experimental range. consequently, the estimated value of the desired parameter at any point in the field can be drawn directly. in figure 3 we represent surface response curves of tpc and tfc for the three factors by an illustration of simultaneous effect of two factors. fig. 3a and fig. 3d illustrate simultaneous effect of time and solid-to-liquid ratio on tpc and tfc. it can be observed from fig. 3a that solid-to-liquid ratio is less significant than other effects. the tpc increases by increasing thesolid-to-liquid ratio or time, but it decreases by increasing the ethanol concentration. fig. 3d shows that tfc increases with increasing the time or solid-to-liquid ratio or ethanol concentration. fig. 4 represents the iso-response curves of tpc and tfc for the three factors. it illustrates the evolution of the tpc and tfc according to two factors simultaneously over the entire experimental range. it can be possible from these curves to draw directly the estimated value of the desired parameter at any point in the field of study. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 187 fig. 3. response surface plots for effects on tpc and tfc from algerian rosmarinus officinalis l. maceration: (a) time vs solid to liquid ratio for tpc, (d) time vs solid to liquid ratio for tfc. fig. 4a shows the pronounced efficiency zone for tpc which is around 2.361 mg gae/g dw. in this zone, the solid-toliquid ratio and ethanol concentration values are ranging from 0.14 g/ml to 0.15 g/ml and from 50% to 55 % respectively. regarding the tfc, the pronounced efficiency zone is around 0.845 mg qe/g dw (fig. 4d). the solid-to-liquid ratio and time values in this case are ranging from 0.145 g/ml to 0.15 g/ml and from 65 hours to 70 hours respectively. the results obtained were in good agreement with the experimental results found in this study. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 188 fig. 4. iso-response curves for efficiency zone of tpc and tfc from algerian rosmarinus officinalis l. maceration: tpc [(a) ethanol concentration vs solid to liquid ratio] and tfc [(d) time vs solid to liquid ratio] 3.4. correlation between the responses of tpc and tfc since experimental conditions, which give an extract with the best tpc, do not givethe highest tfc. the relationships between those responses must be examined more deeply. the correlation coefficient between tpc and tfc on the extract was 0.83, suggesting that approximately 83 % of tpc in the extracts are tfc. this last may be related to the high selectivity of the solvent used in the extraction solution. foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 189 fig. 5. relationship between tpc and tfc of extracts from algerian rosmarinus officinalis l. maceration. 4. conclusion algerian rosmarinus officinalis l. leaf extract was obtained by green extraction process. the selection of the solvent is mainly related to the future use of the extract. ethanol was used as solvent in this study. the effect of the main process variables (time, ethanol concentration, solid-to-liquid ratio) on tpc and tfc has been studied. the characteristic models released from each response are first-order linear models with interactions. the effect of each factor on the two selected responses shows that solid-to-liquid ratio represents the overriding factor for tpc and tfc. time had less significant effects on tpc and tfc respectively. indeed, the best experimental results for tpc and tfc were obtained for 1hour, 0.15 g/ml with 50% and for 72hours, 0.15 g/ml with 80% respectively. the value of tpc and tfc is 2.5 mg gae/g dw and 1 mg qe/g dw. the relationship between tpc and tfc provides a good correlation coefficient (0.83). finally, antioxidant and antibacterial activities of extract will be tested in prospect. 5. acknowledgments the authors thank the colleagues from laboratory for their continuous interest, support and discussion. their insight and expertise have been highly appreciated. 6. references [1]. giacometti j., bursać kovačević d., putnik p., gabric d., bilušić t., kresic g., stulić v., barba f., chemat f., barbosa-cánovas g., & režek jambrak a., extraction of bioactive compounds and essential oils from mediterranean herbs by conventional and green innovative techniques: a review, food research international, 113: 245-262, (2018) [2]. putnik p. & bursać kovačevć d., food safety and protection, v ravishankar rai &jamunaa bai (eds.), boca raton, 728, (2017) foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 190 [3]. franco d., rodríguez-amado i., agregán r., munekata p. e. s., vázquez j. a., barba f. j., & lorenzo, j. m., optimization of antioxidants extraction from peanut skin to prevent oxidative processes during soybean oil storage, lwt food science and technology, 88, 1-8, (2018) [4]. roohinejad s., koubaa m., barba f. leong s., khelfa a., greiner r., & chemat f., extraction methods of essential oils from herbs and spices, essential oils in food processing, 21-55, (2017) [5]. granato d., nunes d. & barba f., an integrated strategy between food chemistry, biology, nutrition, pharmacology, and statistics in the development of functional foods: a proposal, trends in food science & technology, 62: 13-22, (2017) [6]. sueishi y., sue m., & masamoto h., seasonal variations of oxygen radical scavenging ability in rosemary leaf extract, food chemistry, 245: 270-274, (2018) [7]. ribeiro-santos r., carvalhocosta d., cavaleiro c., costa h., albuquerque t. g.,castilho m. c., ... sanches-silva a., a novel insight on an ancient aromatic plant: the rosemary (rosmarinus officinalis l.). trends in food science & technology, 45: 355-368, (2015) [8]. choulitoudi e., ganiari s., tsironi t., ntzimani a., tsimogiannis d., taoukis p., &oreopoulou v., edible coating enriched with rosemary extracts to enhance oxidative and microbial stability of smoked eel fillets, food packaging and shelf life, 12: 107-113, (2017) [9]. karoui r. & hassoun a., efficiency of rosemary and basil essential oils on the shelf-life extension of atlantic mackerel (scomberscombrus) fillets stored at 2°c, journal of aoac international, 100: 335-344, (2017) [10]. craft b. d., kerrihard a. l., amarowicz r., & pegg r. b., phenolbased antioxidants and the in vitro methods used for their assessment. comprehensive reviews in food science and food safety, 11: 148-173, (2012) [11]. anastas p.t., warner j.c., green chemistry: theory and practice, oxford university press, london, (1998) [12]. anastas p.t., zimmerman j.b., desing through the twelve principles of green engineering, environ. sci. technol., 37: 94a101a, (2003) [13]. chemat f., vian m.a., cravotto g., green extraction of natural products: concept and principles, int. j. mol. sci., 13: 86158627, (2012) [14]. singleton v. l. & rossi j. a., colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, american journal of enology and viticulture, 16: 144-158, (1965) [15]. miliauskas g., venskutonis r., & van beek t., screening of radical scavenging activity of some medicinal and aromatic plant extracts, food chemistry, 85: 231-237, (2004) [16]. navarrete a., herrero m., martin a., cocero m.j., & ibanez e., valorisation of solid wastes from essential oil industry, journal of food engineering, 104: 196-201, (2011) [17]. visentín a., cismondi m. & maestri d., supercritical co2 fractionation of rosemary ethanolic oleoresins as a method to improve carnosic acid recovery, innovative food science & emerging technologies, 12: 142-145, (2011) [18]. zhang b., yang r., & liu c., microwave-assisted extraction of chlorogenic acid from flower buds of lonicera japonica thunb, separation and purification technology, 62: 480-483, (2008) [19]. software ‘modde6’. design of experiments and optimization. version 6, may 15, 2001, umetric, www.umetric.com [20]. moreno s., scheyer t., romano c.s., & vojnov a.a., antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition, free radical research, 40: 223-231, (2006) [21]. wojdylo a., oszmianskii j., & czelerys r., abtioxidant activity and phenolic compounds in 32 selected herbs, food chemistry, 105: 940-949, (2001) [22]. dos santos r.d., shetty k., cecchini a.l., & miglioranza l.h., phenolic compounds and total antioxidant activity determination in rosemary and oregano extracts and its use in cheese spread, semina, 33: 655-666, (2012) [23]. yesil-celiktas o., girgin g., orhan h., wicher h.j., bedir e., & verdar sukan f., screening of free radical scavenging capacity and antioxidant activities of rosmarinus officinalis extract with focus on location and harvesting times, european food research and technology, 224: 443451, (2007) http://www.umetric.com/ foodandenvironmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 linda loucif seiad, soraya demim, modeling and optimization of bacs extraction from algerian rosmarinus officinalis l.via green extraction, food and environment safety, volume xix, issue 3 – 2020, pag. 180 191 191 [24]. rodríguez-rojo s., visentin a., maestri d., & cocero m. j., assisted extraction of rosemary antioxidants with green solvents, journal of food engineering, 109: 98-103, (2012) [25]. oliveira g. d., oliveira a. e., conceição e. c., & leles m. i., multiresponse optimization of an extraction procedure of carnosol and rosmarinic and carnosic acids from rosemary, food chemistry, 211: 465-473, (2016) [26]. bucic-kojić a., planinić m., tomas s., bilic m., & velic d., study of solid– liquid extraction kinetics of total polyphenols from grape seeds, journal of food engineering, 81: 236-242, (2006) [27]. amendola d., de faveria d.m. and spigno g., grape marc phenolics: extraction kinetics, quality and stability of extracts, journal of food engineering, 97: 384-392, (2010) [28]. sant'anna v., brandelli a. &marczak l.d.f. and tessaro i.c., kinetic modeling of total polyphenol extraction from grape marc and characterization of the extracts, separation and purification technology, 100: 82-87, (2012) [29]. cacace j., & mazza g, optimization of extraction of anthocyanins from black currants with aqueous ethanol, journal of food science, 68: 240-248, (2006) 1. introduction 4. conclusion articol porumb cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 150 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 150 155 determination of color and anthocyanins in three romanian red wines *cristina ghinea 1 , ana leahu 1 , florin ursache 1 1faculty of food engineering, stefan cel mare university of suceava, romania, cristina.ghinea@fia.usv.ro *corresponding author received 5th july 2019, accepted 27th september 2019 abstract. in this study uv-vis spectrophotometer and cm 700 d spectrophotometer konica minolta colorimeter (cielab method) were used to investigate the anthocyanins content and color of three romanian red wines. the samples were purchased from three different romanian vineyards located in different regions of the country (south east development region (sedr), south-muntenia development region (smdr) and south west oltenia development region (swodr)). in total, nine samples of red wine feteasca neagra, cabernet sauvignon and merlot, the harvest of 2012, were analyzed. during this study chromatic parameters as l *, a *, b *, c * and h0 were determined. the degree of brightness is indicated by the l * parameter and according to our results it is between 0.27 (darkest) for merlot wine from the vineyard located in south west oltenia development region and 15.48 for the feteasca neagra wine from the vineyard located in south-muntenia development region. the results showed that chromatic parameter a, located on the color coordinate red-green, has values ranging from -0.09 (feteasca neagra from smdr) and 1.47 (cabernet sauvignon from smdr). the highest total anthocyanins content of the feteasca neagra wine was recorded in the vineyard located in the swodr (251 mg/l), followed by the vineyard located in the sedr, and the lowest content (97 mg/l) was recorded for the wine from the vineyard located in the smdr. feteasca neagra, cabernet sauvignon and merlot wines have different anthocyanins content from one vineyard to another, demonstrating that the variation of anthocyanins and implicitly variation in wine color is influenced by pedo-climatic conditions in the area, cultivated varieties and vinification technology applied. keywords: anthocyanins, cielab method, red wine, vinification 1. introduction wine is a drink obtained exclusively from the complete or partial alcoholic fermentation of fresh or crushed fresh grapes or of fresh grape must [1]. in 2015, the global grape production was 75 million tons, out of which 71% was used in the wine industry and 29% was consumed as fresh and dried fruits. 127.8 million hl of red wine were produced in the world. this quantity represents 46% of the total wine produced globally. in europe, in 2015, 19.83 million tons of red grapes and 90.59 million hl of red wine were produced. in the same year, revenues of 23.83 billion euros from red winemaking were recorded. among the global largest wine producers, romania was ranked 13th [2], while in the eu was ranked 6th (fig. 1a). the largest wine producers in the eu are france, italy and spain according to [3]. production and consumption of romanian wine between 2011 and 2019 are illustrated in the fig. 1b. it can be seen that the highest production of wine was obtained in 2013 [2]. romanian wine, with an average of 20 l/capita [4], is the second most consumed alcohol after beer (average of 80 l/capita) [5]. the wine quality in eu is classified in http://www.fia.usv.ro/fiajournal mailto:cristina.ghinea@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 151 two categories protected designation of origin (pdo) and protected geographical indication (pgi). fig. 1c illustrates the evolution of romanian wine opening stocks by these two categories. it can be observed that in the period 2010 – 2018 the highest amount of wine was registered in the year 2016, for both wine categories [6]. as it was expected the highest revenue (fig. 1d) from wine production was registered in the year (2013), with the highest amount of wine produced. in 2015, 40 million of euros were registered from romanian red wine production [2]. merlot and cabernet sauvignon are the leading red varieties in romania, while feteasca alba and feteasca regala are the main white varieties [7]. one of the most significant attributes of red wine is color which is formed during winemaking due to the presence of anthocyanins. there are many studies on anthocyanins determination and color investigation of wine. for examples, [8] determined the anthocyanins and color of 173 commercial red wines from pinot noir, merlot, cabernet franc, cabernet sauvignon varieties. organic and inorganic compounds of eight pinot noir red wines were investigated by [9]. they determined anthocyanins and phenolic compounds and also other chemical compounds. anthocyanins and wine color were analyzed by [10] in different stages starting from pressing, to two, six, twelve and thirty-six months of bottle aging. in order to determine the effect of hydroxycinnamic acids on anthocyanin co-pigmentation and color of the merlot wine, fresh and stored for six months, was examined by [11]. parameters like color components are important due to the fact that they are contributors to sensory characteristics and antioxidant properties of wine [12]. quality red wines are wines obtained from black grapes and have outstanding sensory characteristics as compared to other wines. the technological scheme for obtaining red wines is illustrated in the fig. 2. fig. 1. a) wine production in the eu countries; b) production and consumption of romanian wine; c) evolution of romanian wine opening stocks by category; d) romanian wine production value (according to [2, 3, 6]) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 152 fig. 2. technological scheme of obtaining red wines [13] according to [14], the consumption of anthocyanins leads to a lower risk of developing cardiovascular diseases and cancer. the aim of this paper was to investigate the anthocyanins content and color of red wines from three different romanian vineyards. 2. materials and methods the anthocyanins content and color of red wines were investigated by using uv-vis spectrophotometer and cm 700 d spectrophotometer konica minolta colorimeter (cielab method). nine samples of red wine feteasca neagra, cabernet sauvignon and merlot, harvest of 2012, purchased from three different romanian vineyards (south east development region, south-muntenia development region and south west oltenia development region) were analyzed. the color analysis of wine takes into consideration 5 chromatic parameters: l *, a *, b *, c * and h0:  l * = the brightness or brightness of the color corresponds to the light transparency and varies in inverse with the color intensity of the wine. the values are between 0 (black) and 100 (white);  a * = chromatic component of complementary colors, red-green. values are between -100 (green) and +100 (red);  b * = chromatic component of complementary colors, yellow-blue. values are between -100 (blue) and +100 (yellow);  c * = saturation or chromaticity of color corresponding to the dominant wavelength (λ) of the absorbed radiation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 153 the values go from scratch for very low colors and reach 141 for very intense colors.  h0 = azimuth angle, which characterizes the hue / color tone (00-510 red wines, 520-800 purple wines) [15, 16] also used cielab method for determination of red wines color. the cielab parameters are specified by the international organization of vineyard and wine (oiv) to determine the wine color [17]. the total anthocyanins content in wines was determined by applying the method proposed by [18]. anthocyanins content was calculated based on cyanidin-3glucoside. the absorbance of wine samples was measured at 657 nm and at 530 nm using a uv-vis spectrophotometer. in order to correctly read the absorbance, a dilution (50/50) was made for cabernet (from south east development region), cabernet (from south west oltenia development region), merlot (from south east development region) and merlot (from south west oltenia development region). uv/vis spectrophotometry for determination of anthocyanins was also used by [8, 9, 11, 19, 20], whose measurements were performed at 520 nm; while [21-23] applied hplc method to determine these chemical compounds. [24] measured the total anthocyanins content by applying uv/vis spectrophotometry (520 nm), and the individual anthocyanins by using hplc. [25] mention that cyanidin-3glucoside can be determined at 520 nm, but also at 510 or 530 nm depending on the solvent system used. 3. results and discussion the highest total anthocyanins content of the feteasca neagra wine was recorded in the vineyard located in the south west oltenia development region (251 ± 25 mg/l), followed by the wine from vineyard from south east development region, and the lowest content (97 ± 25 mg/l) was recorded in the wine from the vineyard located in the south-muntenia development region (fig. 3). the values of anthocyanins content determined for the cabernet sauvignon were between: 247537 mg/l. 184-500 mg/l anthocyanins were registered for merlot wine. the values obtained by us are similar to those of the literature. the total monomeric anthocyanin content determined by [11] for merlot wine produced in the southern greece was 418.9 ± 4.6 mg/l. the total anthocyanins content for cabernet sauvignon, year 2012, determined by [20] was 341.7±34.32 mg/l for the wine recas wineries and 258.8±10.29 mg/l for the wine from minis wineries. values between 572.8 and 588.9 mg anthocyanin/l wine were obtained for romanian wine merlot, while for cabernet sauvignon there were determined the following ones: 372.7 and 401.2 mg/l, both wines were obtained by [26] from recas vineyard. the total anthocyanins obtained by [27] for cabernet sauvignon, merlot and feteasca neagra wines produced in murfatlar, 2013 harvest, were: 466.37, 259.96, 325.92 mg/l. according to [10], at pressing the wine anthocyanin concentration was 403 mg/l and decreased with aging. the results show that l * parameter which indicates the degree of brightness, is between 0.27 (darkest) for merlot wine from the vineyard located in the swodr and 15.48 for the feteasca neagra wine from the vineyard located in the smdr, while chromatic parameter a, located on the color coordinate red-green, has values ranging from -0.09 (feteasca neagra from smdr) and 1.47 (cabernet sauvignon from smdr). the chromatic parameter b, based on blue-yellow colors, ranges from 0.15 (cabernet sauvignon from vineyard located in the swodr) and 0.33 (fetească food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 154 neagră from the vineyard located in the swodr) (fig. 4). fig. 3. content in anthocyanins of the investigated wine samples fig. 4. values obtained for chromatic parameters: l *, a *, b * 3. conclusion feteasca neagra, cabernet sauvignon and merlot wines have different anthocyanins content from one vineyard to another, demonstrating that the variation in anthocyanins and implicitly variation in wine color is influenced by pedo-climatic conditions in the area, cultivated varieties and vinification technology applied (use of enzymes, maceration conditions and fermentation temperature) and aging. among these factors, maceration conditions have the greatest impact on anthocyanins and sensory characteristics of red wines. 4. references [1]. oiv, the international organization of wine and vine, definitions and descriptions of the vitivinicultural products, basic definition (18/73), i.3.1-1, wines, (2016) [2]. gtk, wine market analysis, (2016), https://gtk.ro/?download=2897 [3]. uesda, wine annual report and statistics, gain report number: it1307, (2013) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 cristina ghinea, ana leahu, florin ursache, determination of color and anthocyanins from three romanian red wines, volume xviii, issue 3 – 2019, pag. 150 – 155 155 [4]. nis, beverage consumption in 2015, national institute of statistics, (2016) [5]. ghinea c., leahu a., life cycle environmental impacts of romanian beer production, sgem2017 vienna green conference proceedings, 17:115-122, (2017) [6]. ec, wine stock declarations 31/7/2018, european commission, (2018) [7]. uesda, wine annual report and statistics 2015, gain report number: it1512, (2015) [8]. cliff m.a., king m.c., schlosser j., anthocyanin, phenolic composition, colour measurement and sensory analysis of bc commercial red wines, food research international, 40:92–100, (2007) [9]. sirén h., sirén k., sirén j., evaluation of organic and inorganic compounds levels of red wines processed from pinot noir grapes, analytical chemistry research, 3:26–36, (2015) [10]. kilmister r.l., mazza m., baker n.k., faulkner p., faulkner m.o., a role for anthocyanin in determining wine tannin concentration in shiraz, food chemistry, 152:475– 482, (2014) [11]. bimpilas a., panagopoulou m., tsimogiannis d., oreopoulou v., anthocyanin copigmentation and color of wine: the effect of naturally obtained hydroxycinnamic acids as cofactors, food chemistry, 197:39–46, (2016) [12]. versari a., parpinello g.p., mattioli a.u., characterisation of colour components and polymeric pigments of commercial red wines by using selected uv-vis spectrophotometric methods, south african journal for enology and viticulture, 28:6-10, (2007) [13]. banu c., handbook of the food industry engineer, vol. ii, technical publishing house, bucharest, (1999) [14]. braga a.r.c., murador d.c., de souza mesquita l.m., de rosso v.v., bioavailability of anthocyanins: gaps in knowledge, challenges and future research, journal of food composition and analysis, 68:31–40, (2018) [15]. almela l., javaloy s., fernandez-lopez j.a., lopez-roca j.m., comparison between the tristimulus measurements yxy and l∗a∗b∗ to evaluate the colour of young red wines, food chemistry, 53:321-327, (1995) [16]. esparza i., santamaria c., calvo i., fernandez j.m., significance of cielab parameters in the routine analysis of red wines, cyta – journal of food, 7:189–199, (2009) [17]. sen i., tokatli f., differentiation of wines with the use of combined data of uv–visible spectra and color characteristics, journal of food composition and analysis, 45:101–107, (2016) [18]. rabino i., mancinelli a., light, temperature and anthocyanin production, plant physiology, 81: 922-924, (1986) [19]. tsanova-savova s., dimov s., ribarova f., anthocyanins and color variables of bulgarian aged red wines, journal of food composition and analysis, 15:647–654, (2002) [20]. nistor e., dobrei a., dobrei a., camen d., mălăescu m., prundeanu h., anthocyanins and phenolics in cabernet sauvignon and pinot noir wines, journal of horticulture, forestry and biotechnology, 19:226229, (2015) [21]. feng h., skinkis p.a., qian m.c., pinot noir wine volatile and anthocyanin composition under different levels of vine fruit zone leaf removal, food chemistry, 214:736–744, (2017) [22]. han f., ju y., ruan x., zhao x., yue x., zhuang x., qin m., fang y., color, anthocyanin, and antioxidant characteristics of young wines produced from spine grapes (vitis davidii foex) in china, food & nutrition research, 61:1339552, (2017) [23]. gabrielyan a., kazumyan k., the investigation of phenolic compounds and anthocyanins of wines made of the grape variety karmrahyut, annals of agrarian science, 16:160– 162, (2018) [24]. dimitrovska m., tomovska e., bocevska m., characterisation of vranec, cabernet sauvignon and merlot wines based on their chromatic and anthocyanin profiles, journal of the serbian chemical society, 78:1309–1322, (2013) [25]. giusti m., wrolstad r.e., characterization and measurement of anthocyanins by uv-visible spectroscopy, current protocols in food analytical chemistry, f1.2.1-f1.2.13, (2001) [26]. lengyel e., oprean l., iancu r., ketney o., tita o., anthocyanins and polyphenols content in red merlot, cabernet sauvignon and pinot noir wines from recas vineyard, acta universitatis cibiniensis series e: food technology, xvi: 51-56, (2012) [27]. artem v., geană e.i., antoce a.o., study of phenolic compounds in red grapes and wines from murfatlar wine center, ovidius university annals of chemistry, 25: 47-52. 48 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, stefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 48 54 sta tis ti ca l e va luat io n o f t he rela t ions hi p betw e en the me ta lli c el em ents th at m igrate fr om a is i30 4 and aisi32 1 s ta inl ess s te e l sam pl es in s im u lant sol u tio ns a t di fferen t im me rs ion t im es *silviu-gabriel stroe1 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229, suceava, romania silvius@fia.usv.ro *corresponding author received february 5th 2014, accepted march 14th 2014 abstract: the aim of this paper was to evaluate the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in acidic food simulant solutions at different immersion times. in this study were used simulant solutions with different concentrations of acetic acid in double distilled water, different stiring environment and different immersion times of metal samples in corrosive solutions. to achieve this purpose, the experimental data were statistically processed by analyzing the correlation between variables based on pearson correlation matrix and analyze relationships between variables by the principal component analysis method (pca). the influence of the immersion time on the dissolution rate of the cr, mn, 56fe and ni metal ions from aisi304 stainless steel samples and the ti, cr, mn, 56fe and ni metal ions from aisi321 stainless steel samples was studied. the results obtained and the conclusions reached, demonstrates that the statistical method used in this study is a useful tool for studying the dissolution phenomena of metal ions in acidic food environments. keywords: stainless steel samples, acetic acid, principal components analysis, correlation matrix, pearson's coefficients 1. introduction in the last decade, globally, the food industry, from processing industry, continuing with supply chain and to the preparation of food, on observed a growing interest for research in the food safety field. in the european union also notes that an integrated approach to this vast field, through monitoring and appropriate action, ensuring at the same time, an efficient operation of the market. thus, a pole of interest, that is the subject of numerous multidisciplinary researches and which gather the food industry technology specialists, specialists in materials engineering and chemists, is the interface processes that occur during the contact of metallic materials on supply chain and food environments. during these stages of processing, the materials of the processing equipments, storage tanks, transmission lines or other materials intended to come into contact with food, are a chemical substances source of food or beverage contaminating. the aim of this paper was to assess the relationship between the metallic elements which migrate from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 49 aisi304 and aisi321 stainless steel samples into acidic food simulant environments, at different immersion times. these studies are a continuation of those presented in a previous paper, where he studied the relationship between the migration of metal ions from metal samples at different concentrations of food simulant solutions [1]. 2. materials and methods 2.1. materials, experimental conditions and statistical methods considering the fact that the most aggressive environments that we encounter in industrial food raw materials processing are acidic environments, were chosen the aisi304 and aisi321 stainless steel grades [2], [3], [4]. in the experimental research, as corrosive solutions, the 3%, 6% and 9% acetic acid solutions were used. the acetic acid is recommended to test the migration of metal ions of the alloys that come into contact with the acidic food environmets [5]. the corrosive environments temperature which was studied the migration behavior of the metallic samples were 22, 28 and 34ºc. the corrosive environments stirring was 0, 125 and 250 rpm. the exposure times of the metal samples were 30, 60 and 90 min. to analyze the chemical composition of corrosive environments, before and after their use in corrosion tests, the mass spectrometry with inductively coupled plasma (icp-ms) was chosen and used, considering the advantages it presents this method over other methods. the experimental data were statistically processed by correlation analysis of the variables based on pearson correlation matrix and analyze relationships between variables by principal component analysis (pca). the evaluation of relationships between metal elements was performed using the trial version of spss v16.0 software. the pearson correlation matrix is a square matrix symmetrical with respect to the main diagonal that highlights the values of the coefficients of correlation (r) of the variable, taking into account two by two. the correlation coefficient expresses the intensity of association between variables and is determined with equation 1. , = , ∙ (1) where, sx and sy are the standard deviations sx,y is the covariance between x and y values calculated using: , = ∑ ( − )( − ) (2) where, n=1…27 (experience number); xi and yi are the variable values of x and y; mx and my are the arithmetic averages strings of the values of the two variables. when the size of the correlation coefficient is closer to the value ±1, is stronger association between variables, and it is closer to 0, the combination is lower. link direction is given by the sign of the correlation coefficient. a positive value of the pearson correlation coefficient indicates a direct relationship between the two variables in the sense that to an increase of the value of a variable corresponds to an increase of the value of the other variable. a negative value of the pearson correlation coefficient indicates an inverse relationship between the two variables in the sense that to an increase in value of the variable corresponds to a decrease in the value of other variables. to take into account the correlation it has to correspond to a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 50 p<0.05 significance level, a level that was chosen at start of statistics study. by applying the technique of the multivariate analysis there is a reduction of the number of variables to a relatively small number of components uncorrelated with each other, called principal components, by maintaining as much as possible the variance of the original data. only major components that have eigenvalues greater than 1 were chosen [6], because they provide more information than the original variables. these eigenvalues measure the amount of variance explained by each principal component. the numbers of principal components have to explain over 70% of the total variance of the data [7]. the experimental data were processed by analyzing the correlation between variables based on pearson correlation matrix and analyze relationships among variables by the principal component analysis method (pca), using spss software v16.0. 3. results and discussion the minimum and maximum value, mean and standard deviation for each of the metal ions of chromium, manganese, iron and nickel migrated from aisi304 stainless steel samples into simulant solutions after 30, 60 and 90 min. are shown in table 1. table 1. specific values of metal ions which released from the aisi304 stainless steel samples in the simulant solutions after 30, 60 and 90 min. metal ions released in solutions * notations used for the metal ion released after 30, 60 and 90 min. calculated value. [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd chromium cr_30 1.00 77.00 10.77 14.54 cr_60 1.00 188.00 17.62 34.69 cr_90 3.00 238.00 29.66 47.88 manganese mn_30 0.72 22.00 2.99 3.92 mn_60 0.92 41.00 4.73 7.75 mn_90 0.82 43.00 7.52 9.09 iron fe_30 10.00 2850.00 307.03 533.30 fe_60 30.00 4450.00 398.51 827.23 fe_90 10.00 3750.00 642.59 823.14 nickel ni_30 3.30 561.00 158.29 212.59 ni_60 3.80 381.00 116.42 144.34 ni_90 5.20 361.00 98.18 114.21 * cr_30, mn_30, fe_30, ni_30 – metal ions of chromium, manganese, iron and nickel that release into simulant solution after 30 min.; cr_60, mn_60, fe_60, ni_60 – metal ions of chromium, manganese, iron and nickel that release into simulant solution after 60 min.; cr_90, mn_90, fe_90, ni_90 – metal ions of chromium, manganese, iron and nickel that release into simulant solution after 90 min. the minimum and maximum value, mean and standard deviation for each of the metal ions of titanium, chromium, manganese, iron and nickel from aisi321 stainless steel samples into simulant solutions after 30, 60 and 90 min. are shown in the table 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 51 table 2. specific values of metal ions which released from the aisi321 stainless steel samples in the simulant solutions after 30, 60 and 90 min. metal ions released in solutions * notations used for the metal ion released after 30, 60 and 90 min. calculated value. [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd titanium ti_30 0.40 2.20 1.03 0.48 ti_60 0.70 4.98 1.77 0.96 ti_90 0.88 8.28 2.43 1.52 chromium cr_30 1.00 21.00 7.25 5.14 cr_60 1.00 24.00 10.51 5.27 cr_90 4.00 49.00 18.66 11.06 manganese mn_30 2.49 5.77 4.34 1.03 mn_60 2.99 11.57 5.99 2.06 mn_90 3.29 19.73 8.91 3.49 iron fe_30 110.00 770.00 350.74 172.73 fe_60 150.00 1070.00 455.18 205.50 fe_90 160.00 1370.00 564.44 245.64 nickel ni_30 7.70 696.00 169.40 229.43 ni_60 10.70 746.00 196.59 256.96 ni_90 14.70 806.00 230.11 289.50 * ti_30, cr_30, mn_30, fe_30, ni_30 – metal ions of titanium, chromium, manganese, iron and nickel that release into simulant solution after 30 min.; ti_60, cr_60, mn_60, fe_60, ni_60 – metal ions of titanium, chromium, manganese, iron and nickel that release into simulant solution after 60 min.; ti_90, cr_90, mn_90, fe_90, ni_90 – metal ions of titanium, chromium, manganese, iron and nickel that release into simulant solution after 90 min. after the immersion time of 30, 60 and 90 minutes in the simulate solutions the correlation coefficient values (table 3) indicate strong correlation (r  0,700), significant at p = 0.01, between the chromium, manganese and iron ions that migrate in corrosive solutions for aisi304. strong correlations between the iron and manganese ions for aisi304 on the same immersion time of 30, 60 and 90 minutes were obtained. table 3. pearson correlation matrix of ions released from aisi304 stainless steel samples into simulant solutions after 30, 60 şi 90 min. cr_30 cr_60 cr_90 mn_30 mn_60 mn_90 fe_30 fe_60 fe_90 ni_30 ni_60 ni_90 cr_30 1.000 cr_60 0.961a 1.000 cr_90 0.914a 0.905a 1.000 mn_30 0.926a 0.971a 0.905a 1.000 mn_60 0.882a 0.940a 0.859a 0.936a 1.000 mn_90 0.872a 0.836a 0.963a 0.845a 0.778a 1.000 fe_30 0.948a 0.970a 0.899a 0.980a 0.929a 0.848a 1.000 fe_60 0.935a 0.981a 0.916a 0.988a 0.938a 0.837a 0.976a 1.000 fe_90 0.537a 0.496a 0.791a 0.544a 0.498a 0.817a 0.523a 0.546a 1.000 ni_30 0.255 0.060 0.009 -0.061 0.025 0.037 0.023 -0.051 -0.159 1.000 ni_60 0.309 0.157 0.085 0.043 0.160 0.075 0.103 0.059 -0.102 0.953a 1.000 ni_90 0.453b 0.359 0.311 0.270 0.425b 0.257 0.329 0.301 0.143 0.754a 0.879a 1.000 bold values represent correlation with significance, a significant correlations at a 0.01 level, b significant correlations at a 0.05 level food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 52 after the immersion time of 30, 60 and 90 minutes in the simulate solutions the correlation coefficient values (table 4) indicate strong correlation (r  0,700), significant at p = 0.01, between the titanium, chromium, manganese and iron ions that migrate in corrosive solutions for aisi321. strong correlations between the iron and manganese ions for aisi321 stainless steel samples on the immersion time of 90 minutes were obtained. table 4. pearson correlation matrix of ions released from aisi321 stainless steel samples into simulant solutions after 30, 60 şi 90 min. ti_30 ti_60 ti_90 cr_30 cr_60 cr_90 mn_30 mn_60 mn_90 fe_30 fe_60 fe_90 ni_30 ni_60 ni_90 ti_30 1.000 ti_60 0.795a 1.000 ti_90 0.561a 0.919a 1.000 cr_30 0.323 0.253 0.168 1.000 cr_60 0.465b 0.282 0.105 0.647a 1.000 cr_90 0.207 0.004 -0.079 0.511a 0.769a 1.000 mn_30 0.294 0.331 0.296 0.408b 0.268 0.013 1.000 mn_60 0.530a 0.607a 0.557a 0.354 0.199 -0.033 0.772a 1.000 mn_90 0.761a 0.825a 0.704a 0.518a 0.392b 0.148 0.549a 0.795a 1.000 fe_30 0.345 0.133 0.072 0.568a 0.356 0.552a 0.369 0.483b 0.487b 1.000 fe_60 0.484b 0.258 0.110 0.673a 0.460b 0.499a 0.463b 0.580a 0.631a 0.931a 1.000 fe_90 0.517a 0.398b 0.282 0.633a 0.461b 0.429b 0.536a 0.680a 0.721a 0.841a 0.940a 1.000 ni_30 0.230 0.516a 0.613a -0.179 0.034 -0.157 -0.107 0.168 0.232 -0.372 -0.335 -0.152 1.000 ni_60 0.240 0.495a 0.594a -0.175 0.036 -0.158 -0.102 0.150 0.213 -0.368 -0.337 -0.157 0.994a 1.000 ni_90 0.233 0.470b 0.557a -0.189 0.044 -0.149 -0.105 0.153 0.204 -0.367 -0.331 -0.160 0.993a 0.995a 1.000 bold values represent correlation with significance a significant correlations at a 0.01 level b significant correlations at a 0.05 level principal components analysis reveals that the first two principal components explain 95.08% of total variance (pc1 = 74.16% and pc2 = 20.92%) (figure 1). regarding the first principal component (pc1), it can be seen that there is very good correlation between the ions of iron, chromium and manganese released from the aisi304 stainless steel samples in the simulant solutions after 30 and 60 min.. these variables are strongly associated with first principal component (pc1). also, from the first principal component (pc1) is remarkable direct correlation between the manganese ions and chromium which migrate after 90 min. of exposure (mn_90 and cr_90). the second principal component (pc2) highlights the difference between the iron and nickel ions that released into simulant solutions after 90 min. (fe_90 and ni_90). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 53 figure 1. relationships between the chromium, manganese, iron and nickel metal ions released from aisi304 stainless steel samples into 3, 6 and 9% ch3cooh solutions at 30, 60 and 90 min. immersion times. the correlation matrix of the metal ions that migrate from aisi321 stainless steel samples into simulant solution after an 30, 60 and 90 min. immersion time (table 4) shows no statistically significant correlation between them. a significant association (r = 0.607, at a p = 0.01 level) resulted between the titanium and manganese ions that migrate into the simulated acetic acid solution after 60 min. increasing the immersion time of 90 min. determines a strong association (r = 0.704, at a p = 0.01 level) among the titanium and manganese ions. a significant association (r = 0.568, at a p = 0.01 level) was obtained between chromium and iron ions released from aisi321 stainless steel samples into the simulated solution an 30 min. immersion time. also, between the chromium and iron ions are moderate association significant at 60 min. immersion time (r = 0.469 at a p = 0.05 level), and at 90 min. immersion time (r = 0.429). significant correlations at p = 0.01 level between manganese and iron ions migrated from stainless steel samples into the simulant solution after 60 min. immersion time (r = 0.580) and after the 90 min. immersion time (r = 0.721) were obtained. figure 2. relationships between the titanium, chromium, manganese, iron and nickel metal ions released from aisi321 stainless steel samples into 3, 6 and 9% ch3cooh solutions at 30, 60 and 90 min. immersion times. it can be said that immersing of the aisi321 stainless steel samples into a simulated solution for 30 min. highlights significant correlation between chromium and iron ions, and that correlations have been obtained between titanium and manganese, chromium and iron, manganese and iron ions that have released from the aisi321 stainless steel samples into simulant solution after 30 min and 90 min. the first two principal components (pc1) and (pc2) (figure 2), explained 97.80% of data variance, with pc1 explaining 66.15%. from the graphical representation of the relationships between metal ions that release from aisi321 stainless steel samples into the simulant solutions on observed along pc1 axis close relationship between titanium, chromium, manganese and iron ions after 30, 60 and 90 min. immersion time. the variables position relative to the first principal component show that there exists a strong direct relationship. since the variables are the same quadrant, the relationship food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 silviu-gabriel stroe, statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54 54 between them is positive. pc2 indicates the association between nickel ions that release into simulated solution at 30, 60 and 90 min. immersion times, and ni_30 variables, ni_60 and ni_90 that are strongly associated with the major component. towards the second principal component (pc2) is shown in the graphic representation the opposition between the iron and nickel ions that released from aisi321 stainless steel samples into simulated solution to a 30, 60 and 90 min immersion time, with negative correlation. 4. conclusion regarding the influence of the immersion time of the metallic samples into acidic simulant solutions, was observed that at 30 min. immersion time, as well an 60 min. immersion time of the aisi304 stainless steel samples occurs a migration phenomenon of the iron, chromium and manganese ions, as compared with nickel ions that migrate after the same immersion time. for all immersion times studied, the iron ions that migrate from aisi321 stainless steel samples into the simulant, compared to the behavior in the same conditions of the nickel ions. the iron ions migrate from aisi321 stainless steel samples into the simulated solution at all three times of immersion forms a distinct group of nickel ions migrate at the same operating parameters. it can be concluded that the extreme positions on the diagram of the principal components of the two groups shows that, from the point of view of the studied variables, between the two groups the greatest differences were obtained. 5. references [1]. stroe s.g., gutt g., mironeasa s., the relationship between metal ions released from aisi304 and aisi321 stainless steels in food simulant solutions at various working parameters, food and environment safety, volume xii, issue 3, p. 265 -270, (2013); [2]. ziemniak , s.e., hanson, m., corrosion behavior of 304 stainless steel in high temperature, hydrogenated water, corrosion science 44, 2209–2230, (2002); [3]. wallinder, d., odnevall wallinder, i., leygraf, c., influence of surface treatment of type 304l stainless steel on atmospheric corrosion resistance in urban and marine environments, corrosion 59 220–227, (2003); [4]. pardo, a., merino, m.c., carboneras, m., viejo, f., arrabal, r., munoz, j., influence of cu and sn content in the corrosion of aisi 304 and 316 stainless steels in h2so4, corrosion science 48 1075–1092, (2006); [5]. food standards agency, uk, migration test protocols for electroplated and dipped metalware intended for food contact (a03007/fs2236), (2003); [6]. gabriel, k.r., the biplot graphic display of matrices with application to principal component analysis, bio. 58, 453-467, (1971); [7]. benzécri, j.p., l’analyse des donnees. la taxionomie, dunod, (1973). 315 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 315 320 consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria * saheed adewale omoniyi 1 , phidelia ramatu waziri-ugwu 2 1department of home science and management, federal university, gashua, yobe state nigeria, omoniyidewale2005@yahoo.com 2department of agricultural economics and extension, federal university, gashua, yobe state nigeria *corresponding author received 14 november 2019, accepted 27th december 2019 abstract: the study investigated the consumers’ knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria. well-structured questionnaire were administered to one hundred and fifty (150) people on information on their demographic characteristics and also on their knowledge about boiled cassava root. the data obtained were analyzed with descriptive statistic. the results showed that majority of respondents are young people (19 – 28 years), male (70.70%), married (61.34%), university graduate (54.67%) and civil servant (72.00%). majority of the respondents (56.00%) are not aware that cassava roots have anti-nutritional factors but also majority (65.20%) of the respondents knew that cassava roots have little nutrient contents. majority of the respondents (54.70%) are consuming boiled cassava root as meal and they did not suffer from any ailment after eating boiled cassava root while minority (5.30%) reported that they suffered stomach ache after consuming it. however, majority (72.00%) of the respondents like eating boiled cassava root with protein based foods such as beans (53.30%), bambara nut (4.00%), pigeon pea (1.30%), eggs (6.70%), groundnut cake (4.00%) and meat (2.70%) while remaining 28.00% stated that they are addicted eating it without protein based foods. the study showed that the majority of the respondents are not aware that cassava roots contained anti-nutritional factors but they aware that it is safe to be consumed. also, majority of the respondents knew that cassava roots have little nutrient contents but consuming it with non-protein foods, and they like to be eating it with protein based foods especially beans. keywords: cassava root, boiling, consumer knowledge, hydrogen cyanide, safety 1. introduction cassava is grown widely in tropic and subtropic countries due to its comestible root [1]. cassava can withstand drought and survive in poor soil [2]. cassava roots have abundant carbohydrate content with lower protein content and other nutrient contents [2]. also, falola [3] reported that cassava roots have high quantity of carbohydrate content with great value of inexpensive calorie. various varieties of cassava roots has been recognized and grouped as either sweet cassava or bitter cassava with respect to linamarin content in the root [4]. anti-nutritional contents in bitter cassava root are higher than in sweet cassava root [5]. hydrogen cyanide is being released during the processing of cassava roots and the degree of removing it is based on methods of processing used [6]. different processing methods such as peeling, pressing, boiling, fermentation, soaking, grating and drying reduce the cyanide level of cassava root to safer level [7], [8]. http://www.fia.usv.ro/fiajournal mailto:omoniyidewale2005@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 saheed adewale omoniyi, phidelia ramatu waziri-ugwu, consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 315 – 320 316 however, montagnac et al. [9] revealed that two or more processing methods are being used to lower the anti-nutritional or increase the quality of cassava products. various authors have reported in literature that boiling majorly reduce anti-nutritional properties in cassava roots. boiling has reported as an important method of lower the cyanide in cassava root [7]. also, omosuli [10] reported that boiling majorly lower the anti-nutritional contents in cassava roots while montagnac et al. [9] revealed that boiling cassava with high volume of water or cutting cassava roots into smaller size improve the effectiveness of boiling method to reduce cyanide. the significant of cassava has improved seriously over three decades [11]. the increase in populations of people in cassava cultivated nations shall keep on supply domestic market for cassava root and its various products [11]. numerous people in underdeveloped nations utilize cassava roots and its various products as principal foods [12]. in nigeria cassava roots are being processed to flour, tapioca, chips, fufu and cassava meal [7]. in nigeria, cassava provides nearly 70% of calorie to more than fifty millions people [13]. gashua is headquarters of bade local government located in north east, yobe state, nigeria. gashua is located in semiarid region of northern part of nigeria and lie between latitude 11º 501 and 12º 551 -north, and longitude 10º 351 and 11º 151east [14]. the food crops being planted in gashua includes millet, groundnut, sorghum, rice, wheat and cowpea [15]. however, probably due to hot weather condition, cassava root is not being cultivated in gashua. thus, it is being purchase from nguru and tulo-tulo in yobe state and also from kwarin kanwa in niger republic, boiled cassava root is only snack of root and tuber crops sold in gashua throughout the year. it is cheaper in price, always available and selling on all streets of gashua. people living in gashua irrespective of age, gender, religious affiliation, educational background and social class consume boiled cassava roots. since boiling reduces the cyanide content of cassava root and boiled cassava root is consumed throughout the year in gashua, there is need to investigate the consumption manner and consumers knowledge about nutrient contents and safety level of boiled cassava root. 2. materials and methods 2.1 study location the study was carried out in gashua, the headquarters of bade local government of yobe state north-east, nigeria. 2.2 methodology well-structured questionnaire were administered to one hundred and fifty (150) respondents. information on their demographic characteristics and also on their knowledge about boiled cassava root sold in gashua, yobe state, nigeria was gathered. data analysis the data were analyzed with descriptive statistic which includes frequency counts and percentages. 3. results and discussion figure 1 shows the frequency and percentage of respondents’ ages that consume boiled cassava root. the ages of respondents are grouped as 19 28 years (34.67%), 29 38 years (32.00%), 39 48 years (21.33%), 4958 years (9.33%) and above 58 years (2.67%). the result showed that majority of ages of respondents that consuming boiled cassava root are the young people (19 – 28 years). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 saheed adewale omoniyi, phidelia ramatu waziri-ugwu, consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 315 – 320 317 52 48 32 14 4 35 32 21 9 3 0 10 20 30 40 50 60 19-28 29-38 39-48 49-58 above 58 f r e q u e n c y a n d p e r c e n t a g e age groups of respondents frequency percentage fig.1. frequency and percentage of age groups of respondents table 1 shows the demographic characteristic of the respondents. the result showed that majority of the respondents were male (70.70%) and married (61.34%). also, majority of the respondents were university graduates (54.67%) and civil servant (72.00%). table 1: demographic characteristics of respondents parameter frequency percentage (%) gender male 106 70.70 female 44 29.30 marital status single 56 37.33 married 92 61.34 separated 2 1.33 educational background university 82 54.67 polytechnic 12 8.00 college of education 16 10.67 college of agriculture 6 4.00 secondary school 34 22.66 occupation civil servant 108 72.00 politicians 4 2.66 business personnel 10 6.67 students 28 18.67 figure 2 shows the frequency and percentage of respondents’ source of safety information for consuming boiled cassava root. majority of the respondents got the information from school (42.70%) while others got their information from radio (12.00%), television (5.30%), newspaper (1.30%), internet (4.00%) and parent (34.70). fig. 2. source of safety information of respodents for consuming boiled cassava root figure 3 shows the frequency and percentage of quality attributes of boiled cassava root preferred by respondents. the quality attributes of boiled cassava root preferred by respondents are taste, texture and aroma. however, majority of the respondents preferred taste (88.00%) while others preferred texture (8.00%) and aroma (4.00%). 132 12 6 88 8 4 0 50 100 150 taste texture aroma f r e q u e n c y a n d p e r c e n t a g e quality attributes of boiled cassava root frequency percentage fig.3. frequency and percentage of quality attributes of boiled cassava root food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 saheed adewale omoniyi, phidelia ramatu waziri-ugwu, consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 315 – 320 318 figure 4 shows the frequency and percentage of food items consumed with boiled cassava root by respondents. however, the food items are non-protein foods which include salt (17.33%), pepper (20.00%), salt/pepper (14.67%) and salt/pepper/maggi (48.00%). 26 30 22 72 17 20 15 48 0 10 20 30 40 50 60 70 80 f r e q u e n c y a n d p e r c e n t a g e foods consumed with boiled cassava root frequency percentage fig.4. frequency and percentage of food items consumed with boiled cassava root table 2 shows the respondents knowledge about boiled cassava root. majority of the respondents (56.00%) are not aware that cassava roots contained anti-nutritional factors. this is in line with thomas and philips [8] who revealed that cassava processors are not aware that cassava roots contained cyanide content. however, out of minority (44.00%) respondents that aware that cassava roots have anti-nutritional factors, most of them got their information from school (32.00%) while others got their information from radio (5.33%), television (1.33%), newspaper (1.33%), internet (2.68%) and parent (1.33%%). majority of the respondents (94.70%) did not suffer from any ailment after eating boiled cassava root. the minority of the respondent (5.30%) reported that they suffered stomach ache after consuming boiled cassava root. table 2 respondents’ knowledge about boiled cassava root question yes no if yes, how? frequency percentage frequency percentage source frequency percentage are you aware that cassava roots have anti-nutritional factors? 66 44 84 56 school 48 32.00 radio 8 5.33 television 2 1.33 newspaper 2 1.33 internet 4 2.68 parent 2 1.33 have you suffered from any ailment after eating boiled cassava root? 8 5.30 142 94.70 stomach ache (ailment) 8 5.30 do you know that cassava roots have little nutrient content? 98 65.30 52 34.70 school 76 50.70 radio 8 5.30 internet 8 5.30 parent 6 4.00 do you like eating it with protein based food? 108 72 42 28 beans 80 53.30 bambara nut 6 4.00 pigeon pea 2 1.30 eggs 10 6.70 groundnut cake 6 4.00 meat 4 2.70 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 saheed adewale omoniyi, phidelia ramatu waziri-ugwu, consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 315 – 320 319 the stomach ache can be attributed to fact that the consumed boiled cassava roots were not thoroughly boiled by the seller. this problem can be overcome by boiling of cassava root thoroughly by the seller as this is in line with ezeigbo et al. [7] who reported that cooking of cassava root for ninety minutes resulted in over 93% decrease in cyanide. 65.20% of the respondents knew that cassava roots have little nutrient contents. the higher percentage is due to the fact that all respondents are literate. the 65.20% respondents got their information from school (50.70%), radio (5.30%), internet (5.30%) and parent (4.00%). since cassava root has little nutrient contents, 72.00% of the respondents like eating boiled cassava root with protein based food while the remaining 28.00% stated that they are addicted to eating it without protein based foods. however, majority (72.00%) of the respondents like eating boiled cassava root with protein based foods. out of 72.00% of the respondents, majority of respondents like eating boiled cassava root with beans (53.30%), while other respondents like eating it with bambara nut (4.00%), pigeon pea (1.30%), eggs (6.70%), groundnut cake (4.00%) and meat (2.70%). since, majority of respondents like eating boiled cassava root with protein based foods especially beans, the combinations of two foods comprises high carbohydrate and protein and this is in agreement with omeire [16] who revealed that proteins of cassava root are rich in arginine but poor in others amino acids, thus it need to be complimentary with protein based foods. also, consumption of boiled cassava root with protein based foods will prevent some protein-deficient diseases as akoja and mohammed [17] reported that the utilization of cassava is limited due to lesser quantity of protein contents and its consumption has been resulted with malnutrition. table 3 shows respondents means of eating, purchasing time and price of boiled cassava root. majority of the respondents (54.70%) are consuming boiled cassava root as meal while remaining 45.30% are consuming it as snack. table 3 respondents means of eating, purchasing time and price of boiled cassava root question means of eating frequ ency percentage snack 68 45.30 meal 82 54.70 time of purchasi ng and eating per day one 134 89.34 two 8 5.33 three 8 5.33 amount bought at a time ₦10 -₦50 ($0.03 -$0.14) 58 38.67 ₦60 -₦100 ($0.17 -$0.28) 48 32.00 ₦110 -₦150 ($0.31 -$0.42) 14 9.33 ₦160 ₦200 ($0.45 -$0.56) 24 16.00 ₦210 -₦250 ($0.58 -$0.69) 6 4.00 this could be attributed to the fact that boiled cassava root can be bought at cheaper price. the respondents are purchasing and consuming boiled cassava root one or two or three times daily with majority (89.34%) consuming it one time daily. the amounts of boiled cassava root bought at a time range from ₦10 ₦250 ($0.03 $0.69). however, majority of the respondents (38.67%) bought boiled cassava root within the range of ₦ 10 ₦50 ($0.03 $0.14) while others are ₦60 ₦100 [$0.17 $0.28] (32.00%), ₦110 ₦150 [$0.31 $0.42] (9.33%), ₦160 ₦200 [$0.45 $0.56] (16.00%) and ₦210 ₦250 [$0.58 $0.69] (4.00%). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 saheed adewale omoniyi, phidelia ramatu waziri-ugwu, consumers knowledge and consumption manner of boiled cassava root sold in gashua, yobe nigeria, food and environment safety, volume xviii, issue 4 – 2019, pag. 315 – 320 320 4. conclusions the study showed that the majority of the respondents are not aware that cassava roots have anti-nutritional factors but they aware that it is safe to be consumed. also, majority of the respondents knew that cassava roots have little nutrient contents, but consuming boiled cassava root as meal with salt, pepper, and maggi which are non-protein foods, and they like to be eating it with protein based foods especially beans. 5. references [1]. lasekan, o., hosnas, r., ng, s., lin, m., azeez, s., teoh, l., gholivand, s. and shittu, r. identification of aromatic compounds and their sensory characteristics in cassava flakes and “garri” (manihot esculenta crantz). cyta – journal of food, 14(1): 154-161, (2016). [2]. aniedu, c. and omodamiro, r. m. use of newly bred β-carotene cassava in production of valueadded products: implication for food security in nigeria. global journal of science frontier research agriculture and veterinary sciences, 12(10): 11-16, (2012). [3]. falola, a. o., olatidoye, o. p., balogun, i. o, and opeifa, a. o. quality characteristics of cookies produced from composite flours of cassava and cucurbita mixta seed. journal of agriculture and veterinary sciences, 3:1-12, (2011). [4]. guédé, s. s., traoré, s. and brou, k. assessment of cyanide content in cassava (manihot esculenta crantz) varieties and derived products from senegal. international journal of nutrition and food sciences, 2(5): 225-231, (2013). [5]. amira, p. o., daramola, a. s. and atolani, s. t. effect of drying on some antinutritional factors present in bitter cassava (manihot utilisima) and sweet cassava (manihot palmata). international journal of engineering science and innovative technology, 3(4): 368-372, (2014). [6]. omeire, g. c., iwe, m. o. and uchechukwu, n. effect of extrusion variables on the hydrogen cyanide and haemagglutinin content of extruded blends of cassava products and african yam bean. food science and quality management, 6: 23-31 6, (2012). [7]. ezeigbo, o. r., ekaiko, m. u. and ibegbulem, z. o. effect of cooking time on starch and cyanide contents of freshly harvested cassava tubers used for tapioca production. british biotechnology journal, 8(4): 1-6, (2015). [8]. thomas, k. a. and philips, o. n. assessment of food safety practices among cassava processors in selected rural communities of oyo state, nigeria. african journal of food, agriculture, nutrition and development, 15(4): 10317-10334, (2015). [9]. montagnac, j. a., davis, c. r. and tanumihardjo, s. a. processing techniques to reduce toxicity and antinutrients of cassava for use as a staple food. comprehensive reviews in food science and food safety, 8: 17-27, (2009). [10]. omosuli, s. v. effects of processing on the chemical and anti-nutritional properties of cassava roots. research and reviews: journal of botanical sciences, 3(2):27-31, (2014). [11]. salata, c. c., leonel, m., trombini, f. r. m. and mischan, m. m. extrusion of blends of cassava leaves and cassava flour: physical characteristics of extrudates. food science and technology campinas, 34(3): 501-506, (2014). [12]. ojo, r. j., obi, n. p., akintayo, c. o. and adebayo-gege, g. i. evaluation of cyanogen contents of cassava and cassava based food products in karu, nasarawa state, north central nigeria. iosr journal of environmental science, toxicology and food technology, 6(1): 47-50, (2013). [13]. adebayo-oyetoro, a. o., oyewole, o. b., obadina, a. o, and omemu, m. a. cyanide and heavy metal concentration of fermented cassava flour (lafun) available in the markets of ogun and oyo states of nigeria. international journal of biological, biomolecular, agricultural, food and biotechnological engineering, 7 (7): 356-359, (2013). [14]. sunama, a. a. a history of bade in the pre-colonial period. boga press, 38 adelaja street, mokola, ibadan, 1-3, (2010). [15]. suleiman, a. s. the great bade emirate history, culture and economic perspective. aje printers nig. limited, garki, abuja, 79, (2007). [16]. omeire, g. c. amino acid profile of raw and extruded blends of african yam bean (sphenostylis stenocarpa) and cassava flour. american journal of food and nutrition, 2(3): 6568, (2012). [17]. akoja, s. s. and mohammed, a. o. evaluation of the proximate, pasting and sensory characteristics of cassava flour (fufu) fortified with pigeon pea flour. pakistan journal of nutrition, 10(2): 124-128, (2011). doi: https://doi.org/10.4316/fens.2021.006 49 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1, 2021, pag. 49 52 good hygiene practices as an important program for safety of final products *ljubica trajkoska1, maja trajanoska1, biljana trajkovska1, ljupche kochoski1, vesna karapetkova-hristova1 1faculty of biotechnical scieneces, university st. kliment ohridski, ljubicaatrajkoska@gmail.com st. partizanska bb,bitola, north macedonia, info@fbn.uklo.edu.mk *corresponding author received 25th october 2020, accepted 30th march 2021 abstract: in order to meet the requirements regarding food safety, in addition to the haccp system, food operators are required to adopt, implement and document good hygiene practice (gmp), good manufacturing practice (gmp), standard operating procedures (sop) and standard sanitary operating procedures (ssop). therefore, the purpose of our research was to determine the importance of implemented hygienic and manufacturing practices in the meat industry in the municipality of prilep. analysis of the sensory, physicochemical and microbiological parameters of water, as well as the presence of listeria monocytogenes on the contact surface and in thin sausage (vacuum packed) was performed. the results of the water analysis show that it is safe and does not lead to additional contamination of sausages. in addition, the absence of listeria monocytogenes is a result of well-established practices regarding the microbiological quality of sausages. keywords:ghp, gmp, security, sausage. 1. introduction today's urbanization conditions, as well as global environmental pollution, make food safety a major global problem [1], while protecting food sources is imperative for protecting human health [2]. in the food sector the implementation of good hygiene practice (ghp) and good manufacturing practice (gmp) ensures the creation and maintenance of favorable production conditions [3], with the aim of improving the safety of the finished product [4]. meat is a food that is an integral part of daily diet, but in well-developed countries its use is limited due to health risks [5], and because it easily becomes subject to microbial contamination [6]. the most important microbiological causes of meat contamination are: listeria monocytogenes, clostridium botulinum, salmonella spp., escherichia coli and yersinia enterocolitica [7]. listeria monocytogenes is the only psychotropic anaerobic/ facultative anaerobic bacterium that causes contamination of thermally unprocessed meat and all processed meat products obtained from it, including readytoeat food [8]. 2. matherials and methods the research was conducted in a meat industry (facility for production of smoked meat products, meat preparations and minced meat) in the prilep region for a period of one year. samples were taken ones a month from contact surface (n=12), finished product (n=12) and water (n=12). the water tests included analyzes of sensory, physicochemical, and microbiological parameters. samples of water were taken in glass sterile bottles. chemical parameters ph (iso 10523: 2012), nitrates (iso 6777: 1993) and mailto:ljubicaatrajkoska@gmail.com mailto:info@fbn.uklo.edu.mk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 ljubica trajkoska, maja trajanoska, biljana trajkovska, ljupche kochoski, vesna karapetkova-hristova, good hygiene practices as an important program for safety of final products, food and environment safety, volume xx, issue 1 – 2021, pag. 49 – 52 50 ammonium ion (iso 7393-3: 1990) were examined. microbiological analysis of e. coli (iso 9308-1), enterococci (iso 78992), pseudomonas aeruginosa (iso 162666) and total number of microorganisms at 22°c (iso 6461-2) and at 37°c (iso 6222) were performed. the methods used are in accordance with rulebook on water safety (official gazette of the republic of macedonia no. 46/2008). analyzes for the presence of listeria monocytogenes on the contact surface and in thin sausage (vacuum packed) were performed using the standard mak en iso 11290-1.2 method. representative samples for examination of the microbiological safety of the contact surface were taken from 50 cm2 of contact surface. for sausages analysis were taken five units (25 g) of one sample. for both analyzes the samples were placed in clean, sterile containers that provide adequate protection against further contamination. 3. results and discussion the quality of water must comply with the standards for safe drinking water, because can be an ingredient in many foods [9]. according to the results shown in table 1, it can be observed that the analyzed parameters are within the permissible limits in accordance with the approval with the authorization [10]. according to [11] the low values obtained for the presence of nitrates and residual chlorine are indicators of good water quality and the absence of pathogenic microorganisms (table 1). table 1 physicochemical and microbiological analysis of water parameters tested (n=12) results output permissible limits ph 7.3 6.5-9.5 nitrates (mg / l) 0.2 50 chlorine (mg / l) 0.3 0.3-0.5 e. coli 0/100 ml 0/100 ml enterococci 0/250 ml 0/250 ml pseudomonas aeruginosa 0/250 ml 0/250 ml total number of microorganisms at 22 °с 5/1 ml 100/1 ml total number of microorganisms at 37 °с 0/20 ml 20/1 ml the most important sensory parameters investigated in water are color, taste, and odor [12]. accordingly, our results indicate that water meets the requirements for use in the food sector as there are no significant changes in it. contact surfaces in food industry are particularly important as a potential source of contamination. the quality, durability and safety of meat products largely depend on cleaning, washing, and disinfecting equipment and contact surfaces [13]. specifically, if the equipment is contaminated with listeria monocytogenes it may be the reason for sausage contamination as a final product during production [14]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 ljubica trajkoska, maja trajanoska, biljana trajkovska, ljupche kochoski, vesna karapetkova-hristova, good hygiene practices as an important program for safety of final products, food and environment safety, volume xx, issue 1 – 2021, pag. 49 – 52 51 table 2 results of the analyzed swabs of the contact surface (slides) and thin sausage (vacuum packed) for the presence of l. monocytogenes parameters tested (n=12) results output l. monocytogenes in contact surface swabs negative l. monocytogenes in thin sausage (vacuum packed) negative the absence of listeria monocytogenes from stainless steel equipment (table 2) was determined; the results indicated a well-implemented ghp [15]. starting from the fact that each meat industry is obliged to make a microbiological analysis on the presence of listeria monocytogenes in the finished product in accordance with the marketing authorization [16], the contaminated meat products with this bacterium represent a serious public health hazard [7]. according to [8] the absence of listeria monocytogenes in the final product is due to the effectiveness of the fermentation, maturation and drying of the products. also, absence of listeria monocytogenes from the equipment (table 2) and appropriate application of the technological procedure decrease in the contamination rate [17]. final products are likely to be contaminated with listeria monocytogenes during storage time if hygienic conditions are unsatisfactory [18] which in turn constitutes a risk of production and sales losses. 4. conclusion in the context of the above results, it can be concluded that well-implemented ghp and gmp in the food industry result in the production of a safe product. in order to get a safe food product we must take care of hygiene throughout the production process. in the case of deviations from the prescribed norms in accordance with the veterinary measures and controls for the protection of public health, a program of corrective action aimed at the introduction and implementation of corrective measures to eliminate the causes of non-compliance should be implemented. 5. references [1]. j. c. h. godfray, p. aveyard, t. garnett, w. j. hall, j. t. key, j. lorimer, t. r. pierrehumbert, p. scarborough, m. spingmann & a. s. jeb. meat consumption, health, and the environment. doi:10.1126/science.aam5324 (2018) [2]. m. minehan. green, healthy and eat meat? (a mixed methods investigation into how meat is used and viewed by meat – eaters in australia) (2013) [3]. asian productivity organization – apo. quality enhance in food processing through haccp. isbn:92-833-7041-4. (2005) [4]. j. knaflewska & e. pospiech. quality assurance systems in food industry and health security of food. acta sci. pol., technol. aliment. 6(2), 75-85, (2007) [5]. j. bereznicka & t. pawlonka. meat consumption as an indicator of economic well– being – case study of a developed and developing economy. acta sci. pol. oeconomia 17 (2), 17-26, (2018) [6]. soepranianondo, к. d. wardhana, budiarto & diyantoro. analysis of bacterial contamination and antibiotic residue of beef meat from city slaughterhouses in east java food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 ljubica trajkoska, maja trajanoska, biljana trajkovska, ljupche kochoski, vesna karapetkova-hristova, good hygiene practices as an important program for safety of final products, food and environment safety, volume xx, issue 1 – 2021, pag. 49 – 52 52 province, indonesia. vet world. 2019; 12(2), 243-248, (2019) [7]. m. pal, f. d. dana, a. patel & y. ayele. microbiological and hygienic quality of meat and meat products. beverage and food world. 45(5) (2018) [8]. n. zdolec, l. kozacinski, m. hadziosmanovic, z. cvrtila, & i. filipovic. inhibition of listeria monocytogenes growth in dry fermented sausages. veterinarski arhiv. 77 (6), 507-514, (2007) [9]. wujie, zhufei & xujing. the influence of water quality on food quality and the treatment of water for food processing. international conference on environmental science and information application technology. 10, 2671-2676, (2011) [10]. official gazette of the republic of macedonia no. 46/2008 (rulebook on water safety) [11]. o. a. fadrian & m. s. mammba. analysis of nitrates and nitrites in some water and factory effluent samples from some cities in swaziland. bull. chem. soc. ethipo. 19(1), 35-44, (2004) [12]. s. grujic, b. odzakovic & m. ciganovic. sensory analysis as a tool in the new product development. proceedings of ii international congress food technology quality and safety – novi sad, serbia, 325-330, (2014) [13]. g. p. crandall, a. c. o`bryan, m. e. martin, h. m. kuefner, s. pendleton, m. e. shannon, a. j. marcy & c. s. ricke. efficacy of cleaning and sanitizing agents against attached listeria monocytogenes on meat slicer components. food protection trends, 32(2), 68-72, (2012) [14]. b. r. tompkin. control of listeria monocytogenes in the food-processing environment. journal of food protection. 65(4), 709-725, (2002) [15]. a. r. bafanda, s. khandi, u. sheikh, a. minhaj khateeb & t. pilli. meat hygiene and associated health hazards awareness among consumers of jammu district of jammu and kashmir. current journal of applied science and technology. 23. 1-11. 10.9734/cjast/2017/36045, (2017) [16]. official gazette of the republic of macedonia no. 100/2013 (rulebook on special requirements relating to microbiological food criteria) [17]. d. thevenot, l. m. delignettemuller, s. christieans & c. vernozyrozand. prevalence of listeria monocytogenes in 13 dried sausage processing plants and their products. international journal of food microbiology. 102, 85-94, (2005) [18]. h. daskalov, j. momfre & n. j. sofos. survival and growth of listeria monocytogenes on sausage formulated with inoculated and stored rework product. food control .17, 981-986, (2006) issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xx, issue 3 30 september 2021 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania microsoft word 9 shevchenko_final.doc 192 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 192 196 desaturation of cultural media in processes of anaerobic fermentation *olexandr shevchenko1, anatoliy sokolenko2, konstantin vasylkivsky2, oleg stepanets2 1 department of processes and apparatus for food production, national university of food technologies 2 department of mechatronics and packaging machinery, national university of food technologies volodymyrska str. 68, kyiv, ukraine, 01601, tmipt@ukr.net, * corresponding author received 5th february 2018, accepted 19th june 2018 abstract: the article deals with the analysis of transients in gas-liquid media with the self-synthesis of the gas phase. it is shown that the restriction of the mass transfer intensity in the culture media of the alcoholic and brewing industries is accompanied by the presence of transients in the state of saturation on со2. it is determined that the level of carbon dioxide accumulation increases due to physical and hydrostatic pressures and the latter one is determined by the geometrical parameters of fermentation vehicles, but the quantitative desaturation per unit volume of the medium does not depend on hydrostatic pressure. quantitative desaturation of co2 depends on the pressure differences in the ingenious volume. it is shown that the restoration of the sorption properties of the liquid phase is possible due to combinations of reduction and subsequent increase of physical pressure. the analytical dependences on determination of interconnections of physical parameters in digestible media are given. keywords: desaturation, gas-liquid medium, gas-retaining ability, mass transfer, hydrostatic pressure, solubility. 1. introduction formulation of the problem. anaerobic fermentation of sugar-bearing media under the action of yeast cells is accompanied by the formation of a new cellular substance, the acceptance and assimilation of substances from the external medium, the splitting and removal of unnecessary and harmful compounds and the transport of substances within the cell. the energy supply of the processes is achieved thanks to the breath that begins with the splitting of glucose. the result of such transformation is the accumulation of ethanol and carbon dioxide in the medium. the presence of ethanol and carbon dioxide is accompanied by an increasing osmotic pressure, which ultimately limits the concentration of ethanol in the range of 8 ... 10% and a maximum of 12% with the use of osmophilic yeast [1-3]. traditionally, these restrictions are associated with ethanol and its osmotic pressure, and the presence of dissolved co2 in the fermented medium is an additive part [4]. in accordance with the van-hoff law and based on the principle of superposition [5], the total osmotic pressure of dissolved substances in the medium is determined by the dependence:       v rt m tm m tm m tm co co al al s s оsm          2 2 , where  tms ,  tmal and  tmсо2 , mass of sugar, alcohol and carbon dioxide respectively as a function of time t; sm , food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olexandr shevchenko, anatoliy sokolenko, konstantin vasylkivsky, oleg stepanets, desaturation of cultural media in processes of anaerobic fermentation, food and environment safety, volume xvii, issue 2 – 2018, pag. 192 – 196 193 alm and 2com the molecular masses of substances respectively in volume v of the solution; r universal gas constant, j / (mol*k); t absolute temperature, k at the end of fermentation when   0еі tm , we have:     v rt m tm m tm co eco al еal osm          2 2 , where еt is the final time of the process;  еco tm 2 the final mean of the solution of carbon dioxide, due to the presence of hydrostatic pressure. calculations show that for alcoholic fermentation, 12% alcohol solution at a temperature t = 303 k creates an osmotic pressure of 6.58 mpa, while 2% solution of carbon dioxide leads to a similar index of 1.07 mpa. for the close values of the molecular weights (46 and 44, respectively), the final difference in osmotic pressure is explained by the limited solubility of co2 in accordance with henry's law, which is reflected by the equation of the material balance:  2соals mmm , by which the main part of carbon dioxide is removed in the form of a dispersed gas phase. the formation of the latter begins when the concentration of co2 reaches saturation constant sс : kpсs  where k is the henry’s constant, depending on the nature of the gas and liquid phases and the temperature of the medium; p partial pressure of the gas phase. under the condition of anaerobic fermentation, the partial pressure is equal to the physical pressure. the latter means the presence of a concentration gradient in connection with hydrostatic pressure. by endogenous processes of synthesis of ethanol and carbon dioxide there are opposite transport flows on the interfacial surface of microorganisms and fluid medium. when there is unbounded solubility of ohhc 52 in water, the saturation of the medium on co2 creates a barrier to mass return and the preconditions for the formation of gas microbubbles. the resistance to the mass transfer in a liquid film at the interface between the liquid and gas phases for the limited soluble gases is the main component [6, 10], and this hypothesis is hypothetically transferred to the surface "cell-medium". transport of co2 through the cytoplasmic and external shell has its own peculiarities and is carried out at the molecular level. at the same level, the penetration through the liquid film into the liquid phase is carried out. at the same time, the state of saturation of the medium is the reason of the resistance to mass transfer and its termination under certain conditions. the course of processes of anaerobic fermentation leads to the conclusion that the state of the liquid phase of the medium is noticeably close to the full saturation and depends on its hydrodynamic state due to the interaction of the dispersed gas and liquid phases. in this case, there is the effect of pressure in the volume of the gas phase, the geometry of the equipment, the hydrostatic pressure, the gas-holding capacity and the intensity of the circulating contours. the purpose of this study is related to the theoretical search for the mutual influence of these parameters for the restoration of sorption properties of culture media due to their forced desaturation. 2. materials and methods theoretical studies and phenomenological conclosions are performed on the basis of laws and principles that are involved in the interaction of chemical and biological processes of gas and liquid phases during food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olexandr shevchenko, anatoliy sokolenko, konstantin vasylkivsky, oleg stepanets, desaturation of cultural media in processes of anaerobic fermentation, food and environment safety, volume xvii, issue 2 – 2018, pag. 192 – 196 194 anaerobic fermentation. they include material and energy balances, pascal and archimedes laws, laws of the continuity of flows, henry, van-hoff [5], gay-lussac laws and le chatelier principle, principles of superposition and the most probable state [7]. the solution of the problem of restoring the sorption properties of culture media in co2 is proposed on the basis of variable pressures [8–9]. 3. results and discussion it is known that the ratio of material flows in the processes of fermentation is determined on the basis of the gay-lussac equation in the form: 88 2 92 2 180 2526126 coohhcohc  let the concentration of dissolved glucose be 160 kg / m3 at the initial fermentation condition, then the total cycle of synthesized co2 is 78.2 kg and ethyl – 81.8 kg. in recalculation on the number of moles of co2 we receive: 3.1777 44 78200 2 сом mol under normal conditions, the volume of gas is: 3.43 10 293314.83.1777 5 2    p rtm v co m3, and the average rate of synthesis of co2 is: 086.1 72 2.782.782  е со tdt dм kg/(hour.m. it is obvious that in the regimes of the lag phase and the digestion, the rate of synthesis is considerably smaller and the state of saturation on со2 is initially achieved on areas with lower hydrostatic pressure with a gradual transition to the full volume. from the beginning of the formation of the dispersed gas phase, it begins transition into a superficial volume of the gas phase with increasing pressure. let’s assume that this pressure in the sealed volume increases from 0.1 to 0.2 mpa, and the increase of saturation constant sс at a temperature of the culture medium of 30 ° c is 1.48 kg / m3 or in moles is 1480: 44 = 33.64 this process is responsible for the areas a-b on the isotherm (fig. 1). forced pressure decrease from 0.2 to 0.1 mpa is accompanied by the release of 1.48 kg of co2 from each cubic metric of the liquid phase, regardless of the value of hydrostatic pressure, since the dependence  рсс ss  is linear. to achieve the desired level of desaturation of the medium in the ingenious gas phase, a pressure of 0.1 mpa is maintained, and it means that the desaturated co2 is taken back from the apparatus (under normal conditions) in volume: 309.0 10 29314.364.33 52   соv m 3/(m3 of the medium). the transition from 2sс to 1sс occurs without going beyond the saturation state, but its positive effect relates to a sharp increase in gas-holding capacity, an increase in the volume of the gas-liquid system and an increase in the dynamics of the hydrodynamic regime. fig. 1. schedule of dependencies sс  рсs in isothermal processes t1 t2 t3 0 р1 p2 p cs а в cs2 cs1 δ с s food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olexandr shevchenko, anatoliy sokolenko, konstantin vasylkivsky, oleg stepanets, desaturation of cultural media in processes of anaerobic fermentation, food and environment safety, volume xvii, issue 2 – 2018, pag. 192 – 196 195 the short-term flow of such transient processes with intensive mass transfer leads the media to the indicator 1sс . at the same time, in an ingenious volume at a pressure of 0.1 mpa the amount of carbon dioxide that remains will be: ingenious2 vм со  , where ρ is the specific mass of со2, kg / m3, which is in moles   ingenious ingenious 3 73.22 44 10 2 v v м псо    at the average rate of synthesis of co2 086.1 2 сом kg/m 3, the recovery of the pressure at 0.2 mpa in the gravity process is achieved in time: 363.1 086.1 48.1 еt hours in this mode, the liquid phase is in saturation, and the dynamics of increase in pressure is reflected by the dependence:     ingenious ingenious73.2244 2 v rtv tm tр co і          where  tm co2 is the amount of synthesized co2 in the function of time. at the same time with the pressure the constant of saturation increases from 1sс to 2sс :                            gh v rtv tm ktkpс co iі   ingenious ingenious n 73.22 44 2 where h is the fluid value of the altitude coordinate in terms of the liquid phase. at constant rate of formation of со2 const dt dм со 2 we have a linear accumulation law: t dt dм м сосо 2 2  . under these conditions, the pressure change is reflected by the linear law (fig. 2). it is important that at the same time, the liquid phase both for the reduction of pressure and its growth is in a state of saturation, which limits the intensity of mass transfer and the final level of accumulation of alcohol. the solution of the problem will be restoring the sorption possibilities of the culture medium due to the rapid increase in pressure in the ingenious volume after the completion of the desaturation phase. such transition leads the system to a state when the fluid concentration of dissolved carbon dioxide is iс ( 2si cc  ). it is obvious that the fluid concentration is a function of time  tсс іi  and during the second phase it is reaching the value 2si cc  . the fulfillment of this condition means the necessity of another reduction of pressure in the system to a magnitude p1. the control over phases is monitored by the values of pressure p1 and p2 with the phase at the point a at the pressure p1 to complete degassing. 4. conclusion the analysis of the transition processes in the gas-liquid media with the self-synthesis of the gas phase allows drawing the following conclusions: fig.2. graphic dependence of the pressure change in the ingenious volume рі м (t) со2 22.73ρrt food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 olexandr shevchenko, anatoliy sokolenko, konstantin vasylkivsky, oleg stepanets, desaturation of cultural media in processes of anaerobic fermentation, food and environment safety, volume xvii, issue 2 – 2018, pag. 192 – 196 196 1. modern technologies of anaerobic fermentation of alcohol and brewing fields are accompanied by the presence of digestible media in conditions which are close to their saturation states on со2. the latter limits the intensity of the mass transfer of carbon dioxide on the surfaces of the phase separation and the final result of the accumulation of ethyl alcohol in the alcohol industry. 2. hydrodynamic modes in the media depend on the gas-holding ability, which in other equal conditions is determined by the ratios of the geometric parameters of the apparatus and media respectively. 3. the levels of co2 accumulation increase due to hydrostatic pressures, but the quantitative desaturation from the unit of the volume does not depend on hydrostatic pressure, but it depends on pressure differences (p2 to p1). 4. restoration of the sorption properties of the culture media is possible only with combinations of sharp reduction in pressure and subsequent increases in pressures in the gas ingenious volume. 5. references [1]. shevchenko o., sokolenko a., vasylkivsky k., vinnichenko i. the qualitative properties of culture media, scientific works of the national university of food technologies, 23(2): 131–139, (2017). [2]. shiyan p., sosnytsky v., oliynychuk s. innovative technologies of the alcohol industry. theory and practice, askania, kyiv, (2009). [3]. shevchenko o., vynnychenko i., stepanets a., boyko o. features of transformations of material and energy flows in fermentation media, scientific works of national university of food technologies, 23(3): 107–115, (2017). [4]. sokolenko a., shevchenko o., makcymenko i. osmotic pressure in fermentation media technologies, ukrainian food journal, 6(1): 134–140, (2017). [5]. holtzer a., holtzer marilyn f., use of the van't hoff relation in determination of the enthalpy of micelle formation, the journal of physical chemistry, 78(14): 1442–1443, (2002). [6]. sokolenko a., mazaraki a., shevchenko o. intensification of heat and mass exchange processes in food technologies: monograph, phoenix, kyiv, (2011). [7]. atkins p., de paula ju., physical chemistry (8th ed.), w.h. freeman and company, (2006) [8]. hohmann s., osmotic adaptation in yeastcontrol of the yeast osmolyte system, international review of cytology, 215: 149–187, (2002). [9]. anderson t.m., industrial fermentation processes, encyclopedia of microbiology (third edition): 349–361, (2009). [10]. hohmann s. osmotic adaptation in yeast control of the yeast osmolyte system, international review of cytology: 149–187, (2002). 14 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 14 24 effect of season on the major physico-chemical parameters of honey product *destaw engidaw 1 , ketema alemayehu 2 , seid mustofa 2 , alebachew tilahun 3 1department of chemistry, college of natural and computational science, debre markos university, debre markos, ethiopia, p. o. box 269, destawtakele@gmail.com 2department of chemistry, college of natural and computational science, wolaitasodo university, wolaitasodo, ethiopia, p. o. box 138. 3school of veterinary medicine, wolaitasodo university, wolaitasodo, ethiopia, p. o. box 138. *corresponding author received 19th november 2019, accepted 30th march 2020 abstract the study was conducted to determine the major physic-chemical parameters and to investigate the effects of season on the quality of honey product collected from dawuro zone, southern nationalities of people region of ethiopia. the contents of physic-chemical parameters within the three honey production seasons ranged as: moisture content, 15-18%; electrical conductivity, 0.27-0.31ms/cm; ph, 4.0 4.5; free acidity, 18.7 24.7meq/kg; lactone acidity, 5.7 8.0meq/kg; hmf content, 16.1 20.3mg/kg; reducing sugars, 70 72g/100g and sucrose content from 2.4 2.8g/100g. this study showed that there were some significant variations for the contents of the different physicochemical parameters between the different honey production districts and seasons. it was confirmed that the results were below the maximum permissible limit to conform to international standards concerning the good quality of honey. although, the honey collected in this area should be consumed as fresh to avoid further contamination. moreover, further studies of other quality parameters of honey are needed. keywords: acidity, dawuro, electrical conductivity, hydroxymethylfural, moisture, sugars and quality 1. introduction honey is a thick, golden liquid produced by industrious bees and saved inside the beehive for eating during times of scarcity[1, 2].it is an ingredient of several industrial products and has been used for a long time because of its antioxidant and antibacterial properties[3-5]. the major ingredients of honey include a mixture of carbohydrates, organic acids, amino acids, proteins, minerals, vitamins and lipids [1, 6,7]. the presence of these major food ingredients in honey made human beings to use it as food sweetening and other human health care. as a result, human beings remove and collect it from the honeycomb and use for different usages without further purification and the quality of honey is unknown. different factors such as nectar source of the plant, the variety of bee, seasonal variation and improper handling and harvesting may contaminate and reduce honey quality[811]. there are different types of honey quality parameters, but in this study moisture content, electrical conductivity, ph and acidity, the content of 5hydroxymethyl-2-furaldehyde (hmf), and reducing sugars and sucrose contents were considered. carbohydrates are the primary components of honey especially for blossom honey glucose and fructose concentrations must http://www.fia.usv.ro/fiajournal mailto:destawtakele@gmail.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 15 be ≥ 60g/100g and sucrose concentration ≤5g/100g[2, 6, 12, 13]. water is the second-highest component of honey and low water content is desirable because honey may begin to ferment and lose its fresh quality if it is above 20% [2, 14]. electrical conductivity is an important criterion to determine the botanical origin of honey and depends on the mineral content, organic acids and proteins of honey [15-17]. naturally, all kinds of honey are acidic generally with the ph values between 3.4 and 6.1[1, 18]. the free acidity of honey was also mostly due to the presence of mineral and organic acids [19, 20]. honey might also contain 5hydroxymethyl-2-furaldehyde (hmf) which is a water-soluble heterocyclic organic compound derived from the acidcatalyzed dehydration of hexose sugars [21-23]. hmf is only present in trace amount in fresh honey and its concentration has been reported to increase with storage and the prolonged heating of the honey [10, 11, 24,25]. the amount of hmf present in honey is the reference used as a guide; the higher the hmf value, the lower the quality of the honey is considered. in dawuro zone there are different apiculture areas with high potential of honey production in the different season especially during october, december and around june but there was no documented data concerning the quality of honey which makes this zone impossible to be competent in the honey commercial market. therefore, the main purposes of this study were determining the quality of honey and to generate quality information that can be used as baseline data to create international market linkage. abbreviations: ec: electrical conductivity; fa: free acid; hmf: 5-hydroxymethyl-2-furaldehyde; la: lactone acidity; snnpr: southern nation nationalities and people region; sd: standard deviation; spss: statistical package for the social sciences 2. materials and methods 2.1. description of the study area the study was conducted in dawuro zone, snnpr, ethiopia with four woredas (districts) namely loma, mareka, tocha and isara that were selected purposefully depending on the areas where the high honey product is available. dawuro zone is located in southern nation nationalities and people regional (snnpr) state of ethiopia with an approximately 500km from addis ababa the capital city of ethiopia and it is found between 6o36' to 7o21' north latitudes and 36o68' to 37o52'east longitudes with an altitude of 500 to 3000meters above sea level. 2.2. sample collection the honey samples were collected in the three different honey production seasons in a year; december (2015), june and october (2016) for each woreda. the collected honey samples were labeled and transported to wolaitasodo university and stored at room temperature for 5 6 months until sampling preparation and analysis. 2.3. preparation and analysis of the honey sample unwanted materials such as wax, sticks, dead bees and particles of combs were removed by straining the samples by clean cloth before analysis. all measurements during each season were carried out in triplicate to determine the physicochemical properties of honey. the moisture content of each honey sample was determined by weighing 5g of the sample on a pre-weighed drying dish and the samples were dried at 105oc to food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 16 constant weight in an oven (dhg-9055a, yunboshi, jiangsu, china)[26]. moisture (%) = 𝑊1−𝑊0 𝑚 𝑥100 where wo is the weight of the empty dish, w1 is the weight of dried sample + dish and m mass of sample taken the electrical conductivity of each sample was measured on a solution of 20% honey dray matter at 20 0c with a previously calibrated conductivity meter (hq40d, hach company, colorado usa). free acid, lactones, and ph were determined by equivalence point titration[14, 27]. five grams of honey [m] were dissolved in a few milliliters of water. the solution was transferred quantitatively to a 50 ml volumetric flask and filled to the mark with water. twenty-five ml of solution was dropped into a 250 ml beaker in the presence of a bar magnet. the initial ph (phi) was recorded and titrated first with the sodium hydroxide solution (up to 10 ml), (free acid determination) then (into the same beaker) with the sulphuric acid solution (up to the second equivalence point) for lactone determination. the free acidity (fa) is expressed in milliequivalents of sodium hydroxide required to neutralize 1 kg of honey. f.a. = v x t x (50/25) x (1000/m) the lactone acidity (la) is expressed in the same units: l.a. = [(10-v) x t 0.05 x v´] x (50/25) x (1000/m) note from the curve the free acidity neutralization volume in ml [v] and the sodium hydroxide excess neutralization volume (corresponding to ph 7) in ml [v´]. the amount of hmf was determined by measuring the uv absorbance of hmf at 284nm by using uv-visible digital spectrophotometer (model 371, electronics india, and new delhi india). to avoid the interference of other components at this wavelength, the difference between the absorbance of a clear aqueous honey solution and the same solution after the addition of bisulfate was determined. the hmf content was calculated after the subtraction of the background absorbance at 336nm [14]. the hmf content of the samples was calculated by the following formula: hmf (mg/kg) = (a284-a336) x149.7x5xd/w where a284 = absorbance at 284nm, a336 = absorbance at 336nm, 149.7 = factor w = weight of sample taken and d= dilution factor, if dilution is necessary the apparent reducing sugars and content of sucrose were determined by the modification of lane-eynonprocedure involving the reduction of soxhlet’s modification of fehling's solution by titration at the boiling point against a solution of reducing sugars in a honey solution using methylene blue as an internal indicator[14, 28]. two grams and six decigrams of honey were weighed and transferred to 500ml volumetric flask. five milliliters of standardized fehling a and b solutions were transferred to a 250ml erlenmeyer, with 7ml of water and 15ml of honey solution. the erlenmeyer was heated and 1ml methylene blue (0.2 %) was added. titration was conducted by adding the diluted honey solution until the indicator was decolorized. determination of sucrose content was carried out by inversion, adding 10ml of diluted hcl, 50ml diluted honey solution and water to a 100ml volumetric flask, heating in a water bath, then cooling and diluting to mark. finally, the lane-enyon method was applied and sucrose content was obtained by difference. the percentage of reducing sugar before and after inversion was calculated as follows; 𝐶 = 2 𝑊2 𝑥 1000 𝑌2 , where c = g reducing sugar before inversion/100g honey food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 17 w2 = weight (g) of honey sample and y2 = volumes (ml) of dilute solution consumed 𝐶′ = 2 𝑊2 𝑥 1000 𝑌2 , where c ′ = g reducing sugar after inversion/100g honey w2 = weight (g) of honey sample and y2 = volumes (ml) of dilute solution consumed the percentage of non-reducing sugar (sucrose) was estimated by subtracting reducing sugar content before inversion from reducing sugar after inversion and then multiplying by a common factor: sucrose content (%) = (𝐶′ − 𝐶)𝑥0.95 2.4. data analysis the data were collected, coded and entered in a microsoft spreadsheet excel and analyzed in spss (version 16). anova was used to assess the association of each season and location on quality parameters. further analysis on differences among the means was determined using the tukey post hoc tests at a 5% significance level. 3. results the results of different physic-chemical parameters are shown in tables 1, 2 and 3 below for mentioned woredas; loma, mareka, tocha and isera during three honey production seasons. table 1 results of the physico-chemical parameters of honey in season one (december 2015) place physicochemical results moisture (%) ec (ms/cm) ph fa (meq/kg) la (meq/kg) hmf (mg/kg) reducing sugar g/100g sucrose g/100g loma 15.1± 0.2 0.31±0.02 4.1±0.1 20.3± 0.6 6.2 ± 0.3 19.0±0.4 73.1±0.5 3.9±0.1 mareka 20.0 ±0.7 0.22±0.02 4.1 ± 0.1 28.3± 0.6 9.9± 0.4 20.2±0.1 69.4±0.4 3.1±0.1 tocha 15.5 ±0.6 0.26±0.03 4.1± 0.1 20.8 ± 0.8 5.7 ± 0.3 18.5±0.3 71.4±0.6 2.2±0.2 isara 15.4± 0.5 0.45±0.03 3.9 ± 0.1 29.2 ± 0.7 10.1± 0.6 23.4±0.6 66.5±0.2 2.2±0.2 results represent the average of three measurements ± sd table 2 results of the physico-chemical parameters of honey in season two (june 2016) place physicochemical results moisture (%) ec (ms/cm) ph fa (meq/kg) la (meq/kg) hmf (mg/kg) reducing sugar g/100g sucrose g/100g loma 17.0± 0.3 0.27 ±0.03 4.3± 0.1 15.0± 0.6 4.2± 0.3 17.7±0.2 69.3±0.5 2.4±0.2 mareka 19.1± 0.4 0.26±0.04 4.2±0.2 23.7±0.7 4.7±0.4 20.0±0.5 69.7±0.2 2.3±0.1 tocha 18.1± 0.4 0.25± 0.04 4.4±0.2 17.2± 0.6 4.5± 0.2 16.1±0.3 66.9± 0.4 1.9±0.1 isara 16.9± 0.4 0.30±0.04 4.1±0.2 30.7±0.5 10.2±0.2 25.7±0.3 73.5 ± 0.2 2.9±0.2 results represent the average of three measurements ± sd table 3 results of the physico-chemical parameters of honey in season three (october 2016) place physicochemical results moisture (%/) ec (ms/cm) ph fa (meq/kg) la (meq/kg) hmf (mg/kg) reducing sugar g/100g sucrose g/100g loma 15.4±0.5 0.29±0.05 4.4±0.3 18.2±0.6 6.7±0.4 16.2±0.2 71.7±0.30 2.9±0.2 mareka 13.7±0.4 0.28±0.04 4.9±0.2 13.3±0.4 4.8±0.1 12.1±0.3 72.1±0.26 2.5±0.2 tocha 17.1±0.1 0.28±0.04 4.3±0.3 25.7±0.6 5.1±0.2 14.6±0.3 68.9±0.48 2.1±0.1 isara 13.5±0.7 0.32±0.04 4.5±0.2 17.5±0.4 6.2±0.3 21.6±0.2 75.4±0.42 3.7±0.2 results represent the average of three measurements ± sd food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 18 in season one (december 2015) the moisture contents of the samples varied from 15.1% in loma to 20.0%in mareka with the significant difference (p = 0) only between mareka and other three places. for the second season (june 2016), the moisture content varied from 16.9% in isara to 19.1% in mareka with significant differences between loma and mareka (p = 0), between mareka and isara (p = 0) and between tocha and isara (p = 0.02). the moisture content of honey during the last production season (october 2016) was ranged from 13.5% in isara to 17.1% in tocha with an insignificant difference (p = 0.95) only between mareka and isara. in season one the electrical conductivity of the samples ranged between 0.22ms/cm in mareka to 0.45ms/cm in isara with insignificant difference between loma and tocha (p = 0.20) and between mareka and tocha (p = 0.23), in season two it was between 0.25ms/cm in tocha and 0.30ms/cm in isara while in the third season electrical conductivity varied from 0.28ms/cm in tocha to 0.32ms/cm in isarain the four studied areas. during season two and three there was no significant difference in electrical conductivity (p≥ 0.4) between the four honey production areas. the ph value of the samples in season one ranged from 3.9 in isara to 4.1 in tocha, during the second season it was between 4.1 in isara and 4.4 in tocha and in the third season it was in the range of 4.3 in tocha to 4.9 in mareka which indicates the honey samples were acidic. the statistical analysis in all seasons showed that there was no significant difference (p ≥ 0.6) in ph value between different districts. the free acidity of the samples in the first season was between 20.3meq/kg in loma and 29.2meq/kg in isara while the lactone content was ranged between 5.7 to 10.1meq/kg. in this season free acidity showed the insignificant difference between loma and tocha (p = 0.81) and between mareka and isara (p = 0.4) while the lactone content showed the insignificant difference between loma and tocha (p = 0.45) and between mareka and isara (p = 0.88). in the second season, free acidity was ranged from 15.0meq/kg in loma to 30.7meq/kg in isara while lactone content was ranged from 4.2meq/kg in loma to 10.2meq/kg in isara. in this season there was a significant difference (p = 0) in lactone content only between isara and the other places. the free acidity of the honey samples in last season was between 13.3meq/kg in mareka and 25.7meq/kg in tocha with no significant difference (p = 0.31) only between loma and isarawhile the lactone content was between 4.8meq/kg in mareka to 6.7meq/kg in loma with no significant difference between loma and isara (p = 0.13) and between mareka and tocha (p = 0.56). the hydroxymethylfurfural(hmf) content of honey samples during the first season ranged between 18.5mg/kg in tocha and 23.4mg/kg in isara, for the second season between 16.1mg/kg in tocha to 25.7mg/kg in isaraand the third season it was ranged from 12.1mg/kg in maraqa to 21.6mg/kg in isara. based on the analysis there was no significant difference (p = 0.36) in hmf content only between loma and tocha for the first season. the content of reducing sugar in the first season was between 66.5g/100g in isara to 73.1g/100g in loma, for the second season it was ranged from 66.9g/100g in tocha to 73.5g/100g in isara with no significant difference (p = 0.54) between loma and mareka and for the last season reducing sugar was ranged from 68.9g/100g in tocha to 75.4g/100g in isarawith no significant difference (p= 0.52) between loma and mareka. the sucrose content during the first season was between 2.2g/100g and 3.9g/100g food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 19 with no significant difference (p = 0.92) between tocha and isara, for the second season it was ranged from 1.9g/100g in tocha to 2.9g/100g in isara with significant differences between loma and tocha (p = 0.02), mareka and isara (p = 0.02) and between tocha and isara(p = 0) whilst for the last season it was ranged from 2.1g/100g in tocha to 3.7g/100g in isara with no significant difference (p = 0.08) between loma and mareka. 4. discussion 4.1. moisture content the moisture content of honey samples in the four different honey production areas in dawuro zone during a year ranged from 15.3% in isara to 17.6% in mareka (table 4) with no significant difference (p ≥ 0.45) between the four honey production areas. table 4 total mean values of physicochemical parameters of honey in four places for dawuro district in a year place physicochemical results moisture (%) ec (ms/cm) ph fa (meq/kg) la (meq/kg) hmf (mg/kg) reducing sugar g/100g sucrose g/100g loma 15.8 0.29 4.3 17.9 5.7 17.6 71.4 3.1 mareka 17.6 0.26 4.4 21.8 6.5 17.4 70.4 2.6 tocha 16.9 0.27 4.3 21.2 5.1 16.4 69.1 2.1 isara 15.3 0.36 4.2 25.8 8.8 23.6 71.8 2.9 results represent the average of the sum of values obtained in the three honey production seasons (season 1, 2 and 3) for each place. based on the collection of the season the moisture content was ranged from 14.9% in season three to 17.8% in season two in a year (table 5) and the moisture content was significantly different (p = 0) only between season two and season three. the study showed that there were some honey production areas and seasons with lower and some with higher moisture contents, which could be due to the difference in the geographical area and climatic conditions. the higher the moisture content would lead to fermentation upon storage by osmotolerant yeasts and also an indicator of honey adulteration[29, 30]. the values in both honey production area and collection of the season showed that the honey samples in dawuro zone have a low possibility of fermentation that fairly agreed with different standards and unions which sets a moisture content below 21% [2, 14, 31]. table 5 total seasonal mean values of honey samples in dawuro district parameter moisture (%) ec (ms/cm) ph fa (meq/kg) la (meq/kg) hmf (mg/kg) reducing sugar g/100g sucrose g/100g season one 16.5 0.31 4.0 24.7 8.0 20.3 70.1 2.8 season two 17.8 0.27 4.2 21.6 5.9 19.9 69.9 2.4 season three 14.9 0.30 4.5 18.7 5.7 16.1 72.0 2.8 results represent the averages of the sum of values obtained in four honey production areas (loma, maraqa, tocha, and isara) for each season. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 20 comparable reports to our study area were given on guji zone of ethiopian honey [32]; with a moisture content of 14.28 15.12%, european atlantic area honey [4]and lazio region honey [33], which had an average moisture content of 17.6% and 16.36% respectively. therefore, the moisture content of our honey samples during the three production seasons and those reports point out good maturity of the samples and were considerably free of fermentation. 4.2. electrical conductivity the mean electrical conductivity values of honey samples in the four production areas varied from 0.26ms/cm in mareka to 0.36ms/cm in isara (table 4) with a significant difference (p ≤ 0.03) between isara and other production areas. these range of electrical conductivity values indicated that the honey samples produced in isara had high mineral, acid or protein contents and the color of the honey samples collected in this area was slightly black which might contribute to high electrical conductivity values[17]. the difference in electrical conductivity in different honey production places was due to the difference in environmental conditions and botanical origin. the average electrical conductivity of honey in terms of production seasons was between 0.27ms/cm in the second honey production season and 0.31ms/cm in the first honey production season (table 5). the post hoc multiple comparison tests (tukey) showed that there was no significant difference (p ≥ 0.28) in electrical conductivity value between the three honey production seasons in dawuro zone. it was observed that the honey samples during the first honey production season showed high electrical conductivity values than the second and third honey production seasons. the electrical conductivity value is important for differentiating honey of different botanical origin and indicated that the honey samples in all production areas and seasons were floral (blossom) types[2, 34]. comparable result to our study area was given in chubut, argentina with an average electrical conductivity value of 0.33ms/cm [35]. in contrary to our study high amounts of electrical conductivity values were reported from harenna forest honey, bale, ethiopia; 0.7ms/cm [28], west shewa zone, oromia region, ethiopia;0.380.65ms/cm [36] and honey from algeria;0.42 0.81ms/cm [37]. 4.3. ph, free and lactone acidity all the honey samples in the study areas were acidic with a ph value generally lying between 4.2 in isara to 4.4 in mareka (table 4) and showed no significant differences (p ≥ 0.5) between all honey production areas. based on the collection season the ph values ranged from 4.0 in the first honey production season to 4.5 in the third production season (table 5) with no significant difference (p = 0.07) between season one and season two. the average free acidity in different woredas ranged between 17.9meq/kg in loma to 25.8meq/kg in isara (table 4) with a significant difference (p = 0.01) between loma and isara. in the same way, the total average lactone acidity was ranged from 5.1meq/kg in tocha to 8.8meq/kg in isara with a significant difference (p ≤ 0.03) between isara and other honey production areas. the average values of free acidity in terms of collection season varied from 18.7meq/kg in the third honey production season to 24.7meq/kg in the first honey production season and average values of lactone content varied from 5.7meq/kg in the third season to 8.0meq/kg in the first season (table 5). based on the tukey test at 0.05 levels, there was a significant difference (p = 0.02) for free acidity and (p = 0.03) for lactone acidity between the first and third honey production seasons. these variations in acidity values have been food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 21 attributed to the difference in harvest season and are fairly below the maximum acceptable limit [2, 20,31]. previous studies carried out on honey quality in tepi areas of ethiopia varied between 3.36 and 4.26 from ph measurements and 18.3 25.3meq/kg from acidity measurements [38] and the study carried out in guji zone of ethiopiashowed an average ph value of 4.45 and 24.08meq/kg in acidity measurements [32]. abroad ethiopia analogous results were reported in turkish blossom honey with a ph of 3.68 4.65, free acidity of 3.86 30.42meq/kg and lactone content of 0.99 9.50meq/kg [39] and for australian honey with a ph of 4.02 4.69, free acidity of 10.25 20.34meq/kg except for the considerably high lactone content of 18.5 33.2meq/kg [40]. our study showed that none of the samples during the three honey production seasons exceeded the limit allowed by international regulations. consequently, the ph and acidity measurements in dawuro district indicated that the honey samples were nearly acidic which can inhibit the growth of microorganisms and implies low fermentation of honey. 4.4. hydroxymethylfurfural (hmf) the mean hmf value in the four honey production areas was ranged from 16.4mg/kg in tocha to 23.6mg/kg in isara (table 4) with a statistically significant difference (p = 0) between isara and the other honey production districts. the average hmf value of honey in terms of production seasons in a year varied from 16.1mg/kg in season three to 20.3mg/kg in season one (table 5) with no significant difference (p = 0.95) between season one and season two. these range of hmf values indicated that the content of hmf in season one was high compared to others. the values both in studied areas and collection season indicated that the hmf content did not exceed the international regulations which set a maximum hmf value of 80mg/kg for tropical countries [2, 31, 34]. the hmf content is an indicator of freshness and in fresh honey its value was very low; 1.89 8.24mg/kg in libyan honey [41]; and 0.84mg/kg in harenna forest honey, bale, ethiopia[28]. this study showed that the hmf value of honey in dawuro zone was considerably high after six months of storage at room temperature (< 25oc) which might be due to environmental, harvesting and storage conditions. previous studies carried out on malaysian honey which were stored for 3 6 months showed hmf content of 2.80 24.87mg/kg,but after 12 24 months storage, its value was increased from 128.19 1131.76mg/kg [24]. these hmf values indicate that honey must be handled under appropriate temperature and storage conditions. although dawuro district of ethiopia is under the tropical country, the hmf values were within the international standard limit and encouraged the quality criteria for honey. 4.5. reducing sugars and sucrose the average reducing sugar content for the different honey production areas varied between 69.1g/100g in tocha to 71.8g/100g in isara (table 4) and the values showed no significant difference between all the study districts (p ≥ 0.2). the average content of reducing sugars in terms of honey collection season varied from 69.9g/100g in season two to 72.0g/100g in season three (table 5) with no significant difference (p ≥ 0.1) between all collection seasons. determination of sucrose content is used to detect adulteration of honey with table sugar or to determine the amount of sucrose naturally found in honey[28]. the content of non-reducing sucrose in the different production areas was varied between 2.1 g/100g in tocha and 3.1g/100g in loma (table 4) and tukey test food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 22 indicated that there were significant differences (p = 0) in sucrose content between loma and tocha and between tocha and isara. sucrose content based on collection season was ranged from 2.4g/100g in season two to 2.8g/100g in season one (table 5). based on the tukey tests, there was no significant difference (p ≥ 0.1) in sucrose content between the three honey production seasons. the low amounts of sucrose content in our study (< 5%) indicated a good maturity of honey and good conversion of sucrose to fructose and glucose. all these sugar measurements met the requirements of blossom kinds of honey and showed the absence of adulteration. the variation in the collection season and region caused changes in the composition of honey [42]however, in our study the variations were not that much necessary. the sugar values of this study area were analogous with other reports such as ethiopian sekota honey; 63.4 71.7 g/100g of reducing sugars and 1.0 5.2g/100 of sucrose content[43] algerian honey; 62.8 70.0g/100g content of reducing sugars and 1.80 2.54g/100g in sucrose content[37]. turkish blossom honey showed 65.63 80.47g/100g of the sum of fructose and glucose but the high amount of sucrose; 2.85 8.44g/100g which could because of inadequate conversion of sucrose to glucose.[39]. european atlantic honey reported a slightly lower sum of fructose and glucose (65.6g/100g) and low sucrose content (0.37g/100g) than our study [4]. 5. conclusion the study showed that the variation in the honey production season and area had some effect on the major physicochemical parameters. however, the physicochemical parameters considered indicated that all the values were within the standard limits given by different organizations. therefore, the honey samples produced from dawuro district, snnpr ethiopia, could conform to international standards concerning a good level of quality which smooth the progress of its national and international commercialization. based on the aforementioned conclusion, the honey produced in this area should be consumed as fresh to avoid further contamination and further studies of other quality parameters of honey are needed. 6. acknowledgments the authors would like to thank wolaita sodo university for every support and mr. merga h/mariam from arba minch university who participated in sample analysis. 7. competing interests the authors declared that they have no competing interests. 8. reference [1]. baglio, e., chemistry and technology of honey production. springer briefs in molecular science, chemistry of foods, catania, italy, 2017. [2]. codex, a., revised codex standard for honey, codex stan. 12–1981, rev. 1 (1987), rev. 2 (2001). 2001: p. 1-8. [3]. alves, a., et al., antioxidant activity, quality parameters and mineral content of portuguese monofloral honeys. journal of food composition and analysis, 2013. 30(2): p. 130138. [4]. escuredo, o., et al., nutritional value and antioxidant activity of honeys produced in a european atlantic area. food chemistry, 2013. 138: p. 851-856. [5]. meo, s.a., et al., role of honey in modern medicine. saudi journal of biological sciences, 2017. 24(5): p. 975-978. [6]. bogdanov, s. and p. gallmann,authenticity of honey and other bee products state of the art : technical-scientific food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 23 information. 2008: eidgenössische forschungsanstalt f. milchwirtschaft. [7]. hermosín, i., r.m. chicón, and m. dolores cabezudo, free amino acid composition and botanical origin of honey. food chemistry, 2003. 83(2): p. 263-268. [8]. anklam, e., a review of the analytical methods to determine the geographical and botanical origin of honey. food chemistry, 1998. 63(4): p. 549-562. [9]. bilisik tosunoglu, a., et al., seasonal variation of collected pollen loads of honeybees (apis mellifera l. anatoliaca). grana, 2008. 47(1): p. 70-77. [10]. fallico, b., et al., effects of conditioning on hmf content in unifloral honeys. food chemistry, 2004. 85(2): p. 305-313. [11]. sahinler, n. and a. gul, effect of heating and storage on honey hydroxymethylfurfural and diastase activity journal of food technology, 2005. 3(2): p. 152-157. [12]. kucuk, m., et al., biological activities and chemical composition of three honeys of different types from anatolia. food chemistry, 2007. 100(2): p. 526-534. [13]. lawal, r., a. k lawal, and j. b adekalu, physicochemical studies on adulteration of honey in nigeria. pakistan journal of biological sciences 2009. 12(15): p. 1080 1084. [14]. bogdanov, s., et al., harmonised methods of the european honey commission. 1997. 28: p. 1-59. [15]. derebas, e., et al., physicochemical and residue analysis of honey from black sea region of turkey. fresenius environmental bulletin 2014. 23(1): p. 10-17. [16]. sancho, m., et al., relationships between electrical conductivity and total and sulphated ash contents in basque honeys. apidologie, 1991. 22(5): p. 487-494. [17]. terrab, a., m.j. diez, and f.j. heredia, palynological, physico-chemical and colour characterization of moroccan honeys: iii. other unifloral honey types. international journal of food science and technology 2003. 38(4): p. 395-402. [18]. pita-calvo, c. and m. vázquez, differences between honeydew and blossom honeys: a review. trends in food science & technology, 2017. 59: p. 79-87. [19]. mondragón-cortez, p., et al., physicochemical characterization of honey from the west region of méxico. cyta journal of food, 2013. 11(1): p. 7-13. [20]. perez-arquillué, c., et al., quality evaluation of spanish rosemary (rosmarinus officinalis) honey. food chemistry, 1994. 51(2): p. 207-210. [21]. pagan-torres, y., et al., production of 5-hydroxymethylfurfural from glucose using a combination of lewis and brønsted acid catalysts in water in a biphasic reactor with an alkylphenol solvent. acs, 2012. 2: p. 930-934. [22]. souza, r.o., et al., 5hydroxymethylfurfural (5-hmf) production from hexoses: limits of heterogeneous catalysis in hydrothermal conditions and potential of concentrated aqueous organic acids as reactive solvent system challenges, 2012. 3: p. 212-232. [23]. yuan, z., et al., catalytic conversion of glucose to 5-hydroxymethyl furfural using inexpensive co-catalysts and solvents. carbohydrate research, 2011. 346: p. 2019–2023. [24]. khalil, d., s.a. sulaiman, and s. gan, high 5-hydroxymethylfurfural concentrations are found in malaysian honey samples stored for more than one year. food and chemical toxicology, 2010. 48: p. 2388–2392. [25]. korkmaz, s.d., ö. kuplulu, and effects of storage temperature on hmf and diastase activity of strained honeys. ankara üniv vet fak derg, 2017. 64: p. 281-287. [26]. khalil, i.m., et al., biochemical analysis of different brands of unifloral honey available at the northern region of bangladesh. journal of medical sciences, 2001. 1(6): p. 385388. [27]. white, j.w., j. petty, and r.b. hager, the composition of honey. ii. lactone content. association of official agricultural chemists, 1958. 41(1): p. 194-197. [28]. belay, a., et al., physicochemical properties of the harenna forest honey, bale, ethiopia. food chemistry, 2013. 141(4): p. 33863392. [29]. azeredo, l.d.c., et al., protein contents and physicochemical properties in honey samples of apis mellifera of different floral origins. food chemistry 2003. 80: p. 249 254. [30]. viuda-martos, m., et al., aroma profile and physico‐chemical properties of artisanal honey from tabasco, mexico. international journal of food science and technology, 2010. 45(6): p. 1111-1118. [31]. qsae, honey specification: ethiopian standard, es 1202:2005. addis ababa, ethiopia:quality and standards authority of ethiopia(qsae). 2005. [32]. tesema, b., honeybee production and honey quality assessment in guji zone, ethiopia. journal of food processing & technology, 2015. 6(11): p. 1-3. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 1 – 2020 destaw engidaw, ketema alemayehu, seid mustofa, alebachew tilahun, effect of season on the majorphysicochemicalparameters of honeyproduct, food and environment safety, volume xix, issue 1 – 2020, pag. 14– 24 24 [33]. conti, m.e., lazio region (central italy) honeys: a survey of mineral content and typical quality parameters. food control, 2000. 11(6): p. 459-463. [34]. bogdanov, s. and p. martin, honey authenticity: a review. swiss bee research centre, 2002: p. 1-20. [35]. aloisi, p., determination of quality chemical parameters of honey from chubut (argentinean patagonia). chilean journal of agricultural research, 2010. 70(4): p. 640-645. [36]. mulugeta, e., et al., physicochemical characterization and pesticide residue analysis of honey produced in west shewa zone, oromia region, ethiopia. american journal of applied chemistry 2017. 5 (6): p. 101-109. [37]. khalil, d., et al., physicochemical and antioxidant properties of algerian honey. molecules, 2012. 17: p. 11199-11215. [38]. yadata, d., detection of the electrical conductivity and acidity of honey from different areas of tepi. food science and technology, 2014. 2(5): p. 59-63. [39]. tornuk, f., et al., quality characterization of artisanal and retail turkish blossom honeys: determination of physicochemical, microbiological, bioactive properties and aroma profile. industrial crops and products, 2013. 46: p. 124-131. [40]. ajlouni, s. and p. sujirapinyokul,hydroxymethylfurfuraldehyde and amylase contents in australian honey. food chemistry, 2010. 119(3): p. 1000-1005. [41]. ahmida, m., et al., physicochemical, heavy metals and phenolic compounds analysis of libyan honey samples collected from benghazi during 2009-2010 food and nutrition sciences, 2013. 4: p. 33-40. [42]. varis, a.-l., j. helenius, and k. koivulehto, composition and properties of finnish honey and their dependence on the season, region, bee race and botanical origin. journal of the scientific agricultural society of finland 1983. 55: p. 451-463. [43]. alemu, t., e. seifu, and a. bezabih,physicochemical properties of honey produced in sekota district, northern ethiopia. international food research journal, 2013. 20(6): p. 3061-3067. 1. introduction 2. materials and methods 2.1. description of the study area 2.2. sample collection 2.3. preparation and analysis of the honey sample 2.4. data analysis 3. results 4. discussion 4.1. moisture content 4.2. electrical conductivity 4.3. ph, free and lactone acidity 4.4. hydroxymethylfurfural (hmf) 4.5. reducing sugars and sucrose 5. conclusion issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xviii, issue 3 30 september 2019 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania review: the main raw materials used for production of 1st and 2nd generation bioethanol 48 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 48 68 review: main raw materials used for production of 1st and 2nd generation bioethanol * vasile-florin ursachi 1 , gheorghe gutt 2 1faculty of food engineering, ”ștefan cel mare” university, suceava, romania, florin.ursachi@fia.usv.ro *corresponding author received 15th february 2020, accepted 25th march 2020 abstract: in recent years, many countries have adopted a strategy to replace conventional fuels which are obtained from petroleum refining with biofuels. this has happened due to rising oil prices, unpredictable depletion of oil reserves, increased greenhouse gas emissions and the negative impact on the environment. bioethanol producțion 1stg has caused competition between fuels and food, because the main raw materials used for are sugar and starch raw materials. the united states is the world's largest producer of bioethanol from corn, followed by brazil which uses sugarcane. in the europe union, the main raw materials for bioethanol production are wheat and sugar beet. therefore, the second generation energy (including bioethanol producțion 2ndg) obtained from lignocellulosic materials (lcm) is gradually attracting global attention. lignocellulosic biomass (lcb) is the most abundant natural organic source, in huge quantities, at low costs and not part of the human food chain. keywords: sugar, starch, celullose, bioethanol, biofuel 1. introduction biomass is considered a renewable energy, an important source of energy and chemical substances for future generations. currently, reports show that worldwide biofuels production from lignocellulosic biomass (lcb) is 30 140 ej, but it could increase to 130 400 ej by 2070. [1]. the international energy agency (2011) estimates by 2050 biofuels will represent 27% of the total fuels used for transport [2]. research is currently underway to convert lignocellulosic biomass (lcb) as efficiently as possible to obtain biofuels [3] and chemical substances [4]. biomass has a low economic value, but it can be capitalized by different treatments: chemical [5], thermal [6] and biological [7]. in the current context of intensive search for energy sources which are environmentally friendly, woody biomass and especially woody wastes which result from forestry activities are of particular interest. a superior valorization of these wastes is that of their conversion into bioethanol. subsequently, there is the possibility that hydrogen and / or synthetic gas can be obtained from bioethanol [8]. bioethanol can be synthesized from various materials that can be classified into three categories [9]: • simple sugars; • starch; • lignocellulose. bioethanol can be obtained from animal feed, corn grains (starch) and sugarcane (sucrose). an efficient source of energy available in huge quantities is lignocellulose. this can be used for bioethanol production, thus becoming more competitive in the future over fossil fuels [10 12]. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 49 2. biofuels biofuels resulting from renewable biomass are becoming more and more affordable as a result and are therefore an important source of renewable energy to replace fossil fuels [13]. according to the legislation in force of the european union, biofuels or other renewable fuels used in the transports sector are intended to replace diesel or gasoline. thus, some objectives could be achieved such as: • fulfilling the commitments on climate change; • security of supply, thus leading to the protection of the environment; • promotion of renewable energy sources. article 2, paragraph (1) lit. (a,b) within the framework of directive 2003/30/ec of the european parliament and of the council of the european union of 8 may 2003, defines [14]: (a) ‘biofuels’ means liquid or gaseous fuel for transport produced from biomass; (b) ‘biomass’ means the biodegradable fraction of products, waste and residues from agriculture (including vegetal and animal materials), forestry and related industries, as well as the biodegradable fraction of industrial and municipal waste; according to art. 2, paragraph (2), lits (a-j) under the same directive, classifies biofuels as follows [14]: (a) ‘bioethanol’: ethanol produced from biomass and/or the biodegradable fraction of waste, to be used as biofuel; (b) ‘biodiesel’: a methyl-ester produced from vegetable or animal oil, of diesel quality, to be used as biofuel; (c) ‘biogas’: a fuel gas produced from biomass and/or from the biodegradable fraction of waste, that can be purified to natural gas quality, to be used as biofuel, or woodgas; (d) ‘biomethanol’: methanol produced from biomass, to be used as biofuel; (e) ‘biodimethylether’: dimethylether produced from biomass, to be used as biofuel; (f) ‘bio-etbe (ethyl-tertio-butyl-ether)’: etbe produced on the basis of bioethanol. the percentage by volume of bio-etbe that is calculated as biofuel is 47 %; (g) ‘bio-mtbe (methyl-tertio-butylether)’: a fuel produced on the basis of biomethanol. the percentage by volume of bio-mtbe that is calculated as biofuel is 36 %; (h) ‘synthetic biofuels’: synthetic hydrocarbons or mixtures of synthetic hydrocarbons, which have been produced from biomass; (i) ‘biohydrogen’: hydrogen produced from biomass, and/or from the biodegradable fraction of waste, to be used as biofuel; (j) ‘pure vegetable oil’: oil produced from oil plants through pressing, extraction or comparable procedures, crude or refined but chemically unmodified, when compatible with the type of engines involved and the corresponding emission requirements. liquid biofuels obtained from lignocellulosic materials (lcm), agricultural residues and other types of residues are produced in low quantities, approximately 1 billion liters in 2014, which means less than 1% of total liquid biofuel production [15]. estimates showed that production will triple, but recently there has been a decrease of investments. currently, the costs of producing biofuels from lignocellulosic biomass (lcb) are much higher than those of conventional liquid biofuels and conventional fuels, but they are expected to become more competitive by 2030 and 2045. recently, progress has been made by obtaining ethanol from a number of cellulose materials (mainly corn stover, corn cobs, straw, and leaves) can allow the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 50 development of this process through practice. because the bioethanol production from these residues is still reduced, the development of the process of obtaining ethanol from cellulosic materials would require a rapid acceleration for the results to be the desired ones. therefore, international renewable energy agency (2016) states that programs for financing, research and development of demonstration projects are needed, as well as financial support for the development of the supply chain with raw materials [16]. for biofuels production can be used different types of raw materials and technologies, therefore they are classified as follows [17, 18]: • first generation biofuels (1stg), • second generation biofuels (2ndg) • third generation biofuels (3rdg) 3. the main raw materials used in the production of first generation bioethanol (1stg) worldwide, the production of first generation bioethanol (1stg) is mainly made from two raw materials, such as corn and sugarcane. bioethanol 1stg is a biofuel produced from agricultural raw materials (figure 1) which contain sugar (sugar, molasses, sugar beet and fruit) or starch (corn, wheat, potatoes, etc.). yeasts have the ability to directly ferment raw materials which contain simple carbohydrates and convert them into 1stg bioethanol. therefore, molasses or sugarcane syrup can be fermented without the need for treatments such as milling, pretreatment, hydrolysis and detoxification (removing of inhibitors which affect the fermentation process). in the case of starchy raw materials, in order to obtain a high yield in bioethanol, it is recommended to apply a series of treatments such as milling, liquefaction and saccharification [19]. figure 1 shows schematically the raw materials used for bioethanol production. the main constituent which is present in cereals is starch (for example, starch content from corn is between 60 70%). this carbohydrate contains 15 25% amylose and 75 85% amylopectin [20]. figure 1. raw materials for bioethanol production (crops) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 51 the main constituent which is present in cereals is starch (for example, starch content from corn is between 60 70%). this carbohydrate contains 15 25% amylose and 75 85% amylopectin [20]. amylose is a polysaccharide which has a linear structure and d-glucose molecules linked to each other by α-1,4 glycosidic bonds. amylopectin is a macromolecule (dp 105 106) which has a branched structure, formed of d-glucose molecules linked by α1,4 and α1,6 glycosidic bonds [21 22]. the starch stored in cereal grains is derived from long chains of glucose molecules, which contain less or more than 1000 glucose molecules/amylose structure and between 1000 – 6000 glucose molecules/ amylopectin structure [23]. table 1 contains the percentage of amylose and amylopectin in different types of starchy raw material. in order to obtain bioethanol, the starch must be converted into glucose syrup. the step of converting the starch into glucose syrup is called enzymatic hydrolysis and is made with the help of α-amylases. the enzymatic hydrolysis of the starch is dependent on its physico-chemical structure and is known as easily hydrolysable starch and difficult hydrolyzable starch (due to the crystalline structure of the granules and requiring a gelatinization process) [27 28]. starchy raw materials are most commonly used for ethanol production in north america and europe. in tropical countries, tubers (e.g. cassava) are used as starch source for bioethanol production [27]. after this step, the mash will be fermented, distilled followed by dehydration to produce anhydrous ethanol [29]. figure 2 shows the chemical structure of starch which is present in different starch raw materials. table 1 amylose and amylopectin content in starchy raw materials [24 – 26, 98, 100] source granule size (µm) amylose (%) dpn am β-lv (%) a amylopectin (%) dpn ap common wheat 1 10 / 15 40 17 29 830 1570 79 85 75-80 13000 18000 durum wheat n.r. 26 28 n.r. n.r. n.r. n.r. rye 1 10 / 10 50 22 26 n.r. n.r. n.r. n.r. triticale n.r. 23 27 n.r. n.r. n.r. n.r. barley n.r. 22 27 1220 1680 76 82 72.5 n.r. oat n.r. 18 29 n.r. n.r. n.r. n.r. rice 3 10 17 29 920 1110 73 87 65-85 2700 12900 corn 5 30 25 28 320 1015 81 84 75-83 9600 15900 sorghum n.r. 22 30 n.r. n.r. 75 n.r. sweet potato n.r. 19 20 3280 76 81.1 n.r. potato 10 100 25 31 4920 68 80 76-83 11200 dpaverage of polymerization degree; amamylose; apamylopectin; βamylolysis limit value; n.r.data not reported. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 52 figure 2. chemical structure of starch (a) glucose unit, (b) amylose and (c) amylopectin [30, 31] table 2 ethanol production of the main raw materials [32 – 34, 38, 106] type of raw material global yield t/ha specific conversion rate to ethanol, l/t annual ethanol yield, l/ha output/ input ratio cost, us$/kg cost of production of anhydrous ethanol us$/l corn 5.62 – 5.86a 350 460 6600 1.34 1.53 0.076 0.2325 sorghum 1.47 – 1.49a 340 340 2040 n.r. 0.149 0.386 sweet sorghum 25 35 68 86 1700 9030 n.r. n.r. n.r. wheat 3.39 3.49a 340 370 1020 3214 2.24 2.84 0.188 0.402 barley n.r. 345 1825 n.r. n.r. n.r. oat n.r. 264 1413 n.r. n.r. n.r. triticale n.r. 368 1757 n.r. n.r. n.r. sugarcane 70 122 68 70 5345 9381 2.5 10.2 0.0100 0.1980 sugar beet 66 78 80 100 5000 6600 1.9 0.170 0.4910 potato 17 20 100 1700 2000 n.r. 0.020 1.330 cassava 20 180 3600 n.r. n.r. n.r. straw 1.93 3.86 170 261 n.r. n.r. n.r. 0.651 n.r.data not reported; a global yield t/ha (2017 2019)[38]; table 2 presents a series of information about the production of ethyl alcohol from different raw materials. table 3 presents the chemical composition of different types of raw materials used for bioethanol production. calculation equations to determine glucose (a), xylose (c) and ethanol yields from these carbohydrates (b, d) [35]: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 53 table 3 chemical composition of raw materials used to obtain bioethanol (% dry matter) type of raw material starch (%) β-g l uc an (%) protein (%) raw fat (%) fiber (%) references corn 71.88± 1.5 0 8.84± 0.35 4.57 ±0.12 2.15± 0.18 [41] corn, waxy 65.3 – 72.9 n.r. 8.8 – 13.7 4.5 – 6.3 1.1 – 1.19a [99] sorghum 78.18 0 7.29 2.49 3.64a [42] sorghum, waxy 68.4 – 72.4 n.r. 9.7 – 12.2 3.3 – 6.8 1 – 1.7 [99] wheat 69.5 0 13.90 3.6 4.5 [42] wheat, hard 68 n.r. 12.1 0.8 0.21 [43] wheat, soft 68.5 n.r. 12.9 0.6 0.19 wheat, waxy 61.9 – 66.5 n.r. 9.4 – 14.2 0.9 – 1.2 0.2 – 0.3 barley, hulled 56.38 4.26 7.92 n.r. n.r. [44] barley hull-less 63.48 4.42 8.41 n.r. n.r. barley, various 50-65 1.9 10.7 8.1 21.2 0.9 3.3 n.r. [45] barley nonwaxy 67.6 6.21 13.6 2.60 n.r. [46] barley high-amylose 65.2 5.5 12.5 2.16 n.r. barley waxy 65.6 6.60 15.5 2.65 n.r. oats, whole 48.2 n.r. 11.3 4.4 13.2a [47] oats groats 57.1 -61.8 4.3-5.8 14.6 19.6 4.6 7.8 2.0a [47, 48] oats, hulled 48.08±0.29 3.15±0.19 10.58±0.67 5.15±0.19 17.63±1.52 [49] oats, hull-less 31.55±3.72 3.29±0.26 15.71±1.10 9.66±1.17 22.97±1.89 rye 55 -65 2 3 10 – 15 2 3 1–3 [50] triticale 67.87 n.r. 10.33 n.r. n.r. [51] rice, brown, long-grain 77.24 n.r. 7.94 2.92 3.5a [52] rice, white, short-grain 79.15 n.r. 6.50 0.52 2.8a rice, white, glutinous 81.68 n.r. 6.81 0.55 2.8 pearl millet 55.36 59.36 n.r. 10.14 11.82 3.62 – 4.46 0.77 – 1.21 [53] cassava 83.42 -87.35 n.r. 1.17 3.48 0.74 – 1.49 3 – 4a [54] sweet potato 68.95±1.75 n.r. 10.25±0.12 1.48±0.08 1.92±0.02 [55] acrude fiber; n.a.data not reported. 3.1. corn (zea mays) corn is a grain belongs to the family grimanaceae [36], subfamily panicoideae maydeae, genus zea and species zea mays [37]. the distribution of the main components of corn grain varies, so most of the starch content is found in the endosperm and is bound to protein (gluten). the oil is present in the germ. the fiber contents are distributed in the endosperm, germ and at the tip of the grain. also, the outer shell of the corn grain (pericarp) has a fiber content made of arabinoxilane and is covered by wax [101]. figure 3 shows the anatomical structure of the corn grain. corn is the most cultivated cereal and ranks 1st in the world. in 2019, about 1.123 billion tons of corn were produced worldwide. of this amount of corn, usa produced 364 million tons, followed by china 257 million tons, brazil 101 million tons and europe 64 million tons [38]. the amount of water required for maize to reach maturity is approximately 560 mm [116]. annually, the maximum concentrations of potassium, phosphate and nitrogen required for optimal corn development are 160 lbs / acre, 70 lbs / food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 54 acre and 140 lbs / acre. therefore, the dosage of these nutrients / acre depends on the concentrations of natural nutrients already existing in the soil. compared to sugar beet, maize requires a double amount of potassium, whereas other nutrients needed are in smaller quantities. however, based on the amount of water needed for irrigation and the nutrients used on an acre surface, sugar beet will result nearly twice as much ethanol / acre. therefore, to produce one liter of ethanol the energy consumed for irrigation and nutrient dosing is much lower for sugar beet compared to corn [117]. the chemical composition of corn grains is an important source for the production of biofuels, especially bioethanol. following the cultivation of corn also results in a series of residues (corn stover, corn cobs, corn roots, leaves) which have various advantages over other energy raw materials [39]. these residues contain cellulose and hemicellulose can be recovered if they are pretreated by hydrolysis techniques. most of the corn is used for animal feed, and the rest is used for population feeding and for other activities [40]. figure 3. anatomical structure of the corn grain [107] 3.2. wheat (triticum spp.) world wheat production ranks 3rd, after corn and rice. overall, wheat production in 2019 was 763 million tons, with 30.9 million tons higher than the 2018 production and only slightly lower than the record production in 2016 (765 million tons) [56]. in canada, europe and australia several varieties of wheat are used as feedstock for ethanol production as fuel. in the united states, there are ethanol factories that ferment wheat starch. in europe and australia, wheat is considered the raw material to expand the fuel ethanol industry. zhao et. al. (2009) [57] analysed 3 different types of wheat (waxy, soft and hard) to see if the chemical composition influences the yield of ethanol. from the obtained data, it was found that the yield is dependent on both total starch and protein content. however, the highest yield in ethanol was obtained from wheat with the highest total starch level. most wheat raw materials have a higher content of starch and protein than corn or sorghum and have a lower fiber content [58 -59]. figure 4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 55 shows the anatomical structure of the wheat grain. the technological scheme for obtaining bioethanol from wheat is similar to other cereals. the main steps are milling, enzymatic hydrolysis (transformation of starch into c6 sugars) and fermentation [60]. then, at a temperature of 32 35° c and a ph of 5.2, the c6 sugars are converted by the yeasts into ethanol. after fermentation, the fermented mash will have an ethanol concentration of 10 15%. the yield in ethanol is about 0.53 gj ethanol / gj wheat (349 liters of ethanol / t wheat) [61], with the possibility to grow to 0.59 gj ethanol / gj wheat in 2020 [62]. figure 4. anatomical structure of the wheat grain [108 109] 3.3. sorghum (sorghum) sorghum is a plant of africa origin which belongs to the poaceae family and is extremely efficient to use of water, carbon dioxide, nutrients and sunlight. in terms of global production, sorghum ranks the 5th after corn, wheat, rice, and barley [63]. in countries such as africa, china and india, sorghum is considered a staple food, whereas in the united states, australia and south america it is primarily used for animal feed [64]. sorghum is cultivated because of its capacity to grow in areas with low precipitation and high temperatures are recorded, such as the semi-arid tropical and subtropical regions of the world, where it is difficult to grow other types of cereals. also, during the dry season in some areas of brazil sorghum is cultivated [65]. in 2019, about 59 million tons of sorghum were produced worldwide. from this amount of sorghum the united states of america produced 9 million tons, followed by nigeria 6.8 million tons, ethiopia and sudan about 5 million tons each, mexico 4.7 million tons and south america 4.5 million tons [38]. sorghum has a relatively low economic value if it is sold directly as fodder cereals [66]. sorghum grains are an important source of carbohydrates and fiber, about 72%. the main constituent is starch, which has properties similar to corn starch. there food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 56 are hundreds of sorghum hybrids available on the market which can be used for bioethanol production. however, these sorghum hybrids have a different chemical composition and can certainly influence the hydrolysis and fermentation [67]. in order to obtain the high yields of ethanol, it is important to know exactly the sorghum hybrid for the ethanol industry, but also the sorghum producers [43]. 3.4. barley (hordeum vulgare) regarding barley production, it is ranked 4th worldwide and cultivated in relatively dry areas [68 70]. in 2019, around 138 million tons of barley were produced worldwide approximately 56 million tons is produced by the european union, followed by russia with 16.7 million tons, canada and australia approximately 8.3 million tons each [38]. barley has not been used for bioethanol production for a long time, because the chemical composition of barley is slightly different from other cereals and bioethanol production is more expensive compared to corn. the first negative factor is the presence of silicates from the hulls of the grain (it represents 2 6% of hull), which is abrasive and causes damage of the equipments for handling and processing of barley grains. for this reason, two strategies were used to change the abrasiveness of barley grains. the first strategy refers to the removal of the hull by abrasive techniques, such as hulling or peeling [72]. this process works well, but some of the starch can be removed, and the ethanol yield is reduced. another strategy was to cultivate other barley hulled varieties. another aspect relates to the fact that most of the barley previously used for bioethanol production had low starch content and low yields of ethanol were obtained. most of the barley used in the past was for food consumption and therefore had low starch content. therefore, new varieties with higher starch content have been created recently for both barley varieties hulled and hull-less [44]. many of these new varieties of barley, especially those hulls-less, have lower fiber content and higher starch content, which are more beneficial for bioethanol production. also, the presence of β-glucans in barley hinders bioethanol production, which is present throughout the grain mass, but especially in the endosperm. β-glucans are water soluble and have β-1.3 and β-1.4 glycosidic bonds. β-glucans in barley or oats have a high nutritional value in human diets, as it has been found that they can reduce ldl levels by 10 15%. the presence of β-glucans in bioethanol production is undesirable because it creates an extremely high viscosity, creating difficulties in mixing, pumping, saccharification and fermentation process [59]. 3.5. oat (avena sativa) oat is a cereal used mainly for animal feed and less used for human nutrition. oat losses are about 2%, but even if it were recovered, about 225 million liters of bioethanol could be obtained, which could replace 161 million liters of gasoline. also, oat straws contain lignin, but especially cellulose and other polysaccharides that can be used for bioethanol production [40]. in 2019, about 21.9 million tons of oat were produced worldwide. from this quantity of oat, european union produced 7.7 million tons, followed by russia 4.7 million tons, north america and oceania about 0.8 million tons each [38]. like barley, oat grains have a similar content of β-glucans (4 6%). however, oats are different from other cereals in their high fat content (4 9%). also, the hull-less oat has higher starch content than food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 57 the hull varieties, thus obtaining a higher percentage of bioethanol [59]. 3.6. rice (oryza sativa) rice is still an important food raw material and ranks 3rd in world grain production. asia is the largest rice producer in the world. in 2019, about 499 million tons of rice were produced worldwide. from this quantity of rice china produced 148 million tons, followed by india 116 million tons, indonesia 36 million tons, thailand 20 million tons, and the usa and brazil each 7 million tons [38]. it is known that the tribes of eastern india and the region of tibet produce an alcoholic drink which is known as "sake". even rice straw, like other agricultural byproducts, can be used as an efficient means of producing alcohol as a fuel [40]. the rice has a high starch level (~75%), has no β-glucans and has a low fiber content. although, barley has been shown to be a cost-effective raw material from which bioethanol can be obtained, however, it was more important to be used it for food consumption. residues from rice fermented mash contain starch (32.6%), protein (55.8%), fat (8.2%) and raw fiber (2.1%) [59]. 3.7. rye (secale cereale) over the last 20 years, there has been a decline in rye production. last year (2019) about 10 million tons of rye were produced worldwide. from this amount of rye, eu produced 6.2 million tons, followed by russia 1.9 million tons, belarus 0.5 million tons, ukraine 0.4 million tons, and turkey 0.3 million tons [38]. traditionally, rye has been used mainly for beer and food consumption. the rye was also used for bioethanol production. however, the use of rye for bioethanol production is modest compared to other raw materials, such as corn and sugarcane. lately, rye has been used as a raw material for bioethanol production, due to the new requirements for the production of renewable fuels. rye and triticale can be considered wheat substitutes for bioethanol production, because the starch content is about the same. [72, 73]. a disadvantage of using rye as a raw material for obtaining bioethanol is the relatively high content of pentosanes and β-glucans. their presence causes a very high viscosity of the mash. compared with barley and oats, the content of β-glucans in rye is approximately 2% [59]. due to the high content of pentosanes and β-glucans, the viscosity is modified with the help of enzymes [74, 75]. the enzymes used to reduce the viscosity are pullulanase, cellulase, xylanase, β-glucosidase, pectinase and proteinase. adding xylanase and pullulanase will increase the concentration of fermentable sugars, improve the fermentation efficiency, and increase the yield of ethanol [74]. 3.8. triticale (triticosecale) triticale is a hybrid between wheat (triticum) and rye (secale) and was created for the fist time in laboratories at the end of the 19th century. this hybrid has some of the rye specific genes and can adapt to environmental conditions which are not favorable to wheat. unlike barley and rye, the triticale mash does not have a high viscosity and therefore does not require an enzyme addition to reduce the viscosity. in sweden, has been study of bioethanol production and it has been concluded that 1 l ethanol, 0.8 kg carbon dioxide and 0.8 kg ddgs result from 2.65 kg of triticale grains [76]. also, several researchers have observed that triticale used for ethanol production have a higher endogenous α-amylase content, thus reducing the amount of enzymes used and thus reducing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 58 production costs [51, 77]. also, triticale contains endogenous glucoamylase or other enzymes [78]. 3.9. pearl millet (pennisetum glaucum) unlike sorghum and corn, pearl millet can grow in semi-arid conditions and is considered as a potential biofuel raw material for these regions. wu et al. analysed four varieties of pearl millet (pennisetum glaucum) to determine of bioethanol yield. in this study it was determined that millet varieties have a starch content between 65.3 70.39% of the dry matter. compared to the dry matter, the protein and fat content is significantly higher than that of corn and ranges from 9.72 to 13.68%, respectively 6.80%. therefore, in the 5 l vessel of a bioreactor were introduced millet mash successively with a raw material concentration of between 20 35% (a total volume of about 4 l), and during the fermentation a series of parameters were monitored. after processing the data was established that bioethanol yield from millet is similar to that of corn [79]. 3.10. sugarcane (saccharum officinarum) sugarcane is a semi-perennial plant and belongs to the poaceae family (grass family) and is specific to tropical and subtropical areas [80]. compared to biomass that is high in starch, bioethanol production from sucrose as raw material does not require a saccharification step, because sugars are easily fermentable and the process of obtaining bioethanol is simple [81]. there are six recognized varieties of sugarcane. s. officinarum is the most common species, has the highest sugar content and is the most suitable in terms of industrial processing. hybrids of s. officinarum were created and selected to be resistant to pests and dryness, to have a high sugar content and to give high biomass yield per hectare [82]. in 2012, worldwide sugarcane production was 1.96 billion tons, out of 26 million hectares of harvested area, and the main producing countries were brazil, india and china, representing 39%, 19 % and 7% respectively [83]. it has been established that 4.8 mha of land suitable for sugarcane cultivation are available in eswatini, malawi, mozambique, south africa, tanzania, zambia and zimbabwe. this area is similar to sugarcane cultivation for ethanol production in brazil. it is equivalent to 2.0% of agricultural land or 2.5% of pasture in these countries [84, 85]. once harvested, the sugarcane should be processed between 24 – 72 hours. the process of sugar extraction consists of several stages. the first step is to crush the stems with specialized rolls to release the juice, followed by the step in which calcium hydroxide (ca(oh)2) is added to precipitate the fiber and the sludge. the solution is filtered and evaporated to concentrate and crystallize the sugar, and then it is removed by centrifugation. the non-crystallized sugar and the salts are concentrated to form the syrup called "black molasses (bsm)" which can subsequently be used as a starting material for ethanol production [86]. comparative studies have been carried out between the mechanized harvesting and the traditional process of manual combustion and cutting with regard to the mineral content of the sugarcane (potassium, calcium, silica, iron and copper). the data obtained showed that following the mechanized harvest the calcium, magnesium and silica content of the sugarcane juice increased by 13%, 32% and 7.6%, respectively, [110, 112]. the presence of these mineral substances can positively or negatively influence the fermentation process. therefore, the presence of magnesium contributes to the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 59 increase of ethanol yield, whereas a high content of copper reduces the yield [111 112]. annually, the sugar industry produces significant quantities of molasses. molasses is the main by-product of sugar and is used mainly as a substrate for yeasts, for the production of bioethanol, biochemicals, but also for animal feed. the molasses has a total residual sugar content between 50 60% (m/v), and its main constituent is sucrose (60%). the largest quantities of molasses are obtained from the processing of sugarcane and sugar beet, which have a total residual sugars content (m/v) of 46% and 48% respectively. following the process of obtaining dried fruit pulps from citrus fruit, molasses has about 45% (m/v) of residual sugars. also, molasses with a concentration of about 43% (m/v) reducing sugars and 73% (m/v) solids results from the process of obtaining glucose from starch. the raw material used to produce glucose from starch is corn and sorghum (starch hirolysis is done with enzymes or acids) [113]. therefore, molasses are a suitable source for bioethanol production. 3.11. sugar beet (beta vulgaris) sugar beet is an important source of sugar in europe and north america. the largest producer of sugar beet is russia, which in 2019 produced about 6.08 billion tons, followed by the us 4.47 billion tons, turkey 2.7 billion tons, ukraine 1.84 billion tons and china 1.32 billion tons [87]. sugar beet has a good yield (25 – 50 tons / acre), grows in the temperate climate area and requires humidity lower than the sugarcane. on average, the yield of ethanol is 25 gallons of ethanol / t of sugar beet. from an economical point of view, bioethanol production from sugar beet is more expensive (applying chemical treatments and higher energy consumption) as opposed to sugarcane. in order to grow to maturity, sugar beet needs about 560 mm of water (efetha, 2008) but also different nutrients such as potassium, phosphate and nitrogen. the maximum concentrations of potassium, phosphate and nitrogen required for optimal development of sugar beet are 80 lbs / acre, 100 lbs / acre and 200 lbs / acre respectively [118]. depending on the type of soil these nutrients are naturally found in different concentrations. therefore, the dosage of these nutrients varies depending on the type of soil [119]. recent studies have shown that the fermentation process can be influenced by both the sugar beet content of sugar beet and the other components in the dry substance. therefore, the yield in bioethanol can be affected by the presence of impurities (potassium, nitrogen and sodium). these impurities can have a negative (inhibitory) effect on saccharomyces cerevisiae and can cause reduced yeast cell growth, reduced glucose consumption and low bioethanol yield. irrational use of fertilizers can result in npk uptake that favors the presence of k, n and na in high root concentration [104]. the chemical composition of beet root consists of 75% water and 25% dry matter. of the total dry matter 75% is carbohydrates and about 5% pulp. the pulp is insoluble in water and consists of cellulose, hemicellulose, lignin and pectin. sugar beet has a sucrose content between 12 20% [105]. several authors have reported that sugar beet is one of the most efficient sources of ethanol / ha. it was determined that the fermentation process of sugar beet can be obtained between 100 and 120 l / t of ethanol (110 l / t, 103.5 l / t [88], 117 l / t respectively [89]. the energy supplied by one tonne of dehydrated sugar beet is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 60 approximately 3.89 gj [90]. ethanol has an energy content of 21.2 mj / l [91], which means that 115 l / t of ethanol obtained from one tonne of sugar beet generates an energy value of 2.44 gj / t sugar beet. according to fao (2008), bioethanol production from sugar beet is of 5,060 l / ha, corn is 1,960 l / ha, wheat is 952 l / ha respectively, considering that on a surface of 1 ha the sugar beet production is 46 t / ha, corn 4.9 t / ha, wheat 2.8 t / ha respectively [92]. 3.12. cassava (manihot esculenta) cassava is an important tropical plant called manioc, sagu, yucca or tapioca. depending on the variety, the cassava root contains approximately 66.72 84.42% starch, 0.74 1.52% protein, 1.08 1.18% fiber, lipids 0.39 0.63%, mineral substances 1.05 2.39%, humidity 5.43 10.87% [93]. compared to potato, rice or corn starches, the properties of cassava starch are different. the cassava starch has a high purity, has a neutral aroma, slight swelling, solubility, high viscosity development and a low tendency to downgrade [102]. it is estimated that in 2018 about world cassava production would be about 277 million tons (equivalent to fresh root), about half a percentage point higher than in 2017. therefore, for 2018 the top 3 most important continents estimates to produce cassava are africa with 160 million tons, in asia at 85.5 30.5 million tons latin america. also, the top 5 most important countries estimated to produce cassava are nigeria 56 million tons, thailand 27 million tons, indonesia 21 million tons, brazil 20.9 million tons and ghana 19.4 million tons [85]. therefore, due to the large quantities produced by cassava yearly, as well as the high carbohydrate composition, this can be an important source for obtaining bioethanol. 3.13. potato (solanum tuberosum) potato (solanum tuberosum) is an annual herbaceous plant that can reach a height of 100 cm. it produces a tuber known as potato and has a high starch content. the potato belongs to the solanaceae family, the genus solanum and includes at least 1000 species, including tomatoes and eggplants. the most cultivated species globally are s. tuberosum andigenum (which is adapted to short-day conditions and is cultivated mainly in the andes) and s. tuberosum chilotanum (potato now cultivated worldwide).throughout the growing period, the potato leaves produce starch that is transported to the ends of its underground stems (or stolons) and can form up to 20 tubers near the soil surface. the potato's chemical composition is 72 75%, starch 16 20%, protein 2 2.5%, fiber 1 1.8%, fatty acids 0.15% [132]. due to the relatively low price, the potato is one of the most consumed foods and is in 3rd place after wheat and rice [133]. this food is easily prepared and has a high nutritional value [134]. in 2017, 388.19 million tonnes were cultivated worldwide. the largest potato producers are china 99.2 million tons, india 48.6 million tons, russia 29.59 million tons, ukraine 22.2 million tons, the united states 20 million tons [135]. european union, in 2018 the cultivation of potatoes was realized on an area of 1.7 million hectares and the production was 51.9 million tonnes. the main potatoproducing countries are: germany (8.9 million tonnes, representing 17.2% of the eu total), france (15.2%), poland (14.3%) and the netherlands (11.6%) [136]. 3.14. sweet potato (ipomoea batatas) sweet potatoes (ipomoea batatas) are the second raw material which has a high content in starch and can be used for bioethanol production. in 2017, the global food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 61 production of sweet potatoes was 113 million tons. china produces 64% of global sweet potatoes, followed by malawi and nigeria [94, 95]. chemical composition of sweet potatoes is 60.1 – 71.4% starch, 4.86 6.53% proteins, 0.56 – 0.76% fats and 1.85 – 2.35% fibers [96]. as compared with cereals, the starch content of potatoes changes during its storage period. it was found that after a storage period of about 6 months and 8 months the potato starch content decreases by 8%, respectively 16.5% [103]. putri et al. managed to obtain ethanol from sweet potato. a yield of 14% was obtained after the end of the 72-hour fermentation period. after 24 hours and 48 hours of fermentation, the yield of ethanol obtained was up to 3.66%, 3.33% respectively [97]. 3.15. agave (agavoideae) in mexico, the juice extracted from the agave stem is used for the production of spirits, due to its high fruit content. this alcoholic beverage is known as tequila and is produced only from agave tequilana weber. another assortment of agave alcoholic beverages is mezcal, which is produced from different agave species (a. angustifolia, a. americana, a. salmiana, etc.). stems from a. salmiana are about two times heavier than a. tequilana. also, the leaves of a. salmiana are more succulent and thicker than those of a. tequilana. once harvested, the agave strains are transported to the factories, where they will be subjected to thermal hydrolysis-assisted and juice extraction (mechanical and / or water-diffused) [120]. following the extraction process, high fructose agave juices are fermented by yeasts, and then the mash is distilled. the solid residues (known as bagasse) as a result of juice extraction have a low sugar content, but they can also be used [120]. agave species have the ability to use water efficiently due to specialized photosynthetic pathways known as crassulacean acid metabolism (cam). this allows them to adapt and growth even when the quantities of water are low. because the temperature is high during the day and the relative humidity is low, the agaves fix co2 at night, thus reducing losses through evapotranspiration [121]. agave is xerophytic perennial plants and can be grown on semi-arid lands on which cannot be cultivated various crops that are used for human or animal feeding. [122]. several researchers found that agave can produce 1stg bioethanol, and production costs, greenhouse gas (ghg) and yields in ethanol are comparable to those of corn, switchgrass and sugarcane [123]. of the species of agave spp. cultivated in the abu dhabi area was obtain an ethanol yield of 6,750 l / ha [124], comparable to palm (3,000 l / ha) [125] or willow (900 l / ha) [126]. agave spp. it is resistant in periods of drought and unlike other plants does not require high nutrient concentrations [127]. however, agave spp. it needs water, and water is a problem in the abu dhabi area because it needs to be desalinated and this process is expensive [128]. in order to solve the problem related to the need of water, consideration was given to growing halophilic plants. for this reason, salicornia [129] is widespread on the abu dhabi coast, and its ethanol yield is about 935 l / ha [130 131]. 4. advantages and disadvantages of bioethanol production 4.1. advantages [114 115] • for the production of bioethanol 1stg, any type of crop (corn, sugar cane, sugar beet, wheat, sorghum, etc.) containing sugar and starch can be used. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 62 • compared to fossil fuels, bioethanol reduces co2 emissions released into the atmosphere following the combustion process. • in contrast to the pollutants emitted from the process of combustion of gasoline, the pollutants resulting from the combustion process of bioethanol are much lower. therefore, the use of ethanol as a biofuel is beneficial for the conservation of the environment because it reduces the destruction of the ozone layer. • mixing bioethanol with gasoline contributes to the reduction of greenhouse gases. • if certain bioethanol accidents or discharges occur, it is biodegradable or may be diluted to non-toxic concentrations. • the raw materials used in the production of bioethanol are considered renewable sources. • the production of bioethanol 2ndg from different types of lignocellulosic materials (lcm) could solve the competition between “biofuel vs food”. 4.2. disadvantages [114 115] • the production of bioethanol from cereal crops or irrigable resources requires large areas of agricultural land, which is why natural habitats such as tropical forests can be destroyed. • from the economic point of view of the bioethanol production, significant profits can be registered, and this can cause the farmers to give up the cereal crops destined to feed the population. by using agricultural land to obtain bioethanol, it can determine the price of food, thus creating a food crisis. • building a bioethanol plant requires large investments. • to obtain high yields of ethanol, specific technical knowledge is required and involves many researches in this field (especially for bioethanol 2ndg). • waste management resulting from the bioethanol production process. 5. conclusion it is very important for biofuel production to get increased in order to replace fossil fuels, which contribute largely to global warming and climate change. worldwide 1stg bioethanol is obtained from raw materials which contain simple sugars or starch which causes competition between "food vs fuel". in food industry, significant quantities of non-food lignocellulosic biomass can be used to produce 2ndg bioethanol. currently, the production of 2ndg bioethanol may not be as advanced as the 1stg, but great progress has been made. every year, huge quantities of lignocellulosic materials (lcm) are generated from agriculture, which instead of being wasted can be converted into 2ndg bioethanol. 6. acknowledgement: this work was supported by “decide development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu / 380/6/13/125031, project co-financed from the european social fund through the 2014 – 2020 operational program human capital” 7. reference [1]. deng y.y., koper m., haigh m., dornburg v., country based assessment oflong-term global bioenergy potential, biomass bioenergy, 74, 253–267, (2015) [2]. iea, international energy agency, technology roadmap biofuels for transport, (2011), https://www.lowcvp.org.uk/assets/reports/biofuels_ roadmap.pdf , accessed at 07.03.2020 [3]. chen w., wu f., zhang j., potential production of non-food biofuels in china. renew. energy, 85, 939–944, (2016) https://www.lowcvp.org.uk/assets/reports/biofuels_roadmap.pdf https://www.lowcvp.org.uk/assets/reports/biofuels_roadmap.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 63 [4]. shen d., jin w., hu j., xiao r., luo k., an overview on fast pyrolysis of the main constituents in lignocellulosic biomass to valuedadded chemicals: structures, pathways and interactions, renew. sustain. energy rev. 51,761– 774, (2015) [5]. lopez f., garcia m.t., feria m.j., garcia j.c., de diego c.m., zamudio m.a.m., diaz m.j., optimization of furfural production by acid hydrolysis of eucalyptus globules in two stages, chem. eng. j., 240, 195– 201, (2014) [6]. le x.t., grace j.r., lim c.j., watkinson a.p., chen h.p., kim j.r., biomass gasification in a circulating fluidized bed, biomass bioenergy, 26, 171–193, (2004) [7]. madhuri n., jisha p.j., balasubramanian v., simultaneous saccharification and fermentation of delignified lignocellulosic biomass at high solid loadings by a newly isolated thermotolerant kluyveromyces sp. for ethanol production, bioresour. technol., 179, 331–338, (2015) [8]. tanksale a., beltramini j.n., lu g.m., a review of catalytic hydrogen production processes from biomass, renew sustain energy rev, 14:166e82, (2010) [9]. balat m., production of bioethanol from lignocellulosic materials via the biochemical pathway: a review, energy convers. manag., 52 (2) 858e875, (2011) [10]. gírio f.m., fonseca c., carvalheiro f., duarte l.c., marques s., bogel-łukasik r., hemicelluloses for fuel ethanol: a review, bioresour. technol., 101 (13) 4775e4800, (2010) [11]. limayem a., ricke s.c., lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects, prog. energy combust. sci., 38 (4) 449e467, (2012) [12]. chiesa s., gnansounou e., use of empty fruit bunches from the oil palm for bioethanol production: a thorough comparison between dilute acid and dilute alkali pretreatment, bioresour. technol., 159 (0) 355e364, (2014) [13]. liew w.h, hassim m.h., ng d.k.s., review of evolution, technology and sustainability assessment of bio-fuels production, j. clean. prod., 71, 11-29, (2014) [14]. directive 2003/30/ec of the european parliament and of the council of the european union of 8 may 2003, https://eur-lex.europa.eu/legalcontent/en/txt/pdf/?uri=celex:32003l0030& from=en , accessed at 07.03.2020 [15]. irena, international renewable energy agency, innovation technology outlook for advanced liquid biofuels (forthcoming). irena, abu dhabi, (2016c) https://www.irena.org//media/files/irena/agency/publication/2016/ire na_innovation_outlook_advanced_liquid_biofu els_2016.pdf , accessed at 08.03.2020 [16]. irena, remap: roadmap for a renewable energy future, 2016 edition. international renewable energy agency (irena), abu dhabi, (2016) https://www.irena.org//media/files/irena/agency/publication/2016/ire na_remap_2016_edition_report.pdf , accessed at 08.03.2020 [17]. singh n.p., singh a., production of liquid biofuels from renewable resources, progress in energy and combustion science 37, 52-68, (2011) [18]. noraini m.y., ong h.c., badrul m.j., chong w.t., a review on potential enzymatic reaction for biofuel production from algae. renewable and sustainable energy reviews, 39 24–34, (2014) [19]. mussatto s.i., dragone g., guimaraes p.m.r., paulo j., silva a., carneiro l.m., roberto i.c., vicente a., domingues l., teixeira j.a., technological trends, global market, and challenges of bio-ethanol production, biotechnology advances, 28 817–830, (2010) [20]. lobo a.r., silva g.m.l., amido resistente e suas propriedades físico-químicas, rev de nutr :16, (2003) [21]. weber f.h, collares-queiroz f.p., chang y.k., caracterização físico-química, reológica, morfológica e térmica dos amidos de milho normal, ceroso e com alto teor de amilose. ciênc tecnol aliment, 29:748–53, (2009) [22]. konik-rose c., thistleton j., chanvrier h., tan i., halley p., gidley m., kosar-hashemi b., wang h., larroque o., ikea j., mcmaugh s., regina a., rahman s., morell m., li z., effects of starch synthase iia gene dosage on grain, protein and starch in endosperm of wheat, theor. appl. genet., 115: 1053–1065, (2007) [23]. cardona c., sanchez o., fuel ethanol production: process design trends and integration opportunities, bioresource technology, 98 2415– 2457, (2007) [24]. ren s., comparative analysis of some physicochemical properties of 19 kinds of native starches. starch/stärke, 68, 1600367, (2017) [25]. srichuwong s., sunarti t.c., mishima t., isono n., hisamatsu m., starches from different botanical sources i: contribution of amylopectin fine structure to https://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex:32003l0030&from=en https://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex:32003l0030&from=en https://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex:32003l0030&from=en https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_innovation_outlook_advanced_liquid_biofuels_2016.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_innovation_outlook_advanced_liquid_biofuels_2016.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_innovation_outlook_advanced_liquid_biofuels_2016.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_innovation_outlook_advanced_liquid_biofuels_2016.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_remap_2016_edition_report.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_remap_2016_edition_report.pdf https://www.irena.org/-/media/files/irena/agency/publication/2016/irena_remap_2016_edition_report.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 64 thermal properties and enzyme digestibility. carbohydr. polym., 60, 529–538, (2005) [26]. bertoft e., review understanding starch structure: recent progress, agronomy, 7, 56; doi:10.3390/agronomy7030056, (2017) [27]. walter m., silva l.p., emanuelli t., amido resistente: características físico-químicas, propriedades fisiológicas e metodologias de quantificação. ciência rural: 35:974–80, (2005) [28]. silva r.m., ferreira g.f., shirai m.a., haas a., scherer m.l., franco c.m.l., demiate i.m., características físicoquímicas de amidos modificados com permanganato de potássio/ácido lático e hipoclorito de sódio/ácido lático, ciênc tecnol aliment:28, (2008) [29]. kumar s., singh n., prasad r., anhydrous ethanol: a renewable source of energy, renewable and sustainable energy reviews, 14 1830–1844, (2010) [30]. pérez, s., & bertoft, e., the molecular structures of starch components and their contribution to the architecture of starch granules: a comprehensive review, starch/staerke, 62(8), 389420, (2010) [31]. alcázar-alay s.c., meireles m.a.a., physicochemical properties, modifications and applications of starches from different botanical sources, food sci. technol, campinas, 35(2): 215-236, (2015) [32]. gawande s. b., patil i. d., utilization of cereal grains for bioethanol production: a critical review, pratibha: international journal of science, spirituality, business and technology (ijssbt), vol. 3, no. 1, issn (print) 2277—7261, (2014) [33]. sanchez o.j., cardona c.a., trends in biotechnological production of fuel ethanol from different feedstocks bio-resource. technol. 99 5270–5295, (2008) [34]. mojović l., pejin d., lazić m., in: bioethanol as fuel state of the art and perspectives, faculty of technology, leskovac, , pp. 1-149 (in serbian), (2007) [35]. schwietzke s., kim y., ximenes e., mosier n., ladisch m., ethanol production from maize, biotechnology in agriculture and forestry, vol. 63, (2009) [36]. eckert c.t., frigo e.p., albrecht l.p., albrecht a.j.p., christ d., santos w.g., berkembrock e., egewarth v.a., maize ethanol production in brazil: characteristics and perspectives, renew. sustain. energy rev., 82, 3907–3912, (2018) [37]. pishgar-komleh s.h., keyhani a., mostofi-sarkari m., jafari a., energy and economic analysis of different seed corn harvesting systems in iran, energy: 43:469–76, (2012) [38]. usda, united states department of agriculture, world agricultural production, foreign agricultural service circular series wap 3-20 march, (2020) https://apps.fas.usda.gov/psdonline/circulars/produc tion.pdf , accessed at 09.03.2020 [39]. ferreira-leitao v., gottschalk l.m.f., ferrara m.a., nepomuceno a.l., molinari h.b.c., bon e.p.s., biomass residues in brazil: availability and potential uses, waste biomass valoriz., 1, 65–76, (2010) [40]. bharathiraja b., jayamuthunagai j., praveenkumar r., vinotharulraj j., vinoshmuthukumar p., saravanara a. bioethanol production from lignocellulosic materials an overview, 28 the scitech journal vol., 01 issue 07, (2014) [41]. shah t.r., prasad, k., kumar p., studies on physicochemical and functional characteristics of asparagus bean flour and maize flour, agroecology and food sciences, isbn: 97881-930585-1-0, (2015) [42]. bolarinwa f., olaniyan s.a., adebayo l.o., ademola a.a., malted sorghum-soy composite flour: preparation, chemical and physico-chemical properties, bolarinwa et al., j food process technol, 6:8, (2015) [43]. zhao r., wu x., seabourn b.w., bean s.r., guan l., shi y.-c., wilson j.d., madl r., wang d., comparison of waxy vs. nonwaxy wheats in fuel ethanol fermentation, cereal chemistry, 86: 145-156, (2009) [44]. griffey c., brooks w., kurantz m., thomason w., taylor f., obert d., moreau r., flores r., sohn m., hicks k., grain composition of virginia winter barley and implications for use in feed, food and biofuels production, journal of cereal science, 51: 41-49 (2010) [45]. macgregor a.w., and bhatty r.s., nonmalting uses of barley, in barley chemistry and technology, eds.pp. 355-418, st. paul: american association of cereal chemists, (1993) [46]. czuchajowska z., klamczynski a., paszczynska b., baik b.k, structure and functionality of barley starches, cereal chem., 75(5):747-754 (1998) [47]. wu w.v., recovery of protein-rich by products from oat stillage after alcohol distillation, journal of agricultural and food chemistry, 38: 588-592, (1990) [48]. doehlert, d.c., mcmullen m.s., hammond j.j., genotypic and environmental https://apps.fas.usda.gov/psdonline/circulars/production.pdf https://apps.fas.usda.gov/psdonline/circulars/production.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 65 effects on grain yield and quality of oat grown in north dakota, crop science, 41: 1066-1072, (2001) [49]. sterna v., zuteb s., brunava l., oat grain composition and its nutrition benefice, agriculture and agricultural science procedia 8 252, (2016) [50]. poutanen k., katina k., heiniö r.l., flavour design novel (bio)processing techniques for flavour design in plant-based foods, second edition, bakery products science and technology, (2014) [51]. kučerova, j.,. the effect of year, site and variety on the quality characteristics and bioethanol yield of winter triticale, journal of the institute of brewing and distilling, 113: 142–146, (2007) [52]. rohman a., helmiyati s., hapsari m., setyaningrum d.l., rice in health and nutrition, international food research journal, 21(1): 13-24 (2014) [53]. malathi d., varadharaju n., gurumeenakshi g., study on nutrient composition of millets and ways to minimize loss during processing and value addition post harvest technology centre agricultural engineering college and research institute tamil nadu agricultural university coimbatore, (641 003, 2014) [54]. emmanuel o.a., clement a., agnes s.b., chiwona-karltun l., drinah b. n., chemical composition and cyanogenic potential of traditional and high yielding cmd resistant cassava (manihot esculenta crantz) varieties, international food research journal, 19(1): 175-181, (2012) [55]. correa d.a., montero castillo p.m., martelo r.j., physicochemical characterization of sweet potato flour from the colombian caribbean, contemporary engineering sciences, vol. 11, 2018, no. 37, 1845 1851 [56]. fao, food and agriculture organization, cereal supply and demand brief, near-record wheat production expected in 2020, ample supplies to help shield food markets from turmoil of coronavirus storm, (2020) [57]. zhao r., ban s.r., wang d., park s.h., schober t.j., wilson, j.d., small-scale mashing procedure for predicting ethanol yield of sorghum grain, journal of cereal science, 49,230238, (2009) [58]. nuez-ortin w.g.n., yu p., nutrient variation and availability of wheat ddgs, corn ddgs, a blend ddgs from bioethanol plants, journal of the science of food and agriculture 89: 1754-1761, (2009) [59]. moreau r., liu ks, kurt a., rosentrater, distillers grains: production, properties, and utilization, (2016) [60]. hendriks a.t.w.m., zeeman g., review pretreatments to enhance the digestibility of lignocellulosic biomass, bioresource technol., 100, 10-18, (2009) [61]. reith j.h., den uil h., van veen h., de laat w.t.a.m., niessen j.j., de jong e., elbersen h.w., weusthuis r., van dijken j.p., raamsdonk l., 12th eur. conf. technol. exhib. biomass for energy, industry and climate protection, amsterdam, ecnrx02-030, (2002) [62]. petrova p., ivanova v. perspectives for the production of bioethanol from lignocellulosic materials, biotechnology & biotechnological equipment, 24:sup1, 529-546, (2010) [63]. shewale s. d., pandit a. b., enzymatic production of glucose from different qualities of grain sorghum and application of ultrasound to enhance the yield, carbohydrate research, 344, pp. 52-60, 2009 [64]. belton p.s., taylor, j.r.n., sorghum and millets: protein sources for africa, trends food science and technology, 15, 94-98, (2004) [65]. smith c.w., frederiksen r.a., sorghum: origin, history, technology, and production, john wiley and sons, new york, (2000) [66]. fang q., hanna m. a., experimental studies for levulinic acid production from whole kernel grain sorghum, bio-resource technology, 81,187-192, (2002) [67]. wang d., bean s., mclaren j., seib p., madl r., tuinstra m., shi y., lenz m., wu x., zhao r., grain sorghum is a viable feedstock for ethanol production, journal of industrial microbiology and biotechnology, 35, 313-320, (2008) [68]. distelfed a., avni r., fischer a.m., senescence, nutrient remobilization, and yield in wheat and barley., j exp bot., 65: 3783-3798, (2014) [69]. hiei y., ishida y., komari t., progress of cereal transformation technology mediated by agrobacterium tumefaciens, front plant sci., 5: 628-638 (2014) [70]. tiefenbacher k.f., chapter two technology of main ingredients—water and flours, wafer and waffle, processing and manufacturing, pages 15-121, (2017) [71]. flores r.a., hicks k.b., wilson j., surface abrasion of hulled and hulless barley: physical characteristics of the milled fractions, cereal chemistry, 84: 485-491 (2007) [72]. ingledew w.m., thomas k.c., hynes s.h., mcleod j. g., viscosity concerns with rye mashes used for ethanol production, cereal chemistry, 76: 459-464, (1999) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 66 [73]. wang s., thomas k.c., sosulski k., ingledew w.m., sosulski f.w., grain pearling and very high gravity (vhg) fermentation technologies for fuel alcohol production from rye and triticale, process biochemistry, 34: 421-428, (1999) [74]. czarnecki z., nowak j., effects of rye pretreatment and enrichment with hemicellulolytic enzymes on ethanol fermentation efficiency, electronic journal of polish agricultural universities, 4(2): l2, (2001) [75]. balcerek m., pielech-przybylska k., effect of supportive enzymes on chemical composition and viscosity of rye mashes obtained by the pressureless liberation of starch method and efficiency of their fermentation, european food research and technology, 229: 141-151, (2009) [76]. fields of energy, sw seed limited. available online: www.swseedco.com. accessed november 2, (2010) [77]. davis-knight h. r., weightman r. m, the potential of triticale as a low input cereal for bioethanol production, adas uk ltd, centre for sustainable crop management, project report no. 434, (2008) [78]. pejin d., mojovic l. j., vucurovic v., pejin j., dencic s., rakin m., fermentation of wheat and triticale hydrolysates: a comparative study, fuel 88, :1625-1628, (2009) [79]. wu x., wang d., bean s.r., wilson j.p, ethanol production from pearl millet using saccharomyces cerevisiae, cereal chemistry 83: 127–131, (2006) [80]. lam e., carrer h., da silva j.a., kole c. (eds.), compendium of bioenergy plants: sugarcane; crc press: boca raton, fl, usa, isbn 1482210584, (2014) [81]. manochio c., andrade b.r., rodriguez r.p., moraes b.s., ethanol from biomass: a comparative overview, renew. sustain. energy rev., 80, 743–755, (2017) [82]. souza g.m., van sluys m.a., sugarcane genomics and biotechnology. in: cortez lab (ed) sugarcane bioethanol for editora edgard blucher ldta, são paulo, (2010) [83]. fao faostat. food and agriculture organization of the united nations. http://faostat.fao.org/, (2013) [84]. fao (food and agriculture organization of the united nations), faostat database, production, crops, www.fao.org/faostat/en/ – data/qc, (2018) [85]. irena, international renewable energy agency, sugarcane bioenergy in southern africa: economic potential for sustainable scale-up, international renewable energy agency, abu dhabi, (2019) [86]. kojima m., johnson t., potential for biofuels for transport in developing countries, the international bank for reconstruction and development/the world bank, energy sector management assistance programme report, october, (2005) [87]. https://knoema.com/atlas/topics/agriculture/c rops-production-quantity-tons/sugar-beetproduction , accessed at 10.03.2020 [88]. shapouri h., salassi m., fairbanks j.n., the economic feasibility of ethanol production from sugar in the united states, joint publication of oepnu, oce, usda, and lsu, (2006) [89]. panella, l., kaffka, s.r., sugar beet (beta vulgarisl) as abiofuel feedstock in the united states. in sustainability of the sugar and sugar-ethanol industries, ed. g. eggleston, 163– 175, (2010) [90]. tzilivakis j., warner d.j., may m., lewis k.a., jaggard k., an assessment of the energy inputs and greenhouse gasemissions in sugar beet (beta vulgaris) production in the uk, agricultural systems, 85: 101–119, (2005) [91]. schmer m.r., vogel k.p., mitchell r.b., perrin,r.k., netenergy of cellulosic ethanol from switchgrass, proceedings ofthe national academy of sciences of usa, 105: 464– 469, (2008) [92]. lee p., sugar beet as an energy crop, sugar tech, 12(3–4):288–293, (2010) [93]. manano j., ogwok p., byarugababazirake g.w., chemical composition of major cassava varieties in uganda, targeted for industrialisation, ournal of food research; vol. 7, no. 1, (2018) [94]. wikipedia, https://en.wikipedia.org/wiki/sweet_potato#produc tion accessed at 10.03.2020 [95]. fao, food and agriculture organization of the united nations, sweet potato production in 2017, world regions/production quantity from pick lists", statistics division (faostat) (2017) [96]. ji h., zhang h., li h., li y, analysis on the nutrition composition and antioxidant activity of different types of sweet potato cultivars, food and nutrition sciences, 6, 161-167, (2015) [97]. putri l.s.e., nasrulloh, haris a., bioethanol production from sweet potato using combination of acid and enzymatic hydrolysis, applied mechanics and materials vols., 110-116 pp 1767-1772, (2012) [98]. delcour j.a., bruneel c., derde l.j, gomand s.v., pareyt b., putseys j.a., wilderjans e., lamberts l., fate of starchin food processing:from raw materialsto http://faostat.fao.org/ https://knoema.com/atlas/topics/agriculture/crops-production-quantity-tonnes/sugar-beet-production https://knoema.com/atlas/topics/agriculture/crops-production-quantity-tonnes/sugar-beet-production https://knoema.com/atlas/topics/agriculture/crops-production-quantity-tonnes/sugar-beet-production https://en.wikipedia.org/wiki/sweet_potato#production https://en.wikipedia.org/wiki/sweet_potato#production food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 67 final food products, annu. rev. food sci. technol, 1:87–111, (2010) [99]. wu x., zhao r., wang d., bean s.r., p.a. seib, tuinstra m.r., campbell m., o'brien a., effects of amylose, corn protein, and corn fiber contentson production of ethanol from starch-rich media, cereal chemistry 83(5), (2006) [100]. zakaria n.h., muhammad n., abdullah m.m.a.b., potential of starch nanocomposites for biomedical applications, international conference on innovative research — icir euroinvent (2017) [101]. vermerris w., nichols n.n, bothast r.j, genetic improvement of corn for lignocellulosic feedstock production, genetic improvement of bioenergy crops, (2008) [102]. zamora l.l., gonzález calderón j.a., trujillo vázquez e., bolaños reynoso e., optimization of ethanol production process from cassava starch by surface response, j. mex. chem. soc., 54(4), 198-203, (2010) [103]. smith t.c., kindred d.r., brosnan j.m., weightman r.m., shepherd m. , sylvester-bradley r., wheat as a feedstock for alcohol production, research review, no. 61, (2006) [104]. gumienna m., szwengiel a., szczepa ska-alvarez a., szambelan k., lasik-kurdy m., czarnecki z., sitarski a., the impact of sugar beet varieties and cultiva-tion conditions on ethanol productivity, biomass bioen-ergy, 85, 228-234, (2016) [105]. marzo c., díaz a.b., caro i., blandino a., status and perspectives in bioethanol production from sugar beet, bioethanol production from food crops (2019) [106]. mcleod j.g., may w.e., salmon d.f., sosulski k., thomas j.b., brown p.d., vera c.l., changes in ethanol production potential due to species, cultivar and location on the canadian prairie, canadian journal of plant science, (2009) [107]. halling p, parrish m., fuglsang c.c., bioenergy advancing grain-based ethanol, novozymes, (2015) [108]. surget a., barron c., 2005. histologie du grain de blé. industrie des céréales 145, 3-7. [109]. barron c., abécassis j., chaurand m., lullien-pellerin v., mabille f., rouau x., sadoudi a., samson m.f., accès à des molécules d’intérêt par fractionnement par voie sèche, innovations agronomiques, 19, 51-62, (2012) [110]. thai c.c.d., bakir h., doherty w.o.s., insights to the clarification of sugar cane juice expressed from sugar cane stalk and trash, j. agr. food chem., 60: 2916-2923, (2012) [111]. costa g.h.g., masson i.s., de freita l.a., roviero j.p., mutton m.j.r., reflects of clarification of sugarcane juice with moringa on inorganic compounds of sugar vhp | reflexos da clarificação do caldo de cana com moringa sobre compostos inorgânicos do açúcar vhp, rev. bras. eng. agric. amb., 19: 154-159, (2015) [112]. alonso-gómez l.a., bello-pérez l.a., four generations of raw materials used for ethanol production: challenges and opportunities, agrociencia, vol.52 n.7 méxico oct./nov. (2018) [113]. senthilkumar v., gunasekaran p., bioethanol from biomass production of ethanol from molasses, in: pandey a, editor, handbook of plant-based biofuels, boca raton, fl, usa: crc press; pp. 73–86, (2009) [114]. chauvet, m. gonzález, r., biocombustibles y cultivos biofarmacéuticos: oportunidades o amenazas, revista el cotidiano, redalyc, 23(147), 51-61, (2008) [115]. nichodemus c.o., bioethanol: production, advantages, disadvantages and environmental impacts, environmentalbiotechnology, (2017) https://www.biotecharticles.com/environmentalbiotechnology-article/bioethanol-productionadvantages-disadvantages-and-environmentalimpacts-3816.html, accessed at 10.03.2020 [116]. kranz l.w., irmak s., van donk s.j., c. yonts d., martin l.d., irrigation management for corn university of nebraskalincoln extension, insitute of agricultural and natural resources, (2008) [117]. bowen e., kennedy s.c., miranda k., ethanol from sugar beets: a process and economic analysis, worcester polytechnic institute, (2010) [118]. efetha a., irrigation management of sugar beets government of alberta: rural and agricultural development, irrigation branch, (2008) [119]. cattanach a.w., dexter a.g., &oplinger, e.s., sugarbeets, alternative field crops manual, (1991) [120]. flores-gómez c.a., escamilla silva e.m., zhong c., dale b.e., da costa sousa l., balan v., conversion of lignocellulosic agave residues into liquid biofuels using an afex™-based biorefnery, biotechnol biofuels, 11:7, (2018) [121]. davis s.c., ming r., lebauer d.s., long s.p., toward systems-level analysis of agricultural production from crassulacean acid metabolism (cam): scaling from cell to https://www.biotecharticles.com/environmental-biotechnology-article/bioethanol-production-advantages-disadvantages-and-environmental-impacts-3816.html https://www.biotecharticles.com/environmental-biotechnology-article/bioethanol-production-advantages-disadvantages-and-environmental-impacts-3816.html https://www.biotecharticles.com/environmental-biotechnology-article/bioethanol-production-advantages-disadvantages-and-environmental-impacts-3816.html https://www.biotecharticles.com/environmental-biotechnology-article/bioethanol-production-advantages-disadvantages-and-environmental-impacts-3816.html food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 vasile-florin ursachi, gheorghe gutt, review: the main raw materials used for production of 1st and 2nd generation bioethanol, food and environment safety, volume xix, issue 1 – 2020, pag. 48 – 68 68 commercial production, new phytol., 208:66–72, (2015) [122]. davis s.c., dohleman f.g., long s.p., the global potential for agave as a biofuel feedstock, gcb bioenergy, 3(1):68–78, (2011) [123]. yan x., tan d.k.y., inderwildi o.r., smith j.a.c., king d.a., life cycle energy and greenhouse gas analysis for agave-derived bioethanol, energy environ sci., 4(9):3110–21, (2011) [124]. sun j., ding s.y., peterson j.d., biological conversion of biomass for fuels and chemicals (explorations from natural utilization system), (2014) [125]. johnson f.x., stockholm environment institute. import substitution and export potential for biofuels trade: the case of ethanol from sugarcane and sweet sorghum, (2006) [126]. zamora d.s., apostol k.g., wyatt g.j., biomass production and potential ethanol yields of shrub willow hybrids and native willow accessions after a single 3-year harvest cycle on marginal lands in central minnesota, usa, (2014) [127]. rios m, kaltschmitt m., bioenergy potential in mexicostatus and perspectives on a high spatial distribution, biomass conversion and biorefinery, 3:239-254, (2013) [128]. azizi k., heidari s., a comparative study on energy balance and economical indices in irrigated and dry land barley production systems, international journal of environmental science and technology, 10:1019-1028, (2013) [129]. buhl p.n., new species of platygastrinae and sceliotrachelinae (hymenoptera, platygastroidea: platygastridae) from australia, international journal of environmental studies, 71(4):425-448, (2014) [130]. brown r.c., introduction to thermochemical technologies for production of fuels and biobased products, center for sustainable environmental technologies iowa state university., (2013) [131]. belasri d., alboainain s. , bastidas-oyanedel j-r., schmidt j.e., assessment of biofuel potential from agave spp. and salicornia in the arid emirate of abu dhabi, advances in environmental and agricultural science, (2015) [132]. food and agriculture organization of the united nations, introducing our special guest, solanum tuberosum, the “humble tuber” that spread from its andean birthplace across six continents, staving off hunger, fuelling economic development and changing the course of world history, international year of the potato, (2008) http://www.fao.org/tempref/docrep/fao/011/i0500e/ i0500e02.pdf , accessed at 11.03.2020 [133]. yamdeu j.h.g., gupta p.h., patel n.j., shah a., talatij g., 2016. effect of storage temperature on carbohydrate metabolism and development of cold-induced sweetening in indian potato (solanum tuberosum l.) varieties, journal of food biochemistry, 40: 71-83, (2016) [134]. lovat c., nassar amk., kubow s., li x.q., donnelly d.j., metabolic biosynthesis of potato (solanum tuberosum l.) antioxidants and implications for human health, critical reviews in food science and nutrition, 56: 2278-2303, (2016) [135]. https://www.potatopro.com/world/pot ato-statistics [136]. https://ec.europa.eu/eurostat/statistics -explained/pdfscache/49931.pdf http://www.fao.org/tempref/docrep/fao/011/i0500e/i0500e02.pdf http://www.fao.org/tempref/docrep/fao/011/i0500e/i0500e02.pdf https://www.potatopro.com/world/potato-statistics https://www.potatopro.com/world/potato-statistics https://ec.europa.eu/eurostat/statistics-explained/pdfscache/49931.pdf https://ec.europa.eu/eurostat/statistics-explained/pdfscache/49931.pdf 1. introduction doi: https://doi.org/10.4316/fens.2021.002 13 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1 2021, pag. 13 20 scientific explanation of composition of acidophilic-whey ice cream, enriched with protein galina polischuk1, oksana kochubey-lytvynenko1, tetiana osmak1, uliana kuzmik1, oksana bass, artur mykhalevych1, * victoria sapiga1 educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str. 68, kyiv, ukraine, vika.sapiga1904@ukr.net *corresponding author received 6th november 2020, accepted 30th march 2021 abstract: the work is devoted to solving the actual problem of the dairy industry complex processing of food raw materials, expanding the range of dairy and milk-containing products of high nutritional value due to enrichment of protein, proand prebiotics, rational use of production resources by reducing the the technological process duration. the aim of the research is to substantiate the composition of a new type of acidophilic-whey ice cream of high nutritional value due to enrichment with a complex of proteins, probiotic sourdough and pectin-containing vegetable paste. the main tasks of scientific work are: development of a scientifically based formulation for a new type of ice cream to expand the range of products that will improve the nutrition structure of the ukraine`s population. the methods used to study the organoleptic and physico-chemical indicators of blends and ice cream are well known and presented in the article. the method of production of sour milk ice cream and the choice of dairy raw materials for obtaining a fermented base was theoretically substantiated, the functional and technological properties of protein concentrates and their compositions in ice cream formulations were studied, the rational content of protein in ice cream was established, also the expediency of using pectin-containing vegetable paste in acidophilic-whey ice cream was proved. keywords: acidophilic-whey ice cream, probiotics, prebiotics, protein, pectin-containing purees 1. introduction ice cream is a product popular with all segments of the population, especially among children. but this dessert product is not low in calories. therefore, nutritionists do not recommend using it for overweight children. the share of sugar in ice cream is quite high from 14 to 30%, and the fat content in ice cream is in the range from 8 to 20%. at the same time, in recent years in the scientific works of many ukrainian and foreign scientists much attention is paid to the production of low-calorie foods [1, 2]. in eu countries, the nutritional value of products indicated on the label is regulated by the relevant regulation [3]. according to this document, a low-fat product should contain no more than 3 g of fat per 100 g of product, and a product with a low sugar content should be 30% less sugar than a similar product with a full content. instead, the range of domestic ice cream is mainly high-fat products of creamy and plombieres groups, although the world's mailto:vika.sapiga1904@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 demand for low-calorie milk ice cream is growing every year. however, in the case of fat content decrease in the milk ice cream, there are defects of consistency in the form of coarse-grained structure and heterogeneous air phase [4]. that is why to prevent such a defect in consistency in milk formulas additionally add moisturebinding components, including proteins and polysaccharides [5]. the range of ice cream with probiotics, non-fat and low-fat in ukraine is extremely limited (yogurt, cottage cheese ice cream), and ice cream enriched with proteins and prebiotics is completely absent [6]. therefore, instead of milk as a dairy base in the composition of ice cream, it is advisable to use fermented probiotic acidophilic yeast secondary dairy resources skimmed milk, buttermilk and whey, which contain biologically complete proteins and can be further enriched with pectin-containing raw materials. in view of the above, research on the expansion of the range of low-calorie ice cream of high nutritional value is a very relevant area of research. 2. matherials and methods 2.1. materials the following raw materials, that met the requirements of current standards, were used for research: whole milk dstu 3662-2018, buttermilk and whey obtained during the production of sweet butter by the method of hfc conversion and natural rennet cheese from whole milk according to dstu 3662-20018, sugar dstu 4623:2006, drinking water gost 287482, skimmed milk powder dstu 4273:2003, buttermilk powder dstu 4555:2006, whey powder dstu 4552:2006, fresh vegetables according to gstu 7033:2009 and dstu 8147:2015, starter on pure cultures of lactobacillus acidophilus ("iprovit"), sodium caseinate (firm "deirico", lutsk casein plant, ukraine) integrated stabilization system cremodan se 406 (firm "danisco", denmark) according to the conclusions of the sanitary-hygienic issued by the central executive body for health care of ukraine. 2.2. methods resistance to melting was determined by a modified method. for research, before sampling, the samples were subjected to short-term warming at a temperature of 20 ºc for 15 minutes. the formed ice cream cylinders with a height of 50 mm and a diameter of 35 mm were placed on perforated plastic stands mounted on tripods. under the tripods were measuring cylinders with a capacity of 100 cm³, into which the liquid fraction flowing from the ice cream samples fell through the funnels. using a special tripod, the digital camera was statically fixed and the samples were photographed at a temperature of 20 ºc for 60 minutes. the time of appearance of the first drop of "melt" and the time of outflow from the ice cream sample of 10 cm³ "melt" were recorded. overrun (s),%, of ice cream was determined by weight and calculated according to the formula: (1) where m m – mass of ice cream blend of a certain volume, g; m the mass of ice cream of the same volume, g. the size of the air bubbles was determined by the method of asrri (all-russian scientific research institute of refrigeration industry). the ice cream sample was applied to the calibrated grid of goryaev's chamber, covered with a cover glass and immediately microscopied at 160 times magnification. the ice crystals melted, but the foam remained, 14 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 because under these conditions, the air bubble shells were not dehydrated. the count was performed in five to seven fields of view. by the number of groups n, the weighted average diameter of the air bubbles for each preparation dav was calculated according to the formula: dav = 3 1 3  n i d in n (2) where i d – the average diameter of air bubbles in each group, μm; i n the number of bubbles in each group; n total amount of bubbles in all groups. organoleptic evaluation of ice cream samples was performed by tasting. determination of organoleptic properties was carried out using qualitative and quantitative methods. qualitative assessment of ice cream was expressed in a verbal description of its taste, smell, structure and consistency, in quantitative assessment organoleptic indicators were reflected in a certain amount of points, according to the 100-point system with the accepted distribution of significance of its individual indicators: taste, smell and aroma 60 points, structure and consistency 30 points, color and appearance 10 points. according to the total number of ice cream was attributed to one of the varieties: extra from 96 to 100 points; the highest from 91 to 95 points; the first from 80 to 90 points. 2.3. preparation of experimental samples separately prepared sour milk base from skimmed milk, buttermilk and whey. for this purpose, dry dairy raw materials was restored in liquid dairy raw materials, the blend was filtered and heated to a temperature of 70±2 ° c, pasteurized at a temperature of 80…85 ° c for 15-20 s, cooled to 40…42 ° c, inoculationed with starter of lactobacillus acidophilus, incubationed for 3-4 h at a temperature of 42…43 ° c to obtain a clot with a ph of not less than 4.1, followed by cooling to a temperature not exceeding 6 ºc. separately in water at a temperature of 40 °c was made stabilizer with sugar in a ratio of 1:3 and the remaining sugar, the blend was pasteurized at a temperature of 82…85 ° c for 15-20 s, cooled to 10 ° c, after-cooled and matured at 4…6 ° c for 4 h. homogenization was excluded from the technological scheme in the absence of fatcontaining components, except for the preparation of the control sample. before freezing, the fermented acidophilic base was mixed with the main mixture. freezing, hardening, re-hardening and storage of ice cream was carried out according to the classical scheme. 2.4. the statistical analysis the obtained measurement results were calculated using standard statistical processing programs microsoft excel. graphical representation of experimental data was performed using microsoft excel. the accuracy of the results was ensured by three to five times the repetition of the experiments. 3. results and discussion samples of sour milk ice cream without filler with mass fraction of fat 2.8% (control) and acidophilic low-fat ice cream based on protein-carbohydrate raw materials (phrm) skimmed milk, buttermilk and whey were prepared according to the typical formulations of yogurt ice cream [7]. it was used starter of direct entering (dvs-starter), so its weight was not taken into account. in order to explain the significant difference in the quality indicators of the studied ice cream samples, the total milk protein content and its percentage in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 dsmr of each ice cream sample were calculated (table 1). table 1. mass fraction of protein in samples of acidophilic ice cream and its percentage content in the dsmr of product,% protein content: sample name ice cream on normalized milk (control) ice cream on skimmed milk ice cream on buttermilk ice cream on whey in ice cream, % 3.23 3.09 2.55 1.28 in dsmr of ice cream,% 37.13 35.52 29.31 14.71 according to table. 1, it becomes clear that in the case of phrm mass fraction of milk protein in ice cream (compared to the control sample) is gradually reduced. in the case of using whey as a milk base, the protein content decreases most significantly from 3.23% (control) to 1.28% (whey ice cream) while a significant shift towards the predominance of dsmr, lactose and mineral salts from 62 , 87, 64,48, 70,69% (for ice cream on normalized milk, skimmed, buttermilk) to 85.29% (for whey ice cream). therefore, whey ice cream itself is most in need of enrichment with milk protein concentrates in the absence of an effective biopolymer a protein that binds free water, structures mixtures and stabilizes the structure of ice cream. at the next stage of work, in order to identify the rational content of protein concentrates in acidophilic-whey ice cream, was investigated the possibility of enriching the product with sodium caseinate (sc), concentrate of whey proteins obtained by ultrafiltration (cwpuf) and soy protein isolate (sp). to enrich the experimental samples of ice cream, the content of milk-protein concentrates in the range from 0.75 to 3.75% with a step of 0.75% was selected. the overrun of acidophilic-whey ice cream with the content of different protein concentrates is shown in figure1. fig. 1 the overrun of acidophilic-whey ice cream, enriched with proteins of various origins according to the results of the study (fig. 1), it was found that the most technologically efficient foaming agent is cwp-uf (78% overrun), and the overrun 20 30 40 50 60 70 80 90 0 0,75 1,5 2,25 3 3,75 cwp-uf sc sp o v e r r u n % mass fraction of protein, % 16 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 of blends with sp and sc was 65 and 60%, respectively. resistance to melting of acidophilic-whey ice cream with the content of various protein concentrates is shown in figure 2. resistance to melting of ice cream with sodium caseinate (fig. 2) was the highest and slightly decreased for the sample with cwp-uf and was the lowest for the sample with soy protein. the average diameter of air bubbles of acidophilic-whey ice cream with the content of different protein concentrates is shown in figure 3. the best dispersing agent that provides a fine distribution of the air phase in ice cream is sc with a slight decrease in this indicator for cwp-uf and for sp (fig. 3). sodium caseinate as a functional and technological ingredient in ice cream mixes improves their structure and consistency, binds part of the free water in the mixes, increases the dispersion of air bubbles. fig. 2 resistance to melting of acidophilic-whey ice cream enriched with proteins of various origins fig. 3 the average diameter of air bubbles of acidophilic-whey ice cream enriched with proteins of different origins thus, according to the results of the analysis of functional and technological characteristics of proteins, it is possible to recommend the mass fraction of cwp-uf (on average 215 uah/kg) at the level of 1.0-1.5%, and the content of sc (275 uah/kg) in the composition of ice cream, due to the manifestation of alkaline taste, despite its other advantages, should be used in an amount not exceeding 0.75%. cheaper soy protein isolate (about 145 uah/kg), as well as cwp-uf, can be recommended in the amount of about 1.01.5%, although this protein is less effective in acidophilic-whey ice cream. therefore, if necessary to enrich acidophilic-whey ice cream with protein in an amount of not less than 2.75%, it is advisable to use the following protein composition: 0.75% sc (the most effective moisturebinding stabilizing high-value protein); 1.0-1.5% cwp-uf (protein with foaming ability); 1.0-1.5% sp (cheap protein with moderate technological activity). protein compositions at the lower and upper limits of the components content are as follows: sc + cwp-uf + sp = 0.75% + 1.0% + 1.0% = 2.75%; sc + cwp-uf + sp = 0.75% +1.0% + 1.5% = 3.25%; sc + cwp-uf + sp = 0.75% + 1.5% + 1.5% = 3.75%. when using the first composition, the protein content in ice cream with the status 20 30 40 50 60 70 80 0 0,75 1,5 2,25 3 3,75 mass fraction of protein, % r e si st a n te t o m e lt in g , m in cwp-uf sc sp 20 25 30 35 40 45 50 55 60 65 70 0 0,75 1,5 2,25 3 3,75 mass fraction of protein, % cwp-uf sc sp t h e a v e r a g e d ia m e te r o f a ir b u b b le s, µ m 17 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 of "protein enriched" will be 4.0%, the second composition 4.5%, and the third 5.0%, which should be indicated in the product label and taken into account in marketing advertising. at the next stage, to give acidophilic-whey ice cream original organoleptic properties, in particular, color, taste and consistency, it was decided to add additional vegetable paste (beetroot + broccoli at a ratio of 1:1), the technology of which was developed at the department of milk and dairy products technology, nuft [8]. due to the probable interaction between milk proteins and soluble pectin of vegetables in the paste [9] it is possible to improve the structure of ice cream, which requires additional confirmation. on the example of the recipe based on fresh cheese whey with the third composition of protein concentrates (sc+cwp-uf+sp = 0.75%+1.5%+1.5% = 3.75%) (table 2) was established rational the content of vegetable paste in the composition of acidophilic-whey ice cream. to do this, was investigated a set of quality indicators of ice cream samples with a mass fraction of paste from 5 to 15%, compared with the sample without vegetable filler (control). table 2. formulations of experimental samples of acidophilic-whey ice cream, enriched with a complex of proteins with different content of vegetable paste, kg per 1000 kg (excluding losses) * formulation component ice cream samples with mass fraction of vegetable paste, % 0 (control) 5 (sample 1) 10 (sample 2) 15 (sample 3) cheese whey (mass fraction of dry matter 6.1%, mass fraction of protin 0.6%, mass fraction of lactose 4.5%) 783.5 (0.47% of protein) 733.5 (0.44% of protein) 683.5 (0.41% of protein) 633.5 (0.38 % of protein) sodium caseinate 7.5 7.5 7.5 7.5 cwp-uf 15.0 15.0 15.0 15.0 soy isolate 15.0 15.0 15.0 15.0 white crystalline sugar 175.0 175.0 175.0 175.0 vegetable puree 50.0 100.0 150.0 stabilization system se 406 4.0 4.0 4.0 4.0 total: 1000.0 characteristics of the finished product mass fraction of dry matter, not less than,%, of them: 26.92 27.56 28.23 28.89 dsmr, not less 9.02 8.66 8.33 7.99 sugar, not less 17.5 17.5 17.5 17.5 protein, not less 4.22 4.19 4.16 4.13 dry matter of vegetables, not less 1.0 2.0 3.0 stabilizer, not less 0.4 0.4 0.4 0.4 * the formulation does not take into account the mass fraction of dvs-starter on pure cultures of lactobacillus acidophilus, in the calculations with the mass of whey indicated the introduction of protein in the percentage of 70+16.64+16.68. 18 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 organoleptic and physico-chemical quality indicators of acidophilic-whey ice cream, enriched with a complex of proteins and vegetable filling, are shown in fig. 4 and 5. fig. 4 – physico-chemical parameters of acidophilic-whey ice cream, enriched with a complex of proteins with different content of vegetable puree fig. 5 score of organoleptic parameters of acidophilic-whey ice cream, enriched with a complex of proteins with different content of vegetable puree 19 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein, food and environment safety, volume xx, issue 1 – 2021, pag. 13 20 15 according to fig. 4, the highest overrun was found at the content of vegetable paste at the level of 10%, which is explained by the rational ratio between the foaming and stabilizing properties of the protein complex and pectin substances. a further increase in the content of the paste probably thickens the mixture too much, which complicates the process of saturating the mixture with air during freezing. the resistance to melting and dispersion of the air phase of ice cream with a increase in the paste content of more than 10% change slightly. organoleptic characteristics of ice cream (fig. 5) confirm the feasibility of adding 10% vegetable paste in formulation of acidophilic-whey ice cream, what significantly improves the taste and smell, turns the product pink, promotes the formation of a light creamy consistency. if the paste content exceeds more than 10%, there is a slight decrease in creaminess also the ice cream is too bright and unnatural. 4. conclusion thus, the expediency of protein complex using in acidophilic-whey ice cream has been scientifically proven, which in combination with vegetable paste due to the complexation between proteins and pectin and in the presence of insoluble dietary fiber of vegetables allows to obtain maximum technological effect. low-fat ice cream, enriched with proteins, has improved organoleptic characteristics, as protein concentrates play the role of mimetics of milk fat. 5. references [1]. swinburn b., caterson i., seidell j., james w.p. diet, nutrition and the prevention of excess weight gain and obesity, public health nutrition: 7(1a), рр.123–146.; [2]. kazakova n.v., tvorogova a.a. influence of sucrose replacement by soluble dietary fibers on physical parameters of ice cream, storage and processing of agricultural raw materials, № 11, 2007, p. 49-51. [3]. regulation (ec) no 1924/2006 of the european parliament and of the council of 20 december 2006 on nutrition and health claims made on foods [4]. polishchuk, g., breus, n., shevchenko, i., gnitsevych, v., yudina, t., nozhechkina-yeroshenko, g., semko, t. determining the effect of casein on the quality indicators of ice cream with different fat content. eastern-european journal of enterprise technologies, 4/11 (106), 2020, p. 24-30. [5]. rudakova t.v., minorova a.v., & narizhny s.a. innovative technologies of ice cream with functional ingredients, (2009). [6]. trubnikova, a., sharakhmatova, t., mamintova, k., & tsupra, o. biotechnological aspects of obtaining a yogurt base for the production of low-lactose ice cream. bulletin of the national technical university "khpi". series: new solutions in modern technologies, (9 (1285)), 2018, 243-255. [7]. arsenyeva t.p. handbook of dairy production technologist. technologies and recipes. vol.4. ice cream st. petersburg: giord, 2002, 184 p.. [8]. sapiga v., sizhko d., polischuk g., osmak t. technological features of milk and vegetable ice cream production, 85th jubilee international scientific conference of young scientists, postgraduates and students "scientific achievements of youth solution problems of human nutrition in the xxi century ", april 11-12, 2019, k .: nuft, part 1., 2019, p. 407. [9]. pavlyuk r. yu., pogarska v.v. innovative technologies of vitamin fruit ice cream with the use of frozen fine additives from vegetable raw materials, east european journal of advanced technologies. 4/10 (64), 2013, p. 57-63. 20 18 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 18 26 study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l. *mirela ardelean 1 , andrei lobiuc 2,3, marian burducea 3 , ciprian mihali 4 , daniela-teodora marți 4 1 „vasile goldiş” western university from arad, plant biotechnology, institute of life science, romania *corresponding author e-mail: mirela.ardelean1@yahoo.com 2 ”stefan cel mare” university, faculty of food engineering, universitatii str. 13, suceava, romania 3 „alexandru ioan cuza” university of iasi, romania, faculty of biology, carol i bd., iasi, romania 4 „vasile goldiş” western university from arad, romania abstract: in this study we aim by using plant biotechnology to study the effect of heavy metals cd and pb on the regeneration capacity of sedum telephium ssp. maximum l. plant species that grows spontaneously in our country and can also be cultivated for ornamental purposes. the results obtained at different concentrations of each metal will be compared to establish, from a morphological point of view, the existence of a dose-response relationship. according to the rules of the usa energy department, the hyperaccumulator plants have to fulfill the following characteristics: rapid growth and large biomass; pest and disease resistance; inedible for humans and animals; easy to harvest; very branched root system; accumulation of different types of heavy metals. following preliminary results (in vitro and ex vitro) we considered that this species fulfils the above conditions. keywords: heavy metals, sedum, in vitro, phytoremediation. 1. introduction plant tissue culture is a convenient laboratory instrument for phytoremediation studies. the most used forms of tissue culture are cell suspensions, calluses and organogenesis [1]. once established, these in vitro cultures can be propagated indefinitely and are available on request [2]. whole plants however, are cultivated in soil or grown in hydroponics, the systems have a limited life span and each individual plant has to be replaced and reestablished after each experiment. thus, the time for investigations can be substantially reduced using tissue culture instead of whole plants [3]. therefore, in this study we aim by using plant biotechnology to study the effect of heavy metals cd and pb on the regeneration capacity of sedum telephium ssp. maximum l. sedum gender belongs to the crassulaceae family [4]; [5] and consists of almost 400 species with succulent leaves. sedum telephium ssp. maximum (l.) krock is frequently spread in the romanian flora as a spontaneous species, as well as an ornamentally cultivated species. more than that, the romanian traditional medicine considers that this plant might have therapeutic (vulnerary, antiseptic, wounds) effects. in the middle of the sixteenth century, hieronymus bock reported that extracts of sedum telephium ssp. maximum were used in the rhine valley to treat internal injuries like lung ulcers [6]. now today, medical researchers isolate the active ingredients from those traditional medicine plants and test their efficacy [13]. in the early 1990's, some researchers in munich have identified two polysaccharides in sedum telephium ssp. maximum that were anti-inflammatory [7]. a few years later, some italian scientists observed the ways in which the polysaccharides and flavonols operated on http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 19 cells during wound healing [14]. the vegetable from the in vitro culture is part of modern biotechnology industry that focuses on various areas, including a special interest in plant biotechnology presents, that in vitro cultivation of physiotherapeutic interest [8]. in general, many herbs micro propagated in vitro were used as starting material in the popular culture media that are filled with bioreactors, and the biomass collected from a number of days in vitro culture pass extraction and condition the compounds of pharmaceutical interest [9]. 2. materials and methods 2.1 plant material, composition of the growth medium and the vitro culture the growth substrate used for all vitro culture experiments was made from agarised murashige skoog (1962) (ms) base medium [10] consisting of macroelements, fe edta and microelements, mineral blend according to the original formulation with the addition of 100 mg/l m-inositol, 30 g/l sucrose and 10 g/l agar-agar [11]. there was no addition of growth regulators (cytokinins or auxins) to this base medium. the added heavy metals were cdso4 and pbcl2. the growth medium variants made during the experiments and presented in the order in which they were performed as well as the concentration of the heavy metals added to the culture medium are shown in table 1. prior to the sterilisation of the growth medium it’s ph was adjusted to 5.5 with hydrochloric acid or sodium hydroxide depending on the basicity or acidity of the final medium [12]. 15 ml of medium were introduced in culture containers made of colorless and thermoresistant glass, 8 cm high and 4 cm in diameter. for autoclaving the containers used in all experiments, after portioning the growth medium, were coverd in aluminium foil. the sterilisation of the containers with the growth media was performed in an autoclave for 30 minutes at 121°c and 1 atm [15]. the plant material used to initiate the vitro cultures was represented by 2 cm long side shoots with 1-2 nodes plus and apical bud, taken from sedum telephium ssp. maximum l plantlets regenerated from the zygotic embryos of seeds germinated for 30 days on murashige skoog (1962) growth medium without growth regulators. the seeds from which the explants germinated were sterilised in a 0.1 % sodium hypochlorite solution diluted with sterile water (1:2) with 2 – 3 drops of tween 20 added to 150 ml disinfectant solution [15]. the containers with the inocula were transfered in the growth chamber, placed on shelves and exposed to a temperature between 230c ± 20c during the light period and 200c ± 20c during dark and a photoperiod of 16 h light/24 h. the light intensity was 1700 lux (osram white fluorescent tubes; wavelength 590nm; dimensions: lx590 mm øx26 mm) [15]. 2.2 statistical methods applied in the interpretation of the results the results were expressed in averages ± standard error. to evaluate the statistically significant differences between the treatments, the averages were compared by variance analysis (anova). the data was tested for the normality and homogeneity of variance using the levene test. when the results were statistically significant, a multiple comparison post-hoc tukey (p ≤0.05) test was used. the software used for statistical analysis was ibm spss v20. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 20 table 1. general scheme for the organisation of vitro culture experiments n r. cr t. experiment type experimental variants code compozițion of murashige – skoog (1962) growth media and the type of heavy metal heavy metal concentration vitro culture duration 1. germination of sedum telephium ssp. maximum l. seeds in aseptic conditions on murashige skoog (1962) culture medium without growth regulators. murashige – skoog (1962) base medium no growth regulators 30 days 2. initiation of sedum telephium ssp. maximum l. vitro cultures from apical cuttings from regenerated plantlets from zygotic embryos on the 30th day of vitro germination on murashige skoog (1962) culture medium. v0 (control) murashige – skoog (1962) base medium no heavy metals 30 days v1 murashige – skoog (1962) base medium with cdso4 50 ppm v2 murashige – skoog (1962) base medium with pbcl2 50 ppm v3 murashige – skoog (1962) base medium with cdso4 25 ppm v4 murashige – skoog (1962) base medium with pbcl2 25 ppm 3. results and discussion the cultivated vitro plantlets were monitored every 7 days for 4 weeks. the biometrization of the qualitative and quantitative characters of vitro plantlets from this study consisted in making the following observations: • measurements on the aerial system (vegetative) of plants made with a ruler (in cm) average stalklet length; number of branches at the base; average length of the branches; number of leaflets; average length of the leaflets; average width of the leaflets. • measurements on the root system of plants made with a ruler (in cm): number of rootlets; total length of the rootlets. the observations made during the 4 weeks of in vitro cultivation on the murashige skoog (1962) (ms) growth medium allowed us to assert that the development of sedum plantlets was positively influenced by the presence of the heavy metals pb (pbcl2) and cd (cdso4) in the growth medium. the average length of the stalklet at 28 days was significantly greater compared to the control (v0) especially for the plantlets grown on murashige skoog (1962) (ms) growth medium supplemented with pb, variant v4 (fig. 1). regarding plant growth through formation of lateral branches, the plants grown on v4 – pbcl2 variant, the concentration of 25 ppm has recorded higher values compared to the v3 – cdso4 variant of the same concentration with both variants having significantly smaller branches compared to the control. however, after 28 days the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 21 vitro plantlets grown on murashige skoog (1962) (ms) medium supplemented with 25 ppm cdso4 (v3) had a greater number of grown leaflets compared to the v4 – pbcl2 variant but significanlty smaller when compared to v0 (fig. 5). the average length of the leaflets for v4 variant has reached the highest value compared to the rest of the experimental variants followed by v2, being able to notice the fact that the addition of 25 ppm pbcl2 to the growth medium has stimulated the formation and development of the leaves. thus, it can be observed that the accumulation of metal stimulates the growth in length of the plantlets, the length and width of the leaves and especially the rhisogenesis (figs. 2, 3, 5 and 8). fig. 1. graphic representation of the average values corresponding to the average length of the stalklet of the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium with no added growth regulators (v0 – control) and of those grown on murashige – skoog (1962) base medium supplemented with cdso4 and pbcl2. note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0,05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 22 fig. 2. morphological aspects after 28 days of in vitro culture of plantlets grown on culture media without heavy metals (v0 – control) and of those grown on media with heavy metals (v1 – v4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 23 note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0.05). fig. 3. graphic representation of the average values corresponding to the number of branches for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0.05). fig. 4. graphic representation of the average values corresponding to the average length of branches for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 24 note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0.05). fig. 5. graphic representation of the average values corresponding to the newly formed leaflets for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0,05). fig. 6. graphic representation of the average values corresponding to the average length of the leaflet for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 25 note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0,05). fig.7. graphic representation of the average values corresponding to the average width of the leaflets for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. note: the values represent the average ± standard error (n = 10). the different letters represent significant differences (p<0,05). fig.8. graphic representation of the average values corresponding to the number of rootlets for the sedum telephium ssp. maximum l. vitro-cultures grown for 7 – 28 days on murashige – skoog (1962) base medium without growth regulators (v0 – control) and of those grown on murashige skoog (1962) (ms) base medium supplemented with cdso4 and pbcl2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 mirela ardelean, andrei lobiuc, marian burducea, ciprian mihali, daniela-teodora marți, study of heavy metals effects on in vitro cultures of sedum telephium ssp.maximum l, food and environment safety, volume xviii, issue 1 – 2019, pag. 18 – 26 26 4. conclusions we consider that our results are in agreement with the data presented in the literature stating that heavy metals can induce the reduction of certain morphological features in cultivated plants such as, in our case, the reduction of the number of leaflets and branches. however, the growth was not slowed down, but on the contrary, the growth medium supplemented with the higher concentration of pbcl2 (v4) has stimulated the plant biomass and the vitro plantlets have not been affected morphologically. this means that the plant has the capacity to tolerate the heavy metal concentrations added to the growth medium and also to accumulate them. 5. acknowledgements this work was supported by the grant “elimination of toxic compounds (pesticides, heavy metals, etc.) from soils through phytoremediation” co -financed by the academy of romanian scientists. 6. references [1]. yao z., li j., xie x., yu c., 2012, review on remediation technologies of soil contaminated by heavy metals, procedia environmental sciences 16 (2012) 722–729. camper nd, mcdonald sk. 1989. tissue and cell cultures as model systems in herbicide research. rev weed sci 4:169–190. [2]. cachiţă, c.d., ardelean, a., crăciun, c., turcuş, v., barbutudoran, l., 2008, the procaine hydrochlorate effect onto the corpuscular anthocyans from the vacuolar sap of different plant cells. in: 14th european microscopy congres, aachen, germany, p. 109 – 110. [3]. ardelean, m., cachiţă-cosma, d., aurel ardelean, tripon, s., 2015. particular changes produced by aphids in wild sedum telephium ssp. maximum l. plants:morphological and anatomical aspects. romanian biotechnological letters vol. 20, no. 3, 2015, pp.10461-10469. [4]. ştefan n., oprea a. 2007. botanică sistematică [systematic botany]. iaşi: publishing house of the univ. “alexandru ioan cuza”, 552 pp. [5]. metcalfe c.r. & chalk l. 1972. crassulaceae, 1: 578581 in anatomy of the dicotyledons. oxford: clarendon press. [6]. chung, k.-t., tit, y.w., cheng, i.w., yao-wen, h., yuan, l., 1998, tannins and human health: a review. critical reviews in food science and nutrition 38 (6), 421 – 464. [7]. mulinacci a.n., vincieri f.f., wagner r. 1993. antiinflammatory and immunologically active polysaccharides of sedum telephium. phytochemistry, 34:1357-1362. [8]. hahn, d.h., 1984. phenols of sorghum and maize: the effect ofgenotype and alkali processing. ph.d. dissertation. texas a&m university, college station, tx. [9]. cachiţă, c.d., crăciun, c., 2004, hiperhidria la vitroculturile de cormofite o boală fiziologică neoplazică. in: lucr. celui de alxii lea simp. naţ. de cult. de ţes. şi cl. vegetale, jibou 5 iunie 2003, intitulat: fitopatologia celulei vegetale în regim de vitrocultură, (ed. coord.) cachiţă, c.d., ardelean, a., fati, v. (edt.) daya satu mare, p. 30 – 42. [10]. murashige, t., skoog, f. 1962. a revised medium for rapid growth and bioassays with tabaco tissue culture. physiol. plant, 15: 473497. [11]. muneer, s., kim, e.j., jeong, s. p., lee.,j,h. 2014. photosynthetic activity under different light intensities in lettuce leaves (lactuca sativa l.) international journal of molecular sciences issn 1422-0067. [12]. manios, t., millner, p.a., stentiford, e.i., 2002. the effect of heavy metals on the total protein concentration of typha latifolia plants, growing in a substarte containing sewage sludge compost and watered with metaliferus wastewater. j. env. sci. health a37 (8), 925/936. [13]. gutt s., guttg., mariana mazareanu, 2010. study on the content of zearalenone from wheat and derivatives, food and environment safety, year ix, no1, 2010, p. 68-73, www.fia.usv.ro. [14]. s. amariei (gutt), e.sănduleac, s. ciornei (ștefăroi), 2013. comparative study of oxidative stability for different types of vegetable oils, food and environment safety, volume xii, issue 2 – 2013, pag. 56-160. [15]. m. ardelean, a. ardelean, i. don, a. lobiuc, m. burducea, 2018. effect of led lighting on growth and phenolic content on in vitro seedlings of ocimum basilicum l. cultivar ,,aromat de buzau”, food and environment safety, volume xvii, issue 1 – 2018, pag. 66 – 73. http://www.fia.usv.ro/ 98 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 22020, pag. 98 115 in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices sarah kehili 1 , mohamed nadjib boukhatem 2,3 *, hussein el-zaeddi 4 , dahbia kellou 5 , amina-bouchra benelmouffok 5 , mohamed amine ferhat 1 , angel a. carbonell-barrachina 4 , william n. setzer 6,7 1 laboratoire de recherche sur les produits bioactifs et valorisation de la biomasse, département de chimie, ecole normale supérieure, kouba, alger, algeria. 2 département de biologie et physiologie cellulaire, faculté des sciences de la nature et de la vie, université – saad dahlab – blida 1, blida, algeria. mail: mn.boukhatem@yahoo.fr 3 laboratoire ethnobotanique et substances naturelles, ecole normale superieure, kouba, alger, algeria. 4 research group « food quality and safety », department of agro-food technology, escuela politécnica superior de orihuela, unviersidad miguel hernández de elche, carretera de beniel, km 3.2, 03312-orihuela, alicante, spain. 5 laboratoire de mycologie, institut pasteur d’algérie, route du petit staouéli, dely-brahim, alger, algeria. 6 department of chemistry, university of alabama in huntsville, huntsville, al 35899, usa. 7 aromatic plant research center, 230 n 1200 e, suite 100, lehi, ut 84043, usa. *corresponding author received 26th march 2020, accepted 28th june 2020 abstract: in the present research, the antifungal and antioxidant activities of mentha piperita essential oil (mpeo) were investigated, and its potential as a natural food preservative in orangina juices was evaluated. the major component was menthol (54.47%). the percentage inhibitions of mpeo were dose dependent with ic50 values of 2.53±1.77 mg/ml in dpph test and 8.24±1.16 mg/ml in metal complexing ability. the microbial inhibition of mpeo was assessed against different food spoiling strains. the mpeo strongly inhibited the growth of rhodotorula sp. and saccharomyces cerevisiae with a diameter of the inhibitory zone (diz) ranging from 17–85 mm at the lower dose (20 µl), and from 35-85 mm at the higher quantity (60 µl). the minimum inhibitory concentration varied from 0.0078 to 0.5% (v/v) for yeasts. in addition, the anti-yeast effectiveness of mpeo alone and in association with moderate heat treatment was investigated in orangina juices. the juices treated with association of mpeo at different doses (1, 2 and 6 µl/ml) and medium heat treatment (80 °c for 2 min) improved the reduction of saccharomyces cerevisiae viability cells. present data confirmed the superior performance of integrated thermal treatment over individual use of peppermint oil for orangina juices preservation. keywords: mentha piperita essential oil, natural food preservative, antimicrobial activity, saccharomyces cerevisiae, menthol, antioxidant activity, orangina juices. 1. introduction abbreviation list bha = butylated hydroxyanisole bht = butylate hydroxytoluene cfu = colony-forming unit diz = diameter of inhibitory zone dpph = 1,1-diphenyl-2-picrylhydrazyl eos = essential oils mic = minimum inhibitory concentration mpeo = mentha piperita essential oil gc-ms = gas chromatography-mass spectrometry hex = hexamidine ic50 = median inhibitory concentration http://www.fia.usv.ro/fiajournal mailto:mn.boukhatem@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 99 nist = national institute of standards & technology rt = retention time ri = retention index sda = sabouraud-chloramphenicol dextrose agar orange juice is healthful and energizing because of its vitamin c (ascorbic acid) quantity, sweet, acidic taste, pleasing color, scented and nutritious. though, even after a few minutes of extraction, the juice starts decaying and its taste, flavor and color get off. this is due to substantial bacterial and fungal charging and enzymatic activity, which spoils the organoleptic and nutritive properties of juice, making it unhealthy for consumers. the main reasons of alteration must be related to the development and growth of pathogens (bacteria, yeast, and fungi), physical and chemical reactions, structural modifications and packing conditions [1,2]. consequently, some processing methods, quickly after the removal of fruit juices, are required to maintain the freshness of juices. for example, freezing, purification, sanitization, and adding of chemical additives are some examples of the current practices applied to accomplish microbiological and chemical stabilities and to control the safety and quality of food and fruit juices [3]. some alternative techniques such as modified co2 atmosphere packaging, ozone treatment, organic acids, irradiation, thermal processing, steam or hot water have been demonstrated to be active for shelf life prolongation in new or processed foods. currently, there is a growing tendency of consuming packed fruit juices as they can be drunk at desire and are simple to bring. nevertheless, fruit juices are processed before packing, and several synthetic and chemical additives are added in order to control their safety and quality. some synthetic preservatives generally uesed in juice are sorbic and benzoic acids and their derivatives, formic acid, formaldehyde, salicylic acid and so2 [4]. in addition, the disputation over the safety of some synthetic and chemical additives has encouraged and prompted the search for their natural alternative compounds. the growing claim for natural preservative molecules has linked in their extended utility. the numerous chemical disinfectants and preservatives are mostly not accepted by users because of their side effects and harmful as well. therefore, natural sanitizers such as vinegar, lemon juice, phytochemicals and essential oils (eos) extracted from medicinal herbs, spices and aromatic plants, not only give flavor and aroma to foods but they also have the benefit of being safe, healthy and natural preservatives [5,6]. natural food preservatives with high antibacterial, antifungal and antioxidant actions that extend the shelf life of juices are appreciated. most medicinal herbs and aromatic plants synthesize and produce antimicrobial biomolecules. these plantbased antimicrobials can be suggested and used as natural food preservatives in fruit juices [7,8]. peppermint (mentha piperita) is a member of lamiaceae family that spreads mostly in the temperate and mediterranean areas of the globe such as algeria. it is considered a rich source of eos, which is commonly used in food production, cosmetic, pharmaceutical and nutraceutical industries. the famous and usually used peppermint is a cultivated natural hybrid of mentha spicata (spearmint) and mentha aquatica (water mint) [9,10]. besides it is used in cosmetics, herbal tea preparations, food industry, and sweets. peppermint was reported as a medicinal and aromatic herb, with an eo having several pharmaceuticals and food uses. the therapeutic and medical food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 100 uses of mentha piperita essential oil (mpeo) comprise anti-inflammatory, antispasmodic, wound-healing, antidiabetic, analgesic and antiemetic applications. in some provinces of the north africa countries, mpeo is used for the treatment of infections, fever, vomiting, nausea, common cold, bronchitis and stimulation of appetite. most recently, mpeo has gained enlarged scientific importance, principally as an antioxidant, analgesic and antibacterial bioactive natural agent [11,12]. however, eos present considerable activity when used in fruit juice matrix, but quantities necessary (33–100 times of in vitro concentrations) are very great [13], and such doses generally exceed the sensory and organoleptic satisfactory ranks. the association of a minor heat processing with different doses of volatile oils can be considered as an important approach to decrease or inhibit bacterial and fungal growth in various food products, eliminating the issues of sensorial impact on juices. consequently, the association of eos with medium heat treatment can be investigated for discovering new active food preservation practices. as per our knowledge, there is no report on the preservation of orangina fruit juices by mpeo which has remarkable antiinflammatory and analgesic properties [11]. therefore, an effort has been made to improve the shelf life of orangina fruit juices with natural phytochemical food additives extracted from peppermint plants. in the current investigation, the effect of mpeo against different food spoiling fungal and yeast species was studied using different in vitro assays (disc diffusion and disc volatilization methods, agar dilution test) as well in orangina fruit juices. also, to decrease the dose of mpeo in the orangina juices, the integrated influence of the mpeo with medium thermal treatment (80 °c for 2 min) was also investigated. the chemical composition profile of mpeo was done by gas chromatography. 2. material and methods 2.1. material 2.1.1. extraction of mentha piperita volatile oil the mpeo used in our research was a commercial sample produced by steam distillation from the aerial part of the plant in industrial conditions (a stainless steel alembic). the mpeo was obtained from extral-bio company (blida, algeria). 2.1.2. food-spoilage microorganisms the in vitro microbial inhibitory action of mpeo was assessed against several mycelial fungi and yeast strains: six candida strains comprising candida glabrata, c. albicans and c. tropicalis; two saccharomyces cerevisiae; two food spoiling aspergillus strains including a. flavus and a. niger and one fusarium sp. strain. such isolates were obtained from food matrix in the laboratory of food quality and microbiology (laboratoire d’hygiène, blida, algeria) and from the mycology laboratory (institute pasteur of algeria, algiers, algeria). these microbial species were identified by standard microbiology assays and stored in sabouraud-chloramphenicol dextrose agar (sda) for yeast and fungi. 2.1.3. chemicals and reagents the following drugs and chemicals were used: dimethyl sulfoxide (dmso), tween 80, gallic acid, butylated hydroxyanisole (bha), l-ascorbic acid (vitamin c), ferrozine™ iron reagent, 1,1-diphenyl-2food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 101 picrylhydrazyl (dpph), iron (ii) chloride (fecl2) were obtained from sigma aldrich (st. louis, mo, usa). the antiseptic solution of isomedine® 0.1% (hexamidine dermal solution, isopharma, algiers, algeria) was used in order to control the sensitivity of tested isolated microorganism strains. 2.2. methods 2.2.1. determination of chemical composition of peppermint volatile oil analysis and identification of the volatile compounds of mentha piperita eo were done using a shimadzu gc-17a gas chromatograph apparatus combined with a shimadzu qp-5050a mass spectrometer detector (shimadzu corporation, kyoto, japan). the gc-ms system has a tracsil meta.x5 (95% dimethylpolysiloxane, and 5% diphenylpolysiloxane) column (60 m×0.25 mm, 0.25 μm film thickness; teknokroma, barcelona, spain). the chromatography analyses were done using helium as the carrier gas at a column flow rate of 0.3 ml/min and a total flow of 3.9 ml/min in a split ratio of 1:200 and the following temperature program: (a) 45 °c for 6 min; (b) increase of 3 °c/min from 45 °c to 210 °c and hold for 4 min; (c) increase of 25 °c/min from 210 °c to 290 °c and hold for 4 min. the temperatures of the detector and injector were 290 °c and 300 °c, respectively. all chemical compounds of mpeo were detected and identified using two different analytical techniques: (1) comparison of experimental retention indexes (ri) with those of the literature and standards; and, (2) mass spectra (nist05 spectral library collection available). only fully identified chemical compounds detected in mpeo are reported in the current research. 2.2.2. determination of in vitro antioxidant activity 2.2.2.1. evaluation of dpph radical scavenging technique dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was evaluated and determined according to wang et al. [14] with some slight modifications, based on the capability of mpeo chemical compounds to neutralize dpph radical. the conversion of the dpph radical into a yellow color reduced form (dpph/h+) is detected immediately. briefly, a series of dilutions of mpeo were done in a pure ethanol solvent. in parallel, the negative control was prepared comprising all reagents except the mpeo. after 25 min incubation period at room temperature in a dark cupboard, the absorbance at 520 nm (maximum absorbance of dpph) was measured and recorded using a spectrophotometer. a blank reading was taken using a covert containing solution without the mpeo, and the absorbance was measured. the dpph free radical scavenging action of selected mpeo concentration was then calculated as percentage inhibition in accordance to the below formula: dpph radical scavenging inhibition % = ( abs blank − abs sample abs blank ) ∗ 100 where abs sample is the optical density or absorbance of dpph free radical with the tested sample and abs blank is the optical density of dpph free radical without mpeo. the in vitro antioxidant property of the mpeo was calculated and expressed as ic50 (median inhibitory concentration), defined as the dose of the mpeo necessary to make a 50% reduction or inhibition in initial dpph solution. ascorbic acid (vitamin c) and bha were used as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 102 positive standards. all these analyses were carried out in triplicate. 2.2.2.2. evaluation of metal complexing activity the complexation of ferrous ions by the mpeo and standards was evaluated and estimated following to the method of ye et al. [15] with slight modifications. mpeo samples were added and mixed to a solution of 2 mm fecl2 (0.05 ml). the reaction was started by adding a quantity of 5 mm ferrozine (0.2 ml) and the combination was shaken strongly and left standing at laboratory temperature for 12 min in a dark cupboard. after the solution mixture had reached equilibrium, the optic density was then measured and recorded by an apparatus of spectrophotometer at 562 nm. all tests and assays were done in triplicate. the percentage inhibition of ferrozine–fe2+ complex creation was calculated following this equation: % inhibition = ( a0 − a1 a0 ) ∗ 100 where a0 was the optical density of the negative control and a1 was the optical density in the presence of the mpeo or standards. the control contains only fecl2 and ferrozine complex formation molecules. gallic acid, bha and vitamin c (ascorbic acid) were used as positive standards. the dose of inhibition of the tested samples was expressed and reported as the percentage of concentration required to do 50% inhibition (ic50). 2.2.3. in vitro antifungal activity of peppermint volatile oil the in vitro fungal inhibitory action of mpeo against several filamentous fungi and yeast strains was done using different methods: disc diffusion, disc volatilization and agar macrodilution assays. 2.2.3.1. agar disc diffusion test agar disc diffusion test was employed for the evaluation and determination of the antifungal property of mpeo [12]. the fungal inoculum of each strain was prepared with fresh cultures by suspending the microorganisms in sterile saline (0.9% nacl). filter paper discs (diameter of 9 mm, schleicher and schull, dassel, germany) were saturated with 3 different quantities (20, 40, and 60 µl) of peppermint eo per disc and positioned on the inoculated plates (sda for fungi and yeast). after maintaining at laboratory temperature for 40 min, the plates were incubated under aerobic conditions for 72 h (yeast) and 5 days (fungi). the fungal inhibitory potential was estimated by calculating the diameter of the inhibitory zone (diz) in millimeters (including disc diameter of 9 mm). antiseptic solution of hexamidine was used as a positive control in order to control the sensitivity of tested isolated microorganisms. 2.2.3.2. vapor diffusion test because eos extracted from aromatic plants are volatile, methods and techniques that test the fungal inhibitory effect of such agents in their vapor phase have been done in this research. a standard experimental setup as published by tyagi et al. [12] was followed with some modifications. the fungal inhibitory potential of mpeo in vapor phase was assessed using the disc volatilization assay at three different amounts (20, 40, and 60 µl per disc). in brief, sda was inoculated over the solidified medium surface with 100 µl of suspension of the mycelial or yeast strains under study. a paper disc was placed on the inside surface of the upper lid and a suitable volume of mpeo was placed on selected paper disc. then, the plate was immediately inverted on top of the lid and closed with parafilm to avoid the runoff of mpeo vapor. plates were incubated under food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 103 aerobic conditions for 72h (yeast) and for 5 days (fungi). the effectiveness of the in vitro fungal inhibitory action of mpeo was calculated and reported by measuring the diz (in millimeters) above the disc. 2.2.3.3. determination of the fungal minimum inhibitory concentration (mic) the agar macro dilution assay was performed as recommended by tyagi et al. [12] with some modifications. all experiments were made in sda medium added with tween 80 (final dose of 0.5% v/v). filamentous fungi and yeast strains were cultured for 24h and geometric dilutions ranging from 2% to 0.007% (v/v) of mpeo were prepared in a culture medium plate, including one growth control (sda + tween 80). petri dishes were incubated under aerobic conditions for 48-72h. the inhibitory effect of fungal growth was detected by the absence of the colonies on the sda medium. the mic values were estimated and expressed as the lowest dose of peppermint eo stopping visible growth of fungal species. 2.2.4. anti-yeast effect of mpeo in orangina juices 2.2.4.1. inoculation of orangina juices with saccharomyces cerevisiae orangina juices were purchased from a local company (djgaguen, blida, algeria). orangina is a lightly carbonated beverage made from carbonated water, orange juice and other citrus juice from concentrate 12%. orangina is sweetened with sugar and natural flavors are added. this beverage contains also synthetic additives such as citric acid (sin330), benzoate sodium (sin211) and potassium sorbate (sin202) as preservatives, and ascorbic acid (sin300) as an antioxidant ingredient. the yeast suspension of saccharomyces cerevisiae was added and then mixed with orangina juices and the inoculated juices were transferred in 100 ml sterilized glass vials. 2.2.4.2. anti-yeast effect of peppermint eo alone tween 80 solution (0.5%) of mpeo was added and then mixed in the inoculated orangina juices at several concentrations (1, 2 and 6 µl/ml). orangina juices sample inoculated with saccharomyces cerevisiae alone was considered as a control group. then, the treated vials were stored at laboratory temperature up to 9 days and juice samples were drawn on 0, 2nd, 3rd, 6th, and 8th day. all treated juice samples were successively diluted in isosaline solution (0.9% nacl) and plated on sda medium. all petri dishes were incubated for 72 h at 25 °c to observe and count the growth and number of yeast colonies that appeared in all plates. the observations were recorded as the number of colonies present in 10 ml of orangina juice samples (cfu/ml). 2.2.4.3. anti-yeast effect of peppermint eo in association with medium thermal treatment a set of inoculated orangina juices vials added with three different concentrations of peppermint eo was exposed to a moderate heat treatment (80 °c) for a short time (2 min). each juice was treated in triplicate. then, the treated vials were deposited at laboratory temperature up to 9 days and orangina juice samples were drawn on 0, 2nd, 3rd, 6th, and 8th day. all treated orangina juices were consecutively diluted in isosaline solution and plated on sda medium. all plates were incubated for 72 h at 25 °c to detect and count the number of yeast colonies that appeared in all plates. the results were calculated and recorded as the number of colonies present in 10 ml of juice sample (cfu/ml). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 104 the effectiveness of the medium thermal treatment (80 °c for 2 min) in association with different doses of peppermint eo was measured and expressed by the variation in log cfu of the inoculated yeast strains with time (up to 8 days). 2.2.5. statistical analyses all the analyses were performed in triplicates to report the results as mean with standard deviation and subjected to one-way analysis of variance (anova) followed by hsd tukey’s post hoc multiple comparison tests to establish whether the differences in experimental results for different samples were significant (p<0.05) or not (p>0.05). the statistical analysis was done using xlstat 2014 software (addinsoft, paris, france). 3. results and discussion 3.1. chemical composition of peppermint volatile oil in the current investigation, the eo from the aerial parts of peppermint (mentha piperita l.) a medicinal and aromatic herb grown in algeria and commonly used in phytomedicine, was extracted using steam distillation method. the determination of the chemical composition profile of mpeo was made by gc-ms, and quantitative and qualitative compositions are reported in table 1 and figure 1. table 1. chemical composition of the essential oil obtained from peppermint (mentha piperita l.) using a steam distillation method. peak no. rt† (min) compound ri, exp. ri, lit. difference area (%) 1 9.017 cis-3-hexen-1-ol 845 849 4 0.01 2 9.716 1-hexanol 860 858 -2 0.01 3 12.624 thujene 918 923 5 0.04 4 13.051 α-pinene 925 921 -4 0.61 5 15.526 sabinene 964 963 -1 0.4 6 15.815 β-pinene 969 964 -5 0.79 7 16.133 1-octen-3-ol 974 978 4 0.1 8 16.674 ß-myrcene 982 983 1 0.07 9 17.28 3-octanol 992 992 0 0.22 10 18.491 α-terpinene 1010 1018 8 0.15 11 19.034 p-cymene 1017 1023 6 0.28 12 19.413 limonene 1023 1027 4 1.44 13 19.637 eucalyptol 1026 1030 4 4.9 14 20.65 trans-ß-ocimene 1040 1043 3 0.05 15 21.455 γ-terpinene 1051 1053 3 0.27 16 22.352 cis-sabinenehydrate 1063 1068 5 0.87 17 24.613 linalool 1095 1098 3 0.29 18 25.171 amyl isovalerate 1102 1108 6 0.09 19 27.617 sabinol 1135 1142 7 0.02 20 28.155 isopulegol 1142 1146 4 0.09 21 28.877 menthone 1152 1155 3 16.75 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 105 22 29.275 menthofuran 1157 1164 7 3.91 23 29.399 isomenthone 1159 1164 5 2.53 24 30.974 menthol 1180 1185 5 54.47 25 34.664 pulegone 1231 1237 6 1.2 26 35.773 piperitone 1246 1250 4 0.46 27 38.472 menthyl acetate 1283 1287 4 6.93 28 47.139 trans-caryophyllene 1408 1411 3 1.75 29 51.138 germacrene d 1469 1467 -2 1.02 30 52.078 ß-elemene 1483 1484 1 0.28 oxygenated monoterpenes 91.64 monoterpene hydrocarbons 5.31 sesquiterpene hydrocarbons 3.05 † rt = retention time; exp. = experimental; lit. = literature; ‡ all compounds were identified using retention indexes (ri) and mass spectra. the main compound identified was menthol (54.47), followed by menthone (16.75%), menthyl acetate (6.93%) and 1.8-cineole (eucalyptol) (4.9%). other chemical compounds were detected but less than 4%. also, mpeo showed a high content of oxygenated monoterpene (93.26%) and low amounts of monoterpene hydrocarbons (2.69%) and sesquiterpene hydrocarbons (3.69%). thus, algerian mpeo extracted by steam distillation may be categorized as a “menthol/menthone chemotype”. fig. 1. the chemical profile of peppermint (mentha piperita l.) essential oil determined by gc-ms. several research groups have analyzed aroma profiles for the various peppermint, which can vary and fluctuate significantly depending on several factors [9,10,12]. experimental findings are in agreement with other published papers, wherein menthone and menthol were the main abundant compounds in eo extracted from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 106 peppermint [16,17]. according to derwich et al. [18], menthone (29%), menthol (5.58%) and menthyl acetate (3.3%) were the major components in peppermint eo from morocco. in turkey, the study of kizil et al. [19] found that (+)-menthol (38%), (-)-menthol (35.6%) and neomenthol (6.7%) were the major compounds of mpeo. remarkably, the mpeo under examination of this research seems to be richer in some important constituents such as menthone, menthol, eucalyptol and oxygenated monoterpenes. a review of the literature accessible on this area shows that several papers have previously been published on mpeo chemical characterization [20]; however, there are no research articles on the chemical profile of mpeo from algerian mitidja area. it has been revealed that the eo distillated from the peppermint leaves grown in unicamp (brazil) is characterized by the dominance of a monoterpenic alcohol (linalool) with a rate of 51%, followed by carvone (23.42%) [21]. in another investigation, the dominant compounds of mpeo from iran were α-terpinene (19.7%) and pipertitinone oxide (19.3%) [22]. several papers have reported that the chemical composition of these mpeos differs in accordance to the countries, or the regions in the same state. these variations seem to depend on several reasons such as climate changes, external environment and other factors such as the method and the period of extraction, collected parts of the plant, irrigation techniques, and phenological transformations [23,24]. 3.2. in vitro antioxidant activity of peppermint eo the in vitro antioxidant effect of mpeo harvested from algeria was investigated using dpph (2,2-diphenyl-1picrylhydrazyl) radical scavenging and ferrous ion complexation assays. the median inhibitory concentrations (ic50) were calculated and values are presented in table 2. table 2. in vitro antioxidant activity of peppermint (mentha piperita l.) essential oil. sample dpph radical scavenging ic50 (mg/ml) complexing power ic50 (mg/ml) mentha piperita essential oil 2.53±1.77 8.24±1.16 positive control (bha) 0.32 ± 0.28 31.22 ± 15.87 positive control (vitamin c) 0.01 ± 0.01 14.19 ± 6.27 positive control (gallic acid) — 31.92 ± 24.18 bha: butylhydroxyanisol; ic50 = median inhibitory concentration 50%; values are given as mean ± sd (n = 3). the decrease ability of dpph free radical was assessed by the reduction in its optical density at 520 nm induced by antioxidant compounds. in the dpph assay, the ic50 value for the mpeo was 2.53±1.77 mg/ml (table 2), indicating a moderate electron transfer capacity for the eo when compared to the standards of bha and ascorbic acid, that presented ic50 values of 0.32±0.28 and 0.01±0.01 mg/ml, respectively. scavenging activity of vitamin c and bha, recognized as powerful antioxidant standards, were comparatively more active than that of peppermint eo. the ferrous complexing capacity test was used to evaluate and confirm the capacity of antioxidant molecules to disrupt the formation of the complexes or to prevent interaction between metal and lipids. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 107 because of the importance of metal complexation as one of the antioxidant mechanisms, the aptitude of the mpeo to compete with ferrozine for iron ions in free solution was evaluated, and the corresponding ic50 values are presented in table 2. unlike the dpph assay, the iron complexing ability of mpeo is more pronounced with an ic50 value of 8.24±1.16 mg/ml, followed by ascorbic acid (14.19±6.27 mg/ml). current findings showed that mpeo has an excellent ferrous ion complexation action. results from current study agreed with those of previous reports in which the in vitro antioxidant power of the mpeo was assessed and linked to the major oxygenated monoterpenes including menthol, menthone, menthyl acetate and 1,8-cineole [25,26]. other mpeo minor chemical compounds that contain molecules in the active methylene group, such as terpinolene, αand γ-terpinene, were also listed and recognized for their powerful antioxidant action, which is equivalent to the positive standard (vitamin e or α-tocopherol) [27]. because the in vitro antioxidant ability of the whole aroma is the consequence of the interaction of all minor and major compounds, it is difficult to attribute the eos antioxidant power to a single molecule, as other mpeo components can contribute showing synergistic, additive or antagonistic effects [28]. 3.3. in vitro anti-yeast and antifungal activity of peppermint volatile oil 3.3.1. agar disc diffusion test the in vitro antifungal effect of peppermint eo was assessed using three different quantities. the resultant diameters of inhibition zones (diz) are presented in table 3. in the current investigation, the inhibitory action of peppermint eo was done against 13 isolates of filamentous fungi and yeast species using the agar diffusion method. as can be seen in table 3, mpeo showed various degrees of in vitro anti-yeast antifungal actions depending on the microorganism strains tested. it is essential to state that in comparison to the positive standard (antiseptic solution of hexamidine), mpeo showed a potent fungal inhibitory action against rhodotorula sp. with a total inhibition zone. the mpeo strongly inhibited the growth of saccharomyces cerevisiae, candida tropicalis (ct1) and c. albicans (ca2) with diz ranging from 13-25 mm at the lower volume of mpeo (20 µl/ml), and from 35-81 mm at the higher amount (60 µl/ml). among the filamentous fungi, aspergillus flavus (af2) was the most susceptible strains; the application of 60 µl of mpeo resulted in a diz of 36 mm. the diz extended with increasing mpeo volume. using 60 µl of mpeo, highest diz was shown by rhodotorula sp. (85 mm), candida albicans (ca2) (81 mm) and c. tropicalis (ct2) (42 mm), in comparison to the positive standard (antiseptic solution of hexamidine). agreeing to the literature, several authors and scientists [29,30] state that the percentage of chemical constituents and the dominant compounds detected in different eos determined the bacterial and fungal inhibitory effect in vitro. the researchers go on to explain the maximum antifungal property [29,31] is caused by chemical elements containing hetero atoms such as oxygen. interestingly, oxygenated monoterpenes and sesquiterpenes were clearly reported as powerful antifungal agents in the chemical composition of different volatile oils, comprising mpeo [32]. furthermore, menthone, menthol and menthyl acetate were shown to display significant fungal and bacterial inhibitory effect against a wide range of pathogens food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 108 and food spoiling microorganisms [33]. nevertheless, as eos have multiple chemical constituents their in vitro fungal inhibitory effect is rather due to synergistic, additive or antagonistic actions of the pure compounds. current research has shown that the volatile oil obtained from the fresh aerial parts of peppermint plant has the potential to be an antifungal agent with a superior activity against a wide variety of food spoilage yeast when compared to synthetic drugs. table 3. in vitro susceptibility of fungal strains to mpeo through liquid and vapor phases in comparison with an antiseptic solution diameter of inhibition zone (diz, mm) † disc diffusion test vapor diffusion test positive control ‡ quantity of mpeo (µl/disc) yeast strains 20 40 60 20 40 60 20 40 60 candida albicans (ca1) 15.6 23.3 28.3 10.3 27.6 35.0 candida albicans (ca2) 13.0 19.5 81.0 16.0 31.0 11.0 15.0 15.0 18.0 candida glabrata (cg1) 12.6 14.6 18.0 20.3 25.0 31.3 22.0 candida glabrata (cg2) 13.5 19.0 21.5 17.0 63.0 candida tropicalis (ct1) 23.6 31.3 35.0 22.3 24.0 30.3 candida tropicalis (ct2) 17.0 32.0 42.0 3.5 13.0 71.0 saccharomyces cerevisiae 25.0 30.0 40.0 11.0 19.0 39.0 rhodotorula sp. 85.0 85.0 85.0 85.0 85.0 85.0 38.0 38.0 40.0 filamentous fungi aspergillus niger (an1) 12.6 15.0 18.6 21.6 28.3 31.3 aspergillus niger (an2) 12.5 16.0 13.0 17.0 21.0 aspergillus flavus (af1) 10.6 18.0 36.0 aspergillus flavus (af2) 14.5 fusarium sp. 10.0 11.6 12.3 19.3 20.3 25.6 † diameter of inhibition zone includes the disc diameter of 9 mm. ‡ antiseptic solution (hexamidine 0.1%) used as a positive control for fungal strains. mpeo: mentha piperita essential oil; (-) no inhibitory activity. 3.3.2. disc volatilization technique the antifungal inhibitory effect of peppermint eo was also evaluated in the vapor phase using the disc volatilization technique (table 3). as observed in the agar disc diffusion method, the diz due to the vapors increased with increasing volumes of the peppermint eo. current data revealed that for the tested fungal strains (candida albicans (ca1), c. glabrata (cg1 and cg2) and c. tropicalis (ct2), the diz resulting from exposure to peppermint eo vapors was higher than that resulting from a similar volume of mpeo in the liquid phase with 60 µl of mpeo per disc. this may be correlated with the difference in the chemical composition of the two phases (liquid and vapor), with the vapor phase being richer in the more volatile chemical elements [12,34]. for example, rhodotorula sp. and candida tropicalis were the most susceptible yeast species to mpeo vapors since a total inhibition zones were generated using 60 μl. nevertheless, no fungal inhibitory activity was noticed in the case of filamentous fungi such as aspergillus niger, a. flavus and fusarium sp. in view of these data, it is surprising that regardless of the publication of several food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 109 original research articles on the antibacterial and antifungal effects of peppermint eo using the agar disc diffusion assay, the in vitro antimicrobial property of the vapor phase of mpeo has been largely ignored. both the liquid oil and oil vapor have been established to have some antifungal activity. the efficacy of mpeo as an antifungal agent against 17 fungal and micromycetal food poisoning, animal, plant, and human pathogens was reported and published by sokovic et al. [35]. peppermint eo presented strong in vitro antibacterial and antifungal activities, higher than bifonazole (commercial fungicide used as a positive control) but lower than that of pure major chemical compound (menthol). in addition, the application of the vapor phase has the supplementary advantages of simplicity of use and avoiding the need for direct interaction with the mpeo. a minor dose of peppermint eo is essential to accomplish the same level of fungal inhibition. the current findings are in conformity with our previous publications on this point [36,37]. 3.3.3. minimum inhibitory concentrations of peppermint eo the mic of mpeo was determined against different food spoiling fungi and yeast species using the agar macrodilution method. the mic values are shown in table 4. table 4. determination of minimum inhibitory concentrations (mic) using the agar dilution method fungal strains mic (% v/v) candida albicans (ca1) 0.125 candida albicans (ca2) 0.25 candida glabrata (cg1) 0.25 candida glabrata (cg2) 0.5 candida tropicalis (ct1) 0.125 candida tropicalis (ct2) 0.5 saccharomyces cerevisiae 0.5 rhodotorula sp. 0.007 aspergillus niger (an1) 1 aspergillus flavus (af2) 0.5 fusarium sp. 0.5 the mpeo exhibited dose-dependent inhibition of the yeast and fungal growth, and the mic varied from 0.125% to 0.5% for candida spp., and from 0.5% to 1% for filamentous fungi. the lowest mic (0.007%) was shown by rhodotorula sp., followed by c. albicans and c. tropicalis (0.125%). the difference in the in vitro fungal inhibitory effect could be linked to different microbial species and also to the chemical profile of the mpeo used. the quantity of major antimicrobial compounds (oxygenated monoterpenes such as menthol and menthone) in mpeo used is higher (61%). the oxygenated monoterpenes can raise the permeability and penetrability of the fungal cell membrane, leading to leakage of the cell substances [9,20]. fungal cell membrane alteration, loss of cytoplasmic constituents and inhibition of respiratory activity due to some oxygenated terpenes (menthol, menthone and menthyl acetate) have been previously reported and published [38]. the existence of oxygenated monoterpenes as major chemical elements could be the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 110 cause for greater in vitro anti-yeast effect of mpeo. furthermore, it has been reported that menthol was the principal inhibitory compound of peppermint volatile oil against the fungus trametes versicolor [39]. this result is consistent with the fact that eo extracted from peppermint grown in brazil, containing high quantities of oxygenated monoterpenes such as linalool (51%), but no menthol, had only a medium fungal inhibitory activity with mic value of 0.6 mg/ml against the strain of candida albicans [40]. the different doses at which mpeo exerted significant in vitro anti-yeast action indicate that there may be possibilities and opportunities for its use as safe and natural food preservative, where a reduction in food spoilage fungi and yeast is necessary. therefore, additional experiments and assays were done to confirm and validate the efficacy of peppermint eo in association with other food preservation technique in orangina fruit juices. 3.4. orangina juices’ preservation using peppermint eo alone or in association with moderate thermal treatment 3.4.1. anti-yeast effect of peppermint eo at different concentrations as peppermint oil was able to inhibit in vitro the growth of several food spoilage yeasts and fungi, it’s potential as a safe food additive in orangina juices was also studied and reported (figure 2). the decrease in yeast viability of the yeast saccharomyces cerevisiae cells due to peppermint eo application in timedependent ways (i.e., 0, 2, 3, 6 and 8 days) and dose-dependent (1, 2 and 6 µl/ml) manner was described and demonstrated. positive control or juice with synthetic antimicrobial additives (sodium benzoate and potassium sorbate)); mpeo: mentha piperita essential oil; cfu: colony-forming unit) fig. 2. variation in viability of yeast strain (saccharomyces cerevisiae) in orangina juices during storage after peppermint eo treatment at various doses (1, 2 and 6 µl/ml). as illustrated in figure 2, a complete growth inhibition of the yeast specie (saccharomyces cerevisiae) was observed and recorded in orangina juices at only higher concentration (6 µl/ml) and only on the 8th day. the viable count of yeast cells in orangina juices increased with the decreasing of peppermint eo dose used. however, the quantity of 1 µl/ml of mpeo did not show a significant reduction 1,54 2,82 0 0.5 1 1.5 2 2.5 3 0 2 4 6 8 lo g c f u /m l days positive control negative control mpeo 6 µl/ml mpeo 2 µl/ml mpeo 1 µl/ml food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 111 in the final number of yeast cells (1.54 log cfu/ml) at the beginning of the experiment as compared to the end of the assay (2.82 log cfu/ml). due to the increasing data about the injurious and dangerous effects of synthetic or chemical food additives, there is an incessant and nonstop pressure from consumers and scientific societies to avoid or diminish the quantity of theses chemical preservatives and ingredients [41] and also to deliver minimally processed food products, without compromising food quality, safety and organoleptic properties. consequently, substitute sources of acceptable and effective natural food preservatives extracted from natural and safe products need to be discovered and investigated, such as eos. for example, rosemary eo has been applied not only as a condiment, but also for its effective antifungal and antioxidant properties. indeed, carnosic acid, one of its major components, is not only an antiseptic and antiviral compound but it also has got a higher antioxidant potential than the powerful food additive antioxidants such as vitamin c, butylate hydroxytoluene (bht) and bha [42]. current data suggested the efficacy of peppermint eo to decrease the yeast load of s. cerevisiae in orangina fruit juices but only at a high concentration. however, this aspect can intensely disturb the physical and chemical properties and sensorial or organoleptic characteristics of the orangina juices. to overcome these concerns, numerous approaches have been studied and investigated for the improvement of antifungal and antibacterial effects of volatile oils in food matrix or systems. to further diminish the necessary mpeo dose for controlling saccharomyces cerevisiae load in orangina fruit juices, the association between peppermint eo and medium thermal treatment was also studied. 3.4.2. anti-yeast effect of peppermint eo in association with medium thermal treatment the cell viable counts of food-spoiling yeast strain (saccharomyces cerevisiae) after exposure to the integrated effect of peppermint eo at three different doses (1, 2 and 6 µl/ml) together with medium thermal treatment (80 °c/2 min) in orangina juices was investigated and noted at different days (i.e. after 0, 2, 3, 6 and 8 days) (figure 3). it is surprising to reveal that in the orangina juices exposed to moderate thermal treatment and mpeo at the doses of 1, 2 and 6 µl/ml, total growth inhibition of the yeast s. cerevisiae was detected after 6 days. additionally, no fungal growth was noticed up to 8 days of storage. hence, the association of medium thermal treatment with mpeo decreased the oil dose requirement to exactly 1/5 of the mic level. the association of moderate thermal treatment with mpeo can offer improved orangina juices preservative. in the current research, the mpeo dose required was decreased by the association with an additional food preservation technique (moderate thermal treatment). this association can be considered as a better method to preserve and control orangina fruit juices from yeast-spoiling contamination without an important influence on the sensory or organoleptic characteristics of the drink. the integration of moderate thermal treatment with mpeo suggests a very valuable synergy, whereby an increase in temperatures during juice storage could improve the vapor phase concentration of chemical volatiles, thereby improving the fungal inhibitory actions for better and superior food preservation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 112 positive control or juice with synthetic antimicrobial additives (sodium benzoate and potassium sorbate)); laeo: mentha piperita essential oil; cfu: colony-forming unit) fig. 3. anti-yeast effect of peppermint eo in association with medium thermal treatment. the synergistic influence of medicinal herbs, aromatic spices and eos on other food preservation matrix, such as mild heat processing, has been also evaluated and reported in the past. essia ngang et al. [43] investigated how to decrease the thermal influence during fruit juice extraction. they showed and proved that pasteurizing pineapple fruit juice at the temperature of 60 °c in presence of coriander eo, dropped the time needed for a 97% reduction of gram-positive bacteria such as listeria monocytogenes compared to juice samples without volatile oils. another study demonstrated that mint, eucalyptus and lemongrass eos functioned in a synergy manner with mild heat treatment to decrease and inhibit totally the yeast growth of s. cerevisiae in mixed juices prepared using different fruits [12]. the combination of peppermint eo with a moderate thermal treatment has never been previously investigated or published for controlling orangina juice yeast spoilage. the current study could be considered as the first report on the potential application or use of peppermint eo as a natural orangina juice preservative in an association with a moderate thermal treatment. 4. conclusion in the current investigation, the significant fungal inhibitory effect of the peppermint volatile oil against several food spoiling fungi and yeast has been assessed using different techniques, in vitro and in a real orangina juice matrix in association with a moderate thermal treatment. the fungal inhibitory action of peppermint oil in orangina juice supports its use in food preservation, because this eo is reported as safe. these data could be considered as a significant platform for the innovation and improvement of active natural food preservatives. 5. acknowledgments the authors would like to thank the laboratory of water microbiology (algérienne des eaux, chiffa, blida, algeria) and the laboratory of food microbiology (laboratoire d’hygiène de blida, blida, algeria)”, especially mr. djamel teffahi and mr. abdenacer -0.5 0 0.5 1 1.5 2 2.5 3 0 1 2 3 4 5 6 7 8 lo g c f u /m l days positive control negative control mpeo 6 µl/ml mpeo 2 µl/ml mpeo 1 µl/ml food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 113 hmida for their help and making the facilities available for carrying out this research. 6. conflict of interest statement the authors have declared that there is no conflict of interest. 7. sources of funding this study did not receive any specific grant from funding agencies in the commercial, public, or not for-profit sectors. 8. references [1]. tournas vh., heeres j., burgess l., moulds and yeasts in fruit salads and fruit juices, food microbiology, 23: 684-688, (2016). https://doi.org/10.1016/j.fm.2006.01.003 [2]. ahmed t., das kk., uddin ma., the microbiological quality of commercial fruit juices-current perspectives, bangladesh journal of microbiology, 35: 128-133, (2018). https://doi.org/10.3329/bjm.v35i2.42643 [3]. rupasinghe hv., yu lj., emerging preservation methods for fruit juices and beverages, food additive, 65-82, (2012). [4]. oladipo ic., adeleke dt., adebiyi ao., the effect of ph and chemical preservatives on the growth of bacterial isolates from some nigerian packaged fruit juices, pakistan journal of biological sciences, 13: 16, (2010). https://doi.org/10.3923/pjbs.2010.16.21 [5]. kapoor ips., singh b., singh s., singh g., essential oil and oleoresins of black pepper as natural food preservatives for orange juice, journal of food processing and preservation, 38: 146-152, (2014). https://doi.org/10.1111/j.1745-4549.2012.00756.x [6]. ekanem jo., ekanem oo., the effect of natural and artificial preservatives and storage temperature on the ph and microbial load of freshly produced apple (malus domestica) juice, agroscience, 18: 16-21, (2019). https://doi.org/10.4314/as.v18i1.3 [7]. pandey ak., kumar p., singh p., tripathi nn., bajpai vk., essential oils: sources of antimicrobials and food preservatives, frontiers in microbiology, 7: 2161, (2017). https://doi.org/10.3389/fmicb.2016.02161 [8]. granata g., stracquadanio s., leonardi m., napoli e., consoli gml., cafiso v., geraci c., essential oils encapsulated in polymer-based nanocapsules as potential candidates for application in food preservation, food chemistry, 269: 286-292, (2018). https://doi.org/10.1016/j.foodchem.2018.06.140 [9]. desam nr., al-rajab aj., sharma m., mylabathula mm., gowkanapalli rr., albratty m., chemical constituents, in vitro antibacterial and antifungal activity of mentha piperita l. (peppermint) essential oils, journal of king saud university-science, 31: 528-533, (2019). https://doi.org/10.1016/j.jksus.2017.07.013 [10]. benzaid c., tichati l., djeribi r., rouabhia m., evaluation of the chemical composition, the antioxidant and antimicrobial activities of mentha piperita essential oil against microbial growth and biofilm formation, journal of essential oil bearing plants, 22: 335-346, (2019). https://doi.org/10.1080/0972060x.2019.1622456 [11]. balakrishnan a., therapeutic uses of peppermint-a review. journal of pharmaceutical sciences and research, 7: 474, (2015). [12]. bardaweel sk., bakchiche b., alsalamat ha., rezzoug m., gherib a., flamini g., chemical composition, antioxidant, antimicrobial and antiproliferative activities of essential oil of mentha spicata l. (lamiaceae) from algerian saharan atlas, bmc complementary and alternative medicine, 18: 201, (2018). https://doi.org/10.1186/s12906-018-2274-x [13]. tyagi ak., gottardi d., malik a., guerzoni me., chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil, lwt-food science and technology, 57: 731-737, (2014). https://doi.org/10.1016/j.lwt.2014.02.004 [14]. wang w., wu n., zu yg., fu yj., antioxidative activity of rosmarinus officinalis l. essential oil compared to its main components, food chemistry, 108: 1019-1022, (2008). https://doi.org/10.1016/j.foodchem.2007.11.046 [15]. ye cl., dai dh., hu wl., antimicrobial and antioxidant activities of the essential oil from onion (allium cepa l.), food control, 30: 48-53, (2013). https://doi.org/10.1016/j.foodcont.2012.07.033 [16]. oh jy., park ma., kim yc., peppermint oil promotes hair growth without toxic signs. toxicological research, 30: 297-304, (2014). https://doi.org/10.5487/tr.2014.30.4.297 https://doi.org/10.1016/j.fm.2006.01.003 https://doi.org/10.3329/bjm.v35i2.42643 https://doi.org/10.3923/pjbs.2010.16.21 https://doi.org/10.1111/j.1745-4549.2012.00756.x https://doi.org/10.4314/as.v18i1.3 https://doi.org/10.3389/fmicb.2016.02161 https://doi.org/10.1016/j.foodchem.2018.06.140 https://doi.org/10.1016/j.jksus.2017.07.013 https://doi.org/10.1080/0972060x.2019.1622456 https://doi.org/10.1186/s12906-018-2274-x https://doi.org/10.1016/j.lwt.2014.02.004 https://doi.org/10.1016/j.foodchem.2007.11.046 https://doi.org/10.1016/j.foodcont.2012.07.033 https://doi.org/10.5487/tr.2014.30.4.297 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 114 [17]. mckay dl., blumberg j b., a review of the bioactivity and potential health benefits of peppermint tea (mentha piperita l.), phytotherapy research, 20: 619-633, (2006). https://doi.org/10.1002/ptr.1900 [18]. derwich e., chabir r., senhaji o., in-vitro antioxidant activity and gc/ms studies on the leaves of mentha piperita (lamiaceae) from morocco, international journal of pharmaceutical sciences and drug research, 3: 130-136, (2011). [19]. kizil s., hasimi n., tolan v., kilinc e., yuksel u., mineral content essential oil components and biological activity of two mentha species (m. piperita l., m. spicata l.), turkish journal of field crops, 15: 148-153, (2010). [20]. fitsiou e., mitropoulou g., spyridopoulou k., tiptiri-kourpeti a., vamvakias m., bardouki h., pappa a., phytochemical profile and evaluation of the biological activities of essential oils derived from the greek aromatic plant species ocimum basilicum, mentha spicata, pimpinella anisum and fortunella margarita, molecules, 21: 1069, (2016). https://doi.org/10.3390/molecules21081069 [21]. sartoratto a., machado a.lm., delarmelina c., figueira gm., duarte mct., rehder vlg., composition and antimicrobial activity of essential oils from aromatic plants used in brazil, brazilian journal of microbiology, 35: 275-280, (2004). https://doi.org/10.1590/s151783822004000300001 [22]. yadegarinia d., gachkar l., rezaei mb., taghizadeh m., astanch sa., rasooli i., biochemical activities of iranian mentha piperita l. and myrtus communis l. essential oils, phytochemistry, 67: 1249-1255, (2006). https://doi.org/10.1016/j.phytochem.2006.04.025 [23]. allali h., chikhi i., dib mea., muselli a., fekih n., meliani n., costa j., antioxidant activity and chemical analysis of mentha spicata cultivated from west northern region of algeria by headspace solid phase microextraction and hydro-distillation, natural products, 9: 258-63, (2013). [24]. laggoune s., öztürk m., erol e., duru me., abaza i., kabouche a., kabouche z., chemical composition, antioxidant and antibacterial activities of the essential oil of mentha spicata l. from algeria, journal of materials and environmental sciences, 7: 4205-4213, (2016). https://doi.org/10.1016/j.foodchem.2007.03.059 [25]. yang sa., jeon sk., lee ej., shim ch., lee is., comparative study of the chemical composition and antioxidant activity of six essential oils and their components, natural product research, 24: 140-151, (2010). https://doi.org/10.1080/14786410802496598 [26]. rozza al., de faria fm., brito ars., pellizzon ch., the gastroprotective effect of menthol: involvement of anti-apoptotic, antioxidant and anti-inflammatory activities, plos one, 9, (2014). https://doi.org/10.1371/journal.pone.0086686 [27]. ruberto g., baratta mt., antioxidant activity of selected essential oil components in two lipid model systems, food chemistry, 69: 167-174, (2000). https://doi.org/10.1016/s0308-8146(99)00247-2 [28]. anthony kp., deolu‐sobogun sa., saleh ma., comprehensive assessment of antioxidant activity of essential oils, journal of food sciences, 77: c839-c843, (2012). https://doi.org/10.1111/j.1750-3841.2012.02795.x [29]. dorman hjd., deans sg., antimicrobial agents from plants: antibacterial activity of plant volatile oils, journal of applied microbiology, 88: 308-316, (2000). https://doi.org/10.1046/j.1365-2672.2000.00969.x [30]. reddy dn., al-rajab aj., chemical composition, antibacterial and antifungal activities of ruta graveolens l. volatile oils, cogent chemistry, 2: 1220055, (2016). https://doi.org/10.1080/23312009.2016.1220055 [31]. lambert rjw., skandamis pn., coote pj., nychas gj., a study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol, journal of applied microbiology, 91: 453-462, (2001). https://doi.org/10.1046/j.13652672.2001.01428.x [32]. knobloch k., pauli a., iberl b., weigand h., weis n., antibacterial and antifungal properties of essential oil components, journal of essential oil research, 1: 119-128, (1989). https://doi.org/10.1080/10412905.1989.9697767 [33]. baratta mt., dorman hd., deans sg., figueiredo ac., barroso jg., ruberto g., antimicrobial and antioxidant properties of some commercial essential oils, flavour and fragrance journal, 13: 235-244, (1998). https://doi.org/10.1002/(sici)10991026(1998070)13:4<235::aid-ffj733>3.0.co;2-t [34]. goñi p., lópez p., sánchez c., gómez-lus r., becerril r., nerín c., antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential https://doi.org/10.1002/ptr.1900 https://doi.org/10.3390/molecules21081069 https://doi.org/10.1590/s1517-83822004000300001 https://doi.org/10.1590/s1517-83822004000300001 https://doi.org/10.1016/j.phytochem.2006.04.025 https://doi.org/10.1016/j.foodchem.2007.03.059 https://doi.org/10.1080/14786410802496598 https://doi.org/10.1371/journal.pone.0086686 https://doi.org/10.1016/s0308-8146(99)00247-2 https://doi.org/10.1111/j.1750-3841.2012.02795.x https://doi.org/10.1046/j.1365-2672.2000.00969.x https://doi.org/10.1080/23312009.2016.1220055 https://doi.org/10.1046/j.1365-2672.2001.01428.x https://doi.org/10.1046/j.1365-2672.2001.01428.x https://doi.org/10.1080/10412905.1989.9697767 https://doi.org/10.1002/(sici)1099-1026(1998070)13:4%3c235::aid-ffj733%3e3.0.co;2-t https://doi.org/10.1002/(sici)1099-1026(1998070)13:4%3c235::aid-ffj733%3e3.0.co;2-t food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sarah kehili, mohamed nadjib boukhatem, hussein el-zaeddi, dahbia kellou, amina-bouchra benelmouffok, mohamed amine ferhat, angel a. carbonell-barrachina, william n. setzer, in vitro growth inhibition of pathogenic and food spoilage yeasts and fungi by peppermint (mentha piperita) essential oil and survival of saccharomyces cerevisiae in fruit juices, food and environment safety, volume xix, issue 2 – 2020, pag. 98 – 115 115 oils, food chemistry, 116: 982-989, (2009). https://doi.org/10.1016/j.foodchem.2009.03.058 [35]. soković md., vukojević j., marin pd., brkić dd., vajs v., van griensven lj., chemical composition of essential oils of thymus and mentha species and their antifungal activities, molecules, 14: 238-249, (2009). https://doi.org/10.3390/molecules14010238 [36]. edris ae., farrag es., antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase, food/nahrung, 47: 117-121, (2003). ttps://doi.org/10.1002/food.200390021 [37]. boukhatem mn., ferhat ma., kameli a., kebir ht., lemon grass (cymbopogon citratus) essential oil as a potent anti-inflammatory and antifungal drugs, libyan journal of medicine, 9: 25431, (2014). https://doi.org/10.3402/ljm.v9.25431 [38]. samber n., varma a., manzoor n., evaluation of mentha piperita essential oil and its major constituents for antifungal activity in candida spp, evaluation, 3, (2014). [39]. matan n., woraprayote w., saengkrajang w., sirisombat n., matan n., durability of rubberwood (hevea brasiliensis) treated with peppermint oil, eucalyptus oil, and their main components, international biodeterioration & biodegradation, 63: 621-625, (2009). https://doi.org/10.1016/j.ibiod.2008.12.008 [40]. duarte mct., figueira gm., sartoratto a., rehder vlg., delarmelina c., anti-candida activity of brazilian medicinal plants, journal of ethnopharmacology, 97: 305-311, (2005). https://doi.org/10.1016/j.jep.2004.11.016 [41]. calo jr., crandall pg., o'bryan ca., ricke sc., essential oils as antimicrobials in food systems–a review, food control, 54: 111119, (2015). https://doi.org/10.1016/j.foodcont.2014.12.040 [42]. de la torre torres je., gassara f., kouassi ap., brar sk., belkacemi k., spice use in food: properties and benefits, critical reviews in food science and nutrition, 57: 10781088, (2017). https://doi.org/10.1080/10408398.2013.858235 [43]. essia ngang jj., nyegue ma., ndoye fc., tchuenchieu kamgain ad., sado kamdem sl., lanciotti r., etoa fx., characterization of mexican coriander (eryngium foetidum) essential oil and its inactivation of listeria monocytogenes in vitro and during mild thermal pasteurization of pineapple juice, journal of food protection, 77: 435-443, (2014). https://doi.org/10.4315/0362-028x.jfp-13323 https://doi.org/10.1016/j.foodchem.2009.03.058 https://doi.org/10.3390/molecules14010238 https://doi.org/10.1002/food.200390021 https://doi.org/10.3402/ljm.v9.25431 https://doi.org/10.1016/j.ibiod.2008.12.008 https://doi.org/10.1016/j.jep.2004.11.016 https://doi.org/10.1016/j.foodcont.2014.12.040 https://doi.org/10.1080/10408398.2013.858235 https://doi.org/10.4315/0362-028x.jfp-13-323 https://doi.org/10.4315/0362-028x.jfp-13-323 1. introduction abbreviation list bha = butylated hydroxyanisole bht = butylate hydroxytoluene cfu = colony-forming unit diz = diameter of inhibitory zone dpph = 1,1-diphenyl-2-picrylhydrazyl eos = essential oils mic = minimum inhibitory concentration mpeo = mentha piperita essential oil gc-ms = gas chromatography-mass spectrometry hex = hexamidine ic50 = median inhibitory concentration nist = national institute of standards & technology rt = retention time ri = retention index sda = sabouraud-chloramphenicol dextrose agar 4. conclusion [14]. wang w., wu n., zu yg., fu yj., antioxidative activity of rosmarinus officinalis l. essential oil compared to its main components, food chemistry, 108: 1019-1022, (2008). https://doi.org/10.1016/j.foodchem.2007.11.046 microsoft word 16_dranca_oroian.doc 246 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 246 251 effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin * florina dranca 1, mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, florina.dranca@fia.usv.ro, *corresponding author received 27th january 2018, accepted 25th june 2018 abstract: this work proposes the use of malus domestica ‘fălticeni’ pomace, resulted from the processing of apples into juice in the geographical area of fălticeni, suceava (romania), as a source for pectin extraction. pectin was extracted from this plant source by using two extractants hydrochloric acid and citric acid, separately at ph 2, solid-to-liquid ratio of 1:20, temperature of 90°c, and extraction time of 120 min. together with acid type, another variable that was varied was the particle size: 200-300 µm, 125-200 µm, and <125 µm. to study the influence of acid type and particle size, pectin was characterized in terms of yield and purity, expressed as uronic acid content. the results showed that acid type and particle size had significant effects on the pectin yield and its uronic acid content. a strong influence of acid type on the extraction yield was recorded by particle sizes of 200-300 µm and 125-200 µm, respectively. regarding the uronic acid content, it was observed that particle sizes of 200-300 µm determined a good purity of the extracted pectin, independent of acid type, while for particle sizes of 125-200 µm and <125 µm, the uronic acid content of pectin was strongly affected by the acid used. the highest pectin yield (21.24%) and uronic acid content (93.90 g/100g) were obtained for citric acid extraction and particle sizes between 125 and 200 µm. keywords: hydrochloric acid, citric acid, pectin recovery, composition. 1. introduction as important components of the plant cell walls, polysaccharides of fruits and vegetables have drawn the interest of researches, manufacturers, and also consumers due of their physical properties and health promoting and diseasepreventing potentials [1], which supported their application in food, pharmaceutical, and cosmetic industries. of the different types of plant polysaccharides, pectin is mostly abundant in the middle lamella layers and the primary cell walls of plants [2,3]. pectin is a complex polymer composed of at least 17 different monosaccharides, with galacturonic acid units linked by α(1→4) linkages (d-gala) being the main constituents, followed by larabinose, d-galactose, l-rhamnose and others [4,5]. structurally, 3 major polysaccharide domains are recognized in the pectic network, i.e. homogalacturonan (hg), which is a linear polymer of d-gala, rhamnogalacturonan i (rg-i), described as a repeating disaccharide of d-gala and lrhamnose, and rhamnogalacturonan ii (rgii), which presents a homogalacturonic backbone with numerous complex side chains containing rhamnose and other neutral sugars [6]. within the d-gala units, the carboxylic or hydroxyl groups may be methyl-esterified and/or o-acetyl-esterified [7]. commercial pectin is extracted from 3 main sources: apple pomace [8], citrus peel [9], and sugar beet pulp [10]. in the last few years several non-conventional sources of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xvii, issue 2 – 2018, pag. 246 251 247 pectin, such as carrot pomace, watermelon rinds, cocoa husks, mulberry branch bark, faba bean hulls, sisal waste, and pumpkin waste have been studied [6]. advances have also been made regarding the extraction of pectin, through the development of some innovative approaches aimed to improve the overall extraction process and the quality of the final product. the innovative techniques applied include subcritical water extraction, ultrasound-, microwave-, and enzyme-assisted extraction [11]. an important tendency observed in the research on the extraction and characterization of pectin is the capitalization of waste streams resulted from processing fruit and vegetables locally. based on this observation, we propose in this study the use of malus domestica ‘fălticeni’ pomace, obtained from processing apples into juice in the geographical area of fălticeni, suceava (romania), as a source for pectin extraction. malus domestica ‘fălticeni’ is an apple hybrid developed through free pollination of the jonathan variety at s.c.d.p. fălticeni in the year 1962. this apple variety was selected as an elite cultivar in 1971; it was approved as a new variety in 1979 and registered in the romanian official catalogue of the varieties of plant species [12]. the enlisting of the variety in the romanian official catalogue was renewed at the end of 2015 [13]. malus domestica ‘fălticeni’ is an autumn variety that is resistant against disease, drought, frost, and other stress factors. according to s.c.d.p. fălticeni, the average production is estimated to 28 t/ha, however, a later study reported a lower average production (18.6 t/ha) [14]. regarding the main characteristics of the fruits, apples of the fălticeni variety are medium sized, of spherical shape, flattened, and have a weight of about 148 g. the best quality of the fruit was recorded from the middle of september until december [12]. this study aims at investigating the potential for capitalization of malus domestica ‘fălticeni’ pomace as a source of pectin, which provides a mean to increase the economic viability of this apple variety. pectin extraction from apple pomace was conducted on a laboratory scale with the purpose of studying the influence of the type of acid (mineral vs. organic) and particle size on the overall process. the extracted pectin was characterized in terms of yield and purity, expressed by the content of uronic acid. 2. materials and methods 2.1. materials apple pomace was collected by processing malus domestica ‘fălticeni’ apples from 2016 harvest, cultivated in the fălticeni area of suceava, romania. the pomace was dried in an oven with air circulation at 60°c until constant weight was achieved and then powdered using a food processor. the obtained powder was passed through an analytical sieve shaker retsch as 200 (retsch gmbh, germany) to separate it on the following particle size intervals: 200300 µm, 125-200 µm, and <125 µm. hydrochloric acid, citric acid, ethyl alcohol, sulfamic acid, potassium hydroxide, sulfuric acid, sodium tetraborate, sodium hydroxide, d-galacturonic acid, and mhydroxydiphenyl were purchased from sigma-aldrich (munich, germany). 2.2. extraction and purification procedure the extraction mixture was prepared by mixing 5 g of apple pomace powder with 100 ml of extraction solvent (solid-to-liquid ratio of 1:20) obtained by adding hydrochloric acid and, respectively, citric acid to distilled water until a ph value of 2 was reached. for each type of acid, 3 separate mixtures, according to the particle size intervals mentioned above, were prepared. to extract pectin, the mixture was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xvii, issue 2 – 2018, pag. 246 251 248 kept in a water bath at the temperature of 90°c for 120 min. after the extraction was completed, the mixture was cooled to room temperature prior to the precipitation and purification steps. pectin was first separated from the remaining solid material by centrifugation at 4000 rpm for 40 min; the resulting supernatant was passed through clean cheesecloth folded 6 times, fitted into the neck of a laboratory bottle with screw cap, and then mixed with ethyl alcohol (>96%, v/v) in a 1:1 ratio (v/v). the content of the bottle was thoroughly mixed and kept at 46°c for 12 h to complete the precipitation. next, the centrifuge was used again to separate the precipitated pectin from the liquid (4000 rpm, 30 min). the wet pectin was washed by ethyl alcohol 3 times and dried at 50°c to a constant weight. 2.3. determination of pectin yield pectin yield was calculated using eq. (1): 100(%) 0  m m yield (1) where: m0 is the weight of dried pectin (g) and m is the weight of dried apple pomace powder (g). [15] 2.4. determination of uronic acid content the uronic acid content of pectin was estimated using the sulfamate/mhydroxydiphenyl method developed by filisetti-cozzi and carpita [16]. for each batch of samples was prepared a standard curve of d-galacturonic acid. sample preparation for the analysis was made according to [17]. briefly, 20 mg of dry pectin were added to 50 ml distilled water (at 40ºc) and mixed using a magnetic stirrer until the sample was completely dispersed. the volume was finally adjusted to 100 ml with distilled water at 40ºc. aliquots of 400 µl from the pectin solutions were placed in glass tubes. to the pectin solution were added 40 µl of 4 m sulfamic acid solution (adjusted to ph 1.6 with saturated solution of potassium hydroxide), followed by 2.4 ml of sulfuric acid containing 75 mm of sodium tetraborate, vigorously vortexing for at least 5 s the content of the tubes in between the addition of the solutions. the samples were place in a 100ºc water bath (boiling) for 20 minutes, and then cooled in an ice bath for 10 minutes. after cooling, 80 µl of mhydroxydiphenyl solution in 0.5% (w/v) sodium hydroxide were added and vortex mixed. between 10 min and 30 min after complete mixture the absorbance was read at 525 nm against the reagent control using a uv-vis-nir spectrophotometer (shimadzu corporation, japan). 3. results and discussion an important observation must be first made regarding the pectin yield achieved through the use of pomace from malus domestica ‘fălticeni’ apples. as fig. 1 shows, the maximum pectin yield was 21.24%. as malus domestica ‘fălticeni’ is an autumn variety (late season), the content of pectin is expected to be high, however, the maximum yield determined for this apple variety exceeds the values reported for a citric acid extraction of pectin from the late season royal (16.65%) and golden (18.79%) apple varieties [18]. 3.1. influence of acid type on pectin yield and purity according to the results presented in fig. 1, pectin yield was significantly influenced by the type of acid used in the preparation of the extraction mixture. as it can be observed, pectin yield varied between 10.41% and 13.51% for the extraction with water acidified with hydrochloric acid and between 15.83% and 21.24% when citric acid was used. the fact that citric acid is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xvii, issue 2 – 2018, pag. 246 251 249 fig. 1 influence of the type of acid on pectin yield more effective in the extraction of pectin was also confirmed by virk and sogi [19], who studied the extraction of pectin from apple (malus pumila cv. amri) peel waste with 0.02m hydrochloric acid, 0.05m hydrochloric acid, and 1% citric acid. another pectin source for which a high extraction efficiency of citric acid was reported is passion fruit peel (passiflora edulis flavicarpa) [20]. in this study, a comparison between the extraction of pectin with three different acids (citric, hydrochloric, and nitric acid) reached the conclusion that although a low ph is fig. 3 influence of particle size on pectin yield fig. 2 influence of the type of acid on the content of uronic acid necessary to improve pectin yield, strong acid solutions could lead to highly soluble smaller pectin molecules due to partial hydrolysis. these pectin molecules may not be precipitated by the addition of alcohol [21], explaining why in this case the extraction using hydrochloric acid gave a pectin yield almost 2 times lower than that obtained with citric acid. the effect of acid type on the uronic acid content of pectin is shown in fig. 2. it can be observed that the use of citric acid determined a higher uronic acid content (84.25-93.90 g/100 g). on the other side, fig. 4 influence of particle size on the content of uronic acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xvii, issue 2 – 2018, pag. 246 251 250 hydrochloric acid produced a larger variation of this chemical parameter, which ranged between 46.82 g/100 g and 86.54 g/100 g. regarding the influence of acid type on the content of uronic acid, it was previously showed that the impact of a strong acid at high temperature seems to combine two simultaneous phenomena: firstly, the release of sugars as product of pectin hydrolysis, and secondly, their degradation under the action of both the acid and the heat [22]. thus, citric acid most likely caused less degradation to the structure of pectin, contributing to a higher uronic acid content. 3.2. effect of the particle size on pectin yield and purity properties of the plant material such as particle size, particle size distribution, and chemical composition have been extensively studied and are known to influence pectin extraction. fig. 3 shows the influence of particle size on the extraction yield, and fig. 4 indicates its impact on the uronic acid content of pectin. while both graphics depict a large influence of the particle size of apple pomace on pectin recovery, the most pronounced variation was observed for pectin yield. for all particle size intervals studied, the lower values (10.41%, 12.85%, and 13.51%) were obtained using hydrochloric acid. citric acid extraction of pectin gave the highest yield (21.24%) when particle size varied between 125 and 200 µm. a similar conclusion was made in a previous study [23], where it was found that the highest average yields of pectin from apple pomace were obtained when the particles was larger than 106 µm and smaller than 250 µm. in a similar manner to the variation of pectin yield, for all particle size intervals, the lower uronic acid contents (86.54 g/100 g, 57.61 g/100 g, and 46.82 g/100 g) resulted by using hydrochloric acid. particle sizes of 200-300 µm determined a good purity of the extracted pectin (uronic acid content above 86 g/100 g), independent of acid type; in the case of particle size intervals of 125-200 µm and <125 µm, the purity of pectin was strongly effected by acid type. whereas citric acid extraction gave good results in terms of pectin purity, the uronic acid content of pectin extracted with hydrochloric acid decreased with the reduction of particle size. the lower purity and yields may be attributed to acid strength and also to the fact that particles that are too small can hinder solvent flow through the matrix due to surface tension of the solvent employed [24]. 4. conclusion considering the high yield of pectin extracted from malus domestica ‘fălticeni’ pomace and its quality, i.e. the high uronic acid content, it can be concluded that this apple variety is a viable source of pectin. the best results for pectin recovery (extraction yield of 21.24% and uronic acid content of 93.90 g/100g) were obtained using citric and apple pomace with particle sizes of 125-200 µm. this statement is in agreement with previous studies in which it was proved that citric acid is a good alternative to the mineral acids usually used in pectin extraction. further research on this pectin source could be focused on the influence of other extraction parameters, the application of non-conventional extraction techniques and the physico-chemical properties of pectin. 5. references [1]. jafari, f., khodaiyan, f., kiani, h., hosseini, s. s., pectin from carrot pomace: optimization of extraction and physicochemical properties, carbohydrate polymers, 157: 1315-1322, (2017) [2]. mellerowicz, e. j., sundberg, b., wood cell walls: biosynthesis, developmental dynamics and their implications for wood properties, current opinion in plant biology, 11(3): 293-300, (2008) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xvii, issue 2 – 2018, pag. 246 251 251 [3]. cosgrove, d. c., comparative structure and biomechanics of plant primary and secondary cell walls, frontiers in plant science, 3: 204, (2012) [4]. kaya, m., sousa, a. g., crépeau, m. j., sørensen, s. o., ralet, m. c., characterization of citrus pectin samples extracted under different conditions: influence of acid type and ph of extraction, annals of botany, 114(6): 1319-1326, (2014) [5]. yapo, b. m., pectic substances: from simple pectic polysaccharides to complex pectins a new hypothetical model, carbohydrate polymers, 86(2): 373-385, (2011) [6]. marić, m., grassino, a. n., zhu, z., barba, f. j., brnčić, m., brnčić, s. r., an overview of the traditional and innovative approaches for pectin extraction from plant food wastes and by-products: ultrasound-, microwaves-, and enzyme-assisted extraction, trends in food science & technology, 76: 28-37, (2018) [7]. chan, s. y., choo, w. s., young, d. j., loh, x. j., pectin as a rheology modifier: origin, structure, commercial production and rheology, carbohydrate polymers, 161: 118-139, (2017) [8]. yates, m., gomez, m. r., martinluengo, m. a., ibañez, v. z., serrano, a. m. m., multivalorization of apple pomace towards materials and chemicals. waste to wealth, journal of cleaner production, 143: 847-853, (2017) [9]. putnik, p., bursać kovačević, d., režek jambrak, a., barba, f. j., cravotto, g., binello, a., shpigelman, a., innovative “green” and novel strategies for the extraction of bioactive added value compounds from citrus wastes a review, molecules, 22(5): 680, (2017) [10]. huang, x., li, d., wang, l. j., characterization of pectin extracted from sugar beet pulp under different drying conditions, journal of food engineering, 211: 1-6, (2017) [11]. adetunji, l. r., adekunle, a., orsat, v., raghavan, v., advances in the pectin production process using novel extraction techniques: a review, food hydrocolloids, 62: 239250, (2017) [12]. s.c.d.p. fălticeni, soiuri omologate de scdp fălticeni, available at http://www.madr.ro/ docs/cercetare/rezultate_activitate_de_cercetare/sc dp_falticeni.pdf [13]. the romanian official catalogue of the varieties of plant species 2016, available at http://istis.ro/image/data/download/catalogoficial/catalog_2016.pdf [14]. grădinariu g., istrate m., dascălu m., valuable apple tree varieties – principal sourse of germoplasma, lucrări ştiinţifice u.ş.a.m.v. bucureşti, seria b, vol. xlv, (2002) [15]. liew, s. q., ngoh, g. c., yusoff, r., teoh, w. h., sequential ultrasound-microwave assisted acid extraction (umae) of pectin from pomelo peels, international journal of biological macromolecules, 93: 426-435, (2016) [16]. melton, l. d., smith, b. g., determination of the uronic acid content of plant cell walls using a colorimetric assay, current protocols in food analytical chemistry, (2002) [17]. miceli-garcia, l. g., pectin from apple pomace: extraction, characterization, and utilization in encapsulating alpha-tocopherol acetate, university of nebraska-lincoln, united states of america, (2014) [18]. kumar, a., chauhan, g. s., extraction and characterization of pectin from apple pomace and its evaluation as lipase (steapsin) inhibitor, carbohydrate polymers, 82(2): 454-459, (2010) [19]. virk, b. s., sogi, d. s., extraction and characterization of pectin from apple (malus pumila. cv amri) peel waste, international journal of food properties, 7(3): 693-703, (2004) [20]. kliemann, e., simas, d., nunes, k., amante, e. r., prudêncio, e. s., teófilo, r. f., amboni, r. d., optimisation of pectin acid extraction from passion fruit peel (passiflora edulis flavicarpa) using response surface methodology, international journal of food science & technology, 44(3): 476-483, (2009) [21]. kalapathy, u., proctor, a., effect of acid extraction and alcohol precipitation conditions on the yield and purity of soy hull pectin, food chemistry, 73(4): 393-396, (2001) [22]. chan, s. y., choo, w. s., effect of extraction conditions on the yield and chemical properties of pectin from cocoa husks, food chemistry, 141(4): 3752-3758, (2013) [23]. canteri-schemin, m. h., fertonani, h. c. r., waszczynskyj, n., wosiacki, g., extraction of pectin from apple pomace, brazilian archives of biology and technology, 48(2): 259266, (2005) [24]. perussello, c. a., zhang, z., marzocchella, a., tiwari, b. k., valorization of apple pomace by extraction of valuable compounds, comprehensive reviews in food science and food safety, 16(5): 776-796, (2017) 68 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 68 73 aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill *adriana dabija1, răzvan pomparău1 1faculty of food engineering, stefan cel mare university of suceava, romania adriana.dabija@fia.usv.ro *corresponding author received 15th december, accepted 5th march 2014 abstract: whey is a valuable by-product resulting from the manufacture of cheese, which in our country is not fully exploited. the composition of the whey will vary depending on the characteristics of the milk from which it is obtained and the cheese manufacturing process. whey contains the most soluble components of milk and casein and fat residue. this paper proposes a simple way to capitalize whey cream goat milk curd with onion and dill. cheese whey cream from goat's milk with onion and dill contains only natural ingredients, whey being obtained after acidification of milk with natural goat curd, onion and dill. the product is an organic functional food, not using any preservatives, additives or genetically modified organisms. the taste was smooth and the soft structure was obtained by slowly heating the whey at a maximum temperature of 80°c. the product was analyzed for sensory, physicochemical points of view and determining the shelf life. finally it was proposed as a way of packaging and marketing the finished product. keywords: serum proteins, immunoglobulins, functional foods, eco-innovative product 1. introduction legends say that the gods of olympus were raised with goat milk, its health benefits being known since antiquity. cheese whey cream with onion and dill focuses and amplifies the healing properties of goat milk [1, 2, 3]. cheese whey cream from goat's milk contains only natural ingredients, whey obtained after acidification of milk with natural rennet kid, onion and dill. no preservatives, additives or genetically modified microorganisms are used. the creamy taste and delicate structure is obtained by slowly heating the whey at a maximum temperature of 80°c [4, 5]. world production of cheese is 17 million tons obtained by processing over a third of world milk production, which results in about 145÷153 x 106 tons of whey. it is estimated that only half the amount of whey produced is used for human food or animal feed, the remainder being discharged through sewage leading to increased environmental pollution [6, 7, 8, 9, 10]. a traditional whey recovery solution is to obtain cheese from whey. cheese whey is obtained both from the production of cheese from cow's milk and from the manufacture of cheese from sheep's milk, as well as the whey from goat's milk cheese. to achieve the cheese whey, whey is heated to a temperature of less than or equal to 80°c and maintained at this temperature, in this case favours the precipitation of most of the serum proteins, which causes the fat from whey, which is accumulated in whey heated surface in the form of agglomerates which are collected in a cedilla, in which after draining the whey, the cheese whey cheese is formed by autopressing [11, 12]. generally, the cheese whey is accumulated in the cedilla food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 adriana dabija, răzvan pomparău, aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill, issue 1 2014, pag. 68 – 73 69 as round balls with a mass of 2-3 kg, the product having a pleasant sweet taste, smooth creamy white texture. fresh cheese whey has about 60% water and 50% fat in dry matter [13, 14, 15]. this paper proposes a study of process obtaining cheese whey cream from goat milk with onion and dill. 2. materials and methods the technological process for obtaining cheese whey cream with onion and dill meet the specific process of obtaining cheese whey, the innovation in this case being the type of milk used, and added ingredients onion and dill. for the above product, the following manufacturing recipe was used (table 1): table 1 manufacturing recipe for 1000 g „cheese whey cream from goat milk with onion and dill” raw material quantity/1000g finished product whey 33 l onion 40 g dill 30 g salt 30 g the raw material used in cheese whey producing is whey, which results in large quantities in the manufacture of cheese via rennet coagulation or by acidification with lactic cultures. the whey obtained by the above processes is characterized by the specific sensorial properties: aspect whey is a yellowish green, opalescent liquid; consistency fluid, viscous not accepted; odour and taste sour, specific to lactic fermentation. the composition of the whey will vary depending on milk characteristics and on the manufacturing process of the main product. the whey contains the most soluble components of milk and casein and fat residue. often, during the manufacturing process, number of substances are added in the milk (calcium chloride, sodium chloride, acid dyes, and so on), which is found in the whey. there are two types of whey: sweet whey, derived from enzymatic coagulation of milk; acidic whey, from the production of fresh or soft cheeses or the production of lactic casein acid whey composition is presented in table 2. table 2 chemical composition (%), ph and protein content of the whey from the milk of goats characteristic quantity [%] dry substance 7.07±0.09 lipids 0.84±0.18 proteins 0.63±0.03 ash 0.57±0.01 lactose 5.02±0.24 ph 6.34±0.29 whey proteins are the most valuable in terms of nutritional value, and are also called fast proteins. they are rich in sulphur amino acids, lysine and tryptophan. serum proteins are those which remain in the whey after precipitation of caseins at ph 4.6. serum proteins (whey) are more water soluble than casein and are more prone to thermal distortions. the distortion cancels their ability to bind water. the following are the main whey proteins: β-lactoglobulin has a molecular weight of 18000 and contains 162 amino acid residues. this protein includes eight genetic variants and represents half of the total whey proteins. the protein exists as the secondary structure and normally exists as a dimmer. at the isoelectric point (ph 3.5 to 5.2) dimmers may be assigned to octomeri, but at ph 3-4 they are dissociated to monomers. milk βlactoglobulin has the ability to stoichiometric fix retinol (vitamin a1) and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 adriana dabija, răzvan pomparău, aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill, issue 1 2014, pag. 68 – 73 70 very strong complex with benzo-(a) pyrene, a potent carcinogen substance; α-lactoalbumine has molecular weight 14000 and contains 123 amino acid residues. this protein has 8 cysteine residues that are involved in the formation of intramolecular-ssconnections. it also contains 4 tryptophan residues. αlactoalbumine has an ordered secondary structure and a spherical, compact tertiary structure. the heat denaturation and ph less than 4, milk αlactoalbumina releases linked calcium; immunoglobulins in colostrum and milk obtained from circulating immunoglobulins in the blood stream. in colostrum and milk are igg immunoglobulins (lg lg g1 and g2), lg and lg m. there are sensitive differences between the content of immunoglobulins in colostrum [11, 12]. onion is a biannual plant of the genus allium, rich in active principles which explain its many therapeutic uses. the onions are found organosulphur compounds, flavonoids, essential oils, organic acids, vitamins (c, b1, b3, b6, b9, pp, e, etc.), carotenes, fitoncide, minerals (calcium, iron, sulphur, iodine, silicon, chromium, magnesium, potassium and so on). the chemical composition of onions is presented in table 3. research has shown that onion genus allium has more effect on the body such as: diuretic properties due to fructosan rich content that has the capacity to produce about 3l urine / day; hypoglycaemic; property to reduce cholesterol and regulate blood pressure; antimicrobial, antifungal, antiinflammatory. dill is an annual plant with a short life of the anatheum genus, species graveolens. it is rich in vitamin a, b, c, chlorophyll, essential oils, and tannins (eg a teaspoon of fennel seeds contain more calcium than a cup of milk). it can stimulate digestion, antiseptic effect, combat hiccups, diuretic, depurative. the essential oil is composed of carvone, felandren, limonene, apiol and terpene hydrocarbons. dill leaves have tonic properties of appetizer and stomachic nature, promoting diuresis and elimination of gas and the fennel grains are strongly carminative, being important for their emmenagogue properties (regulating menstruation). table 3 onion’s chemical composition the chemical composition of dill is presented in table 4. table 4 dill’s chemical composition components quantity/100g carbohidrates, with fiber 7 g 2.1 g lipids 1.1 g proteins 3.5 g tiamine (b1) 0.1 mg riboflavine(b2) 0.3 mg niacine ( b3) 1.6 mg pantotenic acid (b5) 0.4 mg vitamine b6 0.2 mg folic acid (b9) 150 µg vitamine c 85 mg minerals 1.5 g energy value 43 kcal transport. whey is transported by tankers to the factory. the material for these medium composition for100 g energy 3 kcal water 89 % glucids 7.1 % lipids 0.2 % proteins 1.3 % fiber 2.1 % calcium 80 mg magnezium 18 mg potasium 250 mg iron 1 mg vitamins 38 mg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 adriana dabija, răzvan pomparău, aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill, issue 1 2014, pag. 68 – 73 71 means of transport is in many cases, aluminium, stainless steel. quantitative reception. the total amount of whey entering the mill is received quantitatively. this can be done in two ways: volumetric: determining the amount of whey is given taking into account the temperature. this type of measurement can be achieved in two ways: either by hand or by measuring the levels of whey in tanks, and by means of flow meters known as galactometers, the main advantage is to measure the volume and discharge at the same time. gravimetric: is to empty tanks or drums in the basin scale, followed by a readon-screen quantity control. the system of measurement is more accurate although the disadvantage of the batch. quality reception. raw material before entering the factory must be determined based on its quality and to sort, this reception consists of sensorial examination and laboratory tests. heating whey. it is performed in special valves made of steel used in the food industry. heating the whey is carried out at temperatures up to 80-90°c and maintaining this temperature, in this case favours the precipitation of most of the serum proteins that involve whey and fat which accumulates on the surface of the particle resulting in the cheese whey. filtering. made using cloth filters for food industry that separate the cheese from the whey left. cheese whey collection is carried out with a cedilla, which forms spherical pieces with a weight of 1-2 kg. when all of the cheese whey is separated from whey, and hanged onto stainless steel pillars to drain for a few hours. preparation of the mixture. the cheese whey obtained in the desired quantity is dosed according to the recipe and bring in a mixing machine for operation with onion, dill and salt. the mixing takes place until a homogeneous mass is obtained and the secondary raw materials are distributed uniformly throughout the mass of the product. packaging. the type of packaging for this product is in the form of glass bottles with a capacity of 130 g. this type of package has been chosen since: it is insoluble in water and resistant to acids and bases; is chemically inert in contact with food; impermeable to gases, liquids, vapours, scents, micro-organisms; easy to clean; is stiff as keeps its shape, does not contribute to product deterioration. but the greatest advantage of glass packaging is that it does not harm the environment, but can be recycled and reused. storing "cheese whey cream from goat milk with onion and dill" is done in cold storage at a temperature of 4 ± 2°c. delivery of the commercial product made under the law in force with vehicles equipped with refrigerator. 3. results and discussion technical specifications refer to "cheese whey cream from goat's milk with onion and dill" obtained by heating the whey from the production of cheese from goat's milk and adding ingredients. ingredients used to obtain cheese whey cream are: onion, at a rate of 4%; dill, in a proportion of 3%. raw and auxiliary materials used to obtain cheese whey cream, whey and vegetables correspond to product’s technical documents and meet all sanitary and veterinary regulations in force in our country. the product is packaged in jars of 260 g gross weight and net weight of 130 g. sensorial properties are shown in table 5. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 adriana dabija, răzvan pomparău, aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill, issue 1 2014, pag. 68 – 73 72 table 5 sensorial properties of the product " cheese whey cream from goat milk with onion and dill characteristics admission requirements aspect homogeneous colour whitish green consistency creamy taste and odour pleasant, salty enough, characteristic for the added ingredients table 6 physical-chemical properties of the product "cheese whey cream from goat milk with onion and dill" characteristics value analysis method fat/s.u., % min. 62 sr iso 3433:2009 humidity, % max. 59 sr en iso 5534:2004 proteic substances, % min. 10 sr en iso 8968:2004 nacl, % max. 3 sr iso 57651:2008 acidity, °t max. 20 sr iso 6092:2008 minimum durability of the product "cheese whey cream from goat milk with onion and dill" is settled on 14 days of the packaging according to the following criteria: moisture content according to analysis determined that moisture product is 59.01%, which gives a lower shelf-life; acidity: acid was determined at 2 days intervals of the packaging. it was found that it did not change for 14 days from the packing (20°t), but at the 8th determination the acidity reached 25°t (which leads to deterioration of the product). nutritional information per 100 grams of product "cheese whey cream from goat's milk with onion and dill" are:  protein 10.19 g  fat 25.38 g  carbohydrates 11.28g. according to the above equation results: qn = 10.19 · 4.1 + 25.38 · 9.3 + 11.28 · 4.1 qn= 324 kcal/100g 4. conclusions consumption of dairy products in romania is steadily increasing; manufacturers are oriented towards diversification of product assortments, especially traditional cheeses. production and processing of milk and thus the obtaining and processing of whey must strictly comply with legislation. due to its composition, whey is a valuable byproduct that can be exploited by obtaining complex functional foods for human nutrition. taking special care to detail, using raw materials of the highest quality, the paper proposes dairy manufacturers an ecoinnovative product designed to please all consumers. "cheese whey cream from goat's milk with onion and dill" was made from a traditional assortment of cheeses whey, with added onions and dill finally obtaining a finished product with superior quality and nutritional psycho-sensorial properties. another innovative element proposed in the paper is the use of glass jars for packaging because it presents multiple advantages of glass in food packaging. also, by using this type of packaging it increases the shelf life of the product, whey cheeses being products with a low shelf life, susceptible to microbiological contamination due to high water content (about 80 %). 5. references [1]. prazeres, a., et al., cheese whey management. a review, journal of environmental management, vol.110, 48-68, (2012) [2]. spalatelu c., biotechnological valorisation of whey. innovative romanian food biotechnology, 10, 1-8, (2012) [3]. huffman, l.m., processing whey protein for use as a food ingredient. food technology, 5049-5052, (1996) [4]. jelen, p, whey processing, encylcopedia of dairy sciences, vol.4, london academic press, 2739-2751, (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 adriana dabija, răzvan pomparău, aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill, issue 1 2014, pag. 68 – 73 73 [5]. gonzalez sizo, m.i., the biotechnological utilization of cheese whey. a review, bioresource technology, vol.57, 1-11, (1996) [6]. kosseva, m., et al. – use of immobilized biocatalysts in the processing of cheese whey, international journal of biological macromolecules, 45, 437-447, (2009) [7]. pamesar, p., et al. – bioutilisation of whey for lactic acid production, food chemistry, 105, 1-14, (2007) [8]. ozmihci, s., kargi, f. – ethanol production from cheese whey powder solution in a packed bioreactor at different hydraulic residence times, biochemical engineering journal, 42, 180185, (2008) [9]. guimaraes, p., et al., fermentation of lactose to bie-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey, biotechnology advances, vol.28, issue 3, 375-384, (2010) [10]. kosseva, m., et al., use of immobilised biocatalysts in the processing of cheese whey, international journal of biological macromolecules, vol.45, issue 5, 437-447, (2009) [11]. mollea, c., marmo, l., bosco, f., valorisation of cheese whey, a by-product from the dairy industry, edited by innocenzo muzzalupo, isbn 978-953-51-0911-2, (2013) [12]. mann e.j. whey products and their uses, dairy industries international, 13-14, (2000) [13]. gunasekaran s., ko s., xiao l. use of whey proteins for encapsulation and controlled delivery applications, journal of food engineering, 83, 31–40, (2007) [14]. ópez-rubio a., lagaron j.m., whey protein capsules obtained through electro spraying for the encapsulation of bio actives. innovative food science and emerging technologies, 13, 200–206, (2012) [15]. escuma m., et al., whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content, food microbiology, 16, 442-451, (2008) 191 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag.191 200 effect of ripening stage on organic acid profiles and antinutrient contents of three species of wild edible mushroom russula ssp. jaures oscar gbotognon 1 , *michel djary koffi 2 , hubert kouassi konan 1 and eugène jean parfait kouadio 1 1laboratory of biochemistry and food technology, nangui abrogoua university, 02 bp 801, abidjan 02, côte d’ivoire. 2laboratory of biotechnology, felix houphouet boigny university, 22 bp 582 abidjan 22, côte d’ivoire. djaryss@yahoo.fr *corresponding author received 1st september 2019, accepted 28th september 2019 abstract: the present study was aimed at investigating the antinutrient and organic acid contents of three wild edible mushrooms from russula genus as a function of their ripening stage. fresh mushrooms russula lepida, russula mustelina and russula delica were harvested from their natural habit in brobo area’s (7°43'0" n and 4°42'0" w) in center côte d’ivoire. the fresh mushrooms were oven dried and ground to obtain the crude flour. phytate and oxalate contents were investigated using standard colorimetric methods, while organic acid profiles were performed by using hplc analytical methods. the antinutrient composition showed that the greatest content in phytates (1.60±0.10 mg/100 g) and oxalates (5.17±0.01 mg/ 100 g) were observed respectively, in r. lepida and r. mustelina immature fruiting bodies. these values are much lower than the standard safe limits. as regards organic acid profiles, they revealed the presence of at least fourteen organic acids namely benzoic, oxalic, fumaric, succinic, malic, tartaric, ascorbic, citric, lactic, adipic, propionic, formic, shikimic and acetic acids. the main organic acids in r. lepida were lactic (49.70 mg/ 100 g dw, for postmature stage), fumaric (36.00 and 31.22 mg/ 100 g dw, respectively for immature and mature stage), citric (21.60 and 31.60 mg/ 100 g dw, respectively for immature and mature stage) and succinic (21.00 mg/ 100 g dw for post-mature stage) acids. lactic acid (47.90 mg/ 100 g dw) was the dominating organic acid in r. mustelina at immature stage, whereas citric (30.20 mg/ 100 g dw at post-mature stage) and ascorbic acids (21.80 mg/ 100 g dw at post-mature stage) were major in r. delica. r. lepida showed the higher total organic acid contents (ranging from 113.13 mg/ 100 g in immature stage to 103.31 mg/ 100 g in post-mature stage) whatever the ripening stage. as for r. mustelina, the immature stage was better (100.02 mg/ 100 g dw), while r. delica showed better total organic acid content (83.33 mg/ 100 g dw) at post-mature stage. keywords: organic acids, edible mushroom, ripening stage, russula lepida, russula mustelina, russula delica. 1. introduction mushrooms are the best-known example of macrofungi which are wild edible fungi species with large and visible fruiting bodies [1]. mushrooms species having a close relationship with the host (usually a tree) are mycorrhizal (symbiotic). those living on dead organic matter are saprotrophic (saprophytes) while species that live on other species in a nonsymbiotic relationship are parasitic species http://www.fia.usv.ro/fiajournal mailto:djaryss@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 192 [2]. the mycorrhizal species play a vital ecological role through the symbiotic relationships that they form with trees. they enable trees to grow in nutrient-poor soils. for example, the trees of the miombo woodland of central and southern africa and the woodland itself would not exist without their fungal partners [3, 1]. moreover, mushrooms are excellent biodegraders since they decompose organic waste which releases the nutrients in celluloses, hemicelluloses and lignin. they therefore help in cleaning the environment and recycling the nutrients [4]. the increased interest in consumption of mushrooms as food is as a result of their nutritional, antioxidant and therapeutic values [5]. according to boa [1], about 1200 species of fungi are used in eightyfive different countries for their gastronomic value and/or medicinal properties. indeed, wild edible mushrooms are found to be highly rich in proteins, minerals, vitamins, crude fibre and carbohydrate with low fat and oil content [6, 7, 8]. dué et al. [8] reported that they could be used as valuable substitutes for meat or fish especially in developing countries for malnourished children suffering from kwashiorkor (a protein deficiency condition) and for pregnant women. chittaragi and naika [9] argue that mushrooms can provide balancing diet compounds in enough quantities for human nutrition. due to the low fat and oil content, they are recommended as good source of food supplement for patients with cardiac problems or at risk with lipid induced disorders. also, several mushroom species have been described to exhibit varied biological and medicinal properties such as antioxydants, antibacterial, antifungal, antiviral, anti-inflammatory, antimutagenic, antitumoral and antidiabetic activities [10, 11, 12, 13, 14, 15]. therefore, mushrooms have been categorized as therapeutic foods [16]. the bioactive and taste properties of mushrooms are conferred by compounds such as phenolic derivatives and organic acids [17]. phenolic compounds and organic acids (especially malic, tartaric, citric, and succinic acids) may have a protective role against various diseases due to their antioxidant activity [18, 19]. furthermore, organic acids were reported to be responsible of organoleptic characteristics of fruits and vegetables, especially mushrooms [20]. in the center of côte d’ivoire, wild edible ectomycorrhizal mushrooms of russula genus are widely collected and consumed in many households or sold for the improvement of icomes and livelihood of farmers [21]. the species russula lepida, russula mustelina and russula delica were previously investigated for their bioactive compounds and antioxidative properties [22]. considering the importance of organic acids, the present paper focuses on their acid organic profile and antinutrient contents as a function of their ripening stage. 2. materials and methods mushroom collection and preparation of sample fresh mushrooms were harvested at different ripening stages (immature, mature and post-mature) from their natural habit in brobo area’s (7°43'0" n and 4°42'0" w) in côte d’ivoire. taxonomic identification was achieved by dr souleymane yorou nourou (abome calavy university of benin/ munich university of germany), as r. lepida, r. mustelina and r. delica. after collection, mushrooms were placed in a cooler with ice to keep their freshness and then food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 193 transported to the laboratory for flour preparation. fresh mushrooms (500 g) were cleaned to distilled water then, sorted and free from any kind of waste, drained, and dried at 45°c in an oven for 48 h. dried mushrooms were ground using a blender to obtain the crude flours. antinutritional factors analysis the titration method as described by day and underwood [23] was followed. 1 g of sample was weighed into 100 ml conical flask. 75 ml 3 mh2so4 was added and stirred for 1 h with a magnetic stirrer. this was filtered using a whatman no 1 filter paper. 25 ml of the filtrate was then taken and titrated while hot against 0.05 m kmno4 solution until a faint pink colour persisted for at least 30 s. the oxalate content was then calculated by taking 1 ml of 0.05 m kmno4 as equivalent to 2.2 mg oxalate [24]. phytate was extracted according to the procedure described by mohammed et al. [25]. 1.0 g sample was extracted with 3% tri-chloro acetic acid (tca) at 37°c for 45 min. with simple shaking followed by centrifugation and extractation by using anion exchange column. the extracted phytate (0.2 ml) was mixed with 4.6 ml of distilled water and 0.2 ml of chromogenic solution and the tubes were heated in a water bath at 95°c for 30 min, and then were allowed to cool. the developed color was read at 830 nm against blank. standard phytate solution was prepared by dissolving sodium phytate in distilled water to prepare different phytate concentrations as described above in the tested samples. the amount of phytate in the tested samples was expressed as mg phytate/100 g sample. extraction of organic acids the organic acids of each dried sample of mushroom were extracted according hasib et al. [26] method by grinding (waring blendor, polychimie, abidjan, côte d’ivoire) in distilled water (1:10, w/v) and clarified by centrifuging at 4000 rpm for 30 minutes. the supernatant was first filtered through whatmann 4 paper, then through 0.45 μm filter (millipore; sartorius ag, goëttingen, germany) and stored at −20˚c prior further use. hplc analysis of organic acids the separation of the organic acids was carried out as previously reported [22] by using a system consisting of an analytical hplc unit (shimadzu corporation, japan) in conjunction with a column heating device set at 35˚c with the aid of an oven meta therm tm (interchrom, france), with an ions exclusion column icsep ice orh-801 (40 cm × 5 μm, in terchom, france). the system was also coupled to a pump (shimadzu lc-6a liquid chromatograph), a uv detector (shimadzu spd-6a uv spectrophotometric detector) and an integrator (shimadzu chromatopac cr 6a). elution was carried out isocratically with sulphuric acid 0.04 n, at a solvent flow rate of de 0.6 ml/min and detection was performed at 210 nm. organic acids in mushroom extracts were identified by comparing the retention times and spectral data obtained from standards under the same conditions. quantitation was performed by comparing the peak areas with those of the respective external standards. statistical analysis all chemical analyses and assays were performed in triplicate, unless otherwise indicated. results were expressed as mean values ± standard deviation (sd). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 194 analysis of variance (anova) followed by duncan’s test was performed to test for differences between means by employing xlstat 2019 statistical software. significance of differences was defined at the 5% level (p<0.05). principal component analysis (pca) was also used in order to discover relationships between independent variables (mushroom species and organic acids). 3. results and discussion antinutritional factors the phytate and oxalate contents as a function of ripening stage of the three wild edible mushroom russula species are presented in table 1. these results showed that the measured antinutritional parameters decreased significantly during ripening of the different mushroom species. we observed the highest content of phytate (1.60±0.10 mg/100 g) in r. lepida, especially in immature fruiting bodies. gaur et al. [27] reported low values of phytate ranging from 0.11 to 0.19 mg/ 100 g for six selected edible mushrooms in india. however, the phytate contents of the present study were much lower than those found for some cultivated and wild edible mushrooms collected from ethiopia, ranged from 31.3 to 242.8 mg/ 100 g dw [28]. furthermore, the phytate contents of studied russula species are over13 times lower than the standard safe limit of 22.10 mg/ 100 g [29]. this suggest that whatever the ripening stage, the three studied mushroom species could be consumed without risk of toxicity associated with phytate concentration. as regards oxalate, the highest content (5.17±0.01 mg/ 100 g) was estimated in r. mustelina immature fruiting bodies. oxalate contents obtained in this study were higher than that found for the oyster mushroom pleurotus ostreatus (0.41 mg/ 100 g) [30], but quite lower than the tolerable limit of 105 mg/100 g recommended by world health organization. this suggests the safe for consumption of these mushrooms at any ripening stage. moreover, it’s noteworthy that food processes such as boiling or cooking, fermentation and milling could reduce drastically or remove antinutrient elements [31]. table 1. antinutritional factors content as a function of ripening stage of three russula species parameters stage species r. lepida r. mustelina r. delica phytate (mg/100g) i 0.60±0.20ᵃa 1.07±0.02ᵃb 1.60±0.10ᵇc m 0.40±0.30ᵃa 0.56±0.22ᵇa 1.46±0.25ᵃᵇb pm 0.23±0.23ᵃa 0.53±0.15ᵇa 1.20±0.10ᵃb oxalate (mg/100g) i 2.83±0.02ᶜa 5.17±0.01ᶜc 4.34±0.03ᶜb m 2.10±0.01ᵇa 4.13±0.02ᵇc 3.26±0.02ᵇb pm 0.77±0.02ᵃa 3.03±0.01ᵃb 3.08±0.01ᵃc values are mean ± standard deviation of three measurements (n = 3). i: immature; m: mature; pm: post-mature. a,b,cidentical script indicate no significant difference between mean values in line. a,b,cidentical script indicate no significant difference between mean values in column. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 195 organic acids composition organic acids are categorized as important food components for the formation of taste and flavour, and determination of the quality and safety of food and food products [32]. they generate important effects on food such as sensorial, antioxidant and acidifying properties [33]. the hplc-uv analysis (figure not shown) allowed the identification of at least fourteen organic acids namely benzoic, oxalic, fumaric, succinic, malic, tartaric, ascorbic, citric, lactic, adipic, propionic, formic, shikimic and acetic acids. most of these organic acids has already been found in edible mushrooms [34, 35]. results summarized in table 2 indicated that the three mushroom species were characterized by a high variation in their organic acid content. moreover, these organic acid contents were strictly dependent on ripening stage. for the three ripening stages, r. lepida showed the highest total organic acid contents (ranging from 103.31 to 113.13 mg/ 100 g dw), mainly consisting of lactic (49.70 mg/ 100 g dw, for post-mature stage), fumaric (36.00 and 31.22 mg/ 100 g dw, respectively for immature and mature stage), citric (21.60 and 31.60 mg/ 100 g dw, respectively for immature and mature stage) and succinic (21.00 mg/ 100 g dw for post-mature stage) acids. lactic acid is the product of glycolysis under anaerobic condition. in food, it’s characterized as a natural flavour enhancer, mild and lingering [36]. fumaric and citric acids are well known for their antimicrobial and antioxidant properties making them important agents in the prevention of mushroom browning [37]. r. mustelina presented total acid content of 100.02 mg/ 100 g dw at immature stage, with dominating lactic acid (47.90 mg/ 100 g dw), whereas r. delica had a total acid content of 83.33 mg/ 100 g dw at postmature stage. the major acids in r. delica were citric acid (30.20 mg/ 100 g dw at post-mature stage) followed by ascorbic acid (21.80 mg/ 100 g dw at post-mature stage). total organic acid contents found in this study were higher than those found for fustulina hepatica [38]. table 2. organic acids content as a function of ripening stage of three russula species organic acids retention time (min) stage species r. lepida r. mustelina r. delica butyric acid 2.53 i nd nd 0.06±0.01aa m nd 0.05±0.01aa nd pm nd nd nd benzoic acid 5.62 i 4.00±0.02bb nd 0.11±0.01aa m 3.63±0.03aa nd nd pm nd 1.26±0.02aa 3.84±0.01bb oxalic acid 9.66 i 0.62±0.03bc 0.04±0.01aa 0.61±0.01ba m 0.52±0.03bb 0.41±0.01ac 0.61±0.01ca pm 0.06±0.01aa 0.21±0.02bb 1.14±0.01cb fumaric acid 10.48 i 36.00±0.04bb 2.62±0.02ab nd m 31.20±0.02ca 2.14±0.01aa 6.47±0.02bb pm nd 3.10±0.02ac 3.14±0.01aa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 196 succinic acid 11.25 i 5.31±0.02bb 17.70±0.03ac nd m 4.58±0.02ba 0.76±0.02aa nd pm 21.00±0.03cc 1.88±0.3ab 8.16±0.01ba malic acid 12.95 i 3.27±0.01bb nd 0.50±0.01aa m 0.43±0.02aa 2.49±0.02ba 5.19±0.06cb pm nd 2.85±0.02ab 9.82±0.03bc tartaric acid 15.54 i 12.10±0.02bb 10.10±0.03cc 3.76±0.02aa m 11.40±0.03bb 1.29±0.02aa 6.96±0.01bb pm 9.27±0.02ba 9.45±0.01bb 13.10±0.01ac ascorbic acid 16.31 i 7.87±0.02bb 3.48±0.02ab nd m 8.43±0.02bc 0.48±0.35aa nd pm 4.33±0.02aa nd 21.80±0.05ba citric acid 17.7 i 29.60±0.03bb 5.90±0.01ac 30.20±0.01bc m 31.60±0.02cc 1.50±0.02aa 2.46±0.03bb pm 7.94±0.02ca 2.90±0.00bb 2.16±0.02aa lactic acid 20.75 i 6.03±0.03ba 47.90±0.01cc 0.46±0.02aa m 5.98±0.02ba 5.01±0.01aa 11.40±0.01cc pm 49.70±0.02cb 12.10±0.01bb 7.52±0.03ab adipic acid 22.42 i 0.79±0.02cc 0.52±0.03ba 0.02±0.00aa m 0.54±0.02aa 1.13±0.02bb 2.22±0.02cb pm 0.60±0.01ab 1.10±0.01bb nd propionic acid 24.15 i 3.39±0.02cb 1.53±0.03ba 0.94±0.02ab m 4.14±0.02cc 0.41±0.01ab 0.63±0.02ba pm 2.00±0.02ba 0.29±0.01ac 5.45±0.02cc formic acid 28.04 i 1.42±0.03bc 0.24±0.02aa 2.52±0.02cc m 0.99±0.02cb 0.21±0.01aa 0.59±0.02bb pm 0.26±0.02aa 0.51±0.02bb 0.52±0.02ba shikimic acid 34.22 i 2.73±0.02ba 7.24±0.02cb 0.02±0.00aa m 3.78±0.01cb 1.59±0.01aa 1.65±0.02bb pm 5.60±0.02bc 1.85±0.01aa 6.68±0.02cc acetic acid 36.46 i nd 2.75±0.01ac nd m nd 0.49±0.01aa 1.43±0.04ba pm 2.55±0.02ba 0.88±0.02ab nd total i 113.13±0.03 100.02±0.02 39.2±0.02 m 107.22±0.02 17.96±0.01 39.61±0.02 pm 103.31±0.02 38.38±0.03 83.33±0.02 values are mean ± standard deviation of three measurements (n = 3). i: immature; m: mature; pm: post-mature. a,b,cidentical script indicate no significant difference between mean values in line. a,b,cidentical script indicate no significant difference between mean values in column. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 197 the principal component analysis (pca) based on the organic acid contents obtained for the three mushroom species as a function of ripening stage is presented in figure 1. in terms of information, the principal components 1 and 2 contained more than 80 % (81.62) of the total variance explained with eigenvalues for the first two components greater than unity (respectively, 4.226 and 2.922) (table 3). this value of the total variability is relatively significant. also, the kaisermeyer-olkin (kmo) criterion is 0.761 (table 4) suggesting that sampling is acceptable for the validity of pca test in this study. table 3. eigen values of the main components f1 f2 eigenvalues 4.226 2.922 variability (%) 52.262 29.220 cumulated percentage 52.262 81.482 table 4. sampling accuracy rt 0.149 l1 0.770 l2 0.791 l3 0.802 m1 0.657 m2 0.656 m3 0.957 d1 0.855 d2 0.715 d3 0.769 kmo 0.761 the pca analysis (figure 1) makes it possible to distinguish the mushroom species in two large groups. one formed by r. delica and r. lepida and the other represented by r. mustelina. the first one better expresses the axis of the first component (52.41% of the total variance explained) and the second the axis of the second component (29.21% of the total variance explained). the first group is characterized by the relationship r. delica and r. lepida fumaric, citric, shikimic, malic, tartaric, ascorbic, succinic acids. for r. mustelina (which constitutes the second group), pca analysis revealed a close relation with lactic, succinic and tartaric acids. the ripening stages were used as additional variables for the pca analysis (figure 1). results suggest a difference in the influence of the ripening stages on the organic acid contents, contributing in different ways to the constitution the study axes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 198 fig. 1. pca analysis for organic acids content as a function of ripening stage of three russula species 4. conclusion based on the present study, it appears that russula species from côte d’ivoire contained a wide range of organic acids (fourteen). these organic acids are unevenly distributed (with widely varying proportions) according to the species and the ripening stages. r. lepida shows the higher total organic acid contents for all ripening stages mainly consisting of lactic, fumaric, citric and succinic. for r. delica, the major organic acids were citric and ascorbic acids found at post-mature stage, while lactic acid was the dominating acid at the immature stage in r. mustelina. the pca analysis suggests a relationship between r. delica and r. lepida which would forme a group, whereas r. mustelina constitutes another group. 5. references [1]. boa e., wild edible fungi.a global overview of their use and importance to people.non-wood forest products series no. 17. rome: fao, (2004). [2]. kalac p., chemical composition and nutritional value of european species of wild growing mushrooms nova science publishers, (2012). [3]. campbell b., ed. 1996. the miombo in transition: woodlands and welfare in africa. bogor, indonesia, centre for international forestry research. 266 pp, (1996). [4]. oei p., mushroom cultivation: appropriate technology for mushroom growers. mushroom cultivation, 3rd edition, leiden, the netherlands. pp.xii + 429 pp, (2003). [5]. egwim e.c., elem r.c., egwuche r.u., proximate composition, phytochemical screening and antioxidant activity of ten selected wild edible nigerian mushrooms. american journal of food and nutrition, 1(2): 89-94, (2011). [6]. okwulehie i.c., odunze e.i., evaluation of the myco-chemical and mineral composition of some tropical edible food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 199 mushrooms. journal of sustainable agriculture and environment, 6: 163 – 170, (2004). [7]. due e.a., koffi d.m., digbeu y.d., biochemical and functional properties of two wild edible mushrooms (volvariella volvacea and armillaria mellea) consumed as protein substitutes in south côte d’ivoire. journal of chemical, biological and physical sciences, 6(4): 1197 – 1206, (2016) [8]. due e.a., koffi d.m., digbeu y.d., physicochemical and functional properties of flour from the wild edible mushroom termitomyces heimii natarajan harvested in côte d’ivoire. turkish journal of agriculture food science and technology, 4(8): 651 – 655, (2016). [9]. chittaragi a., naika r., physico chemical and primary biochemical studies of hygrocybe cantharellus collected from western ghats region of haniya, shimoga (dist) karnataka. international journal of advances in pharmacy, biology and chemistry, 3(3): 523 – 528, (2014). [10]. garcía-lafuente a., moro c., villares a., guillamón e., rostagno m.a., d’arrigo m., martínez j.a., mushrooms as a source of anti-inflammatory agents. american journal of community psychology, 48: 125–141, (2011). [11]. schillaci d., arizza v., gargano m.l., venturella g., antibacterial activity of mediterranean oyster mushrooms, species of genus pleurotus (higher basidiomycetes). international journal of medicinal mushrooms, 15(6), 591 – 594, (2013). [12]. phan c., lee g., macreadie i., malek s., pamela d., sabaratnam, v., lipid constituents of the edible mushroom, pleurotus giganteus demonstrate anti-candida activity. natural product communications, 8(12): 1763–1765, (2013). [13]. ma k., han j., bao l., wei t., liu h. two sarcoviolins with antioxidative and α‐glucosidase inhibitory activity from the edible mushroom sarcodon leucopus collected in tibet. journal of natural products, 77(4): 942−947, (2014). [14]. amirullah n.a., abidin n.z., abdullah n., the potential applications of mushrooms against some facets of atherosclerosis: a review. food research international, 105: 517–536, (2018). [15]. muszyńska b., grzywaczkisielewska a., kała k., gdulaargasińska j., anti-inflammatory properties of edible mushrooms: a review. food chemistry, 243, 373–381, (2018). [16]. apetorgbor m.m., apetorgbor a.k., obodai m., indigenous knowledge and utilization of edible mushrooms in parts of southern ghana. ghana journal of forestry, 19 & 20: 19 – 34, (2006). [17]. valverde m.e., hernández-pérez t., paredes-lópez o., edible mushrooms: improving human health and promoting quality life. international journal of microbiology, 2015: 376 – 387, (2015). [18]. silva b.m., andrade p.b., valentão p., ferreres f., seabra r.m., ferreira m.a., quince (cydonia oblonga miller) fruit (pulp, peel, and seed) and jam: antioxidant activity. journal of agricultural and food chemistry, 52: 4705 – 4712, (2004). [19]. barros l., pereira c., ferreira i.c.f.r., optimized analysis of organic acids in edible mushrooms from portugal by ultrafast liquid chromatography and photodiode array detection. food analytical methods, 6(1): 309–316, (2013). [20]. vaughan j.g., geissler c.a., in the new oxford book of food plants; oxford university press: new york, p 196, (1997). [21]. koné n.a., yéo k., konaté k.s., linsenmair e., socio-economical aspects of the exploitation of termitomyces fruit bodies in central and southern côte d’ivoire: raising awareness for their sustainable use. journal of applied biosciences, 70: 5580 – 5590, (2013). [22]. kouassi k.a., kouadio e.j.p, djè m., dué a.e., kouamé l.p., edible ectomycorrhizal mushrooms russula spp. of côte d’ivoire: total phenolic content, hplc-profiles of phenolic compounds and organic acids, antioxidant activities. journal of agricultural chemistry and environment, 5: 73 – 84, (2016). [23]. day r.a., underwood a.l., quantitive analysis 5th ed. prentice. hall publication, p. 701, (1986). [24]. chinma c.e., igyor m.a., micronutrients and anti-nutritional contents of selected tropical vegetables grown in southeast, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jaures oscar gbotognon, michel djary koffi, hubert kouassi konan, eugène jean parfait kouadio, effect of ripening stage on organic acid profiles and antinutrient contents of three species of the wild edible mushroom russula ssp., food and environment safety, volume xviii, issue 3 – 2019, pag. 191 200 200 nigeria. nigerian food journal, 25(1): 111– 116, (2007). [25]. mohamed a., perera p.j., hafez y.s., new chromophore for phytic acid determination. cereal chemistry,63(6):475– 478, (1986). [26]. hasib a., jaouad a., mahrouz m., khouili m., hplc determination of organic acids in moroccan apricot. ciencia y tecnología alimentaria, 3, 207 – 211, (2002). [27]. gaur t., rao p.b., kushwaha k.p.s., nutritional and antinutritional components of some selected edible mushroom species. indian journal of natural products and resources, 7(2): 155 – 161, (2016). [28]. woldegiorgis a.z., abate d., haki g.d., ziegler g.r., major, minor and toxic minerals and anti-nutrients composition in edible mushrooms collected from ethiopia. journal of food processing and technology, 6: 430. doi:10.4172/21577110.1000430, (2015). [29]. goud m.j.p., suryam a., lakshmipathi a.v., charya s.m.a., extracellular hydrolytic enzyme profiles of certain south indian basidiomycetes. african journal of biotechnology, 8 (3), 354 – 360, (2009). [30]. duru m., eboagwu i., kalu w., odika p. nutritional, anti-nutritional and biochemical studies on the oyster mushroom, pleurotus ostreatus. ec nutrition 14.1: 36 – 59, (2019). [31]. sandberg a.s., anderine r., hplc method for determination of inositol tri, tetra, pentaand hexaphosphates in food and intestinal contents. journal of food science, 51: 547 – 50, (1986). [32]. yüksel a.k., yüksel m., şat i̇.g., determination of certain physicochemical characteristics and sensory properties of green tea powder (matcha) added ice creams and detection of their organic acid and mineral contents. gida 42(2): 116 – 126, doi: 10.15237/gida.gd16072, (2017). [33]. tormo m., izco j.m., alternative reversed phase high performance liquid chromatography method to analyse organic acids in dairy products. journal of chromatography a, 1033: 305 – 310, (2004). [34]. gąsecka m., magdziak z., siwulski m., mleczek m., profile of phenolic and organic acids, antioxidant properties and ergosterol content in cultivated and wild growing species of agaricus. european food research and technology, doi 10.1007/s00217-017-2952-9, (2017). [35]. magdziak z., siwulski m, mleczek m., characteristics of organic acid profiles in 16 species of wild growing edible mushrooms, journal of environmental science and health, part b, 52: 10, 784 – 789, (2017), doi: 10.1080/03601234.2017.1356676. [36]. jarrett t.n., acids in confections. foodgrade organic acids in confections offer ranges in sourness, intensity and linger; balance sweetness; and round out the taste profile; and perform other functions. the manufacturing confectioner, pp 58-63, (2012). [37]. ribeiro b., andrade p.b., baptista p., barros l., ferreira i.c.f.r., seabra r.m., valentão p., leucopaxillus giganteus mycelium: effect of nitrogen source on organic acids and alkaloids. journal of agricultural and food chemistry, 56: 4769-4774, (2008). [38]. ribeiro b., valentão p., baptista p., seabra r.m., andrade p.b., phenolic compounds, organic acids profiles and antioxidative properties of beefsteak fungus (fistulina hepatica). food chemistry and toxicology, 45: 805–1813, (2007). 1. introduction 4. conclusion 287 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 4 – 2019, pag. 287 297 some quality attributes of sausage roll produced from wheat-tigernut composite flour *emmanuel kehinde oke 1 , michael ayodele idowu 1 , nurat oluwadamilola thanni 1 ,omobolanle omowunmi olorode 2 1department of food science and technology, federal university of agriculture, abeokuta, ogun state, nigeria, kennyoke35@gmail.com 2department of food technology, moshoodabiola polytechnic, ojere, abeokuta, ogun state, nigeria. *corresponding author received 17th september 2019, accepted 27th december 2019 abstract: among the underutilized crops in nigeria is tigernut which could find useful application in baking industry because of its high level of dietary fibre and other inherent properties. this study was carried out to investigate the effects of wheat flour substitution with tigernut flour in sausage roll production. brown variety of tigernut was sorted and dried in a cabinet dryer at 60°c for 72hrs, processed into flour and blended with wheat flour at different ratios of 100:0; 90:10; 80:20; 70:30; 60:40; 50:50, 40:60, 30:70, 20:80, 10:90 respectively. sausage roll was baked from the flour blends and was analyzed for proximate, mineral composition, physical properties (oven spring, weight, volume, specific volume and colour) and sensory evaluation. moisture, crude protein, crude fibre, total ash, crude fat and carbohydrate ranged from 25.43 to 28.14%, 7.09 to7.79%, 1.87 to 2.29%, 1.70 to 1.80%, 8.59 to 9.25% and 51.14 to 55.20% respectively. significant differences (p<0.05) also exist in the mineral composition of the sausage roll. substitution of tigernut flour into wheat flour led to a decrease in oven spring, weight, volume and specific volume of the sausage roll. addition of tigernut flour up to 40% could be acceptable for sausage roll. however, the use of tiger nut flour in sausage roll production improved the protein, fat and mineral content (sodium, calcium and phosphorus) of sausage roll. the lightness, redness and yellowness of the sausage roll samples varied significantly among different levels of substitution. keywords: tigernut flour, wheat flour, sausage roll 1. introduction the problem of malnutrition in africa and how to control it is a major concern of food producers, consumers and processors alike. the situation demands an urgent solution which should be based on the use of locally available crops. exploring the underutilized crops, some of which has been reported to be rich in nutrient and high density with the purpose of enriching the existing food could be a viable means to tackling malnutriton. tigernut (cyperus esculentus l.) is an underused crop which belongs to the divisionmagnoliophyta,classliliopsida,orde r-cyperales and family-cyperaceae(family) and was found to be a cosmopolitan perennial crop of the same genus as the papyrus plant. the plant has several names; this include earth almond and yellow nut grass [1, 2]. in nigeria, three varieties of tigernut are cultivated, this include black, yellow and brown. among these, only the yellow and brown varieties are readily available in the market. it is also known as ‘aya’in hausa, ‘ofio’ in yoruba, ‘akiausa’in igbo.tigernut has been reported to be a rich sources of good quality oil [3, 4] and contain an average amount of protein [5]. it also serve as a source of some useful minerals such as http://www.fia.usv.ro/fiajournal mailto:kennyoke35@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 288 potassium, phosphorus and calcium [6] as well as vitamin e and c [2]. moreover, tigernut has been reported to have higher essential amino acids than those proposed in the protein standard by the fao / who [7] for satisfying adults needs [8]. it has been reported to contain high in dietary fibre content [9] which could be useful in treatment and prevention of certain diseases such as colon cancer [10], coronary heart disease[11], obesity, diabetes, gastrointestinal disoders [12] and losing weight [13]. tigernut seeds are cheap and readily available but grossly underutilized and need more attention because of its nutritional qualities such as high fibre content. recent application of tigernut has been concentrated on tigernut (brown and yellow variety) flour for bread making [14,15], biscuit [16]. however, there is little information on the use of tigernut as composite flour from wheat and brown variety of tigernut for the production of sausage roll. the nutritional content of wheat is low in fibre due to the various processes the whole wheat might have undergone. therefore, the inclusion of tigernut flour (brown variety) would serve as a source of fibre supplement for the production of sausage roll and this could increase its use in food applications with high level of acceptance. therefore, this study evaluated the proximate, mineral composition, physical properties and sensory quality of sausage roll produced from wheat and tigernut flour blends. 2. materials and methods 2.1 materials dry tigernut (brown variety) was locally purchased from kuto market abeokuta, ogun state. wheat flour and other essential ingredients like baking flour, sugar, nutmeg, salt, butter was purchased at kuto market in abeokuta, ogun state. 2.2 tigernut flour preparation the method described by ade-omowaye [14]was used in the preparation of brown variety of tigernut flour. dry tiger nuts (cyperusesculentus) were sorted manually to remove unwanted materials like stones, pebbles and other foreign seeds. the cleaned nuts were dried in a cabinet dryer at 60°c for 72hrs. dried nuts was milled using laboratory mill and the milled sample was sieved (using 250µm screen) to obtain the flour. the tigernut flour was packed and sealed in polyethylene bags at ambient temperature (26±20c) and 760mmhg until further analysis. 2.3 preparation of sausage roll the method described by kohajdova and karovicova[17] with little modification of recipe was used for the preparation of sausage roll. a straight dough process was used for the preparation of sausage roll, recipe such as butter (100g), sugar (1g), salt(1g), water (15ml), baking powder (2g) and tigernut flour was added in appropriate proportion to each of the flour blends and control flour. the substitution of tigernut flour into wheat flour was (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%) for making sausage roll dough. the blends were mixed with ingredients in a spiral mixer (for 5 minutes). water was added to the flour inside the spiral mixer and kneaded for 3 minutes. the dough was scaled and shaped. baking was done at a temperature of 220oc for 13 minutes. 2.4 proximate composition of sausage roll produced from wheat and tigernut flour proximate composition of the sausage roll (moisture content, crude protein, crude fat, total ash and crude fibre) prepared from wheat and tigernut flour blends was analyzed using the method described by [18]. carbohydrate contents of sausage roll samples were calculated by difference food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 289 2.5 mineral composition of sausage roll produced from wheat and tigernut flour the mineral content of the sausage roll was analyzed for potassium (k), calcium (ca), and sodium (na) using the atomic absorption spectrophotometry (aas), after being subjected to dry ashing method. dry ashing was carried out by weighing 1gram of sample into a porcelain crucible, preashed at a temperature of 300ºc and later brought to 600ºc. ashing was done for 2hours in a furnace and then left to cool. 25ml of 3m hcl will be added. the mixture was filtered into a 100ml volumetric flask and diluted to a volume with deionized water. the sample was vortexed for proper mixing, after which the sample was transferred to centrifuge tubes and centrifuged at 3000rpm for 10minutes. the supernatant was decanted into a clean vial for micro and macro determination using atomic absorption spectrophotometry 2.6 physical properties of sausage roll produced from wheat and tigernut flour 2.6.1 determination of oven spring oven spring was determined from the differenced in the height of dough just before and after baking using the method described by idowu et al. [19]. 2.6.2 determination of weight of sausage roll the sausage roll weight was determined using the modified method of shittu et al. [20] the weights of the sausage roll was determined with the aid of weighing balance after cooling and the weight values was recorded for each sample. 2.6.3 determination of volume and specific volume of sausage roll sausage roll volume was measured using the modified method described feili et al. [21], sausage roll volume was determined using sorghum seed displacement method. the sorghum seeds were put into a container to measure the volume and it was measured in a graduate cylinder and mark as v1. thereafter, the sample was placed in the same container and seeds were poured till the test sausage roll is covered. again the sorghum seeds was been measured in another graduated cylinder and mark as v2. the volume of sample was then calculated based on the following equation. sausage volume (ml) = 𝑉1 − 𝑉2 (1) where v1 represents the volume of the sorghum seeds in the empty container (ml), v2 represent volume of the sorghum seeds in the container containing sample (ml). the specific volume was also calculated using the method described by feili et al. [21] as shown in the following equation below: specific volume (cm3/g) = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑆𝑎𝑢𝑠𝑎𝑔𝑒 𝑟𝑜𝑙𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑆𝑎𝑢𝑠𝑎𝑔𝑒 𝑟𝑜𝑙𝑙 2.6.4 determination of density of sausage roll the sausage roll density was determined using method described by feili et al. [21]. the sausage roll density was calculated by dividing the weight of sausage roll obtained by the volume of sausage roll density (g/cm3) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑆𝑎𝑢𝑠𝑎𝑔𝑒 𝑟𝑜𝑙𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑆𝑎𝑢𝑠𝑎𝑔𝑒 𝑟𝑜𝑙𝑙 2.6.5 crumb and crust colour of sausage roll crust and crumb color measurement was measured by the method described by feili et al. [21], minolta chroma meter (cr-410 japan) was used based on (cie) l*a*b* scale. after calibrating the instrument by covering a zero calibration mask followed by white calibration plate, crust and crumb was analyzed by placing them on the petri dish and the image was captured on the samples. the color attributed such as lightness (l*) and (0 = black and 100 = food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 290 white) and chromatically coordinated (a* corresponds to the color range from redgreen coordinates (is given, while + is red)), (b* corresponds to the colour range from blue – yellow coordinates (is blue with + indicating yellowness) was recorded. 2.7.sensory evaluation of sausage roll produced from wheat and tigernut flour the method described by iwe [22] was used. the sensory panel consisted of 50 members who were familiar with the product and were asked to score the bread using a 9-point hedonic scale based on their degree of likeness where 9 = like extremely; 5 = neither like nor dislike; 1 = dislike extremely. sensory quality attributes evaluated were: colour, aroma, taste, texture, appearance and overall acceptability. 2.8 statistical analyses data obtained were subjected to statistical analysis. means, analysis of variance (anova) were determined using spss version 21.0 and the differences between the mean values were evaluated at p<0.05 using duncan’s multiple range test. 3 results and discussion 3.1 proximate composition of sausage roll produced from wheat and tigernut flour the proximate composition of sausage roll produced from wheat and tigernut flour blend is presented in table 1. significant differences (p<0.05) exist in the moisture content of the sausage roll. the moisture content of sausage roll produced wheat and tigernut flour blend from was high and ranged from 25.43 to 28.14%. sausage roll produced from 100% wheat flour had the highest moisture content while sausage roll produced from wheat flour substituted with tigernut at 70% had the lowest moisture content. sanni et al. [23] reported that the lower the moisture content of a product to be stored, the better the shelf stability of such product. high moisture content has been associated with short life as they encourage microbial proliferation that leads to spoilage [24]. the moisture content of wheat-tigernut composite sausage roll was in agreement with the findings of oke et al. [15]. the crude protein content of the sausage roll ranged from 7.09 to 7.79%. sausage roll produced from 100% wheat flour had the highest moisture content while sausage roll produced from wheat flour substituted with tigernut at 70% had the lowest moisture content. the protein content of the wheat-tigernut composite sausage roll observed in this study was moderate; this could be attributed to the level of protein present in wheat and tigernut flour. significant differences (p<0.05) exist in the crude fibre content of wheat-tigernut composite sausage. the crude fibre ranged between 1.87 and 2.29% with sausage roll produced from 100% wheat flour having the lowest crude fibre content while sausage roll produced from wheat flour substituted with tigernut at 90% had the highest crude fibre content. according to ekwe et al. [25] and chugh et al. [26] reported that consumption of food rich in dietary fiber reduces the risk of diabetes mellitus, cardiovascular diseases, constipation, appendicitis, hemorrhoids and colon cancer. the ash content is also a rough estimate of the mineral contents of the samples [27]. the ash content of the sausage ranged from 1.70 to 1.80%. the result shows that sausage produced 100% wheat flour and sausage produced from wheat flour substituted with tigernut at 40% and 80% had the highest ash content when compared with other samples as showed statistically. this suggests that sausage produced from 100% wheat flour and wheat-tigernut flour at 40 and 80% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 291 will be rich in mineral. the crude fat ranged from 8.59 to 9.26%. the high crude fat content obtained in this study could be attributed to the high crude fat content of the tigernut as reported by bamishaiye and bamishaiye [28]and other baking ingredient that was added such as margarine. peter-ikechukwu et al. [29] reported that high level of fat are undesirable in food products because they could lead to rancidity in foods. significant differences (p<0.05) exist in the carbohydrate content of the sausage produced from wheat and tigernut flour. sausage produced from 100% wheat flour and wheat-tigernut flour at 40 and 80% had the lowest carbohydrate content while sausage produced from wheat flour substituted with tigernut at 70% had the highest carbohydrate. the carbohydrate content of the sausage samples substituted with wheat and tigernut flour blends were higher than the sausage produced from 100% wheat flour sample. this may be as a result of the substitution which led to the reduction or changes in the carbohydrate content of the blended sample. table 1 proximate composition of sausage roll produced from wheat and tigernut flour blend (%) wf:tf moisture content crude protein crude fibre total ash crude fat carbohydrate 100:0 28.14±0.39f 7.79±0.11f 1.87±0.01e 1.80±0.00a 9.26±0.12f 51.14±0.62a 90:10 26.79±0.47cd 7.44±0.12cd 2.01±0.01bc 1.70±0.00b 8.84±0.15cd 53.22±0.75cd 80:20 27.09±0.37cde 7.51±0.10cde 2.06±0.01cd 1.75±0.07bc 8.93±0.11cde 52.66±0.66bcd 70:30 25.98±0.18ab 7.23±0.05ab 2.12±0.00ab 1.70±0.00ab 8.59±0.06ab 54.38±0.29ef 60:40 27.49±0.18ab 7.62±0.01def 2.15±0.00cde 1.80±0.00c 9.06±0.02def 51.88±0.09abc 50:50 26.51±0.33bc 7.36±0.08bc 2.24±0.01bc 1.70±0.00ab 8.75±0.10bc 53.44±0.52ef 40:60 26.00±0.15ab 7.24±0.04ab 2.14±0.00ab 1.70±0.00ab 8.60±0.05ab 54.32±0.23ef 30:70 25.43±0.42a 7.09±0.11a 2.21±0.01a 1.65±0.07a 8.42±0.13a 55.20±0.76f 20:80 27.69±0.29ef 7.67±0.07ef 2.25±0.01de 1.80±0.00c 9.12±0.09ef 51.47±0.46ab 10:90 26.90±0.23cd 7.47±0.06cd 2.29±0.01bc 1.70±0.00ab 8.87±0.07cd 52.77±0.37cd mean values with different superscripts within the same column are significantly different (p <0.05) wf: wheat flour, tf: tigernut flour 3.2 mineral composition of sausage roll produced from wheat and tigernut flour the mineral composition of sausage produced from wheat and tigernut flour blend is presented in table 2. significant differences (p<0.05) exist in the mineral composition of the sausage. the sodium and calcium content of the sausage ranges from 102.9 to 552.8mg/100g and 17.06 to 24.26mg/100g respectively. sausage produced from wheat flour substituted with tigernut at food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 292 10% had the highest sodium and calcium content while sausage produced from wheat flour substituted with tigernut at 40% had the lowest sodium and calcium content. sodium maintains proper acidbase balance, assists with transmission of nerve signals and absorption of glucose/other nutrients and aids muscle contraction [30]. calcium is necessary for supporting bone formation and growth, muscle contraction and maintenance of healthy blood pressure [30]. the potassium content of the sausage ranges from 76.15 to 375.3mg/100g. sausage produced from 100% wheat flour had the lowest potassium content while sausage produced from wheat flour substituted with tigernut at 80% had the highest potassium content. potassium is important in the regulation of heartbeat, neurotransmission and water balance of the body. table 2 mineral composition of sausage roll produced from wheat and tigernut flour blend wf:tf na (mg/100g) ca (mg/100g) k(mg/100g) 100:0 273.5±0.01e 22.59±0.01i 76.15±0.07a 90:10 552.8±0.01i 24.26±0.01j 138.1±0.01b 80:20 412.8±0.01h 17.45±0.00b 140.2±0.01c 70:30 102.9±0.01a 21.38±0.03g 173.1±0.01d 60:40 102.9±0.01a 17.06±0.01a 242.7±0.14e 50:50 350.7±0.01g 18.86±0.01d 244.0±0.01f 40:60 288.0±0.01f 21.59±0.01h 263.6±0.00g 30:70 103.7±0.01b 19.26±0.01e 261.2±0.00h 20:80 198.2±0.01d 21.26±0.01f 375.3±0.01i 10:90 194.4±0.00c 17.72±0.01c 363.2±0.00j mean values with different superscripts within the same column are significantly different (p <0.05); wf: wheat flour, tf: tigernut flour, na: sodium, ca: calcium, k: potassium 3.3 physical properties of sausage roll produced from wheat and tigernut flour the physical properties of sausage produced from wheat and tigernut flour blend is presented in table 3. oven spring is an important baking characteristic which measure the height of the dough before baking and height of the dough after baking. the oven spring of sausage produced from wheat and tigernut flour blend ranges from 0.03 to 0.16cm. sausage produced from 100% wheat flour had the highest ovenspring while sausage produced from wheat flour substituted with tigernut at 90% had the lowest ovenspring. the oven spring showed no definite trend but varied significantly (p<0.05) from one another. weight is basically determined by the quantity of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 293 dough baked and the amount of moisture and carbon dioxide diffused out of during baking [20]. the weight of the sausage ranged between 17.00 and 27.50g. sausage produced from 100% wheat flour had the highest weight while sausage produced from wheat flour wheat flour substituted with tigernut at 90% had the lowest weight. it was observed that weight of the sausage decreased as wheat and tigernut flour was added; this can be attributed to lower level of gluten network in the dough and consequently decreased ability for the dough to rise [31, 15]. volume is an important quality characteristic of a baked product. significant differences (p<0.05) exist in the volume of sausage produced from wheat-tigernut composite flour. the volume of the sausage ranges from 45.00 to 175.5cm3. the volume of the control sausage sample and wheat flour substituted with tigernut at 10% was higherthan that of samples that is incorporated with tigernut flour. this effect is probably related to the decreased visco-elasticity of dough resulting from tigernut flour addition. as the level of tigernut flour substitution increases, the volume of the sausage gradually decreased. the decrease in volume could be attributed to poor gas retention which was an indication of the low gluten content of that has weakened the flour due to the addition of tigernut flour. other researchers such as chen et al. [32]; doxastakis et al. [33]; gomez et al. [34] and borchani et al. [35] also observed decreasing volume of different bakery products such as bread, cookies and muffins. table 3 physical properties of sausage roll produced from wheat and tigernut flour blend wf:tf oven spring (cm) weight (g) volume (cm 3 ) specific volume (cm 3 /g) density (g/cm 3 ) 100:0 0.16±0.01 d 27.50±0.71 d 175.0±7.07 g 6.36±0.10 e 0.16±0.00 a 90:10 0.11±0.01 c 27.00±1.41 d 175.0±7.07 g 6.49±0.08 e 0.16±0.01 a 80:20 0.06±0.01 d 27.00±1.41 d 155.0±7.07 f 5.74±0.04 d 0.18±0.01 ab 70:30 0.06±0.01 d 26.00±1.41 d 145.0±7.07 ef 5.58±0.03 d 0.18±0.00 ab 60:40 0.11±0.01 c 25.50±0.71 d 130.0±14.1d e 5.09±0.41 c 0.20±0.01 bc 50:50 0.05±0.01 ab 22.50±0.71 c 115.0±7.07 d 5.11±0.16 c 0.20±0.01 bc 40:60 0.11±0.01 c 20.50±0.71 bc 95.00±7.07 c 4.63±0.18 c 0.22±0.01 c 30:70 0.05±0.01 cb 19.50±0.71 b 75.00±7.07 b 3.80±0.28 b 0.26±0.01 d 20:80 0.05±0.01 ab 18.50±0.71 ab 65.00±7.07 b 3.50±0.25 b 0.29±0.02 d 10:90 0.03±0.01 a 17.00±1.41 a 45.00±7.07 a 2.84±0.20 a 0.38±0.03 e mean values with different superscripts within the same column are significantly different (p <0.05); wf: wheat flour, tf: tigernut flour 3.4 colour attribute of sausage roll produced from wheat and tigernut flour the colour attribute of sausage roll produced from wheat and tigernut flour blend is shown in table 4. colour is an important attribute because it can arouse individual’s appetite. it is one of the parameters used for process control during baking and roasting, because brown pigments appear as browning and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 294 caramelization reactions progress [36]. the colour results in terms of lightness (l*), redness (a*), yellowness (b*). significant (p<0.05) differences existed in lightness (l*), redness (a*) and yellowness (b*) of sausage produced from wheat and tigernut flour blend. lightness, redness and yellowness ranges from 49.14 to 60.39, 3.47 to 6.84 and 12.68 to 22.08 respectively. the colour of the sausage changed to brown as wheat flour is diluted with tigernut flour. this might have been attributed to an increased maillard reaction taking place during baking due to higher lysine content [37]. in the maillard reaction reducing carbohydrates react with free amino acid side chain of protein mainly lysine and lead to amino acid– sugar reaction products (polymerized protein and brown pigments). table 4 colour attribute of sausage roll produced from wheat and tigernut flour blend wf:tf l* a* b* 100:0 60.39±0.13g 3.47±0.00a 18.92±0.29d 90:10 55.26±0.41e 4.76±0.01d 18.71±0.08d 80:20 56.88±0.44f 6.84±0.00f 22.08±0.02e 70:30 52.80±0.82cd 4.36±0.13c 15.21±0.37c 60:40 50.66±0.66b 4.46±0.13c 12.68±0.07a 50:50 51.81±0.50c 6.18±0.18f 15.24±0.22c 40:60 53.21±0.32d 5.33±0.10e 12.84±0.07a 30:70 49.14±0.47a 3.80±0.10b 12.43±0.28a 20:80 53.17±0.29d 3.92±0.00b 15.10±0.07bc 10:90 53.33±0.21d 4.27±0.13c 14.59±0.44b mean values with different superscripts within the same column are significantly different (p <0.05); wf: wheat flour, tf: tigernut flour, l*: lightness, a*: redness, b*: yellowness 3.5 sensory evaluation of sausage roll produced from wheat and tigernut flour the sensory score of sausage roll produced from wheat and tigernut flour blend is presented in table 5. there were significant differences (p<0.05) in all the attributes measured. the colour, aroma, taste, texture, appearance and overall acceptability ranged from 4.07 to 7.83, 4.17 to 7.70, 4.37 to 7.47, 4.03 to 7.57, 3.90 to 7.63 and 4.10 to 7.70 respectively. sausage roll produced from 100% wheat flour had the highest value of likeness for colour, aroma, taste, texture, appearance and overall acceptability while sausage roll produced from wheat flour substituted with tigernut at 90% had the lowest value of likeness for colour, aroma, taste, texture, appearance and overall acceptability. it is observed that up to 40% wheat replacement was accepted (6.47). so addition of tigernut flour up to 40% could be acceptable for sausage roll. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 295 it was observed from the sensory score, preference for wheat-tigernut composite sausage decreased with the amount of tigernut flour substitution in the sausage roll and this is due to the familiarity of the consumers to the 100% wheat flour sausage roll. table 5 sensory score for roll produced from wheat and tigernut flour blend wf:tf colour aroma taste texture appearance overall acceptability 100:0 7.83±1.02f 7.70±0.84a 7.47±1.00fg 7.57±1.25f 7.63±0.81e 7.70±0.84g 90:10 7.33±1.06ef 7.40±0.89fg 7.43±0.70fg 7.10±1.21ef 7.63±0.81e 7.33±0.84fg 80:20 7.60±1.00f 7.67±0.76f 7.67±0.96g 7.27±1.41ef 7.47±0.97e 7.80±1.03g 70:30 7.00±0.83de 6.87±0.82ef 6.87±0.73ef 6.63±0.85e 6.80±0.72d 6.90±0.80ef 60:40 6.63±0.93cd 6.60±0.77de 6.67±0.84e 5.90±1.16d 6.37±0.96d 6.47±0.82de 50:50 6.13±0.90c 6.00±1.23cd 5.90±1.16d 5.77±1.13d 5.77±1.00c 5.97±1.10cd 40:60 5.47±0.82b 5.83±1.31c 5.60±1.42cd 5.37±1.30cd 5.40±1.07bc 5.57±1.10bc 30:70 5.17±1.23b 5.10±1.42b 5.03±1.25bc 4.73±1.23bc 4.93±1.17b 5.03±1.24b 20:80 4.40±1.40a 4.46±1.67a 4.60±1.57ab 4.23±1.59ab 4.33±1.45a 4.33±1.40a 10:90 4.07±1.55a 4.17±1.64a 4.37±1.67a 4.03±1.50a 3.90±1.42a 4.10±1.58a mean values with different superscripts within the same column are significantly different (p <0.05); wf: wheat flour, tf: tigernut flour 4 conclusion this study shows that sausage roll can be produced from wheat and tigernut flour blends.the use of tigernut flour in sausage roll production has the advantage of improving the protein, fat and mineral content (sodium, calcium and phosphorus) of sausage roll. incorporation of tigernut flour led to reduction of sausage roll weight, volume and specific volume. the lightness, redness and yellowness of the sausage roll samples varied significantly among different levels of substitution. tigernut flour could be incorporated up to 40% for the production of sausage roll without affecting the overall acceptability 5 references [1]. odoemelan, s.a.chemical composition and functional properties of conophor nut flour (tetracarpidium conophorum) flour.international journal of food science and technology, 38:729-734, (2003) [2]. belewu, m.a., belewu, k.y. comparative physiochemical evaluation of tigernut, soybean and coconut milk sources. international journal of agriculture and biology, 9: 785-787, (2007) [3]. yeboah, s. o., mitei, y. c., ngila, j. c., wessjohann, l., schmidt, j. compositional and structural studies of the oils from two edible seeds: tiger nut, cyperusesculentum, and asiato, pachira insignis, from ghana. food research international, 47: 259–266, (2012) [4]. dubois, v., breton, s. linder, m., fanni, j., partmentier, m. fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. european journal of lipid science technology, 109: 720 – 73, (2007) [5]. oladele, a.k., aina, j.o. chemical composition and functional properties of flour produced from two varieties of tigernut (cyperusesculentus).african journal of biotechnology, 6:2473-2476, (2007) [6]. bizquert-jimennez, m.horchata and health, healthy properties and in deenfermedades digestive prevention. valencian studies foundation, ministry of agriculture, fisheries and food, (2003) [7]. fao (food and agriculture organization) world health organization. expert consultation energy and protein requirement. geneva, switzerland: world health organization, (1985) [8]. bosch l., alegeria, a., farre, r. rphplc determination of tigernut and orgreat food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 296 amino acid contents. food science technology international, 11: 33-40, (2005). [9]. joy-toran, a., farre-rovira, r. horchata and health, issues nutritional and dietetics.valencia: valencia foundation of advanced studies, department of agriculture, fisheries and food, (2003) [10]. adejuyitan, j.a., otunola, e.t., akande, e.a., bolariwa, i.f.,oladokun, f.m. (2009). some physicochemical properties of flour obtained from fermentation of tigernut (cyperusesculentus) sourced from a market in ogbomoso, nigeria african journal of food science, 3:51 – 55, (2009) [11]. chukwuma, e.r., obioma, n., christopher, o.i. the phytochemical composition and some biochemical effects of nigerian tigernut (cyperusesculentus l. tuber, pakistan journal of nutrition, 9 (7): 709 – 715, (2010). [12]. anderson, j.w., smith, b.m., gustafson, n.j. health benefits and practicalaspects of high fibre diets. american journal of clinical nutrition,59: 1242-1247, (1994) [13]. borges, o. gonclaves, b., sgeoeiro, l., correia, p., silva, a.nutritional quality of chestnut cultivars from portugal. food chemistry, 106: 976 – 984, (2008). [14]. ade-omowaye, b.i.o.,akinwande, b.a. bolarinwa, i.f., adebiyi, a.o. evaluation of tigernut (cyperusesculentus) – wheat composite flour and bread. african journal of food science, 2: 87 – 89, (2008) [15]. oke, e.k., idowu, m.a., sobukola o.p., bakare, h.a. quality attributes and storage stability of bread from wheat–tigernut composite flour. journal of culinary science and technology, 1-14, (2017) [16]. zahra, s.a., ahmed, m.s.h. exploring the suitability of incorporating tiger nut flour as novel ingredient in gluten-free biscuit. polish journal of food nutrition and science,64(1): 27-33, (2014) [17]. kohajdová, z., karovičová, j. influence of hydrocolloids on quality of bakedgoods. acta scientiarium polonorumtechnology. aliment, 7 (2): 43-49, (2008) [18]. aoac.official methods of analysis (17thed.) washington, dc: the association of official analytical chemists, (2000) [19]. idowu, m. a., oni, a., amusa, b. m. bread and biscuit making potentials of somenigerian cocoyam cultivars. nigeria food journal, 14: 1–12, (1996). [20]. shittu t.a., raji, a.o., sanni, l.o. 2006.bread from composite cassavawheatflour: effect of baking timeand temperature on some physical properties of breadloaf: food research international,40: 280290, (2006) [21]. feili, r. wahidu, z., wan, n., wan a., yang. (2013). physical and sensory analysis of high fiber bread incorporated with jackfruit rind flour. food science and technology,12: 30 – 36, (2013) [22]. iwe, m.o. handbook of sensory methods and analysis. enugu, nigeria: rojointcommunication publishers ltd, (2002) [23]. sanni, o.l., adebowale, a.a., filani, t.a., oyewole, o.b., westby, a. quality of flash and rotary dryer dried fufu flour. journal of food agriculture and environment, 4: 74-78, (2006) [24]. ezeama, c.f. food microbiology: fundamentals and applications. natural prints ltd. lagos, (2007) [25]. ekwe, k., nwosu, k., ekwe, c., nwachukwu, l. examining the underexploited values of cocoyam (colocasiaand xanthosoma spp.)for enhanced household food security, nutrition and economy in nigeria. in: jaenicke, h.; ganry, j.; zeledon, a.; hoeschle, i. and kahare, r. (eds.). proceedings of the international symposium on underutilized plants for food security, income and sustainable development.acta horticulture, 86: 71-78, (2009) [26]. chugh, b., singh, g., kumbhar, b. k. development of low-fat soft dough biscuits using carbohydrate-based fat replacers. international journal of food science, article id 576153, 12 pages, (2013) [27]. mepba, h. d., achinewhu, s. n.,aso, s. n.microbiological quality of selected street foods in port harcourt, nigeria. journal of food safety, 27: 208-218, (2007) [28]. bamishaiye, e. i., bamishaiye, o. m. tigernut: as a plant, its derivatives and benefits. african journal of food, agriculture, nutrition and development, 11: 5157–5170, (2011) [29]. peter-ikechukwu, a., osuji, c.m., ihediohamma, n.c., okafor, d.c. &chukwu, m. n. proximate composition and organoleptic characteristics of sausage rolls made from cocoyam and wheat flour enriched food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 emmanuel kehinde oke, michael ayodele idowu, nurat oluwadamilola thanni, omobolanle omowunmi olorode, some quality attributes of sausage roll produced from wheat tigernut composite flour, food and environment safety, volume xviii, issue 4 – 2019, pag. 287 – 297 297 with soybean flour. research journal of food science and nutrition,4(1): 1-11, (2019) [30]. thompson, j. and manore, m. nutrition: an applied approach. pearson education, inc., san francisco, (2005) [31]. aluko r.e., olugbemi l.b. sorghum as a raw material in the baking industry. paper presented at the symposium on the current status and potential of industrial uses of sorghum in nigeria, kano, and 4-6 december, (1989) [32]. chen h., rubenthaler g.l., leung h.k., baranowski j.d.chemical, physical and baking properties of apple fibre compared with wheat and oat bran. cereal chemistry, 65 (3): 244-247, (1988) [33]. doxastakis, g., zafiriadis, i.,irakli, m., tananaki, c. lupin, soya and triticale addition to wheat flour doughs and their effect on rheological properties. food chemistry,77: 219-227, (2002) [34]. gómez m., jiménez s., ruiz e. &oliete b. effect of extruded wheat bran on dough rheology and bread quality. lwt – food science and technology, 44 (10): 2231-2237, (2011) [35]. borchani, c.h., masmoudi, m. besbes, s., attia, h., seroanne, c., blecker, c.h. effect of date flesh fibre concentrate addition on dough performance and bread quality. journal of textural studies, 42 (4): 300-308, (2011) [36]. pereira, d., correia, p.m., guine, r. p. analysis of the physical-chemical and sensorial properties of maria type cookies. acta chimica slovaca, 6: 269–280, (2013) [37]. abdelrahman ragab abdelrahman ahmed.technological and nutritional studies on sweet lupine seeds and its applicability in selected bakery products. ph.d thesis submitted to institute of food technology and food chemistry, technical university of berlin, (2012) 1 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 1 6 the us e of pla nt ma t erials in cu lina ry product s ou t of y e as ted dou gh *oksana arpul1, tetiana sylchuk1, vira kulinich1, olena usatiuk1 1molecular and avantgarde gastronomy, national university of food technologies, ukraine kseniya_arp@mail.ru *corresponding author received 6th january 2015, accepted 16th march 2015 abstract: the article deals with the technology of garlic donuts (pampushkas) enriched with schavnat (hybrid of spinach dock or patience dock and tien shan sorrel) as a source of plant protein. the influence of schavnat on the technological process and the quality of pampushkas was determined. the organoleptic properties and the physical and chemical properties such as titrated acidity, texture, specific volume, dough fermentation property and viscoplastic properties were investigated.we obtained a «special pampushkas with garlic» with a high content in plant proteins which could be recommended for balancing the nutrient composition of population diet. keywords: schavnat, schavnat powder, yeasted dough, garlic donut, pampushka, protein, culinary products. 1. introduction around the world millions of people don’t get enough protein. protein and amino acid deficiencies negatively affect the state of health, human performance and longevity. the ways of protein deficiency recovery are the search of new plant sources of food protein and the development of methods to use them for fortification of massconsumption products. bakery products, unlike other foods, are the daily products, so it is possible to influence on the diet and human health by regulation of their chemical composition [1]. among a large number of protein raw materials schavnat as a culture of new generation deserves attention. schavnat is the species hybrid of spinach dock or patience dock and tien shan sorrel. this new long-term culture was selected in the department of new cultures of m. m. gryshko national botanic garden (ukraine). it contains a large amount of essential nutrients such as ascorbic acid, beta-carotene, minerals, plant protein etc. schavnat is first among the vegetable plants for the content of protein and vitamins in the early phases. this culture is suitable for complex use as food, feed, energy, medicinal and plant for technical use. schavnat as early spring culture is characterized by high ecological plasticity, winter and frost hardiness, productivity, food, feed, technical and medicinal properties in different climatic and geographical zones. its high food and feed properties are noted in the periods of growth, shooting and budformation. during this period the overground mass (12…18 % on a dry matter) contains about 30...40 % of protein, 700 mg% of ascorbic acid and 60 mg% of carotene. the content of nitrogenhttp://www.fia.usv.ro/fiajournal mailto:kseniya_arp@mail.ru food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 1 – 2015, pag. 1 6 2 free extractive substances is between 35 and 55 %, among them about 6...20 % of sugars. the number of lipids in the green mass is negligible (about 2.8...6.2 %). the use of such plant material as schavnat in the technologies of culinary products out of yeast dough is timely and topical. 2. materials and methods 2.1 materials in the laboratory of the department of molecular and avantgarde gastronomy of national university of food technologies (ukraine) the culinary products out of yeast dough such as garlic donuts (pampushkas) were baked. dry schavnat was added in the amount of 0.5…1.0 and 1.5 % of the flour weight for fortification of the products by plant protein. the moisture content of the yeasted dough was 42 %. the fermentation time of dough was 60 min at the temperature of proofer about 30 ± 2 °с. pampushkas were baked at the temperature of 210 to 220 ° c with sprinkling of the baking oven. the weight of dough piece was about 30±1 g each. 2.2 methods the influence of schavnat on the indicators of the technological process and the quality of finished products was investigated. after baking the organoleptic properties and the physical and chemical properties such as titrated acidity, texture, specific volume and moisture content of the finished culinary products were determined. the protein content of schavnat and the culinary products was determined by the modified kjeldahl method. modification of the method was in determination of the nitrogen content directly in the solution of the ashing sample (without pre-distillation) with using indirect hypochlorite and iodometric titration. the mineralizing of the sample was made by the kjeldahl method with the mixture of potassium sulphate and copper (ii) sulphate. peroxide of hydrogen was used for catalyzation the ashing. the titrated acidity of semi-finished product (dough) was determined by the titration method. the viscoplastic properties of dough were investigated by the degree of dough running and the dynamic of specific volume of dough during fermentation. the dough running was determined by the method based on the changes in the diameter of the dough balls with the weigh of 100 g during fermentation at the temperature of 30 ° c for 180 min. the dough portion with the weigh of 50 g was put in the graduated cylinder with a capacity of 250 cm3 and then was put in the thermostat with the temperature of 30 ° c for determination of the dynamic of specific volume. the start value of the specific volume and the changes during fermentation were fixed [2, 3]. 2.3 technology of pampushkas enriched by schavnat the technology of «special pampushkas with garlic» is presented in fig. 1. 3. results and discussion the research of the granulometric composition of dry schavnat was shown that the optimal size is 0.4…0.5 mm for the good organoleptic indicators and minimal protein loss (fig. 1). 23,3 % 34,3 % 30,6 % fraction – 0.7…1.0 mm fraction – 0.6 mm fraction – 0.4…0.5mm fig. 1. dependence of protein content from fractional composition of schavnat powder food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 1 – 2015, pag. 1 6 3 fig. 2. the technological scheme of «special pampushkas with garlic» vegetable oil garlic salt water peeling pulping heating t=100…110 °c cooling t=18…20°c addition grinding gradual blending mixing «garlic sauce» baking to golden crust τ =15…20 min, t=210…220 °c addition of sauce sales pampushky with garlic water salt sugar «lviv» pressed yeast schavnat powder (w=12.4%) wheat flour vegetable oil heating t=35…40°c dilution dilution dilution bolting moistening τ=5…7 min addition mixing of dough to homogeneous consistency τ=7…8 min fermentation τ=60min, t=35…40°c dough piece forming, weight =30±1 g fermentation of dough piece τ=15…20 min, t=35…40 °c yeast dough food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 1 – 2015, pag. 1 6 4 the protein content is directly proportional to the particle size of fractions. thus, the fraction of schavnat with the particle size of 0.4...0.5 mm contains the most amount of protein (34.3%), and the fraction with the largest particle size of 0.7...1.0 mm contains only 23.3% of protein. the schavnat fraction with the smallest particle size should be used for enrichment of pampushkas by protein. the protein content was determined in the finished products with the addition of 0.5%, 1.0% and 1.5% of schavnat powder (fit. 3). the protein content of pampushkas is directly proportional to the additive of schavnat powder. 0,00 2,00 4,00 6,00 8,00 10,00 12,00 control (without schavnat) 0.50% 1.00% 1.50% samples under investigation pr ot ei n, % fig. 3. the protein content in the finished product (pampushkas) with the additive of schavnat powder the finished products with the addition of 0.5% of schavnat powder contained 7.71% of protein, with the addition of 1.0% – 7.98% of protein, with the addition of 1.5% – 11.44% of protein. the research results of organoleptic and technological indicators of the finished products with the additive of schavnat were presented in table. 1. the organoleptic quality of pampushkas did not change significantly with the addition of schavnat in the amount from 0.5 to 1.0% compared to the control, but the colour and surface crust, texture and elasticity were slightly worse with the addition of schavnat in the amount of 1.5%. it was noted the pleasant slightly acid flavour of the finished products. the schavnat additive decreased the specific volume and texture of the finished products (fit. 4). thus, it was established that pampushkas with the addition of schavnat powder in the amount from 3,5 to 13% had the decreased specific volume compared to the control sample (without schavnat). and the texture indicator of pampushkas has been decreasing with the dosage of schavnat powder from 1.05 to 9.17%. 35 45 55 65 75 85 95 105 115 0 30 60 90 fermentation time, min sp ec if ic v ol um e, c m3 control (without schavnat) 0.50% 1.00% 1.50% fig.4. dynamic of specific volume of dough with the additive of schavnat powder the research results were shown that the start value of titrated acidity was increased slightly with the using of schavnat products (fig. 5). 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 control (without schavnat) 0.50% 1.00% 1.50% titrated acidity, degree: a ci tid y, d eg re e start value final value fig. 5. titrated acidity of dough with the additive of schavnat powder food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 1 – 2015, pag. 1 6 5 table 1 the influence of schavnat on the technological process and the quality of pampushkas indicator control dosage of schavnat in the dough, % of flour weight 0.5 1.0 1.5 1 2 3 4 5 dough mixing time, min 8 8 8 8 fermentation time, min 60 60 60 60 proofing time, min 30 30 30 30 baking time, min 25 25 25 25 titrated acidity, degree start value final value 2.2 2.4 2.7 2.9 3.2 3.4 3.6 4.0 finished products – pampushkas titrated acidity, degree 2.0 2.6 2.8 3.4 texture indicator, % 80 79 78 76 specific volume, cm3 3.07 2.89 2.72 2.61 crusts colour golden brown surface crust smooth rough texture medium, uniform compacted elasticity medium medium, compacted flavour inherent to pampushkas sour flavour with increasing the dosage of schavnat powder the dough fermentation property was investigated by the floating dough ball (fig. 6). 0 2 4 6 8 10 12 14 16 18 20 control (without schavnat) 0.50% 1.00% 1.50% samples under investigation fe rm en ta tio n pr op er ty , m in fit. 6. investigation of dough fermentation property with schavnat powder the research results were shown that the incensement of the additive of schavnat powder decreased the dough fermentation property, but within the established norms for bakery products. the dynamic of dough ball running was investigated for establishing the nature of the impact of schavnat on viscoplastic properties of semi-finished products (fig. 7). 60 70 80 90 100 110 120 130 0 30 60 90 fermentation time, min d ia m et er o f d ou gh b al l, m m control (without schavnat) 0.50% 1.00% 1.50% fig. 7. investigation of viscoplastic properties of dough with schavnat powder the dough ball running describes the state of protein and proteinase complex during autolysis. it was found that schavnat improved the viscoplastic properties of dough. the addition of dry schavnat increased the water absorbing capacity and reduced the ability to dough running during fermentation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 oksana arpul, tetiana sylchuk, vira kulinich, olena usatiuk the use of plant materials in culinary products out of yeasted dough, food and environment safety, volume xiv, issue 1 – 2015, pag. 1 6 6 4. conclusion the fortification of food products by schavnat is appropriate and relevant to the development of new products with a high content of plant protein. it was proposed the technology of «special pampushkas with garlic» that could be recommended as a garnish for soups, such as borsch or rassolnik. such fortified pampushkas could balance the food diet by protein of plant origin. 5. references [1] marina n.v., novoselova g.n., shavnin s.a. products with increased biological value out of non-traditional plant raw materials, news of the samara scientific center of the russian academy of sciences, vol. 12, 1(8), 2079-2082 (2010). [2] maría e. matos, teresa sanz, cristina m. rosell. establishing the function of proteins on the rheological and quality properties of rice based gluten free muffins, food hydrocolloids, vol. 35, 3, 150-158 (2014). [3] m. solina, r. l. johnson, frank b. whitfield, effects of soy protein isolate, acidhydrolysed vegetable protein and glucose on the volatile components of extruded wheat starch, food chemistry, vol. 104, 4, 1522-1538 (2010). 287 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 287 296 human health risk assessment of heavy metal contaminants in table salt from nigeria *sunday peter ukwo 1 1department of food science and technology university of uyo, akwa ibom state, nigeria. sonipeter75@gmail.com *corresponding author recieved 20th august 2020, accepted 29th december 2020 abstract table salt has continue to be an important part of human diet in many households around the world. table salt is used for food flavouring, pickling, curing and in several industrial purposes. therefore, the present of contaminants in table salt portends significant risk to human health. the study is aimed at investigating the concentration of heavy metals and human health risk associated with consumption of table salt in nigeria. four refined table salt samples (a, b, c, d) and four samples of unrefined table salt (e, f, g, h) were analysed for the concentration of copper (cu), lead (pb), iron (fe), cadmium (cd) and arsenic (as) using atomic absorption spectrometry. the united state environmental protection agency (usepa) methods were used to estimate hazard indices and cancer risks associated with consuming table salt with these elements. results indicated heavy metal concentrations were significantly higher in unrefined table salt than refined table salt, the estimated daily intake values of cu, fe, and cd were lower than the established provisional tolerable daily intake. the hazard indices did not exceed 1 in all samples of refined and unrefined table salts. however, higher values of hi of pb, cd and as were noted in unrefined table salts. the carcinogenic values of as in refined salt sample b and c and all unrefined table salts were higher than the stipulated one in one million chances which implies that carcinogenic effects were more likely due to consumption of 6.35g/day of salt samples with those contaminants. keywords: table salt, heavy metal, contaminant, health risk, nigeria 1. introduction exposure of humans to environmental hazards especially toxic chemicals occurs spontaneously through different sources. these include the non-occupational exposed populations whose contact and exposure to heavy metal is through food and water. they constitute over 90% of the exposed populations and those whose exposure is as a result of inhalation and dermal contact [1]. food contamination by environmental hazards have continued to be a subject of serious concern to researchers globally particularly due to their deleterious effects on human health. among the chemical contaminants are toxic elements which constitutes a significant food safety risk because of their poor rate of metabolism, potential to bioaccumulate in the biological system resulting from their nonbiodegradable nature and long biological half-lives [2]. although some toxic elements occur naturally in the environment, anthropogenic inputs which originate from various human activities have continued to increase their concentrations [3, 4]. some elements such as lead, cadmium, mercury http://www.fia.usv.ro/fiajournal mailto:sonipeter75@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 288 and arsenic are non-essential and are toxic even at low concentrations. others such as copper and zinc are essential for humans; they play important roles in biological systems. when their intake exceeds specific levels, they result in harmful and toxic effects. these non-essential heavy metals or toxic elements exert harmful effects to cardiovascular, neural, hematopoietic, immunological and gastrointestinal systems [5 6]. edible salts is a frequently used in many homes as an food additive for the purpose of imparting taste to food. it is an essential additive which has been used from prehistoric times for flavouring, pickling and curing meat and fish and for tanning [7]. in addition to salty flavor, it is used as a flavor enhancerand also as a food preservative to inhibit the growth of some spoilage and pathogenic bacteria [6]. sodium chloride content of food grade salt must not be below 97% on dry matter basis while the insoluble dry matter content should not exceed 0.2%. also, with majority of table salt consumed globally coming from mines, it is expected that environmental contaminants such as heavy metal possess a serious health concern to consumers [1]. table salt is consumed daily, hence any contamination, even at low concentrations, could be hazardous to the consumer’s health. therefore, the concentration of heavy metals should be carefully monitored in this food additive [1]. in studies conducted by the fao and who experts on the assessment of several additives in foods and environmental pollutants reported that cadmium, lead and mercury are the major heavy metal implicated in food contamination [8]. also in nigeria, salt deposits are found in many parts of the country and have been exploited traditionally for over 400 years. the process though crude begins with the fetching of the brine from the salt lakes into a mound already built and tilled locally. the brine is then poured into it and the puddle is left under the sun to allow the water to evaporate leaving behind a crust that is rich in crystals. the crust is further broken and process locally into table salt [7]. the direct use of this unpurified or unrefined table salts for culinary and eating purposes have been discouraged by government health agencies and regulatory authorities. this is because of the expected adverse health effect that may occur due to toxic elements contamination which is widespread in the environment [9]. unrefined table salt may contain some of these toxic elements which may create health risks to consumers even at low concentrations due to the daily consumption of salt. in spite of several warnings on the direct use of raw and unrefined table salt by government and regulatory agencies, their availability in the markets proof their deliberate usage [7]. in addition, several studies have shown the presence of heavy metals in both refined and unrefined salts [10, 11, 6]. it is therefore important to conscientiously monitor the concentration of toxic elements in edible salt as well as assess the related risks of dietary intake of these elements in food to human health for the purposes of quality control, food safety and other advisory purposes. there is little or no information about the levels of toxic elements contamination and their associated human health risks from consumption of edible salts produced and sold in nigeria. therefore, this study was conducted to investigate the concentrations of cu, pb, fe, cd, and as in refined and unrefined salts produced and marketed in nigeria. also, the potential human health risks associated with these metals through salt consumption were estimated food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 289 2. materials and methods study area the research was carried out in four locations in the country. these location have history of producing salt traditionally or locally. edible salts are usually produced traditionally from salt lakes, seawater and mines by women from these areas. despite the present of refined table salts in the markets, some residents of these areas forechoose the local or unrefined salt sometimes for several reasons which are cultural and economical related. sample collection four brands (500g) of refined packaged table salts consumed in nigeria were obtained from their distributors in uyo, akwa ibom state in june 2019. the selected brands were checked for their registration with the national agency for food and drug administration and control (nafdac), batch number and expiry date were also confirmed. the refined table salt samples were coded a, b, c, and d. for each brand, three samples with different batch numbers were obtained. also. four samples of locally processed table salt from salt production areas which are mainly found port harcourt, abakaliki, makurdi and jos were obtained from vendors. three samples of 500g were collected from vendors at each location in june 2019. they were also coded as e, f, g and h respectively. the table salts samples obtained were packaged and taken for analysis at the food processing laboratory of the university of uyo. heavy metal analysis heavy metals including copper (cu), lead (pb), iron (fe) arsenic (as) and cadmium (cd) were determined in the refined and unrefined table salt samples using perkin – elmer model 3030 atomic absorption spectrophotometer. the elements were analysed by dissolving 2g of salt samples into 3ml nitric acid this was followed by the addition of 1ml concentrated hydrochloric acid. the solution was diluted with distilled water to 100ml. specific cathode hallow lamp for each element was used to determine their concentration at specific wavelengths [12]. human health risk assessment assessment of human health risk resulting from consumption of salt with heavy metals contaminants were determined based on methods outlined [13]. firstly, the level of exposure resulting from consumption of heavy metal contaminants in salt samples were determined by estimating the daily intake levels; 𝐸𝐷𝐼 = 𝐶𝑥𝐼𝑅𝑥𝐸𝐹𝑥𝐸𝐷 𝐵𝑊𝑥𝐴𝑇. where edi = estimated daily intake (mg/kg – day) ir = ingestion rate of salt (kg/day) c = concentration of chemical contaminant in salt (mg/kg) ef = exposure frequency (days/year) ed = exposure duration (years) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 290 bw = body weight (kg) at = average time (days) non-carcinogenic risk or target hazard quotient values (thq) for each metal were then calculated according to equation below; thq = edi rfd where: thq = non-cancinogenic risk value for heavy metal contaminant. edi = estimated daily intake or exposure rate (mg/kg –day) rfd = reference dose of chemical (mg/kg – day) the calculations were made using the standard assumptions for an integrated risk analysis [14]. for the purpose of this study, the intake rate (ir) of salt in nigeria was assumed to be 6.35g/day [15], average body weight of exposed individual (70kg), exposure frequency (365day/year) while duration of exposure was 70years. the average exposure was calculated as 365days x 70 years. for calculation of target hazard quotient (thq) for each metal, the following reference dose (rfd) [14]. were used; pb = 3.6x10-3 mg/kg-day, cd = 1.0 x 10-3 mg/kgday, as = 3.0 x 10-4 mg/kg-day, fe =7.0x10-1 mg/kg-day and cu = 4.0x10-2 mg/kg-day. the hazard index (hi) is the sum of total hazard quotients; (hi = σthq). the carcinogenic risk (cr) of pb and as were expressed as a product of edi and cancer potency value or cancer slope factor (csf). cr= edi* csf where: cr = life time cancer risk edi = estimated daily intake mg/kg-day csf = cancer slope factor mg/kg-day the cancer slope factor for pb = 8.5x103mg/kg-day; as = 1.5mg/kg-day [14]. data analysis all analyses were carried out in triplicate. the experimental data generated were statistically analyzed using one way analysis of variance. the mean and standard deviation of the data was determined. significant difference between mean were analysed using duncan multiple range test (dmrt). all analyses were done using statistical package for social science (spss) version 20 for windows. statistical test was performed at 5% significant level. 3. results and discussion concentration of heavy metals in edible salt results of heavy metals concentration in various salt samples are shown in fig.1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 291 heavy metal concentrations were significantly higher in local or unrefined table salt samples when compared to those of refined table salt samples. copper concentration in the refined table salt samples ranged from (1.12-1.32 mg/kg) and did not show significant difference among samples, however significant differences (2.21-3.21 mg/kg) existed among the unrefined table salt samples. iron concentration in both local and refined table salts were within the regulatory acceptable limit of son (30 mg/kg) and codex (50 mg/kg) for edible table salt [16, 17]. this may require fortification of salt with iron to increase iron content. cu, copper; pb, lead; fe, iron; as, arsenic; cd, cadmium fig. 1: heavy metals content of refined and local table salts consumed in nigerian lower concentrations copper and iron in foods are very important for human health. copper is a major component of different enzymes involve in energy metabolism, synthesis of haemoglobin, connective tissue and phospholipids while iron is present in cells of living organism and play significant functions in many biochemical processes. iron is the major component of haemoglobin and myoglobin, pigments, cytochromes and other proteins participating in transport, storage and utilization of oxygen [7]. although optimal levels of copper is useful in human body, higher concentrations have been linked to wilson syndrome. the defect is characterized by dysfunction associated with the transmission of copper in the cells. this condition may result in hepatic cirrhosis and degradation of neurons as well as the presence of colourful ring in the cornea of human eye. higher intake of copper can also lead to kidney and brain damage [18, 6]. as was noted for copper, similar variations also existed between the toxic elements such as in lead cadmium and arsenic. lead content of local table salt was much higher (1.36 -1 0 1 2 3 4 5 6 7 a b c d e f g h co n ce n tr a ti o n ( m g /k g ) refined table salts local table salts cu (mg/kg) pb (mg/kg) fe (mg/kg) as (mg/kg) cd (mg/kg) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 292 4.21 mg/kg) where samples e, g and f had values higher than the son and codex acceptable limit of 2.0 mg/kg. values for lead as obtained in this study were comparable to lead concentration in local cooking salts reported at akwana, middle benue trough, in nigeria [19]. it is important to note that nigeria had experienced several cases of lead poisoning in north western and central states of zamfara and niger where over 200 people mostly children had died [20]. also, cadmium concentration in the refined table salts sample were lower than the acceptable limit of 0.5 mg/kg while those of the local table salt samples were higher than maximum permitted limit. the present of arsenic in table salts or any food item is however unacceptable. heavy metals such as lead cadmium and arsenic are absorbed, stored and concentrated in living cells through food chain causing serious toxic effect to humans [21]. they are widespread environmental pollutants which promote serious adverse health effects, especially in young children, pregnant women and other vulnerable groups. they are also implicated in causing several diseases such as osteoporosis and renal failure [22]. their toxicity are due to their ability to replace useful metals in the active sites of enzymes, form complexes and, catalyzes the breakdown of essential metabolites altering their permeability and hindering other elements in their metabolic functions [23]. for instance, lead and cadmium interact with essential elements such as zinc, iron, calcium and copper exerting an inhibitory effects on the activity of enzymes containing this mineral elements. this results in growth failure, improved nutrients tolerance, poor metabolism and absorption of these elements, reduction in plasma ceruloplasmic concentration among others [24, 25]. human health risk assessment estimated daily intake of heavy metals in edible salt the results of the edi for heavy metals through the consumption of local and refined table salts are shown in table 1. in determining the edi for these metals, the mean concentration of each metal as presented in fig.1. and the average consumption rate of salt in nigeria was estimated at 6.35g/day/person [15]. table 1 estimated daily intake (edi) of heavy metals (mg/kg/d) from salt consumption refined table salts local table salts parameters a b c d e f g h cu 0.00013 0.00011 0.00013 0.00011 0.00022 0.00023 0.00031 0.00029 pb 0.000011 0.000023 0.000031 0.000025 0.00024 0.00013 0.00041 0.00038 fe 0.00014 0.00031 0.00080 0.00027 0.00062 0.00061 0.00044 0.00053 cd 0.000034 0.000025 0.000048 0.000044 0.00013 0.00011 0.00013 0.00011 as 0.00000 0.0000029 0.0000048 0.00000 0.000053 0.000049 0.000047 0.000053 this was based on the assumption that the ingested dose of each metal in the salt sample was equal to the absorbed dose and processing had no effect on the metal [13]. in the determination of potential human health risk resulting from daily consumption of edible salt containing these heavy metals, the concept of an estimated daily intake (edi) for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 293 these metals from edible salt have become quite significant. for both the refined and unrefined or local table salts, the levels of edi was consistent with the concentration level of each element present in the salts. the edi values of the refined table salts samples were lower when compared to edi values of the local or unrefined table salts samples. however, the edi values were generally lower than the established provisional tolerable daily intake (ptdi) as outlined by the joint fao/who expert committee on food additives (jecfa) [8]. the edi values for fe and cu for both refined and unrefined edible table salt samples were far below the provisional tolerable daily intake (ptdi) (fe; 0.8 mg/kg/day) and (cu; 0.5 mg/kg/day). similarly, edi values for cd in the refined table salt was far lower when compared with the tolerable daily intake of 0.0008 mg/kg/day. also, according to joint fao/who expert committee on food additives (jecfa) the ptdi for pb and as were no longer considered to be health protective, therefore it was withdrawn. this was on account of several studies not presenting any threshold for the key effects of pb and as, hence it was concluded that it was not possible to establish ptdi that would be considered health protective. therefore, human exposure to lead and arsenic through food need to be seriously discouraged [8]. noncarcinogenic health risk the non-carcinogenic risks resulting from consumption of edible salts with heavy metal contaminants were estimated through the target hazard quotient (thq) and hazard index (hi) as presented in table 2 and 3 respectively. table 2 toxic hazard quotient (thq) values of heavy metals from salt consumption refined table salts local table salts parameters a b c d e f g h cu 0.0032 0.0028 0.0032 0.0028 0.0055 0.0056 0.0078 0.0073 pb 0.0030 0.0060 0.0090 0.0070 0.066 0.067 0.12 0.11 fe 0.0002 0.0004 0.0004 0.0003 0.0089 0.0087 0.0063 0.0076 cd 0.034 0.025 0.048 0.044 0.13 0.11 0.13 0.11 as 0.000 0.0096 0.0026 0.00 0.18 0.17 0.13 0.18 table 3 hazard indexvalues of heavy metals from salt consumption refined table salt local table salt parameters cu 0.012 0.03 pb 0.025 0.36 fe 0.001 0.031 cd 0.150 0.50 as 0.025 0.66 the estimated non carcinogenic risk values were determined on the basis of the reference dose (rfd) for toxic elements [14]. the rfd values represent the estimated daily exposure to which human population may continually be exposed over a life time without any food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 294 appreciable health risk [26]. when the intake or average daily dose (edi) is equal to the reference dose, the non-carcinogenic value or thq is one. when the estimated daily intake (edi) exceeds the reference dose and thus, the thq is greater than one or equal to one, it is likelihood that non-carcinogenic adverse health effects will be observed due to ingestion of 6.35g of salt per day but when thq is less than one its indicate that adverse health effects are not likely to occur due to ingestion of 6.38g of salt per day. therefore a thq value below 1 is an indication of no significant risk of non-carcinogenic effects for the exposed consumers. with the possibility to cumulative and synergic effects of these toxic metals, the thq values of individual metals are summed up to give the hi or the total thq caused by exposure to multiple metals, which is also explained in a similar manner as thq [27]. the thq and hi values of each estimated element did not exceed 1 in all samples of refined and unrefined table salt, suggesting that the exposed population would not experience significant health risks when consuming these individual elements from daily consumption of 6.35g/day of salt. the result as obtained in this study was similar to that reported on edible salt from neighboring ghana [22]. it is worthy to note that, higher thq values is an indication of a higher probability of an exposed risk, even if the thq value was not greater than 1. it is also important note that hazard indices for the unrefined or local table salt was of serious concerned. this is because the consumption of 6.35g of local or unrefined table salt per day accounted for non-carcinogenic risk of 36%, 50% and 66% from pb, cd and as respectively. this suggests the need for consumers to regulate the intake of the unrefined or local table salts as the continuous consumption may result in adverse non-carcinogenic health effect. carcinogenic health risk the carcinogenic risk indices for pb and as in the refined and local and unrefined salt samples are presented in table 4. table 4 carcinogenic index for lead and arsenic from salt consumption refined table salt local table salt parameters a b c d e f g h pb 9.35x10-8 1.96 x10-7 2.60 x10-7 2.10 x10-7 2.04 x10-6 1.0 x10-6 3.40 x10-6 3.21 x10-6 as 0.00 4.35 x10-6 7.20 x10-6 0.00 7.95 x10-5 7.35 x10-5 7.05 x10-5 7.95 x10-5 the result indicated that cancer risk index of pb in the refined salt samples were within the acceptable limit of one in one million (1.0 x 10-6) chances of developing cancer as a consequent of consuming refined salt contaminated with pb [14]. the carcinogenic risk value as obtained in this study was consistent with values reported by weremfo (2019). however, carcinogenic values of pb for the local or unrefined table salt samples were higher than the acceptable limit of 1.0x10-6 chances. also, carcinogenic values of as in refined salt sample b and c and all the local or unrefined table salts were higher than the acceptable limit and according to regulatory directives of the united state of america and european union commission and in this case a consequential risk is deduced [14, 28]. 5. conclusion the study provided a significant information on varying concentrations cu, pb, fe, cd and as in refined and local or unrefined table salt available in nigerian markets. heavy metal concentrations were significantly higher in local or unrefined table salt samples than the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 295 refined table salt samples. cu and fe concentrations in the refined and unrefined table salt samples were far below the stipulated limits of regulatory agencies. the concentration of pb, cd, and as in local or unrefined table were higher than their safety limits as prescribed by son and codex regulations. the estimated daily intake values of cu, fe, and cd were lower than the established provisional tolerable daily intake as outlined by the joint fao/who expert committee on food additives. the thq and hi values of all estimated element did not exceed 1 in all samples of refined and unrefined table salt, suggesting that the exposed population would not experience significant health risks when consuming these individual elements from daily consumption of 6.35g/day of salt. however higher hi of pb, cd and as in the local unrefined table salts indicated a higher probability of exposed risk resulting from cumulative and synergistic effects of these elements. the carcinogenic values of as in refined salt sample b and c and all the local or unrefined table salts were higher than the stipulated one in one million (1.0x10-6) chances which implies that carcinogenic effects were more likely due to consumption of 6.35g/day of salt samples with those contaminants conflict of interest the author declares no conflict of interest 6. references [1]. eftekhari m. h, mazloomi s. m, akbarzadeh m, and ranjbar m, content of toxic and essential metals in recrystallized and washed table salt in shiraz, iran. j environ health sci eng.12(1):10-16, (2014). [2]. censi, p., spoto, s. e., saiano, f., sprovieri, m., mazzola, s., and nardone, g, heavy metals in coastal water systems. a case study from the northwestern gulf of thailand. chemosphere, 64, 1167–1176, (2006). [3]. sarkar, s. k., cabral, h. chatterjee, m. cardoso, i. bhattacharya, a., and satpathy, k. k, biomonitoring of heavy metals using the bivalve mollusks in sunderban mangrove wetland, northeast coast of bay of bengal. (india): possible risks to human health. clean 36: 187-194, (2008). [4]. giri, s., and singh, a. k, human health risk and ecological risk assessment of metals in fishes, shrimps and sediment from a tropical river. int. j. environ. sci. technol. 12:2349–2362 (2015). [5]. çelik, u. and oehlenschläger, j., determination of zinc and copper in fish samples collected from northeast atlantic by dpsav. food chemistry, 87:343-347, (2004). [6]. zarei, m., eskandari, m. h., and pakfetrat, s, determination of heavy metals content of refined table salts. american-eurasian journal of toxicological sciences, 3, 59–62, (2011). [7]. ukwo, s. and edima-nyah, a. micronutrients and heavy metals content of refined and local table salts consumed in some nigerian cities. am. j. innov. res. appl. sci. 2 (4): 193-198. (2016). [8]. fao/who report of the fifth session of the codex committee on contaminant in foods. fao/who, hague, netherlands, 21–25. march 2011. [9]. saturag s, garret, s. sens m. a and sens d. a, cadmium environmental exposure and health outcome. environ. health perspective, 118 (2): 182-190. (2010). [10]. soylak, m., peker, d.s.k., and turkoglu, o., heavy metal contents of refined and unrefined table salts from turkey, egypt and greece. environmental monitoring and assessment, 143 (1–3), 267–272. (2008). [11]. cheraghali, a. m., kobarfard, f., and faeizy, n., heavy metals contamination of table salt consumed in iran. journal of pharmaceutical research, 9 (2), 129–132(2010). [12]. aoac international. official analytical chemistry official methods of analysis: 18th edn.; horwitz w, latimer g. w. eds; aoac international: gaithersburg, md, usa, (2005). [13]. united states environmental protection agency (usepa). guidance for assessing chemical contaminant data for use in fish advisories. volume 2 risk assessment and fish consumption limits, 2,1–383. washington dc usa. pp1-67. (2000). [14]. united state environmental protection agency (usepa). integrated risk information system (iris) (on-line database). washington, dc: office of health and environmental. (2009). [15]. powles, j. fahimi, s. micha, r. khatibzadeh, s. shi, p. ezzati, m. global, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 sunday ukwo, human health risk assessment of heavy metal contaminants in table salt from nigeria, food and environment safety, volume xix, issue 4 – 2020, pag. 287 – 296 296 regional and national sodium intakes in 1990 and 2010: a systematic analysis of 24 h urinary sodium excretion and dietary surveys worldwide. bmj , 3 (12), e003733, (2013). [16]. standard organization of nigeria (son). standard for food grade salt, 168: 27. (1992) available on: http://www.ipan.gov.ng. [17]. codex alimentarius. codex standard for food grade salt. cx stan 150-1985, amended. codex alimentarius (2006). [18]. heshmati, a., vahidinia, a., and salehi, i., determination of heavy metal levels in edible salt. avicenna journal of medical biochemistry, 2, e19836 (2014). [19]. dim l. a. kinyua am., munyithya and adetnnji j, lead and other metals distribution in local cooking salt from fofi salt spring in akwana, middle benue trough, nigeria. j. environmental science and health. 26 (3): 357-365. (1991). [20]. world health organization (who) report on lead poisoning in nigeria. available on: http://reliefweb.int/report/nigeria/lead poisioningnigeria-15-may-2015. [21]. nwochoko n. c, ibiam u. a., agbafor, k. n. and aja, p. m. determination of arsenic, lead cadmium zinc and iodine in some salt samples sold in south-east nigeria and effect of these metals on protein and haemcogtolam content of albino rats. international j. of advanced biological research. 2(4) 599-601 (2012). [22]. weremfo, a. heavy metals in edible salt from ghana with special reference to potential human health risk, toxin reviews, 1-6 (2019). [23]. burger, j. and gochfeld, m., heavy metals in commercial fish in new jersey. environmental research, 99:403-412, (2005). [24]. goyer, r. a. and clarkson, t. w., toxic effects of metals. in: casarett and doull´s toxicology: the basic science of poisons, c. d. klaassen (ed.), mcgraw-hill, ny usa, 811-867, (2001). [25]. carvalho, m. l., santiago, s., and nunes, m. l. assessment of the essential element and heavy metal content of edible fish muscle. analytical and bioanalytical chemistry, 382:426 432. (2005). [26]. saha, n. and zaman, m. r. evaluation of possible health risks of heavy elements by consumption of foods stuffs available in the central market of rajshahi city, bangladesh. environmental monitoring and assessment, 185 (5): 3867-3878. (2013). [27]. united state environmental protection agency (usepa) concepts, methods and data sources for cumulative health risk assessment of multiple chemicals, exposures and effects. washington dc, (2007). [28]. official journal of the european union commission (ojeu). commission regulation (ec) no 1881/2006. setting maximum levels for certain contaminants in foodstuffs, 364: 5–24, (2006). stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiii, issue 1 31 march 2014 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladimir reşitca technical university of moldova, chisinau republic of moldavia alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved title …………………… doi: https://doi.org/10.4316/fens.2021.017 160 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag. 160 164 cleaning the vegetable oil production wastewater with anthracite sergiy boruk 1 , igor winkler 2* , vitaliy mischenchuk 2 1 department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine 2 department of medicinal and pharmaceutical chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine winkler@bsmu.edu.ua *corresponding author received 10th may 2021, accepted 28th june 2021 abstract: the efficiency of anthracite treatment of the wastewaters formed at the sunflower oil production is investigated and discussed in comparison with other water cleaning technologies. it is found that a comparatively small amount of anthracite (10-15 % of the wastewater mass) ensures efficient decontamination and eliminates up to 70 % of the wastewater pollutants. this method does not require any extensive changes in the production technology and can easily be incorporated at the existing production facilities. this efficiency is based on the double-nature structure of the anthracite surface, consisting of the hydrophilic and hydrophobic areas. the former ones ensure a high wettability of the adsorbent and the release of some inorganic ions that provide coagulation of the emulsified contaminants. the latter ones ensure adsorption and extraction of the dissolved and emulsified organic pollutants of the wastewater. the used adsorbent can be filtered out after the extraction and then disposed of in an environmentally safe way through incineration as an admixture to the regular coal fuel. keywords: sunflower oil production; wastewater decontamination; porous coal adsorbents; environment protection 1. introduction food processing is one of the leading branches of the economy in many countries, including ukraine. it produces numerous items with stable high market demand and under competitive conditions. production of vegetable oil (mostly sunflower) is a highly developed part of the food processing in ukraine, with a total of 6.8 million tons of crude vegetable (mostly sunflower) oil produced in 2019 and 6.1 million tons exported to many countries in europe and worldwide [1]. it makes ukraine the world top exporter of these goods [2]. hence, this branch seems very important for the national economy, and the intense functioning of the oil production facilities affects environmental conditions in all regions of ukraine. the equipment used at many oilproduction facilities is quite outdated, causing an increased level of environmental contamination affecting air, water and soils. in the context of water contamination, it should be understood that any oil production facility generates a wide range of contamination agents at all technological stages [3-6]. the suspended tissues of sunflower seeds and oil emulsions are the main water pollution agents generated at such factories. if not extracted or decontaminated, they cause significant water pollution during the processes of their natural decay. it should also be emphasized that some volatile sulfur compounds are http://www.fia.usv.ro/fiajournal mailto:winkler@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 sergiy boruk, igor winkler, vitaliy mischenchuk, cleaning the vegetable oil production wastewater with anthracite, food and environment safety, volume xx, issue 2 – 2021, pag. 160 – 164 161 formed and released into the atmosphere during these processes. it expands the dangerous environmental effects of the oil production industry beyond the area of water pollution. many approaches are applied to oil production wastewater decontamination. the simplest one assumes keeping the wastewaters in an isolated pond until the natural decay processes completed, followed by their gradual discharge into local water bodies. it is an extremely inefficient method that requires a long time and does not ensure sufficient decontamination efficiency. moreover, it does not control the release of environmentally dangerous volatile decay products into the atmosphere. simple filtration, adsorption, electrofiltration, forced oxidation and some other technologies are tested and used widely to increase the efficiency of the oil-production wastewater treatment [4-9]. it should be emphasized that some porous wastes or byproducts of the coal refinery are considered as promising adsorbents for such technologies [5, 7]. based on the nature and properties of the water pollution agents present in this type of wastewater, it is expected that adsorbents with hydrophobic or mosaic surface structure should exhibit a comparatively high pollution extraction activity. it is known that coal-like materials are among such adsorbents [5-7, 10], and therefore, their adsorption performance should be investigated to address the above mentioned issue. in this paper, the results of the oilproduction wastewater treatment by anthracite are reported. this coal material is abundant in ukraine, comparatively inexpensive and easily available. besides, it is environmentally neutral and does not generate any secondary water contamination if used for its treatment. moreover, anthracite can be utilized safely through incineration as a component of the boiler feeding coal mixtures. the wastewater pollution components adsorbed on its surface are organic compounds, and they will also decompose in an environmentally safe way at such incineration. 2. experimental the following types of wastewaters formed at the sunflower oil production factory in chernivtsi, ukraine were used in this investigation: series 1: wastewaters formed at the sunflower seeds grinding; series 2: wastewaters formed at the evaporation of sunflower oil production sludge. the “a” brand ukrainian anthracite with an average ash content of 5.4 % and the moisture content of 2.1 % was used as an adsorbent. the material was ground, and then the 100-250 μm fraction was separated using the appropriate sieves and used in the follow-up adsorption experiments. chemical oxygen demand (cod) was determined by the simplified procedure [11] and used as a characteristic of wastewater contamination because the oxidizable organic components are the most common pollutants of this type of wastewater [4]. besides, the absorbance of the wastewater samples against distilled water was also measured as this parameter depends on the content of the genuinely dissolved or colloid pollutants, not the coarse-dispersed particles. both water quality parameters were determined for the untreated source samples and the same samples after the anthracite treatment. the treatment was performed according to the following procedure. 0.5, 1, 2, 3, 4 or 5 g of the ground anthracite powder were added to 25 ml of the wastewater and left for 24 h. then the suspension was filtered through the paper filter to separate the anthracite adsorbent from water, and the cod index was determined for the filtrates. a control experiment required for the correct cod determination has been performed by the same food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 sergiy boruk, igor winkler, vitaliy mischenchuk, cleaning the vegetable oil production wastewater with anthracite, food and environment safety, volume xx, issue 2 – 2021, pag. 160 – 164 162 procedure but with the distilled water sample. 3. results and discussion a high level of contamination was determined in the initial water samples, and their cod indexes were 2450 mg o/l and 620 mg o/l for series 1 and 2 correspondingly. the absorbance values were comparatively low (0.32 and 0.14, respectively), which means that contamination agents were mostly dissolved or formed stable emulsions. in the case of sample 1, the distinct smell of hydrogen sulfide, manifesting decomposition of the organic pollutants, appeared in two weeks, while for sample 2, it appeared only in four weeks. in our opinion, this difference is caused by the lower contamination of sample 2 and the presence of some trace quantities of the technological gasoline extractant. it acts as a preservative and decelerates the natural bio-decomposition of the organics. the absorbance of both samples increases to 0.72 (sample 1) and 0.45 (sample 2), while their cod indexes decrease to 1920 and 400 mg o/l correspondingly. it can be interpreted as a result of the natural decomposition of the organic substances present in the wastewater samples, which forms the volatile smelling compound leaving the liquid phase. when the anthracite extractant was applied, cod indexes and absorbance of the filtered samples were decreasing substantially because of intense capturing of the oil contaminants on its surface. the dependencies of these parameters on the amount of anthracite are shown in figures 1 and 2. 0 500 1000 1500 2000 2500 0 1 2 3 4 5 adsorbent mass, g c o d , m g o /l fig. 1. dependence of cod indexes on the mass of anthracite added for extraction. ♦ – sample 1; ■ – sample 2. it can be seen that even in the case of highly contaminated sample 1, 3-4 g of anthracite (10-15 wt % from the sample mass) ensure effective decontamination of the sample (elimination of around 70 % of the pollution agents), while in the case of less contaminated sample 2, this effect is reached even for 1-2 g (5-10 %) of the added anthracite. similar results can also be seen for the absorbance of the samples (see fig. 2). further increase in the adsorbent mass does not result in the corresponding increase in the cleaning efficiency. therefore, it can be concluded that 15 % or 10 % of anthracite added to the more or less contaminated samples of the oil-processing wastewaters correspondingly, ensure their efficient decontamination. cod decreases fourfold in the case of more contaminated water, and it halves for less polluted water. it is a promising result that can be achieved food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 sergiy boruk, igor winkler, vitaliy mischenchuk, cleaning the vegetable oil production wastewater with anthracite, food and environment safety, volume xx, issue 2 – 2021, pag. 160 – 164 163 without any additional investment and by using cheap and abundant material. it should also be emphasized that the used anthracite forms a slurry layer on the bottom of the decontamination pond, and this sediment can easily be dried through the periodical draining of the ponds followed by collecting the dried slurry and burning as a fuel admixture it in the regular coal boilers [12-14]. it should also be noted that this process is environmentally friendly and does not result in any significant increase in dangerous gases emissions [15]. the ash formed after such combustion is similar to the conventional coal ash and can be disposed of similarly. 0 0.05 0.1 0.15 0.2 0.25 0.3 0 1 2 3 4 5 adsorbent mass, g a b so rb a n ce fig. 2. dependence of the wastewater absorbance on the mass of anthracite added for extraction. ♦ – sample 1; ■ – sample 2. based on the registered decrease in the wastewater absorbance after its treatment with anthracite, it can be assumed that this material also provides an additional water cleaning effect by releasing the ions followed by the coagulation of the colloid pollutants present in the water. the visual turbidity of both water samples increased after the anthracite was added, and then the coagulated particles were captured by the adsorbent resulting in a decrease in the turbidity (see fig. 2). this result is confirmed by an increase in the wastewater samples conductivity after their treatment with anthracite (see fig. 3). 0 200 400 600 800 1000 1200 0 1 2 3 4 5 ads orbent mas s , g c o n d u c ti v it y , m s /m fig. 3. dependence of the wastewater conductivity on the mass of anthracite added for extraction. ♦ – sample 1; ■ – sample 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 sergiy boruk, igor winkler, vitaliy mischenchuk, cleaning the vegetable oil production wastewater with anthracite, food and environment safety, volume xx, issue 2 – 2021, pag. 160 – 164 164 4. conclusion anthracite, as an adsorbent with the mosaic surface, captures and eliminates up to 70 % of the water contaminants present in the wastewaters formed at the sunflower oil production. this efficiency is based on the synergetic effect of the mineral hydrophilic and coal-like hydrophobic surface domains of the anthracite. due to this surface structure, the anthracite can be added to the wastewater, extract its pollutants, and then be separated by coagulation followed by filtration of the sediment. this technological operation can be performed periodically as the wastewater treatment ponds are filled. even in the case of the highly polluted sunflower grinding wastewater, 1015 mass % of anthracite ensure the abovementioned decontamination efficiency. after the adsorption, the used anthracite can be utilized in an environmentally safe way through its incineration as an admixture to the regular coal fuel. 5. references [1]. output of some types of industrial products in ukraine in 2011-2019. state statistics service of ukraine. http://www.ukrstat.gov.ua/operativ/operativ2006/pr /prm_ric/xls/vppv_2011_2019.xls (accessed on march 02, 2021). [2]. v. shyshkin, o. onyschenko, present state and prospects of agricultural development of ukraine, management and entrepreneurship: trends of development, 3(13), 72-86 (2020). doi: 10.26661/2522-1566/2020-3/13-06 . [3]. b. moss, water pollution by agriculture, phil. trans. r. soc. b, 363, 659-666 (2007). doi: 10.1098/rstb.2007.2176. [4]. s sharma, a. aygun, h. simsek, electrochemical treatment of sunflower oil refinery wastewater and optimization of the parameters using response surface methodology, chemosphere, 249, 126511 (2020). doi: 10.1016/j.chemosphere.2020.126511. [5]. s. v. sverguzova et al, extracting vegetable oils from model waters by sorbent on the base on carbonate sludge. iop conf. ser.: earth environ. sci., 579, 012042 (2020). doi: 10.1088/17551315/579/1/012042. [6]. v. r. vodyanka et al, the use of thiosemicarbazyde in the pressure–driven processes of wastewater treatment, j. water chem. & tech., 33(3), 196–201 (2011). [7]. a. s. makarov et al, utilization of industrial wastewater in production of coal-water fuel, j. water chem. & tech., 36(4), 180–183 (2014). [8]. m.a. de la rubia, f. raposo, b. rincon, r. borja, evaluation of the hydrolytic– acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake, biores. tech., 100(18), 4133-4138 (2009). doi: 10.1016/j.biortech.2009.04.001. [9]. y. saatci, e. i. arsalan, v. konar, removal of total lipids and fatty acids from sunflower oil factory effluent by uasb reactor, biores. tech., 87(3), 269-272 (2003). doi: 10.1016/s0960-8524(02)00255-9. [10]. a. v. artemov, a. v. pinkin, sorption technologies for cleaning the oil contaminated water, water: chem. & ecol., 1(1), 19-25 (2008). [11]. y. avsar, u. kurt, t. gonullu, comparison of classical chemical and electrochemical processes for treating rose processing wastewater, j. hazard. mater., 148(1-2), 340-345 (2007). [12]. b. g. miller, coal-water slurry fuel utilization in utility and industrial boilers, chem. eng. progr., 85(3), 29-38 (1989). [13]. d. o. glushkov, p. a. strizhak, m. yu. chernetskii, organic coal-water fuel: problems and advances (review), therm. eng., 63, 707-717 (2016). [14]. z. meng et al., effect of coal slime on the slurry ability of a semi-coke water slurry, powder tech., 359, 261-267 (2020). [15]. g. s. nyashina, p. a. strizhak, the influence of liquid plant additives on the anthropogenic gas emission from the combustion of coal-water slurries, env. pollut., 242a, 31-41 (2018). http://www.ukrstat.gov.ua/operativ/operativ2006/pr/prm_ric/xls/vppv_2011_2019.xls http://www.ukrstat.gov.ua/operativ/operativ2006/pr/prm_ric/xls/vppv_2011_2019.xls https://doi.org/10.1098/rstb.2007.2176 https://doi.org/10.1016/j.chemosphere.2020.126511 https://doi.org/10.1088/1755-1315/579/1/012042 https://doi.org/10.1088/1755-1315/579/1/012042 1. introduction further increase in the adsorbent mass does not result in the corresponding increase in the cleaning efficiency. therefore, it can be concluded that 15 % or 10 % of anthracite added to the more or less contaminated samples of the oil-processing wastew... fig. 2. dependence of the wastewater absorbance on the mass of anthracite added for extraction. ♦ – sample 1; ■ – sample 2. fig. 3. dependence of the wastewater conductivity on the mass of anthracite added for extraction. ♦ – sample 1; ■ – sample 2. (1) 4. conclusion звіт з ндр 29-81 за 2007 – 2009 р 139 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 2 – 2017, pag. 139 147 the effect of microencapsulation and potato starch on the survival of lactobacillus strains * liliana luca 1 , mircea oroian 1 1faculty of food engineering, stefan cel mare university of suceava, romania lucaliliana@yahoo.com *corresponding author received 15th may 2020, accepted 28th june 2020 abstract: the effectiveness of encapsulating ofactive bacteria in releasing probiotics depends on cell survival. in this study, a prebiotic potato starch was used to evaluate its influence on the viability of probiotics microcapsulated. lactobacilluscasei, lactobacillus rhamnosus and lactobacillus plantarumwas cultured for 16 hours and after that, the cells were added to an emulsion with 1.5% sodium alginate, supplemented with 1% prebiotics and encapsulated. initial tests revealed that the initial culture concentration is not relevant for prebiotic supplementation. the testing of fresh microcapsules recorded a load of 3.31 × 10 ¹³ cfu / g. after 7 days of storage, the probiotics that survived were 2.53 × 10¹¹ colony-forming unit/g (cfu / g) and, after 30 days1.7 × 107cfu / g. the probiotics stored for 7 days at 4 °c were tested for achieving the survival rate of the probiotics in the simulated intestinal juice for 1 hour and it was determined a survival rate of 108cfu / g. the simulation for 4 hours in the intestinal juice reported a survival of 106cfu / g. in conclusion, starch is an effective material for encapsulating lactobacillus cultures. keywords: probiotics, prebiotics, lactobacillus rhamnosus, l.plantarum, l.casei 1. introduction from metchnikof onwards, studies of lactic acid bacteria (lab) began to be increasingly numerous. this group of bacteria are actually natural inhabitants of the human gastrointestinal tract and play an important role in maintaining the microbial ecosystem of the colon. lab are considered extremely advantageous nonpathogenic species for the human population that play important roles in nutrition, metabolism, immunity and defense against pathogens. according to the definition of probiotics given by (fao / who, 2002): living microorganisms that when administered in adequate amounts confer beneficial effects to the host, many species and genera of lab are considered to be probiotics[1].probiotic explorations have led to the development of prebiotics, which are a source of carbon and at the same time bioprotectors for probiotics. the prebiotic sources are represented by vegetables, fruits, grains from which we can mention: starch, inulin, oligofructose, xylose, galactose, etc. although prebiotics are not easily digested in the human intestine, they have a selective role in stimulating the growth or the activity of beneficial bacterial species in the intestine, being nutrients that modify the intestinal microbial flora [2].besides supporting growth, survival of probiotics and suppressing the development of pathogenic micro-organisms, a symbiotic interaction indicates a great potential for improving the efficiency of the functional food class. taking into account the fao/who, 2001recommenddations, that at the time of food consumption the concentrations of live microorganisms must be above 106107cfu g ⁻¹ or ml ⁻¹ then it is necessary to ensure the viability of probiotic strains. this viability may be affected during about:blank about:blank food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 140 processing and storage: temperature, chemical additives, dissolved oxygen, ph, hydrogen peroxide and many others; then after this they will have to survive the storage temperatures, the storage time; and last but not least, it should survive the transit through the digestive tract, low ph values, gastric juice, bile juice, pancreatic juice and intestinal juice, etc.[3].over the years many methods have been conducted in order to protect the probiotics and to improve their survival against the above mentioned harmful factors. one of these methods is the creation by a mechanical process of a physical-chemical barrier around probiotic cells. microencapsulation is a process that tries to improve and maintain the survival of the cells, so that their release to the site of action is in the optimal number for human and animal health. several encapsulation techniques have been developed, of which the most economically accessible and which do not impose harmful solutions and can be performed in both aerobic and anaerobic conditions, is the extrusion encapsulation technique [4-7].at the same time, besides the encapsulation method, an important criterion for the production of resistant probiotic microcapsules is the selection of the appropriate encapsulation material at appropriate concentrations, so that it can resist all harmful factors [8]. therefore, the aim of this study was to evaluate the effect of the addition of an prebiotic (potato starch) in microcapsules produced by the ionic extraction / gelling technique, which contain a mixture of lactobacillus strains(l. casei 431, l. plantarum and l. rhamnosus). evaluation was performed on microcapsules stored at 4 °c for 30 days. 2. materials and methods media and substrates for encapsulation the reagents used were: potato starch soluble (sth), tween 80, de man – rogosa – sharpe (mrs) broth, manganese (ii) sulfate hydrate, potassium chloride, sodium chloride sodium citrate, peptone from casein, meat extract, yeast extract, magnesium sulfate heptahydrate and glucose which werepurchased from sigma-aldrich, saint louis, usa; calcium chloride hex hydrate was purchased from lach-ner company czech republic; sunflower oil, alginic acid sodium salt was purchased from applichem gmbh, mrs agar waspurchased from vwr international bvba/sprl; acetic acidammonium salt, sodium salt trihydrate, sodium phosphate, acetic acid, and sodium phosphate dibasic hydratewere purchased from across organics-spain. bacterial strain and culture conditions the bacterial strains used in this study were lactobacillus plantarum and lactobacillus rhamnosus (bioprox rp 80, france, lactobacillus cells count = 7±2x10e10) and l. casei 431 (christian hansen). from l. casei 431 strain, pure cultures were obtained after 3 reactivations in mrs broth and stored at -20 °c in glycerol.the lyophilized frozen bacterial strains were reactivated 24 hours at 37ocin mrs broth, then for another 24 hours at 37oc in modified mrs broth carbon source, glucose, was replaced with a prebiotic likepotato starchto obtain a densitometry of 5 mcfarland units for l. casei and 10 mcfarland units for the other 2 strains (lactobacillus plantarum and lactobacillus rhamnosus) grown together.these values were determined on a mcfarland densitometer with mcfarland measuring range of 0.3 15.0 at wavelength λ = 565 ± 15 nm. the three strains were inoculated for 24 hours at 37c and raised together in the modified mrs broth media environment. from the latter culture a 16-hour culture was obtained. this one was centrifuged at 2500 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 141 × g for 10 min at 4 °c ± 1 and the pellet was used for micro-encapsulation. encapsulation by extrusion the extrusion technique was used for the preparation of alginate beads. the alginate was added together with 1% prebiotic, 10% vegetable oil in 85% distilled water and mixed using a magnetic stirrer (1400 rpm), with heating until the mixture was completely dissolved. the alginate solution (1.75%) was then autoclaved for 15 minutes at 121°c and allowed to cool to 25 °c. this mixture was stirred at 10000 u/min for 30min. 15 ml of the bacterial pellet was added to the emulsion. this mixture was stirred again for 15 min at 600 rpm / min. then, the emulsion was injected using a peristaltic pump with a power of 3.2 rpm / ml through a needle with a diameter of 0.4 mm. the suspension was dropped into aliquots of sterile calcium chloride solution (2 %) from different heights: 1.5-2.5cm, and 10 15 cm. the droplets formed gel spheres immediately. the beads were allowed to stand for 30 min to harden, then there were harvested using a vacuum pump. the whole procedure was performed using autoclaved (121°c, 15 min) materials and under sterile conditions in a horizontal laminar air-flow cabinet. all solutions and media were prepared with distilled water (aquatron a4000d, cole-parmer ltd) and all glassware and reagents used in the experiment were sterilized before use. viable cell count entrapped within micro-capsules the viability of the lactobacillus strains was determined by homogenizing 0.1 g of fresh microcapsules in 9.9 ml of 1% sterile sodium citrate solution at ph 6.0, by gentle shaking at room temperature for 10-12 minutes. after the release of the cells from the microcapsules,serial dilutions were performed in sterile distilled water and then inoculated in mrs agar. the plates were incubated in anaerobiotic condition at 37 °c for 72 hours. the determination was made in triplicate, and the results were expressed in colony forming units (cfu) / g⁻¹ of microcapsules. bacterial enumeration and survival during storage the microcapsules resulting from the encapsulation process were recovered from calcium chloride, washed and then stored in sealed, sterile petri plates at 4 °c for 30 days. evaluation of survival during 30 days at 4 °c of the lactobacillus (l. casei, l. plantarum, l. rhamnosus)strains in various concentrations of alginate microcapsules and various carbon sources was made at 0, 7, 14, 30 days in triplicate. for the evaluation of the number of viable cells, the protocol described above was followed. the results were expressed as cfu / g of microcapsules. survival of microencapsulated cells to simulated intestinal condition the survival of microencapsulated bacteria was analyzed by counting the viable cells after treatment with simulated intestinal condition.simulated intestinal juice was prepared by dissolving bile salts in intestinal solution (0.65% nacl, 0.0835% kcl, 0.022% cacl2 and 0.1386% nahco3) ph 7.5 to final concentrations of 0.3% [9]. for thisanalysis it wasused beads which were stored for 7 days at 4 °c.allthedeterminationwere made in triplicate andincubated at 37 °c for 60, 120, 180and 240 min. aftereachincubation, survival of microencapsulatedcells was immediately counted by pour plating on mrs agar following the protocol described above. microencapsulation efficiency (me %) andencapsulation yield (ey%) microencapsulation efficiency is the survival rate of microorganisms during the microencapsulation process and calculated according to: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 142 me % = n1 / n0 x 100 (1) as proposed by martinet et al. [10], where n 1 is the number of viable cells (log cfu g-1) released from the microcapsules and n 0 is the number of viable cells (log cfu g 1) in the cell concentrate used for microencapsulation. the encapsulation yield (ey) was calculated according to the formula used by [9, 11, 12]: ey = log10n1 / log10n0 x 100 where log10n0 represents the number of viable cells trapped in the capsule and log10n1represents the amount of free viable cells added to the emulsion during the encapsulation process and the result of the equation is expressed as number of cfu / ml. this formula, ey, represents a criterion for measuring how the encapsulation process influences the number of viable cells [12]. characterization of the microcapsules the dimensions and the morphological characterization of a number of 50 randomly chosen micro-capsules were performed using optical microscopy and electron microscopy. the surface morphology of the capsules was determined by scanning electron microscope (sem). statistical analysis the results were computed, the means submitted to a normality test and reported as the means and standard deviations. all the variables were submitted to a two-way anova analysis followed by tukey’s multiple comparisons test with a significance level of 5% (p < 0.05). 3. results and discussions morphological characterization of micro particles the characterization of the particle morphology helped us to a better understanding of the possible morphological or structural changes of the microcapsules during the microencapsulation process. the size of the microcapsules can be influenced by factors such as: alginate concentration, calcium chloride concentration, needle diameter, pump pressure, distance between the needle and calcium chloride solution.following the study of the morphology of the microcapsules with the help of the optical microscope, it was observed different forms of microcapsules resulted from the microencapsulation process but the most abundant were the round ones. it was also observed that the vast majority are not adherent to each other and the dimensions are much smaller than 1 mm than in other studies such as those conducted by mokarram et al., [13]; hansen et al. [14] who obtained microcapsules with dimensions > 1mm using the same encapsulation technique and the same material for the capsule matrix.at the same time, the study of the morphology of the micocapsules was also performed with the help of the scanning electron microscope where it was also observed that there are different forms (figure 1 – a,b, c;) but very few adhesions between them. with the help of electron microscopy it was observed that on the surface of the microcapsules, from the study, there are no free lactobacillus cells, which leads to the idea that the methodology approached for microencapsulation is improved. evaluation of the mean diameter and distribution of microparticles size figure 2 shows the dimensional distributions of the microcapsules that have sth in their matrix.on the other hand, the histogram shows the presence of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 143 two well-separated groups of sizes, one corresponding to the dimensions between 450-500μm and the other between 550600μm. according to table 1 it can be seen that the most common are microcapsules with size 483μm.factors such as stirring speed, the concentration of surfactant or the ratio between water and oil (v / v) can influence the distribution and the size of the microcapsules. the capsule compression can also be explained by the inner water partial expulsion due to addition of the cellular pellet in the emulsion and the expulsion of the emulsion in calcium chloride with gel layer forming[10, 15]. table1. beads size expressed as mean of 50 microcapsules ± standard deviation. size in micrometers (μm) mean 504.1 ± 119 median 497.5 mode 483 minimum 259 maximum 920 count 50 comparing with other studies, it was found that the method approached in this study is improved because smaller microcapsules were obtained. for example lenton et al., [16] obtained an average size for microcapsules of 2.9 mm using also microencapsulation by extrusion; then in another study in which a needle similar to the one in the present study was used, muthukumarasamy et al., [17] obtained an average size of 2.37 mm; in another study it has obtained an average of the microcapsules 1.86mm [18] obtained by microencapsulation by extrusion. fig. 2. particle size distribution expressed as percentage of total particles. (n =50) viable cell count and microencapsulation efficiency and yield to determine the efficiency of the matrix with the chosen prebiotic, the way the probiotic cells were protected during encapsulation, i.e. the efficiency of encapsulation, was analyzed. table 2 shows the results obtained for the starch microcapsules and it can be observed that they have an optimal encapsulation efficiency of 100%.the results showed an encapsulation efficiency of over 100%, this occurs due to the fact that the capsules are a suitable medium for the growth of encapsulated bacteria. this percentage, over 100% is due to the fact that the matrix of the capsules contains prebiotics and that the enumeration of the encapsulated bacteria was performed approximately 2 hours after encapsulation. in other studies it was observed that after 4-6 hours from encapsulation the growth rates for probiotic strains of lactobacillus plantarum and l. rhamnosus or l. casei can be about 1 log [19, 20]. analyzing other authors such as raddatz et al. [8] who used as an encapsulation technique internal emulsification / gelling, they 0 5 10 15 20 25 30 35 40 100 200 300 400 500 600 700 800 950 % the size of the microcapsules with starch in μm histogram food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 144 obtained an ey% between 82.65% and 91.24%; and the authors jantarathin et al. [21] using the extrusion technique with l. acidophilus and using as material for the encapsulation matrix sodium alginate and inulin had an ey% = 88.19%.other authors obtained an even lower ey between 43% and 50 % using the internal gelling technique for the encapsulation of bifidobacterium bifidumf-35[22]. it is very important to maintain cell viability following the encapsulation process regardless of the type of encapsulation. a b c fig.1. scanning electron microscope photographs of fresh beads. a) bead with sth; b, c) surface of a bead with sth food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 145 table 2. number of cells entrapped compared to initial number of cells and encapsulation yield probiotic population before encapsulation (cfu/ml) encapsulated probiotic population (cfu/g) ey %= log10n1/log1 0 n0 2.40e+13±4.6 3.31e+13±2.7 101,04 viability of encapsulated bacteria during storage the cell viability of starch microcapsules stored in the refrigerator for 30 days at 4° c is shown in figure 3. the viability of the cells between the values obtained at the same time was statistically analyzed and it was found that there are significant differences (p <0.05) and these differences persisted over the 30 days. if in the first 7 days after encapsulation a reduction of 2.0 log cfu / g was observed, in the next 7 days the decrease of the cellular viability of the microcapsules was fig.3. stability of microparticles of lactobacillus during 30 days at 4ºc smaller, by 1.45 log cfu / g, so the cellular viability decreased by approximately 3.53 log cfu / g from time 0.therefore, the viability decreased in the first 7 days by 0.77% and between 7 and 14 days of storage the percentage was much higher, 3.68%.after 21 days of storage, a decrease in cell viability of 2.04 log cfu / g in microcapsules was observed compared to day 14 and a decrease of 5.57 log cfu / g compared to the time of encapsulation. this decrease will not be the same after 30 days when the reduction was only 0.64 log cfu / g compared to the previous period.after 30 days, the survival rate of lactobacillus cells in the microcapsules indicates a decrease of 6.21 log cfu / g. data obtained attest to the fact that the presence of a carbon source in the microcapsule matrix offers the possibility of the existence of a metabolic activity of probiotic cells at 4 °c.the decrease in cell viability is not due to the encapsulation process, but we believe that some of the main causes would be the time, the consumption of the nutrient substrate, the presence of compounds resulting from the metabolic process, such as metabolic acids and bacteriocins.on the other hand, at each sampling the petri dishes were opened, which could have led to the increase of humidity and implicitly to the increase of water activity and here the presence of residual water from microcapsules must be taken into account [14].humidity is also known to have a negative effect on cell viability [23]. analyzing other studies that used the same encapsulation method but different strains (l. gasseri and bifidobacterium bifidum),it was found that a decrease in viability was reported in the first 11 days of 4.11 cfu log / ml; and after 14 days they observed no survival [11].in order to survive and reach the intestine at the site of action in sufficient quantities to facilitate colonization, a large number of the initial cells must be trapped in the microcapsules. tabel 3 shows the cell viability following the microcapsules of the simulated intestinal conditions. it was found that after 4 hours cell viability falls within the limits recommended by the 1.00e+06 3.25e+07 1.06e+09 3.43e+10 1.12e+12 3.63e+13 1.18e+15 fresh beads 7 days 14 days 21 days 30 days c fu / g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 146 fao. however, in order to survive the passage through all the dull conditions of the digestive tract and to benefit from a beneficial action, the cellular concentration can be increased during encapsulation.this would lead to an increased number of bacterial survivors at the end of incubation in simulated gastric conditions. table 3. the effect of exposure to simulated intestinal juice on the survival of microcapsulated cells number of cells entrapped (log cfu/g) time (min) simulated intestinal juice 0 days 7 days 60 120 180 240 13.52 ± 1.27 11.40 ± 0.426 7.93 ± 0.561 7.74 ± 0.343 7.72 ± 0.621 6.27 ± 0.987 4. conclusions the present study showed that the microencapsulation of lactobacillus strains with a combination of alg (1.5%) and starch (1%) ensures a good protectionof probiotic bacteria during the 30 days of storage as well as in simulated intestinal conditions and at the same time it improves their viability. the results of this study also indicate that starch microcapsules can be used as a micrometric delivery vehicle due to an improved encapsulation method. investigating the stability of these microcapsules in food models during storage is an area that requires further research. 5. acknowledgements this research work was carried out with the support of decide -development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu / 380/6/13/125031, project co-financed from the european social fund through the 2014 – 2020 operational program human capital. 6. reference [1]. lin x., xia y., yang y., wang g., zhou w., ai l., robiotic characteristics of lactobacillus plantarum ar113 and its molecular mechanism of antioxidant. lwt, liu, c., kolida(2020) [2]. kerry r. g., patra j. k., gouda s., park y., shin h. s., das g., benefaction of probiotics for human health: a review. journal of food and drug analysis, 26(3), 927, (2018) [3]. martín m. j., lara-villosladaf., ruiz m. a., morales m. e.,microencapsulation of bacteria: a review of different technologies and their impact on the probiotic effects. innovative food science & emerging technologies,27,15, (2015) [4]. qaziyani s. d., pourfarzad a., gheibi s., nasiraie l. r.,effect of encapsulation and wall material on the probiotic survival and physicochemical properties of synbiotic chewing gum: study with univariate and multivariate analyses. heliyon, 5(7), e02144, (2019) [5]. burgain j., gaiani c., linder m., scher j., encapsulation of probiotic living cells: from laboratory scale to industrial applications. journal of food engineering, 104(4), 467-483, (2011) [6]. li h., zhang t., li c., zheng s., li h., yu j. development of a microencapsulated synbiotic product and its application in yoghurt. lwt, 122, 109033, (2020) [7]. xavier-santos d., bedani r., lima e. d., saad, s. m. i.,impact of probiotics and prebiotics targeting metabolic syndrome. journal of functional foods, 103666, (2019) [8]. raddatz g. c., poletto g., de deus c., codevilla c. f., cichoski a. j., jacoblopes e., de menezes c. r., use of prebiotic sources to increase probiotic viability in pectin microparticles obtained by emulsification/internal gelation followed by freeze-drying. food research international, 130, 108902, (2020) [9]. rather s. a., akhter r., masoodi f. a., gani a., wani s. m.,effect of double food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 liliana luca, mircea oroian, the effect of microencapsulation and potato starch on the survival of lactobacillus strains, food and environment safety, volume xix, issue 2 – 2020, pag. 139– 147 147 alginate microencapsulation on in vitro digestibility and thermal tolerance of lactobacillus plantarum ncdc201 and l. casei ncdc297. lwt-food science and technology, 83, 50-58, (2017) [10]. martin m. j., lara-villoslada f., ruiz m. a., morales m. e.,effect of unmodified starch on viability of alginateencapsulated lactobacillus fermentum cect5716. lwt-food science and technology, 53(2), 480486(2013) [11]. chávarri m., marañón, i., ares, r., ibáñez, f. c., marzo, f., &del carmen villarán, m.,microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions. international journal of food microbiology, 142(1-2), 185-189, (2010) [12]. picot a., lacroix c., encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. international dairy journal, 14(6), 505-515, (2004) [13]. mokarram r. r., mortazavi s. a., najafi m. h., shahidi f., the influence of multi stage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice. food research international, 42(8), 1040-1045, (2009) [14]. hansen l. t., allan-wojtas p. m., jin y. l., paulson a. t., survival of caalginate microencapsulated bifidobacterium spp. in milk and simulated gastrointestinal conditions. food microbiology,19(1), 35-45, (2002) [15]. sánchez m. t., ruiz m. a., lasserrot a., hormigo m., morales m. e.,an improved ionic gelation method to encapsulate lactobacillus spp. bacteria: protection, survival and stability study. food hydrocolloids, 69, 67-75, (2017) [16]. nualkaekul s., lenton d., cook m. t., khutoryanskiy v. v., charalampopoulos d., chitosan coated alginate beads for the survival of microencapsulated lactobacillus plantarum in pomegranate juice. carbohydrate polymers, 90(3), 1281-1287, (2012) [17]. muthukumarasamy p., holley r. a.,microbiological and sensory quality of dry fermented sausages containing alginate microencapsulated lactobacillus reuteri. international journal of food microbiology, 111(2), 164-169.75, (2006) [18]. valero-cases e., frutos, m. j.,effect of different types of encapsulation on the survival of lactobacillus plantarum during storage with inulin and in vitro digestion. lwt-food science and technology, 64(2), 824-828, (2015) [19]. luca l., oroian m., lobiuc a., the prebiotic potential of some carbohydrates on the growth of lactobacillus plantarum and lactobacillus rhamnosus. food & environment safety, 18(2), (2019) [20]. luca l., oroian m., the impact of potential prebiotics inulin, oligofructose and potato starch on the growth of lactobacillus casei. agrolife scientific journal, 8(1), 153-159, (2019) [21]. jantarathin s., borompichaichartkul c., sanguandeekul r., microencapsulation of probiotic and prebiotic in alginate-chitosan capsules and its effect on viability under heat process in shrimp feeding. materials today: proceedings, 4(5), 6166-6172, (2017) [22]. zou q., zhao j., liu x., tian f., zhang h. p., zhang h., chen, w., microencapsulation of bifidobacteriumbifidum f‐ 35 in reinforced alginate microspheres prepared by emulsification/internal gelation. international journal of food science & technology, 46(8), 16721678, (2011) [23]. heidebach t., först p., kulozik u., influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. journal of food engineering, 98(3), 309-316, (2010) effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin 82 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2-2019, pag.82 88 optimization of sequential ultrasound-assisted extraction – heating treatment to obtain pectin from malus domestica ‘fălticeni’ pomace *florina dranca 1 , mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, florina.dranca@usm.ro, *corresponding author received 15th march 2019, accepted 25th june 2019 abstract: in this study, a sequential ultrasound-assisted extraction – heating treatment (uaeh) was applied for pectin extraction from malus domestica ‘fălticeni’ pomace. the extraction procedure involved a sonication at 20 khz and 100% amplitude for 30 min (solid-to liquid ratio of the apple pomace-water mixture of 1:10, ph of 1.8), followed by a heating treatment at different temperatures (70, 80 or 90 °c) and time (60, 120 or 180 min). it was observed that with the increase of temperature and time, pectin yield and the degree of esterification increased. at the same time, the increase of temperature determined a decrease of the galacturonic acid content of the extracted pectin. a boxbehnken response surface design was used to optimize the effects of process variables on the pectin yield and physico-chemical properties. the optimum conditions to obtain a maximum yield of 6.85%, with a 69.694 g/100 g galacturonic acid content and 80.09% degree of esterification of the extracted pectin were the temperature of 85.71 ºc and 147 min heating time. keywords: ultrasound, heating, pectin yield, composition. 1. introduction pectin is a complex and heterogeneous polysaccharide molecule found in the middle lamella and primary cell walls of higher plants [1]. structurally, pectin is a polymer composed of at least 17 different monosaccharides interconnected through more than 20 different linkages, having a backbone that consists primarily of a linear chain of α-(1→4)-d-galacturonic acid (gala) units interrupted by occasional lrhamnose (l-rha) residues [2]. the major pectic substructures are homogalacturonan (hg), rhamnogalacturonan i (rg-i), and rhamnogalacturonan ii (rg-ii). the hg substructure has a backbone formed of dgala units which may be methyl esterified at c-6 and/or o-acetylated at o-2 and/or o3 [3]. rg-i, the second major substructure, is composed of a repeating [→2)-α-l-rha(1→4)-α-d-gala-(1→] disaccharide in which the l-rha residues can be substituted and may vary among plants [4]. rg-ii, a very complex branched substructure occurring with much less frequency than rg-i, has highly conserved side chains containing different sugars and derivatives [5]. pectin structure is described as consisting of a hg backbone that has rg-i and rg-ii attachments, however, recent direct visualization of the molecular structure of the polysaccharide contributed to a new alternative structure characterized by the presence of hg as side chains of rg-i [6]. commercially, pectin is extracted from two main sources, namely citrus peel and apple pomace. the sources for pectin extraction are the focus of numerous recent studies that investigated other agriculture byproducts and wastes containing important http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 83 amounts of pectic polysaccharides. among waste streams from fruits and vegetables processing, sugar beet pulp, berry pomace, potato pulp, cocoa husks, mulberry branch bark, faba bean hulls, pistachio green hulls, pomegranate peels, pumpkin, banana peel and mango peel were studied [7]. apart from achieving waste valorization, these other sources can also provide pectins with diverse structures and functional properties. the utilization of a suitable method for pectin extraction is necessary in order to maximize the extraction yield and obtain a good product quality. the methods applied to extract pectin from different plant sources can be distinguished by the approach taken: on one side, conventional techniques such as extraction with mineral acids offer the advantage of an increased pectin yield, and on the other side several innovative techniques bring advantages such as the shorter extraction time, lower energy consumption, and therefore reduced impact on the environment. the innovative extraction techniques are microwave-, ultrasound-, enzyme-assisted extraction, subcritical water extraction, and combined techniques such as ultrasound-microwave assisted extraction, ultrasound-assisted heating extraction and enzymatic-ultrasonic extraction. this study aimed at evaluating the use of a sequential ultrasound-assisted extraction – heating treatment (uaeh) process to isolate pectin from apple (malus domestica ‘fălticeni’) pomace. the extraction of pectin from this plant source provides a mean to increase the economic viability of malus domestica ‘fălticeni’, an apple hybrid that is native to the geographical area of fălticeni, suceava county (romania), where it is primarily cultivated and also processed into juice in small scale plants. the effects of the combined extraction technique were evaluated in terms of pectin yield and galacturonic acid content and degree of esterification of the extracted pectin. 2. materials and methods 2.1. materials apple pomace was obtained after the extraction of juice from malus domestica ‘fălticeni’ apples. the apples were from 2016 harvest, and were cultivated in the fălticeni area of suceava, romania. the pomace was dried in an oven with air circulation at 60 °c to a constant weight then powdered in a food processor. the resulting powder was passed through an analytical sieve shaker retsch as 200 (retsch gmbh, germany), and pomace with 125-200 µm particle sizes was used in the extraction process. citric acid, ethyl alcohol, sulfamic acid, potassium hydroxide, sulfuric acid, sodium tetraborate, sodium hydroxide, hydrochloric acid, d-galacturonic acid, and mhydroxydiphenyl were purchased from merck kgaa (germany). 2.2. extraction and purification procedure the extraction mixture was prepared by mixing 10 g of apple pomace powder with 100 ml of distilled water acidified to a ph of 1.8 with citric acid (solid-to-liquid ratio of 1:10, w/v). the ultrasound-assisted extraction (uae) was performed with an ultrasonic device (sonopuls hd 2070, bandelin, germany), operated at 20 khz, and equipped with a flat tip probe (ke 76, bandelin, germany) that was submerged at 15 mm depth into the extraction mixture. sonication was carried out for 30 min at 100% amplitude. following the uae, the samples were placed in a water bath and exposed to a heating treatment in the following conditions: temperature of 70, 80 or 90 °c and time of 60, 120 or 180 min (table 1). after the sequential ultrasound-assisted extraction – heating treatment (uaeh), the mixture was cooled to room temperature prior to precipitation and purification. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 84 extract was first separated from the remaining solid material by centrifugation at 4000 rpm for 40 min, after that it was passed through a clean cheesecloth (folded 6 times) fitted into the neck of a laboratory bottle with screw cap, and then mixed with ethyl alcohol (>96%, v/v) in a 1:1 ratio (v/v) to precipitate pectin. the bottles were tightly closed, and then the content was thoroughly mixed and kept at 4-6 °c for 12 h to complete the precipitation. next, pectin was separated from the liquid by centrifugation (4000 rpm, 30 min), was washed 3 times by ethyl alcohol, and dried in a hot air oven at 50 °c to a constant weight. 2.3. determination of pectin yield pectin yield was calculated using eq. (1): 100(%) 0  m m yield (1) where: m0 – weight of dried pectin (g) and m – weight of dried apple pomace powder (g) [8]. 2.4. determination of galacturonic acid content the galacturonic acid content (gala) of pectin was determined in triplicate by the sulfamate/m-hydroxydiphenyl method developed by filisetti-cozzi and carpita [9]. sample preparation was made according to [10], as follows: 20 mg of dry pectin were added to 50 ml of distilled water (at 40 ºc) and mixed using a magnetic stirrer until completely dispersed. after that, the volume was adjusted to 100 ml with distilled water at 40 ºc. aliquots of 400 μl from the pectin solutions were placed in glass tubes, to which were added 40 μl of 4 m sulfamic acid solution (adjusted to ph 1.6 with saturated solution of potassium hydroxide), followed by 2.4 ml of sulfuric acid containing 75 mm of sodium tetraborate. the mixture was hydrolyzed in a 100 ºc water bath for 20 minutes, and then cooled in an ice bath for 10 minutes. after cooling, 80 μl of mhydroxydiphenyl solution in 0.5% (w/v) sodium hydroxide were added and the content was vortex mixed. between 10 min and 30 min after complete mixture the absorbance was read at 525 nm against the reagent control using a uv-vis-nir spectrophotometer (shimadzu corporation, japan). for each batch of samples, a calibration curve of d-galacturonic acid was performed. 2.5. determination of degree of esterification the degree of esterification (de) of pectin was determined in triplicate by the titrimetric method described by franchi et al. [11], as follows: 50 mg of pectin were dissolved in 10 ml of boiled distilled water, then the resulting solution was titrated with 0.1 n naoh using phenolphthalein as indicator; the volume of sodium hydroxide used for titration, v1, was recorded. after titration, 20 ml of 0.5 m naoh were added and the solution was kept under continuous stirring at 400 rpm for 30 min to achieve saponification. then, 20 ml of 0.5 m hcl were added to neutralize the solution and a final titration with naoh was made (v2). the degree of esterification was calculated using eq. (2): 100(%) 21 2    vv v de (2) where: v1 – volume of sodium hydroxide used for the first titration (ml) and v2 – volume of sodium hydroxide used for the second titration (ml). 2.6. experimental design and statistical analysis a two factors, three levels box-behnken response surface experimental design (bbd), with two repetitions at the center points, was employed to investigate the individual and interactive effects of process variables – temperature (x1) and time (x2) – on pectin yield and its galacturonic acid content and degree of esterification. the independent variables and their coded food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 85 levels are presented in table 1. experimental data was analyzed using design expert 11 (trial version) (state-ease inc., minneapolis, usa). 3. results and discussion 3.1. model fitting and statistical analysis the experimental results of uaeh extraction of pectin from malus domestica ‘fălticeni’ apple pomace using two-factorthree level bbd are shown in table 2. analysis of variance (anova) was used to examine the statistical significance of the model terms and the results are listed in table 3. the model f-values and the associated low p-values (p < 0.05) implies the developed model was significant and indicates that most of the variation in the response can be explained by the regression equation. the high value of r2 clearly stated that the quadratic model can explain and predict most of the variation of pectin yield (r2=0.9591), galacturonic acid content (r2=0.9317) and degree of esterification (r2=0.9263). the square polynomial equations that describe the combined effect of temperature (x1) and time (x2) on the extraction yield (eq. (3)), galacturonic acid content (eq. (4)) and degree of esterification (eq. (5)) are shown below: y (%) = 4.073 + 2.445 × x1 + 1.134 × x2 + 0.886 × x1 × x2 + 2.008 × x1 2 + 0.018 × x2 2 (3) gala (g/100 g) = 71.218 – 4.6 × x1 + 2.178 × x2 + 5.547 × x1 × x2 – 0.925 × x1 2 – 4.9 × x2 2 (4) de (%) = 89.95 – 5.36 × x1 – 4.011 × x2 – 2.615 × x1 × x2 – 7.4 × x1 2 + 0.135 × x2 2 (5) table 1. independent variables and levels used for box-behnken design variables levels -1 0 1 x1: temperature, ºc 70 80 90 x2: time, min 60 120 180 table 2. box-behnken design with experimental and predicted values run independent variables measured response predicted response x1 x2 y (%) gala (g/100 g) de (%) y (%) gala (g/100 g) de (%) 1 80 120 3.49 70.55 90.65 4.07 71.22 89.95 2 80 180 5.87 68.88 88.56 5.23 68.50 86.07 3 70 180 3.54 65.09 85.49 3.90 66.63 86.65 4 70 60 3.10 71.55 91.47 3.41 73.37 89.44 5 70 120 4.31 78.26 87.04 3.64 74.90 87.91 6 90 60 6.31 53.93 85.81 6.52 53.06 83.95 7 80 60 3.48 65.08 90.21 2.96 64.14 94.10 8 80 120 3.49 70.55 90.65 4.07 71.22 89.95 9 90 180 10.30 69.66 69.37 10.57 68.51 70.70 10 90 120 9.01 70.55 76.66 8.53 71.22 77.19 x1 – temperature, x2 – time; y – pectin yield, gala – galacturonic acid content, de – degree of esterification. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 86 table 3. analysis of variance (anova) of quadratic model source sum of squares degree of freedom mean square f-value p-value (a) pectin yield, % model 56.47 5 11.29 18.77 0.0070 temperature 35.89 1 35.89 59.66 0.0015 time 7.73 1 7.73 12.85 0.0231 temperature × time 3.15 1 3.15 5.23 0.0842 r² 0.9591 adjusted r² 0.9080 (b) galacturonic acid content, g/100 g model 342.27 5 68.45 10.92 0.0190 temperature 127.39 1 127.39 20.32 0.0108 time 28.47 1 28.47 4.54 0.1001 temperature × time 123.08 1 123.08 19.63 0.0114 r² 0.9317 adjusted r² 0.8464 (c) degree of esterification, % model 426.96 5 85.39 10.05 0.0220 temperature 172.38 1 172.38 20.29 0.0108 time 96.56 1 96.56 11.37 0.0280 temperature × time 27.35 1 27.35 3.22 0.1472 r² 0.9263 adjusted r² 0.8341 fig. 1. response surface plots showing the effect of process variables on pectin yield (a), galacturonic acid content (b) and degree of esterification (c) 3.2. effect of process variables according to the data presented in table 2, pectin yield varied between a minimum value of 3.10% (temperature of 70 ºc, 60 min heating time) and a maximum of 10.30% (temperature of 90 ºc, 180 min heating time). fig. 1a shows that an increase of temperature and time led to a substantial increase of pectin yield; this increase was more pronounced when the temperature was higher than 80 ºc. both process variables had a significant influence on yield (p < 0.05), however, the interaction between them did not significantly influence this parameter (p > 0.05), as indicated by the results of the analysis of variance (table 3). some previous studies had a similar observation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 87 regarding the positive effects of high temperatures and longer exposure to heating on pectin extraction yield from pomegranate peel [12], banana peel [13], apple pomace [14], and lemon by-product [15]. the galacturonic acid content (fig. 1b) of the extracted pectin, which had values between 53.93 g/100 g (temperature of 90 ºc, 60 min heating time) and 78.26 g/100 g (temperature of 70 ºc, 120 min heating time), was significantly influenced by temperature and the temperature-time interaction (p < 0.05), as seen in table 3. the evolution of this parameter was opposite to that reported by previous studies, where the increase of gala at high temperatures was associated with a substantial hydrolysis of pectin neutral sugars found in the rhamnogalacturonic regions [12,16]. in the case of this study, the heating treatment applied after uae might have determined some degradation of pectin structure, which was more pronounced at the highest temperature and the shortest heating time. the evolution of the degree of esterification presented in fig. 1c shows that higher extraction temperature and prolonged time led to a decrease of this parameter to 69.37% (temperature of 90 ºc, 180 min heating time, table 2), while lower temperature and heating time was correlated with an increased de (91.47% at a temperature of 70 ºc, 60 min heating time, table 2). a similar observation was made regarding the de of pectin extraction from pomegranate peel [12] and banana peel [13], and was ascribed to the contributing effect of harsher extraction conditions on the deesterification of the polygalacturonic acid chain [17]. however, it is possible that harsher treatments may also extract more strongly bound pectins, possibly of lower degree of esterification, as stated previously [18]. overall, all extracted pectin samples were of high de and this psychochemical parameter was significantly influenced (p < 0.05) by temperature and time, but not by the interaction of these two process variables (p > 0.05). 3.3. optimization of extraction parameters the objective of optimization was to find out the uaeh conditions which give the maximum extraction yield of pectin and the highest galacturonic acid content and degree of esterification. the optimum extraction conditions were a temperature of 85.71 ºc and time of 147 min, and the maximum yield of pectin achievable in these conditions was 6.85%, with a 69.694 g/100 g gala and de of 80.09%. 4. conclusion in this study, sequential ultrasound-assisted extraction – heating treatment was optimized for the extraction of pectin from malus domestica ‘fălticeni’ pomace. two factors at three levels box-behnken response surface experimental design were successfully used to optimize and study the individual and interactive effect of process variables such as temperature and time on the extraction yield of pectin and its galacturonic acid content and degree of esterification. from the experimental results, the change of temperature was found to influence both the extraction yield and the physico-chemical properties of pectin, while the variation of time strongly affected pectin yield and its degree of esterification. for the optimum extraction conditions (85.71 ºc, 147 min) it was estimated a maximum pectin yield obtained by uaeh of 6.85%, with 69.694 g/100 g gala and de of 80.09%. 5. acknowledgement this work was supported by a grant of the romania national council for higher education funding, cnfis, project number cnfis-fdi-2019-0600. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 florina dranca, mircea oroian, effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin, food and environment safety, volume xviii, issue 2 – 2019, pag. 82 – 88 88 6. references [1]. naraian, r., gautam, r. l. penicillium enzymes for the saccharification of lignocellulosic feedstocks, new and future developments in microbial biotechnology and bioengineering, elsevier, 121-136, (2018) [2]. kaya, m., sousa, a. g., crépeau, m. j., sørensen, s. o., ralet, m. c., characterization of citrus pectin samples extracted under different conditions: influence of acid type and ph of extraction, annals of botany, 114(6): 1319-1326, (2014) [3]. voragen, a. g., coenen, g. j., verhoef, r. p., schols, h. a., pectin, a versatile polysaccharide present in plant cell walls, structural chemistry, 20(2): 263-275, (2009) [4]. harholt, j., suttangkakul, a., scheller, h. v., biosynthesis of pectin, plant physiology, 153(2): 384-395, (2010) [5]. maxwell, e. g., belshaw, n. j., waldron, k. w., morris, v. j., pectin–an emerging new bioactive food polysaccharide, trends in food science & technology, 24(2): 64-73, (2012) [6]. round, a. n., rigby, n. m., macdougall, a. j., morris, v. j., a new view of pectin structure revealed by acid hydrolysis and atomic force microscopy, carbohydrate research, 345(4): 487-497, (2010) [7]. grassino, a. n., barba, f. j., brnčić, m., lorenzo, j. m., lucini, l., brnčić, s. r., analytical tools used for the identification and quantification of pectin extracted from plant food matrices, wastes and by-products: a review, food chemistry, 266: 47-55, (2018) [8]. liew, s. q., ngoh, g. c., yusoff, r., teoh, w. h., sequential ultrasound-microwave assisted acid extraction (umae) of pectin from pomelo peels, international journal of biological macromolecules, 93: 426-435, (2016) [9]. melton, l. d., smith, b. g., determination of the uronic acid content of plant cell walls using a colorimetric assay, current protocols in food analytical chemistry, (2002) [10]. miceli-garcia, l. g., pectin from apple pomace: extraction, characterization, and utilization in encapsulating alpha-tocopherol acetate, university of nebraska-lincoln, united states of america, (2014) [11]. franchi, m. l., marzialetti, m. b., pose, g. n., cavalitto, s. f., evaluation of enzymatic pectin extraction by a recombinant polygalacturonase (pgi) from apples and pears pomace of argentinean production and characterization of the extracted pectin, journal of food process technology, 5(8): 1-4, (2014) [12]. pereira, p. h. f., oliveira, t. í. s., rosa, m. f., cavalcante, f. l., moates, g. k., wellner, n., azeredo, h. m., pectin extraction from pomegranate peels with citric acid, international journal of biological macromolecules, 88: 373-379, (2016) [13]. emaga, t. h., ronkart, s. n., robert, c., wathelet, b., paquot, m., characterisation of pectins extracted from banana peels (musa aaa) under different conditions using an experimental design, food chemistry, 108(2): 463-471, (2008) [14]. garna, h., mabon, n., robert, c., cornet, c., nott, k., legros, h., paquot, m., effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol, journal of food science, 72(1): c001-c009, (2007) [15]. masmoudi, m., besbes, s., chaabouni, m., robert, c., paquot, m., blecker, c., attia, h., optimization of pectin extraction from lemon by-product with acidified date juice using response surface methodology, carbohydrate polymers, 74(2): 185-192, (2008) [16]. vriesmann, l. c., teófilo, r. f., de oliveira petkowicz, c. l., optimization of nitric acid-mediated extraction of pectin from cacao pod husks (theobroma cacao l.) using response surface methodology, carbohydrate polymers, 84(4): 1230-1236, (2011) [17]. joye, d. d., luzio, g. a., process for selective extraction of pectins from plant material by differential ph, carbohydrate polymers, 43(4): 337342, (2000) [18]. de roeck, a., sila, d. n., duvetter, t., van loey, a., hendrickx, m., effect of high pressure/high temperature processing on cell wall pectic substances in relation to firmness of carrot tissue, food chemistry, 107(3): 1225-1235, (2008) 1. introduction 14 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 14 22 intensification of the heat transfer through corrugated wall *donka stoeva1 1university of food tehnologies, mafi department, 26 maritsa blvd; 4000 plovdiv; bulgaria bodurova@gmail.com *corresponding author received february 17th 2014, accepted march 14th 2014 abstract: the corrugation of the smooth steel tubes increases the heating surface per length unit. the advantages compared to smooth pipe bodies are the lower relative mass at same thermal power and the smaller volume. the aim of the present research is to trace the temperature alteration near to a specially corrugated wall with baffles. the baffles have variable step towards the flow direction at two different heights. we observe developed turbulent air flow at temperature 353к (80oc) through a corrugated tube with inside diameter d=0.08m and length 0.2m. the tube wall is heated to 453k (180oc). the heat transfer between the tube and the flowing fluid has been modulated at two different corrugation grades of the tube – х1 and х2 – equation (9) and with four different reynolds numbers. key words: cfd, boundary layer , surface baffles, turbulization, circulation zone. 1. intorduction the intensity of the heat transfer can be influenced by alteration of the geometric dimensions of the channel, alteration of the velocity of the heat carrier and the form of the heat transfer surface, which defines the temperature field [1, 2, 3]. one of the ways for intensification of the heat flows is by increasing the velocity of the heat carrier or by corrugating the surfaces. when flowing onto hard wall there is a boundary layer forming. it is the main thermal resistant. the thicker the boundary layer is, the lower the heat transfer is [4, 5, 6]. the decreasing of the boundary layer thickness and the increasing of the transfer coefficients for moment and heat is the essence of the heat transfer intensification. most profitable hydrodynamic regime regarding the heat transfer is the transitional and turbulent regime in the boundary layer. this can be achieved by artificial turbulization of the flow in the boundary layer or destroying of the boundary layer near the wall. this enforces the application of artificial methods for intensification of the heat transfer. the methods for intensification of heat transfer are divided in two groups: passive and active. passive methods are the ones that do not need direct use of outer energy source. active are those, who use outer source. there are two methods used for intensification of the heat transfer: increase of the heat flow, regardless of the costs and increase of the heat flow at defined power of the heat carrier [1, 2, 3, 4]. the most frequently used passive method is intensification through surface baffles, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 15 such as rough surfaces and equally distributed roughness [5, 6]. the configuration of the baffles is chosen is such a way, so that it destroys the viscosity sub-layer and increases the turbulence near the wall [7, 9, 10]. one of the most important conditions for choosing of method is the hydrodynamic structure of the flow in which we need alteration in the temperature field distribution. knowing this structure, we can reduce the areas in which the increase of the intensification of the turbulent pulsations will make greatest influence on the heat transfer intensification [1, 7, 8, 9]. the analysis and the visualization of the flow in such tubes are of great importance because they have wide application in the heating equipment and many other spheres. the aim of the present research is to trace the temperature alteration near to a specially corrugated wall with baffles. the baffles have variable step towards the flow direction at two different heights. 2. materials and methods the model researches are carried out at four different reynolds numbers (re). we have used the equation for fluid movement for turbulent regime (equation 1) and we have added two equations for kinetic energy (equation 2)– k and kinetic energy dissipation (equation 3) 22ji i ti t i j j i i k i j uu u vk k k u v ( ) ( v ) v( ) x x x x x x x                        (1)  2k cvt  (2) k c x u x u x u v k c x v v xx u j i i j j i t iv t ii i 2 21 )()(                          (3) the constants are: 1 2 1 2 2 1 92 1 44 1 92 1 0 9 . c . ; c . ( . a a )      (4) for solving the heat transfer system of equations we add the equation for energy of the following type: eff j j eff hj ( e ) .( v( e p )) .( k t h j ( )) s t                  (5) where effk is the effective conductivity, tk is the turbulent conductivity defined by the used turbulent model, and jj  is a diffusion of the flow type j . the first three nominals at the right side of the equation present the energy transfer through conductivity, the specific diffusion and the viscous dissipation. hs includes heat from chemical reactions and others, if such are defined. 2 2 p v e h     (6) where the enthalpy h for an incompressive fluids is calculated by equation (7) : food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 16 j jj p h y h    (7) in the above equations jy is the mass fraction of the type j . t j p , jtref h c dt  (8) where tref =298.15к for the two-dimension case the mash contains 16500 cells, 33350 faces and 16581 knots as a preliminary test for independence of the solution from the mash thickness has been performed [3, 4, 9, 10]. we observe developed turbulent air flow at temperature 353к (80oc) through a corrugated tube with inside diameter d=0.08 m and length 0,2 m. the tube wall is heated to 453k (180o c). the heat transfer between the tube and the flowing fluid has been modulated at two different corrugation grades of the tube – х1 and х2 – equation (9) and with four different reynolds numbers (we achieve that by setting four different initial velocities at the boundary conditions – equation (10)). the graphic relations are shown for central section in the geometric body and for the outgoing section. 1 1 0 002 0 025 0 08 h . x . d .    ; 22 0 004 0 05 0 08 h . x . d .    (9) 1 1 5 0 75 0 08 3353 1 1 7894 10в х v .d . . re . .       22 5 1 0 08 4470 8 1 7894 10в х v .d . re . .       33 5 2 0 08 8941 5 1 7894 10в х v .d . re . .       544 5 2 8 0 08 0 125 10 1 7894 10в х v .d . . re . .        (10) fig. 1 geometric model of observed area from the corrugated tube with threshold height h1=2 mm. the steps between two thresholds are different. for the first drawing (fig 1) the height of the threshold is h1=2 mm. the dimensionless ration between the height of the threshold and the distance between the two thresholds calculated by equation (11) is: 1 1 1 0 045 22 5 0 002 p , y , h ,    ; 22 2 0 085 42 5 0 002 p , y , h ,    (11) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 17 fig. 2 geometric model of observed area from the corrugated tube with threshold height h2=4 mm on (fig.2) the height of the threshold is h2=4 mm. the distance between the thresholds is different. the dimensionless ration between the height of the threshold and the step p – equation (12) is: 1 3 1 0 045 11 25 0 004 p . y . h .    ; 24 2 0 085 21 25 0 004 p . y . h .    (12) 3. results and discussions -0,07 -0,06 -0,05 -0,04 -0,03 350 360 370 380 390 400 410 420 430 440 450 y-coo rdina te (m ) t x 1 =0.025 t x 2 =0.05 t em pe ra tu re [k ] twall=453k,ta ir=353k, re 1 =355 3.1 fig.3 temperature gradient in cross-section in the center for re1=3353.1 at two different thresholds х1 and х2 -0,07 -0,06 -0,05 -0,04 -0,03 340 360 380 400 420 440 460 t em pe ta tu te [k ] y-coordinate ( m) re 4 =0.125×105; x 1 =0.025 re 4 =0.125×105; x 2 =0.05 fig. 4. comparison of the temperature gradient in cross-section at re4=0.125×105 at two different thresholds х1 and х2 -0,07 -0,06 -0,05 -0,04 -0,03 340 360 380 400 420 440 460 y-coordinate ( m) te m pe ta tu te [k ] re4 x 1 =0.025 re4 x 2 =0.05 re1 x 1 =0.025 re1 x 2 =0.05 fig. 5 temperature gradient at the two heights of the threshold х1 and х2 for re1=3353.1 and re4 =0.125×105 the change in the flow character near the wall reflects in the distribution of the temperature and velocity towards the flow direction and perpendicular to the flow. the temperature profile change is most sensitive at the higher threshold (х2=0.05) at the same reynolds number. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 18 -0,070 -0,065 -0,060 -0,055 -0,050 340 360 380 400 420 440 460 t em pe ra tu re [ k ] y-coordinate [m] re1=3353.1; re2=4470.8 re3=8941.5; re4=0.125×10 5 fig.6. influence of the reynolds number on the temperature distribution at cross direction at х1. on figure 6 we can follow the influence of the reynolds criteria on the temperature distribution at geometric model with relative height of the threshold – х1=0.025. by increasing the value of re the temperature profile is changed as it nears the wall. the temperature profile at re4 is nearest to the wall, which means that at high re values there will be the best heat transfer and the heat transfer surface will used in the most effective way. the observation of the location and the size of the circulation zone give opportunity for optimization of the shape and geometrical parameters of the threshold. after modulation and analysis of the circulation zone we can estimate the best solution for maximal effective heat transfer, which is the final goal of the numerical modulation. every observation is performed after testing of the calculating mash for adequacy and after proving the independence of the solutions from the thickness of the calculating mash. fig.7 constant temperature lines – isotherms, x1=0.025 and re1=3353.1 fig.8 constant temperature lines isotherms, x1=0.025 and re1=3353.1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 19 lines with constant temperature – isotherms have been calculated and drawn. the visualization of the isotherms near the flowed wall is of great interest regarding the intensification. the isotherms are drawn for equal temperature intervals – by 20 degrees. the most intensive heat transfer we have near the wall, not in the centre of the flow that is why it is important to show the temperature distribution exactly at this place. after comparison of the isotherms (fig. 8 and fig. 9) at the two heights of the thresholds we can make a conclusion for the temperature gradient alteration. we can see that at the higher threshold the higher temperature isotherms are further from the wall. the prove for that statement is the visible difference in the temperature profiles near the wall (fig. 11 and fig. 12) at the two heights of the threshold and at same reynolds number. this means that at higher threshold there is better heat transfer. the increase of the threshold dimension can not be done infinitely and there is a maximal value, up to which it can be increased. fig. 9 constant temperature lines isotherms, x2=0.05 and re1=3353.1 fig.10 constant temperature lines – isotherms, x1=0.025 and re4=0.125×105 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 20 fig. 11 constant temperature lines – isotherms, x1=0.025 and re4=0.125×10 5 fig. 12 constant temperature lines – isotherms, x2=0.05 and re4=0.125×10 5 we can see on figures 7, 11 and 12 the simultaneous impact of the higher threshold as well as of the higher reynolds number. the correct choice of these two significant factors defines the good heat transfer between the heated surface and the cool fluid. at the exit on fig. 12 there is the smallest cold sector, which means that the best heat transfer will be achieved if the height of the threshold is 4mm and re4=0.125×105. by comparison of the shape of the temperature profiles we can estimate the influence of the corrugation and the velocity of the fluid on the temperature distribution in y direction. at the highest values of the reynolds number (re4) we have the highest value of the temperature of the heated fluid (air) at the outlet in perpendicular to the flow direction. we achieve better heat transfer at higher values of reynolds number. if we combine enough high threshold and velocity of the flowing fluid at the outlet we can achieve the least cold zone. that is the important role of the calculating hydrodynamics and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 21 the methods for modulation of such processes – because these modern calculation tools help making the right choice for the best solution amongst the many variants of the given problem. fig.13 influence of the threshold height on the temperature distribution we can see on fig. 13. the lines with the equations, which describe them for the size of the zone with temperature 353к. at the high threshold (х2) at the outlet the section with temperature 353к is 0.0026m and at the lower threshold (х1) it is 0.012m. we have mathematically described the dimensions of the zones with temperature 353к. the cold zone at the higher threshold is smaller, which means there is a better mixing and turbulization of the flow. 4. conclusion on the base of the result, we can make the following conclusions: 1. the uneven step of the baffles leads to better picture of the wall flowing. 2. by increasing the criteria of re we reduce the size of the non-flowing zone after the baffles, which leads to increasing the heat transfer. 3. the increasing of the temperature under the influence of the baffles is highly expressed in the direction of the flow. 4. the higher the threshold – the better the heat transfer. 5. there is a better heat transfer at higher reynolds number values. 6. if we combine high enough threshold and velocity of the fluid at the outlet, we achieve the smallest cold zone. 7. this research can be used for finding optimal values of the reynolds number regarding the conditions for heat transfer. 5. references [1]. kostov p., k. atanasov, n. krastev, d. angelova. „universal velocity profiles of twisted injected jet“, scientific works tome lx “food science, technology and processes – 2013“ 18-19 october 2013, plovdiv, pp.1531-1535. (in bulgarian) [2]. kostov p, krastev n, angelova d, observation of injected radial jet at isothermal conditions – national conference with international participation “machine science”2012 – sliven. heat technology 3, year 3, book 1 issn 13142550 pp. 8-11. (in bulgarian) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 donka stoeva , intensification of the heat transfer through corrugated wall, volume xiii, issue 1 2014, pag. 14 – 22 22 [3]. kostov p., krastev n., angelova d., dimitrov i. „estimation of temperature fields from flat-flame burner”, xvii scientific conference with international participation emf 2012. pp. 86-90 http://www.tusofia.bg/faculties/emf/confer/documents/tom_1.pdf (in bulgarian) [4]. bergles, a. e. exhft for fourth generation heat transfer technology, exp. thermal fluid sci., vol. 26, nos. 2-4, pp. 335-344, (2002). [5]. bergles, a. e. exhft for fourth generation heat transfer technology, exp. thermal fluid sci., vol. 26, nos. 2-4, pp. 335-344, (2002). [6]. catchpole, j.p., drew, b.c.e., evaluation of some shaped tubes for steam condensers, steam turbine condenser, report of a meeting at nel, glasgow, uk, pp. 68-75, (1974). [7]. manglik, r.m., bergels, a. e, enhanced heat transfer in the new millenium: a review of the 2001 literature, thermal fluids and thermal processing laboratory, laboratory report № tftpl-eb01, university of cincinnati, oh, (2002). [8]. v. zimparov, enhancement of heat transfer by a combination of a single – start spirally corrugated tubes with a twisted tape, experimental thermal and fluid science 25 (2002) 535-546. [9]. v. zimparov, prediction of friction factors and heat transfer coefficients for turbulent flow in corrugated tubes combined with twisted tape inserts. part 2: heat transfer coefficients, international journal of heat and mass transfer 47 (2004) 385-393. [10]. ő. ağra, h. demir, numerical investigation of heat transfer and pressure drop in enhanced tubes, international communications in heat and mass transfer 38 (2011) 1384-1391. effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin 210 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 3 2020, pag. 210 218 analysis for the determining factors of the evolution of hepatitis a viral in suceava county, romania, in the period 2018 2019 paraschiva tomăscu 1 , matei jarcău 2, mihaela jarcău3, micşunica rusu 3 , maria poroch – seriţan 3* 1omnis dental med s.r.l., suceava, romania 2iuliu haţieganu university of medicine and pharmacy, cluj-napoca, romania 3faculty of food engineering, stefan cel mare university, suceava, romania, mariap@fia.usv.ro, *corresponding author received 15th july 2020, accepted 21st september 2020 abstract: the evolution of the hepatitis a virus is an important public health problem. this study was conducted in 2018 2019 and provides an analysis of the determinants regarding the evolution of hepatitis a virus (hav) in suceava county, romania. the determining factors for the study were the subjects' place of residence and the source of the outbreak. it turned out that the main factor the type of infectious outbreak is mainly represented by variable educational communities and amounts to 56%, and the variability of home contacts is 42%. the distribution of the determining factor, according to which the source of the outbreak selects water as the predominant variable is 62%, followed by food 26%. these factors may play a significant role in the spread of hepatitis a among vulnerable populations. keywords: hepatitis a viral, place of residence, household type, the type of outbreak, source of outbreak 1. introduction hepatitis a virus (hav) infection is still a major public health problem worldwide [1]. from the point of view of clinical manifestations, it varies from asymptomatic, subclinical forms of the disease, to forms with medium, severe or even fulminant evolution with acute hepatic insufficiency. globally, only 1.5 million clinical cases of hav are reported annually, while the infection rate is much higher [2]. in extremely endemic countries, almost all children become infected at an early age with mostly asymptomatic exposure, but acquire lifelong immunity [3, 4]. in 2017, 30 eu / eea countries reported to tessy (the european surveillance system a system for collecting, analyzing and disseminating data on communicable diseases) a number of 29307 confirmed cases (41% in romania and 9% in bulgaria). cases occurred in all age groups, mainly in the 5 14 years (39%) and 25 44 years (19%) age groups. cases in men were more common than in women, especially in the 15 24 and 25 44 age groups (male / female ratio of 1.3 and 1.2, respectively). the majority of reported infections were those with familial transmission (91%). for travel-related cases, the most common destinations were syria, morocco and turkey [4, 5]. in 2018, 4560 suspected cases of hav were registered nationally, of which 4436 cases were confirmed. county dsps reported, in 2018, 157 outbreaks of acute hepatitis a viral, with a total number of 1799 cases of hepatitis a viral, of which http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 211 1456 cases were registered in the age group of 0 14 years. of the 157 outbreaks: 89 were community outbreaks, 60 were family outbreaks and 8 were community outbreaks [6]. hepatitis a virus (hav) belongs to the genus hepatovirus, a separate genus in the family picornaviridae and it is small (27 nm), without an envelope, and consists of a single-stranded rna with a positive polarity, including about 6500 nucleotides, which synthesizes a protein from cleavage of which by protease 3c results in structural proteins v1 v4 [7, 8]. infection with hav occurs via person-toperson contact and is mainly acquired through faecal-oral transmission resulting from exposure to contaminated water and food [9] and physical agents [10]. hav is stable at low ph, therefore gastric acidity does not destroy its viability [11]. it is resistant to drought, survives to frost, persists for months in fresh or salt water, which is why contaminated and eaten raw mollusks can transmit the virus. it maintains its infectivity in food for a few days. it is inactivated at temperatures above 60ºc, as well as formalin, ethanol, ultraviolet networks. the stability of the virus at various temperature and ph conditions allows it to spread and persist in the external environment with the possibility of spreading the infection [8, 12]. the population incidence of hav infection is related to socio-economic conditions including density of housing, sanitation, quality of water [13] and income. overall improvements of such conditions worldwide are leading to a shift of susceptibility to infection from early age to young and even older adults [14]. the aim of the present study conducted in 2018 2019 was to analyze the determinants of the evolution of hav in suceava county, romania, as these factors are plausible to have consequences on the prevalence in susceptible people. 2. materials and methods 2.1. materials our study was performed on 50 subjects diagnosed with hepatitis a viral residing in suceava county, with the date of clinical onset between 2018-01-01 and 2019-12-31. the epidemiological study of acute hepatitis a viral (hva) was observational longitudinal. for each registered case, the sex, the age at onset of the jaundice syndrome, the place of residence, the type of household (apartment or courtyard house), the type of outbreak of infection, identified by epidemiological investigation were recorded as follows: (a) contact in the household with a hepatitis a patient; (b) attending an educational community (from kindergarten to university) or a community at work, if these circumstances had occurred within a period of up to 45 days before onset, the source of the infection (food, water, fruit and water + fruit) as well as the clinical manifestations of hepatitis a virus infection. 2.2. statistical analysis the programs used in the statistical analysis in order to validate and correlate the data from the study were:  microsoft excel;  spss 20.0 [15]. we analyzed the following outcome determining factors: sex, age, place of residence, household type, type of outbreak, source of outbreak, clinical manifestations. each variable (factor) was analyzed using the following tests: pearson correlation to calculate statistical correlations; nonparametric chi-square test (χ²-test); one sample t-test; significance threshold. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 212 statistical differences are obvious if the significance threshold is equal to p < 0.05, and the correlations are valid when the value of r tends to 0.9 1 when the value of r tends to 0 or -1 the correlations between the variables are negative [16]. 3. results and discussion 3.1. the sex of the subjects from the distribution by sex (figure 1) it is found that females predominate (58% f) compared to males (42% m), but there are no major statistical differences between the two sexes (p = 0.2). figure 1. the distribution by sex of subjects infected with hepatitis a virus hepatitis a virus (hav) infections are associated with considerable morbidity and mortality. from an epidemiological point of view, acute viral hepatitis a (hva) shows wide variations in terms of endemicity, age at the time of infection and frequency of the disease. the clinical course of the disease may be influenced by the age and immune status of the host, ranging from asymptomatic infection to fulminant hepatitis [4, 17]. 3.2. the age of the subjects by distribution, we see that the highest incidence of viral hepatitis a infection in children aged 5 years old is 10%. subjects 7, 12 and 15 years old, respectively, make up 8% in each group; at the age of 2 years old, 10 years old, 13 years old and 17 years old, 6% each, and at the age of 1 year, 9 years old, 14 years old, 18 years old, 21 years old and 25 years old, the incidence is 4% each. the least affected subjects were 3, 6, 11, 28, 29, 31, 37, 40, and 45 years old, which accounted for 2% (figure 2). the age of onset of the hepatitis a virus is also changing in adolescents and adults. this has led to a more symptomatic disease, because hepatitis a infection in children is usually asymptomatic, children under 6 often have no symptoms, this aspect being known as the paradox of the epidemiology of hepatitis a. the occurrence of this infection in older age groups may increase the severity of the disease and increase the risk of mortality [18]. 3.3. place of residence of the 50 subjects, it can be seen that only 14% (7 subjects) come from urban areas and a proportion of 86% (43 subjects) from rural areas (figure 3). what makes the difference between rural and urban areas is the accessibility to information. people in the country are more prone to infections due to lack of education and lack of running drinking water, but also inadequate treatment of the water system sewerage. water transmits hav, both by consuming it and by swimming in contaminated pools, unhygienically maintained and insufficiently chlorinated. consumption of contaminated water usually causes epidemic explosive episodes, when a significant share of receptors is involved, but its intervention in inducing sporadic cases cannot be denied. surface, groundwater or shallow waters are contaminated, with the possibility of discharging household fecal residues [19]. 3.4. household type according to the household type determining factor, we observe the same food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 213 percentage distribution as the place of residence, so that 14% (7 subjects) live in an apartment building and 86% (43 subjects) in a courtyard building (figure 4). figure 2. age distribution of subjects infected with hepatitis a viral figure 3. place of residence distribution of subjects infected with hepatitis a viral figure 4. household type of subjects infected with hepatitis a viral food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 214 there is a close link between the place of residence and the type of household, so that people living in the courtyard house and rural area have a higher probability of infection due to lack of drinking water [13] or by infecting their own wells with animal manure and lack of food hygiene. the indirect mode of transmission is the most common and involves the circulation of hav through water, food, various objects (dishes, toys, toiletries, etc.) or hands, contaminated with feces. food contaminated by humans, flies, cockroaches, during their processing, processing or storage, can contribute to indirect transmission. this type of transmission is involved in the consumption of unprocessed food [9]. of particular importance is the "seafood", which concentrates large amounts of hav, their consumption can cause epidemics of various sizes, for example the shanghai epidemic of 1988, with over 300000 diseases [8, 19]. the favorable factors of the epidemiological process of hav are important in the evolution of morbidity, on the first places being placed the economic level and the deficient living conditions, the agglomeration, the promiscuity that facilitates and amplifies the interhuman contamination. these conditions, associated with deficiencies in hygiene and water and food supply [20] are factors that determine an increased frequency of hav infections in preschool and school communities, construction sites, work camps, etc. natural calamities such as floods, earthquakes, as well as wars, severe economic crises, pilgrimages, certain rituals, are other factors that favor-stimulate the epidemiological process of hav [7]. 3.5. the type of outbreak according to the variable type of outbreak, we observe an approximately equal proportion in terms of the nature of the outbreak, so that 56% (28 subjects) acquired the infection in educational communities or in the workplace and 42% (21 subjects) in household contacts due to ingestion of contaminated food or water (figure 5) [13, 21]. 3.6. source of outbreak the determining factor represented by the source of the outbreak indicates the percentage as follows: 62% (31 subjects) became infected as a result of consuming contaminated water, 26% (13 subjects) as a result of ingesting contaminated food and the remaining 12% (6 subjects) due to an uncertain variable contaminated (unwashed) fruit or polluted water with which the eaten fruit was washed (figure 6). hav being transmitted [22] primarily by faecal-oral route, either by person-toperson contact or by ingestion of contaminated food or water, contamination with hav of a foodstuff may occur at any time during cultivation, harvesting, processing, preparation and distribution of the preparation. recognizing food-based transmission using routine surveillance data can be difficult, as there are cases where patients may have difficulty remembering their food history in 2 to 6 weeks before illness. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 215 figure 5. the type of outbreak of subjects who were infected with hepatitis a viral figure 6. source of outbreak of subjects who were infected with hepatitis a viral 3.7. clinical manifestations from the distribution of subjects it was found that 68% (34 subjects) of subjects had abdominal pain, 62% (31 subjects) had a decrease in appetite and vomiting, 60% (30 subjects) had nausea, 54% (27 subjects) had headache and jaundice and 52% (26 subjects) had diarrhea (figure 7). the onset of the disease is usually sudden with fever, malaise and abdominal discomfort. jaundice is the predominant symptom. symptoms can last from one to two weeks to several months. in 15% (7 subjects) of cases, recurrent hepatitis lasting up to one year occurs. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 216 figure 7. clinical manifestations of subjects who were infected with hepatitis a viral the determinants of the evolution of hepatitis a viral are represented by the place of residence of the subjects and the source of the outbreak, so that a close link was found in the case of infections between the two variables of the two factors. according to the percentage distribution, 86% (43 subjects) of the infected subjects live in rural areas. from the application of non-parametric tests (chi-square (χ²-test)), but also of parametric ones (one-sample test) it is found that there are statistical differences between the two place of residence of the subjects (p < 0.05). the distribution by type of household shows that those who live in the courtyard house predominate 86% (43 subjects). by applying the non-parametric tests (chisquare (χ²-test)), but also the parametric ones (one-sample test), differences are found from a statistical point of view between the two types of housing (p < 0.05). by applying the chi-square test (χ²-test) we notice that there are statistical differences between the variables of the type of infection outbreak (p < 0.05). the predominant variable of the type of infection outbreak was represented by the educational communities or in the workplace communities 56% (28 subjects). we can therefore conclude that more than half of these subjects became infected with the hepatitis a virus in the educational community (kindergarten and up to university) or in a community at work, if these circumstances had occurred in a interval up to 45 days before onset. the distribution on the determining factor the source of outbreak highlights as predominant the variable water with 62% (31 subjects) followed by the variable food with 26% (13 subjects). it is found by applying non-parametric tests (chi-square (χ²-test)), but also parametric ones (onesample test) that there are differences from a statistical point of view between these variables of the source of outbreaks (p < 0.05) applying the pearson correlation it was found that there is a correspondence between the place of residence and the source of outbreak (r = 0.2), but also between the place of residence and the type of outbreak (r = 0.4), which means that the place of residence can influence both sources, as well as the types of outbreak. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 217 given that the sources of outbreak were represented by the variables food / fruit and water, they will be correlated with the appearance of symptoms in the subjects. the strongest correlation found was between source and nausea symptom (r = 0.5). there was a correlation for each of the symptoms and the source of outbreak, but these are not all statistically significant. the only symptoms that were statistically suggestive were nausea (p < 0.001) and abdominal pain (p = 0.008). the present study aims to highlight the importance of prevention in areas at high risk of endemicity at regional level by implementing information systems on risk exposure for viral hepatitis a infection and the consequences that society may have by the outbreak. 4. conclusion in conclusion, this study provides a summary of the evaluation on the evolution of hepatitis a viral in suceava county in the period 2018 2019, depending on the determining factors. hepatitis a viral is a major public health problem and it is plausible that the determinants have consequences for the prevalence in susceptible individuals. the subjects' place of residence and the source of the outbreak are the determining factors of the study. the determining factor the type of outbreak is predominantly represented by the variable educational communities 56% (28 subjects), while the variable household contacts is 42% (21 subjects). the distribution of the determining factor, the source of the outbreak, highlights as predominant the variable water with 62% (31 subjects) followed by food with 26% (13 subjects). reducing the transmission of viral hepatitis a can be achieved by defining and raising awareness of specific critical points of infection and requires a good understanding of how population-related disease / diseases caused by pathogens with "gateway" to the digestive tract can occur. the hygienic, sanitation, decontamination; mass vaccination campaigns; avoiding the transmission through water or food contaminated with hav, achievable by ensuring the potability of the water and the observance of the hygienic conditions in the public alimentation units; population education on hygienic skills in families, the community, which requires a hygienic culture and continuous training are important strategies in preventing and combating the emergence and spread of hav. 5. references [1]. joo il k., yun s.k., young k.j., oh s.k., yeon s.k., yang s.k., duck j.c., și ju h.k., factors influencing the severity of acute viral hepatitis a, korean journal hepatology, 16(3): 295– 300, (2010) [2]. who position paper on hepatitis a vaccines. wkly epidemiol rec, 87:15, (2012) [3]. chakravarti a., bharara t., epidemiology of hepatitis a: past and current trends, 2019 https://www.intechopen.com/books/hepatitis-a-andother-associated-hepatobiliarydiseases/epidemiology-of-hepatitis-a-past-andcurrent-trends accesat 11/07/2020 [4]. franco e., meleleo c., serino l., sorbara d., zaratti l., hepatitis a: epidemiology and prevention in developing countries, world j hepatol, 4(3): 68-73, (2012) [5]. dsp suceava, ministry of health, national institute of public health, regional center for public health, timisoara, campaign "world hepatitis day" (zmh) 2019 july 28, (dsp suceava, ministerul sănătăţii, institutul naţional de sănătate publică, centrul regional de sănătate publică, timişoara, campania “ziua mondială de luptă împotriva hepatitei” (zmh) 28 iulie 2019) https://www.dspsv.ro/uploads/promovareasanatatii/ hepatita%202019/analiza_hepatite_2019.pdf, accesat 11/07/2020 [6]. ministry of health, national institute of public health, activity report 2018, (ministerul sănătăţii, institutul naţional de sănătate publică, raport de activitate 2018) https://www.insp.gov.ro/index.php/informatiihttps://www.intechopen.com/books/hepatitis-a-and-other-associated-hepatobiliary-diseases/epidemiology-of-hepatitis-a-past-and-current-trends%20accesat%2011/07/2020 https://www.intechopen.com/books/hepatitis-a-and-other-associated-hepatobiliary-diseases/epidemiology-of-hepatitis-a-past-and-current-trends%20accesat%2011/07/2020 https://www.intechopen.com/books/hepatitis-a-and-other-associated-hepatobiliary-diseases/epidemiology-of-hepatitis-a-past-and-current-trends%20accesat%2011/07/2020 https://www.intechopen.com/books/hepatitis-a-and-other-associated-hepatobiliary-diseases/epidemiology-of-hepatitis-a-past-and-current-trends%20accesat%2011/07/2020 https://www.dspsv.ro/uploads/promovareasanatatii/hepatita%202019/analiza_hepatite_2019.pdf https://www.dspsv.ro/uploads/promovareasanatatii/hepatita%202019/analiza_hepatite_2019.pdf https://www.insp.gov.ro/index.php/informatii-publice/send/7-informatii-publice/722-raport-insp-2018 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 paraschiva tomăscu, matei jarcău, mihaela jarcău, micşunica rusu, maria poroch – seriţan, analisis for the determining factors of the evolution of hepatitis a viral in suceava country, romania, in period 2018 – 2019, food and environment safety, volume xix, issue 3 – 2020, pag. 210 218 218 publice/send/7-informatii-publice/722-raport-insp2018, accesat 11/07/2020 [7]. feinstone s.m., kapikian a.y., purcell r.h., detection by immune electron microscopy of a viruslike antigen associated with acute illness, science, 182, 1026 – 1036, (1973) [8]. ivan a., treatise on the epidemiology of communicable diseases, polirom publishing house, iaşi, (2002), (tratat de epidemiologie a bolilor transmisibile, editura polirom, iaşi, (2002)) [9]. amariei s., gutt g., poroch-serițan m., vizitiu a., biocip optic pentru avertizarea degradării cărnii de porc și de vită, ro patent 128634 (b1), 2017/11/29 [10]. sattar s.a., jason t., bidawid s., farber j. foodborne spread of hepatitis a: recent studies on virus survival, transfer and inactivation. can j infect dis, 11:159–163, (2000) [11]. scholz e., heinricy u, flehmig b., acid stability of hepatitis a virus, j gen virol., 70 (pt 9):2481-5, (1989)) [12]. babeş v., berca c., acute viral hepatitis. diagnostic, etiological and epidemiological guide, facla publishing house, timişoara, 159 – 171, (1988), (hepatitele virale acute. ghid diagnostic, etiologic şi epidemiologic, editura facla, timişoara, 159 – 171, (1988)) [13]. bârlă f., ciornei (dudici) l., poroch – seriţan m., microbial contamination of the drinking water distribution system in suceava country, romania – three years of monitoring (2010-2012), journal of faculty of food engineering, ştefan cel mare university of suceava, romania, volume xii, issue 4, 316 – 321, (2013) [14]. lemon s.m., ott j.j., van damme p., shouval d., type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention, journal of hepatology, 68, 167–184, (2018) [15]. www.ibm.com/support/docviw.wss?uid=swg24038510, accesat 11/05/2020 [16]. spinei l. lozan o., badan v., biostatistics, “nicolae testemitanu” state university of medicine and pharmacy; school of management in public health, chisinau, (2009), (biostatistica, universitatea de stat de medicină şi farmacie „nicolae testemiţanu”; şcoala de management în sănătate publică, chişinău, (2009)) [17]. horvat m. m., phd thesis – abstract, clinical immunological correlations in the evolution of acute viral hepatitis, “iuliu haţieganu” university of medicine and pharmacy cluj-napoca, doctoral school, (teză de doctorat – rezumat, corelaţii clinico-imunologice în evoluţia hepatitelor acute virale, universitatea de medicină şi farmacie “iuliu haţieganu” clujnapoca, școala doctorală), accesat 11/07/2020 [18]. michaelis k., poethko-müller c., kuhnert r., stark k., faber m., hepatitis a virus infections, immunisations and demographic determinants in children and adolescents, germany, scientific reports, 8:16696, doi:10.1038/s41598018-34927-1, (2018), www.nature.com/scientificreports, accesat 11/07/2020 [19]. hutin y.j., pool v., cramer e.h., nainan o.v., weth j., williams i.t., goldstein s.t., gensheimer k. f., bell b.p., shapiro c.n., alter m.j., margolis h.s., a multistate foodborn outbreak of hepatitis a; n. engl. j. med., 340, 595 – 602, (1999) [20]. bârlă f., poroch – seriţan m., săvuţ (straton) v.d., incidence of food contamination with staphylococcus aureus in suceava county, romania, journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1, 89 – 94, (2013) [21]. fiore a.e., hepatitis a transmitted by food, clinical infectious diseases, 38 (5), 705–715, (2004) [22]. hu x., collier m.g., xu f., hepatitis a outbreaks in developed countries: detection, control, and prevention, foodborne pathogens and disease, 17(3), 166 – 171, (2020) https://www.insp.gov.ro/index.php/informatii-publice/send/7-informatii-publice/722-raport-insp-2018 https://www.insp.gov.ro/index.php/informatii-publice/send/7-informatii-publice/722-raport-insp-2018 https://pubmed.ncbi.nlm.nih.gov/?term=scholz+e&cauthor_id=2550575 https://pubmed.ncbi.nlm.nih.gov/?term=heinricy+u&cauthor_id=2550575 https://pubmed.ncbi.nlm.nih.gov/?term=flehmig+b&cauthor_id=2550575 http://www.-ibm.com/support/docviw.wss?uid=swg24038510 http://www.-ibm.com/support/docviw.wss?uid=swg24038510 http://www.nature.com/scientificreports https://pubmed.ncbi.nlm.nih.gov/?term=nainan+ov&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=weth+j&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=williams+it&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=goldstein+st&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=gensheimer+kf&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=bell+bp&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=shapiro+cn&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=alter+mj&cauthor_id=10029643 https://pubmed.ncbi.nlm.nih.gov/?term=margolis+hs&cauthor_id=10029643 1. introduction issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xviii, issue 1 31 march 2019 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania microsoft word 00 primele pagini 4_2021 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xx, issue 4 31 december 2021 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 52 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 1 2019, pag. 52 59 research on the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips alina kovtun 1 ,* vladimir kovbasa 1 , vitaliy pichkur 1 1national university of food technologies, volodymyrska str. 68, kyiv, ukraine kovtunav@ukr.net *corresponding author received 3th february 2019, accepted 28th march 2019 abstract: the paper considers the current state and prospects of enrichment of formed potato chips with bran, pulp and cryopowder containing from 14 to 40% of food fibres. the importance of the use of food fibres, which is connected with the prevention of alimentary-dependent diseases, has been substantiated. the optimum size of the degree of grinding of rye bran, barley bran, pumpkin seeds pulp, broccoli cryopowder and red beetroot cryopowder and their influence on rheological parameters of semi-finished products have been determined. the influence of temperature and time on the change of maximum viscosity of model solutions of potato semi-finished potato products has been investigated. key words: chips, food fibre, semi-finished products 1. introduction formed potato chips (fpc) are widely popular among the population of many countries of the world. at the same time, they have a number of disadvantages, including imbalance in chemical composition, high energy value, and low amount of biologically active substances. it is known that fpc contain a high content of fats, carbohydrates and low content of proteins, dietary fibres, unsaturated fatty acids, vitamins. in connection with this, in recent years the trend is aimed at the production of new types of fpc with increased nutritional value, by increasing the number of required functional ingredients [1]. the improvement of the nutritional value of final product can be achieved by using raw materials that contain a significant amount of useful nutrients for the human body. we propose to use raw materials of the secondary flour-mill production in the formulation of fpc such as: rye bran (rb), barley bran (bb), as well as pumpkin seeds pulp (psp) and vegetable powders, namely broccoli cryopowder (bc) and red beet cryopowder (rbc) which today are relatively cheap and are available on the market in the necessary quantity by sources of food fibres (ff). they are isolated from plant material: cereals, fruit, nuts, root crops, berries and are divided into soluble and insoluble [2, 3, 4, 5]. insoluble ff are not digested in the upper digestive tract and fall into the thick intestine almost unchanged, improving its motor skills. they carry out probiotic functions, providing bifidobacteria with active growth, suppressing the negative mailto:kovtunav@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 53 and pathogenic microflora in the intestine, strengthening immunity [6]. functions of soluble fibres provide important mechanisms associated with the prevention of food-borne diseases. they improve lipid metabolism, contribute to the decrease of triglycerides and low density lipoproteins, slow the formation of fatty deposits in the liver, improve the action of liver enzymes, assimilation of calcium, and reduce the risk of tumours of the intestine. due to their functional properties, ff are recognized to be a necessary component of human food. in addition, ff contain in their composition a significant amount of minerals and vitamins of group b. daily norm of intake of ff to the human body is of 25-40g. however, in the diet of modern humans, the content of food fibres is almost three times less than the required norm. this problem can be solved by enriching popular products by food fibres, which directly relate to fpc. bran, pulp and cryopowders are promising raw materials for enriching fpc because they contain from 14 to 40% of the dietary fibres. it will allow using a small amount of their dosage to the mass of the product to provide 10-15% of the daily requirement of the human body of this nutrient [7, 8]. 2. materials and methods in our study, potato cereals (bikrampur potato flakes ind.ltd dhanmondi 120 dhala «bangladesh», rb, bb, psp (“agrosilprom”), bc and rbc (“gamma”) were used [1, 9]. the purpose of this work was to investigate the rheological characteristics of semi-finished product of fpc (potato dough) with the addition of rb, bb, psp, bc and rbc. the fractional composition of the raw material was investigated using the mastersizer micro laser granulometer. the durability of the structure of the semifinished samples and control ones were in the formulation rb, bb, psp, bc and rbc was determined on penetrometer “pioneer – 1”. rheological indicators of model solutions of experimental samples were determined on "reotest-2". the maximum viscosity was determined on the "brabender" amilograph [10]. 3. results and discussion structural and mechanical properties of semi-finished products of fpc and organoleptic parameters of finished fpc are significantly influenced by the degree of milling of raw materials. according to the manufacturer, most particles of rb, bb and rsp used in our studies had an average size of 365, 385 and 351 microns [1]. therefore, in order to ensure the homogeneity of the mixture of components of semi-finished products, we carried out an additional milling of rb, bb and rsp using a mechanical mill, followed by sifting on a laser granulometer to the proportion of potato cereals size (pc) and cryopowders. the obtained results are presented in the table 1. when analysing the obtained data, it can be concluded that the average size of particles of raw materials should be in the range of 30 to 100 microns in order to ensure the homogeneity of the mixture in the formulation of fpc. large particles make structure of the dough semi-finished product worse, and also the organoleptic characteristics of the finished product. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 54 тable 1 disperse composition of crushed bran, pulp and cryopowders c o n te n t, % faction, microns 0…10 10…20 20…30 30…40 40…60 60…80 80…100 100…140 140і≥ pc 0.3 10.4 37.8 49.2 2.3 rb 8.5 15.5 63.9 12.1 bb 7.9 20.7 58.7 12.7 rsp 5.1 22.4 51.3 17.4 3.8 – rbc 1.2 5.4 44.7 31.2 9.8 2.9 – bc 0.9 20.1 40.5 27.9 8.1 2.5 – there were created recipes, in which the pc is mixed with rb, bb, rsp, bc, and rbc in a ratio (4:1), since ratios (0.5:1), (1:1), (2:1), and (3:1) deteriorate the organoleptic characteristics of the finished product. experimental samples of semifinished fpc were prepared with a mass fraction of moisture of 42%. a recipe of chips, which included potato cereals, salt and water, was chosen as a control sample. in order to study the structural and mechanical parameters of dough semiproducts at a ratio (4:1), the definition of penetration rates was performed. the results of the research are presented in fig. 1 fig. 1 indicators of the penetrometer pioneer-1 of semi-finished experimental samples in the ratio of potato cereals to additional raw materials as 4:1 from the obtained data we see that for the same amount and temperature of the added water, semi-finished products with rb, bb, and rsp have less stable structure as compared with the control and with samples where cryopowders were used. the obtained results can be explained by different water absorption capacity of raw materials. previously, we have investigated that bran and pulp absorb 1.7-2 and 0.9-1.3 times more water than cryopowders. obviously, this is due to their fractional 130 140 150 160 170 180 potato cereals (control) potato cereals + rye bran potato cereals + barley bran potato cereals + pumpkin seeds pulp potato cereals + broccoli… potato cereals + red beet cryopowder units of device food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 55 composition, and also because bran and pulp contain more ff, and cryopowders contain more pectin substances. investigation on viscosity of model systems of experimental samples provides necessary information on the strength of interaction between raw material molecules and the structure they form. the main structure former of semi-finished fpc is potato starch of potato cereals. we studied the curves of the flow and viscosity of the model samples according to which it is possible to assert the strength of the intermolecular bonds of potato starch of potato cereals in the model solutions of semi-finished fpc with rb, bb, rsp, bc and rbc with a concentration of dry substances of 16%. their rheological curves have been constructed (fig. 2.3). fig. 2 curves of flow of model solutions of samples of semi-finished products of fpc fig. 3 curves of viscosity of model solutions of samples of semi-finished products of fpc 0 100 200 300 400 0 1002003004005006007008009001000110012001300140015001600 ε , s1 p, pа potato cereals + red beet cryopowder potato cereals + broccoli cryopowder potato cereals + rye bran potato cereals 0 50 100 150 200 250 300 350 400 0 500 1000 1500 potato cereals potato cereals + barley bran potato cereals + pumpkin seeds pulp potato cereals + red beet cryopowder potato cereals + broccoli cryopowder potato cereals + rye bran p, pа η , p а ·s food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 56 rheological curves of viscosity of model samples are characteristic to structured systems. the systems we are investigating have anomalous viscosity, which varies depending on the speed of the cylinder rotation and the tension of displacement. in the analysis of rheological curves, the values of the anomaly of viscosity η0-ηm were determined, which characterizes the strength of the coagulation structures that are formed in the system. with insignificant rates of displacement rate, the anomaly of viscosity of the pc is quite pronounced, with an increase in the rate of displacement, the anomaly of viscosity practically does not change (fig. 4). fig. 4 diagram of values of anomalies of viscosity η0-ηm of model solutions of semi-finished products of fpc when comparing the values of the "viscosity anomaly" (η0-ηm) for the studied samples, it was found that a control sample has the strongest coagulation structure, which can be explained by a greater number of intermolecular bonds produced by potato starch as compared to model samples whose structure was made by rb, bb and rsp. even smaller values of η0-ηm were shown with vegetable powders. the obtained results are likely to indicate that the molecules of the added raw material do not form strong intermolecular bonds with each other and reduce the viscosity of the system accordingly, by reducing the number of contacts between starch molecules. the difference in data between samples with bran and vegetable powders can be explained by different chemical composition and the degree of dispersion of raw materials, which is higher in vegetable powders (table 1), respectively, the area of contact with starch molecules, will also increase. the strength of the formed structural framework decreases in experimental samples, which can be confirmed by the tension diagram of the practically destroyed structure рm (fig. 5) obtained during the analysis of rheological curves. 0 50 100 150 200 250 300 350 potato cereals (control) potato cereals + barley bran potato cereals + rye bran potato cereals + pumpkin seeds… potato cereals + red beet… potato cereals + broccoli… η0-ηm, pа∙s food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 57 fig. 5. tension of practically destroyed structure pm of model solutions of samples of semifinished products of fpc the obtained data partly explain the penetration data the reduction of the strength of the structure of semi-finished formed potato chips when incorporated in their formulation of raw materials rich in food fibres. the change in the condition and properties of the raw material under investigation when heated, which contains starch in its composition, is critical during the heat treatment of semi-finished products. taking into account the specifics of the chemical composition and properties of the investigated raw materials, it was necessary to investigate the process of starch gelatinization, which is contained in the investigated semi-finished products of fpc. to characterize the change in the consistency of the mixture of pc with bran, pulp and cryopowders, an amylogram of model solutions during heating was analysed (fig. 6). fig. 6. maximum values of viscosity of model solutions of semi-finished products of fpc 0 200 400 600 800 1000 1200 potato cereals (control) potato cereals + barley bran potato cereals + rye bran potato cereals + pumpkin seeds… potato cereals + red beet… potato cereals + broccoli… pm, pа food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 58 the obtained figures show that the combination of pc with rb, bb, rsp does not affect the time when the maximum viscosity of the gelatinized mixture is reached, but lower the temperature of the gelatinization for rb, bb, rsp by 10, 13, 11.5%, respectively, in comparison with the control sample. bran and pulp are practically insoluble in hydro colloidal mixtures, but do not form viscous solutions. due to the large size of the molecules and the increased amount of carboxyl groups in their composition, they are capable to retain a significant amount of moisture by thickening the semifinished product of fpc. bc and rbc are a complex mixture of colloidal structured polysaccharides consisting of galacturonic acid polymers having pentose and hexose branching. the combination of pc with cryopowders does not significantly reduce time to reach the peak and the temperature of the gelatinization, but significantly increases the viscosity of the suspension due to the presence of a large number of polysaccharides and pectin substances that are capable to increase the viscosity of solutions when dissolved in water with increasing temperature. 4. conclusion in the manufacture of semi-finished formed potato chips using rb, bb, rsp, bc, rbc, the chemical composition of the investigated raw material, its fractional composition and homogeneity of the particles, and also the ability to absorb water during the formation of semifinished potato dough influence on its the structural and mechanical properties. in our opinion, the strength of the formed coagulation structures of molecules of potato starch is likely to be influenced by the degree of milling, and therefore in samples with cryopowders, the degree of milling of which is greater, the viscosity of the system will decrease. the heating process indicates an increase in the viscosity of the system in cryopowder samples compared to samples with bran and pulp, which obviously can be explained by the difference in the chemical composition of the prescription components, namely, an increase in the amount of pectin substances in vegetable cryopowders, in comparison with bran and pulp in which the greater predominates amount of ff. the degree of water absorption of the formulation components will determine the strength of the structure of the semi-finished formed potato chips, confirming the penetration data. 5. references [1]. ts 15.8-24239651-007: 2007 «vegetable fiber». [2]. marina m.v., novoselova g.n., shavnin s.a. products of increased biological value from non-traditional vegetable raw materials, news of samara scientific center of the russian academy of sciences, ed. 12.1 (8): 2079-2082, (2010). [3]. ts u 02070938-004-98 «cryopowders of vegetable food». [4]. sipos p., horváth m., adácsi c., horváth b., babka b., győri z. enrichment of pasta products using beetroot, food and environment safety, 16(4): 209-215, (2017). [5]. codină g.g., atudorei d., cimpoi a., mironeasa s., todosi-sănduleac e. quality evaluation of wheat-pumpkin-golden flaxseed composite bread, food and environment safety, 16(2): 61-70, (2017). [5]. kricman e.s. new generation of food fibers, nutritional ingredients, 1: 28, (2008). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 1 – 2019 alina kovtun, vladimir kovbasa, vitaliy pichkur, research of the effect of non-transitional raw materials on the rheological indicators of the semi-finished products of formed potato chips, food and environment safety, volume xviii, issue 1 – 2019, pag. 52 – 59 59 [6]. lysiy o.v., zapototska o.v., pichkur v.ya., grabovskaya o.v. investigation of rheological properties of various types of modified starch, ukrainian food journal, 3: 35-38, (2012). [7]. sirokhman i.v., zavgorodnya v.m. commodity study of functional food products: a training manual. k.: csl, 544, (2008). [8]. spirichev v.b. theoretical and practical aspects of modern vitaminology, questions of nutrition, 5: 12-19, (2005). [9]. drobot v.i., arsenyeva l.yu., bilyk o.a. laboratory workshop on the technology of bakery and macaroni production. kyiv: center for educational literature, 341, (2008). microsoft word 12 trud -zora_corrected 26 aprilie.doc 213 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 213 223 positive impact of frequent milk and dairy products consumption on bone mineral density of over 50-aged macedonian women *zora uzunoska1, tanja kalevska1, viktorija stamatovska1, daniela nikolovska nedelkoska1, natalija trajceska1, daniela belichovska2, katerina belichovska3 1"st. kliment ohridski" university in bitola, faculty of technology and technical sciences nutrition, dimitar vlahov bb, 1400 veles, republic of macedonia 2mit university, faculty of environmental resources management, blvd. treta makedonska brigada bb, 1000 skopje, republic of macedonia 3" st. cyril and methodius university in skopje, faculty of agricultural sciences and food, blvd. aleksandar makedonski bb, 1000 skopje, republic of macedonia zora_51@hotmail.com *corresponding author received 19th february 2018, accepted 26th june 2018 abstract: a segment of our research was focused on examining the impact of the frequency of milk and dairy products’ (mdp) consumption on bone mineral density (bmd) of macedonian female population. bmd was measured by dexa-densitometer in 210 females at the public institution “borka taleski” in prilep and a questionnaire about their dietary habits was performed. females were categorized into 3 groups according to mdp consumption: those who consume mdp on a daily base; 3 5times/week; or 1 2 times/ week, and those who did not consume mdp were a reference category. data analysis was performed by multiple regression analysis (r), while the significance was determined by p<0.05. the results indicated that daily consumption of milk and dairy products was most beneficial for bmd of the females, then 3 5 times/ week, and the worst impact on bmd was present in females of 1 2 times/ week mdp consumption. females on a daily mdp consumption had in average 0.17 g/cm2 significantly higher bmd as compared to non consumers. females of 3 5 times / week mdp consumption had an average 0.08 g/cm2 significantly higher bmd as compared to non consumers. females of 1 2 times/ week mdp consumption had 0.02 g /cm2 insignificantly lower bmd as compared to non consumers. the results strongly suggest that daily and /or 3 5 times/ week mdp consumption had a significant positive effect on bmd of females and can be considered as a natural benefactor in osteoporosis prevention. keywords: bone mineral density, milk and dairy products, osteoporosis, female population 1. introduction the mode of nutrition is a significant factor of bone mineral density preservation and bone health in humans. optimal early growth is supported by adequate nutrition which has positive and lasting effects on bone mineralization, such as breast milk in the diet has a vital importance for the child. bmd reflects bone strength through the content of calcium, which is particularly present in milk and dairy products. densitometry is a sensitive and specific method by which a healthy bone, osteopenia (mild bone loss, which occurs asymptomatic) or osteoporosis (expressed bone loss, which can cause symptoms and fracture diathesis) can be determined [1-5]. osteoporosis is defined as a progressive systemic-skeletal disease, characterized by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 214 a disorder of bone mineral microanatomy, low bone mass and mineral density, causing brittle bone fragility and increasing susceptibility to fracture. this disease of today's civilization, with greater incidence in developed western countries occurs more frequently in women after menopause (postmenopausal osteoporosis) and in elderly (senile osteoporosis), but may result in significant morbidity and mortality in men as well [6]. it is estimated that osteoporosis affects over 100 million people worldwide, and 22 million women and 5.5 million men suffer from osteoporosis in the european union [7]. an individual reaches the peak maximum of the bone mass (density) about 30 years of age. furthermore, the rate of bone loss is slowly increasing, while the bone buildup rate decreases. whether a person will develop reduction of bmd to osteopenia or osteoporosis depends on thickness of the bones in early life, peak bmd, gender, age, lifestyle, nutrition, climatic conditions, etc. [8]. in the wide spectrum of risk factors for osteoporosis, nutritional factors play a significant role and at the same time provide an opportunity for intervention in preventive purposes and / or slowing down the process of bone loss [9 11]. numerous studies have suggested that the consumption of milk and dairy products (cheese, yogurt, and other fermented dairy products) have a significantly positive impact on bmd of adolescents [12], females and males, on bmd of postmenopausal women [13 14] and older women [15]. some studies confirm the positive correlation of calcium intake through mdp consumption and bmd [16 17]. conversely, some studies have not established a correlation between calcium intake through mdp consumption and bone health [18 20]. osteoporosis, which can lead to disability and fracture diathesis over human's lifetime and to life quality impairment of elderly is in the focus of the scientific interest, in order to identify all possible risk factors for occurrence of osteoporosis and to recommend an adequate preventive measures, especially in the field of nutrition as a variable category, which may have a dual role, to represent a potential risk factor or benefactor. there are no data investigating the influence of certain type of nutrition on bmd of females in republic of macedonia. therefore, the aim of this study was to investigate the impact of mdp frequency consumption on bmd of the female macedonian population, i.e. to determine whether the frequent mdp consumption can have a beneficial effect on bmd of females and can be suggested as a nutritional natural factor in osteoporosis prevention and/or in slow downing the bone loss. 2. materials and methods the investigation was conducted on 210 women who came for examination in public health institution "borka taleski" in prilep. bmd was measured with a densitometer (dexa), and a questionnaire about dietary habits was used. females were categorized into 4 groups by age (40 49; 50 59; 60 69 and over 69 years); and in 4 subgroups according to the manner of consumption of mdp: those who consume daily; 3 5 times / week; and 1 2 times/ week. referent category was the group of respondents who did not consume mdp. during the processing of the obtained data, both types of expression of the values of bone mineral density were used, i.e. g/cm2 and "t-score", statistic analysis and graphic display. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 215 data analysis was performed by statistical program "statistica 7.1 for windows"and "spss statistics 17.0". in the series with numerical marks (age), descriptive statistics was produced (mean; std. deviation; ± 95.00% ci; minimum; maximum). the distribution of data was tested with: kolmogorov-smirnov test; lilliefors test; and shapiro wilks test (p). the relationship between bone mineral density as dependent phenomena and age and the frequency of mdp consumption as independent phenomena has been investigated with multiple regression analysis (r). the significance was determined by p <0.05. 3. results and discussion bone mineral density levels in terms of the frequency of mdp consumption are shown in table 1. the results show that the percentage of the normal bmd level is higher in females of daily mdp consumption, and of 3 5 times/week consumption as compared to those who consume 1 2 times/week or do not consume mdp at all (33.3%; 25% v. 17 %; 10%). in addition to the favorable effect of the daily consumption of milk and dairy products, the percentages of severe form of osteoporosis was much lower in everyday mdp consumers as compared to non consumers (6.8% v. 30%). there are studies suggesting that dairy products, especially those with low fat, did not increase the risk of cardiovascular disease. rozenberg at al. [20], claim that the intake of dairy products, 3 times a day, was solely beneficial for bone health. another study [21] suggested that consumption of milk and dairy products had a beneficial effect on bone, cardio, metabolic, cognitive and digestive health of the individual. due to the nutritional profile and the current awareness of mdp benefits, the guidelines of several countries recommend 1 3 cups of dairy products per day. table 1 bmd levels in terms of frequency of mdp consumption bone mineral density frequency of mdp consumption normal level osteopenia osteoporosis severe form of osteoporosis total daily 14 33.3% 14 33.3% 12 26.6% 3 6.8% 43 100% 3-5 times/week 14 25% 20 35% 12 20% 12 20% 58 100% 1-2 times/week 14 17% 39 47.6% 14 17.1% 15 18.3% 82 100% no consumers 3 10% 5 20% 11 40% 8 30% 27 100% the study of wadolowska et al. [13] suggested that consumption of dairy products in elder women did not lead to significant difference of bmd (p> 0.05) when comparted to those who did not consume. however, in pre-school girls and adolescents a significantly positive difference (p < 0.05) was obtained. the results shown on table 2 relate to the investigated relationship between bmd ing/cm2 as a dependent phenomena and the frequency of mdp intake (daily & 3 5 times/ week & 1 2 times/week & non consumers) as independent phenomena. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 216 table 2 bone mineral density & frequency of mdp consumption frequency of mdp consumption beta* std. err. of beta b** std. err. of b t (206) plevel intercept 0.88 0.03 29.08 0.000 daily 0.40 0.09 0.17 0.04 4.44 0.000 3-5 times/ week 0.20 0.10 0.08 0.04 2.09 0.04 1-2 times /week -0.05 0.10 -0.02 0.03 -0.47 0.64 beta*the correlation between bmd in gr/cm2 and mdf consumption b** bmd in gr/cm2 in the investigated ratio for r = 0.42 and p < 0.001 (p = 0.000), a medium strong significant correlation was determined. no consumers group was a reference category. the highest influence was present in daily mdp consumers (beta = 0.40), then 3 5 time/ week (beta = 0.20, while the weakest impact was noted in 1 2 times/week consumers (beta = -0.05). females on daily mdp consumption had an average of 0.17 g/cm2 (b = 0.17) higher bmd compared to no consumers, which was statistically significant p < 0.001 (p = 0.000). females on 3 5 times/week mdp consumption have an average of 0.08 g/cm2 (b = 0.08) significantly higher bmd compared to no consumers, for p < 0.05 (p = 0.04). however, females on 1 2 times/week mdp consumption had an average of 0.02 g/cm2 (b = -0.02) insignificantly lower bmd compared to no consumers, for p > 0.05 (p = 0.64). these results are in line with the findings of sato et al. [22] suggesting that more frequent milk consumption was associated with lower bone turnover, higher bmd and higher bone micro architecture index in the population of older men with relatively low calcium intake in japan. sahni et al. [23] proved that the consumption of milk and yoghurt was associated with higher bmd, but not with fractures of the hips. the tables below show the correlation of frequency of mdp consumption and bmd of the four age groups of females (aged 40 49 50 59, 60 69 and over 69). 3.1. 40 – 49aged females there was no significant difference for p > 0.05 (p = 0.62) in bmd values in females aged 40 49 years in terms of daily mdp consumption, 3 5 times/ week, 1 2 times/week basis and non consumers (1.14 g/cm2 v. 0.97 g/cm2 v. 1.07 g/cm2 v. 0.97 g/cm2) –figure 1. fig. 1. consumption of mdp and bmd in a multiple comparison of p values in respect of bmd of females who consumed mdp on a daily base, 3 5 times /week, 1 2 times/ week, and non consumers there was no significant difference for p > 0.05. the results shown in table 3 relate to the examined relationship between bmd (g/cm2) as a dependent phenomenon and mdp consumption (daily & 3 5 times/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 217 week & 1 2 times/week & non consumers) & the age of women as independent phenomenon. in the investigated ratio for r = 0.81 and p > 0.05 (p = 0.08) a strong non-significant correlation was found. women who did not consume milk and dairy products were taken as the reference category. a more significant influence on the investigated ratio had the age (beta = 0.94), than the daily mdp consumption (beta = 0.76), 1 2 times/week consumption (beta = 0.20), and the worst impact had 3 5 times/week mdp consumption (beta = -0.08). with each increase in the age by one year, bmd decreased for 0.08 g/cm2 (b = -0.08) significantly for p < 0.05 (p = 0.01) /at constant values of the other parameters. in this age group, no statistically significant difference of bmd between the subgroups of the examinees in the context of the consumption of mdp, i.e. between the daily consumers, 3 5 times/week, 1 – 2times/week consumers, and non consumers was found at constant values of other parameters. namely, females who regularly consume mdp had an average of 0.35 g/cm2 (b = 0.35) which was slightly higher bmd for p >0.05 (p=0.06) as compared to non consumers. females of 3 5 times/week mdp consumption had an average of 0.04 g/cm2 (b = -0.04) insignificantly lower bmd than no consumers for p > 0.05 (p = 0.78). females of 1 2 times/week mdp consumption had for 0.11 g/cm2 (b = 0.11) insignificantly higher bmd compared to no consumers, for p > 0.05 (p = 0.48). females 40 49 years old are still protected by estrogens, despite of estrogens fluctuations, which have a positive impact on their bmd. these results suggest that other factors, such as genetics, peak bone mass, physical activity, and climatic conditions have a greater impact on bmd of this age group. soroko et al. [24] suggested that mdp daily consumption of milk in youth and adulthood is associated with improved bmd in older women. table 3 bmd/age & frequency of mdp consumption beta std.err. of beta b std.er r.of b t (206) plevel intercept 4.40 0.99 4.40 0.003 age -0.94 0.29 -0.08 0.02 -3.30 0.01 daily 0.76 0.35 0.35 0.16 2.19 0.06 3-5 times/ week -0.08 0.29 -0.04 0.12 -0.29 0.78 1-2 times/ week 0.20 0.27 0,11 0.15 0.74 0.48 3.2. 50-59-aged females there was a significant difference for p < 0.001 (p = 0.0002) in bmd values in this age group of 50 59 according to mdp consumption subgroups: on daily basis, 3 5 times/week, 1 2 times/week and non consumers (1.08 g/cm2 v. 1.00 g/cm2 v. 0.88 g/cm2 v. 0.86 g/cm2) figure 2. fig. 2. consumption of mdp and bmd females who regularly consume mdp (figure 2) had a significantly higher bmd as compared to females of 1 2 times/week consumption for p < 0.01 (p = 0.006); and compared to non consumers for p < 0.05 (p = 0.04). in addition, women who consume mdp 3 5 times/week had a significantly higher bmd than those of 1 2 times/week food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 218 consumption for p < 0.01 (p=0.005); but the difference in bmd between 1 2 times/week mdp consumers and non consumers was not significant for p > 0.05 (p = 0.08). this result may be explained by the assumption that non-dairy respondents have a better bmd peak, or a compensated calcium intake through other types of food such as fruits, vegetables and/or a better lifestyle, which suggest further research. there was no significant difference in bmd among women who daily consume mdp and those with 3 5 times/week consumption for p > 0.05 (p = 1.00); between those who consume mdp 3 5 times/week and non consumers for p > 0.05 (p = 0.08); and between subgroups of those who consume mdp products 1 2 times/week and non consumers for p > 0.05 (p = 1.00). the results shown in table 4 relate to the examined relationship between bmd (g/cm2) as a dependent phenomenon and mdp consumption (daily & 3 5 times/week & 1 2 time/week & no consumers) & the age of women as independent occurrences. in the investigated ratio for r = 0.52 and p < 0.001 (p = 0.0009), a mean strong significant correlation was determined. female non consumers were taken as the reference category. the strongest influence on this ratio had a daily mdp consumption (beta = 0.48), then 3 5 times/week (beta =0.41), then 1 2 times/week consumption (beta = 0.06) and the lowest influence had the age (beta = -0.03). in the case of unchanged values of other parameters, the following results were obtained. females of daily mdp consumption had an average of 0.23 g/cm2 (b = 0.23), i.e. significantly higher bmd for p < 0.01 (p = 0.002) as compared to non consumers. similar results were obtained in females who consumed mdp 3 5 times/week, on average they had 0.14 g/cm2 (b = 0.14) significantly higher bmd for p < 0.05 (p = 0.02) compared to non consumers. however, bmd difference was insignificant for p >0.05 (p = 0.72) in females who consume 1 2 times/week compared to non consumers, they had 0.02 g/cm2 (b = 0.02) higher bmd than non consumers. with each increase in age for a year bmd decreased by approximately 0.002 g/cm2 (b = -0.002), which is insignificant for p > 0.05 (p = 0.7). table 4 bmd / age & mdp consumption beta std.err. of beta b std.err. of b t (206) p-level intercept 0.95 0.35 2.71 0.009 age -0.03 0,11 -0.002 0.01 -0.27 0.79 daily 0.48 0.15 0.23 0.07 3.28 0.002 3-5 times/week 0.41 0.17 0.14 0.06 2.42 0.02 1-2 times/ week 0.06 0.18 0.02 0.05 0.37 0.72 the results suggest that in postmenopausal period, the frequent mdp consumption has a positive impact on bmd of those females. these results correspond to the research of young at al. [25] and their study of the prevalence of osteoporosis over the level of intake of certain food in postmenopausal koreans which indicated the beneficial effect of food containing sufficient amounts of calcium such as dairy products, algae, fish, fruits, which support calcium absorption and might prevent osteoporosis later in life. 3.3. 60-69-aged females there was a significant difference for p < 0.01 (p = 0.004) of bmd in females aged 60 69 years in terms of mdp consumption on daily basis, 3 5 times/week, 1 2 times/week and non food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 219 consumers (1.14 g/cm2 v. 0.93 g/cm2 v. 0.86 g/cm2 v. 0.97 g/cm2 respectively). the results are shown in figure 3. the results of figure 3 showed that only women who consumed mdp on a daily basis had significantly higher bmd for p < 0.05 (p = 0.02) compared to 3 5 times/week mdp consumers; and to 1 2/ week mdp consumers for p < 0.01 (p = 0.007). it is interesting to note that females who daily consume mdp did not have a significantly higher bmd for p > 0.05 (p = 0.87) compared to non consumers. perhaps the reason for this result is the fact that women who do not consume milk and dairy products meet the needs for calcium through the consumption of fruits, fish and vegetables. consumption of mdp 3 5 times/week, was not associated with a significantly higher bmd, compared to females of 1 2 times/week consumption; and to no consumers for p > 0.05 (p = 1.00). also, non consumers had insignificantly higher bmd for p > 0.05 (p = 1.00) compared to those of 1 2 times/week. fig.3. consumtion of mdp and bmd the results presented in table 5 refer to the examined relationship between bmd in g/cm2 as a dependent phenomenon and frequency of mdp consumption (daily & 3 5 times/week & 1 2 times/week & no consumers) & the age of women as independent phenomena. the examined ratio for r = 0.50 and p < 0.01 (p = 0.009) showed a medium strong significant correlation. as a reference category, non consumers were taken into consideration. the highest influence on the ratio had a daily mdp consumption (beta = 0.39), than 1 2 times/week mdp consumption (beta = -0.19), than 3 5 times/week mdp consumption (beta = -0.07) and the weakest influence had the age of female (beta = 0.06). at unchanged values of other parameters, females of a daily mdp consumption had an average of 0.18 g/cm2 (b = 0.18) higher bmd compared to no consumers, which was insignificant for p > 0.05 (p = 0.12). 1 2 times/week mdp consumers had an average 0.10 g/cm2 (b = -0.10) insignificantly lower bmd compared to non consumers for p > 0.05 (p = 0.39) as well as 3 5 times/week mdp consumers had an average of 0.03 g/cm2 (b = -0.03) insignificantly lower bmd compared to non consumers for p > 0.05 (p = 0.80). with each age increase in a year, bmd increased insignificantly by a mean of 0.004 g/cm2 (p = 0.004), for p > 0.05 (p = 0.68) / under unchanged values of other parameters. table 5. bmd / age & mdp consumption 3.4. females over 69 years there was a significant difference for p<0.01 (p = 0.004) of bmd in patients beta std.err. of beta b std.err .of b t (206) plevel intercept 0.70 0.66 1.06 0.29 age 0.06 0.14 0.004 0.010 0.41 0.68 daily 0.39 0.24 0.18 0.11 1.60 0.12 3-5 times/ week -0.07 0.26 -0.03 0,11 -0.26 0.60 1-2 times /week -0.19 0.22 -0.10 0.12 -0.86 0.39 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 220 over 69 years in terms of mdp consumption, daily 3 5 times/week, 1 2 times/week and non consumers (0.95 g/cm2 v. 0.94 g/cm2 v. 0.84 g/cm2 v. 0.80 g/cm2 ) figure 4. women over age of 69, who daily consume mdp had significantly higher bmd for p < 0.05 (p = 0.03) than female non consumers. there was no significant difference in bmd between women of daily mdp consumption and those who consume 3 5 times a week – p > 0.05 (p = 1.00), or 1 2 times a week for p > 0.05 (p = 0.06). also, there was no significant difference in bmd among women who consume mdp 3 5 times/ week compared to those who consume 1 2 times/week for p > 0.05 (p = 0.17); and in relation to non consumers for p > 0.05 (p = 0.07). in addition, no difference was found in bmd between two subgroups of consumers: 1 2 /weeks and non consumers for p > 0.05 (p = 1.00) fig. 4. consumption of mdp and bmd the results shown in table 6 refer to the examined relationship between bmd g/cm2 as a dependent phenomenon and frequency of mdp consumption (daily & 3 5 times/week & 1 2 times/week & non consumers) & the age of women as independent phenomena. in the investigated ratio for r = 0.44 and p < 0.01 (p = 0.002), a moderately strong correlation was found. as the reference category, female non consumers were taken into account. the greatest impact on the examined ratio had everyday mdp consumption (beta = 0.45), than 3 5 times week consumption (beta=0.39), than 1 2 times/week mdp consumption (beta = 0.14) and the weakest impact had the age (beta = 0.07). table 6. bmd / age & mdp consumption in the case of unchanged values of other parameters, the following results were obtained: females who daily consumed mdp had an average of 0.15 g/cm2 (b = 0.15) significantly higher bmd for p < 0.01 (p = 0.003) compared to non consumers. females who consumed mdp 3 5 times/week had an average of 0.14 g/cm2 (b = 0.14) significantly higher bmd compared to non consumers for p < 0.01 (p = 0.005). females of 1 2 times/week mdp consumption had an average of 0.04 g/cm2 (b = 0.04) insignificantly higher bmd for p > 0.05 (p = 0.37), compared to no consumers. with each increase in age for a year, bmd increased insignificantly by an average of 0.002 g/cm2 (b = 0.002) for p > 0.05 (p =0.49) / under unchanged values of other parameters. beta std.err. of beta b std.err. of b t (206) plevel intercept 0.62 0.25 2.44 0.02 age 0.07 0.10 0.002 0.003 0.70 0.49 daily 0.45 0.14 0.15 0.05 3.12 0.003 3-5 times/ week 0.39 0.14 0.14 0.09 2.86 0.005 1-2 times/ week 0.14 0.15 0.04 0.04 0.90 0.37 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 221 the results from this age group of females over 69, suggested that daily and 3 5 times/week mdp consumption had a favorable effect on bmd and can be treated as natural factor in prevention of osteoporosis and in the fight against the dramatic decline of bmd to the fracture threshold. these results correspond with the epidemiological study of nguyen et al. [26] where 1075 women and 696 men, with an average age of 69 +/6 years were studied in order to investigate the relationship of osteoporosis (body mass index) with calcium intake through food and physical activity. the authors concluded that an adequate intake of calcium through food and a physically active lifestyle in the late decades of life can reduce the the risk of osteoporosis and thereby improve the quality and, perhaps, the quantity of life in the elder population. the meta-analysis of 11 studies of measuring bmd, bone mineral content and bone metabolism markers performed by ma et al. [27] suggested that intake of milk, significantly reduced bone loss by inhibiting bone metabolism. conflicting results were obtained by tai et al. [28] who concluded that increased calcium intake through food, or by calcium supplements can cause a slight nonprogressive increase of bmd, which can prevent a significant bone reduction and the risk of fractures. 4. conclusion daily consumption of milk and dairy products has a significant positive impact on the bone mineral density of the female population. however, when the females were categorized into 4 age groups, the results were different. namely, in women aged 40 49 years, bmd values did not associate with the frequency of consumption of milk and dairy products. at this age, physiological mechanisms are probably able to prevent the occurrence of osteoporosis or other factors than the consumption of milk and dairy products are involved. a particularly beneficial effect on bmd has everyday mdp consumption and 3 5 times/week mdp consumption in age groups of women of 50 59 and over 69 years, which suggests that the frequent consumption of dairy products is a natural nutrition protective factor which can play a significant role in the prevention of osteoporosis and consequent dramatic loss of bmd to fracture threshold in older women over 50 years of age. also, the results suggest that women who do not consume milk and dairy products might regulate the intake of calcium by other types of food. further research is needed to confirm these findings. 5. references [1]. johnell o., gullberg b., allander e., kanis j.a., the apparent incidence of hip fracture in europe: medus study group, osteoporos int. 2, pp. 298-302, (1992). [2]. sanders k.m., nicholson g.c., ugoni a.m., seeman e., pasco j.a., kotowicz m.a., fracture rate lower in rural than urban communities: the geelong osteoporosis study. j epidemiol community health, 56, pp. 466-470, (2002). [3]. blake g.m., chinn d.j., steel s., patel r., panayiotou e., thorpe j., fordham j.n., a list of device-specific thresholds for the clinical interpretation of peripheral x-ray absorptiometry examinations, osteoporosis int. 16, pp. 2149-2156, (2005). [4]. bouxsein m.l., palermo l., yeung c., black d.m., digital x-ray radiogrammetry predicts hip, wrist and vertebral fracture risk in elderly women: a prospective analysis from the study of osteoporotic fractures. osteoporos int. 13, pp. 358-365, (2002). watts n.b., fundamentals and pitfalls of bone densitometry using dual-energy x-ray absorptiometry (dxa) osteoporos int. 15, pp. 847-854, (2004). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 222 [5]. halling a., persson g.r, berglund j., johansson o., renvert s., comparison between the klemetti index and heel dxa bmd measurements in the diagnosis of reduced skeletal bone mineral density in the elderly, osteoporos int. 16, pp. 999-1003, (2005). [6]. hernlund e., svedbom a., ivergård m., compston j., cooper c., et al., osteoporosis in the european union: medical management, epidemiology and economic burden. a report prepared in collaboration with the international osteoporosis foundation (iof) and the european federation of pharmaceutical industry associations (efpia). arch osteoporos. 8 (1-2), 136, (2013). [7]. https://nos.org.uk/media/1622/all-aboutosteoporosis-august-2016. pdf . [8]. moschonis g., katsaroli i., lyritis g.p., manios y., effects of a 30-month dietary intervention on bone mineral density: the postmenopausal health study, br j. nutr. 104, pp. 100 -107, (2010). [9]. jakobsen a., laurberg p., vestergaard p., andersen s., clinical risk factors for osteoporosis are common amongelderly people in nuuk, gree nland. int j circumpolar health 72, pp. 195-196, (2013). [10]. rizzoli r., dairy pro ducts, yogurts, and bone health, am j clin nutr. 99 (5), 1256s-1562s, (2014). [11]. kim s.h., kim w.k., kang m.h., effect of milk and milk products on physical growth and bone mineral density in korean adolescents, nutr res pract. 7 (4), pp. 309-314, (2013). [12]. shin s., joung h., a dairy and fruit dietary pattern is associated with a reduced likelihood of osteoporosis in korean postmenopausal women, br j. nutr. 110 (10), pp. 1926-1933, (2013). [13]. wadolowska l., sobas k., szczepanska j.w., slowinska m.a., czlapka matyasik m., niedzwiedzka e., dairy products, dietary calcium and bone health: the possibility of prevention of osteoporosis in women: the polish experience, nutrients. 5 (7), pp. 2684-2707, (2013). [14]. eysteinsdottir t., halldorsson t.i., thorsdottir i., sigurdsson g., sigurðsson s., harris t., launer l.j., gudnason v., gunnarsdottir i., steingrimsdottir l., milk consumption throughout life and bone mineral content and dessity in elderly men and women, osteoporo s int. feb 25 (2), pp. 663-672, (2014). [15]. lowe n.m., ellahi b., bano q., bangash s.a., mitra s.r., zaman m., dietary calcium intake, vitamin d status, and bone health in postmenopausal women n in rural pakistan, j health popul nutr. oct 29 (5), pp. 465470, (2011). [16]. włodarek d., głąbska d., kołota a., adamczyk p., czekajło a., grzeszczak w., drozdzowska b., pluskiewicz w., calcium intake and osteoporosis: the influence of calcium intake from dairy products on hip bone mineral density and fracture incidence a population-based study in women over 55 years of age, public health nutr. feb, 17 (2), pp. 383-389, (2014). [17]. feskanich d., willett w.c., colditz g.a., calcium, vitamin d, milk consumption, and hip fractures: a prospective study among postmenopausal women. am j clin nutr. feb, 77 (2), pp. 504-511, (2003). [18]. harter d.l., busnello fm., dibi r.p., stein a.t., kato s.k ., vanin c.m., association of low bone mass and calcium and caffeine intake among perimenopausal women in souther n brazil: cross-sectional study, sao paulo med j. 131 (5), pp. 315-322, (2013). [19]. rasi-uusi k., kärkkäinen m.u., allardt-lamberg c.j., calcium intake in health ma intenance a systematic review, fo of nutr res. may, 16; 57, (2013). [20]. rozenberg s., body j.j., bruyère o., bergmann p., brand m.l., cooper c., devogelaer j.p., gielen e., goemaere s., kaufman j.m., rizzoli r., reginster j.y., effects of dairy products consumption on health: benefits and beliefs a commentary from the belgian bone club and the european society for clinical and economic aspects of osteoporosis, osteoarthritis and musculoskeletal diseases, calcif tissue int. jan, 98 (1), pp. 1-17, (2016). [21]. hess m.j., jonnalagadda s.s., slavin l.j., dairy foods: current evidence of their effects on bone, cardiometabolic, cognitive, and digestive health, comprehensive reviews in food science and food safety , march, 15 (2), pp. 251-268, (2016). [22]. sato y., iki m., fujita y., tamaki j., kouda k., yura a., moon j.s., winzenrieth r., iwaki h., ishizuka r., amano n., tomioka k., okamoto n., fujiwara-kyo osteoporosis risk in men (formen), kurumatani n., greater milk intake is associated with lower bone turnover, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 2 – 2018 zora uzunoska, tanja kalevska, viktorija stamatovska, daniela nikolovska nedelkoska, natalija trajceska, daniela belichovska, katerina belichovska, frequent milk and dairy products consumption has a positive impact on bone mineral density of macedonian women over age of 50, food and environment safety, volume xvii, issue 2 – 2018, pag. 213 – 223 223 higher bone density and higher bone microarchitecture index in a population of older japanese men with relatively low dietary calcium intake ) study , osteoporos int 26, pp. 1585-1594, (2015). [23]. sahni s., tucker k.l., kiel d.p., quach l., casey v.a., hannan m.t., milk and yogurt consumption are associated with higher bone mineral density, but not with hip fracture: the framingham offspring study , arch osteoporos, 8: 119, (2013). [24]. soroko s., holbrook t.l., edelstein s., barrett-connor e., lifetime milk consumption and bone mineral density in older women, ame rican journal of public health, aug, 84 (8), pp. 1319-1322, (1994). [25]. suk-young l., sang-wha l., tserendejid z., so-yeon j., go g.e., prevalence of osteoporosis according to nutrient and food group intake levels in korean postmenopausal women: using the 2010 national health and nutrition examination survey nutr res practice, oct, 9 (5), pp. 539-546, (2015). [26]. nguyen tv center j.r., eisman j.a., osteoporosis and elderly men and women: effects of dietary calcium, physical activity, and body mass index, j bone miner res, 15, pp. 322-331, (2000). [27]. ma d.f et al., milk intake increases bone mineral content by inhibiting bone resorption: meta-analysis of randomized controlled trials, clinical nutrition espen, february, 8 (1), pp. e1e7, (2013). [28]. tai v., leung, w., gray a., reid i.r., mark j., bolland m.j., calcium intake and bone mineral density: systematic review and metaanalysis, bmj, 351, (2015). doi: https://doi.org/10.4316/fens.2021.020 182 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 182 188 physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques *deffan zranseu ange bénédicte1, yapi jocelyn constant1, fagbohoun jean bedel2, masse diomandé1, beugre grah avit maxwell1 1 agrovalorization laboratory, jean lorougnon guédé university, daloa, (ivory coast) bp 150, daloa, (ivory coast), dezranbe@yahoo.fr 2 laboratory of biochemistry-genetics, peleforo gon coulibaly university, korhogo, ivory coast bp 1328 korhogo, ivory coast. *corresponding author received 24th may 2021, accepted 5th september 2021 abstract: in côte d’ivoire it is difficult to keep vegetables and fruits, especially eggplant, and this is due to a lack of proper storage techniques. the few processes used are expensive and inaccessible to most of the population. the purpose of this study is to determine appropriate eggplant conservation techniques. the aim is to determine some of the physico-chemical parameters including dry matter, oil content, protein and sugar content at different types of conservation (respective immersion in vinegar, kitchen salt and wood ash). open air storage was used as a control. analyzes were performed weekly during ten (10) weeks of storage. the results showed that the dry matter content reached the maximum values of 8.45% to 17.43% in saline preservation and 8.45% to 16.56% in ambient, the highest sugar content was observed in vinegar-based preservatives, 4.16, 4.33 and 4.66% in sugars total, and at the level of reducing sugars the highest levels are observed at the level of conservation with ash for a value of 3,90%. the highest protein concentrations were observed in vinegar 2.33% and ash 2.55%. however, the highest level of oil is observed in ash conservation 0.61% to twenty-four (24) days of preservation, the ash method also reveals the highest levels of minerals throughout conservation. the results therefore indicate that vinegar and ash conservation at the method level is positively different from that of the ambient environment and salt. keywords: preservation, methods, ash, vinegar, salt 1. introduction grown for its fruit and consumed as a fruit vegetable, eggplant is produced throughout the tropical regions of africa, latin america and asia for the exploitation of its edible fruits [1]. these are ellipsoidal, ovoid or fusiform globose berries, which also have therapeutic virtues : sedative, carminative, anti-colic, antibacterial, antihypertensive, anti-tetanic [2]. eggplant is consumed even less in northern europe and north america. it remains an asian vegetable with a production of 93% of the world and marginally mediterranean [1]. in côte d’ivoire, its cultivation is carried out throughout the territory, using generally traditional techniques. despite the economic and food potential of african eggplant, little work has been done to improve the production techniques of this crop in côte d’ivoire. if all peri-urban and rural production in the country is able to fully satisfy local demand, the surplus crop is often wasted by lack of knowledge of good processing, conservation and storage http://fens.usv.ro/index.php/fens mailto:dezranbe@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 183 techniques. several research studies have been carried out on the conservation of our food. however, apart from the studies carried on the appearance of the fruit during its preservation [3], no less expensive preservation process has been put in place which could guarantee the availability and the physico-chemical quality of eggplants during the off-season or throughout the year. the rare conservation techniques encountered are mostly artisanal and based on dehydration of vegetable however, these methods not only cause the loss of many volatile or thermosensitive compounds, but also affect the organoleptic quality of fruit; this drastically lowers their market value. conventional methods in vogue (controlled atmosphere, use of additives cold conditioning, etc.) remain quite expensive and inaccessible to the majority of ivorian consumers and producers. this study was proposed with a view to finding solutions to this problem. this will identify conservation processes that will allow the physicochemical properties of eggplants to be maintained at lower costs. 2. materials and methods materials the plant material concerned the eggplant (solanum aethiopicum l.), cultivated on an experimental plot, carried out at the jean lorougnon guédé university in daloa, côte d’ivoire. method sampling healthy plants with no history of disease were selected and the species of eggplant used in this study was carefully harvested. carefully transported to the laboratory, the selected fruits are stored in vinegar, kitchen salt, and wood ash. open air storage will be used as a control treatment. for each treatment, four (04) batches of 500 grams of eggplants (including one batch per preservation technique) were treated. also per batch, three (3) samples were formed thus giving twelve (12) samples per treatment. analyzes (treatments) were carried out every three (3) days during one (1) month of storage. the treatments will be named t0, t1, t2, t4, t5, t6, t7, t8, t9 and t10 for the thirty (30) days of conservation. a total of one hundred and twenty (120) samples will be analyzed during this study. physico-chemical analysis dry matter and ash levels were determined using bipea methods [4]; the protein content was determined using the aoac method [5] using kjedhal; fat content according to bipea [4] using soxhlet; the total sugar content according to dubois and collaborators [6] using phenol and reducing sugar levels according to benfeld [7] using 3,5-dinitro salicylic acid (dns). statistical analyses the data analysis was carried out using microsoft office excel 2007 and statistica 7 software, to determine the standard averages and deviations. onefactor variance analysis (anova) was performed on the data to determine significant differences between averages during conservation. then, the statistical differences were highlighted by the duncan test at the threshold of α = 0.05. 3. results and discussion determination of dry matter content dry matter levels generally increase throughout the storage except for the control (table 1). values increase continuously from 8.45 to 17.53% (cooking salt), to 12.01% (vinegar) and to 13.96% (ash). the method of preservation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 184 with salt significantly reveals the highest values; then come the conservation with ash and then vinegar (table 1). table 1 change in dry matter ratio treatments witness cooking salt vinegar ash p intra processing t0 8.45±0.76 ba 8.45±0.76 aa 8.45±0.76 aa 8.45±0.76 aa t1 9.88±0.21 ca 9.22±0.37 aa 8.70±0.84 aa 9.08±0.96 aa 0.080 t2 14.01±1.12 eb 9.83±1.62 aa 9.01±0.21 aa 9.33±0.44 aa 0.002 t3 16.56±1.08 fc 12.33±0.87 bb 10.33±0.96 ba 11.66±0.87 ba 0.038 t4 15.94±0.26 fb 13.42±1.25 bab 11.25±1.00 ba 12.25±1.11 ba 0.001 t5 15.03±0.33 ac 14.66±1.06 abc 10.95±0.36 ba 12.88±0.86 bcb 0.014 t6 11.10±0.86 da 16.10±1.92 bcc 11.43±1.03 ba 13.33±1.17 bcb 0.024 t7 10.26±0.33 cda 15.88±1.65 bcc 12.19±0.76 bcb 13.20±1.37 bcb 0.031 t8 6.13±0.11 aa 16.35±0.75 cd 11.88±0.66 bb 14.11±1.16 cdc <0.001 t9 6.35±0.43 aa 17.33±1.18 cd 12.10±1.03 bcb 14.53±0.93 cdc <0.001 t10 5.42±0.55 aa 17.53±0.96 cd 12.01±1.10 bb 13.96±0.65 bcc <0.001 p intra matérial <0.001 0.044 0.047 0.038 determination of oil content eggplant is found to be low in fat. however, variations were observed during storage with the highest t2 values of 0.51% (control), t4 values of 0.42% (salt), t5 values of 0.55% (vinegar) and then 0.61% for ash at t8. the highest rate, however, was observed preservation with ash (table 2). table 2 change in oil content treatments witness cooking salt vinegar ash p intra processing t0 0.23±0.00 ea 0.23±0.00 aa 0.23±0.00 aa 0.23±0.02 aa t1 0.28±0.01 fb 0.22±0.01 aa 0.23±0.02 aa 0.20±0.01 aa 0.040 t2 0.51±0.03 ic 0.33±0.02 bcb 0.26±0.01 ba 0.33±0.01 bb 0.002 t3 0.36±0.00 gb 0.33±0.00 bca 0.33±0.00 ca 0.36±0.00 cb 0.028 t4 0.44±0.02 hb 0.42±0.02 db 0.45±0.01 eb 0.35±0.01 bca 0.042 t5 0.23±0.01 ea 0.33±.02 bcb 0.55±0.03 fd 0.44±0.03 dc <0.001 t6 0.20±0.00 da 0.40±0.00 db 0.43±0.01 ec 0.43±0.02 dc 0.024 t7 0.16±0.00 ca 0.30±0.00 bb 0.49±0.04 fc 0.49±0.03 dec 0.021 t8 0.13±0.01 ba 0.35±0.01 cb 0.38±0.02 dc 0.61±0.01 cdd <0.001 t9 0.11±0.00 aa 0.33±0.02 bcb 0.51±0.03 fc 0.53±0.03 dec 0.037 t10 0.11±0.00 aa 0.33±0.03 bcb 0.43±0.01 ec 0.46±0.00 dd <0.001 p intra matérial <0.001 0.008 <0.001 0.001 determination of raw the content of mineral ash also in small amounts in eggplant (<0.5%), shows different variations throughout conservation. the highest value (0.26%) was observed at t3 preservation in ash. they are also raised at t2 in vinegar and salt, respectively (0.21%) and (0.23%), and then (0.24%) at the t4 control level. the ash-based method, however, reveals the highest rates throughout the shelf-life (table 3). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 185 table 3 change in raw ash content treatments witness cooking salt vinegar ash p intra processing t0 0.15±0.00 da 0.15±0.00 ba 0.15±0.00 ba 0.15±0.00 aa t1 0.12±0.00 ba 0.22±0.00 ded 0.15±0.01 bb 0.18±0.00 bc <0.001 t2 0.21±0.01 ea 0.23±0.00 eb 0.21±0.01 da 0.23±0.01 db 0.042 t3 0.16±0.01 db 0.13±0.01 aa 0.13±0.00 aa 0.26±0.00 ec <0.001 t4 024±0.01 fc 0.12±0.00 aa 0.15±0.00 bb 0.25±0.01 dec 0.014 t5 0.13±0.00 ca 0.16±0.01 bb 0.15±0.00 bb 0.18±0.01 bc <0.001 t6 0.20±0.01 eb 0.18±0.01 cb 0.13±0.00 aa 0.23±0.01 dc 0.004 t7 0.16±0.00 da 0.18±0.00 cb 0.19±0.01 cdbc 0.20±0.00 cc 0.011 t8 0.10±0.00 aa 0.15±0.00 bb 0.20±0.00 dd 0.18±0.01 bc <0.001 t9 0.12±0.00 ba 0.23±0.01 ec 0.18±0.01 cb 0.23±0.01 dc <0.001 t10 0.12±0.00 ba 0.21±0.01 dc 0.13±0.00 ab 0.24±0.00 dd <0.001 p intra matérial <0.001 0.001 <0.001 0.008 nb: the assigned values of the same letter in lowercase and bold are not significantly different between retention times. the assigned values of the same uppercase letter are not significantly different between retention modes determination of protein content protein levels generally increased significantly from t0 to t3 (from 1.55 to 1.96%) and decreased to 1.22% in the control. variations are rather different in other conservation methods. indeed, the levels increase from 1.55 to 1.92 % (t4), 2.33 % (t3), 2.25 % (t4) respectively for the storage using salt, vinegar and ash. the highest protein concentrations are observed significantly in vinegar and ash preservatives (table 4). table 4 change in protein content treatments witness cooking salt vinegar ash p intra processing t0 1.55±0.06 ca 1.55±0.06 aba 1.55±0.06 aa 1.55±0.06 aa t1 1.88±0.11 dec 1.52±0.03 aa 1.70±0.04 bb 1.88±0.09 bc 0.022 t2 1.81±0.05 dea 1.71±.12 bca 2.01±0.11 db 1.83±0.14 bab 0.041 t3 1.96±0.10 eb 1.83±0.03 ca 2.33±0.13 ec 2.16±0.07 cc 0.002 t4 1.84±0.02 da 1.92±0.15 dab 1.95±0.03 cb 2.25±0.11 dc <0.001 t5 1.73±0.10 dab 1.66±0.06 ba 1.83±0.10 bcb 1.88±0.06 bb 0.013 t6 1.60±0.01 ca 1.70±0.02 bb 2.13±0.03 dd 1.93±0.10 bc <0.001 t7 1.46±0.10 bca 1.88±0.11 cdbc 2.19±0.16 dec 2.00±0.13 bcc 0.001 t8 1.53±0.13 ca 1.65±0.05 ba 1.88±0.13 bcb 2.11±0.11 cc <0.001 t9 1.33±0.02 aa 1.53±0.10 abb 2.18±0.06 ded 1.93±0.03 bc <0.001 t10 1.22±0.11 aa 1.48±0.06 ab 2.01±0.10 dc 2.06±0.15 bcc 0.021 p intra matérial 0.001 <0.001 <0.001 0.018 determination of sugar content total sugar content t3 is the storage time when the sugar concentrations are higher. thus, the values are 4.16, 4.33 and 4.66%, respectively in the control, the vinegar and ash method. concentrations observed at the saline method are rather lower. the values then food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 186 vary inhomogeneously and significantly at the level of the four (4) storage methods up to t10. the highest levels of total sugars are observed in vinegar and ash preservatives (table 5). table 5 change in total sugar content treatments witness cooking salt vinegar ask p intra processing to 3.95±0.11 ea 3.95±0.11 da 3.95±0.11 da 3.95±0.11 ba t1 3.88±0.22 eb 3.22±0.13 ca 3.70±0.14 cdb 3.83±0.16 bb 0.040 t2 4.01±0.26 efa 3.83±0.22 da 4.01±0.21 da 3.93±0.14 ba 0.052 t3 4.16±0.33 efb 3.33±0.17 ca 4.33±0.26 eb 4.66±0.37 dbc 0.038 t4 3.74±0.21 ea 3.42±0.25 ca 4.25±0.10 eb 4.25±0.11 cb 0.026 t5 3.83±0.10 eb 3.66±0.06 cda 3.95±0.16 db 3.88±0.20 bb 0.044 t6 2.60±0.20 db 2.10±0.12 aa 3.43±0.23 cc 3.33±0.17 ac 0.020 t7 1.46±0.10 aba 2.88±0.11 bcb 4.19±0.26 dec 4.20±0.30 cc 0.001 t8 1.53±0.09 ba 2.35±0.15 abb 3.88±0.33 cdc 4.11±0.16 bcc 0.003 t9 2.03±0.26 ca 2.33±0.18 abab 2.48±0.16 bb 3.53±0.33 abc <0.001 t10 1.33±0.11 aa 2.53±0.26 bc 2.01±0.10 ab 3.96±0.15 bd <0.001 p intra material <0.001 0.001 <0.001 0.014 reducing sugar content the reduced sugar levels of the eggplant studied are similar to those of total sugar. however, the highest values observed during retention are 2.83% (t3), 3.66% (t2), 3.75% (t4), 3.90% (t7); in the control and in the methods of conservation based on salt, vinegar and ash, respectively. the control records the lowest concentrations at t7 (1.00%). ash and vinegar get the highest levels of reducing sugars during conservation (table 6). table 6 variation in reducing sugar content treatments witness cooking salt vinegar ash p intra processing to 2.10±0.03 ba 2.10±0.03 ca 2.10±0.03 ba 2.10±0.03 aa t1 2.90±0.12 ec 2.10±0.10 ca 2.60±0.10 cbc 2.83±0.21 cc <0.001 t2 3.53±0.14 db 2.83±0.11 ea 3.41±0.23 db 3.43±0.24 deb 0.043 t3 3.66±0.23 dbc 2.03±0.18 ba 3.30±0.13 db 3.88±0.17 ec <0.001 t4 2.44±0.21 cdb 2.12±0.05 bca 3.75±0.10 ec 3.75±0.11 ec 0.026 t5 2.73±0.09 deab 2.66±0.16 da 3.05±0.28 dc 2.88±0.12 cb 0.002 t6 2.10±0.16 bcab 1.80±0.12 aa 2.33±0.23 cb 2.50±0.19 bb 0.012 t7 1.00±0.02 aa 2.01±0.10 bb 3.49±0.12 cc 3.90±0.22 ed <0.001 t8 1.03±0.01 aa 1.95±0.15 abb 2.98±0.23 cdc 3.81±0.17 ed <0.001 t9 1.77±0.03 ba 1.66±0.13 aa 2.00±0.11 bb 2.43±0.23 bc <0.001 t10 1.01±0.06 aa 1.83±0.12 ac 1.41±0.13 ab 3.02±0.26 cd <0.001 p intra matérial 0.018 <0.001 <0.001 0.034 nb: the assigned values of the same letter in lowercase and bold are not significantly different between retention times. the assigned values of the same uppercase letter are not significantly different between retention modes increases in the dry matter rate indicate that the methods used would be favorable. this would be linked to the evaporation phenomenon (more intense with the saline food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 187 method) in dry storage cases and then to the phenomenon of osmosis during storage in the vinegar. decreases in the dry matter rate in the control would be the cause of decay observed beyond the twelve (12) days of storage. extending the green lifespan of eggplants in our study suggests that different treatments would positively alter the interactions of fruits with the surrounding environment [3]. the observed fat levels would indicate that oil would not be the main energy reserve in eggplants due to the low levels observed. however, their variations during conservation would be related to their uses in either catabolic activities resulting in the formation of carbohydrates or lipogenesis [8]. low ash levels in eggplants also show higher values in the ash-based method. the constitution of the conservation environment may be at the origin of this difference [9]. the minerals would come from the wood ash used. the different variations observed are due to the use of different ash constituents in biochemical activities during conservation [10]. the variations in the different percentages of protein observed could be due to the different hydrolysis and metabolic reactions. synthetic responses would be earlier in open-air and vinegar preservation where aqueous medium (vinegar) would promote the resumption of metabolic activities [11]. rather than in drier environments, however, the high values observed in ash conservation may be due to the involvement of the constituent mineral elements in different synthesis reactions. the total sugar concentrations falling at the saline method could be related to the different interactions between the fruit and the medium. according to the work of el jaafari [12], salinity led to the formation of aggregates in soil nutrients, resulting in pseudocimentation of the environment, severely impeding synthesis. differences in the proportions of sugars observed at all times in other conservation conditions would be related to the different postharvest metabolic reactions that take place during conservation [13]. retention in vinegar as well as the mineral elements that make up wood ash would further enhance glucid syntheses. studies show that eggplant fruits are mainly reduced sugars. according to lewis [14], the major source of fructose in the plant is sucrose, which, after cleavage, releases equimolecular amounts of fructose and glucose. this carbohydrate source would be quickly catabolized at the harvest of fruits, resulting in a high concentration of reducing sugars, which constitute the crossroads of most biochemical activities. sucrose and polysaccharides are used as precursors of synthesis, by conversion to glucose and fructose [8]. thus the different variations observed would be the result of reactions of neoglucogenesis and/or glycolysis [15]. vinegar and ash would be more beneficial for neoglucogenesis or non-reducing sugar cleavage activities, hence higher levels. 4. conclusion the objective of this study was to highlight the impact of different conservation methods on the green life of eggplants. to achieve this, the physicochemical characteristics were carried out on the fruits. the results therefore indicate that vinegar and ash conservation at the method level is positively different from that of the ambient environment and salt. however, further studies on organoleptic tests could clearly differentiate between these two conservation methods. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell, physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques, food and environment safety, volume xx, issue 3 – 2021, pag. 182 – 188 188 5. references [1] chen w.. wu z.. su m.. zhu j. (2000). post-harvest technology. banana transport and marketing in china. agrociencia. 36(2): 169-180. [2] lépengue a.. mouaragadja i.. dick e.. m’batchi b. & aké s. (2010). improving the shelf life of plantains at room temperatures. int. j. biol. chem. sci.. 4(3): 730737. [3] lepengué a.. mouaragadja i.. dick e.. mbatchi b.. ake s.. (2012). trial to improve the shelf life of eggplants at room temperatures in gabon. int. j. biol. chem. sci. issn 1991-8631. 6(2): 792-798. [4] bipea. (1976) recueil des méthodes d’analyse des communautés européennes. [5] aoac (2000). association of official analytical chemical; official methods of analysis. 14th ed. washington d.c usa. 201 p. [6] dubois m.. gilles k.. hamilton j.. rebers p. et smith f.. (1965) colorimetric methods for determination of sugars and related substances. anal. chem. 28 : 350356. [7] bernfeld p. 1955. amylase β and α. (assay method). in enzymology i. colowick and kaplan. eds.academic press. new york. pp. 149154. [8] assa r. (2007) diagnosis of the peasant coconut grove of the ivorian coast: physicochemical study. microbiological and organoleptic of the water and the kernel of the fruits of four cultivars of the coconut palm (cocos nucifera l.) according to the stages of maturity. single doctorate thesis from the university of cocody (ivory coast). 188 p. [9] lester r. & a. seck. (2004). solanum aethiopicum l. in : grubben g. j. h. et o. a. denton (eds.). ressources végétales de l’afrique tropicale 2. légumes. fondation prota. backhuys publishers. wageningen. pays bas : pp 530 536. [10] pettigrew w. (2008) potassium influences on yield and quality production for maize. wheat. soybean and cotton. physiology plant. 133. 670-681. [11] goffner d.. cazalir r.. percie c.. calmas j. & cavalie g. (1988) 14c photoassimilate partitioning in developing sunflower seeds. journal of experimental botany. 39: 1411-1420 [12] el jaafari s. (1993) contribution to the study of the biophysical and biochemical mechanisms of drought resistance in wheat. doctorate. faculty of agronomic sciences of gembloux. belgium. 214p. [13] pech o.. may a.. muller h.. vieth a.. solte m & ell c. (2002). journal of experimental botany 53 (367). 201-206. [14] lewis d. (1984) physiology and metabolism of alditols. in lewis d.h.. ed. storage carbohydrates in vascular plants. cambridge. uk. cambridge university press. 157–179. [15] duangsrisai s.. yamada k.. shiratake k.. kanayama y. & yamaki s.. (2007) properties of sorbitol dehydrogenase in strawberry fruit and enhancement of the activity by fructose and auxin. journal of the japanese society for horticultural science 77. 318–323. doi: https://doi.org/10.4316/fens.2021.009 68 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2021, pag. 68 80 use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria) *aissam ghrieb 1 , fethi baali 2 , chemceddine fehdi 2 , azzedine hani 3 , hicham chaffai 3 & larbi djabri 3 1 department of earth sciences, faculty of exact sciences, science of nature and life, university of larbi tebessi, tébessa, algeria. aissam.ghrieb@univ-tebessa.dz 2 water and environment laboratory, department of earth sciences, faculty of exact sciences, science of nature and life, university of larbi tebessi, tébessa, algeria. fethi.baali@univ-tebessa.dz , fehdi@yahoo.fr 3 laboratory of water resource and sustainable development (redd), department of geology, faculty of earth sciences, badji mokhtar university, annaba, algeria. haniazzedine@yahoo.fr , hichamchaffai@yahoo.fr , djabri_larbi@yahoo.fr corresponding author received 3rd january 2021, accepted 30th march 2021 abstract the water supply environment in seybouse valley (north east of algeria) is sensitive and fragile as the aquifer is highly vulnerable to various sources of pollution, must recognize the pollution sources and water quality integration. so, there is a need for a better knowledge and understanding of the water pollution determinants to meet the domestic, agricultural and industrial uses. the pollution of this ground water was determined by total dissolved solids (tds). this represents the salinity of freshwater and originate from natural sources, sewage, urban, runoff, industrial wastewater and chemicals. based on cause-and-effect relationships, the driver–pressure–state–impact–response (dpsir) plan was used to establish indicators for an integrated water resource management approach to water quality in the semi-arid mediterranean region. the aim of this work is to determine the most pressing pollution source of seybouse valley. with this intention, the artificial neural network (ann) models were used to model and predict the relationship between groundwater quality with point and diffuse pollution sources determinants. the selected variables were classified and organized using the multivariate techniques of hierarchical cluster analysis (hca), factor analysis (fa), principal components and classification analysis (pcca). it was concluded that the industrial wastewater that is the most pressing pollution source followed by seawater intrusion. keywords: dpsir model; ann; multivariate techniques; seybouse valley. 1. introduction two recent problems in water systems planning have been a shortage of water resources and optimal control. population development, the loss of available water supply, changes in lifestyle, the rising rate of use, climate change, and a number of other factors have made usable water a major concern for the future [1]. the seybouse valley is currently facing the problem of water shortage. over the next twenty years, the demand for drinking water is expected to increase by 20% [2]. the establishment of sustainable water management, both qualitatively and quantitatively, is therefore imperative for the future of the population of the seybouse valley, but also of ecosystems, and mailto:aissam.ghrieb@univ-tebessa.dz mailto:fethi.baali@univ-tebessa.dz mailto:haniazzedine@yahoo.fr mailto:hichamchaffai@yahoo.fr mailto:djabri_larbi@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 69 conditions economic and social development. the seybouse valley is also confronted with urban, industrial and agricultural pollution. this pollution comes from the various agglomerations, factories and agricultural areas located on both banks of the wadi. the pollution has reached a high degree. 4.5 million m3 are discharged annually into the river, of which 3 million m3 are used oils [3]. the insufficient treatment of wastewater, the proliferation of urban waste, atmospheric pollution and various industrial pollution significantly affect the health of citizens with adverse consequences on lowincome groups living in slums or in areas unattractive (proximity to landfills, wastewater discharge areas, etc.). the rehabilitation of the seybouse valley can be a good example of integrated management, since it would include aspects of protection of surface and underground resources, rationalization of domestic, agricultural and industrial uses, but also the fight against pollution and protection of the environment. only an integrated approach offers the possibility of managing these resources while respecting the natural environment, the interests of citizens and those of economic actors. the research envisaged responds to this need for integration, from the location and evaluation of underground resources, to the protection of their quality, but while evaluating the possible harmful effects. they thus contribute to the implementation of the current sustainable development policy, which is deployed through a number of directives, including that on water. but beyond that, they are part of the plan adopted at the johannesburg sustainable development summit. the development of a model to predict understanding would help to manage water resources effectively. there are different methods for data analyses, such as statistic techniques. prediction of water quality parameters necessitates a thorough examination of the various processes that can influence water quality, as well as the creation of mathematical or deterministic models based on the data collected [4]. second, developing data driven models using information and collected data is an essential tool. the study area climate is semi-arid mediterranean; the annual rainfall is varies between 700 and 900 mm. the potential evapotranspiration is closely linked to the temperatures. the annual average of the evapotranspiration ranges from 1000 to 2000 mm [5]. the conservation and management of these resources is becoming increasingly necessary as the world's population and industrial demands grow. the intensive agricultural activities in the plain of annaba (algeria) induce the increase of the risk of the fresh water degradation [6]. in fact, uncontrolled high pumping rates of water causes modification of the natural flow system and induces seawater flow from the coast making the deterioration of the water table quality [7]. there are many water quality parameters, but tds is an important water quality parameter especially in reservoirs. in fact, tds can be considered a pollutant for this reason, it is vital to have information about the existing situation, seasonal variations and expectations of the future situation of the parameter [4]. domestic solid waste, domestic wastewater of the municipalities, industrial wastewater, pesticides, organic fertilizers, chemical fertilizers, petrol stations, carbon dioxide (co2) and seawater intrusion are the main pollution point sources of the seybouse valley. the coastal aquifer system supplies the water demands of municipalities, several food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 70 villages, thousands of hectares of agricultural lands, and several industries. ever increasing population, resulting in a high in domestic water demands as well as industrial demand including but not limited to development of agricultural networks, and inter-basin water transfers, darken the outlook for water quality of the seybouse valley. the principal purpose of the study is to develop ann model studying the relation between total dissolved solids from coastal aquifer (represented by tds (mgl−1) and the main pollution sources. understanding spatial relations between hydrological variables and salinity of groundwater can contribute in an integration of water resources management. this research might be considered as one of the few contributions in qualitative modelling of the relation between groundwater salinity and the pollution pressure variables in spatial scale using ann. 2. materials and methods 2.1. study area the seybouse river basin is situated in algeria's northern east, and the coastal portion is the western part of the annaba plain. because of the length of its course, the number of tributaries, and the size of its basin, the seybouse is one of algeria's most significant rivers. the seybouse river basin is divided into three sections: the high plains (high seybouse), the mean seybouse, and the maritime seybouse. the latter denotes the research field. about 36°30' and 37° north latitude and 7°30' and 7°55' east longitude, the research region is situated in north-east algeria. with an area of 103 km², the research area is part of the seybouse river basin, and its natural boundaries are the mediterranean sea in the north, the eastern extension of the cheffia numidian mountains in the south, and the edough metamorphic complex and the fetzara lake in the west, finally, the eastern extension of the annaba-bouteldja plain and the mounts of nador n'bail in the east. as seen in the diagram, the research area includes the bulk of annaba city and the western portion of el taref city (fig.1). fig. 1 study area site the study area is divided into 31 main sectors: annaba, el bouni, kherraza, boukhadra, berkazerga, bouzaaroura, essarouel, sidi salem, oued ennil, sidi amar, hadjar eddis, derradji redjem, merzoug amar (el gantra), bergouga, el hadjar, el kerma, chabi larbi, el heraicha, ain berda, el harrouchi, ain sayd, medjez rassoul, salmoune hachemi, labidi mohamed, drean, ain allam, djenane el chouk, fedaoui moussa, chbaita mokhtar, zourami ali, chihani bachir. 2.2. geological and hydrogeological framework the studies realized in the region there are two types of formations [8], one metamorphic presented by the eddough massif and the other sedimentary occupying the totality of annaba plains. (fig.2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 71 the sediments are heterogeneous with numerous alternations of sandy clays, sands and gravels beds. two main aquifers are distinguished [9]. aquifers, both shallow and deep: the gneiss altered; dune massif, the dunes, and the water current; and recent alluvium constitute a surface aquifer that stretches across the whole plain of annaba and flows through the surface silts (fig.3) the deep aquifer is captive and composed of gravel; its roof is made up of various textures (clay and clay loam, clay and sand); the main aquifer of the basin is built in permeable sediments (mio-pliocène), is made up mostly of pebble, sand, and clay along the wadis, and has stronger hydraulic properties; and becomes free at the region. drean is a character in the drean franchise. the superficial layer overtakes the gravel layer at this stage (drean), and there is the risk of a phenomenon leakage between the two layers [10]. these aquifers are connected by a semi-permeable and/or impermeable intermediate layer, forming a single two-level aquifer. rainfall and runoff of seybouse wadi water from further south feed the aquifer. the need to protect this aquifer's supplies is critical becauseit is the primary source of water for human use. fig. 2 annaba plain geological sketch map [11], amended 2010. fig. 3 hydrogeological cup through the plain of annaba [12], amended 2010). 1: pebbles and gravels, 2: sand, 3: numidian clay, 4: cenomanian marl and marly limestone, 5: plio-quaternary detrital clays, 6: metamorphic formations, 7: fault, 8: drilling; source: own elaboration 2.3. dpsir analysis approach the water framework directive (wfd) 2000/60/ec clearly sets the basis and principles for effective protection of groundwater, internal, transitional and coastal waters at the river basin scale. several approaches and methodologies have been proposed for improving water resource management at this scale, with the driver-pressure-state-impact-response (dpsir) approach [13], being one of the most widely used in the context of integrated water resource management [14]. a new computational water interconnected model based on cause-effect relationships has been created to address the life cycle of water resource management. the new conceptual model is based mainly on three decisive categories: (1) the natural system, which is of critical significance for the water available quantities and qualities, (2) the human system, which determines the use of water and the pollution of the resource, (3) the institutional and management system must balance consideration of the natural and human systems and their interdependencies. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 72 the three systems are divided into five categories based on cause-effect dpsir framework for development of water related variables. the variables reflect and translate the water sustainability concepts, preventive and ecosystem approaches. the five categories are: socio-economic aspects, anthropogenic pollution pressures, state of water quality, public health and ecological impacts and the institutional responses. the human system is explained by the socio-economic, anthropogenic pressures and public health variables. the natural water system is presented by the state of water, and ecological impacts and the institutional system is reflected by the institutional responses. the driver-pressure-state-impactresponse (dpsir) framework was chosen as a well-established framework for developing the possible variables under five categories that are separated into a collection of most important variables that represent essential aspects of water supplies. following the categories are: d: driving forces are underlying socioeconomic and sectoral factors influencing a variety of relevant variables. drivers produce a series of pressures and are quantified by aggregated data, population, tourism, agriculture water consumption etc; p: pressure indicators describe the variables which directly cause environmental problems, the consequences of the driving forces on water supplies are manifested as pressures. pressures weaken the condition of water supplies and have an effect on both them and humans; s: state indicators illustrate the existing conditions and the observable changes of the environment (chemical characteristics); i: impact indicators describe the ultimate effects of changes of state on the human and ecosystems; and r: response indicators present the efforts of the administration and policy making level (decision makers, management) the measures taken to improve the state of the water resources. in the european research project eurocat, which seeks to achieve integrated catchment and coastal zone management, the driving powerpressure stateimpactresponse (dpsir) system has been chosen to study all regional water catchments. [13]. it will help forecast how potential socio-economic developments in water catchments will affect water quality, allowing policy responses to be developed to mitigate the stresses caused by those drivers and the impacts of such pressures on water quality [15, 16]. developed metrics for an effective and operational decision support mechanism for efficient use of water supplies at the catchment level using the dpsir method for environmental cause-effect relationships. the new conceptual water integrated model was applied to the life cycle of water resources management in the seybouse valley. the research's key goals were to: – characterize the efficient variables of water sector management and identify the geographical areas under water stresses; – establish prediction relationships between water abstraction from the coastal aquifer and water quality state; – group municipalities according to their water-related variables; – make proposals for reform, including new ideas for preserving natural water supplies as sources of supply for current and future generations [1]. 3. results and discussions 3.1. statistical analysis for the normality tests and multivariate statistical analyses–hierarchical cluster analysis (hca) and principal component food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 73 analysis (pca)–as well as neural networks, the statistical program statistica (version 8; 2008) are used [17]. ten pollution variables (solid waste, domestic wastewater, industrial wastewater, pesticides, organic fertilizers, chemical fertilizers, petrol stations, carbon dioxide (co2), seawater intrusion and total dissolved solids (tds) were utilized in the statistical analyses). 3.1.1. analysis with artificial neural networks (ann) to define and prioritize the efficient variables of pollution pressure categories, ann models were used. the use of ann models does not include linearity or normality of data. as a consequence, there is no need for data transformation [18]. total dissolved solids data and pollution parameters were applied to create the ann model. the pollution parameters were: domestic solid waste (solid waste), domestic wastewater (dom wastw), industrial wastewater (ind wastw), pesticides (pest), organic fertilizers (org fert), chemical fertilizers (chem fert), petrol stations (petrol stat), carbon dioxide (co2), seawater intrusion (seawater intr) and total dissolved solids (tds). the pollution pressure variables were considered as the possible input variables whilst the target output variable was the total dissolved solids (tds). the variables representing the pollution pressure variables were considered as the possible input variables whilst the target output variable was the total dissolved solids (tds) [18]. the mlp network can be represented by the following compact form: {tds}= ann [solid waste, dom wastw, ind wastw, pest, org fert, chem fert, petrol stat, co2, seawat intr] a schematic diagram of this network is given in (fig. 6). fig. 6 mlp network (three layers), pollution pressure variables mlp (3 and 4 layers), rbf, grnn, and linear networks are the forms of networks considered. several networks were examined during the study [1]. the best optimal ann model found is mlp (3 layers) with 4 hidden nodes (fig. 6) and a minimal error of 25.56 compared with the other types of ann networks table1. the model has very good performance in verification with regression ratio (s.d. ratio) of 0.01919 and the correlation coefficient is higher than 87% for training, 99% for verification and testing (table 1) which shows an excellent agreement between the actual observed and predicted tds (fig. 7). the ann sensitivity analysis of pollution pressure variables in both training and verification phases (table 2) indicates that solid waste and domestic wastewater, industrial wastewater, carbon dioxide and seawater intrusion are the most important and pressing pollution sources. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 74 the ann model removed four input variables due to their low sensitivity which are pesticides, organic fertilizers, chemical fertilizers, petrol stations. table 1 statistical regression parameters for the target output (tds)-pollution pressure variables tr. tds ve. tds te. tds data mean 2209.906 2620.833 2142.188 data s.d. 1091.389 1231.062 954.9432 error mean -112.8458 -16.76796 115.2068 error s.d. 595.6944 23.62893 129.9397 abs. error mean 404.7289 22.67534 116.7138 rms error 594.5 25.56 156.6 s.d. ratio 0.545813 0.01919 0.1360706 correlation 0.8789501 0.9998636 0.9907989 legend: tr: training, ve: verification, te: testing fig. 7 predicted tds vs observed tds table 2 sensitivity analysis of independent input variables— pollution pressure variables verification phase are industrial wastewater, seawater intrusion, carbon dioxide (co2), solid waste, domestic wastewater. the ann model removed four input variables – pesticide, organic fertilizers, chemical fertilizers, petrol stations because of its low sensitivity. the results of the ann model are shown in (table 3). table 3 ranking of input pollution pressure variables tests of normality (pollution pressure) the normality tests are used in addition to the graphical normality evaluation [19]. the kolmogorov-smirnov (k-s) test, lilliefors corrected k-s test, and shapiro-wilk test are the most common methods used to determine normality [20]. the most efficient measure is the shapiro–wilk test [21]. the shapiro-wilk test is recommended by some researchers as the best alternative for determining data normality [22]. tests of normality of the pollution category variables the pressure variables “p” for the study area are summarized in (table 3). according to the shapiro-wilk test, shows that all the variables have non-normal distribution of data. solid waste dom wast w ind wast w co2 seawater intr rank 4 5 1 3 2 error 725.203 656.0755 801.8969 728.4742 730.8205 ratio 1.167607 1.056309 1.291088 1.27013 1.176651 rank 4 5 1 3 2 error 396.4911 238.3604 670.42 631.0517 604.3308 ratio 1.386904 0.8337713 2.345092 2.0198 2.113915 ind wastw seawater intr co2 solid waste dom wastw rank 1 2 3 4 5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 75 table 4 tests of normality of pollution pressure variables n maxi k-s lilliefors p w p dom wastw 31 0.389100 p < .01 p < .01 0.325455 0.000000 solid waste 31 0.245586 p < .05 p < .01 0.667737 0.000000 pest 31 0.187130 p < .20 p < .01 0.912573 0.015067 org fert 31 0.150107 p > .20 p < .10 0.925074 0.032257 chem fert 31 0.134144 p > .20 p < .20 0.912688 0.015171 petrol stat 31 0.370968 p < .01 p < .01 0.445230 0.000000 ind wastw 31 0.330812 p < .01 p < .01 0.695460 0.000001 co2 31 0.231724 p < .05 p < .01 0.799566 0.000051 seawater intr 31 0.283124 p < .01 p < .01 0.766539 0.000013 tds 31 0.251339 p < .05 p < .01 0.828221 0.000180 3.1.2. correlation matrix analysis for testing the relation between any two variables using the logarithm (log), the pearson’s correlation coefficient was used to show the interrelationship and coherence pattern among pollution pressure parameters [23]. the correlation coefficient values of the analyzed pollution pressure parameters are given in (table 5). correlation matrix showed inter-parameter. strong (p < 0.01) and significant correlation (p < 0.05) were observed in the pollution parameters. log(domestic wastewater) has significant and positive linear relationships with log(petrol stations), log(carbon dioxide), log(industrial wastewater), and log(seawater intrusion). increase in domestic solid waste is associated with the increase in wastewater generation since the garbage produced at the screens in the treatment facilities are transferred to the solid waste sanitary landfills. domestic wastewater increases with the increase in the industrial wastewater generation since the industrial facilities are connected to the urban wastewater systems immediately after pre-treatment. the increase in the domestic wastewater generation indicates rise in groundwater abstraction and thus increase in seawater intrusion. log(pesticides) has significant and positive linear relationships with log(organic fertilizers), log(chemical fertilizers). the use of pesticides is always associated with organic fertilizers and chemical fertilizers since they are applied for the same agriculture land but with different proportions. log(industrial wastewater) has a positive linear relationship with log(seawater intrusion), log(carbon dioxide) and log(total dissolved solids). increase of industrial wastewater generation is caused by more use of groundwater which results in lowering the water table and seawater intrusion. the log(total dissolved solids), had a positive strong correlation with log(carbon dioxide), log(industrial wastewater), and moderate correlation with log(petrol stations). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 76 table 5 pearson correlation matrix of pollution pressure variable note: significant values (at p 0.05) are in bold, n=31 (casewise deletion of missing data) table 6 factor-variable correlations (factor loadings), pollution pressure variables variable factor 1 factor 2 dom wastw -0.70 0.39 solid waste -0.39 0.83 indwastw -0.88 -0.19 co2 -0.83 -0.20 seawater intr -0.79 -0.01 tds -0.83 -0.30 *pest 0.49 -0.02 *org fert 0.62 0.03 *chem fert 0.73 0.07 *petrol stat -0.75 0.27 expl.variance 5.57 1.43 propretotale 0.55 0.14 in bold: loadings are >.700000 active and supplementary variables *supplementary variable (underlined loadings are >.700000) 3.1.3. factor analysis (fa) the global pcca of the set of data related to "sources of pollution" demonstrate that the first two factors (f1 and f2) provide a justified inertia of 73,6 % that appears rather good, having an average inertia, a considerable number (31) of analyzed samples and (10) variables taken into consideration; knowing that the cumulated percentage goes gradually towards 100%. (table 6) presents variances of factors and their loadings from variables. the first factor corresponds to the largest eigenvalue (5,57) and accounts for approximately 56,89% of the total variance. it is most correlated with the variables domestic wastewater, industrial wastewater, carbon dioxide, seawater intrusion, total dissolved solids and petrol stations (negative correlation) and chemical fertilizers (positive correlation). the second factor corresponding to the second eigenvalue (1,43) accounts for 16,66% of the total variance. it is highly correlated with solid waste (positive correlation). (fig. 8(a) and 8(b)) displays coordinates for the two factors. the graph shows a unit circle with active variables that were used to compute the current factor solution and a supplementary variable that was only mapped into the coordinate system defined by the factors. because the current analysis is based on correlations, the highest dom wast w soli d wast pest org fert chem fert petr ol stat ind wastw co2 seawat er intr tds dom wastw 1.00 solid wast 0.36 1.00 pest -0.26 -0.25 1.00 org fert -0.31 -0.29 0.89 1.00 chem fert -0.35 -0.30 0.75 0.89 1.00 petrol stat 0.83 0.37 -0.36 -0.41 -0.47 1.00 ind wastw 0.53 0.18 -0.29 -0.42 -0.63 0.61 1.00 co2 0.46 0.21 -0.47 -0.64 -0.65 0.53 0.66 1.00 seawater intr 0.54 0.22 -0.47 -0.51 -0.61 0.68 0.66 0.50 1.00 tds 0.33 0.20 -0.44 -0.57 -0.66 0.41 0.73 0.75 0.55 1.00 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 77 possible factor coordinate (variable-factor correlation) is 1.0, and the number of all squared factor coordinates for a variable (squared correlations between the variable and all factors) cannot be greater than 1.0. the circle will show you (on a scale) how well each variable is defined by the current set of variables (the closer a variable in this plot is located to the unit circle, the better is its representation by the current coordinate system). based on the magnitudes of the factor coordinates (variable-factor correlations) for the variables in the analysis, in (figure 8(a)), it is highly correlated with the variables of industrial wastewater, domestic wastewater, seawater intrusion, co2, tds and the supplementary variable petrol stations (negative correlation) on the other hand the factor 1 is positively correlated with pesticides, chemical fertilizers and organic fertilizers. this factor can be characterized by pollution of domestic origin to which is added pollution of industrial origin, also marked by agricultural pollution indicators (pesticides, chemical fertilizers and organic fertilizers). in the plan of individuals, (figure 8 (b)), the municipalities of el bouni, sidi salem, sidi amar and el hadjar are distinguished by seawater intrusion, industrial wastewater, air pollution and therefore by high tds values. the municipality of annaba is characterized by the large quantities of domestic wastewater due to the density of the population. the municipality of berka zarga, which houses the largest landfill in the sector, is characterized by solid waste. finally, the municipalities of el kerma, el heraicha, ain allam, djenane el chouk, el harouchi, ain sayd and zourami ali, are characterized by agricultural pollution. fig. 8(a) projection of the variables on the factor-plane (i–ii); and (b) projection of the cases on the factor-plane (i–ii) 3.1.4. hierarchical cluster analysis (hca) hca was used for organize observations and variables in the same category of the data set, into more meaningful groups sharing close and similar characteristics. transformed variables were standardized; complete linkage of tree clustering was selected so that euclidean distance between two clusters is determined by the distance of the furthest cases of these two clusters. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 78 the r-mode cluster analysis was applied to predict pollution variables groupings dendrogram chart has been obtained and the results are shown in (fig.9(a)) and classified into two dissimilar clusters. cluster 1 included two subgroups. the first formed by tds and co2, it can be labelled as water quality. solid waste and domestic wastewater with the same weight to industrial wastewater, form the second subgroup indicating anthropogenic pollution by industrial and urban discharges to which add contamination by solid waste, following the infiltration into the ground of water laden with pollutants from landfills, after leaching. cluster 2 contained only seawater intrusion; it expresses the pollution of groundwater by seawater. the spatial similarities and site grouping among the sampling points were identified using q-mode cluster analysis. in a sample cluster, a particular group/class exhibits identical characteristics in relation to the analyzed parameters. the 31 municipalities sites for fall into two dissimilar clusters (fig.9(b)). the first cluster (right) consists of coastal municipalities or located a short distance from the sea; where seawater intrusion is felt such as annaba, sidi salem. the second cluster of municipalities contains el bouni, boukhadra, bouzaaroura, oued ennil, berka zerga, essarouel, sidi amar, bergouga, chabi larbi, hdjar eddis, derradji redjem, el gantra, el hadjar, el heraicha, el kerma, chbaita mokhtar, zourami ali, drean, ain allam, djenane el chouk, fedaoui moussa, chihani bachir, kherraza, el harrouchi, ain sayd, medjez el rassoul, labidi mohamed, salmoune hachemi and ain berda. the first cluster of municipalities is associated with the first group of variables, while the second cluster of municipalities is associated with the second group of variables, based on the magnitudes of the linkage distances. as a result, the first cluster of municipalities is classified as the "seawater intrusion" cluster, while the second cluster is known as the "anthropogenic pollution" cluster. fig. 9(a) cluster analysis results for variables state of water quality, and (b) hierarchical tree for cases state of water quality complete linkage euclidean distances. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (northeastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 79 4. conclusion this paper presents integrated approaches for characterizing pollution pressures of groundwater in the seybouse valley (north east of algeria). based on gis and dpsir methods; and the major objective of the study was to establish a modelling relationship between total dissolved solids and pollution determinants, and characterize their priorities. to this end, the effective variables have been characterized and prioritized using multi-criteria analysis with ann. the selected variables have been classified and organized using the multivariate techniques of cluster analysis (hca), principal component and classification analysis (pcca) and factor analysis (fa). the conclusions of data analysis using the techniques of ann, basic statistics and multivariate can be summarized as follows: the data of total dissolved solids (tds) and pollution pressures were applied to create the ann model to assist water planners and managers in the seybouse valley to better understand the pollution determinants influencing the attractiveness of groundwater users. the selection and prioritization of effective pollution parameters indicate that this model is a useful tool to devise priority interventions, optimizing the limited available financial resources, towards to provision of appropriate quantities of water of suitable quality. all water policy and management responses are significant. sustainable coastal aquifer management must take into consideration technical engineering as well as managerial interventions such that top priority should be given to the industrial wastewater treatment that is the most pressing pollution source followed by seawater intrusion. where majority of the industries do not have wastewater treatment plants and the river and aquifers are the direct receptors of their effluents. the municipalities of el bouni, sidi salem, sidi amar and el hadjar are distinguished by seawater intrusion, industrial wastewater, air pollution and therefore by high tds values. the municipality of annaba is characterized by the large quantities of domestic wastewater due to the density of the population. the municipality of berka zarga, which houses the largest landfill in the sector, is characterized by solid waste. finally, the municipalities of el kerma, el heraicha, ain allam, djenane el chouk, el harouchi, ain sayd and zourami ali, characterized by agricultural pollution. the main sources of pollution are related to anthropogenic activities: agriculture, socioeconomic activities, industrial activities, cattle farming and urbanization. as a result, water quality is affected by both point and diffuse sources of pollution. 5. abbreviation solid waste: domestic solid waste, tons.d-1 dom wastw: domestic wastewater, hm3.y-1 ind wastw: industrial wastewater, hm3.y-1 pest: pesticides, tons.y-1 org fert: organic fertilizers, tons.y-1 chem fert: chemical fertilizers, tons.y-1 petrol stat: petrol stations co2: carbon dioxide, ppm food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri, use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria), food and environment safety, volume xx, issue 1 – 2021, pag. 68 – 80 80 seawater intr: seawater intrusion, hm3.y-1 tds: total dissolved solids, mg.l-1 6. acknowledgments the authors would like to thank dr lamine sayad badji mokhtar annaba university for every support and discussion. 7. references [1]. ghrieb, a. baali, f. fehdi, ch. hani, a. chaffai, h. & djabri, l, on the use of gis and dpsir methods to analyse water quality in seybouse valley (north east of algeria), journal of biodiversity and environmental sciences (jbes), (2019). 114-125. [2]. aoun sebaiti, b. optimized management of water resources from a coastal aquifer application to the annaba plain (northeastern algeria) phd thesisuniv of lille, (2010). p113–121–134. [3]. aoun-sebaiti b, hani a, djabri l, chaffai h, aichouri i, bougherira n, simulation of water supply and water demand in the valley of seybouse (east algeria). desalination and water treatment. taylor & francis, (2013). doi: 10.1080/19443994, 855662, 1-6. [4]. asadollahfardi g, water quality management. assessment and interpretation. springer briefs in water science and technology. springer, (2015). doi 10.1007/978-3-662-44725-3. [5]. djabri, l. fehdi, ch. hani, a. bouhsina, s. nouiri, i. djouamaa, m.c. boch, a.p. & baali, f, climate change and water resources: seasonal hydrochemical changes of water from alluvium aquifers: drean-annaba aquifer case study (ne algeria), (2015). book id: 326568–1–en. chapter 17. [6]. halimi, s. baali, f. kherici, n. zairi, m. & bouhsina, s. irrigation and risk of saline pollution. example: groundwater of annaba plain (north east of algeria), (2016) v12n6p241. [7]. kouzana, l. ben mammou, a & gaaloul, n, seawater intrusion and salinization in a coastal water table (korba, cap-bon, tunisia), geo-eco-trop, (2007). 31: 57-70. [8]. hammor, d, from pan miocene. 600 million years polycyclic evolution in the massive edough (north east algeria). traced by petrology, tectonics and geochronology (u/pb, rb/sr, sm/nd and 39ar/40ar). thesis of ustlanguedoc, univ. montpellier ii, (1992). p 205. [9]. lamouroux, c. & hani, a. identification of groundwater flow paths in complex systems aquifer. hydrol. processes (2006). 20, 2971–2987. [10]. kherici, n, vulnerability to chemical pollution of groundwater systems superposed layers in industrial and agricultural annaba mafragh the east algerian community. phd thesis-univ of annaba, (1993). p28–34. [11]. gaud, b, hydrogeological study system annaba bouteldja. knowledge synthesis and research of modelling conditions. a.n.r.h. report (unpublished), annaba, vol 2, 230p. 10 boards, (1976). [12]. hani, a, methodological analysis of the structure and anthropogenic processes: application to water resources in a mediterranean coastal basin phd thesis-univ of annaba, (2003). 214p. p18–33 [13]. european environmental agency (eea), the dpsir framework used by the eea, (2014). available online: http://glossary.eea.europa.eu//terminology/sitesearc h?term=dpsir. [14]. mattas, c. voudouris, k. & panagopoulos, a. integrated groundwater resources management using the dpsir approach in a gis environment: a case study from the gallikos river basin, north greece. 6, 1043–1068, (2014). [15]. cave, r.r. ledoux, l. turner, k. jickells, t. andrews, j.e.& davis, h, the humber catchment and its coastal area: from uk to european perspectives. the science of the total environment, article in press, (2003). doi 10.1016/s0048-9697(03)00093-7. [16]. jeunesse, i.l. rounsevell, m. & vanclooster, m, delivering a decision support system tool to a river contract: a way to implement the participatory approach principle at the catchment scale? journal of physics and chemistry of the earth, article in press. volume 28, (2003). issues12–13, 547–554. [17]. statistica. electronic manual statsoft, inc. statistica data analysis software system, (version 8; 2008). [18]. jalala, s, characterizing the multi-criteria parameters of integrated water management model in the semi-arid mediterranean region: application to gaza strip as a case study. phd thesis-univ of lille, (2005). p98–108–110. [19]. iahs-international association of hydrological sciences. international hydrology today, (2003). [20]. elliott,ac. & woodward, wa. statistical analysis quick reference guidebook with spss examples. 1st ed. london: sage publications, (2007). [21]. oztuna, d.elhan, ah.&tuccar, e, investigation of four different normality tests in terms of type 1 error rate and power under different distributions. turkish journal of medical sciences, (2006). 36(3), 171–176. [22]. yap, b.w. &sim, c.h, comparisons of various types of normality tests, journal of statistical computation and simulation, (2011). 81, 12, 2141–2155. [23]. thode, hj. testing for normality, new york: marcel dekker, (2002). https://www.researchgate.net/profile/larbi_djabri https://www.researchgate.net/profile/larbi_djabri https://www.researchgate.net/profile/chemseddine_fehdi https://www.researchgate.net/profile/azzedine_hani https://www.researchgate.net/profile/saad_bouhsina https://www.researchgate.net/profile/f_baali https://www.researchgate.net/profile/f_baali https://www.researchgate.net/profile/f_baali https://www.researchgate.net/profile/f_baali https://www.researchgate.net/profile/f_baali http://glossary.eea.europa.eu/terminology/sitesearch?term=dpsir http://glossary.eea.europa.eu/terminology/sitesearch?term=dpsir https://www.sciencedirect.com/science/journal/14747065/28/12 https://www.sciencedirect.com/science/journal/14747065/28/12 fig. 8(a) projection of the variables on the factor-plane (i–ii); and (b) projection of the cases on the factor-plane (i–ii) 87 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 87 93 total phenolics of fresh and frozen minor berries and their antioxidant properties *ana leahu1, mircea oroian1, sorina ropciuc1 1food engineering faculty, stefan cel mare university of suceava, romania analeahu@fia.usv.ro *corresponding author received 10th february, accepted 19th march 2014 abstract: the aim of this work is revealing the minor berries biological abundance (bilberries and cowberry), fruits which can be found a lot in bucovina’s forests and are used at their real value. the biochemical characteristic studied in order to prove their strong antioxidant potential are the content of ascorbic acid, total polyphenol, antiradical activities, brute protein, humidity and dry substance of fresh and frozen fruits of bilberries (vaccinium myrtillus l.) and cranberries/lingonberry (vaccinium vitis-idaea). the content of total phenolic compounds was measured in methanol fruits’ extracts as well as their radical scavenging activity. bilberries were highest in total phenolics and antioxidant activities, whereas cranberries contained the lowest quantity of phenolics, corresponding to 4.68 mg gae/kg.it was studied the evolution of ascorbic acid during storage lengthy freeze fruit for 6 and 12 months at -25°c. in conclusion at the end of frozen storage period, ascorbic acid content and total poliphenols remained significantly unchanged compared with the values measured just after the freezing process. keywords: lingonberries, ascorbic acid, dpph, frozen storage, loss, total poliphenols. 1. introduction bilberry and lingonberries are one of the most important shrubs that grow in our forests and mountain hill and mountain meadows, across the carpathians, especially on shady slopes and humid forests. berries have a more pronounced flavor than cultivated fruits, with sugar content higher than vitamins constitute a valuable food for human consumption. the genus vaccinium belongs to the family ericaceae and consists of more than 150 species. some plants of this genus bear edible fruits that have attracted the attention of the industry for the production of juices and jams. like other fruits, bilberry contains microand macronutrients such as vitamins, minerals, and fiber; however, their biological properties have been largely attributed to their high levels of various phenolic compounds [1-3]. dried blueberries give a tea; have been used as a folk remedy to fight against dysentery and diarrhea. also, stalks and leaves are commonly used for tea, in order to reduce blood glucose and combating of diabetes, with similar effects to those of insulin. bilberry anthocyanidins present in fruits develop a protective role against radiation are chemoprotective [4]. many studies have shown a lower incidence of cancer as a result of dietary antioxidant that neutralizes free radicals multiple. elements of phenolic constituents of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 88 berries are designed to protect the body from oxidative stress caused by free radicals [5]. eating cranberries or cranberry products based prevents urinary tract infections, the active principle is been identified type-a proanthocyanidins (pac), [6]. blueberries are commercialized in different ways mainly as fresh or frozen products [1]. iwona scibisz, 2007 investigate the effect of freezing and long-term frozen storage on total phenolic, total anthocyanin contents and antioxidant activity of highbush blueberries (vaccinium corymbosum l. accordind iwona scibisz, 2007 research, at the end of frozen storage period, antioxidant activity remained significantly unchanged compared with the values measured just after the freezing process [7]. cranberries or blueberries red (vaccinium vitis-idaea) is a shrub 10-40 cm, which grows at high altitudes in the mountain meadows and clearings. cranberries are composed of water (88%), organic acids (including salicylate), fructose, vitamin c (high levels, i.e., 200 mg/kg of fresh berries), flavonoids, anthocyanidins, catechins, and triterpinoids [8]. lingonberry (vaccinium vitis-idaea l.) is rich in antioxidants such as vitamins c, a, and e (tocopherol) and especially polyphenols [9]. anthocyanin pigments are responsible for the red color and are part of the complex antioxidants. lingo berries contained only cyanidin based anthocyanin and three anthocyanins in order of elution (% proportion of individual anthocyanins) were found: cyanidin-3-galactoside (79%), cyanidin-3-glucoside (10%), and cyanidin3-arabinoside (11%), [10]. cranberries/lingonberry (v. vitis-idaea) are preserved well in fresh water at a temperature of 10-18°c and for a year, because they contain substances such as benzoic acid which are natural preservatives and can be eaten as sauce, jam, sorbet, juice and pickled [11]. it is also used in the preparation of a wine. cranberry tea (leaves and stems) is effective action in combating diseases of the kidney and urinary tract. cranberry juice, predominantly in the form of a juice cocktail drink with approximately 25% cranberry juice, has been the traditional choice of most women seeking to prevent lower urinary tract infections [8]. hoda m. eid et al., 2010 elucidate the mechanism of action of one of the berries of vaccinium vitis idaea, to treat symptoms of diabetes. they findings indicate that quercetin and quercetin 3-oglycosides are responsible for the antidiabetic activity of v. vitis crude berry extract [11]. the impact of bioprocessing on lingonberry flavour was studied by viljanen, kaarina, et al. 2014. enzyme treatment is a potential tool to decrease naturally acidic flavour of lingonberry. fermentation, especially with yeast, could be an interesting new approach to increase the content of natural preservatives, such as antimicrobial benzoic acid [12]. the antioxidant properties of red cranberry are reported in the literature, and red berries have the highest antioxidant activity among many other fruits [13-14]. studies with lingonberry (v. vitis-idaea) also associated health-promoting effects such as the decrease in proliferation of cancer cells by the fruit extract [15-16]. from the research by mane carine, et al. 2011, the use of lingonberry extract as a dietary supplement may be considered in the future to improve the antioxidant activity in human health. antioxidants in red cranberry composition have effects for the entire cardiovascular system and the property to restore normal levels of cholesterol [17]. the aim of this work was to determine total phenolic content (tpc), antioxidant activity (taa) and investigate how freezing and long-term storage can affect food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 89 the ascorbic acid content (aac) in blueberries and lingo berry. 2. materials and methods 2.1. samples and storage conditions collection of fruits was carried out manually at the time of full maturity. bilberry and cranberries was collected at the site pojorâta. fresh undamaged berries were frozen and stored at -20°c for twelve months. the fruits were defrosted and mashed before chemical analyses. 2.3. chemical analysis the determination of moisture and volatile matter content in kernel was effectuated according to the european standard en iso 665/2000 by the drying process in a drying chamber at the temperature of 103 °c. total ash composition was obtained by calcinations of 5g of sample at 600 °c for 240 min. total soluble solids (tss) was assayed using the refractometric method, with an abbe refractometer and corrected to the equivalent reading at 200c (aoac, 1995). protein content was analyzed by kjeldahl method. ascorbic acid determination was done on acid extracts of samples. extraction of ascorbic acid from samples the extracts were obtained following rodriguez-saona and wrolstad (2001) modified protocol: 4 gram of samples was extracted with 12 ml of acidified solutions (perchloric acid and ophosphoric acid 1%) using a ceramic mortar and a pestle. the residue was re-extracted until the extraction solvents remained colorless (the total solvent volume was 50 ml).the extract was filtered through a whatman no. 1 filter paper. the extracts were kept at -20°c until further analysis. ascorbic acid separation, identification and dosage the ascorbic acid in the samples was separated, identified and dosed in a hplc shmadzu system coupled with uv–vis detector (dad). zorbax c18 column (5μm, 250x4,6) was used. the column was eluted in isocratic system with a mobile phase consisted of phosphate buffer ph = 3.5 (tfa): solution 0.02 m/l of monopotassium phosphate and orthophosphoric acid 10%, adjusted to ph = 3.5. at a flow rate of 0, 6 ml/min. the chromatograms were registered at 245 nm. for ascorbic acid identification standard lascorbic acid (sigma 99% standard l ascorbic acid) was used. for dosage of ascorbic acid in the samples, a calibration curve was constructed using dilutions of standard l-ascorbic acid in bidistilled water. the content of total polyphenolic compounds in fruits methanol extracts diluted 1/10 was determined by folinciocalteu method. for the preparation of the calibration curve 0.5 ml aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 μm/ml aqueous gallic acid solution were mixed with 10.0 ml folin-ciocalteu reagent (diluted ten-fold) and 1.0 ml sodium carbonate (20.0%) and the volume made up to 10.0 ml with h2o. the absorption was read after 2 h at 25°c, at 760 nm. total phenols were determined as gallic acid equivalents on a dry weight (mg gae g-1 fw). 2,2-di (4-tert-octylphenyl)-1pycrilhydra-zyl (dpph) scavenging capacity assay the method used for determining the antioxidant activity of berries extracts is based on scavenging 2,2-di (4-tertfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 90 octylphenyl)-1-picrylhydrazyl (dpph) radicals. the berries samples aliquot (0.5 ml) was added to freshly prepared dpph reagent. after incubating for 5 min, the absorbance of the resulting solutions was measured at 517 nm using a spectrophotometer. the control was conducted in the same manner, except that distilled water was used instead of sample. the ic50 is the concentration of an antioxidant that is required to quench 50% of the initial dpph radicals under the experimental conditions given. the dpph scavenging capacity assay value is calculated according to the formula: ic50 % = [1 – (asamples/ acontrol)] × 100. where acontrol is the absorbance of the control, and asample is the absorbance of the sample. ph value was measured in macerate of edible fruits by direct insertion of electrode (rimpapa, 2007). acidity was determined by titrating samples with 0.01m naoh solution up to ph 8.2, and was expressed as citric acid per 100 g samples. 2.4. statistical analysis the correlation and linear regression analyses were performed using microsoft office excel 2007. statistical analysis was performed with student’s t-tests. differences with p values of less than 0.05 were considered statistically significant. the experiments were performed in triplicates and repeated two times. 3. results and discussion the results obtained from proximate composition of berries samples were shown in table 1. moisture content was higher in bilberry (vaccinium myrtillus l.) (85.8%) and lower in cranberries (vaccinium vitisi claea l.) (57.28%). ash content.this analysis showed ash content of 1.457% for cranberries and 1.331% indicating the presence of minerals and heavy metals. total soluble solids is specific to each specy, but it can be also used as an indicator for ripeness stage. the potential effectiveness of fruit supplements in promoting various aspects of health depends upon their botanical and chemical composition and on the concentrations of active ingredients that they contain [18]. ascorbic acid content in this study the wild species v. myrtillus had the highest amount of vitamin c (404.7 mg/kg fw). similar amounts of vitamin c were observed in cranberries species vaccinium vitisi claea l. (361.42 mg/kg fw), respectively. rop o., 2010 et al. reported lower values in different cultivars of fruit european cranberrybush that ranged from 1.01 to 1.64 grams/kg fm, [19]. total phenols. comparison between different fruits showed broad variations in both phenolic content and antioxidant activity as estimated by the scavenging 2,2-di (4-tert-octylphenyl)-1picrylhydrazyl (dpph) radicals. bilberry identified to have high phenolic content and high antioxidant activity. this is in line with the findings of milivojevic j. et al. 2012, which examined and quantify important tasteand healthrelated compounds in vaccinium berries. however, other authors have measured total polyphenol contents in in wild bilberry and 2 commercial blueberry cultivars and quoted a range from 3.87 mg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 91 gae/g fw to 1.50 mg of gae/ g fresh weight, depending on the cultivar [20]. due to its high contents of polyphenolics ranged from 6.80 to 8.29 grams of gallic acid/kg fm, as recently reported by rop o., 2010, for fruit of european cranberrybush [19]. dpph scavenging capacity assay the antioxidant capacities of cranberry juice and three extracts isolated from frozen cranberries containing anthocyanins, water-soluble and apolar phenolic compounds, was evaluated by caillet, s et al., 2011. according to this, the juice exhibited a much lower antioxidant activity, compared to the cranberry extracts, especially when compared with the extract containing water-soluble compounds which the extraction conditions were similar to those used to obtain the juice [16]. the ph and acidity. in a table 1 results showed that measured ph value was highest in bilberry, and furthermore decreased in cranberries. rimpapa et al., 2007 reported that fruit acidity depended directly on the concentration of aliphatic and aromatic organic acids in the fruits [18]. table 1 concentrations of moisture, ash, tss%, brute protein, ascorbic acid, tp, ic50, ph and titrable acidity of minnor berries samples moisture % ash % 1tss % brute protein % s.u. 2aa mg/kg *fw 3tp (mg gae /kg) 4ic50 ph 5ta (g of citric acid/100g bilberry vaccinium myrtillus l. 85.8 1.331 6.8 5.72 404.7 5.87 0.33 3.12 0.55 cranberries vaccinium vitisi claea l. 57.28 1.457 15.3 3.7 361.42 4.68 0.52 2.72 1.94 *fw – fresh weight of fruits abbreviations: 1tss-total soluble solids (0brix), 2aa-ascorbic acid mg/kg, 3tp mg gae/kg – total polyphenols expressed as gallic acid equivalents, 4ic50 = free radical scavenging activities, 5ta-titrable acidity the contents of the basic nutritional compounds (moisture, total soluble solids, protein) was higher in vaccinium fruit, but not statisticaly significant (table 2). we registered a good correlation between the content of phenolics and the antioxidant potential. in our study, the long-term frozen storage of bilberry and cranberries did not induce significant changes in vitamin c content (figure 1). table 2 univariate analysis of biochemical indicators studied groups average standard deviation moisture 71.54 20.17 ash 1.395** 0.09 tss 11.05 6.01 protein 4.71 1.43 aa 383.06 30.60 tp 5.275* 0.84 ph 1.525 1.69 ta 1.245 0.98 * indicates significant differences at p < 0.05 ** indicates significant differences at p < 0.01 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 92 iwona scibisz and marta mitek [2007] showed similar antioxidant activity losses for frozen highbush blueberries stored for 6 months at -35°c. comparing the evolution of the ascorbic acid of the two vaccinium fruits, one can say that after 12 month of storage at -20ºc the values of this index was reduced by 4.46% in bilberries and by 4.64% in cranberries. in cranberries, after 12 month of frozen storage, the poliphenols content content decreased by 11.53% (figure 2). pearson correlation coefficients were calculated to reveal the relationship between the vitamin c, total polyphenols content and time of freezing. the correlation value for the parameters ascorbic acid and total phenols measured in the selected fruits is 0.984, showing that there is strong correlation between these two parameters (table 3). table 3 correlation matrix for the variables measured in vaccinium fruits: tp (mg gae/100g) and aa (mg /kg) 4. conclusions vaccinium fruits are rich in antioxidant activity. the contents of polyphenols, ascorbic acid and the antioxidant activity are significantly higher in bilberry vaccinium myrtillus l. during frozen storage, the biggest losses of ascorbic acid were recorded in cranberries. the evolution of the poliphenols concentration in the two types of vaccinium fruits, in all cases, greater decreased during frozen storage. the work should contribute to the popularization of this minor berries as a promising fruits in human nutrition and the high antioxidant activity that guarantees the best nutritional results. time aa losses aa tp losses tp time 1 aa -0.165 1 losses aa 0.980 -0.086 1 tp -0.305 0.984 -0.243 1 losses tp 0.870 0.119 0.948 -0.052 1 fig. 1 ascorbic acid concentrations of bilberry and cranberries during frozen storage fig. 2 total phenols of of bilberry and cranberries during frozen storage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ana leahu, mircea oroian, sorina ropciuc, total phenolics of fresh and frozen minor berries and their antioxidant properties, issue 1 2014, pag. 87 93 93 5. references [1]. lohachoompol v., srzednicki g. and craske j., the change of total anthocyanins in blueberries and their antioxidant effect after drying and freezing, journal of biomedicine and biotechnology: 5 248–252, (2004). [2]. min-sheng su, po-jung chien, antioxidant activity, anthocyanins, and phenolics of rabbiteye blueberry (vaccinium ashei) fluid products as affected by fermentation, food chemistry 104 182–187,(2007). [3]. schreckinger m.e., lotton j., lila m.a., and gonzalez de mejia e., berries from south america: a comprehensive review on chemistry, health potential, and commercialization, j med food 13 (2), 233–246, (2010). [4]. koca i., karadeniz b., antioxidant properties of blackberry and blueberry fruits grown in the black sea region of turkey, scientia horticulturae 121, 447–450, (2009). [5]. qi you, baowu wang, feng chen, zhiliang huang, xi wang, pengju g. luo, comparison of anthocyanins and phenolics in organically and conventionally grown blueberries in selected cultivars”, food chemistry 125, 201– 208, (2011). [6]. davidson e., benno f. zimmermann b. f., e. jungfer and s. chrubasikhausmann, prevention of urinary tract infections with vaccinium products, phytotherapy research, volume 28, issue 3, pages 317–474, (2014). [7]. scibisz i., mitek m., the changes of antioxidant properties in highbush blueberries (vaccinium corymbosum l.) during freezing and long-term frozen storage, acta sci. pol., technol. aliment. 6(4), 75-82 (2007). [8]. hisano m., h. bruschini, a. c. nicodemo, m. srougi, review cranberries and lower urinary tract infection prevention, clinics ; 67(6): 661-667, http://www.producao.usp.br/handle/bdpi/40398 (2012). [9]. mane, c., loonis, m., juhel, c., dufour, c., & malien-aubert, c., food grade lingonberry extract: polyphenolic composition and in vivo protective effect against oxidative stress. journal of agricultural and food chemistry, 59(7), 3330-3339 (2011). [10]. lee, j., & finn, c. e., lingonberry (vaccinium vitis-idaea l.) grown in the pacific northwest of north america: anthocyanin and free amino acid composition. journal of functional foods, 4(1), 213-218, (2012). [11]. hoda mohamed mahmoud eid, antidiabetic activity of vaccinium vitis-idaea, a medicinal plant from the traditional pharmacopeia of the james bay cree, these, université de montréal, faculté de médicine, ttps://papyrus.bib.umontreal.ca/xmlui/handle/1866/ 8327, (2010). [12]. viljanen, k., heiniö, r. l., juvonen, r., kössö, t., & puupponen-pimiä, r., relation of sensory perception with chemical composition of bioprocessed lingonberry. food chemistry, 157, 148-156 (2014). [13]. heinonen, m., antioxidant activity and antimicrobial effect of berry phenolics–a finnish perspective. molecular nutrition & food research, 51(6), 684-691, (2007). [14]. mcdougall, g. j., ross, h. a., ikeji, m., & stewart, d., berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro. journal of agricultural and food chemistry, 56(9), 3016-3023, (2008). [15]. olsson, m. e.; gustavsson, k. e.; andersson, s.; nilsson, a.; duan, r. d., inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels. j. agric. food chem., 52, 7264– 7271, (2004). [16]. caillet, s., côté, j., doyon, g., sylvain, j. f., & lacroix, m., antioxidant and antiradical properties of cranberry juice and extracts. food research international, 44(5), 14081413, (2011). [17]. zafra‐stone, s., yasmin, t., bagchi, m., chatterjee, a., vinson, j. a., & bagchi, d., berry anthocyanins as novel antioxidants in human health and disease prevention. molecular nutrition & food research, 51(6), 675-683, (2007). [18]. rimpapa, z., toromanovic, j., tahirovic, i., šapčanin, a., & sofic, e., total content of phenols and anthocyanins in edible fruits from bosnia. bosnian journal of basic medical sciences, 7(2), 119-122, (2007). [19]. rop, o., reznicek, v., valsikova, m., jurikova, t., mlcek, j., & kramarova, d., antioxidant properties of european cranberrybush fruit (viburnum opulus var. edule). molecules, 15(6), 4467-4477, (2010). [20]. milivojevic j., vuk maksimovic, dragisic maksimovic j., radivojevic d., milena poledica, sezai erci̇şli̇, a comparison of major tasteand health-related compounds of vaccinium berries, turk j biol 36, 738-745, (2012). 170 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 2 2020, pag. 170 175 impact of washing solutions on chemical composition and physico-chemical properties of surimi-like mechanically deboned turkey meat *oleg galenko1, svitlana schuler1, vadim bezpalko1, ostap gasyuk1 1faculty of technology meat and meat products, national university of food technologies, 68, volodymyrska str., kyiv, 01601, ukraine, galen@i.ua *corresponding author received 12th april 2020, accepted 27th june 2020 abstract: the impact of washing solutions on mechanically deboned turkey meat (mdm) was evaluated with the aim to produce surimi-like material using solutions of succinic (ph 5.9) and lactic acid (ph 5.8). inorganic phosphoric acid was found to be the strongest ionization constants, with tartaric, then citric, malic, lactic and succinic acids. in washed samples found that moisture content at the end of the study was 87.6%–92.7%, fat 1.04%–3.2%, protein 17.4–18.2%, ash 0.079–0.086%. the number of washing significantly influenced the increase in moisture content and the reduction of fat content. the moisture content increased from 65.0% (p <0.05) for unwashed masses to 69.3–74.1% after the second washing and to 76.2–79.7% after the third washing. obtained surimi-like material may have a moisture content in the range of 73 to 80%, but 78% moisture content is technologically the best. during the experimental studies of turkey mdm it was found that the best indicators were achieved for minced meat washed with 0.3% succinic acid at ambient temperature of 15°c, 1:4 ratio of turkey mdm to acid solution, 20 min mixing time and 20 min centrifugation. surimi-like material washed with succinic acid had higher ph and moisture retention capacity, and showed better colour changes than unwashed turkey mdm and other surimi-like materials. this study demonstrates that succinic acid solution provides the achievement of desired surimi-like material with the best technological quality and low fat content. keywords: mechanically deboned turkey meat, washing liquids, surimi-like material. 1. introduction shortage of domestic fresh meat (mainly beef and pork), fast growth, high reproducibility, productivity and viability of farm poultry makes it necessary to develop and apply innovative scientificbased processing [1]. resource-saving technologies of poultry processing industry provide for complex processing of poultry [5]. conservation and rational use of poultry meat in food technologies through introduction of deep industrial processing in order to obtain a wide range of products for various purposes is an urgent nationwide problem [3]. one of the raw materials for meat processing industry,which is widely used due to its high adaptability, high protein content, low cost, is mechanically deboned turkey meat (mdm) [1]. mechanically deboned poultry meat is poultry meat obtained by mechanically separating soft tissues from the bones of poultry carcasses or parts thereof, in which calcium content is higher than that in chopped meat [2]. mdm, in compliance with standards, should have a paste-like viscous consistency, be pink or red, and have fresh smell. mdm should not contain bones over 0.75 mm in size, salt, sodium nitrite, animal and vegetable materials, except http://www.fia.usv.ro/fiajournal mailto:galen@i.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk, impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat, food and environment safety, food and environment safety, volume xix, issue 2 – 2020, pag. 170 175 171 poultry, foreign impurities and other additives [6]. in many countries production of mechanically deboned meat should meet very stringent requirements. one of them, for example, is to limit the shelf life of bones used to produce mdm no more than 24 hours at 0°c or 8 days frozen [7]. mdm are made from chicken or turkey carcasses. they are treated with circular saws and pneumatic tools. quite often, separators are used to break bones and divide them into components (soft and hard) [2]. as a result, there is a half-dry bone mass and a mass of muscle tissue which looks like pasty stuffing. it also includes fat, tendons, skin and connective tissues. such product is used immediately after boning [8]. the use of mdm both as minced meat and as a recipe component has a number of negative aspects. among them are the reduction of oxidative and microbiological stability, specific fat shine and red color (from pink to dark red), which is due to technological factors of production and biochemical properties of this raw material due to the transition of bone marrow lipids and hemoproteins [9]. on this basis, the urgent task is to improve the technology of mdm through the use of stabilizing, antioxidant additives and other technological operations, which will increase functional, technological and consumption properties of such meat systems [3]. not well-researched there are regulations for washing cycles, temperature for washing liquids, setting the modes of centrifugation, optimal introduction of surimi-like material in the formulation to create a product with a prolonged shelf life [2, 10]. the purpose of the research is to theoretically substantiate and experimentally confirm the effectiveness of washing of mdm with various washing liquids (organic acids lactic and succinic, deionized water, sodium chloride, sodium bicarbonate solution, potassium phosphate buffer solution) adhesive ability (surimilike material). poultry deboning and further processing of poultry meat lead to accumulation of a large number of by-products from the less noble parts (neck, wing tips, skeleton and back bones, skin), the commercial cost of which is low. the process of mechanical deboning is associated with problems such as darkening and odor, and thus is not suitable for long-term storage [6, 7, 8]. to overcome this problem, there is a need to re-use mechanically deboned meat in a process that reduces darkening, bitter taste and odor. thus, surimi processing was proposed [8, 9]. surimi is the original japanese term referring to white, tasteless and odorless myofibril preparation obtained from mechanically deboned fish meat, washed separately with water and mixed with cryoprotectants [11]. when protein concentration is obtained by washing meat other than fish, the product is known as surimi-like material [12]. washing meat with low marketing value is thought to contribute to increased raw material value [8]. the surimi-like product was made by using different types of meat, such as [15], beef [14], pork [13], lamb [12], heep [11], laying hens [10], ducks [9, 8], chicken meat [7, 8, 9]. surimi-like material has been found to have very good technological properties, above all high moisture retention capacity and good thermal gelation [8]. it is not associated with intense sensory taste or odors. however, the final quality of surimi like material obtained from washed meat is not always identical, depending on raw material and recovery parameters such as the number of washing cycles, type of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk, impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat, food and environment safety, food and environment safety, volume xix, issue 2 – 2020, pag. 170 175 172 washing solutions and meat / water ratio [11]. in addition to water, sodium chloride (nacl), sodium bicarbonate and sodium phosphate buffer [9] have been proposed as useful washing solutions for treatment of surimi-like material. thus, information on the use of surimi-like meat material can be found in several literature sources. such studies include pork hot dogs with mechanically separated surimi-like material [8], beef heart with surimi-like material [14], pork sausage with surimi-like material [13], crab sticks with surimi – like poultry material [6], pork pies with surimi-like pork material [11], burgers with surimi-like duck material [9] and mechanically deboned chicken meat with surimi-like material [8]. processing of turkey meat to surimi condition will facilitate the use of byproducts from less noble parts of turkey for production of high-quality industrial products, as well as will prevent odors and darkening during long-term storage. 2. materials and methods frozen and cooled turkey mdm was taken as raw material for research. turkey mdm was washed with organic acid solutions at a concentration of 0.3%. the experiment was conducted three times with 1: 2 and 1: 4 ratio of turkey mdm / acid, in solutions of organic acids with stirring the solutions in the range from 10 to 20 minutes [8]. centrifugation from 10 to 20 min was performed to separate soluble proteins, connective tissue, bone tissue, cell membranes and storage of neutral lipids, as well as to reduce liquid content. physico-chemical methods were used during the study. determination of amino acid composition was conducted in accordance with the method of ion exchange chromatography. quality and quantitative determination of components consisted in dividing of them into separate components after the hydrolysis of proteins and determination of their quantitative estimation with the help of automatic analyzer of amino acids as т339, on polystyrene sulfonate ion exchange resins of "ostion lj anb" in licitrate buffer one column mode. the elutions of amino acids from a column conduct in turn by li by citrate buffers from рн 2,75±0,01; рн 2,95±0,01; рн 3,2±0,02; рн 3,8±0,02; рн 5,0±0,2. amino acids rectifying with the help of solution of ninhydrin on a running photometer at a length of waves by 560 nm. the results of detection was registered oneself by a variplotter on a paper in form the peaks of absorption of light of ninhydrin-positive substances in an eluate, that in number in direct ratio concentrations of this substance in solution. correlation of solution of ninhydrin reagent and eluents is 1 to 2; temperature of thermostatic t1=38,5 °c ; t= 65 °c. the prototype was diluted in licitrate buffer by ph 2,2±0,02 and inflicted on a ion exchange column with the help of metering device. the quantitative estimation of хроматограм of preproduction model settles accounts in relation to standard mixture of amino acids of firm віоrad [10]. amino acid score expected according to the certificate scale of fао/who [4, 6]. the choice of acid for studies was focused on ionization constants. ionization constant is a specific equilibrium constant in chemistry that shows the degree of dissociation of hydrogen ions (h +) with acids in solution. strong acids dissociate almost completely and, accordingly, have large values of acidity constant (ka> 1). weak acids dissociate incompletely and have the value of ka << 1 [11]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk, impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat, food and environment safety, food and environment safety, volume xix, issue 2 – 2020, pag. 170 175 173 since the difference in ka value between the weakest and the strongest acids is several orders of magnitude, the ionization constant is usually denoted by the inverse of its decimal logarithm (pka). thus, the purpose of this research was to develop surimi like material from turkey mdm by studying the effect of washing solutions on its composition, technological and functional properties [4]. 3. results and discussion 3.1. study of raw materials and ionization constants of acids. data in table 1 show that inorganic phosphoric acid is the strongest in terms of ionization constants, tartaric, then citric, malic, lactic, succinic acids are the strongest organic acids [2]. acetic is the weakest acid. lactic acid is monobasic, malic and succinic two-basic, citric three-basic acids. succinic, tartaric and malic are related acids. malic acid is derivative of succinic acid. all these acids are good preservatives. they can wash out various substances from the product and can enter into complexing reactions [5]. table 1 acid ionization constants that are widely used in food industry acids ionization constant ка рка = –log citric 7.4 · 10–4 3.13—4.55 2.2 · 10–5 4.0 · 10–7 apple 3.5 · 10–4 3.46—5.05 8.9 · 10–6 succinic 1.6 · 10–5 4.21—5.63 2.3 · 10–6 lactic 1.5 · 10–4 3.83 acetic 1.74 · 10–5 4.76 phosphoric 7.1 · 10–3 2.15 tartaric 1.3 · 10–3 2.89 3.2. effect of selected wash solutions on surimi-like material. washing solutions with different ph media were used: succinic acid (5.9); lactic acid (5.8) [4]. during preparation of surimi like material the ph and buffering agents nature of the washing solution play an important role not only in product stability but also from a technological point of view [8]. number of washings significantly influenced the increase of moisture content and the reduction of fat content. the moisture content increased from 65.0% (p <0.05) for unwashed product to 69.3– 74.1% after the second washing and to 76.2–79.7% after the third washing. the number of washings increases moisture content [6]. it has been found that surimilike material can have a moisture content in the range of 73 to 80%, but 78% moisture content is technologically the best. centrifugation on a centrifuge clmn – 24 helped to reduce water content, to separate soluble proteins, bones tissues, skin, connective tissues, cell membranes and to preserve neutral lipids [12]. this process food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk, impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat, food and environment safety, food and environment safety, volume xix, issue 2 – 2020, pag. 170 175 174 reduces pigment content to the acceptable for turkey mdm level. in the process of washing we obtained surimi-like material and studied its physico-chemical parameters (table 2). table 2 influence of solutions on physico chemical indicators of surimi-like material obtained from mechanically deboned turkey meat table 2 shows chemical composition of washed turkey mdm. no significant interaction effect was found between the detergent solution and the number of washings for any of the estimated parameters [12]. however, the number of washings had significant effect on increase of moisture content and reduction of fat and protein content. obviously, washing increased humidity (from 60.0% to 87.6% -92.7%) and reduced fat content (from 19.8% to 1.04% -3.2 %). moisture content in the washed samples increases due to the concentration of protein and reduction of its interaction with other components, such as fat, thus increasing its hydration [11]. fat removal is crucial in preparation of surimi-like material since fat is likely to be oxidized and protein denaturation will increase during frozen storage [9, 13]. in addition, lipids tend to oxidise in surimilike material with high fat content, which worsens aroma due to rancidity. thus, fat removal improves the quality of surimilike material and demonstrates inhibition of lipid oxidation [2]. as reported in previous studies [1, 8, 2], fat content in surimi-like products can reach 3% due to the fat content of the raw materials used. according to fatty and sarcoplasmic proteins are removed in the process of efficient washing. however, sequential washing decreases the amount of crude protein in the end surimi-like product. protein content increased compared to the reference (turkey mdm) sample. ash content (mean value 0.084) had no effect (p> 0.05) on washing and did not differ (p> 0.05) from unwashed turkey mdm. this is not consistent with other studies [3, 6] that reported significant decrease of this component after successive washings. during experimental studies of turkey mdm we found that the best quality indicators are obtained in mdm washed with 0.3% succinic acid at a temperature of 15, 1: 4 ratio of turkey mdm to acid solution, 20 minutes duration of stirring and 20 minutes duration of centrifugation. as a result of washing the product looks very similar to white poultry meat, which is due to the fact that washing significantly tones up light and discolors red colours. 4. conclusion turkey mdm is a potentially available raw material for production of surimi-like product obtained in the course of washing. washing solution/meat t℃ τ (min) ratio (turkey mdm/ acid) moisture content, % fat content, % protein content, % ash, % turkey mdm 63.0 19.8 12.4 0.096 lactic acid 0.3% 15 10 1:2 87.6 3.2 17.6 0.086 lactic acid 0.3% 15 20 1:4 92.7 2.1 17.4 0.091 succinic acid 0.3% 15 10 1:2 89.6 2.93 18.2 0.079 succinic acid 0.3% 15 20 1:4 91.8 1.04 18.0 0.083 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 oleg galenko, svitlana schuler, vadim bezpalko, ostap gasyuk, impact of washing solutions on chemical composition and physicochemical properties of surimi-like mechanically deboned turkey meat, food and environment safety, food and environment safety, volume xix, issue 2 – 2020, pag. 170 175 175 functional properties of turkey mdm can be improved by processing it into surimilike material. three times washing of meat was sufficient to effectively remove fat, maintain stable protein content and achieve high functional and textural properties. washing allowed us to get surimi-like material with high technological properties. thus, based on our research, succinic acid is the best washing solution since it allows to get surimi-like material with the highest technological quality for further use in production of low-fat meat products. the influence of washing liquids and cycles on the yield and recovery of protein from the gray-like material of mdm turkey is established. the best results are obtained when washing with sodium bicarbonate after the second wash (protein yield 37.3% recovery 38.6%). 5. references [1]. ramadhan k., huda n., ahmad r., effect of number and washing solutions on functional properties of surimi-like material from duck meat/ j food sci technol 51(2):256–266, (2014). [2]. ismail i., huda n., ariffin f., quality characteristics of spent duck sausages containing surimi like material substitution during refrigerated storage. international journal of poultry science, 13(4): 228–239, (2014). [3]. armando abel massingue, robledo de almeida torres filho, paulo rogério fontes, susuana asaam, maria emília de sousa gomes, alcinéia de lemos souza ramos, eduardo mendes ramos, effect of washing cycles and solutions on chemical composition and physicochemical properties of the surimi‐like material from mechanically deboned turkey meat. journal of food processing and preservation: 1–11, (2019). [4]. zabolotna o., ivanova t., peshuk l., fiziko – himichni pokazniki m’yasa ptici mehanichnogo obvalyuvannya, promitogo riznimi organichnimi kislotami [physical and chemical characteristics of poultry meat mechanically washed with various organic acids]. naukovi praci nuht [scientific works nuht]. т., № 5: 44-57, (2017). [5]. melnyk o., radzievska i., galenko o., peshuk l., investigation of vegetable oils to oxidative degradation of varying degrees of saturation with tocopherol, carpathian journal of food science and technology, 10 (3): 164-171, (2018). [6]. cortez‐vega w., fonseca g., prentice c., optimization of parameters for obtaining surimi‐like material from mechanically separated chicken meat using response surface methodology. journal of food science and technology, 52(2): 763–772, (2013). [7]. froning g., mechanical deboning of poultry and fish. advances in food research, 27: 109–147, (1981). [8]. cortez‐vega w., pizato s., prentice c., nutritional quality evaluation of surimi and kamaboko obtained from mechanically separated chicken meat. nutrition and food science, 44(6): 483–491, (2014). [9]. ng x. y., huda n., thermal gelation properties and quality characteristics of duck surimi‐like material (duckrimi) as affected by the selected washing processes. international food research journal, 18(2): 731–740, (2011). [10]. peshuk l., galenko o., sergina v., technology for meat gerodietetic products, ukrainian journal of food science, 3 (1): 16-26, (2015). [11]. stangierski j., zabielski j., grześ b., modification of functional quality of raw myofibril preparation obtained from waterwashed mechanically recovered chicken meat. european food research and technology, 236(3): 449–458, (2013). [12]. tina n., nurul h., ruzita a., surimi‐like material: challenges and prospects. international food research journal, 17(3): 509– 517, (2010). [13]. ramadhan k., nurul n., ruzita a., physicochemical and sensory characteristics of burger made from duck surimi‐like material. poultry science, 91: 2316–2323, (2012). [14]. jin r., meng r., zhang h., yang x., wu z., effects of different deodorizing processes on the off‐odour compounds and gel properties of common carp surimi. international journal of food science and technology., 53(9): 2045–2053, (2018). [15]. choi y.m., choe j.h., cho d.k., kim b.c., practical use ofsurimi‐like material made from porcine longissimus dorsi muscle for the production of low‐fat pork patties. meat science, 90(2): 292–296, (2012). 1. introduction 286 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 2018, pag. 286 299 determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia rehrahie mesfin 1,3 , getnet assefa 2 and fassil assefa 3 1holetta agricultural research center, p.o. box 2003, addis ababa, ethiopia, phone: +251-112370300; e-mail: rehrahiemesfin@gmail.com 2addis ababa university (aau), science faculty, department of microbial, cellular and molecular biology, p.o.box 1176, addis ababa, ethiopia, phone: +251-111-239472 e-mail: asefafasil2013@gmail.com 3ethiopian institute of agricultural research (eiar), livestock research directorate office, p. o. box 2003, addis ababa, ethiopia, phone +251-116-454432; e-mail: getnetassefa@yahoo.com *corresponding author received 25 december 2017, accepted 13rd september 2018 abstract: aflatoxin b1 (afb1) in feeds and aflatoxin m1 (afm1) in milk were studied in holetta, bishoftu and hawassa, ethiopia using enzyme-linked immunosorbent assay (elisa). concentrate feeds were more contaminated (7.67 ± 0.80 μg/kg) than roughage feeds (0.41 ± 0.14 μg/kg); hay was more contaminated (0.72 ± 0.25 μg/kg) than straw (0.05 ± 0.05 μg/kg) and oilseed cake based concentrate feeds were more contaminated (13.09 ± 1.12 μg/kg) than concentrate feeds without oilseed cake (2.78 ± 0.66 μg/kg). fifty percent of the feed samples contained 0 µg/kg aflatoxin and 69% and 31% of them fulfilled the permissible limit of the eu (5μg/kg) and the usa (20 μg/kg) for feeds respectively. the average afb1 of feeds in bishoftu, holetta and hawassa were 9.76µg/kg, 6.33µg/kg and 1.19µg/kg, respectively. the afb1 of feeds in dairy producers, feed manufacturers and feed retailers were 9.35 ± 1.04µg/kg, 7.50 ± 1.43 µg/kg and 6.91 ± 1.09µg/kg respectively. twenty nine percent of the milk samples had aflatoxin content of 0 μg/l and; 58% and 42% of them fullfilled the eu (0.05μg/l) and the u.s.a.(0.5 μg/l) permissible limits respectively. further studies are required by using other techniques such as hplc, gc and multi-mycotoxin assay using lc-ms-ms. the contribution of too acidic and too alkaline ph on aflaoxin reduction should also be studied. awareness creation is required to feed processers, feed traders and dairy producers along the feedmilk production and marketing chain. to minimize the incidence of aflatoxin, feed processers, feed traders and dairy producers should employ better feed/grain storage practices. key words: safety; concentrate; hay; straw; holetta; bishoftu; hawassa 1. introduction agriculture in ethiopia is an important component of rural livelihoods which creates job opportunity for > 85% of the population. it is a major source of food, income, raw materials for domestic industries and foreign exchange 1. ethiopia has diverse agro-ecologies suitable for production of different livestock species and contributes to the national economy in terms of food security, employment, draught power and foreign currency 2. livestock resources in the country are also the sole source of livelihoods for about 10 million pastoral communities 3. ethiopia has good potential for dairy production. with the growing trend of urbanization, tourism industry and rising of incomes, there is an increasing demand for production of safe and better quality milk 4. however, the quality and safety of milk is influenced by different feed contaminants. mycotoxins are among the many contaminants that affect the quality of feeds. aflatoxins are http://www.fia.usv.ro/fiajournal mailto:rehrahiemesfin@gmail.com mailto:asefafasil2013@gmail.com mailto:getnetassefa@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 287 among the many members of mycotoxin that contaminate food and feedstuffs. cereals and oilseed grains and their by products are regularly contaminated with fungi occurring as plant pathogens in the field or developing during storage, transportation and during feeding 5. the aflatoxin group found in feeds consists of b1, b2, g1 and g2 and aflatoxin b1 is the most common, carcinogenic and potent 6. aflatoxin m1 is hydroxylated aflatoxin b1 released in milk when dairy cows consume aflatoxin contaminated feed. livestock species exposed to aflatoxin contamination experiences higher susceptibility to infectious diseases and reduce their yield of meat, milk and egg 7. in human beings, aflatoxin is carcinogenic and poses to liver cancer. epidemiological studies carried out in china, kenya, mozambique, philippines, swaziland, thailand and south africa showed, around 100.000 new cases of hepatocellular carcinoma attributable to aflatoxin exposure 8. in children it poses to stunted growth and susceptibility to infectious diseases 7. children living in high aflatoxin exposure zones in malawi were found to lose 22% less height than children in low-exposure zones 8. aflatoxin is potent and the largest outbreak reported in the world was in 2004 when 317 cases were attributed to 125 deaths due to consumption of aflatoxin contaminated maize in kenya 9. in indonesia, philippines and thailand, 5% of the aflatoxin contaminated maize and peanuts were discarded 7; in 1980s’ in malawi, the share of groundnut exports had collapsed from 64% to 0.2% 8 (rios et al., 2013). past research efforts in ethiopia had paid little attention to feed quality particularly to aflatoxins. since feed is an integral part of the food chain, it needs assesemnt for its quality. the current study therefore was undertaken to detect and quantify the contamination level of aflatoxin in dairy feeds and milk following the production, processing, marketing and utilization of feeds. 2. materials and methods description of study locations the study was undertaken in the central highlands of ethiopia particularly in western shoa (holetta), eastern shoa (bishoftu) and southern shoa (hawassa). the study sites were located on the important feed and milk production chain of the country and represent the different temperature and humidity conditions of the central highlands. holetta is locateed in western shoa, representing the cooller (dega) climatic regions of the country and is situated at 38° 30`e, 9° 3`n and 45 km west of addis ababa and lies at an elevation of 2400 m.a.s.l. the annual rainfall is 1066 mm and the average annual minimum and maximum temperatures were 6° and 22°c respectively 10. holetta could represent sub humid areas of the country. within holetta, there were different sampling sites including welmera, ejere (addis alem), dendi (ginchi), ambo and guder. bishoftu (debre-zeit) is considered to have relatively warmer (weynadega) climatic condition and dry in its humidity level. it is located at 8o50 to 8o53 latitude and 38o55 to 38o59 longitude. it is located 45 km east of addis ababa, at an altitude of 1600-2400 m a.s.l. 11. the mean minimum and maximum temperature are 10.9 and 27.28 °c, respectively. the mean annual rainfall and relative humidity are 56.81mm and 65.92% respectively 12. hawassa is located in the southern nations, nationalities and peoples (snnp) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 288 and sidama zone of southern ethiopia, 285 km far from addis ababa in the great rift valley. it is situated at an altitude of 1708 m, latitude 7°3′n and longitude of 38°28′e. the average annual rainfall was 1100 mm and average low and high temperature of 12.6 co and 27.3 co respectively 13. tabour sub-city and monopol areas in hawassa town were considered as sampling sites. within each study location, dairy feed chain actors representing feed processors, feed retailers and dairy producers were delineated and the study locations are demonstrated in the following maps. fig 1. map of holetta in western shoa fig 2. map of bishoftu eastern shoa fig 3. map of hawassa description and collection of feeds samples feed and milk samples were collected based on purposive sampling and a total of 205 samples consisting of 160 feeds (115 concentrate feed and 45 hay) and 45 milk were collected from all study locations. the collected samples from the three study locations include: 73 samples containing 15 hay, 43 concentrate feed and 15 milk (holetta), 63 samples containing 15 wheat straw, 33 concentrate feed and 15 milk (bishoftu), 69 samples consisting of 15 hay, 39 concentrate and 15 milk (hawassa). hundred ml of fresh milk samples were collected in ice box from each household from the morning milking and stored in a refrigerator. season of sampling for feeds was in the dry and warmer season of april to june and milk samples were collected from september to november 2014. the collected samples were dairy feeds commonly utilized for dairy cattle which included roughage (basal feed) and concentrate (supplemental feed). the concentrate feeds included by-products from agro-industry of flour milling byfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 289 products such as wheat bran and wheat middling’s, by-products from oilextracting factories including noug seed cake, cotton seed cake and linseed cake. majority of the concentrate feed samples were mixture of wheat bran, oilseed meals and byproducts of cereals, pulses which were categorized in to three: oilseed cake based concentrate feed, concentrate feeds without oilseed cakes and pure wheat bran. the oilseed cake based concentrates included wheat bran, wheat middlings and either or mixture of noug seed cake, linseed cake or cotton seed cake. the concentrate mixture feeds without oilseed cakes included wheat bran, wheat middlings and other products of pulses and cereal by products. the roughage feeds samples were categorized in to hay, wheat straw and straw of haricot bean. majority of market oriented dairy producers in ethiopia mainly utilize hay as roughage feed and different concentrate feeds as supplemental feeds. description of target people the study has examined aflatoxin situation of dairy feeds and milk along the production, processing, marketing and consumption chain. the target people considered for this study included dairy feed processors, feed retailers and dairy producers. feed processors are individuals that owe flour milling and oil extracting factories and engaged in production of concentrate feeds. they produce, sell and distribute flour milling by products and oilseed meals as concentrate feeds for dairy cattle feeding. the second target people were feed retailers that buy concentrate feed from feed processors and sell to dairy producers. the third group of people was market oriented urban smallholder milk producers having crossbred dairy cows and buy concentrate feeds from feed retailers to supplement dairy cows and sell their milk to consumers. sample preparation and laboratory investigations sample preparations including drying, chopping, milling and packing and labeling processes were carried out at holetta agricultural research center and laboratory investigation for aflatoxin was carried out in kenya, naivashsa dairy research coordination center. extraction process of feed samples extraction process of feed samples was carried out according of the protocol provided by (helica biosystem inc.). twenty g of each feed sample was transferred into 250 ml erlenmeyer flasks containing 100 ml of 70% methanol and allowed to digest for 30 minutes. the particulate matter was filtered with whatman ♯1 filter paper no 541 or 542 and the filtrate was transferred into 100 μl centrifuge tubes for determination of aflatoxin. assay process of feed samples assay process was performed based on the protocol provided by the manufacturer 14 using the commercial enzyme-linked immunosorbent assay (elisa) based test kits and micro plate elisa reader of model biotech usa. hundred µl of the sample solutions or standards were added in to each dilution wells containing 200 µl of assay diluient and the mixture was mixed using the multi-channel priming micro pipette. then, hundred µl of the mixture was transferred in to each of the antibody coated micro wells and incubated for 15 minutes at room temperature. after adding 200 µl of horseradishperoxidase (hrp) as a conjugate, incubation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 290 continued further for 30 minutes. then the liquid was poured out of the wells and wells were washed 5 times using distilled water and the bound substances was stacked to the antibody coated micro wells. wells were tapped face down on a layer of paper towel to remove the residual wash solution. next, hundred µl of tetramethylbenzidine (tmb) as enzyme substrate solution was added in to each well and incubated for 5 minutes at room temperature in the dark. then 100 µl of acidic stop solution was added which changed the chromagen color from blue to yellow. the optical density (od) of each samples in the micro well was read using elisa reader at 450 nm and the concentration of afb1 (µg/kg) was directly measured from the standard curve that was constructed with known concentration of afb1 reagent grade standards. assay process of milk samples the laboratory investigation for afm1 was carried out at naivashsa dairy research coordination center, nairobi, kenya. the standards were presented in homogenized skim milk (milk plasma). an aliquot of unprocessed raw fatty milk was placed at refrigeration temperature overnight to allow the fat globules to rise to the surface in a natural “creaming” effect. the upper fatty layer was removed by aspiration and the lower plasma layer was used for the assay. the aflatoxin standards were: 0.0 pg/ml, 5.0 pg/ml, 10.0 pg/ml, 25.0 pg/ml and 100.0 pg/ml. the reagents were brought from refrigeration temperature to room temperature before starting the assay. using a new pipette tip for each, 100 µl aliquots of standards and samples were dispensed into the appropriate wells. this was incubated at room temperature for 2 hours. the contents of the wells were discarded into an appropriate receptacle. the micro wells were washed by filling each with pbstween wash buffer and were decanted into an appropriate receptacle. this was repeated for three washings. the wells were tapped face down on a layer of absorbent towel to remove residual wash buffer. two hundred µl of conjugate was added to each well and incubated at room temperature for 15 minutes. then hundred µl of enzyme substrate was placed to each micro-well and incubated at room temperature for 5 minutes in the dark. next, hundred µl of stop solution was added in the same sequence and at the same pace as the substrate was added. the optical density (od) of the standards and the samples were read on each well with a micro plate reader using a 450nm filter. a standard curve was run for quantification of aflatoxin concentration for each feed sample. the graph plotted was based on the optical density (y-axis) and the aflatoxin concentration of the standards (x-axis). the concentration of aflatoxin m1 was measured in picogram per milliliter (pg/ml). the manual used for determination of aflatoxin in milk was 15. study design and statistical analysis the design of the study was completely randomized design (crd) and the statistical model was yi= µ + li + ei where, yi = level of aflatoxin µ = the overall mean li = effect of location ei = the error term the data was subjected to analysis of variance using the general linear model procedures of statistical analysis system 16. different statistical analysis including food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 291 descriptive statistics and t-test were employed to determine the differences. 3. results and discussion level of aflatoxin in feeds the detection and level of aflatoxin in this study showed that 81 feed samples (50.6%), did not contain any level of the toxins; whereas 29 of the feed samples (18.1%) and 50 samples (31.3%) contained aflatoxins within the eu permitted level of 5μg/kg, and the u.s.a. permitted level of 20 μg/kg afb1, respectively (table 3.2). this result was similar to the afb1 level of feeds reported in portugal almost all (98%) of the feed samples fulfilled the permissible limits of the e.u. and the u.s.a 17. the aflatoxin status and level of feeds in this study was much lower than the previous report in the country where the contaminated level was in a range of 7-419 μg/kg 18. the results of this study is in contrary with the afb1 level of feeds reported in india, kenya and previous authors in ethiopia in which 17% and 15%, 46% and 45% of the feed samples did not fulfill the u.s.a afb1 level recommended for feeds (table 1). the reason for the difference of afb1 in feeds between ethiopia and kenya; as well as ethiopia and india could be due to the difference in feed type used in ethiopia (wheat by-products based feeds) and other african countries and india (by-products of maize and groundnut based feeds) which are the most susceptible crops to afb1 contamination as compared to wheat-based feeds 19. table 1. afb1 (μg/kg) in feeds in this study & other countries in comparison with eu and usa permissible limits (%) country n range (μg/kg) 0 level eu standard usa standard > usa standard reference ethiopia 160 0-20 50.6 68.7 31.3 0 this study portugal 312 5.1-74 0 84 14 2 18 india 40 1.8-245 28 13 55 17 21 kenya 144 1-9661 0 27 54 46 22 india 356 46 na 39 15 23 ethiopia 156 7-419 0 5 50 45 19 n= number of samples, na=not available afb1 mean, range and percent of feed samples belonging to different level of aflatoxin the afbi distribution showed variations among the different feed types (table 2). accordingly, concentrate feeds contained significantly (p<0.05) higher afb1 (7.67μg/kg) contents than roughage feeds (0.41μg/kg). among the concentrate feeds, oil seed cake based concentrate feed contained significantly (p<0.05) higher afb1 (13.09μg/kg) than those without oilseed cake and grass hay showed significantly (p<0.05) higher afb1 (0.72 μg/kg) level than the other roughage feed types. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 292 table 2. afb1 mean, range and percent of feed samples belonging to different aflatoxin categories type of feed n level of aflatoxin (μg/kg) range (μg/kg) mean (μg/kg) 0 0.1 5 5.1 20 percent roughage feeds 45 21.9 6.3 0.63 0-0.55 0.41 ± 0.14f grass hay 25 33.3 22.2 2.2 0-5.5 0.72 ± 0.25e wheat straw 15 31.1 2.2 0 0-1 0.05 ± 0.05g harricot bean 5 11.1 0 0 0 0 concentrate feeds 115 6.3 2.5 22.5 0-20 7.67 ± 0.8b with oil seed cake 47 8.7 3.5 31.3 0-20 13.09 ± 1.12a without oil seed cake 68 33.9 8.7 14.8 0-20 2.78 ± 0.66c wheat bran 44 26.1 6.1 4.4 0-20 2.15d mean 5.63 n= number of samples, ls-means with different superscripts among three consecutive feeds in the last columns were significantly different. the lowest afbi was recorded from wheat straw (roughage feed) and wheat bran (concentrate feed) with afbi of 0.05μg/kg and 2.15μg/kg, respectively. interestingly, no aflatoxin was detected from the haricot roughage feed that may be associated with the limited number of samples tested for afbi.the pattern of afb1 contamination in roughage feeds and concentrate feeds was similar to the report of 22. who showed that concentrate feeds (47.841 μg/kg), contained almost 10 times more afbi than the roughage feeds (5.14 g/kg) indicating concentrate feeds are more susceptible to afb1 contamination than roughage feeds. this may be due to the fact that concentrate feeds are rich in nutritional composition than roughage feeds which can provide adequate substrate for growth and reproduction of fungi. this is justified by the fact that, compared to grass hay which contains 59 g cp/kg feed, noug and noug based concentrate feeds contained cp contents of 312 g/kg and 251.3g/kg, respectively 23, 24 also reported that food stuffs with high fat content are good substrates for aflatoxin producing fungi and are more susceptible to afb1contamination as compared to other dairy feeds 25; 18. it was reported that the first effect of mold on feeds is utilization of nutrients for their metabolism and propagation which results in decreased nutritional value of feeds. the energy, crude protein and crude fat values of moldy feeds were decreased by 5%, 6% and 63% respectively and dietary fats are affected more extensively than proteins or carbohydrates and they are decreased 37% to 40% after 25 days of storage or 52% to 57% after 50 days of storage 26. the result showed that higher proportion (22%) of the roughage feed samples contained 0μg/kg of afb1 with fewer samples (0.63%) with afb1 level in the range of 5.1-20 μg/kg. on the contrary, higher proportions (22.5%) of the concentrate feed samples contained afb1 in the range of 5.1-20 μg/kg, and fewer proportions (6.3%) of the concentrate feed samples were free from aflatoxin contamination similarly, higher proportions (31.3%) of afb1 was detected in the range of 5.1-20 μg/kg, from concentrate feeds containing oilseed cake compared to the lower proportions (14.8%) detected from the same concentrate without fortification with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 293 oil seed cake (table 2). the overall mean and range of afb1 in feeds in this study was 5.63 μg/kg and 0-20 μg/kg respectively which was lower than the mean and range afb1 of feeds (25.53 μg/kg and 0.54-204.72 μg/kg) reported in chennai, india 27. level of aflatoxin in different feeds sampled from different study locations the average afb1 content of feeds in bishoftu (9.76µg/kg) was significantly (p<0.05) higher than the afb1 content of feeds in holetta (6.33µg/kg) and this then was significantly higher than that of hawassa (1.19µg/kg) (table 3) in which case, it had fulfilled the eu standard of 5μg/kg 28. the afb1 contamination of feeds in bishoftu (9.76 µg/kg) was higher than the average afb1 of holetta and hawassa (3.8 µg/kg) which had similar trend with report of 19 in that afb1 level in bishoftu (156 µg/kg) was higher than the average afb1 content of other study locations (sendafa, sululta and addis ababa) which was 133 µg/kg. the lowest afb1 content of feeds observed in hawassa in this study was mainly due to the short storage duration (<1 month) of dairy feeds practiced in that area. the roughage feeding practice in urban dairying in hawassa was mainly based on cut and carry system collected from protected areas like universities and prison stations and roughage was not stored not for more than 10 days. in addition, the concentrate feeds belonging to feed manufacturers and feed retailers in hawassa was mainly (80%) wheat bran with some mixture of linseed cake and other home produced grain by products from barley and pulses which are relatively less susceptible to aflatoxin contamination 29. table 3. level of aflatoxin (µg/kg) in different feeds by study locations type of feed study location holetta % bishoftu % hawassa % no of samples 58 48 54 roughage feeds grass hay 1.07 ± 1.4a 13.8 0.13 ± 1.7a 1.9 wheat straw 0.07± 1.4a 2.1 concentrate feeds with oil seed cake 10.29± 1.1b 29.3 17.5 ± 1.3a 35.4 8.13 ± 2.8b 7.4 without oil seed cake 5.24 ± 1.3b 24.1 9.64 ± 1.5a 20.8 2.0 ± 1.6b 11.1 wheat bran 3.61 ± 2.1ab 10.3 6.43 ± 2.1a 8.3 0.81 ± 1.0b 9.3 mean afb1 6.33 ± 1b 9.76 ± 1.3a 1.19 ± 1.1c storage time (month) 3-6 77.5 3-6 66.6 < 1 month 29.7 % percentage of aflatoxin contamination for each type of feed under each study location ls means with different superscripts between columns were significantly different food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 294 though there was no significance difference among roughage feeds across the study locations, afb1 level of wheat straw in bishoftu had the lowest concentration (0.07µg/kg) as compared to afb1 content of hay in holetta (1.07µg/kg) and hawassa (0.13µg/kg) (tabe 3). the lowest afb1 level in wheat straw is related to its poor nutritional composition 30:19-20 which makes it less vulnerable to fungal contamination. the data also showed that the afb1 in concentrate feeds fortified with oilseed cakes was significantly (p<0.05) higher (17.50 ± 1.3 µg/kg) in bishoftu compared to the same type of feed in holetta with afb1 content 10.29 ± 1.1 µg/kg and afb1 of 8.13 ± 2.8 µg/kg at hawassa respectively. similarly, the highest afb1 content of concentrate feeds without oilseed cakes was observed in bishoftu (9.64 ± 1.5µg/kg) as compared to holetta (5.24 ± 1.3µg/kg) and hawassa (2.0 ± 1.6µg/kg). similar pattern of afb1 contamination was also observed on other feed types in bishoftu sampling sites (table 3). in general, the highest afb1 content in feeds observed in bishoftu might be due to the relatively higher temperature which could be aggravated by the relatively longer storage duration (temperature 250c and storage duration 3-6 month) employed in this area as compared to holetta (temperature 220c and storage duration 3-6 month) and hawassa (temperature 270c and storage duration < 1 month). aflatoxin contamination level of feeds across the feed value chain the afb1 contamination level of feeds belonging to dairy producers was significantly (p<0.05) higher (9.35 ± 1.04µg/kg) than feed retailers (6.91 ± 1.09 µg/kg) and was also higher than the afb1 content of feeds in feed manufacturers (7.50 ± 1.43 µg/kg) (table 4). regarding the study locations, the afb1 content of feeds produced by feed manufactures in bishoftu (12.45 ± 3.25μg/kg) was significantly higher than that of holetta (7.92 ± 1.84μg/kg) and hawassa (2.14 ± 2.42 μg/kg), respectively. similarly, the afb1 content of feeds handled by feed retailers in bishoftu (15.40 ± 2.01μg/kg) was significantly higher than that of holetta (5.33 ± 2.02μg/kg) and hawassa (0μg/kg), respectively. the trend was also the same for afb1 content of feeds handled by dairy producers across the study locations (table 4). table 4. afb1 (μg/kg) contamination of feeds across the feed value chain dairy feed chain actors n study location mean afb1 holetta bishoftu hawassa feed processors 29 7.92 ± 1.84ab 12.45 ± 3.25a 2.14 ± 2.42c 7.50 ± 1.43ab feed retailers 41 5.33 ± 2.02b 15.40 ± 2.01a 0.00 c 6.91 ± 1.09bc dairy producers 45 11.37 ± 1.88ab 15.18 ± 1.88a 1.5 ± 1.30c 9.35 ± 1.04a overall 115 ls-means with different superscripts between rows were significantly different (last column); ls-means with different superscripts between columns were significantly different (excluding last column); n=number of samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 295 the high afb1 contamination level of feeds across the feed value chain was in the order of dairy producers > feed manufacturers > feed retailers (table 4). the reason for higher level of afb1 in concentrate feeds handled by urban smallholder dairy producers might be due to the poor storage situation employed by them. the lower contamination level of afb1 of concentrate feeds administered by feed retailers could be related to the small scale of holding and shorter duration of storage because of high market demand. for example in hawassa, concentrate feeds in feed retailers were not stored for more than a week. 31: 219-226 in their study of afb1 in animal feeds in kenya reported that contamination levels for urban smallholder dairy, feed manufacturers and feed retailers respectively was 20.48 ± 29.8μg/kg, 20.62 ± 15.62 and 22.38 ± 17.78 which was much higher than the results found in this study. level of aflatoxin m1 (afm1) in milk across study locations the overall (average) afm1 contamination of milk in this study was in the range of 0-0.146; with a mean afm1 of 0.054 µg/l which was different from the average and range of afm1 (0.41μg/l and 0.028-4.98 μg/l) in milk samples reported in ethiopia by 18. the data also showed variations in afm1 content of milk samples at different experimental sites (table 5). accordingly, milk samples collected from bishoftu contained relatively higher afm1 (0.088 μg/l), followed by milk samples with afm1 contents of (0.057 μg/l and (0.017 μg/l collected from holetta and hawassa sampling sites, respectively (table 5). although all samples were within the eu and usa permitted levels, the quality of milk seemed to be better at hawassa than the other experimental sites which may be associated with the lower afm1 content of feeds in the area (table 5). table 5. pattern of aflatoxin contamination of milk samples at different sampling sites sampling site mean (µg/l) range (µg/l) free from afb1 (n & %) eu standard (0.05 µg/l) (n & %) us standard 0.5μg/l (n & %) bishoftu 0.088 0-0.1403 2 (13) 4 (27) 11 (73) holetta 0.057 0.0015-0.146 0 (0) 4 (27) 11 (73) hawassa 0.017 0-0.11 11(73) 15 (100) 0 (0) quality status (overall) 0.054 0-0.146 29% 58% 42% level of aflatoxin in milk in this study in comparison with other countries level the quality of milk in terms of afltoxin contamination in this study was compared with other countries (table 6) and the afm1 contamination level was slightly higher than the afm1 of milk studied in turkey where 39% of the samples were free from aflatoxin, and 61% of the samples were within the eu stringent standard reported by 32. it is interesting to note that the aflatoxin level of milk in this study was in the "low " category (within the stringent eu standard) similar to most reports from different countries, especially from egypt 33; 34; iran 35 36; kenya 31; india 37 sri lanka 38 (table 6). the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 296 afm1 content of milk in this study was lower than the previous results reported in ethiopia 18, in pakistan 39. however the quality of milk in terms of the level of afm1 in milk was lower than the milk samples reported from iran 36 and 32 tukey (table 6). table 6. level of afm1 in milk in this study in comparison with other countries (2009-2016) country afm1 in milk (μg/l) reference mean range statius ethiopia 0.054 0–0.146 low this study ethiopia 0.41 0.028-4.98 medium 18 pakistan 0.38 0.01–0.76 medium 39 india 0.5 medium 40 iran 0.0021 0.131 low 35 egypt 0.05 low 33 egypt 0.063 0.002-0.11 low 34 sudan 0.5 medium 41 thailand 0.070 low 34 kenya 0.064 low 31 india 0.046 low 37 sri lanka 0.04 medium 38 iran 0.0004 low 36 turkey 0.0023 low 32 low within the eu standard; medium within the usa standard the overall afb1 and afm1 levels in feeds and milk in the current study was 5.63µg/kg and 0.054µg/l respectively and the transfer rate of afb1 in feeds to afm1 in milk was 0.054/5.63 x 100 = 0.96% which was similar to the findings of 42 with a transfer rate of 1-2 %. 4. conclusions in this study, half of the feed samples were free from aflatoxin contamination; whereas the remaining were within the stringent eu and the more permissible usa standards. the pattern of aflatoxin distribution amongst the different feed sources, showed that concentrate feeds such as oil seed cakes contained more afb1, followed by cereal byproducts, roughage feeds (grass hay and straws) respectively. the data also showed difference in aflatoxin content amongst sampling sites in that feeds from bishoftu contained more aflatoxin, followed by feeds from holetta and hawassa, respectively. the aflatoxin contamination level was in the order that feeds belonging to dairy producers contained significantly higher afb1 than feed retailers, followed by feed manufacturers. all variations in aflatoxin contents amongst feeds and sampling sites were due to different nutritional contents of feeds and the storage situations by the value chains actors in different areas. regarding milk samples, aflatoxin was not detected from a reasonable number of samples (29%); whereas 58% of the samples were within the stringent eu standard of (0-0.05); and 42% were within the usa standard set for milk samples indicating that the milk samples were within the permissible level of aflatoxin in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 297 milk. the data also showed the conversion rate of afltoxin from feeds to milk was within the values computed for other milk samples elsewhere. the milk quality of the present study were within the "low category" comparable to studies from several countries. in general, the afm1 contamination level of feeds and milk samples collected from the study locations were in the order of hawassa < holetta < bishoftu. 5. recommendations further studies are required along the feed and milk production and marketing chains using other techniques such as hplc, gc and multi-mycotoxin assay using lc-msms by considering different storage conditions such as use of palate, ventilation, and duration of feed storage on aflatoxin and nutrient contents of feeds. the contribution of extreme acidic and extreme alkaline ph conditions on aflaoxin reduction should also be studied. the contribution of cooking, roasting and boiling of agricultural products on reduction of aflatoxin contamination should also be studied. since, proper handling and management is a key issue for minimizing aflatoxins contaminations on grains and feeds, people engaged in crop production, feed processing, feed marketting and feed utilization should employ better agricultural practices, better grain/feed storage practices and hygienic feeding practices. there is a need to educate and train farmers, feed traders and feed manufacturers on good agricultural practices and good storage practices to produce grains and feeds with low levels or exempt from aflatoxin contamination. special care and attention is required in storing feeds and grains during rainy season. feed manufacturers should employ proper screening of spoiled grains and feed ingredients before milling and processing. in addition, dairy producers should follow better feeding practices such as screening of moldy feeds before offering to livestock species. 6. acknowledgements the authors like to thank the east african agricultural productivity project (eaapp) for covering cost of the research and the management of holetta agricultural research center for facilitating the research activities. the authors would like to thank the experts and development agents of livestock agencies at zonal and district level of west shoa, east shoa and hawassa, ethiopia, for cooperation during sample collection. we are also thankful to the experts and laboratory technicians of the naivasha dairy research center of excellence, kenya for allowing undertaking the laboratory analysis. finally i declare that this article is part of my phd study [43]. 7. references 1. berhanu kuma, fekede feyissa and kedir nesha, gender based analysis of livestock production systems at kuyu wereda in north shao zone, ethiopia. in: institutional arrangements and challenges in market-oriented livestock agriculture in ethiopia, pp. 88-100, (tamrat degefa and fekede feyissa, eds). proceedings of the 14th annual conference of the ethiopian society of animal production (esap) held in addis ababa, ethiopia, september 5-7, 2006 part ii: technical papers, (2006) 2. hadera gebru, role of livestock in food security and food self sufficiencyin the highland production system. in: livestock in food security – roles and contributions, proceedings of 9th annual conference of the ethiopian society of animal production (esap). 433 pp. 30-31august 2001, addis ababa, ethiopia, (2002) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 298 3. little, p., smith, k., cellaruis, b., coppock d., barrette, c. avoiding disaster, diversification and risk management among east african herders. development and change. 32 (30): 401-433, (2001) 4. gian, n. f., promoting milk quality in smallholders’ cooperatives: evidence from ethiopia. in:institutional rrangements and challenges in market-oriented livestock agriculture in ethiopia, pp. 69-82, (tamrat degefa and fekede feyissa, eds). proceedings of the 14th annual conference of the ethiopian society of animal production (esap) held in addis ababa, ethiopia, september 5-7, 2006 part ii: technical papers. addis ababa, ethiopia, (2006) 5. abdullah, a.n., lkhalaf1, khaled, o.a., sman and ahmed, s.k., monitoring of aflatoxins and heavy metals in some poultry feeds. african journal of food science, 4(4): 192-199, (2010) 6. reddy, s.v, waliyar, f., properties of aflatoxin and its producing fungi. aflatoxin. int. crops res. inst. for the semi-arid tropics, (2000) 7. bhat, r.v. and vasanthi, s. mycotoxin food safety risk in developing for food, agriculture and environment. food safety in food security and food trade. international food policy research institute (ifpri), (2003) 8. rios, l.d., gokah, i.b., kauma, b.c., matumba, l., njoroge, s. and chinseu, a., advancing collaboration for effective aflatoxin control in malawi. malawi program for aflatoxin control (mapac), lilongwe, malawi, (2013) 9. lewis, l., onsongo, m., njapau, h., schurz-rogers, h., luber, g., nyamongo, s. j., baker, l., dahiye, a.m, misore, a, kevin, d.r, and the kenya aflatoxin investigating group, aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central kenya. environmental health perspective, 113 (12): 1763-1767, (2005) 10. fekede feyissa, getinet asefa, lulseged g/hiwot, muluneh minta and tadesse t/tsadik, evaluation of napir grassvetch mixture to improve total herbage yield in the central highlands. in: the role of agricultural universities/colleges in transforming animal agriculture in education, research and development in ethiopia: challenges & opportunities, pp. 155-164, (tamrat degefa and fekede feyissa, eds). proceedings of the 13th annual conference of the ethiopian society of animal production (esap) held in addis ababa, ethiopia, august 25-27, 2004 part ii: technical papers, (2004) 11. samuel menbere, azage tegegne and hegde, b.p., feed resources availability, utilization and management in smallholder livestock production system in yerer watershed of ada liben district. in: commercialization of livestock agriculture in ethiopia, pp 61-74, (tamrat degefa and fekede fetissa, eds). proceedings of the 16th annual conference of the ethiopian society of animal production (esap) held in addis ababa, ethiopia. october 8-10, 2008. part ii technical papers, (2009) 12. metrology office, debrezeit agricultural research center, bishoftu, ethiopia, (2016) 13. https://en.wikipedia.org/wiki/awasa, (2016) 14. helica biosystems inc., aflatoxin b1 elisa quantitative catalog ♯ 41bafl01b196 www.helica.com, california, usa., (2015) 15. helica biosystems inc., aflatoxin m1 elisa quantitative catalog # 961aflm01m96www.helica.com california, usa, (2015) 16. sas (statistical analysis systems), sas institute version 9.0. sas inc., cary, north carolina, usa, (2002) 17. martins, h.m., mendes guerra, m.m., almeida bernardo, f.m., occurrence of aflatoxin b1 in dairy cows’ feed over 10 years in portugal (1995-2004). rev. iberoam micol 24:69-71, (2007) 18. dawit gizachew, barbara szonyi, azage tgegne, jean hanson and delia grace, aflatoxin analysis of dairy feeds in the greater addis ababa milk shed, ethiopia. food control, 59:773-779, (2016) 19. idris, y. m. a., mariod, a. a., elnour, i. a., and mohamed, a. a., determination of aflatoxin levels in sudanese edible oils. food chem. toxicol. 48(8-9): 25392541, (2010) 20. vijayasamundeeswari,a., mohankumar, m., kartikeyan, m. vijayanandrja, s., paranidaharan, v., velazhahan, r., prevalence of aflatoxin b1 in pre and post harvest maize kernels, food products, poultry and livestock feeds in tamil nadu, india. journal of plant protection research, 49(2): 220224, (2009) 21. senerwa, d.m., prevalence of aflatoxin in feeds and cow milk from five countries in kenya. african journal of food, agriculture, nutrition and development, 16(3): 11005-11021, (2016) https://en.wikipedia.org/wiki/awasa http://www.helica.com/ http://www.helica.com/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 3 – 2018 rehrahie mesfin, getnet assefa, fassil assefa, determination of aflatoxin in dairy feeds and milk in some selected areas of ethiopia, food and environment safety, volume xvii, issue 3 – 2018, pag. 286 – 299 299 22. makau, c.m., matofari, j.w., muliro, p.s., bebe, b.o., aflatoxin b1 and deoxynivalenol contamination of dairy feeds and presence of aflatoxin m1 contamination in milk from smallholder dairy systems in nakuru, kenya. international journal of food contamination, 3(6) pp 1-15, (2016) 23. rehrahie mesfin and ledin, i., biological and economical evaluation of feeding urea treated teff and barley straw based diets to crossbred dairy cows in the highlands of ethiopia. msc thesis. swedish university of agricultural sciences, department of animal nutrition and management, uppsala, sweden, (2001) 24. ephrem guchi, implication of aflatoxin contamination in agricultural products. american journal of food and nutrition, 3(1): 12-20, (2015) 25. makun, h. a., dutton, m.f., njobeh, p.b., gbodi,t.a. and ogbadu, g.h., aflatoxin contamination in foods and feeds: a special focus on africa, trends in vital food and control engineering, ayman, a.e. (ed.), isbn: 978-953-51-0449-0, (2012) 26. dicostanzo, a. murphy, m., strategies for feeding mycotoxin and mold contaminated grains to cattle. beef cattle feed lot nutrition, university of minoseta extension, (2012) 27. ramesh, j., sarathchandra, g. and sureshkumar, v., analysis of feed samples for aflatoxin b1 contamination by hptlc a validated method. international journal of current microbiology and applied science, 2(5): 373-377. 28 fao (2004). worldwide regulations for mycotoxins in food and feed in 2003. food and nutrition paper 81, rome, (2013) 29. adebayo-tayo, b.c. and ettah, a.e., microbiological quality and aflatoxin b1 level in poultry and livestock feeds. nigerian journal of microbiology, 24(1): 2145-2152, (2010) 30. schiere, j. b. and ibrahim, m. n. m., practical methods of urea treatment. feeding of urea ammonia treated rice straw, a compilation of miscellaneous reports produced by the straw utilization project (sri lanka), pudoc, wageningen, pp 19-20, (1989) 31. kang, e.k. and lang, k.a., aflatoxin b1 and m1 contamination of animal feeds and milk from urban centers in kenya. african health sciences, 9(4): 219-226, (2009) 32. aksoy, a., yavuz, o., guvenc, d., das, y.k., terzi, g. suleyman celik, s., determination of aflatoxin levels in raw milk, cheese and dehulled hazelnut samples consumed in samsun province, turkey, (2010) 33. amer, a.a. and ibrahim, m.a.e., determination of aflatoxin m1 in raw milk and traditional cheeses retailed in egyptian markets journal of toxicology and environmental health sciences, 2 (4): 50-53, (2010) 34. ghareeb, k., elmalt, l.m., awad, w.a., bohm, j., prevalence of aflatoxin m1 in raw milk produced in tropical state and imported milk powder. j.vet. anim. sci. 3(1-2): 1-4, (2013) 35. rokhi, m. l., determination of aflatoxin m1 levels in raw milk samples in gilan, iran. advanced studies in biology, 5(4): 151-156, (2013) 36. norian, r., mahmoudi, r., porfarzaneh, a., mashatian, f., ata kaboudari, a., faezeh, s., mahalleh, r.p., katiraee, f., determination of aflatoxin m1 levels in raw milk samples using elisa and high-performance liquid chromatography in qazvin, iran journal of mycology research, 2(1):41-48, (2015) 37. iqbal, s.z., asi, m.r. and ariño, a., aflatoxin m1 contamination in cow and buffalo milk samples from the north west frontier province (nwfp) and punjab provinces of pakistan, food additives & contaminants: part b, 4 (4): 282-288, (2011) 38. pathirana, u.p.d., wimalasiri, k.m.s., silva, k.f.s.t. and gunarathne, s.p., investigation of farm gate cow milk for aflatoxin m1. tropical agricultural research, 21(2): 119 – 125, (2010) 39. jawaid, s., talpur, f.n., nizamani, s.m., afridi, h.i., contamination profile of aflatoxin m1 residues in milk supply chain of sindh, pakistan. toxicology reports, 2:1418-1422, (2015) 40. lunden, h., what is milk? aflatoxin and antibiotic residues in cow’s milk in assam, north east india. msc thesis, swedish university of agricultural sciences, uppsala, sweden, (2015) 41. suleiman, s. e., and abdalla, m. a., presence of aflatoxin m1 in dairy cattle milk in khartoum state-sudan. international journal of scientific & technology research (2) 4: 22778616, (2013) 42. van egmond, h.p., aflatoxin m1: occurrence, toxicity, regulation. in: van egmond h. p. (ed). mycotoxins in dairy products. london and new york: elsevier applied science, (1989). [43]http://publication.eiar.gov.et:8080/xmlui/bitstream/ha ndle/123456789/2227/rehrahie%20phd%20thesis%20f inal.pdf?sequence=1&isallowed=y http://publication.eiar.gov.et:8080/xmlui/bitstream/handle/123456789/2227/rehrahie%20phd%20thesis%20final.pdf?sequence=1&isallowed=y http://publication.eiar.gov.et:8080/xmlui/bitstream/handle/123456789/2227/rehrahie%20phd%20thesis%20final.pdf?sequence=1&isallowed=y http://publication.eiar.gov.et:8080/xmlui/bitstream/handle/123456789/2227/rehrahie%20phd%20thesis%20final.pdf?sequence=1&isallowed=y the study was undertaken in the central highlands of ethiopia particularly in western shoa (holetta), eastern shoa (bishoftu) and southern shoa (hawassa). the study sites were located on the important feed and milk production chain of the country and ... 341 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 3 -2018 , pag. 341 351 pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon *jean sonchieu 1 , john fru nsoh 1 , caroline nain waingeh 2 1department of social economy and family management, higher technical teacher training college, the university of bamenda, po. box 39 bamenda, cameroon; jsonchieu@yahoo.fr, 1department of social economy and family management, higher technical teacher training college, the university of bamenda, po. box 39 bamenda, cameroon; frunsoh2005@yahoo.com 2food technology and post-harvest laboratory, institute of agricultural research for development (irad) bambui, p o box 51, bamenda, cameroon; nainkain@yahoo.com *corresponding author received 3rd may 2018, accepted accepted 19th september 2018. abstract: safety of bread sold in bamenda municipality has been always problematic because of the poor hygienic practices of sellers. this study aimed at assessing the microbial load of bread sold in bamenda municipality and pesticides exposure of sellers. ninety samples of bread were randomly collected from standard bakeries, local bakeries and roadside bread vendors; they were analyzed for total viable count, coliforms and yeast and molds (fungi) using the routine analytical method described by the american public health association. the exposure of some bread vendors to pesticide shops was evaluated using questionnaires. as results, the total viable count ranged from 3.09 x 103 to 2.57 x 105cfu/g, coliform count ranged from 2.27 x 101 to 1.18 x 103cfu/g, while yeast and molds count ranged from 1.32 x 103 to 2.67 x 106cfu/g. bread from roadside vendors was the most contaminated with molds, while the standard bread was the most contaminated with bacteria and coliforms. the local bread was the least contaminated. sixteen bread sellers counted around pesticides shops presented ailments (headache, eye irritation, etc.). bread sold in bamenda is contaminated by microbes and some vendors are exposed to pesticides. key words: bread, bacteria, coliform, molds, pesticides 1. introduction bakery products are the most important staple foods in most countries and cultures. these bakery products and cereals are a valuable source of nutrients in our diets [1]. bread is one of such bakery products, well priced for its taste, aroma, and texture. it is a staple food prepared by baking dough of flour and water [2]. bread is universally accepted as a very convenient form of food that is important to all populations. it is a good source of nutrients such as macronutrients (carbohydrates, proteins, and fats) and micronutrients (minerals and vitamins) that are essential for human health [3]. the basic criteria for the quality of bread and other bakery products are safe with optimum sensory properties. bread is one of the most ancient of human foods and it is produced with the help of microorganisms [4]. it is often spoiled within 48 hours of production, causing significant loss to producers and consumers. by its nature, bread is liable to spoilage at several stages of production to just before consumption. bread spoilage is a metabolic process that causes bread to be undesirable for human http://www.fia.usv.ro/fiajournal mailto:jsonchieu@yahoo.fr mailto:frunsoh2005@yahoo.com mailto:nainkain@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 342 consumption due to changes in sensory characteristics. contaminated bread may be physically safe, but may cause illness because of the presence of pathogens or toxins while changes in texture, smell, or appearance cause them to be rejected [5]. bread spoilage can result from physical, chemical or microbial activity. however, hocking argues that bread spoilage due to microbial activity, in particular mold growth is of major economic importance. [6] microbes including bacteria, mold, yeast and others grow on metabolizing bread to cause severe damage [7]. microbial species can be controlled by improving sanitary conditions at bakeries and sale points, as well as incorporating different acids and their salts in bread formulations provide the bread protection against rapid growth of mold and bacteria [8]. contamination is the presence of something harmful in food or drink that creates a risk of illness, injury or discomfort. bread can be contaminated by chemicals, dirt, pests, pets, waste food or small objects, microbes. chemicals are used in food production, from the farm where they are grown and during processing or manufacturing. dirt or small objects can find their way in to food via transportation of the food, handling and in the home [9]. bread spoilage causes a reduction in the quality of bread. this reduction in the quality is identified by deterioration in the physical, chemical and or sensory properties. contaminated bread causes poisoning which symptoms are vomiting, diarrhea, nausea and headache [10]. a number of molds produce toxin (mycotoxins) that stay in the food as part of the production process. some chemicals can even find their way in to food (bread) accidentally. growth of microorganisms (viruses, bacteria, parasites and molds) causes food spoilage or possible poisoning [7]. at time selling points are closer to agrochemical shops exposing the products and the sellers since various pesticides have high vapor pressure [11]. it has been reported that, the sales conditions by sellers or dealers in pesticides are not adequate to minimize intoxication or poisoning in bamenda [10]. pesticides are handled carelessly with bare hands which can lead to poisoning through the mouth (when hands are used for eating). retailing and mixing are done with no personal protective equipment and outside where bread vendors stand. rooms are not properly ventilated, chemicals may be inhaled and this can cause building effects leading to long term chronic diseases for sellers and for people around. . the aim of this study was to assess the microbiological load of bread produced and distributed in bamenda municipality, and to estimate, according to the proximity of selling site to pesticides shops, the exposure of sellers to pesticides. 2. materials and method 2.1 sample collection a total of ninety bread samples, three from each sampling site were collected from ten standard bakeries, ten local bakeries and ten roadside vendors in the bamenda city area. the maximum number of samples collected in each session was ten for duration of three months (one sampling per month). the bread samples were each put in a sterile stomacher bag, labeled with the names of the bakeries from which they were collected and placed in an airtight cooler (flask) to prevent cross contamination and contamination in the cause of transportation in view of stopping microorganism’s multiplications. immediately after collection, the bread samples were transported to the microbiology unit of the food technology food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 343 and post-harvest laboratory (ftphl) of the institute of agricultural research for development (irad) bambui for microbiological analysis. 2.2 preparation of sample upon arrival at the laboratory, the bread samples were removed from the cooler (flask) but still kept in the stomacher bags and stored in a refrigerator for one night. ten grams of each bread sample was cut with knives (stainless steel) and weighed on a top loader electric balance (mettler ae200), and placed in sterile stomacherblending bags labelled with codes. the knives used had been swabbed with 70% alcohol. ninety milliliter (90ml) of 0.1% peptone solution (diluents) was added to the bread sample in each stomacher-blending bag and blended for fifteen (15) minutes in a stomacher (stomacher model 400 circulator®, seward-england) rotating at three hundred and sixty revolutions per minute. 2.3 microbiological analysis microbiological analyses were carried out following routine analytical method described by the american public health association [12]. 2.3.1 preparation and plating of samples peptone was supplied in powder form. 0.1 % of peptone solution was prepared and dispensed in 90 ml and 9 ml amounts respectively, in to universal dilution bottles and sterilized in an autoclave (dixon’s surgical instrument® ltd u.k), for 15 minutes at a temperature of 1210c. the work bench was swabbed with 70% ethyl alcohol and bunsen burners were lit to provide a sterile working environment. small glassware was autoclaved (dixon surgical instrument ltd u.k), at a temperature of 1210c for 20 minutes before use. previously washed petri dishes were sterilized by heating in a vacuum oven at 1200c for three hours and allowed to cool still wrapped or in their canisters to be removed just before use. without opening the petri dishes, they were labelled externally using a permanent marker, with the sample code number, dilution factor and the plating date. the 0.1% peptone solution already distributed into universal dilution bottles were arranged according to the number of dilutions to be made. 2.3.2 preparation of serial dilution ten folds dilutions of bread samples were made by aseptically transferring 1 ml of the blended bread sample in to 9 ml of the sterile 0.1% peptone solution in universal dilution bottles to give a 10-2 dilution [13]. further ten folds dilutions of up to 10-4 were made by transferring 1 ml of successive dilutions into 9 ml of 0.1% peptone solution. all work was done close to the flame of a bunsen burner to avoid eventual contamination. 2.3.3 enumeration of total viable bacteria the enumeration of total viable bacteria was done using nutrient agar (na). this culture medium was prepared and sterilized in an autoclave (dixon’s surgical instrument ltd. u.k.) according to the manufacturer’s instruction. it was prevented from solidifying by placing it in a water bath (haake wb 20) at a temperature of 450c. 1 ml of the 10-3 and10-4 dilutions of each sample was aseptically plated in sterile petri dishes initially labeled with the sample code, culture medium, and plating date. the plating was done using the pour plate technique. about 20 ml of na was added, allowed to cool and solidify before incubating at 370c for two days. the petri dishes were placed up-side-down in an incubator (hearson®) to enable the viable bacteria to grow to the bottom of the petri dish to ease counting since the viable bacteria are aerobic. at the end of the second day, the total colonies of viable bacteria were counted. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 344 2.3.4 enumeration of coliforms the enumeration of coliforms was done using macconkey agar (mca). this culture medium was prepared and sterilized in an autoclave (dixon’s surgical instrument ltd. u.k.) according to the manufacturer’s instruction. it was prevented from solidifying by placing it in a water bath (haake wb® 20) at a temperature of 450c. 1 ml of the 10-1 and 10-2 dilutions of each sample was aseptically plated in sterile petri dishes initially labelled with the sample code, culture medium, and plating date. the plating was done using the pour plate technique. about 20 ml of mca was added, allowed to cool and solidify before incubating at 370c for two days. the petri dishes were placed up-side-down in an incubator (hearson®) to enable the coliforms to grow to the bottom of the petri dish to ease counting since coliforms are aerobic. at the end of the second day, the total colonies of coliforms were counted. 2.3.5 enumeration of yeasts and molds the enumeration of yeast and molds was done using sabouraud dextrose agar (sda). this culture medium was prepared and sterilized in an autoclave (dixon’s surgical instrument ltd. u.k.) according to the manufacturer’s instruction. it was prevented from solidifying by placing it in a water bath (haake wb20) at a temperature of 450c. 1 ml of the 10-3 and 10-4 dilutions of each sample was aseptically plated in sterile petri dishes initially labeled with the sample code, culture medium, and plating date. the plating was done using the pour plate technique. about 20 ml of sda was added, allowed to cool and solidify before incubating at 280c for three days. the petri dishes were placed upright in an incubator (hearson®) to enable the yeast and molds to grow to the bottom of the petri dish to ease counting since yeast and molds are anaerobic. at the end of the third day, the total colonies of yeasts and molds were counted. in all cases colonies were counted manually on an electric lit background of a gallenkamp colony counter. the positions of colonies counted were marked externally on the petri dish with a permanent marker to avoid re-counting. only plates containing between 30-300 colony forming units were considered during counting. plates having 30-300 colony forming units were chosen because this range is considered statistically significant. if there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. 2.4. exposure of bread sellers to pesticides a list of agro-chemical shops in bamenda municipality was gotten from the regional phytosanitary intervention brigade. since the population of this study was not vast, a systematic sampling method was used in which all the 16 shops agro-chemical shops were investigated, surveyed and questionnaires were administered to 16 bread sellers whose selling points were closer (less than 5 (five) meters distant) from the pesticides shops. 2.5 data analysis the microbial counts observed on bread samples were transformed using log10 transformation. the transformed data was subjected to analysis of variance (anova) to determine if there were significant differences between mean counts in colony forming unit per gram (cfu/g) for the different bread samples. significantly different means were separated by the duncan’s multiple range test (dmrt) at a probability level of 95%. all analyses were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 345 done with the help of statistical package for social sciences (spss) version 16. the results from exposure will be expressed in terms of percentages and frequency. 3. results and discussion 3.1. microbial counts on bread samples from standard bakeries 3.1.1 total colony count of bacteria the minimum total colony count of bacteria (table 1) was 3.09 x 103 cfu/g of bread while the maximum was 3.46 x 104 cfu/g of bread. the total colony count of bacteria in the bread from standard bakeries were not significantly different 3.2 microbial counts on bread samples from local bakeries 3.2.1 total colony count of bacteria there was no significantly different (p>0.05) in the total colony count of bacteria in bread samples from local bakeries (p>0.05). the minimum total colony count of bacteria was observed in bread samples from bakery bs20 (8.91 x 103cfu/g) whereas the maximum total colony count of bacteria was observed in bread samples from bakeries bs12 and bs14 (1.99 x 104cfu/g) (table 2). 3.2.2 coliforms the values for the coliform counts in the bread samples from local bakeries were significantly difference (p<0.05). the minimum coliform count was recorded by bread samples from bakery bs19 (2.86 x 101cfu/g) while the maximum coliform count was recorded by bread samples from bakery bs15 (1.18 x 103cfu/g) (table 2). coliform counts on bread samples from bakeries bs11, bs12, bs14, bs16, bs17, and bs20 were not significantly different. (p>0.05). however the minimum colony count of bacteria was recorded by bakery bs4 (3.09 x 103cfu/g) while the maximum colony count of bacteria was recorded by bakery bs2 (3.46 x 104cfu/g). 3.1.2 coliforms there were significant differences in the coliform count. the lowest coliform count (table 1) was observed on bread samples from bakery bs5 (2.27 x 101cfu/g) while the highest coliform count was observed on bread samples from bakery bs8 (3.46 x 102cfu/g). coliform counts on bread samples from bakeries bs1, bs2, bs3, bs5, bs7, and bs9 had no significant difference. but amongst them bs5 had the least coliform count (2.27 x 101cfu/g) and bs2 had the highest coliform count (5.79 x 101cfu/g). coliform counts of bread samples from bakeries bs4, bs6, bs8, and bs10 equally had no significantly different (p>0.05). 3.1.3 yeast and molds the yeast and molds counts were significantly different (p>0.05) (table 1). the minimum yeast and count was recorded in the samples from bakery bs5 (1.32 x 103cfu/g) while the maximum yeast and mold count was recorded by samples from bakery bs9 (2.69 x 104cfu/g). yeast and molds counts recorded in bread samples from bakeries bs1, bs2, bs3, bs4, bs6, bs7, bs8, and bs10 had no significantly different (p>0.05). however, amongst them bakery bs7 had the least yeast and mold count (1.63 x 103cfu/g) while bakery bs10 had the highest count (1.22 x 104cfu/g). however samples from bakery bs16 recorded the minimum coliform count (3.16 x 102cfu/g) amongst them while samples from bakery bs12 recorded the maximum coliform count (4.26 x 102cfu/g). the samples from bakeries bs13, bs18, and bs19 did not have any significant difference (p>0.05) in their coliform count. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 346 table 1 total colony count of microorganisms on bread from standard bakeries roadside vendor bacteria (maximum limit: 105 cfu/g) coliforms (maximum limit: 102 cfu/g) yeast and molds (maximum limit: 104 cfu/g) bs1 2.39 x 104 ± 9.46a 4.78 x 101 ± 3.29a 5.79 x 103 ± 9.46ab bs2 3.46 x 104 ± 3.04a 5.79 x 101 ± 1.62a 6.45 x 103 ± 7.38ab bs3 4.36 x 103 ± 1.37a 5.01 x 101 ± 2.22a 1.99 x 103 ± 3.36ab bs4 3.09 x 103 ± 4.64a 3.33 x 102 ± 1.13b 2.99 x 103 ± 4.36ab bs5 5.01 x 103 ± 1.35a 2.27 x 101 ± 2.44a 1.32 x 103 ± 3.56a bs6 1.25 x 104 ± 2.02a 2.83 x 102 ± 1.13b 1.01 x 104 ± 1.43ab bs7 3.46 x 103 ± 14.55a 2.43 x 101 ± 2.25a 1.63 x 103 ± 5.87ab bs8 6.16 x 103 ± 1.27a 3.46 x 102 ± 1.15b 1.16 x 104 ± 3.12ab bs9 1.28 x 104 ± 3.22a 3.60 x 101 ± 1.72a 2.69 x 104 ± 4.98b bs10 2.63 x 104 ± 3.22a 3.16 x 102 ± 1.25b 1.22 x 104 ± 1.33ab means along the same column with different superscripts are significantly different (p < 0.05). 3.2.3 yeast and molds there was no significant difference in the yeast and mold counts of bread samples from local bakeries p>0.05). the minimum yeast and molds count was 2.45 x 103cfu/g recorded by bread samples from bakery bs15 while the maximum yeast and molds count was 1.98 x 104cfu/g recorded by bread samples from bakery bs14 table 2 total colony count of microorganisms on bread from local bakeries roadside vendor bacteria (maximum limit: 105 cfu/g) coliforms (maximum limit: 102 cfu/g) yeast and molds (maximum limit: 104 cfu/g) bs11 1.25 x 104 ± 1.15a 3.83 x 102 ± 1.16b 7.13 x 103 ± 1.51a bs12 1.99 x 104 ± 1.25a 4.26 x 102 ± 1.25b 1.12 x 104 ± 2.10a bs13 1.31 x 104 ± 1.74a 3.26 x 101 ± 2.78a 6.23 x 103 ± 1.94a bs14 1.99 x 104 ± 1.55a 3.38 x 102 ± 1.15b 1.98 x 104 ± 5.56a bs15 1.09 x 104 ± 1.67a 1.18 x 103 ± 1.18c 2.45 x 103 ± 5.97a bs16 1.69 x 104 ± 1.35a 3.16 x 102 ± 1.25b 4.82 x 103 ± 10.6a bs17 1.58 x 104 ± 1.21a 3.36 x 102 ± 1.03b 9.69 x 103 ± 1.39a bs18 1.23 x 104 ± 1.97a 4.39 x 101 ± 1.43a 4.78 x 103 ± 1.62a bs19 9.33 x 103 ± 1.60a 2.86 x 101 ± 1.4a 7.85 x 103 ± 3.28a bs20 8.91 x 103 ± 1.60a 3.80 x 102 ± 3.89b 5.79 x 103 ± 10.28a means along the same column with different superscripts are significantly different (p < 0.05). 3.3 microbial counts on bread samples from roadside vendors 3.3.1 bacteria the bacterial counts were significantly different (p<0.05). the highest bacteria count was observed from bread samples from vendor bs22 (2.57 x 105cfu/g) and the lowest bacterial count was observed on bread samples from vendor bs23 (3.16 x 104cfu/g) (table 3). the bacteria count of bread samples from vendors bs21 and bs24 were not significantly different (p>0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 347 3.3.2 coliforms the coliform counts of bread samples from roadside vendors were significantly different (p>0.05). the highest count was recorded by bread samples from vendors bs30 (8.31 x 102cfu/g) and the lowest coliform count was recorded by bread samples from vendors bs23 (8.44 x101cfu/g) (table 3). 3.3.3 yeast and molds the yeast and molds count were significantly different (p>0.05). the highest yeast and molds count was observed on bread samples from vendor bs29 (2.67 x 106cfu/g) while the lowest count was recorded by bread samples from vendor bs25 (3.16 x103cfu/g) as showed in table 3. 3.4 total colony count of microorganisms on different categories of bread comparing the general total count of microorganisms in the different categories, it was observed that the bacteria count in the standard bakery category was significantly different (p>0.05) from those of local bakeries and roadside vendors (table 4).the lowest bacteria count of 9.36 x103cfu/g was recorded by standard bakery while the highest bacteria count was recorded by roadside vendors (8.02 x 104cfu/g). coliform counts were significantly different (p>0.05). standard bakeries had the coliform count (8.83 x 101cfu/g) while roadside vendors recorded the highest coliform count (3.64 x 102cfu/g). table 3 total colony count of microorganisms on bread from roadside vendors roadside vendor bacteria (maximum limit: 105 cfu/g) coliforms (maximum limit: 102 cfu/g) yeast and molds (maximum limit: 104 cfu/g) bs21 1.73 x 105 ± 1.01e 4.33 x 102 ± 1.04c 5.79 x 104 ± 1.03d bs22 2.57 x 105 ± 1.06g 4.43 x 102 ± 1.04c 2.34 x 105 ± 1.00h bs23 3.16 x 104 ± 1.08a 8.44 x 101 ± 1.26a 6.21 x 103 ± 1.03b bs24 1.81 x 105 ± 1.00e 3.28 x 102 ± 1.04b 1.41 x 105 ± 1.02f bs25 3.23 x 104 ± 1.07a 3.38 x 102 ± 1.07b 3.16 x 103 ± 1.08a bs26 1.20 x 105 ± 1.01d 3.18 x 102 ± 1.08b 1.41 x 105 ± 1.02f bs27 4.07 x 104 ± 1.05b 3.68 x 102 ± 1.04bc 6.86 x 104 ± 1.02e bs28 2.18 x 105 ± 1.01f 3.54 x 102 ± 1.02bc 1.53 x 105 ± 1.01g bs29 5.88 x 104 ± 1.03c 6.65 x 102 ± 1.33d 2.67 x 106 ± 1.07i bs30 2.04 x 105 ± 1.01f 8.31 x 102 ± 1.04e 4.97 x 104 ± 1.04c means along the same column with different superscripts are significantly different (p < 0.05). table 4 general total colony count of microorganisms on bread from the different categories of bread distributors category bacteria (maximum limit: 105 cfu/g) coliforms (maximum limit 102 cfu/g) yeast and molds (maximum limit 104 cfu/g) standard bakeries 9.36 x 103 ± 2.44a 8.83 x 101 ± 3.41a 3.03 x 103 ± 4.79a local bakeries 1.35 x 104 ± 1.33b 2.01 x 102 ± 3.75b 3.83 x 103 ± 3.89a roadside vendors 8.02 x 104 ± 2.51b 3.64 x 102 ± 1.80c 7.71 x 104 ± 6.56b means along the same column with different superscripts are significantly different (p < 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 348 yeast and molds counts for standard and local bakeries were significantly different (p>0.05) from that of roadside vendors. the lowest yeast and molds count was recorded by standard bakery (3.03 x 103cfu/g) while the highest was recorded by roadside vendors (7.71 x 104cfu/g). 3.5 ailment and poisoning cases among bread’s sellers closest to pesticide shops the breads sellers presented varied ailments including headaches, weakness, skin irritation, eye irritation, nose irritation, throat irritation and difficulty in breathing (table 5). the range of symptoms presented by the sellers was 1-6 per seller; so one seller may present only one ailment and another up to six ailments. eye and nose irritations are the most frequent ailments: 53% and 66% respectively while difficulty in breathing is the least. many cases of poisoning have been reported by breads sellers themselves during the investigation. these cases were experienced by themselves or by a colleague. these include skin irritation, death and others, as illustrated in table 4. out of the sampled population, only 37.5% of sellers have actually witnessed cases of poisoning in terms of skin burns, death, food poisoning, collapse, and respiratory problems among themselves or among neighboring pesticides sellers. death was the most prevalent case as a result of suicide or through accidental inhalation. table 5 ailments among bread sellers working nearest pesticides shops ailments number of sellers (n) number of sellers with ailment (%) number of poisoning found (%) headache 16 7 (28.1) weakness 16 4(21.8) skin irritation 16 5(28.1) eye irritation 16 9(53.1) nose irritation 16 11(65.6) throat irritation 16 6(37.5) difficulty in breathing 16 1(6.3) skin burn 16 9(12.5) death 16 7(18.8) others 16 9(6.2) 3.6 discussion viable bacteria, coliforms and fungi (yeast and molds) were detected in all the samples. the high level of total viable bacterial count observed on bread samples from roadside vendors could be associated to poor hygienic standards often characterizing roadside vendors and poor storage conditions. in a related research conducted by isong et al. in abak local government area in nigeria, readyto-eat breads from hawkers were evaluated for similar microbiological quality [13]. their report was in line with that of bryan et al. who concluded that the general state of inadequate hygiene and sanitation could account for organisms in ready-to-eat food [14]. although the bacteria count observed in bread samples from roadside vendors was high, it did not exceed the standard recommended limits of bacterial contamination for readytoeat foods set by the international commission on microbiological specification for food food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 349 which is x 105cfu/g of food for total bacteria plate count [15]. thus all the bread samples in this study could be termed safe because the bacterial count for all the samples were less than x105cfu/g [16]. according to hocking, depending on the product, a high standard plate count indicates that the product might have been prepared under poor hygienic conditions or stored inappropriately [6]. thus when assessing foods for standard plate count, the processing and / or ingredients present in the foods need to be considered. though the results did not reveal the different types of bacteria associated with the bread samples, it was clear that the bacteria colony count was a mixed flora since the counts were less than 105cfu/g. any count above this level implies there is usually a predominant organism [17]. the coliform counts were significantly different (p<0.05) for the bread samples from standard bakeries, local bakeries, and roadside vendors in the bamenda city area. found values of coliform counts fall within the range of international microbiological standard units of coliforms in foods (102 cfu/g). in a related study conducted in nigeria, a similar coliform count was recorded in the cooling step in bakery production [18]. majority of food contamination involving coliforms are due to improper handling arising from contact with handlers, their feces or objects contaminated with feces which is a very common occurrence amongst local baked food handlers and retailers [19, 20]. coliform bacteria will not likely cause an illness. however, their presence indicates that disease-causing organisms (pathogens) could be in the system [21]. thus, it could be concluded that the presence of the coliform bacteria on bread in the bamenda city area is a consequence of contamination which occurred after the baking process because coliform germ being asporulated and acapsulated, they do not resist high temperatures and are entirely destroyed in the baking process [22]. since coliform presence on food sample is a serious quality concern as most of the bread sold in bamenda city is consumed unsterilized, producers need to handle their products under suitable hygienic conditions to avoid high levels of microbial contamination and possible health risks. the analysis revealed that the yeast and molds counts were significantly different (p<0.05) for the bread samples from standard bakeries, local bakeries, and roadside vendors in bamenda city area. the yeast and molds count on bread from roadside vendors was higher than that on bread from local bakeries and standard bakeries). this is on line with the work done by isong et al. in a similar research conducted in nigeria [13]. the acceptable limit of yeast and molds in ready-toeat food is <104cfu/g [23]. thus bread samples from standard and local bakeries had yeast and molds count that were within the acceptable limits in ready-to-eat food. however, the yeast and molds count in bread sample from roadside vendors (hawkers) was higher and out of the acceptable limit of yeast and molds for ready-to-eat food. such high levels of yeast and molds on bread should be worrying because several food borne molds and possibly yeast may be a potential hazard to human or animal health because of their ability to produce toxic metabolites known as mycotoxins. certain food borne molds and yeast may also be a hazard because of their ability to elicit allergic reactions and even cause infections. although most food borne fungi are not infectious, some species can cause infections, especially to vulnerable population groups like the aged, individuals receiving chemotherapy or antibiotic treatment [24]. the pesticides sold are mostly liquid. according to their properties they are colorless or colored (yellow). globally, they have a strong odor, are flammable and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 350 volatile. consequently they are able to affect respiratory, cardiac, central nervous, liver, kidney, and reproductive systems. their main routes of entering the body are through inhalation, ingestion, skin penetration or eyes irritation. the effects on the respiratory system are very obvious since most of them are fumigants [25]. the symptoms evaluated in this study were only mild symptoms, as mentioned by beti, which may make a worker feel uncomfortable: eyes can water and get red, and itchy; skin can get red burns and itchy; dizziness, faintness, blurry vision, vomiting; coughing , fainting ,very bad headaches ; wheezing or trouble ; drooling; small pupils of the eyes[26]. all of these symptoms where found amongst sellers interviewed. subsequently, they witness the effect of those chemicals on their health. severe symptoms which may be cold, flu, or heat exhaustion where not evaluated. however, very severe pesticides poisonings cases leading to death were testified by respondents. these accidents generally happened when opening containers, handling or mixing chemicals and when gases leak from containers [27]. increasing distances, prevention and prompt cleaning of spills and suitable emergency procedures which are important control measures were poorly respected. henry’s law constant, vapor pressure, and volatilization are all properties responsible for the movement of chemicals from the surface into the atmosphere. at room temperature, pvp values can range from 10-s to 300 mm of hg (mercury). consequently, symptoms observed reflect characteristics of some of those products with high vapor pressure such as glyphosate which releases carbon monoxide, toxic gas, after combustion [28]. 4. conclusions the aim of this study was to evaluate the prevalence of microbes on bread produced and marketed in the bamenda city area of the north west region of cameroon. the results revealed the prevalence of bacteria, coliform, and yeast and molds on bread samples from standard bakeries, local bakeries and roadside vendors. thus, regular checks by sanitary inspectors are encouraged to check the microbial quality of most of the bread in the bamenda city area. the ailments presented by bread sellers whose selling points are nearest to pesticide shops are nose irritation, throat irritation, headaches, weakness, skin irritation, eye irritation, and difficulty caused by pesticide breathing. 5. acknowledgments the authors are grateful to the administration of irad bambui centre for facilitating the work in the microbiology unit of the food technology and post-harvest laboratory 6.references [1]. saranraj, p., and geetha, m., microbial spoilage of bakery products and its control by preservatives.international journal of pharmaceutical & biological archives, 3 (1): 38-48, (2012). [2]. osuji, c. m., importance and use of additives in breadmaking. a paper presented at a training workshop on the use of cassava/wheat composite flour and non-bromate additives for making bread and other confectionaries. held at michael okpara university of agriculture, umudike, (2006). [3]. oluwajoba, s. o., malomo, o., ogunmoyela, o. a. b., dudu, o. e. o., and odeyemi, a.,. microbiological and nutritional quality of warankashi enriched bread. journal of microbiology, biotechnology and food science, 2(1): 42-68, (2012) [4]. prescott, l. m., harley, s. p., and klein, d. a., microbiology, 4th edition. mcgraw hill, inc. new york, 907-910, (1999) [5]. burkepile, d. e., parker, j. d., woodsen, c. b., mills, h. j., kubanek, j., sobecky, p. a., and hay, m. e., chemically mediated completion between microbes and animals: microbes as consumers in food webs. ecology, 87:2821-2823, (2006). [6]. hocking, a. d., foodborne microorganisms of public health significance (6th edition: waterloo: australian institute of food science and technology, (2003). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii , issue 3 – 2018 jean sonchieu, john fru nsoh, caroline nain waingeh, pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon, food and environment safety, volume xvii, issue 3 – 218 , pag. 341 – 351 351 [7]. doyle, m. e. microbial food spoilage-losses and control strategies. fri brifings, 1-18, (2007). [8]. frias, i., alvarez, r., and sierra, a., aspectosbromatologicos y toxicologicos de los conservantes benzoic y sorbicoalimentaria, 6: 109114, (1996). [9]. perraton, g., food and technology.book 2.jacaranda, melbourne, australia, 356p, (2010). [10]. lasslett, s., and collins, c., food for vce unit 3 & 4. cambridge, melbourne, 2001., (2010). [11]. allan g.g., carroll j.p., controlled release delivery of agrochemicals : looking back and looking forward. in: research and development of controlled release formulations of pesticides volume i. proceedings of a seminar organized by the joint fao/iaea division of nuclear techniques in food and agriculture and held in vienna, 6-9 september 1993. iaea, vienna, 1994; iaea-tecdoc-768, issn 1011-4289 printed by the iaea in austria. 1322, (1994) [12]. apha, a compadium of methods for microbiological examination of foods. american public health association, washington d. c, usa, 4th edition (2001), (2001). [13]. isong, n. b., akpan, m. m., udota, h. i., and barber, l., antimicrobial and microbial assessment of breads in abak local government area, nigeria, west africa. journal of microbiology and biotechnology research, 3(3): 155-159, (2013). [14]. bryan, f. l., microbiological quality of bread in nigeria.journal of food protection, 59 (3):287-294, (1988). [15]. international commission on microbiological specification of food (icmsf), microorganism in food 7 microbiological testing in food safety management. new york: academy publisher, 70-80. (1998). [16]. gilbert, r. j., guidelines for the microbiological quality of some ready –to-eat food samples at the point of sale. communicable disease and public health, 3: 163-167, (2000). [17]. health protection agency (hpa), guidelines for assessing the microbiological safety of ready-toeat foods: london: health protection agency, (2009). [18]. daniyan, s. f., and nwokwu, o. e., enumration of microorganisms associated with the different stages of bread production in futamin bakery, nigeria. international research journal of pharmacy, 2(7): 88-91, (2011). [19]. hobbs, b. c., and robert, d., food poisoning and food hygiene.6th edition. central public health laboratory, london. 160-170, (1998). [20]. rowe, m. t., predictive microbiology: uses for assessing quality and safety of dairy products. journal of industrial microbiology, 12: 330 – 336, (1993). [21]. washington state department of health (wsdh) coliform bacteria in drinking water and food-stuff, (2015). www.doh.wa.gov. (accessed: 15 / 04 / 2017). [22]. viosencu, d., and misca, c., the microbiological parameters in technological process of bread production. agroalimentary process and technology, 11(2): 475 – 480, (2005). [23]. heath, h., mckenzie, h., and tully, l. food solution, food and technology. unit 3and 4.2nd edition.pearson , melbourne 2010, (2008). [24]. philip, b., mislivec, c., and stack m. e. enumeration of yeast and molds, and preparation of toxins. (2000). www.fo.org /docreplx5636e/htm. [25]. national park service (nps), physical properties and health effects of pesticides used on national park service collections. nps museum management program, 1849 c street nw (nc 230), washington, dc 20240; (202) 343-8142, p. 24, 2001,url: http://www.cr.nps.gov/museum/publications/conserve ogram/ (august, 2017). [26]. beti t., pesticides safety handbook -curriculum development and reproduction made possible by grants from the niehs (p01es09601) and us epa (r826886), (2002). [27]. who (world health organization), children's health and environment: a review of european environment agency and the who regional, (2010). [28]. clark d. j., physico-chemical properties and environmental fate of pesticides environmental protection agency department of pesticides regulation environmental monitoring and pest management brahch 1020 n street, sacramento, california 95814, eh 94-03 office for europe environmental issue. report no 29 luxembourg: office for official publications of the european communities, 223p, (1994). http://www.doh.wa.gov/ http://www.fo.org/ http://www.cr.nps.gov/museum/publications/conserveogram/ http://www.cr.nps.gov/museum/publications/conserveogram/ pesticide exposure of bread sellers and microbial safety of bread sold in bamenda, cameroon the aim of this study was to assess the microbiological load of bread produced and distributed in bamenda municipality, and to estimate, according to the proximity of selling site to pesticides shops, the exposure of sellers to pesticides. 2.2 preparation of sample 2.3 microbiological analysis 2.3.1 preparation and plating of samples 2.3.3 enumeration of total viable bacteria 2.3.4 enumeration of coliforms 2.3.5 enumeration of yeasts and molds 3. results and discussion 3.1. microbial counts on bread samples from standard bakeries 3.1.1 total colony count of bacteria the minimum total colony count of bacteria (table 1) was 3.09 x 103 cfu/g of bread while the maximum was 3.46 x 104 cfu/g of bread. the total colony count of bacteria in the bread from standard bakeries were not significantly different 3.2 microbial counts on bread samples from local bakeries 3.2.1 total colony count of bacteria 3.2.2 coliforms 3.1.2 coliforms 3.1.3 yeast and molds 3.2.3 yeast and molds 3.3.1 bacteria 3.3.2 coliforms 3.3.3 yeast and molds 3.6 discussion 4. conclusions 6.references issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xx, issue 1 31 march 2021 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) ispublished by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails:fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de josuniversityof galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare universityof suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. klimentohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of foodscience, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 contents: 1. amino-acid content in grain protein of tetraploid opaque-2 maize andrei palii, grigorii batîru 1 7 2. chemical mutagenesis in the currant (ribes l.) and gooseberry (grossularia mill.) selective breeding igor e. buchenkov 8 -13 3. intensification of the heat transfer through corrugated wall donka stoeva 14 22 4. the influence of processing on phytic acid content in some wheat products marcel avramiuc 23 27 5. research concerning physico-chemical and microbiological characteristcs of quinoa, dried milk and oat bran yogurt mihaela adriana tiţa, ovidiu tiţa, adriana dabija, alexandra majdik 28 33 6. ion -sele ctiv e el ect r od es based on ca l ci um h y dr o xyl a pati t e as a t ool fo r ana lysis of v a ri ou s env i ron men ta l ob je cts, fo od a nd ra w ma t er i als volodymyr diichuk, mariia vorobets, igor kobasa 34 37 7. m ulticr it eria ev alua ti on of muni cipal so lid wast e ma nag em en t sc en a ri os : ca se study i a si, r om ani a cristina ghinea, hans th. a. bressers, maria gavrilescu 38 47 8. statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times silviu-gabriel stroe 48 54 9. influence of microwave drying on the content of photosynthetic pigments in spinach ramona mihaela zavada, ana-maria roşu, neculai doru miron, gheorghe surpateanu, ileana denisa nistor 55 59 10. dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova 61 -67 11. aspects concerning the obtainment of cheese whey cream from goat’s milk with onion and dill adriana dabija, răzvan pomparău 68 73 12. veg e table fa ts in flu en ce on th e p hysic oche mica l a nd sens orial p rop e rties o f “t elem ea ” che es e mircea oro ian, a n a l eah u 74 79 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 13. heavy metals in tobacco sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi 80 86 14. total phenolics of fresh and frozen minor berries and their antioxidant properties ana leahu, mircea oroian, sorina ropciuc 87 93 15. polyamine variation in raw ground pork and beef meat octavian baston, octavian barna 94 98 16. author instructions i v 17. subscription information vi microsoft word 01 cuprins 2_2021 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 contents: 1. characterization of pasta from heat moisture treated wheat flour and grape peels mădălina iuga, silvia mironeasa 91 100 2. phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria dimitar dimitrov, tatyana yoncheva, vanyo haygarov, anatoli iliev 101 112 3. study on extraction and purification of trans-resveratrol from grape cane-a review, alina lenuța crăciun, gheorghe gutt 113 127 4. migration of melamine and formaldehyde from kids' bamboo dinner sets svetla petrova petrova , valentina christova – bagdassarian 128 136 5. development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke 137 148 6. the effects of sugar and fat substitution on the textural properties of the pie dough dana huțu, sonia amariei 149 159 7. cleaning the vegetable oil production wastewater with anthracite sergiy boruk, igor winkler, vitaliy mischenchuk 160 – 164 8. effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue 165 171 9. author instructions i v 10. subscription information vi effect of acid type and particle size on the yield and purity of apple (malus domestica ‘fălticeni’) pomace pectin doi: https://doi.org/10.4316/fens.2021.031 282 journal homepage: http://fens.usv.ro/index.php/fens/index journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 282 288 study on alcohol consumption among young people in suceava county, romania mihaela jarcău 1 , * maria poroch – seriţan 1 , matei jarcău 2 1faculty of food engineering, stefan cel mare university, suceava, romania, mariap@fia.usv.ro 2iuliu haţieganu university of medicine and pharmacy, cluj-napoca, romania *corresponding author reeived 17th june 2021, accepted 28th september 2021 abstract: there is expedient evidence showing that differences in adolescent alcohol consumption and other risk-behaviour depend on many factors, but an important role is the habits acquired in the family or group of friends. the current study reveals the type of consumer of the analyzed group. the results showed that the percentage of alcohol abstinence among adolescents was higher than those who are at risk of developing serious medical problems due to alcohol consumption. in order for a person to become alcohol-dependent, there are several factors that must be analyzed: the psychological field, the amount of alcoholic beverages available to consumers, the great variety, social pressure (in our civilizations there are no events that are not lived in around alcoholic beverages holidays, family reunions), genetic predisposition. keywords: alcohol; adolescent; risk-behaviour, age, drugs, covid-19 1. introduction the period traversed by all mankind, starting with 2020, due to the pandemic caused by the covid-19 virus, is one that involves major transformations in terms of habits and behaviours related to a healthy life. the online posting of many activities has led to forced isolation, with major implications for the development of a young person [1]. among the substances that are used very often, in the erroneous attempt to overcome a difficult situation, alcohol is the most used. alcoholism is considered as the third public health problem by the world health organization. research on alcoholism is increasingly focused on the study of alcohol behaviour, predisposing factors, alcohol dynamics, the body's reaction to alcohol intoxication, often requiring multi disciplinary teams. the consequences of alcohol consumption are both from the point of view of the person directly involved and from the harmful consequences borne by people who are part of alcoholics' close relatives. the somatic diseases that this pathology involves, the frequency of the severe forms that patients face has major implications in society. in romania, according to data from the 1994 survey, alcohol consumption was recognized by 56.2% of people aged 15 and over interviewed. the frequency of alcohol consumption is higher in males (74.2%) than in females (39.7%). the modal age group, as with smoking, is 25-44 years old, with 66.3% (84.1% in men and 49.4% in women), followed by 45 64 years and the elderly (65 years and over). regarding the type of consumption, the daily consumption was declared by 3.7% of http://fens.usv.ro/index.php/fens/index mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 283 the interviewees, the occasional consumption of 36.9% and once or 2 3 times / week, of 15.6%. according to the alliance for the fight against alcoholism and drug addiction, romanians are currently among the largest alcohol consumers in europe, with an average consumption of more than 18.5 liters per person per year, compared to nearly 15 liters per person in the european union, and there are approximately two million people who consume excessive alcohol on a national level. 2. materials and methods the study group consists of young pupils and students, volunteers in this research. data processing was performed based on a questionnaire applied to the group, using the methodology of descriptive statistics for quantitative characteristics, tables and graphical illustrations. young people are a population group that is of interest due to their role in society, as well as the longterm impact of acquired behaviours. 3. results and discussion alcohol consumption was recognized by 78.8% of the people examined (fig. 1). fig. 1. the structure of the population group studied according to the “age” characteristic depending on the domicile, 59.3% of young people from urban areas participated, 40.7% from rural areas, of which 69.7% were female, 29% male, 1.4% did not want to declare membership. depending on the level of education, 53.1% were students, 44.1% students, 2.8% master students. alcoholism is a complex situation both psychologically, socially and biologically of the somatic and mental alterations that result from this intoxication. the approach to alcoholism from a medical perspective began in 1849 with magnus huss [2 5]. the following information was provided in the questionnaire regarding the measures of the beverages, namely: 1 drink = 12 g pure alcohol = 1 beer (330 ml) = 1 glass of wine (125 ml) = 1 shot of spirits (40 ml) depending on the type of consumption, 27.6% stated that they had never consumed, 47.6 per month or less, 18.6% 2 4 times per month, 2.8% 2 3 times per week, and 3.4% 4 or more times a week (table 1 and fig. 2). table 1. type of alcohol consumption alcohol consumption type number people % consume never 400 28.1 monthly or less frequently 690 47.3 2 4 times a month 270 18.5 2-3 times a week 40 2.7 4 or more times a week 50 3.4 total 1450 100.0 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 284 fig. 2. type of alcohol consumption (% of alcohol users) regarding the number of drinks in an ordinary day, it can be seen from fig. 3 that 84.9% consume one or two drinks, but there are also 2.1% who consume ten or more. fig. 3. a type of alcohol consumption (number of drinks) it is found that the frequency of consumption of at least 6 alcoholic beverages on a single occasion, 74.0% answered "never", 21.8% answered "monthly or less often", "monthly" 2.1% and "weekly" the same percentage, 2.1%, none claimed to consume "daily or almost daily" (see fig. 4). fig. 4. type of alcohol consumption (frequency with which at least 6 alcoholic beverages are consumed on a single occasion) analyzing the answers regarding the implications of alcohol consumption, as can be seen in table 2, those who are answering "never" are over 70%, to all aspects pursued. table 2. implications of alcohol consumption in daily activities no a) b) c) d) e) question % consume % consume % consume % consume % consume 4. 95.2 3.4 1.4 0 0 5. 97.9 1.4 0.7 0 0 6. 95.2 3.4 0.7 0 0.7 7. 88.3 11 0.7 0 0 8. 90.3 8.3 0.7 0.7 0 the legend: 4. how often have you noticed in the last year that you can't stop drinking once you start? 5. how often in the last year have you failed to do what you were expected to do because of drinking? 6. how often in the last year did you need to drink your first drink in the morning to recover from a heavy drunkenness? 7. how often in the last year have you had a feeling of remorse or guilt after drinking? 8. how often in the last year have you been unable to remember what happened last night because you drank? a) never 0 b) monthly or less frequently 1 c) monthly 2 d) weekly 3 e) daily or almost daily 4. the consumption of alcohol is one of the main risk behaviours among adolescents [6 7]. alcohol can be a part of the adolescents maturing process and also the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 285 start to consumption of other substances [8 9]. alcohol consumption makes adolescents vulnerable by developing maladaptive behaviour, being also causes of violence, accidents, crime, emotional instability, depression, social exclusion and suicide [10]. despite obvious risks and adverse outcomes, alcohol consumption is still increasing among adolescents in some european countries [11, 12]. to the question “you or someone else was injured as a result of drinking?”, 97.9% answered “no”, 1.4% with “yes, but not in the last year”, and 0.7% “yes, but in the last year". fig. 5. the impact of alcohol consumption (if someone has been injured due to alcohol consumption) to the question “was a relative, friend, doctor, or other person worried about your drinking or advised you to reduce it?” 94.5% answered “no”, 3.4% with “yes, but not in the last year”, and 2.1% “yes, but in the last year". fig. 6. the impact of alcohol consumption on those around the consumer analyzing the answers of those who said that people around them are worried about alcohol consumption, 55.6% are men and 44.4 are women. of these men respondents, 40% have a declared consumption of 4 or more times a week, 60% a declared consumption of 2 4 times a month, while of the total number of respondent women who bring concern to those close to them due to their alcohol consumption, all people reported monthly or less frequent consumption. regardless of the speed of evolution, the addicted subject will present disorders immediately after the cessation of alcohol consumption, disorders that attenuate if consumption is resumed, thus creating a vicious circle of addiction [13]. this stage is not yet the stage of true pathological complications, but the stage of addictive alcoholism that will cause disruptions in family, professional and social life in general [14]. alcoholism complications include: neurological, visceral liver complications are the most common, with the risk occurring at a consumption of 80g of pure alcohol per day, and cirrhosis occurring in 15 years if this consumption increases and exceeds 160g of pure alcohol per day. gastritis, duodenitis, pancreatitis, ethyl myocarditis, and metabolic disorders are also common. alcoholism in the mother has an effect on the fetus (high risk of dwarfism, mental weakness, microcephaly, abnormalities). alcoholism is a risk factor for five cancer sites: the oral cavity, the pharynx, the larynx, the esophagus, and the liver [15, 16, 17, 18], with smoking increasing the risk. alcohol consumption has both medical and social consequences. reduced productivity due to absenteeism, accidents, poor work performance, and increased costs for health, social welfare, and criminal justice systems, deteriorating health, increasing mortality, particularly by increasing the frequency of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 286 premature deaths (8 10% of all deaths recorded between the ages of 16 and 74 are attributable to alcoholism), the burden of health services (6 20% of hospitalizations), health problems include: hypertension, cardiovascular disease, diabetes, cancer [19, 20, 11], cirrhosis, mental illness, addiction. alcoholism is also a major risk factor for accidents, regardless of the type of accident; it is also involved in many public policy issues (crime, violence) and is the leading cause of dissent, domestic violence, and child abuse. fig. 7. distribution of scores obtained table 4. interpretation of questionnaire results interpretation of questionnaire results 0 7 consumption that is safe for health. it is recommended to inform about the health effects of alcohol consumption. 8 15 risky consumption: although alcohol consumption may not be a problem at present, the amount and frequency of consumption put the consumer at risk in the future. it is recommended to consult a doctor. 16 19 harmful consumption: alcohol consumption is currently associated with negative effects on health. it is recommended to consult a doctor. ˃ 20 extremely harmful consumption: alcohol consumption is currently associated with serious negative effects on health. urgent consultation with a doctor is recommended. 4. conclusion from the interpretation of the results we can state that: 94.7% have a score between 0 and 7, which corresponds to consumption that is not dangerous for health. it is recommended to inform about the health effects of alcohol consumption. 4.9% have a score between 8 and 15, which corresponds to a risky consumption: although alcohol consumption may not food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 287 cause problems at present, the amount and frequency of consumption put the consumer at risk in the future. it is recommended to consult a doctor. 0.4% have a score between 16 and 19, which corresponds to harmful consumption: alcohol consumption is currently associated with negative effects on health. it is recommended to consult a doctor. no person is recorded with a corresponding consumption ˃ 20 extremely harmful consumption: alcohol consumption is currently associated with serious negative effects on health. urgent consultation with a doctor is recommended. in a future paper we aim to analyze the dependence of alcohol consumption in young people on the structure of the family, and alcohol consumption in the family. 5. references [1] usher k., durkin j., bhullar n., international journal of mental health nursing (2020) 29, 315–318 editorial the covid-19 pandemic and mental health impacts [2] fitzpatrick m. (2004), risk factors. lancet.; 363(9407): 500. [3] okoro c.a, brewer r.d, naimi t.s, moriarty d.g, giles w.h, mokdad a.h. (2004), binge drinking and health-related quality of life; do popular perceptions match reality? am j prev med.; 26(3): 230-233. [4] rehm j, room r, graham k, monteiro m, gmel g, sempos c.t. (2003), the relationship of average volume of alcohol consumption and patterns of drinking to burden of disease: an overview. addiction.; 98(9): 1209-1228. [5] stein cj, colditz ga. (2004), modifiable risk factors for cancer. br j cancer.; 90(2): 299-303. [6] brener, n.d.; grunbaum, j.a.; kann, l.; mcmanus, t.; ross, j. (2004), assessing health risk behaviors among adolescents: the effect of question wording and appeals for honesty. j. adolesc. health, 35, 91–100. [7] miller, j.w.; naimi, t.s.; brewer, r.d.; jones, s.e. (2007), binge drinking and associated health risk behaviors among high school students. pediatrics; 119, 76–85. [8] demoss, b.c., (1992), gateway drugs. am. fam. phys., 46, 666–668, 673. [9] joyce, c.; kemp, w. (1972), from soft to hard drugs—progression, regression or digression. in drug abuse current concepts and research; charles c. thomas publishing ltd.: springfield, il, usa; pp. 243–250. [10] kaess, m.; brunner, r.; parzer, p.; carli, v.; apter, a.; balazs, j.a.; bobes, j.; coman, h.g.; cosman, d.; cotter, p. (2013), risk-behaviour screening for identifying adolescents with mental health problems in europe. eur. child adolesc. psychiatry, 23, 611–620. [11] bellis, m.a.; morleo, m.; hughes, k.; downing, j.; wood, s.; smallthwaite, l.; cook, p.a. (2010), a cross-sectional survey of compliance with national guidance for alcohol consumption by children: measuring risk factors, protective factors and social norms for excessive and unsupervised drinking. bmc public health, 10, doi:10.1186/1471-2458-10-547. [12] hibell, b.; guttormsson, u.; ahlström, s.; balakireva, o.; bjarnason, t.; kokkevi, a.; kraus, l. (2009), the 2007 espad report. substance use among students in 35 countries; the swedish council for information on alcohol and other drug: stockholm, sweden. [13] nagy k, barabas k, nyari t. (2004), attitudes of hungarian healthcare professional students to tobacco and alcohol. eur j dent educ.; suppl 4: 32-35. [14] centers for disease control and prevention (cdc). alcohol use among adolescents and adultsnew hampshire, 1991-2003. mmwr morb mortal wkly rep. 2004; 53(8): 174-175. [15] aronson k., (2003), alcohol: a recently identified risk factor for breast cancer. cmaj., 168 (9): 1147-1148. [16] brennan p, lewis s, hashibe m, bell d.a, boffetta p, bouchardy c, caporaso n, chen c, coutelle c, diehl s.r, hayes r.b, olshan a.f, schwartz s.m, sturgis e.m, wei q, zavras a.i, benhamou s. (2004), pooled analysis of alcohol dehydrogenase genotypes and head and neck cancer: ahuge review. am j epidemiol; 159(1): 1-16. [17] burger m, mensink g, bronstrup a, thierfelder w, pietrzik k. (2004), alcohol consumption and its relation to cardiovascular risk factors in germany. eur j clin. nutr.; 58(4): 605614. [18] etique n, chardard d, chesnela, merlin jl, flament s, grillier-vuissoz i. (2004), ethanol stimulates proliferation, eralpha and aromatase expression in mcf-7 human breast cancer cells. int j mol med.; 13(1): 149-155. https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=durkin%2c+joanne https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=bhullar%2c+navjot food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 mihaela jarcău, maria poroch – seriţan, matei jarcău, study on alcohol consumption among young people in suceava county, romania, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 282 – 288 288 [19] castellsague x, quintana m.j, martinez m.c, nieto a, sanchez m.j, juan a, monner a, carrera m, agudo a, quer m, munoz n, herrero r, franceschi s, bosch f.x. (2004), the role of type of tobacco and type of alcoholic beverage in oral carcinogenesis. int j cancer; 108(5): 741-749. [20] chung w. (2004), type of alcoholic beverage and high-risk drinking: how risky is beer drinking in korea? krousel-wood ma, muntner p, he j, whelton pk. primary prevention of essential hypertension. med clin north am.; 88(1): 22338. [21] maekawa s.j, aoyama n, shirasaka d, kuroda k, tamura t, kuroda y, kasuga m. (2004), excessive alcohol intake enhances the development of synchronous cancerous lesion in colorectal cancer patients. int j colorectal dis.; 19(2): 171-175. 1. introduction 237 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 3 2020, pag. 237 259 physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review * vasile-florin ursachi 1 , gheorghe gutt 1 1faculty of food engineering, ”ștefan cel mare” university, suceava, romania, florin.ursachi@fia.usv.ro *corresponding author received 27th august 2020, accepted 26st september 2020 abstract: in the last decade, environmental protection is one of the major challenges. it is necessary to ensure the protection of the environment and the conservation of natural resources, in accordance with the requirements of a sustainable economic and social development. the most important impact of modern human activities is the release of large amounts of different compounds after fossil fuels burning; these compounds are responsible for increasing of greenhouse gases (ghg) concentrations in the atmosphere. the depletion of fossil fuels and necessity to increase energy reserves, especially for the propulsion of transport, contributed to search and use of alternative fuels. partially or completely substitution of gasoline with bioethanol is an alternative method to reduce ghg emissions. currently, biofuels (first generation) are produced from sources used to feed the population. the competition food vs. biofuel could be solved if biofuels were obtained from renewable resources such as lignocellulosic biomass (lcb). second-generation biofuels are obtained from raw materials such as agricultural residues (straws, sugarcane bagasse, corn stalks and cobs) and forestry residues (sawdust, bark, branches, etc.) which do not interfere with global food production. in 2019, the main producers of bioethanol were usa, brazil and eu which produced about 54%, 30% and 5% respectively of the worldwide bioethanol. this paper reviews one of the most important steps of bioethanol production which is the pretreatment of lcb. numerous pretreatments are available, as follows: physical, chemical, physico-chemical, biological and combined pretreatments. the combined pretreatments were found to be more effective when compared to single pretreatments, and there is a wide range of combinations that can be applied in the future. keywords: cellulose, hemicellulose, lignin, pretreatment, bioethanol 1. introduction the term biofuel often refers to liquid or gaseous fuels that are used in the transport sector and are obtained mainly from biomass. the main characteristics of biofuels are related to sustainability, reduction of greenhouse gas emissions, development of economic, social and agricultural sector, and food security [1]. in the last century, increasing of global energy consumption has implicitly led to increasing in co2, so2 and nox emissions due to the burning of fossil fuels which is the main cause of air pollution [2]. the reduction of fossil fuel deposits, but also their negative effects on the environment led to the exploration of alternative energy resources which are environmentally friendly [3,4]. regarding the sources of bioenergy, lignocellulosic biomass (lcb) is an important raw material that can be used for biofuels production and also for extraction of high value compounds [5]. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 238 annually, large amounts of lcb are generated, and these include forest, agricultural and agro-industrial residues that can be capitalized in bioethanol production [6]. the most important polymers present in lcb are cellulose (32% – 51%), hemicellulose (19% – 35%) and lignin (10% – 30%) [7,8]. compared to agricultural biomass (ab), the physical properties but also the chemical composition of wood biomass (wb) are different. wb has a more pronounced recalcitrance than microbial and enzymatic actions when compared to ab [9]. currently, critical concerns are focused on the sustainability of bioethanol production, as it is obtained mostly from cereal crops that contain starch and sugar. for this reason, the irrational use of these crops can create competition between food and biofuels [10,11]. the main advantage of lignocellulosic materials (cml) are that they are renewable sources that do not compete with food for human consumption [12-14], thus using these resources may avoid food security issues [6]. other advantages include the extraction of high value substances [15] and the relatively low cost of processing, which is cheaper than that of crude oil [8,16]. however, lcb also has an important disadvantage that refers to its complex structure, which is resistant to chemical and enzymatic degradation [17]. therefore, in order to modify the physicochemical properties of the lignocellulosic matrix, various pretreatment methods must be applied to the lcb; these pretreatments are considered to be expensive [18-20]. the aim of this review was to identify the physical and chemical methods of pretreatment of lcb and establish which of these pretreatment methods can disrupt the complex structure of lcms and remove lignin most efficiently. the pretreatment process conditions must to be given special attention because at this step the selection of the best choice can lead to a significant increase in the yield of fermentable sugars and also reduce the formation and release of toxic compounds. 2. overview of sources and bioethanol production 2.1. clasification of biofuels biofuels are classified into two broad groups: primary and secondary. primary biofuels are used in crude form for heating, cooking or electricity production. secondary biofuels are products resulting from biomass processing and can be used for transport or various industrial processes. depending on the raw material and the technology used for the production of secondary biofuels, they divide into three subgroups: first generation, second generation and third generation (figure 1) [21-24]. 2.2. global ethanol production renewable fuels association (rfa) argues that the largest worldwide producer of ethanol is the us (corn), followed by brazil (sugar cane). in 2018, the united states and brazil produced about 16.1 billion gallons and 7.95 billion gallons, respectively (28%). this means that these two countries produced aproximatively 84% of global ethanol production [25]. figure 2 shows the global ethanol production from 2007 to 2018. 2.3. structure of lignocellulosic biomass cellulose is a linear polymer composed of d-glucose units linked by β-1,4 glycosidic bonds. the hydroxyl groups of each glucose unit form intraand intermolecular hydrogen bonds and give the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 239 cellulose chain a rigid and partially crystalline structure. this crystallinity indicates that the structure of cellulose is more orderly, but limits the action of enzymes during saccharification [27-28]. by removing water from each molecule of glucose, long chains of cellulose that contain 5000 10000 units of glucose are formed. fig 1. clasification of biofuels [21-24] fig 2. global ethanol production [26] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 240 the basic unit of cellulose consists of two units of anhydrous glucose, also called cellobiose units [29]. hemicellulose is a carbohydrate that contains different types of sugars: with 5 carbon atoms (β-d-xylose, α-l-arabinose and rhamnose) and with 6 carbon atoms (β-d-glucose, β-d-mannose and α-dgalactose [30]. lignin is the second most abundant biopolymer of lcb, after cellulose. in combination with hemicellulose it is distributed around the cellulose fibers in both the primary and secondary cell walls. lignin has three basic monomers: pcoumaryl alcohol, coniferyl alcohol and sinapyl alcohol [31]. figure 3 shows the structure of lcb. the chemical composition of lcms used for production of second-generation bioethanol is shown in table 1. . fig 3. structure of lcb [32] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 241 table 1 the chemical composition of lcms biomass cellulose (%) hemicellulose (%) lignin (%) extractable substances (%) ash (%) references agricultural biomass/residues corn stalks 38.0 26.0 18.5 6.0 5.1 [33] corn cobs 36.75 ± 0.54 29.98 ± 3.60 23.13 ± 3.40 6.76 ± 1.52 0.95 ± 0.03 [34] wheat straws 43.1 27.7 17.5 5.5 5.3 [35] rice straws 35.63% 18.06% 31.97% n.a 10.24% [36] barley straws 33.25 20.36 17.13 5.64 2.18 [37] rye straws 35.8 14.5 3.5 n.a n.a [38] triticale straws 33 23 29 n.a 3 [39] oat straws 37.60 23.34 12.85 7.11 2.19 [37] sorghum straws 35.87 26.04 7.52 n.a n.a [40] sugarcane bagasse 45.5 ± 1.1 27.0 ± 0.8 21.1 ± 0.9 2.2 ± 0.1 4.6 ± 0.3 [41] rapeseed 37.0 19.6 18.0 19.7 5.7% [42] canola straws 42.39 16.41 14.15 7.56 2.10 [37] cotton stalks 31.1 10.7 27.9 9.0 6.0 [43] hemp 74 18 13 n.a n.a [44] hemp fiber 56.1 58.7 10.9 14.2 6 4.3 [45] kenaf 31 – 57 21.5 – 23 15 – 19 n.a n.a [46] jute 72 13 13 n.a n.a [44] sisal 73 13 11 n.a n.a grape stalks 16.7 ± 0.2 18.0 ± 0.2 2.6 5.7 19.2 24.2 ± 0.5 22.6 n.a [47] nut shells 25 – 30 25 – 30 30 – 40 n.a n.a [48,49] coconut 33.29 ± 0.09 33.61± 0.07 19.87 ± 0.08 1.27± 0.05 5.5± 0.05 [50] coir 43 <1 45 n.a n.a [44] banana waste 13.2 14.8 14 n.a n.a [51] grasses miscanthus 41.9 20.6 23.4 3.7 3.0 [52] switch grass 34.645 23.5 31.4 12.0-21.0 20.9 [49,53] forestry biomass/ residues hardwoods quercus robur 48–49 18–22 29–34 n.a n.a [54] fagus sylvatica 47–48 18–22 30–35 n.a n.a populus tremula 48–49 21–25 26–31 n.a n.a eucalyptus gigantea 49 23 22 n.a n.a [55,56] alnus rubra 44 30 24 n.a n.a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 242 softwoods picea abies 42 20 27 n.a 8 [39] abies alba 35–37 24–26 37–41 n.a n.a [54] tillia cordata 48–51 20–22 27–32 n.a n.a salix 37.1 17.8 27.0 3.8 1.1 [52] larix occidentalis 48 17 27 n.a. n.a. [55,56] pinus sylvestris 32.5 50 24 39.7 16.3 20 n.a. n.a. [57,58] pseudotsuga menziesii 44.0 11.0 27.0 n.a. n.a. [58] other residues newspaper 0-55 25-40 18-30 5-8 n.a. [48,59,60] waste papers from chemical pulps 60-70 10-20 5-10 n.a. 2 n.a. – not analyzed 3. the impact of pretreatments on lcms the pretreatment step has an important role in the biofuel production process because by pretreating lcb there can be obtained yields of up to 90% as compared to 20% in the case of untreated lcb. [61]. the pretreatment step was introduced to separate lcb into the main constituent biopolymers and to facilitate hidrolysis. the pretreatment step should allow an easy recovery of lignin and other nonfermentable constituents that can be used for the synthesis of other chemical compounds [62,63]. the pretreatment methods used for bioethanol production from lcb are shown in figure 4. 3.1. physical methods for pretreatment of lcb physical pretreatments include processes such as mechanical, pressure, microwave, ultrasonication, pyrolysis, pulsed electric field, etc. 3.1.1. mechanical pretreatment mechanical pretreatments of lcb include chipping, grinding and milling. these methods are used for releasing biomass fragments with small particle size, disruption of cell structure, decreasing the crystallinity of cellulose in biomass, and to facilitate further chemical and biological treatments [64]. for raw materials, a certain pretreatment method is required to minimize substrate degradation and improve carbohydrates yield [61,63]. grinding biomass facilitates the access of enzymes and steam. the energy consumed to reduce the particle size represents approximately 30% of the total energy consumption of the process. the extractable substances can be removed using steam (~160 °c) [61]. mechanical pretreatment of lcms is an important step in the technological process of obtaining biofuels because it contributes to improving bioconversion by reducing cellulose crystallinity, particle size, degree of polymerization [65], particle density and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 243 distribution, while increasing enzymatic accessibility and transformation of lcms. [66]. lcm has a complex composition, thus different types of mills are used to decompose and reduce its crystallinity. the most common types of mills are ball mills, centrifugal mills, colloidal mills, hammer mills, knife mills, pin mills and vibratory mills [67]. milling is used to reduce the crystallinity and size of lcb particles and can result in particles with a size of 0.2 mm [68]. mani et al. [69] used a hammer mill with a screen opening of 0.8 mm, 1.6 mm and 3.2 mm and determined the specific energy consumption for grinding wheat straws, barley straws, corn stover and switchgrass that had a humidity of 8.3 – 12.1 %wb, 6.9 – 12.0 %wb, 6.2 – 12.0 %wb and 8.0 – 12.0 %wb, respectively. the average specific energy consumption for wheat straws, barley straws, corn stover and switchgrass was 11.36±1.02 – 51.55±2.93 (kwh t−1), 13.79±0.18 – 99.49±7.35 (kwh t−1), 6.96±0.75 – 34.30±1.47 and 23.84±0.63 – 62.55±0.63 (kwh t−1), respectively [69]. bitra et al. [70] directly measured the mechanical energy used by the knife mill to reduce the size of switchgrass, wheat straw and corn stover. in the case of the knife mill, for a screen size of 25.4 mm and an optimum speed of 250 rpm, the optimum feed speed obtained was 7.6, 5.8 and 4.5 kg/min, the corresponding total specific energies were 7.57, 10.53, and 8.87 kwh/mg, and the efficient specific energies were 1.27, 1.50 and 0.24 kwh/mg for switchgrass, wheat straw and corn stover, respectively. energy use ratios were determinated and were, as follows: 16.8%, 14.3% and 2.8% for switchgrass, wheat straw and corn stover, respectively [70]. fig. 4. pretreatments applied to lcb [49,68,71]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 244 3.1.2. microwave pretreatment microwave irradiation is considered an alternative method to conventional heating [72] and present interest in different domains. the use of microwave irradiation has some advantages such as reduced process energy requirements, uniform and selective processing and the ability to start and interrupt the process instantly [73]. there are numerous studies that have shown that microwave irradiation could contribute to the disruption of the complex structure of lcms [74] and facilitate their enzymatic hydrolysis [75]. combined pretreatments (microwave irradiation + chemical pretreatment) can also be applied to lcms, and can contribute to the acceleration of the chemical reaction rate [76]. also, microwave irradiation reduces time, and the severity of liquid ionic and alkali pretreatment [77,78]. ma et al. [79] pretreated the rice straws using microwave irradiation with a maximum power of 800 w. the optimal conditions identified were a microwave power (mp) of 680 w, irradiation time (it) of 24 min and substrate concentration (sc) of 75 g/l. under these optimal conditions, cellulose saccharification (cs), hemicellulose saccharification (hs) and total saccharification (ts) reached 37.8%, 20.2% and 31.8% with increased rates of 30.6%, 43.3% and 30.3% as compared to the straw of raw rice. therefore, microwave irradiation is an effective pretreatment method and could disrupt the silicified waxed surface, decompose the complex structure of lignin-hemicellulose, and partially remove silicon and lignin thus facilitating the action of cellulases [79]. in the study conducted by liu et al. [80] on poplar sawdust (80 mesh) it was applied a combined pretreatment using as solvent choline cloride/oxalic acid dihydrate (chcl/oa) deep eutectic (des) with ph= 1.31 + microwave treatment and solid toliquid ratio of 1:20. 80% of total lignin was removed from the samples pretreated only with chcl/oa after being maintained for 9 hours at 110 °c, while the same results were obtained by applying microwave irradiation for 3 minutes at 800 w [80]. chen et al. [81] analyzed the impact of microwave-assisted (10 %wt solid loading) pretreatment using a radiation power of 800 w, temperature of 152 °c, and time of 45 s on corn stover, switchgrass and miscanthus. after pretreatment, significant amounts of lignin and xylan were identified in the liquid fraction. the lignin content removed from corn stover, switchgrass and miscanthus was 79.60%, 72.23% and 65.18%, respectively [81]. 3.1.3. ultrasonic pretreatment the use of ultrasound is an effective method for separating constituents from lcms. ultrasonic treatment is based on the working principle of the acoustic cavity, which is described as spontaneous formation, growth and subsequent collapse of the microsize cavities/bubbles caused by the propagation of ultrasonic waves in the liquid medium. the implosion of these cavities generates high temperatures and pressure gradients locally for microsecond conditions, creating the effect of hot-spot in the liquid [82,83]. esfahani et al. [84] pretreated sugarcane bagasse (particle size >1, 1-0.5, 0.5-0.18, <0.18 mm) using ultrasound-assisted diluted h2so4 pretreatment (20 khz, 50, 80, 120 and 200 w; 0, 1, 3 and 5% (v/v) h2so4) for 0, 60, 12 and 180 s, respectively. the most significant impact was recorded when the ultrasound power was 120 w [84]. yuan et al. [85] have applied an ultrasound-assisted organic solvent pretreatment to delignify poplar wood at 20 khz, 570 w and 25 °c for 30 min using three organic solvents – 95% ethanol, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 245 methanol, and dioxane. following the application of ultrasound-assisted organic solvents pretreatment, approximately 25.7% of the original lignin was extracted [85]. sun et al. [86] have pretreated wheat straws using ultrasound-assisted alkali pretreatment at 20 khz and 100 w with 0.5 m koh at 35 °c for 2.5 h, and after 35 min of ultrasound irradiation approximately 8.4% of lignin was extracted [86]. 3.1.4. pulsed electric field (pef) pretreatment pulsed electric field (pef) is a very short non-thermal treatment (from a few microseconds to a few milliseconds) with a pulse amplitude from about 300 v/cm to 20-40 kv/cm. by comparison to other treatments, the damage caused to cell membranes or tissue matrix is lower. in other words, pef penetrates the biological membrane which temporarily or permanently loses its semi-permeability [87]. the electrical permeability of biological membranes is called electroporation and can be reversible or irreversible [88]. electrical permeability of different species of switchgrass and wood chips using pef can be applied to facilitate the hydrolysis of cellulose to glucose in order to obtain fuels [89]. almohammed et al. [90] analyzed the impact of pulsed electric field intensity e and duration tpef on the expression kinetics of dissolved substances in sugar beet tails (sbt). in regards to the intensity and optimal duration of pef, it was established that e = 450 v/cm and tpef= 10 ms corresponded to an energy input q= 1.91 wh/kg, as the yield of dissolved substances increased from 16.8% to 79.85% by comparisson to untreated sbt. also, the liquid fraction resulting from the pef pretreatment was more concentrated (10% vs. 5.2%) and implicitly higher sucrose content was obtained (8.9 °s compared with 4.5 °s in the juice from untreated sbt). therefore, it was found that by applying the pef pretreatment it would be achieved an ethanol content of 6.1% v/v, as compared to 2.95% v/v for the untreated sbt [90]. kumar et al. [89] investigated the impact of pef on untreated and treated samples of switchgrass using 1000, 2000 and 5000 pulses of 2.5, 5, 8 and 10 kv/cm with a pulse width of 100 μs and a frequency of 3 hz, and samples of untreated and treated wood chips (southern pine), for which they applied 1000 and 2000 pulses of 1 kv/cm and 1000, 2000, and 5000 pulses of 10 kv/cm, the pulse width and frequency being similar. to indicate the impact of pef on internal diffusion in the tissues of the samples, the absorption of a neutral red dye c15h17cln4 (mw ∼ 289) was studied. in the case of switchgrass samples, no structural changes were recorded at low field intensities up to 5 kv/cm. changes in the structure were recorded at field intensities of 2000 and 5000 pulses of 8 kv/cm and 10 kv/cm, respectively. changes were observed for wood chips treated at 10 kv/cm [89]. 3.2. chemical pretreatments 3.2.1. acid pretreatment compared to the alkali pretreatment which removes more lignin, the acid pretreatment removes more hemicellulose, while cellulose and lignin fractions are less affected [90,92]. regarding the acid pretreatment of lignocellulosic biomass, mineral acids (hcl, hno3, h2so4 and h3po4) and organic acids (e.g. ch2o2, c2h4o2, c3h6o2 and c4h4o4) can be used successfully [93]. as their use affects the environment, it is necessary to find food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 246 pretreatment techniques to optimize yields and reduce costs [94]. table 2 presents different acids used for pretreatment of lcms. table 2 different acids used for pretreatment of lcms type of acid lcm process conditions reference hcl corn stover 1 % (w/w) hcl at 100 130 °c for 20 40 min [95] grass 1 % (w/w) hcl boiled at 100 °c for 30 min [96] hno3 corn stover 0.2 1 % (w/w) hno3 at 120, 140 and 160 °c for 1, 5.5 and 10 min [97] oat hulls 4 % (w/w) hno3 at 94 96 °c for 4 h [98] sugarcane bagasse 1 % (w/v) hno3 autoclaved at 121 °c for 30 min [99] h2so4 sugarcane bagasse 2 6% (w/w) naoh at 100 128 °c for 0 300 min [100] corn stover 0.71 1.41% (w/w) h2so4 at 165–195°c for 2.9 – 12.2 min [101] wheat straws 0.75 – 2.25% (v/v) h2so4 at 120, 140 and160 °c for 10, 20 and 30 min [102] rice straws 0.5% (w/v) h2so4 autoclaved at 120 °c (15 lb pressure) for 60 min [103] sugarcane bagasse 0.5 3% (w/v) h2so4 at 112.5 157.5 °c for 5 35 min [104] h3po4 corn stover 0.16 – 1.84 % (v/v) h3po4 at 126.36 – 193.63 °c for 1.59 – 18.41 min [105] wheat bran 0.5 3% (w/v) h3po4 at 150 210 °c for 5 20 min [106] sugarcane bagasse 1 % (w/w) h3po4 at 170 and 180 °c for 4 h [107] eucalyptus benthamii 1% (w/w) h3po4 at 180 200 °c for 5 15 min [108] ch2o2 scots pine sawdust 0.5 – 2.5% (w/v) h2so4 at 100, 120, or 140 °c for 1, 1.5, and 2 h [109] 15 – 40% (w/v) ch2o2 at 100, 120, or 140 °c for 1, 1.5, and 2 h c2h2o4 corn cob 0.015 – 0.037 g/g c2h2o4 at 120 180 °c for 10 90 min [110] yellow poplar sawdust 24 – 139 mm c2h2o4 at 160 °c for 2 58 min [111] 3.2.2. alkali pretreatment alkali pretreatment is based on the use of hydroxides such as naoh, koh, ca(oh)2 and nh4oh for the pretreatment of lignocellulosic biomass, cellulose swelling, partial decrystallization of cellulose [112-115] and partial removal of hemicellulose [115-116]. by applying the alkali pretreatment, lignin can be extracted; this is the basis of the pulping process in order to obtain high quality paper (kraft process) [91-92]. most studies were performed on the impact that naoh has on the complex structure of lcb and it was found that this hydroxide can remove lignin and facilitate the activity of cellulolytic enzymes [117]. numerous lcms were subjected to alkali pretreatment methods and these include corn stover, sugarcane bagasse, wheat straws, rice straws, switchgrass, and sawdust [117-119]. table 3 presents different hydroxides used for pretreatment of lcms. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 247 table 3 different hydroxides used for pretreatment of lcms type of hydroxide lcm process conditions reference naoh corn stover 0.25, 0.5, 1 % (w/v) naoh at 21 °c for 3, 6, 9 h [120] wheat straws 0.25 1.5% (w/v) naoh was at 121 °c/15 psi for 0.5 2.5 h [121] sugarcane bagasse 3% naoh and thermostated in oven at 121 °c, 60 min [122] sugarcane tops 3% (w/w) naoh and thermostated at 121, °c (15 lb pressure), 60 min [123] cotton stalk 1, 2, 3, 4, 5% (w/w) naoh at 120, 150,180, 200 °c, 45 min [124] spruce sawdust 3%, 7%, and 10% (w/w) naoh at 60 °c for 0.5, 1, and 2 h 7% naoh (w/w) at −20 °c and 121 °c for 0.5, 2, and 24 h [125] bamboo 2% naoh at 120, 140,160, 180 °c, 60 min [126] koh switchgrass 0.5, 1.0, 2.0% koh at 21°c for 6, 12, 24, 48 h 0.5, 1.0, 2.0% koh at 50°c for 6, 12, 24 h 0.5, 1.0, 2.0% koh at 121°c for 0.25, 0.5, 1.0 h [127] ca(oh)2 corn stover 0.0 0.30 g ca(oh)2 (g/dry biomass) at 120 °c for 5 h [128] poplar 0.1 0.3 g ca(oh)2 (g/dry biomass) at 60 – 250 °c for 0.25 24 h [129] newspaper 0.05 0.3 g ca(oh)2 (g/dry biomass) at 60 – 150 °c for 1 24 h nh4oh corn stover 0.5 50.0 wt.% nh4oh at 30 °c for 4 12 weeks [130] wheat straws 6.2, 15.4, 24.6 and 30.8% (w/v) nh4oh at 20, 32.2, 50, 67.8 and 80 °c for 6, 14.5, 27, 39.5 and 48 h [131] 3.2.3. ozonolysis pretreatment ozone (o3) is considered a strong oxidant and has high solubility in water. it converts to oxygen and has a strong affinity for c-c double bonds in the structure of lignin as opposed to carbohydrates where these bonds are missing. for this reason, ozone can be used for the pretreatment of different agricultural residues and energy crops. the most used ozone pretreatment method is the one made in a fixed bed reactor (with humidity of 20-40%) for 60-180 min, under room conditions [132]. even if the ozonolysis is exothermic, different pressures and temperatures can be applied [133]. by applying ozone pretreatment, approximately 50% of the lignin present in lcb is depolymerized and removed [134], and the ph of lcb drops to 2-3. by increasing the ph it was observed that the depolymerization of lignin is reduced [133]. travaini et al. [132] reported that ozone pretreatment of sugarcane bagasse slightly reduced carbohydrates, with cellulose and xylan recovery rates being greater than 92%. in this study the following parameters were varied: 1.37 ± 0.03 3.44 ± 0.11% (v/v) o3, humidity 28 ± 0.11 80 ± 0.32% (w/w), and ozonolysis time 45 ± 0.02 195 ± 0.02 min. also, ozonolysis facilitated the enzymatic hydrolysis obtaining the yields of glucose and xylose [132]. in the study by garcia-cubero et al. [133] the ozonolysis pretreatment was applied on wheat straws, rye straws, oat straws, barley food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 248 straws using 2.7% (w/w) o3 and 40% (w/w) humidity under room conditions [133]. 3.2.4. organosolv pretreatment organic solvents are used to extract/remove lignin from lcb before performing enzymatic hydrolysis of the cellulose fraction. in the case of pretreatment with organosolv, single organic solvent or different ratios of organic solvents/water can be used. it was found that in order to increase the solubilization rate of lignin and hemicellulose and their removal it is recommended to use an acid as catalyst to facilitate the enzymatic hydrolysis of the cellulose fraction. the most commonly used organic solvents for the pretreatment of lcms are ethanol, methanol, acetone and ethylene glycol [135] and the maximum temperature at which they can be used can range up to 200 °c. in some cases it is not necessary to use maximum temperatures, however, depending on the type of lcm lower temperatures can be applied alongside an acidic catalyst [12]. because the solvent used in the pretreatment of lcm can have inhibitory effects on the enzymatic hydrolysis and fermentation steps, it must be separated and recycled. [14]. table 4 presents different organosolv and catalysts used for pretreatment of lcms. table 4 different organosolv and catalysts used for pretreatment of lcms [68,136] type of organosolv lcm process conditions references 60% ethanol corn stover n-propylamine at 140°c for 40 min [137] 60% ethanol corn stalk 4% naoh at 110°c for 90 min [138] 25% ethanol wheat straws 1% h2so4 at190°c for 60 min [139] 50% ethanol 0.35% h2so4 at 180°c for 40 min [140] 60% ethanol 0.29% h2so4 at 190°c for 60 min [141] 45% ethanol rice straws 1% h2so4 at 180°c for 30 min [142] 65% ethanol 1.1% h2so4 at 170°c for 60 min [143] 50% acetone barley straws 0.5% h2so4 at 140°c for 20 min [144] 50% ethanol 1.6% fecl3 at 170°c for 60 m in [145] 25% butanol sorghum bagasse 0.5% h2so4 at 200°c for 60 min [146] 50% ethanol sweet sorghum 1% h2so4 ar 140°c for 30 min [147] 50% ethanol sugarcane bagasse 1.25% h2so4 ar 175°c for 60 min [148] 60% ethanol 0.025% fecl3 at 160°c for 72h [149] 70% glycerol at 220°c for 120 min [150] 60% ethanol bamboo at 160°c 60 min [151] 56% glycerol eucalyptus wood at 200°c for 69 min [152] 25% ethanol 1% ch3cooh at 200 °c for 60 min [153] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 249 3.2.5. ionic liquids (ils) ionic liquids (ils) are considered to be environmentally friendly molten salts and are part of a new class of solvents that have high polarity, low melting point, nonvolatility and designability [154-157]. regarding the pretreatment of lignocellulosic biomass ils were successfully used as solvents for lignin extraction and crystallinity reduction of carbohydrates [158-159]. ils offer more attractive features when compared to conventional methods [160]. the physicochemical properties of the il, the reaction time and temperature, the ratio between biomass and il, the type of biomass and the humidity of the sample are the criteria that must be taken into account when selecting the type of il used for the lcm pretreatment [161]. compared to conventional methods, ils have numerous attractive features [160]. for lcb pretreatment, ils should have the following properties [160,162]: ability to dissolve lcb at low temperatures; chemical stability; low viscosity; easy to regenerate and recycle; cost-effective and easy to process; absence of toxicity during enzymatic hydrolysis and microbial fermentation steps. numerous studies have shown that higher conversion and/or yields of intermediates can be obtained if metal or acid catalysts are also used alongside ils [163]. the most representative ils containing organic cation salts are nitrate [no3]-, hexafluorophosphate [pf6] -, alkylimidazolium [r1r2im] +, alkylpyridinium [rpy]+, methanesulfonate (mesylate) [ch3so3] -, trifluoromethane sulfonate [cf3so3] -, tetraalkylammonium [nr4] +, or tetraalkylphosphonium [pr4] + and anions, and bis(trifluoromethanesulfonyl) imide [tf2n] -. there are also salts of chloride, iodine and bromide [164]. table 5 presents different ionic liquids and catalyst used for pretreatment of lcms. table 5 different ionic liquids (ils) and catalysts used for pretreatment of lcms [160] type of acid lcm process conditions reference 1-butyl-3methylimidazolium chloride corn stalk hcl at 100 °c for 0.5 h [165] hcl at 100 °c for 5.5 h rice straws hcl at 100 °c for 7.5 h pine wood hcl at 100 °c for 0.8 h bagasse hcl at 66 °c for 1 h 1-butyl-3methylimidazolium bromide corn stalk hcl at 100 °c for 1 h 1-allyl-3-methylimidazolium chloride hcl at 100 °c for 1.5 h 1-hexyl-3 methylimidazolium chloride hcl at 100 °c for 20 h 1-ethyl-3methylimidazolium acetate rice straws and cassava pulp at 25 120 °c for 24 h [166] 1-ethyl-3methylimidazolium diethyl phosphate 1,3-dimethylimidazolium methyl sulfate n-methylmorpholine-noxide spruce and oak 6 %, 90–130 c, 1–3 h [167] spruce and birch chips 6 %, 130 c, 1–5 h [168] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 250 3.2.6. deep eutectic solvents (des) recently, deep eutectic solvents (des) have attracted more and more attention and are considered to be alternative ils or in other words are considered to be green solvents that have physico-chemical properties similar to ils. in most cases, des contain two or three components that are linked by a hydrogen bond thus resulting an eutectic mixture with a melting point lower than each individual component. [169,170]. usually, below 100 °c des are in liquid form. compared to ils, des are biodegradable and the production costs are lower [169]. the technology for obtaining des refers to mixing a quaternary ammonium salt with a metal salt or hydrogen bonding donor (hbd) which can create a complex with the halogen ion of the quaternary ammonium salt [171]. zhang et al. pretreated 0.3 g of corncob with a des that was prepared by mixing choline chloride (chcl) with carboxylic acid (monocarboxylic and dicarboxylic) or polyalcohol at 90 °c for 24 h [172]. xu et al. pretreated corn with an acid des consisting of choline chloride: formic acid (chcl: ch2o2) and obtained noteworthy results in terms of removal of hemicellulose and lignin [173]. also, pan et al. [174] pretreated 10 g of rice straw with 200 g chcl/urea and transferred the mixture to 500 ml erlenmeyer flasks. then, the contents were stirred and maintained at 110 °c and 130 °c for 4 h, 6 h and 8 h, respectively [174]. jablonský et al. [175] pretreated wheat straw with six types of des using different ratios of choline chloride with urea, malic, lactic, malonic, lactic, and oxalic acid. 2.5 g of wheat straw were pretreated with individual des at a ratio of 1:20 (w/w) for 24 hours at 60 °c; for choline chloride and urea and choline chloride and malic acid the temperature was 80 °c [175]. 4. advantages and disadvantages of physical and chemical pretreatments regarding the technological process of bioethanol production from lcm, selection of the pretreatment type specific to each lcm is very important because this step has a great impact on all subsequent steps (hydrolysis and fermentation) [176]. therefore, the choice of pretreatment should be made carefully in the process of obtaining bioethanol because the pretreatment also affects the cost of the next steps of operation and refers to the determination of compounds that cause inhibition of fermentation, enzyme hydrolysis rates and enzyme dosages alongside other factors that may influence the fermentation process. table 6 shows the main advantages and disadvantages of the most common pretreatment technologies used for the conversion of lcb to bioethanol [177]. 5. conclusion the growing need for energy worldwide and environmental pollution must lead us to focus on the exploitation of lignocellulosic biomass, which is a renewable source that is widely available and relatively inexpensive. in order to convert lcms to bioethanol, their complex structure must first be fractionated as much as possible. this can be done only by correctly choosing from the various pretreatment technologies available, which include biological, mechanical, chemical and various other combined methods. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 251 table 6 advantages and disadvantages of the most common pretreatment technologies used for the conversion of lcb to bioethanol [177,178] pretreatment increases accessible surface area cellulose decrystallization hemicellulose solubilization lignin removal lignin structure modification production of toxic compounds mechanical +++ +++ 0 0 0 0 irradiation +++ +++ + +++ +++ + acid +++ 0 +++ ++ +++ +++ alkali +++ +++ ++/+++ +++ +++ + ozonolysis ++ ++ ++/+++ +++ ++ + organosolv ++ +++ ++/+++ ++ ++/+ ionic liquids ++ +++ +++ ++/+++ ++ ++/+ (+++) high effect; (++) moderate effect; (+) low effect; (0) no effect physical pretreatment methods, and especially mechanical ones, reduce the crystallinity and particle size and cause an increased contact surface with the pretreatment agent. however, this process generates high energy consumption and therefore high costs. in regards to the application of chemical pretreatment methods, the use of chemicals such as acids, alkalis, ozone or organic solvents can effectively remove lignin, thus facilitating the enzymatic hydrolysis of cellulose. we cannot name a method that is the best choice and can be applied among all types of lignocellulosic biomass. the choice of pretreatment method depends for the most part on the type of lcb and therefore each pretreatment method has its own effects on the cellulose, hemicellulose and lignin fractions. 6. reference [1].reijnders, l. (2006). conditions for the sustainability of biomass based fuel use. energy policy, 34(7), 863-876. [2].demirbas, a. (2007). progress and recent trends in biofuels. progress in energy and combustion science, 33(1), 1-18. [3].bhatia, l., johri, s., & ahmad, r. (2012). an economic and ecological perspective of ethanol production from renewable agro waste: a review. amb express, 2(1), 1-19. [4].mussatto, s. i. (2016). a closer look at the developments and impact of biofuels in transport and environment; what are the next steps?. biofuel research journal, 3(1), 331-331. [5].pereira, s. c., maehara, l., machado, c. m. m., & farinas, c. s. (2015). 2g ethanol from the whole sugarcane lignocellulosic biomass. biotechnology for biofuels, 8(1), 44. [6].go, a. w., conag, a. t., igdon, r. m. b., toledo, a. s., & malila, j. s. (2019). potentials of agricultural and agro-industrial crop residues for the displacement of fossil fuels: a philippine context. energy strategy reviews, 23, 100-113. [7].van maris, a. j., abbott, d. a., bellissimi, e., van den brink, j., kuyper, m., luttik, m. a., pronk, j. t. (2006). alcoholic fermentation of carbon sources in biomass hydrolysates by saccharomyces cerevisiae: current status. antonie van leeuwenhoek, 90(4), 391-418. [8].ge, x., chang, c., zhang, l., cui, s., luo, x., hu, s., li, y. (2018). conversion of lignocellulosic biomass into platform chemicals for biobased polyurethane application. in advances in bioenergy (vol. 3, pp. 161-213). elsevier. [9].zhu, j. y., & pan, x. j. (2010). woody biomass pretreatment for cellulosic ethanol production: technology and energy consumption evaluation. bioresource technology, 101(13), 49925002. [10]. cherubini, f. (2010). the biorefinery concept: using biomass instead of oil for producing energy and chemicals. energy conversion and management, 51(7), 1412-1421. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 252 [11]. hlpe, 2013. biofuels and food security. a report by the high level panel of experts on food security and nutrition of the committee on world food security, rome 2013. http://www.fao.org/3/ai2952e.pdf [12]. sun, y., & cheng, j. (2002). hydrolysis of lignocellulosic materials for ethanol production: a review. bioresource technology, 83(1), 1-11. [13]. pimentel, d., marklein, a., toth, m. a., karpoff, m. n., paul, g. s., mccormack, r., krueger, t. (2009). food versus biofuels: environmental and economic costs. human ecology, 37(1), 1. [14]. thompson, p. b. (2012). the agricultural ethics of biofuels: the food vs. fuel debate. agriculture, 2(4), 339-358. [15]. taherzadeh, m. j., & karimi, k. (2008). pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. international journal of molecular sciences, 9(9), 1621-1651. [16]. breaking the chemical and engineering barriers to lignocellulosic biofuels, & huber, g. w. (2008). breaking the chemical and engineering barriers to lignocellulosic biofuels: next generation hydrocarbon biorefineries. washington, dc: national science foundation, chemical, biogengineering, environmental and transport systems division. [17]. barakat, a., de vries, h., & rouau, x. (2013). dry fractionation process as an important step in current and future lignocellulose biorefineries: a review. bioresource technology, 134, 362-373. [18]. saha, b. c. (2005). enzymes as biocatalysts for conversion of lignocellulosic biomass to fermentable sugars. handbook of industrial biocatalysis, 1-12. [19]. hernández-beltrán, j. u., lira, h. d., omar, i., cruz-santos, m. m., saucedo-luevanos, a., hernándezterán, f., & balagurusamy, n. (2019). insight into pretreatment methods of lignocellulosic biomass to increase biogas yield: current state, challenges, and opportunities. applied sciences, 9(18), 3721. [20]. aftab, m. n., iqbal, i., riaz, f., karadag, a., & tabatabaei, m. (2019). different pretreatment methods of lignocellulosic biomass for use in biofuel production. in biomass for bioenergy-recent trends and future challenges. intechopen. [21]. nigam, p. s., & singh, a. (2011). production of liquid biofuels from renewable resources. progress in energy and combustion science, 37(1), 52-68. [22]. alam f., date a., rasjidin r., mobin s., moria h., baqui, a. (2012), biofuel from algaeis it a viable alternative?, procedia engineering, vol. 49: 221-227. [23]. noraini, m. y., ong, h. c., badrul, m. j., & chong, w. t. (2014). a review on potential enzymatic reaction for biofuel production from algae. renewable and sustainable energy reviews, 39, 24-34. [24]. dragone g., fernandes b., vicente a.a., teixeira j.a. (2010), third generation biofuels from microalgae in current research, technology and education topics in applied microbiology and microbial biotechnology, mendez-vilas a (ed.), formatex, 1355-1366. [25]. renewable fuels association, et al. powered with renewed energy: 2019 ethanol industry outlook. rfa, washington, dc, 2019. [26]. renewable fuels association. http://www.ethanolrfa.org/resources/industry/statist ics/#1454098996479-8715d404-e546 [27]. hall, m. p., bansal, lee, j.h., realff, m.j., bommarius, a.s., (2010). cellulose crystallinitya key predictor of the enzymatic hydrolysis rate, febs j 277 (2010) 1571. [28]. chauve, m., barre, l., tapinlingua, s., silva perez dd, decottignies, perez, d.s., et al., (2013). evolution and impact of cellulose architecture during enzymatic hydrolysis by fungal cellulases, adv. biosci. biotechnol. 04 (2013) 1095. [29]. mohan d., pittman c.u., steele p.h., (2006). pyrolysis of wood/biomass for bio-oil: a critical review. energy fuels 2006;20:848–89. [30]. frederick jr., w.j., lien, s.j., courchene, c.e., demartini, n.a., ragauskas, a.j., lisa k., (2008). production of ethanol from carbohydrates from loblolly pine: a technical and economic assessment, bioresource technology 99 (2008) 5051–5057. [31]. boerjan w., ralph j., baucher m. (2003). lignin biosynthesis. annu rev plant biol 2003;54:519–46. [32]. biswas, r., uellendahl, h., & ahring, b. k. (2015). wet explosion: a universal and efficient pretreatment process for lignocellulosic biorefineries. bioenergy research, 8(3), 1101-1116. [33]. cheșcă, a. m., tofănică, b. m., puiţel, a. c., nicu, r., & gavrilescu, d. (2018). environmentally friendly cellulosic fibers from corn stalks. environmental engineering and management journal, 17(7), 1765-1771. http://www.fao.org/3/a-i2952e.pdf http://www.fao.org/3/a-i2952e.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 253 [34]. sánchez, c., serrano, l., andres, m. a., & labidi, j. (2013). furfural production from corn cobs autohydrolysis liquors by microwave technology. industrial crops and products, 42, 513519. [35]. puitel, a. c., tofanica, b. m., & gavrilescu, d. (2012). environmentally friendly vegetal fiber based materials. environmental engineering and management journal, 11(3), 651-659. [36]. kaur, a., & kuhad, r. c. (2019). valorization of rice straw for ethanol production and lignin recovery using combined acid-alkali pretreatment. bioenergy research, 12(3), 570-582. [37]. adapa, p., tabil, l., & schoenau, g. (2009). compaction characteristics of barley, canola, oat and wheat straw. biosystems engineering, 104(3), 335-344. [38]. sun, j. x., xu, f., geng, z. c., sun, x. f., & sun, r. c. (2005). comparative study of cellulose isolated by totally chlorine‐free method from wood and cereal straw. journal of applied polymer science, 97(1), 322-335. [39]. teghammar, a., karimi, k., horváth, i. s., & taherzadeh, m. j. (2012). enhanced biogas production from rice straw, triticale straw and softwood spruce by nmmo pretreatment. biomass and bioenergy, 36, 116-120. [40]. cardoso, w. s., tardin, f. d., tavares, g. p., queiroz, p. v., mota, s. s., kasuya, m. c. m., & queiroz, j. h. d. (2013). use of sorghum straw (sorghum bicolor) for second generation ethanol production: pretreatment and enzymatic hydrolysis. química nova, 36(5), 623-627. [41]. de moraes rocha, g. j., martin, c., soares, i. b., maior, a. m. s., baudel, h. m., & de abreu, c. a. m. (2011). dilute mixedacid pretreatment of sugarcane bagasse for ethanol production. biomass and bioenergy, 35(1), 663670. [42]. lu, x., zhang, y., & angelidaki, i. (2009). optimization of h2so4-catalyzed hydrothermal pretreatment of rapeseed straw for bioconversion to ethanol: focusing on pretreatment at high solids content. bioresource technology, 100(12), 3048-3053. [43]. silverstein, r. a., chen, y., sharma-shivappa, r. r., boyette, m. d., & osborne, j. (2007). a comparison of chemical pretreatment methods for improving saccharification of cotton stalks. bioresource technology, 98(16), 3000-3011. [44]. mwaikambo, l. y., & ansell, m. p. (2002). chemical modification of hemp, sisal, jute, and kapok fibers by alkalization. journal of applied polymer science, 84(12), 2222-2234. [45]. rachini, a., le troedec, m., peyratout, c., & smith, a. (2009). comparison of the thermal degradation of natural, alkali‐treated and silane‐treated hemp fibers under air and an inert atmosphere. journal of applied polymer science, 112(1), 226-234. [46]. li, x., tabil, l. g., & panigrahi, s. (2007). chemical treatments of natural fiber for use in natural fiber-reinforced composites: a review. journal of polymers and the environment, 15(1), 25-33. [47]. spigno, g., maggi, l., amendola, d., dragoni, m., & de faveri, d. m. (2013). influence of cultivar on the lignocellulosic fractionation of grape stalks. industrial crops and products, 46, 283-289. [48]. howard, r. l., abotsi, e. l. j. r., van rensburg, e. j., & howard, s. (2003). lignocellulose biotechnology: issues of bioconversion and enzyme production. african journal of biotechnology, 2(12), 602-619. [49]. kumar, a. k., & sharma, s. (2017). recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review. bioresources and bioprocessing, 4(1), 7. [50]. xu, c., zhu, s., xing, c., li, d., zhu, n., & zhou, h. (2015). isolation and properties of cellulose nanofibrils from coconut palm petioles by different mechanical process. plos one, 10(4), e0122123. [51]. monsalve, j. f., medina de perez, v. i., & ruiz colorado, a. a. (2006). ethanol producction of banana shell and cassava starch. dyna, 73(150), 21-27. [52]. grams, j., kwapińska, m., jędrzejczyk, m., rzeźnicka, i., leahy, j. j., & ruppert, a. m. (2019). surface characterization of miscanthus× giganteus and willow subjected to torrefaction. journal of analytical and applied pyrolysis, 138, 231-241. [53]. sathitsuksanoh, n., zhu, z., wi, s., & percival zhang, y. h. (2011). cellulose solvent‐based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass. biotechnology and bioengineering, 108(3), 521-529. [54]. popescu, m. c., popescu, c. m., lisa, g., & sakata, y. (2011). evaluation of morphological and chemical aspects of different wood species by spectroscopy and thermal methods. journal of molecular structure, 988(1-3), 65-72. [55]. rowell, r. m., pettersen, r., han, j. s., rowell, j. s., & tshabalala, m. a. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 254 (2005). cell wall chemistry. handbook of wood chemistry and wood composites, 2. [56]. pettersen, r.c. (1984). the chemical composition of wood. in: the chemistry of solid wood, rowell, r. m., editor, advances in chemistry series 20, american chemical society. [57]. cuilty, k. r., ballinascasarrubias, l., de san miguel, e. r., de gyves, j., robles-venzor, j. c., & gonzález-sánchez, g. (2018). cellulose recovery from quercus sp. sawdust using ethanosolv pretreatment. biomass and bioenergy, 111, 114-124. [58]. isikgor, f. h., & becer, c. r. (2015). lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. polymer chemistry, 6(25), 4497-4559. [59]. anwar, z., gulfraz, m., & irshad, m. (2014). agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. journal of radiation research and applied sciences, 7(2), 163-173. [60]. fatma, s., hameed, a., noman, m., ahmed, t., shahid, m., tariq, m., tabassum, r. (2018). lignocellulosic biomass: a sustainable bioenergy source for the future. protein and peptide letters, 25(2), 148-163. [61]. hamelinck, c. n., van hooijdonk, g., & faaij, a. p. (2005). ethanol from lignocellulosic biomass: techno-economic performance in short-, middle-and longterm. biomass and bioenergy, 28(4), 384-410. [62]. palonen h., (2004) role of lignin in enzymatic hydrolysis of lignocellulose, vtt technical research, espoo, p 11-22, p 26-32, 2004; [63]. maties m.i., balcu i., segneanu a.e., macarie c., mirica m.c., fitigau f. (2011). “bioetanol”, editura “mirton” 2011; [64]. alvira, p., tomás-pejó, e., ballesteros, m., & negro, m. j. (2010). pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. bioresource technology, 101(13), 4851-4861. [65]. amin, f. r., khalid, h., zhang, h., u rahman, s., zhang, r., liu, g., & chen, c. (2017). pretreatment methods of lignocellulosic biomass for anaerobic digestion. amb express, 7(1), 72. [66]. barakat a., chuetor s., monlau f., solhy a., rouau x., (2014). eco-friendly dry chemo-mechanical pretreatments of lignocellulosic biomass: impact on energy and yield of the enzymatic hydrolysis. applied energy, 113, 97– 105, doi:10.1016/j.apenergy.2013.07.015. [67]. cheng, j.j., timilsina, g.r., (2011). status and barriers of advanced biofuel technologies: a review, renewable energy 36 (2011) 3541–3549. [68]. baruah, j., nath, b. k., sharma, r., kumar, s., deka, r. c., baruah, d. c., & kalita, e. (2018). recent trends in the pretreatment of lignocellulosic biomass for valueadded products. frontiers in energy research, 6, 141. [69]. mani, s., tabil, l. g., & sokhansanj, s. (2004). grinding performance and physical properties of wheat and barley straws, corn stover and switchgrass. biomass and bioenergy, 27(4), 339-352. [70]. bitra, v. s., womac, a. r., igathinathane, c., miu, p. i., yang, y. t., smith, d. r., sokhansanj, s. (2009). direct measures of mechanical energy for knife mill size reduction of switchgrass, wheat straw, and corn stover. bioresource technology, 100(24), 65786585. [71]. sasmal, s., & mohanty, k. (2018). pretreatment of lignocellulosic biomass toward biofuel production. in biorefining of biomass to biofuels (pp. 203-221). springer, cham. [72]. hu, z., & wen, z. (2008). enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment. biochemical engineering journal, 38(3), 369-378. [73]. datta, a. k. (2001). fundamentals of heat and moisture transport for microwaveable food product and process development. food science and technology-new yorkmarcel dekker-, 115-172. [74]. xiong, j., ye, j., liang, w. z., & fan, p. m. (2000). influence of microwave on the ultrastructure of cellulose i. journal of south china university technology, 28(1), 84-89. [75]. azuma, j. i., tanaka, f., & koshijima, t. (1984). enhancement of enzymatic susceptibility of lignocellulosic wastes by microwave irradiation. j. ferment. technol.;(japan), 62(4). [76]. caddick, s. (1995). microwave assisted organic reactions. tetrahedron, 51(38), 1040310432. [77]. boonsombuti, a., luengnaruemitchai, a., & wongkasemjit, s. (2013). enhancement of enzymatic hydrolysis of corncob by microwaveassisted alkali pretreatment and its effect in morphology. cellulose, 20(4), 1957-1966. [78]. aguilar-reynosa, a., romani, a., rodriguez-jasso, r. m., aguilar, c. n., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 255 garrote, g., & ruiz, h. a. (2017). microwave heating processing as alternative of pretreatment in second-generation biorefinery: an overview. energy conversion and management, 136, 50-65. [79]. ma, h., liu, w. w., chen, x., wu, y. j., & yu, z. l. (2009). enhanced enzymatic saccharification of rice straw by microwave pretreatment. bioresource technology, 100(3), 1279-1284. [80]. liu, y., chen, w., xia, q., guo, b., wang, q., liu, s., yu, h. (2017). efficient cleavage of lignin–carbohydrate complexes and ultrafast extraction of lignin oligomers from wood biomass by microwave‐assisted treatment with deep eutectic solvent. chemsuschem, 10(8), 1692-1700. [81]. chen, z., & wan, c. (2018). ultrafast fractionation of lignocellulosic biomass by microwave-assisted deep eutectic solvent pretreatment. bioresource technology, 250, 532537. [82]. sawant, s. s., anil, a. c., krishnamurthy, v., gaonkar, c., kolwalkar, j., khandeparker, l., pandit, a. b. (2008). effect of hydrodynamic cavitation on zooplankton: a tool for disinfection. biochemical engineering journal, 42(3), 320-328. [83]. subhedar, p. b., & gogate, p. r. (2016). use of ultrasound for pretreatment of biomass and subsequent hydrolysis and fermentation. in biomass fractionation technologies for a lignocellulosic feedstock based biorefinery (pp. 127-149). elsevier. [84]. esfahani, m. r., & azin, m. (2012). pretreatment of sugarcane bagasse by ultrasound energy and dilute acid. asia‐pacific journal of chemical engineering, 7(2), 274-278. [85]. yuan, t. q., xu, f., he, j., & sun, r. c. (2010). structural and physico-chemical characterization of hemicelluloses from ultrasoundassisted extractions of partially delignified fastgrowing poplar wood through organic solvent and alkaline solutions. biotechnology advances, 28(5), 583-593. [86]. sun, r., & tomkinson, j. (2002). comparative study of lignins isolated by alkali and ultrasound-assisted alkali extractions from wheat straw. ultrasonics sonochemistry, 9(2), 85-93. [87]. zimmermann, u. (1986). electrical breakdown, electropermeabilization and electrofusion. in reviews of physiology, biochemistry and pharmacology, volume 105 (pp. 175-256). springer, berlin, heidelberg. [88]. miklavčič, d., šemrov, d., mekid, h., & mir, l. m. (2000). a validated model of in vivo electric field distribution in tissues for electrochemotherapy and for dna electrotransfer for gene therapy. biochimica et biophysica acta (bba)-general subjects, 1523(1), 73-83. [89]. kumar, p., barrett, d. m., delwiche, m. j., & stroeve, p. (2011). pulsed electric field pretreatment of switchgrass and wood chip species for biofuel production. industrial & engineering chemistry research, 50(19), 10996-11001. [90]. almohammed, f., mhemdi, h., & vorobiev, e. (2016). pulsed electric field treatment of sugar beet tails as a sustainable feedstock for bioethanol production. applied energy, 162, 49-57. [91]. galbe, m., & wallberg, o. (2019). pretreatment for biorefineries: a review of common methods for efficient utilisation of lignocellulosic materials. biotechnology for biofuels, 12(1), 1-26. [92]. yoon, s. y., kim, b. r., han, s. h., & shin, s. j. (2015). different response between woody core and bark of goat willow (salix caprea l.) to concentrated phosphoric acid pretreatment followed by enzymatic saccharification. energy, 81, 21-26. [93]. behera, s., arora, r., nandhagopal, n., & kumar, s. (2014). importance of chemical pretreatment for bioconversion of lignocellulosic biomass. renewable and sustainable energy reviews, 36, 91-106. [94]. solarte-toro, j. c., romerogarcía, j. m., martínez-patiño, j. c., ruiz-ramos, e., castro-galiano, e., & cardona-alzate, c. a. (2019). acid pretreatment of lignocellulosic biomass for energy vectors production: a review focused on operational conditions and techno-economic assessment for bioethanol production. renewable and sustainable energy reviews, 107, 587-601 [95]. zu, s., li, w. z., zhang, m., li, z., wang, z., jameel, h., & chang, h. m. (2014). pretreatment of corn stover for sugar production using dilute hydrochloric acid followed by lime. bioresource technology, 152, 364-370. [96]. yang, g., & wang, j. (2019). ultrasound combined with dilute acid pretreatment of grass for improvement of fermentative hydrogen production. bioresource technology, 275, 10-18. [97]. kim, i., seo, y. h., kim, g. y., & han, j. i. (2015). co-production of bioethanol and biodiesel from corn stover pretreated with nitric acid. fuel, 143, 285-289. [98]. skiba, e. a., budaeva, v. v., baibakova, o. v., zolotukhin, v. n., & sakovich, g. v. (2017). dilute nitric-acid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 256 pretreatment of oat hulls for ethanol production. biochemical engineering journal, 126, 118-125. [99]. ascencio, j. j., chandel, a. k., philippini, r. r., & da silva, s. s. (2019). comparative study of cellulosic sugars production from sugarcane bagasse after dilute nitric acid, dilute sodium hydroxide and sequential nitric acidsodium hydroxide pretreatment. biomass conversion and biorefinery, 1-10. [100]. bustos, g., ramírez, j. a., garrote, g., & vázquez, m. (2003). modeling of the hydrolysis of sugar cane bagasse with hydrochloric acid. applied biochemistry and biotechnology, 104(1), 51-68. [101]. schell, d. j., farmer, j., newman, m., & mcmillan, j. d. (2003). dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor. applied biochemistry and biotechnology, 105(1-3), 69-85. [102]. baboukani, b. s., vossoughi, m., & alemzadeh, i. (2012). optimisation of diluteacid pretreatment conditions for enhancement sugar recovery and enzymatic hydrolysis of wheat straw. biosystems engineering, 111(2), 166-174. [103]. kshirsagar, s. d., waghmare, p. r., loni, p. c., patil, s. a., & govindwar, s. p. (2015). dilute acid pretreatment of rice straw, structural characterization and optimization of enzymatic hydrolysis conditions by response surface methodology. rsc advances, 5(58), 4652546533. [104]. canilha, l., santos, v. t., rocha, g. j., e silva, j. b. a., giulietti, m., silva, s. s., ... & carvalho, w. (2011). a study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid. journal of industrial microbiology & biotechnology, 38(9), 1467-1475. [105]. avci, a., saha, b. c., dien, b. s., kennedy, g. j., & cotta, m. a. (2013). response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production. bioresource technology, 130, 603-612. [106]. nair, r. b., lundin, m., brandberg, t., lennartsson, p. r., & taherzadeh, m. j. (2015). dilute phosphoric acid pretreatment of wheat bran for enzymatic hydrolysis and subsequent ethanol production by edible fungi neurospora intermedia. industrial crops and products, 69, 314-323. [107]. nieves, i. u., geddes, c. c., miller, e. n., mullinnix, m. t., hoffman, r. w., fu, z., ingram, l. o. (2011). effect of reduced sulfur compounds on the fermentation of phosphoric acid pretreated sugarcane bagasse by ethanologenic escherichia coli. bioresource technology, 102(8), 5145-5152. [108]. castro, e., nieves, i. u., mullinnix, m. t., sagues, w. j., hoffman, r. w., fernández-sandoval, m. t., ingram, l. o. (2014). optimization of dilute-phosphoric-acid steam pretreatment of eucalyptus benthamii for biofuel production. applied energy, 125, 76-83. [109]. rusanen, a., lappalainen, k., kärkkäinen, j., tuuttila, t., mikola, m., & lassi, u. (2019). selective hemicellulose hydrolysis of scots pine sawdust. biomass conversion and biorefinery, 9(2), 283-291. [110]. lee, j. w., rodrigues, r. c., kim, h. j., choi, i. g., & jeffries, t. w. (2010). the roles of xylan and lignin in oxalic acid pretreated corncob during separate enzymatic hydrolysis and ethanol fermentation. bioresource technology, 101(12), 4379-4385. [111]. jeong, s. y., & lee, j. w. (2016). optimization of pretreatment condition for ethanol production from oxalic acid pretreated biomass by response surface methodology. industrial crops and products, 79, 1-6. [112]. harmsen, p. f. h., huijgen, w., bermudez, l., & bakker, r. (2010). literature review of physical and chemical pretreatment processes for lignocellulosic biomass (no. 1184). wageningen ur-food & biobased research. [113]. cheng, y. s., zheng, y., yu, c. w., dooley, t. m., jenkins, b. m., & vandergheynst, j. s. (2010). evaluation of high solids alkaline pretreatment of rice straw. applied biochemistry and biotechnology, 162(6), 1768-1784. [114]. ibrahim, m. m., el-zawawy, w. k., abdel-fattah, y. r., soliman, n. a., & agblevor, f. a. (2011). comparison of alkaline pulping with steam explosion for glucose production from rice straw. carbohydrate polymers, 83(2), 720-726. [115]. mcintosh, s., & vancov, t. (2010). enhanced enzyme saccharification of sorghum bicolor straw using dilute alkali pretreatment. bioresource technology, 101(17), 6718-6727. [116]. sills, d. l., & gossett, j. m. (2011). assessment of commercial hemicellulases for saccharification of alkaline pretreated perennial biomass. bioresource technology, 102(2), 13891398. [117]. zhao, y., wang, y., zhu, j. y., ragauskas, a., & deng, y. (2008). enhanced enzymatic hydrolysis of spruce by alkaline food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 257 pretreatment at low temperature. biotechnology and bioengineering, 99(6), 1320-1328. [118]. liang, y., siddaramu, t., yesuf, j., & sarkany, n. (2010). fermentable sugar release from jatropha seed cakes following lime pretreatment and enzymatic hydrolysis. bioresource technology, 101(16), 64176424. [119]. park, j. y., shiroma, r., al-haq, m. i., zhang, y., ike, m., arai-sanoh, y., tokuyasu, k. (2010). a novel lime pretreatment for subsequent bioethanol production from rice straw–calcium capturing by carbonation (cacco) process. bioresource technology, 101(17), 6805-6811. [120]. zhang, x., xu, j., & cheng, j. j. (2011). pretreatment of corn stover for sugar production with combined alkaline reagents. energy & fuels, 25(10), 4796-4802. [121]. han, l., feng, j., zhang, s., ma, z., wang, y., & zhang, x. (2012). alkali pretreated of wheat straw and its enzymatic hydrolysis. brazilian journal of microbiology, 43(1), 53-61. [122]. janu, k. u., sindhu, r., binod, p., kuttiraja, m., sukumaran, r. k., & pandey, a. (2011). studies on physicochemical changes during alkali pretreatment and optimization of hydrolysis conditions to improve sugar yield from bagasse. [123]. sindhu, r., kuttiraja, m., binod, p., sukumaran, r. k., & pandey, a. (2014). physicochemical characterization of alkali pretreated sugarcane tops and optimization of enzymatic saccharification using response surface methodology. renewable energy, 62, 362-368. [124]. binod, p., kuttiraja, m., archana, m., janu, k. u., sindhu, r., sukumaran, r. k., & pandey, a. (2012). high temperature pretreatment and hydrolysis of cotton stalk for producing sugars for bioethanol production. fuel, 92(1), 340-345. [125]. trevorah, r. m., & othman, m. z. (2015). alkali pretreatment and enzymatic hydrolysis of australian timber mill sawdust for biofuel production. journal of renewable energy, 2015. [126]. kuttiraja, m., sindhu, r., varghese, p. e., sandhya, s. v., binod, p., vani, s., sukumaran, r. k. (2013). bioethanol production from bamboo (dendrocalamus sp.) process waste. biomass and bioenergy, 59, 142-150. [127]. sharma, r., palled, v., sharmashivappa, r. r., & osborne, j. (2013). potential of potassium hydroxide pretreatment of switchgrass for fermentable sugar production. applied biochemistry and biotechnology, 169(3), 761-772. [128]. kaar, w. e., & holtzapple, m. t. (2000). using lime pretreatment to facilitate the enzymic hydrolysis of corn stover. biomass and bioenergy, 18(3), 189-199. [129]. chang, v. s., nagwani, m., kim, c. h., & holtzapple, m. t. (2001). oxidative lime pretreatment of high-lignin biomass. applied biochemistry and biotechnology, 94(1), 1-28. [130]. li, x., & kim, t. h. (2011). low-liquid pretreatment of corn stover with aqueous ammonia. bioresource technology, 102(7), 47794786. [131]. li, y., merrettig‐bruns, u., strauch, s., kabasci, s., & chen, h. (2015). optimization of ammonia pretreatment of wheat straw for biogas production. journal of chemical technology & biotechnology, 90(1), 130138. [132]. travaini, r., otero, m. d. m., coca, m., da-silva, r., & bolado, s. (2013). sugarcane bagasse ozonolysis pretreatment: effect on enzymatic digestibility and inhibitory compound formation. bioresource technology, 133, 332-339. [133]. garcía-cubero, m. t., coca, m., bolado, s., & gonzález-benito, g. (2010). chemical oxidation with ozone as pretreatment of lignocellulosic materials for bioethanol production. chemical engineering, 21. [134]. kojima, y., & yoon, s. l. (2008). improved enzymatic hydrolysis of waste paper by ozone pretreatment. journal of material cycles and waste management, 10(2), 134. [135]. zhao, x., cheng, k., & liu, d. (2009). organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. applied microbiology and biotechnology, 82(5), 815. [136]. zhang, z., harrison, m. d., rackemann, d. w., doherty, w. o., & o'hara, i. m. (2016). organosolv pretreatment of plant biomass for enhanced enzymatic saccharification. green chemistry, 18(2), 360-381. [137]. tang, c., chen, y., liu, j., shen, t., cao, z., shan, j., ... & ying, h. (2017). sustainable biobutanol production using alkalicatalyzed organosolv pretreated cornstalks. industrial crops and products, 95, 383392. [138]. tang, c., shan, j., chen, y., zhong, l., shen, t., zhu, c., & ying, h. (2017). organic amine catalytic organosolv pretreatment of corn stover for enzymatic saccharification and high-quality lignin. bioresource technology, 232, 222-228. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 258 [139]. vergara, p., wojtusik, m., revilla, e., ladero, m., garcia-ochoa, f., & villar, j. c. (2018). wheat straw fractionation by ethanol-water mixture: optimization of operating conditions and comparison with diluted sulfuric acid pretreatment. bioresource technology, 256, 178-186. [140]. salapa, i., katsimpouras, c., topakas, e., & sidiras, d. (2017). organosolv pretreatment of wheat straw for efficient ethanol production using various solvents. biomass and bioenergy, 100, 10-16. [141]. wildschut, j., smit, a. t., reith, j. h., & huijgen, w. j. (2013). ethanol-based organosolv fractionation of wheat straw for the production of lignin and enzymatically digestible cellulose. bioresource technology, 135, 58-66. [142]. asadi, n., & zilouei, h. (2017). optimization of organosolv pretreatment of rice straw for enhanced biohydrogen production using enterobacter aerogenes. bioresource technology, 227, 335-344. [143]. sannigrahi, p., pu, y., & ragauskas, a. (2010). cellulosic biorefineries—unleashing lignin opportunities. current opinion in environmental sustainability, 2(5-6), 383-393. [144]. salapa, i., topakas, e., & sidiras, d. (2018). simulation and optimization of barley straw organosolv pretreatment. industrial crops and products, 113, 80-88. [145]. kim, y., yu, a., han, m., choi, g. w., & chung, b. (2010). ethanosolv pretreatment of barley straw with iron (iii) chloride for enzymatic saccharification. journal of chemical technology & biotechnology, 85(11), 1494-1498. [146]. teramura, h., sasaki, k., oshima, t., kawaguchi, h., ogino, c., sazuka, t., & kondo, a. (2018). effective usage of sorghum bagasse: optimization of organosolv pretreatment using 25% 1-butanol and subsequent nanofiltration membrane separation. bioresource technology, 252, 157-164. [147]. ostovareh, s., karimi, k., & zamani, a. (2015). efficient conversion of sweet sorghum stalks to biogas and ethanol using organosolv pretreatment. industrial crops and products, 66, 170-177. [148]. mesa, l., gonzález, e., ruiz, e., romero, i., cara, c., felissia, f., & castro, e. (2010). preliminary evaluation of organosolv pre-treatment of sugar cane bagasse for glucose production: application of 23 experimental design. applied energy, 87(1), 109-114. [149]. zhang, h., zhang, s., yuan, h., lyu, g., & xie, j. (2018). fecl3-catalyzed ethanol pretreatment of sugarcane bagasse boosts sugar yields with low enzyme loadings and short hydrolysis time. bioresource technology, 249, 395401. [150]. sun, s., sun, s., cao, x., & sun, r. (2016). the role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. bioresource technology, 199, 49-58. [151]. mou, h., & wu, s. (2017). comparison of hydrothermal, hydrotropic and organosolv pretreatment for improving the enzymatic digestibility of bamboo. cellulose, 24(1), 85-94. [152]. romaní, a., ruiz, h. a., teixeira, j. a., & domingues, l. (2016). valorization of eucalyptus wood by glycerol-organosolv pretreatment within the biorefinery concept: an integrated and intensified approach. renewable energy, 95, 1-9. [153]. teramoto, y., lee, s. h., & endo, t. (2009). cost reduction and feedstock diversity for sulfuric acid-free ethanol cooking of lignocellulosic biomass as a pretreatment to enzymatic saccharification. bioresource technology, 100(20), 4783-4789. [154]. lee, j. m. (2011). solvent properties of piperidinium ionic liquids. chemical engineering journal, 172(2-3), 1066-1071. [155]. lee, j. m., ruckes, s., & prausnitz, j. m. (2008). solvent polarities and kamlet− taft parameters for ionic liquids containing a pyridinium cation. the journal of physical chemistry b, 112(5), 1473-1476. [156]. mäki-arvela, p., anugwom, i., virtanen, p., sjöholm, r., & mikkola, j. p. (2010). dissolution of lignocellulosic materials and its constituents using ionic liquids—a review. industrial crops and products, 32(3), 175201. [157]. lee, j. m., & prausnitz, j. m. (2010). polarity and hydrogen-bond-donor strength for some ionic liquids: effect of alkyl chain length on the pyrrolidinium cation. chemical physics letters, 492(1-3), 55-59. [158]. fu, d., mazza, g., & tamaki, y. (2010). lignin extraction from straw by ionic liquids and enzymatic hydrolysis of the cellulosic residues. journal of agricultural and food chemistry, 58(5), 2915-2922. [159]. lee, s. h., doherty, t. v., linhardt, r. j., & dordick, j. s. (2009). ionic liquid‐mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. biotechnology and bioengineering, 102(5), 1368-1376. [160]. nguyen, t. a. d., kim, k. r., han, s. j., cho, h. y., kim, j. w., park, s. m., ... & food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 vasile-florin ursachi, gheorghe gutt, physical and chemical pretreatments used for bioethanol production from lignocellulosic biomass review, food and environment safety, volume xix, issue 3 – 2020, pag. 237 259 259 sim, s. j. (2010). pretreatment of rice straw with ammonia and ionic liquid for lignocellulose conversion to fermentable sugars. bioresource technology, 101(19), 7432-7438. [161]. zakrzewska, m. e., bogelłukasik, e., & bogel-łukasik, r. (2010). solubility of carbohydrates in ionic liquids. energy & fuels, 24(2), 737-745. [162]. holm, j., & lassi, u. (2011). ionic liquids in the pretreatment of lignocellulosic biomass. rijeka, croatia: intech open access publisher. [163]. binder, j. b., & raines, r. t. (2010). fermentable sugars by chemical hydrolysis of biomass. proceedings of the national academy of sciences, 107(10), 4516-4521. [164]. tadesse, h., & luque, r. (2011). advances on biomass pretreatment using ionic liquids: an overview. energy & environmental science, 4(10), 3913-3929. [165]. li, c., wang, q., & zhao, z. k. (2008). acid in ionic liquid: an efficient system for hydrolysis of lignocellulose. green chemistry, 10(2), 177-182. [166]. weerachanchai, p., leong, s. s. j., chang, m. w., ching, c. b., & lee, j. m. (2012). improvement of biomass properties by pretreatment with ionic liquids for bioconversion process. bioresource technology, 111, 453-459. [167]. shafiei m, karimi k, taherzadeh mj (2010) pretreatment of spruce and oak by nmethylmorpholine-n-oxide (nmmo) for efficient conversion of their cellulose to ethanol. bioresour technol 101:4914–491 [168]. lennartsson pr, niklasson c, taherzadeh mj (2011) a pilot study on lignocelluloses to ethanol and fish feed using nmmo pretreatment and cultivation with zygomycetes in an air-lift reactor. bioresour technol 102:4425–4432 [169]. zdanowicz, m., wilpiszewska, k., & spychaj, t. (2018). deep eutectic solvents for polysaccharides processing. a review. carbohydrate polymers, 200, 361-380. [170]. dominguez de maría, p. d., & maugeri, z. (2011). ionic liquids in biotransformations: from proof-of-concept to emerging deep-eutectic-solvents. current opinion in chemical biology, 15(2), 220-225. [171]. smith, e. l., abbott, a. p., & ryder, k. s. (2014). deep eutectic solvents (dess) and their applications. chemical reviews, 114(21), 11060-11082. [172]. zhang, c. w., xia, s. q., & ma, p. s. (2016). facile pretreatment of lignocellulosic biomass using deep eutectic solvents. bioresource technology, 219, 1-5 [173]. xu, g. c., ding, j. c., han, r. z., dong, j. j., & ni, y. (2016). enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation. bioresource technology, 203, 364-369. [174]. pan, m., zhao, g., ding, c., wu, b., lian, z., & lian, h. (2017). physicochemical transformation of rice straw after pretreatment with a deep eutectic solvent of choline chloride/urea. carbohydrate polymers, 176, 307314. [175]. jablonský, m., škulcová, a., kamenská, l., vrška, m., & šima, j. (2015). deep eutectic solvents: fractionation of wheat straw. bioresources, 10(4), 8039-8047. [176]. yang, b., & wyman, c. e. (2008). pretreatment: the key to unlocking low‐cost cellulosic ethanol. biofuels, bioproducts and biorefining: innovation for a sustainable economy, 2(1), 26-40. [177]. lebaka, v. r. (2013). potential bioresources as future sources of biofuels production: an overview. biofuel technologies: recent developments, 223-258. [178]. tomás-pejó, e., alvira, p., ballesteros, m., & negro, m. j. (2011). pretreatment technologies for lignocellulose-tobioethanol conversion. in biofuels (pp. 149-176). academic press. 267 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 267 274 effect of rosemary extract on thermal stability of sunflower oil *artan gashi 1 , mila arapcheska 1 , fatos rexhepi 2 1 faculty of biotechnical sciences, “st. kliment ohridski“university, bitola, republic of north macedonia 2 department of technology, university of mitrovica “isa boletini” ukshin kovaqica, mitrovica, 40000, kosovo agashi02@hotmail.com *corresponding author received 22nd september 2020, accepted 29th december 2020 abstract: the purpose of this study was to evaluate the effect of rosemary extract (rosemarinus officinalis l.) on thermal stability of sunflower oil. samples of sunflower oil and sunflower oil containing 0.1% rosemary extract were subjected to three successive cycles of thermal heating at burning point for 8 hours, followed by cooling at room temperaturefor16 hours. after each cycle, samples of oils were taken for analysis ofdensity, viscosity and ftir spectroscopy measurements. analyzed parameters were used as indicators of structural changes in analyzed samples of oils. according obtained results the addition of rosemary extract in sunflower oil effectively protect its triglyceride structure and level of unsaturation during thermal heating and increase its thermal stability. performed ftir spectroscopy has indicated the role of rosemary extract as a natural protective agent. samples of sunflower oil with rosemary extract have shown improved thermal stability compared with pure sunflower oil. keywords:sunflower oil, thermal stability, rosemary extract, natural antioxidants,ftir spectroscopy 1. introduction vegetable oils, especially cold pressed are an essential component of a healthy diet. polyunsaturated fatty acids contained in vegetable oils are important substances from physiological and nutritional points of view. despite their health benefits, they can also be considered as a reason of instability of oils during the technological processes of their production, thermal heating, during cooking or during the storage [1]. double bounds present in chemical structure in edible oils make them prone to oxidative reactions, which are major cause of deterioration of oils during the period of heating. thermal heating processes may compromise oil`s stability, causing modifications in the chemical structure and organoleptic properties. moreover, these processes result in the formation of secondary, potentially toxic compounds [2-4]. diets in developed countries contain substantial quantities of oils subjected to different processing and heating treatments [5]. high temperature treatment such as deep frying oil is one of the most common http://www.fia.usv.ro/fiajournal mailto:agashi02@hotmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 268 food processing methods used worldwide. the quality parameters of the edible oils such as: taste, texture, shelf life and nutritional composition are affected as a result of complex chemical reactions i.e. oxidation, hydrolysis, and polymerization, which are occurred during the high temperature heating process [6]. heating in the presence of air cause oxidative and thermal degradations in polyunsaturated triacylglycerolsin the oils, and induce the formation of polar compounds and polymers [5]. the changes linked to the process of heating itself, depends of: temperature, duration of heating, type of heating (continuous or discontinuous), type of used oil, its initial quality, unsaturation level, antioxidant content and presence of additives, etc. [7, 8]. lipid oxidation occurring in food products is responsible for rancid odors and flavors of the products, and leads to consequent decrease in their nutritional value and safety, and is one of the major concerns in food technology. the problem of ensuring a high quality of lipids and lipid-containing products and prolonging their storage time is directly associated with their optimum stabilization by addition of suitable antioxidants. this is very important from aspect of human health protection, and also it is economically important [9, 10]. due to their ability to protect foods containing oils and fats, antioxidants play an important role in food processing [2]. they are added to food in order to retard, reduce or prevent oxidative deterioration. in food industry it is not unusual the use of synthetic antioxidants. however, the utilization of some of them has been questioned because of their toxicity [11]. according to recent literature data there concerns about the safety and health risks associated with the use of some of synthetic antioxidants. for this reason, the use of herbs and spices to inhibit the development of oxidative reactions in food systems and improve the oxidative stability of food has recently become popular. plants, including herbs and spices, are known to have many phytochemicals that could be potential sources of natural antioxidants, which can retard oxidative rancidity via the following pathways: capturing of free radicals, decomposing/deoxidizing peroxides, and scavenging oxygen [9,12, 13]. мany species of the lamiaceae family have strong antioxidant capacity. rosemary (rosmarinus officinalis l.) extract has potentanti oxidant activity and it is widely used in the food industry. its antioxidant activity has been associated with the presence of several phenolic compounds: diterpenes, triterpenes, carnosic acid, carnosol, rosmarinic acid, rosmanol,rosmariquinone, rosmaridiphenol and flavonoids [14-16]. the effects of high temperatures on vegetable oils with high contents of unsaturated fatty acids, especially polyunsaturated fatty acids are major concern both for product quality and for human health. in the diet, sunflower oil is widely used, and it is a valuable source of essential linoleic acid [17]. it is one of the most important vegetable oils employed for deep-frying. because of higher content of unsaturated fatty acids, especially polyunsaturated fatty acids, sunflower oil is more susceptible to oxidation. the purpose of this study was to evaluate the effect of rosemary extract on thermal stability of sunflower oil. 2. matherials and methods 2.1. samples sunflower oil was purchased from local market in kosovo. according to the manufacturer`s specifications, the oil was free from antioxidants. the samples were prepared as: sunflower oil (control) and sunflower oil with 0.1% rosemary extract. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 269 the samples were subjected to three successive cycles of thermal heating at burning point for 8 hours, followed by cooling at room temperature for 16 hours. after each cycle, samples of oils were taken for analysis of density, viscosity and fourier transform infrared (ftir) spectroscopy measurements. analyzed parameters were used as indicators of structural changes in analyzed samples of oils. 2.2. chemicals folin ciocalteu reagent, dpph (2, 2diphenyl-1-picrylhydrazyl), gallic acid and sodium carbonate were purchased from sigma – aldrich. gallic acid stock solution in ethanol (1 mg/ml) was prepared and used for the preparation of working standard solutions necessary for creation of calibration curve. 2.3. characterisation of rosemary extract rosemary extract was obtained from rosemary (rosemarinus officinalis l.) leaves by steam distillation. determination of total phenolic content of rosemary extract the total phenolic content in the rosemary extract was determined using folin–ciocalteu reagent [16]. a volume of 1 ml of rosemary extract solution, prepared in ethanol at a concentration of 0.1 mg/ml, was mixed with 7.5 ml folin–ciocalteu reagent diluted 10 times. the mixture was left 5 min at room temperature before mixing with 7.5 ml of 60 mg/ml of aqueous sodium bicarbonate (na2co3) solution. the prepared mixture was placed at room temperature at darkness for 2 h. the absorbance of the sample was measured at 725 nm using spectrophotometer (jasco v630). the concentration was determined using gallic acid as reference and the result is expressed as mg of gallic acid equivalents per gram extract (mg gae/g). antioxidant activity of rosemary extract– the antioxidant activity of rosemary extract was determined using dpph (2, 2diphenyl-1-picrylhydrazyl). for this purpose, 0.3 ml aliquot of rosemary extract was mixed with freshly prepared 1.5 ml of dpph solution in ethanol (0.1 mmol/l). the solution was stored at 25 °c for 30 min at dark and then the absorbance was measured at 517 nm by uv-vis spectrophotometer. the control was conducted in the same manner, except that distilled water was used instead of sample (rosemary extract). the ic50 is the concentration of an antioxidant that is required to inhibit 50% of the initial dpph radicals under given experimental conditions. the dpph scavenging capacity assay value was calculated according to the formula (1): ic50 % = [1 – (asamples / acontrol)] × 100 (1) 2.4. density and viscosity measurements after each cycle of thermal heating, samples of oils (with and without rosemary extract) were taken for analysis of viscosity and density. density of oil samples was measured by pycnometer using 10 ml of sample at 25°c, and viscosity was measured using ostwald viscometer (astm d445 england). 2.5. ftir measurements an irafinity-1 shimadzu fourier transform infrared (ftir) spectrophotometer equipped with a dlatgs (deuterated l-alanine triglycene sulfate) detector and calcium fluoride caf2 as transparent window, managed by the ir software was used to acquire ftir spectra of analyzed samples of sunflower oil (with and without rosemary extract). samples for analysis were taken after each cycle of thermal heating. spectra were recorded in the spectral region from 4000 to 1000 cm-1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 270 with a resolution of 4 cm-1. ftir spectral bands were assigned to specific vibrations based on literature data and software spectral library. 3. results and discussion in recent years, natural antioxidants from plant origin have attracted considerable attention due to their beneficial functional and nutritional effects including antioxidant activity. since ancient times, herbs have been used not just for food flavorings, but also for its medicinal antiseptical, and preservative properties. preservative effects of herbal extracts suggest the presence of antimicrobial and antioxidant constituents in their composition [18, 19]. in this study, rosemary extract was obtained by steam distillation from rosemarinus officinalis l. leaves. the physicochemical properties of the obtained extract are presented in table 1. the density of the exract was 0.87 (g/cm3), and viscosity 67±2 mp. the folin-ciocalteu assay was used for determination of the total phenolic content, which in the analysed rosemary extract was 21.6±0.5 mg gae/g. the antioxidant activity is defined as capacity of antioxidants to protect a biological system against the harmful effects of oxidative processes. the antioxidant activity of analysed extract was 76±2.5%. physicochemical characterization of rosemary extract indicates high content of polyphenols compounds as well as high antioxidant activity of analyzed extract, and obtained results are in accordance with the literature data [20]. table 1. physicochemical parameters of rosemary extract physicochemical parameters rosemary extract density (g/cm3) 0.87 viscosity (mp) 67±2 polyphenols (mg gae/g) 21.6±0.5 antioxidant activity (%) 76±2.5 effect of rosemary extract on thermal stability of sunflower oil was studied through analysis of density, viscosity, and ftir measurements. analyzed parameters were used as indicators of structural changes in analyzed samples of oils during their thermal heating. comparative analysis of parameters between samples of two types of oil, sunflower oil (sfo) and sunflower oil with rosemary extract (sfo + re) was done. density changes during the heating of sunflower oil samples with and without rosemary extract are presented in figure 1. differences are very clear between the samples. during the heating of oil several reactions are occurred. these reactions lead to decomposition of natural constituents into oxidative and nonoxidative degradation products that can polimerize, which later results with density increase [21]. the viscosity changes of analyzed samples of oil during the thermal heating are presented in figure 2. according obtained results during the first heating cycle, decrease of viscosity was noted in both type of oil (sfo and sfo + re). this should be due to the weakening of the intermolecular forces between oil components which lead to the oil degradation. in the second and third heating cycles an increase of viscosity values was noted. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 271 fig. 1. density changes in sunflower oil (sfo) and sunflower oil with 0.1% rosemary extract (sfo + re) during the thermal heating fig.2. viscosity changes in sunflower oil (sfo) and sunflower oil with 0.1% rosemary extract (sfo + re) during the thermal heating according literature data high temperature process such as frying produces compounds (polymers) with high molecular weight. polymerization products decrease the stability of frying oils and can be used to measure oil fry life [22]. results of this study indicate that the increase of oil viscosity is a consequence of increased amount of polymerization compounds. the changes in density and visosity values are more rapidly in the pure sunflower oil (sfo) compared with mixture (sfo + re) (figure 1 and figure 2), this is probably due to the effect of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 272 polyphenols content and antioxidative activity of rosemary extract on higher thermal stability of the mixture oil. another procedurе used in this study for evaluation of chemical changes in analysed samples of oils (sfo and sfo+re) during the thermal heating was ftir spectroscopy. the characteristic functional group used in this research and their wavenumbers are presented in table 2. ftir spectral bands were assigned to specific vibrations based on software spectral library and literature data [23, 24]. table 2. ftir absorbance bands and their characteristic functional groups wavenumbers cm-1 characteristic group and mode of vibration 3008 =c–h stretching symmetric vibration of the cis double bonds 2925 −ch(ch2) asymmetric stretching vibration of the aliphatic ch2 group 2854 −ch(ch2) symmetric stretching vibration of the aliphatic ch2 group 1745 −c = o stretching vibration of ester carbonyl functional groups of triglycerides ftir characterization of analysed samples of oils during the first heating cycle is presented in figure 3. obtained ftir spectra peaks are common for the majority of vegetable oils. intesity of peak at 3007cm-1 can be interpreted by the unsaturation level. analysis of ftir spectra based on carbonyl group at 1745cm-1 indicate triglycerides as mayor compounds in anlysed oil`s samples. fig. 3. ftir spectra of sunflower oil and sunflower oil with 0.1% rosemary extract after 8 hours of thermal heating food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 273 fig. 4. intesity ratio at 3008/2854 cm-1 during the thermal heating of sunflower oil (sfo) and sunflower oil with 0.1% rosemary extract (sfo + re) in this study as an indicator for the chemical changes in analysed samples of oil during thermal heating the intensity ratio of selected peaks is used (figure 4). oxidative reactions, are major cause of deterioration of vegetable oils during the period of heating. usually as a result of oxidation, double bonds are converted in single. the decreasy of ratio intensity 3008/2854 cm-1 means conversion of double bonds in single. according obtained results rapid decrease of ratio 3008/2854 cm-1 was noted in sunflower oil, especially during the first heating cycle, which was not the case for sunflower oil with rosemary extract (the decrease was more slowly). 4. conclusion the results obtained in the present study showed that the rosemary extract is a promising source of natural antioxidants for vegetable oils. it can protect the oils against oxidative processes, even at high temperatures. according obtanied results rosemary extract was more effective in delaying the oxidation of sunflower oil. samples of sunflower oil with rosemary extract have shown improved thermal stability compared with pure sunflower oil. in general rosemary extract in sunflower oil effectively protect its triglyceride structure and level of unsaturation (with minor structural changes). ftir spectroscopy used in this study was proved to be suitable method for the analysis of structural changes vegetable oil during heating. this methodology brings advantages over conventional laboratory procedures and allows rapid, specific, noninvasive analyses independent of number of chemicals and without requirement of special sample preparation. 5. references [1]. vaskova h., buckova m., thermal degradation of vegetable oils: spectroscopic measurement and analysis, procedia engineering, 100: 630–635, (2015) [2]. casarotti s., jorge n., antioxidant activity of rosemary extract in soybean oil under thermoxidation, journal of food processing and preservation. 38: 136–145, (2014) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 artan gashi, mila arapcheska, fatos rexhepi, effect of rosemary extract on thermal stability of sunflower oil, food and environment safety, volume xix, issue 4 – 2020, pag. 267 274 274 [3]. cordeiro a.m.t.m., medeiros m.l., santos n.a., soledade l.e.b., pontes l.f.b.l., souza a.l., queiroz n., souza a.g., rosemary (rosmarinus officinalis l.) extract. thermal study and evaluation of the antioxidant effect on vegetable oils. journal of thermal analysis and calorimetry, 113:889-895, (2014) [4]. shahidi f., zhong y., lipid oxidation and improving the oxidative stability. chemical society reviews, 39: 4067–79, (2010) [5]. sanchez-muniz f.j., oils and fats: changes due to culinary and industrial processes, international journal for vitamin and nutrition research, 76 (4): 230–237, (2006) [6]. zhang q., saleh a.s., chen j., shen q., chemical alterations taken place during deep-fat frying based on certain reaction products: a review, chemistry and physics of lipids 165: 662– 681, (2012) [7]. dobarganes c., márquez-ruiz g., possible adverse effects of frying with vegetable oils, british journal of nutrition, 113 suppl 2: s49-s57, (2015) [8]. choe e., min d.b., chemistry of deep-fat frying oils, journal of food science, 72 (5): 77-86, (2007) [9]. turan s., effects of some plant extracts on the oxidative stability of canola oil and its purified triacylglycerols, journal of food quality, 37 (4): 247–258, (2014) [10]. yanishlieva n.v., marinova e., pokorný j., natural antioxidants from herbs and spices, european journal of lipid science and technology, 108: 776–793, (2006) [11]. gordon m.h., l kouiimska l., the effects of antioxidants on changes in oils during heating and deep frying, journal of the science of food and agriculture, 68: 347353, (1995) [12]. yang y., song x., sui x., qi b., wang z., li y., jiang l., rosemary extract can be used as a synthetic antioxidant to improve vegetable oil oxidative stability, industrial crops and products, 80: 141–147, (2016) [13]. sayyad r., jafari s., ghomi m., thermoxidative stability of soybean oil by natural extracted antioxidants from rosemary (rosmarinus officinalis l.), international journal of food properties, 20 (2): 436–446, (2017) [14]. filip s., hribar j., vidrih r., influence of natural antioxidants on the formation of trans fatty-acid isomers during heat treatment of sunflower oil, european journal of lipid science and technology, 113: 224-230, (2011) [15]. senanayake s.p.j.n., rosemary extract as a natural source of bioactive compounds. journal of food bioactives, 2: 51–57, (2018). [16]. chammema n., saoudi s., sifaoui i., sifi s., de person m., abderraba m., moussa f., hamdia м., improvement of vegetable oils quality in frying conditions by addingrosemary extract, industrial crops and products, 74: 592–599, (2015) [17]. fan l., eskin n.a.m., the use of antioxidants in the preservation of edible oils. in handbook of antioxidants for food preservation, shahidi, f., eds; woodhead publishing, 373-388, (2015) [18]. erkan n., ayranci g., ayranci e., antioxidant activities of rosemary (rosmarinus officinalis l.) extract, blackseed (nigella sativa l.) essential oil, carnosic acid, rosmarinic acid and sesamol, food chemistry, 110 (1): 76–82 (2008) [19]. taghvaei m., jafari s.m., application and stability of natural antioxidants in edible oils in order to substitute synthetic additives, journal of food science and technology, 52(3): 1272–1282, (2015) [20]. efsa panel on food additives and nutrient sources added to food (efsa ans panel). refined exposure assessment of extracts of rosemary (e 392) from its use as food additive, efsa journal, 16: e05373 (2018) [21]. xu y., qian s., liu q., wang z., oxidation stability assessment of a vegetable transformer oil under thermal aging, ieee transactions on dielectrics and electrical insulation, 21, (2): 683–692 (2014) [22]. kumar d., singh a., tarsikka p.s., interrelationship between viscosity and electrical properties for edible oils, journal of food science and technology, 50, (3): 549– 554, (2013) [23]. vlachos n., skopelitis y., psaroudaki m., konstantinidou v., chatzilazarou a., tegou e., applications of fourier transform–infrared spectroscopy to edible oils, analytica chimica acta. 573–574: 459–465, (2006) [24]. rexhepi f., surleva a., hyseni a., bruçi m., kodraliu b., comprehensive investigation of thermal degradation characteristics and properties changes of plant edible oils by ftir-spectroscopy, acta chem. iasi, 27 (2): 263–286, (2019) issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xxi, issue 1 31 march 2022 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania doi: https://doi.org/10.4316/fens.2021.035 322 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 322 332 monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater toufik djebassi 1 , ilhem abdeslam 2 , hassen djabari 3 , amor hamad 4 , * chemseddine fehdi 5 1,2,4,5 laboratory of water and environment, université larbi tébessi, tébessa 12 000 algeria, djebassi.toufik@univ-tebessa.dz, abdeslam.ilhem@yahoo.fr, hsamir2001@gmail.com, fehdi@yahoo.fr 5 departement of geology, université kasdi merbeh ouergla, 30 000 algeria, d_hacene@yahoo.fr *corresponding author received 10th july 2021, accepted 20th december 2021 abstract: the study area is characterized by low rainfall and a significant outcrop of salt formations, which significantly compromises the use of water for consumption and other purposes (agriculture and industry). groundwater contamination affects both water quality and human health. hence it is essential for continuous monitoring of the quality of groundwater so that contamination can be minimized. application of pollution index of groundwater (pig) to the problem of estimating groundwater contamination with respect to their drinking water quality standards has been done by the investigation of 58 water sample. the computed values of pollution index of groundwater (pig) for the study area vary from 0,457 to 8.190. spatial variation map has been prepared using gis. the variation map shows that most of the study area accounts for high and very high pollution zones, which is mainly due to geogenic, anthropogenic factors and also it is compounded due to mining activities. geochemical ratios (na+: cl-, hco3 : cl-, na+: ca2+ and mg2+: ca2+) also form the quantitative basis of the index. this manuscript synthesizes a set of data that allowed us to make a classification of the contamination zones, using pig tool, of the study area. keywords: drinking-water, pollution index of groundwater, semi-arid area, tebessa, algeria. 1. introduction water is considered as rare, precious and indispensable source in many domains, such as: industry, agriculture, construction, etc. water chemistry can be used to help understand effects natural and human activities. the hydrochemical composition of water inside an aquifer is normally controlled by elements such as: lithology, weathering, dissolution, precipitation or ion exchange. the algerian climate is a transitional climate. it varies from the mediterranean type in the north to the desert type in the sahara. the climatic conditions and the lithology of arid zones are contributing in the modification of the physic-chemical properties of the waters. in this context, several researches have been conducted on groundwater to assess the hydro chemicalprocesses, groundwater quality and water suitability for the irrigation of this region. they have shown that groundwater hydrochemistry depends ontwo important resources: natural and anthropogenic processes. in this context, several researches have been conducted on groundwater to assess the hydrochemicalprocesses, groundwater quality and water suitability for the irrigation of this region. they have shown that groundwater hydrochemistry depends mailto:djebassi.toufik@univ-tebessa.dz mailto:abdeslam.ilhem@yahoo.fr mailto:hsamir2001@gmail.com mailto:d_hacene@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 323 on two important resources: natural and anthropogenic processes [1] and [2]. tebessa basin is among the semi-arid areas, where the increased forage yield is the main source of water consumption in drinking and irrigation. nevertheless, the northern part of the region is an area of active and abundant mines which constitute about 83% of the total algerian iron production [3]. due to its abundance and availability nexus the water source static levels, their geological constituents cause realistic challenge on groundwater quality. moreover, its ecological function has great impact on public health and on flora diversity [4]. the increasing population as well as industrial and agricultural demands renders the protection and preservation of groundwater resources all the more important. the aim of the present paper is to characterize the groundwater quality by using a pollution index of groundwater (pig) for groundwater contamination quantificationin the study area (figure 1), and the determination of pollution zones for proposing remedial solutions at a specific site. fig. 1. geographic situation of the study area 2. characteristics of the study area the tebessa basin is an agricultural region located in the north east of algeria, within the semi-arid zones of the country and characterized by a precipitation of less than 400 mm per year (figure1). the basin of tebessa is a flat plain surrounded by several mountains with different altitudes such as: djebel troubia, djebel serdiess in the west, by djebel kouif and bouremane in the east, and by djebel dyr and belkfif in the north and by djebel doukkane, tezbent and essen to the south, respectively, as it described by the figure 2. the hydrogeological system of tebessa basin consists of mio-plio-quaternary and cretaceous aquifer. the superficial unit of the mio-plio-quaternary is constituted from t u n i s i a morocco a l g e r i a l i b y a m e d i t e r ra nean s e a tébessa tébessa hammame t bekkaria food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 324 unconsolidated materials, consisted by actual and recent alluvial deposits, conglomerates, gravels and sandstones, which form an alluvial aquifer. fig. 2. natural boundaries of the study area middle cretaceous lowcretaceous trias wadi roads cities actually alluvial continental quaternary continental pliocen continental miocen loweocen high cretaceous fig. 3. geological map of the study area food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 325 the cretaceous units forming a series of anticlines and synclines.the stratigraphic sequence is presented in the form of alternation of carbonated formations of limestones, marly-limestones and argillaceous marls forming a very important karstic aquifer [5] [6]. it is noted the presence of triassic evaporitic outcrops (clay and gypsum), which stand above the eastern borders of the study area (figure 3). 3. hydrochemical data 3.1 field work a survey was carried in tebessa basin, and 58 samples were collected from open dug wells during february 2020 (fig.4). water samples were collectedin clean polythene bottles, washed thoroughly with dilute nitric acid then rinsed with distilled water, and again rinsed with representative water samples. the physical parameters of the water sources, such as ph, electrical conductivity (ec), and total dissolved solids (tds) were measured in the field. 3.2 laboratory work the chemical parameters of the groundwater samples like major cations, calcium (ca++), magnesium (mg++) were determined by edta titration method. sodium (na+) and potassium (k+) were determined by flame photometric method. anions like bicarbonate (hco3 -) were measured by titration to the methyl orange end point. the amount of chloride (cl-) present in groundwater samples was determined by titration and precipitation of agcl until silver chromate appears. sulfate (so4 2-) was determined by precipitation of baso4 and then measuring the absorbency with spectrophotometer. trace metals namely iron (fe), copper (cu), lead (pb) and manganese (mn) were analyzed using atomic absorption spectrophotometer (aas).organic matter such as phosphate (po4), nitrite (no2), ammonium (nh4) and nitrate (no3) was measured by the phenol disulfonic acid method. fig. 4. map of sampled wells – tebessa basin wells temporarywadi permanent wadi basin food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 326 the analyzed data (table 1) has been used in the computations. the standards for drinking purposes as recommended by who and bis have been considered for the calculation of pollution index of ground water (pig) [7]. there are different steps for computing pig which includes assigning a weight to each chemical parameter according to its relative importance in the overall quality. table 1. chemical composition of groundwater in the tebessa basin water qualitymeasure units minimum maximum moyenne standard deviation ph 6.140 8.220 7.186 0.300 tds mg/l 6.070 11550.000 3345.357 2619.367 rs mg/l 420.000 18501.000 2511.847 2670.277 ca mg/l 39.360 793.280 258.251 169.925 mg mg/l 13.520 243.870 86.878 57.135 na mg/l 28.000 1560.000 375.441 395.859 k mg/l 1.000 38.000 4.271 5.382 cl mg/l 40.000 2025.000 448.475 451.330 so4 mg/l 22.000 3820.000 851.966 806.846 no3 mg/l 0.000 275.000 47.746 54.847 hco3 mg/l 30.500 939.400 298.475 128.108 4. computation of pollution index of groundwater (pig) pig is an arithmetic scale, quantifying the degree of water contamination. it represents a complex influence of individual water quality measures on general water quality of aquifer. the steps to compute pig is given as follows: 4.1 relative weight (rw) a relative weight (rw) for each chemical parameter is assigned a weight age by keeping its impact on human health into consideration. the range of numerical magnitude of relative weight ranges from 1 to 5 (table 2). table 2. weighting scheme for drinking water quality standard with respect to water quality measures water quality measure units relative weight (rw) weightparameter (wp) drinking water quality standard (ds) * ph 5 0.139 7.5 tds mg/l 5 0.139 500 ca mg/l 2 0.056 75 mg mg/l 2 0.056 30 na mg/l 4 0.111 200 k mg/l 1 0.028 10 cl mg/l 4 0.111 250 so4 mg/l 5 0.139 150 no3 mg/l 5 0.139 45 hco3 mg/l 3 0.083 300 sum ∑ 36 1.000 *davis and dewiest (1966), holden (1970), and bis (2003) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 327 for instance, the value of 5 of the rwis assigned to ph, tds, no2, nh4, and no3; 4 to na+, cl-, po4, fe, mn and pb; 3 to hco3 -; 2 to ca++ and mg++ and 1 to k+. the lower values of rwindicate lesser impact of respective chemical parameters on health and higher values have more impact over human health. 4.2 computation of weight parameter (wp) weight parameter is the ratio of rwof every water quality measure to the sum of all relative weights. weight parameter helps to know about the relative share of each water quality measure on overall water quality. the wpis given by the equation: wp= rw/ σrw (1) 4.3 status of concentration (sc) the sc is computed by dividing the concentration (c) of each water quality measure of every water sample by its respective drinking water quality standard. (ds): sc= c / ds (2) 4.4 overall water quality (ow) the overall water quality is computed by taking the product of each water quality measure with its corresponding status of concentration. ow reflects overall water quality and also enables to understand the nature of weight parameter with respect to concentration of each water quality measure. ow is calculated by: ow= wp * sc (table 3). (3) 4.5 pollution index of groundwater (pig) pollution index of groundwater is calculated by adding all values of ow contributed by measures ofall water quality of each sample of groundwater. pig is given by: pig = σow (4) 4.6 pig classification the classification of pig is based on water quality standard for drinking purpose. pig classification could also be used in the assessment of groundwater contamination. when both the values of quality of particular water sample and concentration of water quality measure are same then their impact on health could be insignificant. with an account of this, when the pig value is less than 1.0, it could be considered as a non-pollution index and when pig exceeds more than 1.0, it is probably dueto the contribution of additional concentrations of water quality measures into groundwater by the direct pollution of the aquifer. the intensity of pig is as follows: pig values pig intensity pig<1.0 insignificant pollution 1.0<pig<1.5 low pollution 1.5<pig<2.0 moderate pollution 2.0<pig<2.5 high pollution pig>2.5 very high pollution when ow is more than 0.1 (which represents 10% of 1.0 of pig value), the relative contribution of concentration of water quality measure of each water sample is taken into consideration. this gives an idea on impact of pollution on groundwater. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 328 5. results and discussion 5.1 groundwater movement and evolution the piezometricmap shows that the direction of groundwater movement must be moving as represented in fig. 5 from the east towards the center in one path and the other path is from the western part towards the center part.groundwater movement show that the tebessa basin is divided in two aquifer system, at the east this system is recharged through carbonate formations outcropping from bouromane mountains and in the west part this aquifer system have a direct alimentation from meastrichtian fracturated limestone from hammamet and doukkane mountains. 5.2 water quality the value of physical parameters (ph, ec and tds) of the water samples collected from tebessa basin changes from 6.9 to 7.6, 1500 to 3320 μs / cm and 1060 to 1960 mg/l, with mean values of 7.39, 2652.73 μs / cm and 1489.09 mg/l respectively (table 1). the concentrations (mg/l) of major cations (ca2+, mg2+, na+ and k+), major anions (hco3 -,cland so4 2-),and organic matter (no3 -) are in the range of (39.36 to 793.28,13.52 to 243.87, 28 to 1560 and 1.00 to 38.00), (30.50 to 939.4, 40.00 to 2025.00 and 22 to 3820), and ( 0 to 275).statistical parameter of the analytical results of groundwater is given in table 1. the calculated values of pig in our study region are between 0,457 to 8.190 (figure 6). according to the classification of pig, 22.41 % of the all groundwater samples which represent moderate to high pollution zone and about 43.10% as very high pollution zone. spatial distribution map of zones of pig has been prepared using gis (map-2). the variation map (figure 7) depicts a pollution zone insignificant observed in the extreme southern part, this part of the study area is characterized by a very high fig. 5.piezometric map –tebessabasin food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 329 topography (upstream area). low pollution zone is observed and clearly dominate the central part. moderate pollution zone is spread in the eastern, western and northern parts of the study area. high pollution zone is observed in the northern and western part. very high pollution zone is spread in the extreme northern part, where the topography is low (downstream area). thus, the spatial distribution map of pig zones shows a gradual increasing of pig values from upstream to downstream. fig. 7. spatial distribution of groundwater pollution zones in the case study area (tebessa basin) based on pig food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 330 this suggests a progressive increase of pollution from insignificant level to significant level. insignificant pollution zone is principally characterized by the following values: tds (0.167), ph (0.133), and so4 2-, (0.14) corresponding to ow values more than 0.1 (table 3). the rest of water quality measures, ca2+ (0.081), mg2+ (0.057), na+ (0.033), k+ (0.003), cl(0.038), hco3 (0.075), and no3 (0.091), are considered as principal contributors under natural conditions, because their ow values are less than 0.1. ph variation from 6.14 to 8.22 (table 1) is due to the concentration of hco3 -.the groundwater aquifer system is in permanent contact to soil co2, this last one coming, generally,from organic matter and root respiration. the co2 combined to the rainwater infiltration form bicarbonates-, leading to a mineral dissolution [8]. all dissolved ions in the groundwater area direct result of the total dissolved salts tds [9]. in the low pollution zone,the water quality measures, ph (0.135), tds (0.251), so4 2-, (0.25) and no3 (0.1),ow values are more than 0.1 (table 3). they have higher ow values compared with those values of the water quality measures ca2+ (0,096), mg2+ (0.083), na+ (0.07), k+ (0.01), cl(0.06) and hco3 -(0.09) .weathering of gypsum is the main source of so4 2-, in the study area [10]. ion exchange and precipitation of caco3 are also the mainly causes for increased ca2+ [11] and [12]. anthropogenic sources (chemical fertilizers, irrigation return flows, poor drainage conditions and leakage of septic tanks), lead to the presence no3 in the groundwater. the water quality measures, ph (0.13), tds (0.38), ca2+ (0.16), mg2+ (0.13), so4 2-, (0.45) and no3 (0.13), show ow values more than 0.1 in the moderate pollution zone (table 3), due to the geological and anthropogenic origin. they have more ow values compared with those values of na+ (0.08), k+ (0.02), cl(0.1) and hco3 (0.1) in the same pollution zone (table 3). the higher values of ow in the groundwater samples are observed in the very high pollution zone compared with those of water quality measures in the moderate to high pollution zone (table 3). the value of ow of no3 is more than 0.1 in the high pollution zone, which is probably due to role of anthropogenic activities on the groundwater aquifer system in the high pollution zone. furthermore, the ow value of bicarbonatesisless than that of cl-, while that of sodium is less than that of chloride. this confirms that the geogenic source is the main detrimental factor in the control of groundwater quality. in the insignificant, low, moderate, high pollution zones and the very high pollution zone (figure 8), the geochemical ratio of na+-clis more than one (1.00 to 1.45) this is probably due to the meteoric source of groundwater in the very old zones. the ratio hco3 -: clis more than unity in the insignificant (1.87) and low (1.41) pollution zones and is less than unity in the moderate (0.92), high (0.40) and very high pollution (0.19) zones (figure 8). this is due to a meteoric source of groundwater in the old part of the aquifer and dissolution of triasic formations in the shallow zones. the ratio hco3 -:clis rapidly dropped to 0.19 in the very high pollution zone.this suggest that the groundwater is mainly influenced by evaportitic formations in the very high pollution zone. the ratios na+ : ca2+ are observed to be more than unity (1.11 to 1.53) for high and very high pollution zone (figure 9),this suggests a precipitation of caco3 and less than one (0.5 to 0.8) in the insignificant, low and moderate pollution zone (figure 9), showingthe geogenicorigin (influence of triassic formations). the ratios mg2+: ca2+isless than one (0.235 to 0.288) in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 331 pollution zones (figure 9). this is due probably to caco3 precipitation and ionic exchanges. however, thevalue of the mg2+: ca2+ ratio is almost similar in all pollution zones. this shows the sameorigins of mg2+ and ca2+ ions, as is also observed from the ratio of na+: cl(figure 8).the differences in the geochemical ratios explain the different factors controlling concentrations changes of water quality measures in all zones (table 3). fig. 8. ratios of na + : cl and hco3 : cl with respect to pollution zones fig.9. ratios of mg 2+ : ca 2+ and na + : ca 2+ with respect to pollution zones food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi, monitoring of groundwater quality in a semi-arid region, tebessa basin (north-east of algeria): using pollution index of groundwater, food and environment safety, volume xx, issue 4 – 2021, pag. 322 – 332 332 6. conclusion this paper presents integrated approaches for characterizing hydrochemistry and suitability of groundwater quality in the tebessa basin (north east of algeria). based on pig method; and the major objective of the study was to disseminate the groundwater contamination zonesand characterize the status of concentrations of water quality measures with respect to their water quality standards. this study comes as the first attempt in applying pig tool to assist water planners and managers in the study area to better understand the water quality determinants influencing the attractiveness of groundwater users. the proposed index computed from the study area varies from 0.46 to 8.19. the distribution of pollution zones, in the study area, suggests that the geogenic origin (triasic evaporate formations) is the main controlling factor of groundwaterquality, but it can be changed by the influences of some anthropogenic activities. as the index is calculated and validated, the study throws light to undertake remedial solutions at anysite to control the activity of pollution. 7. acknowledgments the authors would like to thank, the technical staff at the laboratory of water and environment in tebessa university, algeria, for their help and support during the realisation of this research work. we would like to thank pr. subbarao n (india), pr. salameh. e (jordan) and pr. audra. ph (france). 8. references [1] salameh e. 2001. sources of water salinities in the jordan valley area, jordan. actahydrochim .hydrobiol. 6-7: 329-362. [2]. fehdi ch. boudoukha. a. rouabhia aek. salameh. e. 2011. origin of groundwater salinity in the morsott-el aouinet basin, northeastern algeria: hydrochemical and environmental isotopes approaches. desalination and water treatment journal, desalination publications. doi: 10.5004/dwt.2011.12050. [3]. djebassi t. et al 2020. hydrochemical quality of the tebessa alluvial aquifer and the vulnerability to triasic formations of djebissa and belkefif mountains (n-e of algeria). international journal of ecology and development.vol 35, issue number 3. [4]. rouabhia aek. baali f.fehdi ch. kherici n. djabri l2008. hydrochemical and isotopic investigation of a sandstone aquifer groundwater in a semi arid region, el ma el abiod, algeria. environmental geology, springer-verlag, berlin; heidlerg. doi 10.1007/s00254-008-1451-5 [5]. vila, j.m. 1980. la chaîne alpine de l’algérie orientale et des confinsalgéro-tunisiens. thèse de doctorates-sciences, université pierre et marie curie, paris vi. [6]. fehdi ch, rouabhia aek , mechai a, debabza m, abla kh, voudouris k. 2014. hydrochemical and microbiological quality of groundwater in the merdja area, tebessa, northeast of algeria. applied water science, doi: 10.1007/s13201-014-0209-3 [7]. hem jd. 1991. study and interpretation of the chemical characteristics of natural water. scientific publishers: jodhpur, india; 2254. [8]. mishra p.c and patel r.k. 2011.study of the pollution load in the drinking water of rairangpur, a small tribal dominated town of north orissa, indian j. environ. ecoplan. 5(2), 293 298. [9] holden, ws. 1970. water treatment and examination. j & churchill publishers: london. [10].fehdi ch. rouabhia aek. baali f 2008. the hydrogeochemical characterization of morsott-el aouinet aquifer, northeastern algeria. environmental geology, springer-verlag, berlin; heidlerg. doi 10.1007/s00254-008-1667-4. [11]. subba rao n 2012. pig: a numerical index for dissemination of groundwater contamination zones. hydrological process 26: 3344–3350 [12]. subba rao n, et al 2018.geochemical characteristics and controllingfactors of chemical composition of groundwater in a part ofguntur district, andhra pradesh, india. applied water science. 8: 227. https://doi.org/10.1007/s13201018-0864-x 1. introduction 6. conclusion doi: https://doi.org/10.4316/fens.2021.013 113 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag. 113 127 study on extraction and purification of trans-resveratrol from vine waste a review * alina lenuța crăciun 1 , gheoghe gutt 1 1faculty of food engineering, ștefan cel mare university, suceava, romania, alina.craciun@usm.ro, *corresponding author received 25th april 2021, accepted 28th june 2021 abstract: resveratrol (3, 4', 5-trihydroxystilbene) is a natural phenolic compound synthesized by plants in response to various stressful circumstances (mechanical injury, siege of bacteria or fungi and uv exposure). commercial resveratrol supplements on the market are mostly extracted from japanese knotweed. as it grows even in heavily polluted environments and the presence of emodin, which is a compound that may have laxative effects and is found in partially purified extracts, additional concerns about the safety of these commercial preparations were raised. an alternative source of resveratrol intensely studied in recent years is vine waste, with recent studies showing high concentrations of resveratrol in extracts. in the last years, the researcher’s attention is focused on obtaining an efficient method of extraction and purification of this compound from the vine waste to show that this material can be used as a raw material for production of commercial supplements of resveratrol. keywords: trans-resveratrol; vine waste; purification; supplements; health 1. introduction human food was composed since the paleolithic era of products of plant origin and hunted, this dividing the tribes into gatherers and hunters. over time, human has evolved and thus his diet has evolved, becoming more and more diversified, especially with the domestication of animals and the cultivation of land. if in the recent years the possibility of having many foods meant wealth, now people's preferences tend towards a healthier lifestyle and diet [1]. although the general tendency is to have a lifestyle as healthy as possible, food cannot stop the oxidative processes that take place in the body and cause aging or disease. recent studies focus on obtaining compounds that slow down oxidative processes, using substances of plant origin with high availability [2]. resveratrol (3, 4', 5-trihydroxystilbene) is a natural phenolic compound of plant origin. it belongs to the category of stilbenes, which are secondary metabolites of plants with a "stilbene skeleton" comprising two aromatic rings linked to an alkene [3]. it was found that some plants synthesize resveratrol in response to various stressful circumstances (damage, exposure to high doses of ultraviolet radiation, fungal parasites, etc.). it is found in both stereoisomeric forms (cis in very small amounts and trans which is the most present) and is fat-soluble [4]. this compound is found in grapes (only in the skin and in different amounts depending on the variety, region and exposure to fungal infections), hazelnuts, and berries of the http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 114 family vaccinum (red and black currants, blueberries, cranberries) [5]. resveratrol is found in different amounts in grapes and vines, the largest amount being in the stem, followed by grape skin, seeds and pulp [6]. the first article on resveratrol appeared in 1939 [7]. the presence of resveratrol in red wine was first discovered by researchers at cornell university, siemann and creasy in 1992 [5]. this finding linked the compound to the famous "french paradox", a term derived from epidemiological research that showed that the mortality rate due to cardiovascular disease was significantly lower in the french population compared to other european countries, despite their higher cholesterol and saturated animal fat intake [8]. subsequent research has suggested that the lower risk of cardiovascular disease may be attributed to the habit of consuming red wine more often among the french [9]. the product is already available on the market in various forms. the market for this supplement is constantly growing because demand is higher than supply, and this product has uses in the pharmaceutical, cosmetic and food industries. due to the growing demand for resveratrol supplements, the interest of researchers in the extraction and purification of resveratrol from vineyard waste has increased, so this new opportunity for waste recovery was extensively studied in recent years [10]. the purpose of this work was to highlight the progress and summarize the data obtained by researchers in the extraction and purification of resveratrol from vine wastes and thus show that this material can be used as a raw material for production of commercial supplements of resveratrol. 2. resveratrol and its characteristics table 1. shows beside identification elements and technical security data, the physicochemical properties required in extraction, detection and purification of resveratrol. table 1. identification elements, physicochemical properties and technical safety data of resveratrol [11] resveratrol identification elements other names trans-3,5,4′-trihydroxystilbene 3,4 ′, 5-stylbenetriol trans-resveratrol (e)-5-(p-hydroxystyryl) resorcinol (e)-5-(4-hydroxystyryl)benzene-1,3-diol cas number 501-36-0 physicochemical properties chemical formula c14h12o3 molar mass 228.25 g/mol appearance white powder with slightly yellowish hues boiling point 528 °c melting point 261-263 °c, (502-505 °f, 534-536 k) solubility in water 0.03 g/l solubility in dimethylsulfoxide 16 g/l solubility in ethanol 50 g/l uv-vis (λmax) 304 nm (trans-resveratrol, in aqueous solution) 286 nm (cis-resveratrol, in aqueous solution) technical security data risk phrases r36 – irritating to eyes safety phrases s 26 – in case of contact with eyes, rinse immediately with plenty of water and seek medical advice lethal dose ld50 5.29 g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 115 3. effects of resveratrol to human body resveratrol has a lot of beneficial effects on human health, these being shown in fig.1 and detailed below. fig.1. the beneficial effects of resveratrol [1] 3.1. slowing down the aging process one of the most sought after properties of any chemical supplement is its ability to rejuvenate cells and tissues to extend their lifespan. some of the early evidence for resveratrol's ability to extend cell life comes from the work of howitz et al. in 2003, who show that resveratrol in the yeast saccharomyces cerevisiae has the effect of increasing the stability of genomic dna through various signals, resulting in an overall life extension of 70%. these results prompted further research into the anti-aging effect of the compound and its adaptation to treat agingrelated disorders such as dementia or alzheimer's disease [12]. a study by kumar et al. in 2006 found that a 20 mg/kg body dose of resveratrol has the ability to regenerate depleted areas of hippocampal tissue in the brains of older mice [13]. another study conducted by preliminary tests of 20000 compounds and studies in mice showed that resveratrol has the most promising life-extending abilities [2]. these findings have brought resveratrol to the attention of researchers, highlighting its potential to be developed as a therapeutic agent for neurodegenerative diseases related to aging. 3.2. the antioxidant activity of resveratrol the best known health benefit of resveratrol is its ability to act as an antioxidant. being a phenolic compound, resveratrol can act against free radicals and also as a promoter of cellular antioxidant enzymes such as glutathione, peroxidase, glutathione-transferase and glutathione reductase to induce neutralization of radical peroxides [14]. based on the in vitro study of shang et al. since 2009, a resveratrol antioxidant anti-inflammatory protection against infections prevents aging reduces obesity antiviral protection against ischemia cancer protection cardioprotector neuroprotective food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 116 reaction mechanism has been proposed for the antioxidant activity of resveratrol, galvinoxyl radicals and 2,2-diphenyl-1picrilhydrazyl (dpph) [15]. other in vitro studies that mimic physiological systems and biological systems such as mouse pheochromocytoma cell lines have shown that resveratrol is a potent antioxidant compared to a number of typical food additives such as tocopherols, butylated hydroxyanizole (bha), butyl hydroxytoluene (bht) and vanillin [16]. because of the findings that suggest that reactive oxygen species are linked to cardiovascular disease, various cancers, diabetes, and periodontal disease, the antioxidant activity of resveratrol makes this compound a potential therapeutic agent against these diseases [17]. 3.3. inhibition of enzymes responsible for skin aging and pigmentation the main enzymes responsible for aging, pigmentation and dull appearance of the skin are tyrosinase, hyaluronase, collagenase and elastase. resveratrol was found to inhibit the activity of these enzymes and the results showed that its effect is concentration-dependent [2]. 3.4. antibacterial effects of resveratrol 3.4.1. effect against propionibacterium acnes the appearance and progression of acne is due to p. acnes, which is part of the resident microbiota in the hair follicles of human skin. p. acnes multiplies using sebum as a source of nutrients and produces lipase. it degrades sebum triglycerides, releases fatty acids and induces a series of inflammatory reactions. white blood cells "wander" and infiltrate the dermis and release the inflammatory factor. it irritates the skin and accelerates the cornification of the epidermis with inflammation [18]. by performing an assay on an agar culture medium to which resveratrol was added, it was shown that it controls the multiplication of p. acnes starting at a concentration of 300 µg/ml or higher [2]. 3.4.2. effect against helicobacter pylori helicobacter pylori is a bacteria that causes gastric ulcer and stomach cancer and lives in the stomach. the japanese have high degrees of infection with h. pylori, as over 50% of the population over 60 is affected. the antibacterial activity of resveratrol is effective against h. pylori, and was confirmed by in vitro tests [2]. 3.5. antifungal effects of resveratrol according to kuc's definition, phytoalexins are antimicrobial compounds synthesized and accumulated by plants in response to biotic or abiotic elicitors [19]. in the woody material of some vitis species, resveratrol and its derivatives can accumulate up to 700 mg/g and are involved in the prevention of wood degradation and in the resistance of plants to pathogens. the antifungal properties of resveratrol have been demonstrated using bioassays. langake et al. reported reduced fungal toxicity to resveratrol with the effective dose required for mortality of 50%, a dose between 71 and 200 mg/l on p. viticola spores and b. cinerea conditions [20]. radial growth of b. cinerea and p. viticola fungal mycelium on agar substrates decreased in the presence of a resveratrol dose of 50 to 100 ppm. adrian et al. observed an inhibition of b. cinerea germination in the presence of 7x10-4m resveratrol [21]. 3.6. anti-inflammatory activity inflammation, the body's response to toxins and exogenous infections, is triggered by reactive oxygen species and could lead to tissue damage and atherosclerosis [22]. the antioxidant activity of resveratrol helps reduce inflammatory responses by eliminating reactive oxygen species. however, some food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 117 studies suggested that resveratrol is also able to regulate inflammatory responses by disrupting the associated biochemical pathways [22]. such a pathway involves the activation of a transcription factor known as nf-κb, which induces the expression of cellular signaling proteins (cytokines) to initiate the inflammatory response in target tissues. resveratrol inhibits the nf-κb pathways, resulting in decreased inflammatory signaling [23]. therefore, the compound is useful in decreasing the inflammatory response triggered by various signals. 3.7. anti-carcinogenic effect the anti-cancer activity of resveratrol is an area that was extensively studied over the last decade. many research results suggest that this compound has pleiotropic functionality against cancer. the antioxidant and anti-inflammatory actions of resveratrol help prevent such damage to the genetic material. in addition, many studies demonstrate that this compound plays a key role in regulating the transduction pathways of multiple signals to inhibit the progression of mutations and cancer cells [24]. the work conducted by yi et. al. since 2013 demonstrated the ability of the compound to cause cell cycle arrest by regulating the expression of transcription factors involved in the phase of dna synthesis in the cell cycle, even in highly aggressive malignancies such as anaplastic thyroid carcinoma and colon cancer, which are less receptive to conventional methods of treatment [24]. in addition, research results have shown that resveratrol is able to induce the expression of proteins associated with cellular apoptosis signaling pathways to cause programming of cell death in postcancer cell lines. the results also indicate that this compound has a minimal effect on healthy cell lines for inducing apoptosis. therefore, resveratrol has the potential to be used as a selective chemotherapeutic agent for cancer [25]. 3.8. cardioprotective effects the cardioprotective effects of resveratrol were discovered based on research that showed an inverse correlation between red wine consumption and the incidence of cardiovascular disease. scientists have discovered a phenomenon called the "french paradox", which is based on the hypothesis that a moderate consumption of red wine reduces the occurrence of cardiovascular disease. all this is due to the inhibitory effect on cell membrane lipid peroxidation and protection against the oxidation of low-density lipoproteins (ldl), which influence the reduction of hdl cholesterol concentration [26]. 3.9. protection against diseases of the nervous system one of the reasons for diseases of the nervous system is the deposition of βamyloid in the brain, which does not allow the proper functioning of nerve cells, preventing their communication. this leads to memory loss or impaired cognitive function in humans, a disease also called alzheimer's. there is more research on the use of resveratrol as a compound that helps treat alzheimer's disease, given that this compound limits the function of β-amyloid proteins and stimulates their decay as short polypeptides and amino acids through the proteasome mechanism. this promising result can help inhibit or slow down the development of these disorders [27]. 3.10. beneficial effects on the symptoms of diabetes resveratrol was found to increase insulin sensitivity in mice on a high-calorie diet, increase lipid metabolism in diabetic mice, reduce nerve pain, and protect neurons [2]. 4. recovery of vine waste for resveratrol extraction food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 118 because resveratrol is a natural phytoalexin, it is synthesized by a multitude of plants, about 700 species. the amount of resveratrol synthesized by plants varies widely, and only a few plants are of interest for its extraction and purification to obtain chemical supplements. [1] the best known plants with a significant content of resveratrol are the vine (vitis vinifera) and the japanese knotweed (polygonum cuspidatum) [28]. recent studies in the field have brought to light other plants containing resveratrol, such as several species of spruce (picea mariana, picea abies, picea glauca, picea sichensis), hazelnuts (arachis hypogaea), legumes (cassia sp., eucalyptus globules) and others [29]. many of the commercial preparations of resveratrol are extracted from the root of the japanese knotweed (polygonum cuspidatum), and vary widely in degree of purity. some studies showed that an additional compound such as emodin is present in unpurified or partially purified resveratrol extracts, and could have a laxative effect [30]. because the plant thrives even in environments heavily polluted with heavy metal-contaminated soils, cellular uptake of these contaminants by root tissues raises additional concerns about the safety of commercial resveratrol preparations obtained from the roots of this plant [31,32]. another representative source for the extraction of this valuable compound is the waste from the wine industry, which abounds in this compound. recent studies have shown the presence of this compound in significant quantities in various byproducts from the wine industry, which include: wood waste from the grooming of vines and pomace and bunches resulting from winemaking technology [33]. viticulture is one of the most important agricultural activities in the world, with approximately 7.4 million hectares cultivated globally in 2018, of which 4.3 million hectares are cultivated in europe. the first country is represented by spain, with an area of 969,000 hectares, followed by china with 875,000 hectares and france with 793,000 hectares [34]. the annual average of by-products resulting from the cleaning of the vine is 1.3 kg of wood/live log, which results in 2x107 tons of wood harvested annually in the world. currently, the only use of this waste is as fertilizer for vines, by grinding and distribution on vineyards [35]. the chemical composition of the vine shoots is characterized by a fraction of holocellulose, with a content of approximately 55.1% (31.9% α-cellulose and 23.2% hemicellulose), and another fraction of lignin, which represents about 38.5%. the most important fraction is the minor fraction, represented by phenolic and volatile compounds, those with a greater importance for the food, pharmaceutical and cosmetic industry, being the most intensively studied stilbene group [35]. due to the growing demand for resveratrol for food supplements, researchers in many countries have turned their attention to waste resulting from winemaking technology. the resveratrol content of the vine has been studied extensively in recent years by researchers in various countries. aavikasar et al. determined the resveratrol content of vineyard waste from an estonian wine region in three different seasons in 2002. these were analyzed immediately after harvest, and after prior maceration with 80% ethanol, 80% methanol and 96.6 % ethanol the values were between 0.1-2.9 mg/g dry weight (d.w.) [33] the study conducted on the variety vitis amurensis by chinese researchers investigated the content of resveratrol in different plant tissues at different times. although this study found that the content of resveratol was different depending on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 119 the anatomical part of the plant and varied depending on the season, the values are considerably lower than the values obtained from the analysis of vine waste. rhizomes have the highest content of resveratrol (92.0-165.7 mg/kg d.w.), followed by roots (76.1-123.4 mg/kg d.w.), grape skin, (30.6-63.2 mg/kg d.w.), shoots (5.3-18.9 mg/kg d.w.), seeds (20.4-36.5 mg/kg d.w.) and leaves (1.3-10.3 mg/kg d.w.) the resveratrol content begins to increase in the summer months, reaching a maximum in autumn, and then decreases with the temperature [36]. the difference in resveratrol concentration in the different anatomical parts of the vine was also determined by researchers in hungary. from the data obtained it was observed that the highest concentration of resveratrol is present in the stem (344.5 ± 32.2 mg/kg d.w.), followed by buds (183.3 ± 10.4 mg/kg d.w.), root (113.0 ± 6.2 mg/kg d.w.), grape skin (113.0 ± 6.2 mg/kg d.w.), bunches (17.43 ± 2.34 mg/kg d.w.), carcasses (2.96 ± 0.29 mg/kg d.w.) and seeds (0.78 ± 0.02 mg/kg d.w.) [37]. the resveratrol content of wine waste was also analyzed by researchers in turkey. they determined the resveratrol content of 10 different grape varieties by analyzing samples resulting from 60% ethanol extraction, and reported values ranging from 0.95 ± 0.08 to 3.94 ± 0.21 g/100 g d.w. [38]. as studies show, resveratrol is present in all component parts of the vine, but in different amounts. vine waste can be a viable source for the extraction of resveratrol, both due to the high concentration of resveratrol and also due to the fact that they are economically advantageous. 5. optimization of methods for the extraction of resveratrol from wine waste the extraction efficiency can be influenced by a multitude of factors such as: the drying time and temperature of the samples, the degree of crushing, the extraction temperature and time, and the type of solvent and procedure used. for this reason, numerous studies were aimed at finding an optimal method of transresveratrol extraction. in the research conducted in the czech republic by ivo soural et al. 25 different extraction methods were compared by varying these factors [39]. grinding of samples in the case of extractions with cut samples and grinded samples, higher yields of trans-resveratrol were always obtained in the case of grinded samples [39]. solvents used methanol is a better solvent than acetone for resveratrol extraction, the biggest difference being visible in the case of samples that were cut into pieces, where the amount of resveratrol extracted was considerably higher [39]. according to literature, the solvents with a high degree of extraction of resveratrol are ethanol and methanol, with an optimal concentration of 80% [29]. the only disadvantage of these solvents is that they have low selectivity, so alongside resveratrol are extracted other compounds that can hinder its further purification. an alternative to these solvents could be diethyl ether, which is a more selective solvent that, however, gives a lower resveratrol extraction yield [40]. extraction temperature the yield of resveratrol obtained by maceration of samples at room temperature was lower when using acetone than methanol. when using methanol as solvent the concentration of resveratrol was close to that obtained when macerating at higher temperatures [39]. multiple extractions multiple extractions were performed by soxhlet extraction using methanol. the first extraction step food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 120 (with 10 cycles) had much higher yields compared to the second, third, fourth and fifth steps. the yields of all three compounds up to the second stage were below 5%, and the yields of the third and subsequent steps were below 1% [39]. in the case of conventional maceration at room temperature with 80% methanol for 72 hours, the yield of resveratrol in the second extraction was 80% lower than in the first extraction [33]. extraction procedure in the study conducted by manuela m. et al., 2018 on wine waste from 2 different varieties, the efficiency of 3 different extraction methodologies was compared: conventional extraction, microwaveassisted extraction and supercritical aqueous extraction; the results were compared based on the phenolic composition of the extracts and antioxidant capacity. the optimal parameters for each method were established using the literature in the field [41]. the experimental data obtained showed that the best extraction yield of resveratrol was obtained by microwave-assisted extraction, followed by conventional extraction and supercritical aqueous extraction (136 ± 7 > 133 ± 6>9.39 ± 0.47 mg/100 g d.w.) between the first two methods a small difference in yield was observed [41]. due to the fact that resveratrol is susceptible to photochemical isomerization, the extraction methods used must involve solvents and temperatures that do not affect the final compound. the temperature stability of resveratrol was studied by researchers in spain for microwave-assisted extraction. for the tests, a standard solution of resveratrol was placed in the extraction vessel with 100% ethanol, and then the degree of recovery of resveratrol from these samples was analyzed at temperatures of 50 °c, 75 °c, 100 °c, 125 °c, and 150 °c. the degree of resveratrol recovery was around 90% for all temperatures [3]. the extraction methodologies performed and the solvents used can be very critical for the quality of the final compound. in the case of resveratrol, it is susceptible to photochemical isomerization [42]. thus, the method of extracting resveratrol must include protective measures, such as the absence of light, the use of an inert atmosphere or the addition of an antioxidant with higher antioxidant properties than resveratrol. in recent years, the development and use of an extractive method has become increasingly popular because it can obtain products with higher yields and better quality [43]. recent studies have highlighted the possibility of extracting stilbene, including resveratrol, from the vine by supercritical aqueous extraction. in the study by julein g. et al. a supercritical aqueous extraction at 160 °c for 5 min led to the recovery of 1.30 g/kg dry weight resveratrol from shoots, 2.11 g/kg d.w. strain resveratrol and 1.52 g/kg d.w. resveratrol from the roots [44]. another alternative method of extracting resveratrol from wine waste could be extraction with low polarity water under pressure. trans-resveratrol was extracted using this method by varying the extraction parameters. the best yield (3.40 mg/g d.w.) was obtained when using 25% ethanol at 105 °c with a flow rate of 1 ml/min or 15% ethanol at 105 °c with a flow rate of 5 ml/min. an increase of temperature above 105 °c or decrease of the ethanol concentration below 25% negatively affected the resveratrol concentration [42]. 6. vine waste processing to improve resveratrol content recent publications argued that resveratrol also accumulates in the post-pruning period of vine shoots. in the study conducted in france by houille b. et al. it was shown that the concentration of transresveratrol can increase up to 40-fold food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 121 during long-term storage of vine shoots [45]. in the study by philipp e. et al. in germany, an increase in resveratrol concentration was studied depending on the shelf life of the shoots. in the first 6 months after storage, a major increase (between 400% and 1400%) of transresveratrol was recorded, resulting in values between 2096 mg/kg dry weight for the regent variety and 752 mg/kg d.w. for the cabernet sauvignon variety [35]. another study by spanish researchers on vine shoots of the varieties airèn and cencibel showed an increase in the concentration of trans-resveratrol and other non-volatile phenolic compounds in samples kept for one month, 3 months and 6 months [35]. in the airèn variety the concentration was 77.10 ± 11.29 mg/kg d.w. after one month, 151.60 ± 9.42 mg/kg d.w. after 3 months and 170.44 ± 3.82 mg/kg d.w. after 6 months. in the cencibel variety the concentration was 50.41 ± 2.74 mg/kg d.w. after one month, 224.83 ± 29.64 mg/kg d.w. after 3 months and 227.00 ± 6.03 mg/kg d.w. after 6 months [35]. in the study by billet k. et al. in france, it was analyzed the increase in the concentration of resveratrol in harvested shoots by exposure to biotic stress caused by botrytis cinerea infection, by short-term exposure to uv light, and by mechanical stress generated by cutting shoots into sections of different lengths. after the first two attempts, no significant increase in the concentration of trans-resveratrol in the vine shoots was observed. in the third experiment the shoots were cut into sections of 0.2 cm, 0.5 cm, 1 cm, 5 cm and 10 cm and the results are presented in the figure 2.. [47] after only two weeks, cutting into 0.5 cm sections allowed for optimal accumulation of resveratrol, which is much faster when compared to previous data reporting the highest accumulation in 6 weeks [45], 8 weeks [47] and 20 weeks [46]. fig.2. accumulation of trans-resveratrol in vine shoots by storage time [46] a study conducted in chile showed a significant increase in the concentration of resveratrol, up to 5 times, of pinot noir vine residues harvested in 2012, after 2 months of storage at room temperature in the laboratory. during the storage period (8 months after cutting) the transresveratrol content increased significantly until the 2nd month, in the 3rd month a slight decrease began and then remained almost constant (fig. 3). in the case of the shoots remaining on the plant, only a slight increase of the resveratrol concentration was observed (fig. 4) [48]. 7. potential techniques and approaches for the development of a method for the purification of resveratrol from wine waste in recent years, with the discovery of high resveratrol content in wine waste, a number of techniques have been investigated for the isolation and purification of resveratrol from these extracts [49]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 122 fig. 3 concentration of stilbenes (mg/kg d.w.) from the 2012 pinot noir vine branches left on the plant [48] fig. 4 concentration of stilbenes in pinot noir vines from 2012, after cutting them from the plant (mg/kg d.w.) [48] the purification of resveratrol from wine waste extracts cannot be carried out by a single process, thus for the highest possible purification it is necessary for the extract to be processed by a sequence of steps and processes [50]. in the study by aaviksaar et al. attempts were made to extract and purify resveratrol from wine waste. in the preparative purification experiment, a mixture of vine powder from 4 different varieties, hasaine sladki, jubilei novgoroda, marechal joffre and zilga was used [33]. in the first phase, the extract was purified by separation from the brown impurities. for this purpose, diethyl ether was added to the extraction solution of 96.6% ethanol in a ratio of 1: 4 diethyl ether ethanol, because the dry extract obtained with 80% ethanol solution is not soluble in diethyl ether. following the extraction with the obtained solvent, a slightly separable precipitate of brown impurities appeared in the amount of 30% of the extract. as there were no components with optical activity at 306 nm in the precipitate, the described diethyl ether treatment proved to be an effective method for lowering the impurity content of the extract without affecting the extraction yield of resveratrol. the next technique used to purify resveratrol was to separate it by column chromatography with polyamide support. following hplc analysis of the extract resulted from purification (fig. 5), 93.8% purity resveratrol was obtained, with a recovery rate of 77% [33]. fig. 5. reverse phase column hplc profile of purified trans-resveratrol ethanolic solution from vine strains by ethanolic extraction, diethyl ether treatment and column chromatography on woelm polyamide (93.8% purity) [33]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 123 a method for the preparative separation and purification of resveratrol from vine roots by flash chromatography was developed by yangji w et al. the solution resulting from the alcoholic maceration with 70% v/v methanol was partially purified with 3% sodium bicarbonate solution and ethyl acetate using a separatory funnel. the obtained extract was further subjected to the purification process by flash chromatography in countercurrent. for this procedure, a phase solvent system composed of chloroform, methanol, n-butanol and water in a ratio of 4:3:0.05:2 v/v was used. the fractions of the separated peaks were collected manually according to the chromatographic profiles. all experiments were performed at room temperature. the resulting fractions were further separated by high performance liquid chromatography using a reverse phase c18 column. the obtained resveratrol fraction had a purity of 97.89%, according to the chromatogram in fig. 6 [51]. fig. 6. hplc chromatogram and uv spectrum of the resveratrol fraction (purity 97.89%) [51] another study by al-jumaily et al. recommends the use of column chromatography as a process for the partial purification of extracts from wine waste. for this purpose, the partial purification of the extracts was made in an open glass column filled with special g60 silica gel filler. the extract was dissolved in 1-2 ml of methanol and the mobile phase consisted of benzene: methanol: acetic acid 20:4:1. the elution was collected in separate tubes, each tube being filled with 3 ml of eluent. all fractions were tested with 1% ferric chloride solution as a colorimetric test for phenol identification. only positive results were collected and dried under vacuum in a rotary evaporator [52]. specialized packing materials, such as sephadex lh-20, have been frequently used in purification studies of natural substances. sephadex lh-20 possesses a large number of hydroxyl groups in its polymeric structure, which allows a high degree of interaction with compounds from complex mixtures [39]. the work of guder et al. demonstrated that resveratrol can be isolated in pure form from grape extracts on a sephadex lh-20 column. the suggested purification method involves an initial step on a silica gel column, followed by separation on a sephadex lh-20 column to obtain a pure resveratrol fraction. despite the need to collect a large number of column fractions before eluting the target compound, this approach resulted in high purity and high yield of resveratrol from primary grape extracts [53]. using the techniques of extraction and purification of resveratrol food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 124 from viticultural waste described in the literature, a partial purification of up to 93.8% [aa, '03] or 97.89% [ya, '14] was obtained. in the study conducted by piyaratne s. in 2018 on the extraction and purification of resveratrol from black spruce bark, a purification of resveratrol of 99% was obtained. various techniques, such as liquid-liquid extraction, column chromatographic separation with multiphase silica gel (methanol, chloroform, ethyl acetate, formic acid), borate complex extraction, ion exchange column chromatography, column with sephadex lh-20, acetylation and diacetylation of extracts, and purification on reversedphase column chromatography with c18 coated silica were applied in this study [54]. these tests resulted in a purification scheme comprising the following steps: liquid extraction with sodium bicarbonate, open column chromatography on silica gel with a special silica gel g60 fill, sephadex lh-20 column chromatography with dichloromethane and methanol and reverse phase column chromatography with dichloromethane and ethyl acetate. these steps were similar to the procedures described in attempts to purify resveratrol from vine waste [17]. fig. 7. hplc-uv chromatograms obtained at 306 nm (maximum absorption for the transresveratrol isomer), 286 nm (maximum absorption for the cis-resveratrol isomer) and 206 nm (maximum absorption for many other phenolic compounds) for the extracts of purified resveratrol from black spruce bark [17] the results (fig.7.) indicate a clean peak of trans-resveratrol and a low peak of cisresveratrol. therefore, the trans-resveratrol resulting from the purification method proposed by piyaratne s. meets the purity level of the products available on the market. by combining and using these processes on vineyard waste it could be possible to purify resveratrol up to 99%. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 125 8. conclusion studies in the field showed the beneficial effect of resveratrol on human health. thus, the interest of researchers in obtaining high-purity commercial resveratrol preparations has increased. vine waste could be used to obtain resveratrol due to the large amount it contains and the low purchase price. according to recent studies, the amount of resveratrol can increase up to 40 times in the vine waste after keeping them for 2 months at room temperature. thus, they could be a very good source for the extraction of this compound for the pharmaceutical, food and cosmetics industry. regarding the extraction conditions, the experimental data showed that the most recommended solvents for the extraction of resveratrol from wine waste are ethanol and methanol in 80% concentration. the extraction temperature may adversely affect the extraction yield or may expose the compound to chemical isomerization. however, as the increase in temperature does not result in a significant increase of the extraction yield, it would be advisable to work with temperatures that have no effect on the chemical stability of the samples. good yields, close to those obtained by other techniques were also achieved by macerating the samples with alcoholic solutions at 50 °c. the extraction and purification techniques of resveratrol from wine waste described in the literature achieved a partial purification of up to 93.8% or 97.89%. the method of purification of resveratrol from wine wastes could be further improved to obtain a purification of resveratrol up to 99% by combining multiple processes in a succession of steps. 5. references [1]. lester p., enrique e., resveratrol in health and disease, university of southern california school of pharmacy, los angeles, california, (2006 [2]. oryza oil & fat chemical co, tokyo, japan, resveratrol for anti-ageing, antioxidation, neuron protection & metabolic syndrome, (2008) [3]. pineiro z., marrufo-curtido a., vela c., palma m., microwave-assisted extraction of sttilbenes from woody vine material, food and bioproducts processing 103, pg. 1-26 (2017) [4]. brezoiu, a. m., matei, c., deaconu, m., stanciuc, a. m., trifan, a., gasparpintiliescu, a., & berger, d., polyphenols extract from grape pomace. characterization and valorisation through encapsulation into mesoporous silica-type matrices, food and chemical toxicology, 133, 110787. (2019) [5]. sieman e.h., creasy l., concentration of phytoalexin resveratrol in wine, am. journal, 49:52, (1992) [6]. he, y. z., wang, b., zhuang, y., & lu, y. p. study on separation and purification of resveratrol in wine grape residue with aqueous two phase extraction method. in advanced materials research (vol. 550, pp. 1743-1746). trans tech publications ltd (2012) [7]. takaoka m., resveratrol a new phenolic compound from veratrum grandi-florum, j.chem. soc, japain, 1090:1100 (1939) [8]. richard j.l., cambien f., ducimetière p., epidemiologic characteristics of coronary disease in france, nouv. press. med., (1981) [9]. chadwick d.j., goode i.a., alcohool and cardiovascular diseases, novartis foundation symposium (1998) [10]. teixeira, a., baenas, n., dominguez-perles, r., barros, a., rosa, e., moreno, d. a., & garcia-viguera, c.. natural bioactive compounds from winery byproducts as health promoters: a review. international journal of molecular sciences, 15(9), 15638-15678. (2014) [11]. https://www.caymanchem.com/msdss/706 75m.pdf [12]. howitz k., bietterman k.j., cohen y.h., lamming d.w.,wood j., zipkin r.e., chimg p., kisielewski a, zhang l.l., brandy s., sinclair d.a., small molecule activators of sirutuins extend saccharomyces cerevisiae lifespan, nature 425, 191:196, (2003) [13]. kumar v., pandy a., jahan s., shukla r.k., kumar d., srivastava a., https://www.caymanchem.com/msdss/70675m.pdf https://www.caymanchem.com/msdss/70675m.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 126 singh s., rajpurohit c, yadav s., khanna z.k., differential responses of transresveratrol on proliferation of neuronal progenitor cells and aged rat hippocampal neurogenesis, sci.rep., (2016) [14]. lastra c.a., villegas i, resveratrol as an atioxidant and pro-oxidant agent: mechanisms and clinical implications, biochem. soc. trans.35, 1156:1160, (2007) [15]. shang v.j., quan y.p., liu x.d., dau f, shang x.l., jia w.q., liu q., fang j.g., zhau b., radical-scavenging activity and mechanism of resveratrol-oriented analogues: influence of the solvent, redical, and substitution, journal org. chem. 74, 5025-5031, (2009) [16]. murcia m.a., marti’nez-tome m., antioxidant activity of resveratrol compared with common food additives, j. food prot. 64, 379-384, (2001) [17]. piyorotne s.p., extraction and purification of (e)-resveratrol from the bark of maine’s native spruces, university of maine, (2018) [18]. silva, v., igrejas, g., falco, v., santos, t. p., torres, c., oliveira, a. m., & poeta, p., chemical composition, antioxidant and antimicrobial activity of phenolic compounds extracted from wine industry by-products. food control, 92, 516-522 (2018). [19]. kuc j, phytoalexins, stress metabolism, and disease resistance in plants, annu rev phytopathol 33, 275–297, (1995). [20]. langcake p, disease resistance of vitis spp. and the production of the stress metabolites resveratrol, e-viniferin, a-viniferin and pterostilbene, physiol plant pathol 18, 213–226, (1981). [21]. adrian m, jeandet p, veneau j, weston la, and bessis r, biological activity of resveratrol, a stilbenic compound from grapevines, against botrytis cinerea, the causal agent for gray mold, j chem ecol 23(7), 1689– 1702 (1997) [22]. mittal m., siddiqui m.r., tran k., reddy s.p., malik a.b., reactive oxygen species in inflammation and tissue injury, antioxidant redox sigmal 20, 1126-1167, (2014) [23]. tian y., wang j., zhang l., xu j., wang k., li d., resveratrol supplements inhibited the nf-kb inflammation pathway through activating ampkα-sirt 1 pathway in mice with fatty live, mol. cel biochem. 422, 75-84 (2016) [24]. yiu x.m., jaskula-sztul r., ahmed k., harrisan a.d., kunnimalaiyaan m, chen h., resveratrol induces differentiation markers expression in anaplastic thyroid suppresses cell growth, mol.cancer ther. 12, 1276-1287, (2013) [25]. shankar s., soddiqui i., srivastava r., molecular mechanisms of resveratrol (3,4,5-trihydroxy-stilbene) and its interaction with tnf-related apoptosis inducind ligand (trail) in androgen-insensitive prostate cancer cells, moll. cell. biochem. 304 273-285, (2007) [26]. fabjanowicz m.,platka-wasylka j., namiesnik j., detection, identification and determination of resveratrol in wine. problems and chalenges, trends in analytical chemistry103, 2133, (2018) [27]. vidavalur r., otani h., singal p., máulik n., significance of wine and resveratrol in cardiovascular disease: french paradox revisited, exp. clin. cordial. 11, 217-225 (2006) [28]. wang, d. g., liu, w. y., & chen, g. t. a simple method for the isolation and purification of resveratrol from polygonum cuspidatum. journal of pharmaceutical analysis, 3(4), 241-247 (2013). [29]. garcia l., garcia r., pocheca g., sutili f., de souza f., mansur e., leal i., optimized extraction of resveratrol from arachis repens hancera by ultrasound and microwave: a correlation study with the antioxidant properties and phenol contents, the scient. world j., id 5890897, (2016) [30]. srinivas g., babykutty s., sothiadevan p., srinivas p., molecular mechanism of emodin action: transition from laxative ingredient to an antitumor agent, med. res. 2715, 591-608 (2007) [31]. berchová-bimová k., soltysiak j., vach m., role of different taxa and cytotyses in heavy metals absorption in knotweeds (fallopia), sci. agric. 45,. 11-18 (2014) [32]. billet, k., houillé, b., besseau, s., mélin, c., oudin, a., papon, n.& lanoue, a., mechanical stress rapidly induces e-resveratrol and e-piceatannol biosynthesis in grape canes stored as a freshly-pruned byproduct, food chemistry, 240, 1022-1027. (2018) [33]. nonomura, s., kanagawa, h., & makimoto, a.. chemical constituents of polygonaceous plants. i. studies on the components of ko-j o-kon.(polygonum cuspidatum sieb. et zucc.). yakugaku zasshi, journal of the pharmaceutical society of japan, 83, 988. (1963) [34]. aavikasar a., haga m., pursa t., roasto m., tsoupras g., purification of resveratrol from vine stems, proc. estonian acad, sci, chem. 52, pg 155-164 (2003) [35]. http://oiv.int/public/medias/6782/oiv-2019statistical-report-on-world-vitiviniculture.pdf [36]. cebrián, c., sánchez-gómez, r., salinas, m. r., alonso, g. l., & zalacain, a. effect of post-pruning vine-shoots http://oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf http://oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 alina lenuța crăciun, gheorghe gutt, study on extraction and purification of trans-resveratrol from grape cane a review, food and environment safety, volume xx, issue – 2021, pag. 113 – 127 127 storage on the evolution of high-value compounds, industrial crops and products, 109, 730-736 (2017) [37]. ji, m., li, q., ji, h., & lou, h. investigation of the distribution and season regularity of resveratrol in vitis amurensis via hplc–dad–ms/ms, food chemistry, 142, 61-65 (2014) [38]. németh g., hegyi o., dunai a., kocsis l., stilbenes in the different organs of vitis vinifera cv. merlot grafted on tk5bb rootstock, oeno one, 51,. 323-328, (2017) [39]. çetin, e. s., altinöz, d., tarçan, e., & baydar, n. g.. chemical composition of grape canes. industrial crops and products, 34(1), 994-998. (2011) [40]. soural i., vrchatová n., tríska j., balík j., horník s., curinová p., sykora j., various extraction methods for obtaining stilbenes from grape cane of vitis vinifera l., molecules journal (2015) [41]. bravo, m. n., feliciano, r., silva, s., coelho, a. v., boas, l. v., & bronze, m. r. analysis of trans-resveratrol: comparison of methods and contents in muscatel fortified wines from setúbal region in portugal. journal of food composition and analysis, 21(8), 634-643, (2008). [42]. moreira m., fatima barroso m., porto v., ramalhosa m.j., švarc-gajić j., estevinho l., morais s., deleruematos c., ”potential of portuguese vine shoot wastes as naturalresources of bioactive compounds”, science of total envioment 634, 831842, (2018) [43]. karacabey e., mazza g., bayindirli l., artik n., extraction of bioactive compounds from milled grape canes (vitis vinifera) using a pressurized low-polarity water extractor, food bioprocess technol 5, 359371, (2012) [44]. ju, y., zhang, a., fang, y., liu, m., zhao, x., wang, h., & zhang, z. phenolic compounds and antioxidant activities of grape canes extracts from vineyards. spanish journal of agricultural research, 14(3), 18(2016). [45]. gabaston j., leborgne c. , valls j., renouf e., richard t., waffoteguo p. , mérillon j., ” subcritical water extraction of stilbenes from grapevine by-products: a new green chemistry approach” industrialcrops&products 126, 272-279, (2018) [46]. houillé b., besseau s., courdavault v., oudin a., glévarec g., delanoue g., guérin l., simkin a.j., papon n., clastre m., giglioliguivarc’h n., lanoue a., byosynthetic origin of resveratrol acummulation in grape canes during pastharvest storage, j. agric. food chem. 63, 1631-1638, (2015) [47]. billet, k., houillé, b., besseau, s., mélin, c., oudin, a., papon, n., & lanoue, a. mechanical stress rapidly induces eresveratrol and e-piceatannol biosynthesis in grape canes stored as a freshly-pruned byproduct. food chemistry, 240, 1022-1027. (2018). [48]. vergara, c., von baer, d., mardones, c., wilkens, a., wernekinck, k., damm, a., winterhalter,p. stilbene levels in grape cane of different cultivars in southern chile: determination by hplc-dad-ms/ms method, journal of agricultural and food chemistry, 60(4), 929–933, (2012) [49]. gorena, t., saez, v., mardones, c., vergara, c., winterhalter, p., & von baer, d. „influence of post-pruning storage on stilbenoid levels in vitis vinifera l. canes”, food chemistry, 155, 256–263, (2014) [50]. bavaresco, l., cantu, e., fregoni, m., & trevisan, m. constitutive stilbene contents of grapevine cluster stems as potential source of resveratrol in wine. vitisjournal of grapevine research, 36(3), 115. (2015). [51]. rayne, s., karacabey, e., & mazza, g. grape cane waste as a source of transresveratrol and trans-viniferin: high-value phytochemicals with medicinal and antiphytopathogenic applications. industrial crops and products, 27(3), 335-340. (2008). [52]. yangji wep, pi li1, liyan ma, jingming lp, separation and purification of four stilbenes from vitis vinifera l. cv. cabernet sauvignon roots through high-speed counter current chromatograph ,centre for 'viticulture and enology, college of food science and nutritional engineering, china agricultural university, beijing, 100083, p.r. china (2014) [53]. al-jumaily, e. f., hamid, g. s., & ali, k. f., synthesis and total phenol content of new resveratrol derivative. open j adv drug deliv, 2(3), 320-329 (2014) [54]. güder, a., korkmaz, h., gökce, h., alpaslan, y.b., alpaslan, g. isolation, characterization, spectroscopic properties and quantum chemical computations of an important phytoalexin resveratrol as antioxidant component from vitis labrusca l. and their chemical compositions. spectrochim acta mol biomol spectrosc 133:378-395. (2014) [55]. karacabey, e., mazza, g., bayindirli, l., & artik, n. extraction of bioactive compounds from milled grape canes (vitis vinifera) using a pressurized low-polarity water extractor. food and bioprocess technology, 5(1), 359-371. (2012). 1. introduction 8. conclusion 55 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 55 59 influence of microwave drying on the content of photosynthetic pigments in spinach ramona mihaela zavada1,2, ana-maria roşu2*, neculai doru miron2, gheorghe surpateanu2, *ileana denisa nistor2 1"gheorghe asachi" technical university, faculty of chemical enginering and environmental protection, 71a, d. mangeron rd., 700050 iasi, romania, ramona_mihaela2008@yahoo.com 2“vasile alecsandri”university bacau, faculty of enginering, ica department, 157, calea marasesti, 600115 bacău, romania, denisanistor@yahoo.com, ana.rosu@ub.ro *corresponding author received 2nd march 2014, accepted 28th march 2014 abstract: drying is the process of removing the moisture in the product up to certain threshold value by evaporation. in this way, the product can be stored for a long period, since the activities of the microorganisms, enzymes or ferments in the material are suppressed via drying.spinach leaves (spinacia oleracea) were dried using two different methods: hot air and microware drying. the aim of this study was to determine the effects of the microwave output power on drying time, moisture content, moisture ratio and the dried product quality in terms of chlorophyll content. 10 g of leaf samples were dried in an oven at 1000c to asses their initial moisture content. six different microwave output powers (120, 280, 340, 460, 600 and 700 w) were investigated in drying experiments at constant sample loading. microwave drying period lasted between 3 and 10 minutes. increasing the microwave output power resulted in a considerable decrease in drying time. the content of chlorophylls was colorimetrically determined by measuring absorbance in the absorbance maximum for these pigments. the chlorophylls were extracted using acetone which was proven to be one of the most efficient solvents. results showed that the optimum microwave power for drying in terms of chlorophyll content is 340w. keywords: microwave drying, chlorophyll content, drying rate 1. introduction spinach (spinacia oleracea l.) is a cool season annual vegetable. leaf vegetables, including spinach, compose an important group of plant foods. spinach has a high content of vitamins, carotenoids, chlorophylls, organic acids and alkaline mineral constituents as well as antioxidants [1, 2]. it is a popular vegetable that is eaten raw, boiled or baked into various dishes. spinach is low in calories and is a good source of ascorbic acid [3]. spinach is a vegetable which rapidly perishes after harvest and which is consumed only in the product season. drying is the one of the storage methods, which has the capability of extending the consumption period of spinach, yet maintaining its vitamin content. drying is one of the oldest methods of food preservation and represents a very important aspect of food processing. food drying is a traditional method of food preservation which is also used for the production of special foods and food ingredients [4]. drying not only affects the water content of the product, but also alters other food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ramona mihaela zavada, ana-maria roşu, neculai doru miron, gheorghe surpateanu, ileana denisa nistor, influence of microwave drying on the content of photosynthetic pigments in spinach, issue 1 2014, pag. 55 – 59 56 physical, biological and chemical properties such as enzymatic activity, microbial spoilage, viscosity, hardness, aroma, flavor and palatability of foods [5]. natural drying (drying in the shade) and hot air drying are still most widely used methods to produce dried foodstuffs. natural drying has many disadvantages due to the inability to handle the large quantities and to achieve consistent quality standards [6]. hot-air drying leads to serious injuries such as the worsening of the taste, nutritional content and color of the product, decline in the water absorbance capacity, density and shifting of the solutes from the internal part of the drying material to the surface, due to the long drying period and high temperature [7-12]. compared to hot air drying, microwave energy applications in the drying of vegetables have several advantages including the shortening of drying time, a homogenous energy distribution on the material and formation of suitable dry product characteristics due to the increase in temperature in the centre of the material. among the other benefits of using microwave drying are inhibition of high surface temperatures, continuation of the product respiration, lowered product temperatures when combined with vacuum drying, reduction in the loss of watersoluble components and energy savings [13]. the aim of this study was to determine the microwave drying characteristics of spinach and to compare hot-air drying to microwave drying which reduces drying time considerably. also the effect of these drying techniques on the chlorophyll content of spinach was determined. 2. experimental 2.1. materials fresh spinach leaves were purchased from the local market. they were washed and stored at 40c in refrigerator for about one day for equilibration of moisture. before drying experiments, the samples were taken out of the refrigerator and leaves were separated from stems and then weighed. 2.2. methods 2.2.1. microwave drying a programmable domestic microwave oven with maximum output of 700w was used in the drying experiments. the oven was operated at microwave output powers of 120, 280, 340, 460, 600 and 700 w. five grams of spinach leaves were spread evenly on a glass microwave oven plate and processed until the leaves were completely dried. the mass of the samples was measured every 1 minute during the drying procedure using a digital balance. the following common semi-empirical equations (eq. (1) and (2)) was used to describe the thin layer drying kinetics of spinach leaves [14, 15], where mr is the moisture ratio and dr is drying rate; mt is the moisture content at a specific time (g water·g dry base-1); me is the equilibrium moisture content was assumed to be zero for microwave drying; m0 is the initial moisture content (g water·g dry base-1); mt+dt is the moisture content at t+dt (g water·g dry base-1) and t is drying time (minutes). the equilibrium moisture content (me) was assumed to be zero for microwave drying [16, 23]. ܴܯ = ெ೟ିெ೐ ெబିெ೐ (1) ܴܦ = ெ(೟శ೏೟)ିெ೟ ௗ೟ (2) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ramona mihaela zavada, ana-maria roşu, neculai doru miron, gheorghe surpateanu, ileana denisa nistor, influence of microwave drying on the content of photosynthetic pigments in spinach, issue 1 2014, pag. 55 – 59 57 2.2.2. quantification of photosynthetic pigments pigments were extracted from the dried materials using acetone, as extraction solvent. the ratio dried material: solvent was of 1:10. quantification of photosynthetic pigments was performed on a thermospectronic genesys 20. the corrected equations for the quantitative evaluation of chlorophyll a and b in acetone were used as previously determined in other studies [17, 26, 30]. 3. results and discussion the moisture content versus time curves for microwave drying of spinach leaves as influenced by microwave output power are shown in figure 1. figure 1. drying curves of spinach leaves under various microwave output powers the time required from the lowering of moisture content of spinach levels to 0,1 level varied between 3 and 11 minutes depending on the microwave power level. as the microwave output power was increased, the drying time of leaves was significantly reduced. similar findings were reported by several authors [18-21]. by working at 700w instead of 120w, the drying time is shortened by 75%. the drying rate was calculated as the quantity of moisture removed per unit time, per dry matter. the drying rate curves for spinach dried at different microwave output powers are given in figure 2. the moisture content of the material was very high during the initial phase of the drying which resulted in a higher absorption of microwave power and higher drying rates due to the higher moisture diffusion. as the drying progressed, the loss of moisture in the product caused a decrease in the absorption of microwave power and resulted in a fall in the drying rate. higher drying rates were obtained at higher microwave output powers. thus, the microwave output power had a crucial effect on the drying rate. similar findings were reported in previous studies [16, 21, 22]. figure 2. drying rate curves for spinach leaves under various microwave output powers the drying rate decreased continuously throughout the drying period. it is obvious from figure 2 that the constant rate period was absent; all drying process of spinach took place in falling rate period. these results are in good agreement as compared to the earlier studies of various vegetables [23-25]. figure 3 shows the moisture ratio versus the drying time through the semi-empirical equations (1). figure 3. moisture ration versus time curves for spinach leaves under various microwave output powers 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 9 10 11 drying time, min m oi st ur e co nt en t ( db ), kg [w at er ]/k g [d ry m at er ia l] 120 w 280 w 340 w 460 w 600 w 700 w 0 0,5 1 1,5 2 2,5 3 3,5 4 0 1 2 3 4 5 6 7 8 9 10 11 12 moisture content, (db) d ry in g ra te 120 w 280 w 340 w 460 w 600 w 700 w 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 1 2 3 4 5 6 7 8 9 10 11 drying time, min m oi st ur e ra ti o 120 w 280 w 340 w 460 w 600 w 700 w food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ramona mihaela zavada, ana-maria roşu, neculai doru miron, gheorghe surpateanu, ileana denisa nistor, influence of microwave drying on the content of photosynthetic pigments in spinach, issue 1 2014, pag. 55 – 59 58 ultraviolet-visible spectroscopy was used for the quantification of photosynthetic pigments from dried material depending on the microwave output power. figure 4 shows the dependence between the microwave output power and the content of chlorophylls of the dried material. the highest concentration of chlorophylls is obtained for the samples dried at 336w. results showed that a prolonged application of microwave radiation determined a decrease in chlorophyll content. also the content of chlorophylls is also diminished when a high microwave radiation is applied to samples. figure 4. chlorophyll content dried spinach leaves under various microwave output powers the values for chlorophyll content in the samples dried using microwave radiation varied from 3,75mg/g dried material. the chlorophyll content in the samples dried using hot-air was 2,48mg/g dried material. this value can be explained by the fact that photosynthetic pigments are highly susceptible to degradation during thermal treatment. the hot-air drying of spinach involved a 2 hours thermal treatment which determined a much pronounced degradation of chlorophylls. 4. conclusion microwave drying techniques can greatly reduce the drying time of biological materials without quality degradation [13, 16, 21, 2729]. compared with hot-air drying microwave drying offers opportunities as energy saving, precise process control, uniform energy and high thermal conductivity to the inner sides of the material, space utilization, sanitation, and high quality of the finish product. it also reduces the drying time and prevents food from decomposing [31, 32]. in the present study the microwave drying period of spinach varied from 3 to 10 minutes depending on the microwave powers. drying time decreased considerably with increased microwave output power. therefore microwave output power had a crucial effect on the drying rate. the chlorophyll contents varied from 3,76 to 5,84mg/g dried material. the highest value was obtained for the sample dried at 340w. also the lowest values for chlorophyll content, 2,48mg/g dried material, was obtained for hotair dried material. as an overall conclusion, compared to hot-air drying, the use of microwave rays in the drying of products has become widespread because it minimizes the decline in quality and provides a reduced drying time. 5. references [1] jaworska g., kmiecik w., maciejaszek i., comparison of the quality of canned spinach (spinacia oleracea l.) and new zealand spinach (tetragonia expansa murr). electronic journal of polish agricultural universities (ejpau) food science and technology, 4(2), (2001). [2] lisiewska z., gębczyński p., bernaś e., kmiecik w., retention of mineral constituents in frozen leaf vegetables prepared for consumption, journal of food composition and analysis, 22, 218–223, (2009). [3] toledo m.e.a., ueda y., imahori y., ayaki m., l-ascorbic acid metabolism in spinach (spinacia oleracea l.) during postharvest storage in light and dark. postharvest biology and technology, 28, 47–57, (2003). [4] maroulis z.b., saravacos g.d., food process design (1st ed.). new york, usa: marcel dekker, 243–244, (2003). [5] barbosa-canovas g.v., vegamercado h. dehydration of foods, 1st ed., new york, united states of america, 29, (1996). [6] soysal y., oztekin s., comparison of seven equilibrium moisture content equations for some medicinal and aromatic plants, journal of c hl or op hy ll s co nc en tr at io n (m g/ g ) 0 1 2 3 4 5 6 7 120w 280w 336w 462w 595w microwave output power food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 ramona mihaela zavada, ana-maria roşu, neculai doru miron, gheorghe surpateanu, ileana denisa nistor, influence of microwave drying on the content of photosynthetic pigments in spinach, issue 1 2014, pag. 55 – 59 59 agricultural engineering research, 78(1), 57–63, (2001). [7] bouraout m., richard r., durance t., microwave and convective drying of potato slides. journal of food process engineering, 17, 353–363, (1994). [8] drouzas a.e., tsami e., saravacos g.d., microwave/vacuum drying of model fruit gels. journal of food engineering, 39(2), 117–122, (1999). [9] feng, h., & tang, j. microwave finish drying of diced apple slices in a spouted bed. journal of food science, 63(4), 679–683, (1998). [10] lin t. m., durance t. d., seaman c.h., characterization of vacuum microwave air and freeze dried carrot slices. food research international, 4, 111–117, (1998). [11] maskan m., drying, shrinkage and rehydration characteristics of kiwifruits during hot air and microwave drying. journal of food engineering, 48(2), 177–182, (2001). [12] yongsawatdigul j., gunasekaran s., microwave-vacuum drying of cranberries: part ii. quality evaluation., journal of food processing and preservation, 20, 145–156, (1996). [13] torringa e., esveld e., scheewe i., van den berg r., bartels p., osmotic dehydration as a pre-treatment before combined microwave-hot-air drying of mushrooms. journal of food engineering, 49, 185–191, (2001). [14] sharma g.p., prasad s., drying of garlic (allium sativum) cloves by microwave-hot air combination. journal of food engineering, 50, 99–105, (2001). [15] soysal y., microwave drying characteristics of parsley. biosystems engineering, 89(2), 167–173, (2004). [16] maskan m., microwave/air and microwave finish drying of banana. journal of food engineering, 44, 71–78, (2000). [17] porra r.j., the chequered history of the development and use of simultaneous equations for the accurate determination of clorophylls a and b. photosynthesis research, 73, 149-155, (2002). [18] al-duri b., mcintyre s., comparison of drying kinetics of foods using a fan-assisted convection oven. journal of food engineering, 15, 139–155, (1992). [19] prabhanjan d.g., ramaswamy h.s., raghavan g.s.v., microwave assisted convective air drying of thin layer carrots. journal of food engineering, 25, 283–293, (1995). [20] drouzas a.e., schubert h., microwave application in vacuum drying of fruits. journal of food engineering, 28, 203–209, (1996). [21] funebo t., ohlsson t., microwaveassisted air dehydration of apple and mushroom. journal of food engineering, 38(3), 353–367, (1998). [22] sharma g.p., prasad s., drying of garlic (allium sativum) cloves by microwave-hot air combination. journal of food engineering, 50, 99–105, (2001). [23] akpinar e. k., mathematical modelling of thin layer drying process under open sun of some aromatic plants. journal of food engineering, 77, 864–870, (2006). [24] bimbenet j. j., daudin j. d., wolff e., air drying kinetics of biological particles, in proceedings of the fourth international drying symposium, kyoto, japan, 249–256, (1984). [25] senadeera w., bhandari b.r. young g., wijesinghe b., influence of shapes of selected materials on drying kinetics during fluidized bed drying. journal of food engineering, 58, 277–283, (2003). [26] rosu a.m., nistor i.d., miron n.d., popa m.i., cojocaru r.m., ultrasoundassisted extraction of photosynthetic pigments from dried dill (anethum graveolens). food and environment safety – journal of faculty of food engineering, stefan cel mare university – suceava, volume xi, issue 4, 5-9, (2012). [27] ren g., chen f., drying of american ginseng (panax quinquefolium) roots by microwave–hot air combination. journal of food engineering, 35, 433–443, (1998). [28] moreno j., chiralt a., escriche i., serra j.a., effect of blanching/osmotic dehydration combined methods on quality and stability of minimally processed strawberries. food research international, 33, 609–616, (2000). [29] nindo c.i., sun t., wang s.w., tang j., powers j.r., evaluation of drying technologies for retention of physical quality and antioxidants in asparagus (asparagus officinalis l.). lebensmittel wissenschaft und technologie, 36, 507–516, (2003). [30] cojocaru r.m., popa m.i., surpateanu g., rosu a.m., miron n.d., nistor i.d., study concerning the extraction od photosynthetic pigments from spinach, buletinul institutului politehnic din iasi, tomul lviii (lxii), fasc.3, sectia chimie si inginerie chimica, 101109, (2012). [31] decareau r.v., microwaves in the food processing industry 1st ed., orlando, united states of america: academic press, 102, (1985). [32] zhang m., tang j., mujumdar a. s., wang s., trends in microwave-related drying of fruits and vegetables. trends in food science and technology, 17(10), 524–534, (2006). 7 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 7 14 yogu rt s e nrich ed w i th pea pr o te i n *zapryana denkova1, emil filipov1, bogdan goranov1, iliyan dobrev1, velichka yanakieva1 1 university of food technology, department of microbiology, zdenkova@abv.bg *corresponding author received 19th february 2015, accepted 23rd march 2015 abstract: with the increasing cost of animal breeding, vegetable proteins seem to be a good alternative to this problem. the ability of three yogurt starters with probiotic strain lactobacillus delbrueckii ssp. bulgaricus to develop in cow’s milk, enriched with pea protein, was examined. the three starters developed in cow’s milk, enriched with pea protein, the concentration of the active cells reaching 1013 1014 cfu/cm3. yogurts containing different percentages of pea protein (0-10%) with the selected starters were prepared. the yogurt obtained with starter m and starter s using cow’s milk, enriched with 2 – 4% pea protein had the best organoleptic properties so they were stored at 4 ± 2°c for 25 days. there were no significant changes in the concentration of viable cells and the titratable acidity. the obtained yogurts enriched with pea protein are new dietary probiotic foods. along with the inherent prebiotic ingredients of pea, significant amount of useful microflora (1013-1014cfu/cm3) enters the human gastrointestinal tract, which is necessary to restore and maintain the balance of the gastrointestinal microflora, which is essential for human health. keywords: lactobacillus, pea protein, pea milk, starter, yogurt 1. introduction the widespread use of canned foods in modern man‘s diet, the massive use of antibiotics and other antimicrobial agents, the permanent stress and poverty, the deteriorating environmental situation led to deterioration of health. all these factors have direct effects on the balance of the gastrointestinal microflora. to restore and maintain the balance of the microflora in the stomach and intestines beneficial lactobacilli should be taken in with food. the consumption of lactic acid products introduces live beneficial bacteria necessary for the normalization of microflora in the gastrointestinal tract. this equilibrium is influenced by various factors of the medium (physical, chemical, biological). its violation leads to disruption of the functions not only of the digestive system, but also of the related organs and systems. the restoration and maintainance of the balance of the microflora in the gastrointestinal tract is achieved through introduction of beneficial microorganisms (lactobacilli and bifidobacteria) by consumption of concentrates of viable cells called probiotics or functional foods. moreover, in order to carry out their inherent preventive role, the concentration of viable cells of lactobacilli and bifidobacteria should exceed 108 cfu/g/cm3. generally, probiotic bacteria are included in the composition of yoghurts and other fermented yogurt beverages [1-4]. nowadays, non-dairy probiotic preparations imported into beverages or applied in the form of tablets, capsules, http://www.fia.usv.ro/fiajournal mailto:zdenkova@abv.bg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n k o v a, e mil f i l i po v , b og da n g o r a no v, il i ya n d ob r e v , ve lic h ka y an ak ie v a, yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nm e nt sa f e t y, v o l u me x iv , i s s ue 1 – 2 0 15 , pa g . 7 1 4 8 lyophilized preparations are gaining increasing popularity [5, 6]. soy milk is a good substrate for the development of lactobacilli and bifidobacteria with probiotic properties. by culturing of symbiotic combinations or single strains of lactobacilli, streptococci, denkova et al., 2001 [7] obtained fermented soy foods, such as soy yogurt, soy acidophilic yogurt (lactobacillus acidophilus) and soy bifid yogurt (bifidobacterium bifidum), and soy yogurt beverages (acidophilic-bifid beverage). the concentration of active cells exceeds 109cfu/cm3 and the storage life of soy fermented foods is more than 20 days in a refrigerator (4±2ºc). the authors found that the aroma substances , such as lactic, acetic, tartaric and malic acid, acetaldehyde, diacetyl, generated by the metabolism of lactobacilli and bifidobacteria removed soy offflavor and soy fermented foods receive consumers‘ acceptance. pea beans contain small amounts of carbohydrates, dietary fiber, lipids, saturated fatty acids, but the predominat substances are starch and valuable vegetable protein, vitamins (β-carotene, vitamins a, e, h, pp, b vitamins, vitamin c (in a raw state)), mineral elements such as iron, zinc, copper, manganese, aluminum, boron, molybdenum, fluorine, vanadium, nickel, titanium, silicon, lead, selenium, zirconium, cobalt, chromium, potassium, phosphorus, sulfur, chlorine, calcium, magnesium, sodium, i.e., possesses rich mineral composition. carbohydrates are less in quantity, while starch is more in mature pea beans. all substances in pea beans have beneficial impact on the health of the body. peas are useful in anemia and obesity, improves the condition of the liver, kidneys, cardiovascular system. green peas exhibit antiseptic activity. the cellulose in the pea beans helps to purify the stomach and intestines of slag; nicotinic acid (vitamin pp) maintains the level of cholesterol and reduces the risk of oncologic diseases; thiamine (vitamin b1) improves the functioning of the brain; vitamin h (biotin) exhibits antioxidant properties, regulates the level of blood sugar and stimulates the activity of the digestive and nervous system. the reduction of the amount of cow's milk as well as the increasing cost of animal breeding leads to seeking new ways to develop functional foods. proteins of plant origin offer a good alternative for solving this problem. there has been considerable success in replacing part of cow's milk with soy milk in the production of a number of products currently available on the market [8]. inspired by this success, researchers continue exploring the possibilities for substitution of cow‘s milk with other vegetable proteins. currently, another vegetable attracts the attention of researchers pisum sativum [9]. several processes for the extraction of the gliadin and glutenin (main pea proteins) from the whole pea beans can be used. one of them is water-vapor extraction of the pea beans to obtain pea milk rich in bioactive substances. but the most commonly used method is the grinding of the pea beans and concentrating the protein by drying to obtain an isolate which is subsequently resuspended to obtain „pea-milk“. pea protein isolate has a number of beneficial functional qualities such as good solubility in water, stability at high temperatures, good foaming capacity and high oil in water emulsifying power [10] and good stability in terms of shearing and retorting [11, 12]. then pea milk is mixed with the skimmed cow's milk at different ratios, and digested by microorganisms as a new raw material/substrate. from an economic perspective pea protein costs are almost half as much as milk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n k o v a, e mil f i l i po v , b og da n g o r a no v, il i ya n d ob r e v , ve lic h ka y an ak ie v a, yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nm e nt sa f e t y, v o l u me x iv , i s s ue 1 – 2 0 15 , pa g . 7 1 4 9 protein and 25% less than soy protein. in addition, the price of pea protein is relatively stable and resistant to fluctuations [12]. bacteria from the genus lactobacillus are be able to use fractions of the pea protein hydrolysates deleterious for other bacteria as substrates independently from the state of their existence. therefore, the consumption of foods enriched with pea protein may beneficially affect the human organism [13]. the purpose of the present research is to explore the possibilities of enriching yogurt foods with pea protein and obtaining of fermented probiotic foods. 2. materials and methods strains and media three yogurt starters containing different probiotic strain of lactobacillus delbrueckii ssp. bulgaricus were used in the present research: starter m, starter d and starter s. the used media were  skim milk with titratable acidity of 16-18ºt;  laptg10-broth;  laptg10-agar;  medium for detection of molds and yeasts in milk and dairy products at 25°c according to iso 6611/2004;  baird parker agar base (staphylococcus aureus);  medium for detection and isolation of salmonella sp. by horizontal process according to bs en iso 6579/2003;  chromocult tbx-agar (e. coli);  pca agar (medium for the determination of tba (total bacterial abundance) according to bs iso 6610:2002;  pea milk. preparation of pea milk raw peas (pisum sativum l.) were ground to flour in the laboratory mill. samples (35 g) of ground pea flour were extracted with 140 ml of 50 mm tris–hcl (ph 8.8) for 1 h at 4 °c with constant agitation and subsequently centrifuged (20 000 g, 20 min). the extraction was repeated twice. the supernatant, containing albumins and globulins, was dialysed at 4 °c for 48 h against distilled water and later on lyophilised [13, 14]. pea isolate, containing 81.34% pea protein, was obtained and was used to prepare 10% pea milk. determination of the titratable acidity ten сm3 of each sample were mixed with 20 сm3 of distilled water. the titratable acidity was determined by titration of each sample with 0.1 n naoh using phenolphthalein as an indicator until the appearance of a pale pink colour persisting over 1 min. one torner degree (°т) corresponds to 1 сm3 0.1 n naoh, needed for the neutralisation of an equivalent amount of organic acid, contained in 100 сm3 of the cultural medium [15]. determination of the concentration of viable cells appropriate serial dilutions in saline solution of the obtained yogurts were prepared and the spread plate method was applied. 0.1 cm3 of the last three dilutions was used to inoculate laptg10-agar (for the enumeration of lactobacilli and streptococci) or the respective elective solid medium (for the enumeration of the specific microorganisms). the inoculated petri dishes were incubated for 3 days at optimum temperature for the growth of the respective microorganisms until the appearance of countable single colonies (frank & yousef, 2004). the count of the colonies was then used to estimate the number of bacteria in the original sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n k o v a, e mil f i l i po v , b og da n g o r a no v, il i ya n d ob r e v , ve lic h ka y an ak ie v a, yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nm e nt sa f e t y, v o l u me x iv , i s s ue 1 – 2 0 15 , pa g . 7 1 4 10 organoleptic assessment was performed in accordance with bds 15612-83 statistical analysis data from triplicate experiments were processed using ms office excel 2003 software, at level of significance of p<0.05. 3. results and discussion the ability of yogurt starters with probiotic strain lactobacillus delbrueckii ssp. bulgaricus to develop in cow’s milk, enriched with pea protein, was examined. pea milk cannot be used for the preparation of foodstuffs alone, due to the high content of starch in it. thus, extraction of pea protein which has good solubility and can be used in food production was required for the preparation of 10% pea milk. at that concentration of pea protein and below, pea cow’s milk with increased density was obtained, but no layering of the milk during storage was detected. at concentrations of pea protein more than 10% there was clear layering of the pea milk during storage. the microbiological status of the pea isolate (unflavored) was examined. the results of these experimental tests are shown in table 1. table 1 microbiological status of the pea isolate tba, cfu/g specific microorganisms, cfu/g molds and yeasts, cfu/g e.coli (tbxagar) st.aureus salmonella sp. 4.6х102 <100 not found not found <100 two groups of bacteria were found in the pea isolate – sporeforming and nonsporeforming bacteria. the presence of sporeforming bacteria proved that pasteurization is not an appropriate method for sterilization of the pea milk obtained using the pea isolate. therefore the sterilization of the obtained pea milk is done at 121°c and 1 atm in order for the safety of the food products to be guarranteed. cow‘s milk containing 4% pea isolate was used for the primary screening for the starters able to grow in cow‘s milk enriched with pea protein. the inoculated milk substrates were incubated at 41±1ос for 4 – 5 hours until the coagulation of the yogurts. the microbial purity of the cow‘s milk enriched with pea protein was determined (table 2). table 2 microbiological status of the cow‘s milk enriched with pea protein tba specific microorganisms, cfu/cm3 molds and yeasts, cfu/cm 3 e.coli (tbx -agar) st. aureus salmonella sp. <1 <10 not found not found <10 the three starters developed in cow’s milk, enriched with pea protein, and produced lactic acid. the titratable acidity of the medium was within the range of 45-70°t. the concentration of the active cells reached 1013 1014 cfu/cm3 (fig. 1). fig. 1. concentration of viable cells and titratable acidity of non-traditional yogurts, obtained with cow’s milk, enriched with 4% pea protein. incubation – 4 – 5 hours (until coagulation) at 41±1ос food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n k o v a, e mil f i l i po v , b og da n g o r a no v, il i ya n d ob r e v , ve lic h ka y an ak ie v a, yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nm e nt sa f e t y, v o l u me x iv , i s s ue 1 – 2 0 15 , pa g . 7 1 4 11 the ratio between streptococcus thermophilus and lactobacillus delbrueckii ssp. bulgaricus was from 1:1 to 1:3 which indicates that the yogurts bear a considerable amount of the probiotic strain lactobacillus delbrueckii ssp. bulgaricus mz2. yogurts containing different percentage of pea protein (0-10%) with the selected starters were prepared. the fermentation process was conducted at 41±1°c for 4-17 часа until coagulation (table 3, fig. 2, fig. 3, fig. 4). table 3. organoleptic evaluation of the yogurts, obtained with cow‘s milk enriched with pea protein starter pea isolate, % organoleptic evaluation microscopic pattern starter m 0 % yogurt with distinct yogurt flavor; low density texture best yogurts 2% and 4% pea isolate rich microscopic pattern; cocci:rods = 1:1 starter m 2 % denser in texture, pleasant yogurt flavour, no pea off flavor rich microscopic pattern; cocci:rods = 1:1 starter m 4 % dense in texture, soft yogurt flavour, no pea off flavor rich microscopic pattern; cocci:rods = 2:1 starter m 6 % dense in texture, weak yogurt flavour, strong pea off flavor rich microscopic pattern; cocci:rods = 3:1 starter m 10 % blunt, no yogurt flavour, pea off flavor poor microscopic pattern; cocci:rods = 1:1. very long rods. starter s 0 % yogurt with typical yogurt flavour best yogurts 2% and 4% pea isolate rich microscopic pattern; cocci:rods = 1:50 starter s 2 % denser in texture, pleasant yogurt flavour, no pea off flavor rich microscopic pattern; cocci:rods = 1:1 starter s 4 % denser in texture than the previous variant, distinct yogurt flavour, no pea off flavor rich microscopic pattern; cocci:rods = 2:1 starter s 6 % denser in texture than the previous variant, very weak yogurt flavour, weak pea off flavor rich microscopic pattern; cocci:rods = 2:1 3:1 starter s 10 % blunt, strong pea off flavor rich microscopic pattern; cocci:rods = 50:1 long chains of cocci and rods starter d 0 % blunt, weak yogurt flavour poor microscopic pattern; cocci in doubles and chains starter d 2 % dense in texture, weak yogurt aroma, no pea off flavor poor microscopic pattern; cocci in doubles and chains, no rods starter d 4 % dense creamy texture, very weak yogurt flavour, no pea off flavor poor microscopic pattern; cocci in doubles and chains, no rods starter d 6 % dense creamy texture, weak rancid flavour poor microscopic pattern; cocci in doubles and chains, no rods starter d 10 % blunt, no yogurt flavour, pea off flavor poor microscopic pattern; too elongated cells, organized in long chains food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n ko v a, e mil f i l i po v , b og da n g or a n o v, i liya n d o b r e v , ve lic h ka y a n ak ie va , yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 7 1 4 12 a) b) fig. 2. concentration of viable cells (a) and titratable acidity (b) of pea yogurt, obtained with starter d. incubation – 4 – 5 hours (until coagulation) at 41±1ос a) b) fig. 3. concentration of viable cells (a) and titratable acidity (b) of pea yogurt, obtained with starter m. incubation – 4 – 5 hours (until coagulation) at 41±1ос a) b) fig. 4. concentration of viable cells (a) and titratable acidity (b) of pea yogurt, obtained with starter s. incubation – 4 – 5 hours (until coagulation) at 41±1ос the yogurt obtained with starter m and starter s using cow’s milk, enriched with 2 – 4% pea protein had the best organoleptic properties. yogurts with the selected concentrations of pea protein for each starter (starter m and starter s) were prepared and stored at 4 ± 2°c for 25 days. there were no significant changes in the concentration of viable cells and the titratable acidity (fig. 5, table 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n ko v a, e mil f i l i po v , b og da n g or a n o v, i liya n d o b r e v , ve lic h ka y a n ak ie va , yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nme nt sa f e t y, v o l ume x iv , is s ue 1 – 2 0 15 , pa g . 7 1 4 13 fig. 5. change in the concentration of viable cells of pea yogurts, obtained with starter m and starter s, during storage at 4±2ос for 25 days. table 4. change in the titratable acidity of pea yogurts, obtained with starter m and starter s, during storage at 4±2ос for 25 days starter pea isolate, % day 1 day 5 day 10 day 15 day 20 day 25 starter m 0 % 86 86,25 87.20 85.02 84.55 84.75 starter m 2 % 68 67,55 67.45 61.60 66 65 starter m 4 % 66 56,05 67.93 65.50 65.80 66.5 starter s 0 % 82 65,08 72.28 77.90 83.13 82.18 starter s 2 % 74 74,90 69.68 71.70 72.20 72.68 starter s 4 % 60.5 60,15 60.73 60.7 60.33 61.75 the obtained yogurts enriched with pea protein are new dietary probiotic foods. along with the inherent prebiotic ingredients of pea, significant amount of useful microflora (1013 1014cfu/cm3) enters the human gastrointestinal tract. it is necessary to restore and maintain the balance of microflora in the digestive tract, which is essential for human health. 4. conclusion yogurt starters able to grow in cow‘s milk enriched with pea protein were selected. the optimal percentage of pea isolate in the milk substrate for the preparation of pea yogurts was determined to be 2% and 4%. the obtained pea yogurts had high concentration of viable cells (10131014cfu/cm3) and moderate titratable acidity and the changes in these values during their 25-day storage are insignificant. therefore they can be used as functional foods, providing the body with the necessary amount of useful microflora to carry out its inherent preventive effect. moreover, these new products containing pea protein have a number of other beneficial effects on different systems of the human organism, thus ensuring its resistance to the negative factors of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 z ap r ya na d e n k o v a, e mil f i l i po v , b og da n g o r a no v, il i ya n d ob r e v , ve lic h ka y an ak ie v a, yog u r t s e nr i c h e d wi t h p ea p r ot ei n, fo o d a nd e nvi r o nm e nt sa f e t y, v o l u me x iv , i s s ue 1 – 2 0 15 , pa g . 7 1 4 14 environment, including prevention of infections. 5. references [1]. laroia s, martin jh, effect of ph on survival of bifidobacterium bifidum and lactobacillus acidophilus in frozen fermented dairy desserts. cultured dairy prod. j. 26:13–21 (1991). [2]. hagen m, narvhus ja, production of ice cream containing probiotic bacteria. milchwissenschaft 54:265–268 (1999). [3]. denkova r, yanakieva v, denkova z, urshev z, goranov b, sotirova e, identification and examination of some probiotic properties of lactobacillus plantarum f3. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava 11 (4):22 – 29 (2012). [4]. denkova r, ilieva s, nikolova d, evstatieva y, denkova z, yordanova m, yanakieva v, antimicrobial activity of lactobacillus plantarum x2 against pathogenic microorganisms. bulg. j. agric. sci. 2 (19):108– 111 (2013). [5]. schrezenmeir j, de vrese m, probiotics, prebiotics, and synbioticsd approaching a definition. am.j. clin. nutr., 77, 361–364 (2001). [6]. berni-canani r, cirillo p, terrin g, cesarano l, spagnuolo mi, de vicenio a, albano f, passariello a, de marco g, manguso f, guarino a, probiotics for treatment of acute diarrhea in children: randomized clinical trail of five different preparations. bmj, 335–340 (2007). [7]. denkova z, murgov i, soy fermented foods. interobmen ltd, plovdiv (bg) (2001). [8]. canabady-rochelle ls, mellema m, physical–chemical comparison of cow's milk proteins versus soy proteins in their calcium binding capacities. colloids and surfaces a: physicochemical and engineering aspects 366 (1– 3):110-112 (2010). [9]. adebiyi ap, aluko re, functional properties of protein fractions obtained from commercial yellow field pea (pisum sativum l.) seed protein isolate. food chemistry 128 (4):902908 (2011). [10]. donsi f, senatore b, huang q, ferrari g, development of novel pea proteinbased nanoemulsions for delivery of nutraceuticals. journal of agricultural and food chemistry 58(19):10653–10660 (2010). [11]. sullivan f, strategic analysis of the european pea market (2010). [12]. kent rm, doherty sb, probiotic bacteria in infant formula and follow-up formula: microencapsulation using milk and pea proteins to improve microbiological quality. a review. food research international 64:567–576 (2014). [13]. świątecka d, świątecki a, kostyra h, marciniak-darmochwał k, kostyra e, the impact of pea protein hydrolysates on bacterial physiological activity— an in vitro study. international journal of food microbiology 140:263–270 (2010). [14]. weiss w, vogelmeier c, görg a, electrophoretic characterization of wheat grain allergens from different cultivars in bakers' asthma. electrophoresis 14 (8):805–816 (1993). [15]. denkova z, development and application of probiotics. dsc thesis. university of food technologies, plovdiv, bulgaria (bg) (2005). 80 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 80 86 heavy metals in tobacco *sonia amariei1, cristina-elena hretcanu1, gheorghe gutt1, alexandra agachi1 1faculty of food engineering, stefan cel mare university of suceava, romania gutts@fia.usv.ro *corresponding author received 25th february 2014, accepted 15th march 2014 abstract: heavy metals in cigarettes were determined considering that the number of smokers of both sexes is growing. besides air, water, food raw materials, foodstuff, dietary supplements, the human body can accumulate heavy metals from tobacco. tobacco samples from romanian and ukrainian market were analyzed using inductively coupled plasma mass spectrometry (icpms). the results showed that the romanian samples have lower content of heavy metal than the ukrainian samples. tolerable weekly intake of cadmium was over in the case of two samples for minimum and maximum consumption and in the case of three samples for maximum consumption. key words: tobacco, heavy metals, risk assesment, tolerable weekly intake. 1. introduction it is estimated that the actual number of substances in tobacco varies between 2000 and 3000, half of them existing in tobacco and the remainder resulting from the conversion into chemical processes that occur from burning tobacco. hundreds of additives to improve the taste, odor, flavor of different varieties of tobacco are added to these. besides these substances carcinogen acting radioactive isotopes (pb210, po201, k30 etc.) have been found in tobacco. an important factor in heavy metal uptake is the soil composition. the chemical composition of soil, ph, the humus content influenced pb and cd accumulation in leaves [1], [2], [3]. these elements were captured mainly by tobacco plants from radioactive substances forming the environment fund. the humus content influenced pb and cd accumulation in [1]. industrial emissions, phosphate fertilizers containing cadmium [2], [3], insecticides based on heavy metals or metallic compounds of cadmium, mercury, lead, arsenic constitutes a health hazard to the consumer [5], [6], [7]. cigarette smoking and tobacco chewing are a major source of cadmium exposure [8], [9]. cigarette smoke is a very dangerous source of poisoning with cd for both active smokers and passive ones. to highlight the heavy metals content in cigarettes 14 varieties of cigarettes, both romanian and ukrainian market, were analyzed. 2. matherials and methods 2.1 materials tobacco samples are presented and coded in table 1. food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 81 table 1. codification of samples sample code sample cigarettes country of origin 1 kent nanotek neo romania 2 kent nanotek ukraine 3 kent 8 romania 4 kent clik romania 5 winston blue romania 6 winston balanced blue ukraine 7 φэct ukraine 8 mallboro red romania 9 malboro gold romania 10 monte carlo red ukraine 11 pall mall ukraine 12 pall mall 3tek charcoal filter romania 13 l&m red label romania 14 l&m tune slims romania 2.2 sample preparation sample preparation is carried out in accordance with the standard sr en iso 14082:2003, determination of trace elements by atomic absorption spectrometry after ashing. moisture content of tobacco samples was determined by oven drying method. ash content for each sample was determined by ashing in the furnace nabertherm p330. dissolving of ash is carried out according to sr en iso 14082:2003. ash of cigarettes taken from each sample was dissolved in 5 ml of hydrochloric acid concentration of 6 mol / l, the acid is evaporated in a water bath, and the residue was dissolved in a volume of 10 ml of nitric acid 0.1 mol / l 2.3 reagents all solutions were prepared with reagent grade chemicals and ultra-pure water (18 mω cm). nitric acid and hydrochloric acid were purchased from sigma aldrich. 2.4. apparatus the analysis of samples was performed with mass spectrometry inductively coupled plasma icp-ms agilent technologies 7500 series precisely to 10-12 2.5 calculation of results concentration (c) of the heavy metals in samples is expressed in g/g sample and is calculated using the formula: c m v a  (1) where: a concentration value measured by the device, [ppb]; v volume of acid dissolving the sample [ml]; m mass of sample mineralized, [g]. 2.6 assesment of risk the estimated daily intake (eh s) was calculated for heavy metals and compared with tolerable daily intake (td / s). the data is based on the assumption that body weight is 60 kg: edi=(cxfdc)/bw (2) where: c the concentration of contaminant (µg/g), fdc stand for tobbaco daily consumption (g/d) bw the body weight (kg) [10]. the current tolerable weekly intake (twi) of 2.5 µg/kg body weight (b.w.) for cadmium is established by the joint fao/who expert committee on food additives (jecfa) in 2010, and the contam panel of european food safety authority efsa reassessed the twi in 2011 and concluded that the twi of 2.5 µg/kg b.w. is still appropriate. food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 82 2.6. statistical analysis all analyses were carried out in triplicates with replication. the mean and standard deviation of the data obtained were calculated. principal component analysis (pca) was used to aggregate variables obtained from the amount of heavy metals (corresponding to a daily consumption of minimum 10 and maximum 20 cigarettes per day) into a smaller number of orthogonal factors. principal component analysis was carried out with the software unscrambler x 10.1 (camo, norway). 3. results and discussions samples were always analyzed in triplicates. ash content and moisture of the samples is shown in figure 1. fig. 1 ash content and moisture of cigarettes samples heavy metal analysis was performed using icp-ms device. taking into account the masses of samples and using equation (1), the amount of heavy metals corresponding to a daily consumption of minimum 10 and maximum 20 cigarettes per day was calculated [8]. as shown in table 3, the content of the elements as, pb, hg is not exceeded even a consumption of 20 cigarettes per day, conclusions drawn also by other authors after analyzing a number of samples of tobacco [11-12]. food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 83 tabel 2 elemental concentrations of samples sample kent nanotek neo ro 10/20 cigarettes [ppb] kent nanotek ru 10/20 cigarettes [ppb] kent 8 10/20 cigarettes [ppb] kent clik 10/20 cigarettes [ppb] winsto n blue ro 10/20 cigarettes [ppb] winston balanced blue ru 10/20 cigarettes [ppb] φэct ru 10/20 cigarettes [ppb] mallboro red 10/20 cigarettes [ppb] malboro go ld 10/20 cigarettes [ppb] monte carlo red ru 10/20 cigarettes [ppb] pall mall ru 10/20 cigarettes [ppb] pall mall 3tek charcoal filte r ro 10/20 cigarettes [ppb] l&m red label 10/20 cigarettes [ppb] l&m tune slims 10/20 cigarettes [ppb] li 7 12.98943 / 25.97887 11.72699 / 23.45398 14.38711 / 28.77422 11.13898 / 22.27796 15.36353 / 30.72706 13.79028 / 27.58055 9.93768 / 19.87535 11.00166 / 22.00332 6.65879 / 13.31757 17.11050/ 34.22100 3.63803/ 7.27606 10.91738/ 21.83477 18.31804/ 36.63608 9.25871/ 18.51742 b 11 14.17029 / 28.34058 7.94209/ 15.88418 7.81884/ 15.63767 8.14046/ 16.28093 10.88631/ 21.77261 8.71705/ 17.43409 5.93768/ 11.87535 6.33753/ 12.67505 5.96270/ 11.92540 8.20324/ 16.40649 16.65138/ 33.30276 4.80487/ 9.60973 13.17226/ 26.34452 3.16007/ 6.32014 mg 24 2639.32049/ 5278.64098 2428.12823/ 4856.25646 2223.97476/ 4447.94953 2022.57439/ 4045.14879 1662.9682/ 3325.93656 1727.00644/ 3454.01289 2350.14164/ 4700.28329 1986.70781/ 3973.41562 1174.1528/ 2348.30575 2422.20182/ 4844.40363 2113.52288/ 4227.04576 1899.64521/ 3799.29042 1479.84667/ 2959.69334 1585.94211/ 3171.88423 al 27 903.87404 / 1807.74808 1791.72699/ 3583.45398 750.67598/ 1501.35196 1124.50120/ 2249.00239 282.33912/ 564.67824 206.56122/ 413.12244 165.77904/ 331.55807 375.38571/ 750.77142 192.14605/ 384.29209 149.27048/ 298.54097 1219.09712/ 2438.19423 655.1444/ 1310.28890 471.94796/ 943.89592 378.47017/ 756.94034 k 39 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ca 43 11412.8858 / 22825.77170 10566.7011/ 21133.40228 9361.19874/ 18722.3974 7763.08289/ 15526.1657 6929.9047/ 13859.8094 8329.23257/ 16658.4651 8962.03966/ 17924.07932 7843.57940/ 15687.1587 4083.2676/ 8166.53533 8783.79757/ 17567.59514 6327.32090/ 12654.64180 6699.44247/ 13398.88495 4772.91207/ 9545.82414 5772.29770/ 11544.59539 cr 53 2.19598/ 4.39196 1.36505/ 2.73009 0.96890/ 1.93781 1.66458/ 3.32915 1.12257/ 2.24514 0.77329/ 1.54657 1.08782/ 2.17564 0.90197/ 1.80394 0.72235/ 1.44471 0.73462/ 1.46924 1.89544/ 3.79089 0.77040/ 1.54080 0.51109/ 1.02219 0.54637/ 1.09273 mn 55 113.09302/ 226.18604 98.42813/ 196.85626 78.40243/ 156.80487 73.64041/ 147.28081 89.53139/ 179.06279 41.69889/ 83.39777 98.11898/ 196.23796 88.52362/ 177.04723 37.54925/ 75.09850 90.38874/ 180.77747 81.10670/ 162.21339 62.43284/ 124.86569 52.95621/ 105.91242 46.64796/ 93.29592 fe 56 381.18914/ 762.37829 380.55843/ 761.11686 241.09959/ 482.19919 243.98586/ 487.97173 201.01814/ 402.03629 168.71705/ 337.43409 231.16147/ 462.32295 491.33634/ 982.67268 34.14762/ 68.29525 218.34507/ 436.69013 228.26862/ 456.53725 196.65484/ 393.30968 101.05703/ 202.11407 153.57354/ 307.14708 co 59 0.87632/ 1.75264 0.79214/ 1.58428 1.51307/ 3.02614 0.59628/ 1.19257 0.53909/ 1.07819 0.49561/ 0.99121 0.66062/ 1.32125 0.58509/ 1.17019 0.26661/ 0.53323 0.68666/ 1.37333 0.55335/ 1.10670 0.57070/ 1.14141 0.44488/ 0.88977 0.44743/ 0.89486 ni 60 3.02465/ 6.04931 1.98552/ 3.97104 3.67283/ 7.34565 1.77859/ 3.55718 1.90576/ 3.81151 1.47627/ 2.95255 1.88102/ 3.76204 1.49537/ 2.99074 0.99816/ 1.99632 2.10183/ 4.20365 1.89544/ 3.79089 1.78409/ 3.56817 0.99895/ 1.99791 1.12227/ 2.24454 cu 63 15.26828/ 30.53657 11.72699/ 23.45398 9.88058/ 19.76115 9.88485/ 19.76970 12.23078/ 24.46156 7.69772/ 15.39543 17.41643/ 34.83286 7.91597/ 15.83195 24.12661/ 48.25322 16.70238/ 33.40475 6.89901/ 13.79802 4.73391/ 9.46782 4.72761/ 9.45522 5.27171/ 10.54341 zn 66 24.03149/ 48.06298 19.85522/ 39.71044 17.57548/ 35.15097 16.64576/ 33.29153 15.14163/ 30.28325 11.24780/ 22.49561 17.67705/ 35.35411 17.32732/ 34.65464 11.29498/ 22.58997 13.87614/ 27.75227 14.87822/ 29.75645 12.77243/ 25.54486 9.98955/ 19.97909 9.30301/ 18.60602 ga 69 3.70831/ 7.41661 3.08170/ 6.16339 0.85624/ 1.71248 1.24273/ 2.48546 1.13562/ 2.27124 0.38664/ 0.77329 0.30595/ 0.61190 0.59340/ 1.18680 0.19701/ 0.39401 0.27548/ 0.55096 0.65220/ 1.30439 1.51039/ 3.02078 0.25555/ 0.51109 0.75310/ 1.50620 as 75 0.60079/ 1.20157 0.49638/ 0.99276 0.63091/ 1.26183 0.51305/ 1.02611 0.36549/ 0.73098 0.60926/ 1.21851 0.70255/ 1.40510 0.65274/ 1.30548 0.28894/ 0.57788 0.78563/ 1.57127 0.59105/ 1.18211 0.49671 0.99341 0.34847/ 0.69695 0.36917/ 0.73833 se 82 br 79 22.58131/ 45.16263 14.68459/ 29.36918 8.90041/ 17.80081 13.56744/ 27.13488 10.44250/ 20.88500 16.28588/ 32.57176 13.48442/ 26.96884 7.35818/ 14.71635 1.97006/ 3.94011 6.12182/ 12.24365 8.15245/ 16.30490 9.02179/ 18.04359 3.02010/ 6.04019 3.54400/ 7.08801 ag 107 60078.7238/ 120157.447 37228.5418/ 74457.0837 2280.2417/ 4560.48341 7120.8165/ 14241.6330 cd 111 8701.05656/ 17402.1131 9720.78594/ 19441.5718 9689.0491/ 19378.0982 5472.5800/ 10945.1601 4568.5941/ 9137.1883 5858.2308/ 11716.4616 19943.3427/ 39886.6855 6883.4559/ 13766.9119 656.68505/ 1313.37011 10815.2229/ 21630.44587 7744.82829/ 15489.65658 3345.15966/ 6690.31931 3368.56778/ 6737.13556 sn 118 ce 140 191257.509/ 382515.019 205418.821/ 410837.642 168228.93/ 336457.863 136039.22/ 272078.440 155749.90/ 311499.804 71845.342/ 143690.685 93280.4532/ 186560.906 129741.27/ 259482.554 43761.491/ 87522.9839 73788.3889/ 147576.777 152165.494/ 304330.989 100679.168/ 201358.3375 71227.78488/ 142455.5697 65445.95393/ 130891.9078 pt 195 0.00021/ 0.00041 0.00012/ 0.00025 0.00007/ 0.00014 0.00011/ 0.00023 0 0 0 0.000024/ 0.000047 0 0 0.00011/ 0.00022 0.000051/ 0.000101 0 0 au 197 0.00056/ 0.00112 0.00141/ 0.00281 0.00025/ 0.00050 0.00025/ 0.00050 0.00035/ 0.00070 0.00009/ 0.00019 0.00008/ 0.00016 0.00017/ 0.00033 0.000066/ 0.000131 0.000031/ 0.000061 0.00036/ 0.00071 0.00002/ 0.00004 0.00028/ 0.00056 0.00028/ 0.00056 hg 202 0.00601/ 0.01202 0.00786/ 0.01572 0.00160/ 0.00319 0.00026/ 0.00052 0.00269/ 0.00539 0.06950/ 0.13900 0.00030/ 0.00061 0.03869/ 0.07738 pb 208 bi 209 0.05652/ 0.11303 0.01030/ 0.02060 0.01947/ 0.03894 0.00727/ 0.01455 0.00559/ 0.01117 0.00408/ 0.00815 0.00519/ 0.01038 0.00330/ 0.00660 0.00234/ 0.00468 0.00253/ 0.00506 0.01455/ 0.02910 0.00373/ 0.00746 0.00451/ 0.00901 0.00174/ 0.00348 u 235 0.05179/ 0.10358 0.02482/ 0.04964 0.01915/ 0.03831 0.01938/ 0.03876 0.02741/ 0.05482 0.01640/ 0.03281 0.01700/ 0.03399 0.02492/ 0.04985 0.00867/ 0.01734 0.01530/ 0.03061 0.02446/ 0.04891 0.01723/ 0.03447 0.02672/ 0.05343 0.02215/ 0.04430 per day levels were calculated considering a consumption of minimum 10 cigarettes and maximum 20 cigarettes food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 84 in contrast, the cd content is exceeded in the case of three samples for a minimum consumption of 10 cigarettes per day, and the content is exceeded for five samples at a consumption of 20 cigarettes per day, figure 2. fig. 2 comparative analysis of the cd content in romanian and ukrainian cigarettes principal component analysis was carried out according to the moisture content, ash and mineral concentrations in different samples of cigarettes. the scores of samples in the reduced space and the influence of chemical composition of the main component analysis are presented in figure 3 and figure 4. pc-1 (65%) -0.2 -0.1 0 0.1 0.2 0.3 p c2 (2 6 %) -0.2 -0.1 0 0.1 0.2 scores 1 2 3 4 5 6 7 8 9 10 11 12 13 14 food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 85 fig. 3 principal component analysis in different samples of cigarettes, according to the moisture and ash this analysis identifies some kinds of cigarettes chemically similar. principal component analysis was performed to assess the overall effect of chemical composition on the origin of cigarettes. principal component 1 (pc1) explained 65% of variance, while component (pc2) explains 26% of variation, the overall percentage of variation of the two main components being 91% (figure 4). fig. 4 principal component analysis in different samples of cigarettes, according to the mineral concentrations component by pc1 distinguishes samples according to the content in ag, bi, u235 while pc2 component distinguished samples according to the content in as, mn and cd. the hg and al concentrations and proximity to the origin of the coordinates indicates that these parameters are not useful in the total variation. component pc2 distinguishes types of cigarettes in two categories: on the left side there are romanian cigarettes, while on the right side there are ukrainian cigarettes. cigarettes kent nanotek neo romania are in discordant note to the other romanian cigarettes being placed close to the ukrainian cigarettes because of metal content. 4. conclusion the metals cu, ni, cr, se, hg, pb were found to be neglijible. for cadmium, twi was exceeded for kent clik cigarettes of 2.52 times, at a rate of 10 cigarettes per day, and of 5.10 times at a consumption of 20 cigarettes per day. in the case of φэct cigarettes, twi has been exceeded of 1.86 times at a consumption of 20 cigarettes per day. the penetration of toxic elements in the body is influenced by the moisture content of tobacco [9]. in the case of a high tobacco moisture more and more water vapor are generated which allows a drive of several toxic substances to the mouth end of the cigarette. smoking of the last third of cigarette lead to increasing the ingestion of toxic substances from its total content determined. food and environment safety journal of faculty of food engineering, stefan cel mare university suceava volume xiii, issue 1 – 2014 sonia amariei, cristina-elena hretcanu, gheorghe gutt, alexandra agachi, heavy metals in tobacco, issue 1 2014, pag. 80 86 86 5. references [1]. zaprjanova penka, ivanov krasimir, angelova violina, dospatliev lilko, relation between soil characteristics and heavy metal content in virginia tobacco, world congress of soil science, soil solutions for a changing world, 1 – 6 august 2010, brisbane, australia. published on dvd, 205-208 [2]. pelit füsun okçu, demirdöğen ruken esra, henden emür, investigation of heavy metal content of turkish tobacco leaves, cigarette butt, ash, and smoke, environ monit assess (2013) 185:9471–9479 [3]. zhang y., yang x., zhang s., tian y., guo w., the influence of humic acids on the accumulation of lead (pb) and cadmium (cd) in tobacco leaves grown in different soils, journal of soil science and plant nutrition, 2013, 13(1), 43-53 [4]. lugon-moulin n., ryan l., donini p., rossi l., cadmium content of phosphate fertilizers used for tobacco production, agron. sustain. dev. 26 (2006), 151–155 [5]. beccaloni eleonora, vanni fabiana, beccaloni massimiliano, carere mario, concentrations of arsenic, cadmium, lead and zinc in homegrown vegetables and fruits: estimated intake by population in an industrialized area of sardinia, italy, microchemical journal 107, (2013) 190–195 [6]. musharraf syed ghulam, shoaib muhammad, siddiqui amna jabbar, najamul-haq muhammad, aftab ahmed, quantitative analysis of some important metals and metalloids in tobacco products by inductively coupled plasma-mass spectrometry (icp-ms), chemistry central journal, (2012) [7]. r.s. pappas, s.b. stanfill, c.h. watson, d.l. ashley, analysis of toxic metals in commercial moist snuff and alaskan iqmik , journal of analytical toxicology, vol. 32, may 2008, p.281-290 [8]. ashraf muhammad waqar, levels of heavy metals in popular cigarette brands and exposure to these metals via smoking, the scientific world journal, (2012), article id 729430, p.1-5 [9]. prabhakar v, jayakrishnan g, nair sv, ranganathan b, determination of trace metals, moisture, ph and assessment of potential toxicity of selected smokeless tobacco products, indian journal of pharmaceutical sciences, volume75, issue 3, 9 (2013), 262-269 [10]. yared beyene yohannes, yoshinori ikenaka, shouta m.m. nakayama, aksorn saengtiencha, kensuke watanabe, mayumi ishizuka, organochlorine pesticides and heavy metals in fish from lake awassa, ethiopia: insights from stable isotope analysis, chemosphere 91 (2013), 857–863 [11]. lazarević konstansa, nikolić dejan, stošić ljiljana, milutinović suzana, videnović jelena, bogdanović dragan, determination of lead and arsenic in tobacco and cigarettes: an important issue of public health, cent eur j public health (2012); 20 (1): 62–66 [12]. dhaware dhanashri, deshpande aditi, khandekar r.n., chowgule rohini, determination of toxic metals in indian smokeless tobacco products, the scientific world, 9 (2009), 1140–1147 doi: https://doi.org/10.4316/fens.2021.024 211 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 211 223 hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria) salim boubelli 1,2 , nabil bougherira 2 , djamel rihia 3 , *hicham chaffai 2 1faculty of sciences and technology, university of ghardaïa, airport road, bp 455 ghardaïa 47000, algeria. 2laboratory of water resource and sustainable development (redd), department of geology, faculty of earth sciences, university badji mokhtar, bp 12, 23000 annaba, algeria. hichamchaffai@yahoo.fr 3department of earth sciences and the universe, university of jijel, algeria. *corresponding author received 27th july 2021, accepted 28th september 2021 abstract: the study of the hydrogeochemical characteristics of the groundwater samples made it possible to assess the impact of this process on the degradation of the quality of borehole water in the province of sebseb. samples taken from 10 boreholes across the study region were analyzed for the determination of physicochemical characteristics such as ph, electrical conductivity, temperature, bicarbonates, chlorides, sulphates, nitrates, ammonium, calcium, magnesium, sodium, potassium and total dissolved solids (tds). examination of the variation in the hydrogeochemical composition of groundwater indicates high concentrations for all parameters (anions and cations) except potassium, sodium and electrical conductivity. the hydrogeochemical evolution of groundwater in sebseb province was mainly controlled by the dissolution / precipitation of carbonate, sulphate and chloride minerals. the dissolution of minerals may explain the chemical characteristics of the waters studied. the waters of the sebseb region undergo deterioration in their quality and are no longer drinkable from a physicochemical point of view. the result of the saturation index calculation showed that most of the water samples were saturated or supersaturated with respect to carbonate minerals (calcite, dolomite and aragonite) and sulphate minerals (anhydrite and gypsum). understanding the hydrogeochemical process can strengthen their protection for a sustainable use of these groundwater resources. keywords: hydrogeochemistry, physico-chemical quality of water, groundwater, sebseb, south algeria. 1. introduction water is a very important component of the earth's environment. throughout human history, water has always supported life and served communities. however, the importance of the quality of the available water cannot be overestimated [1]. as a result, groundwater is not entirely pure as it generally contains mineral ions [1], [2]. the type and concentration of these dissolved minerals can affect the usefulness of groundwater for different purposes [3]. the geochemistry of groundwater is currently the subject of specific programs in many countries, particularly with a view to determining the geochemical background [4]. often, the composition of water does not represent a state of equilibrium but a step along a succession of reactions leading to a state of global equilibrium [5]. different waters collected in a given aquifer can represent stages of water-rock interaction along the same http://fens.usv.ro/index.php/fens mailto:hichamchaffai@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 212 evolutionary process. in fact, the quality of groundwater is also deteriorating due to climate change resulting in low precipitation and high evapotranspiration [6]. the quality of groundwater in an area depends largely on natural processes, such as the dissolution and precipitation of minerals, the velocity of groundwater, the quality of recharge water, and the interaction with other types of aquifers. the alteration and erosion of hydrographic basins are anthropogenic inputs, in particular urbanization, agricultural and industrial activities [7-9]. among the natural factors, the geology of the watershed, the degree of chemical alteration of the different types of rocks and inputs from sources other than the water-rock interaction play an important role in the control of water chemistry [10], [11]. over the past decades, numerous studies have been published on the main ions, trace elements and potentially harmful elements in water, providing information on possible geological and anthropogenic influences on aquifers [1214]. in the sebseb region, a desert province, groundwater has been the only resource for human consumption and agricultural needs since very ancient times [15]. the main objective of this work is to provide information on the geochemistry of the waters of the region studied, to describe the variations in the concentration of major elements and some nutrients, to understand the mechanisms responsible for these variations in order to determine the physicochemical quality of these waters, considered as the only resource used in drinking water in the province of sebseb. 2. matherials and methods 2.1. study area the commune of sebseb is part of the province of ghardaïa; it is located 60 km from the main city and 663 km south of the capital algiers. its geographical position gives it an influence of the saharan climate, arid and hot (fig. 1). the m'zab chebka is made up of a multitude of small wadis born from the junction of several small ravines. the sebseb valley is the widest among these wadis and is recognized for its groundwater potential. fig. 1. situation map of the study area food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 213 2.2. geological overview the geological synthesis of this region of southern algeria shows that the sebseb valley rests on a formation that belongs to the continental quaternary. this structure is in the form of beds of wadis, depressions and dunes. it is located at the western edges of the secondary sedimentary basin of the sahara, on a large subhorizontal plateau of limestone massifs of turonian age. the thickness of these limestone massifs intersected by soundings is of the order of 110 meters. under the turonian limestones, we cut an impermeable layer of 220 meters formed of green clay and marl rich in gypsum and anhydrite. it is attributed to the cenomanian. the albian stage is represented by a large mass of fine sands, sandstone and green clays. it is home to considerable water resources, and its thickness is around 300 meters (fig. 2). quaternary alluvium formed by sands, pebbles and clays line the bottom of the valleys of the wadis of the ridge, their thickness varies from 20 to 35 meters [16]. quaternary alluvium formed by sands, pebbles and clays line the bottom of the valleys of the wadis of the ridge, their thickness varies from 20 to 35 meters [16]. 2.3. sample status and analysis data sampling was carried out on wells in the town of sebseb (fig. 3). the results of 10 samples analyzed during the observation period of september 2017 are reported in table 1. chronostratigraphy hydrogeological unit depth lithostratigraphy lithology quaternary water table 20 m 120 m 520 m 700 m 1100 m 2000 m mio-pliocene tertiary tablecloth of the terminal complex (ct) sand tablecloth waterproof evaporite eocene secondary limestone layer (senonian-eocene)) carbonate eocene carbonate senonian waterproof senonian lagoon turonian tablecloth turonian waterproof cénomanian vraconian continental intercalaire tablecloth (ci) albien tablecloth albian aptian barremian fig. 2. lithostratigraphic column from the ghardaïa region (m’zab valley) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 214 fig. 3. sampling point inventory map table 1 data from analyses carried out in the region of sebseb sampling points p1 p2 p3 p4 p5 p6 p7 p8 p9 p10 parameters t°c 22.60 22.50 22.40 22.10 22.60 16.20 18.20 21.10 17.50 16.90 ph 7.64 7.61 7.66 7.62 7.52 7.43 8.27 8.41 7.48 7.90 cond. 25°c µs/cm 1514.00 1445.00 1520.00 1551.00 1487.00 1529.00 1480.00 1554.00 1465.00 1522.00 hco3 (mg/l) 156.16 200.08 148.40 161.04 139.08 143.96 287.92 305.00 139.08 278.16 cl 273.70 251.01 260.93 268.02 249.59 276.53 266.61 226.90 255.26 218.39 so4654.76 670.83 629.26 522.67 590.16 535.02 416.90 461.76 535.02 449.43 ca++ 190.78 181.16 192.38 184.37 185.97 139.47 134.67 153.91 107.41 157.11 mg++ 95.26 79.70 81.65 89.42 86.51 32.07 88.45 71.93 46.65 77.76 na+ 183.33 155.56 166.67 161.11 194.28 167.00 163.63 180.00 162.00 168.18 k+ 9.00 10.00 10.00 17.00 7.50 6.20 10.00 8.75 10.00 6.25 no314.81 14.54 15.38 8.37 11.55 12.14 10.25 17.52 10.28 12.12 nh4+ 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.26 tds 880.00 840.00 880.00 904.00 864.00 904.00 858.00 901.00 866.00 882.00 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 215 3. results and discussion 3.1. chemical facies given the predominance of ions (na+, cl-, ca++ and so4 --) in the water of the aquifer sampled, a representation of the chemical facies in the na+/ca++ and cl-/so4 - diagram (fig. 4), shows the existence of two characteristic poles of the water table considered. these two poles are represented by the sulphate-sodium facies and the other, sulphate-calcium. fig. 4. geochemical facies of surface waters (reports: (na + /ca ++ cl /so4 -) 3.2. saturation index the saturation index is calculated by the debye-huckel law (1923): is = [log (q)] / [log (kps)] of minerals dissolved in water (table 2). the steady state of this index is in the range of -0.5 to +0.5. chloride (cl-) ion only precipitates at higher ionic strengths, so it is a good indicator of the state of concentration of water. the order of the elements classified by molality is not always the same because the elements do not concentrate in proportion to the concentration factor. the contents of some elements increase while others decrease [18]. 3.3. concentration diagram the results of analyzes are represented in concentration diagrams with the logarithm of the molalities (meq.l-1) on the ordinate and the logarithm of the concentration factor on the abscissa. table 2 variation in saturation indices of some minerals and co2 partial pressure sample aragonite calcite dolomite anhydrite gypsum halite sylvite co2 nh3 o2 log pco2 p1 2.64 2.79 5.34 1.19 1.50 -3.35 -4.07 -0.96 -8.76 -37.47 -2.52622 p2 2.69 2.83 5.36 1.17 1.48 -3.47 -4.08 -0.81 -8.78 -37.62 -2.38902 p3 2.65 2.80 5.28 1.19 1.50 -3.41 -4.04 -1.00 -8.72 -37.45 -2.56591 p4 2.65 2.80 5.34 1.12 1.43 -3.40 -3.77 -0.93 -8.75 -37.72 -2.49377 p5 2.49 2.63 5.00 1.16 1.47 -3.35 -4.18 -0.89 -9.05 -37.95 -2.46042 p6 2.24 2.39 4.14 0.99 1.37 -3.37 -4.14 -0.77 -9.45 -40.56 -2.39469 p7 3.30 3.45 6.75 0.85 1.22 -3.38 -3.93 -1.38 -8.55 -36.48 -2.89894 p8 3.49 3.64 7.02 0.94 1.27 -3.43 -4.11 -1.51 -8.43 -34.90 -2.99827 p9 2.17 2.32 4.30 0.89 1.26 -3.42 -3.98 -0.82 -3.42 -39.89 -2.44733 p10 3.01 3.16 6.02 0.95 1.32 -3.46 -4.21 -0.99 -7.58 -38.42 -2.55658 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 216 the water concentration factor (fc) can be estimated from the ratio between the cl content of a water and the minimum value of the clcontent from the database. this allows a relative classification of the samples with respect to each other [17]. 3.3.1. concentration diagram of the major elements the sulphated facies is dominant in the groundwater collected at sebseb. the high concentration of so4 is possibly due to the dissolution of anhydrite and gypsum which are marked by a supersaturated saturation index (fig. 5). these two minerals can come from the dissolution of marl rich in gypsum and anhydrite. the hco3 bicarbonate content decreases faster than the concentration factor. this is due to the precipitation of carbonate minerals. the concentration of carbonate alkalinity in the waters of the region may be due to the dissolution of limestones present in the study area. the evolution of the co3 ion is mainly due to the presence of the latter in the compounds of aragonite caco3, calcite caco3 and dolomite camg (co3)2 which are marked by a supersaturated saturation index (table 2). the evolution of na+2 starts from the log (fc) close to 0 up to 0.06. after that, the sodium molalities are decreasing to the value log (fc) = 0.07. then, it increases a second time from the log (fc) and becomes greater than 0.07. the strong sodium molalities can be explained by a simultaneous dissolution of chlorides and sodium from the same origin; we are talking about halite which is undersaturated. magnesium mg+2 and calcium ca+2 are marked by weak molalities; their evolution with the concentration factor seems to be identical. this can be explained by the common origin of the latter two; we are talking here about the dissolution of dolomite camg (co3)2, the mineral is marked by the greatest saturation index. when the water concentrates, the calcium contents increase, the alkalinity (alc) increases and then decreases according to the law of residual alkalinity. as long as the waters are undersaturated with respect to calcite, ca+2 and alc can increase simultaneously. when equilibrium with this mineral is reached, the ca+2 content increases and the alkalinity decreases because ca+2 > alc, residual alkalinity: positive calcite [19]. potassium shows very low concentrations. however, a significant increase is noted in some samples (participation in probable sylvite precipitation (kcl) then a contribution of this element which increases its concentration), and a decrease for other samples. but in general, the evolution of potassium remains very slow than the concentration factor [20]. fig. 5. concentration diagram of major elements food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 217 3.3.2. nutrient concentration diagram for no3 nitrates, there is a stabilisation in the evolution of the latter with the concentration factor (fig. 6). the nh4 + ammonium content decreases slightly with respect to the concentration factor. this can be explained by the transformation of ammonium into nitrite and then into nitrate. in an oxidizing medium, ammonium is transformed into nitrites and then nitrates, which induces oxygen consumption [21]. the presence of ammonium in water is believed to be related to the denitrification process by oxygen consumption and its concentration increases when the water contains a low o2 content and a high co2 content. fig. 6. nutrient concentration diagram 3.3.3. carbonate concentration diagram carbon dioxide co2 is marked by a perfect evolution with the concentration factor. this evolution may depend on a chemical reaction between calcium and bicarbonates, which induces the composition of calcite and the release of carbon dioxide (fig. 7). alkalinity is conservative in the sense that it is not affected by the gain or loss of co2, the dissolution or precipitation of a strong acid or strong base salt [22], [23]. however, it can be modified by the precipitation or dissolution of weak acid or base salts. saturation indices of the main carbonate minerals (calcite, dolomite and aragonite) show that they are in equilibrium. this state of carbonate minerals indicates that the water has undergone significant changes as a result of co2 degassing. this induces precipitation of some of the carbonate minerals [24]. in natural solutions, interactions with minerals are the main controls on alc and calcium, with calcite being the first mineral involved [25]. the calco-carbonic equilibrium reaction can be written: caco3(s) + co2(g) + h2o ↔ ca +2 + 2hco3 the hco3 bicarbonate content decreases faster than the concentration factor. this may be due to collusion with calcium to give calcite as shown in the previous equation or the dissolution of bicarbonates which gives carbonate ions co3 -2 and hydrogen ions, explained by the following equation: hco3 ↔ h++ co3 -2 the decrease in molalities of the co3 ion and mainly due to the collusion of the latter with calcium to give calcite: ca+2 + co3 -2 ↔ caco3 the reaction can also combine a hydrogen ion to give hco3 bicarbonates. fig. 7. carbonates concentration diagram food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 218 3.3.4. water-calcite balance diagram we plot the log line: (ca 2+ ) + 2ph + log (h20) + log (pc02) = 9.76 over-saturation increases when estimated pco2 decreases (fig. 8) a phenomenon that has been already observed in literature [26-29]. sebseb's groundwater is therefore supersaturated with respect to calcite (fig. 8). fig. 8. water-calcite balance diagram 3.3.5. water-dolomite balance diagram in the same way as for calcite, by plotting the log line (ca2+) + log (mg2+) +4ph as a function of 2.log (pco2). with log: (ca 2+ ) + log (mg 2+ ) + 4ph=19.2-2.log (pco2) the remarks are generally the same for saturation with respect to calcite and dolomite. all samples are in a state of over-saturation (fig. 9). it can be concluded that saturation with carbonates (calcite and dolomite) is a factor in controlling water mineralization. in this case, saturation is the limiting factor. fig. 9. water-dolomite balance chart 3.3.6. mineral stability index according to ph the saturation indices of carbonates (calcite, dolomite and aragonite) increase steadily with ph, but this is not the case for sulphates, which remain almost stable and no longer increase with ph (fig. 10a). the alkalinity produced by the dissolution of carbonates is the main cause of the increase in ph [20]. fig. 10. variation in the stability index minerals by ph food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 219 the si of carbonate and sulphate minerals follows a positive linear trend, which suggests that the mineral phases are changing with a similar trend following the same hydrogeochemical process (figs. 10b, 10c). 3.3.7. log (pco2) and is relationship of minerals the log (pco2) vs is bonds of carbonates (calcite, dolomite and aragonite) show that all the samples are in equilibrium (supersaturated) and tend to precipitate (fig. 11). sulphates (anhydrite and gypsum) are also in a state of equilibrium and can precipitate. on the other hand, the log bonds (pco2) with the main chlorinated minerals (halite and stylites) show that these waters are under saturated, which induces dissolution of these minerals. 3.4. physico-chemical water quality the purpose of the physico-chemical analysis is to demonstrate that the wells studied have concentrations that meet drinking water standards or not. the results were compared with the values prescribed in water quality standards in world health organization (who, 2017). 3.4.1. physical parameters the ph depends on the origin of the water, the geological nature of the substrate and the watershed crossed [30]. in the case of the study region, the ph values of the groundwater do not show significant variations, they all comply with the potability standard (tab. 3). the temperature and the total dissolved solids (tds) also show values which remain acceptable in terms of drinking water standards. fig. 11. relation log (pco2) minerals si table 3 water potability standards (2017) therefore, electrical conductivity provides information on the degree of mineralization of water. the waters of the controlled points are mineralized (fig. 12), with values that oscillated between 1445 and 1554 μs / cm. these high values seem to result from the leaching of the reservoir rock in which the water resides. these waters, compared to who standards for drinkability with respect to conductivity, are considered drinkable. parameters who standards parameters who standards ph ≥ 6,5 et ≤ 9 k+ (mg/l) 12 ce (µs/m) 1000 hco3(mg/l) / tds (mg/l) < 600 cl(mg/l) 250 ca+2 (mg/l) 200 so4(mg/l) 500 mg+2 (mg/l) 150 no3(mg/l) 50 na+2 (mg/l) 200 nh4+ (mg/l) 0.2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 220 fig. 12. water quality in relation to physical parameters 3.4.2. cations calcium is generally the dominant element in drinking water and its content varies mainly according to the nature of the terrain crossed, such as limestone or gypsum facies [31]. the high calcium contents could be attributed to the dissolution of sulphated and carbonated minerals, which are supersaturated in the waters of the region, which induces a non drinkability of these waters with respect to calcium. magnesium mg2+ comes from carbonic acid attack of magnesium rocks and the dissolution of magnesium in the form of carbonates and bicarbonates [32]. it is marked by concentrations above the drinking water standards with the exception of the p6 and p9 wells, which remain in compliance with the drinking water standards with respect to mg2+. in unpolluted groundwater without contact with evaporite, the sodium content na2+ is between 1 and 20 mg / l [33]. the formations in the region are rich in evaporite, which explains the high sodium values despite the fact that these waters remain in compliance with admissible standards for drinking water. potassium is generally the least abundant major element in water (fig. 13). the water of the region has a potability vis-à-vis this element. potassium, in our case comes from the dissolution of sylvite (kcl). 3.4.3. anions sulphates (so4 -) show values greater than the guide value (500 mg / l) of the standard relating to the quality of water intended for drinking water supply, with the exception of three wells, which is the case of wells p7, p8 and p10. the very high presence of sulphates is due to the richness of the geological formations of the region in gypsum (fig. 14). fig. 13. water quality with respect to cations water too rich in chlorides is laxative and corrosive; the concentration of chlorides in the water also depends on the terrain crossed [34]. based on the results of analyzes carried out on the water samples, the chloride contents food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 221 greatly exceed the standards for drinking water; they mainly result from the dissolution of halite and sylvites. the bicarbonate content in groundwater depends mainly on the presence of carbonate minerals in the soil and aquifer, as well as the co2 content of the air and soil in the catchment area [33]. fig. 14. water quality with respect to anions in the water analyzed, concentrations above the drinking water standards are observed for the p7, p8 and p10 wells and the other wells comply with the drinking water standards. the high values are probably due to the circulation of these waters in the aquifer reservoir through the limestone formations which characterize the region. 3.4.4. nutrients nitrates are present in water by leaching nitrogenous products from the soil, by decomposing organic matter or synthetic or natural fertilizers [35]. ammonium nh4 + is the most toxic form of nitrogen and the presence of ammoniums in large quantities is indicative of contamination by releases of human or industrial origin [20]. nitrates and ammonium are low concentrations and are good for human consumption. in addition to these two elements, except the p10 well is marked by an ammonium concentration higher than the drinking standards, this is probably due to the use of chemical fertilizers in agriculture in the vicinity of this well (fig. 15). fig. 15. water quality with respect to nutrients 4. conclusion groundwater is an essential water resource for drinking water supply in the sebseb region. in this study, the saturation index was used to assess the hydrogeochemical evolution of groundwater in this area. chemical analysis showed that na2+, ca2+, cland so4 2were the dominant ions in all groundwater samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 222 the results of the calculations of this index showed that most of the groundwater samples were saturated or supersaturated with regard to carbonate minerals (calcite, dolomite and aragonite) and sulphate minerals (anhydrite and gypsum). all groundwater samples were undersaturated for chlorinated minerals (halite and sylvite). the hydrogeochemical analysis of the groundwater in the study area was mainly marked by the alteration of rocks, which is to say by the dissolution of carbonate and sulphated and even chlorinated minerals. the dissolution of these minerals increases the concentration of chemical elements and almost all become higher than the standards of drinkability requiring treatment and demineralization of these waters before consumption. 5. acknowledgments the authors wish to express their gratitude to the laboratory of water resources and sustainable development (redd) under the aegis of the general directorate of scientific research and technological development (dgrsdt), ministry of higher education and scientific research for its contribution to the realization of this study. 6. references [1]. oyedotun, t. d. t.; obatoyinbo, o., hydro-geochemical evaluation of groundwater quality in akoko north west local government area of ondo state, nigeria. ambi-agua, taubaté, v. 7, n. 1, p. 67-80, (2012) http://dx.doi.org/10.4136/ambi-agua.851 [2]. okagbue c. o., hydrology and chemical characteristics of surface and groundwater resources of the okigwi area and environs, imo state, nigeria. in: ofoegbu, c.o. (ed.). groundwater and mineral resources of nigeria. braunschweig: vieweg and sohn, p. 3–16, (1988) [3]. boyle d. r., application of groundwater geochemistry in mineral exploration. in: berry, m. j. (ed.). current activities forum 1988, dartmouth: geological survey of canada, (1988) [4]. barbier j., chery l., relation entre fond géochimique naturel et teneurs élevées en métaux lourds dans les eaux (antimoine, arsenic, baryum, chrome, nickel, plomb, zinc). application (auvergne et limousin) et validation. rapport brgm r39544, 47 p, (1997) [5]. helgeson harold c., evaluation of irreversible reactions in geochemical processes involving minerals and aqueous solutions—i. thermodynamic relations, geochimica et cosmochimica acta, volume 32, issue 8, p. 853877, (1968), issn 0016-7037, https://doi.org/10.1016/0016-7037(68)90100-2 [6]. dixit s., gupta s. k., & tiwari s., “nutrients overloading of a freshwater lake in bhopal, india,” electronic green journal, vol. 21, pp. 2–6, (2005) [7]. nag s. k., & das s., “quality assessment of groundwater with special emphasis on irrigation and domestic suitability in suri i & ii blocks, birbhum district, west bengal, india,” american journal of water resources, vol. 2, no. 4, pp. 81– 98, (2014) [8]. shigut d. a., liknew g., irge d. d., & ahmad t., “assessment of physico-chemical quality of borehole and spring water sources supplied to robe town, oromia region, ethiopia,” appl water sci, vol. 7, no. 1, pp. 155–164, (2017) [9]. schuh w. m., klinekebiel d. l., gardner j. c., & meyar r. f., “tracer and nitrate movement to groundwater in the northern great plains,” journal of environmental quality, vol. 26, no. 5, pp. 1335–1347, (1997) [10]. singh s. k., sarin m. m., & francelanord c., “chemical erosion in the eastern himalaya: major ion composition of the brahmaputra and δ13c of dissolved inorganic carbon,” geochimica et cosmochimica acta, vol. 69, pp. 3573–3588, (2000) [11]. krishna kumar s., chandrasekar n., seralathan p., prince s., & godson n. s. m., “hydrogeochemical study of shallow carbonate aquifers, rameswaram island, india,” environmental monitoring and assessment, vol. 184, no. 7, pp. 4127– 4138, (2012) [12]. potter p. e., “petrology and chemistry of modern big river sands,” the journal of geology, vol. 86, no. 4, pp. 423–449, (1978) [13]. datta p. s. & tyagi s. k., “major ion chemistry of groundwater in delhi area: chemical weathering processes and groundwater flow regime,” journal of the geological society of india, vol. 47, pp. 179–188, (1996) [14]. fantong w. y., satake h., ayonghe s. n., aka f. t., & asai k., “hydrogeochemical http://dx.doi.org/10.4136/ambi-agua.851 https://doi.org/10.1016/0016-7037(68)90100-2 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 salim boubelli, nabil bougherira, djamel rihia, hicham chaffai, hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 211 – 223 223 controls and usability of groundwater in the semiarid mayo tsanaga river basin: far north province, cameroon,” environmental geology, vol. 58, no. 6, pp. 1281–1293, (2009) [15]. guessoum h., benbrahim f., halilat m. t., laouar f., bensalama m & darem s., pollution biologique des eaux phréatiques de la région de ghardaia (cas de sebseb). journal of advanced research in science and technology, 2(2), p. 246-257, (2015) [16]. a.n.r.h., rapport technique, ‘inventaire des forages d’eau et enquête sur les débits extraits de la wilaya de ghardaïa’, secteur de ghardaïa, 88p, (2011) [17]. barbièro l., vallès v., aspects géochimiques de l’alcalinisation des sols dans la vallée du dallol bosso. cah.orstom, sér, pédol, vol xxvii, n° 2, p. 143-152, (1992) [18]. nezli i. e., achour s., djabri l., approche géochimique des processus d’acquisition de la salinité des eaux de la nappe phréatique de la basse vallée de l’oued m’ya (ouargla). larhyss journal, issn 1112-3680, n° 06, p. 121-134, (2007) http://larhyss.net/ojs/index.php/larhyss/article/view/ 89 [19]. ouardi j., elghmari a., valles v., benaabidate l., caractérisation géochimique et statistique des eaux souterraines du périmètre irrigué du tadla (maroc). revue ljee, share.ensh.dz, p. 38-46, (2014) [20]. boubelli s., chaffai h., sakaa b., djidel m., kherici n., hydrogeochemical characterization and assessment of the quality of surface waters of oued saf-saf (north-east algeria). j. bio. env. sci. 12(2), p. 168-178, (2018) https://goo.gl/bszzn8 [21]. gaujous d., la pollution des milieux aquatiques: aide-mémoire. edition technique et documentation, lavoisier-paris. 220p, (1995) [22]. stumm w. & morgan j.j., aquatic chemistry. wiley-interscience, new-york, 780p, (1981) [23]. bourrie g., relation entre le ph, l'alcalinité, le pouvoir tampon et les équilibres de co2 dans les eaux naturelles. science du sol, 3, p. 141-159, (1976) [24]. de montety v., radakovitch o., vallet coulomb c., blavoux b., hermititte d., valles v., origin of groundwater salinity and hydrogeochemical processes in a confined coastal aquifer. case of the rhône delta (southern france). appl.geochem. 23: p. 2337-2349, (2008) [25]. ribolzi o., valles v., barbiero l., contrôle géochimique des eaux par la formation de calcite en milieu méditerranéen et en milieu tropical : arguments d'équilibre et argument de bilan. science du sol, 31 (1/2), p. 77-95, (1993) [26]. al-droubi a., géochimie des sels et des solutions concentrées par évaporation. modèle thermodynamique de simulation. application aux sols salés du tchad. strasbourg : institut de géologie – université louis-pasteur, sciences géologiques, mémoire, 46, 184 p, (1976) [27]. dosso m., géochimie des sols salés et des eaux d'irrigation. aménagement de la basse vallée de l'euphrate en syrie. thèse doct. ing. institut national polytechnique, toulouse, 181p, (1980) [28]. gac j.y., al-droubi a., paquet h., tardy y., chemical model for origin of elements in salts and brines during evaporation of waters. application to some saline lakes of tibesti and chad. in: 2end symposium on the origin and distribzition of elements. paris, unesco. ahrens ed. pergamon press, p. 149-158, (1977) [29]. valles v., modélisation des transferts d'eau et de sels dans un sol argileux : application au calcul des doses d'irrigation. sci. géol., mém 79, 148p, (1987) [30]. bermond r. & vuichaard r., les paramètres de la qualité des eaux. documentation française, paris, 179p, (1973) [31]. rodier j., bernard l., nicole m & coll., l’analyse de l’eau, 9e édition. dunod (éditeur), paris, france, 1579p, (2009) [32]. bermond r. et perrdon c., paramètres de la qualité des eaux. ministère de l’environnement paris, 259p, (1979) [33]. förstner u., wittmann g. t. w., metal pollution in the aquatic environment. springer-verlag berlin heidelberg, doi: 10.1007/978-3-642-69385-4, 488p, (1981) [34]. tarik a., qualité physico-chimique de l’eau de boisson et la solubilité de certains médicaments utilisés chez la volaille dans certaines région du maroc. thèse de doctorat vétérinaire iav hassan ii, rabat-maroc, 183p, (2005) [35]. samake h., analyse physico-chimique et bactériologique au l.n.s des eaux de consommation de la ville de bamako durant la période 2000 et 2001, 77p, (2002) http://larhyss.net/ojs/index.php/larhyss/article/view/89 http://larhyss.net/ojs/index.php/larhyss/article/view/89 https://goo.gl/bszzn8 1. introduction 111 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 2 2019, pag. 111 122 polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction methods * abel inobeme 1,2 , alexander ikechukwu ajai 2, abdullahi mann 3, yahaya ahmed iyaka4 1department of chemistry, edo university iyamho, edo state, nigeria, inobeme.abel@edouniversity.edu.ng, 2department of chemistry,federal university of technology, minna, niger state nigeria *corresponding author received 15th april 2019, accepted …june 2019 abstract: the presence of polycyclic aromatic hydrocarbons (pahs) and heavy metals in smoked food in appreciable amount is an issue of concern in recent times. the present study focuses on the pahs and heavy metal contents of smoked beef. the effect of three solvent combinations (n-hexane, dichloromethane and n-hexane:dcm) and two extraction methods (soxhlet and sonication) on pahs contents were also assessed. gc-ms was used for the quantification of pahs while heavy metal contents were analyzed using atomic absorption spectrometer. the concentrations of metals were in the general order: fe > zn> mn > pb > cu > cd. the content of metals was generally lower than the permissible limits based on international standard. sonication method gave the highest yield of total pahs (45.15µg/kg) while n-hexane registered the highest extraction efficiency amongst solvents. the concentration of pahs ranged between 0.09 9.90 µg/kg. benzo[a] pyrene exceeded the 5.0µg/kg maximum limit in some cases. it is necessary to continually check the concentrations of pahs and heavy metals in food. keywords: smoking, extraction, heavy metals, beef, polycyclic aromatic hydrocarbons 1. introduction meat and other food substances have been preserved through smoking for so long. originally the purpose of heat treatment and smoking was to preserve food, partly by reducing the moisture content and also through the transfer of anti-microbiological components from smoke to food. currently smoking is primarily used to achieve the characteristic taste and appearance of smoked food, with preservation playing a minor role [1]. pahs are present in smoked food as they are formed during the smoking process [2]. the process of grilling meat, fish or other food with intense heat over a direct flame results to fat dripping on the hot fire and yielding flame containing a number of pahs. these chemicals adhere to the surface of the food. the more intense the heat the more the pahs formed [3]. the main source of human exposure to pahs is food, and this accounts for more than 90% of total exposure. pahs account for 4 of the top 10 most hazardous substances on the 2010 agency of toxic substances and disease registry priority list of hazardous substances. the pivotal adverse effect resulting from exposure to pah is carcinogenicity. increased incidences of lung, skin, and bladder cancers are associated with occupational exposure to pahs [4]. several polycyclic aromatic hydrocarbons (pahs) are among the most potent carcinogens known to exist, producing tumours in some organisms through single exposures to microgram quantities. levels as high as 200 μg/kg have been found for individual pahs in http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=25 http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=25 http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=25 http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=25 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 112 smoked fish and meat samples. for instance, in barbecued meat, 130 μg/kg has been reported whereas the average background values are usually in the range of 0.01 to 1 μg/kg in uncooked foods [5]. the joint fao/who expert committee on food additives (jecfa) conducted a comprehensive risk assessment of pah in 2005 [2]. jecfa recommended keeping the content of pahs in food low. the presence of pahs in the environment and the potential for these contaminants to be present in food has resulted in the need for their safety concern in human health. extraction methods and solvents have been reported to affect significantly the yield of pahs in environmental matrices hence give different results on extent of contamination by pahs [6]. there are very limited studies on pahs and heavy metal content barbecue beef, chicken and fish obtained from minna, niger state, nigeria. the presence of heavy metals in processed meat, fish and other grilled foods have also been reported. some of these metals are present in concentrations beyond permissible limit in some available studies [7]. heavy metal constitutes one of the major groups of environmental contaminants. heavy metals when released in the environment are toxic to humans and animals. however, some heavy metals are beneficial to the body. the largest manmade sources of these metals include combustion of fossil fuels and industrial processes [3]. some of the anthropogenic sources of pahs are mobile while others are stationary [4]. the properties of the individual pahs depend on the number of hydrocarbon rings. pahs are generally lipophilic, which means they dissolve poorly in water but well in fats and oils [4]. they have low vapour pressure, relatively high melting and boiling points due to their high molecular masses. most pah can be photo-oxidized and degraded to simpler substances [8]. pah typically occur as mixtures in food, with variation in the toxicity (or potency) of individual compounds; some compounds are genotoxic and/or carcinogenic or neither [9]. pah formation during charcoal grilling was shown to be dependent upon the fat content of the meat, the time of cooking and the temperature [4]. heavy metals are naturally-occurring elements that have high atomic numbers and densities higher than the density of water by at least five times. the term “heavy metal” refers to any metallic element that has relatively high density and is poisonous or toxic even at low concentration [9]. “heavy metal” is a general collective term, which applies to the group of metals and metalloids with atomic density greater than 4g/cm3. however, being a heavy metal has little to do with density but concerns chemical properties. heavy metals include cadmium (cd), zinc (zn), mercury (hg), arsenic (as), silver (ag), lead (pb) chromium (cr), copper (cu), iron (fe) and platinum group elements [10].heavy metals are not biodegradable and are widely distributed in the environment [11]. heavy metals are naturally present in the environment, however, metals such as arsenic, nickel, mercury, cadmium, and lead do not have any known or reported biochemical importance and their appreciable concentration could constitute significant lethal hazards [12]. considering the need to provide an additive input on existing data base on pahs and heavy metals in barbecue beef, it is paramount to assess the effect of various methods of extraction and extraction efficiency of solvents on the pahs content of smoked beef and also investigate the heavy metal contents. 2. materials and methods food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 113 sampling method raw beefs were collected from twelve different selling points (n=12) within minna in niger state from chanchaga, bosso, tunga and maitubi. they were split into two and a part smoked in a local kiln while the other was kept to check for background concentrations. smoking parameters and sample pretreatment samples collected were cut into smaller sizes and smoked using charcoal. the smoking temperature was 200ºc. smoking was done for 2 hours. smoking temperature was measured using a noncontact infra-red thermometer. the samples were packed in aluminum foil, placed in polyethylene bags and then transported to the laboratory in line with ec directives [13]. the samples were kept in the oven and dried for at 40ºc for two days. the dried samples were grounded using mortar and pestle. preparation of standard solutions and quantification of pahs and metals quantification of pahs was done using external standard method. a calibration mixture containing soxhlet extraction and the 16 polycyclic aromatic hydrocarbons reference standard was used. solutions of concentrations 5, 10, 25, 50 and 100 µg/ml were used to make a five point calibration curve in line with [14]. quality control quality assurance procedures and precautions were carried out. all materials used for processing were screened with nitric acid and acetone. computation of limit of detection (lod) and limit of quantification (loq) were done using linear regression method in line with [15]. recovery studies were carried out by spiking samples with standard concentrations of mixed pahs and reanalyzing them at two concentration levels in line with [4]. determination of physicochemical parameters determination of physicochemical parameters (moisture, ash and fat contents) was done in line with [16]. moisture content was determined in oven at 105ºc. extraction of samples and clean-up extractions were done using three solvent combinations (n-hexane, dcm and mixture of both). soxhlet extraction and ultrasonic extraction were carried out in line with the method reported by [17] with slight modifications. the clean-up was performed using activated florisil (magnesiumsilicate) and anhydrous na2so4. determination of heavy metal concentration digestion of the ground beef (fresh and smoke) was done using nitric acid (hno3) and perchloric acid (hclo3) in line with [18]. dilution of the solution was done to reach the mark of 50ml and heavy metal quantification was carried out using atomic absorption spectrophotometer (aas). statistical analysis analysis of variance (anova) was performed on pah concentration data using spss software. the significant statistical level was set at p<0.05. 3. results and discussion table 1 presents the qualitative and quantitative parameter of the pahs investigated. the lod, loq and rsd values are also shown for the sixteen pahs. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 114 table 1 qualitative and quantitative parameters of pahs analyzed compound r2 rsd lod(µg/kg) loq(µg/kg) naphthalene 0.99 1.92 0.01 0.02 naphthalene, 2 methyl 0.99 0.98 0.09 0.10 biphenylene 0.97 1.34 0.01 0.02 acenapthene 0.99 1.90 0.09 0.13 fluorene 0.99 2.10 0.05 0.10 phenanthrene 0.96 1.45 0.08 0.12 anthracene 0.99 2.11 0.07 0.09 fluoranthene 0.97 1.89 0.08 0.10 pyrene 0.98 1.80 0.05 0.08 benz [a] anthracene 0.99 0.88 0.09 0.11 chrysene 0.96 1.23 0.11 0.16 benzo [b] fluoranthene 0.98 1.87 0.09 0.10 benzo [a] pyrene 0.99 0.99 0.09 0.11 indeno [1,2,3, cd] pyrene 0.08 1.47 0.08 0.10 dibenz [a,h] anthracene 0.99 1.89 0.02 0.09 benzo [g,h,i] perylene 0.97 2.10 0.08 0.10 table 2 gives the result for the percentage recoveries of pahs using the two different extraction methods and solvent combinations. the percentage recoveries ranged from 76.60 to 99.90%. sonication method using n-hexane as extractant showed the highest percentage recovery. table 2 pahs recoveries using various extraction methods and extractants pahs soxhlet extraction sonication extraction n-hexane dcm hexane:dcm n-hexane dcm hexane:dcm napthalene 92.30 76.90 80.95 98.10 88.90 93.40 naphthalene,2-methyl 90.65 81.90 77.30 97.00 87.50 95.60 biphenylene 87.11 79.80 80.60 89.20 87.70 90.10 acenaphthene 80.40 80.40 89.40 96.50 90.60 89.90 fluorene 90.17 77.60 91.30 97.00 88.90 94.67 phenanthrene 88.20 87.90 90.40 95.30 87.40 92.30 anthracene 90.40 90.10 88.20 96.50 90.80 93.80 fluoranthene 79.86 78.98 90.11 96.50 92.10 89.89 pyrene 90.10 81.20 79.30 98.10 89.80 91.20 benz[a] anthracene 91.10 89.80 90.20 99.90 95.30 96.10 chrysene 80.40 79.87 88.70 89.90 90.20 91.80 benzo[b]fluoranthene 78.86 80.10 83.20 89.80 87.60 80.89 benzo [a] pyrene 89.89 90.20 91.80 97.80 89.80 90.96 indeno [1,2,3 – cd]pyrene 90.60 76.80 90.10 96.60 91.20 93.80 dibenz[a,h]anthracene 91.15 89.90 90.80 97.90 90.70 94.90 benzo[ghi]perylene 98.70 79.80 91.70 98.70 93.20 93.80 the results of some physico-chemical parameters, polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, chicken and fish from minna in niger state are reported and discussed below. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 115 table 3 presents the ash, moisture and fat contents of the raw and smoke beef. smoke beef had a lower moisture content (9.10%) which could be attributed to the loss of water during smoking. the implication of this is that smoking decreases moisture in food thereby increasing their shelf life. higher fat content was observed in fresh beef sample. the dripping of fat during smoking could be responsible for the lower fat content of the smoke beef. the ash content where 2.47 and 3.20% for fresh and smoke beef respectively. table 3 physico-chemical properties of samples analyzed (%) fresh smoke fat content 13.90±0.30 10.80 moisture content 11.20±0.23 9.10 ash content 2.47±0.20 3.20 results are expressed as mean±sd for triplicate determination table 4 shows the pahs content of fresh beef using soxhlet extraction method. the total pahs obtained were 9.60, 7.53 and 8.09 µg/kg respectively for n-hexane, dcm and n-hexane: dcm respectively. the concentration of b[a] p ranged from 0.51 to1.03 µg/kg. this shows that the content of b[a] p, commonly described as the marker pah was generally lower than the permissible limit of 5.0µg/kg based on international standards. the significant difference at p<0.05 observed in most of the compounds above shows that the various extractants differ appreciably in their extraction potentials. this may be related to the inherent chemical and physical properties of the solvents such as polarity and volatility [19]. table 4 pahs content of fresh beef using soxhlet extraction method pahs n-hexane dcm hexane: dcm ml napthalene nd nd 0.37±0.14 5.00 (ec, 2014) naphthalene, 2-methyl 0.28±0.09b 0.27±0.14ab 0.25±0.23a biphenylene nd nd nd acenaphthene nd nd nd fluorene 0.19±0.11a 0.43±0.01b 0.52±0.13c phenanthrene 0.37±0.05b 0.37±0.15b 0.32±0.09a anthracene nd 0.27±0.11b 0.13±0.12a fluoranthene 0.88±0.13a 0.98±0.01a 1.24±0.09b pyrene 2.53±0.23b 2.61±0.12b 1.69±0.12a benz[a] anthracene 2.19±0.14b 1.36±0.09a 1.32±0.23a chrysene 0.13±0.08 nd nd benzo[b]fluoranthene nd nd nd benzo [a] pyrene nd 1.03±0.12b 0.51±0.03a indeno [1,2,3 –cd]pyrene 1.28±0.09c 0.67±0.04a 1.14±0.07b dibenz[a,h]anthracene 1.75±0.09b nd 0.61±0.01a benzo[ghi]perylene nd nd nd pahs16 9.60 7.53 8.09 results are expressed as m±sd for triplicate determinations. values with same superscript letters on same row do not differ significantly at p< 0.05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 116 the pahs content of fresh beef using sonication method is depicted in table 5. with the exception of naphthalene which was detected in the combined mixture of the two solvents, the first four pahs were not detected in the sample analyzed. the concentration of the pahs ranged from 0.06 to 3.09 µg/kg. the highest content was pyrene in n-hexane solvent, while the least was anthracene (0.06 ug/kg). the content of the total pahs in fresh samples using sonication method of extraction range from 6.6 to 9.98 µg/kg. the concentrations of pahs in fresh beef samples were generally low. this is in line with findings from other researchers that the formation of these compounds occurs during processing of this meat as the fats drip into the flame [8]. table 5 pahs content of fresh beef using sonication method (𝛍𝐠/𝐤𝐠) pahs n-hexane dcm n-hexane: dcm ml napthalene nd nd 0.56±0.21 5.00(ec, 2014) naphthalene, 2-methyl nd nd nd biphenylene nd nd nd acenaphthene nd nd nd fluorene 1.68±0.03c 0.48±0.13a 0.66±0.12b phenanthrene 1.51±0.01c 0.62±0.07b 0.42±0.23a anthracene 0.06±0.02a 0.09±0.01ab 0.12±0.09b fluoranthene 0.95±0.01b 1.18±0.21c 0.29±0.01a pyrene 2.49±0.08a 3.09±0.09b 2.57±0.26a benz[a] anthracene nd nd 0.88±0.01 chrysene nd 0.14±0.12 nd benzo[b]fluoranthene 0.20±0.01a 0.99±0.16b nd benzo [a] pyrene 1.27±0.13b 1.13±0.09a nd indeno [1,2,3 –cd]pyrene nd 0.67±0.11b 0.49±0.10a dibenz[a,h]anthracene 1.82±0.09c 1.14±0.09b 0.61±0.09a benzo[ghi]perylene nd 0.39±0.01 nd pah16 9.98 9.92 6.6 results are expressed as m±sd for triplicate determinations. values with same superscript letters on same row do not differ significantly at p< 0.05. pahs content in smoked beef using soxhlet extraction method is presented in table 6. there were significant differences in the concentrations of the pahs obtained from the smoked samples in the various solvents of interest. the concentration of b[a]p ranged from 1.01 to 1.70 μg/kg. when compared to the fresh samples, the values obtained for the different extractants are relatively higher. the significant differences observed amongst most of the values reflect the different extraction efficiencies of the various extractants employed. these are higher than the maximum limit of 5.0µg/kg. in a related study [20] reported 0.015, 0.013 and 0.056 as the concentration of pyrene, fluorene and naphthalene respectively. in a study on pahs content of grilled meat and chicken [21] reported that benzo(a)pyrene concentration in charcoal grilled chicken ranged from 0.49-7.20µg/kg and 2.01 µg/kg mean concentration. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 117 table 5 pahs content of smoked beef using soxhlet method (𝛍𝐠/𝐤𝐠) pahs n-hexane dcm hexane: dcm ml napthalene 2.53±0.17c 0.60±0.01a 2.05±0.09b 5.00 (ec, 2014) naphthalene, 2-methyl 0.58±0.03b 2.23±0.24c 0.35±0.12a biphenylene 0.22±0.01a 0.29±0.01ab 0.34±0.01b acenaphthene 0.46±0.01b 0.27±0.06a 0.89±0.12c fluorene 9.83±0.26b 7.10±0.56c 6.01±0.18a phenanthrene 9.42±0.29b 8.20±0.11b 4.50±0.24a anthracene 0.20±0.01ab 0.17±0.01a 0.23±0.01b fluoranthene 1.65±0.19b 1.31±0.16a 2.65±0.11c pyrene 7.17±0.24c 4.80±0.03a 5.48±0.28b benz[a] anthracene 3.94±0.14b 2.14±0.08a 3.99±0.03b chrysene 0.36±0.01b 0.19±0.12a 0.15±0.01a benzo[b]fluoranthene 1.32±0.08b 1.37±0.08b 1.20±0.12a benzo [a] pyrene 1.01±0.12a nd 1.70±0.03b indeno [1,2,3 –cd]pyren 2.96±0.09b 1.86±0.14a 3.33±0.12c dibenz[a,h]anthracene 2.44±0.17a 2.71±0.67ab 2.85±0.14b benzo[ghi]perylene 0.71±0.01 nd nd pah16 42.80 33.04 35.72 results are expressed as m±sd for triplicate determinations. values with same superscript letters on same row do not differ significantly at p< 0.05. table 6 shows the content of pahs in smoked beef using sonication method of extraction. the content of the pahs were observed to have increased appreciably in the beef samples on smoking. this confirms the findings from other studies [22]. table 6 pahs content of smoked beef using sonication method (𝛍𝐠/𝐤𝐠) pahs n-hexane dcm hexane: dcm ml napthalene 3.73±0.89b 1.38±0.10a 1.30±0.07a 5.00 (ec, 2014) naphthalene, 2-methyl 0.51±0.01a 0.49±0.01a 0.53±0.03a biphenylene 0.29±0.03a 0.59±0.06b nd acenaphthene 0.46±0.12a 0.54±0.01b 0.46±0.16a fluorene 8.70±0.09c 8.03±0.12b 5.48±0.12a phenanthrene 9.50±0.13b 9.90±0.09b 8.10±0.18a anthracene 0.22±0.06a 0.20±0.01a 0.37±0.04b fluoranthene 2.06±0.06a 2.77±0.10c 2.10±0.09b pyrene 6.50±0.19a 7.64±0.23b 6.71±0.06a benz[a] anthracene 2.76±0.11c 2.28±0.13a 2.98±0.11b chrysene 0.21±0.08b 0.34±0.02c 0.13±0.03a benzo[b]fluoranthene 1.63±0.06c 1.01±0.05b 0.72±0.01a benzo [a] pyrene 1.75±0.01a 3.82±0.23b 1.54±0.14a indeno [1,2,3 –cd]pyrene 3.90±0.19c 2.25±0.05a 2.96±0.02b dibenz[a,h]anthracene 1.63±0.07a 2.97±0.23b 1.84±0.01a benzo[ghi]perylene 1.19±0.01a 0.85±0.01a 0.93±0.01a pah16 45.04 45.15 36.15 results are expressed as m±sd for triplicate determinations. values with same superscript letters on same row do not differ significantly at p< 0.05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 118 it was also reported by [3] that the formation of pahs in food is favored by high temperature cooking processes. these concentrations were slightly above the limit of 5.0ug/kg and their continuous consumptions is unsafe for humans. the concentrations of pahs obtained is lower than 0.12mg/kg reported by [20] for b(a)p in their study on sardine. sonication method was observed to be more efficient in the yield of pahs when compared to the conventional soxhlet extraction method. the combined solvents had the least total pahs of 36.15µg/kg. fig. 1: pah4 content in smoked beef using sonication extraction (𝛍𝐠/𝐤𝐠) figure 1 gives the pah4 content in various extractants in fresh beef using sonication method of extraction. n-hexane had the highest (11.25 μg/kg) while dcm had the least (5.45 μg/kg). the content of pah4 in all the extractants were lower than the maximum limit of 30 μg/kg specified by international standards. the result shows that the pah4 content increased on smoking while for fresh sample the content varied from 0.88 to 2.93 μg/kg. a concentration range of 3.38 to 20.06 μg/kg was obtained by [23] for the concentration of pah4 in a related study on smoked meat. fig. 2: pah4 content in smoked beef using soxhlet extraction (𝛍𝐠/𝐤𝐠) the content of pah4 in smoked beef using soxhlet extraction method is shown in figure 2. the concentrations were 6.63, 3.7, 7.04 and 30 μg/kg for n-hexane, dcm, n-hexane and maximum limit respectively. the values obtained where generally higher when compared to the pah4 content in the fresh beef sample. this observed difference can be attributed to the formation of pahs during smoking. fig. 3: percentage of various classes of pahs based on carcinogenicity present in smoked beef the percentage of various classes of pahs based on carcinogenicity present in 0 5 10 15 20 25 30 n-hexane dcm hexane: dcm ml 11.25 5.45 5.41 30 p a h 4 ( u g /k g ) extractants 0 5 10 15 20 25 30 6.63 3.7 7.04 30 p a h 4 ( u g /k g ) 0 20 40 60 80 n-hexane dcm both 5.8 8.46 4.26 80 73.82 76.61 4.5 6.1 5.8 group 1 group 2a group 2b group 3 p e rc e n t (% ) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 119 smoked beef is presented in figure 3 above. group 2a pahs were mostly present in all the extractants. their values ranged from 73.82 to 80%. n-hexane had the highest while dcm had the least. group 1 pahs had the least percentage composition. group 1 pahs are those known with established evidences of carcinogenity. this is no doubt the most dreaded. the content in the smoked beef was the lowest (4.26 to 5.80%) of the total pahs. benzo [a] pyrene is the only pah that fits into this class hence was long used as a reference for assessing the presence of other pahs. the highest class of pah were those considered to be possibly carcinogenic. table 7 heavy metal contents in beef (mg/kg) metal fresh smoked ml source fe 110.56±1.91 118.11±1.01 300 who, 2014 mn 6.98±0.01 6.20±0.15 15.00 who, 2008 zn 17.64.±0.19 19.07±0.17 40.00 fao, 1978 cu 0.75±0.24 0.81±0.51 5.00 ec, 2006 cd 0.24±0.03 0.27±0.01 0.50 who, 2014 pb 0.91±0.37 1.17±0.34 2.00 who, 2014 results are expressed as mean±sd. table 7 shows the heavy metal content in the fresh and smoked beef. the concentration of cd ranged from 0.24 to 0.27 mg/kg for the fresh and smoked beef respectively. there was increase in the concentration of all the metals except manganese on smoking. the concentrations of the metals were generally lower than the maximum limits based on international standards. the concentration of fe was observed to be affected most by the smoking process. the higher values of iron in smoke beef may be related to the additives added or the interaction with the metal gauze. iron plays a vital role in animal and plants physiology [24]. the concentration of mn did not differ significantly between raw and smoke. the slight decrease in the concentration of mn was from 6.98 to 6.82 on grilling. manganese is recognized as an essential trace element. exposure to high concentration is however toxic to health. the detrimental effect of manganese to health is more targeted to the central nervous system and the brain [25].the concentrations of mn were safe for humans when compared for to the maximum limit of 15.00mg/kg reported by [26]. manganese is an essential trace element that is normally present in all mammalian tissues in concentrations ranging from 0.3 to 2.9 μg/g [27]. the content from this study was however lower than 3.06 mg/kg and maximum 6.141 mg/kg reported by [28] in a related study. slight drop in the content of zn was also observed in the beef and fish samples on smoking. the content of zn in the fish and beef sample was however considered safe with respects to the maximum limit of 40.00 mg/kg by fao, 1978. in a related study, [24] obtained 4.95–48.23 mg.kg-1 for zn in some fresh chicken and turkey meat. a mean levels of 28.53± 3.39 mg.kg-1 zn in lean meat of poultry was reported by [29] in lahore. the change in concentrations of copper was not significant in the smoked fish and beef. these values were however lower than the permissible limit of 5.0mg/kg for copper in smoked food as reported by [13]. [30], in his study also reported that the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 120 heavy metals concentration in the fresh and smoked beef samples did not differ significantly ( p> 0.05 ) from each other. cows and other animals also feed directly on these crops with their pesticide residues. in a related study, [24] reported 0.01–5.15 mg.kg-1 as the content of cu in fresh turkey. cd is one of the most toxic heavy metal and has no known function in biological system. cd did not differ significantly between the fresh and smoked samples. a slight increase by 0.04 was observed in smoked beef. [24] obtained 0.01–5.68 mg.kg-1 as the range of cd in selected fresh meat. the presence of cd in raw beef could be from contaminated feed, water sources etc. also the relatively higher content of cd in smoked sample could result loss of water, exposure to contaminants during grilling and presence of additives. much of the cadmium which enters the body by ingestion comes from terrestrial foods [27]. the concentration of pb was observed to increase on smoking. lead is a very toxic metal and has the potential of binding with enzymes and other vital cellular components thereby causing damage to vital body organs like brain and kidney [29]. like cadmium, lead is highly toxic and has the tendency to accumulate in tissues of organisms as they are consumed [28]. pb is dispersed over long distances through air and water usually in areas with high industrial plants [27]. in a related study, [30] reported a rise in the concentration of pb from 0.07 in fresh beef to 2.24mg/kg on smoking. table 8 inter-elemental correlation coefficient of metal in smoked beef cu mn zn fe cd pb cu 1 0.55 0.52 0.78* 0.55 0.79* mn 1 0.90** 0.49 0.91** 0.73 zn 1 0.72* 0.67 0.89** fe 1 0.79* 0.61 cd 1 0.92** pb 1 *correlation is significant at the 0.05 level (2 tailed) ** correlation is significant at the 0.01 level (2 tailed) table 8 shows the inter-elemental correlation coefficient among the heavy metals studied. there was strong positive association among cu with fe and pb, mn with zn and cd, and cd with pb. strong positive association may suggest a close source between such metals. 4. conclusion the study shows that the concentration of pahs in smoked beef was significantly higher than the raw beef which confirms that pahs formation occurs due to the smoking process. sonication method was observed to be more efficient in the recovery of pahs. n-hexane was found to be better than dcm and the mixture of both solvents in pahs extraction. 5. acknowledgments the authors are sincerely grateful to the laboratory technologists of jawura environmental services limited for their assistance. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 121 6. references [1]..ahmed e. o., adamu h. t., & mohammed k. e., investigating the quality changes of hot smokedclariaslazera at refrigeratedtemperature ( 5 ± 1 ° c ). journal of agriculture and food science, 1(3): 27–32, (2013) [2].mariusz s., kubiaka b., & magdalena p., the level of chosen polycyclic aromatic hydrocarbons (pahs) in meat products smoked by using an industrial and a traditional method. polish journal of natural sciences, 30(2):137–147, (2015) [3].isioma t., ozekeke o., & lawrence, e., human health risk assessment of polycyclic aromatic hydrocarbons (pahs) . journal chemical science international. 3 (9): 93 – 100, (2017) [4].guo j.y., wu f.c., zhang, l., liao, h.q., zhang r.y., li w., zhao x.l., chen, s.j., & mai, b.x. screening level of pahs in sediment core from lake hongfeng, southwest, (2011) [5].gomez-guillen m.c., gomez-estaca j., gimenez b., & montero p. , alternative fish species for cold-smokingprocess.international journal of food science technology, 44,1525-1535, (2009) [6].yusa v., pardon p. & pastor a., application of accelerated solvent extraction followed by gel performance chromatography and high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons in musseltissue. food additives & contaminants, 22:5, (2005) [7].ujowundu c.o., ihekweazu k., alisi, c., ujowundu f., & igwe c., procarcinogens: polycyclic aromatic hydrocarbons and heavy metal content in some locally processed foods in south eastern nigeria. british journal of applied science & technology, 4(1):249-260, (2014) [8].ajai a.i., suleiman m.a.t., dauda b.e.n., sadiku o.s., &aberuagba f., effect of extraction methods on the polycyclic aromatic hydrocarbons content of smoked catfish species in niger state of nigeria.jordan journal of biological sciences, 5(1):271 – 80, (2012) [9].iyaka, y. a., abubakar u.&adeniran, m. a. total and plant available contents of copper and zinc in urban cultivated rice farmlands. journal of applied chemical science international. 3 (3): 93 – 100, (2015) [10]. nwude d.o., okoye p.a.c. & babayemi, j.o., assessment of heavy metal concentrations in the liver of cattle at slaughter during three different seasons. res. j. environ. sci., 5: 288-294, (2011) [11]. inobeme a., ajai a.i, mann a., iyaka y.a . determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken. fia.usv.ro/fiajournaljournal of faculty of food engineering, 4(2):395 403, (2018) [12]. rajeshkumar, x. l. bioaccumulation of heavy metals in fish species from the meiliang bay, taihu lake, china sivakumar.toxicology reports journal homepage: www.elsevier.com/locate/toxrep5, 288-295, (2018). [13]. european council.summary and conclusions of the sixty-fourth meeting of the joint fao/who expert committee on food additives expert committee on food additives [internet]. (2005) [14]. singh l., & agarwal t. polycyclic aromatic hydrocarbons in diet: concern for public health. trends in food science & technology, 79, 160–170. (2018) doi:10.1016/j.tifs.2018.07.017 [15]. shrivastava a, & gupta v.b. methods for the determination of limit of detection and limit of quantitation of the analytical methods. chron young sci, 2:21-5, (2011) availablefrom: http://www.cysonline.org/text. asp?2011/2/1/21/79345 [16]. tajudeen a. a., chijioke o., olasupo, s. b. review article polycyclic aromatic hydrocarbons (pahs) in some smoked foodstuffs in lagos state, southwest, nigeria. science journal of chemistry; 5(3): 3135, (2017) [17]. zarith s.a b. & mohd y. determination of heavy metal accumulation in fish species in galas river, kelantan and beranang mining pool, selangor.international conference on environmental forensics.procedia environmental sciences, 30 :320 – 325, (2015) doi: 10.1016/j.proenv.2015.10.057 \ [18]. lage m.a., yusty c., & cortizo j.l. supercritical fluid extraction and highperformance liquid chromatography – fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetableoil.food control, 16, 59-64, (2005) [19]. yurchenko s., & mölder u., the determination of polycyclic aromatic hydrocarbons http://www.elsevier.com/locate/toxrep5 http://www.cysonline.org/text.asp?2011/2/1/21/79345 http://www.cysonline.org/text.asp?2011/2/1/21/79345 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 2 – 2019 abel inobeme , alexander ikechukwu ajai, abdullahi mann, yahaya ahmed iyaka, polycyclic aromatic hydrocarbons and heavy metals in smoked beef: effect of solvents and extraction method, food and environment safety, volume xviii, issue 2 – 2019, pag. 111 – 122 122 in smoked fish by gas chromatography mass spectrometry with positive-ion chemical ionization. j. foodcomposit. anal. 18, 857-869, (2005). [20]. ahmed h., mona k., ashraf m., & elmarsafy e. estimated daily intake and health risk of polycyclic aromatic hydrocarbon by consumption of grilled meat and chicken in egypt.international journal of current microbiology and applied sciences, 5(2), 435-448, (2016) [21]. akpambang, v. o. e., purcaro, g., lajide, l., amoo, i. a., conte, l.s., moret, s.,. polycyclic aromatic hydrocarbons in some nigerian smokedplantfoods.frontiers in food& nutrition research, 1(1).1-5, (2015) [22]. mariusz s., kubiaka b., & magdalena p. the level of chosen polycyclic aromatic hydrocarbons (pahs) in meat products smoked by using an industrial and a traditional method. polish journal of natural sciences, 30(2), 137–147, (2015) [23]. iwegbue c. m. a., g. e. nwajei & iyoha e. h. heavy metal residues of chicken meat and gizzard and turkey meat consumed in southern nigeria, bulgarian journal of veterinary medicine, 11 (4): 275−280, (2008). [24]. osman a., alaa g.m. & werner k. (2010). water quality and heavy met.al monitoring in water, sediments and tissues of the african catfish clarias gariepinus from the river nile, egypt. journal of environmental protection, 1, 389400. [25]. kalogero p., sotirios k., aikaterini s., stella a., manos d., michael s., heavymetals in raw, fried and grilled mediterranean finfish and shellfish, food and chemical toxicology, 50 (10): 37023708. (2012) [26]. parekhan a., banaz r., dler m. s., assessment of heavy metals in livers of cattle and chicken by spectroscopic method. journal of applied physics (iosr-jap) 6 : 23-26,(2014) [27]. rehman k., shahla a., ansar m., syed m., mian muhammad n. assessment of heavy metals in different tissues of broilers and domestic layers, global veterinaria 9 (1): 32-37, (2012) [28]. mariam i., iqbal s., & nagra s.a., distribution of some trace and macro minerals in beef, mutton and poultry. international journal of agriculture & biology, 6 (2), (2004) [29]. federick, a., mireku d, & huda n.(2018). assessment of selected heavy metal concentration in fresh and grilled beef a case study in eastlegon, ghana. int j onehealth, 4:40-44. [30]. inobeme a., ajai a.i, mann a., iyaka y.a . determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken. fia.usv.ro/fiajournaljournal of faculty of food engineering, 4(2):395 403, (2018) 1. introduction 4. conclusion 116 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 116 121 the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds *marcel avramiuc1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received april 27th 2014, accepted may 25th 2014 abstract. the influence of wheat seeds viability on phenolic content, conductivity and sugars efflux as well as the relationships between these physiological and biochemical indices was the purpose of this paper. the biological material, supplied by suceava agricultural research and development station, was represented by six different seed samples of wheat (triticum aestivum l. spp. aestivum) belonging to a local cultivar, kept different periods in uncontrolled storage conditions. it has determined: seeds viability (germination capacity %) as well as conductivity, transmembrane sugars effllux, total phenolic content (tpc) before germination and tpc post germination, evaluating the relationships between these indices, too. both before and post germination, the seed samples with higher viability have recorded higher total phenolic content values, compared to samples with low viability. the germination process has increased the total phenolic content values with much higher percentages in samples with risen viability. the correlations between viability and total phenolic content, determined before and post germination, have shown positive correlations significant for relationship viability tpc post germination. regarding the seminal exudate, the conductivity values have fluctuated, the highest value being in seed sample with lowest viability. the sugars efflux have steadily registered increasing values once with seed viability decreasing. the correlation between viability and conductivity, as well as between viability and transmembrane sugars efflux in the six wheat samples, have shown negative correlations, significant for relationship viability sugars efflux. keywords: wheat, viability, total phenolic content, conductivity, sugars efflux 1. introduction the seeds viability (germination capacity %) plays an important role in the lives of all plants, where the seed is a new beginning or the life cycle continuing in a new body. but the viability depends on many factors such as: pedo-climatic conditions during seed growth and development, the biocompounds content and quality of these seeds, the coatings integrity (cell membranes), the conditions and duration of grains storage etc. in turn, the viability of seeds can influence physiobiochemical processes in the seed and during plant forming, including the concentration of some nutrients or other compounds (antioxidants) which shows importance for diet and human health. wheat is an important component of the human diet, and therefore it may be an important source of phenolic antioxidants [1], a wide variation of phenolic content being in wheat cultivars [2]. important macronutrient sources that contain a wide range of antioxidant compounds are especially whole-grain cereals [3]. according to hyun young kim et al. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 marcel avramiuc, the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds, volume xiii, issue 1 2014, pag. 116 – 121 117 (2013), cereals are rich in phenolic compounds, vitamin e, γ-oryzanol, and carotenoids, and contain small quantities of phytoestrogens and flavonoids. in this paper it has searched in what extent the viability can influence the phenolic content, conductivity and sugars efflux in wheat seeds, as well as the relationships between these physiological and biochemical indices. 2. materials and methods the biological material, supplied by suceava agricultural research and development station, was represented by six different seed samples of wheat (triticum aestivum l. spp. aestivum) belonging to a local cultivar. except the sample w1, whose seeds were harvested in 2013, the other ones have included seeds from the same cultivar, but with different storage periods in uncontrolled conditions, as follows: w2 (4 years), w3 (3 years), w4 (9 years), w5 (13 years), and w6 (10 years). for viability (germination %) assessment there were used 100 seeds for each sample. the distilled water, which imbibed a special filter paper put into petri boxes, represented the germination medium. in the first stage, the seeds were soaked in water (t = 15 17°c) for 4 hours, and water was changed 3 times in the first 24 hours, after each change seeds being left without water for 2 h. in the next 24 hours the temperature was maintained at 16°c, and further at 20°c by the end of the trial (7 days). there were considered germinated the seeds whose rootlets were at least 1 mm length [5]. to determine total phenolic contents (tpc) in wheat samples, first it was obtained an extract for each seeds sample, weighing each 1 g of grain, which were finely ground and subjected to extraction with a mixture methanol and water (80/20), by stirring, centrifuging and recovering the supernatant [3]. the estimation of total phenolic contents in seed extracts was carried out through a colorimetric assay, by measuring its reducing capacity with folin-ciocalteu reagent. tpc was expressed as mg gallic acid equivalent/g seed (gae/g seed). for this purpose, it has generated a standard curve (fig.1) representing the absorbance values of gallic acid standard solutions in relation to their concentrations [6]. fig. 1. standard curve for tpc using gallic acid the seed exsudate electrical conductivity was evaluated using a conductometer supplied by hanna instruments. each sample, containing 30 seeds, has been introduced into berzelius tumbler with 50 ml distilled water and incubated at 25°c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 marcel avramiuc, the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds, volume xiii, issue 1 2014, pag. 116 – 121 118 (in the dark). after 6 hours, the distilled water, containing the seed exudate, was collected for measuring its conductivity. the results have been reported as s/g (ohm/g), seeds being previously weighed [7]. the total sugars dosing in exsudate has been accomplished of 1 g seeds, which were incubated in 10 ml distilled water at 25°c (in the dark). after 6 hours there were dosed the total sugars, using a method based on reducing of picric acid to picramic acid by monoglucides [8, 9]. the results were expressed as g sugars at 1g of seed. each determination was made in four replicates, and their data were statistically processed. in order to calculate differences between results, it was used the analysis of variance, the differences at p < 0.05 being considered significant. 3. results and discussion in the table 1 are rendered the mean values of viability and total phenolic content (tpc) in six wheat samples. table 1 comparative mean values of viability and tpc in wheat seed samples wheat samples viability (g%) tpc (mg gae/g seed) before germination post germination w1 96±1 1.38±0.29 2.24±0.08 w2 85±1 1.42±0.18 2.32±0.20 w3 74±2 0.94±0.06 1.72±0.12 w4 66±3 1.16±0.21 1.46±0.07 w5 58±1 0.82±0.03 0.92±0.02 w6 37±2 0.65±0.05 0.70±0.09 note: g = germination percentage; tpc = total phenolic content as seen in the table 1, the highest viability (g%) has recorded the sample w1, whose seeds were harvested in 2014, and the lowest one the sample w6, with seeds stored for 10 years in uncontrolled conditions. small viabilities were also recorded the samples w4 and w5, kept in uncontrolled stored conditions for 9, and 13 years. as to the mean values of tpc before germination, these ones ranged between 0.65 ± 0.05 mg gae/g (w6) and 1.42 ± 0.18 mg gae /g (w2). it is found that in seed samples with lower viability (w3-w6) tpc values (determined before germination) were smaller, with percentages between 18.9 22.4 (w4) and between 112.3 118.4 (w6), compared to the samples w1 and w2 (p < 0.05). the determination of tpc in germinated seeds has shown values between 0.70 ± 0.09 (w6) and 2.32 ± 0.20 (w2). in post germination also, in seed samples with less germination capacity (w3-w6) tpc values were lower, with percentages between 30.2-34.8% (w3), and between 220 231.4% (w6), as compared to samples w1 and w 2 (p < 0.05). comparing tpc values before and after germination, indicates that the largest percentage increases were recorded, in order, to samples w3 (82.9%), w2 (63.4%) and w1 (62.3%), and the lowest ones in samples w6 (7.7%) and w5 (12.2%). searching the antioxidant activities of winter cereal crops before and after germination, hyun young kim et al. (2013) have found a significant increasing (30,6%) of the total phenolic content in wheat seeds from 1.57 mg/g gae (pregermination) to 2.05 mg/g gae (postgermination). the phenolic content increased significantly during wheat germination for up to 7 days [10]. according to li, shewry and ward (2008), the phenolic acids are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 marcel avramiuc, the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds, volume xiii, issue 1 2014, pag. 116 – 121 119 found in cereal grains in free, soluble conjugate and insoluble bound forms. the increase in phenolic compounds in the post-germination winter cereals can be explained as an increase in the amount of free forms from hydrolysis due to the dismantling of the cell wall during germination [4]. it is possible that lower values of tpc in seed samples with lower viability to have as cause the reduced activity of some enzymes (eg. hydrolases), which release phenolic acids from conjugated soluble forms, as a result of transformations occurring by longer and in bad conditions seeds keeping. the correlations between viability (g%) and tpc, determined before germination (tpcbg), and between viability and tpc, determined post germination (tpcpg) have shown positive correlations (fig. 23), with r2 = 0.8207 (for the relationship v–tpcbg) and r2 = 0.91 (for the relationship v–tpcpg). the table 2 reproduces the mean values of viability and exudate indices in wheat seeds. y = 0.0135x + 0.124 r2 = 0.8207 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 0 20 40 60 80 100 120 viability (g%) t p c b ef or e ge rm in at io n (m g g a e /g ) tpc bg linear y = 0.0306x 0.561 r2 = 0.91 0 0,5 1 1,5 2 2,5 0 20 40 60 80 100 120 viability (g%) t p c p os t ge rm in at io n (m g g a e /g ) tpc pg linear fig. 2. linear regression for the correlation between viability (g%) and tpcbg (mg/g seed) fig. 3. linear regression for the correlation between viability (g%) and tpcpg (mg/g seed) table 2 comparative values of viability, conductivity and sugars efflux in wheat seeds wheat samples viability (g%) conductivity (s/g) sugars efflux (g/g) w1 96±1 24.13±1.2 152.50±5.2 w2 85±1 29.87±0.9 237.35±3.8 w3 74±2 23.20±3.2 270.28±2.9 w4 66±3 28.45±1.5 298.65±11.3 w5 58±1 28.10±0.7 325.16±7.4 w6 37±2 47.35±2.8 462.70±9.7 note: g = germination percentage as can be seen from the table, the mean values of the seeds conductivity were between 23.20 ± 1.2 s/g (w3) and 47.35 ± 2.8 s/g (w6). a low value was recorded by w1 (24.13 ± 1.2 s/g), while samples w2, w4 and w5 showed close values, ranging between 28 and 30 s/g. the transmembrane efflux sugars had average values between 152.50 ± 5.2 g/g (w1) and 462.70 ± 9.7 g/g (w6). compared to conductivity values, which have fluctuated, but have had the highest value in the seeds sample with the lowest viability, the total content of sugars (sugars efflux) in the seeds exudate has steadily registered values increasing once with decreasing seed viability (p < 0.05). the correlations between viability (g%) and conductivity (c), and between viability and transmembrane sugars efflux (se) in the six wheat seed samples food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 marcel avramiuc, the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds, volume xiii, issue 1 2014, pag. 116 – 121 120 (fig. 4-5), have indicated negative correlations, with r2 = 0.9754 (for the relationship v–se) and r2 = 0.6262 (for the relationship v–c). y = -0.3349x + 53,406 r2 = 0.6262 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 120 viability (g%) c o nd u ct iv ity ( μs /g ) c (μs/g) linear (c y = -4.8975x + 630.66 r2 = 0.9754 0 50 100 150 200 250 300 350 400 450 500 0 20 40 60 80 100 120 viability (g%) s ug ar s ef flu x (μ g/ g) se (μg/g) linear (se fig. 4. linear regression for the correlation between viability (g%) and conductivity (μs/g seed) fig. 5. linear regression for the correlation between viability (g%) and sugars efflux (μg/g seed) using the tpc values, determined before seed germination (tpcbg), the conductivity (c), and the transmembrane efflux of sugars (se) there were calculated the correlations between these indices, which were negative, with r2 = 0.336 (for the relationship tpcbg–c) and r2 = 0.7532 (for the relationship tpcbg–se). 4. conclusions analysing six wheat seed samples (belonging to a local cultivar), with various storage periods, as to relationships between viability (germination %), total phenolic content (tpc), conductivity and sugars efflux, there were found different values of these indices as well as negative or positive correlations, more or less significant, depending on seeds viability. both before and post germination, the seed samples with higher viability have recorded higher total phenolic content values, compared to samples with low viability. the germination process has increased the total phenolic content values with much higher percentages in samples with risen viability. the correlations between viability and total phenolic content determined before and post germination have shown positive correlations significant for relationship viability – tpc post germination. regarding the seminal exudate, the conductivity values have fluctuated, the highest value being in seed sample with lowest viability. the sugars efflux has steadily registered increasing values once with seed viability decreasing. the correlation between viability and conductivity, as well as between viability and transmembrane sugars efflux in the six wheat samples, has shown negative correlations, significant for relationship viability – sugars efflux. 5. acknowledgments many thanks to dr. eng. dumitru bodea, director of suceava agricultural research and development station, for the biological material supplied. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 marcel avramiuc, the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds, volume xiii, issue 1 2014, pag. 116 – 121 121 6. references [1] hernández lia, afonso desirée, rodríguez elena m., díaz carlos, phenolic compounds in wheat grain cultivars, plant foods for human nutrition, ©springer science business media, lc 2011 10.1007/s 11130-0110261-1 (2011) [2] li l, shewry p., ward j., phenolic acids in wheat varieties in the health grain diversity screen, j agric food chem., 56:9732–9739 (2008) [3] adom, k. k., liu r. h., antioxidant activity of grains, j. agric. food chem., 50, 6182–6187, (2002) [4] hyun young kim, sang hoon lee, in guk hwang, et al., antioxidant and antiproliferation activities of winter cereal crops before and after germination., food sci. biotechnol., 22(1):181-186 doi 10.1007/s10068013-0025-9 (2013) [5] ellis r.h., hong t.d., roberts e.h., handbook of seed technology for genebanks, international board for plant genetic resources, vol. ii, rome (1985) [6] moore jeffrey and yu liangli (lucy), methods for antioxidant capacity estimation of wheat and wheat-based food products in: wheat antioxidants, edited by liangli yu, published by john wiley & sons, inc., hoboken, new jersey, 147-150 (2008) [7] hosnedl, v., behal, j., horcicka, p., problems of using conductometric tests for pulse seed, quatrieme rencontre internationale sur les semences, angers, france, 20-22 jullet 1992, vol 3, 969-973 (1993) [8] schell, h., biochemistry, edit. did. şi ped., bucureşti, 53-80 (1980) [9] rajan katoch, analytical techniques in biochemistry and molecular biology, springer science business media, llc, 233 spring street, new york, ny 10013, usa, 73-74 (2011) [10] cevallos-casals b.a., cisneroszevallos l., impact of germination on phenolic content and antioxidant activity of 13 edible seed species., food chem., 119: 1485-1490 (2010) issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xix, issue 2 30 june 2020 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare university of suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 192 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 3 2020, pag. 192 198 effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage *kitisart kraboun 1 , putkrong phanumong 1 , kamonwan rojsuntornkitti 2 , teeraporn kongbangkerd 2 , mayura thongchuang 3 , ithinath tantivitittapong 4 , pornrak chowvanayotin 4 1division of food safety management and technology, department of science, faculty of science and technology, rajamangala university of technology krungthep, bangkok 10120, thailand. 2department of agro-industry, faculty of agriculture, natural resources and environment, naresuan university, phitsanulok 65000, thailand. 3department of chemistry, faculty of science, ramkhamhaeng university, bangkok 10240, thailand. 4division of television and radio broadcasting technology, department of science, faculty of science and technology, rajamangala university of technology krungthep, bangkok 10120, thailand. kitisart.k@mail.rmutk.ac.th *corresponding author received 5th may 2020, accepted 29th july 2020 abstract: this research aimed to study the effect of pectin and xanthan gum on the monascus pigment turbidity, dpph radical scavenging ability, ph and sensory evaluation of monascus-pigment extract beverage (mpeb). the pectin and xanthan gum contents used in the beverage were 0.50 – 0.70 % w/v and 0.01 – 0.03 % w/v, respectively. the results showed that the addition of 0.60 % w/v of pectin in mpeb showed the highest values of the turbidity of monascus pigment (od660 = 2.59), dpph radical scavenging ability (45.68 %) and sensory evaluation ( >8 of all attributes). keywords: pectin; xanthan gum; monascus-pigment extract beverage 1. introduction presently, functional foods are highly popular products on the market such as baby foods, baked goods and cereals, dairy foods, confectionery, snacks, meat products, spreads, and beverages [1]. in particular, the advantages of functional beverages are the following: (1) a lot of bioactive compounds; (2) convenience and possibility to meet consumers’ demands for container contents, sizes, shapes, and appearances; (3) ease of distribution and better storage for refrigerated and shelfstable products [2]. there are beverages produced from herbs and fruit pulps indicating highly potential antioxidant activities. for examples, awe et al. [3] documented the antioxidant benefits of a novel functional beverage obtained from hibiscus sabdariffa extract (hse), cocoa and ginger since hse extracts contain the high amounts of protein and other nutrients required for good health [4]. moreover, lu et al. [5] suggested that a durian pulp drink obtained from a combination of fermentation of bifidobacterium animalis subsp. lactis or lactobacillus casei and yeast williopsis saturnus could be a novel non-dairy durian-based functional beverage to deliver probiotics. to date, monascus-pigment extract from monascal rice has never been used as a functional drink. thus, the research of a monascuspigment extract beverage (mpeb) is interesting; this beverage is expected to be an alternative drink to lower cholesterol in blood and a novel anti-oxidative drink. monascus pigment is comprised of many kinds of cholesterol-lowering statins and pigments produced by monascus sp. this http://www.fia.usv.ro/fiajournal mailto:kitisart.k@mail.rmutk.ac.th food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 193 pigment is used as a medicine to the potent cholesterol-lowering, antiatherosclerotic drug lovastatin, a 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase inhibitor [6]. monascus red pigment, obtained from monascus fermentation is interesting as indicated by high antioxidant activities from in vitro experiments, such as inhibition of peroxidation, reducing power, scavenging ability on dpph radicals and chelating ability on fe2+ as well as used in food as a coloring agent in fish and meat [7, 8, 9, 10]. commonly, the destabilizing problems of beverages are the coalescence and the sedimentation of food particles or bioactive compounds which are causes of unacceptable consumer [11]. the pigments are well known as low water solubility [12]. from this standpoint, the expected drawbacks found in mpeb are the phenomena of the coalescence and the sedimentation of water insoluble compositions around neck of beverage bottle. there are the researches applying the hydrocolloids, such as xanthan gum and pectin, to solve the dispersion in many products. zecher and van coillie [13] reported that the contents of xanthan gums in the range of 0.001 – 0.5 % were used in drinks made with citrus and in fruitflavored drinks to create a satisfactory texture and as a stabilizer for odor and flavor. xanthan gum improved the insoluble components suspended in the sample at low ph, which the components would be dissolved quickly and completely. lucey et al. [14] also mentioned that whey separation was significantly decreased by increased pectin concentration. therefore, the effects of pectin and xanthan gum might be expected to occur as the functional properties on the stability of mpeb. the aims of this work were to evaluate the turbidity, dpph radical scavenging ability, ph, and sensory evaluation of mpeb formulated by pectin and xanthan gum. 2. matherials and methods 2.1 chemicals 2,2-diphenyl-1-picrylhy-drazyl (dpph) and ethanol were analytical reagent grade and obtained from sigma–aldrich (st. louis, mo). pectin and xanthan gum were of food grade and purchased from daejung (shiheung, south korea). 2.2 monascal rice preparation about 100 g of rice seeds ( oryza sativa) and 10 ml of distilled water were transferred into a 500 ml flask. the mixture was sterilized by an autoclave at 121 °c for 15 min and then left until cool down. a 5 ml of 106 spores/ml spore suspension of m. purpureus tistr 3090 obtained from actively growing slants in sterile water was inoculated into sterilized waxy corn and incubated at 25 °c for 12 days [9]. then, the product was dried in an oven at 40 °c for 24 h and its moisture content was 7.12 + 1.22 %. a fine powder (20 mesh) was obtained using a mill (retsch ultracentrifugal mill and sieving machine, haan, germany). 2.3 preparation of monascus extract from angkak the extraction method described by yang et al. [7] was used with some modifications. a 10 g sample was extracted in a shaker with 100 ml of ethanol at 170 rpm for 24 h and the solution was filtered through whatman no.4 filter paper. the residue was then extracted with two additional 100 ml portions of ethanol as described above. the combined ethanolic extracts were then evaporated at 40oc to dryness. the dried food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 194 extract was kept in -20oc until used for monascus-extract beverage preparation. 2.4 monascus-extract beverage preparation (mpeb) the monascus extract was diluted with drinking water at a ratio of 0.5 : 100 (w/v). then, the contents of pectin ( 0.50, 0.60 and 0.70 % w/v) and xanthan gum ( 0.01, 0.02 and 0.03 % w/v) were added in the mixtures and mixed for 5 min using a mixer. mpebs containing both pectin and xanthan gum were pasteurized at 60 °c for 20 min., whereas pectin and xanthan gum were not added to the control sample. mpebs added with pectin and xanthan gum after pasteurization were then poured and packed in polypropylene bottles, and they were kept in 4oc until the analysis. 2.5 turbidity the turbidity of mpeb was determined in duplicate by diluting each emulsion in deionized water (1:1000) and measuring the absorbance at 660 nm against distilled water using a spectrophotometer (uv 2100, unico, england) [15]. 2.6 ph measurements the ph of the samples was measured by using a ph meter (thermo orion model 420, usa). the measurement was carried out in triplicate [16]. 2.7 dpph free radical scavenging assay the dpph free-radical scavenging activity was determined by the method described by kraboun et al. [9] two hundred and fifty microliters of the beverages in ethanol solution (5 mg/ml) was added to 250 µl of 5.07 × 10-4 m dpph ethanol solution. the reaction mixture was incubated in the dark at room temperature; the absorbance was measured at 517 nm after 15 min. the absorbance of a blank sample containing the same amount of solvent was also measured. dpph radical scavenging activity was calculated by using the following equation: scavenging activity (%) = abs blank – abs sample × 100% abs blank 2.8 sensory evaluation twenty-six males and forty-four females of undergraduates from the division of food safety management and technology, between the ages of 18 and 24, were panel participants. the samples were coded with three digits and panelists were instructed to evaluate the appearance, odor, texture, flavor and overall score using a nine-point hedonic scale ranging from "1=extremely dislike" to "9 =extremely like" according to the method of sudha et al. [17]. 2.9 statistical analysis all determinations were performed in triplicate and results were expressed as the mean + standard deviation calculated using spreadsheet software microsoft excel. this was carried out in a completely randomized design (crd) which the data were analyzed by an analysis of variance (p<0.05) and means were compared with duncan’s multiple range test. the results were processed by spss 16.0 (spss inc., chicago, il, usa) for windows. 3. results and discussion 3.1 turbidity and ph of monascuspigment extract beverage (mpeb) turbidity is an expression of the optical property that causes light to be scattered and absorbed rather than transmitted with no change in direction or flux level through the sample. for turbidity analysis for a cloudy beverage, the beverage should have high turbidities due to indicating the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 195 stability of beverage or absence of ringing resulting from raised flocculation and coalescence of water insoluble compositions around the neck of the beverage bottle [18]. according to figure 1, the results showed that the turbidity of mpebs increased with percentage of pectin which mpeb supplemented with 0.60 % w/v of pectin gave the highest turbidity (od660 = 2.59). however, increasing percentages of xanthan gum affected decreased turbidity of the beverages. this is in agreement with mirhosseini et al. [19], who reported that the addition of pectin to the orange beverages affected more physical stability and cloudiness than those added with other hydrocolloids. buffo et al. [20] demonstrated the balance of van der waals, electrostatic and polymeric sterics as the main droplet interactions playing a key role in the stability of beverage. this aggregation phenomenon of the water insoluble pigments of monascus may be due to the change in refractive index of the pigment phase and aqueous phase. therefore, addition of a high content of pectin is needed in the formulation in order to reduce ring formation phenomenon [21]. the changes of ph in mpebs are shown in figure 1 and were significant difference (p<0.05), which were in the range of 3.92 – 5.43. the addition of xanthan gum contributed to higher ph values of mpebs than that of pectin. this is agreement with rothschild and karsenty [22], who studied the low turbidity of pasteurized beverages during storage. it indicated that the loss of turbidity during storage was from a high acidity condition. hence, this factor would be resolved by storing the products in nearneutral ph. fig. 1 turbidity and ph of mpeb added with various concentrations of pectin and xanthan gum 3.2 dpph free-radical scavenging ability of monascus-pigment extract beverage (mpeb) the dpph free-radical scavenging ability of mpeb is shown in figure 2. there were a relationship between dpph free-radical scavenging ability and turbidity of mpebs food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 196 (figure 1 and 2). as pectin results, the higher 0.60 % w/v of pectin indicated decreased dpph free-radical scavenging ability; however, increasing the contents of xanthan gum affected decreasing dpph free-radical scavenging ability. it might be possible that the better dispersion of the pigment in mpeb added with 0.60 % w/v of pectin led to the better dispersion of monacolin k and/or other antioxidants (phenolic compounds) throughout within the bottle, which this cause affected the good detection for dpph free-radical scavenging ability. moreover, the addition of high pectin contents in mpeb (0.60 % w/v) increased the antioxidant activity by the nonpectins (phenols and proteins) obtained from pectin extraction [23]. therefore, investigating the capacity might be from antioxidant crude pectic polysaccharides from plant cell wall matrix (i.e., bioaccessibility). raw polysaccharide extracts have been shown to possess the higher antioxidant activity than purified extracts [24]. these results agree with those of wang and lu [25], who found that the pectin has been shown to exhibit higher anti-dpph activities (greater than 50%) at a concentration of 1 mg/ml. pectin fractions showed to inhibit xanthine oxidase (xo) and scavenge reactive oxygen species (ros). inhibition of xo may be mediated by pectic polysaccharide chains, whereas protein and phenolic contaminants were suggested to provide the scavenging of ros. these data demonstrated each constituent of pectin fractions to be important for implementation of antioxidant effect in the gastrointestinal milieu. fig. 2 dpph free radical scavenging ability of mpebs added with various concentrations of pecin and xanthan gum 3.3 sensory evaluation of monascuspigment extract beverage (mpeb) the results of a sensory evaluation using the 9-point hedonic scale are shown in table 1. the results of sensory testing for appearance, color, taste, odor and overall acceptability showed significant difference (p<0.05) between mpebs containing pectin and xanthan gum. therefore, the types of stabilizer affected these sensory food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 197 parameters. the sensory quality of mpeb added with pectin at 0.60 % w/v reached the highest scores of all attributes. whereas, the scores of all attributes decreased when concentration of xanthan gum increased. this is in agreement with enriquez and flick [26] affirmations, who reported that pectin was used as an odor and flavor stabilizer in the drinks because it provides good texture and odor. table 1 sensory evaluation of monascus-pigment extract beverage (mpeb) added with various concentrations of pectin and xanthan gum attribute hydrocolloid concentration (% w/v) appearance color odor taste overall acceptability pectin 0.50 5.67±1.47c 5.83±1.20c 6.33±1.60e 6.87±1.63e 5.57±1.67c 0.60 8.57±1.65d 8.70±1.57d 8.70±0.91f 8.97±1.18f 8.83±1.08d 0.70 4.50±1.07b 5.07±1.38b 5.87±0.97d 6.83±2.05e 5.17±1.45b xanthan gum 0.01 5.50±1.77c 5.17±1.55b 5.27±2.16c 5.40±1.52d 5.13±1.22b 0.02 4.77±1.30b 4.23±1.73a 4.43±0.93b 4.57±1.61c 5.14±.79b 0.03 4.67±1.07b 4.13±1.35a 4.07±1.41a 4.10±1.71b 5.10±1.44b control (no addition of hydrocolloid) 4.10±1.39a 4.03±1.74a 4.02±1.27a 3.47±1.85a 3.93±1.63a different letters indicate significant differences in treatment means from three determinations (p≤0.05). 4. conclusion the mpeb supplemented with 0.60 % w/v of pectin gave the highest turbidity, dpph free-radical scavenging ability and sensory attributes. however, the changes of ph in all mpeb formulae were significantly different (p<0.05), which were in the range of 3.92 – 5.43. therefore, the use of pectin and xanthan gum could play an important role in the dispersion of monascus pigment in the beverage product. 5. acknowledgements this work was funded by rajamangala university of technology krungthep. moreover, the authors would like to thank all staffs of the faculty for maintenance and operation of the scientific laboratory and equipment. 6. references [1] ofori, j.a. & hsieh, y.h.p. novel technologies for the production of functional foods. in bagchi, d. bagchi, m. moriyama, h. & shahidi, f. (eds.). bio-nanotechnology: a revolution in food, biomedical and health sciences (pp. 143–62). new york : john wiley and sons, (2013). [2] kausar, h., saeed, s., ahmad, m.m. & salam, a. studies on the development and storage stability of cucumber-melon functional drink. journal of agricultural research, 50: 239248, (2012). [3] awe, f.b., fagbemi, t.n., ifesan, b.o.t. & badejo, a.a. antioxidant properties of cold and hot water extracts of cocoa, hibiscus flower extract, and ginger beverage blends. food research international, 52: 490-495, (2013). [4] adanlawo, i.g. & ajibade, v.a. nutritive value of the two varieties of roselle (hibiscus sabdariffa) calyces soaked with wood ash. pakistan journal of nutrition, 5: 555-557, (2006). [5] lu, y., putra, s.d. & liu, s.q. a novel non-dairy beverage from durian pulp fermented with selected probiotics and yeast. international journal of food microbiology, 265: 1-8, (2018). [6] panda, b.p., javed, s. & ali, m. optimization of fermentation parameters for higher lovastatin production in red mold rice through coculture of monascus purpureus and monascus ruber. food and bioprocess technology, 3: 373378, (2010). [7] yang, j.h., tseng, y.h., lee, y.l. & mau, j.l. antioxidant properties of methanolic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 kitisart kraboun, kamonwan rojsuntornkitti, teeraporn kongbangkerd, mayura thongchuang, putkrong phanumong, effect of xanthan gum and pectin on stabilization and antioxidant activity of monascus-pigment extract beverage, food and environment safety, volume xix, issue 3 – 2020, pag. 192 198 198 extracts from monascal rice. lwt-food science and technology, 39: 740-747, (2006). [8] kongbangkerd, t., tochampa, w., chatdamrong, w. & kraboun, k. enhancement of antioxidant activity of monascal waxy corn by a 2-step fermentation. international journal of food science and technology, 49: 17071714, (2014). [9] kraboun, k., tochampa, w., chatdamrong, w. & kongbangkerd, t. effect of monosodium glutamate and peptone on antioxidant activity of monascal waxy corn. international food research journal, 20: 623-631, (2013). [10] kraboun, k., phanumong, p., tochampa, w., jittrepotch, n., rojsuntornkitti, k., chatdamrong, w. & kongbangkerd, t. impact of in vitro digestion phases on antioxidant properties of monascal waxy corn from 2-step fermentation. journal of microbiology, biotechnology and food sciences, 7: 454-456, (2018). [11] piorkowski, d.t. & mcclements, d.j. beverage emulsions: recent developments in formulation, production, and applications. food hydrocolloids, 42: 5-41, (2014). [12] dufosse, l., galaup, p., yaron, a., arad s.m., blanc, p., murthy, k.n.c. & ravishankar, g.a. microorganisms microalgae as sources of pigments for food use: a scientific oddity or an industrial reality?. trends in food science and technology, 16: 389-406, (2005). [13] zecher, d. & van coillie, r. cellulose derivatives. in imeson, a. (ed.). thickening and gelling agents for food (pp. 40-65). london : blackie academic and professional, (1992). [14] lucey, j.a., tamehana, m., singh, h. & munro, p.a. stability of model acid milk beverage: effect of pectin concentration, storage temperature and milk heat treatment. journal of texture studies, 30: 305-318. (1999). [15] gharibzahedi, s.m.t., mousavi, s.m., khodaiyan, f. & hamedi, m. optimization and characterization of walnut beverage emulsions in relation to their composition and structure. international journal of biological macromolecules, 50: 376–384, (2012). [16] zhang, x., kong, b. & xiong, y.l. production of cured meat color in nitrite-free harbin red sausage by lactobacillus fermentum fermentation. meat science, 77: 593-598, (2007). [17] sudha, m.l., baskaran, v. & leelavathi, k. apple pomace as a source of dietary fiber and polyphenols and its effect on the rheological characteristics and cake making. food chemistry, 104: 686–692, (2007). [18] hernandez, e., baker, r.a. & crandall, p.c. model for evaluating turbidity in cloudy beverages. journal of food science, 56, 747-750, (1991). [19] mirhosseini, h., tan, c.p. & taherian, a.r. effect of glycerol and vegetable oil on physicochemical properties of arabic gumbased beverage emulsion. european food research and technology, 228: 19-28, (2008). [20] buffo, r., reineccius, g. & oehlert, g. factors affecting the emulsifying and rheological properties of gum acacia in beverage emulsions. food hydrocolloids, 15: 53-66, (2001). [21] nasrabadi, m.n., goli, s.a.h. & nasirpour, a. stability assessment of conjugated linoleic acid (cla) oil-in-water beverage emulsion formulated with acacia and xanthan gums. food chemistry, 199: 258–264, (2016). [22] rothschild, g. & karsenty, a. cloud loss during storage of pasteurized citrus juices and concentrates. journal of food science, 39: 10371041, (1974). [23] kostalova, z., hromadkova, z., ebringerova, a., polovka, m., michaelsen, t.e. & paulsen, b.s. polysaccharides from the styrian oil–pumpkin with antioxidant and complement–fixing activity. industrial crops and products, 41: 127–133, (2013). [24] wikiera, a., mika, m., starzynskajaniszewska, a. & stodolak, b. application of celluclast 1.5l in apple pectin extraction. carbohydrate polymers, 134: 251-257, (2015). [25] wang, x. & lu, x. characterization of pectic polysaccharides extracted from apple pomace by hot–compressed water. carbohydrate polymers, 102, 174–184, (2014). [26] enriquez, l.g. & flick, g.j. marine colloids. in charalambous, g. & doxastakis, g. food emulsifiers: chemistry, technology, functional properties and applications. amsterdum : elsevier science publishing company inc, (1989). 1. introduction microsoft word 01 cuprins 3_2022 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 contents: 1. extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou 198 208 2. study of heat treatment effect on the biological value of pine nuts kernels artur mykhalevych, uliana kuzmyk 209 217 3. comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei 218 229 4. haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu 230 236 5. evaluation of the quality of wheat with added improvers amelia buculei, anca gatlan, adriana cojocariu 237 246 6. texture profile changes of amaranth seeds subjected to sous vide treatment ovidiu procopeţ, mircea oroian 247 256 7. influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes ana batariuc, ionica coţovanu, silvia mironeasa 257 264 8. characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez 265 272 9. author instructions i v 10. subscription information vi doi: https://doi.org/10.4316/fens.2022.003 28 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 28 32 2 carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay *veronica tanasa 1,2 , madalina doltu 2 , dorin sora 2 , radu i. tanasa 3 , narcisa babeanu 1 1university of agronomical sciences and veterinary medicine in bucharest, romania 2institute of research and development for industrialization and marketing of horticultural products horting, 5n drumul gilaului, district 4, 042159, bucharest, romania 3national institute of research “cantacuzino”, 103 splaiul independentei, district 5, 050096, bucharest, romania *vero.tanasa@yahoo.co.uk received 26th january 2022, accepted 25th march 2022 abstract: carbendazim is a fungicide that is not approved for use in the european union, but is used in some other countries to preserve agricultural crops. enzyme-linked immunosorbent assay (elisa) become a viable alternative to traditional instrumental methods in pesticides analysis, for screening purpose. carbendazim residues were determined in two green lettuce cultivars, from the experimental lots, harvested at different times after the treatment with 0.1 % carbendazim solution, by an elisa carbendazim commercial kit and the values were compared with those obtained by high performance liquid chromatography (hplc). the results of elisa and hplc analysis of carbendazim residues in the two green lettuce cultivars were well correlated (pearson correlation coefficient r=0.8925). carbendazim residues in green lettuce/vegetables can be determined with high reliability by commercial elisas with results well correlated with those obtained by hplc, with an additional sample preparation step involving solid phase extraction, in order to reduce matrix effects; the assay can be used for carbendazim screening of many vegetable/fruit samples in shorter time and with inexpensive/cheaper equipment. keywords: carbendazim, green lettuce, elisa, hplc. 1. introduction the concern about the food safety with reference to pesticide residue levels and the appropriate analysis methods is constant [1-3]. carbendazim (metil nbenzimidazol-2-carbamat) belongs to carbamate pesticide class and is a fungicide used in fruit and vegetable growing and viticulture. this pesticide is not approved for the use in the european union [4], but it is used in some other countries to preserve agricultural crops. carbendazim causes embryotoxicity, apoptosis, teratogenicity, infertility, hepatocellular dysfunction, endocrine disrupting effects, disruption of haematological functions, mitotic spindle abnormalities mutagenic and aneugenic effects [5]. carbendazim residues are detected by high performance liquid chromatography (hplc) with uv [6,7], fluorescence detector [8] or by liquid chromatography-mass spectrometry [9]. but simple, high-throughput and fast analytical techniques with low cost are in great need as an alternative to traditional instrumental methods for screening purposes [10]. and so immunoassays become a viable alternative to traditional instrumental methods, especially enzymelinked immunosorbent assay (elisa), characterized by increased specificity and mailto:*vero.tanasa@yahoo.co.uk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay, food and environment safety, volume xxi, issue 1 – 2022, pag. 28 32 29 sensitivity, simple operation, the possibility of analyzing a large number of samples in a short time and low costs per analysis. itak et al. [11] developed a competitive elisa for the quantitation of benomyl (as carbendazim) and carbendazim in water. this immunoassay can also be used for the determination of carbendazim in fruit juice. williams et al. [12] reported that some commercial elisa kits designed to detect pesticide residues in water can, with care and experience, be successfully applied as a screening technique for residues in food extracts. a modified enzyme immunoassay method has been developed to determine methyl 2-benzimidazole carbamate in fruit juices and concentrates and the agreement between this method and high-performance liquid chromatography was good [13]. the aim of our study was to determine carbendazim residues in two green lettuce cultivars, from the experimental lots, harvested at different times after the treatment with 0.1% carbendazim solution, by an elisa carbendazim commercial kit and to compare the results with values obtained by high performance liquid chromatography. 2. materials and method 2.1. field trial the experience was conducted into the department of horticultural cultures in protected areas, horting institute, bucharest, romania, during the vegetation period of the vegetable plants grown in a block type cold greenhouse. the biological material used was represented by two green lettuce cultivars: allegiance and lollo bionda, intended for fresh consumption. the treatment with 0.1% carbendazim solution (prepared in the chemistry lab) was achieved by sprinkling the plants during the vegetation period. the samples were collected at different intervals after carbendazim treatment (3; 8; 13 days) and kept at -200c before being analysed. 2.2. reagents and standards maxsignal® carbendazim elisa test kit was purchased from bioo scientific (austin, texas, usa). carbendazim (97 %) was purchased from sigma-aldrich (darmstadt, germany). a carbendazim stock solution (250 µg/ml) was prepared in methanol and used for the preparation of working standard solutions necessary for calibration curve (1; 2.5; 5; 7.5; 10; 12.5 and 15 µg/ml) in hplc. all other reagents used were p.a. grade and solvents were hplc grade. 2.3. analytical procedure analysis of carbendazim by elisa maxsignal® carbendazim elisa test kit is a competitive enzyme immunoassay for the quantitative analysis of carbendazim in honey, juice, meat, rice/feed. vegetables are complex matrices, so a response enhancement could appear in elisa. for this reason we first clean up the vegetables extracts by solid phase extraction. samples were well blended and 10 g sample were extracted in 20 ml methanol. the extracts were filtered through whatman no. 1 filter papers (ge healthcare life sciences whatman); the filtrates were cleanup on oasis mcx cartridges (waters, ireland) following the manufacturer instructions, then were concentrated to 1 ml using a turbovap lv equipment (caliper lifesciences, usa). the concentrated filtrates were prediluted 1:100 with phosphate-buffered saline (ph 7.2), then to 1:500 with sample extraction buffer f, and after used in elisa according to the protocol described by kit manufacturer. samples and calibration standards were analyzed in duplicate. absorbance was recorded on a microplate reader ez read 400 research (biochrom, massachusetts, usa) with a 450 nm filter. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay, food and environment safety, volume xxi, issue 1 – 2022, pag. 28 32 30 analysis of carbendazim by hplc samples were well blended and 10 g sample were extracted in 20 ml methanol. the extracts were filtered through whatman no. 1 filter papers (ge healthcare life sciences whatman); the filtrates were cleanup on oasis mcx cartridges (waters, ireland) following the manufacturer instructions, then were concentrated using a turbovap lv equipment (caliper lifesciences, usa), so that the injected volume contained an amount of carbendazim within the linear range of the diode array detector. finally the samples were filtered through 0.45 μm syringe filters (thermo scientific, usa) prior to injection. the chromatographic separation was performed using a lichrocart purospher rp-18 column (250 * 4 mm), with 5 μ particle size (merck kgaa, germany) and the mobile phase consisted of water and methanol (25:75 by volume) under isocratic chromatographic conditions, with a flow rate of 1 ml/min. the column temperature was set at 200c. carbendazim was detected at 286 nm by the diode array detector [14]. samples were analyzed in duplicate.the data acquisition and processing have been done with the chromquest 4.2. software (thermo electron corporation, usa). we calculated the average, standard deviation, and then calculated the pearson correlation coefficient (r) to express the correlation between the concentration of carbendazim by elisa and hplc. the value of r ranges from -1 to +1. the closer |r| is to 1, the stronger is the correlation between the variables [15]. the results were statistically processed using graphpad prism (version 8.0.2, graphpad software inc., san diego, ca) [16]. 3. results and discussion in elisa the standard calibration curve was prepared in the concentration range of 0.5 to 12.0 ng/ml (0.5; 1.5; 3.0; 6.0; 12.0 ng/ml) and the correlation coefficient was 0.9982 (fig. 1). an enzyme-linked immunosorbent assay kit was used for the analysis of carbendazim residues in fruit and vegetables [17]. the authors observed a response enhancement in elisa due to the sample matrix and controlled this issue using a strawberry sample extract free of carbendazim for standard preparation. in this study we attenuated interferences in elisa response by cleaning up the vegetables extracts using solid phase extraction. fig. 1. maxsignal carbendazim elisa test kit standard curve carbendazim residues were detected (at concentrations >0.001 mg/kg) in 8.3% of 20469 vegetable samples from 31 chinese provinces, analyzed between 2014 and 2016 [18]. carbendazim was found most often in cowpeas, celery, beans with pods, lettuces, cucumbers, and leeks. lettuces contained the highest mean carbendazim concentration. carbendazim residues in green lettuce samples collected at different post treatment days are presented in table 1. for both cultivars carbendazim residues in samples collected on 3, 8 respectively 13 days after treatment with 0.1% carbendazim solution were between 0.21 and 3.05 mg/kg. the results of elisa and hplc analysis of carbendazim residues in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay, food and environment safety, volume xxi, issue 1 – 2022, pag. 28 32 31 the two green lettuce cultivars were well correlated (r=0.8925) (fig. 2). fig. 2. correlation between carbendazim concentrations as determined by elisa and hplc in green lettuce (n=18, pearson correlation coefficient r=0.8925) mountfort et al. [17] reported a correlation coefficient of 0.91 between elisa and hplc for the analysis of carbendazim residues in fruit and vegetables. our results for both cultivars on 3 and 8 days are similar with those obtained by bhattacherjee et al. [19] in mango fruits pulp after pre-harvest application of carbendazim at the rate of 0.1% (2.24 mg/kg on 4 days, respectively 1.23 mg/kg on 9 days), but the values obtained on 13 days were lower (0.51 mg/kg on 15 days in mango fruits pulp at the rate of 0.1%). table 1. carbendazim residues in allegiance and lollo bionda cultivars determined by elisa and hplc carbendazim residues (mg/kg)±sd (n=3 samples/cultivar/test) time interval post carbendazim treatment 3 days 8 days 13 days cultivar elisa hplc elisa hplc elisa hplc allegiance 3.05±0.62 2.43±0.31 1.21±0.15 1.05±0.15 0.21±0.03 0.22±0.04 lollo bionda 2.33±0.42 2.29±0.40 1.53±0.31 1.28±0.38 0.29±0.07 0.27±0.04 sd=standard deviation 4. conclusion carbendazim residues in green lettuce/vegetables can be determined by maxsignal carbendazim elisa test kit, with an additional sample preparation step involving solid phase extraction. the results of elisa and hplc analysis of carbendazim residues in the two green lettuce cultivars correlated well (pearson correlation coefficient r=0.8925). in addition, elisa technique implies shorter analysis time than chromatographic method, that is very important for screening of a large number of fresh vegetables/fruits that are perishable. elisa also implies smaller solvent volumes, less expensive and sophisticated equipment. 5. acknowledgments this work was supported by the romanian ministry of education and scientific research executive agency for higher education, research, development and innovation funding (uefiscdi), under the national r&d&i plan ii partnering program, grant pn ii-ptpcca-2013-4-0128, contract no. 147/2014 to m.d. and r.i.t. some expenditure was supported by the doctoral school in engineering and plant and animal resources management of the university of agronomical sciences and veterinary medicine in bucharest, for v.t. and n.b. all the authors declare no conflict of interest. 6. references [1]. fernandes v.c., domingues v.f., mateus n., delerue-matos c., determination of pesticides in fruit and fruit juices by chromatographic methods. an overview, journal of chromatographic science, 49: 715-30, (2011) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu, carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay, food and environment safety, volume xxi, issue 1 – 2022, pag. 28 32 32 [2]. carvalho f.p., pesticides, environment, and food safety, food and energy security, 6(2): 48–60, (2017) [3]. lazić s., šunjka d., jovanov p., vuković s., guzsvány v., lc-ms/ms determination of acetamiprid residues in sweet cherries, romanian biotechnological letters, 23(1): 13317-26, (2018) [4]. commission regulation (eu) no 559/2011 of 7 june 2011 amending annexes ii and iii to regulation (ec) no 396/2005 of the european parliament and of the council as regards maximum residue levels for captan, carbendazim, cyromazine, ethephon, fenamiphos, thiophanatemethyl, triasulfuron and triticonazole in or on certain products [5]. singh s., singh n., kumar v., datta s., wani a.b., singh d. et al., toxicity, monitoring and biodegradation of the fungicide carbendazim, environmental chemistry letters, 14(3): 317-29, (2016) [6]. paranthaman r., sudha a., kumaravel s., determination of pesticide residues in banana by using high performance liquid chromatography gas chromatography –mass spectrometry, american journal of biochemistry and biotechnology, 8(1): 1-6, (2012) [7]. phansawan b., prapamontol t., thavornyutikarn p., chantara s., mangklabruks a., santasup c., a sensitive method for determination of carbendazim residue in vegetable samples using hplc-uv and its application in health risk assessment, chiang mai journal of science, 42(3): 681-90, (2015) [8]. hu y., yang x., wang c., zhao j., li w., wang z., a sensitive determination method for carbendazim and thiabendazole in apples by solidphase microextraction−high performance liquid chromatography with fluorescence detection, food additives and contaminants part a., 25(3): 314-19, (2008) [9]. blasco c., fernandez m., pico y., font g., manes j., simultaneous determination of imidacloprid, carbendazim, methiocarb and hexythiazox in peaches and nectarines by liquid chromatography–mass spectrometry, analytica chimica acta, 461 (1): 109-16, (2002) [10]. li y.f., sun y.m., beier r.c., lei h.t., gee s., hammock b.d. et al., immunochemical techniques for multianalyte analysis of chemical residues in food and the environment: a review, trends in analytical chemistry, 88: 25-40, (2017) [11]. itak j.a., selisker m.y., jourdan s.w., fleeker j.r., herzog d.p., determination of benomyl (as carbendazim) and carbendazim in water, soil, and fruit juice by a magnetic particlebased immunoassay, journal of agricultural and food chemistry, 41: 2329-32, (1993) [12]. williams k.j., thorpe s.a., reynolds s.l., the use of elisa for the determination of pesticide residues in food, international journal of environmental analytical chemistry, 65:149-52, (1996) [13]. bushway r.j., savage s.a., ferguson b.s., determination of methyl 2benzimidazolecarbamate in fruit juices by immunoassay, food chemistry, 35: 51-58, (1990) [14]. tanasa v., doltu m., sora d., tanasa r.i., babeanu n., validation of a high performance liquid chromatography method for carbendazim residues quantification in tomatoes, food and environment safety, 17(3): 327-31, (2018) [15]. ellison s.l.r., barwick v.j., duguid farrant t.j., practical statistics for the analytical scientist: a bench guide, 2nd edition. lgc limited, published by the royal society of chemistry, cambridge, uk, (2009), pp. 92-113 [16]. welcome to the prism 8 user guide https://www.graphpad.com/guides/prism/8/userguide/index.htm (accessed 21 january 2022) [17]. mountfort k.a., reynolds s.l., thorpe s.a., white s.n., comparison of elisa and hplc techniques for the analysis of carbendazim and thiabendazole residues in fruit and vegetables, food and agricultural immunology, 6: 17-22, (1994) [18]. xu x., chen j., li b., tang l., carbendazim residues in vegetables in china between 2014 and 2016 and a chronic carbendazim exposure risk assessment, food control, 91: 20-5, (2018) [19]. bhattacherjee a.k., pandey b.k., om prakash, persistence and dissipation of carbendazim residues in mango fruits after preand post-harvest applications, journal of food science and technology, 46(4): 347-49, (2009) https://www.graphpad.com/guides/prism/8/user-guide/index.htm https://www.graphpad.com/guides/prism/8/user-guide/index.htm применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 contents: 1. dynamics of springy systems of packaging lines for food products anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko 298 313 2. extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin mariana spinei , mircea oroian 314 321 3. monitoring of groundwater quality in a semi-arid region, tebessa basin (northeast of algeria): using pollution index of groundwater toufik djebassi, ilhem abdeslam, hassen djabari, amor hamad, chemseddine fehdi 322 332 4. estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs) fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar , asma remil, mohammed benali 333 342 5. survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide 343 351 6. analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria omotade ogunremi, enobong ukpong, aima nosa 352 359 7. production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku 360 370 8. the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara 371 379 9. potential of romanian vine wastes as a natural resource of trans-resveratrol alina lenuța crăciun, gheorghe gutt 380 391 10. microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria bukola caroline ogunyemi, adewale kayode ogunyemi, simeon kolawole odetunde, adesola oluwafunmilayo olalekan, kabir olusegun akinyemi, bamidele abiodun iwalokun 392 402 11. blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization ana leahu , cristina ghinea, sorina ropciuc 403 410 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 12. nutritional characterization of the peripheral layers of barley grains cultivated in algeria rawda benguella, samira meziani, hayet mehida, yamina belkessam, kouider sennous, abassia demmouche 411 415 13. author instructions i v 14. subscription information vi title …………………… 176 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix , issue 2 – 2020 , pag. 176 179 total phenolic contents of some natural fruit juices and nectars from romanian market *veronica tanasa 1 1institute of research and development for industrialization and marketing of horticultural products horting, 5n drumul gilaului, district 4, bucharest, romania vero.tanasa@yahoo.co.uk *corresponding author received 23th december 2019, accepted 25th june 2020 abstract: total phenolic contents (tpc) of some natural fruit juices and nectars from the romanian market were determined immediately after opening and after three days of refrigeration (4-80c). the highest tpc were revealed in aronia juice and black currant nectar (110.80 respectively 120.69 mg gae/100 ml). the lowest tpc were registered in peach and apricot nectars (18.17 respectively 24.91 mg gae/100 ml). after three days of refrigeration, there were significant differences in tpc for three samples:apple juice, blueberry juice and peach nectar. the natural fruit juices and nectars tested are a good source of antioxidants (especially aronia, blueberry and black currant). keywords: total phenols, fruit juices, nectars. 1. introduction phenolic compounds are bioactive secondary metabolites that occur in a variety of fruits, vegetables, nuts, seeds and flowers. beneficial effects of phenolic compounds on human health are known, as they exhibit anti-carcinogenic, anti-ulcer, antithrombotic, anti-inflammatory, immunomodulating, antimicrobial, vasodilatatory and analgesic effects [1-4]. fruits and vegetables are an important dietary source of phenolic compounds. phenolic content and antioxidant activity in food items of vegetal origin remain a subject of interest for many researchers [57]. regarding the daily intake of fruit, romania is at the bottom of the list (42%), while the eu average is 64% [8]. that is why natural fruit juices and nectar could be an alternative to fresh fruits, especially for the children that do not like eating fruits. the aim of this study was to determine total phenolic contents of some natural juices and nectars from the romanian market. 2. materials and method 2.1. samples natural fruits juices and nectars free of food additives and preservatives were purchased from supermarkets: aronia, apple, raspberry and blueberry juices, black currant, apricot and peach nectars. fruits juices and nectars were obtained by soft evaporation in vacuum at 600c. 2.2. reagents and standards folin ciocalteu reagent, gallic acid and sodium carbonate were purchased from sigma. gallic acid stock solution in ethanol (1 mg/ml) was prepared and used for the preparation of working standard solutions necessary for calibration curve. http://www.fia.usv.ro/fiajournal mailto:vero.tanasa@yahoo.co.uk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix , issue 2 – 2020 veronica tanasa, total phenolic contents of some natural fruit juices and nectars from romanian market, food and environment safety, volume xix , issue 2 – 2020, pag. 176 179 177 2.3. total phenolic content (tpc) was determined by folin-ciocalteu assay [9]. samples were filtered through whatman no. 1 paper. absorption spectrum of reduced folin-ciocalteu reagent was recorded with a uv vis able jasco spectrophotometer v-550 (fig. 1), and the absorbtion maximum was registered at 755 nm. the standard calibration curve was prepared in the concentration range of 100 to 500 µg gae/ml and the correlation coefficient was 0.999800 (fig. 2). results are reported at gallic acid equivalent (gae) and were statistically processed using graphpad prism (version 7.00, graphpad software inc., san diego, 2007). 3. results and discussion total phenolic contents of seven romanian natural fruit juices and nectars were determined immediately after opening (t0) and after three days of refrigeration at 480c (t3), according to shelf life after opening indicated by producers (table 1). the highest tpc were revealed in aronia juice and black currant nectar (110.80 respectively 120.69 mg gae/100 ml). the lowest tpc were registered in peach and apricot nectars (18.17 respectively 24.91 mg gae/100 ml). tpc in apple juice was comparable with that obtained by wern et al. [10] in fresh apple juice (44.82 mg gae/100 ml) and by herken & guzel [11] in commercial apple juice (2.3 mm gae/l). total phenol values for apricot and peach nectars were lower than those obtained by herken & guzel [11] in commercial juices (3.0 respectively 3.4 mm gae/l). tpc in black currant nectar and raspberry were lower than values reported by jakobek et al. [12] (2770.94 respectively 1234.27 mg gae/l), and by piljac-zegarac et al. [13] (1919.8 mg gae/l for black currant juice). also the value obtained for blueberry juice was lower than those obtained by piljaczegarac et al. [13] in industrial juice (1795.5 mg gae/l). tpc in aronia juice was lower than values obtained by tolic [14] (11093; 9339; 8834 mg gae/l) in fresh juice made in laboratory between 2012 and 2014. these differences could be explained by fruit cultivar, culture technology, weather conditions and juice manufacture methods used. after three days of refrigeration, there were significant differences in tpc for three samples: apple juice, blueberry juice and peach nectar. fluctuations (increase) in tpc in fruits and vegetables juices during refrigeration were reported also by others authors. piljaczegarac et al. [13] observed that all dark fruit juices exhibited fluctuations in total phenol values with a marked increase after 48 hours in refrigerated storage, and a greater overall total phenol content in 5/6 studied juices after 29 days. and kim [15] reported fluctuations in total phenol values in green vegetables juices with maximum values at 21 days in shinsenocho branch and kale branch juices. folin-ciocalteu reagent reacts with many reducing substances. increases in tpc values for two samples (apple juice and peach nectar) during refrigeration could be explained by some juices chemical composition change. also decrease in tpc during refrigeration was reported. kim [15] observed a decreasing tendency of total polyphenolic contents of leaf parts after 4 days (shinsenocho leaf) or 7 days (kale leaf). decrease in tpc in blueberry juice may be due to enzymatic / nonenzymatic degradation. more investigations are necessary on fruit juices available on the market. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix , issue 2 – 2020 veronica tanasa, total phenolic contents of some natural fruit juices and nectars from romanian market, food and environment safety, volume xix , issue 2 – 2020, pag. 176 179 178 0.54 0.66 0.55 0.6 0.65 700 850750 800 abs wavelength [nm] fig. 1. absorption spectrum of the reduced folin -ciocalteu reagent 0 0 . 6 6 4 8 2 0 . 2 0 . 4 0 . 6 0 5 4 02 0 0 4 0 0 a b s c o n c . µg gae/ml fig. 2. folin ciocalteu gallic acid standard curve table 1. total phenolic contents of some natural fruits juices and nectars from romanian market (folin-ciocalteu assay) sd=standard deviation (n=3); t0 = immediately after bottles opening; t3= after three days of refrigeration (4-80c); gae= gallic acid equivalent; *p<0.05. sample tpc (mg gae/100 ml)±sd t0 t3 aronia juice 110.80±30.33 112.25±23.60 apple juice* 48.51±4.40 54.91±7.99 raspberry juice 33.44±8.24 34.92±8.29 blueberry juice* 95.54±12.65 91.91±7.83 black currant nectar 120.69±66.47 120.39±35.23 apricot nectar 24.91±7.88 27.15±12.06 peach nectar* 18.17±0.56 20.50±4.4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix , issue 2 – 2020 veronica tanasa, total phenolic contents of some natural fruit juices and nectars from romanian market, food and environment safety, volume xix , issue 2 – 2020, pag. 176 179 179 4. conclusion the results showed that natural fruit juices and nectars are a good source of antioxidants (especially aronia, blueberry and black currant). after three days of storage in refrigerator, there were significant differences in tpc for three samples: apple juice, blueberry juice and peach nectar. 5. acknowledgments this work was supported by horting institute, internal project no. 1415/28.09.2018. the author declares no conflict of interest. 6. references [1]. pandey k.b., rizvi s.y., plant polyphenols as dietary antioxidants in human health and disease, oxidative medicine and cellular longevity, 2 (5): 270-278, (2009) [2]. andujar i., recio m.c., giner r.m., rios j.l., cocoa polyphenols and their potential benefits for human health, oxidative medicine and cellular longevity, 2012: 1-23, (2012) [3]. khurana s., venkataraman k., hollingsworth a., piche m., tai t.c., polyphenols: benefits to the cardiovascular system in health and in aging, nutrients, 5: 3779-3827, (2013) [4]. gorzynik-debicka m., przychodzen p., cappello f., kubanjankowska a, gammazza a.m., knap n., wozniak m., gorska-ponikowska m., potential health benefits of olive oil and plant polyphenols, international journal of molecular sciences, 19: 547-559, (2018) [5]. karakaya s., el s.n., taç a.a., antioxidant activity of some foods containing phenolic compounds, international journal of food sciences and nutrition, 52: 501-508, (2001) [6]. nowak d., gośliński m., wojtowicz e., comparative analysis of the antioxidant capacity of selected fruit juices and nectars: chokeberry juice as a rich source of polyphenols, international journal of food properties, 19(6): 1317-1324, (2016) [7]. ljevar a., ćurko n., tomašević m., radošević k., gaurina srček v., kovačević ganić k., phenolic composition, antioxidant capacity and in vitro cytotoxicity assessment of fruit wines, food technology and biotechnology, 54 (2): 145-155, (2016) [8]. https://www.euronews.com/2019/04/01/eu-fruitand-vegetable-consumption-how-much-does-yourcountry-eat [9]. waterhouse a.l., determination of total phenolics, in current protocols in food analytical chemistry, wrolstad, r.e. et al (eds.), john wiley & sons inc.: i.1.1.1-i.1.1.8, (2002) [10]. wern k.h., haron h., keng c.b., comparison of total phenolic contents (tpc) and antioxidant activities of fresh fruit juices, commercial 100% fruit juices and fruit drinks, sains malaysiana, 45(9): 1319–1327, (2016) [11]. herken e.n., guzel s., total antioxidant capacity and total phenol contents of selected commercial fruit juices in turkey, international journal of food properties, 13:1373–1379, (2010) [12]. jakobek l., seruga m., medvidovickosanovic m., novak i., anthocyanin content and antioxidant activity of various red fruit juices, deutsche lebensmittel-rundschau / 103. jahrgang, heft 2: 58-64, (2007) [13]. piljac-zegarac j., valek l., martinez s., belscak a., fluctuations in the phenolic content and antioxidant capacity of dark fruit juices in refrigerated storage, food chemistry, 113: 394–400, (2009) [14]. tolić m-t, krbavčić i.p., vujević p., milinović b., jurčević i.l., vahčić n., effects of weather conditions on phenolic content and antioxidant capacity in juice of chokeberries (aronia melanocarpa l.), polish journal of food and nutrition sciences, 67(1): 67–74, (2017) [15]. kim s.y., fluctuations in phenolic content and antioxidant capacity of green vegetable juices during refrigerated storage, preventive nutrition and food science, 20(3): 169–175, (2015) 6. references 395 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 42018, pag. 395 403 determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken * abel inobeme 1 , alexander ikechukwu ajai 2 , abdullahi mann 2 , yahaya ahmed iyaka 2 1department of chemistry, edo university iyamho, edo state, nigeria, inobeme.abel@edouniversity.edu.ng 2department of chemistry, federal university of technology, minna, niger state, nigeria, talk2alexajai@gmail.com *corresponding author received 16th october 2018, accepted 20th december 2018 abstract: the purpose of this work is to investigate the polycyclic aromatic hydrocarbons (pahs) and heavy metal contents of barbecue fish, beef and chicken obtained from minna, niger state, using standard procedures. extraction of pahs was done using sonification method. n-hexane, dichloromethane (dcm) and mixture of both (1:1) were used as extractants. pahs content was analysed using gc-ms while the heavy metal analysis was done using aas. the results obtained from the study showed that grilled chicken had the highest content of total pahs (214.41µg/kg) while beef had the least (25.71µg/kg). 45 to 98% of the fractions of pahs observed in the samples were 4 and 5 member rings. benzo (a) pyrene was detected in all the samples analysed, its concentrations varied from 1.82 to 12.89 µg/kg. the concentrations of pahs in the grilled samples were generally higher than the control. the efficiency of the solvents from this study were in the order: n-hexane > (nhexane: dcm) > dcm. with the exception of pb which had a concentration of 3.17 mg/kg to 4.68 mg/kg, other metals investigated were within safe limit based on international standard. the concentrations of the metals in chicken and beef samples were in the general order: fe>pb>cu>mn. there is need to continually check the contents of pahs and heavy metals in barbecue food since they have bio-accumulative tendencies and are deleterious to health. keywords: barbecue; heavy metals; polycyclic aromatic hydrocarbons 1. introduction the demand for fish, beef and chickens as well as their products is increasing in most countries, especially in view of their significant nutritional values. they are major components of human diets and supply essential amino acids and minerals [1]. however, some of these food substances get contaminated during processing either by heat treatment and other exposure processes. these contaminants such as polycyclic aromatic hydrocarbons (pahs), polycyclic amines and heavy metals have lethal effects. this has raised an increasing concern about the quality and safety of foods in several parts of the world [2]. some of the contaminants even when present in trace amount have significant ecological impacts due to their ability to enter the food chain and bioaccumulate in the tissues of living organisms [3]. pahs are known to constitute one of the prominent classes of hazardous pollutants. of primary concern is the fact that some of them are carcinogenic, teratogenic and mutagenic [4]. pahs are group of organic compounds containing two or more fused aromatic rings made up of carbon and hydrogen atoms. their formation occurs during processing of coal, crude oil and natural gas, as well as incomplete combustion of coal and other organic substances including food [5]. the presence of these compounds in processed meat and other http://www.fia.usv.ro/fiajournal mailto:inobeme.abel@edouniversity.edu.ng mailto:talk2alexajai@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 396 food substances has become a subject of much concern in the recent years [6]. the concentration of pahs determined in a food matrix could be affected by processing method, extraction techniques as well as the efficiency of the solvent employed. various studies have reported the presence of pahs in processed food substances in different parts of the world [7]; but much has not been done on barbecue fish, beef and chicken obtained from minna and its environs in niger state, nigeria. thus the aim of this study is to determine the pahs and heavy metals content of barbecue fish, chicken and meat obtained from minna in niger state. 2. materials and methods 2.1 sampling method a total of six (6) samples were used for the purpose of this study. at each of the sampling point, five (5) subsamples were collected and homogenised so as to ensure a representative sampling. the samples collected from each area include beef, chicken (agric. type) and fish (catfish clarias gariepinus) processed using charcoal grilling. fresh samples were also collected and used as control. the samples were packed in aluminium foil, placed in polyethylene bags and then transported to the laboratory for pre-treatment and subsequent analyses [8]. 2.2 sample pre-treatment the samples were dried in an oven at low temperature of 105°c for eight hours. they were then homogenised using a mortar and pestle and stored in a refrigerator at 4°c prior to extraction and other analyses. 2.3 preparation of pahs standard and calibration curve a mixture of 16 pahs reference standards was purchased from supleco inc., usa. five standard solutions each containing 1.0, 2.0, 5.0, 10.0 and 20.0 cm3 of 500 mg/l of each standard pah was made up to 100cm3 with dichloromethane. these were transferred into a capped and sealed vial until ready for analysis. calibration curve of pahs was obtained by running standards of 5, 10, 25, 50 and 100 mg/l. a calibration curve was obtained by analysing each of the standard pahs solutions prepared with gc/ms. the concentration of each pah in the sample was determined in line with procedure reported by [9]. 2.4 determination of selected physicochemical properties some physicochemical parameters (ash, moisture and fat contents) were determined in line with [10]. 2.5 ultrasonic assisted extraction of samples the ultrasonic extraction was carried out in line with the method reported by [9] with slight modifications. the extract was then filtered and concentrated using a rotary evaporator under controlled vacuum. 2.6 silica gel column clean-up the clean-up was performed in line with the method reported by [11] using activated florisil (magnesium silicate) and anhydrous na2so4. the eluate was collected into an evaporating flask and rotary evaporated to dryness. the clean-up procedure was repeated using the other solvents (n-hexane and dcm) as eluents. 2.7 determination of heavy metal concentration the determination of selected heavy metals (mn, zn, cu, pb, cr and cd) was done using the method reported by [12]. analysis of variance (anova) was performed on pah concentration data using spss software. the significant statistical level was set at p<0.05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 397 3. results and discussion 3.1 physico-chemical properties table 1 physico-chemical properties of analysed samples fish chicken beef fat content (%) 16.40±0.70 27.30±0.20 20.81±1.01 moisture content (%) 25.15±1.21 15.30±1.90 29.40±0.99 ash content (%) 2. 20±0.93 1.62±0.12 1.41±1.20 results expressed as mean±sd for triplicate determinations. table 1 shows the physico-chemical parameters of the samples analysed. the fat contents were 16.40±0.70, 27.30±0.20 and 20.81±1.01% for fish, chicken and beef respectively. barbecue beef had the highest moisture content (29.40±0.99%﴿ while chicken had the least (15.30±1.90%). moisture content relates to the freshness and stability of food for long time storage [13]. food samples with very low moisture content are less prone to microbial attack hence less perishable [14]. the ash content of the samples in this study was generally low (1.41 to 2.20%﴿ which relates to the mineral compositions of the sample. ash is a measure of the inorganic components in food sample after removal of water and organic matter. fish had the highest ash content (2.20±0.93%﴿ while beef had the least. fat content determination is basic to pahs analysis, since the dripping of fat into the flame is one of the mechanisms of pahs formation. chicken had the highest fat content (27.30±0.20%﴿ while fish had the least (16.40 ±0.70%﴿. the low value of fat observed in some of the samples may be attributed to the fact that the samples were not analysed the same day of collection thereby resulting to the oxidation of fat. the difference in some of these parameters with findings from other studies can be attributed to geographical areas, sex, amongst others [15]. 3.2 polycyclic aromatic hydrocarbon contents of the samples most of the lower members pahs were not detected in the fish samples (table 2). this observation may be connected with the higher volatility of pahs of lower molecular weight. the highest amount of benzo (a) pyrene was obtained from nhexane (12.89 µg/kg) and the mixture of the two solvents (12.79 µg/kg). this implies that n-hexane is a better extraction solvent for b(a)p using sonification method. n-hexane extract gave a higher total pahs content for fish sample (99.27µg/kg) than dcm (33.05µg/kg). the total pahs obtained from fish using nhexane was about thrice that of dcm. this shows that n-hexane is more efficient for pahs extraction. the concentration of b(a)p ranged from 0.99±0.31 to 12.89±0.13 µg/kg. the values for n-hexane and combined solvent extracts are higher than 0.63±0.57µg/kg reported by [16] for b(a)p in a related study. the total pahs obtained is higher than a maximum of 14.95mg/kg documented by [17] in charcoal broiled beef burger. the concentration of b(a)p in dcm extract is however lower than 5.0µg/kg based on [17] standard. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 398 table 2 pahs content of grilled fish (µg/kg) using different extractants pahs dcm n-hexane dcm: n-hexane (1:1) control 1. napthalene nd nd nd nd 2. napthalene, 2-methyl nd nd nd nd 3. biphenylene nd nd nd nd 4. acenapthene nd nd nd nd 5. fluorene nd 16.01±0.13b 1.59±0.09a nd 6. phenanthrene 1.16±0.18a 7.01±0.91b 7.01±0.10b nd 7. fluoranthene 1.06±0.12a 1.12±0.08a 1.17±0.11a nd 8. pyrene 1.19±0.80a 4.18±0.10b 4.18±0.14b 0.98±0.91c 9. benz [a] anthra 1.51±0.12a 11.69±0.16b 11.73±0.04b 3.10±0.32a 10. triphenylene 1.74±0.10a 1.21±0.09a 1.21±0.21a 2.01±0.19a 11. benz[b] flu. 7.95±0.18b 12.39±0.11c 12.39±0.09c 5.98±0.12a 12. benzo [a] pyrene 1.92±0.13a 12.89±0.13b 12.79±0.11b 0.99±0.31a 13. indeno [1,2,3]pyrene 5.12±1.07a 10.11±0.30b 10.11±0.08b 2.98±0.08a 14. dibenz [a,h] anthrh 6.29±0.10a 17.19±0.05b 17.11±0.10b nd 15. benzo [ghi] peryle 5.11±0.11a 5.49±0.81a 5.49±0.59a nd ʃ pah 33.05 99.27 84.78 16.04 results are expressed as mean ± sd. values with same superscript on same row do not differ significantly at p< 0.05. nd: not detected. fig.1 presents the sum total fraction of pahs from the grilled fish. medium molecular weight (mmw) pahs (4 and 5 ring members) constitute majority (61.09 to 71.46%) of the total pahs. fig.1 the fractions of total pahs in grilled fish using different extractants lmw: low molecular weight (2 and 3 rings pahs); mmw: medium molecular weight (4 and 5 rings pahs); hmw: high molecular weight (6 rings). studies on carcinogenicity of pahs have shown that medium and higher molecular weight pahs (4 to 6) rings are more carcinogenic when compared to lower molecular weight members [18]. also dcm showed better efficiency in the extraction of six ring pahs (30.95%) when compared to n-hexane (15.25%). table 3 gives the pahs content of the barbecue chicken using various extractants. the total pahs concentration in chicken ranged from 165.63 to 214.41µg/kg. the highest for total pahs was observed in the extract of the combined solvents. n-hexane gave a higher yield of total pahs (185.63µg/kg) 0 20 40 60 80 dcm n-hexane dcm:n-hexane lmw mmw hmw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 399 when compared to dcm (165.63µg/kg). while individual solvents did not extract naphthalene and 2-methylnaphthalene, but with a combination of the extracting solvents, concentrations of 1.08±0.23 to 2.16±0.10 µg/kg were obtained. the least amount was in the control (1.01µg/kg) the pahs contents obtained here are comparable to the findings of [19] in their study on smoked fish from southern nigeria. the concentrations of pahs obtained is lower than 0.12mg/kg reported by [20] for b(a)p in their study on sardine. table 3 pahs content of grilled chicken (µg/kg) using different extractants pahs dcm n-hexane dcm: nhexane (1:1) control 1. napthalene nd nd 1.08±0.23a nd 2. napthalene, 2methyl nd nd 2.16±0.10a nd 3. biphenylene 1.26±0.09a nd 16.11±0.06b nd 4. acenapthene 2.66±0.01a 13.39±0.89b 13.41±0.12b nd 5. fluorene 3.57±0.11a 11.89±1.03b 11.85±0.09b nd 6. phenanthrene 4.94±0.01a 6.91±0.10a 5.89±0.92a nd 7. fluoranthene 1.19±0.08a 3.73±0.04b 3.77±0.10b nd 8. pyrene 50.91±0.01b 76.41±0.33c 76.39±2.07c 1.01±0.01 a 9. benz [a] anthracene 8.19±0.15c 3.09±0.09b 3.09±0.02b 0.99±0.21 a 10. triphenylene 1.78±0.91a 4.01±0.02b 6.74±0.03c 0.04±0.03 a 11. benzo [b] fluoranthene 10.39±0.01b 15.01±0.12c 11.51±0.10bc 2.97±0.01a 12. benzo [a] pyrene 16.11±0.10c 11.01±0.07b 10.61±0.07b 3.02±0.10 a 13. indeno[1,2,3,-cd] pyre 9.04±1.09b 8.02±1.01b 11.59±0.11c 4.98±0.01a 14. dibenz [a,h] anthracene 24.79±0.09c 19.11±0.10b 28.11±0.11c 7.01±0.10a 15. benzo [ghi] perylene 30.81±0.12c 13.11±0.09b 12.11±0.09b 5.99±0.03a ʃ pah 165.63 185.68 214.41 26.01 results are expressed as mean ± sd. values with same superscript on same row do not differ significantly at p< 0.05. nd; not detected fig.2 shows the distribution of pahs extracted from chicken in terms of the number of rings present. n-hexane was more effective in extracting 45 ring pahs (71.28%) compared to dcm (68.43%), mixture of both solvents gave the least (62.14%). dcm however gave the highest yield for 6 ring members (dibenz(a,h)anthracene and benzo[ghi]perylene). fig. 2. the fractions of total pahs in grilled chicken using different extractants 0 20 40 60 80 dcm n-hexane dcm: n-hexane lmw mmw hmw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 400 the pahs content in barbecued beef is presented in table 4. the first five members were not detected in the beef extract. indeno [1,2,3-cd] a six ring pah was present in highest concentration among other pahs (4.88 ±0.06 to 8.79±0.13µg/kg). the concentration of b(a)p in the grilled samples ranged from 1.48±0.11 to 5.62±0.11µg/kg. benzo (a) pyrene (b(a)p) is considered as a biomarker hence it presence is used in assessing other pahs in the environment [19]. the concentration of dibenzo(a,h)anthracene and fluorene observed here are higher than 0.0099mg/kg to 0.104 mg/kg reported by [21] in roasted meat. they are however lower when compared to 1.34 to 5.56 µg/kg reported by [22] in a similar study at port harcourt. the presence of pyrene and benzo(a) pyrene in all the extract is in agreement with findings of [23]. table.4 pahs content of grilled beef (µg/kg) using different extractants pahs dcm n-hexane dcm: nhexane (1:1) control 1. napthalene nd nd nd nd 2. napthalene, 2-methyl nd nd nd nd 3. biphenylene nd nd nd nd 4. acenapthene nd nd nd nd 5. fluorene nd nd nd nd 6. phenanthrene 1.08±0.71a 1.11±0.09a nd 0.49±0.01a 7. fluoranthene nd 1.07±0.55a nd 1.01±0.03a 8. pyrene 7.01±0.011c 1.01±0.06a nd 0.01±0.21b 9. benz [a] anthracene 1.29±0.07a 2.54±0.10a 1.59±0.01a 0.98±0.11a 10. triphenylene 2.36±0.02a 2.66±0.09a 2.49±0.10a 1.02±0.15b 11. benzo [b] fluoranthene 1.65±0.06a 4.31±0.05b 1.88±0.07a 1.03±0.31a 12. benzo [a] pyren 1.48±0.11a 1.82±0.10a 5.62±0.11b 1.01±0.23a 13. indeno [1,2,3, -cd] p 4.88±0.06a 7.89±0.07b 8.79±0.13b nd 14. dibenz [a,h] anthrace 5.95±0.11a 6.96±0.12a 6.06±0.09a 2.03±0.01a 15. benzo [ghi] perylene nd 5.49±1.08a nd nd ʃ pah 25.71 34.86 26.52 7.58 fig. 3. fractions of total pahs in grilled beef using different extractants the fractions of pahs extracted from beef in terms of molecular weight and ring number is shown in fig. 3 the fraction of lower molecular weight pahs in all the 0 20 40 60 80 100 dcm n-hexane dcm: n-hexane lmw mmw hmw food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 401 beef extracts where insignificant (0.00 to 4.20%). 4 and 5 rings pahs constituted the major components of the total pahs. these groups appear to be of more concern due to their carcinogenic risk. dcm gave 76.81% of the 4-5 rings compared to 58.43% from n-hexane. presence of 4-5 member rings above 50% of total pahs shows dominance of combustion. 3.3 heavy metal concentration table 5 shows the concentrations of the metals in the samples. chromium was not detected in all the samples analysed. [24] reported the concentration of chromium as 0.43 to 1.22mg/kg in his study on barbecued food. the concentration of fe ranged from 2.52±0.036 to 3.52±0.100mg/kg. barbecued beef had the highest amount while the least was in fish. contamination by fe and other metals may occur during processing of meat and other foods through contact with processing materials and penetration through surfaces exposed to surrounding environmental contaminants [25]. the concentration of fe from this study is in consonance with 2.13 to 4.65mg/kg reported by [24] in a similar study. mn had the least concentration among the elements analysed. the least content was in barbecued chicken. earlier studies have attributed the presence of some toxic metals to some of the additives and spices which some barbecued meat sellers add prior to processing [26].the highest amount of copper was in beef (2.97±0.35mg/kg), which was significantly higher than those of fish and chicken. the high content of copper may be related to environmental contamination due to exposure in the course of heating alongside background content. [21] in a related study on heavy metals in meat reported a significant increase in the concentration of metals after grilling, which he attributed to the method employed in cooking. [25] in a related study highlighted the content of copper as 0.15 to 0.35mg/kg. pb had the highest concentration which ranged from 1.17±0.100 to 2.68±0.037mg/kg. pb content varied significantly among the samples studied. fish sample had the highest content of pb. the concentration of pb in all samples from the present study is higher than the permissible limit of 0.5mg/kg in meat [8]. the value from the present study is also higher than 0.02mg/kg reported by [27] in roasted meat and 0.125 mg/kg documented by [26] in a related study. table 5 heavy metals content of analysed samples (mg/kg﴿ metals fish chicken beef permissible limit cr nd nd nd 1.0 (usda, 2006) fe 2.52±0.036a 2.58±0.047a 3.52±0.100b 43 (who, 2011) mn 0.51±0.015b 0.28±0.019a 0.48±0.015b 5.5 (who, 2011) cu 0.94±0.045a 0.95±0.050a 2.97±0.350b 10 (who, 2011) pb 2.68±0.037c 1.57±0.011b 1.17±0.100a 0.10(who, 2011) result expressed as mean ± sd for triplicate determinations. values on same row with same superscript do not differ significantly at p<0.05. 3.4 inter-elemental correlation coefficient there was a strong positive association between fe and cu (0.99) using spearman coefficient (table 6). fe correlated negatively with pb. a weak positive association (0.35 to 0.38) was observed among most of the other metals considered. the positive association among some of the metals shows their common origin. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 402 table 6 interelemental correlation coefficients in the samples cr fe mn cu pb cr 1 fe 1 0.35 0.99** -0.73 mn 0.35 1 0.38 0.37 cu 0.99** 0.38 1 -0.72 pb -0.73 0.37 -0.72 1 **. correlation is significant at the 0.01 level (2-tailed). 4. conclusion findings from this study have reveal that 4 to 5-membered ring pahs made up major fraction (60 to 80%) of the total pahs from the barbecued fish, chicken and beef. with a few exceptions, the efficiency of solvents in extraction of total pahs from the present study could best be arranged in the order: n-hexane> mixture (1:1) >dcm. the mean concentrations of pahs in the sample were in the following order: chicken>fish>beef. the concentration of b(a)p in some of the samples analysed exceeded the maximum acceptable limit of 5µg/kg (efsa, 2008). for heavy metal analysis, fe had the highest concentration in chicken and beef while mn had the least in all the samples analysed. 5. acknowledgments the authors are sincerely grateful to the laboratory technologists of the department of chemistry, federal university of technology, minna and the staff of central research laboratory, university of lagos, nigeria. 6. references [1]. olatunde s.o., olalekan s.f., beatrice o.o., bhekumusa j.x., benzo[a]pyrene and benzo[k]fluoranthene in some processed fish and fish products. international journal of environmental research and public health, issn 1660-4601, (2017) [2]. world health organization, polycyclic aromatic hydrocarbons. who food additives series 55: safety evaluation of certain contaminants in food. international programme of chemical safety (ipcs), world health organization, geneva, 563-743, (2006) [3]. froehner s., maceno m., machado k., predicting bioaccumulation of pahs in the trophic chain in the estuary region of paranagua, brazil. environmental monitoring and assessment, 174 (14):135-145, (2011) [4]. anila i.k., analysis of 18 polycyclic aromatic hydrocarbons in soil using the quechers method. thermo fisher scientific publications, runcorn, cheshire, uk. pg.1, (2014) [5]. ahmed e.o., adamu h.t., mohammed k.e., investigating the quality changes of hot smoked clariaslazera at refrigerated temperature ( 5 ± 1 ° c ). journal of agriculture and food science, 1(3):27–32, (2013) [6]. h. martins, forthcoming european pah legislation. santra bulletin, santra technology centre, usa.pg.1. [7]. vincent v., thierry s., fabrice m., bruno l.b., carole p., determination of pahs profiles by gc-ms/ms in salmon processed by four coldsmoking techniques. food additives and contaminants, 24(7):744-756, (2015) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xvii, issue 4 – 2018 abel inobeme , alexander i. ajai , abdullahi mann , yahaya a. iyaka, determination of polycyclic aromatic hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, food and environment safety, volume xvii, issue – 2018, pag. 395 – 403 403 [8]. ec, summary and conclusions of the sixtyfourth meeting of the joint fao/who expert committee on food additives expert vitamins in plasma. nutrition research, 24:435–446. (2005) [9]. tajudeen a.a., chijioke o., olasupo s.b., review article polycyclic aromatic hydrocarbons (pahs) in some smoked foodstuffs in lagos state, southwest, nigeria. science journal of chemistry, 5(3):31-35. (2017) [10]. aoac. official method of analysis (18th ed.). association of official analytical chemists international, maryland, usa. (2005) [11]. l.m. palm, m. a. gorleku, o. yeboah, w.j. qusie, characterization of polycyclic aromatic hydrocarbons (pahs) present in smoked fish from ghana. advance journal of food science and technology, 3(5), 332-338, (2011) [12]. a. federick, and a. kumah and s. bright, assessment of the presence of selected heavy metals and their concentration levels in fresh and grilled beef/guinea fowl meat in the tamale metropolis, ghana. science alert. (2015) [13]. m. q. jihad, s. m. ayman, and f. a. ali, nutritive value of seven varieties of meat products (sausage) produced in jordan. pakistan journal of nutrition, 8,332-334, (2009) [14]. c.ogbonnaya, m.s. ibrahim, effects of drying methods on proximate compositions of catfish (clariasgariepinus). world journal of agricultural sciences, 5(1),114-116 (2009) [15]. s. ande, l. leke, i. eneji, and s.yakubu, proximate analysis of smoked and unsmoked fish (cat and tilapia) in ombi river lafia nasarawa state nigeria. elixir food science, 53, 11801-11803, (2012) [16]. y.a. elhassaneen, the effects of charcoal-broiled meat consumption on antioxidant defence system of erythrocytes and antioxidant. toxicology reports, 3,45–51, (2004) [17]. european food safety authority polycyclic aromatic hydrocarbons in food scientific opinion of the panel on contaminants in the food chain (question n° efsa-q-2007-136) adopted on 9 june 2008, the efsa journal (2008) 724, 1-114 , (2008) [18]. silva bo, adetunde ot, oluseyi to, olayinka ko, alo bi, effects of method of smoking on the levels of pahs (polycyclic aromatic hydrocarbons) in some locally consumed fishes in nigeria. afr. j. of food sci., 5: 148 – 155, (2011) [19]. s. yurchenko, u. molder, the determination of polycyclic aromatic hydrocarbons in smoked fish by gas chromatography mass spectrometry with positive-ion chemical ionization. j. food composit. anal. 18, 857-869, (2000) [20]. t., isioma, o. ozekeke & e.lawrence, human health risk assessment of polycyclic aromatic hydrocarbons (pahs) in smoked fish species from markets in southern nigeria. toxicology reports, 4,55–61, (2017) [21]. j.c. akan, f.i. abdulrahman, o.a sodipo, y.a. chiroma, distribution of heavy metals in the liver, kidney and meat of beef, mutton, caprine and chicken from kasuwanshanu market in maiduguri metropolis, borno state, nigeria. research journal of applied sciences, engineering and technology, 2(8), 743-748, (2010﴿ [22]. j. d. nwineewii, and f. kpee, polycyclic aromatic hydrocarbons (pahs) concentrations in roasted plantain and fish from port harcourt city, rivers state, nigeria. chemsci rev lett, 6(23), 1356-1362, (2017﴿ [23]. n. omodara, a.o. obijole, e.a. adekunbi, h.o. oloyele, a. oyebade, polyaromatic hydrocarbons concentration in roasted plantain, roasted meat and smokked fish in ondo state. international journal of innovative studies research, 3(1), 27-31, (2015) [24]. t.nagwa, e. mahmoud, some heavy metals residues in chicken meat and their edible offal in new valley. 2nd conference of food safety, suez canal university, faculty of veterinary medicine, 1, 53 60, (2015﴿ [25]. a. benouadah, , a. diafat, b. djellout, assessment of trace heavy metals contents of chicken from algeria .international journal of plants, animals and environmental science, 5, (2), 2231-4490, (2015) [26]. a. iweala, o.olugbuyiro, m.durodola, m. fubara, k. okoli, metals contamination of food and drinks consumed in ota, nigeria. research journal of environmental 1. introduction 4. conclusion microsoft word 5 rehrahie_perception_corectat 31 iulie.doc 154 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xvii, issue 2 2018, pag. 154 164 perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in some selected areas of ethiopia *rehrahie mesfin1.3, getnet assefa2, fassil assefa3 1holetta agricultural research center, p.o. box 2003, addis ababa, ethiopia, e-mail: rehrahiemesfin@gmail.com 2ethiopian institute of agricultural research (eiar), livestock research directorate office, p. o. box 2003, addis ababa, ethiopia 3addis ababa university (aau), science faculty, department of microbial, cellular and molecular biology, p.o.box 1176, addis ababa, ethiopia received 28th november 2017, accepted 25th june 2018 abstract: the study was undertaken in western, eastern and southern shoa ethiopia with the aim of assessing the handling and storage practices of feeds and risks related to livestock water along the feed production, marketing and utilization chain. a total of 180 individuals (feed processors=33, feed retailers=51, smallholder dairy producers=96) participated in the study. because of improper harvesting and stacking, 91% of farmers encountered mold in roughage feeds. most of the farmers provide light-moldy feeds to livestock by mixing with uncontaminated ones. about 67% and 33% of the interviewed farmers utilized extreme moldy feeds for firewood and damping respectively. farmers perceived industrial effluent and leech to be as a safety risk to livestock water. all the interviewed farmers from eastern shoa were aware of industrial effluents as a problem for dairy production; and 66% and 34% of the respondents from eastern and western shoa respectively identified leech to be a safety problem to livestock water. about 69% of the farmers used bucket for scooping from water bodies to exclude leech from being consumed by animals; and 50% of them treated animals with chopped tobacco and onion as indigenous knowledge. feed processors=64%, feed retailers=82% and dairy producers=56% did not store their feed on raised floor indicating that there is probability of mold formation in stored feeds. about 67% of the feed processors, 73% feed retailers and 58% of the dairy producers stored concentrate feeds for shorter period of time (1 month). keywords: concentrate feed, dairy producers, questionnaire, storage time, value chain. 1. introduction in ethiopian agriculture, livestock has a 40% share of agricultural production and contributes to 13-16% of the total gross domestic product (gdp) 1. at household level, livestock production has become important for the livelihood of pastoralists, agro-pastoralists, and smallholder farmers 2. the economic benefits gained from export of livestock and livestock products in 2015/2016 were estimated at 37.5 million us dollar 3. although the country poses the highest number of livestock species in africa, the annual per capita consumption of milk in 1996 for example was of 17 kg which was lower than that of per capita milk consumption of other south and central african countries and developed countries which was of 26 kg and 200 kg respectively 4. despite some improvements on export of livestock and livestock products in recent years, productivity and commercialization of the livestock sub-sector is still very low food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 155 2 5.this is because livestock production is mainly undertaken by small-holding rural producers and pastoralists using inefficient local breeds depending on poor quality pastures and lack of veterinary services 4. moreover, the peri-urban and urban livestock production system is suffering from inadequate supply of animal feeds due to the underdeveloped agri-food chain and other structural problems 4. livestock production in the country is mainly dependent upon basal roughage feeds resources including natural pasture, hay and crop residues whereas peri-urban and urban dairy producers use supplemental feed including agroindustrial by-products such as wheat bran, noug seed meal, linseed meal, cottonseed meal and non-conventional concentrate feeds such as poultry liter and home produced by products of cereals and pulses (rehrahie, unpublished data). for many years now, a lot of attention has been given to increase the quantity of feed through research in ethiopia 6 7, however, there is a dearth of information on the quality and safety status of feed stuffs that determine the quality, safety of livestock products (milk, meat and egg). the ever-growing movement of food through international trade also necessitates guarantying the quality and safety of imported, exported and locally produced food products 8. the main factors that affect feed and food safety is categorized into biological, chemical and physical contaminants 9. feed and water sources are contaminated with chemicals such as heavy metals, and toxins and compromise the quality and safety of dairy products through the food chain. the biological contaminants, mainly the filamentous fungi grow on agricultural products and cause postharvest deterioration in cereals, oilseeds and legumes and by producing the chemical byproducts known as mycotoxins 10. the fungus that is grown in the field is further flourish at harvest, storage and processing 11 12. under optimum condition (moisture of > 13% and temperature of 400f-1000f), mold spores germinate, increase in number and consequently utilize the nutrients in grains and feeds and can reduce the nutritional quality of grains and feeds 13. 14 reported that storage fungi had adverse impact in reducing the carbohydrate content of sorghum grain in eastern harerghie, ethiopia. apart from that, supply of moldy feeds results in reduced digestibility 13, reduced feed intake, nutrient intake, weight gains and milk production 15. a production performance lose of 5-10% was also observed with feeding moldy feeds to livestock even in the absence of mycotoxins 13. there are different stakeholders in the value chain of dairy production systems which include small-scale peasant farmers, peri-urban and urban milk producers, feed processors, and distributors. the experience and indigenous knowledge of these partners, together with the monitoring and quantitative analysis of physical, chemical, and biological contaminants, is very vital to improve the handling and management of feeds in order to ensure the safety of dairy products. such studies can be used as benchmarks to scale up dairy production in the country. in this study attempt was made to assess feed storage practices, and problems associated with safety of feeds and water on livestock species based on the experiences and perception of stakeholders including feed processers, feed retailers, smallholder urban dairy producers and farmers. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 156 2. materials and methods 2.1. study location the study was carried out in western shoa (welmera/holetta), addis ababa, eastern shoa (akaki, gelan and ada) and hawassa. addis ababa and holetta are located at 38° 30`e, 9° 3`n. ada and hawassa are located in the great rift valley at latitude of 8o50 to 8o53 and longitude of 38o55 to 38o59 at an altitude of 1708 m.a.s.l and 1600-2400 m.a.s.l respectively. hawassa is located 285 km of addis ababa, south ethiopia. the average annual rainfall was 1100 mm and average low and high temperature of 12.6 oc and 27.3 oc respectively 16. map of the study locatons are indicated in fig. 1, 2 ,3 and 4. fig 1. map of holetta in western shoa fig 3. map of addis ababa 2.2. data collection and analysis the study was conducted using a semistructured questionnaire involving a total of 180 feed processors, feed retailers, urban smallholder dairy producers and rural farmers in central ethiopia (welmera, addis ababa, akaki, gelan, ada and hawassa) on feed storage fig 2. map of bishoftu in eastern shoa fig 4. map of hawassa methods and observed risks on livestock species that have been exposed to contaminated feeds and water. the collected data (qualitative) were organized and analyzed using descriptive statistics available in ibm 17. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 157 3. results and discussions 3.1. problems of mold in animal feeds the experiences of farmers on occurrence of moldy feeds, suitable seasons for mold formation and risks of moldy feeds to livestock species are presented in table 1. according to the interviewed respondents 91% (78) of them recognized the presence of mold problem mainly in roughage feeds including crop residues and hay. according to these respondents, mold formation occurred mainly during the rainy season or when roughage feeds are stacked in the absence of proper drying during harvest or because of bad baling style that can promote transfer of rain water. table 1 response of farmers on occurrence of mold in feeds, severe moldy season and problems of moldy feeds to livestock farmer's response farmer's response farmer's response suitable season for mold formation n % problems of moldy feeds to livestock n % manageme nt of moldy foods n % rainy season 32 100 illness 52 59 damping 9 33 dry season 0 0 feed rejection 22 25 firewood 18 67 total 32 100 abortion 13 15 total 27 100 death 1 1 total 88 100 those farmers who encountered mold in their feeds admitted that they provide lightly contaminated moldy feeds with livestock species by mixing them with fresh feeds which is in agreement with the practices employed in many countries 18. however, 33% of the interviewed farmers completely discarded/dumped the extremely moldy feeds in landfills, and 67% of the respondents used moldy feeds for firewood implying that farmers have recognized the disadvantage of feeding moldy feeds to livestock species. in the situations when animals were fed with moldy feeds, farmers observed problems with their livestock such as illness (59%), feed rejection (25%), abortion (15%) and death (1%). the observation of farmers was similar to the bad effect of mold indicated in 19 where feed rejection and reduced feed intake were some of the associated problems of moldy feeds to livestock species. it was also reported that moldy feeds affected the production performance of cattle because of deterioration of nutrients in the mold contaminated feeds. apart from any toxin effects, the mold itself caused production losses 20. livestock producers in minoseta reported that the animals given mold contaminated feeds encountered health problems such as reduced feed intake, diarrhea, reduced weight gain, abortions or death 20. 3.2. farmers’ perception on causes and effects of contaminated water the experience of farmers in relation to the causes and effects of contamination/ pollution of livestock water is presented in table 2. accordingly, the majority (80%) of the interviewed farmers had knowledge on contamination of livestock water with undesirable substances and parasites in their surroundings and recognized the two major contaminants of livestock water. one of them was the effluents discharged from industries particularly in eastern shoa, and the other was the problem of the parasitic leech (lymnatis nilotica) that naturally infest water sources during dry season which was commonly observed in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 158 eastern and western shoa. it was reported that scarcity and poor quality of water in relation to leech infestation was reported by farmers as third most important problems (27%) of livestock production in dendi area, ethiopia next to feed and animal diseases 21. high prevalence of leech infestation was observed in the dry season because of reduced flow, quantity and run of water into water sources as well as because of washing and bathing closer to livestock watering points 21. report from interview of farmers in sodo district snnp, also revealed that 95% of the respondents rated leech as a major animal health problem 22. whereas, in northern ethiopia, alamata, infestation of livestock water with leech was identified as a last risk among the major cattle health problems and verified, leech, as blood sucking parasite attach the pharynx part of the oral cavity while animals drink water and was responsible for loose of large amount of blood within short period of time resulting in anemia 23. 24 in libya also reported the exposure of different livestock species to leech infestation with increased rate observed in cattle 24. table 2 farmer's perception on causes and risks of contaminated water causes of water contamination by location farmers’ response farmers’ response risks related to leech contaminated water farmers’ response % risks related to effluent contaminated water n o % no % leech in holetta 34 diaharrea 6 20 illness and water rejection 37 76 leech in bishoftu 66 bad smell, water rejection, skin itching & emaciation 22 73 death 8 16 effluent in holetta 0 coughing 2 7 milk reduction 4 8 effluent in bishoftu 100 total 30 100 total 49 100 almost all of the interviewed farmers from eastern shoa have observed the effluents released from different factories in the surrounding water bodies that have become a risk to health of livestock species. about 66% of the interviewed farmers from eastern shoa admitted that the parasite leech (lymnatis nilotica) was the second important parasite in livestock water. however, the interviewed farmers from holetta did not mention pollution of water because of effluents released from factories; but 34% of the respondents from holetta identified that leech was the major contaminant of livestock water. almost all of the farmers in eastern shoa had the perception of the different risks related to drinking of effluent contaminated water to livestock species, and 73% of them associated the contamination with commonly observed problems of bad smell, water rejection, emaciation and skin itching, followed by diarrhea (20%) and coughing (7%). likewise, the majority (76%) of the farmers in both locations agreed that food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 159 illness (nose bleeding), reduced body weight and water rejection were commonly associated with leech contamination of water followed by death (16%) and milk reduction (8%). the health problems mentioned by the interviewed farmers in the present study was similar to the reports of 25 in that bleeding and reduced appetites were the commonly observed problems in cows which consumed leech contaminated water in iran. 3.3. indigenous practices employed to overcome leech infested water the farmers also had the traditional knowledge and experience of applying different traditional practices to control contamination of leech in water bodies and mouth of the animal (table 3). among the interviewed farmers the highest proportion (69%) of them used bucket for selectively scooping water from the water body and excluding the ingestion of leech by the animals. farmers also employed immersion of endod in water bodies as a second (13%) important leech minimizing practice. this was scientifically proved by a study undertaken in sodo, guragie zone of ethiopia, in that application of endod (phytolacca dodecandra) in water bodies resulted in 97-100% reduction in streams used for livestock drinking 22. farmers also practiced different strategies to remove leech from the mouth of animals of which 50% of them used to drench chopped tobacco and or onion to livestock species. according to the respondents, 32% of them physically removed the leech from the animal by hand which was similar to the practices and experience of leech removal employed in iran 25. in this study, 10% and 8% of the respondents respectively solved the problem by drenching tablet and restriction of animals from drinking water for one day to facilitate release of leech in the mouth of the animal so as to be able easier to remove it from the mouth. to prevent contamination of livestock water with leech, the farmers in west shoa (dendi), ethiopia employed different practices such as separating human and livestock water points, washing and bathing areas and fencing livestock water points 21. table 3 indigenous practices to overcome leech infested water practice to prevent leech from being consumed by animals % practice to kill leech entered in animal body % immersion of stalk of noug and linseed 6 drenching chopped tobacco and/or onion 50 immersion of endod (phytolacca dodecandra) 13 drenching tablet 10 bucket watering 69 withdrawal of water 8 use of other alternative water sources 12 taking away from mouth 32 total 100 total 100 3.4. storage methods of concentrate feeds in feed processing factories in this study, different stakeholders involved in the feed production, marketing and utilization chain were interviewed to evaluate the storage methods of concentrate feeds. accordingly, the majority of the feed manufacturers (64%) stored their concentrate feed on cement floor without using palate (table 4). palate is any material (minimum of 50 cm height) placed on a floor over which concentrate feed is stored to avoid contact between the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 160 concentrate feed and the floor to minimize the transfer of moisture. it was observed that some feed manufacturers left some space between two adjacent stored concentrate feeds and between the stored feed and the wall and the roof to allow ventilation implying that they have good understanding of benefit of air circulation in stored feeds. they have similar understanding with people in developed countries in that grain storage facilities in the u.s. have fans installed for ventilation and temperature control in the stored grain 15. table 4 storage methods of concentrate feeds in feed processing factories (% and n) study location storage method holetta bishoftu hawassa total on cement floor with no palate 61 (11) 50 (3) 78 (7) 64 (21) on cement floor with palate 33 (6) 17 (1) 11 (1) 24 (8) on soil floor with no palate 0 (0) 17 (1) 0 (0) 3 (1) on soil floor with palate 6 (1) 0 (0) 0 (0) 3 (1) missing 0 (0) 17 (1) 11 (1) 6 (2) total 100 (18) 100 (6) 100 (9) 100 (33) consecutive figures in each cell are percent and number of respondents respectively among the interviewed feed manufacturers in this study, the majority (72%) of them preferred using palate to store only wheat flour whereas, 28% of the interviewees used palate equally for both wheat flour and wheat bran. 3.5. storage duration of concentrate feeds in feed manufacturers the storage duration of concentrate feeds by feed manufacturers is presented in table 5. accordingly, the majority (67%) of the interviewed feed manufacturers stored their concentrate feeds for shorter duration (a maximum of 1 month) which is related to the higher market demand for concentrate feeds in the country. the result also showed that 24 % of the feed manufacturers kept their concentrate feed for duration of 6-8 months, particularly during the rainy seasons when alternative feed resources such as green grass were available and resulting in decreased market demand and increased storage time of concentrate feeds. table 5 storage duration of concentrate feeds in feed manufacturers (% and n) study location storage duration (month) holetta bishoftu hawassa total 1 61 (11) 50 (3) 89 (8) 67 (22) 2 3 6 (1) 0 (0) 0 (0) 3 (1) 4 6 6 (1) 0 (0) 0 (0) 3 (1) 6 8 28 (5) 33 (2) 11 (1) 24 (8) missing 0 (0) 17 (1) 0 (0) 3 (1) total 100 (18) 100 (6) 100 (9) 100 (33) consecutive figures in each cell are percent (%) and number of respondents (n) respectively food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 161 3.6. storage methods of concentrate feeds in feed retailers the storage style of concentrate feeds in feed retailers is presented in table 6. the majority of the feed retailers (82%) in the study locations stored their concentrate feed on cement floor without using palate. the situation was worse than that of feed manufacturers and feed processors where 64% of the owners stored their concentrate feed on cement floor without using palate. among these feed retailers the majority were in hawassa (93%) followed by holetta (84%) and bishoftu (71%). table 6 storage methods of concentrate feeds in feed retailers (% and n) study location feed storage styles holetta bishoftu hawassa total on cement floor with no palate 84 (16) 71 (12) 93 (14) 82 (42) on cement floor with palate 5 (1) 6 (1) 0 (0) 4 (2) on soil floor with no palate 0 (0) 24 (4) 7 (1) 10 (5) on soil floor with palate 5 (1) 0 (0) 0 (0) 2 (1) missing 5 (1) 0 (0) 0 (0) 2 (1) total 100 (19) 100 (17) 100 (15) 100 (51) consecutive figures in each cell are percent (%) and number of respondents (n) respectively 3.7. storage duration of concentrate feeds in feed retailers the storage durations of concentrate feeds in feed retailers is shown in table 7. the majority of them (73%) in the study locations on average stored their feed for a shorter period of time (1 month). this was because of the higher demand of concentrate feeds by livestock producers. the feed retailers that stored their concentrate feeds for very short duration of time (1 month) was lower in holetta (47%) than the feed retailers from bishoftu (82%) and hawassa (93%). table 7 storage duration of concentrate feeds in feed retailers (% and n) study location feed storage duration (month) holetta bishoftu hawassa total 1 47 (9) 82 (14) 93 (14) 73 (37) 1 2 0 (0) 6 (1) 0 (0) 2 (1) 2 – 3 5 (1) 0 (0) 0 (0) 2 (1) missing 47 (9) 12 (2) 7 (1) 24 (12) total 100 (19) 100 (17) 100 (15) 100 (51) consecutive figures in each cell are percent (%) and number of respondents (n) respectively 3.8. storage methods of concentrate feed in dairy producers the storage methods of concentrate feeds in smallholder dairy producers is shown in table 8. the majority (56%) of the smallholder dairy producers stored on cemented floor with no palate, followed by 16% of the dairy producers that stored their feed on soil floor lined with certain sheet such as plastic or cloth of which the majority were dairy producers from holetta (27%). majority of the dairy producers from bishoftu (76%) and hawassa (73%) stored their concentrate feeds on cemented floor without palate. the present study has also shown that 64% of the feed processor (table 5.4), 82% of feed retailers (table 7) and 56% of dairy producers (table 9) have not used palate for placing their food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 162 concentrate feeds indicating, there is probability of mold formation in stored concentrate feeds. although no information was available showing the bad effect of the traditional storage and handling methods of livestock feeds in ethiopia and abroad, few survey studies were conducted in the country to evaluate traditional grain storage practices and structures (differing from region to region) on nutrient quality of cereal grains. accordingly, storing sorghum for longer duration deteriorated the nutritional composition 14; 26. about 16% lose in grains (by weight) in the pastoralist areas of ethiopia were also recorded because of deteriorations in nutrient composition of stored sorghum in relation to mold contamination 27. according to 28, quality and nutrient deterioration was observed in stored maize in farmers employing traditional grain storage practices. the author identified mold and insects to be the two major factors causing nutrient deterioration in grains and observed moisture and temperature as ideal medium for mold growth. according to 29, 50% loss in the yield of sorghum grain was recorded because of insect pests in south western ethiopia. table 8 storage methods of concentrate feed in dairy producers (% and n) study location feed storage methods holetta bishoftu hawassa total on cemented floor with no palate 21 (7) 76 (25) 73 (22) 56 (54) on cemented floor with palate 0 (0) 0 (0) 10 (3) 3 (3) on soil floor with no palate 3 (1) 15 (5) 7 (2) 8 (8) on soil floor lined with certain sheet 27 (9) 9 (3) 10(3) 16 (15) missing 49 (16) 0 (0) 0 (0) 17 (16) total 100 (33) 100 (33) 100 (30) 100 (96) consecutive figures in each cell are percent (%) and number of respondents (n) respectively 3.9. storage duration of concentrate feeds in dairy producers the storage durations of concentrate feeds in smallholder dairy producers is presented in table 9. due to increased price of concentrate feeds these days, the majority of dairy producers (58%) stored their concentrate feed for a shorter period of about 1-2 weeks. table 9 storage duration of concentrate feeds in dairy producers (% and n) study location storage duration (week) holetta bishoftu hawassa total 1 0 (0) 0 (0) 25 (10) 10 (10) 1 2 30 (10) 33 (11) 40 (16) 58 (37) 3 4 24 (8) 42 (14) 20 (8) 12 (30) missing 46 (15) 24 (8) 15 (6) 31 (29) total 100 (33) 100 (33) 100 (40) 100 (106) consecutive figures in each cell are percent (%) and number of respondents (n) respectively 4. conclusions in this study, the majority of farmers had the knowledge on the occurrence, causes and effects of mold in feeds and, farmers dwelling in the industrial zone of eastern shoa mainly in gelan area were aware of the pollution problems of livestock water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 163 caused by released effluents from different factories. they were also aware of leech problem in livestock water use, and some also had indigenous knowledge on how to protect and treat their livestock from the scourge of the leech. under these circumstances, they were also conscious of the pollution problems on the health and productivity of their livestock.the majority of the dairy producers, feed retailers and feed manufacturers did not use palate to store concentrate feed indicating that there was the possibility of mold occurrence in concentrate feeds. further research needs to be undertaken along the feed production, processing, marketing and utilization chain, on the effects of different storage conditions by considering factors such as use of palate, ventilation and time of feed storage on mold formation and nutrient quality of feeds using larger sample size. 5. references 1 zinash, s., alemu, y. contribution of animal science research to food security. proceedings of the 9th ethiopian society of animal production (esap). addis ababa, ethiopia. 31-45, (2001). 2 negassa, a., rashid, s., gebremedhin, b. livestock production and marketing in ethiopia. ethiopian support strategy program ii (essp ii) working paper 26. washington, d.c. ifpri. (2011). 3 inter-governmental authority on development (igad). the contribution of livestock to the ethiopian economy-part ii. igad livestock policy initiative, igad lpi working papers, no 02-11, (2017). 4 azage, t., alemu, g. prospects for periurban dairy development in ethiopia. available at: http://hdl.handl.net/10568/50997/, (1998). 5 alemie, a., lemma, z. contribution of livestock sector in ethiopian economy: a review. advances in life science and technology, 29: 79-90, (2015). 6 dereje, d., debela, k., wakgari, k., zelalem, d., gutema, b., gera, l., adugna, t. assessment of livestock production system and feed resources availability in three villages of diga district ethiopia. research program on integrated systems for the humid tropics. ilri, (2014). 7 endale, y., abule, e., lemma, f., getnet, a. feed resources and its utilization practices by smallholder farmers in meta-robi district, west shoa zone, oromiya regional state, ethiopia. academic research journal of agricultural science and research, 4(4): 124-133, (2016). 8 fao/who. food safety risk analysis a guide for national food safety authorities. fao food and nutrition paper 87, rome, italy, (2006). 9 feed industry haccp auditor manual. version 3, office of the texas state chemist, texas, (2011). 10 mold fact sheet. cornell cooperative extension, new york times magazine, cornel university, (2001). 11 iheshiulor, o., esonu, b.o., chuwuka, o.k., omede, a.a., okoli, i.c., ogbuewu, i.p. effects of mycotoxins in animal nutrition: a review. asian j. anim. sci., 5(1): 1933, (2011). 12 suleiman, s. e., and abdalla, m. a. presence of aflatoxin m1 in dairy cattle milk in khartoum state-sudan. international journal of scientific & technology research, (2)4: 2277-8616, (2013). 13 tarr, b. managing the effects of molds and mycotoxins in ruminants. nutrition news and information update, shur-gain, nutrico canada inc., (2006). 14 mashlla, d. grain storage method and their effects on sorghum grain quality in harerghe, ethiopia. department of ecology and crop production science, doctoral thesis, swedish university of agricultural university, uppsala, sweden, (2004). 15 thomas, j.w., hjubik, j.g., patric, h.l., meerdink, g., marczewski, a.e. how to handle mold feed problems. msu ag facts, extension buletin e, 1670, (1983). 16 https://en.wikipedia.org/wiki/awasa, (2017). 17 statistical procedures in social sciences (spss). spss for windows version 20, statistical package for social sciences, us, (2011). 18 shewmaker, g. what do you do with moldy hay? extension dairy specialist, university of idaho extension, (2009). 19 westway feed products. mold inhibition: factors and options. e-z glo feed mill products (2013). 20 dicostanzo, a., murphy, m. strategies for feeding mycotoxin and mold food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume vii, issue 2 – 2018 rehrahie mesfin, getnet assefa and fassil assefa, perception of farmers, feed processors and feed retailers on contaminants of livestock feed and water in central highlands of ethiopia, food and environment safety, volume xvii, issue 2 – 2018, pag. 154 – 164 164 contaminated grains to cattle. beef cattle feed lot nutrition, university of minoseta extension, (2012). 21 belay, d., getachew, e., azage, t. and hedge, b. farmers perceived livestock production constraints in ginchi watershed area: results of participatory rural appraisal. international journal of livestock production, 4(8): 128-134, (2013). 22 tadesse, e., getnet, a., mesfin, s. control of aquatic leeches (lymantis nylotica) using phytoloca dodecandra (endod) in sodo district, guragie zone, southern nations, nationalities and peoples region, ethiop. vet. j., 14(2): 125-135, (2010). 23 yohannes, t. major animal health problems of market oriented livestock development in alamata wereda. doctor of veterinary medicine (dvm) thesis, debre zeit, ethiopia, (2007). 24 negm-eldin, m.m., abdraba, a.m., benamer, h.e. the first reported leech infestation by limnatis nilotica (savigny 1822) of farm animals in libya. rabat serie zoologie, 4:3336, (2013). 25 bahmani, m., efekhari, z., mohsezadeghan, a., ghotbian, f., alighazi, n. leech (limnatis nilotica) causing respiratory distress in a pregnant cow in ilam province in iran. comp clin. pathom., 21: 501-503, (2012). 26 abebe, h.g. and bekele, h. farmers ‘post harvest grain management choices under liquidity constraints and impending risks. implications for achieving food security objectives in ethiopia. poster paper prepared for presentation at international association for economists conference, gold coast, australia, (2006). 27 hodgson, r.j. the southern sidamo rangeland project 1985-1988. perspectives on development interventions and extension. unpublished monograph, (1990). 28 dubale, b. factors affecting quality of grain stored in ethiopian traditional storage structures and opportunities for improvement. international journal of sciences: basic and applied research (ijsbar), 18(1): 235-257, (2014). 29 chemeda, a., abush, t., zekarias, s. and habte, j. survey on farmer’s perception and management practices of insect pests on traditional stored sorghum in south western ethiopia. crop protection 26, 1817-1825, (2007). doi: https://doi.org/10.4316/fens.2021.038 360 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 360 370 production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes sunday sunday sobowale 1 , *olawale paul olatidoye 2 habibat oluwatoyin animashaun 3 , ayinde lukman odunmbaku 3 , 1mountain top university, department of food science and technology, college of basic and applied sciences, km 12, lagos-ibadan expressway, ogun state, nigeria 2department of food technology, yaba college of technology, p.m.b. 2011, yaba, lagos state, nigeria, waleolatidoye@gmail.com 3department of food technology, moshood abiola polytechnic, abeokuta, ogun state, nigeria. *corresponding author received 25th may 2021, accepted 20th december 2021 abstract: alcoholic drinks produced from cassava, maize and plantain flour using local hydrolyzed enzyme and imported enzymes were evaluated and compared in this study. about 2 kg of each flour substrate was divided into two portions; a portion was hydrolyzed using imported enzymes (α-amylase and amylo-glucosidase) while the other portion was processed with local hydrolyzed enzyme (malt) for production of alcoholic drink. physicochemical, color and sensory attributes of the alcoholic drink samples were examined. results showed that there were significant differences (p<0.05) in the physicochemical and color profile of the samples. alcoholic drink produced from plantain with the minimum degree brix liberated the highest alcohol content, followed by cassava and maize. generally, low lightness, negative a* (greenness) and b* (blueness) confers alcoholic drinks with grey color appearance. sensory test of the alcoholic drinks were all acceptable by the panelists. thus, cassava, maize and plantain could serve as a good substrate for the production of alcoholic drinks. keywords: colour profile, sensory evaluation, physicochemical properties, alcoholic drink 1. introduction cassava (manihot esculenta crantz) is one of the leading food and feed plants in the world [1]. it is the most important source of calories in the tropics after rice and corn, providing energy nourishment for more than half a billion people worldwide [2]. maize (zea mays l.) is an important cereal grain providing nutrients for humans and animals and serving as a basic rawmaterial for the production of starch, oil and protein, alcoholic beverages and food sweeteners [3]. maize compares well with other cereals as energy source; it is rich in vitamin b and minerals such as phosphorus, magnesium, manganese, zinc, iron and small amounts of potassium [4]. plantain (musa paradisiaca) have a high carbohydrate content and low fat content and is traditionally grown in west africa for food and used as part of local staple diets or processed into flour that can be stored for later use [5]. alcoholic beverages are integral to daily life; alcohol has been consumed since prehistoric periods. various types of local, traditional and indigenous alcoholic beverages are produced from local food stuffs using traditional and indigenous techniques [6]. in nigeria, there has been local production of ethanol from cereal and other starch substrate. fermented products like burukutu from maize and pito from millet or guinea-corn (percentage alcohol varies from 3-6% alcohol by volume (abv); palm wine, tapped from raffia and oil palm trees (3-4% alcohol by volume abv) and gin like distillates ogogoro, kinkana, kai http://www.fia.usv.ro/fiajournal mailto:waleolatidoye@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 361 kai and apetesi (ranging from 40-80% alcohol by volume abv) have been prepared an drunk in nigeria [7]. in developing countries including nigeria, indigenous food processing techniques such as fermentation and traditional distillation of alcoholic drinks from starch materials has continuously been practiced by vast ethnic groups with locally available equipments, utensils, substrates and poor processing condition which pose challenges for commercialization[8]. the consumption pattern of the alcoholic drinks is still at household in the rural settings [6]. also of concern are health problems like food poisoning and food intoxication owing to lack of regulated processing standards and poor acceptable hygienic and content specifications [9]. harnessing laboratory pilot scale production of alcoholic drinks from starch substrates is still limited due to non-readily available hydrolyzing enzymes. hence, malting of local cereals could as well provide an alternative means of obtaining hydrolyzing enzymes. the knowledge of physicochemical properties, color profile and sensory characteristics of alcoholic drinks could give health wise insight of behavioural pattern of compositing compounds, improved final product quality and safety as well as positioning alcoholic drinks for potential commercialization. considering the highlighted possibilities, an investigation was therefore geared towards the evaluation and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally sourced and imported enzyme. 2. materials and methods materials: matured cassava roots, wholesome maize, and unripe plantain fruits were purchased from kuto market (7.5 on, 4.5 oe), abeokuta, nigeria. hydrolyzing enzymes (α-amylase and amylo-glucosidase) were sourced at enzymology laboratory of the department of biotechnology, fiiro, nigeria. brewer’s yeast was purchased from a commercial dealer at ojota, lagos, nigeria. all analytical chemical used were of analytical grade. preparation of cassava flour the fresh cassava roots were processed into flour using the method described by [10]. 2.5 kg of fresh cassava roots were peeled manually with a sharp knife, washed and grated in a locally fabricated mechanical grater. it was thereafter packed into hessian sack and dewatered by pressing in a mechanical press to dewater the mesh. the dewatered lump was pulverized with hands and sifted on a local raffia made sieve of mesh (0.3cm x 0.3cm) mounted on a rectangular wooden frame 40 cm2 to remove the fibres. the sifted cassava meal obtained was allowed to dry in oven dryer at 60 oc for 24 h. the dried meal was milled and packaged in high density polyethylene film and kept under refrigerated storage until ready for further analysis. preparation of maize flour the method of [11] was used for the preparation of maize flour. 2.5 kg of wholesome maize kernels were sorted, cleaned by winnowing and thereafter passed through discs of hammer mill to crush grains, and coarse maize flour was obtained. preparation of plantain flour the preparation of plantain flour was carried using the method of [12] with modification. 2.5 kg of unripe plantain fruits were washed with distilled water, peeled and sliced to about 5 mm using a stainless steel knife. the slices were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 362 subsequently steamed for 5 min to inactivate enzymes. subsequently, the pulp was drained and dried in an oven drier at 60 °c for 24 h, after which the dried plantain slices were milled into flour using attrition mill, sieved and packed in high density polythene bags. preparation of malt for local hydrolyzed enzyme the preparation of sorghum malt was done using the modified method described by [13]. 1.5 kg of grains was steeped in 3 l of distilled water after prior sterilization with sodium hypochlorite solution (containing 1% available chlorine for 20 min). steeping lasted for 24 h. drained grains were thereafter germinated for 96 h in germination trays in an atmosphere of near water saturation. grains were turned every 12 h and watered by spraying with 60 ml distilled water at 24 h intervals, germinating grains were withdrawn and immediately kiln-dried. kilning was done for 24 h at 40 °c in a forced draught oven. the malt obtained was ground using a laboratory blender and thereafter packaged. preparation of alcoholic drinks using imported enzyme and local hydrolyzed enzyme the method of [10, 14,] were employed for the preparation of alcoholic drinks from cassava, maize and plantain flour using imported enzyme and local hydrolyzed enzyme with modification, as presented in figure 1. 1 kg of each flour substrate was stirred in 5000 ml of water to form slurry. the mixture was gelatinized at 70 oc for 20 min until smooth gel was formed and thereafter 4 ml of α-amylase was added to the gelatinized starch and incubated at 70 oc for 60 min. 4 ml of aqueous solution of amylo-glucosidase was then added to the liquefied sample at 50 oc and incubated at 70 oc for 60 min. while for local hydrolyzed enzyme; 250 g of malt was added to the gelatinized mash, stirred and the mixture was incubated at 60 oc for 5 h. the mixture (for both imported and local hydrolyzed enzyme) was sieved using a sieving cloth after the hydrolysis. the sieved sweet hydrolysate wort was boiled at 70 oc for 5 min to arrest further enzyme action. fermentation and distillation process of hydrolysate wort the fermentation and distillation process of hydrolysate wort obtained using imported and local hydrolyzed enzymes were carried out according to the method of [10]. fermentation yeast inoculum (100 ml of 15% yeast inoculum (15 g of dry brewer’s yeast rehydrated in 100 ml of distilled water at 37 °c for 10 min) was added to the hydrolysate wort (at 30 oc) before anaerobic incubation for 5 days at 28-30 °c. the crude ethanol obtained was filtrated and distilled at temperatures between 78-80 oc using distillation apparatus (setup). the resulting product was an alcoholic drink from different flour substrates (figure 1). physicochemical properties of alcoholic drinks determination of total titratable acidity the total titratable acidity of the samples was determined according to the method illustrated by [15]. 250 ml beaker was filled with 100 ml distilled water and a few drops of phenolphthalein indicator were added and mixed. the mixture was titrated with 0.1n naoh to a pale pink color. 10 ml of alcoholic drink sample was measured into the mixture and titrated against 0.1 naoh to a pale pink color which persisted for at least 30 s. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 363 where; n = normality, t = titre value, s = sample volume and 75 = constant determination of ph the method of [15] was used for determination of ph. hanna digital bench top ph meter equipped with an electrode probe was used for the determination. the electrode probe was calibrated and then placed into a beaker containing the alcoholic drink sample to obtain the ph readings. determination of total soluble solids total soluble solids of the alcoholic drink samples were determined according to the method of [15]. a hand held refractometer (atago's master series, japan) was used. the refractometer was standardized by placing a drop of distilled water on the prism and placed such that it allowed entry of sunlight into the prism. the eye-piece was used to observe the standardization after adjusting the coarse and fine adjustment knob properly. a drop of the sample was placed on the prism of refractometer and the lid was closed. the readings were taken by the graduated mark which indicated the total soluble solid values of the samples and was recorded in obrix. determination of density the method described by [16] was used for the determination of density. an empty graduated cylinder was weighed and the mass recorded. 100ml of each sample was poured into the cylinder and the mass was also recorded. the volume of the sample was gotten by subtracting the mass of the sample from the mass of the cylinder. hence, the density was calculated as: density = mass/volume (g/cm3) determination of alcohol content the alcohol content of the drink samples was determined using the method of [17]. this was established by combining the results of two simple test measurements, that of a refractometer (to record sugar content) and a hydrometer (to determinate the specific gravity). the alcohol content (%) was calculated according to the equation below; where r = refractometer reading, s.g = specific gravity colour profile of the alcoholic drinks the colour profile of indigenous alcohol drinks were obtained using the method described by[18] with the use of adobe photoshop 6.0 software, normalized to l*lightness, a*redness/greenness and b* yellowness/blueness according to following equations (3-5). the colour difference between the indigenous alcohol drinks was determined by taking the euclidean distance between them, according to [19] as shown in equation (6): food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 364 flour sample (cassava/maize/plantain flour) gelatinization (70oc for 20 min) liquefaction liquefaction and saccharification (addition of α-amylase at 70oc for 60 min) (addition of malt at 60oc for 5 hour) saccharification (addition of amylo-glucosidase at 70oc for 60 min) filtration boiling (at 70oc for 5 min) hydrolysate wort cooling (30-33oc) fermentation (addition of yeast and left to ferment for 5 days at 28-30oc) crude alcohol filtration distillation (at 78-80oc) alcoholic drink fig.1. flowchart for the production of alcoholic drink from cassava, maize and plantain flour source: iita (2005) and ocloo and ayernor [10] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 365 sensory evaluation of the alcoholic drinks sensory evaluation was conducted using the modified method of [15]. 50 semi trained panelists made up of student in department of food technology, moshood abiola polytechnic was used. 25 ml each of the alcoholic drinks samples were presented to the panelists and evaluated based on attributes such as color, aroma, appearance, sensation and overall acceptability on a nine (9) point hedonic scale where (1)dislike extremely and (9) like extremely. panelists were instructed to chew cracker biscuits and water between testing of samples. each panelist was provided with enough privacy to avoid biased assessment. statistical analysis all the experimental data were analyzed in triplicate and subjected to analysis of variance (anova) to determine significant differences (p<0.05) using spss 22 software (ibm, usa), while means were separated using duncan multiple range test [20]. 3. results and discussion physicochemical properties of alcoholic drinks produced from plantain, cassava and maize flour the physicochemical properties of alcoholic drinks produced from plantain, cassava and maize flour showed significant differences (p < 0.05) with the exception of density (table 1). earlier study reported that total titratable acidity and ph values are critical parameters to determine the flavor and shelf life of indigenous fermented beverages [21]. table 1 physicochemical properties of alcoholic drinks produced from plantain, cassava and maize flour sample total titratable acidity (%) ph total soluble solid (obrix) density (ml/m3) alcohol content (%) a 3.00f (0.06) 2.30a (0.00) 0.20ab (0.14) 0.99a (0.00) 5.03b (1.05) b 2.08d (0.03) 2.45ab (0.07) 0.10a (0.00) 0.99a (0.00) 4.93b (0.00) c 0.42a (0.05) 2.75c (0.07) 0.15a (0.07) 1.00a (0.00) 3.28a (0.90) d 1.32c (0.05) 2.60bc (0.14) 0.25ab (0.07) 0.99a (0.00) 4.61ab (0.46) e 2.82e (0.11) 2.45ab (0.07) 0.40b (0.14) 0.99a (0.00) 4.06ab (0.10) f 0.96b (0.08) 2.55bc (0.07) 0.20ab (0.00) 0.99a (0.00) 4.24ab (0.16) mean values with different subscripts within a column are significantly different (p < 0.05). a – alcoholic drink produced from plantain flour using imported enzyme, b alcoholic drink produced from plantain flour using local hydrolyzed enzyme, c alcoholic drink produced from cassava flour using imported enzyme, dalcoholic drink produced from cassava flour using local hydrolyzed enzyme, e alcoholic drink produced from maize flour using imported enzyme, falcoholic drink produced from maize flour using local hydrolyzed enzyme the alcoholic drinks produced in this study confirms inverse relationship between total titratable acidity and ph value, i.e. increase in total titratable acidity was relative to decrease in ph value and vice versa. this observation is in agreement with studies food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 366 conducted on some indigenous fermented beverage such as distilled local liquor [21, 22] and tchapalo [23]. in addition, lower percentage of total titratable acidity was estimated in alcoholic drinks produced from cassava flour while that from plantain flour indicated a higher percentage. fermentable sugar naturally present in plantain flour might have favoured rapid metabolism processes to give rise to higher lactic acid [17]. on the other hand, the ph of the alcoholic drinks from plantain and maize flour using local hydrolyzed enzyme demonstrated higher values (less acidic) whereas the use of imported enzyme gave least ph (more acidic). in comparison with previous research studies, higher ph conferring less acidic medium and total titratable acidity values have been detailed on traditional beverages from cassava drink (ph 3.7), maize drink (ph 4.1), plantain drink (ph 3.3) [6] and other liquors [24,25]. in respect to the type of enzyme used, imported enzyme influenced higher total titratable acidity than its’ local hydrolyzed substitute enzyme except for the alcoholic drink produced from cassava. the reduced enzymatic activity of the malt could be attributed to higher substrate concentrations of the flour samples for enzymatic hydrolysis [26, 27]. total soluble solids reflect the available sugar content in a medium and forms part of the important indexes during fermentation process to influence the percentage of alcohol content in alcoholic beverages [23]. the total soluble solid of the alcoholic drinks revealed maize flour (using imported enzyme) gave the maximum obrix level while the minimum degree brix was obtained in plantain drink (using local hydrolyzed enzyme). as with total titratable acidity, imported enzyme influenced higher total soluble solid that the local hydrolyzed enzyme except for the alcoholic drink produced from cassava which shows an inverse relationship. the maximum degree brix observed possibly showed that not all dissolved solids were available for metabolism during fermentation [23]. several authors demonstrated significant correlation between total soluble solid and alcohol content and thereafter suggested that the quantity of alcohol liberated in a fermenting medium depends on the utilization of available soluble solids [15, 23]. these premises are in agreement with the observation of this study, alcoholic drink produced from plantain with the minimum degree brix liberated the highest alcohol content, followed by cassava and maize. similar practical example was established in indigenous distilled liquor (areki), where the observed least total dissolved solids liquor was proportional to higher alcohol content [23]. nonetheless, the light alcohol contents (3.28-5.03%) observed for the drinks produced was in conformity with other local alcoholic beverages reported by several authors; tella: 2-6% [28], maize and banana extract: 3.32-4.63% [17], banana alcohol: < 8% v/v [29], cassava spirits: 7.8-26.3% [6] and kachasu from maize meal: 9-41% v/v [30]. the density of a liquid sample is defined as mass of the liquid per volume [31]. irrespective of the fermenting substrate source (plantain, cassava and maize flours) and type of hydrolyzing enzymes (local and imported) used, the density showed similar values with no significant differences (p < 0.05). in essence, the constant values suggested that the alcoholic drink density was not influenced by and with type of hydrolyzing enzymes. yohannes et al.[31] deduced that density is not an accurate parameter to measure ethanol concentration. however, the ≤ 1 ml/m3 observed for the alcoholic drinks is comparable to similar range previously reported by [16,32] for alcoholic fruit beverages. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 367 color profile of alcoholic spirit drinks produced from plantain, cassava and maize flour the color profile of alcoholic drinks produced from plantain, cassava and maize flour is presented in table 2. reports on local drinks have shown that color test is one of the most preferred quality attribute of beverages [33]. alcoholic drink produced from cassava flour using imported enzyme gave the maximum lightness. similar trend was observed for plantain flour while for alcoholic drink produced from maize flour, local hydrolyzed enzyme gave increased lightness. these variations in lightness could be due to concentration of white mucilage present in the alcoholic drinks after distillation process [34]. the negative a* (greenness) was observed in all the produced alcoholic drinks. similar greenness (-4.30-2.22) was reported for an alcoholic fermented plum beverage [35]. the imported enzyme might have influenced higher greenness than substrate sources as depicted by the values. in respect to negative b* (blueness), the substrate sources had greater effect, wherein, plantain flour alcoholic drink was observed with the highest blueness, followed by cassava and maize flour. the negative values of a* and b* indicated that all the samples were more red and yellow but the yellow parameters showed a higher values which is also event in the values of ∆e which are very close. according to [34], generally low red/green axis and yellow/blue axis on the color plane confers fermented product appearance tending towards grey color. table 2 color profile of alcoholic drinks produced from plantain, cassava and maize flour sample l* a* b* ∆e a 7.51b (0.02) -0.32c (0.01) -0.96a (0.01) 169.71b (0.00) b 7.39a (0.04) -0.44b (0.02) -0.95a (0.02) 169.70a (0.00) c 8.84f (0.02) -0.45ab (0.00) -0.88b (0.01) 169.73c (0.00) d 8.32e (0.03) -0.43b (0.01) -0.67c (0.00) 169.73c (0.00) e 7.66c (0.01) -0.31c (0.01) -0.46d (0.00) 169.73c (0.01) f 7.89d (0.00) -0.48a (0.02) -0.44d (0.02) 169.73c (0.00) mean values with different subscripts within a column are significantly different (p < 0.05). l* lightness, a* redness/greenness, b* yellowness/blueness, ∆e color difference. a – alcoholic drink produced from plantain flour using imported enzyme, b alcoholic drink produced from plantain flour using local hydrolyzed enzyme, c alcoholic drink produced from cassava flour using imported enzyme, dalcoholic drink produced from cassava flour using local hydrolyzed enzyme, e alcoholic drink produced from maize flour using imported enzyme, falcoholic drink produced from maize flour using local hydrolyzed enzyme the color difference within the alcoholic drink showed that cassava and maize flour drinks had same differences but the plantain flour gave the least difference. the total colour change (∆e*) was parameter considered for the overall colour difference evaluation among the alcoholic drinks produced. it could be suggested that the color difference was affected by the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 368 substrate source rather than the hydrolyzing enzymes. sensory evaluation of alcoholic drinks table 3 indicates the sensory evaluation of alcoholic drinks produced from plantain, cassava and maize flour. the multi-sensory experiences including appearance, color, aroma, taste etc. just before or during food/beverage intake depicts the product sensorial attributes [36]. the sensory evaluation of the alcoholic drinks revealed slight significant differences (p<0.05). the appearance of alcoholic drink from plantain flour using imported enzyme had the highest value while cassava flour produced with local hydrolyzed enzyme gave the least value. for aroma, highest score was adjudged to alcoholic drink produced from cassava drink using imported enzyme while plantain flour with same processed enzyme has least score. based on overall acceptability, alcohol drinks produced using imported enzyme was rated the most acceptable whereas the least rating was scored by same substrate using malt. table 3 sensory evaluation of alcoholic drinks produced from plantain, cassava and maize flour sample appearance color aroma taste overall acceptability a 8.00b 7.60ab 6.90a 7.10ab 7.65ab b 7.75b 7.85ab 7.10a 6.95ab 7.35a c 7.80b 8.05b 8.15b 7.75b 8.15b d 6.85a 7.00a 7.15a 6.55a 7.10a e 7.20ab 7.30ab 7.35ab 7.00ab 7.40a f 7.50ab 7.75ab 7.05a 6.85ab 7.25a mean values with different subscripts within a column are significantly different (p < 0.05). a – alcoholic drink produced from plantain flour using imported enzyme, b-alcoholic drink produced from plantain flour using local hydrolyzed enzyme, c alcoholic drink produced from cassava flour using imported enzyme, d-alcoholic drink produced from cassava flour using local hydrolyzed enzyme, e-alcoholic drink produced from maize flour using imported enzyme, falcoholic drink produced from maize flour using local hydrolyzed enzyme 4. conclusion the drinks produced from plantain, cassava and maize flour revealed closely related low acidity, soluble solid, density and alcohol content which imply good substrate for the production of alcoholic drinks. inverse trends relationship between total titratable acidity and ph value was confirmed. higher percentage of acidity in plantain drink informed better alcoholic flavor than the other alcoholic drinks. significant differences (p < 0.05) in color profile of the alcoholic drinks produced from cassava, maize and plantain flour depend on substrate source; the low lightness, negative a* (greenness) and b* (blueness) confers alcoholic drinks with grey color appearance. however consumer preference differs when choosing the most preferred alcoholic drink. alcoholic drink produced from cassava using imported enzyme was highly rated in color, aroma, taste and overall acceptability compared to that produced from maize and plantain flour. further studies should investigate the volatile components of the alcoholic drinks in order to enumerate possible toxic elements or health benefiting compounds that might have liberated during processing. 5. acknowledgments the provision of conducive atmosphere by the management of moshood abiola food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 369 polytechnic, abeokuta, nigeria is duly acknowledged. 6. disclosure statement no potential conflict of interest was reported by the authors. 7. informed consent written informed consent was obtained from all study participants. 8. references [1]. lawrence, j. h., & moore, l. m., united states department of agriculture plant guide. cassava manihot esculenta crantz. usda: washington, dc, http://plants.usda.gov/plant guide/doc/pg_maes.doc (accessed march 24, 2008) (2005) [2]. murugan, k., yashitha, k. s., & al‐sohaibani, s., detoxification of cyanides in cassava flour by linamarase of bacillus subtilis km05 isolated from cassava peels. african journal of biotechnology, 11, 7232-7237(2012) [3]. fao (1992). maize in human nutrition. fao food and nutrition series no.25 1-160. rome: fao. [4]. léder, i., sorghum and millets, in cultivated plants, primarily as food sources. in encyclopedia of life support systems (eolss), (ed.) g füleky (developed under the auspices of the unesco, eolss publishers, oxford, uk (2004) [5]. dadzie, b. k. (1995). cooking qualities of black sigators resistant plantain hybrids. infomusa, 4(2), 7-9. [6]. kubo, r., funakawa, s., araki, s., & kitabatake, n., production of indigenous alcoholic beverages in a rural village of cameroon. journal institute of brewing, 120, 133-141(2014) [7]. obot, i. s., nigeria: alcohol and society today. addiction, 102, 519-522 (2007) [8]. lee, m., regu, m., & seleshe, s., uniqueness of ethiopian traditional alcoholic beverage of plant origin, tella. journal of ethnic foods, 2, 110-114(2005) [9]. von rudloff, a., mogkatlhe, l., & mookodi, g., botswana: summary of nationwide research, in international center for alcohol policies (ed.), producers, sellers, and drinkers: studies of non-commercial alcohol in nine countries. in global actions on harmful drinking; icapmonograph: washington, dc, usa, 13-18(2012). [10]. ocloo, f. c. k., & ayernor, g. s., physical, chemical and microbiological changes in alcoholic fermentation of sugar syrup from cassava flour. african journal of biotechnology, 7(2), 164168(2008) [11]. bothast, r. j. & schlicher, m. a. biotechnological processes for conversion of corn into ethanol. applied microbiology and biotechnology, 67, 19-25. (2005) [12]. ojure, m. a., & quadri, j. a. (2012). quality evaluation of noodles produced from unripe plantain flour. international journal of research and reviews in applied science, 13, 740-752. [13]. coulibaly, a., kouakou, b., & chen, j. extruded adult breakfast based on millet and soybean: nutritional and functional qualities, source of low glycemic food. journal of nutrition and food sciences, 2(7), 1-9.(2012). [14]. iita (2005). integrated cassava project. promoting the cassava industry. www.cassavabiz.org/postharvest/ ethanol01.htm. [15]. wireko-manu, f. d., agyare, a. m., agbnenorhevi, j. k., & oduro, i., development and quality assessment of cassavasweet potato non-alcoholic beverage. moj food processing and technology, 2(3), 1-5(2016) [16]. ebana, r.u.b., edet, u. o., anosike, k. i., etok, c. a., & kanu, t.o. (2019). nutritional analysis and wine production potentials of telfairia occidentalis (fluted pumpkin) leaves and cucumis sativus l. (cucumber) using baker’s and palm wine yeast strains. world news of natural sciences, 22, 12-30.(2019). [17]. pauline, m., alexandre, o., andoseh, b. k., abeline, m. t. s., & agatha, t., production technique and sensory evaluation of traditional alcoholic beverage based maize and banana. international journal of gastronomy and food science, 10, 11-15(2017) [18]. yam, k. l., & papadakis, s. e. a simple digital imaging method for measuring and analyzing color of food surfaces. journal of food engineering, 61, 137-142.(2004) [19]. mariscal, m., & bouchon, p., comparison between atmospheric and vacuum frying of apple slices. food chemistry, 107(4), 1561-1569(2008) [20]. steel, r. g. d., & torrie, j. h., principles and procedure of statistics. (2nd edition). mc graw-hill book co; new york (1980) [21]. worku, b. b., gemede, h. f., & woldegiorgis, a. z. nutritional and alcoholic contents of cheka: a traditional fermented beverage in southwestern ethiopia. food science and nutrition, 6, 2466-2472.(2018). http://www.cassavabiz.org/postharvest/%20ethanol01.htm food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 sunday s. sobowale, olawale p. olatidoye, habibat o. animashaun, ayinde l. odunmbaku, production and comparative assessment of alcoholic drinks produced from cassava, maize and plantain flour using locally produced and imported enzymes, food and environment safety, volume xx, issue 4 – 2021, pag. 360 – 370 370 [22]. alemayehu, h. g. physico-chemical characterization of commercial local alcohol beverages available in south nations, nationalities and people’s regional state, ethiopia. international journal of chemtech research, 11(8), 227-231.(2018) [23]. attchelouwa, c. k., aka-gbézo, s., n’guessan, f. k., kouakou, c. a., & djè, m. k. (2017). biochemical and microbiological changes during the ivorian sorghum beer deterioration at different storage temperatures. beverages, 3(43), 1-13. [24]. bawari, r. a. indigenous processing methods and raw materials of keribo: an ethiopian traditional fermented beverage. journal of food resource science, 2(1), 13-20.(2013) [25]. teshome, d. a., rainer, m., noel, j., schüßler, g., fuchs, d., bliem, h. r., & günther, b. k., chemical compositions of traditional alcoholic beverages and consumers’ characteristics, ethiopia. african journal of food science, 11(7), 234-245(2017) [26]. mojovic, l.,nikolic, s., rakin, m., & vukasinovic, m., production of bioethanol from corn meal hydrolyzates. fuel, 85, 17501755(2006) [27]. ugwu, o. d., hydrolysis of cassava starch by malted acha (digitari aexilis) for bioethanol production. m.sc. thesis, university of nigeria, nsukka, 70(2013) [28]. lee, m., regu, m., & seleshe, s., uniqueness of ethiopian traditional alcoholic beverage of plant origin, tella. journal of ethnic foods, 2, 110-114(2005) [29]. prabir, d., sudipta, d., soumitra, b., & saikat, m., production of banana alcohol and utilization of banana residue. journal of research and engineering technology, 02(10), 15(2013) [30]. motlhanka, k., zhou, n., & lebani, k., microbial and chemical diversity of traditional non-cereal based alcoholic beverages of sub-saharan africa. beverages, 4(36),1-25.(2008) [31]. yohannes, t., melak, f., & siraj, k. preparation and physicochemical analysis of some ethiopian traditional alcoholic beverages. african journal of food science, 7(11), 399403.(2013) [32]. ogodo, a. c., ugbogu, o. c., ugbogu, a. e. and ezeonu, c. s., production of mixed fruit (pawpaw, banana and watermelon) wine using saccharomyces cerevisiae isolated from palm wine. springer plus journal, 4, 683 (2018) [33]. wireko-manu, f. d., ellis, w. o., & oduro, i., production of non-alcoholic beverage from sweet potato (ipomoea batatas l.). african journal of food science, 4(4), 180-183 (2010) [34]. oyedokun, j., badejo, a. a., & enujiugha, v. n. comparison of nutrient and color changes and sensory characteristics of bacillus subtilis and spontaneous fermented parkia biglobosa seed. proceedings of nigeria institute of food science & technology (nifst), 4th nifst western chapter regional food science and technology summit refosts/ annual general meeting, june 6th-7th 2018, federal university of technology, akure, nigeria.(2018) [35]. williams, g., van wyk, j., & hansmann, c., the development of alcoholic fermented beverages utilizing plums with the application of innovative beverage fermentation technology. south african journal of enology and viticulture, 37(2), 199-210 (2016) [36]. redondo, n., gómez-martínez, s., & marcos, a., sensory attributes of soft drinks and their influence on consumers' preferences. food and function, 5, 16861694(2014) 127 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 127 133 in flu ence of di ffer ent c he m ical a ge nt s on the a dul te re d m ilk phys ica l pro per ties corre ct ion *mircea oroian1, daniel bernicu1 1food engineering faculty, stefan cel mare university of suceava, m.oroian@fia.usv.ro *corresponding author received april 7th 2014, accepted may 7th 2014 abstract: in the milk industry, one of the most common frauds is the mixing of milk with water with the goal of improving the quantity. after the water adding to the milk, the counterfitert adds different substances to bring the physical properties in the right range. the aim of this study is to evaluate the influence of adulteration agents on the milk physical properties. for this purpose, the milk was adultered with different percentages of water (0, 5, 10 and 20 % respectively). the milk adultered with 20 % water was mixed with four substantces in order to bring the density and crioscopic temperature in the right range. the substantces used for the density and crioscopic temperature corrections were: nacl, nh4cl, nh4no3 and ch4on2. the nacl, nh4cl, nh4no3 and ch4on2 were mixed with the milk substituted with 20% eater in three different levels (0.25, 0.5 and 1% respectively in the case of nacl, and 0.05, 0.10 and 0.20% respectively in the case of the other adulterants). all the four chemical substances brought the density and crioscopic temperature in the normal range but in different percentages. keywords: milk, adulteration, density, crioscopic temperature 1. introduction one of the most consumed food products in the world is the bovine’s milk. the bovine’s milk contains 3-4% fat [1], and its fat contains 98% triacylglycerols and 2% of other lipids such as diacylglycerols, phospholipids and cholesterol [2]. the milk industry challenges a growing number of frauds, because of the high nutritional value of milk, that have an effect on the process of these products [3]. the principal adulteratios in the case of milk are: the milk dilution with water, fat partial or total removal, skimmed milk powder addition, milk from other species addition, chemical substances addition for neutralisation and conservation, fertilizers addition and dyes addition, respectively [4, 5]. another fraudulent practise in the milk industry happened in 2007 and 2008 when it was found melamine into the milk. these incidents happened in china [6, 7]. the milk density is the ratio of its mass and volume, and is expressed into g/cm3. the milk density is influenced by the temperature, species, diet, the milk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 128 chemical composition, adulteration and cow’s diseases. the cow milk density is ranging between 1.029 – 1.033 g/cm3. in the case of water adding into the milk, the density is decreasing, while in the case of fat removal the density is increasing [4]. another important physical parameter of milk is crioscopic temperature. the crioscopic temperature is defined as the temperature where the milk is freezing. the value of crioscopic temperature is influenced by: the solubile substances concentration (lactose, mineral salts, nitrogen substances which form the neproteic nitrogen), substances added for the acidity reduction (carbonates), substances added for the dry matter increasing, water adding to the milk. the crioscopic temperature ranges between -0.512 and -0.560 °c. the crioscopic temperature should be corrected in function of milk acidity [4]. in the case of milk substition with water are added different substances (nacl, nh4cl, nh4no3 and ch4on2) in order to correct their physical parameters [8]. the aim of this study is to evaluate the influence of adulteration agents on the physical properties of milk. 2. materials and methods 2.1 materials milk (3.5 % fat), distilled water, nacl, nh4cl, nh4no3 and ch4on2. 2.2. methods density determination the milk density was measured using the picnometer method. crioscopic temperature determination the crioscopic temperature was measured using the cryostar i device. 2 ml of sample was placed into the device till the crioscopic temperature is achieved and displayed on the device display. 2.3. milk adulteration the milk was adultered with distilled water in different percentages (0, 5, 10 and 20% respectively). the sample with 20% water was mixed with different concentractions of nacl, nh4cl, nh4no3 and ch4on2. 3. results and discussions 3.1. the water influence on the milk physical properties it is well known that if water is added to the milk, its density is decreasing with the increasing of the water percentage added. in our case the adding of water, in different percentages (ranging from 0 to 20%) decreased the magnitude of the density. in the table 1 is presented the milk density of the milk and of the adulterated samples. table 1. milk density evolution with different percentages od water sample density (g/cm3) blanck 1.030 milk with 5% water 1.027 milk with 10% water 1.025 milk with 20% water 1.021 the substition of milk with water is making that the density to not be in the normal range. if the milk is substituted with 20 % water, the density is decreasing with 0.87 %. the crioscopic temperature of the adulterated milk increased with the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 129 increasing of the water percentage. the increasing of the crioscopic temperature is caused by the dilution of the chemical parameters concentrations. in the table 2 is presented the milk crioscopic temperature of the milk and of the adulterated samples. table 2. milk crioscopic temperature evolution with different percentages od water sample crioscopic temperature (°c) blanck -0.5398 milk with 5% water -0.5220 milk with 10% water -0.4837 milk with 20% water -0.4296 from the data presented in the table 2 we can see that the sample adultered with 5% water has the crioscopic temperature in the normal range, while in the case of the samples with 10 and 20 % water, the crioscopic temperature is not the right range. the addition of 20% water is increasing the crioscopic temperature of milk with 25.65%. 3.2. the influence of nacl on the physical properties of milk adulterated with 20 % water the nacl is added into the milk for masking the water adding into the milk and for the correction of the density. in this paper, it was added nacl to the milk adultered with 20% water in different concentrations: 0.25, 0.5 and 1 % respectively in order to achieve the optimum quantity for the density and crioscopic temperature correction. in the figure 1 is presented the density of the sample adultered with 20% water in which was added different concentrations of nacl. fig. 1. the density of milk adultered with 20% mixed with different concentrations of nacl, a: milk with 20% water, b: milk with 20% water with 0.25% nacl, c: milk with 20% water with 0.50% nacl, d: milk with 20% water with 1.00% nacl the addition of nacl into the adultered milk increased the density, but the quantity of nacl needed for achieving the right density is lower than the quantity studied in the present paper. the quantity should be around 0.1% nacl. the addition of 1% nacl increased the density with 5.38%. fig. 2. the crioscopic temperature of milk adultered with 20% mixed with different concentrations of nacl: a: milk with 20% water, b: milk with 20% water with 0.25% nacl, c: milk with 20% water with 0.50% nacl, d: milk with 20% water with 1.00% nacl 0.95 1 1.05 1.1 a b c d density (g/cm3) sa m pl e -3 -2 -1 0 a b c d crioscopic temperature (°c) sa m pl e food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 130 in the figure 2 is presented the crioscopic temperature of the sample adultered with 20% water in which was added different concentrations of nacl. the addition of nacl into the adultered milk decreased the crioscopic temperature, but the quantity of nacl needed for achieving the right crioscopic temperature is lower than the quantity studied in the present paper. the quantity should be around 0.1% nacl, like in the case of the density. the addition of 1% nacl decreased the crioscopic temperature with 477.4 %. 3.3. the influence of nh4cl on the physical properties of milk adulterated with 20 % water the nh4cl is added, like nacl too, into the milk for masking the water adding into the milk and for the correction of the density. in this paper, it was added nh4cl to the milk adultered with 20% water in different concentrations: 0.05, 0.10 and 0.20 % respectively in order to achieve the optimum quantity for the density and crioscopic temperature correction. in the table 3 is presented the density of the sample adultered with 20% water in which was added different concentrations of nh4cl. the addition of nh4cl into the adultered milk increased the density, it seems that the addition of 0.05% is bringing the milk density into the desired range. the others percentages studied were to big for bringing the density in the normal range. the addition of 0.20% nh4cl increased the density with 1.37%. table 3. the density of milk adultered with 20% mixed with different concentrations of nh4cl sample density (g/cm3) milk with 20% water 1.021 milk with 20% water with 0.05% nh4cl 1.031 milk with 20% water with 0.10% nh4cl 1.034 milk with 20% water with 0.20% nh4cl 1.035 in the table 4 is presented the crioscopic temperature of the sample adultered with 20% water in which was added different concentrations of nh4cl. table 4. the crioscopic temperature of milk adultered with 20% mixed with different concentrations of nh4cl sample crioscopic temperature (°c) milk with 20% water -0.4296 milk with 20% water with 0.05% nh4cl -0.5869 milk with 20% water with 0.10% nh4cl -0.6687 milk with 20% water with 0.20% nh4cl -0.9050 the addition of nh4cl into the adultered milk decreased the crioscopic temperature, but the quantity of nh4cl needed for achieving the right crioscopic temperature is lower than the quantity studied in the present paper. the quantity should be around 0.03% nh4cl. the addition of 0.20 % nh4cl decreased the crioscopic temperature with 110.66 %. 3.4. the influence of nh4no3 on the physical properties of milk adulterated with 20 % water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 131 the nh4no3 is added, like nacl and nh4cl too, into the milk for masking the water adding into the milk and for the correction of the density. in this paper, it was added nh4no3 to the milk adultered with 20% water in different concentrations: 0.05, 0.10 and 0.20 % respectively in order to achieve the optimum quantity for the density and crioscopic temperature correction. in the figure 3 is presented the density of the sample adultered with 20% water in which was added different concentrations of nh4no3. fig. 3. the density of milk adultered with 20% mixed with different concentrations of nh4no3, a: milk with 20% water, b: milk with 20% water with 0.05% nh4no3, c: milk with 20% water with 0.10% nh4no3, d: milk with 20% water with 0.20% nh4no3 the addition of nh4no3 into the adultered milk increased the density, it seems that the addition of 0.05 and 0.10% nh4no3 is bringing the milk density into the desired range. in the case of 0.20% nh4no3 the value of the density is appropriated to the normal value. the addition of 0.20% nh4no3 increased the density with 1.27%. in thefigure 4 is presented the crioscopic temperature of the sample adultered with 20% water in which was added different concentrations of nh4no3. fig. 4. the crioscopic temperature of milk adultered with 20% mixed with different concentrations of nh4no3: a: milk with 20% water, b: milk with 20% water with 0.05% nh4no3, c: milk with 20% water with 0.10% nh4no3, d: milk with 20% water with 0.20% nh4no3 the addition of nh4no3 into the adultered milk decreased the crioscopic temperature, it seems that the addition of 0.05% of this adulterant brings the value in the normal range. the addition of 0.20 % nh4no3 decreased the crioscopic temperature with 73.16 %. 1.01 1.02 1.03 1.04 a b c d density (g/cm3) sa m pl e -0.8 -0.6 -0.4 -0.2 0 a b c d crioscopic temperature (°c) sa m pl e food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 132 3.5. the influence of ch4on2 on the physical properties of milk adulterated with 20 % water the ch4on2 is added, like nacl, nh4no and nh4cl too, into the milk for masking the water adding into the milk and for the correction of the density. in this paper, it was added ch4on2 to the milk adultered with 20% water in different concentrations: 0.05, 0.10 and 0.20 % respectively in order to achieve the optimum quantity for the density and crioscopic temperature correction. in the table 5 is presented the density of the sample adultered with 20% water in which was added different concentrations of ch4on2. table 5. the density of milk adultered with 20% mixed with different concentrations of ch4on2 sample density (g/cm3) milk with 20% water 1.021 milk with 20% water with 0.05% ch4on2 1.032 milk with 20% water with 0.10% ch4on2 1.034 milk with 20% water with 0.20% ch4on2 1.035 the addition of ch4on2 into the adultered milk increased the density, it seems that the addition of 0.05 ch4on2 is bringing the milk density into the desired range. in the case of 0.10% ch4on2 the value of the density is appropriated to the normal value. the addition of 0.20% ch4on2 increased the density with 1.35%. in the table 6 is presented the crioscopic temperature of the sample adultered with 20% water in which was added different concentrations of ch4on2. table 6. the crioscopic temperature of milk adultered with 20% mixed with different concentrations of ch4on2 sample crioscopic temperature (°c) milk with 20% water -0.4296 milk with 20% water with 0.05% ch4on2 -0.4916 milk with 20% water with 0.10% ch4on2 -0.5410 milk with 20% water with 0.20% ch4on2 -0.6794 the addition of ch4on2 into the adultered milk decreased the crioscopic temperature, it seems that the addition of 0.10% of this adulterant brings the value in the normal range. the addition of 0.20 % ch4on2 decreased the crioscopic temperature with 58.14 %. 4. conclusions the milk adulteration is one of the common adulterations of food products. the substitution of milk with water is leading to the modification of the physical parameters. in this paper we studied the influence of different adulterants on the physical properties of the milk substituted with 20% water. the substances used for the density and crioscopic temperature are useful for the correction of the two parameters. all the four adulterants (nacl, nh4cl, nh4no3 and ch4on2) brought the density and the crioscopic temperature in the normal range. for the corrections of the density is needed 0.1% nacl or 0.05% ch4on2, 0.05% nh4no3 or 0.05% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 m ir ce a or oi an , d a nie l b e rn ic u, i n fl u e nc e of d if f e r e n t ch em i c a l ag e nt s on t h e ad ul t er ed m i l k p h y si ca l p r o pe r t i e s c o r r e ct i on , issue 2 2014, pag. 127 133 133 nh4cl. in the case of the crioscopic temperature is needed 0.1% nacl or 0.10% ch4on2 or 0.05% nh4no3 or 0.05% nh4cl for the corrections. the addition of nacl in the milk is not leading to a health issue, but the other three substances must not be presented in the milk. 5. references [1] botaro, b. g., lima, y. v. r., aquino, a. a., santos, m. v., effect of betalactoglobulin polymorphism and seasonality on bovine milk composition, journal of dairy research, 75, 176-181, (2008) [2] garcia, j. s., sanvido, g. b., saraiva, s. a., zacca, j. j., cosso, r. g., eberlin, m. n., bovine milk powder adulteration with vegetable oils or fats revealed by maldi-qtof ms, food chemistry, 131(2), 722-726, (2012) [3] calvano, c. d., de ceglie, c., aresta, a., facchini, l. a., zamboni, c. g., maldi – tof mass spectrometric determination of intact phospolipids as markers of illegal bovine milk adulteration of high-quality milk, analytical and bioanalytical chemistry, 405, 1641-1649, (2013) [4] banu, c., bulancea, m., ianiţchi, d., bărăscu, e., stoica, a., industria alimentară între adevăr şi fraudă, ed. asab, bucureşti, (2013) [5] kartheek, m., smith, aa., muthu, a.k., manavalan, r., determination of adulterants in food: a review, journal of chemical and pharmaceutical research, 3, 629-636, (2011) [6] moore, j. c., spink, j., lipp, m., development and application of a database of food ingredient fraud and economically motivated adulteration from 1980 to 2010, journal of food science, 77, 118-126, (2012) [7] santos, p. m., pereira-filho, e.r., rodriguez-saona, l. e., rapid detection and quantification of milk adulteration using infrared microscpectroscopy and chemometrics analysis, food chemistry, 138(1), 19-24, (2013) [8] oroian, m., autentificarea produselor alimentare şi depistarea falsurilor, ed. performantica, iaşi, (2014) doi: https://doi.org/10.4316/fens.2021.021 189 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3-2021, pag. 189 196 extraction and characterization of resveratrol from vegetable waste from vines *petronela gabriela nistor 1 , sonia amariei 2 1faculty of food engineering, ”stefan cel mare„ university, suceava, românia, nistor.gabi.petronela.96@gmail.com, *corresponding author received 20th june 2021, accepted 25th september 2021 abstract: vine shoots resulting from vine pruning can become valuable products of interest for many industries. research conducted to date showed that resveratrol is found in many parts of the vine. the aim of the present study was to determine the resveratrol content in romanian vine sprouts samples by direct injection into the hplc instrument coupled with diode array detector after being filtered through 0.45 μm ptfe membrane filters. the resveratrol content varied from 5.44 mg/100 g to 10.27 mg/100 g in ethanolic extracts. for aqueous extract, the resveratrol was found in only two samples in very low concentration. thise paper aimed to contribute to the research on the capitalization of residual products from viticulture industry. keywords: sprouts, antioxidant capacity, hplc, polyphenols, trans-resveratrol 1. introduction resveratrol is one of the most well-known and studied polyphenols today. resveratrol is a compound belonging to the stilbenoid class [1-2]. this compound generates so many benefits for the body that one can talk about a miracle of nature. resveratrol has many biological properties, but the most common biological activity is antioxidant activity. these properties have been used to protect cells against oxidative stress [3-4]. this polyphenols has antibacterial activity, has an anti-inflammatory role, fights diabetes, has an anticancer effect and fights against aging [5]. the importance of this compound is highlighted in several industries, including the food industry, the pharmaceutical industry and the medical industry[6].with a very wide range of biological activities on human body, alternatives are currently being sought out to obtain resveratrol, even from vegetable waste resulting from pruning[7-8]. the waste resulting from the pruning of the vine is a rich source of compounds with phytochemical value, but these are often not recovered[9-10].during the summer cuttings, a considerable amount of green mass is removed, such as portions of different sizes of shoots and leaves[1112].resveratrol is available in various concentration in these by-products of pruning, and can be used in various fields of interest for this compound [13].among the many benefits to human health the following are included: the fight against free radicals through its antioxidant activity, protecting the heart and cardiovascular system, activating the action of sirtuins that fight against aging, increasing nitric oxide production, preventing platelet aggregation, causing http://fens.usv.ro/index.php/fens food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 190 blood clots in the blood vessels[14], decrease in the frequency of polydipsia and preliphagia in patients with diabetes [15]. 2. matherials and methods the trans-resveratrol standard (99 gc) was purchased from sigma-aldrich (germany), and methanol and acetonitrile were purchased from merk (merk kgaa, darmstadt, germany). in order to extract resveratrol, vine shoots harvested from a crop located in mălini, suceava country, were used.the plant material was dried in an oven at 45 οc and grounded with a centrifugal mill. to determine resveratrol, 1 g of vine powder was mixed in 6 vials with 20 ml of ethanol (for each vial separately), and in another 6 vials the mixture was made with 20 ml of water. the concentration of ethyl alcohol used was 80%. the 12 vials were exposed to ultrasound treatment at diffrent amplitudes (30, 40, 50, 60, 70, 99%), at a temperature above 50 οc, pulsation of 0:0, for 30 minutes. after the ultrasound treatment, the samples were centrifuged for 10 minutes at 3200 rpm and filtered using qualitative filter paper and stored at -18οc. 2.1 determination of the total polyphenol content by folin ciocâlteu method thus, in 12 test tubes labeled according to the established codes, the following substances were added: 0.2 ml of vine mixture; 2 ml of folin ciocâlteu reagent (1:10 g/g) and 1.8 ml of na2co3 (7.5%). the samples obtained were mixed very well and stored in the dark for 30 minutes. the absorbance was measured at a wavelength of λ=750 nm, using the uvvisnir 3600 shimadzu spectrophotometer (shimadzu corporation, japan). 2.2 determination of the antioxidant activity of the vine extract by the dpph method the measurement of the antiradical capacity of the vine shoots was performed by using the stable free radical dpph. the following substances were added to 13 tubes: 0.5 ml of extract, 0.5 ml of 80% methanol and 5 ml of dpph reagent.the tubes were shaken very well and stored in the dark for 30 minutes. the wabsorbance of the samples was measured using a uv-vis-nir 3600 shimadzu spectrophotometer, at the wavelenght of λ=517 nm. the following formula was used to determine the antioxidant activity of vine shoots: 𝑅𝑆𝐴% = 𝐴𝑏𝑙𝑎𝑛𝑘 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑙𝑎𝑛𝑘 × 100 where rsa is the radical scavening activity, asampleis the absorbance of the analyzed sample at 517 nm and ablankis the absorbance of the blank at 517 nm. 2.3 determination of resveratrol by high performance liquid chromatography in order to perform the analysis, a set of dilutions were prepared from the stock solution: 0.025; 0.05; 0.075; 0.1; 0.5; 1; 2.5; 5; 10; 20 and 25 mg/l, respectively. the purified and filtered samples were injected into the hplc device in an amount of 8 μl, and for analysis an spdm-20 a diode array detector was used. separation was performed on a phenomenez kinetex 2.6 μm biphenyl 100 å hplc column 15x4.6 mm thermostated at 200 οc. elution was performed with a solvent system consisting of pure water (a) and acetonitrile (b). the solvent flow rate was of 0.5 ml/min. 2.4 determination of ash content of vine shoots food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 191 to determine the ash content, two samples were tested in parallel. in two clean, dry and previously weighed porcelain crucibles, a quantity of 5 g of vine grind was introduced, in a layer as uniform as possible. after weighing, the crucibles were placed in the calcination furnace. the calcination was performed at a temperature of 550 οc, for 9 hours, with the temperature being gradually raised in the calcination furnace. the ash content determined was expressed as percentage to two decimal places and calculated according to the formula: 𝐴𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡, % = 𝑚2 − 𝑚 𝑚1 − 𝑚 𝑥100 where m2 is the crucible weight with calcined sample, m1 is the crucible weight with uncalcined sample and the m is the weight of the empty crucible. 2.5 determination of mineral content by energy dispersive x-ray spectrometry in order to performe this analysis, a shimadzu x-ray energy dispersive spectroscope (edx; shimadzu corporation, japan) equipped with test tanks and a pc with the edx software application was used. to determine the mineral content of the vine shoots, two samples were calcined in parallel in the calcination furnace, thus finally as a percentage, the calcination being done automatically using the edx software application. 2.6 determination of copper by flame atomic absorption spectroscopy the device used for analysis was a flame atomic absorption spectrophotometer aa shimadzu (shimadzu corporation, japan), and the wave length at which the copper was read was 324.07 nm. after tracing the calibration curve, the absorbance of two samples of the vine extract was measured. a cu cavity cathode lamp was used as the radiation source.the winlab 32 application automatically calculated the copper concentrations. the concentration determined as mg/l was multiplied by the corresponding dilution. 3. results and discussion 3.1 total polyphenol content the calculation of the total polyphenols in the vine shoots was performed using the calibration curve, and the results were expressed as mg gallic acid (gae)/100 g vegetable product (sample) (fig. 1). fig. 1 calibration curve for total polyphenol content in vine shoots 0.092 0.269 0.511 0.754 1.01 1.907 y = 0,0095x + 0,0295 r² = 0,9987 0 0.5 1 1.5 2 2.5 0 25 50 75 100 125 150 175 200 225 a b so r b a n c e 7 5 0 n m concentration mg/l food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 192 the results of the total polyphenol content of the first 6 vine samples in which water was used as solvent are shown in fig. 2. the figure shows that the highest total polyphenol content was determined in sample 6, which was obtained by ultrasound treatment at 70% amplitude for 30 minutes, and the value of the resveratrol content was 62.80 mg gae/100 g. samples that were exposed to ultrasound at amplitudes of 50, 40 and 30% had average values of the content of total polyphenols of 30.84, 33.11 and 31.20 mg gae/100 g vegetable product.another value that was close to the maximum value of sample 6 was that determined in sample 2, which was extracted at an ultrasound amplitude of 60% and had a polyphenol content of 47.70 mg gae/100 g plant product.figure 2 shows that the sample that was extracted at 99% ultrasound amplitude had the lowest value of total polyphenol content, which was of 29.05 mg gae/100 g plant product.regarding the total polyphenols content that was determined in the case of ethanolic extractions, in was observed that these values were higher, as follows: for samples c (50:30), d (40:30), e (30:30), f (70:30) and b (60:30) the values of total polyphenol content were 49.50 mg gae/100 g; 55.06 mg gae/100 g; 57.36 mg gae/100 g; 62.80 mg gae/100 g and 67.52 mg gae/100 g (fig. 3). fig. 2 total polyphenol content of vine extracts (aqueous extraction) fig. 3 total polyphenol content of vine extracts(ethanolic extraction) by analyzing the results obtained for the two types of extraction, it can be concluded that the samples in which ethanol was used as solvent, had higher 29.05 47.70 30.84 33.11 31.20 62.80 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 pb 1 (99:30) pb 2 (60:30) pb 3 (50:30) pb 4 (40:30) pb 5 (30:30) pb 6 (70:30) 38.47 67.52 49.50 55.06 57.36 62.80 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 pb a (99:30) pb b (60:30) pb c (50:30) pb d (40:30) pb e (30:30) pb f (70:30) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 193 values of the total polyphenol content than the samples in which water was used as solvent. results closer to the total polyphenol content determined in romanian vine shoots were obtained by llobera, in 2012 [6].following the extractions performed with ethanol/water solvent and acetone/water, the highest value was identified in the shoots samples coming from a vine crop on the island of mallorca, spain (38,4 mg gae/100 g vegetable product). the total polyphenol content of 29.4 mg gae/100 g plant product was identified in the 80% ethanol extraction of red grape shoots, and 22.9 mg gae/100 g was determined in the acetone extractions 70% and 80% ethanol of the shoots, respectively. 3.2 antioxidant activity by the dpph method regarding the antioxidant activity, in the case of aqueous extraction the highest percentages were identified for samples 4 and 5 (27.56%), followed by sample 3 (27.01%). the lowest value was obtained for sample 2, and it was 25.46% (fig. 4). the antioxidant activity of the samples in which ethanol was used as solvent showed the highest value in sample c (28.18%). samples d and f also presented high values, which were of 28.11% and 28.14%. the lowest antioxidant activity was determined in the case of sample b, and it was 27.97% (fig. 5). fig. 4 antioxidant capacity of vine shoots studied using the dpph method, %(aqueous extract) fig. 5 antioxidant activity of vine shoots studied using the dpph method, %(ethanolic extract) 26.11 25.46 27.01 27.56 27.56 26.42 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 pb 1 (99:30) pb 2 (60:30) pb 3 (50:30) pb 4 (40:30) pb 5 (30:30) pb 6 (70:30) 27.97 27.94 28.18 28.11 27.97 28.14 27.80 27.85 27.90 27.95 28.00 28.05 28.10 28.15 28.20 pb a (99:30) pb b (60:30) pb c (50:30) pb d (40:30) pb e (30:30) pb f (70:30) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 194 3.3 resveratrol content determined by hplc the standard calibration curve obtained showed high degrees of linearity (r>0.99). the concentration was determined from the equation of the curve in the samples of vine shoots: y=117661x, and the absorbance at which the resveratrol content was measured was 306 nm (fig. 6). fig. 6 calibration curve for resveratrol content expressed as mg/l the concentration of trans-resveratrol varied from 5.44 mg/100 g to 10.27 mg/100 g for ethanolic extracts, but in the case of aqueous extractsresveratrol was determined only in two samples, where its concentration was of 0.11 mg/100 g(sample 6) and 0.67 mg/100 (sample 2). the highest trans-resveratrol content was cuantified in sample a (10.27 mg/100 g plant product), followed by sample f (9.36 mg/100 g). mean values of trans-resveratrol content were identified in samples b (5.44 mg/100 g), c (5.80 mg/100 g ), d (6.09 mg/100 g), and e (5.50 mg/100 g).resveratrol concentration similar to those determined in the analyzed vine shoots was also obtained by ghaewalova et al., 2018 [7]. the trans-resveratrol content of the shoots in the czech republic varied between 3.13 mg/100 g and 13.23 mg/100 g. 3.4 ash content of vine shoots following the calculation performed for the two samples of vine shoots, an ash content of 2.7035 was obtained in the case of the first sample and 2.894 g for sample number 2. 3.5 minerals concentration in vine shoots a light-dispersive x-ray spectrometer, the shimatzu 900 hs model, was used to analyze the ash samples. the concentrations of minerals determined are presented in table 1. by analyzing the results it can be observed that in the two samples the following elements were found: calcium (ca), potassium (k), phosphorus (p), sulfur (s), iron (fe), scandium (sc), manganese (mn), strontium (sr), zinc (zn), chromium (cr) and copper (cu). among the studied elements, calcium (ca) had the highest value of 52.37% for sample 1 and 55.70% for sample 2. another element that predominated in vine shoots was potassium (k) with the values of 35.53% and 33.08% respectively, followed by phosphorus (p) and sulfur (s), for whichthe recorded values were 6.86% and 3.00% and 6.29%, 2.56% for sample 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 195 table 1 concentration of metals selected from samples 1 and 2 of vine shoots analyte sample 1 sample 2 ca 52.375 % 55.701 % k 35.537 % 33.081 % p 6.861 % 6.290 % s 3.002 % 2.564 % fe 0.688 % 0.658 % sc 0.680 % 0.811 % mn 0.363 % 0.385 % sr 0.253 % 0.250 % zn 0.130 % 0.127 % cr 0.062 % 0.080 % cu 0.049 % 0.054 % 3.6 copper content of vine shoots the calculation of the copper content in the vine shoots was performed using the calibration curve, and the results were expressed as mg cu/l. the absorbance of copper was read at the wavelength λ=324,87 nm (fig. 8). for the first sample a copper content of 0.5675 mg/l was determined, and for the second sample the copper content was 1.2569 mg/l. the copper concentration obtained from the analysis of the vine shoots from suceava county proved to be relatively lower than the concentration from the shoots from other areas of the country. the values recorded for the copper concentration in the vine shoots coming from the iași vineyard varied in a rather large range (25.17 mg/kg – 37.47 mg/kg), by comparisson to those obtained in the murfatlar vineyard, where in the vine shoots it was determined a copper content of 45.84 mg/kg for the variety sauvignon blanc, and the ștefănești argeș vineyard (fetească regală 38.47 mg cu/kg) and târnavelor vineyard where a copper content of 31.84 mg cu/kg was determined in shoots of the variety sauvignon blank[8]. fig. 7 – absorption spectrum for copper 4. conclusion the experimental results showed that the extraction of total polyphenol was influenced by the type of solvent used and the conditions of the ultrasound treatment applied. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 petronela gabriela nistor, sonia amariei, extraction and characterization of resveratrol from vegetable waste from vines, food and environment safety, volume xx, issue 3 – 2021, pag. 189 – 196 196 it was found that following aqueous extraction, the values recorded for the content of total polyphenols were much lower than those obtained in the case of ethanolic extraction.the antioxidant activity of the studied vine shoots proved to be relatively lower, with the values recorded for aqueous extraction varying in a range between 25.46 and 27.56%, and those determined for ethanolic extractions from 27.96% to 28.18%.when compared to previous studies, these values were found similar to those obtained for vine shoots in the czech vineyard.the highest tras-resveratrol content was identified in sample a (10.27 mg/100 g plant product), followed by sample 7 with a content of 9.36 mg/100 g, and the mean values were identified in samples b (5.44 mg/100 g), c (5.80 mg/100 g), d (6.09 mg/100 g) and e (5.50 mg/100 g). the conclusion drawn from this analysis was that even in the case of resveratrol the, ethanolic extraction was much more advantageous than water extraction.the mineral content determined indicated that the samples of vine shoots represented a matrix rich in calcium, potassium and phosphorus, the most abundant being calcium with a value between 52.37% and 55.70%. the copper concentration obtained from the analysis of the vine shoots from suceava county proved to be relatively lower by comparison to the concentrations detected in the vine shoots from other areas of the romania. 5. references [1]. triska j., houska m., physical method of resveratrol induction in grapes and grape products –a review,czech i. food sci., 30: 489-502, (2012). [2]. keylor m. h., matsuura b. s., stephenson c. r. j., chemistry and biology of resveratrol-derived natural products, chemical reviews, 115: 8976-9027, (2015). [3]. fabjanowicz m., plotkawysylka j., namiesnik j., detection, identification and determination of resveratrol in wine. problems and challenges, trends in analytical chemistry, 103: 21-33, (2018). [4]. kiselev k. v., perspectives for production and application of resveratrol, applied microbiology and biotechnology, 90: 417-425, (2011). [5]. salehi b., mishra a. p., nigam m., sener b., kilik m., sharifi-rad m., fokou p. v. t., martins n., sharifi-rad j., resveratrol: a double-edged sword in health benefits, biomedicines, 6: 2-13, (2018). [6]. llobera a., study on the antioxidant activity of grape stems (vitis vinifera). a preliminary assessment of crude extracts, food and nutrition sciences,3: 500-504, (2012). [7]. ghaerwalova l., hutar d., masak j., kolouchova i., antioxidant activity and phenolic content of organic and conventional vine cane extracts, food analysis, food quality and safety, 36 (4): 289-295, (2018). [8]. https://docplayer.ro/145228002-microsoftpowerpoint-prezentare-faza-v-aderpptx.htmlacccessed june. 24, 2021. [9]. yang t., wang l., zhu m., zhang l., properties and molecular mechanisms of resveratrol: a review, pharmazie, 70: 501-506, (2015). [10]. silva p., sureda a., tur j., a., andreoletti p., cherkaoui-malki m., latruffe n., how efficient is resveratrol as an antioxidant of the mediterranean diet, towards alterations during the aging process?, free radical reasearch, 53 (1): 1101-1112, (2019). [11]. senila b., neag e., torok i., cadar o., kovacs e., țenu i., roman c., vine shoots waste – new resources for bioethanol production, romanian biotechnological letters, 25 (1): 1253-1259, (2020). [12]. ratz-lyko a., and arct j., resveratrol as an active ingredient for cosmetic and dermatological applications: a review, journal of cosmetical laser therapy, 2-8, (2018). [13]. mironeasa i., and mironeasa s., application of heat moisture treatment in wheat pasta production, food control, 128: 2-18, (2021). [14]. lai j., chiu j., m., chiou r. y. y., fresh preservation of alfa sprouts and mushroom slices by soaking with thymol and resveratrol solution, food sciences and nutrition, 5: 776-783, (2017). [15]. david g., michel j., gastaldi e., gontard n., angellier-coussy h., how vine shoots as fillers impact the biodegradation of phbv-based composites, international journal of molecular science, 21 (228): 2-13, (2020). https://docplayer.ro/145228002-microsoft-powerpoint-prezentare-faza-v-ader-pptx.html https://docplayer.ro/145228002-microsoft-powerpoint-prezentare-faza-v-ader-pptx.html https://docplayer.ro/145228002-microsoft-powerpoint-prezentare-faza-v-ader-pptx.html 1. introduction применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 contents: 1. influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread iryna medvid, olena shydlovska, tetiana ishchenko 172 181 2. physico-chemical parameters of eggplants (solanum aethiopicum l.) according to conservation techniques deffan zranseu ange bénédicte, yapi jocelyn constant, fagbohoun jean bedel, masse diomandé, beugre grah avit maxwell 182 188 3. extraction and characterization of resveratrol from vegetable waste from vines petronela gabriela nistor, sonia amariei 189 196 4. healthy food for teenagers part 2: meat analysis for hormonal drugs marianna barus, vita antsupova, yuliia lomakina 197 204 5. physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation vlasios goulas, polina aresti 205 210 6. hydrogeochemical characterization and quality of groundwater in the sebseb region, ghardaïa province (southern algeria) salim boubelli, nabil bougherira, djamel rihia, hicham chaffai 211 223 7. cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria sofia bahroun , samira gheid, warda boumaraf 224 234 8. evaluation of surface water quality and contamination status of the zeremna valley sub-basin in the skikda region (north-eastern of algeria) nadjet zair, badra attoui, abd elmonem miloudi, abd erahman khachkhouche 235 246 9. extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani 247 255 10. antioxidants’ effect on the storage of dairy products with high-fat content olena grek, tetiana pshenychna, mariia nikolaieva 256 261 11. evaluation of mexa dam water quality in el tarf region (extreme north-east algeria) samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, 262 – 273 12. determination of trace levels of nickel(ii) by cyclic voltammetry with spes from food products liliana anchidin-norocel, sonia amariei, gheorghe gutt 274 281 13. study on alcohol consumption among young people in suceava county, romania mihaela jarcău, maria poroch – seriţan, matei jarcău 282 – 288 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 14. removal of toxic compounds from natural extracts using green techniques francisco lorca, david quintín, maría-dolores lópez, presentación garcía 289 297 15. author instructions i v 16. subscription information vi 69 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 69 75 physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder * amal messaoudi 1 , djamel fahloul 1 1department of food technology, food sciences laboratory (lsa), hadj lakhdar batna 1 university, batna, algeria,messaoudi_amel@live.fr, *corresponding author received 8th march 2020, accepted 30th march 2020 abstract: bread is the principal cereal product. date pomace is a by-product of the date syrup industry. the main objective of this work is to evaluate physico-chemical and sensory properties of barley bread supplemented by freeze dried date pomace powder of garn ghzel date variety. barely bread was prepared after incorporation of 0, 5, 10, 15 and 20% of freeze dried date pomace powders. the results show that the rate of incorporation of date pomace powder has a significant effect on physico-chemical (thickness, weight before and after cooking, volume, specific volume, moisture, dry matter, ash, ph, wet gluten, dry gluten and color) and the sensory properties of barley bread in terms of appearance/shape, crust, brown color, chewing, date aroma, general acceptability. all barley bread samples were acceptable; however, the samples fortified by 20% of freeze dried date pomace powders are the most preferred. findings showed that good and acceptable barley bread could be produced using freeze dried date pomace powder at different levels. keywords: barely bread, freeze drying, date pomace powder, physico-chemical properties, sensory properties. 1. introduction bakery products are consumed all over the world [1]. baked products are gaining popularity because of their availability, ready to eat convenience and reasonably good shelf life [2]. common bakery products include biscuit, muffin, cake, bread, pastries and pies [3]. bread is one of the commonest staple food eaten world over [4]. it is the major baked product that is widely acceptable and consumed as a very convenient form of food worldwide; however, there are increasing demands toward consumption of high fiber breads due to their health prompting properties. it is a good source of nutrients, such as macronutrients (carbohydrates, protein and fat) and micronutrients (minerals and vitamins) that are all essential for human health [5]. barley (hordeum vulgare) is a widely consumed cereal important for human nutrition. approximately 80-90% of the barley harvest is used as animal feed and to produce malt. however, barley is now receiving renewed interest as an ingredient in functional foods due to its high concentrations of bioactive compounds such as β-glucans and polyphenols. additionally, consumption of barley is associated with lower total and serum cholesterol, improved postprandial glucose and insulin response and reduced incidence of heart disease and cancer [6]. date pomace is a by-product of date syrup industry [7]. however, information on incorporation of date pomace powder in barley bread is unusual. http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 70 the use of fruit and vegetable by-products as natural food additives has recently been suggested, due to their high content of polyphenols, carotenoids, dietary fiber, or other bioactive compounds [8]. the objectives of this work were to study the valorization of date pomace powder of garn ghzel date variety by incorporation into barley bread product and to determine its physico-chemical and sensory properties. 2. materials and methods preparation of date pomace powder the mixture was stirred for 20 min at 60°c in a water bath, (dk-420, china). finally, the juice was extracted through a muslin cloth. left material after juice extraction is called date pomace. it was freeze dried in a laboratory freeze dryer model (ld 28 christ beta plus, germany) for 24 hours at a pressure and temperature of 0.12 mbar and -40°c, respectively. samples were ground and sieved in a sieve of 1 mm diameter [7]. barley bread preparation the barley bread formula was adapted from the recipe of ndangui (2015) [10] with some modifications. barley bread formula was shown in table 1. date syrup was prepared according to alfarsi (2003) [9] method with a slight modification: a chopper was used to cut dates into pieces of 1 cm, and then mixed with an equal amount of distilled water. table 1. enriched barley bread formulation ingredient (g) control bbdpp5 bbdpp10 bbdpp15 bbdpp20 barley flour 100 95 90 85 80 date pomace powder 0 5 10 15 20 yeast 3.0 3.0 3.0 3.0 3.0 salt 2.0 2.0 2.0 2.0 2.0 margarine 5 5 5 5 5 water 60 60 60 60 60 control, bbdpp5, bbdpp10, bbdpp15 and bbdpp20 barley bread: prepared with 0%, 5%, 10%, 15% and 20% of date pomace powder, respectively. physicochemical properties of barley bread according to mohd. jusoh et al., (2008) [11] with some modifications, the thickness of barley bread was measured using a digital vernier caliper with a precision of up to 0.01 mm. the bread weight was determined according to olubunmi et al., (2015) [5] with some modifications, weighing the bread before and after cooking, using the laboratory scale (kern, imlab france) and the readings recorded in grams. the barley bread volume was determined by using rapeseed displacement method. this was done by loading millet grains into an empty box with calibrated mark until it reached the marked level and unloaded back. the bread sample was put into the box and the measured millet was loaded back again. the remaining millet grains left outside the box was measured using measuring cylinder and recorded as loaf volume in cm3 [5]. the flow chart for the production of barley bread is presented in fig. 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 71 fig.1. flow chart for the production of barley bread based on freeze dried date pomace powder the specific volume (volume to mass ratio) (cm3/g) was thereafter calculated. the specific volume (volume/mass ratio) (cm3/g) was calculated as follows: (1) according to sassi (2008) [12] with some modifications, gluten extraction was obtained by mechanically kneading flour dough and washing with a buffered nacl solution and then squeezing and weighing the residue (aacc 38.12, icc standard 137). gh% = pg × 10 (2) gh: wet gluten (retained) pg: gluten weight. the total gluten was dried in a hot plate for 4 minutes and then weighed [12]. the color of barely bread was determined using minolta chroma meter. color value is based on l* (lightness–darkness), a* (redness–greenness) and b* (yellowness– blueness) according to the method of rosa et al. (2015) [13] with some modification. sensory evaluation the determination of acceptability level was based on appearance/form, crust, brown color, chewiness, date aroma and overall acceptability were conducted using a 9-point hedonic scale (9=liked very much, and 1= disliked very much) [5]. statistical analysis all analysis was performed with three repetitions, while color with six and the results were expressed as the mean ± standard deviation (sd). results were submitted to the analysis of variance (anova) and the means were compared with tukey (hsd) test using xl stat 2016. differences were considered to be significant at p<0.05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 72 3. results and discussion physico-chemical characteristics of barley bread changes in barley bread enriched with date pomace powder are shown in table 2. table 2. physico-chemical characteristics of barley bread prepared with date pomace powder control bbdpp5 bbdpp10 bbdpp15 bbdpp20 thickness (cm) 6.4±0.4a 5.73±0.23ab 5.33±0.23b 5.33±0.23b 5.07±0.23b pre-cooking weight (g) 160.21±0.55c 170.85±0.05a 159.64±0.02d 161.32±0.00b 153.27±0.06e post-cooking weight (g) 130.35±0.32e 132.97±0.02c 140±0.00a 136.12±0.08b 130.99±0.18d volume (cm3) 44.75±0.25bc 44.33±0.58c 44.08±0.14c 45.5±0.5ab 46±0.00a specific volume (cm3/g) 0.35±0.00b 0.33±0.00c 0.31±0.00d 0.33±0.00c 0.35±0.00a moisture (%) 40.81±0.96b 39.90±0.52b 45.64±1.98a 41.75±0.30b 40.23±1.02b dry matter (%) 59.19±0.96a 60.10±0.52a 54.36±1.98b 58.25±0.30a 59.77±1.02a ash (%) 3.81±0.00a 2.97±0.01a 2.05±0.00a 1.51±0.01a 0.93±1.16a ph 6.03±0.02ab 5.94±0.02c 5.94±0.02c 6.04±0.04a 5.97±0.02bc wet gluten (%) 29.77±0.23a 27.63±0.13b 24.87±0.10c 19.32±0.06d 14.72±0.20e dry gluten (%) 9.09±0.8a 8.24±0.3b 8.16±0.04b 5.65±0.12c 4.41±0.08d control, bbdpp5, bbdpp10, bbdpp15 and bbdpp20: prepared with 0%, 5%, 10%, 15% and 20% replacement of barley flour with date pomace powder, respectively. results are expressed as mean values of three determinations ± sd; analysis of variance (anova) p<0.05 with tukey (hsd) tests. the thickness of enriched barley bread decreases with the increase in the rate of incorporation of freeze-dried date pomace powder, it is higher for the control with a value of 6.4 ± 0.4 cm and lower for bbdpp20 with 5.07 ± 0.23 cm. significant differences observed between enriched breads weight (before and after cooking) with values varied from 153.27 ± 0.06 to 170.85 ± 0.05g and from 130.35 ± 0.32 to 140 ± 0.00g respectively. the volume of the loaves varies from 44.75 ± 0.25 to 50.67 ± 0.58 cm3 and the specific volume ranges from 0.31±0.00 to 0.35±0.00 (cm3/g). according to ndangui (2015) [10], bread volume variation can be attributed mainly to different levels of carbon dioxide production and the degree of starch gelatinization. the results obtained are similar to those of ndangui (2015) [10], for which the volume of bread decreases with the increase in the sweet potato flour substitution rate. the water content of barley breads enriched with freeze-dried date pomace powder ranges from 39.90 ± 0.5 to 45.64 ± 1.98%. these results are superior to those of obiegbuna et al. (2013) [4] who studied the influence of sugar replacement by date pulp flour on physicochemical, organoleptic properties and on bread preservation. the difference in results is due to the difference in the preparation of bread. according to the results obtained the ash content decreases with the increase in the concentration of date pomace powder (dpp) from 3.81 ± 0.00 to 0.93 ± 1.16%. obiegbuna et al. (2013) [4] obtained similar results. the ph of different breads varies from 5.94 ± 0.02 to 6.04 ± 0.04, the results obtained is in agreement with those reported by semić et al. (2010) [14]. according to ouazib (2017) [15], the gas retention capacity depends on the quantity and especially the quality of the gluten to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 73 form a paste that can retain the released co2. wet gluten extracted from wheat flour is a viscoelastic substance consisting mainly of the insoluble fraction of proteins (gliadins and glutenins). it has properties of extensibility, elasticity and toughness that can influence the behavior of the pasta in production and the quality of the finished product (bread, biscuit, ...) [12]. the wet and dry gluten of barley breads produced is high for control with 29.77 ± 0.23 and 9.09 ± 0.8% and low for bbdpp20 with 14.72 ± 0.20, 4, 41 ± 0.08%. these results are consistent with those obtained by sassi (2008) [12] for whole semolina and halaby et al. (2014) [16] for different wheat flour. the breads enriched with different concentrations of date pomace powder (5-20%) are characterized by: a proportional difference in the different brightness (l*), redness (a*) and yellowing (b*) parameters of different fortified breads (fig. 2); fig. 2. color measurements of barley bread product with different proportions of date pomace powders: 0% (control), 5% (bbdpp5), 10% (bbdpp10), 15% (bbdpp15) and 20% (bbdpp20) the results obtained are influenced by the amount of date pomace powder incorporated and the color of the barley flour. these values are compared with those of ishida and steel (2014) [17] for bread samples available on the brazilian market. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 74 sensory evaluation web diagram for mean sensory scores of enriched date pomace powder barley bread was presented (fig. 3). according to the sensory evaluation, the bbdpp20 sample has the highest score (appearance/shape, crust, brown color, chewingness and date aroma). the global acceptability of all samples followed the same trend. thus, the sample containing 20% of lyophilized date pomace powder is the most preferred. fig. 3. diagram for mean sensory scores of date pomace powder barley bread with different proportions of date pomace powders: 0% (control), 5% (bbdpp5), 10% (bbdpp10), 15% (bbdpp15) and 20% (bbdpp20) 4. conclusion the results of this study showed that it is possible to develop barley bread with garn ghzel date pomace powder. the incorporation of freeze dried date pomace powder in the formulation of barley bread was found to improve physicochemical and sensory properties of samples. barley bread prepared at incorporation rates of 5%, 10%, 15% and 20% are all acceptable in terms of global acceptability, appearance/shape, crust, brown color, chewiness and date aroma. barley bread with 20% of date pomace powder was the most preferred. 5. acknowledgements the authors thank all the staff of food sciences laboratory (lsa), department of food technology, hadj lakhdar batna 1 university. 6 . references [1]. lu t.m., lee ch.ch., mau j.l., lin sh.d., quality and antioxidant property of green tea sponge cake, food chemistry, 119: 1090-1095, (2010). [2]. vijayakumar m.c., peter d., bobde h., john s.m., quality characteristics of cookies prepared from oats and finger millet based composite flour, engineering science and technology: an international journal, 3: 677-683, (2013). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 amal messaoudi, djamel fahloul, physico-chemical and sensory properties of barley bread enriched by freeze dried date pomace powder, food and environment safety, volume xix, issue 1 – 2020, pag. 69 – 75 75 [3]. paul p., bhattacharyya s., antioxidant profile and sensory evaluation of cookies fortified with juice and peel powder of fresh pomegranate (punica granatum), international journal of agricultural and food science, 5: 85-91, (2015). [4]. obiegbuna j.e., akubor p.i., ishiwu c.n., ndife j., effect of substituting sugar with date palm pulp meal on the physicochemical, organoleptic and storage properties of bread, african journal of food science, 7: 113-119, (2013). [5]. olubunmi i.ph., babatunde k.s., bolanle o.o., seyioba s.o., taiwo l.t., olukayode o.a., nwankego e.g., quality evaluation of fibreenriched bread, international journal of nutrition and food sciences, 4: 503508, (2015). [6]. tanzila h., mudasir a., farooq a.m., adil g., umera b., physicochemical and pasting properties of barley/wheat flour blends and the physical, baking and sensory characteristics of cakes, nutrafoods, 17: 41-45, (2018). [7]. messaoudi a., fahloul d., physicochemical and sensory properties of pancake enriched with freeze dried date pomace powder, annals. food science and technology, 19: 59-68, (2018). [8]. prokopov t., goranova zh., baeva m., slavov a., galanakis ch.m., effects of powder from white cabbage outer leaves on sponge cake quality, international agrophysics, 29: 493-500, (2015). [9]. al-farsi m.a., clarification of date juice, international journal of food science and technology, 38: 241-245, (2003). [10]. ndangui ch. b., sweet potato flour production and characterization (ipomoeabatatas.lam): optimization of breadmaking technology. phd thesis in food biotechnology and processes, lorraine and marien ngouabi universities, 151p, (2015). [11]. mohd. jusoh y.m., chin n.l., yusof y.a., abd. rahman r., bread crust thickness estimation using l a b colour system, pertanika journal of science & technology, 16: 239-247, (2008). [12]. sassi kh., contribution to the study of the adaptation of durum wheat cultivars (triticum durum desf.) to organic farming: grain yield, stability and technological and nutritional quality. phd thesis in agricultural sciences, university of 7 november in carthage, national agronomic institute, tunisia, 171p, (2008). [13]. rosa c.s., tessele k., prestes r.c., silveira m., franco f., effect of substituting of cocoa powder for carob flour in cakes made with soy and banana flours, international food research journal, 22: 21112118, (2015). [14]. semić a., oručević s., bauman i., muminović š., spaho n., klepo b., effects of increasing sourness of bread dough on bread quality, 5th international congress flour‐bread ʹ09, 7th, croatian congress of cereal technologists, 416-424, (2010). [15]. ouazib m., effect of treatments on the nutritional and functional parameters of chickpea produced locally: impact on the rheological, physicochemical and sensory properties of chickpea bread. phd thesis in food science, university of a.mira-bejaia, algeria, 153p, (2017). [16]. halaby m.s., farag m.h., gerges a.h., potential effect of date pits fortified bread on diabetic rats, international journal of nutrition and food sciences, 3: 49-59, (2014). [17]. ishida p.m.g., steel c.j., physicochemical and sensory characteristics of pan bread samples available in the brazilian market, food science and technology, campinas, 34: 746754, (2014). 1. introduction 156 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 156 166 activity of selected mixed starter cultures for specific production of three main types of attieke in côte d’ivoire *jean-paul koffi maïzan bouatenin 1 , alfred kohi kouame 1 , theodore n’dede djeni 1 , marcellin koffi dje 1 1faculty of food sciences and technology, university of nangui abrogoua, 02 bp 801 abidjan 02, côte d’ivoire, bouateninkoffi@gmail.com *corresponding author received 30th june 2019, accepted 27th september 2019 abstract: in this study, mixed potential starter cultures have been set up to control the fermentation of cassava dough for the production of each type of attieke in côte d'ivoire. the results showed that potential starter culture from ebrie was the most acid with ph and total titratable acidity values respectively of 4.68 and 3.22±0.2 % after 24 hours fermentation. also several organic acids were detected in the potential starter cultures, lactic and acetic acids being predominant. lactic acid content reached 670±19.4 mg/100g in inoculum from adjoukrou group, 580±9 mg/100g in and 778±13 mg/100g in those from ebrie group. for acetic acid, its highest rate (347±14 mg/100g) was obtained in alladjan inoculum and the lowest (195.2 ±14 mg/100g) in ebrie’s one after 24 hour fermentation. fumaric, citric, tartaric and propionic acids have not been detected. volatile substances (acetaldehyde, ethanol) were produced throughout the fermentation by the potential starter cultures formed. the microbial analysis showed that highest loads were represented by lactic acid bacteria with values of 7.2 ± 0.8.107 cfu / g; 2.9 ± 0.7.107 cfu / g and 7.6 ± 1.106 cfu / g respectively in the doughs inoculated with the selected starters adjoukrou, alladjan and ebrie. the production of attieke specific to each ethnic group with potential starter cultures showed that these attieke were of acceptable quality. the importance of mixed potential starter cultures formed could be necessary to standardize at small and industrial scale the process of production each type of attieke in côte d’ivoire. keywords: starter cultures, organic acids, standardization, attieke 1. introduction in côte d’ivoire, cassava roots are processed into about ten dishes of which the most known are attoukpou, gari, placali and attieke [1]. the major fermented plant food of the country is attieke. it is a steamed granular cassava (manihot esculenta crantz) meal ready-to-eat, couscous-like product, with slightly sour taste and whitish colour [2]. in the past, this food was prepared and consumed exclusively by the adjoukrou, ebrie, alladjan, avikam and ahizi peoples of côte d'ivoire. the processing of cassava into attieke requires several difficult steps. generally, roots are peeled, cut into pieces and then washed three times with fresh water. before milling, 5% 10% (w/w) of inoculum, 10% (v/w) water and about 0.1% (v/w) of palm oil are added and the pieces are ground into a fine paste and left to ferment for about 12 to 15 hours at room temperature (30˚c 37˚c). after fermentation, the dough is continuously pressed and then sieved and granulated. the resulting grains are dried in the sun for a few minutes to half an hour. then, the fibres and dirt are removed by sprinkling after drying. finally, the grains are steamed for about 20 to 25 minutes. [3]. the attieke obtained is packed in plastic http://www.fia.usv.ro/fiajournal mailto:bouateninkoffi@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 157 bags, hermetically sealed for sale on local and international markets. but, the production of high quality attieke is often associated with specific locations and specific ethnic groups in côte d'ivoire, thus generating several types of attieke found on various markets with specific characteristics and differently appreciated by consumers. the ethnic groups adjoukrou, ebrie and alladjan are recognized as the best producers and consumers of attieke in côte d'ivoire. besides these groups, their products are also consumed on a large scale in urban areas, particularly in abidjan. yet, the fermentation of cassava during the production of attieke requires the use of a traditional ferment. the method of preparing this ferment varies according to ethnic groups [4]. however, this ferment is the main source of microorganisms active in the fermentation of dough. fermentation relies on the autochthonous microbial populations to start the process. spontaneous fermentations typically result from the competitive activities of a variety of autochthonous and contaminating microorganisms. thus, those best adapted to the conditions during the fermentation process will eventually dominate. however, initiation of a spontaneous or back slopping process takes a relatively long time, with a high risk for failure. failure of fermentation processes can result in spoilage and/or the survival of pathogens, thereby creating unexpected health risks in food products [5, 6]. thus, in a framework to develop starter culture for controlled fermentation and production of regional specific attieke, with greater consistency in quality and safety, the use of starter cultures is recommended, as it would lead to control and optimisation of the fermentation process in order to alleviate the problems of variations in organoleptic quality and microbiological stability observed attieke in côte d'ivoire. many previous studies were carried out before putting the establishment of starter cultures specific to the three main types of attieke in côte d'ivoire. these are studies conducted by [7] based on screening microorganisms for their abilities to produce α-amylase, β-glucosidase, pectinase and their rate acidification. these strains used, have been characterized and identified by morphological, biochemical as well as molecular approaches [8, 9]. the optimization of α-amylase production by selection of microorganisms have been realized by [10] in the same way, use of starter cultures of lactic acid bacteria, yeasts, bacilli and moulds in the fermentation of cassava dough for attieke (an ivoirian fermented food) preparation [11] . from these studies, the controlled mixed starter cultures have been shown to be the most interesting technology profiles for any application at small or large scale. therefore, this study was carried out to provide sufficient knowledge about the final biochemical and microbiological characteristics of potential starter cultures in order to control and standardize the cassava fermentation process for each types of attieke produced in côte d'ivoire. 2. materials and methods 2.1. cassava roots the cassava roots used for this study were of the iac variety harvested from a field at nangui abrogoua university in abidjan. 2.2. strains used strains used in this study were isolated and pre-selected from three different types of ready to use cassava traditional inoculum, with regard to ethnical groups food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 158 (ebrie, adjoukrou and alladjan) which produce them in small-scale attieke production in the three main processing zones (abidjan, dabou and jacqueville) in côte d’ivoire (table 1). these strains were previously characterized and tested for their technological properties, interesting for fermentation [7-11]. table 1 potential starter cultures specific to the three main types of attieke in côte d’ivoire. strains have been preselected with regards to their origin and characterized, tested for their technological properties interesting for cassava fermentation in our previous studies origin of starter potential starter cultures codes of potential starter cultures adjoukou labx2bx5lvx14mx4 ss.ad alladjan laby9-by4lvy3 my2 ss.al ebrie labz46-bz15-lvz18-mz4 ss.eb ss.ad: mixed culture of labx2 (leuconostoc mesenteroides ssp mesenteroides), bx5 (bacillus amyloliquefaciens), lvx14(candida tropicalis) and mx4(rhizopus oryzae) selected as microbials component of potential starter adjoukrou ss.al: mixed culture of laby9 (lactobacillus plantarum), by4 (bacillus amyloliquefaciens), lvy3 (candida tropicalis) and my2 (aspergillus oryzae) selected as microbials component of potential starter alladjan ss.eb: mixed culture of labz46 (lactobacillus plantarum), bz15 (bacillus subtilis), lvz18 (candida tropicalis) and mz4 (rhizopus oryzae) selected as microbials component of potential starter ebrie 2.3. preparation of microbials suspensions and potential starter cultures for attieke production 2.3.1. preparation of the microbial suspension the strains that constitute each mixed starter culture as described in the table 1, has been stored at -80°c in sterile cryotubes containing appropriate broth medium with 20% (v/v) glycerol until needed. lab were cultivated by streaking on mrs agar (conda, spain) and incubated anaerobically at 30°c for 24 h. bacilli were cultivated by streaking on plate count agar containing 2% of soluble starch and incubated at 30°c for 24 h. yeasts and moulds were cultivated by streaking on sabouraud chloramphenicol agar and incubated at 30°c for 72 h. after incubation, a colony was picked from each pure culture plate, grown successively in appropriate broth before centrifugation at 7500 g for 15 min. the pellet was washed in peptone physiological salt solution, centrifuged again, redistributed in peptone physiological salt solution and the diluted to yield an optical density equivalent between 5 and 7 mac farland (mcf), equivalent to 106 cells/ml. the mix of the different single suspensions (100 ml of lab suspension, of 100 ml of bacilli suspension, 100 ml of yeasts suspension and 100 ml of moulds suspension) constitutes the suspension of a potential starter specific to a type of attieke. 2.3.2. preparation of selected starters for attieke production the starter selected was obtained by peeling the roots and cooking them for 20 min. after cooling to 30°c, 250 g of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 159 cooked roots were crushed in each stomacher sachet containing 100 ml of inoculum (suspension of a potential starter) at 106 cells/ml. the cassava dough obtained was fermented for 24 hours at room temperature (28°c±2). after 24 hours, the dough obtained was the selected starter (mixed), typical to each traditional ferment. all fermentations were conducted for 24 hours and samples were taken every 6 hours for biochemical and microbiological analysis. after all the analyses, the preparation of attieke from each selected starter was carried out using the artisanal attieke preparation method described by [4] in côte d'ivoire. 2.4. biochemical and microbial analyses 2.4.1. biochemical analysis forty grams (40 g) of starter cultures samples selected were ground in 300 ml of distilled water in a porcelain mortar and then centrifuged at 4000 tours/min for 30 min. the ph was determined on 50 ml of the supernatant using a ph-meter (p107 consort). total titratable acidity (tta) was determined by titrating 30 ml of supernatant used for ph determination against 0.1 m naoh using phenolphthalein as indicator. tta was calculated as percentage of lactic acid. water-soluble carbohydrates were determined by the phenol sulphuric acid method according to [12] and the values were expressed in g/100 g of fresh matter, while the reducing sugars were quantified as described by [13] and expressed in mg/100 g of fresh matter. organic acids of samples were before extracted and then analyzed by high performance liquid chromatography using an ion exclusion orh-801 column (300 mm×6.5 mm) (interchrom, france) as achieved by [14]. running conditions were: mobile phase, h2so4 40 mmol.l-1; flow rate, 0.8 ml.min-1; wave length, 210 nm; room temperature (25 °c). the separated components were detected by an uv spectrophotometric detector (spd-6a, shimadzu corporation, japan). volatile compounds were determined by gas chromatography (shimadzu cg-14a, shimadzu corporation, japan) following the method used by [15]. 2.4.2. microbial analysis microbial analyses were carried out to determine the microbiological loads of inoculum samples. preparation of stock solutions, inoculation of agar plates, cultivation and quantification of microorganisms were carried out according to [16]. for all determinations, 10 g of samples were homogenized in a stomacher with 90 ml of sterile peptoned buffered water (aes laboratoire, combourg france). tenfold serial dilutions of stomacher fluid were prepared and spread plated to determine microorganism counts. yeasts and moulds were enumerated on plates of sabouraud chloramphenicol agar (fluka, bochemica 89579, sigma-aldrich chemie gmbh, inda) incubated at 30 °c for 4 days. bacili species were enumerated on plates mossel agar (aes laboratoire, combourg france) after incubation at 30°c for 2 days. enumeration of lab was carried out using plates of de man, rogosa and sharpe (mrs) agar (merck, darmastadt, germany) which were incubated under anaerobic conditions (anaerocult a, merck) at 37° c for 72 h. 2.5. statistical analysis one-way analyses of variance based on duncan multiple tests with significant level α=0.05 were performed in order to compare biochemical and microbial characteristics samples and also food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 160 to determine significant differences between potential starter cultures types. the software used for the statistical evaluation was xlstat (addinosoft inc.). 3. results and discussion the present study on potential starter cultures specific to the three main types of attieke (adjoukrou, alladjan and ebrie) was performed firstly to establish a scientific database of characteristics of these potential starter cultures, and secondly to produce attieke with the potential culture starters set up to ensure the quality of each type of attieke in côte d'ivoire. during various fermentation tests made, there was a significant acidification resulting in a rapid reduction in the ph during the first twelve (12) hours, an increase in the titratable acidity rate and a significant production of organic acids, particularly lactic and acetic acids. all these inocula were acid with different degrees of acidity. potential starter culture from ebrie is the most acid with ph and total titratable acidity values respectively of 4.68 and 3.22±0.2 %. these values were respectively 4.26 and 2.3±0.2 % for potential starter culture from adjoukrou group and of 4.49 and 2.09±0.1 for alladjan type after 24 hours fermentation (fig. 1). fig.1. ph (a) and titratable acidity (b) of cassava doughs inoculated with the three types of starters selected during fermentation. ss.ad: selected starter adjoukrou, ss.al: starter selected alladjan, ss.eb: selected starter ebrie also, as it could be seen in the figure 2, in the potential starter cultures, lactic and acetic acids were predominant. lactic acid content reached 670±19.4 mg/100g in inocula from adjoukrou group, 580±9 mg/100g in and 778±13 mg/100g in those from ebrie group (fig. 2a). for acetic acid, its highest rate (347±14 mg/100g) b a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 161 was obtained in alladjan inocula and the lowest (195.2 ±14 mg/100g) in ebrie’s one after 24 hours fermentation (fig.2b). indeed, this acidification is attributed to the lab that colonizes the selected starters set up. a c fig. 2. production of organic acids by the selected starters in the various cassava doughs in fermentation. lactic acid (a); acetic acid (b); ascorbic acid (c). ss.ad: selected starter adjoukrou, ss.al: starter selected alladjan, ss.eb: selected starter ebrie these results were in agreement with those of [17, 18]. these authors attribute to lactic bacteria the main role of rapid acidification of food products. indeed, the high activity of producing lactic acidity or titratable acidity is related to the diversity of microorganisms with important enzymatic activities (α-amylase, β-glucosidase and pectinase). it could be suggested a cometabolism between yeasts and other microorganisms especially lactic acid bacteria, whereby bacteria produce acid in the medium for yeast growth then these yeasts provide vitamins and other growth factors for bacteria. thus, it could be said that starters set up have a microflora b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 162 capable for the production of attieke. in addition, [14] stated that the high content of lactic acid and acetic acid in the fermented dough would clearly result in a safe action against pathogens. here, the presence of pathogens has not been sought. however, also high content of organic acids, especially acetic acid, could have an adverse effect on the sensory properties of the finished product [4]. acidification was more intense in the dough inoculated with these starters selected than that established by [14] during his study on the performance of traditional ferments adjoukrou, allandjan and ebrie. the acidification contributes to the improvement of the taste, the texture and the life time of the final product, it could be stated that the organoleptic qualities of the finished product in each selected starter will be improved more than those in each traditional ferment. these results are consistent with those [19, 20]. acidification is an essential parameter for the organoleptic characteristics of fermented foods. although organic acids, mainly lactic and acetic acids are the main metabolites of cassava fermentation, it is also obvious that their synthesis by microorganisms is possible thanks to the degradation of cassava constituents (starch, soluble sugars, etc.). this degradation of soluble sugars in the doughs inoculated by selected starters was total after 18 hours of fermentation except for the selected starter ebrie which degrades the reducing sugars by 14 ± 0.008% after 12 hours of fermentation (fig. 3). a b fig. 3. utilization of sugars during fermentation of cassava doughs inoculated with the three types of starters selected. total sugars (a); reducing sugars (b). ss.ad: starter selected adjoukrou, ss.al: starter selected alladjan, ss.eb: starter selected ebrie in fact, the evolution of sugar content is the result of two phenomena: the consumption of sugars by microorganisms and the degradation of complex sugars into simple sugars. according to [21] the degradation of complex sugars into fermentable sugars leads to an increase in sugars in the culture medium. for [22], the variation of sugars in the medium is due to the amylolytic activity of fermentative microorganisms. the metabolic activity of these fermentative microorganisms would also lead in addition to the production of organic acids, to the production of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 163 precursors of flavoring compounds such as amino acids that could be either deaminated or decarboxylated to form aldehydes. and these aldehydes would be oxidized or reduced to alcohols [23, 24]. some food flavor compounds have also been detected in the different fermenting doughs. this was ethanol, acetaldehyde and methylethylketone (mek) (fig. 4). during fermentation, large quantities of acetaldehyde were produced by isolates of the selected starter ebrie with contents of 60±2 mg/100g and 68±3 mg/100g after 12 and 18 hours of fermentation, (fig. 4a). in contrast to acetaldehyde, the quantity of methylethylketone observed in fermented dough was irregular with a high concentration of 9.4±0.94 mg/100g produced by alladjan starter isolates after 12 hours of fermentation (fig. 4b). on the other hand, a large content of ethanol (5±0.15 mg/100g) was produced by the ebrie starter isolates after 12 hours of fermentation (fig. 4c). a b c fig. 4. production of volatile compounds by the selected starters in the different fermenting cassava doughs. acetaldehyde (a), methylethylketone (b) and ethanol (c). ss.ad: selected starter adjoukrou, ss.al: starter selected alladjan, ss.eb: selected starter ebrie a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 164 the production of these compounds was irregular during fermentation. yeast is believed to be the basis of ethanol production [24]. the presence of these volatile compounds depends on several factors such as yeast seeding rate, yeast strain, medium composition, fermentation temperature and aeration [25]. thus, isolation and enumeration of microorganisms revealed that lactic acid bacteria, bacilli, yeasts and moulds in varying proportions (fig. 5). the flora, more abundant was represented by lactic acid bacteria with loads of 7.2 ± 0.8.107 cfu/g; 2.9 ± 0.7.107 cfu/g and 7.6 ± 1.106 cfu/g respectively in the doughs inoculated with the selected starters adjoukrou, alladjan and ebrie after 18 hours of fermentation(fig. 5a). at the same time, average loads of bacilli were respectively 1.3 ± 0.7.106 cfu/g, 9.9 ± 0.9.105 cfu/g and 9.1 ± 0.2.104 cfu/g (fig. 5b). loads of yeasts and moulds were also significantly different (p <0.05) among all these potential starter cultures. a b c d fig. 5. microbial load in cassava doughs inoculated with the three types of starters selected during fermentation. lactic acid bacteria (a), bacillus (b), yeast (c) and mould (d). ss.ad: selected starter adjoukrou, ss.al: starter selected alladjan, ss.eb: selected starter ebrie food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 165 this microbial load used during the degradation of soluble sugars remains lower than that obtained by [26] during traditional fermentation. this indicates that the presence and multiplication of these microorganisms with known properties lead to significant changes in each dough controlled by a mixed starter. thus, in all the analyses, each mixed ferment was used to prepare a typical attieke for each ethnic group. these attieke in doughs inoculated with the selected starters adjoukrou, alladjan and ebrie were of acceptable quality and appearance similar to traditional attieke. so, the formulation of a mixed starter from a traditional ferment makes it possible to obtain a microflora capable of degradation of cassava starch and the production of organic acids and volatile compounds necessary for the attieke production and flavor. 4. conclusion the comparative study of the performance of the three (3) starters of attieke (selected starters adjoukrou, alladjan and ebrie) clearly indicates that the evolution of physico-chemical and microbiological parameters of the dough inoculated with the selected starters adjoukrou and alladjan were similar except the ethanol content that was higher in the dough inoculated with the alladjan starter selected during the first six (6) fermentations. on the other hand, these two (2) doughs differ from the inoculated dough with the selected starter ebrie by its high titratable acidity rate and its high acetaldehyde content. after having carried out all analyses, each mixedferment was used to prepare a typical attieke for each ethnic group. these attieke in doughs inoculated with the selected starters adjoukrou, alladjan and ebrie were of acceptable quality and appearance similar to traditional attieke. 5. acknowledgement the authors gratefully acknowledge attieke producers which had freely accepted to participate in this study. there is no conflict of interests concerning this manuscript. 6. references [1]. caransa a., simplified economical cassava starch process, starch, 32: 48-54, (1980) [2]. djeni n.t., nguessan k.f., toka d.m., kouame k.a., dje k.m., quality of attieke (a fermented cassava product) from the three main processing zones in cote d'ivoire, food research international, 44: 410-416, (2011) [3]. heuberger c., cyanide content of cassava and fermented products with focus on attiéké and attiéké garba. ph.d thesis, swiss federal institute of technology, zurich. 126 p, (2005). [4]. kouadio n.a., mosso k., kouakou k., angbo s.f., etude comparative des méthodes traditionnelles de la préparation de l'attieke dans le sud de la cote d'ivoire, cahiers de la recherche scientifique et technique, 108 : 703706, (1991). [5]. sanni a.i., onilude a.a., oguntoyinbo f.a. optimization of process conditions for owoh, a fermented cotton seed condiment, advances in food science, 20: 163-167, (1998) [6]. holzapfel w.h., appropriate starter culture technologies for small-scale fermentation in developing countries, int. j. food microbiol, 75: 197-212 (2002) [7]. bouatenin k.m.j-p., djeni n.t., aka s., brou k., dje k.m., the contribution of microorganisms to the fermentation of cassava dough during attiéké processing in côte d’ivoire, food, 6:54-68, (2012) [8]. bouatenin k.m.j-p., djeni t.n, ouassa t., zinieu e., menan h., dje k.m., characterization and enzyme activities of microorganisms from a traditional cassava starter used for the production of adjoukrouattieke (cote d’ivoire), j. food technol, 11: 4-13, (2013) [9]. djeni n.t., bouatenin k.m.j-p, assohoun n.m.c., toka d.m., menan e.h., dousset x., dje k., biochemical and microbial food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 jean-paul koffi maïzan bouatenin, alfred kohi kouame, theodore n’dede djeni, marcellin koffi dje, activity of selected mixed starter cultures for specific production of the three main types of attieke in côte d’ivoire, food and environment safety, volume xviii, issue 3 – 2019, pag. 156 – 166 166 characterization of cassava inocula from three main attieke production zones in côte d’ivoire, food control, 50: 133-140 (2015) [10]. bouatenin k.m.j-p., djeni n.t., kakou a.c., menan e.h., dje k.m., optimisation de la production de l’ α-amylase par les microorganismes isolés des ferments traditionnels de manioc provenant de trois zones de production de l’attiéké en côte d’ivoire, european scientific journal, 12: 259-272, (2016) [11]. bouatenin k.m.j-p., djeni n.t., kouame k.a., tra bi y.c., menan e.h., dje k.m., use of starter cultures of lactic acid bacteria, yeasts, bacilli and moulds in the fermentation of cassava dough for attieke (an ivoirian fermented food) preparation, international journal of innovation and applied studie, 21: 48-58, (2017) [12]. dubois m, gilles k.a., hamilton j.k., rebers p.a., smith f., colorimetric method for determinations of sugars and related substances, analytical chemistry, 280: 350-356 (1956) [13]. bernfeld d., amylase β et α, in methods in enzymology 1, s. p. colowick and n. o. k., academic press, inc, new york, 149-154, (1955) [14]. djeni n.t, n’guessan k.f., dadie a.t., dje k.m., impact of different rates of a traditional starter on biochemical and microbiological changes during the fermentation of cassava for attiéké production, food, 2 : 145-151, (2008) [15]. djeni n.t., typologie de l’attiéké de trois zones de production de côte d’ivoire et analyse des propriétés des levains traditionnels utilisés pour sa préparation, thèse unique de doctorat, universite´ abobo-adjamé, 170 p, (2009). [16]. coulin p., farah z., assanvo j., spillman h., puhan z., characterisation of the microflora of attiéké, a fermented cassava product during traditional small-scale production, international journal of food microbiology, 10: 131-136, (2006) [17]. regez p.f., schmidt-lorenz w., identification des bactéries lactiques isolées de la pâte de manioc et du pain de manioc (chikwangue) au zaïre, lebensmittel technologie, 21: 66-69, (1988) [18]. sawadogo-lingani h., diawara b., traoré a.s., jakobsen m., technological properties of lactobacillus fermentum involved in the processing of dolo and pito, west african sorghum beers, for the selection of starter cultures, journal of applied microbiology, 104: 873-882, (2008). [19]. montet d., loiseau g., zakhiarozis n., microbial technology of fermented vegetables. in microbial biotechnology in horticulture, (r.c. ray and o.p. ward, eds.), science publishers inc., enfield n.h. 1: 309–343, (2006) [20]. panda s.h., parmanick m., ray r.c., lactic acid fermentation of sweet potato (ipomoea batatasl.) into pickles. journal of food processing and preservation. 31: 83–101, (2007) [21]. okafor n., ijioma b., oyolu c. studies on the microbiology of cassava retting for fufu production, j. appl bacteria, 56: 1-13, (1984) [22]. giraud e., brauman a., keleke s., gosselin l., raimmult m., contrôle de la fermentation du manioc pour un meilleur gari : utilisation d'un starter de lactobacillus plantarum à activité linamarase et amylase, transformation alimentaire du manioc, éditions orstom, p353-365, (1995) [23]. gobbetti m., corsetti a., rossi j., the sourdough microflora. interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates, appl. microbiol. biotechnol, 41: 456– 460, (1994) [24]. hansen a., hansen b., flavour of sourdough wheat crumb. z. lebensm. unters. forsch. 202: 244–249, (1996) [25]. moll m. bieres et al. technique & documentation-lavoisier, paris. 515p, (1991) [26]. assanvo j.b., agbo g.n., behi y.n., coulin p., farah z., microflora of traditional starter made from cassava for ‘attiéké’ production in dabou (côte d’ivoire) , food contro,. 17: 37-41, (2006) 4. conclusion применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 contents: 1. some physicochemical and organoleptic properties of the short dough with gooseberry powder sergiy boruk, igor winkler 5 12 2. scientific explanation of composition of acidophilic-whey ice cream, enriched with protein galina polischuk, oksana kochubey-lytvynenko, tetiana osmak, uliana kuzmik, oksana bass, artur mykhalevych, victoria sapiga 13 20 3. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation sunday ukwo, mfoniso udo, ekefre ekong 21 34 4. protozoan distribution in farmed cyprinid fish from macedonia dijana blazhekovikj dimovska, stojmir stojanovski 35 42 5. provision of children’s tourist groups with functional drinks kristina belinska 43 48 6. good hygiene practices as an important program for safety of final products ljubica trajkoska, maja trajanoska, biljana trajkovska, ljupche kochoski, vesna karapetkova-hristova 49 52 7. life cycle assessment of wastewater from dairy industry diana cornelia adamovici costea, cristina ghinea 53 60 8. evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats) samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga 61 67 9. use of artificial neural networks and multivariate statistical analysis for modeling the pollution pressure of water resources in the seybouse valley (north-eastern algeria) aissam ghrieb, fethi baali, chemceddine fehdi, azzedine hani, hicham chaffai, larbi djabri 68 80 10. effect of fermentation on the quality of flour and biscuit from cashew kernel flour bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi 81-91 11. author instructions i v 12. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 23 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 23 27 the influence of processing on phytic acid content in some wheat products *marcel avramiuc1 1faculty of food engineering, stefan cel mare university of suceava, romania avramiucm@fia.usv.ro *corresponding author received january 7th 2014, accepted february 26th 2014 abstract. the influence of wheat processing (milling, fortification, bread making) on phytic acid content in flour and bread was the purpose of this paper. the biological material was wheat (triticum aestivum l.) flour of types 550, 680, 1350, fortified wheat flour (3 mg of ferrous sulfate per 100 g) from the same types, and bread made from these flours (simple and fortified ones). wheat grains processing (milling, flour fortification with iron, and bread making) has influenced the phytic acid content in flour and bread. of the three types of wheat flour (550, 680 and 1350), the flour with largest degree of extraction (type 1350) has recorded the highest value of the phytic acid. the phytic acid content in all three types of unfortified flour was higher than in fortified ones. as compared to unfortified versions, the biggest drop in phytic acid content was recorded in fortified flour type 550, followed by flour type 680, and flour type 1350. the highest content of phytic acid was recorded in the bread made with flour type 1350. in the case of bread made from fortified flour (with fe), the phytic acid content has increased with flour extraction degree rising. the phytic acid content in all bread samples made from unfortified flour was higher than in bread made from fortified ones. one can notice that during bread making, the phytic acid content of flour has decreased in all cases reaching much lower within fortified bread coming from flour with lower degree of extraction. keywords: phytic acid, wheat, iron, fortified flour, bread 1. introduction the phytic acid (inositol hexaphosphate or phytate) is a major component of all plant seeds and has been an important dietary component for humans since ancient times [1,2]. in cereal grains, most of the minerals form salts with phytic acid that act as a storage compound of phosphorous [3]. phytate is deposited in globoids from the aleurone cell layer of the grain [4]. phytic acid can exist as free acid, phytate, or phytin depending on the physiological ph and the metal ions presen [5,1]. according to casey and walsh (2004), the phytases are enzymes that catalyses the degradation of phytate to lower inositol phosphates and free inorganic phosphorous is formed depending on extend of the enzyme activity. cereal phytase is present with varying activity in all unprocessed cereal grains, and the activity in rye is high compared to other cereals [7,3]. some operations of cereals processing, such as grains soaking at temperatures over 45°c (full meal, flours with different extraction degrees, bran), under optimum conditions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 marcel avramiuc, the influence of processing on phytic acid content in some wheat products, volume xiii, issue 1 2014, pag. 23 – 27 24 for the specific phytase, e.g. ph 6 for rye grains phytase [8,3] can result in a strong hydrolysis of phytate and a significant increase in the bioavailability of mineral elements. phytate hydrolysis occurs in all different stages of bread preparation, and depends on the type of bread that is prepared [9]. in this paper it has investigated the influence of some wheat processing (milling, fortification, bread making) on phytic acid content in flour and bread. 2. experimental 2.1. research materials in this paper, the biological material was wheat (triticum aestivum l.) flour of types 550, 680, 1350, and fortified wheat flour from the same types; bread made from flour of types 550, 680, 1350 (simple and fortified). for the fortification process there were used 3 mg of ferrous sulfate per 100 g flour with continuous mixing for 15 minutes. afterwards, the obtained mixture was kept for 5 hours in a sealed container at a temperature of 30ºc. the recipe for preparing a bread sample was: 150 g flour (simple or fortified by case), 6 g of yeast, 125 ml of water and 1 g of salt. the fermentation temperature was 32ºc, the dough ph was adjusted to 4.5, and the fermentation time was 2 hours. 2.2. research methods the phytic acid determination (mg/100 g) was done using garcia-villanova method, based on treating the sample with a ferric salt and titration with sulfosalicylic acid (in the presence of edta solution as an indicator) of fe3+ ions precipitated along with phytic acid [10]. four replicates for each determination represented the data of experiments, which were statistically processed, the analysis of variance being used to calculate differences between results. the differences at p< 0.05 were considered significant. 3. results and discussion in the table 1 are reproduced the phytic acid values in analyzed wheat flour samples. table 1 the phytic acid concentration in unfortified and fortified wheat flour with different degrees of extraction samples test wf type 550 wff type 550 wf type 680 wff type 680 wf type 1350 wff type 1350 phytic acid (mg/100 g) 578±15.3 542±4.8 605±12.2 573±13.1 658±8.9 630±11.2 wf=wheat flour; wff= wheat flour fortified (3 mg feso4 per 100 g flour) as seen in the table 1, in the unfortified flour types the highest content of phytic acid (inositol phosphates) was recorded in flour type 1350, ie with 13.8% more than flour type 550, and with 8.8% more than flour type 680 (p˂0.05). the obtaining way of the flours has directly influenced the phytic acid values, so the flour with largest degree of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 marcel avramiuc, the influence of processing on phytic acid content in some wheat products, volume xiii, issue 1 2014, pag. 23 – 27 25 extraction has recorded the highest value of this biochemically index too. the phytic acid content is higher in whole grain and whole meal flour compared to flour with low extraction rate [11]. also in the case of the fortified flours with fe, the phytic acid content has increased with flour extraction degree rising. so, the flour type 1350 has recorded the highest value of the analyzed index, with 16.2% more than flour type 550 and with 9.9% more than flour type 680 (p˂0.05). the phytic acid content in all three types of unfortified flours was higher than fortified ones, because the presence of iron ions in fortified flours has led to the phytin content increase, and correspondingly to the content reducing the of phytate (phytic acid). from the table 1 it can see that, as compared to unfortified versions, the biggest drop in phytic acid content was recorded in fortified flour type 550 (6.2%), followed by flour type 680 (5.3%) and flour type 1350 (4.2%) (p˂0.05). one can notice that the flour fortification has led to a greater decrease in the phytates concentration. the lower extraction degree of fortified flour the greater decrease of phytates was. in the table 2 are reproduced the phytic acid values in bread samples. table 2 the phytic acid concentration in bread obtained from unfortified and fortified wheat flour with different degrees of extraction samples test bread from wf type 550 bread from wff type 550 bread from wf type 680 bread from wff type 680 bread from wf type 1350 bread from wff type 1350 phytic acid (mg/100 g) 201±3.8 68±0.7 398±9.3 248±2.5 627±11.7 530±10.1 wf=wheat flour; wff= wheat flour fortified (3 mg feso4 per 100 g flour) as seen in the table 2, in bread samples made with unfortified flours the phytic acid content was different depending on the type of flour. the highest content of phytic acid was recorded in the bread made with flour type 1350: ie with 212% more than bread made with flour type 550, and with 57.5% more than bread made with flour type 680 (p˂0.05). in the case of breads made from fortified flours (with fe), the phytic acid content has increased with flour extraction degree rising. so, the bread made from flour type 1350 has recorded the highest value of the phytic acid, with 826.5% more than flour type 550 and with 113,7% more than flour type 680 (p˂0.05). the phytic acid content in all bread samples made from unfortified flours was higher than breads made from fortified ones. from the table 2 it can see that, as compared to unfortified versions, the biggest drop in phytic acid content was recorded in fortified bread coming from flour type 550 (195.6%), followed by bread from flour type 680 (60.5%) and bread from flour type 1350 (18.3%) (p˂0.05). the flour fortification has led to a greater decrease of the phytates concentration in bread samples. the lower extraction degree of flour the greater decrease of bread phytates was. comparing the data from the two tables, it can be seen that during bread processing the phytic acid content of flours food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 marcel avramiuc, the influence of processing on phytic acid content in some wheat products, volume xiii, issue 1 2014, pag. 23 – 27 26 has decreased in all cases (p ˂ 0.05), reaching much lower:with 187.5% in unfortified bread and with 697% in fortified one (the both made from flour type 550), with 52% in unfortified bread and with 131% in fortified one (the both made from flour type 680) and with 4.9% in unfortified bread and with 18.8% in fortified one (the both made from flour type 1350). these data show that, compared with phytic acid, the phytin (formed by iron fortification) was hydrolyzed more intense by phytases (derived from wheat or yeast). the reduction of phytate content during bread making depends on phytase action, which is influenced by several factors: the degree of flour extraction, the proofing time and temperature, the acidity of the dough, the yeast, enzymes added to the dough and the presence of calcium salts [12,3]. in our experiments, the dough ph was 4.5, and the fermentation temperature was 32ºc. it seeems that during bread making, the ph would be the most important factor reducing the content of phytic acid because the phytic acid in doughs with ph 4.3–4.6 is more effectively reduced than in doughs with higher ph [13]. according to garcia-estepa et al. (1999), another factor that reduces phytic acid content in bread is baking temperature, since within roasted sliced french toast 86% of phytic acid was destroyed. fermentation and bread making reduce phytic acid content of cereals with percentages between 8.9% in the wholemeal breads (unleavened) and 66-100% in white breads [15,16,17]. 4. conclusions the processing of wheat grains (milling, flour fortification with iron, and bread making) has influenced the phytic acid content in flour and bread. the flours extraction degree has directly influenced the phytic acid values. of the three types of wheat flour analyzed (550, 680 and 1350) the flour with largest degree of extraction (type 1350) has recorded the highest value of the phytic acid. the phytic acid content in all three types of unfortified flour was higher than in fortified ones. as compared to unfortified versions, the biggest drop in phytic acid content was recorded in fortified flour type 550, followed by flour type 680, and flour type 1350. the lower extraction degree of fortified flour the greater decrease of phytates was. in bread samples made with unfortified flour, the phytic acid content was different depending on the type of flour. the highest content of phytic acid was recorded in the bread made with flour type 1350. in the case of bread made from fortified flour (with fe), the phytic acid content has increased with flour extraction degree rising. the phytic acid content in all bread samples made from unfortified flour was higher than in bread made from fortified ones. the flour fortification has led to a greater decrease of the phytates concentration in bread samples. the lower extraction degree of flour the greater decrease of bread phytates was. one can notice that during bread making, the phytic acid content of flour has decreased in all cases reaching much lower within fortified bread coming from flour with lower degree of extraction. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 marcel avramiuc, the influence of processing on phytic acid content in some wheat products, volume xiii, issue 1 2014, pag. 23 – 27 27 5. references [1]. dendougui f., schwedt g. in vitro analysis of binding capacities of calcium to phytic acid in different food samples, european food research and technology © springer-verlag 2004 10.1007/s00217-004-0912-7 original paper, published online: 27 july 2004 [2]. kamao m., tsugawa n., nakagwa k. j nutr sci vitaminol 46:34–41 (2000) [3]. nielsen m.m., damstrup m.l., thomsen a.d., rasmussen s.k., hansen å. phytase activity and degradation of phytic acid during rye bread making, european food research and technologyzeitschrift für lebensmittel untersuchung und forschung a © springerverlag 2006 10.1007/s00217-006-0397-7 original paper, published online: 26 july 2006 [4]. lott j.n.a., ockenden i., raboy v., batten g.d. a global estimation of phytic acid and phosphorus in crop grains, seeds, and fruits. in: reddy n.r., sathe s.k. (eds) food phytates. crc press, boca raton, pp 7–24 (2002) [5]. oatway l., vasanthan t., hels j.h. food rev int 17(4):419–431 (2001) [6]. casey a., walsh g. j biotechnol 110:313– 322 (2004) [7]. greiner r., egli i. j agric food chem 51:847–850 (2003) [8]. greiner r., konietzny u., jany k.d. j food biochem 22:143–161 (1998) [9]. reddy n.r., pierson m.d., salunkhe d.k. legume-based fermented foods. crc press, boca raton florida (1986) [10]. bordei d. (coord.), bahrim g., pâslaru v., gasparotti c., elisei a., banu i., ionescu l., codină g quality control in the bakery industry. analytical methods. ed. academica, galaţi, pp: 236-237 (2007) [11]. garcía-estepa r.m., guerrahernández e., garcía-villanova b. food res int 32:217–221 (1999) [12]. türk m., sandberg a.-s. j cereal sci 15:281–294 (1992) [13] fretzdorff b., brümmer j.-m. cereal chem 69:266–270 (1992) [14]. garcia-estepa r.m, guerrahernandez e., garcia-villanova b. phytic acid content in milled cereal products and breads. food research international, 32, 217-221 (1999) [15]. reddy n.r., pierson m.d., sathe s.k., salunkhe d.k. phytates in cereals and legumes. crc press inc., boca raton, florida (1989) [16]. mckenzieparnell j.m., davies n.t. destruction of phytic acid during home breadmaking. food chem. 22:181–192 (1986) [17]. sathe s.k., venkatachalam m. influence of processing technologies on phytate and its removal in: food phytates, ed. by rukma reddy, shridhar k. sathe, crc press llc, 2000 n.w. corporate blvd., boca raton, florida 33431 (2002) doi: https://doi.org/10.4316/fens.2022.022 230 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 230 236 haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market *olaniyi alaba olopade 1 , henry eyina dienye 1 , kingsley kenechukwu agbo 1 1faculty of agriculture, university of port harcourt, choba, east-west road, pmb 5323, port harcourt, rivers state, nigeria, olaniyi.olopade@uniport.edu.ng *corresponding author received 7th april 2022, accepted 29th september 2022 abstract: this study aimed to investigate the haematological profile of clarias gariepinus after stress from transportation and handling in the market. c. gariepinus mixed sexes and siblings reared in the same fish tank were subjected to transportation and market stressors associated with transportation and handling in the market. ten fish were collected at random at successive time intervals at the fish farm (a), another immediately after arriving at the choba market (b), and then after 3 hours (c), 6 hours (d), and 9 hours (e) at the same location. the results revealed that red blood cell (rbc) levels were higher in the non-stressed group (group a) than in the test populations (groups b-e).the differences in values were not statistically significant (p<0.05). haemoglobin levels were consistent with the rbc. the non-stressed group had the highest value of 14.87±0.81 g/dl. mean values of the platelets ranged from 195.00±12.50 ×10 /l to 242.33±12.99 ×10 /l for all the samples, and this parameter showed a significant difference. the mean packed cell volume (pcv) values differed significantly between different population groups. there was no statistically significant difference (p<0.05) in neutrophils, lymphocytes, or monocytes values, but eosinophils showed a significant difference (p <0.05) across all treatments. the glucose mean values did not differ significantly across the populations, with the lowest value of 24.3±52.71g/dl recorded for the control relative to the test populations. it can be concluded that acute stress alters blood parameters in fish, and all the haematological parameters determined in the present study are important stress markers. keywords: blood, glucose, stressors, haematological parameters, aquaculture. 1. introduction in nigeria catfish accounts for roughly 64% of aquaculture fish production [1], with production put at 189,562 tonnes in 2017 [2]. increased production of clarias gariepinus, one of the clarrid species, was supported by consumer and farmer acceptance of the fish due to beneficial characteristics such as fast growth and high market demand. [3]. at current levels of production, there is a need for permanent exchange relationships to exist between producers and consumers. the availability of fish to consumers at the right time and place necessitates an efficient marketing system. as such, the marketing system must develop well to provide the necessary services to ensure the machineries for enhancing marketing efficiency are put in place. there is a consumer perception that live fish are healthier than fresh or frozen products, and many consumers perceive that live fish are superior to frozen in terms of taste, freshness, and nutritional value. as a result, catfish are frequently distributed and sold live from farm gates to mailto:olaniyi.olopade@uniport.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 237 231 designated fish markets in the majority of cities and towns across the country, where the fish are sold to restaurants, processors, and final consumers. [4]. during transport and marketing, fish are exposed to a multitude of stressors such as loading density, handling stress, water movement, noise, thirst, hunger, malnutrition, vibrations, poor water conditions, pain, injury, discomfort, disease, restriction of normal behaviors, fear, and suffering [5]. exposure to such stressors simultaneously or in rapid succession may induce severe physiological stress [5]. fish struggle during harvest has been shown to cause rapid decreases in post-mortem muscle ph, affecting fillet quality through loss of fillet firmness and increased fillet gaping. [6]. therefore, since the transport of live fish to markets some miles away is stressful for fish, it deserves special attention. knowledge on stress in fish following transport and handling in the market is important to delineate the health status and quality of fish and provide a future understanding of stress impacts that could modify the physiological state of the fish. it is well known that haematological and serum biochemical changes are termed as pre-requisite to determine the health status of fish [7]. among haematological indices, blood cells are one of the best indicators of body health [8]. furthermore, blood glucose is a marker of the metabolic rate of the stress response [9]. the majority of past research on stress physiology in fish during the last few decades has focused on laboratory experiments, which enforce scientific control and a highly controlled setting in a laboratory. this study was designed to determine the physiological response of c. gariepinus to acute stress of transportation and handling in the field to reflect real-life conditions. 2. matherials and methods adult catfish (c. gariepinus) in excellent health status, (mean weight 973 ± 3.34 g and mean length 32 ± 00.3 cm mixed sexes) were purchased from a commercial fish farm in ahoada and choba market, rivers state, nigeria. they were siblings reared in the same fish tank. at the farm, the catfish are starved prior to transportation to the market a distant of about 47.8km. food is being withheld for 24 h. the experiment was carried out in the rainy season (june 2019). the following experimental protocol was used: fish were divided into six groups (groups a, b, c, d, e and f), fish of group a were not subjected to stress (control group), while those of group b were subjected to acute handling and transportation stress. group c, d, e and f were subjected to transportation stress and acute handling in the market. at the time of sampling, ten fish were collected at random at a successive time intervals at the fish farm (a), another immediately after arriving at the choba market (b) and then 3 hours (c), 6 hours (d), 9 hours (e) at the same location, to determine the effects of stress associated with transportation and handling in the market on the haematology of fish. blood samples were collected using 5ml syringe, and transferred to test tubes after each collection and immediately put on melting ice. the experiment was conducted in triplicates for each category. samples were stored on ice for immediate analysis in the laboratory. laboratory experiment blood collection and processing blood was drawn from the posterior caudal vein using needle and syringes according to [10] and 2ml was decanted in edta bottles for the assessment of haematological parameters, while another food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 237 232 2ml was decanted into heparinized bottles for the bll determination. whole blood (50µl) was stained for enumeration of red and white blood cells [11]. blood smears were air-dried for five minutes, fixed in absolute methanol, and stained for 60 seconds in giemsa stain. haematological analyses red blood cells (rbc) and white blood cells (wbc) were counted in a neubauer chamber; packed cell volume (pcv) by the microhematocrit technique; and haemoglobin level (hb) by the cyanomethemoglobin method. haematological indices were computed using standard formulae while glucose was measured in the laboratory using an electronic blood glucose meter. data analysis statistical analysis of the data was performed using a statistical package for the social sciences (spss v16.0). a oneway anova was applied to understand the difference in haematological parameters between and among groups of fish, and significance was set at 0.05 3. results and discussion the red blood parameters profile of c. gariepinus exposed to handling, transportation, and market stressors is summarized in table 1. the red blood cell (rbc) levels of the stressed groups were generally lower than those of the nonstressed group (group a). the highest value of 24.17±17.97 (×10^12) was recorded for the non-stressed group (group a), while the values recorded for stressed groups ranged from 4.73±0.12 (×10^12) to 5.90±0.21 (×10^12). the variations in values, however, were not statistically significant (p<0.05). stress caused by capture, handling, and sampling procedures was observed to cause intraspecies haematological value variation [12]. the haemoglobin mean values ranged between 10.13±0.30 g/dl and 14.87±0.81 g/dl with the non-stressed (group a) having the highest (14.87±0.81 g/dl) relative to the test populations (groups b-e). the mean platelet values for all samples were extremely high, as indicated in table 1, ranging between 195.00±12.50 ×10 /l recorded for nonstressed and 242.33±12.99 ×10 /l (group e), and this parameter showed a significant difference. haemoglobin values were consistent with red blood cell. the highest value of 14.87±0.81 g/dl was recorded for the non-stressed group. these changes were attributed to a direct or feedback response to structural damage to the rbc membrane, which resulted in haemolysis and impaired haemoglobin synthesis, as well as stress-related rbc release from the spleen [13,15]. the mean packed cell volume (pcv) values decreased sharply from the non-stressed group (44.67±2.40 %) to 40.00±1.15 % (group b) and then decreased gradually to 30.33±0.88 % (group e). hattingh & van pletzen [14] reported a decrease in t he fish blood haemoglo bin concentrat io n and pcv. this is consistent with the findings of [15] studies, which show that the park cell value is lower in the non-stressed group compared to the stressed group. as follows, four different types of white blood cells were identified in the peripheral blood of c. gariepinus: lymphocytes, neutrophils, eosinophils, and monocytes (figures 1, 2, 3, 4, and 5). there were no statistically significant (p<0.05) in the values of neutrophils, lymphocytes, and monocytes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 237 233 table 1. red blood parameters profile of c. gariepinus exposed to handling transportation and market stressors parameters initial stage groupa landing site group b 3 hours after landing group c 6 hours after landing group d 9 hours after landing group e hgb (g/dl) 14.87±0.81 a 13.33±0.38 ab 12.23±0.79 b 11.33±0.69 bc 10.13±0.30 c rbc (×10^12) 24.17±17.97 5.90±0.21 5.50±0.36 4.97±0.26 4.73±0.12 plat.(×10^9) 195.00±12.50 b 194.00±15.95 b 217.00±10.69 ab 230.00±8.14 ab 242.33±12.99 a pvc (%) 44.67±2.40 a 40.00±1.15 ab 36.67±2.40 b 34.00±2.08 bc 30.33±0.88 c pcv: packed cell volume, hgb: haemoglobin, rbc: red blood cell, plat: platelet mean in same row with different superscripts are significantly different (p<0.05). fig. 1. white blood cell of c. gariepinus exposed to handling transportation and market stressors fig. 2: lymphocyte of c. gariepinus exposed to handling transportation and market stressors food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 237 234 fig. 3. neutrophils of c. gariepinus exposed to handling transportation and market stressors fig. 4: eusynophils of c. gariepinus exposed to handling transportation and market stressors fig.5: monocyte of c. gariepinus exposed to handling transportation and market stressors the numbers of eosinophils were significantly (p <0.05) in all the groups. the mean values of white blood cells and neutrophils followed the same pattern, with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 237 235 the non-stressed group having the lowest values compared to the stressed groups, while the mean value of lymphocytes decreased gradually from the non-stressed group to the stressed groups. from the present studies, the mean values of white blood cells and neutrophils in the nonstressed population had the lowest values compared to the test population. this is in agreement with the report of [16] that changes in the composition of circulating white blood cells are more reliable indicators of chronic crowding stress. stress or glucocorticoid treatment specifically causes increases in neutrophil numbers (neutrophil) and decreases in lymphocyte numbers (lymphopenia or lymphocytopenia) [17]. the non-stressed group had the highest mean values in terms of lymphocytes and then decreased gradually in test populations (groups b– e). leucocyte profiles have recently been considered significant in assessing stress [18], and the reduction in the number of lymphocytes is generally considered a stress response [19]. the percentages of eosinophilis and monocytes in the contents of the white blood cells differed widely among the populations, with eosinophilis showing significantly lower values (p <0.05). an absolute monocytes high can also be a response to stress, chronic infections or autoimmune disorders, while too much tension and anxiety can lead to higher eosinophilic inflammation. eosinophils numbers decreased below the reference range, or eosinopenia, may occur as part of a stress response but is a fairly nonspecific response. the blood glucose values of c. gariepinus in this study is presented in table 2. there were differences in the values blood glucose (p>0.05). the glucose values ranged from 24.3±52.71g/dl to 41.75±3.2 g/dl with the lowest value recorded in the non-stressed group (group a) and the highest value of 41.75±3.2 g/dl recorded in the stressed groups (group e). vijayan et al. [20] reported that increased or decreased glucose levels are considered a signs of stress. according to seibel et al. [21] stress-induced change in muscle activity accelerate the conversion of glucose to lactate (or alternatively, to pyruvate) by anaerobic glycolysis. table 2 plasma glucose values of c. gariepinus exposed to handling transportation and market stressor parameter initial stage group a landing site group b 3 hours after landing group c 6 hours after landing group d 9 hours after landing group e glucose (g/dl) 24.3±52.71 32.00±3.85 36.91±3.91 38.32±0.37 41.75±3.22 mean in same row with different superscripts are significantly different (p<0.05) 4. conclusion the results obtained during the examination of the haematological profile of c. gariepinus after stress from transportation and handling in the market reveal that blood biochemical parameters in fish change with acute stress. as a result, proper training in handling fish during transportation and marketing is required because these activities cause stress responses in fish, which have an impact not only on the welfare of the animals but also on the quality of the fish products. 5. acknowledgments authors would like to thank staffs of the department of fisheries, university of port harcourt for their help at the lab as well as in the field. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 olaniyi alaba olopade, henry eyina dienye, agbo kingsley kenechukwu, haematological profile of catfish (clarias gariepinus burchell, 1822) following the stress associated with transportation and handling in the market food and environment safety, volume xxi, issue 3 – 2022, pag. 230 – 236 236 6. references [1]. fao. world aquaculture performance indicators (wapi): information, knowledge and capacity for blue growth. rome. fao (2018). url: http://www.fao.org/documents/card/en/c/i962 2en [2]. fao. fishery and aquaculture country profiles: the federal republic of nigeria. fisheries and aquaculture department. rome fao (2017). url: http://www.fao.org/fishery/facp/nga/en [3]. dauda a. b., natrah i., karim m., kamarudin m. s. & bichi a. h., african catfish aquaculture in malaysia and nigeria: status, trends and prospects. fish aqua journal, 9,237, 2018. doi:10.4172/21503508.1000237. [4]. igoni-egweke q. n., analysis of value addition in commercial catfish (clarias gariepinus and heterobranchus spp.) production in rivers state nigeria (doctoral dissertation). federal university of technology, owerri, 2018. [5]. http://futospace.futo.edu.ng/xmlui/bitstream/h andle/123456789/1894/igoni.pdf?sequence= 1&isallowed=y. [google scholar]. [6]. mcewen b. s. & wingfield j. c., the concept of allostasis in biology and biomedicine. horm behav., 43:2–15, 2003. [7]. berg t., erikson u. & nordtvedt t. rigor mortis assessment of atlantic salmon (salmo salar) and effects of stress. j. food sci. 62, 439–446, 1997. [8]. okomoda v.t., koh i.c.c. hassan a. amornsakun t. & shahreza m.s. hematological parameters of pure and reciprocal crosses of pangasianodon hypophthalmus (sauvage, 1878) and clarias gariepinus (burchell, 1822). comparative clinical pathology 27:549–554, 2018. doi: 10.1007/s00580-017-2623-z. [9]. ahmed i., sheikh z.a. comparative study of hematological parameters of snow trout schizopyge plagiostomus and schizopyge niger inhabiting two different habitats. european zoological journal 12-19, 2020. doi.org/10.1080/24750263.2019.1705647 [10]. davis k. b. management of physiological stress in finfish aquaculture. n am j aquacult 68(2):116-121, 2006. [11]. schmitt c. j., blazer v. s., dethloff g. m., tillitt d. e., gross t .s., bryant jr. w. l., deweese l. r., smith s. b., goede r. w., bartish t. m. & kubiak t. j., biomonitoring of environmental status and trends (best) program: field procedures for assessing the exposure of fish to environmental contaminants. information and technology report usgs/brd-1999-0007. u.s. geological survey, biological resources division, columbia, 68 pp, 1999. [12]. shaw a.f., a direct method for counting the leucocytes, thrombocytes and erythrocytes of bird’s blood, journal of pathology bacteria, 33, 833-835, 1930. [13]. bauck g. r. & ball r. c., influence of capture methods on blood characteristics and mortality in the rainbow trout (salmo gairdneri), translantic american fisheries society, 95(2), 170-176, 1966. [14]. shah s. l., haematological parameters iu tench, tinca tinca after short term exposure to lead. journal of applied toxicology, 26 (3), 223228, 2006. [15]. hattingh j. & van pletzen a. j. j., the influence of capture and transportation on some blood parameters of fresh water fish. comp. biochem. phys., 4ta, 607-609, 1974. [16]. gabriel u. u., akinrotimi o. a. & eseimokumo f., haematological responses of wild nile tilapia oreochromis niloticus after acclimatation to captivity. jordan journal biol sci., 4,225-30, 2011. [17]. pickering a. d. & pottinger t. g., crowding causes prolonged leucopenia in salmonid fish, despite interrenal acclimation, journal of fish biology, 30: 701–712, 1987. [18]. davis a. k., maney d. l. & maerz j. c., the use of leukocyte profiles to measure stress in vertebrates; a review for ecologists. functional ecology 22, 760-772, 2008. [19]. chen w. h., sun l.t., tsai c. l., song y. l. & chang c. f., “cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin m, and leukocyte phagocytosis in tilapia.” general and comparative endocrinology 126: 90100, 2002. [20]. pickering a. d. & pottinger t. g., cortisol can increase the susceptibility of brown trout, salmo trutta l., to disease without reducing the white blood cell count, journal of fish biology, 27, 611-619, 1985. [21]. vijayan m. m, ballantyne j. s. & leatherland j. f., high stocking density alters the energy metabolism of brook charr, salvelinus fontinalis. aquaculture, 88:371–81, 1990. doi: 10.1016/0044-8486(90)90 162-g. [22]. seibel h, baßmann b. & rebl a., blood will tell: what hematological analyses can reveal about fish welfare. front. vet. sci. 8, 616955, 2021. doi: 10.3389/fvets.2021.616955 http://www.fao.org/documents/card/en/c/i9622en http://www.fao.org/documents/card/en/c/i9622en http://www.fao.org/fishery/facp/nga/en http://futospace.futo.edu.ng/xmlui/bitstream/handle/123456789/1894/igoni.pdf?sequence=1&isallowed=y http://futospace.futo.edu.ng/xmlui/bitstream/handle/123456789/1894/igoni.pdf?sequence=1&isallowed=y http://futospace.futo.edu.ng/xmlui/bitstream/handle/123456789/1894/igoni.pdf?sequence=1&isallowed=y http://scholar.google.com/scholar_lookup?hl=en&publication_year=2018&author=qn.+igoni-egweke&title=analysis+of+value+addition+in+commercial+catfish+%28clarias+gariepinus+heterobranchus+spp.%29+production+in+rivers+state 1. introduction 4. conclusion 94 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 94 98 polyamine variation in raw ground pork and beef meat octavian baston1, *octavian barna1 1dunarea de jos university of galati, 111, domneasca street, 800201, galati, romania, octavian.baston@ugal.ro , octavian.barna@yahoo.com *corresponding author received 9th march, accepted 19th march 2014 abstract: samples of fresh ground beef and pork meat were studied for biogenic amine content. the initial amount of putrescine, cadaverine, spermidine and spermine was studied. the initial amount of cadaverine and putrescine were very low or not detected in both raw ground beef and pork meat. the initial amount of spermidine for ground pork meat was 5.56 ± 0.18 mg/kg dry weight and for ground beef meat was 4.32 ± 0.53 mg/kg dry weight. the initial amount of spermine was much higher than spermidine. the spermine in fresh ground pork meat was 26.33 ± 0.72 mg/kg dry weight and in fresh ground beef was 32.08 ± 0.17 mg/kg dry weight. at aerobically refrigeration storage for ten days, the amount of putrescine and cadaverine increased. the spermidine and spermine amounts slightly varied compared with putrescine and cadaverine along refrigeration storage of ground meats. putrescine and cadaverine can be used as spoilage indicators for ground beef and pork meat stored in a refrigerated state for a longer time. key words: consumer health, shelf life, romanian traditional dishes. 1. introduction biogenic amines can be produced by biosynthesis in the living organisms or by microbial activity. putrescine, cadaverine, spermidine and spermine are polyamines that are found in eukaryote cells. spermidine and spermine are biogenic amines that occur naturally in living organisms and are present after the organisms are dead. because they are charged molecules, they bind to deoxyribonucleic acid (dna), ribonucleic acid (rna), and proteins. kusano [1] states that spermidine and spermine are essential regulators of growth, gene transcription, and ribosome-mediated translation. so, these two polyamines can be found in ground meat since they are located in cells and tissues. usually, spermidine amount in fresh beef and pork meat is up to 5 mg/kg. spermine amount in fresh beef and pork meat is higher compared with spermidine content, ranging between 20 and 40 mg/kg [2]. putrescine and cadaverine are biogenic amines that are considered products of alteration by microbial activity. they derive from free amino acids by decarboxilation. cadaverine is produced from lysine amino acid and putrescine from ornithine amino acid [3]. these two biogenic amines, in certain amount, influence negatively the sensorial characteristics of foods. their names are associated with unpleasant odor and with meat alteration. the putrescine and cadaverine amount found in fresh meat is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 octavian baston, octavian barna, polyamine variation in raw ground pork and beef meat, issue 1 2014, pag. 94 98 95 low, usually not detected or around the limit of detection of the equipment. the increased levels of the biogenic amines are due to microbial activity. the microbial activity in meat is due to poor hygiene, possibly while slaughtering, cutting and mincing in case of ground meat. ground meat is used in romanian dishes in combination with vegetables, pasta, cheese, eggs, or as meatballs. we mention some of the romanian traditional dishes such as „mici”, which are minced meat rolls, „sarmale” made from ground meat with rice wrapped in wine or pickled cabbage leaves, sausages, „chiftele” or meatballs, „perisoare” or meatballs for soup, all of them using ground meat. therefore, the polyamine content of the main ingredient (ground meat) is very important in terms of food innocuousness and human health. the research aims are: to assess the initial levels of putrescine (put), cadaverine (cad), spermidine (spd) and spermine (spm) in fresh raw ground meat. to determine biogenic amine levels in refrigerated raw ground meat stored under aerobically conditions for ten days. 2. materials and methods sampling and refrigeration raw ground beef and pork meat were purchased directly from butcheries. all samples were put into an ice box for transportation to the laboratory. the samples were purchased from the same traders as ready to use fresh ground meat, on the production day. they were refrigerated at 4 ± 2 °c for ten days using the dbk386 wd (beko, turkey) refrigerator. the samples were analyzed every two days. all the samples were stored aerobically, in plastic bags, packed since they were purchased from butcheries. the samples needed for analyses were taken aseptically from the original package. chemical analyses the chemical determ inations were made on the same day of the purchase. in total we analyzed 21 samples of ground pork meat and 19 samples of ground beef meat. the biogenic amine determinations were made in triplicates. chemical analyses of meats were made on 12 samples, in triplicates. raw protein content was determined by digestion, distillation and titration by kjeldahl method according to aoac 984.13 method [4] using udk 130 d distilling unit (velp scientifica, italy). fat amounts were determined by soxhlet extraction with solvent according to aoac 945.16 method [4]. ash contents were determined following aoac 942.05 method [4] using fb 1300 furnace (barnstead, usa). moisture levels were determined by air drying in the oven at 100 °c according to aoac 950.46 method [4] using ule 400 oven (memmert, germany). all the reagents used for ground meat analysis were analytical grades. the determination of biogenic amine amounts using high performance liquid chromatography was performed according to the method described in baston [5]. the calibration curves for biogenic amines are linear, having r2 as follows: putrescin r2 = 0.9987; cadaverine r2 = 0.9985; spermidine r2 = 0.9981; spermine r2 = 0.9983. the concentration of each biogenic amine was expressed in mg/kg dry weight. all the reagents were chromatographical grades. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 octavian baston, octavian barna, polyamine variation in raw ground pork and beef meat, issue 1 2014, pag. 94 98 96 statistical data treatment the statistic analysis was made using microsoft office excel to determine the mean and standard deviations. 3. results and discussion a very important indicator about the biogenic amine levels in raw ground beef and pork meat is the chemical structure of the products. therefore, we analyzed the meats and the results are presented in table 1. table 1 chemical parameters of raw ground beef and pork meat product protein (g %) total fat (g %) ash (g %) water (g %) raw ground pork meat 15.83 ± 1.22 20.88 ± 2.62 1.04 ± 0.86 62.25 ± 7.77 raw ground beef meat 17.60 ± 3.40 17.34 ± 4.10 0.91 ± 0.73 64.22 ± 5.05 the results represent means ± standard deviation the chemical analysis of ground meats cannot be compared with the chemical composition of pork or beef meats because it depends on the fattening state of the animal, age, anatomical area used for mincing and the fat amount added to the ground meat composition. it has been acknowledged that ground meats have a higher amount of fat and a smaller amount of protein. generally, our average values for chemical parameters can be compared with the ones of a fat top round of beef and fat ham for pork [6]. protein amount is the only important parameter for our study because the proteins have amino acids that can be broken to biogenic amines by spoilage microbiota. in our case, cadaverine and putrescine are the biogenic amines. figure 1 presents the putrescine variation in raw ground meats. initially, the putrescine content of ground beef is below the detection limit. ground pork meat has an amount of 0.10 ± 0.09 mg/kg. on the second day of refrigerated storage, the putrescine amount of ground beef is still under the detection limit, while in pork ground meat it increased to 0.69 ± 0.61 mg/kg. beginning with the fourth day of storage, the putrescine in ground pork and beef meats is increasing. this increase is due to microbial decarboxilation of free ornithine. fig. 1 putrescine variation in ground meats. as can be seen in figure 2 the cadaverine amount of raw ground beef meat in the first four days of determination is below the detection limit. cadaverine was found in pork meat on the second day of refrigeration to be under 1 mg/kg. after the second day of refrigeration, the cadaverine amount has increased in both types of meats studied. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 octavian baston, octavian barna, polyamine variation in raw ground pork and beef meat, issue 1 2014, pag. 94 98 97 . fig. 2 cadaverine variation in ground meats. the highest amount of biogenic amines was registered on the tenth day of storage in ground pork meat. spermidine was found in small amounts in both types of ground meats. fig. 3 spermidine variation in ground meats. the initially registered spermidine values were of 5.56 ± 0.18 mg/kg in ground pork meat and of 4.32 ± 0.53 mg/kg in ground beef meat. since spermidine is an amine produced by the body, it is found in tiny amount in pork and beef meat, as other researchers stated in their works [4,7]. after ten day-storage the spermidine in ground pork meat varied between 4.5 and 6 mg/kg. the amount of spermidine in ground beef meat was smaller than that determined in ground pork meat and it varied depending on refrigerated storage from 4.12 to 4.9 mg/kg (as mean values). the variation of spermidine is due to its production from spermine and its use as carbon and nitrogen source by spoilage bacteria [7,8]. high amount of spermine was found initially in ground pork and beef meats as compared with spermidine. the following spermine values were obtained: 26.33 ± 0.73 mg/kg in ground pork meat and 32.08 ± 0.17 mg/kg in ground beef meat respectively. the spermine variation after ten day refrigeration storage is low, values between 25.01 ± 2.47 and 26.33 ± 0.73 mg/kg being registered in ground pork meat and 31.0 ± 0.84 to 32.19 ± 0.68 mg/kg in ground beef meat respectively. the spermine variation in beef and pork meats is due to its production from putrescine and its transformation to spermidine [9,7]. in beef and pork meats spermine can be found in a large range, from 13.3 to 47.5 mg/kg in beef and from 14.5 to 70.3 mg/kg in pork meat [2]. fig. 4 spermine variation in ground meats. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 octavian baston, octavian barna, polyamine variation in raw ground pork and beef meat, issue 1 2014, pag. 94 98 98 4. conclusion the polyamine values registered by our research comply with the limits established in the literature. the smaller amounts of some polyamines found in ground meats are due to many factors of influence. one of them is the chemical composition of ground meats with reference to protein amount. and if this is low, then the polyamines that can be produced by microorganisms will be low, too. meat hygiene is also very important because the initial contamination and the type of microorganism influence the polyamine type and amount. moreover, polyamine amounts in ground meats are influenced by the age of the animal, fat addition or contained by the meat, and the anatomical area used. 5. references [1] kusano t., berberich t., tateda c., takahashi y., polyamines: essential factors for growth and survival, planta, 228(3). 367-381, (2008). [2] kalač p., biologically active polyamines in beef, pork and meat products: a review, meat science, 73(1), 1-11, (2006). [3] segal r., biochimia produselor alimentare, ed. academica, galaţi, 360 p, (2006). [4] association of official analytical chemists (aoac), official methods of analysis (18th ed.), washington dc, (2005). [5] baston o., stroia a.l., moise d., barna o., validation study of a hplc method able to measure biogenic amines in chicken meat, annals of the university dunarea de jos of galati fascicle vi-food technology, 1(31), (2008). [6] banu c., calitatea şi analiza senzorială a produselor alimentare, bucureşti, agir, 183 p, (2007). [7] min j.s., lee s. o., jang a., jo c., park c.s., lee m., relationship between the concentration of biogenic amines and volatile basic nitrogen in fresh beef, pork, and chicken meat, asian australasian journal of animal sciences, 20(8), 1278, (2007). [8] dasu v.v., nakada y., ohnishikameyama m., kimura k., itoh y., characterization and a role of pseudomonas aeruginosa spermidine dehydrogenase in polyamine catabolism, microbiology, 152(8), 22652272, (2006). [9] chen m.t., lin y. s., tsai h. t., kuo h.l., efficiency of hurdle technology applied to raw cured meat (si-raw) processing, asian-aust. j. anim. sci, 15(11), 1646-1652, (2002). 219 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 3 2020, pag. 219 227 occurrence and assessment of physical contaminants based on food recalls in canada *mohd taufiq mohd khairi 1 , sallehuddin ibrahim 1 , mohd amri md yunus 1 , mahdi faramarzi 1 , jaysuman pusppanathan 2 , azwad abid 1 1school of electrical engineering, faculty of engineering, universiti teknologi malaysia, johor, malaysia. 2sport innovation and technology centre (sitc), institute of human centred engineering (ihce), school of biomedical engineering & health sciences, faculty of engineering, universiti teknologi malaysia, johor, malaysia. taufiq_khairi@yahoo.com *corresponding author received 25th march 2020, accepted 14th september 2020 abstract: this paper investigates the association between types of food products, physical contaminants and year using food recalls dataset obtained from the canadian food inspection agency (cfia) between 2014 and 2019. in the period of studies, a total of 269 foods under the category of physical hazards were recalled. chi-square per cell test was used to deeply analyse the contingency table of the investigated topic categories. the results show that there is no association between the year and the number of food recalls by food products and year by physical contaminants type. however, the results indicated that there is an association between the food products and physical contaminants type. in particular, there were significant numbers of insects being found in grain and fruits/vegetables products with 15 and 53 cases, respectively. plastic and bone fragments were significantly found in dairy and meat, poultry and seafood products, respectively with 9 and 15 cases. glass was significantly found in wine and beverage (6 cases) and other food product (9 cases). plastic material was highly detected in candy and confectionery product with 9 cases. the sources of the physical contaminants have been analysed, together with the precautionary measures that must be taken. findings from this study provide the food industry with essential information. an understanding and analysis of physical hazards is critical for companies in order to restructure their food safety policies and technologies. keywords: physical contaminants, food recall, food safety, foreign bodies 1. introduction food contaminated by hazardous agents may endanger the consumer’s safety. among the classifications of hazards there are chemical, biological and physical ones [1]. chemical hazards include water, pesticides and food additives. viruses (hepatitis a and rotavirus), parasites (e.g. trichinella spiralis and cryptosporidium parvum) and harmful bacteria (bacillus cereus and salmonella) are biological hazards [2]. on the other hand, physical hazard in food may refer to a foreign body that existed in a food product [3,4]. objects such as metal fragments, glass, plastic pieces, stones, insects and wood debris are among frequent type of objects that have been found in food [5,6]. the existence of a foreign body may harm the consumer if it is accidentally being consumed. it can cause choking and internal injuries especially in the abdomen and intestines when it is ingested. as a consequent, surgery needs to be carried out to remove the foreign body [7]. a foreign body might accidentally enter food at any stage of the food chain such as processing, http://www.fia.usv.ro/fiajournal mailto:taufiq_khairi@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 220 packaging and distribution of food. food and drugs administration (fda) has regulated that the unwanted object in food that has a length of 7 mm to 25 mm is considered as a foreign body [8]. the food industry makes numerous efforts to avoid unwanted foreign objects in food. several technologies and techniques have been applied to detect foreign bodies such as a metal detector, magnet, x-ray, ultrasound, near-infrared, terahertz and surface penetrating radar [9–11]. although these methods are available, there are still cases of food recalls by food agencies. some manufacturers do not use these technologies due to cost constrain. in addition, the weaknesses of each technique have restricted their applications and abilities to detect all types of foreign bodies. recall notifications have to be made when the foods are believed to be contaminated and may harm the consumers. the food recalls were usually conducted by the food manufacturer or distributor. it also can be requested by government authorities or agencies around the world. the food recalls statistics is annually published based on the data collected from reports or complaints from a variety of sources such as manufacturers, retailers, government agencies and consumers. several authors used the database in their research primarily for analysis of microbial and chemical hazard [12–15]. although there have been numerous studies conducted on high-risk pathogenic threats such as salmonella, e. coli, and listeria, to our knowledge still there is a lack of research in food recalls focusing on physical hazards which are equally important to address. food recalls analysis in food industry was performed in recent studies by potter et al. [16] and page [17] where the results revealed that the number of recalls regarding the physical hazard is less frequent than biological and chemical hazards. therefore, the study and analysis on physical hazard is important, rendering possible the use as references for manufacturers to implement the hazard analysis and critical control point (haccp) program in order to predict the causes and implement preventive measures related to type of particular physical hazards and a particular type of food. food recalls can cause significant economic losses in food industry and consumers also could lose their confidence in that product [18–20]. in this sense, the aim of this study is to provide a descriptive statistics of food recalls cases based on physical contamination occurring in canada from the year 2014 to 2019. 2. matherials and methods 2.1 sampling the data were collected from the canadian food inspection agency (cfia) website (http://www.inspection.gc.ca). the website showed that the food that had been recalled was due to biological, chemical and physical hazards. all notifications that were recorded in the website under the physical hazard category were extracted for the period from january 1, 2014 to december 31, 2019. within the records, the year, the food product category and type of foreign bodies were classified and tabulated in tables. all the degree classes of health risk which are class i, class ii and class iii are considered in the sampling data. 2.2 statistical analysis the association between the year and the number of food recalls by product category and types of foreign bodies were evaluated using the global chi-square test and chihttp://www.inspection.gc.ca/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 221 square per cell test [21]. the tests had been used in previous food safety studies for dairy foods [22] and moldy foods [23]. in this study, the test has been carried out to determine whether there are statistically significant differences between different types of food products, different types of foreign objects and year. the statistical processing was performed using xlstat 2019.2 software (adinsoft, paris, france) and the statistical level significance was set at α = 0.05. 3. results and discussion in the period of 2014 2019, a total of 269 foods from cfia under the category of physical hazard were recalled where the distribution by year is shown in figure 1. the data show that the year 2016 has the highest number of food recalls with 58 cases. the trend of graph also revealed that the number of recalls have increased from the year 2014 to 2016 before it fell consecutively in 2017 and 2018. in 2019, the recalls number increased with 43 cases. the total of food recalls has been sorted into food categories as shown in the figure 2. the statistic shows that the fruit and vegetable products have the highest number of recall notifications with 82 cases, followed by meat, poultry and seafood products (50 cases), other food products (35 cases), grain and cereals (29 cases) as well as candy and confectionary (20 cases). other food products comprise foods such as soup, sauce, vinegar and baby food. fig. 1: number of foods recalls due to physical hazards from the year 2014 to 2019 table 1 and table 2 show the quantitative data from 2014 to 2019 for types of food products and types of foreign bodies, respectively. as computed with global chisquare test, there was no association between the year and the type of food product (observed χ2 = 47.909; critical χ2 = 72.153; p = 0.707) as well as types of foreign bodies (observed χ2 = 56.453; critical χ2 = 72.153; p = 0.383). the results show that the p-value for both tables are greater than the significance level α = 0.05. in table 1, the chi-square test per cell shows similar citations for all types of food products except nut and bakery products throughout the years of study. a lower citation was observed in 2015 for breads/bakery products. in 2018 and 2019, lower citation was demonstrated for nut. high citations were revealed for breads and bakery with 7 cases in 2019. overall, there was no significant difference in citations for all food products except for nut and breads/bakery products. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 222 fig. 2: number of foods recalls due to physical hazards for different food products the results of chi-square test per cell for the types of foreign bodies from 2014 to 2019 are shown in table 2. metal item indicated that it was the most significantly cited in 2014 and has a lower frequency of citation in 2015. it can also be observed that the item plastic did not reveal any significant differences of citations during the period of study. glass and insects are noticeably less cited in 2017 and 2015, respectively. fewer citations are also observed for bone in 2018 and rubber items in 2014 and 2019. wood item indicated that it was significantly less cited in 2017 and 2018, while stone item shows the same results in 2014, 2016 and 2017. finally, there was no significant difference in citations for unknown object throughout the years of study. overall, food recalls caused by insects had shown the highest notification in 5 years with 81 cases, followed by metal (56 cases), plastic (50 cases), unknown object (25 cases) and glass (22 cases). the unknown object is considered as a solid object whose material cannot be identified. table 1 distribution of foreign bodies based on product type from the year 2014 to 2019 product type years 2014 2015 2016 2017 2018 2019 total grain and cereals 2 7 10 3 3 4 29 fruit and vegetables 13 8 18 20 14 9 82 dairy 1 5 2 7 3 3 19 meat, poultry and seafood 9 11 6 10 5 9 50 nut 1 1 1 2 0(-)* 0(-)* 7 breads and bakery 2 0(-)* 5 2 1 7(+)* 17 wine and beverages 1 2 2 2 2 1 10 candy and confectionery 1 3 2 7 3 2 20 other food products 5 4 12 4 4 8 35 total 35 41 58 57 35 43 269 * the effect of the chi-square per cell. (+) or (-) indicates that the observed value is higher or lower than the expected theoretical value. significance level, α < 0.05. 29 82 19 50 7 17 10 20 35 0 20 40 60 80 100 n u m b e r o f fo o d r e c a ll s food products food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 223 table 2 distribution of types of foreign bodies from the year 2014 to 2019 foreign bodies years 2014 2015 2016 2017 2018 2019 total metal 13(+)* 4(-)* 14 7 8 10 56 plastic 8 11 8 14 4 5 50 glass 3 4 5 1(-)* 5 4 22 insects 6 7(-)* 20 23 13 12 81 bone 1 3 2 5 0(-)* 6 17 rubber 0(-)* 3 2 1 1 0(-)* 7 wood 1 2 2 0(-)* 0(-)* 1 6 stone 0(-)* 1 0(-)* 0(-)* 1 3 5 unknown 3 6 5 6 3 2 25 total 35 41 58 57 35 43 269 * the effect of the chi-square per cell. (+) or (-) indicates that the observed value is higher or lower than the expected theoretical value. significance level, α < 0.05 in table 3, a global chi-square test revealed that there is an association between food products and types of foreign bodies (observed χ2 = 190.512; critical χ2 = 103.010; p < 0.0001). as computed, the p-value is lower than the significance level α = 0.05. the results of chi-square per cell test for product and foreign body types are demonstrated in table 3. the statistics revealed that grain and cereal products have low citation for wood item with no recall notification. concerning fruits and vegetables, it is observed that insects are significantly more cited, and the other items show fewer citations except rubber and the unknown object. dairy product presented a higher frequency of citation for plastic item and fewer citations for insects, bone, rubber, wood and stone. the citations for meat, poultry and seafood product are significantly higher for bone and significantly lower for glass, insects and stone. there is no recall notification regarding nut product for metal, glass, bone, rubber, wood, stone and unknown objects. for breads and bakery products, lower citations are observed for bone, rubber, wood, stone and unknown objects. wine and beverages are markedly significantly higher for glass item and there are no complaints regarding plastic, bone, rubber, wood and stone. candy and confectionery show low citation for glass, insects, bone and stone items. finally, other food products exhibited a higher frequency of citations for glass items and fewer citations for bone and rubber items. the correlation between food products and the types of foreign bodies is interesting to analyse. overall, insect items have the most recall notifications among food products, followed by metal and plastic items with 69, 46 and 45 cases, respectively. all types of foreign bodies have been reported in the period of studies except for wood in grain and cereals products. insects such as ant, flies and larvae show the most recalls in the fruit and vegetable products with 47 cases. this is not surprising because insects are usually attracted to this kind of products. sometimes, there are hidden or trapped in the product which is difficult to detect. visual inspection systems such as food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 224 conventional x-ray has difficulty in detecting insects because it cannot distinguish the difference between the density of the product and insects [24]. terahertz and near-infrared techniques could be utilized to detect insects as reported in [25–27]. this kind of contamination has to be prevented and removed since it may bring along pathogens and germs. table 3 distribution of physical hazards incidence according to product and foreign bodies types product types foreign bodies metal plastic glass insects bone rubber wood stone unknown total grain and cereals 6 2 2 10 2 1 0(-)* 1 1 25 fruits and vegetables 7(-)* 5(-)* 2(-)* 47(+)* 0(-)* 3 0(-)* 0(-)* 9 73 dairy 5 8(+)* 1 1(-)* 0(-)* 0(-)* 0(-)* 0(-)* 3 18 meat, poultry and seafood 13 10 0(-)* 1(-)* 9(+)* 2 2 0(-)* 4 41 nut 0(-)* 3 0(-)* 2 0(-)* 0(-)* 0(-)* 0(-)* 0(-)* 5 breads and bakery 3 5 1 1 0(-)* 0(-)* 0(-)* 0(-)* 0(-)* 10 wine and beverages 1 0(-)* 5(+)* 1 0(-)* 0(-)* 0(-)* 0(-)* 2 9 candy and confectionery 6 7 0(-)* 0(-)* 0(-)* 1 1 0(-)* 1 16 other food products 5 5 7(+)* 6 0(-)* 0(-)* 2 1 3 29 total 46 45 18 69 11 7 5 2 23 226 * the effect of the chi-square per cell. (+) or (-) indicates that the observed value is higher or lower than the expected theoretical value. significance level, α < 0.05. for dairy products, plastic is the most frequent foreign body that leads to recall notifications with 8 cases, followed by metal with 5 cases. plastic fragments may exist through packing debris, equipment and pallets. several techniques that can be used to detect plastic materials are the filtering method [28], ultrasound [29] and hyperspectral imaging [30]. the conventional x-ray may not be appropriate to utilize since x-ray has a limitation in detecting low-density materials like insects and plastic. however, li et al. [24] and einarsdóttir et al. [31] have proposed a solution on this issue by introducing the polycapillary x-ray lens and grating-based multimodal x-ray imaging. metal has been mostly found in meat, poultry and seafood products which subsequently lead to recall warning. the existence of metal might be due to fragments from chopping tools and machine equipment parts [32]. bone had been observably getting more citations in this type of food product where the source might come from the raw materials itself such as bones in fish or meat. it may due to the imperfect separation process between fish/meat and bone. metal and bone items could be distinguished using visual inspection such as near-infrared (nir) spectroscopy [33] and hyperspectral imaging (hsi) technique [34]. the presence of metal also can be prevented by utilizing low cost techniques such as metal or magnet detection system [35]. the food recalls for nut and bakery products is mostly due to plastic debris with 3 and 5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 225 cases have been reported, respectively. whereas, broken glass was frequently found in wine and beverages as well as other food products. it may be due to broken containers and jars that are used to fill up the product [36]. imaging techniques such as thermal imaging [37], ultrasound [38] and terahertz [39] are suitable to be applied for monitoring this kind of products since it is non-invasive and suitable be use in the production line. with regard to candy and confectionery product, plastic and metal materials had contributed to the food recall with 7 and 6 cases, respectively. these items probably originated from the packaging material and machinery parts. early detection and removal of foreign bodies are important to maintain good manufacturing practice. preventive measures for the presence of foreign bodies can be divided into three main categories stages; plant/raw material, processing and final product. pest control implementation can prevent pesticides from attacking the raw materials [40]. the use of technology such as sieving and filtration system to detect foreign bodies can be applied in food plant and food processing environment. periodic training should be given to employees in handling the process of food production and monitor by supervisors [41]. employees must be always reminded to practice good personal hygiene such as keep hand’s clean, short fingernails, avoid wearing jewellery and wearing clean clothes. food products are usually packed and located in the final production line. therefore, visual inspection technologies such as x-ray, hyperspectral and ultrasound imaging systems are best suited to be utilized to ensure food is completely free from foreign materials [42]. the imaging systems have several advantages such as non-invasive and non-destructive, able to operate in real-time and provide high imaging resolution. however, not all companies especially in small scale industry are afforded to utilize it due to the high cost [43]. 4. conclusion the findings show that food recalls due to the incidence of physical contaminants is relatively small compared to biological and chemical contaminants; still, it should not be neglected. food recall is a vital action to ensure that all the affected products are removed from the market as soon as possible. detection techniques especially visual inspection play important role in ensuring the highest safety and quality level regardless of the food product type. authorities need to make periodic inspections to ensure that the manufacturers comply with the good manufacturing practice (gmp) and standard operating procedure (sop) for handling food. the food adulteration due to foreign bodies can be avoided with the cooperation and endeavour of all the parties involved. 5. acknowledgments the authors would like to acknowledge the assistance of the ministry of higher education malaysia under the mybrain15 program and universiti teknologi malaysia for providing the research grants 15h85 and 4j255 which enabled this research to be carried out. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 226 6. references [1] a. liu, l. shen, y. tan, z. zeng, y. liu, c. li, food integrity in china: insights from the national food spot check data in 2016, food control, 84: 403–407, (2018). [2] n.a. abdul-mutalib, a.n. syafinaz, k. sakai, y. shirai, an overview of foodborne illness and food safety in malaysia, int. food res. j, 22(3): 896–901, (2015). [3] m.c. edwards, m.f. stringer, t.b. in f.s. group, observations on patterns in foreign material investigations, food control, 18(7): 773– 782, (2007). [4] c.a. batt, chemical and physical hazards in food, ref. modul. food sci, 1–2, (2016). [5] i. djekic, d. jankovic, a. rajkovic, analysis of foreign bodies present in european food using data from rapid alert system for food and feed (rasff), food control, 79: 143–149, (2017). [6] e.c. mattos, v.s.m.g. daros, r. dal col, a.l. nascimento, occurrence of foreign matter in food: applied identification methodassociation of official agricultural chemists (aoac) and food and drug administration (fda), int. j. biol. biomol. agric. food biotechnol. eng, 10(3): 101–105, (2016). [7] a.r. olsen, regulatory action criteria for filth and other extraneous materials. i. review of hard or sharp foreign objects as physical hazards in food., regul. toxicol. pharmacol. 28(3): 181–189, (1998). [8] fda compliance policy guidance, cpg sec. 555.425 foods, adulteration involving hard or sharp foreign objects, 1–3, (2005). [9] m. graves, a. smith, b. batchelor, approaches to foreign body detection in foods, trends food sci. technol, 9: 21–27, (1998). [10] d. peariso, preventing foreign material contamination of foods, blackwell publishing, iowa, usa, 2006. [11] m.t.m. khairi, s. ibrahim, m.a.m. yunus, m. faramarzi, noninvasive techniques for detection of foreign bodies in food: a review, j. food process eng, 41(6): 1–20, (2018). [12] s. wong, d. street, s.i. delgado, k.c. klontz, recalls of foods and cosmetics due to microbial contamination reported to the u.s. food and drug administration, j. food prot, 63(8): 1113–1116, (2000). [13] x. shang, g.t. tonsor, food safety recall effects across meat products and regions, food policy. 69: 145–153, (2017). [14] r. teratanavat, n.h. hooker, understanding the characteristics of us meat and poultry recalls: 1994-2002, food control, 15(5): 359–367, (2004). [15] s. paramithiotis, e.h. drosinos, p.n. skandamis, food recalls and warnings due to the presence of foodborne pathogens — a focus on fresh fruits, vegetables, dairy and eggs, curr. opin. food sci, 18: 71–75, (2017). [16] a. potter, j. murray, b. lawson, s. graham, trends in product recalls within the agri-food industry: empirical evidence from the usa, uk and the republic of ireland, trends food sci. technol, 28(2): 77–86, (2012). [17] e.t. page, trends in food recalls: 2004-13, eib-191, u.s. dep. agric. econ. res. serv. (2018). [18] v.f. pozo, t.c. schroeder, evaluating the costs of meat and poultry recalls to food firms using stock returns, food policy, 59: 66–77, (2016). [19] o. unsal, m.k. hassan, d. zirek, product recalls and security prices: new evidence from the us market, j. econ. bus. 93: 62–79, (2017). [20] d. kong, l. shi, z. yang, product recalls, corporate social responsibility, and firm value: evidence from the chinese food industry, food policy, 83: 60–69, (2018). [21] r. symoneaux, m. v. galmarini, e. mehinagic, comment analysis of consumer’s likes and dislikes as an alternative tool to preference mapping. a case study on apples, food qual. prefer, 24(1): 59–66, (2012). [22] r.s. aguiar, e.a. esmerino, r.s. rocha, t.c. pimentel, v.o. alvarenga, m.q. freitas, m.c. silva, a.s. sant’ana, a.c.o. silva, a.g. cruz, physical hazards in dairy products: incidence in a consumer complaint website in brazil, food control, 86: 66–70, (2018). [23] j.g. lemos, m.v. garcia, r. de o. mello, m.v. copetti, consumers complaints about moldy foods in a brazilian website, food control, 92: 380–385, (2018). [24] f. li, z. liu, t. sun, y. ma, x. ding, confocal three-dimensional micro x-ray scatter imaging for non-destructive detecting foreign bodies with low density and low-z materials in food products, food control, 54: 120–125, (2015). [25] g.j. kim, j.-i. kim, s.-g. jeon, j. kim, k.k. park, c.-h. oh, enhanced continuous-wave terahertz imaging with a horn antenna for food food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 mohd taufiq mohd khairi, sallehuddin ibrahim, mohd amri md yunus, mahdi faramarzi, jaysuman pusppanathan, azwad abid, occurrence and assessment of physical contaminants based on food recalls in canada, food and environment safety, volume xix, issue 3 – 2020, pag. 219 – 227 227 inspection, j. infrared, millimeter, terahertz waves, 33(6): 657–664, (2012). [26] g. ok, h.j. kim, h.s. chun, s.w. choi, foreign-body detection in dry food using continuous sub-terahertz wave imaging, food control, 42: 284–289, (2014). [27] k. bhuvaneswari, p.g. fields, n.d.g. white, a.k. sarkar, c.b. singh, d.s. jayas, image analysis for detecting insect fragments in semolina, j. stored prod. res, 47(1): 20–24, (2011). [28] fda administration, hazards & controls guide for dairy foods haccp, (2007). [29] m.t.m. khairi, s. ibrahim, m.a.m. yunus, m. faramarzi, ultrasonic tomography for detecting foreign objects in refrigerated milk cartons, int. j. dairy technol, 71(4): 1–7, (2018). [30] a.a. gowen, c.p. o’donnell, p.j. cullen, g. downey, j.m. frias, hyperspectral imaging an emerging process analytical tool for food quality and safety control, trends food sci. technol, 18(12): 590–598, (2007). [31] h. einarsdóttir, m.j. emerson, l.h. clemmensen, k. scherer, k. willer, m. bech, r. larsen, b.k. ersbøll, f. pfeiffer, novelty detection of foreign objects in food using multi-modal x-ray imaging, food control, 67: 39–47, (2016). [32] e.d. van asselt, h.j. van der felsklerx, h.j.p. marvin, h. van bokhorstvan de veen, m.n. groot, overview of food safety hazards in the european dairy supply chain, compr. rev. food sci. food saf, 16: 59–75, (2017). [33] p. pallav, g.g. diamond, d.a. hutchins, r.j. green, t.h. gan, a nearinfrared (nir) technique for imaging food materials, j. food sci, 74(1): 23–33, (2009). [34] s.c. yoon, k.c. lawrence, d.p. smith, b. park, w.r. windham, bone fragment detection in chicken breast fillets using transmittance image enhancement, trans. am. soc. agric. biol. eng. 51(1): 331–339, (2008). [35] m.c. edwards, detecting foreign bodies in food, woodhead publishing limited, cambridge, england, (2004). [36] e. haeggstrom, m. luukkala, ultrasound detection and identification of foreign bodies in food products, food control, 12: 37–45, (2001). [37] l. senni, m. ricci, a. palazzi, p. burrascano, p. pennisi, f. ghirelli, online automatic detection of foreign bodies in biscuits by infrared thermography and image processing, j. food eng, 128: 146–156, (2014). [38] p. pallav, d.a. hutchins, t.h. gan, air-coupled ultrasonic evaluation of food materials, ultrasonics, 49: 244–253, (2009). [39] c. jördens, m. koch, detection of foreign bodies in chocolate with pulsed terahertz spectroscopy, opt. eng, 47(3): 37003, (2008). [40] k. v. kotsanopoulos, i.s. arvanitoyannis, the role of auditing, food safety, and food quality standards in the food industry: a review, compr. rev. food sci. food saf, 00: 1–16, (2017). [41] m. neio demirci, j.m. soon, c.a. wallace, positioning food safety in halal assurance, food control, 70: 257–270, (2016). [42] q. chen, c. zhang, j. zhao, q. ouyang, recent advances in emerging imaging techniques for non-destructive detection of food quality and safety, trends anal. chem, 52: 261– 274, (2013). [43] m. edwards, other significant hazards: physical hazards in foods, in: encycl. food saf., elsevier ltd., 117–123, (2014). 1. introduction 4. conclusion 34 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xii, issue 1 – 2014, pag. 34 37 ionse lec tiv e e le c tro des ba se d on cal cium hyd roxyl apa t it e as a tool fo r analys is of v ar ious en viron m enta l objec ts , food a nd r aw mat er i al s volodymyr diichuk1, mariia vorobets1, *igor kobasa1 1yu. fedkovych national university of chernivtsy, ukraine, imk-11@hotmail.com * corresponding author received february 19th 2014, accepted accepted march 24th 2014 abstract: a method of the potentiometric determination of ca2+ and hpo42ions in some food and raw products using the original ion-selective electrodes with calcium hydroxylapatite has been developed. the plasticized ion-selective electrodes were constructed of calcium hydroxylapatite on pvc substrate. investigation of the electrode characteristics proved the linear parts on the ca2+ and hpo42v-a dependencies. the slope coefficients also were calculated for the both ions. preliminary treatment of the electrodes in the ca2+solutions results in significant changes in the both slope coefficients, which approach to the theoretical values. keywords: food; environmental objects; ion-selective electrodes; calcium hydroxylapatite 1. introduction deep modernization of the food processing branch is required in order to enhance its competitive advances and to mitigate consequences of the economical crisis, industrial depression and decrease in the labour productivity. a series of actions such as economical changes along with technical reequipment, development of resource and energy-saving technologies and processes, ensuring thorough control of the food and raw materials quality should be taken to support this strategy. various physico-chemical and biological methods are widely involved in such analytical technologies. potentiometry of the environmental objects, food and raw materials quality using the ion-selective electrodes is an advanced and powerful method of investigation. it is express and accurate method that is capable to work with samples from one-two tenth to 1 ml, usually no sample pretreatment is needed, the method can be applied directly to the turbid mixtures, viscous substances without filtration, distillation or extraction. this way, the method is highly suitable to be employed in the automatic and well-controllable mode [1, 2]. however, new solid-ion selective electrodes with high endurance and stable output signal should be designed in order to perform analytical determination of some compounds including ions ca2+ and hpo42-. one of the advantageous methods of synthesis for such electrodes presupposes formation of the electroactive layer directly from calcium hydroxylapatite in course of its synthesis. calcium hydroxylapatite (ca-hoa) can be found in nature and also synthesized. this is the basic phosphate са10(ро4)6(он)2, it food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 volodymyr diichuk, mariia vorobets, igor kobasa, ion-selective electrodes based on calcium hydroxylapatite as a tool for analysis of various environmental objects, food and raw material, volume xiii, issue 1 2014, pag. 34 – 37 35 is non-toxic and almost dissoluble compound [3]. this paper deals with investigation of possible utilization of the hydroxylapatite as the electrode-active substrate for designing of the ion-selective electrodes. 2. experimental a method of chemical depositions from the solutions of calcium nitrate and ammonia hydrophosphate at ph=9-10 according to reaction 10са(no3)2 + 6(nн4)2нрo4 + 8nн4он = са(рo4)6(он)2↓ + 20nн4no3 +6н2o has been used to synthesize the stoichiometric calcium hydroxylapatite. the crystals growth and deposition took place gradually then the sediment was washed by water, then ethanol and dried at 105 0c. the electrode has been made using pvc as substrate. this substance was dissolved in cyclohexanone. then the powder of pure ca-hoa was mixed with the 10 % solution of pvc to form a suspension with the ratio ca-hoa:pvc = 1:50. this suspension was applied on the graphite rod and then dried at the room temperature during a day and night. if required, the above operations could be repeated several times. a series of the sample solutions containing of ca(no3)2 and (nh4)2hpo4 with concentrations of the both compounds ranged from 10-5 to 10-1 mol/dm3 has been prepared to investigate some electro-analytic parameters of the system. two types of the electrodes were used to find a pattern of dependence of the cahoa electrode’s potential on concentration of ions ca2+ and hpo4 2-. first type of electrode was untreated while the second one was kept in the solution of ca(no3)2 and (nh4)2hpo4 with concentrations of the compounds 10-3 mol/dm3 for a day and night prior the investigation. 3. results and discussion it is known [4] that the electrode-active compounds should comply with some requirements: have as low as possible value of solubility product; exhibit ionic type of conductance and be non-toxic and easily accessible. ca-hoa comply with all these requirements: its solubility product is 1.0· 13410 [5], high ionic conductivity was reported in [6] while easy and cheap syntheses were also shown. toxicity investigation of the substance proved its complete safety [7]. it can be expected that the cahoa based electrode should be sensitive to the both types of ions: calcium and phosphates. that is why these ions were selected to find experimentally a character of the electrode potential dependence on the ions’ concentration. dependence for the first type electrode (without preliminary keeping in the solutions) on ca2+ concentration is shown in fig. 1. three different parts with different slopes can be identified in this line. the first and the third parts exhibit low slope meaning that the electrode potential depends on ca2+ concentration insignificantly within the corresponding ranges of ca2+ concentrations. -5 -4 -3 -2 -1 0,12 0,14 0,20 0,22 0,24 lg[ca 2+ ] e, v 1 2 figure 1. dependence of the equilibrium potential of the plasticized electrode on concentration of ca2+: 1 – type 1 electrode (untreated); 2 – type 2 electrode (pretreated). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 volodymyr diichuk, mariia vorobets, igor kobasa, ion-selective electrodes based on calcium hydroxylapatite as a tool for analysis of various environmental objects, food and raw material, volume xiii, issue 1 2014, pag. 34 – 37 36 the second part lies from concentrations 10-4 to 10-2 and exhibits the slope coefficient 0.0145 v/decade, which is close to the theoretical value 0.01475 v/decade calculated for the 4-charge ions. since the experimental dependence was determined for the double-charged ions ca2+, the slope coefficient had to be close to the value 0.0295 v/decade. the significant discrepancy fixed in our experiments can be caused by the effect of some side ions on the electrode potential. -5 -4 -3 -2 -1 0,10 0,12 0,14 0,16 0,20 0,22 0,24 lg[hpo 4 2-] e, v 1 2 figure 2. dependence of the equilibrium potential of the electrode on concentration of hpo4 2-: 1 – type 1 electrode (untreated); 2 – type 2 electrode (pretreated in the 10-3 mol/dm3 solution of ca(no3)2). an ion-selective electrode can usually be kept before the usage in the solution containing the respective ions in order to enhance its characteristics [8]. the curve 2 in fig. 1 represents results of investigation of such pretreated ion-selective electrode after the day and night long keeping in the 10-3 mol/dm3 solution of ca(no3)2. the curve shows a dependence of the electrode potential on ca2+ concentration. it can be seen that the slope coefficient of the curve 2 is different and equal to 0.022 v/decade within 10-5–10-3 mol/dm3 (compare with 0.0145 v/decade for the untreated electrode). therefore, it can be concluded that pretreatment of ca-hoa electrode makes an effect on its slope coefficient bringing it closer to the theoretical value for the double-charged ions (0.0295 v/decade). fig. 2 represents similar dependencies of the electrode potential on the phosphate ions concentration. this dependence has two parts for the untreated electrode (fig. 2, curve 1). the electrode potential changes insignificantly within the first part (concentration of hpo4210-5–10-3 mol/dm3) and slope coefficient is 0.0035 v/decade. in the second part ranged from 10-3 to 10-1 mol/dm3 hpo42-, the slope coefficient is 0.015 v/decade. similarly to the previous case, these values are different from the theoretical value for the doubleand triple charged ions (0.0295 v/decade and 0.0197 v/decade respectively). table 1 linear ranges in dependencies of the ca-hoa electrode potentials on concentrations of ca2+ and hpo4 2and corresponding slope coefficients (s) potential determinating ion ca-hoa-electrode untreated after pretreatment (kept in 10-3 mol/dm3 ca(no3)2 concentration range, mol/dm3 s, v/decade concentration range, mol/dm3 s, v/decade ca2+ 10-4–10-2 0.0145 10-5–10-3 0.0220 hpo4210-3–10-1 0.0150 10-3–10-1 0.0225 the curve 2 represented in fig. 2 represents a dependence of the electrode potential on concentration of hpo42for the pretreated electrode. it can be seen that this dependence is linear within the range of hpo4 2concentration 10-3–10-1 mol/dm3 and the slope coefficient is 0.0225 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xii, issue 1 – 2014 volodymyr diichuk, mariia vorobets, igor kobasa, ion-selective electrodes based on calcium hydroxylapatite as a tool for analysis of various environmental objects, food and raw material, volume xiii, issue 1 2014, pag. 34 – 37 37 v/decade (compare with 0.015 v/decade for the untreated electrode). therefore, the electrode pretreatment influences its characteristics in the solutions of ca2+ and hpo42and brings values of the slope coefficients closer to the theoretical magnitudes. another samples of the plasticized cahoa electrode have been pretreated in the 10-3 mol/dm3 solution of (nh4)2hpo4 according to the above mentioned methodics. no influence of such pretreatment on the electrode characteristic in solutions of ca2+ and hpo42was registered. it can be summarized, that this method of potentiometric determination of ca2+ and hpo42ions concentration can be applied to find contents of these ions in some natural objects, raw and food samples. the plasticized electrodes based on the pvc substrate and ca-hoa has been developed and their electrode characteristics for the ca2+ and hpo42solutions were found, (table 1). 4. conclusion untreated electrodes show the linear dependence of the electrode potential on concentration of ca2+ ranged from 10-4 to 10-2 mol/dm3 with the slope coefficient 0.0145 v/decade. after pretreatment in the ca2+ solution, the same electrode also showed the linear dependence with the slope coefficient 0.022 v/decade within the ca2+ concentrations range 10-5–10-3 mol/dm3. similar parameters for the hpo42solutions were 0.015 v/decade within 10-3–10-1 mol/dm3 (untreated electrode) and 0.0225 v/decade within 10-3–10-1 mol/dm3 (treated electrode). therefore, ca-hoa can be used in the ionselective electrodes for the food and raw materials analysis methods. 5. references [1]. darst r., ion-selective electrodes. mir, moscow. (1972). [2]. electroanalytical methods. theory and practice. (ed.: f. scholz). binom, moscow. (2006). [3]. orlovsky v., sukhanova g., ezhova z.h., rodicheva g., hydroxylapatite bioceramics. bulletin of the ussr chemical society, 36, 683-688 (1991). [4]. vlasov y., bychkov. e., a composition of the chalcogenic membrane for determination of cadmium ions. journal of analytical chemistry, 40, 1067-1070 (1985). [5]. zhybarev a, e. zharykov, ahmetshin. e., a method of synthesis of hydroxylapatite. russian federation patent 2505479. [6]. kibatltsev v., komarov v., express synthesis of hydroxylapatite crystals. journal of inorganic chemistry, 25, 565-567 (1980). [7]. filippenko v., zyman z., mezentsev v., use of the hydroxylapatite ceramics in the musculoskeletal systems surgery. orthopaedics, traumatology and prostetics, 3, 141-142 (2000). [8]. lakshminarayanh n., membrane electrodes. khimiya, leningrad (1979). microsoft word 8_104 euloge adjou_corrected 2 doi: https://doi.org/10.4316/fens.2021.018 165 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 22021, pag. 165 171 effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br *euloge s. adjou1, t. u. makosso antoine allavo1, oscar akotowanou1, jasmine a. kogbeto1, audray vodounou1, dominique c.k. sohounhloue1 1laboratory of study and research in applied chemistry, university of abomey-calavi, polytechnic school of abomey-calavi, 01, p. o. box 2009, cotonou. eulogesenan@yahoo.fr. * corresponding author received 23th may 2021, accepted 29th june 2021 abstract: the present study aims to investigate the effect of essential oil of pimenta racemosa on the microflora affecting the marketable quality of african mustard obtained from fermented seeds of parkia biglobosa. essential oil of pimenta racemosa was extracted by hydrodistillation. different types of african mustard were produced by the modification of the traditional processing technology and the introduction of a step of essential oil adjunction during the process. three different essential oil concentrations (1.0, 2.0 and 3.0 µlg-1) were investigated. microbiological analyzes were performed in order to evaluate the quality of the african mustard produced. results obtained revealed that the essential oil has inhibition property against the growth of total flora and fungi affecting marketable quality of african mustard during eight (08) days of storage, at the concentrations tested. however, bacterial growth was continuous and exponential in control samples. this supports the antimicrobial properties of the leaves of pimenta racemosa and its traditional use as food ingredient. it is therefore suggested that further studies be carried out to isolate, purify and identify all active compounds present in all part of this plant. key words: pimenta racemosa, parkia biglobosa, african mustard, essential oil, biological activities 1. introduction the availability of good urban food is a major challenge facing african cities nowadays. the problem of urban food supply will intensify in the next future, given the "explosive" growth of urban poles, the rural exodus and the evolution of food behaviors associated with urbanization and the rapid adoption of imported food products [1]. however, the food supply of cities does not depend solely on the good articulation of agricultural production with the areas of consumption. it also depends on the urban food organization, which is complex due to the multiplicity of relationships and interactions between socio-economic actors, institutions and market conditions [1]. in this case food industry also plays a preponderant role through agro-food processing. this sector involves a majority of women who know certain traditional skills that they value. traditionally fermented condiments come from a very old empirical knowledge [2]. it is a process of natural fermentation of protein and oil seeds including those of parkia biglobosa, adansonia digitata, arachis hypogeae, hibiscus sabdariffa and glycine max [2]. these fermentations lead to the involvement of lactic acid bacteria, yeasts and molds, which, through their numerous lytic activities, modify the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 166 biochemical, nutritional and sensory characteristics of transformed seeds [3]. these sensory modifications participate in the typicality of these african mustards. in benin, african mustard commonly called ‘’ afitin ’’ is a traditional condiment produced from fermented seeds of néré (parkia biglobosa) by different sociocultural and sociolinguistic groups [4]. in addition, according to gutierrez et al. [1], it is accepted in public opinion that this traditional condiment would have medicinal properties, which also explains the high consumption rate observed. but, because of their high water content and the presence of nutrients, traditional condiments provide a favorable environment for the development of microorganisms. however, during fermentation, lactic acid bacteria produce many natural antimicrobial compounds, such as lactic acid, acetic acid, formic acid, and caproic acid, carbon dioxide, hydrogen peroxide, diacetyl, ethanol and bacteriocins [5]. indeed, the production of organic acids and antimicrobial compounds during fermentation leads to a decrease in ph that makes difficult the development of pathogenic bacteria [5]. in opposition, the development of fungi affecting marketable quality has been reported in fermented food products by degnon et al. [4]. moreover, there is nowadays more restrictions imposed by international food organism on the use of chemical synthesis food preservatives, which increasingly encourage the use of essential oils in food preservation [6]. their applications as preservatives in food or antiseptics and disinfectants have been widely investigated [7]. some components present in essential oils are classified as generally recognized as safe substances and therefore can be used to prevent postharvest growth of native and contaminant bacteria. then many areas of food science are concerned about the use of essential oils. in food products, essential oils have been used in bakery [8], cheese [9], meat [10], and fruit [11], among others. the advantage of essential oils is their bioactivity in the vapor phase, a characteristic that makes them useful as possible fumigants for stored commodity protection. then, plants belonging to myrtaceae family like pimenta racemosa have retained the attention of researchers, not only because of their high diversity and their distribution around the world, but also for their variable use in popular medicines to treat diseases [12]. in benin, the leaves of this plant are used in culinary preparations where the species is known as ‘‘laurel leaf’’. thus the present study aims to investigate the effect of essential oil of pimenta racemosa on the microflora affecting the marketable quality of african mustard obtained from fermented seeds of parkia biglobosa. 2. materials and methods 2-1-collection of plant leaves plant materials used for essential oil (eo) extraction were fresh leaves from pimenta racemosa. plants were collected at abomey-calavi (southern benin) and identified at the benin national herbarium, where voucher specimens are deposited. 2-2-essential oil extraction the eo tested was extracted by the hydrodistillation method using clevenger-type apparatus. the oil recovered was dried over anhydrous sodium sulfate and stored at 4 °c until it was used [13]. 2-3-local mustard production and experimental process the choice of production technology as well as the selection of women who participated in the present study were made thanks to a prospecting mission carried out in partnership with ‘’solidarités entreprises nord-sud’’ (sens-benin). the technology of local mustard food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 167 production used in this experimentation can be described as follows: p. biglobosa seeds are collected and cleaned. then they were cooked. the cooked seeds are then shelled to separate the seed coat from the cotyledons. then comes the step of rinsing with water to rid the cotyledons of their already softened shell. after selective sorting, the cotyledons are rinsed again and soaked for hours. they then undergo a second cooking. after cooling, they are fermented for 12 hours at room temperature. the cotyledons thus fermented are then salted and ground to obtain a paste which is packaged in the form of balls (figure 1). the local mustards thus produced are divided into four batches called a, b, c and d. the first three batches received the respective doses of 1.0, 2.0 and 3.0 µl.g-1 of essential oil. the last batch (d) did not receive any essential oil and is considered as a control. the batches of local mustards thus treated are stored at 25 °c. periodic sampling followed by inspections and analyzes made it possible to follow the evolution of the microbial flora during storage. fig. 1. process diagram of local mustard production seeds collection cleaning fermentation cooking soaking cooking shelling rinsing shorting rinsing local mustard salting grinding food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 168 2-4-microbiological analysis to 25 g of each sample, 225 ml of peptone water was added and homogenized. from the initial concentration, appropriate decimal dilutions were prepared and aliquots were plated in duplicates on various media. plate count agar was used for the total bacterial count. plates were incubated at 30°c for 72 h. after incubation, the number of colonies was tracked using a colony counter. the number of bacteria expressed as colony forming units per gram (cfu/g) was then determined by calculation, considering the factors of dilution. the method used for detection of fungi in samples was performed using dilution plating method. 10 g of each sample were separately added to 90 ml of sterile water containing 0.1% peptone water. this was thoroughly mixed to obtain the 10-1 dilution. further tenfold serial dilutions up to 10-4 were made. one milliliter of each dilution was separately placed in petri dishes, over which 10–15 ml of potato dextrose agar with 60 µg/ml of chloramphenicol (pdac) was poured. the plates were incubated at 28 ± 2°c for 7 days [14]. all media used for microbiological analysis were prepared as indicated by the manufacturer. 2-5-statistical analyses the data generated from these studies were analyzed using statistical analysis software (sas) and systat 5.05. [15]. 3. results and discussion figures 2 and 3 present the results obtained during the evaluation of the effect of the essential oil of pimenta racemosa in the preservation of african mustard obtained from the fermented seeds of parkia biblobosa. these results indicate that the bacteria constituting the total flora of african mustard are sensitive to the essential oil of pimenta racemosa (figure 2). indeed, the results of monitoring the evolution of the total flora during storage, showed a significant decrease in the microbial quantum in the african mustard samples that received the different concentrations of the essential oil, during the first eight days of storage. this antimicrobial effect of the essential oil depends on the dose tested because, at a dose of 3µl.g-1, the evolution of the total flora in the product is greatly slowed down, compared to the control samples (without essential oil). on the other hand, after eight days of storage, there is a gradual resumption of the growth of the total flora. these results are also in agreement with those obtained during the monitoring of the evolution of the fungal flora in the samples of african mustard supplemented with essential oil of pimenta racemosa at different concentrations (figure 3). indeed, during the first eight days of storage, a significant decrease of fungal growth is also observed in samples with different doses of the essential oil of pimenta racemosa, when compared to the control samples (without essential oil). these results indicated that the essential oil of pimenta racemosa has an inhibitory activity on the microflora of african mustard at the concentrations tested. however, the resumption of microbial growth after eight days of conservation, could be related to the volatile nature of the essential oil. the obtained results confirm the antibacterial properties of the essential oil of pimenta racemosa, as reported by several research works, such as adjou et al. [12] and ismail et al., [16]. food processing makes use of various unit operations and technologies to convert relatively perishable and typically inedible raw materials into more useful shelf-stable and palatable foods or potable beverages [17]. processing also contributes to food security by minimizing waste and losses in the food chain and by increasing food availability and marketability. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 169 fermentation bioprocess is the major biotechnological application in food processing. it is often one step in a sequence of food processing operations which may include cleaning, size reduction, soaking and cooking (figure1). fermentation bioprocessing uses microbial inoculants to enhance properties such as the taste, aroma, shelf-life, safety, texture and nutritional value of foods. in the present study, microbes associated with the raw food material such as seeds of parkia biglobosa and the processing environment, serve as inoculants in this spontaneous fermentations. several studies have also reported the high antimicrobial potential of this essential oil. indeed, the antimicrobial activity of pimenta racemosa oil, could be due to the presence in the oil of the main component with high antimicrobial activity such as eugenol [12]. indeed, eugenol is a remarkably versatile molecule incorporated as a functional ingredient in numerous products and has found application in the pharmaceutical, agricultural, fragrance, flavour, cosmetic and various other industries. in addition, it is widely used in agricultural applications to protect foods from microorganisms during storage. fig. 2. total bacterial count of african mustard samples during conservation fig. 3. evolution of fungal flora count in african mustard samples during conservation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 170 4. conclusion this research underlined the antimicrobial properties of essential oil of pimenta racemosa against microflora affecting marketable quality of african mustard. results indicate that the bacteria and fungi constituting the total flora of african mustard are very sensitive to the essential oil of pimenta racemosa. developing biocides from this essential oil would be also contributive for a lasting solution to the many problems posed by the use of synthetic antimicrobial products. however, in relation to the composition of foods, further investigations are also necessary to identify the conditions that maximize their antimicrobial activity without detrimental effects on the organoleptic properties of the african mustard. 5. acknowledgments the authors are grateful to ‘’solidarités entreprises nord-sud’’ (sens-benin) for their support. 6. references [1]. gutierrez m.l., maizi p., nago c.m., hounhouigan d.j., production et commercialisation de l'afitin dans la région d'abomey-bohicon au bénin. montpellier: cerna, cnearc, cirad, (2000). [2]. azokpota p., houngbo h.y., akissoe n.h., aptitude stabilisatrice des conservateurs traditionnels de l'afitin, condiment africain à base de graines de néré (parkia biglobosa jack. p. br). cah agric 20: 494-9, (2011). [3]. ibeabuchi j.c., olawuni i.a., iheagwara m.c., ojukwu m., ofoedu c.e., microbiological evaluation of ‘iru’ and ‘ogiriisi’ used as food condiments. journal of environmental science, toxicology and food technology, 8: 45-50, (2014). [4]. degnon r.g., konfo c.t.r., aboudou k., bagbonon y. m. g. g., influence de la variété sur les caractéristiques microbiologiques, physico-chimiques et sensorielles du afitin, un condiment produit à base de graines de soja au bénin. international journal of innovation and applied studies 29(4) : 11531160, (2020). [5]. mouroufie, a.k.j., eudes s.k.p.a.n., kouakou a.c., kouakou e.k.v., kati-coulibaly, s., review on the study of health effects of some fermented foods consumed on côte d'ivoire. global journal of biology, agriculture, health sciences 7 (3): 1-9, (2018). [6]. adjou e.s., kouton s., dahouenon-ahoussi e., soumanou m.m., sohounhloue d.c.k., effect of essential oil from fresh leaves of ocimum gratissimum l. on mycoflora during storage of peanuts in benin. mycotoxin res. 29: 29–38, (2013). [7]. yehouenou b., noudogbessi j.p., sessou p., wotto v., avlessi f., sohounhloué c.k.d., etude chimique et activités antimicrobiennes d’extraits volatils des feuilles et fruits de xylopia aethiopica (dunal) a. rich. contre les pathogènes des denrées alimentaires. j soc ouest-afr de chim 29:19–27 (2010). [8]. nielsen p.v., rios r., inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging with special emphasis on mustard essential oil. int. j. food microbiol. 60, 219–229, (2000). [9]. vazquez b.i., fente c., franco c.m., vazquez m.j., cepeda a., inhibitory effects of eugenol and thymol on penicillium citrinum strains in culture media and cheese. int. j. food microbiol. 67, 157–163, (2001). [10]. quintavalla s., vicini l., antimicrobial food packaging in meat industry. meat sci. 62 : 373–380. (2002). [11]. lanciotti r., gianotti a., patrignani f., belleti n., guerzoni m.e., gardini f., use of natural aroma compounds to improve shelf life and safety of minimally processed fruits. trends food sci. technol. 15: 201–208. (2004). [12]. adjou, e.s., degnon, r.g., dahouenon-ahoussi e., sohounhloue, d. c. k., improvement of fermented fish flour quality using essential oil extracted from fresh leaves of pimenta racemosa (mill.) j. w. moore. natural product and bioprospecting 7(4): 299– 305, (2017). [13]. de billerbeck v.g., roques c.g., bessiere j.m., fonvieille j.l., dargent r., effect of cymbopogon nardus (l) w. watson essential oil on the growth and morphogenesis of aspergillus niger. can j microbiol 47:9–17, (2001). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 euloge s. adjou, t. u. makosso antoine allavo, oscar akotowanou, jasmine a. kogbeto, audray vodounou, dominique c.k. sohounhloue, effect of essential oil of pimenta racemosa on microflora affecting quality of african mustard obtained from fermented seeds of parkia biglobosa jack. p. br, food and environment safety, volume xx, issue 2 – 2021, pag. 165 – 171 171 [14]. rampersad f., laloo s., laborde a., maharaj k., sookhai l., teelucksingh j., reid s., bacteriological quality of raw oysters in trinidad and the attitudes, knowledge and perceptions of the public about its consumption. in epidemiological infection 123, 241-250, (1999). [15]. ogbeibu a.e., biostatistics, a practical approach to research and data handling (mindex publishing company ltd., benin city), p. 264, (2005). [16]. ismail m.m., samir r., saber f.r., ahmed s.r., farag m.a., pimenta oil as a potential treatment for acinetobacter baumannii wound infection: in vitro and in vivo bioassays in relation to its chemical composition. antibiotics 9: 679-695, (2020). [17]. barrett t., fang p., swaminathan b., amplification methods for detection of food-borne pathogens. in h. lee, s. morse & o. slovak, eds. nucleic acid amplification techniques: application to disease diagnosis, boston, usa, eaton publishing, 171–181, (1997). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2022 contents: 1. influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value anastasiia shevchenko, svitlana litvynchuk 5 14 2. waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium raghul m 15 21 3. contribution to geospatial classification by using quality indices for irrigation water in arid zones, case of ouargla region, south east algeria med hicham boutelli, aziez zeddouri, tayeb boulmaiz 22 39 4. physicochemical and sensory characterization of five promising aromatic rice accessions in benin valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou 40 49 5. fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh 50 58 6. charcoal-based conservation methods’ impact on some functional properties of flours of three plantain varieties (musa sp.) loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio 59 70 7. effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus) olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere 71 78 8. author instructions i v 9. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2022 doi: https://doi.org/10.4316/fens.2021.041 392 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 392 402 microbiological, physicochemical, nutritional and antinutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria *bukola caroline ogunyemi 1 , adewale kayode ogunyemi 2 , simeon kolawole odetunde 3 , emmanuel olukayode olumuyiwa 4 , adesola oluwafunmilayo olalekan 5 , kabir olusegun akinyemi 6 , bamidele abiodun iwalokun 7 1department of chemical sciences, yaba college of technology, yaba, lagos state, nigeria, bukolaalagbada@gmail.com 2department of microbiology, university of lagos, akoka, lagos state, nigeria. 3department of biological sciences, school of pure and applied sciences, lagos state polytechnic, ikorodu, lagos state,, nigeria. 4department of biological sciences, college of natural and applied sciences, bells university of technology, ota, ogun state, nigeria. 5molecular biology and biotechnology unit, department of medical laboratory science, college of medicine, university of lagos. lagos state, nigeria. 6department of microbiology, lagos state university, lagos state, nigeria. 7molecular biology division, nigerian institute of medical research (nimr), yaba, lagos state, nigeria. *corresponding author received 3rd august 2021, accepted 23rd december 2021 abstract: kunun zaki is a traditionally fermented cereal-based beverage produced locally and generally consumed throughout nigeria. this study aimed to evaluate the microbiological, physicochemical, nutritional and anti-nutritional content of kunun zaki samples sold in various markets in lagos, nigeria using standard methods. the total bacteria and coliform count in kunun zaki ranged from 12 x 106 39 x 106 cfu/ml for bacteria and 3 x 105 17 x 105 cfu/ml for coliform respectively, while mould and yeast had total counts of 5 x 104 19 x104 cfu/ml and 1.0 x 104 9 x 104 cfu/ml respectively. the predominant bacterial species isolated were staphylococcus aureus, e.coli, klebisella sp., bacillus sp. streptococcus sp., citrobacter feundii and pseudomonas aeruginosa. the predominant fungal species isolated from the samples were fusarium sp., aspergillus parasiticus, aspergillus niger, candida albicans, penicillium sp. and saccharomyces cerevisiae. the ph, titratable acidity and specific gravity of the samples ranged from 3.87-4.28, 0.18-0.43 g/l and 0.736-0.75 respectively. the proximate analysis revealed that kunu samples contained 0.3-0.6% protein, 0.4-1.1% fat, 1.3-1.62 ash, 12.8-20.4% carbohydrate and moisture content ranging from 76.75-84.45%. the results for the mineral content analysis showed the presence of phosphorus, calcium, potassium, copper, manganese and magnesium. this study revealed that kunun zaki obtained from different markets in lagos, nigeria contained pathogenic microorganisms that pose threats to public health. awareness campaign, training of local producers, and monitoring of compliance with safety standards are required to reduce risk of contamination. keywords: kunun; coliform; proximate; bacteria; fungi; mineral. 1. introduction fermented cereal-based non-alcoholic beverages like kunun zaki have formed an essential part of the dietary lifestyle of many adult and children in developing countries [1]. kunun zaki, is a highly nutritious, refreshing and filling cerealbased beverage which is widely consumed in nigeria due to its sweet, creamy, mailto:bukolaalagbada@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 393 refreshing taste and consistency [2]. kunun zaki has been reported to contain a variety of nutrients including carbohydrates, proteins, vitamins and several amino acids [3]. the traditional production of kunun zaki majorly requires the use of various grains, which include sorghum (sorghum bicolor), pearl millet (pennisetum glaucum), rice (oryza sativa), fonio (digitaria exilis) and maize (zea mays) [4]. spicy ingredients such as chili pepper, ginger and cloves are also used in addition to sweet potatoes, malted cereals as well as saccharifying agents to improve aroma, taste, nutritional quality and medicinal values of kunun zaki [5,6]. according to adeleke and abiodun [7], the appearance of kunun zaki is milky cream and is usually consumed few hours after its production. the method of processing kunun zaki generally varies based on the producer's household, personal interests, taste and cultural preferences. this results in a difference in the presentation, appearance, taste, flavor, consistency and overall quality of kunun zaki [8]. due to the traditional method of preparation of kunun zaki, the ingredients used are neither quantified nor standardized, therefore the risk of contamination is very high [9]. kunun zaki and other cereal-based products have been reported to contain probiotic bacterial communities including bifidobacterium spp., lactobacillus brevis, weissella confusa, streptococcus lutetiensis, streptococcus gallolyticus [10,11]. several scientific studies have evaluated the microbial and nutritional composition of kunun zaki in various parts of nigeria with different results. according to osuntogun and aboabo [12], “kunun zaki contains lactic acid bacteria (lab) such as lactobacillus spp., streptococcus spp. and leuconostoc spp. which can cause food borne illnesses”. findings have shown that non-alcoholic beverages consumed in different parts of nigeria contained anti-nutritional factors such as phytate and trypsin inhibitors, which may negatively affect their nutritional values [13,14]. presently, there is a high intake of non-alcoholic beverages in different parts of lagos, nigeria, and some of these beverages are produced under unhygienic conditions [15]. this suggests that safety and nutritional values associated with the consumption of kunun zaki may be compromised. hence, the present study aimed to evaluate the microbiological, physico-chemical, nutritional and antinutritional qualities of kunun zaki sold in lagos, nigeria. 2. materials and methods sample collection collection of kunun zaki samples were collected randomly at thirteen (13) major markets in lagos state, nigeria. a total of one hundred (100) samples of kunun zaki were collected, stored in sterile bottles and transferred to the laboratory for further evaluation. however, the samples were stored at 4º c and analyzed within 6 h after collection on sampling days twice per week for 1 month. figure 1 shows the satellite image of sampling points in lagos state, nigeria. microbiological analysis one milliliter (1.0 ml) of kunun zaki sample aseptically taken and introduced into 9 ml sterile water previously autoclaved at 121ºc and 15psi for 15 min to make 1:10 dilution. this was followed by 6 serial 10-fold dilutions (10-1 – 10-7) with 1 ml of each dilution mixed with molten agar (45ºc) to determine microbial count. nutrient agar, macconkey agar, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 394 potato dextrose agar were used for total bacterial count, total coliform count and total fungal count respectively. malt extract agar supplemented with streptomycin was used for total yeast count. plates for total and coliform count determination were incubated at 37°c for 48 h, while those for total and yeast count determination were incubated at 28 ºc for 72 h [16]. all experiments were performed in triplicates with mean count calculated and measured as mean colony forming unit (cfu/ml) of kunun zaki [17]. colonial, morphological and biochemical characterization of bacteria isolates were carried out using standard microbiological techniques according to bergey’s manual of determinative bacteriology [18]. fungal identification and enumeration was based on their colony elevation, colour, texture, shape and arrangement of conidia (spherical or elliptical, unicellular or multicellular), branched or unbranched mycelia, presence or absence of cross walls (whether septate or non-septate) and others. they were enumerated according to ‘illustrated manual on identification of some seed borne fungi’ [19] and ‘illustrated genera of imperfect fungi’ [20]. determination of ph, titratable acidity and specific gravity the ph, titratable acidity and specific gravity of the kunun zaki samples were determined according to the method of association of official analytical chemists (aoac) [21]. the titratable acidity was expressed as % lactic acid present in the sample. determinations were done in triplicates and mean values were calculated. proximate and mineral analysis moisture content, crude fat, protein and carbohydrate content were determined according to aoac methods [21]. ‘atomic absorption spectrophotometer’ (aas) was used to determine three mineral contents (calcium, copper, iron, and magnesium) of the samples, while other minerals (potassium, phosphorus) were determined using‘flame photometry’according to aoac [21]. determinations were performed in triplicates and mean values were calculated. anti-nutritional factors determination the levels of phytate and trypsin inhibitor in the samples were determined using the colorimetric method [22,23]. determinations were done in triplicates and mean values were calculated and expressed as mg per 100g (mg/100g) sample. statistical analysis data obtained in this study were subjected to one-way analysis of variance (anova) graph pad prism® software package, version 5.0 (graphpad software inc., san diego, ca, usa). 3. results and discussion the microbial counts in kunun zaki are presented in table 1. the total bacteria and coliform count in kunun zaki ranged from 12 x 106 39 x 106 cfu/ml for bacteria and 3.0 x 105 17 x 105 cfu/ml for coliform respectively. total count of mould ranged from 5.0 x 104 19 x104 cfu/ml while total yeast count ranged from 1.0 x 104 9 x 104 cfu/ml.the results showed that ojuelegba had the highest bacteria count (39 x 106 cfu/ml) followed by oyingbo (35 x 106 cfu//ml) while yabatech had the least count (12 x 106 cfu/ml). the samples from oyingbo had the highest fungi count (19 x 104 cfu/ml) followed by orile (17 x 104 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 395 cfu/ml) while unilag had the least fungi count (5 x 104 cfu/ml). bariga had highest yeast count (9 x 104 cfu/ml) while surulere had the least yeast count (1.0 x 104 cfu/ml). the occurrences of bacteria and fungi species were presented in figures 2 and 3. bacillus sp. had the highest occurrence (27.14%) followed by e.coli (22.86%) while citrobacter feundii had the least occurrence (5.71%). whereas saccharomyces cerevisiae had the highest occurrence (61.72%) followed by candida albicans (25.31%). penicillium sp. (1.23%) had the least occurrence. from this present study, the results indicate that kunun zaki presented high microbial counts. coliforms and e. coli are known as indicators of faecal contamination in food products. according to who guidelines for drinking water [25], indicator organisms must not be present (=0 cfu/100ml). the coliform counts obtained in this study are far higher than the 104 cfu/g taken as tolerable in foods in developed countries [26]. studies have shown that escherichia coli, clostridium, staphylococcus, campylobacter, and vibrio are some of the common bacteria that cause food-related illness with severe implications [27]. the presence of coliform and e. coli in the kunun zaki indicates fecal contamination that may be due to unhygienic practices during or after processing the beverage [28]. poor water quality, an unsanitary environment, low quality of materials, unclean equipment, stock containers, and improper handling by vendors could be the cause of microbial contamination in all kunun zaki samples [29, 30]. ekanem et al. [31] previously reported that “samples of kunnu had total colony counts ranging from 0.5×105 to 3.2 ×105, 0.5×105 to 3.2 ×105, 5.2×105 to 8.0×105 (cfu/ml) for coliform bacteria, heterotrophic bacteria and heterotrophic fungi respectively”. efiuwevwere and akoma [32] also reported the occurrence of high microbial communities in kunu-zaki samples produced in jos metropolis. anumudu and anumudu [33] found the following occurrence (%) for nine genera of bacteria in kunnu: “staphylococcus sp. (16.66%), e. coli (13.33%), citrobacter sp. (13.33%), proteus sp. (10.00%), serratia sp. (10.00%), lactobacillus sp. (10.00%), salmonella sp. (10.00%), streptococcus sp. (10.00%) and enterobacter sp. (6.66%)”. table 1 microbial counts in the kunun zaki samples. sampling site bacterial counts (x 106 cfu/ml) coliform counts (x 105 cfu/ml) fungi counts (yeast) (x 104 cfu/ml) fungi counts (mould) (x 104 cfu/ml) jibowu 18 3 2 7 surulere 15 6 1 9 tejuosho 20 8 2 11 orile 34 11 3 17 obalende 19 9 nyg 10 iyanaipaja 42 12 4 15 ojuelegba 39 16 6 13 unilag 16 5 nyg 5 yabatech 12 4 nyg 6 saboyaba 26 7 5 16 oyingbo 35 17 7 19 lagos island 17 8 8 8 bariga 31 16 9 15 nyg-no yeast growth seven (7) bacteria species were isolated and identified (e. coli (3 strains), klebisella sp. (5 strains), staphylococcus aureus (5 strains), bacillus sp. (3 strains), streptococcus sp. (3 strains), citrobacter feundii (3 strains), and pseudomonas aeruginosa (3 strains)). six (6) fungal species were isolated and identified as aspergillus parasiticus, aspergillus niger, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 396 fusarium sp., penicillium sp., candida albicans and saccharomyces cervisae (table 2 and 3). the result from this study is consistent with previous findings [34], which reported the isolation of staphylococcus aureus, shigella sp., streptococcus sp., salmonella sp., e. coli, lactobacillus sp., klebsiella sp., citrobacter sp. in samples of kunu zaki produced in kwara state, nigeria. adeyemi and umar [1] had earlier reported the presence of lactobacillus plantarum, leuconostoc mesenteriodes, mucor spp., rhizopus spp. and saccharomyces cerevisiae in kunun zaki prepared with a combination of sorghum and millet; while osuntogun and aboaba [12] isolated lactobacillus, streptococcus, aspergillus and penicillium. however, findings from olasupo et al. [35], have confirmed the presence of lactic acid bacteria as the only test organism (including lactobacillus salivarius, lactobacillus casei, lactobacillus acidophilus, lactobacillus jensenii, lactobacillus cellobiosus and lactobacillus plantarum) found in kunun zaki. adebayo et al. [3] isolated ‘aspergillus niger, rhizopus stolonifer, aspergillus nidulans and aspergillus flavus from kunun zaki. the results from this study indicates the presence of contaminants, which may be due to unhygienic practices or low production quality [3, 33, 36]. the consumption of such contaminated beverage may pose serious health risks to the consumers. proximate analysis as presented in table 4, the result of the proximate analysis in this study showed that kunun zaki from different locations of lagos metropolis contained 0.3-0.6% protein, 0.41.65% fat, 1.3-1.62% ash and 12.8-20.4% carbohydrate with moisture content ranging from 76.75-84.45%. the result of the proximate analysis in this present study are similar to a previous study by onyeleke and shittu [37] who reported that kunun zaki sold in minna contain 0.33% protein, 1.0% fat, 1.52 ash and 12.2% carbohydrate. previous report by essien et al. [38] described that the loss of protein during processing may be responsible for the low protein content observed in the drinks. fig.2: occurrence of bacteria species in the kunun zaki beverage obtained from different markets fig.3: occurrence of fungi species in the kunun zaki beverage obtained from different markets food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 397 table 2 morphology and biochemical tests of isolates from kunun zaki products isolates 1 2 3 4 5 6 7 8 9 10 identification bc1 rod + + + + + + e.coli bc2 rod + + + -+ + + klebsiella sp. bc3 coccus + + + + + + + staphylococcus aureus bc4 rod + + + + + + + bacillus sp. bc5 coccus + + + + + + + staphylococcus aureus bc6 rod + + + -+ + + klebsiella sp. bc7 rod + + + + + + citrobacter freundii bc8 rod + + + + pseudomonas aeruginosa bc9 coccus + + + + + + streptococcus sp. bc10 rod + + + + + + citrobacter freundii bc11 rod + + + + + + + bacillus sp. bc12 coccus + + + + + + streptococcus sp. bc13 rod + + + + + + e.coli bc14 rod + + + + pseudomonas aeruginosa bc15 coccus + + + + + + + staphylococcus aureus bc16 rod + + + -+ + + klebsiella sp. bc17 rod + + + + + + + bacillus sp. bc18 coccus + + + + + + + staphylococcus aureus bc19 rod + + + -+ + + klebsiella sp. bc20 rod + + + + + + citrobacter freundii bc21 coccus + + + + + + + staphylococcus aureus bc22 rod + + + + + + e.coli bc23 rod + + + + pseudomonas aeruginosa bc24 rod + + + -+ + + klebsiella sp. bc25 coccus + + + + + + streptococcus sp. 1-shape; 2-gram status; 3-growth in air; 4-catalase; 5-urease; 6-oxidase; 7-glucose; 8-maltose; 9-arabinose; 10sorbitol, +=positive, -=negative table 3 microscopic and macroscopic characteristics of fungi isolates colony colour colony diameter (mm) conidia colour conidiophore colour conidiophore surface texture phialides identification black 33±1.4 brown to black brown very rough, globose biseriate aspergillus niger dark green 28±2.2 brown colourless distinctly rough, globose uniseriate aspergillus parasiticus pinkish white 32±0.9 light brownish orange rough-walled, with an apical swelling biseriate fusarium sp. black 25±1.3 green blue fuzzy, smooth uniseriate penicillium sp. smooth creamy colonies 40±2.2 brush-like white white biseriate candida albicans cream 18±1.2 white white white biseriate saccharomyces cerevisiae food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 398 table 4 proximate analysis of kunun zaki products obtained from various lagos markets proximate contents moisture content fat ash protein cho jibowu 76.75±1.3 1.63±0.02 1.5±0.05 0.25±0.05 12.8±0.10 surulere 79.47±0.36 1.42±0.01 1.3±0.25 0.6±0.10 17.2±0.10 tejuosho 81.72±0.32 1.59±0.01 1.38±0.03 0.37±0.01 20.2±0.05 orile 79.31±0.39 1.48±0.03 1.62±0.03 0.45±0.01 18.3±0.10 obalende 82.38±0.72 1.57±0.06 1.38±0.010 0.33±0.07 17.6±0.50 iyana-ipaja 78.63±1.30 1.65±0.03 1.6±0.10 0.5±0.10 19.4±0.10 ojuelegba 78.52±0.49 1.63±0.06 1.48±0.09 0.45±0.05 18.4±0.10 unilag 81.16±0.02 1.57±0.06 1.6±0.10 0.37±0.03 17.3±0.05 yabatech 82.13±0.33 1.54±0.01 1.5±0.10 0.42±0.02 18.8±0.40 sabo-yaba 84.45±0.21 1.61±0.02 1.3±0.10 0.6±0.10 19.3±0.40 oyingbo 83.29±0.81 1.54±0.02 1.55±0.11 0.3±0.01 20.4±0.70 lagos island 78.24±0.03 1.43±0.02 1.5±0.10 0.4±0.05 19.8±0.25 bariga 83.77±0.06 1.61±0.20 1.4±0.10 0.5±0.05 20.3±0.10 cho-carbohydrate; s.i units for all parameters are in % physicochemical analysis the physicochemical properties of kunun zaki are presented in table 5. the ph value of the kunun zaki samples ranged from 3.874.28 which shows that the samples are acidic. studies have shown that the ph value of a food is considered a measure of microbial spoilage. table 5 physicochemical characteristics of kunun zaki samples sold in lagos, nigeria sampling site ph titratable acidity specific gravity jibowu 3.87±0.05 0.24±0.05 0.745±0.01 surulere 4.03±0.01 0.18±0.01 0.738±0.02 tejuosho 4.1±0.05 0.36±0.03 0.75±0.01 orile 4.16±0.02 0.32±0.02 0.742±0.02 obalende 3.97±0.05 0.43±0.01 0.745±0.015 iyana-ipaja 4.23±0.18 0.28±0.01 0.741±0.00 ojuelegba 4.27±0.05 0.31±0.06 0.739±0.00 unilag 4.12±0.05 0.18±0.01 0.738±0.00 yabatech 4.28±0.03 0.24±0.01 0.745±0.00 sabo-yaba 4.05±0.25 0.32±0.01 0.743±0.00 oyingbo 4.2±0.06 0.37±0.01 0.748±0.00 lagos island 4.15±0.03 0.41±0.02 0.736±0.00 bariga 4.27±0.01 0.28±0.05 0.739±0.00 microorganisms have been reported to have optimum ph requirements and so the higher the ph of a food, the more prone it is to microbial spoilage. this indicates kunun zaki as a food with high acid content (<4.6) may therefore be more resistant to microbial spoilage [39]. the ph values recorded in this study were lower than the previously reported values of 4.70-5.75 [40], but comparable to 3.34.3 reported by elmahmood and doughari [41]. in this study, the titratable acidity (tta) of kunun zaki ranged from 0.18 to 0.43 of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 399 lactic acid. this result is consistent with previous studies by adebayo et al. [3] and essien et al. [38]. since high acidity and low ph can be attributed to the absorption of amino acids and the release of organic acid, the acidity of kunun zaki may be as a result of lactic acid production during fermentation [42]. microorganisms utilize carbohydrates and this contributes to the production of more hydrogen ions, which signify increased production of acid in the beverage [43]. in this study, the specific gravity of the kunun zaki samples ranged from 0.736-0.75. this is lower than the values (1.02-1.06) reported by omowayetaiwo and oluwamukomi [44]. mineral composition the results obtained for the mineral content of kunun zaki are presented in table 6. in this study, the kunun zaki samples contained p (176.4-278mg/100g), ca (290.7-427.2mg/100g), k (155203mg/100g), cu (0.1-0.9mg/100g), mn (0.4-1.5mg/100g) and mg (90.2112.8mg/100g). the mineral content of kunun zaki recorded in this study is similar to a previous study by nkama et al. [45]. minerals are nutritionally important components in food and they are essential for health and cellular functions in the body [12]. in this study, calcium had the highest value followed by phosphorus, potassium, magnesium and manganese while copper was the least abundant in kunun zaki samples. this is consistent with previous findings by ofudje et al. [33] who observed that kunun zaki has high content of calcium and low content of copper and manganese. the results for potassium, magnesium and copper are similar to results from previous study by omowayetaiwo and oluwamukomi [44]. table 6 mineral composition ofkunun zaki products obtained from various lagos markets sampling site p (mg/100g) ca (mg/100g) k (mg/100g) cu (mg/100g) mn (mg/100g) mg (mg/100g) jibowu 180.4±0.10 320.7±0.20 155±2.0 0.5±0.10 0.7±0.10 90.5±0.02 surulere 176.4±0.20 290.7±0.10 160±2.0 0.3±0.20 1.2±0.15 107.2±0.10 tejuosho 205.1±0.00 308.5±0.10 172±1.0 0.2±0.1 1±0.11 98.2±0.20 orile 240±1.0 407.5±0.02 159±2.0 0.6±0.1 1.1±0.16 91.5±0.40 obalende 195±2.0 370.3±0.3 163±3.0 0.1±0.01 0.9±0.01 93.7±0.10 iyana-ipaja 187.4±0.20 350.2±0.2 174±1.0 0.35±0.12 1.25±0.06 90.2±0.20 ojuelegba 195±2.0 410.7±0.1 180±0.58 0.51±0.06 1.3±0.06 95.1±0.05 unilag 230.5±0.10 345.1±0.03 203±1.0 0.27±0.07 0.8±0.10 108.4±0.10 yabatech 240±2.0 290.7±0.2 165±1.0 0.4±0.03 0.7±0.11 112.8±0.10 sabo-yaba 278±2.0 350.2±0.1 190±1.0 0.22±0.01 1.2±0.20 102.9±0.79 oyingbo 251.8±0.10 380±2.0 185±1.0 0.3±0.10 1.5±0.10 94.2±1.2 lagos island 270.4±0.10 345±1.0 175±2.0 0.4±0.09 0.7±0.20 90.8±1.6 bariga 265±2.0 427.2±0.10 200±2.0 0.9±0.20 0.4±0.10 91.4±0.3 p-phosphorus; ca-calcuim; k-potassium; cu-copper; mn-manganese; mg-magnesium food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 400 anti-nutritional contents of kunun zaki samples the result of the anti-nutritional contents (phytate and trypsin) of kunun zaki samples are presented in table 7. the phytate content of the samples ranged from 0.093mg/100g to 0.27mg/100g, while a range of 0.058mg/100g to 0.081mg/100g was obtained for trypsin inhibitor. according to food and agriculture organization (fao) [46], anti-nutritional factors are generally defined as naturally occurring substances present in grains and other substances present in grain as a result of fungal or other environmental contamination. the reductions in the concentration of anti-nutritional factors in several grains have been attributed to the use of different processing techniques including germination, fermentation, soaking and cooking [47]. adelekan et al. [12] reported a reduction in the concentration of trypsin inhibitor from 0.067 mg/100 g in unmalted kunun zaki to 0.057 mg/100 g in malted kunun zaki and similar occurrence was found in the result of the phytic acid. studies have shown that some anti-nutritional factors affect the nutritional value of grains, while others have significantly serious consequences [46]. table 7 anti-nutritional contents of kunun zaki samples sold in lagos metropolis. sampling site phytate (mg/100g) trypsin (mg/100g) jibowu 0.15±0.01 0.068±0.01 surulere 0.17±0.01 0.072±0.01 tejuosho 0.093±0.03 0.061±0.001 orile 0.14±0.04 0.069±0.003 obalende 0.27±0.05 0.08±0.01 iyana-ipaja 0.22±0.03 0.065±0.005 ojuelegba 0.18±0.01 0.073±0.002 unilag 0.14±0.02 0.072±0.002 yabatech 0.12±0.01 0.074±0.002 sabo-yaba 0.2±0.01 0.062±0.001 oyingbo 0.18±0.01 0.058±0.005 lagos island 0.17±0.01 0.073±0.001 bariga 0.13±0.02 0.081±0007 4. conclusion the study showed that kunun zaki contains many essential nutrients, such as carbohydrates, protein, and fat. in addition, kunun zaki also contains essential mineral elements necessary for growth and development. the presence of contaminants in the kunun zaki samples, shows that the vended kunun zaki had been prepared under low hygienic condition and may pose serious health risk to consumers. hence, improving the nutritional value and microbial quality of locally produced kunun zaki has become imperative. 5. references [1]. adeyemi i.a, umar s. effect of method of manufacture on quality characteristics of kunun zaki; a millet-based beverage. nigeria food journal. 12: 944-947. (1994). [2]. obadina a.o., oyewole o.b., awojobi t.m. (2008). effect of steeping time of milled grains on the quality of kunnu-zaki (a nigerian beverage). ajfs 2: 033-036. (2008) [3]. adebayo g.b., otunola g.a., ajao t.a. physicochemical, microbiological and sensory characteristics of kunu prepared from millet, maize and guinea corn and stored at selected temperature. adv j food sci technol. 2(1): 41-46, (2010) [4]. ahmed e.u., musa n., ngoddy p.o. sensory attributes of extruded cereal legume blends of instant kunnu-zaki beverage analogue proceedings on the second annual nifsi conference. pp: 5-6, (2003). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 401 [5]. makinde f., oyeleke o. effect of sesame seed addition on the chemical and sensory qualities of sorghum based kununzaki drink. afr j food sci technol. 3:204–212, (2012). [6]. olaoye o.a., ubbor s.c., uduma e.a. determination of vitamins, minerals, and microbial loads of fortified nonalcoholic beverage (kunun zaki) produced from millet. food sci nutr. 4(1):96–102. https://doi.org/10.1002/fsn3.267, (2015) [7]. adeleke r.o., abiodun o.a. physicochemical properties of commercial local beverages in osun state, nigeria. pakistan j nutr. 9 (9):853-855, (2010) [8]. adelekan a.o., alamu a.e., aris n.u., adebayo y.o., dosa a.s. nutritional, microbiological and sensory characteristics of malted soy-kunu zaki: an improved traditional beverage. adv microbiol. 3:389-397, (2013). [9]. aboh m.i., oladosu .p. microbiological assessment of kunnu-zaki marketed in abuja municipal area council (amac) in the federal capital territory (fct). nigeria. african j microbiol res. 8 (15):1633-1637, (2014). [10]. crittenden .r., karppinen s., ojanen s., tenkanen m., fagerström r., mättö j., saarela m., tiina s., mattila‐sandholm t., poutanen k. (2002). in vitro fermentation of cereal dietary fibre carbohydrates by probiotic and intestinal bacteria. j sci food agric. 82:781-789, (2002). [11]. oguntoyinbo f.a., tourlomousis p., gasson m.j., narbad a. analysis of bacterial communities of traditional fermented west african cereal foods using culture independent methods. int j food microbiol. 145: 205-210, (2011). [12]. osuntogun b., aboabo o.o. microbiological and physiochemical evaluation of some non-alcoholic beverages. pakistan j nutr. 2(3):21-33, (2006). [13]. omemu a.m., okafor u.i., obadina a.o., bankole m.o., adeyeye s.a.o. microbiological assessment of maize ogi cofermented with pigeon pea. food sci nutr. 6. 10.1002/fsn3.651, (2018). [14]. ajayeoba t.a., dula s., ijabadeniyi o.a. properties of poly-γ-glutamic acid producing-bacillus species isolated from ogi liquor and lemon-ogi liquor. front microbiol. 10:771, (2019) [15]. gbadamosi a., iwaloye o., bamber d. an exploratory study of students' consumption of non-alcoholic beverages in nigeria: a qualitative perspective. nutr food sci. 39:609618, (2009). [16]. fawole m.o., oso b.a. laboratory manual of microbiology. 5th edition, spectrum books limited, ibadan, pp.22-23, (2007). [17]. harrigan w.f., mccance m.e. laboratory methods in food and dairy microbiol. 8th (edn.), academic press inc, london, pp. 7-23, (1990). [18]. holt j.g., kreig n.r., sneath, p.h.a., stanley j.t., william s.t. bergey’s manual of determinative bacteriology. baltimore, usa: william and wikins 213pp, (1994). [19]. kulwant s.j.c. an illustrated manual on identification of some seed borne aspergilli, fusaria, penicillia and their mycotoxins. hellerup, denmark. danish government institute of seed pathology for developing countries and department of biotechnology, the technical university of denmark, (1991). [20]. barnett h.l., hunter b.b. illustrated genera of imperfect fungi. 3rd edition, burgess publishing co., minneapolis, 241 pp, (1972). [21]. association of official analytical chemists (aoac). official methods of analysis 20th edition. washington d.c, (2005). [22]. haug w., lantzsch h.j. sensitive method for the rapid determination of phtytate in cereals and cereal products. j sci food agric. 34:1423–1426. doi:10.1002/jsfa.2740341217, (1983) [23]. hou h.j., chang k.c. (2004). storage conditions affect soybean color, chemical composition and tofu qualities. journal of food processing and preservation, 28:473-488, (2004). [24]. center for food safety (cfs). microbiological guidelines for food (for ready-toeat-food in general and specific food items). center for food safety, 1-46, (2014). [25]. world health organization (who). guidelines for drinking-water quality; fourth edition incorporating the first addendum: geneva, switzerland, 2017; p. 149 (2017). [26]. cooke e.m., gibson g.l. essential clinical microbiology. new york, ny: john wiley and sons ltd. intestinal diseases; pp. 16–21, (1990). [27]. amoah d. microbial risk assessment of mixed vegetable salads from selected canteens in the kumasi metropolis, ghana. pp 41-56, (2014). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 b.c. ogunyemi, a.k. ogunyemi, s.k. odetunde, e.o. olumuyiwa, a.o. olalekan, k.o. akinyemi, b.a. iwalokun, microbiological, physicochemical, nutritional and anti-nutritional evaluation of locally made non-alcoholic kunun zaki beverage sold in lagos state, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 392 – 402 402 [28]. khan m.m., islam m.t., chowdhury m.m.h., alim s.r. assessment of microbiological quality of some drinks sold in the streets of dhaka university campus in bangladesh. int. j. food contam. 2, 4, (2015). [29]. auad l.i., ginani v.c., stedefeldt e., nakano e.y., nunes a.c.s., zandonadi r.p (2019). food safety knowledge, attitudes and practices of brazilian food truck food handlers. nutrients, 11, 1784, (2019). [30]. nemo r., bacha k., ketema t. microbiological quality and safety of some-street vended foods in jimma town, southwestern ethiopia. afr. j. microbiol. res. 11, 574–585, (2015). [31]. ekanem j.o., mensah b.j., marcus n.s., ukpe b.a. (2018). microbial quality and proximate composition of kunu drinks produced and sold in ikot ekpene metropolis, akwa ibom state, nigeria. j appl sci environ mgt. 22 (11):1713–1718, (2018). [32]. efiuwevwere b.j.o., akoma o. (1995). the microbiology of kunuzaki a cereal beverage from northern nigeria during the fermentation (production) process. j microbial biotechnol.11:491-493. (1995). [33]. anumudu i.c., anumudu c.k. bacteriological quality of kunu-zaki sold on the streets of owerri metropolis, nigeria. af j bio sc.1(1): 18-22. (2019). [34]. edem v.e., elijah a.i., umoh s.h. (2017). microbial quality of kunun-zaki sold in eiyenkorin, kwara state, nigeria. nigerian j agric food environ.13 (2), 112-116, (2017). [35]. olasupo n.a., olukoya d.k., odunfa s.a. identification of lactobacillus species associated with selected african fermented foods. zeitschrift fur naturforschung c-a j biosci. 5 (2):105-108, (1997). [36]. ofudje e.a., okon u.e., oduleye o.s., williams o.d. proximate, mineral contents and microbial analysis of kunu-zaki (a non-alcoholic local beverage) in ogun state, nigeriaj adv biol biotechnol. 7(1): 1-8, (2016). [37]. onyeleke s.b., shittu a. (2005). quality food assessment of kunnu-zaki sold in minna. proceedings science and technology” handbook of cereal of the biotechnology society of nigeria (10 annul science and technology (crc press) 99 2 edition th conference) minna, niger state (2005). [38]. essien e., monago c., edor e.a. evaluation of the nutritional and microbiological quality of kunnu-zakin (a cereal based nonalcoholic beverage) in rivers state, nigeria. the internet journal of nutrition and wellness 10: 1-10, (2011). [39]. babajide j.m., olaluwoye a.a., taofik shittu t.a., adebisi m.a. physicochemical properties and phytochemical components of spiced cucumber-pineapple fruit drink. nigerian food j. 31: 40– 52, (2013). [40]. amusa n.a., ashaye o.a. effects of processing on nutritional, microbiological and sensory properties of kunnu-zaki (a sorghum based nonalcoholic beverage) widely consumed in nigeria. pakistan j nutr. 8(3):288-292, (2009). [41]. elmahmood a.m., doughari j.h. microbial quality assessment of kunu-zaki beverages sold in griei town of adamawa state, nigeria. african j food sci. 20 (2): 011-015, (2017). [42]. ashiru a.w., olaleye o.o., egbenni p.o. occurrence of pathogenic organisms in kunun drink sold within lagos metropolis, proceedings of the 27th annual nifst conference. pp. 84, (2003). [43]. fasoyiro s.b., babalola s.o., owosibo s.b. chemical composition and sensory qualities of fruit flavoured roselle (hibiscus sabdariffa) world j agric res. 1 (2):161-164, (2005). [44]. omowaye-taiwo o.a., oluwamukomi m.o. physical and nutritional composition of instant kunun-zaki powder obtained by three drying methods. fjrs 1:114-122, (2015). [45]. nkama i., agarry o.o., akoma o. sensory and nutritional quality characteristics of powdered ‘kunun zaki’: a nigerian fermented cereal beverage. african j food sci. 4(6):364 –370, (2010). [46]. food and agricultural organization (fao) of the united nations. food and agricultural organization (fao) of the united nations, sorghum and millets in human nutrition (fao food and nutrition series, no. 27), rome. isbn 92-5-103381-1, (1995). [47]. adeyemo s.m., onilude a.a., olugbogi d.o. reduction of anti-nutritional factors of sorghum by lactic acid bacteria isolated from abacha an african fermented staple. front sci. 6(1): 25-30, (2016). 1. introduction 147 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, stefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 147 153 evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees *silviu-gabriel stroe1 1stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229, suceava, romania *silvius@fia.usv.ro *corresponding author received may 16th 2014, accepted june 7th 2014 abstract: the objective of this work was to assess through statistical methods the relationship between metallic ions migrated from aisi304 and aisi321 stainless steel food samples into acid simulant solutions depending on the stirring degrees of corrosive environments. principal component analysis method (pca) was used to fulfill the purpose aimed at. the metallic samples were immersed into corrosive solutions at diferent stirring degrees: 0 r/min (stationary environment), 125 r/min and 250 r/min. acetic acid solutions in bidistilled water were used as food simulant environment. after conducting the migration tests, the concentration of cr, mn, fe and ni ions migrated from aisi304 stainless steel samples into corrosive solutions and ti, cr, mn, fe and ni ions concentration migrated from aisi321 stainless steel samples were analyzed. these concentrations were analyzed by mass spectrometry with inductively coupled plasma (icp-ms). in order to characterize the diffusion processes occurring under accelerated corrosion, the experimental data obtained were statistically processed in two steps: analysis of the correlation between variables based on pearson’s correlation matrix and analysis of relationships between variables through the principal component analysis. pca method has identified the two significant principal components that explain more than 93% of the original data variance. significant correlations between the metallic ions migrated into corrosive solutions stirred at 125 and 250 r/min were found. keywords: stainless steel, food simulant solution, pearson correlation matrix, principal components analysis 1. introduction the study on the diffusion of metallic elements in foods is an area of interest for many scientists and technologists in this field. a statistical method frequently used to study the phenomenon of diffusion is principal component analysis (pca). principal components analysis is a multivariate analytical statistical technique that can be applied in qualitative and quantitative analysis in order to reduce the dependent variables to a smaller number of variables based on correlations between the initial data [1]. therefore, many researches have been undertaken to explain this complex phenomenon, often leading to the alteration of the final quality of food products [2], [3], [4], [5]. this paper carries on previous studies in the field: over the last two years we have published other studies on the relationship between food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 148 metallic ions from metal samples at different working parameters [6], [7]. the objective of this work was to assess through statistical methods the relationship between the metallic ions migrated from aisi304 and aisi321 stainless steel food samples into acid simulant solutions depending on the stirring degrees of corrosive environments. 2. materials and methods taking into account that the most aggressive environment found in the food industry is the acidic environment, acetic acid solutions in bidistilled water were used in the present research. the metal samples were made of aisi304 (x5crni18-10) and aisi321 (x6crniti18-10) stainless steel food grade, with a side of 40 mm. the argument of choosing the two grades of stainless steel is that aisi304 stainless steel grade is the most used of the 300 series, in that they have a good corrosion resistance in a very wide range of food environments. compared to aisi304 stainless steel, aisi321 stainless steel has in its composition added titanium (ti), metallic element that has the ability to prevent the precipitation of chromium carbides, thus eliminating the risk of corrosion at the grain boundaries. the metal samples of two stainless steel grades were tested under the same working conditions. the experimental conditions are shown in table 1. mass spectrometry with inductively coupled plasma method (icp-ms agilent 7500) was used to analyse the chemical composition of corrosive environments, before and after performing the corrosion tests. in order to characterize the diffusion processes occurring under accelerated corrosion, the experimental data obtained were statistically processed in two steps: analysis of the correlation between variables based on the pearson correlation matrix, and analysis of the relationships between variables through the principal component analysis, using the trial version of spss v16.0 software. table 1. experimental conditions of accelerated migration tests experimental conditions levels solution conc., [%, wt] 3 6 9 testing temperature, [ºc] 22 28 34 immersion time, [min.] 30 60 90 stirring degree, [r/min] 0 125 250 the pearson correlation matrix was performed using the values of the stirring dregree variable of corrosive solutions. the adequacy of variables was verified in order to apply the principal component analysis. for this purpose the kaisermeyer-olkin (kmo) statistic test and bartlett test of sphericity were applied. by applying the pca method, a reduction of the number of variables to a smaller number of components, unrelated to each other, called principal components, was obtained. the first principal component extracted (pc1) represents the linear combination of variables that take the maximum possible variance of the original data, and the second principal component (pc2) takes a smaller variance compared to the first component. the number of components chosen have to explain more than 70% the total variance of the data [8]. 3. results and discussion minimum and maximum values, mean values and standard deviation values for food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 149 each of ti, cr, mn, fe and ni metal ions migrated from aisi304 and aisi321 stainless steel samples into the simulant solutions stirred at 0, 125 and 25 r/min are shown in table 2 and 3. table 2. specific values of metal ions that migrate from aisi304 stainless steel into the simulated acid solutions stirred at 0, 125 and 250 r/min metal ions migrated into solutions * notations used for metal ions released into solutions stirred at 0, 125 and 250 r/min calculated value, [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd chromium cr_0 1.00 20.00 8.22 5.30 cr_125 1.00 79.00 13.40 15.04 cr_250 3.00 238.00 36.44 56.34 manganese mn_0 0.72 14.67 2.74 2.79 mn_125 1.02 16.00 3.76 3.25 mn_250 1.42 43.00 8.74 11.36 iron fe_0 10.00 560.00 192.22 156.21 fe_125 10.00 1950.00 328.88 366.23 fe_250 50.00 4450.00 827.03 1148.21 nickel ni_0 3.30 411.00 99.62 139.60 ni_125 5.30 531.00 120.37 168.59 ni_250 5.90 561.00 152.91 177.87 * cr_0, mn_0, fe_0, ni_0 – metal ions of chromium, manganese, iron and nickel migrated into simulant solution at 0 r/min; cr_125, mn_125, fe_125, ni_125 – metal ions of chromium, manganese, iron and nickel migrated into simulant solution at 125 r/min; cr_250, mn_250, fe_250, ni_250– metal ions of chromium, manganese, iron and nickel migrated into simulant solution at 250 r/min; table 3. specific values of metal ions migrated from aisi321 stainless steel into the simulated acid solutions stirred at 0, 125 and 250 r/min metal ions migrated into solutions * notations used for metal ions released into solutions stirred at 0, 125 and 250 r/min calculated value, [ppb] minimum value, xmin maximum value, xmax average value, x standard deviation, sd titanium ti_0 0.58 3.50 1.66 0.91 ti_125 0.50 5.28 1.81 1.09 ti_250 0.40 8.28 1.77 1.56 chromium cr_0 1.00 24.00 7.51 5.48 cr_125 2.00 37.00 12.00 7.84 cr_250 4.00 49.00 16.92 10.52 manganese mn_0 2.49 14.57 6.01 2.72 mn_125 2.69 16.57 6.69 3.14 mn_250 2.93 19.73 6.54 3.32 iron fe_0 110.00 770.00 361.11 176.33 fe_125 130.00 1370.00 518.14 282.55 fe_250 190.00 870.00 491.11 174.80 nickel ni_0 7.70 716.00 167.81 222.26 ni_125 9.70 756.00 204.62 267.34 ni_250 13.70 806.00 223.66 286.09 *ti_0, cr_0, mn_0, fe_0, ni_0 – metal ions of titanium, chromium, manganese, iron and nickel migrated into simulant sol. at 0 r/min; ti_125, cr_125, mn_125, fe_125, ni_125 – metal ions of titanium, chromium, manganese, iron and nickel migrated into simulant solution at 125 r/min; ti_250, cr_250, mn_250, fe_250, ni_250 – metal ions of titanium, chromium, manganese, iron and nickel migrated into simulant solution at 250 r/min; food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 150 the correlation matrix of the variables studied ions migrated from aisi304 samples into solutions shows significant positive correlations between some of the variables (table 4). a moderate correlation (r = 0.499), significant at p = 0.01, between the chromium and nickel ions migrated in stationary corrosive environment (0 r/min), was obtained. strong correlations (r > 0.7, p = 0.01) were obtained between chromium and manganese ions, between manganese and iron ions, at 125 and 250 r/min., according to table 4. table 4. pearson correlation matrix of ions migrated from aisi304 stainless steel into the simulated acid solutions stirred at 0, 125 and 250 r/min cr_0 cr_125 cr_250 mn_0 mn_125 mn_250 fe_0 fe_125 fe_250 ni_0 ni_125 ni_250 cr_0 1.000 cr_125 0.534a 1.000 cr_250 0.039 0.173 1.000 mn_0 0.282 0.395b 0.236 1.000 mn_125 0.482b 0.876a 0.275 0.561a 1.000 mn_250 0.053 0.227 0.973a 0.236 0.353 1.000 fe_0 0.301 0.520a 0.637a 0.364 0.622a 0.657a 1.000 fe_125 0.364 0.898a 0.211 0.417b 0.854a 0.274 0.635a 1.000 fe_250 0.016 0.369 0.843a 0.299 0.448b 0.888a 0.649a 0.468b 1.000 ni_0 0.499a 0.162 -0.160 0.190 -0.036 -0.241 -0.026 -0.059 -0.216 1.000 ni_125 0.515a 0.211 -0.159 0.195 -0.004 -0.236 -0.027 -0.014 -0.202 0.993a 1.000 ni_250 0.508a 0.193 0.099 0.257 0.015 0.006 0.126 -0.018 -0.017 0.962a 0.962a 1.000 bold values represent correlation with significance, asignificant correlations at a 0.01 level, bsignificant correlations at a 0.05 level table 5. pearson correlation matrix of ions migrated from aisi321 stainless steel into the simulated acid solutions stirred at 0, 125 and 250 r/min. ti_0 ti_125 ti_250 cr_0 cr_125 cr_250 mn_0 mn_125 mn_250 fe_0 fe_125 fe_250 ni_0 ni_125 ni_250 ti_0 1.000 ti_125 0.719a 1.000 ti_250 0.266 0.823a 1.000 cr_0 0.468b 0.530a 0.318 1.000 cr_125 0.364 0.459b 0.308 0.551a 1.000 cr_250 0.421b 0.373 0.138 0.434b 0.901a 1.000 mn_0 0.700a 0.549a 0.149 0.609a 0.565a 0.582a 1.000 mn_125 0.705a 0.654a 0.297 0.600a 0.535a 0.569a 0.906a 1.000 mn_250 0.444b 0.833a 0.833a 0.549a 0.533a 0.381b 0.491a 0.645a 1.000 fe_0 0.554a 0.419b 0.105 0.530a 0.557a 0.674a 0.671a 0.594a 0.303 1.000 fe_125 0.720a 0.459b 0.009 0.471b 0.430b 0.595a 0.791a 0.829a 0.311 0.767a 1.000 fe_250 0.458b 0.539a 0.348 0.476b 0.600a 0.735a 0.641a 0.654a 0.545a 0.786a 0.670a 1.000 ni_0 0.106 0.556a 0.727a 0.077 -0.014 -0.216 0.030 0.027 0.464b -0.230 -0.331 -0.105 1.000 ni_125 0.019 0.435b 0.611a 0.041 -0.016 -0.214 0.038 -0.009 0.383b -0.241 -0.364 -0.104 0.964a 1.000 ni_250 0.032 0.461b 0.636a 0.048 -0.002 -0.201 0.018 -0.014 0.397b -0.238 -0.368 -0.101 0.973a 0.996a 1.000 bold values represent correlation with significance, asignificant correlations at a 0.01 level, b significant correlations at a 0.05 level food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 151 the correlation matrix between ions migrated from aisi321 stainless steel samples into the simulant solution stirred at 0, 125 and 250 r/min (table 5) shows significant correlations between some variables. thus, significant correlations were obtained between: titanium and chromium ions (r = 0.468), titanium and manganese ions (r = 0.700), titanium and iron ions (r = 0.554) migrated into the simulant solutions stirred at 0 rpm; titanium and chromium ions (r = 0.459), titanium and manganese ions (r = 0.654), titanium and iron ions (r = 0.459), titanium and nickel ions (r = 0.435) migrated into the simulant solutions stirred at 125 r/min; titanium and manganese ions (r = 0.833) and titanium and nickel ions (r = 0.636) migrated into the simulant solutions stirred at 250 r/min; chromium and manganese ions and between chromium and iron migrated into the simulant solution stirred on all three levels; manganese and iron ions migrated into the simulant solution stirred on all three levels. by applying the method of principal component analysis and the graphical representation of principal components, allows the visualization of the relationship between variables and identification the possible groups. figure 1. pca loadings plot for the cr, mn, fe and ni ions migrated from aisi304 stainless steel samples into the simulated acid solutions. symbols and definition: ○ stirring degree 0 r/min; stirring degree 125 r/min; ∆ stirring degree 250 r/min. after carrying out the statistical data analysis on aisi304 stainless steel grade, the first two principal components explain 94.38% of the original data variance, pc1 food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 152 = 87.53 % and pc2 = 6.85%, according to figure 1. the graphical representation shows that the ions contained in aisi304 stainless steel migrated into simulant solution can form three distinct groups, each group containing the same ions: manganese, iron and chromium the groups being differentiated only by the stirring degree of the solution. figure 1 presents along the first principal component (pc1) that there is a strong association between the manganese, iron and chromium ions, migrated into the simulant solution stirred at 125 r/min (encircled in red). these variables indicate a strong correlation with pc1. the second principal component (pc2) is strongly correlated with the manganese, iron and chromium ions migrated into the simulant solution stirred at 250 r/min (encircled in blue). the second principal component (pc2) highlights the contrast between the iron and nickel ions migrated into the simulant solution at 0 and 250 r/min. figure 2. pca loadings plot for the ti, cr, mn, fe and ni ions migrated from aisi321 stainless steel samples into the simulated acid solutions. symbols and definition: ○ stirring degree 0 r/min; stirring degree 125 r/min; ∆ stirring degree 250 r/min the correlation between the metal ions migrated from the aisi321 stainless steel samples into the simulant solution stirred at 0, 125 and 250 r/min is shown in figure 2. the first two principal components explain 93.18% of the original data food and environment safety journal of faculty of food engineering, ştefancel mare university – suceava volume xiii, issue 2 – 2014 silviu-gabriel stroe, evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees, food and environment safety, volume xiii, issue 2 – 2014, pag 147 153 153 variance, pc1 = 64.78 % and pc2 = 28.4%. regarding the first principal component (pc1), one can notice a very good correlation between the manganese, iron and chromium ions migrated into thew simulant solution stirred at 0, 125 and 250 r/min (encircled in red). these variables are strongly correlated with the first principal component (pc1). the second principal component (pc2) distinguishes between the nickel ions migrated into simulant solution stirred at 0, 125 and 250 r/min as well as the iron ions migrated under the same stirring conditions (encircled in blue). 4. conclusion after carrying out the statistical processing of experimental data, it can be concluded that the principal component analysis technique is an essential tool to analyze relationships between metal ions migrated from metal samples into acid simulant solutions. in the solution stirred at 125 r/min as well as in the solution stirred at 250 r/min, the metal ions migrated from the aisi304 stainless steel samples into simulant solutions are the chromium, iron and manganese ions as compared to the nickel ions migrated in the same stirring degrees. iron, chromium and manganese ions migrated into the simulant solution stirred at 125 r/min, form a distinct group from that of iron, chromium and manganese ions migrated into the solution stirred at 250 r/min. at all three stirring degrees, the iron ions are the ones which migrated from aisi321 stainless steel samples, as compared with the nickel and titanium ions. 5. references [1]. lawless, h.t., heymann, h., sensory evaluation of food: principles and practices. new york: chapman & hall, . p. 608, (1998); [2]. roots, o., zitko, v., principal component analysis of polychlorinated biphenyls and polycyclic aromatic hydrocarbons in seals, estonian environmental research centre, 10617 tallinn, estonia fisheries and oceans canada, marine environmental sciences, canada, экологическая химия, 2003, 12(1): 57–66,(2002); [3]. varzakas, t. h. et al., theoretical and experimental approaches towards the determination of solute effective diffusivities in foods. enzime and microbial technology, v. 37, n.1, p. 29-41, (2005); [4]. dos santos, j. s., dos santos, m. l. p., conti, m. m., comparative study of metal contents in brazilian coffees cultivated by conventional and organic agriculture applying principal component analysis, journal of the brazilian chemical society, vol.21 no.8, são paulo, (2010); [5]. ghosh, d., chattopadhyay, p., application of principal component analysis (pca) as a sensory assessment tool for fermented food products, journal of food science and technology, 49(3): 328–334, (2012); [6]. stroe, s. g., gutt, g., statistical study of the dependence between concentration of metallic elements migrated from stainless steel grade aisi321 and working parameters, food and environment safety, volume 12, issue 2, (2013); [7]. stroe, s. g., statistical evaluation of the relationship between the metallic elements that migrate from aisi304 and aisi321 stainless steel samples in simulant solutions at different immersion times, food and environment safety, volume xiii, issue 1 – 2014, pag. 48 54, (2014); [8]. benzécri, j.p., l’analyse des données. la taxionomie, paris: dunod, (1973). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 contents: 1. silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini 5 15 2. structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds) meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi 16 27 3. carbendazim residues quantification in green lettuce by enzyme-linked immunosorbent assay veronica tanasa, madalina doltu, dorin sora, radu i. tanasa, narcisa babeanu 28 32 4. influence of the addition of chamomile on the content of tannins in tea anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets 33 38 5. compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851) rasheed olatunji moruf, mosunmola florence akinwunmi 39 46 6. effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka 47 61 7. evaluation and comparison of groundwater vulnerability to pollution by the drastic and god methods: a case of wadi nil alluvial plain (jijel, ne algeria) souhil mahdid, nabil chabour, taha hocine debieche, abdelmalek drouiche, faouzi zahi, séverin pistre 62 75 8. quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea 76 81 9. bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche 82 93 10. preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) karima ould yerou, fatima zohra el kadi, khedoudja kanoun, hadj mostefa khelladi, hanane benzahia, sara bekhti 94 106 11. author instructions i v 12. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county 116 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 22020, pag. 116 121 study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county *sonia amariei 1 , alina mihalcea 1 1faculty of food engineering,”stefan cel mare” university, suceava, romania, sonia@usm.ro, alina.filip@fia.usv.ro *corresponding author received 15th april 2020, accepted 28th june 2020 abstract: the purpose of the study is to evaluate the contamination with mycotoxins of the corn cultures in the county of bacau, in correlation with the geographical and agro-climatic conditions of this area. romania is one of the largest corn producers from the european union and harvested almost 10746.4 thousand of tons in 2016, 14326.1 thousand of tons in 2017, 18353 thousand of tons in 2018, 16990 thousand of tons in 2019, over 1.5 million tons going into exports in countries both from the eu and outside. the main mycotoxins identified in the corn cultures in the county of bacau are: aflatoxins, deoxynivalenol, zearalenone, toxin t2 and ht2, fumonisins. the study aimed to determine the quantity of ochratoxin and zearalenone from corn kernels harvested in the county of bacau, the identified mycotoxins do not exceed the maximum limits according to reg. (ce) 1881/2006, which regulates the maximum limits for certain contaminants in food products. keywords: mycotoxins, corn, ochratoxin, zearalenone 1. introduction molds are biological agents that cause significant damage to cereal crops. the fungi that produce mycotoxins are divided into 2 groups: those that invade before harvest, called field fungi, and those that appear after harvest, called deposit fungi. cereal contamination occurs in the field under the influence of agro-climatic conditions that are favorable to fungal attack and mycotoxin production. the appearance of extreme meteorological phenomena (high temperature, drought, heavy rains), non-compliance with agricultural technologies, negatively affects the quantity and quality of crops, favoring the appearance of fungi and the production of mycotoxins. ochratoxins are part of the group of nephrotoxic mycotoxins and are produced by fungi belonging to the group of penicillium and aspergillus. ochratoxin a is a phenylalanine derivative resulting from the formal condensation of the amino group of l-phenylalanine with the carboxy group of (3r)-5-chloro-8hydroxy-3-methyl-1-oxo-3,4-dihydro-1h2-benzopyran-7-carboxylic acid (ochratoxin alpha).the main toxic components isolated in a. ochraceus strain extracts were ochratoxin a and ochratoxin b [1]. the optimal temperature for ochratoxin production on natural environments (corn, wheat, rice) varies between 20 and 28°c, and the optimum humidity of 18-19% [1]. zearalenone is a resorcyllic acid lactone, with a chemical structure similar to steroid hormones, produced by fungi of the type fusarium (f. roseum, f. tricinctum, f. oxysporum, f. moniliforme, f. culmorum, f. graminearum) [1]. zearalenone contamination is closely related to http://www.fia.usv.ro/fiajournal mailto:sonia@usm.ro mailto:mariap@fia.usv.ro https://pubchem.ncbi.nlm.nih.gov/compound/phenylalanine https://pubchem.ncbi.nlm.nih.gov/compound/amino https://pubchem.ncbi.nlm.nih.gov/compound/l-phenylalanine https://pubchem.ncbi.nlm.nih.gov/compound/%283r%29-5-chloro-8-hydroxy-3-methyl-1-oxo-3%2c4-dihydro-1h-2-benzopyran-7-carboxylic%20acid https://pubchem.ncbi.nlm.nih.gov/compound/%283r%29-5-chloro-8-hydroxy-3-methyl-1-oxo-3%2c4-dihydro-1h-2-benzopyran-7-carboxylic%20acid https://pubchem.ncbi.nlm.nih.gov/compound/%283r%29-5-chloro-8-hydroxy-3-methyl-1-oxo-3%2c4-dihydro-1h-2-benzopyran-7-carboxylic%20acid https://pubchem.ncbi.nlm.nih.gov/compound/%283r%29-5-chloro-8-hydroxy-3-methyl-1-oxo-3%2c4-dihydro-1h-2-benzopyran-7-carboxylic%20acid https://pubchem.ncbi.nlm.nih.gov/compound/ochratoxin%20alpha food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sonia amariei, alina mihalcea, study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county, food and environment safety, volume xix, issue 2 – 2020, pag. 116 121 117 substrate moisture, drying and product storage techniques. balzer et al. (1976) showed that the humidity of corn in 1974 was very high, it was strongly invaded by gibberella zeae (f. graminearum), zearalenone being found in more than 50% of the analyzed samples [1]. the study verifies the corn crops in bacau county regarding the content of ochratoxin a and zearalenone. 9 collection points were established for the investigation of the two mycotoxins: onesti, bacau, racaciuni, damienesti, calugareni, sascut, buhusi, margineni, orbeni. the elisa test is a commonly used immunoassay method to rapidly monitor mycotoxins. it is commonly used by agrifood laboratories [2, 3, 4]. there are commercially available kits for all regulated mycotoxins; they contain microtiter elisa plates that have welldefined applicability, analytical range and validation criteria [3, 5, 6]. there are several commonly available elisa formats, but the predominant form is the competitive one [7]. the competitive format is characterized by the fact that the signal intensity is inversely correlated with the antigen concentration in the sample [8, 9]. the classic competitive format is to immobilize the antigen standard on the plate surface. then, an incubation of the antibodies directed against the target mycotoxin with the sample takes place. the antigens in the sample will compete with those immobilized for binding to these antibodies. after the washing step, the analyte-bound antibodies are washed [8]. direct detection uses a primary antibody labeled with an enzyme that reacts with the antigen, while a secondary antibody labeled with the enzyme is used for indirect detection of the primary antibody [9]. in the competitive inhibition format, competition occurs between unlabeled antigens in samples and enzymelabeled antigens (enzyme conjugate) for binding to an antibody directed against the target mycotoxin. in this format, the plate can be coated with capture antibodies with affinity for the analyte or for a primary antibody [8, 10]. for both types of competitive assays it is similar to add a suitable substrate which is allowed to incubate so that the enzyme which has conjugated to the antibody or antigen (classical or inhibitory format, respectively) acts and produces changes in a given parameter [10, 11, 9]. the last step of all assays is to add a stop solution that causes the reaction between the enzyme and the substrate to stop. the signal strength weakens as the antigen concentration of the sample increases, as a higher amount of analyte results in either fewer enzyme-labeled antibodies bound to the antigen adsorbed on the plate (classical format) or fewer enzyme-labeled antigens bound to the antibody on the test plate [11, 8, 9]. the advantages of elisa include, in addition to the specificity of antibodyantigen binding, a relatively low detection limit (lod), a high sample yield with low sample volume, minimal cleaning procedures and ease of application [3, 5]. 2. matherials and methods 2.1 materials bacău county is located in the eastern part of the country, occupying an area of 6621 km, about 2.8% of romania's area. the climatic regime of bacău county is an example of a gradual transition from the pronounced continental climate in the east to the moderate one in the west. characteristic for bacău county is the insular distribution of temperatures, conditioned by the specificity of the relief steps. the pedogenetic conditions led to the formation of various soils, on the territory of bacau county, the brown and brown argiloiluvial soils predominated, which had humus content of 1-5%. they food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sonia amariei, alina mihalcea, study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county, food and environment safety, volume xix, issue 2 – 2020, pag. 116 121 118 ensure good average fertility for agricultural land. cereals are a group of cultivated plants belonging to the poaceae family that includes: wheat, corn, rye, barley, rice, sorghum, millet, oats [12]. one of the most important grains is corn (zea mays). it is a plant with a high stem, broad leaves, monoecious asexual flowers arranged separately: the male ones in terminal panicles, the female ones on a thick spadix. it will form corn cobs after fruiting [13]. 2.2. reagents elisa kits produced by prognosis biotech greece were used to determine ochratoxin a and zearalenone, which comply with the specifications of en iso 14675: 2003. bio-shield ochratoxin a, b2448 / b2496, bio-shield zon, b2748 / b2796 are immunoassay methods that determine ochratoxin a, zearalenone in grains, ears, grains and other products including food for animals. the elisa kit contains all the reagents needed for the immunoassay method. other equipment used: fritsch pulverisette 14 rapid laboratory mill, kern precision balance, plt 2000-3dm, used for weighing (with a precision of 10-3 g) of the samples to be studied, elisa line consisting of: biomerieux tecan a-5082 washer washer 430, biomerieux shaker 50x incubator, biomerieux reader 230s reader, used for homogenization, incubation, sample reading. 2.3. methods the elisa test produced by prognosis biotech is based on the principles of the linked enzyme immunoassay test. the wells of the microtiter strips are coated with mycotoxin-specific antibodies. the toxins are extracted from the ground sample with 70% methanol. mycotoxin standards or samples and the conjugate (detection solution) are added to the lined wells. the conjugate binds to the binding sites of the lined antibodies that are not already occupied by the mycotoxin in standards or samples. any unbound conjugate in the detection solution is removed in a wash step. a chromogenic substrate is added to the wells, which leads to the progressive development of a bluecolored complex with antibody detection. the development of the color is then stopped by the addition of acid, which turns yellow in the resulting final product. the measurement is made photometrically at 450 nm and the intensity of the colored complex produced is indirectly proportional to the mycotoxin concentration present in the samples and standards. 3. results 3.1. ochratoxina a table 1 presents the results of the tests performed for the identification and dosing of ochratoxin in the maize grains from the mentioned areas. undetectable values (<lod) were recorded in all 10 samples taken to investigate the presence of ochratoxin a. each analysis was performed in triplicate. 3.2. zearalenone zearalenone contamination is low in field grains and increases in storage conditions with humidity greater than 30% –40%. [14]. a tolerable daily intake for zearalenone of 0.25 g/kg b.w./day was established by the european food safety authority. [15] currently, the limits for zen in cereals vary between countries and range from 50 to 1000 pg/kg [16]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sonia amariei, alina mihalcea, study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county, food and environment safety, volume xix, issue 2 – 2020, pag. 116 121 119 table1. ochratoxin a in corn grains from different areas of bacau county samples place of sampling lod (μg/kg) loq (μg/kg) the result (μg/kg) maximum allowed limit (μg/kg) 1 bacau 1.054 1.755 <1.054(undetectable) 5 2 onesti 1.054 1.755 <1.054(undetectable) 5 3 racaciuni 1.054 1.755 <1.054(undetectable) 5 4 damienesti, village calugareni 1.054 1.755 <1.054(undetectable) 5 5 damienesti 1.054 1.755 <1.054(undetectable) 5 6 buhusi 1.054 1.755 <1.054(undetectable) 5 7 margineni 1.054 1.755 <1.054(undetectable) 5 8 sascut 1.054 1.755 <1.054(undetectable) 5 9 sascut 1.054 1.755 <1.054(undetectable) 5 10 orbeni 1.054 1.755 <1.054(undetectable) 5 lodlimit of detection (the lowest quantity of a substance that can be distinguished from the absence of that substance with a stated confidence level loqlimit of quantification (the limit at which the difference between two distinct values can be reasonably discerned) the maximum limits allowed for zen should be in the range of 100-200 µg/kg in processed cereals, 75 µg/kg for processed cereals, 20 µg/kg in processed cereal foods and 50 µg/kg in cereal snacks according to the laws in eu [17]. risk assessments were performed based on exposure to zen given in france, germany, finland, china and india. in only a few cases, it has been found that the possible zen contribution exceeds the tdi and almost all studies have agreed that the majority of the population does not exceed the tdi value given by the eu [18]. table 2. zearalenone in corn grains from different areas of bacau county sample no. place of sampling lod (μg/kg) loq (μg/kg) the result (μg/kg) maximum allowed limit (μg/kg) 1 bacau 2.061 2.457 <2.061(undetectable) 350 2 onesti 2.061 2.457 <2.061(undetectable) 350 3 racaciuni 2.061 2.457 2.68 350 4 damienesti, village calugareni 2.061 2.457 2.68 350 5 damienesti 2.061 2.457 3.8 350 6 buhusi 2.061 2.457 2.68 350 7 margineni 2.061 2.457 <2.457(unquantifiable) 350 8 sascut 2.061 2.457 <2.457(unquantifiable) 350 9 sascut 2.061 2.457 14.13 350 10 orbeni 2.061 2.457 <2.457(unquantifiable) 350 https://en.wikipedia.org/wiki/confidence_interval food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sonia amariei, alina mihalcea, study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county, food and environment safety, volume xix, issue 2 – 2020, pag. 116 121 120 fig.1. zearalenone content in the tested samples analyzing the results (fig. 1), we observe that 5 samples have undetectable (<lod, represented by blue) and non-quantifiable (<loq, represented by green) values, 5 samples have numerical values (represented by red): 2.68 μg / kg for the sample collected from racaciuni, 2.68 and 3.8 μg / kg for the sample collected from damienesti, 2.68 μg / kg for the sample collected from buhusi, 14.13 μg / kg for the sample collected from sascut. zearalenone was present in 50% of the samples, in 50% of the analyzed samples and it was below the limit of detection, quantification (fig.2). fig.2. share of samples in which zearalenone was identified 4. conclusions the presence of both ochratoxins and zearalenones was found in the analyzed corn samples. the determined quantities were far below the limit allowed by the legislation. in 50% of the analyzed samples, these mycotoxins were below the limit of detection, quantification. considering the accentuated climatic changes, the pluviometric regime of the area and the fact that the analyzed raw material is a basic one in animal feeding, but also the basis for a large number of food products, the systematic analysis of the presence of mycotoxins and the factors that favor their appearance is absolutely necessary. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 sonia amariei, alina mihalcea, study on ochratoxin a and zearalenone content in corn grains from different areas of bacau county, food and environment safety, volume xix, issue 2 – 2020, pag. 116 121 121 5. references [1] coman i., popescu o., mycotoxins and mycotoxicosis, ceres, 82-99, (1985). https://www.madr.ro/culturi-decamp/cereale/porumb.html https://www.agro.basf.ro/ro/stiri/fermier-inromania/cultura-porumb-rotatie-semanatingrijiretratamente-recoltare.html https://www.prognosis-biotech.com/ [2] pereira, v.l.; fernandes, j.o.; cunha, s.c. mycotoxins in cereals and related foodstuffs: a review on occurrence and recent methods of analysis. trends food sci. technol. 36, 96–136 (2014) [3] xie, l.; chen, m.; ying, y. development of methods for determination of aflatoxins. crit. rev. food sci. nutr. 56, 2642–2664 (2016) [4] dzuman, z.; zachariasova, m.; lacina, o.; veprikova, z.; slavikova, p.; hajslova, j. a rugged high-throughput analytical approach for the determination and quantification of multiple mycotoxins in complex feed matrices. talanta 121, 263–272 (2014) [5] venkataramana, m.; chandranayaka, s.; prakash, h.s.; niranjana, s.r. mycotoxins relevant to biowarfare and their detection. toxinology 1–22 (2014) [6] anfossi, l.; giovannoli, c.; baggiani, c. mycotoxin detection. curr. opin. biotechnol. 37, 120–126 (2016) [7] santos pereira, c.; c. cunha, s.; fernandes, j.o. prevalent mycotoxins in animal feed: occurrence and analytical methods. toxins 11, 290 (2019) [8] robinson, r.; pellenz, s. an introduction to elisa (part 2). available online: https://www.antibodiesonline.com/resources/17/1464/an-introduction-toelisa-part-2/. [9] bio-rad laboratories elisa basics guide; bio-rad laboratories: kidlington, uk, 2017; pp. 1–40. [10] thermo fisher scientific overview of elisa. available online: https://www.thermofisher.com/pt/en/home/lifescience/protein-biology/protein-biologylearningcenter/protein-biology-resourcelibrary/pierceprotein-methods/overviewelisa.html#2 [11] r-biopharm ag. good elisa practice manual; r-biopharm ag: darmstadt, germany, (2016) [12] l. oprica, food biochemistry, ed. tehnopress, 251-261, (2011) [13] sava d, botanical atlas, north's star, 165167, (2018) [14] tola, m.; kebede, b. occurrence, importance and control of mycotoxins: a review. cogent food agric. 2, 1–12 (2016) [15] ünüsan, n. systematic review of mycotoxins in food and feeds in turkey. food control 97, 1–14 (2019) [16] zinedine, a.; soriano, j.m.; moltó, j.c.; mañes, j. review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. food chem. toxicol. 45, 1–18 ( 2007) [17] european food safety authority. scientific opinion on the risks for human and animal health related to the presence of modeified forms of certain mycotoxins in food and feed. efsa j. 9, 2197 (2011), [18] rai, a.; das, m.; tripathi, a. occurrence and toxicity of a fusarium mycotoxin, zearalenone. crit. rev. food sci. nutr. 59, 1–20 (2019) https://www.madr.ro/culturi-de-camp/cereale/porumb.html https://www.madr.ro/culturi-de-camp/cereale/porumb.html https://www.agro.basf.ro/ro/stiri/fermier-in-romania/cultura-porumb-rotatie-semanat-ingrijiretratamente-recoltare.html https://www.agro.basf.ro/ro/stiri/fermier-in-romania/cultura-porumb-rotatie-semanat-ingrijiretratamente-recoltare.html https://www.agro.basf.ro/ro/stiri/fermier-in-romania/cultura-porumb-rotatie-semanat-ingrijiretratamente-recoltare.html https://www.prognosis-biotech.com/ https://www.thermofisher.com/pt/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierceprotein-methods/overview-elisa.html#2 https://www.thermofisher.com/pt/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierceprotein-methods/overview-elisa.html#2 https://www.thermofisher.com/pt/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierceprotein-methods/overview-elisa.html#2 https://www.thermofisher.com/pt/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierceprotein-methods/overview-elisa.html#2 https://www.thermofisher.com/pt/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierceprotein-methods/overview-elisa.html#2 1. introduction 4. conclusions 201 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 201 207 existing regulations on different markets regarding the obtaining and marketing of foods with benefits for health * micşunica rusu 1 1 faculty of food engineering, stefan cel mare university of suceava, romania rusum@fia.usv.ro *corresponding author received 28th august 2019, accepted accepted 27th september 2019 abstract: the issue of population health is a global problem, and because of that, the calling for food with health benefits has become a concern for many consumers, but also for various institutions that legislate or with interest in managing the health of population. the first researches in this field, applied as a national policy, were carried out in japan, starting with 1984, the japanese introduced “foods for specified health uses” (foshu) products, for which there are special labeling rules. products have diversified, appearing, over time, alongside foshu, other categories of food with health benefits: foods with nutrient function claims (fnfc) and foods with function claims (ffc). to encourage the choice of healthy foods, but also to prevent the use of false messages, ambiguous or misleading, many countries have adopted legislation that ensures correct information and consumer safety. so, in the european union (ue), were adopted and applies, regulation (ec) no 1924/2006 on nutrition and health claims made on foods and regulation (eu) no 1169/2011, on the provision of food information to consumers, also in the usa, fda applies the nutrition labeling and education act of 1990 (nlea), dietary supplement health and education act of 1994 (dshea), food and drug administration modernization act of 1997 (fdama). keywords: health claims; nutrition claims; foshu; fnfc; regulation (ec) no 1924/2006; nlea; dshea; fdama. 1. introduction foods with health benefits have become a concern for many consumers [1]. this was due to several converging factors, like: awareness of the deterioration of the population's health, increasing the number of people who "prescribe" their own medication [2], increased levels of information from health authorities and the mass media, on the link between diet and health, outstanding scientific advances in nutrition research, the competitive food market etc. [3], [4]. foods with health benefits carry messages (claims) that convey, in a certain way, information that links product consumption to health or well-being. according to “codex alimentarius”, a health claim represents „any representation that states, suggests, or implies that a relationship exists between a food or a constituent of that food and health” [5]. to be effective, developing food with health benefits must be based on appropriate regulations [6]. in countries where there is specific legislation on foods with health benefits, they are subject to a rigorous approval process before marketing [7]. the first country that paid special attention to the obtaining and marketing of these products was japan. the products have been called “foods for specified health uses” (foshu), and at http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 202 international level, they have been called functional foods [8]. functional foods can be identified on the market by two distinct elements: (i) the classical food form, not the pills; (ii) the presence on the packaging of a message that has to make a positive correlation between the food/food component and an improvement in a body function or a reduction in the risk of certain diseases [9], [10]. this messages (phrases or representations, including pictures, graphs, or symbolic representations, in any form) the health claims must have a solid scientific basis, not misleading to win and maintain consumer confidence) and be well understood by the average consumer [11]. a health claim, by definition, has two essential components: (1) a substance (either a food, a food component or a food supplement ingredient) and (2) a disease or condition related to health [12]. there are wide differences between the ways to regulate on the use of health claims in different countries and regions of the world [13]. so, some countries have an institution that regulates health claims (the ministry of health, labor and welfare – in japan; the food and drug administration in the us; the state food and drug administration (sfda) in china etc.), other countries regulates through private companies (uk and sweden), and other have decided to cooperatively develop regulations on health and nutrition claims (european union, australia, new zealand). 2. japan japan is the country that recognizes foods with health benefits as a distinct category, and the japanese market for these foods is the largest and most innovative in the world. the food category, which appeared on the japanese market in 1984 and was thought to have a beneficial impact on the health of consumers, was named “foods for specified health uses” (foshu). according to the definition, foshu refers to foods containing ingredients with health functions and for which there are officially approved claims for physiological effects in the human body (health claims). foshu products are intended to be consumed to maintain / promote health or to be used by people who wish to control their health [14]. it is possible to delimit several categories of foods with health benefits on the japanese market (table 1); for each category there are clear indications of information (claims) that can be displayed on the packaging [15]. (a) the foshu category, comprising: 1. "normal" foshu refers to foods containing ingredients with health functions and for which there are officially approved health claims. foshu limits health claims to fourteen formulations, and requires extensive clinical trials [16], [3]. 2. qualified foshu are foods with a role in health that is not proven with scientific evidence at the same level as “normal” foshu, or foods with a certain effectiveness but without effective determination of the mechanism of action of the food components for the function concerned; 3. standardized foshu are foods with sufficient approvals and that have accumulated sufficient scientific evidence, for which standards and specifications are established, according to the legislation specific to this group of foods; 4. reduction of disease risk foshu are the foods for which the claim allowed when the disease risk reduction is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 203 clinically and nutritionally determined for the target ingredient. 5. for the latter three subgroups, there are facilities for approval, for applicants [15]. (b) foods with nutrient function claims (fnfc) can be manufactured and distributed without further authorization or notification from the japanese government. for these, standards and specifications for indicating nutritional functions [15] are established. standards and specifications refer to: the amount of nutrient in the products, which must be less than the values specified by the recommended daily intake; nutrient claims; warnings. (c) foods with function claims (ffc) are introduced on the japanese market in 2015 and allow for a more accessible and faster registration process than for foshu [17]. ffc monitoring is carried out by the governmental organization consumer affairs agency (caa), unlike the other categories, which are regulated by mhlw. for ffc, any health claim may be used. the new rules have made the claims environment less complex, widening the market at the same time and creating healthy competition. in line with the ffc guidelines, manufacturers have to provide the caa with scientific information on the safety and functional character of these products [18]. manufacturers are also required to provide the health claim that will be included on the label [19]. table 1 categories of products with a beneficial effect on health, in japan medicines fhc (food with health claims) another foods foshu (food for specified health uses) fnfc (foods with nutrient function claims) ffc (foods with functional claims) individual approval system standard regulatory system notification 1991 2001 2015 3. european union (eu) from the documents which govern health food and information on food labels on european markets can be listed: a) regulation (eu) no 1169/2011, on the provision of food information to consumers; b) regulation (ec) no 1924/2006 on nutrition and health claims made on foods, including food supplements. the stated objective of this regulation is to ensure the effective functioning of the internal market in terms of the use of these two categories of claims, while providing a high level of consumer protection [20]. according to regulation (rec) no 1924/2006, in the eu, are used on food: (i) nutrition claims, (ii) health claims, (iii) mentions on the reduction of disease risk, and (iv) children's development and health claims [21]. (i) nutrition claims. rec no 1924/2006 defines the nutrition claim and stipulates that only the nutrition claims listed in the regulation are allowed, provided that they comply with the established conditions of use [22]. (ii) health claims, under the rec, means any claim that states, suggests or implies that there is a relationship between a food food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 204 category, a food product or one of its constituents, and health. health claims are adopted by the european council (ec) after consultation of the european food safety authority (efsa), according to article 13 of the rec 1924/2006, and shall form the consolidated list, which shall be completed continuously, in accordance with the procedure referred to in article 25 (2) of that regulation [21]. the consolidated list includes the wording of the claims and the conditions applicable to them, together with all restrictions [23]. the consolidated health claims database, according to article 13, contained 4637 mentions from efsa, in 2011 [24]. from 2011 to august 2017, according to article 13, paragraph 5 of rec 1924/2006, efsa received 48 applications, 13 were withdrawn, 27 were adopted, the others being under review [25]. in order to support the preparation and submission of applications for authorization, efsa initially prepared two guides: scientific and technical guidance for the preparation and presentation of an application for authorisation of a health claim [26] and general scientific guidance for stakeholders on health claim applications, followed by many others, in order to help the industry in substantiating health claims. on the packaging of the food product that have on health claims must be specified: the quantity of food, the form of consumption, possible warnings, restrictions of use and instructions for use [26]. it is important that the consumer can consume enough food, as part of a balanced diet, to achieve the claimed effect [27]. efsa established, based on evidence from different studies, conditions for the use of health claims for the main groups of functional compounds (in table 2 are presents the conditions for the use of health claims related to fat and fatty acids – according efsa – [28]). table 2 conditions for the use of health claims assessed by the efsa, depending on the type of fat and composition in fatty acids fat type function usage conditions linoleic acid cholesterol management ensuring at least 15% of the recommended daily intake, of 10 g linoleic acid/day; alpha-lipoic acid (ala) cholesterol management ensuring at least 15% of the recommended daily intake, of 2g ala/day; plant sterols cholesterol management 0,8 g/zi; docosahexaenoic acid / eicosapentaenoic acid (dha / epa) blood triglycerides management 2 4g/day; blood pressure management 3 g/day; heart health 250 mg/day; dha eyesight 250 mg/day; brain function 250 mg/day; (iii) claims on reduction of disease risk. in accordance with article 14 (1) (a), were been endorsed by 2009, three claims to the reduction of disease risk: plant sterols and heart disease; plant stanols and heart disease; chewing gum sweetened with 100% xylitol and dental caries or cavities [29]. medical claims for foods (claims that express, imply or suggest that the product has properties to treat, prevent or cure diseases) are prohibited by european labeling rules [21]. in order for a product to be able to bear a medical claim, that product must be classified as a medicine. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 205 c) eu directive 2002/46 regulates nutritional supplements. these are, legally, considered foods not medicines or an intermediate category [30], so all health and nutrition claims that can be used for food, can also be used for nutrition supplements [31]. 4. usa in the us, three categories of claim may be used under the law: (1) nutrition claims; (1) health claims and (3) claims related to a function-structure link [32]. the responsibility for these claims rests with: (i) the manufacturer; (ii) the food and drug administration (fda), the institution regulating the production and marketing of food, and medicines in the united states and (iii) the federal trade commission, in the case of advertising [12]. in the usa are used: 1. nutrition claims are regulated by the nutrition labeling and education act (nlea), in 1990. 2. health claims. there are three ways the fda exercises its oversight on the use of health claims on labels [33; 34]: authorized health claims, according to nlea and dietary supplement health and education act (dshea), from 1994. these regulations characterize a relationship between a food, component of a food or ingredient of a food supplement, and the risk of a particular disease. the fda authorizes these types of health claims based on an extensive analysis of the scientific literature, following the submission of a claim for a health claim [35]. health claims based on statements of authorities, according to food and drug administration modernization act (fdama), issued in 1997. under this law, a health claim can be approved for a food on the basis of an "authorized statement" of a scientific institution of usa, with official responsibility for the protection of public health or research, directly related to human nutrition, such as the national institutes of health, the national academy of sciences, the center for disease control and prevention [35]. qualified health claims, according “consumer health information for better nutrition initiative”, of 2003, that allows the fda to recognize these claims when there is a clear relationship between a food / component of the food or the food supplement and reducing the risk for a disease or for maintaining health status [36]. authorizations for claims via nlea or fdama require rigorous and timeconsuming evidence, based on a number of rigid standards. the fda has recognized the need for health claims based on less scientific evidence when they are not misleading consumers. as a result, the agency has established intermediate procedures through which qualified health claims for conventional foods and supplements can be used. 3. structure-function claims are used on conventional food labels and on food supplements [37]. the structure-function claims may also describe a benefit in the context of disease due to the deficiency of a nutrient (such as vitamin c and scurvy). the manufacturer is responsible for ensuring the accuracy and authenticity of these claims; they are not pre-approved by the fda, but must be truthful and not misleading. manufacturers of dietary supplements using structure-function claims labels must submit a notification containing that claim to the fda at least 30 days before the food supplement is marketed [38]. 5. conclusions foods with health benefits are primarily intended to improve the general health and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 206 wellbeing of population. these foods bear messages (claims) that convey, in a certain way, information that links product consumption to health or well-being. of particular interest in this direction was japan, initially, through the introduction of foshu products (considered as a basis in the development of functional foods), but also the us and the eu, through the specific legislation adopted. communicating health claims has an important impact on consumers’ attitudes, acceptance and acquisition. 6. references [1]. schmidt, h., chronic disease prevention and health promotion, public health ethics: cases spanning the globe, public health ethics analysis, 3: 19-27, (2016); [2]. martirosyan d., singh j., a new definition of functional food by ffc: what makes a new definition unique?, functional foods in health and disease, 5: 209-223, (2015); [3]. dhiman a., vaibhav w., arun n., introduction to the functional foods, in introduction to functional food science, 2nd ed. richardson, (2014); [4]. abuajah c.i., ogbonna a.c., osuji c.m., functional components and medicinal properties of food: a review, j food sci technol, 52(2): 2522-2529, (2015); [5]. fao. guidelines for use of nutrition and health claims (cac/gl 23-1997), available at http://www.fao.org/ag/humannutrition/3244409f5545b8abe9a0c3baf01a4502ac36e4.pdf, accessed: 06.06.19; [6]. martirosyan d., singharaj b., health claims and functional food: the future of functional foods under fda and efsa regulation, functional foods for chronic diseases, 1st edition, food science publisher, 410-424, (2016); [7]. bigliardia b., galatib f., innovation trends in the food industry: the case of functional foods, trends in food science & technology, 31(2): 118-129, (2013); [8]. siro i., kapolna e., kápolna b., lugasi a., functional food. product development, marketing and consumer acceptance a review, appetite, 51: 456-467, (2008); [9]. aggett p.j., antoine j.m., asp n.g., bellisle f., contor l., cummings j.h., howlett j., müller d.j.g., persin c., pijls l.t.j., rechkemmer g., tuijtelaars s., verhagen h., passclaim: process for the assessment of scientific support for claims made on foods. consensus on criteria, european journal of nutrition, 44 (suppl 1): i/5–i/30, (2005); [10]. yasmeen r., fukagawa n., wang t., establishing health benefits of bioactive food components: a basic research scientist’s perspective, current opinion in biotechnology, 44: 109-114, (2017); [11]. hieke s., cascanette t., pravst i., kaur a., trijp h., verbeke w., grunert k., the role of health-related claims and symbols in consumer behaviour: the clymbol project. agro food industry hi tech., 27: 26-29, (2016); [12]. fda, code of federal regulation title 21 food and drugs, subpart a general requirement for health claims (21 cfr 101.14), available at: https://www.accessdata. fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr =101.14, accessed: 06.06.18; [13]. pappalardo g., lusk , j., the role of beliefs in purchasing process of functional foods, food quality and preference, 53: 151-158, (2016); [14]. nielsen k., health beneficial consumer products status and trends, in book: developing food products for consumers with specific dietary needs, woodhead publishing series in food science, technology and nutrition, 15-42, (2016); [15]. mhlw, food with health claims, food for special dietary uses and nutrition labeling, available at: http://www.mhlw.go.jp/english/topics/foodsafety/fh c/, accessed: 20.12.2018; [16]. ohama h., ikeda h. moriyama h., health foods and foods with health claims, toxicology, 221: 95-111, (2006); [17]. maeda-yamamoto m., development of functional agricultural products and use of a new health claim system in japan, trends in food science & technology, 69, part b: 324-332, (2017); [18]. kamioka k., tsutani h., origasa t., quality of systematic reviews of the foods with function claims registered at the consumer affairs agency web site in japan: a prospective systematic review, nutrition research, 40: 21-31, (2017); [19]. caa, system of “foods with function claims”…questions??, available at https://www.wysiwyg.co.jp/service/systematicrevie w/systematicreview-e, accessed: 20.10.2018; food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 micşunica rusu, existing regulations on different markets on the obtaining and marketing of foods with benefits for health, food and environment safety, volume xviii, issue 3 – 2019, pag. 201 207 207 [20]. santeramo f.g., carlucci d., de devitiisa b., emerging trends in european food, diets and food industry, food research international, 104: 39-47, (2018); [21]. ue, regulation (ec) no 1924/2006 of the european parliament and of the council of 20 december 2006 on nutrition and health claims made on foods, official journal of the european union, 18: 3-18, (2007); [22]. pravst i., functional foods in europe: a focus on health claims, in: scientific, health and social aspects of the food industry, ed. b. valdez, (2012); [23]. efsa, final scientific and technical guidance for applicants for preparation and presentation of the application for authorisation of a health claim, available at http://www.efsa.europa.eu/efsa/scientificpanels/ nda/efsa_locale-178620753812_ guidance467.htm, accessed: 20.10.2014; [24]. efsa, "general function" health claims under article 13, available at https://www.efsa.europa.eu/en/topics/topic/generalfunction-health-claims-under-article-13, accessed: 20.11.2016; [25]. osborn s., morley w., developing food products for consumers with specific dietary needs, 1st edition, woodhead publishing, 25-48, (2017); [26]. efsa, scientific and technical guidance for the preparation and presentation of a health claim application, efsa journal, 9: 12-22, (2011); [27]. gilsenan m.b., nutrition & health claims in the european union: a regulatory overview, trends in food science & technology, 22(10): 536-542, (2011); [28]. efsa, scientific opinion on the substantiation of health claims related to vitamins, minerals and omega-3 fatty acids (id 1, 2, 3, 4, 5, 7, 10, 11, 12, 111, 112, 168, 173, 174, 179, 184, 201, 210, 217, 362, 372, 717, 1464, 1515, 2872, 2874, 3094, 3095, 4279, 4280, 4281, 4282, 4284, 4285, 4286, 4287, 4289, 4291, 4292, 4708) pursuant to article 13(1) of regulation (ec) no 1924/2006, efsa journal, 9: 207077, (2011); [29]. shimizu t., health claims regulations and scientific substantiation of functional foods and the international comparison, clinical aspects of functional foods and nutraceuticals, crc press, (2014); [30]. ce, directive 2002/46/ec of the european parliament and of the council of 10 june 2002 on the approximation of the laws of the member states relating to food supplements, official journal, 183: 0051 – 0057, (2002); [31]. verhagen h., vos e., francl s., heinonen m., van loveren h., status of nutrition and health claims in europe, archives of biochemistry and biophysics, 50: 6-15, (2010); [32]. fda, label claims for conventional foods and dietary supplements, available at: http://www.fda.gov/food/ingredientspackaginglab eling/labelingnutrition/ucm1 11447.htm, accessed: 28.07.16; [33]. fortin n., food regulation: law, science, policy and practice, wiley-blackwell, chichester, uk, (2008); [34]. bagchi d., nutraceuticals and functional foods regulations in the united states and around the world,. toxicology, 221: 1 – 3, (2006); [35]. fda, label claims for conventional foods and dietary supplements, available at https://www.fda.gov/food/labelingnutrition/ucm111 447.htm., accessed: 28.07.16; [36]. fda, qualified health claims: letters of enforcement discretion, available at: https://www.fda.gov/food/ingredientspackaginglabe ling/labelingnutrition/ ucm072756.htm, accessed: 28.07.16; [37]. fda, regulation. u.s. food and drug administration. guidance for industry: evidencebased review system for the scientific evaluation of health claims – final, available at http://www.fda.gov/food/guidanceregulation/gui dancedocuments regulatoryinformation/labelingnutrition/ucm0733 32.htm, accessed: 23.07.2017. [38]. lalor f., wall, p.g., health claims regulations: comparison between usa, japan and european union, british food journal, 113(2): 298313, (2011). issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xxi, issue 2 30th july 2022 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 297 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 297 306 quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize *abiodun aderoju adeola 1 , dorcas olawunmi olayioye 2 , alexander adedolapo oluyombo 3 , babatunde adebiyi olunlade 4 1 food and nutrition research programme, institute of food security, environmental resources and agricultural research, federal university of agriculture, p. m. b. 2240, abeokuta, ogun state, nigeria. orcid https://orcid.org/0000-0003-0761-3507 2 department of food science and technology, federal university of agriculture, p. m. b. 2240, abeokuta, ogun state, nigeria. 3 department of food science and technology, federal university of agriculture, p. m. b. 2240, abeokuta, ogun state, nigeria. orcid https://orcid.org/0000-0002-8535-5948 4 department of food science and technology, bowen university, iwo, nigeria *corresponding author received 2 nd july 2020, accepted 29 th december 2020 abstract: protein and vitamin a deficiencies are two public health concerns in nigeria. this study investigated the quality attributes of biscuits prepared from yellow fleshed cassava flour (yfcf) and quality protein maize flour (qpmf). the proximate, mineral and antinutrient composition, β-carotene content and color attributes of the composite flour and biscuits were determined. data were subjected to analysis of variance and the means separated by duncan’s multiple range test. the protein and βcarotene contents of the flour blends ranged from 5.69 to 8.54% and 1.45 to 2.76 µg/g, respectively. on the other hand, the biscuits contained 11.38 to 14.6% protein and 1.35 to 1.82 µg/g β-carotene. the protein, ash, crude fiber, spread ratio and mineral composition of biscuits increased as qpmf increased. the hardness of the biscuits decreased with increase in qpmf. furthermore, the mean sensory scores of the biscuits revealed that they were liked moderately. however, the most acceptable biscuit was the one prepared from 20% yfcf and 80% qpmf.this study has demonstrated the possibility of producing acceptable biscuit from quality protein maize, which could be used in school feeding programmes in nigeria. keywords: biscuit, nutrient composition, quality protein maize, sensory attributes, yellow fleshed cassava 1. introduction biscuits are popular food items among nigerians, may be as a result of their work schedule where there is little or no time to cook main dishes. biscuit was reported as being the most consumed snack among young nigerians [1]. the versatility of biscuit thus makes it a suitable vehicle in ameliorating cases of malnutrition in nigeria. the major raw material in biscuit manufacture is the flour. the conventional flour in biscuit manufacture is the one obtained from wheat. however, flours obtained from other crops are being used, depending on such factors as availability, affordability, accessibility and nutritional need [2, 3]. type of flour has been reported to influence the quality attributes of biscuit. for example, agiriga and iwe [1] reported that composite flour of cassava, groundnut and corn starch affected the thickness and spread ratio of cookies. many authors also reported that flours affected the functional, acceptability and nutritional value of biscuits [3, 4, 5, 6]. http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 298 cassava (manihot esculentacrantz) has been recognised as the world’s sixth most important crop (after wheat, rice, maize, potato, and barley) and the most widely cultivated root crop in the tropics [7]. nigeria is the world’s largest producer of cassava roots. its potential in improving food insecurity and socioeconomic wellbeing of nigerian citizens have been documented [7]. cassava root, though rich in starch, is poor in protein [8]. new varieties of cassava with low in cyanide content, drought-resistant, high yielding, diseaseand pest-resistant, and early maturing have been developed. of particular note is the yellow fleshed cassava (or vitamin a biofortified cassava) variety which is of relevance in solving the endemic vitamin a deficiency in nigeria [9]. the typical composition of yellow fleshed cassava flour is 23.18 to 53.56% starch, 0.62 to 1.74% crude fibre, 0.93 to 1.85% ash, 0.146 to 0.877 µg/g β-carotene, 2.43 to 3.4 mg/kg hcn [9, 10]. these varieties could supply about 25% of daily vitamin a requirements of children and women [9]. cassava flour has been advocated as an ideal substitute to wheat flour in the production of pastries due to its availability and low cost of production [8, 11].previous studies [12,13] have shown that 40% partial substitution of wheat flour with cassava flour for bread production is possible while complete 100% substitution of wheat flour with cassava for biscuit manufacture has been reported by oyewoleet al [14]. in terms of cultivation areas and total production, maize (zea mays) has been reported as the third most important cereal (after wheat and rice) in the world [3]. it is a major food commodity for the teeming population of developing countries. varieties of maize include pop, flint, dent, floury and sweet cultivars. quality protein maize (qpm) is a biofortified cultivar with twice the amount of lysine and tryptophan than the conventional maize and a protein bio-availability that rivals milk casein [15, 16, 17]. average composition of qpm has been given as 10% moisture, 10% protein, 3.06% crude fat, 1.2% ash, 2.38% crude fibre and 85.75% carbohydrate [18]. the total phenolic content and antioxidant activity of qpm are 1.38 mg gaeg-1 and 68% dpph inhibition, respectively. biscuits have been produced from qpm-wheat, qpm-wheat-oat-maizesoybean, qpm-pumpkin leaves-cocoa powder flour blends [18, 19, 20, 21]. 2. materials and methods 2.1 materials the yellow fleshed cassava flour (yfcf) was obtained from the institute of food security, environmental resources and agricultural research, federal university of agriculture, abeokuta, nigeria. the cassava variety used in preparing the flour was tms 01/1412. qpm grains (art 98/swi-y) were purchased from the seed store of the institute of agricultural research and training, obafemi awolowo university, nigeria. sugar, salt, margarine, sodium bicarbonate (baking powder), milk (skimmed milk powder), eggs, and vanilla flavour were sourced from a local market in ogun state. 2.2 preparation of quality protein maize flour (qpmf) qpmf was prepared according to the method described by mesfin and shimelis[22]. sorting was done by removing the undesirable seeds and other foreign materials from qpm by the principle of flotation. then the seeds were sundried and dry milled. the resultant flour was sieved with a mesh size of 250µm, and packaged in a polyethylene bag. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 299 2.3 formulation of blends the composite blends were formulated (table 1) after a preliminary screening experiment using paired preference test. the upper limit of substitution of yfcf with qpmf was set at 80% due to the results and observations obtained from the preliminary experiment. 2.4 preparation of biscuit the composite flour and the other ingredients (as presented in table 2) were weighed using the ingredient formulation by oyewoleet al [14]. the main stages of the biscuit preparation were mixing, rolling and cutting. baking was done at 160 oc for 20 minutes. 2.5 determination of proximate composition the proximate composition of the composite flours and biscuits was determined using the methods stated by aoac [23]. moisture content was determined using a digital moisture analyser in which about 0.5 g of ground sample was spread in the drying pan after cleaning and tarring the weight of the pan. the soxhlet method of solvent extraction was used to determine the crude fat content. the weight of the filter paper was noted and about 2 g of ground sample was measured into the filter paper. the summation of the weights of the filter paper and sample was denoted as w1.the filter paper was well tied to avoid the spilling out of the content. then the filter paper was put into the thimble. the thimbles with the samples were attached to the extraction apparatus. then diethyl ether was poured into a boiling flask after which set up of the extraction apparatus was done. the apparatus hot plate was set on to allow for the extraction process to take place for minimum of 6 h. then the recycling process made by the diethyl ether was stopped to let the solvent to evaporate from the boiling flask with the extract. in the process, the evaporated solvent was recovered in the apparatus. the filter paper was removed from the thimble and was allowed to dry in the oven for 30 min at 150°c to evaporate the remaining solvent in the sample. then, it was removed from the oven, cooled in a desiccator for 30 min. thereafter, the filter paper was weighed and recorded as w2. the percentage fat was then calculated. then, 20 ml of sulphuric acid was added to the samples in the digestion tubes and about half of a kjeldahl tablet was added to facilitate the digestion process by catalysing and raising the boiling point temperature of sulphuric acid. after this, the mixture was placed in the digester at a temperature of about 370 °c for 1.5 h. on completion of the digestion the digested sample was cooled and made up to 100 ml by adding distilled water. about 20 ml of the solution was then poured into the distillation flask and distillation was done using 40% naoh. the ammonia was then distilled into a receiving flask that contained solution of excess boric acid (4%) for reaction with ammonia. titration was carried out with 0.1 n of hcl using methyl red as an indicator. in determining the crude fibre content of sample, about 1.0 g of defatted samples was measured and transferred into a roundbottomed flask. then 50 ml of trichloroacetic acid was added to the sample in the round-bottomed flask and boiled. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 300 table 1. composite blends of yellow fleshed cassava flour and quality protein maize flour flour blends yfcf (%) qpmf (%) control 100 0 cm-1 90 10 cm-2 70 30 cm-3 50 50 cm-4 30 70 cm-5 20 80 yyfcf= yellow fleshed cassava flour; qpmf= quality protein maize flour table 2. biscuit ingredients ingredients quantity flour 200g fat 100g salt 0.2g vanilla flavor 0.2g baking powder 2g powder milk 3 tablespoons egg 1 whole granulated sugar 70g source: oyewole et al (1996) 3. results and discussion 3.1 quality attributes of composite flour of yellow fleshed cassava flour and quality protein maize flour the quality attributes of the composite flour are presented in table 3. the moisture, fat, protein, crude fibre, ash and carbohydrate of the blends ranged from 9.25 to 10.04%, 0.80 to 3.87%, 5.9 to 8.54%, 1.20 to 3.30%, 0.78 to 1.22%, and 75.98 to 81.02%, respectively. the moisture contents of the blends were lower than 14% recommended for safe storage of flour foods [30]. similar ranges of values were reported by thariseet al[30], for cassava-based composite blends. the protein, crude fibre, ash and mineral contents of the blends increased while the carbohydrate energy and cyanide contents decreased as the maize flour increased. the hydrogen cyanide, which decreased as the maize flour increased in the blend, ranged between 1.08 and 1.62 mg/kg. the cyanide content ranged from 1.08 to 1.62 mg/kg and increased as qpmf increased in the blend. the β-carotene contents of the composite flour ranged from 1.40 to 1.87 µg/g, and decreased with increase in qpmf. the quantity of β-carotene in the biscuit samples is much lower than the daily recommended values of 750µg and 400 to 600 µg for women and children, respectively [10]. the lightness (l*), redness (a*) and yellowness (b*) increased as the content of qpmf increased in the blends. this observation contradicts the findings of bolade and adeyemi [31] who reported that the l* value of cassava-maize flour blend increased with increase in cassava flour. the difference may be due to the varieties of maize and cassava used in the two studies. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 301 table 3. quality attributes of composite flour of yellow fleshed cassava and quality protein maize quality attributes cm-1 cm-2 cm-3 cm-4 cm-5 proximate composition (%) moisture 10.03b 10.04b 9.81b 9.26a 9.25a fat 0.80a 3.87e 3.13c 1.97b 3.43d protein 5.9ab 6.35ab 6.79bc 7.66cd 8.54d crude fibre 1.20a 1.90ab 2.65bc 3.15c 3.30c ash 1.04ab 0.78a 1.14b 1.22b 1.20b carbohydrate 81.02b 77.05a 76.48a 76.73a 75.98a mineral composition (mg/100g) calcium 0.10a 1.17b 1.46c 1.67d 1.92e sodium 0.29a 0.41c 0.37b 0.42d 0.47e magnesium 0.21a 0.22b 0.24c 0.26d 0.27e zinc 0.11a 0.12b 0.13c 0.16d 0.17e iron 0.11a 0.12b 0.17c 0.17d 0.21e energy (kcal/g) 354.90a 368.48b 361.21ab 355.34a 367.80b β-carotene (µg/g) 1.87b 1.58ab 1.45a 1.51a 1.40a hcn (mg/kg) 1.62d 1.60d 1.41c 1.30b 1.08a colour l* 91.43d 87.30bc 85.15bc 82.39ab 79.33a a* -0.005a 0.085ab 0.135ab 0.370bc 0.560c b* 12.40b 15.50c 18.82d 23.62e 25.22f cm-1= 90% cassava flour + 10% qpm; cm-2=70% cassava flour+ 30% qpm; cm-3=50% cassava flour+50% qpm; cm-4=30% cassava flour + 70% qpm; cm-5=20% cassava flour + 80% qpm. means with same superscripts within a row are not significantly different at p > 0.05 3.2 physical and chemical attributes of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize the physical and chemical attributes of the biscuits are presented in table 4. the weight, diameter, thickness and spread ratio of the biscuits, which varied significantly, were 6.64 to7.93g, 4.51to 4.56 cm, 0.622 to 0.767 cm, and 5.93 to 7.33, respectively. the weight, thickness and diameter of the biscuits increased as the proportion of qpmf increased in the flour blend. similar trend was reported by previous workers [20, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 302 25, 21]. this may be due to the additional protein from qpmf. proteins (especially the polar amino acids) have been reported to enhance binding of biscuit components due to their high water absorption capacity [21]. bolade and adeyemi [31] reported that the water holding capacity of maize flour (2.09 g/g) was higher than cassava flour (1.87 g/g). the authors further reported that the water holding capacity of maize-cassava flour blend decreased with increase in the level of cassava flour. except for the biscuits prepared from flour blend containing 20% yellow fleshed cassava and 80% quality protein maize, other samples had spread ratio higher than the control. the spread ratio has been referred to as spread in another publication [32]. it depends on the available moisture to act as solvent and the dough strength, among other factors. increased availability of water has been reported to result in a decrease in dough viscosity and increase in spread ratio [32]. increased spread is generally desirable in biscuit making; exception however is ginger nut biscuit where increased thickness (height) is required to develop its desirable porous and soft crumb [32]. the spread ratio tended to increase with increase in qpmf. significant (p < 0.05) differences occurred in the colour attributes of the biscuit samples. there was a decrease in the lightness (l* value) and increase in the redness (a* value) and yellowness (b* values) as the level of qpmf increased in the blend. the textural properties of a food refer to those properties impacted by the structural elements of the food. they are objectively measured by the sense of touch in relation to the deformation, disintegration and flow of food under a force and are functions of time, mass and distance [33]. changes in ingredients and processing can cause variations in texture [34]. the peak force of the qpm-based biscuits was lower than the control. taiwo et al [21] reported similar results. the high the peak force the harder the cookies; harder cookies are undesirable [35]. there were significant (p < 0.05) differences in the proximate composition of the biscuits. the moisture contents of the biscuit samples were between 5.57 and 6.51%, which is higher than the level (1.60 to 1.19%) reported by taiwo et al [21] and the 5% limit given by the nigerian industry standard. the importance of moisture content in the storability of biscuit cannot be overemphasized as it affects the biochemical activity of foods. the range of values of protein, moisture, ash, fat, fibre and carbohydrates of the biscuit samples were 11.38 to 14.67%, 1.57 to 2.51%, 0.66 to 1.48%, 25.10 to 28.22%, 1.60 to 2.89% and 52.59 to 58.45%, respectively. the protein, ash, fat and crude fibre contents of the biscuits increased while the moisture and carbohydrate decreased with increase in qpmf level. similar results were reported by olapade and ogunade [36] and amir et al [37]. foods containing high amount of protein, fat, ash and crude fibre tended to have lower amount of carbohydrate [21]. kaur et al [18] associated increase in spread ratio with increase in crude fibre. the authors further submitted that high-fibre flours produce weaker dough than refined flours, which results in greater flow of cookie dough and larger cookie diameter. there were significant (p < 0.05) differences in the mineral composition of the biscuits. the mineral contents increased with increase in qpmf level. the energy content decreased the qpmf level increased. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 303 table 4. physical and chemical attributes of biscuits prepared from biofortified cassava-maize composite flour quality attributes cm-1 cm-2 cm-3 cm-4 cm-5 control* physical attributes weight (g) 6.88b 6.64a 7.18c 7.43d 7.93e 7.34d diameter (cm) 4.51a 4.56c 4.53b 4.55bc 4.55bc 4.51a thickness (cm) 0.639ab 0.622a 0.645c 0.767b 0.767b 0.675c spread ratio 7.05c 7.33d 6.74b 7.04c 5.93a 6.67b colour l* 58.82d 55.95c 50.82b 51.24b 46.79a 63.82e a* 7.23b 7.75c 9.06d 8.91d 10.32e 5.59a b* 21.75c 21.94e 21.24b 22.83d 20.87a 21.74c peak force (n) 58.65e 32.70d 29.27c 13.19a 22.10b 93.10f proximate composition (%) moisture 6.01c 5.74ab 5.81ab 5.57a 5.93c 6.51d fat 25.65a 28.22a 25.10a 27.20a 26.83a 27.81a protein 12.92b 12.48b 11.38a 14.67c 14.67c 12.92b crude fibre 1.60a 2.89c 2.15ab 2.15ab 2.50bc 1.60a ash 0.82a 0.66a 1.10b 0.82a 1.48c 1.10b carbohydrate 57.01ab 54.01ab 58.45b 53.58ab 52.59a 54.06ab mineral composition (mg/100g) calcium 0.85b 0.87c 1.46d 1.67e 1.92f 0.77a sodium 0.29b 0.41d 0.37c 0.42e 0.47f 0.23a magnesium 0.21b 0.22b 0.24bc 0.26c 0.27d 0.16a zinc 0.11b 0.12bc 0.13c 0.16d 0.17d 0.09a iron 0.11a 0.12a 0.17b 0.17b 0.21c 0.10a energy (kcal/g) 510.53a 519.92a 505.26a 517.82a 510.48a 518.21a β-carotene (µg/g) 0.045a 0.819b 1.45c 2.82a 2.17d 1.51c hcn (mg/kg) 1.41a 1.19b 1.22b 1.12b 1.09b 1.62a cm-1= 90% cassava flour + 10% qpm; cm-2=70% cassava flour+ 30% qpm; cm-3=50% cassava flour+50% qpm; cm-4=30% cassava flour + 70% qpm; cm-5=20% cassava flour + 80% qpm. means with same superscripts within a row are not significantly different at p > 0.05, *biscuit prepared from 100% biofortified cassava flour however, except for the two samples obtained from yfcf:qpmf (70:30) and yfcf:qpmf (20:80), there was no significant (p > 0.05) difference between the energy content of the qpm-based biscuits and control. the low energy value of the control compared to the other flour blends could be as a result of the higher protein and fat contents of qpm-based biscuits since dietary fat is the most concentrated source of energy. the βcarotene content of the biscuits, which varied significantly from 1.08 to 1.62 µg/g, decreased with increase in qpmf. there was a reduction in the hydrogen cyanide content of the biscuit samples. the hydrogen cyanide content ranged between 1.09 and 1.62 mg/kg. 3.4 sensory attributes of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize in terms of colour, no significant (p > 0.05) difference was observed between the control and the biscuit samples containing ≤ 50% maize flour (table 5). there was no significant (p > 0.05) difference in the taste, crunchiness and aroma of biscuits prepared from the composite flour and 100% cassava flour. on the other hand, biscuits prepared from composite flour containing ≥ 50% maize flour were rated higher than control in terms of overall acceptability. however, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 304 biscuit prepared from composite flour of 20% cassava and 80% high quality protein maize were the most accepted. olapade and ogunade [36] also reported that the addition of maize flour enhanced the sensory attributes of crunchy snacks made from sweet potato flour. table 5. sensory attributes of biscuits prepared from composite flour yellow fleshed cassava and quality protein maize quality attributes cm-1 cm-2 cm-3 cm-4 cm-5 control* colour 6.86a 7.32ab 7.32ab 7.54b 7.58b 6.74a taste 6.40a 6.88a 7.20a 6.68a 6.74a 6.94a crunchiness 6.38a 6.72a 6.82a 6.78a 7.12a 6.64a aroma 6.54a 7.00a 7.00a 7.16a 6.86a 6.62a overall acceptability 6.70c 7.02c 7.34b 7.40a 7.42b 7.08d cm-1= 90% cassava flour + 10% qpm; cm-2=70% cassava flour+ 30% qpm; cm-3=50% cassava flour+50% qpm; cm-4=30% cassava flour + 70% qpm; cm-5=20% cassava flour + 80% qpm. means with same superscripts within a row are not significantly different at p > 0.05, *biscuit prepared from 100% biofortified cassava flour 4. conclusion the study investigated the nutrient composition, physical and sensory attributes of biscuit from composite flours of yellow fleshed cassava flour and quality maize. the protein, fat and crude fibre and mineral (ca, na, mg, zn and fe) contents of the biscuit samples increased with increased substitution with quality maize flour. the study has indicated that acceptable and nutritious biscuit could be obtained from composite flour of 20% yellow fleshed cassava and 80% quality maize flour. 5. references [1]. agiriga, a.n., iwe, m.o. physical properties of cookies produced from cassava –groundnut-corn starch blenda response surface analysis. nigerian food journal, 26(2), (2008). available at: https://doi.org/10.4314/nifoj.v26i2.47441. accessed: 01.02.2020. [2]. olapade, a.a., adeyemo, a.m. evaluation of cookies produced from blends of wheat, cassava and cowpea ours. international journal of food studies,3: 175-185, (2014). [3]. adeyeye, s.a.o., akingbala, j.o. quality, functional, and sensory properties of cookies from sweet potato–maize flour blends. journal of culinary science & technology, 14(4): 363–376, (2016). [4]. oluwole, o.b., karim, o. r. production of biscuits from bambara, cassava and wheat flours. journal of raw materials research, 2(1): 34-38, (2005). [5]. bala, a., gul, k., riar, c.s. functional and sensory properties of cookies prepared from wheat flour supplemented with cassava and water chestnut flours. cogent food & agriculture, (2015). available at: https://doi.org/10.1080/23311932.2015.1019815. accessed: 01.02.2020. [6]. ikuomola, d.s., otutu, o.l.,oluniran, d.d.quality assessment of cookies produced from wheat flour and malted barley (hordeum vulgare) bran blends. cogent food & agriculture,3, (2017). available at: https://doi.org/10.1080/23311932.2017.1293471. accessed: 02.04.2020. [7]. otekunrin, o.a., sawicka, b. cassava, a 21st century staple crop: how can nigeria harness its enormous trade potentials. acta scientific agriculture, 3: 194-202, (2019). https://doi.org/10.1080/23311932.2015.1019815 https://doi.org/10.1080/23311932.2017.1293471 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 305 [8]. falade, k.o., akingbalaj.o.improved nutrition and national development through the utilisation of cassava in baked foods. in: robertson, g.l. and lupien, j.r. (eds): using food science and technology to improve nutrition and promote national development. usa: international union of food science & technology, (2008). [9]. aniedu, c., omodamiro, r.m. use of newly bred pro-vitamin a cassava in production of value-added products: implication for food security in nigeria. global journal of science frontier research, 12: 10-16, (2012). [10]. eleazu, c.o., eleazu, k.o. determination of the proximate composition, total carotenoids, reducing sugar and residual cyanide levels of flours of 6 new yellow and white cassava (manihot esculenta crantz) varieties. american journal of food technology, 7(10): 642-649, (2012). [11]. ogunjobi, m.a.k., ogunwolu, s.o. physicochemical and sensory properties of cassava flour biscuits supplemeted with cashew apple powder. journal of food technology, 8 24-29, (2010). [12]. kim, j.c., de ruiter, d. bread from nonwheat flours. food technology, 22(7): 867-878, (1968). [13]. eggleston, g., omoaka, p.e., arowosegbe, a.c. flour, starch and composite bread-making quality of various cassava varieties. journal of the science of food and agriculture, 62: 49-59, (1993). [14]. oyewole, o.b., sanni, l.o., ogunjobi, m.a. production of biscuits using cassava flour. nigerian food journal, 14: 25-30, (1996). [15]. nuss, e.t., tanumihardjo, s.a. quality protein maize for africa: closing the protein inadequacy gap in vulnerable populations. advances in nutrition, 2: 217-224, (2011). [16]. gunaratna, s., moges, d., de groote, h. biofortified maize can improve quality protein intakes among young children in southern ethiopia nilupa. nutrients 11: 192, (2019). available at: https://doi.org/10.3390/nu11010192. accessed: 05.03.2020. [17]. motukuri, s.r.k. quality protein maize: an alternative food to mitigate protein deficiency in developing countries. intechopen, (2019). available at: https://doi.org/10.5772/intechopen.89038. accessed: 02.04.2020. [18]. kaur, j., kaur, a., singh, j. nutritional evaluation and utilisation of composite whole flours for making functional cookies rich in β-glucan and isoflavones. british food journal, 119(4): 909-920, (2017). [19]. gupta, h.o., singh, n.n. preparation of wheat and quality protein maize basedbiscuits and their storage, protein quality and sensory evaluation. journal of food science and technology –mysore, 42(1): 43-46, (2005). [20]. giwa, e.o., ikujenlola, a.v. quality characteristics of biscuits produced from composite flours of wheat and quality protein maize. african journal of food science and technology,1(5): 116119, (2010). [21]. taiwo, k.a., raji, o.i., ikujenlola, v.o., adebayo, w.a. production of biscuits from quality protein maize enriched with dried fluted pumpkin leaves and cocoa powder. in: american society of agricultural and biological engineers annual international meeting, proceedings of 2018 asabe annual international meeting. detroit, michigan, (2018). [22]. mesfin, w., shimelis, a. effect of soybean/cassava flour blend on the proximate composition of ethiopian traditional bread prepared from qpm. african journal of food, agriculture, nutrition and development, 13(4):7985-8003, (2013). [23]. aoac. official methods of food analysis. washington, dc: association of official analytical chemists, (2010). [24]. james,c.s. analytical chemistry of foods. new york, usa: blackie academic & professional, (1996). [25]. tesfaye, b. development of biscuits from wheat, qpm and carrot composite flours. m.sc. thesis, addis ababa institute of technology, ethiopia, (2010). [26]. kimura, m., amaya, r. harvestplus handbook for carotenoid analysis.brazil:harvestplus technical monographs series 2, (2004). [27]. sarkiyayi, s., abdullahi, h.s. chemical evaluation of meat pies sold in some selected bakeries in mimeta metropolis, adamawa state, nigeria. direct research journal of agriculture & food science, 3(3): 57-61, (2015). [28]. aacc. approval method of the american association of cereal chemist. method 10-52micro sugar-snap cookie method. usa: aacc, (1995). [29]. da silva, p.,, moreira, r. vacuum frying of high-quality fruit and vegetable based snacks. lwtfood science and technology41: 1758-1767, (2008). doi:10.1016/j.lwt.2008.01.016. https://doi.org/10.3390/nu11010192 https://doi.org/10.5772/intechopen.89038 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade, quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize, food and environment safety, volume xix, issue 4 – 2020, pag. 297 – 306 306 [30]. tharise, n., julianti, e., nurminah, m. evaluation of physico-chemical and functional properties of composite flour from cassava, rice, potato, soybean and xanthan gum as alternative of wheat flour. international food research journal, 21(4) 1641-1649, (2014). [31]. bolade, m.k., adeyemi, i.a. functionality enhancement of composite cassava flour in the production of maize tuwo (a nonfermented maize-based food dumpling). food bioprocess technology, 5 1340–1348, (2012). [32]. filipčev, b., šimurina, o., bodrožasolarov, m. impact of buckwheat flour granulation and supplementation level on the quality of composite wheat/buckwheat ginger-nut-type biscuits. italian journal of food science,27: 495504, (2015). [33]. jisha, s., padmaja, g., sajeev, m.s.nutritional and textural studies on dietary fibreenriched muffins and biscuits from cassava-based composite flours. journal of food quality, 33 79-99, (2010). [34]. gaines, c.s. objective assessment of cookie and cracker texture. in: faridi, h. (ed): the science of cookie and cracker production, 455-495. new york, usa: chapman & hall, (1994). [35]. laukova, m., minarovicˇova, l., karovicova, j., kohajdova, z. quality evaluation of sweet potato powder-enriched cereal products. food science and technology international, 25(6): 523-532, (2019). [36]. olapade, a.a., ogunade, o.a.production and evaluation of flours and crunchy snacks from sweet potato (ipomea batatas) and maize flours. international food research journal,21(1): 203-208, (2014) [37]. amir, b., mueen-ud-din, g., abrar, m., mahmood, s., nadeem, m., mehmood, a. chemical composition, rheological properties and biscuits making ability of composite flours from maize, sorghum and wheat. journal of agroalimentary processes and technology, 21(1) 28-35, (2015). doi: https://doi.org/10.4316/fens.2021.028 256 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 – 2021, pag. 256 261 antioxidants’ effect on the storage of dairy products with high-fat content olena grek 1 , *tetiana pshenychna 1 , mariia nikolaieva 1 1educational and research institute of food technology, national university of food technologies, 01601, volodymyrska str.68, kyiv, ukraine. tanya5031@ukr.net *corresponding author received 18th june 2021, accepted 28th september 2021 abstract: the article presents the research results of the antioxidants’ effect of various origins on the storage of dairy products with high-fat content. the quality change of such products was monitored by the oxidation product content. the antioxidants’ effect of natural origin and human-made origin on the change in the peroxide and acid numbers in spreads with food fibers during storage period for 15 and 30 days at a temperature of (20±2) ° c and (0...5) °c has been determined. a significant increase in the peroxide number occurs at a temperature of (20±2) °c. it has been established, that after 6 storage days under the given conditions, the indicator for the control sample increases 4 times, while for products with high-fat content with the addition of antioxidants "novasol cof" and "grindox 109" – 1.2 times. on the 12th storage day, the peroxide number in the spread without additives increased by 8.65 times, in the spread with the introduction of the natural flavoring agent "caraway – elite flavor" – by 7.15, with the antioxidants "novasol cof" and "grindox 109" – by 4.45 and 4 times, respectively. there was a similar trend towards an increase in the acid number for the research samples of products with high-fat content. after 12 storage days, a significant increase in the value of the acid number has been found in the samples of spreads without antioxidants – 13 times, with the introduction of the natural flavoring agent "caraway – elite flavor" – 7.8 times. low acid number values are observed in the samples with the addition of the antioxidants "novasol cof" and "grindox 109", in comparison with other spreads. the obtained results are the basis for the development of recommendations for extending the storage life of products with high-fat content. keywords: product with high-fat content, antioxidant, spread, storage period, peroxide number, acid number. 1. introduction modern industrial milk processing is a large complex of consistently interconnected chemical, physicochemical, microbiological, biochemical, thermophysical, and other labor-intensive and specific technological processes. they focused on the production of dairy products containing all the constituents of whole milk or its components. manufacturers desire to improve sensory properties and physicochemical indicators, to achieve safety and products profitability in order to increase the competitiveness of the brand name sometimes leads to a change in traditional production methods, composition rationalization, and the use of other measures, in which economic feasibility does not always positively correlate with quality indicators, food and biological value of products. relevant in the dairy industry is the simultaneous provision of traditional production methods using modern equipment and the development of new technologies for highquality dairy products with a http://fens.usv.ro/index.php/fens mailto:tanya5031@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 olena grek, tetiana pshenychna, mariia nikolaieva, antioxidants' effect on the storage of dairy products with high-fat content, food and environment safety, volume xx, issue 3 – 2021, pag. 256 261 257 multicomponent composition and an extended storage life [1-2]. despite the current trends towards a decrease in the calorie content of dairy products, there is an expansion of the assortment range of products with a highfat content – spreads and butter with fillers, drinking cream, sour cream products with non-dairy fats, desserts with a fat mass fraction of 15.0 to 46.0 % based on cottage cheese (chocolate glazed curd bar, pastes, sweet masses, cakes, creams). extending the storage life of the abovementioned products is a priority task, as the composition combination and the addition of the ingredients that are not traditional for products with high-fat content leads to defects and spoilage. to prevent the above-mentioned processes, it is advisable to use antioxidants capable of reacting with free radicals and thus breaking the oxidation chain and prolonging the storage life [3-4]. the aim of this work is to study the antioxidants’ effect of various origins on the storage of products with a high-fat content – spreads with food fibers. according to dstu 4445: 2005, a spread is a food fat product (water-in-fat emulsion), which consists of milk and vegetable fat with a total fat mass fraction of 50 % to 85 % and in which the milk fat fraction is not less than 25 % of total fat, with a heavy or soft consistency. spreads can be manufactured with food additives, fillers, and vitamins. during spreads’ storage, vegetable and animal fats included in its composition, under the influence of atmospheric oxygen, light, especially direct sunlight or ultraviolet, moisture, gradually acquire an unpleasant taste and smell [5]. the reason for fatty foods' spoilage is the development of oxidative and enzymatic processes. thus, the oxidative destruction of the fatty components in spreads, first of all, leads to the formation of peroxides, aldehydes, ketones, low molecular weight acids, and other compounds. fats and oils, which contain desaturated fatty acids (linolenic, linoleic, oleic acids), when exposed to air, dissolve their components, including oxygen, and oxidize. as a result, the nutritional and biological value of fats decreases, and they may be unsuitable for consumption [6]. 2. matherials and methods the object of research is products with high-fat content – spreads with food fibers and antioxidants. considering theoretical information, an antioxidants’ mixture based on vegetable fats: natural origin – "novasol cof" or human-made origin – "grindox 109", natural flavoring agent "caraway elite aroma" (tu u 24.622961668-007: 2007) based on the aromatic oil, have been used to extend the storage life of the spreads. "novasol cof" (belagroleks, belarus) is a highly effective liquid antioxidant, the main active substances of which are ascorbic acid (vitamin c) (e 300) and tocopherol (vitamin e) (e 306) [7-8]. "grindox 109" (danisco, denmark) is a mixture of antioxidant substances: butylhydroxyanisolum (e-320) (10%), butylhydroxytoluenum (e-321) (10%), propyl gallate (e 310) (6%), citric acid with propylene glycol, food emulsifier and rapeseed oil (68%) as a basis [9]. the natural flavoring agent technology of caraway aromatic oil has been developed by nuft scientists. this technology provides for vacuum fractionation of raw materials, obtaining fractions with desired aromatic properties. as the caraway aromatic oil is insoluble in water, therefore, the dosage is carried out with a 40 % alcohol-water solution. the chemical composition is represented by the following main components: βmyrcene, limonene, α-terpineol, carvone, p-cymene, cis-limonene oxide, translimonene oxide, caryophilene, etc. it is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 olena grek, tetiana pshenychna, mariia nikolaieva, antioxidants' effect on the storage of dairy products with high-fat content, food and environment safety, volume xx, issue 3 – 2021, pag. 256 261 258 known that this compound's class is extremely reactive due to the presence of active chemical centers (and bonds, carbonyl groups). these substances can play the role of antioxidants, as they are among the class of the same compound as vitamin e [10]. obtaining research samples of spreads with food fibers to determine the antioxidants’ effect of various origins on the storage life of products with high-fat content – spreads with food fibers, research samples have been developed using a technology based on the separate preparation of a milk-fat emulsion (oil (15.0 %), сіtгі-fі (0.3 %), skim milk), and mixing with cream [11]. the food fibers' addition is caused by their high fat-binding capacity, emulsifying, structure-forming properties. the next technological operations were the antioxidants introduction into the emulsion, namely "novasol cof"– 0.04 %, or "grindox 109" – 0.05 % or a natural flavoring agent based on aromatic oils "caraway – elite aroma" – 0.05 % (the quantity is chosen according to the manufacturers’ recommendations), mixing until complete dissolution, emulsification and pasteurization, intensive thermomechanical processing. during the last operation, the liquid fat/water emulsion is converted to a reverse type water/fat emulsion system. the finished samples were packed in 100 g parchment paper. spreads were stored with no exposure to light for 15 days at a temperature of (20±2) °с and for 30 days at (0...-5) °с. control samples were spreads with food fiber without antioxidants. the quality change in spreads with food fibers during the storage period was controlled by the content of oxidation products (peroxide number) and fat hydrolysis (acid number). the peroxide number characterizes the content of primary oxidation products in the fat – peroxides and hydroperoxides, which have almost no effect on the sensory characteristics of the product but indicate significant changes in the research object [12]. therefore, by determining the peroxide compounds accumulation during the storage period of the fat base of the spread, it is possible to establish its resistance to oxidation long before the change in its sensory characteristics. determination of peroxide and acid numbers in products with high-fat content to determine the peroxide number in spreads with food fibers, 1 g of spread was weighed into a conical flask with a ground stopper with an accuracy of 0.2 mg. the spread was melted in a water bath and 10 cm3 of chloroform and 10 cm3 of glacial acetic acid were added along the wall, washing off the residues. then 0.5 cm3 of a saturated freshly prepared aqueous solution of potassium iodide was introduced. the flask was closed with a stopper, the contents were mixed and left in a dark place for 3 min. then 100 cm3 of water was added, to which 1 cm3 of 1 % starch solution was previously added, and titrated by 0.01 n thiosulfate solution until the blue color disappeared. peroxide number (x), mmol 1/2 o/kg, was calculated by the formula: where x – the peroxide number, mmol 1/2 o/kg; v1 – the amount of 0.01 n thiosulfate solution, which was used for titration during the main experiment with a fat weight, cm3; v – the amount of 0.01 n thiosulfate solution, which was used for titration of the sample without fat, cm3; g – fat weight, g; 0.00127 – the gram number of iodine, equivalent to 1 cm3 of 0.01 n thiosulfate solution. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 olena grek, tetiana pshenychna, mariia nikolaieva, antioxidants' effect on the storage of dairy products with high-fat content, food and environment safety, volume xx, issue 3 – 2021, pag. 256 261 259 to determine the acid number, a spread with food fibers weighing 5 g was introduced into a conical flask with a volume of 100 cm3, heated in a water bath at 45...50 °c until melting, and dissolved in 20 cm3 of a neutralized mixture of ethyl alcohol and diethyl ether (1:1). then 3 drops of 1 % phenolphthalein solution were added and, with constant stirring, titrated with an aqueous solution of 0.1 n alkali (naoh). the titration was finished when a pink color appeared and did not disappear within 1 min. the acidity in the spread with food fibers was calculated by multiplying the amount of 0.1 n alkali solution, which was used to neutralize milk fat, by 2 [13]. 3. results and discussion the antioxidants’ effect of various origins on the change in the peroxide number of products with high-fat content – spreads with food fibers at a temperature of (20±2) °с and (0...-5) °с is shown in figure 1. according to the research results presented in figure 1, the auto-oxidation process of the fatty base of the spreads at different temperatures was different. a significant increase in the peroxide number occurs at a temperature of (20±2) °c. thus, after 6 storage days under the given conditions, the indicator for the control sample increases 4 times, while for products with high-fat content with the addition of antioxidants "novasol cof" and "grindox 109" – 1.2 times. intensive peroxides accumulation in spreads with food fibers has been found on the 12th storage day. in particular, the peroxide number in the spread without additives increased by 8.65 times, in the spread with the introduction of the natural flavoring agent "caraway – elite flavor" – by 7.15, with the antioxidants "novasol cof" and "grindox 109" – by 4.45 and 4 times, respectively. in the control sample, the primary product accumulation of fat oxidation reached a maximum value of 20.3 1/2 o/kg after 12 storage days. by the end of the storage period (15 days), the peroxide numbers in products with high-fat content decreased slightly in comparison with the corresponding values of this indicator on the 12th day. this is due to the subsequent transformation of primary oxidation products into secondary ones. а) b) fig. 1. the antioxidants’ effect of various origins on the change in the peroxide number of spreads with food fibers during the storage period at different temperatures: a) t = (20±2) °с and b) t = (0... -5) °с food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 olena grek, tetiana pshenychna, mariia nikolaieva, antioxidants' effect on the storage of dairy products with high-fat content, food and environment safety, volume xx, issue 3 – 2021, pag. 256 261 260 in this period, the difference between the peroxide numbers in the control sample and spreads with antioxidants of various origins ranged from 1 to 2.7 %, respectively (when stored at a temperature of (20±2) °c). so, the highest antioxidant activity during the storage period of spreads at a temperature of (20±2) °с was shown by antioxidants of natural origin "novasol cof" and human-made origin "grindox 109". the value of peroxide number is less than 10 mmol 1/2 o/kg, which is the maximum permitted concentration according to the requirements of regulatory documents [14]. the antioxidants added to the product slowed down the course of oxidative processes in comparison with the control sample. it is known, as a result of hydrolysis, free fatty acids are disengaged, the content of which characterizes the acid number. by the value of the latter, the fat quality can be determined, an increase in acidity indicates spoilage. the change in the acid number of products with high-fat content – spreads with food fibers, depending on the antioxidants type during storage period at temperatures (20±2) °с and (0...5) °с is depicted in figure 2. a) according to the research results (fig. 2 a), the amount of free fatty acids in the spreads after 3 storage days at a temperature of (20±2) °с was insignificant. with an increase in the storage duration of the spreads, the acid number of their fat base – a mixture of milk fat and oil – gradually increased, most intensively in the control sample. the increase in the acid number on the 9th storage day at a temperature of (20±2) °с is noticeable only in the spread without antioxidants – by 6.25 times, while in the spreads with the addition of the natural antioxidant "novasol cof" the amount of free fatty acids increased by 1, 3 times. a similar value is observed in the sample with the addition of the human-made antioxidant "grindox 109" (an increase in the acid number by 1.25 %). after 12 storage days, a significant increase in the value of the acid number has been found in the samples of spreads without antioxidants – 13 times, with the introduction of the natural flavoring agent "caraway – elite flavor" – 7.8 times. regarding the samples of spreads, which were stored at a temperature of (0...-5) °с (fig. 2 b), a similar tendency towards an increase in the acid number has been observed. b) fig. 2. the antioxidants’ effect of various origins on the change in the acid number of spreads with food fibers during the storage period at different temperatures: a) t = (20 ± 2) °с and b) t = (0... -5) °с food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 olena grek, tetiana pshenychna, mariia nikolaieva, antioxidants' effect on the storage of dairy products with high-fat content, food and environment safety, volume xx, issue 3 – 2021, pag. 256 261 261 however, due to low temperatures, the growth dynamic of this indicator is slow. thus, the largest amount (0.18 mg koh/g) of free fatty acids in the spread without antioxidants has been revealed on the 20th storage day. low acid number values have been observed in the samples with the antioxidants’ addition of natural origin and human-made origin "novasol cof" and "grindox 109", compared to other products with high-fat content. 4. conclusions while investigating the antioxidants’ effect of various origins on the storage of products with high-fat content – spreads with food fibers for peroxide and acid numbers, it has been established that the most active are antioxidants of humanmade origin "grindox 109". by choosing antioxidants, it is possible to significantly reduce unwanted oxidative and hydrolytic changes in the fatty base of the spreads. food storage at low temperatures inhibits these changes and, as a result, reduces the rate of radical reactions. in addition, it is possible to combine milk and vegetable fats with food fibers. investigation of the intensity level of spoilage processes can be used to regulate the storage life of other dairy products with a multicomponent composition and high-fat content. 5. references [1] grek e., krasulya e., savchenko a., petrina a., fatty acid composition of dairy fat products of vegetable origin. ukrainian journal of food science. 2(1): 14-21, (2014). [2] comert e. d., gokmen v., evolution of food antioxidants as a core topic of food science for a century. food research international, 105: 76-93, (2018). [3] grek o. v. the use of antioxidants for dairy products with high-fat content. dairy industry, 2: 2830, (2013). [4] carocho m., morales p., ferreira i. antioxidants: reviewing the chemistry, food applications, legislation and role as preservatives. trends in food science & technology, 71: 107-120, (2018). [5] mendez-cid f. j., centeno j. a., martinez s. carballo j., changes in the chemical and physical characteristics of cow’s milk butter during storage: effects of temperature and addition of salt. journal of food composition and analysis, 63: 121-132, (2017). [6] o' dwyer s. p., o'beirne d., ni eidhin d., henessy a. a., o' kennedy b. t., formation, rheology and susceptibility to lipid oxidation of multiple emulsions (o/w/o) in table spreads containing omega-3 rich oils. lwt food science and technology, 51(2): 484-491, (2013). [7] khaneghah a. m., an overview on some of important sources of natural antioxidants. international food research journal, 23: 928-933, (2016). [8] nechaev a. p., nikolaeva y. v., pilipenko o. v., dubrovin g. a., samoilov a. v., ways to improve oxidation resistance of low fat spreads using natural antioxidants in micellised form. food industry, 3: 11-14, (2018). [9] savchenko o. a., grek o. v., petrina a. b., topchiy o. a., krasulya o. o., technologies of products with modified fat composition: realities and trends. monograph k.: "komprint", isbn 978-966-929-915-4, 250 p. (2018). [10] lorenzo j. m., munekata e. s., gomez b., barba f. j., mora l., perezsantaescolastica c., toldra f., bioactive peptides as natural antioxidants in food products. trends in food science & technology, 79: 136-147, (2018). [11] grek o. v., krasulya o. o., the influence of complex antioxidants on the storage life of dairy products. products & ingredients, 8(83): 43-44, (2011). [12] podkovko o. a., rashevskaya t. a. research of antioxidant properties of oil paste with cryopowder from table beet. food industry, 3: 81-86, (2015). [13] darmawan m. a., muhammad b. z., harahap a. f., ramadhan m. y., sahlan m., supriyadi t., adb-azizd s., gozan m. reduction of the acidity and peroxide numbers of tengkawang butter (shorea stenoptera) using thermal and acid activated bentonites. heliyon, 6. (12): (2020). doi.org/10.1016/j.heliyon.2020.e05742 [14] dstu 4445:2005 spreads and fat blends. general specifications, state committee for technical eegulation and consumer policy, 25 p. (2006). 321 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 321 -336 the potential of electronic noses in quality and freshness prediction of fresh and stored meat products review * leonard fweja 1 1faculty of science, technology and environmental studies, the open university of tanzania, p.o box 23409, dar es salaam, tanzania, lfweja@yahoo.com *corresponding author received 27th november 2019, accepted 27th december 2019 abstract: the present review aimed at evaluating the potential of e-noses in real-time quality and freshness monitoring and evaluation of a variety of fresh meat products (beef, pork, chicken and fish) in view of their inherent differences. electronic noses (e-noses) have been demonstrated to have great potential in food quality monitoring and prediction. several experimental trials on e-noses have been conducted to assess their ability in quality screening of food products. in this review, the success and shortcomings of the e-nose performances are discussed including the previous and current advancement. a narrative literature review approach was used to critically evaluate literature which waz summarized with conclusions drawn and the inconsistence / drawbacks in the functionality of enoses in quality monitoring revealed. key words: e-noses, food quality screening, meat products 1. introduction meat and fish are among the highly perishable foods with a very short shelf life especially when handled inappropriately. poor handling subjects these perishable food items to accelerated spoilage. semeano [1] indicated that fish products are exceedingly perishable and sensitive to microbial growth, and the risks linked with the consumption of food contaminated with harmful bacteria are considerable. according to chamhuri and batt [2] food quality is usually related to freshness, food safety and nutritional value. other researchers [3] reported that once the quality of food is lessened because of the low level of freshness, purity factor, safety and nutrition, it can contribute to health risks. proper monitoring of quality is therefore an important element in ensuring food quality and safety of consumers and it is even more critical when dealing with highly perishable food materials. the situation can even be further aggravated due to improper handling during transport, selling or storage. conventional quality control methods are available which include microbiological and physical or chemical techniques. although efficient and accurate, these methods have some common shortcomings, such as high costs of execution, extended time of analysis, low samples throughput, dependence on greatly qualified labor force, inapplicability for online production monitoring [4] and destructiveness of investigated products [5]. on the other hand, zhang et al. [6] described end-results of sensory analysis, an alternative method to rely on http://www.fia.usv.ro/fiajournal mailto:lfweja@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 322 individual’s skills; as such it is prone to frequent errors. other major drawbacks of this method are the elevated cost of training the human panel, low degree of reproducibility of evaluation, and standardization of the measurement [5]. falasconi et al. [4] documented several chemical methods for examining meat quality such as total volatile basic nitrogen (tvbn), ph, triphenyl tetrazolium chloride (ttc), and test paper which also require extended time, are expensive with inadequate precision [6]. due to their compositional nature, meat and fish can quite easily (especially in case of poor handling) reach a spoilage point. according to benabdellah et al. [7] the point of spoilage is described by the upper limit of acceptable bacterial level, undesirable odor or aspects for consumption. the familiar characteristics associated with spoilage are foul odor and gas production [7]. in view of the challenges associated with the conventional methods and a very short shelf life of meat products, researchers have attempted to develop quick simple alternatives, easy to use non-destructive devices for real-time monitoring and evaluation of food freshness and quality. these attempts include mimicking the human nose system to identify spoiled products based on their odor [5] that is an electronic nose (e-nose). e-nose, a device performing odor analysis takes its name from the human nose, and the process is known as machine olfaction [5]. the chemical and biosensor technologies have emerged as valuable contenders for food quality control due to their simplicity of use, low cost, rapidity, and good correlation with sensory panels [4]. enoses rely on an array of semi selective gas sensors and a suitable data processing method (pattern classification algorithm/ technique) [4, 5, 8] capable of measuring and identifying volatiles which contribute to specific odors [5, 8]. e-noses are advocated to present rapid and accurate way of sensing the incidence of food contaminant bacteria with minimal or no sample preparation [4]. therefore, this present review paper aims to bring to light an overview of current results achieved by applying e-noses in quality evaluation, highlighting their success, strength and drawbacks, scope of use and their potential in quality monitoring of meat products with great inherent differences. 2. description of meat products and enose 2. 1 fish freshness parameters generally, fish freshness quickly degrades due to enzymatic decomposition of adenosine triphosphate after death. gradually, reductive gaseous species which are the main components of the odour resulting from fish decay such as volatile sulfur compounds, volatile acidic compounds, volatile carbonyl compounds and volatile basic nitrogen compounds are released during the decomposition of fish. dimethylamine (dma) and trimethylamine (tma) are some of the representative odours which are produced during fish decomposition [9]. ólafsdóttir [10] on the other hand described the characteristic odour changes due to the formation of volatile compounds, like alcohols, aldehydes, ketones, esters, sulfur compounds and amines, by specific spoilage organisms (sso) as key determinants of chilled fish quality. 2.2 meat freshness parameters according to rivai et al. [11] aroma, color, texture and taste can be used to identify the freshness level of meat. the freshness and quality of slaughtered chicken meat depend on complex processes which are associated with different factors including food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 323 the bacteria present on the carcass prior to slaughter, the efficiency of processing, and the conditions and time of storage, which permit propagation prior to consumption and enzyme reactions [12]. the taste quality of meat is affected by volatile organic compound (voc) content. aroma or smell of meat is produced by complex mixture of voc coming from a diverse chemical reaction in meat. the modern categorization of meat freshness is based on the total amount of volatile basic nitrogen (tvb-n) found in meat. tvb-n is the quantity of nitrite material that is purified from vapor or gas in meat under alkalization conditions. tvb-n includes all the nitrogen content that can form ammonia under these conditions. on the basis of tvb-n, fresh meat is defined as meat containing tvb-n lesser than 15 mg / 100 g of meat. semi fresh meat has tvbn between 15-30 mg / 100g of meat, and altered bacon may contain over 30 mg / 100 g of meat [11]. 2.3 meat decomposition products meat is composed of water, protein, fat, and some carbohydrates; enzymes and bacteria will decompose some meat components into volatile gases. the protein will decompose into ammonia, hydrogen sulfide, and ethanethiol; fat decomposes into aldehydes and aldehyde acid; and carbohydrate decomposes into alcohol, ketone and carboxylic acid. in the process of spoilage, these gases’ concentrations will increase exponentially [13]. 2.4 general description of e-nose an e-nose is an analytical instrument designed to act-like human nose [11]. it is used for sensing food freshness by examining gaseous properties [14]. it consists of an array of chemical sensors matched with an appropriate data processing technique, capable of measuring and recognising volatile compounds which contribute to odours [8]. the configuration of an e-nose mimics the human sense of smell, learning the functions of evaluating, recognizing and identifying volatile chemicals to a higher accuracy [6]. human olfaction relies on the chemical interaction of volatile odour compounds and neurons in the nasal cavity. the signals generated by neurons are conveyed to the brain for further recognition of a specific substance. in machine olfaction, the sensors are comparable to human neurons and the pattern-matching algorithm is similar to the brain’s recognition process [5]. the enose technology is founded on the absorption and desorption of volatile chemicals onto a matrix of sensors [6]. the two major parts of an e-nose are the sensing system and the pattern recognition system. sensing systems may consist of arrays or chain of diverse sensing elements (e.g. chemical sensors), in which every sensing element measures the different properties of the chemicals tested [11]. unlike gas sensors which use specific sensors for measuring single attributes and quality and safety in the food and beverage sector, information can be also obtained from arrays of non-specific sensors with partly overlapping selectivity and treating the data obtained with pattern recognition software [15]. an e-nose is a substitute to single gas sensors which characterize complex mixtures that are more suitably recognized by an overall fingerprint rather than by the individual identification of their constituents [15]. these sensors show physical and chemical interactions with the chemical compounds when they flow over or are in contact with the sensors [14]. the response of a sensor is usually measured as the change of some physical parameters, e.g. conductivity or current [8] or change of electrical resistance once they get in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 324 touch with gas with the varied odors and aromas [6]. the responses of all sensors form a response pattern that can be learned by a computer [8]. e-nose technology is particularly of use as a speedy measurement technique for food which has been kept for an extended time and gases may present risk to the human olfactory cells [6]. the distinguishing flavor of volatile compounds, namely finger-print, may offer information about safety and specific characteristics of food. a number of volatile compounds can originate from biochemical processes of food, as a result of technological treatments or product age / storage. undesirable smells (off-flavours) may comprise of substances originating from the metabolism of spoilage microorganisms, bacteria and fungi, which may naturally or accidentally contaminate the products before or during its production [4]. 2.5 e-nose sensors working principle the design of an e-nose includes the design of a matrix of chemical sensors, for example, gas sensors and development of a pattern-recognition algorithm. the sensor matrix sniffs the vapor from a sample and provides a set of measurements; the pattern-recognizer compares the pattern of measurements with the stored patterns of known materials. gas sensors tend to have very broad selectivity, responding to many different substances. this, in fact, it is a disadvantage in most applications, but it is a definite advantage in e-nose. each sensor in a matrix may respond to a given chemical and these responses will be different [6]. fig. 1 shows the sketch of an e-nose system with several sensors arranged in a cabin and constructed into a sensor matrix. flavor is detected by the sensor matrix and electric signals are generated. signals are filtered and amplified by an adjustment circuit board (acb), and converted from analog to digital signals by a/d transfer card and the digital signals are analyzed by a computer [6]. fig. 1: electronic nose system (adopted from zhang et al., 2008) 2.6 types of gas sensors there are several types of gas sensors that have been developed which are currently available. gas sensors are distinguished on the basis of their materials or forming materials [11] such as metal oxide semiconductor (mos), conducting polymer (cp), metal oxide semiconductor field effect transistor (mosfet), and piezoelectric sensors [5, 11]. in gas sensing, two kinds of piezoelectric sensors are utilized, the surface acoustic wave (saw) device and the quartz crystal microbalance (baw) [14]. mos type gas sensor is one type of sensors that is most widely used to build e-nose systems. this is due to high sensitivity and relatively cheap price [11]. 2.6 application of e-noses in a variety of fields e-noses have been applied in various food domains including food quality control [7], process monitoring, food freshness evaluation, shelf-life assessment, authenticity determination [5], product traceability [8], quality estimation of ground meat, beer production, prediction of the degree of musty odor in cereal and many more [3]. it is also used to classify the quality of stored grain, analysis of water and wastewater, screen roasting process, detect and diagnose pulmonary food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 325 infections (e.g., pneumonia) and ulcers through breath tests, test freshness of fish and fruit, control the manufacture of cheese, sausage, beer and bread, detect bacterial growth in meat and vegetables [6]. the largest market for e-nose however, is the food industry particularly for quality monitoring or grading of food, beverage and fruits, inspection of food packaging materials [8]. other documented applications are reflected in medicine, agriculture, environment, perfumes, drinks [7] diagnosing disease, water quality, air pollution as well as liquid chemical concentration [3] and many more [15]. 3. review and discussion of e-nose experimental trials in meat products as indicated earlier, e-noses have wide applications in several industries, however, in the present review the focus is confined on the experimental results of e-nose performance in quality evaluation of fish and meat products (beef, pork, chicken and fish). several studies have been made to evaluate its (e-nose) applicability in quality evaluation. li [8] used a freshsense enose with an array of four electrochemical gas sensors (co, h2s, so2 and nh3) to evaluate the freshness of redfish (sebastes marinus) maintained in ice and modified atmosphere packaging (map). the results of e-nose measurements were compared with the results of sensory evaluation (qi scores). the results indicated different responses and selectivity to the volatile compounds tested (both in standard compounds and red fish storage experiment). the co sensor was the only sensor that responded to ethanol, while tma was the only one detected by the nh3 sensor. the so2 was only sensitive to dimethyl disulfide (dmds). the results also showed that both co and h2s sensors responded to acetaldehyde and all the sensors responded to dmds. the co sensor was more sensitive to acetaldehyde than to ethanol. all the sensors were more sensitive to dmds aqueous solution than any other compounds and dmds in oil. they related the sensors’ sensitivities to dmds insolubility in water which accounts to its much higher concentrations above the aqueous phase than the oil phase. this implies that the solubility of volatiles compounds may affect the head space concentrations of volatile compounds and hence the sensitivity of some gas sensors. solubility dilutes the concentration so that it reduces the sensor sensitivity. other researchers [19] similarly reported the possibility of solubility of odorant to affect the sensitivity of the sensor since insolubility increases head space concentration of the odorant. the variation in solubility characteristics among volatile compounds emanating from different sources could thus have an implication on the functionality of the sensors. the higher sensitivity of co sensor to acetaldehyde than that of ethanol at same concentrations was attributed to the lower boiling point of acetaldehyde than ethanol which implies its higher vapour pressure in the headspace. it was also documented that the minimum concentration the sensor can measure depends on the nature of the odorant. according to available information [20] the composition of volatile compounds in the end product is affected by different factors: breed, sex, diet and age of an animal; conditions and process of slaughter; duration and conditions of meat storage; type of muscle; preparation of meat and type of additives applied as well as conditions of heat treatment (cooking, roasting, smoking). in the red fish storage experiment [8], the co sensor showed the highest response among sensors for all the samples stored under various conditions (ice, ice-map and map-ice). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 326 a slower spoilage rate reflected by lower intensities of sensors’ response was observed in map stored red fish compared with other storage conditions. the freshsense measurements were generally in agreement with the results of sensory evaluation (qi scores). the conclusion was that the sensors showed good selectivity, sensitivity and repeatability to standard compounds that are representative of spoilage compounds in fish [8]. his results are supported by ólafsdóttir [10] who in exploring the potential of e-nose (equipped with co, nh3, h2s and so2 sensors) in quality evaluation of different species of fish and fish products (capelin, cod, haddock, red fish and cold smoked salmon) documented the sensors selective responses to characteristic odours emanating from volatile compounds. co sensor was responsive of the formation of alcohols, aldehydes and esters whereas nh3, h2s and so2 sensors were sensitive to amines and sulfur compounds. the discrimination demonstrated by gas sensors [8] on the four standard compounds that are representative of spoilage compounds in fish (tma, ethanol and acetaldehyde, dmds) implies the dependence on the functionality of gas sensors (in terms of selectivity and sensitivity) on the aroma / odour compositions of volatile compounds. more gas sensors means that the portability of e-nose is reduced. comparable results were reported by other researchers [6] who demonstrated varied responses of dissimilar sensors to different gases, implying that each sensor has different output voltages for each gas. an integration of several sensors into a matrix could thus widen the scope of detection and selectivity of the system at the expense of an error inherent in a single sensor. although this is advantageous as it broadens the gas sensors selectivity, thus responding to several different substances, it may also affect its ability to discriminate the pattern of volatiles compounds due to their overlap. another study [6] determined freshness of beef by using six mos sensors based on various gases volatilized from beef stored at 20°c and relative humidity of 60% for days varying from 1 to 6. the results of beef freshness were evaluated by sensor measurement and confirmed by sensory evaluation. the results showed that dissimilar sensors can respond to different gases, implying that each sensor has different output voltages for each gas. the odor of beef originates from a mixture of gas compounds. the results demonstrated an increase in the output voltages of sensors with the stored time of beef sample, except for one sensor (taguchi gas sensor, tgs2442) which reflected its inability to evaluate beef freshness. it was concluded that a few sensors have no reaction to fresh beef but have extreme reaction to decomposed beef. however, a sensor matrix constructed from sensors that react differently to beef freshness could enhance reliability and sensitivity of detection due to their sensitivity to different gases. on the other hand, the inability of certain sensors (tgs2442) to evaluate fish freshness quality in either fresh or spoiled form [6] indicates insensitivity of some sensors regardless of the age of the food or storage duration. this can further be explained by the existing data [10] which demonstrated the responsiveness of co sensor to the formation of alcohols, aldehydes and esters and the responsiveness of nh3, h2s and so2 sensors to amines and sulfur compounds. this suggests that the functionality of gas sensors in quality prediction may also depend on odour source, type and composition of the emanating odours. the odour of food items is based on a mix of several volatile / gas compounds, the use of an array of gas food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 327 sensors (as opposed to a single gas sensor) can enhance its effectiveness and performance. this can be achieved by widening the scope of reaction through exploiting and integrating the capability of each gas sensor into a single working unit. it has also been observed that [6] some sensors are inactive / have no reaction to fresh beef but they have extreme reaction to the decomposed beef. this indicates the reliance of some sensors on the concentration of odorous compounds which increases with storage and decomposition level. this suggests early stages of spoilage might not be recognized though it is not always the case. therefore, a proper selection of suitable and appropriate gas sensors could help in reducing or alleviating such shortcomings. an extra experimental trial [7] was done to detect spoiled meat and its degradation level using an e-nose equipped with five mos gas sensors (tgs and mq) and humidity and temperature sensor (dht11). mq stands for mĭngăn qǐ lai (in chinese) which means sensitive to gas. durations and gases given off by decayed meat were determined by pca (principal components analysis) for classification and dfa (discriminate factorial analysis) for dating and for the identification of unknown sample. the temporal response of the sensor array in the presence of two types of beef samples during 11 days showed the detection of various organic solvent vapours by different sensors and concluded that the output voltage of each sensor increased between the third and fifth day. classification by pca method indicated three well separated groups namely chicken free, chicken about to rot and rotten chicken. the identification results by dfa method indicated defined separation of three groups, with distinct grouping of classes around their centers of gravity with 82.29.5% success of classification. the results also showed the possibility of identification between two types (beef and chicken) that are nearly of the same odor at rottenness by dfa technique with a percentage of 78.88%. another separate study [16] examined the freshness of beef and poultry slices, and plaice and salmon fillets using a handy and simplified e-nose system called mastersense equipped with four mos sensors. the sample’ freshness was examined based on total viable count and the results were used to categorize freshness to develop grouping models by the k-nearest neighbours’algorithm and partial least square discriminant analysis. both established models showed sensitivity and specificity with estimation greater than 83.3% and 84.0%, respectively. the comparison of the prediction ability of the two classification algorithms using mcnemar’s test, gave comparable values. based on the results, it was concluded that the mastersense e-nose system operating with the k-nearest neighbours’ model provides the most suitable freshness assessment of meat and fish [16]. other researchers [21] identified extended settling time of some sensors as a potential drawback particularly of methods that rely on sensors to achieve a steady state which is a disadvantage for applications needing a fast response. however, the same researchers [21] further indicated that the shortcoming can be avoided by using a fast classification algorithm that does not rely on the sensors to attain a steady state, but it rather depends on transient information from the response characteristic of the sensor once exposed to an analyte. this could possibly explain the results reported by other researchers, too [16]. another study [9] used a multiple sensor system consisting of an oxidationreduction (orf) gas sensor, a hydrogen sulfide (hs) gas electrode and an ammonia (nh3) gas electrode to measure the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 328 gaseous species released, that is, dma and tma which are produced during fish decomposition. the response of sensor system to odors of salmon and sardine was also determined during fish decay and compared with a sensory evaluation. the results showed a lack of response by hs sensor to dma and tma, but the orfsensor and nh3 sensor were effective for the detection of volatile basic nitrogen compounds, which constitute one of the odour materials released during fish decomposition. the potential change for orf-sensor and nh3 sensor corresponded to the difference in odour concentration. the results reflected the effectiveness of the two sensors in detecting volatile basic nitrogen compounds, which form one of the odour materials released during fish decay. the reliability assessment results of the system against sensory evaluation based on salmon fish, indicated that eight participants assessed one day sample as having undergone putrefaction which coincided with the nh3 sensor response. this suggests that the nh3 sensor can efficiently determine salmon condition. in the second day, all panelists declared alteration of salmon sample. for sardine, all ten panelists declared one day sample as unaltered. however, in the second day, only seven panelists declared the sample as unaltered. but after two and half days all the participants declared the sample altered. during this storage experiment all sensors registered, small electric potential changes up to the second day. for the orf-sensor, increase in electric potential was registered in the two and a half day sample. after the third day, a gradual increase in potential changes was registered for all sensors. the results generally indicated that sensory test corresponded well with potential changes of sensors. the reliability assessment using colony count results was evaluated based on bacterial counts n fish meat; that is, freshness n = 107 cells/g which is commonly defined as a stage of putrefaction. when the judgment by the sensory test panel was "no putrefaction", the sample n was about104 cells/g. the n of samples, declared as putrefied by the sensory test, was about 105 107cells/g, n = 107 cells/g which is commonly defined as a stage of putrefaction. on the basis of the colony count results, it was concluded that the validity of the sensory evaluation was objectively assessed and the potentiometric gas sensor system is efficient for fish freshness estimation. responsiveness of the hs sensor to dma and tma and the responsiveness of the or-sensor and nh3 sensor to volatile basic nitrogen compounds, released during fish decomposition demonstrate that dissimilar sensing elements measures dissimilar properties of the chemicals measured as such any chemical or gaseous vapor exposed to a sequence of sensors will generate a pattern distinctive of the gas [11]. the other study [5] used a mos based enose to measure the smell signature in two meat foods (beef and fish) stored at room temperature and identified the decayed products. they separated the samples into two groups: fresh beef with decayed fish and fresh fish with decayed beef. in addition, they tested the e-nose using three pattern classification algorithms including artificial neural network (ann), support vector machine (svm) and k-nearest neighbor (knn)), and evaluated them on the basis of accuracy, sensitivity and specificity. the gas array sensor used consisted of eight mos sensors. their experimental results for the rotten beef with fresh fish demonstrated that out of 684 samples, the system correctly categorized 670 (ann), 639 (svm), and 639 (knn) samples as rotten beef and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 329 incorrectly identified 156 (ann), 20 (svm), and 0 (knn) rotten beef samples as fresh beef. as a result, ann had the uppermost sensitivity of 97.2% but the lowest specificity of 69.59% whereas knn had the highest specificity of 100% and a reasonable sensitivity of 93.42%. similarly, for the rotten fish with fresh beef group, knn did significantly better than ann and svm. additionally, the accuracy rate was 85% for ann, 94.5% for svm, and 96.2% for knn, which indicated that knn algorithm has the highest performance accuracy. the results generally showed that knn is a more reliable technique than svm or ann due to its relatively higher values of sensitivity and specificity for both kinds of samples. furthermore, knn had the uppermost accuracy rate which is a sign that it has the highest performance. it was thus identified as the most suitable pattern classification algorithm (pca). on the other hand, the three dimensions projection of the pca results clarified almost 95% of the variation in the data, 61.61% for pca1, 25.55% for pca2 and 7.3% for pca3. they concluded that the proposed e-nose system is a very efficient tool for food inspections. the variations in sensitivity, specificity and accuracy of the pca in recognizing the alterations in these meat products implies that the efficiency of the e-nose system is influenced by several factors including the type of pca and the type of gas sensors. this is supported by available data [21] which indicated that the shortening of the settling time required by sensors to achieve a steady state can be achieved by means of a fast classification algorithm. nonetheless, the performance of pca can also be affected by the original or source of the odour. for example, while ann was highly sensitive to rotten beef with fresh fish, knn had the uppermost sensitivity and reasonable specificity to rotten fish with fresh beef. this suggests the need for integration of an array of gas sensors with a wide scope of selectivity coupled with appropriate pattern classification algorithm. nonetheless, the use of great numbers of sensors could affect the e-nose portability and onsite application. alternatively, the development of a variety of e-noses meant for localized use for particular meat and fish products could address this limitation. according to other researchers [7], odour classification relies on the constructed database that characterizes the different odour footprints. as such the design and development of enose system can vary depending on the differences in odour foot prints. this suggests the difficulty that could be faced in designing and developing a gas sensing system that can universally be used due to the complexity of meat volatile compounds. in the other experimental trial [11], a sensor system was applied into a raspberry pi supplied with three mos gas sensors and one color sensor as the freshness level identifier tools of meat. pattern recognition power-driven by an artificial neural network (ann) was utilized to identify meat’s freshness. three levels of freshness were tested (fresh meat, semi altered and altered meat). the voltage value of the gas sensor in clean air was employed as a reference of the created enose system [11]. during measurement, the voltage values of three gas sensors form a pattern that represents the state of a fresh meat. the voltage values of gas sensor when compared with that of clean air did not show significant difference. they attributed this to the condition of fresh meat that does not give out a vicious smell. further testing with semi altered meat sample (i.e. meat with unpleasant odor, but with less concentrated aroma) indicated an increase in the voltage values on the three sensors as compared with the results of fresh meat sample. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 330 the results generally demonstrated responses of the three sensors to the aroma produced by meat during its decay process. nonetheless, half rotten meat was at times identified as rotten meat which was attributed to their tiny dissimilar patterns. the identification error was linked with unequal environmental condition in which the meats undergo dissimilar decomposition. the other identified challenge concerned the measuring of freshness level in ambient air, in which case the air condition will affect the measurement. they concluded that the system had 100% degree of success in identifying fresh meat and non-fresh meat (i.e. halfrotten and rotten meat). nonetheless, the results implies the accuracy of the sensors can well be realized when the spoilage stages differs greatly. in the other research [17] the use of e-nose in assessing the freshness of shrimp (penaeus vanmamei) stored at 5°c was studied. e-nose was used alongside sensory evaluation and tvbn. the output voltage of 6 gas sensors were evaluated using pca and the three principal components of pca achieved accounted for 86.97% of total variation, and they were applied to develop a model to estimate the shrimp freshness with fisher liner discriminant. the discriminant rates were 98.3% for 120 modeling sample data, and 91.7% for 36 testing sample data. it was concluded that the model could be definitely used to evaluate the freshness of shrimp with better accuracy. other researchers [19] on the other hand examined quality variation in fresh chicken meat (based on volatile fatty acids vfa) during storage using the mos sensor system and assessed against the results of traditional chemical measurements. the variations of vfa in chicken meat (stored at +4°c) during the aging process determined by traditional chemical analysis method showed that both meat samples kept fresh for the first 4 days and only after this time the spoilage of thigh meat was noticed i.e. vfa began to increase. minced chicken breast meat (that is less fatty) started to spoil after 5 aging days and it was totally spoiled one day later than the thigh meat. the initial ph values were 6.02 and 6.21 for chicken breast and chicken thigh, respectively, which fall within the limits of 5.6 and 6.2 indicating product freshness. though the ph of chicken meat increased with time and is considered as one of the important parameters for evaluation of freshness, results did not give any clear and consistent information about spoilage progression. the ph did not show any significant correlation with the signals of sensors. they attributed this inconsistence in ph meat freshness evaluation to its variation dependence on many factors. on the other hand, the relations between the responses of ch, nh3, and o3 e-nose sensors and vfa concentration indicated that the best results were obtained using ch sensor. the highest correlation was recorded between ch sensor output signals in the fresh chicken meat samples gauged by e-nose and vfa concentration values measured using a traditional chemical method (r2 = 0.89). similarly assessment of the freshness of chilled yellow chicken meat using both traditional methods namely sensory evaluation, adenosine triphosphate (atp) and inosine monophosphate (imp) contents and an enose was made [12]. on the basis of the results it was concluded that traditional methods and e-nose technology are appropriate for evaluating freshness and enose is suggested to evaluate freshness of chicken meat due to superior speed, simplicity, and accuracy. evaluation of the response of mos sensors to specific chicken odour [19] in which one of the test samples was the meat of yellow chicken food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 331 that was fed by maize, the results revealed that a specific breeding method affects taste, colour and odour of such type of chicken meat. this suggests that in the case of a wide variability of localized bred based foods, functionality of e-noses could be affected (that is, could be place bound, confined or localized) due to the effects of traits on scent signature. in another study [14] researchers made an electronic device integrated with biosensors that can detect food spoilage. they used sensors that can measure different parameters of food like ph, moisture and ethanol level. the device consisted of a microcontroller arduino uno, bluetooth module, electrical and bio sensor like ph sensor, moisture sensor and gas sensor. they examined freshness of a variety of foods based on the depicted trend of the studied parameters with passage of time which indicated good to use [14]. previous researchers [18] investigated changes in volatile compounds and the effects of different temperatures on the development of volatile compounds during storage of capelin by gas sensor measurements, sensory evaluation, and total volatile bases (tvb) analysis. the enose consisted of an array of eight electrochemical gas sensors and a temperature sensor. capelin samples were stored at 5 and 0°c for 8 days, and the effect of 0.2% added acetic acid was assessed at 5°c. the results showed that co and so2 sensors from dräger and the nh3 sensor from city technology were most sensitive toward the standard compounds tested and also showed the most characteristic responses to the capelin headspace. the highest intensity of signals, with early detection of spoilage was indicated by nh3 sensor among the three sensors (co, nh3 and so). the correlation analysis of results between tvb measurements of capelin and the response of the nh3 and co sensor to capelin headspace in the 0°c samples was greater for the nh3 sensor (r0°c = 0.98), than for the co sensor (r0°c = 0.92). the differences in sensitivity were attributed to dissimilar sensitivity to different compounds e.g. co sensor is insensitive toward amine. during the final few days of storage, the responses of the sensors increased more than the tvb values. the observation was associated with poor sensor selectivity and ability to detect a wide range of several very volatile degradation compounds which get formed at advanced phases of spoilage. this further suggests the need of integrating several sensors. the enhanced response of the sensors in advanced spoilage lowers the correlation between sensors and tvb values in the 5°c series (r5°c = 0.89 for the nh3 sensor and r5°c = 0.90 for the co sensor). the descriptive sensory evaluation of the odor of capelin indicated the coincidence of the first spoilage and stale odors with tvb values of around 40-50 mg of n/100 g and when the odor of the capelin was putrid and the tvb values had increased to about 80 mg of n/100 g. the results of the gas sensor measurements demonstrated good reproducibility, and their responses correlated well with classical tvb measurements. the calibration of sensors in the study [18] was done using different standards for each sensor; ethanol represented alcohols used to calibrate the co sensor, dimethyl disulfide for sulfur compounds to calibrate the so2 sensor, and tma for amines to calibrate the nh3 sensor. this implies lack of a standardized calibration procedure. other researchers [13] monitored the pork meat freshness at four distinct grades (high, medium, low, and spoilage) which were established using the sensor value and model equation. a single gas sensor supported with temperature sensor and humidity sensor as auxiliary sensors and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 332 rfid (radio frequency identification) tag were used to establish the food poisoning index. temperature, humidity, and gas concentration of the meat stored in environmental conditions, were compared to establish the relationship between meat freshness and the sensor signal. the reliability of the system was confirmed by performing experiments on pork and comparing to the experimental results of an e-nose (nh3 sensor output) and total volatile basic nitrogen (tvbn) measurements (table 1). the researchers’ [13] confirmation of the output voltage of the sensor for each freshness grade by experimentation is described in table 1 from the experimental data. the spoilage detection by e-nose against tvbn measurements are reflected in table 1. the spoilage detection by nh3 sensor output – mv coincided with the spoilage established by the tvbn value of >25 mg/100 g. table 1: description of meat freshness and comparison of the experimental gas sensor results with the existing method freshness status nh3 sensor output mv content tvbn (mg/100g) high < 630 very fresh 10.95 medium 630-730 can be eaten 10.95 – 16.94 low 730-100 food poisoning may occur 16.94 25 spoilage >1000 spoilage >25 source: eom et al. (2014) in the table 2 it is indicate a comparison of the rfid technology experimental results with other method (e-nose and tvbn). the experiment confirmed similar experimental results (table 2) as for the existing methods. table 2 comparison of the experimental results with the existing methods (in days) status proposed system (day) electronic nose (day) tvbn (day) high < 1.25 < 1 < 0.9 medium 1.253.12 1 – 3.3 0.9 – 3 low 3.12-6.96 3.3 7 3-7 spoilage >6.96 > 7 > 7 source: eom et al. (2014) by combining the rfid technology in their trial with a gas sensor, temperature sensor, and humidity sensor, they established the relationship between meat freshness and gases released. these results [13] suggest that the accuracy and reliability of the sensor in freshness quality prediction can be further improved if complemented with temperature and humidity sensors since the temperature and humidity of the storage environment has a direct relationship with the freshness of the sample under consideration. according to documented data [22] rfid systems are favoured over other identification and inventory techniques mostly due to their non-line-ofsight operation and capability to function in adverse environments. this is attributed to the rfid sensors combination with several measured parameters from the resonant sensor antenna with multivariate data analysis and deliver a unique ability of sensing with rejection of environmental interferences with a single sensor. more or less similar advancements in enose technology have been demonstrated in some trials by other researchers [7]. they detected spoiled meat and its degradation level using an e-nose equipped with metal oxide-based gas sensors supported by humidity and temperature sensors. classification by pca method indicated well separation into three distinct groups namely chicken free, chicken to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 333 become rotten and rotten chicken. also in another study [1] tilapia fish spoilage was monitored based on the application of a single gas sensitive gel material coupled to an optical e-nose. the changes in the optical signal produced by the gas sensor, once exposed to the headspace of fish samples stored at ambient situation, were correlated with the mesophilic bacteria counts during storage time. the results of the microbial examination were consistent with the results achieved by the optical gas sensor. the amplified relative response of the sensor matched the increase in bacterial counts in fish, implying that the sensor was detecting specifically the volatile compounds produced by the microorganisms that caused the fish to deteriorate. the results demonstrated that a single optical sensor was efficient in monitoring the quality of fish. the largest change in the optical signal happened exactly while the biggest increase in bacterial counts was examined and reached the limit to declare the fish’s unsuitability for human consumption (above 6 7 log cfu/g). however, the decline in the baseline of the optical signal showed structural alteration in the sensing material, which may imply the need to apply it as a non-reusable component of the sensing device. they considered the observation as a weakness attenuated by the low cost of the sensing film. overall they conclude that the good correlation between changes in optical sensor signals and the extent of mesophilic bacterial growth evidences its potential application for tilapia fish freshness monitoring. these results [13] suggest that the accuracy and reliability of the sensor in freshness quality prediction can be further improved if complemented with temperature and humidity sensors since the temperature and humidity of the storage environment has a direct relationship with the freshness of the sample under consideration. in view of the available data [22] rfid systems are favoured over other identification and inventory techniques mostly due to their non-line-of-sight operation and capability to function in adverse environments. this is attributed to the rfid sensors combination with several measured parameters from the resonant sensor antenna with multivariate data analysis and deliver a unique ability of sensing with rejection of environmental interferences with a single sensor. though the results from the experimental trials indicate the high potential of e-noses in freshness quality evaluation, however, the method can only be effectively used when benchmarked against other methods which could act as reference points for quality assessment. nevertheless, the sensor output (mv) levels established by other researchers [13] to define the degree of freshness (high, medium, low and spoilage) can however enable the usage of e-noses independent of the reference method. this enhancement of e-noses with auxiliary sensors and rfid (radio frequency identification) tag could possibly explain these advancements. the existing data indicate that sensors have dissimilar reactions to different gases/ volatile compounds to be detected which can also be influenced by other external factors such as breeding [19]. the efficiency of the gas sensors could thus depend on the uniqueness of each system to particular odour foot prints. it can thus be deduced that the data on aroma composition which are reflective of the properties of meat products are the most desirable information for an efficient design and development of effective gas sensing systems. the available literature [9] shows that some sensors such as quartz-resonator sensors and oxide semiconductor sensors food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 334 are operated in the high-temperature range of 300°c to 400°c and their responses are simply affected by humidity. it is also documented [22] that mos sensors operate at temperatures between 200°c and 650°c as the organic volatiles transmitted to the sensing surface need to be oxidized which results in the alterations of electrical resistance. on the other side it is reported [9] that a potentiometric gas electrode is operated at room temperature and it is not affected by humidity. the variations in responses of gas sensors due to the influence of humidity and temperature could further imply localization of the application of gas sensors in quality estimation unless if equipped with self-calibration and selfcorrection for variable ambient conditions. moreover, the recognition by e-nose depends on the pattern-recognizer comparison between the pattern of measurements and the stored patterns of known materials. unlike gas sensors which use specific sensors to measure particular single attributes and quality and safety in the food and beverage industry, information can also be obtained from arrays of nonspecific sensors with partially overlapping selectivity and treating the data obtained with pattern recognition software [15]. a recent study [11] that identified three levels of freshness (fresh meat, half-rotten meat, and rotten meat) using an e-nose, employed the voltage value of the gas sensor in clean air as a reference / for calibration of the created e-nose system [11]. other researchers [18] on the other hand in their measurements by means of standard compounds demonstrated that the sensitivities of identical sensors are dissimilar thus new gas sensors have to be calibrated. nonetheless, specific validation procedures for different sensors for specific usage have been lacking. furthermore, [18] they demonstrated the use of different standards for each sensor, where ethanol was chosen to represent alcohols to calibrate the co sensor, dimethyl disulfide for sulfur compounds to calibrate the so2 sensor, and tma for amines to calibrate the nh3 sensor. however, little has been documented in subsequent trial studies on calibration procedures, implying the need for development of standardized calibration procedures. 4. conclusions the available literature data on e-nose experimental trials indicate the high potential of e-noses in freshness quality evaluation; however, the method can effectively be used when benchmarked against other methods which act as reference points for quality assessment. the use of the e-nose without relying on reference methods requires the establishment of threshold levels of volatile compound detection which can define the level of freshness or spoilage or else be used alongside other conventional methods. recent development however suggests the sensor output (mv) levels can define the degree of freshness of meat items depending on design modifications. although, the efficiency and effectiveness of e-noses depend on the type, selectivity and sensitivity of an array of gas sensors, however they can further be enhanced if coupled with appropriate pattern classification algorithms. nonetheless, the use of great numbers of sensors could affect the e-nose portability and onsite application. alternatively, the development of a variety of e-noses meant for localized use for particular meat products could address this limitation. this is due to the fact that odour classification relies on the constructed database that characterizes different odor footprints. as such the design and development of an e-nose food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 335 system can vary depending on the differences in odour foot prints. this suggests the difficulty that could be faced in designing and developing a gas sensing system that can be universally used due to the complexity of meat volatile compounds. this may further be explained by the sensors’ dissimilar reactions to different gases/ volatile compounds to be detected which can also be influenced by other external factors such as breeding method which affects taste, colour and odour of meat items which reflects the effect of traits on scent signature. in addition, though the concentration threshold is an important element in the detection and sensitivity of e-noses to volatile compounds, but also the temperature at which volatilization occurs, that is, the lower the boiling point the higher the sensitivity and vice versa. it can thus be deduced that data on aroma composition which are reflective of the properties of meat products are the most desirable information on an efficient design and development of effective gas sensing systems. furthermore, the functionality of e-noses can be further improved if equipped with self-calibration and self-correction for variable ambient conditions. . 5. competing interest the author declares that has no competing interests. 6. references [1]. semeano ats, maffei df, palma s, li rwc, franco bdgm, roque aca, gruber j. tilapia fish microbial spoilage monitored by a single optical gas sensor, food control, 72-75, (2018) [2]. chamhuri n, batt p. consumer perception of food quality in malyasia. british food journal; 117: 1168-1187, (2015) [3]. mohamed rr, yaacob r, mohamed ma, dir tat, rahim fa. food freshness using electronic nose and its classification method: a review. international journal of engineering & technology, 7: 49-53, (2018) [4]. falasconi m, concina i, gobbi e, sberveglieri v, pulvirenti a, sberveglieri g. electronic nose for microbiological quality control of food products. international journal of electrochemistry, article id 715763, (2012) http://dx.doi.org/10.1155/2012/715763 [5]. hasan n, ejaz n, ejaz w, kim hs. meat and fish freshness inspection system based on odor sensing. sensors 12, 15542-15557, (2012); doi:10.3390/s121115542 [6]. zhang z, tong j, chen d, lan y. electronic nose with an air sensor matrix for detecting beef freshness. journal of bionic engineering 5:67-73, (2008) [7]. benabdellah n, hachami k, bourhaleb m, benazzi n. identification of two types of rotten meat using an electronic nose for food quality control. international journal on smart sensing and intelligent systems, 10: 673-695, (2017) [8]. li., x. electronic nose to monitor the freshness of red fish (sebastes marinus) stored in ice and modified atmosphere packaging (map). final project dalian fisheries university, china, (2000) [9]. kaneki n, tanaka h, kurosaka t, shimada k, asano ya. measurement of fish freshness using potentiometric gas sensor. sensors and materials, 15: 413-422, (2003) [10]. ólafsdóttir g. volatile compounds as quality indicators in chilled fish: evaluation of microbial metabolites by an electronic nose. thesis for the degree of doctor of philosophy, faculty of science university of iceland, (2005) [11]. rivai m, budiman, f, purwanto d, simamora j. meat freshness identification system using gas sensor array and color sensor in conjunction with neural network pattern recognition. journal of theoretical and applied information technology, 96: 3861 – 3872, (2018) [12]. lu w, wu y, guo q, ren l, zhu p, xu l, chang g, chen g. establishment of a freshness-evaluating standard for chilled yellow chicken meat. food anal. methods doi 10.1007/s12161-017-0815-4, (2017) [13]. eom k, hyun k, lin s, kim j. the meat freshness monitoring system using the smart rfid tag. international journal of distributed sensor networks, article id 591812, (2014). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 leonard fweja, the potential of electronic noses in quality and freshness prediction of fresh and stored meat products, food and environment safety, volume xviii, issue 4 – 2019, pag. 321 336 336 [14]. shahzad n, khalid u, iqbal a, meezan-ur-rahman. efresh – a device to detect food freshness. international journal of soft computing and engineering (ijsce), 8:1-4, (2018) [15]. actia (association de coordination technique pour i’industrie agro alimentaire, france). draft final report synthesis of the roadmaps for the implementation of microsystems in the food and beverage sector, (2013). [16]. grassi s, benedetti s, opizzio m, nardo e, buratti ss. meat and fish freshness assessment by a portable and simplified electronic nose system (mastersense). sensors 19: 3225, (2019) [17]. du l, chai c, guo m, lu x. a model for discrimination freshness of shrimp. sensing and bio-sensing research 6: 336-32, (2015) [18]. ólafsdóttir g, martinsdóttir e, jónssón eh. rapid gas sensor measurements to determine spoilage of capelin (mallotus villosus). j. agric. food chem., 45: 2654-2659, (1997) [19]. raudiene e, gailius d, vinauskiene r, eisinaite v, balciunas g, dobiliene j, tamkute l (2018). rapid evaluation of fresh chicken meat quality by electronic nose. food technology and economy, engineering and physical properties. czech j. food sci., 36: 420–426, (2018). [20]. kosowska m, majcher ma, fortuna t. volatile compounds in meat and meat products. food sci. technol (campinas), 37, (2017) [21]. längkvist m, coradeschi s, loutfi a, rayappan j. fast classification of meat spoilage markers using nanostructured zno thin films and unsupervised feature learning. sensors, 13: 1578-1592, (2013) [22]. kant r, prabhakar pk, samadder sand, srivastav pp. radio frequency based sensor: an innovative detection tool for food spoilage. the pharma innovation journal; 6: 8086, (2017). http://dx.doi.org/10.1016/j.sbsr.2015.11.001 http://dx.doi.org/10.1016/j.sbsr.2015.11.001 doi: https://doi.org/10.4316/fens.2021.003 21 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1 2021, pag. 21 34 nutrient content of rhynchophorus phoenicis and its application in complementary food formulation *sunday ukwo1, mfoniso udo2, ekefre ekong1 1department of food science and technology, university of uyo, uyo, nigeria 2department of home economics, nutrition and dietetics, university of uyo, uyo, nigeria * sonipeter75@gmail.com received 29th january 2021, accepted 30th march 2021 abstract: palm weevil (rhynchophorus phoenicis) is an edible insect that is highly exploited as food. this study investigated variability in nutritional content during three developmental stages: early larva (esl), late larva (lsl) and adult stage (ads) and application of early larva stage in the formulation of complementary foods from the blend of broken rice and soybean flour at different proportions: a (100:0), b (94.5:5.5), c (89.5:10:5), (85.3:14.7), e (75.5:24.5) and f (70:30). samples were assessed for proximate and mineral content while anti-nutritional factors and sensory analysis were conducted on the complementary food samples. standard methods of analysis were used in all determinations. results of proximate analysis indicated protein and lipid content were significantly different (p<0.05) among the developmental stages. higher lipid content were noted in early larva (49.59%) and late larva stage (47.06%). the early larva stage was superior in ash and energy content while adult weevil had the highest percentage of protein (36.19%), carbohydrate (9.46%), fibre (9.43%) as well as micro mineral of zn and fe. proximate composition of complementary foods indicated a significant (p<0.05) increase in the values for moisture, protein, lipid, ash and fibre content as the percentage of palm weevil flour increases in the formulated food samples. the recommended dietary levels for zn and fe in complementary foods were met with addition of 24.5% and 30% palm weevil flour without any further fortification. antinutritional factors present in all complementary food samples were within safe levels. sensory results showed that sample e with 24.5% and sample f with 30% palm weevil flour were more acceptable in all the sensory attributes determined. key words: insect, broken rice, soybean, developmental stage, palm weevil 1. introduction feeding a growing world population require an increase in food production. this will inevitably place more pressure on already limited resources especially those associated with raising livestock. livestock and fish are important sources of protein in most countries of the world and their production accounts for 70% of all agricultural land use [1]. with their global demand expected to more than double between 2000-2050 (229milion tons to 465million tons), meeting this demand will require innovative solutions [2]. the potential of using insect to help meet rising demand for meat products and to replace fish meal and oil is enormous. in order to guarantee food security and preserved the natural habitats, several studies with focus mailto:sonipeter75@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 22 on diversification of food sources has been carried out and systematically investigated widely, with the utilization of insects for nutrition being suggested as a possible solution [3, 2]. hence, more interests are currently being channeled toward entomophagy. this novel potential strategy can radically solve the problem of malnutrition and other nutritional deficiencies as well as improve the overall food and nutrition security, particularly in the developing regions like sub-saharan african that are the worst hit. this is because different studies have shown that entomophagy contributes effectively to nutrition and health, the environment, and livelihoods of those involved in the edible insects value chain [2]. as reported by food and agricultural organization, over 2 billion people globally consume insects as an integral part of their diets [2] and from study carried out by jongema [4] about 2000 species of edible insects have so far been documented and it expected to increase as research on insect as food progresses and when this is compare to traditional livestock, edible insect production has the potential to positively contribute to environmental sustainability due to lower resource requirements, feed-conversion rates, and greenhouse gas emissions[5, 6]. with respect to locally-sourced food ingredients in low and middle income countries (lmics), where the challenges of malnutrition is highest, edible insects can provide the much needed essential nutrients necessary to supplement dietary quality and provide diversity among individuals who naturally consume cerealbased foods [7]. nonetheless, studies on nutritional quality of edible insect shows substantial differences in content within the same species and between species [8] and this variability is attributed to the diverse nature of insect species. the nutritional quality of insect also depends on the antinutritional content such phytate, thiaminase as well as vary according to transitional stages of development, origin of the insect and their feeding pattern [9]. also, the nutritional value changes according to the processing methods applied before consumption. according to payne et al. [7], insect nutritional composition showed high diversity between species. for instant, the nutrient value score of crickets, palm weevil larvae and mealworm was significantly healthier than that of beef and chicken and none of the tested insects were statistically less healthy than meat. studies on the development and production of complementary foods with animal products as major ingredients have not been widely reported. existing studies are based on the use of fish and other seafood products [10, 11] with very little or no study reported on the use of edible insects. presently there is a growing international interest in promoting diets composed of traditional and local food sources during complementary feeding regime [12, 13]. this is with the hope of combating malnutrition in young children through the use of local food ingredients such as insects processed using improved indigenous technologies specially in developing countries [14]. also, complementary foods in developing countries have mainly been cereal based [14]. it is until recently that in africa, supplementation of cereals with protein rich plant sources, soya bean, groundnut [15, 16], cowpeas [17, 18, 19], pigeon peas [20], common bean [18] and bambara nuts [17] have been tested. as a result, the world food programme (wfp) have continue to promote and encourage fortified flour blends containing soy bean in management of mild condition of malnutrition especially in children [21]. also, corn-soy-blend plus (csb+) is an example of a fortified flour blend commonly used for supplementary feeding programme in management of mild food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 23 malnutrition among children under five years old. edible palm weevil (rhynchophorus phoenicis) is a major pests of raffia palms, oil palms and coconut palms. although they are seen to be destructive, their nutritional potential have made them very valuable to man. they highly valued and exploited in many cultures including southern nigeria. rhynchophorus spps are found in a wide range of geographical regions of the world such as asia, africa and southern america and presently, they are available in the wild and cultured in warehouses and laboratories [22]. because of their nutritional quality, accessibility, simple rearing technique and rapid growth rates, insects offer a cheap and efficient means of checking protein deficiency among the low and middle income families by providing emergency food and improving the quality of traditional diets among vulnerable people. the objective of this study is to examine the influence of transitional stages of development on the nutritional potential of edible palm weevil rhynchophorus phoenicis. this study also aimed at developing and evaluating complementary foods based on edible palm weevil to combat malnutrition in nigeria. 2. materials and methods material procurement samples of edible palm weevil at various stages of development (adult weevil, larva and late larva stages) were harvested from raffia palm tree (raffia hookeri) at ikpe anang in essien udim local government area. they were collected alive while the late larva stage was in the cocoon. broken rice grains were obtained from at a local rice mill that processes rice planted and harvested by the local farmers in ini local government area while soybean were bought from akpan andem market in uyo, akwa ibom state, nigeria . sample preparation the adult palm weevils were de-winged while the cocoon were opened to remove the later stages larva. they were killed by asphyxiation in a freezer and dried at 50 °c for 48 h [23]. part of the palm weevil samples were oven dry to a constant weight and then grounded to a flour for complementary food formulation. the broken rice grains (5 kg) and (3 kg) soybean seeds were properly cleaned and sorted to remove stones, dirt, chaff and other extraneous matters before they were used for further processing. the broken grains of rice obtained were washed with portable water and allowed to dry gradually in an oven for 6 hours at 600c. the dried grains were milled into fine flour and packaged in an airtight container. the soybean seeds were also washed with portable water and oven dried for 40 minutes at 600c. the dried soybean seeds were then roasted at 1200c for 2 hrs then decorticated and winnowed, then milled and sieved to obtained soy bean flour. the flours were then stored at room temperature (27± 20 c) till used. food formulations composite flour was formulated by mixing 70g rice and 30g of soybean flours. five (5) formulations of complementary foods were generated by replacing 5.5, 10.5, 14.7, 24.5, 30.0g of the composite flour with palm weevil larva flour while sample without palm weevil larva component serves as a control (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 24 the complementary foods were formulated with the intention of meeting the specifications for complementary food protein-energy composition as outlined by lutter and dewey [24]. broken rice flour served as the main source of carbohydrates; soybean flour as a source of protein, sugars; palm weevil grub as a source of protein, lipids, minerals like, iron, zinc, vitamins e and bvitamins; soybean oil to supplement the source of fat and improved organoleptic properties of the final product; while sugar was to enhance taste and energy content of the product [25]. table 1 complementary food formulation sample code composite flour (g) palm grub flour (g) sugar (g) soybean oil (ml) a 100 0 10 2 b c 94.5 89.5 5.5 10.5 10 10 2 2 d 85.3 14.7 10 2 e 75.5 24.5 10 2 f 70.0 30 10 2 production process food formulations were thoroughly mixed in a rotary mixer (philips type hr 1500/a, holland) to produce homogenous complementary food formulations. they were processed by extrusion cooking and ground into flour that could be reconstituted in to porridge as recommended by codex [26]. extrusion was carried out using a locally fabricated extruder. the foods were produced in six batches. different batches were sampled, homogenised and laboratory samples taken. methods of analysis proximate and caloric value and mineral analysis were carried out on the three developmental stages of edible palm weevil (adult weevil, early larva and late larva stages) and in the formulated food products. anti-nutrient composition and sensory analysis were conducted on the finished products only. determination of proximate composition and calorific value the flour samples were analysed for their moisture, crude protein, crude fat, ash and crude fibre contents according to the method described by aoac [27]. carbohydrate was determined by difference i.e. % carbohydrate = 100 – (% moisture + % crude protein + % crude fat + % ash + % crude fibre). the calorific value was calculated using atwater factor: energy value = (crude protein x 4) + (crude fat x 9) + (carbohydrate x 4). determination of mineral content techniques of ultraviolet – visible spectrophotometry, atomic absorption spectrophotometry and flame photometry were used for analysis of selected mineral content of edible palm weevil samples and the formulated complementary food samples. standard stock solutions of mineral element to be analysed were prepared, diluted to the corresponding working solution for recovery experiment according to the methods outlined by onwuka [28]. the wet ashing method as outlined by the author was used to determine the concentration of metallic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 25 element in food samples. the method of preparation and digestion procedure for biological sample was also employed. determination of sodium and potassium content was done through the use of jenway flame photometer at the wavelength of 589nm and 767nm for potassium and sodium respectively, while analysis for other mineral elements were carried out using a perkin – elmer model 3030 atomic absorption spectrophotometer (aas) using their respective lamp and wavelength. analysis of phosphorus was done using uv – visible spectrophotometer [27]. determination of anti-nutritional factors anti-nutritional factors such as saponins, phytic acid, tannins, and oxalate were determined on the formulated complementary food samples only. saponins were determined using the method described by aoac [27]. phytic acid concentration was determined using the method described by wheeler, and ferrel [29]. the method of allen et al., [30] was used for the determination of tannins level in food samples while the concentration of oxalate in the samples was determined using the method of onwuka [28]. sensory evaluation of complementary food sensory characteristics of the complementary food were evaluated for different sensory attributes by twenty (20) semi trained panelist. all the panelists were briefed before the commencement of the evaluation process. sensory attributes considered included appearance, taste, flavour, mouthfeel, consistency, aftertaste and overall acceptability. the rating were on a ninepoint hedonic scale ranging from 9 (like extremely) to 1(dislike extremely) according to ihekoronye and ngoddy [31]. all panelists were provided with water at room temperature to rinse their mouth between evaluations. statistical analysis statistical package for social science (spss, version 23) was used for statistical analysis. the differences between samples in each parameter tested was done using one way analysis of variance (anova) and new duncan’s multiple range test was used when the analysis of variance indicates a significant difference in their means. a significant level of p<0.05 was used throughout the study. 3. results and discussion proximate composition and calorific value of developmental stages of edible palm weevil the result the proximate composition and calorific value of the three developmental stages of edible palm weevil is presented in table 2. the result indicated a higher lipid content in the early stage (49.59%) and the late larva stage (47.06%) when compare to that of the adult weevil (40.20%). the early larva stage was also superior in ash and energy content. also, calorific value, protein and lipid content indicated significant differences (p<0.05) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 26 among the developmental stages of the edible palm weevil however the adult weevil had the highest percentage of protein (36.19%), carbohydrate (9.46%) and fiber content (9.43%). the trend of result obtained in this study was similar to values reported by omotoso and adedire, [32]. the lipid content is responsible for its high palatability of the larva stages of edible palm weevil when consumed. the present of lipid in food is necessary for the proper cellular activities and structural functions of the body. values for lipid as reported in this study was higher than those reported by singh [33] on insects such as caterpillar and termite. also higher protein content that was noted in the adult weevil could be attributed the variable feeding pattern. the adult is exposed to other sources of diets apart from the palm. as noted by teffo et al [34], edible insect have shown to have a higher protein content and quality on mass basis when compared to some animal and plant foods therefore the consumption of palm weevil provide the essential amino acid needed for building body tissue and metabolic functions. table 2 proximate composition (% dry weight) and calorific value of developmental stages of edible palm weevil stages of development parameters esl lsl ads lipid 49.58a±0.15 47.06b±1.12 40.20c±1.02 protein 34.63c±1.05 36.06b±0.05 36.19a±0.02 carbohydrate 5.41b±0.02 7.18a±0.03 8.56a±0.15 ash 5.80a±0.11 3.82b±0.01 5.62a±1.00 fiber 4.50b±0.02 5.62b±1.01 9.43a±1.00 calorific value(kj/100g) 2515.14a±1.53 2474.30b±1.68 2248.15c±1.23 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05). esl=early stage larva, lsl= late stage larva, ads= adult stage higher fibre noted in the adult weevil could be attributed to the presence of chitin that constitute fibrous materials. the adult weevil has wing with rigid body structure which provide the beetle covering and body protection. the presence of fibre in diet is significant in easing bowel movement because of its ability to bind water and thus soften stool [1]. mineral content of edible palm weevil the mineral content of the developmental stages of edible palm weevil is shown in table 3. the edible palm weevil had a significant amount of macro elements of sodium, calcium, magnesium, potassium and phosphorus. the highest values of na (62.14 mg/100g), k (95.63 mg/100g), mg (132.00 mg/100g) and p (105.12 mg/100g) were noted in early larva stage while least values of 42.62 mg/100g 89.71 mg/100g 103.30 mg/100g 85.21 mg/100g were recorded for na, k, mg, and p respectively. however, the highest concentration of ca 62.13 mg/100g was noted in the adult palm weevil. values obtained for the micro mineral of zn and fe in the three developmental stages did food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 27 not indicate any significant differences (p˂0.05). however, adult stage recorded highest values of 3.03 mg/100g and 6.05 mg/100g in zn and fe respectively. edible palm weevil. the present of macro element in edible palm weevil have been reported different authors [34, 32, 35] and values obtained in this study was similar to values reported. the present of these mineral elements in the edible palm weevil makes it an important food ingredient toward the eradication of various nutritional deficiencies. sodium and potassium are the major electrolytes needed for intercellular ion balance, osmo-regulation and transportation across cells and membranes. the present of higher concentration of potassium when compared to sodium makes edible palm weevil very useful to human body when consumed as food. table 3 mineral content (mg/100g) of developmental stages of edible palm weevil stages of development parameters esl lsl ads sodium (na) 62.14a±1.10 61.59a±0.15 43.62b±1.02 potassium (k) 95.63a±0.05 90.14b±0.15 89.72b±0.02 calcium (ca) 56.24b±1.15 42.46c±0.03 62.13a±0.15 magnesium (mg) 123.12a±2.16 106.27b±5.05 103.30c±2.00 phosphorus (p) 105.00b±5.02 102.0a±10.01 85.21c±5.02 zinc (zn) iron (fe) 3.02a±0.10 6.02a±0.05 2.96a±0.05 5.95a±0.01 3.03a±0.02 6.05a±1.00 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05). esl=early stage larva, lsl= late stage larva, ads= adult stage calcium and magnesium formed a significant part of human bones as well as prosthetic group in enzyme that hydrolyses and transfer phosphate groups, consequently, they are essential in energy requiring chemical reactions in human body. phosphorus is usually found in equal amount in human system where they served as key structural element. the function of zinc in human body is that zn forms an integral part of a number of metalloenzymes and as a catalyst in regulating the activity of specific zinc dependent enzyme. iron is present in cells of living organism and plays a vital role in several biochemical reactions. most of iron is present in the haemoglobin and myoglobin, pigments, cytochromes and other proteins participating in transport, storage and utilization of oxygen [32, 7, 36] proximate composition and calorific value of complementary food table 4 shows the result of proximate composition of complementary food formulated from blends of broken rice, soybean and edible palm weevil larva food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 28 flour. there was a significant (p<0.05) increase in the recorded values for moisture, protein, lipid, ash and fibre content as the percentage of palm weevil flour increases in the formulated food samples. protein content ranged from 15.99% in sample a to 25.10% in sample f. the lipid content in the formulated food samples ranged from 4.36% in samples a to 11.07% in sample e. the ash content also varied from 4.84% in ample a to 6.30% in sample f. fibre content of the formulated food ranged from sample a (1.20%) to sample f (2.40%). however, there was a significant reduction (p<0.05) in the moisture and carbohydrate contents. moisture content decreases from 9.10% in sample a to 5.10% in sample f while carbohydrate content also decreases from 64.50% for sample a to 50.02% for sample f. the addition of palm weevil flour led to a significant increase on the calorific value which varied from 1530 kj/100g for sample a to 1687 kj/100g) for sample f. table 4 proximate composition(%) and calorific value of complementary food complementary foods parameters a (100:0) b (94.5:5.5) c (89.5:10.5) d (85.3:14.7) e (75.5:24.5) f (70:30) moisture 9.10a±0.01 7.52b±0.02 7.13bc±0.03 6.30c±0.01 6.13cd±0.01 5.10d±0.02 cho 64.50a±1.00 60.62a±0.05 58.05c±0.01 55.70d±0.03 51.05c±0.02 50.02c±0.01 protein 15.99e±0.11 18.46d±0.02 20.36c±0.01 22.00b±0.05 24.32a±0.05 25.10a±0.02 lipid 4.36d±0.02 6.63cd±0.11 7.25c±0.12 8.26b±0.12 10.15ab±0.20 11.07a±0.01 fiber 4.84d±0.05 5.07c±0.02 5.38c±0.01 5.58b±0.11 6.14a±.0.02 6.30a±0.05 ash 1.20d±0.01 1.70c±0.01 1.83c±0.01 2.15b±0.12 2.21b±0.02 2.40a±0.02 calorific value (kj/100g) 1530f±0.05 1590e±1.00 1601d±0.05 1625c±0.08 1657b±0.05 1687a±2.01 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05). cho: carbohydrate the moisture content of any food product is an integral factor when considering food quality characteristics of that product. as reported by folake and bolanle [37], moisture content of a food affects its stability and overall quality. the moisture content of formulated complementary food samples were within the acceptable limit of not more than 10% [38]. the carbohydrate in the breakfast cereal is attributed to the high proportion of broken rice which serves as the principal ingredient in this formulations. rice is a very rich carbohydrate source responsible for the bulk of daily calories for many humans and animals [39], and it is an important source of dietary energy. carbohydrate content of this product fell within the recommended levels of carbohydrate in complementary foods as outlined by lutter and dewey [24] as well as values reported by kinyuru et al [25] on insect fortified complementary foods in kenya and higher when compared to values reported by parker et al [40] from ghana. variability of protein and lipid content of the complementary food samples resulted from the use of varying degrees of soy flour and palm weevil flour in different formulations. soy flour is a rich source of protein while palm weevil flour contain moderate protein but higher in lipid content. values for protein as obtained in this study was higher than the recommended levels in complementary food while lipid content was within the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 29 recommended levels as outlined by lutter and dewey [24]. as noted by parker et al [40], palm weevil offers an essential amount of nutrients which can feasibly be added into various nutrition intervention programmes to address various health and nutrition deficiencies. moreover, there is evidence of an improved nutrient content of the formulated complementary food samples. mineral content of formulated complementary food table 5 shows the concentration of mineral elements in rice/ soybean complementary food fortified with palm weevil flour. values obtained from the analysis of the complementary food samples for the macro elements (na, k, ca, mg and p) indicated an increased in all the macro element concentrations as the percentage of palm weevil flour increases in the rice/soybean flour blends. values obtained for macro elements indicated significant difference (p˂ 0.05) in all instances. result showed that na concentration increased from 8.12 mg/100g in sample a to 27.15 mg/100g in sample f, k increased from 2.54mg/100g in sample a to 12.34 mg/100g in sample f, ca level increased from 10.25 mg/100g to 25.70 mg/100g in sample f. the concentration of mg also increased from 5.62 mg/100g in sample a to 12.38 mg/100g in sample f while that of p increased from1.91 mg/100g in sample a to 5.50 mg/100g in sample f. the concentrations of the trace elements ( zn and fe) analysed also indicated an increased as the percentage of palm weevil flour increases in the rice/soybean flour blends. the concentrations of zn and fe increased from 0.84 mg/100g and 1.52 mg/100g in sample a to 4.42 mg/100g and 6.62 mg/100g in sample f mg/100g respectively. values as obtained for the trace elements zn and fe are within the recommended levels of 4.6 mg/100g and 9.3 mg/100g respectively stipulated by fao/who [41]. however, values for the macro elements were similar to values obtained in an insect fortified complementary foods in ghana [40] and still below the recommended daily allowance of these elements [42]. as suggested by kinyuru et al [25], mineral premix is need to increase mineral bioavailability in formulated complementary foods. the result as obtained for zn and fe shows that it is possible to achieve the recommended dietary level of these important mineral elements using local ingredients without resorting to fortification. it is expected that the fe and zn bioavailability especially in the formulated complementary foods would have been higher if the soybean seed were germinated and the level of polishing of rice grain reduced table 5 mineral content (mg/100g) of formulated complementary food complementary foods parameters a (100:0) b (94.5:5.5) c (89.5:10.5) d (85.3:14.7) e (75.5:24.5) f (70:30) na 8.12f±0.05 12.25e±1.50 14.90d±1.00 17.20c±0.05 22.80b±1.00 27.15a±0.05 k 2.54d±0.05 5.31cd±0.01 6.41c±0.02 9.23b±0.01 12.26a±0.01 12.34a±0.02 ca 10.25d±0.01 12.18c±0.05 14.41bc±0.05 16.93b±0.01 23.51ab±0.01 25.70a±0.05 mg 5.62d±0.05 8.29cd±0.01 9.92c±0.02 10.15b±1.10 12.21a±0.10 12.38a±0.05 p 1.91d±0.01 3.54c±0.02 3.88b±0.01 3.98b±0.03 5.41a±.0.01 5.50a±0.01 zn 0.84c±0.01 3.91b±0.05 3.98b±0.02 4.12ab±0.10 4.40a±0.01 4.42a±0.01 fe 1.52d±0.01 3.21c±.02 3.38bc±0.01 4.09b±0.02 6.22a±0.01 6.60a±0.02 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 30 anti-nutritional content of formulated complementary food the anti-nutrient content of complementary foods formulated from rice/soybean four blends fortified with palm weevil flour is shown in table 6. significant differences (p < 0.05) exist between all samples in terms of their antinutritional components. the results indicated a decreased in the concentration of saponin, tannin and oxalate as the percentage of palm weevil flour increases in the complementary foods samples. however, the concentration of phytate increases as the percentage of palm weevil in the complementary food increased. saponin concentration decreased from 3.61% in sample a to 2.51% in sample f. the concentration of tannin and oxalate also decreased from 5.24 mg/100g and 4.93 mg/100g in sample a to 3.82mg/100g and 2.01mg/100g in sample f respectively. in addition, phytate concentration increased from 11.05 mg/100g in sample a to 17.30 mg/100g in sample f. saponins are present in food products containing soybean and other legumes. they are not regarded as true antinutrient because they known to help lower risk of high cholesterol and cancer thereby controlling blood sugar [43]. however, the present of saponins in food imparts bitter taste and astringency resulting from poor organoleptic properties of the food product. saponins are also implicated in the inhibition of various enzymes thereby negatively impacting on protein digestion and assimilation. literature has shown that phytic acid forms complexes with minerals such as calcium, iron, magnesium zinc and protein and influence their solubility and bioavailability. the phytate content of all samples were below the 25 mg/100g permissible level [44]. high phytate content of the complementary food samples could be attributed to the present of broken rice in the formulations. tannins are anti-nutrient that form complex with protein resulting in insoluble precipitate of proteins in the gut thereby reducing digestibility of proteins in foods. apart from saponins, tannins are known to cause an astringent reaction in the mouth and make food unpalatable. tannins also interfere with digestibility and absorption of mineral nutrients such as iron and zinc. table 6 anti-nutritional content of formulated complementary food complementary foods parameters a (100:0) b (94.5:5.5) c (89.5:10.5) d (85.3:14.7) e (75.5:24.5) f (70:30) saponins(%) 3.61a±0.01 3.58a±0.02 3.27b±0.01 3.07b±0.01 2.58c±0.02 2.51c±0.03 phytate(mg/100g) 11.05d±1.20 11.65cd±1.00 12.27c±1.20 13.50b±2.00 16.78a±2.00 17.30a±1.22 tannins(mg/100g) 5.24a±0.02 5.21a±0.01 5.18a±0.02 5.10a±0.01 3.90b±0.05 3.82b±0.05 oxalate(mg/100g) 4.93a±1.00 4.85a±0.05 3.71b±1.00 3.60b±0.03 2.18c±0.01 2.01c±0.02 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 31 the present of oxalates in large doses in human food causes gastrointestinal disorders. oxalates form complexes with calcium, magnesium and iron leading to the formation of insoluble salts and resulting in the formation of oxalate stones [45]. it has also been known to negatively affect the absorption of potassium and sodium in human body. the antinutritional factors present in all the complementary food samples were at safe levels necessary to prevent all these effects from taking effect consequent upon the consumption of the products . sensory properties of formulated complementary food the mean scores obtained from sensory analysis of complementary foods produced from broken rice/ soybean flour blends fortified with palm weevil flour is presented in table 7. the mean scores obtained from the assessment of the appearance of the complementary food samples indicated a decreased in appearance as the proportion of palm weevil flour increases in the blends. the mean scores for other attributes such as taste, after taste, flavour, mouthfeel, consistency and overall acceptability increased as the proportion of palm weevil flour the blends increases. sample a(100:0) which had no palm weevil flour in the blend was the best in appearance. sample e(75.5:24.5), recorded the best scores for after taste, mouthfeel consistency and overall acceptability while sample f(70:30) had the highest scores for taste and flavour. sample e and f were more acceptable in all sensory attributes determined except in appearance only. this may be due to higher proportions of palm weevil flour in those food samples table 7: sensory properties of formulated complementary food complementary foods parameters a (100:0) b (94.5:5.5) c (89.5:10.5) d (85.3:14.7) e 75.5:24.5) f (70:30) appearance 8.12a±0.05 8.07a±2.10 7.59b±2.00 7.23c±1.05 5.36c±1.60 5.10cd±1.21 taste 6.58b±1.00 6.30bc±1.50 6.21c±1.02 6.53b±1.00 7.01a±0.05 7.04a±0.05 after taste 6.12b±0.05 6.10b±0.07 6.00bc±0.25 5.86c±1.01 7.10a±0.01 7.05a±1.23 flavour 7.45c±1.20 7.46c±0.50 7.55bc±1.00 7.62b±0.05 7.89a±1.50 7.96a±1.61 mouthfeel 6.63e±0.05 6.71d±1.00 6.85c±1.30 6.88c±1.00 7.21a±.1.21 7.00b±0.01 consistency 6.84d±0.06 6.91c±0.05 6.98bc±0.01 7.12b±1.10 7.40a±1.00 7.38a±0.04 overall acceptability 7.11b±0.01 7.10b±2.00 7.03c±0.05 7.19ab±1.21 7.24a±0.42 7.21a±1.00 *values are means ± sd of triplicate determination means in the same row with different superscript are significantly different at (p <0.05). 4. conclusion edible palm weevil (rhynchophorus phoenicis) is an edible insect that is highly valued and exploited by locales due to their nutritional potential. the present food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 32 study established the variability in nutritional content of developmental stages of edible palm weevil and it application in the formulation of complementary foods from broken rice and soybean flour blends. the proximate composition and calorific value of the three developmental stages of edible palm weevil indicated a higher lipid content in the early stage and late larva stages of development when compare to that of the adult weevil. the early larva stage was also superior in ash and energy content. however the adult weevil had the highest percentage of protein, carbohydrate and fiber content .the edible palm weevil had a significant amount of macro elements of sodium, calcium, magnesium, potassium and phosphorus in the three developmental stages while values obtained for the micro mineral of zinc and iron indicated that adult stage recorded highest values. the present of these micro minerals in the edible palm weevil makes it an important food ingredient toward the eradication of various nutritional deficiencies. results of proximate composition of complementary food formulated from blends of broken rice, soybean and edible palm weevil larva flour indicated that moisture, protein, lipid, ash and fibre content increased as the percentage of palm weevil flour increases in the formulated food samples. protein and carbohydrate content of complementary foods samples as obtained in the study was higher than the recommended levels while lipid content was within the acceptable limits. values obtained from the analysis of the complementary food samples for the macro elements (na, k, ca, mg and p) indicated an increased in all the macro element concentrations as the percentage of palm weevil flour increases in the rice/soybean flour blends. however values were far below the recommended daily allowance (rda). the result for zn and fe at 30% of palm weevil flour impute in the broken rice and soybean flour blends gave an acceptable levels of zn and fe which indicated that it is possible to achieve the recommended dietary level of these important mineral elements using local ingredients without resorting to fortification. the anti-nutritional factors present in all the complementary food samples were at safe levels necessary to prevent negative health effects consequent upon the consumption of the products. sensory results showed that sample e with 24.5% and sample f with 30% palm weevil flour were more acceptable in all sensory attributes determined except in appearance only. this may be attributed to higher proportions of palm weevil flour in the blends. 5. references [1]. food and agricultural organisation (fao). promises and challenge of informal food sector in developing countries. food and agriculture organisation. rome (2007). [2]. van huis, a.; van itterbeeck, j.; klunder, h.; mertens, e.; halloran, a.; muir, g.; vantomme, p. edible insects: future: prospects for food and feed security, 1st ed.; food and agriculture organization of the united nations: rome, italy, (2013). [3]. belluco, s., c. losasso, m. maggioletti, c. c. alonzi, m.g. paoletti, and a. ricci.. edible insects in a food safety and nutritional perspective: a critical review. compr. rev. food sci. food safety. 12:296–313, (2013) available at https://doi.org/10.1111/1541-4337.12014 [4]. jongema, y. worldwide list of recorded edible insects. wageningen (nl): wageningen university & research; [cited 2017 jun 04]. 100. available from http://www.wur.nl/en/expertiseservices/chair-groups/plant-sciences laboratory-of-entomology/edible insects/worldwide-species-list.htm. [5]. oonincx d. g, van itterbeeck, j. heetkamp, m. j, van den brand h, van loon j. j, van huis a. an exploration on greenhouse gas and ammonia production by insect species suitable for animal or human consumption. plos one; 5(12):e14445.(2010). https://doi.org/10.1111/1541-4337.12014 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 33 [6]. van huis a, oonincx d. g.. the environmental sustainability of insects as food and feed. a review agron sustain dev.;37:43 (2017). [7]. payne c, scarborough p, rayner m, nonaka k. 2016. are edible insects more or less ‘healthy’ than commonly consumed meats? a comparison using two nutrient profiling models developed to combat overand under nutrition. eur j clin nutr. 70(3):285–91 (2016) available at https://doi doi: 10.1038/ejcn.2015.149. [8]. rumpold b, schlüter o. nutritional composition and safety aspects of edible insects. mol nutr food res. 57(5): 802–23.(2013). [9]. finke m. d. complete nutrient content of four species of feeder insects. zoo biol. ;32(1):27– 36 (2013). [10]. haug, a. christophersen, o. a., kinabo, j., kaunda, w. and eik, l. o.,. use of dried kapenta (limnothrissa miodon and stolothrissa tanganicae) and other products based on whole fish for complementing maize-based diets. african journal of food agriculture, nutrition and development 10: 2478-2500 (2010) [11]. mosha, t. c. e. and bennink, m. r., protein digestibility-corrected amino acid scores, acceptability and storage stability of ready-to-eat supplementary foods for pre-school age children in tanzania. journal of the science of food and agriculture 85: 1513-1522 (2005) available at https://doi: 10.1002/jsfa.2147 [12]. briend, a. and darmon, n. determining limiting nutrients by linear programming: a new approach to predict insufficient intakes from complementary foods. pediatrics 5: 1288-1290. (2000) [13]. onimawo, l. a., akubor, i. p. food chemistry. integrated approach with biochemical background, ambik press ltd. 222-235 (2005). [14]. michaelsen, k. f., hoppe, c., roos, n., kaestel, p., stougaard, m., lauritzen, l., molgaard, c., girma, t. and friis, h., choice of foods and ingredients for moderately malnourished children 6 months to 5 years of age. food and nutrition bulletin 30: 343s404s .(2009) [15]. anigo, k. m., ameh, d. a., ibrahim, s. and danbauchi, s. s.,. nutrient composition of complementary food gruels formulated from malted cereals, soybeans and groundnut for use in north-western nigeria. african journal of food science 4: 65-72. (2010). [16]. martin, h., laswai, h. and kulwa, k.,. nutrient content and acceptability of soybean based complementary food. african journal of food nutrition and development 10: 2040-2049. (2010). [17]. inyang, u. e. umoh, h. i. and ukwo, s. p.. effect of co-fermentation duration on the nutritional composition and anti-nutritional contents of sorghum-cowpea flours and sensory properties of their gruels. iosr journal of environmental science, toxicology and food technology 13(2): 56-64. (2019) available at https:// doi:10.9790/24021302015664www.iosrjournals.org. [18] muhimbula, h. s., abdulsudi, i. and kinabo, j., . formulation and sensory evaluation of complementary foods from local, cheap and readily available cereals and legumes in iringa, tanzania. african journal of food science 5: 26-31. (2011). [19]. onyeike, e. n., ayalogu, e. o., and okaraonye, c. c. nutritive value of the larvae of raphia palm beetle (oryctes rhinoceros) and weevil (rhyncophorus pheonicis). journal of the science of food and agriculture, 85, 1822– 1828 (2005). [20]. asma, m. a., babiker, f. e. and tinay, a. h., . development of weaning food from sorghum supplemented with legumes and oil seeds. food and nutrition bulletin 27(1): 26-34 (2006) [21]. world food programme. (wfp) handbook: fortified blended food – good manufacturing practice and haccp principles (a handbook for processors in partnership with the un world food programme). wfp, rome, italy (2010) . available at: http://foodquality. wfp.org. [22]. okoli, c. olodi, w. b. ogbuewu, p. aladi, n. o. and okoli, c. g. . nutrient composition of african palm grub (rhynchophorus phoenicis) larvae harvested from raphia palm trunk in the niger-delta swamps of nigeria. asian journal of biological sciences. 12 (2):.284-290. (2019).available at https://doi: 10.3923/ajbs.2019.284.290. [23]. womeni h. m, tiencheu b, linder m, chouatcho nabayo e, tenyang n, tchouanguep mbiapo f, villeneuve p, fanni j, parmentier m. nutritional value and effect of cooking, drying and storage process on some functional properties of rhynchophorus phoenicis. international journal of life science and pharma research, 2: 203-21 (2012). [24]. lutter, c. k. and dewey, k. g.,. proposed nutrient composition for fortified complementary foods. journal of nutrition 133: 3011s-3020s (2003). [25]. kinyuru, j. n. konyole, s. o. onyango-omolo, s, a. kenji, g. m. . onyango, v.o. owino b.o. owuor, b.b. estambale and roos, n.. nutrients, functional properties, storage stability and costing of complementary foods enriched with either termites and fish or commercial micronutrients. journal of insects as food and feed 1 (2): 149 158 https://doi.org/10.1038/ejcn.2015.149 https://doi.org/10.1002/jsfa.2147 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 sunday ukwo, mfoniso udo, ekefre ekong. nutrient content of rhynchophorus phoenicis and its application in complementary food formulation, food and environment safety, volume xx, issue 1 – 2021, pag. 21 – 34 34 (2015). available at https://doi 10.3920/jiff2014.0011. [26]. codex alimentarius. codex standard for food grade salt. cx stan 150-1985, amended. codex alimentarius (1991). [27]. official methods of analysis, (aoac) 18th edition association of official analytical chemists, washington d.c, usa. 56-68 (2010). [28]. onwuka, g. i. food analysis and instrumentation. theory and practices. napthali prints, lagos, nigeria 140-176 (2018). [29]. wheeler, e. l., ferrel, r. e., . a method for phytic acid determination in wheat fractions. journal of cereal chemistry, 45:312-316 (1971). [30]. allen, s. e., grimshaw, h. m., parkinson, j. a., quarmby, c.. chemical analysis of ecological materials. ed. s. e. allen. blackwell sci. pub., oxford, london, edinburgh, melbourne, 565 (1984) [31]. ihekoronye, a. i., ngoddy, p, o.. integrated food science and technology for the tropics. 1st ed. macmillan publishers. 261, 265, 291 (1985). [32]. omotoso o.t. nutritional quality, functional properties and anti-nutrient compositions of the larva of cirina forda (westwood) (lepidoptera: saturniidae). journal of zhejiang university science b 7: 51-55 (2007). [33]. singh, p. the rearing of beneficial insects. new zealand entomologist, 7, 304–310 (2004). [34]. teffo, l. s. ton, r. b. and eloff, j. n. preliminary data on the nutritional composition of edible sting-bug consumed in lipopo province south africa. s. afr. j of sci, 103: 434436 (2007). [35]. okaraonye c. c, ikewuchi j. c.. rhynchophorus phoenicis (f) larva meal nutritional value and health implications. j biol sci. 8(7):1221–1225 (2008). [36]. elemo, b. o., elemo, g. n., makinde, m. a. erukainure, o. l. chemical evaluation of african palm weevil, rhynchophorus phoenicis larva as a food source”.journal of insect science (11): 146, 15362442 (2011). available at . https:// doi:10.1673/031.011.14601. [37]. folake, o. s. and bolanle, o. o.. chemical analysis and sensory evaluation of ogi enriched with soybeans and crayfish. nutr. food sci. 36(4): 214-217 (2006). [38]. alawode, e. k., idowu, m. a., adeola, a. a., some quality attributes of complementary food produced from flour blends of orange flesh sweet potato, sorghum, and soybean. croatian journal of food science and technology, 9(2): 122-129 (2017). https://doi.org/10.17508/cjfst.2017.9.2.06 [39]. ryan, e. p. bioactive food components and health properties of rice bran. j. amer. veter. med. assoc. 238:593600 (2011). https://doi.org/10.2460/javma.238.5.593. [40]. parker ,m. e. zobrist, s. lutterodt, h. e. asiedu,c. r. donahue, c. edick, c. mansen, k. pelto, g. milani, p. soor, s. laar, a. and engmann, c. m.. evaluating the nutritional content of an insect-fortified food for the child complementary diet in ghana. bmc nutrition 6(7): 1-11 (2020) available at https://doi.org/10.1186/s40795-020-0331-6 [41]. food and agriculture organisation/world health organisation (fao/ who). human energy requirements. joint fao/who expert consultation. fao, rome, italy (2004). [42]. food and agriculture organisation/world health organisation. (fao/ who) vitamin and mineral requirements in human nutrition. joint fao/who expert consultation. who, geneva, switzerland (2002). [43]. iwe, m. o. the science and technology of soybean. 1st edition. rejoint communication services limited. enugu nigeria. 97 (2003). [44]. nagel, r., living with phytic acid. the weston a. price foundation wincosin ave., washington d.c. (2010). http://www.weston aprice.org/food-features/living-with –phytic-acid [45]. owino, v.o., sinkala, m., amadi, b., tomkins, a.m. and filteau, s.m.,. acceptability, storage stability and costing of αamylase-treated maize-beans-groundnutsbambaranuts complementary blend. journal of the science of food and agriculture 87: 1021-1029 (2007). available at https://doi:10.1002/jsfa.2799 https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1673%2f031.011.14601?_sg%5b0%5d=tev3cmcrjip2wkv5dlpjicq286_w5df-b9w4adwcta9oj41v8iqpasvhyil0_ifao8xqcwxhlvgj76xsbfk3lfd4tw.cshakutuykdh5nkoe1d9sdsnm-gm1cxcq0iu4lxg9xxokdp44vwmtmpvpyeusziknqj4pl4b5inrwqcckbfjwq https://doi.org/10.17508/cjfst.2017.9.2.06 https://doi.org/10.2460/javma.238.5.593 https://doi.org/10.1002/jsfa.2799 223 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 32019, pag. 223 234 effects of wet ageing of beef muscle gluteus medius on textural and technological parameters *elena todosi sănduleac 1 , gheorghe gutt 1 1food engineering faculty,ştefan cel mare university of suceava, romania, elena.sanduleac@fia.usv.ro *corresponding author received 28th may 2019, accepted 27th september 2019 abstract: the aim of this study was to observe the effects of wet aging time of beef muscle gluteus medius on the textural and technological parameters. meat samples (400 g) were aged by wet ageing process for 21 days and were periodically analyzed at 2, 7, 12, 17 and 21 days respectively. the ph value registered a slight increase during the 21 days of ageing. cooking loss increased over the 21day ageing period by 1.03%. the ageing time affected the instrumental colour measurements. the cie-lab parameters have changed slightly through in an increase in lightness, l*, and yellowness, b*, but they did not affect redness, a*. the color difference, δe*, between the control samples and the aged samples was highlighted, both in raw and cooked meat samples. mechanical tests were performed on both raw and thermally treated samples. the results of warner bratzler mechanical test showed decreases as compared with control samples (p <0.05). the tpa parameters such as: hardness, resilience, springiness, adhesiveness, chewiness, gumminess and cohesiveness) were signifficaly modified (p <0.05), during 21 days of wet ageing. keywords: beef meat, gluteus medius, tenderness, color, textural properties, cooking loss 1. introduction meat is an important source of valuable quality protein, whose amino acid composition compensates for the deficiencies of other food raw materials. another important nutritional characteristic of the meat is its content in absorbable iron, zinc, selenium and in vitamins of group b, thiamine, riboflavin, niacin and vitamin b12, with an essential role in healthy nutrition [1]. tenderness is one of the important assessment indices of beef quality. the meat industry aims for reliable meat quality monitoring through out the production process in order to guarantee a high quality of the final product [2],[3]. for this reason, the meat sector is interested in technology for accurate, fast and non-destructive determination of quality attributes. both chemical (ph, color and fat content) and physical parameters (tenderness and juiciness) contribute to the meat appearance and eating experience and, therefore, to the final consumer acceptability [4]. ageing is defined as storing meat for a certain period of time to improve its eating quality attributes, including tenderness, flavor, and juiciness [5],[6]. wet ageing is performed on vacuum pre-packed meat in plastic sheets and maintained at refrigeration temperature. the advantages of wet aging process are: it is less expensive; it allows a faster distribution in the marketing units and ensures protection against microbiological contamination [7]. controlling the key factors that influence the rate and extent of aging impacts is http://www.fia.usv.ro/fiajournal mailto:elena.sanduleac@fia.usv.r food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 224 highly relevant to the development of effective strategies to improve meat quality and value. meat tenderness is described as the most important factor influencing consumer satisfaction, which has led to a great deal of research on understanding and measuring beef tenderness [8]. tenderness can be evaluated using objective methods, instrumental or sensorial methods with trained panels, or subjective methods with a consumer panel [9]. warner–bratzler shear test was one of the first instrumental methods developed to measure beef tenderness and, despite criticism, has remained the most commonly used globally [10]. the wbsf protocol measures the amount of force required to shear across entire muscle fibers. this method requires cooking the meat at an internal temperature of 71° c, cooling it at room temperature and cutting the samples into a round shape with a diameter of 1.27 cm. the cutting knife speed is 200-250 mm / min until complete shearing of the samples, perpendicular to the orientation of the muscle fibers [9]. two main components of meat, myofibres and connective tissue, are considered to be the main determinants of the shear force deformation curve [11]. a shear force curve always has a maximum positive peak shear force, but peaks of less importance are also, but not always, observed before and after maximum positive peak shear force. mf-sf and ct-sf is myofibrillar/ connective tissue peak shear forces (fig. 1) [11], [12]. fig.1. example of a shear curve measured with a texture analyser [11], [12]. tpa is still widely used by industry and academic research to characterize the texture of solid and semisolid foods. the method is simply a double compression, which compresses the food material twice to a limited extent to avoid structural damage to the material [13]. texture profile analysis (tpa) can be performed on either a raw sample or heat treated by boiling. the results of the tpa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 225 analysis may be significant in terms of hardness, succulence and chewiness, compared to the warner-bratzler test, where the elasticity is more significant than the texture parameter of meat [14]. in figure 2. is present the texture parameters according to texture profile analysis (tpa) [15]. fig.2. load diagram in two cycles used for texture profile analysis (tpa) [15]. the compression parameters obtained with tpa have been employed by many authors to evaluate meat products [16], [17], [18]. 2. matherials and methods 2.1. materials beef meat (gluteus medius) containing: 73.25 % moisture, 23.9 % protein, 2.29% fat, and 0.54% ash, and 5.69 ph) was purchased from a local slaughterhouse. at 12 h postmortem, gluteus medius (gm) muscles were excised and six 2.5 cm steaks were obtained. each meat sample was individually vacuum-packaged using vacuum sealer, 4.5 l/min, 120w, peach ph310, produced by peach industrial, romania, and assigned to aging times of 0, 2, 7, 12, 17 and 21 days at 4-5°c, to develop a wide range of possible tenderness. after ageing, at the specified time intervals, each sample was analyzed both raw and heat treated. the heat treatment was performed on in batchesin a 71 °c water bath for 35 min. [19]. meat samples were placed in polyethylene bags and cooked until reached a temperature of 71 °c. when the end-point temperature has been obtained, samples were removed from the water bath and cooled for 15 min. (adapted from honikel, 1998) [20]. the sample coding according to meat aging duration is presented in table 1. table 1. sample coding, according to meat aging duration meat aging duration 0 days 2 days 7 days 12 days 17 days 21 days sample code control m1 m2 m3 m4 m5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 226 2.2.methods 2.2.1. ph-analysis the changes in ph during wet ageing were monitored using a ph portable f2 standard mettler toledo device.the ph values of each sample were measured in a homogenate prepared with 5 g of raw meat sample and distilled water. all determinations were performed in triplicate. 2.2.2. cooking loss cooking loss was calculated as the percent weight difference between raw and cooked samples relative to the weight of raw samples according to dominguez et al., usind equation 1 [21]. 𝐶𝑜𝑜𝑘𝑖𝑛𝑔 𝑙𝑜𝑠𝑠(%) = 𝑅𝑎𝑤 𝑚𝑒𝑎𝑡 𝑤𝑒𝑖𝑔ℎ𝑡(𝑔)−𝐶𝑜𝑜𝑘𝑒𝑑 𝑚𝑒𝑎𝑡 𝑤𝑒𝑖𝑔ℎ𝑡(𝑔) 𝑅𝑎𝑤 𝑚𝑒𝑎𝑡 𝑤𝑒𝑖𝑔ℎ𝑡(𝑔) × 100 (1) 2.2.3.cie-lab color determination the color of the raw and heat treated meat was measured by means of the cie-lab method, where l* (100 = white, 0 = black) represents the brightness, and a* (+ red, green) and b* (+ yellow, blue) are color parameters. the determinations were performed using the konica minolta chroma meter cr-400 device. lightness (l*), redness (a*), and yellowness (b*) values were recorded. the hue angle (h°), color difference (δe*) and chroma difference (δc*), were calculated as follows equations [22]: h° = arctan (b⁎/a⁎) (2) δe⁎ = (δl⁎2 + δa⁎2 + δb⁎2)1/2 (3) δc⁎ = (δa⁎2 + δb⁎2)1/2 (4) color analysis is enhanced by increasing the number of measurements in a sample [23], therefore, three determinations were made at random on the surface of the measuring muscle. the color values for the three measurements were averaged and the average of each value was recorded for statistical analysis. 2.2.4. warnerbratzler shear force the analyses were carried out on raw and cooked samples according to amsa guidelines [9]. samples were sheared perpendicular to the fibre direction using the perten tvt 6700, from perten instruments fitted with a 20 kg tension/compression load cell and cross head speed of 1,5 mm/s. maximum peak force recorded during the test was reported as shear force (wbsf). seven cores per sample were analysed and following exclusion of the highest and lowest values, the average of five measurements was recorded for each sample. the average peak sheer force of the seven cores was used for statistical analyses. 2.2.5. texture profile analysis (tpa) using a texture analyzer, perten tvt 6700, from perten instruments, the tpa parameters of raw and cooked meat beef was evaluated as described by bourne (1978) [24]. the characterization of the texture profile was achieved using thetexturometer perten tvt 6700 and software, texcalc 5. briefly, three cylinders of 1 cm height and 1,27 cm diameter were prepared from every sample. a double compression cycle test was performed up to 50% compression of the original portion height with an aluminium cylinder probe of 2 cm diameter. a time of 5 s was allowed to elapse between the two compression cycles. force–time deformation curves were obtained with a 20 kg load cell applied at a cross-head, speed of 2 mm/s. the data obtained from tpa curve were used for the calculation of textural parameters. among the tpa parameters, hardness is expressed as maximum force food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 227 for the first compression. adhesiveness is expressed as negative force area for the first bite or the work necessary to pull the compressing plunger away from the sample. springiness is calculated as the ratio of the time from the start of the second area up to the second probe reversal over the time between the start of the first area and the first probe reversal. cohesiveness is a measure of the degree of difficulty in breaking down the samples internal structure. gumminess and chewiness have been reported as products of hardness, cohesiveness. chewiness is calculated as hardness × cohesiveness × springiness. resilience reflects the redeformation capacity of samples tissue after penetration. the texture analysis conditions were used as described by ben slima et al. (2017) [25], texture parameters (hardness, springiness, adhesiveness, chewiness, and cohesiveness) were quantified during 21 days of storage at 45°c. the force versus time/displacement plots were used to calculate tpa values, such as: hardness, chewiness, gumminess, adhesiveness, springiness and cohesiveness [24]. 2.2.6. statistical analysis the statistical analysis was made using microsoft excel 2007 and unscrambler x 10.1 software system (camo, norway). 3. results and discussion 3.1. ph and cooking loos results the cooking loss results and the variation of the ph of the meat samples during the ageing time, are shown in table 2. table 2. ph and cooking loss results samples control m1 m2 m3 m4 m5 ph 5.690±0.1 5.720±0.12 5.780±0.1 5.820±0.2 5.820±0.1 5.920±0.15 cooking loss(%) 25.754±0.2 25.758±0.3 25.988±0.22 26.031±0.3 26.349±0.12 26.776±0.25 figure 3 (a) shows the evolution of meat ph over the 21 days of aging, and the cooking loss (b) measured at the specified intervals: 0, 2, 7, 12, 17 and 21 days. the ph value registered a slight increase between 5.69 and 5.92 during the 21 days of maturation. small differences indicate ph stability during wet ageing, except for day 21, where a slight increase in ph was observed (table 2), which is in acord to minh ha et al. [26]. in figure 3(b) is registred the values of cooking loss of meat samples after cooking method. the difference between the cooking loss of the control sample and the meat sample aged 21 days it was of 1.03%. although the cooking losses show no clear trend, all samples increased in their percentage of moisture lost during cooking over the total ageing period. an increase in cooking loss was reported in their study and wyrwisz et al. [27]. the increments of cooking loss are due to the degradation of proteins in the aging process [28]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 228 fig.3. evolution of ph on the meat during aging period (a) and cooking loss (%) (b). lopes et al. studied the influence of cooking methods on amino acid and mineral content of veal and reported that cooking method affected the chemical composition and nutritional value of meat [29]. therefore, is registered the slight increase of the cooking loss on the meat wet aged for 21 days, this increase has been ascribed to the denaturation of intramuscular collagen or changes in the myofibrilar structure due to cooking. 3.2. cie-lab color parameters 5.550 5.600 5.650 5.700 5.750 5.800 5.850 5.900 5.950 control m1 m2 m3 m4 m5 5.690 5.720 5.780 5.820 5.820 5.920 25.200 25.400 25.600 25.800 26.000 26.200 26.400 26.600 26.800 control m1 m2 m3 m4 m5 25.754 25.758 25.988 26.031 26.349 26.776 ph a ) ) b cooking loss (%) https://www.sciencedirect.com/science/article/pii/s0023643816304297?via%3dihub#bib27 https://www.sciencedirect.com/topics/food-science/veal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 229 cie-lab color parameters of the raw meat samples are shown in table 3. table 3. the cie-lab color parameters of raw meat samples sample color parameters l * a * b * δc* h° δe* control 38.640±0.10 16.686±0.12 9.216±0.10 19.57±0.20 28.91±0.12 m1 40.938±0.21 16.544±0.16 9.296±0.20 18.98±0.17 29.33±0.22 2.30±0.12 m2 41.274±0.31 16.406±0.21 9.614±0.21 19.02±0.14 30.37±0.18 2.68±0.14 m3 41.676±0.13 16.226±0.16 9.700±0.14 18.90±0.21 30.87±0.19 3.11±0.20 m4 42.204±0.14 16.328±0.17 9.902±0.12 19.10±0.18 31.23±0.10 3.65±0.14 m5 42.434±0.20 16.155±0.12 9.987±0.15 18.99±0.15 31.72±0.12 3.91±0.10 the a*, b*, hue angle and chroma of beef meat were influenced by the ageing period, showing a trend for the colour of muscles to become lighter (p < .0001), more yellow (p < .001) and more saturated (p < .0001) over time. ageing time affected the instrumental colour measurements of the wet aged raw samples through in an increase in lightness, l*, and yellowness, b* , but did not affect redness, a*.color difference, δe* , between control meat sample and sample aged for 21 days, it was of 3,91 value. changes in color parameters during wet ageing were also reported by dikeman et al. [30]. hue angle, h°, which has a strong correlation with visual colour, represents the meat colour change from red to yellow and a larger hue angle value generally indicates a shift to lower redness and higher yellowness [31]. the increased beef color values after aging are related to the influence limited by the enzymatic activity during vacuum aging, which resulted in a deeper o2mb layer that is created in the presence of air oxygen, and blooming process occurs faster and more intensively [32]. moreover, a higher lightness l*, after aging, can be explained by the proteindegradation process during aging, leading to weakening of the protein structures, which results in higher light dispersion, thus increasing the lightness of meat [33]. the color parameters of the cooked meat samples are shown in table 4. table 4. the cie-lab color parameters of cooked meat samples sample color parameters l * a * b * δc* h° δe* control 36.598±0.20 7.098±0.22 14.424±0.10 16.08±0.12 63.80±0.20 m1 36.233±0.12 6.752±0.18 14.478±0.20 15.98±0.20 65.00±0.18 0.51±0.10 m2 35.948±0.14 6.338±0.18 14.802±0.22 16.10±0.14 66.82±0.10 1.07±0.12 m3 35.698±0.20 5.888±0.22 15.52±0.18 16.60±0.10 69.22±0.10 1.86±0.10 m4 35.670±0.12 5.618±0.14 16.204±0.18 17.15±0.12 70.88±0.14 2.49±0.12 m5 34.122±0.12 4.867±0.10 16.333±0.10 17.04±0.14 73.41±0.12 3.84±0.20 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 230 ageing time affected the instrumental colour measurements of the wet aged cooked samples through in an decrease in lightness, l*, and redness, a*. yellowness, b*, recorded a slight increase. color difference, δe*, between control meat sample and sample aged for 21 days, it was of 3.84 value. 3.3. warner bratzler shear force measurement the textural properties of the effect of wet aging duration of meat on the warner bratzler parameters of raw meat samples can be observed in table 5. table 5. warner bratzler shear force of raw and cooked meat samples samples control m1 m2 m3 m4 m5 wbsf( n)raw 13.100±0.1 12.17±0.13 10.95±0.14 9.869±0.1 8.676±0.1 8.218±0.1 wbsf( n) cooked 45.692±0.20 37.957±0.16 36.059±0.24 33.362±0.2 31.983±0.25 29.580±0.20 the warner–bratzler shear force values ranged from 13.100 n (first days aged) to 8.218 n (21-days aged) (table 5). between control sample and 21th day of ageing, wbsf values significantly decreased in beef muscle, gluteus medius (p < 0.05). for wbsf, both factors (ageing time and temperature) affected the results. fig.4. the correlations between wbsf of de raw and cooked meat samples and time wet ageing figure 4 shows the correlation between wbsf of de raw and cooked meat samples, and time ageing. thus, the wbsf of the beef muscle reached a good correlations, r2 = 0.983, for the raw sample and r2 = 0.898 for the cooked sample. cooking causes heat-induced denaturation of both myofibrillar proteins and connective tissue. koohmaraie et al, have been reported to reach maximum tenderness at approximately 11 to 14 days of ageing beef meat, they tend to experience a much smaller decrease in shear force values compared with game meat. y = -1.0283x + 14.097 r² = 0.9894 y = -2.8907x + 45.89 r² = 0.904 .000 5.000 10.000 15.000 20.000 25.000 30.000 35.000 40.000 45.000 50.000 0 2 7 12 17 21 raw sample cooked sample wbsf(n) time ageing (days) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 231 thus, the higher degree of tenderization in eland muscles compared to beef may support the conclusion that game species may have increased proteolytic enzyme activity and a more efficient proteolytic system; an aspect that warrants further research [34]. 3.4. tpa parameters results the textural properties of the effect of wet aging duration of meat on the tpa parameters of raw meat samples can be observed in table 6. table 6. tpa parameters of gluteus medius muscles on raw and cooked meat samples tpa parameters* samples control m1 m2 m3 m4 m5 raw cooked raw cooked raw cooked raw cooked raw cooked raw cooked h (n) 15.36 23.13 13.73 22.79 12.49 20.53 12.74 19.67 10.92 18.47 9.66 17.96 r 2.29 2.25 2.35 2.55 2.39 2.98 2.78 3.00 2.80 3.01 2.85 3.07 a(n*s) -0.06 -0.001 -0.08 -0.001 -0.11 -0.002 -0.12 -0.002 -0.13 -0.004 -0.19 -0.004 s 1.000 1.000 0.998 0.998 0.999 0.998 0.997 0.997 0.996 0.997 0.996 0.996 co 0.443 0.566 0.436 0.582 0.435 0.601 0.434 0.636 0.439 0.630 0.394 0.644 g (n) 6.810 13.10 5.995 13.26 5.430 12.35 5.535 12.30 4.789 11.64 3.809 11.58 ch(n) 6.810 13.10 5.988 13.24 5.429 12.32 5.519 12.26 4.774 11.60 3.796 11.53 *h: hardness, r: resilience, a: adhesineness, s: springiness, co: cohesiveness, g: gumminess and ch: chewiness as it can be seen, all the textural parameters (hardness, resilience, chewiness, gumminess, adhesiveness, springiness and cohesiveness) were affected by the wet aging duration of meat. the hardness for raw meat samples varied from 15.356 n to 9.66 n, for the cooking meat varied from 23.13 n to 17.96 n, thus registering a significant decrease (p = 0.05). the resilience of the meat samples, both raw and cooked ones, registered slight increases, from 2.29 and 2.83 for raw meat and from 2.25 to 3.07 for cooked meat. the modifications for adhesiveness and springiness are insignificant, the variations of this parameters being very small. the values for cohesiveness were between 0.443 and 0.394, for the raw meat samples, and from 0.566 to 0.644 for the cooked meat samples. another important texture parameter in meat products targeted at older consumers is chewiness. wet ageing had significantly reduced chewiness (p = 0.05) compared with control sample. furthermore, trends for gumminess were the same as for chewiness values, and depend slightly from hardness. some studies indicated that hardness accounted approximately 40.6 and 45.7 in tenderness and overall palatability [35]. furthermore, they also noted that tpa springiness and chewiness were highly related to intramuscular fat content and consequently tenderness and juiciness. to elucidate our experimental design which examined texture profiles as a function of different ageing days, springiness, cohesiveness and chewiness were also determined, and the data was extensive explored during data mining process. springiness of meat is probably associated to fibre swelling and diameter [36]. 3.5. statistical analysis tables 7 and 8 show the pearson correlations between the wet ageing duration and the textural parameters obtained by the tpa and warner bratzler methods. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 232 table 7. pearson correlation between the wet aging time and textural parameters of raw meat samples parameters time(days) wbsf(n) h(n) r a(n*s) s co g(n) ch(n) time(days) 1.000 wbsf(n) -0.992 1.000 h(n) -0.956 0.959 1.000 r 0.948 -0.948 -0.835 1.000 a(n*s) 0.952 -0.933 -0.959 0.853 1.000 s -0.895 0.896 0.799 -0.970 -0.808 1.000 co -0.704 0.643 0.750 -0.566 -0.866 0.567 1.000 g(n) -0.947 0.941 0.994 -0.820 -0.978 0.789 0.817 1.000 ch(n) -0.948 0.942 0.994 -0.823 -0.978 0.792 0.816 1.000 1.000 h: hardness, wbsf: warner bratzler shear force, r: resilience, co: cohesiveness, g: gumminess and ch: chewiness values varying between different timesof wet ageing were used to perform the correlation analysis (p< 0.05). the statistical analysis showed significant negative correlations (p< 0.05) between time ageing and: wbsf (coefficient0.992), hardness tpa (coefficient -0.948), springiness (coefficient -0.895), cohesiveness (coefficient -0.704), chewiness (coefficient -0.948) and gumminess (coefficient -0.947), and positive correlations (p< 0.05) of resilience (coefficient 0.948) and adhesiveness (coefficient 0.952). table 8. pearson correlation between the wet aging time and textural parameters of cooked meat samples parameters time(days) wbsf(n) h(n) r a(n*s) s co g(n) ch(n) time(days) 1.000 wbsf(n) -0.916 1.000 h(n) -0.973 0.913 1.000 r 0.853 -0.942 -0.886 1.000 adh(n*s) 0.980 -0.885 -0.930 0.799 1.000 spg -0.936 0.991 0.943 -0.929 -0.888 1.000 co -0.687 0.633 0.667 -0.568 -0.794 0.586 1.000 g(n) -0.909 0.853 0.914 -0.803 -0.943 0.843 0.912 1.000 che(n) -0.910 0.856 0.915 -0.806 -0.944 0.846 0.910 1.000 1.000 h: hardness, wbsf: warner bratzler shear force, r: resilience, co: cohesiveness, g: gumminess and ch: chewiness values varying between different time of wet ageing were used to perform the correlation analysis (p< 0.05) for the cooked meat samples, the statistical analysis showed significant negative correlations (p<0.05) between time ageing and wbsf (coefficient-0.916), hardness tpa (coefficient -0.973), hardness warner bratzler (coefficient – 0.951), springiness (coefficient -0.936), cohesiveness (coefficient -0.687), chewiness (coefficient -0.910) and gumminess (coefficient -0.909), and positive correlations (p <0.05) of resilience (coefficient 0.853) and adhesiveness (coefficient 0.980). resilience can be used as detection indicator for beef freshness in storage. this study provides a method for rapid detection of beef freshness, and provides the basis for beef fresh keeping during production and transportation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 233 4. conclusion this study evaluated the effects of wet ageing on raw and cooked beef meat, gluteus medius during 21 days of storage at 4-5 °c as regards textural, physicochemical and color parameters.in conclusion we can remark that the anticipation of textural parameters in noncooked, raw and cooked muscle has become more important for the consumers in terms of meat choice and satisfaction. the current data indicated that the magnitude of thermal toughening greatly varied between beef muscles where hardening of muscle samples by heating was more obvious.therefore, the current data indicated that estimation of meat texture from raw material to cooked meats varies depending on the muscle type and its interaction with wet ageing day. in addition, the results mirror the importance of wet ageing time for objective measurements which ultimately estimate sensory tenderness and other quality traits. 5. references [1].mcneill,s.h., van elswyk, m.e., meat: role in the diet, reference module in food science encyclopedia of food and health, 693–700, (2016). [2].damez, j. l., clerjon, s., meat quality assessment using biophysical methods related to meat structure, meat science, 80(1), 132–149, (2008). [3].troy, d. j., kerry, j. p., consumer perception and the role of science in the meat industry, meat science, 86(1), 214-226, (2010). [4].van beersa, r., kokawa, m., aernouts, b., watté, r., de smet, s., saeys,w., evolution of the bulk optical properties of bovine muscles during wet aging , meat science, 136, 50-58, (2018). [5]. lee, h. j., choe, j., kim, k. t., oh, j., lee, d. g., kwon, k. m., choi, y.i., jo, c., analysis oflow-marbled hanwoo cow meat aged with different dry-aging methods, asian-australian journal of animal science, 30, 1733–1738, (2017). [6].oh, j., lee, h. j., kim, h. c., kim, h. j., yun, y. g., kim, k. t., choi, y.i., jo, c., the effects of dry or wet aging on the quality of the longissimus muscle from 4year-old hanwoocows and 28-month-old hanwoo steers, animal production science, (2017). [7].jiménez-colmenero, f., carballo, j., cofrades, s., healthier meat and meat products their role as functional foods, meat science, 59, 5– 13, (2001). [8]. ramos, e.m., gomide, l.a.m., avaliação da qualidade de carnes: fundamentos e metodologias (1 ed.). viçosa: editora ufv, (2007). [9].amsa, research guidelines for cookery, sensory evaluation, and instrumental tender-ness measurements of fresh meat.chicago: american meat science association (amsa) & national live stock and meat board, (1995). [10].wheeler, t.l., shackelford, s.d., johnson, l.p., miller, m.f., miller, r.k., koohmaraie, m., a comparison of warner– bratzler shear force assessment within and among institutions. journal of animal science, 75(9), 2423–2432, (1997) [11].møller, a. j., analysis of warner–bratzler shear pattern with regard to myofibrillar and connective tissue components of tenderness, meat science, 5, 247–260, (1980). [12]. girard, i., bruce, h.l., basarab, j.a., larsen, i.l., aalhus, j.l., contribution of myofibrillar and connective tissue components to thewarner–bratzler shear force of cooked beef, meat science, 92, 775-782, (2012). [13]. bourne, m. c., food texture and viscosity (2nd ed). new york: academic press, (2002). [14]. ruiz de huidobro, r., miguel, e., blázquez, b., onega, e., a comparison between two methods (warner–bratzler and texture profile analysis) for testing either raw meat or cooked meat, meat science, volume 69, issue 3, 527-536, (2005). [15]. amariei, s., gutt, g., oroian, m. a., sănduleac, e., paduret, s., automated process for achieving food texture profile, patent invention, osim file a130706/2015. [16]. houben, j. h., van't hooft, b. j., variations in product-related parameters during the standardised manufacture of a semi-dry fermented sausage, meat science, 69(2), 283–287, (2005). [17]. hoz, l., d’arrigo, m., cambero, i., ordóñez, j. a., development of ann-3 fatty acid and α-tocopherol enriched dry fermented sausage, meat science, 67(3), 485–495, (2004). [18]. romero de ávila, m.d., cambero, m.i., ordóñez, j., a., lorenzo de la hoz, l., herrero, a.m., rheological behaviour of commercial cooked meat products evaluated by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 3 – 2019 elena todosi sănduleac, gheorghe gutt,effects of wet ageing time of beef muscle gluteus medius on textural and technological parameters, food and environment safety, volume xviii, issue 3 – 2019, pag. 223– 234 234 tensile test and texture profile analysis (tpa), meat science, 98, 310-315, (2014). [19]. holman, b.w.b., alvarenga, t.i.r.c., van de ven, r.j., hopkins, d.l., a comparison of technical replicate (cuts) effect on lambwarner–bratzler shear force measurement precision, meat science, 105, 93-95, (2015). [20]. honikel, k.o., reference methods for the assessment of physical characteristics of meat, meat science, 49 (4), 447-57, (1998). [21]. dominguez, r., gómez, m., fonseca, s., lorenzo, j. m., influence of thermal treatment on formation of volatile compounds, cooking loss and lipid oxidation in foal meat, lwtfood science and technology, 58, 439–445, (2014). [22]. grigelmo-miguel, n., abadíasserós, m.i., martín-belloso, o., characterisation of low-fat high-dietary fibre frankfurters, meat science, 52 (3) , 247-256, (1999). [23]. alcalde, m.j., negueruela, a.i., the influence of final conditions on meat colour in light lamb carcasses, meat science, 57, 117-123, (2001). [24]. bourne, m.c., texture profile analysis, food technology, 32, 62-66, (1978). [25]. ben slima, s., ktari, n., trabelsi, i., triki, m., feki-tounsi, m., moussa, h., makni, i., herrero, a., jiménezcolmenero, f., ruiz-capillas perez, c., ben salah, r., effect of partial replacement of nitrite with a novel probiotic lactobacillus plantarum tn8 on color, physico-chemical, texture and microbiological properties of beef sausages, lwt food science and technology, 86, 219–226, (2017). [26]. ha, m., mcgilchrist, p., polkinghorne, r., huynh, l., galletly, j., kobayashi, k., nishimura, t., bonney, s.,.kelman, k.r., warner, r.d., effects of different ageing methods on colour, yield, oxidation and sensory qualities of australian beef loins consumed in australia and japan, food research international,125,108528, (2019). [27]. wyrwisz, j., moczkowska , m., kurek, m.a., karp, s., atanasov, a.g., wierzbicka, a., evaluation of wbsf, color, cooking loss of longissimus lumborum muscle with fiber optic near-infrared spectroscopy (ftnir), depending on aging time, molecules 24(4), 757, (2019) [28]. moczkowska, m.; półtorak, a.; wierzbicka, a., the effect of ageing on changes in myofibrillar protein in selected muscles in relation to the tenderness of meat obtained from cross-breed heifers, international journal of food science and technology, 52, 1375–1382, (2017) [29].lopes, a.f., alfaia, c.m., partidario, a.m., lemos, j.p., prates, j.a., influence of household cooking methods on amino acids and minerals of barrosa-pdo veal, meat science, 99, 38-43, (2015) [30]. dikeman, m.e., obuz , e., gök, v., akkaya , l., stroda, s., effects of dry, vacuum, and special bag aging; usda quality grade; and end-point temperature on yields and eating quality of beef longissimus lumborum steaks, meat science, 94 (2), 228-233, (2013) [31]. brewer, m.s., zhu, l.g., bidner, b.,meisinger, d.j., mckeith,f..k., measuring pork color: effects of bloom time, muscle, ph and relationship to instrumental parameters, meat science, 57 (2), 169-176, (2001) [32]. węglarz, a., effect of pre-slaughter housing of different cattle categories on beef quality, animal science papers and reports, 29, 43–52, (2011) [33]. wyrwisz, j., moczkowska, m., kurek, m., stelmasiak, a., półtorak, a., wierzbicka, a., influence of 21 days of vacuum-aging on color, bloom development, and wbsf of beef semimembranosus, meat science, 122, 48–54, (2016) [34]. koohmaraie, m., kent, m.p., shackelford, s.d., veiseth, e., wheeler, t.l., meat tenderness and muscle growth: is there any relationship?, meat science, 62, 345-352, (2002) [35]. caine, w.r, aalhus, jl, best, d.r, dugan, m.e.r, jeremiah, l.e., relationship of texture profile analysis and warner-bratzler shear force with sensory characteristics of beef rib steaks, meat science, 64:333–9, (2003) [36]. palka, k., henry, d. ,changes in texture, cooking losses, and myofibrillar structure of bovine m. semitendinosus during heating, meat science, 51, 237–43, (1999) https://www.sciencedirect.com/science/article/pii/s0023643817305509#! https://www.sciencedirect.com/science/article/pii/s0023643817305509#! https://www.sciencedirect.com/science/article/pii/s0023643817305509#! https://www.sciencedirect.com/science/article/pii/s0023643817305509#! https://www.sciencedirect.com/science/article/abs/pii/s0309174013000430#! https://www.sciencedirect.com/science/article/abs/pii/s0309174013000430#! https://www.sciencedirect.com/science/article/abs/pii/s0309174013000430#! https://www.sciencedirect.com/science/article/abs/pii/s0309174013000430#! https://www.sciencedirect.com/science/article/abs/pii/s0309174013000430#! https://www.sciencedirect.com/science/journal/03091740/94/2 https://www.sciencedirect.com/science/article/abs/pii/s0309174000000899#! https://www.sciencedirect.com/science/article/abs/pii/s0309174000000899#! https://www.sciencedirect.com/science/article/abs/pii/s0309174000000899#! https://www.sciencedirect.com/science/article/abs/pii/s0309174000000899#! https://www.sciencedirect.com/science/article/abs/pii/s0309174000000899#! https://www.sciencedirect.com/science/journal/03091740 https://www.sciencedirect.com/science/journal/03091740/57/2 1. introduction doi: https://doi.org/10.4316/fens.2022.030 313 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 313 320 comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines monique t. estrella 1 , aileen fritz t. samson 1 , *rafael a. espiritu 1 1department of chemistry, de la salle university, 2401 taft avenue, 0922 manila, philippines rafael.espiritu@dlsu.edu.ph *corresponding author received 11th october 2022, accepted 28th december 2022 abstract: chicken meat consumption remains high in the philippines, and consequently, annual chicken production has risen steadily over the years. although most of the chicken meat available in the market are sourced from factory farms, there has been growing popularity of free-range chicken production, largely driven by its perceived health benefits and sanitation concerns in factory farms, among others. in this work, we aimed at comparing the trace metal (copper, iron, lead, manganese, and zinc) content of free-range and factory-farmed broiler chicken meat that are being sold in laguna, philippines. the samples were subjected to acid digestion and analysis was done using atomic absorption spectrometry. results showed an average concentration between 0.17 – 0.24 mg/kg, 2.22 – 2.34 mg/kg, 0.12 – 0.15 mg/kg, 0.95 – 1.32 mg/kg, and 1.81 – 1.96 mg/kg for copper, iron, lead, manganese, and zinc, respectively. statistical analysis using one-way anova and tukey multiple comparison test (p < 0.05) showed no significant differences in the trace metal content of free-range and factory-farmed chicken meats, suggesting that in terms of the levels of these trace metals, not much differentiates the two varieties. analysis of the health hazards, reported as target hazard quotient (thq) and hazardous index (hi), associated with the consumption of the said chicken breast meat indicated no potential health risks. keywords: atomic absorption spectroscopy, free-range chicken, factory-farmed chicken, trace metal analysis 1. introduction livestock production accounts for about 40% of the gross value of agricultural products worldwide and is the fastest growing sector in agriculture, according to food and agriculture organization (fao) of the united nations [1]. the fao also notes that of the major source of animal protein, poultry ranks at the top, estimated to account approximately 39% of global meat production in 2019 which is projected to increase to 50% by 2025, and with global per capita consumption increasing fivefold since the 1960s [2, 3]. in the philippines, the situation is not much different, with volume of chicken production in 2018 recorded at 1.8 million metric tons, with around 80% being factory-farmed broilers, and a nationwide annual per capita consumption of around 13 kg, just behind pork meat [4, 5] free-range practice in the philippines, consisting mostly of native chickens raised in backyard farms, has increasingly gained popularity, which follows the global trend of more health-conscious consumers and their preference for organic chicken meat products [6]. chickens reared through the free-range method have more freedom to forage for themselves rather than be dependent on feeds, with consequent health benefits of having more crude protein and lower fat content than their mailto:rafael.espiritu@dlsu.edu.ph food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 – 320 314 commercial broiler counterparts [7]. these traits, among others, justify the premium price of free-ranged chickens. nevertheless, numerous reports over the years showed that free-range chickens are also exposed to various anthropogenic activities that ultimately result in undesirable contaminants in their meat such as antimicrobial-resistant bacteria, parasites, and other organic pollutants, e.g. dioxins [8–10]. heavy metals and trace elements also accumulate in chicken tissues, especially under factory-farming conditions, arising from extensive use of formulated animal feed and feed additives [11]. in some cases, worse contamination, e.g. with lead, was even associated with free-range chickens [12, 13]. these accumulated trace metals can then be passed on and in turn accumulate in animals on top of the food chain via biomagnification, and potentially cause chronic toxicity and serious health effects in humans. therefore, as a an important health concern, trace metal content in chicken meat and meat products derived from them have been investigated extensively in various countries [13, 14]. these imply that the quality, and therefore, health benefits associated with, and concerns raised against free-ranged and factory-farmed chickens, respectively, also greatly depend on the local environment where such practices are carried out. with these in mind, and the seemingly sparse studies available on the differences between free-range and factory-farmed chickens in the philippines, in this study, we aimed to determine and compare the trace metal content of free-range and commercial factory-farmed chicken meat that are being sold in supermarkets in laguna province, the philippines. results from this research could contribute to the human exposure assessment associated with the intake of these metals from chicken meat. the results could also serve as a focal point for further research on the factors soil quality, environmental pollution, chicken feed, and breeding area location that may have contributed to the levels of these metals in the chicken meat test samples. finally, in conjunction with other data, the consumers might be provided with the necessary information to aid them in their decision-making process in the context of the financial and health considerations concerning free-ranged and factory-farmed chickens. 2. matherials and methods sample preparation whole chicken from two commercial brands that sell both free-range (with explicit labeling) and factory-farmed varieties were used in this study, designated either as fr or ff, respectively. the samples were bought from supermarkets in sta. rosa city, laguna, in the philippines. for each variety, four samples of whole chicken were chosen in random order. only the breast part of the chicken was used for the analysis. the separated meat/muscle from each variety were combined separately, and 10 replicates were prepared for each sample by weighing approximately 60 grams of chicken meat into an evaporating dish. these samples were dried in the oven at 100°c for 24 hours. immediately after drying, the samples were ashed in a furnace for 3 – 4 hours at 400°c. two grams of ash for each sample were accurately weighed, transferred to 250-ml erlenmeyer flasks, and were treated with 20 ml of 12 m hydrochloric acid. the samples were then heated under the hood at medium heat for 30 minutes, or until the solution turned into a yellowish color and there was an appearance of white smoke. afterwards, 30 ml of distilled water were added to each sample. the solution was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 – 320 315 then filtered into a 50 ml volumetric flasks and diluted accordingly [15]. trace metal analysis standard solutions of varying concentrations were prepared from the 1000 ppm stock solutions of metal standards (sigma-aldrich). the concentration range of prepared standard solutions was from 0.1 ppm to 10 ppm. analysis of the trace metals was performed using a shimadzu atomic absorption spectrophotometer (aa – 6300) using an air-acetylene gas mixture, and with the following settings: fuel gas flow rate of 1.8 – 2.2 l/min, support gas flow rate of 15 l/min, burner height between 7 – 9 mm, and slit width between 0.2 – 0.7 nm. the detection wavelengths used for copper, iron, lead, manganese, and zinc were 324.7 nm, 248.3 nm, 217.0 nm, 279.5 nm, and 213.9 nm, respectively. estimated daily intake (edi) of trace metals the edi of the different trace metals (cu, fe, pb, mn, and zn) was expressed as mg/kg body weight/day that depended both on the average concentration of the metals and chicken meat consumption. this parameter was calculated based on the following equation [16]: where mc is the average concentration of the metal in the chicken meat (mg/kg), fdc is the average chicken meat consumption in the region (g/person/day), and bw is the average body weight of a child (30 kg) or adult (60 kg). based on government statistics [17], the average per capita chicken meat consumption in laguna, philippines stands at 26 g/day. target hazard quotient (thq) the non-carcinogenic and carcinogenic health risks associated with chicken meat consumption was assessed based on the thq. this parameter is expressed as a ratio of a determined dose of a pollutant to that of a reference dose level, where a value of less than 1 implies that the exposed population is unlikely to experience adverse health effects. thq was determined using the following equation [18]: where efr is the frequency of exposure (365 days/year), ed is the duration of exposure (70 years average lifespan), fdc is the food ingestion rate (g/person/day), mc is the average metal concentration in chicken meat (mg/kg), rfd is the oral reference dose (mg/kg/day), bw is the child (30 kg) or adult (60 kg) average body weight, and at is the average exposure time (365 days/year and assuming 70 years as the average lifespan). the rfd consensus values are as follows [18]: 0.04, 0.7, 0.004, 0.14, and 0.3 mg/kg/day for cu, fe, pb, mn, and zn, respectively. hazardous index (hi) to evaluate the cumulative health risk of consuming the trace metals analyzed in this study, the hi was calculated based on the summation of all the thq values for the analyzed trace metals using the following equation [18]: an hi value of less than 1 is interpreted as having no hazard, between 1 and 10 is considered moderate hazard, while a value food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 – 320 316 greater than 10 suggests high risk or hazard. statistical analysis determination of significant difference in the mean concentration values were carried out by one-way anova and tukey multiple comparisons test (p < 0.05) using origin pro 8. 3. results and discussion the bird population is especially susceptible to the effects of anthropogenic activities on the environment. in line with this, it was identified that factors such as eating habits, growth, age, and breeding may influence metal concentration in the species [19]. although some heavy metals are identified to be biologically important trace elements, the toxic effects brought upon by consumption beyond the recommended limits continue to be a concern globally [20]. in this regard, consumers have turned to patronize freerange meat products since these are deemed to be a safer and healthier option. nevertheless, there is still a lack of data to back these claims in the philippine setting, since comparative analysis on the trace metal content of poultry products, specifically between commercial factoryfarmed broilers and free-range chickens, remain very limited. fig. 1. average concentration of various trace metals from the breast part of free-ranged (fr) and factory-farmed (ff) chickens. using atomic absorption spectroscopy, we quantified the trace metal content of chicken breast meat samples from two brands of free-range (fr) and factoryfarmed (ff) chickens. the average metal concentration generally followed the following trend: fe > zn > mn > cu > pb (figure 1). as the first four elements are known to have essential biological role in various organisms, while pb is a toxic contaminant, these results agree with previous findings showing the higher content of these essential metals in the analyzed chicken tissues [21]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 320 317 iron and zinc, with the former being one of the most abundant transition metal elements, are known to have extensive physiological roles such as in aerobic respiration, dna synthesis and repair, and cell death. as such their higher concentration in an organism, relative to other essential metals, are expected. overexposure to these two metals may lead to tissue damage, inflamed pancreas, anemia, and acute renal failure, to name a few. in our work, we found an average fe concentration of 2.22 and 2.34 mg/kg in fr and ff samples, respectively. similarly, a comparable concentration of zn was found in the fr and ff samples, with mean value of 1.81 and 1.96 mg/kg, respectively. the higher content of these two metals in chicken meat relative to the others analyzed in this study was consistent with previous reports showing a similar trend [14, 18, 21]. manganese is an essential element in various organisms, and its deficiency in vertebrates is known to cause skeletal and reproductive abnormalities while overexposure to this metal has been linked to neuropsychiatric disorders [16]. the average concentration of manganese found in this study was 0.95 and 1.32 mg/kg in the fr and ff samples, respectively (fig. 1). copper is an essential element that has important role as co-factor in various enzymes that are part of many physiological processes such as cellular respiration and energy production and utilization [22]. in this study, the average concentration of cu was determined to be 0.17 and 0.24 mg/kg in fr and ff samples, respectively. these values were lower than those detected from chicken muscles in other studies, which reported mean cu content of more than 0.44 mg/kg [14, 18, 21]. table 1. estimated daily intake of trace metals. free-ranged (fr) and factory-farmed (ff) chickens. lead is one of the most toxic heavy metals, and prolonged exposure to this contaminant is often ascribed to varying physiological and pathological effects. this element affects almost all organs in the body, although it has its most profound deleterious consequence in the nervous system [23]. similar to previous reports [14, 18, 21], the concentration of pb in chicken breast found in this study was the lowest among the metals tested, with average value of 0.12 and 0.15 mg/kg in fr and ff samples, respectively. estimated daily intake, edi (mg/kg bw/day) fr ff fr ff adult child cu 0.075 0.10 0.15 0.21 fe 0.96 0.97 1.93 1.94 mn 0.41 0.57 0.83 1.15 pb 0.052 0.066 0.10 0.13 zn 0.79 0.85 1.58 1.70 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 320 318 fig. 2. hazard quotient (thq) of trace metals in adults and children via consumption of breast meat of free-ranged (fr) and factory-farmed (ff) chickens. fig.3. hazard index (hi) for adults and children via consumption of breast meat of free-ranged (fr) and factory-farmed (ff) chickens. statistical analysis of the results (p < 0.05) indicated no significant differences between the two types of chicken samples in all the metals tested. this indicates that as far as this aspect is concerned, consuming a free-range chicken is not much different from the more common factory-farmed variety. to assess the health risks associated with prolonged consumption of chicken muscle, the estimated daily intake (edi), target hazard quotient (thq), and hazard index (hi) were calculated. meat consumption is a known way by which humans are exposed to different pollutants/ contaminants, and toxicity ultimately depends on the daily intake by individuals of contaminated foodstuffs. the edi values are summarized in table 1 and comparing these with the rfd for each of this metal, all except pb exceeded the oral reference dose. it is important to point out, however, that consumption more than the designated rfd for a specific metal does not automatically translate to deleterious health effects, as this only taken to mean that a conservative reference oral dose has been exceeded [18]. indeed, the computed thq values for all metals are extremely small (less than 0.04), indicating that consumption of chicken breast meat from both free-ranged and factory-farmed animals will unlikely result in adverse health effects to the exposed population (figure 2). furthermore, the hi, which takes into account the cumulative risks associated with the five metals tested, had values less than 0.10 (figure 3) indicating no potential hazard with consumption of chicken breast meat. it is worth mentioning that although thq and hi values were very low, the risk is higher in children primarily due to a lower body weight and prolonged exposure since childhood increases the chances of developing acute and chronic toxic effects. furthermore, it is also important to note that the study only considered chicken breast meat samples, but consumption of other organs like gizzard and liver, where these and other toxic metals bioaccumulate [16, 18], as well as meat products from other animals could increase the risk of biomagnification and thus could potentially introduce health risks in the consuming population. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 320 319 4. conclusion in this study, chicken breast meat samples from free-ranged and factory-farmed chickens were compared for their trace metal content. the determined average concentration ranged from 0.17 – 0.24 mg/kg, 2.22 – 2.34 mg/kg, 0.12 – 0.15 mg/kg, 0.95 – 1.32 mg/kg, and 1.81 – 1.96 mg/kg for copper, iron, lead, manganese, and zinc, respectively. statistical analysis using one-way anova and tukey multiple comparison test showed no significant difference between any of the metals from the two types of chicken (p < 0.05), indicating no added benefit of eating the free-range variety, at least in the context of this study. analysis of the potential health hazards from consuming chicken muscle, quantified using parameters such as thq and hi, showed that consuming this meat product is unlikely to result in deleterious health effects for the exposed population. 5. acknowledgments the authors are grateful to the de la salle university chemistry instrumentation laboratory. 6. references [1]. animal production | fao | food and agriculture organization of the united nations, (available at http://www.fao.org/animalproduction/en/). [2]. gateway to poultry production and products | food and agriculture organization of the united nations, (available at http://www.fao.org/poultry-productionproducts/en/). [3]. fao, “oead-fao agricultural outlook 2016-2025” (2016), , doi:10.1787/19991142. [4]. philippines statistics authority, chicken situation report (2019), (available at https://psa.gov.ph/sites/default/files/chicken_ipr _jan_dec2013.pdf). [5]. oecd, “meat consumption (indicator)” (2020), , doi:10.1787/fa290fd0-en. [6]. f. cobanoglu, k. kucukyilmaz, m. cinar, m. bozkurt, a. catli, e. bintas, comparing the profitability of organic and conventional broiler production. rev. bras. ciência avícola. 16, 403–410 (2014). [7]. m. sumague, o. del rosario, w. tan, d. santiago, f. flores, a. algar, l. mopera, v. dia, l. collado, physicochemical composition and functional properties of native chicken meats. philipp. j. sci. 145, 357–363 (2016). [8]. b. c. fielding, a. mnabisa, p. a. gouws, t. morris, antimicrobial-resistant klebsiella species isolated from free-range chicken samples in an informal settlement. arch. med. sci. 8, 39–42 (2012). [9]. x. c. liu, y. he, d. g. han, z. c. zhang, k. li, s. wang, l. x. xu, r. f. yan, x. r. li, detection of toxoplasma gondii in chicken and soil of chicken farms in nanjing region, china. infect. dis. poverty. 6 (2017), doi:10.1186/s40249-017-0277-3. [10]. g. schoeters, r. hoogenboom, contamination of free-range chicken eggs with dioxins and dioxin-like polychlorinated biphenyls. mol. nutr. food res. 50, 908–914 (2006). [11]. h. poulsen, zinc and copper as feed additives, growth factors or unwanted environmental factors. j. anim. feed sci. 7, 135–142 (1998). [12]. e. j. grace, g. r. macfarlane, assessment of the bioaccumulation of metals to chicken eggs from residential backyards. sci. total environ. 563–564, 256–260 (2016). [13]. j. yabe, s. m. m. nakayama, y. ikenaka, k. muzandu, k. choongo, g. mainda, m. kabeta, m. ishizuka, t. umemura, metal distribution in tissues of freerange chickens near a lead-zinc mine in kabwe, zambia. environ. toxicol. chem. 32, 189–192 (2013). [14]. y. hu, w. zhang, g. chen, h. cheng, s. tao, public health risk of trace metals in fresh chicken meat products on the food markets of a major production region in southern china. environ. pollut. 234, 667–676 (2018). [15]. v. l. m. medenilla, n. adri, r. a. espiritu, trace metal analysis of organic vegetables sold in some supermarkets in manila, philippines. carpathian j. food sci. technol. 10, 185–193 (2018). [16]. n. bortey-sam, s. m. m. nakayama, y. ikenaka, o. akoto, e. baidoo, y. b. yohannes, h. mizukawa, m. ishizuka, human health risks from metals and metalloid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 monique t. estrella, aileen fritz t. samson, rafael a. espiritu, comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines, food and environment safety, volume xxi, issue 4 – 2022, pag. 313 320 320 via consumption of food animals near gold mines in tarkwa, ghana: estimation of the daily intakes and target hazard quotients (thqs). ecotoxicol. environ. saf. 111, 160–167 (2015). [17]. philippines statistics authority, consumption of selected agricultural commodities in the philippines, (available at https://psa.gov.ph/sites/default/files/2015-2016 csac vol2.pdf). [18]. e. t. ogbomida, s. m. m. nakayama, n. bortey-sam, b. oroszlany, i. tongo, a. a. enuneku, o. ozekeke, m. o. ainerua, i. p. fasipe, l. i. ezemonye, h. mizukawa, y. ikenaka, m. ishizuka, accumulation patterns and risk assessment of metals and metalloid in muscle and offal of free-range chickens, cattle and goat in benin city, nigeria. ecotoxicol. environ. saf. 151, 98–108 (2018). [19]. j. kim, s. k. park, t. h. koo, trace elements and pollutants concentrations in shorebirds from yeongjong island, korea in the east asian-australian migration flyways. ecotoxicology. 16, 403–410 (2007). [20]. j. o. duruibe, m. o. c. ogwuegbu, j. egwurugwu, “heavy metal pollution and human biotoxic effects” (2007). [21]. s. a. abduljalee, m. shuhaimi-o, a. babji, assessment of trace metals contents in chicken (gallus gallus domesticus) and quail (coturnix coturnix japonica) tissues from selangor (malaysia). j. environ. sci. technol. 5, 441–451 (2012). [22]. who, trace elements in human nutrition and health (1996), (available at https://www.who.int/nutrition/publications/micr onutrients/9241561734/en/). [23]. a. l. wani, a. ara, j. a. usmani, lead toxicity: a review. interdiscip. toxicol. 8, 55– 64 (2015). 1. introduction 4. conclusion 25 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 1 2020, pag. 25 39 antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains *hayet messaoui 1 , salima roudj 1 , nour-eddine karam 1 1laboratory of microorganisms biology and biotechnology, department of biotechnology, faculty of natural sciences and life, oran1 university ahmed benbella, b.p. 1524, el m’naouer, 31000 oran, algeria. mess_hayet@yahoo.fr *corresponding author received 29th februaruy 2020, accepted 30th march 2020 abstract: the present study investigated the antioxidant and antibacterial properties of hydrolysates obtained from bovine whey proteins, after hydrolysis with lactobacillus plantarum strains isolated from ewe milk and lactobacillus brevis isolated from camel milk. the aim here was to valorize whey proteins and to constitute local collection of bacteria, with potentially interesting technological and biological features. the protein hydrolysis was determined by degree of hydrolysis (dh) and electrophoresis sds–page, and then optimized using temperature, ph, cells/whey proteins ratio and time incubation. antioxidant activity of hydrolysates was evaluated by 2,2-diphenyl-1-picrylhydrazyl (dpph) and nitric oxide (no) free radicals scavenging methods. the antibacterial activity was examined by the agar well diffusion assay. the results revealed that lb. plantarum lbbs2 and lb. plantarum lbm2 exhibited an optimum of hydrolysis of whey proteins at ph 7, at 30 °c, with (2/1) as ratio cells/whey proteins and after 48 h of incubation. under these conditions, the hydrolysates exhibited an antibacterial activity against gram positive and gram negative bacteria, including some pathogenic and spoilage microorganisms. meanwhile, scavenging radical properties by the same hydrolysates reached a maximum after 24 h. these results suggest that lb. plantarum lbbs2 and lb. plantarum lbm2 could be used as new potential adjunct bacteria with interesting proteolytic activity. moreover, milk whey which is the main by-product of food industry could be used for the production of molecules with a promising interest as antioxidants and antibacterials. keywords: lactobacillus plantarum, milk whey, hydrolysates, degree of hydrolysis, electrophoresis sds–page. 1. introduction lactic acid bacteria (lab) are widely utilized in the fermented food industry for their technological and health-promoting features, such as improving the sensorial properties of fermented products, as well as providing special characteristics to foods particularly the release of compounds with health-promoting effects [1]. lab have an efficient proteolytic system comprised of a cell envelope proteinase (cep) which initiates protein degradation, a transport system and various intracellular peptidases. in this regard, lab are able to degrade milk proteins [2, 3]. lactobacillus spp. are greatly employed in the food industry as starter cultures for the manufacture of fermented foods and beverages or as probiotics for incorporation into functional foods [4]. whey is the principal by-product of the food industry. it represents about 85–90% of milk volume and retains approximately 55% of milk nutrients [5]. previously, whey was considered as important http://www.fia.usv.ro/fiajournal mailto:mess_hayet@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 26 pollutant of the dairy industry, not only due to its great organic load, but also because of its important volume [6]. in fact, continued growth in the production of dairy products, especially cheese, has seen a concomitant increase in the volume of whey worldwide (currently >200 million tonnes per year) [7]. whey is a rich mixture of secreted proteins with wide variety of functional characteristics for biological, nutritional, and food purposes. it contains 20% of total milk proteins. nowadays, the perception of whey as a pollutant has changed with the discovery of its functional and bioactive properties, being considered as an additional product of cheese manufacture [5]. among the food proteins, most studies have been performed on whey proteins due to their high nutritional value, inexpensiveness, diverse functional properties and widespread application in food industry [8, 6]. oxidative stress, the increased production of reactive oxygen species (ros) and free radicals are incriminated in many degenerative diseases like cancer, atherosclerosis and diabetes [9]. improvement of the body’s antioxidant defences through food supplementation would appear to a practical initiative to reduce the level of oxidative stress. as obvious antioxidant feature of whey protein hydrolysates was detected in recent studies, it was good for their application in food industry as functional materials [9, 10]. furthermore, the presence of pathogenic and spoilage microorganisms in foods, combined with new consumer interest resulting in restrictions on the use of traditional antimicrobial chemical agents, puts pressure on food manufacturers to develop new preservatives [11]. during the past decade, growing attention has been focused on the production of antimicrobial peptides derived from whey proteins in order to reduce the need for antibiotics [8, 12]. various protein hydrolysates obtained through enzymatic catalysis demonstrate biological activities, which are usually associated with bioactive peptides. thus, production of hydrolysates can be an interesting approach to add value to whey. in fact, the bioactive peptides are inactive while encrypted in the sequence of original protein but can be released by: a) hydrolysis by digestive enzymes, b) by proteolytic microorganisms, and c) by the action of plant or microbial proteases [13]. in this context, protein-rich by-products might be utilized to produce bioactive peptides using specific protease producing microorganisms. to our knowledge, no study has been carried out on the antioxidant and antibacterial properties of bovine whey protein hydrolysates generated by ewe milk and camel milk lactobacillus strains. therefore, the present work was undertaken to study and compare, antioxidant and antibacterial activities of whey protein hydrolysates obtained with autochthonous lactobacillus strains isolated from ewe milk and camel milk. 2. materials and methods bacterial strains and growth conditions nine lactobacillus plantarum strains (coded lbm1, lbm2, lbs1, lbsc1, lbsc2, lbbs1, lbbs2, lbbg1, lbbg2), one strain of lactobacillus paracasei subsp. paracasei coded lbs2 isolated from ewe milk and one strain of lactobacillus brevis coded chtd27 isolated from camel milk, were supplied by the laboratory of microorganisms biology food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 27 and biotechnology, oran1 university (algeria). ewe milk was collected in different regions of algeria, and camel milk in tindouf area (southwest, algeria). the lab strains were maintained in reconstituted skimmed milk (10%) at 20 °c. standard cultures were prepared by inoculation of de man, rogosa and sharpe medium (mrs, fluka, saint louis, mo) broth at ph 5.7 with the frozen stocks followed by incubation at 30 °c for 18 h. qualitative determination of proteolytic activity the proteolytic activity of strains was estimated using agar medium with reconstituted skimmed milk (10% w/v) at a final concentration of 3%. a loopful of lactic strain cultures (optical density od600nm = 1) was spotted on skimmed milk agar and incubated at 30 °c for 48 h. proteolytic activity was detected by the occurrence of clear zones around the colonies [14]. preparation of whey proteins whey proteins were prepared from collected cow milk as reported by neyestani et al. [15] with some modifications. in brief, cream was skimmed from fresh cow milk by centrifugation (5000 g, 30 min, 4 °c) and the top lipid layer removed. the process was repeated three times. the casein was precipitated and separated from whey by acidification with 1 n hcl at ph 4.6. the precipitate was then removed by centrifugation (5000 g, 30 min, at 4 °c). the clear straw colored whey was obtained as a supernatant and filtered through whatman filter paper n°1 (whatman ltd., maidstone, uk). whey globulins were eliminated by precipitation with ammonium sulfate at 58% saturation (2.3 m) and at 4 °c in 24 h. the supernatant obtained from this step contained αlactalbumin (α-lb), β-lactoglobulin (β-lg), bovine serum albumin (bsa) and lactoferrin (lf). ph was adjusted to 7.0 using diluted 1 n naoh solution. the whey proteins were dialyzed against distilled water, lyophilized, stored at 20 °c and referred to as cow whey proteins (cwp). the purity of cwp was checked using sds-page. preparation of whey protein hydrolysates whey protein hydrolysates (wph) were prepared as described by pescuma et al. [16] with slight modifications. lactobacillus strains were inoculated in mrs broth medium with 5 mm cacl2. the inoculum was cultured in 300 ml mrs at 30 °c for 18 h. cells were collected by centrifugation (5000 g, 10 min, 4 °c) at the exponential growth phase (od600nm= 1), washed twice with 0.85% (w/v) saline solution supplemented with 10 mm cacl2, and suspended in 100 mm sodium phosphate (ph 7.0) with 5 mm cacl2. cell suspensions were kept at 37 °c for 30 min for amino acid starvation; non-proliferating cells of strains were incubated with 1% cwp (w/v) in a cells/cow whey proteins ratio (c/cwp) of 2/1 (v/v). cwp were previously dissolved in 100 mm sodium phosphate (ph 7.0) supplemented with 5 mm cacl2 and heated at 80 °c for 30 min (to avoid microbial contamination of whey). the cells-whey proteins mixture was incubated for 24 h at 37 °c; samples were withdrawn and then centrifuged (5000 g, 10 min, 4 °c). wph were analyzed by estimation of hydrolysis degree and by sds–page. determination of the tca-soluble proteins food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 28 the trichloroacetic acid (tca)-soluble proteins of the wph were determined by the method of folin-ciocalteu [17]. an aliquot of the hydrolysates was added to an equal volume of tca (12% w/v). the mixture was incubated for 30 min at room temperature then centrifuged (5000 g, 15 min, 4 °c). an aliquot (500 μl) of the supernatant (wph) was added to 2500 μl of naoh (0.2 m) and 250 μl of folinciocalteu reagent diluted twice with distilled water. the reaction mixture is stirred and left at room temperature for 10 minutes. the blue color that develops is measured spectrophotometrically (optizen, 2120 uv, mecasys co., ltd, korea) at 750 nm against blank (mixture without hydrolysate). the results were expressed as μg of tyrosine per ml referring to a standard curve of tyrosine (0–50 µg/ml). determination of the hydrolysis degree (dh) the degree of hydrolysis (dh) is defined as the percentage of peptide bonds cleaved. it was determined by using a modification of the method described by boudrant and cheftel [18] and was defined as follows, expressed as per cent: where dt corresponded to the amount of tca soluble peptides after whey protein hydrolysis expressed as tyrosine; d0 corresponded to soluble peptides of unhydrolysed whey proteins expressed as tyrosine; dmax corresponded to the maximum amount of tca soluble peptides expressed as tyrosine and determined after hydrolysis of the whey protein solution by hcl (6 n) at 110 °c for 24 h [19]. electrophoresis (sds-page) the samples from cwp hydrolysis were examined by polyacrylamide-sds gel electrophoresis as described by laemmli [20] using page mini vertical protein electrophoresis system (cleaver scientific ltd, uk). the polyacrylamide gels consisted of 17% resolving gel and 5% stacking gel. t0, t24h samples for each strain as well as sample of cwp non hydrolysed and the molecular weight marker (protein marker standard 11-190 kda, new england biolabs inc., ipswich, ma, usa) were loaded separately at a total running time of 3 h at 120 v. the gels were fixed in a solution of 12% tca for 15 min and then stained by using coomassie brilliant bleue r–250 stain solution (merck, saint louis, mo) for 5 h with 2–3 changes of the destaining solution. optimization of the hydrolysis conditions hydrolysis of cwp by the strains was conducted as described by pescuma et al. [16] under different conditions of temperature, ph, c/cwp ratio and time hydrolysis. incubation temperature effect the effect of incubation temperature was determined in 10 mm phosphate buffer (ph 7.0) in the range of 30 to 45 °c with a c/cwp ratio of 2/1 (v/v). samples were withdrawn after 24 h and 48 h of incubation and then centrifuged (5000 g, 10 min, 4 °c). the hydrolysates have been analyzed by estimation of hydrolysis degree and by sds–page. ph effect the effect of ph on the hydrolysis of cwp was examined in the ph range of 6 to 8 by using different buffers: acetate buffer 0.2 m, phosphate buffer 0.2 m, tris (hydroxymethyl) aminomethane-hcl buffer 0.2 m. the cells-whey protein food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 29 mixtures were incubated at 30 °c with a c/cwp ratio of 2/1(v/v). samples were pulled out after 24 h, 48 h of incubation, centrifuged (5000 g, 10 min, 4 °c) and analyzed. c/cwp ratio effect the enzymatic process was carried out at three different c/cwp ratios (1/1, 1/2 and 2/1). the relationship between the hydrolysis of cwp by the strains and the cells/whey proteins ratio was studied at 30 °c and ph 7. samples were withdrawn after 24 h and 48 h of incubation, centrifuged (5000 g, 10 min, 4 °c) and then analyzed. antioxidant activity by dpph radical scavenging assay the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity of the hydrolysates was determined according to the method described by lin et al. [10] with minor modifications. at first, 2744 µl of methanolic dpph solution (0.2 mm) were added to 56 µl aliquot of hydrolysate solution or phosphate buffer (control). the mixture was shaken vigorously and kept under dark at ambient temperature for 30 min. absorbance of each sample and control was read at 517 nm against blank by using a uv-visible spectrophotometer (optizen, 2120 uv, mecasys co., ltd, korea). the antioxidant activity of hydrolysates was estimated by comparison with ascorbic acid which is known as natural antioxidant. the dpph radical scavenging activity, expressed as the percentage of inhibition, was calculated by the following equation: where ac is the absorbance of the control and as is the absorbance of the sample. nitric oxide radical scavenging activity the effect of hydrolysates on nitric oxide (no) radical scavenging was investigated by using a sodium nitroprusside (snp) generating no system. briefly, 100 mm of snp was dissolved in phosphate buffered saline (pbs) ph 7.4. the tested hydrolysates were added to snp (10 mm) in pbs in a final volume of 2 ml and then incubated at 25 °c for 150 min. a control experiment without tested hydrolysates was conducted in an identical manner. after incubation, 1.0 ml of samples was diluted with 1.0 ml of griess reagent (1% sulphanilamide and 0.1% naphthyletylenediamine dihydrochloride in 2% h3po4) [21]. ascorbic acid was used as a positive control. the mixtures were kept under dark at room temperature for 30 min, and then the absorbance measured at 540 nm against the corresponding blank solutions. the percentage of no inhibition was calculated by the following equation: where ac is the absorbance of the control and as is the absorbance of the sample. assay for antibacterial activity the antibacterial activity was tested by using a well diffusion method in agar [22]. the indicator strains used to detect the antibacterial activity were staphylococcus aureus 25923, pseudomonas aeruginosa 27853, escherichia coli 25922 (from pasteur institute, algeria), ten (10) bacteria: salmonella typhi, serratia marcescens, proteus mirabilis, citrobacter freundii, klebsiella pneumoniae, acinetobacter baumanii, bacillus subtilis, bacillus cereus, enterobacter aerogenes and enterococcus faecium (isolated and identified at our laboratory). to check the antibacterial activity, target strains were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 30 grown in nutrient broth overnight at 37 °c. the plates were further overlaid with soft nutrient agar (0.75% w/v agar) inoculated at 1% (v/v) with cultures of indicator strains adjusted to od600nm= 1. wells of 5 mm diameter were cut with sterile well borer into these agar plates. 50 μl of wph (t0, t24h and t48h of each strain) previously filtered through 0.22 μm pore-size filters were placed into each well. plates were stored at 4 °c for overnight to permit diffusion of samples, and then incubated at 37 °c for 24 h. the appearance of a clear halo around the wells was measured and presented as inhibition zones (mm). statistical analyses: all experiments were carried out in triplicate. the results were expressed as mean ± standard error (se). mean separation and significance were analyzed using the ibm spss® software (spss statistical software, inc., chicago, il, usa). one-way analysis of variance (anova) was carried out to discriminate among the means. tukey and studentnewman-keuls procedures were used as post-hoc tests. differences between samples were considered as significant at p < 0.05. 3. results and discussion proteolytic activity of strains the proteolytic systems of lactic acid bacteria are important means of making free amino acids and peptides from milk proteins for growth. in this study a total of 11 lactobacillus strains were screened for proteolysis. the results show that all the strains exhibited proteolytic activity (figure 1). proteolytic activity has been reported several times for lab, including enterococcus, lactobacillus, lactococcus, and pediococcus [23]. the lactobacillus genus, produce proteases that can hydrolyze αand β-casein thus, having a direct effect on the biochemistry of dairy products. furthermore, the hydrolysis of proteins in milk (i.e., casein, β-lg, and αlb) produces small peptides, which have been suggested to present biological activities [24]. however, the strains were different in the efficiency proteolysis. the h/c ratio varies from 1.24±0.028 (lb. paracasei subsp. paracasei lbs2) to 2.65±0.028 (lb.brevis chtd27) and 2.71±0.042 (lb. plantarum lbbs2) which represent the highest values (p˂0.05) (figure 1). in fact, variability in proteolysis is commonly reported for isolates of lactic acid bacteria from natural sources [25]. each microorganism used in food fermentation has different capacities to produce enzymes that hydrolyze proteins in different conditions [26]. based on this screening, the strains lb. plantarum lbm2, lb. plantarum lbbs2 from ewe milk, and lb.brevis chtd27 from camel milk which have displayed the greatest proteolytic activity among the strains tested (p˂0.05), were further subjected to whey protein hydrolysis and whether potentially healthful bioactive peptides could be produced. ability of strains to hydrolyse whey proteins the hydrolysis reaction was carried out on sodium phosphate at 37 °c, ph 7.0, with ratio c/cwp of 2/1 for 24 h. to compare the extent of hydrolysis, whey protein hydrolysis in terms of degree of hydrolysis was determined by measuring amount of amino acids and peptides liberated, using the folin-ciocalteu reagent. the results showed that cwp were degraded by all the strains studied; however, the efficiency of hydrolysis was strain dependant. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 31 fig. 1. proteolytic activity of whole cells on milk-agar (3%) medium (a); halo/colony values (b) obtained at 30 °c after 48 h of incubation. bars represent the standard error from triplicate determinations. values with different letters (a-g) indicate results significantly different between the strains (p < 0.05). as shown in figure 2, the dh value in wph generated by lb. plantarum lbbs2 (24.92%±0.61) was significantly higher than lb. plantarum lbm2 (16.51%±0.39) and lb. brevis chtd27 (10.46%±0.48) which showed the lowest rate of hydrolysis (p<0.05). fig 2. dh values of the hydrolysates obtained by the strains at 37 °c, ph 7 with ratio c/cwp (2/1) after 24 h of incubation. bars represent the standard error from triplicate determinations. values with different letters (a-c) indicate significant differences between hydrolysates (p<0.05). previous research specified that the activity of proteinases and peptidases varied among lactic acid bacteria species [27, 28]. nevertheless, in previous research, the strain lb. brevis chtd27 from camel milk which is weakly proteolytic on whey proteins, was able to cleave to a greater extent caseins. this better performance on caseins could be due to protease specificity of this bacterium [29]. electrophoresis sds-page revealed different peptide profiles from cwp hydrolysis according to the strain tested. the electrophoresis diagram (figure 3) results of cwp (lane 2) showed four bands i.e. lf (88,000 kda), bsa (69,000 kda), βlg (18,400 kda) and α-lb (14,300 kda). hydrolysis revealed a decrease in protein intensity at t24h compared to t0 for the strains lb. plantarum lbm2 (lanes 5, 6) and lb. plantarum lbbs2 (lanes 7, 8) but no changes were observed in the intensity of the proteins for lb. brevis chtd27 (lanes 3, 4). these data were consistent with earlier results of proteolytic activity by assessment of dh. several studies revealed that an increase in dh of the proteins decreased their molecular size by creating small peptides [9, 24]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 32 fig 3. sds-page analysis of cwp hydrolysed by the strains at 37 °c, ph 7 with ratio c/cwp (2/1) after 24 h of incubation. (1) molecular weight marker ; (2) non hydrolysed cwp ; (3) and (4) hydrolysates obtained with the strain chtd27 at t0, t24h respectively ; (5) and (6) hydrolysates obtained with the strain lbm2 at t0, t24h respectively ; (7) and (8) hydrolysates obtained with the strain lbbs2 at t0, t24h respectively. optimization of hydrolysis of whey proteins by the strains in order to enrich hydrolysates with potentially bioactive peptides, cwp hydrolysing ability of the strains was optimized under different conditions. results proved that hydrolysis temperature, ph, c/cwp ratio and incubation time had a great effect on the dh of whey proteins. the strains exhibited activity at temperatures ranging from 30 °c to 40 °c with an optimum at 30 °c (p<0.05) (table 1). guo et al. [28] also reported an optimum temperature of 30 °c for hydrolysis of whey proteins by lb. helveticus lb13. this could be explained by the fact that an overly high temperature might have caused proteinase denaturation and lowered enzyme activity. the effect of ph on the hydrolysis was examined for the ph range 6 to 8. activity was maximal at ph 7 (p<0.05). pan et al. [30] also optimized the proteolytic conditions of lb. helveticus jcm1004 at ph 6.5–7.0. the ratio c/cwp of 2/1 revealed optimum hydrolysis of whey proteins by the strains (p<0.05). the increase of c/cwp ratio resulted in an increase in dh of whey proteins. the results could be due to greater hydrolysis of the proteins when more proteases were added [31]. the incubation time also influenced the degradation extent of whey proteins which was maximal after 48 h (p<0.05). similar results were observed for the strains lb. acidophilus crl 636, lb. delbrueckii subsp. bulgaricus crl 656 and streptococcus thermophilus crl 804 when the incubation period on whey protein concentrate was extended [32]. under optimal conditions, the dh values in whey protein hydrolysates obtained with lb. plantarum lbbs2 and lb. plantarum lbm2 were significantly higher than the strain lb. brevis chtd27 which showed the lowest rate of hydrolysis (p<0.05). analysis by sds-page revealed different profiles (data not shown) according to the strains and parameters studied. the peptides pattern produced from cwp hydrolysis differed among the lactobacillus strains used, suggesting different protease specificities. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 33 table 1. effect of temperature, ph, ratio cells (c)/cow whey proteins (cwp) and time incubation on hydrolysis of cwp by the strains chtd27, lbm2 and lbbs2, expressed by measurement of dh (%) (mean ± standard error, n=3). chtd27 lbm2 lbbs2 24 h 48 h 24 h 48 h 24 h 48 h 30 °c 18.01±0.47b 28.66±1.42a 38.33±0.56d 64.71±0.75a 39.80±1.04cd 82.24±0.47a 37 °c 10.80±0.85c 19.49±0.85b 17.19±1.41g 38.98±0.75d 34.88±0.75e 51.27±0.56b 40 °c 06.37±0.47d 12.93±1.04c 11.95±0.56i 16.21±0.47g 22.93±1.13i 38.16±0.47d 45 °c 04.08±0.47d 06.71±0.37d 10.80±0.75i 15.06±0.94gh 14.89±0.66k 30.62±0.47f ph 6 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 00.00±0.00l 00.00±0.00l ph 7 18.83±1.70b 31.12±1.89a 38.00±0.66d 58.32±0.37c 35.21±0.85e 80.28±0.94a ph 7.5 00.00±0.00e 00.00±0.00e 21.12±0.38f 33.58±1.41e 27.51±0.28h 41.33±0.66c ph 8 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 19.32±0.65j 35.38±0.94e c/cwp 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 0.00±0.00l 00.00±0.00l c/2cwp 00.00±0.00e 06.38±0.85d 13.42±0.85hi 20.79±1.23f 16.53±0.37k 28.98±0.85fg 2c/cwp 16.21±2.08b 28.33±0.8a 39.15±0.37d 60.77±0.56b 36.03±0.28e 81.09±0.66a different letters (a, b…) in hydrolysates of the same strain indicate significant differences at different conditions of hydrolysis (p < 0.05). results were related to the degree of hydrolysis of cwp upon hydrolysis by the strains. as depicted in figure 4, at 30 °c, ph 7, ratio c/cwp (2/1) and at 48 h of hydrolysis, the strain lb. plantarum lbbs2 exhibited the most important activity. the protein bands became lighter at t48h resulting in higher hydrolysis. furthermore, small fragments appeared with molecular weights lower than lf and bsa. this is due to the production of some peptides obtained after cwp hydrolysis. the strain lb. plantarum lbm2 showed less pronounced hydrolysis than lb. plantarum lbbs2, whereas the strain lb.brevis chtd27 revealed thicker protein bands which are indicatives of lower hydrolysis. these results point to a different proteolytic behavior for the strains. antioxidant properties of whey protein hydrolysates (wph) the results indicated that all wph reduced the dpph radical to a yellow-coloured compound, manifestly because of the dpph radical accepting an electron or hydrogen to become a stable molecular. however, antioxidant capacity was strainspecific and reached to maximum at 24 h of hydrolysis compared with intact whey proteins (t0) for all the strains. as shown in figure 5, wph derived from lb. plantarum lbbs2 resulted in significantly slightly higher antioxidant capacity (65.98%±0.11) compared to antioxidant activity of ascorbic acid (62.16%±2.69), followed by lb. plantarum lbm2 (55.59%±1.30), whereas wph from lb.brevis chtd27 exhibited the lowest food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 34 (33.99%±2.42) antioxidant capacity (p< 0.05). fig. 4. sds-page analysis of cwp hydrolysed by the strains at optimal conditions: 30 °c, ph 7, with ratio c/cwp (2/1) after 48 h of incubation. (1) non hydrolysed cwp; (2) and (3) hydrolysates obtained with the strain chtd27 at t0, t48h respectively; (4) and (5) hydrolysates obtained with the strain lbm2 at t0, t48h respectively; (6) and (7) hydrolysates obtained with the strain lbbs2 at t0, t48h respectively. the results of no radical scavenging assay were similar to those of dpph radical scavenging activity. at 24 h of hydrolysis, wph from lb. plantarum lbbs2 showed significantly the highest antioxidant activity (57.41%±1.52) similar to ascorbic acid (61.71%±0.98), followed by lb. plantarum lbm2 (48.29%±0.60), while wph from lb.brevis chtd27 displayed the lowest radical scavenging properties (37.61%±1.10) (p<0.05) (figure 5). it was demonstrated that lb. plantarum strains screened from conventional fermented food possess several functional features, including antioxidant properties [33]. the results observed in our experiments are in accordance with those reported by lin et al. [10] who found that the antioxidant activity of whey protein concentrate solution was improved by enzymatic hydrolysis. this was in agreement with observations of other results that indicated a higher dpph radical scavenging activity for an enzymatically-prepared whey protein hydrolysates than whey protein isolates [34]. the increased antioxidant activity of samples could be attributed to the hydrolysis of proteins/peptides by the proteases of lactobacillus strains. non hydrolysed whey proteins showed a relatively low antioxidant activity. these results relating to the antioxidant activity of whey proteins are consistent with previous studies which reported that whey is essentially consisting of polar antioxidant compounds [35]. however, the radical scavenging activity of hydrolysates decreased for all strains upon further hydrolysis and was not directly connected to dh after 24 h (figure 5). this suggests that high dh may result in further degradation of antioxidant peptides into short peptides with less activity or none [24]. these results were in agreement with the findings of virtanen et al. [9] on 25 strains of lab which showed that the radical scavenging activity is more dependent on specific proteolytic enzymes of bacterial strains than on high proteolytic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 35 state of strains. the ability of protein hydrolysates to inhibit damaging changes caused by lipid oxidation seems to be related to the nature and the composition of the various peptide fractions produced, depending on the protease specificity [36]. antioxidative properties of the peptides are related to their composition, structure, and hydrophobicity [6]. according to the results obtained in our work, the development of antioxidative activity was strain dependent and generally enhanced during hydrolysis but was not directly correlated with hydrolysis time [9]. fig. 5. antioxidant activities of cwp hydrolysates expressed as percentage inhibition of dpph (a), and no (b) radicals. error bars represent the standard error of the mean of triplicate experiments. values with different letters (a-f) indicate results significantly different (p < 0.05). antibacterial activity as depicted in table 2, no inhibitory activity was observed nor for nonhydrolysed whey proteins (t0) nor for the hydrolysates at t24h for all the strains. nonethless, hydrolysates obtained after 48h from the strains lb. plantarum lbm2 and lbbs2 exhibited growth inhibition against the target strains tested except bacillus subtilis that was not affected. salami et al. [8] noted that hydrolysis of cow whey proteins showed a tendency for increased antimicrobial potential when compared to non-hydrolysed whey proteins. furthermore, the substrate specificities of ceps determine the pools of generated peptides, which are responsible for the functional activities exerted [24]. wph derived from the strain lb. plantarum lbm2 showed antagonistic effects against 7 tested strains (2 gram positive and 5 gram negative), wph from lb. plantarum lbbs2 were able to inhibit 6 target microorganisms (1 gram positive and 5 gram negative), whereas hydrolysates obtained with lb. brevis chtd27 were unable to inhibit the growth of the thirteen target bacteria assayed. furthermore, the highest inhibition halo zones were obtained with wph from lb. plantarum lbbs2. this could be related to its proteolytic activity which was significantly the most important (p < 0.05) (table 2). molecular distribution profiles obtained at optimal conditions showed that hydrolysates obtained by the strains lb. plantarum lbbs2 and lbm2 which exhibited antibacterial inhibition also possessed a higher proportion of peptides, in contrast with the strain lb. brevis chtd27 (figure 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 36 table 2. antibacterial activity of hydrolysates obtained from the strains at t0, t24h and t48h against gram positive and gram negative bacteria. samples tested microorganisms chtd27 lbm2 lbbs2 t0 t24h t48h t0 t24h t48h t0 t24h t48h gram + staphylococcus aureus 25923 na na na na na na na na 12.95±0.086a bacillus subtilis na na na na na na na na na bacillus cereus na na na na na 07.60±0.17d na na na enterococcus faecium na na na na na 08.94±0.20c na na na gram pseudomonas aeruginosa 27853 na na na na na 07.8±0.11d na na na acinetobacter baumanii na na na na na na na na 09.36±0.069c escherichia coli 25922 na na na na na 07.86±0.054d na na na salmonella typhi na na na na na 06.85±0.17e na na na proteus mirabilis na na na na na na na na 09.15±0.11c citrobacter freundii na na na na na 07.96±0.16d na na na klebsiella pneumoniae na na na na na na na na 10.71±0.23b serratia marcescens na na na na na 09.55±0.11c na na 12.35±0.13a enterobacter aerogenes na na na na na na na na 12.84±0.12a results are expressed as the mean of the diameter of the inhibition zone in mm±standard error, n=3. different letters (a-e) indicate significant differences between hydrolysates (p<0.05). na: no antibacterial activity thus, the pattern of antibacterial activity observed for lb. plantarum lbbs2 and lb. plantarum lbm2 strains could be associated with their proteolytic activity on whey proteins, since most antibacterial capacity accords with the greatest proteolytic activity and the increase of peptides production. pellegrini et al. [37] reported that proteolytic digestion of bovine β-lg by trypsin generates four peptide fragments with bactericidal activity. nevertheless, the most studied antimicrobial peptides are the lactoferricins derived from lf. the antimicrobial activity of lactoferricin seems to be related with the net positive charge of the peptides. these cationic peptides kill sensitive microorganisms by increasing cell membrane permeability [6]. indeed, the difference in amino acid sequence, charge distribution, net charge, size, and amphipathicity as well as secondary structure could be responsible for the differential behavior of antibacterial peptides against bacteria [38]. 4. conclusion in this study, whey proteins extracted from cow milk were used in order to analyze their degradation by strains of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 37 lactobacillus isolated from ewe milk and camel milk. weak hydrolysis of whey proteins was noted for the camel milk strain lb. brevis chtd27. however, interesting rate of hydrolysis was observed for two ewe milk strains lb. plantarum lbbs2 and lb. plantarum lbm2, suggesting the potential interest of these strains. optimum of hydrolysis of bovine whey proteins was obtained at ph 7, 30 °c, with ratio cells/whey proteins of (2/1) and after 48 h of incubation. hydrolysates derived from lb. plantarum lbbs2 and lb. plantarum lbm2 exhibited antioxidant and antibacterial activities suggesting that proteases of these strains have the ability to release bioactive peptides from cow whey proteins. at optimal conditions, hydrolysates from the strains lb. plantarum lbbs2 and lb. plantarum lbm2 have displayed a large activity spectrum towards different gram positive and gram negative bacteria. however, the highest antioxidant activity was obtained from these same hydrolysates at 24 h. based on these interesting properties, whey proteins could be used for the production of molecules with a promising interest as antibacterials and antioxidants. the strains lb. plantarum lbbs2 and lb. plantarum lbm2 could represent promising candidates to be used as potential food biopreservatives. meanwhile, additional studies are needed to prove the safety of these strains, to identify and characterize peptides produced and nutraceutical properties. 5. references [1]. das d. & goyal, a., lactic acid bacteria in food industry. microorganisms in sustainable agriculture and biotechnology, springer, pp. 757772, (2012). [2]. hebert e. m., mamone g., picariello g., raya r. r., savoy g., ferranti p., addeo f., characterization of the pattern of αs1and β-casein breakdown and release of a bioactive peptide by a cell envelope proteinase from lactobacillus delbrueckii subsp. lactis crl 581. applied and environmental microbiology, 74(12): 3682-3689, (2008). [3]. do carmo a., da silva d., de oliveira m., borges a., de carvalho a., de moraes c., genes involved in protein metabolism of the probiotic lactic acid bacterium lactobacillus delbrueckii ufv h2b20. beneficial microbes, 2(3): 209-220, (2011). [4]. leroy f. & de vuyst l., lactic acid bacteria as functional starter cultures for the food fermentation industry. trends in food science and technology, 15(2): 67-78, (2004). [5]. smithers g. w., whey and whey proteinsfrom ‘gutter-to-gold’. international dairy journal, 18(7): 695-704, (2008). [6]. mann b., athira s., sharma r., kumar r., sarkar p., bioactive peptides from whey proteins. whey proteins, elsevier, pp. 519547, (2019). [7]. smithers g. w., whey-ing up the optionsyesterday, today and tomorrow. international dairy journal, 48: 2-14, (2015). [8]. salami m., moosavi-movahedi a. a., ehsani m. r., yousefi r., haertle t., chobert j-m., razavi s. h., henrich r., balalaie s., ebadi s. a., pourtakdoost s., niasari-naslaji a., improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis. journal of agricultural and food chemistry, 58(6): 3297-3302, (2010). [9]. virtanen t., pihlanto a., akkanen s., korhonen h., development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. journal of applied microbiology, 102(1): 106-115, (2007). [10]. lin s., tian w., li h., cao j., jiang w., improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. international journal of food science and technology, 47(10): 2045-2051, (2012). [11]. elbarbary h. a., ejima a., sato k., generation of antibacterial peptides from crude cheese whey using pepsin and rennet enzymes at various ph conditions. journal of the science of food and agriculture, 99(2): 555-563, ( 2019). [12]. dullius a., goettert m. i., de souza, c. f. v., whey protein hydrolysates as a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 38 source of bioactive peptides for functional foodsbiotechnological facilitation of industrial scale-up. journal of functional foods, 42: 58-74, (2018). [13]. korhonen h., pihlanto a., bioactive peptides: production and functionality. international dairy journal, 16(9): 945-960, (2006). [14]. fransen n., o'connell m., arendt e., a modified agar medium for the screening of proteolytic activity of starter cultures for meat fermentation purposes. international journal of food microbiology, 36(2-3): 235-239, (1997). [15]. neyestani t. r., djalali m., pezeshki m., isolation of α-lactalbumin, βlactoglobulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. protein expression and purification, 29(2): 202-208, (2003). [16]. pescuma m., hebert e. m., haertle t., chobert j-m., mozzi f., de valdez g. f., lactobacillus delbrueckii subsp. bulgaricus crl 454 cleaves allergenic peptides of β-lactoglobulin. food chemistry, 170: 407-414, (2015). [17]. folin o. & ciocalteu v., on tyrosine and tryptophane determinations in proteins. journal of biological chemistry, 73(2): 627-650, (1927). [18]. boudrant j. & cheftel c., continuous proteolysis with a stabilized protease. ii. continuous experiments. biotechnology and bioengineering, 18(12): 1735-1749, (1976). [19]. baek h. & cadwallader k. r., enzymatic hydrolysis of crayfish processing by‐products. journal of food science, 60(5): 929935, (1995). [20]. laemmli u. k., cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature, 227(5259): 680-685, (1970). [21]. sumanont y., murakami y., tohda m., vajragupta o., matsumoto k., watanabe h., evaluation of the nitric oxide radical scavenging activity of manganese complexes of curcumin and its derivative. biological and pharmaceutical bulletin, 27(2): 170-173, (2004). [22]. juillard v., laan h., kunji e., jeronimus-stratingh c. m., bruins a. p., konings w. n., the extracellular pi-type proteinase of lactococcus lactis hydrolyzes betacasein into more than one hundred different oligopeptides. journal of bacteriology, 177(12): 3472-3478, (1995). [23]. garcia-cano i., rocha-mendoza d., ortega-anaya j., wang k., kosmerl e., jimenez-flores r., lactic acid bacteria isolated from dairy products as potential producers of lipolytic, proteolytic and antibacterial proteins. applied microbiology and biotechnology, 103: 5243-5257, (2019). [24]. kliche t., li b., bockelmann w., habermann d., klempt m., de vrese m., wutkowski a., clawin-raedecker i., heller k. j., screening for proteolytically active lactic acid bacteria and bioactivity of peptide hydrolysates obtained with selected strains. applied microbiology and biotechnology, 101(20): 76217633, (2017). [25]. abdelgadir w. s., hamad s. h., moller p. l., jakobsen m., characterisation of the dominant microbiota of sudanese fermented milk rob. international dairy journal, 11(1-2): 6370, (2001). [26]. gurung n., ray s., bose s., rai v., a broader view: microbial enzymes and their relevance in industries, medicine, and beyond. biomed research international, 2013, (2013). [27]. shihata a. & shah n., proteolytic profiles of yogurt and probiotic bacteria. international dairy journal, 10(5-6): 401-408, (2000). [28]. guo z., wang j., yan l., chen w., liu x-m., zhang h-p., in vitro comparison of probiotic properties of lactobacillus casei zhang, a potential new probiotic, with selected probiotic strains. lwt-food science and technology, 42(10): 1640-1646, (2009). [29]. bounouala f. z., roudj s., karam n-e., recio i., miralles b., casein hydrolysates by lactobacillus brevis and lactococcus lactis proteases: peptide profile discriminates strain-dependent enzyme specificity. journal of agricultural and food chemistry, 65(42): 9324-9332, (2017). [30]. pan d., luo y., tanokura m., antihypertensive peptides from skimmed milk hydrolysate digested by cell-free extract of lactobacillus helveticus jcm1004. food chemistry, 91(1): 123-129, (2005). [31]. holzapfel w. h., haberer p., geisen r., bjorkroth j., schillinger, u., taxonomy and important features of probiotic microorganisms in food and nutrition. the american journal of clinical nutrition, 73(2): 365s-373s, (2001). [32]. pescuma m., hebert e. m., mozzi f., de valdez g. f., functional fermented wheyfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 hayet messaoui, salima roudj, nour-eddine karam, antioxidant and antibacterial activities of bovine whey proteins hydrolysed with selected lactobacillus strains, food and environment safety, volume xix, issue 1 – 2020, pag. 25 – 39 39 based beverage using lactic acid bacteria. international journal of food microbiology, 141(1-2): 73-81, (2010). [33]. tang w., xing z., li c., wang j., wang y., molecular mechanisms and in vitro antioxidant effects of lactobacillus plantarum ma2. food chemistry. 221: 1642-1649, (2017). [34]. peng x., kong b., xia x., liu q., reducing and radical-scavenging activities of whey protein hydrolysates prepared with alcalase. international dairy journal, 20(5): 360-365, (2010). [35]. restani p., velona t., plebani a., ugazio a., poiesi c., muraro a., galli c. l., evaluation by sds‐page and immunoblotting of residual antigenicity in hydrolysed protein formulas. clinical and experimental allergy, 25(7): 651-658, (1995). [36]. kenny o., fitzgerald r., o’cuinn g., beresford t., jordan k., growth phase and growth medium effects on the peptidase activities of lactobacillus helveticus. international dairy journal, 13(7): 509-516, (2003). [37]. pellegrini a., dettling c., thomas u., hunziker p., isolation and characterization of four bactericidal domains in the bovine βlactoglobulin. biochimica et biophysica acta (bba)-general subjects, 1526(2): 131-140, (2001). [38]. brandelli a., daroit d. j., correa a. p. f., whey as a source of peptides with remarkable biological activities. food research international, 73: 149-161, (2015). 1. introduction 4. conclusion in this study, whey proteins extracted from cow milk were used in order to analyze their degradation by strains of lactobacillus isolated from ewe milk and camel milk. weak hydrolysis of whey proteins was noted for the camel milk strain lb. brevis cht... [1]. das d. & goyal, a., lactic acid bacteria in food industry. microorganisms in sustainable agriculture and biotechnology, springer, pp. 757-772, (2012). doi: https://doi.org/10.4316/fens.2021.036 333 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 333 342 estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs) *fatima zohra el kadi 1 , khedoudja kanoun 2 , asma maaz 3 , fatima zohra sarsar 1 , asma remil 1 , mohammed benali 1 1 biotoxicology laboratory, biology department , faculty of natural sciences and life ,djillali liabés university of sidi-bel-abbés, bp n°. 89 sidi bel abbes 22000 algeria, elkadifatimazohra@yahoo.fr 2 laboratory of molecular microbiology health and proteomics, biology department , faculty of natural sciences and life ,djillali liabés university of sidi-bel-abbés, bp n°. 89 sidi bel abbes 22000 algeria. 3 medical analysis central laboratory of the mother and child of sidi bel abbes hospital *correspaonding author received 26th july 2021, accepted 20th december 2021 abstract: the objective of this study was to evaluate the effects of subchronic exposure to nambs (90 days), on the quantitative hemogram and the thymus histology spleen and stomach in wistar rats. the study involved 24 female wistar rats, receiving increasing concentrations of 0.25%, 1% and 4% of nambs in drinking water, for 90 days compared to the control group receiving drinking water without nambs. at the end of the experiment, the control and experimental rats were sacrificed, the blood and organs were removed and the hematological parameters were assayed. the thymus, spleen and stomach were used for histological study. in all cases a value p <0.05 was considered significant. the results obtained show that, the group receiving 4% of nambs showed a significant decrease in the number of red blood cells and hemoglobin (p< 0.05), an increase in the number of white blood cells (p< 0.01) and the number of platelets were observed. the doses of 1% and 4% cause statistically significant changes of the erythrocyte indices (mcv, mchc, mchc); and of the leukocyte formula (lymphocytes, monocytes, granulocytes), in comparison with the control group. hyperplasia of the spleen and stomach in animals treated with 1% and 4% of nambs, was observed in comparison with those of control animals. in addition, histological examination revealed hyperplasia of the white pulp and inflammation of the spleen, in rats treated with 4% of nambs and inflammation of the gastric mucosa, in the groups receiving 1% and 4% of nambs dose. the results obtained indicate that, the subchronic ingestion of nambs above the adi, seems to cause alterations in some hematological parameters such as splenic and gastric histo-alterations in wistar rats. abbreviations: mchc: mean corpuscular hemoglobin concentration, mch: mean corpuscular hemoglobin, mcv: mean corpuscular volume, nambs: sodium metabisulfite, adi: acceptable daily intake. keywords: hemogram, histophysiology, sodium metabisulfite, subchronic, toxicity, wistar rat 1. introduction omnipresent in industrial food, additives are found in our plates in ever-increasing quantities. since sea salt, the first chemical preservative for meat and fish since antiquity, the substances used by humans to protect or improve his food have multiplied [1]. sulphites are used in food as additives and can also be found in some medicines [2,3].the european union has classified them as preservatives and assigned them an "e" code ranging from e 220 to e 228 [4,5]. sulfites are the result of the fermentation process, and therefore they form naturally in foods such as garlic and onions [6]. sulphites are generated in vivo by the http://www.fia.usv.ro/fiajournal mailto:elkadifatimazohra@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 334 degradation of sulfur amino acids, such as methionine and cysteine. they can also be generated by neutrophils polynuclear [7, 8]. in addition, many mammalian cells produce sulfites from h2s [9]. similarly, sulfites are allergenic additives, to which the majority of asthmatics are sensitive [10]. the sulfites adi (expressed as so2) is 0.7 mg / kg of body weight [11]. they are mainly metabolized in the liver by sulfite oxidase; to give sulfates [12], which are excreted in the urine [13]. this enzyme is present in the mitochondria and in most tissues [14]. the organs with the highest sulfite-oxidase activity are the liver, kidneys, and heart, while the lowest are the brain, spleen, lungs, and testes [14,15]. the sulfites toxicity in mammals has been studied by many researchers. these studies have described the sulfites toxicological effects on animal organs with in-vivo and in-vitro tests [16, 17, 18, 19, 20, 21, 22, 23]. our previous work has shown that the subchronic intake of 1% and 4% nambs seems to alter immune function, biochemical: (calcium, uric acid, urea, creatinine, transaminases), hematological: (hemoglobin, red blood cells and white blood cells) and physiological parameters in the wistar rat [24]. another complementary study performed by el kadi et al, 2017[25], shows the effect of sodium metabisulphite in the genesis of an oxidative stress state, characterized by an overproduction of mda (malondialdehyde), a significant decrease in the antioxidant enzymes activities of (gpx, sod, cat): (glutathione peroxidase, superoxide dismutase, catalase) and thiol groups in the stomach and the spleen of the wistar rat. in this work, we are interested in studying the effect of this synthetic preservative in doses (0.25%, 1%, 4%), that can be provided via the diet on certain haematological parameters and on the histological structure of certain target organs (thymus, spleen and stomach). 2. materials and methods 2.1. materials 2.1.1. chemical material the chosen test substance is sodium metabisulfite, which was procured from biochem chemopharma international, with a purity level of 95%. the substance appears as a very fine white powder with a pungent odor and easily soluble in water. the specifications of this product are listed in (table 1). 2.1.2. choice of the administered dose the administration mode of nambs is in drinking water solution, to avoid partial destruction of thiamine in the diet by so2 before ingestion. the orally consumption of 0.25% nambs is equivalent to 72 mg/kg of so3 -2, with these data, the world health organization (who) has established an acceptable daily intake which is 0.7 mg/kg of body weight taking into account, the safety factor 100 [27]. the chosen doses in this study are based on the value of the adi [28]. in addition, these doses were selected with reference to certain studies, which analyze the sulfites toxicity at high doses [29, 30]. 2.1.3. experimental groups and treatment the experiments were carried out on 24 rats of wistar strain provided by the breeding center of the algiers pasteur institute. they are 8-week-old female rats, weighing an average (130.41±22.68gr), food and water were provided ad libitum. the animals were divided into 4 lots at a rate of 6 rats per lot. each lot received the test substance at different doses for 90 days, by varying the concentration of the prepared solution. the different lots were distributed as follows: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 335  1st control lot: rats receiving water without nambs.  2nd lot: rats receiving a dose of 0.25% of nambs = adi.  3rd lot: rats receiving a dose of 1% nambs = 4 times the adi.  4th lot: rats receiving a dose of 4% nambs = 16 times the adi. table 1 sodium metabisulfite e223 specifications, according to european commission directive 2008/84/ec [26] 2.2. methods 2.2.1. hematological parameters at the end of the experiment (d 90), the animals were anaesthetized under urethane, after 16 hours of fasting. blood was collected by puncture in the abdominal aorta. the blood was collected in edta (ethylen-diamino-tetracetic-acid) tubes. the analyses of the haematological parameters were performed on the same day of the collection (mcv: mean corpuscular volume, mchc: mean corspuscular haemoglobin concentration, mch: mean corpuscular haemoglobin, plt: platelets, lym: lymphocytes, mon: monocytes, gra: granulocytes) using an advia 60 ct (bayer dc brand) automated system. 2.2.2. histological parameters from the first minutes after the animal's sacrifice, the organs (thymus, spleen and stomach) were carefully removed and rinsed with 0.9% of nacl. quickly, part of the thymus, spleen and stomach were immersed in the 10% buffered formalin solution for their fixation for histopathological examination. tissue sections (3µm thick) were cut and stained with hematoxylin and eosin; and observed under an olympus microscope (zeiss, axiostar plus, oberkochen, germany). 2.2.3. statistical analysisthe results are expressed as means and their standard error (x±es). the comparison of means is performed using the t "student" test. the differences are considered significant at p < 0.05. 3. results and discussion 3.1. hematological study the results obtained reflect a certain form of hematopoiesis dysfunction, by a quantitative disturbance of the circulating blood. these results may lead us to suggest that, sulfites are capable of exerting continuous pressure on the hematopoiesis specifications specifications biochem chemopharma name of the product chemical name synonyms ue reference code cas einecs chemical formula molecular weight purity (na2s2o5) insoluble material heavy metals (expressed in pb) chloride iron arsenic synthetic food preservative sodium metabisulfite disodium pentaoxodisulfate; sodium pyrosulfite; sodium disulfite e223 7681-57-4 231-673-0 na2 s2 o5 190.11 min 95%. max 0.005% max 0.0005% max 0.005% max 0.0005% max 0.0001 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 336 and thereby cause hemopathy. according to our results, the analysis of the haemogram varied significantly (fig.1). fig 1 : serum levels of mcv, mchc and mch of control and treatment groups ** p<0.01 and ***p<.001: significant difference from controls. cg: control group; 0.25% g: group receiving 0.25% nambs; 1%g: group receiving 1% nambs; 4%g: group receiving 4% nambs. the evaluation of erythrocyte indices: mcv, mchc and mch showed statistically significant changes, when compared to the control lot; mcv indicates the volume of red blood cells and, is used to diagnose and classify anemia. a mcv>94fl/l reflects a macrocytic anemia observed in vitamin b12 deficiency and hepatopathies [31]. our study showed an increase in mcv value in the group treated with 1% nambs; and a decrease in the group receiving 4% of the test substance. the mchc measures the quantity, more precisely the mass of hemoglobin contained in a red blood cell. the results obtained were correlated with that of the mcv. this parameter increased at a dose of 1% and decreased at a dose of 4%. the mch corresponds to the hemoglobin charge per 100 ml of blood. it is the ratio of hemoglobin concentration to hematocrit. the mch is significantly increased in 1% g (fig.1). results indicate that the hemoleukocyte formula showed a decrease in lymphocytes; and an increase in monocytes and granulocytes in the group receiving the dose of 4% of nambs (fig.2). fig 2 : serum levels of lymphocytes, monocytes and granulocytes of control and treatment groups ** p<0.01 and ***p<.001: significant difference from controls. cg: control group; 0.25% g: group receiving 0.25% nambs; 1%g: group receiving 1% nambs; 4%g: group receiving 4% nambs. 0 20 40 60 80 100 c g 0,25% g 1% g 4% g lymphocytes % monocytes % granulocytes % food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 337 hyperleukocytosis may be the result of the immune system activation in response to infection, inflammation, and necrosis [32], other situations may also cause changes in white blood cell counts (wbcs) such as, emotional stress and dehydration [33]. these results correspond to the work carried out by etlik et al , 1997 [34], which indicates a statistically significant increase, in the number of leukocytes in 7 rats, exposed to (26.6 mg/m3) of sulphur dioxide 1 h/d for 45 days. moreover, till et al, 1972 [35], detected hyperleukocytosis in wistar rats receiving 6% of nambs for 56 days. 0 200 400 600 800 1000 1200 1400 1600 1800 cg 0,25% g 1% g 4% g p la te le ts ( 1 0 * 9 /l ) ** fig 3 : serum levels of platelets of control and treatment groups ** p<0.01 and ***p<.001: significant difference from controls. cg: control group; 0.25% g: group receiving 0.25% nambs; 1%g: group receiving 1% nambs; 4%g: group receiving 4% nambs. the platelet count revealed a very significant increase in 4% g (fig.3), which can be explained by a bone marrow disorder. similarly, this increase indicates a hypercoagulability risk. it has been shown that the bone marrow can be a target for certain toxins that can destroy its cells, resulting in a decrease in the number of red blood cells [36]. our results are in agreement with those carried out by gunnison et al , 1981 [37],who demonstrated that rats fed with a diet containing 6% of nambs for 21 days, became severely anemic, and this was due to the destruction of cyanocobalamin (b12 vitamin), in the diet or colon. likewise, roberts and buidinsky, 2000 [36] carried out a study in wistar rats, receiving high dietary levels of nambs (0%-8 %) for 10-56 days. anaemia was produced at the 2% dose. several inhalation studies show the oxidative effects on erythrocytes of sulfur dioxide exposure. according to dikmenoglu et al ,1991 [38], an increase in lipid peroxidation of erythrocytes was reported in 12 guinea pigs, exposed to 26.6 mg/m3of sulphur dioxide for 30 days. our results are not similar to those of etlik et al, 1997 [34], which indicate a significant increase in methemoglobin and sulfhemoglobin ratios, lipid peroxidation and increased erythrocyte fragility, as well as a statistically significant increase in erythrocyte count, hematocrit and hemoglobin in 7 rats exposed to 26.6 mg/m3 of sulfur dioxide 1 h/d for 45 days. 3.2. histological observation 3.2.1. thymic parenchyma the different doses of nambs do not cause any histological alteration of the *** food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 338 thymic parenchyma compared to the control group (fig .4). fig .4: histological sections of the thymus of control and experimental wistar rats (g×10). a: control; b: 0.25% of nambs; c: 1% of nambs; d: 4% of nambs cz : cortical zone (cortex); mz : medullary zone (medulla); ct: conjunctive tissue; cs : conjunctive septum; tl: thymic lobule. 3.2.2. splenic parenchyma administration of the 0.25% and 1% nambs doses during the 90 days didn’t cause any changes or alterations in the tissue architecture of the spleen. however, rats’s splenic tissue receiving 4% of nambs showed white pulp hyperplasia and polymorphic inflammatory infiltrates (fig.5). fig.5: histological sections of the spleen of control and experimental wistar rats a: control (g×10); b: 0.25% of nambs (g×10); c: 1% of nambs (g×10); d: 4% of nambs (g×40). cc: conjunctive capsule; rp: red pulp; wp: white pulp; ct: conjunctive tract with trabecular vein; ip: inflammatory process. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 339 3.2.3. gastric parenchyma histological examination confirms the presence in the gastric mucosa of the small inflammatory areas of animals treated with 1% of nambs. at the highest dose (4%), we will find the same lesions described above but with more inflammatory foci (fig.6). fig.6: histological sections of the gastric mucosa of control and experimental wistar rats a: control (g×10); b: 0.25% of nambs (g×40); c: 1% of nambs (g×40); d: 4% of nambs (g×40). mm: muscular mucosa; sm: submucosa with vessels; fg : fundic glands; ip : inflammatory process. the effect of nambs on biological parameters was considered as an indicator of tissue damage. the histological study didn’t reveal any tissue damage of thymus. on the other hand, inflammatory areas with white pulp hyperplasia (due to hyperfunction) were observed in the spleen of treated rats with 4% of nambs. in addition, inflammation of the stomach in the 1% and 4% dose groups were observed. these tissue disturbances reflect the appearance of an imbalance between the organism's antioxidant and pro-oxidant systems in favor of the latter. this imbalance confronts the organism to super exposure to reactive oxygen species, which always manifests itself by a decrease in tissue levels of free radical scavengers coumpounds such as, glutathione [39]. thus, free radicals can be diffused in the cytoplasm and across membranes to attack cellular components [40]. the attack of organic components cells (lipids, proteins, carbohydrates), allows the rapid transmission of free radicals and triggers severe pathologies up to animal death [41]. this confirms our results, particularly with regard to the inflammatory states observed in the splenic and gastric parenchyma of the animals. moreover, the sulfite oxidase (sox) activity in mammalian tissues shows a very significant difference in the same species. for example, the liver, kidney and heart tissues have high sox activity, while the brain, spleen and testes have very low activity [14, 15]. several researchers have intensively studied the gastrotoxic effect of sulfites. beems et al , 1982 [42], showed that, administration of 6% of sodium metabisulfite in male wistar rats for 12 weeks caused hyperplasia of the fundic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 340 mucosa glands. similarly, till et al , 1972 [35] concluded that, the incorporation of nambs (≥1%) in the diet of wistar rats for 8 weeks caused glandular hyperplasia, hemorrhage, ulceration and stomach necrosis with inflammation of stomach. according to hui et al , 1989[28], gastric lesions are noted in both (normal and sulfite oxidase-deficient) receiving 350 mg/kg bw/day of sulfur dioxide equivalent for 3-5 weeks; gastric lesions are more severe and more frequent in sulfite oxidase deficient rats. 4. conclusion in conclusion, the toxicity of nambs is dose-dependent, including all damage appearing with the highest dose (above the adi). all these results suggest that nambs has a very high toxic potential, because it disrupts some important biological functions such as hematopoiesis and gastric and splenic histological structure. 5. acknowledgments the authors declare that they have no conflicts of interest in relation to this article. 6. references [1].bourrier t., intolerances and allergies to colorants and additives. revue française d’allergologie et d’immunologie clinique, 46 (2):68-79. https://doi.org/10.1016/j.allerg.2005.12.002 , (2006). [2]. efsa., opinion of the scientific panel on dietetic products, nutrition and allergies on a request from the commission relating to the evaluation of allergenic foods for labelling purposes (request nº efsa-q-2003-016) (adopted on 19 february 2004) .the efsa journal, 32:1-197 .doi: https://doi.org/10.2903/j.efsa.2004.32 ,(2007). [3].taylor s l., bush r k., and nordlee ja., sulfites, in metcalfe d d., sampson h a., and simon ra ., food allergy : part 4 adverse reactions to foods and food additives. wiley-blackwell. 4th edition. isbn:978-1-44435816-2.section 29 : pp 353-368, (2011). [4].denil m., and lannoye p., guide to food additives precautions to be taken, paris. frisonroche, 2nd édition. pp 1-164, (2004). [5].jecfa., directory of standards for food additives. http://www.fao.org/ag/agn/jecfaadditives/ search.html? lang = fr ,(2009). [6]. mazza g., special crops rich in bioactive compounds beneficial to health, food research program, agriculture and agri-food. http://res2.agr.ca/parc-crapac/pubs/bioactives_f.htm , (2001). [7].mitsuhashi h., nojima y., tanaka t., ueki k., maezawa a., yano s., and naruse t., sulfite is released by human neutrophils in response to stimulation with lipopolysaccharide. j leukoc biol, 64 (5):595-599. doi: 10.1002/jlb.64.5.595, (1998). [8].wexler.,p., encyclopedia of toxicology. 2nd edition, (eds) elsevier.2000 pages, (2005). [9].mitsuhashi h., yamashita s., ikeuchi h., kuroiwa t., kaneko y., hiromura k., kazue u., and yoshihisa n., oxidative stress-dependent conversion of hydrogen sulfide to sulfite by activated neutrophils. shock, 24 (6):529-534. doi: 10.1097/01.shk.0000183393.83272.de, (2005). [10]. vally h., and thompson pj., role of sulphite in wine induced asthma: single dose and cumulative dose studies. thorax,56 (10) :763-769. doi: 10.1136/thorax.56.10.763 , (2001). [11].fao/who., expert committee of food additives sulfur dioxide.<http:// www.inchem.org/documents/jecfa/jeceval/jec_2215 .htm> accessed 12.11.07, (2007). [12]. constantin d., bini a., meletti e., moldeus p., monti d., and tomasi a., age-related differences in the metabolism of sulphite to sulphate and in the identification of sulphur trioxide radical in human polymorphonuclear leukocytes. mech ageing dev, 88 (1-2):95-109.doi: 10.1016/0047-6374(96)017289, (1996). [13].lauwerys r., irritating and asphyxiating gases and vapors. toxicologie industrielle et intoxications professionnelles, 3th ed. paris, masson, pp 388-391, (1990). [14]. cabre f., marin c., cascante m., and canela ei.,occurence and comparison of sulfite oxidase activity in mammalian tissues. biochem med metab biol, 43 (2):159-162.doi: 10.1016/0885-4505(90)90021-r, (1990). https://doi.org/10.1016/j.allerg.2005.12.002 http://res2.agr.ca/parc-crapac/pubs/bioactives_f.htm https://pubmed.ncbi.nlm.nih.gov/?term=naruse+t&cauthor_id=9823763 https://doi.org/10.1002/jlb.64.5.595 https://pubmed.ncbi.nlm.nih.gov/?term=nojima+y&cauthor_id=16317383 https://pubmed.ncbi.nlm.nih.gov/?term=nojima+y&cauthor_id=16317383 https://doi.org/10.1097/01.shk.0000183393.83272.de https://doi.org/10.1136/thorax.56.10.763 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 341 [15]. woo w h., yang h., wong kp., and barry h., sulphite oxidase gene expression in human brain and in other human and rat tissues. biochem biophys res commun, 305 (3) :619-623. https://doi.org/10.1016/s0006-291x(03)00833-7 , (2003). [16]. yargiçoglu p ., agar a., gümüslü s., bilmen s., and oguz y., age-related alterations in antioxidant enzymes, lipid peroxide levels, and somatosensory-evoked potentials: effect of sulfur dioxide. arch environ contam toxicol, 37 (4):554-560. doi: 10.1007/s002449900552,(1999). [17]. agar a., küçükatay v., yargiçoglu p., aktekin b., kipmenkorgun s., gümüslü d., and apaydin c., the effect of sulfur dioxide inhalation on visual evoked potentials, antioxidant status and lipid peroxidation in alloxan-induced diabetic rats. arch environ contam toxicol , 39 (2) :257-264. doi: 10.1007/s002440010103 , (2000). [18]. gümüslü s., bilmen s., korgun d k., yargiçoglu, p., and agar a., age-related changes in antioxidant enzyme activities and lipid peroxidation in lungs of control and sulfur dioxide exposed rats. free radic res,34(6) :621-627.doi: 10.1080/10715760100300511 ,(2001). [19]. meng z., oxidative damage of sulfur dioxide on various organs of mice: sulfur dioxide is a systemic oxidative damage agent. inhal toxicol, 15(2):181-195.doi: 10.1080/08958370304476, (2003). [20]. aydin s., yargicoglu p., derin n., aliciguzel y., abidin i., and agar a., the effect of chronic restraint stress and sulfite on visual evoked potentials (veps): relation to lipid peroxidation. food chem toxicol, 43(7) :10931101. doi: 10.1016/j.fct.2005.02.014 ,(2005). [21] .elmas o., aslan m., çaglar s., derin n., agar a., alicigüzel y., and yargiçoglu p., the prooxidant effect of sodium metabisulfite in rat liver and kidney. regul toxicol pharmacol, 42(1) :77-82. doi: 10.1016/j.yrtph.2005.01.010, (2005). [22].meng z., and liu y., cell morphological ultrastructural changes in various organs from mice exposed by inhalation to sulfur dioxide. inhal toxicol,19 (6-7) :543-551. doi: 10.1080/08958370701271373, (2007). [23]. li j., meng z., the role of sulfur dioxide as an endogenous gaseous vasoactive factor in synergy with nitric oxide. nitric oxide, 20 (3) :16674. doi: 10.1016/j.niox.2008.12.003, (2009). [24]. el kadi fatima z., benali a i., benali m., and belbraouet s., effect of sodium metabisulphite on blood metabolic status of wistar rats .food nutr sci, 5(15) :15291537.doi : 10.4236/fns.2014.515165 ,(2014). [25]. el kadi fatima z., boufadi m y., meziani s., sarsar f z., djebara s., dra a g., benali a i., mansour i ., and benali m., effect of sodium metabisulfite on lipid peroxidation and enzyme activities in adult rat stomach and spleen . south asian j exp biol,7(1) :1-8 .doi : http://sajeb.org/index.php/sajeb/article/view/20369, (2017). [26].directive 2008/84 / ce of the european commission of august 27, 2008 establishing specific purity criteria for food additives other than colorings and sweeteners. http://data.europa.eu/eli/dir/2008/84/oj. [27].nair b., and elmore ar., cosmetic ingredients review expert panel. final report on the safety assessment of sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite. int j toxicol, 22 (2):6388 .doi: 10.1080/10915810305077x, (2003). [28].hui jy., beery j t., higley n a., and taylor s l., comparative subchronic oral toxicity of sulphite and acetaldehyde hydroxysulphonate in rats. food chem toxicol, 27(6):349-359.doi: 10.1016/0278-6915(89)901403, (1989). [29].derin n., akpinar d., yargicoglu p., agar a., and aslan m., effect of alphalipoic acid on visual evoked potentials in rats exposed to sulfite. neurotoxicol teratol, 31(1): 3439.doi: 10.1016/j.ntt.2008.08.002, (2009). [30]. ercan s., ozturk n., celik-ozenci c., gungor ne., and yargicoglu p., sodium metabisulfite induces lipid peroxidation and apoptosis in rats gastric tissue. toxicol ind health, 26 (7):425-431.doi: 10.1177/0748233710369665, (2010). [31]. balcells a., laboratory examinations for the practitioner, masson edition, paris, 612 pages, (1998). [32].marieb e., essentials of human anatomy and physiology (9th éd.), san francisco (ca), pearson/benjamin cummings, 632 pages, (2009). [33]. pagana k d., pagana t j ., and mac donald s ., mosby’s canadian manual of diagnostic and laboratory tests (1st ed. canadienne), toronto, elsevier mosby, 1368 pages, (2013). [34].etlik o., tomur a., tuncer m., ridvanagaol ay., and andac o., protective effect of antioxydant vitamins on red blood cell lipoperoxydation induced by so2 inhalation. j basic clin physiol pharmacol, 8 (12):31-430.doi: 10.1515/jbcpp.1997.8.1-2.31, (1997). [35]. till h.p., feron v j., and de groot a p., toxicity of sulfite .i. long term feeding and https://doi.org/10.1016/s0006-291x(03)00833-7 https://pubmed.ncbi.nlm.nih.gov/?term=yargi%c3%a7o%c4%9flu+p&cauthor_id=10508905 https://pubmed.ncbi.nlm.nih.gov/?term=a%c4%9far+a&cauthor_id=10508905 https://pubmed.ncbi.nlm.nih.gov/?term=g%c3%bcm%c3%bc%c5%9fl%c3%bc+s&cauthor_id=10508905 https://pubmed.ncbi.nlm.nih.gov/?term=bilmen+s&cauthor_id=10508905 https://pubmed.ncbi.nlm.nih.gov/?term=o%c4%9fuz+y&cauthor_id=10508905 https://pubmed.ncbi.nlm.nih.gov/?term=yargi%c3%a7o%c4%9flu+p&cauthor_id=11697037 http://dx.doi.org/10.4236/fns.2014.515165 http://data.europa.eu/eli/dir/2008/84/oj food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 fatima zohra el kadi, khedoudja kanoun, asma maaz, fatima zohra sarsar, asma remil, mohammed benali, estimation of hematopoitic and histopathological disorders in wistar rats subchronously exposed to food additive e223 (nambs), food and environment safety, volume xx, issue 4 – 2021, pag. 333 – 342 342 multigeneration studies in rats. food chem toxicol, 10 (3): 291-310.doi : https://doi.org/10.1016/s0015-6264(72)80250-5, (1972). [36].roberts a., and buidinsky j r ., hematotoxicity : chemically induced toxicity of the blood .in : principles of toxicology , environmental and industrial applications .edited by williams pl., james rc., and roberts sm, second edition , wiley interscience publication , 603 pages,(2000). [37]. gunnison af., sulfite toxicity: a critical review of in vitro and in vivo data. food chem toxicol, 19:667-682. doi :https://doi.org/10.1016/0015-6264(81)90519-8 ,(1981). [38]. dikmenoglu n., baskurt o.k., and levi e., how does sulphur dioxide affect erythrocyte deformability? .clin. hemorheol. microcirc, 11(5):497-499.doi:10.3233:/199111526, (1991). [39].halliwell b., and chirico s. , lipid peroxidation: its mechanism, measurement, and significance. am j clin nutr, 57(5):715s-725s.doi: 10.1093/ajcn/57.5.715s,(1993). [40].cossu c., doyotte a., jacqui m c., and vasseur p., mechanisms of formation of reactive oxygen species. biomarkers of oxidative stress in aquatic animals. in biomarqueurs en écotoxicologie. aspects fondamentaux. l. [41]. neuzil j., gebicki j m., and stocker r., radical-induced chain oxidation of proteins and its inhibition by chain breaking antioxidants. biochem j, 293 (pt 3):601-606. doi: 10.1042/bj2930601 ,(1993). [42].beems rb., spit bj., koeter hb., feron vj., nature and histogenesis of sulfiteinduced gastric lesions in rats. exp mol pathol, 36(3):316-325. https://doi.org/10.1016/00144800(82)90061-2, (1982). https://doi.org/10.1016/s0015-6264(72)80250-5 https://doi.org/10.1016/0015-6264(81)90519-8 https://dx.doi.org/10.1042%2fbj2930601 https://doi.org/10.1016/0014-4800(82)90061-2 https://doi.org/10.1016/0014-4800(82)90061-2 1. introduction 4. conclusion 38 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 38 47 mu lt icrit e ri a eva lua t io n o f m un ici pa l s o l id w ast e man agem ent sc e nar io s: cas e s t udy ias i, r om ania *cristina ghinea1,2, hans th. a. bressers3, maria gavrilescu1,4 1“gheorghe asachi” technical university of iasi, faculty of chemical engineering and environmental protection, department of environmental engineering and management, 73 prof. dr. doc. dimitrie mangeron street, 700050 iasi, romania; cbghinea@yahoo.com 2stefan cel mare university of suceava, faculty of food engineering, 13 universitatii street, 720229 suceava, romania, cbghinea@yahoo.com 3university of twente, faculty of management and governance, twente centre for studies in technology and sustainable development, p.o. box 217, 7500 ae enschede, the netherlands 4academy of romanian scientists, 54 splaiul independentei, ro-050094 bucharest, romania *corresponding author received december 17th 2013, accepted february 24th 2014 abstract: solid waste management is of great interest as a topical issue discussed and reviewed internationally. interdisciplinary studies have been conducted worldwide to assess sustainable solid waste management. the main objectives of a sustainable waste management should be: preservation of natural resources and energy, minimizing pollution and environmental impacts, establishing a high quality performance of the environment. due to its complexity and the uniqueness, each waste management system is designed to satisfy particular objectives, such as waste policies and specific environmental targets (restricted to site specific constraints such as: waste composition, generation rates, geographical origin, installed treatment capacity, waste management technologies, stakeholders preferences). sustainable management of waste can be accomplished using different types of models, which are in fact decision support models such as multicriteria evaluation models. the multicriteria evaluation can rank different waste management systems considering various evaluation criteria in order to choose the most suitable system. in this purpose we have proposed different scenarios as alternatives to the municipal solid waste management system existent in iasi in 2009. multicriteria decision making approach was emphasized and applied for the evaluation of waste management scenarios, since it enables a complex, integrated and logical framework. this way we are able to identify and characterize the interactions and interdependence among factors, structured hierarchically or like a network to deal with dependence and feedback. keywords: ahp, models, solid waste, sustainability 1. introduction waste management is a major problem for all communities in the world and in particular for the eu countries, since different actors involved (policymakers, industry, and municipalities) are facing a lack of methodology and software for defining, evaluating, optimizing or adapting their waste treatment decision and for meeting the progress targets set at the eu level [1]. one of the most relevant objectives is to reduce the amount of waste generated. however, these efforts are still very limited, especially in some south-eastern european countries, like romania, with a mix of results and relatively few efforts food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 39 that have been made to regulate the management of various categories of waste (for example, organic materials that usually comprise over 50% of the total waste generation in the cities) [1]. although romania is an eu member state, and the european policy in the area of solid waste management should be implemented as soon as possible, these changes are still difficult to be made [1]. the solid waste management (swm) is a complex multidisciplinary issue since a waste management system must cover all activities: collection, transport, and waste treatment processes [1-5]. municipal and industrial wastes should be managed according to the solid waste management hierarchy for a healthy environment (fig. 1) [1, 2]. waste hierarchy does not attempt to assess environmental impacts for a waste management system and does not take into account any local conditions which may significantly change the environmental consequences [6]. economic, environmental, technical, social and legal issues must be considered in planning, selection, design and implementation of solid waste management systems [1, 7, 8]. over the years different methods have been developed to assist the decision factors in the selection of the appropriate municipal solid waste management (mswm) systems in such a way to ensure sustainable development and solve environmental problems associated to waste generation [1, 7, 9, 10]. one of the very fast growing areas is considered multicriteria decision analysis (mcda) developed for supporting decision makers to make suitable choice in different situations [1], [11-12]. it is considered that mcda methods can help in selecting the best compromise alternatives [11]. this method has an important applicability in the waste management area and can be considered a valuable tool for decision makers [1], [1315]. there are several methods available to solve multicriteria decision making problems: analytic network process (ahp) developed by saaty [16], preference ranking organisation method for enrichment evaluations (promethee) developed by brans [17], electre (elimination et choix traduisant la realité/the elimination and choice translating reality) developed by bernard roy [18], technique for preference by similarity to the ideal solution (topsis), multicriteria decision aiding hybrid algorithm (thor) developed by gomes [19] etc. these methods were used in several disciplines as well as in environmental engineering. in this paper we have applied a multicriteria evaluation method (ahp), considering three criteria (environmental, economic and technical criteria) to evaluate the municipal solid waste management system existent in iasi, romania (in 2009) and alternatives that we have proposed. 2. experimental waste management in iasi, romania the waste composition consists in organic waste (biowaste: food waste and garden waste, generated in gardens and streets from plants and grass) which represents the fraction with the major percentage (approximately 50%) followed by paper and cardboard, plastic, glass, metals (table 1)[20]. until 2009 the mixed waste collected in iasi was landfilled at the nonconform tomesti landfill which was closed in 2009. landfill from tomesti continues to be an important source of pollution for soil and groundwater, because the leachate collection is improper. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 40 figure 1. classic and reversed management hierarchy with the most and the least favourable option [1]. table 1 waste composition in iasi in 2008 under the long term investment plan [20] waste fraction (%) urban rural paper and cardboard 7.68 6 glass 4.35 3.5 metals 1.78 2.5 plastic 6.17 5.5 textiles 3.16 2.5 biodegradable waste 47.15 64 wood 1 0.5 others 28.71 15.5 leachate may migrate into soil and groundwater and can generate risks for the environment and human health [21]. from 2009 a new landfill was built according with the legislation and put into operation namely tutora landfill. in tutora landfill the collection and treatment of leachate is carried out according to the law. also the collection of landfill gas is going to be set up. in 2009 a sorting station at tutora was put into operation with a capacity of 29,000 t/y and a composting station was at that time under construction [1, 20, 21, 22]. in march 2012 was started composting process also at this point return manually by operators windrows with pyramidal shape, a length of 30 m, height 2 m and width 3 m, wetting is made by the operator (waste of the windrow are 100% vegetable) in october 2012 was made windrows with green waste and household waste (25-30%) [1]. in the municipal waste stream the organic waste consists mainly of organic household waste and garden waste [23]. both biowaste from households and garden waste are suitable for composting. another biological process used for treatment of organic waste is anaerobic digestion in which the organic wastes are decomposed by microorganisms in anaerobic conditions. the main products of the anaerobic digestion are: biogas which can be used to produce electricity and compost. 3. results and discussion application of analytic network process for evaluation of waste management scenarios in the ahp method the most important components of a problem are arranged into a hierarchical structure similar to a family tree [13]. ahp is based on the utility function that aggregates different criteria food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 41 into one global criterion [8]. the ahp method was applied for: the evaluation and choice of the best solid waste management alternative (system) [13, 15], evaluation of the implementation of weee management systems [24], for assessing the collection of municipal solid wastes and comparing different cases [14], assessment of scenarios on thermal processing of infectious hospital wastes [25], for landfill site selection [26], for supplier evaluation and selection in a steel manufacturing company [27] etc. analytic hierarchy process is a support for decision making with regard to complex sustainability issues and can help to recognize and define a problem in detail. it can be used to compare the impacts of alternative policies generated by other tools like: physical assessment tools, modelling tools and environmental appraisal tools [28, 29]. analytic hierarchy process (ahp) was developed and introduced by saaty (1980) and is one of the widely methods used in multicriteria decision making (mcdm) problems [29, 30]. the analytic hierarchy process (ahp) is a multi-criteria decision making method, which helps the decision-maker to solve problems based on the evaluation of a set of criteria [24, 31, 32]. the ahp methodology compares criteria or alternatives with respect to a criterion, in a natural, pairwise mode allowing structuring, measuring and/or synthesis [33]. ahp is based on: decomposition, comparative judgement and hierarchic composition or synthesis of priorities [33, 34]. it is a method that decomposes a complex problem into a multi-level hierarchical structure of objectives, criteria, sub-criteria, and alternatives [35] (fig. 2). basically, the ahp method consists of four main steps [30]: structuring the hierarchy of criteria and alternatives for evaluation; establishing a pair-wise comparison to assess the decision makers evaluation; prioritising of criteria and alternatives using eigenvector method; synthesizing the priorities of alternatives according to the criteria to rate the alternatives for performance score calculation. 3.1. goal of the study the aim of this study is to analyze and evaluate different scenarios of municipal solid waste management and alternatives that could be implemented by considering economic, environmental and technical criteria in an integrated manner. in this purpose we have applied the analytic hierarchy process (ahp) methodology which include different types of methods (rating and raking methods, pairwise comparisons) to evaluate the four proposed mswm scenarios 3.2. establishing of criteria and subcriteria indicators for the evaluation of the waste management system, the three criteria selected include different indicators [1] (fig. 3): economical criteria (c1): economical indicators (costs (c.1.1), benefits (c1.2), market prospect of products (c1.3), land requirement (c1.4)), environmental criteria (c2): environmental indicators (acidification (c2.1), eutrophication (c2.2), climate change (c2.3), human toxicity (c2.4), photochemical ozone formation (c2.5), wastewater (c2.6), water consumption (c2.7), noise pollution (c2.8)), technical criteria (c3): technical indicators (existing experience reliability (c3.1), adaptability to local conditions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 42 (c3.2), energy consumption (c3.3), energy production (c3.4)). the investment costs, operating and maintenance costs, administrative and other costs of municipal solid waste management scenarios were calculated and are expressed in euro per tone of waste treated (€/t). investments costs are for: sorting 122 €/t, composting 159 €/t, anaerobic digestion 370 €/t and the operating and maintenance costs: sorting 30.72 €/t, composting 30.3 €/t, anaerobic digestion 70.1 €/t [1]. the benefits were calculated considering considering three types of benefits: economic benefits, environmental and social benefits [1]. the environmental criteria such as acidification potential (expressed in kg so2 eq.), eutrophication potential (expressed in kg 3 4po eq.), global warming potential (expressed in kg co2 eq.), human toxicity potential (kg dcb eq., dichlorbenzol), and photochemical ozone creation potential (expressed in kg ethene eq.) were calculated using life cycle assessment methodology [1]. the indicators indicator existing experience – reliability and adaptability to local conditions were estimated using a qualitative scale from 0100, where 100 is the score where a facility is fully accepted, while 0 is the lowest social acceptance degree for a certain facility in the case of first indicator and for the second indicator 100 is the score when a facility is fully adaptable, while 0 denotes the lowest adaptability to local conditions. 3.3. setting of alternatives: municipal solid waste management scenarios for iasi the first scenario includes mixed waste collection, landfilling with collection of biogas and treatment of leachate new landfill was established in 2009, according to landfill directive provisions [36]. scenarios 2-4 are alternatives to the waste management system including various methods for treatment/elimination of waste (fig. 3): scenario 2: composting of biodegradable waste and landfilling; scenario 3: sorting of recyclable waste, composting of biodegradable waste and landfilling; scenario 4: sorting of recyclable waste, composting and anaerobic digestion of biodegradable waste, and landfilling. 3.4. application of rating and raking methods and pairwise comparisons for evaluation of scenarios –results rating and raking methods and pairwise comparisons were applied for evaluation of municipal solid waste management scenarios. the steps that we performed applying ranking method are: setting of the decision elements (indicators) list; each expert involved in the evaluation was asked to put the list of decision elements in order of importance using saaty scale (table 2); calculation of ranking votes sum for every economic, environmental and technical criteria for each scenario evaluated according to table 3; the relative weight of each criterion calculated according with table 4. also was performed the following phases of the rating method: -to each decision element was given a rating or a percentage score between 0 and 100; the sum of rating votes for every economic, environmental and technical criteria for each scenario evaluated were calculated according to table 3; also the relative weights of each criterion were calculated according with table 4. results obtained from both calculation methods were combined (according with table 5) in order to obtain the combined weight. combined weights for each food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 43 criterion were calculated by averaging the relative weights determined for both the ranking and rating techniques. the next step is represented by scoring. a score is given for each criteria or indicator, which reflects the performance. combined weights were multiplied by the score in order to establish the final score for each indicator. final score of the analysis is calculated by taking the sum of the final scores obtained for each criteria and dividing it by 100. the score obtained for each scenario were: s1 2.02 s2 2.99 s3 3.53 s4 3.17 a score of three or better is acceptable: at or above the norm for good operations in the region (table 6). figure 2. proposed hierarchy for municipal solid waste management system [1] a) b) c) d) figure 3. scenarios proposed for the analysis of msw management in iasi, romania: a) scenario 1, b) scenario 2, c) scenario 3, d) scenario 4, ts – temporary storage; ct collection and transport; l landfilling; lgc landfill gas collection; lc leachate collection; e engine; lt -leachate treatment; mt mechanical treatment; c composting; m maturing; sa soil application; b bio-filter; s – sorting; f – fermentation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 44 table 2 scale of relative importances [16, 29, 37] intensity of importance definition/judgment explanation/significance 1 equal importance two actions contribute equally to the objective 3 weak importance of one over other experience and judgment slightly favour one activity over another 5 essential or strong importance experience and judgment strongly favour one activity over another 7 demonstrated importance an activity is strongly favoured and its dominance demonstrated in practice 9 absolute importance the evidence favouring one activity over another is of the highest possible order of affirmation 2,4,6,8 intermediate values between the two adjacent judgment when compromise is need table 3 calculation of the sum of experts votes [1] criteria rank rating c1 n1 m1 c2 n2 m2 . . . . . . nk mk  in , i=1,…, k  im , i=1,…,k table 4 calculation of relative weights [1] criteria relative weight relative weight c1 11 100*/ ann i  11 100*/ bmm i  c2 22 100*/ ann i  22 100*/ bmm i  . . . . . . kik ann  100*/ kip bmm  100*/   100ia , i=1,…,k 100 ib , i=1,…,k table 5 calculation of combined weight [1] criteria combined weight c1   111 2/ rba  c2   222 2/ rba  . . .   kkk rba  2/ 100 ir , i=1,…,k food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 45 table 6 the score scale [1] score description 0 not an applicable criteria or indicator 1 extremely weak performance; strongly unfavourable 2 poor performance; unfavourable; may be the norm for the region, but major improvement needed 3 acceptable; at or above the norm for good operations in the region 4 very favourable performance; well above the norm for the region, but still needing improvement to be state of the art 5 ‘state of the art’ in region; clearly outstanding performance which is way above the norm for the region scenarios s4, s3, had a good score while s1 and s2 performing a little below what is considered good operational regional standard (poor performance). the pairwise comparison method involves one-on-one comparisons between each of the indicator [39]. pairwise comparisons was performed following these steps: comparative judgements on the relative importance of each pair of indicators in terms of the measured criterion were made; comparative matrixes of the criteria for each scenario were determined; relative weights of the indicators in each matrix were determined in three steps according to pairwise comparisons methodology; the vector of priorities (vp) for each matrix was obtained after determining the matrices by multiplying the n elements in each row and taking the n-th root resulting in a column vector which was normalized; the consistency of the results can be determined by the calculation of the eigenvalue max , when each matrix is multiplied by its vector of priorities vp [38]; the final max was determined by summing the components in the resulting vector and dividing the results by n. in this case final eigenvalue is almost equivalent to the n number of alternatives 4n . this indicates that the results have a very high consistency since the closer the max is to n, the more consistent are the results of the pairwise comparison for the criteria. final criteria for each scenario using the pairwise comparison were determined by multiplying the calculated weights with the score given to each indicator. the final score obtained for each scenario was: s1 1.93 -s22.61 s3 3 s4 3.23 consistency index (ci) was also calculated for each matrix: the ci values were below the tolerance consistency index of 0.10 which means tolerable limits of inconsistency ( 045.01 sci ; 0045.02 sci ; 001.03 sci ). scenario s4 is the most suitable alternative to the municipal solid waste management system, followed by scenarios s2, s3. in rank order of the alternatives, the weight of criteria has a big influence. in order to be able to make the final decision the decision makers must know the degree of reliability of the results [39]. 4. conclusions the evaluation was performed by applying the rating, ranking and the pairwise comparisons methods using the software media offered by excel and matlab. results highlighted that scenario 4 is the most suitable alternative to the municipal solid waste management system in terms food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 46 of environmental, economic and technical aspects followed by scenarios 2 and 3. future studies will be performed since other alternatives can be also proposed and evaluated and can be chosen other criteria such social criteria in order to be much closer to the real situation. 5. acknowledgment this work was supported by eurodoc “doctoral scholarships for research performance at european level” project, financed by the european social found and romanian government; university of twente, twente centre for studies in technology and sustainable development (cstm) and by a grant of the romanian national authority for scientific research, cncs – uefiscdi, project number pnii-id-pce-2011-3-0559 idei project, contract 265/2011. 6. references [1]. ghinea c., waste management models and their application to sustainable management of recyclable waste, phd thesis, gheorghe asachi technical university of iasi, romania, (2012) [2]. banar m., cokaygil z., ozkan a., life cycle assessment of solid waste management options for eskisehir, turkey, waste management, 29. 54-62, (2009) [3]. eriksson o., carlsson reich m, frostell b., bjorklund a., assefa g., municipal solid waste management from a systems perspective, journal of cleaner production, 13. 241-252, (2005) [4]. kollikkathara n., feng h., stern e., a purview of waste management evolution: special emphasis on usa, waste management, 29. 974-985, (2009) [5]. schiopu a.m. apostol i., hodoreanu m., gavrilescu m., solid waste in romania: management, treatment and pollution prevention practices, environmental engineering and management journal, 5. 451-465, (2007) [6]. kirkeby j.t., modelling of life cycle assessment of solid waste management systems and technologies, phd thesis, institute of environment & resources, technical university of denmark, (2005) [7]. ghinea c., gavrilescu m., decision support models for solid waste management – an overview, environmental engineering and management journal, 9. 869-880, (2010) [8]. ozkan a., banar m., acar i.p., sipahioğlu a., application of the electre iii method for a solid waste management system, anadolu university journal of science and technology – a applied sciences and engineering, 12. 11-23, (2011) [9]. morrissey a., browne j., waste management models and their application to sustainable waste management, waste management, 24. 297-308, (2004) [10]. ness b., urbel-piirsalua e., anderbergd s., olssona l., categorising tools for sustainability assessment, ecological economics, 60. 498-508, (2007) [11]. behzadian m., kazemzadeh r.b., albadvi a., aghdasi m., promethee: a comprehensive literature review on methodologies and applications, european journal of operational research, 200. 198-215, (2010) [12]. balkwaste, report on the criteria for the assessment of alternative technologies version 2 –august (2011), on line at: http://www.balkwaste.eu/?page_id=21, (2011) [13]. garfì m., tondelli s., bonoli a., multi-criteria decision analysis for waste management in saharawi refugee camps, waste management, 29. 2729-2739, (2009) [14]. karagiannidis a., xirogiannopoulou a., perkoulidis g., moussiopoulos n., assessing the collection of urban solid wastes: a step towards municipality benchmarking, water, air, and soil pollution, 4. 397-409, (2004) [15]. marković d., janošević d., jovanović m., nikolić v., application method for optimization in solid waste management system in the city of niš, facta universitatis, series: mechanical engineering, 8. 63-76, (2010) [16]. saaty t.l., the analytic hierarchy process: planning, priority, setting, resource allocation, mcgraw hill, new york, (1980) [17]. brans j.p., “l’ingénierie de la décision: élaboration d’instruments d’aide à la décision. la méthode promethee”, presses de l’université laval, (1982) [18]. roy b., classement et choix en présence de points de vue multiples (la méthode electre), la revue d'informatique et de recherche opérationelle (riro), 8. 57-75, (1968) [19]. gomes c.f.s., thor – um algoritmo híbrido de apoio multicritério à decisão para processos decisórios com alternativas discretas, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 cristina ghinea, hans th. a. bressers, maria gavrilescu, multicriteria evaluation of municipal solid waste management scenarios: case study iasi, romania, issue 1 2014, pag. 38 – 47 47 tese de doutorado, engenharia de produção, coppe-ufrj, (1999) [20]. iasi county council, long-term investment plan for the period 2008-2038 on integrated waste management, on line at: http://www.icc.ro/activitate/mediu/masterp/master plan.pdf, (2009) [21]. schiopu a.m., robu b.m., apostol i., gavrilescu m., impact of landfill leachate on soil quality in iasi county, environmental engineering and management journal, 8. 11551164, (2009) [22]. iasi county council, report on the study of environmental impact assessment, integrated waste management system in iasi, (2011) [23]. andersen j.k., composting of organic waste: quantification and assessment of greenhouse gas emissions, phd thesis, department of environmental engineering, technical university of denmark, (2010) [24]. ciocoiu c.n., colesca s.e., burcea s., an ahp approach to evaluate the implementation of weee management systems, recent researches in environment, energy planning and pollution. proceedings of 5th wseas international conference on waste management, water pollution, air pollution, indoor climate (wwai '11), 223-238, (2011) [25]. karagiannidis a., papageorgiou a., perkoulidis g., sanida g., samaras p., a multi-criteria assessment of scenarios on thermal processing of infectious hospital wastes: a case study for central macedonia, waste management, 30. 251-262, (2010) [26]. guiqin w., li q., guoxue l., lijun c., landfill site selection using spatial information technologies and ahp: a case study in beijing, china, journal of environmental management, 90. 2414-2421, (2009) [27]. tahriri f., rasid osman m., ali a. yusuff r.m., esfandiary a., ahp approach for supplier evaluation and selection in a steel manufacturing company, journal of industrial engineering and management, 1. 54-76, (2008) [28]. kasperczyk n., knickel k., the analytic hierarchy process, on line at: www.ivm.vu.nl/en/images/mca3_tcm53161529.pdf, (2005) [29]. triantaphyllou e., mann s.h., using the analytic hierarchy process for decision making in engineering applications: some challenges, international journal of industrial engineering: application and practice, 2. 35-44, (1995) [30]. tarmudi z., abdullah m.l., tap a.o.m., evaluating municipal solid waste disposal options by ahp-based linguistic variable weight, matematika, 26. 1-14, (2010) [31]. ishizaka a., labib a., analytic hierarchy process and expert choice: benefits and limitations, or insight, 22. 201-220, (2009) [32]. vachajitpan p., measurement scales and derivation of priorities in pairwise and group decision making, mcdm, whistler, canada, (2004) [33]. forman e.h., gass s.i., the analytic hierarchy process – an exposition, on line at: www.johnsaunders.com/papers/ahpexpo.pdf, (2000) [34]. saaty t.l., fundamentals of decision making, rws publications, pittsburgh, usa, (1994) [35]. sharma m.j., moon i., bae h., analytic hierarchy process to assess and optimize distribution network, applied mathematics and computation, 202. 256-265, (2008) [36]. ec council directive, council directive 1999/31/ec of 26 april 1999 on the landfill of waste, official journal of the european communities, l 182/1, 16.7.1999, brussels. [37]. mendoza g.a., macoun p., prabhu r., sukadri d., purnomo h., hartanto h., guidelines for applying multicriteria analysis to the assessment of criteria and indicators, center for international forestry research, jakarta, indonesia, (1999) [38]. chang s. y., bindiganaville k., lca and multicriteria evaluation of solid waste recycling, environmental informatics archives, 3. 118-129, (2005) [39]. aragones-beltran p., mendoza roca j.a., bes-pia a., garcia-melon m., parra-ruiz e., application of multicriteria decision analysis to jar test results for chemicals selection in the physical-chemical treatment of textile wastewater, journal of hazardous materials, 164. 288-295, (2009) doi: https://doi.org/10.4316/fens.2022.012 116 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 116 128 functional and technological properties of oat beta-glucan in acidophilic-whey ice cream *artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak educational and research institute of food technologies, national university of food technologies, 01601, 68 volodymyrska str., kyiv, ukraine, artur0707@ukr.net *corresponding author received 13th may 2022, accepted 25th july 2022 abstract: the purpose of the work is to study the functional and technological properties of oat beta-glucan both individually and as part of acidophilic-whey ice cream. the research methods, that were used, are well known and have relevant references in the work. at the first stage of the experiment, the functional and technological properties of oat beta-glucan from two manufacturers were studied: «amulyn» (china) and «danson» (china). according to the results of sensory evaluation, beta-glucan «danson» received 8.75 points, and «amulyn» 10 points. it was found that the water holding capacity of beta-glucan «amulyn» is 15.45 % higher than with beta-glucan «danson», and fat binding capacity is higher by 8.61 %, which is probably due to the different degree of purification of these additives. therefore, a more effective beta-glucan «amulan» was chosen for further use in acidophilic-whey low-fat ice cream. at the second stage of scientific work the influence of oat beta-glucan «amulyn» on the quality of acidophilic-whey low-fat ice cream was studied. it was found that beta-glucan in the amount of 0.75 % increases the indicator of overrun more than twice compared to the control sample without beta-glucan. beta-glucan in a certain amount also significantly improves the organoleptic characteristics of ice cream, increases resistance to melting and ensures uniform distribution of the air phase throughout the volume of the product, as evidenced by microstructural analysis of its samples. a significant effect of beta-glucan on the dynamic viscosity coefficient of ice cream mixes has been established. the detected effect allows to reduce the ripening process of ice cream mixtures with betaglucan in the amount of 0.75 % by 2 hours, provided that the recommended viscosity of these systems is achieved. thus, the expediency of using oat beta-glucan as an effective functional and technological ingredient in acidophilic-whey ice cream is scientifically substantiated. keywords: β-glucan, fermented ice cream, whey, viscosity, overrun, microstructure. 1. introduction in modern human life and activity, it is important to increase the biological value of functional foods, which play a key role in improving overall health and preventing a number of diseases. fermented milk products contain all the nutrients of milk, and in the presence of natural probiotic cultures help strengthen the body's resistance [1,2]. recently, scientists are paying more attention to studying the properties and possibilities of using the acidophilic bacillus lactobacillus acidophilus, in particular in the technology of fermented dairy products such as beverages, including on the base of buttermilk, whey and fermented milk ice cream [3-5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 117 fermented ice cream is a product made from whey and/or starter on a pure cultures of lactic acid bacteria with or without the addition of aromatic substances [6]. its production at domestic enterprises in ukraine is extremely limited, and its most popular type is yogurt ice cream. at the same time, even according to the interstate gost 32929-2014 "fermented ice cream" [7], adopted for use in the postsoviet countries, including ukraine, fermented ice cream is intended to be made on the basis of not only yogurt and curd, but also kefir, acidophilin, airan, rhazhenka, sour clotted milk, cultured cream, cultured baked milk and kumys, which is not taken into account by domestic producers. in addition, the schemes of fermented ice cream production described in the standard technological instruction [8] are very complex. ice cream made by this technology has low resistance to melting, overrun and "empty" taste, which emphasizes the need to improve the technology and composition of fermented ice cream, in particular acidophilic-whey. in order to avoid the above defects in the ice cream producers add technological ingredients that act as a thickener and moisture-binding agent, structure the mixture at the stage of ripening and allow to obtain ice cream with high resistance to melting, overrun and original taste [9-11]. due to their high molecular weight, betaglucans are better thickeners than other polysaccharides, such as starch, inulin, dextrin, xylan, pectin preparations, fiber [12,13]. the use of beta-glucans can reduce the use of stabilizing substances in the prescription composition of ice cream, which is consistent with the concept of healthy eating [14-16], as well as give lowfat ice cream creamy taste, as this additive mimics fat in low-fat and non-fat products. nevertheless, the number of published research results on the use of beta-glucan in the production of ice cream, in particular low-fat dairy, is limited [17]. the effect of polysaccharide content and structure, including highly purified beta-glucan preparations, on the functional properties of low-fat ice cream has never been thoroughly studied. the aim of the research was to study the functional and technological properties of oat beta-glucan both individually and as part of acidophilic-whey ice cream. to achieve this goal, the following tasks were set: 1. to study the functional and technological properties of oat beta-glucan from different manufacturers and choose the most appropriate for further use in formulation of acidophilic-whey ice cream. 2. investigate the effect of oat beta-glucan on the resistance to melting, overrun and dispersion of the air phase in acidophilic-whey ice cream. 3. identify the features of structuring mixtures of low-fat ice cream with beta-glucan. 2. matherials and methods raw materials for research dry demineralized whey with a degree of demineralization of 90 % (tm "dairy alliance", ukraine), with a mass fraction of moisture 3.0 %, protein 10 %, fat 1.0 %, lactose 80 % was selected for research. a single-strain probiotic culture of l. acidophilus (tm "danisko", denmark) was used as a starter, that intended in the production of dairy products and cheeses. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 118 to study the technological parameters of beta-glucans in order to compare and select the best for further use in the composition of acidophilic-whey ice cream were selected oat beta-glucan from two manufacturers: «amulyn» (china), «danson» (china). the recipe composition of the studied samples of ice cream is due to the following requirements: dry matter content should be from 23.79 to 24.09 %, which is typical for the chemical composition of acidophilic-whey ice cream with low fat content; mass fraction of fat 2.0 %, which corresponds to its content in low-fat ice cream; mass fraction of dry non-fat residue 6.79 %, which ensures the completeness of the milk taste in ice cream with low fat content; mass fraction of sugar 17.0 %, which provides the traditional degree of sweetness and maintains the dry matter content in ice cream not less than 23.5 %; mass fraction of the stabilizator from 0.1 to 0.3 %, which corresponds to its recommended content for this type of ice cream in accordance with the manufacturer's recommendations; mass fraction of oat beta-glucan from 0.25 to 1.0 %, which corresponds to the research of other scientists [18-20]. preparation of samples the following samples were made: 1. control without beta-glucan, mass fraction of stabilizer (guar gum) 0.4 %. 2. sample 1 parts of beta-glucan 0.25 %, mass fraction of stabilizer (guar gum) 0.3 %. 3. sample 2 parts of beta-glucan 0.5 %, mass fraction of stabilizer (guar gum) 0.2 %. 4. sample 3 parts of beta-glucan 0.75 %, mass fraction of stabilizer (guar gum) 0.1 %. 5. sample 4 parts of beta-glucan 1.0 %, without stabilizer. preparation of samples was carried out as follows. starter activation. weighed with an open fire scalpel lyophilized starter on the basis of lactobacillus acidophilus at the rate of 0.5 g per 1 liter of mixture. the weighed starter was added to the pre-calculated amount of milk that was pasteurized at a temperature of 84…88 ℃ for 3…5 min and cooled to a fermentation temperature of 38…42 ℃. the duration of fermentation was 6…8 hours before reaching the acidity of not more than 5.0 ph. preparation of the mixture. dry components (demineralized dry whey, sugar, stabilizer, beta-glucan) were weighed according to recipes on laboratory scales to the nearest 0.01 g. the components were mixed and distilled water was added. after this mixture was pasteurized at the temperature of 84…88 ℃ for 3...5 min and cooled to 38…42 ℃. pre-activated starter was added. the fermented mixture was subjected to ripening for 6…8 years until the acidity is not higher than 5.0 ph. the fermented mixture was cooled to a temperature of 2…6 ℃, kept for at least 2 hours and freezed in a freezer for making ice cream "frosty icm-15a" with a power of 50 rpm for 45 minutes. storage. ice cream samples were cooled and stored in a freezer "caravell" a / s (denmark) at a temperature of minus 21…23 ° c in the continuation of the study. samples of the same chemical composition were made at least 3 times. research methods organoleptic evaluation of beta-glucan. the product was mixed and determined the color, structure, consistency, the presence of dense lumps that do not fall apart when lightly tapped and foreign particles. when mixing, it was necessary to pay attention to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 119 the presence or absence of product compaction and signs of caking. to determine the color of beta-glucan (particle size up to 1 mm), it was placed in a clean dry container at a layer height of at least 2 mm and evaluated in high light conditions. odor, taste and aroma were analyzed immediately after consistency assessment. the evaluation was carried out on a scale of six people. the maximum value of the score for each of the indicators was 5 points. based on the results, a histogram was constructed. the general complex indicator of organoleptic evaluation [21] of dry ingredients (beta-glucan) can be represented as a set of the following characteristics: appearance (k1), color (k2), taste (k4), odor (k4). the overall complex indicator of organoleptic evaluation was determined by the equation: where k is the overall complex indicator; m1, м2, м3, м4 are importance factors of each of the groups of indicators k1, к2, к3, к4. fat binding capacity (fbc) of betaglucan was determined by centrifugation method [22]. after receiving the results, the values of the fbc, %, were calculated according to the formula: , where a is a weight of test tube with test material and bound oil, g; b is the weight of the test tube with the test material, g; c is a sample of experimental material, g water holding capacity (whc) of betaglucan was determined by centrifugation method [22]. whc, %, was calculated by the formula: , where m is the mass of the sample, g; m1 is the mass of the test tube with the dry sample, g; m2 is mass of test tube with wet sample, g. studies of fat binding capacity and water holding capacity were conducted on the basis of milk and dairy technology department of the national university of food technologies using a laboratory centrifuge «orbit» (clu-1) at a speed of 1370 rpm. to determine the coefficient of dynamic viscosity [23], a hepler viscometer with a set of 6 glass and metal beads of different diameters was used. the calculation of the coefficient of dynamic viscosity of the mixture µ (mpa·s) was performed by the formula: , where k is the constant of the ball, mpa*cm3/g; ρ1 and ρ2 are respectively, the density of the materials of the ball and the mixture, g/cm3; t is the duration of the ball between the ring marks, s. resistance to melting was determined by a modified method [24], according to which the time of appearance of the first drop of "melt" and the time of leakage from the sample of ice cream 10 cm³ "melt" were recorded. the size of air bubbles was determined by the method of vnihi (all-russian research institute of refrigeration industry) [25], according to which an ice cream sample was applied to the calibrated grid of goryaev's chamber, covered with a cover glass and immediately microscopied at 160 times magnification. overrun (s), %, of ice cream was determined by weight method [26]. organoleptic evaluation of ice cream was performed by tasting by a commission of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 120 10 people with an assessment of compliance with the requirements of dstu 4733: 2007 (state standard of ukraine) [27], as well as a 10-point scale, according to which the maximum number of points for taste and smell is 6, for consistency is 3, for color and appearance is 1. statistical processing after laboratory experiment was performed using microsoft excel. graphical representation of the numerical data of the experiment was performed using the program mathcad 15. to ensure the accuracy of the experimental results, experiments were performed three to five times under the same conditions. 3. results and discussion investigation of functional and technological properties of oat beta-glucan at the first stage of the research, a comparative analysis of oat beta-glucan indicators of two manufacturers, whose products are presented on the ukrainian market of food additives, was conducted. beta-glucan «danson» (china) and «amulyn» (china) were selected for the study. organoleptic evaluation of these additives is given below (table 1). table 1. organoleptic evaluation of beta-glucan indicator sample 1 (danson) sample 2 (amulyn) appearance and color the surface is homogeneous, smooth and clean. the color is white with a cream tinge, uniform throughout the mass. the surface is homogeneous, smooth and clean. the color is cream, uniform throughout the mass. taste, smell and aroma no foreign odors, but there is a taste of oatmeal. clean, without foreign tastes and odors. structure and consistency homogeneous, without mechanical inclusions, with single lumps. homogeneous, without lumps and mechanical inclusions. based on the results of organoleptic evaluation of beta-glucan from two manufacturers, a histogram was constructed, which is presented in figure 1. fig.1 histogram of organoleptic evaluation of beta-glucan food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 121 based on the results of organoleptic evaluation, the overall complex indicator of organoleptic evaluation of beta-glucan of two manufacturers was calculated. the results are shown below (table 2). table 2. overall complex indicator of organoleptic evaluation of beta-glucan indicator coefficient of importance sample № 1 (danson) 2 (amulyn) appearance 1,0 4 5 smell 0,25 1,25 1,25 taste 0,25 1 1,25 color 0,5 2,5 2,5 overall complex indicator (k) 2,0 8,75 10 according to the results of the calculation of the overall complex indicator of betaglucan (sample № 1) received 8.75 points, and sample № 2 10 points. the difference in estimates may indicate a higher degree of purification of beta-glucan «amulyn» (no oatmeal taste), as well as possible noncompliance with the technological process of production beta-glucan «danson» (the presence of single lumps). the next step was to investigate the fbc and whc of beta-glucan in order to determine the most effective additive for its further use in the composition of acidophilic-whey ice cream. the results obtained are shown below (table 3) table 3. the results of the study of fat binding capacity and water holding capacity of beta-glucan, % indicator sample № 1 (danson) 2 (amulyn) whc 76.4±1,8 88.2±2,1 fbc 32.04±0,8 34.8±0,7 (р ≥ 95 %, n = 3) in accordance with those specified in table 3 data, it is noticeable that the whc for sample № 2 is higher than in sample № 1, by 15.45 %, and fbc by 8.61 %. the higher whc and fbc of «amulyn» betaglucan compared to «danson» betaglucan is probably due to the higher degree of purification of the first additive according to the manufacturer's specification, which requires further study. the obtained data are correlated with the indicators of other researchers who studied oat beta-glucan both individually and as part of food products [28,29]. according to the results of the study of functional and technological properties of beta-glucan of two manufacturers, betaglucan «amulyn» was chosen as more technologically effective for further use in the composition of acidophilic-whey ice cream. investigation of the effect of oat beta-glucan on the quality of acidophilic-whey ice cream to conduct the study, experimental samples of ice cream were made and their food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 122 physicochemical and organoleptic characteristics were studied. indicators of overrun and resistance to melting of ice cream are shown in figure 2. fig. 2 overrun and resistance to melting of ice cream with beta-glucan and without (control) the overrun of the control sample is 21.6 %, the addition of beta-glucan in the amount of 0.25 % (sample № 1) leads to an increase in overrun by 18.1 %, and a dose of 0.5 % of beta-glucan (sample № 2) at 57.7 %. with a beta-glucan content of 0.75 % (sample № 3), the overrun increases by 217.6 %. however, a further increase in the dose of beta-glucan to 1.0 % (sample 4) has a negligible effect on the overrun of the ice cream and leads to a compaction of the mass, which impairs the organoleptic characteristics of the product. it should be noted that the mass fraction of stabilizer (guar gum) in the composition of ice cream is gradually reduced from 0.4 % for control to 0.1 % for sample № 3, and in sample № 4 it is absent. at the same time, the revealed properties of beta-glucan allow to significantly reduce the use of stabilizing substances in ice cream and achieve a high technological effect. resistance to melting during the accumulation time of 10 cm3 of "melt" also increases steadily from 47.3 min (control) to 72.1 min (sample № 4). this confirms the technological possibility with the help of beta-glucan to purposefully influence the indicators of overrun and resistance to melting of ice cream. the next step was to study the microstructure of ice cream samples for a more detailed study and explanation of the effect of oat beta-glucan on overrun and resistance to melting. the microstructure of the samples is shown in figure 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 123 control 1 2 3 4 fig. 3 microstructure of ice cream as can be seen in fig. 3, the content of beta-glucan at the level of 0.75 % provides the most uniform distribution of the air phase inside the ice cream, which has a positive effect on its consistency. the phenomenon of "cluster formation" from air bubbles due to their aggregation was also observed, which can be explained by the specific interaction between beta-glucan macromolecules in aqueous medium [30], which should be further studied. the addition of beta-glucan in the amount of 0.25…0.5 % does not ensure uniform distribution of the air phase in full, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 124 sometimes large air bubbles are observed. at the same time, increasing the dosage to 1.0 % leads to an excessive increase in the viscosity of the ice cream mixture and, as a result, complicates its saturation with air during freezing. as can be seen in fig. 3 (sample № 4), in some places there are gaps at the junction of air bubbles, which further adversely affects the consistency of the product. in order to study the taste properties of experimental samples of acidophilic whey ice cream with beta-glucan and without it, an organoleptic evaluation was performed. the results of organoleptic evaluation of ice cream are shown in table 4. table 4. organoleptic evaluation of acidophilic whey ice cream with beta-glucan and without (control) indicato r sample № control 1 2 3 4 color light yellow, yellow, saturated yellow, uniform throughout the mass taste and smell too sweet taste with a slight sourness, with noticeable ice crystals sweet taste with a slight sourness moderately sweet taste with a slight sourness moderately sweet taste with a slight sourness and extraneous aftertaste without foreign odors consist ence homogeneou s mass with ice crystals, that melts quickly homogene ous mass with single ice crystals that melt quickly homogene ous, whipped mass нomogene ous, creamy mass too dense mass quantitative assessment of organoleptic characteristics of ice cream was expressed on a 10-point scale and is presented in figure 4. fig. 4 organoleptic evaluation of ice cream on a scale food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 125 from the above data of organoleptic evaluation, it can be concluded that the dose of beta-glucan at 0.75 % provides the optimal combination of physicochemical and organoleptic parameters of acidophilic-whey ice cream, which allows to obtain a product with excellent consumer properties. investigation of the features of structuring mixtures of low-fat ice cream with beta-glucan at the last stage of scientific research, the effect of oat beta-glucan on the structuring of ice cream mixes was studied. the control mixture and the mixture with the addition of beta-glucan in the amount of 0.75 %, which was selected as the most technologically feasible according to the results of the previous stage of the experiment. since the recommended values of the dynamic viscosity coefficient of milk ice cream mixtures are not less than 140 mpa*s [23], this minimum allowable value was chosen as a criterion of efficiency when studying the process of structuring mixtures with beta-glucan. to establish rational modes of maturation of mixtures of the developed type of ice cream, the values of effective viscosity were measured at variable parameters: at temperatures in the range of 0…8 ⁰с in steps of 2 ⁰с; the duration of ripening was varied in the range from 0 to 10 hours in steps of 2 hours. the dependence of the dynamic viscosity of the control and test ice cream mixes on the temperature and duration of ripening is illustrated in figure 5. а b fig. 5 dynamic viscosity of ice cream mixes under variable ripening modes: a control mix, b mix of new type of ice cream (p ≥ 95%, n = 3). according to fig. 5, rational modes of ripening of acidophilic-whey ice cream mixtures were established. thus, the achievement of the minimum required value of the dynamic viscosity at 0 ºc for control was observed after 2 h, and the mixture of acidophilic-whey ice food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 126 cream with oat beta-glucan was already structured without aging, immediately after cooling. in the case of compliance with the recommended temperature from 2 to 6 ⁰c, the duration of exposure to the control mixture of ice cream should be at least 4 hours, and for the mixture of a new type of ice cream at least 2 hours. it is proved that it is possible to reduce the duration of ripening of ice cream of the developed composition by 2 hours, compared to the control sample of the classic composition, which will be taken into account in the technological scheme of its production. for both samples, the ripening temperature of 8 ºc does not allow to reach the recommended value of the dynamic viscosity of ice cream mixes. 4. conclusion for use in acidophilic whey low-fat ice cream, technologically effective oat beta-glucan of the trademark «amulan» was chosen. beta-glucan «amulyn» in the amount of 0.75 % increases the overrun of ice cream more than twice compared to the control sample without beta-glucan, as well as improves organoleptic performance, increases resistance to melting and ensures uniform distribution of the air phase in the finished product. beta-glucan in the amount of 0.75 % significantly affects the coefficient of dynamic viscosity of ice cream mixes, which allows to reduce by 2 hours the ripening of such mixes with beta-glucan. the prospect of further research is to determine the foaming properties of oat betaglucan in order to explain the effect of "cluster formation" in the air phase of ice cream, which was detected during microscopy. 5. acknowledgments the work was performed at the national university of food technologies within the state research «implementation of resource-saving methods of modification of functional and technological characteristics of whey in the technology of food products» (state registration number: № 0120u100868) and «scientific substantiation of resource-efficient food technologies, enriched with polyfunctional ingredients» (state registration number: № 0120u102556). 6. references [1]. a. solomon, development of technology for fermented sour milk desserts enriched with bifidobacteria and biologically active substances, 15 (102), colloquiumjournal, (2021), 61-64. https://doi.org/10.24412/25206990-2021-15102-62-64. [2]. m. garcía-burgos, j. morenofernández, m. j. alférez, j. díaz-castro, i. lópez-aliaga, new perspectives in fermented dairy products and their health relevance. 72, journal of functional foods, (2020), 104059. https://doi.org/10.1016/j.jff.2020.104059. [3]. j. r. wilburn, e. p. ryan, fermented foods in health promotion and disease prevention: an overview. fermented foods in health and disease prevention, (2017), 3-19. https://doi.org/10.1016/b978-012-802309-9.00001-7. [4]. m. i. torino, g. font de valdez, f. mozzi, biopolymers from lactic acid bacteria. novel applications in foods and beverages. 6, frontiers in microbiology, (2015), 834. https://doi.org/10.3389/fmicb.2015.00834. [5]. a. baschali, e. tsakalidou, a. kyriacou, n. karavasiloglou, a. l. matalas, traditional low-alcoholic and nonalcoholic fermented beverages consumed in european countries: a neglected food group. 30 (1), nutrition research reviews, (2017), 1-24. https://doi.org/10.1017/s0954422416000202. [6]. r.h. davidson, s.e. duncan, c.r. hackney, w.n. eigel, j.w. boling, probiotic culture survival and implications in fermented frozen yogurt characteristics. 83 (4), journal of dairy science, (2000), 666–673. https://doi.org/10.3168/jds.s0022-0302(00)74927-7. [7]. gost 32929-2014 (mezhgosudarstvennyj standart) «morozhenoe kislomolochnoe. tehnicheskie uslovija» [fermented ice cream. specifications] (in russian). https://doi.org/10.3389/fmicb.2015.00834 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 127 [8]. tipova tehnologіchna іnstrukcіja z virobnictva moroziva molochnogo, vershkovogo, plombіru; plodovo-jagіdnogo, aromatichnogo, shherbetu, l'odu; moroziva z kombіnovanim skladom sirovini [typical technological instructions for the production of ice cream: milk, cream, plombir, fruit and berry, aromatic, sherbet, ice, with a combined composition of raw materials]: ttі 31748658-1-2007 do dstu 4733:2007, 4734:2007, 4735:2007. kiїv: asocіacіja ukraїns'kih virobnikіv «ukraїns'ke morozivo ta zamorozhenі produkti». 2007. 100 s (in ukrainian). [9]. j. cheng, y. ma, x. li, t. yan, j. cui, (2015). effects of milk protein-polysaccharide interactions on the stability of ice cream mix model systems. 45, food hydrocolloids, (2015), 327-336. https://doi.org/10.1016/j.foodhyd.2014.11.027. [10]. r. k. goraya, m. singla, u. bajwa, a. kaur, s. pathania, impact of sodium alginate gelling and ingredient amalgamating order on ingredient interactions and structural stability of ice cream. 147, lwt, (2021), 111558. https://doi.org/10.1016/j.lwt.2021.111558. [11]. r.b. meneses, m.s. silva, m.l.g. monteiro, m.h.m. rocha-leão, c.a. contejunior, effect of dairy by-products as milk replacers on quality attributes of ice cream. 103 (11), journal of dairy science, (2020), 10022-10035. https://doi.org/10.3168/jds.2020-18330. [12]. a. ahmad, f.m. anjum, t. zahoor, h. nawaz, z. ahmed, extraction and characterization of β-d-glucan from oat for industrial utilization. 46 (3), international journal of biological macromolecules, (2010), 304–309. https://doi.org/10.1016/j.ijbiomac.2010.01.002. [13]. d. el khoury, c. cuda, b. l. luhovyy, g. h. anderson, beta glucan: health benefits in obesity and metabolic syndrome. journal of nutrition and metabolism, (2012), 851362. https://doi.org/10.1155/2012/851362. [14]. z. burkus, f. temelli, (2000). stabilization of emulsions and foams using barley β-glucan. 33, food research international, (2000), 27–33. https://doi.org/10.1016/s0963-9969(00)00020-x. [15]. a. m. h. abdel-haleem, r. a. awad, some quality attributes of low-fat ice cream substituted with hulless barley fl our and barley β-glucan. 52 (10), journal food science & technology, (2015), 6425–6434. https://doi.org/10.1007/s13197-015-1755-x. [16]. m. aljewicz, e. tonska, j. juskiewicz, g. cichosz, the influence of product acidity and βglucans isolated from various sources in the mineral composition and the mechanical and microstructural properties of the femur in growing wistar rats. 44, journal of functional foods, (2018), 191–200. https://doi.org/10.1016/j.jff.2018.02.035. [17]. v. sapiga, g. polischuk, m. buniowska, i. shevchenko, t. osmak, enzymatic destruction of protopectin in vegetable raw materials to increase its structuring ability in ice cream. 10 (4), ukrainian food journal, (2021), 691–702. https://doi.org/10.24263/2304974x-2021-10-4-5. [18]. m. shoukat, a. sorrentino, (2021), cereal β-glucan: a promising prebiotic polysaccharide and its impact on the gut health. 56 (5), international journal of food science & technology, 2088-2097. https://doi.org/10.1111/ijfs.14971. [19]. m. aljewicz, a. florczuk, a. dąbrowska, influence of β-glucan structures and contents on the functional properties of low-fat ice cream during storage. 70 (3), polish journal of food and nutrition sciences, (2020), 233-240. https://doi.org/10.31883/pjfns/120915. [20]. f. shibani, s. asadollahi, m. eshaghi, the effect of beta-glucan as a fat substitute on the sensory and physico-chemical properties of low-fat ice cream. 1 (1), journal of food safety and processing, (2021), 7184. [21]. o.v. kochubej-litvinenko, a.v. timchuk, t.g. osmak, tehnologії konservuvannja ta zberіgannja molochnih produktіv: laboratornij praktikum dlja zdobuvachіv osvіtn'ogo stupenja «bakalavr» specіal'nostі 181 «harchovі tehnologії» osvіtn'oprofesіjnoї programi «harchovі tehnologії ta іnzhenerіja» dennoї formi navch. [technologies of preservation and storage of dairy products: laboratory workshop for applicants for the degree of "bachelor" specialty 181 "food technology" of educational and professional program "food technology and engineering" of full-time education], k.: nuht, (2020), 93 s (in ukrainian). [22]. m. klimenko, і. kishen''ko, o. grek, viznachennja vodota zhiroutrimuval'noї zdatnostі harchovoї sirovini [determination of water holding and fat binding capacity of food raw materials]. 3, tavrіjs'kij vіsnik, herson, (1998), s. 176-177 (in ukrainian). [23]. t. p. arsen''eva, spravochnik tehnologa molochnogo proizvodstva. t.4. morozhenoe [handbook of dairy production technologist. v. 4. ice cream], spb: giord, (2003), 184s (in russian). [24]. g.e. polіshuk, formuvannja skladnih dispersnih sistem moroziva molochnogo z natural'nimi komponentami: dis. … doktora tehn. nauk: 05.18.04 «tehnologіja m j̀asnih, molochnih produktіv ta produktіv z gіdrobіontіv [formation of complex dispersed systems of milk ice cream with natural components: dis. … doctor of technical sciences: 05.18.04 «technology of meat, dairy products and products from hydrobionts]. k., (2013), 248 s (in ukrainian). [25]. g. n. krus'', a. m. shaligіna, z. v. volokitina, metody issledovanija moloka i https://doi.org/10.1016/j.ijbiomac.2010.01.002 https://doi.org/10.1016/s0963-9969(00)00020-x https://doi.org/10.1016/j.jff.2018.02.035 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream, food and environment safety, volume xxi, issue 2 – 2022, pag. 116 – 128 128 molochnyh produktov [methods of research of milk and dairy products]. m.: kolos, (2002), 368 s (in russian). [26]. g. polischuk, o. kochubeilytvynenko, t. osmak, u. kuzmyk, o. bass, a. mykhalevych, v. sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein. 20 (1), food and environment safety, 2021, 13-20. [27]. v. sapiga, g. polischuk, t. osmak (fedchenko), a. mykhalevych, m. maslikov, scientific explanation of the composition and technological modes of manufacture of dairy ice cream with vegetable puree. 7 (1), ukrainian journal of food science, (2019), p. 83-91. https://doi.org/10.24263/23101008-2019-7-1-10. [28]. s. berggren, water holding capacity and viscosity of ingredients from oats: the effect of b-glucan and starch content, particle size, ph and temperature (dissertation), (2018), retrieved from http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-70544. [29]. s. karp, j. wyrwisz, m. a. kurek, the impact of different levels of oat β-glucan and water on gluten-free cake rheology and physicochemical characterisation. 57 (10), journal of food science and technology, (2020), 3628–3638. https://doi.org/10.1007/s13197-020-04395-5. [30]. a. m. h. abdel-haleem, r.a. awad, some quality attributes of low-fat ice cream substituted with hulless barley fl our and barley β-glucan. 52 (10), journal food science & technology, (2015), 6425–6434. https://doi.org/10.1007/s13197-015-1755-x. issn 1583-2295 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xix, issue 4 31 december 2020 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientificjournalfoodandenvironmentsafety (issn 2068 – 6609) ispublished byuniversity publishinghouse of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails:fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishinghouse of suceava. allrightreserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de josuniversityof galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare universityof suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. klimentohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare universityof suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of foodscience, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 contents: 1. evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire armel f. zoro, lessoy t. zoue, sébastien l. niamke 99-108 2. ev alu ati on of th e t ox ico l og i ca l sa fet y of a freez e -d ri ed p robi otic p rep a ra ti on zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova 109 115 3. the influence of viability on phenolic content, conductivity and sugars efflux, and the relationships between these indices in wheat seeds marcel avramiuc 116 121 4. biogenic amine amount in ground pork and beef meat octavian baston, octavian barna 122 126 5. in flu en ce o f di ffe rent che mi cal ag en ts on the a dult e red milk ph ysica l pr ope rt ies co rr e cti on mircea oroian, daniel bernicu 127 133 6. importance of local cereal flours in the diet habit of consumers in the district of abidjan cote d’ivoire yevi d. nguessan, micael e. bedikou, rose-monde megnanou, sébastien l. niamke 134 146 7. evaluation of the relationships between metallic ions migrated from aisi304 and aisi321 stainless steel samples into food simulant solutions at various stirring degrees silviu-gabriel stroe 147 153 8. types of synonymy in food engineering terminology alice iuliana rosu 154 159 9. new starches from improved yellow colored cassava: enzymatic and acid sturdiness and potential of industrial utilization ginette g. doué1 , micaël e. bédikou , gisèle a. koua, sévérin k. kra , sébastien l. niamké 160 168 10. the prosp ects of sa fe ty a nd mi crobi ol ogi cal stab il i ty imp r ov em ent o f bak ery p roduc ts by u sin g ph yt o-a ddit iv es tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko 169 178 11. author instructions i v 12. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 articol porumb doi: https://doi.org/10.4316/fens.2021.007 53 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 12021, pag. 53 60 life cycle assessment of wastewater from dairy industry diana cornelia adamovici costea 1 , *cristina ghinea 1 1faculty of food engineering, stefan cel mare university of suceava, romania, cristina.ghinea@fia.usv.ro *corresponding author received 9th march 2021, accepted 30th march 2021 abstract. in this study, life cycle assessment (lca) was used to investigated the environmental impacts associated with dairy wastewater treatment. the composition of dairy wastewater was investigated by applying various water quality methods. indicators like ph, suspension, chemical oxygen demand (cod), biochemical oxygen demand (bod), extractable substances, detergents, sulfates (so4 2-), nitrates (no3 -), nitrites (no2 -), n, p and others were determined. also, the composition of treated water, at the exit of the treatment plant, was established. monitoring of the effluent composition was performed for three years, the determinations being performed every month in triplicate. for the environmental inventory analysis, the average of the values obtained for quality indicators in 2015 was considered, the year in which the highest values for the quality indicators were registered. gabi software was used for the environmental impact calculation. results showed that treatment of dairy wastewater influences mostly the eutrophication potential. other impact categories are mainly influenced by electricity production for consumption in the wastewater treatment process. it was observed that acidification potential has the highest value, followed by the global warming potential, the photochemical ozone formation potential, and human toxicity potential, respectively. keywords: dairy, environmental impacts, life cycle, milk 1. introduction dairy industry includes various processes, from raw milk pasteurization to production of different food products based on raw milk transformation (yoghurt, cheese, cream, butter, and various dessert types) [1, 2]. annually, approximately 850 million tons of raw milk are produced globally [3], while, for example, in the eu in 2019, 158.2 million tons were produced, of which 1.122 million tons in romania [4]. in milk processing, water is used in every step included in the technological process, and considerable water volumes are consumed in washing operations [5, 6]. it is estimated that for every liter of processed milk, between one and five liters of wastewater are generated [7]. but, other authors [8] have reported that for one liter of processed milk, between 0.2 and 10 l of wastewater are generated. the waters resulting from the dairy industry are characterized by a high content of organic matter, nutrient and extreme ph variations, and if these waters are not treated properly, serious environmental issues can occur [1, 9]. the discharge of this wastewater, without proper treatment or without being treated would lead to rapid depletion of dissolved oxygen from water bodies, due to high organic load leading to the destruction of aquatic life and environmental damage [1]. the environmental impacts associated with wastewater treatment can be determined by applying life cycle assessment (lca). widely used in the field of wastewater treatment [10, 11], this tool could provide useful information for: improvement of the wastewater plant operation, comparison of different alternative systems, development http://www.fia.usv.ro/fiajournal mailto:cristina.ghinea@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, food and environment safety, volume xx, issue 1 – 2021, pag. 53 60 54 of new technologies or nutrient recycling [12 -14]. there are some recent studies based on assessment of dairy effluents from environmental point of view, for example: stanchev et al. [6] evaluated the environmental performance of anaerobic treatment of dairy effluents; elginoz et al. [15] investigated the environmental impacts associated with production of volatile fatty acids from dairy wastewater; queiroz et al. [16] determined the environmental impacts associated with application of aquatic macrophytes for phytoremediation of dairy effluents. the aim of this study was to investigate the environmental impacts associated with treatment of wastewater generated from a dairy factory in romania. 2. case study the wastewater treatment plant, considered in this study, includes the following stages (fig. 1): particle separation, standardization, flocculation and coagulation, primary and secondary decantation, biological phase, flotation and sludge collection. the dairy effluents composition is determined at the entrance to the station and at the exit. wastewater quality indicators, maximum permitted concentrations according to [17] and analysis methods used in this study are presented in table 1. 3. lca methodology 3.1. goal, system boundaries, functional unit the goal of this study was to determine the environmental impacts of wastewater from dairy industry by applying lca methodology, in accordance with [18, 19]. the specific objectives of this study are: performing an inventory analysis on the use of materials and resource consumption with wastewater treatment; identification of the environmental impacts of the researched system. system boundaries: in general, in lca studies the construction, operation and demolition phases are within the system limit, but this study is limited only to the operation phase. fig. 1. the flow diagram of treatment processes food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, food and environment safety, volume xx, issue 1 – 2021, pag. 53 60 55 table 1. wastewater quality indicators and analysis methods no. quality indicators allowed values [17] methods of analysis used in this study 1 ph (ph unit) 6.5-8.5 sr iso 10523: 2012. water quality. determination of ph 2 suspension (mg/l) 60 sr en 872: 2005 water quality. determination of suspended solids content. filtration method on fiberglass filters 3 chemical oxygen demand cod (mg o2/l) 125 is015705: 2002 (e). water quality. determination of chemical oxygen consumption 4 biochemical oxygen demand bod (mg o2/l) 25 sr en 1899-2 / 2002. determination of biochemical oxygen consumption after n days (bodn). part 2: method for undiluted samples 5 extractable substances (mg/l) 20 method 1664 b. determination of extractable substances 6 detergents (mg/l) 0.5 sr en 903/2003. water quality. determination of anionic surfactants by measuring the methylene blue index mbas 7 sulfates (so4 2-) (mg/l) 600 stas 8601-70. surface water and wastewater. determination of sulfates 8 ammonium (nh4 +) (mg/l) 2 sr iso 11905. water quality. determination of nitrogen 9 total phosphorus (p) (mg/l) 2 sr en iso 6878/2005. water quality. determination of phosphorus. ammonium molybdate spectrometric method. 10 total nitrogen (n) (mg/l) 10 sr iso 11905. water quality. determination of nitrogen 11 filtered residue (mg/l) 2000 stas 9187/84. surface water, groundwater. residue determination 12 nitrates (no3 -) (mg/l) 25 sr iso 7890-3 / 2000. water quality. determination of nitrate content. sulfosalicylic acid spectrometric method 13 nitrites (no2 -) (mg/l) 1 sr iso 11905. water quality. determination of nitrogen 14 chlorides (cl-) 500 stas 8663-70. surface water and wastewater. determination of chlorides the functional unit of the study allows the reporting of all data collected in the inventory phase and is the basis for comparison for the wastewater treatment systems. the functional unit adopted in this study is the volume of treated wastewater (m3) in a day in the treatment plant (fig. 1). 3.2. inventory analysis the volume of water entering in the treatment plant is represented by a minimum volume of 191.357 m3/day and a maximum of 287.193 m3/day. there is an installation for measuring the flows of treated water discharged into the emissary. the water norm for the main products of manufacture is for 1000 l milk/day. from fig. 2a, which presents the volume of wastewater discharged from a dairy plant in the period 2015-2017, it can be observed that the volume decreased over time due to the fact that the largest amount of wastewater was recirculated. in fig. 2b is illustrated the variation over time of the filterable residue amount per l of wastewater discharged from the dairy plant considered in this study. it can be seen that the values have increased over time. in accordance with [17] on the setting of pollutant loading limits for wastewater discharges to natural receptors, the indicators followed largely fall within these limits. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, food and environment safety, volume xx, issue 1 – 2021, pag. 53 60 56 fig. 2. a) volume of wastewater discharged (thousand m3) from dairy industry; b) filterable residue indicator (mg/l) the values of the bod/cod ratio assigned for the wastewater at the station exit during 2015-2017 indicate the presence of easily biodegradable organic substances. according to fig. 3a cod values are higher and a decrease of them is observed over time. exceptions are ammonium and sulfates, which exceed the maximum permitted concentrations in 2015 (fig. 3b). also, an exception are extractable substances, phosphorus and detergents, which have exceeded the maximum permitted concentrations in 2015 (fig. 3c). from fig. 3b it can be seen that the values decreased over time for the indicators nh4 +, n and so4 2-, while for chlorides there was a lower value in 2016 compared to 2015, after which there was again an increase in the concentration of chlorides emitted in 2017. from fig. 3c it can be seen that there has been a decrease in concentration over time for the following quality indicators: extractable substances, phosphorus, detergents, while for indicators no3 and no2 there is a decrease in emitted concentrations recorded in 2016, followed by an increase in 2017. the values of the indicators determined after collecting and analyzing the samples from the water body, in which the treated wastewater was discharged, are illustrated in fig. 4. from fig. 4a it can be observed that the values of the cod indicator decreased in 2016 and 2017, compared to the increase registered in 2015. from fig. 4b it can be seen that the values of the indicators followed are within the allowed values. from fig. 4c it can be seen that the values of the no3 indicator increase in 2015 and decrease in the following years. the energy consumption of the treatment plant is 450 kwh/month, 5400 kwh/year. the treatment plant also uses 40 kg fecl3/year, 80 kg polyelectrolyte /year, 36 kg naoh/koh/year, 36 kg hno3/year, 24 kg h3po4/year, 36 kg h2o2/year, 48 kg polymers/year. the amount of sludge produced is 1500 kg/year. there are also produced 200 kg/year of plastic and cardboard packaging and 50 kg glass waste /year, solid waste 3 m3/month, sand 80 kg/month, grease 40 kg/month. the sludge from the treatment plant is dehydrated and temporarily stored in the transport container, until it is picked up by authorized operators. it can be used in agriculture, land improvement, wet oxidation and gasification. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, food and environment safety, volume xx, issue 1 – 2021, pag. 53 60 57 fig. 3. quality indicators of wastewater from a dairy factory between 2015-2017: a) biochemical oxygen demand (bod) and chemical oxygen demand (cod); b) ammonium (nh4 +), total nitrogen (n), sulfates (so4 2-), chlorides (cl-); c) nitrates (no3 -), nitrites (no2 -), phosphorus (p), extractable substances, synthetic detergents fig. 4. quality indicators values of the water collected from the water body in which the treated water is discharged: a) biochemical oxygen demand (bod) and chemical oxygen demand (cod); b) ammonium (nh4 +), total nitrogen (n), sulfates (so4 2-), chlorides (cl-); c) nitrates (no3 -), nitrites (no2 -), phosphorus (p) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, volume xx, issue 1 – 2021, pag. 53 60 58 3.3. life cycle impact assessment, results and discussion gabi software was used to obtain the environmental impacts of wastewater treatment plant. cml 2001-2016 and edip 2003 methods were selected for the assessment. the limits of the system have a major influence on the results of life cycle assessment studies. if the impact values obtained are positive, this mean a negative impact on the environment. fig. 5 show values for all impact categories due to electricity consumption, especially. the process itself influences the eutrophication potential the most and the other impact categories the least. from fig. 5 it can be seen that the acidification potential has the highest value, followed by the global warming potential, the photochemical ozone formation potential, and human toxicity potential, respectively. it can be observed that the lowest value was obtained for the eutrophication potential. results showed that the values obtained for the potential eutrophication impact category are negative for the stages of primary settling, secondary settling, flotation and biological stage (-1.24e-05 kg po4 3-eq.; -9.26e-06 kg po4 3-eq.; 6.73e-06 kg po4 3-eq.; -1.21e-05 kg po4 3eq.) which means environmental savings. instead, for the mechanical stage the value is positive which means negative impact on the environment. the wastewater treatment plant from a dairy plant is designed to reduce the impact on the environment. by implementing all the measures imposed by environmental protection programs and by adopting the most modern technologies, the impact on the environment will be insignificant. an impact on the environment could be possible due to accidental discharges of pollutants into natural receptors due to improper operation of the biological wastewater treatment stage, but this would only happen in the event of a malfunction. from the specific results on the impact obtained with the help of gabi software, it can be said that the process itself influences the eutrophication potential the most and the other impact categories the least. the use of wastewater instead of drinking water for non-potable industrial use should be improved in the treatment system. wastewater can be reused after the application of a tertiary treatment consisting of several stages, including reverse osmosis [20]. fig. 5. normalized values of environmental impacts obtained by applying: a) cml2001-2016 (ap acidification potential, ep eutrophication potential, gwp global warming potential, htp human toxicity potential, pocp photochemical ozone formation potential, pe persons equiv.) and b) edip 2003 (apacidification potential, gwglobal warming, pofh photochemical ozone formation-impact on human and material health, pofv photochemical ozone formation impact on vegetation, te terrestrial eutrophication) methods food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, volume xx, issue 1 – 2021, pag. 53 60 59 another improvement to the treatment system would be the recognition of sludge as a resource and not as waste. using sludge as a source of energy and resource recovery is a good alternative for its management, given the requirements of legislation and the principles of the circular economy. recognition of sludge as a resource, not as waste, has led researchers to consider recovering valuable components from sludge, such as carbon and nutrients. a sustainable solution can be energy production from wastewater sludge [21]. 4. conclusions life cycle assessment has proved to be a useful tool that can be used to assess the environmental impact of wastewater treatment systems. the composition of wastewater from a dairy factory is quite complex, as it contains milk residues, dairy products, by-products, detergents and other components. due to mechanical, chemical and biological processes, wastewater will be treated in a percentage of over 80%. the biological methods used to treat wastewater are the most effective and economical for removing organic substances. in accordance with [17] on the setting of pollutant loading limits for wastewater discharges into natural receptors, the indicators pursued largely fall within these limits. given the entire life cycle of wastewater treatment systems and the relative contribution of each phase, inventory analyzes were carried out on the use of materials, resource consumption and the impact on the environment associated with wastewater treatment. after environmental impact evaluation, it can be concluded that some environmental savings are performed due to the fact that the values for the potential eutrophication impact category are negative for the primary settling, secondary settling, flotation and biological stage stages. according to the results obtained, energy is the main contributor to the impact categories of abiotic depletion and global warming. the results indicated that the process itself mainly influences the eutrophication potential. 5. references [1]. slavov a.k., general characteristics and treatment possibilities of dairy wastewater – a review, food technology and biotechnology, 55:14–28, (2017) [2]. marazzi f., bellucci m., fantasia t., ficara e., mezzanotte v., interactions between microalgae and bacteria in the treatment of wastewater from milk whey processing, water, 12:297, (2020) [3]. ghinea c., leahu a., life cycle assessment of fermented milk: yogurt production, ovidius university annals of chemistry, 31:49-54, (2020) [4]. eurostat, milk and milk product statistics, (2021), https://ec.europa.eu/eurostat/statisticsexplained/inde x.php/milk_and_milk_product_statistics#milk_pro duction [5]. sarkar b., chakrabarti p.p., vijaykumar a., kale v., wastewater treatment in dairy industries – possibility of reuse, desalination, 195:141–52, (2006) [6]. stanchev p., vasilaki v., egas d., colon j., ponsá s., katsou e., multilevel environmental assessment of the anaerobic treatment of dairy processing effluents in the context of circular economy, journal of cleaner production, 261: 121139, (2020) [7]. queiroz r.c.s., andrade r.s., dantas i.r., ribeiro v.s., rodrigues l.b., almeida neto j.a., use of native aquatic macrophytes in the reduction of organic matter from dairy effluents, international journal of phytoremediation, 19(8):781-788, (2017) [8]. akansha j., nidheesha p.v., gopinath a., anupama k.v., suresh kumara m., treatment of dairy industry wastewater by combined aerated electrocoagulation and phytoremediation process, chemosphere, 253: 126652, (2020) [9]. sivrioğlu o., yonar t., determination of the acute toxicities of physicochemical pretreatment and advanced oxidation processes applied to dairy effluents on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 diana cornelia adamovici costea, cristina ghinea, life cycle assessment of wastewater from dairy industry, volume xx, issue 1 – 2021, pag. 53 60 60 activated sludge, journal of dairy science, 98:2337-2344, (2015) [10]. corominas l., foley j., guest j.s., hospido a., larsen h.f., morera s., shaw a., life cycle assessment applied to wastewater treatment: state of the art, water research, 47:5480-5492, (2013) [11]. corominas l., byrne d.m., guest j.s., hospido a., roux p., shaw a., short m.d., the application of life cycle assessment (lca) to wastewater treatment: a best practice guide and critical review, water research, 184:116058, (2020) [12]. bai s., wang x., zhang x., zhao x., ren n., life cycle assessment in wastewater treatment: influence of site-oriented normalization factors, life cycle impact assessment methods, and weighting methods, rsc advances, 7:26335, (2017) [13]. lam k.l., zlatanović l., van der hoek j.p., life cycle assessment of nutrient recycling from wastewater: a critical review, water research, 173:115519, (2020) [14]. zhang y., zhang c., qiu y., li b., pang h., xue y., liu y., yuan z., huang x., wastewater treatment technology selection under various influent conditions and effluent standards based on life cycle assessment, resources, conservation and recycling,154:104562, (2020) [15]. elginoz n., atasoy m., finnveden g., cetecioglu z., ex-ante life cycle assessment of volatile fatty acid production from dairy wastewater, journal of cleaner production, 269:122267, (2020) [16]. queiroz r.c.s., maranduba h.l., hafner m.b., rodrigues l.b., neto j.a.a., life cycle thinking applied to phytoremediation of dairy wastewater using aquatic macrophytes for treatment and biomass production, journal of cleaner production, 267:122006, (2020) [17]. decision no. 352/2005 regarding the modification and completion of the government decision no. 188/2002 approving some norms regarding the discharge conditions in the aquatic environment of the waste waters, normative on the establishment of limits for pollutant loading of industrial and urban waste water to evacuation to natural receptors, ntpa-001/2005, (2005) [18]. iso 14040:2006, environmental management life cycle assessment principles and framework, geneva, international standardization organization, (2006) [19]. iso 14044:2006, environmental management life cycle assessment requirements and guidelines, geneva, international standardization organization, (2006) [20]. pintilie l., torres c.m., teodosiu c., castells f., urban wastewater reclamation for industrial reuse: an lca case study, journal of cleaner production, 139: 1-14, (2016) [21]. teodosiu c., gilca a.f., barjoveanu g., fiore s., emerging pollutants removal through advanced drinking water treatment: a review on processes and environmental performances assessment, journal of cleaner production, 197: 1210-1221, (2018) doi: https://doi.org/10.4316/fens.2022.019 198 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 198 208 extraction of natural antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study * linda loucif seiad 1 , soraya demim 1 , mohamed loucif seiad 2 , hassina boussak 3 , souad hammadou 4 1 department of chemistry, faculty of sciences, university m’hamed bougara, boumerdes 35000, algeria, loucifsiad_linda@univ-boumerdes.dz, louciseiadl@yahoo.fr 2 center for development of advanced technologies, baba hassen, algiers, algeria, 3 coatings laboratory, materials and environment, university m’hamed bougara, boumerdes 35000, algeria, 4petrochemical synthesis laboratory, fhc-umbb, university m’hamed bougara, boumerdes 35000, algeria, * corresponding author received 29th july 2022, accepted 29th september 2022 abstract: the objectif of this study was to improve extraction of natural antioxidants (nas) found in algeria rosmarinus officinalis l. using cleaner extraction processes. to verify the single factors effects, a factorial design (fd) was used to study the impact of the main process variables (time, solid-to-liquid ratio, and temperature) on extraction efficiency. total polyphenol content (tpc), total flavonoids content (tfc) and antioxidant activity (aa) were measured to control the extraction process efficiency on different experimental conditions. the best experimental result was obtained at 30°c for 30 min and 0.05 g/ml. the value of tpc, tfc and aa was respectively, 121.80 mg gae/g dw, 44.01 mg qe/g dw and 96.36 %. the correlation between tpc, tfc and aa remains unclear. the extraction of polyphenols was shown to obey first order kinetic with a 0.045 min-1coefficient for the fast period and 0.05 min-1 for the slow period. finally, the plant morphology investigated by sem revealed physical changes on plant material. keywords: rosmarinus officinalis l., nas, modeling, kinetic. 1. introduction in food industry, synthetic antioxidants (sas) [butylated hydroxytoluene (bht), butylated hydroxyanisole (bha), tertiary butyl hydroquinone (tbhq)...] have been used for a long time as food’s preserved [1]. however, these products cause many problems to the food quality (degradation) and the human health when consumed. many recent studies have focused on their high toxicity [2]. moreover, research was conducted to replace these products with substances derived from plants. for these reasons, scientists aimed to substitute sas by natural antioxidants (nas). they are extracted from plants by cleaner extraction processes. the major classes of compounds with antioxidant activity are: vitamins (c and e), carotenoids, flavonoids, phenolic acids, tannins, lignans and stilbenes [3]. nas can be extracted from plants and substitute the synthetic antioxidants, source of toxicity [4]. among these plants, rosemary, which is very widespread plant in the mediterranean region was used as food preservative [5]. for these reasons, some studies on this spice and its antioxidant properties were conducted and reported. several techniques were used to extract nas from plants. these include conventional and non-conventional extractions: solid-liquid extraction, heated reflux extraction, ultrasoundassisted extraction, microwave-assisted extraction, supercritical fluid extraction, accelerated, and batch extraction technique. the latter is preferred because it always gives good outcome. mailto:loucifsiad_linda@univ-boumerdes.dz mailto:louciseiadl@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 199 the aim of this work was the valorization of algerian plant rosmarinus officinalis l. and improvement of the extraction process using hydroalcoholic mixture. only a few papers about the plant used in this work are available, which motivated the actual study. furthermore, another motivation for the study was the scarce information on the application of design of experiment (doe) in the polyphenols extraction from rosmarinus officinalis l., and also the impact of extraction on plant microstructure. in this study, factorial design (fd) was used to verify the single factors effect’s (time, solid-to-liquid ratio and temperature) on extraction efficiency. tpc, tfc and aa were measured to control the efficiency of extraction process on different experimental conditions. the interactions between factors were determined using rsm. the extraction kinetics was also determined. finally, the morphology of plant was analyzed using sem before and after extraction. the effect of plant microstructure on the extraction mechanisms was discussed in detail in this paper. 2. matherials and methods 2.1. characterization of plant matrix rosmarinus officinalis l. was collected in algiers (algeria) during the month of march. to preserve the molecules' greatest integrity, the leaves were cut off the stems and let to dry at room temperature for three weeks. the dried leaves were finely powdered in a mortar with a sieve of 1.12 mm after being crushed into little pieces. the powder was stored protected from light and moisture for subsequent use. 2.2. reagents and chemicals folin-ciocalteu reagent, gallic acid quercetine and (dpph) (2, 2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate) were purchased from sigma–aldrich. aluminum chloride was obtained from merck. additionally, etoh of 96% purity (rieldel-de haen), meoh (sigmaaldrich), xylen (cheminova prs) and sodium carbonate (bhd chemicals ltd) were used. 2.3. analysis 2.3.1. total polyphenols content according to singleton et al. [6], tpc was determined by folin-ciocalteu and expressed as gallic acid equivalents (mg gae/g dw). using a spectrophotometer (uv-vis secomam s250), absorbance was measured at 760 nm. the results were calculated using a standard gallic acid curve of (1–30 μg/ml). 2.3.2. total flavonoids content the miliauskas technique [7] was used to measure tfc, which was expressed as quercetin equivalents (mg qe/g dw). a spectrophotometer (uv-vis secomam s250) was used to detect absorbance at 430 nm. the results were calculated using a standard curve of quercetin (1-40 μg/ml). 2.3.3. dpph scavenging assay the ability of the extracts to scavenge dpph radical was assessed spectrophotometrically as described by que et al. [8]. 2.3.4. morphology analysis the samples were observed using a scanning electron microscope (jeol jsm 6360lv) with a maximum resolution and voltage (50 nm, 30kv). these observations serve to locate precisely the sites producing active molecules and to visualize any modification after extraction. 2.4. experimental design and statistical analysis food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 200 fd was used to study the effects of three variables: extraction time (x1), solid-toliquid ratio (x2) and temperature (x3). the low and high levels each factor in the experimental design are showed in table 1. the variables responses (tpc, tfc and aa) were expressed individually as a function of the independent variables. the experimental design presented eight (8) combinations (table 2), including three replicates of the central point in order to estimate pure error and to assess the fit lack of the proposed models. all the experiments were performed randomly. a first-order polynomial equation (1) was used to express tpc (y1), tfc (y2) and aa (y3) of rosemary as a function of independent variables as-follow: 𝑌𝑖 = β0 + β1𝑋1 + β2𝑋2 + β3𝑋3 + β12𝑋1𝑋2 + β13𝑋1𝑋3 + β23𝑋1𝑋2 (1) where yi represents the response variables, β0 is the model constant, βi and βij are the linear and interaction coefficients respectively. xi and xj are the levels of the independent variables. the model appropriateness was also evaluated by the coefficient model (r2) and adjusted coefficient model (r2adj) and also by the statically significance model (p-value model) properly tested by anova. 3. results and discussion 3.1 choice of solvent nas are usually extracted by conventional solvent extraction methods. ethanol (etoh) was used instead of methanol due to its high toxicity. moreover, etoh has been classified since 2012 as gras (generally recognized as safe) by the fda (food and drug administration, usa). however, it is not recommended to use it alone when extracting nas from plants because it causes the deshydratation of vegetable cells. however, nas are usually linked to sugar. etoh can be blended with water in any proportion. studies showed that tpc yield increased by increasing water content [9]. etoh (80 %) was selected as solvent in the actual study (batch extraction). 3.2. fitting model three factors that may affect the experimental responses were selected as independent variables at three levels. the minimum, central, and maximum levels for each factor and the different independent variables of the extracts are listed in table 1. the experiments were performed according to the design of experiments shown in tables 2. the same table displays observed and predicted responses on two levels. statistical analysis of experimental data was performed by using the statgraphics plus software version 5.1. table 1. coded/real levels used in factorial design of algerian rosmarinus officinalis l. batch extraction. coded levels -1 0 +1 time (min) (x1) 30 60 90 solid-to-liquid ratio (g/ml) (x2) 0.05 0.1 0.15 temperature (°c) (x3) 30 45 60 table 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 201 factorial design of three variables with their experimental and predicted responses of algerian rosmarinus officinalis l. batch extraction. run coded variable tpc (mg gae/gdw) tfc (mg qe/gdw) aa (%) x1 x2 x3 experimental predicted experimental predicted experimental predicted 1 +1 +1 +1 80.65 88.53 19.13 18.86 92.68 95.92 2 -1 +1 +1 34.40 31.33 12.43 12.46 95.08 96.30 3 +1 -1 +1 110.06 106.99 40.20 40.23 53.01 54.23 4 -1 -1 +1 95.45 103.33 42.10 41.83 48.83 52.07 5 +1 +1 -1 20.91 17.84 13.98 14 47.27 48.49 6 -1 +1 -1 12.15 20.03 11.73 11.46 91.14 94.38 7 +1 -1 -1 55.12 62.99 38.85 38.58 51.00 54.24 8 -1 -1 -1 121.80 118.73 44.01 44.03 96.36 97.58 9 0 0 0 76.51 68.72 25.71 27.68 79.01 74.15 10 0 0 0 70.21 68.72 29.01 27.68 81.06 74.15 11 0 0 0 78.64 68.72 27.31 27.68 80.20 74.15 the results obtained indicate that the levels of tpc ranged from 12.15 mg gae/g dw to 121.80 mg gae/g dw. the best results related to the lower levels were observed in run 8 at 121.80 mg gae/g dw followed by 110.06 mg gae/g dw on run 3. the levels of tf ranged from 12.43 mg qe/g dw to 44.01 mg qe/g dw. the best results were observed in run 8 at 44.01 mg qe/g dw followed by 42.10 mg qe/g dw in run 4. for aa the level ranged from 47.27 % to 96.36 %, the best results were observed also in run 8 at 96.36 % followed by 95.08 % in run 2. overall, run 8 gave the best values for the three responses. it is about 121.80 mg gae/g dw for polyphenols, 96.36% for aa and for flavonoids a value of 44.01 mg qe/g dw. these results indicate the presence of high content of polyphenols. similar results were obtained using the same reagent on turkish rosemary [10], and it varied from 147.3 to 34.1 mg gae/g dw. the authors pointed out that the extract composition changes according to many factors (e.g., the type of sample, location, time, etc). the physicochemical nature of the individual polyphenols in the extract has probably more contribution to the aa against the tpc. we noted that some researchers have shown lower phenolic contents of rosemary extract [11]. the results for tpc, tfc and aao tudied by analysis of variance (anova) were summarized in table 3. for tpc, four effects have p-values less than 0.05, indicating that they are significantly different from zero at the 95% confidence level. for tfc and aa, two effects and four effects have p-values less than 0.05. in the present study, the values of r2 were 96.4%, 99.7% and 96.1% for tpc, tfc and aa respectively. the values of r2adj were 90.9 %, 98.9% and 90.3% for tpc, tfc and aa respectively. the durbin-watson (dw) values for tpc, tfc and aa were 1.9, 1.96 and 1.56 respectively. since the dw value is greater than 1.4, there is no serious auto correction in the residuals. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 202 table 3. analysis of variance (anova) for tpc, tfc and aa of algerian rosmarinus officinalis l.batch extraction. variable response source of variation sum of square df mean square f-ration p-value tpc x1 1.08045 1 1.08045 0.01 0.9264 x2 6863.23 1 6863.23 61.38 0.0014* x3 1528.49 1 1528.49 13.67 0.0209* x1 x2 1433.27 1 1433.27 12.82 0.0232* x1 x3 1763.59 1 1763.59 15.77 0.0165* x2 x3 356.445 1 356.445 3.19 0.1487 tfc x1 0.446512 1 0.446512 0.29 0.6168 x2 1455.03 1 1455.03 956.28 0.0000* x3 3.49801 1 3.49801 2.30 0.2040 x1 x2 32.04 1 32.04 21.06 0.0101* x1 x3 7.43051 1 7.43051 4.88 0.0916 x2 x3 5.13601 1 5.13601 3.38 0.1400 aao x1 955.938 1 955.938 24.53 0.0077* x2 740.548 1 740.548 19.01 0.0121* x3 1.83361 1 1.83361 0.05 0.8389 x1 x2 3.23851 1 3.23851 0.08 0.7874 x1 x3 1035.35 1 1035.35 26.57 0.0067* x2 x3 1125.04 1 1125.04 28.88 0.0058* the effect of extraction factors is presented in figure 1. pareto chart traduces anova for operating parameters and their combinations. it shows their own effects from the most to the least significant level at 5%. the vertical line indicates the statistical level. figure 1a shows that for tpc the interaction of x2 has a higher effect, followed by the interaction of (x1/x3), the effect of x3 and of (x1/x2) respectively. the effect of the extraction factors on tfc is presented in figure 1b. this figure shows that one parameter (x2) has a significant effect. figure1c shows that for aa, the interaction of (x2/x3) has a higher effect. it is followed by the interaction of (x1/x3), the effect of x1 and of x2 respectively. regarding the tpc and tfc, the solid-toliquid ratio has a higher effect. for aa, the solid-to-liquid ratio has also an effect but it is not the higher effect. the solid-to-liquid ratio has an effect because increasing the solvent proportion leads to increase of the concentration gradient, which increases the diffusion of solid compounds in the solvent. in the actual condition, temperature had an effect for tpc extraction. this result was confirmed by many authors [12]. increasing temperature accelerates mass transfer, which increases cellulose activity and polyphenols solubility [12]. some previous work has food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 203 shown that extraction temperature was the main parameter. the increase of temperature implies directly an increase of diffusion coefficient and solubility of the compounds. up the temperature of 50 °c phenolic compounds can be degraded completely]. the time had no significant effect on tfc and aa but not on tpc. fig. 1. pareto chart for tpc, tfc and aa effects from algerian rosmarinus officinalis l. batch extraction (a): pareto chart for tpc, (b): pareto chart for tfc and (c): pareto chart for aa. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 204 the fitted mathematical models (y1, y2, and y3) for tpc, tfc and aa with 95% for coefficient level are given in eq 2, 3 and 4 respectively: 𝑌1 = 68.72 + 0.37𝑋1 − 29.29𝑋2 + 13.82𝑋3 + 13.38𝑋1𝑋2 + 14.85𝑋1𝑋3 + 6.68𝑋2𝑋3 (2) 𝑌2 = 27.68 + 0.24𝑋1 − 13.43𝑋2 + 0.66𝑋3 + 2.00𝑋1𝑋2 + 0.96𝑋1𝑋3 + 0.80𝑋2𝑋3 (3) 𝑌3 = 74.15 − 10.93𝑋1 + 9.62𝑋2 + 0.48𝑋3 − 0.64𝑋1𝑋2 + 11.37𝑋1𝑋3 + 11.86𝑋2𝑋3 (4) the values of r2 for tpc, tfc and aa were 96.4%, 99.7% and 96.1% respectively. it indicates that there is a good correlation between the experimental and predicted data. 3.3. correlation between the responses of tpc and tfc figure 2 does not show a relationship between aa, tpc and tfc. this result is in agreement with investigations on aa of plant extracts by other authors [13]. it is due probably to synergetic effects between the extracted products. figure 3 does not show a relationship between tpc and tfc. however, the correlation between tpc, tfc and aa remains unclear. the recovery of aa may indicate that this is only partially related to the compounds observed here. it may also indicate the presence of other chemicals involved in its activity. antioxidants present in rosemary extracts are not limited only to polyphenols [14]. moreover, it is important to consider the occurrence of synergism between the chemical compounds in the extract. aa dependes on chemical structure and the interaction between antioxidant substances [15]. previous studies indicated that the correlation degree of aa depends not only on the tpc, but also on the extracts composition. fig. 2. aa plotted according to tpc and tfc of extracts from algerian rosmarinus officinalis l. batch extraction. fig. 3. relationship between tpc and tfc of extracts from algerian rosmarinus officinalis l. batch extraction. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 205 thus, kinetic examination is required to generate detailed data, which could be used for comparison of the efficiency of various extraction methodologies and for the engineering of improved extraction processes. we will examine the kinetics of polyphenols in next section. 3.4. kinetics studies to identify the model that better described polyphenols extraction. concentration values were plotted as a function of t. the best model fitted to the extraction kinetics using non-linear regression was described by the following equation: [y = a(1 − e−bx)] (5) this suggested that the extraction of polyphenols as a function of t can be adequately predicted by equation (6). it represents first-order kinetics [16, 17]. [ ct = c∞(1-e −kt)] (6) where 𝐶𝑡 is the concentration of total polyphenols at time t, 𝐶∞ the final concentrarion of total polyphenols, and k is the apparent first-order rate constant of extraction. the extraction process follows first-order kinetic, with a fast period from 0 to 20 min and a slow period from 20 to 100 min of extraction. when ln[c∞/(c∞ − c)] is plotted against time (figure 4), the points fall on two interesting straight lines, the first with a relatively steep slope and the second with a relatively shallow one. the points of intersection of ln[c∞/(c∞ − c)] vs t plot the fast and the slow stages are termed transition points. fig. 4. kinetics of the fast and the slow stage of tpc extraction and respective constants of algerian rosmarinus officinalis l. batch extraction. indeed, the coefficient at the fast period was 0.045 min-1 and of 0.050 min-1 for the slow period. 3.5. morphology algerian rosmarinus officinalis l. leaves morphologies were analyzed by sem food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 206 (quanta 200, fei company; france) before and after extraction processes, as illustrated in figure 5 and figure 6 respectively. it can be clearly observed a change in the morphological structure of the plant. fig. 5. sem images of fresh algerian rosmarinus officinalis l. leaf before batch extraction process these observations show the presence of many non-glandular trichomes commonly called hair-bearing. some of them are unicellular, simple and conical in shape. others are multicellular and branched [18, 19]. however, only glandular trichomes can synthesize and contain the essential oil which can also have antioxidant activities. after extraction, physical changes are observed on the plant material as seen on figure 6. secretarial channels micrograph shows that extraction process was enough to cause damage on cells and their walls. we observe a bursting of the walls of the cuticle of glandular hair and a dehydration of the leaves. this can be explained by a rise in temperature in the in-situ cellular structures. it induced an increase of internal pressure of these cells and caused their auto destruction by emptying them of their contents. the changes observed for extraction process are clearly showing that cells were damaged during extraction process. similar effects were reported in the literature [20]. fig. 6. sem images of fresh algerian rosmarinus officinalis l. leaf after batch extraction process doi: https://doi.org/10.4316/fens.2022.019 207 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 198 208 4. conclusion algerian rosmarinus officinalis l. leaves was extracted in batch mode with grassolvent (ethanol 80%). the selection of solvent is mainly related with the future use of the extract. the effect of the main process variables (time, solid-to-liquid ratio, temperature) on the yield was studied. the characteristic models released from each response are first-order linear models with interactions. the investigation of the effects of each factor on the three selected responses shows that solid-toliquid ratio presents the overriding factor for two responses (tpc, tfc). indeed, the best experimental result was obtained at 30°c for 30 min and 0.05 g/ml. the value of tpc, tfc and aa was respectively 121.80 mg gae/g dw, 44.01 mg qe/g dw and 96.36%. the correlation between tpc, tfc and aa remains unclear. the polyphenols extraction follows first order kinetic with coefficient of 0.045 min-1 for fast period and 0.05 min-1 for the slow period. the morphology investigations by sem revealed physical changes on plant material. antibacterial activity of extracts will be tested in prospect. 5. acknowledgements the authors thank the colleagues from laboratory for their continuous interest, support and discussion. their insight and expertise have been highly appreciated. many thanks also to a. naitbouda for his semsupport. 6. references [1]. franco, d., rodríguez-amado, i., agregán, r., munekata, p. e. s., vázquez, j. a., barba, f. j., & lorenzo, j. m. optimization of antioxidants extraction from peanut skin to prevent oxidative processes during soybean oil storage. lwt, 88: 1-8, (2018) [2]. moghaddam, m., miran, s.n.k., pirbalouti, a. g., mehdizadeh, l., & ghaderi, y. variation in essential oil composition and antioxidant activity of cumin (cuminum cyminum l.) fruits during stages of maturity. industrial crops and products, 70: 163169, (2015) [3]. acosta-estrada, b. a., gutiérrezuribe, j. a., & serna-saldívar, s. o. bound phenolics in foods, a review. food chemistry, 152: 46-55, (2014) [4]. granato, d., nunes, d. s., & barba, f. j. an integrated strategy between food chemistry, biology, nutrition, pharmacology, and statistics in the development of functional foods: a proposal. trends in food science & technology, 62: 13-22, (2017). [5]. sebranek, j.g., sewalt, v.j.h., robbins, k., & houser, t.a. comparison of a natural rosemary extract and bha/bht for relative antioxidant effectiveness in pork sausage. meat science, 69: 289-296, (2005) [6]. singleton., v. l. & rossi., j. a. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. american journal of enology and viticulture, 16: 144-158, (1965) [7]. miliauskas, g., venskutonis, p. r., & van beek, t. a. screening of radical scavenging activity of some medicinal and aromatic plants. food chemistry, 85: 231-237, (2004) [8]. que, f., mao, l., & pan, x. antioxidant activities of five chinese rice wines and involvement of phenolic compounds. food research international, 39: 581-587, (2006) [9]. zhang, b., yang, r., & liu, c. z., microwave-assisted extraction of chlorogenic acid from flower buds of lonicera japonica thunb. separation and purification technology, 62: 480483, (2008) [10]. yesil-celiktas, o., girgin, g. o. z. d. e., orhan, h., wicher, h.j., bedir, e., & verdar sukan, f. screening of free radical scavenging capacity and antioxidant activities of rosmarinus officinalis extract with focus on https://citeweb.info/19650000007 https://citeweb.info/19650000007 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 linda loucif seiad, soraya demim, mohamed loucif seiad, hassina boussak, souad hammadou, extraction of naturals antioxydants from algerian rosmarinus officinalis l. with gras-solvent and kinetic study, food and environment safety, volume xxi, issue 3 – 2022, pag. 198 – 208 208 location and harvesting times. european food research and technolo9gy, 224: 443-451, (2007) [11]. wojdylo, a., oszmianskii, j., & czelerys, r. antioxidant activity and phenolic compounds in 32 selected herbs. food chemistry, 105: 940-949, (2007) [12]. d’alessandro, l. g., kriaa, k., nikov, i., & dimitrov, k. ultrasound assisted extraction of polyphenols from black chokeberry. separation and purification technology, 93: 42-47, (2012) [13]. herrero, m., plaza, m., cifluentes, a., & ibáñez, e. green process for the extraction of bioactives from rosemary: chemical and functional characterization via ultraperformance liquid chromatography-tandem mass spectrometry and in-vitro assays. journal of chromatography a, 1217: 2512-2520, (2010) [14]. ibarra, a., cases, j., bily, a., he, k.., bai, n., roller, m., coussaert, a., & ripoll, c. importance of extract standardization and invitro/ex vivo assay selection for the evaluation of antioxidant activity of botanicals: a case study on three rosmarinus officinalis l. extract. journal of medicinal foods, 13: 11671175, (2010) [15]. georgetti, s. r., casagrande, r., souza, c.r.f., oliviera, w. p., & fonseca, m. j. v. spray drying of the soybean extract: effects on chemical properties and antioxidant activity. lwtfood science and technology, 41: 1521-1527, (2008) [16]. herodez, s.s., hadolin, m., škerget, m., & knez, z. solvent extraction study of antioxidants from balm (melissa officinalis l.) leaves. food chemistry 80: 275-282, (2003) [17]. spiro, m., & jago, d.s. kinetics and equilibria of tea infusion. part 3. rotating disc experiments interpreted by steady–state model. journal of the chemical society, farady transactions, 78: 295-305, (1982) [18]. marin, m., koko, v., duleticlausevic, s., marin, p. d., rancic, d., & dajic-stevanovic, z. glandular trichoms on the leaves of rosmarinus officinalis: morphology, stereology and histochemistry. south african journal of botany, 72: 378-382, (2006) [19]. bottega, s., & corsi, g. structure, secretion and possible functions of calyx glandular hairs of rosmarinus officinalis l. (labiatae), botanical journal. of the linnean society, 132: 325-335, (2000). [20]. pare, j.r.j. & belanger, j.m.r. instrumental method in foods analysis; elseiver science, eds, amsterdam, isbn: 9780080534770, (1997). 1. introduction 2.3.4. morphology analysis the samples were observed using a scanning electron microscope (jeol jsm 6360lv) with a maximum resolution and voltage (50 nm, 30kv). these observations serve to locate precisely the sites producing active molecules and to visualize any modification a... doi: https://doi.org/10.4316/fens.2021.011 91 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag. 91 100 characterization of pasta from heat moisture treated wheat flour and grape peels *mădălina iuga 1 , silvia mironeasa 1 1faculty of food engineering, ştefan cel mare university, suceava, romania madalina.iuga@usm.ro *corresponding author: received 7th may 2021, accepted 29th june 2021 abstract: pasta nutritional and functional value can be enhanced by incorporating fiber-rich ingredients such as grape peels and/or by applying hydrothermal treatments to wheat flour. the purpose of this study was to characterize dough rheological behavior, chemical and sensory charateristics of pasta from heat moisture treated (hmt) wheat flour, native wheat-grape peels composite flour and treated wheat-grape peels flour mix. the results revealed that grape peels addition increased the nutritional value of pasta by raising the ash, fiber and polyphenols content, while hmt determined higher resistant starch values. the elastic and viscous moduli of dough increased when hmt was applied to wheat flour and/or grape peels were incorporated. the addition of grape peels led to higher glass transition temperature, while an opposite effect was observed for hmt. both grape peels addition and hmt caused lower maximum creep and recovery compliances. acceptable sensory scores were obtained for all the analysed samples, pasta from treated wheat grape peels composite flour was characterized as nutritious, fruity, sourish, pasta from treated wheat flour as elastic, gummy, sweet, pleasant, while pasta from native wheat-grape peels was characterized as satiating, fragile, innovative, sourish. these results evidenced the opportunity to increase the functional and nutritional value of pasta by applying physical modification of flour and/or by incorporating grape peels as a source of dietary fiber and bioactive compounds. keywords: wheat, grape peels, pasta, heat moisture treatment, rheology, sensory profile. 1. introduction consumer preferences for food are constantly changing as society evolves and globalization occur. vinification generates valuable byproducts that can be introduced in various forms into human nutrition. their use has an advantage from both economic and environmental point of view. recently, researchers have turned their attention to the development of new products by incorporating functional ingredients such as grape peels [1, 2]. grape peels are considered sources of dietary fiber [3], being in the same time rich in phenolic compounds with antioxidant activity [4]. consumption of foods with high amounts of fibers and antioxidants can reduce the occurrence of chronic diseases such as constipation, some cancers, cardiovascular diseases [5, 6]. some studies revealed the influence of grape by-products on pasta quality. gaita et al. [1] revealed that polyphenolics and anioxidant activities of wheat pasta enriched with grape peels were higher compared to the control, acceptable sensory characteristics being observed at levels up to 6%. the results obtained by sant’anna et al. [7] showed a decrese of the sensory scores of fettuccini pasta when grape pomace was incorporated. in the study of mironeasa et al. [8] on the effects of grape peels on wheat bread dough rheology it is http://www.fia.usv.ro/fiajournal mailto:madalina.iuga@usm.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 92 stated that the viscoelastic moduli and the gelatinization temperature increased, depending on the addition level and particle size. starch-based foods are known to posess high glycemic index, which can lead to obesity and / or diabetes [9]. reducing the glycemic index of foods can be achieved by introducing whole grains and pseudo-cereals, by using phenolic compounds that reduce the digestibility of starch [9] or by modifying flour by hydrothermal treatment in order to increase the content of slowly digestible starch and resistant starch, and to decrease digestible starch content [10, 11, 12]. hydrothermal treatments applied to wheat flour produce a reorganization of starch amylose and amylopectin chains, which leads to changes in crystallinity of the granules, viscosity, thermal and gelatinization properties and also digestibility. all these changes, along with protein denaturation that occur during hydrothermal treatment [13] and interactions between components, result in rheology and texture of final product modification [10, 11, 14]. heat moisture treatment (hmt) supposes starch or flour treatment at high temperatures (50 < x < 120 °c) and low moisture (10 < x < 35 %) for a given time [15]. hmt caused the decrease of wheat noodles slowly digestible starch and rapid digestible starch, while resistant starch contents were higher than of the native [16]. chandla et al. [17] presented improved sensory characteristics of noodles made of hmt amaranth and corn starch compared to the native, mostly in terms of flavor, taste and overall acceptability. the study conducted by lazaridou et al. [18] on wheat-barley rusk dough revealed that hmt caused an increase of elastic (g') and viscous (g") moduli, while the compliances (j) were lower compared to the control. the aim of this study was to characterize dough and pasta products made of heat moisture treated wheat flour, wheat-grape peels composite flour and treated wheatgrape peels composite flour in terms of dough rheology, pasta chemical and sensory characteristics. 2. materials and methods materials, treatment conditions and pasta production grape peels were obtained after manual separation from dry pomace of feteasca regala variety, then they were grinded and sieved to particle size < 180 μm. wheat flour of 650 type was treated according to the methodology presented in the literature [19]: moisture content was increased by adding corresponding amount of water in small portions with continuous stirring, then the samples were kept for 24 h in hermetically sealed containers for moisture equalization. flour was treated in a closed system for the given time and temperature, and after cooling, it was grinded and sieved to particle size < 300 μm. dough was made by mixing flour with appropriate quantities of water to achieve 40% dough moisture in a kitchen aid mixer (whirlpool corporation, usa). after 15 min of resting, pasta modeling was done on a kitchen aid accessory for short pasta. drying was made firstly at room temperature for 1/2 h, then at 40 °c for 1 h, at 80 °c for 2 h and again at 40 °c for 2 h [20]. the samples considered in this study were obtained after previous optimizations. control sample was made of untreated wheat flour, oth sample was made of wheat flour treated at 85.30 °c, for 1 h and 26.80 % moisture [21], ops was formed of native wheat flour with 4.62 % grape peels [22] and otps sample was made from trated flour at 64.35 °c, for 3 h and 26.65 % moisture mixed with 4.94 % grape peels [23]. dough rheology in order to characterize dough behavior during processing, dynamic oscillation and creep-recovery tests were applied using a thermo-haake mars 40 rheometer food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 93 (karlsruhe, germany) with parallel plates of 40 mm diameter, at a gap of 3 mm. the dough was laminated to a thickness of 3 mm and allowed to rest for 1 hour before testing to relax internal strain. then, the sample was placed in the measuring system and kept for 120 s at 20 °c before testing to allow relaxation and temperature stabilization. excess dough was removed and a layer of vaseline was applied to the exposed edge to protect it from moisture loss during testing. before evaluating the variation of the viscoelastic moduli with frequency, the limits of the linear viscoelastic region (lvr) were established by applying a strain range from 0.1 to 100 pa, at a constant oscillation frequency of 1 hz. the variation of the elastic (g′) and viscosous (g″) modulus with frequency was evaluated by applying a constant strain of 15 pa (in the lvr) and a frequency range from 0.1 to 20 hz. in order to evaluate the behavior of pasta dough at heating, the dynamic oscillation tests were applied at a constant frequency of 1 hz, and the temperature was varied from 20 to 100 ºc with a heating speed of 4ºc / min. the elastic (g′) and viscoscous mdoulus (g″) were recorded. the initial glass transition temperature (ti) determined at the minimum value of g′ and the glass transition temperature (tg) determined when the maximum value of g″ was reached were identified [24]. creep and recovery tests allowed the evaluation of compliance (j) at a constant temperature of 20 ºc and a strain of 50 pa as follows: the strain was applied for 60 s, then it was removed to allow the sample to recover for 180 s. burgers mathematical model was applied [8, 25] for the experimental data obtained in the creep phase (equation 1) and in the recovery phase respectively (equation 2). (1) (2) where: jco (pa -1) is the instantaneous compliance in the creep phase, jro (pa -1) is the instantaneous compliance in the recovery phase, jcm (pa -1) is the retarded elastic compliance in the creep phase, jrm (pa -1) is the retarded elastic compliance in the recovery phase, t (s) is the phase time, λc, λr (s) are the retarded time in creep and recovery phase, μco (pa·s) is the shear viscosity at time 0, jmax (pa -1) is the maximum compliance during creep phase, jr (pa-1) is the equilibrium compliance calculated as the sum of jro and jrm. also, dough recovery capacity (jr/jmax) was determined. pasta chemical composition pasta moisture, ash, protein and lipids were measured according to sr en iso 712/2010, sr iso 2171/2002, sr en iso 20483/2007, and sr 91/2007 respectively standards. fiber content was measured by nir spectrometry (foss 6500 nir device, silver springs, sua) and the carbohydrates were calculated by difference. megazyme kit was used to measure resistant starch (rs), slowly digestible starch (sds) and rapid digestible starch (rds) of boiled pasta. the principle of determination consisted of samples digestion with αamylase and amyloglucosidase for a specific time and spectrophotometric quantification of the glucose resulted. the procedure described by melili et al. [26] was used for extract preparation in order to quantify total polyphenols: grinded sample (2 g) was extracted with methanol (80%) in a sonication bath at 37 ºc and 45 hz for 40 min. after filtration, the extract (0.20 ml) was mixed with 0.80 ml distilated water, folin-ciocalteu reagent (0.50 ml) and sodium carbonate 20% (2.50 ml). the absorbance was read after 40 min of resting in the darkness at 725 nm on an uv–vis– nir shimadzu 3600 (tokyo, japan) spectrophotometer. the content of total polyphenols was calculated from a calibration curve with gallic acid (r2 = 0.99). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 94 sensory profile of pasta pasta sensory profile was established according to the descriptive method check all the attributes (cata) described by bustos et al. [27], with some modifications. a panel of 9 semitrained judges evaluated fresh boiled pasta in one session. the coded samples were randomly presented, water being provided for mouth cleaning. the terms used for characterization were previously discussed and established in the training session. texture was characterized by the following attributes: grainy, pasty, sticky, al dente, seed, soft, gummy, elastic or fragile; color was evaluated as unpleasant or pleasant; flavor was characterized by sweet, back flavor, sourish, flavored, fruity or bitter; general aspect attributes used were healthy, tempting, tasty, light, natural, satiating, pleasant, artificial or nutritious. global acceptability was evaluated on a scale from 1 to 9 points. statistical analysis all the measurements were performed in triplicate. tukey post-hoc test was applied in order to underline significant differences (p < 0.05) between samples. the data of cata analysis were processed using principal corespondence analysis (pcoa). in order to identify the relationships between characteristics, principal component analysis was applied. all the statistical analysis were made using xlstat for excel 2021 version (addinsoft, new york, sua) software. 3. results and discussion chemical composition pasta proximate composition is presented in table 1. significant higher (p < 0.05) ash content was observed for ops and otps, samples containing grape peels. the obtained results underlined the positive effects of hmt and grape peels addition on fiber content which was enhanced compared to the control. the carbohydrates were lower in pasta from treated wheat flour (oth) and pasta with grape peels (ops) which could led to lower glycemic index. these results are due to the nutrients intake of grape peels, similar findings being reported by acun and gül [28] for baked products supplemented with grape pomace, seeds, seedless pomace. table 1. chemical and sensory characteristics of pasta characteristic control oth ops otps moisture* (%) 11.54 ± 0.03a 7.02 ± 0.12d 11.27 ± 0.07b 8.40 ± 0.03c ash* (%) 0.56 ± 0.02b 0.58 ± 0.07b 0.71 ± 0.08ab 0.75 ± 0.06a lipids* (%) 0.11 ± 0.01c 0.31 ± 0.05a 0.19 ± 0.02b 0.27 ± 0.01a proteins* (%) 12.33 ± 0.09b 12.59 ± 0.09ab 12.68 ± 0.09a 12.42 ± 0.16ab fiber* (%) 0.02 ± 0.00c 1.55 ± 0.05b 1.45 ± 0.05b 2.35 ± 0.05a carbohydrates* (%) 75.44 ± 0.10b 77.95 ± 0.19a 73.71 ± 0.21c 75.81 ± 0.15b rs* (%) 0.88 ± 0.00d 1.56 ± 0.03c 1.61 ± 0.00b 2.24 ± 0.01a sds* (%) 5.87 ± 0.14b 3.38 ± 0.00a 6.59 ± 0.32d 4.67 ± 0.17c rds* (%) 23.80 ± 0.04b 26.80 ± 0.00d 18.29 ± 0.08a 23.18 ± 0.11c tp* (μg gae / g) 93.00 ± 0.83b 96.41 ± 3.01b 129.89 ± 2.38a 124.43 ± 1.89a global acceptability 8.41 ± 0.04a 8.00 ± 0.00b 7.78 ± 0.03c 7.55 ± 0.05d *the results are expressed to sample weight as it is, rs – resistant starch, sds – slowly digestible starch, rds – rapid digestible starch, tp – total polyphenols, oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels, a-d means followed by different letters in the same row are significantly different (p < 0.05). resistant starch (rs) has been proved to be higher in products from heat moisture treated starch or flour [29], which is in agreement with our results. the increase of rs could be due to starch structure reorganization during hmt and/or to the starch-polyphenols and polyphenols inhibition effects on digestive enzymes [12, 30]. slowly (sds) and rapid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 95 digestible (rds) starch decreased when hmt was applied compared to the control, similar trend being reported by li et al. [16] for noodles from hmt wheat flour. as it was expected, no significant changes (p > 0.05) of total polyphenols (tp) were observed between control and sample from treated flour (oth), while the addition of grape peels resuted in significant (p < 0.05) higher tp contents (table 1). gaita et al. [1] also reported increases of tp and antioxidant activity of pasta as the grape peels level was higher. it was stated that the polyphenols from grape pomace presented high bioaccesibility and availability for the intestinal absorption [31], the metabolism of phenolic compounds being directly influenced by hydrogen, ionic, covalent, and hydrophobic linkages with proteins [1]. dough rheology frecuency sweep test revealed that the elastic and solid-like character of all pasta dough samples since g' > g" (figure 1). fig.1. variation of the elastic (g') and viscous (g") moduli with frequency: oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels the addition of grape peels and wheat flour hmt determined higher elastic and viscous moduli compared to the control, probably due to the starch and proteins modifications during treatment and to the fiber content of grape peels which may compete with gluten for water [8, 29]. the highest impact on g' and g" was observed for pasta made from hmt flour which may be related to the formation of protein and starch aggregates, leading to solubility and interactions between components changes [12]. the variation of g' and g" with temperature is showed in figure 2. fig. 2. variation of the elastic (g') and viscous (g") moduli with temperature: oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels grape peels and hmt determined higher g' and g" values compared to the control. in the first heating step, a decrease of g' up to a certain temperature value could be due to the protein denaturation phenomenon which led to water absorption capacity changes [8]. in the next step, a rapid increase of g' until the maximum gelatinization temperature was reached, followed by a decrease was observed which can be related to the gelatinization process that occurs at about 50 °c. dough samples made from hmt wheat showed different behavior in the second heating step, showing an increase of g' and g" at the end of the test, at temperature > 90 °c. this trend could be explained by the gliadin content of the samples [32]. heat moisture treatment showed a lowering effect on flour onset gelatinization temperature (ti) and glass transition (figure 3). on the other hand, grape peels addition resulted in higher ti values compared to the control. these g ' , g " ( p a ) f (hz) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 96 changes may be due to the interactions between starch components and lipids and/or to the bonds formed during hmt, similar findings being reported by collar and armero [33]. fig.3. glass transition parameters of flours: ti – onset temperature, tg – glass transition (peak) temperature, oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels, a-d means followed by different letters in the same row are significantly different (p < 0.05) creep and recovery tests could provide information about dough rigidity [8]. dough compliances evolution in time is presented in figure 4. hmt and grape peels addition determined higher resistance to deformation showed by the decrease of creep compliances compared to the control. similar trend was reported by mironeasa et al. [8] for bread dough enriched with 7 and 9 % grape peels. fig. 4. creep and recovery of pasta dough: oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels lower values for burger’s parameters were obtained for samples from treated wheat flour and/or with grape peels (table 2). compared to the control, the instantaneous (jco, jro) and the retarded compliances (jcm, jrm) for both creep and recovery phases were lower for oth, ops and otps, indicating lower instant and retarded deformation which may be led to dough water content of the treated and/or enriched samples [34]. table 2. burger’s model parameters parameter control oth ops otps creep phase jco  10 5 (pa-1) 3.89 ± 0.31a 0.72 ± 0.01c 2.82 ± 0.23b 1.17 ± 0.04c jcm  10 5 (pa-1) 10.01 ± 0.01a 3.51 ± 0.20b 9.06 ± 0.86a 4.51 ± 0.35b c (s) 33.20 ± 1.64 bc 43.94 ± 1.21a 30.82 ± 1.97c 36.28 ± 0.73b co  10 -6 (pa  s) 1.08 ± 0.06c 3.64 ± 0.21a 1.51 ± 0.16c 2.86 ± 0.38b jmax  10 5 (pa-1) 14.24 ± 0.79a 3.39 ± 0.10c 10.81 ± 0.81b 4.91 ± 0.36c recovery phase jro  10 5 (pa-1) 4.03 ± 1.33a 1.84 ± 0.00b 2.92 ± 0.20ab 1.67 ± 0.28b jrm  10 5 (pa-1) 5.59 ± 1.15a 0.67 ± 0.00b 4.50 ± 0.54a 1.93 ± 0.06b r (s) 42.53 ± 11.90 a 9.89 ± 0.00b 45.78 ± 2.00a 37.40 ± 2.24a jr  10 5 (pa-1) 9.61 ± 0.31a 2.51 ± 0.00d 7.42 ± 0.50b 3.60 ± 0.35c jr/jmax (%) 67.51 ± 4.27 a 74.19 ± 2.32a 68.68 ± 1.02a 73.33 ± 1.71a oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels, jco – instantaneous compliance for creep phase, jro instantaneous compliance for recovery phase, jcm retarded elastic compliance for creep phase, jrm retarded elastic compliance for recovery phase, t – phase time, λc, λr – retardation time, μco – zero shear viscosity, jmax maximum creep compliance obtained at the end of the creep phase, jr – recovery compliance calculated as sum of jro and jrm , jr/jmax – dough recovery capacity, a-d means followed by different letters in the same row are significantly different (p < 0.05) t e m p e ra tu re ( °c ) ti tg 100.00 80.00 60.00 40.00 20.00 0.00 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 97 dough recovery capacity given by the ratio between jr and jmax was higher for pasta dough made from treated wheat flour and/or with grape peels addition compared to the control. the results are in agreement with those reported by mironeasa et al. [8] for dough with different levels and particle sizes of grape peels. the recovery capacity may provide information on dough macrostructure. for example, increased recovery capacity could be due to the presence of small molecules in dough system, while small values could be associated with large molecules [35]. sensory profile of pasta both hmt and grape peels addition in wheat pasta resulted in lower global acceptability (table 1). sant’anna et al. [7] also reported lower sensory characteristics score when grape pomace was incorporated in fettuccini pasta. another study showed that the appearence of noodles made of hmt amaranth starch presented lower values compared to the samples from native starch [17]. pasta attributes resulted from cata analysis are presented in figure 5. the first dimension (f1) explained 56.71 % and the second one (f2) 29.65 % of total inertia. fig. 5. principal corespondence analysis (pcoa) results for pasta sensory attributes: oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels control sample was characterized as light, gourmet, tempting, while oth made of treated wheat flour was identified as being elastic, gummy, sweet, soft. both samples containing grape peels were described as being nutritious, fruity, sourish, fragile, innovative, with pleasant color and satiating, being positionated closely one to each other. grape peels were responsible for the back flavor identified by the panelists probably due to the polyphenol’s presence. relationships between characteristics principal component analysis bi-plot exhibiting relationships between characteristics and samples is shown in figure 6. the first principal component (pc1) explained 47.10 % of the total variance, while the second one (pc2) explained 40.92 %. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 98 fig. 6. principal component analysis bi-plot: oth – sample from heat moisture treated wheat flour, ops – sample from wheat-grape peels composite flour, otps – sample from treated wheat flour and grape peels, rs – resistant starch, sds – slowly digestible starch, rds – rapid digestible starch, tg – glass transition temperature, tp – total polyphenols, jr – recovery compliance, jmax – maximum creep compliance pc1 was associated with rds, moisture, jmax, carbohydrates, lipids, while pc2 was associated with rs, ash, tp fibers contents and global acceptability. rds was strongly correlated with moisture content (r = 0.96, p < 0.05), while rs content was positively correlated to fibers (r = 0.98, p < 0.05), which was in agreement with the statement that rs behavior in the digestion system is similar to that of fibers [36]. moisture was significantly (p < 0.05) related to the creep and recovery complinances (r = 0.97), underlying the influence of water on dough rheological behavior previously established [34]. pasta acceptability was negatively correlated with the rs content (r = 0.97, p < 0.05) and with fibers (r = 0.96, p < 0.05), these results supporting gaita et al. [1] findings which showed that the sensory scores were directly influenced by grape peels level and fibers presence. 4. conclusion heat moisture treatment and grape peels can be useful techniques for pasta nutritional and functional value increase. hmt determined higher resistant starch values, while the addition of grape peels resulted in higher pasta fiber and polyphenols contents compared to the control. dough rheology showed significant changes when hmt was applied and/or when grape peels were added, viscous and elastic modulus showing higher values. dough resistance to deformation increased when hmt and/or grape peels were used, while the recovery capacity was improved. the sensory profile revealed that pasta from treated wheat flour was associated with elastic, sticky, gummy texture, while the samples containing grape peels were described as nutritious, fruity, sourish, satiating, innovative and fragile. these results evidenced the opportunity to use hmt and/or grape peels in pasta production. 5. acknowledgment this work was supported by romania national council for higher education funding, cnfis, project number cnfisfdi-2021-0357. 6. references [1]. gaita, c., alexa, e., moigradean, d., filomena, c., poiana, m.-a. (2020), food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 99 designing of high value-added pasta formulas by incorporation of grape pomace skins, romanian biotechnological letters, 25(3): 1607–1614, (2020) https://doi.org/10.25083/rbl/25.3/1607.1614 [2]. bender, a. b. b., speroni, c. s., salvador, p. r., loureiro, b. b., lovatto, n. m., goulart, f. r., lovatto, m. t., miranda, m. z., silva, l. p., penna, n. g., grape pomace skins and the effects of its inclusion in the technological properties of muffins, journal of culinary science and technology, 15(2): 143–157, (2017). https://doi.org/10.1080/15428052.2016.1225535 [3]. zhu, f., du, b., zheng, l., li, j., advance on the bioactivity and potential applications of dietary fibre from grape pomace, food chemistry, 186: 207–212, (2015). https://doi.org/10.1016/j.foodchem.2014.07.057 [4]. tang, g. y., zhao, c. n., liu, q., feng, x. l., xu, x. y., cao, s. y., meng, x., li, s., gan, r. y., li, h. bin., potential of grape wastes as a natural source of bioactive compounds, molecules, 23(10): 1–20, (2018). https://doi.org/10.3390/molecules23102598 [5]. kendall, c. w. c., esfahani, a., jenkins, d. j. a., the link between dietary fibre and human health, food hydrocolloids, 24: 42-48, (2010). https://doi.org/10.1016/j.foodhyd.2009.08.002 [6]. rasines-perea, z., ky, i., cros, g., crozier, a., teissedre, p.l., grape pomace: antioxidant activity, potential effect against hypertension and metabolites characterization after intake. diseases, 6(60): (2018). https://doi.org/10.3390/diseases6030060 [7]. sant’anna, v., christiano, f. d. p., marczak, l. d. f., tessaro, i. c., thys, r. c. s., the effect of the incorporation of grape marc powder in fettuccini pasta properties, lwt food science and technology 58(2): 497–501, (2014). https://doi.org/10.1016/j.lwt.2014.04.008 [8]. mironeasa, s., iuga, m., zaharia, d., mironeasa, c., rheological analysis of wheat flour dough as influenced by grape peels of different particle sizes and addition levels, food and bioprocess technology, 12(2): 228–245, (2019). https://doi.org/10.1007/s11947-018-2202-6 [9]. guzar, i., effect of starch-polyphenol interactions on starch hydrolysis. ontario, canada: university of guelpf, (2012). [10]. collar, c., armero, e., impact of heat moisture treatment and hydration level on physico-chemical and viscoelastic properties of doughs from wheat-barley composite flours, european food research and technology, 244(2): 355–366, (2017). https://doi.org/10.1007/s00217-017-2961-8 [11]. lawal, o. s., studies on the hydrothermal modifications of new cocoyam (xanthosoma sagittifolium) starch, international journal of biological macromolecules, 37(5): 268–277, (2005). https://doi.org/10.1016/j.ijbiomac.2005.12.016 [12]. chen, x., he, x., fu, x., huang, q., in vitro digestion and physicochemical properties of wheat starch/flour modified by heat-moisture treatment, journal of cereal science, 63: 109– 115, (2015). https://doi.org/10.1016/j.jcs.2015.03.003 [13]. cetiner, b., acar, o., kahraman, k., sanal, t., koksel, h., an investigation on the effect of heat-moisture treatment on baking quality of wheat by using response surface methodology, journal of cereal science, 74: 103–111, (2017). https://doi.org/10.1016/j.jcs.2017.01.002 [14]. lan, h., hoover, r., jayakody, l., liu, q., donner, e., baga, m., … chibbar, r. n., impact of annealing on the molecular structure and physicochemical properties of normal, waxy and high amylose bread wheat starches, food chemistry, 111(3): 663–675, (2008). https://doi.org/10.1016/j.foodchem.2008.04.055 [15]. alcazar-alay, s.c., almeida meireles, m.a., physicochemical properties, modifications and applications of starches from different botanical sources, journal of food science and technology, 35(2): 215-236, (2015). https://doi.org/10.1590/1678-457x.6749 [16]. li, m., liu, c., zheng, x., li, l., bian, k., physicochemical properties of wheat flour modified by heat-moisture treatment and their effects on noodles making quality, journal of food processing and preservation, 44(9): 1–9, (2020). https://doi.org/10.1111/jfpp.14590 [17]. chandla, n. k., saxena, d. c., singh, s., processing and evaluation of heat moisture treated (hmt) amaranth starch noodles; an inclusive comparison with corn starch noodles, journal of cereal science, 75: 306–313, (2017). https://doi.org/10.1016/j.jcs.2017.05.003 [18]. lazaridou, a., marinopoulou, a., biliaderis, c. g., impact of flour particle size and hydrothermal treatment on dough rheology and quality of barley rusks, food hydrocolloids, 87: 561–569, (2019). https://doi.org/10.1016/j.foodhyd.2018.08.045 [19]. collar, c., armero, e., impact of heat moisture treatment and hydration level on physico-chemical and viscoelastic properties of https://doi.org/10.25083/rbl/25.3/1607.1614 https://doi.org/10.1080/15428052.2016.1225535 https://doi.org/10.1016/j.foodchem.2014.07.057 https://doi.org/10.3390/molecules23102598 https://doi.org/10.1016/j.foodhyd.2009.08.002 https://doi.org/10.3390/diseases6030060 https://doi.org/10.1016/j.lwt.2014.04.008 https://doi.org/10.1007/s11947-018-2202-6 https://doi.org/10.1007/s00217-017-2961-8 https://doi.org/10.1016/j.ijbiomac.2005.12.016 https://doi.org/10.1016/j.jcs.2015.03.003 https://doi.org/10.1016/j.jcs.2017.01.002 https://doi.org/10.1016/j.foodchem.2008.04.055 https://doi.org/10.1590/1678-457x.6749 https://doi.org/10.1111/jfpp.14590 https://doi.org/10.1016/j.jcs.2017.05.003 https://doi.org/10.1016/j.foodhyd.2018.08.045 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 mădălina iuga, silvia mironeasa, characterization of pasta from heat moisture treated wheat flour and grape peels, food and environment safety, volume xx, issue 2 – 2021, pag. 91 – 100 100 doughs from wheat-barley composite flours, european food research and technology, 244(2): 355–366, (2018). https://doi.org/10.1007/s00217-017-2961-8 [20]. bergman, c., gualberto, d., weber, c., development of a hightemperature-dried soft wheat pasta supplemented with cowpea (vigna unguiculata (l) walp) cooking quality, color, and sensory evaluation, cereal chemistry, 71(6): 523–527, (1994). [21]. iuga, m., mironeasa, s., application of heat moisture treatment in pasta production, food control, 128: 108176, (2021). https://doi.org/10.1016/j.foodcont.2021.108176 [22]. iuga, m., mironeasa, s., use of grape peels by-product for wheat pasta manufacturing, plants, 10(5): 926–943, (2021). https://doi.org/10.3390/plants10050926 [23]. iuga, m., mironeasa, s., simultaneous optimization of wheat heat moisture treatment and grape peels addition for pasta making, lwt food science and technology, 150: 112011, (2021). https://doi.org/10.1016/j.lwt.2021.112011 [24]. pereira, p. m., oliveira, j. c., measurement of glass transition in native wheat flour by dynamic mechanical thermal analysis (dmta), international journal of food science and technology, 35(2): 183–192, (2000). https://doi.org/10.1046/j.13652621.2000.00289.x [25]. moreira, r., chenlo, f., torres, m. d., prieto, d. m., influence of the particle size on the rheological behaviour of chestnut flour doughs, journal of food engineering, 100(2): 270–277, (2010). https://doi.org/10.1016/j.jfoodeng.2010.04.009 [26]. melilli, m. g., pagliaro, a., scandurra, s., gentile, c., di stefano, v., omega-3 rich foods: durum wheat spaghetti fortified with portulaca oleracea, food bioscience, 37: 100730, (2020). https://doi.org/10.1016/j.fbio.2020.100730 [27]. bustos, m. c., paesani, c., quiroga, f., león, a. e. technological and sensorial quality of berry-enriched pasta, cereal chemistry, 96(5): 967–976, (2019). https://doi.org/10.1002/cche.10201 [28]. acun, s., gül, h., effects of grape pomace and grape seed flours on cookie quality, quality assurance and safety of crops and foods, 6(1): 81–88, (2014). https://doi.org/10.3920/qas2013.0264 [29]. iuga, m., mironeasa, s., a review of the hydrothermal treatments impact on starch based systems properties, critical reviews in food science and nutrition, 60(22): 3890– 3915, (2020). https://doi.org/10.1080/10408398.2019.1664978 [30]. sun, l., miao, m., dietary polyphenols modulate starch digestion and glycaemic level: a review, critical reviews in food science and nutrition, 8398: 1–15, (2019). https://doi.org/10.1080/10408398.2018.1544883 [31]. gomes, t. m., toaldo, i. m., haas, i. c. da s., burin, v. m., caliari, v., luna, a. s., de gois, j. s., bordignonluiz, m. t., differential contribution of grape peel, pulp, and seed to bioaccessibility of micronutrients and major polyphenolic compounds of red and white grapes through simulated human digestion, journal of functional foods, 52: 699–708, (2019). https://doi.org/10.1016/j.jff.2018.11.051 [32]. madeka, h., kokini, j. l., changes in rheological properties of gliadin as a function of temperature and moisture: development of a state diagram, journal of food engineering, 22(1–4): 241–252, (1994). https://doi.org/10.1016/0260-8774(94)90033-7 [33]. collar, c., e. armero., value-added of heat moisture treated mixed flours in wheat based matrices: a functional and nutritional approach, food and bioprocess technology, 11 (8): 1536–51, (2018). https://doi:10.1007/s11947-018-2125-2 [34]. ronda, f., pérez-quirce, s., angioloni, a., collar, c., impact of viscous dietary fibres on the viscoelastic behaviour of gluten-free formulated rice doughs: a fundamental and empirical rheological approach, food hydrocolloids, 32(2): 252–262, (2013). https://doi.org/10.1016/j.foodhyd.2013.01.014 [35]. weegels, p.l., orsel, r., van de pijpekamp, a.m., lichtendonk, w.j., hamer, r.j., schofield, j.d., fundamental properties of low mr wheat proteins. ii. effects on dough properties. journal of cereal science, 21: 117–126, (1995). [36]. barros, j. h. t., telis, v. r. n., taboga, s., franco, c. m. l., resistant starch: effect on rheology, quality, and staling rate of white wheat bread, journal of food science and technology, 55(11): 4578–4588, (2018). https://doi.org/10.1007/s13197-0183393-6 https://doi.org/10.1007/s00217-017-2961-8 https://doi.org/10.1016/j.foodcont.2021.108176 https://doi.org/10.3390/plants10050926 https://doi.org/10.1016/j.lwt.2021.112011 https://doi.org/10.1046/j.1365-2621.2000.00289.x https://doi.org/10.1046/j.1365-2621.2000.00289.x https://doi.org/10.1016/j.jfoodeng.2010.04.009 https://doi.org/10.1016/j.fbio.2020.100730 https://doi.org/10.1002/cche.10201 https://doi.org/10.3920/qas2013.0264 https://doi.org/10.1080/10408398.2019.1664978 https://doi.org/10.1080/10408398.2018.1544883 https://doi.org/10.1016/j.jff.2018.11.051 https://doi.org/10.1016/0260-8774(94)90033-7 https://doi:10.1007/s11947-018-2125-2 https://doi.org/10.1016/j.foodhyd.2013.01.014 https://doi.org/10.1007/s13197-018-3393-6 https://doi.org/10.1007/s13197-018-3393-6 1. introduction issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volumexix, issue 3 30 september 2020 available online http://www.fia.usv.ro/fiajournal © publisedby universitypublishinghouse of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: indexcopernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiuneacolegiultehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analeleuniversitatii stefan cel mare suceava, sectiuneaingineriealimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering(issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made byniculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientificjournalfoodandenvironmentsafety (issn 2068 – 6609) ispublished byuniversity publishinghouse of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails:fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishinghouse of suceava. allrightreserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare universityof suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de josuniversityof galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tytonbiosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefancel mare universityof suceava, romania călina petruța cornea university of agronomic sciencesandveterinarymedicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agriculturalsciencesandveterinarymedicinetimisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. klimentohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of natureresearch center, vilnius, lithuania ana leahu ştefancel mare universityof suceava, romania anna maraz corvinusunversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of foodscience, usa ileana denisa nistor vasilealecsandriuniversity of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agriculturalsciencesandveterinarymedicinetimisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de josuniversityof galați, romania adrian riviş banat university of agriculturalsciencesandveterinarymedicinetimisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarianuniversity of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences andveterinarymedicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania 298 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xviii, issue 42019, pag. 298 307 physico-chemical parameters of romanian raspberry honey * daniela pauliuc 1 , mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, *daniela_pauliuc@yahoo.com, *corresponding author received 15th october 2019, accepted 19th december 2019 abstract: six samples of raspberry honey collected from different regions of romania were analysed in order to confirm that they are classified as monofloral honey. melissopalynological analysis, alongside the determination of physico-chemical parameters moisture, ph, free acidity, electrical conductivity, hydroxymethylfurfural (hmf) content, color, total polyphenols content, flavonoids content, dpph radical scavenging activity were chosen as methods of analysis that give indications regarding the botanical origin of honey.the results of the melissopalynological analysis showed that all honey samples had a percentage of rubus idaeus pollen grains above the minimum of 45%, which was required in order to classify the samples as monofloral honey. the values determined for ph (4.01-4.31), free acidy (20.1-42.1 meq/kg) and electrical conductivity (0.36-0.52 ms/cm) confirmed that the samples were of pure raspberry honey. in the case of moisture content, one honey sample exceeded the moisture content set by codex alimentarius (20%). the limit set for hmf content was not exceeded by any raspberry honey sample, for which values of 6.13-26.79 mg hmf/kg were determined. raspberry honey had high total polyphenols content (8.11-12.86 gae/100 g), flavonoids content (25.36-41.35 mg qe/100 g) and dpph radical scavenging activity (58.1-94.35%). these results contribute to the knowledge of the chemical compositions and physical parameters of romanian honeys. keywords: honey, authentication, physico-chemical properties 1. introduction honey is a natural produce widely consumed for its taste and nutritional value, but also for its health benefits [1]. honey is one of the few natural foods offered today. clinical studies on honey have shown that it has a broad spectrum of bioactive activity such as anticarcinogenic, anti-inflammatory, antithrombotic, and analgesic; natural honey consumption also contributes to a reduced risk of cardiovascular disease, being associated with a decrease in body weight [2]. romania is a country with an old tradition in beekeeping. the national institute of statistics reported that honey production in romania is about 18000 tonnes per year, of which 85% is exported [3]. in romania, climatic conditions and honey bees are favourable for beekeeping, which presents a real advantage. since 1961, romania has been present in international honey production statistics, the percentage of contribution to european honey production ranged from 6.6% (in 1961) to 13.5% (1977) [4]. according to the fao stat data, the contribution of romania to the production of honey in europe in the year 2017 was of 24611 tonnes, which represents about 6.36% of the total european production. variations in honey production are closely linked to the climatic conditions and vegetative cycles of the plants that the bees feed on. however, despite these variations romania and bulgaria together with italy and spain remain important suppliers of organic honey. in developed european countries, http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 299 intensive use of pesticides has led to the premature death of hundreds of thousands of bees, and consequently decreased production [4]. in romania, there are not many types of monofloral honey, and of the produced honey types the most predominant ones are acacia honey (robinia pseudoacacia), tilia (tilia sp.), rape (brassica napus var. oleifera) and sunflower (helianthus annuus). other types of honey are also found, but these originate from plants with a relatively small geographical area such as raspberry honey (rubus idaeus), mint honey (mentha spp.) or thyme honey (thymus serpyllum).the botanical origin of honey has a great influence on honey quality. the composition of honey depends on the floral source used by bees to collect the nectar, the seasonal and environmental factors, as well as processing [5]. the botanical source of honey is closely linked to its price and, therefore, in order to increase the profit, the producers tend to adulterate it. equally important is the determination of the geographical origin, which is an important parameter in terms of honey differentiation and commercial value. depending on the geographical origin, the environment and the area where the hives are located, honey can acquire different characteristics and properties [1] . the aim of this study was to analyze the physicochemical parameters (melissopalynological analysis, color, ph, free acidity, electrical conductivity, moisture, hydroxymethyl furfural content, total polyphenol content, flavonoids content, and antioxidant activity) of raspberry honey in order characterize it and confirm that is monofloral honey. 2. materials and methods 2.1. samples six samples of raspberry honey from 2017 and 2018 were purchased from romanian beekeepers. all samples were liquefied at 50°c and homogenized to carry out the analysis. 2.2. melissopalynological analysis 10 grams of honey were mixed with 40 ml of distilled water, and the resulting mixture was centrifuged at 4500 rpm for 15 min. after carefully removing the supernatant, the residue was re-dissolved and centrifuged for other 15 min. the sediment was spread on a microscopic slide and the pollen granules were counted by a light microscopy (motic x 40) [6], [7]. physicochemical analysis the physico-chemical analyses were performed according to the analytical methods harmonized by the international honey commission [8]. honey samples were analyzed to determine 9 physicochemical characteristics: moisture (abbe refractometer, leica mark ii plus), electrical conductivity (portable conduct meter hq14d, hach, usa), ph (ph meter mettler toledo fivego, mettler toledo, sua), free acidity (titroline easy, schott instruments, germany), color (photometer pfund, hanna instruments hi 96785 and a portable chromameter, cr-400, konica minolta, japan), hydroxymethyl furfural (hmf) content (spectrophotometer uvvis-nir schimadzu uv-3600, schimadzu corporation, japan,) total polyphenols content (uv-nir spectrometer ocean optics hr4000cguv-nir, sua), flavonoids content (uvnir spectrometer ocean optics hr4000cg-uv-nir , sua) and dpph radical scavenging activity. moisture the moisture content was analyzed by refractometry using an abbe refractometer (leica mark ii plus). in order to measure the moisture content the samples were initially liquefied at a temperature of 50ºc. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 300 the refractometer was regularly calibrated with distilled water and the measurement was performed at a temperature of 20ºc. water content (%) was then obtained from the chataway table [9] . ph the ph was measured on a 10% honey solution with a mettler toledo fivego (mettler toledo, sua) ph-meter. free acidity the free acidity of honey is a measure of the content of all free acids, expressed in milli equivalents/kg honey and was determined by titrimetric method. 10 g honey were dissolved in 75 ml of carbon dioxide-free water, then the ph was measured and the solution was titrated with 0.1m sodium hydroxide solution with titroline easy device (schott instruments, germany) to a ph of 8.30. calculation was made using the following equation: free acidity= ml of 0.1m naoh x 10(1) hmf content the hydroxymethyl furfural (hmf) content was determined using the method proposed by [10]. honey samples were divided into two clarified aliquots. water was added to one, and a solution of sodium bisulphite to the other, considered the blank solution. sodium bisulphite breaks the conjugated double bond responsible of hmf absorbance. the absorbance at λ = 284 nm and λ = 336 nm was then read for the sample solution against the blank solution using a uv-vis-nir 3600 spectrophotometer (schimadzu corporation, japan). the results were expressed as mg/kg. color the color was measured with two instruments: a honey color photometer pfund (hanna instruments hi 96785) and a portable chromameter (cr-400, konica minolta, japan). the cie l*a*b* coordinates were measured with a portable chromameter (cr-400, konica minolta, japan, where l* is the luminance component (ranging from 0 to 100), while a* and b* are colour coordinates related with the red/green and yellow/blue, respectively. electrical conductivity the portable conduct meter hq14d (hach, usa) was used to measure the electrical conductivity of a solution of 20 g dry honey dissolved in 100 ml distilled water. the result was expressed in millisiemens per centimetre (ms·cm-1) determination of total phenolic content the total content of polyphenols (tpc) was determined using the folin-ciocalteu method [11],[12]. the results were expressed as mg gallic acid equivalent (gae)/100 g of honey [13]. the method proposed by biesaga et al. [14] was used to determine tpc and sample preparation was made, as follows: 1 g of honey sample were extracted with 5 ml of 40% methanol/acidified water (v/v, ph = 2, adjusted with hcl). then, the samples were stirred for 15 min with a magnetic stirrer. 0,2 ml of extract were mixed with 2 ml of folin-ciocalteu reagent previously diluted in a ratio of 1:10 with distilled water and 1,8 ml of na2co3 solution (7.5%, w/v). after incubating in the dark for 30 min, the absorbance of all samples were measured at 750 nm with a ocean optics hr4000cg-uv-nir spectrometer (sua). a calibration curve prepared with gallic acid (0-1000 mg/l) was used. flavonoids 5 ml of extract obtained according to the method proposed by biesaga et al. [14] was mixed with 300 µl of nano2 5% and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 301 300 µl of alcl3. after 5 minutes in the dark the samples mix with 2 ml of naoh 1n and after another 6 minutes in the dark, the absorbance of all samples was measured at 510 nm with an ocean optics hr4000cg-uv-nir spectrometer (sua). dpph assay the dpph (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of honey samples was determined as described by brand-williams et al. [15]. sample preparation was made, as follows: 1 g of honey sample was dissolved in 5 ml of methanol 40% (v/v, with acidified water) and filtered. the absorbance of the solution was measured at 515 nm using a ocean optics qe65000 spectrometer (sua). the results were expressed as % ddph using the formula: % dpph= , (2) where: a0= the dpph absorbance, a1= the sample absorbance 3. results and discussion pollen analysis pollen analysis is a method developed and proposed by the international bee botanical commission (ibbc) in 1970 and revised in 1978 [16]. this method consists of identifying the pollen granules by microscopic analysis in order to determine the plants visited by bees during honey production [17]. honey is classified as monofloral honey when at least 45% of the pollen grains belong to a single plant species and this category of honey is the most preferred by consumers for its specific aroma, taste and biological properties [18], [19]. the rubus idaeus pollen was predominant in all samples, accounting for more than 45% pollen grains. therefore, the studied samples correspond to monofloral rubus honeys. escuredo et al. [20] reported a variation in the percentage of rubus pollen from 46.4 to 91.3% when they studied thirty-three honey samples that were collected from galicia (northwest spain). moisture content according to codex alimentarius [25] moisture content should be lower than 20%. honey samples that do not meet this criterion may be unstable during storage [21], the moisture content of honey being a very important factor in determining quality and stability [22]. the moisture content is largely related to the harvest, season and the maturity of the honey in the hive [23]. during storage, low moisture may lead to the development of caramelization and the maillard reaction, while higher water content may cause honey to ferment and acetic acid to form [24]. the moisture content of the analyzed samples ranged from 17.32 % to 20.12%. the results were in accordance with the values reported by sohaimy et al. [22] when analyzing honey from egypt. when analyzing acacia and tilia honey, oroian et al. [3] reported similar results for moisture content. sakač et al. [21] reported a moisture content in the range of 13.2– 21.3% and 10.2–24.1% for acacia honey and sunflower respectively, which means that some samples exceeded the limit (>20%) [25]. ph the ph of the raspberry honey samples ranged from 4.01 to 4.31. the values were similar to those reported by white [26], who reported ph values that ranged from 3.2 to 4.50. escuredo et al. (2013) [27] reported ph values between 3.50 and 5 for honey from north-western spain, while terrab et al. [28] reported ph values between 3.56 and 4.79 in a study of spanish thyme honey [23]. the ph values are of particular importance during the extraction and storage of honey as they influence the texture, stability and storage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 302 time; the average value reported by terrab et al. [28] was 4.2. free acidity the free acidity of honey depends on the organic acids produced from nectar during the ripening by glucose oxidase. geographical origin and harvest season are some factors that influence total acidity [29]. the free acidity of raspberry honey analyzed in this study ranged from 20.1 to 42.1 meq/kg. moujanni et al. [30] reported in their study that free acidity showed values between 14.07 and 32.29 meq/kg, and all the samples are below the legal acidity limit, which states no more than 50 meq/kg for the free acidity [31]. sakača et al. [21] reported in a previous study that the honey with the lowest acidity was acacia honey, with an average value of 9.77 ± 2.08 meq/kg, while honeydew was characterized by a higher mean value (19.3 ± 1.88 meq/kg), which does not differ from sunflower honey (19.1 ± 3.19 meq/kg). hmf content hmf is a parameter that indicates the degree of freshness of honey and consequently its degree of deterioration. the causes of honey deterioration could be the strong or prolonged thermal treatment and the inadequate storage conditions [32]. the maximum level of hmf allowed in honey is 40 mg/kg [25] and the raspberry honey samples do not exceed the hmf limit (6.13-26.79 mg hmf/kg). color honey color is one of the most important sensory parameters for consumers. the color of honey depends on the type of honey and certain chemical characteristics such as mineral content [33].the transition elements existing in honey may form complexes with organic honey compounds that tend to be highly colored and thus affect the color of the honey [34]. changes in honey color appear slowly during storage or rapidly during heat treatment as a result of the maillard reaction [35]. table 1 physical-chemical parameters of raspberry honey raspberry honey parameter sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 f -ratio l* 35.41bc 38.77a 36.4b 34cd 33.16d 28.86e 50*** h*ab 64.42d 74.94b 65.19d 69.73c 83.67a 82.71a 315*** c*ab 23.65bc 25.44a 24.93ab 20.64d 25.03ab 23.18c 14** color (mm pfund) 73.5a 64c 74.5a 68.5b 39e 49d 913*** ph 4.04a 4.31a 4.21a 4.29a 4.01a 4.1a 0.79ns free acidity (meq/kg) 23.7c 24.3c 20.1d 25.4c 42.1a 28.2b 144*** electrical conductivity (ms/cm) 0.367c 0.524a 0.382c 0.427b 0.449b 0.528a 41*** moisture (%) 17.32c 17.44c 18.2bc 17.92c 20.12a 18.92ab 9** hmf (mg/kg) 21.4c 6.58d 26.79b 4.94d 46.4a 6.13d 533*** total phenolic content (mg gae/ 100 g) 24.58a 25.72a 22.32b 14.48e 16.22d 16.4c 122*** flavonoids content (mg qe/100 g) 25.36c 35.29b 41.35a 39.54a 26.29c 33.69b 98*** dpph 81.99b 94.35a 75.77c 92.13a 58.1e 71.99d 165*** food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 303 the color of raspberry honey ranged from extra light amber (39 mm pfund) to light amber (74.5 mm pfund). escuredo et al. [20] reported that according to the obtained results, it seems that rubus honey is characterized by an amber color. the honey samples analyzed in their study had an amber color (mean value of 94 mm pfund), although they varied from light amber (39 mm deep) to dark amber (150 mm deep). the color of honey depends on various factors, but mainly on its floral origin [36]. all honey samples showed similar lightness values (26.52-38.77). electrical conductivity electrical conductivity is an important physical parameter used to authenticate unifloral honey types. electrical conductivity is a factor integrated in the new international standards regarding the differentiation between honeydew and flower honey; the specific electrical conductivity ranges for mixed honey between 0.5-0.8 ms/cm, while a value lower than 0.5 ms/cm indicates pure floral honey, with numerous exceptions [37].a value greater than 0.80 ms/cm is not acceptable for a floral honey because this is specific to honeydew [3]. in this study, 2 samples had values higher than 0.5 ms/cm, while the others had an electrical conductivity between 0.36 and 0.45 ms/cm, which confirm that the samples are pure monofloral honey. kaskoniene et al. [38] confirmed that floral honey has a lower electrical conductivity than that of honeydew and thus this parameter represents a quality indicator that serves as a mean to distinguish between honeydew and floral honey [3]. the value of the electrical conductivity increases with the increase of the ash and acid content of honey [39]. total phenolic content the functional properties of honey are related to the amount of natural antioxidants from pollen collected by bees and other floral nectars [40]. the constituents of honey that are responsible for the antioxidant effects are: flavonoids, phenolic acids, ascorbic acid, catalase, peroxidase, carotenoids, products resulting from the maillard reaction [41], hydroxycarnic acids, and anthocyanins [42]. phenolic acids and flavonoids have been extensively investigated in honey [43] and are used to evaluate honey quality. the antioxidant activity is correlated with the total concentration of phenols, confirmed for seven types of honey that come from italy [44] and for four types from romania [36]. in another study on portuguese honey it was shown that polyphenols in honey are responsible for its antimicrobial effects [45]. the total phenolic content (tpc) of raspberry honeys ranged from 8.11 to 12.86 mg of gallic acid equivalent (gae)/100 g of honey, the average value being 19.95 mg gae/100 g.in the case of irish polifloral honeys tpc ranged from 2.59 to 81.10 mg gae/100 g of honey (n=124, mean ± sd=23.84 ± 13.07) [46]. the variation of tpc in different types of honey depends on their floral origin, kaškonienė et al. [47] considered that heather honey has a higher average tpc (20.12 ± 0.55 mg/100 g) compared to rape honey. the difference in values can be attributed to several factors, including the geographical origin, the “purity” of the honey and the storage conditions [46]. gül & pehlivan [48] reported an average total phenolic content (tpc) of honey samples of 185.81 ± 13.01 mg gae/100 g honey, but this ranged from 470.70 ± 7.43 to 34.37 ± 0.44.the highest phenolic content of samples was found in parsley, rhododendron, carob, and chestnut honey samples as 470.70 ± 7.43, 408.35 ± 4.71, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 304 336.31 ±3.91 and 327.60 ± 0.88 mg gae/100 g, respectively; and the lowest phenolic content was found in wild mint (34.37 ± 0.44 mg gae/100g) and acacia (51.91 ± 1.32 mg gae/100 g). flavonoids content the amount of flavonoids in honey can reach up to 6 mg/kg, while their quantity is much higher in pollen (0.5%) and propolis (10%); according to literature, the most frequent flavonoids found in honey are pinocembrin, apigenin, campferol, quercetin, pinobanksin, luteolin, galangin, hesperetin, and isorhamnetin [13]. a large number of flavonoids, such as pinobanksin, pinocembrin and chrysin, which are found in honey, do not come from the nectar of flowers, but from propolis [49]. in general, the flavonoid content was higher than that of phenolic acids. the flavonide content of raspberry honey varied between from 25.36 to 41.35mg qe/100 g, with an average value of 33.58 mg qe/100 g. tanleque & escriche [50] reported that the highest level of flavonoids was identified in samples from nampula with a value of 30.45 mg/100 g, followed by sofala (19.54 mg/100 g), zambezia (15.22 mg/100 g) and manica (12.95 mg/100 g). dpph assay the dpph method with the stable organic radical 1,1-diphenyl-2-picrylhydrazyl is used for determination of free radical scavenging activity, usually expressed as ic50, the amount of antioxidant necessary to decrease the initial concentration of dpph by 50%. raspberry honey from this study has a dpph value between 58.1 and 94.35%, with an average value of 79%. bertoncelj et al. [51] reported results showing that monofloral honey, acacia and lime had lower antioxidant activities, as these samples had higher ic50 values. their ic50 values were of 53.8 and 28.8 mg/ml, respectively, and were significantly higher (p <0.05) than the ic50 values of other types of honey. chestnut and multifloral honey had ic50 values from 7.2 to 10.7 mg/ml. for pine honey sample was determined a value of 65.52 mg/ml, while the antioxidant activity of citrus honey sample was 2.01 mg/ml [48]. noor et al. [52] found similar results (2.85–39.86 mg qea/100 g) in a variety of honey samples across pakistan. 4. conclusion the physico-chemical characterization of raspberry honey samples collected from different areas of romania in the years 2017 and 2018 was performed to examine the quality of honey samples and to determine any similarities and differences that may be attributed to the botanical origin of honey. all the investigated honey samples (6 samples) met the quality criteria examined (moisture, ph, free acidity, hmf content, color and electrical conductivity), except for a sample that in the case of moisture content exceeds the allowed limit. the pollen analysis together with the physicochemical parameters indicates that the honey samples analyzed were monofloral raspberry samples. in the raspberry honey an average value of 19.95 mg gae/100 g was obtained for the total polyphenol content. in terms of flavonoid content, the average value was higher; respectively 33.58 mg qe/100 g. raspberry honey from this study had high antioxidant activity, with an average value of 79%. 5. acknowledgment this paper was supported by the romania national council for higher education funding, cnfis, project number cnfisfdi-2019-0600. 6. references food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 305 [1]. soares, s., amaral, j. s., oliveira, m. b. p., & mafra, i.,a comprehensive review on the main honey authentication issues: production and origin, comprehensive reviews in food science and food safety, 16(5), 1072-1100, (2017) [2].yaghoobi, n., al-waili, n., ghayourmobarhan, m., parizadeh, s. m. r., abasalti, z., yaghoobi, z., & saloom, k. y., natural honey and cardiovascular risk factors; effects on blood glucose, cholesterol, triacylglycerole, crp, and body weight compared with sucrose, the scientific world journal, 8, 463469, (2008) [3]. oroian, m., amariei, s., rosu, a.,& gutt, g., classification of unifloral honeys using multivariate analysis, journal of essential oil research, 27(6), 533-544, (2015) [4]. isopescu, r. d., josceanu, a. m., colta, t., & spulber, r., romanian honey: characterization and classification, honey analysis, 27, (2017) [5].kivrak, ş., & kivrak, i̇., assessment of phenolic profile of turkish honeys, international journal of food properties, 20(4), 864-876, (2017) [6].von der ohe, w., oddo, l. p., piana, m. l., morlot, m., & martin, p., harmonized methodsofmelissopalynology, apidologie, 35(suppl. 1), s18-s25, (2004) [7]. louveaux, j., maurizio, a., & vorwohl, g., commission internationale de botanique apicole de l'uisb: les méthodes de la mélisso-palynologie, apidologie, 1(2), 211227,(1970) [8]. harmonised methods of the international honey commission, (2008) [9]. bogdanov, s., lullmann, c., mossel, b. l., d'arcy, b. r., russmann, h., vorwohl, g. & flamini, c., honey quality, methods of analysis and international regulatory standards: review of the work of the international honey commission,mitt. lebensm. hyg., 90, 108-125, (1999) [10]. white, j. j., spectrophotometric method for hydroxymethylfurfural in honey, journalassociation of official analytical chemists, 62(3), 509-514 work of the international honey commission, mitt. lebensm. hyg., 90, 108-125, (1979) [11]. singleton, v. l., orthofer, r., & lamuela-raventós, r. m.,analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. in methods in enzymology (vol. 299, pp. 152-178), academic press, (1999) [12]. yorulmaz, h. o., & konuskan, d. b., antioxidant activity, sterol and fatty acid compositions of turkish olive oils as an indicator of variety and ripening degree, journal of food science and technology, 54(12), 4067-4077, (2017) [13]. beretta, g., granata, p., ferrero, m., orioli, m., &facino, r.m.,anal. chim. acta 533, 185–191. doi:10.1016/j.aca.2004.11.010, (2005) [14]. biesaga, m., & pyrzyńska, k., stability of bioactive polyphenols from honey during different extraction methods, food chemistry, 136(1), 46-54, (2013) [15]. brand-williams, w., cuvelier, m. e., & berset, c. l. w. t., use of a free radical method to evaluate antioxidant activity, lwt-food science and technology, 28(1), 25-30, (1995) [16]. louveaux, j., maurizio, a., & vorwohl, g.,methods of melissopalynology, bee world, 59(4), 139-157, (1978) [17]. pires, j., estevinho, m. l., feás, x., cantalapiedra, j., & iglesias, a.,pollen spectrum and physico‐chemical attributes of heather (erica sp.) honeys of north portugal,journal of the science of food and agriculture, 89(11), 1862-1870, (2009) [18]. consonni, r., & cagliani, l. r.,recent developments in honey characterization, rsc advances, 5(73), 59696-59714, (2015) [19]. siddiqui, a. j., musharraf, s. g., & choudhary, m. i., application of analytical methods in authentication and adulteration of honey,food chemistry, 217, 687-698, (2017) [20]. escuredo, o., silva, l. r., valentão, p., seijo, m. c., & andrade, p. b., assessing rubus honey value: pollen and phenolic compounds content and antibacterial capacity,food chemistry, 130(3), 671-678, (2012) [21]. sakač, m. b., jovanov, p. t., marić, a. z., pezo, l. l., kevrešan, ž. s., novaković, a. r., & nedeljković, n. m., physicochemical properties and mineral content of honey samples from vojvodina (republic of serbia), food chemistry, 276, 15-21, (2019) [22].el sohaimy, s. a., masry, s. h. d., & shehata, m. g., physicochemical characteristics of honey from different origins, annals of agricultural sciences, 60(2), 279-287, (2015) [23]. karabagias, i. k., badeka, a., kontakos, s., karabournioti, s., & kontominas, m. g., characterization and classification of thymus capitatus (l.) honey according to geographical origin based on volatile compounds, physicochemical parameters and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 306 chemometrics, food research international, 55, 363-372, (2014) [24]. chirifie, j., zamora, m. c., & motto, a., the correlation between water activity and % moisture in honey: fundamental aspects and application to argentine honeys, journal of food engineering, 72, 287–292, (2006) [25].codex alimentarius commission,codex alimentarius: fats, oils and related products (vol. 8), fao, (2001) [26]. white, j. w., minerals in honey. honey, a comprehensive study, 157-206, (1975) [27]. escuredo, o., míguez, m., fernández-gonzález, m., & seijo, m. c., nutritional value and antioxidant activity of honeys produced in a european atlantic area, food chemistry, 138, 851–856, (2013) [28]. terrab, a., recamales, a. f., hernanz, d., & heredia, f. j., characterisation of spanish thyme honeys by their physicochemical characteristics and mineral contents,food chemistry, 88(4), 537-542, (2004) [29]. de oliveira alves, r. m., production and marketing of pot-honey. in pot-honey (pp. 541-556), springer, new york, ny, (2013) [30].moujanni, a., partida, l., essamadi, a. k., hernanz, d., heredia, f. j., & terrab, a., physicochemical characterization of unique unifloral honey: euphorbia resinifera,cyta-journal of food, 16(1), 27-35, (2018) [31]. council, e. u., council directive 2001/110/ec of 20 december 2001 relating to honey, official journal of the european communities l, 10, 47-52, (2002) [32]. önür, i̇., misra, n. n., barba, f. j., putnik, p., lorenzo, j. m., gökmen, v., & alpas, h., effects of ultrasound and high pressure on physicochemical properties and hmf formation in turkish honey types, journal of food engineering, 219, 129-136, (2018). [33].gonzález-miret, m. l., terrab, a., hernanz, d., fernández-recamales, m. á., & heredia, f. j., multivariate correlation between color and mineral composition of honeys and by their botanical origin,journal of agricultural and food chemistry, 53(7), 2574-2580, (2005) [34]. harris, e. d.,minerals in food: nutrition, metabolism, bioactivity, destech publications, inc., (2014) [35]. da silva, p. m., gauche, c., gonzaga, l. v., costa, a. c. o., & fett, r., honey: chemical composition, stability and authenticity,food chemistry, 196, 309-323, (2016) [36]. al, mărghitaş l., daniel, d., moise, a., bobis, o., laslo, l., & bogdanov, s., physico-chemical and bioactive properties of different floral origin honeys from romania,food chemistry, 112(4), 863-867, (2009) [37]. saxena, s., gautam, s., & sharma, a., physical, biochemical and antioxidant properties of some indian honeys, food chemistry, 118(2), 391-397, (2010) [38]. kaškonienė, v., venskutonis, p. r., & čeksterytė, v., carbohydrate composition and electrical conductivity of different origin honeys from lithuania, lwt-food science and technology, 43(5), 801-807, (2010) [39].bogdanov, s., & martin, p., honey authenticity. mitteilungen aus lebensmitteluntersuchung und hygiene, 93(3), 232-254, (2002) [40].schramm, d. d., karim, m., schrader, h. r., holt, r. r., cardetti, m., & keen, c. l., honey with high levels of antioxidants can provide protection to healthy human subjects, journal of agricultural and food chemistry, 51(6), 1732-1735, (2003) [41]. gheldof, n., & engeseth, n. j., antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples, journal of agricultural and food chemistry, 50(10), 3050-3055, (2002) [42]. sergiel, i., pohl, p., & biesaga, m., characterisation of honeys according to their content of phenolic compounds using high performance liquid chromatography/tandem mass spectrometry., food chemistry, 145, 404-408, (2014) [43]. kečkeš, s., gašić, u., veličković, t. ć., milojković-opsenica, d., natić, m., & tešić, ž., the determination of phenolic profiles of serbian unifloral honeys using ultra-highperformance liquid chromatography/high resolution accurate mass spectrometry, food chemistry, 138(1), 32-40, (2013) [44]. rosa, a., tuberoso, c. i. g., atzeri, a., melis, m. p., bifulco, e., & dessì, m. a., antioxidant profile of strawberry tree honey and its marker homogentisic acid in several models of oxidative stress, food chemistry, 129(3), 10451053, (2011) [45]. estevinho, l., pereira, a. p., moreira, l., dias, l. g., & pereira, e., antioxidant and antimicrobial effects of phenolic compounds extracts of northeast portugal honey, food and chemical toxicology, 46(12), 3774-3779, (2008) [46]. kavanagh, s., gunnoo, j., passos, t. m., stout, j. c., & white, b., physicochemical properties and phenolic content of honey from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xviii, issue 4 – 2019 daniela pauliuc, mircea oroian, physicochemical parameters of romanian raspberry honey, food and environment safety, volume xviii, issue 4 – 2019, pag. 298 – 307 307 different floral origins and from rural versus urban landscapes,food chemistry, 272, 66-75, (2019) [47]. kaškonienė, v., maruška, a., kornyšova, o., charczun,n., ligor, m., & buszewski, b., quantitative and qualitative determination of phenolic compounds in honey,cheminė technologija, 52(3), 74-80, (2009) [48].gül, a., & pehlivan, t., antioxidant activities of some monofloral honey types produced across turkey, saudi journal of biological sciences, 25(6), 1056-1065, (2018) [49]. tomás‐barberán, f. a., martos, i., ferreres, f., radovic, b. s., & anklam, e., hplc flavonoid profiles as markers for the botanical origin of european unifloral honeys, journal of the science of food and agriculture, 81(5), 485-496, (2001) [50]. tanleque-alberto, f., & escriche, i., antioxidant characteristics of honey from mozambique based on specific flavonoids and phenolic acid compounds,journal of food composition and analysis, 103377, (2019) [51]. bertoncelj, j., doberšek, u., jamnik, m., & golob, t., evaluation of the phenolic content, antioxidant activity and colour of slovenian honey,food chemistry, 105(2), 822-828, (2007) [52]. iftikhar, f., mahmood, r., islam, n., sarwar, g., masood, m. a., & shafiq, h. physicochemical analysis of honey samples collected from local markets of rawalpindi and islamabad, pakistan,american journal of biochemistry, 4(2), 35-40, (2014) 1. introduction author instructions food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xxii, issue 1 2023 i author instructions submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a published lecture or academic 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of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 354 363 recovery of hesperidin from lemon by-products using green techniques francisco lorca 1 , david quintín 1 , *presentación garcía 1 1national technological centre for the food and canning industryctnc-. c/concordia, s/n, 30500, molina de segura, murcia, spain, sese@ctnc.es *corresponding author received 2nd november 2022, accepted 28th december 2022 abstract: the food industry is the main producer of food waste. therefore, the development of methodologies to reuse and make the most of it is of great interest. food industry waste can contain compounds with added value that, if properly extracted and used, can be applied to the development of healthy foods (clean label), nutraceuticals, senior food, cosmetics, etc. the revaluation of by-products from the citrus industry will make it possible to reduce the large volume of lemon waste, reducing the cost of waste management and obtaining compounds of interest from them. in this work, different alternative methodologies to the use of organic solvents have been used for the extraction of hesperidin in lemon by-products, including thermal, enzymatic and ultrasound treatment and purification adsorption processes with resins, granular activated carbon and zeolite were optimized. the optimum hesperidin extraction (86371 ± 422 mg gae/g dry extract) was achieved using an enzymatic pre-treatment, cellulase enzyme (validase trl®-sigma) at 0.01% of weight of the lemon peel by-product, treatment time of 60 min, and water/citrus by-product ratio of 3:1 w/w, followed by a purification adsorption/desorption process through purolite pad610 resin. keywords: green techniques, citrus by-product, extraction, lemon extract, ultrasound treatment, enzymatic treatment, adsorption, desorption, phenolic compounds recovery. 1. introduction for several decades, agro-industrial waste has been a focus of attention for researchers worldwide. industrial and agricultural wastes are materials in solid or liquid state that are generated from the direct consumption of primary products or their industrialization, and that are no longer useful for the process that generated them, they are susceptible of use or transformation to generate another product with economic value, of commercial and/or social interest [1]. the valorisation of byproducts of the food industry is presented as a sustainable alternative within the european framework, considering the serious environmental problem it causes, where 1.600 mt of waste generate 3.3 billion tons of co2 equivalent of greenhouse gases released into the atmosphere per year. [2]. the current need for a sustainable food chain requires the implementation of a circular economy approach in the processing industries. the main focus of this approach is to revalorize the discarded parts of vegetables due to their high content of bioactive compounds, that can be used for application in different industries, due to their antioxidant, antimicrobial and functional power. [3,4]. mailto:sese@ctnc.es food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 355 recently, food by-products such as citrus (genus citrus l.) by-products have been studied for their content of other non-nutritional compounds with potential biological properties, such as flavonoids, carotenoids, vitamins, and minerals, which may help reduce the risk of many chronic diseases (cardiovascular and age-related degenerative diseases) [5]. the most abundant flavonoid in citrus has been identified as hesperidin [6] also well known for its antifungal activity [7] cardiovascular beneficial effects [8], antioxidant [9] and anticancer [10] properties. numerous applications as food ingredient or preservative to extend shelf life, however, functional properties are also for its special consideration too. [11-12] citrus by-products are composed of about 50% by weight of matter, consisting of rind, seeds, pulp and peel [13]. different studies use green methodologies for the extraction of flavonoids such us hesperidin from lemon residues, including the use of enzymes, thermal treatments and ultrasound technology [14]. with the appropriate enzymes it is possible to break the structure of the cell membranes to provoke the release of the citrus flavonoids such us hesperidin. essential oils have been extracted from citrus peels (orange and lemon) using enzymatic treatments with cellulases. the yield obtained showed an increase from 2 to 6 [15-17]. the ultrasound technology is based in cells disruption by cavitation. as a result, internal diffusion, and the transfer of compounds of interest from agri-food waste to extract is increased. the use of ultrasound-assisted techniques favours the extraction yield. the extraction of flavonoids from citrus peels was studied, reducing the extraction time and temperature when ultrasonic technology was used. [1821]. adsorption processes are an optimal technology for the recovery of bioactive compounds. this is due to the chemical affinity with compounds in the surface area, selectivity and the facility of regeneration of resins [22]. for the elution of compounds of interest, one of the best eluents is ethanol. [23, 24] in this study, different green extraction and purification technologies, including the use of enzymes, thermal treatments and ultrasound technology, alternative to the conventional ones, were developed to obtain extract rich in hesperidin and polyphenols from lemon by-products. the purification of the extract was carried out by adsorptiondesorption processes with resins, granular activated carbon (gac) and zeolite. compounds of interest such as hesperidin and total polyphenolic compounds were characterised in lemon extracts obtained. 2. materials and methods 2.1 plant material the lemon (citrus limon (l.) burm. f) by-products composed of lemon peels were supplied by several companies in murciaspain along march and april of 2022. the lemon peels were transported directly to lab within no more than 1 hour after their generation and stored at -18 + 1ºc until extraction experiments were conducted. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 356 2.2 adsorbent materials granular activated carbon (gac), particle ranging from 0.5-3.13 mm, was supplied by zeocat (barcelona, spain) as for zeolite adsorbent, with a 0.6-1.5 mm particle size, was supplied by panreacapplichem (barcelona, spain). the polymethacrylic resin crosslinked with divinylbenzene (pad610), polydivinybenzene resin (pad900) and polymethacrylic resin (pad950) were supplied by purolite (hangzhou, china). table 1 shows the technical description of the adsorbent materials used. table 1. physical properties of the adsorbents used (supplier's information). resin matrix *surface area (m 2 ·g -1 ) *pore diameter (å) *pore volume (mg·l 1 ) specify gravity particle size (mm) hydrophobicity pad610 pmc-dvb1 490 300 1.2 1.1 0.35-1.2 moderate pad900 pdvb2 850 220 1.9 1.02 0.35-1.2 moderate pad950 polymethacrylic 450 120 0.6 1.1 0.35-1.2 moderate zeolite aluminosilicate 70-80 4-8 n.d 0.85 0.6-1.5 low cag carbon n.d n.d n.d n.d 0.5-3.13 low *measured by nitrogen adsorption 1 pmc-dvb: polymethacrylics cross-linked with divinylbenzene 2 pdvb: polydivinylbenzene n.d: not determined elution of the compounds of interest adsorbed on the resin surface was carried out by desorption process with ethanol (etoh) 96% scharlau (barcelona, spain). 2.3 experimental design in this study three techniques were applied to look into the impact of hesperidin and polyphenolic compounds. the best technology was then applied for further investigation to determinate the best flavonoids and hesperidin adsorbent materials with the aim to purify the extracts increasing the recovery yield. concentration of the hesperidin and total polyphenols in the extracts were compared for identification of suitable conditions for maximum recovery yield from lemon peels. the figure 1 shows the experimental design. 2.4 extraction of phenolic compounds using different techniques. extracts of interest were prepared with three different treatments as follow: aqueous treatment (we): water was added to lemon by-product in a 3:1 (w/w) proportion. it was them heated up to 100ºc and constantly stirred for 60 minutes. enzymatic treatment (ee): the lemon by-product was mixed with water 1:3 (w/w). a cellulase enzyme (validase trl®-sigma) was added at 0.01% of weight of the lemon peel by-product and the mixture was kept stirred in constant agitation at 25 °c for 60 minutes. then the enzyme was deactivated heating the mixture at 100 °c. this methodology was described by the enzyme supplier. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 357 fig. 1. experimental design ultrasound treatment (us): according with the procedure described by saifullah et al (2020) [25], the lemon by-product was mixed with water 1:3 (w/w). an ultrasonic treatment in a uip500hdt equipment (hielschergermany) with a power of 164 w, amplitude of 100 % with constant agitation at 75 °c for 60 minutes was applied to the mixture. all the samples obtained through the we, ee and us treatment were centrifugated (centrífuga heraeus labofuge 200, thermo scientific, massachusetts, eeuu) at 11.200xg for 20 min, filtered (whatman nº. 42) and lyophilised using coolvacuum lyoepic85 (coolvacuum technologies, granollers, spain). all the experimental processes were conducted on triplicate. these conditions were defined in preliminary tests, considering the sample characteristics and the limitation of the apparatus. moreover, recommendations of granone et col (2022) [26], saifullah et al (2020) [25], si-yu lu et al (2021) [27] and menezes-barbosa el al. [28] were followed. 2.5 purification of the extract using different adsorbers materials. batch adsorption experiments were carried out according to the methodology described by drevelegka et al (2020) [29]. prior to every experiment, the adsorber materials and resins were pretreated following the suppliers’ recommendations. the lemon by-product was processed through the enzymatic treatment (ee), the liquid obtained after centrifugation and filtration steps (as described in point 2.4) was mixed with 5 different adsorbates 10:1 (w/w): resin pad610 (purolite), resin pad900 (purolite), resin pad950 (purolite), zeolite (zeocat) and granular activated carbon (gac) (panreac applichem). it was kept in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 358 contact with the adsorbates under constant agitation for 30 minutes at room temperature. after that the adsorbated hesperidin and polyphenols were eluted with etoh 96% 1:1 (w/w). finally, the solvent was evaporated from the purified extracts in a vacuum evaporator (evaporator r-200, buchi, switzerland). all the experiments were carried out in triplicate. 2.6 determination of total polyphenols the total polyphenols content was determined using folin-ciocalteu method. folin-ciocalteu reagent (fcr) was prepared from water (90 ml) and folin-ciocalteu stock solution (10 ml). a dilution was made by mixing with fcr (5 ml), 5% sodium carbonate solution (4 ml) and the dissolved sample and kept in the dark for 1 hour. after 1 hour, the sample was measured in a spectrophotometer (uvmini 1240 shimadzu, kuoto, japan) with a wavelength of 765 nm. the units used to express the total polyphenols were mg gallic acid equivalents per 1 g dry matter (mg gae/kg dm). [30]. 2.7 determination of limonin limonin was analysed on a liquid chromatography hplc system with detector photodiode array detector pad (agilent 1100, santa clara, eeuu): wavelength 207 nm, with a mobile phase a of 55% water and a mobile phase b of 45% acn, a flow rate of 1 ml/min, an injection volume of 20ml, a c-18 column with a temperature of 45 °c according to can liu et al. [31] 2.8 determination of hesperidin hesperidin was analysed on a hplc systems equipped with a pda (photodiode array detector) (agilent 1100, santa clara, eeuu) with fixed wavelength at 260 nm. the mobile phase that was used consisted of two solvents: water (a) and acetonitrile (b), both containing 0.1% acetic acid (v/v). for elution, a gradient elution with a sample injection volume of 5 μl was used with a kinetex c18 column (100 × 4.6 mm, 2.6 µm, 100 å, phenomenex torrance, ca, usa) with the following parameters: 1 min (10% b), 2 min (20% b), 4 min (30% b), 5 min (90% b), 8 min (10% b) at a temperature of 52 °c with a mobile phase flow rate of 1.0 ml/min. the parameters used were proposed by chaves et al [32]. 2.9 data processing the experiments were performed in triplicate as were the analytical determinations. the values presented are the mean values obtained together with the standard deviation (sd). 3. results and discussion effect of the we, ee and us treatments in the extraction of hesperidin and polyphenols from the lemon peels. table 2 reported the values of hesperidin and polyphenols in the three lyophilized lemon extracts. table 2 illustrated the concentration of compounds of interest.: hesperidin and polyphenols. highlighting that the highest concentration of hesperidin (17689.5 mg/kg) and the total polyphenols (59606.1 mg gae/kg) were obtained when the ee treatment food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 359 was used. compared to the other extractions techniques (we and us), ee produces an extract of higher quality in shorter processing times [33]. table 2. effect of we, ee and us treatment in the extraction of hesperidin and polyphenols for the lemon peels. we ee us hesperidin, (mg/kg) 14676.9 ± 164 17689.5 ± 187 9377.3 ± 85 total polyphenols, (mg gae/kg dm) 23936.9 ± 254 59606.1 ± 425 22816.6 ± 241 results are expressed as mean values of triplicates ± s.d. we: aqueous treatment ee: enzymatic treatment us: ultrasound treatment several experiments have used enzymes to increase the concentration of polyphenols in various vegetables, including citrus fruits [34]. enzymes can be used with other extraction processes, such as ultrasound, thereby increasing the yield of polyphenols and making the processes more costeffective. [35]. purification of hesperidin and polyphenols from lemon peel by adsorption/desorption process. the enzymatic treatment was found to be the most profitable technique for the recovery of hesperidin and polyphenols. it was chosen as a pre-treatment for further purification through adsorption/desorption process. so as to increase hesperidin and polyphenols concentration in lemon extracts, the efficiency of several adsorbent materials were compared. after performing the purification procedure as described in the apart 2.5, the concentration of hesperidin and polyphenols were determined for each dry extract obtained. table 3 shows the results of the analysis of the hesperidin and polyphenols after the purification of the liquid obtained after the treatment of the lemon peel with cellulose with different adsorbent materials. the best adsorbent for obtaining an extract with a high concentration of total polyphenols was the resin pad900, this effect may be due to the higher affinity of the polyphenols for the surface of this resin [36]. cifuentescabezas et al. [37] investigated the application of mn200, mn202 pad900 and pad950 for polyphenol retention using olive mill wastewater and found that the most optimal resin for this application was pad900 resin. the adsorbent material with the best performance for obtaining extract rich in hesperidin was resin pad610, with the highest pore size (table 1), resulted more efficient for the adsorption of compounds with a big structure like hesperidin. when zeolite and cag were used in the adsorption processes, low concentrations of hesperidin and total polyphenols were obtained because food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 360 citrus polyphenols have a strong hydrophilic characteristic [38-39]. table 3. concentration of hesperidin and polyphenols in purified lemon extracts. resin pad610 resin pad900 resin pad950 zeolita gac determination of compounds interest hesperidin, (mg/kg dm) 86371.2 ± 422 33912.9 ± 205 31552.8 ± 251 18300.5 ± 116 6200.8 ± 74 total polyphenol, (mg gae/kg dm) 136670.2 ± 712 152616.8 ± 781 142728.1 ± 765 61752.9 ± 463 28722.3 ± 297 results are expressed as mean values of triplicates ± s.d. 4. conclusions in the present work, the efficiency of treatment with different technologies for the extraction of hesperidin and polyphenols from lemon waste was studied. the technologies were based on water heat treatment, enzymatic treatment and ultrasound treatment. the enzymatic treatment with cellulose was showed the best results in terms of recovery hesperidin and polyphenols. in addition, extracts with a high concentration of hesperidin and polyphenolic compounds were obtained through adsorption-desorption processes as a purification method with resins. the resins tested were showed good affinity for the citrus phenolic compounds, presenting high percentages of adsorption/desorption. the main conclusion is extract rich in hesperidin compound of high purity could be obtained. in summary, this work has shown an optimized methodology to revalue lemon peels by-products using enzymatic technology as a treatment. regarding the procedure cellulase at a concentration of 0.01 % w/w was first added, following with the addition of water in a proportion of 3/1 (w/w) and , time of extraction of 60 min. after enzymatic extraction, the hesperidin and the polyphenols were purified by divinylbenzene resin (pad610 purolite), with an optimum hesperidin concentration of 86371.2 ± 422 mg/kg dm. a strategy applied to the integral use of food lemon by-products based on obtaining bioactive substances of interest was proposed. to this end, different environmentally friendly methodologies that require a reduced consumption of solvents and energy have been researched. these applied treatments are consider as green extraction, as it eliminates the use of environmentally harmful organic solvents, uses natural resources that companies have as waste and a final product with a quality concentration of interest compounds is obtained. in future research we will optimize the process conditions to industrial scale up. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 361 5. acknowledgments this work was supported by agromatter project – establecimiento de una red cervera para el desarrollo de materiales técnicos altamente sostenibles derivados de subproductos o residuos de la industria agrícola y de las operaciones de conservación de espacios naturales. (reference: cer-20211013) from ministerio de ciencia e innovacióncdtispain. in addition, the author francisco lorca salcedo would like to acknowledge ctnc for the individual phd grant (reference: organic chemistry university of murcia). funding: this research was funded by agromatter project – establecimiento de una red cervera para el desarrollo de materiales técnicos altamente sostenibles derivados de subproductos o residuos de la industria agrícola y de las operaciones de conservación de espacios naturales. (reference: cer20211013) from ministerio de ciencia e innovación-cdtispain. conflicts of interest: the authors declare no conflict of interest. 6. references [1] dong-shin, k., sang-bin, l. kinetic study of subcritical water extraction of flavonoids from citrus unshiu peel, separation and purification technology. volume 250, 2020, 117259. [2] fernandes, f.; farias. d.; nerinuma, i.; pastore, g. polyphenols and their applications: an approach in food chemistry and innovation potential. food chemistry. 2021. 338, 127535. [3] din, l.; antonini, s. agro-industrial wastes as sources of bioactive compounds for food and fermentation industries. cienc. rural. 2020. 50, 50. [4] cruzado, m.; cedrón, j. nutracéuticos, alimentos funcionales y su producción. rev. química pucp. 2012. 2, 33– 36. [5] reynoso-camacho, r.; rodríguez-villanueva, l. d.; sotelo-gonzález, a. m.; ramosgómez, m.; pérez-ramírez, i. z. citrus decoction by-product represents a rich source of carotenoid, phytosterol, extractable and nonextractable polyphenols. food chemistry. 2021. 350, 129239. [6] liu y., benohoud, m., hubert j., yamdeu, g., gong, y., orfila, c. green extraction of polyphenols from citrus peel byproducts and their antifungal activity against aspergillus flavus, food chemistry: x, volume 12, 2021, 100144. [7] mahato, n., sinha, m., sharma, k., koteswararao, r., & cho, m. h. modern extraction and purification techniques for obtaining high purity food-grade bioactive compounds and value-added co-products from citrus wastes. 2019. foods, 8(11). [8] pla-pagà, l., valls, r.m., pedret, a., calderón-pérez, l., llauradó, e, companys, j., domenech-coca, c., canela, n., del bas, j.m, caimari, a., puiggròs, f., covas, mi, arola, ll., solà, r. effect of the consumption of hesperidin in orange juice on the transcriptomic profile of subjects with elevated blood pressure and stage 1 hypertension: a randomized controlled trial (citrus study), clinical nutrition, volume 40, issue 12, 2021, pages 5812-5822. [9] zheng, m., lu s., xing, j., enhanced antioxidant, anti-inflammatory and αglucosidase inhibitory activities of citrus hesperidin by acid-catalyzed hydrolysis, food chemistry, volume 336, 2021, 127539. [10] pandey, p., khan, f. a mechanistic review of the anticancer potential of hesperidin, a natural flavonoid from citrus fruits, nutrition research, volume 92, 2021, pages 21-31. [11] panwar, d.; saini, a.; panesar, p. s.; chopra, h. k. unraveling the scientific perspectives of citrus by-products utilization: progress towards circular economy. trends in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 362 food science & technology. 2021. 111, 549562. [12] espinosa-paez, e., hernandezluna, c. e., longoria-garcía, s., martínez-silva, p.a., ortizrodríguez, i., villarreal vera, m. t., & cantú-saldaña, c. m. pleurotus ostreatus: a potential concurrent biotransformation agent/ingredient on development of functional foods (cookies). 2021. lwt, 148, article 111727. [13] russo, m.; bonaccorsi, i.; torre, g.; sarò, m.; dugo, p.; mondello, l. underestimated sources of flavonoids, limonoids and dietary fibre: availability in lemon's by-products. journal of functional foods. 2014. 9, 18-26. [14] wedamulla, n.f., fan m., youngjin choi, eun-kyung kim, citrus peel as a renewable bioresource: transforming waste to food additives, journal of functional foods, volume 95, 2022, 105163, [15] gligor. o.; mocan, a.; moldovan, c.; locatelli, m.; crișan, g.; ferreira, i. c. f. r. enzyme-assisted extractions of polyphenols – a comprehensive review. trends in food science and technology. 2019. 88, 302-315. [16] chávez-gonzález, m. l.; lópezlópez, l. i.; rodríguez-herrera, r.; contrerasesquivel, j. c.; aguilar, c. n. enzyme-assisted extraction of citrus essential oil. chem. pap. 2016. 70, 412–417. [17] sowbhagya, h. b.; chitra, v. n. enzyme-assisted extraction of flavorings and colorants from plant materials. crit. rev. food sci. nutr. 2010. 50, 146–161. [18] khandare, r. d.; tomke, p. d.; rathod, v. k. kinetic modeling and process intensification of ultrasound-assisted extraction of d-limonene using citrus industry waste. chemical engineering and processing process intensification. 2020. 159, 108181. [19] dzah, c. s.; duan, y.; zhang, h.; wen, ch.; zhang, j.; chen, g.; ma, h. the effects of ultrasound assisted extraction on yield, antioxidant, anticancer and antimicrobial activity of polyphenol extracts: a review. food bioscience. 2020. 35, 100547. [20] wang, w.; ma, x.; xu, y.; cao, y.; jiang, z.; ding, t.; ye, x.; liu, d. ultrasoundassisted heating extraction of pectin from grapefruit peel: optimization and comparison with the conventional method. food chem. 2015. 178, 106–114. [21] ma, y. q.; chen, j. c.; liu, d. h.; ye, x. q. simultaneous extraction of phenolic compounds of citrus peel extracts: effect of ultrasound. ultrason. sonochem. 2009. 16, 57– 62. [22] víctor-ortega, m.d., airadorodríguez, d. (2018) revalorization of agro-industrial effluents based on gallic acid recovery through a novel anionic resin. process saf. environ. 115, 17-26. [23] gu, h., chen f., zhang, o., zang, j. application of ionic liquids in vacuum microwave-assisted extraction followed by macroporous resin isolation of three flavonoids rutin, hyperoside and hesperidin from sorbus tianschanica leaves,journal of chromatography b, volume 1014. 2016. pages 45-55. [24] felisberto dos santos, f-k., moraes de rezende, c., florêncio da veiga júnior, f. macroporous polymeric resins as a tool to obtain bioactive compounds in food and food-waste: a review, journal of food composition and analysis, volume 114. 2022. 104703. [25] saifullah, md.; maccullum, r.; mccluskey, a.; vuong, q. comparison of conventional extraction technique with ultrasound assisted extraction on recovery of phenolic compounds from lemon scented tea tree (leptospermum petersonii) leaves. helyon. 2020. 6, 4, e03666. [26] granone, l. i.; hegel, p. e.; pereda, s. citrus fruit processing by pressure intensified technologies: a review. the journal of supercritical fluids. 2022. 188, 105646. [27] lu, s.; chu, y.; sridhar, k.; tsai, p. effect of ultrasound, high-pressure processing, and enzymatic hydrolysis on carbohydrate hydrolyzing enzymes and antioxidant activity of lemon (citrus limon) flavedo. lwt. 2021. 138, 110511. [28] menezes-barbosa, p.; roggia ruviaro, a.; mateus martins, i.; alves macedo, j.; lapointe, g.; alves macedo, g. enzyme-assisted extraction of flavanones from citrus pomace: obtention of natural compounds with antivirulence and anti-adhesive effect against salmonella enterica subsp. enterica serovar typhimurium. food control. 2021. 120, 107525. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 francisco lorca, david quintín, presentación garcía, recovery of hesperidin from lemon by-products using green techniques, food and environment safety, issue 4 – 2022, pag. 354 363 363 [29] drevelegka, i; m. goula, a. recovery of grape pomace phenolic compounds through optimized extraction and adsorption processes. chemical engineering and processing process intensification. 2020. 149, 107845. [30] singleton, v. l.; orthofer, r.; lamuela-raventos, r. m. analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteau reagent. meth. enzymol. 1999. 299, 152–178. [31] liu, c.; liu, j.; rong, y.; liang, n.; rong, l. aqueous extraction of limonin from citrus reticulate blanco. czech j. food sci. 2012. 30, 4, 364-368. [32] o. chaves, j.; l. sanches, v.; viganó, j.; m. de souza, l.; mariana c. de souza, m.; c. da silva, l.; acunha, t.; h. faccioli, l.; a. rostagno, m. integration of pressurized liquid extraction and in-line solidphase extraction to simultaneously extract and concentrate phenolic compounds from lemon peel (citrus limon l.). food research international. 2022. 157, 111252. [33] pyrzynska, k. hesperidin: a review on extraction methods, stability and biological activities. nutrients. 2022. 14, 2387. [34] kelly, n.p.; kelly, a.l.; o'mahony, j.a. strategies for enrichment and purification of polyphenols from fruit-based materials. trends in food science & technology. 2019. 83, 248-258. [35] nadar, s. s.; rao, p.; rathod, v. k. enzyme assisted extraction of biomolecules as an approach to novel extraction technology: a review. food research international. 2018. 108, 309–330. [36] namasivayam, c.; muniasamy, n.; gayatri, k.; rani, m.; ranganathan, k. removal of dyes from aqueous solutions by cellulosic waste orange peel. bioresour. technol. 1996. 57, 37–43. [37] cifuentes-cabezas, m.; sanchezarévalo, c. m.; mendoza-roca, j. a.; vincent-vela, m. c.; álvarezblanco, s. recovery of phenolic compounds from olive oil washing wastewater by adsorption/desorption process. separation and purification technology. 2022. 298, 121562. [38] farooque, s., rose, p. m., benohoud, m., blackburn, r. s., & rayner, c. m. (2018). enhancing the potential exploitation of food waste: extraction, purification, and characterization of renewable specialty chemicals from blackcurrants (ribes nigrum l.). journal of agricultural and food chemistry, 66(46), 12265–12273 [39] mahato, n., sinha, m., sharma, k., koteswararao, r., & cho, m. h. (2019). modern extraction and purification techniques for obtaining high purity food-grade bioactive compounds and value-added co-products from citrus wastes. foods, 8(11). doi: https://doi.org/10.4316/fens.2021.014 128 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag. 128 136 migration of melamine and formaldehyde from kids' bamboo dinner sets svetla petrova petrova 1 , *valentina christova – bagdassarian 2 1food composition and consumer products, national center of public health and analyses, sofia, bulgaria, s.chavdarova@ncpha.government.bg 2food composition and consumer products, national center of public health and analyses, sofia, bulgaria, v.hristova@ncpha.government.bg *corresponding author received 12th march 2021, accepted 28th june 2021 abstract: in recent years, products resembling the composition and / or appearance of plastic materials have been available on the market, which are actually made of a material containing various fillers and additives, such as melamine-formaldehyde resin, bamboo flour and other substances. they are advertised as natural bamboo, environmentally friendly and safe. the migration of melamine and formaldehyde from melamine-formaldehyde resin to food is possible. melamine and formaldehyde are authorized substances in the eu for use in food contact materials under regulation (eu) 10/2011. in 2019, melamine was classified as a possible human carcinogen by the iarc. it is toxic and increases the risk of kidney cancer. in 2012, formaldehyde was classified as carcinogenic to humans by the iarc. it could damage the central nervous system and especially the upper colic of the visual nervous system. the purpose of this study is to determine whether there is a migration of melamine and formaldehyde from kids' bamboo dinner sets offered on the bulgarian market and thus to prove the presence of melamine-formaldehyde resin in their composition. a total of 24 samples 6 bamboo sets, consisting of 4 items bowls, cups, plates and utensils (fork and spoon) were analyzed. standardized methods hplc-uv dd cen/ts 13130-27:2005 for melamine and spectrophotometric dd cen/ts 13130-23:2005 for formaldehyde were used. migration of melamine and formaldehyde from all tested products was found. melamine migration exceeds the specific migration limit (sml) in 62% of products, and the highest value reaches 2.6 times sml. all results of formaldehyde are within the eu legislation permissible values. the material from which the bamboo products are made must be classified as plastic, as it contains melamineformaldehyde resin. the results also show significant inhomogeneity of the composite material both within a batch and between batches. keywords: food contact materials, specific migration, melamine, formaldehyde, bamboo sets 1. introduction bamboos (bambuseae) are a subfamily bambusoideae of the flowering perennial evergreen plant in the grass family poaceae. their homeland is asia. about 91 genera and 1000 species of bamboo are known [1]. bamboo is a fast-growing grass and can be cut without killing the plant. the chemical composition of bamboo determines its properties and uses. over 90% of the total mass of bamboo consists of cellulose, hemicellulose and lignin, and the remaining up to 10% are resins, tannins, waxes and inorganic salts. http://www.fia.usv.ro/fiajournal mailto:s.chavdarova@ncpha.government.bg mailto:v.hristova@ncpha.government.bg food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 129 bamboo is used as a material for making various dishes and utensils. its environmental characteristics are the main reason bamboo kitchen utensils have become popular and widely used. they are ideal for picnics, camping and travel. they are sought after for their strength, durability and biodegradability. they are more durable than wood and plastic. in addition, they are smooth and light, more resistant to heat and water. they do not absorb odors and are easy to clean. bamboo dishes are easy to colour and decorate. this makes them attractive to consumers, giving a natural touch to any kitchen. in recent years, items resembling the composition and/or appearance of plastic materials have been available on the market. these items are similar to kitchen melamine dishes, but are more matte. these include various bowls, cups, plates and cutlery. some of them consist of melamine plastic as the main structural component, but also contain fillers ground bamboo ("bamboo-melamine"), corn flour and other similar ingredients. other items may include bamboo fibers and polymer resin as a binder to form a composite product. a number of such bamboo-melamine food contact items are sold with the claim of being recyclable and natural. they are labeled and marketed as "biodegradable", "environmentally friendly", "organic" or "natural" or even in some cases "100% bamboo" (figure 1). fig.1. "bamboo" dishes offered in a large food chain in fact, so-called "bamboo" kitchen utensils are made of a material containing various fillers and additives, such as melamine-formaldehyde resin, bamboo and/or corn flour and other substances often not described on the label, so their composition is unknown. 2. melamine-formaldehyde resin melamine-formaldehyde resin is a polymeric material (figure 2), which is obtained in the process of polymerization of melamine and formaldehyde. melamine-formaldehyde resin is used as a filler in bamboo tableware. migration of melamine and formaldehyde into the food could occur through use of such items in microwave ovens or due to cooker. such migration could also be the result of production problems and defects, e.g. incomplete polycondensation during polymer production, injury and/or scratching of surfaces during molding. fig. 2. preparation of melamine-formaldehyde resin a study by the federal institute for risk assessment in germany (bfr) shows that the use of melamine tableware at high temperatures and heating in microwave ovens leads to a risk of increased migration of melamine and formaldehyde. factors favoring the increase of migration are scratching, disruption of the surface of the kitchenware and their use in an acidic environment [2]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 130 3. legislation bamboo is not included in the list of permitted monomers, but it is a natural polymeric substance. the use of natural additives, such as bamboo in a plastic matrix, may not necessarily pose a direct health risk [3]. however, migration of melamine and formaldehyde is possible from dishes containing melamineformaldehyde resin [2]. melamine (fcm № 239) and formaldehyde (fcm № 98) are authorized substances in the eu [3] for use in the manufacturing of plastic food contact materials. however, they are subject to restrictions, with specific migration limits (smls) of 15 mg/kg for formaldehyde (expressed as total formaldehyde and hexamethylenetetramine) and 2.5 mg/kg for melamine [4]. the requirement (see: article 3 (2) of regulation (ec) № 1935/2004 [5]) is that the labeling, advertising and presentation of a material or article does not mislead consumers. therefore, manufacturers guarantee the actual composition of kitchen utensils through correct labeling and advertising in their marketing. due to the increased number of reports of high levels of migration of formaldehyde and melamine from melamine plastic products, commission regulation (eu) № 284/2011 [6] has been introduced. this regulation lays down specific conditions and detailed procedures for the import of plastic melamine kitchenware originating in the people's republic of china and hong kong. since the beginning of 2020, the rasff (rapid alert system for food and feed) has received reports of established high levels of migration of melamine and formaldehyde from bamboo products intended for food contact, mostly imported from china (table 1). the migration of melamine of 28.5 mg/kg (reported by germany) exceeds 11.4 times the specific migration limit of the monomer. migration of formaldehyde of 269.0 mg/kg (reported by switzerland) is 18 times the sml for formaldehyde. these data raise concern about the items available on the european market. table 1. notification for migration of melamine and formaldehyde according to rasff the system for the period january october 2020. country month 2020 bamboo item country of origin melamine,m g/kg formaldehyde, mg/kg germany april set of utensils france 28.5 12.2 may cups china via netherlands 4.3 15.7 october bowls austria 4.2 239.0 poland january bowls china 188.2 czech republic may kids’ bamboo dinner sets china 5.5 23.4 france february kids’ bamboo dinner sets china via belgium 4.71 19.2 lithuania september bamboo cups for travel china via poland 2,52 switzerland september bamboo bowls hong kong 269.0 portugal april bowls china 3.81 may bowls china 5.5 16.37 greece march food boxes china 16.7 48.4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 131 4. melamine and formaldehyde characteristics, health risks melamine (c3h6n6) (fig. 3) is an aromatic compound known as iupac 2,4,6-triamino-1,3,5-triazine. it is a colorless crystal, insoluble in water and slightly soluble in most organic solvents. fig. 3. chemical structure of melamine melamine shows low acute toxicity in experiments with animals. the potential risk to human and animal health is associated with melamine’s ability to form uric acid complexes, leading to the formation of crystals in the urine and severe kidney damage. melamine is toxic to the kidneys and increases the risk of kidney cancer. it is harmless in low single doses, but at high levels and in combination with its analogues such as cyanuric acid, the health risk becomes significant [7]. melamine has adverse effects on the male reproductive system, including testicles and sperm morphology at doses between 5 and 25 mg/kg body weight [8]. in 2019, melamine was classified as a probable human carcinogen (group 2b) by iarc [9]. no evidence of genotoxicity in vitro and in vivo and teratogenic effects have been recorded. formaldehyde (fig. 4) hcho is a colorless gas with a pungent, irritating odor. its iupac name is methanal. it is soluble in water and in most organic solvents. fig.4. chemical structure of formaldehyde it enters into the body by inhalation or using an oral or dermal route. it is degraded in the liver to methyl alcohol and formic acid. a single intake of small amounts of formaldehyde does not cause acute effects. formaldehyde damages the central nervous system and especially the superior colliculi (“little hills”) in the visual nervous system. this is due to its metabolites formic acid and methyl alcohol. it has mutagenic and carcinogenic effects [10]. in 2012, formaldehyde was classified as carcinogenic to humans (group 1) by iarc [10]. bamboo tableware were analyzed based on the procedures described in the guidelines of the european reference laboratory for food contact materials (eurl-fcm) [11, 12]. the conditions for migration testing time, temperature regime, model solutions are selected according to regulation (ec) 10/2011 [4]. 5. matherials and methods reagents and equipment the reagents used are presented in table 2. the equipment for melamine analysis includes:  ltrasonic bath with timer and heater ultrasonic usc200th;  liquid chromatograph hp agilent – series 1050;  column: reverse phase lichrosorb 250 х 4.6 (5µm);  uv detector hp agilent series 1050. the equipment for formaldehyde analysis includes:  water bath, with constantly controlled temperature;  spectrophotometer "spekol 11", manufactured by carl zeiss jena. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 132 table 2. reagents used for melamine for formaldehyde  melamine standard reference material (2,4,6-triamino-1,3,5-triazine), cas. № 10878-1);  acetic acid, 96%, honeywell (fluka);  acetonitrile – gradient grade for hplc,  sodium dihydrogen phosphate monohydrate (nah2po4.h2o) to prepare a buffer for the mobile phase  formaldehyde certified reference material (formaldehyde solution, cas. № 50-00-0);  acetic acid, 96%, honeywell (fluka);  sulfuric acid – 95-97%, honeywell (fluka)  chromotropic acid (c10h6na2o8s2.2h2o), merck description of the samples all samples were purchased from different retail stores and are representative of different batches. the test was performed on 6 bamboo sets. each set consists of 4 items 6 bowls, 6 cups, 6 plates and 6 pairs of cutlery (fork and spoon), a total of 24 samples, shown in figure 5 to figure 7. the products are labeled as made of bamboo, environmentally friendly and biodegradable. the description indicates their origin (china) and declares that they are dishwasher safe. sample preparation each item was analyzed 6 times and the average value was calculated (n = 6). the food simulant used to extract the two monomers is a 3% acetic acid solution (model solution b [4]). each sample was filled with the model solution up to 0.5 cm below the rim and exposed to it. the items are covered with watch glass and sealed with parafilm to prevent evaporation of the food simulant during their stay in the thermostat at a controlled temperature of 70⁰c for 2 hours. the simulants from the first and second tests were discarded. the food simulant from the third exposure was analyzed for melamine and formaldehyde migration. fig.5. image of the set "panda" bowl, cup, plate, utensils (fork and spoon) fig.6. image of the set "cow" bowl, cup, plate, utensils (fork and spoon) fig.7. image of the set "boy" bowl, cup, plate, utensils (fork and spoon) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 133 analytical instrumental methods hplc-uv standardized method dd cen/ts 13130-27:2005 “materials and articles in contact with foodstuffs. plastics substances subject to limitation. determination of 2,4,6-triamino-1,3,5triazine in food simulants” was used to quantify migrated melamine in 3% aqueous acetic acid [13]. the analytical conditions for melamine analysis are as follows:  mobile phase phosphate buffer with ph 6.5: acetonitrile = (80: 20);  flow rate – 1.2 ml/min;  uv detection at λ = 230 nm;  injection volume – 50 µl. a standardized spectrophotometric method dd cen/ts 13130-23:2005 “materials and articles in contact with foodstuffs. plastics substances subject to limitation. determination of formaldehyde and hexamethylenetetramine in food simulants” was used to quantify formaldehyde in 3% aqueous acetic acid [14]. the analytical conditions for formaldehyde analysis are as follows: exactly 8 ml of 75% sulfuric acid and 1 ml of chromotropic acid were added to 1 ml of sample, followed by heating in a water bath at 60 °c for 20 minutes and cooling to room temperature for 1 hour. the extinction of the obtained color compound is determined spectrophotometrically at a wavelength λ = 574 nm. the analytical parameters for both methods are presented in table 3: table 3. analytical parameters parameter melamine formaldehyde limit of detection (lod), mg/l 0.5 0.005 limit of quantification (loq), mg/l 1.0 1.5 working range, mg/l (1.0 ÷ 8.0) (1.5 ÷ 30.0) correlation coefficient r2 = 0.9996 r2 = 0.9976 6. results and discussion the analyzed items of the two sets of "panda" indicated migration of melamine, which exceeds the limit value of 2.5 mg/kg according to the european legislation 1.6 times. the highest obtained average is (4.14 ± 0.07) mg/kg, in bowl "panda 1". all items in the same sets show a migration of formaldehyde but below the specific migration limit of 15 mg/kg according to european legislation. the highest mean value of (6.49 ± 0.01) mg/kg was obtained with the same bowl "panda 1". the data obtained for the panda bamboo sets are presented in figures 8 a) and b). fig.8a. results for set "panda 1" fig.8b. results for set "panda 2" food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 134 for the sets "cow", melamine migration was again reported for all products. the highest mean value obtained is (5.12 ± 0.004) mg/kg for the plate "cow 2", up to twice exceeding the permissible level by 2.5 mg/kg. all items in the same sets show a migration of formaldehyde. the highest mean value is (5.91 ± 0.01) mg/kg obtained for the bowl "cow 2". all results for formaldehyde migration are below the permissible level of 15 mg / kg according to european legislation. the data obtained for the cow bamboo sets are presented in figures 9 (a) and (b). fig. 9a. results for set "cow 1" fig.9b. results for set "cow 2" for „boy“ bamboo sets, melamine migration was again reported for all products. the highest mean value obtained is (6.55 ± 0.26) mg/kg, for the bowl "boy 1", exceeding the permissible level up to 2.6 times. figure 10 shows the chromatograms of the highest value obtained of melamine and melamine standard solution. migration of formaldehyde was detected in all sets "boy". all formaldehyde migration results are below the permissible level of 15 mg / kg according to european legislation. the highest mean value is (10.40 ± 0.01) mg/kg obtained from the same bowl "boy 1". the data obtained for the boy bamboo sets are presented in figures 11 a) and b). fig.10. chromatograms of the highest migration value obtained and melamine standard solution food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 135 fig. 11a. results for set "boy 1" fig. 11b. results for set "boy 2" in all bamboo products studied, migration of both monomers forming melamine formaldehyde resin melamine and formaldehyde was found. this indicates that the material from which they were made should be classified as plastic. the results obtained for both melamine and formaldehyde are scattered in a wide concentration range. this shows the inhomogeneity of the plastic material from which the individual items in the different batches are made. these results confirm the impossibility of monitoring the quality of imports of such products. figure 12 presents the summarized results of the performed analyzes for migration of melamine and formaldehyde by sets. in 50% of the sets, the migration of melamine exceeds the specific migration limit for the monomer up to 1.6 times. no migration of formaldehyde above the legal requirement of 15 mg/kg has been identified. fig.12. results for bamboo sets 7. conclusion migration of melamine and formaldehyde from all tested products has been proven. melamine migration exceeds the specific migration limit (sml) in 62% of products and the highest value reaches 2.6 times sml. all formaldehyde results are within the permissible values in european legislation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 svetla petrova petrova, valentina christova – bagdassarian, migration of melamine and formaldehyde from kids' bamboo dinner sets, food and environment safety, volume xx, issue 2 – 2021, pag. 128 – 136 136 the material from which the bamboo products are made should be defined as plastic, as it contains melamineformaldehyde resin. plastic products labeled as made of bamboo create a misconception in the consumer about them being "made of natural materials", "environmentally friendly", "biodegradable". in fact, although they contain bamboo fibers, they are made of plastic and are not completely biodegradable. 8. references [1]. zhang q. s., jiang s. x., tang y. y. industrial utilization on bamboo: technical report no. 26. the international network for bamboo and rattan (inbar), people‟s republic of china., 206, (2002). isbn 962-85124-7-1. retrieved from https://www.inbar.int/wpcontent/uploads/2020/05/1489455793.pdf [2]. bfr, release of melamine and formaldehyde from dishes and kitchen utensils, 2011. retrieved from https://mobil.bfr.bund.de/cm/349/release_of_melam ine_and_formaldehyde_from_dishes_and_kitchen_ utensils.pdf [3]. summary of discussions of the expert working group on food contact materials on the use and placing on the market of plastic food contact materials and articles containing ground bamboo or other similar constituents, brussels, (2019). retrieved from https://ec.europa.eu/food/sites/food/files/safety/doc s/cs_fcm_bamboo_wg-201906.pdf [4]. commission regulation (eu) no 10/2011 of 14 january 2011 on plastic materials and articles intended to come into contact with food. [5]. regulation (ec) no 1935/2004 of the european parliament and of the council of 27 october 2004 on materials and articles intended to come into contact with food and repealing directives 80/590/eec and 89/109/eec. [6]. commission regulation (eu) no 284/2011 of 22 march 2011 laying down specific conditions and detailed procedures for the import of polyamide and melamine plastic kitchenware originating in or consigned from the people’s republic of china and hong kong special administrative region, china. [7]. hsieh t. j., hsieh p. c., tsai y. h. et al., melamine induces human renal proximal tubular cell injury via transforming growth factor-β and oxidative stress, toxicol sci., 130(1):17 – 32, (2012). [8]. yang y. et al., acute low-dose melamine affects hippocampal synaptic plasticity and behavior in rats, toxicol lett, 214:63 – 68, (2012). [9]. some chemicals that cause tumours of the urinary tract in rodents, iarc monographs on the evaluation of carcinogenic risks to humans, 119:274, (2019). [10]. chemical agents and related occupations, iarc monographs on the evaluation of carcinogenic risks to humans, 100f:628, (2012). [11]. technical guidelines on testing the migration of primary aromatic amines from polyamide kitchenware and of formaldehyde from melamine kitchenware, 1st edition [in support of commission regulation 284/2011 laying down specific conditions and detailed procedures for the import of polyamide and melamine plastic kitchenware originating in or consigned from people's republic of china and hong kong special administrative region, china] eur24815 en 2011, (2011). retrieved from: https://ec.europa.eu/jrc/en/publication/eurscientific-and-technical-research-reports/technicalguidelines-testing-migration-primary-aromaticamines-polyamide-kitchenware-and [12]. eu guidelines on conditions and procedures for the import of polyamide and melamine kitchenware originating in or consigned from people’s republic of china and hong kong special administrative region, china european commission., health and consumers directorategeneral directorate, esafety of the food chain e6innovation and sustainability, version 1 (2011). retrieved from https://ec.europa.eu/food/sites/food/files/safety/doc s/cs_fcm_legis_china_guidelines_import-polymidemelamine.pdf [13]. dd cen/ts 13130-27:2005. materials and articles in contact with foodstuffs. plastics substances subject to limitation. determination of 2,4,6-triamino-1,3,5-triazine in food simulants. [14]. dd cen/ts 13130-23:2005. materials and articles in contact with foodstuffs. plastics substances subject to limitation. determination of formaldehyde and hexamethylenetetramine in food simulants. https://www.inbar.int/wp-content/uploads/2020/05/1489455793.pdf https://www.inbar.int/wp-content/uploads/2020/05/1489455793.pdf https://mobil.bfr.bund.de/cm/349/release_of_melamine_and_formaldehyde_from_dishes_and_kitchen_utensils.pdf https://mobil.bfr.bund.de/cm/349/release_of_melamine_and_formaldehyde_from_dishes_and_kitchen_utensils.pdf https://mobil.bfr.bund.de/cm/349/release_of_melamine_and_formaldehyde_from_dishes_and_kitchen_utensils.pdf https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_bamboo_wg-201906.pdf https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_bamboo_wg-201906.pdf https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-research-reports/technical-guidelines-testing-migration-primary-aromatic-amines-polyamide-kitchenware-and https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-research-reports/technical-guidelines-testing-migration-primary-aromatic-amines-polyamide-kitchenware-and https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-research-reports/technical-guidelines-testing-migration-primary-aromatic-amines-polyamide-kitchenware-and https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-research-reports/technical-guidelines-testing-migration-primary-aromatic-amines-polyamide-kitchenware-and https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_legis_china_guidelines_import-polymide-melamine.pdf https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_legis_china_guidelines_import-polymide-melamine.pdf https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_legis_china_guidelines_import-polymide-melamine.pdf 1. introduction 7. conclusion doi: https://doi.org/10.4316/fens.2023.004 40 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 40 49 physicochemical and sensory characterization of five promising aromatic rice accessions in benin *valère dansou 1 , paul ayihadji ferdinand houssou 1 , yannick sourou rosanoff kouke 2 , abel bodéhoussè hotegni 1 , warou arnold cospe sagui 1 , kowiou aboudou 1,3, hugue zannou 1 1agricultural and food technology program of the agricultural research center of agonkanmey of the national institute of agricultural research of benin (inrab), vadansou@gmail.com 2rice research program of the south agricultural research centre of the national institute of agricultural research of benin (inrab) 3department of food technology engineering of the polytechnic school of abomey calavi of the university of abomey calavi (uac) *corresponding author received 9th october 2022, accepted 28th march 2023 abstract: rice research program of the national institute of agricultural research of benin (inrab) has selected five romising aromatic rice accessions in terms of yield performance, resistance to diseases and climate change but their quality is not yet known for a better utilization, hence the objective of this present study. thus, five promising aromatic rice accessions ara-1, ara-2, ara-5, ara-18 and ara-23 and two released aromatic rice varieties used as check (ir 841 and briz 10b) were collected in three localities (covê, glazoué and malanville). physico-chemical and nutritional characteristics of the rice samples were evaluated using standard methods. the sensory quality was evaluated by a panel of rice farmers. results showed that the ash contents of white rice varied significantly (p<0.05) whatever accessions and highest values was obtained for accessions ara-18 (0.70%) and ara-23 (0.76%) and the lowest for ara-1 (0.51%) and ir 841 (0.51%). for parboiled rice, ara-18 (0.82%) and ara-23 (0.85%) presented the highest ash contents while (0.55%) and ara-5 (0 .66%) the lowest one. protein contents varied from 7.50% to 8.76% for white rice and from 9.5% to 10.9% for parboiled rice. parboiled type of accessions ara-1 (3), ara-2 (3), ara-5 (4) and ara-23 (2) are characterized by high gelatinization temperature (75oc -79oc) like that of parboiled type of briz 10b (2). aromatic rice accessions and checks were characterized by high level of homogeneity rate (>85%) and low (0.1 to 10%) uniformity presence of starch as well as white and parboiled rice. no significant difference (p>0.05) was observed between the swelling rates of the white and parboiled rice samples. parboiled cooked rice (ara-2 and ara-5) and white cooked rice (ara-2, ara-18 and ara-5) were respectively most appreciated by 70% and 65% of the panelist compared to the parboiled and white cooked check varieties, above all because of their high roma, attractive colour, non-sticky appearance and very pleasant taste. keywords: aromatic rice accessions, white rice, parboiled rice, characterization, qualities, benin. 1. introduction in the context of promotion of local rice in benin, more than thirty rice varieties including high yielding varieties (ir 841 and nerica l20) have been developed by research institute at various levels [1]. despite the extensive research on common high-yielding rice varieties with successful results, consumers have an increasing preference for flavored rice [1]. to address this consumer preference, recent studies conducted in 2018 by the rice research program of national institute of agricultural research of benin (inrab), have selected 28 aromatic rice accessions mailto:vadansou@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 41 and five of them look like promising in terms of yield, disease resistance and climate change. however, the processing and storage suitability of these aromatic rice accessions are not sufficiently elucidated. therefore, it is necessary that physico-chemical and organoleptic tests must be conducted on those five promising aromatic rice accessions selected before their releasing. present study was initiated in this perspective with the objective of evaluating the physicochemical, nutritional and organoleptic qualities of five promising new aromatic rice accessions in benin. 2. matherials and methods plant material the plant material used consisted of five promising aromatic rice accessions, namely ara-1, ara-2, ara-5, ara-18 and ara-23 and two checks rice varieties (ir 841 and briz 10b), collected in three localities (covê, glazoué and malanville). studies were carried out on the parboiled and white rice type of each rice sample. parboiling of aromatic rice accessions is a hydrothermal treatment following the method written by authors of [2]. physicochemical and nutritional analysis of new fragrant rice accessions parameters such as moisture content, alkaline digestibility, water absorption index, homogeneity rate, swelling rate, total ash content and protein content were determined on each type of rice using the methods described by authors of [3]. moisture content was determined by drying the sample in a thermostatically controlled oven to constant weight. five grams (5.0 g) of the sample was accurately weighed into a previously cleaned, dried and weighed glass crucible. the crucible with its content was put into the oven at 103°c for 12 h. the sample was then cooled in a desiccator and weighed. the loss in weight expressed as a percentage of the initial weight of the sample gives the moisture content of the sample. for the determination of ash content, 2.0 g sample was weighed into a previously dried and weighed porcelain crucible. the crucible with its content was placed in a furnace preheated to 600°c for 2 h. the sample was allowed to cool in the furnace to 250°c. the crucible and the ash were then transferred into an oven at 100°c for 30 min cooling. after this period, the crucible with its content was cooled in a desiccator. the crucible with its content was weighed. the weight of the ash was expressed as a percentage of the initial weight of the sample. protein content was determined by microkjeldahl method and it was calculated by multiplying total nitrogen content by a factor of 5.95 to convert nitrogen content into protein content of rice. for water absorption index and swelling rate, 1 g of starch was deposited in a centrifuge tube and 25 ml of distilled water at 60°c was also added. subsequently, the blend was centrifuged at 4500 rpm for 15 min. the supernatant was immediately discarded in previously dry beakers at 60°c and weighed. then, the supernatant was dried at 70°c for 12 hours after weighing the time beaker to obtain the weight of soluble starch portion. the centrifuge tubes weighed to obtain the weight of the gel. participatory evaluation of organoleptic quality of promising aromatic rice accessions with rice farmers after processing rice samples, sensory tests were conducted by a panel of 15 rice farmers using the appropriate quality attributes/criteria. the assessment of different rice type (paddy, parboiled and white rice, parboiled and white cooked rice) was done on the basis of the quality criteria. paddy rice was assessed on the basis of quality attributes/parameters such food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 42 as appearance/colour, size, length, fill rate, impurity rate. while the milled forms of white and parboiled rice were evaluated using quality attributes such as: appearance, hull breakage, presence of white core, presence of black spot and overall acceptability. finally, the cooked forms of parboiled and non-parboiled (white) rice were evaluated with respect to colour/appearance, aroma, taste, chewability, stickiness and overall acceptability. statistical analysis the data were processed using microsoft excel 2013 spreadsheet software and spss v21.0. one-way analysis of variance (anova) was performed for means comparison. the student newman-keuls multiple comparisons test were performed in order to verify weather eventual postural variations in the different mandibular positions were statistically significant 3. results and discussion physico-chemical characteristics of white and parboiled aromatic rice accessions in the white rice, it was observed that the ash content varied significantly (p<0.05) from 0.31% (check 2) to 0.76% (ara-23). the high content was observed for accessions ara-18 (0.70%) and ara-23 (0.76%), followed by accessions ara-1 (0.51%) and check 1 (0.51%) (table 2). accessions ara-2 (0.57%) and ara-5 (0.54%) showed a highest ash content than check 1 (0.51%) and check 2 (0.31%). therefore, four aromatic rice accessions ara-18, ara-23, ara-2 and ara-5 have good nutritional quality compared to the check variety due to their higher ash content. these values are lower than those found by [4] for the cargo or whole rice who reported that the majority of the mineral elements were concentrated in the bran of the rice. compared to the parboiled type, the results show that the ash contents of parboiled aromatic rice accessions varied significantly (p<0.05) (table 1). indeed, the aromatic rice accessions ara-2 (0.79%), ara-18 (0.82%) and ara-23 (0.85%) showed high ash contents compared to the check variety 1 (0.55%) and check variety 2 (0.73%), ara-1 (0.73%) and ara-5 (0.66%). this difference is believed to be due to the parboiling process, which causes the migration of nutrient values of rice from the husk to the endosperm. the same observations were made by several authors [2,5,6] who reported that parboiling process improves the nutritional quality of rice withing variety. regarding protein content, it was found that content of the white rice type varied from 7.50% (ara-23) to 8.76% (check 2). statistical analysis showed a significant difference (p<0.05) in protein content between three aromatic rice accessions and check varieties. the aromatic rice accessions ara-1 (7.67%), ara-2 (7.81%), and ara-23 (7.50%) had lower protein contents than those obtained by check 1 (8.40%) and check 2 (8.76%) (table 2). while accessions ara-5 (8.10%), ara-18 (8.36%) showed similar protein levels to check 1 (8.40%) and check 2 (8.76%). despite the fact that three aromatic rice accessions had low protein contents compared to the check varieties, these values were higher than those obtained by [7] who found lower protein contents in aromatic rice varieties in ghana such as royal feast (5.3%), marshall (5.9%) and jasmin (5.8%). similarly, for the parboiled rice samples, statistical analysis showed a significant difference (p<0.05) in protein content between three aromatic rice accessions and the check varieties. the aromatic rice accessions ara-1 (9.67%), ara-2 (9.81%), and ara-23 (9.50%) had lower food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 43 protein contents than the check 1 (10.6%) and check 2 (10.76). while the rice accessions ara-5 (10.7%), ara-18 (10.9%) showed similar protein levels to those obtained by the two check varieties. apart from accessions ara-1, ara-2 and ara-23, the other accessions as well as the two check varieties had higher protein contents than those reported by [8]. the alkaline digestibility of the five white aromatic rice accessions and the two check varieties showed is less than four (table 2). these results indicate the balance within the rice starch between the number of long and short amylopectin chains [9]. these results also indicate that white aromatic rice samples are characterized by a high gelatinization temperature (75 °c 79 °c). research has shown that this high gelatinization temperature can be explained by the presence of disulphidlinked proteins in the rice that would reduce the swelling of the rice during heat treatment [10]. in the parboiled type, the accessions ara1 (3), ara-2 (3) and ara-23 (2) were also characterized by less than 4 alkaline digestibility like that of check 2 (2). while accessions ara-5 (4), ara-18 (4.5) showed average digestibility like that of the check1 (5.5) (table 1). alkaline digestibility is a very important rice parameter that correlates directly with the gelatinization temperature of cooked rice and the amount of amylopectin contained in its starch [11]. this lower alkaline digestibility of parboiled rice accessions indicates that they have a high gelatinization temperature [12]. this high gelatinization temperature would imply the use of more fuel during the cooking process of the rice, and therefore more expense for the processors, whereas accessions with medium alkali digestibility would require less fuel. the homogeneity rate and chalkiness (presence of starchy endosperm) were also measured on the accessions. in the white rice samples, accessions ara-1 (96.96%), ara-2 (93.51%), ara-5 (96.71%), ara18 (95.39%) and ara-23 (95.46%) had respectively a high homogeneity rate (>85%), as those of check1 (97.25%) and check 2 (95.52%) (table 2). all the white rice samples analyzed had chalkiness score equal to 1. it means that the percentage of starchy endosperm in the rice grains varies from 0.1 to 10%. the homogeneity parameters was important to appreciate quality because the more homogeneous a rice is, the more it is appreciated by consumers. results obtained showed the high homogeneity rate of the five (05) aromatic rice accessions mean that they will be highly appreciated by consumers like the check varieties. the low percentage of chalkiness observed with all white rice samples is similar to those obtained by [13] during the analysis of rice varieties in benin. the same trends for homogeneity and chalkiness parameters are obtained for the parboiled rice samples with higher values for homogeneity rate (table 1). these results obtained for uniformity show that the five aromatic rice accessions had good quality similar to check varieties. the low percentage of chalkiness observed for all rice samples is certainly related to the intensity of temperature during the rice production cycle from filling to grain maturity stage [14]. the analysis of the swelling rate of the aromatic white rice samples revealed that no significant difference (p>0.05) was observed between ara-1 (3.35) and check 1 (3.49) samples in one hand and ara-2 (4.08), ara-5 (4.24), ara-18 (4.32), ara-23 (4.62) and check 2 (4.17) samples on the other hand. however, the swelling rate of the ara-1 and check 1 rice samples is lower than that of ara-2, ara-5, ara-18, ara-23 and check 2 (table 2). from these results, it appears food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 44 that the aromatic rice accessions ara-2, ara-5, ara-18, ara-23 and check 2 contain more amylose than the other rice samples. the same observations were made for the unparboiled rice samples. according to [4], there is a positive correlation between the swelling rate of rice and the amylose content of rice. the results on the water absorption rate of the white rice samples showed that there is a significant difference (p<0.05) between the five aromatic rice accessions and the check varieties. the accessions ara-2 (4.30%), ara-5 (4.37%), ara-18 (4.13%) and ara-23 (3.97%) showed similar water uptake to that of check 2 (4.23%) (table 2). while the accession ara-1 (2.90%) showed a similar water uptake rate as check 1 (3.08%). according to [15], the more the rice grain is characterized by a hard texture, the more it will tend to absorb water. thus, the aromatic rice accessions ara-2, ara-5, ara-18 and ara-23 would be genetically characterized by a hard texture like that of check 2. for the parboiled rice samples, a significant difference (p<0.05) was also observed between the five parboiled aromatic rice accessions and the check varieties. thus, the aromatic rice accessions ara-2 (4.43%), ara-5 (4.23%), ara-18 (4.53%), and ara-23 (4.17%) showed high water uptake compared to check 2 (4.27%) (table 3). while ara-1 (3.43%) showed a similar water uptake rate as check 1 (3.25%) (table 1). the high water uptake of rice samples ara-2, ara-5, ara-18, ara23 and control 2 could be explained by the fact that they are characterized by harder grains than the other rice samples [16,17]. the harder the rice grain, the more water it will tend to absorb during cooking [16]. compared to the cooking time of white rice, there was a significant variation (p<0.05) between the cooking time of the different aromatic rice accessions. the accessions ara-2 (17.17 min), ara-5 (18.83 min) and ara-23 (17.67 min) showed a high cooking time compared to check 1 (15.33 min) and check 2 (14.17min). while the rice accessions ara-1 (14.17min) and ara-18 (12 min) showed a lower cooking time than check 1 (15.33 min) (table 2). for the parboiled type, the rice accessions had an average cooking time of 15.76 min (table 1). no significant difference (p>0.05) was observed in the cooking time of accessions ara-2 (20 min), ara-5 (20.7 min) and ara-23 (23.17 min) on the one hand, and accessions ara-1 (15.83 min), ara-18 (15.83 min), check 1 (18.83 min) and check 2 (17.17 min) on the other hand. the small difference in cooking time between the five aromatic rice accessions and the check varieties could slightly increase the production costs for the processor. sensory characteristics of promising aromatic rice accessions the sensory test showed that the accession ara-23 is more appreciated the panelist (figure 1) compared to the two check varieties. panelists reported that this accession has a very good appearance, medium grain rice (length and width highly appreciated) and very clean. check 1 (ir 841), the most popular aromatic rice variety produced in benin, takes second place because of similar traits with ara23. check 2 is the least appreciated variety because it was not well filled, that’s related to a water deficit during production. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 45 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 46 fig. 1. panelist preferences for paddy rice from promising aromatic rice accessions and two check (ir 841 and briz 10b) figure 2 shows the results of appreciation of the uncooked form of white rice just after hulling. it can be seen that the aromatic rice accessions ara-5 and ara-2 are more preferred (70% of panelist) because these accessions have more perceptible aroma than the other accessions. samples ara-1, check 1 and check 2 are the least preferred. fig. 2. tasters' preferences for uncooked parboiled rice from new fragrant rice accessions and two check (ir 841 and briz 10b) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 47 compared to the uncooked parboiled form (figure 3), panelist (75%) preferred the accessions ara-5 and ara-23 compared to the other accessions, mainly because of the absence of black spots and the clarity of the rice. panelist reported that these two accessions more closely look like ‘’arosso rice’’, which is the commercial name of a most appreciate parboiled rice imported and sold in benin. fig. 3. panelist’s preferences for uncooked parboiled aromatic rice accessions and two check (ir 841 and briz 10b) for cooked white rice, aromatic rice accessions ara-2, ara-18 and ara-5 are most preferred by panelist (65%) because of their low stickiness, strong aroma expression, good taste and easy chewiness, unlike the other accessions which are less appreciated (figure 4). fig. 4. panelist's preferences for cooked white rice of aromatic rice accessions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 48 compared to the parboiled type, accessions ara-5 and ara-2 are the most appreciated by panelist (70%) because they are less sticky and have a better taste (figure 5). fig. 5. panelist's preferences for parboiled rice of aromatic rice accessions and two check (ir 841 and briz 10b) 4. conclusion the promising rice accessions (ara-1, ara-2, ara-5, ara-18 and ara-23) show very good physico-chemical and nutritional characteristics compared to the check used (ir841 and briz-10b). all these white and parboiled type of aromatic rice accessions, as well as the check varieties, have a high degree of uniformity (>80%), low alkaline digestibility and no chalkiness. the ara-2 and ara-5 accessions were found to be more preferred than the checks, mainly because of the strong aroma expression, attractive color, non-sticky appearance and very pleasant taste. 5. acknowledgments we would like to thank national institute of agricultural research of benin (inrab), for his support and guide. 6. references [1]. konnon d.d., sotondji cs, adidehou ya. rapport de l’etude d’etat des lieux de la filière riz au bénin en 2014; rapport final, crp-b: composition 97p (2014) [2]. houssou a. f. p., tchatcha d., mensah, g.a, kaboré, a., futakuchi, k., traoré, k., moreira, j., and diagne a. suitability for parboiling of rice varieties from benin through assessing the soaking temperature and rice quality. european scientific journal, edition vol.12, no.21 issn: 1857 7881 (print) e issn 18577431 doi: 10.19044/esj. 2016.v12n21p144-158. (2016b) [3]. houssou a. f. p., gankoué, y. b., kaboré, a., futakuchi, k., traoré, k., moreira, j., diagne a., manful, j. and mensah, g.a . comparison of parboiled and white rice obtained from ten varieties cultivated in benin. international food research journal issn 19854668 (print), issn 22317546 (online) vol.23 (6) p 2479-2486 (2016a) [4]. juliano, b.o. rice in human nutrition. (fao food and nutrition series. no. 26) isbn 925-103149-5 p 162. (1993) [5]. islam m.m., kayesh e. zaman e. haque m.m. evaluation of rice (oryza sativa l.) genotypes for drought tolerance at germination and early seedling stage. the agriculturists 16(1) :44-54. (2018) [6]. zohoun e. v., ndindeng k.s. soumanou m.m., tang e.n., bigoga j., manful j., sanyang s., akissoe n. h, futakuchi k. appropriate parboiling steaming time at atmospheric pressure and variety to produce rice with weak digestive properties. food sci nutr. 1-8. doi: 10.1002/fsn3.617. (2018) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 valère dansou, paul ayihadji ferdinand houssou, yannick sourou rosanoff kouke, abel bodéhoussè hotegni, warou arnold cospe sagui, kowiou aboudou, hugue zannou, physicochemical and sensory characterization of five promising aromatic rice accessions in benin, food and environment safety, volume xxii, issue 1 – 2023, pag. 40 – 49 49 [7]. diako c, sakyi-dawson e, bediako-amoa b, saalia f.k. and manful j.t. cooking characteristics and variations in nutrient content of some new scented rice varieties in ghana, annals. food science and technology, vol 12 (1), 39-44. (2011) [8]. zohoun e. v., erasmus n. t., mohamed m. s., manful j., akissoe n. h., bigoga j., koichi futakuchi, ndindeng k.s. a., physicochemical and nutritional properties of rice as affected by parboiling steaming time at atmospheric pressure and variety. food sci nutr. 2018;6: 638-652. (2017) [9]. umemoto t, terashima k, nakamura y, satoh h. lerences in amylopectin structure between two rice varieties in relation to the e وٴects of temperature during graln͟filllng starch-stärke 51: 58-62. (1999) [10]. derycke, v., vandeputte, g. e., vermeylen, r., de man, w., goderis, b., koch, m. h. j., & delcour, j. a. starch gelatinization and amylose-lipid interactions during rice parboiling investigated by temperature resolved wide angle x-ray scattering and differential scanning calorimetry. journal of cereal science, 42, 334-343. https://doi.org/10.1016/j.jcs.2005.05.002. (2005) [11]. fan c. c. yu xq, xing y. z., xu c. g., luo l. j. нe main ejects epistatic ejects and environmental interactions of qtls on the cooking and eating quality of rice in a doubled-haploid line population. нeoretlcal and applied genetics 110: 1445-1452. (2005) [12]. waters dl, henry rj, reinke rf, fitzgerald ma. gelatinization temperature of rice explained by polymorphisms in starch synthase. plant biotechnology journal 4: 115-122. (2006) [13]. fofana m., wanvoeke, j., manful, j., futakuchi, k., van mele, p., zossou, e., bléoussi, t.m.r. effect of improved parboiling methods on the physical and cooked grain characteristics of rice varieties in benin. international food research journal. 18, pp 697703. (2005) [14]. petrov m. and faure j. the scent of rice: how to recognize and characterize aromas? agriculture et développement n° 9 – march. (1996). [15]. pandey v. and shukla a. acclimation and tolerance strategies of rice under drought stress. rice sc. 22(4):147-161. http://dx.doi.org/10.1016/j.rsci.2015.04.001. (2015) [16]. migan s., evaluation of the quality of some local varieties of parboiled rice: physical, nutritional and sensory aspects. end of training thesis for the diploma of design engineer of food technology, epac-uac.68p (2011) [17]. griffiths g. atungulu, r.e. kolb, j. karcher z. mohammadi shad, postharvest technology: rice (2019). https://doi.org/10.1016/j.jcs.2005.05.002 http://dx.doi.org/10.1016/j.rsci.2015.04.001 1. introduction sensory characteristics of promising aromatic rice accessions the sensory test showed that the accession ara-23 is more appreciated the panelist (figure 1) compared to the two check varieties. panelists reported that this accession has a very good appearance, medium grain rice (length and width highly appreciat... for cooked white rice, aromatic rice accessions ara-2, ara-18 and ara-5 are most preferred by panelist (65%) because of their low stickiness, strong aroma expression, good taste and easy chewiness, unlike the other accessions which are less appreciate... 4. conclusion doi: https://doi.org/10.4316/fens.2022.006 47 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 47 61 effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds *pius ifeanyi okolie 1 , emilymary chima okolie 2 , sarah abiola ogundimu 1 , kudirat aina agboola 1 and maureen chinwe ojinnaka 3 1 federal university of agriculture (funaab), abeokuta, ogun state, nigeria, okoliepi@funaab.edu.ng, 2national biotechnology development agency of nigeria, lugbe, fct-abuja, 3michael okpara university of agriculture (mouau), umudike, abia state, nigeria, *corresponding author received 9th december 2021, accepted 20th march 2022 abstract: african yam bean seeds (sphenostylis sternocarpa) iru-like condiment was produced with 0.005 and 0.0075 g broth/g seed different concentrations of bacillus amyloliquefaciens culture. the sample fermentation was conducted at 35 °c for 5 days. proximate composition, water absorption capacity, oil absorption capacity, ph, and titratable acidity of the fermented samples were assessed using standard methods. proximate composition of fermented african yam bean (ayb) condiment with 0.005 g broth/g seed inoculum concentration as determined were crude protein (6.86%-7.74%), crude fat (1.37%-1.71%), crude fiber (2.73%-4.81%), ash content (1.02%-1.59%), moisture content (9.71%-14.92%), dry matter (85.10%-90.28%), carbohydrate (70.43%-77.16%). the ph, total titratable acidity, water absorption capacity (wac), and oil absorption capacity (oac) of the fermented condiment were 6.76-7.60 ml, 0.07-0.09 ml, 0.50%-0.92%, and 0.55%-0.65% respectively. the fermented condiment produced with 0.0075 g broth/g seed inoculum concentration had its proximate composition as determined as crude protein (8.10%-8.53%), crude fat (1.81%-2.12%), crude fiber (2.83%-3.73%), ash content (1.23%-1.42%), moisture content (8.97%-12.81%), dry matter (87.19%-90.91%), carbohydrate (72.22%-76.13%) while its ph, total titratable acidity, wac and oac ranged as 7.10-8.76 ml, 0.06-0.11 ml, 0.38%-0.81% and 0.54%-0.84% respectively. the sensory acceptability scores reveal condiment samples from 0 hours as the best-preferred sample produced from the use of the inoculum concentrations. the study showed that ayb seeds condiments produced were significantly different in terms of different concentrations of starter culture used. although the single starter culture did not deliver acceptable products during the fermentation, it played a few parts within the product quality. keywords: inoculum concentration, alkaline fermentation, nutritional enhancement, starter culture 1. introduction numerous underutilized leguminous seeds abound in the tropical regions of african countries and adequate characterization of these seeds has not been fully evaluated. exploration for possible food product development from these underutilized food sources has been the focus of research interest aimed at alleviating food security problems in the african continent. in nigeria, there has been an increasing shift in the utilization of these underutilized leguminous seeds to produce food condiments, a high protein-rich product. protein from fermented leguminous plant seeds is an imperative wholesome nutrient source to people of most african nations [1]. traditional fermented vegetable about:blank food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 48 proteins are routinely used as seasonings and condiments in african and asian diets. these matured food varieties are attractive considering the benefits of further developed flavor, nutritive worth, and edibility over unfermented products [2, 3]. nutrient bioavailability due to fermentation entails an increase in the essential amino acids and vitamins and a reduction in the availability of antinutrients. some researchers have reported that fermented foods are associated with greater digestibility, increased flavors, and aromas as well as can impart some health-promoting significances [4, 5, 6]. condiments also add to individual calorie and protein consumption and are frequently utilized as a modest meat substitute by low-pay families in the developing world [7]. african yam bean seed (sphenostylis sternocarpa) is an underutilized plant species in most west african countries. african yam bean, also known as wild yam bean is an annual crop that belongs to the leguminous family and sub-family of papilonacea sp. it is a promising raw material for condiment production. its nutritional composition is like that of most edible legumes such as soya bean, locust bean seed, and bambara nut seeds. it has been reported to contain about 21.10% protein, 5.70% crude fiber, 74.10% carbohydrate, 3.20% ash, 8.5% moisture, and 8.25% fat [8]. several reviews have been done on the biochemistry and nutritional aspect of unfermented african yam bean seeds. research studies on ayb seed have focused mainly on the utilization of the unfermented seeds as part of composite flour in confectioneries [9, 10, 11, 12, 13]. ayb seeds have limited utilization as food and food ingredients due to the presence of anti-nutrients such as tannin, phytic acid, saponin, and as well as its hard-to-cook phenomena. despite the high nutritive value of the african yam bean seed, there is still a dearth of information on the utilization of fermented seeds for condiment production. the objective of this study is to evaluate the effect of the different concentrations of pure culture of bacillus amyloliquefaciens on the physiochemical properties of “iru”type condiments produced from african yam bean seeds under controlled fermentation. 2. materials and methods source of raw materials african yam bean seeds (sphenostylis stenocarpa) used for this experiment were collected from the isi-gate market in umuahia, abia state. the seeds were characterized as varieties tss-5 and tss45 at iita, ibadan. the seeds were collected in polythene bags, aerated for 24 h, and stored in the refrigerator (6± 2 °c) until use. bacterial culture characterization and purification starter culture (bacillus amyloliquefacien) was obtained from natural fermented ayb seeds. the organism was characterized genotypically using combined molecular techniques of pcr amplification of 16s23s rdna intergenic transcribed spacer (its-pcr) gene region together with repetitive sequence-based pcr (rep-pcr) and 16s rrna gene sequencing to generate a unique molecular sequence for the isolate. the sequence was subjected to a blasting program on the ncbi site for isolate identity. the isolate was maintained on a tryptone soy agar (tsa; oxoid cm131, basingstoke, uk) slant in the refrigerator until use. preparation of starter inoculum culture. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 49 starter culture preparation was conducted according to the modified method of [14]. glycerol stored pure culture of bacillus amyloliquefacien strain was plated on tryptone soy agar (tsa; oxoid cm131, basingstoke, uk) plate and incubated overnight at 30 ± 2 °c. the bacillus strain was subcultured in 50 ml of tryptone soy broth (tsb; oxoid cm129, basingstoke, uk) in 100 ml conical flask and incubated at 30 ± 2 °c for 24 hours with an incubator shaker. the turbid cultures were centrifuged with a refrigerated centrifuge (centurion, scientific ltd, uk) for 10mins at the speed of 10,000rpm. cell pellets were harvested and re-suspended in 5 ml of 0.9% sterile normal saline (containing 9 g l-1 nacl; ph 7.0). the number of viable cells in the resuspension was determined with a spectrophotometer (t60uv, pg instruments ltd, uk) at a wavelength of 540 nm and diluted it to 0.5 mcfarland standard. the inoculum volume needed to inoculate 200 g of sterile ayb seeds to achieve the required concentration of 0.05 and 0.075 g broth/ g seed (containing 1.5x 108 cells per ml), was calculated. preparation of controlled fermented ayb seeds condiment the modified method of [8] was used in the laboratory fermentation of ayb seeds under controlled conditions. sorted african yam bean seeds were thoroughly washed and soaked in tap water for 6 h. the cotyledons were obtained by removing the seed coat by pressing the seeds between fingers. approximately 100 g of the dehulled seeds were weighed and added into six 250 ml conical flasks. sterilization of the seeds in the conical flask was done with an autoclave for 15 minutes at 121 °c. the sterile seeds were allowed to cool to room temperature and thereafter, inoculated with the bacterial culture. the inoculation was done carried out with different concentrations (0.005 g broth/g seed and 0.0075 g broth/g seed) of bacillus amyloliquefacien containing 1.5 x 108 cfu/ml. the samples were fermented thereafter in a fermenter (incubator) at 30 ± 2 °c for 120 hours. analysis of the microbial growth during fermentation total viable bacteria during fermentation stages were enumerated by pour plate method with tryptone soya agar (tsa, oxoid cm0131, basingstoke, hampshire, uk). samples (1g) of fermenting condiments from different inoculum concentrations were homogenized using 9ml of 0.9% of normal saline diluent in a stomacher bag. replicate portions of tenfold serial dilutions of samples were made for all samples collected for the two inoculum concentrations at different fermentation intervals (0h, 24h, 48h, 72h, 96h, and 120h). after serial dilution, 0.2 ml each of the serially diluted sample solution was pipetted into appropriately labeled petri dish and autoclaved tsa media was poured aseptically into the different labeled petri dishes. the plates were swirled gently in a planar circular motion to ensure uniform distribution and the media were allowed to solidify and incubated at 34°c for 24 hours. after incubation, colony growth was observed, and colony count was conducted. microbial count (cfu/g) = colony count × volume of inoculum × dilution factor × correction factor. analysis of the physicochemical properties of fermented condiment fermenting samples were drawn at every 24 h intervals for enumeration of microbial load and determination of ph, titratable acidity, water absorption capacity, temperature, and oil absorption capacity. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 50 some portions of the sample were ovendried at 60 °c for 2-3h, ground to powder using kenwood blender, and stored in sealable nylon bags at 4° c for proximate composition analysis. proximate composition analysis of the proximate composition of the condiment samples for moisture, ash, total fat, crude fiber, and crude protein was carried out using the standard methods of the association of official analytical chemists [15]. carbohydrate content was calculated by difference. titratable acidity the total titratable acidity was assessed as described by [16]. two (2) g of fermenting sample mash was homogenized with 20 ml with sterile distilled water. the sample mixture was filtered, and 10 ml of the filtrate containing 2 drops of 1% phenolphthalein solution as an indicator was titrated against 0.1 mmol l-1 naoh solution at ph 8.1, and the mean titer value was taken. the titratable acidity of the sample was calculated as the percentage of lactic acid. one milliliter of 0.1 mmol l-1 naoh solution was taken as equivalent to 0.009 g lactic acid. ph ten (10) g of fermenting otiru samples were taken aseptically and homogenized in 40 ml deionized water. buffer calibrating solutions of ph 4 and ph 7 were used for initial calibration of the meter before sample readings were taken. (r1 – 02895 hanna, italy) [17]. temperature the temperature of the fermenting otiru at the various period of evaluation was determined by inserting a sterile thermometer (sanitized by swabbing with 95 % ethanol) into each of the samples. the mercury-in-glass thermometer was used. triplicate determinations were made in all cases [17]. water/oil absorption capacity water absorption capacity and fat absorption capacity of the fermenting samples were measured as recorded by [18]. five grams of fermenting sample was ground to make slurry in 15 ml distilled water in determining the water absorption capacity (oil in the case of oil absorption capacity); the slurry was subjected to 20 min centrifugation at the speed of 4,000rpm. the free water (oil) after centrifugation was decanted, and the amount absorbed by the sample was calculated by difference. sensory evaluation sensory evaluation was conducted on the laboratory-produced condiment from african yam beans seed with different inoculum concentrations and a fresh market sample of “iru” (which served as a control sample). the sensory evaluation was done by 50 untrained panelists made up of male and female students selected from the federal university of agriculture, abeokuta, ogun state. the panelists were to access the appearance, color, texture, aroma, and overall acceptability of the fermented condiment using a 9-points hedonic scale ranging from like very much (9) to dislike very much (1) as described by [19]. statistical analysis all analyses were performed in triplicate. data were analyzed using spss (version 25) analysis of variance (anova) and means separation was done with duncan’s multiple range test at p ≤0.05. 3. results and discussion food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 51 bacteria isolation and identification presumptive b. amyloliquefacien was isolated during the natural fermentation of african yam bean seed condiment. the organism was examined to be grampositive rods, oxidase-negative, and catalase-positive. the isolate was also able to degrade citrate as their only carbon source. the isolate was able to hydrolyze starch and degrade casein and had a facultative mode of growth. molecular characterization using combined techniques of its-pcr, rep-pcr, 16s rrna gene sequencing, and sequence blasting, identified the isolate as b. amyloliquefacien dsm7 (t) with a 98.84 % sequence match and accession number of fn597644. table 1 shows the result of the preliminary biochemical examination of the isolate. table 1: biochemical, morphological and molecular characterization of bacillus amyloliquefacien characteristics/ biochemical test observation gram stain positive oxidase test negative catalase test positive starch hydrolysis positive casein degradation positive sugar fermentation: glucose positive with gas lactose positive sucrose positive growth between 20 and 50 °c positive colony morphology slimy, big, circular, entire, pulvinate, dull, rough, opaque cell morphology rods presumptive identity bacillus amyloliquefacien molecular identification (its-pcr/rep-pcr/16s rrna gene pcr): b. amyloliquefacien dsm7 (t) (fn597644) (98.84% matched) microbial analysis of controlled fermented ayb condiment from the first and second concentration of bacillus amyloliquefaceins table 2 shows the result of the microbial load in the fermenting samples from the two inoculum concentrations. the result indicates that there was maximum growth of bacillus amyloliquefaceins in the controlled fermented samples. sample batch fermented with 0.005 g broth/g seed concentration of the inoculum recorded 2.1×109 cfu/g at 96 hours fermentation and 2.42 ×107 cfu/g at 72 hours. the microbial load increased as fermentation progressed from 24 hours to 48 hours but decreased at the 72 hour period. the highest cfu/g in the 0.0075 g broth/g seed concentration fermented sample batch was observed at 48 hours with 5.4×1010 cfu/g and the least microbial load was recorded at 24 hours with 3.5× 108 cfu/g. the microbial load decreased slightly to 6.5 × 109 cfu/g as the fermentation progressed to 72 hours. it was observed that microbial growth increases with fermentation time. the rate of microbial development was observed to be comparatively higher in the ayb condiment samples fermented with 0.0075 g broth/g seed concentration of the inoculum starter. this higher fermentation rate could be attributed to the higher concentration of the starter. table 2 shows that approximately the same volume of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 52 microbial load obtained in 48 hours of fermentation of the ayb condiment batch fermented with 0.005 g broth/g seed concentration of the inoculum starter was recorded in sample batch fermented with 0.0075 g broth/g seed concentration of the inoculum starter at 24 hour of fermentation. table 2: results for microbial load analysis fermentation time 0.005 g broth/g seed concentration 0.0075 g broth/g seed concentration 0 hour nd 1.5 × 108 24 hours 3 × 107 3.5 × 108 48 hours 3.6 × 108 5.4 × 1010 72 hours 2.42 × 107 6.5 × 109 96 hours 2.1 × 109 nd 120 hours 3.7 × 108 1.32 × 1010 values are mean of triplicate determination keys: cfu/g = colony forming unit per gram. nd =not determined effects of fermentation on proximate composition of african yam bean seed condiment. table 3 shows the effect of fermentation with 0.005 g broth/g seed of inoculum concentration on the proximate composition of fermented ayb condiment. condiment samples from 72 hours and 120 hours intervals had no significant difference in terms of their moisture content and dry matter. however, the mean values for moisture content and dry matter from other fermentation times were significantly different (p ≥0.05). the percentage moisture content value of the sample was highest at 24 hours (14.92 %) but this decreased as fermentation progressed until 9.71% at 96 hours fermentation. the value for the moisture content increased finally to 10.95 % at 120 hours. the dry matter content increased significantly from 24 hours to 96 hours fermentation. the highest was 85.1% at 24 hours and 90.28% at 96 hours. there was a reduction in fat content when compared to the raw sample. the values were significantly different (p ≥0.05) except for the samples 48 hours and 96 hours fermentation intervals. the raw sample had 3.85% while the fat content between the unfermented and fermented ayb seeds ranged between 1.37% 1.71%. this decrease in percentage fat content could be attributed to the utilization of fat by the microorganism [20]. there was a significant difference in crude ash content between raw seed and unfermented ayb seed as well as fermented ayb seeds. there was an increment when compared to the raw sample. the raw seed was 1.19% and the highest value obtained from the unfermented seed (ufs) was 1.59%. it was noticed that the value for the fermenting samples decreased as fermentation time increases. this is similar to the report of [21] during the production of a condiment from arachis hypogaea linn. this observation might be as a result of the de-hulling process of the beans since a higher percentage of the fiber and ash was concentrated on the seed hull which was removed during processing. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 53 crude fiber content was highest at 24 hours with 4.81% and lowest at 120 hours with 2.73%. the values were significantly different (p ≥0.05). it was noticed that fiber content reduced as fermentation time increased, and this agrees with the result reported by [22] that fermented foods from legumes contain low fiber. the protein content decreased when compared to raw samples. the highest protein value of the fermented sample was 7.74% at 96 hours and the lowest was 6.86% at 0 hours. the values were significantly different (p ≥0.05). the observed reduction in value may be as a result of the breakdown of protein molecule structure in the seed leading to the release of ammonia which forms an excellent source of nitrogen for the growth of the fermenting organism [23, 20]. the carbohydrate content was highest at 120 hours with 77.16% and ufs with 70.43%. the values were significantly different (p ≥0.05). there was an increase in the percentage carbohydrate content when compared to that of the raw sample. this difference could be attributed to the inability of the fermenting organism to degrade carbohydrates [20]. the result of the proximate composition of fermented condiment samples inoculated with 0.0075 g broth/g seed inoculum concentration shows that moisture content was highest at 96 hours with 12.81% and lowest at 8.97% at 24 hours (table 4). the values were significantly different (p ≤ 0.05). there was an increase in moisture content as fermentation time increases for 3 consecutive days (24 hours – 96 hours). this could be caused by the hydrolytic decomposition of the fermenting beans which had been earlier reported by [24]. there was a significant decrease in dry matter content from 24 hours to 96 hours of fermentation. the mean values for dry matter from the different fermentation times were significantly different (p ≤ 0.05). the highest value was 91.00% at 24 hours and the lowest was 87.19% at 96 hours. there were significant differences in the fat content of samples fermented with 0.0075 g broth/g seed inoculum concentration across the different fermentation times. there was a reduction in fat content when compared to the value from the raw ayb seed sample. the raw sample had 3.85% while the highest of the fermented seed had 2.12%. this decrease in the percentage of fat content could be caused by the increased lipolytic enzyme activities by the microorganism. fermenting organisms with high lipolytic enzymes potentials are good in degrading fat components into glycerol and fatty acid [23]. crude ash content increased when compared to the raw ayb seeds. the raw seed was 1.19% and the highest value of the fermented seed was 1.42 at 24 hours. the values were significantly different (p ≥0.05). it was noticed that the values decreased as fermentation time increases. this trend is similar to the report of [21] during the production of a condiment from locust beans. this observation might be a result of the de-hauling process of the beans since a higher percentage of the fiber and ash were concentrated on the seed hull which was removed during processing. crude fiber content was highest at 120 hours with 3.73%. the values were significantly different among samples from different fermentation times. it was noticed that fiber content increased as fermentation time increased from 48 hours – 120 hours. the protein content decreased when compared to raw samples. there was no significant difference between fermented samples from 48 hours and 72 hours in their protein content. however, the highest protein content value of 8.68% (24 hours) was observed among the fermented samples while the unfermented food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 54 (ufs) recorded 8.1%. the observed reduction in value between the raw ayb seeds and the fermented samples could be due to the release of nitrogen from the free ammonia produced during the hydrolysis of protein components in the seed [23, 20]. the carbohydrate content was highest at 48 hours with 76.13% and lowest at 96 hours with 72.22%. the samples were significantly different. ezekiel et al., [20] had a similar result while working on fermented cotton seeds for owoh condiment production. observed changes in percentage carbohydrate content as the fermentation time changes aligned with the results of [25] on fermented cowpea and millet. table 3: results for proximate analysis for 0.005 g broth/g seed concentration of the starter culture fermentation time moisture content (%) dry matter content (%) fat content (%) ash content (%) crude fiber (%) crude protein (%) carbohydrate (%) ufs 12.16d ± 0.01 87.88c ± 0.03 1.59c±0.04 1.59bc±0.03 3.84e±0.02 6.86a ± 0.02 74.52c ± 0.09 24 hours 14.92f ± 0.08 85.10a ± 0.07 1.45b+0.01 1.23d ± 0.01 4.81f ±0.04 7.18c ± 0.05 70.43b ± 0.04 48 hours 10.92c ± 0.02 89.09d ± 0.03 1.69d±0.01 1.20d ± 0.02 3.46d±0.01 6.97b ± 0.02 75.83d ± 0.15 72 hours 10.57b ± 0.32 89.42e ± 0.32 1.37a±0.01 1.02a ± 0.04 2.90b±0.01 7.47d ± 0.01 76.66e ± 0.03 96 hours 9.71a ± 0.23 90.28f ± 0.02 1.71d±0.01 1.15c ± 0.01 2.89b±0.07 7.74e ± 0.02 76.75e ± 0.04 120 hours 10.59b ± 0.09 89.47e ± 0.09 1.61c±0.02 1.09b ± 0.02 2.73a±0.01 6.87a ± 0.02 77.16f ± 0.01 raw seed 12.48e ± 0.06 87.54b ± 0.05 3.85c±0.00 1.19d ± 0.01 3.25c±0.02 14.59f ± 0.02 64.63a ± 0.01 values are the mean of triplicate determination. mean values with different superscripts within the same column are significantly different (p ≤ 0.05). ufs: unfermented african yam bean seed sample table 4: result for proximate analysis for 0.0075 g broth/g seed concentration of the starter culture. values are the mean of triplicate determination. mean values with different superscripts within the same column are significantly different (p ≤ 0.05). ufs: unfermented african yam bean seed sample changes in the chemical composition of african yam bean seed condiment table 5 shows changes in the physiochemical composition of african yam bean seed fermented with 0.005 g broth/ g seed concentration of the starter culture. the fermenting time had a significant difference in the ph of the samples. the ph value ranged from 6.83% to 7.60% in the samples fermented with fermentation time moisture content (%) dry matter content (%) fat content (%) ash content (%) crude fiber (%) crude protein (%) carbohydrate (%) ufs 11.17e ± 0.07 88.86c ± 0.06 1.81a ± 0.01 1.24b ± 0.01 2.83a ± 0.03 8.10a ± 0.02 74.86d ± 0.01 24 hours 8.97a ± 0.06 91.00f ± 0.05 2.12e ± 0.05 1.42e ± 0.02 3.05c ± 0.03 8.68e ± 0.04 75.80e ± 0.21 48 hours 9.09b ± 0.03 90.91f ± 0.03 2.00d ± 0.02 1.37d ± 0.01 2.97b ± 0.02 8.45c ± 0.02 76.13f ± 0.04 72 hours 9.47c ± 0.08 90.57e ± 0.08 1.92c ± 0.01 1.35c ± 0.00 3.25d ± 0.04 8.42c ± 0.03 75.68e ± 0.07 96 hours 12.81g ± 0.02 87.19a ± 0.02 1.86b ± 0.02 1.23b ± 0.01 3.57e ± 0.01 8.29b ± 0.02 72.22b ± 0.01 120 hours 10.34d ± 0.06 89.68d ± 0.06 2.10e ± 0.01 1.34c ± 0.01 3.73f ± 0.01 8.53d ± 0.02 73.96c ± 0.01 raw seed 12.48f ± 0.06 87.54b ± 0.05 3.85f ± 0.00 1.19a ± 0.01 3.25d ± 0.02 14.59f ± 0.02 64.63a ± 0.11 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 55 0.005 g broth/g seed concentration of the starter. a rise in ph tending towards alkalinity was noticed in the fermented african yam bean seed as the fermentation time increases. this increase in ph can be explained as due to the proteolytic activity of b. amyloliquefaciens leading to the production of amines and free ammonia as a result of degradation of protein of the raw material. the produced ammonia gives the condiment its strong pungent and ammoniacal smell. the observed rise in ph agrees with the findings of some other researchers during fermentation of soybeans, kariya seed, tamarind seed, and some vegetable proteins [26, 24, 20, 1, 16]. table 5: changes in the physicochemical properties of fermented condiments with concentrations of b. amyloliquefaciens. fermentation time ph wac (ml) oac (ml) bafs1 bafs2 bafs1 bafs2 bafs1 bafs2 ufs 6.83a ± 0.05 7.10a ± 0.10 0.80d ± 0.00 0.56b± 0.04 0.55a ± 0.01 0.80c± 0.06 24 hours 6.76a ± 0.05 7.26b ± 0.05 0.50a ± 0.05 0.81c ± 0.09 0.57a± 0.00 0.84c± 0.04 48 hours 7.53c ± 0.05 7.46c ± 0.05 0.58ab ± 0.06 0.57b ± 0.02 0.65a ± 0.14 0.63ab± 0.08 72 hours 7.36b ± 0.05 8.76d ± 0.05 0.92e±0.08 0.54b ± 0.04 0.57a ± 0.03 0.70b ± 0.04 96 hours 7.23b ± 0.05 8.23d ± 0.05 0.62bc ± 0.02 0.57b ± 0.09 0.57a± 0.03 0.58ab ± 0.11 120 hours 7.60c ± 0.00 8.33e ± 0.05 0.70c ± 0.04 0.38a ± 0.09 0.65a ± 0.10 0.54a± 0.00 values are the mean of triplicate determination. mean values with different superscripts within the same column are significantly different (p ≤ 0.05). bafs1: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.005 g broth/g seed; bafs2: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.0075 g broth/g seed; ufs: unfermented african yam bean seed sample. wac = water absorption capacity. oac = oil absorption capacity. figure 1 reveals the result of the percentage changes in the titratable acidity of the ayb condiment. a gradual decline in the titratable acidity was noticed in the samples fermented with 0.005 g broth/g seed concentration of the starter. this decline in value was from 0.09% to 0.07% as the fermentation time increases. this drop-in titratable acidity could be attributed to the effect of a rising in ph value observed at the end of fermentation [20, 27]. samples from 24h, 72h, 96h, and 120h fermentation times had no significant difference at p≥0.05 among them. these samples were significantly different from samples from 0h and 48h which were significantly the same. the titratable acidity results of samples from inoculation with 0.075 g broth/g seed concentration of the starter were significantly different concerning fermentation time. the titratable acidity decreases as the fermentation time increases and ranges from 0.11% to 0.06%. the titratable acidity from this concentration had an inverse relationship with the ph. the ph of the samples increases towards alkalinity with increasing fermentation time. the ph ranged from 7.10% to 8.33%. the temperature of the fermenting samples was not significantly different for samples inoculated with 0.005 g broth/g seed concentration of the starter. the starter culture concentrations had no significant effect on the temperature of the samples. the mean temperature value ranged from 31.33 °c to 37.8 °c and 30 °c to 39 °c for batches of samples with 0.005 and 0.0075g broth/g seed concentrations respectively. the temperatures increase as the fermentation time increases irrespective of the inoculum concentration. there was a significant difference in the water absorption capacity of the ayb seeds fermented with 0.005g broth/g seed concentration. the wac was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 56 lowest at 24 hours (0.50 ml) and highest at 72 hours (0.92 ml) this indicates that wac increases with fermentation time. however, for the samples fermented with 0.0075g broth/g seed concentration, there was no significant difference between the unfermented seeds and the fermented samples from 48 hours, 72 hours, and 96 hours fermentation times concerning their water absorption capacity. the wac was lowest at 120 hours (0.38 ml) and highest at 24 hours (0.81ml). there was no significant difference in the oil absorption capacity among all the ayb seeds samples fermented with for 0.005g broth/g seed concentration the oac for this concentration had the lowest at 24 hours (0.57 ml) and highest at 48 hours and 120 hours (0.65 ml) while for 0.0075g broth/g seed concentration, the oac was lowest at 120 hours (0.54 ml) and highest at 24 hours (0.84 ml). this shows that oac increases with fermentation time for the first concentration and vice versa for the second concentration. figure 1: change in percentage of lactic acid of ayb condiment samples fermented with 0.005 and 0.0075 g broth/ g seed concentrations of b. amyloliquefaciens effects of fermentation on mineral composition of controlled fermented african yam bean condiment the result of the mineral composition of the fermented condiment produced with different inoculum concentrations of the starter culture was presented in table 6. the sodium content of the controlled fermented condiment was between 2.16 2.87 mg/100g and 3.16 4.04 mg/100g for inoculum concentrations of 0.005 and 0.0075 g broth/g seed respectively. there was a reduction in the sodium content value of the fermented condiment when food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 57 compared to the raw ayb seed sample with 4.87 mg/100g. the mean values for the sodium content of the condiment across the fermentation time were significantly different (p ≤ 0.05). the mean values for the magnesium content from the different inoculum concentrations were significantly different (p ≤ 0.05). the magnesium content ranged from 152.98 160.79 mg/100g for samples fermented with inoculum concentrations of 0.005 g broth/g seed and 181.45 216.77 mg/100g for samples fermented with inoculum concentrations of 0.0075 g broth/g seed. there was an increase in value when compared to the raw sample (158.79 mg/100g). the phosphorus content of the fermented condiment was significantly different (p ≤ 0.05) for both inoculum concentrations. the values for the phosphorus content were between 216.79 – 281.45 (mg/100g) and 291.50 – 324.50 (mg/100g) for the samples fermented with 0.005 g broth/g seed and 0.0075 g broth/g seed concentration of bacillus amyloliquefaciens starter respectively. there was an increase in phosphorus content when compared to the raw sample with 277.43 (mg/100g). the increment in the phosphorus content from this study agrees with the research findings of [28, 29] on the production of dawadawa condiments from bambara groundnut and assessment of volatile flavor compounds from “ugba” fermentation respectively. the mineral contents determined were significantly affected with regards to the fermentation time and inoculum concentration. table 6: results for the mineral composition of controlled fermented ayb condiment fermentation time sodium (mg/100g) magnesium (mg/100g) phosphorus (mg/100g) bafs1 bafs2 bafs1 bafs2 bafs1 bafs2 ufs 2.87f ± 0.02 3.67b ± 0.28 157.07c ± 1.48 188.72d ± 0.03 281.45g±.00 296.54d ± 0.03 24 hours 2.16a ± 0.00 4.04f ± 0.04 152.98a ± 0.21 206.11e ± 0.00 242.11b±.00 301.65e ± 0.02 48 hours 2.67d ± 0.00 3.78c ± 0.00 161.09e ± 0.01 186.45c ± 0.00 273.12e±.00 324.50g ± 0.00 72 hours 2.48b ± 0.00 3.16a ± 0.00 156.82c ± 0.03 216.77g ± 0.01 216.79a±.01 287.67b ± 0.01 96 hours 2.61c ± 0.00 3.83d ± 0.01 154.15b ± 0.00 181.45b ± 0.00 261.31d±.01 291.50c ± 0.01 120 hours 2.81e ± 0.00 3.89e ± 0.00 160.79e ± 0.02 212.67f ± 0.02 258.30c±.00 312.53f ± 0.02 raw seed 4.87g ± 0.01 4.87g ± 0.01 158.79d ± 0.00 158.79a ± 0.00 277.43f±.01 277.43a ± 0.01 values are the mean of triplicate determination. mean values with different superscripts within the same column are significantly different (p ≤ 0.05). bafs1: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.005 g broth/g seed; bafs2: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.0075 g broth/g seed; ufs: unfermented african yam bean seed sample effects of fermentation on antinutritional factors in controlled fermented ayb condiment table 7 shows the result of the antinutritional factors of the fermented ayb seeds condiment produced with different inoculum concentrations of the starter culture. there was a significant difference in the phytate content as fermentation time increases. the phytate content ranged between 2.20% 2.88% and 0.34% 3.46% for inoculum concentrations of 0.005 and 0.0075 g broth/g seed respectively. the result indicates a significant (p ≤ 0.05) reduction in the concentrations of antinutrients food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 58 examined as the fermentation increases. the observation is in line with the trend of findings by [5] on the assessment of the antinutrient contents present in hura crepitans seed. however, the mean values for phytate content observed in this study were higher when compared to the report of [30] on some varieties of velvet beans (mucuna spp). tannin content was between 4.58% and 5.13% for 1st concentration and it was between 4.81% and 5.68% for 2nd concentration. these values are higher than the result of 0.80% reported by [31] for mucuna pruriens. the values were significantly different (p> 0.05). trypsin inhibitor content was between 0.13% 0.17% and 0.24% 0.38% for 0.005 and 0.0075 g broth/g seed respectively. the mean values for the trypsin inhibitor content of the fermented ayb seeds from the two inoculum concentrations were significantly different (p ≤ 0.05) concerning the fermentation time. there was a general reduction in the anti-nutritional content when compared to the raw sample. table 7: result for anti-nutritional components of controlled fermented ayb condiment fermentatio n time phytate (%) tannins (%) trypsin inhibitor (%) bafs1 bafs2 bafs1 bafs2 bafs1 bafs2 ufs 2.61d ±0.00 0.34c ± 0.01 5.13 c±0.02 5.46d ± 0.01 0.15 d ± 0.00 0.31d ± 0.00 24 hours 2.20a ±0.01 3.19a ± 0.03 4.80 b±0.05 4.81a ± 0.00 0.13 a ± 0.00 0.28b ± 0.00 48 hours 2.83e ±0.01 3.66e ± 0.01 4.62 a±0.01 5.36c ± 0.03 0.14 c ± 0.00 0.24a ± 0.00 72 hours 2.26c ±0.01 3.46d ± 0.01 4.76 b±0.10 5.64e ± 0.02 0.17 f ± 0.00 0.30c ± 0.00 96 hours 2.22b ±0.00 3.27b ± 0.06 4.88 b±0.10 5.19b ± 0.01 0.13 b ± 0.00 0.34e ± 0.00 120 hours 2.88f ±0.00 3.28b ± 0.00 4.58 a±0.06 5.68f ± 0.00 0.16 e ± 0.00 0.38f ± 0.00 (raw seed) 4.87g ±0.01 4.87f ± 0.01 5.90d±0.01 5.90g ± 0.01 0.40g ± 0.00 0.40g ± 0.00 values are the mean of triplicate determination. mean values with different superscripts within the same column are significantly different (p ≤ 0.05). bafs1: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.005 g broth/g seed; bafs2: condiment produced from fermented african yam bean seeds using a single starter culture of bacillus amyloliquefacien inoculated at a concentration of 0.0075 g broth/g seed; ufs: unfermented african yam bean seed sample sensory properties of fermented condiments table 8 shows the sensory score for the controlled fermented ayb condiment from different concentrations of bacillus amyloliquefaceins. it was observed that for the 0.005 g broth/g seed inoculum concentration fermented samples, the 0hour sample had the most preferred appearance with values of 7.62 while the least preferred was the 96 hours fermented sample with values of 5.96. for the sample batch fermented with 0.0075 g broth/g seed inoculum concentration, the 0 hours and control samples had the highest value with 7.62 and the lowest was 6.06 respectively. the highest recorded value for color was 7.28 from the 0-hour sample and the least was 6.32 at 48 and 96-hour fermentation for the samples fermented with 0.005 g broth/g seed inoculum concentration and the least was 6.32 at 72hour fermentation for the batch fermented with 0.0075 g broth/g seed inoculum concentration. the 24-hour sample had the highest value for texture, 7.14 at 24-hour fermentation and the least was 1.83 at 96 hours for the 0.005 g broth/g seed inoculum concentration fermented samples while for the sample batch fermented with 0.0075 g broth/g seed inoculum concentration, the 24-hour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 59 sample was mostly preferred with values 6.82 and the least preferred had values 6.18 at 120-hour fermentation. the samples with the highest values for aroma from the 0.005 g broth/g seed inoculum concentration fermented batch were samples from 0 and 24-hour fermentation, both values were 6.78 and the least was 6.10 at 120 hours fermentation while for the batch fermented with 0.0075 g broth/g seed inoculum concentration, the highest value was also 6.78 from the 0-hour sample and the least was 6.28 from 24hour fermentation. the samples that were generally accepted for both concentrations were samples from 0 and 24 hours of controlled fermentation. the values were 7.32 and 7.22 for the batch fermented with 0.005 g broth/g seed inoculum concentration and 7.32 and 7.04 for the samples fermented with 0.0075 g broth/g seed inoculum concentration. this showed that even at the different levels of concentration of b. amyloliquefacein in the samples, 0 and 24 hours were more preferred in terms of appearance, color, texture, and aroma. 4. conclusion fermented condiments form an important nutrient-enhancing component of the diet in most african countries. the main effect of the fermentation time and inoculum concentration as well as their interaction effects on the physicochemical and proximate properties of the fermented african yam bean seed condiment shows that the physicochemical properties of fermented condiment were affected by inoculum concentration and fermentation time. the proximate composition of fermented ayb was lower than the raw seed which may be as a result of the low concentration of microorganisms added to the samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 60 the inoculum concentration had a significant effect on the mineral and antinutrient factors of the fermented condiment. the mineral content was increased, and anti-nutritional factors were reduced. 5. acknowledgments the authors acknowledge the support of the management of biotechnology centre, federal university of agriculture, abeokuta, ogun state, nigeria in the provision of laboratory equipment for this research work. conflict of interest: the authors declare no conflict of interest 6. references [1]. fawale, o.s., gbadamosi, s.o., ige, m.m. & kadiri, o. effects of cooking and fermentation on the chemical composition, functional, and antinutritional properties of kariya (hildergardia barteri) seeds. wiley food science and nutrition, 5(6): 1106-1115, (2017). [2]. qin p., wang t. & luo. y. a review on plant-based proteins from soybean: health benefits and soy product development, journal of agriculture and food research, 7: 100265, (2022), https://doi.org/10.1016/j.jafr.2021.100265. [3]. parkouda, c., nielsen d.s., azokpota p., ouoba l.i.i., amoa-awua w.k., thorsen l., hounhouigan j.d., jensen j.s., tano-debrah k. & diawara b. the microbiology of alkaline fermentation of indigenous seeds used as food condiments in africa and asia. critical reviews in microbiology 35(2):139–56, (2009). [4]. adewumi, g.a., health-promoting fermented foods. in: melton, l., shahidi, f., & varelis, p. (eds.), encyclopedia of food chemistry, 3: 399–418. elsevier. isbn: 9780128140260 (2019). [5]. melini, f.; melini, v.; luziatelli, f.; ficca, a.g. & ruzzi, m. health-promoting components in fermented foods: an up-to-date systematic review. nutrients, 11: 1189. (2019). https://doi.org/10.3390/nu11051189 [6]. adebo, o.a. african sorghum-based fermented foods: past, current and future prospects. nutrients 12: 1111. (2020), https://doi.org/10.3390/nu12041111 [7]. ahaotu, i., anyogu a., njoku o.h., odu n.n., sutherland j.p., & ouoba l.i.i. molecular identification and safety of bacillus species involved in the fermentation of african oil beans (pentaclethra macrophylla benth) for production of ugba. international journal of food microbiology. 162 (1): 95–104, (2013). [8]. jeff-agboola, y.a. microorganisms associated with natural fermentation of african yam bean (sphenostylis sternocarpa harms) seeds for the production of condiment. research journal of microbiology. 2(11): 816-823, (2007). [9]. adebo, j.a.; njobeh, p.b.; gbashi, s.; oyedeji, a.b.; ogundele, o.m.; oyeyinka, s.a. & adebo, o.a. fermentation of cereals and legumes: impact on nutritional constituents and nutrient bioavailability. fermentation, 8: 63. (2022). https://doi.org/10.3390/fermentation8020063 [10]. idowu, a. chemical composition and sensory and pasting properties of blends of maizeafrican yam bean seed. journal of nutritional health and food science. 3: 1-6, (2015). [11]. inyang, u.e. & eyo, e.a. effect of soaking african yam bean seeds in unripe plantain peel ash solutions on the nutrients, anti-nutrients, and functional properties of the flour. journal of food and nutrition sciences. 3(4), pp. 147-151, (2015). [13]. yusufu, p.a., netala, j. & opega, j.l. chemical, sensory and microbiological properties of cookies produced from maize, african yam bean and plantain composite flour. indian j nutri.; 3(1), 122, (2016). [12]. okoye, j.i. & obi, c.d. chemical composition and sensory properties of wheatafrican yam bean composite flour cookies. discourse journal of agriculture and food sciences, vol. 5(2): 21-27, (2017). [14]. omodara t. r & aderibigbe e. y. effects of the use of starter culture on the quality of fermented parkia biglobosa, international journal of bio-technology and research (ijbtr), vol. 3, issue 4, 33-40, (2013). [15]. aoac international. official methods of analysis, 20th edn. (on-line). aoac international, rockville, md (2016). [16]. olagunju, o.f., ezekiel, o.o., ogunshe, a.o., oyeyinka, s.a. & ijabadeniyi, o.a. effects of fermentation on proximate composition, mineral profile and about:blank food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 pius ifeanyi okolie, emilymary chima okolie, sarah abiola ogundimu, kudirat aina agboola, maureen chinwe ojinnaka, effects of bacillus amyloliquefaciens culture concentrations on microbial load, physicochemical and sensory properties of “iru”-type condiment from african yam bean (sphenostylis stenocarp) seeds, food and environment safety, volume xxi, issue 1 – 2022, pag. 47 – 61 61 antinutrients of tamarind (tamarindus indica l.) seed in the production of daddawa-type condiment. lwt food science and technology, 90, 455-459, (2018). [17]. ojinnaka, m.c. & ojimelukwe, p.c. study of the volatile compounds and amino acid profile of bacillus fermented castor oil bean condiment. journal of food research, 29(1), 192203, (2012). [18]. chandra, s. & samsher s. assessment of functional properties of different flours. african j. of agric, 8(38): 4849-4852, (2013). [19]. stone h., bleibaum r.n., & thomas h.a. affective testing. in: stone h., bleibaum r.n., & sidel j.l. (eds) sensory evaluation practices, 4th edn. elsevier academic press, pp 306–309. (2012). [20]. ezekiel, o.o., ogunshe, a.a., & jegede, d.e. controlled fermentation of cotton seeds (gossypium hirsutum) for owoh production using starter cultures. nig. food j., 33, 54-60, (2015). [21]. chukwu, m., nwakodo, c.s., ndulaka, j.c. & nwokocha, n.j. production and proximate composition of ogiri-ahuekere (arachis hypogaea linn) seed condiment. research journal of agriculture and environmental management, 7(1): 007-017, (2018). [22]. ahaotu, n.n., echeta, c.k., bede, n.e., awuchi, c.g., anosike, c.l., ibeabuchi, c.j., & ojukwu, m. study on the nutritional and chemical composition of “ogiri” condiment made from sandbox seed (hura crepitans) as affected by fermentation time. gsc biological and pharmaceutical sciences, 11(2), 105–113, (2020). https://doi.org/10.30574/gscbps.2020.11.2.0115 [23]. diether, n.e. & willing, b.p. microbial fermentation of dietary protein: an important factor in diet–microbe–host interaction. microorganisms, 7(1): 19, (2019). https://doi.org/10.3390/microorganisms7010019 [24]. odunfa, s.a. microbiological and toxicological aspects of fermentation of castor oil seeds for ogiri production. journal of food science, 50(6), 1758-1759, (1981). [25]. adebiyi j.a., obadina a.o., adebo o.a. & kayitesi e. comparison of nutritional quality and sensory acceptability of biscuits obtained from native, fermented, and malted pearl millet (pennisetum glaucum) flour, food chemistry, 232, 210-217, (2017). [26]. dakwa, s., sakyi-dawson, t., diako, c., annan, n.t., & amoa-awua, w.k. effects of boiling and roasting on the fermentation of soybean into dawa dawa (soy-dawa dawa). int. j. food microbiol. 10: 69-82, (2005). [27]. oluseyi, e.o. & temitayo, o.m. "chemical and functional properties of fermented, roasted and germinated tamarind (tamarindus indica) seed flours", nutrition & food science, vol. 45 no. 1, pp. 97-111, (2015). [28]. adebiyi, j.a., njobeh, p.b. & kayitesi, e. assessment of nutritional and phytochemical quality of dawadawa (an african fermented condiment) produced from bambara groundnut (vigna subterranea), microchemical journal, 149: 104034, (2019). https://doi.org/10.1016/j.microc.2019.104034. [29]. nwokeleme, c. o., & ugwuanyi, j. o. evolution of volatile flavour compounds during fermentation of african oil bean (pentaclethra macrophyllabenth) seeds for “ugba” production. international journal of food science, 2015, 1–8, (2015). [30]. tuleun, c.d., carew, s.n. & patrick, j.a. proximate and anti-nutritional factors profile of some varieties of velvet beans (mucuna spp) found in benue state of nigeria. proceedings of the 13th annual conference of animal science association of nigeria (asan), abu, zaria. pp. 550-553, (2008). [31]. akinmutimi, a.h. effect of cooking period on the nutrient composition of velvet beans (mucuna pruriens). proceedings from the 32nd annual conference of nigeria society for animal production, university of calabar. pp. 223-236, (2007). about:blank about:blank about:blank 1. introduction 4. conclusion звіт з ндр 29-81 за 2007 – 2009 р doi: https://doi.org/10.4316/fens.2022.004 33 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xхi, issue 1 2022, pag. 33 38 influence of the addition of chamomile on the content of tannins in tea anna makarenko 1 , *mariia vorobets 1 , iryna kondrachuk 1 , anastasiia sachko 1 , heorhii vorobets 2 1educational and scientific institute of biology, chemistry and bioresources yuriy fedkovych chernivtsi national university, ukraine, *m.vorobets@chnu.edu.ua, 2educational and scientific institute of physical, technical and computer sciences yuriy fedkovych chernivtsi national university, ukraine *corresponding author received 19th november 2021, accepted 25th march 2022 abstract: in this work, the influence of chamomile on the content of tannin in the blended green and black teas is investigated by the titrimetric method, which relies on the assessment of tannin oxidation by potassium permanganate with the indicator indigo carmine. the standard procedure of titration was altered in such a way that the dilution of filtrate used during the investigation was decreased by 10 times. this modification made it possible to use the regular 250 ml flasks instead of big and inconvenient porcelain bowls. this way, identification of the point of equivalence became easier and more accurate. application of this modified method to the determination of tannin in a series of blended teas showed results that are in good agreement with those obtained by the standard methods. they proved that the content of tannin in tea does not depend on the added chamomile and, therefore, this herb does not depress the medical value of the investigated blended teas. keywords: tannins, blended tea, chamomile, medical values, modified titration, permanganatometry 1. introduction tea is one of the most common beverages in the world, which has unique taste and specific organoleptic properties. it relieves fatigue and headaches, increases mental and physical activity, adsorbs harmful substances and removes them from the body [1, 2]. the chemical composition of tea leaves is very diverse and according to the latest data [3], tea contains about 500 different substances. tea leaves polyphenols have antiviral, antitumor and anti-oxidant activity. they are characterized by antiinflammatory, antimicrobial, hemostatic, antispasmodic, antioxidant properties. having the ability to balance human blood sugar levels, they can be used for the prevention and treatment of type ii diabetes [4, 5]. there is no universal method of quantitative analysis of tannins, because tannins are a mixture of different classes of polyphenols that have different structures. the basic methods, which often used for quantitative determination of tannins are the next – gravimetric, photocolorimetric, spectrophotometric, titrimetric, nephelometric, tensiometric and others [3, 5–14]. determination by gravimetric analysis is often performed by deposition. quantitative determination of tannins by gravimetric method is based on their precipitation by gelatin, heavy metal ions, skin powder. the disadvantage of this method is the long-term definition. the titrimetric method is based on the oxidation of tannins by potassium permanganate in the presence of the indigo carmine indicator [15]. the disadvantage is the use of a large volume of water during the titration, which is carried out in a porcelain cup for evaporation and mixing – with a glass rod creates signifihttp://www.fia.usv.ro/fiajournal mailto:m.vorobets@chnu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xхi, issue – 2022 anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets, influence of the addition of chamomile on the content of tannins in tea, food and environment safety, volume xxi, issue 1 – 2022, pag. 33 – 38 34 cant inconvenience of fixing the equivalence point. using various additives – berries, leaves, herbs, etc., tea can be enriched with vitamins and beneficial properties. chamomile is one of the most valuable medicinal plants, which constantly attracts the attention of researchers. to date, scientists have substantiated the possibility of using herbal collection, one of the components of which is chamomile, in anticancer therapy [16]. tannins and catechins, as well as rutin (vitamin p) regulate the process of collagen formation in blood vessels, which helps to normalize blood pressure. tea is the main source of these substances for the human body [7, 17]. therefore, the study of the content of tannins in various teas, in particular blended with medicinal plants, is quite relevant. the purpose of the work is to improve the standard method of titrimetric determination of tannin content in tea; determine how the different content of chamomile supplement in blended teas based on black / green will affect the concentration of tannins in them. 2. materials and methods the tea samples which were used for research were: greenfield golden ceylon (1st grade, black leaf tea, loose, origin – ceylon, country of origin – ukraine) and green ahmad tea chinese green tea (leaf tea, chinese). as a supplement, medicinal chamomile was used – pjsc "liktravy", ukraine. for the experiments, 2 series of blended samples based on only black or only green (black / green) tea with different wt. % of chamomile additive were prepared in parallel (table 1). black / green tea without the addition of chamomile and chamomile without tea were used as control samples. to determine the content of tannins in the studied samples of blended teas, a standard method of permanganotometric titration in the presence of an indigo carmine indicator was chosen [15]. table 1 test samples № sample 1 2 3 4 5 6 7 8 tea (black / green) wt. % 100 90 80 60 50 40 20 0 chamomile wt.% 0 10 20 40 50 60 80 100 preparation of 0.1 % solution of indigo carmine: 0.5 g of finely ground pure indigo carmine was dissolved in 25 cm3 of chemically pure concentrated sulfuric acid (ρ = 1.84 g/cm3); the volume was adjusted to 500 cm3 by gradually pouring the solution into distilled water. the resulting solution was filtered through a pleated filter. preparation of 0.1 mol/dm3 of potassium permanganate solution: kmno4 weighing 1.58 g was weighed on technical scales. after complete dissolution of the drug, the contents of the flask (500 cm3) were adjusted to the mark with distilled water. the normality of the prepared solution was established after 10 – 12 days. preparation of exactly 0.1 mol/dm3 of oxalic acid h2c2o4 solution: on analytical scales weighed the exact portion of recrystallized oxalic acid weighing 1.5750 g, transferred to a volumetric flask with a capacity of 250 cm3, dissolved it in distilled water, adjusted to the mark with distilled water. standardization of kmno4 solution: to 10 cm3 of freshly prepared oxalic acid solution was added 15 cm3 of sulfuric acid solution (1 part of concentrated h2so4 : 5 parts of h2o), heated to a temperature of 70 – 80 °c, immediately titrated. at the end of the titration, the solution turns pink, which does not disappear for 1 – 2 minutes. titration was performed three times, finding the arithmetic mean value of the volfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xхi, issue – 2022 anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets, influence of the addition of chamomile on the content of tannins in tea, food and environment safety, volume xxi, issue 1 – 2022, pag. 33 – 38 35 ume of titrant consumed. the normality of the kmno4 solution ( 4kmno n ) was calculated by the formula: 4 422422 4 kmno ochoch kmno v vn n   , where 422 och n – normality of the h2c2o4 solution, mol/dm3; 422 och v – volume of 0.1 mol/dm3 of h2c2o4 solution (10 cm 3); 4kmno v – volume of kmno4 solution used on titration, cm3. according to the standard method, the determination of the content of tannins is carried out starting with the grinding of a tea sample in a porcelain mortar; 2.5 ± 0.0002 g of crushed tea is weighed on clear glass and transferred to a 250 cm3 flask. then 200 cm3 of boiling distilled water is poured into the flask and placed in a water bath for 45 minutes. the resulting extract is filtered into a volumetric flask with a capacity of 250 cm3. the content of the flask is cooled to 20 °c and made up to the mark using distilled water. using a pipette, 10 cm3 of the filtrate is taken and placed in a cup for evaporation, which is pre-poured with 750 cm3 of water and 25 cm3 of 0.1 % solution of the indigo carmine indicator. potassium permanganate is titrated using the 0.1 mol/dm3 solution with constant stirring with a glass rod. during this, the blue color of the liquid gradually changes through blue-green, dark green, light green, yellowgreen, and turns yellow with a golden tinge. completion of titration is determined by the disappearance of the green hue and the appearance of a pure stable yellow color. the volume of 0.1 mol/dm3 of potassium permanganate consumed solution is fixed in cm3. titration is performed three times and the arithmetic mean of the consumed titrant volume is calculated. similarly, the blank titration of water with indigo carmine 0.1 mol/dm3 solution of potassium permanganate is performed. the content of tannins (x) in % is determined by the formula: %;100 004157,0)( 1 1     mv vaa x where x is the tannins content, %; a – volume of 0.1 mol/dm3 of kmno4 solution, used on titration of prepared of tea extract, cm3; a1 – volume of 0.1 mol/dm 3 of kmno4 solution used on titration of water with indigo carmine, cm3; 0.004157 – the amount of tannins that is oxidized 1 cm3 0.1 mol/dm3 solution kmno4, g; m – the weight of a portion of dry tea, g; v – volume of the obtained tea extract, cm3; v1 volume of tea extract taken for research, cm3. 3. results and discussion as mentioned above, the standard method for the determination of tannins in tea by the permanganate method is not very convenient due to such features as the addition of 750 cm3 of distilled water, use in the titration of large porcelain cups and mixing their contents with a glass rod. we modified the methodology. in particular, it is proposed to reduce the volume of distilled water by 10 times, which allows using ordinary titration flasks with a capacity of 250 cm3 instead of large and uncomfortable porcelain cups and eliminates the need to mix the contents of the flask with a glass rod.the results of titration of extracts of some test samples obtained by standard and modified methods are presented in table 2. table 2 the volume of 0.1 n kmno4 solution (ml) used during the titration of some test samples by standard and modified methodology sample №1 №3 № 4 № 5 standard methodology 2.6 2.3 1.9 1.7 modified methodology 2.6 2.3 1.9 1.7 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xхi, issue – 2022 anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets, influence of the addition of chamomile on the content of tannins in tea, food and environment safety, volume xxi, issue 1 – 2022, pag. 33 – 38 36 comparative analysis of the results (table 2) shows that the values of the volumes of titrant spent during titration by standard and modified methods are absolutely the same. in addition, as the water volume decreases, it is easier to observe a clear change in color from blue to straw yellow, i.e. the identification of the equivalence point is more accurate. determination of tannins content in the studied samples was performed using a modified method. the obtained experimental results are presented in fig. 1. the data show that with decreasing wt. % of green / black tea in blended samples with chamomile medicinal content of tannins decreases. in samples № 5 (50 wt. % green tea and 50 wt. % chamomile) and № 6 (40 wt. % green tea and 60 wt. % chamomile) the content of tannins is the same. test sample № 7 (20 wt. % green tea and 80 wt. % chamomile) and control sample № 8 (100 wt. % chamomile) contain 2.9 wt. % tannins (fig. 1, curve 1). in samples № 6 (40 wt. % black tea and 60 wt. % chamomile), № 7 (20 wt. % black tea and 80 wt. % chamomile) and in the control sample № 8 the tannins content is almost the same and equal 2.6 – 2.7 wt. % (fig. 1, curve 2). there is a pattern: in blended samples with a low content of green / black tea, the value of the tannin content is approximately 3 wt. %. in general, regarding the effect of the addition of chamomile on the content of tannins in blended black / green teas, it can be stated that it does not depend on the content of chamomile. approximation of experimental data shows a linear dependence of tannin content on wt. % of tea in blended samples y = kx + a, where a = 0 (fig. 1): based on green tea y = 0,104x; based on black – y = 0,065x. however, when the content values are less than 30 wt. % of black / green tea, there is a deviation from these linear dependences in the form of some "background values" of tannin content in the range of 2.6 – 2.9 wt. %. if these background values were characteristic of chamomile, they should be manifested at low wt. % chamomile in blended samples. but under such conditions, the linear approximations would be different from the zero value of the parameter a, in particular: on the basis of green tea y = 0.074x + 2.92; based on black – y = 0.039x + 2.6. according to the literature on the chemical composition of chamomile [3, 9], tannins are absent. therefore, in our opinion, it is incorrect to list the amount of potassium permanganate used by wt. % tannins in blended samples with a content of chamomile drug more than 70 wt. %. fig. 1. dependence of tannins content in blended teas on the wt. % of tea: 1 – green tea; 2 – black tea; 3 – sensitivity limit of the method; lines on experimental graphs – linear approximation of theoretical models food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xхi, issue – 2022 anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets, influence of the addition of chamomile on the content of tannins in tea, food and environment safety, volume xxi, issue 1 – 2022, pag. 33 – 38 37 most likely, the reason for such results is the relatively low sensitivity of the permanganatometric method to determine the content of tannins in tea, which, according to the literature, is equal to 3 wt. % (fig. 1, line 3). the content of 2.9 wt. % tannins in the control sample № 8 can be explained by the fact that potassium permanganate oxidizes not only polyphenols, but also simple phenolic compounds and other reducing agents of non-phenolic nature, in particular organic acids, vitamin c and a number of compounds contained in chamomile. theoretical modeling and quantitative evaluation of the content (wt. %) of tannins in samples of black or green tea without the addition of chamomile were performed to test this hypothesis. the theoretically calculated values correlate well with the values of tannins content found experimentally in the blended samples (fig. 2, fig. 3). fig. 2. comparative diagram of tannins content in samples № 2–7 (blended black tea), calculated theoretically and found experimentally fig. 3. comparative diagram of tannins content in samples № 2–7 (blended green tea), calculated theoretically and found experimentally 4. conclusions it was found that the addition of chamomile to black or green tea does not affect the tannin content in them. therefore, it is possible to make blended teas with different content of chamomile, which will have food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xхi, issue – 2022 anna makarenko, mariia vorobets, iryna kondrachuk, anastasiia sachko, heorhii vorobets, influence of the addition of chamomile on the content of tannins in tea, food and environment safety, volume xxi, issue 1 – 2022, pag. 33 – 38 38 medicinal properties without losing their properties, i.e. to expand the range of teas. the standard procedure of titration was altered in such a way that the dilution of filtrate used during the investigation was decreased by 10 times. this modification made it possible to use the regular 250 ml flasks instead of big and inconvenient porcelain bowls. this way, identification of the point of equivalence became easier and more accurate. the proposed method for determining the tannin content in blended black and green teas has been tested. the results obtained by standard and modified methods are exactly the same. 5. references [1]. e. melnyk. research of different varieties of tea and their physiological effect on the human body. scientific-practical conference. zaporizhzhia, ukraine, 160-163, (2019). [2]. ya. yashin, a. yashin. tea. the chemical composition of tea and its impact on human health. moscow: translit, 160 p. (2010). [3]. т. denysenko. spectrophotometric determination of polyphenols using heteropolycomplexes of dawson structure. dis. cand. chem. sciences: 02.00.02. odessa, ukraine, 162 p. (2016). [4]. s. kel, v. zolotopup, v. sharipova, o. kuzmenko. study of the composition and properties of tea. clarification of the impact of tea on human health. scientific-practical conference. kharkiv, ukraine, 34-41, (2019). [5]. o. mazulin, t. balanchuk, g. mazulin. investigation of tannins content in grass of thistle species (carduus l.) collection of scientific works of employees nmape named after p. shupyk, ukraine, 79-86, (2017). [6]. g. zatolokina, o. rudakova. comparative analysis of the chemical composition of medicinal plant raw materials of chinese camellia. scientific-practical conference. kharkiv, ukraine, 26-29, (2019). [7]. t. denisenko, s. gatsenko. tasting evaluation and research of physical and chemical indicators of tea quality. visnyk of chernihiv state technological university. series: technical sciences. chernihiv, ukraine, 2 (57), 35-41, (2012). [8]. h. sakakibara, y. honda, s. nakagawa. simultaneous determination of all polyphenols in vegetables, fruits and tea. journal of agricultural and food chemistry. washington, usa, 3 (51), 571-581, (2003). [9]. v. kislichenko, i. zhuravel, s. marchyshyn and others. pharmacognosy: textbook for students of higher pharmaceutical education. edditor v. kislichenko. kharkiv: nuph: golden pages, ukraine, 736 p. (2015). https://gnosy.nuph.edu.ua/wpcontent/uploads/2020/10/фармакогнозія_2015.pdf [10]. e. ryabinina. comparison of chemicalanalytical methods for determining tannins and antioxidant activity of plant raw materials. analytics and control. ekaterinburg, russia, 2 (15), 202-204, (2011). [11]. h. sakakibara [et al.]. simultaneous determination of all polyphenols in vegetables, fruits, and teas. journal of agricultural and food chemistry. washington, usa, 3 (53), 4290-4302, (2005). [12]. l. rybak, o. konovalova, o. kolyadych. comparative study of polyphenols in different species of geranium (geranium l.) by permanganatometric titration and spectrophotometric method. pharmaceutical journal. kyiv, ukraine, 6, 44-47, (2010). [13]. a. fedosov, v. kislichenko, o. novosel. determination of the quantitative content of the sum of phenolic compounds in artichoke inflorescences, leaves and garlic bulbs. medical and clinical chemistry. ternopil, ukraine, 1 (20), 100104, (2018). [14]. g. vorobets, m. solomiychuk, a. zelya, v. strebezhev, m. vorobets, v. buchakchiysky [et al.]. methods and computerized means of identification and rapid analysis of carcinogenic components in bioactive media and materials. problems of informatics and computer engineering: proceedings of the viii international scientific-practical conference. chernivtsi, ukraine, 66-67, (2019). [15]. gost 19885-74 [restriction 1994-11-12 removed]. (interstate standard). tea. methods for determining the content of tannin and caffeine. publishing house of standards. moscow, russia, 8 p. (1995). [16]. a. makarenko, m. vorobets, i. kondrachuk, a. sachko. influence of the addition of chamomile on the content of tannin in tea. the international conference “biotechnologies, present and perspectives”. suceava, romania, 61, (2021). [17]. k. khanbabaee, t. ree. tannins: classification and definition. natural product reports. 18, 641-649, (2001). https://gnosy.nuph.edu.ua/wp-content/uploads/2020/10/фармакогнозія_2015.pdf https://gnosy.nuph.edu.ua/wp-content/uploads/2020/10/фармакогнозія_2015.pdf doi: https://doi.org/10.4316/fens.2021.025 224 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 224 234 cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria *sofia bahroun 1 , samira gheid 2 , warda boumaraf 3 1faculty of natural and life sciences, laboratory of biodiversity and ecosystem pollution, chadlibendjedid university, el tarf, algeria, sofiabahroun@gmail.com, 2faculty of natural and life sciences, laboratory of biodiversity and ecosystem pollution, chadlibendjedid university, el tarf, algeria, gheidsamira@gmail.com, 3faculty of natural and life sciences, chadlibendjedid university, el tarf, algeria, wardaboumaraf1@gmail.com, *corresponding author received 23th august 2021, accepted 29th september 2021 abstract: nowaday, water is threatened by pollution due to a similar degradation of hydric resources. the knowledge about water quality is an essentialcondition allowing the establishment of a management system, that will contributeto guarantee water supply in the future. the purpose of this work is to determine the physico-chemical and bacteriological quality of spring water, takenfrom the sub-basin level of the kallewadi in el tarf region, located in the extreme north east of algeria. in order to monitor its water quality, we have chosen four sampling sources distributed in the kallewadi sub-basin. the sampling was carried out during a period of four months extending from february to may 2019. the results show that all physicochemical parameters meet the current standards. as for the bacteriological analyses, they show the absence of all pathogenic germs.the calculation of microbiological contamination index confirms the absence of degradation in water quality inwhich contamination is null.the results are visualized by using gis via the realization of thematic map, which were elaborated to have an idea about the quality of the studied spring water; these maps show an excellent quality. key words: sub-basin, wadi, kalle, sources, bacteriological, index, gis, map, excellent quality. 1. introduction water is a natural resource that is becoming increasingly scarce. it is part of the patrimony of a nation and also of humanity. the protection, enhancement and development of resources that can be used within the framework of natural balances are of general interest in the world. we must ensure the respect of this noble principle and consider water as an economic and social good, whose access is a right for each person. water is necessary for the development of all life forms, animal or plant. it is essential to ensure preservation of this vital supply from the qualitative and quantitative viewpoints. in order to do this, we must ensure sustainable management of this resource and preserve the environment, in which water is in continuous interaction. for several decades, in algeria, animal, agricultural, industrial and domestic dejections have contributed, more and more to rivers and groundwater pollution than elsewhere. http://fens.usv.ro/index.php/fens mailto:gheidsamira@gmail.com food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 225 in recent years, a lot ofresearches have focused on the study of groundwater and surface water resources quality. the present work comes in addition to other works and deals with the physicochemical and bacteriological quality of spring water taken from the kallewadi subbasin in el tarf region. the latter, mainly agricultural region is located in the extreme north-east of algeria. 1.1. geographic location of the study area the study area which is the kallewadi coastal sub-basin coded as 03-18 [1 2], extends from 8°13ʹ3ʺ to 8°41ʹ4ʺ east longitude, and from 36°46ʹ43ʺ to 36° 56ʹ33ʺ north latitude. it is limited (fig. 01): fig. 1. map of the coastal sub-basinof the kallewadi (03-18). from north by the mediterranean sea; and from east by the algerian and tunisian frontiers; from the west by ouedmafragh coastal sub-basin 03-17, andfrom south by ramel souk 03-16 coastal sub-basin.it covers an area of 432.2 km². the study area covers the eastern part of el tarfcity, divided into six communes, five of which are partially included in the kallewadi sub-basin, and one is entirely included. the available statistics announce 56,487 inhabitants in the whole of kallewadisubbasin, 2018 estimate according to the 2008 census [3]. this population is strongly concentrated in the most important settlements [4]. the main elements that constitute the relief of the kallewadisub-basin are [5]. a dune cordon, small eminences of sandstone relief of low altitudes, as well as a set of hills not exceeding 600 m, two alluvial plains swampy, the western slopes of mountains going from north to south and finally, the northern slope of jebel kourima with kef hammam of 561 m. the geological study of the kallewadi subbasin has allowed to the following results[6], an allochthonous set, which includes two complexes; the flysch complex with micro breccia (where its lower bedding outcrops only in the food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 226 southeastern part of el aioun), and the numidian complex, which is characterized by its upper sandstone bedding whose structures have a simple internal layout with a gentle slope (almost horizontal).and an autochthonous post-nape complex, which contains the miocene water table that develops in its large part in el aioun sector, covered by quaternary deposits [7]. from the hydrogeological viewpoint, two formations are observed in the region: permeable and low permeable formations. sandstones, numidian clays, as well as clays and silts belong to the low permeable formations. their permeability is low [5]. given the geological structure of the watershed, the supply of aquifers in the watershed is from surface water of the northern and southern slopes as well as from wadi el eurg, wadi el haut and direct rainfall on slopes that feel the same low-permeability bedrock as that of the aquifers. the study of sub-catchment soils allows us to determine two soil types, the zonal soils, (which are mainly dependent on the weather), and the azonal soils, (which are independent of weather) [5]. the hydrographic network of the subcatchment includes all the streams draining the territory of the catchment. it contains all the canals and streams leading to the main stream called in our case wadi (fig. 2). the sub-catchment presents two major streams that run all year round (oued el haut, 14 km long, and oued eleurg, 10 km long) [5]. the sub-basin is characterized by a mediterranean climate with abundant rainfall during the wet season and cold months and by a drought during the summer [8]. fig. 2. map of the hydrographic network of the coastal sub-basin of kallewadi. 2. material and methods 2.1. framework of the study this study took place at the hygiene laboratory service of prevention and water bacteriological analysis in the public health institution of proximity (boussebsi omar), at the public multiservices clinic (saidimousa called khalifa) in el kala, el tarf city. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 227 2.2. period and type of study this study was carried out on the waters of four sources, located in the sub-basin of kallewadi. these waters were analyzed in the laboratory of hygiene service of prevention and bacteriological analysis of waters, during the months of february, march, april and may throughout the yearof 2019, at a rate of two sampling per month. the purpose of this work is to gain insight into the microbiological quality of these spring waters through a microbiological index during 2019. 2.3. presentation of the sampling stations for the achievement of this study, four sources have been chosen whose waters are destined for human consumption such as (fig. 3): the source of melloul; the source of siporex; the source of ainbargougaya; the source of segleb, their geographical distribution is summarized in tab. 1. fig. 3. map of sampling sources. 2.3.1. the source of melloul the melloul spring, located 2 km north of the souarekh commune, which is a natural state uncovered and coming from an important phreatic grounwater. its catchment by drilling gives birth to the wadimelloul. it constitutes an important irrigation perimeter of the region. the catchment is connected by a high-pressured pipe which arrives to a 2 x 500 l reservoir, supplying the locality of melloul with a flow of 250 l/s. 2.3.2. the source of siporex located at 300 m from the melloul nape, it is a catchment that supplies the siporex factory and somedwellings. 2.3.3. the source of segleb located 4 km east of souarekh commune, this spring contains several aquatic plants that can adversely affect water quality and can be exposed to runoff. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 228 2.3.4. the source of ainbargougaya ainbargougaya is located 7 km from souarekh, and 3 km north of el aiounecommune, on the national road 44 towards tunisia. its catchment and its adduction include gravitational runoffs from the mountains to a catchment chamber of 100 m length, fitted out in a public fountain of a rectangular shape with an average flow of 100 l/s. table 1. inventory of sampling sources with names and coordinates. source number name of sampling source coordinates (degree minute) altitude (m) 01 melloul 3653.784n0832.606e 32 02 siporex 3653.576n0833.017e 29 03 segleb 3654.621n0836.241e 120 04 ainbargougaya. 3650.952n0835.402e 308 2.4. material the following materials have been used during the field work. multiparameteroffield series u-50, horiba; sterile bottle of 250 ml for water sampling; a cooler to transport the samples; digital camera sony dsc-w220; and a gps to record the coordinates of each point. 2.4.1. sampling samples are collected in bottles and previously subjected to rigorous cleaning and sterilization to eliminate thebacteria.the sample from each source must be collected in a bottle. each sample is accompanied by an information card on which is noted: the origin of water (water source) ; the exact address of the sampling site; and the date of sampling. 2.4.2. sampling technique for the water sampling necessary to bacteriological analysis, bottles out of pyrex glass were preferably used, provided with a broad collar and a stopper with metal screw. the sampling techniques varied according to the purpose and the nature of water to be analyzed. 2.4.3. transportation and storage in order to prevent that the initial germ content of the water is not likely to be modified in the bottle, all the analyses were carried out as quickly as possible. the evolution is difficult to predict and depends on many factors: temperature, bacterial competition of the presented species, and chemical composition of the water. to this effect, the circular of january 21, 1960, related to the methods of bacteriological analyses of water supply specifies that « if the period of the transportation exceeds 1 hour, and if the outside temperature is higher than 10°c, the samples will be transported in coolers whose temperature must be between 4 and 6°c ». even in such conditions, bacteriological analysis must begin within a maximum of 8 hours after the sample is collected. if in exceptional cases, the analysis must be postponed, the samples must be stored at 4 °c [9]. this means thateach time the samples are taken; the bottles are clearly labeled, stored in a refrigerated cooler at 4°c and to be transported to the laboratory [10]. 2.5. methods the analysis of the studied waters and the monitoring of detectable compounds were carried out using the following techniques: food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 229 2.5.1. in situ measurement of physicalchemical parameters the parameters recorded in situ are:potential of hydrogen (ph), temperature (t), electrical conductivity (ec), dissolved oxygen (do), redox potential(eh), turbidity and salinity. the measuring apparatus is a multi-parameter field meter series u-50, horiba. it is equipped with a probe that simultaneously displays the results of 11 indication parameters such as potential of hydrogen, redox potential, dissolved oxygen, conductivity, salinity, total dissolved solids, seawater specific gravity, temperature, turbidity and depth. 2.5.2. research method and enumeration of microbiological parameters in this study, three microbiological parameters have been chosen such as: total germs, faecal coliforms, research and numbering of faecal streptococci. during each trip, we had a sample consisting of 4 samples to undergo a bacteriological analysis at the laboratory.the bacteriological analysis aims to highlight the presence of germs, based on the search and counting of these in the samples to be analyzed. the searched germs are: the total germs, the faecal coliforms, the search and the enumeration of the faecal streptococci. the search and the enumeration of the indicators of contamination are done according to two methods of choice:  either by filtration on 0.45 µm membrane in solid medium by supposing the availability of a filtration ramp,  or in a liquid medium on bcpl by npp technique (most probable number). the membrane filtration technique was chosen. to characterize the microbiological contamination at the level of studied spring water, we preceded according to a method the calculation of the iqm [11 12]. the microbiological contamination index method is presented below [12]. they include five quality classes corresponding to the generally accepted colors (fig. 4). null contamination low contamination moderate contamination strong contamination very strong contamination fig. 4. class of alteration. the principle is to divide the values of the bacteriological parameters into five classes (tab. 2), then to determine, from one'sown measurements, the corresponding class number for each parameter and then to take the average. table. 2. quality grid (imc) parameter class total coliforms/ml faecal coliforms/ml faecal streptococci/ml 5 4 3 2 1 <2000 2000-9000 9000-45000 45000 360000 >360000 <100 100-500 500-2500 2500 20000 >20000 <5 5-10 10-50 50 500 >500 𝐼𝑀𝐶 = (𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑙𝑖𝑓𝑜𝑟𝑚𝑠 + 𝐹𝑎𝑒𝑐𝑎𝑙 𝐶𝑜𝑙𝑖𝑓𝑜𝑟𝑚𝑠 + 𝐹𝑎𝑒𝑐𝑎𝑙 𝑆𝑡𝑟𝑒𝑝𝑡𝑜𝑐𝑜𝑐𝑐𝑖) 3⁄ imc of 5.0 to 4.6: null microbiological contamination; imc of 4.5 to 4.0: low microbiological contamination; imc of 3.9 to 3.0: moderate microbiological contamination; imc of 2.9 to 2.0: strong microbiological contamination; imc of 1.9 to 1.0: very strong microbiological contamination. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 230 3. results and discussion 3.1. physico chemical parameters (in situ measurement) the physic chemical parameters measurement resultsof the studied spring water are reported in tab. 3. they are compared to the algerian standards relating to water quality used for water production meant for human consumption. table. 3. averages results of the of physico-chemical parameters of spring water during the year 2019 parameter source t (°c) ph eh (mv) turbidity (ntu) do (mg/l) salinity (g/l) conductivity (µs/cm) ainbargougaya 17.83 5.7 50 1 9.92 0.144 220 segleb 18.72 6.63 -4 0 9.22 0.473 739 melloul 20.53 7.07 -30 0 9.82 0.008 150 siporex 19.42 7.02 -28 0 9.7 0.010 180 3.1.1. temperature he curve shape shows that the temperature is particularly high in melloul spring; this is explained by the shallow waters that are directly influenced by the air temperature. it is noted that the temperatures at the level of all springs do not exceed the algerian standard which is lower than 25 °c [13]. in table. 3 we present the obtained results which indicate a water temperature that varies between a minimum of 17.83 °c at the source of ainbargougaya and a maximum of 20.53 °c at the source of melloul. 3.1.2. potential of hydrogen ph values measurements recorded in spring water shows that the measured ph at all springs, ranges between 5.70 at the level of ainbargougaya and 7.07 at the level of melloul, which generally translates a character of slightly acidic to neutral water. table. 3 illustrates that ainbargougaya and segleb waters are marked by an acidic ph, probably due to contamination bynitrogenous compounds. acidic water is in effect aggressive (corrosive) and can liberate the constituent metals of the pipes, such as iron, copper, lead, nickel, chrome and zinc. 3.1.3. turbidity in table. 3. we present the values of turbidity fluctuate between 0 and 1 ntu. this variation shows that the studied source water have clear water [14], therefore they meet the standards to be drinkable. 3.1.4. electrical conductivity the measurement of the conductivity allowed us to appreciate very quickly, but very approximately the mineralization. in table. 3 , we present the data analysis that shows a minimum value of 150 µs/cm recorded at the level of melloul, and a maximum of 739 µs/cm marked at the level of segleb spring, probably due to water runoff. 3.1.5. dissolved oxygen through the obtained results, we have seen that the studied sources present a good oxygenation. table. 3 shows that the values oscillate between a minimum of 9.22 mg /l in segleb source and maximum of 9.92 mg /l at the source of ainbargougaya, so the spring waters of the sub-basin in kallewadi have a good oxygenation and of an excellent quality. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 231 3.1.6. redox potential among the values of eh measured in source water and according to table. 3, we note amaximum of 50 mv recorded at ainbargogaya and a minimum of -30 mv recorded at melloul. the eh-hp diagram in figure. 5 shows that for most sources, the environment is reducing and stable except for ainbargougaya source, where the environment is always stable but oxidizing. this is explained by the high contribution of dissolved oxygen. fig. 5. evolution of the different sources from eh to ph. 3.1.7 salinity the measurements of salinity in source water showed values between 8 mg/l at the level of melloul and 473 mg/l at the level of segleb (tab. 3). this high value is mainly due to erosion of the lithospheric rocks, produced by runoff and groundwater. all along their path, salts are carried away by water and accumulate. 3.2 bacteriological parameters 3.2.1. application of the microbiological contamination index method for a more significant interpretation, we have chosen two results from the different bacteriological parameters of each month for all sources [15] (tab. 4). according to table. 4 and the results of 13/02/2019 during the month of february. wehave noticed that at the level of all the sources, the microbiological contamination is null.according to the results of 28/02/2019, we noticed a null contamination but with an early more or less high of total coliforms at the level of siporex and segleb spring water, with respectively 5 and 6 germs/100 ml, seen the norms of who. this does not translate a danger but rather a water to be watched. during the month of march and following the results of 12/03/2019, the contamination index is null but with anincrease of total coliforms at the melloul level. the latter is a natural state (not covered). for the 26/03/2019, the contamination is null, with anelevation of total coliforms at segleb and ainbargougaya level whose early is 9 germs/100ml, respectively, due to runoff and gravity runoff of the mountains. for the month of april and according to the results of 09/04/2019, we note a null contamination consequently water of good bacteriological quality. the same remark for the results of 24/04/2019. finally, during the month of may and according to the results of 16/05/2019 and 30/05/2019. the quality of source water is still excellent following the contamination index, which is null, but the melloul source presents high early total coliforms with 9 germs/100 ml.the water of this source coming from an important phreatic groundwater. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 232 table 4. bi-monthly values of indices and classes of contamination for spring waters. month date sources tc/ml fc/ml fs/ml index contamination f e b r u a r y 1 3 /2 /2 0 1 9 siporex segleb ainbargougaya melloul 0.00 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0,00 0.00 5 5 5 5 null null null null 2 8 /2 /2 0 1 9 siporex segleb ainbargougaya melloul 0.05 0.06 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null m a r c h 1 2 /0 3 /2 0 1 9 siporex segleb ainbargougaya melloul 0.03 0.01 0.01 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null 2 6 /0 3 /2 0 1 9 siporex segleb ainbargougaya melloul 0.01 0.09 0.09 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null a p r il 0 9 /0 4 /2 0 1 9 siporex segleb ainbargougaya melloul 0.03 0.02 0.01 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null 2 4 /4 /2 0 1 9 siporex segleb ainbargougaya melloul 0.00 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null m a y 1 6 /5 /2 0 1 9 siporex segleb ainbargougaya melloul 0.03 0.03 0.03 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null 3 0 /5 /2 0 1 9 siporex segleb ainbargougaya melloul 0.03 0.03 0.03 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5 5 5 5 null null null null this groundwater probably is contaminated by the waters of wadimelloulwhich feeds the groundwater in the period of low waters. in conclusion, this does not translate as a danger but rather a water to be watched. in addition, bacteriological analyses reveal the total absence of faecalcoliforms and faecal streptococci during our entire study period in all sources. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 233 3.2.2. establishment of the spring water quality map for 2019 for the establishment of the quality map of source waters, we chose the method of the microbiological contamination index [12]. this method reflects the reality of contamination rates (fig. 6).in order to have a more meaningful result, the average of different concentrations of the four months for all sources was chosen. fig. 6. source water quality map for 2019 the map clearly indicates an excellent quality of source water in the kallewadisub-basin, marked by null microbiological contamination. the low values of this index are explained by a very low total coliform count and the total absence of faecal coliforms and faecal streptococci in the studied sources. 4. conclusion the present work was devoted to the study of the physic chemical and bacteriological quality of source waters at the level of the kallewadisub-catchment, in the region of el tarf. according to our analyses and according to the parameter indicators of groundwater quality spring water indicate that: the temperatures at the level of all sources don’t exceed the algerian standard, which is lower than 25 °c. the values of the ph recorded in the source waters are slightly acidic to neutral. the values of the turbidity show that the studied source waters have clear water. thedata of analysis show an important conductivity at the level of segleb source, probably caused by the runoff water. the studied sources present a good oxygenation, the values of eh measured in the source waters present a maximum of 50 mv translating an oxidizing environment, and a minimum of30 mv interpreting a reducing location. finally, the measurements of salinity in the source waters showed a maximum of 473 mg/l. on the basis of bacteriological results, the analyses reveal the total absence of faecal coliforms and faecal streptococci during the whole study period in all sources. in fact, the results clearly establish the aptitude of all spring waters for drinking water supply and require a monitoring, because of alteration by total coliforms in all sources. according to the microbiological contamination index and during all our period of study, the contamination is null. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xx, issue 3 – 2021 sofia bahroun , samira gheid, warda boumaraf, cartography of the microbiological quality index of spring waters in the kalle wadi sub-basin in el tarf region, extreme north east of algeria, food and environment safety, volume xx, issue 3 – 2021, pag. 224 – 234 234 it emerges that the studied water sources is of an excellent bacteriological quality. in perspective, it would be judicious to develop and elaborate a strategy of monitoring and protection of the sub-basin source in kallewadi, based on a permanent follow-up of the physicochemical and bacteriological parameters to identify the problem of the risk which is in relation with water. 5. references [1]. bahroun s., environmental rejection objectives for pollutants in the receiving environment and optimization of self-purifying power: case of wadikébirest (northeast algeria). phd thesis, faculty of earth sciences, university of annaba, algeria, (2016), 220. [2]. hydrographic basin agency., constantinoisseybouse-mellegue, the agency's workbooks, book number 4, (2000), 75. http://abhcsm.dz/telechargement/cahiersagence/04 cotiers01.pdf [3]. bordj a., physical-chemical and bacteriological quality of source waters in the oued la kalle sub-basin, extreme north-east algeria, master's thesis, chadlibendjedid university, el tarf, (2019), 46. [4]. bahroun s., chaib w., the quality of surface waters of the dam reservoir mexa, northeast of algeria, journal of water and land development, 34 (vii–ix), (2017), 11-19. http://www.itp.edu.pl/wydawnictwo/journal/34_201 7_vii_ix/bahroun%20chaib.pdf. [5]. raachi m., preliminary study for an integrated management of the resources of the basin of lake tonga in north-east algeria, dissertation presented as a partial requirement for the master's degree in geography, université du québec à montréal,(2007), 188. https://archipel.uqam.ca/736/1/m10067.pdf. [6]. haouli z., inventory and highlighting of pollutants, toxic potentials of leach water from oumteboul and azzaba mines on water resources quality, phd thesis, faculty of earth sciences . university badjimokhtar annaba,(2017), 116.https://biblio.univ-annaba.dz/wpcontent/uploads/2018/06/these-haouli zouina.pdf. [7]. bentouili m.y., inventory and water quality of the springs of el kala national park (n.est algeria), magister thesis, badjimokhtar university annaba,(2007), 9-13. https://biblio.univannaba.dz/wpcontent/uploads/2014/06/bentouili.pdf [8]. samraouiet de belair g., the wetlands of eastern numidia: assessment of knowledge and management perspectives. synthesis (special issue) n°4, (1998), 19. https://www.researchgate.net/publication/29102425 4_les_zones_humides_de_la_numidie_orientale_ bilan_des_connaissances_et_perspectives_de_gesti on/link/5ce2d091299bf14d95aa8703/download. [9]. rodier j., legube b., merlet n., l'analyse de l'eau, 9th edition, ed. dunod, paris,(2009), 1579. [10]. rodier j., water analysis. natural water, waste water, sea water, 5th edition ed. dunod, paris, (2005), 1383. [11]. leclercq l., running waters: characteristics and means of study, in wetlands, proceedings of the colloquia organized in 1996 by the ministry of the walloon region in the framework of the world year of wetlands, legs, walloon region, dgrne,(2001), 67-82. [12]. bovesse m., depelchin a., cartography of the pollution of the watercourses of the province of namur: bacteriological analyses, final report (january 1979 january 1980), (1980), 25. [13]. afnor., collection of french standards: water quality. 3 edition, (1999). [14]. joel g., drinking water quality, techniques and responsibilities, paris,(2003), 143. [15]. kherifi w., pollution and protection of the waters of lake mellah (north-east algeria) urban discharges: proposal for a principal collector, phd thesis, faculty of engineering sciences,university badjimokhtar annaba, (2016), 284. https://biblio.univ-annaba.dz/wpcontent/uploads/2019/05/these-kherifi-wahida.pdf. https://archipel.uqam.ca/736/1/m10067.pdf https://biblio.univ-annaba.dz/wp-content/uploads/2018/06/these-haouli%20zouina.pdf https://biblio.univ-annaba.dz/wp-content/uploads/2018/06/these-haouli%20zouina.pdf 74 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 74 79 veg eta ble fa ts influ en ce on the phy s i co -ch emi cal a nd sens or ial proper tie s of “ tel em ea” ch eese *mircea oroian1, ana leahu1 1faculty of food engineering, stefan cel mare university of suceava, romania m.oroian@fia.usv.ro *corresponding author received 17th february, accepted 24th march 2014 abstract: the aim of this paper is to study the influence of the milk fat substitution with vegetable fats into the “telemea” cheese, a romanian traditional cheese. the milk fat was substitute with vegetable fats in two percentages (50 % and 75%). the three samples have been made at industrial scale and their properties have been analysed from physicochemical (acidity, ph, fat, fat acidity) and sensorial properties: texture (consistency, compactness, firmness, cohesiveness, creaminess, gumminess and cheese severity), the taste (sweet, bitter, salty and sour), colour, smell, the exterior aspect and the section aspect. the vegetable fats are rich in vitamin e and carotenoids, vitamin a precursors; the three class of substances being natural antioxidants, which protect the organism from the free radical actions. the vegetable fats are the most stables fats used in food industry, keeping their properties for a greater time. the addition of vegetable fats to the “telemea” cheese do not change noticeable the values of the physicochemical and sensorial properties of cheese. keywords: milk, vegetable fats, substitution physicochemical, sensorial properties 1. introduction the “telemea” cheese is a type of cheese fermented into brine. cheese is a concentrated source of nutrients that come from milk used in it manufacture. the main nutrients are represented by milk casein, minerals, lipids and fat-soluble vitamins. “telemea” cheese is a nutritious and versatile food that can play an important role because it is a balanced nutritional food. actually this cheese is a product that is "popular" because of its positive and healthy image appreciated by consumers as having beneficial effects on health. this cheese, in many ways, it is an ideal food: nutritional flexible in use and application, the touch is appreciated by a large number of consumers. the product is reducing the illness risk, and it is considered relatively safe. the cheese is an unfermented or fermented products formed, in principal consisting casein which forms a protein matrix where are embedded fats, lactose, mineral salts and vitamins [1]. nowadays there is a growing tendency to consume low-fat cheeses around the word. despite the known benefits for human health, the reduction of the lipid content has a negative effect on the sensory characteristics of food products. consequently, some strategies can be used in the cheese-making process in order to improve the texture of low fat cheeses, being one example the addition of microparticulated whey proteins as fat replacers [2]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 m ir ce a o ro i an , ana l e a h u, ve g et ab l e f at s i n f lu e nc e on t he ph y s i c och e m i c al a nd s e ns o ri a l p r o pe r t i e s o f “ te l em e a ” ch e e s e, issue 1 2014, pag. 74 – 79 75 the contribution of the fat replacer to improve the textural and functional properties of cheeses, however, varies. such compounds can be derived from carbohydrate, protein, lipid and synthetic chemical groups. the use of carbohydrate based fat replacers in foods is of particular interest as they appear to closely impart the desired effects lost after the removal of fat. this occurs due to the bulking effect associated with moisture retention [3,4]. sensory evaluation is one means of measuring and quantifying the relationship between the sensory characteristics of food and its consumer references. techniques such as descriptive sensory analysis, cluster analysis and external preference mapping have been applied to cheese study. descriptive sensory analysis is a research tool to characterize the aromas and flavors in cheese [5,6]. the aim of this study is to find the influence of the milk fat substitution with vegetal fats for the production of “telemea” cheese by physicochemical a sensorial point of view. 2. materials and methods 2.1. materials milk, vegetable fats, streptococcus termophilus and lactobacillus bulgaricus lactic bacteria, purac 80 from enzymes and derivates, piatra neamt, romania. naoh, sulphuric acid, izoamilic acid reagents for purchased from sigma aldrich 2.2. experimental the samples have been prepared as follows: sample a – blanck sample made using milk, sample b – sample with 50% milk fat and 50% vegetable fats, and sample c – 25 % milk fat and 75 % vegetable fats. 2.3. cheese preparation the milk was pasteurized at 72 c for 2030 minutes, after it was added the lactic acid (purac 80) and lactic bacteria (streptococcus termophilus and lactobacillus bulgaricus). the coagulation process has been made into coagulation tub using chi-max rennet. the mix is kept for 30-40 minutes into the tub. after the coagulation takes place, the curd is divided into pieces. 2.4. physicochemical determinations the fat content has been measured using iso 1211/fil 1:2010 method [7], the moisture content has been determined using the oven method, the acidity was measured using an titrimetric method (with 0.1 n naoh), the fat acidity index was measured using a titrimetric method (with 0.1 n naoh), the ph was measured using a ph meter, 2.5. sensorial analysis the sensorial analysis is the only method used for appreciate the quality of “telemea” cheese. the three samples have been analysed by 5 tasters. the method used for the sensorial analysis consisted into unitary point scale. the sensorial properties analysed were: texture (consistency, compactness, firmness, cohesiveness, creaminess, gumosity and cheese severity), the taste (sweet, bitter, salty and sour), smell, the exterior aspect and the section aspect. 2.6. statistical analysis the statistical analysis was made using the next software packs: excel 2007. the variables were weighted with the inverse of the standard deviation of all objects in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 m ir ce a o ro i an , ana l e a h u, ve g et ab l e f at s i n f lu e nc e on t he ph y s i c och e m i c al a nd s e ns o ri a l p r o pe r t i e s o f “ te l em e a ” ch e e s e, issue 1 2014, pag. 74 – 79 76 order to compensate for the different scales of the variables. 3. results and discussions in table 1 are presented the physicochemical parameters analysed for the milk and for the three “telemea” cheese products made. comparing to the parameters of the milk used for the cheese production, cheese parameters have not suffered noticeable modifications. table1. physicochemical parameters of milk and “telemea” cheese ph acidity (°t) fat (%) fat acidity mg koh/g milk 6.57 15 3.2 11.2 sample a 5.10 170 42.8 14.5 sample b 5.23 180 42.4 13.4 sample c 5.26 170 42.2 14.5 as shown in the table above, all the parameters have not changed noticeable after the cheese processes occurred. the few changes were made to the parameter values indicating acidity (ph, acidity) changes due to the activity of lactic acid bacteria starter cultures who consumed milk lactose turning it into lactic acid, thus decreasing ph and increases acidity within certain limits. different additives applied to samples of “telemea” cheese influenced fermentative processes, but and the proteolysis, lipolysis, fermentation, protein coagulation, without changes in the physicochemical and sensorial properties of the product. regarding ph changes, for each sample analysed, decreases with the increasing of the acidity due to the metabolism of lactic acid by the bacteria from selected cultures. the decrease of ph was due to the effects of additives on the metabolism of lactic acid bacteria present in the starter culture which was inoculated into the milk. the acid value of the fat is an indicator of lipolysis, a greater acid number would indicate a high amount of free fatty acids in the final product, so a higher degree of glyciredes dissociation under the influence of the lipolytic enzymes present in the product. the taste, colour, consistency, flavour, whey elimination and section aspect of the three samples analyzed into the table 2. the taste of the cheese have not changed due to the substitution of the milk fat with vegetable fat, the taste is corresponding to the cheese. table 2. taste, colour, consistency, whey elimination and section aspect of the three samples of ‘telemea” cheese taste colour consistency flavour whey elimination section aspect sample a good, specific to cheese from cow milk white firm, is breaking easy without shatters good without whey uniform sample b good whiteyellow compact, elastic good without whey with random holes sample c good, salty whiteyellow firm, is breaking easy without shatters good without whey uniform with random holes food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 m ir ce a o ro i an , ana l e a h u, ve g et ab l e f at s i n f lu e nc e on t he ph y s i c och e m i c al a nd s e ns o ri a l p r o pe r t i e s o f “ te l em e a ” ch e e s e, issue 1 2014, pag. 74 – 79 77 regarding to the colour of the samples, it seems that the substation of the fat is change the colour, appearing yellow tones. the yellow tones come from the yellow pigments presented into the vegetable fats. the consistency is firm, compact, without shatters, the flavour is characteristics, neither one of the samples present whey elimination, while the section aspect is uniform, just the samples with vegetable fats present random holes. in the table 3 are presented the consistency, compactness, firmness, cohesiveness, creaminess, guminess and cheese severity of the “telemea” cheese samples. table 3. consistency, compactness, firmness, cohesiveness, creaminess, guminess and cheese severity of the “telemea” cheese samples sample consistency compactness firmness cohesiveness creaminess guminess cheese severity a 24.0 16 14.4 8.8 8.2 2.6 2.2 b 26.4 18.4 12 8.8 7.4 2.2 2.2 c 24.0 14.4 13.2 9.2 7.0 2.6 2.4 it cannot be observed differences according to the degree of substitution of the milk fat with vegetable fats between the consistency, compactness, firmness, cohesiveness, guminess and cheese severity of the “telemea” samples. in the figure 1 is presented the radial representation of consistency, compactness, firmness, cohesiveness, guminess and cheese severity of the “telemea” samples. fig.1. radial representation of consistency, compactness, firmness, cohesiveness, creaminess, guminess and cheese severity of the “telemea” cheese samples asample a, bsample b, csample c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 m ir ce a o ro i an , ana l e a h u, ve g et ab l e f at s i n f lu e nc e on t he ph y s i c och e m i c al a nd s e ns o ri a l p r o pe r t i e s o f “ te l em e a ” ch e e s e, issue 1 2014, pag. 74 – 79 78 for the gustatory evaluation we used the method of characteristics profile that includes a number of shades which states taste intensity using the following scale: 0 not noticeable, 1 to be significant 2 moderately perceptible 3 strong noticeable. the five panelists tried successively three samples of “telemea” cheese, so specifying the intensity of each sensation (components) taste: sweet, bitter, sour, salty using the scale of 0-3; the results are presented in table 4. table 4. gustatory properties of “telemea” cheese sample sweet bitter sour salty a 1 1 1.4 2.8 b 0.8 1 1.2 2.4 c 1.2 1 1.4 2.4 the sweet, bitter, sour and salty scores of the “telemea” cheese do not change noticeable. in figure 2 is presented the radial representation of the scores for sweet, bitter, sour and salty. fig.2. radial representation of the “telemea” cheese gustatory properties, asample a, bsample b, csample c the odor characteristic of the “telemea” cheese is of lactic fermentation and diacetyl flavor. in evaluating the odor and flavor of the cheese was used the rank ordering method which consists of: classifying samples by the intensity of the sensory characteristics, each taster evaluates the samples placed in an order determined performing a preliminary classification; all the results are processed in the same time. the smell and aroma scores of the “telemea” cheese are presented in the table 5. table 5. smell and aroma of “telemea” cheese sample smell and taste a 2.6 b 2.6 c 2.2 the substitution of the milk fat with vegetable fats does not influence the smell and aroma scores. in the case of the samples with 50% milk fat and 50% vegetable fats the smell and the taste obtains a lower score. in the figure 3 is presented the radial representation of the smell and aroma of “telemea” cheese. fig. 3. radial representation of smell and aroma of “telemea” cheese asample a, bsample b, csample c to assess the exterior aspect of the “telemea” cheese it was used the rank food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 m ir ce a o ro i an , ana l e a h u, ve g et ab l e f at s i n f lu e nc e on t he ph y s i c och e m i c al a nd s e ns o ri a l p r o pe r t i e s o f “ te l em e a ” ch e e s e, issue 1 2014, pag. 74 – 79 79 ordering method. the results are presented into table 6. table 6. exterior aspect of “telemea” cheese sample exterior aspect a 2.6 b 2.6 c 2.2 just in the case of the sample with 25% milk fat and 75% vegetable fats has a lower score for the exterior aspect. 4. conclusions the influence of substitution of milk fats with vegetable fats for the production of “telemea” cheese can be considered a good for its production. the physicochemical parameters of the three samples do not change noticeable with the degree of substitution. the samples maintained the same physicochemical parameters. from the sensorial points of view, the five tasters do not felt high differences between the samples. in conclusions the “telemea” cheese obtained with vegetable fats presents the same properties like the authentic one. 5. references [1] banu, c., milk industrial processing, ed. tehnica, bucharest, (1998) [2] zisu, b., shah, n. p., textural and functional changes in low-fat mozzarella cheeses in relation to proteolysis and microstructure as influenced by the use of fat replacers, preacidification and eps starter, international dairy journal, 15, 957-972, (2005) [3] fennema, o. r., food chemistry (3 ed.). new york: marcel dekker, inc., (1996) [4] meza, b. e., verdini, r. a., rubiolo, a. c., temperature dependency of linear viscoelastic properties of a commercial low-fat soft cheese after frozen storage, journal of food engineering, 109, 475-481, (2012) [5] drake, m. a., mcingvale, s., gerard, p. d., cadwallader, k. r., civile, g. v. development of a descriptive language for cheddar cheese. journal of food science, 66, 1422–1427, (2001) [6] zhang, x. y., guo, h. y., zhao, l., sun, w. f., zeng, s. s., lu, x. m., cao, x., ren, f. z., sensory profile and beijing youth preference of seven cheese varieties, food quality and preference, 22, 101-109, (2011) [7] iso 1211/fil 1:2010, milk, fat content determination doi: https://doi.org/10.4316/fens.2022.015 152 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 152 170 morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria olanike maria buraimoh1,5 temitope olawunmi sogbanmu2, olusola abayomi ojoomoniyi3, olumide afolabi4 , chinwe mary ganobi1 1department of microbiology, faculty of science, university of lagos, akoka, lagosnigeria. 2ecotoxicology and conservation unit, department of zoology, faculty of science, university of lagos, akoka, lagos nigeria. 3 department of microbiology, lagos state university, ojo 102101 lagosnigeria, olusola.ojo-omoniyi@lasu.edu.ng 4 department of biological sciences, faculty of basic and applied sciences, university of africa, toru-orua, bayelsa – nigeria. 5microbial diversity, bioinformatics and biotechnology research group, tetfund centre of excellence on biodiversity conservation and ecosystem management (tcebcem), university of lagos, lagos-nigeria. *corresponding author received 15th april 2022, accepted 25th july 2022 abstract: this study aimed to evaluate and characterize the pathogenic fungi as well as the influence of effluent from the kara abattoir on the ogun river, nigeria. in this study, the fungal population of the abattoir effluent, surface water and a species of fish (chrysichthys nigrodigitatus) from the ogun river were sampled and examined during the 2018 dry and rainy seasons using morphological, biochemical and molecular methods. several pathogenic fungi were characterized both in the effluent and fish gut during both seasons. penicillium sp., fusarium oxysporum, and some other unknown species were found in the fish gut during the rainy season. rhodotorula mucilaginosa was found both in the fish gut and effluent during the rainy season. talaromyces sp., was found in the fish gut and surface water during the rainy season. aspergillus flavus, aspergillus sp., talaromyces sp. were found in the fish gut and effluent during the dry season. penicillium citrinum was found in the fish gut and surface water during both seasons. saccharomyces sp., candida albicans, rhizopus stolonifer, mucor sp. and an unknown fungus were found in all three samples during both seasons. only aspergillus niger was present in the surface water during both seasons. the results of this study showed that anthropogenic activities at kara abattoir adversely impacts the ogun river, hence constituting potential environmental and public health risks. strategic advocacy campaigns, strong evidence informed policies or regulations, as well as provision of adequate facilities for effluent treatment are recommended to mitigate the current non-sustainable trends at the kara abattoir in ogun state, nigeria. this will support the efforts towards achieving the united nations sustainable development goal 3 (good health and wellbeing) and 14 (sustain life below water). key words: abattoir, contamination, effluent, fungi, river, sustainable development 1. introduction the impact of wastewater on receiving aquatic ecosystems in developing countries have been variously studied and adjudged to contribute immensely to poor water quality [1]. in developing countries, such effluents which arise from industries, domestic sources, aquaculture and slaughterhouses, among others are often discharged untreated into the receiving surface waters [2]. although, there are regulations and guidelines which set limits for effluent physico-chemical properties before discharge, attendant adverse impact still persists [2]. the direct nature of effluent inputs into recipient aquatic ecosystems can help in determining the source, nature of effluent and, potential adverse biological effects in receiving aquatic environments. the current study brings to fore the impact of activities at the mailto:olusola.ojo-omoniyi@lasu.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 153 kara abattoir (a popular slaughterhouse in lagos, southwest, nigeria), on the receiving aquatic ecosystem (ogun river in particular). the ogun river which is located in southwest nigeria, rises from oyo state near shaki at coordinates 8°41'0'n 3°28'0'e, flows through ogun state and discharges into the ikorodu axis of the lagos lagoon at coordinates 6.745589°n 3.34259°e (fig. 1). it is a source of water for drinking, cooking and bathing for the oyo as well as ogun state communities living close to the river. fig. 1: map of the ogun river showing settlement southwest nigeria (source: [3]) the kara abattoir is one of the approved centres for slaughtering and trading of cows in nigeria. alani et al. [1] estimated that more than half of the meat consumed daily in lagos comes from kara abattoir. the abattoir industry is an important segment of the livestock industry which provides meat (an important protein rich food item) for many nigerians and also provides jobs and income for many. lagos state has a population of over 20 million people. hence, it is expected that there would be a high demand for meat from such a large population. one of the major facilities meeting that demand is the kara abattoir. on a daily basis, animals are slaughtered and packaged at the abattoir; a process which involves butchering, removal of animal hides, intestine management, rendering, trimming and cleaning [4]. this process generates large volumes of wastewater. abattoir wastewater usually comprise oil, blood, organic and mineral solids, salts and chemicals added in the handling process [5, 6]. abattoir wastewater has also been shown to contain microorganisms such as bacteria and fungi capable of causing diseases in living organisms [7, 8, and 9]. the lack of facilities for effective wastewater management coupled with ignorance on the shortand long-term consequences of improper disposal of the wastewater are potential cursors of indiscriminate discharge of untreated wastewater into the ogun river. the consequences of this environmentally unsustainable practice, though considered food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 154 easy and convenient are multifaceted. the wastewater alters the physical, chemical and biological characteristics of the water. these changes disrupt the aquatic ecosystem and can lead to significant environmental and public health hazards [8]. researchers have reported high presence of microbes and faecal coliforms, as well as unfavourable physical and chemical characteristics such as high turbidity, low dissolved oxygen, high biological and chemical oxygen demand and high nutrient status in the ogun river around the kara abattoir and elsewhere [1, 2, 10, and 11]. while some researchers have reported bacterial contamination of the ogun river as a result of activities at the kara abattoir [11], this study aimed to isolate and identify indigenous fungi in the abattoir effluent, the ogun river surface water at the point of receipt of the effluent from the kara abattoir and in the gut of dominant fish (chrysichthys nigrodigitatus) sample from the ogun river near the abattoir. the results from this study would identify any potential risk posed to fisheries and humans based on the nature of fungi identified as well as contribute to the holistic assessment of the abattoir and its receiving water body in order to proffer targeted interventions to promote good health and wellbeing (united nations sustainable development goal (un sdg) 3) and sustain life below water (unsdg 14) from any fungal contamination at the ogun river from the kara abattoir effluents. 2. materials and methods sample collection the abattoir wastewater samples were collected at the point of its discharge into the ogun river with a sterile glass container. the surface water samples were collected at some distance away from where the abattoir wastewater was discharged into the river with a sterile glass container. live fish samples (chrysichthys nigrodigitatus) were also collected from the same points as the surface water. the samples were then properly labelled and placed in a cooler containing ice cubes which was transported to the laboratory at the department of microbiology, university of lagos, nigeria. the samples were collected during 2018 dry and rainy seasons. serial dilutions of each of the effluent and water samples were done aseptically in the laboratory. the fish sample was dissected and the gut was removed aseptically. approximately, 1 g of the gut was weighed and placed in 9 ml of sterile distilled water and shaken thoroughly, then serial dilution was carried out on the mixture up to 10 -5 dilution. thereafter, 10-2 and 10-3 dilutions were inoculated using spread plate method onto potato dextrose agar (pda) plates in duplicates. the plates were incubated at 27°c (for both the dry and rainy season samples) for a period of five (5) days. thereafter, the resulting moulds and yeast were sub-cultured on sabouraud dextrose agar and left for another five (5) days at the same temperature [12]. media preparation potato dextrose agar (pda) and sabouraud dextrose agar (sda) were prepared following manufacturer’s instruction. all microbiological media were sterilized at 121ºc for 15 minutes [12]. morphology and biochemical characterization of isolates macroscopic and microscopic examination of isolates were determined following standard methods [12, 13]. dna extraction and amplification of the its gene dna extraction a measurement of 80 mg (wet weight) of fungal cell that had been re-suspended in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 155 up to 200 µl of deionized water to a zr bashingbeadtm lysis tube was obtained. it was secured in a bead beater fitted with a 2.0 ml tube holder assembly (scientific industries’ disruptor genietm, cat. no. s6001-2 from zymo research corp.) and processed at maximum speed for 5 minutes. the zr bashingbeadtm lysis tube was centrifuged in a micro-centrifuge at ≥10,000 x g for 1 minute. up to 400 µl supernatant was transferred to a zymospintm iv spin filter (orange top) in a collection tube and centrifuged at 7,000 rpm (7,000 x g) for 1 minute. the base of the zymo-spintm iv spin filter was snapped off prior to use. 1,200 µl of fungal dna binding buffer was added to the filtrate in the collection tube of step four. thereafter, 800µl of the mixture from step five was transferred to a zymo-spintm iic column in a collection tube and centrifuged at 10,000 x g for 1 minute. the flow through was discarded from the collection tube and step six was repeated. then, 200 µl dna per-wash buffer was added to the zymo-spintm iic column in a new collection tube and centrifuged at 10,000 x g for 1minute. thereafter, 500 µl fungal dna wash buffer was added to the zymo-spintm iic column and centrifuged at 10,000 x g for 1 minute. the zymo-spintm iic column was transferred to a clean 1.5 ml micro-centrifuge tube and 100µl dna elution buffer was added directly to the column matrix. it was then centrifuged at 10,000 x g for 30seconds to elute the dna [14]. pcr amplification of the its gene and sequencing polymerase chain reaction (pcr) was carried out to amplify the its gene of the fungal isolates using the primer pair its-1 (5'-tccgtaggtgaacctgcgg-3') and its-4 (5'tcctccgcttattgatatgc-3'). the pcr reaction was carried out using the solis biodyne 5× hot firepol blend master mix. pcr was performed in 25 µl of a reaction mixture, and the reaction concentration was brought down from 5× concentration to 1× concentration containing 1× blend master mix buffer buffer (solis biodyne), 1.5 mm mgcl2, 200 µm of each deoxynucleoside triphosphates (dntp)(solis biodyne), 25pmol of each primer (biomers, germany) and 2 unit of hot firepol dna polymerase (solis biodyne). additional taq dna polymerase was incorporated into the reaction mixture to make a final concentration of 2.5 units of taq dna polymerase, proofreading enzyme, 2µl of the extracted dna, and sterile distilled water was used to make up the reaction mixture. thermal cycling was conducted in an eppendorf vapo protect thermal cycler (nexus series u.s.a.) for an initial denaturation of 95°c for 15 minutes followed by 35 amplification cycles of 30 seconds at 95°c; 1 minute at 58°c and 1 minute 30 seconds at 72°c. this was followed by a final extension step of 10 minutes at 72°c. the amplification product was separated on a 1.5% agarose gel and electrophoresis was carried out at 80v for 1 hour 30 minutes. after electrophoresis, dna bands were visualized by ethidium bromide staining. 100bp dna ladder was used as dna molecular weight standard [14, 15]. biochemical identification of yeast isolates biochemical tests which include gram reaction, carbohydrate fermentation and germ tube test (gtt) were used to characterize yeast isolates following the methods [12, 13]. germ tube test (gtt) this is a test used for differentiating candida albicans from other yeast species, when candida albicans is grown in human food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 156 or sheep serum at 37°c for 3 hours, they form a germ tube which can be detected with a wet koh film as filamentous outgrowth extending from their cells. approximately, 0.5mls of human serum was placed in testtubes, a little inoculum was then placed into it using a well flamed inoculating loop. the tubes were then incubated at 37°c for 3 hours after which slides were made then viewed under the optika microscope camera (magnification × 100  × 400) and results were taken [16]. data analysis in order to identify organisms, dna sequence data generated from this study was blasted against its/16s rrna type strain database on ncbi. threshold for identification was set with reference to [17], with modification to accommodate species with high congeneric sequence divergence range. an isolate was assigned to species level if the best matching reference species showed ≥98% homology and the next best matching reference species showed at least 0.8% less sequence homology. a strain or isolate was assigned to genus level when there was 95% to 98% homology to the best matching species, or when more than one sequence entry of several species from the same genus showed ≥98% homology. for sequences with 95% homology to the best match, an assessment of the congeneric sequence divergence range was carried out by blasting an arbitrarily chosen reference sequence of a type organism for the genus of interest against the type organisms’ its/16s rrna gene sequence database. the sequence divergence observed in this blast hits was used to adjudge the congeneric sequence divergence range and this range was used to determine the plausibility of the identification for sequences with 95% homology to blast’s best match. where homology fell below 95% and without the support of congeneric sequence divergence value, identification was considered unsuccessful. phylogeny was constructed to support the identification by its sequence using fast minimum evolution algorithm as deployed on ncbi and visualized on mega11 [18]. the counts of species and genus were utilized as an estimate of species and genus diversity respectively. in the estimation of the species diversity, unidentified isolates were considered as different from the identified species and therefore counted as different species. in the estimation of the genus diversity, as opposed to the treatment in the estimation of species diversity, unidentified isolates were removed from the analysis. other descriptive and inferential statistics were carried out on r using the follow r packages: base [19], reshape2 [20], dplyr [21], ggplot2 [22], ggpubr [23], qpcr [24], knitr [25, 26 and 27]. the data wrangling was achieved using reshape2 and dplyr; inferential statistics were carried out on r base; and data was visualized using ggplot2, ggpubr, qpcr and knitr. 3. results after five (5) days of incubation of the pda plates which was inoculated with diluted effluent, surface water and fish gut samples (obtained both in the wet and dry seasons), the isolates described in table 1 were observed. the moulds isolated were identified based on colonial morphology and microscopy at the end of the experiment, they were; aspergillus niger, aspergillus flavus, and other aspergillus sp., fusarium oxysporum, penicillium sp., rhizopus stolonifer and mucor sp. (table 2). the yeast isolates were identified based on biochemical test and colony morphology to be rhodotorula sp., saccharomyces sp. and candida albicans (table 3). pcr was done on six (6) isolates (3 from each season) in order to amplify their its gene (figure 2). the fungal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 157 species identified in the study are listed in table 4. in the abattoir effluents, saccharomyces sp., candida albicans, rhizopus stolonifera and mucor sp. were observed in both dry and wet seasons. aspergillus flavus was observed in the effluents only in the dry season while rhodotorula mucilaginosa was observed only in the wet season. in the surface water, saccharomyces sp., candida albicans, rhizopus stolonifera, mucor sp. penicillium citrinum and aspergillus niger were found present both in the dry and wet seasons. talaromyces sp. was found in the surface water only during the wet season. in the gut of fish samples collected from the ogun river surface water, saccharomyces sp., candida albicans, rhizopus stolonifera, mucor sp., penicillium citrinum and talaromyces sp. were identified both in the dry and wet seasons. fungal species found only in the dry season were aspergillus flavus and aspergillus sp. while penicillium sp., rhodotorula mucilaginosa, fusarium oxysporum and penicillium sp. were identified from the fish gut only in the wet season. during the dry season it was observed that two (2) isolates that were present in the fish gut were absent in the effluent. also, four (4) isolates that were in the fish gut and effluent, were absent in the surface water. more isolates were obtained in the fish than the effluent and surface water for both seasons. during the rainy season about seven (7) isolates were present in the fish gut which were not found in the effluent and surface water. there was only one (1) isolate that was present in the surface water and absent in both the fish gut and abattoir effluent for both the rainy and dry season. only one (1) isolate was peculiar to the fish gut and effluent during the rainy season. for the molecular identification of selected isolates, quality genomic dna was extracted from pure cultures and successful pcr amplification of the its region was achieved. sequences of the identified isolates have been submitted to the genbank and the accession numbers are provided in table 4. identification by blast (homology search) was supported by fast minimum evolution phylogenetic analysis (figure 3a and 3b) and congeneric sequence divergence range (table 5 and 6). isolates r9 and r62 possessed sequence homologies of 99.13% and 98.73% to rhodotorula mucilaginosa and aspergillus welwitschiae, respectively (table 4). however, these isolates did not fulfil the second condition for allocation to species: difference of at least 0.8% between the query sequence’s homology to the best match and its (query sequence) homology to the second-best match. the second-best match to r9 is rhodotorula alborubescens jcm 5352 (nr_153197.1) with a homology of 99.07%, and homology difference of 0.06% between the best and the second-best match. the uncertainty in identification created by this circumstance can also be seen in the phylogeny (figure 3b (d)), where r9 clusters with both r. mucilaginosa and r. alborubescens. similarly, the difference between the homology of r62 to a. welwitschiae cbs 139.54 (nr_137513.1) and its homology to aspergillus tubingensis nrrl 4875 (nr_131293.1) is 0.7% and it is also observed that r62 clusters with a. welwitschiae, a. foetidus and a. niger on the phylogeny (figure 3b [d]) this suggests that the its region alone cannot sufficiently identify these isolates to the species level. isolates c56 and r4 presented lower sequence homologies than the set threshold of 95% for genus level identification (table 4). c56 presented a homology of 93.03% to penicillium citrinum; the best match from the blast search. to justify the allocation of this isolate to the genus penicillium, the congeneric its sequence food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 158 divergence range for the genus penicillium was assessed through blast search: an independent blast search of genbank’s type organisms its database was carried out, using the standard organism p. citrinum nrrl 1841 (accession number: nr_121224.1) as the query its sequence. the result (table 5) showed that p. nothofagi cbs 130383 (nr_121518.1), the tenth blast hit, has a sequence similarity of 87.89% to p. citrinum nrrl 1841. this suggests a congeneric sequence divergence range of ~12% and therefore justifies the allocation of c56, which has a sequence divergence of ~7% to p. citrinum nrrl 1841, to the genus penicillium. similar analysis was carried out with respect to r4 which has a similarity of ~93% with talaromyces annesophieae and talaromyces domesticus, using talaromyces domesticus nrrl 58121 (accession number: nr_171608.1) as the query its sequence. the result (table 6) reveals that talaromyces unicus cbs 100535 (nr_157429.1), the 100th blast hit, has a sequence similarity of 84.97% with t. domesticus nrrl 58121. this suggests a congeneric sequence divergence range of ~15%, and thus justifies the allocation of r4, which has a sequence divergence of ~7% form t. domesticus nrrl 58121, to the genus talaromyces. a comparison of the identification by the different methods, showed slight disparity in the identification by morphology and identification by molecular method. species diversity is higher in the wet than in the dry season (figure 3a & amp; b). the fish gut (fg) presented the highest species diversity (figure 4a & amp; c. similar patterns were observed in the genus diversity, with fish gut possessing the highest genus diversity among studied locations and wet season presenting a higher diversity than dry season (figure 4 d, e &amp; f). however, none of the observed values were statistically significant at p = 0.05. aspergillus flavus and two other unidentified species of aspergillus (c55 and c58), were unique to the dry season, while fusarium oxysporum, rhodotorula sp., two unidentified species of talaromyces (r4 and r57) and three unidentified isolates (r6, r7 and r32) were unique to the wet season. no genus is unique to the dry season, meanwhile fusarium, rhodotorula and talaromyces were isolate from only the wet season (table 10). no unique species was isolated from the abattoir effluent (ef), meanwhile an unidentified species of aspergillus (r62) was found unique to the surface water (sw), while the fish gut present 5 unique species which include fusarium oxysporum, one unidentified species of talaromyces (r4) and three unidentified isolates (r6, r7 and r3). no unique genus was found in the abattoir effluent and surface water, while fusarium is the only identified unique genus isolated from the fish gut (table 10). comparatively, the fish gut showed marked difference from the abattoir effluent and surface water, in its fungal flora. common genera isolated from the wet and dry seasons were: aspergillus, candida, mucor, penicillium, rhizopus and saccharomyces which include candida albicans and rhizopus stolonifera. c53 is an unidentified isolate that is common to all locations and seasons. common genera isolated from all sampled locations (abattoir effluent, shallow water and fish gut) are: aspergillus, candida, mucor, rhizopus and saccharomyces. these also include candida albicans and rhizopus stolonifera (tables 1, 3 & 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 159 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 160 table 2 identification of fungal isolates based on microscopy and colonial morphology isolate microscopy image colony images identification of isolates based on microscopy and colonial morphology 1-r4 penicillium sp. 2-r6 unknown species 3-r7 unknown species 4-r12 fusarium oxysporum 5-r18 penicillium sp. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 161 6-r32 unknown species 7-c45 aspergillus flavus 8-r47 rhizopus stolonifera 9-r48 not available 10-c55 aspergillus sp. 11-c56 and r56 penicillium citrinum food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 162 12-c58 and r57 talaromyces sp. 13-c61 aspergillus niger 14-c62 aspergillus flavus 15-r62 aspergillus niger table 3 biochemical characterization of yeast isolates samples gram stain gf sf lf gtt probable identity 1-r2 +ve +ve +ve +ve -ve saccharomyces sp. 2-r9 +ve -ve -ve -ve -ve rhodotorula mucilaginosa 3-r11 +ve +ve +ve +ve +ve candida albicans 4-c34 +ve +ve +ve +ve -ve saccharomyces sp. 5-c53 +ve +ve +ve +ve -ve unknown species. 6-r61 +ve +ve +ve +ve -ve saccharomyces sp. legend: gfglucose fermentation; sfsucrose fermentation lflactose fermentation gttgerm tube test +ve = positive -ve = negative food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 163 table 4 molecular identification of selected isolates. isolate identification sequence accession numbers description percentage identity reference strain isolate c53 no identification c56 penicillium sp. (closest species: penicillium citrinum) **93.03% nr_121224.1 mk542006 c58 aspergillus sp. (closest species: 1. aspergillus austwickii or 2. aspergillus aflatoxiformans) 97% 1. nr_171607.1 2. nr_171606.1 mk542007 r4 talaromyces sp. (closest species: 1. talaromyces annesophieae or 2. talaromyces domesticus) **92.88% 1. nr_170732.1 2. nr_171608.1 mk542008 r9 rhodotorula sp. (closest species: rhodotorula mucilaginosa) *99.13% nr_073296.1 mk542009 r62 aspergillus sp. (closest species: aspergillus welwitschiae) *98.73% nr_137513.1 mk542010 **identification requires congeneric divergence range support. *the difference in percentage similarity between best and second-best matches is lower than 8% table 5 assessment of the congeneric sequence divergence range in penicillium sp. blast hit position description (its region from type material) accession percentage identity 1 penicillium citrinum nrrl 1841 nr_121224.1 100.00% 2 penicillium hetheringtonii cbs 122392 nr_111482.1 99.40% 3 penicillium malacaense nrrl 35754 nr_121344.1 93.43% 4 penicillium terrigenum cbs 127354 nr_121515.1 88.40% 5 penicillium copticola cbs 127355 nr_121516.1 88.25% 6 penicillium gallaicum cbs 167.81 nr_103657.2 88.03% 7 penicillium raphiae cbs 126234 nr_121511.1 88.24% 8 penicillium anatolicum nrrl 5820 nr_121225.1 88.13% 9 penicillium argentinense cbs 130371 nr_121523.1 87.99% 10 penicillium nothofagi cbs 130383 nr_121518.1 87.89% table 6 assessment of the congeneric sequence divergence range in talaromyces sp. blast hit position description (its region from type material) accession percentage identity 1 talaromyces domesticus nrrl 58121 nr_171608.1 100.00% 2 talaromyces annesophieae cbs 142939 nr_170732.1 99.45% 3 talaromyces pratensis nrrl 62170 nr_165529.1 99.26% 4 talaromyces adpressus cbs 140620 nr_171595.1 98.90% 5 talaromyces malicola nrrl 3724 nr_165531.1 98.89% 96 talaromyces tiftonensis nrrl 62264 nr_155920.1 84.41% 97 talaromyces endophyticus accc 39141 nr_171599.1 87.71% 98 talaromyces yelensis cbs 138209 nr_145183.1 84.99% 99 talaromyces emodensis cbs 100536 nr_137077.1 84.88% 100 talaromyces unicus cbs 100535 nr_157429.1 84.97% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 164 table 7 fungal isolates from kara abattoir effluent, fish gut and surface water of ogun river fungi isolate dry season wet season et fg sw et fg sw saccharomyces sp. + + + + + + talaromyces sp. (r4) + rhodotorula sp. + + candida albicans + + + + + + fusarium oxysporum + aspergillus flavus + + rhizopus stolonifera + + + + + + mucor sp. + + + + + + aspergillus sp. (c55) + + penicillium sp (c56) + + + + aspergillus sp. (c58) + + talaromyces sp. (r57) + + aspergillus sp. (r62) + + r6 (unknown) + r7 (unknown) + r32 (unknown) + c53 (unknown) + + + + + + legend: eteffluent fgfish gut swsurface water + present absent fig. 2. dna extraction and amplification lane 1= dna marker lane 2 = negative control lane 3= c53 lane 4= c56 lane 5 = c58 lane 6 = r4 lane 7= r9 lane 8= r62 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 165 fig. 3a. molecular identification of selected fungal isolates: a. isolate c56, b. isolate c58 fig. 3b. molecular identification of selected fungal isolates: c. isolate r4, d. isolate r9, e. isolate r62. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 166 legend: eteffluen, fgfish gut, swsurface water fig. 4. genus and species diversities: a. species diversities in different seasons disaggregated by location, b. species diversities in different seasons, c. species diversities in different locations, d. genus diversities in different seasons disaggregated by location, e. genus diversities in different seasons, f. genus diversities in different locations. 4. discussion the results from this study showed that pathogenic fungi of different species were present in the kara abattoir effluent as well as in the surface water and fish gut from the ogun river. the presence of fungi in kara cow market effluent was also reported [9], who reported the presence of aspergillus niger, fusarium sp. and trichoderma sp. in the kara cow market effluent. [5] reported the presence of different species of fungi, namely trichoderma sp. trichophyton sp. aspergillus sp. and scedosporium sp. in wastewater from lafenwa abattoir in abeokuta, ogun state nigeria. aspergillus sp., coccidioides sp., trichophyton sp. and scedosporium sp. were also reported to be present in surface water receiving lafenwa abattoir wastewater [5]. this corroborated the findings of this study. in this study, there were similarities and variations in the fungal species found in the effluent, in the surface water and those found in the gut of the fish samples. also, there were variations in the fungi species identified during rainy and dry seasons. the results showed that greater species variation was observed during the rainy season than during the dry season. this suggested climatic influence on fungal population dynamics. increase in fungi variety during rainy season can be explained by the fact that the lower temperatures and higher humidity during rainy seasons favour propagation and growth of fungi in the respective media. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 167 abattoir effluents are rich in nutrients and organic matter, hence the presence of microbes such as bacteria and fungi which facilitate biodegradation of organic molecules. some of these fungi are known to be pathogenic and can cause various types of diseases. the release of untreated effluent into the ogun river exposes the water and the living organisms in it to various chemical and biological agents of pollution already present in the wastewater. this practice constitutes several environmental and public health hazards. rhodotorula mucilaginosa, for example was observed in this study to be present in the effluent and fish gut during the wet season. rhodotorula sp. are considered an emerging pathogen and were reported as the main cause of invasive fungal infections by non-candida yeasts in china [28, 29]. rhodotorula sp. were also reported as the most common microorganism isolated from the hands of hospital employees and patients and the third most commonly isolated yeast from blood cultures [29, 30]. rhodotorula sp. have also been reported to cause meningeal, skin, ocular, peritoneal and prosthetic joint infections in humans whether immune-compromised or not [31]. aspergillus flavus were also identified in the effluents and in the fish gut during the dry season while aspergillus niger was observed during the wet season. asperillus flavus is well known to produce aflatoxins (a toxic carcinogen) in the seeds of crops before and after harvest. consequently, when spores of a. flavus are inhaled by humans, this leads to aspergillosis (a health condition often characterised by lung infections and allergic reactions) especially in people with weak/suppressed immunity [32]. aspergillus niger also produces mycotoxins (fumonisins and ochratoxins) which have been reported to be carcinogenic. when surface water becomes contaminated with such pathogenic fungi, they constitute risks to the living organisms in the water, to man and animals dependent on the water body, and even to crops (if such water is used in irrigation). for example, if such contaminated water is used for irrigation, crops become exposed to fungal contamination with consequences on food security and safety. the problem of plant pathogen in irrigation water has been noted as a growing challenge. pathogens in irrigation water are often the primary/sole source of inoculum for diseases in crops [33]. the channels through which diseases can be spread from a fungi polluted water body to other living organisms are numerous (it could be through direct consumption by man/animals, consumption of contaminated fish /other aquatic organisms, use in irrigation of crops, domestic uses, and so on). it was also observed from the study that the batch slaughter system (in which animals are killed and processed sometimes on the bare floor or on corrugated roofing sheets) employed in kara abattoir is highly unhygienic and not environment friendly. a more eco-friendly slaughter systems should be adopted in the abattoir. in a study carried out previously to compare contamination levels in three different abattoir systems (namely: line, slaughter slab and batch), their findings proved that the batch slaughter system was least healthy/hygienic as it brought greater levels of contamination to the floor, butchering knives, workers’ hands and effluent water. high level of contamination in the batch slaughter system is due to the fact that the system compromises standard operating procedures for running abattoirs recommended by the world health organisation [34]. the release of kara abattoir wastewater into the ogun river has gone on for years despite several reports emphasising the negative effects of such practice [1, 9 and 11]. a careful analysis of the issue reveals that ignorance on the part of people, weak food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 168 policies, and poor infrastructure/facilities have caused this ecologically harmful practise to go on unhindered. to ensure meat is provided for people without threatening public health directly/indirectly through the process, we recommend the strategic advocacy/enlightenment, strong policy/regulation, and state of the art facility/infrastructure for abattoir management. first, enlightenment campaigns should be designed and executed to reach the people carrying out these activities as knowledge on abattoir best practises and on the harms caused by bad practises would stimulate a willingness to change and adjust; such willingness is an important precursor for change. also, since people’s practice are usually guided by set policies/standards, government must ensure that policies based on scientific evidence are crafted and implemented to regulate the slaughter houses and ensure adherence to best practises. it is important that government provide strong policies/regulations that compels provision of sufficient facilities/infrastructures that guarantees sustainable waste management development. facilities such as slaughter floor, lairage, chiller, tripe room, power, water supply, effluent disposal, hide and skin processing should be provided so that the abattoir can function in harmony with the sustainable development goals. lack of adequate facilities and poor management of abattoirs besides leading to environmental pollution could also lead to meat contamination and occupational hazard to the workers on the site [35]. it must also be stated that though sufficient facility, sound policies and strong advocacy require funds to institute, such cost must be seen as an investment because it yields tremendous gains for man and the environment. besides preventing negative outcome in the environment and health, proper abattoir management has the potential of also yielding more financial gains and making abattoir management more economically and socially sustainable (thus cutting across the three arms of sustainability: planet, profits and people)[36]. meanwhile, by properly managing waste and integrating secular economic principles in abattoir management, maximal value will be obtained from every by-product/left-over of the abattoir processes. hence, there is need to overlook the short-term cost of eco-friendliness and focus on the long-term gain which far outweighs the cost [36, 37]. 5. conclusion in conclusion different species of pathogenic fungi were isolated from kara abattoir wastewater, ogun river surface water and fish from the river. some of the fungi species identified in this study were aspergillus flavus, aspergillus niger, rhodotorula mucilaginosa, saccharomyces sp., candida albicans, rhizopus stolonifera and mucor sp., penicillium sp., fusarium oxysporum, talaromyces sp. and penicillium citrinum. many of these species are known to cause various forms of diseases in living organisms, thus making the river unsafe for those dependent on it for various activities. in order to remedy this situation and protect the environment in this and other similar cases we recommend strategic advocacy campaign on eco-friendliness and harmony with nature. 6. statements and declarations funding: the authors declare that no funds, grants, or other support were received during the preparation of this manuscript. this is a self-funded research by the listed authors. competing interests: the authors have no competing interest to disclose because this study was self-sponsored by all the listed authors as a collaborative study. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 169 authors did not receive support from any organization for the conduct of the study and the submitted manuscript. author contributions: all authors contributed to the study conception and design as well as preparation, data collection and analysis. all authors contributed equally to all the previous versions of the manuscript. all authors read and approved the final manuscript. dataset: all dataset are available in genbank and ncbi repository, the accession numbers for isolates are displayed and trackable. (www.ncbi.nlm.nih.gov/genome). ethical consent: ethical approval is not required for this type of work in nigeria, since we did not test any material on human and animal subjects. consent to participate: the consent to participate in this study was given by each author. consent to publish: the consent of each author was obtained prior to the commencement of the publishing process. 7. references [1]. alani, r., alo, b., ukoakonam, f. preliminary investigation of the state of pollution of ogun river at kara abattoir, near berger, lagos. international journal of environmental science and toxicology, 2(2): 11-23. (2014). [2]. olaniran, e. i., sogbanmu, t. o., saliu, j. k. biomonitoring, physico-chemical and biomarker evaluations of abattoir effluent discharges into the ogun river from kara market, ogun state, nigeria using clarias gariepinus. environmental monitoring and assessment 191: 44. (2019). [3]. fashae, o. a., faniran, a. downstream morphologic characteristics of alluvial section of lower river ogun, nigeria. j. environmental geography 8(1-2): 1-10. (2015). [4]. elemile, o. o., raphael, d. o., omole, d. o., oloruntoba, e. o., ajayi, e. o., ohwavborua, n. a. assessment of the impact of abattoir effluent on the quality of groundwater in a residential area of omu‑aran, nigeria. environmental sciences europe, 31:16. (2019). [5]. adebowale, o. o., adeyemo, o. k., jayeola, a. o., ojo, o. e., adebowale, o., kehinde o. o., kperegbeyi, e. a. microbial characterization of the waste water from a major abattoir and its receiving surface water in abeokuta, nigeria. proceedings of the xvth international congress of the international society for animal hygiene, vienna, austria, 3-7 july 2011, 3: 1131-1134. . (2011). [6]. akange, e. t., chaha, j. a., odo, j. i. impact of wurukum abattoir effluent on river benue nigeria, using macroinvertebrates as bioindicators. int. j. aquac, 6(22):1–11. (2016). [7]. adesemoye, a., opere, b., makinde, s. microbial content of abattoir wastewater and its contaminated soil in lagos, nigeria. african journal of biotechnology 5:1963-1968. (2006). [8]. nafarnda, w. d., ajayi, i. e., shawulu, j. c., kawe m. s., omeiza, g. k., sani, n. a., tenuche, o. z., dantong, d. d., tags, s. z. bacteriological quality of abattoir effluents discharged into water bodies in abuja, nigeria. international scholarly research notices (isrn) vet. sci. volume 2012 article id: 515689. (2012). [9]. sogbanmu, t. o., sosanwo, a. a., ugwumba, a. a. a. histological, microbiological, physicochemical and heavy metals evaluation of effluent from kara cow market, ogun state, nigeria in guppy fish (poecilia reticulata). the zoologist 17: 54-61. (2019). [10]. balogun, s., ayangbenro, a., ogunsanya, g., azeez, a., muonaka, c., ihongbe, m. bacteriological pollution indicators in ogun river flowing through abeokuta metropolis. journal of science and technology 36: 54-63. (2016). [11]. adesina, a. o., ogunyebi, a. l., fingesi, t. s., oludoye, o. o. assessment of kara abattoir effluent on the water quality of ogun river, nigeria. journal of applied sciences and environmental management 22(9):1465-1470. (2018). [12]. gerhardt, p., murray, r. g. e., costilow, r. n., nester, e.w., wood, w. a., krieg, n. r., phillips, g. b. manual of methods for general bacteriology. american a. society for microbiology (1981) pp. 524. [13]. smith, g. smith’s introduction to industrial mycology 7th edition (eds.) a. h. s. onions, d. allsopp, h. o. w. http://www.ncbi.nlm.nih.gov/genome https://www.cabdirect.org/cabdirect/search/?q=do%3a%22animal+hygiene+and+sustainable+livestock+production.+proceedings+of+the+xvth+international+congress+of+the+international+society+for+animal+hygiene%2c+vienna%2c+austria%2c+3-7+july+2011%2c+volume+3%22 https://www.cabdirect.org/cabdirect/search/?q=do%3a%22animal+hygiene+and+sustainable+livestock+production.+proceedings+of+the+xvth+international+congress+of+the+international+society+for+animal+hygiene%2c+vienna%2c+austria%2c+3-7+july+2011%2c+volume+3%22 https://www.cabdirect.org/cabdirect/search/?q=do%3a%22animal+hygiene+and+sustainable+livestock+production.+proceedings+of+the+xvth+international+congress+of+the+international+society+for+animal+hygiene%2c+vienna%2c+austria%2c+3-7+july+2011%2c+volume+3%22 https://www.cabdirect.org/cabdirect/search/?q=do%3a%22animal+hygiene+and+sustainable+livestock+production.+proceedings+of+the+xvth+international+congress+of+the+international+society+for+animal+hygiene%2c+vienna%2c+austria%2c+3-7+july+2011%2c+volume+3%22 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojo-omoniyi, olumide afolabi, chinwe mary ganobi, morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag. 152– 170 170 eggins. publ. pitman press, bath. sjöstrom, (1981). pp.125-152. [14]. guter, r. l., koshland jr. d. e. the molecule of the year. science, 246: 1543-1544. (1990). [15]. solanki, g. polymerase chain reaction. international journal of pharmaceutical research 2(3):98-102. (2012). [16]. pincus, d. h., orenga, s., chatellier, s. yeast identification-past, present and future methods. medical mycology 45(2):97210. (2007). [17]. wang, x., fu, y. f., wang, r. y., li, l., cao, y. h., chen, y. q., zhu, l. p. identification of clinically relevant fungi and prototheca species by rrna gene sequencing and multilocus pcr coupled with electrospray ionization mass spectrometry. plos one, 9(5): e98110. (2014). [18]. tamura, k., stecher, g., kumar s. mega11: molecular evolutionary genetics analysis version 11. molecular biology and evolution 38:3022-3027. (2021). [19]. r core team r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url https://www.r-project.org/. (2021). [20]. wickham, h. reshaping data with the reshape package. journal of statistical software, 21(12): 1-20. url http://www.jstatsoft.org/v21/i12/. (2007). [21]. wickham, h., françois r., henry, l., müller, k., dplyr: a grammar of data manipulation. r package version 1.0.7. https://cran.r-project.org/package=dplyr (2021). [22]. wickham, h. ggplot2: elegant graphics for data analysis. springer-verlag new york. (2016). [23]. kassambara, a. ggpubr: 'ggplot2' based publication ready plots. r package version 0.4.0. https://cran.r-project.org/package=ggpubr (2020). [24]. spiess, a. qpcr: modelling and analysis of real-time pcr data. r package version 1.4-1. https://cran.r-project.org/package=qpcr (2018). [25]. xie, y. knitr: a comprehensive tool for reproducible research in r. in: victoria stodden, friedrich leisch and roger d. peng, (editors) implementing reproducible computational research. chapman and hall/crc. isbn 9781466561595. (2014). [26]. xie, y. dynamic documents with r and knitr. (2nd edition). chapman and hall/crc. isbn 978-1498716963. (2015). [27]. xie, y. knitr: a general-purpose package for dynamic report generation in r. r package version 1.36. (2021). [28]. xiao m, chen sc-a, kong f, fan x, cheng j-w, hou x, et al. five-year china hospital invasive fungal surveillance net (chifnet) study of invasive fungal infections caused by non-candidal yeasts: species distribution and azole susceptibility. infect. drug resist. 11:1659–67. (2018). [29]. jarros, i. c., veiga, f. f., corrêa, j. l., barros, i., gadelha, m. c., voidaleski, m. f., pieralisi, n., pedroso, r. b., vicente, v. a., negri, m., svidzinski, t. microbiological and virulence aspects of rhodotorula mucilaginosa. excli journal, 19: 687–704. (2020). [30]. gomez-lopez, a., mellado, e., rodriguez-tudela, j. l., cuencaestrella, m. susceptibility profile of 29 clinical isolates of rhodotorula spp. and literature review. j antimicrob. chemother. 55: 312–316. (2005). [31]. fernanda, w., luciano, g. epidemiology of rhodotorula: an emerging pathogen. interdisciplinary perspectives on infectious diseases, 465717:7. (2012). [32]. centers for disease control and prevention fungal diseases: aspergillosis. retrieved on august 10, 2021 from: https://www.cdc.gov/fungal/diseases/aspergillosis/i ndex.html. (2021). [33]. hong, c. x., moorman, g. w. plant pathogens in irrigation water: challenges and opportunities. critical reviews in plant sciences, 24: 181-208. (2005). [34]. ayode, o., olayioye, e. o. microbiological assessment of housekeeping practices and environmental impact of selected abattoirs in lagos and ogun states of nigeria. journal of applied biosciences. 99: 9363 – 9372. (2016). [35]. cook, e. a. j., de glanville, w. a., thomas, l. f., kariuki, s., bronsvoort, b. m., fevre, e. m. working conditions and public health risks in slaughterhouses in western kenya. bmc public health 17: 14. (2017). [36]. united nations environment program making peace with nature: a scientific blueprint to tackle the climate, biodiversity and pollution emergencies. https://www.unep.org/resources/making-peacenature (2021). [37]. u.s environmental protection agency. science and ecosystem support division (sesd) operating procedure waste water sampling. athens, georgia https://www.epa.gov/sites/default/files/201506/documents/wastewater-sampling.pdf (2013). https://www.r-project.org/.%20(2021) http://www.jstatsoft.org/v21/i12/.%20(2007) https://cran.r-project.org/package=dplyr https://cran.r-project.org/package=ggpubr https://cran.r-project.org/package=qpcr https://www.cdc.gov/fungal/diseases/aspergillosis/index.html https://www.cdc.gov/fungal/diseases/aspergillosis/index.html https://www.unep.org/resources/making-peace-nature https://www.unep.org/resources/making-peace-nature https://www.epa.gov/sites/default/files/2015-06/documents/wastewater-sampling.pdf https://www.epa.gov/sites/default/files/2015-06/documents/wastewater-sampling.pdf doi: https://doi.org/10.4316/fens.2021.010 81 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2021, pag. 81 90 effect of fermentation on the quality of flour and biscuit from cashew kernel flour *bleou jean jaurès touzou (1) , doudjo soro (2) , soronikpoho soro (1) , kouadio ernest koffi (1) 1 laboratory of biochemistry and food science, training and research unit biosciences, félix houphouëtboigny university of abidjan, ufhb 01 bp v 34 abidjan, côte d’ivoire, bleoutz@gmail.com 2 food science and environmental chemical processes, polytechnic national institute, félix houphouët-boigny (inphb), bp 1313 yamoussoukro, côte d’ivoire. *corresponding author received 28th december 2020, accepted 30th march 2021 abstract: the purpose of this paper is to investigate the effect of fermentation on the quality of cashew kernel flour for biscuit making. fermented and unfermented cashew kernels flours produced were used to substitute wheat flour. functional properties of flours were evaluated. chemical and sensory characteristics of various biscuits obtained were used to compare them. fermentation improved water absorption capacity, foaming and emulsification properties but reduced (p < 0.05) oil absorption capacity of cashew kernel flour. the analysis of nutritive values showed that fermentation increased the protein and fat contents respectively from 34.10±0.41 to 38.67±0.4 and from 28.16±0.07 to 31.02±0.18. substitution of wheat flour by cashew kernels flours increase protein, fat, fiber and ash contents of biscuits. biscuits from cashew kernel flour overall acceptability decreased with fermented cashew kernel flour incorporation. negative correlation was observed between brown colour score and overall acceptability also between aroma (cashew kernel aroma and fermented cashew kernel aroma) scores and overall acceptability of biscuits. panelists accepted regarding overall acceptability, biscuits containing 100 % unfermented cashew kernel flour and those containing 40 % of fermented cashew kernel flour. substitution of wheat flour by unfermented cashew kernel flour and 40 % by fermented cashew kernel flour would improve nutritive values of biscuits and cashew kernel consumption locally. keywords: fermentation, cashew kernels flours, substitution, quality, biscuits. 1. introduction biscuits are a widely consumed food produced from wheat flour, sugar and fat [1]. however, the wheat imported by subsaharan african countries is expensive and the proteins, fibers and minerals content of the wheat flour commonly used (t45) for biscuit production is relatively low. research is being carried out to reduce the cost of biscuit production and to improve the functionality of biscuits by modifying their nutritional composition. this involves the use such as of non-wheat flour to improve nutritive quality of biscuits and overcome the high cost problems of wheat flour faced by importing countries whose climates are unfavorable to wheat growing [2, 3]. acceptable biscuits have been produced from blends of fonio, wheat and cowpea; millet and pigeon pea; wheat and plantain; maize and pigeon pea [4; 5; 6; 7]. however, increasing the levels of cashew nut paste in the biscuits resulted in a significant decrease in the sensory attribute scores of the biscuits [8]. [9] also made similar findings. cashew nuts are the fruit of the cashew tree (anarcadium occidentale) which is a widely grown in tropical region. cashew trees are widely grown in several regions of côte d’ivoire, which is the world's largest producer of raw cashew nuts with 25 % of world market share [10, 11]. mailto:bleoutz@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 82 fermentation helps enhance the taste and aroma of food products [12] this study assessed the effects of fermentation on the quality of flour prepared from cashew kernel. the quality of biscuits developed from composite flours containing cashew kernel fours and wheat flour were also investigated. 2. material and methods material the small pieces of cashew kernel were obtained by mechanical shelling at the fassou cashew factory based in yamoussoukro (côte d'ivoire). food ingredients including wheat flour, sugar, margarine, eggs, and baking powder were purchased from a supermarket in yamoussoukro (côte d’ivoire). methods production of unfermented cashew kernel flour defatted cashew kernel flour was produced according to the modified method proposed by [13]. the cashew kernels were cleaned of plant debris and unhealthy kernels. the oil of the previously treated kernels was extracted twice with n-hexane at a ratio of 1: 1 (w / w). the oilcake were dried at 70 ° c in an oven for 12 hours in order to remove traces of hexane before being reduced to flour using a hammer mill containing a 150 μm mesh. the resulting flour was stored in polyethylene bags at room temperature. production of fermented cashew kernel flour the sorted kernels are fermented using the modified method described by [14]. almonds (1 kg) are boiled at 100° c with distilled water (w / v) for 30 minutes. they are wrapped in plantain leaves to be fermented for 72 hours. the fermented kernels are dried in a ventilated oven at 65 ͦ c for 48 hours. the fermented kernels oil was then extracted twice with n-hexane at a ratio of 1: 1 (w / w) as previously described. the oilcake were dried at 70 ° c in an oven for 12 hours in order to remove traces of hexane before being reduced to flour using a hammer mill containing a 150 μm mesh. the flour is stored in polyethylene bags at room temperature. preparation of composite flour composites flours are prepared by substituting the wheat flour with unfermented cashew kernels flour or fermented cashew kernels flour in the ratio of 100:0, 80:20, 60:40, 40:60, 20:80 and 100:0 w/w. functional properties analysis of flours bulk density was determined by the method described by [15]. the water absorption capacity (wac) was determined by the method of [16]. the oil absorption capacity (oac) was determined according to the method of [17]. emulsion activity (ea) was carried out according to the method described by [18]. the foam capacity (fc) was determined by the method of [19]. biscuit production the recipe adopted after preliminary experimentation was as follows: flour (200 g), margarine (100 g), powdered sugar (100 g), whole egg, baking powder (4 g), liquid milk (100 g/ l) (50 ml). the margarine and the powdered sugar was creamed together at medium speed until light and fluffy appearance is formed. the flour is added to the cream and then homogenized. the egg and liquid milk were added to the cream. the flour previously mixed with baking powder and then sieved is introduced last. the ingredients are mixed and the dough obtained is prepared using a pocket. the dough pieces are placed on baking paper in trays and baked for 15 minutes in an oven preheated to 180 ° c. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 83 nutritional composition of biscuits the moisture content and fat content of the various biscuits samples were performed using [20]. the crude protein and fat contents were respectively determined by estimating the nitrogen content using the kjedahl and using the soxhlet apparatus methods [21]. ash and carbohydrate were respectively analysed using the method of [22] and [23]. biscuit samples were analysed for fiber content according to the method described in [24]. energy values were determined by methods described by [25]. sensory evaluation the descriptive test of the biscuits was carried out using the method described by [26]. fifteen (15) trained panelists composed of women and men judged the samples. the method consists to quantify the appropriate descriptors (colour, texture, taste and aroma) according to a category 9 scale. all the instructions were provided to panelists before evaluation [27]. biscuits were also evaluated by 60 panelists for overall acceptability on a 9-point hedonic scale (1 – extremely dislike, 2 – dislike very much, 3 – dislike moderately, 4 – dislike slightly, 5– neither like nor dislike, 6 – like slightly, 7 – like moderately, 8 – like very much, 9 – extremely like) [27]. different types of biscuit samples were presented in random order coded with random three-digit numbers on white plastic dishes, and served at the day of manufacture as well as under normal illumination (daylight). water was provided to the panelists in order to cleanse their palates in between every sample. statistical analysis statistical analysis was carried out using statistica 7.1. soft software. the newmankeuls multiple means comparison test was used to verify differences between the samples. p < 0.05 was set as the criterion of significance. 3. results and discussion the effect of cashew kernel fermentation and wheat flour substitution by cashew kernel flours on the functional properties is presented in table 1. water absorption capacity (wac), oil absorption capacity (oca), emulsifying activity (ea) and foaming capacity (fc) were significantly differs in the types of flours (table 1). fermentation of cashew kernel for 72 h decreased the bulk density of the cashew kernel flour. [28] reported that bulk density depends on the combined effects of interrelated factors such as the intensity of attractive inter-particle forces, particle size, and number of contact points. the result is similar to that of [29] who reported that germination and fermentation leads to decrease in the bulk density of foods. the decrease in bulk density of the fermented flour would improve the fluidity of the slurry. fermentation would therefore be a useful method for the preparation of weaning feeds [30]. fermentation significantly (p < 0.05) increased the water absorption capacity of cashew kernel flour. also, wheat flour substitution by cashew kernels flours increased water absorption capacity of composite flours. the amount and nature of hydrophilic constituents such as proteins and to some extent the ph value favorably influence the water absorption capacity of a flour [31]. the correlation analysis revealed in table 2 supported that the proteins content and water absorption capacity were highly positive correlated (r = 0.98 p < 0.05). proteolytic activity during fermentation may have increased the polar groups, which improved hydrophilicity of the cashew kernel flour proteins. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 84 in contrast, fermentation decreased significantly (p > 0.05) the oil absorption capacity of cashew kernel flour. table 1 functional properties of flours values are means of triplicate replications. means within a column with different superscript were significantly (p < 0.05) different. ucf: unfermented cashew kernel flour; ucf80: 20 wheat flour + 80 unfermented cashew kernel flour; ucb60: 40 wheat flour + 60 unfermented cashew kernel flour; ucb 40: 60 wheat flour + 40 unfermented cashew kernel flour; ucb20: 80 wheat flour + 20 unfermented cashew kernel flour; fcb: fermented cashew kernel flour; fcb80: 20 wheat flour + 80 fermented cashew kernel flour; fcb60: 40 wheat flour + 60 fermented cashew kernel flour; fcb40: 60 wheat flour + 40 fermented cashew kernel flour; fcb20: 80 wheat flour + 20 fermented cashew kernel flour; wf: wheat flour. the result however agrees with the work of [32] who observed a significant decrease in oil absorption capacity of defatted coconut (cocos nucifera l.) flour with increase in fermentation time. the decrease observed could be attributed to the increase in the fat content during the fermentation process [33]. unfermented and fermented cashew kernel flours has a higher oil absorption capacity than wheat flour. the higher oil absorption capacity suggest that these flours would be useful in formulation of foods such as biscuits and cakes where oil absorption capacity is an important consideration. resullts (table 1) showed that fermented cashew kernel flour had high emulsion activity (ea). fermentation had significantly increased (p < 0.05) the emulsion activity. the emulsifying activity is the work of soluble proteins, which, present at the water and oil interface, promote the formation of emulsions [34]. the fermentation would therefore make the proteins more soluble, thus promoting the formation of more emulsions in the fermented cashew kernel flour. these results are in agreement with the work of [35] who observed an increase in emulsifying capacity during the fermentation of sorghum flour. wheat flour substitution by cashew kernels flours increased emulsion activity of composite flours. the emulsifying activity has utility in the preparation of pastry and bakery products [36]. the increase in foaming capacity is thought to be due to the reduction of the surface tension of proteins because of the hydrolysis of proteins into amino acids during fermentation [37]. foams are used to improve texture, consistency and appearance of foods. wf 0.63±0.05 i 109.75±1.5 a 89.52±0.9 a 48.27±0.7 a 35.7±0.05 a ucf20 0.59±0.01 h 110.68±0.59 b 98.75±0.1 c 49.59±0.09 b 40.75±0.01 b ucf40 0.56±0.08 fg 116±1.2 c 110.03±0.88 e 51.36±0.1 d 46.37±0.02 d ucf60 0.51±0.02 e 128.21±1.3 f 118.79±0.64 g 53.28±0.19 e 52.78±0.07 f ucf80 0.47±0.09 cd 133.12±0.5 g 126.28±0.55 i 55.32±0.25 g 59.07±0.05 h ucf 0.44±0.04 b 144.01±0.11 i 135.87±0.83 k 58.57±1.23 i 68.23±0.03 j fcf20 0.57±0.07 gh 120.75±1.15 d 96.17±0.4 b 51.19±0.23 c 42.83±0.06 c fcf40 0.54±0.03 f 123.27±1.26 e 107.22±0.7 d 54.97±0.07 f 47.66±0.06 e fcf60 0.48±0.05 de 139.33±1.24 h 111.98±0.95 f 56.79±0.13 h 54.17±0.06 g fcf80 0.45±0.04 bc 148.19±1.1 j 116.07±0.9 h 60.88±0.35 j 64.57±0.03 i fcf 0.4±0.06 a 154.83±0.89 k 123.4±0.28 j 64.81±1.85 k 72.3±0.03 k flours samples bulk density (g/ml -1 ) wac (% ) oac (% ) ea (% ) fc (% ) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 85 table 3 shows positive correlation between proteins and fibers contents and functional properties (wac, oac, ea and fc). these positive correlations indicate that incorporation rates of cashew kernels flours increase significantly (p < 0.05) functional properties of composite flours. table 2 correlation coefficients among functional properties analysis of flours, nutritional composition and sensory evaluation of biscuits. correlations in bold are significant at the 0.05 level the results in table 3 show a significant difference (p < 0.05) between the nutritional characteristics of biscuits containing cashew kernels (unfermented cashew kernels flour or fermented cashew kernels flour) and biscuits made from 100 % wheat flour nutritional values. indeed, the incorporation rates of cashew kernels flours allow an increase in proteins, lipids, ash and fibers content. it is well documented that cashew kernel is a major source of proteins, fat, ash and fiber [38; 39]. the inclusion of cashew kernel flours to the formulation decreased the carbohydrate content with regard to the control sample. table 3 proximate composition of biscuits values are means of triplicate replications. means within a column with different superscript were significantly (p < 0.05) different. wb: wheat flour biscuit; ucb20: 80 %wheat flour + 20 % unfermented cashew kernel flour biscuit; ucb40: 60 % wheat flour + 40 % unfermented cashew kernel flour biscuit; ucb60: 40 % wheat flour + 60 % unfermented cashew kernel flour biscuit; ucb80: 20 % wheat flour + 80 % unfermented cashew kernel flour biscuit; ucb: unfermented cashew kernel flour biscuit; fcb20: 80 %wheat flour + 20 %fermented cashew kernel flour biscuit; fcb40: 60 %wheat flour + 40 % fermented cashew kernel flour biscuit; fcb 60: 40 %wheat flour + 60 % fermented cashew kernel flour biscuit; fcb80: 20 %wheat flour + 80 % fermented cashew kernel flour biscuit; fcb: fermented cashew kernel flour biscuit. sample s moisture (g/100g) proteins (g/100g) fat (g/100g) carbohydrate (g/100g) ash (g/100g) fiber (g/100g) energy (kcal/100g) wb 3.40±0.02 a 12.77±0.23 a 10.36±0.09 a 0.98±0.15 a 71.48±0.57 k 1.39±0.2 a 430.28±0.3 a ucb20 3.53±0.08 b 16.35±0.09 b 12.18±0.05 b 1.42±0.04 c 66.51±0.11 j 1.91±0.2 d 441.35±0.32 b ucb40 3.86±0.04 d 18.53±0.25 d 15.53±0.10 d 1.90±0.08 f 60.16±0.2 h 2.49±0.1 g 454.51±0.2 d ucb60 4.06±0.02 f 25.25±0.07 f 20.52±0.35 f 2.38±0.05 i 47.62±0.39 f 3.04±0.3 i 476.74±0.14 f ucb80 4.23±0.06 g 29.60±0.11 h 26.88±0.20 h 2.84±0.19 j 36.39±0.45 d 3.63±0.6 j 506.16±0.1 h ucb 4.35±0.01 i 34.10±0.41 g 28.16±0.07 i 3.32±0.15 k 30.11±0.24 b 3.99±0.4 k 510.26±0.34 i fcb20 3.73±0.05 c 17.79±0.37 c 13.97±0.09 c 1.23±0.07 b 63.27±0.34 i 1.65±0.1 b 450.03±0.2 c fcb40 3.96±0.07 e 23.15±0.10 e 17.83±0.24 e 1.51±0.12 d 53.54±0.16 g 1.87±0.2 c 467.17±0.19 e fcb60 4.27±0.09 h 26.93±0.29 g 22.55±0.15 g 1.77±0.2 e 44.46±0.28 e 2.19±0.3 e 488.55±0.4 g fcb80 4.52±0.03 j 33.93±0.17 i 27.97±0.06 j 2.05±0.24 g 31.52±0.11 c 2.43±0.1 f 513.52±0.1 j fcb 4.73±0.05 k 38.67±0.4 k 31.02±0.18 k 2.33±0.16 h 23.26±0.13 a 2.74±0.1 h 526.86±0.28 k parameters proteins carbohydrates fat fibres wac oac ea fc brown colour firmness crispness aroma oveall accep proteins 1.00 carbohydrates -0.99 1.00 fat 0.98 -0.99 1.00 fiber 0.62 -0.67 0.74 1.00 wac 0.98 -0.97 0.95 0.55 1.00 oac 0.83 -0.86 0.90 0.95 0.77 1.00 ea 0.84 -0.81 0.77 0.22 0.87 0.46 1.00 fc 0.98 -0.98 0.99 0.74 0.95 0.89 0.78 1.00 brown colour 0.81 -0.77 0.71 0.15 0.86 0.43 0.79 0.70 1.00 firmness -0.88 0.90 -0.91 -0.75 -0.86 -0.89 -0.62 -0.87 -0.62 1.00 crispness -0.91 0.92 -0.92 -0.64 -0.91 -0.82 -0.65 -0.88 -0.81 0.92 1.00 aroma 0.95 -0.93 0.92 0.57 0.96 0.77 0.77 0.91 0.89 -0.84 -0.93 1.00 overall accep -0.94 0.92 -0.88 -0.37 -0.97 -0.62 -0.91 -0.88 -0.93 0.76 0.87 -0.94 1.00 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 86 at the same incorporation rate of cashew kernel flour, biscuits containing fermented cashew kernel flour and those containing unfermented cashew kernel flour have significantly different. fermentation helps to increase significantly (p < 0.05) proteins content. this trend is similar to the observations of [40] who found a significant increase in proteins content of soybean (glycine max), cowpea (vigna unguiculata) and ground bean (macrotyloma geocarpa) after fermentation. this increase could be attributed to the increase in microbial mass during fermentation, causing extensive hydrolysis of proteins to amino acids and other simple peptides [41]. the crude fibers decrease from 20.16 to 17.20 g / 100 g after fermentation. this could result from the degradation of fibers by microorganisms [42]. on the other hand, the content of carbohydrates and fiber are reduced by fermentation. the sensory characteristics of a product are an important aspect in the overall acceptability of that product. figures 1 and 2 shows the sensory profiles of the unfermented cashew kernels flours biscuits and the fermented cashew kernel flour biscuits, respectively. the browning of the biscuits is significant (p < 0.05) more noticeable in the case of biscuits based on fermented cashew kernels than with the biscuits based on unfermented cashew kernel flour. biscuits made from fermented or unfermented cashew kernel flours have a significantly higher intensity of brown colour (p < 0.05) than that of the control biscuit (100 % wheat). the perception of brown colour in biscuits increases significantly with the rate of substitution of wheat flour by cashew kernel flours (fermented cashew kernel flour or unfermented cashew kernel flour). a significant and positive correlation (r = 0.81) between protein content and biscuits brown colour intensity is observed. the proteins in the presence of reducing sugars, by the maillard reaction favors the production of browner biscuits [43]. the browning of biscuits decreases the colour acceptability score. fig. 1. sensory profile of biscuits made from unfermented cashew kernel flour and wheat flour results are presented as the mean (n = 15).wb: wheat flour biscuit; ucb20: 80 %wheat flour + 20 % unfermented cashew kernel flour biscuit; ucb40: 60 % wheat flour + 40 % unfermented cashew kernel flour biscuit; ucb60: 40 % wheat flour + 60 % unfermented cashew kernel flour biscuit; ucb80: 20 % wheat flour + 80 % unfermented cashew kernel flour biscuit; ucb: unfermented cashew kernel flour biscuit. 0 1 2 3 4 5 6 7 8 colour (brown) texture (firmness) crispness taste (sugary) taste (cashew kernel) aroma (cashew kernel) wb ucb20 ucb40 ucb60 ucb80 ucb food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 87 fig. 2. sensory profile of biscuits made from fermented cashew kernel flour and wheat flour results are presented as the mean (n = 15). wb: wheat flour biscuit; fcb20: 80 %wheat flour + 20 %fermented cashew kernel flour biscuit; fcb40: 60 %wheat flour + 40 % fermented cashew kernel flour biscuit; fcb 60: 40 %wheat flour + 60 % fermented cashew kernel flour biscuit; fcb80: 20 %wheat flour + 80 % fermented cashew kernel flour biscuit; fcb: fermented cashew kernel flour biscuit. the descriptors “cashew kernel taste” and “cashew kernel aroma” are significantly (p < 0.05) perceived by the panelists when tasting the biscuits produced from 60 % substitution rate for wheat flour by unfermented cashew kernel flour. while the descriptors "fermented taste" and "fermented aroma" are significantly, (p < 0.05) perceived from 40% substitution rate of wheat flour by fermented cashew kernel flour. biscuits based on unfermented cashew kernel flour and those based on fermented cashew kernel flour show no significant difference (p < 0.05) in firmness and crispness. wheat flour biscuits have the highest intensity of firmness and crispness. it is also observed that the perception of the intensity of the crispness and the firmness of biscuits based on composite flours (cashew kernel / wheat) decreases due to the substitution of wheat flour by kernel flours of cashew (fermented and unfermented cashew kernel flour). composite flour biscuits are significantly (p < 0.05) less firm from 60 % substitution rate for wheat flour with cashew kernel flours. the results (table 4) showed that cashew kernel fermentation and the substitution of wheat flour for cashew kernels flours influences negatively overall acceptability of biscuits made from composite flours. this decrease in overall acceptability could be explained by the hedonic characteristics (colour, aroma, taste) of cashew kernels flours. this interpretation could further be supported by the results obtained from the correlation analysis. as shown in table 2, brown colour, cashew kernel aroma and fermented aroma negatively correlated with overall acceptability (r = -0.93 and r = -0.94, respectively 0 1 2 3 4 5 6 7 8 9 colour (brown) texture (firmness) crispness taste (sugary) taste (fermented) aroma (fermented) wb fcb20 fcb40 fcb60 fcb80 fcb food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 88 table 4 overall acceptability of biscuits biscuit samples overall acceptability wb 8.28±0.95g ucb20 8.23±1,3g ucb40 8.20±1.18g ucb60 7.58±0.97f ucb80 7.51±0.84ef ucb 7.44±1.11e fcb20 7.57±1.52f fcb40 6.72±1.03d fcb60 4.82±1.25c fcb80 4.75±1.26b fcb 4.68±1.47a means (n = 60) within a column with different superscript were significantly (p < 0.05) different. wb: wheat flour biscuit; ucb20: 80 %wheat flour + 20 % unfermented cashew kernel flour biscuit; ucb40: 60 % wheat flour + 40 % unfermented cashew kernel flour biscuit; ucb60: 40 % wheat flour + 60 % unfermented cashew kernel flour biscuit; ucb80: 20 % wheat flour + 80 % unfermented cashew kernel flour biscuit; ucb: unfermented cashew kernel flour biscuit; fcb20: 80 %wheat flour + 20 %fermented cashew kernel flour biscuit; fcb40: 60 %wheat flour + 40 % fermented cashew kernel flour biscuit; fcb 60: 40 %wheat flour + 60 % fermented cashew kernel flour biscuit; fcb80: 20 %wheat flour + 80 % fermented cashew kernel flour biscuit; fcb: fermented cashew kernel flour biscuit. sensory attributes decrease when chickpea flour is added to bread [44]. in addition, fermentation induced alteration in food thereby the protein and fat may be degraded with consequent effect upon food flavor and texture [45]. with the cashew kernel, the panelists have depreciated these changes. there is no significant difference at the 5 % threshold between biscuits prepared with 80 % wheat flour and 20 % unfermented cashew kernel flour (ucb20), biscuits with 60% wheat flour and 40 % unfermented cashew kernel flour (ucb40) and biscuits prepared with 100 % wheat flour (wb). 4. conclusion the substitution of wheat flour for cashew kernels flours has improved the protein, ash fiber content, and energy value of biscuits. the incorporation of 20 % and 40 % unfermented cashew kernel flour could enrich the biscuits without significantly affecting their overall acceptability. the members of the panel accepted biscuit produced with unfermented cashew kernel flour and biscuits produced with composite flour containing 40 % fermented cashew kernel flour. the consumption of these biscuits could reduce the cost of importing wheat, increase the processing level of cashew at the local level and helped to fight malnutrition in africa. 5. references [1]. o’briena c. m., chapmanb d., neville d. p., keogh m. k., arendt e. k., effect of varying the micro-encapsulation process on the functionality of hydrogenated vegetable fat in short dough biscuits. fd. res. int., 36:215–221, (2003). [2]. ayo j. a., nkama i., adewori r. , physicochemical, invitro digestibility and organoleptic evaluation of “acha”-wheat biscuit supplemented with soybean flour, nig. fd. j., 25(2):77-89, (2007). [3]. akubor p. i., protein contents, physical and sensory properties of nigerian snack food (cake, chin-chin and puff-puff) prepared from cowpeawheat flour blends, int. j. fd. sci. tech., 39(4):419424, (2004). [4]. mcwatters k. h., ouedraogo j. b., resurreccion a. v. a., hung y. phillips v., physical and sensory characteristics of sugar cookies containing mixtures of wheat, fonio (digitaria exilis) and cowpea (vigna unguiculata) flours, int. j. food sci. tech., 38:403-410, (2003). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 89 [5]. eneche e. h., biscuit making potentials of millet/pigeon pea flour blends, plant foods hum. nutr., 54: 21-27, (2003). [6]. mepba h. d., eboh l., nwaojigwa s. u., chemical composition, functional and baking properties of wheatplantain composite flours, afr. j. food agric. nutr. dev., 7(1): 1-22, (2007). [7]. echendu c. a, onimawo i. a., somtochi a., production and evaluation of doughnuts and biscuits from maizepigeon pea flour blends, nig. food j., 22: 147-153, (2004). [8]. ojinnaka m. c. & agubolum f. u., nutritional and sensory properties of cashew nutwheat based cookies, american journal of food and nutrition, 33: 127-134, (2013). [9]. aroyeun s. o., utilization of cashew kernel meals in the nutritional enrichment of biscuit, african journal of food science, 3(10): 316-319, (2009). [10]. faostat. cashew nut production quantities by country, (2015). [11]. cca (conseil coton anacarde en côte d’ivoire), publié le lundi 15 février 2016. [12]. koko a. c., konan a., tetchi f., assidjo e. & amani g. quality of fermented cassava flour processed into placali. international journal of biology chemestry science, 6(1): 415420, (2012). [13]. sze-tao k. w. c. & sathe s. k., functional properties and in-vitro digestibility of almond (prunusdulcis l) protein isolate, food chemistry, 69: 15316, (2004). [14]. ijarotimi o. s., oluwalana i. b. & ogunedojutimi m. o., nutrient composition, functional, sensory and microbial status of popcorn based (zea may everta) complementary foods enriched with cashew nut (anacardium occidentale l.) flour, african journal of food science, 12(5): 6424-6446, (2012). [15]. chau c. f. & huang y. l., comparison of the chemical composition and physicochemical properties of different fibers prepared from the peel of citrus sinensis l. cv. liucheng, journal of agricultural food and chemistry, 51(9): 2615-2618, (2003). [16]. philips r. d., chinnan m. s., branch a. l., miller j. & mcwatters k. h., effects of pretreatment on functional and nutritional properties of cowpea meal, journal of food science, 53: 805–809, (1988). [17]. sosulski f. w., the centrifuge method for determining flour absorption in hard red spring wheat, cereal chemistry, 39: 344-350, (1962). [18]. neto v. q., narain n., silvia j. b. & bora p. s., functional properties of raw and heatprocessed cashew nut (anarcadium occidentale l.) kernel protein isolate, nahrung, (45): 258-262, (2001). [19]. bencini m. c., functional properties of drum-dried chickpea (cicer arietinum l.) flours, journal of food science, 51(6), 1518-1521, (1986). [20]. aoac, official methods of analysis of the association of official analytical chemists, 15 th edition, virgina, 217 p, (1999). [21]. aoac, official methods of analysis of the association of official analytical chemists, 15th edition, washington dc, 1230 p, (1990). [22]. aoac, official method of analysis of association of official analytical chemists, 17th edition, gaithersburg, 2200 p, (2000). [23]. aoac, official method of analysis of the association of official analytical chemist, 5th edition, aoac press, arlington, virginia, usa, (2005). [24]. van soest p. s., use of detergents in the analysis of fibrous feeds iia rapid method for the determination of fiber and lignin, journal of association of official analytical chemistry, 46: 829-835, (1963). [25]. atwater w. & rosa e., a new respiratory calorimeter and the conservation of energy in human body, ii-physical, 9: 214-251, (1899). [26], [27]. meilgaard m., civille g. v. & carr b. t., sensory evaluation techniques. crc press, boca raton. 281 p, (1999). [28] peleg m., physical characteristics of food powders, inphysical properties of foods (eds. m. peleg and e. b. bagley), 293-324, (1983). [29]. onimawo i. a., akubur p. i., food chemistry .ambik press ltd, (2005). [30]. ariahu c. c., ukpabi u. & mbajunwa k. o., production of african breadfruit (treculia africana) and soybean (glycine max) seed based food formulations, plant foods hum. nutri., 54: 192-206, . (1999). [31]. onimawo i. a. & akubor p. i., food chemistry (integrated approach with biochemical background, 2 nd edition joytal printing press, agbowo, ibadan, nigeria, (2012). [32], [33]. igbabul b. d., amove. j. & twadue i., effect of fermentation on the proximate composition, antinutritional factors and functional properties of cocoyam (colocasia esculenta) flour, african journal of food science and technology, 5(3): 67-74, (2014). [34]. moure a., sineiro j., dominguez h. & parajo j. c., functionally of oil seed protein products: a review, food research international, 38(9): 945-963, (2006). [35]. abd elmoneim o. e., schiffler b. & bernhardt r., effect of fermentation on the functional properties of sorghum flour, food chemistry, 92(1): 1-5, (2004). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 bleou jean jaurès touzou, doudjo soro, soronikpoho soro, kouadio ernest koffi, effect of fermentation on the quality of flour and biscuit from cashew kernel flour, food and environment safety, volume xx, issue 1 – 2021, pag. 81 – 90 90 [36]. akubor p. i. & badifu g. l. o., chemical composition, functional properties and baking potential of african breadfruit kernel and wheat flour blends, international journal of food science & technology, 39: 223-229, (2004). [37]. sosulski f. w., garratt m. o. & slinkard e. a., functional properties of instit. food sci. technol. j., 19:66 – 69, (1976). [38]. freitas j. b., naves m. m. v., chemical composition of nuts and edible seeds and their relation to nutrition and health, rev. nutr. campinas, 23(2): 269-279, (2010). [39]. akinhanmi t. f., atasie v. n., chemical composition and physicochemical properties of cashew nut (anacardium occidentale) oil and cashew nut shell liquid, j. agric. food environ. sci. 2(1): 1-10, (2008). [40]. egounlety m., production properties and utilization of mould fermented foods from soybean (glycine max. m.) cowpea (vigna unguiculata l. walp) and ground bean (macrotyloma geocarpa h.). ph.d thesis. university of ibadan, nigeria. pp 2128, (1994). [41]. igbabul b. d., amove j., twadue i., effect of fermentation on the proximate composition, antinutritional factors and functional properties of cocoyam (colocasia esculenta) flour, afr. j. food sci. technol. 5(3): 67-74, (2014). [42]. babalola r. o., giwa o. e., effect of fermentation on nutritional and anti-nutritional properties of fermenting soy beans and the antagonistic effect of the fermenting organism on selected pathogens, international research journal of microbiology, 3(10): 333-338, (2012). [43].mannay.s shadaksharaswany c. m. (2005) foods: facts and principes, 2nd edition. new age international ltd. publishers. new delhi, india, (2005). [44]. man s., păucean a., muste s., & pop a., effect of the chickpea (cicer arietinum l.) flour addition on physicochemical properties of wheat bread, bulletin of university of agricultural sciences and veterinary medicine cluj-napoca. food science and technology, 72(1), 41-49, (2015). [45]. komolafe i. o. cereal crops and their food in tropical, afr. j. agri 62: 105-116, (2002). применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 contents: 1. use of artificial neural networks for modeling inflow and outflow and salinity of lake fetzara in the region annaba (ne algeria) zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai 107 115 2. functional and technological properties of oat beta-glucan in acidophilic-whey ice cream artur mykhalevych, victoria sapiga, galina polischuk, tatiana osmak 116 128 3. diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria christiana n. opara, uchenna c. okpe, christian k. anumudu 129 142 4. microbial generation of silver nanoparticles by synthesizing enterococcus faecalis strain mi103 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi and matthew olusoji ilori 143 151 5. morphological, biochemical and molecular identification of autochthonous fungal population in slaughterhouse effluent, surface water and fish gut from the ogun river, nigeria olanike maria buraimoh, temitope olawunmi sogbanmu, olusola abayomi ojoomoniyi , olumide afolabi , chinwe mary ganobi 152 170 6. evaluation of soil salinity of the fetzara lake region (north-east algeria) faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel 171 184 7. influence of somatic cell count in raw milk on the quality of beaten cheese ljubica trajkoska, andrej stojkoski, biljana trajkovska, ljupche kochoski 185 189 8. nutritional and biological value of mushroom snacks inna bobel, greta adamczyk , nataliia falendysh 190 197 9. author instructions i v 10. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 doi: https://doi.org/10.4316/fens.2022.023 237 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 237 246 evaluation of the quality of wheat with added improvers * amelia buculei1, anca gatlan1, adriana cojocariu2 1faculty of food engineering, university stefan cel mare of suceava, romania, ameliab@fia.usv.ro, 2 sc mopan sa, suceava, romania *corresponding author received 15th august 2022, accepted 27th september 2022 abstract: one of the main indicators of quality of grain flours is the enzyme content. the most important enzymes on the wheat flour are the  amylase and the  amylase. unique for wheat flour dough are its rheological properties, namely elasticity and extensibility, which are largely due to the properties of gluten formed during kneading and how it interacts with other components of flour and product ingredients. in the present paper are presented the experimental results regarding the falling index hagberg and determination the main rheological properties tenacity, extensibility, elasticity, and baking strengthfor differently wheat flour types produced, from 2021 production. the research is important for specialists in the field, in terms of the need to apply the corrections that must be made to flour in the technological process of baking, in order to obtain quality products. the main objective of this work was to improve the knowledge regarding the qualitative analysis of wheat and to improve the quality of flour by adding improvers in the field of the variability of the quality parameters of wheat, which is an annoying feature of the romanian crops. keywords: corrections, enzyme, baking, technological process 1. introduction the rheological properties of the dough play an important role in the production processes, in which the dough is subjected to the action of forces that cause tension and cause deformation. they express the deformation over time of the dough under the action of the external forces exerted on it. the dough possesses both the properties of a viscous liquid and those of a solidelastic, being considered viscoelastic.[1] in romania, the quality assurance of bakery products is a priority because many food producers consider the use of new and innovative enzymes that have a long history of study and use. when an enzyme is added, it is often destroyed during the heat-baking process. enzymes are intended for a certain function, thus eliminating undesirable effects [2]. wheat flour consists mainly of starch (ca. 70–75%), water (ca. 14%), and proteins (ca. 10–12%). the typical levels of amylose and amylopectin in starch are 25– 28% and 72–75%, respectively [3]. in some countries, such as the u.s., france, spain and england to make quality bakery products, industrial-grade fungal proteolytic preparations containing amylases are also used [4]. physicochemical characteristics of seventy wheat flours of different species namely einkorn (triticum monococcum), spelt (triticum spelta) and common wheat (triticum aestivum) were analyzed using different standard methods. the wheat grains were analyzed for moisture, ash, protein, wet gluten, sedimentation index, ph, acidity, fat, starch, falling number, damage starch and glutograph parameters stretching and relaxation. the relationship between physicochemical characteristics and wheat samples were analyzed using the principal component analysis. for almost all the physicochemical data except moisture and damage starch were obtained food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 238 significant differences between species [5]. as one of important consumable raw materials in our daily lives, wheat flour provides numerous nutrients, such as carbohydrates, protein, and minerals. however, the quality and safety of wheat flour products are sometimes challenged by the inferior quality parameters and adulteration, which cannot be easily detected and pose risks to human health [6,7]. the main quality parameters of wheat flour include its chemical composition, which is related to the moisture, protein, ash, and wet gluten contents of the flour, and technological parameters, such as the sedimentation value, falling number and rheological properties of wheat flour dough [8, 9]. a large variation in correlations between rheological properties of wheat dough and baking performance of the resulting breads were explored. many factors in addition to rheological properties should be considered to develop a predictive model that could be used in the bake industry [10]. the bread obtained has a slower aging rate due to better gelatinization of starch and shortening of amylopectin chains that have a slower tendency to retrograde. also, low doses of α-amylase improve core elasticity and porosity structure [11]. high doses, however, reduce the elasticity of the core and increase its stickiness, an effect seen at much lower doses of bacterial amylase than with malt or fungal amylase. this is explained by the higher thermostability of bacterial α-amylase and therefore the longer duration of action in the dough on the one hand, but also by the degree of polymerization of the formed dextrins, of 25 – 35. the addition of amylase alters the rheological qualities of the dough. damaged starch grains absorb more water than intact grains [12, 13]. the alveographer provides information on the biaxial stretching rheological properties of the dough at constant hydration. the method is based on the tensile strength of a dough sheet maintained at rest for 20 minutes after which it is subjected to an air stream at constant pressure until it breaks. the air pressure inside the bubble is recorded until the bubble breaks and graphically extrapolated as a curve whose characteristics render the dough resistant to deformation. [14]. high w flour is high gluten-containing flour, which is most suitable for prolonged leavening [15]. gluten, which is the protein part of a meal, is able to absorb water once and a half of its weight, so the stronger the flour, the higher the concentration of protein. as scientific objectives this paper aims to: description of the influence of amylases and parameters determined by means of the alveographer on the finished product; evaluation of the behavior of alphaamylase before and after the addition of the improver; evaluation of the influence of the rheological behavior of the prepared dough made with the help of the alveographer chopin; evaluation of changes in porosity, core elasticity and acidity in both breeder and non-added bread. 2. matherials and methods the research focused on determining the hagberg drop index and determining the main rheological properties toughness, extensibility, elasticity and baking resistance to different types of wheat flour from wheat production in 2021. the analyzed material is represented by the nine flour samples (five samples of white flour 650; 2 samples of black flour 1350 and two samples of whole flour 1500) after the addition of the enzyme preparation belpan mopa. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 239 3. results and discussion determination falling number. the results obtained from the determination of alphaamylase activity for the 9 samples with the addition of belpan mopa and the nine samples of unadded flour are shown in figure 1. a decrease in the drop index may be observed in the nine flour samples (five samples of white flour 650; 2 samples of black flour 1350 and two samples of whole flour 1500) after the addition of the enzyme preparation belpan mopa. in the case of white flour samples 650 after the addition of the enzyme preparation, the drop index of values between: 220÷280 the amylase activity of flour is normal; 290 the amylase activity of flour is weak, and the bread obtained from them is not developed, has small volume and dry core. in the case of samples of black flour 1350 after the addition of the enzyme preparation, the drop index of values between: 220 the amylase activity of the flour is normal; 290 the amylase activity of the flour is poor, and the bread obtained from it is not developed, has small volume and dry core. the variation of falling number index for both non-improver and breeder-added flour can be seen in fig.1. determination of rheological indicators using the alveographer. the quality of the dough after kneading was also determined with the help of the alveographer. the alveographer measures three parameters, namely: the maximum pressure required for the deformation of the sample, which represents the tenacity of the dough; the length of the curve, which represents the extensibility of the dough; these two parameters must be in a balance. the most important parameter is w, which shows the strength of the dough after kneading. the graphical representation of the values of the parameters of the nine flour samples can be found in figure 2 (a-i). according to banu et al. (2000) the values of an alveogram for flour intended for bread are within the ranges: p 60÷75mm; l 130÷150 mm; g 25÷30; p/l 0,55÷0,65 and w 200 10÷j. the values for parameter p are between 78÷103 mm for white flour 650 that is, they are higher and do not fall within the range specified by banu c. there are no values for whole flour 1500 that are lower than this range; only the values for black flour 1350 fall. in the case of flour with a w greater than 250 as in white flour 650, the protein network is large and has as a consequence the ability to produce a highly leavened product. in the case of flour intended for bread it is recommended that w should have values between 170÷250 10 "j", and in the case of analyzes carried out in this study only one type of white flour 650 falls . fig.1. variation of falling number index for both non-improver and breeder-added flour belpan mopa food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 240 a. the alveogram f. 650 (lot 1) b. the alveogram f. 650 (lot 2) c. the alveogram f. 650 (lot 3) d. the alveogram f. 650 (lot 4) e. the alveogram f. 650 (lot 5) f. the alveogram f. 1350 (lot 1) g. the alveogram f. 1350 (lot 2) h. the alveogram f. 1500 (lot 1) i. the alveogram f. 1500 (lot 2) fig. 2. variation of rheological indicators food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 241 sensory analysis the sensory quality of the bread is shown in table 3 and in figures 3,4,5, you can see the outer appearance, in the section, but also the porosity of the core. figura 3. white flour bread 650 with added belpan mopa fig. 4. white flour bread 1350 and 1500 with the addition of belpan mopa fig. 5. white flour bread 650 with no enzyme preparation added determination of core porosity porosity of bread (figure 6) is an important physico-chemical parameter in the sensory analysis of a bakery product which provides information not only on the total volume of pores in the analyzed core but also on the degree of its assimilation by analyzing the structure and thickness of the pores, their uniformity. the results obtained from the determination of porosity are listed. both porosity and elasticity are expressed as a percentage, the higher their value, the better the quality of the bakery products is considered and the more appreciated by consumers. adding the percentage of enzymatic preparation to the dough resulted in an increase in the porosity of the bread represented in figure 6. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 242 table. 3. sensory analysis of bread product quality indices characterization of finished products no addition characterization of finished products with the addition of belpan mopa 1. form and volume long shape, characteristic of loaf but flattened, with low volume long shape, characteristic of loaf, symmetrical, well developed, unflattened or bulging 2. color and appearance of the crust the crust is beautifully browned, characteristic color of the assortment, the surface of the shell is smooth, glossy, without cracks or malfunctions, the shell is crispy;cracks; products with uniform unshelled shell, characteristic color, smooth peel surface, no cracks; 3. the degree of ripening, the condition and appearance of the core well-baked products, when striking the shell produces a specific, clear sound, characteristic of well-baked products, the core is elastic, when pressing with the finger returns to the original shape and when cutting with the knife the core does not stick to the blade; well-baked products, when striking the shell produces a specific, clear sound, characteristic of well-baked products, the core is elastic, when pressing with the finger returns to the original shape and when cutting with the knife the core does not stick to the blade; 4. core porosity and structure pori the pore structure is fine, uniform, with a maximum of 3 gaps in the section; the pore structure is fine, uniform, with a maximum of 2 gaps in the section; 5. flavor the product has flavor (smell) pronounced, characteristic of fermented and well-baked bread. 6. taste products with characteristic taste, weak sour-sweet, without foreign taste. 7. foreign bodies the products do not have any foreign bodies . 650 650 650 650 650 1350 1350 1500 1500 porosity without addition 70 74 73 75 73 67 73 70 68 porosity with added belpan mopa 78,8 82,9 77,3 77,9 76 75,4 78,7 81 78,4 0 10 20 30 40 50 60 70 80 90 p or os it y (% ) fig. 6. variation of porosity determination of the elasiticity of the core. the elasticity of the core is based on determining the compressibility and relaxation of the core by pressing under certain conditions and measuring the height it returns to after removing the pressing force. the elasticity of the bread core improves but the differences are not large as shown in figure.7. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 243 fig.7. variation in core elasticity determination of the acidity of the bread.the acidity of the bread by the tritrimetric method has the following values given in fig.8. the variation in acidity is not significant with added enzymatic bread compared with non-added bread. the values are normal and fall within those mentioned in the literature and their variation can be seen in figure 8. fig.8 variation of acidity 650 650 650 650 650 1350 1350 1500 1500 core elasticity without addition 93 89 90 88 90 87 89 86 90 core elasticity with added belpan mopa 96 95 91 95 95 95 95 95,3 96 78 80 82 84 86 88 90 92 94 96 98 e la st ic ity ( % ) 650 650 650 650 650 1350 1350 1500 1500 acidity without addition 3,1 2,4 3 2,5 3,3 6,7 6 7 6,8 acidity with added belan mopa 2,6 2,9 2,3 2,9 3 7 6,3 7,8 6,6 0 1 2 3 4 5 6 7 8 9 a ci di ty (d eg re es o f a ci di ty /1 00 g pr od uc t) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 244 from the sensory analysis of bread obtained from flour 650; 1350 and 1500 (for which the color of the peel, the appearance of the peel, the porosity of the core, the smell, taste and general acceptability were assessed), it can be concluded that all samples are acceptable for consumption. the ripening test for the improved bread produced well-developed products which do not present irregularity in the exterior appearance, pores well defined, but in the case of those without a breeder, a flattening of the bread volume was observed, but the appearance of the peel and the core were appropriate as seen in fig. 9. fig.9. bread without breeder (flattened) at the porosity and elasticity level, no large differences were observed as a result of the graphical representation of the values obtained from analyzes carried out. the porosity of the samples can be seen in figure 10(a-c). a. the porosity of the improved white cloth b.7. porosity of white bread without breeder c. the porosity of the improved black stalk fig. 10 (a-c). the structure of the bread with added/no breeder adios food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 245 4. conclusion bread traditionally obtained without the addition of improvers at the industrial level imposes difficulty because it has a sticky dough that is difficult to handle on the lines, it imposes a certain time of leavening that differs from one batch of flour to another.at industrial novel no one assumes the creation of a new way of working according to the quality of flour that is different when supplying a new batch of flour. it is therefore necessary to add improvers to regulate the quality of the flour in order not to impose difficulties in processing, but the addition of these improvers can also cause disadvantages when it is not dosed correctly. in order to avoid this, it is necessary to carry out analyzes for the respective batch of flour with different quantity of breeder to choose the appropriate dosage according to the quality of the flour concerned. in the case of small businesses, the working times for each bread processing operation can be changed according to the parameters that flour has if it is intended to obtain traditional products that are currently in high demand. from the analyzes carried out with the help of the alveographer, it is noted that the most important parameter is the so-called force index w which must have values between 170÷250 in order to obtain a quality bread, values higher than 250 are addressed to products with long leavings, pastry. 6. references [1]. zhang s., liu s.,shen l., shujuan c., he l., liu a., application of near-infrared spectroscopy for the nondestructive analysis of wheat flour: a review, (2022) [2]. parenti, o., guerrini l., mompin, s.b., toldr m., zanoni, b., the determination of bread dough readiness during kneading of wheat flour: a review of the available methods. j. food eng. 309, 110692 https://doi.org/10.1016/j. jfoodeng.2021.110692 (2021) [3]. goesaert h., brijs k., veraverbeke w s ., courtin cm., gebruers k., trends in food science & technology, volume 16, issues 1–3, january– march 2005, pages 12-30, https://doi.org/10.1016/j.tifs.2004.02.011, (2005) [4]. leonte m., consumer perception and attitudes, journal, 3: 42–50, (2000) [5]. golea c.m, codina, g., oroian m., prediction of wheat flours composition using fourier transform infrared spectrometry (ft-ir) food control volume 143, , 109318 https://doi.org/10.1016/j.foodcont.2022.109318, (2023) [6]. stojceska v., butler f., investigation of reported correlation coefficients between rheological properties of the wheat bread doughs and baking performance of the corresponding wheat flours, trends in food science & technology volume 24, issue 1, pages 13-18, https://doi.org/10.1016/j.tifs.2011.09.005, (2012) [7]. della valle g. chiron g., cicerelli l., kansou k., katina k., ndiaye a, basic knowledge models for the design of bread texture trends food sci technol https://doi.org/10.1016/j.jfoodeng.2015.10.010, (2014) [8]. wang x., liang y.,wang q.,zhang x., wang j., effect of low-sodium salt on the physicochemical and rheological properties of wheat flour doughs and their respective gluten, journal of cereal science volume 102, 103371, (2021). [9]. kofi s.,kudadam koresi j., hensel o.,schurm b.,pawelzik e., rheological properties of dough and bread quality characteristics as influenced by the proportion of wheat flour substitution with orange-fleshed sweet potato flour and baking conditions lwt, volume 147, 111515, https://doi.org/10.1016/j.lwt.2021.111515, (2021). [10]. yang x., wu l., zhu z.,ren g.,liu s., variation and trends in dough rheological properties and flour quality in 330 chinese wheat varieties, the crop journal, pages 195-200, https://doi.org/10.1016/j.cj.2014.04.001, (2014) [11]. li x., xu p., ling j. y., farinograph quality number (fqn): a new index for rheological property measurement on dough with farinograph, j. chin. cereal oil assoc. 17 18–22 (in chinese with english abstract) (2002). [12]. hyun j., maeda t., morita n., effect of fungal α-amylase on the dough properties and bread quality of wheat flour substituted with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 buculei amelia, gatlan anca, cojocariu adriana, evaluation of the quality of wheat with added improvers, volume xxi, food and environment safety, issue 3 – 2022, pag. 237 – 246 246 polished flours food research international volume 39, issue 1, , pages 117-126 ,(2006) https://doi.org/10.1016/j.foodres.2005.06.008 [13]. harris r .h., jesperson e., a study of the effect of various factors on the swelling of certain cereal starches, journal of colloid science, volume 1, issue 6, december 1946, pages 479-493, (1946), https://doi.org/10.1016/00958522(46)90056-6, [14]. schlegel r. j., hicks, j. a., amdt j., king l. a. thine own self: true self-concept accessibility and meaning in life. journal of personality and social psychology, 96, 473-490, (2009), https://doi.org/10.1037/a0014060 [15]. rp borwankar și cf shoemaker, rheology of foods , elsevier science, (2013), pp. 92, isbn 978-1-4832-9258-8 doi: https://doi.org/10.4316/fens.2021.022 197 journal homepage: http://fens.usv.ro/index.php/fens faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3-2021, pag. 197 204 healthy food for teenagers part 2: meat analysis for hormonal drugs *marianna barus 1 , vita antsupova 2 , yuliia lomakina 1 1bukovinian state medical university, chernivtsi, ukraine, barus.m@bsmu.edu.ua 2bohomolets national medical university, kyiv, ukraine, vitaantsupova@gmail.com *corresponding author received 7th july 2021, accepted 15th september 2021 abstract: considering the widespread use of hormonal drugs in animal husbandry, the problem of their possible content in adolescents' food becomes urgent. the remains of synthetic steroid hormones that enter the human body along with meat can have a negative effect on the body that develops. the main goal of our work was to study meat from urban markets for the content of synthetic hormone residues in it. the article presents the results of the study for 2019-2020. from three organized markets in the city of chernivtsi, 23 samples were tested for the content of hormonal drug residues. for the study of each sample, 6 standard qualitative reactions were carried out. a total of 138 tests were carried out. the results showed that, on average, over the period studied, only 22% of the meats tested were not contaminated with hormone residues. most of the meat samples tested contained residues of synthetic hormonal drugs: 75% in 2019 and 82% in 2020. the most popular meat varieties among bukovina adolescents (pork and chicken) contained residues of synthetic hormonal preparations in 100% and 71% of the samples, respectively. this may indicate an increase in the likelihood of eating unhealthy meat with all the negative consequences for the teenager's body. given the small number of samples, the data obtained are preliminary and require further study. keywords: meat, hormonal drugs, steroid hormones, quality response, sensitivity threshold. 1.introduction the main structural elements of all body tissues are proteins. almost half of a person's protein needs is from meat. meat is a valuable food product, so nowadays the consumption of animal products has increased significantly. to meet consumer demand in agriculture, synthetic steroid hormones are widely used to stimulate the growth and muscle mass of animals. this creates the risk of concomitant hormones entering the human body through meat, milk, fish, eggs. the developing organism has the greatest risk from exposure to various biologically active substances [1-3]. hormones – biologically active, chemically diverse substances produced by the endocrine glands in small quantities. the term "hormone" was coined by bayliss and starling in 1905 when they studied the effects of secretin. currently, more than one and a half hundred hormones from various multicellular organisms have been described and isolated. there are many classifications of hormones. given the topic of our study, we give a classification adopted in pharmaceutical chemistry. according to this classification, all hormones are chemically divided into two groups. the first group includes hormones – amino alcohols, amino acids, polypeptides, proteins and compounds http://fens.usv.ro/index.php/fens mailto:barus.m@bsmu.edu.ua mailto:vitaantsupova@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 198 related to them in chemical structure (hormones of the cerebral layer of the adrenal glands, pituitary gland, thyroid and parathyroid glands, pancreas). the second group of hormones has a steroidal structure, they include hormones of the cortical layer of the adrenal glands (corticosteroids), sex hormones (androgens, estrogens, progestogens) [4]. hormones are carried with the blood and regulate all vital processes in the body: metabolism; morphogenesis; sexual development and reproductive function; adaptation to the conditions of existence (reactions of adaptation of the organism). this regulation is due to a biological feedback mechanism. there is also a close relationship between hormones and the nervous system, which is also bilateral. all hormones have common properties: high biological activity (hormones show their effect in very low concentrations); specificity of action (each hormone causes strictly specific corresponding reactions of organs and tissues); distance of action (hormones have their effect on the metabolism of organs and tissues located at a distance from the place of their formation); high selectivity of action (hormones show their effect only on the target organs sensitive to them, whose cells have specific protein receptors for this hormone). excess or deficiency of hormones is equally dangerous. hormonal disorders are the cause of failure of the physiological state of the body and the development of relevant diseases [5-7]. the unique properties of hormones have prompted the creation of a large number of hormonal drugs – drugs that contain hormones or hormonoids are similar in pharmacological effects to hormones. for medical purposes, hormones are secreted from the endocrine glands of animals, it can be both individual substances and total biopreparations. synthetic and semisynthetic analogues of hormones are also used. there are hormone – like substances of plants (phytohormones). these drugs are widely used in medical practice, veterinary medicine, various fields of agriculture. for example, sex hormones, their synthetic analogues and anabolic steroids: estradiol, testosterone, progesterone, trenoboloneacetate are used to stimulate meat and dairy productivity of poultry, laying birds. hormonal drugs, in addition to the positive effects, have a very dangerous negative effect. for example, due to the great economic effect in animal husbandry, thyrostatics, which block thyroid hormones, are often used illegally. these substances disturb the heat balance of the animal's body, which provides high weight gain, the animals become hydrated (such meat is advantageous to sell raw). at the same time, they have a very negative effect on the human thyroid gland, which can lead to mental retardation, reproductive dysfunction. sexual dysfunction and even cancer can be caused by steroid hormones. therefore, the use of hormonal drugs, which by a similar effect outweigh natural hormones more than 100 times, requires strict control, and their impact on the development of the body – further careful study [2, 4, 8]. one of the most difficult tasks is to provide the population with food. nutrition from birth to the last day of human life affects our body. nutrient ingredients enter the human body with food and are converted in the process of metabolism as a result of complex biochemical transformations into structural elements of cells. a balanced diet provides our body with plastic material and energy, creates the necessary physiological and mental performance, and determines the health, activity and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 199 life expectancy, the ability to perceive. nutrition is an important factor in determining the health of a nation. hormonal drugs are widely used in modern animal husbandry for various purposes. as a result, meat sold or processed contains substances which, if constantly supplied, present a serious danger to human health [9]. hormones, especially steroids, which are stored in meat for a long time, can withstand deep freezing and high temperatures. together with food, they are easily absorbed in the intestines, enter the bloodstream and interfere with the work of organs and systems. pregnant women, children, and adolescents are most prone to this effect. the adolescent's body is actively growing, and this happens under the influence of the endocrine system. it is her delicate work that ensures the harmonious development of both the physical and the mental. even small hormonal changes at this age can have consequences. residual amounts of hormonal drugs in meat significantly disrupt hormonal processes, causing serious illness. for example, eating meat that contains male and female sex hormones can cause premature puberty in adolescents or lead to malformation. steroids can act as a factor in the development of obesity in children. synthetic estrogens have been shown to be the most dangerous steroids for human health. these substances are carcinogenic and can stimulate the development of hormonally active tumors of the breast [10]. in violation of the terms of use of drugs, as well as the shutter speed of animals to excrete a stimulant from the body, harmful compounds remain in the meat. to prevent the negative effects of the use of bioactive substances, there are mandatory conditions for keeping animals before slaughter until the complete elimination of hormonal drugs from their body, but these conditions are not always met[6]. 2. materials and methods meat is one of the most popular foods. for a healthy diet, information about the presence of hormones in the meat we eat is very relevant. the purpose of our work was to investigate the content of hormone residues in meat from the city markets; justify the need to monitor meat for steroid hormone residues as a guarantee of safe and healthy nutrition for adolescents. 23 samples of meat from the markets of chernivtsi were tested for the presence of residues of hormonal drugs using standard qualitative reactions in 2019-2020 years. to study each sample, 6 qualitative reactions for the presence of hormones were performed. a total of 138 qualitative tests for hormonal drugs were performed. for qualitative reactions, a concentrate extract from the test meat sample was used, which was prepared for each sample separately. weighing the prepared minced meat according to state standard, weighing 5 g was transferred to a conical flask with 20 ml of twice boiled distilled water; with triple stirring, boiled for 15 minutes the resulting extract was filtered. hormones are chemical compounds that have certain chemical functional groups, each of which can be determined by an appropriate qualitative reaction. due to the fact that the amino acids that are part of the animal protein have identical functional groups with hormones, the protein was denatured by boiling the filtrate for ten minutes. natural hormones undergo chemical transformations in living organisms and in their usual form can not be manifested by qualitative reactions. also, for each of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 200 qualitative reactions there is such a thing as a "threshold of sensitivity", which directly depends on the concentration of the substance being determined. it is known that in the studied samples the initial concentration of synthetic hormones is much lower than the concentration of these substances in animals [8]. that is why when studying the filtrate of one sample in the experimental series of qualitative reactions in some cases, we observed a positive analytical effect of "+" (positive reaction) – the formation of sediment, gas evolution, turbidity, etc .; and in others these signs were absent, which was interpreted as a negative analytical effect "" (negative reaction).the lowest concentration of hormone that can be detected is the open minimum, which is less than one microgram in the minimum volume of the extremely dilute solution [11]. qualitative analysis of steroid hormones in the studied samples was carried out taking into account the chemical classification according to standard methods. the structural basis of steroid hormones is the skeleton of the hydrocarbon cyclopentaneperhydrophenanthrene (fig. 1.).a common reaction for all steroid hormones and their synthetic analogues is the reaction with concentrated sulfuricacid. . fig. 1. general formula of steroid hormones reaction i. when dissolved in concentrated sulfuric acid and heated, the substance gives a specific color, sometimes fluorescence, with the subsequent addition of water, chloroform, iron (iii) solution of ammonium sulfate color changes, there is a specific fluorescence. steroid hormones that have a keto group in position 3(fig. 2.), enter a substitution reaction with hydroxylamine hydrochloride, phenylhydrazine,2,4dinitrophenylhydrazine, isoniazid – precipitation with a characteristic melting point is observed or a characteristic color appears(yellow,orange-red). fig. 2. reactions of steroid hormones that have a keto group food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 201 reaction іі. for the identification of hormones having hydroxy groups in positions 3 or 17, the formation of esters is often used: acetates, benzoates. the reaction is carried out in 80% acetic acid, and gently concentrated sulfuric acid is added – an orange color appears at the phases boundary; the upper phase is characterized by yellow-green fluorescence. the esterification reaction (fig. 3.), resulting in the formation of an ester group, which can be detected using a hydroxamic test (fig. 4.). fig. 3. esterification reactions fig. 4. hydroxamic test reaction ііі. hydrolysis of hormones with potassium hydroxide in ethanol produces estradiol, which precipitates when diluted with water and acidified with hydrochloric acid. reaction іv. steroid hormones are dissolved in sulfuric acid and added to ammonium sulfate solution (iii). the precipitate is reddish-brown; after dilution with water, a pink-red precipitate precipitates. reaction v. the extract concentrate of hormones is dissolved in chloroform, a solution of fecl3 and pyridine are added. there is a blue color that turns green from the excess pyridine: the phenolic groupalso, iron trichloride can act as an oxidant for alcohol hydroxyl (fig. 5.). fig. 5. oxidation reaction of alcohol hydroxyl reaction vi. when added to the test sample isoniazid in the presence of hydrochloric acid, a yellow color appears [11-12]. 3. results and discussion to study the presence of hormonal drugs in meat at random in the markets of chernivtsi, 12 samples of different types of meat were selected (2019). we performed 72 qualitative reactions to the content of hormonal drugs in the studied samples, 13 of which showed a positive effect, ie 18% of specific reactions had a sufficient threshold of sensitivity to detect hormonal drugs in the studied meat samples (table 1). tests for synthetic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 202 hormones showed that only in three samples (№1 rabbit; №5 veal; №8 chicken) all qualitative reactions were negative, ie it can be assumed that 25% of the studied meat did not contain synthetic hormones. qualitative reactions gave a positive analytical effect in 9 samples, which indicates the presence of hormonal drugs in 75% of the studied meat. table 1 the results of the study of the presence of hormonal drugs in meat products from the markets of chernivtsi for 2019 year № meat sample kind of meat sampling market reaction number і іі ііі іv v vi 1 rabbit lower 2 chicken main + 3 pork main + + 4 chicken main + 5 veal central 6 pork central + 7 chicken central + 8 chicken lower 9 veal main + + 10 veal central + + 11 pork central + 12 pork lower + + notes:і; іі; ііі; іv; v; vi – see materials and methods; «+» – positive reaction; «-» –negative reaction. in order to determine the trend of contamination of meat with hormonal drugs, similar studies were conducted in 2020 (table 2). 66 qualitative reactions to the content of hormonal drugs in the test samples were performed, 14 of which showed a positive effect, which is 21% of the total. compared to 2019, we see an increase in this indicator by 3%. in 2020, only in two samples (№1 chicken; №2 rabbit) all qualitative reactions were negative. it can be assumed that 18% of the studied meat did not contain synthetic hormones, and 82% (9 samples) were contaminated with hormonal drugs. table 2 the results of the study of the presence of hormonal drugs in meat products from the markets of chernivtsi for 2020 year № meat sample kind of meat sampling market reaction number і іі ііі іv v vi 1 chicken central 2 rabbit main 3 chicken lower + + 4 veal central + 5 pork central + 6 veal central + + 7 chicken main + 8 pork lower + 9 pork main + + 10 veal central + + 11 pork central + + notes: see table 1. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 203 compared to 2019, we see an increase in this indicator by 7%. this may indicate an increase in the use of hormonal drugs in livestock and an increase in the likelihood of purchasing unsafe meat. analyzing the results for the entire period of research (fig. 6), we can conclude that about 78% (18 of 23 samples) of the studied meat – contaminated with residues of hormonal drugs. steroid hormones are more likely to enter the body of a teenager along with meat. synthetic steroid hormones can adversely affect the sexual development of adolescents, be a cause of obesity in children.during the experiment, it was found that only 22% of meat (5 of 23 samples: rabbit, chicken and veal) were uncontaminated by hormonal drugs. the most commonly used type of pork in 100% (8 samples) contained residues of hormonal drugs. hormone contamination of chicken was detected in 71% (5 samples out of 7). veal samples contained hormones in 83% (5 samples out of 6). 100% (2 samples) of rabbit had no residues of hormonal drugs. fig. 6.the content of residues of synthetic hormones in samples of popular meat varieties given the small number of samples, the data obtained are pilot in nature and require further study. 4.conclusion 78% of the meat tested is contaminated with hormonal residues. there is a tendency to increase the content of synthetic hormones in meat from the markets of chernivtsi. 22% of the studied meat (rabbit, chicken and veal) does not contain residues of hormonal drugs. the most common types of meat: 100% of the tested pork samples contain residues of hormonal drugs; 71% of chicken contains residues of synthetic hormones. hormone steroids can get into the body of a teenager along with meat. the content of harmful substances in meat does not depend on its type. the obtained data are of a pilot nature and require further research. 5.references [1] belachew b. hirpessa, beyza h. ulusoy, canan h. hormones and hormonal anabolics: residues in animal source food, potential public health impacts, and methods of 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% rabbit veal chicken pork food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 marianna barus, vita antsupova, yuliia lomakina, healthy food for teenagers part 2: meat analysis for hormonal drugs, food and environment safety, volume xx, issue 3 – 2021, pag. 197 – 204 204 analysis. journal of food quality, volume, 2020: 1-12, (2020) [2] tan x.-t., li z.-m., deng l.-g., zhao s.-c., wang m.-l. analysis of 13 kinds of steroid hormones in raw milk using modified quechers method combined with uplcqtofms. journal of integrative agriculture, xv, 9: 2163-2174, (2016) [3] ahmedin a. musa. effect of different packaging methods on consumers eating quality of beef, journal of faculty of food engineering, ştefan cel mare university of suceava, romania, xviii, 3: 235-245, (2019) [4] donovan c. if fda does not regulate food, who will? a study of hormones and antibiotics in meat production, american journal of law and medicine, xxxxi, 2-3: 459-482, (2015) [5] us–fda, tolerances for residue of new animal drugs in food, in animal drugs, feeds, and related products, implantation or injectable dosage form new animal drugs, code of federal regulation (cite: 21cfr 522),volume 6, fda, silver spring, md, usa, (2017) [6] us–fda, “implantation or injectable dosage form new animal drugs, zeranol,” in animal drugs, feeds, and related products, code of federal regulation (cite 21cfr 556.760), volume 6, fda, silver spring, md, usa, (2017) [7] fsis, united states national residue program for meat, poultry, and egg products 2015 residue sampling plans, united states department of agriculture food safety and inspection service (usda-fsis), office of public health science, washington, dc, usa, (2015) [8] cac, maximum residue limits (mrls) and risk management recommendations for residues of veterinary drugs in foods, codex alimentarius commission (cac), mrl 2, cac, japan,1-84, (2017) [9] kaabia z., dervilly-pinel g., popot m.a., baily-chouribberry l., ploul p., bonnaire y., le bizeci b. monitoring the endogeneous steroid profile disruption in urine and blood upon nandrolone administration: an efficient and innovative strategy to screen for nandrolone abuse in entire male horses. drug test anal., 6: 376-388, (2014) [10] yucel u., atasoy n. determination ofsome anabolic hormone residues in cattle meat consumption in van, turke, international journal of advanced research, vi, 8: 129-139, (2018) [11] bezugliy p. a. pharmaceutical chemistry, vinnytsia: the new book, 560, (2006) [12] state pharmacopoeia of ukraine, 556, (2015) healthy food for teenagers part 2: meat analysis for hormonal drugs 188 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 188 – 192 the eff e ct of ult ras on ic d ur ing nac l a nd k c l meat b r ining *olga drăghici1, alina toader1, daniela oana andrăşescu1 1faculty of agricultural sciences, food industry and environmental protection “lucian blaga” university, 5-7 ion raţiu street, 550012, sibiu, romania, e-mail: olga.draghici@ulbsibiu.ro *corresponding author received august 19th 2014, accepted september 24th 2014 abstract: the main purpose of the paper is the comparative study of the salting process for pork and beef, in brine of nacl and kcl at different concentrations, using ultrasonic treatment in order to reduce the time needed salting process (for salting). ultrasounds accelerates the salting process, due to increased mass transfer, through the generation and collapse of microscopic bubbles in a liquid medium, but also due to changes in the structure of muscle tissue.the study was conducted on pork m. longissimus lumborum and on bovine muscle psoas major. the samples were submerged in brine for 48 hours, at different concentrations (5%, 10%, 15% nacl and kcl respectively), and then the salt content was determinated. the samples were maintained for 30 minutes in an ultrasonic bath. it was noted that the results are higher at ultrasonic treatment, in the case of pork preserved in brine nacl, respectively for beef in kcl solution. the potassium ion produces an enhanced efficacy of salting process only at low concentrations of brine. also ultrasonic treatment acts positively for the diluted solutions. results showed that in general ultrasound accelerates the process of salting meat. keywords: nacl, kcl, salting, meat, ultrasound 1. introduction salting meat is an important step, during meat processing. sodium chloride influences the conservation, the flavor and texture of meat products. its effect of conservation is mainly due to the water activity reduction capacity [1]. moreover, salt affects some chemical or biochemical phenomena such as proteolysis, lipolysis, or oxidation of lipids that contribute to the development of texture and flavor specific to meat [2]. a low levels of salt in meat products, raises issues in terms of the perception of salinity. also the characteristic flavor intensity decreases [3]. however, a high consumption of sodium chloride is associated with the occurrence of hypertension [1]. this fact has promoted the current trend to reduce the intake of sodium by partial or total replacement of sodium ion, with other ions such as potassium, calcium or magnesium [4,5,6,7]. most researchers believe that the most effective substituent as potassium chloride, although it has some disadvantages: in high doses gives the product a metallic taste, astringent and bitter, but modern methods allow to eliminate these inconvenient [8]. on the other hand, the salting can be made with dried salt or with brine by injecting it or by immersion of the pieces of meat in the brine. this latter option is rarely used due to the long time of salting. to increase the mass transfer, can be used ultrasound (us) [9, 10]. ultrasonic waves occur at frequencies higher than 20 khz and can be classified according to their frequency into three groups as follows: power us at the frequencies less than 100 khz, high frequency for frequencies greater than 100 khz and less than 1 mhz and diagnostic us for frequencies between 1 and 500 mhz. in salting meat is using frequencies from 20 to 100 mhz, this interval being food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 o l ga d r ăg h ic i , ali n a t o a d er , d anie la o a na a n dr ăş e s cu , t he e ff e c t of u lt r a s o ni c d ur i ng n ac l a nd k c l m e a t b r i n i n g, fo o d a nd e nv ir o nm e nt s a f e t y, v o l ume x i i i, i ss ue 3 – 2 01 4 , p a g. 18 8 -19 2 189 specific in cases of concern on physical or chemical phenomena [11]. a sound wave in a liquid medium produces a cyclic sequence of compression and expansion. during the phase of expansion, in the liquid medium occur bubbles and due to the lower pressure inside them, these bubbles are filled with gas and vapour. when the bubbles reach a critical size, depending on the wave frequency and the pressure inside the bubbles is much smaller than the outside, occurs a collapse and liquid jets enter into the space that was occupied by bubbles. for this reason, in the space that was occupied by bubbles and in the layer closer, it is found an increase in temperature and pressure. subsequently the formation and expansion of bubbles is resumed. so, we can say that the effect of ultrasound is due of generation and collapse processes, of cavitation bubbles, which determine the appearance of microcurrents [12]. shortening the process of salting meat immersed in the presence of ultrasound, can be explained in conclusion by these mechanisms [13]. thus, the main objective of this work is to provide further information on nacl and kcl migration within the meat upon salting with or without ultrasonic treatment. 2. experimental this study was realized on pork longissimus lumborum and m. psoas major bovine muscle, commercially purchased on the day of analysis and maintained at 4°c until analysis. subsequently it was partitioned, resulting pieces with identical shapes and weights (10 g). these were maintained in various concentrations of nacl brine, respectively kcl in tables 1-2. ultrasound treatment was achieved by keeping the samples for 30 minutes in an ultrasonic bath (elma 60 h). all samples were kept for 48 hours in the corresponding solution at a temperature of 4°c, then were maintained in distilled water for 20 seconds [14] and the salt content was determinated by the method sr iso 1841:2000 [15]. table 1 preparing pork samples samples analyzed sample pork 10 g /sample pp1 +50 ml solution nacl 5% pp2 +50 ml solution nacl 10% pp3 +50 ml solution nacl 15% pp4 +50 ml solution nacl 5% + us* pp5 +50 ml solution nacl 10% + us pp6 +50 ml solution nacl 15% + us pp7 +50 ml solution kcl 5% pp8 +50 ml solution kcl 10% pp9 +50 ml solution kcl 15% pp10 +50 ml solution kcl 5% + us pp11 +50 ml solution kcl 10% + us pp12 +50 ml solution kcl 15% + us * us sonication table 2 preparing beef samples samples analyzed sample beef 10 g /sample pb1 +50 ml solution nacl 5% pb2 +50 ml solution nacl 10% pb3 +50 ml solution nacl 15% pb4 +50 ml solution nacl 5% + us* pb5 +50 ml solution nacl 10% + us pb6 +50 ml soluție nacl 15% + us pb7 +50 ml soluție kcl 5% pb8 +50 ml soluție kcl 10% pb9 +50 ml soluție kcl 15% pb10 +50 ml soluție kcl 5% + us pb11 +50 ml soluție kcl 10% + us pb12 +50 ml soluție kcl 15% + us * us sonication in order to compare between them the results obtained for nacl and kcl brine, it was preferred the percentage concentration of chlorine from meat samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 o l ga d r ăg h ic i , ali n a t o a d er , d anie la o a na a n dr ăş e s cu , t he e ff e c t of u lt r a s o ni c d ur i ng n ac l a nd k c l m e a t b r i n i n g, fo o d a nd e nv ir o nm e nt s a f e t y, v o l ume x i i i, i ss ue 3 – 2 01 4 , p ag . 18 8 -19 2 190 3. results and discussion figures 1 and 2 shows the concentration of chlorine ions in the pork, respectively beef, maintained under the conditions indicated in tables 1 and 2. it can be seen, both for the pork and for beef when brine was obtained with nacl, respectivley with kcl, the ultrasound treatment had a beneficial effect, favoring the salting. in order to highlight the effect of ultrasound treatment was done the ratio of chloride ion concentrations from the samples meat, held in the same type of brine without or with ultrasound (fig. 3, 4). all ratios have values higher than one, mostly around 1.5 and for beef that was in 5% kcl solution, this ratio reaches 2.03. it also is found that the results are higher for pork, maintained in brine of nacl, respectively in the case of beef in kcl solution. not the least (finally) it may be noted that in general, the effect of ultrasound treatment is greater for dilute solutions. these results strengthens the hypothesis previously issued namely that ultrasound leads to accelerated salting procces, due to increased mass transfer in the samples analyzed, by the implosion of microscopic bubbles due to their propagation [16-18]. figure 1. variation of concentration in chloride ion for samples pp1-pp12 pork, maintained in brine for different concentration (5%, 10%, 15%) of nacl, respectively kcl. figure 2. variation of concentration in chloride ion for samples pb1-pb12 beef, maintained in brine for different concentration (5%, 10%, 15%) of nacl, respectively kcl. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 o l ga d r ăg h ic i , ali n a t o a d er , d anie la o a na a n dr ăş e s cu , t he e ff e c t of u lt r a s o ni c d ur i ng n ac l a nd k c l m e a t b r i n i n g, fo o d a nd e nv ir o nm e nt s a f e t y, v o l ume x i i i, i ss ue 3 – 2 01 4 , p a g. 18 8 -19 2 191 figure 3. ratio of the concentration of chloride ions from the samples of pork maintained in the same type of brine with / without ultrasound. figure 4. ratio of the concentration of chloride ions from the samples of beef maintained in the same type of brine with / without ultrasound. on the other hand, one possible explanation is that at the level of ultrastructure and microstructure of muscle tissue, during treatment with ultrasonic may occur modifications which favoring the salting process. thus, reynolds et al. (1978) identified ruptures in the thick fibers and increase in size of endomysum [19]. similar observations were made by vimini et al. (1983) [20]. subsequently, siró et al. (2009), noted a thickening of myosin filaments and ruptures at the level of z line due to treatment with ultrasound, thus increasing the space between the fibers [17]. these changes at the muscle structure due to the effects of ultrasound, however are not supported by all researchers. mcdonnell (2014) by histological studies could not identify changes in the meat subjected to ultrasound treatment in comparison with the blank [21]. comparing the effect of na ions and k on the salting process it is found to be variable. the ions of k favors salting process only at low concentrations. in the case of pork preserved in brine 5% and subjected to ultrasonic treatment, cl ions concentration values were 1.91% in nacl solution and 2.20% in kcl solution. for beef, the highest variation has been reported in favor of k for ultrasound treatment in 5% concentration, namely 1.77% in nacl solution, respectively 2.66% in kcl solution. barat et al (2014) studying the kinetics of the salting process, they have noted that the presence of kcl in brine of nacl, in proportion 50%, increases the salting speed, and their explanation was that the addition of kcl, decreases the ph from 9.9 to 6.2. the same authors, after some mathematical calculations observes that the diffusion coefficient is higher for ion of k, than for ion of na, in brine concentration 15% [22]. 4. conclusion in the pork and beef preserved in brine, intensifies the procces of salting using ultrasound treatment. from the analyses performed, result that at low concentrations of brine, the effect is more intense. moreover it was evidenced that the variation of ions cl concentration was higher for beef in brine of kcl. pork has a higher concentration of chloride ions in brine of nacl. thus was presented a possibility to shorten the time required salting process using ultrasonic treatment and its effects during brining on meat. 1,00 1,50 2,00 2,50 p b 4 / p b 1 p b 5 / p b 2 p b 6 / p b 3 pb 10 / p b 7 pb 11 / p b 8 pb 12 / p b 9 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 o l ga d r ăg h ic i , ali n a t o a d er , d anie la o a na a n dr ăş e s cu , t he e ff e c t of u lt r a s o ni c d ur i ng n ac l a nd k c l m e a t b r i n i n g, fo o d a nd e nv ir o nm e nt s a f e t y, v o l ume x i i i, i ss ue 3 – 2 01 4 , p a g. 18 8 -19 2 192 5. references [1]. ruusunen m., puolanne e., reducing sodium intake from meat products. meat sci., 70(3): 531-41, doi: 10.1016/j.meatsci. 2004.07.016, (2005). [2]. armenteros m., aristoy m.c., barat j.m., toldrá f., biochemical changes in dry-cured loins salted with partial replacements of nacl by kcl. food chemistry, 117(4), 627-63, (2009). [1]. gonçalves a.a., development of smoked chicken product with low-sodium. estudos tecnológicos, 5(3): 431-439, (2009). [2]. paulsena m.t., nys a., kvarberg r., hersleth m., effects of nacl substitution on the sensory properties of sausages: temporal aspects. meat sci., 98: 164–170, (2014). [3]. baker i.a., alkass j.e., saleh h.h., biochemical, microbial and sensory changes in lamb patties salted with partial replacement of nacl and kcl during storage. j. anim. sci., 1(2): 27-31, (2012). [4]. us institute of medicine, strategies to reduce sodium intake in the united states. in j. e. henney c.l. taylor, boon c.s. (eds.), institute of medicine (us) committee on strategies to reduce sodium intake, (2010). [5]. zhao j., claus j.r., influence of partial or complete replacement of sodium with potassium chloride blends on protein extraction and meat quality of sausages 59th international congress of meat science and technology, 1823rd august2013, izmir, turkey, (2013). [6]. berry d., reducing salt. meat & poultry magazine, 59, 2:64-68, (2013). [7]. welti-chanes j., velez-ruiz j.f., transport phenomena in food processing, crc press, 265-278, (2002). [8]. jambrak a.r., herceg z., subaric d., babic j., brncic m., brncic s.r., bosiljkov t., cvek d., tripalo b., gelo j., ultrasound effect on physical properties of corn starch. carbohydrate polymers, 79, 91– 100, (2010). [9]. bermúdez-aguirre d., mobbs t., barbosa-cánovas g.v., ultrasound applications in food processing, hao feng, gustavo barbosa-cánovas, jochen weiss, ultrasound technologies for food and bioprocessing, springer science & business media, 65-106, (2010). [10]. taner b., aslihan d., ultrasound in food technology, handbook on applications of ultrasound, sonochemistry for sustainability, edited by ackmez mudhoo, crc press,163-182p (2011). [11]. garcía-pérez j.v., de prados m, pérez-muelas n., cárcel j.a., benedito j., ultrasonic characterization of pork meat salting. iop conf. ser.: mater. sci. eng. 42, doi:10.1088/1757-899x/42/1/012043, (2012). [12]. ozuna c., puig a., garcía-pérez j.v., mulet a., cárcel j.a., influence of high intensity ultrasound application on mass transport, microstructure and textural properties of pork meat (longissimus dorsi) brined at different nacl concentrations. j. food eng. 119: 84–93, (2013). [13]. sr iso 1841:2000 [14]. mcdonnell c.k., allen p., morin c., lyng j.g., the effect of ultrasonic curing on meat protein and water–protein interactions in meat. food chemistry, 147: 245– 251, (2013). [15]. siró i., vén, c., balla, c., jónás, g., zeke i., friedrich l., application of an ultrasonic assisted curing technique for improving the diffusion of sodium chloride in porcine meat, j. food eng., 91: 353–362, (2009). [16]. cárcel j.a., benedito j., bon j., mulet a., high intensity ultrasound effects on meat brining. meat sci., 76: 611–619, (2007). [17]. reynolds j.b., anderson d.b., schmidt g.r., theno d.m., siegel d.g., effects of ultrasonic treatment on binding strength in cured ham rolls. j. food sci., 43: 886–869, (1978). [18]. vimini r.j., kemp j.d., fox j.d. effects of low frequency ultrasound on properties of restructured beef rolls. j. food sci., 48: 1572– 1573, (1983). [19]. mcdonnell c.k., allena p.c. morinc, lyngb j.g., the effect of ultrasonic salting on protein and water–protein interactions in meat. food chemistry, 147(15): 245–251, (2014). [20]. barata j.m., baigtsa d., aliñoa m., fernándezb f.j., pérez-garcíac v.m., kinetics studies during nacl and kcl pork meat brining, j. food sci., 106 (1): 102–110, (2011). 148 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 2 2020, pag. 148 155 organic acids and physico-chemical parameters of romanian sunflower honey *daniela pauliuc1, mircea oroian1 1faculty of food engineering, stefan cel mare university, suceava, romania daniela_pauliuc@yahoo.com, *corresponding author received 5th aprilie 2020, accepted 28th june 2020 abstract: the samples of sunflower honey collected from romania were analyzed in order to characterize them from the physico-chemical point of view. melissopalynological analysis, together with the determination of organic acids and physico-chemical parameters (color, moisture, free acidity, ph, electrical conductivity, hydroxymethylfurfural (hmf) content, dpph radical scavenging activity) were chosen as methods for honey characterization. the results of the melissopalynological analysis showed that all honey samples had a high percentage of helianthus annuus pollen grains and these results help to classify the samples as monofloral honey. the values determined for electrical conductivity (314.82 440.55 µs/cm), color (32.87 47.52 mm pfund), ph (3,65 4.34) confirmed the purity of the samples. in the case of moisture content, all the sunflower honey samples complied with the limit imposed by codex alimentarius (20%). the hmf content ranged between 2.66 and10.96 mg hmf/kg,which showed that the samples were fresh and had a hmf value below the limit of 40 mg hmf/kg. the dpph radical scavenging activity ranged between 60.24 and 76.95 %. gluconic acid was the organic acid predominant in all honey samples. these results led to an accurate classification of the analyzed honey according to its botanical origin, namely sunflower honey. keywords: honey, organic acids, physico-chemical properties 1. introduction honey has got well-known functional properties that determine its role in preventing disease, apart from being recognized as a natural product with multiple nutritional values. its properties and also its extraordinary medicinal values can be guaranteed only if the product is authentic [1]. in romania, the arable land used for agriculture represents over 60% of the total land of the country. among crops, oilseeds (mainly brassica napus and helianthus annuus) account for about 10%. other crops of importance for the country production include wheat, soybean, sugar beet, vegetables and vineyards. flora and agricultural production determines which the most common types of romanian honey are; these include acacia, sunflower, lime, raspberry, mint, chestnut, heather, honeydew and polyfloral honey. sunflower honey is produced in large quantities in romania because sunflower crops have been present in this region for a long time. sunflower honey is an assortment of monofloral honey from romania and it is widely consumed [2]. honey is well known to consumers as a sweetener, but it is also a high quality food that is rich in beneficial substances (sugars, phenolic compounds, proteins, carotenoids, enzymes, vitamins, minerals, organic and free amino acids) essential for ensuring balanced biological processes.therefore, the functional potential of honey has recently been throughly investigated [3]. some studies could easily demonstrate the health benefits of honey of different botanical origin [4],[5], as well as the correlation between its benefitial effects on http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 149 human health and the levels of bioactive compounds, particularly polyphenols and phenolic compounds, contained by honey. natural honey is a sweetener that presents high dietary value and an exclusive flavor character and therefore, it is more expensive than other sweeteners. this makes honey a frequent target of adulteration with the purpose of gaining increased economic profit. when compared to the adulteration of synthetic sweeteners, the most common fradulent practices in honey are relatively easy to achieve, but at the same time they can be difficult to detect. considering that honey is the third most adulterated food product, authenticity of honey is of global concern for both commercial producers and consumers [6]. honey is increasingly valuable for our diet due to its medicinal benefits, as it also has a high content of natural antioxidants. honey also contains a small proportion of organic acids (0.5%), which can be used as indicators of deterioration, freshness, purity and authenticity. organic acids are the result of aerobic and anaerobic fermentation and they are responsible for the special flavors of honey [7]. the aim of this study was to analyze the organic acids content and the physicochemical parameters (melissopalynological analysis, moisture, ph, color, free acidity, electrical conductivity, hydroxymethylfurfural content, and antioxidant activity) of romanian sunflower honey. 2. materials and methods 2.1. samples six samples of sunflower honey from 2018 from romania were liquefied at 50 °c and homogenized to carry out the analysis. 2.2. melissopalynological analysis pollen grains from sunflower honey samples were counted from a sediment spread on a microscopic slide using the light microscope (motic ×40) [8], [9]. this sediment was obtained from 10 g of honey dissolved in 40 ml of distilled water. the resulting mixture was centrifuged at 4500 rpm for 15 min. the supernatant was removed, the residue obtained was redissolved and centrifuged for another 15 minutes. [8], [9]. 2.3. physico-chemical analysis the physico-chemical analyses were performed for sunflower honey samples according to the analytical methods harmonized by the international honey commission [10]. the physico-chemical characteristics analyzed for honey samples were the following: 2.3.1.moisture content the abbe refractometer leica mark ii plus (leica, usa) was used to determine the moisture content for liquefied sunflower honey samples. liquefaction took place at a temperature of 50 ⁰c. the chataway table [11] was used to calculate water content (%). 2.3.2. electrical conductivity electrical conductivity was measured using a portable conductometer hq14d, hach (usa). 2.3.3. ph ph meter mettler toledo fivego, mettler toledo, (sua) was used to analyze the ph of samples. 2.3.4. free acidity free acidity was measured using titroline easy, schott instruments (germany). 2.3.5. color color was analyzed using the photometer pfund, hanna instrumets hi 96785 and the portable chromameter cr-400, konica minolta, (japan). 2.3.6. hydroxymethylfurfural (hmf) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 150 hmf content was determined with a spectrophotometer uv-vis-nir schimadzu uv-3600, (schimadzu corporation, japan) using the white method[12]. 2.3.7. dpph assay the determination of 1,1-diphenyl-2picrylhydrazyl (dpph) radical scavenging activity was made, as follow: 1 g of honey was dissolved in 5 ml of methanol 40% (v/v, with acidified water) and stirred for 15 min with a magnetic stirrer [13]. then, 35 µl of honey solution were mixed with 250 µl of dpph. the absorbance was measured using a qe65000 spectrometer (ocean optics, sua) at 515 nm. the results were obtained using the formula in eq. 1: % dpph = 100 0 1 0          a a a , (1) (3) where a0is the dpph absorbance, a1 is the sample absorbance. 2.4.determination of organic acids 0.5 g of sunflower honey was mixed with 2.5 ml of 4% metaphosphoric acid (w/v) and then were vortexed. they were determined according to the method proposed by özcelik et al. [14]. 3. results and discussion pollen analysis pollen analysis is the traditional method proposed by the international bee botanical commission (icbb) in 1970 [11]. using the microscopic analysis we can determine the plants visited by bees during honey production [15]. the helianthus annuus pollen was predominant in all the samples, accounting for more than 45% pollen grains (50-92 % pollen grains). oroian & ropciuc [16] reported in a different study made on sunflower honey that their samples had around 60% of pollen belonging to helianthus annuus. moisture content processing and storage conditions influence the moisture content of honey along with the relative moisture in the region of honey origin [17]. the honey samples that have a high moisture content may be unstable during storage. thus, the moisture content is used as an indicator of honey quality and stability, and codex alimentarius has set an allowable limit of 20% [18, 19]. the moisture content of the analyzed samples ranged from 16.23 % to 20.39%. these results were in accordance with the values (17.3%) reported by oroian et al.[20] when analyzing sunflower honey. in serbian sunflower honey the moisture content varied between 15.5 and 20.63% [21]. do nascimento et al.[22] reported for forty-nine monofloral honey samples a variation of moisture content from 16.4% to 19.4%. ph normally the ph of honey is between 3.5 and 5.5 due to the presence of organic acids that give both the aroma of honey and protection against microbial damage [6]. ph values contribute to the identification of honey botanical origin [23]. ph of the sunflower honey samples ranged from 3.65 to 4.34. kukurová et al. [24] reported for sunflower honey a ph of 3.7 and terrab et al.[25] reported ph values between 3.56 and 4.79 in a study of spanish thyme honey. manzanares et al. [26] reported values between 3.70 and 5.15 for one hundred eighty and two samples of honey samples from tenerife. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 151 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 152 free acidity the analysis of free acidity is preferred to determine the freshness of honey. along with the deterioration of honey, the value of free acidity increases as a result of the fermentation of sugars into organic acids. thus, free acidity depends directly on the organic acids present in honey, but also on the harvesting season as well as on the geographical origin [17]. 50 milliequivalents of acid per 1,000 grams is the maximum limit allowed by legislation for free acidity [27].the free acidity of sunflower honey analyzed in this study ranged from 15.94 to47.32 meq/kg. matović et al. [28] reported for serbian honey a value of 23.64% and geană et al. [29] reported for romanian sunflower honey a free acidity value of 35.1 %. hmf content alongside free acidity, the analysis of hmf content is a parameter used to indicate the degree of freshness of honey. 40 mg/kg is the limit imposed by the legislation for hmf. the samples that exceed this value have been subjected to strong or prolonged heat treatment or they have been improperly stored [30], [18], [27]. all the honey samples analyzed in this study had a low hmf content. matović et al.[28] reported a value of 2.69 mg/kg for serbian honey samples and yardibi & gumus [31] reported an average hmf content of 7.02 mg/kg in the honey samples analyzed in their study. color the first quality attribute evaluated by consumers is the color of honey that must fall within a range expected and accepted by consumers [32],[33]. color is often used to distinguish variations that may be due to geographical origin, botany or production particularities [34]. by visual color analysis of honey, the botanical origin of this product, as well as the degree of heat treatment and the presence of flaws such as fermented products can be assessed [35]. the color of sunflower honey ranged from white (32.87 mm pfund) to extra light amber (47.52 mm pfund). kadar et al.[36] reported for sunflower honey a value of 58.11 mm (light amber color).juan-borrás et al.[37] reported for romanian sunflower honey a value of 51 mm and the value of 66.7 mm was reported for sunflower honey from spain. all honey samples showed similar lightness values (28.5-49.53). electrical conductivity electrical conductivity is a parameter that provides information on the botanical origin of honey. electrical conductivity is positively correlated with the ash content and acidity due to the presence of ions, acids and organic proteins [38]. minerals are brought in honey primarily with pollen, so the electrical conductivity correlates with the pollen content of monofloral honey [39], which determines the botanical origin [40]. as the values in table 1 show, the sunflower honey samples analyzed had an electrical conductivity less than 500 µs/cm, therefore these samples were of pure floral honey. devillers et al. [41] reported for sunflower honey a mean value of 306.2 µs/cm. in our study the values ranged between 314.82 to 440.55 µs/cm. kádár et al. [36] reported for sunflower a value of 420 µs/cm and for acacia honey 220 µs/cm. matović et al.[28] reported for serbian honey a value of 220 µs/cm. dpph assay the dpph test was used because high dpph purification activity provides the sample with a superior antioxidant activity [42]. in this study the sunflower honey had a dpph value between 60.24 and 76.95 %.đogo mračevića et al.[43] reported for sunflower honey values between 33.18% and 40.18 %.romanian sunflower honey had a higher antioxidant activity than those in serbia investigated by đogo mračevića. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 153 organic acids content organic acids are very important because they contribute to the preservation and assessment of the sensory properties of honey [17]. gluconic acid was the predominant acid in all the sunflower honey samples analyzed in this study. the maximum gluconic acid content was determined in sample 2 (5.62 g/kg) and the lowest value in sample 1 (3.99 g/kg). sahin et al. [44] identified gluconic acid as the predominant acid in all the honey samples that they studied, and the maximum gluconic acid content was reported in chestnut honey (8.90 g/kg). formic, acetic propionic and succinic acids were also present in all sunflower honey samples in this study, but in smaller quantities. sahin et al. [44] reported the presence of formic acid in almost all the samples. in their study, suarez-luque et al.[45], reported a low concentration of citric, malic, succinic and fumaric in eucalyptus honey samples and a high concentration in chestnut honey. 4. conclusion the physico-chemical characterization, pollen analysis and organic acids content of sunflower honey samples collected from romania were performed to examine the quality of honey samples. these analyses were used to determine the botanical origin of honey. the six honey samples met the quality criteria examined (electrical conductivity, color, moisture, ph, free acidity, and hmf content), and together with the pollen analysis and organic acids content indicated that the honey samples analyzed were monofloral sunflower samples. 5. acknowledgment this paper was supported by “decide development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu / 380/6/13/125031, project co-financed from the european social fund through the 2014 2020 operational program human capital”. 6. references [1]. ansari, m. j., al-ghamdi, a., khan, k. a., adgaba, n., el-ahmady, s. h., gad, h. a., ... & kolyali, s., validation of botanical origins and geographical sources of some saudi honeys using ultraviolet spectroscopy and chemometric analysis. saudi journal of biological sciences, 25(2), 377-382, (2018). [2]. isopescu, r. d., josceanu, a. m., colta, t., & spulber, r., romanian honey: characterization and classification. honey analysis, 27, (2017) [3]. das, a., datta, s., mukherjee, s., bose, s., ghosh, s., & dhar, p., evaluation of antioxidative, antibacterial and probiotic growth stimulatory activities of sesamum indicum honey containing phenolic compounds and lignans. lwtfood science and technology, 61(1), 244-250, (2015). [4]. gašić, u., šikoparija, b., tosti, t., trifković, j., milojković-opsenica, d., natić, m., & tešić, ž., phytochemical fingerprints of lime honey collected in serbia. journal of aoac international, 97(5), 1259-1267, (2014). [5].da silva, i. a. a., da silva, t. m. s., camara, c. a., queiroz, n., magnani, m., de novais, j. s., & de souza, a. g.. phenolic profile, antioxidant activity and palynological analysis of stingless bee honey from amazonas, northern brazil. food chemistry, 141(4), 3552-3558, (2013) [6]. siddiqui, a. j., musharraf, s. g., & choudhary, m. i., application of analytical methods in authentication and adulteration of honey, food chemistry, 217, 687-698, (2017) [7].mato, i. – huidobro, j. f. – simollazono, j. – sancho, m. t.: analytical methods for the determina-tion of organic acids in honey. analytical chemistry, 36, pp. 3–11, (2006) [8]. von der ohe, w., oddo, l. p., piana, m. l., morlot, m., & martin, p., harmonized methods of melissopalynology, apidologie, 35(suppl. 1), s18-s25, (2004) [9]. louveaux, j., maurizio, a., & vorwohl, g., commission internationale de botanique apicole de l'uisb: les méthodes de la mélisso-palynologie, apidologie, 1(2), 211-227, (1970) [10]. harmonised methods of the international honey commission, (2008) [11]. bogdanov, s., lullmann, c., mossel, b. l., d'arcy, b. r., russmann, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 154 h., vorwohl, g. & flamini, c., honey quality, methods of analysis and international regulatory standards: review of the work of the international honey commission, mitt. lebensm. hyg., 90, 108-125, (1999) [12]. white, j. j., spectrophotometric method for hydroxymethylfurfural in honey, journalassociation of official analytical chemists, 62(3), 509-514 work of the international honey commission, mitt. lebensm. hyg., 90, 108-125, (1979) [13]. brand-williams, w., cuvelier, m. e., & berset, c. l. w. t., use of a free radical method to evaluate antioxidant activity, lwt-food science and technology, 28(1), 25-30, (1995) [14].özcelik, s., kuley, e., & özogul, f. formation of lactic, acetic, succinic, propionic, formic and butyric acid by lactic acid bacteria. lwt, 73, 536-542, (2016). [15].pires, j., estevinho, m. l., feás, x., cantalapiedra, j., & iglesias, a., pollen spectrum and physico‐chemical attributes of heather (erica sp.) honeys of north portugal, journal of the science of food and agriculture, 89(11), 1862-1870, (2009) [16].oroian, m., & ropciuc, s. honey authentication based on physicochemical parameters and phenolic compounds. computers and electronics in agriculture, 138, 148-156, (2017). [17].da silva, p. m., gauche, c., gonzaga, l. v., costa, a. c. o., & fett, r., honey: chemical composition, stability and authenticity, food chemistry, 196, 309-323, (2016) [18].codex alimentarius commission,codex alimentarius: fats, oils and related products (vol. 8), fao, (2001) [19].el sohaimy, s. a., masry, s. h. d., & shehata, m. g. physicochemical characteristics of honey from different origins. annals of agricultural sciences, 60(2), 279-287, (2015). [20].oroian, m., amariei, s., rosu, a.,& gutt, g., classification of unifloral honeys using multivariate analysis, journal of essential oil research, 27(6), 533-544, (2015) [21].lazarević, k. b., andrić, f., trifković, j., tešić, ž., & milojkovićopsenica, d., characterisation of serbian unifloral honeys according to their physicochemical parameters. food chemistry, 132(4), 2060-2064, (2012). [22].do nascimento, k. s., sattler, j. a. g., macedo, l. f. l., gonzález, c. v. s., de melo, i. l. p., da silva araújo, e., ... & de almeida-muradian, l. b. phenolic compounds, antioxidant capacity and physicochemical properties of brazilian apis mellifera honeys. lwt, 91, 85-94, (2018). [23]. sanz, m. l., gonzalez, m., de lorenzo, c., sanz, j., & martinezcastro, i. a contribution to the differentiation between nectar honey and honeydew honey. food chemistry, 91(2), 313-317, (2005). [24].kukurova, k., karovièová, j., kohajdova, z., & bilikova, k. authentication of honey by multivariate analysis ofits physico--chemical parameters. journal of food & nutrition research, 47(4). (2008). [25].terrab, a., recamales, a. f., hernanz, d., & heredia, f. j. characterisation of spanish thyme honeys by their physicochemical characteristics and mineral contents. food chemistry, 88(4), 537-542, (2004). [26].manzanares, a. b., garcía, z. h., galdón, b. r., rodríguez-rodríguez, e. m., & romero, c. d. physicochemical characteristics and pollen spectrum of monofloral honeys from tenerife, spain. food chemistry, 228, 441-446. (2017). [27].council, e. u., council directive 2001/110/ec of 20 december 2001 relating to honey, official journal of the european communities l, 10, 47-52, (2002) [28].matović, k., ćirić, j., kaljević, v., nedić, n., jevtić, g., vasković, n., & baltić, m. ž., physicochemical parameters and microbiological status of honey produced in an urban environment in serbia. environmental science and pollution research, 25(14), 1414814157, (2018). [29].geană, e. i., ciucure, c. t., costinel, d., & ionete, r. e. evaluation of honey in terms of quality and authenticity based on the general physicochemical pattern, major sugar composition and δ13c signature. food control, 109, 106919,(2020). [30].önür, i̇., misra, n. n., barba, f. j., putnik, p., lorenzo, j. m., gökmen, v., & alpas, h., effects of ultrasound and high pressure on physicochemical properties and hmf formation in turkish honey types. journal of food engineering, 219, 129-136, (2018). [31].furkan yardibi, m., & gumus, t. some physico‐chemical characteristics of honeys produced from sunflower plant (helianthus annuus l.). international journal of food science & technology, 45(4), 707-712, (2010). [32].dominguez, m. a., & centurión, m. e., application of digital images to determine color in honey samples from argentina. microchemical journal, 118, 110-114, (2015). [33].gonzález-miret, m. l., terrab, a., hernanz, d., fernández-recamales, m. á., & heredia, f. j., multivariate correlation between color and mineral composition of honeys and by their botanical origin. journal of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 daniela pauliuc, mircea oroian, physicochemical parameters of romanian sunflower honey, food and environment safety, volume xix, issue 2 – 2020, pag. 148 – 155 155 agricultural and food chemistry, 53(7), 2574-2580, (2005). [34].hutchings, j., ronnierluo, m., & ji, w. food appearance and expectations. color in food: technological and psychophysical aspects, 3-10, (2012). [35].doner, l. w. honey. encyclopedia of food sciences and nutrition, 3125-3130, (2003). [36].kádár, m., juan-borrás, m., hellebrandova, m., doménech, e., &escriche, i., differentiation of acacia, sunflower and tilia honeys from different countries based on sugar composition, physicochemical and color parameters. bulletin of university of agricultural sciences and veterinary medicine cluj-napoca. agriculture, 67(2), (2010). [37].juan-borrás, m., domenech, e., hellebrandova, m., & escriche, i. effect of country origin on physicochemical, sugar and volatile composition of acacia, sunflower and tilia honeys. food research international, 60, 86-94, (2014). [38].yücel, y., & sultanog, p. characterization of honeys from hatay region by their physicochemical properties combined with chemometrics. food bioscience, 1, 16-25, (2013). [39].kaškonienė, v., venskutonis, p. r., & čeksterytė, v., carbohydrate composition and electrical conductivity of different origin honeys from lithuania. lwt-food science and technology, 43(5), 801-807, (2010). [40].terrab, a., gonzález, a. g., díez, m. j., & heredia, f. j., characterisation of moroccan unifloral honeys using multivariate analysis. european food research and technology, 218(1), 88-95, (2003). [41].devillers, j., morlot, m., phamdelegue, m. h., & dore, j. c. classification of monofloral honeys based on their quality control data. food chemistry, 86(2), 305-312, (2004). [42].beretta, g., granata, p., ferrero, m., orioli, m., & facino, r. m., standardization of antioxidant properties of honey by a combination of spectrophotometric/luorimetric assays and chemometrics. analytica chimica acta, 533(2), 185-191, (2005). [43].mračević, s. đ., krstić, m., lolić, a., & ražić, s., comparative study of the chemical composition and biological potential of honey from different regions of serbia. microchemical journal, 152, 104420, (2020). [44]. sahin, f. t. s. k. h., & erim, e. u. f. b. evaluation of organic acid, saccharide composition and antioxidant properties of some authentic turkish honeys. journal of food and nutrition research, 50(1), 33-40, (2011). [45].suarez-luque, s., mato, i., huidobro, j. f., simal-lozano, j., & sancho, m. t., rapid determination of minority organic acids in honey by high-performance liquid chromatography. journal of chromatography a, 955(2), 207-214, (2002). 1. introduction title …………………… doi: https://doi.org/10.4316/fens.2021.001 5 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2021, pag. 5 12 some physicochemical and organoleptic properties of the short dough with gooseberry powder sergiy boruk 1 , igor winkler 2* 1 department of chemical analysis, food safety and testing, institute of biology, chemistry and bioresources, yu. fedkovych national university of chernivtsi, 2 kotsyubynsky st., chernivtsi, 58012, ukraine 2 department of medicinal and pharmaceutical chemistry, bucovina state medical university, 2 teatralna sq., chernivtsi, 58002, ukraine winkler@bsmu.edu.ua *corresponding author received 21th december 2020, accepted 20th march 2021 abstract: gooseberry powder can be used as an admixture to the confectionery short dough to improve its healthy food qualities and to increase the content of minerals and vitamins. rheological, optical and some organoleptic properties of the dough were investigated for different contents of the powder. the viscosity of the gooseberry containing dough was found lower than that for the no gooseberry samples and, it was revealed that no optically active components moved from this admixture to the dough bulk. structuring of the gooseberry-containing dough was weaker than for the no-gooseberry materials and, in the former case, it took longer to restore the dough structure after it was damaged by the mechanical stress. therefore, an overall fluidity of the gooseberry dough is some higher than that for the pure samples. the integral analysis of the organoleptic parameters of the pastry baked of the dough with different contents of gooseberry shows that no more than 2-3 % of the gooseberry powder should be added to the recipe to ensure a healthier consumption of the pastry and to preserve its consistency, taste and appearance. keywords: short dough; gooseberry; viscosity; organoleptic parameters; healthy food qualities 1. introduction confectionery is a wide group of popular food products manufactured under different recipes, by various production technologies and possessing different consumerattractive qualities. even though it cannot be considered as an essential food, the demand for this group of products is quite high and stable. besides, confectionery is also important in the context of providing the human organism with energy and some vital components [1-5]. dough products have several hundreds of types of items and are the most widespread and popular confectionery goods among different age groups. the short dough is made of flour, sugar, fats, some additional flavoring and aromatic additives [1]. this is quite a popular type of food embracing around a quarter of the dough confectionery market and known as a product with a significant energy value because of a high content of sugar and fat. on the other hand, the nutritional value of shortbread is rather low because its mineral and vitamins compositions are not balanced. this problem can be eliminated or mitigated by adding some components rich of the vegetable fibres and bioactive compounds [3]. this way, the nutritional quality of shortbread http://www.fia.usv.ro/fiajournal mailto:winkler@bsmu.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 6 confectionery can be increased since such components provide immunity stimulation and are physiologically active. moreover, they can also decrease the energy value of the confectionery to make it healthier and less fattening [5]. gooseberry is a widely spread herb with bright, tasty and healthy fruits. it can be used as an additive to the shortbread to improve its quality, taste and consumer attractiveness in the abovementioned context [6,7]. in this paper, we report the results of the investigation of the influence of gooseberry powder on the rheological characteristics of the short dough, its organoleptic and some other characteristics. 2. experimental all investigations were carried out with the dough kneaded according to the traditional technology [1] using a mixture of the top grade flours of various ukrainian brands obtained from a regular grocery store and, when needed, fine powder of the unbranded dried gooseberry fruits obtained from the same store. optical properties of the dough suspensions were investigated using the centrifugate separated from the dough after the 15 minutes long rotation at 1000 s-1. the spectral properties of the centrifugate were investigated using the sf-2 spectrophotometer by lomo (st. petersburg, russia). in all cases, the absorbance of the filtrate was determined against distilled water. all rheological investigations were performed using the cylindrical rotary viscometer “rheotest-2” by veb mlw (leipzig, germany) and the experimental guidelines attached to the device [8-12]. the gooseberry powder is a side component in the dough and, therefore, it will affect an inter-particle interaction between the particles of other dough components resulting in some changes in its viscosity and fluidity. on the other hand, some compounds can potentially be washed-out from the gooseberry powder during the kneading and transfer into the bulk of the dough. all these questions had to be addressed by the above mentioned experimental investigations. 3. results and discussion it has been found that no gooseberry components were washed from its powder and transferred in the dough bulk. this result is grounded on the constancy of spectra recorded for the centrifugates obtained from the dough with and without an admixture of gooseberry (fig. 1). as seen from the fig.1, the pattern and peaks position for both samples are similar while some raise in the absorbance in the case of gooseberry-containing dough is caused by the natural decrease in the liquid transparency resulted by adding some more solute. therefore, it seems that the optically active gooseberry components stay inside the powder, and none of them move to the dough bulk. the dough is a complex and multicomponent heterogeneous system [9,10] and any change in its composition can lead to unwanted shifts in its physicochemical parameters. the static and dynamic viscosity is an important characteristic of dough since it is usually transported at the bakeshops by various screw conveyors and other equipment, where these parameters are critical for effective functioning. as seen from the results of rheological investigations (see fig. 2), the effective viscosity of the dough is decreasing with increase in the displacement speed but, in case of the gooseberry-containing dough, it always stays below than that for the control dough across the entire range of the displacement speeds covered by this investigation. as the displacement speed raises, the displacement forces make an initially chaotic distribution of the suspension particles more ordered by rearranging and lining them up along the flux direction. on the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 7 other hand, the interparticle aggregation is also weakening, and both these processes contribute to a decrease of the dough viscosity with raise in the displacement speed as seen in fig. 2. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 200 300 400 500 600 700 wave length, nm a b so rb a n ce , co n v . u n it s fig. 1. absorbance spectra of the centrifugates obtained from the samples of dough with (■) and without (▲) a 5 % admixture of the gooseberry powder. it should also be mentioned that the dough viscosity is decreasing with a raise in the gooseberry powder content (see fig. 3). this effect is caused by a weakening of the interparticle aggregation because of the introduction of the foreign gooseberry powder which cannot take part in structuring processes running in the dough with the flour particles [8, 9]. the higher the content of the gooseberry powder, the more defective and less rigid structure is formed in the dough, which evidences itself through a decrease in its viscosity. the ability of the dough to restore its pseudo-solid structure after it was destructed mechanically is one of the important operational characteristics since this parameter controls porosity of the baked pastry and smoothness of the technological transportation of the dough [1,8]. it has been found (see fig. 2) that the damaging and then destruction of the pseudosolid structure of the dough occurs at the application of the shifting deformation caused by rotation of the viscometer cylinders [12]. as a result, at some rotation speed, a dependence of the dough viscosity on the rotation intensity interrupts, and it becomes almost independent on the rotation as it is characteristic for a newtonian liquid. one can see that the dough turns into a newtonian liquid at the rotation speed of around 100 s-1 for both dough samples (a further increase in the speed has only a minimal effect on the dough viscosity). as external mechanical stress is decreasing, the dough structure is gradually restoring and it will return to the initial pseudosolid state. however, this process occurs not exactly the way as it did at the increase of the rotation speed. therefore, a loop of hysteresis will appear for the dynamic dependence of the dough viscosity on the rotation speed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 8 0 2 4 6 8 10 12 14 16 18 20 0 100 200 300 400 500 600 700 800 displacement speed, s -1 v is co si ty , p a * s fig. 2. dependence of the short dough viscosity on the displacement speed for the 5 % content of the gooseberry powder (■) and for the control sample (▲, no gooseberry). these dependencies for the gooseberryfree and gooseberry-containing dough are shown in fig. 4 and 5 respectively. as the hysteresis loops seem different (the one for the gooseberry dough is wider than that for the control gooseberry-free sample), it can be concluded that the gooseberry powder actually influences the dough viscosity and its ability to restore the structure after mechanical stress. a comparison between fig. 4 and 5 proves that a viscosity of the no-gooseberry dough is some higher than that for the gooseberry consisting sample at the same rotation speed and, on the other hand, in the former case, it restores sooner than in the latter one (compare the width of the loops shown in fig. 4 and fig. 5). this relaxing character of the gooseberry powder can be caused by a difference into the surface hydrophilicity of the regular dough particles and the particles of gooseberry. this difference results in weaker bonding between these two types of the particles and, therefore, decreases viscosity and makes the particles’ ability to bond into a structure weaker. both types of dough behave like the nonnewtonian systems since their effective viscosity within the laminar regime is not a constant, and it depends on the mechanical shifting force. there are two processes: structure deformation and restoration running simultaneously in the dough being affected by any mechanical stress, and the effective viscosity is the parameter representing a balance between them. the higher the content of gooseberries in the dough, the more the structure of the dough is shifted towards destruction, and the higher is the fluidity of the system. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 9 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 0 1 2 3 4 5 6 gooseberry powder content, % v is co si ty , p a * s fig. 3. dependence of the dough viscosity on the gooseberry powder content at the displacement speed (rotation) 27 s -1 . 0 4 8 12 16 20 0 100 200 300 400 500 600 700 800 rotation speed, s -1 v is co si ty , p a * s fig. 4. the hysteresis loop for the short dough without an admixture of gooseberry. upper line (▲) corresponds to the viscosity at an increase of the rotation speed and the lower line (■) – at the decrease. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 10 0 4 8 12 16 20 0 100 200 300 400 500 600 700 800 rotation speed, s -1 v is co si ty , p a * s fig. 5. the hysteresis loop for the short dough with 5 % of gooseberry. upper line (▲) corresponds to the viscosity at an increase of the rotation speed and the lower line (■) – at the decrease. overall consumer qualities of the pastry were investigated by the following organoleptic parameters: appearance (shape, color, crust thickness, presence and width of the crust cracks); conditions of the pastry body (baking degree and uniformity, porosity, the occurrence of the dough voids; the occurrence of insufficient baking or under-kneaded spots); consistency; taste and smell (whether they inherent in the pastry, are there any side flavors or smells). four samples obtained by the addition of 1, 2, 3 and 5 % of gooseberry and one control sample consisting of no gooseberry were baked for this organoleptic parameters investigation. all pastry was baked using the traditional technology of the short dough baking [3, 4]. in case of the gooseberryconsisting dough, this powder was added to the mixture together with the flour and before adding of the egg-sugar-butter component. table 1 shows the overall organoleptic parameters obtained from the blind tests involving 27 independent volunteers of both genders enrolled among the 18-21 years old students of yu fedkovych university of chernivtsi studying food technologies. all of them tasted different numbered pastry samples being not informed of their composition. the following three conditions were considered at the selection of the most optimal gooseberry content in the short dough: an amount of the powder should be sufficient to provide some improvement in the nutritional value, rheological characteristics of the dough shouldn’t get significantly worsen, and an appearance of the pastry (crust and the pastry body color) should stay almost unchanged. basing on this approach, the following conclusion can be drawn from the abovementioned organoleptic tests: all organoleptic characteristics of the pastry with 1% of gooseberry stay the same or close to those of the control samples containing no gooseberry. however, no significant improvefood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 11 ment of the nutritional quality can be expected in this case since the content of gooseberry is rather low; all organoleptic characteristics also stay almost unchanged when the content of gooseberry reaches 2% although this amount is still considered as sufficient to improve the nutritional quality of pastry; as the nutritional qualities get further improvement for the pastry containing 3% of gooseberry, its organoleptic qualities turn worse; finally, the above tendency continues for the pastry containing 5% of gooseberry and its organoleptic qualities go far below the appropriate standards. table 1 comparative characteristics of some organoleptic parameters of the pastry consisting or not consisting some gooseberry powder parameter control (no gooseberry) pastry with 1 % of gooseberry pastry with 2 % of gooseberry pastry with 3 % of gooseberry pastry with 5 % of gooseberry appearance smooth crust, some small cracks, uniform color smooth crust, some small cracks and few surface dots uniform crust, some small cracks and few surface dots uneven crust with some cracks and well-seen inclusions of gooseberry uneven crust, many cracks, non-uniform coloring and well-seen inclusions of gooseberry color light brown, uniform light brown, uniform light brown, some notuniform light brown, uneven dark-brown, uneven crust color light yellow, uniform light yellow, with few inclusions of gooseberry light yellow, with some gray tone gray yellow, with some inclusions of gooseberry gray yellow with numerous inclusions of gooseberry consistency brittle, with little pores brittle, with little pores brittle, with little pores brittle, with little pores but denser than before brittle, with little pores but denser than before smell inherent in pastry inherent in pastry inherent in pastry inherent in pastry with some bitter note inherent in pastry with a distinct bitter note taste inherent, sweet, no side smacks inherent, sweet, no side smacks inherent, sweet, no side smacks inherent, sweet with light bitterness inherent, sweet with a distinct bitterness pastry body conditions well baked, no under-kneads well baked, no underkneads well baked, no underkneads well baked, no underkneads well baked, no underkneads food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue – 2021 sergiy boruk, igor winkler, some physicochemical and organoleptic properties of the short dough with gooseberry powder, food and environment safety, volume xx, issue 1 – 2021, pag. 5 – 12 12 therefore, it can be concluded that 2-3% of gooseberry seems the most optimal content of the gooseberry powder. this addition provides some appreciable improvement of the nutritional qualities while the organoleptic parameters stay almost unchanged. 4. conclusion it has been shown that the gooseberry powder can be used as an additive to some confectionery and pastry to improve their nutritional and healthy food qualities. an admixture of gooseberry ensures some decrease in the dough structuring ability and makes it more fluid. these effects should be taken into consideration at the construction and operation of the dough transportation and mechanical kneading equipment. no extra compounds exhibiting an optical activity are realized from the gooseberry powder into the dough bulk, therefore, the above physicochemical changes are caused by the presence of the gooseberry particles distributed among the flour and other components of the dough. since gooseberry causes some deterioration of the pastry organoleptic properties, it is not recommended to exceed 2-3% of its content. if this requirement is observed, some improvement of the nutritional qualities is achieved while the pastry appearance, taste, color and other properties remain almost unchanged. 5. references [1]. a. v. pavlov, a digest of the recipes of the dough confectionery and pastry, hydrometeoizdat, moscow (1998) (in russian) [2]. n. yeshak, sensory evaluation and nutritional value of balady flat bread supplemented with banana peels as a natural source of dietary fiber, annal. agri. sci., 61(2), 229-235 (2016) [3]. a. volkova et al., innovative technologies of using promising phyto-fortificants in bakery products of high nutritional value, iop conf. ser.: earth environ. sci., 403, 012119 (2019) [4]. m. bijlwan et al, recent developments in dough based bakery: a mini review, the pharma innov. j., 8(5), 654-658 (2019) [5]. m. b. yerkebaev, fundamentals of the food rheology. textbook, atu, almaty (2006) (in russian) [6]. g. griffith, prelude to good health: the organic wild gooseberry diet, archway publishing, usa (2017) [7]. p. combris et al, improvement of the nutritional quality of foods as a public health tool, public health, 125(10), 717-724 (2011) [8]. r. borwankar, c. f. shoemaker (eds), rheology of food, elsevier applied science uk, london (1992) doi: 10.1016/c2009-0-01131-3 [9]. m. f. kravchenko, o. l. romanovska, s. d. boruk, rheological properties of the biscuit dough made of the flour “health”, sci. bull. national food. uni., 21(5), 69-74 (2015) (in ukrainian) [10]. m. f. kravchenko, o. l. romanovska, s. d. boruk, a comparative analysis of the rheological characteristics of the mixtures containing cacao and carob, sci. bill. chernivtsi uni., series chemistry, 753, 41-45 (2015) (in ukrainian) [11]. s boruk, o. romanovska, o. gerych, structuring in the disperse systems of semolina and quinoa, proc. of lviv chem. readings, f29 (2017) (in ukrainian) [12]. veb mlw: guidelines to rheotest-2. http://www.pochva.com/?content=3&book_id=131 1 accessed nov. 23, 2020. https://doi.org/10.1016/c2009-0-01131-3 http://www.pochva.com/?content=3&book_id=1311 http://www.pochva.com/?content=3&book_id=1311 1. introduction it should also be mentioned that the dough viscosity is decreasing with a raise in the gooseberry powder content (see fig. 3). this effect is caused by a weakening of the interparticle aggregation because of the introduction of the foreign gooseberry ... the ability of the dough to restore its pseudo-solid structure after it was destructed mechanically is one of the important operational characteristics since this parameter controls porosity of the baked pastry and smoothness of the technological tran... as external mechanical stress is decreasing, the dough structure is gradually restoring and it will return to the initial pseudo-solid state. however, this process occurs not exactly the way as it did at the increase of the rotation speed. therefore, ... fig. 2. dependence of the short dough viscosity on the displacement speed for the 5 % content of the gooseberry powder (■) and for the control sample (▲, no gooseberry). this relaxing character of the gooseberry powder can be caused by a difference into the surface hydrophilicity of the regular dough particles and the particles of gooseberry. this difference results in weaker bonding between these two types of the par... fig. 3. dependence of the dough viscosity on the gooseberry powder content at the displacement speed (rotation) 27 s -1. fig. 5. the hysteresis loop for the short dough with 5 % of gooseberry. upper line (▲) corresponds to the viscosity at an increase of the rotation speed and the lower line (■) – at the decrease. four samples obtained by the addition of 1, 2, 3 and 5 % of gooseberry and one control sample consisting of no gooseberry were baked for this organoleptic parameters investigation. all pastry was baked using the traditional technology of the short dou... 4. conclusion применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 contents: 1. tilia honey’s fructose, glucose and sucrose content prediction using ft-ir spectra with partial least squares regression, daniela pauliuc, paula ciursa, florina dranca, sorina ropciuc, mircea oroian 260 266 2. effect оf rosemary extract on thermal stability оf sunflower oil artan gashi, mila arapcheska, fatos rexhepi 267 274 3. aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri 275 286 4. human health risk assessment of heavy metal contaminants in table salt from nigeria sunday ukwo 287 296 5. quality characteristics of biscuits prepared from composite flour of yellow fleshed cassava and quality protein maize abiodun aderoju adeola, dorcas olawunmi olayioye, alexander adedolapo oluyombo, babatunde adebiyi olunlade 297 306 6. brix production from cassava peels and optimization of bioethanol synthesis using saccharomyces cerevisiae ihuoma offor-emenike, vincent ibekwe, campbell akujobi, wesley braide 307 315 7. comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okekeagulu and chizoba chizea 316 322 8. a rapid method of determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc alina lenuța crăciun , gheoghe gutt 323 331 9. author instructions i v 10. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 doi: https://doi.org/10.4316/fens.2021.041 403 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 403 410 blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization *ana leahu 1 , cristina ghinea 1 , sorina ropciuc 1 1stefan cel mare university of suceava, romania, faculty of food engineering, 13th university street, suceava, romania, analeahu@fia.usv.ro *corresponding author received 5th september 2021, accepted 20th december 2021 abstract: this work aimed at optimizing the extraction of total phenolic (tp) and total monomeric anthocyanin (tma) content for the development of a potentially functional wild blueberry smoothie (bp) with apple puree (ap), apple juice (aj) and sea buckthorn puree (sbj) in various proportions using conventional extraction (ce) and advanced extraction techniques [ultrasound-assisted (uae)]. there were also analyzed the rheological behavior and the color attributes (lightness and redness respectively) of smoothie samples. s7 smoothie sample [30 ap/30 aj/20 sbj/20 bp (uae)] provided a beverage with significant higher amount (21.40%) of anthocyanins (tma) compared to sample s10 [30 ap/30 aj/20 sbj/20 bp (ce)] in which the extraction of anthocyanins was by the classical method. further, different color attributes, particularly brightness (l *), redness (a *) and yellow (b *) decreased considerably for the samples with 15 and 20% addition of blueberry puree. therefore, ultrasound-assisted extraction had a significant effect on the amount of anthocyanins extracted, compared to the classical extraction method. keywords: fruit smoothies; ultrasound; extraction; anthocyanin content; cielab method. 1. introduction although, it is recognized that a healthy diet can reduce the risk of many common chronic diseases, the prevalence of dietrelated diseases is still increasing. unhealthy eating habits can affect nutrient intake, and these habits include: lack of natural foods or eating too many foods and beverages that are low in fiber or high in fat, salt and / or sugar. nutritional status is very important to maintain a strong immune system against the coronavirus disease 2019 (covid-19) pandemic, so there is great interest in foods rich in substances that help strengthen the body's immune system. various studies have shown changes in eating habits, consuming food of a poorer quality and a decrease in fruit consumption during the quarantine period [1-3]. smoothies are blended beverages containing fruits, vegetables and liquids such as water, milk, fruit juices and yogurt. wild blueberries, sea buckthorn and apples are fruits native to northern bukovina, and are an important part of the usual diet and have practically identical historical uses and known health benefits. the use of both berries and apples has its roots in food traditions, which used fresh or sun-dried fruit to make juices, syrup, jams, cakes, dairy products or season food. the presence of micronutrients and phytochemical compounds, such as vitamins, fibers and phenolic compounds (anthocyanin and non-anthocyanin compounds), with nutritional and functional properties, that justify the growing interest in these berries, not only mailto:analeahu@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 404 for food applications, but also in the pharmaceutical industry [4]. bilberries (vaccinium myrtillus l.), belong to the most popular fruits, are a dark blue fruit and an interesting source of phenolic compounds, mainly anthocyanins, sugars, vitamins, fibers, minerals, making them valuable to the food, pharmaceutical industries, or they can be used in the development of functional foods [5, 6]. the content of anthocyanins in bilberries varies between 1017 mg per 100 g fresh weight, and 1610 mg/l to 5963 mg/l in juice [7]. blueberry with tall (cultivated) shrub is a branched, bushy subshrub, which reach maturity after about 6-8 years from planting. there are many varieties of blueberries early, seasonal and late varieties. wild blueberries have a more pronounced aroma than cultivated fruits; they are shrubs that grow in non-forest habitats and mountain hills, over the carpathians, especially on shady slopes [8]. the fruits of sea buckthorn (hippophaë l., family eleagnaceae) are berries of orange to red colour and have the taste sour, bitter and astringent; they have a special aroma, which cannot be compared with the aroma of any other fruit [9]. the presence of total phenolic compounds and tocopherols in sea buckthorn fruits [10, 11], vitamin c and minerals [12] antioxidant activity and flavonoids [13], which are considered as major preventive agents against several degenerative diseases, with healthpromoting effects, have been reported. sea buckthorn berries are also used in traditional medicine and has become an ingredient in natural products for food, pharmaceutical and cosmetic products. the apple is a well-known fruit, and a major fruit crop in eurasia, that belongs to the species malus domestica from the rosaceae family. thousands of apple varieties are grown worldwide and have multiple uses in the food industry (dried, juices, purees, cider, ingredients from various preparations), but can also be used fresh or in various forms of preservation [14]. nowadays, the apple are considered to be a good source of a large number of bioactive compounds, including phenolic compounds, flavanols, and anthocyanins, vitamins (ascorbic acid), minerals [15,16], that ensure nutritional and healthpromoting effects as an important part of our daily food. apple is a widely consumed and epidemiological studies show that eating apples reduces the risk of of colorectal cancer [17], cardiovascular disease [18] asthma and diabetes [19]. ivanovic et al. [20] studied the ultrasoundassisted extraction on the colour stability and the total content of anthocyanins (tac) of the blackberry fruit purée, cultivar “čačanska bestrna” widely grown in serbia. the greatest increasing of anthocyanin content was obtained for extract isolated at 40 0c after 30 min of sonication (1.38 g/100 g dw) [20]. the effects of ultrasonic treatment (for 15, 30 and 60 minutes at 25 0c, frequency 40 khz, 130 w) have already been described for freshly squeezed chokanan mango juice, in which was observed after sonication, an increase in brightness (l*) and a decrease in redness (+ a*) and yellowing (+ b*) was observed [21]. in a recent study, potter et al. [22] tested a beverage of blueberry-soy with 12% blueberry juice and 2.8% soy protein isolate, and this new formula represented a great source of anthocyanins. thus, new smoothie products rich in bioactive compounds can be developed to meet the requirements of consumers while facing the modern lifestyle. the aim of this paper is to obtain new formulas for smoothie sources of natural antioxidants, with special sensory, biochemical and textural properties. https://www.sciencedirect.com/topics/chemistry/phenolic-compound https://www.sciencedirect.com/topics/food-science/bioactive-compound food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 405 2. materials and methods plant material and sample preparation fruits of bilberries (vaccinium myrtillus l.) and sea buckthorn (h. rhamnoides l.) were harvested optimally ripe, in suceava county, romania. fruits were cleaned and then stored at 4 0c until analysis; the juice was extracted by cold pressing. apples malus domestica ‘fălticeni’ were obtained from a local retailer. the fruits were blended in a homogenizer. smoothie composition by weight was whole apple, apple juice, sea buckthorn, and blueberry mixed immediately after obtaining, in appropriate proportions (50/30/10/10, 40/30/15/15 and 30/30/20/20). finally, seven different products were obtained: four semi products (100% apple puree (ap); 100% apple juice (aj); 100% sea buckthorn puree (sbj); 100% blueberry puree (bp) and three smoothie’s samples [s5 50ap/ 30aj/ 10sbj/ 10bp; s6 40ap/ 30aj/ 15sbj/ 15bp; s7 30ap/ 30aj/ 20 sbj/ 20sb. total monomeric anthocyanin determination extraction procedure 5 g smoothie samples were extracted with 10 ml of 80% methanol aqueous solution containing 1 g / 100 g hcl in an ultrasonic bath for 30 minutes 20 khz, as described before by dranca and oroian [24] with a slight modification. a conventional 1-hour extraction was performed at room temperature with a magnetic stirrer. the beverage samples were then centrifuged for 10 min at 10000 rpm. after extraction, the whole extracts were filtered through a filter paper and placed in a volumetric flask (25 ml) and the volume was completed with the 80 % methanol. total monomeric anthocyanin (tma) content in smoothie samples was determined by uv–vis spectrophotometer, (ocean optics, largo, fl, usa) according to leahu et al. [23]. absorbance was measured at 530 and 657 nm, and tma content was expressed as cyanidin-3-glucoside (mg/g) using the following eq. (1): (1) where: mw= 449.1, a net= a530− 0.25 a657, df-dilution factor, v-total volume (ml), wt-sample weight (g) the total phenolic content of smoothie methanol extract was measured, by using the folin-ciocalteu method [8]. the data was reported as mg gallic acid equivalents (gae)/100 g fresh-weight (fw). brix degree determination this method evaluates the total soluble sugar (tss) content, by measuring index of refraction with a digital handheld brix hanna hi96801. the refractive index was recorded and converted to °bx. colour measurement colour components of obtained products, were measured using the cielab-system. l*, a* and b* (where: l* (100 = white; 0 = black), a* (+ red, – green), b* (+ yellow; – blue), with a minolta chroma meter cr-200 (tristimulus method) [23]. sensory evaluation taste, flavor, color, sweetness, acidity, and overall acceptability on a 9-point hedonic scale were estimated in the fruit smoothie samples compared to the control fruits puree by twenty panelists (12 females, 8 males) untrained assessors between 18 and 50 years of age. following the processing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 406 smoothie samples were coded and served at room temperature (aprox 20 0c) in aliquots of 20 ml together with non-salted table biscuits and still water (bucovina, romania) to panelists for evaluation of sensory attributes. statistical analysis the obtained data were analyzed with minitab 17 statistical software. data were reported as means ± standard deviation. in order to observe if there is any correlation between the parameters pearson correlation was used. 3. results and discussion effect of ultrasound on the total monomeric anthocyanin content the anthocyanin contents of the apple peel extract, s1 (5.84 mg/ g fresh peels) were measured (figure 1). the results showed that the total anthocyanin content increased after 30 min of sonication using the 20 khz system, compared to the conventional extraction control samples. the blueberry puree (37.56 mg cyanidin 3-glucoside/g fresh weight) had a similar content to the s7 (30/30/20/20) smoothie (32.89 mg cyanidin 3-glucoside/ g fresh weight), because it has the highest addition of blueberries. total monomeric anthocyanin (tma) content of s7 30/30/20/20 smoothie sample increased by 14.43 % at 20 khz (figure 1), 30 min sonication compared to the conventional s10 extraction sample. ultrasound assisted extraction (uae) of red raspberry puree prepared from crushed berries was recently observed by golmohamadi et al. [25]. total monomeric anthocyanin content of red raspberry puree increased by 12.6% at 20 khz and by 6.7% at 490 khz after 10 min sonication (p < 0.05) [25]. fig. 1: anthocyanin content of smoothie samples. results are the means of three analyses and bars show the standard error s1 100% apple puree (ap); s2 100% apple juice (aj); s3 100% sea buckthorn puree (sbj); s4 100% blueberry puree (bp); s5 smoothie sample 50/30/10/10 (uae); s6 smoothie sample 40/30/15/15 (uae); s7 smoothie sample 30/30/20/20 (uae); s8 smoothie sample 50/30/10/10 (ce); s9 smoothie sample 40/30/15/15 (ce); s10 smoothie sample 30/30/20/20 (ce). values are mean ± standard deviation (n = 3). means that do not share a letter (a-j) are significantly different (p ≤ 0.05). total phenolic content smoothie samples were analyzed for their total phenolic content using folin– ciocalteu assay. the total phenolic compounds (tp) values in the smoothie extracts analyzed were in the range 186.16 – 376.3 mg gae/100 g fw fresh weight (figure 2). among all the samples analyzed, the s7 sample revealed the highest tp concentration at 376.3 mg gae/100 g fw. total soluble sugar (tss) content s2 100% apple juice (aj) showed an initial tss of 12.8 0bx, which tended to decrease in the smothie samples. however, s6 (40 ap/30 aj/15 sbj/15 bp) smoothie samples showed the highest tss (12.6 ° bx) and were not significantly different from the control samples. the total soluble solids content, or sugar index, is an important indicator of quality for fruit and vegetable smothie drinks [26]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 407 fig. 2: total phenolic compounds content of smoothie samples. results are the means of three analyses and bars show the standard error s1 100% apple puree (ap); s2 100% apple juice (aj); s3 100% sea buckthorn puree (sbj); s4 100% blueberry puree (bp); s5 smoothie sample 50/30/10/10 (uae); s6 smoothie sample 40/30/15/15 (uae), s7 smoothie sample 30/30/20/20 (uae); s8 smoothie sample 50/30/10/10 (ce); s9 smoothie sample 40/30/15/15 (ce); s10 smoothie sample 30/30/20/20 (ce). values are mean ± standard deviation (n = 3). means that do not share a letter (a-j) are significantly different (p ≤ 0.05). fig. 3: content of total soluble sugar (tss) of smoothie samples s1 100% apple puree (ap); s2 100% apple juice (aj); s3 100% sea buckthorn puree (sbj); s4 100% blueberry puree (bp); s5 smoothie sample 50/30/10/10; s6 smoothie sample 40/30/15/15; s7 smoothie sample 30/30/20/20. values are mean ± standard deviation (n = 3). means that do not share a letter (a-d) are significantly different (p ≤ 0.05). color measurement in the cie l*a*b* system in general, the acceptability of a product is influenced by color. color properties of smoothie samples are illustrated in figure 4. fig. 4: color parameters of smoothie samples. different lowercase letters (a–f) show significant differences between the groups (p < 0.05) l* values, indicating the luminosity level, ranged from 24.80±0.01 to 55.03±0.02. in the case of a* the samples presented values situated in the pozitive region (ranged between 3.29±0.03 and 22.04±0.03), more towards red and in the case of b* they were situated in the positive region (7.98±0.02 47.42±0.06), more towards yellow. sensory evaluation members of the sensory panel evaluated a total of seven smoothie products (four semi products and three smoothie beverages). the sensory scores of the four semi products (100% apple puree (ap); 100% apple juice (aj); 100% sea buckthorn puree (sbj); 100% blueberry puree (bp) and three smoothie samples are presented in figure 5. in general, all samples received high scores of preferences and all smoothie samples were characterized by fruit flavour, smoothness and sweetness. the s5 smoothies sample, which is the sample with the smallest addition of sea buckthorn, received the highest overall impression score, along with the highest taste and texture scores (figure 5). smoothies s7 was defined as dense, astringent which could result from the presence of fiber with high water holding capacity and apparent viscosity. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 408 fig. 5: sensory evaluation of the smoothie samples s1 100% apple puree (ap); s2 100% apple juice (aj); s3 100% sea buckthorn puree (sbj); s4 100% blueberry puree (bp); s5 smoothie sample 50/30/10/10; s6 smoothie sample 40/30/15/15; s7 smoothie sample 30/30/20/20. pearson correlation and principal component analysis from table 1, it can be observed that the pearson correlation between tma and tp is 0.517 which indicates that is a moderate positive relationship between these two variables and a strongly negative relationship between tma and l* (−0.709) and b* (-0.617) (p-value < 0.05). a strongly positive relationship can be observed between tp and tss (0.733) and strongly negative relationship between tp and b* (-0.898) and l* (-0.881) (p-value < 0.05). strongly negative relationship was observed between tss and color parameters. table 2 show the results obtained after performing the analysis of the principal components and it was observed that the first two components have eigenvalues greater than 1. table 1 correlation matrix (pearson correlation) tma tp tss l* a* tp 0.517 tss -0.012 0.733 l* -0.709 -0.881 -0.585 a* 0.254 -0.032 -0.439 0.22 b* -0.617 -0.898 -0.654 0.992 0.283 table 2 principal component analysis: tma, tp, tss, l * , a * , b * eigenvalue 3.7777 1.4716 0.6156 0.103 0.0318 0.0002 proportion 0.63 0.245 0.103 0.017 0.005 0 cumulative 0.63 0.875 0.977 0.995 1 1 eigenvectors variable pc1 pc2 pc3 pc4 pc5 pc6 tma -0.318 -0.571 -0.401 -0.602 0.186 -0.12 tp -0.483 -0.053 0.385 0.269 0.737 0.032 tss -0.372 0.45 0.475 -0.615 -0.234 -0.015 l* 0.504 0.089 0.18 -0.216 0.353 -0.731 a* 0.127 -0.679 0.647 0.049 -0.32 -0.004 b* 0.507 0.003 0.122 -0.37 0.375 0.671 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 409 the first component has a proportion of 0.63, which means that it explains 63% of the variability in the data and the second component has 24.5% and can both be included in the evaluation. the other components have lower proportions and may not be important enough to be included into the evaluation. the first component has a positive association with l* (0.504) and b* (0.507) and large negative association with tp, while the second component has a large negative association with tma (−0.571) and a* (−0.679). there is a statistically significant association between the indicators. 4. conclusion the investigation of the smoothie samples revealed a special biochemical and sensory properties. this indicates that apple, blueberry and sea buckthorn are a promising source of health-promoting biochemical compounds with antioxidant activity. these results illustrate that the use of ultrasound-assisted extraction is suitable for the extraction and quantification of anthocyanin compounds and total polyphenols to improve the quality of fruitbased beverages for consumers. 5. references [1]. sidebottom c., ullevig s., cheever k., zhang t., the effect of covid19 pandemic and quarantine period on physical activity and dietary habits of college-aged students, sports medicine and health science, 3: 228-235, (2021) [2]. sidor a., rzymski p., dietary choices and habits during covid-19 lockdown: experience from poland, nutrients, 12(6):1657, (2020) [3]. di renzo l., gualtieri p., cinelli g., bigioni g., soldati l., attinà a., de lorenzo a., psychological aspects and eating habits during covid-19 home confinement: results of ehlc-covid-19 italian online survey, nutrients, 12(7): 2152, (2020) [4]. pires t.c., caleja c., santosbuelga c., barros l., ferreira i.c., vaccinium myrtillus l. fruits as a novel source of phenolic compounds with health benefits and industrial applications-a review, current pharmaceutical design, 26(16):1917-1928, (2020) [5]. stajčić s.m., tepić a.n., đilas s.m., šumić z.m., čanadanović-brunet j.m., ćetković, g. s., ... tumbas v.t., chemical composition and antioxidant activity of berry fruits, acta periodica technologica, (43): 93-105, (2012) [6]. tadić v.m., nešić i., martinović m., rój e., brašanac-vukanović s., maksimović s., žugić a., old plant, new possibilities: wild bilberry (vaccinium myrtillus l., ericaceae) in topical skin preparation, antioxidants, 10(3): 465, (2021) [7]. müller d., schantz m., richling e., high performance liquid chromatography analysis of anthocyanins in bilberries (vaccinium myrtillus l.), blueberries (vaccinium corymbosum l.), and corresponding juices, journal of food science, 77(4): c340-c345, (2012) [8]. leahu a., oroian m., ropciuc s., total phenolics of fresh and frozen minor berries and their antioxidant properties, food and environment safety journal, 13(1): 87-93, (2016) [9]. gâtlan a.m., gutt g., sea buckthorn in plant based diets. an analytical approach of sea buckthorn fruits composition: nutritional value, applications, and health benefits, international journal of environmental research and public health, 18(17): 8986, (2021) [10]. madawala s.r., brunius c., adholeya a., tripathi s.b., hanhineva k., hajazimi e., landberg r., impact of location on composition of selected phytochemicals in wild sea buckthorn (hippophae rhamnoides), journal of food composition and analysis, 72:115-121, (2018) [11]. guo r., guo x., li t., fu x., liu r.h., comparative assessment of phytochemical profiles, antioxidant and antiproliferative activities of sea buckthorn (hippophaë rhamnoides l.) berries, food chemistry, 221: 997-1003, (2017) [12]. ercisli s., orhan e., ozdemir o., sengul m., the genotypic effects on the chemical composition and antioxidant activity of sea buckthorn (hippophae rhamnoides l.) berries grown in turkey, scientia horticulturae, 115(1): 27-33, (2007) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 a. leahu, c. ghinea, s. ropciuc, blueberry, sea buckthorn fruits and apple beverage: biochemical and sensorial characterization, food and environment safety, volume xx, issue 4 – 2021, pag. 403 – 410 410 [13]. périno-issartier s., abert-vian m., chemat f., solvent free microwave-assisted extraction of antioxidants from sea buckthorn (hippophae rhamnoides) food by-products, food and bioprocess technology, 4(6): 1020-1028, (2011) [14]. cornille a., antolín f., garcia e., vernesi c., fietta a., brinkkemper o., roldán-ruiz i., a multifaceted overview of apple tree domestication, trends in plant science, 24(8): 770-782, (2019) [15]. vieira f.g.k., borges g.d.s.c., copetti, c., di pietro, p. f., da costa nunes, e., & fett, r., phenolic compounds and antioxidant activity of the apple flesh and peel of eleven cultivars grown in brazil, scientia horticulturae, 128(3): 261-266, (2011) [16]. kalinowska m., bielawska a., lewandowska-siwkiewicz h., priebe w., lewandowski w., apples: content of phenolic compounds vs. variety, part of apple and cultivation model, extraction of phenolic compounds, biological properties, plant physiology and biochemistry, 84: 169-188, (2014) [17]. jedrychowski w., maugeri u., popiela t., kulig j., sochacka-tatara e., pac a., musial a., case–control study on beneficial effect of regular consumption of apples on colorectal cancer risk in a population with relatively low intake of fruits and vegetables, european journal of cancer prevention, 19(1): 42-47, (2010) [18]. chai s.c., hooshmand s., saadat r.l., payton m.e., brummel-smith k., arjmandi b.h., daily apple versus dried plum: impact on cardiovascular disease risk factors in postmenopausal women. journal of the academy of nutrition and dietetics, 112(8): 1158-1168, (2012) [19]. boyer j., liu r.h., apple phytochemicals and their health benefits, nutrition journal, 3(1): 1-15, (2004) [20]. ivanovic j., tadic v., dimitrijevic s., stamenic m., petrovic s., zizovic i., antioxidant properties of the anthocyanincontaining ultrasonic extract from blackberry cultivar “čačanska bestrna”, industrial crops and products, 53: 274-281, (2014) [21]. santhirasegaram v., razali z., somasundram c., effects of thermal treatment and sonication on quality attributes of chokanan mango (mangifera indica l.) juice, ultrasonics sonochemistry, 20(5):1276-1282, (2013) [22]. potter r.m., dougherty m.p., halteman w.a., camire m.e., characteristics of wild blueberry–soy beverages, lwt-food science and technology, 40(5): 807-814, (2007) [23]. leahu a., ghinea c., oroian m.a., damian c., determination of essential and toxic elements, ascorbic acid content and color of different leaves in two cabbage varieties, ovidius uni ann chem, 29(2):110-116, (2018) [24]. dranca f., oroian m., optimization of ultrasound-assisted extraction of total monomeric anthocyanin (tma) and total phenolic content (tpc) from eggplant (solanum melongena l.) peel, ultrasonics sonochemistry, 31: 637-646, (2016) [25]. golmohamadi a., möller g., powers j., nindo c., effect of ultrasound frequency on antioxidant activity, total phenolic and anthocyanin content of red raspberry puree, ultrasonics sonochemistry, 20(5):1316-1323, (2013) [26]. managa m.g., akinola s.a., remize f., garcia c., sivakumar d., physicochemical parameters and bioaccessibility of lactic acid bacteria fermented chayote leaf (sechium edule) and pineapple (ananas comosus) smoothies, frontiers in nutrition, 8:120, (2021) 1. introduction extraction procedure total phenolic content fig. 5: sensory evaluation of the smoothie samples doi: https://doi.org/10.4316/fens.2021.032 289 journal homepage: http://fens.usv.ro/index.php/fens/index journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 289 297 removal of toxic compounds from natural extracts using green techniques *francisco lorca 1 , david quintín 1 , maría-dolores lópez 1 , presentación garcía 1 1aei ctnc national technological centre for the food and canning industry, murcia, spain, tecnología1@ctnc.es *corresponding author received 17th august 2021, accepted 28th september 2021 abstract: the aim of the present study was compare two natural bio adsorbent materials with different commercial adsorbents, such as granular activated carbon (cag) and zeolite, for the elimination of pesticides in extracts of interest. two natural bio adsorbents were developed from rice husk and almond skin. artichoke and lemon extracts were obtained from artichoke by-product and citrus by-product respectively. the trend is supported by the potential of these food wastes, which could be used for the production of valued products. in fact, peels, seeds, stems, or vegetable pulps are considered raw materials to obtain bioactive ingredients with multiple applications, mainly for the production of food ingredients, cosmetics, or nutraceuticals. furthermore, bioactive compounds of vegetable wastes have demonstrated antioxidant and antimicrobial actions. the results obtained show the effectiveness of the different adsorbents in the adsorption of pesticides that artichoke and lemon extracts contain. adsorbents from almond and rice by-product were effective, reducing in case of almond skin adsorbent by half the concentration of metalaxyl and reducing a large amount of azoxystrobin and difenoconazole. rice bio-adsorbent was less effective than the other adsorbates even though it was more effective than zeolite. the use of agri-food waste allows solving not only an environmental problem of pollution of high importance, but also a problem of management of large amounts of waste generated in the agri-food industry contributing to the promotion of circular economy. two adsorbents have been developed from almond and rice by-products which are effective for the elimination of different toxic compounds through adsorption processes such as pesticides. keywords: pesticides, green tehcniques, adsorbents, natural extracts, organic compound. 1. introduction nowadays, consumption of fruit and vegetable is very widespread as they provide health benefits. these benefits are partly due to the organic compounds that products contain. these bioactive molecules have antioxidant, antithrombotic, anti-inflammatory, and antidiabetic properties, among others [1–4]. artichoke (cynara scolymus l.) is an herbaceous perennial plant belonging to cynara genus and asteraceae family. commonly known as globe artichoke, it is traditionally consumed in the mediterranean diet. their cultivation is considered an important activity of the agro-economy of the semediterranean countries [5,6]. the mediterranean region has an annual production of about 770,000 tons. the edible portion of this plant includes the receptacle of immature flowers and the inner bracts, named “capitula” or heads. during the artichoke processing, the http://fens.usv.ro/index.php/fens/index mailto:tecnología1@ctnc.es food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 290 residues, principally external leaves or stems, represent approximately 60–80% of the total harvested plant material, which is translated in more than 460,000 tons of wastes generated annually. nevertheless, these by-products possess a great content of bioactive phenolic compounds, inulin, fibers, and minerals [7–11]. this information highlighted the significance of the evaluation of artichoke wastes for the extraction of bioactive compounds. despite its content in these interesting compounds, it is the presence of bioactive compounds that has aroused greater interest, especially phenolic compounds [12, 13]. the interest in their phytochemicals has been linked to various pharmacological activities exerted on humans. therefore, the artichoke has been used for medicinal purposes since antiquity, being considered a functional food [14, 15, 16, and 17]. consequently, the revalorization of food by-products as a bioactive source material has experienced a great growth due to the economic and environmental benefits that it produces. in the case of artichoke, recent studies based their research on external bracts, leaves and floral stems by-products, which are considered the principal discarding parts of the artichoke processing because they are not suitable for human consumption [18]. the processing of citrus fruits in the agroindustry has different phases from the collection of raw materials to obtaining the desired products. during this process, a significant amount of waste is generated. the nature of the mentioned waste is lowquality fruits and, especially, parts of the fruits without commercial value (crust) removed during the transformation process. the huge volume of citrus waste is not been managed and eliminated using advanced scientific and technical approaches, but it is mainly done in an inadequate way implying negative environmental effects. discarded parts of citrus fruits have a very high content of cell wall polysaccharides that are source of dietary fiber and constitute up to now the only raw material for the production of pectin used by the food industry as stabilizers or gelling agents. this trend is supported by the potential of these food wastes, which could be used for the production of valued products. in fact, peels, seeds, stems, or vegetable pulps are considered raw materials to obtain bioactive ingredients with multiple applications, mainly for the production of food ingredients, cosmetics, or nutraceuticals. furthermore, bioactive compounds of vegetable wastes have demonstrated antioxidant and antimicrobial actions in developed food additives used for conservative purposes the current need of a sustainable food chain demands an implementation of a circular economy approach in the processing industries. the major focus of this approach is to revalorize the discarding parts of vegetables due to their great contents in bioactive compounds [19, 20]. the pesticide-based treatments that are applied to fruit and vegetables are present in the by-products generated after industrial processing. these by-products that are currently used for their revaluation through the extraction of compounds of interest give rise to the dragging of these pesticides and other toxic substances such as heavy metals and mycotoxins, concentrated in the extracts, making their use as ingredients in the food, nutraceutical and cosmetic industry unfeasible because these substances are potentially toxic to humans being related to various diseases including food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 291 cancer and decreased fertility, among others [21-22]. the use and extraction of compounds of interest from fruit and vegetable waste reduces the risk of accumulation of waste in the environment, preventing the negative effect on the environment of waste treatment such as incineration or landfilling, as well as the cost and investment of labor in the process. the adsorption of toxic compounds on the surface of adsorbent materials is presented as a promising, effective, sustainable and low-cost measure, compared to other types of treatments, looking for low-cost and environmentally friendly adsorbents obtained from biomass. [23-25]. the possibility of using agri-food waste as potential precursors for the preparation of adsorbents provides the opportunity to have a wide range of adsorbents, because they are generated on a large scale and have a low cost compared to commercial adsorbents. the use of bio-adsorbents allows solving not only an environmental problem of pollution of high importance, but also a problem of management of large amounts of waste generated in the agri-food industry contributing to the promotion of circular economy. therefore, in this study, bioadsorbents have been developed from agrifood residues for the elimination of pesticides in extracts containing compounds of interest and the result of adsorption has been compared with commercial adsorbates such as granular activated carbon (gac) and zeolite. the selection of appropriate substrates in adsorption systems for the decontamination of pesticides from aqueous streams is of utmost importance. drobeket al. studied a natural adsorber from rice by-product showing that it has a significantly high potential to adsorb the pesticides atrazine and imidacloprid. al-qodah et al. carried out the adsorption of the pesticides deltamethrin and lambda-cyhalothrin using ash from shale oil as adsorbent, proving to be very effective and very low cost, compared to other metallic adsorbers and activated carbon. currently, the scientific community focuses on adsorption as a promising, effective, sustainable and low-cost measure, compared to other types of treatment, seeking low-cost and environmentally friendly adsorbents obtained from biomass [28-30]. gupta et al publish a study that reveals an alternative approach to the management of fruit residues with the production of value-added products and their use of residues as a bio adsorbent. the aim of the present study was comparenatural bio adsorbent materials with different commercial adsorbents, such as granular activated carbon (cag) and zeolite, for the elimination of pesticides in extracts of interest. 2. materials and methods adsorbent materials adsorption experiments were performed with granular activated carbon adsorbent, with granular size range 0,5-3,13 mm, and zeolite adsorbent with granular size range 0,5-3,13 mm,were supplied by panreacapplichem. production of different bio-adsorbent materials two bio-adsorbent were performed in adsorption experiments. the natural adsorbent of almond by-product and rice by-product were activated by treating almond skin and rice husk through a process of washing, and activation with solution of 2% of h2o2thus obtaining two natural adsorbents. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 292 extracts of interest extracts of interest were prepared as follows: artichoke or lemon by product were mixed with wáter 1:1 proportion. the mixture was heated at 100ºc and have been kept in constant agitation for 1 hours. the different mixture have been filtered, the treated aqueous extract has been frozen and dehydrated through a lyophilization process. use of adsorbent in the food laboratory scale system 200 grams of the different adsorbates (cag, zeolite, almond and rice adsorbent) have been mixed with 1 liter of the aqueous extract of artichoke orlemon extract and have been kept in constant agitation for 3 hours. after 3 hours, the different adsorbates have been filtered, the treated aqueous extract has been frozen and dehydrated through a lyophilization process. hplc analysis the determination of pesticides were carried out by the liquid chromatography technique coupled to mass spectrometry with a triple quadrupole lc-ms / ms qqq analyzer (quechers method). the application range is established between the limit of quantification (0.01 mg / kg) and the maximum limit (2 mg / kg). for pesticides with mrls greater than 2 and less than or equal to 6 mg / kg, the maximum limit will be 7 mg / kg. this procedure is based on the sanco document “guidance document on analytical quality control and validation procedures for pesticides residues analysis in food and feed”, standard une-en 15662, une-en 12393-1 / 3 (61,62) and directive 96 / 23 / ec (63). two stock solutions of each of the specified pesticides will be prepared from commercial certified active ingredients coded by the laboratory, using acetonitrile as solvent. an internal standard (istd), triphenyl phosphate, will be prepared from its commercial certified active material, also coded by the laboratory, using acetonitrile as a solvent in the same way. from this stock standard, a 1 ppm concentration is prepared, which will be the one added to each of the dilutions of the calibration line, to have a constant istd concentration of 0.05 ppm in each of the points on the line. from said stock solutions, two multipatterns will be prepared in acetonitrile, the analytes of which will be found at a concentration of 1 ppm. with one of the multipatterns, dilutions with white matrix extracted in acetonitrile will be prepared, from which we will obtain the calibration line. the response factor (fr) of each point of the function will be calculated, according to the following expression: fr=(a_pesticide⁄aistd)/(c_pesticide⁄cistd) next, the mean and coefficient of variation of the response factors obtained will be calculated, which must be ≤ 20%. both the calibration line and the correlation coefficient, r, the fr, mean and coefficient of variation of said response factors are calculated by the agilent mass hunter workstation software. the minimum quantity of sample necessary to carry out the test will be 250g. the concentrations of the pesticides in the test sample will be calculated by introducing the ratio of areas between the active matter and the istd detected in the sample, in the calibration line. this process is carried out automatically by the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 293 equipment software, agilent mass hunter workstation. statistical analysis all determinations and experiments were performed in triplicate andthe presented results are the average values of three determinations and standard deviation 3. results and discussion content of pesticides and organic compounds of interest in artichoke extract and lemon extract table 1 presents thevalues of pesticides and organic compounds obtained in artichoke extracts. table 1. content of pesticides and organic compounds of interest in artichoke. results are expressed as mean values of triplicates ± s.d. compounds of interest (mg/kg) chlorogenic acid 25359 ± 378 cynarine 19232 ± 138 pesticides (mg/kg) azoxystrobin 0.42 ± 0.01 difenoconazole 12.2 ± 0.1 dimethoate 2.8 ± 0.1 metalaxyl 59.5 ± 1.6 table 1 shows that the artichoke extract has compounds of interest such as caffeic acid, chlorogenic acid and cynarin.with respect to its pesticide content, the presence of azoxystrobin, difenoconazole, metalaxyl and dimethoate were detected. table 2 presents the values of pesticides and organic compounds obtained in lemon extracts. table 2 shows that the lemon extract has compounds of interest such as hesperidin and limonin.with respect toits pesticide content, the presence of imazalil and pyrimetanilwere detected. table 2. content of pesticides and organic compounds of interest in lemon extract. results are expressed as mean values of triplicates ± s.d. compounds of interest (mg/kg) hesperidin 16478 ± 178 lemonin 2110 ± 44 pesticides (mg/kg) imazalil 11.2 ± 0.5 pyrimetanil 1.3 ± 0.1 adsorption process in order to comparethe effectiveness of the different adsorbents, artichoke and lemon extract were treated with bio adsorbents, zeolite andactivated carbon. after performing the adsorption process for the reduction of pesticides with the different adsorbates using the procedure described above, different pesticides food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 294 and compounds of interest were analized. table 3 shows the values of pesticides and organic compounds of interest in artichoke extract after adsorption process with bio-adsorbents, activated carbon and zeolite adsorbents. table 3. content of pesticides and organic compounds of interest in artichoke results are expressed as mean values of triplicates ± s.d. no treatment zeolite gac adsorbent rice adsorbent almond determination of compounds interest (mg/kg) chlorogenic acid 25359 ± 378 22947 ± 316 18566 ± 122 24186 ± 178 21512 ± 164 cynarine 19232 ± 138 18394 ± 101 15081± 92 16654 ± 133 14914 ± 145 pesticide determination (mg/kg) azoxystrobin 0.42 ± 0.01 0.32 ± 0.04 0.029 ± 0.01 0.53 ± 0.09 0.12 ± 0.03 difenoconazole 12.2 ± 0.1 10.5 ± 0.8 0.44 ± 0.11 6.7 ± 0.2 1.5 ± 0.1 dimethoate 2.8 ± 0.1 2.8 ± 0.1 1.1 ± 0.2 2.4 ± 0.3 2.2 ± 0.1 metalaxyl 59.5 ± 1.6 46.1 ± 1.3 2.5 ± 0.3 50.0 ± 2.5 25.4 ± 2.1 granular activated carbon was the most efficient adsorbate.adsorbents from almond and rice by-product were effective. reducing in case of almond skin adsorbent by half the concentration of metalaxyl and reducing a large amount of azoxystrobin and difenoconazole. rice bio-adsorbent was less effective than the other adsorbates even though it was more effective than zeolite. partly reducing difenoconazole and metalaxyl. the artichoke extracts obtained preserved the compounds of interest in similar concentrations and pesticides were greatly reduced. table 4 shows the values of pesticides and organic compounds of interest in lemon extract after adsorption process with bio-adsorbents. activated carbon and zeolite adsorbents. table 4. content of pesticides and organic compounds of interest in lemon extract. results are expressed as mean values of triplicates ± s.d. no treatment zeolite gac adsorbent rice adsorbent almond determination of compounds interest (mg/kg) hesperidin 16478 ± 178 15020 ± 125 16909 ± 82 15156 ± 96 16703± 120 limonin 2110 ± 44 1505 ± 38 958 ± 28 1486 ± 31 461 ± 11 pesticide determination (mg/kg) imazalil 11.2 ± 0.5 5.9 ± 0.3 8.9 ± 0.2 6.8 ± 0.2 pyrimetanil 1.3 ± 0.1 1.5 ± 0.2 0.48 ± 0.03 1.0 ± 0.1 0.24 ± 0.06 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 295 almond bio-adsorbent was the most effective in completely eliminating imazalil and greatly reduced pyrimetanil. with respect to the other adsorbents used greatly reduced pesticides. the lemon extracts obtained preserved the compounds of interest in similar concentrations. 4. conclusion the use of agri-food waste allows solving not only an environmental problem of pollution of high importance. but also a problem of management of large amounts of waste generated in the agri-food industry contributing to the promotion of circular economy. in addition. the elimination of toxic compounds in extracts with large concentrations of compounds of interest is one of the key points for the use of these extracts in the food. nutraceutical and cosmetic industry due to the toxicity of these compounds to health. two adsorbents have been developed from almond and rice by-products which are effective for the elimination of different toxic compounds through adsorption processes such as pesticides. recovering extracts containing compounds of interest by reducing the concentration of these toxic compounds. 5. acknowledgments this research was financially supported by region of murcia development agency. 6. references [1]. shirahigue. l.d.; ceccatoantonini. s.r. agro-industrial wastes as sources of bioactive compounds for food and fermentation industries. cienc. rural 2020. 50. 50. [2]. muñoz-jauregui. a.m.; ramosescudero. f. componentes fenólicos de la dieta y sus propiedades biomedicinales— phenolicscompoundsofthediet and hisbiomedicinalproperties. horiz. méd. 2007. 7. 23–38. [3]. bataglion. g.a.; da silva. f.m.a.; eberlin. m.n.; koolen. h.h.f. determination of the phenolic composition from brazilian tropical fruits by uhplc-ms/ms. food chem. 2015. 180. 280–287. [4]. bataglion. g.a.; da silva. f.m.a.; eberlin. m.n.; koolen. h.h.f. simultaneous quantification of phenolic compounds in buriti fruit (mauritia flexuosal.f.) by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. food res. int. 2014. 66. 396–400. [5]. sonnante. g.; pignone. d.; hammer. k. the domestication of artichoke and cardoon: from roman times to the genomic age. ann. bot. 2007. 100. 1095– 1100. [6]. abu-reidah. i.m.; arraezroman. d.; segura-carretero. a.; fernandez-gutierrez. a. extensive characterisation of bioactive phenolic constituents from globe artichoke (cynara scolymus l.) by hplc-dad-esi-qtof-ms. food chem. 2013. 141. 2269–2277. [7]. lattanzio. v.; kroon. p.a.; linsalata. v.; cardinali. a. globe artichoke: a functional food and source of nutraceutical ingredients. j. funct. foods 2009. 1. 131–144. [8]. el-mesallamy. a.m.d.; abdelhamid. n.; srour. l.; hussein. s.a.m. identification of polyphenolic compounds and hepatoprotective activity of artichoke (cynara scolymus l.) edible part extracts in rats. egypt. j. chem. 2020. 63. 2273–2285. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 296 [9]. sanchez-rabaneda. f.; jauregui. o.; lamuela-raventos. r.m. identification of phenolic compounds in artichoke waste by highperformance liquid chromatography—tandem mass spectrometry. j. chromatogr. a 2003. 1008. 57–72. [10]. pandino. g.; lombardo. s.; mauromicale. g. globe artichoke leaves and floral stems as a source of bioactive compounds. ind. crop. prod. 2013. 44. 44–49. [11]. orlovskaya. t.v.; luneva. i.l.; chelombit’ko. v.a. chemical composition of cynara scolymus leaves. chem. nat. compd. 2007. 43. 239–240. [12]. shallan. m.a.; ali. m.a.; meshrf. w.a.; marrez. d.a. in vitro antimicrobial. antioxidant and anticancer activities of globe artichoke (cynara cardunculus var. scolymus l.) bracts and receptacles ethanolic extract. biocatal. agric. biotechnol. 2020. 29. 101774. [13]. kuki´c. j.; popovi´c. v.; petrovi´c. s.; mucaji. p.; ciri´c. a.; stojkovi´c. d.; sokovi´c. m. antioxidant and antimicrobial activity of ´ cynara cardunculus extracts. food chem. 2008. 107. 861–868. [14]. lattanzio. v.; kroon. p.a.; linsalata. v.; cardinali. a. globe artichoke: a functional food and source of nutraceutical ingredients. j. funct. foods 2009. 1. 131–144. [crossref] [15]. el-mesallamy. a.m.d.; abdelhamid. n.; srour. l.; hussein. s.a.m. identification of polyphenolic compounds and hepatoprotective activity of artichoke (cynara scolymus l.) edible part extracts in rats. egypt. j. chem. 2020. 63. 2273–2285. [16]. aguilera. y.; martin-cabrejas. m.a.; gonzalez de mejia. e. phenolic compounds in fruits and beverages consumed as part of the mediterranean diet: their role in prevention of chronic diseases. phytochem. rev. 2016. 15. 405–423. [crossref] [17]. shen. q.; dai. z.; lu. y. rapid determination of caffeoylquinic acid derivatives in cynara scolymus l. by ultra-fast liquid chromatography/tandem mass spectrometry based on a fused core c18 column. j. sep. sci. 2010. 33. 3152–3158. [crossref] [18]. rejeb. i.b.; dhen. n.; gargouri. m.; boulila. a. chemical composition. antioxidant potential and enzymes inhibitory properties of globe artichoke by-products. chem. biodivers. 2020. 17. e2000073. [19]. mármol. i.; quero. j.; jiménezmoreno. n.; rodríguez-yoldi. m.j.; ancín-azpilicueta. c. a systematic review of the potential uses of pine bark in food industry and health care. trendsfoodsci. technol. 2019. 88. 558–566. [crossref] [20]. cruzado. m.; cedrón. j. nutracéuticos. alimentos funcionales y su producción. rev. química pucp 2012. 2. 33– 36. [21]. leonel. a.c.. bonan. r.f.. pinto. m.b.. kowalski. l.p.. perez. d.e. the pesticides use and the risk for head and neck cancer: a review of case-control studies. med. oral. patol. oral. 23962. (2020). [22]. hu. y.. zhang. y.. vinturache. a.. wang. y.. shi. r.. chen. l. effects of environmental pyrethroids exposure on semen quality in reproductive-age men in shanghai. china. chemosphere. 245. 125580. (2020). [23]. berrazoum. a.. marouf. r.. ouadjenia. f.. schott. j.bioadsorption of a reactive dye from aqueous solution by municipal solid waste. biotechnology reports. 7. 44–50. (2015). [24]. mateen f.. javed i.. rafique u.. tabassum n.. sarfraz m.. safi sz. new method for the adsorption of organic pollutants using natural zeolite incinerator ash (zia) and its application as an environmentally friendly and costeffective adsorbent. desalination and water treatment. 57(14): 6230-6238. (2016). [25]. mallek m.. chtourou m.. portillo m.. monclús h.. walha k.. ben salah a. granulated cork as biosorbent for the removal of phenol derivatives and emerging contaminants. journal of environmental management. 223: 576-585. (2018). [26]. drobek m.. figoli a.. santoro s.. navascués n.. motuzas j.. simone s.. algieri c.. gaeta n.. querze l.. trotta a.. barbieri g.. mallada r.. julbe a.. drioli e. (2015). pvdf-mfi mixed matrix membranes as vocs adsorbers. microporous and mesoporous materials. v 207. pp 126-133. [27]. al-qodah z.. shawaqfeh a.t. lafi w.f. (2007). adsorption of pesticides food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 francisco lorca, david quintín, maría-dolores lópez, presentación garcía, removal of toxic compounds from natural extracts using green techniques, food and environment safety, food and environment safety, volume xx, issue 3 – 2021, pag. 289 – 297 297 from aqueous solutions using oil shale ash. desalination. v 208. i 1–3. pp 294-305. [28]. berrazoum. a.. marouf. r.. ouadjenia. f.. schott. j. (2015). bioadsorption of a reactive dye from aqueous solution by municipal solid waste. biotechnology reports. 7. 44–50. [29]. mateen f.. javed i.. rafique u.. tabassum n.. sarfraz m.. safi sz.. yusoff i.. asrhaf m.. 2016. new method for the adsorption of organic pollutants using natural zeolite incinerator ash (zia) and its application as an environmentally friendly and costeffective adsorbent. desalination and water treatment. 57(14): 6230-6238. [30]. mallek m.. chtourou m.. portillo m.. monclús h.. walha k.. ben salah a.. salvadó v.. 2018. granulated cork as biosorbent for the removal of phenol derivatives and emerging contaminants. journal of environmental management. 223: 576-585. [31]. gupta. k.. kumar. v.. tikoo. k.b.. kaushik. a.. singhal. s.. 2020. encrustation of cadmium sulfide nanoparticles into the matrix of biomass derived silanized cellulose nanofibers for adsorptive detoxification of pesticide and textile waste. chem. eng. j.385. 123700. 76 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 12020, pag. 76 83 research on the functional-technological properties of protein complex and natural color in the composition of restructured ham products iryna shevchenko 1 , viktoriya zhuk 1 , galyna polishchuk 1 , *tatyana osmak 1 1educational and research institute of food technology, national university of food technologies, kyiv, ukraine, osmaktg@ukr.net *corresponding author received 10th march 2020, accepted 30th march 2020 abstract: the biopolymer complex of thermostable animal proteins (sodium caseinate, dry buttermilk, blood plasma vepro 75 psc), which have functional and technological properties, including stabilizing ability in the composition of restructured ham products, has been developed. the results of research confirmed the advantages of using the developed biopolymer complex, which consists of increasing water retention capability, fat-holding capacity and stabilize the physicochemical parameters of meat products the possibility of replacement of nitrite used to form color characteristics of meat systems with nitrate in the composition of vegetable raw materials at combination with nitrite-reducing microorganisms has been studied. the vegetable concentrate obtained from sweet potato potato and the starter culture staphylococcus carnosus were used as dye. the possibility of stabilizing the color characteristics of meat raw materials in the process of pickling has been proved by applying a new kind of natural color stabilizer. the results of the study have practical importance and will help to expand the range of restructured ham products with natural ingredients. keywords: biopolymer complex, meat systems, proteins, sweet potato, staphylococcus carnosus starter culture, coloring, restructured ham product 1. introduction increasing demand of protein products and necessity to provide balanced diet of the population led to the occurrence and rapid development of new directions in the production of meat products. a significant potential resource for food proteins is milk protein concentrates and blood plasma proteins, which are used irrationally in the conditions of tough saving high value animal proteins [1]. at the same time, the creation of restructured ham products with high biological value requires the study of the color formation of meat products [2]. it is possible to use nitrate from vegetable raw materials in combination with nitritereducing microorganisms which are able to fully compensate the action of sodium nitrite as a color stabilizer for meat products. quite a large amount of nitrite is present in vegetables, so in sweet potato the nitrite concentration can reach 15002800 mg / kg. that is, the juice of the sweet potato and its dry concentrates with a high content of nitrates can replace traditional sodium nitrite [3]. in order to develop foods of high biological value that meet the requirements of food security and to counteract the negative impact of sodium nitrite on http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 77 health, we investigated the possibility of replacing sodium nitrite with powdered juice of sweet potato in the presence of nitrite-reducing bacterial culture in order to color the restructured ham products low in myoglobin [4]. 2. materials and methods materials the dye used was purchased from chr. hansen company, obtained on the basis of a combination of sweet potatoes hansen™–coloring fruitmax®. sweet potato powder has a dark red color, well soluble in water, the ph of 10% solution is 2.7 3.4. the dye fully complies with eu foodstuffs regulation eu / 178/2002 with the latest changes, 1881/2006 / eu, which sets the maximum level for the content of certain food ingredients. staphylococcus carnosus was used as the nitriding starter culture. methods determination of the total pigment content was carried out according to the conventional method, which is based on the extraction of pigments of meat first with water and then with hydrochloric acid acetone, followed by photocolorimetry of the extract at a wavelength of 540 nm relative to hydrochloric acid acetone [5]. determination of the content of nitrosopigments was carried out by a method based on the extraction of nitrosopigments with a water solution of acetone, followed by determination of the optical density of solutions on a spectrophotometer at a wavelength of 540 nm relative to 80% water solution of acetone [5]. the content of nitrosopigments relative to the total number of pigments was calculated by the formula 𝑁𝑆 = 𝐸540 𝑁𝑂 𝐸540 𝑃𝑃 100 where ns content of nitrosopigments, %; 𝐸540 𝑁𝑂 the optical density of the nitrosopigment solution; 𝐸540 𝑃𝑃 the optical density of the solution containing all the pigments present in the product. color characteristics were determined in the colorimetric system dcielab: d65-10 using the demo paint program and the spectra of the spectrophotometer x-rite ci 7600 / ci 7800 with annex for solids. qualitative and quantitative composition of the amino acid mixture is determined by comparing the standard chromatogram and the studied mixtures of amino acids and calculating the peak area of each amino acid. amino acid fast the ratio of the content of the essential amino acid product to the corresponding content an indispensable amino acid "ideal protein" on the fao / who scale (food and agriculture organization, in conjunction with the world health organization): af = eaapr / eaae where af amino acid rate,%; eaapr the content of the essential amino acid in the product, g / 1 g protein; eaaep the content of the "essential protein" of the "essential protein" on the fao / who scale, g / l protein. standard research methods were used to determine the organoleptic, physicochemical parameters of model meat systems and restructured ham. the penetration voltage was determined using a ulab3-31m penetrometer, the depth of the indenter immersion in the test sample at a temperature of 20°c. three measurements were made on the open surface of the sample at a distance of at least 10 mm from the edge of the product and at the maximum distance from the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 78 points of other measurements, so that the deformed part of the surface did not enter the measurement area, then the penetration value was converted to the value of the penetration voltage.the water activity (aw) of model minced meat systems and meat chopped semi-finished products was determined using a rotronic hygro palm23 analyzer [5]. moisture content was determined according to dstu iso 1442:2005 meat and meat products. moisture content test method (control method) (iso 1442: 1997, idt). mass fraction of fat according to dstu iso 1443: 2005 meat and meat products. the method of determining the total fat content. mass fraction of protein by the kjeldahl method "agricultural food products. general guidelines for the determination of nitrogen content by the kjeldahl method (iso 1871:1975, idt)". organoleptic indices of cutlets according to dstu 4823.2:2007 meat products. organoleptic evaluation of quality indicators. general requirements. moisture-absorbing capacity (mac) and moisture-holding capacity mhc were determined by centrifugation [5]. moisture-absorbing capacity of model meat systems (mac, %) was evaluated by the method of grau r. and hamm r. in the modification of v.p. volovinsky and b.i. kelman by the ratio of mass fractoin of free and bound moisture [5]. materials. preparation of experimental samples a mixture of animal protein (sodium casein, buttermilk, dry pork blood plasma (75 psc) blood plasma at a ratio of 1: 1: 1) in the amount of 3% was added to replace meat raw materials in model meat systems made from low-fat pork (50%) and turkey meat (50%). as a control system was selected a protein-free meat system and colored with sodium nitrite in an amount 0.0075% sodium. the obtained samples of forcemeat were mixed at 12 ° c for 15 min, formed in the form of loaves, left at 10 ° c for 180 min to undergo the reaction of reduction of nitrate to nitrite and heat treatment according to the standard technological scheme for cooked meat products. the statistical analysis all the analytical determinations were performed at least three times and the value reported for determined characteristics was the average value ± of the standard deviation (s.d). the statistical analysis was performed at the microsoft excel statistical software version 2010. 3. results and discussion the ratio of amino acids in a food product and diet (amino acid formula) of essential amino acids is important for ensuring protein synthesis in the body. tryptophan is taken as the unit in this ratio; the amount of other essential amino acids should be 34 times more. this is such an important factor that in a number of diseases mixtures of free amino acids or their mixtures in the form of hydrolysates of natural raw materials are used, for example, in enteral and parenteral nutrition. naturally, the special enrichment of meat products, usually containing 15 ... 20% protein, even with some amino acids, will position them as therapeutic and prophylactic. one such solution is the use of biopolymer complexes based on functionaltechnological proteins in the composition of restructured ham products, which are capable of providing a stable quality level of biologically complete meat products food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 79 in order to substantiate the composition of restructured ham products of higher biological value, by the method of calculation of amino acid score, the authors selected and balanced the amino acid composition of the biopolymer complex with animal proteins (sodium casein, buttermilk, blood plasma 75psc in ratio 1:1:1), that made it possible to approximate it by the content of essential amino acids to the standard fао/who. according to the results of the calculation, the amino acid composition of the biopolymer complex was selected and balanced:sodium casein, buttermilk, blood plasma 75psc in ratio 1:1:1), that made it possible to approximate it by the content of essential amino acids to the standard fао/who. (table 1) table 1. amino acid composition of the biopolymer complex of blood plasma proteins, sodium caseinate and buttermilk in a ratio of 1:1:1 name of amino acid amino acid composition, g / 100 g protein amino acid score,%, of protein vepro 75 ps c sodium caseinat e buttermil k protein mixtur e fао/ wh o vepro 75 ps c sodium caseinat e buttermil k protein mixtur e valine 4.1 6.2 9.2 6.5 5.0 82.0 124.0 184.0 130.0 isoleucine 3.4 4.5 7.9 5.3 4.0 60.0 112.5 197.5 132.5 leucine 6.7 7.3 16.7 10.2 7.0 95.7 104.3 238.6 145.7 lysine 5.7 7.4 14.2 9.1 5.5 103. 6 134.5 258.2 165.5 methionine + cystine 4.6 2.1 4.7 3.8 3.5 131.4 60.0 134.3 108.6 threonine 4.2 3.2 8.7 5.4 4.0 105.0 80.0 217.5 135.0 tryptophan 1.2 1.2 15.9 6.1 1.0 120.0 120.0 1590 610.0 phenylalanin e + tyrosine 7.0 4.2 16.3 9.2 6.0 116.7 70.0 271.7 153.3 sum 36.9 36.1 93.6 55.6 36.0 animal proteins selected as the biopolymer complex (sodium caseinate, buttermilk and blood plasma vepro 75 psc) are thermostable functional proteins that have the ability to stabilize meat systems. in order to develop recommendations for the use of animal protein biopolymer complex as a functional and technological ingredient in the introduction in formulation of restructured ham of lean pork and turkey meat, the chemical composition and functional technological properties (ftp) of model meat systems with different level of its use were discovered. the development of a rational composition of restructured ham products of higher biological value was carried out by computer optimization based on the chemical composition of the recommended ingredients and the results of studies of their functional technological properties (ftp). moisture retaining ability (mra) and fat-holding capacity (fhc) were chosen as a function of the target, which is a criterion for the stability of moisture and fat retention in meat systems. knowledge of ftp is necessary for the rational processing of raw meat. they make it possible to predict and directively regulate the quality characteristics of finished products. the results of the studies are presented in table 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 80 table2. chemical composition and functional technological properties of model meat systems (n=3; р ≥ 95, mean ±sd) indicator the content of the biopolymer complex of animalproteins, % 0 (control sample) 2 3 4 moisture content, % 69.25±2.94 70.08±2.87 70.45±2.95 71.36±2.92 protein content, % 16.39±0.74 17.24±0.63 18.26±0.67 18.35±0.64 fat content, % 13.66±0.56 11.95±0.48 10.65±0.54 9.56±0.51 ash percentage by weight of dry substance ,% 0.70±0.001 0.73±0.001 0.73±0.001 0.73±0.001 рн 5.9±0.21 6.03±0.22 6.04±0.20 6.04±0.21 mra, % 67.90±2.76 74.70±2.54 75.1±2.78 75.79±2.63 fhc, % 68.00±2.15 74.52± 2.96 74.63±2.79 74.57±2.78 output, % 112.35±3.17 121.14±3.27 125.19±3.49 125.61±3.46 thermal treatment losses, % 11.66±0.43 9.51±0.41 7.19±0.39 7.05±0.39 residual content nano2×10 -3 3.92±0.02 3.56±0.02 3.24 ±0.02 3.17±0.02 cutting effort, па×10-5 1.79±0.03 1.71±0.03 1.63±0.02 1.62±0.05 cut-off voltage, kp 21.35±0.67 20.54±0.59 20.07±0.62 19.93±0.59 according to the table 2, introduction of the biopolymer complex of animal proteins in the amount of 3% in the composition of the restructured ham of lean pork and turkey meat increases mra of model meat systems by 10.1 ... 11.6%, and fhc by 9.5 … 9.7%, which certainly contributes to the improvement of the structure of restructured ham. introduction the biopolymer complex of animal proteins in the amount of 3% to the formulation of restructured ham of low-fat pork and turkey meat as a functional and technological ingredient allowed to increase their biological value by the content of essential amino acids by 13.2%, compared to the control. the results of the studies are presented in table 3. table 3. amino acid composition of restructured ham name of 
 aminoacids control: ham “for breakfast” dstu 4668: 200 the prototyp standard (chicken egg protein). g / 100 content of amino acids. g /100 g protein: essential : 40.59±1.81 45.94 ±1.23 36 valinе 5.15±0.15 5.78 ±0.31 5.00 isoleucinе 4.29 ±0.21 4.92 ±0.18 4.00 leucinе 7.65 ±0.32 9.02 ±0.29 7.00 lysine 7.73 ±0.31 8.75 ±0.29 5.50 methionine + cystin 3.63 ±0.15 3.89 ±0.37 3.50 threonin 4.25 ±0.20 4.64 ±0.56 4.00 tryptophan 1.16 ±0.05 1.32 ±0.19 1.00 phenylalanine + tyrosine 6.73 ±0.24 7.62 ±0.49 6.00 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 77 table 3 data indicate that the test and control samples of ham do not contain limiting amino acids. among all prototypes of heat-treated restructured ham, the lowest score was obtained by the control sample, which was characterized by insufficient juiciness, fragility of the structure, lower output and higher by 2.15-4.61% losses in heat treatment. the best qualitative characteristics (high juiciness, denser and more elastic consistency) are characteristic of prototypes with biopolymer complex of animal proteins. the use of the developed biopolymer complex of animal proteins in the amount of 3% in the composition of restructured ham of lean and turkey pork allows to obtain a functional product with the best organoleptic and technological properties, and balanced in chemical composition and biologically complete meat quality level. at the next stage of the research, the coloring properties of the fruitmax® vegetable concentrate were investigated in the presence of a nitride-reducing starter culture of staphylococcus carnosus, whose activity manifests itself at a ph higher than 5.5 and a temperature above 8 °c. an analysis of the literature data [6, 7, 8, 9, 10] and our own experimental studies confirmed that the formation of coloring of meat products begins in the process of pickling. the reaction of formation of nitrosopigments intensively proceeds at ph 5.5… 6.0. at meat ph greater than 6.0, the nitrosomyoglobin (nomb) reaction occurs at a slower rate. temperature has a significant influence on the color formation of meat products. in the process of traditional pickling and cold smoking, about 40 ... 50% of nomb nitrosomyoglobin is formed. it has been found that the use of the sweet potato juice powder fruitmax® in the amount of 0.30, 0.35 and 0.40% in the presence of 0.025% staphylococcus carnosus contributes to the accumulation of no-pigment in an amount that is typical for color formation in the presence of sodium nitrite about 47.83… 48.23% to the total pigment (table 4). usage of fruitmax® in the amount of 0.35 ... 0.40% leads to a more intensive formation of nitrosopigments and, as a consequence, to a lower content of residual nitrite in the product. table 4. influence of powdered juice of sweet potato on nitrosation in restructured ham (n=3; р ≥ 0.95) amount of powdered sweet potato in the meat system,% common pigments, units of optical densit no-pigment content, % to the total pigmen number residual nitrite, mg / 100 0 (control sample nаno2) 0.634 ± 0.017 48.23 ± 0.22 3.26 ± 0.03 0,30 0.583 ± 0.019 47.53 ± 0.20 3.12 ± 0.05 0,35 0.621 ± 0.013 47.82 ± 0.21 3.10 ± 0.03 0,40 0.634 ± 0.017 47.83 ± 0.22 3.06 ± 0.03 the identity of the content of nitrosopigments in the test specimens, compared to the control, and characterized by the conversion of nitrate from vegetable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 82 raw materials by nitro-reducing microorganisms to nitrite, which interacts with myoglobin of meat. the result is a no-pigment that gives the meat products typical pink-red color. the use of stained ham products in the presence of 0.025% staphylococcus carnosus in the replacement of sodium nitrite for the formation of color of the ham products also reduces the residual content of sodium nitrite in the product and avoids the accumulation of carcinogens. spectrophotometric measurement of the coloring of restructured ham products was performed using sweet potato juice powder in combination with nitrate-reducing microorganisms (table 5). table 5. spectral characteristics of the coloring of restructured ham products nr. crt. name of dy concentration % color coordinatescielab l (light shade intensity) а (intensity of red color) b (intensity of yellow color) 1 sodium nitrite 0.0075 67.93 5.20 16.00 2 juice powder sweet potato 0.2 0.2 % 77.99 4.32 15.80 3 juice powder sweet potato 0.3 0.3 % 68.45 5.21 15.14 4 juice powder sweet potato 0.4 0.4 % 68.59 5.23 14.94 in table 5 are presented spectral characteristics of the restructured ham have shown that, in light shade intensity, the test specimens are inferior to the control due to the natural coloration of pork and turkey meat. however, against the background of high enough indicator intensity of red color and a low value of "yellowness", the test specimens have a typical color of meat products pink and red, which confirms the feasibility of using in their composition a natural dye powder juice of sweet potato fruitmax®, in the amount of 0.3… 0.4%, in combination with nitrate-reducing microorganismsthe prospect of further research is to develop basic formulations of restructured ham products of high nutritional value with protein complex and natural dye, as well as to clarify the technological modes of their production. 4. conclusion the biopolymer complex of animal proteins in restructured ham products exhibits functional technological properties, which is confirmed by the improvement of qualitative and structuralmechanical parameters, reduction of losses during heat treatment and increase of output of finished meat products. combinational variation in the composition of restructured ham products is promising and needs further improvement in order to obtain products of high nutritional and biological value. using juice powder of sweet potato fructmax® for color formation of the restructured ham in the presence of staphylococcus carnosus in the replacement of sodium nitrite allows reducing the residual content of sodium nitrite in a product with low myoglobin content in meat raw materials and avoids the accumulation of carcinogens. 5. references [1]. rogov i. food chemistry. book. 1 (proteins: structure, functions, role in nutrition), m.: koloss, 853 p, (2007). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 1 – 2020 iryna shevchenko, viktoriya zhuk, galyna polishchuk, tatyana osmak, research of functionaltechnological properties of the protein complex and natural color in the composition of restructured ham products, food and environment safety, volume xix, issue 1 – 2020, pag. 76 – 83 83 [2]. salavatulin r. rational use of raw materials in sausage production. st. petersburg: zao trade house george, 236 p., (2005). [3]. feiner h. meat products. scientific bases, technologies, practical recommendations. publ: profession, 720 p., (2010). [4]. tarte r. ingredients in the production of meat products. properties, functionality, applied. publ: id profession, 464 p., (2015). [5]. kyshenko i., starchova v., goncharov g. meat and meat products technology. kiev: nukht, 367 p., (2010). [6]. vedernikova i. operation of colorforming compositions based on the preparation of hemoglobin: diss. cand. tech sciences: 05.18.04. m., 253 p., (2004). [7]. kasyanov g. theoretical bases of formation of color characteristics of meat paste, food industry, n. 4, p. 24 – 28, (2000). [8]. mokeeva a. dyes from natural raw materials to improve the color and quality of food, nutritional ingredients. raw materials and additives, n. 1, p. 18 – 19, (2001). [9]. ivanov s., kishenko i., kryzhova yu. research of quality indicators of the raw material base of the meat processing industry of ukraine, maisto chemijair technologija. mokslo darbai proceedings (food chemistry and technology), n. 1, t. 47, p. 35-43, (2013). [10]. zhuk v., shevchenko i., polishchuk g. pasca m. color-correcting compositions of meat systems with a low content of hemoglobin-containing raw materials, scientific bulletin lnuvmb imeni s.z. gzhytsʹkoho. series: food technology, vol. 21, n 91, p.136-142, (2019). 1. introduction 4. conclusion 228 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefancel mare university of suceava, romania volume xix, issue 3 2020, pag.228 -236 physicochemical and color evaluation of confectionery mousses raluca olimpia zimbru1, sergiu pădureţ1*, sonia amariei1 1faculty of food engineering, stefan cel mare university of suceava, 13 university str. 720229, suceava, romania, sergiu.paduret@fia.usv.ro *corresponding author received17th july 2020, accepted 21st september 2020 abstract: in this study was investigated the effect of raw material and the production process on the physicochemical composition and color of confectionery mousses. for this approach, two different mousses’ production procedures were used and the chemical composition (fat, moisture, protein content and soluble substances concentration), physical parameters (overrun, density, specific volume), and color were determined. the dairy cream mousse samples (m1 and m3) presented a higher fat content (33.68% and 30.57%), while the protein content varied from 0.9% to 3.42%, being influenced by the raw materials used in the production process. color measurements indicated that the mousses based on vegetable cream were whiter with a brightness (l*) ranging between 90.04 – 94.25 and a higher whiteness index. the results of one factor analysis of variance — anova showed that the color difference between mousse samples is statistically significant (p<0.001). keywords: overrun, color, mousse, density, aeration 1. introduction confectionery represents a class of food products with unique and special sensory properties characterized by a high energy value. the romanian confectionery market is growing, due to the changes in consumption habits, but on the other hand, the increase of local competition and the international trends also contribute to this growth [1]. aerated desserts are widespread and show an important market perspective, as a function of consumers’ comportment, which has a special interest for lighter and healthier food products [2]. the production of aerated dairy confections is difficult and delicate, demanding thorough acquaintance of the formation and stabilization of food foams, the utilization of functional ingredients such as emulsifiers, stabilizers and also knowledge about the interaction and interference of the process parameters on the characteristics of the finite product [3]. the introduction and fixing of air bubbles or other gases in dairy desserts produce unique texture characteristics or sensory properties, reduces the density of the products [4], and decrease the product’s caloric content and the production costs since less raw material are used [5]. besides mechanical whipping processes in which rotor–stator air dispersing devices are used [6], the aerated structure of dairy desserts can be performed also by mixing the food matrix with gas under high pressure; a suitable pressure being between 2 – 6 bar. this process presents the advantage that the re-coalescence of the air/gas bubbles in the apparatus is reduced. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 229 however, it also has some disadvantages related to the bubble's expansion after leaving the device such as (i) a small interior pressure (laplace pressure) which leads to a more easily deformable product, the modulus of deformability, and respectively the foam stiffness is reduced, and the aerated product decreases its ability of shape retention; (ii) the products’ creaminess decreases due to the fact that large size of the air/gas bubbles reduces the interface of the bubbles thus diminishing the continuous phase immobilization capability and accordingly resulting faster drainage losses; (iii) bubbles expansion can lead to foam lamella rupture resulting even larger disproportionate bubbles, a process also called ostwald ripening [7]. for the aeration process, in addition to air, it is recommended to use gases without smell, taste, non-toxic and which allow controlled aeration [5]. among the used gases (air, steam, carbon dioxide, nitrogen or nitrous oxide) [8], the carbon dioxide presents some important features such as a good fat solubility, which makes it less predisposed to escape from the aerated fat food matrix during cold-stamping, than other gases less soluble in fat, such as nitrogen [5]. the mousses are defined as aerated confectionery products or overrun milkbased products with a stabilized aerated structure that nowadays is produced on an industrial scale and is gaining space in the dessert market [2]. according to grassler 2001, the overrun food products are understood to be products that have been subjected to an increase in volume by overrunning (from 80% to 120%). additionally, mousses contain sugar, a flavoring component, cream, and a gelling or a thickening agent [9]. the most popular mousse flavor is chocolate, cocoa followed by orange, strawberry, and lemon [2]. when it comes to overrun food products (aerated products) like chantilly, mousses, whipped cream, marshmallow, or ice cream an important aspect is represented by the product stability, which is closely connected to the product microstructure, and more exactly, to the gas/air voids dimension, distribution and also volume fraction [10]. the structure of aerated whipped products is primarily affected by the continuous phase properties, like viscosity, density or surface tension and also by the process parameters such as overrun, whipping time, velocity, whipping head geometry, power input, temperature, and static pressure [7]. according to mcclements 2016, food foams with the smallest air/ gas bubbles and uniform distribution of them show an improved texture, being creamier and more appealing to consumers and presenting higher stability [11, 12]. given that aerated confectionery products are complex food systems, represents also a significant category of food materials [13] and diverse types of aerated confections have been designed to develop novel products properly adapted to consumer preferences, using air, as a free ingredient [14]; therefore, this study aims to evaluate the influence of the raw materials and the production process on the physicochemical composition and color of confectionery mousses. 2. materials and methods preparation of mousse samples. for experimental mousse samples were used vegetable and dairy cream, white chocolate, sugar, and vanilla; following two different procedures as presented in figures 1 and 2. the samples aeration was made by the whipping process at 600 rpm [15] for 5 minutes, using a kitchenaid planetary mixer with 6 wire whip attachment [8]. the m1 and m2 mousse samples were made by the first procedure (figure 1), where half of the dairy cream/vegetable cream (62.5 g) was heated to boiling and mixed with chocolate (50 g), food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 230 and the other half was whipped, finallybeing combined and put into parallelepiped shapes. the m1 represents the dairy cream mousse samples, while the m2 represents the vegetable cream samples. the m3 and m4 mousse samples were made by the second procedure (figure 2). m3 represents the dairy cream mousse samples, while the m4 represents the vegetable cream mousses. a small amount of dairy cream/vegetable cream was heated and infused with vanilla (1g), while the remaining dairy cream/vegetable cream was whipped with sugar and finally mixed together. fig. 1. the process of obtaining the m1 and m2mousses samples fig. 2. the process of obtaining the m3 and m4 mousses samples physicochemical analysis. the moisture content of mousse samples was performed through the oven drying method at 103±2 °c [16], while the protein content was measured using the kjeldahl assay and a conversion factor of 6.38 (equivalent to 0.156 g nitrogen per gram of protein) [17]. the total fat content of mousse samples was measured by soxhlet assay, using petroleum ether as solvent [18]. the results of protein and fat analysis were expressed as a percent of wet basis. the acidity was measured by direct titration with 0.1 n naoh and the results were expressed as thorner degrees (ºth) [19]. all reagents used for mousses analysis were of analytical grade. the ph values were measured with a mettler toledo ph-meter and the water activity aw was determined with aqualab lite water activity meter. the soluble substances concentrations (expressed as °brix) were measured with leica mark ii plus refractometer. in addition to chemical composition, the overrun parameter of mousse samples was also calculated. the overrun characterizes 10g 40 g 5g food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 231 the mousse samples in terms of air volume incorporated and fixed in the product structure. overrun was evaluated for each mousse using the following equation (eq. 1) [20, 12]. 100   ww wwwu overrun (1) for overrun calculation, the volume corresponding to the weight of unwhipped mousse (wu) is the same as the volume corresponding to the weight of whipped mousse (ww). more accessible parameters like density and specific volume were also measured [21]. color evaluation. the color properties were evaluated with acr-400 chroma meter from konica minolta (konica minolta, japan). also for color measurements, the commission internationale de l’eclairage (cie) l*a*b* uniform color space assay was applied, where: l* represents the mousse brightness (0 represents black and 100 white), a* is the red–green color parameter (+a* represents red and a* green) and b* is the yellow–blue color parameter (+b* represents yellow and b* blue). the other color parameters like color difference (δe*), hue angle or tone (h0), chroma or color intensity (c*), whiteness index (wi), and yellowness index (yi) were also calculated (eq.2-6) [22-24]. δe* = [(δl2) + (δa2) + (δb2)]1/2 (2) where: δl* is the brightness difference between two samples, δa* is the difference between red-green color parameters and δb*is the difference between yellow-blue color parameters. h0=tan-1 (b*/ a*) (3) (4) c*= 2*2* ba  (5) wi=   2*2*2*100100 bal  (6) the mousse samples were stored at 4-6 °c until they were analyzed and the results represent the average of three measurements. statistical analysis. the obtained values for mousses analysis were submitted to analysis of variance anova by statgraphics centurion xvi (trial version). thestatistical significance was set at α = 0.05. 3. results and discussion physicochemical analysis. in table 1 are shown the physicochemical analysis of moussesamples, respectively the fat content, protein content, moisture, soluble substances concentration, water activity, acidity, ph, overrun, density, and specific volume. the m1 mousse sample presents a high-fat content (33.68%), followed by the m3 sample (30.57%); both m1 and m3mousses being produced with dairy cream. unlike m1 and m3, the m2 and m4 samples showed a lower fat content, ranging between 24 28%, due to the use of vegetable cream, which had a lower fat content. the samples’ protein content varies from 0.9% to 3.42%, depending on the raw materials used in the production process; the m1 samples presented the highest values, while the m4 samples presented the lowest values. the fat and protein content has an important role in food products aeration, initially, the air/gas bubbles being stabilized by the proteins and fixed by fat globules [4, 25]. the * *86.142 l b yi  food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 232 moisture contentof mousse samples ranged from 40.84% to 59.98%, for the m3 and m4 samples, produced by the second procedure (figure 2), were observed the highest concentration. in the case of soluble substances concentration and total acidity, it can be observed that m1 and m2 samples, produced by the first procedure (figure 1), had the highest values, whereas the ph values were close to the neutral zone (6.34-6.03). when it comes to confections, water activity has a significant contribution to quality evaluation, product safety, processing, shelf life, texturaland also sensory characteristics [27]. the water activity of mousse samples was in the same range, between 0.93 and 0.97, which is similar to those reported by schmidt & fontana 2020 [28]. table 1. physicochemical properties of mousses samples sample m1 m2 m3 m4 f – ratio p–value fat [%] 33.68 (0.57)a 28.16 (0.42)c 30.57 (0.38)b 24.38 (0.81)d 399.6 p < 0.001 protein [%] 3.42 (0.25)a 1.90 (0.32)b 2.04 (0.22)b 0.9 (0.34)c 15.2 p < 0.01 moisture [%] 40.84 (0.25)c 37.58 (0.37)d 54.29 (1.21)b 59.98 (1.54)a 405.5 p < 0.001 brix [ºb] 21.50 (0.50)b 32.00 (0.80)a 11.50 (0.45)d 13.00 (0.50)c 259.1 p < 0.001 acidity [ºth] 23.50 (1.20)a 20.50 (0.45)b 15.50 (0.55)c 11.00 (0.75)d 97.2 p < 0.001 aw 0.93 (0.02)c 0.94 (0.01)bc 0.96 (0.02)ab 0.97 (0.01) a 4.0 p > 0.05 ph 6.03 (0.07)d 6.18 (0.05)c 6.28 (0.01)b 6.34 (0.02)a 91.5 p < 0.001 overrun [%] 17.51 (3.22)d 89.13 (3.55) b 59.02 (2.02)c 236.76 (2.10)a 2019.8 p < 0.001 density [g/cm3] 0.997 (0.04)a 0.618 (0.05)c 0.641 (0.03)b 0.311 (0.01)d 9378.5 p < 0.001 specific volume [cm3/g] 1.002 (0.04)d 1.617 (0.05)b 1.559 (0.03)c 3.205 (0.01)a 1865.8 p < 0.001 different lowercase letters (a–d) in a row show significant differences between the groups (p<0.05). the overrun parameter of whipped mousse gives information on the amount of air/gas incorporated in the product [12], and according to camacho 1998[29], the overrun of aerated confectionery products can be improved using hydrocolloids substances like carrageenan, gelatin, or locust bean gum. the highest overrun was recorded by vegetable cream mousses (m4236.76% and m2-89.13%), while the m1 sample presented the lowest value (17.51%). furthermore, van aken 2001[15] reported that the overrun parameter of whipped dairy cream is influenced by fat concentration. according to campbell 1999 [20], dairy products such as different types of ice cream, mousses, beating cream, or frozen desserts include commonly from 15% to 60% air, offering a textural variety. the overrun results of analyzed mousses were similar to those reported for whipped cream (70150%), ice cream (40%), or cake (200250%), [20]. the introduction of air reduced the mousses’ density, which varied between 0.311 g/cm3 and 0.997 g/cm3, while de specific volume increased. in the case of mousses’ physicochemical properties, the anova analysis highlighted a difference between samples at a level of p < 0.01 for protein, fat, moisture, soluble substances concentration, porosity, overrun, density and specific volume, which means that the procedures food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 233 used as well as the raw materials lead to different samples in terms of physical and chemical composition. color evaluation. the anova of color parameter values of the analyzed mousses is shown in table 2. color is the quality component evaluated instantaneously by consumers, being decisive to product acceptance or rejection. foods color is given by the biochemical, chemical, microbial, and physical modifications which occur during processing, growth, ripening process, maturation, heat treatments, postharvest handling, represents an important quality characteristic and determines the consumers choice and preferences [30]. table 2. anova of mousses’colorparameters sample m1 m2 m3 m4 f – ratio p– value l* 82.72(0.84)c 90.04(1.05) b 91.06(0.87) b 94.25(0.89) a 46.3 p < 0.001 a* -7.55 (0.10) d -6.01 (0.08) b -6.63 (0.11) c -5.65 (0.12) a 189.4 p < 0.001 b* 25.36(0.28) a 15.39(0.09) c 17.25(0.26) b 9.79(0.12) d 2828.3 p < 0.001 c 26.46(0.24)a 16.53(0.05)c 18.48(0.28)b 11.30(0.17)d 2732.8 p < 0.001 h° 106.57(0.38)c 111.34(0.39)b 111.01(0.02)b 119.97(0.21)a 1084.6 p < 0.001 yi 43.81(0.93)a 24.42(0.43)c 27.06(0.15)b 14.84(0.10)d 1578.8 p < 0.001 wi 68.38(0.66)d 80.68(0.59)b 79.45(0.12)c 87.23(0.70)a 564.3 p < 0.001 δe m1 12.46 (0.30)b 12.02(1.31)b 19.53 (1.44)a 41.9 p < 0.01 m2 2.61 (0.92)b 6.63 (0.79)a 32.49 p < 0.01 m3 8.17 (0.98) different lowercase letters (a–d) in a row show significant differences between the groups (p<0.05). for color measurements, the (commission internationale de l’eclairage) cie l*a*b* method was used and it can be observed that the brightness (l*) of mousses varies from 82.72 to 94.25, the mousse samples with chocolate presented lower values whilethe m4 sample was the whitest. according to pathare 2013, food color can be associated with other quality characteristics such as nutritional properties, sensory characteristics, some defects and can help to manage them immediately [30]. as the data shows the yellow–blue color parameter b* had positive values which mean that the samples have a yellowish tint. the samples based on dairy cream and chocolate showed higher values ranged between 25.36 and 15.39; instead, the vegetable mousse samples exhibited lower values for the b*-color parameter and higher values for whiteness index (wi) and hue angle or tone (h0). according to pathare2013, a higher tone or hue angle value corresponds to a lesser yellow characteristic [30]. in the case of a* color food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 234 parameter (red–green axis), all values are in the negative zone; lower values being observed for dairy cream-based mousses (m1 and m3 samples). the results of one factor analysis of variance anova showed that the color difference between mousse samples is statistically significant (p<0.001). the mousses’ chroma value varies from 11.30 to 26.46 and a higher value of chroma corresponds to a more intense color of mousse samples perceived by the human eye. the samples produced by the first procedure with dairy cream presented a high value of chroma and also a high yellowness index (yi 43.81). the total color difference represents a measure of the modulus of the distance vector between the initial color values and the actual color coordinates [31] and indicates the magnitude of color difference between dairy and vegetable cream mousses produced through two different procedures and different raw materials. according to adekunte 2010, if δe>3 the color difference isvery distinct, if 1.5<δe<3 the color difference between samples is distinct, and if δe<1.5 the color difference is barely perceptible [32]. as regarding the color difference (δe*) of mousses samples it can be observed that almost all values are greater than 6.63, the color of mousses samples being significantly different (p < 0.01). a small color difference (2.61) was recorded between m2 and m3 mousses. 4. conclusion the mousse samples produced with dairy cream showed higher fat and protein content than the vegetable cream mousses. as regarding the amount of air incorporated and fixed in the product it can be observed that vegetable cream mousses presented higher values for overrun parameter and lower values for density measurements, resulting that these samples (m2 and m4) had a larger amount of embedded air than those based on dairy cream. additionally, the mousses produced through the second procedure had the highest concentration of moisture while the highest concentration of soluble substances was observed for mousses made through the first procedure. the color was influenced by the raw materials used, a more yellow tint was recorded for the dairy cream mousse samples, which exhibit higher values of b* color parameter, color intensity (c*) and also yellowness index. whereas the vegetable cream mousses were whiter with higher values of brightness, of whiteness index and lower b* color parameter. 5. references [1]. stanciu, s., confectionery sector in romania: market overview, education excellence and innovation management through vision 2020, 3783-3795, (2020). [2]. aragon-alegro, l. c., alegro, j. h. a., cardarelli, h. r., chiu, m. c., and saad, s. m. i., potentially probiotic and synbiotic chocolate mousse. lwt-food science and technology, 40(4), 669-675, (2007). [3]. cardarelli, h. r., aragon‐alegro, l. c., alegro, j. h., de castro, i. a., and saad, s. m., effect of inulin and lactobacillus paracasei on sensory and instrumental texture properties of functional chocolate mousse. journal of the science of food and agriculture, 88(8), 1318-1324, (2008). [4]. zimbru, r. o., pădureţ, s., and amariei, s., aerated confectionery: physicochemical and textural evaluation. food and environment safety journal, 19(2), (2020). [5]. gustav, t., castrillon, p. m., and hauger, r. u.s. patent no. 10,178,870. washington, dc: u.s. patent and trademark office, (2019). [6]. zimbru, r. o., pădureţ, s., and amariei, s., effect of aeration on physicochemical, color and texture characteristics of confectionery foams. ukrainian food journal, 9(1), (2020). food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 235 [7]. müller-fischer, n., and windhab, e. j., influence of process parameters on microstructure of food foam whipped in a rotor– stator device within a wide static pressure range. colloids and surfaces a: physicochemical and engineering aspects, 263(1-3), 353-362, (2005). [8]. pădureţ, s., zimbru, r. o., and amariei, s., texture and rheological evaluation of aerated confectionery, ovidius university annals of chemistry, volume 31, number 1, pp. 60 65, (2020). [9]. grassler, w., and wild, m, u.s. patent no. 6,231,902. washington, dc: u.s. patent and trademark office, (2001). [10]. miquelim, j. n., lannes, s. c., and mezzenga, r., ph influence on the stability of foams with protein–polysaccharide complexes at their interfaces. food hydrocolloids, 24(4), 398405, (2010). [11]. mcclements, d. j., food emulsions: principles, practices, and techniques. crc press. new york, usa, (2015). [12]. duquenne, b., vergauwen, b., capdepon, c., boone, m. a., de schryver, t., van hoorebeke, l., andde block, j., stabilising frozen dairy mousses by low molecular weight gelatin peptides. food hydrocolloids, 60, 317-323, (2016). [13]. mezzenga, r., schurtenberger, p., burbidge, a., and michel, m., understanding foods as soft materials. nature materials, 4(10), 729-740, (2005). [14]. narchi, i., vial, c., and djelveh, g., effect of protein–polysaccharide mixtures on the continuous manufacturing of foamed food products. food hydrocolloids, 23(1), 188-201, (2009). [15]. van aken, g.a., aeration of emulsions by whipping, colloids and surfaces a: physicochemical and engineering aspects 190,333354, (2001). [16]. nielsen, s.s., (ed.), food analysis new york: springer, pp. 139-141, (2010). [17]. sr en iso 8968-2:2002, determination of nitrogen content. block digestion method. [18]. aoac international, official methods of analysis, 20th edn., aoac international, rockville, md, 2016. [19]. tyl, c., sadler, g.d., ph and titratable acidity. in: nielsen s. (eds), food analysis, food science text series, springer, cham, pp. 389–406, (2017). [20]. campbell, g. m., and mougeot, e., creation and characterisation of aerated food products. trends in food science and technology, 10(9), 283-296, (1999). [21]. sereno a. m., silva m. a., mayor l., determination of particle density and porosity in foods and porous materials with high moisture content. international journal of food properties, 10(3), 455-469, (2007). [22]. jung h., sato t., comparison between the color properties of whiteness index and yellowness index on the cielab. textile coloration and finishing, 25(4), pp. 241–246, (2013). [23]. pădureț s., oroian m., gutt g., amariei s., evaluation of strawberry texture in close relation with their anisotropy, international journal of food properties, 20(2), pp. 247–259, (2017). [24]. hsu, c. l., chen, w., weng, y. m., and tseng, c. y., chemical composition, physical properties, and antioxidant activities of yam flours as affected by different drying methods. food chemistry, 83(1), 85-92, (2003). [25]. rodriguez-aguilera, r., oliveira, j. c., montanez, j. c., and mahajan, p. v., effect of modified atmosphere packaging on quality factors and shelf-life of mould surface-ripened cheese: part ii varying storage temperature. lwt-food science and technology, 44(1), 337-342, (2011). [26]. shahidi, f. (ed.)., bailey’s industrial oil and fat products, edible oil and fat products: processing technologies (vol. 5). john wiley and sons, (2005). [27]. fontana, a., water activity for predicting quality and shelf life. manufacturing confectioner, 85(11), 45, (2005). [28]. schmidt, s. j., and fontana jr, a. j. e., water activity values of select food ingredients and products. water activity in foods: fundamentals and applications, 573-591, (2020). [29]. camacho, m. m., martineznavarrete, n., and chiralt, a., influence of locust bean gum/lambda-carrageenan mixtures on whipping and mechanical properties and food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xix, issue 3 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, physicochemical and color evaluation of confectionery mousses, food and environment safety, volume xix, issue 3 – 2020, pag. 228 236 236 stability of dairy creams. food research international, 31, 653e658, (1998). [30]. pathare, p. b., opara, u. l., and al-said, f. a. j., colour measurement and analysis in fresh and processed foods: a review. food and bioprocess technology, 6(1), 3660, (2013). [31]. patras, a., brunton, n. p., tiwari, b., and butler, f., stability and degradation kinetics of bioactive compounds and colour in strawberry jam during storage. food and bioprocess technology, 4, 1245–1252, (2011). [32]. adekunte, a. o., tiwari, b. k., cullen, p. j., scannell, a. g. m., and o’donnell, c. p., effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice. food chemistry, 122(3), 500-507, (2010). 169 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 169 178 the pros pec ts of sa fety a nd m i crob iol ogical s tab il ity im prov em e nt o f baker y pro du c ts by us in g phy to add it ives tatyana lebedenko1, antonina yegorova1, *viktoriia kozhevnikova1, yekaterina korolenko1 1odessa national academy of food technologies, ukraine, kozhevnikova-viktoriya@inbox.ru *corresponding author received june 17th 2014, accepted june 27th 2014 abstract: in this article the main problems of bakery product quality and safety formation have been examined; it has been noted that one of the main objectives of the industry is implementation of measures of bread ropiness and molding prevention. on the basis of analyzing the main methods of product microbiological stability improvement during storage that include the usage of artificial preservatives or sourdough with specifically cultivated fermentative microflora and evaluating their effectiveness, the importance of search for alternative solution for problems of baking industry has been demonstrated. in this aspect especially interesting are aromatic and medicinal plants known for their antiseptic, antioxidant, and other properties capable of stabilizing the product quality. the main goal of this work is the study of a number of phyto-additives and their impact on pathogenic microorganisms and fermentative microflora of semi-products, their biotechnological properties, and finished product quality. the usage of phyto-additives as extracts has been proposed. it has been determined that hop, st john’s wort, and ashberry extracts have pronounced antiseptic properties towards microbiological pathogens of bakery products. the sage, marigold, peppermint, and chamomile extracts have been effective against mold fungi. all extracts have positive effect on intensity of fermentation process where the best results has been achieved by using cinnamon rose, haw, ashberry, and hop extracts. the consumer properties of wheat bread with extracts added judged by volume, texture, and shape stability for almost all samples have been either improved or identical to check sample. their microbiological stability has been considerably improved. keywords: extract, aromatic and medicinal plants, bread ropiness, molding 1. introduction the bakery products have a special place in nutrition of population of many countries, and are consumed daily regardless of the customers’ age, health state, and lifestyle. they are the constant source of many nutrients and biologically active substances (bas), have high assimilability, and are capable of regulating the activity of gastrointestinal tract [1]. however the potentially harmful substances of chemical or microbiological nature can enter the human organism with bakery products presenting a serious danger to health. those are mainly the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 170 compounds accumulated because of violating the rules of growing and storage of raw materials, those used to solve the technological problems of baking industry as well as those produced during bread production and storage. among these are toxicological agents, radionuclides, polycyclic substances, pesticides, components of mineral fertilizers, products of lipid oxidation and pest vital functions. the problem of raw material contamination by highly toxic, mutagenic, and cancerogenic mycotoxins produced by mold fungi has escalated worldwide [2, 3]. the concern of consumers and nutritionists specializing in product safety has also been caused by the presence of a wide spectrum of additives statistically confirmed to be used by more than 80 % of bakeries in order to improve the quality of bread and pastries [4]. they are used in order to solve the technological problems of forming the product of necessary quality under the condition of considerable fluctuations of raw materials (mainly flour) properties, implementation of accelerated technologies, and introduction of new sources of bas with low technological properties. besides, the fact of bread being one of the products with high acrylamide content [5] is a cause of concern since acrylamide formed during baking as a result of a reaction of asparagine with sugar has cancerogenic and genotoxic properties. with the implementation of accelerated technologies of bread production and introduction of high amounts of reducing sugars the acrylamide content of the finished product increases [6]. one of the main problems leading to deterioration of consumer qualities and safety of wheat bakery products and economical losses for manufacturers is a low microbiological stability of finished products during storage. the frequent cases of bread ropiness caused by sporeforming microorganisms bacillus subtilis and bacillus mesentericus and molding caused by the growth of fungi of aspergillus, mucor, penicillum, and rhizopus genus [7, 8]. the situation escalation in the last years has been caused by a number of factors. firstly, the tendency of manufacturers to save time, energy, material, and labor resources leads to implementation of accelerated technologies with shortened duration of technological processes and mainly dough fermentation. this leads to the decrease of growth and fermentative activity of lactic acid bacteria that are the antagonists of undesirable microflora of flour semiproducts. secondly, complying with modern requirements of physiological properties and nutritional value of bread, the manufacturers expand the raw materials base introducing the ingredients potentially more microbiologically contaminated. all of this including the increase of amounts of highly contaminated wheat flour escalates the problem of bakery products microbiological spoilage (fig. 1). in order to improve the biological stability of bread products during storage the chemical preservatives are mainly used, however, many of them inhibit the fermentative activity of lactic acid bacteria as well. this creates difficulties connected to flavor, aroma, and texture formation of finished product, ensuring the safety of produce, and its staling rate. the efficient technological solution of this problem is the usage of wheat sourdough with specifically cultivated microflora. however, it complicates the technological process considerably due to continuity of sourdough maintenance and its quick contamination by foreign microflora that deteriorates their biotechnological properties due to the lack of aseptic conditions in bakeries. thus the pursuit of alternative quick and effective solutions for baking industry food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 171 problems including the methods of improving bread products safety and imparting necessary physiological properties is relevant. fig. 1. the problems of formation and improvement of bread microbiological stability [2, 3, 7-10] food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 172 in this aspect especially interesting are the aromatic and medicinal plants known for their antiseptic, antioxidant, and other properties capable of stabilizing the product quality as well as their tonic, therapeutic, and prophylactic properties. nowadays these plants are considered as the main source of parapharmaceuticals – minor components with physiologically active properties, capable of regulating organism functions, and showing immunomodulating, antimutagenic, protective, adaptogenic, and therapeutic effects [11]. the value of aromatic and medicinal plants as raw material for food industry is in the variety of chemical composition and biochemical properties that ensures the complex formation of different technological and physiological properties. the usage of phyto-additives containing phenolic, tanning, pectic agents, phytoncides, vitamins, and wide range of major and minor dietary elements in baking industry is promising in order to improve the biotechnological properties of semi-products, regulate the dough structural-mechanical properties, modify the intensity of technological process, and stabilize the product quality while decreasing the usage of artificial food additives [12, 13]. besides, nowadays there is a wide search for methods of decreasing the acrylamide content in products including bread showing the perspective of using the phyto-additives with high polyphenol, organic acids, and vitamin b3 content as well as those intensifying the alcoholic and lactic acid fermentation. due to their inclusion into the formulation the structure and content of asparagine – the precursor of acrylamide – in baking semiproducts changes which leads to lower rate of acrylamide formation and correspondingly to decrease of its content in finished products [3, 14]. thus the usage of phyto-additives should allow the manufacturers to provide the population with wholesome high-quality produce in increasingly more difficult conditions of industry complying with high demands to assortment, consumer properties, safety, biological activity, and nutritional value. 2. materials and methods the goal of our work was the study of antiseptic properties of such phytoadditives as hop (humulus lupulus) cones, pelleted hop of ua-aroma sort, st john’s wort (hypericum perforatum), chamomile (matricaria chamomilla), marigold (calendula officinalis), peppermint (mentha × piperita), lemon balm (melissa officinalis), sage (salvia officinalis), redhaw hawthorn (craetegus sanguinea), cinnamon rose (rosa majalis), and mountain ash (sorbus aucuparia), and the research of their effect on biotechnological properties of fermentative microflora and finished product quality. the technology of extract preparation for fruit phyto-additives (haw, rose, and rowan) includes their grinding, bolting, steeping for 1 hour with material to extractant ratio of 1:10, and extraction with the same ratio at 100 °c for 30 min [15]. hop extraction conditions were material to extractant ratio – 1:100, temperature – 100 °c, duration – 90 min [16]. the preparation of the other phyto-additives included their grinding, bolting, and extraction with material to extractant ratio of 1:20 at 90-100 °c for 30 min. the moisture content was determined by the drying process in a drying chamber. the particle size was determined by sifting through specific sieves. total soluble solids (tss) content was determined by refractometric method. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 173 ph value of the extracts was measured by direct insertion of electrodes. the activity of chosen extracts towards pathogenic microflora inducing the spoilage of bread products was measured by method of diffusion into agar medium. the test-cultures were the specimen of microflora found in bread semi-products – hay bacillus (bac. subtilis), mold of aspergillus and penicillium genus found in grain, wild yeast, and lactic acid bacteria lactobacillus plantarum. the inoculated medium was cultivated for 48 hours at 30±1 °c. the antimicrobial activity was determined by the diameter of inhibition zone of test-culture colonies. the rising power was determined by standard method which included measuring the time a 10 g dough pellet submerged in water at 30±1 °c needed to break surface. acidity was determined by titrating samples with 0.1n naoh solution. the bread quality was determined by the following characteristics: moisture content, titrated acidity, loaf specific volume (volume to weight ratio), shape stability (height to diameter ratio), and porosity (pore to crumb volume ratio). in order to determine the resistance to microbiological spoilage, bread samples were packed into polythene bags and stored at 30±1 °c for 96 hours, controlling the appearance of first signs of microbiological spoilage. 3. results and discussion the results of experimental bread making (changes of wheat bread texture, crumb color, and flavor brought by introduction of powdered phyto-additives) indicate the merit of using water extracts instead (table 1). water was chosen as an extractant because it is a base material for bread industry included in almost all formulations; it is versatile, accessible, and easy to use. table 1 properties of phyto-additives and their extracts properties h op (c on es ) p el le te d ho p st j oh n’ s w or t (h er b) c ha m om ile (b lo ss om s) m ar ig ol d (b lo ss om s) l em on b al m ( he rb ) p ep pe rm in t ( he rb ) sa ge (l ea ve s) r ed ha w h aw th or n (f ru it ) c in na m on r os e (f ru it ) m ou nt ai n as h (f ru it ) phyto-additive moisture content, % 10.9 9.2 12.2 7.8 9.9 12.4 13.3 12.8 12.6 13.8 16.4 phyto-additive particle size, less than, mm 2.2 – 2.2 2.2 2.2 2.2 2.2 2.2 0.29 0.329 0.329 extract tss, % 0.6 0.7 2.6 1.8 2.1 2.4 2.6 2.9 4.8 5.6 5.1 extract рн value 5.79 5.90 5.97 6.89 6.55 6.68 6.55 6.77 5.18 4.24 5.32 according to the experimental data (table 2) hop, st john’s wort, and ashberry extracts exhibited pronounced antiseptic properties towards pathogenic microorganisms causing bread spoilage. the extract of hop cones was the most effective at inhibiting the foreign microflora while the fermentative microorganisms showed resistance to it. the hop antiseptic properties are due to food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 174 high content of bitter acids, essential oils, and tanning agents [17] transferred to extract and isohumulone formed during extraction [16, 18]. ashberries contain considerable amounts of sorbic (natural preservative) as well as succinic, tartaric, malic, and ascorbic acids [19]. st john’s wort due to high content of phytoncides, tanning agents, flavonoids, and essential oils inhibited the growth of pathogenic microorganisms as well as wild yeast that might compete with fermentative microflora and lower the biotechnological properties of baking semi-products [20]. however, ashberry and st john’s wort unlike hop extracts had negative effect on lactic acid bacteria metabolism. the sage, peppermint, and chamomile extracts were effective against mold fungi. the haw and rose extracts exhibited no bactericidal or fungicidal effect. table 2 antiseptic activity of phyto-extracts extract of: diameter of inhibition zone of test-culture colonies, mm lactobacillus plantarum wild yeast bacillus subtilis aspergillus penicillium hop cones 13.5±0.5 11,3±0,5 15.8±0.5 pelleted hop 11.8±0.5 10,6±0,5 12.3±0.5 st john’s wort 11.8±0.5 11.0±0.5 13.2±0.5 11,1±0,5 14.5±0.5 chamomile 11,7±0,5 12.6±0.5 marigold 11.8±0.5 lemon balm peppermint 12,4±0,5 13.6±0.5 sage 13,7±0,5 13.9±0.5 haw cinnamon rose ashberry 10.8±0.5 14.6±0.5 10.0±0.5 11.4±0.5 note: "-" – no inhibition zone the study of antiseptic properties of phytoextracts showed the necessity of researching their effect on alcoholic and lactic acid fermentation during preparation of bread semi-products, specifically sponges with 70±2 % moisture content. the preliminary studies had determined the rational dosage of phyto-extracts that didn’t lower the wheat bread quality. thus the rose extract content should not be more than 15 % to flour mass; however the full replacement of water (60 % to flour mass) with haw extract showed no negative effect. the maximum dosage of hop extracts was 30 % to flour mass, for sage, st john’s wort, peppermint, and lemon balm extracts it was 5-10 %, however on the upper limit bread crumb gained light grey color. since the weakening effect of chamomile and marigold extracts on gluten had been determined, they may be included in the formulation in the amount of up to 20 % to flour mass, if the flour is strong or medium strong. the changes of rising power (the intensity of alcoholic fermentation) and acidity (the intensity of lactic acid fermentation) of traditional sponge with phyto-extracts included in the formulation in aforesaid dosages have been presented on figure 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 175 0 5 10 15 20 25 30 35 40 45 0 60 120 180 240 300 fermentation duration, min r is in g p o w er , m in 0 1 2 3 4 5 6 t itr at ed a ci d ity , ° 0 5 10 15 20 25 30 35 40 45 0 60 120 180 240 300 fermentation duration, min r is in g p ow er , m in 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 t itr at ed a ci d ity , ° 0 5 10 15 20 25 30 35 40 45 0 60 120 180 240 300 fermentation duration, min r is in g p ow er , m in 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 t itr at ed a ci d ity , ° 0 5 10 15 20 25 30 35 40 45 0 60 120 180 240 300 fermentation duration, min r is in g p o w er , m in 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 ti tr at ed a ci d ity , ° fig. 2. the effect of phyto-extracts on rising power (a) and acidity (b) of liquid sponges: 1 – control sample; with extracts of: 2 – rose; 3 – haw; 4 – ashberry; 5 – sage; 6 – st john’s wort; 7 – chamomile; 8 – marigold; 9 – peppermint; 10 – lemon balm; 11 – hop cones; 12 – pelleted hop the data analysis determined that water extracts of hop, st john’s wort, and ashberry in chosen dosages had selective antiseptic properties namely capacity for inhibiting pathogenic microflora that fermentative microorganisms were resistant to. all phyto-extracts had positive effect on alcoholic fermentation intensity though the best results were reached using hop, rose, haw, and ashberry extracts. this could be explained by their high content of nutrients, organic acids, vitamins, and major and minor dietary elements, deficient in wheat flour semi-products, especially high-grade, and necessary for yeast metabolism. the complex estimation of phyto-extract technological properties was achieved by experimental baking (table 3). the obtained results confirmed the effectiveness of using the water phytoextracts to improve the quality of bread products and their microbiological stability during storage. thus the consumer qualities of wheat bread (specific volume, shape stability, and porosity) with extracts for almost samples were either improved or analogous to check sample. at the same time their microbiological stability improved. even the rose and haw extracts that demonstrated no antiseptic properties (table 2) allowed improving the microbiological stability of finished products. most likely, it was caused by higher amounts of substances inhibiting the growth of foreign microflora produced by lactic acid bacteria as a result of more intense fermentation (fig. 2). 1а 2а 3а 4а 4b 3b 2b 1b 1а 1b 1а 1b 8а 5а 6а 7а 5b 6b 7b 8b 9b 9а 10а 10b 11а 11b 12b 12а 1а 1b food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 176 table 3 the effect of phyto-extracts on bread quality and microbiological stability samples bread quality properties m oi st ur e co nt en t, % t it ra te d ac id it y, ° sp ec if ic v ol um e, g/ cm 3 sh ap e st ab ili ty , н /d p or os it y, % f ir st s ig ns o f m ol di ng f ir st s ig ns o f br ea d ro pi ne ss rose extract check sample 42.3 2.2 3.03 0.47 70.9 after 72 h after 48 h 15 % to flour mass 42.5 2.9 3.44 0.50 76.2 – – ashberry extract check sample 42.9 2.2 2.92 0.52 70.0 after 72 h after 48 h 15 % to flour mass 43.0 2.4 3.13 0.59 77.4 – – haw extract check sample 42.8 2.2 2.93 0.45 73.2 after 72 h after 48 h 60 % to flour mass 43.0 2.7 3.38 0.50 81.1 after 96 h – sage extract check sample 42.4 2.2 2.84 0.54 70.0 after 72 h after 48 h 5 % to flour mass 42.4 2.3 3.18 0.58 74.5 – – st john’s wort extract check sample 42.2 2.2 2.94 0.50 71.4 after 72 h after 36 h 5 % to flour mass 42.5 2.4 3.46 0.54 75.5 – – pelleted hop extract check sample 43.0 2.8 2.92 0.47 68.0 after 48 h after 36 h 30 % to flour mass 42.8 3.1 3.30 0.50 73.2 – – hop cone extract check sample 43.0 2.8 2.92 0.47 68.0 after 48 h after 36 h 30 % to flour mass 43.1 3.3 3.37 0.54 73.7 – – chamomile extract check sample 42.8 2.4 2.90 0.47 67.4 after 72 h after 48 h 10 % to flour mass 43.1 2.9 2.96 0.41 71.3 – after 72 h marigold extract check sample 42.6 2.3 2.98 0.49 68.1 after 72 h after 48 h 10 % to flour mass 42.9 2.4 2.95 0.50 67.9 after 96 h after 96 h peppermint extract check sample 42.4 2.2 3.06 0.54 70.0 after 72 h after 48 h 5 % to flour mass 43.1 2.9 3.29 0.51 75.2 – – lemon balm extract check sample 42.4 2.2 3.06 0.54 70.0 after 72 h after 48 h 5 % to flour mass 43.0 2.7 3.12 0.53 73.2 – after 96 h note: “-“ – no signs of microbiological spoilage 4. conclusion thus the results of studying the antiseptic properties of phyto-extract and their effect on biotechnological properties of semiproducts and bread quality demonstrate the merit of their usage in baking industry to solve a number of problems. thus during the processing of raw materials with high microflora contamination from our point of view the most effective would be hop, sage, and st john’s wort extracts. in order food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 177 to improve the microbiological stability of buns and pastries containing ingredients with high nutritional value that might need concealment flavor-wise it would be worthwhile to use peppermint and lemon balm extracts. the rose, haw, and ashberry extracts would be useful in accelerated technologies in order to intensify the fermentative process and improve the finished product quality and microbiological stability as a result. in order to control the biotechnological properties of sourdough, liquid yeast, and sponges, the hop extracts might be useful due to their selective antiseptic properties. their usage should allow controlling the species composition of fermentative microflora in flour semi-products, regulating their biotechnological properties, and improve the bread quality as a result. the new plant ingredients are interesting from the viewpoint of their usage to improve the nutritional value and physiological properties of bakery products. the high content of pectins, polyphenols, and organic acids is the evidence of their protective, anticarcinogenic, and antioxidant properties. thus their further study as ingredients for baking industry is important in order to impart the specific physiological properties on finished products as well as lower their acrylamide content. the diversity of ukrainian aromatic and medicinal herbs, the growing demands to nutritional properties of food products, and complicated conditions of bread industry demonstrate the merits of researching the natural potential of phyto-additives, their physiological and technological properties in order to find more effective, simple, and safe solution to the problems of baking industry and improve the quality of finished products. 5. references [1]. tsyprian v.i., hygiene of nutrition, book 2, medicine, kiev, 544 p, (2007) [2]. machihina l.i., alekseieva l.v., lvova l.s., ecological safety of grain and grain by-products during storage and processing, storage and processing of agricultural raw materials, 12, 37-40, (2008) [3]. holynko o.n., levitskaia n.p., the problems of safety and quality of bread products, nutrition problems, 1, 16-26, (2013) [4]. tiurina ye.b., the market of food additives for baking industry, food ingredients. raw materials and additives, 1, 19, (2010) [5]. acrylamide. the toxicological, epidemiological, and dietary exposure evaluation of compounds on the agenda, evaluation of certain food contaminants: sixty-fourth report of the joint fao/who expert committee on food additives, geneva, 9-20, (2005) [6]. code of practice for the reduction of acrylamide in foods, codex alimentarius, cac/rcp 67-2009, http://www.codexalimentarius.org/download/standa rds/11258/cxp_067e.pdf. last accessed: 07.28.2014 [7]. bykovchenko t.v., kostiuchenko m.n., volohova l.t., the modern system of bread ropiness prevention and ensuring the microbiological purity of bakery products, breadmaking of russia, 2, 26-27, (2013) [8]. kosovan a.p., the general idea and systematic ecologization of bread industry, storage and processing of agricultural raw materials, 12, 34-36, (2008) [9]. sidorova o.a., berdyshnikova o.n., the chemical methods of improving the microbiological purity of bakery products, baking industry, 2, 29-37, (2011) [10]. berdyshnikova o.n., sidorova o.a., the influence of sourdoughs cultivated on different nutrient mediums on ensuring the microbiological safety of bread products, baking industry, 5-6, 10-14, (2011) [11]. kovaliov v.m., pharmacognosy with the basics of plant biochemistry, prapor, kiev, 705 p, (2007) [12]. nielsen p.v., rios r., inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 2 – 2014 tatyana lebedenko, antonina yegorova, viktoriia kozhevnikova, yekaterina korolenko, the prospects of safety and microbiological stability improvement of bakery products by using phyto-additives, food and environment safety, volume xiii, issue 2 – 2014, pag. 169 – 178 178 in active packaging, with special emphasis on mustard essential oil, international journal of food microbiology, 2-3, 219-229, (2000) [13]. iorgacheva ye.h., lebedenko t.ye., potential of medicinal and aromatic plants to increase the quality of wheat bread, easterneuropean journal of enterprise technologies, 2/12(68), 101-108 [14]. bahriantseva o.v., shatrov h.n., khotimchenko s.a., about the question of necessary implementation of measures of lowering the acrylamide content in food products, the materials of xii all-russian congress of dietitians and nutritionists “nutrition and health”, 10-11, (2010) [15]. lebedenko t., kozhevnikova v., novichkova t., prospects of improvement of accelerated bread technologies by usage of dogrose and hawthorn, technology audit and production reserves, 3, 8-11, (2013) [16]. lebedenko t.ye., sokolova n.yu., polyfunctional usage of hop in bread industry, food industry: science and technology, 1, 40-50, (2014) [17]. liashenko n.i., biochemistry of hop and hop by-products, polesie, zhitomir, 385, (2002) [18]. kuznetsova ye.a., koriachkina s.a., khmeleva ye.v., the usage of hop extract in order to improve the microbiological safety of grain during its pre-processing for preparation of grain bakery products, fundamental research, 12, 100-103, (2010) [19]. nilova l., dubrovskaia n., new additive for bakery products is a powder from graded ashberry, bread products, 11, 52-53, (2008) [20]. paschenko l.p., kolomnikova ya.p., bread ropiness. new methods of diagnosis and prevention, bread business, 3, 32-35, (2009) doi: https://doi.org/10.4316/fens.2022.026 265 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 265 272 characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis *mădălina ungureanu-iuga 1 , víctor d. ávila akerberg 2 , tanya m. gonzález martínez 3,4 1faculty of food engineering, stefan cel mare university of suceava, 13, universităţii street, suceava, c.p. 720229, romania, madalina.iuga@usm.ro 2universidad autónoma del estado de méxico, instituto de ciencias agropecuarias y rurales, campus universitario “el cerrillo”, a.p. 435, toluca, estado de méxico, c.p. 50200, méxico 3 faculty of sciences, national autonomous university of méxico (unam), av. universidad 3004, copilco universidad, coyoacán, c.p. 04510 ciudad de méxico, méxico 4 mexican agrobiodiversity project, national council for knowledge and use of biodiversity (conabio) *corresponding author received 25th august 2022, accepted 29th september 2022 abstract: tortillas are one of the most important foods for the mexican’s nutrition. the industrialization of the manufacturing process led to changes in final product quality. the aim of this study was to group and to identify the best tortilla from tlazala, mexico market. ten industrial and artisanal samples were analyzed from chemical, textural and sensory point of view. taking into account these characteristics, the samples were grouped by using hierarchical cluster analysis. the differences between groups were interpreted by mean comparison and analysis of variance. three groups resulted, the first composed of machine made tortillas, the second from 100% maize grain nixtamalized in situ and the third comprised blue tortilla mechanically made from maize grain. the best characteristics were observed for the second group, mostly composed of artisanal tortillas. the results showed that the best tortilla is traditionally made, from 100% local maize grain, using a wood griddle. keywords: tortillas, cluster analysis, physico-chemical properties, sensory characteristics. 1. introduction maize products have a big importance in people from latin-american diet. maize tortillas are ones of the principal foods in mexico, being consumed in both urban and rural areas [1]. in 2010, the annual consumption in urban areas was about 56.7 kg per capita, while in the rural areas was 79.5. tortillas consumption brings an intake of carbohydrates, fibers, proteins, minerals and some vitamins [2]. nowadays people are most interested in organic and traditional foods, this tendency increasing in the last 10 years [3]. the traditional maize tortillas production implies grains nixtamalization which means maize grains cooking with a slaked lime solution. this process has many benefits from both nutritional and sensory point of view, the grains becoming softer and easy to ground, the nutritional value being higher, the aroma being improved and the mycotoxins effects being decreased [1]. an increased maize consumption led to an industrialization of tortilla making processes which implies autoclaving and dough drying to obtain the nixtamalized industrial maize flour. this process modification had some consequences for http://www.fia.usv.ro/fiajournal mailto:madalina.iuga@usm.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 266 the final product quality, since the aroma, texture and nutritional value are changed [1,4]. a study about the maize origin for tortilla making in lakes pátzcuaro and zirahuén basins showed that for home made tortillas people use mostly local maize, while for those made with machines the maize comes from other regions of mexico or they use industrial nixtamalized flour [5]. the characteristics of tortillas depend on the maize grain quality. for example, the starch content of maize grains directly influences dough moisture, texture, rheology and tortillas shelf-life. thus, the origin of maize and physicochemical characteristics affect final product properties [6]. furthermore, the nixtamalization process conditions may also change tortillas quality, the texture, color and sensory profile of tortillas being determined by the solubilisation and diffusion processes that occur during lime cooking [4]. tortillas are one the most important daily food for mexican people. thus, the final product quality knowledge may be of interest for both consumers and producers to choose properly or to improve product’s characteristics. in this regard, the aim of this study was to characterize tortillas from tlazala, mexico market, to compare and to group them depending on the chemical, textural and sensory properties. 2. materials and methods 2.1. materials: tortillas samples fresh tortillas were acquired from market at specialized shops called tortillerías in tlazala town, mexico and were kept in polyethylene bags at 4°c until analyses were performed or until drying at 55°c for 24 h and milling with a tomas willy mill with a 0.84 mm sieve. samples codes and characteristics are presented in table 1. five tortillas types from different places were analyzed. from the industrial ones made up with machines, two were mixes of industrial maize flour and maize nixtamalized (tmx) in situ, two made of maize from other regions (tmz), two only of industrial maize flour (tmn) and one of blue maize from another region (tma). all the artisanal tortillas (ta) were made of maize nixtamalized in situ, ta1 and taa being cooked on gas hob, while ta2 was baked using a wood hob. 2.2. methods 2.2.1. chemical characteristics moisture content was determined according to the aoac (1999) [7] method 925.098; ash content by method 923.03; fat content by soxhlet extraction in petroleum ether (method 920.39c; proteins content by kjeldahl method. fibers contents (neutral and acid detergent) were achieved using an ankom 200 fibers analyzer [8]. sugars contents were calculated by difference. the extract for polyphenols and antioxidant activity determination was prepared by methanol 80% extraction as it is described by hernandez-martinez et al. (2016) [9]. polyphenols content was determined by folin-ciocalteu method, according to hernandez-martinez et al. (2016) [9] and the antioxidant activity expressed as inhibition percent (ic50) was measured by the dilution method described by iora et al. [10], lower values indicating high antioxidant activity. ph was determined according to the method described by serena-saldivar (2012) [11], by using an oakton con 510 ph-meter. all measurements were performed at least in triplicate. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 267 2.2.2. textural parameters tortillia’s hardness was evaluated by using a ta.tx plus texture analyser. according to the method described by vaca-garcía et al. [12] a spherical probe perforated the sample pieces of 6.5 cm diameter hold by two metallic plates until they broke, at a speed of 10 mm s-1. for each fresh sample nine measurements were performed. 2.2.3. sensory characteristics the sensory characteristics of fresh tortillas samples were evaluated by nineteen panelists with a 15 points scale, as it is described by bejosano et al. (2005) [13]. the studied properties were color uniformity, surface uniformity, moisture, opacity, maize smell, lime smell, fermented smell, acid taste, salted taste, sweet taste, and lime taste. also, the subjective textural characteristics of tortillas samples were achieved according to the method described by morten meilgaard, gail vance civille, and thomas carr [14]. thus, roughness, elasticity, hardness, masticability, moisture absorption and tooth packing characteristics were evaluated. a rolability test was performed by rolling the sample around a 13 mm diameter dowel and oticing the appearance of breakings [13]. 2.2.4. statistics a hierarchical cluster analysis (hca) using average linkage between groups was performed to see how the tortillas samples can be grouped and characterized, according to the chemical, textural and sensory characteristics that they present. to avoid the effects of high differences in mass concentration values the data were z-transformed. groups of tortilla samples were formed by using the squared geometric euclidian distance (eq. 1) [15]: (1) where xi and xi’ are objects in the multidimensional space. table 1. tortillas samples characteristics sample purchase place maize source technology observations tmx1 industrial maize flour and white maize machine tmx2 tortillería industrial maize flour and yellow maize machine tmz1 tortillería white maize from the north of mexico machine tmz2 tortillería white maize from sinaloa/hidalgo machine preservatives addition tmn1 tortillería industrial maize flour machine tmn2 tortillería industrial maize flour machine ta1 particular house white maize from specialized shops artisanal wheat flour is added ta2 particular house local white maize, from tlazala region artisanal tma tortillería blue maize from the north of mexico machine taa particular house blue maize from specialized shops artisanal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 268 the results were presented as a dendrogram comprising a hierarchical structure. the differences between clusters (significant at p < 0.05) were evaluated by applying to the cluster solution an analysis ofvariance (anova) [16]. for the calculations spss 13.0 (trial version) software was used. 3. results and discussion the ten tortillas samples were grouped as function of the chemical, textural and sensory characteristics, as it is shown in the dendogram presented in figure 1. according to the resulting dendrogram (figure 1), the samples form three groups as it follows: the first group is composed of tmn1, tmn2, tmx1, tmx2 and tmz1 which are made with machines, the second group comprises ta1, ta2, taa and tmz2 which contain only maize grains nixtamalized in situ and tma which is mechanically made of maize grains nixtamalized in situ form the third group.  fig.1. dendrogram for cluster analysis of tortillas table 2. tortillas groups sensory characteristics characteristic group minimum maximum mean standard deviation color uniformity 1 10.78 12.22 11.33a 0.72 2 5.67 9.67 7.00c 1.81 3 9.56 9.56 9.56b surface uniformity 1 8.00 11.78 9.84a 1.41 2 6.56 11.78 8.36c 2.33 3 9.00 9.00 9.00b moisture sensory 1 6.22 8.22 7.22b 0.92 2 8.11 9.33 8.42a 0.61 3 4.33 4.33 4.33c opacity sensory 1 7.67 9.22 8.53c 0.75 2 6.00 11.89 9.69b 2.61 3 12.78 12.78 12.78a maize smell 1 4.89 7.67 6.40b 1.13 2 7.67 10.00 8.78a 0.96 3 7.33 7.33 7.33c lime smell 1 5.78 9.89 8.24a 1.54 2 3.56 7.89 4.83c 2.07 3 5.44 5.44 5.44b fermented smell 1 3.00 4.56 3.80b 0.66 2 2.11 3.67 2.86a 0.81 3 2.89 2.89 2.89a acid taste 1 1.89 4.00 2.96a 0.86 2 2.22 2.89 2.53b 0.31 3 1.67 1.67 1.67c salted taste 1 2.44 3.44 3.02a 0.44 2 2.78 3.67 3.28a 0.41 3 2.67 2.67 2.67b sweet taste 1 1.67 4.00 3.09b 1.00 2 3.78 5.44 4.67a 0.90 3 2.89 2.89 2.89c lime taste 1 5.11 11.11 7.80a 2.39 2 3.44 6.22 4.50b 1.24 3 4.33 4.33 4.33b mean values followed by different letters are significantly different (p < 0,05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 269 differences regarding tortillas samples characteristics between groups were observed (tables 2, 3 and 4). according to the results obtained from the anova, the variables of color uniformity, moisture sensory determined, maize smell, rolability, protein content and ph are significant (p < 0.05) in relation to the cluster membership, while the others are not (p > 0.05). as it is shown in table 2, tortillas from the first group have the most uniform color and surface, are the less opaque, have the lowest maize smell and the higher lime smell and taste and the highest fermented and acid taste compared to the other groups, probably due to the fact that they are made with machines which may affect product quality. the second group of tortillas is made from maize grains nixtamalized in situ and is characterized by lower color and surface uniformity, higher moisture, maize smell, sweet and salted taste and lowest lime smell and taste, fermented smell and acid taste. the last group formed by tma tortilla from blue maize is characterized by lower moisture level, higher opacity, lower sweet and lime taste. the results are consistent with those obtained by mendez-albores et al. (2012) [17] for maize tortillas and by hernández-martínez et al. (2016) [9] for blue maize tortillas. regarding the texture (table 3), the first group of tortillas has the highest elasticity and lower hardness measured by both subjective and objective methods. table 3. tortillas groups textural characteristics characteristic group minimum maximum mean standard deviation roughness 1 4.56 5.39 5.02b 0.32 2 2.56 5.83 4.38c 1.40 3 7.22 7.22 7.22a rolability 1 10.44 13.89 12.31b 1.30 2 11.56 14.11 12.92a 1.11 3 7.56 7.56 7.56c elasticity 1 5.78 7.06 6.54a 0.49 2 5.00 7.89 5.97c 1.31 3 4.78 4.78 4.78b hardness sensory 1 3.67 7.33 4.97c 1.59 2 3.78 5.33 4.64b 0.81 3 8.22 8.22 8.22a masticability 1 7.56 10.44 8.96b 1.31 2 9.67 10.78 10.33a 0.54 3 8.22 8.22 8.22c moisture absorption 1 8.20 11.11 9.66b 1.29 2 8.89 11.11 9.67b 1.00 3 11.11 11.11 11.11a tooth packing 1 6.00 8.78 7.44b 1.09 2 6.78 9.44 7.89a 1.13 3 4.44 4.44 4.44c firmness 1 2.86 4.64 3.75c 0.83 2 3.71 6.94 4.87a 1.46 3 4.61 4.61 4.61b mean values followed by different letters are significantly different (p < 0,05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 270 the chemical characteristics for the three groups are presented in table 4. tortillas from the first group are characterized by the lowest protein, carbohydrates, polyphenols contents and antioxidant activity, while the fnd, ash and water content are higher than of those from the other groups. tortillas from the second group present the highest protein, fad contents and antioxidant activity and lowest lipids content and ph. the third group is characterized by the highest lipids, carbohydrates and polyphenols levels and the lowest fnd, fad, ash and water contents. these results are in agreement with those reported by valderrama-bravo et al. (2017) and hernandez-martinez et al. (2016). the chemical characteristics of the final product are related to the maize variety, nixtamalization conditions (temperature, time, lime quantity) and production process (manual, mechanic) [18]. table 4. tortillas groups chemical characteristics characteristic group minimum maximum mean standard deviation proteins 1 7.51 8.10 7.75b 0.23 2 8.05 9.52 9.07a 0.68 3 7.84 7.84 7.84b lipids 1 1.03 1.44 1.19a 0.15 2 0.73 1.13 0.96b 0.19 3 1.23 1.23 1.23a carbohydrates 1 32.44 41.07 36.90c 3.60 2 38.60 43.95 40.99b 2.34 3 43.02 43.02 43.02a fnd 1 14.25 33.96 19.16a 8.35 2 8.32 27.01 18.55b 8.14 3 17.58 17.58 17.58c fad 1 0.61 1.02 0.78b 0.15 2 0.54 1.83 1.20a 0.54 3 0.40 0.40 0.40c ash 1 1.28 3.35 2.38a 0.75 2 1.41 2.14 1.66b 0.32 3 1.64 1.64 1.64b water content 1 47.95 55.58 51.57a 3.37 2 43.72 51.00 47.31b 3.04 3 45.58 45.58 45.58c poliphenols 1 0.17 0.38 0.25c 0.08 2 0.24 0.84 0.51b 0.25 3 0.65 0.65 0.65a ic50 1 63.58 150.18 98.67a 36.61 2 54.00 108.48 79.91c 22.94 3 95.22 95.22 95.22b ph 1 8.70 11.08 10.27a 0.93 2 7.33 9.64 8.21c 1.04 3 8.76 8.76 8.76b mean values followed by different letters are significantly different (p < 0,05). cluster analysis and groups characterization revealed that the artisanal tortillas and those made only of maize grains nixtamalized in situ have better characteristics than theindustrial ones which contain industrial maize flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 271 4. conclusion tortillas characteristics influence consumer’s purchase decision and preferences, being important also for producers. the industrialization tendency in the food processing field can affect the quality of the final product. the artisanal tortilla were found to have higher nutritional value compared to those made in tortillerias, raised values of proteins, fibers and poliphenols being identified. thus, the appearance of tortillerías where tortillas are mechanically made and the use of industrial maize flour instead of maize grains nixtamalized in situ is related to changes of product’s quality. the studied tortillas marketed in tlazala, mexico studied can be grouped as function of their chemical, textural and sensory characteristics. the group with the best characteristics was made of mostly artisanal tortillas, all the comprising sample being made of 100% maize grain nixtamalized in situ. the main limitations of this study are related to the small area from which the samples were collected. further research regarding the preferences of consumers and chemical particularities of tortillas from many regions of mexico are needed to better complete the view of mexican’s consumer behavior. 5. acknowledgment this work was supported by romania national council for higher education funding, cnfis, project number cnfis‐fdi‐2022‐0259. we would like to thank baciliza quintero salazar, research professor at uaem, for supporting and advising the sensory analysis methodology. 6. references [1]. martínez-velasco, a., alvarezramirez, j., rodríguez-huezo, e., meraz-rodríguez, m., vernon-carter, e.j. and lobato-calleros, c., effect of the preparation method and storage time on the in vitro protein digestibility of maize tortillas. journal of cereal science, 84, 7-12, (2018). [2]. astier, m., odenthal, g., patricio, c. and ramírez, q.o., handmade tortilla production in the basins of lakes pátzcuaro and zirahuén, mexico, journal of maps, 15(1), 52-57, (2019). [3]. gallegos-hernández, b.p., pérezvillarreal, h.h., barahona, i., mayettmoreno, y., analysis of the intrinsic signals, extrinsic signals and the expected quality of the organic tortilla to assess its purchasing intentions. cogent business & management, 5, 1548548, (2018). [4]. ruiz-gutiérrez, m.g., quinteroramos, a., meléndez-pizarro, c.o., talamás-abbud, r., barnard, j., márquez-meléndez, r., lardizábalgutiérrez, d., nixtamalization in two steps with different calcium salts and the relationship with chemical, texture and thermal properties in masa and tortilla, journal of food process engineering, 35, 72–783, (2011). [5]. orozco-ramírez, q., barrerabassols, n., astier, m., masera, o., el sistema maíz-tortilla en el estado de michoacán. in j. j. seefoó luján and n. m. keilbach (eds.), ciencia y paciencia campesina: el maíz en michoacán; seefoó-luján, jj, keilbach, n., eds, 119-136, (2010). [6]. valderrama-bravo, c., domínguez-pacheco, a., hernándezaguilar, c., zepedabautista, r., del real-lópez, a., pahuaramos, m.e., arellano-vázquez, j.l., moreno-martínez, e., physical and chemical characterization of masa and tortillas from parental lines, crosses, and one hybrid. international agrophysics, 31, 129-138, (2017). [7]. association of official analytical chemists (aoac), official methods of analysis of the aoac (16th edition), washington d.c., united states of america, (1999). [8]. ankom, analytical methods, (2019), accessed on 10 november 2022 at: https://www.ankom.com/analytical-methodssupport/fiber-analyzer-a200. https://www.ankom.com/analytical-methods-support/fiber-analyzer-a200 https://www.ankom.com/analytical-methods-support/fiber-analyzer-a200 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 20221 mădălina ungureanu-iuga, víctor d. ávila akerberg, tanya m. gonzález martínez, characterization of tortillas marketed in tlazala, mexico by using hierarchical cluster analysis, food and environment safety, volume xxi, issue 3 – 2022, pag. 265 – 272 272 [9]. hernández-martínez, v., salinasmoreno, y., ramírez-díaz, j.l., vásquezcarrillo, g., domínguez-lópez, a., ramírez-romero, a.g., color, phenolic composition and antioxidant activity of blue tortillas from mexican maize races. cyta – journal of food, 14(3), 473-481, (2016). [10]. iora, s.r.f., maciel, g.m., zielinski, a.a.f., da silva, m.v., de a. pontes, p.v., haminiuk, k.w.i, granato, d., evaluation of the bioactive compounds and the antioxidant capacity of grape pomace. international journal of food science and technology, 50(1), 62-69, (2014). [11]. serena-saldívar, o., cereal grains laboratory reference and procedures manual, crc press taylor & francisc group, llc, usa, (2012). [12]. vaca-garcía, v.m., martínezrueda, c.g., mariezcurrena-berasain, m.d., domínguez-lópez, a., functional properties of tortillas with triticale flour as a partial substitute of nixtamalized corn flour. lwt-food science and technology, 44, 1383-1387, (2011). [13]. bejosano, f.p., joseph, s., lopez, m.l., kelekci, n.n., waniska, r.d., rheological and sensory evaluation of wheat flour tortillas during storage. cereal chemistry, 82(3), 256-263, (2005). [14]. morten meilgaard, d.s., gail vance civille, b.s., thomas carr, m.s., sensory evaluation techniques (4th edition), crc press, new york, usa, (2007). [15]. astel, a., astel, k., tsakovski, s., biziuk, m., obolewski, k., glińskalewczuk, k., ... timofte, c.m., comprehensive comparison of a few variants of cluster analysis as data mining tool in supporting environmental management. environmental engineering & management journal (eemj), 15(6), (2016). [16]. popa, m., multivariate statistics applied in pshychology, polirom, iasi, romania, (2010). [17]. méndez-albores, a., martínezmorquecho, r. a., moreno-martínez, e. and vázquez-durán, a., technological properties of maize tortillas produced by microwave nixtamalization with variable alkalinity. african journal of biotechnology, 11(85), 15178-15187, (2012). [18]. iuga, m., ávila akerberg, v. d., gonzález martínez, t. m., mironeasa, s., consumer preferences and sensory profile related to the physico-chemical properties and texture of different maize tortillas types. foods, 8(11), 533, (2019). 1. introduction 21 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 12015, pag. 21 29 screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food regina ekoua krabi1 , antoine allah assamoi1, fafadzi ayawovi ehon1, *sébastien lamine niamke1 1faculty of biotechnologies, ufr biosciences, university félix houphouët-boigny 22 bp 582 abidjan 22, abidjan, côte d’ivoire niamkes@yahoo.fr *corresponding author received february 13rd 2015, accepted march 27th 2015 abstract: the purpose of this paper is to resolve unpredictable quality of attiéké by the selection of lactic acid bacteria (the predominant microorganism of cassava fermentation) as microbial starter culture. thus, 237 lactic acid bacteria were isolated from traditional cassava starter and their acidification activity was evaluated using mrs-soluble starch broth. also, their abilities to produce αamylase, pectinase, cellulase, phytase, tannase and β-glucosidase were studied. among the isolated strains, 126 were lactococci and 111were lactobacilli. the first acidifying group (34.24 % of isolates) decreases the ph of 1 unit after 6 h of fermentation and the second acidifying group (20.10 %) after 12 h of fermentation. the latter group (45.66 %) consists of isolates which did not decrease the ph of 1 unit less than 12 h of fermentation. among faster acidifying lab, 22.67 % were able to produce amylase, 2.67 % showed pectinase production capacity, 5.33 % cellulase and 8 % were able to produce β-glucosidase. these 17 isolates could be preselected as potential starter for cassava dough fermentation into attiéké production. keywords: lactic acid bacteria, acid production, enzyme production, cassava fermentation, attiéké. 1. introduction cassava (manihot esculenta crantz) is an important staple food grown in tropical countries. it has important agronomic advantages such as high yields in poor soils, resistance to drought and diseases, storability in the soil after maturity and high yield of starch, in comparison with over starchy crops such as yam [1]. however, cassava is faced with two major problems, namely rapid postharvest deterioration [2] and toxicity due to cyanogenic glucosides [3, 4]. to avoid these problems, cassava roots are processed by several ways. over 90 % of the cassava processed in africa is used for human nutrition as fermented products [5]. so fermentation is the most popular method used for cassava process. more, the fermentation process has been shown to be a suitable method to enhance the safety, organoleptic and nutritional quality of many cassava-derived foods [6-7]. in côte d’ivoire, attiéké is the most fermented cassava product that is widely consumed [8]. it is also known in neighbouring countries [9]. attiéké is a steamed granular cassava meal ready-toeat, couscous-like product, with slightly sour taste and whitish colour [10]. attiéké is a typically ivorian food which was originally prepared and consumed exclusively by ethnic groups living in the lagoon area such as ebrié, adjoukrou, http://www.fia.usv.ro/fiajournal mailto:niamkes@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 22 alladjan, avikam... this food, a simple dish as only a product of self-consumption, attiéké has now acquired the statute of annuity product, nourishing big urban markets. thus, there has been a change from family production to commercial production stimulated by the increasing urban demand and the formation of small cooperatives [11]. attiéké is produced by women according to a traditional technology requiring several hard operations [12]. to obtain attiéké, cassava roots are peeled, cut into pieces and washed. before grinding, 10% of a traditional starter, and 0.1% of palm oil are added and the pieces are ground to a fine paste, which is placed in large bowls. the dough is left to ferment for about 15 to 24 h at ambient temperature. after fermentation, the dough is placed in a jute bag and pressed continuously by a hand press. the pressed cake is then passed through sieve for sifting. the grains are formed by shaking and rotating the powder in a large bowl. then, the grains are sundried. after drying, fibres and dirt are removed by sprinkling the grains, which the grains are poured into the sieve for steaming with boiling water. traditional starter used for attiéké production is obtained after 2 to 3 days of spontaneous fermentation of cassava roots (raw or cooked), thus colonized by wide variety of microorganisms. this starter is the main source of microorganisms necessary for the fermentation of cassava dough during attiéké process. among microorganisms isolated of traditional starters, lactic acid bacteria (lab) are predominant [13-14]. lab are mainly responsible for the acidification of cassava dough [15-16-17]. lab can further contribute to the reduction of cyanogenic glucosides [18-2], to development of taste, flavor, and texture of fermented food [19]. in fact, quality of attiéké, like fermented food in africa, is unpredictable because of unregulated conditions and sensory fluctuations. characteristic of each attiéké and the difference between them are probably due to the difference in their traditional starters used to conduct the fermentation [10]. various investigations on the microbiology and biochemistry of cassava fermentation and attiéké production have been done. to date, investigations aimed to standardize attiéké process. it is in this context that our study was performed to select lab with suitable technological properties as potential starter to control the cassava dough fermentation into attiéké. 2. matherial and methods 2.1. material the biological material used in this study was constituted by already traditional cassava (manihot esculenta crantz) starter, which was obtained after 2 to 3 days of spontaneous fermentation of boiled cassava roots. 2.2. sampling traditional starters were taken in small scale attiéké production in fourteen processing zones (yopougon, adjamé, treichville, marcory, koumassi, cocody, port-bouët, abobo, attécoubé, dabou, jacqueville, anyama, bingerville and grand bassam). on each processing zone, 3 producers were randomly selected and about 100 g of the traditional ferment by producer were taken. samples were collected by scraping the surface of traditional inocula. then, samples were put in plastic bags (stomacher) and immediately transported to the laboratory for microbial analysis. 2.3. isolation of lactic acid bacteria all samples were mixed and divided into 3 parts. preparation of stock solutions, 10 g of each part were aseptically homogenized food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 23 in a stomacher with 90 ml of sterile peptone buffered water (oxoid, basingstoke, united kingdom). stock solutions were incubated at 30 °c for 18 h. isolation of lab were carried out using plates of de man rogosa and sharp agar (mrs), mayeux, sandine eliker agar (mse), bile esculin azide agar (bea) and m17 agar (all from oxoid, basingstoke, hampshire, uk). these media were used as describe previously by kostinek et al. [17] to obtain the widest possible species variety of lab associated to the fermentation of cassava. these different media were supplemented with 0.1 % of nystatin to inhibit fungal growth. plates were streaked and incubated anaerobically at 30 °c for 48 h. after incubation, presumptive lab were identified as gram positive, oxidase negative and catalase negative and 5 colonies were randomly picked from agar plates. after picking, strains were grown in the same type of medium from which they were isolated, and streaked out repeatedly to check for purity. stock cultures of the isolates were stored in mrs broth containing 20 % of glycerol at -80 °c. 2.4. fermentative type and acidification capacity of isolates fermentative type of lab was determined by the ability of strains to produce gas from glucose by using mrs agar supplemented with 0,005 % of bromocresol. medium was put in tube and sterilized for 15 min at 121°c. after cooling, each strain was cultivated at 30 °c for 48 h. a negative control was carried out in the same conditions but not inoculated with strains. the ability of each strain to produce acid is assessed by the change of the medium colour comparatively to the negative control. presence of gas at the bottom of the tube indicates heterofermentative lab, otherwise (absence of gas) the strain is homofermentative lab. 2.5. study of rate of acidification of isolates fermentation was carried out with each isolate by using a modified mrs broth with 2 % of soluble starch as substrate. broth was distributed in tubes (5 ml by tube) and autoclaved at 121°c for 20 min. inoculum of each strain was obtained as following: a loopful of each isolate collected from a mrs agar plate was cultured in 5 ml of mrs broth and incubated at 30°c for 18h; 100 µl of microbial culture (od600nm = 1) transferred in 5 ml of mrs-soluble starch broth and incubated at 30 °c for 24 h. every 3 h, biomass was measured by turbidity using a spectrophotometer. also, the ph was measured with ph-meter (hanna) and titratable acidity by titration with naoh (0.1 n) using phenolphthalein as indicator. 2.6. screening of enzymatic activities screening of α-amylase producing lab the ability of the isolates to produce amylase was performed as described by sanni et al. [20]. many spots of each isolate were made with an inoculation needle on modified mrs agar without glucose but with 2 % of soluble starch (w ⁄ v) as the only carbon source. the plates were incubated at 30°c for 48 h in an anaerobic jar. after incubation, the culture plates were flooded with lugol’s iodine and a colourless area around the growth indicated a positive test. screening of pectinase, cellulase, phytase and tannase producing lab the ability of the isolates to produce pectinase, cellulase, phytase and tannase was detected as described by sanni et al. [20], but with slight modification. thus, the soluble starch was replaced by pectin, carboxy-methyl-cellulose, phytic acid and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 24 tannic acid as only carbon source for research of pectinase, cellulase, phytase and tannase, respectively. for phytase and tannase media, 3 % of agar was used. all plates were incubated at 30 °c for 48 h in an anaerobic jar. after incubation, lugol’s iodine was used for the revelation of pectinase, cellulase and phytase production. for tannase production, appearance of clear zone around the growth attests a positive test. screening of β-glucosidase producing lab the screening of β-glucosidase activity was tested according to the method of weagant et al. [21] using 4-nitrophenyl-βd-glucopyranoside, a linamarin analogue, as substrate. the medium for testing βglucosidase activity was prepared by adding 0.1 g of 4-nitrophenylβ-dglucopyranoside (merck, darmstadt, germany) to 100 ml nah2po4 (0.666 m; ph 6) (merck, darmstadt, germany) and filter-sterilized (millipore filter of 0.2 microns). the test culture was grown on mrs for 24 h at 30 °c. colonies were picked from the plates using a sterile loop and were emulsified in trypton-salt (mcfarland turbidity standard no. 3). thereafter, 0.75 ml of culture was added to 0.25 ml of the test medium and was incubated at 30 °c for 12 h. after incubation, 1 ml of sodium carbonate (1 m) was added to the medium and the change of colour (from colourless to yellow) indicated a production of βglucosidase. 3. results and discussion results of lab isolation are given in table 1. in our study, different media were used to isolate the widest possible species of lab associated with fermentation of cassava. a total of 237 strains characterized as gram positive, oxydase negative and catalase negative were isolated. among them, 97 were isolated from mrs, 25 from mse, 70 from bea and 45 from m17. the results show that number of lactococci (126) with a percentage of 53.16 % was high than number of rods (111) with 46.84 %. the high number of cocci has been previously reported by coulin et al. [14] who found a highest growth of cocci in these traditional inocula as well at the start of fermentation of cassava dough in attiéké process. however, several investigations showed that lactobacilli (lactobacillus plantarum) were the most predominant lab in cassava fermentation [3-13-16-17]. this is probably a result of differences in geographic regions, products and the practiced production technologies in the different studies. in our case, the high number of cocci can be explained by three most of these media wich were specific of cocci. thus, bea is a medium specific for enteroccocus, mse for leuconostoc and m17 for steptococcus. this study also revealed those traditional starters were dominated by homofermentative lab (89.03%) which convert carbohydrates exclusively into lactic acid. but the finding of heterofermentative lab confirms that spontaneous fermentation of cassava is heterofermentative type. more, heterofermentative lab produce also acetic acid and other volatiles compounds which contribute to aroma and flavour of fermented products by formation of lactic acid, acetoin, acetaldehyde, diacetyl, peptides and amino acid which are precursors of aromas [22]. homofermentative cocci (41 strains) isolated on bea agar may be enterococcus. enterococcus strains are often isolated from cassava fermentation. these microorganisms play an important role in conservation and bacteriological quality of food and their nutritional and organoleptic properties [23]. however, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 25 these are markers of fecal contamination (enterococcus faecalis and enterococcus faecium) and are considered as hygienic indicator in the manufacturing process of foods. thus, their use as starter is controversial [23]. none heterofermentative cocci was isolated in mse medium. it is may be that there is no leuconostoc in our samples because the occurrence of leuconostoc is high at the beginning of fermentation while our samples were collected at the end of fermentation i.e the third day during the spontaneous fermentation of cassava roots into traditional starters. table 1 lactic acid bacteria isolated from traditional starters of attiéké morphology of bacteria media of isolation total mrs mse bea m17 cocci fermentative type homofermentative 47 10 41 21 126 heterofermentative 2 0 3 2 rod fermentative type homofermentative 38 14 21 19 111 heterofermentative 10 1 5 3 total 97 25 70 45 237 mrs: de man, rogosa and sharpe; mse: mayeux sandine eliker: bea: bile esculine azide among 237 presumptive lab isolated, 18 failed to grow upon further propagation. thus, 219 strains were used for acid production. the acidification activity was measured by the change in ph (δph) during time and change of ph was calculates as δph= phat timephzero time. the initial ph of mrs-soluble starch broth used to evaluate the acidifying activity of isolates was 6.2. for this study, the variation of ph of 1 unit (δph=1) was taken as reference to evaluate acidifying activity of isolates. thus the isolates were clustered into 3 groups (table 2). at 6 h of fermentation, 75 (34.24 %) isolates of lab were able to decrease the ph of 1 unit. they were clustered in group 1, the faster acidifying lab, with ph value ranging between 3.93 and 5.20. the second group, the medium acidifying lab that registered 44 isolates (20.10 %) decreased the ph of 1 unit at 12 h of fermentation with ph values between 4.79 and 5.18. finally, the third group (slow acidifying lab), registered isolates which couldn’t decrease the ph of 1 unit in less than 12 h of fermentation. this cluster with mean of ph of 5.33 and mean titratable acidity of 0.35 % was constituted by 100 isolates (45.66 %). a good ph reduction is important to reduce the levels of contaminating microorganisms present on the raw materials, utensils and the environment which can compete with the starters for nutrients [24]. in this study, fast acidifying lab showed a high property of acidification and confirmed their major role of acidification in attiéké production. they acidify cassava dough by production of organic acids which lowered the ph. some isolates were able to produce lactic acid at rate of 0.72 % and decrease the ph to 3.93 at 6 h of fermentation. our results demonstrated also that acidifying activity of lab in cassava dough takes place mainly at the beginning of fermentation (6 12 h). in this time, majority of lab (54.33 %) which is clustered in faster and medium acidifying lab produced high amount of lactic acid. after 12 h of fermentation, decreasing of ph is weak. lab remaining contribute to maintain the environment acid by the weak production of lactic acid after 12 h of fermentation. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 26 table 2 characteristics of isolates according to their rates of acidification fast acidifying lab medium acidifying lab slow acidifying lab δph>1 at 6 h δph>1 at 12 h δph<1 after 12 h number of isolates (%) 75 (34.24 %) 44 (20.10 %) 100 (45.66 %) mean of ph 5.01±0.2 5.14±0.12 5.33±0.27 ph min 3.93 4.79 4.76 ph max 5.20 5.18 6.2 mean of ta (%) 0.400±0.104 0.377±0.067 0.351±0.08 ta min (%) 0.275 0.228 0.171 ta max (%) 0.720 0.570 0.581 δph: variation of ph; ta: titratable acidity for the screening of their enzymes activities, only the 75 fast acid producer strains were tested for amylase, pectinase, cellulase, phytase and tannase production. all isolates were able to grow in different media of enzymes activities. however, after revelation, none isolate showed phytase and tannase activities because of absence of colorless area around the spot. contrariwise, 17 were able to produce amylase, in which 6 were able to produce β-glucosidase (lab 4, lab 16, lab 68, lab 113, lab 127 and lab 251). four strains were able to produce cellulase (lab 16, lab 171, lab 210, and lab 245) while 2 strains (lab 72 and 114) were able to produce pectinase (table 3). table 3 enzymes production by lactic acid bacteria enzymes number of isolates tested number of lab producing enzymes percentage amylase 75 17 22.67 pectinase 75 2 2.67 cellulase 75 4 5.33 tannase 75 0 0 phytase 75 0 0 βglucosidase 75 6 8 furthermore, to be selected as pure microbial starter for cassava dough fermentation, enzymes synthesis is required. ability of isolates to produce amylase is important because cassava roots are composed of more than 80 % starch. the hydrolysis of starch must provide simple sugars easily metabolizable into lactic acid, which is expected to increase the content of this acid in cassava dough. in addition, this facilitates the digestibility of starchy food. however, production of amylase was a rare trait among lab. thus, only 22.67 % of our isolate were able to produce amylase. as to kostinek et al. [17] and edward et al. [25], none amylolytic lab was found during their studies for the selection of lab strains as potential pure microbial starter for cassava dough fermentation into gari. the breaking down of the coarse texture of the cassava roots was critical for the softening of cassava tissue, which is necessary for the quality improvement of cassava product [26]. this action is achieved by microorganisms which produce pectinase and cellulase in cassava dough. cassava roots itself contains linamarase which hydrolysis linamarin when the plant cells are disrupted. however, the endogenous linamarase of cassava is insufficient for a complete detoxification of cassava [27]. so, microbial β-glucosidase activities are also necessary to detoxify the cassava dough by the hydrolysis of linamarin present in the plant cells. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 27 among 75 lab isolated with important acid production, 17 were preselected as potential starter for attiéké production on the basis of their biochemical properties. biochemical properties considered were fast acid production, α-amylase, βglucosidase, cellulase, and pectinase activities. the results of the preselected starter are shown in table 4. table 4 preselected lab as starters 4. conclusion this is an essential preliminary work to select pure microbial starter for the control of cassava dough fermentation into attiéké. in regarding to interesting technological properties (acidification activity and enzymes synthesis), 17 presumptive lab strains have been preselected for further studies. thus, the next step will be a more detailed analysis and identification of these microorganisms. their mixer in pilot scale fermentation of cassava dough is also necessary to finally select the ones which can induce an optimal and constant desirable physico chemical and organoleptic quality of attiéké properties. 5. references [1]. kouadio n.a., kouakou, k.e., angbo, s.f., mosso k., etude comparative des methods traditionneles de la preparation de l’attiéké dans le sud de la côte d’ivoire. cahier de la recherche scientifique et technologique, 108 : 703-708, (1991) [2]. westby a., cassava utilization, storage and small-scale processing. in cassava: biology, production and utilization ed. hillocks, r.j., tresh, j.m. and bellotti, a.c., 281–300. wallingford, uk: cab international, (2002) [3]. amoa-awua w.k.a., appoh f.e., jakobsen m., lactic acid fermentation of cassava dough into agbelima. international journal of food microbiology, 31: 87–98, (1996) [4]. kimaryo v.m., massawe g.a., olasupo n.a., holzapfel w.h., the use of a starter culture in the fermentation of cassava for the production of ‘‘kivunde’’, a traditional tanzanian code of isolates α-amylase β-glucosidase cellulase pectinase lab 4 + + lab 16 + + + lab 19 + lab 20 + lab 21 + lab 34 + lab 68 + + lab 69 + lab 72 + + lab 113 + + lab 114 + + lab 127 + + lab 140 + lab 171 + + lab 210 + + lab 245 + + lab 251 + + food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 28 food product. international journal of food microbiology, 56: 179–190, (2000) [5]. mensah p., fermentation – the key to food safety assurance in africa? food control, 8: 271 278, (1997) [6]. cooke r.d., twiddy d.r. reilly p.j.a., lactic acid fermentation as a low-cost means of food preservation in tropical countries, fems microbiology reviews, 46: 369–379, (1987) [7]. oyewole o.b., lactic fermented foods in africa and their benefits. food control, 8: 289– 297, (1997) [8]. kakou a.c., optimisation des conditions d’application d’une methode de conservation longue durée de la pate de manioc (manihot esculenta crantz) en vue d’améliorer la qualité alimentaire de l’attiéké et du placali. thèse de troisième cycle en biochimie-microbiologie, université de cocody, abidjan – côte d’ivoire, 123 p, (2000) [9]. firmin a., optimisation of traditional fermentation of cassava. tropical science, 38: 220223, (1995) [10]. djeni n.t., n’guessan k.f., toka d.m., kouame k.a., dje k.m., quality of attieke (a fermented cassava product) from the three main processing zones in côte d’ivoire. food research international, 44: 410–416, (2011) [11]. diop a., l’attiéké dans la région d’abidjan, analyse économique de la filière traditionnelle à travers quelques types d’organisation (adjoukrou, ebrié, attié). thèse de doctorat troisième cycle en economie rurale. université de cocody, abidjan, côte d’ivoire. 146 p, (1992) [12]. toka d.m., djeni n.t., dje k.m., improved process of cassava processing into “attieke”, a traditional food product of côte d’ivoire. international journal of food engineering, 4: 1–10, (2008) [13]. assanvo b., agbo g.n., behi y., coulin p., farah z., microflora of traditional starter made from cassava for “attieke” production in dabou (côte d’ivoire), food control, 17: 37–41, (2006) [14]. coulin p., farah z., assanvo j., spillmann h., puhan z., characterisation of the microflora of ‘attieke¢, a fermented cassava product during traditional small-scale preparation, international journal of food microbiology, 106: 131–136, (2006) [15]. bouatenin k.m.j.p., djeni n.t., aka s., brou k., dje k.m., the contribution of microorganisms to the fermentation of cassava dough during attiéké processing in côte d’ivoire, food, 6: 58-64, (2012) [16]. padonou s.w., nielsen d.s., akissoe n.h., hounhouigan j.d., nqgo m.c., jakobsen m., development of starter culture for improved processing of lafun, an african fermented cassava food product, journal of applied microbiology, 109: 1402–1410, (2010) [17]. kostinek m, specht i., edward v.a., pinto c., egounlety m., sossa c., mbugua s., dortu c., thonart p., taljaard l., mengu m., franz c.m.a., holzapfel w.h., characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures, international journal of food microbiology, 114: 342-351, (2007) [18]. giraud e., gosselin l., raimbault m., degradation of cassava linamarin by lactic acid bacteria, biotechnology letters, 14: 593-598, (1992) [19]. ayad e.h.e., nashat s., el-sadek m., metwaly h., el-soda m., selection of wild lactic acid bacteria isolated from traditional egyptian dairy products according to production and technological criteria, food microbiology, 21, 715-725, (2004). [20]. sanni, a.i., morlon-guyot j., guyot j.p., new efficient amylase-producing strains of lactobacillus plantarum and lact. fermentum isolated from different nigeran traditional fermented foods, international journal food microbiology, 72: 53–62, (2002). [21]. weagant s.d., feng p., stanfield j.t., yersinia enterocolitica and yersinia pseudotuberculosis, in: bacteriological analytical manual online, us food and drug administration, center for food safety and applied nutrition. http://www.cfsan.fda.gov/ (2001). consulted 07/05/2014. [22]. raimbault m., importance des bactéries lactiques dans les fermentations du manioc. in transformation alimentaire du manioc. ed. : agbor e. t., brauman a., griffon d., trèche s. 1995, éditions orstom, 259-275, (1995) [23]. aguilar-galvez a., duboisdauphin r., destain j., campos d. thonart p., les entérocoques: avantages et inconvénients en biotechnologie (synthèse bibliographique). biotechnology agronomy society environment, 16: 67-76, (2012) [24]. holzapfel w.h., appropriate starter culture technologies for small-scale fermentation in developing contries, international journal of food microbiology, 75: 197-212, (2002) http://www.cfsan.fda.gov/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 regina ekoua krabi, antoine allah assamoi, fafadzi ayawovi ehon, sébastien lamine niamke, screening of lactic acid bacteria as potential starter for the production of attiéké, a fermented cassava food, food and environment safety, volume xiv, issue 1 – 2015, pag 21 – 29 29 [25]. edward v.a., egounlety m., huch m., zyl p.j. v., singh s., nesengani n.d., haakuria v.m., franz c.m.a.p., isolation and screening of microorganisms from a gari fermentation process for starter culture development, african journal of biotechnology, 11: 12865-12877, (2012) [26]. amoa-awua w.k.a., jakobsen m. role of bacillus species in the fermentation of cassava, journal of applied bacteriology, 79: 250256, (1995) [27]. ikediobi c.o., onyike e., linamarase activity and detoxification of cassava (manihot esculenta) during fermentation for gari production, agricultural and biological chemistry, 46: 16671669, (1982) doi: https://doi.org/10.4316/fens.2021.019 172 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 172 181 influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread *iryna medvid 1 , olena shydlovska 1 , tetiana ishchenko 1 1department of hotel-restaurant busines, national university of food technologies, volodymyrska str. 68, kyiv, 01601, ukraine, medvidrina@gmail.com, *corresponding author received 31th march 2021, accepted13th september 2021 abstract: the article investigates the influence of low-fat lecithin, sunflower oil, and dry egg white on the rheological properties and microstructure of gluten-free rice dough and quality indicators of ready-made bread. to determine the effectiveness of the use of these emulsifiers, the bread technology with implementation of enzyme modificated (by amylolytic enzymes) flour starch was used. to assess the rheological state of the dough, its viscoplastic properties (shear stress and effective viscosity of the test systems) were analyzed using a rotary viscometer. the dough microstructure was examined by microscopy. traditional methods to determine physical and chemical indicators of ready-made bread quality were used. the results showed that the emulsifiers implementation changed the behavior of rice dough, and had the positive effect on the qualitative (specific volume and porosity). it has been found that the use of a combination of emulsifiers from low-fat lecithin, sunflower oil, and dried egg white in rice bread technology in combination with enzymatic modification of flour starch significantly improves the rheological properties and structure of gluten-free dough and provides high indicators of specific volume and porosity of ready-made products. keywords: gluten-free bread, rice dough, sunflower oil, lecithin, dried egg white. 1. introduction the priority task of the food and restaurant industries in today's conditions is to expand the sector of production of products for special dietary consumption [1], intended for the treatment and prevention of food-borne diseases. one of the common ones is celiac disease, which occurs in 1-3 % of the world’s population [1, 2]. it’s the immune-mediated reaction to gluten of genetically predisposed people [3], which is accompanied by damage and inflammation of the small intestinal mucosa and malabsorption of many important nutrients such as iron, folic acid, fat-soluble vitamins, calcium [4, 5]. despite the progress in the understanding of the pathogenesis of celiac disease and the development of new therapy methods, nowadays, only the strict gluten-free lifetime diet is the safest and the most effective method of patients’ treatment, which means constant removal of gluten from the diet [1, 3]. generally, the term "gluten" combines the prolamins of some cereals that contain specific toxic oligopeptide sequences – wheat gliadin, rye secalin, and barley hordein [4]. the increasing incidence of celiac disease is leading to an increase in demand for gluten-free foods [3], so the development of new technologies is an extremely important issue. the preparation of gluten-free bread is a complicated technological task, as http://fens.usv.ro/index.php/fens mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 173 prolamins play a key role in ensuring the unique structural and mechanical properties of the dough [1, 6, 7]. as a basis for the production of non-gliadin bread, rice flour is becoming increasingly popular. this raw material is a source of vegetable protein, complete in amino acid composition, contains sodium, potassium, phosphorus, magnesium, vitamins b1, b2, and pp, a high amount of easily digestible carbohydrates, has a mild taste and white color [8, 9]. however, gluten absence in the rice flour leads to the preparation of dough, which has reduced structural and mechanical properties, dense porosity of the crumb of the ready-made product, and other quality defects [10]. the analysis of existing scientific research shows that to improve the quality of gluten-free bread made of rice flour, the use of ingredients, imitating functional properties of gluten was suggested by many scientists [7, 11]. to this end, it is important to include in the formulation of hydrocolloids (gums of plant origin, microbial polysaccharides, cellulose and its derivatives, modified starch) [2, 3, 1214], proteolytic enzymes and transglutaminase [8, 15], emulsifiers (datem, sodium stearoyl lactylate, lecithin) [9], raw materials with high protein content (isolates and concentrates) [16] and combinations. to prevent the loss of carbon dioxide bubbles formed during dough fermentation and their coalescence, the use of lecithins as structural agents is promising, which not only act as emulsifiers but are also a valuable source of phospholipids that are important physiologically [17]. the molecule of lecithin is hydrophilic (polar) on the one hand and hydrophobic (nonpolar) on the other, which improves the efficiency of its use in combination with fats of vegetable, animal origin or shortening [4]. this leads to better plasticization of gluten-free bread, reduction of adhesion, and, consequently, sticking of the dough to the working parts of equipment and its losses during the bread production. taking into consideration the fact, that patients with celiac disease typically have low protein intake, it is promising to model the viscoelastic behavior of gluten in the dough and improve the texture of the final product by using raw materials high in protein, which also improves the nutritional value of products [3]. the functional ingredient with a structureforming effect is dry egg white, the properties of which can be considered as a display of three molecular aspects: hydration, emulsifying, and hydrodynamic (rheological) properties [18]. rice flour is characterized by a high content of starch (75-81 %), a small amount of mono-, disaccharides (0.6-0.7 %), which are essential for the yeast metabolism and activity during fermentation, and low activity of its own αand β-amylases. due to this, rice flour is a potential raw material for modifying its carbohydrate composition using amylolytic enzymes, the substrate of which is starch. in order to enrich the dough with sugars, we selected α-amylase of fungal origin and glucoamylase, the joint use of which catalyzes the hydrolytic decomposition of glucoside bonds of starch molecules with the formation of maltose, glucose, and dextrins of different molecular weights. according to the results of previous studies, it was determined that the use of enzymatic modification of rice flour starch in the preparation of the dough contributes to a more intense course of microbiological processes, which is displayed in the activation of gas and acid accumulation [19]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 174 since research on improving the properties of dough and the quality of gluten-free bread remains a topical issue, the aim of this article was to analyze the effect of a combination of lecithin emulsifiers, sunflower oil, and dried egg white on rheological properties, and microstructure of rice dough using an enzymatic modification of flour starch with the help of fungal α-amylase and glucoamylase and quality indicators of ready-made products. 2. matherials and methods 2.1. raw matherials the rice flour (8 g/100 g of protein, 80.3 g/100 g of starch, 0.7 g/100 g monoand disaccharides and humidity 11.5 %) produced by “cascade” llc, ukraine; pressed baker's yeast (lesaffre, ukraine); salt (artemsil, ukraine) and tap one water were used in the preparation of gluten-free bread. hydrolysis of rice flour starch was performed using amylolytic enzymes: fungal α-amylase alphamalt vc 5000 sn (muhlenchemie, germany) with an activity of 5000 skb/g, the optimal operating conditions of which ph 4.7, temperature 37-40 ºc; glucoamylase glucomil (germany), the activity of which is 500 amg/g, optimal ph 3.0-5.5, temperature 40-64 ºc. the citric acid (mriya, ukraine) was also used in the studies. low-fat sunflower lecithin (dniprotechnologies research and production center, ukraine) with a phospholipid content insoluble in acetone, 96.5 %, was used as a structuring ingredient; refined sunflower oil (sandrade, ukraine); dry egg white (ovostar union, ukraine) with a mass fraction of protein substances of 85 % and a solubility of 93.7 %. 2.2. methods enzymatic modification of rice flour starch since the temperature and acid optimums of fungal α-amylase and glucoamylase differ, the modification of the carbohydrate composition of rice flour was performed at a temperature of 40 ºc and ph 4.7, which are optimal for both enzymes. in order to maintain the appropriate ph of the environment, citric acid was used in an amount of 0.065 % by weight of flour. αamylase (0.005 % by weight of flour) and glucoamylase (0.03 % by weight of flour) were pre-dissolved in the water at a temperature of 25-30 ºc at hydro module 10. to carry out the enzymatic modification, a mixture of 50 % of rice flour from its prescription amount, citric acid, enzymes, and water was hydrolyzed in an electric thermostat at 40 °c for 120 min until the accumulation of sugars in it 5.9 %. the humidity of the flour mixture was 65 % [19]. the dough making and bread baking the following dosages of ingredients (g per 100 g of rice flour) were used during the preparation of the dough: low-fat sunflower lecithin (1 g), refined sunflower oil (3 g), dry egg white (4 g), compressed yeast (3 g) and salt. (1.2 g). after fermentation, lecithin, oil, and dry egg white were added to the obtained hydrolyzate from rice flour, dispersing it for 5-7 min at a rotational speed of the working body of the food processor (electrolux, sweden) of 100-120 min-1. to prepare the dough with a humidity of 53 %, a modification of the steamless method was used. yeast suspension, salt solution and the second part of rice flour according to the recipe were added to the rice flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 175 hydrolyzate with the structure-forming ingredients dispersed therein. the dough was kneaded on a food processor with replaceable nozzles (electrolux, sweden) for 8-10 min. then the dough pieces were formed by hand and subjected to fermentation in steel molds for baking in a stand (unox, italy) for 50 min at a temperature of 30-32 ºc and a relative humidity of 75-80 %. baking was performed at a temperature of 180 ºc in a humidified baking cabinet (unox, italy) during 25-30 min. the ready-made bread weighing 400-450 g was cooled for 180 min. to compare the effect of amylolytic enzymes and structure-forming ingredients (using their concentrations mentioned above) on the properties of gluten-free dough and the quality of rice flour bread, 4 samples of test systems were prepared: control (without additives); with enzymes; with enzymes, lecithin and oil; with enzymes, lecithin, oil and dry egg white. rheological (viscous-plastic) properties of gluten-free dough rheological properties of the dough after kneading and after 50 min of fermentation were determined at a temperature of 25 ºc on a rotary viscometer "reotest-2" (germany), which is equipped with replaceable cylinders with different diameters: external – s and internal – s2. samples of the semi-finished product with a moisture content of 70 % weighing 20-25 g were placed in the measuring system of the device and read α at 12 shear rates in mode a, each of which corresponds to a shear deformation constant or shear rate dr (s-1): 1a – 1; 2a – 1.8; 3a – 3; 4a – 5.4; 5a – 9; 6a – 16.2; 7a – 27; 8a – 48.6; 9a – 81; 10a – 145.8; 11a – 243; 12a – 437.4. the increase in the shear rate occurs when the speed of rotation of the inner measuring cylinder by switching the gearbox. the shear stress (τ, pa) was calculated using the instrument scale readings (α) and the constant of the inner cylinder (ᴢ), which for s2 is 5.91 pa according to the following equation: (1) when calculating the effective viscosity (ƞ, pa*s), the data of shear stress (τ, pa) and shear rate (dr, s -1) were taken into account. equation [20] was used to determine this indicator: (2) based on the obtained results, viscosity curves (dependence of viscosity on shear stress) ƞ = f(τ) were constructed. to characterize the change in the effective viscosity of the dough systems during maturation, the degree of dilution (k, %) was determined by the following equation: (3) gluten-free dough microstructure studies of the microstructure of gluten-free rice dough were performed using an electronic scanning microscope jsm-6060 la (jeol, japan), equipped with an energy dispersion spectrometer oxford x max-80t. after 50 min of fermentation, the test specimens were frozen, dried under vacuum, subjected to fracture, sprayed with ash on the fracture area of 5 mm. after that, they were examined at a magnification of 1000 times and the most expressive areas were photographed. physical and chemical quality indicators of gluten-free bread the determination was performed not earlier than 3 hours after baking of all samples. the specific volume of bread was calculated as the ratio of the volume it occupies to its weight (cm3/g). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 176 porosity of the finished products was determined at a zhuravlev device in line with a known procedure [19]. the results of experimental studies were statistically processed using the standard microsoft office software package. 3. results and discussion 3.1 characteristics of the rheological properties of gluten-free dough rheological properties of gluten-free dough are decisive in the formation of qualitative characteristics of finished bakery products. their adjustment allows to influence such indicators of bread quality as specific volume and porosity, which in general characterizes the consumer value of products. information on the influence of ingredients used in rice bread technology on the rheological behavior of the dough is given by the analysis of viscoplastic properties of dough systems after kneading (a) and after 50 min of fermentation (b) on a rotary viscometer reotest-2 (figure 1). a) b) fig. 1. rheological viscosity curves (a) after kneading and (b) at the end of dough fermentation it was determined that right after kneading the dough (fig. 1, a) the value of the effective viscosity for the samples using the enzymatic modification of rice starch decreases in comparison to the control. it’s explained by dehydrating properties of monoand disaccharides formed during the hydrolytic decomposition of starch under the action of α-amylase and glucoamylase. additional application of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 177 low-fat lecithin together with sunflower oil reduced the viscosity of the dough system with enzymes compared to the sample without emulsifier. this was facilitated by the ability of the oil, adsorbed on the surface of starch grains and protein micelles, to prevent swelling of these colloids and increase the content of the liquid phase of the dough. the introduction of dry egg white into the dough causes an increase in its effective viscosity in relation to the sample with enzymes, lecithin and oil, which is probably the result of the interaction of the protein matrix of flour with the active groups of animal protein molecules. analyzing the rheological viscosity curves at the end of the dough fermentation (fig. 1, b), it was found that the effective viscosity of all test samples decreases due to the hydrolysis of dough biopolymers. however, determining the degree of dilution (k, %) of test systems (table 1) indicates a slowdown in the relaxation of their structure during maturation. table 1 the degree of dilution of the dough systems during the maturation period name of the sample effective viscosity, pa*s the degree of dilution (k, %) ƞ after kneading the dough ƞ at the end of dough fermentation control (without additives) 15.96 13.00 23.0 with enzymes 13.89 11.82 18.0 with enzymes, lecithin and oil 10.64 9.16 16.0 with enzymes, lecithin, oil and dried egg white 11.53 10.22 12.8 it was found that the use of enzymatic modification of flour starch in rice bread technology reduced the degree of dough thinning by 21.7 % compared to the control, and additional application of lecithin with sunflower oil – by 30.4 %. the results can be explained by the deepening of colloidal processes in the dough, which are probably related to the ability of dextrins formed due to the action of α-amylase on starch, to bind a significant amount of free moisture; partial complexation of surfactants with flour components, as well as high activity of unsaturated fatty acids of vegetable oil in the formation of complexes with proteins and starch polysaccharides. the use of dry egg white as a structuring agent in combination with enzymes, lecithin and oil reduced the degree of dilution of the dough by 1.7 times compared to the control, from which we can conclude that the use of a combination of these ingredients increased the structural viscosity of gluten-free rice dough, i. e. improving its rheological properties. 3.2 gluten-free dough microstructure during dough preparation, flour biopolymers undergo significant structural and conformational changes, which are accompanied by swelling, peptization, and enzymatic cleavage of the components [21]. a more thorough explanation of the effect of amylolytic enzymes, lecithin, oil and dry egg white on the structural and mechanical properties of gluten-free rice dough can be obtained by microscopy (figure 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 178 analysis of the obtained microphotographs showed that samples of rice dough with used enzyme modificated flour starch (fig. 2, b, c, d) were characterized by the presence of smaller grains of starch compared to the control, on their surface in some places spots similar to deepening are visible, which is probably due to the hydrolytic action of α-amylase and glucoamylase. the more continuous structure of the studied dough samples than the control can be explained by the better intensity of microbiological processes during maturation, as well as the interaction of incomplete products of hydrolysis of starch – dextrins, with moisture and other components of the dough system. a) b) c) d) fig. 2. the microstructure of the dough: a – control (without additives), b – with enzymes, c – with enzymes, lecithin and oil, d – with enzymes, lecithin, oil and dry egg white the dough microstructure with lecithin and oil in combination with amylolytic enzymes (fig. 2, c) was more homogeneous and monolithic in comparison to samples without emulsifier and fat, which can be explained by wrapping of starch and its hydrolysis products with fat pellicle and possible formation of complexes with molecules. the results from the study of the dough microstructure with additional introduction of dry egg white (fig. 2, d) indicated that its colloidal solution promoted the adhesion of starch grains and other components in the dough system, forming conglomerate protein-polysaccharide complexes, which creates a more developed dough structure during the fermentation period. thus, the results of studies of the rheological properties and microstructure of gluten-free rice dough with the studied structuring agents indicate an improvement in its structure, which will improve the quality of ready-made products, in particular the specific volume and porosity. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 179 3.3 the research of physical and chemical indicators of gluten-free bread quality important criteria for assessing the impact of raw ingredients on the quality of finished products are the specific volume and porosity of the bread, which reflects the ratio of pore volume to total crumb volume. the digestibility of bread is also related to the amount of porosity [20]. the results of the study of these physical and chemical quality indicators for gluten-free rice bread using amylolytic enzymes, lecithin, oil and dry egg white in its technology are presented in table 2. table 2 physical and chemical quality indicators of gluten-free bread quality indicators gluten-free rice bread samples control (without additives) with enzymes with enzymes, lecithin and oil with enzymes, lecithin, oil and dried egg white specific volume, cm3/g 1.31 1.51 1.97 2.53 change in control, % +15.3 +50.4 +93.1 porosity, % 36.7 41.4 51.5 65.5 change in control, % +12.8 +40.3 +78.3 it was found that the most pronounced effect on the specific volume and porosity of the ready-made products has a joint use in their technology of lecithin, oil and dried egg white in combination with the modification of rice flour starch with amylolytic enzymes. thus, the use of a combination of these structuring ingredients increased the specific volume of bread by 93.1 %, while the porosity of the crumb was improved by 78.3 % in comparison to the control sample. so, the analysis of the results of the quality of rice bread samples confirms the patterns obtained during the study of the rheological properties and of the dough microstructure. 4. conclusion according to the results of the research, the expediency of modifying the carbohydrate composition of rice flour with the help of amylolytic enzymes and the joint use of low-fat lecithin, sunflower oil and dried egg white as structureforming ingredients in the technology of gluten-free bread. their positive effect on the rheological properties and microstructure of the dough was determined. particularly, the decrease of the effective viscosity and the degree of dilution of the dough systems with the introduction of the suggested components in relation to the control without additives, which can be noted as an improving effect. due to the lower viscosity of the dough, the carbon dioxide bubbles that are the part of the dispersed phase expand more while baking, and the ready-made products are characterized by higher values of specific volume and porosity. ensuring the required quality of rice bread using enzymes, lecithin with oil and dry egg white is also due to a significant improvement in the structural and mechanical properties of the dough examined by microscopy.taking into consideration the high efficiency of these structuring ingredients in combination with enzymatic modification of flour starch to improve the properties of the dough and quality indicators of readyfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 180 made products, bread technology with their use can be implemented in the baking industry and in the production conditions of restaurants. 5. references [1]. ronda f., pérez-quirce s., villanueva m., rheological properties of gluten-free bread doughs: relationship with bread quality. advances in food rheology and its applications, ahmed j., ptaszek р., basu s., eds., woodhead publishing: kidlington, united kingdom: 297-334, (2017) [2]. sabanis d., tzia с., effect of hydrocolloids on selected properties of gluten-free dough and bread, food science and technology international, 17(4): 279-291, (2011) [3]. cappa с., lucisano м., mariotti м., influence of psyllium, sugar beet fibre and water on gluten-free dough properties and bread quality, carbohydrate polymers, 98: 1657-1666, (2013) [4]. pruska-kedzior а., kedzior z., goracy м., pietrowska к., przybylska а., spychalska к., comparison of rheological, fermentative and baking properties of gluten-free dough formulations, eur food res technol, 227: 1523-1536, (2008) [5]. zhang d., mu t., sun h., comparative study of the effect of starches from five different sources on the rheological properties of gluten-free model doughs, carbohydrate polymers, 176: 345355, (2017) [6]. mezaize s., chevallier s., le-bail а., de lamballerie м., gluten-free frozen dough: influence of freezing on dough rheological properties and bread quality, food research international, 43: 2186-2192, (2010) [7]. torbica а., hadnaԁev м., dapčević т., rheological, textural and sensory properties of gluten-free bread formulations based on rice and buckwheat flour, food hydrocolloids, 24: 626-632, (2010) [8]. hatta e., matsumoto k., honda y., bacillolysin, papain, and subtilisin improve the quality of gluten-free rice bread, journal of cereal science, 61: 41-47, (2015) [9]. nunes m.h.b., moore m.m., ryan liam a.m., arendt e.k., impact of emulsiwers on the quality and rheological properties of gluten-free breads and batters, eur food res technol, 228: 633-642, (2009) [10]. rostamian м., milani j. m., maleki g., physical properties of gluten-free bread made of corn and chickpea flour, international journal of food engineering, 10(3): 467-472, (2014) [11]. tsatsaragkou k., yiannopoulos s., kontogiorgi a., poulli e., krokida m., mandala i., effect of carob flour addition on the rheological properties of gluten-free breads, food and bioprocess technology, 7(3): 868-876, (2013) [12]. lazaridou а., duta d., papageorgiou м., belc n., biliaderis c.g., effects of hydrocolloids on dough rheology and bread quality parameters in gluten-free formulations, journal of food engineering, 79: 1033-1047, (2007) [13]. witczak м., juszczak l., ziobro r., korus j., influence of modified starches on properties of gluten-free dough and bread. part i: rheological and thermal properties of gluten-free dough, food hydrocolloids, 28: 353-360, (2012) [14]. hryshchenko a.m., udvorheli l.i., mykhonik l.a., kovalevska ye.i., research of structural and mechanical properties of exalbuminous dough with guar gum and xanthan gum, food science and technology, 1(10): 63-65, (2010) [15]. shanina o.m., lobacheva n.l., zverev v.o., the effect of transglutaminase enzyme on the properties of flour proteins, easterneuropean journal of eenterprise technologies, 5/11(71): 28-33, (2014) [16]. ziobro r., witczak т., juszczak l., korus j., supplementation of gluten-free bread with non-gluten proteins. effect on dough rheological properties and bread characteristic, food hydrocolloids, 32: 213-220, (2013) [17]. medvid і., shydlovska о., dotsenko v., the use of sunflower lecithin in the technology on gluten-free bread with enzymatic modification of flour starch, food and environment safety, 17(4): 352-362, (2018) [18]. dotsenko v., medvid і., shydlovska о., ishchenko т., studying the possibility of using enzymes, lecithin, and albumen in the technology of gluten-free bread, eastern-european journal of enterprise technologies, 1/11(97): 42-51, (2019) [19]. medvid і., shydlovska о., dotsenko v., influence of fermentative modification of rice flour starch on bread quality for patients with celiac disease, ukrainian food journal, 6(4): 632-647, (2017) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 iryna medvid, olena shydlovska, tetiana ishchenko, influence of the combination of emulsifiers on the properties of rice gluten-free dough and the quality of bread, food and environment safety, volume xx, issue 3 – 2021, pag. 172 – 181 181 [20]. drobot v.i., yurchak v.g., bilyk o.a., bondarenko y.v., gryschenko a.m., zinchenko i.m., kambulova y.v., makhynko v.m., mykhonik l.a., falendish n.o. techno chemical control of raw materials and bakery and pasta products, drobot, v. i., ed., condor publisher: kyiv, 972 р., (2015) [21]. pivovarov o.a., mykolenko s.y., tishchenko g.p., microstructural features of the dough based on solutions exposed to contact nonequilibrium plasma, food science and technology, 1(18): 67-70, (2012) 1. introduction 4. conclusion according to the results of the research, the expediency of modifying the carbohydrate composition of rice flour with the help of amylolytic enzymes and the joint use of low-fat lecithin, sunflower oil and dried egg white as structure-forming ingredie... 122 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 22020, pag. 122 130 aerated confectionery: physico-chemical and textural evaluation raluca olimpia zimbru1, *sergiu pădureţ1, sonia amariei1 1faculty of food engineering, stefan cel mare university of suceava, romania sergiu.paduret@fia.usv.ro *corresponding author received 15th april 2020, accepted 25th june 2020 abstract: the effect of raw materials and production process on stress relaxation texture parameters (hardness, total mechanical work, elastic mechanical work, deformability modulus, relaxation and relaxation work) and physico-chemical properties (protein content, fat content, dry matter, soluble substances concentration, aw, density and specific volume) of aerated confections were investigated in this study. the aeration process of confectionery samples was based on mechanical beating, using a six wire whip. according to pearson’s correlation the samples’ hardness and relaxation were positively influenced by the dry matter content (r = 0.943*) and density (r = 0.997**), whereas the specific volume had a negative effect (r = -0.982**). the highest soluble substances concentration was recorded by the vegetable cream aerated samples; the anova factorial analysis showed a significant difference, p < 0.001. keywords: stress-relaxation, hardness, confectionery, density, deformability modulus. 1. introduction there are many food products which contain air bubbles or other gases distributed in a liquid or solid viscoelastic mass, such as: confectionery products or other desserts, ice cream, beating cream or bread dough. the appearance, textural characteristics, sensory properties and shelf life of these products are strongly affected by the dimensions and concentration of bubbles and it is important to prevent bubbles’ properties changes over time [1]. aerated confections have air trapped and dispersed as small bubbles throughout the food matrix, thus the density of these products is lower than that of nonaerated food products and also are frequently more brittle. as a means to characterize these products, density’s measurements are often used, which can vary greatly from 1.1 g/ cm3 (nougat) to 0.2 g/ cm3 (marshmallows) [2]. the gases of greatest relevance used in food products’ aeration are represented by: air, carbon dioxide, steam, nitrogen, nitrous oxide or oxygen, applied separately or in different combinations [3]. in the case of aerated confectionery products the air is trapped in the product structure and can accelerate the oxidative changes like fats rancidity, vitamins or flavor oxidation and thereby reduce the products’ shelf life [4]. this process could be diminished or even stopped by substituting the air, during the whipping stage, with another gas like carbon dioxide, nitrogen or nitrous oxide. furthermore, it should be mentioned that these aeration agents can also provoke some tainting to the products; the aeration agents based on nitrogen and carbon dioxide can dissolve in the fats and carbon http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 123 dioxide is water soluble, which can then determine a modification in the products’ flavor [5]. texture is extremely important to products acceptability, especially when it comes to confectionery and the textural parameters of aerated confections are influenced to a large extent by the air/gas content, although the characteristics of the continuous phase imparts some particular properties, too [4]. for the mechanical beating process various type of whip wire have been developed to facilitate aeration [6]; first large air bubbles are incorporated in the food matrix, but in order to form a stable aerated product, these large air bubbles must be broken up into smaller ones. this occurs in turbulent flow during the first phase of mechanic beating followed by the second phase of beating when the air bubbles are coated by a thin layer of fat globules [7]. to the authors knowledge there are few studies based on the evaluation of aerated confectionery products’ texture. therefore, the purpose of this research was to study the influence of the raw materials and the production process on the stress-relaxation texture parameters and also to characterize the main physicochemical composition of the aerated confections. 2. matherials and methods experimental samples preparation. for the aeration process was used a six wire whisk whip, the speed was set at an approx. 600 rotations/min and the beating time was 5 minutes. the raw materials used in the production process were: white chocolate (callebaut, belgium, 36 % fat), sugar, yolk, milk, gelatin, 25 % fat vegetable cream for a1 and a3 samples and 32 % fat dairy cream for a2 and a4 samples. the production of aerated confectionery samples followed the procedure described by zimbru 2020 [8], thus a1 and a2 samples were produced by incorporation of the mixture of chocolate, sugar, yolk, milk and gelatin in the whipped vegetable or dairy cream, while the a3 and a4 samples were produced by the incorporation of the chocolate, sugar, yolk and gelatin one at a time in the whipped vegetable or dairy cream. physico-chemical analysis. total protein content of aerated samples was determined by kjedahl method [9], the dry matter was measure by oven drying method [10] and the fat content was carried out using a soxhlet extractor [11]. for water activity (aw) was used a water activity meter (aqualab lite) and for the concentration of soluble substances measurements was used a leica mark ii plus refractometer. also density and specific volume were determined [12]. all reagents used were of analytical grade. texture evaluation. the stress-relaxation compression test was performed at room temperature (approx. 20 ºc) on cubic samples (30 mm) using a mark 10-esm301 texture analyzer (mark 10 corporation, usa). the compression probe was flat (ø 50 mm), the test speed was set at 10 mm/min and the trigger force was set at 0.05 n. the sample loading was established at 10 mm and the relaxation time was 200 seconds. the mesuregauge software was used for load (n), deformation (mm) and time (s) recording at a reading rate of 10 points per second. resulting load deformation and load time curves were used for the evaluation of textural parameters like: hardness (h, n), total mechanical work (wt, j) calculated as the total area under the loading curves, mechanical work of elastic deformation (we, j) calculated as the area under the unloading curves, the loading modulus of deformability (ed, pa) [13], relaxation (r, n/s) calculated as the differences of initial load of relaxation (fi) and the final load of relaxation (ff) reported on relaxation time (trelaxation, s), food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 124 (eq.1). the relaxation work (rw, n·s) was calculated as the area under the relaxation curves [14]. (1) statistical analysis. the results were subjected to analysis of variance anova by statgraphics centurion xvi (trial version); the statistical significance being set at α = 5%, while pearson correlation was calculated with spss 13.0 (spss inc. chicago, il). 3. results and discussion physico-chemical analysis. the physicochemical analysis of aerated confectionery samples, respectively the protein, fat, dry matter, brix concentration, water activity, density and specific volume are presented in table 1 and we can observe that the protein content ranged between 9.55 % 11.12 %, the anova analysis highlighted this difference based on the ingredients used at a level of p< 0.05. fat content ranged between 15.35 % and 20.18 %; the aerated samples based on dairy cream, a2 and a4, presented a higher fat content than a1 and a3 samples, which contain whipped vegetable cream. one way anaova highlighted this difference at a level of p< 0.001. proteins and fats are of great importance in the aeration process of confectionery products; in the first phase initial large air bubbles are stabilized by proteins and as these bubbles are broken into smaller sizes they are covered by a film of fat globules [15; 7]. the dry matter content of aerated confectionery samples varied considerably from 52.38 % to 57.72 %, a4 sample had the highest content and according to nardozza, 2011 [16] in the case of some food products the dry matter content influences texture properties. when it comes to confections, another important parameter is represented by water activity which has an important role in quality control, establishing the shelflife of the products and influences texture and sensory parameters [17]. water activity of aerated confections is between 0.826 0.848 and according to subramaniam, 2016 [2] foods that show a water activity value greater than 0.750 are not stable against microbial spoilage. the highest soluble substances concentration was recorded by the vegetable cream aerated samples, while the dairy cream aerated samples showed smaller values; the anova factorial analysis showed a significant difference (p < 0.001). fixing air into the product structure results in a decrease of density, obtaining a lighter product in terms of calories [18]. the samples’ density ranged between 0.400 and 0.780 g/cm3, a1 and a2 samples showing smaller values than a3 and a4 samples; thus, the one step incorporation (first procedure) of the other ingredients in whipped vegetable or dairy cream introduced and fixed a larger amount of air in the product structure. in addition to a low density, aerated confections show a more fragile texture and a shorter shelf life due to the oxidative changes of fats (fats rancidity) or vitamins [5]. as regarding the specific volume of the analyzed samples, it was between 1.28and 2.50 cm3/g; the a1 and a2 aerated samples presenting higher values than a3 and a4 samples. the specific volume results were similar to those reported for marshmallow (2.2cm3/g), whipped cream (1.7-2.5 cm3/g), cake batter (1.25-1.8 cm3/g) or aerated chocolate bar (1.25-1.45cm3/g), but smaller than those reported for meringue (5.5-6 cm3/g) and sponge cake (3-4 cm3/g) [18]. 200 fi ff relaxationt f r     food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 125 table 1. physico-chemical parameters of aerated confectionery samples different lowercase letters (a–d) in a row show significant differences between the groups (p<0.05). ns – not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001. texture properties measurements. figure 1a and 1b shows the stress relaxation and loading unloading compression curves of aerated confectionery samples, of which two were produced with whipped dairy cream and two with vegetable cream, following two different procedures. there was observed that the aerated samples exhibit both elastic and viscous behavior, namely a viscoelastic solid behavior, with a decrease of stress (required to maintain the strain) during time (figure 1b). based on loading unloading (load-travel) and stress relaxation curves were calculated the texture parameters of aerated samples, as shown in the table 2. hardness (h) values were calculated as the force measured at the end of loading and express in newton; lower values were measured for a1 (1.92 n) and a2 (2.7 n) samples, which were produced by homogenization of the mixture formed from other raw materials in beating vegetable or dairy cream. furthermore a1 and a2 samples showed lower density values which means that these samples are more aerated, containing a larger amount of air in the structure, leading to lower hardness, while the a3 and a4 samples showed higher hardness and density values which corresponds to a less aerated structure. according to campbell, 2016 [3] the mechanical strength of solid aerated foods depends on the amount of air incorporated into the food matrix. in the same way, total mechanical work of compression was higher in the case of a3 and a4 aerated samples, while for a1 and a2 samples presented smaller values. the mechanical work of elastic deformation (we) represents the recoverable energy invested in the sample deformation [19] and varies from 0.771 to 3.348 ·10-3 joules, the highest value being observed for a4 and the lowest for a1 sample. the modulus of deformability (ed) is a measure of food material stiffness [13] and it was calculated as the slope of the stress strain curves (figure 1 a), being associated with hardness texture parameter [20]. as the data shows, the modulus of deformability was higher (15.615 kpa) for aerated confectionery samples with high hardness. sample protein fat dry matter brix aw density specific volume [%] [%] [%] ºb [g/cm3] [cm3/g] a1 11.12a (0.70) 15.35c (0.40) 52.38d (0.20) 25.96a (0.28) 0.836a (0.20) 0.400c (0.02) 2.50a (0.15) a2 9.55b (0.35) 20.10a (0.52) 55.01c (0.24) 24.58b (0.22) 0.826a (0.15) 0.520b (0.05) 1.92b (0.17) a3 10.83a (0.53) 16.42b (0.35) 56.13b (0.19) 26.14a (0.20) 0.845a (0.17) 0.771a (0.07) 1.30c (0.12) a4 9.70b (0.40) 20.18a (0.50) 57.72a (0.28) 24.80b (0.50) 0.848a (0.21) 0.780a (0.06) 1.28c (0.10) f – ratio 6.66 59.30 285.82 22.68 0.13 32.65 56.25 p-value < 0.05 < 0.001 < 0.001 < 0.001 > 0.05 < 0.001 < 0.001 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 126 fig. 1. loading unloading (a) and stress relaxation (b) curves of aerated confectionery samples: a1 (), a2 (), a3 (), a4 () a) b) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 127 from table 2, it can be seen that the relaxation, r, and the relaxation work, wr, of the aerated confectionery samples were about 0.0049 0.0111 n/s and 233.5 607.8 n·s, which were smaller to those reported by gutt,2014 [14] for fresh meat (0.0113 – 0.044 n/s). the samples produced by second procedure (a3 and a4), which involved the incorporation of the raw materials one at a time in the beating vegetable or dairy cream presented a higher density associated with a less aerated structure and showed also a higher relaxation and higher relaxation work, respectively. table 2. texture parameters of aerated confectionery samples texture parameters / sample a1 a2 a3 a4 hardness h [n] 1.92 2.7 4.38 4.680 total mechanical work wt [·10-3 j] 11.56 10.37 23.28 19.830 elastic mechanical work we [·10-3 j] 0.771 1.703 2.053 3.348 modulus of deformability ed [kpa] 6.406 9.009 14.614 15.615 relaxation r [n/s] 0.0049 0.0068 0.0099 0.0111 relaxation work wr [n·s] 233.5 339.6 574.6 607.8 pearson correlation. table 3 shows the pearson correlation matrix between physico-chemical properties and stress – relaxation texture parameters of confectionery samples. as regarding the physico-chemical properties a negative pearson correlation was recorded between fat and protein content (r = -0.996**) and also between fat content and brix concentration (r = -0.956*). the specific volume of the aerated samples was negatively correlated with dry matter (r = 0.956*) and density (r = -0.988**). another correlation was observed between dry matter and density (0.931*). according to pearson correlation matrix the stressrelaxation texture parameters are highly positively connected between them (hardness with modulus of deformability, relaxation and relaxation work; elastic mechanical work being correlated with relaxation). the samples’ hardness was highly positively influenced by the dry matter content (0.943*) and density (0.997**); agreeing with fontana, 2005 [17], while the specific volume had a negative effect on this texture parameter (r = -0.982**). furthermore, the dry matter content positively influenced the we, ed, r and wr values and density was correlated with ed, r, wr and wt. another pearson correlation was recorded between specific volume and ed, r and wr stressrelaxation texture parameter. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 128 t a b le 3 . p e a r so n c o r r e la ti o n b e tw e e n p h y si c o -c h e m ic a l a n d t e x tu r e p a r a m e te r . * . c o rr e la ti o n i s si g n if ic a n t a t th e 0 .0 5 l e v e l (1 -t a il e d ). * * . c o rr e la ti o n i s si g n if ic a n t a t th e 0 .0 1 l e v e l (1 -t a il e d ). p ro te in p , f a t -f , d ry m a tt e r d m , b ri x c o n c e n tr a ti o n b , w a te r a c ti v it y a w , d e n si ty d , s p e c if ic v o lu m e s v , h a rd n e ss h , t o ta l m e c h a n ic a l w o rk w t, e la st ic m e c h a n ic a l w o rk w e , m o d u lu s o f d e fo rm a b il it y e d , r e la x a ti o n r , r e la x a ti o n w o rk w r. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 129 4. conclusion the analyzed aerated confectionery products presented both elastic and viscous behavior, respectively a viscoelastic solid behavior, with a decrease of load during time. the a2 and a4 samples based on dairy cream presented the highest fat content, while the highest soluble substances concentration was recorded by the vegetable cream aerated samples (a1 and a3); the anova factorial analysis showed a significant difference (p < 0.001). the stress -relaxation texture parameters of aerated confections were influenced by the amount of air fixed into the product structure (density) and also by dry matter content. as regarding the samples preparation the one step ingredients’ incorporation procedure introduced and fixed a larger amount of air, resulting in a decrease of density, a more fragile texture and also a decrease of relaxation and relaxation work. 5. references [1]. kulmyrzaev a., cancelliere c., and mcclementsd. j., characterization of aerated foods using ultrasonic reflectance spectroscopy. journal of food engineering, 46(4), 235-241, (2000). [2]. subramaniamp., the stability and shelf life of confectionery products. in the stability and shelf life of food (pp. 545-573). woodhead publishing,(2016). [3]. campbell g. m., aerated foods. in encyclopedia of food and health, elsevier inc, pp. 51-60 (2016). [4]. hartel r. w., joachim h., andhofberger, r., other ingredients. in confectionery science and technology (pp. 151-185). springer, cham,(2018). [5]. subramaniam p., andwareing p. (eds.). the stability and shelf life of food. woodhead publishing, (2016). [6]. getz c. a., smith g. f., tracy p. h.,prucha m. j., instant whipping of cream by aeration. journal of food science, 2(5), 409-428, (1937). [7]. van akeng. a., aeration of emulsions by whipping. colloids and surfaces a: physicochemical and engineering aspects, 190(3), 333-354,(2001). [8]. zimbru r.o.,pădureţs., amarieis., effect of aeration on physicochemical, color and texture characteristics of confectionery foams, ukrainian food journal, volume 9 issue 199110, (2020). [9]. sr en iso 8968-2:2002, determination of nitrogen content. block digestion method. [10]. sr iso 673-1996, milk, cream and evaporated milk. determination of total solids content. [11]. aoac international, official methods of analysis, 20th edn, aoac international, rockville, md, (2016). [12]. sereno a. m., silva m. a.,mayor l., determination of particle density and porosity in foods and porous materials with high moisture content. international journal of food properties, 10(3), 455-469,(2007). [13]. bourne m., food texture and viscosity: concept and measurement. elsevier, pp. 1-324, (2002). [14]. gutt g., pădureţ s., amariei s., plesca, m., physical and texture parameters used in the analysis of meat freshness. journal of agroalimentary prosesses and technologies, 20(3), 257-262, (2014). [15]. han j., zhou x., cao j., wang y., sun b., li y.,zhang l., microstructural evolution of whipped cream in whipping process observed by confocal laser scanning microscopy. international journal of food properties, 21(1), 593-605, (2018). [16]. nardozza s., gamble j., axten l. g., wohlers m. w., clearwater m. j., feng j., harker f. r., dry matter content and fruit size affect flavour and texture of novel actinidia deliciosa genotypes. journal of the science of food and agriculture, 91(4), 742-748,(2011). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 2 – 2020 raluca olimpia zimbru, sergiu pădureţ, sonia amariei, aerated confectionery: physicochemical and textural evaluation, food and environment safety, volume xix, issue 2 – 2020, pag. 122 130 130 [17]. fontanaa., water activity for predicting quality and shelf life. manufacturing confectioner, 85(11), 45,(2005). [18]. campbell g. m.,mougeot e., creation and characterisation of aerated food products. trends in food science & technology, 10(9), 283-296,(1999). [19]. barbosa-cánovas g. v., (ed.),food engineering-volume ii. eolss publications,(2009). [20]. serna cock l., torres valenzuela, l. s.,ayala aponte a., changes in mechanical properties of minimally-processed yellow pitahaya treated with 1-mcp. dyna, 79(174), 71-78,(2012). 1. introduction 4. conclusion 199 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 3 2020, pag. 199 209 impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8 edwige larissa koffi 1 , *thierry yapo monnet 1 , kouassi hubert konan 1 , n’guessan jean parfait eugène kouadio 1 1 laboratoire de biocatalyse et des bioprocédés, université nangui abrogoua, 22 bp 801 abidjan 22, côte d’ivoire, monnet_tieri@yahoo.ca, *corresponding author received 2nd june 2020, accepted 14th september 2020 abstract: the purpose of this paper has been to study the antioxidant activities and phytochemical compounds at different stages of maturity from papaya (carica papaya l. var solo 8) peel, pulp and seeds in order to understand the correlation throughout these parameters. thereby, three parts of papaya such as peel, pulp and seeds were sampled fresh, dried in an oven at 45 °c for 48 hours, ground and analyzed according to standard procedures. carica papaya l. var solo 8 peels, seeds and pulps were specifically rich in phytochemical content around 1/8 and ¼ advanced stage of maturity before decreasing at advanced maturity stage. results showed high level of total phenolic (434.36 ± 0.10 mg (gae)/100 g dw), flavonoids (23.74 ± 0.01 mg (qe)/100 g dw) and tannins (89.33 ± 0.06 mg (tae)/100 g dw) respectively in seeds and peels. the dpph, frap and abts assays of methanolic extracts from this papaya (carica papaya l. var solo 8) showed that the antioxidant activity measured was high for the peels followed by pulp and seeds at immature stage. these data indicated that the whole parts of this papaya (carica papaya l. var solo 8) could constitute a potential good source of natural antioxidant for local population. keywords: antioxidant activity, papaya variety solo 8, phytochemical composition, maturity stage 1. introduction papaya is compared with other fruit such as the banana or the apple, it has been found a good natural source of macronutrients (carbohydrates and proteins) and macronutrients (vitamin a and vitamin c) 1. there is also a strong relationship between the intake of these antioxidant-containing plants and reduced mortality caused by diseases 2. indeed, natural antioxidants warrant further scientific scrutiny given their activity against free radicals, which contribute to chronic degenerative diseases 3. medicinal plants play important roles in preventing various diseases and have received much attention from many researchers over the last few decades. studies on the antioxidant contents of fruits and vegetables are increasing because natural antioxidant consumption has been found to be related to decreased risk for cancer and heart diseases 4. papaya contains a broad spectrum of phytochemicals including enzymes (in the latex), carotenoids (in fruits and seeds), alkaloids (in leaves), phenolics (in fruits, leaves, and shoots), and glucosinolates (in seeds and fruits). http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 200 during maturation, several variations (biochemical, physiological, and structural) take place and determine the fruit’s quality 5. to our knowledge there are no reports in one study about the influence of maturity stages on phytochemical content and antioxidant activity from different parts of c. papaya l. var solo 8. so, the goal of this work was to study the differences in amount and composition of phenolic compounds, total carotenoids, vitamin c and to estimate the antioxidant activities of c. papaya l. var solo 8 fruit harvested in côte d’ivoire with respect to different stages of maturity (immature, 1/8 advanced, 1/4 advanced and advanced) and fruit parts (peel, pulp and seeds). the result of this study will form the basis of advising consumers and the biological world, when and how best to utilize this fruit. 2. material and methods biological material biological material is composed of the peel, epicarp and seeds of harvested carica papaya l. var solo 8 at four stages of maturity which are immature, 1/8 advanced, 1/4 advanced and advanced yellow skin (figures 1 to 3). these stages follow those proposed by yao 6 to investigate pectinolytic activities. the fruits were harvested from a farm near thomasset (azaguié), a village located at about 50 km north of felix houphouet boigny airport, abidjan, cote d’ivoire. immature 1/8 advanced 1/4 advanced advanced fig. 1. carica papaya l. var solo 8 peels at different stages of maturity immature 1/8 advanced 1/4 advanced advanced fig. 2. carica papaya l. var solo 8 pulps at different stages of maturity immature 1/8 advanced 1/4 advanced advanced fig. 3. carica papaya l. var solo 8 seeds at different stages of maturity food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 201 methods collection and sampling from different parts of papaya the sampled papaya fruits were previously washed and rinsed with bleach and separated according to the stage of maturity. the peel, pulp and seeds were then separated from each other using a knife, peeler, and spatula. a quantity of 500 g of each part of the papaya (peel, pulp, and seeds) at the various stages of ripeness was spread out on aluminium film and then dried in a ventilated memmert oven at 45 °c for 2 days. after drying, the parts were crushed in a mill type mill ika (germany/deutschland). the resulting grind of each sample formed the papaya powder (flour) used at different analyses. these grindings were then packaged in glass bottles which were previously dried in an oven at 45 °c and then stored in a desiccator. phytochemical composition extraction of phenolic compounds extraction of phenolic compounds was determined using singleton et al. method 7. a sample (10 g) of fine dried papaya sample flour was extracted by stirring with 50 ml of methanol 80 % (v/v) at 25 °c for 24 hours and filtered through whatman n°4 paper. the residue was then extracted with two additional 50 ml portions of methanol. the combined methanolic extracts were evaporated at 35 °c (rotary evaporator heildolph laborata 4003 control, schwabach, germany) until 25 ml, prior to phenolic compound contents determination. determination of total phenolic compounds content contents of total phenolic compounds were estimated according to the folinciocalteu method 7. a volume of 1 ml of methanolic extract of each sample was added to 1 ml of folin-ciocalteu solution in a test tube. after 3 minutes, 1 ml of 20 % sodium carbonate solution was added to the mixture and adjusted to 10 ml with distilled water. the mixture was let to stand at room temperature in a dark environment for 30 min. absorbance was measured against the blank reagent at 725 nm. gallic acid was used for the calibration curve with a concentration range of 50 1000 μg/ml. results were expressed as mg gallic acid equivalent (gae)/100g dw (dry weight). determination of tannins: tannin content was determined using the method described by bainbridge et al. 8. a volume of 1 ml of each methanolic extract was collected and mixed with 5 ml of reaction solution [vanillin 0.1mg/ml in sulphuric acid 70 % (v/v)]. the mixture could stand at room temperature in a dark environment for 20 min. the absorbance was measured at 500 nm against a blank (without extract). tannic acid was used for the calibration curve with a concentration range of 0 100 μg/ml. the results were expressed as mg of tannic acid equivalents (tae)/100 g dw. determination of total flavonoid content the total flavonoids content was determined using the dowd method 9. 5 ml of 2 % aluminum trichloride (alcl3) in methanol was mixed with the same volume of the methanolic extract solution (0.4 mg/ml). after ten minutes the absorbance was measured at 415 nm using perkinelmer uv-vis lambda. blank sample consisting of a 5 ml extract solution with 5 ml methanol without alcl3. the total flavonoid content was determined using a standard curve with catechin (0 – 100 mg/l) as the standard. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 202 total flavonoids content is expressed as mg of catechin equivalents (ce)/100 g dw. determination of total carotenoid content (tcc) and estimation of vitamin c the tcc and vitamin c was determined using the spectrophotometric method as described by these authors 10. optical density was measured at 450 nm and 491 nm respectively in a spectrophotometer with the appropriate blank (pe:de: 2:1, v, v). the amount of carotenoid and vitamin c in mg/100 g of dry weight (dw) was calculated using lambertbeer equation (as described in rocheford’s lab protocol). antioxidant activities dpph assay the antioxidant activity of the extracts was evaluated by dpph radical scavenging assay which was described by 11. dpph is solubilized in absolute ethanol to have a solution of 0.3 mg/ml. different ranges of concentrations in the order of the microgram of each extract are prepared (2 mg/ml, 1 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml). absolute ethanol was used as the dilution solvent. in dry, sterile tubes, 2.5 ml of extract solution to be analyzed and 1 ml of dpph solution are added. after stirring, the tubes are placed in the dark for 30 min and the absorbance of the mixture is measured at 517 nm against a blank of 2.5 ml pure ethanol and 1 ml dpph solution. the percentage of inhibition of dpph radical formation is calculated as follows according to the formula: i = [(ab – as) / ab] × 100 with i, the percentage of inhibition, ab, the absorbency of blank as, the absorbance of the sample. abts assay the trolox equivalent antioxidant capacity (teac) test described in 11 was used to assay the antioxidant activities. the abts + radical cation was produced by mixing a solution of abts (8 mmol. l-1) and a solution of k2s2o8 (3 mmol. l-1) (1 / 0.5, v / v). the mixture was then incubated for 16 h in the dark at room temperature (25 °c). then, 0.1 ml (standard or extract) diluted in methanol (1/10, v / v), was added to 3.9 ml of the diluted abts solution. the mixture was vigorously vortexed for 2 min following by incubation for 6 min in the dark at room temperature (25 °c). the absorbance of the mixture was read in the jasco v-530 uvvisible spectrophotometer at 734 nm. the results were expressed in μmol. g-1 te (trolox equivalents). the percent degradation of abts by trolox was compared to that of the sample. percentage degradation a (%) of abts was expressed by using following mathematical formula, a(blank) = blank absorbance after incubation. a(extract) = extract absorbance at 734 nm after incubation frap assay reducing power determination the ferric reducing capacity of extracts was investigated by using the potassium ferricyanide-ferric chloride method 11. briefly, 0.2 ml of each of the extracts at different concentrations, 2.5 ml of phosphate buffer (0.2 m, ph 6.6), and 2.5 ml of potassium ferricyanide k3fe(cn)6 (1 %) were mixed and incubated at 50 °c for 20 min, to reduce ferricyanide into ferrocyanide. the reaction was stopped by adding 2.5 ml of 10 % (w/v) trichloroacetic acid followed by centrifugation at 1000 rpm for 10 min. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 203 finally, 2.5 ml of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of fecl3 (0.1 %) and the absorbance was measured at 700 nm. statistical analysis all the chemical analyses and assays were performed in triplicate, unless otherwise indicated. the results were expressed as mean values • ± standard deviation (sd). analysis of variance (anova) followed by duncan’s test was performed to test the differences between means by using kyplot (version 2.0 beta 15, c1997-2001, koichi yoshioka) statistical software. 3. results and discussion phenolic contents estimation of polyphenol, flavonoids, and tannins phenolic compounds, as secondary metabolites, are a large group of molecules widely distributed in fruits and vegetables. they are considered the main actors for the antioxidant capacity of plants and have also many benefits for human health, as free radical scavenger. recently, universal importance is given to the identification of maturity stages with the best levels of polyphenols targeting increased functional properties of fruits and vegetables. but it is hard to estimate the real content of the phenolic compounds, due to the fact that the phenolic content of fruits and vegetables is largely influenced by many factors, such as biotic and abiotic stress, senescence, cultivar, tissue, harvesting time, post-harvest treatment and also extraction techniques 11. the maturity stage is an important factor that influences the compositional quality and the quantity of fruits. table 1 shows the content of estimated polyphenols, flavonoids, and tannins in different parts of carica papaya l. var solo 8 fruits harvested at several stage of maturity. polyphenols: the levels of total phenolic content (tpc) in the evaluated parts of the papaya fruits varied significantly from 149.85 to 434.36 mg gae / 100 g of dry weight (dw). the seeds contained the highest phenolic content (434.36 ± 0.10), followed by the peel (350.34 ± 0.68) and the pulp (254.41 ± 0.04) at immature to 1/8 advanced stage. obviously, seeds have higher content at the stage of ¼ advanced maturity followed by peel and pulp with a peak at 1/8 and ¼ of advanced maturity, respectively. decrease is observed for all the parts of fruits at advanced maturity stage. nonetheless, in comparison with several studies, carica papaya linn var. solo 8 seemed to have the highest level of total phenolic compound (12; 13 ). these high contents of phenolic compounds found in papaya (carica papaya l. var solo 8) could constitute interesting data for population nutrition since it is well-known that these bioactive compounds found in human diet act as the antioxidant compounds and play a role in stabilizing lipid peroxidation 14. flavonoids: the peel and seeds have important level respectively at 1/8 advanced maturity and immature stage, followed by pulp at ¼ advanced maturity one. accumulation of flavonoids in investigated parts from papaya fruits ranged from 9.46 to 23.74 mg (qe) / 100 g dw. a high level of flavonoids was determined in the peel accounting for 23.74 ± 0.01, followed by 23.28 ± 0.01 and 16.61 ± 0.05 mg (qe) / 100 g dw in the seeds and the pulp, respectively. the lowest flavonoid accumulation was determined for the pulp (9.46 ± 0.02 mg (qe) / 100 g dw) at immature stage. however, no significant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 204 difference was observed at risk (p < 0.05) between peel and seeds flavonoids content at 1/4 advanced maturity. considering these data, consuming pulp at immature stage is not interesting due to the known beneficts of flavonoids. fortunately, people consume papaya pulp beyond the immature stage. it is well known that flavonoids endow a wide range of pharmacological and biochemical properties, such as antimicrobial activities, anti-inflammatory, and inhibition of platelet aggregation 14. there was a significant variation in the accumulation of total flavonoids. tannin: the condensed tannin amount determined was significantly different through the maturity stages besides the investigated parts in the fruits of carica papaya l. var solo 8. high level was observed for the peel (89.33 ± 0.06 at ¼ advanced maturity) and the seeds (87.92 ± 0.02 at immature stage) with no significant difference at the level p < 0.05. tannin content of the peel remains interesting during the maturation process (immature to advanced mature) accounting from 75.05 to 56.70 mg (tae) / 100 g dw. whereas the peel, pulp and seeds have lower tannin levels at ¼ and advanced maturity stage with 18.32 to 13.09 and 17.73 mg (tae) / 100 g dw, respectively. indeed, regardless of fruit parts and maturity stage, our results confirmed that fruits of c. papaya were an excellent source of phenolic compounds when comparing to other varieties, maradol variety 15. table 1 polyphenol, flavonoid and tannin compounds in c. papaya l. var solo 8 parameters analyzed parts of the fruits stage of maturity immature 1/8 advanced 1/4 advanced advanced polyphenols peel 235 ± 1.01d 350.34 ± 0.68g 266.74 ± 3.24e 261.23 ± 1.15e pulp 149.85 ± 0.75a 203.03 ± 0.48b 254.41 ± 0.04e 218.74 ± 0.42c seeds 211.73 ± 0.11c 240.44 ± 1.51d 434.36 ± 0.10h 339.2 ± 0.23f flavonoids peel 19.79 ± 0.05d 23.74 ± 0.01f 21.62 ± 0.01e 19.18 ± 0.03d pulp 9.46 ± 0.02a 16.32 ± 0.02c 16.61 ± 0.05c 12.29 ± 0.01b seeds 23.28 ± 0.01f 18.93 ± 0.01d 21.57 ± 0.02e 19.82 ± 0.05d tannins peel 75.05 ± 0.03g 79.70 ± 0.17g 89.33 ± 0.06h 56.70 ± 0.05d pulp 25.98 ± 0.07c 68.61 ± 0.02f 18.32 ± 0.01b 13.09 ± 0.01a seeds 87.92 ± 0.02h 69.29 ± 0.01f 62.29 ± 0.01e 17.73 ± 0.02b values are expressed as mean ± standard deviation (n = 3). means with different letters within a column were significantly different at the level p < 0.05. total carotenoids and vitamin c contents estimation of total carotenoid and vitamin c c. papaya l. var solo 8 is a red-fleshed papaya. during fruit maturity process, the most visible change of papaya is the color of exocarp turning from green to yellow whilst the pulp and seeds changing from food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 205 white to orange red and black (figures 1 to 3). the content of major carotenoids was determined during the ripening of papaya fruit (table 2). a large amount of carotenoid, as well as a small amount of lycopene was detected in the peel. the highest content was observed in the peel (93.50 ± 0.41 mg /100 g dw) and the pulp (91.50 ± 0.64 mg /100 g dw) followed by the seeds (72.49 ± 0.61 mg /100 g dw) at ¼ , immature and 1/8 advanced stage of maturity, respectively. an important content of ß-carotene, as well as small amount of lycopene were detected in the pulp at advanced stage of maturity. the content of lycopene increased, while and the content of ß-carotene decreased in the peel during papaya ripening. the highest content was 8.03 ± 0.27 mg /100 g dw for the pulp at ¼ advanced stage of maturity whereas the lowest amount occurred at advanced stage of maturity with the seeds accounting 0.03 ± 0.03 mg /100 g dw. the results were in contrast with those obtained by the variety maradol by some authors 16, 17, 18. indeed, ß-carotene accumulation from c. papaya l. var solo 8 is still higher than lycopene, which content remained important 19. the positive health benefits of lycopene contained in different fruits and vegetables have been widely reported, including reduction of cardiovascular problems 20. therefore, the possible benefits of papaya consumption fruit could be compared to those reported by other vegetables rich in lycopene such as tomatoes. however, the concentration of other phytochemical present in these products that contribute to health needs is to be considered. vitamin c, measured by spectrophometer at 491 nm, both in the peel, the pulp and the seeds was higher in peel than in the pulp at ¼ advanced maturity stage, and seeds at 1/8 advanced maturity (table 2). significant differences (p ≤ 0.05) were found in vitamin c between different parts. the largest amount of vitamin c in the peel was 22.5 ± 0.04 mg/100 g dw (at ¼ advanced maturity) and it remained stable. however, pulp had lower vitamin c values with 07.50 ± 0.02mg/100 g dw (at 1/8 advanced maturity satge) papaya fruit. in previous reports, papaya vitamin c content was higher than in this study and ranged from 60 to 84 mg/100 g 21. solo type (kapoho or sunrise) papayas obtained from retail markets were analyzed in those studies. the content of vitamin c could vary, mainly because of the type of fruit cultivation, type of soil, weather and level of fruit ripeness 21. vitamin c or ascorbic acid is a powerful hydrosoluble antioxidant that protects body against oxidative stress, due to its ability of trapping hydroxyl and superoxide radicals. in addition, a regular daily intake of vitamin c ranged from 250–500 mg reduces oxidative damage by removing free radicals 22 antioxidant activities in this study, three complementary tests were used to assess the antioxidant activity of carica papaya l. var solo 8 from different fruit parts harvested at several stages of maturity: dpph free radicalscavenging activity, trolox equivalent antioxidant capacity (teac) and reducing power assays. antioxidant activity is a complex procedure usually happening through several mechanisms and is influenced by many factors, which cannot be fully described with one single method. therefore, it is essential to perform more than one type of antioxidant capacity measurement to consider the various mechanisms of antioxidant action [11; 12]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 206 estimation of dpph as summarized in table 3, the rank order of antioxidant potency was the same for all assays, namely stage, in decreasing order, immature, followed by 1/8 advanced, ¼ advanced, and advanced stage of maturity. all the extracts were able to reduce the stable, purple coloured radical dpph into yellow-coloured dpph-h. the water extract obtained from seeds at a concentration of 2 mg/ml exhibited a percentage inhibition of 90.03 ± 0.45 % at ¼ advanced stage of maturity. on the other hand, the lowest capacity to reduce dpph was observed in pulp water extract (i = 23.01 ± 0.44 %) by immature stage. table 2 total carotenoid and vitamin c compound in c. papaya l. var solo 8 values are expressed as mean ± standard deviation (n = 3). means with different letters within a column were significantly different at the level p < 0.05. table 3 effet of maturity stage on antioxidant measured by dpph assays from different parts of c. papaya l. var solo 8 stages of maturity evaluated parts of fruits dpph c 0.0625 c 0.125 c 0.25 c 0.5 c 1 c 2 immature peel 31.52 ± 0.66c 36.43 ± 0.63e 44.06 ± 0.88i 49.10 ± 0.57k 65.25 ± 0.45m 84.01 ± 0.93p pulp 23.01 ± 0.44a 31.64 ± 0.33d 39.22 ± 0.49f 42.33 ± 0.38h 60.17 ± 0.48n 81.05 ± 0.61o seeds 29.29 ± 0.68b 34.26 ± 0.74d 41.09 ± 0.14g 45.22 ± 0.43 j 62.01 ± 0.97 n 82.53 ± 0.55o 1/8 advanced peel 38.22 ± 0.61d 41.1 ± 0.37e 49.08 ± 0.32h 53.72 ± 0.39i 69.25 ± 0.28k 85.66 ± 0.58n pulp 36.16 ± 0.83c 39.26 ± 0.83d 45.17 ± 0.53f 49.26 ± 0.66h 54.11 ± 0.57i 72.24 ± 0.49l seeds 31.63 ± 0.57a 35.07 ± 0.95b 46.01 ± 0.89g 46.29 ± 0.78g 64.21 ± 0.76j 80.24 ± 0.52m 1/4 advanced peel 35.45 ± 0.35b 37.74 ± 0.49c 41.26 ± 0.75e 47.18 ± 0.34g 50.02 ± 0.63i 60.21 ± 0.89k pulp 35.47 ± 0.71b 39.32 ± 0.76d 41.44 ± 0.98e 46.74 ± 0.64g 50.01 ± 0.34i 55.17 ± 0.51j seeds 34.19 ± 0.51a 39.04 ± 0.61d 48.09 ± 0.45h 50.03 ± 0.59i 68.29 ± 0.65l 90.03 ± 0.45m advanced peel 28.01 ± 0.58a 30.56 ± 0.36b 40.61 ± 0.59f 45.29 ± 0.48i 48.76 ± 0.44k 58.06 ± 0.75o pulp 35.47 ± 0.71d 39.32 ± 0.76e 41.44 ± 0.98g 46.74 ± 0.64j 50.01 ± 0.34k 55.17 ± 0.51n seeds 32.28 ± 0.40c 36.11 ± 0.31i 39.88 ± 0.66e 43.21 ± 0.14h 48.68 ± 0.61k 52.24 ± 0.48m trolox 45.43 ± 0.66a 59.76 ± 0.11b 69.43 ± 0.27c 82.01 ± 0.19d 85.08 ± 0.51e 92.29 ± 0.41f tests: n=3; means ± standard deviation with different lowercase letters on the same line are significantly different at p<0.0 5 in duncan's test. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 207 table 4 effet of maturity stage on antioxidant measured by abts assays from different parts of c. papaya l. var solo 8 stages of maturity evaluate d parts of fruits abts c 0.0625 c 0.125 c 0.25 c 0.5 c 1 c 2 immatur e peel 44.62 ± 0.58c 49.34 ± 0.38e 56.03 ± 0.92h 61.9 ± 0.74i 64.19 ± 0.94j 70.22 ± 0.46k pulp 41.24 ± 0.16b 47.01 ± 0.47d 49.36 ± 0.28e 54.29± 0.28g 60.74 ± 0.45i 61.02 ± 0.18i seeds 40.97 ± 0.29a 44.19 ± 0.98c 46.21 ± 0.11d 51.11 ± 0.24f 57.62 ± 0.49h 60.17 ± 0.90i 1/8 advance d peel 47.21 ± 0.80c 49.64 ± 0.51e 54.33 ± 0.92g 59.88 ± 0.17j 63.74 ±0.54m 66.14 ± 0.99o pulp 45.41 ± 0.91b 49.35 ± 0.77e 51.86 ± 0.26f 56.67 ± 0.42h 61.59 ± 0.80l 64.28 ± 0.23n seeds 44.82 ± 0.85a 48.23 ± 0.76d 54.18 ± 0.14g 57.71 ± 0.46i 60.07 ± 0.30k 61.47 ± 0.31l 1/4 advance d peel 46.90 ± 0.14a 47.67 ± 0.34b 49.53 ± 0.43e 53.92 ± 0.12f 57.09 ± 0.89i 61.78 ± 0.26l pulp 46.67 ± 0.32a 49.09 ± 0.19c 53.21 ± 0.84f 57.10 ± 0.13i 58.41 ± 0.27j 60.01 ± 0.20k seeds 47.59 ± 0.56b 48.54 ± 0.42c 54.02 ± 0.24g 55.61 ± 0.42h 57.29 ± 0.15i 58.94 ± 0.13j advance d peel 49.75 ± 0.47f 47.19 ± 0.27d 49.21 ± 0.26f 54.39 ± 0.25j 57.43 ± 0.26k 59.07 ± 0.26l pulp 44.82 ± 0.39b 46.66 ± 0.28c 48.64 ± 0.36e 50.42 ± 0.59g 52.61 ± 0.37i 54.18 ± 0.81j seeds 43.56 ± 0.50a 46.15 ± 0.20c 48.38 ± 0.27e 50.02 ± 0.11g 51.84 ± 0.29h 52.24 ± 0.27h trolox 75.15 ± 0.05 a 79.76 ± 0.09b 84.56 ± 0.13c 89.10 ± 0.19d 90.21 ± 0.21e 92.08 ± 0.17e tests: n=3; means ± standard deviation with different lowercase letters on the same line are significantly different at p<0.05 in duncan's test. estimation of abts the method described gives a measure of the antioxidant activity of the range of peel, pulp and seeds at different stages of maturity, fruit extract antioxidants, determined by the decolorization of the abts, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm. the results given in the table 4 remain like dpph inhibition. peel had the highest level of abts antioxidant (70.22 ± 0.46 %) at immature stage. infact the used methods have different reaction mechanisms 23. for instance, dpph and abts assays are based on electron and h atom transfer, while the frap assay is based on electron transfer reaction 23; 24. however, the three methods clearly indicated that the studied plants possess considerable antioxidant and antiradical activities. furthermore, well-pronounced correlations were observed between these methods which confirm that the three assays were all suitable and reliable to assess the total antioxidant capacities of plant extracts. estimation of frap as depicted in table 5, immature stage of fruits showed the highest radical te values. at all extract concentrations, peel had the highest te value where it ranks a percentage inhibition of 87 ± 7.79 %. the lowest value of 28.60 ± 1.60 % was shown by seeds. frap assay measures the tendency of an antioxidant which acts as a reducing agent and brings a single electron to the fe3+ in a redox-linked colorimetric reaction based on the breaking of the free-radical chain in order to stabilize and finish the radical chain reactions 24. this will cause an increase in absorbance; however, it is limited to only hydrophilic antioxidants or water-soluble antioxidants 25. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 208 table 5 effet of maturity stage on antioxidant measured by frap assays from different parts of c. papaya l. var solo 8 stages of maturity evalua ted parts of fruits frap c 0.0625 c 0.125 c 0.25 c 0.5 c 1 c 2 immature peel 42.00 ± 1.40f 49.00 ± 3.40h 54.20 ± 2.03j 60.80 ± 4.13k 79.60 ± 2.36m 87.00 ± 7.79n pulp 32.00 ± 0.03b 36.40 ± 2.49c 40.20 ± 1.11e 53.20 ± 5.33i 60.40 ± 3.67k 60.80 ± 6.69k seeds 28.60 ± 1.60a 33.80 ± 5.07b 36.60 ± 3.55c 48.20 ± 5.18g 60.60 ± 2.43k 64.60 ±1.70l 1/8 advanced peel 38.80 ± 0.12c 44.80 ± 3.94e 49.60 ± 2.83g 59.40 ± 4.39h 66.20 ± 2.80i 73.40 ±5.19l pulp 38.60 ± 2.20c 38.00 ± 2.30c 40.40 ± 3.36d 48.20 ± 5.29f 68.80 ± 5.29j 66.80 ± 3.8i seeds 34.00 ± 0.13a 35.40 ± 5.60b 36.60 ± 4.01b 40.60 ± 2.10d 69.40 ± 2.50k 74.40 ±4.10m 1/4 advanced peel 38.20 ± 2.80d 43.40 ± 4.93g 50.20 ± 6.82j 57.80 ± 2.50l 60.80 ± 3.22m 69.60 ±5.05p pulp 36.80 ± 4.19b 41.60 ± 3.33f 45.00 ± 5.44h 54.60 ± 4.52k 67.40 ± 7.27o 64.20 ±5.38n seeds 33.40 ± 5.60a 37.00 ± 3.12c 40.00 ± 3.89e 46.80 ± 6.29i 79.80 ± 9.34q 79.40 ±6.82q advanced peel 37.80 ± 3.82c 42.60 ± 3.90e 49.20 ± 7.07g 54.00 ± 5.24h 57.80 ± 8.77i 66.80 ± 6.88l pulp 35.60 ± 4.10b 39.40 ± 5.26d 46.80 ± 6.35f 49.00 ± 6.42g 63.60 ± 3.55k 62.20 ±7.03j seeds 32.20 ± 4.99a 35.00 ± 3.94b 38.40 ± 5.07d 41.80 ± 4.71e 64.20 ± 3.40k 51.60 ± 5.12g trolox 40.21 ± 0.09 a 55.43 ± 0.04b 70.55 ± 0.07c 81.78 ± 0.31d 85.77 ± 0.29e 90.13 ±0.52f 4. conclusion the results presented in this work clearly demonstrate that the amounts of phenolic compounds and the antioxidant capacities of papaya (carica papaya l. var solo 8) were affected by maturation stages. it was shown that peel has a notable antioxidant activity at immature stage and it is found to be a good source of antioxidants. these findings confirm the antioxidant potential of papaya (carica papaya l. var solo 8) and increase focus on the impact on health promoting antioxidative compounds in all parts of the fruit during the four maturation stages. the papaya (carica papaya l. var solo 8) peel flour could be used as a potential source for functional food ingredients and, in addition, it could be further processed into therapeutic functional food products, at immature stage. however, pulp and seeds are good for consumers from 1/8 to ¼ of advanced maturation. 5. references [1]. peterson r.n., cherry j.p. simmons j.g., composition of pawpaw (asimina triloba) fruit. northern nut growers association annual report., 73: 97-107, (1982) [2]. halliwell b., gutteridge j.m., cross c.e., free radicals, antioxidants, and human disease: where are we now? j. lab. clin. med., 119(6) : 598, (1992) [3]. bray t.m., dietary antioxidants and assessment of oxidative stress. nutrition, 16(7-8): 578, (2000) [4]. temple n.j., antioxidants, and disease: more questions than answers. nutr. res., 20(3): 449-459 (2000) [5]. siddiqui md.w., momin c.m., acharya p., kabir j., debnath m.k., dhua r.s., dynamics of changes in bioactive molecules and antioxidant potential of capsicum chinense jacq. cv. habanero at nine maturity stages. acta physiologiae plantarum, 35, 1141– 1148, (2013) [6]. yao n.b., conservation de la papaye (carica papaya l. var solo 8) par le contrôle du niveau de maturité à la récolte et des activités biochimiques. thèse de doctorat: biochimie des aliments. université nangui abrogoua, côte d’ivoire, p 188, (2013) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 3 – 2020 edwige larissa koffi , thierry yapo monnet, kouassi hubert konan, n’guessan jean parfait eugène kouadio, impact of maturity stage on antioxidant activity and phytochemical properties of different parts from pawpaw: carica papaya l. var solo 8, food and environment safety, volume xix, issue 3 – 2020, pag. 199 209 209 [7]. singleton v.l., orthofer r., lamuela-raventos r.m., analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. methods enzymol, 299: 152-178, (1999) [8]. bainbridge z., tomlins k., willings k., westby a., methods for assessing quality characteristics of non-grain starch staple. part 4 advanced methods. national resources institute, university of greenwich, uk isbn 085954-400-, 43-79, (1996) [9]. meda a., lamien c.e., romito m., millogo j., nacoulma o.g., determination of the total phenolic, flavonoid and proline contents in burkina fasan honey, as well as their radical scavenging activity. food chem, 91: 571-577, (2005) [10]. desai a.p., desai s., uv spectroscopic method for determination of vitamin c (ascorbic acid) content in different fruits in south gujarat region. int j environ sci nat res. 21(1): 556055, (2019) [11]. tlili n., mejri h., nasri n., yahia y., saadaoui e., rejeb s., khaldi a., phytochemicals and antioxidant activities of rhus tripartitum (ucria) fruits depending on locality and different stages of maturity. food chem 160 98– 103, (2014) [12]. dada f.a., nzewuji f.o., esan a.m., oyeleye s.i., adegbola v.b., phytochemical and antioxidant analysis of aqueous extracts of unripe pawpaw (carica papaya linn.) fruit’s peel and seed, ijrras, 27 (3): 68-71, (2016) [13]. maisarah a.m, asmah r., fauziah o., proximate analysis, antioxidant and antiproliferative activities of different parts of carica papaya j nutr food sci, 4:2, (2014) [14]. adingra k.m-d., konan k.h., kouadio e.j.p., tano k., phytochemical properties and proximate composition of papaya (carica papaya l. var solo 8) peels, turkish journal of agriculture, food science and technology, 5(6): 676-680, (2017) [15]. adetuyi f.o., iroaye a.o., babatunde o.m., effiong g.j., the influence of storage of pawpaw carica papaya fruit on the bioactive components, antioxidative properties, and inhibition of fe2+ induced lipid peroxidation of water-extracts of pawpaw seed. pak. j. sci. ind. res. ser. b: biol. sci. 59(3) 146-154 (2016) [16]. shen y.h., yang f.y., lu b.g., zhao w.w., jiang t., feng l., chen x.j., ming r., exploring the differential mechanisms of carotenoid biosynthesis in the yellow peel and red flesh of papaya bmc genomics 20:49, (2019) [17]. rivera-pastrana d.m., yahiab e.m., gonz´alez-aguilara g.a., phenolic and carotenoid profiles of papaya fruit (carica papaya l.) and their contents under low temperature storage. j sci food agric 90:2358– 2365, (2010) [18]. gayosso-garcía sancho l.e., yahia e.m., gonzález-aguilar g.a., identification and quantification of phenols, carotenoids, and vitamin c from papaya (carica papaya l., cv. maradol) fruit determined by hplcdad-ms/ms-esi, food research international 44: 1284–1291, (2011) [19]. kimura m., rodriguez-amaya d.b., yokoyama s.m., cultivar differences and geographic effects on the carotenoid composition and vitamin a value of papaya. lebensmittelwissenschaft & technologie 25, 415–418, (1991) [20]. somanah j., bourdon e., bahorun t., extracts of mauritian carica papaya (var. solo) protect sw872 and hepg2 cells against hydrogen peroxide induced oxidative stress j food sci technol 54(7):1917–1927, (2017) [21]. wall m.m., ascorbic acid, vitamin a, and mineral composition of banana (musa sp.) and papaya (carica papaya) cultivars grown in hawaii, journal of food composition and analysis 19: 434–445 (2006) [22]. tariq s.a., role of ascorbic acid in scavenging free radicals and lead toxicity from biosystems. molecular biotechnology, 37,62-65, (2007) [23]. prior r.l., wu x., schaich k., “standardized meathods for the determination of antioxidants capacity and phenolics in food and dietary supplements,” j. agric. food chem., vol. 43, pp. 401– 403, (2005) [24]. huang d., ou b., prior r.l., “the chemistry behind antioxidant capacity assays,” journal of agricultural and food chemistry, vol. 53, no. 6, pp. 1841–1856, (2005) [25]. tyug t.s., prasad k.n., ismail a., “antioxidant capacity, phenolics and isoflavones in soybean byproducts,” food chem., vol. 123, pp. 583–589, (2010) 1. introduction 4. conclusion 316 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 316 322 comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production *godfrey onuwa 1 , anthony mgbojikwe 1 , bassey emmanuel 2 , kingsley okeke-agulu 2 and chizoba chizea 3 1department of agricultural extension and management, federal college of forestry, jos, plateau state, nigeria. onuwag@gmail.com 2montanne research station, forestry research institute of nigeria, jos, plateau state, nigeria. 3department of agricultural technology, federal college of forestry, jos, plateau state, nigeria. *corresponding author received 17nd july 2020, accepted 29th december 2020 abstract: the purpose of this paper is to analyze the potentials of soybean and sunflower feed rations on the growth parameters of broilers. the study was carried out at the department of agricultural extension and management, federal college of forestry, jos, plateau state. one hundred (100) day old broiler chicks were purchased and divided into two (2) groups (a and b) with three (3) replicates in each group. group a were fed with soybean feed ration, while group b was fed with sunflower feed ration over a period of eight (8) weeks. descriptive, proximate analysis and statistical ratios were used in analyzing data for the study. feed a is composed of soybean meal (55%), groundnut cake (40%) and yellow maize (15%), while feed b is composed of sunflower (55%) groundnut cake (40%) and yellow maize (15%). the result of the proximate analysis indicated that each feed ingredients contained crude protein, crude fiber, ash and fat in significant quantities. the survival rate for the duration of the feeding trials for treatment b was significant at 97%. the estimate of daily weight gain was significant for treatment b from week 1 to week 4 and week 5 to week 8 with an index of 0.205 and 0.537 respectively. also, the feed conversion ratio for treatment b was significant. while the protein efficiency ratio was significant for treatment a. the implication of these findings suggests that sunflower was a very palatable poultry feed ration, with significant effects on the growth parameters of broilers. keywords: feed conversion ratio, feed intake, survival rate, weight gain. 1. introduction the history of poultry feeding is one that has undergone drastic changes, particularly in nigeria. in time past poultry birds were left to scavenge on the leftovers from the farms, kitchen and table and their production capacities were low. the output of these birds in terms of meat and egg production was low due largely to inadequate nutrition [1]. to improve productivity poultry birds require adequate feed and nutrients. the rapid development and application of technology in the area of poultry production have revolutionized and transformed it into a major occupation [2]. a good quality feed is one that is formulated to provide the appreciable levels of nutrients for the poultry birds to which it will be fed. it must be formulated bearing in mind the age and type of bird and also the purpose of production [3]. however, feeding of poultry commercially is complicated since unlike ruminants they require all the essential amino acids and vitamins of the b-complex group besides the other organic and inorganic nutrients. feed being a primary expenditure in poultry production, represents 60%-65% of the absolute cost of production. poultry feeds are frequently affected by price changes and supply deficits which further http://www.fia.usv.ro/fiajournal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 317 constitute constraints in broiler production soybean and groundnut cake have traditionally been the main protein source in the formulation of poultry feed. this however has continued to increase in price and reduced in availability having a resultant increase in the cost of production. this can be attributed to poor harvest, competition between man and animal and increased cost of producing these crops therefore, necessitating the need to look into other ways or other protein sources that can be used to formulate feed, notably the use of sunflower meal. therefore, this study was undertaken to assess potentials of sunflower meal as an alternative protein source for feed formulations in broiler production. 2. matherials and methods 2.1. study area the study was carried out at the department of agricultural extension and management, federal college of forestry, jos north local government area of plateau state, nigeria. it is situated on latitude 9o50 and 10o05”n and longitude 8050” and 8055e, it covers a land area of about 3,365 square kilometers [4]. jos has an altitude of about 1,200 meters above sea level and average annual rainfall ranges from 870mm to 1,55mm. monthly average temperature ranges between 17oc minimum and 38oc maximum [4]. 2.2. analytical procedure and techniques the sunflower used in ratio formulation was procured from zaria, kaduna state, while the other materials including the soybean cake, groundnut cake, maize, bone meal, salt, additives and cereal offal were obtained from markets in jos metropolis. the actual feed formulation was carried out at the premises of the federal college of forestry, jos. descriptive, proximate analysis and statistical ratios were used in analyzing data for the study. the proximate composition of feeds includes crude protein (cp), crude fiber (cf), ash and amino acid (fat) contents in the rations. these components may be of interest for product development, quality control (qc) or regulatory purposes. proximate analysis stands for a method, which determines the values of the macronutrients in feed samples. in general, those values are being declared as nutritional facts shown usually on the labels of the final (end) food products, but they are also being determined during the production process. the primary objectives of proximate analyses are to measure the amount of each individual component in the feedstuff. the components measured are the nutrients. depending on the specific analysis, the analysis will measure the total amount of the nutrient class or the amount of a specific nutrient within the nutrient class. for example, one analysis will measure the total amount of protein and another analysis will measure the individual amino acids in the feedstuff. the laboratory-based estimations provide an accurate measure of nutrient quantity. however, the laboratorybased estimations do not provide nutrient utilization information. the initial procedures used to estimate the quantity of an individual component of a feedstuff are the procedures of the proximate analysis system. each of the procedures provides a quantitative assessment. the system is a series of chemical analyses to estimate the quantity of individual components of a feedstuff. fig.1. proximate analysis system food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 318 figure 1 is a brief diagram of the proximate analysis system.the initial procedure of proximate analysis is to determine the dry matter (dm) content of the feedstuff. the dry matter procedure removes the free water from the sample. to perform the procedure the original sample is weighed, the weighed sample is placed in a 105 degree celsius oven for 1216 hours, and the sample is reweighed. the dry matter and moisture content of the feedstuff are calculated. the second procedure of proximate analysis is to determine the crude protein (cp) content of the feedstuff. the crude protein procedure estimates the quantity of protein and non-protein nitrogen compounds in the feedstuff. the kjeldahl method is used to measure the nitrogen content of the sample. the nitrogen content is converted to a crude protein estimate under the assumption all proteins contain approximately 16% nitrogen. the procedure consists of boiling a dried sample in sulfuric acid, the sample is then diluted with water and neutralized with sodium hydroxide, and finally, the sample is distilled and the distilled ammonium is titrated with a known concentrate of sulfuric acid. to determine crude protein content, the nitrogen value from the procedure is multiplied by 6.25. the procedure introduces two primary sources of error. the procedure incorrectly assumes all nitrogen-containing compounds are proteins and all nitrogen sources are 16% nitrogen. the crude fat or ether extract (ee) procedure estimates the quantity of lipids. to perform the procedure, the dried samples are ground and extracted with an organic solvent for 4 hours and the remaining residue is dried and weighed. ether extract is the calculated as the difference between the original sample and the ether extract residue. the main source of error is the ether extract procedure incorrectly assumes all substances soluble in diethyl ether are lipids. the ash procedure estimates the quantity of inorganic material or minerals, known as ash, in the feedstuff. to perform the procedure, the sample is heated at 500600 degrees celsius for 2-4 hours and the remaining residue is weighed. the remaining residue is ash. the crude fiber (cf) procedure estimates hemicellulose, cellulose, lignin, pectin, and other components associated with the fibrous carbohydrates in feedstuffs. the process attempts to simulate the ph-related digestive processes in the stomach and the small intestine of the animal. the process does not account for enzymatic digestion. the sample is prepared by performing the dry matter and subsequently the ether extraction procedure on the sample. to perform the procedure the residue is boiled in a sulfuric acid solution for 30 minutes and rinsed, and then boiled in a sodium hydroxide solution for 30 minutes and rinsed. finally, the residue is dried, weighed, ashed, and reweighed. the crude fiber content is estimated as the difference between the pre-ash weight and the postash weight. the crude fiber procedure has many sources of error and, therefore, is the most unsatisfactory principle of proximate analysis. the primary source of error is that the acidic and basic solutions solubilize some of the true fiber; therefore the procedure underestimates true fiber content of the feedstuff. another source of error is proteins bound to insoluble fiber fractions are included in the crude fiber content, thus overestimating crude fiber. the inaccuracy of the procedure is the reason laboratories have replaced the crude fiber analysis with the detergent fiber system. the final component of proximate analysis is nitrogen-free extract (nfe). the nitrogen-free extract estimates nonfibrous carbohydrates, such as sugars and starches. the nitrogen free extract determination is the only estimate of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 319 proximate analysis determined by a calculation of a difference versus chemical analysis followed by appropriate calculations. the calculation for nitrogen free extract is: % nfe = 100 % – (% ee + % cp + % ash + % cf). as nitrogen-free extract is calculated by difference, all the errors associated with proximate analysis are additive in the estimate of nitrogen-free extract. the estimations of carbohydrates in the proximate analysis system, i.e. crude fiber and non-fiber extracts, especially for fibrous feedstuffs, have been replaced with the estimations provided by the detergent fiber system. one hundred (100) day old broiler chicks were obtained and grouped into two (2) treatments (a and b) with three (3) replicates in each treatment. broiler chicks subjected to treatments a were fed with soybean meal over the period of the feed trials, while the ones subjected to treatment b was fed with sunflower meal over the period of the feed trials. the feed intake, mortality and growth parameters [5] were estimated. at the end of all the trials, the following growth parameters were determined; a. feed intake b. broiler survival (mortality rate) c. daily weight gain = final weight – initial weight/56 days..... (1) d. food conversion ratio (fcr) = dry feed consumed/broiler weight gain..... (2) e. protein efficiency ratio (per) = broiler weight gain/dietary protein consumed..... (3) 3. results and discussion 3.1. comparative analysis of feed ration composition the results in table 1 revealed the compositions of the feed rations. feed a was composed of soybean meal (55%), groundnut cake (40%) and yellow maize (15%) and feed b was composed of sunflower (55%) groundnut cake (40%) and yellow maize (15%). both feed rations indicated progressive increase in the growth parameters of broiler production over the period of 8 weeks. this result corroborates with the findings of [6] and [7], who also reported similar feed ration compositions. table 1 comparative analysis of feed ration composition source: experimental data, 2018 3.2. proximate analysis of feed ingredients table 2 shows the mean value of proximate analysis of feed ingredients; soybean cake, sunflower meal, groundnut cake and yellow maize. the following results were obtained for soybean (44.9% crude protein, 5.5% crude fiber. 6.0% ash and 3.5% fat) sunflower (42% crude protein, 12% crude protein feed a % feed b % 1. 2. 3. soybean groundnut yellow maize 55 40 15 sunflower groundnut yellow maize 55 40 15 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 320 5.8% ash and 7% fats) groundnut cake (43% crude protein, 3.8% crude fiber, 5.5% ash and 9.17% fats) and yellow maize (11.7% crude protein, 10.5% crude fiber, 0.8% ash and 1.7% fat), implying that the feed materials contained all the nutrient requirements for broiler production, this corroborates with the findings of [8]; [9]; [10]; [11] and [12] who also reported similar results on proximate analysis of poultry rations. table 2: mean values of proximate analysis of feed ingredients on dry weight basis (%) ingredients crude protein crude fiber ash fat soybean cake sun flower meal groundnut cake yellow maize 44.90 42.00 43.00 11.70 5.50 12.00 3.81 10.60 6.00 5.80 5.50 0.80 3.50 7.00 9.17 1.70 3.3. comparative analysis of feed intake and growth parameters the result in table 3 reveals the quantity of feed intake and growth parameters of the broilers over a period of 8 weeks. feed a: at week 1 4 the estimated amount of feed consumed was 25kg, survival rate was 95%, daily weight gain was 0.201kg, feed conversion ratio was 4.174 and protein efficiency ratio was estimates as 0.575. at week 5 8 the estimated amount of feed consumed was 50kg, survival rate was 95%, daily weight gain was 0.525kg, feed conversion ratio was 0.896 and protein efficiency ratio estimate was 0.861. feed b: at week 1 to 4 the estimated amount of feed consumed was 25kg, survival rate was 97%, daily weight gain was 0.205kg, feed conversion ratio was 4.331 and protein efficiency ratio was estimates as 0.563. at week 5 to 8 the estimated amount of feed consumed was 50kg, survival rate was 97%, daily weight gain was 0.537kg, feed conversion ratio was 0.915 and the estimate for protein efficiency ratio was 0.841. the survival rate of broilers for the duration of the feeding trial for treatment b was significant and estimated as 97%. the estimate of daily weight gain was significant for treatment b from weeks 1 4 and 5 8 with an index of 0.205 and 0.537 respectively. also, feed conversion ratio was significant for treatment b from weeks 1 4 and 5 – 8; the estimates were 4.331 and 0.915 respectively. however, the protein efficiency ratio was significant for treatment a from weeks 1 4 and 5 – 8, with an index of 0.575 and 0.861 respectively. the implication of these findings suggests that sunflower feed ration was a more palatable poultry feed ration, with significant effects on the growth parameters of broilers. this result corroborates with the findings of [13]; [14]; [15]; and [16], who also reported similar effects on the growth parameters of broilers using similar feed ration compositions. table 3: comparative analysis of feed intake and growth parameters of broilers under feed trials growth parameters weeks 1 – 4 weeks 5 – 8 feed a feed b feed a feed b feed intake (kg) survival rate (%) daily weight gain (kg) food conversion ratio protein efficiency ratio 25 95 0.201 4.174 0.575 25 97 0.205 4.331 0.563 50 95 0.525 0.896 0.861 50 97 0.537 0.915 0.841 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 321 source: experimental data, 2018 fig. 2. comparative analysis of feed intake and growth parameters of broilers under feed trials 4. conclusion this paper analyzed the potentials of soybean and sunflower feed rations on the growth parameters of broilers. broiler chicks were purchased and divided into two (2) treatments (a and b). treatment a was fed with soybean feed ration, while treatment b was fed with sunflower feed ration over the period of the feed trials (8 weeks). the survival rate, daily weight gain and feed conversion ratio were significant for the broilers in treatment b. however, the protein efficiency ratio was significant for the broilers in treatment a. the broilers in treatment b responded favorably to sunflower feed rations. the implication of these findings suggests that sunflower was a very palatable poultry feed ration, with significant effects on the growth parameters of broilers. both feed rations indicated progressive increase in the growth parameters of the broilers over the period of the feed trials. the result also suggests that sunflower feed ration can be administered as a replacement or alternative for soybean feed ration in broiler ration formulation. from the results obtained, the following policy recommendations are suggested; multiplication of sunflower plants at commercial scale. adoption and utilization of sunflower in poultry (broiler) feed formulations. more research work on the nutrient component of sunflower seeds in feed rations should be carried out. 5. acknowledgments author a and author b designed the study, handled the computation of the statistical ratios and wrote the protocol and first draft of the manuscript. author b and author c managed the analyses of the study. author d and author e managed the literature searches. all authors read and approved the final manuscript. 6. references [1]. chesso, c.h., poultry production in warm wet climate. world’s poultry science journal, 15: 326-329, (2001). [2]. ravindram, h. and blair, r., feed resources for poultry production in asia and the pacific plant protein sources. world’s poultry science journal, 48: 205-231. (2005). [3]. babu, a.m.p. and deregowda, g., effect of fiber degrading enzymes in diet on performance of broilers. indian journal of poultry science. 32: 207-211. nonstarch polysaccharide degrading enzymes in poultry science journal 157: 251-263, (1997). [4]. national bureau of statistics (nbs), the nigerian statistical fact sheets on economic and social development. abuja: national bureau of statistics, 121pp., (2007). [5]. thornley, j.h.m and france, j., mathematical models in agriculture. 2nd edition, cabi publishing, wallingford uk: 923pp., (2007). [6]. u.s. department of agriculture, agricultural research service. usda national nutrient database for standard reference, release 22. in: nutrient data laboratory. (2009).http://www.nal.usda.gov/fnic/foodcomp/d ata/sr15/dnload/pk260w32.exe. accessed 11 sep 2010. [7]. ditta, y.a. and king, a.j. recent advances in sunflower seed meal as an alternate source of protein in broilers. [8]. world's poultry science journal, 73(3): 527-542, (2017). [9]. vivereos, a. and elice, r. sunflower seed meal, sunflower oil and full–fat sunflower seeds, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 godfrey onuwa, anthony mgbojikwe, bassey emmanuel, kingsley okeke-agulu and chizoba chizea, comparative analysis of growth parameters of soybean and sunflower feed rations in broiler production, food and environment safety, volume xix, issue 4 – 2020, pag. 307 – 322 322 hulls and kernels for broilers, poultry science manual, 71 (5): 905 918, (1998). [10]. mbajiorgu, c. a., ng’ambi, j. w. and norris, d. d. voluntary feed intake and nutrient composition in chickens. asian journal of animal and veterinary advances. 6(1): 20-28, (2011). [11]. velmurugu, r. poultry feed availability and nutrition in developing countries. food and agriculture organization of the united nations poultry development review. massey university, palmerton north, new zealand, (2012). al707e00pdf. [12]. scanes, c. g., brant, g. and ensminger m. e. poultry science. upper saddle river, new jersey, usa, pearson prentice hall, (2004). [13]. prabakran, j. j. , dhanapal, s. analysis of proximate composition and aflatoxins of some poultry feeds. asian journal of biotechnology. 1(3): 104-110, (2009). [14]. paradis, g.s., ranga reddy, p. , narahari, d. effect of sunflower oil cake supplementation in broiler diets. indian journal of poultry science, 37: 285-288 (2002). [15]. ramesh, k. s. nutritive value of sunflower seeds in broiler ration. m.v.sc. thesis, tamil nadu veterinary and animal sciences university, chennai, india, (2000). [16]. rama, r.s.v., raju, m.v.l.n., shailaja, s.m. and krishna, m, o. effect of supplemental enzymes in diets containing yellow maize and pearl millet (pennisetum typhoides) as the principal source of energy in broiler chicken. animal nutrition and feed technology, 4: 101111, (2004). [17]. rama, r.s.v., prashanth, k., paul, s.s., raju, m. v. l. n., nagalakshmi, d., prakash, b. evaluation of feeding value of combination of alternate protein sources in white leghorn layers. british poultry science, 61(6): 710-718, (2020). 1. introduction 4. conclusion stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiii, issue 3 30 september 2014 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladimir reşitca technical university of moldova, chisinau republic of moldavia alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved doi: https://doi.org/10.4316/fens.2023.012 122 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 122 134 chemical composition of animal feed and its influence on the milk quality * julijana tomovska 1 , ilmije vllasaku 2 , elena josevska 3 1faculty of biotechnical science, university ,,st. kliment ohridski”, bitola, north macedonia, dzulitomovska@yahoo.com, dzulijana.tomovska@uklo.edu.mk 2ubt higher education institution, kalabrija nn, 10000 prishtina, kosovo, 3faculty of biotechnical science,university ,,st. kliment ohridski”, bitola, north macedonia, *corresponding author received 1st february 2023, accepted 28th june 2023 abstract: knowledge of the chemical composition of animal feed is important for the normal health of dairy cows and the high production of quality milk. the research presents an examination of the chemical composition of animal feed shown in percentage such as: fodder crop alfalfa hay and straw which are a by-product of wheat and two types of fodder mixes (fm). as for the research the following nutrients were examined: protein, crude fiber, fat, moisture, ash and their total values, as well as nitrogen-free extractive substances (nes) in 3 samples of each animal feed from 3 different farms (a, b, c). the highest total value for all nutrient compounds was obtained in alfalfa, at farm a =65.91%. the lowest total value was obtained in straw used in farm a=53.78%. the total value of nes in all three farms was 34.78%. namely, according to the results farm a=38.54% used fm2, however farm b=39.13%, and farm c=42.15 % used fm1. analyses of chemical composition of the milk (dry matter, fat, protein, lactose and density) provide a conclusion indicating that the values from the three farms appear to be similar, the milk sample from farm b has the highest value of dry matter with 8.22% and fat 3.65%. the milk sample from farm c appears to have the highest value of protein with 3.20% and a density of 28.56%. the aforesaid refers to the fact that the feed mixture used for dairy cows contain at least 18-22% protein, which is ideally balanced feed in order to obtain a daily production of over 30 liters. the use of alfalfa and straw enriches the animal feed with proteins and influences the milk quality. keywords: animal feed, fodder mixture, alfalfa hay, wheat straw, chemical compounds. 1. introduction nutrients are part of the digestible animal feed which are important for the animal organism. they serve to satisfy physiological needs, maintain basal metabolism and reproduction, as well as the productive needs during milk production. the animal feed aimed for the dairy cows is mostly of vegetable origin and partially of mineral origin in order to provide calcium and phosphorus, which are two basic mineral substances for normal health and high milk production. when feeding dairy cows, it is of a particular importance to provide sufficient quality feed with all the necessary nutrients, in proper quantity and ratio for their normal utilization. the diet of dairy cows is quite complex and specific. quantity and quality of animal feed and water are by far the most important for the high production of milk, meat and eggs, but also for the health of farm animals [1]. fodder crops are the basic food for feeding farm animals and mostly grown in livestock-developed countries, as they represent low-priced source of food and give large yields. forage crops in our country are grown in small areas. thus, in 2012 fodder crop covered 12% of the sown area in r. macedonia. this represents a http://www.fia.usv.ro/fiajournal mailto:dzulitomovska@yahoo.com mailto:dzulijana.tomovska@uklo.edu.mk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 123 very low share in contrast to countries with developed animal husbandry, in which fodder crops cover 40% of the sown areas. alfalfa has the largest share from fodder crops in our country with 55%, and the production is of a great importance because it is characterized by a high protein and according to some researches the presence of the other fodder crops is less represented [2]. the chemical composition and nutritional value of animal feed depend on many factors, however the most important ones are: the type of fodder crops, the conditions in which the fodder crops are grown (climate, soil, fertilize), the harvesting stages, the method of canning, the storage conditions, the method of use, etc. differences in the chemical composition and general nutritional value of the forage crops used as feed for dairy cows should be known in order to be able to balance the nutrient and energy requirements for each category of livestock in order to meet the physiological needs [3, 4, 5]. in addition to the type and genetic predisposition of animals for higher milk production, suitable and balanced nutrition is crucial. the nutritional requirements of dairy cows are mainly met through the use of fodder crops in fresh or canned state rather than as waste from the food industry (beet chips, bran, beer dregs) and other agro-industrial products [6]. alfalfa (medicago sativa l.) is one of the most important fodder crops and is a valuable leguminous crop used to feed livestock in the dried state and in the form of hay. blue alfalfa as a fodder crop mostly grown in areas where there are irrigation conditions is the most cultivated variety. alfalfa as a forage crop in our country is used in the form of hay and slightly in its green state because it causes bloat in cows and due to this it is not recommended for a green diet [7]. alfalfa as animal feed is used as fresh and canned. fresh green alfalfa previously processed by mowing and drying is used as an animal feed for ruminants. grazing alfalfa is not practiced due to the cause of bloat in ruminants, and therefore if used fresh it has to undergo a process of drying for a day after mowing and further on to proceed to harvesting and given as feed. in our country preparing hay from alfalfa is not a common practice. it is mostly used as hay and grown on several continents and in more than 80 countries covering an area of over 35 million hectares [8]. alfalfa is a rich source of protein, flavonoid antioxidants, mainly apigenin, tricyclic lutein and glycoside and phenolic compounds that have anti-inflammatory effects acting as an antioxidant and neuroprotector [9]. the root system of alfalfa is well developed and penetrates to a depth of more than 7m and it absorbs more minerals and vitamins than other plants, therefore it is called "the king of all forage crops". alfalfa as a forage crop also characterized by having positive health effects. it is believed that alfalfa has a direct effect on reducing blood cholesterol in cows and control of hormones [10]. the nutritional value of alfalfa is great and it represents the best quality coarse food, because it is rich in protein, crude protein content (18 22 %) and crude fiber (25 35 %). alfalfa, in addition to raw proteins, crude fiber, minerals and vitamins, contains a large amount of saponins that are not desirable for daily intake in livestock nutrition. it is believed that feeding dairy cows with alfalfa could produce over 8 liters of milk per day [11]. wheat straw (triticum vulgare) which is used by some farmers as animal feed (as in the case of one farm in our research), provides physical satiety to cows, and the needs of easily digestible nutrients are provided through the use of alfalfa hay and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 124 fodder mixtures. wheat straw is quite rich in cellulose and hemicellulose, which is characterized by low digestibility, and the presence of a large amount of lignin contributes to this matter. straw is a natural fiber that can survive for many thousands of years and remains of straw are found since the ancient egyptians [12]. straw indicates a large presence of many chemical elements (21), such as k, ca, mg, p, zn, fe, e.t.c., crude protein and non-nitrogen extractive substances (nes) [13]. wheat straw contains several organic chemical compounds, such as carbohydrates (cellulose, hemicellulose, lignin), proteins, minerals (calcium and phosphorus), silica, detergent fibers, acidifiers and ash [14]. according to the analysis by korr (2017) it is shown that straw contains a small amount of protein, nutrients, calcium and phosphorus from peas, while larger amount of potassium is contained in oat straw. he analyzed the chemical composition of several types of straw such straw from barley, wheat, peas, cinnamon and lentils, as well as different chemical composition of indigestible proteins, processed nutrients, calcium, phosphorus and potassium [15, 16]. fodder mixtures (fm) or concentrates used to feed dairy cows, are composed of grain fodder crops (corn, barley, bran, sunflower and soybean meal), vitamin supplement (premix) and mineral substances. depending on the type and category of farm animals, fodder mixtures concentrates are characterized by a high content of light digestible proteins and carbohydrates, minerals, vitamins and have high energy value. by using the fodder mixtures, the daily meal of dairy cows is balanced with all the necessary nutrients. macedonian name of the concentrate is kmk-18 as the concentrate contains 18% of crude protein. the content of crude proteins in different fodder mixtures is varaible and depends on the type and category of animals which they are intended for. the feed mixtures produced in doo "agroinvest", 2017, which have their own catalog number are produced according to the animal feed regulations of the republic of macedonia [17]. fodder mixtures provide dairy cows with additional energy because the bulk food consumed by cows (alfalfa hay and wheat straw) cannot meet the nutrient and energy needs for high milk production. fodder mixtures for dairy cows should be balanced carefully in terms of composition and content of nutrients that are essential for good health and high production of quality milk. fodder mixtures for dairy cows should ensure high efficient digestion of food and utilization of nutrients by the organism for higher productivity. the latest studies show that the higher milk production is a result of the large intake of easily digestible nutrients consumed through feed mixtures. the mobilization of the body energy is a result of the body fat and this efficient part (i.e. the usage of the energy) is not being increased during the lactation [18]. to obtain optimal milk production, rations should be balanced well in terms of the content of all nutrients. for economical and profitable milk production, the daily ration of dairy cows should be composed of high-quality coarse feed and feed mixtures for dairy cows, ahdb dairy (2018) [19]. according to rumsey (1980) forage for dairy cows should meet energy and protein requirements for maintenance, milk production, growth and reproduction [20]. forages for dairy cows are mainly a source of easily digestible energy and protein, but they usually contain a large amount of minerals and other important nutrients that cannot be fulfilled through roughage forage [21, 22]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 125 composition of the concentrates kavanagh (2016), examined the composition of the concentrates and their characteristics [23]. according to the chemical composition, feed mixtures for dairy cows contain water, crude proteins, different types of carbohydrates, crude fiber, fats, minerals and vitamins [7]. in the daily meal of dairy cows, it is necessary to add a certain amount of nonprotein nitrogen, which serves as food for the microorganisms in the rumen of the cows [4]. when it comes to the nutrition of farm animals, the largest number of scientific papers on the topic refer to the qualitative and quantitative needs, and as a result, the successful application is seen in the production of milk, meat, etc. in order to get a clear picture of the nutrient needs of the animal organism, a good knowledge of the feed used in the diet of farm animals, its chemical composition and digestibility and utilization of nutrients is necessary. proteins as nutrients participate in the building of skeletal muscles, and as structural elements of tissues built from a large number of amino acids [24]. milk is a colloidal solution of proteins, milk fat, milk sugar and mineral substances. in our country, the most cultivated breed is the black and white breed, which are represented in almost all small and large farms. the black and white breed of cows have successfully acclimatized in our country and bred in all regions of our country. it is characterized by good milk yield, but lately, to improve milk yield, it has crossed with holstein friesian breed. the quality of milk from the black and white breed is standard and it is greatly influenced by the diet of the cows and the use of fodder crops. the quality of the milk also depends on the cow’s lactation period. cows that are at the beginning of lactation have milk with a lower fat content, and at the end of lactation the fat content increases. the use of coarse fodder for feeding cows increases fat content, while concentrated feed mixes reduce it. the type of forage has little effect on protein content and no influence on lactose content [25, 26]. milk as a product of the mammary gland is characterized by a certain nutritional value, but it also contains a large amount of other biostimulating substances that make it an indispensable food for all age categories of people [27, 28]. nutrition of dairy cows according to numerous studies, the amount of milk per dairy cow is a breed characteristic that largely depends on the rearing conditions (accommodation, roughage quality and the quality of forage compounds for dairy cows). under normal housing conditions and quality coarse feed, a large production of milk of excellent quality can be expected [29]. the chemical composition of milk varies greatly among different types of animals, according to numerous studies carried out by feskanich et al., (2011), cow's milk contains an average of 3.4% protein, 3.6% milk fat and 4 .6% lactose, 0.7%, minerals (92) with an energy value of 66 kcal of energy in 100 grams [30]. breed as genetic factors have their own positive influence on the content of milk fat in milk and as a result there are breeds of cows that give milk with higher fat content (jersey and holstein). the content of milk fat in cows that are at the beginning of lactation is lowest, and at the end of lactation it is the highest. during the summer, cows give milk with a lower content of milk fat as a consequence of the high temperatures, and during the winter with a higher content of milk fat [25]. however, different breeds of cows have different chemical composition of milk and differ in the average values of the components. cows of the black and white breed have food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 126 the lowest milk fat and protein content, while the holstein-friesian breeds have milk with the highest milk fat content [31]. with the latest research on the chemical composition of milk, especially on the amino acid composition of proteins, new amino acids and other biostimulating substances have been discovered, which are quite significant especially in terms of the quality of milk (although they are present in traces) [32]. 2. materials and methods as a research material for this study, sampes for animal feed and samples of raw milk have been examined. the test material taken from three farms (a, b, c) randomly selected are from different regions of the r. macedonia. the samples of animal feed included two types of concentrates (fm1, fm2), produced by doo "agroinvest" fodder mixture for dairy cows with crude protein (kmk18%), alfalfa hay and straw, [17]. farm a used alfalfa, straw and both types of concentrate. farm b used alfalfa, both the same types of concentrate, and farm c used only alfalfa and one type of concentrate. in order to get a complete picture of the state of transfer of nutrients from animal feed to milk, we also provide the following data on the feeding and milking of cows. the cows are of the holstein-frisein breed and are indigenous. feeding was performed three times a day in the morning with concentrate, at lunch with alfalfa and straw, while in the evening with concentrate as well. milking was done by machine. the main objective of this research is the analysis of animal feed and milk, in order to see the influence of the chemical composition of animal feed on the quality of milk. the moisture, proteins, crude fiber, ash and fat we examined in animal feed, and in fresh milk we examined the dry matter, protein, fat, lactose, milk density and relative volume mass/(g/cm3) [5]. 2.1 methods for determining the chemical composition of animal feed determination of proteins was perforemed by the kjeldahl method and determination of total fats by the soxlet method, with diethyl ether extraction. determination of total mineral substances (ash) was done by the gravimetric method. the principle of this method is based on burning the sample at a temperature of 600 ˚c and measuring the resulting residue. determination of the amount of water (moisture) was carried out by the gravimetric method. the principle is based on drying the sample at a temperature of 105 ˚с to a stable weight. the determination of the amount of raw fiber was based on treating 1 g of the sample with sulfuric acid and sodium hydroxide, and the rest separated by filtering and collecting from the cloth in a porcelain pot of known weight [7, 33, 35]. 2.2 methods for determining the chemical composition of milk gerber's method of fat in milk was used for milk fat determination. this method was firstly proposed by dr. gerber in 1892 and later applied and renamed in acidobutrymetric method because it uses sulfuric acid and works in butyrometers. determination of nitrogen substances such as proteins, and the total nitrogen (n, nitrogenium) were examined according the kjeldahl method. determination of milk sugar – lactose was done by iodometric method with reagents: fehling's solution a blue solution of copper (ii) sulfate; fehling's solution b – colorless aqueous solution of potassium sodium tartrate (known as rochell's salt); strong alkali (usually naoh); 0.5% starch solution dissolved in water; 2% alcoholic solution of phenolphthalein and n/10 solution of iodine. determination of dry food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 127 matter was done by drying milk at a temperature of 1050c to constant weight. determination of the density of milk was performed with lactoscan. the specific gravity of milk varies and depends on the quantitative ratio of its components, such as mineral substances and lactose. if there is fat in the milk with the same ratio of other ingredients, it will show a lower specific gravity and vice versa. the content of all parameters is expressed in percentage [7, 33, 35]. 3. results and discussion the results of analysis of chemical composition (moisture, protein, crude fiber, ash and fat) and their average value in the feed such as concentates (fm1, fm2), alfalfa hay and straw from farms a, b and c are shown in figures 1, 2, 3. from the figures we observed the total highest average values for all chemical compounds in alfalfa from farm a (65.91%), while for alfalfa from farm b and farm c they are equal to 65.22%. the lowest total value for the chemical composition was measured in the straw from farm a, 53.78%. for nitrogen-free extractives, we have the highest average values in concentrates fm1 from farm a (61.33 %) and fm2 from farm b (61.29 %). concentrates, alfalfa hay and wheat straw is a feed that has been applied in the winter period and is different from the feed that cattle consume when they are on pastures, fresh and green. this indicates that the chemical composition of animal feed in the winter period greatly affects the quality of milk in contrast to food in the summer period. the nutritional value of the types of food will depend on several factors, but the first of all on the content of nutrients, which, in turn, will depend on the utilization of the organism itself [3, 4]. chemical composition / % farm a , nes = (nitrogen-free extractive substances) fig. 1 chemical analysis in animal feed of farm а food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 128 chemical composition / % of farm b, nes = (nitrogen-free extractive substances) fig. 2 chemical analysis in animal feed of farm b chemical composition / % farm c , nes = (nitrogen-free extractive substances) fig. 3 chemical analysis in animal feed of farm c the average values of chemical composition of alfalfa from the three farms are shown in figure 4. according to the given results it can be observed that the highest value for water (moisture) in alfalfa was measured from farm b (10.82 %), and the lowest from farm a (9.76 %). for protein, the highest value in alfalfa from farm b of (13.93 %), and the lowest from farm a (7.80 %), for crude fiber, the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 129 highest value in alfalfa from farm b (38.44 %), the lowest from farm c (35.70 %). for ash, the highest value in alfalfa from farm a (4.80 %), and the lowest for alfalfa from farm b (13.3 %), and for fat, the highest value in alfalfa from farm a (2.24 %) and the lowest from farm c (1.64 %). all chemical composition has high value in alfalfa hay from farm b. nes / (%) = 100 – water (%) – protein (%) – fiber (%) – ash (%) – fat (%) fig. 4 chemical composition of animal feed alfalfa from the three farms calculated average values for chemical composition of concentrates (fm1, fm2) from the three farms are shown in figure 5. according to the results above it is seen that the highest value for water (moisture) was measured in fm1 (11.17%) and in fm2 (11.15%) from farm a, lower values in farm b, fm2 (10.32%) and fm1 (10.49%), while the lowest value is from farm c (10.60%) regarding the moisture. for proteins, the highest values were measured in fm1 from farm b (14.86%), then in farm a in fm2, 14.62% and 14.26% were measured in fm1, while lower values were measured in farm c (6.14%). for raw fiber, high values were measured in the concentrate from farm c (10.45%), then in farm a (8.17%), while a lower average value was measured for farm b in fm2 (7.35%) and in the fm1 (7.54 %). a high average value for ash was calculated in the concentrate from farm c (4.16 %), then in fm2 (3.43 %) and fm1 (3.29 %) from farm b, and the lowest values were calculated in fm1 (2.41 %) and fm2 (2.26 %) from farm a. the content of fat in fm2 (3.43%), and in fm1 (3.29%) farm b, were measured as well. the lowest values in fm1, (2.41%) and in fm2 from farm a, (2 .26 %). 3.24% fat was measured in the concentrate from farm c. for the total value of all chemical compounds (water, protein, crude fiber, ash, fat) it can be said that the highest values were measured in the concentrate from farm c (42.51%), followed by farm b (39.13%), while the lowest total value was calculated in fm1 from farm a (38.54 %). the highest percentage of moisture and protein was measured in alfalfa from farm c, the highest percentage of fiber was found in farm b, while the presence of ash and fat was the mostly concentrated in alfalfa from farm a. the values for the chemical composition of animal feed and the formation of the necessary compounds food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 130 for the growth and development of alfalfa depend on the composition of the soil, irrigation, climate and other factors [39]. fodder research and extension policies in terms of better quality seeds, seed rate, improved agronomic practices and improved inputs (fertilizers, water and pesticides) [39]. wheat straw (triticum vulgare) was only detected in farm a. it is a secondary product and is rich in proteins and minerals, such as phosphorus, potassium, etc., [8]. nes / (%) = 100 – water (%) – protein (%) – fiber (%) – ash (%) – fat (%) fig. 5 chemical composition of concentrate feed mixture from three farms in figure 6 presented results of analysis the chemical composition of the milk obtained from the three farms regarding the dry matter, fat, protein, milk lactose and milk density. the results of the dry matter are similar in the milk from the three farms: in farm b is 8.22%, in farm c is 8.21%, while in farm a is 7.98%. the values for dry matter are lower, because the tests were done on skim milks and the value obtained from the dry matter tested on skim milks is added to the value of fat and their sum gives the total value of dry matter for raw milks. the concentration of milk fat is similar, in farm b the high values were measured (3.65 %), while in farm c (3.32 %), and in farm a, the values were lower (3.20 %). approximate value was measured for protein in milk from the three regions. the highest values for lactose were measured in farm b (4.49%), and the lowest in the milk sample from farm a (4.38%). for milk density, it is also noted that the highest values were measured in farm c (28.56%), followed by the milk sample from farm b (28.49%), and the lowest in the milk sample from farm a (28.05%). regarding the chemical composition of animal feed, there are many different theses and recommendations due to the content of 18 to 22% crude protein and 2535% crude fiber compared to maturity. alfalfa also contains saponins, which are not desirable for daily feeding of livestock [11, 37]. according to research by ishler et al., (2006), concentrates are mainly a source of energy and a source of proteins and they also contain minerals and other important nutrients that can’t be fulfilled by feed from forage crops. similar findings done food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 131 by ishler et al. are also confirmed by the research in this paper [22, 38]. this means that the fodder mixture has 18% protein and is ideally balanced in order to obtain an optimal level of energy, for higher productivity in cows, with a daily milk production of over 30 liters. the chemical composition of this fodder mixture provides the necessary levels of nutrients and energy, thus enabling optimal production from the cow, while at the same time it were characterized by high quality of milk production in terms of fat and protein level [17, 34]. results from figure 6 of the chemical composition of milk can be noted that the parameters dry matter (8.22 %), fat (3.65 %) and lactose (4.49 %) dominate in the milk sample from farm b, while in the milk sample from farm c is dominated by protein (3.10 %) and milk density (28.56 %). the lowest values for the chemical composition of milk were measured in milk the sample from farm a. fig. 6 chemical composition of milk from three farms our results compared with those already published by the research as of feskanich et al., (2011), according to which cow's milk contains on average (3.4%) protein (3.6%), fat (4.6%), lactose (0.7%), mineral and supplies 66 kcal of energy per 100 grams, appear to be similar with only decimal minimum deviations observed [30]. examinations of the chemical composition of milk correspond to the latest research from cornell university (2018) in the paper "composition of milk": where the raw cow's milk contains: water 87.3% (85.5-88.7%), fat 3.9 % (2.4 – 5.5 %), protein 3.25 % (2.3 – 4.4 %), casein 2.6 % (1.7 – 3.5 %), serum proteins, small proteins, carbohydrates 4.6% (3.8 – 5.3%) [32]. according to the results from the milk analysis, the content of dry matter, fat, protein and milk lactose, the chemical composition of the milk from the three farms appears to be quite similar, and the results from this study are consistent with the results of research by douglas (2014). dry matter (8.22 %), fat (3.65 %) and lactose (4.49 %) dominate in the milk sample from farm b, while the milk sample from farm c dominated in protein (3.10 %) and milk density (28.56 %). the lowest values are registered for the chemical composition of milk measured in milk of farm a [35, 37]. 4. conclusion as from chemical analysis the animal feed which is characterized with high content of crude protein (18 22 %) and crude fiber (25 35 %) [11, 36, 37], the obtained results (7.8 14 %) and (35.5 38.5%) are respectively low due to the additional usage of feed mixture produced by a domestic company according to the feed regulation of r. macedonia. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 132 the use of daily meal in the three farms was different, farm a used 20 kg per day, farm b 28 kg, and farm c 31 kg per day. the best chemical composition is the feed from farm b. the lowest values for the chemical composition of milk were measured in the milk sample from farm a, although used in the largest amount of meal. the food for dairy cows should be of known quality, with a known chemical composition and the daily ration should provide all the necessary nutrients for good health and high milk production. from the chemical analysis of the milk, it is well established that the diet with fodder crops has a great influence on the milk quality. the use of different types of fodder increases fat content. feed mixtures reduce it and have limited effect on protein content and have no effect on lactose content. the milk that contains the least fat (3.65%) is from farm a, while the least of the protein (3.10%) is registered in the milk sample from farm c. our research correspondts with numerous studies on the topic due to the fact that the amount of milk per dairy cow is a characteristic of the breed that largely depends on the rearing conditions (accommodation, quality of roughage and quality of forage compounds for dairy cows). 5. references [1] learner guide, primary agriculture nqf intermediate animal nutrition, level 4 unit standard no: 116282, version 01 date: july 2006, agriseta (2006). http://www.agriseta.co.za/downloads/learningmate rial/116282_lg.pdf, 18.02.2018. [2] sreten a., aleksandar u., sonja s., srecko gj., lubomir d., trajan k., vladimir v., elena e., usaid, study on the adaptation of animal production in the republic of macedonia according to climate change with an action plan skopje: network for rural development of the republic of macedonia, (2014). [3] caisin l., vasile h., vasile v., chemical composition and nutritional value of the fooder grown in the conditions of the republic of moldava, scientific papers, series d. animal science,vol. lvi, (2012). [4] alberta agriculture and rural development (ard), nutrition and management: feeding lightweight calves, (2009d). (accessed jul, 5th, 2012). http://www1.agric.gov.ab.ca/$department/deptdocs. nsf/all/beef11678, 26.09.2018. https://open.alberta.ca/publications/4429907 [5] aoac official method 950.02, animal nutrition group, (2000 2010). animal feed. preparation of sample, 2012. http://www.grains.kstate.edu/extension/doc/procedu res/animal-feed-fiber-procedures.pdf, 16.06.2018. [6] sarwar m., shahzad m. a., nisa m., bhatti s. a., tauqir n. a., enhancing buffalo productivity through usage of low quality feed stuffs. institute of animal nutrition and feed technology, university of agriculture, faisalabad, pakistan, the journal of animal and plant sciences, 22 (3 suppl.), pp. 128-132. issn: 10187081, (2012). [7] petre р i., тatajana p., romina k. practicum in fodder production. st. cyril and methodius university, faculty of agriculture, skopje, 91-117, 2000; 2011. [8] jasmina r., dejan s., d., jordan m., alfalfa, most important perenial forage legume in animal husbandry. biotechnol. anim. husb., 25, pp. 465475, (2009). [9] stochmal a., oleszek w., seasonal and structural changes in flavones in alfalfa (medicago sativa) aerial parts, int. j. food agric. environ., 5, pp. 170-174, (2007). [10] choi k. c., hwang j. m., bang s. j., kim b. t., kim d. h., chae m., lee s. a., chae m. j., kim, d. h., lee, j. c., chloroform extract of alfalfa (medicago sativa) inhibits lipopolysaccharide-induced inflammation by downregulating erk/nfb signaling and cytokine production, j. medic. food, 16, pp. 410-420, (2013). [11] fao. selected indicators of food and agriculture development in the asia – pacific region 2000-2010, food and agriculture organization of the united nations, regional office for asia and the pacific, bangkok, (2011). [12] muhammad z. a., azeem m. k., ikram s., akhtar a., shafique z., abdul m., small farmers perceptions regarding improved fooder and forage varieties: results of participatory on farm research, pakistan j. agric. res. vol. 25 no. 4, 295-306, (2012). [13] ivana p., zeljka b. m., ţ., аlan a., chemical composition of straw as an alternative material to wood raw material in fibre isolation, drvna industrija, 67 (2), pp. 119-125, (2016). [14] tehmina s. k., umarah m., wheat http://www.agriseta.co.za/downloads/learningmaterial/116282_lg.pdf http://www.agriseta.co.za/downloads/learningmaterial/116282_lg.pdf http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://animalsciencejournal.usamv.ro/index.php/scientific-papers/10-articles-2013/156-chemical-composition-and-nutritional-value-of-the-fodder-grown-in-the-conditions-of-the-republic-of-moldova http://www1.agric.gov.ab.ca/%24department/deptdocs.nsf/all/beef11678 http://www1.agric.gov.ab.ca/%24department/deptdocs.nsf/all/beef11678 https://open.alberta.ca/publications/4429907 http://www.grains.kstate.edu/extension/doc/procedures/animal-feed-fiberhttp://www.grains.kstate.edu/extension/doc/procedures/animal-feed-fiberhttp://www.grains.k-state.edu/extension/doc/procedures/animal-feed-fiber-procedures.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 133 straw: a pragmatic overvie, current research journal of biological sciences, 4 (6), pp. 673-675, (2012). [15] korr j., ag-info centre, alberte and rural development. using straw in cattle rations – frequently asked questions. published on november 20, 2003. last review/revised on september 21, (2017). (https://www1.agric.gov.ab.ca/$department/deptdoc s.nsf/all/faq7594 [16] juanita k., ag-info centre, alberta agriculture and rural development, (2017). https://www1.agric.gov.ab.ca/$department/deptdoc s.nsf/all/faq7594 [17] catalog for fodder mixtures, doo "agroinvest" ai-zp-sd-02, (2017). http://www.agroinvest.com.mk/content/catalogs/8animal-feed-1.pdf [18] gordon f. j., patterson d. c., yan t., porter m. g., mayne c. s. and unsworth e. f., the influence of genetic index for milk production on the response to complete diet feeding and the utilization of energy and nitrogen, animal science, 61, pp. 199-210, (1995). [19] ahdb dairy, concentrate feeds, agricultural drought impact summer 2018 industry briefing agriculture and hirticulture development, uk, (2018). [20] rumsey g. l., aquaculture development and coordination programme. fish feed technology, chapter 10. antioxidants in compounded feeds, tunison laboratory of dish nutrition, food and agriculture organization of the united state, fao, cortland, new york, (1980). [21] ferris c. p., patterson d. c., gordon f. j. and kilpatrick d. j, the effect of concentrate feed level on the response of lactating dairy cows to a constant proportion of fodder beet inclusion in a grass silage-based diet. 2003 blackwell publishing ltd. grass and forage science, 58, pp. 17-27, (2003). [22] ishler v. a., adams r.s., heinrichs a. j., varga g. a. concentrates for dairy cattle, college of agricultural sciences • cooperative extension, department of dairy and animal science the pennsylvania state university, das 94-06, pp.1-6, (2006). http://www.dairyweb.ca/resources/uswebdocs/c oncentrates.pdf. [23] siobhan k., feeding the dairy cow (concentrates) teagasc agriculture and food authority, chapter 35, section 6, pp. 214-220, (2016). https://www.teagasc.ie/media/website/animals/dair y/feedingdiarycowconcentrate.pdf. https://www.teagasc.ie/animals/dairy/ [24] zivojin m., veljko p., stocna hrana, privredni pregled, beograd, (1978). [25] jones, c. m., and heinrichs a. j., milk components: understanding milk fat and protein variation in dairy herd, description of variation found in production of milk components, factors that contribute to this variation, and strategies to improve component production. pennstate extension, 8/17/2018. https://extension.psu.edu/milk-componentsunderstanding-milk-fat-and-proteinvariation-inyour-dairy-herd [26] guide to good dairy farming practice, fao animal production and health issn 8, 1810-0708. fao animal production and health guidelines guide to good dairy farming practice published by food and agriculture organization of the united nations and international dairy federation rome, (2011). [27] from feed to milk: understanding rumen function part ii: feed and feed nutrients for dairy cattle, the pensilvania state university, 1996. https://www.topsoils.co.nz/wpcontent/uploads/2014/09/from-feed-to-milkunderstanding-rumen-function-penn-state pdf. [28] walter l. h., milk composition and characteristics, milk composition & synthesis, university illinois, urbana, champaign, (2009). [29] richard o. k, david c. c., livestock feeds and feeding (6th edition), university of pennsylvania extension. ,, monitoring dairy heifer growth‖, (2010). [30] feskanich d., willett w. c., stampfer m. j., colditz g. a., milk, dietary calcium, and bone fractures in women: a 12-year prospective study. am j public health, 87 (6), pp. 992-7 (1997). [31] balley k. e., jones c. m., heinrichs a. j., economic returns to holstein and jersey herds under multiple component pricing, j. dairy sci., 88, pp. 2269-2280, (2005). [32] cornell universitety, department of animal sciences. college of agriculture and life sciences, dairy extension, food safety laboratory and milk quality improvement program, composition of milk ithaca, new york, pp.1-5, 2018. https://foodsafety.foodscience.cornell.edu/mqip/ (https://dairyextension.foodscience.cornell.edu/sites /dairyextension.foodscience.cornel l.edu/files/shared/composition%20of%20milk.pdf) . [33] rulebook on sampling methods and methods for physical, chemical and microbiological analyzes of kmk 18% "official magazine of sfrj", no. 15 p. 423–427, (1987). http://www.slvesnik.com.mk/issues/e4f1e3658001 4f1e8eaf4011aa24349b.pdf [34] khalili h., sairanen a, effect of concentrate type on rumen fermentation and milk production of cows at pasture. animal feed science https://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/faq7594 https://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/faq7594 https://www1.agric.gov.ab.ca/%24department/deptdocs.nsf/all/faq7594 https://www1.agric.gov.ab.ca/%24department/deptdocs.nsf/all/faq7594 http://www.dairyweb.ca/resources/uswebdocs/concentrates.pdf http://www.dairyweb.ca/resources/uswebdocs/concentrates.pdf https://www.teagasc.ie/media/website/animals/dairy/feedingdiarycowconcentrate.pdf https://www.teagasc.ie/media/website/animals/dairy/feedingdiarycowconcentrate.pdf https://www.teagasc.ie/animals/dairy/ https://extension.psu.edu/milk-components-understanding-milk-fat-and-protein-variation-in-your-dairy-herd https://extension.psu.edu/milk-components-understanding-milk-fat-and-protein-variation-in-your-dairy-herd https://extension.psu.edu/milk-components-understanding-milk-fat-and-protein-variation-in-your-dairy-herd https://extension.psu.edu/milk-components-understanding-milk-fat-and-protein-variation-in-your-dairy-herd https://www.topsoils.co.nz/wp-content/uploads/2014/09/from-feed-to-milk-understanding-rumen-function-penn-state https://www.topsoils.co.nz/wp-content/uploads/2014/09/from-feed-to-milk-understanding-rumen-function-penn-state https://www.topsoils.co.nz/wp-content/uploads/2014/09/from-feed-to-milk-understanding-rumen-function-penn-state https://foodsafety.foodscience.cornell.edu/mqip/ https://dairyextension.foodscience.cornell.edu/sites/dairyextension.foodscience.cornell.edu/files/shared/composition%20of%20milk.pdf https://dairyextension.foodscience.cornell.edu/sites/dairyextension.foodscience.cornell.edu/files/shared/composition%20of%20milk.pdf https://dairyextension.foodscience.cornell.edu/sites/dairyextension.foodscience.cornell.edu/files/shared/composition%20of%20milk.pdf http://www.slvesnik.com.mk/issues/e4f1e36580014f1e8eaf4011aa24349b.pdf http://www.slvesnik.com.mk/issues/e4f1e36580014f1e8eaf4011aa24349b.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 julijana tomovska, ilmije vllasaku, elena josevska, chemical composition of animal feed and its influence on the milk quality, food and environment safety, volume xxii, issue 2 – 2023, pag. 122 – 134 134 and technology 84, pp.199-212, (2000). [35] 2012-26 rulebook on the special requirements for safety and hygiene and the manner and procedure of performing official controls of milk and milk products, agency for food and veterinary, skopje (2012-2026). http://fva.gov.mk/mk/zakon-bezbednosthranata/pravilnik/2012-26-pravilnik-posebnitebarana-bezbednost-higiena-nachinot-postapkatavrshene-sluzhbenite-kontroli https://www.agriclinics.net/samp eprojects/5.pdf [36] ben s., project report on dairy farming (cow), benny maliyekkal kundannur po wadakkanchery, thrissur 8979725166 id : kl200000024, nodal training institute rars pattambi, agri clinics & agri business center scheme sponsored by ministry of agriculture & farmers welfare, government of india national institute of agricultural extension management (manage), hyderabad & nabard www.agriclinics.net (2019 – 2020). [37] agricultural production statistics 2000–2020 fao, faostat analytical brief series no. 41. rome, (2022). https://www.fao.org/3/cb9180en/cb9180en.pdf https://www.fao.org/food-agriculturestatistics/data-release/crop-livestock-and-food/en/ [38] virginia i., and gabriella v., carbohydrate nutrition for lactating dairy cattle, pennsilvania state university, college of agricultural sciences • cooperative extension. dairy&animal science department of dairy and animal science, the pennsylvania state university 324 henning building university park, pa 16802, adapted from nutrient requirements of dairy cattle, 2001 nrc and field experience, (2018). https://extension.psu.edu/carbohydrate-nutritionfor-lactating-dairy-cattle https://www.nutritime.com.br/arquivos_internos/art igosbk/chonutrition.pdf [39] sarwar m., khan m. a., igbal z., feed resources for livestock in pakistan. status paper, intl. j. agri. biol., 4, pp. 186-192, (2002b). http://fva.gov.mk/mk/zakon-bezbednost-hranata/pravilnik/2012-26-pravilnik-posebnite-barana-bezbednost-higiena-nachinot-postapkata-vrshene-sluzhbenite-kontroli http://fva.gov.mk/mk/zakon-bezbednost-hranata/pravilnik/2012-26-pravilnik-posebnite-barana-bezbednost-higiena-nachinot-postapkata-vrshene-sluzhbenite-kontroli http://fva.gov.mk/mk/zakon-bezbednost-hranata/pravilnik/2012-26-pravilnik-posebnite-barana-bezbednost-higiena-nachinot-postapkata-vrshene-sluzhbenite-kontroli http://fva.gov.mk/mk/zakon-bezbednost-hranata/pravilnik/2012-26-pravilnik-posebnite-barana-bezbednost-higiena-nachinot-postapkata-vrshene-sluzhbenite-kontroli https://www.agriclinics.net/samp%20eprojects/5.pdf https://www.agriclinics.net/samp%20eprojects/5.pdf https://www.fao.org/3/cb9180en/cb9180en.pdf https://www.fao.org/food-agriculture-statistics/data-release/crop-livestock-and-food/en/ https://www.fao.org/food-agriculture-statistics/data-release/crop-livestock-and-food/en/ https://extension.psu.edu/carbohydrate-nutrition-for-lactating-dairy-cattle https://extension.psu.edu/carbohydrate-nutrition-for-lactating-dairy-cattle https://www.nutritime.com.br/arquivos_internos/artigosbk/chonutrition.pdf https://www.nutritime.com.br/arquivos_internos/artigosbk/chonutrition.pdf 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2021.004 35 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1 – 2021, page 35 – 42 protozoan distribution in farmed cyprinid fish from macedonia *dijana blazhekovikj dimovska 1 , stojmir stojanovski 2 1faculty of biotechnical sciences, university „st. kliment ohridski”, bitola, n. macedonia 2hidrobiological institute, ohrid, n. macedonia dijanablazekovic@yahoo.com *corresponding author received 17th november 2020, accepted 30th march 2021 abstract: the main purpose of this research was to analyze protozoan distribution in farmed cyprinid fish from macedonia which can have a negative economic impact in developing countries where aquaculture activities contribute to food production. the parasitological examinations were performed on the four fish species from the most significant and larger cyprinid aquaculture facilities in macedonia. a total of 1134 fish samples were examined, from which parasite infestation with protozoa was determined in 533 fish, with a total prevalence of 47.002 % and a mean intensity of 6.492. eight representatives of protozoan parasites in farmed cyprinid fish were identified in this study: apiosoma piscicola, chilodonella hexasticha, myxobolus müelleri, myxobolus encephalicus, thelohanellus nikolskii, ichthyophthirius multifiliis, trichodina sp. (in common carp), and trichodina sp. (in grass carp). the finding of chilodonella hexasticha in common carp (cyprinus carpio) as a first record for macedonian waters, is first published in this paper. keywords: cyprinid aquaculture, protozoan parasites, prevalence, mean intensity 1. introduction according to [1], parasitic protozoan diseases are responsible not only for great losses to the commercial fishing industry, but also for a negative economic impact in developing countries where aquaculture activities contribute to food production to the need of the population. from [2] and [3] found that ecto and endoparasitic protozoa are some of the most dangerous threats to the fish population. according to the authors, even a moderate infection with these organisms can cause fatal disease, because the infected fish lose their appetite and stop consuming food. [4] reported that most of the protozoa live in commensalism with their hosts, while some species have adapted to parasitic relationships to fish, representing their ectoparasites. from [5] stated that parasitic protozoa of at least seven different types have been identified in fish, including endo and ectoparasites with a wide range of life cycles and pathology. [6] reported that in many cases, certain protozoa species cause secondary infections with other pathogens, such as viruses, fungi, and bacteria. at the same time, they are one of the most dangerous parasitic groups that are likely to cause more disease in the fish population than any other parasite species. 2. materials and methods samples of four fish species including common carp (cyprinus carpio), grass carp (ctenopharingodon idella), silver mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 36 carp (hypophtalmichthys molitrix), and bighead carp (hypophtalmichthys nobilis) from the most significant and larger cyprinid aquaculture facilities in macedonia, including fish ponds and cage farms on reservoirs, were examined for parasitological investigations. this study was carried out by seasons, in a period of three years. the specimens were placed in plastic tanks with fishpond water and immediately transferred to the research laboratory or were inspected on the spot. during the dissection, gills, fins and skin were examined using stereomicroscopes „zeiss”stemi dv4 and „mbs 10”, as well as microscopes „zeiss”primovert and „reichart”, at the department for fish diseases in hydrobiological institute ohrid (macedonia). all parasites found in each fish were identified and enumerated. during the study period, data on parasite species were categorized according to the season, including prevalence and mean intensity. classical epidemiological variables (prevalence and mean intensity) were calculated according to [7]. the parasite specimens were identified using reference keys of [8]. 3. results and discussion during the parasitological examinations of the fish from the most significant and larger cyprinid aquaculture facilities in macedonia, a total of 1134 fish samples were examined, from which parasite infestation with protozoa was determined in 533 fish, with a total prevalence of 47.002 % and a mean intensity of 6.492 (table 1). table 1. total prevalence and mean intensity with representatives of protozoa in cyprinid aquaculture facilities in macedonia parasites number of examined fish number of infected fish mean intensity prevalence protozoan species 1134 533 6.492 47.002 % whereby from the representatives of protozoa, 8 species were identified: apiosoma piscicola, chilodonella hexasticha, myxobolus müelleri, myxobolus encephalicus, thelohanellus nikolskii, ichthyophthirius multifiliis, trichodina sp. (in common carp) and trichodina sp. (in grass carp). based on the total number of fish examined (1134), the highest prevalence with protozoan parasites is confirmed with trichodina sp. (in common carp) (30.159%), followed by apiosoma piscicola (6.996%), ichthyophthirius multifiliis (4.233%), thelohanellus nikolskii (3.086%), myxobolus encephalicus (0.794%), trichodina sp. (in grass carp) (0.794%), chilodonella hexasticha (0.441%) and myxobolus müelleri (0.441%).the highest mean intensity with protozoan parasites is determined with ichthyophthirius multifiliis (26.479), followed by trichodina sp. (in grass carp) (7.556), trichodina sp. (in common carp) (4.567), apiosoma piscicola (4.241), myxobolus encephalicus (4.000), myxobolus müelleri (3.400), thelohanellus nikolskii (2.444) and chilodonella hexasticha (1.800) (table 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 37 table 2. mean intensity and prevalence with determined protozoan parasites in cyprinid aquaculture facilities in macedonia parasite species mean intensity prevalence (%) 1 apiosoma piscicola 4.241 6.996 2 chilodonella hexasticha 1.800 0.441 3 myxobolus müelleri 3.400 0.441 4 myxobolus encephalicus 4.000 0.794 5 thelohanellus nikolskii 2.444 3.086 6 ichthyophthirius multifiliis 26.479 4.233 7 trichodina sp. (cyprinus carpio) 4.567 30.159 8 trichodina sp. (ctenopharingodon idella) 7.556 0.794 fig. 1. mean intensity with determined protozoan parasites in cyprinid aquaculture facilities in macedonia fig. 2. prevalence with determined protozoan parasites in cyprinid aquaculture facilities in macedonia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 38 table 3. an infestation of protozoan parasites during the seasons season number of examined fish number of infected fish % of infected fish spring 278 162 58.273 summer 279 109 39.068 autumn 289 103 35.640 winter 288 159 55.208 representatives of protozoa most often occur during spring (in 58.273% of examined fish), then in winter (55.208%), summer (39.068%), and less in autumn (35.640%). out of the total number of examined fish, 533 fish were infected with protozoan parasites. by seasons, the number of infected fish is spring 162; summer 109; autumn 103 and winter 159 fish (table 3). table 4. prevalence (p) and mean intensity (i) with protozoan parasites in cyprinid aquaculture facilities in macedonia, by seasons parasite species spring summer autumn winter i p (%) i p (%) i p (%) i p (%) apiosoma piscicola / / / / / / 4.241 6.966 chilodonella hexasticha 1.800 0.441 / / / / / / myxobolus müelleri 3.400 0.441 / / / / / / myxobolus encephalicus 4.000 0.794 / / / / / / thelohanellus nikolskii / / 2.444 3.086 / / / / ichthyophthirius multifiliis 12.556 0.794 26.706 2.998 50.00 0.441 / / trichodina sp. (cyprinus carpio) 4.552 11.816 4.667 3.439 4.404 7.848 4.725 7.055 trichodina sp. (ctenoph. idella) / / / / 7.556 0.794 / / during our research, we noticed that the dynamics of parasite occurrence in fish varies depending on the parasite species and the season. during the three-year research: 5 protozoan parasites species were identified in the spring, as follows: chilodonella hexasticha, myxobolus müelleri, myxobolus encephalicus, ichthyophthirius multifiliis, and trichodina sp. (in common carp); 3 protozoan parasites species were found in the summer: thelohanellus nikolskii, ichthyophthirius multifiliis, and trichodina sp. (in common carp); 3 protozoan parasites species were found in the autumn: ichthyophthirius multifiliis, trichodina sp. (in common carp), and trichodina sp. (in grass carp); 2 protozoan parasites species were found in the winter: apiosoma piscicola and trichodina sp. (in common carp). the highest prevalence (11.816 %) is confirmed with trichodina sp. (in common carp) in spring, while the lowest (0.441 %) with chilodonella hexasticha and myxobolus müelleri in spring, as well as, ichthyophthirius multifiliis in autumn. the highest mean intensity (50.000) is determined with ichthyophthirius multifiliis in autumn, while the lowest (1.800) with chilodonella hexasticha in spring (table 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 39 table 5. total prevalence and mean intensity with determined protozoan parasites in common carp (cyprinus carpio) parasite species number of infected fish mean intensity prevalence (%) 1 apiosoma piscicola 79 4.241 8.246 2 chilodonella hexasticha 5 1.800 0.522 3 myxobolus muelleri 5 3.400 0.522 4 myxobolus encephalicus 9 4.000 0.939 5 thelohanellus nikolskii 35 2.440 3.653 6 ichthyophthirius multifiliis 34 26.706 3.549 7 trichodina sp. 342 4.567 35.699 *тhe calculations are based on the total number of examined samples of common carp (cyprinus carpio) (958) table 6. total prevalence and mean intensity with determined protozoan parasites in grass carp (ctenopharyngodon idella) parasite species number of infected fish mean intensity prevalence (%) 1 ichthyophthirius multifiliis 14 25.929 18.421 2 trichodina sp. 9 7.556 11.842 *тhe calculations are based on the total number of examined samples of grass carp (ctenopharyngodon idella) (76) table 7. total prevalence and mean intensity with determined protozoan parasites in bighead carp (hypophtalmichthys nobilis) parasite species number of infected fish mean intensity prevalence (%) 1 ichthyophthirius multifiliis 5 50.00 9.434 *тhe calculations are based on the total number of examined samples of bighead carp (hypophtalmichthys nobilis) (53) by fish species, the highest prevalence (35.699 %) in common carp is confirmed with trichodina sp., while the lowest (0.522 %) with chilodonella hexasticha and myxobolus müelleri. the highest mean intensity (26.706) is determined with ichthyophthirius multifiliis, while the lowest (1.800) with chilodonella hexasticha (table 5). in grass carp, the highest prevalence (18.421 %) and mean intensity (25.929) are determined with ichthyophthirius multifiliis (table 6). ichthyophthirius multifiliis is the only protozoa found in bighead carp with a prevalence of 9.434 % and a mean intensity, 50.000 (table 7). in this paper, for the first time, we published the result for the first finding of chilodonella hexasticha in common carp in macedonia. at the same time, common carp is a new host for the chilodonella hexasticha in macedonian waters. in our research, chilodonella hexasticha was found on the gills of 5 specimens of common carp in a fish farm in dolneni, in spring. the prevalence with chilodonella hexasticha in common carp was is 0.522 %, while the mean intensity is 1.800 (table 5). the body of chilodonella hexasticha is typically oval or leaf-shaped, dorsoventrally compressed, and is characterized by the presence of a groove at the end of the front of the body. it has a length of 38.4 (28.5 48.3) μm and a width of 31.7 (24.2 -39.2) μm. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 40 cytoplasm is ventrally granulated. the cytostome occurs in front of the naked area (zone without cilia). it leads to visible cytopharynx. the cytopharynx is protruding and secured with 8 to 10 visible cuticular bands, forming a funnel-shaped tube with a curved inner end. the cytopharynx may be slightly extruded and may form and destroy epithelial cells. two contractile vacuoles are present. the macronucleus is round, 15 (13.5 16.5) μm long, and 12.7 (11.2 14.2) μm wide. the micronucleus is located near the macronucleus and has a length of 4.3 (3.5 5.1) μm and a width of 3.5 (2.5 4.4) μm. according to literary reviews from the neighboring countries, the presence of chilodonella hexasticha was established in waters in serbia by [9]. according to the world's literary reviews, data on the appearance of representatives of the genus chilodonella in common carp in fishponds in iraq were published by [10], [11] and [12]. [13] found huge mortality in juveniles and adult carp in fishponds in sri lanka as a result of the presence of this protozoan. the protozoan chilodonella hexasticha is the causative agent of the parasitic disease chilodonellosis that attacks the skin, fins, and gills of common carp fry and fingerling up to 1 year of age. adult fish appear as carriers of the parasite. in [14] it stated that these cilia are morphologically well adapted to adhesion to the body surface and gills of fish and have a rapidly projected cytostomal opening. therefore, they are obligate parasites in fish, causing direct damage by disrupting epithelial cells. [15] reported that infestations with chilodonella hexasticha have been identified in both warm-water and cold-water fish worldwide, causing high mortality. in severe infestations with this parasite, the fish are weakened and dark discoloration is observed on the skin. [16] considered that as a result of the parasitism of chilodonella hexasticha, severe damage to the gills occurs in the form of epithelial hyperplasia, which disrupts the fine respiratory epithelium, leading to fish mortality. one of the symptoms of this disease is the swimming of fish on the surface of the water due to a lack of oxygen. gray-blue deposits consisting of mucus and necrotic epithelial cells appear on the skin, fins, and gills. as a result, the fish itches from objects in the water, without taking food. mortality depends on the fish size and the infestation degree. fig.3 apiosoma piscicola in common carp (cyprinus carpio) (original) fig. 4. myxobolus encephalicus in common carp (cyprinus carpio) (original) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 41 fig.5. trichodina sp. in grass carp (ctenopharyngodon idella) (original) fig.6. trichodina sp. in common carp (cyprinus carpio) (original) 4. conclusion eight representatives of protozoan parasites in farmed cyprinid fish were identified in this study: apiosoma piscicola, chilodonella hexasticha, myxobolus müelleri, myxobolus encephalicus, thelohanellus nikolskii, ichthyophthirius multifiliis, trichodina sp. (in common carp), and trichodina sp. (in grass carp). in cyprinid aquaculture facilities, based on the total fish examined, the highest prevalence with protozoan parasites is confirmed with trichodina sp. (in common carp) while the lowest with chilodonella hexasticha (0.441%) and myxobolus müelleri (0.441%). the highest mean intensity is determined with ichthyophthirius multifiliis (26.479), while the lowest with chilodonella hexasticha (1.800). the finding of chilodonella hexasticha in common carp (cyprinus carpio) as a first record for macedonian waters, is first published in this paper. in our research, infestation with chilodonella hexasticha was determined in fish with reduced general resistance (unfavorable hygienic conditions, overpopulation, poor nutrition, skin injuries) in early spring, which correlates with literature data that the optimum temperature for chilodonella development is about 10°c. during the warm, summer months this parasite does not occur. it is most often observed in young carp in winterings or in spring when the fish are kept in these facilities for too long time. during this period, the fish are weakened and the low water temperatures are favorable for the development and multiplication of the parasite. representatives of the protozoa were identified in all seasons, but the highest infestation was observed during spring and winter. during our research, we found that protozoa represent a huge problem in cyprinid aquaculture facilities, where high temperatures and organic matter content accelerate the life cycle of parasites and support their spread. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 dijana blazhekovikj – dimovska, stojmir stojanovski, protozoan distribution in farmed cyprinid fish from macedonia, food and environment safety, volume xx, issue 1 – 2021, pag. 35 – 42 42 5. references [1] bondad-reantaso g. m., subasinghe r. p., arthur r. j., ogawa k., chinabut s., adlard r., tan z., shariff m. (2005): disease and health management in asian aquaculture, veterinary parasitology, 132, pp. 249 – 272. [2] sterud e., simolin p., kvellestad a. (2003): infection by parvicapsula sp. (myxozoa) is associated with mortality in sea caged atlantic salmon salmo salar in northern norway. diseases of aquatic organisms, 54(3): 259-63. [3] enayat s.r. mohamed m. el-naggar, nagwa m. ali (2008): “trichodinid ectoparasites (ciliophora: peritrichida) infecting the nile cichlid fishes sarotherodon galilaeus and tilapia zillii at dakahlia province, egypt”. egypt j. zool., 51: 199 -219. [4] hristovski m., stojanovski s. (2005): biologija, odgleduvanje i bolesti na ribite, nacionalen forum za zastita na zivotnite na makedonija, skopje. [5] woo p.t. (2006): fish diseases and disorders, vol. 1: protozoan and metazoan infections. cab international, london: pp.791. [6] el-tantawy s.a.m, el-sherbiny h.a.e. (2010): some protozoan parasites infecting catfish clarias gariepinus inhabiting nile delta water of the river nile, dakahlia province, egypt, journal of american science; 6 (9). [7] bush a.o., lafferty k.d., lotz j.m. and shostak a.w. (1997): parasitology meets ecology on its own terms: margolis et al. revisited. j. parasitology., 83: 575583. [8] lom j., dykova i. (1992): protozoan parasites of fishes. elsevier, amsterdam and london. pp. 315. [9] cakić p. (1992): paraziti riba u vodama sjeniţko-pešterske visoravni i mogucnosti njihovog suzbijanja. doktorska disertacija. veterinarski fakultet univerziteta u beogradu. katedra za parazitologiju. [10] ali n. m., salih n. e., abdul-ameer k. n. (1988): protozoa and crustacea infesting three species of carp raised in ponds in iraq, journal of biological science research, vol. 19 no. 2, pp. 387-394. [11] al-marjan s.n. karwan, abdullah m.a. shamall (2008): some ectoparasites of the common carp (cyprinus carpio) in ainkawa fish hatchery, erbil province, the 2nd kurdistan conference on biological sciences j. duhok univ. vol.12, no.1 (special issue), pp. 102-107. [12] mansoor n.t., al-shaikh s.m.j. (2010): protozoans infection of cyprinus carpio l. from bab al-muatham fish markets, baghdad city, iraqi journal of veterinary medicine vol. 34, no. 1. [13] subasinghe r. p. (1992): hatchery diseases of freshwater fish in sri lanka. in, diseases in asian aquaculture 1, edited by m. shariff, r. p. subasinghe and j. r. arthur, asian fisheries society, philippines. [14] reda e. s. a. (2011): a review of some ecto-and endo protozoan parasites infecting sarotherodon galilaeus and tilapia zillii from damietta branch of river nile, egypt, journal of american science; 7 (3). [15] van as j. g., basson l. (1988): the incidence and control of fish ectoparasitic protozoa in south africa. technical communication, no. 211 republic of south africa. [16] paperna i., van as j.g. (1983): the pathology of chilodonella hexasticha (kiernik). infection in cichlid fishes. j. fish. biol. 23: 441450. 1. introduction doi: https://doi.org/10.4316/fens.2022.027 273 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 273 291 functional, pasting and sensory properties of complementary food from orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour using response surface methodology omobolanle omowunmi olorode 1 , *emmanuel kehinde oke 2 , pius alaba agbebi 3 , adebola atinuke alabi 3 , femi fidelis akinwande 4 1department of food technology, moshood abiola polytechnic, abeokuta, nigeria 2department of food science and technology, federal university of agriculture, abeokuta, nigeria, kennyoke35@gmail.com 3department of hospitality, leisure and tourism, moshood abiola polytechnic, abeokuta, nigeria 4department of food technology, yaba college of technology, lagos, nigeria *corresponding author received 2nd september 2022, accepted 13th december 2022 abstract: this study investigated the functional, pasting and sensory properties of complementary food from blends of orange fleshed sweet potato, roasted african yam bean and tigernut composite flour using response surface methodology. orange fleshed sweetpotato root, roasted african yam bean and tigernut seed were blended into composite flours based on d-optimal mixture design resulting into fourteen experimental runs. the orange fleshed sweetpotato, roasted african yam bean and tigernut flour blends were analyzed for their functional and pasting properties using standard laboratory procedures. the composite flours were optimized using d-optimal of the mixture design. the optimized composite flours were used to prepare complementary food and sensory properties (taste, colour, aroma, thickness and overall acceptability) were evaluated using nine point hedonic scale of preference. the bulk density, water absorption capacity, swelling power, dispersibility, least gelation capacity and wettability ranged from 0.82-0.85g/ml, 300.00-359.00%, 6.00-6.51%, 60.20-65.99%, 2.72-3.69% and 22.71to 40.81% respectively. significant differences (p<0.05) were observed in the pasting properties of orange fleshed sweetpotato, roasted african yam bean and tigernut flour blends. however optimized complementary prepared from the different composite flours were all accepted by the panelists except complementary food prepared from 82.95% orange fleshed sweetpotato, 10.26% roasted african yam bean and 6.78% tigernut composite flour blend. keywords: complementary food, orange fleshed sweet potato, roasted african yam bean, tigernut flour 1. introduction complementary foods are foods given to infants in addition to breast milk when breast milk nutrients become insufficient for their energy and growth needs [1, 2]. in developing west african countries such as nigeria, the introduction of complementary food typically starts between the ages of four to six months (i.e. after six months of exclusive breast feeding). the complementary food is usually in form of semi-liquid porridge prepared from staple cereals such as sorghum, maize and millet or tubers such sweet potato, cocoyam, irish potato etc. it has however been observed that during this period, infants between the ages of 611 months are given complementary foods two-four times daily which make it the major source of nutrients for the infants. complementary feeding is needed for both nutritional and developmental reasons, and is an important phase in the transition from milk feeding to family foods. the development of nutritious complementary foods from local and readily accessible raw materials has received considerable attention in many developing countries [2]. this is in response to the need of a highly nutritious food for infants. mailto:kennyoke35@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 274 orange fleshed sweetpotato is a conventionally bio-fortified crop with βcarotene contents of 30 – 100 ppm [4]. orange fleshed sweetpotato has considerable antioxidant activity and can potentially improve vitamin a level in growing children in developing countries [5, 6, 7]. it is rich in carotenoids and βcarotene (containing 24.2 – 73.9mg/100 g of β-carotene on dry basis), a precursor of vitamin a [8] which protects the body from several diseases, such as night blindness and other serious diseases. orange fleshed sweetpotato roots are source of fibre, starch, sugar, vitamin a, iron, calcium and several other minerals [9, 7]. african yam bean (ayb) (sphenostylis stenocarpa) has been observed by researchers [10, 11] to be one of the less utilized legumes that are gradually going into extinction; this was reported to be probably due to the long cooking time and its taste. it was however suggested to be used in the preparation of other plant based products which can be imitated milk, milk shake, flours, candy, gruel and cheese. adewale et al. [12] and idowu [11] observed and reported that the vast genetic and economic potentials of ayb have been recognized most especially in reducing malnutrition among africans but the crop has not received adequate research attention. ayb is highly nutritious with protein, mineral and fibre contents. its protein content is reported to be similar to that of some commonly consumed legumes and its amino acid (such as lysine, tryptophan) is comparable and even better than those of cowpea, soy bean and pigeon pea according to [13]. ade-omowaye et al. [14] and soetan et al. [15] reported that ayb contain several bioactive compounds such as polyphenols and flavonoids which are beneficial to consumers for the maintenance of a good and healthy lifestyle. tigernut (cyperus esculentus) is a high dietary fibre tuber crop which could be utilized in the treatment and prevention of many disease including colon cancer, coronary heart diseases, heart diseases, obesity and gastro intestinal disorder as reported by [16] and [17]. its flour has been observed to be rich source of quality oil, vitamin e and some useful minerals such as iron and calcium which are essential for body growth and development [18, 19]. due to its high amino acids contents, tigernut has inherent nutritional and therapeutic advantage which could serve as a good alternative to cassava in baking [20] and other food processes. in many developing countries, traditional weaning foods are prepared mainly from cereals such as maize, sorghum [21] and millet. these cereal crops have been reported to be poor in protein quantity and quality. it has also been reported by [22] and [23] that high cost, viscous nature of commercially available complementary foods and poor hygiene of food handlers are the major constraints in the provision of complementary food of adequate nutrients for children. it is, therefore necessary to study ways and means of developing inexpensive but equally nutritious complementary food that may be within the reach of the under privilege, using locally available cereals, legumes and easy means of technologies in its production. the formulation of nutritious complementary foods from local and readily available raw materials such as orange flesh sweetpotato, african yam bean and tigernut is expected to provide protein based weaning foods that are of high quality and inexpensive. hence, the overall objective of this study was to determine the functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato, roasted african yam bean and tigernut food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 275 composite flour using response surface methodology 2. materials and methods materials orange fleshed sweetpotato roots were obtained from the research farm of federal university of agriculture, abeokuta while african yam bean and tigernut (yellow variety) were purchased from kuto market, abeokuta, ogun state, nigeria production of orange fleshed sweetpotato flour the orange fleshed sweetpotato flour was produced according to the standard method of singh et al. [24] described by alawode et al. [2]. the orange fleshed sweetpotato tubers were peeled and cut into thin slices manually with sharp knives. the sweetpotato slices were soaked in 1% potassium metabisulphite solution for a period of five minutes to avoid browning and to also intensify its colour. they were dried in a cabinet dryer set at 55 °c for 24 hours. the dried orange fleshed sweet potato chips were then milled into flour using the laboratory hammer mill and the milled flour sieved using 250 μm screen to obtain the flour. the flour was packaged in a sealed plastic film and stored in a properly closed plastic container till further use. production of roasted african yam bean flour the modified method described by idowu [11] and okolie et al. [25] was used for roasted african yam bean seed. the african yam bean seeds were cleaned to remove extraneous elements such as stones, deformed bean and pest-infested beans. the seeds were washed and steeped in tap water for 72 hours and dehulled. the dehulled seeds were dried in the cabinet dryer at 60°c for 24 hours and then roasted using a gas cooker until the seeds were cracked. the seeds were dry milled after cooling into flour using the laboratory hammer mill and then sieved with 250 μm screen. the flour was packaged in an air tight plastic film and stored in a tightly closed plastic container until further use. production of tigernut flour tigernut flour was produced by the method described by ade-omowaye et al. [26] and oke et al. [27]. after removing stones, pebbles, and other unknown materials from the tigernut (cyperus esculentus), they were washed under running tap water. the cleaned nuts were then dried in a cabinet dryer at 60°c for 72 hours. the dried nuts were milled in a laboratory hammer mill and sample was sieved (using 250μm screen) to obtain the flour. the tigernut flour was packed and sealed in a airtight polyethylene bags until further use. flour blends formulation d-optimal mixture design was used for the design of experiment with three independent variables, including orange fleshed sweetpotato flour, roasted african yam bean flour and tigernut flour using design expert (version 6.0.8). the levels of each variable were established based on a series of preliminary experiments resulting in a total of 14 experimental runs (table 1). flour from orange fleshed sweetpotato, roasted african yam bean and tigernut were blended together using phillips blender (model hr-1702) and packaged in polythene bags at ambient temperature (26±2 °c) until further analysis. functional properties of orange fleshed sweetpotato-roasted african yam beantigernut composite flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 276 bulk density the bulk density of the composite flour was determined using the method described by akpapunarn and markakis [28] and ohizua et al. [29]. approximately ten grams of the composite flour was adequately weighed into a 50 ml graduated measuring cylinder. the composite flour was packed by tapping the cylinder gently on the bench top up to 10 times from a height of 5cm. the volume of the composite flour was thereafter recorded. table 1: orange fleshed sweetpotato, roasted african yam bean and tigernut formulations using d-optimal mixture design runs ofsp rafb tf 1 80.00 15.00 5.00 2 82.50 10.00 7.50 3 80.83 10.83 8.33 4 80.00 10.00 10.00 5 83.33 10.83 5.83 6 80.83 13.33 5.83 7 85.00 10.00 5.00 8 85.00 10.00 5.00 9 80.00 10.00 10.00 10 80.00 15.00 5.00 11 80.00 12.50 7.50 12 80.00 12.50 7.50 13 82.50 12.50 5.00 14 81.67 11.67 6.67 ofsp: orange fleshed sweetpotato, rafbroasted african yam bean, tf: tigernut flour swelling power (sp) the swelling power was determined using the method described by oke et al. [17]. composite flour of absolutely one gram was weighed into a 50ml centrifuge tube. after which 50ml of distilled water was added and mixed gently together. the mixture was heated in a water bath at 90 ºc for 15 minutes with gentle stirring to prevent clumping of the flour. thereafter, the tube containing the paste was centrifuged at 3,000rpm for 10 minutes using a centrifuge machine. the supernatant was decanted immediately after centrifuging; the weight of the sediment was taken and recorded. the moisture content of sediment gel was then determined to get dry matter content of the gel. water absorption capacity (wac) the method described by onwuka [30] was used for the determination of the water absorption capacity of the composite flour. about one gram of the composite flour was weighed into a 15 ml centrifuge tube and suspended in 10 ml water. it was vigorously shaken on a platform tube rocker for 1 minute at room temperature. the sample was allowed to stand for about 30 minutes and centrifuged at 1200 x g for 30 minutes. the volume of free water was then read directly from the centrifuge tube. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 277 dispersibility the method described by kulkarni et al. [31] and ohizua et al. [29] was adopted for the determination of the dispersibility of the composite flour. about ten grams of each sample was suspended in 200 ml measuring cylinder and distilled water was added up to 100 ml mark. it was then stirred vigorously and allowed to settle for 3 hr. the volume of the settled particles was thereafter recorded and subtracted from 100. the difference was reported as percentage dispersibility. wettability the method described by onwuka [30] and iwe et al. [32] was adopted for the determination of the wettability of the composite flours. one gram of each flour sample was weighed and added into a 25 ml graduated measuring cylinder of 1cm diameter. finger was then placed over the open end of each cylinder, inverted and clamped at a height of 10cm from the surface of a 600 ml beaker containing 500ml distilled water. the finger was then removed and the test sample was allowed to dump. the value of the wettability was recorded as the time required for the sample to become completely wet. least gelation concentration (lgc) this was determined according to the method described by coffman and garcia [33]. sample suspensions of between 220% (w/w) were prepared in distilled water and the dispersions were transferred into a test tube. it was heated in a boiling water bath for about 1 h, followed by a rapid cooling in a cold water bath. the test tubes were further cooled at 4 ºc for 2 h. the least gelation concentration of the sample was determined as the concentration when the sample from the inverted test tube did not slip or fall. pasting properties of orange fleshed sweetpotatoroasted african yam beantigernut composite flour these were determined with the aid of a rapid visco analyzer (rva instrument). absolutely three grams of each sample were weighed into a dried empty canister and 25ml of distilled water was dispensed into the canister containing the sample, the suspension was mixed properly to avoid lumps formation and the canister was fitted into the rapid visco analyzer. a paddle was placed into the canister and immediately automatically plotting the characteristic curve as the process proceeds. the parameters estimated in the process were peak viscosity, setback viscosity, final viscosity, trough, breakdown viscosity, pasting temperature and time to reach peak viscosity. preparation of orange fleshed sweetpotatoroasted african yam bean complementary food complementary foods from orange fleshed sweetpotatoroasted african yam beantigernut composite flour were prepared according to the method described by opara et al. [34] with little modification in the addition of boiled water. complementary foods in form of gruels were prepared by reconstituting 60 g of each of the composite flours in 100 ml of clean water. about 60 ml of boiled water was added to the suspension to produce hot gruel, and this was brought to a boil for 5 min, cooled, and served to a panel of 25 members. sensory evaluation of complementary food from orange flesh sweetpotato african yam bean-tigernut food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 278 complementary foods in form of gruels were served to 25 members of panel comprising majorly of mothers among students and members of staff of moshood abiola polytechnic, abeokuta. they were asked to score the gruels on a 9-point hedonic scale of preference where 9 = like extremely; 5 = neither like nor dislike; 1 = dislike extremely. sensory qualities evaluated were: taste, colour, aroma, thickness and overall acceptability [35] statistical analysis data except sensory scores were obtained in triplicate and subjected to statistical analysis using spss version 21.0. the means, analysis of variance and the difference between the mean values were evaluated at p<0.05 using duncan multiple range test. the investigation of the optimization procedure was done using design expert version (6.0.8) software which was used to predict the effects of the variation of composite flour. the statistical parameters, including the adjusted coefficients of determination (adjusted r2), coefficient of determination (r2) and regression (f value) were used to evaluate and select the best-fitting mathematical method [36]. the design was expressed by the polynomial regression equation to generate the model as shown below: yi = βₒ + β1x1 + β2x2 + β3x3 + β4x4 − β11x12 + β22x2+ β33x32 + β44x42 + β12x1x2+ β13x1x3 + β14x1x4 + β23x2x3 + β24x2x4 + β34x3x4 where yi is the predicted response; βₒ, βi, and βii are the linear coefficient and interaction coefficient. the suitable polynomial equations for the design, such as linear, quadratic, or special cubic models, were chosen according to the fittest model. in order to view the interaction of the response variables and the factor variables, the mixture design space and three-dimensional (3d) surface plots of the fitted polynomial regression were generated. 3. results and discussion functional properties of orange fleshed sweetpotatoroasted african yam beantigernut composite flour functional properties of orange fleshed sweetpotatoroasted african yam beantigernut composite flour are shown in table 2. bulk density depicts the behaviour of the material in dry mixes and is an important parameter that can determine packaging requirement of the product [37, 38]. the bulk density of the orange flesh sweetpotato african yam bean-tigernut composite flour ranged from 0.82 to 0.85g/ml. as presented in the regression table (table 3), the interaction effect of orange flesh sweetpotato, roasted african yam and tigernut flour were not significantly (p>0.05) affected which might be due to the differences in the particle size since the composite flours were prepared using the same processing equipment. however, adebowale et al. [39] and adegunwa et al. [40] reported that bulk density of flour is usually affected by its particle size and also by density of the flour blend which always have significant application in packaging, transportation and raw material handling. the low bulk density of the composite flour observed in this study would be of advantage in the formulation of complementary food. this is due to the fact that low density has been reported [41, 2] to be of advantage in the formulation of easily digestible food. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 279 table 2: functional properties of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour ofsp rafb tf bulk density (g/ml) water absorption capacity (%) swelling capacity (%) dispersibility (%) least gelation capacity (%) wettabil ity (sec.) 80.00 15.00 5.00 0.841g 306.50b 5.67c 63.78d 3.55d 37.61e 82.50 10.00 7.50 0.848h 329.50de 5.73d 64.78f 3.36c 28.75c 80.83 10.83 8.33 0.834d 359.00g 5.82e 63.98d 2.82a 22.81a 80.00 10.00 10.00 0.821a 334.50e 5.09b 65.99g 3.15bc 26.69b 83.33 10.83 5.83 0.834d 340.50f 6.19g 60.20a 3.69d 40.81f 80.33 13.33 5.83 0.836e 300.00a 5.00a 63.98d 3.05b 29.58c 85.00 10.00 5.00 0.834d 318.50c 5.79e 65.19c 3.23bc 32.63d 85.00 10.00 5.00 0.834d 318.50c 5.79e 65.19c 3.23bc 32.63d 80.00 10.00 10.00 0.821a 334.50e 5.09b 65.99g 3.15bc 26.69b 80.00 15.00 5.00 0.841g 306.50b 5.67c 63.78d 3.55d 37.61e 80.00 12.50 7.50 0.834d 327.50d 5.05ab 64.38e 2.72a 22.71a 80.00 12.50 7.50 0.834d 327.50d 5.05ab 64.38e 2.72a 22.71a 82.50 12.50 5.00 0.828c 305.00ab 6.51h 62.49b 3.62d 37.39e 81.67 11.67 6.67 0.825b 320.50c 6.12f 63.40c 3.19bc 36.60e mean values with different superscripts within the same column are significantly different (p <0.05); ofsp: orange fleshed sweetpotato flour, rafb-roasted african yam bean flour, tf: tigernut flour, table 3: regression coefficient of functional properties of orange fleshed sweetpotato-roasted african yam beantigernut composite flour parameters bulk density wac swelling capacity dispersibility least gelation capacity wettability a (x1) 0.88 320.26 5.80 62.89 3.28 33.39 b(x2) 4.88 303.20 5.55 64.01 3.52 36.81 c(x3) 0.87 337.12 5.15 66.01 3.12 25.86 ab -8.47 -25.06 2.73 -6.12 0.94 20.99 ac 1.23 52.51 1.87 -2.66 0.69 8.05 bc -8.30 38.73 -1.31 -2.38 -2.70* -34.96 r2 0.4432 0.6570 0.7263 0.6958 0.8650 0.7867 adj r2 0.095 0.4426 0.5552 0.5057 10.26 0.6534 f-value 1.27 3.06 4.25 3.66 0.7807 5.90 *significant at p<0.05; a (x1) – orange fleshed sweetpotato flour, b (x2)-roasted african yam bean flour, c (x3) – tigernut flour, wac: water absorption capacity; amain effect of orange fleshed sweetpotato flour, b-main effect of roasted african yam bean flour, cmain effect of tigernut flour, abinteraction effect of orange fleshed sweetpotato and roasted african yam bean flour, acinteraction effect of orange fleshed sweetpotato and tigernut flour, bcinteraction effect of roasted african yam bean and tigernut flour, r2coefficient of determination the surface plot for the effect of bulk density on orange fleshed sweetpotato roasted african yam bean-tigernut composite flour is illustrated in figure 1. water absorption capacity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour ranged from 300.00 to 359.00%. the regression coefficient is shown in table 3 where it was observed that the quadratic model developed for water absorption capacity has the coefficient of determination (r2) of 0.6570 and f-value of 3.06. the regression coefficient showed that the main effect and the interaction effect of orange fleshed sweetpotato, roasted african yam bean and tigernut flour were not significantly (p>0.05) affected. figure 2 shows the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 280 response surface plots for the water absorption capacity of composite flour from orange flesh sweetpotato, roasted african yam bean and tigernut flour. water absorption capacity signifies the ability of a product to associate with water under limited water conditions [42]. the values obtained for water absorption capacity of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour varies from each other, this might be due to the differences in the protein and starch contents of the flour blends [43, 44]. however, fekria et al. [45] reported that flours with high water absorption capacity have more hydrophilic constituents such as polysaccharides. swelling power (sp) is the ability of the flour to absorb water and hold it in the swollen flour granule [38]. the swelling capacity of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour ranged between 6.00 and 6.51%. as shown in the regression table, the quadratic model developed for swelling capacity has the coefficient determination (r2) of 0.7263 and f-value of 4.25. the regression table revealed that when tigernut flour was held constant, increase in orange flesh sweetpotato and roasted african yam bean caused an increase in the swelling capacity; this could be due to high amylose content likely to be present in the starch granules of orange fleshed sweetpotato flour. this implies that the composite flour will produce a thick viscous gruel in the preparation of complementary food [2]. the surface plot for the effect of swelling power on orange flesh sweetpotato-roasted african yam bean-tigernut composite flour is illustrated in figure 3. dispersibility is the ability of the flour blends to reconstitute in water. the dispersibility of composite flour from orange fleshed sweetpotato-roasted african yam bean x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 0.224825 1.38943 2.55403 3.71864 4.88324 b ul k d en si ty x1 (80.00) x2 (15.00) fig. 1: 3d surface plot showing the effect of flour blends on bulk density of orange fleshed sweetpotato roasted african yam bean-tigernut composite flours x1 (85.00) x2 (5.00) x3 (15.00) x3 (10.00) 296.676 309.036 321.395 333.754 346.113 w a c x1 (80.00) x2 (10.00) fig. 2: 3d surface plots showing the effect of flour blends on water absorption capacity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 4.99415 5.36669 5.73923 6.11177 6.48431 s we lli ng c ap ac ity x1 (80.00) x2 (15.00) fig. 3: 3d surface plot showing the effect of flour blends on swelling capacity of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 281 tigernut ranged between 60.20 and 65.99% the regression of coefficient has shown in table 3 shows that the coefficient of determination (r2) and f-value for dispersibility is 0.6958 and 3.66 respectively. the regression table shows that the main and the interaction effect were not significantly (p>0.05) affected on dispersibility of the flour blends. according to kulkarni et al. [31] and alawode et al. [2] the higher the dispersibility, the better the flour reconstitutes in water. however, the values of dispersibility obtained in this study for orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour are high, this implies they will easily reconstitute to give fine consistency of dough during mixing [39; 29] the surface plot for the effect of dispersibility on the composite flour is presented in figure 4. the least gelation concentration is a measure of the minimum amount of flour blends that is needed to form a gel in a measured volume of water [29]. the least gelation capacity of orange fleshed sweetpotato-roasted african yam beantigernut composite flour ranged from 2.72 to 3.69%. the quadratic model developed for the least gelation capacity has the coefficient of determination (r2) and fvalue of 0.8650 and 10.26 respectively. it was observed from the regression table that the interaction effect of african yam bean and tigernut flour had a negative significant (p<0.05) effect on the least gelation capacity of the composite flour. when tigernut was held constant, an increase in orange fleshed sweetpotato and roasted african yam bean caused an increase in the least gelation capacity. this could be as a result of roasted african yam bean which serve as a protein source, increases the protein concentration thereby enhancing the interaction among the binding forces which in turn increases the gelling ability of the flour [46]. however, it was reported by adebowale et al. [47] that gelation takes place more readily at higher protein concentration because of greater intermolecular contact during heating and that high protein solubility is always necessary for gelation. figure 5 shows the surface plot for the effect of least gelation capacity on the orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour. wettability is the measure of the rate at which flour samples get wetted in water. the wettability of orange fleshed sweetpotato-roasted african yam beantigernut composite flour ranged from 22.71 to 40.81%. the regression coefficient presented in table 3 shows that the quadratic model developed for wettability has the coefficient of determination (r2) of 0.7867 and f-value of 5.90 respectively. the interaction effect of roasted african yam bean and tigernut flour were significantly (p<0.05) affected negatively as shown in table 2. wettability of flour is an important indicator of instant characteristics of dried flours. in this study, it was observed that the wettability of the composite flour samples was observed to be high which is an indication of a slight reduction in instant characteristic of the flour as reported by udensi et al. [48] to be an indication for change in flour characteristic. figure 6 shows the surface plot for the effect of wettability on the orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour pasting properties of orange fleshed sweetpotato-roasted african yam beantigernut composite flour the pasting properties of orange fleshed sweetpotato-roasted african yam beantigernut composite flour are presented in table 4. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 282 x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 61.6468 62.7366 63.8263 64.9161 66.0058 d is pe rs ib ili ty x1 (80.00) x2 (15.00) fig. 4: 3d surface plot showing the effect of flour blends on dispersibility of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 2.63531 2.93706 3.23881 3.54056 3.8423 l gc x1 (80.00) x2 (15.00) fig. 5: 3d surface plot showing the effect of flour blends on least gelation capacity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour x1 (85.00) x2 (5.00) x3 (15.00) x3 (10.00) 21.7367 27.165 32.5934 38.0217 43.4501 w et ta bi lit y x1 (80.00) x2 (10.00) fig. 6: 3d surface plot showing the effect of flour blends on wettability of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour the peak viscosity of orange fleshed sweetpotato-roasted african yam beantigernut composite flour ranged from 718.00 to 864.5rvu. from the regression table 5, the quadratic model developed for peak viscosity has the coefficient determination (r2) of 0.9621 and f-value of 40.92 respectively. the regression table shows that interaction effects of orange fleshed sweetpotato and roasted african yam bean, roasted african yam bean and tigernut flour had a negative significant (p<0.05) effect on the peak viscosity of the composite flour. when tigernut flour was held constant, increase in orange fleshed sweetpotato and roasted african yam bean caused a decrease in the peak viscosity. peak viscosity is indicative of the strength of the pastes which are formed from gelatinization during processing in food application [49]. peak viscosity is often correlated with final product quality. it is the maximum viscosity developed during or soon after heating [50, 42]. it reflects the ability of starch to swell freely before their physical breakdown [51]. it has been suggested that high peak viscosity contributes to good texture of paste, which basically depends on high viscosity and moderately high gel strength [50]. the relatively high peak viscosity exhibited by orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour is an indication that the flour may be suitable for products requiring high gel strength and elasticity. figure 7 shows the surface plot for the effect of peak viscosity on the orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour trough is the minimum viscosity after the initial peak and occurs after the commencement of the sample cooling [42]. the trough viscosity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour ranged from 713.00 to 840.00rvu. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 283 table 4: pasting properties of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour ofsp rafb tf peak (rvu) trough (rvu) breakdown (rvu) final viscosity (rvu) setback (rvu) peak time (mins.) pasting temp (ºc) 80.00 15.00 5.00 864.50c 840.00c 24.50b 1188.50c 348.50c 5.73ab 84.85a 82.50 10.00 7.50 823.50bc 804.50bc 19.00ab 1131.00bc 326.50abc 5.34a 83.98a 80.83 10.83 8.33 788.50abc 783.00abc 5.00ab 1100.00abc 317.00abc 5.74ab 84.83a 80.00 10.00 10.00 779.50abc 778.00abc 1.50a 1087.50abc 309.50abc 6.47bc 85.65a 83.33 10.83 5.83 818.50bc 798.00bc 20.00ab 1129.50bc 331.00bc 5.87abc 84.00a 80.33 13.33 5.83 776.50abc 770.50abc 6.00ab 1085.00abc 314.50abc 6.47bc 85.70a 85.00 10.00 5.00 838.50bc 811.00bc 26.50b 1159.50c 347.50c 5.33a 85.68a 85.00 10.00 5.00 838.50bc 811.00bc 26.50b 1159.50c 347.50c 5.33a 85.68a 80.00 10.00 10.00 779.50abc 778.00bc 1.50a 1087.50abc 309.50abc 6.47bc 85.65a 80.00 15.00 5.00 864.50c 840.00c 24.50b 1188.50c 348.50c 5.73ab 84.85a 80.00 12.50 7.50 718.00a 713.00a 5.00ab 1001.00a 288.00a 6.70c 84.38a 80.00 12.50 7.50 718.00a 713.00a 5.00ab 1001.00a 288.00a 6.70c 84.38a 82.50 12.50 5.00 799.50abc 793.50abc 6.00ab 1108.00abc 314.50abc 6.34bc 85.60a 81.67 11.67 6.67 752.50ab 742.50ab 10.00ab 1036.00ab 294.00ab 6.60c 85.18a mean values with different superscripts within the same column are significantly different (p <0.05); ofsp: orange fleshed sweetpotato flour, rafb-roasted african yam bean flour, tf – tigernut flour, temp-temperature the regression coefficient shown in table 5 revealed that the quadratic model developed for trough has the coefficient of determination (r2) of 0.9452 and f-value of 27.62 respectively. the regression table shows that interaction effects of orange fleshed sweetpotato and roasted african yam bean, roasted african yam bean and tigernut flour were significantly (p<0.05) affected negatively. figure 8 shows the response surface plots for the trough viscosity of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour. the high trough viscosity obtained in this study is an indication that the paste can withstand breakdown during cooling because oke et al. [17] reported that the higher the trough viscosity, the greater the ability of the paste to withstand breakdown during cooling breakdown is a measure of the ease with which the swollen granules can be disintegrated [52]. the breakdown of composite flour from orange fleshed sweetpotato-roasted african yam beantigernut ranged between 1.50 and 26.50rvu. from the regression table, the regression of coefficient shows that the coefficient of determination (r2) and fvalue for breakdown is 0.9703 and 52.50 respectively as shown in table 5. the regression shows that interaction effects of orange fleshed sweetpotato and roasted african yam bean, roasted african yam bean and tigernut flour were significantly (p<0.05) affected negatively while interaction effect of orange fleshed sweetpotato and tigernut flour was significantly (p<0.05) affected positively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 284 increase in orange fleshed sweetpotato and roasted african yam bean led to decrease in the breakdown when tigernut flour was held constant. table 5: regression coefficient of pasting properties of orange fleshed sweetpotato-roasted african yam beantigernut composite flour parameters peak trough breakdown final viscosity setback peak time pasting temperature a (x1) 839.26 811.13 27.12 1160.04 347.89 5.34 85.49 b(x2) 863.15 839.30 23.86 1187.70 348.39 5.74 84.94 c(x3) 782.77 781.51 1.26 1092.56 311.04 6.39 85.66 ab -210.37* -134.12* -74.65* -264.11* -127.93* 3.77* 2.30 ac 83.35 59.16 25.78* 65.03 7.93 -2.27* -6.29* bc -411.59* -379.73* -32.01* -538.75* -158.89* 2.46* -2.52 r2 0.9621 0.9452 0.9703 0.9407 0.9343 0.8904 0.6321 adj r2 0.9384 0.9110 0.9517 0.9037 0.8933 0.8219 0.4021 f-value 40.92 27.62 52.50 25.40 22.76 13.00 2.75 *significant at p<0.05; a (x1) – orange fleshed sweetpotato flour, b (x2)-roasted african yam bean flour, c (x3) – tigernut flour, wac: water absorption capacity; amain effect of orange fleshed sweetpotato flour, b-main effect of roasted african yam bean flour, cmain effect of tigernut flour, abinteraction effect of orange fleshed sweetpotato and roasted african yam bean flour, acinteraction effect of orange fleshed sweetpotato and tigernut flour, bcinteraction effect of roasted african yam bean and tigernut flour, r2coefficient of determination x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 716.137 753.108 790.079 827.049 864.02 p ea k x1 (80.00) x2 (15.00) fig. 7: 3d surface plot showing the effect of flour blends on peak viscosity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour x1 (85.00) x2 (5.00) x3 (15.00) x3 (10.00) 713.49 746.736 779.982 813.228 846.474 t ro ug h x1 (80.00) x2 (10.00) fig. 8: 3d surface plot showing the effect of flour blends on trough viscosity of orange fleshed sweetpotato-roasted african yam beantigernut composite flour however, adebowale et al. (47) and oke et al. (17) reported that the higher the breakdown in viscosity, the lower the ability of starch sample to withstand heating and shear stress during cooking. this implies that large values of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour in this study indicate little breakdown of sample starches [42]. figure 9 shows the response surface plot for the effect of breakdown viscosity on the orange fleshed sweetpotato-roasted african yam beantigernut composite flour the final viscosity indicates the consistency at which the cooked paste of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 285 the flours is likely to be consumed [52]. the final viscosity of composite flour from orange fleshed sweetpotato-roasted african yam bean-tigernut ranged between 1001.00 and 1188.50 rvu. the quadratic model developed for the final viscosity content has the coefficient of determination (r2) and f-value of 0.9407 and 25.40 respectively. it was observed from the regression table that the interaction effects of orange fleshed sweetpotato and roasted african yam bean, roasted african yam bean and tigernut flour were significantly (p<0.05) affected negatively. when roasted african yam bean was held constant, increase in orange fleshed sweetpotato and tigernut flour caused an increase in the final viscosity. the variation in the final viscosity for orange flesh sweetpotato-african yam bean-tigernut composite flour might be due to the sample kinetic effect of cooling on viscosity and the re-association of starch molecules in the samples [53]. the final viscosity is an important parameter in predicting the final textural quality of food [54]. figure 10 shows the response surface plot for the effect of final viscosity on composite flour from orange fleshed sweetpotato-roasted african yam beantigernut. the setback viscosity is an index of retrogradation or re-ordering of the starch molecules. it has been correlated with texture of the food products [55; 17]. the setback of composite flour from orange fleshed sweetpotato-roasted african yam bean-tigernut ranged between 288.00 and 348.50rvu. from the regression table, the quadratic model developed for setback has the coefficient of determination (r2) of 0.9343 and fvalue of 22.76 as shown in table 5. the interaction effects of orange fleshed sweetpotato and roasted african yam bean, roasted african yam bean and tigernut flour were significantly (p<0.05) affected negatively as shown in table 5. at a constant african yam bean, while orange flesh sweetpotato and tigernut flour increases, the setback increases. the setback value obtained in this study was high, this implies the higher the setback value, the higher the rate of retrogradation [47]. soluble amylose is largely responsible for retrogradation during setback. the response surface plot for setback on composite flour from orange fleshed sweetpotato-roasted african yam bean-tigernut is depicted in figure 11. peak time is the time at which the peak viscosity occurred in minutes and it is a measure of the cooking time of the flour [17]. the peak time of composite flour from orange fleshed sweetpotato-roasted african yam bean-tigernut ranged between 5.33 and 6.70mins. the response surface plots for peak time on orange fleshed sweetpotato-african yam bean-tigernut composite flour are shown in figure 12. the regression table revealed that increase in orange fleshed sweetpotato and roasted african yam bean when tigernut flour was held constant which led to increase in the peak time of orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour, which is an indication of more processing time in the composite flour. the pasting temperature is the temperature at which the viscosity starts to rise [49]. the pasting temperature of composite flour from orange fleshed sweetpotato-roasted african yam beantigernut ranged between 83.98 and 85.68 ºc as shown in table 4. from the regression table (table 5), the quadratic model developed for pasting temperature has the coefficient of determination (r2) of 0.6321 and f-value of 2.75. the interaction effect of orange fleshed sweetpotato and tigernut flour had a negative significant (p>0.05) effect on the pasting temperature of orange fleshed food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 286 sweetpotato-roasted african yam beantigernut composite flour. at an increase in orange flesh sweetpotato and african yam bean and at a constant tigernut flour, the pasting temperature increase which is an indication of the gelatinization time during processing [17]. a higher pasting temperature implies higher water binding capacity and higher gelatinization [56]. the response surface plots for pasting temperature on orange fleshed sweetpotato-roasted african yam beantigernut composite flour are shown in figure 13. x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 0.571087 8.6308 16.6905 24.7502 32.81 b re ak d ow n x1 (80.00) x2 (15.00) figure 9: 3d surface plot showing the effect of flour blends on breakdown viscosity of orange fleshed sweetpotato-roasted african yam beantigernut composite flour x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 1001.32 1048.66 1096 1143.34 1190.69 f in al v is co si ty x1 (80.00) x2 (15.00) figure 10: 3d surface plot showing the effect of flour blends on final viscosity of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour x1 (85.00) x2 (5.00) x3 (15.00) x3 (10.00) 287.843 304.575 321.307 338.039 354.771 s et ba ck x1 (80.00) x2 (10.00) figure 11: 3d surface plot showing the effect of flour blends on setback viscosity of orange fleshed sweetpotato-roasted african yam beantigernut composite flour x1 (85.00) x2 (5.00) x3 (15.00) x3 (10.00) 4.82822 5.30182 5.77542 6.24903 6.72263 p .t im e x1 (80.00) x2 (10.00) figure 12: 3d surface plot showing the effect of flour blends on peak time of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 287 x1 (85.00) x2 (10.00) x3 (10.00) x3 (5.00) 83.8077 84.4252 85.0427 85.6602 86.2777 p .t e m p x1 (80.00) x2 (15.00) figure 13: 3d surface plot showing the effect of flour blends on pasting temperature of orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour optimization of orange fleshed sweet potato, roasted african yam bean and tigernut composite flour for complementary food the composite flour was optimized based on some important properties attributed to complementary food. bulk density, water absorption capacity, swelling capacity, dispersibility, least gelation capacity, wettability, peak, trough, breakdown, final viscosity, setback, peak time and pasting temperature were the major quality parameters studied in this research which were also the criteria based on desirability concept as well as their main quality parameters serving as the constraints to process optimization. the solution to the optimized orange flesh sweet potato, african yam bean and tigernut composite flour for complementary food is presented in table 6 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 288 sensory score for complementary food from optimized orange fleshed sweetpotato-roasted african yam beantigernut composite flour sensory scores for optimized and control complementary food quality attributes (taste, colour, aroma, thickness and overall acceptability) are shown in table 7. there were significant (p<0.05) differences in all the attributes evaluated. the scores for taste, colour, aroma, thickness and overall acceptability ranged from 6.15 to 6.60, 6.60 to7.10, 5.25 to 6.60, 5.70 to 7.00 and 5.65 to 7.30 respectively. complementary food prepared from 100% orange fleshed sweetpotato flour and optimized complementary food prepared from 81% orange fleshed sweetpotato flour, 10% roasted african yam bean flour and 8.85% tigernut flour had the highest degree of likeness followed by complementary food prepared from 81.06% orange flesh sweetpotato flour, 10% african yam bean flour and 8.94% tigernut flour while complementary food prepared from 82.95% orange fleshed sweetpotato flour, 10.26% roasted african yam bean flour and 6.78% tigernut flour had the lowest degree of likeness. hence optimized complementary prepared from orange fleshed sweetpotato-african yam beantigernut composite flour were all accepted by the panelist except complementary food prepared from 82.95% orange fleshed sweetpotato flour, 10.26% roasted african yam bean flour and 6.78% tigernut flour. 4. conclusion this study revealed that characterization of orange fleshed sweetpotato, roasted african yam bean and tigernut composite flour was significantly affected by the functional and pasting properties. however, acceptable complementary food can be obtained at an optimized condition of 81.15% orange fleshed sweetpotato, 10% african yam bean and 8.85% tigernut flour. 5. acknowledgments the authors acknowledged the support of the department of food technology, moshood abiola polytechnic, abeokuta, nigeria and department of food science and technology, federal university of agriculture, abeokuta, nigeria for allowing the research team to utilize their laboratories for this research work. 6. declaration of interest no potential conflict of interest was reported by the authors concerning this work. table 7: sensory score for complementary food from optimized orange fleshed sweetpotato-roasted african yam bean-tigernut composite flour ofsp rayb tf taste colour aroma thickness overall acceptability 100 0 0 6.55a 7.10a 6.30b 6.85b 7.30ac 80.00 14.88 5.12 6.60a 6.75a 6.60b 7.00b 6.65ac 81.15 10.00 8.85 6.15a 6.60a 6.05ab 6.90b 7.00bc 82.95 10.26 6.78 6.20a 6.70a 5.25a 5.70a 5.65a 81.06 10.00 8.94% 6.25a 6.60a 6.45b 6.20ab 6.30ab mean values with different superscripts within the same column are significantly different (p<0.05); ofsp: orange fleshed sweetpotato flour, rafb-roasted african yam bean flour, tf – tigernut flour food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 289 7. references [1]. onyekwere, e. u. development of maizebambara groundnut complementary foods fortified with foods rich in calcium, iron, zinc and provitamin a. m.sc. thesis, department of food science and technology, university of nigeria, nsukka, (2007) [2]. alawode, e.k., idowu, m.a., adeola, a.a., oke, e.k. & omoniyi, s.a. some quality attributes of complementary food produced from flour blends of orange flesh sweetpotato, sorghum, and soybean. croatian journal of food science and technology, 9 (2), 122 – 129, (2017) [3]. who. global strategy for infant and young child feeding world health organization, geneva pp 1-9, (2004) [4]. harvest plus. disseminating orangefleshed sweetpotato: uganda country report. washington, d.c.: harvest plus, (2012) [5]. hotz, c.c., loechl, a., de brauw, p., eozenou, d., gilligan, m., moursi, b., munhaua, p., van jaarsveld, a., carriquiry, j.v. & meenakshi. a largescale intervention to introduce orange sweet potato in rural mozambique increases vitamin a intakes among children and women. british journal of nutrition, 108, 163-176, (2012) [6]. laurie, s., faber, m., adebola, p. & belete a. biofortification of sweetpotato for food and nutrition security in south africa. food research international, 76, 962-970 (2015) [7]. browser, t.j., ojwang, f., sahs, r., & brandenberger, l. promotion of orange flesh sweetpotato by demonstration of acceptance and food product development. african journal of food science, 11(12), 383-388 (2017) [8]. alam, m.k., rana, z.h. & islam, s.n. comparison of the proximate composition, total carotenoids and total polyphenol content of nine orange-fleshed sweetpotato varieties grown in bangladesh. foods, 5(3), 64-65 (2016) [9]. oloo, b.o., shitandi, a., mahungu, s., malinga, j.b. & ogata, b.r. effects of lactic acid fermentation on the retention of betacarotene content in orange fleshed sweetpotatoes. international journal of food studies, 3, 13-33 (2014) [10]. klu, g.y.p., amoatey, h.m. bansa, d. & kumaga, f.k.. cultivation and uses of african yam bean (sphenostylis stenocarpa) in the volta region of ghana. journal of food technology african, 6, 74–77 (2001) [11]. idowu, a. o. nutrient composition and sensory properties of kokoro (a nigerian snack) made from maize and african yam bean flour blends. international food research journal, 22, 739–744 (2015) [12]. adewale, b.d., kehinde, o.b., aremu, c.o popoola, j.o. & dumet, d.j. seed metrics for genetic and shape determination in african yam bean. african journal of plant science, 4(4), 107–115 (2010) [13]. uguru, m.i. & madukaife, s.o. studies on the variability in agronomic and nutritive characteristics of african yam bean (sphenostylis stenocarpa hochst. ex. a. rich harms). plant product resource journal, 6, 10-19 (2001) [14]. ade-omowaye, b.i.o., tucker, g.a. & smetanska, i. nutritional potential of nine underexploited legumes in southwest, nigeria. international food research journal, 22, 798-806 [15]. soetan, k. o., olaiya, c. o. & karigidi, k. o. comparative in vitro antioxidant activities of six accessions of african yam beans (sphenostylis stenocarpa l.). annals food science & technology, 19 (2018) [16]. anderson, j.w., baird, p., davis, r.h., ferreri, s., knudtson, m. & koraym, a. health benefits of dietary fibre. nutrition review, 67, 188–205 (2009) [17]. oke, e.k, idowu, m.a. & omoniyi, s.a. proximate, functional, pasting and rheological properties of wheattigernut composite flour. annals food science and technology, 17(2), 411420 (2016) [18]. oladele, a. k., & aina, j. o. chemical composition and functional properties of flour produced from two varieties of tiger nut (cyperus esculentus). african journal of biotechnology, 6, 2473–2476 (2007) [19]. oke, e.k., idowu, m.a., adeola, a.a., abiola, t.o. & adeniji, i.o. evaluation and storage studies of sausage roll produced from wheat-tigernut flour blends. croatian journal of food technology, biotechnology and nutrition, 14 (3-4), 82-90 (2019) [20]. awolu, o.o., ifesan, b.o.t.,sodipo, m. a., ojewunmi, m.e., arowosafe, c.f. & oladeji, o. a. optimization of nutritional and pasting properties of rice sweetpotato based composite flour for biscuit production. applied tropical agriculture, 22(2), 143-149 (2017) [21]. gernah, d.i., ariahu, c.c. & ingbian, e.k. effects of malting and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 290 fermentation on some chemical and functional properties of maize (zea mays). american journal of food technology, 6 (5), 404 – 412 (2011) [22]. ashworth, a. & draper, a. the potential of traditional technologies for increasing the energy density of weaning foods. a critical review of existing knowledge with particular reference to malting and fermentation. who/cbdedp/92.4 (1992) [23]. gernah, d.i., ariahu, c.c. & ingbian, e.k. nutritional and sensory evaluation of food formulations from malted and fermented maize (zea mays) fortified with defatted sesame (sesamun indicum l.) flour. african journal of food, agriculture, nutrition and development, 12 (6), 6615 – 6631(2012) [24]. singh, s., ria, c.s., & saxena, d. c. effect of incorporating sweet potato flour to wheat flour on the quality characteristics of cookies. african journal of food science, 2, 65-7 (2008) [25]. okolie, p.i., coker, m.t., okolie, e.c., oke, e.k., agboola, k.a. & abayomi, s.o. physiochemical and quality evaluation of kokoro (a maize-based snack) made from blends of yellow maize, fermented african yam bean and rice flour. applied food research, 2, 100104 (2022) [26]. ade-omowaye, b.i.o., akinwande, b. a., bolarinwa, i. f., & adebiyi, a.o. evaluation of tigernut (cyperus esculentus)–wheat composite flour and bread. african journal of food science, 2, 87–89 (2008) [27]. oke, e.k., idowu, m.a., sobukola, o.p. & bakare, h.a. quality attributes and storage stability of bread from wheat–tigernut composite flour. journal of culinary science and technology, 1-14 (2017) [28]. akpapunarn, m.a. & markakis, d. physico-chemical and nutritional aspects of cowpea flour. journal of food science, 46, 972-973 (1981) [29]. ohizua, e.r., adeola, a.a., idowu, m.a., sobukola, o.p., afolabi, t.a., ishola, r.o., ayansina, s.o., oyekale, t.o. & falomo, a. nutrient composition, functional, and pasting properties of unripe cooking banana, pigeon pea, and sweetpotato flour blends. food science and nutrition, 00, 1-13 (2017) [30]. onwuka, g. i. food analysis and instrumentation: theory and practice. lagos, nigeria: naphtali prints (2005) [31]. kulkarni, k.d., kulkarni, d.n., & ingle, w.m. sorghum malted and soybean weaning food formulation: preparation, functional properties and nutritive value. food nutrition bulluletin, 13, 322–329 (1991) [32]. iwe, m.o., michael, n., madu, n.e., obasi, n.e. & onwuka, g.i. physicochemical and pasting properties of high quality cassava and wheat flour blends. agrotechnology, 6, 167 (2017) [33]. coffman, c.w. & garcia v.v. functional properties and amino acid content of protein isolate from mung bean flour. journal of food technology, 12, 473-484 (1977) [34]. opara, b.j., uchechukwu, n., omodamiro, r. m., & paul, n. production and sensory evaluation of food blends from maizeplantain-soybean as infant complementary food. journal of stored products and postharvest research, 3 (6), 75-79 (2012) [35]. iwe, m.o. hand book of sensory methods and analysis rojoint communication publishers ltd. enugu. pp.1-50 (2002) [36]. zainol, s., basri, m. & basri, h.b. formulation optimization of a palm-based nanoemulsion system containing levodopa. international journal of molecular sciences, 13(10), 13049–13064 (2012) [37]. mohammed, t.k., zhu, k., issoufou, a., fatmata, t. & zhou, h. functionality, in vitro digestibility and physicochemical properties of two varieties of defatted foxtail millet protein concentrates. international journal of molecular sciences, 10, 5224-5238 (2009) [38]. oke, e.k., idowu, m.a., sobukola, o.p. & bakare, h.a. nutrient composition, functional, physical and pasting properties of yellow yam (dioscorea cayenensis) and jackbean (canavalia ensiformis) flour blends. carpathian journal of food science, 12(5), 52-71(2020) [39]. adebowale, a.a., sanni, l.o. and fadaunsi, e. l. functional properties of cassava – sweet potato starch blend. proceeding of the 32nd annual conference of nigerian institute of food science and technology 304-305 (2008) [40]. adegunwa, m.o., adeniyi, o.d., adebowale, a.a. & bakare, h.a. quality evaluation of kokoro produced from maize-pigeon pea flour blends. journal of culinary science and technology, 13(3), 200-213 (2015) [41]. akpata, m.i., & akubor, p.i. chemical composition and selected functional properties of sweet orange (citrus sinesis) seed flour. plant food human nutrition, 54, 353-362 (1999) [42]. adegunwa, m.o., adelekan, e.o., adebowale, a.a., bakare, h.a. & alamu, e.o. evaluation of nutritional and functional properties of plantain (musa paradisiaca l.) and tigernut (cyperus esculentus l.) flour blends for food formulations. cogent chemistry, 3, 1383707 (2017) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande, functional, pasting and sensory properties of complementary food from orange fleshed sweetpotato roasted african yam bean-tigernut composite flour using response surface methodology, food and environment safety, issue 4 – 2022, pag.273 -291 291 [43]. omoniyi, s.a., awonorin, s.o., idowu, m.a. & adeola, a.a. physicochemical and functional properties of sweetpotatosoybean flour blends. applied tropical agriculture, 21 (2), 84-88 (2016) [44]. khuthadzo, n., mpho, e.m. & henry, s. physicochemical and functional properties of chemically pretreated ndou sweetpotato flour. hindawi international journal of food science, article id 4158213 (2019) [45]. fekria, a.m., isam, a.m.a., suha, o.a. & elfadil, e.b. nutritional and functional characterization of defatted seed cake flour of two sudanese groundnut (arachis hypogaea) cultivars. international food research journal, 19, 629-637 (2012) [46]. lawal, o.s. functionality of african locust bean (parkia biglobossa) protein isolate: effects of ph, ionic strength and various protein concentrations. food chemistry, 86, 345– 355(2004) [47]. adebowale, a. a., sanni, l.o. & awonorin, s. o. effect of texture modifiers on the physiochemical and sensory properties of dried fufu. food science and technology international, 11, 373–382 (2005) [48]. udensi, e.a., oselebe, h.o. & iweala, o.o. the investigation of chemical composition and functional properties of water yam (dioscorea alata): effect of varietal differences. pakistan journal of nutrition, 7(2), 342–344 (2008) [49]. eke-ejiofor, j. physico-chemical and pasting properties of starches from cassava, sweet potato and three leaf yam and their application in salad cream production. international journal of biotechnology and food science, 3(2), 23-30 (2015) [50]. linus-chibuezeh, a, iwe, m.o., ngadi, m., asumugha, v.u & obasi, n.e. evaluation of some physicochemical and pasting properties of three improved cassava varieties available in the southeast of nigeria. imperial journal of interdisciplinary research (ijir), 3 (9), 641-650 (2017) [51]. sanni, l.o., kosoko, s.b., adebowale, a.a. & adeoye, r.j. the influence of palm oil and chemical modification on the pasting and sensory properties of fufu flour. international journal of food properties, 7(2), 229–237 (2004) [52]. oppong, d., arthur, e., kwadwo, s. o., badu, e., & sakyi, p. proximate composition and some functional properties of soft wheat flour. int. journal of innovative research in science, engineering and technology, 4, 753–758 (2015) [53]. nwokeke, b., adedokun, i. & osuji, c. effect of blending on the proximate, pasting and sensory attributes of cassava-african yam bean fufu flour. international journal of scientific and research publications, 3(8), 1-7 (2013) [54]. otegbayo, b., aina, j., aseidu, r. & bokanga, m. pasting characteristics of fresh yams (dioscorea spp) as indicators of textural quality in a major food product‘pounded yam’. food chemistry, 99, 663-669(2006) [55]. michiyo, w., tomoko, m., kikuchi, t., hiroshi, k. & naofumi, m. application of pre–germinated brown rice for breadmaking. cereal chemistry, 3, 293 -317 (2004) [56]. numfor, f.a., walter, w.m. & schwartz, s.j. effect of emulsifiers on the physical properties of native and fermented cassava starches. journal of agriculture and food chemistry, 44, 2595-2599 (1996) doi: https://doi.org/10.4316/fens.2023.008 79 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 79 88 chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania *maria poroch seriţan1, paula maria greculeac (galan)2, dumitriţa – sabina dobrincu1, mihaela jarcău1, alexandra-lăcrămioara matei1, tudorița chicaroș1 1faculty of food engineering, stefan cel mare university of suceava, mariap@fia.usv.ro, 2mihai cristea plant genetic resources bank suceava, romania *corresponding author received 5th may 2023, accepted 25th june 2023 abstract: the aim of our study was to characterize beans (phaseolus vulgaris l.), one of the most important legumes at the international level, by examining their physical and biochemical properties, to highlight the importance of preserving local bean varieties in romania, beans which are kept in the collection of the mihai cristea suceava plant genetic resources bank. local cultures of phaseolus vulgaris, have been best preserved, especially in the maramureș area, followed by suceava area. based on all 28 samples from the common bean germplasm collection, the mean values for seed size characteristics were seed length (l) 14.64 ± 2.24 mm and seed width (w) 8.93 ± 1.51 mm. the average weight of the 1,000-seed characteristic was 521.34 g, with the minimum and maximum values ranging from 136.96 to 1,045 g for all 28 samples. the highest coefficient of variation was calculated for 1,000seeds weight (39.9 %) and the lowest for l/w (13.2 %). the protein content determined for 16 samples from the common bean germplasm collection was 23.79 ± 2.49 g/ 100 g of dry matter. the amount of protein varies between 18.84 g/ 100 g of dry matter (sample f23) and 26.69 g/ 100 g of dry matter (sample f27). the free amino acid content varies between 0.56 g and 1.29 g/100 g of dry matter, and the boiling time between 35 and 80 minutes. boiling time is dictated by the variety of beans, but a very interesting thing, observed from the analyses carried out, is that the boiling time varies inversely with the percentage of protein. so, in sample f27 we have a boiling time of 35 minutes and a protein content of 26.69 g/ 100 g of dry matter. the sample with the highest protein content has the lowest boiling time. at the same time, sample f19 has a boiling time of 80 minutes and a protein content of 19.44 g/ 100 g of dry matter. keywords: the cooking time, free amino acids, local populations, quantitative seed descriptors. 1. introduction the total disappearance of many plant species is one of the many consequences of anthropogenic activities, some of which have devastating environmental effects. throughout its existence, humanity has used some 10,000 species of crop plants, but according to the latest reports from the food and agriculture organisation of the united nations (fao) [1], 90% of food production is currently based on 120 species. the advent of industrialised agriculture has led to a drastic reduction in biodiversity and a marked process of genetic erosion. modern varieties have been developed at the expense of the old and local varieties of crops [2]. at the national level, the most critical situation is observed in flax and hemp (fibre) growing, where local varieties have almost disappeared. phaseolus vulgaris crops are among the best preserved, especially in the maramures area, followed by suceava county. to preserve the agricultural genetic heritage on farms in romania, specific governmental and political measures are needed, as well as the elaboration of a national food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 80 conservation plan, involving farmers and small agricultural producers [3, 4]. local varieties, which are in danger of extinction, are an invaluable source of plant genetic resources and are used to generate new varieties and hybrids with higher productivity than local varieties, resulting in sustainable agriculture and food security [5]. the fao considers the common bean, or phaseolus vulgaris l., the most important vegetable in the world [1]. this particular importance for human nutrition is due to its chemical composition and also to its use as green pods, green and dried beans, bean meal, leaves, husks, and straw as animal feed. the chemical composition is mainly influenced by the state of growth of the bean (green, dry) and its variety; other factors that may influence it are climatic and soil conditions [3, 6]. carbohydrates are found in the highest concentration in dried beans, ranging from 50 to 60 g/ 100 g of the dry matter [7, 8]. among the polysaccharides, starch is the most abundant molecule. with 23-33% protein in dry matter, beans are an excellent source of protein and play an important role in human nutrition. protein is rich in essential amino acids such as lysine, arginine, and tryptophan [8, 9]. the fat content is low, about 2g/100 g of dry matter. most lipids are phospholipids. beans are an important source of unsaturated fatty acids. linoleic acid is the predominant unsaturated fatty acid in beans [8, 10]. beans are an important source of minerals and vitamins, far exceeding the amounts provided by grains [8, 11]. area of distribution of the genus phaseolus vulgaris and cultivated area beans are grown all over the world where the soil and climate conditions are suitable for the plant's growth and development. summer temperatures should be above 15oc and minimum rainfall 150 mm, up to 45 500 northern latitudes. in the 2,000s, the main bean producers were the united states, china, france, russia and senegal [12]. in our country, between 1975 and 1977, beans were grown on an area of 83,700 ha, with a production per hectare of 600 kg [13, 14]. the last national statistics for bean production were made in 2017, with a total production of 16,125 tonnes and an average production of 1,402 kg/ha, with the highest production in the north-eastern part of romania [15]. the aim of our study was to determine the physical and biochemical characteristics of local populations of phaseolus vulgaris, maintained in the collection of the mihai cristea suceava plant genetic bank, in order to raise awareness of the importance of preserving local bean varieties in romania, as a valuable source of qualitative and quantitative nutrients reaching the consumer. 2. materials and methods plant material twenty-eight common bean accessions, of which fourteen white common bean accessions, seven black common bean accessions and seven reddish-brown common bean accessions, (figure 1), were made available for the study by the plant genetic resources bank "mihai cristea" suceava [3]. thus, the plant genetic resources bank of suceava is responsible for the ex-situ conservation of plant resources. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 81 figure 1. photo examples for common bean accessions (phaseolus vulgaris l.) groups distribution according to seed color table 1. samples, sample registration number, locality (village, county) where collected, year of collection, species and colour sample name nr. acces locality year varieties colour f1 svgb-5794 botiza, mm 1991 var. compressus white bean f2 svgb-1988 bosanci, sv 1988 var. nanus white bean f3 svgb-13339 vama, sv 2000 var. compressus white bean f4 svgb-14022 cupșeni, mm 2001 var. ellipticus white bean f5 svgb-13753 călinești, mm 2001 var. vulgaris white bean f6 svgb-13954 lăpuș, mm 2001 var. vulgaris white bean f7 svgb-14128 cernești, mm 2001 var. vulgaris white bean f8 svgb-14150 tg. lăpuș, mm 2001 var. vulgaris white bean f9 svgb-13977 suciu de jos, mm 2001 var. compressus white bean f10 svgb-14092 dealu mare, mm 2001 var. compressus white bean f11 svgb-13936 mocira, mm 2001 var. compressus white bean f12 svgb-5714 botiza, mm 1991 var. coccineus white bean f13 svgb-14046 peteritea, mm 2001 var. compressus white bean f14 svgb-5791 botiza, mm 1991 var. cocineus white bean f15 svgb-13775 săpânța, mm 2001 var. vulgaris reddish-brown bean f16 svgb-15959 bistra, mm 2003 var. vulgaris reddish-brown bean f17 svgb-13752 călinești, mm 2001 var. vulgaris reddish-brown bean f18 svgb-13685 lunca de la tisa, mm 2001 var. vulgaris reddish-brown bean f19 svgb-13674 bistra, mm 2001 var. vulgaris reddish-brown bean f20 svgb-13778 sarasău, mm 2001 var. vulgaris reddish-brown bean f21 svgb-15919 plopișor, mm 2003 var. vulgaris reddish-brown bean f22 svgb-15964 ieud, mm 2003 var. vulgaris black bean f23 svgb-14003 groșii țibleșului, mm 2001 var. vulgaris black bean f24 svgb-8759 cupșeni, mm 1993 var. vulgaris black bean f25 svgb-10423 moisei, mm 1994 var. vulgaris black bean f26 svgb-13639 repedea, mm 2001 var. ellipticus black bean f27 svgb-13687 lunca de la tisa, mm 2001 var. vulgaris black bean f28 svgb-13777 săpânța, mm 2001 var. vulgaris black bean legend: mm – maramures county, sv – suceava county at present, the bank's collection contains about 18,000 unique varieties at +40c and 200c respectively. common bean accessions were collected from 21 villages in two counties maramureș and suceava, (table 1, figure 2), of the north northfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 82 west region of romania, from people's gardens, representing local populations, seventeen of the species phaseolus var. vulgaris, two common bean accessions var. coccineus, six common bean accessions var. compressus, one common bean accessions var. nanus, two common bean accessions var. ellipticus. samples were collected in different years and analyses were performed on the original sample, preserved at +40c in table 1. fig. 2. map of the north north-west region of romania presenting the common bean sampling villages (counties maramures and suceava) the sampling area of a common bean accession is located in the extreme northnorth-western part of romania, at the border with hungary (n-w) and ukraine (n) and covers the administrative territory of maramures-mm and suceava sv counties. the climate of the area is temperate continental, with oceanic influences in the west and continental influences in the east. these influences are reflected in seasonal temperatures (january february, tmin = -30.0 0c; july august, tmax = 39.0 0c) and rainfall (february march, pmin = 32.6 mm; may june pmax = 205.4 mm). seed evaluation using quantitative characteristics quantitative seed descriptors included the evaluation of the following five characteristics: seed length (l) [mm]; seed width (w) [mm]; seed length/width ratio (l/w); 1,000 (for common bean) seed weight [g] [16]. sample preparation broken and damaged beans, as well as foreign materials, were removed. then, the samples were ground in a mill to obtain a fine and homogeneous powder using an ika a11 analytical mill (sigmaaldrich, germany), and maintained at 18°c until analysis. protein quantity protein content was determined using the micro-kjeldahl method and a conversion factor of 6.25 [17, 18]. the total amino acids were verified the method based on the interaction of the primary amine group present in amino acids with ninhydrin. reaction product of ninhydrin with primary amino groups produces the coloured chromophore called as rheumann’s purple [19]. the absorbance was read at 530 nm with a t70+ uv/vis spectrometer (pg instruments ltd, united kingdom), using deionized water as the blank and 0.5 cm optical path. the and free amino acids were expressed as amino nitrogen [%], using a calibration curve in the range of 0.1...0.25 mg tyrosine/ml of a freshly prepared tyrosine solution. the results will be expressed in percentages for the analysed product and are calculated using equation 1: amino nitrogen % = 𝐺∙𝑑 𝑚∙100 ∙ 100 (1) https://www.sigmaaldrich.com/catalog/product/aldrich/z341789?lang=en&region=us https://www.sigmaaldrich.com/catalog/product/aldrich/z341789?lang=en&region=us food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 83 where, g = the amount of amino nitrogen or tyrosine determined from the calibration curve, [mg]; m = the quantity of product taken into analysis, [mg]; d = the dilution used. the cooking time to determine the cooking time, the beans were placed in water at 100°c and the time elapsed from that moment until the texture of the bean softened was timed. bean texture was analysed subjectively by sticking a needle into the bean. 3. results and discussion seed characterization of common bean accessions (phaseolus vulgaris l.) a total of four seed morphological characteristics, i.e., quantitative (seed length l; seed width w; seed length/width ratio l/w and 1,000 seed weight) were evaluated. in the upper part of table 3 are presented summary statistics for four quantitative common bean seed characteristics. the mean values for seed size characteristics based on all 28 accessions in common bean germplasm collection were for l 14.64 ± 2.24 mm, and for w 8.93 ± 1.51 mm (see table 3). the minimum and maximum values ranged between 10.01 and 19.5 mm for l, and 6.07 and 12.95 mm for w. for characteristic1,000 seed weight the mean value of all 28 accessions was 521.34 ± 208.19 g, while the minimum and maximum values ranged between 136.96 and 1,045 g. the highest coefficient of variation was calculated for the 1,000 seed weight (39.9%) and the lowest for l/w (13.2%). as discussed by rana et al. [20] for 4,274 common bean accessions conserved in indian gene bank, l ranged 5.0 – 20.3 mm, w 2.0 – 12.0 mm and 100 seed weight 3.5 – 96.3 g. similarly, kara et al. [21] reported for 12 registered turkish common bean accessions genotypes l 9.1 – 17.8 mm, w 5.8 – 10.0 mm, t (seed thickness) 4.6 – 6.0 mm and 100 seed weight 18.0 – 65.6 g. as discussed by giurcă [22] for 9 common beans accessions originating from northern romania and western ukraine, l ranged 11.8 – 18.0 mm, w 7.4 – 9.7 mm, t (seed thickness) 4.4 – 6.9 mm and 100 seed weight 34.3 – 54.2 g. logozzo et al. [23] evaluated 533 accessions of the european common bean germplasm and reported accessions with l 12.0 – 13.9 mm (35.5%), w 7.1 – 8.0 mm (33.0%) and t 5.0 – 5.9 mm (37.1%) were the most frequent. as discussed by sinkovič et al. [16] for 953 common bean accessions conserved in slovene gene bank at the agricultural institute of slovenia, being part of the national plant gene bank, l (range 7.3 – 27.2 mm); t (range 4.2 – 11.0 mm); w (range 0.3 – 16.5 mm); l/w (range 0.4 – 2.6 mm); w/t (range 0.6 – 2.2 mm); and 100 for common (range 19.3 – 98.4 mm) bean seed weight. table 3. summary statistics for four quantitative seed characteristics in accessions of common (phaseolus vulgaris l.) characteristics range mean ± sd cv (%) seed length (l) [mm] 10.01 – 19.5 14.64 ± 2.24 15.3 seed width (w) [mm] 6.7 – 12.95 8.93 ± 1.51 16.9 seed length/width ratio (l/w) 1.2973 – 2.1847 1.66 ± 0.22 13.2 1,000 seed weight [g] 136.96 – 1,045 521.34 ± 208.19 39.9 all seed characteristics measured quantitatively showed wide range of variation among all common bean accessions evaluated. frequency distribution graphs of 28 common bean accessions for quantitative seed characteristics are shown in figure 3. based on quantitative measurements, the common bean accessions were classified according to the l into two groups, i.e., medium, and large. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 84 the first group included accessions with medium seeds measuring l from 10.0 to 15.0 mm (16 accessions or 57.14 %); and the second group accessions with large seeds and l > 15.0 mm (12 accessions or 42.86 %), (figure 3a). from the point of view of the width of the accessions, the samples are divided into two broad and narrow groups. the first group included broad-seeded accessions measuring w from 8.925 to 12.95 mm (11 accessions or 39%); and the second group of accessions with narrow seeds w < 8,925 mm (17 accessions or 60.71 %), (figure 3b). similarly, the common bean accessions were classified according to the 1,000 seed weight into three groups, i.e., low-weight, mediumweight and high-weight. (a) (b) (c) fig. 3. frequency distribution of 28 common bean accessions (phaseolus vulgaris l.) for quantitative seed characteristics ((a) seed length; (b) seed width; (c) – 1,000 seed weight) low-weight seeds group included common bean accessions with 1,000 seed weight < 500.0 g (19 accessions or 67.85 %); the medium-weight seeds group accessions with 1,000 seed weight measuring from 500.0 to 1,000.0 g (8 accessions or 28.57 %); and the high-weight seeds group accessions with 1,000 seed weight >1,000 g (1 accessions or 3.57 %), (figure 3c). protein quantity beans are rich in protein (21 – 25 %) [18, 24], carbohydrate (56 %), dietary fibre, and are a good source of antioxidants [25], as well as vitamins (vitamin b1 (0.5 mg %), vitamin b2 (0.4 mg %), niacin (3.4 mg %), vitamin c (20 mg %)) and minerals (potassium (1,770 mg %), calcium (195 mg %), phosphor (420 mg %), iron (7 mg %), nickel, cuprum, cobalt) [8, 11, 24, 26]. unlike cereal proteins, the proteins present in beans accessions are considered to be the range of meat protein, with a range of 20 – 30 % proteins. beans are rich in globulins which constitute 50 – 70 % of the total proteins along with a considerable amount of glutelins [27] (20 – 30 %), prolamin (2 – 4 %) and free amino acids (5 – 9 %) [28]. a glycoprotein, phaseolin consisting of neutral sugars especially mannose is also found in beans accessions. beans accessions are also rich source of essential amino acids including lysine which is deficient in cereals [29]. the amount of protein in fresh beans averages 23 g / 100 g of dry matter, as the authors mentioned above reported. according to the determination of the amount of protein in beans that have been kept in the bank's collection since 2001, high percentages can be seen in table 4 and in the graphical representation in figure 4. the mean values for the amount of protein based on 16 accessions in the common bean germplasm collection were 23.79 ± 2.49 g/ 100 g of dry matter. the amount of protein ranged from 18.84 g/ 100 g of dry matter (sample f23) to 26.69 g/ 100 g of dry matter 1 3 4 0 8 5 4 1 0 2 0 5 10 11 12 13 14 15 16 17 18 19 20n o . o f a cc e ss io n s seed length [mm] 1 8 8 6 3 0 2 0 2 4 6 8 10 7 8 9 10 11 12 13 n o . o f a cc e ss io n s seed width [mm] 1 7 11 7 1 1 0 5 10 15 200 400 500 800 1000 1200 n o . o f a cc e ss io n s 1,000 seed weight [g] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 85 (sample f27). twelve samples (f1 – f3, f7, f12, f14, f16, f20, f21, f22, f24, f25) were not representative in terms of protein and amino acid content, respectively cooking time; and they were not represented graphically (figure 4, figure 5, figure 6, figure 7). the difference between the minimum and maximum values is quite large and can be explained by differences at the molecular level, in the genes that determine the amount of protein. of course, the phenotypic and genotypic values of the quantitative trait are determined by the amount of improvement, the deviation due to dominance, the deviation due to the interaction of the non-allelic or epistatic genes and the environmental deviation of sample f23 from groșii țibleșului, mm and sample f27 from lunca de la tisa, mm. table 4. summary statistics for protein quality in accessions of common (phaseolus vulgaris l.) characteristics range mean ± sd cv (%) protein content [g/100 g of dry matter] 18.84 – 26.69 23.79 ± 2.49 10.5 free amino acids [g/100 g of dry matter] 0.56 – 1.29 1.019 ± 0.21 20.8 the cooking time [min.] 35 80 52.37 ± 12.93 24.7 fig. 4. the variation on protein content of 28 common bean accessions figure 5 shows the variation in protein content of the 16 common bean accessions (phaseolus vulgaris l.) according to three varieties: var. compressus, var. vulgaris and var. ellipticus. in the case of var. vulgaris the variation in protein content is significant between 18.84 g/ 100 g of dry matter (sample f23) and 26.69g/ 100 g of dry matter (sample f27), while in var. compressus is insignificant between 24.42 g/ 100 g of dry matter (sample f10) and 26.64 g/ 100 g of dry matter (sample f13). fig. 5. the variation on protein content of common bean accessions (phaseolus vulgaris l.) 25.42 26.26 21.9 22.85 24.92 24.42 25.63 26.64 25.48 24.76 24.02 19.44 18.84 20.55 26.69 22.94 0 5 10 15 20 25 30 f4 f5 f6 f8 f9 f10 f11 f13 f15 f17 f18 f19 f23 f26 f27 f28 p r o te in c o n te n t [% ] sample number 20.55 0 10 20 30 f4 f9 f10 f11 f13 f5 f6 f8 f15 f17 f18 f19 f23 f27 f28 f26 var. compressus var. vulgaris var. ellipticus food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 86 the content, figure 6, in free amino acids varies between 0.56 g/ 100 g of dry matter (sample f13) and 1.29 g/ 100 g of dry matter (sample f9), with an average of 1.019 ± 0.21 g/ 100 g of dry matter, and cooking time between 35 and 80 minutes, figure 7. the cooking time is determined by the variety of beans, but a very interesting fact, observed in the analysis, is that the cooking time varies inversely with the percentage of protein. so, in sample f27 we have a boiling time of 35 minutes and a protein content of 26.69 g/ 100 g of dry matter. the sample with the highest protein content has the lowest boiling time. at the same time, sample f19 has a boiling time of 80 minutes and a protein content of 19.44 g/ 100 g of dry matter. fig. 6. the variation on free amino acid content of bean samples (phaseolus vulgaris l.) fig. 7. the variation on the cooking time of common bean accessions (phaseolus vulgaris l.) research over time has shown that boiling time is not influenced by specific genes, but more by storage conditions and storage time [30]. however, in a recently published study, cichy k. a et al., 2015 [31] uses an experiment to show that specific genes can further affect boiling time. on the bean pv06 chromosome, three genes encoding udp-glucosyl transferases, a family of genes involved in flavonoid synthesis and pigment development, were found to be of great interest, as bean colour was correlated with the cooking time. other candidate genes for bean cooking time, also located on chromosome pv06, are atchx3 and 1.14 1.26 0.7 1.07 1.29 1.12 1.27 0.56 1.01 1.08 1.1 0.97 1.05 1.01 1.02 0.66 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 f4 f5 f6 f8 f9 f10 f11 f13 f15 f17 f18 f19 f23 f26 f27 f28 a a [ % ] sample number 47 45 55 64 52 43 43 36 48 50 60 80 62 75 35 43 0 10 20 30 40 50 60 70 80 90 f4 f5 f6 f8 f9 f10 f11 f13 f15 f17 f18 f19 f23 f26 f27 f28 c o o k in g t im e s [m in .] sample number food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 87 atchx4, which are related to ion exchange at the molecular level, h+ and ca2+. there is evidence that the ca2+ ion leads to an increase in cooking time. akond et al., 2011 [32] also show that white beans boil faster than coloured beans due to a lower content of phenolic compounds. the longest cooking time was found in the white cranberry bean [33]. in our case, sample f13 (36 minutes) boiled very quickly, being white, and sample f19 (80 minutes) boiled the hardest, being reddish-brown. but in our case we have an exception, sample f27, which boiled the fastest (35 minutes), being black in colour, (figure 7). 4. conclusion beans (phaseolus vulgaris l.) are the most important legume internationally, being a valuable source of protein, starch, fibre, antioxidants, minerals and vitamins [34, 35, 36]. following the determinations, we obtained the following: the mean values for seed size characteristics based on all 28 accessions in common bean germplasm collection were for l 14.64 ± 2.24 mm and for w 8.93 ± 1.51 mm. protein content ranged from 18.84 26.69 g/ 100 g of dry matter. total amino acid content ranged from 0.56 1.29 g/ 100 g of dry matter. the sample with the highest protein content has the lowest boiling time. 5. acknowledgments thanks for the collaboration to the biologist silvia străjeru, from „mihai cristea” plant genetic resources bank suceava, 1 mai str., no. 17, suceava, romania. 6. references [1]. food and agriculture organization of the united nations (fao.org) [2]. giuliani a., developing markets for agrobiodiversity. security livelihoods in dryland areas, biodiversity international, rome, italy, (2007) [3]. banca de resurse genetice vegetale „mihai cristea” suceava (svgenebank.ro) [4]. străjeru s., ibănescu m., constantinovici d., landrace inventories: nees and methodologies. european cooperative programme for plant genetic resources. rome, italy, biodiversity technical bulletin, 15: 137-142, (2009) [5]. pardey p. g., skovmand b., taba s., van dusen m. e., wright b. d., the cost of conserving maize and wheat genetic resources ex situ. in farmers gene banks and crop breeding: economic analyses of diversity in wheat maize and rice. 35-56, (m. smale, ed.) kluver acad. publ., (1998), springer, (2000) [6]. audu s.s., aremu m.o., effect of processing on chemical composition of red kidney bean (phaseolus vulgaris l.) flour, pakistan journal of nutrition, 10: 1069-1075, (2011) [7]. ovando-martinez m., luis, a., bello-pereza, l. a., whitney, k., osoriodiaz, p. senay, s., starch characteristics of bean (phaseolus vulgaris l.) grown in different localities. carboh. polym., 85: 54–64, (2011) [8]. hayat i., ahmad a., masud t., ahmed a., bashir s., nutritional and health perspectives of beans (phaseolus vulgaris l.): an overview, critical reviews, food science and nutrition, 54: 580–592, (2014) [9]. butt m. s., batool r., nutritional and functional properties of some promising legume protein isolates, pak. j. nut., 9 (4): 373–379, (2010) [10]. yoshida h., tomiyama y., mizushina y., characterization in the fatty acid distributions of triacylglycerols and phospholipids in kidney beans phaseolus vulgaris l., j. food lipids., 12: 169–180, (2005) [11]. welch r. m., house w. a., beebe s., cheng z., genetic selection for enhanced bioavailable levels of iron in bean (phaseolus vulgaris l.) seeds, j. agric. food chem., 48: 3576– 3580, (2000) [12]. ciofu r., stan n., popescu v., chilom p., apahidean s., horgoș a., berar v., lauer k. f., atanasiu n., tratat de legumicultură, ed. ceres, bucurești, (2004) [13]. krauss s.l., he t.h., rapid genetic identification of local provenance seed collection https://www.fao.org/home/en https://www.fao.org/home/en https://svgenebank.ro/ https://svgenebank.ro/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș, chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania, food and environment safety, volume xxii, issue 2 – 2023, pag. 79 – 88 88 zones for ecological restoration and biodiversity conservation, j. nat. conserv., 14: 190-199, (2006) [14]. anuarul statistic al r.s.r., 1978, https://adt.arcanum.com/ro/collection/anuarulstatis ticalrepubliciisocialisteromania/ [15]. institutul național de statistică, https://insse.ro/cms/ [16]. sinkovič l., pipan b, sinkovič e., meglič v., morphological seed characterization of common (phaseolus vulgaris l.) and runner (phaseolus coccineus l.) bean germplasm: a slovenian gene bank example, hindawi biomed research international, 2019(2):1-13, (2019) [17]. horwitz w., latimer g. w., official methods of analysis of the association of official analytical chemists. (18th ed.). gaithersburg: adac, (2010) [18]. rezende a. a., pachecd m. t. b., nunes da silva v. s., ferreira t. a. p. c., nutritional and protein quality of dry brazilian beans (phaseolus vulgaris l.), food sci. technol, campinas, 38(3): 421-427, (2018) [19]. gupta c., puri r., hussain a., jain s., development and validation of ninhydrin based colorimetric spectrophotometric assay for determination of pregabalin in different dissolution mediums, eurasian j anal chem, 8 (2): 90-98, (2013) [20]. rana j. c., sharma t. r., tyagi r. k., chahota r., gautam n. k., singh m., sharma p., ojha s., characterisation of 4274 accessions of common bean (phaseolus vulgaris l.) germplasm conserved in the indian gene bank for phenological, morphological and agricultural traits, euphytica, 205(2): 441 – 457, (2015) [21]. kara m., sayinci b., elkoca e., öztürk i., özmen t. b., seed size and shape analysis of registered common bean (phaseolus vulgaris l.) cultivars in turkey using digital photography, tarim bilimleri dergisi, 19(3), 219 – 234, (2013) [22]. giurca d. m., morphological and phenological differences between the two species of the phaseolus genus (phaseolus vulgaris and phaseolus coccineus), cercetari agronomice in moldova, 42(2): 39 – 45, (2009) [23]. logozzo g., donnoli r., macaluso l., papa r., knüpffer h., zeuli p. s., analysis of the contribution of mesoamerican and andean gene pools to european common bean (phaseolus vulgaris l.) germplasmand strategies to establish a core collection, genetic resources and crop evolution, 54(8): 1763– 1779, (2007) [24]. pârvu c, universul plantelor, editura asab, bucureşti, (2006) [25]. luthria d. l., pastor-corrales m. a., phenolic acids content of fifteen dry edible bean (phaseolus vulgaris l.) varieties, journal of food composition and analysis, 19(2-3): 205-211, (2006) [26]. campos-veja r., loarca-pina g., domah b. d., minor components of pulses and their potential impact on human health. food research international, 43(2): 461-482. (2010) [27]. slupski j., effect of cooking and sterilization on the composition of amino acids on immature seeds of flageolet beans (phaseolus vulgaris l.) cultivars, food chem., 121:1171-1176, (2010) [28]. ma y., bliss f.a., seed proteins of common bean, crop sci., 17:431-437, (1978) [29]. alice a. k., pandey a. k., singh s., chakma j., talha ansari m.d., shulee ariina m.m., nutritional profile of french bean: a mini review, international journal of chemical studies, 7(1): 282-284, (2019) [30]. arruda b., guidolin a. f., coimbra m., luís j., battilana j., environment is crucial to the cooking time of beans, ciência e tecnologia de alimentos, 32(3): 573-578 (2012) [31]. cichy k. a, wiesinger j. a, mendoza f. a, genetic diversity and genome-wide association analysis of cooking time in dry bean (phaseolus vulgaris l.), theor appl genet, 128(8): 1555-67, (2015) [32]. akond a., khandaker l., berthold j., gates l., peters k., delong h., hossain k., anthocyanin, total polyphenols and antioxidant activity of common bean, am j food technol, 6: 385–394, (2011) [33]. elsadr h.t., wright l.c., pauls k.p., bett k.e., characterization of seed coat post harvest darkening in common bean (phaseolus vulgaris l.), theor appl genet, 123: 1467–1472, (2011) [34]. broughton w. g., hernández g., blair m., beebe s., gepts p., vanderleyden j., beans (phaseolus spp.) model food legumes, plant and soil, 252(1): 55– 128, (2003) [35]. aguilera y., estrella i., benitez v., esteban r. m., martín-cabrejas m. a., bioactive phenolic compounds and functional properties of dehydrated bean flours, food research international, 44(3): 774–780, (2011) [36]. pedrosa m. m., cuadrado c., burbano c., muzquiz m., cabellos b., olmedilla-alonso b., asensio vegas c., effects of industrial canning on the proximate composition, bioactive compounds contents and nutritional profile of two spanish common dry beans (phaseolus vulgaris l.), food chemistry, 166(1): 68–75, (2015) https://adt.arcanum.com/ro/collection/anuarulstatisticalrepubliciisocialisteromania/ https://adt.arcanum.com/ro/collection/anuarulstatisticalrepubliciisocialisteromania/ https://insse.ro/cms/ https://www.semanticscholar.org/author/r.-chahota/36233223 https://www.semanticscholar.org/author/n.-k.-gautam/35205146 https://www.semanticscholar.org/author/mohar-singh/2110448087 https://www.semanticscholar.org/author/p.-sharma/2112484185 https://www.semanticscholar.org/author/s.-ojha/2112651212 https://pubmed.ncbi.nlm.nih.gov/?term=cichy+ka&cauthor_id=26003191 https://pubmed.ncbi.nlm.nih.gov/?term=wiesinger+ja&cauthor_id=26003191 https://pubmed.ncbi.nlm.nih.gov/?term=mendoza+fa&cauthor_id=26003191 doi: https://doi.org/10.4316/fens.2021.033 298 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 298 313 dynamics of springy systems of packaging lines for food products anatoly sokolenko 1 , *oleksandr shevchenko 2 , oleg stepanets 1 , serhiy but 3 , anastasiia shevchenko 4 1 department of mechatronics and packaging technics, national university of food technologies, volodymyrska str., 68, kyiv, ukraine, 01601, mif63@i.ua 2 department of processes and apparatus for food production, national university of food technologies, volodymyrska str., 68, kyiv, ukraine, 01601, tmipt@ukr.net 3 department of technology of canning, national university of food technologies, volodymyrska str., 68, kyiv, ukraine, 01601 4 department of bakery and confectionery goods technology, national university of food technologies, volodymyrska str., 68, kyiv, ukraine, 01601, nastyusha8@ukr.net *corresponding author received 24th may 2021, accepted 20th december 2021 abstract: the purpose of the study was to develop mathematical models and proposals for improving the technology and equipment for the supply of roll packaging materials. the analysis of features of systems for transportation of packing materials, dynamics of transient processes, research methods concerning movements of rolled materials, features of loadings in systems of cyclic action with springy elements are resulted in the article. it is shown that the modern theoretical basis of synthesis of technological machines on the basis of use of film rolled materials combines possibilities of the account of technological requirements, indicators of high productivity, neighborhood conditions, restriction of power parameters. mathematical formalizations concerning the ratios of force factors in systems of transportation of loads of cyclic action at the levels of external force actions, generated friction forces, geometrical, kinematic and dynamic parameters were obtained. mathematical models concerning runaway modes and estimation of stiffness of springy bonds, on the basis of film materials, methods of estimation of system parameters were offered. synchronized combination of operations, the use of optimal laws of movement of working bodies, limitation of dynamic loads was carried to the positive directions of creating efficient technological machines and lines. it is proposed to determine the ratio of the run-out time of rolls of variable mass by the ratio of the squares of the initial and final radii. the solution of the problem of kinetic energy recovery in systems of periodic action is simultaneously connected with the restriction of the non-uniformity of the drive of machines. keywords: dynamics, springy connections, packaging lines, transient processes, mechanical systems. 1. introduction intensive innovations in food production technologies are accompanied by the introduction of the latest equipment in product packaging lines. this applies to packaging made of flexible, semi-rigid and rigid materials. an important component of modern packaging lines is that in many cases they contain raw materials and equipment for the manufacture of blanks, the work of which is synchronized with the subsequent operations of packaging, sealing, design and creation of group packaging. the list of modern flexible packaging materials satisfies the packaging mailto:tmipt@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 299 industry of any society at the state level, reflecting the development of the economy, agricultural sector, social development. packaging materials in combination with physico-chemical methods of food stabilization and packaging technologies provide regulatory requirements for warehousing, transportation, storage and sale of products. a promising direction in the development of modern food production is the concentration of production capacity and increasing the range of products in different types and sizes of packaging. the latter means the feasibility of using systems with universal flexible equipment for reconfiguration. the dynamics of development of production of packaging materials and technological equipment is approaching the exponential law, which has the prospect of abrupt transformations rather than evolutionary changes. expectations of important and fleeting changes are projected on the basis of the development of scientific base and empirical achievements in existing technologies, as well as on the basis of modern combination of mechanics and electronics, which are accompanied by the possibility for implementing high-precision laws in dynamics with load and energy costs. such a dualism of the final result is possible on the basis of in-depth assessments in the course of transients and established modes of operation under continuous and discrete conditions. hence, the issues of synthesis of packaging equipment are shifted in the areas of universality, reliability, accuracy of operation of working bodies, economy modes, economic efficiency. however, the synthesis of modern highperformance machines is accompanied by the need to overcome the barrier of natural contradiction, on the other side of which there are dynamic loads. high productivity of the car means necessity to carry out the set movements in limited time with considerable accelerations and inertial loadings. the use of technological machines of discrete action leads to the need to overcome such contradictions inherent in the systems for the supply of roll packaging materials. the presence of rolls with significant masses in them leads to complications of the modes of stabilization of the movements of the film canvas and their tensions at distances between the rolls and the leading working bodies. a feature of the providing systems is the use of friction drives in various forms. this applies to the mechanisms of rotation of the rolls, regulation of their runoff modes, the device of compensation and damping devices and devices for driving film canvas. the inability to achieve synchronization in the modes of run-out of the leading masses with the working bodies and the driven masses of the rolls led to the use of compensation and damping devices, which divide the system into almost two dynamically independent parts. in connection with this division, there is a need for in-depth analysis of the supply systems of rolled packaging materials in order to stabilize the kinematic parameters in their double parts, the tension of the film canvas in the areas between compensation and shock working devices, improving synchronization modes and increasing friction systems. the purpose of the study was to develop mathematical models and proposals for improving the technology and equipment for systems for the supply of roll packaging materials. based on the analysis of their construction, the following research objectives were formulated:  to perform analysis and generalization, which relates to the features food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 300 of the systems of movement of rolled packaging film materials;  to develop a mathematical apparatus for describing transients in systems for moving film materials, separated by compensation and depreciation devices;  to develop recommendations for limiting the dynamic components of film flow loads, the possibilities of kinetic energy recovery and limiting the unevenness of the stroke in the drives of the equipment. the dynamics of transients in technological machines continues to be an important component, thanks to which scientists have information on loads, power supply, uniformity of machine systems. of course, in-depth load forecasting applies to complex engineering structures, but in conditions of non-stationary external influences, it is advisable to supplement them with the volume of operational data [1]. the result of this approach was not only the prediction of the dynamic behavior of loads, but also an accurate assessment of their statistical characteristics with data on capacity. the direction of achieving accuracy in the performance of technological operations involved the use of special measures at the level of dynamic compensation of the reactions of systems exposed to environmental vibrations and other influences [2]. the accuracy of the manufacture of special purpose parts required special monitoring of transients, which was realized due to the known relationship between power consumption and power performance. however, the manifestations of the present control devices and the effects of transmissions led to errors in determining the actual force. the calculation of a twisted model based on the effective power signal to compensate differences was proposed [3]. the creation of methods for the study of transients was being actively improved. the study of the mechanical system with hydrodynamic power transmission under monoharmonic loading, which was carried out by introducing a stationary calculation of an immeasurable mathematical model was an example [4]. for this purpose, a modified method of harmonic balance was used. the effects of radial forces and machining were accuracy combined on the basis of the equivalent contact load model and hertz's theory [5] led to estimation of errors in the manufacture of details. inertial loads corresponded to transients and their values had significant manifestations in high-speed machines. at high speeds and accelerations, the relationships between the subsystems became more complicated, which required the use of a new research method. it was shown [6] that at limited speeds and accelerations inertial forces had little effect on system performance. this means the expediency of their restrictions in certain ranges. in the study [7] the evaluation of the effects of machine vibration isolation on the dynamic loads of bearings with the determination of the range of dynamic reactions and the possibilities of their limitations was made. dynamic prediction of the characteristics of a 5-dof hybrid machine based on a scale model taking into account deformations is an example of the possibilities of analyzing systems taking into account changes in mass and stiffness [8]. the transition processes of start and idling of machines related to energy loads and energy costs within the interests and balances of generation and consumption systems [9, 10] proposed a procedure for limiting peak loads by implementing certain laws. energy saving technologies logically food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 301 extend to various areas of technological machines [11] and the stability of multimachine power systems [12]. increased energy losses were related to cyclic machines, which are largely represented in the packaging lines. peculiarities of the course of transients in these cases did not allow to use the properties of dynamic systems based on the control effects of flywheels or flywheel masses [13]. however, the driven masses of technological carousel machines were commensurate with the reduced driving masses, which accordingly determined the potentials of their kinetic energy and the feasibility of organizing recuperative regimes. in connection with this, the regulation of the movement of machines and the kinematics of movements of the working bodies in combination provided a double result in terms of limiting the dynamic loads and energy consumption. 2. materials and methods methods of mathematical modeling of physical and mechanical processes of interactions of working bodies of the equipment with film material streams, kinematic and dynamic synthesis of elements of systems, mathematical and statistical analysis of results of experiments were used. methods of mathematical programming using universal engineering-mathematical complex matlab were used for carrying out mathematical calculations and results of experiments. 3. results and discussion 3.1. the results of the study of the system for providing rolled packaging materials. dynamics of driven masses of rolls in technical generalization, it was considered that the devices for providing rolled packaging materials were represented by three groups [14]. these included groups of main, auxiliary and additional working bodies. the main group was represented by roll holders, devices for moving materials and equipment for cutting and trimming material. auxiliary working bodies provided accumulation and amortization of materials, braking of rolls, braking and stopping of film streams and the direction of their movements. additional working bodies controlled the flows of materials and products, modes of unwinding of rolls, stocks of film in a roll, adjustment of kinematic parameters, automatic filling of the film and providing of the film from various rolls. the working bodies of the main and auxiliary groups directly interacted with the rolls and film canvas, and the bodies of the additional group performed control, blocking, out-of-cycle, regulating and other similar operations [15, 16]. their functional tasks were to control the operation of devices for providing packaging materials and other devices of packaging machines, which had malfunctions of the film supply system. an important dynamic component of the supply system of rolled packaging materials was a roll holder with a roll of film material. the task entrusted to the roll holder was to ensure the appropriate positioning of the roll in the specified coordinates with the possibility of rotational movement in the mode of winding the film. the relatively significant mass of the roll holder and the roll in dynamic models of the system led to the fact that they were collectively attributed to the driven masses, which in transients determined a significant proportion of dynamic loads [17]. obviously, the design of the roll holder was directly related to the weight of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 302 roll and to some extent with the mode of operation of the packaging line. on the one hand, this formulated the requirement to limit the weight of the roll holder, but on the other hand, the design must have ensured the reliability of the specified coordinates and position of the roll and the possibility of its rapid replacement. the driving factor in the transition process was the tension of the film material, which depended on its load on the working body, its law of motion and the ability to develop and transmit force to the film canvas. obviously, from the point of view of the interests of finding the optimal parameters of the components of the system, a positive result was quite achievable, but it corresponded to the stabilized values of the driving and driven masses and stiffness. if with respect to the driving mass and stiffness such a condition was acceptable, then with respect to the driven mass, its changes by an order of magnitude or more were predicted. the last conclusion was connected with change of weight of a roll and its diameter at the beginning and at the end of winding film from it. the maximum and minimum values of the moments of inertia of the combination of the roll holder and the roll meant the effects on the maximum and minimum values of the springy loads of the film canvas. in cyclically operating equipment, transient modes of acceleration and run-out accompanied the entire technological time of operation of the equipment and this was the reason for finding opportunities to stabilize the dynamic parameters. to confirm this position, we turned to the comparison of the moments of inertia of the driven mass in the range of changes in the radius of the roll from the initial  ir to the final  fr at    fi rr  . under such conditions we have:       2 2 ii i rm і  ;       2 2 ff f rm і  ; (1) since not only radii but also masses will be variable  im and  fm as the initial and final, respectively. let's display changes of masses through geometrical parameters and specific weight ρ of a film material:    ii vm  ;    ff vm  , (2) where  iv and  fv –the initial and final volumes of the roll, respectively, which were determined by the dependences:    нrv ii 2  ;    нrv ff 2  (3) the corresponding substitution give the value:    нrm ii 2  ;    нrm ff 2  , (4) where н – roll height. then,    нrі ii 4 5.0  ;    нrі ff 4 5.0  (5) then the ratio    fi іі is:         4 4 f i f i r r і і  (6) if the value   2 1 mkgі i  is accepted than the ratio    fi іі will be displayed in the following row: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 303  fr 0.5  ir 0.4  ir 0.3  ir 0.2  ir    fi іі 16 39.06 123.46 625 the given number of numerical values showed extremely powerful changes of values of the moment of inertia of the roll. the decrease in the moment of inertia of the roll indicated that as such a transformation occurred, there was a corresponding decrease in the dynamic components of the loads, which should be evaluated by a positive result. however, the reduction of the specified moment of inertia was accompanied by the same intense limitation of the run-out time at a constant run-out time of the driving mass or its programmed kinematic run-out braking. such an imbalance should be assessed by additional dynamic perturbation, which will result in destabilization of the tension of the film canvas. let's compare the changes related to the processes of running out at radius values  ir і  fr . reducing the mass of the roll and, at the same time, its moment of inertia meant, other things being equal, a decrease in the level of kinetic energy at the beginning of the run. let the displacement of the film canvas during the cycle time to be a value с . then the average speed in movement is: ссс tv  . (7) the average angular velocity of the roll corresponds to this average value : rvсс  (8) since the average velocity was stabilized, this means that the average angular velocity will be variable as the changes of the radius of the roll from  ir to  fr . so at a radius  ir the value will be smaller and, conversely, as r approaches the value  fr , the angular velocity  f increases. then the kinetic energy of the system within the changes from  ir to  fr will be:       2 5.0          i c ii r v іе ;       2 5.0          f c ff r v іе (9) then the ratio of kinetic energies expended in the run-out mode to overcome the moments of friction forces in the supports of the roll holder is:             2 2 i f f i f i r r і і е е  (10) but since it is shown that         4 4 f i f i r r i i  , then         2 2 f i f i r r е е  . the equation which corresponds to the regime of the run-out mode of the rollholder-roll system in the general case: і м res  (11) or for comparable conditions we have:      i ires i і м  ;      f fres f і м  (12) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 304 the change of resistance moments from the value  iresм to  fresм is associated with a decrease in the mass of the roll. in the first approximation, we assumed that these moments of resistance were proportional to the masses:    iires k mм  ;    ffres kmм  (13) then:      i i i і km  ;      f f f і k m  (14) hence the integration was obtained under the initial conditions   0it ,   ci vx  :       t і km v i i ci  ;       t і km v f f cf  (15) since the values   0i and   0f correspond to the end of the run-out, we determine the end time of the run:      i ic iend km іv t  ;      f fc fend k m іv t  (16) substitution of values       2 2 i ii r mі  and       2 2 f ff r mі  led to the form:     k rv t ic iend 2 2  ;     k rv t fc fend 2 2  (17) then the ratio of run-out time at the beginning and end of the technological process is:         2 2 f i fend iend r r t t  (18) however, the formation of packaging from rolled packaging material was accompanied by important compliance with minor changes in the tension of the film. in this regard, in any highperformance packaging system with periodic or continuous operation there was provided the function of depreciation and accumulation of film material. the presence in the machines of the linear type of areas of packing, packaging, sealing, marking led to the need to take into account their features, stiffness, variable laws of motion of the driving masses, the presence of shock modes and loads. 3.2. transients of interaction of driving and driven masses the rules developed on the basis of the law of conservation of energy allowed to make transitions from physical systems to their models on the principles of equivalence of kinetic energies (reduction of masses), instantaneous powers (reduction of forces and moments of forces) and potential energies of deformations (reduction of rigidities) [18]. the presence of springy bonds in the form of film canvases, tapes, belts in the packaging lines means the expediency of using linear models. in fig. 1 a two-mass scheme is shown, which corresponds to the case with the absence of technological or non-technological gaps. in this case, the springy bond with stiffness c can be loaded to the value of the resistance force pres applied to the driven mass or unloaded. in the first case, the system startup is modeled in one stage, and in the second in two stages. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 305 in the one-stage case, the driving mass m1 and the driven mass m2 simultaneously begin to move from the moment of application of the driving force to the driving mass with the load of the springy connection in addition by the forces of inertia. the load condition of the springy bond to the value of psp = pres in the systems of film movement was achieved through the use of compensation devices. fulfillment or non-fulfillment of this condition, or the presence of a technological gap δ (fig. 2) lead to a different number of stages of dynamic interaction of the masses. the sequence of stages is as follows. the case of a system with a technological gap when δ ≠ 0 and psp = 0: the overlap of the gap δ due to the movement of the leading mass m1 under the action of pdr corresponds to the first stage: .11 drрхm  (19) as a result of integration of condition (1) we have the coordinate of movement x1, the speed 1х of the driving mass with a sweep in time: ; 2 2 1 1 t m р х dr  , 1 1 t m р х dr  (20) final parameters of the first stage and time of its course:   ;1 fх    ; 2 2 1 1 f dr f t m р х      . 2 11 dr fі f р хm t  (21) the movement of the driving mass at the first stage had no counteraction, which under certain conditions may be accompanied by the achievement of a constant value of speed. in the case of using in the drives of the driving masses of motors with rigid characteristics, this can lead to shock loads at the next stage. at the second stage, the springy element was loaded to the value of pres by moving the driving mass and the stationary driven mass. the equation of motion 1m is written in the form: ,111 схрхm dr  (22) c x2 m2 рres x1 m1 рdr δ fig. 2. scheme of a two-mass model with a technological gap food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 306 and the solution of the differential linear equation of the second order is carried out under the initial conditions:        .;0;0 111 і f іі i іі i іі i хххt   (23) herewith ,cossin 11 1 c р t m с bt m с ах dr  (24) where a and b are the integration constants that are determined after substituting the initial conditions (5). from here: ; c р в dr    . 1 1 с m ха і f  (25) then by substituting the constants of integration we finally get:   .cossin 11 1 11 c р t m с c р t m с с m хх drdrі f   (26) at the second stage, the springy force was determined by the dependence:   .cossin 11 111 drdr і fsp рt m с рt m с сmхсхр   (27) the substitution psp = pres corresponds to the completion of the second stage. then:       .cossin 1 1 1 1 11 dr іі fdr іі f і fres рt m с рt m с сmхр   (28) the value   іі ft1 from condition (28) was determined by iterations or using computer programs, according to which making the corresponding substitutions   іі fх1 and   іі fх1 were defined as the initial conditions of the third stage. regarding it, it is written:  ;2111 ххсpxm dr  (29)   .2122 respххсxm  (30) the last two conditions are a general system of differential equations, which determines the motion of masses m1 and m2 at the given level. transition from the real system to the given scheme of preliminary analysis with definition of driving and driven weights in packing machines by a link of reduction m1 it is expedient to appoint a working body taking into account weights of the drive, and the driven weight m2 selects a roll of packing material. in this case, the system has intermediate masses of rollers, the packaging material between the driving and driven masses, elements of the compensation mechanism and so on. the combined actions of intermediate and main masses are estimated in the assumption that their effects are synchronous. obviously, for limited values of the stiffness of springy bonds, this assumption is not true. however, it meets the worst working conditions of springy connections, which should provide protection against overload. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 307 taking into account, that the intermediate masses have limited values compared to the driving and driven masses and the fact that the errors lead to increased loads and strength conditions, it is possible to come to the perception of such an assumption [19]. however, there is another borderline approach, at which the influence of intermediate masses is proposed to be neglected. formal rules for transforming the system of differential equations (29) (30) allow to write the following:   ; 1 21 1 1 m p хх m с x dr  (31)   . 2 21 2 2 m p хх m c x res  (32) subtracting from condition (31) equation (32), we have:     . 21 21 21 21 m p m p хх m c m c хх resdr            (33) since the task of the study was to determine the loads of the springy bond psp, it was advisable to make the transition to the equation of springy forces, multiplying all the components from conditions (33) by the given stiffness from the system:     . 21 21 21 21                   m p m p схх m c m c сххс resdr (34) since   spрххс  21 and   spрххс   21 , we have: . 2121                   m p m p сp m c m c p resdr spsp  (35) the solution of the last condition is in the form: ,cossin 21 211          m p m pc tbtаp resdrsp   (36) where . 21 m c m c  (37) the integration constants a1 and b1 are searched for under the initial conditions of the kinematic parameters:             .0;0;;;0 22111  ii іі fi res ii хххх с p хt  (38) then the initial conditions for the values of springy forces are:        ;21 resiiisp pххсp  (39)         .121 іі fiiisp хсххсp    (40) substitution of the initial condition psp(i)=pfres leads to the value: . 21 21          m p m pc рв resdr res  (41) to determine the integration constant a1, the differentiation of condition (36) is performed: ,sincos 11 tbtаpsp    (42) from this it was obtained     .; 1 111   іі fіі f хc aaхc    (43) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 308 then we finally have:   .cossin 21 2 21 2 1                           m p m pc t m p m pc pt хc p resdrresdr res іі f sp       (44) from the latter condition it is seen that the total load of the springy bond is determined by the sum of static and two dynamic components. this means that the function рsp = рsp(t) will have extrema. to determine them, it is necessary to find the value of the text the time of their achievement. after differentiation of the function (44) and equating the result to zero, we obtain:   . 1 21 2 1                            m p m pc p хc arctgt resdr res іі f ext  (45) the found value of text allowed to define extreme loading:   .cossin 21 2 21 2 1                           m p m pc t m p m pc pt хc p resdr ext resdr resext іі f extsp       (46) it is obvious that the obtained value of psp.ext allowed to determine the achievement of the condition of the strength of the canvas of springy bonding, for example, in the form:   ,. t extsp t f p   (47) which corresponds to the condition of tensile strength, where f is the crosssectional area of the canvas; [σ]t is the allowable tensile strength of the material. it is obvious that the factors influencing the achievement of the strength condition are the values of psp.ext and f, which are variables. so if necessary there is a possibility in some step mode to choose the increased width or thickness of canvas or it is a possibility of decrease psp.ext. however, it should be taken into account that the increase in the cross-sectional area of the canvas requires a new recalculation of the value of the extreme load due to the increase in stiffness. the latter was determined by the properties of the canvas material and geometric parameters in the form: mн еf с ,   (48) where е – the modulus of springiness of the packaging material, pа; ℓ – the length of the springy ligamentу, m. the latter dependence followed from the formulation of the parameter c, because the stiffness was determined by the ratio of the force action on the sample to the amount of deformation caused by the action of this force. since the force action was the value of psp.ext, and the value of the absolute deformation according to hooke's law was: , ef р   (49) then .  efр с    (50) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 309 in our case, the basis for the recalculation was an increase in the parameter f, and therefore it was necessary to re-determine psp.ext and re-recalculate the strength condition. it was possible that obtaining the final result could require recalculation. in addition to the above reason, there was another caveat in such actions, which concerned the features of packaging lines in the modeling of the dynamics of force. the model in equations (29) (30) indicated the lack of consideration of the distributed mass of the flexible connection, which was due to one of the assumptions that the mass of the springy connection was limited and neglected. at the same time, condition (44), although it provided an answer to the question of the value of springy loading, but it was almost impossible to assess the factors of influence and prospects for load limitation. a certain exception applied to the driving forces of pdr and the forces of resistance pres, because in the amplitude of the cosine component and in the component of static load they were written in numerators. this confirms the possible phenomenological conclusion about the increase in psp at their growth. analysis of the amplitude of the sinusoidal dynamic component indicated the importance of the influence of velocity   іі fх1 and stiffness c, although the influence of this component was also veiled by the parameter ω, which reflected the natural circular frequency of oscillations of the dynamic system. taking into account the fact that it was investigated equipment of cyclic action, the comparison of natural and external frequency of influences was relevant. evaluating the stiffness of polymer packaging films, the characteristics of polyethylene and polyethylene terephthalate materials as examples were taken into account. ranges of parameters, such as thickness and modulus of springiness of material, were accepted in calculations. in the generalized estimate of the ranges of specific stiffness c, the thickness of the film materials was considered to be the root cause, without rejecting the effects of variable values of density, because in the classical sense, the modulus of springiness reflected the physical properties of materials. in addition, the length of the section ℓ was an important factor in destabilizing the stiffness values. the significant influence of the length of the film section in the case of changes  in the modes of interaction between the driving and driven mass indicated that the dynamic loads in the form of psp will be different. in addition, the natural oscillation frequencies of the system will be variable. in figures 3 and 4 the graphical dependences c = c (ℓ) are shown, from which it follows that as the total value of ℓ decreases, the influences in values с increase. this means that with changes in the values of δc in response to changes in length ℓ dynamic models became nonlinear, with increasing stiffness the amplitudes of the dynamic components increased and there was an effect on the static component of the springy bond load. in addition, from the formula (44) follows the presence of the initial velocity of the third stage of the driving mass in the amplitude of the sinusoidal component. it was the final velocity at the previous stage   іі fх , and therefore the worst situation in terms of springy bond loads corresponded to the shock interaction, when it was achieved   vхх іііі f  max1 during the selection of the gap δ. recourse to such a case led to the condition: ,sin 1 2 ressp рt m с сmvр  (51) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 310 at this condition the springy load consisted of dynamic and static components. the maximum value of the dynamic component was reached at time tm: , 2 2 c m tm   (52) mathematical formalization (51) in contrast to the previously obtained equation (44) allowed to unambiguously assess the role of influencing factors. thus, with respect to the amplitude of the dynamic component, the presence of three factors such as velocity v, driven mass m2 0 500000 1000000 1500000 2000000 1 2 3 4 5 6 7 8 9 10 с, n/m ℓ, m polyethylene film δ = 300 μm, е = 1000 мpа polyethylene terephthalate film δ = 350 μm, е = 5500 мpа fig. 4. the dependence of the stiffness of dynamic systems on the length of the flexible connection for larger values of film thickness 0 500 1000 1500 2000 2500 3000 3500 1 2 3 4 5 6 7 8 9 10 с, n/m ℓ, m polyethylene film δ = 1 μm, е = 100 мpа polyethylene terephthalate film δ = 1 μm, е = 3400 мpа fig. 3. the dependence of the stiffness of dynamic systems on the length of the flexible connection for limited values of film thickness food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 311 and stiffness c of the elastic element was clearly defined. however, the structure of the amplitude of the dynamic sinusoidal component in model (44) formally coincided with (51), because we had:   , 21 21 mm mсm ха іі f    (53) where complex m1m2/(m1+m2) acted as a reduced mass of the system. comparing the two models, it should be concluded that the second model gave slightly more rigid results for psp, but calculations for them showed deviations in the range of 5 ... 8%, which in the descriptions of the dynamics of machine units was considered acceptable. however, from the point of view of the use of influence parameters in the interests of variations of psp values, there were certain limitations. thus, the kinematic parameter of the velocity of the driving mass corresponded to a given system performance and its limitations were not always possible. the value of the driven mass was also technologically determined, and the reduction in stiffness could turn the system into a weakly damped, the disadvantage of which may have been violations in positioning and the need for dampers. these three parameters of influence were dynamic factors, the change of which required additional analysis. this was all the more relevant due to the fact that they could be variable in technological use. for example, the fastening of the packagepallet with a stretchable film was accompanied by a variable speed of winding the film from the spool. the speeds changed periodically from minimum to maximum and again to minimum, which was accompanied by uneven tension of the film with the prospect of destruction of the packagepallets during transportation. in addition, the length of the film changed at limited values of this parameter and hence the variable stiffness in the hopping mode, followed by a nonlinear change. the driven mass of a roll with a film will be also variable, however these changes occur extended in time. 4. conclusion modern theoretical basis for the synthesis of technological machines based on the use of film roll packaging materials combined the ability to take into account technological, economic requirements, high productivity, neighborhood conditions, energy savings, restrictions on dynamic loads and more. achieving the combination of these requirements was largely due to the use of flexible connections directly between the working bodies of technological machines and in the packaging lines. synchronized combination of operations, the use of optimal laws of movement of working bodies, high-precision fabrication of elements of kinematic pairs and parts, limitation of dynamic loads, kinetic energy recovery measures were among the positive areas of creating efficient technological machines and lines of them. limiting the mutual influences on the performance of individual machines and lines in general due to intermediate compensators was a logical direction of synthesis of systems, but some caveats relate to the growth of material, economic and energy costs in the modes of their creation and operation. energy costs in the systems of movement of film, roll packaging materials are associated with the work of driving forces against friction and to create flows of kinetic energy of moving masses. unwinding of film rolls was accompanied by changes in their moments of inertia in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 312 cycles from the beginning to the end of their use by two orders of magnitude. the ratio of the run-out time of rolls of variable mass was determined by the ratio of the squares of the initial and final radii. the presence in the lines of movement of film materials compensation and damping devices led to their division into almost two dynamically independent parts. a variable nature of the moment of inertia of the roll with the film had significant destabilizing effect on the dynamics of the system. in this regard, it is advisable to create a compensator-regulator of the moment of inertia of the roll and the roll holder. the solution of the problem of kinetic energy recovery in systems of periodic action is simultaneously connected with the restriction of the unevenness of the drives of machines. in systems with a flexible bond of film material, the run-out mode of the driving mass is controlled, and the run-out of the driven mass can be controlled, provided that the acceleration by breaking it by module is greater than the acceleration module of the run-out of the driving mass. otherwise, the springy connection between the working body and the driven mass is lost and there is a need to use a compensator-regulator. mathematical formalizations of interactions at the level of two-mass models in the stepwise mapping of kinematic and dynamic parameters, including with the use of equations of springy forces for the possibility of achieving strength conditions for flexible connections, have been developed. a method for estimating the stiffness of springy bonds based on film materials taking into account their geometric parameters is proposed. it is shown that the variable transient value of the springy bond length acts as a dynamic exciter of the system. 5. references [1]. sun w., shi m., zhang c., zhao j., song x., dynamic load prediction of tunnel boring machine (tbm) based on heterogeneous insitu data, automation in construction, 92: 23-34, (2018). dоі:https://doi.org/10.1016/j.autcon.2018.03.030 [2]. boschetti g., caracciolo r., richiedei d., trevisani a., model-based dynamic compensation of load cell response in weighing machines affected by environmental vibrations, mechanical systems and signal processing, 34(1-2): 116-130, (2013). dоі:https://doi.org/10.1016/j.ymssp.2012.07.010 [3]. klocke f., döbbeler b., goetz s., estimation of the load torque in a hobbing machine using effective power signals, procedia manufacturing, 18: 43-49, (2018). dоі:https://doi.org/10.1016/j.promfg.2018.11.006 [4]. murin j., a machine aggregate with hydrodynamic power transmission at periodic loading, mechanism and machine theory, 36(1): 77-92, (2001). dоі:https://doi.org/10.1016/s0094-114x(00)00029x [5]. du x., chen b., zheng z. investigation on mechanical behavior of planetary roller screw mechanism with the effects of external loads and machining errors, tribology international, 154: 1066-1089, (2021). dоі:https://doi.org/10.1016/j.triboint.2020.106689 [6]. zhang t., zhang d., zhang z., muhammad m. investigation on the load-inertia ratio of machine tools working in high speed and high acceleration processes, mechanism and machine theory, 155, (2021). dоі:https://doi.org/10.1016/j.mechmachtheory.2020 .104093 [7]. golec z., cempel c. machine vibroisolation and dynamic loads of bearings, mechanical systems and signal processing, 4(5): 367-375, (1990). dоі:https://doi.org/10.1016/0888-3270(90)90021-c [8]. song y., wu j., yu g., huang t. dynamic characteristic prediction of a 5-dof hybrid machine tool by using scale model considering the geometric distortion of bearings, mechanism and machine theory, 145, (2020). dоі:https://doi.org/10.1016/j.mechmachtheory.2019 .103679 [9]. voet h., garretson i., falk b., schmitt r., linke b. peak power load and energy costs using the example of the startup and idling of a grinding machine, procedia cirp, 69: 324-329, (2018). https://www.sciencedirect.com/science/article/abs/pii/s0926580517307914?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0926580517307914?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0926580517307914?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0926580517307914?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0926580517307914?via%3dihub#! https://www.sciencedirect.com/science/journal/09265805 https://doi.org/10.1016/j.autcon.2018.03.030 https://www.sciencedirect.com/science/article/abs/pii/s0888327012002737?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0888327012002737?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0888327012002737?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0888327012002737?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0888327012002737?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0888327012002737 https://www.sciencedirect.com/science/article/pii/s0888327012002737 https://www.sciencedirect.com/science/article/pii/s0888327012002737 https://www.sciencedirect.com/science/article/pii/s0888327012002737 https://www.sciencedirect.com/science/journal/08883270 https://www.sciencedirect.com/science/journal/08883270 https://doi.org/10.1016/j.ymssp.2012.07.010 https://www.sciencedirect.com/science/article/pii/s2351978918313210?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2351978918313210?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2351978918313210?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2351978918313210 https://www.sciencedirect.com/science/article/pii/s2351978918313210 https://www.sciencedirect.com/science/journal/23519789 https://www.sciencedirect.com/science/journal/23519789 https://doi.org/10.1016/j.promfg.2018.11.006 https://www.sciencedirect.com/science/article/abs/pii/s0094114x0000029x?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x0000029x https://www.sciencedirect.com/science/article/pii/s0094114x0000029x https://www.sciencedirect.com/science/article/pii/s0094114x0000029x https://www.sciencedirect.com/science/journal/0094114x https://doi.org/10.1016/s0094-114x(00)00029-x https://doi.org/10.1016/s0094-114x(00)00029-x https://www.sciencedirect.com/science/article/abs/pii/s0301679x20305144?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0301679x20305144?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0301679x20305144?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0301679x20305144 https://www.sciencedirect.com/science/article/pii/s0301679x20305144 https://www.sciencedirect.com/science/article/pii/s0301679x20305144 https://www.sciencedirect.com/science/article/pii/s0301679x20305144 https://www.sciencedirect.com/science/journal/0301679x https://www.sciencedirect.com/science/journal/0301679x/154/supp/c https://doi.org/10.1016/j.triboint.2020.106689 https://www.sciencedirect.com/science/article/pii/s0094114x20303128?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x20303128?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x20303128?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x20303128?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x20303128 https://www.sciencedirect.com/science/article/pii/s0094114x20303128 https://www.sciencedirect.com/science/article/pii/s0094114x20303128 https://www.sciencedirect.com/science/journal/0094114x https://www.sciencedirect.com/science/journal/0094114x https://www.sciencedirect.com/science/journal/0094114x/155/supp/c https://doi.org/10.1016/j.mechmachtheory.2020.104093 https://doi.org/10.1016/j.mechmachtheory.2020.104093 https://www.sciencedirect.com/science/article/abs/pii/088832709090021c?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/088832709090021c?via%3dihub#! https://www.sciencedirect.com/science/article/pii/088832709090021c https://www.sciencedirect.com/science/article/pii/088832709090021c https://www.sciencedirect.com/science/journal/08883270 https://www.sciencedirect.com/science/journal/08883270/4/5 https://doi.org/10.1016/0888-3270(90)90021-c https://www.sciencedirect.com/science/article/abs/pii/s0094114x19322852?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0094114x19322852?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0094114x19322852?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0094114x19322852?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0094114x19322852 https://www.sciencedirect.com/science/article/pii/s0094114x19322852 https://www.sciencedirect.com/science/article/pii/s0094114x19322852 https://www.sciencedirect.com/science/journal/0094114x https://doi.org/10.1016/j.mechmachtheory.2019.103679 https://doi.org/10.1016/j.mechmachtheory.2019.103679 https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2212827117308132 https://www.sciencedirect.com/science/article/pii/s2212827117308132 https://www.sciencedirect.com/science/article/pii/s2212827117308132 https://www.sciencedirect.com/science/journal/22128271 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anatoly sokolenko, oleksandr shevchenko, oleg stepanets, serhiy but, anastasiia shevchenko, dynamics of springy systems of packaging lines for food products, food and environment safety, volume xx, issue 4 – 2021, pag. 298 – 313 313 dоі:https://doi.org/10.1016/j.procir.2017.11.044 [10]. dai y., zhao p. a hybrid load forecasting model based on support vector machine with intelligent methods for feature selection and parameter optimization, applied energy, 279, (2020). dоі:https://doi.org/10.1016/j.apenergy.2020.115332 [11]. ganapathi raju n.v., radhanand a., balaji kumar k.n., pradeep reddy g., sampath krishna reddy p. machine learning based power saving mechanism for fridge: an experimental study using gismo iii board, materialstoday: proceedings. (2020). dоі:https://doi.org/10.1016/j.matpr.2020.08.387 [12]. devarapalli r., bhattacharyya b., kumar sinha n., dey b. amended gwo approach based multi-machine power system stability enhancement, isa transactions. (2020). dоі:https://doi.org/10.1016/j.isatra.2020.09.016 [13]. sokolenko а., stepanets о., pryhodii d. regulation machine running, food industry, 21: 155-163, (2017). doi:http://dspace.nuft.edu.ua/jspui/bitstream/12345 6789/26422/1/%e2%84%9621.pdf [14]. gavva o., bespalko a., volchko a., kokhan о. packing equipment. kyiv: iac "packaging". (2010). [15]. gavva o., maslo m., yarovyy v. devices for feeding rolled packaging material, packaging, 5: 46-54. (2003). [16]. gavva о., volchko a., maslo m. devices for forming packaging from thermowelded roll materials, packaging, 1: 30-32. (2005). [17]. maslo m., gavva o. structural elements of transport systems of packing equipment, packing, 2: 44-46. (2006). [18]. pavlov s. improvement of methods of calculation of packing equipment of food productions with working elements of variable rigidity: abstract dis. ... cand. tech. sciences. kyiv: nuft. (2012). [19]. sokolenko a., vasylkivsky k., yakymchuk m. synthesis of machines of packing and energy saving lines, packaging, 3: 5355. (2012). https://doi.org/10.1016/j.procir.2017.11.044 https://www.sciencedirect.com/science/article/abs/pii/s0306261920308448?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0306261920308448?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0306261920308448 https://www.sciencedirect.com/science/article/pii/s0306261920308448 https://www.sciencedirect.com/science/article/pii/s0306261920308448 https://www.sciencedirect.com/science/article/pii/s0306261920308448 https://www.sciencedirect.com/science/journal/03062619 https://doi.org/10.1016/j.apenergy.2020.115332 https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s2214785320362593 https://www.sciencedirect.com/science/article/pii/s2214785320362593 https://doi.org/10.1016/j.matpr.2020.08.387 https://www.sciencedirect.com/science/article/abs/pii/s0019057820303906?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0019057820303906?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0019057820303906?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0019057820303906?via%3dihub#! https://www.sciencedirect.com/science/article/abs/pii/s0019057820303906?via%3dihub#! https://www.sciencedirect.com/science/article/pii/s0019057820303906 https://www.sciencedirect.com/science/article/pii/s0019057820303906 https://www.sciencedirect.com/science/journal/00190578 https://doi.org/10.1016/j.isatra.2020.09.016 http://dspace.nuft.edu.ua/jspui/bitstream/123456789/26422/1/%e2%84%9621.pdf http://dspace.nuft.edu.ua/jspui/bitstream/123456789/26422/1/%e2%84%9621.pdf [1]. sun w., shi m., zhang c., zhao j., song x., dynamic load prediction of tunnel boring machine (tbm) based on heterogeneous in-situ data, automation in construction, 92: 23-34, (2018). dоі:https://doi.org/10.1016/j.autcon.2018.03.030 [2]. boschetti g., caracciolo r., richiedei d., trevisani a., model-based dynamic compensation of load cell response in weighing machines affected by environmental vibrations, mechanical systems and signal processing, 34(1-2): 116-130, (2013). dоі:https://doi.org/10.1016/j.ymssp.2012.07.010 [3]. klocke f., döbbeler b., goetz s., estimation of the load torque in a hobbing machine using effective power signals, procedia manufacturing, 18: 43-49, (2018). dоі:https://doi.org/10.1016/j.promfg.2018.11.006 [4]. murin j., a machine aggregate with hydrodynamic power transmission at periodic loading, mechanism and machine theory, 36(1): 77-92, (2001). dоі:https://doi.org/10.1016/s0094-114x(00)00029-x [5]. du x., chen b., zheng z. investigation on mechanical behavior of planetary roller screw mechanism with the effects of external loads and machining errors, tribology international, 154: 1066-1089, (2021). dоі:https://doi.org/10.1016/j.triboint.2020.106689 [6]. zhang t., zhang d., zhang z., muhammad m. investigation on the load-inertia ratio of machine tools working in high speed and high acceleration processes, mechanism and machine theory, 155, (2021). dоі:https://doi.org/10.1016/j.mechmachtheory.2020.104093 [7]. golec z., cempel c. machine vibroisolation and dynamic loads of bearings, mechanical systems and signal processing, 4(5): 367-375, (1990). dоі:https://doi.org/10.1016/0888-3270(90)90021-c [8]. song y., wu j., yu g., huang t. dynamic characteristic prediction of a 5-dof hybrid machine tool by using scale model considering the geometric distortion of bearings, mechanism and machine theory, 145, (2020). dоі:https://doi.org/10.1016/j.mechmachtheory.2019.103679 [9]. voet h., garretson i., falk b., schmitt r., linke b. peak power load and energy costs using the example of the startup and idling of a grinding machine, procedia cirp, 69: 324-329, (2018). dоі:https://doi.org/10.1016/j.procir.2017.11.044 [10]. dai y., zhao p. a hybrid load forecasting model based on support vector machine with intelligent methods for feature selection and parameter optimization, applied energy, 279, (2020). dоі:https://doi.org/10.1016/j.apenergy.2020.115332 [11]. ganapathi raju n.v., radhanand a., balaji kumar k.n., pradeep reddy g., sampath krishna reddy p. machine learning based power saving mechanism for fridge: an experimental study using gismo iii board, materialstoday: proceedings. (2020). dоі:https://doi.org/10.1016/j.matpr.2020.08.387 [12]. devarapalli r., bhattacharyya b., kumar sinha n., dey b. amended gwo approach based multi-machine power system stability enhancement, isa transactions. (2020). dоі:https://doi.org/10.1016/j.isatra.2020.09.016 [13]. sokolenko а., stepanets о., pryhodii d. regulation machine running, food industry, 21: 155-163, (2017). doi:http://dspace.nuft.edu.ua/jspui/bitstream/123456789/26422/1/%e2%84%9621.pdf [14]. gavva o., bespalko a., volchko a., kokhan о. packing equipment. kyiv: iac "packaging". (2010). [15]. gavva o., maslo m., yarovyy v. devices for feeding rolled packaging material, packaging, 5: 46-54. (2003). [16]. gavva о., volchko a., maslo m. devices for forming packaging from thermowelded roll materials, packaging, 1: 30-32. (2005). [17]. maslo m., gavva o. structural elements of transport systems of packing equipment, packing, 2: 44-46. (2006). [18]. pavlov s. improvement of methods of calculation of packing equipment of food productions with working elements of variable rigidity: abstract dis. ... cand. tech. sciences. kyiv: nuft. (2012). [19]. sokolenko a., vasylkivsky k., yakymchuk m. synthesis of machines of packing and energy saving lines, packaging, 3: 53-55. (2012). doi: https://doi.org/10.4316/fens.2021.029 262 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 262 273 evaluation of mexa dam water quality in el tarf region (extreme north-east algeria) *samira gheid 1 , sofia bahroun 2 , amina benabdallah 2 , abdelhak gheid 3 1 department of biology, faculty of life and natural sciences, university of chadli bendjedid, el-tarf, 36000, algeria. samiragheid@yahoo.fr 2 bpe laboratory, faculty of life and natural sciences, university of chadli bendjedid, bp 73, el-tarf, 36000, algeria. sofiabahroun@gmail.com , benabdallahamina@hotmail.fr 3 water and environment science and technology laboratory, faculty of life and natural sciences, university of mohamed-cherif messaadia, souk ahras, 41000. algeria. hakgheid@yahoo.fr *corresponding author received 8th june 2021, accepted 29th september 2021 abstract: the water is a precious natural resource and vital for the living beings, the increasing water demand for irrigation or feeding, have made necessary the construction of a great number of dams that require an excellent physicochemical quality. the objective of our work is to evaluate the seasonal water quality of mexa dam (eastern algeria) during four years 2016, 2017, 2018 and 2019. the present work aims to establish a physicochemical characterization to determine the parameters affecting the water quality, such as temperature, conductivity, turbidity, hydrometric title, salinity, complete alkalimetric title, calcium, magnesium, bicarbonate, chloride, iron, ph, dry residue (dr), organic matter (om), nitrate, nitrite, ammonium, biological oxygen demand (bod5) and chemical oxygen demand (cod). the results of the water physico-chemical analyses of this dam show that the majority of the obtained values are in conformity with standards, thus the water can directly be used for irrigation. according to nahr grid, the water quality records of mexa dam show a medium quality for the majority of the studied parameters. the level of nitrogenous matter (nh4 +, no2 -) in the water is high during 2016 and 2017, this pollution is caused by the oxidation of nitrogenous organic matter. keywords: mexa dam, eastern algeria, physicochemical parameters, irrigation water, drinking water. 1. introduction water is a raw resource, partially renewable, like air, it is considered vital to human life. it is one of the main factors in the organization and development of territories [1]. the protection of water resources is one of the most important concerns of any environmental policy [2], it is a strategic global investment that should essentially be managed within the framework of a sustainable development policy [3]. in algeria, water is an increasingly precious resource, the competition between agriculture, industry and daily population uses accessing to a limited water supplies is already affecting the development efforts of many countries [4]. important investments have been made in the construction of the dams, especially in the eastern part of the country. dams offer many benefits to our society, such as the provision of drinking and irrigation water [5]. however, these surface waters are sensitive to all kinds of pollution and are often of bad quality [6]. they may contain significant amounts of natural organic matter such as humic substances, but also other compounds from various pollutant wastes or intensive agricultural practices [7]. these events, able to affect the water quality, require a rigorous control mailto:samiragheid@yahoo.fr mailto:sofiabahroun@gmail.com mailto:benabdallahamina@hotmail.fr mailto:hakgheid@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 263 of the systems, which is of great interest to ensure the correct performance of these systems. hence, this work aims to evaluate the quality of mexa dam water located in el tarf province (north-east of algeria), based on the monitoring of physicochemical parameters for both seasons (rainy and dry), during four years (from 2016 till 2019). 2. materials and methods the studied area concerns the mexa dam, located in the municipality of bougous near the algerian-tunisian border, 8 km from the chief town of el tarf municipality, and 71 km from annaba city by the national road n°44 [8]. fig. 1. geographical location of the maxa dam [8]. the region has a great agricultural vocation, farming sector provides most of permanent and seasonal jobs [9], it is characterized by a wide variety of cultures (citrus, pear, pomegranate, medlar and quince trees, sorghum, melon, watermelon, and other vegetables) [10]. industry, sensu stricto, is mainly inexistent in the basin, as well as in the whole province [11]. sampling: sampling campaigns of mexa dam waters were conducted for a period of four years (2016, 2017, 2018, and 2019), by monthly sampling for each year. the survey was conducted on the raw water from the dam site [12-13]. surface water samples were collected directly with polyethylene bottles, by sampling at a depth of 15-30 cm from the water surface, avoiding edge effects (oxygenation too close to the surface, solid matter suspension too close to the bottom), facing the water current. the samples were kept at a temperature of 4°c. chemical analyses were conducted in the laboratory where the major elements analyses were performed on the filtered samples [14]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 264 table 1. the different methods used for physicochemical parameters analysis parameters methods in situ analysis (physical parameters) ph, temperature, electrical conductivity, turbidity and dissolved oxygen electro-metric method with combined electrode in vitro analysis (chemical parameters) hydrometric title full alkalimetric title calcium and magnesium iron bicarbonate chloride ammonium, nitrite, nitrate, phosphate bod5, cod volumetric method volumetric method flame spectrometer spectrometric method volumetric method mohr method flame spectrometer [15]. spectrometric method measured using an oxitop is12 dam water quality measurement the study focused on the in situ measurement of ph and dissolved oxygen with the determination of other elements in the laboratory (dry residue, om, bod5, cod, po4 3-, no2 -, nh4 + and no3 -). the assessment of water quality is based on the comparison of various analyzed chemical elements (often called parameters) to standards or thresholds (table 2). all the threshold values for the considered parameters constitute a quality grid, through which it would be possible to assign a quality class to the dam water [1617]. for a better illustration of the different water pollution levels, we have assigned a color to each class according to the grid used by the nahr (national agency of hydraulic resources):  the class of good water quality is represented in blue.  the class of medium water quality is represented in green.  the class of bad water quality is represented in yellow  the class of very bad water quality is represented in red. table 2. water quality grid approved by nahr (the national agency of hydraulic resources). parameters unit quality scales good medium bad very bad ph 6.5 8.5 6.5 8.5 8.5 9 >9 et <6.5 dissolved o2 % 100 90 90 50 50 30 < 30 ammonium nh4 + mg/l 0 0.01 0.01 0.1 0.1 3 > 3 nitrate no3 mg/l < 10 10 20 20 40 > 40 nitrite no2 mg/l 0 0.01 0.01 0.1 0.1 3 > 3 bod5 mg/l < 5 5 10 10 15 > 15 cod mg/l < 20 20 40 40 50 > 50 po4 -3 mg/l 0 0.01 0.01 0.1 0.1 3 > 3 om mg/l < 5 5 10 10 15 > 15 dr mg/l 300 1000 1000 1200 1200 1600 > 1600 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue … – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 265 3. results and discussion the temperature: the seasonal temperature changes are represented in figure 2. fig. 2: seasonal temperature changes these findings indicate that temperature degrees vary with the seasons, the highest values observed during the dry season and the lowest ones were recorded during the rainy season, which does not exceed the standards of potability during the four years (2016, 2017, 2018 and 2019) [18]. the recorded temperature reached the maximum value in 2018, with an average of 25°c during the dry season and its lowest point in 2017 with an average of 15,46°c, during the rainy season (fig. 2). according to [8], the water temperature of mexa dam fluctuated between 10°c, in the rainy season, and 30°c in the dry season, from 2010 to 2012; hence this variation is mainly seasonal. these results are close to those recorded by [6], at the dam of foum el-khanga (souk ahras, algeria), and remain lower than those reported by [19], at the sidi chahed dam (morocco). the value of this parameter is generally influenced by the ambient temperature [20]. it obviously varies according to the season, the geological nature and the depth of the water (even superficial or deep water) [21]. the electrical conductivity (ec) fig. 3: seasonal electrical conductivity changes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 266 according to the results obtained from the raw water conductivity of mexa dam measured during the period (2016-20172018-2019), varied from 308 μs/cm to 515 μs/cm (fig. 3). we noticed that all the conductivity values recorded during these years are lower than that given by the algerian standard of potability, fixed at 2800 μs/cm [18]. in addition, the conductivity variation is caused by the presence of ions in the medium, which are mobile in an electric field [15]. this mobility depends on the nature of the dissolved ions and on their concentrations. usually, electrical conductivity increases with the solution ion concentration and temperature [22]. our findings are consistent with those obtained by [23], at the mexa and cheffia dam, however these values are significantly inferior to those recorded by [6], at the foum el-khanga dam (souk ahras, algeria), and by [24], at the bir m'chergua dam (tunisia) the turbidity: fig. 4. seasonal turbidity changes. these results exceed the algerian standards with an average of 178.56 ntu (fig. 4), during the rainy season in 2016, since the studied dam is located in a very densely wooded area with a high vegetation cover, which increases the values of turbidity. in this case, the elevation could be mainly of organic nature, and may therefore induce a significant increase in the chlorine amounts during dam water treatment. the obtained results are comparable to those performed by [25], at the mexa and cheffia dams. during the dry season, turbidity is low compared to the rainy season, where the inflow of runoff increases the turbidity of water [26]. the latter is caused by the presence of suspended matter such as mineral substances (sand, clays or silts), organic matter (decomposed plants, suspended plankton). the evaluation of these matters abundance measures its degree of turbidity [27]. hydrometric title: ht the hydrometric title values of water are expressed in mg/l of caco3, the lowest rates are recorded in 2017, during the dry season and in 2018, during the rainy season, in the investigated waters. this parameter presents a great variation, it decreases in dry period and increases in rainy one, this variation could be related to the lithological nature of the aquifer formation, particularly to its composition in terms of magnesium and calcium [28food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 267 29]. the hydrometric title of natural water depends on the geological structure of the crossed soils [30]. fig. 5. seasonal hydrometric title changes. salinity: salinity measurements revealed values ranging from 0.15% recorded in 2019, to 0.25% in 2016, for the rainy season (fig. 6). these values are lower than that required by the algerian standard of 1.5% [18]. this salinity pattern is closely related to the freshwater inflow, and to the low water evaporation which increases the salt concentration in the water. the salinity levels are lower than those of moroccan lakes, as reported by [31] at hassan ii lake tank, [32] at hassan dakhil lake, [33] at mansour eddahbi lake tank. fig. 6. seasonal salinity changes. complete alkalimetric title (cat): according to the obtained results, the cat varies between means of 87.98 mg/l registered in rainy period during 2017 and 108.47 mg/l recorded in 2018 in dry period. therefore the results do not exceed the algerian standards of 500 mg/l [18]. the cat findings of our raw water are much higher than those reported by [6] for the waters of the foum el-khanga dam, (souk-ahras, algeria). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 268 fig. 7. seasonal complete alkalimetric variation. major elements: calcium (ca2+): the obtained results of calcium content ranged from a minimal mean of 42.48mg/l in the rainy season in 2018 to a maximal mean of 59.95mg/l observed in 2016, for the dry season. the calcium concentration is lower, which means that these waters are less impacted by the dissolution of carbonate and gypsum creations. in contrast to other researches, these concentrations are lower than those reported by [6] at the foum elkhanga dam (algeria) and [23] at bir m'chergua dam (tunisia). fig. 8. seasonal calcium changes. the magnesium ( mg2+): according to these results, magnesium is the lowest major element in the dam water, among the other studied components. the magnesium contents are generally lower than the norm, they vary between 13.6 mg/l and 5.47 mg/l. these contents turn out to be lower than those found by [6] at foum elkhanga dam (algeria). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 269 fig. 9. seasonal magnesium changes. bicarbonates (hco3 -): according to figure 10, the bicarbonate concentrations analysis of the dam waters is about 132.38mg/l recorded in dry period and 92.39mg/l noted in rainy period in 2018.the concentrations show significant variations indicating a heterogeneity of these bicarbonates sources. similar bicarbonate contents were recorded in 2010 by [8]. nevertheless, our results still lower than those reported by [24] of bir m'chergua dam (tunisia). fig. 10. seasonal bicarbonates variation. chloride (cl-): based on the obtained results (fig. 11), the chloride concentrations in the dam waters are mostly significant. in dry periods, they vary between 46.14 mg/l (2016) and 38.18mg/l (2017), while in the rainy ones, the fluctuation are between 47.13 mg/l (2016) and 35.45 mg/l (2019). we noticed that the chloride levels in the water of mexa dam do not exceed the world health organization standard (250mg/l). these findings are in agreement with those reported by [31] at the hassan ii dam (morocco). the origins of the chlorides in the waters are the numidian clays, marls and clayey marls of lutetian age [34]. on the other hand, [35] have mentioned that chloride ions, at a concentration higher than 250 mg/l affect the water flavor, which could lead to a water quality deterioration. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 270 fig. 11. seasonal chloride changes. total iron fe2+ : the figure 12 illustrates the concentration changes of iron ions in the mexa dam waters, these contents are low, they vary between a mean of 0.13mg/l (dry season 2016) to 0.51 mg/l (rainy season 2018). the presence of iron could be due to agricultural soil leaching and the local geological setting (ferromagnesian richness in the sediments) [36]. fig. 12. seasonal iron changes. seasonal changes in the water quality of the dam over four years: according to the water quality data (fig. 13), we note that mexa dam waters are of medium quality for the majority of parameters, the ph of mexa dam raw water exhibits a range of 7.20 to 8.03, which does not exceed the standard of potability, therefore reflecting a medium quality. these results are consistent with those previously reported in the same study area [23-8] and in the dam of foum el-khanga (souk ahras, algeria) [6]. the waters of this dam are charged in nh4 + and no2 during 2016 and 2017, this elevation is due to the oxidation of nitrogenous organic matter and the extensive use of fertilizers as well as the inputs of pollutants from urban wastewater discharges and the eutrophication process [37]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 271 the improvement of the water quality of this dam requires treatments, in order to reduce the content of nh4 + and no2 [17]. in 2018-2019, we have an improvement of the water dam quality, this could be due to the rainfull period which lead to a high water dam level amongseasons. legends good medium bad very bad fig. 13. seasonal means quality sheet for mexa dam water (2016-2019). 4. conclusion water quality analyses of the mexa dam reveal that the water is poorly mineralized, with high values of electrical conductivity (near 515 μs/cm), calcium (59.95 mg/l), magnesium (13.6 mg/l), bicarbonate (132.38mg/l), chlorides (47.13 mg/l) and iron (0.51 mg/l). the chemical quality parameters are all lower than the who potability standards. the mexa dam water quality records exhibit a medium quality for the majority of the studied parameters. the content of nitrogenous matter (nh4 +, no2 -) in the water is high during 2016 and 2017, this pollution is due to nitrogenous organic matter oxidation. therefore, in order to improve water quality and reduce these high levels of nutrients, it is necessary to consider the installation, in upstream, of small pretreatment stations. 5. acknowledgement the present work was supported by dgrsdt (general directorate of scientific research and technological development-algeria), from the ministry of higher education and scientific research (mesrs). this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. 2016 rainy season dry season 2017 rainy season dry season 2018 rainy season dry season 2019 rainy season dry season ph dr om physico-chemical parameters cod bod5 o2 organic matter parameters nh4 + no3 no2 nitrogenous matter parameters po4 3 phosphorous matter parameters food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 272 6. references [1]. dovonon l., chemical quality of groundwater in the municipality of dassa-zoumé (benin): health impacts of non-standard fluoride levels and treatment trials with calcined bovine bone, phd thesis, fast, university of abomeycalavi, 2011, p. 206. [2]. attoui b., toumi n., messaouidi s., benrabah s., degradation of water quality: the case of plain west of annaba (northeast algeria), no. 31, journal of water and land development, 2016, p. 3–10. doi 10.1515/jwld-20160031. [3]. allalgua a., ayari at., boutmedjet s., znati n., kaouachi n., boualleg c., boucenna i., bensouilah m., physicochemical and bacteriological characterization of the raw and drinking waters of the dam ain dalia (souk-ahras). 1st national seminar on health and bio-surveillance of aquatic ecosystems. university of souk ahras, algeria, 2014, pp. 8. [4]. pdare. master plan for the development of water resources, summary report of the cheliff hydrographic region, 2009, 266 pages. [5]. derdous o., djemili l., bouchehed h., tachi s. e., a gis based approach for the prediction of the dam break flood hazard – a case study of zardezas reservoir “skikda, algeria”, no. 27, journal of water and land development, 2015, p. 15–20. doi 10.1515/jwld-20150020. [6]. allalgua a., kaouachi n., boualeg c., ayari a., physico-chemical characterization of the water from the foum el-khanga dam (souk-ahras region, algeria), vol.13, no.12, european scientific journal edition, issn: 1857 – 7881 (print) e issn 18577431. april 2017. [7]. achour s., effect of chlorination, flocculation and adsorption processes on the evolution of organic and mineral compounds in natural waters, phd thesis. hydraulic sciences, university of tizi-ouzou, algeria, 2001, 231 p. [8]. bahroun s., chaib w., the quality of surface waters of the dam reservoir mexa, northeast of algeria, no. 34 (vii–ix): 11–19, journal of water and land development, 2017, pl issn 1429-7426. [9]. ouelmouhoub s., multi-use management and conservation of the forest heritage: case of the subaeryes of el kala national park (algeria). no. 78, thesis of science ciheam-iam montpellier, france, 2005, p. 46-54. [10]. sadoune a., vulnerability and assessment of water resources in the extreme northeast of algeria, msc thesis. faculty of earth sciences, university of annaba 2012, pp. 99. [11]. saadali b., derradji e.f., saboua t., remita r., zahi f., impact of the anthropogenic activity on the degradation of the environment and water's quality: el kala national park (northeast of algeria), no. 1, vol. 30, synthesis of science and technology, 2015, p. 6675. [12]. aelb. loire water agency. sample collection in the river. sampling techniques for physicochemical analysis, isbn 978-2-916869-001, technical guide. brittany, 2003, pp. 60. [13]. aqua-ref guide to technical prescriptions for the physico-chemical monitoring of aquatic environments: sampling in watercourses (waters and sediments) in continental environments, 2011, pp. 32. [14]. rodier j., analysis of the water natural water, waste water, sea water, 8ème edition, ed. dunod, paris, 2006. [15]. rodier j., legube b., merlet n. water analysis, 9ème édition, dunod paris, 2009, p. 1217, 1242, 1293, 1294, 1332. [16]. touhari., study of the water quality in the north west of algeria, phd thesis, national school of hydraulics, blida, algeria, 2015, pp 80-118. [17]. touhari f., mehaiguene m., messelmi h., evolution of dam water quality in the haut cheliff basin, vol 02, no 01, sargen, janvier 2018, pp 41-57. [18]. jora., 2011. executive decree no. 11-125 of march 22, 2011 relating to the quality of water for human consumption, n° 18, official journal of the algerian republic, 23th march 2011. [19]. mehanned s., chahlaoui a., zaid a., samih m., chahboune m. typology of the physic-chemical quality of the waters of the dam sidi chahed-morocco, sci. 5 (5) 1633-1642, j. mater. environ, 2014. [20]. ghazali d., zaid a. study of the physicochemical and bacteriological quality of the waters of the ain salama-jerri spring (region of meknes morocco), larhyss journal, issn 1112-3680, n° 12, janvier 2013, pp. 25-36. [21]. rodier j., legube b., merlet n., coll., water analysis, 9th edition, edition dunod, paris, 2005, 1529p. [22]. dib i., the impact of agricultural and urban activity on the quality of groundwater in the gadaine-ain yaghout plain (east of algeria), master's thesis in hydraulics, hydro-technical construction and environment, faculty of engineering sciences, hydraulics department, hadj lakhdar university, batna, algeria, 2009, 127 p. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 samira gheid, sofia bahroun, amina benabdallah, abdelhak gheid, evaluation of mexa dam water quality in el tarf region (extreme north-east algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 262 – 273 273 [23]. harrat n., the quality of surface waters in eastern algeria and their reactivity to chlorine, thesis of magister, university of annaba, algeria, 2007. [24]. ismail y., benalaya a., mchabet m., physico chemical characterization of waters of dam bir m'chergua its affluents (tunisia), v international congress on renewable energies and the environment. sousse, tunisia, 2010, pp. 10. [25]. harrat n., achour s., physico-chemical pollution of dams waters in the el tarf region. impact on chloration, n°8, larhyss journal, 2010, 47-54. [26]. ahonon a., assessment of the physicochemical and bacteriological quality of surface water in the mountainous areas of south-west togo: case of the canton of lavie, international master thesis in environment water and health, lome university, 2011, 42 p. [27]. jemali a., kefati a., reuse of wastewater in morocco. water demand management forum, department of development and irrigation management/ madref/ rabat, mars 2002. [28]. derwich e., benaabidate l., zian a., sadki o., belghity d., physico-chemical characterization of the waters of the alluvial aquifer of haut sebou downstream of its confluence with oued fes, larhyss journal, n° 08, 2010, 101-112. [29]. ghazali, d., zaid, a. physico-chemical characterization of water from the ain salamajerri source (meknes region), volume 4, n° 120106, science lib editions mersenne, 2012, 13 p. [30]. belghiti m. l., chahlaoui a., bengoumi d., elmoustaine r., study of the physicochemical and bacteriological quality of the groundwater of the plio-quaternary water table in the region of meknes (morocco), n°14, larhyss journal, issn 1112-3680, 2013, pp. 21-36. [31]. chahboune m., chahlaoui a., zaid a., ben moussa a., contribution to the physicochemical characterization of reservoir waters of lake hassan ii dam, n° 14, larhyss journal, 2013, pp. 61. [32]. ouhmidou m., chahlaoui a., physicochemical quality of the water from the hassan addakhil errachidia-morocco dam, volume 5, n ° 130908, science lib editions mersenne, issn 2111-4706, 2013, 09-23. [33]. sadani m., ouazzani n., mandi l., impact of drought on changes in water quality in lake mansour eddahbi (ouarzazate, morocco), vol. 17, n° 1, revue des sciences de l'eau journal of water science, 2004, p. 69-90. [34]. bahroun s., environmental rejection objectives for pollutants in the receiving environment and optimization of self-purifying power: case of wadi kébir est (northeast algeria, phd thesis. faculty of earth sciences, university of annaba, algeria 2016, pp. 220. [35]. andrews j e., brimblecombe p., jickells tim d., liss peter s., reid brian., an introduction to environmental chemistry, 2nd edn. john wiley & sons, isbn1444312375, 9781444312379, 2009, 320 pages. [36]. kaimoussi a., study of the variability of the accumulation of heavy metals in the different compartments (sediment, molluscs and algae) of the coast of the el jadida region, 3rd cycle thesis, chouaib doukkali university, faculty of science. el jadida. 1996, 147 p. [37]. de villers j., squilbin m., yourassowsky c., physicochemical and chemical quality of surface water, brussels institute for environmental management, 2005. [38]. who: world health organization. water, hygiene, and sanitation technical sheets in emergency situations. measuring chlorine levels in water supply systems, 2004. evaluation of mexa dam water quality in el tarf region (extreme north-east algeria) *samira gheid1, sofia bahroun2, amina benabdallah2, abdelhak gheid3 sampling: 3. results and discussion the temperature: the seasonal temperature changes are represented in figure 2. the electrical conductivity (ec) fig. 6. seasonal salinity changes. doi: https://doi.org/10.4316/fens.2022.001 5 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 5 15 silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage *amel alayat 1,2 , zineddine boumedris 1,2 , sana benosmane 2,3 , amira atailia 2,4 , nesrine hacini 4 1lafe laboratory, faculty of life and natural sciences, chadli bendjedid university, el tarf, algeria, amel.alayat@yahoo.com, 2 ltc laboratory, faculty of life and natural sciences, badji mokhtar university, annaba, algeria 3 faculty of sciences and technology, university of 08 may 1945 guelma, algeria 4 lfee laboratory, faculty of life and natural sciences, chadli bendjedid university, el tarf, algeria. *corresponding author received 16 november 2021, accepted 25th march 2022 abstract: using two wheat (triticum durum) cultivars (cvs. vitron and simeto) hydroponic solution experiments were conducted in order to study the genotypic variation in tolerance to nacl toxicity and to investigate effect of silicon supplied to the nutrient solution on wheat plants grown at salt stress. the experiment was a 2×2 factorial arrangement with two levels of nacl in nutrient solution, 0 and 100 mm, and two levels of silicon (si) in nutrient solution, 1 and 2 mm, as na2sio3.9h2o. silicon supplementation has an important role in alleviating salinity injury, however, the definite mechanisms stay scantily understood, and must be examined. the role of silicon application in improving growth, maintaining water status and alleviating oxidative injury of salt affected wheat plants were studied. indeed, our results indicate salinity induced in wheat plants a notable increase in oxidative biomarkers, reduced plant growth and produced less dry matter content than those without nacl. however, the reductions of seedling height, dry biomass, and soluble content were greatly alleviated due to si addition to the culture solution. thus, the beneficial effects of si on oxidative biomarkers contents under nacl stress were genotype-dependent. the beneficial effect of si on alleviating oxidative stress was much more pronounced in vitron (salt tolerant cultivar) than in simeto (salt sensitive cultivar). keywords: alleviation, silicon, salinity, wheat cultivars, membrane permeability, tolerance, oxidative stress. 1. introduction in arid and semi-arid ecosystems, marked by severe and frequent droughts, soil salinization is one of the main factors limiting plant development. salinity is a major abiotic stress reducing the yield of a wide variety of crops all over the world [1]. projections for 2050 show a further increase in the scale and impact of this environmental threat [2-3]. the drastic impacts of salinity stress on plant development are attributable to a hyperosmotic and ionic imbalance with excessive-production of reactive oxygen species (ros) that severely hamper several physiological and biochemical pathways and molecular changes including chlorophyll depletion, lipid peroxidation, nucleic acid mutilation, and reduction in cell membrane fluidity and selectivity [46]. salinity can be minimized through methods cultivating saline soils is selecting salt-tolerant species; however, profit to costs of cultures has been extremely restricted, since salt-tolerant genes are governed by several features, and their mailto:amel.alayat@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 6 simultaneous selection is not a simple task [7]. additionally, enhancing stress tolerance through substances application such as silicon may prove a promising approach for sustainable farming in an area under salinity stress conditions [8-9]. silicon has been appraised for its beneficial properties in alleviating abiotic stresses such as drought and salinity [10], and it increases the yields of a diverse range of crops under stress conditions, including: rice, wheat, sugarcane, soybean, apples [11-19]. silicon considerably functions in improving photosynthesis and ion homeostasis, activation of antioxidant capacity, regulation of genes required in diverse physiological processes, production of secondary metabolites, and the outcomes of si foliar spraying, such as enhanced plant development, and biomass production have been previously reported [18-19]. improving plant stress tolerance through si supplementation is associated with enhanced antioxidant capacity [17, 19, 9]. additionally, si application can attenuate salt injury by decreasing sodium uptake via the formation of na–si complexes in roots or by partially blocking the transpiration bypass flow, thereby increasing the potassium/sodium ratio [20, 18, 9]. a short-term experiment with two wheat cultivars (vitron and simeto) was conducted to study the effectiveness of silicon in mitigating the adverse effects of salinity and to investigate possible mechanisms of silicon enhancement of salt tolerance in wheat. one of these cultivars (vitron) was chosen because it is considered to be relatively salt tolerant, the other on account of its sensitivity to salinity. 2. matherials and methods plant growth conditions and treatments seeds of two wheat (triticum durum desf.) cultivars, vitron (salt-tolerant) and simeto (salt-sensitive), were surface sterilized with hgcl2 (1.0 g/l) for 5 min, rinsed thoroughly with distilled water. the seeds were submerged indeionized water in the dark overnight and germinated in sterilized moist quartz sand in a controlled chamber at 22°c/18°c (light/dark temperatures) respectively, with photoperiod of 16h light/8h dark and light. twelve-day-old uniform seedlings (second leaf stage) were transplanted on to 3l pots. the pots were covered with polystyrol-plate with seven evenly spaced holes and placed in a greenhouse, in each hole two seedlings were located. the composition of the basic nutrient solution was (mg l-1): (nh4)2so4 48.2, mgso4 65.9, k2so4 15.9, kno3 18.5, ca (no3)2 59.9, kh2po4 24.8, fe citrate 6.8, mncl2.4h2o 0.9, znso4.7h2o 0.11, cuso4.5h2o 0.04, h3bo3 2.9, h2moo4 0.01. the solution was continuously aerated with air pumps and renewed every four days. half strength nutrient solution was applied for the first four days and then changed to complete nutrient solution for two weeks. thereafter, sodium chloride (nacl) and silicon (as na2sio3.9h2o) were added to the nutrient solutions to form six treatments with six replications for each treatment as following: (1) control (basal nutrient), (2) 1 mm si (basal nutrient+ 1 mm si), (3) 2 mm si (basal nutrient + 2 mm si), (4) nacl (100 mm), (5) nacl + 1 mm si (100 mm nacl +1mm si) and (6) nacl + 2 mm si (100 mm nacl +2 mm si). the ph of culture solution in each pot was adjusted to 5.7 every other day with 1m hcl or naoh as required. determination of plant growth growth traits were measured in terms of plant height, shoot and root dry weight after 30 days of treatment. ten plants from each replication of all treatments were sampled and measured by a centimeter food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 7 scale, and then the plants were separated into shoots and roots, before fresh weights were recorded. relative water content (rwc) between four to six samples (replications) are taken from a single treatment. each sample is placed in a pre-weighed airtight vial. in the laboratory vials were weighed to obtain fresh leaf sample weight (fw), after which the sample was immediately hydrated to full turgidity for 4 h under normal room light and temperature. samples were rehydrated by floating on deionized water in a closed petri dish. after 4 h the samples were taken out of water and were well dried of any surface moisture quickly and lightly with filter paper and immediately weighed to obtain fully turgid weight (tw). samples were then oven dried at 80°c for 24 h and weighed to determine dry weight (dw). all weighing were done to the nearest mg. [21]. calculation: membrane permeability, lipid peroxidation and hydrogen peroxide determinations electrolyte leakage was used to assess membrane permeability. this procedure was based on lutts et al. 1996 [22]. electrolyte leakage was measured using an electrical conductivity meter. leaf samples of one randomly chosen plant per replicate were taken from the youngest fully expanded leaf and cut into 1 cm segments. leaf samples were then placed in individual stoppered vials containing 10ml of distilled water after three washes with distilled water to remove surface contamination. these samples were incubated at room temperature (ca. 25 °c) on a shaker (100 rpm) for 24 h. electrical conductivity (ec) of bathing solution (ec1) was read after incubation. the same samples were then placed in an autoclave at 120 °c for 20 min and the second reading (ec2) was determined after cooling solution to room temperature. the electrolyte leakage was calculated as ec1/ec2 and expressed as percent. lipid peroxidation and hydrogen peroxide determinations: the level of lipid peroxidation in plant tissues was expressed as 2-thiobarbituric acid (tba) reactive metabolites, mainly malondialdehyde (mda) and was determined according to hodges et al. (1999) [23]. fresh samples (leaves) of around 0.5 g were homogenized in 4.0 ml of 1% trichloroacetic acid (tca) solution and centrifuged at 10,000×g for 10 min. the supernatant was added to 1 ml 0.5% (w/v) tba made in 20% tca. the mixture was heated in boiling water for 30 min and the reaction was stopped by placing the tubes in an ice bath. the samples were centrifuged at 10,000×g for 10 min and the absorbance of the supernatant was recorded at 532 nm. correction of non-specific turbidity was made by subtracting the absorbance value read at 600 nm. the level of lipid peroxidation was expressed as nmol/g fresh weight, with a molar extinction coefficient of 0.155/mm/cm. the hydrogen peroxide (h2o2) contents in the leaves were assayed according to the method of velikova et al. (2000) [24]. leaves were homogenized in ice bath with 0.1% (w/v) tca. the extract was centrifuged at 12,000×g for 15 min, after which to 0.5 ml of the supernatant was added 0.5 ml of 10 mm potassium phosphate buffer (ph 7.0) and 1 ml of 1 m ki and the absorbance was read at 390 nm. the content of h2o2 was given on a standard curve. statistical analysis all values reported in this study are the mean of at least three replicates. for each parameter, data were subjected to a oneway anova analysis. differences food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 8 between means were evaluated for significance by using tukey’s (hsd) test (minitab software version 16.0). 3. results growth parameters effect of exogenous silicon treatment on the plant growth under nacl stress is presented in table 1. reductions in length roots in the nacl treatment were 30% and 36% compared to control with no salt added and in the absence of supplementary si, for salt-tolerant (vitron) and sensitive (simeto) cultivars, respectively. moreover, salinity stress considerably decreased length leaves showed 19% and 21 % in both cultivars grown at high salinity compared to control values. when nacl was absent from the nutrient solution, si significantly increased plant height at 2 mm level and 1 mm level relative to the control. indeed, higher si level (2mm) had greater alleviating effect of nacl toxicity than lower si level (1 mm). table 1: effects of silicon (si) application methods on length roots and leaves of two wheat cultivars under salinity stress conditions treatments root length (cm) leaves length (cm) vitron control(c) 9.66 c 17.98 c c + si 1 10.59 b 18.1 b c+ si 2 11.65 a 18.48 a salinity (s) 6.73 f 14.5 f s+ si 1 7.38 e 16.24 e s + si 2 simeto control (c) 8.3 d 8.25 c 16.70 d 16.78 c c + si 1 9.42 b 17.25 b c+ si 2 10.21a 17.76 a salinity (s) 5.26f 13.12 f s+ si 1 6.85e 14.05 e s+ si 2 7.16d 14.98 d the same letters after the data within a column indicates there was no significant difference at a 95% probability level (tukey’s test) between treatments (p < 0.01). relative water content (rwc) and electrolyte leakage (el) relative water contents (rwc) were lower in both cultivars grown at high salinity compared to control values; lowest values were in simeto (table 2). on another note, under saline conditions si application mitigated the decreased relative water content levels in the salinity treatments. the effects of nacl and si application on the electrolyte leakage content of wheat plants are presented in table 2. the results showed that wheat plants under salinity displayed a noteworthy increase in electrolyte leakage content relative to the control group. the salinity treatment impaired membrane permeability by increasing electrolyte leakage. however, silicon application to the nacl treated plants partially maintained membrane permeability but only fully restored in to control levels in the salt tolerant cultivar (vitron) with high concentration si2+ salinity (100 mm nacl). lipid peroxidation (mda) and hydrogen peroxide (h2o2) contents food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 9 oxidative damages to cell membrane lipids were evaluated by estimating the malondialdehyde (mda) production. the stress induced by salinity accelerated mda production that increased by 57% and 69% compared to the control with salt tolerant (vitron) and sensitive (simeto) cultivars, respectively (table 3). in contrast, si addition decreased mda production. this mitigating effect was observed in the salinized wheat plants receiving si application, as evidenced by 56% and 34% decline in mda production respectively, relative to salt affected plants. table 2: electrolyte leakage (el) and relative water content (rwc) in two wheat cultivars grown in nutrient solution with or without nacl and supplementary si treatments cultivars vitron simeto relative water content (%) electrolyte leakage (%) relative water content (%) electrolyte leakage (%) control (c) 92.60 c 22.67 d 91.45 c 23.56 d c + si 1 93.22 b 20.78 e 92.87 b 21.74 e c+ si 2 94.01 a 18.89 f 93.05 a 20.05 f salinity (s) 87.06 f 46.08 a 85.67 f 51.35 a s+ si 1 90.0 e 37.14 b 87.65 e 45.33 b s+ si 2 91.44 d 29.06 c 89.78 d 38.64 c the same letters after the data within a column indicates there was no significant difference at a 95% probability level (tukey’s test) between treatments (p < 0.01). the h2o2 contents in both the two cultivars of wheat plants are shown in table 3. the results show that salinity increased hydrogen peroxide production in the wheat plants of two genotypes relative untreated plants. however, si application methods reduced nacl induced h2o2 accumulation in the shoots relative to salt affected plants. this outcome was notable with treatment s+ si 2 that attenuated the impact of salinity, causing a 43% and 38% decline in h2o2 production in the salt tolerant cv. and the salt sensitive cv. respectively relative to plants grown under salinity only. simeto (salt sensitive cultivar) had consistently and significantly higher mda and h2o2 contents regardless of the stress treatment or stress duration. table 3: effects of silicon (si) application methods on the malondialdehyde (mda) and hydrogen peroxide (h2o2) concentrations of two wheat cultivars under salinity stress conditions the same letters after the data within a column indicates there was no significant difference at a 95% probability level (tukey’s test) between treatments (p < 0.01). treatments cultivars vitron simeto malondialdehyde (nm/gfw) hydrogen peroxide (μm/gfw) malondialdehyde (nm/gfw) hydrogen peroxide (μm/gfw) control (c) 5.61 d 15.69 d 4.79 d 18.97 d c + si 1 3.59 e 14.21 e 3.83 e 16.24 e c+ si 2 3.01 f 12.99 f 3.26 f 13.05 f salinity (s) 12.94 a 48.13 a 15.71 a 55.78 a s+ si 1 9.51 b 35.73 b 12.33 b 45.01 b s+ si 2 8.25 c 27.45 c 10.23 c 34.33 c food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 10 4. discussion in this experiment, it has been shown that salt stress in wheat causes significant reductions in length root and shoots growth. plant development was drastically suppressed in stress conditions. comparable results were observed in salinity affected plants [6, 7, 25, 26]. inhibition of plant growth under saline conditions may either be due osmotic reduction in water availability or to excessive ion na and cl, accumulation in plant tissues [27]. indeed, the reduction in growth related attributes may be due to impaired cell development resulting from growth hormone effectively, leading to a reduction in cell turgor, cell volume, and ultimately cell growth, and it may also be due to the blocking up of conductive tissue vessels; hence, blocking every translocation that passes through these tissues [28]. moreover, the present results showed that plant growth parameters of wheat plants were increased by si addition under non-nacl stress condition, proving the beneficial effect of si. similarly, many researchers reported beneficial effects of silicon in many crops [29-32]. the positive impacts of si on plant growth may be due to the increased antioxidant capacity [19], and k+ absorption, which increase the number of chloroplasts per cell, and leaf area [33]. salinity stress significantly reduced relative water content, thereby significantly increasing water saturation deficiency. indeed, salinity leads to a reduction in the capacity of plants to absorb water, a drop in leaf water and osmotic potentials [34-35]. salinity tolerance levels vary greatly between plants [36]. the study revealed that the relative water content of durum wheat plants decreases significantly in sodium chloride-treated plants, compared to control plants. thus, according to various authors, salt stress results from the disruption of the water, mineral and carbon nutrition functions of plants [37-38]. the relative water content is often considered as an excellent indicator of the water status of the plant. it is linked to the plants capacity to maintain a level of hydration of the tissues in order to guarantee the continuity of its metabolism or metabolic activity [6]. on another note, under saline conditions, si application mitigated the decreased relative water content. also, this induced that the osmolyte accumulation was associated with the increasing osmotic adjustment capacity relative to that of the control plants [7-39]. this may be related to limiting water loss and optimising the hydromineral nutrition of plants by decreasing excessive transpiration, as excess transpiration leads to stomatal closure and decreased photosynthesis [1140]. electrolyte leakage is one of the majorly used indicators in detecting the severity of salinity-induced injuries [17]. in the current study, electrolyte leakage increased significantly with salinity. the significant increase in electrolyte leakage observed could indicate that salt stress affected the membrane integrity and stability of the stressed plants. similar results were obtained by lutts et al. (1996); kaya et al. (2002); tuna et al. (2007) and farouk et al., 2020 [22, 41, 42, 7]; who reported that high salt concentration can affect the membrane permeability of rice, strawberry and wheat varieties, respectively. the dysfunction of cell membrane permeability can be expressed by the increased rate of electrolyte leakage. the cellular membrane dysfunction due to stress is well expressed in its increased permeability for ions and electrolytes, which can be readily measured by the efflux of electrolytes [22]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 11 in addition, in our work, we also found that the application of silicon decreased the rate of electrolyte leakage in both salt-stressed and unsalted plants. several similar works have been reported [43-46]; suggesting that silicon could restore membrane permeability and maintain membrane integrity [32]. the cell membrane can be the target of reactive oxygen species. ros primarily target the lipids present in cell and subcellular membranes. lipid peroxidation induces a change in membrane fluidity and permeability [47]. malondialdehyde, the oxidation product of lipid membranes, accumulates when plants are exposed to oxidative stress. mda concentrations are considered as an indicator of lipid peroxidation after abiotic stress [48]. the determination of its concentrations is used as a reliable tool to detect lipid peroxidation [49-51]. thus, our results showed that sodium chloride induced a very highly significant increase in mda concentrations in the leaves of stressed durum wheat plants. daud et al. (2015) [52], also recorded that mda level increases in cotton (gossypium hirsutum l.) subjected to salt stress. this may be due to lipid peroxidation; and thus membrane destabilization due to the high production of ros causing oxidative damage. indeed, the overproduction of ros causes lipid peroxidation which leads to the formation of degradation products such as alkanes and aldehydes (malondialdehyde) [53]. the most detrimental effect of ros in plants is lipid peroxidation, which can lead to membrane disruption [54-55]. in contrast, the addition of both concentrations of silicon to saltstressed wheat plants decreased mda concentrations in the leaves of durum wheat plants. this indicates that silicon reduces lipid peroxidation by improving membrane permeability in salt-stressed plant cells. similar results have been reported [56-58]. as for hydrogen peroxide (h2o2), we noted a clear increase in the leaves of durum wheat plants treated with sodium chloride. this may be related to oxidative damage to the membrane. our results also show that the accumulation of h2o2 is more noted in the leaves of treated plants compared to controls. salinity inducedphytotoxicity is strongly associated with ros accumulation [7]. similarly, salt stress drastically induced h2o2 accumulation, leading to increased mda production that destroys cellular membranes and upsets regular cellular processes [16, 17, 59]. hydrogen peroxide can be derived from the dismutation reaction of superoxide anion by sod [6061]. it can also be caused by the alteration of electron transport in the photosynthetic and respiratory chains [62-63]. this increase could be explained by the important role that h2o2 plays in oxidative stress signaling [64]. h2o2 can be diffused over relatively long distances causing changes in the redox status of surrounding tissues and cells or at relatively low concentrations where it triggers an antioxidant response. thus, h2o2 acts as a signal molecule that alerts the cell to the presence of environmental stress [65]. hydrogen peroxide (h2o2) can function as a secondary messenger at low concentrations but becomes toxic at high concentrations [66]. furthermore, in our work we noticed that pretreatment with silicon significantly decreased the production of hydrogen peroxide (h2o2) in leaves of nacl-treated durum wheat plants. the decrease of h2o2 synthesis after silicon application has been shown by many authors [27, 67, 68, 69, 17, 19]. indeed, it has been suggested that pretreatment of plants with silicon could significantly improve the defense capacity against oxidative damage induced by salt stress toxicity. these findings are parallel to previous reports, wherein si treatment effectively attenuated the undesirable food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 12 effects of oxidative stress on the cell membrane by acting as a ros scavenger [18-59]. 5. conclusion these results showed existence of a variation in tolerance to salinity between the durum wheat cultivars vitron and simeto. higher nacl sensitivity of simeto was associated with increased levels of oxidative biomarkers mda and h2o2. in conclusion, suitable application of silicon concentration in the plant-growing medium with nacl could alleviate salinity induced toxicity in wheat plants by relieved oxidative damages through biological processes, including, improving growth of shoots and roots, maintaining balance water status, reducing electrolyte leakage and alleviating oxidative injury, thereby resulting in a considerable reduction in ros-induced oxidative biomarkers in both salt-sensitive and tolerant cultivars. 6. references [1]. tester m., and davenport r., na+ tolerance and na+ transport in higher plants. annals of botany, 91, (2003), 503-527. http://dx.doi.org/10.1093/aob/mcg058. [2]. shrivastava p., kumar r., soil salinity: a serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. saudi journal of biological sciences volume 22, issue 2, (2015), 123-131. https://doi.org/10.1016/j.sjbs.2014.12.001. [3]. hassani a., azapagic a., shokri n., predicting long-term dynamics of soil salinity and sodicity on a global scale proc. natl. acad. sci., 117, (2020), pp. 33017-33027. [4]. parida a.k., das a.b., salt tolerance and salinity effects on plants: a review. ecotoxicol. environ. saf. 60, (2005), 324–349. https://doi.org/10.1016/j. ecoenv.2004.06.010. [5]. foyer c.h., reactive oxygen species, oxidative signaling and the regulation of photosynthesis. environ. exp. bot. 154, (2018), 134–142. https://doi.org/10.1016/j. envexpbot.2018.05.003. [6]. ren j., ye j., yin l., li g., deng x., wang s., exogenous melatonin improves salt tolerance by mitigating osmotic, ion, and oxidative stresses in maize seedlings. agronomy 10, 663, (2020).https://doi.org/10.3390/agronomy10050663. [7]. farouk s, elhindi khalid m., alotaibi majed a., silicon supplementation mitigates salinity stress on ocimum basilicum l. via improving water balance, ion homeostasis, and antioxidant defense system. ecotoxicology and environmental safety, 206, (2020). https://doi.org/10.1016/j.ecoenv.2020.111396. [8]. bahcesular b., yildirim e.d., karaçocuk m., kulak m., karaman s., seed priming with melatonin effects on growth, essential oil compounds and antioxidant activity of basil (ocimum basilicum l.) under salinity stress. ind. crops prod., 146, (2020). https://doi.org/10.1016/j.indcrop.2020.112165. [9]. souri z., khanna k., karimi n., ahmad p., silicon and plants: current knowledge and future prospects. j. plant growth regul. (2020). https://doi.org/10.1007/s00344-020-101727. [10]. thorne s.j., hartley s.e., maathuis f.j.m., is silicon a panacea for alleviating drought and salt stress in crops?. front. plant sci., 11, (2020), pp. 1-16, 10.3389/fpls.2020.01221 [11]. ma j.f., takahashi e., soil, fertilizer, and plant silicon research in japan. soil, fertil. plant silicon res. japan, 107–180, (2002), 10.1016/b978-044451166-9/50007-5. [12]. korndörfer g.h., lepsch i., effect of silicon on plant growth and crop yield. stud. plant sci., 8, (2001), pp. 133-147, 10.1016/s09283420(01)80011-2. [13]. wijaya k.a., effects of si-fertilizer application through the leaves on yield and sugar content of sugarcane grown in soil containing abundant n. agric. sci. proc., 9, (2016), pp. 158162, 10.1016/j.aaspro.2016.02.111. [14]. pati s., pal b., badole s., hazra g.c., and mandal b., effect of silicon fertilization on growth, yield, and nutrient uptake of rice. commun. soil sci. plant anal. 47, (2016), 284–290. doi: 10.1080/00103624.2015.1122797. [15]. artyszak a., effect of silicon fertilization on crop yield quantity and quality-a literature review in europe plants, 7 (2018),p. 54, 10.3390/plants7030054. [16]. farouk s., and al-amri s.m., exogenous zinc forms counteract nacl-induced damage by regulating the antioxidant system, osmotic adjustment substances and ions in canola http://dx.doi.org/10.1093/aob/mcg058 https://www.sciencedirect.com/science/article/pii/s1319562x14001715#! https://www.sciencedirect.com/science/article/pii/s1319562x14001715#! https://www.sciencedirect.com/science/journal/1319562x https://www.sciencedirect.com/science/journal/1319562x/22/2 https://doi.org/10.1016/j.sjbs.2014.12.001 https://doi.org/10.1016/j.%20ecoenv.2004.06.010 https://doi.org/10.1016/j.%20envexpbot.2018.05.003 https://doi.org/10.1016/j.%20envexpbot.2018.05.003 https://doi.org/10.3390/agronomy10050663 https://doi.org/10.1016/j.ecoenv.2020.111396 https://doi.org/10.1016/j.indcrop.2020.112165 https://doi.org/10.1007/s00344-020-10172-7 https://doi.org/10.1007/s00344-020-10172-7 https://doi.org/10.3389/fpls.2020.01221 https://doi.org/10.1016/b978-044451166-9/50007-5 https://doi.org/10.1016/s0928-3420(01)80011-2 https://doi.org/10.1016/s0928-3420(01)80011-2 https://doi.org/10.1016/j.aaspro.2016.02.111 https://doi.org/10.3390/plants7030054 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 13 (brassica napus l. cv pactol) plants. j. soil sci. plant nutr. 19 (4), (2019), 887–899. https://doi.org/10.1007/s42729-019-00087-y. [17]. abdelaal k.a.a., mazrou y.s.a., hafez y.m., silicon foliar application mitigates salt stress in sweet pepper plants by enhancing water status, photosynthesis, antioxidant enzyme activity and fruit yield. plants (9), 733, (2020). https://doi.org/ 10.3390/plants 9060733. [18]. al murad m., khan a., muneer s., silicon in horticultural crops: cross-talk, signaling, and tolerance mechanism under salinity stress. plants 9, 460, (2020). https://doi. org/10.3390/ plants9040460. [19]. farouk s., omar m.m., sweet basil growth, physiological and ultrastructural modification, and oxidative defense system under water deficit and silicon forms treatment. j. plant growth regul.(2020).https://doi.org/10.1007/s00344-02010071-x. [20]. coskun d., deshmukh r., sonah h., menzies, j.g., reynolds o., ma j.f., kronzucker h.j., belanger r.r., the controversies of silicon’s role in plant biology. new phytol. 221, (2018), 67–85. https://doi.org/10.1111/nph.15343. [21]. barr h.d., weatherley p.e., a reexamination of the relative turgidity technique for estimating water deficit in leaves. aust. j biol. sci. 15, (1962), 413–428. [22]. lutts s., kinet, j.m., bouharmont, j., nacl-induced senescence in leaves of rice (oryza sativa l.) cultivars differing in salinity resistance. ann. bot. 78, (1996), 389–398. [23]. hodges, d.m., delong, j.m., forney, c.f., prange, r.k., improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. planta 207, (1999), 604–611. [24]. velikova v., yordanov i., edreva a., oxidative stress and some antioxidant systems in acid rain-treated bean plants-protective role of exogenous polyamines. plant. sci, 151, (2000), 5966. [25]. sofy m.r., elhindi k.m., farouk s., alotaibi m.a., zinc and paclobutrazol mediated regulation of growth, upregulating antioxidant aptitude and plant productivity of pea plants under salinity, (2020), plants 1197. https://doi.org/10.3390/ plants9091197. [26]. al-garni s.m.s., khan m.m.a., bahieldin a., plant growth-promoting bacteria and silicon fertilizer enhance plant growth and salinity tolerance in coriandrum sativum, journal of plant interactions, 14, (2019), 386396, doi: 10.1080/17429145.2019.1641635 [27]. gunes a., inal a., bagci eg., coban s., sahin o., silicon increases boron tolerance and reduces oxidative damage of wheat grown in soil with excess boron. biol. plant., 51, (2007), 571– 574. [28] queiros f., rodrigues j.a., almeida j.m., almeida d.p., fidalgo f., differential responses of the antioxidant defense system and ultrastructure in a salt-adapted potato cell line. plant physiol. biochem. 49 (12), (2011), 1410– 1419. https://doi.org/ 10.1016/j.plaphy.2011.09.020. [29]. kaloterakis n., h.van delden s., ,hartley s., b.de deyn g., silicon application and plant growth promoting rhizobacteria consisting of six pure bacillus species alleviate salinity stress in cucumber (cucumis sativus l). scientia horticulturae, 288, 15, (2021),110383.https://doi.org/10.1016/j.scienta.202 1.110383. [30]. yin j., jia j., lian z., hu y., guo j., huo h., zhu y., gong h., silicon enhances the salt tolerance of cucumber through increasing polyamine accumulation and decreasing oxidative damage ecotoxicol. environ. saf., 169, (2019), pp. 8-17, 10.1016/j.ecoenv.2018.10.105 [31]. wang s., liu p., chen d., yin l., li h., deng x., silicon enhanced salt tolerance by improving the root water uptake and decreasing the ion toxicity in cucumber. front. plant sci., 6, (2015), pp. 1-10, 10.3389/fpls.2015.00759. [32]. zhu z., wei g., li j., qian q., yu j., silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of saltstressed cucumber (cucumis sativus l.). plant sci. 167, (2004), 527–533. [33]. taiz l., zeiger e., plant physiology. sinauer associates inc, sunderlands, usa, (2010). [34]. joseph b., and jini d., development of salt stress-tolerant plants by gene manipulation of antioxidant enzymes. asian journal of agricultural research 5 (1), (2011), pp.17-27. [35]. hamdia m., shaddad m.a.k., doaa m.m, mechanisms of salt tolerance and interactive effects of azospirilum brasilence inoculation on maize cultivars grown under salt stress conditions. plant growth regulation. v 44, n°02, (2004), p 165-174. (10). [36]. rabie g.h., almadini a.m., role of bioinoculants in development of salt-tolerance of vicia faba plants under salinity stress. african journal of biotechnology.,4, (2005), 210–222. [37]. levitt j., responses of plants to environmental stresses: water, radiation, salt and https://doi.org/10.1007/s42729-019-00087-y https://doi.org/10.1007/s00344-020-10071-x https://doi.org/10.1007/s00344-020-10071-x https://doi.org/10.1111/nph.15343 https://doi.org/10.1080/17429145.2019.1641635 https://www.sciencedirect.com/science/article/pii/s0304423821004908#! https://www.sciencedirect.com/science/article/pii/s0304423821004908#! https://www.sciencedirect.com/science/article/pii/s0304423821004908#! https://www.sciencedirect.com/science/article/pii/s0304423821004908#! https://www.sciencedirect.com/science/article/pii/s0304423821004908#! https://www.sciencedirect.com/science/journal/03044238 https://www.sciencedirect.com/science/journal/03044238/288/supp/c https://doi.org/10.1016/j.scienta.2021.110383 https://doi.org/10.1016/j.scienta.2021.110383 https://doi.org/10.1016/j.ecoenv.2018.10.105 https://doi.org/10.3389/fpls.2015.00759 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 14 other stresses, academic press, new york, (1980), pp. 365-488. [38]. levigneron a., lopez f., berthomieu p., casse-delbart f., les plantes face au stress salin. cahiers agriculture. france ; 4, (1995), 263-273. [39]. chen d., wang s., yin l., deng x., how does silicon mediate plant water uptake and loss under water deficiency?. front. plant sci., 9, (2018), pp. 1-7, 10.3389/fpls.2018.00281. [40]. ahsan-farooq m., shafaqat a., amjad h., wajid i., khalid m., zafar i, alleviation of cadmium toxicity by silicon is related to elevated photosynthesis, antioxidant enzymes ; suppressed cadmium uptake and oxidative stress in cotton. ecotoxicology and environmental safety, 96, (2013), 242–249. [41]. kaya c., ak, b.e., higgs d., murilloamador b., influence of foliar applied calcium nitrate on strawberry plants grown under salt stress conditions. aust. j. exp. agric. 42, (2002), 631– 636. [42]. tuna a.l., kaya c., higgs d., murillo-amador b., aydemir s., girgin a.l., silicon improves salinity tolerance in wheat plants, environmental and experimental botany elsevier., 121, (2007), 1-7. [43]. liang y.c., shen q.r., shen z.g., ma t.s., effects of silicon on salinity tolerance of two barley cultivars. j. plant nutr. 19, (1996), 173– 183. [44].abassi m., mguis k., bejaoui z., albouchi a., morphogenetic responses of populus alba l. under salt stress j. for. res., 25, (2014), 155-161. [45]. xu j.w., huang x., lan h.x., zhang h.s., huang j., rearrangement of nitrogen metabolism in rice (oryza sativa l.) under salt stress. plant signal behav, 11 (3), (2016), p. e1138194. [46]. li j.h., shakeel a.a., liu m.r., niu j.h, wang r., song j.x., et al., modulation of morpho-physiological traits of leymus chinensis (trin.) through exogenous application of brassinolide under salt stress. the journal of animal and plant sciences, 25, (2015), 1055-1062. [47]. pamplona r., portero-otín m., riba d., requena j. r., thorpe s.r., lópez-rentel m., knight m.r., oxidative stress-induced calcium signaling in arabidopsis. plant physiol., 135, (2004), 1471-1479. [48]. ding h.d., wan y.h., qi n.m., zhu w.m., yang x.f., shao y.c., effects of cd2+ and zn2+ stress on antioxidant enzyme system of tomato seedlings. acta. agr. shangai, 20, (2004), 79-82. [49]. taulavuori k., sarala m., taulavuori e., growth responses of trees to arctic light environment. progress in botany, 71, (2001), 157-168. [50]. loureiro j., rodriguez e., dolezel j., santos c., flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of dna content. annals of botany, 98, (2006), 515–527. [51]. yadav s.k., cold stress tolerance mechanisms in plants. agron. sustain. dev. 30, (2010), 515–527. doi:10.1051/agro/2009050. [52]. daud m.k., quiling h., lei m., ali b., zhu s.j., ultrastructural, metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress, chemosphere, vol. 120, (2015), 309-320. [53]. ferrat l., pergent-martini c., roméo m., assesssment of the use of biomarkers in aquatic plants for the evaluation of environmental quality : application to seagrasses. aquat. toxicol, 65, (2003), 187–204. [54]. timbrell j., principles of biochemical toxicology, 4th ed. informa healthcare, (2009). [55]. wahsha m., al-jassabi s., azirun m., abdul-aziz k., biochemical screening of hesperidin and naringin against liver damage in balb/c mice exposed to microcystin-lr. middle east. journal of scientific research, 6 (4), (2010), 354-359. [56]. song a., li p., li z., fan f., nikolic m., liang y., the alleviation of zinc toxicity by silicon is related to zinc transport and antioxidative reactions in rice. plant and soil, 344, (2011), 319333. [57]. tripathi p., tripathi r.d., singh r.p., dwivedi s., goutam d., shri m., chakrabarty d., silicon mediates arsenic tolerance in rice (oryza sativa l.) through lowering of arsenic uptake and improved antioxidant defence system. ecol. eng. 52, (2013), 96–103. [58]. alzahrani y., kuşvuran a., alharby h.f., kuşvuran s., rady m.m., the defensive role of silicon in wheat against stress conditions induced by drought, salinity or cadmium. ecotoxicol. environ. saf., 154, (2018), 187–196. 10.1016/j.ecoenv.2018.02.057 [59]. siddiqui m.h., alamri s., alsubaie q.d., ali h.m., khan m.n., al-ghamdi a., ibrahim a.a., alsadon a., exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings. nitric oxide biol. chem. 94, (2020), 95– 107.https://doi.org/10.1016/j.niox.2019.11.002. [60]. cakmak i., possible role of zinc in protecting plant cells from damage by reactive https://doi.org/10.3389/fpls.2018.00281 https://doi.org/10.1016/j.niox.2019.11.002 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 amel alayat, zineddine boumedris, sana benosmane, amira atailia, nesrine hacini, silicon enhances the salt tolerance of two wheat cultivars through decreasing oxidative damage, food and environment safety, volume xxi, issue 1 – 2022, pag. 5 – 15 15 oxygen species. new phytologist, 146, (2000), 185–205. [61] mishra s., srivastava s., tripathi r.d., govindarajan r., kuriakose s.v., prasad m.n.v., phytochelatin synthesis and response of antioxidants during cadmium stress in bacopa monnieri l. plant physiol. bioch, 44, (2006), 25-37. [62]. dixit v., pandey v and shyam r, differential oxidative reponses to cadmium in roots and leaves of pea (pisum sativum cv. azad). j. exp. bot, 52, (2001), 1101-1109. [63].gomes-junior ra, c.a. moldes, f.s. delite, p.l. gratao, p. mazzafera, p.j. lea, r.a. azevedo. nickel elicits a fast antioxidant response in coffea arabica cells. plant physiol. biochem., 44, (2006), pp. 420-429. [64]. neil s., desikan r., hancock j,. hydrogen peroxide signalling. curr. opin plant. biol., 5, (2002), 388-395. [65]. rentel m., and knight m.r., oxidative stress-induced calcium signaling in arabidopsis. plant physiol. 135, (2004), 14711479.. [66]. dat j.f., lopez-delgado h., foyer c.h., scott i.m., effects of salicylic acid on oxidative stress and thermotolerance in tobacco. journal of plant physiology, 156, (2000), 659-665. [67]. li l., zheng c., fu y., wu d., yang x., shen h., silicate-mediated alleviation of pb toxicity in banana grown in pbcontaminated soil. biol. trace elem. res., 145, (2012), 101–108. [68]. tripathi d.k., singh v.p., kumar d., chauhan d.k., rice seedlings under cadmium stress : effect of silicon on growth, cadmium uptake, oxidative stress, antioxidant capacity and root and leaf structures. chem. ecol., 28, (2012), 281–291. [69]. liu j., zhang h., zhang y., chai t., silicon attenuates cadmium toxicity in solanum nigrum l. by reducing cadmium uptake and oxidative stress. plant physiol. biochem., 68, (2013), 1–7. 1. introduction 5. conclusion doi: https://doi.org/10.4316/fens.2022.008 76 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 76 81 quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas *maria poroch seriţan 1 , mihaela jarcău 1 , narcisa mihaela cornea 2 1faculty of food engineering, stefan cel mare university, suceava, romania; mariap@fia.usv.ro, 2sc laropharm srl, sos. alexandriei 145 a, bragadiru, ilfov, romania; *corresponding author received 31th january 2022, accepted 30th march 2022 abstract: many studies have proved that bioactive components of silybum marianum powder have excellent hepatoprotective action. the silymarin is a mixture of mainly three flavonolignans, for example, silybin, silydianin and silychristin, with silybin being the most active. the active substances work upon the membrane of the liver cell preventing its destruction and favoring the reconstruction of the cell which is already in the process of destruction. in this study, we measured the quantitative of total silymarin in the powder of silybum marianum from various geographical areas. results showed that silymarin content varied according to the geographical location. the quantitative dosing of the silymarin extract existing in the silybum marianum powder samples from the analyzed geographical areas was performed by the uv spectrophotometric method on soxhlet extraction and they fall within the parameters described in the european pharmacopoeia. keywords: bioactive components, hepatoprotection, uv spectrophotometric method 1. introduction according to the most recent statistics on medicinal and aromatic plants, statistical analysis shows a significant increase in the consumer interest of natural products in recent years, although, units that make pharmaceutical, cosmetic, and non-toxic products are excluded [1]. according to studies, people prefer natural products because they provide health benefits. natural products, in general, are more widely available, have fewer side effects, are not addictive, are easily assimilated, can be combined with other drugs and diets, and are much more affordable [2]. people are increasingly turning to herbal treatments, teas, and other herbal products and by-products. in recent years, the cultivation of medicinal plants has become a good business opportunity due to the low costs, high yield, and easily bearable initial costs. natural product demand is constantly increasing, both in the domestic and international markets [3]. the romanian flora includes approximately 3.700 species recognized as having properties on a national level, with approximately 370 species having pharmacodynamic effects. recently, the production and marketing of medicinal plants derived from natural vegetation have emerged as a major economic concern, providing income not only to producers but also to processors [4]. the cardui mariae fructus is used to treat hepatitis, cirrhosis, pancreatitis, diabetes, and chronic digestive diseases [5, 6]. for silybum marianum gaerth plants, staggered fruit ripening is characteristic, the most valuable being in the central anthodes compared to those on the lateral sprouts [7]. the plant contains saponosides, volatile oil. the fruits contain silymarin (consisting http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea, quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas, food and environment safety, volume xxi, issue 1 – 2022, pag. 76 81 77 of flavonolignans silychristin, silydianin, silybin, isosilybin), betaine hydrochloric acid, amino acids (1-cysteine, glycine, 1glutamic acid, d-1-2-amino-butyric acid, d1-leucine, tyramine), lipids (3-4%), polyhydroxyphenylchromone, fumaric acid, etc [8]. silymarin, a mixture of flavonolignans (figure 1), is the active component isolated from the seeds and fruits of the plant (silychristin, silydianin, silybin a, silybin b, isosilybin a, isosilybin b, taxifolin) [9, 10]. fig. 1. chemical structures of the main active constituents in silybum marianum, the flavonolignans the fruits have therapeutic value in both human and veterinary medicine. the active ingredients are eupeptic, tonic-bitter and hepatoprotective. the anti-hepatotoxic, protective, and curative effects on the liver have been confirmed [11]. the active substances act on the liver membrane, preventing the cell from destroying itself. it encourages the regeneration of liver cells, which are already being destroyed. silymarin is non-toxic and does not have any side effects. recommended in liver diseases (hepatitis, cirrhosis, liver failure), poisoning with α-amanitin and phalloidin in poisonous fungi, in indigestion [12, 13, 14]. mechanism of action silymarin hepato protective effects are purportedly accomplished via several mechanisms; these include:  antioxidation;  inhibition of lipidperoxidation;  stimulation of ribosomal rna polymerase and subsequent protein synthesis, leading to enhanced hepatocyte regeneration;  enhanced liver detoxification via inhibition of phase i detoxification;  enhanced glucuronidation and protection from glutathione depletion;  anti-inflammatory effects, including inhibition of leukotriene and prostaglandin food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea, quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas, food and environment safety, volume xxi, issue 1 – 2022, pag. 76 81 78 synthesis, kupffer cell inhibition, mast cell stabilization and inhibition of neutrophil migration;  slowing or even reversing of fibrosis by reduction of the conversion of hepatic stellate cells into myofibroblasts;  anticarcinogenesis by inhibition of cyclin-dependent kinases and discontinuance of cancer cell growth;  silymarin is also found to have immunomodulatory effects on the diseased liver [15]. adverse effects  silymarin has very low toxicity and has been shown to possess a good safety profile. at high doses, a laxative effect is observed due to increased bile flow.  adverse effects related to the intestinal gastrointestinal tract such as: dyspepsia, bloating, nausea and diarrhea.  serious adverse effects, which are rare, include gastoenteritis associated with collape and allergy [15]. the goal of this study was to perform the quality control on the finished product, milk thistle (silybum marianum) seed powder from various geographical areas, using analysis to verify the product conformity in terms of silymarin content dosing parameters. 2. matherials and methods silybum marianum seed powder from various geographical areas (southeast europe, south asia, central europe, and east asia) was purchased in the domestic market from specialty stores. each of the six powder samples was subjected to three determinations. the extraction of silymarin from weaving seed powder is a two-step process: first, the weaving seed powder is degreased with n-hexane for 4 hours, followed by the actual extraction with the soxhlet extractor for 8 hours with ethyl acetate, from which a fine yellow powder is obtained after evaporation at a maximum temperature of 50°c. the drying loss is carried out in weighing vials, placed in the oven at 105°c for 3 hours until a constant mass and is calculated according to equation 1: loss on drying, % = 100 (1) where: ampoule mass containing the sample, expressed in g; dry ampoule mass, expressed in g; sample mass, expressed in g [16]. the dosage of silymarin extracted from milk thistle (silybum marianum) seed powder, by uv spectrometric method, consists in the following procedure: in a 100 ml bale weigh the equivalent of 0.1 g of silymarin (according to ph. eur. add 50 ml of methanol, ultrasound for 30 minutes, then make up to volume with the same solvent. 1 ml of the solution obtained is diluted to 100 ml with the same solvent. the absorbance was read at a wavelength of 287 nm, and the reference is methanol. the calculation formula used for silymarin dosing is according to equation 2: silymarin, % (relative to the dry matter) = (2) where: – the value of the sample solution at an absorbance of 287 nm; – 402.53 (specific absorbance); mp – the amount of substance to be analyzed in g; u – loss on drying [16]. 3. results and discussion the rich chemical composition of the plant depends on a large number of factors: the type and conditions of cultivation, harvest time, climate, species and the method used for harvesting [17, 18]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea, quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas, food and environment safety, volume xxi, issue 1 – 2022, pag. 76 81 79 fig. 2. loss on drying of silymarin extract following the determination of the drying loss, figure 2, visible differences were found between the determined samples. eligibility conditions: loss on drying [%], maximum 7% [19]. for sample 4 and sample 6 in central europe, the highest values of drying loss were obtained, respectively 3.17% for sample 6 and 3. 21% for sample 4. this is directly related to the results from the parameter dosage. the amount of silymarin (%) being the lowest, figure 3, respectively 89.5% for the silymarin extract obtained for sample 6 and 85.62% for the silymarin extract obtained from sample 4. eligibility conditions: silymarin dosage [%] (relative to dry matter) = minimum 80% [19]. fig. 3. dosing of silymarin from powder of silybum marianum seed food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea, quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas, food and environment safety, volume xxi, issue 1 – 2022, pag. 76 81 80 from this, we can conclude that in countries where the climate is richer in rainfall, it directly influences the amount of active substance in plant material. for this reason, when we talk about cultivated medicinal plants, the interaction between vegetation factors must be taken into account, so as to ensure an optimal ratio between the pedoclimatic conditions and the biological requirements of the medicinal plants. situations where climatic conditions generally lead to an increase in the production of plant biomass, which is not important for the cultivation of medicinal plants, to the detriment of the content of active substances, should be avoided. of interest to medicinal plants is the amount of active substances. 4. conclusion the flavonolignans silybin, silydianin and silychristin are the most active of the three flavonolignans that make up silymarin. we measured the quantity of total silymarin in silybum marianum powder from diverse geographical areas in this study. the content of silymarin varied depending on the geographic area, according to the findings. quantitative dosing by uv method of the silymarin extract existing in the samples from the geographical areas analyzed falls within the parameters described in the european pharmacopoeia (admissibility conditions > 80%). silymarin extracts obtained from areas richer in precipitation are quantitatively poorer in silymarin compared to areas with higher temperatures and less precipitation. 5. references [1] roman gh. v., toader m., epure l.i., ion v., basa a. gh., cultivarea plantelor medicinal şi aromatice în condiţiile agriculturii ecologice, editura ceres, bucureşti, (2009); [2] bojor o., popescu o., fitoterapie tradițională și modernă, ediția a iii-a, editura fiat lux, bucurești, (2005); [3] ilea (zăvoi) s. v., caracterizarea unor plante medicinale autohtone şi a unor bioproduse derivate cu acţiune hepatoprotectoare, rezumat al tezei de doctorat, cluj-napoca, (2011); [4] ilie t., minoiu m., plante medicinale miraculoase din flora româniei, editura artmed, bucureşti, (2004); [5] shaker e., mahmoud h., mna s., silymarin the antioxidant component and silybum marianum extracts prevent liver damage, food and chemical toxicology, 48, 803–806, (2010); [6] stoica v., căpraru s., piţigoi d., ionică c., diculescu m., evaluation of clinical and biological effects of silymarin versus silymarin +astragalus in chronic liver disease, practica farmaceutic, 7(2), (2014); [7] musteaţă g., armurariu – silybum marianum (l.) gaerth, plante medicinale valoroase, chişinău, p. 6-11, (2002); [8] aziz m., saeed f., ahmad n., ahmad a., afzaal m., hussain s., mohamed a. a., alamri m. s., anjum f. m., biochemical profile of milk thistle (silybum marianum l.) with special reference to silymarin content, food science&nutrition, 9 (1), 244-250, (2021), https://doi.org/10.1002/fsn3.1990; [9] omar a.a., hadad g.m., bad rj.m., first detailed quantification of silymarin components in the leaves of silybum marianum cultivated in egypt during different growth stages, acta chromatographica, 2(43), 463–474, (2012), https://doi: 10.1556/achrom.24.2012.3.9; [10] kurkin v. a., antioxidant activity of flavolignans from silybum marianum (l). gaertn. fruits, research and reviews: journal of pharmacognosy and phytochemistry, 4(1), (2016); [11] kshirsagar a., ingawale d., ashok p., vyawahare n., silymarin: a comprehensive review, phcog rev., 3(5), 126134, (2009); [12] surai p. f., silymarin as a natural antioxidant: an overview of the current evidence and perspectives, antioxidants, 4, 204-247, (2015), https://doi:10.3390/antiox4010204; [13] ishii y., okada y, matsuoka s., shen m., yui k., yagi m., yonei y., effect of supplement containing silybum marianum extract, soy extract, collagen peptide, bifidobacteria and apple extract on skin: a randomized placebocontrolled, double-blind, parallel group comparative clinical study, glycative stress research; 3(3): 156-171, (2016); [14] zhu s. y., jiang n., tu j., yang j., zhou y., antioxidant and antiaging activities of silybum marianum protein hydrolysate in mice https://www.libraronline.ro/autor/gheorghe-valentin-roman--i4096 https://www.libraronline.ro/autor/maria-toader--i4097 https://www.libraronline.ro/autor/lenuta-iuliana-epure--i4098 https://www.libraronline.ro/autor/viorel-ion--i4099 https://www.libraronline.ro/autor/adrian-gheorghe-basa--i4100 https://www.librarie.net/cautare-rezultate.php?editura_id=58 https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=aziz,+maryam https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=aziz,+maryam https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=saeed,+farhan https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=saeed,+farhan https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=ahmad,+nazir https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=ahmad,+aftab https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=ahmad,+aftab https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=afzaal,+muhammad https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=afzaal,+muhammad https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=hussain,+shahzad https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=hussain,+shahzad https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=mohamed,+abdellatif+a https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=mohamed,+abdellatif+a https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=mohamed,+abdellatif+a https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=alamri,+mohamed+s https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=alamri,+mohamed+s https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=anjum,+faqir+muhammad https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=anjum,+faqir+muhammad https://doi.org/10.1002/fsn3.1990 https://doi.org/10.1002/fsn3.1990 https://doi.org/10.1002/fsn3.1990 https://doi.org/10.1002/fsn3.1990 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 maria poroch seriţan, mihaela jarcău, narcisa mihaela cornea, quantitative research on the active component of silymarin in silybum marianum seed powder from various geographical areas, food and environment safety, volume xxi, issue 1 – 2022, pag. 76 81 81 treated with d-galactose, biomed environ sci, 30(9): 623-631, (2017); [15] dixit n., baboota s., kohli k., ahmad s., ali j., silymarin: a review of pharmacological aspects and bioavailability enhancement approaches, indian j. pharmacol, 39(4), 172-179, (2007); [16] metodă dezvoltată şi validată de departamentul de controlul calităţii, laropharm ce se desfăşoară la standardele cerute de regulile de bună practică de fabricaţie şi a regulilor de bună practică de laborator, method developed and validated by the quality control department, laropharm, which is carried out to the standards required by the rules of good manufacturing practice and the rules of good laboratory practice (2018), https://www.laropharm.ro/servicii/controlul-calitatii [17] elwekeel a., elfishawy a., zids. a., silymarin content in silybum marianum fruits at different maturity stages, journal of medicinal plants research, 7(23), 1665-1669, (2013); [18] giannakoudakis d.a., hosseinibandegharaei a., tsafrakidou p., triantafyllidis k.s., kornaros m., anastopoulos i., aloe vera waste biomassbased adsorbents for the removal of aquatic pollutants: a review. j environ manag, 227:354– 364, (2018), https://doi.org/10. 1016/j.jenvman.2018.08.064; [19] european pharmacopoeia (ph. eur.) 10th edition. https://www.edqm.eu/en/europeanpharmacopoeia-ph-eur-10th-edition (accessed on 1.09.2021). https://www.laropharm.ro/servicii/controlul-calitatii https://doi.org/10.%201016/j.jenvman.2018.08.064 https://doi.org/10.%201016/j.jenvman.2018.08.064 https://www.edqm.eu/en/european-pharmacopoeia-ph-eur-10th-edition https://www.edqm.eu/en/european-pharmacopoeia-ph-eur-10th-edition 1. introduction the goal of this study was to perform the quality control on the finished product, milk thistle (silybum marianum) seed powder from various geographical areas, using analysis to verify the product conformity in terms of silymarin content dosing parame... 4. conclusion stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xiii, issue 2 30 june 2014 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava editor-in-chief: sonia gutt (amariei) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania thiery abraham enzymes & derivates, bruxelles, belgium marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey lidija basa university of ljubljana, slovenia iulian bobe tyton biosciences, darien , usa marija bodroža-solarov university novi sad, institute for food technology, serbia jános csapó kaposvár university, faculty of animal science, hungary cristina-gabriela constantinescu (pop) ştefan cel mare university of suceava, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa georg gutt ştefan cel mare university of suceava, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania ştefania iordache valahia university of târgovişte, romania heinz – dieter isengard hohenheim university, germany süleyman kaleli sakarya university, medical faculty, department of medical biology, sakarya turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania anna maraz corvinus unversity of budapest, hungary norbert kreuzinger vienna university of technology, institute for water quality, resources and waste management, austria angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa christian neususs technical university of aalen, germany regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia vladimir reşitca technical university of moldova, chisinau republic of moldavia alice roşu ştefan cel mare university of suceava, romania sorin sabau tokai university, faculty of biological science, sapporo, japan osman kamil sag piri reis maritime university, istanbul, turkey jurgen schram krefeld university, germany rodica sturza technical university of moldova, chisinau republic of moldavia yarema tevtul yuriy fedkovych chernivtsi national university, ukraine gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia lester wilson iowa state university, usa igor winkler yuriy fedkovych chernivtsi national university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, ştefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia gutt, gutts@fia.usv.ro assoc. prof. ph.d, eng. gabriela constantinescu (pop), gabriela.constantinescu@fia.usv.ro assoc. prof. ph.d cristina hretcanu, cristina.hretcanu@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro gutts@fia.usv.ro, cristina.hretcanu@fia.usv.ro, gabriela.constantinescu@fia.usv.ro – the postal address: faculty of food engineering, stefan cel mare university suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved 15 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 1 2015, pag. 15 20 specifics of air conditioning in the germination process of malt *serhyi but 1 1national university of food technology, 68 volodymirska st, kyiv, ukraine bilyklena@gmail.com *corresponding author received 1st february 2015, accepted 25th march 2015 abstract: information is provided on the specifics of stabilizing the temperature of germinated malt in the spring-summer and winter seasons. the situation is complicated by the rated values of the grain mass temperatures ranging between 12 and 18º c, which are rather far from the ambient temperatures in the specified seasons with maintained relative humidity of air used for aeration. the possibility and advisability of using exhaust air masses for recuperation are estimated; calculation formulas are provided concerning the organization of recuperation modes in the aeration processes. information regarding the thermodynamic air preparation in the winter by using open steam and the results of calculations in support of advisability of energy conservation through recuperation are provided. the thermodynamic peculiarities of preparing the air for aeration with the need to achieve the rated temperature and the relative humidity φ=100 % are demonstrated. keywords: germination, grain, malt, heat, air, aeration, temperature, humidity, recuperation. 1. introduction the synthesis of advanced technology systems requires a comprehensive analysis in the courses and transformations of material and energy flows with the organization of their interaction by using appropriate information systems. such analysis may be useful even for quite simple systems, or rather for the traditional and conventional technologies. the latter entirely concerns the processes of grain mass germination in the production of malts. the presence of biochemical processes creates conditions and physical consequences, to which the germinated grain aeration system should respond. biochemical changes trigger the release of heat and carbon dioxide. the secondary effects are the reduced relative humidity of the air that passes through the grain mass and the drying of the latter. however, it is a process disturbance, as grain humidity and temperature must be stabilized [1-3]. in view of the above, the purpose of this study is to overcome the thermodynamic incompatibility regarding the requirements to the interaction of grain masses and air streams for aeration. http://www.fia.usv.ro/fiajournal mailto:bilyklena@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 serhyi but, specifics of air conditioning in the germination process of malt, food and environment safety, volume xiv, issue 1 – 2015, pag. 15 – 20 16 2. matherials and methods the study is a theoretical one and is based on information about biochemical transformations in grain mass and the processes of energy and mass interchange with the aeration air [4-9]. 3. results and discussion even a cursory analysis of air preparation processes leads to the conclusion about the diversity of problems and solutions regarding the winter and summer seasons. for the summer season, the mixing of fresh and recuperative air flows can ensure a nominal result in a considerable range of temperatures. the temperatures of recirculation air tr.a.being entirely predictable, it is possible to determine the required ratios of their volumes with variable values of the fresh air temperatures tf.a. suppose we have .... araf vv , (1) where vf.a. і vr.a. are volumes of fresh and recuperative air. considering the latter dependence, the temperature of the selected volumes mixture will be found according to the formula   .... ....1 afar araf m tt tt t      . (2) by using the above formula it is possible find the ratio of air volumes, which will satisfy the condition of obtaining the rated mixture temperature: ...... ...... arafafm armaraf tttt tttt    . (3) where f .а.t – the temperature of the fresh air; r.а.t – the temperature of regenerative air. to assess the importance of using recuperative modes let us refer to the conditions of aeration air preparation in the winter season, the fresh air temperature being tf.a. = –20 °с. calculations will be done for the volume vf.a. = 1000 m3, with its final temperature being 16 °с. in this case the amount of heat energy to be transmitted to the air in the heater will be: q = vf.a.сf.a (16 – (– 20)) = 46,740.4 kj. the further part of calculations will be done for the conditions of grain germination in 8 drums, each containing 8,000 kg of green malt. the level of aeration for each drum is about 10,000 m3 of air per hour. then, in the absence of recirculation, the energy consumption under the conditions formulated above will amount to: 600,467 000,1 000,10  qqd kj/h, and q8d = 8qd = 8 · 467,600 = 800,740,3 kj/h for eight drums, respectively. with the calorific value of natural gas amounting to 41,000 kj/m3, the gas consumption will be: 114 8.0000,41 800,740,3 41000 8     б g q v m3/h, where η is the efficiency factor of the air heating system. under the value ε = 1 gas consumption will be reduced by half, while a further increase in recirculation will improve the results. the above part of calculations to find out the energy consumption for heating up the air has been performed in accordance with the provisions of thermodynamics. however, we do not take into account the process requirement of bringing the relative fresh air humidity to 100 percent. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 serhyi but, specifics of air conditioning in the germination process of malt, food and environment safety, volume xiv, issue 1 – 2015, pag. 15 – 20 17 we will consider the specifics of the course of operations by using diagram i–d (fig. 1). the initial temperature t1 and the relative humidity φ1 of air on the diagram correspond to point 1, which also corresponds to the absolute moisture content d1. the phase of air heating through heat-exchange surface is carried out provided that d = const = d1. if we assume that point 2 in terms of the previously accepted value corresponds to t2 = 16°c, then the further isenthalpic saturation process leads to point 3 on the diagram, which corresponds to an isotherm with the temperature t3. it means that the task will fail through the above combination of heating and cooling. at the same time, a full-fledged option would be a semi-stage or even a single-stage process, which, however, will require preliminary determination of the positions of points 2 and 3. fig. 1. diagram i–d representing the single-stage air heating and humidification mode with the values t3 = trated and φ = 100 % the calculations should reasonably begin with finding the position of point 3 on the curve φ = 100 %, while the second coordinate is the rated temperature isotherm t3 (fig. 2). fig. 2. diagram i–d representing the two-stage air preparation process also, the absolute moisture content of the air d1 should be known. we can find the position of point 2 and the corresponding temperature t2, to which air should be heated in the single-stage mode, by moving along the isoenthalpy from point 3 to the intersection with the ordinate, which corresponds to d1. the two-stage process is reflected in the diagram provided in fig. 2. in this case, the fixed points on the diagram are points 1 and 5, while the positions of points 2 or 2' and 4 or 4' are variable. however, the choice of particular laws in the air heating and humidifying processes does not influence the overall energy consumption results, as they are theoretically determined by the dependence     ,....)()( ...... afafif afnafaf rvdd ttcvq   (4) where d(i) and d(f) are the initial and final absolute air humidity. φ2 φ=100 % i d d1 d3 t1 t2 t3 2 1 φ1 3 φ4 φ=100 % i d d1 d3 d3 ' d5 t1 t5 t2' t2 t3 2' 2 і2 φ2 3' φ1 1 4 t4 5 4' 3 і4 і2' food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 serhyi but, specifics of air conditioning in the germination process of malt, food and environment safety, volume xiv, issue 1 – 2015, pag. 15 – 20 18 fig. 3. diagram i–d representing the air and steam mixing process based on the performed analysis we may conclude that it is possible and reasonable to combine fresh air heating and humidification operations by introducing steam into the latter. the course of such a process may be represented by the same diagram i–d in fig. 3. the transition from point 1 to point 2 may coincide with or exceed the position of the curve φ = 100%. however, the final temperature t(f) = ti must be reached along with the full steam saturation of air. it is obvious that the course of the process and its final results depend inter alia on steam parameters. in this case the following correlation must be met:   steamsteamifafaf міttcvq  )()(.... , (5) where мsteam is the steam mass supplied to the process, and іsteam is the steam enthalpy. hence, from the perspective of achieving the rated temperature, the steam flow must be as follows:   steam ifafaf steam і ttcv м )()(....   . (6) however, three conditions may arise, in particular:   ....)()( afafifsteam vddм  ; (7)   ....)()( afafifsteam vddм  ; (8)   ....)()( afafifsteam vddм  ; (9) condition (7) corresponds to the flows ratio, under which one portion of steam will be condensed, while the other portion will become part of the air saturated to the extent of φ = 100 %. whereas expression (8) involves the case of steam fully becoming part of the air, the rated air temperature mode is achieved in the situation corresponding to expression (9). however, the relative humidity in this case is φ < 100 %. in the latter case complete saturation of the air with moisture should be achieved by spraying water of rated temperature. it is obvious that in most cases a necessity arises to heat up the air and to increase its enthalpy in the winter time. for humid air, the latter shall be found based on the superposition principle, under which sadah ііі  .... , (10) where ..adі is dry air enthalpy, and іs is steam enthalpy. substitution of values ..adі and sі leads to the form  ttсі рah 93.1500,2..  , kj/kg. (11) fig. 4 scheme for sequencing the operations in diagram i–d since winter time is characterized by low values of absolute moisture content, air saturation up to φ = 100% and an increase in temperature are simultaneously і(f) φ=100 % i d d(i) d(f) t(f) t(i) 2 1 і(i) φ=100 % i d d(f) d(i) d2 tsup. t(i) t(f) 1 4 φ1 2 is і3 3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 serhyi but, specifics of air conditioning in the germination process of malt, food and environment safety, volume xiv, issue 1 – 2015, pag. 15 – 20 19 performed operations. thus the total energy consumption is determined by the difference of air enthalpies under final and initial thermodynamic parameters. air conditioning in the summer period has some distinctions, due to which the specifics and sequence of operations for changing the thermodynamic parameters should be considered. the coordinates of the point of initial thermodynamic parameters are determined by the values of temperature and relative humidity (fig. 4). thus the final values are also known and constitute the values t(f) = 10 °с and φ = 100 %. suppose that the specified coordinates and the relevant parameters in the diagram correspond to points 1 and 3, while point 2 corresponds to the parameters of fully saturated humid air with φ = 100 %. if saturation is taken as the first operation, its completion provides only for the rated relative humidity, while the rated temperature is not reached. to provide for the latter, it is necessary to continue cooling the saturated humid air. the diagram (fig. 4) shows that the subsequent transformation of the thermodynamic parameters of saturated humid air involves a decrease in the absolute moisture content from d2 to d(f). it is obvious that, from a physical point of view, this means the presence of a steam condensing mode, the energy cost of which will be the condensation heat extraction and 323221 ііі;0і   . (12) the result of this process will be a state of thermodynamic equilibrium of the humid air with parameters of point 3, while the load on the refrigeration unit will correspond to the value 32і  . in the diagram, air cooling without prior moisture saturation corresponds to the segment 1–4, and point 4 corresponds to temperature t4 > t3 and enthalpy і4 > і3. here we have ;ііі 4141   (13) 3434 ііі   , (14) while the load on the air cooling system is 3134413441.у.х ііііііііq   . (18) as і1 = і2 in the isenthalpic process, the choice in favour of the first or second option does not affect the result regarding the cooling system load. however, if there is no difference regarding the material balance, there are some distinctions concerning the heat balance. the cooling process following the arc 2-3 on the curve φ = 100 % must be accompanied by moisture condensation in the amount of d2 – d3. at the same time, the process of condensation requires the relevant conditions, condensation centers, energy security, creating an interphase surface, etc. given the brevity of the said process and the above conditions, it may be concluded that the air stays in a metastable state, i.e. in a state of oversaturation with moisture. the interaction of such air with the grain mass having the temperature 2-3° c higher should not be accompanied by condensation. however, as the air temperature increases, the level of thermodynamic disequilibrium is reduced and thus the drainage capacity is lowered. 4. conclusion based on the analysis of air preparation for germinated grain aeration in the production of malt the following conclusions can be made: 1. preparation of air flows for germinated grain aeration in the summer and winter seasons has significant differences not only in terms of the input food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 serhyi but, specifics of air conditioning in the germination process of malt, food and environment safety, volume xiv, issue 1 – 2015, pag. 15 – 20 20 thermodynamic parameters but also regarding the techniques of achieving the specified output parameters; 2. in the winter period, it is reasonable to heat up the incoming flows of fresh air by using live steam; 3. the thermodynamic inconsistency of air in terms of temperature and relative humidity may be limited by increasing the intensity of aeration. the use of recuperation modes used in grain aeration processes reduces energy consumption by 50 %. the results and recommendations may be applied to various types of malt houses. 5. references [1]. kunz b. technology malt and beer. trans. with him., st. petersburg title: 912 p., (2001). [2]. demirbas a. combustion characteristics of different fuels, progress in energy and combustion science, 300: 219-230, (2004) [3]. pіdlіsny v., sokolenko a., shevchenko o. osoblivostі pіdgotovki povіtrya, nutritive promislovіst, 11: 13-14 (2007). [4]. domarecki v. investigation of the processes of production of malt for the purpose of intensification and creation of high vehicles. abstract. dis. for the degree of doctor of science k., 1979. 40 p. [5]. kovbasa v., shevchenko a., poddubny v., et al. process improvement of air conditioning systems germination malt // grain storage and processing. 2007. № 10. s. 34-36. [6]. kolotusha p. technology malt. m.: the institute of system studies in education. 1993. 542 p. [7]. podlesniy v. determination of thermal and material flows in the process of germination malt // food industry, 2008. № 6. s. 90-92. [8]. domarecki v., hivrich b., shovels t. investigation of the influence processes solodoroschennya on quality of soybean lipids // proceedings uduht. k.: uduht, 2000. № 6. s. 87-88. [9]. shevchenko o. pіdgotovka povіtrya in processes virobnitstva malt // nutritive prom-st. 2003. № 2. s. 66-69. doi: https://doi.org/10.4316/fens.2021.008 61 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1 2021, pag. 61 67 evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l) : bran oats and whole grain (black and prevision oats) *samira meziani 1 , noreddine menadi 1 , hayet mehida 2 , soheila ougad 2 , souad saidani 2 , lahouaria labga 2 1 faculty of life science and nature. laboratory of biotoxicology. djillali liabes. sidi-bel-abbes, algeria, meziani_samira@yahoo.fr 2 faculty of life science and nature. department of biology. djillali liabes university. algeria *corresponding author received 13th december 2020, accepted 30th march 2021 abstract: the objective of our work is to study the composition of total polyphenols, flavonoids and the evaluation of the antioxidant power present in whole prevision oats, whole black oats and prevision oat bran and black oat bran. oats are a whole grain cereal with the most beneficial nutritional value; its regular consumption would be particularly beneficial in reducing the risk of cardiovascular disease. good source of soluble fiber (beta-glucans), oat bran is the husk of the oat grain, it has many medicinal properties since it is able to regulate the metabolism of carbohydrates in people with diabetes, to greatly lower blood cholesterol levels, to prevent colon cancer and to fight against constipation by capturing water and thus facilitating transit. oat bran is also widely used in the diet for its appetite suppressant action. the results obtained show that the total polyphenols represent respectively values of 0.205 ± 0.0131 mg (eag) / g and 0.17 ± 0.014 1mg (eag) / g for prevision whole oat and whole black oats, 0.203 ± 0.005 mg (eag) / g, 0.216 ± 0.0117 mg /eag for prevision oat bran and black oat bran. for flavonoids, whole oats prevision has a rate of 0.192 ± 0.371 mg eq / g, 0.093 ± 0.006 mg eq / g for whole black oats, and 0.201 ± 0.025 mg eq / g for black oat bran and 0.071 ± 0.020 mgeq / g for the prevision oat bran. the results of the antioxidant power reveal that the black oat bran has a high antioxidant activity (ic50 = 2.95mg / ml) followerd by prevision oat bran (ic50 = 6.15mg / ml) whole oats prevision (ic50 = 6.37mg / ml) and whole black oats (ic50 = 11 mg / ml). keywords: oat bran, whole oats, polyphenols, flavonoids, antioxydant activity 1. introduction the poaceae play a role at the global level by producing the basis of valuable food, historically, the fruits of various cereals (wheat, rye, corn, barley, oats, rice and millet) have been one of the most important sources of food human and animal, both as fodder and as a nutritious grain, in terms of quantity oats rank among the first food products, it is one of the most important cereal crops in the world [1]. in recent years, public attention has been particularly focused on the health and well-being of the inhabitants, through the consumption of a healthy diet rich in cereals. oats are rich in protein, fat, starch and phenolic compounds concentrated in the peripheral layer of bran [2]. oats (avena sativa l.) is considered a nutritional food of interest due to its high fiber content. consumption of oats is believed to have various health benefits such as hypocholesterolemic and anticancer properties, oats have also recently been considered suitable in the diet of mailto:meziani_samira@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 62 celiac disease patients. oat-based bread [3] is considered to be an important value in combination with functional proteins, βglucan and phytochemicals such as linked glycerol [2], ester linked alkyl conjugates [4], may have beneficial effects on human health. there are also fat, vitamins and minerals present in a considerable quantity [5]. they are functional foods known to provide a beneficial effect on the health of the consumer, decrease the risk of various diseases, the valuable physiological and nutritional attributes of oats by β-glucans and other components of dietary fiber, rich in tocopherol. the level of a natural antioxidant has generated an increased demand for oats in human nutrition [6]. oats differ from other grains in that they are generally eaten as a whole grain, including the bran, as a result, the antioxidant-rich part of the grain is retained, including the phenolic compounds, which in oats are mainly represented by phenolic acids and avenanthramides [7]. oats have various health benefits, cholesterol-lowering and anticancer drugs, recommended for patients with celiac disease. additionally, it could be added to certain food products such as bread, cookies, and baby food [8]. the proteins, fibers, minerals and phenolic compounds present in whole oatmeal of which 100 g have almost 20% protein and more than 50% of carbohydrates, 15% of a soluble fiber [9, 10]. the objective of the present study was to determine the specific natural compounds of oats, to compare the phytochemical content in particular of the total phenolic content (tpc) of this species and to investigate the antioxidant power between prediction oats and oats black. this will help to reflect and reiterate the nutritional benefits of oats for consumers and the algerian market. 2. matherials and methods 2.1. plant material the objective of this experimental study is to evaluate the level of flavonoids and total polyphenols, and to investigate the antioxidant power between two varieties of oats (prevision oats and black oats). the plant material of this study includes two varieties of oats: oats prevision provided by technical institute of field crops (itgc) of and black oats provided by national center for the control and certification of seeds and plants (cncc) of the west region of algeria. the results are given in (table 1). 2.1.1. sample preparation method whole oats: to obtain whole oats, the oat varieties were crushed using an electric grinder and the mash obtained was stored in paper bags at room temperature, in a place dry and protected from moisture and light until use. 20g of each sample is macerated in 100 ml of 70% ethanol, after the sample is stirred for 24 hours. after stirring, the solutions were separated by filtration. then undergo evaporation using a rotary evaporator at 45°c, which removes the solvent in vacuo. finally, drying in the oven. oat bran: oat bran is obtained after separating the bran from the grain manually. table 1 characteristics of the varieties varieties characteristics p r e v is io n o a t origin : sidi bel abbes type : pure line half spreed tillering, low glaucescnece, high thousand grain weight, high yield origin : tiaret b la c k o a t type : pure line half spreed tillering, low glaucescnece, high thousand grain weight, high yield food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 63 2.2. methods 2.2.1. the determination of total polyphénoles in whole oat and oat bran extracts of the two varieties studied is carried out according to the method of folin ciocalteu [11]. the reagent used consists of a mixture of phosphotungstic acid (h3pw12o40) and phosphomolybdic acid (h3pmo12o4) of yellow color. the principle of the method is based on the oxidation of phenolic compounds by this reagent, which results in the formation of a new complex of metal oxides of tungsten and molybdenum in blue color. the intensity of the coloring is proportional to the amount of polyphenols present in the plant extracts [12]. 200 μl of each extract, 800 μl of sodium carbonate solution (na2co3) and 1 ml of folin-ciocaltaeu reagent are added to a hemolysis tube, after shaking the tubes are incubated for 30 min. reading this fact has an absorbance of 765nm using a uv spectrophotometer (the level of polyphenols was expressed in microgram equivalent of gallic acid per milligram of dry extract (mg eag/mg es). calibration was carried out in parallel under the same operating conditions using gallic acid. 2.2.2. the determination of flavonoids the method for determining flavonoids is based on the ability of these compounds to form chromogenic complexes with aluminum chloride (alcl3) [13]. 400 μl of the extract, 2 ml of distilled water and 120 μl of sodium nitrite (nano2) are mixed in a hemolysis tube, after 5 min 120 μl of aluminum chloride (alcl3) at 10% are added after 6 min 800 μl of sodium hydroxide naoh at 4% are added, the volume is adjusted to 5 ml with distilled water after stirring the mixture is incubated for 15 min protected from light the reading this fact has an absorbance of 510 nm using a spectrophotometer. the concentration of flavonoids is calculated by referring to a calibration curve obtained using quercitrin. the concentrations are expressed in mg quercitrin equivalent (mg eq q). 2.2.3 anti-free radical activity with dpph the evaluation of the antioxidant power of the various extracts is carried out by the dpph test which is considered as a relatively stable free radical, the principle of this method is based on the measurement of the scavenging of free radicals of dpph (1,1-diphenyl-2picrylhydrazyl purple in color). in the presence of so-called antioxidant molecules, dpph is transformed into its reduced form (diphenyl picryl-hydrazine: yellow in color), which leads to a decrease in absorbance [14]. the discoloration of dpph is directly proportional to the ability of bioactive molecules to reduce it. according to the protocol described by sanchez-moreno (1998) and agrawal (2011). the dpph solution is prepared by dissolving 4 mg of this product in 100 ml of methanol. 50 μl of each methanolic solution of the extracts at different concentrations are added to 1.95 ml of dpph solution. the mixture is left in the dark for 30 min and the absorbance reading is taken against a blank at 517 nm [15]. statistical analysis all results were performed in three replicates. the results are expressed as average ± standard deviation. the data (the averages plus at least standard deviation as well as representations) are statistically analyzed using the statistical software excel 2010. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 64 3. results and discussion 3.1. dry extract yield the extraction yield is calculated by the following formula (falleh et al, 2008). r (%) = (m ext/m samp) x 100 r : is the yield in % m ext : is the mass of the extract after evaporation of the solvent in mg. m samp : is the dry mass of the plant sample in mg. the yield of dry extract is calculated by adding 20g of whole oats and 10g of oat bran . 3.2. result of total phenolic content and flavonoid the total polyphenolic content of each extract was calculated from the calibration curve y = 8.721x-0.002, where y is the absorbance and x the concentration expressed in milligrams gallic acid equivalent per gram of dry extract (mg eag / g es) optical density measurement was performed at a wavelength of 765 nm. the results obtained from whole oat and oat bran extracts (pwo, bwo, pob, bob) analyzed reveal that bob is richer in total polyphenols with a content of 0.216 ± 0.0117 (mg eag / g es), than pob with a content of 0.203 ± 0.005 (mg eag / g es), and pwo is richer in total phenolic compounds 0.205 ± 0.013 (mg eag / g es) than bwo 0.107 ± 0.014 (mg eag / g es) .(table 2). flavonoids are the most important polyphenolic, the flavonoid content of each extract was calculated from the calibration curve y = 4.6153x-0.0118, where y is the absorbance and x the concentration and expressed in milligrams equivalent of quercetin per gram of dry extract (mg eq / g es, optical density measurement was performed at wavelength of 510 nm. the results obtained from the oat extracts (pwo ,bwo,pob,bob) analyzed reveal that the pwo contains a flavonoid content of 0.192 ± 0.371 (mg eq/ges) higher than that of bwo 0.093 ± 0.006 (mg eq/ges), and bob is rich in flavonoids of 0.201 ± 0.025 (mg eq/g es) when supplied to the pob 0.071 ± 0.020 (mg eq/ges) (table 2). the results obtained for the determination of total polyphenols and flavonoids reveal that the extracts of whole oats and oat bran of the two varieties (prevision and black) contain high levels of total polyphenols and flavonoids. the polyphenol content of the different varieties of oats is higher due to their flavonoid content. the rate of phenolic content of whole oat varies from 0.35-0.90 mg / g. according to a study made by[16]. by comparing our results obtained by intake with those of [17], that the total phenolic content varies from one variety to another, these differences may be due to genetic differences, methods of test, oat varieties used, degree of ripeness, extraction solvents, and environmental conditions. it has been shown that most of the ingredients including dietary fiber, minerals, tocopherols and phenolic compounds are concentrated on the bran layer, while most starch is mainly distributed on the endosperm of the kernels [17]. from the final results obtained and the differences observed in phenolic compounds and flavonoids for the two varieties of oats cultivated in sidi bel abbes and tiaret, suggest that there may be many factors that influence the content of whole grain phenolic compounds, including variety (genetics) and differences in agro-climatic. heat treatments are used in the case of grains treated before consumption (drying, steaming and boiling water), these processes have an impact on the content of phenolic compounds. although these processes should decrease the content of phenolic compounds, they can have a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 65 favorable effect on the bioaccessibility and bioavailability of these elements, the majority of these compounds are closely related to the cellulosic matrix. table 2 dry extract yield in %,total phenolic content in mg eag /g , flaonoids in mgeq /g. parameters dry extract yield % total phenolic flavonoids content varieties content (mg eag /g) (mgeq/g) pwo 9.5 0.205±0,013 0.192±0.371 bwo 7.9 0.107±0,014 0.093 ±0.006 pob 11.4 0.203±0,005 0.071 ±0.020 bob 4.2 0.216±0,0117 0.201 ±0.025 pwo : prevision whole oat bwo : black wholeoat pob : prevision oat bran bob : black oat bran 3.3. anti-free radical activity with dpph the results of the anti-free radical activity or the inhibition of free radicals are expressed as a percentage of inhibition (i%) estimated according to the equation below i (%) = (abs (control)abs (ext)/ abs control* 100 i %: percentage of anti-free radical activity abs control :absorbance of the dpph solution at time 0 abs ext: abosrobance of the extract . the ic50 (50% inhibitory concentration), also called ec50 (efficient concentration 50), is the concentration of the sample tested necessary to reduce 50% of the dpph radical. the ic50s are calculated graphically by percentages of inhibition as a function of the different concentrations of the extracts tested [19], a low value of the ic50 indicates a strong antioxidant activity. by comparing the results of ic 50 of the varieties of whole oats and oat bran with that of ascorbic acid (reference antioxidant) we see that the ic 50 of the varieties of whole oats and oat bran are higher than that of ascorbic acid with values of 11 mg / ml for bwo and 6.37 mg / ml for pwo, 6.15 mg / ml for pob and 2.95 mg / ml for bob by intake at 0.0295 mg / ml for ascorbic acid. so the antioxidant capacity of ascorbic acid is higher than that of whole oat and oat bran varieties, because the antioxidant capacity of a compound is higher when its ic50 value is small. then we see that the bob has a higher antioxidant power when compared to the pob and when compared to the bwo and pwo. fig 1: histogram of the values of the inhibitory concentrations 50 of the different varieties in mg/ml food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 66 the results observed for whole oats and its by-products (oat bran) showed a high antioxidant capacity compared to the results found by [17]. the extraction procedures, the agronomic conditions or of geographical origin can explain this increase. in addition, the antioxidant activity is a powerful scavenger of free radicals which can be exerted by modulating the defenses by the action of phenolic compounds according to a study carried out by [18]. 4. conclusion oat grains are a good source of protein, fiber, vitamins and certain natural compounds with important phytochemical properties such as (polyphenols and flavonoids). the regular consumption of oats in whole grain and the by-product of this species (oat bran) would be particularly beneficial in reducing the risk of cardiovascular disease has many medicinal properties and therapeutic since it is able to regulate the metabolism of carbohydrates in people with diabetes, and thus facilitating the intestinal transit. in this study, black oat bran is considered to be an important source of phenolic compounds. the varietals differences may be due to genetic differences, methods of test, oat varieties used, degree of ripeness, extraction solvents, and environmental conditions. this work deserved to be depth studied, particularly with regard to their effects on human health. 5. acknowledgments the authors acknowledge the university of sidi bel abbes, biology department at sidi bel abbes for the financial support of this research. sm wrote the manuscript and supervised in final reviewer the manuscript; nm and so participated in the experiment of this study; hm supervised data analysis; ss and ll for vegetal materials and designed for english grammar correction. 6. references [1]. hansen jm., the earliest seed remains from greece: palaeolithic through neolithic at franchthi cave.berichte der deutschen botanischen gesellshaft, 91,39–6, (1978). [2]. gray da., clarke mj., baux c., bunting jp., salter am, antioxidant activity of oat extracts added to human ldl particles and in free radical trapping assays. j. cereal sci;36(2):209-18. doi:10.1006/jcrs.2001.0456, (2002). [3]. ahmad a., anjum fm., zahoor t., nawaz h., ahmed z., extraction and characterization of oat β-glucan for industrial use. intj biol macromo; 46: 304-309, (2010). [4]. daniels dg, martin hr, antioxidants in oats: monoesters of caffeic and ferulic acids. j sci food agric; 18: 589-595, (1967) [5]. head ds., cenkowski s., arntfield s., henderson k, oatmeal superheated steam treatment. lwt, food sci technology; 43: 690– 694, (2010) [6]. zwier, p., k.oats.in: wrigley c., corked h., and walker c., (eds) encyclopedia of grain science. oxford: elsevier. international federation of societies of cosmetic chemists, (2004) [7]. peterson dm., oat antioxidants. j. cereal sci. 33: 115-129, (2001) [8]. rasane p., jha a., sabikhi l., kumar a., unnikrishnan vs, nutritional advantages of oats and opportunities for its processing as value added foods a review. j. food sci. technol, 52 (2):662-75. doi:10.1007/s13197-013-1072-1, (2015) [9]. czerwinski j., bartnikowska e., leontowicz h., lange e., leontowicz m., katrich e., trakhtenberg s., and gorinstein s, oat (avena sativa l.) and amaranth (amaranthus hypochondriacus) meals positively affect plasma lipid profile in rats fed cholesterol containing diets. journal of nutritional biochemistry, 15, 622-629, (2004) [10]. marlett ja, comparisons of dietary fiber and selected nutrient compositions of oat and other grain fractions. in: wood pj (ed) oat bran, (1993) [11]. boizot n., and charpentier jp, rapid method for evaluating the content of phenolic compounds in the organs of a foliage tree. the inra techniques book. pp 79-82. (2006) [12]. pascal ribéreau-gayon, phenolic compounds in plants (1968) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1– 2021 samira meziani, noreddine menadi, hayet mehida, soheila ougad, souad saidani, lahouaria labga, evaluation of phenolic compound and antioxidant capacity of two varieties of oats (avena sativa l): bran oats and whole grain (black and prevision oats), food and environment safety, volume xx, issue 1 – 2021, pag. 61 – 67 67 [13]. zhishen j, mengcheng t and jianming w, the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chemistry, 64, 555559, (1999) [14]. mansouri a., guendez e., kokkalou e. and kefalas p, phenolic profile and antioxidant activity of algerian ripe date palm (phoenix dactylifera).food.chem.89:411-420, (2005) [15]. sánchez‐moreno josé a., larrauri fulgencio saura‐calixto, journal of the science of food and agriculture, (1999). [16]. belobrajdic et bird, most abundant phytomicronutrients according to the cereal considered, (2013) [17]. chao chen., li wang., ren wang., xiaohu lu.,oyongfu li., juan li., yanan li., zhengxing che, (phenolic contents, cellular antioxidant activity and antiproliferative capacity of different varieties of oats, (2018). [18]. madhujith h., et shahidi f., antioxidant and anti-oliferative property of certain cultivars of barley (hordeum vulgarae l.) and their potential for inhibiting the oxidation of low density lipoprotein (ldl) cholesterol, j. agric. food. chem. 55 (13): 5018–5024, (2007). [19]. molyneux p.,the use of the stable free radical diphenylpicrylhydrazyl (dpph) for estimating antioxidant activity, (2004) 1. introduction doi: https://doi.org/10.4316/fens.2021.037 343 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 343 351 survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis janet olaitan 1 , *temilade ozabor 1 , abideen wahab 1 and odunola oluwajide 1 1faculty of basic and applied sciences, department of microbiology,osun state university, osogbo, nigeria praise.ozabor@uniosun.edu.ng *corresponding author received 1st september 2021, accepted 28th december 2021 abstract: the purpose of this paper is to investigate the different yeast species in dairy products that can be harnessed industrially for biotechnological functions. eighteen (18) different yeast species were obtained from thirty (30) different dairy products namely: yogurt, milk and cheese. ph, temperature and ethanol tolerance were conducted using standard methods while fungi isolation was done using culturedependent methods. the antibacterial activity and ftir analysis the yeast species were also done using standard methods. result obtained showed that s. cerevisiae was the predominant fungi isolated (19.11%) while chrysosporiumspp and c. fimetiwere the least with 1.47% occurrence each. all isolates showed moderate antibacterial activity on test pathogens. s. cerevisiae, c. krusei, c.glabrataand k. fragilis were analzyed for functional groups with the corresponding peaks 3439.19, 3437.26, 3439.19, 3431.48, 3417.98 and 3448.84(cm-1) respectively. all possess hydroxyl (o-h) and methylene functional group (c-h) while only d. hansenii(c-c) had an additional alkenyl group. in conclusion, representative isolate analyzed by ftir can be employed industrially for fermentation processes while d. hanseniiis a promising organism for plastics degradation/ bioplastic production. keywords: milk, yoghurt, cheese, antibacterial activity, ethanol tolerance, s. cerevisiae, d. hansenii 1. introduction dairy products such as milk, yoghurt and cheese (wara) are protein rich substrates in which probiotic yeasts can be easily isolated. literature has reported that milk is one of the most satisfactory single food that can be prepared and consumed by humans. it contains proteins, vitamins, fats, carbohydrate and inorganic salts [1]. the proteineous nature of milk makes it a suitable growth medium for different types of organisms [2]. cheese is one of the numerous food products that can be obtained by processing of milk and [3] has documented that about one-third of the total volume of milk produced in african is been used for cheese production. according to [4], there are more than 1000 varieties of cheese produced globally, each having a unique flavour, taste and form. there are four major ingredients involved in the production of cheese which are: milk, rennet, microorganisms and salt. soft cheese can be used as a drug for certain infections and possesses a strong inhibitory activity against diarrhea causing infections [5], [6]. however, if milk is not properly pasteurized, it can become a vehicle for pathogenic microorganisms when processed into cheese and other delicacies. yeast species have the ability to ferment lactose sugar and assimilate various acids such as citric, succinic and lactic acids [7]. the various impacts of probiotic yeast species in food processing involves: production of cheese, bread, and milk products, biopreservation which prevents the spoilage of food products as well as biofortification of foods [8], [9], [10]. yeasts can also be used in the production of biofuel [11]. fermented milk can be consumed as food beverages whose market and storage values are better improved than the raw milk. in addition, the major locally produced traditionally fermented milk products in nigeria are wara and nunu which are made by using fresh milk collected from cow, sheep or goat. the african soft cheese (wara) is a good source of nutrients mailto:praise.ozabor@uniosun.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 344 such as protein, fat, calcium, iron, phosphorus, vitamins and essential amino acids [12]. wara (local cheese) has higher moisture content than the hard cheese which makes it to have a lower shelf life due to spoilage by microorganisms. it can also be deep fried in groundnut oil for preservation over a long period of time. it can also be eaten in various forms in different african regions either as a raw cheese, flavoured snack, fried cake, meat substitute in dishes as well as filling in sandwiches [13]. the concentration of lactic acid bacteria in yogurt is higher than that of yeasts [14]. yeasts have the ability to boost the immune system, induce anti-tumor effects, anti-cholestrolemic effect and also help to stabilize the growth of lactic acid bacteria. yeasts are also leavening agents which can either be chemical or biological in nature. microorganisms such as saccharomyces cerevisiae can produce carbon dioxide from sugar utilization [15]. they can be used during fermentation process for baking and brewing. however, in brewing, alcohol is released by microorganisms which is of utmost need in the maturation and development of fermentation flavor [16]. 2. materials and methods sample collection a total sample of 30 dairy products (10 soft cheeses (wara); 10 evaporated liquid milk and 10 yogurt samples) were purchased from the following local markets: oja oba, igbona, orisunmibare and a supermarket (ace), osogbo, osun state, nigeria. the cheeses were collected into a sterile low density polythene bag and transported immediately to the microbiology laboratory of osun state university for further studies. preparation of samples and ph measurement a 5.0 ml each sample was aseptically withdrawn using a clean pipette and transferred into a test tube containing 5.0ml of 0.1 % sterile peptone water. ten-fold serial dilutions of each samples was prepared and 0.2ml of the appropriate dilutions (10 -3 and 10 -5 ) were spread plated onto potato dextrose agar (pda) containing 100µg of chloramphenicol/ml. plates were incubated at 30°c for 5-7days and purified by sub-culturing to obtain pure isolates. the pure cultures were stored in the refrigerator as stock on potato dextrose agar slants. ph measurement the ph of the samples were taken using ph meter (orion ph meter (model 310, orion research inc., beverly, ma) which was first standardized with buffer solutions of ph 4, 7 and 9. 5g of the samples were pipetted out aseptically and transferred into test tubes. 20 ml of sterile distilled water was added and the mixture was shaken for 30 minutes on the rotary orbital shaker. the ph of the suspension was then determined by inserting the electrode of the ph meter into the solution and the ph values were read when the reading was stable [17]. characterization and identification of isolates the cultural characteristics of the isolates on the potato dextrose agar (pda) plates were observed by staining the cultures with lactophenol blue on a sterile grease-free glass slide and placed under the microscope to view. aplexopoulos (fungi compendium) was used to study properties such as the elevation, surface and colour were viewed and recorded [18]. thermotolerance test the ability of yeast isolates to grow at higher temperatures was characterized by culturing on potato dextrose agar and incubated at varying degrees of temperature (30, 45 and 65 0 c) for 72 hours [19] ethanol tolerance tests the isolated yeast species were grown on potato dextrose broth containing varying ethanol concentrations (5, 10 and 15%) in order to determine their ethanol concentration level. in-vitro production of inhibitory metabolites in-vitro antimicrobial activity of the screened yeast isolates were tested against test pathogenic microorganisms (escherichia coli atcc 43816, staphylococcusaureus nctc 6571, klebsiella pneumoniae atcc 25922, proteus mirabilis atcc 7002and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 345 corynebacterium diphtheriae atcc 13813) using the agar well diffusion technique [20]. 24 hr old cultures of test organisms and antimicrobial producing yeasts isolates were aseptically transferred into sterile nutrient broth and incubated at 37 o c for 24 hr. the turbidity was adjusted to macfarland standard and the numbers of cells were confirmed using the spectrophotometer. the promising antimicrobial producing yeast isolates were seeded on the surface of sterile mueller hinton agar (mha) plates using sterile swab sticks. wells of 6 mm diameter were bored into the agar plates. the yeasts isolates were centrifuged for 20 minutes at 6,000 rpm and 70μl, 80μl, 90μl and 100 μl of the supernatants (which contains active metabolites) was pipetted into the wells and incubated at 30 0 c for 24-48 hr. the zones of inhibition around the test organisms were measured and recorded appropriately. test was conducted in triplicates and the mean value was recorded using statical analysis. ftir (fourier transform infrared spectroscopy) representative isolates namely:m. furfur, d. hansenii, s. cerevisiae, c. krusei, c.glabrataand k. fragilis were further analysed using the fourier transform infrared spectroscopy in order to know the functional groups that these yeasts belong to, thus giving an insight to the biotechnological exploitations of these yeasts. the method of [21] was used with slight modifications. 3. results and discussion the ph of yogurt, milk and cheese ranged from 3.21-4.47, 5.30-6.60 and 5.63-5.87 respectively. the lowest ph (3.21) was recorded from yogurt while the highest (6.60) was recorded from hollandia milk. similar findings were reported by [22], [23] and [24] who recorded ph value range of 4.16-2.20, 2.00-5.50 and 1.30-7.80 from yeast species isolated from: egyptian karish cheese, cereal based nigerian traditional fermented beverages namely (burukutu, kunu-zaki and ogi) and whole grain millet sourdoughs respectively. the ability of the yeast species to grow at low acidic ph confers microbial stability on the food. according to [25], the ability of moulds and yeasts at ph range of 2-8 demonstrates the ability of the yeasts to eliminate spoilage microorganisms, thus creating a conducive environment for the growth of food grade microorganims. the total count of fungi species from each of the dairy sample ( yogurt, milk and cheese) is presented in table 1 above. s. cerevisiae was the predominant fungiisolated (19.11%), followed by b. bassianaandd. hansenii(8.82%); m. furfur and b. hawaliensis(7.35%); a. kalrae, c. kruseiandp. purpurogen(5.88%);c. bantiana, k. fragilisandm. ferrugineum(4.4.1%); p. funiculosum, b. ranarum, c. glabrata, f. oxysporumandn. dimidiatum(2.94%) whilechrysosporiumspp and c. fimetiwere the least isolated with 1.47% occurrence each. the predominance of the yeast s. cerevisiaein yogurt and cheese had been earlier reported in literature according to the works of [26], [27], [28] from amasi of cow milk, sameel made of cow, goat, camel or sheep milk, chal made of camel milk and sourdough made of millet respectively. however, it is noteworthy to mention that some of the isolated yeast species are normal flora of the skin, the calotropis procera leaf (used as coagulant for cheese production), utensils used, food handlers as well as the environment (air, water and soil) such as: b. bassiana, b.hawaliensis, c. glabrata, chrysosporiumspp, c. krusei, f. oxysporum, c. fimeti, c. bantiana, m. ferrugineum and p. funiculosum. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 346 table 1 total count of isolated yeasts from each dairy sample presumptive organisms tem eam tac total arthrographiskalrae 2 0 2 4 basidiobolusranarum 0 2 0 2 beauveria bassiana 2 2 2 6 bipolarishawaliensis 2 0 3 5 candida glabrata 0 0 2 2 candida krusei 0 0 4 4 chaetomium fimeti 0 1 0 1 chrysosporiumspp 0 1 0 1 cladophialophorabantiana 0 3 0 3 derbaryomyceshansenii 1 0 5 6 fusarium oxysporum 0 2 0 2 kluyveromyces fragilis 1 0 2 3 malassezia furfur 1 0 4 5 microsporumferrugineum 0 3 0 3 neoscytalidiumdimidiatum 0 2 0 2 pencilliumpurpurogen 4 0 0 4 penicillium funiculosum 0 2 0 2 saccharomyces cerevisiae 5 0 8 13 total 18 18 32 68 key: tem= yogurt; eam= milk; tac= cheese table 2 gives the information on the ethanol and temperature tolerance of the isolated yeast species. all the isolates were subjected to growth at 5, 10 and 15% respectively and the result showed that they all grew well at 5 and 10% while arthrographis kalrae, basidiobolus ranarum, candida glabrata, candida krusei, kluyveromycesfragilis fusarium oxysporumand microsporumferrugineum did not grow at 15% ethanol according to table 2. this denotes that these yeast species cannot tolerate high ethanol concentration as the growth of organisms at high ethanol concentration show to be species dependent. furthermore, ethanol tolerance of the yeast isolates shows that they can be used as starter cultures in alcohol production because the fermentation process will be stopped if the organisms cannot tolerate the ethanol produced which is an indication that the yeast cell walls can withstand osmotic stress according to the reports of [29],[30], [31] and [32]. key: gt= gentamycin(control) table 2 ethanol and temperature tolerance of isolated yeasts ethanol concentration temperature range presumptive organisms 5 % 1 0 % 1 5 % 4 5 0c 3 7 0c 3 0 0c arthrographiskalrae + + + + + basidiobolusranarum + + _ + + + beauveria bassiana + + + + + + bipolarishawaliensis + + + + + + candida glabrata + + + + + candida krusei + + + + + chaetomium fimeti + + + + + + chrysosporiumspp + + + + + + cladophialophorabantiana + + + + + + derbaryomyceshansenii + + + + + + fusarium oxysporum + + _ + + + kluyveromyces fragilis + + + + + malassezia furfur + + + + + + microsporumferrugineum + + + + + neoscytalidiumdimidiatum + + + + + + pencilliumpurpurogen + + + + + + penicillium funiculosum + + + + + + saccharomyces cerevisiae + + + + + + food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 347 table 3 antibcaterial activity of isolated yeast species against pathogenic test organisms by measuring zone of diameter (mm) values are means ± standard deviation of duplicates at p < 0.05. in addition, growth at 30, 37 and 45oc was also carried out on all the isolates and the result showed that there was a 100% growth in all the three (3) subjected temperatures. [33] had earlier documented that the ability of yeast species to grow within a wide temperature range (30-45oc) confirms a vast difference in their thermostability, which is a suitable quality to be considered for use in fermentation processes. the antibacterial effect (table 3) of the s. cerevisaie, c.krusei, c. glabrata, m. furfur, d. hansenii and k fragilis againstp.mirabilis, s. aureus, k. pneumoniae, e. coli and c. diphtheriae showed these corresponding zone of inhibition (mm):9.92, 9.00, 11.20, 13.99 and 9.69; 9.45, 10.00, 10.90, 10.20 and 10.00; 9.64, 8.67, 13.01, 9.33 and 9.54; 9.88, 12.34, 9.67, 9.61 and 9.70; 9.27, 9.90, 9.45, 10.01 and 9.41; 9.11, 9.62, 9.21, 9.23 and 9.22 respectively. however, [33] had ealier documented high antibacterial activity of c. intermedia, c. kefyrand c. lusitaniae againsts. aureus, e.coli while they documented moderate activity of c. tropicalis, c. lusitaniae and s. cerevisiae against e. coli. in addition, they also documented low activity of c. intermedia, c. kefyr,c.lusitaniae, c. tropicalis and s. cerevisiae against pseudomonas aeruginosa. furthermore, the work of [34] also support the claims of this study. six (6) representative fungi in this study were further analyzed using fourier transform infrared spectroscopy (ftir) to further identify the yeast fungi species (figures 1-2). the six fungi are: d. hansenii, m. furfur, s. cerevisiae, c. krusei, c.glabrata and k. fragilis with the corresponding peaks: 3439.19, 3437.26, 3439.19, 3431.48, 3417.98 and 3448.84(cm1) respectively. they all have stretch type of vibration in their ftir spectra reading and single intensity. all possess hydroxyl and methylene functional group (table 4) while only d. hansenii has an additional alkenyl group. hydroxyl group o-h consists of one atom of oxygen covalently bonded to one atom of hydrogen (alcohols and carboxylic acid) and methylene group comprises of two hydrogen atoms bounded to one carbon atom (methyl chloride, methyl alcohol/methanol) while alkenyl belongs to the vinyl group (vinyl chloride, a precursor to polyvinyl chloride) which is a functional group formed by removing a hydrogen atom yeast isolates p. mirabilis s. aureus k. pneumoniae e.coli c. diphtheriae gentamycin (30µg) s. cerevisiae 9.92±0.03 9.00±1.13 11.20±0.01 13.99±1.23 9.69±0.09 18.00±0.01 c. krusei 9.45±0.17 10.00±1.02 10.90±0.11 10.20±1.06 10.00±0.03 22.00±0.11 c. glabrata 9.64±0.03 8.67±1.03 13.01±0.33 9.33±1.09 9.54±0.03 21.00±0.21 m. furfur 9.88±0.09 12.34±1.32 9.67±0.11 9.61±1.32 9.70±0.03 21.00±0.33 d. hansenii 9.27±0.06 9.90±1.06 9.45±0.03 10.01±1.44 9.41±0.03 18.00±0.10 k. fragilis 9.11±0.04 9.62±0.02 9.21±0.22 9.23±0.05 9.22±0.03 16.00±0.30 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 348 from an alkene. in this study, the result obtained shows that d. hansenii, s. cerevisiae, c. krusei, c. glabrata and k. fragilis can be employed on an industrial scale fermentation for the production of alcohols, methyl chloride methyl alcohol (which can be used as an anti-freeze) and methanol based on the ftir analysis of their functional group. table 4 ftir spectra showing the different functional groups of the yeast species s/ n types of vibration absorption, cm-1 ,class of compounds and chemical formula intensity derbaryomyce shansenii malassezia furfur saccharomyces cerevisiae candida krusei kluveromyce s fragilis candidagla brata 1. stretch 3439.19 (hydroxyl group) o-h 3437.26 (hydroxyl group) o-h 3439.19 (hydroxyl group) o-h 3431.49 (hydroxyl group) o-h 3443.84 (hydroxyl group) o-h 3417.95 (hydroxyl group) o-h single 2. stretch 2908.75 (methylene group) c-h 2929.97 (methylene group) c-h 2960.83 (methylene group) c-h 2366.74 (methylen e group) c-h 2935.76 (methylene group) c-h 2943.47 (methylene group) c-h single 3. stretch 2353.23 (methylene group) c-h 2675.36 (methylene group) c-h 2677.29 (methylene group) c-h 2000.25 (methylen e group) c-h 2360.95 (methylene group) c-h 2677.29 (methylene group) c-h single 4. stretch 1635.69 (alkenyl group) c-c 2376.38 (methylene group) c-h 2000.25 (methylene group) c-h 2007.96 (methylene group) c-h 2362.88 (methylene group) c-h single food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 349 fig. 1: ftir spectra generated froms. cerevisiae fig.2: ftir spectra generated from d. hansenii furthermore, d. hansenii possesses an additional functional group (c-c) that makes a potential source for the production of polyvinyl chloride which is a bioplastic. interestingly, there is limited information about it. [35], [36] also obtained similar results using ftir to analyze the functional groups of s. cerevisiae. 4. conclusions in conclusion, the results obtained from this study shows that d. hansenii and s. cerevisiae are potential yeasts that can be used as starter cultures for industrial fermentation processes of foods and beverages. more interestingly, both yeast species can also for employed for bioplastic production as the ftir analysis has confirmed this. there, it is recommended that further studies should be carried to optimize the bioplastic production processes. 5. references [1]. uzeh r.e., ohenhen r. e., rojugbokan a. k. (2006). microbiological and nutritional qualities of dairy products: nono and wara. nature and science, 4(3): 37 – 40. [2]. akabanda, f., owusukwarteng j., glover j. k., tanodebrah, k., glover, r.l.k., nielsen, d.s. and jespersen, l. (2013). taxonomic and molecular characterization of lactic acid bacteria and yeasts in nunu, a ghanaian fermented milk product. food microbiology. 3: 277-283. [3]. fusco, v., chieffi, d., fanelli, f., logrieco, a.f., cho, g., kabisch, j., bohnlein, c. and franz, c.m. (2020). microbial quality and safety of milk and milk products in the 21st century. comprehensive reviews in food science and food safety. 19(4): 2013-2049. [4]. cogan tm (2000): cheese microbiology. in p. f. fox, t. guinee, t. m. cogan, & p. l. h. mcsweeney (editions). fundamentals of cheese science. gaithersburg: aspen publishers. [5]. alakeji, t.p., ,banwo, k., ogunremi, o.r. and sanni, a.i. (2015). functional properties of yeasts isolated from some nigerian traditional fermented foods. journal of microbiology, biotechnology and food science. 4(5): 437-444. [6]. olorunfemi ob, adebolu tt, adetuyi fc (2006): antibacterial activities of micrococcus lactis strains isolated from nigerian fermented cheese whey against diarrhoea-causing organisms. research journal of biological sciences 1: 24-27 [7]. lopandic, k., zelger, s., banszky, l.k., eliskases-lechner, f. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 350 and prillinger, h. (2006). identification of yeasts associated with milk products using traditional and molecular techniques.food microbiology. 23: 341350. [8]. breuer, u and harms, h. (2006). debaryomyceshansenii an extremophilic yeast with biotechnological potential. yeast journal 23: 415-437. [9]. romano, p., capece, a. and jespersen, l. (2006). taxonomic and ecological diversity of yeasts in food and beverage. in: querol a, fleet gh (editions) yeasts in food and beverages. springerverlag, berlin. [10]. chanchaichaovivat a, ruenwongsa p, panijpan b. (2007). screening and identification of yeast strains from fruits and vegetables: potential for biological control of postharvest chilli anthracnose (colletotrichum capsici). biological control. 42:326–335. [11]. mohd ak, loh sk, nasrin a, astimar a, rosnah ms (2011). bioethanol production from empty fruitbunches hydrolysate using saccharomyces cerevisiae. research journal environmental science. 5(6):573-586. [12]. banwo, k., sanni, a. and tan, h. (2014). technological properties and probiotic potential of enterococcus faecium strains isolated from cow milk. journal of applied microbiology. 114: 229-241. [13]. olasupo, n.a., schillinger, u. and holzapfel, w. (2001). studies on some technological properties of predominant lactic acid bacteria isolated from nigerian fermented food. food biotechnology. 15(3): 157-167. doi: 10.1081/fbt-100107627. [14]. karaduman, a., ozaslan, m., kilic, i.h., oguzkan, s.b., kurt, b.s. and erdogan, n. (2017). identification using maldi-tof mass spectrometry of lactic acid bacteria isolated from noncommercial yogurts in southrn anatolia, turkey. international microbiology. 20(1): 2530. [15]. el-sheikha, a.f. and montet, d. (2016). fermented foods-artisan household technology to biotechnology era. fermented foods, part 1: biochemistry and biotechnology. pg 1 [16]. balarabe, m.m., sani, s.d. and orutokan, a.a.(2017). sreening of fermentative potency of yeasts isolates from indigenous sources for dough leavening. international journal of microbiology and biotechnology. 2(1): 12-17. [17]. daniyan s. y., abalaka m. e., momoh j. a. and adabara n. u. (2011). microbiological and physicochemical assessment of street vended soybean cheese sold in minna, nigeria. international journal of biomedical and advance research, 2(1): 25 31. [18]. kurtzman, c.p., fell, j.w. and robert, v. (2011). methods for isolation phenotypic characterization and maintainance of yeasts. in: kurtzman cp, fell jw, boekhout t. editors. the yeast a taxonomic study 5th edition elsevier. [19]. thais, m., guimares dg, moriel ip, machado, cyntia mt, f. (2006) isolation and characterization of saccharomyces cerevisae strain of winey interest. brazilianjournal of pharmacology science. 42:119-126. [20]. hatoum, r., labrie, s. and fliss, i. (2012). antimicrobial and probiotic properties of yeasts: from fundamental to novel applications. frontiers in microbiology 3 (2):421-431. [21]. mihoubi, w., sahli, e., gargouri, a. and amiel, c. (2017). ftir spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over-expression and its suppression by nigella sativa extracts. plos one. 12(7): 1-16 [22]. ogunremi, o.r., sanni, a.i. and agrawal. (2015). probiotic potentials of yeasts isolated from some cereal-based nigerian traditional fermented food products. journal of applied microbiology. 119: 797-808. [23]. adisa, a.m., ifesan, b.o.t., enujiugha, v.n. and adepeju, a.b. (2020). microbiological and probiotic assessment of yeast isolated from wholegrain millet sourdoughs. journal of advances in microbiology. 20(1): 1-10. [24]. abdelatif, s.s., elsayed, s.m., bahout, a.a. and bayoumi, a.m. (2016). studies on beneficial yeasts isolated from some egyptian dairy products. zagazigveterinay journal. 44(1): 75-84. [25]. adebolu, t.t. and ademulegun, o.h. (2005). effect of cheese whey on diarrhoea causing bacteria in southwestern nigeria. nigeria bioscience research communications. 16(1): 57-60 [26]. gadaga, t.h., mutukumira, a.n. and narvhus, j.a. (2000). e numeration food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 janet olaitan, temilade ozabor, abideen wahab, odunola oluwajide, survey and fourier transform infrared microscopy (ftir) analysis of yeasts isolated from dairy products in osogbo metropolis, food and environment safety, volume xx, issue 4 – 2021, pag. 343 –351 351 and identification of yeasts isolated from zimbabwean traditional fermented milk. international dairy journal. 10(6): 456-466. doi: 10.1016/s0958-6946(00)00070-4 [27]. al-otaibi, m. m. (2012). isolation and identification of lactic acid bacteria and yeasts from sameel milk: asauditraditionalfermentedmilk. international journal of dairyscience. 7(1): 73– 83.doi:10.3923/ijds.2012.73.83. [28]. younis, g., awad, a., dawod, r.e. and yousef, n.e. (2017). antimicrobial activity of yeasts against some pathogenic bacteria. veterinary world. 10(8): 979-983. [29]. alloysius, c.o., ositadinma, c.u., amadike, e.u. and chukwuma, s.e. (2015). production of mixed fruit (pawpaw, banana and water melon) using saccharomyces cerevisiae isolated from palm wine. african journal of food science. 10: 1-11. [30]. gomar-alba, m.a., angelesmorcillo, p. and merceclli, d.o. (2015). response of cells to high glucose involves molecular and physilogical difference when comapred to other osmostress conditions. fems: yeast resistance. 15(5): 20-39. [31]. techaparin, a., thanonkeo, p. and klanrit, p. (2017). high-temperature ethanol production using thermotolerant yeast newly isolated from greater mekong subregion. brazillian journal of microbiology. 48(3): 461-475. [32]. fadahunsi. i.f., akoja, a.d.. and ozabor, p.t. (2020). characterization of indigenous yeast species for pineapple wine production. carpathian journal of food science and technology. 12(5): 109-121 [33]. yam, b.z., khomeiri, m., mahounak, a.s. and jafari, s.m. (2015). isolation and identification of yeasts and lactic acid bacteria from local traditonal fermented camel milk chal. journal of food processing technology. 6:460-471. [34]. fakruddin, m., hossain, m.n. and ahmed, m.m. (2017). antimicrobial and antioxidant activities of s.cerevisiae ifst062013, a potential probiotic. bmc complementary and alternative medicine. 17(4): 1591-1599. [35]. keller, m.(2010). the science of grape vines agronomy and physiology. san deigo academic pess. (chapter 2). [36]. qvirist, l.a., defilippo, c., strati, f., stefanini, i., sordo, m., andlid, t., felis, g.e., mattarelli, p. and cavalieri., d. (201 6). isolation , identification and characterization of yeasts from fermented goat milk of the yaghr valley in tajikistan. frontiers in microbiology. 7(1): 117. 109 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 109 115 eval ua t ion o f the to xi co logic al s afe ty o f a fre e zedr ie d prob iot ic pr ep ar at ion *zapryana denkova1, ivan murgov1, rositsa denkova1, rositsa enikova2 1university of food technology, department of microbiology, bulgary zdenkova@abv.bg 2national center of public health and analyses, department of microbiology, bulgary *corresponding author received 19th june, accepted 29th june 2014 abstract: a combination consisting of selected probiotic strains lactobacilli and bifidobacteria from the national bank for industrial microorganisms and cell cultures (nbimcc), bulgaria was developed. during their joint cultivation the strains demonstrated their ability to multiplicate, accumulating high concentrations of viable cells. the toxicogenity of the symbiotic combination was examined by testing the freeze-dryed substance, containing lactobacilli and bifidobacteria, on biological objects (white mice). each experimental animal was injected intraperitoneally with 5.108cfu viable cells daily in the duration of 10 days. after the experimental period a post-mortem examination was conducted to analyze the safety of the substance of probiotic lactobacilli and bifidobacteria, which is the basis for the preparation of probiotic combinations. the absence of toxicogenity of the freeze-dried substance of probiotic lactobacilli and bifidobacteria is proven by the lack of any pathological changes in the health status, damages to the parenchymal organs, the peritoneum or the omentum that would otherwise indicate toxicity. keywords: probiotic, toxicology, safety, lactobacillus, bifidobacterium 1. introduction probiotics are biologically active preparations containing high concentrations of viable cells of bifidobacteria and lactobacilli as well as products of their metabolism that confer a health benefit to the host when administered in adequate amounts [1, 2]. there is growing scientific evidence for the number of beneficial effects of live cells of bifidobacteria and lactobacilli on a variety of gastrointestinal, immunological and metabolic conditions [3]. many strains have obtained the generally recognized as safe (gras) status or have been determined to be safe for their traditional food uses [4]. studies have also demonstrated that most lactobacillus species show no pathogenecity or acute oral toxicity to animals [5, 6] or humans [7, 8]. although there is a reasonable number of well characterized probiotic strains available for commercial use around the world [9, 10, 11], the isolation and characterization of new strains is still desirable. although traditional lactobacillus strains have an excellent history of safe use in the formation of dairy products and other foods and some have “generally recognized as safe” (gras) status [12], newly isolated organisms often have no previous history of food product use and do not necessarily share the gras status of traditional lactobacillus strains. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 110 therefore, it is necessary to conduct the safety assessment on any new strain or combination of strains prior to being added into foods or used as a dietary supplement [13]. in general, in vitro data alone is not sufficient to describe strains as probiotics, but they still remain valuable and can provide scientific insight into characteristics of probiotic organisms. therefore, the selection of suitable strains may be further refined by undertaking a series of in vitro experiments [14]. additionally, in vitro tests are still recommended by the fao/who [15] for an initial screening of potential probiotic bacteria. in addition to the traditional selection procedures, in recent years, knowledge on intestinal microbiota, nutrition, immunity and mechanisms of action has increased dramatically and can now be combined with genomic data to allow the isolation and characterization of new targetor site-specific probiotics [16]. in order for the newly isolated and characterized probiotic strains to be included in the composition of probiotic preparations, both in vitro and in vivo testings need to be conducted. the aim of the present work was to investigate in vivo the toxicogenity of a developed freeze-dried probiotic preparation containing lactobacilli and bifidobacteria in experimental animals mice. 2. materials and methods preparation of the material for toxicogenity testing. the probiotic strains lactobacillus delbrueckii ssp. bulgaricus nbimcc 3706, lactobacillus acidophilus nbimcc 2416, lactobacillus plantarum nbimcc 2415, bifidobacterium bifidum nbimcc 3601 were propagated in a fermenter, concentrated and freeze-dried in compliance with standard operating procedures (sop) and quality control (qc) procedures at probiotical s.p.a (novara, italy). microbiological analyses were done and bacterial culture purity was confirmed immediately after production, at study baseline and throughout the study. the strains were grown in the optimal medium for the optimal time needed for each strain to accumulate 1010cfu/cm3 and the single strain cultural suspensions were freezedried. the freeze-dried single strain products of the four probiotic strains lactobacillus delbrueckii ssp. bulgaricus nbimcc 3706, lactobacillus acidophilus nbimcc 2416, lactobacillus plantarum nbimcc 2415, bifidobacterium bifidum nbimcc 3601 were mixed in a ratio of 1 : 1 : 1 : 1 in order to obtain the material for toxicogenity testing. thus the toxicogenity of the four probiotic strains in the composition of the tested material can be determined simultaneously. toxicogenity testing – study design. the freeze-dried probiotic substance of lactobacilli and bifidobacteria (lactobacillus delbrueckii ssp. bulgaricus nbimcc 3706, lactobacillus acidophilus nbimcc 2416, lactobacillus plantarum nbimcc 2415, bifidobacterium bifidum nbimcc 3601), was examined in order to determine the concentration of viable cells of lactobacilli according to the iso 7889 and bs 10945-91 standards in sterile milk to determine the most probable number according to mccrady. the material for toxicogenity testing was diluted ten times in sterile, inert diluent. the toxicogenity analysis was conducted by parenteral (intraperitoneal) daily injection in white mice for a period of 14 days. the white mice were injected intraperitoneally with 0,5 cm3 of the suspension of the diluted material in one and the same place the inguinal canal – in order to obtain comparable results. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 111 experimental groups. the following groups of experimental animals were used in the experiment: 1. control group untreated 10 mice (5 male and 5 female); 2. experimental group treated with diluted material 20 mice (10 male and 10 female). both groups of experimental animals were grown in standard mode with experimental housing food and water ad libitum and were observed (general physical appearance, body weight, food consumption and abnormalities) daily for 10 days prior to the experiment. post-mortem examination. animal use was approved by iacuc. all mice were humanely sacrificed at the end of the test and a complete necropsy was conducted. after the experimental period post-mortem examination in „laboreks” ad laboratory „pathomorphology” was conducted. a macroscopic examination of the internal organs liver, spleen, kidneys and adrenals – was done. stomach, small and large intestine, spleen, liver, kidney, adrenals, peritoneum and omentum samples were histologically tested 10 days after the injection of lactic acid bacteria in the peritoneal cavity of the treated animals. after fixing the tissues in 4% neutral formalin the samples were incorporated into paraffin cubes. the obtained microscopic preparations were stained with hemalaun eosin. a transmission electronic microscopic examination on liver, mediastinum and omentum from control and experimental animals was conducted. the abdominal wall from the application site from the experimental animals was also observed. 3. results and discussion suitable strains of lactobacilli and bifidobacteria for incorporation in starter cultures for fermented milk products, probiotics and probiotic foods and drinks were chosen from the national bank for industrial microorganisms and cell cultures (nbimcc), bulgaria. they have the ability to reproduce in the model conditions of digestion, to synthesize lactic and other organic acids, bacteriocins, which inhibit the growth of pathogens, causing toxemia, toxic fungal infections [18]. they also allow the accumulation of high concentrations of viable cells in the processes of fermentation, immobilisation and freeze-drying and also retain viability under storage conditions. the experiment was performed using the prepared freeze-dried substance of lactobacilli and bifidobacteria (consisting of the strains lactobacillus delbrueckii ssp. bulgaricus nbimcc 3706, lactobacillus acidophilus nbimcc 2416, lactobacillus plantarum nbimcc 2415, bifidobacterium bifidum nbimcc 3601) for the production of probiotic preparations and the used biological objects were white mice. after ten-day monitoring, postmortem examination was conducted including macroscopic examination, histological findings and a transmission electronic microscopic (tem) study. there were no statistically significant differences in body weights and in weekly food consumption between the treatment groups and the control group. no treatment-related adverse effects and no deaths were observed. macroscopic examination the internal organs liver, spleen, kidneys and adrenals showed no visible changes under examination. their shape, size and color were unchanged. the stomach, the small and the large intestines were whitish pink, the serous was smooth and shiny. there were no obviously enlarged peyer's patches. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 112 peritoneum and the omentum had smooth surfaces without adhesion and nodule formation. histological findings the stomach, small and large intestine, spleen, liver, kidney, adrenals, peritoneum and omentum samples were histologically tested. the study of these eight organs was conducted 10 days after the infiltration of lactic acid bacteria in the peritoneal cavity of the treated animals. no changes in the parenchymal organs were established in all the animals included in the study (from both groups – experimental and control) except for the vascular changes indicating acute venous stasis (reaction to euthanasia). in the experimental group some changes in the serous and the subserous layer of the examined organs were observed proliferation of histiocytic elements and appearance of macrophages (fig. 1). fig. 1. tem: a fragment of a spleen and adipose tissue. magnification 25x. subserous proliferation of macrophages, some of which have the characteristics of giant macrophages. adipose tissue with more extensive proliferation of histiocytes and macrophages. the process was localized and it was less pronounced in the subserous, while larger clusters were found on the serous surface. in the adipose tissue of the peritoneum and the omentum, similar clusters of the described cellular elements were observed as well (fig. 2). fig. 2. tem: adipose tissue. treated group. magnification 4000. adipocytes, bacteria at different depths in the cell. the relatively poor bacterial cell proliferation is due to the rapid elimination of lactic acid bacteria. it is believed that this reaction is a response to the injected lactic acid bacteria (+ inert solvent, which would produce the same response). the observed changes are protective and physiological a „foreign body” reaction. in some animals of both sexes two types of bacteria were histologically established one morphological type of lactic acid bacteria probably cocci (fig. 3) and lactobacilli (fig. 4, fig. 5). fig. 3. tem: adipose tissue. tested group. magnification 4000. presence of cocci-shaped bacteria. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 113 most likely, these bacteria are the lactobacilli introduced but it is possible that some of them endogenously penetrate from the intestinal tract. fig. 4. tem: adipose tissue from the omentum.. magnification 63x. macrophages with ingested cocci in the cytoplasm. fig. 5. tem: adipose tissue. magnification 40x. presence of lactobacilli. electron microscopic examination the ultrastructural study was conducted on liver, mediastinum and omentum from control and experimental animals. the abdominal wall from the injection site from the experimental animals was also observed. the tissues were fixed in glutaraldehyde and samples were prepared for tem examination. liver there were no differences between control and experimental groups. the ultrastructure of hepatocytes nuclear apparatus, mitochondria, granular endoplasmic reticulum, smooth endoplasmic reticulum, golgi apparatus was unchanged. in some control and experimental animals tiny fatty droplets were found. no proliferation of kupfer’s cells in the sinuses was found and the µw were normal. bile ducts showed no pathological changes. a characteristic distinguishing feature of murine liver is the presence of multiple argirophilic granules in the hepatocytes (fig. 6a, fig. 6b, fig. 6c, fig. 6d). fig. 6a. tem: liver. control. magnification 4000. hepatocyte with a nucleus with two nucleolus. single fatty droplets, normal cellular ultrastructure. fig. 6b. tem: liver. control. magnification 4000. two border hepatocytes. many argirophilic granules. mediastinum, omentum in two of the experimental animals groups of two types fully preserved bacteria were found in the striae fat cells. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 114 fig. 6c. tem: liver. tested group. magnification 4000. four border hepatocytes, argirophilic granules, preserved ergastoplasm. fig. 6d. tem: liver. tested group. magnification 5000. a hepatocyte, a smooth sinus, unactivated kupfer’s cell. section of the abdominal wall the examination of the abdominal wall of the experimental animals showed no evidence of lactic acid bacteria (fig. 7a, fig. 7b). fig. 7a. tem: abdominal wall. magnification 9000. longitudinal cut. striated muscles without ultrastructural changes. fig. 7b. tem: abdominal wall. magnification 9000. transverse section. striated muscles without ultrastructural changes. it has been strongly recommended to confirm the safety of any newly isolated stain for developing combinations of strains before their incorporation into food products. as many lactobacillus and bifidobacterium strains have the gras classification due to their long history of safe use in food production (donohue and salminen, 1996), the studies demonstrated that most of the lactobacillus and bifidobacterium species show no pathogenecity or acute oral toxicity to animals [5, 6, 18] or humans [7, 8] which is consistent with our results. 4. conclusion during the whole ten-day experimental period of the tested freeze-dried substance of lactobacilli and bifidobacteria (lactobacillus delbrueckii ssp. bulgaricus nbimcc 3706, lactobacillus acidophilus nbimcc 2416, lactobacillus plantarum nbimcc 2415, bifidobacterium bifidum nbimcc 3601), the control and the experimental animals showed no changes in their behavioral responses. there were no pathological changes in the health status or the mortality rate. the histomorphologic and electron microscopic studies suggest the following conclusion: the only histomorphologic change was the physiological response of the tissue to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 zapryana denkova, ivan murgov, rositsa denkova, rositsa enikova, evaluation of the toxicological safety of a freeze-dried probiotic preparation, volume xiii, issue 2 2014, pag. 109 – 115 115 injection injury – a “foreign body” response. there was some evidence of local penetration of lactobacilli and cocci-shaped bacteria from the intestinal tract of the experimental animals, which were confirmed by electron microscopic observation. there was no evidence for parenchymal organ, peritoneum and omentum damages indicating toxicity. the overall conclusion for the freezedried substance of probiotic lactobacilli and bifidobacteria is the absence of toxicogenity. 5. acknowledgements the examined freeze-dried substance of probiotic lactobacilli and bifidobacteria was the basis for the development of a series of the probiotic preparations „enterosan”, bulgarian patent №103295, [19]. 6. references [1]. fao/who. health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. report of e joint fao/who expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. ftp://ftp.fao.org/es/esn/food/probio_report_en.pdf (2001). [2]. wohlgemuth s, loh g, blaut m, recent developments and perspectives in the investigation of probiotic effects. international journal of medical microbiology 300: 3–10 (2010). [3]. marteau p, probiotics in clinical conditions. clinical reviews in allergy and immunology 22, 255–273 (2002). [4]. efsa scientific committee, introduction of a qualified presumption of safety (qps) approach for assessment of selected microorganisms referred to efsa. efsa journal 587: 1–16 (2007). [5]. monose h, igara m, era t, fukuda y, yamada m, ogasa k, toxicological studies on bifidobacterium longum bb-536. pharmacometrics 17: 881–887 (1979). [6]. donohue d, deighton m, ahokas j, salminen s, toxicity of lactic acid bacteria. in: salminen, s., von wright, a. (eds.), lactic acid bacteria. (marcel dekker inc., ny) pp. 307–317 (1993). [7]. saxelin m, rautelin h, salminen s, makela p, safety of commercial products with viable lactobacillus strains. infectious disease in clincal practice 5: 331–335 (1996). [8]. kailasapathy k, rybka s, l. acidophilus and bifidobacterium spp. – their therapeutic potential and survival in yoghurt. australian journal of dairy technology 52: 28–35 (1997). [9]. ezendam j, van loveren h, probiotics: immunomodulation and evaluation of safety and efficacy. nutrition reviews 64: 1 – 14 (2006). [10]. reid g, jass j, sebulsky m, mccormick j, potential uses of probiotics in clinical practice. clinical and microbiology reviews 16: 658 – 672 (2003). [11]. santosa s, farnworth e, jones p, probiotics and their potential health claims. nutrition reviews 64: 265 – 274 (2006). [12]. donohue d, salminen s, safety of probiotic bacteria. asia pa cific journal of clinical nutrition 5: 25–28 (1996). [13]. jones m, martoni c, di pietro e, simon r, prakash s, evaluation of clinical safety and tolerance of a lactobacillus reuteri ncimb 30242 supplement capsule: a randomized control trial. regulatory toxicology and pharmacology 63: 313–320 (2012). [14]. reid g, the importance of guidelines in the development and application of probiotics. current pharmaceutical design 11: 11 – 16 (2005). [15]. fao/who. guidelines for the evaluation of probiotics in food. food and agriculture organization of the united nations and world health organization working group report. [16]. gueimonde m. & s. salminen, new methods for selecting and evaluating probiotics. digestive and liver disease 38: 242 – 247 (2002). [17]. denkova z, murgov i, slavchev a, selection of lactobacilli with probiotic properties. bulgarian journal of agricultural science 12: 689706 (2006). [18]. jia x, wang w, song y, li n, a 90-day oral toxicity study on a new strain of lactobacillus paracasei in rats. food and chemical toxicology 49: 1148–1151 (2011). [19]. denkova z, murgov i, enterosan probiotics: protecting the health of modern man. biotechnology & biotechnological equipment 19/1: 188 – 192 (2005). doi: https://doi.org/10.4316/fens.2021.039 371 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 371 379 the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera *anthony chukwuka mgbojikwe 1 , samuel k vihi 1 , adeola o. olanihan 1 , kingsley i. okeke-agulu 1 , jude o.okpara 2 1agricultural extension and farm management department, federal college of forestry, forestry research institute of nigeria, jos, plateau state, nigeria, anthonymgbojikwe@fcfjos.edu.ng 2federal college of animal health and production technology, national veterinary research institute, vom, plateau state, nigeria *corresponding author received 18th october 2021, accepted 27th december 2021 abstract: the purpose of this paper is to evaluate the effect of the aqueous leaf extract of moringa oleifera extract on coccidiosis of chicken. two hundred and forty (240) broiler birds were experimentally infected with eimeria tenella and treated with the aqueous leaf extract of m. oleifera at different concentrations (50mg/ml, 100mg/ml, 200mg/ml and 0%). the birds were divided into four groups t1, t2, t3 and t4 with three replicates in each group. t1, t2, t3 and t4 had percentage mortality rate of 16.7%, 8.3%, 5.0% and 91.7%, respectively. duncan count was carried out at the end of the experiment for the scoring of haemorrhagic lesions. analysis of variance (anova) showed that there is a significant difference (p>0.005) between the different levels of inclusion of aqueous leave extract of m. oleifera as was shown in the lesion score. from the study, it is suggested that m. oleifera has great potentials in being used to control coccidiosis in birds given that t3 had the lowest mortality rate while t4 (control) had the highest mortality rate. vitamin and mineral element analysis showed that m. oleifera contains vitamins a, b1, b2, b3, e and c. the minerals m. oleifera contains include calcium, copper, iron, magnesium, phosphorus, potassium and zinc. the phytochemical analysis and thin layer chromatography of the powdered leaf of m. oleifera revealed the presence of alkaloids, yohimbin; tannins, catechin and epicatechin; saponins, β-hederin; glycosides, kaempferol and quercetin; flavonoids, chlorogenic acid, hyperoside, rutosid, rahmnetin and isorahmnetin and terpenoids, oleanoic acid and β-sitosterol, which have all been shown to have retrogressive effect on microorganisms. keywords: coccidiosis, eimeria tenella, moringa oleifera, phytochemistry, thin layer chromatography 1. introduction avian coccidiosis remains one of the most common diseases of poultry industry all over the world. it constitutes an important wet season hazard to poultry farmers in nigeria [1]. coccidiosis caused by eimeria species cause huge economic losses in poultry and this includes the costs for treatment of birds, reduced productivity and losses due to mortality of birds [2]. since the introduction of various anticoccidial drugs and vaccines however, medications with these drugs have been fairly effective in preventing serious outbreaks of the disease; the reason for the emergence of resistant strains of coccidian parasites [3]. besides the scarcity of livestock drugs [1], access to anticoccidial drugs which is difficult especially to the rural poultry farmers hence the urgent need to develop alternative drugs for the purpose of curing coccidiosis in poultry. this necessitated the need to look into other ways of controlling this disease notably the use of herbs or plants, such as m. oleifera which has been acclaimed both http://www.fia.usv.ro/fiajournal mailto:anthonymgbojikwe@fcfjos.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 372 scientifically [4] and by traditionalists to have curative abilities for several diseases of both man and animals. therefore, the use of this plant extracts as medicine may alleviates these difficulties, as they are not natural products but may comprise new therapeutic molecules to which resistance has not yet developed [5]. 2. methodology 2.1. collection of moringa oleifera leaves the green leaves of m. oleifera was collected from the eto-baba, angwanrukuba area of jos, plateau state. the plant was identified using stock number ah05 in the herbarium unit of the forestry research institute of nigeria, federal college of forestry, jos. the leaves were then air dried at room temperature, grinded into powder and stored in a cool dry place. 2.2. preparation of plant extract the aqueous leaf extract of m. oleifera was prepared by dissolving the dried pulverized plant in ethanol at the ratio of 1:3, left to stand for 24hr at room temperature and filtered using whatman’s no. 1 filter paper. the filtrate was then poured into stainless steel plates and dried in hot water bath. the dried extract of m. oleifera was then stored in a dessicator. 2.3. preparation of test samples three (3) separate test samples were prepared by dissolving the dried extract of m. oleifera in distilled water (50mg/ml, 100mg/ml, and 200mg/ml), and stored in a refrigerator at 4oc for 48 hr. 2.4. phytochemical screening of powdered plant parts of moringa oleifera phytochemicals such as alkaloid, tannin, glycoside, resin, flavonoid and saponin were analyzed for based on standard acceptable scientific method [6]. 2.4.1. test for alkaloids 0.5g of the dried pulverized m. oleifera was dissolved 5mls of 1% aqueous hcl on a steam bath. this was filtered and 1ml of the filtrate treated with a few drops of draggendorffs reagent and 1ml of a second portion of the filtrate treated with wagner’s reagent; the formation of precipitates indicated the presence of alkaloid. 2.4.2. test for tannins 0.5g of the dried pulverized m. oleifera was dissolved in 10ml of distilled water. this was filtered and 2ml of 5% fecl3 added to the filtrate. a deep green coloration showed the presence of tannins. a second portion of the filtrate was treated with a 2ml of iodine solution. a faint bluish coloration confirmed the presence of tannins. 2.4.3. test for saponins 0.5g of the dried pulverized m. oleifera was measured, placed into a test tube, 5mls of distilled water added and shaken thoroughly. the formation of froth which persists on warming indicated the presence of saponins. 2.4.4. test for flavonoids 0.5g of the dried pulverized m. oleifera was measured, placed in a test tube and dissolved in 2mls of dilute naoh solution. three drops of concentrated h2so4 were then added. the appearance of a colorless solution indicated the presence of flavonoids. 2.4.5. test for resins 0.5g of the dried pulverized m. oleifera was measured and placed in a test tube and 5mls of boiling ethanol added. the solution was then filtered using a whatman’s no. 1 filter paper and the filtrate diluted with 4mls of 1% aqueous hcl. the formation of a heavy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 373 resinous precipitate indicated the presence of resins. 2.4.6 test for glycosides 0.5g of the dried pulverized m. oleifera was dissolved in 10mls of distilled water. the solution was filtered and 2mls of the filtrate hydrolyzed with a few drops of concentrated hcl. the mixture was then alkalized with a few drops ammonia solution. five (5) drops of this solution was added to 2mls of benedict’s qualitative reagent and boiled. a reddish-brown precipitate showed the presence of glycosides. 2.4.7 test for tannins a 2g of dried pulverized leaf of m. oleifera was treated with potassium ferric cyanide and ammonia solution. a deep red color indicated the presence of tannins. 2.5. chromatographic screening of phytochemicals in powdered plant parts of m. oleifera the phytochemicals that were qualitatively extracted were then assayed chromatographically using the solvent system ethylacetate-methanol-water in the ratio of 100:13.5:10 [7]. 2.5.1. preparation of chromatoplates for thin layer chromatographic analysis five (5) chromatoplates were thoroughly washed with water and allowed to dry. the plates were further cleaned with acetone and set on the plate holder. 50g of silica gel powder was dissolved in 110mls of deionized water, the mixture corked and fixed on an action wrist shaker, balanced with 50mls of water and shaken for 30 minutes. the mixture was then applied on the plates and drawn from one to the other with a spreader at a thickness of 0.25mm and allowed to dry for 30 min. 2.5.2. extraction of active principles from dried pulverized leaves of moringa oleifera. the phytochemicals identified were extracted and prepared for chromatographic screening using a standard acceptable scientific method [7]. i. alkaloids: 1g of the dried pulverized m. oleifera was mixed thoroughly with 1ml of 10% ammonia solution and extracted by shaking for 5 minutes with 5mls of methanol at 60°c on a water bath, filtered and the filtrate used for the chromatography. ii. saponins: 1g of the dried pulverized m. oleifera was shaken for 15 minutes with 20mls of chloroform and filtered. the filtrate was then evaporated to dryness and residue dissolved in 2mls of chloroform/methanol (1:1). the mixture was then used for the chromatography. iii. flavonoids: 1g of the dried pulverized m. oleifera was extracted with 10mls of methanol for 5 minutes on a water bath at 60°c and filtered. the filtrate was used for the chromatographic screening. iv. glycosides: 2g of dried pulverized m. oleifera leaf was extracted by heating for 15 min under reflux with 30 ml 50% ethanol, with the addition of 10ml 10% lead-(ii)-acetate solution. after cooling and filtration, the solution was extracted by shaking with three 15ml quantities of dichloromethane/isopropanol (3:2); shaking must be gentle to avoid emulsion formation. the combined lower phases were filtered over anhydrous sodium sulphate and evaporated to dryness. the residue was dissolved in 1ml dichloromethane/isopropanol (3:2) and used for chromatographic screening v. terpenoids: 1g of dried pulverized m. oleifera leaf was extracted for 15 min with 15ml methanol under reflux. the filtrate was evaporated to 3ml and 30µl was used for the chromatographic screening. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 374 vi. tannins: samples were prepared by diluting the crude extracts of chloroform, acetone, methanol and water with 1g of dried pulverized m. oleifera leaf and then applied using 1-10μl volumes to the origins of a tlc plate 2cm above its bottom with the help of capillary tubes. 2.5.3. activation of chromatoplates the chromatoplates were set in the hot air oven and activated at 110°c for 30 minutes, removed and allowed to cool to room temperature. 2.5.4. application of extracts on chromatoplates. each of the extracts were spotted on the chromatoplates on a line from the lower edge (origin) of the plates with a spacing of 2-3cm in-between. 2.5.5. saturation of the chromatographic tank the chromatographic tank was saturated for 1 hour with the developing solvent chroloform/ methanol (85:15) for alkaloids; chloroform/methanol/water (64:50:10) for saponins; n-butanol/glaciaacetic acid/water (40:10:50) for flavonoids and chloroform/methanol (95:5) for the tannins, respectively. the spotted plate was then placed in the chromatographic tank containing the solvent after petroleum jelly (vaseline) had been applied on its edges and the lid covered. when the solvents reached the score line or solvent front, the plate was removed and reactivated in an oven at a temperature of 110°c for 10 minutes. 2.5.6. visualization and identification of separated components the chromatographic film was set in the ultra violet light machine and viewed under the short wave length. the individual components were identified by their characteristic colors and their rf values. the rf values obtained were compared with those of known standard in a standard atlas. rf = distant moved by the solute distant moved by the solvent 2.6. vitamin and mineral element analysis various vitamins and some macro and micro elements were determined after ashing and digestion, using atomic absorption spectrometer (model dwaa4530f) according to the method described by the association of the official analytical chemist [8]. 2.6.1. ashing and digestion a 2g of the dried pulverized m. oleifera leaves were weighed into a crucible and ashed in a murfle furnace preheated to 600°c for 4 hours. the crucible was then transferred directly to a desicator and allowed to cool. the various ash from different parts of the plant were then separated and treated with a few millilitres of hcl, few drops of concentrated hno3 and boiled. these were then cooled and filtered. the filtrate made up to 10mls in a standard volumetric flask with deionized water. these solutions were used for the determination of cations. 2.6.2. determination of cations seven (7) cations; sodium, calcium, magnesium, potassium, phosphorus, selenium and lead were determined with the aid of atomic absorption spectrometer. the results were converted from ppm to mg per 100g using the formular: mg/100g = conc.(ppm)×soln.vol×100 10 wt. (g)2 1 2.7. experimental organisms the experimental organism (eimeria tenella) was sourced from the parasitology division of the national veterinary food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 375 research institute vom, plateau state nigeria. 2.8experimental birds two hundredand forty-day-old broilers chicks were brooded for three weeks within which routine management practices were observed. at three (3) weeks of age, the birds were divided into four (4) groups (t1, t2, t3 and t4) with three (3) replicates in each group. 2.8.1. experimental infection with eimeria tenella and treatment with the plant extract. challenge of birds with e. tenella was carried out at 30 days of age for two consecutive days and on observation of clinical signs, treatment with the plant extract commenced. to groups t1, t2 and t3 were administered 50mg/ml, 100mg/ml and 200mg/ml of the aqueous leaf extract of moringa oleifera for five (5) days, respectively. to group t4, no treatment was administered (control). 2.8.2. performance parameters. the birds were observed daily for clinical signs. morbidity and mortality were recorded daily in each sub-group. necropsies were carried out on the birds that died during the experiment. seven days after the challenge, the evaluation of caecal lesions was carried out on fifteen birds from each group. a lesion score was assigned from 0 to 4, where 0 corresponds to normal status with no gross lesion, 1 to small scattered petechiae, 2 to numerous petechiae, 3 to extensive petechiae and 4 to extensive haemorrhage that gives a dark color to the caecal intestine [9]. 2.9 statistical analysis data was subjected to analysis of variance (anova) using statistical package for social science (spssltd, working, surrey, uk). the significant treatment effect was discussed at probability significant means and was separated using the duncan’s new multiple range test and test was considered significant at a probability of p<0.05. 3. result and discussion m. oliefera has been reported to have a strong antioxidant effect against prostrated band skin cancers, an anti-tumor and an anti-aging substance [10]. furthermore, m. oliefera modulates anaemia, high blood pressure, liver and kidney problems. m. oliefera has strong anti-inflammatory properties and ameliorating rheumatism, joint pain, arthritis and lupus. the plant is effective against digestive disorders including colitis, diarrhea, and flatulence (gas), ulcer or gastritis. m. oliefera is an anti-bacterial and anti-viral agent; it is effective against urinary tract infection, detoxifer and outstanding immune builder [11]. it is used in many countries to treat malnutrition and malaria [11]. moringa oleifera is widely regarded by water purification experts as one of the best hopes for reducing the incidence of the water borne diseases. m. oleifera is claimed to be the most nutrient-rich plant yet discovered [12]. 3.1. phytochemical screening the dried powdered leaves of m. oliefera was subjected to phytochemical screening for various phytochemicals according to the method [6]. m. oliefera leaf contains tannins, saponins, flavonoids, glycoside and alkaloids highly present, and terpenoids moderately present (table 1). tannins is used in styptic preparations which produce contractions of blood vessels; stopping bleeding having the quality of retaining hemorrhages when applied to the bleeding part. saponins have the property of causing haemolysis of cells even at low dilution, tends to be deposited food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 376 on the surface of cells with which they come in contact and are not absorbed by the normal epithelium of the alimentary canal. the presence of saponin in the plant is a demonstration of the fact that the plant may have expectorant actions which are very useful in the management of inflammation of the upper respiratory tract in addition to its cardio-tonic properties as reported by [13] and [6]. alkaloid which represents the active principle of vegetable drugs, are alkaline in reaction and richly combine with acids, forming salts soluble in water. some drugs may contain more than alkaloid and the actions of these may be antagonistic to each other. alkaloid produces analgesic, antiinflammatory and adaptogenic effects which help to develop resistance against disease and endurance against stress [14]. glycosides have a tendency to block the conduction of the electrical impulse that causes contraction as it passes from the atria to the ventricles of the heart. cardiac gylcosides also have a tendency to produce an abnormal cardiac rhythm by causing electrical impulses to be generated at points in the heart other than the normal pace marker region, the cells that rhythmically maintain the heartbeat [15]. flavonoids are used as a supplement which reduces the symptoms of hemorrhoids. a number of flavonoids have been shown to have anti-inflammatory effects and to strengthen blood vessels. flavonoids have been investigated for possible antiinflammatory effects and anti-viral properties. chlorogenic acid is one of the most important flavonoids in the field of pharmacologyhas been shown to have antibacterial, antimutagenic, antitumor, and antiviral activities, plus antioxidant and clastogenic activities. the transisomer acts as an insect oviposition stimulant, and it may also reduce larval growth [16]. diverse functional roles of terpenoids have been critically studied and wellaccepted now. some of them include natural flavor additives for food or fragrances in perfumery and in traditional and alternate medicines used in aromatherapy. terpenoids have been extensively studied for its effect in the prevention and treatment of cancer. illustratively, taxol derivative (paclitaxel and docetaxel) are among the widely used drugs in cancer chemotherapy. other important therapeutic uses of terpenoids include antimicrobial, antifungal, antiviral, antihyperglycemic, antiinflammatory, antioxidants, antiparasitic, immunomodulatory, and as skin permeation enhancer [17]. table 1 phytochemicals found in m. oliefera leaves. phytochemicals concentration alkaloids +++ saponins +++ tannins +++ terpenoids ++ glycosides +++ flavonoids +++ key: = negative + = mildly present ++ = moderately present +++ = highly present 3.2. chromatographic screening of m. oleifera result after the chromatographic screening of the leaves of m. oleifera showed alkaloid, yohimbin with rf value 0.80; saponin, ᵦ-hederin with rf value 0.80; flavonoids, chlorogenic acid, hyperoside, rutosid, rahmnetin and isorahmnetin with rf values 0.75, 0.60, 0.39, 0.64 and 0.09, respectively; glycoside (cardiac) kaempferol and quercetin with rf values 0.12 and 0.63, respectively; tannin catechin and epicatechin with rf value 0.20 and 0.36, respectively; terpenoids oleanic acid and β-sitosterol with rf values 0.39 and 0.52, respectively; saponin β-hederin food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 377 with rf value0.80. the chromatogram did not indicate any spot for bitter principles (table 2). table 2 chromatogram of active principles present in m. oleifera leaf chemical group rf value possible identity alkaloids 0.80 yohimbin saponins 0.80 β-hederin tannins 0.20 0.36 catechin epicatechin ` terpenoids 0.39 0.52 oleanoic acid β-sitosterol glycosides 0.12 0.63 kaempferol quercetin flavonoids 0.75 chlorogenic acid 0.60 hyperoside 0.39 0.64 0.09 rutosid rahmnetin isorahmnetin 3.3. vitamin and mineral element analysis of m. oleifera leaf water soluble vitamins such as vitamins a, b1, b2, b3 and c were observed in both the fresh and dried leaves of m. oleifera. fat soluble vitamins observed include, vitamins a and e (table 3). the minerals m. oleifera contains include calcium, copper, iron, magnesium, phosphorus, potassium and zinc. it was observed that the concentrations of the vitamins were exponentially increased in the dried m. oleifera leaf which may be as a result of the loss of moisture content in the dried leaves. this result agrees with the result of [12], howbeit with variations in some of the figures recorded. these variations may be as a result of differences in the locations where the plants were harvested. [18] reported that the chemical composition of a plant species is affected by geographical location; factors such as soil type, soil nutritional content and climate have been implicated in altering the concentrations of chemicals found in plants. 0 500 1000 1500 2000 2500 fresh leaves (mg) dried leaves (mg) vitamin a vitamin b1 vitamin b2 vitamin b3 vitamin c vitamin e minerals calcium copper iron magnesium phosphorus fig. 1: vitamins and minerals present in m. oleifera leaf 3.4. statistical analysis from table 4, the null hypothesis was rejected and accepts alternative hypothesis (ha) because the figure calculated is greater than the figure tabled from the anova result. this indicates that there is a significant difference between the different levels of inclusion of aqueous leave extract of m. oleifera used in the experiment. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 378 table 4 analysis of variance (anova) of the degree of hemorrhage of birds, experimentally infected with emieria tenella sov ss df ms fratio ftab total treatment error 29.3 29.24 0.06 11 3 8 2.66 9.75 0.0075 1300 4.066 source: field survey, 2018. fcal>ftab 3.5. performance of birds infected with eimeria tenella and treated withmoringa oleifera table5 shows the mortality rate in percentage of birds infected with eimeria tenella. in treatment 4 (t4) which is the control, had the highest mortality of 91.7% while treatment 1 (t1) and 2 (t2) has 16.7% and 8.3%, respectively while treatment 3 (t3) had 5.0%; the lowest mortality rate. the result shows that the birds that were treated with 200mg/ml of the aqueous leaf extract of m. oleifera responded more positively to the treatment compared to other levels of concentration. mortality result showed that a farm can have a 100% mortality rate when there is a coccidiosis outbreak. table 5 percentage mortality of birds experimentally infected with eimeria tenella and treated with different concentrations of aqueous leaf extract of m. oleifera. treatments number of birds mortality percentage (%) mortality t1 (50mg/ml) 60 10 16.7 t2 (100mg/ml) 60 5 8.3 t3 (200mg/ml) 60 3 5.0 control (0mg/ml) 60 55 91.7 source: field survey, 2018. 4. conclusion from the experiment, it has been shown that m. oliefera has great potentials being used in the treatment and control of cocciodiosis of birds. the phytochemicals identified has been shown to have a wide range of health benefits which amongst others include antioxidant effects, modulation of detoxification enzymes, stimulation of the immune system, modulation of hormone metabolism and antiviral/ antibacterial effect. these phytochemicals have also been shown to help in slowing the aging process and reduce the risk of many disease [19]. the m. oleifera plant is the most inexpensive and credible alternative to providing good nutrition. m. oleifera is the most nutrient-rich plant yet discovered. m. oleifera is an extraordinary plant because all parts of the tree are edible, but the most amazing aspect is its exceptionally high nutritional value. m. oliefera provides a rich and rare combination of nutrients, amino acids, antioxidants, anti-aging and inflammatory properties used for nutrition and healing. the leaves of the m. oliefera tree are an excellent source of vitamin a (four times the amount in carrots), the raw leaves are rich in vitamin c (seven times the amount in oranges), and they are also a good source of vitamin b and other minerals. the leaves are also an outstanding source of calcium (four times the amount in milk), protein (twice the amount in milk), and potassium (three time the amount in bananas). the content of iron is very good as well and the leaves have purportedly been used for treating anemia in the philippines. the content of amino acids such as methionine and cystine is also high. carbohydrates, fats and phosphorous content are low making this one of the finest plant foods to found. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 anthony chukwuka mgbojikwe, samuel k. vihi, adeola o. olanihan, kingsley i. okeke-agulu, jude o.okpara, the evaluation of the anticoccidial properties of aqueous leaf extract of moringa oleifera, food and environment safety, volume xx, issue 4 – 2021, pag. 371 – 379 379 5. acknowledgements  the herbarium unit, federal college of forestry, jos plateau state, nigeria.  the parasitology division, national veterinary research institute, vom, nigeria.  biochemistry division, national veterinary research institute, vom, nigeria. 6. references [1] chah, k.f. and n.e. nweze 2001): antibiotics use in poultry production in nsukka, southeastern nigeria, and prod. 25: 303-306. [2] umaya, r. s. and parvatham, r. (2012). protective efficacy of moringa oleifera during aflatoxin exposure in broiler.res. j biotech, 2012; 7. [3] helmuth, r. and d. protz. (2004). how to modify conditions limiting resistance in bacteria in animal and other reserviours.clin. infect. and dis. 24 (suppl.): pp.136-139 [4] ola-fadunsins, d. and ademola i.o. (2013). direct effect of moringa oleifera lam (moringaceae) acetone leaf extract on broiler chickens naturally infected with eimeria species. trop anim health prod, 2013; 45:1423-1428. [5] adamu, m., and chaiwat b. (2013): protective effects of moringa stenopetala leaf supplemented diets on eimeria tenella infected broiler chickens in dedre zeit, central, ethiopia.kasetstart j. (nat. sci.) 47: 398-406. [6] trease, g. e. and evan, w.c. (2000). in: a textbook of pharmacognosy, 13th edition, bailiere tindall ltd; london. pp 795. [7] wagner, h., bladt, s. and zgainski, e. m. (1984). in: plant drug analysis. a thin layer chromatography atlas. berlin heidelberg springer-verlag. 319 pp [8] aoac (1975): official methods of analysis. association of official analytical chemists. washington, d.c, usa, pp 450 [9] johnson j. and reid w. m. (2001). anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chicken, exp. parasitol. 28 (1970) 99-102. [10] osila, k. (2012). harnessing the potentials of moringatree.posted in the tide news online, feb 8th, 2012. [11] dahot, m. u. (2003).antimicrobal activity of small protien of moringa oleifera leaves. j. islam. acad. sci., 11:1, 27-32. [12] ram, c. d., sheo, d. m., brijendra, k. p., narendra, b. and manisha, g. (2011). moringa: the herbal gold to combat malnutrition. chronicles of young scientists. vol. 2. issue 3. https://www.researchgate.net/publication/2164631 90_moringa_the_herbal_gold_to_combat_malnutr ition [13] frantisek, s. s. (2002): the natural guide to medicinal herbs and plants. tiger barks institute twickenham, u.k. pp. 21-70. [14] gupta, s.s. (2007). prospects and prospective of natural products in medicine indian journal of pharmacology, 26:1-12 [15] clark a.m. (2009). national products as resources for new drugs, pharm. res., 3, 113311411 [16] jeffery, b. h. and herbert, b. (1993). phytochemical dictionarya hand book of bioactive compounds from plants. taylor and francis limited pp. 475. [17] priyanka, p. b. and pathik s. b. (2013). terpenes: chemistry, biological role, and therapeutic applications. natural products pp 2665-2691. [18] mgbojikwe, l. o. (2000). acaricidal properties of the aqueous stem bark extract of adenium obesum. pp.151-152. [19] palombo, e. a. (2006). phytochemicals from traditional medicine plants used in the treatment of diarrhoea: mode of action and effects on intestinal function.phyother research. 2006 sep, 20 (9): 717–24. https://www.researchgate.net/publication/216463190_moringa_the_herbal_gold_to_combat_malnutrition https://www.researchgate.net/publication/216463190_moringa_the_herbal_gold_to_combat_malnutrition https://www.researchgate.net/publication/216463190_moringa_the_herbal_gold_to_combat_malnutrition issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xxi, issue 4 31st december 2022 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania doi: https://doi.org/10.4316/fens.2022.011 107 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 107 115 use of artificial neural networks for modeling inflow and outflow and salinity of lake fetzara in the region-annaba (ne algeria) *zahra bouhali 1 , larbi djabri 1 , hamza bouguerra 1 , fatma trabelsi 2 , azzedine hani 1 , hicham chaffai 1 1 faculty of earth sciences, badji mokhtar university, annaba, algeria. zara.bou5@gmail.com 2 medjez elbab engineering school, djendouba university. tunisia *corresponding author received 8th december 2021, accepted 25th june 2022 abstract: lake fetzara is one of the important lakes in the northeast of algeria; the water supplying this lake comes from different precipitation and wadis. moreover, meboudja wadi constitutes the drainage channel. the water of the lake and the underlying groundwater is exposed to excessive overuse; which seriously threatens the hydrological and ecological balance. the overexploitation is explained by the increase in water mineralization, which poses a risk of soil salinization. to this end, this article deals with the subject of current salinity and predict its evolution over time by means of the modeling of the artificial neural network (ann), according to the period of low water and the period of high water. the ann were trained using three different algorithms: the scaled conjugate gradient back propagation (scg) algorithm and one step secant back propagation (oss) algorithm and quasi-newton algorithm (bfgs). the performance results indicate that the three algorithms provided satisfactory simulations according to the determination coefficient (r2) and the performance criteria of the mean square error (rmse), with priority to the bfgs algorithm; where the coefficient of determination using the bfgs algorithm varies between 69.5% and 95.3%. the bfgs method presents better results in order to design appropriate institutional mechanisms, capable of leading to the protection of the quality of these resources essential to the promotion of sustainable development. keywords: inflow, outflow, drainage, salinity, oued, water table, artificial neural network. 1. introduction water is necessary element for the life of human beings, fauna and flora. the quality of groundwater has been degraded due to climate change, the intensive use of fertilizers, domestic wastewater and various discharges from factories rejected directly into the streams; particularly in the wadis feeding the lake fetzara. [1] the hydrographic network of the lake fetzara is composed of four streams, which are: the mellah stream, with a length of nearly 8 km, the el hout stream, with a length of 10 km, the zied stream, with a length of about 10.5 km and finally the bou messous stream which has a length of 9 km. all these streams flow into fetzara lake with a very irregular regime, torrential in winter and dry in summer. [2] the degradation of quality and quantity threatens hydrological and ecological balance. the increase of water mineralization leads to the risk of soil salinization. [2] to this end, this article deals with the subject of current salinity and predict its evolution over time by means of the modeling of the artificial neural network (ann) according to the period of low and http://www.fia.usv.ro/fiajournal mailto:zara.bou5@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 108 high water and weekly measurement of the flow of wadis along two hydrological cycles. regarding the provision of water resources and the management of application, it is undeniable to predict the quality of groundwater, various factors contributes to the variation in water quality. their uncertainly inherent in weight, as more than one variable effects water quality. the inhomogeneity the environment reflect the prediction of quality and approaches into complexity. the salinity parameter is one of the fundamental parameters for potable and agricultural water. [3] this is directly related to the conductivity of the water and its dissolved salt (sodium chloride, magnesium chloride, magnesium sulfate, etc.) and the quality of potable water. salinity was examined in this study as such a suitable approach to examine the behavior of groundwater by model system; it is necessary to know the mechanism of quality fluctuations over time in order to have an idea about the situation of the water table and the quantity of accessible water. [4] artificial neural network are “the black box” non-linear mathematical models capable of establishing relationships between inputs and outputs of a system.[5] ann is considered practical substitutes for regression and empirical models to predict the behavior of water ressources; due to their temporal reliability and adaptability to unforeseen changes. these are applied not only in qualitative forecasting, but also in forecasting the situation and the volume of groundwater, much of the modeling has been done in this regard. [6] as for prediction based on the neural network, [6] 2. materials and methods 1.2. description of study area lake fetzara is cosedered as wetland. its location is about 18 km southwest from the chief state of annaba (daïra and municipality of berrahal), it covers 18.600hectares. [7] several small tows border it: to the north, the municipality of berrahal, to the south the municipalities of eleulma (crosseb by elhout wadi) and cheurfa, to the east the small villages of elgantra and zied wadi. (figure 1) lake fetzara has been listed on the ramsar list of wetlands of international importance since 2002. its large extent and relatively temporary nature make it a representative wetland of the mediterranean region [7]. the hydrological function of the lake contributes to flood control, as well as to the recharge of the water table. lake fetzara stretches 17km length and 13km width. the open water area, which depends almost exclusively on the intensity of rainy season, occupies an area of more than 5800hectares. in addition, there are several thousand hectares of flood plains forming vast wet meadows. [7] 2.2. analytical method to ensure rigorous monitoring of the quality and quantity of water entering and leaving the lake, weekly monitoring of the flow of the wadis during two hydrological cycles (2016/17) and (2017/18) as well as seasonal piezometric campaigns has been realized. for sampling, the sites chosen are in near-urban areas and nearby the industrial zone of the municipality of berrahal. the number of the wells selected is 39 wells. in-situ parameters were measured using multi-parameter band hanna instruments inc (made in romania). the main elements presents in the water analyzed using a metrohm 883 basic ic plus ion chromatography apparatus (made in courtaboeuf, france) with a conductometric detector. prior to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 109 their chemical analysis. the water samples are filtered through a nylon membrane (0.22 µm). fig. 1. geographic location of fetzara lake in the wilaya of annaba. algeria. (design by p. pentsch) [8] the mobile phase used is mixture of nahco3 (168mg) and na2co3 (678mg) dissolved in two liters of ultra-pure water. the injection rate is 0.7ml/min and the volume injected is 20 µl. the chromatograph was calibrated using five standard solutions containing a concentration of cations or anions ranging from 1 to 50mg/l. the prediction of salinity of groundwater is done using three different artificial network algorithms. 2.3. data analysis the data set is arranged into two layers: the first one is the training part (72% of the data) the second one is used for model validation (28% of data). several tests were made to ensure that all training and validation modules were examined in a trial and error manner, in order to obtain better results. in order to detect model perturbation, a portion of data is used for training and another portion is reserved for testing the performance of the model in order to decide to stop training with optimal hidden nodes [9]. this stop is made when the model validation error starts to increase. [10] the conductivity is directly proportional to the water mineralization; [11] for this food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 110 purpose, we have seven descriptor parameters: conductivity, chloride, sulphates, bicarbonates, calcium, magnesium and sodium. the sampling date is from the 08/11/2016 to 15/05/2018. simple descriptive statistic of the raw data is shown in (table 1). the correlation corresponding to the statistical data collected from the sampling of 39 wells in the region surrounding lake fetzara and the wadis that feed it, shows that the most significant correlation with the conductivity parameter is observed among the other parameters, in which the correlation was established through the software statistica 8 (table 2) table 1. statistical description of parameters. variables training dataset validation dataset min max mean sd cv min max mean sd cv ec [μs/cm] 281 6800 1789.75 1225.67 0.68 276 2440 1093.43 621.71 0.57 cl [mg/l] 85.2 2996.2 675.02 523.60 0.78 35.5 1065 402.02 268.04 0.67 so4 2 [mg/l] 19.2 725.3 231.11 133.25 0.58 19.2 528 171.29 106.88 0.62 hco3 [mg/l] 23.18 1284.53 357.88 209.51 0.59 34.77 614.88 301.55 140.96 0.47 ca 2+ [mg/l] 33.6 672 167.33 106.71 0.64 43.2 348.8 139.36 77.89 0.56 mg 2+ [mg/l] 1.92 886.4 63.87 89.85 1.41 4.8 176.64 32.38 30.24 0.93 na + [mg/l] 46 2622 382.33 390.66 1.02 23 575 196.02 126.12 0.64 selection of the input combination: table 2. correlation matrix (pearson) between variables. variables ec cl so4 2 hco3 ca 2+ mg 2+ na + ec 1 0.941 0.474 0.500 0.748 0.465 0.789 cl 0.941 1 0.409 0.407 0.746 0.464 0.776 so4 2 0.474 0.409 1 0.216 0.359 0.296 0.425 hco3 0.500 0.407 0.216 1 0.274 0.331 0.504 ca 2+ 0.748 0.746 0.359 0.274 1 0.250 0.492 mg 2+ 0.465 0.464 0.296 0.331 0.250 1 0.419 na + 0.789 0.776 0.425 0.504 0.492 0.419 1 different combination of inputs used in the modeling: no. combination 01 cl 02 cl& na+ 03 cl-, na+ & ca2+ 04 cl-, na+, ca2+ & hco3 05 cl-, na+, ca2+, hco3 & so4 2 06 cl-, na+, ca2+, hco3 -, so4 2& mg2+ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 111 training algorithms the performance of the neural artificial network was constructed in order to determine which algorithm would be the most effective in predicting the salinity of groundwater in the region by the following algorithms: bfgs: quasi-newton back propagation (brayden 1970; fletcher 1970; goldfarb 1970; shanno 1970). scg: scaled conjugate gradient back propagation oss: one-step secant back propagation 3. results and discussion in this research, we created a model to predict the total variables of groundwater salinity based on different water quality states of lake fetzara and the underlying aquifer. the artificial neural network is used to estimate the relationship between different variables. the results of the prediction show that the neural network approach has good and wide applicability for modeling the salinity of waters of the region. (figure 2) presents the calibration of predicted salinity according to the bfgs, scg and oss algorithms with the observed salinity by anno5. . (2a) (2b) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 112 fig. 2c. fig. 2. observed and predicted salinity ''ec'' according to bfgs (2a), scg (2b) and oss (2c) algorithms using ann05 in the validation phase. the (figure 3) presents the calibration of the values of observed salinity simulated by the bfgs, scg and oss algorithms and those of the predicted salinity by anno6 for each water point in the validation phase. the error graph indicates the observations of the observed salinity values with the predicted salinity values in the trainig phase (figure 4). note that r2 is a measure of the rate of explanation of phenomenal reality by the model adopted. the predicted salinity versus the observed salinity with the best improvement of the region’s water is well presented in anno6. note that r2 is around 0.9 for calibration and validation, which proves that the model retains the statistical characteristics linked to the values observed with almost unchanged coefficients of variation. by comparing between the model’s values by ann with experimental data (table 3) reveals that the mlp model (bfgs) (provides values: r2=0.99, rmse= 114.98 for the training phase and r2=0.95, rmse=138.41 for the validation phase) is the best model. in this study we use the artificial neural network to estimate groundwater salinity based on other quality analisis parameters. 4. conclusion lake fetzara is the main source of irrigation for the surrounding town. [13] the deterioration of the supply and the quality of its waters can cause irreparable damages to the environment. monitoring to predict future conditions needs precise models. these tasks are the foundation for planning to conserve and manage critical resources. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 113 fig. 3. observed and predicted salinity ''ec'' according to bfgs, scg and oss algorithms using ann06 in the validation phase. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 114 fig. 4. observed versus predicted salinity by bfgs, scg and oss algorithms using ann05 and ann06 models for training phase. this study consists of the use of three autonomous hybrid algorithms to determine and predict the salinity of the lake and the water of the underlying aquifer. we used the method of artificial neural network: multilayer perceptron (mlp) with three algorithms (bfgs, scg and oss). the collected weekly flow measurement and physico-chemical analyzes of the water over two hydrological cycles were used to construct several sets of combinations of variables as inputs for the model construction and evaluation. qualitative and qualitative criteria were applied to validate and compare the models. the results reveal that the most suitable model for modeling the salinity of groundwater in a future studies is the mlp (bfgs), where the rmse=114.98 and the coefficient of determination r2=0.99 for the training phase and rmse=138.41, r2=0.95 for the validation. the analysis of the data acquired and the impact of the environmental factors show the qualitative and quantitative degradation of the inputs water of the lake. to this end, it is recommended to carry out geophysical studies to determine the extension of the underlying aquifer and to carry out regular measurement campaigns in order to encourage organic farming in vulnerable sectors. 5. acknowledgment we would like to thank dr lamine sayad monitor at badji mokhtar university, annaba, algeria, for his support and guide. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 zahra bouhali , larbi djabri, hamza bouguerra, fatma trabelsi, azzedine hani, hicham chaffai, modeling inflow and outflow and salinity of lake fetzara (ne algeria),food and environment safety, volume xxi, issue 2 – 2022, pag. 107 – 115 115 6. references [1]. habes s, djabri l, hani a, bouhsina s & mudry j. quantification of inputs and dewatering of a lake: case of the fetzara lake. north-east of algeria, rev. sci. technol., synthèse (2011) [2]. habes s, djabri larbi., chemical characteristics of a lake belonging to the humid ecosystems of the north algeria, case of the fetzara lake. north-east of algeria. phd thesis in hydrogeology, faculty of earth sciences, department of geology, badji mokhtar university, annaba. algeria. (2013) [3]. boudjemaa s., mapping of soilvegetation relationships in a salty environment (fetzara lake), magister thesis, faculty of sciences, department of biology. badji mokhtar university. annaba. algeria, (2010) [4]. hakim khelfaoui., hicham chaffai., azzedine hani., rabah laouar., impacts des rejets industriels sur les eaux de la région de berrahal (nord est algérien), rev. sci. technol., synthèse. (2012) [5]. p. abbasi maedeh, n. mehrdadi, g.r. nabi bidhendi, h. zare abyaneh, application of artificial neural network to predict total dissolved solids variations in groundwater of tehran plain, iran. , international journal of environment and sustainability, (2013) [6]. hani a., lallahem s., mania j., djabri l, the use of finite difference and neural network models to evaluate the impact of underground water overexploitation”. hydrol process 20:4381– 4390. doi:10.1002/hyp.6173. (2006) [7]. boumezbeur a., fact sheet on ramsar wetlands, chott zaherz chergui general directorate of forests. (algeria), (2003) [8]. http://journals.openedition.org/mediterrane e/docannexe/image/8077/img-2.jpg [9]. brown r. et al., transitioning to water sensitive cities: historical, current and future transition states, 11th international conference on urban drainage, united kingdom. (2008) [10]. braddock rd., kremmer ml., sanzogni l., feed forward artificial neural network model for forecasting rainfall, environmetrics journal. (1997) [11]. bouguerra hamza, tachi salah-eddine, derdous oussama, bouanani abderrazak, khanchoul kamel. suspended sediment discharge modeling during flood events using two different artificial neural network algorithms, springer international publishing, (2019) [12]. kaveh ostad-ali-askari., mohammad shayannejad., hossein ghorbanizadeh-kharazi., “artificial neural network for modeling nitrate pollution of groundwater in marginal area of zayandeh-rood river, isfahan, iran “, ksce journal of civil engineering. (2017) [13]. mellouk k, aroua n., the fetzara lake, a fragile wetland threatened by the urban growth of annaba city (north-east algeria) (2015) [14]. kahoul m., derbal n., alioua a., ayad w., assessment of the physical-chemical quality of well water in the region of berrahal (algeria), larhyss journal. (2014) [15]. bachir sakaa., hicham chaffai., azzedine hani., ann’s approach to identify water demand drivers for saf-saf river basin, journal of applied water engineering and research. (2020) [16]. salim heddam & ozgur kisi, extreme learning machines: a new approach for modeling dissolved oxygen (do) concentration with and without water quality variables as predictors, springer. (2017) javascript:; javascript:; javascript:; javascript:; javascript:; 1. introduction 275 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 275 286 aquifer suceptibility and agricultural activities effect on water quality in semiarid zone: el ma labiod plain, north east of algeria *samoun fatah 1 , layachi gouaidia 2 , chemsedine fehdi 1 , mohamed laid hemila 2 and larbi djabri 3 1 water and environment laboratory (lee), 2faculty of natural and life sciences, larbi tebessi university, route de constantine, tebessa, algeria 12002: fatah.samoun@univ-tebessa.dz*, gouaidial@yahoo.fr, fehdi@yahoo.fr , hemilamohamedlaid@yahoo.fr , 3department of geology, badji mokhtar university – b.p.12, annaba, 23000 algeria: djabri_larbi@yahoo.fr *corresponding author received 22th july 2020, accepted 29th december 2020 abstract: for more than three decades, many agrarian systems have been intensified in an attempt to reduce the widening gap in food deprivation. the concern to feed the population has meant that the consequences have not been well studied, especially the degradation of groundwater, which was not yet among the issues discussed in developing countries. the study of piezometric measurements shows that the groundwater level of the plain of el ma labiod decreases on average by one meter per year. the chemical analysis shows that few of the 40 representative water points sampled, in the study area, exceed drinking water standards for nitrate and chloride concentrations, making 5 wells unsuitable for drinking water production and 8 others unfit for human consumption without prior treatment. it was found that nitrate was the main component that degrades the quality of groundwater. the application of the drastic and god methods to estimate the susceptibility of aquifers has made it possible to highlight three classes of vulnerability from very low to moderate. the mapped results using gis data and arcgis tools show a large similarity in the spatial distribution of vulnerability classes and the alteration of water quality. the purpose of this study is to take stock of the state of groundwater and create a basic document for decisionmaking to ensure healthy environment development in the study area. keywords: el ma labiod, groundwater vulnerability, drastic, god, arcgis, water quality seqeau. 1. introduction food needs are the main factor which induces the extension and intensification of agricultural practices; intensification which is done by providing enough water for irrigation and non-organic nitrogen fertilizers and pesticides. following a spread, the fraction of pesticides and fertilizers that reaches the ground contaminates the groundwater with water infiltration [1]. contamination is defined as the presence of something harmful in food or drink that creates a risk of illness, injury or discomfort [1]. the increasing urbanization, industrialization, agriculture and climate change make the quantity and quality of water available rapidly diminishing [2]. indeed, in the semiarid zone, where surface http://www.fia.usv.ro/fiajournal mailto:fatah.samoun@univ-tebessa.dz mailto:fatahabder@gmail.com* mailto:fatahabder@gmail.com* mailto:gouaidial@yahoo.fr mailto:fehdi@yahoo.fr mailto:hemilamohamedlaid@yahoo.fr mailto:djabri_larbi@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 276 water is very insufficient, if not null, the irrigation water and all other needs are withdrawn from a groundwater reducing its reserves by overexploitation. this contributes, in the long term, to the depletion of the water resource [3] so the knowledge of regional hydrogeological circumstances [4] becomes a priority. phosphorus and nitrogen-based pollutants are a persistent problem of surface and groundwater pollution in rural and urban environments, from point and diffuse sources. diffuse sources are mainly of agricultural origin [5]. all groundwater is vulnerable! [6]. therefore, agricultural system must be continually evaluated and corrected during this transitional period which lasts. the compromise "food production environmental impact" must not trample health standards, especially those relating to the air near homes or to water intended for human and animal consumption. drinking water becomes increasingly scarce under inefficient management in a semiarid environment [7], thus it is imperative to protect the public health by ensuring water and its housing environment safety [8]. the awareness of the vitality of this resource imposes some precautionary measures the first of which is the evaluation of the current quality of groundwater and the vulnerability of the aquifer. this study was undertaken due to the lack of recent studies on the effects of strengthening agricultural practices on the quality and quantity of groundwater in the study area. the last study dates from 2006 [9]. the algerian legislation concerning the standards of drinking water is, apart from a few elements, very close to the lists of those issued by the who [10, 11]. the aim of this paper is to check the water quality, applying the “seq-eau” (water quality assessment system) to 40 representative sampled wells in the studied area and to compare its spatial distribution with the vulnerability map of the aquifer under el ma labiod plain. groundwater vulnerability maps are expected to draw the attention of land-use planners and decision-makers to the effects to groundwater of human activities on the soil [12] and allow the district to readily assess potential groundwater quality impacts of future changes in land use [13]. the analysis of groundwater is centered on its components, as they are the main indicators of the social and environmental problems [14] and the aquifer vulnerability assessed by two intrinsic overlaid methods is mapped using arcgis tools in gis environment. 2. matherials and methods 2.1 presentation of the study area: el ma labiod plain is situated at 15 kilometers south of tebessa-town in the northeast of algeria. it is limited by the parallels 35°05’ and 35°25’ north and by meridians 7°57’ and 8°25’ east (figure1). the studied watershed covers 582.33 square kilometers. the elevation from sea level goes from 1650 m in the north west (dj. doukane) to 980 m at the outlet. the plain presents an average slope of 3%. it has a semiarid climate and an enough developed drainage which streams feed the big watershed of melghigh which concentrates southward. the studied zone is an unconfined covered aquifer in a negative form with a miocene filling of sand and clayey sandstone in abnormal contact with a cretaceous border. the alluvial deposit thickness varies from 0 to 10 meters [9]. the geo-electric study titled “geophysical prospecting of the el ma labiod basin” carried out by ugf brno (geophysical and drilling unit, non-destructive reconnaissance office) in 1971 for the state secretariat of hydraulics. (unpublished report). geophysical unit, archives of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 277 anrh tebessa, shows two pairs of faults which limit the aquifer area in the plain. the lithology and the structural built share the plain in water-bearing and non-waterbearing zones and give the watershed varied hydrogeological characteristics. with almost 400 wells and drillings the wound is exploited in more than 25 million of cubic meters per year [15]. the aquifer of el ma labiod provides the sole resource to cover all water needs (drinking, industrial and irrigation) for more than 70% of the population of tebessa region. fig.1 geographic setting of study area the plain of el ma labiod presents a negative water balance despite the reported contributions from the karstified limestone of the north east and south west limits as in [9] confirmed by boughanem m. in his master work (2011). referring to data from the last three decades, the piezometric level decreases, in the study area, with an average of 1m per year. 2.2. aquifer vulnerability assessment: both of used methods; drastic [16], [17], [13] and [18] and god [10], [19], [20] and [21] have been widely applied around the world and became notorious. the requested parameters for the drastic method and the god method, their sources, their mode of acquisition, the formulas to calculate the vulnerability indexes and their mapping modes are summarized in the respective operating diagrams figure 2 and figure 3. 2.2.1. drastic method: drastic is the acronym formed by the initials of the seven parameters required by the method [16]: “d” for depth to water table, “r” for net recharge, “a” for aquifer media, “s” for soil media, “t” for topography, “i” for the impact of unsaturated zone and “c” for hydraulic conductivity. the drastic groundwater vulnerability index (drastic gvi) is obtained by summing the parameters as following: drastic gvi = dr.dw + rr.rw + ar.aw + sr.sw + tr.tw + ir.iw + cr.cw or: food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 278 (1) fig.2 the operating diagram of drastic method where: “i” ϵ {d, r, a, s, t, i, c} the seven parameters. “ir” and “iw” are respectively the rated and the weighted parameters in each measured area, the first evaluates the state of the parameter on a scale of 1 to 10 and the second represents the weight assigned by the authors to each parameter according to its importance in the drastic gvi formula (1). 2.2.1. god method: god is the acronym formed by the initials of the three required parameters [19]; [20]: “g” for groundwater occurrence, “o” for overall lithology; aquiperm – aquitard and “d” for depth to water table. the god vulnerability index (god gvi), is obtained by multiplying the three parameters between them as in formula (2) [19]. (2) where: cg, co and cd are the respective rates assigned to parameters g, o and d. its map is shown in figure 7b. 2.3. data acquisition: each datum was acquired at its source: the depth to the water table “d” was directly measured with a piezometric probe. the net recharge “r” was calculated applying the formula (3) of tixeront berkaloff [9] in thornthwaite's water balance for four decades (1975 to 2015): (3) with: -r as a net recharge, -p as precipitation, -etp as a potential evaporationtranspiration and -etr as a real evaporation-transpiration. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 279 the saturated media and unsaturated aquifer media “a” and “i” were evaluated applying authors’ tables [16] according to their lithology using logs of wells and drills from archives of national agency of hydraulic resources (anrh) and water fig.3 the operating diagram of god method direction of wilaya (dew) and geoelectric data from anrh archives. the soil media “s” was directly evaluated, supported by land use scheme obtained from the local subdivision of agriculture. the slope (topography) "t" map was prepared with arcgis using a global mapper-v18 exported shape file of an strm gdem v2 worldwide elevation data (1 arc-second resolution). the hydraulic conductivity “c” was determinate through a falling head permeameter [22] with 12 samples of materials extracted during new drilling and from other representative points applying the formula (4): (4) where: -t1 is the necessary time that water goes from level “h0” to “h1” in the tube with diameter “a” through the cylinder with “a” diameter and “l” thickness. the coordinates of the water points were determined by garmin “nuvi” personal gps, and confirmed by an imagery world satellite image downloaded through "popular sources" then reported on a map at 1/50.000. 2.3.1 the drastic gvi assessment: the six parameters d, r, a, s, i and c: the collected data compiled under excel, were rated and weighted [16], then interpolated and cut through the study area limits by arcgis tools in 2d analysis. (figures: 4a, 4b, 4c, 4d, 4f and 4g.). the “t” slope map (figure 4e) was directly drawn by the “slope” function in 3d analysis of arc map using the tin created with global mapper v. 18 tools. table 1 shows the drastic parameters ranges in field their rates and weights. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 280 the limits of the study area, data required to download image and elevation data map, were extracted from the topographic map of el ma labiod sheet n° 235. the drastic vulnerability index map was obtained by applying the formula (1) with maps of rated parameters as shown in formula (5): (5) where: m is used for mapped item. table 1 the rated and weighted parameters of the drastic method. parameter state and measures rates weighted rates depth “d” 18 – 30,5m and d >30,5 m 1 – 3 and 6 5 to 15 and 30 net recharge “r” 10 mm/y 1 4 aquifer media “a” sand, gravel, clayey sandstone, marl and limestone 3 to 10 9 to 30 soil media “s” thin, sand, gravel and clayey 7 to 10 14 to 20 slope “t” 0 18% 3 to 10 3 to 10 vadose zone media “i” sand, gravel, clayey sandstone, marl and limestone 7 to 10 28 to 40 hydraulic conductivity “c” (0.13 and 65) *10-5 m/s 1 to 6 3 to 24 the seven maps were summed cell-by-cell [16] using the “raster calculator” of "map algebra” in “spatial analyst tools” of arcgis 10.3.1. (figure 2). the table 2 shows the ranges for the drastic vulnerability index classification [23]. table 2 drastic vulnerability index ranges for classification value 0 – 75 75 – 100 100 125 125 – 150 150 – 204 state very low low medium high very high 2.3.2the god gvi assessment: table 3 shows the ranges of classification for the groundwater vulnerability index assessed by god method [19]. table 3 the rated parameters of god method parameter rate ranges state rated process (g)groundwater occurrence 0 – 1 unconfined (covered) 0.6 – 1 field and well logs (o) overall lithology 0.4 to 1 sands, gravel, sandstones and limestone 0.5 – 0.7 well logs and geophysics sheets (d) depth to water table 0.4 to 1 28,6 m – 82,8 m 0.5 – 0.6 piezometric probe the god gvi map was produced by similarity to the drastic gvi map using the formula (6) as shown in figure 3: (6) where: m is used for mapped item. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 281 table 4 shows the classification ranges of god gvi [19]. water quality assessment: the seq-eau system (water quality standards for natural water bodies) used to estimate the quality of groundwater in the plain of el ma labiod (groundwater quality assessment system) adopted by abh (hydrographic basin agency) for the constantine region, seybousse and mellègue [24]. table 4 god vulnerability index ranges classification. value 0 0 0.1 0.1 0.3 0.3 0.5 0.5 0.7 0.7 – 1.0 state none negligible low moderate high extreme table 5 shows the ranges for eachparameter for classification of water quality according to seq-eau abh (constantineregionseyboussemellegue). table 5 the parameters and classes of seq-eau deterioration by mineralization: parameters unit conductivity µs/cm [180 – 400] ] 400 – 2500] <180 or ]2500 – 4000] >4000 chlorides mg/l <25 200 / >200 sulfates mg/l <25 250 / >250 deterioration by nitrogenous materials except nitrates: parameters unit ammonium mg/l <0,05 0,5 4 >4 nitrites mg/l <0,05 0,1 0,7 >0,7 deterioration by nitrates: parameters unit nitrates mg/l <25 50 100 >100 legend color meaning safe water : don’t need any treatment drinkable but would be better if treated not drinkable without treatment unfit for human drink even after treatment 3. results and discussion 3.1. drastic parameters maps: the seven maps induced by the seven rated and weighted parameters are shown in figure 4. figure 5 shows the histogram of god and drastic gvi class areas in km2 through the studied watershed and figure 6 shows the areal rates (%) of gvi classes referred to the plain area (watershed without mountains area) and to the aquifer area (plain without aquitard area). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 282 fig.4 maps of the seven parameters overlaid classes in el ma labiod plain: a – depth map “d” for drastic method. b – net recharge map “r” for drastic method. c – aquifer medium map “a” for drastic method. d – soil map “s” for drastic method. e – slope map “t” for drastic method. f – impact of vadose zone “i” for drastic method. g – hydraulic conductivity map “c” for drastic method. fig.5 the areas of gvi classes in the studied watershed in km 2 . fig.6 the areal rates (%) of gvi classes referred to the plain area and to the aquifer area. 3.2. drastic gvi map: the drastic groundwater vulnerability index, which ranges from 28 to 124 highlights three levels of susceptibility: referring to the studied zone, it shares the plain of el ma labiod in very low vulnerability 85,9%, low vulnerability 13,76%, and medium vulnerability 0,34%. its map is shown in figure 7a. -referring to the aquifer area, the low vulnerability class covers 56% around the central axe of exploited part of the plain, and the moderate one covers 1.34% in two little areas. the eastern one is probably due to the high value of “c” induced by the longtime of over pumping and the relatively low value of the depth to the water table. 3.3. god gvi map: the god vulnerability index ranges from 0 to 0.43, and highlights three levels of susceptibility. it divides the plain of el ma food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 283 labiod as follows: none to negligible vulnerability 72%, low vulnerability 26.1% and moderate vulnerability 1.9%. its map is shown in figure 7b. referring to the of aquifer area, the low vulnerability range occupies practically the complete aquifer area with 1.3% of the very low class and 7.6% for the moderate one with two little areas at the north which don’t rise in drastic gvi map. these are situated in non-covered sand dunes with clean sand. the extend areas are so large because the range of parameter d (0 to 100 m) which covers all measured water points. 3.4. water quality: table 6 shows the distribution (in %) of the 40 water points sampled across the quality classes according to the seq-eau system. 3.5. findings: nitrates, with five water points unsuitable for drinking water production and eight others unfit for human consumption without prior treatment, are practically the cause of deterioration in the quality of el ma labiod groundwater. three of the four water points which appear with a high level of chlorides are among those which are unsuitable due to nitrates and the fourth one is a 32 m deep well which has a water height of less than two meters above a clay substratum. thus, the map of nitrate concentrations reclassified according to the seq-water margins had been used as a map of water quality. table 6 water points classified according to the seqeau system deterioration by mineralization: parameters conductivity / 100% / / chlorides / 90% / 10% sulfates 2.5% 97.5% / / deterioration by nitrogenous materials except nitrates: parameters ammonium / 100% / / nitrites 95% 5% / / deterioration by nitrates: parameters nitrates 22,5% 45% 20 % 12,5 % 3.6. maps confrontation: the comparison of the two groundwater vulnerability indexes (gvi) maps themselves and with he map of nitrate concentrations in the groundwater of the of el ma labiod plain shows: •a perfect spatial concordance of the maximums of the three items. •that both gvi share the study area in three classes and went to moderate one. •the space occupied by the gvi assessed by the god method for the moderate and low classes is greater and cover integrally those resulting from drastic method. fig. 7 confrontation of vulnerability indexes and nitrate concentration maps: a– map of gvi assessed with the drastic method. bmap of gvi assessed with the god method. cmap of nitrate concentrations in the groundwater of the plain of el ma labiod. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 284 •the low gvi class of both methods includes all nitrate values over 30 mg/l and all high values of the red quality class are located in the moderate gvi class areas. the difference observed in these spatial extents (figure 5) is probably due to the operating mode of the two assessing approaches: •the parameter d covers more important fields of rated depth, up to 100 m, for god method, whereas drastic method is limited to the first 30 meters. •the rating systems use different scales and number of parameters. •the calculation methods of gvi are different. •the spatial distribution of values that exceed the drinking water standards confirms the results obtained by the two used methods to assess the vulnerability of the aquifer. 4. conclusion the effect of agricultural activities on groundwater is no longer to be proven; the level of the water concerned is seriously reduced by irrigation needs and its quality is deteriorated by pollutants from the fields. referring to data from the last three decades, the water table level is decreasing in the study area, with an average of 1m per year, despite the inflow from the north and the south west limestone borders. the contamination of certain areas of el ma labiod plain is concrete and it exceeds drinking standards, with five water points unsuitable for drinking water production and eight others unfit for human consumption without prior treatment. the analysis of the samples designates nitrates and the agricultural activities as an essential cause of the water quality deterioration in el ma labiod plain. both resulting drastic and god vulnerability index maps are spread over three levels with a larger "susceptibility” areas for the god method which has a "d" parameter ranging from 0 to 100 m. thus, the god method is better suited to the arid and semi-arid climate zones which are characterized by deep water tables. the comparison of the map of the nitrate concentrations with drastic and god gvi maps shows a perfect match of the respective maximum values confirming the preferential pathways for surface contaminants to reach the water table. whatever the gap between the needs and the availability, consideration must be given to the safety of other vital resources and the environment before the start of any agricultural or industrial food production project. 5. acknowledgments we sincerely appreciate the director of the ferphos mining complex and the laboratory staff for their welcome and assistance despite their busy schedule, and mister fathallah of the food expertise laboratory for the additional analysis and availability. we express our gratitude to: the mayor of el ma labiod and his assistant mosbahi djamel for the support on the ground during the reconnaissance trips. we also thank the staff of the local subdivision of agriculture, the anrh (hydraulic agency) and the dwe (water direction) of tébessa for the documentation and clarifications. we have a sincere thought for the late professor rouabhia a. who left us his precious pioneering job in the field. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 285 6. references [1]. sonchieu j., frusoh j., waingeh c. n., pesticide exposure of bread sellers and microbial safety of bread sold sold in bamenda, cameroon, food and environment safety, 3: 341351, (2018). [2]. anyanwu e. d., emeka c. s., application of water quality index in the drinking water quality assessment of southeastern nigeria river, food and environment safety, 4:308-314, (2019). [3]. notarnicola b, tassielli g, renzulli p. a., castellani v, serenella s., environmental impacts of food consumption in europe, journal of cleaner production xxx: 1-13, (2016). [4]. vrba j., groundwater for emergency situations – unesco-ihp vi project. the united nations educational, scientific and cultural organization ihp/2007/gw-15 © unesco (2007). [5]. oprea-gancevici d. i., savu c., ground water quality of domestic wells in cajvana town, food and environment safety, 1: 36-45, (2016) [6]. wireman m., ground water vulnerability an overview of concepts and assessment methodologie, usepa denver, co. 7th national water monitoring conference – denver, co (2010). https://acwi.gov/monitoring/conference/2010/a6/a 6_wireman%20nwqmc2010_revforweb.pdf [7]. ghalmi s., fehdi c., baali f., contamination of groundwater and sediments with heavy metals caused by abundant mine: a case study of northern basin of tebessa, algeria. international journal of ecology and development 1: 25 – 42, (2019) [8]. guerrad c., fehdi c, management and arrangement of water resources in semi-arid region of wadi mellegue in algeria. international journal of ecology and development 3: 19 29, (2018) [9]. rouabhia a., vulnerability and risk of pollution of groundwater in the miocene sands of the plain of el ma labiod north east algeria. doctoral thesis university badji mokhtar, annaba, algeria. 195p (2006). (in french). http://biblio.univ-annaba.dz/wpcontent/uploads/2014/06/doctoratrouabhia22.pdf [10]. jora, executive decree no. 14-96 of march 4, 2014 on the quality of water for human consumption. official journal of the algerian republic n°13, 14 – 17 (2014). (in french and arabic). [11]. who, guidelines for drinking-water quality 4th ed. who library cataloguing-in-publication data. who press, geneva switzerland (2011). https://apps.who.int/iris/bitstream/handle/10665/44 584/9789241548151_eng.pdf?sequence=1 [12]. ibrahim m., koch b., assessment and mapping of groundwater vulnerability using sar concentrations and gis: a case study in almafraq, jordan. journal of water resource and protection 7: 588-596, (2015) doi.org/10.4236/jwarp.2015.77047. [13]. abdeslam, i., fehdi, c., djabri, l., application of drastic method for determining the vulnerability of an alluvial aquifer: morsott-el aouinet north east of algeria: using arcgis environment. energy procedia 119, 308-317, (2017). [14]. briciu a. e., costan (briciu) l. a., ionesei v. a., gîza i.c., șchiopu c. m.,water chemical analysis off wells in three settlements of suceava plateau romania, food and environment safety, 17(1): 87 – 96, (2018). [15]. djebari h., resources and test of integrated groundwater management in the el ma labiod basin w. from tébessa, northeast algeria. magister thesis, university badji mokhtar, annaba, algeria.164p, (2011). (in french). [16]. aller, l., lehr, j.h., petty. r., bennett, t., drastic: a standardized system to evaluate groundwater pollution potential using hydrogeologic settings. national water well association, worthington ohio, united states of america. 641p, (1987). [17]. ragoi i.j., popa r., vulnerability assessment of a shallow aquifer situated in danub’s plain (oltenia-region, romania) using different overlay and index methods. chapter 6 in book groundwater vulnerability assessment and mapping: iah-selected papers volume 11. published october 18, 2019 by crc press 260 p. [18]. barbulescua a., assessing groundwater vulnerability: drastic and drastic-like methods: a review. mdpi water journal.water 2020, 12, 1356; doi: 10.3390/w12051356 [19]. foster, s.s. hirata, r.c.a., groundwater pollution risk assessment; a methodology using available data. pan american center for sanitary engineering and environmental sciences (cepis). 3rd printing. lima, perou. 80p. [20]. knouz, n., plain, t., cartography of intrinsic aquifer vulnerability to pollution using god method: case study beni amir groundwater, tadla, morocco. journal of materials and environmental sciences 8(3) 1046-1053. (2017) [21]. nistor m. m., vulnerability of groundwater resources under climate change in the pannonian basin, geo-spatial information science, 4: 345-358, (2019) doi: 10.1080/10095020.2019.1613776 https://acwi.gov/monitoring/conference/2010/a6/a6_wireman%20nwqmc2010_revforweb.pdf https://acwi.gov/monitoring/conference/2010/a6/a6_wireman%20nwqmc2010_revforweb.pdf http://www.ceser.in/ceserp/index.php/ijed/issue/view/582 http://www.ceser.in/ceserp/index.php/ijed/issue/view/582 http://www.ceser.in/ceserp/index.php/ijed/issue/view/582 http://biblio.univ-annaba.dz/wp-content/uploads/2014/06/doctorat-rouabhia22.pdf http://biblio.univ-annaba.dz/wp-content/uploads/2014/06/doctorat-rouabhia22.pdf http://biblio.univ-annaba.dz/wp-content/uploads/2014/06/doctorat-rouabhia22.pdf https://apps.who.int/iris/bitstream/handle/10665/44584/9789241548151_eng.pdf?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/44584/9789241548151_eng.pdf?sequence=1 https://doi.org/10.1080/10095020.2019.1613776 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 samoun fatah, layachi gouaidia, chemsedine fehdi, mohamed laid hemila, larbi djabri, aquifer susceptibility and agricultal activities effect on water qualityin semiarid zone: el ma labiod, north east of algéria, food and environment safety, volume xix, issue 4 – 2020, pag. 275 – 286 286 [22]. state of california, method of test for permeability of soils. california test 220. department of transportation. transportation laboratory 5900 folsom boulevard sacramento, california 9: 5819-4612. (1998). http://www.dot.ca.gov/hq/esc/ctms/pdf/ct_220.pdf [23]. gouaidia l., influence of lithology and climatic conditions on the variation of the physical and chemical parameters of water from water table in semi arid zone: case of the water table of meskiana northeast algeria.doctoral thesis, university badji mokhtar, annaba, algeria. 199p. (2008). (in french). http://biblio.univ-annaba.dz/wpcontent/uploads/2014/06/these-gouaidia-.pdf [24]. arab s., bouchelouche d., hamil s., arab a., application of water quality index for surface water quality assessment boukourdane dam, algeria. advances in sustainable and environmental hydrology, hydrogeology, hydrochemistry and water resource, 18: 308 – 314, (2019). http://www.dot.ca.gov/hq/esc/ctms/pdf/ct_220.pdf http://biblio.univ-annaba.dz/wp-content/uploads/2014/06/these-gouaidia-.pdf http://biblio.univ-annaba.dz/wp-content/uploads/2014/06/these-gouaidia-.pdf 1. introduction 2. matherials and methods doi: https://doi.org/10.4316/fens.2022.031 321 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 321 332 development of a new device for applying atmospheric pressure cold plasma on foods *tamer akan 1 , sehban kartal 2 , sühendan mol 3 , serap coşansu 4 , şehnaz yasemin tosun 3 , didem üçok alakavuk 3 , şafak ulusoy 3 , hande doğruyol 3 , kamil bostan 5 1 department of physics, faculty of science, eskişehir osmangazi university, eskişehir-turkey, akan@ogu.edu.tr 2 department of physics, faculty of science, i̇stanbul university, i̇stanbul-turkey 3 department of fisheries and seafood processing technology, faculty of aquatic sciences, istanbul university, i̇stanbul-turkey, 4 department of food engineering, engineering faculty, sakarya university, sakarya-turkey, 5 department of gastronomy and culinary arts, faculty of fine arts, i̇stanbul aydın university, i̇stanbul-turkey *corresponding author received 30th september 2022, accepted 28th december 2022 abstract: the studies on cold plasma, a non-thermal food processing technology, have been recently focused on extending the shelf life of foods and controlling pathogenic microorganisms since its antimicrobial activities were recognized. atmospheric pressure cold plasma is a cost-effective and user-friendly technique since it does not require a vacuum environment and apparatus. in this study, a new device that produces atmospheric pressure cold plasma in large volumes has been designed and produced to treat various types and properties of food. the system can operate at three different modes, producing direct (device mode-i) and indirect (device mode-ii) volume dielectric barrier discharge (vdbd) and surface dielectric barrier discharge (sdbd) plasma (device mode-iii). the system can be operated consecutively without any problems. the cold plasmas produced in three different modes were applied to different food types, and the foods were examined whether cold plasmas caused a noticeable change in sensory properties. the reactions caused by the system under different operating conditions were measured by optical emission spectroscopy, and the chemical interactions that the foods might be exposed to during plasma treatment were determined. the types of plasma particles that can be effective in food-plasma applications were discussed. the sensory analysis results showed that various foods such as chicken, fish, apple, and milk could be treated with atmospheric pressure cold plasma without causing significant changes in their sensory properties. keywords: cold plasma; dielectric barrier discharge; food safety; sensory; quality. 1. introduction plasma, referred to as the fourth state of matter, occurs when high energy is applied to a gas. in other words, when any gas is energized, it ionizes, and free electrons and ion groups are formed. this state, generally named ionized gas, is called 'plasma' if it has a certain ionization rate. in the plasma state of matter, neutral atoms and molecules, ionized atoms and molecules, excited atoms and molecules, high-energy excited (metastable) atoms and molecules, electrons, radical particles (such as o, oh, no), and photons are formed. moreover, significant amounts of ultraviolet (uv) light and ozone are also produced. these plasma particles with different properties are used very effectively in lighting, medicine, food, and agriculture, as well as coating and electronic technologies. it has been observed that cold plasmas have antibacterial effects without producing any heat and are also effective on different microstructures such as fungi and biofilms food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 322 [1]. plasma can sterilize the air in air conditioners. additionally, it can be used to treat bacterial and viral infectious diseases and to sterilize blood. it is also used in treating burns and wounds, oraldental health and dental caries, different types of cancers, ulcers such as corneal ulcers, in the coagulation of blood for seamless surgery and various skin improvements such as wrinkles and brown spots. the recent application areas of cold plasmas are stem cells, cell mutation, reproductive cells, and neurology [2]. in recent years, it has been observed that cold plasma inactivates bacteria and viruses with the effect of oxygen and nitrogen radicals, uv radiation and charged particles. thus, the studies have focused on using plasmas to produce safe food with high quality and extended shelf life. it has been reported that cold plasma can inhibit pathogen growth in cereals and vegetables [3], reduce spoilage microflora [4], decontaminate surfaces [5], delay metabolic activity [6], reduce peroxidase [7] and lipase – lipoxygenase activities [8]. in animal-based foods, cold plasma reported to inhibit pathogens [9-11], extend microbiological shelf life [12-15], and help to maintain quality [16-17]. cold plasmas are considered strong alternatives to traditional food preservation methods due to their advantages, such as being effective at low, providing powerful antimicrobial effect, not producing toxic by-products, not causing significant damage to food products and being low cost [18]. in addition, plasma is a harmless and environmentally friendly technology without requiring any chemicals and leaving toxic residues [19]. dielectric barrier discharge (dbd) is a system that successfully produces cold plasma and is safe for the people who apply it [20]. however, the equipment and treatment conditions must be validated on food products and target organisms to be commercially applicable [21]. it is important to investigate the effects of cold plasma technology in foods because it represents a potential technology for surface decontamination, shelf life extension, pathogen control and quality improvement. for the in vitro studies, it may be sufficient to generate cold plasma in a very small area, but for food studies, it is necessary to create it in an area where food can be placed comfortably. this study aimed to develop a plasma system with a sufficient volume suitable for treating foods with different types and sizes. a cheap, user-friendly and practical cold plasma device to be used in industrial applications was intended. 2. materials and methods 2.1. atmospheric pressure cold plasma device the system designed and manufactured in this study consists of 2 cm thick polyethylene material. the system is 16 cm in height, 16 cm in width, 80 cm in length, with an internal volume of approximately 15 liters. a 4 mm-thick plexiglass cover that can move within the channels opened in the main body is placed on the front side of the chamber. the food can be placed in the chamber for cold plasma treatment by opening and closing this cover. the device has been designed and manufactured to provide three different cold plasma environments. a direct plasma field (device mode-i) is formed on the right side of the device, and an indirect plasma field (device mode-ii) is formed on the left side; meanwhile, helium gas is added into the chamber. the system has two copper electrodes with a width of 12 cm, length of 36 cm, and thickness of 1 mm. the electrode on the bottom base is placed in a plexiglass material (14 mm in width, 38 mm in length and 4 mm in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 323 thickness) to minimize the accumulation of charge on the system. helium gas is filled from the back of the system between two parallel-placed copper electrodes. surface dielectric barrier discharge (device mode-iii) is created by removing the upper electrode of the device and replacing it with another electrode, consisting of a one mm-thick copper cable placed zig-zag on the plexiglass material. the dimensions of this electrode are 12 cm  40 cm  4 mm. additionally, an alternating current (ac) power supply (30 kv and 20 khz) was designed, produced, and used in the study. a 1000x high voltage probe was used for oscilloscope measurement of this power supply used during the study. 2.2. sensory analysis of cold plasmaapplied foods our device is designed and produced to expose foods to cold plasma to extend their shelf life or prevent pathogen growth. regardless of the provided effect on shelf life or pathogen control, consumer acceptance of the product is the most important criterion for using relevant technology. for this reason, it is of great importance to know whether there is a sensory loss in foods exposed to cold plasma produced by our device. for this, different types of foods (chicken, fish, apple, and milk) were exposed to cold plasma for 2 minutes by the three different modes of our device and the effect of the process was examined by sensory analyzes. ten panelists experienced in sensory evaluation participated in the sensory analysis. food samples were coded with randomly generated 3-digit numbers and presented to the panelists in white plates. the sensory panel was carried out under daylight in a wellventilated room. it was ensured that panelists made the sensory evaluation with a physical distance to prevent their interaction. color, odor, texture, and taste parameters were evaluated. for the taste parameter, the cooking process of the chicken meat (breast) and fish samples (red mullet, mullus barbatus linnaeus, 1758) was done in odorless glass jars using the bain-marie method. the apple (malus domestica, borkh., 1803) and milk (uhtcow milk) samples were tested at room temperature, without any heating process. the classification proposed by [22] was used in sensory evaluation (9: excellent, 8: very good, 7: good, 6: above average, 5: moderate, 4: below average, 3: bad, 2: very bad, 1: extremely bad). during the sensory panel, the panelists also asked, "would you buy this food?" the options "i definitely don't buy (1 point), i probably don't buy (2 points), i may or may not buy (3 points), i probably buy (4 points), i buy definitely (5 points)" options were presented [23-24]. this test aimed to determine whether the cold plasma application significantly changes the purchasing preference of the consumer. sensory evaluations were also performed on control samples that were not treated with plasma. the spss program (spss 16.00, chicago, il, usa) was used for the statistical analysis. the significance level was chosen as p < 0.05. 3. results and discussion 3.1. atmospheric pressure cold plasma device the pressure of the gas used for plasma production is the most important parameter affecting the plasma temperature. it is quite easy to produce cold plasma even when a voltage of 1-5 kv is applied as direct current (dc) at pressures as low as 10-2 mbar. however, at least 20-30 kv high voltages are needed to produce plasma at high pressures around 1 atmosphere. these high voltages result in the occurrence of thermal plasma. the electrode design and the voltage frequency http://en.wikipedia.org/wiki/carl_linnaeus http://researcharchive.calacademy.org/research/ichthyology/catalog/getref.asp?id=2787 https://en.wikipedia.org/wiki/moritz_balthasar_borkhausen food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 324 used to produce cold plasma at high pressures are important [25]. the electrode design of the system produced in this study and the applied voltage frequency constitute the original aspect of the study. the system has been specially designed to produce cold plasma at atmospheric pressure in an area large enough for food applications. in the dielectric barrier discharge, one of the electrodes is covered with an insulator, preventing the generation of secondary electron emission in dc glow discharges. thus, the formation of thermal plasma is prevented. accordingly, our system produces dielectric barrier discharge (dbd) between the electrodes, thanks to the insulating material coating of the lower electrode. the electrodes of our device prevent secondary electron emission, and the type of voltage we use is another important parameter that prevents the heating of the plasma. the production of cold plasma at atmospheric pressure is highly dependent on the type of gas used and the electrode design of the power source [10]. since the effects of different types of gas on foods can vary, the system is designed to work by sending gas from outside. %99.999 pure helium can be sent to our device using the gas flow meter with sockets and a pneumatic hose from the he gas tank regulated with a regulator. the gas flow meter is mounted on the system and can measure the gas flow between 0-15 liters/minute. when the average gas flow is set as 8-10 liters/minute, the distance between the electrodes as 5-6 cm and the electrodes are powered, a visible plasma is generated (figure 1). the plasma formed between the copper plates on the right side of the device can be applied directly to the food placed in this area (direct plasma field, device mode-i). the plasma products formed can be transported to the left side with the help of the fan in the middle of the system and can be applied indirectly to the food placed on this side of the device (indirect plasma field, device mode-ii). the helium gas, continuously sent to the system, also supports the transport of the particles produced by the plasma generated between the electrodes from the right to the left side (figure 1). thus, the plasma particles indirectly affect the food placed in the area far from the electrodes on the device’s left side. indirect plasma application is defined as exposing the sample to the indirect effect of plasma by leaving a distance between the sample and the plasma source. it is stated that the effects will be different from that of the direct plasma [26-27]. plasma produces many different particles such as invisible electrons, ions and radical particles, and photons. when plasma is applied indirectly to a food sample, the effects of invisible particles produced in the plasma are in question. determining the indirect effect of cold plasma is important for industrial applications. because generating the plasma in large areas is more expensive due to high energy requirements. when plasma is applied to large-sized foods in commercial applications, each part of the food may not fit in the plasma field (a large fish, food parcel, fish box, etc.). so, it is important to know the effects provided by the indirect plasma to understand the effects on the food parts that do not directly enter the plasma field. our device works with helium gas in device mode-i (direct plasma) and device mode-ii (indirect plasma). since gas-operated systems may not be preferred in industrial applications due to additional costs and the need to use gas, the other mode (device mode-iii) was designed since it works with atmospheric air. accordingly, the upper electrode of the device is removed, and another electrode is placed on it, on which a copper cable of 1 mm thickness is placed in a zig-zag food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 325 shape.this mode of our device (device mode-iii) creates surface dielectric barrier discharge (sdbd) with atmospheric air, thanks to this specially designed electrode (figure 2). figure 2a shows the electrode design, figure 2b shows its assembled state, and figure 2c shows the surface dielectric barrier discharge plasma formed in purple color. fig.1. large volume atmospheric pressure cold plasma system, direct (device mode-i) and indirect (device mode-ii) cold plasma fields 2. a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 326 2. b 2. c fig. 2. device mode-iii, surface dielectric barrier discharge (sdbd) with atmospheric air (2a. electrode design of surface dielectric barrier discharge, 2b. mounted electrode on the system, 2c. surface dielectric barrier discharge plasma formed in purple color) 3.2. application of cold plasma to various foods with the designed device apple, fish (red mullet), milk and chicken (breast) were exposed to cold plasma produced by different device modes. their sensory characteristics such as taste, smell, texture, and color were evaluated. as a result of all cold plasma applications (direct-i, indirect-ii, sdbd-iii), it was observed that all food samples preserved their sensory-like properties. none of the cold plasma treatments caused a significant change in the sensory scores of the samples, and all foods retained their sensory acceptability (table 1). table 1. sensory evaluation of foods treated with cold plasma by three different modes of the device sensory test scores (mean ± sd) control direct plasma indirect plasma sdbd plasma apple 8.16±0.37 a 8.16±0.37 a 8.00±0.50 a 8.00±0.50 a fish (red mullet) 7.28±0.84 a 7.56±0.71 a 7.32±0.95 a 7.28±0.94 a milk 8.32±0.56 a 8.36±0.49 a 8.36±0.49 a 8.24±0.66 b chicken (breast) 7.76±0.52 a 7.68±0.56 a 7.64±0.57 a 7.60±0.58 ab a,b: the same letters on the same row indicate that the statistical difference is not significant (p ≤0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 327 panelists were also asked, "would you buy this product?". all cold plasma-treated foods were evaluated as "probably" or "definitely" would buy. thereby, it has been concluded that plasma-treated foods kept their marketable preferable properties (figure 3). none of the panelists gave the answers "i definitely don't buy", "i probably don't buy" or "i may or may not buy". when the food is placed between the electrodes, the uv light, ozone, radical oxygen (o), radical hydroxyl (oh), excited and ionized particles and electrons produced by the plasma affect it. the high electromagnetic field applied between the electrodes is also effective on food. particles produced in plasma, especially uv light, ozone, radical oxygen (o), radical hydroxyl (oh) and excited particles, create an important antibacterial effect on the food with which they interact [28]. therefore, the use of cold plasma to increase the shelf life of various foods or to control pathogens constitutes a new and important area of study. although the applied process will provide significant benefits in terms of food safety, quality or shelf life, food preference for the consumer is usually based on sensory properties, and consumer acceptance is indispensable for the food to be sold. for this reason, it is important whether cold plasma causes a noticeable sensory change in the food to which it is applied. fig. 3. response of panelists to the question "would you buy this product?" in literature, there are few studies on sensory changes of foods treated with cold plasma. chiper & al. [29] stated that the cold plasma applied to the fish in the package filled with ar/co2 mixture deteriorates the sensory quality as the processing time increases but did not share the sensory analysis findings. kim & al. [30] observed significant reductions in sensory parameters of bacon treated with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 328 plasma for 5 and 10 minutes. on the other hand, chen & al. [31] reported positive effects of atmospheric cold plasma treatment (1 min.) on the sensory properties of chub mackerel. choi & al. [16] applied cold plasma to dried squid for 1, 2, and 3 minutes and stated that there was no significant difference in sensory scores after treatment. in another study, no change in sensory properties was reported in frozen pork meats treated with cold plasma, while plasma application caused some sensory changes in fresh samples [32]. in our study, the cold plasma applied by three different modes of our device for 2 minutes did not cause a noticeable change in the sensory properties of apple, chicken, fish, and milk. therefore, our device can successfully apply atmospheric pressure cold plasma to different food types. 3.3. plasma characterization of atmospheric pressure cold plasma system reactive oxygen species (ros) and reactive nitrogen species (rns) are called free radicals. both ros and rns are key regulators of processes such as metabolism, growth, development, and cell death. the production of various ros and rnss has been confirmed to have antibacterial activity. plasma can produce ros/rns in the presence of atmospheric oxygen and nitrogen. ultraviolet (uv) radiation, charged particles, energetic ions, reagents such as ros and rns can be generated using air as the working gas. among ros, ozone, atomic oxygen, singlet oxygen, superoxide, peroxide, and hydroxyl radicals are thought to participate in bacterial inactivation [28]. the generation of cold plasma in the environment can trigger oxidation, nitration, hydrolysis, and amination reactions that cause bacterial death [33]. plasma generates reactive oxygen and nitrogen species, disrupting the membrane integrity and thus damaging the bacteria [34]. the particles produced in the plasma generated by our device are circulated in the chamber with a fan. in this manner, the food placed far from the plasma generating area can still be exposed to the plasma products. analyzing the properties of the plasma system and the types of plasma particles is a crucial process to determine the operating conditions and possible effects of plasma particles on food samples [35]. thus, the plasma reactions that lead to positive effects in the applied food can be detected. by finding the most efficient plasma reaction, the system can be used very efficiently. suppose any particle in the plasma creates a positive change in sample quality dominantly. in that case, plasma production conditions can be adjusted to produce more particles of that type. if any particle produces an undesirable effect on the sample, it can be minimized by adjusting the system parameters. therefore, a spectroscopic study was observed to determine the plasma particle types produced by our system. table 2 presents the main chemical reactions and particle types produced by any plasma system operating with helium gas at atmospheric pressure. although it may be known from the reaction rate coefficients which of these reactions occur, they may differ according to the characteristics of the plasma system. for this reason, spectroscopic measurements of the produced plasma are decisive about the reactions in the plasma system [35]. optical emission spectroscopy results from our atmospheric pressure cold plasma system provide information about these reactions and particle types. in figure 4, the optical emission spectrum of the helium plasma produced at atmospheric pressure without any food in the system is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 329 given. the spectrum was taken with the ocean optics usb2000+ device. when helium gas plasma is produced at atmospheric pressure with our device, transitions at 380 (n2), 391 (n2 +), 399 (n2), 405 (n2), 419, 427 (n2 +), 434, 470, 587 (he), 668 (he), 707 (he), 728 (he) ve 777 (o) nm wavelengths are observed (figure 4). table 2. important reactions that can occur in helium plasma at atmospheric pressure [35]. interaction reaction interaction reaction helium atom interactions he + e he* + e he + e he+ + 2e nitrogen interactions n2 + + en2 he+ + n2  n + + n + he he+ + n2  n2 + + he he2 + + n2  n2 + + he + he he* + n2  n2 + + he + e he2 * + n2  n2 + + 2he + e he2 + + n2  n2 + + he2 * n+ + e+ he  n + he helium excited atom interactions e+ he*  he + e he* + e he+ + 2e he* + 2he  he2 * + he he* + he*  he2 + + e he* + he*  he+ + he + e he2 * + m  2he + m 2he*  he2 + + e 2he2 *  he2 + + 2he + e no interactions no+ he  no + he + e no+ + o3 + he  no + o2 + o + he helium ion interactions he+ + e+ e he + e he+ + e+ e he* + e he2 + + e+ e he + he + e he2 + + e he* + he he2 + + e he + he oxygen interactions e+ o2 + he  o2 + he o+ o2 + he  o3 + he o+ + n2 + he  no + + n + he o + o + he  o2 + he he+ + o2  o + + o + he he+ + o2  o2 + + he o + o2 + he  o3 + he o+ + e+ he  o + he water interactions h2o + e  h + oh + e h2o + o *  2oh he* + h2o  oh * + h + he he* + h2o  o * + h2 + he he* + h2o  oh + h * + he the excited particle transitions were given on the corresponding peaks. these transitions are standard and are provided collectively at nist (www.nist.gov). as expected, since helium plasma is generated in the atmospheric environment, a large number of nitrogen reactions occur, and therefore nitrogen ions and excited particles are present in the plasma. helium peaks appear at standard wavelengths as expected. at 777 nm, radical oxygen is seen. in this case, the food treated in our atmospheric pressure cold plasma device will interact with the nitrogen, helium, and radical oxygen atoms. http://www.nist.gov/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 330 fig. 4. the optical emission spectrum of atmospheric pressure cold plasma system helium plasma our atmospheric pressure cold plasma device uses atmospheric air as gas to produce surface dielectric barrier discharge (sdbd) at the device mode-iii. in this case, the optical emission spectrum from the plasma is given in figure 5. as seen in figure 5, the produced atmospheric pressure air plasma emits radiation at 297 nm wavelength of no gas; and 315 nm, 337 nm, 353 nm, 357 nm, 374 nm, 379 nm, 398 nm and 405 nm wavelengths of the nitrogen molecule. as is seen, almost all of them are below 400 nm, which can be considered ultraviolet radiation. our system produces ultraviolet with very high antibacterial activity at device mode-iii, working with atmospheric air. in addition, when the system operates using helium gas (device modes i and ii), radical oxygen with a very high antibacterial activity is formed (777 nm) (figure 4). when the system works with atmospheric air at device mode iii, it produces no, a radical with high antibacterial activity (figure 5). in both cases, all these formations are important for sterilization and inactivation. the results achieved in food applications can be evaluated in terms of interaction with the particle types produced by the plasma. the system can work for 30 minutes without interruption, and it can work throughout the day if 15-minute applications are made consecutively. while our device was operating in all three modes, measurements were made with an ozonometer, and it was determined that the created plasma contained ≥1 ppm ozone. considering ozone above one ppm is enough for antibacterial effect [36], one may conclude that the efficacy of cold plasma may increase due to this high amount of ozone. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 331 fig. 5. the optical emission spectroscopy in a surface dielectric barrier discharge (sdbd) plasma system 4. conclusion a new device that produces cold plasma at atmospheric pressure with a sufficient volume has been designed and manufactured to apply cold plasma to foods. the system can produce volume dielectric barrier discharge and surface dielectric barrier discharge plasmas. the device can be operated in three different modes. these three modes of the device can provide direct or indirect plasma application to foods, work with helium gas or create plasma from atmospheric air without needing an additional gas. the system can work for 30 minutes without interruption, and it can work throughout the day if 15-minute applications are made consecutively. in this study, by taking the optical emission of the plasmas produced in the device, the effective plasma types produced in the plasma are revealed, and their possible effects in food applications are discussed. treatment of various foods such as chicken, fish, apple and milk with atmospheric pressure cold plasma applied by three different modes of our device for 2 minutes did not cause any significant change in sensory characteristics. it was concluded that this system would be a suitable device for applying plasma to foods to control pathogen growth or extend shelf life. 5. acknowledgment this study was supported by tübi̇taktovag 1001 project numbered 219o283. the authors would like to thank tübi̇tak for this support 6. conflicts of interest the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constituted as a conflict of interest regarding the publication of this paper. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan, development of a new devıce for applyıng atmospherıc pressure cold plasma on foods, food and environment safety, volume xxi, issue 4– 2023, pag. 321 – 332 332 7. references [1] h. shintani, a. sakudo, p. burke, g. mcdonnell, experimental and therapeutic medicine, 1, 731, (2010). [2] t. akan, in plasma technologies, ed by b. i̇brahimoğlu: plasma medicine, (ürün, ankara, 2014), p.199 [in turkish]. [3] d. ziuzina, s. patil, p.j. cullen, k.m. keener, p. bourke, p., food microbiology, 42, 109 (2014). [4] n.n. misra, k.m. keener, p. bourke, j.p. mosnier, p.j. cullen, journal of bioscience and bioengineering, 118(2), 177 (2014). [5] n.n misra, s. patil, t. moiseev, p. bourke, j.p. mosnier, k.m. keener, p.j. cullen, journal of food engineering, 125, 131 (2014). [6] s. tappi, a. berardinelli, l. ragni, m.d. rosa, a. guarnieri, p. rocculi, innovative food science and emerging technologies, 21, 114 (2014). [7] s.k. pankaj, n.n. misra, p.j. cullen, innovative food science and emerging technologies, 19, 153 (2013). [8] h. tolouiea, m.e. mohammadifara, h. ghomic, a.s. yaghoubid, m. hashemie, innovative food science and emerging technologies, 47, 346 (2018). [9] d.d. jayasena, h.j. kim, h.i. yong, s. park, k. kim, w. choe, c. jo, food microbiology, 46, 51 (2015). [10] b. kim, h. yun, s. jung, y. jung, h. jung, w. choe, c. jo, food microbiology, 28, 9 (2011). [11] h. lee, h.i. yong, h.j. kim, w. choe, s.j. yoo, e.j. jang, c. jo, biotechnology, 25(4), 1189 (2016). [12] a. bauer, y. ni, s. bauer, p. paulsena, m. modic, j.l. walshb, f.j.m. smulders, meat science, 128, 77 (2017). [13] j. wang, h. zhuang, a. hinton j. zhang, food microbiology, 60, 142 (2016). [14] i. albertos, a.b. martin-diana, p.j. cullen, b.k. tiwaric, k. shikha ojhac, p. bourkeb, c. álvarezc, d. ricoa, innovative food science and emerging technologies, 44, 117 (2017). [15] i. albertos, a.b. martin-diana, p.j. cullen, b.k. tiwaric, k. shikha ojhac, p. bourkeb, d. ricoa, innovative food science and emerging technologies, 53, 85 (2019). [16] s. choi, p. puligundla, c. mok, food science and biotechnology, 26(4), 1137 (2017). [17] s. choi, p. puligundla, c. mok, lwt food science and technology, 75, 323 (2017). [18] n.n. misra, c. jo, trends in food science & technology, 64, 74, (2017). [19] m. gavahian, y.h. chua, a.m. khaneghahb, f.j. barbac, n.n. misra, trends in food science & technology, 77, 32 (2018). [20] s. förster, c. mohr, w. viöl, surface & coatings technology, 200, 827 (2005). [21] j. wa, j. coventry, p. swiergon, p. sanguansri, c. versteeg, trends in food science & technology, 20, 414 (2009). [22] h. stone, j.l. sidel, sensory evaluation practices, (elsevier, 2004). p. 377. [23] m.c.a. silva, j.s.f. leite, b.g. barreto, m.v.a. neves, a.s. silva, k.m. viveiros, r.s.f.t. passos, n.p. costa, r.v. silva, c.p. cavalheiro, lwt food science and technology, 137, 110409 (2021). [24] s.d. dutcosky, análise sensorial de alimentos / sensory analysis of foods, (curitiba; champagnat; 3 ed; 2011) p.426. isbn-10: 8554945476 [25] t. akan, a. çabuk, journal of electrostatics, 72, 218 (2014). [26] g. fridman, a. d. brooks, m. balasubramanian, a. fridman, a. gutsol, v. n. vasilets, h. ayan, and g. friedman, plasma processes and polymers, 4, 370 (2007). [27] m. laroussi, ieee transactions on plasma science, 37, 714 (2009). [28] h. kandemi̇r, f.a. kandemi̇r, b. güler, a. gürel, journal of agricultural faculty of bursa uludag university, 35(1), 217 (2021). [29] a.s. chiper, w. chen, o. mejlholm, p. dalgaard, e. stamate, plasma sources science and technology, 20, 025008 (2011). [30] h.j. kim, h.i. yong, s. park, w. choe, c. jo, current applied physics, 13, 1420 (2013). [31] j. chen, s.z. wang, j.y. chen, d.z. chen, s.g. deng, b. xu, journal of the science of food and agriculture, 99, 39 (2019). [32] s. choi, p. puligundla, c. mok, annals of microbiology, 66, 685 (2016). [33] m. bencina, m. resnik, p. staric, i. junkar, molecules, 26, 1418 (2021). [34] p. brun, g. bernab, c. marchiori, m. scarpa, m. zuin, r. cavazzana, b. zaniol, e. martines, journal of applied microbiology, 125, 398 (2018). [35] k.r. stalder, r.j. vidmar, g. nersisyan, w.g. graham, journal of applied physics, 99, 093301 (2006). [36] y. alparslan, t. baygar, d. yildiz, gıda teknolojileri elektronik dergisi, 7(3), 24 (2012). microsoft word 2_91 dimitrov_final (2) doi: https://doi.org/10.4316/fens.2021.012 101 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag. 101 112 phenolic content and atioxidant activity of red grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria *dimitar dimitrov1, tatyana yoncheva1, vanyo haygarov1, anatoli iliev1 1department of selection, enology and chemistry, institute of viticulture and enology, pleven, bulgaria, ilv@el-soft.com dimitar_robertov@abv.bg *corresponding author received 9 march 2021, accepted 27 june 2021 abstract: a study to determine the phenolic content and antioxidant activity of grapes from red international (cabernet sauvignon), local (gamza) and hybrid (rubin) grapevine varieties grown in the region of central northern bulgaria was conducted. the phenolic content of the grapes, the phenolic content and antioxidant activity of their must were determined. rubin shown the best content of total phenolic compounds (tpc), flavonoid phenolic compounds (fpc), non-flavonoid phenolic compounds (npc) and anthocyanins in the structural elements of the cluster. this hybrid variety also shown the highest content of tpc in the must (0.74±0.00 g/l), followed by gamza, and the lowest tpc was found in the cabernet sauvignon – control variety (0.25±0.00 g/l). the highest amount of anthocyanins was found in rubin grape must (19.91±0.48 mg/l). the dynamics of increasing of the anthocyanin content in the must of the studied varieties followed the order international variety (cabernet sauvignon) < local variety (gamza) < hybrid variety (rubin). in the red varieties, the grape must of the rubin hybrid shown significant and the highest antioxidant activity of the three studied varieties. it was four times higher than that found in the cabernet sauvignon control. a correlation between tpc, anthocyanin content and antioxidant activity in rubin was found. this proved the close connection between the phenolic composition and the antioxidant capacity of the variety. the study proved that the grape must of the two studied hybrid and local varieties shown high biological potential and activities. keywords: antioxidant activity, grapes, phenols, free radicals, grapevine varieties. 1. introduction antioxidants are a complex of substances a component of the foods composition, or artificially added to them. their main function is to neutralize oxidative processes in the human body. the product of these processes are the so-called reactive oxygen species ros (free radicals). by binding to various molecules in the body, ros can initiate serious damages to cellular structures, leading to the formation of many diseases atherosclerosis, rheumatoid arthritis, cancer forms [1-2]. the antioxidant effect of grapes is mainly due to its phenolic complex. phenolic compounds are localized in the solid parts skins, bunches, seeds (source of flavonoid compounds) and fleshy part (source of non-flavonoid compounds) [3]. flavonoid phenolic compounds are represented by catechins, proanthocyanidins, anthocyanins, flavones, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 102 flavonones and flavonols, while nonflavonoids include benzoic and cinnamic acids [4]. the synthesis, distribution and accumulation of phenolic compounds in grapes are highly dependent on a number of factors: the genotypic potential of the variety to accumulate phenolic substances [5-6], complex environmental influences [7], the presence of water, temperature and active access of the vine to sunlight [8] strong sun exposure is directly proportional to the degree of phenolic accumulation in the grapes [9]. the total content of phenolic compounds in red varieties occupies the range 1000.00 5000.00 mg/l [10]. a study of the phenolic composition of red grapes (cabernet sauvignon, merlot, rubired and petit syrah) grown in mexico found a total phenolic content of 321.90 mg gae. 100g-1 (cabernet sauvignon) to 607.60 mg gae. 100g-1 (rubired) [11]. the antioxidant activity of grapes is closely correlated with its phenolic composition. this activity plays a significant role in reducing the oxidation of ldl (low-density lipoproteins), a major factor in atherogenesis (accumulation of cholesterol plaques in blood vessels) [1213]. grapes phenols have been shown to inhibit proliferation and apoptosis in in vitro prostate cancer cell lines [14] and exhibit strong antibacterial, antiviral, anticancer and anti-inflammatory properties [15-16]. the aim of the present study was to determine the phenolic composition and antioxidant activity of grapes from red international (cabernet sauvignon), local (gamza) and hybrid (rubin) grapevine varieties grown in the region of central northern bulgaria. 2. matherials and methods grapevin varieties the study was conducted at the institute of viticulture and enology (ive) pleven (central northern bulgaria). the object of the study was grapes, harvest 2020, of three red grapevine varieties – international, local and hybrid: cabernet sauvignon red grapevine variety, originating form the gironde region of france [17]. it is distributed in all wine-growing countries around the world. for the region of pleven, bulgaria it ripens in the second half of september. it has very good fertility, relatively droughtresistant and cold-resistant. the grapes have a high sugar accumulation (21-24%) at relatively high titratable acidity (6.5-9.0 g/l). the wines are characterized by an intense ruby red color, specific for the variety aroma and dense taste. they are suitable for aging, during which they develop their characteristics [18-19]. gamza red local grapevine variety. gamza is distributed in hungary, croatia and serbia [20-22]. in hungary, it is the main variety for the production of red table wines [23-24]. it is also widespread in small areas in ukraine, france, romania and some regions in asia [25-28]. it has been grown in bulgaria since ancient times, but has a local distribution. it was most widespread in the regions of veliko tarnovo, sevlievo, svishtov, pleven, lovech, vidin and others [29]. late ripening variety, the grapes ripens in the second half of september. the variety has a high fertility, susceptible to fungal diseases and low winter temperatures. the grapes have a sugar accumulation of 1921% and titratable acids of 5.9-8.9 g/l. the wines are characterized by bright red ruby color, delicate fruity aroma, young wines [18,19,30]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 103 rubin a hybrid red grapevine variety. it was obtained by intraspecific hybridization by crossing nebbiolo x syrah [31] and approved in 1961. it was included in the official variety list of bulgaria in 2012. the variety is medium ripening, and for the region of pleven ripens in the first half of september. it has a good fertility. it is not resistant to fungal diseases, with weak resistance to low winter temperatures. the variety has a high sugar accumulation capacity of 22-24% and more, with titratable acids of 5.5-6.0 g/l. the grapes are suitable for the production of dry and dessert wines, with an intense dark red color, high extractivity and harmonious taste [18-19]. determination of the phenolic content of grapes and must from the studied varieties to study the phenolic content of the grapes by varieties, at the onset of technological maturity and after harvesting, their content in the structural elements of the cluster bunches, seeds, skins, berries were analyzed. for this purpose, an average sample of grapes of each variety (2 kg) was selected, from which 5 g of bunches (dried and cut into segments), 5 g of seeds (pre-ground in a mortar), 5 g of skins (predried) and 10 g whole berries (pre-torn in a mortar and with crushed seeds) were weighed. the prepared and weighed quantities were transferred to erlenmeiler flasks. extractant c2h5oh / hcl (1% v/v) was used for extraction [32]. to determine the phenolic content of the grapes (in the liquid phase, after the extraction) and the phenolic content of the must (separated immediately after crushing of the grapes) the following indicators were analyzed [32]: • total phenolic compounds (tpc), g/l gallic acid singleton et rossi method with folin-chiocalteu reagent • flavonoid phenolic compounds (fpc), mg/l catechin equivalent sommers method • non-flavonoid phenolic compounds (npc), mg/l caffeic equivalent sommers method • anthocyanins, mg/l method of singleton et rossi by changing the ph determination of antioxidant (dpph •) activity of grapes and must the antioxidant activity was determined according to the method of wang et al. (1996) [33], as antiradical activity against the stable product dpph• (2,2-diphenyl-1-picrylhydrazyl) (sigma aldrich, germany). the values of the molecular light absorption (spectrophotometrically at a wavelength of 515 nm) of the control and experimental sample, noted by ac and ae, respectively, were measured. measurements were made at reaction times of 5 and 15 minutes from the time of the reagents mixing. the antiradical activity was calculated by the formula: ааr = 102.(аc аe).ак-1, % (1) statistical processing statistical data processing was performed, including determination of standard deviation (± sd), with three repetitions for each analysis. the determination of the indicator was realized with the ms excel 2007 from the microsoft package (microsoft corporation, usa). 3. results and discussion phenolic content of grapes the content of total phenolic compounds, flavonoid phenolic compounds, non flavonoid phenolic compounds and anthocyanins in the structural elements of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 104 the cluster berries, skins, bunches, seeds was studied. the results are presented in table 1. the tendency in the content of the studied components was their increase in the order of whole berries < skins < bunches < seeds. there were significant differences in the phenolic content of the structural elements of the cluster with tpc. the highest tpc were contained in the berries (0.55 ± 0.00 g/l), skins (1.18 ± 0.01 g/l and seeds (3.00 ± 0.07 g/l) of the rubin variety, and in the skins they were almost twice as much as in the other varieties. gamza bunches were with the highest content of tpc (1.89 ± 0.00 g/l) compared to cabernet sauvignon and rubin. the fpc of rubin variety was the highest in the berries (1854.55 ± 4.19 mg/l), skins (2465.66 ± 2.63 mg/l) and seeds (6893.61 ± 8.22 mg/l). in the bunches of gamza variety fpc were the highest (3247.98 ± 3.76 mg/l). rubin berries contain fpc twice as much as cabernet sauvignon (779.76 ± 3.45 mg/l) and four times as much as gamza (398.33 ± 0.82 mg/l). in the skins of rubin fpc were twice as much as cabernet sauvignon (1001.25 ± 2.53 mg/l) and three times more than gamza (841.12 ± 3.75 mg/l). in the bunches and seeds of cabernet sauvignon variety fpc were the lowest, respectively 1575.71 ± 0.55 mg/l and 4348.75 ± 11.93 mg/l. the differences in the content of npc in the different structural parts of the cluster in the three varieties were also significant. their quantity was significantly higher in the berries (133.76 ± 0.28 mg/l), skins (245.66 ± 0.29 mg/l) and seeds (482.04 ± 0.48 mg/l) of the rubin variety. with the exception of berries, cabernet sauvignon shown the lowest content of npc in the other parts. in the skins they were three times, and in the bunches two times lower than in rubin. gamza variety has the lowest npc only in the berries (47.02 ± 0.34 mg/l). table 1. phenolic content of grapes from the studied red grapevine varieties structural elements of the cluster indicators units cabernet sauvignon gamza rubin whole berries tpc g/l g.a. 0.37±0.02 0.24±0.00 0.55±0.00 fpc mg/l cat. equiv. 779.76±3.45 398.33±0.82 1854.55±4.19 npc mg/l caf. equiv. 62.38±10.44 47.02±0.34 133.76±0.28 anthocyanin s mg/l 154.23±0.64 47.27±3.52 147.40±0.16 skins tpc g/l g.a. 0.59±0.02 0.49±0.00 1.18±0.01 fpc mg/l cat. equiv. 1001.25±2.53 841.12±3.75 2465.66±2.63 npc mg/l caf. equiv. 80.31±0.43 111.14±0.31 245.66±0.29 anthocyanin s mg/l 388.75±1.51 254.19±0.44 596.90±2.22 bunches tpc g/l g.a. 1.04±0.00 1.89±0.00 1.76±0.01 fpc mg/l cat. equiv. 1575.71±0.55 3247.98±3.76 2983.93±1.15 npc mg/l caf. equiv. 98.92±0.25 172.89±0.68 161.87±0.43 seeds tpc g/l g.a. 2.15±0.01 2.66±0.05 3.00±0.07 fpc mg/l cat. equiv. 4348.75±11.93 5286.14±27.10 6893.61±8.22 npc mg/l caf. equiv. 234.11±1.07 245.82±1.33 482.04±0.48 in the red grapevine varieties, the content of anthocyanins is of major importance and it is an important characteristic for the variety. in all three varieties, their content in the skins was several times higher than in the whole berries. the content in the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 105 skins is especially important, because during the alcoholic fermentation as a result of ongoing extraction processes anthocyanins pass into the grape must. they are the main component determining the color properties of the wines. rubin skins has the best stock of anthocyanins (596.90 ± 2.22 mg/l), followed by cabernet sauvignon (388.75 ± 1.51 mg/l), and the lowest was in gamza (254.19 ± 0.44 mg/l). the trend in berries, however, was different. the highest content of anthocyanins was reported in cabernet sauvignon (154.23 ± 0.64 mg/l), and the lowest, as well as in the skins in the variety gamza (47.27 ± 3.52 mg/l). phenolic compounds tpc, fpc, npc and anthocyanins in grape must of the studied varieties the phenolic complex is a major factor for the biological value of the grapes and wine, determining their antioxidant activities. tpc, fpc, npc and anthocyanins were identified in the grape must of the studied varieties. the data for tpc in grape must of the studied varieties are presented in figure 1. fig. 1. total phenolic compounds (tpc) in grape must of the studied varieties the lowest content of tpc in the must of the control variety cabernet sauvignon (0.25 ± 0.00 g/l) was found. the results indicated that the local variety gamza (under the soil and climatic conditions of central northern bulgaria) shown a higher potential to accumulate tpc. they were found in an amount of 0.36 ± 0.00 g/l. very high levels of tpc shown the third studied variety the intraspecific hybrid rubin. tpc of 0.74 ± 0.00 g/l was found in its grape must. they were almost three times higher than those found in the control variety cabernet sauvignon. the higher level of phenolic compounds in its must is an indirect evidence of its biological potential. the obtained results regarding the content of fpc in grape must of the three studied varieties are presented in figure 2. from the results for fpc in the grape must of the three studied red grapevine varieties, a concentration in the control international variety cabernet sauvignon of 373.69 ± 3.91 mg/l was established. fpc in the control exceeded the established amount in the other studied variety the local gamza. the content of fpc in its must was 129.41 ± 7.20 mg/l. the highest levels on this indicator shown the hybrid variety rubin. the concentration of fpc in it (832.49 ± 12.37 mg/l) was more than twice in comparison of that found in the must of the cabernet sauvignon control variety. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 106 the data on the established content of nonflavonoid phenolic compounds in the grape must of the studied varieties are presented in figure 3. the highest concentration of npc was found in the control cabernet sauvignon (162.91 ± 1.57 mg/l). the local red variety gamza shown the lowest concentration of npc (116.76 ± 1.45 mg/l). second, after the control, by the presence of npc was the hybrid rubin. the npc content of 131.57 ± 2.37 mg/l was found in its must. anthocyanins are red pigments in red grapevine varieties localized in their skins [3]. in the grapes they are contained in an amount of about 600.00 mg/l, in the wine they are extracted from 50 70% of this amount [3]. fig. 2. content of fpc in grape must of the studied varieties fig. 3. content of npc in grape must of the studied varieties due to the weaker contact during crushing of the grapes (as the first phase of technological processing) in the fresh grape must, their quantities are usually low. the results obtained for their content in the grape must of the red varieties are presented in figure 4. from the obtained results it was clear that the international control variety cabernet sauvignon (9.01 ± 0.33 mg/l) was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 107 characterized by the lowest content of anthocyanins. in the local variety gamza, the amount of these phenolic components in fresh grape must was higher (11.92 ± 0.07 mg/l) than that found in the control. it was evident from the results that rubin shown the highest concentration of anthocyanins (19.91 ± 0.48 mg/l) from the three studied varieties, and it exceeded more than twice that found in the cabernet sauvignon control. in practice, a smooth dynamics of increase in the content of anthocyanins was established in the direction: introduced variety (cabernet sauvignon, control) < local variety (gamza) < hybrid variety (rubin). fig. 4. content of anthocyanins in grape must of the studied varieties the main characteristic (as a biological effect) of anthocyanins is the proven in vitro antioxidant activity [34]. the obtained results indicated that from the three studied red grapevine varieties grown in the town of pleven, central northern bulgaria (harvest 2020) potential, in terms of anthocyanin accumulation, was the hybrid variety rubin. the content of anthocyanins is directly related to the antioxidant capacity. antioxidant activity (as antiradical activity against a stable dpph radical) in grape must of the studied red varieties the antioxidant activity is one of the main factors determining the biological potential of the grapes. the obtained data on the antioxidant activity (dpph •) in grape must of the international (cabernet sauvignon), local (gamza) and hybrid (rubin) red grapevine varieties are presented in figures 5, 6 and 7. the highest antioxidant activity in the grape must of the red varieties was found in the hybrid variety rubin. at te = 600.00 mg/l and a reaction time of 5 min, it shown antioxidant activity of 95.15 ± 0.005 %. at a reaction time of 15 minutes, the activity of the must from this variety had a minimal change. a minimal increase was reported 95.17 ± 0.01 %. at te = 400.00 mg/l there was a decrease in the antioxidant capacity of rubin. at a reaction time of 5 min, an activity of 71.92 ± 0.03 % was detected. at a reaction time of 15 minutes it was almost identical 71.69 ± 0.005 %. rubin was followed by the local variety gamza. its antioxidant activity, however, was many times lower than rubin's. gamza grape must at te = 600.00 mg/l and a reaction time of 5 min shown 36.34 ± 0.14 % antioxidant activity. as the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 108 reaction time increased the percentage of free radical elimination (dpph •) increased too, with an activity of 39.75 ± 0.04%. in this variety with a decrease in the concentration of the total extract was registered decrease in the antioxidant activity. thus, at te = 400.00 mg/l and a reaction time of 5 min, 22.70 ± 0.11 % elimination of dpph was found. when the reaction time was increased to 15 min, a slight increase in the antioxidant activity of gamza was found 25.96 ± 0.06 %. fig. 5. antioxidant activity of red international variety cabernet sauvignon at te = 600.00 mg/l and te = 400.00 mg/l *te – total extract fig. 6. antioxidant activity of red local variety gamza at te = 600.00 mg/l and te = 400.00 mg/l *te – total extract food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 109 fig. 7. antioxidant activity of red hybrid variety rubin at te = 600.00 mg/l and te = 400.00 mg/l *te – total extract the lowest reported antioxidant activity was observed in the control introduced cabernet sauvignon variety. at te = 600.00 mg/l and a reaction time of 5 min, its must shown only 20.34 ± 2.10 % elimination of the free dpph radical. at a reaction time of 15 minutes, the same extract shown activity close to that recorded at 5 minutes of reaction 20.70 ± 0.10 %. at te = 400.00 mg/l and a reaction time of 5 min, the antioxidant activity of cabernet sauvignon was 20.19 ± 0.08 %. as the reaction time increased to 15 minutes, it increased slightly, reaching a value of 21.26 ± 0.10 %. apparently from the results obtained for the antioxidant effect of the control variety cabernet sauvignon was that it retained an almost constant anti-radical capacity, which was not depended on the amount of extract in the must. however, cabernet sauvignon grape must shown the lowest ability to eliminate dpph • compared to the other two studied varieties. the grape must of the rubin hybrid shown four times higher antioxidant activity than that of cabernet sauvignon. gamza's must also shown a higher antioxidant capacity than cabernet sauvignon, but the difference between them was significantly smaller. the results for the antioxidant activity of grape must in red varieties fully correlated with the established content of total phenolic compounds (tpc) in them. the following dependencies have been identified: tpc = 0.25 g/l (control, international variety cabernet sauvignon) < tpc = 0.36 g/l (local variety gamza) < tpc = 0.74 g/l (hybrid variety rubin) aa (control, international variety cabernet sauvignon) < aa (local variety gamza) < aa (hybrid variety rubin) the data clearly outline the confirmed relationship between the phenolic composition of grapes and its radical scavenging ability. the hybrid variety rubin shown an extremely high potential for antioxidant properties, which was significantly distinguishable compared to the other two studied varieties. in addition, rubin dominated cabernet sauvignon control over this specific activity. there was also a correlation between the antioxidant activity of the must of red food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 110 varieties and their anthocyanin content. it was lowest in the cabernet sauvignon control, increased in the local gamza variety, and was found in the highest amount in the rubin hybrid. the anthocyanins are compounds with high antioxidant capacity and the present study empirically confirms the relationship between them and the antioxidant capacity of red grapevine varieties. 4. conclusion as a result of the conducted research the following conclusions could be made: • the phenolic content of the varieties in terms of tpc, fpc, npc and anthocyanins in the structural elements of the cluster increased in the order of whole berries < skins < bunches < seeds. • rubin has the best stock of tpc, fpc, npc and anthocyanins. the tpc and fpc of the variety were mostly in the berries, skins and seeds, as in the skins they were almost twice as much as in the other varieties. the amount of npc in all structural elements of the cluster of rubin was the highest (except for the bunches). the skins of this variety has the best stock of anthocyanins, followed by cabernet sauvignon, and the lowest was in gamza. in the berries, the highest content of anthocyanins was reported in cabernet sauvignon. • the tpc of the grape must from the studied red grapevine varieties was the highest in the hybrid variety rubin (0.74 ± 0.00 g/l). it was followed by the local gamza in this indicator, and the lowest amount of tpc was found in the control international variety cabernet sauvignon (0.25 ± 0.00 g/l). the rubin hybrid demonstrated significant and high potential to accumulate phenols at the conditions of central northern bulgaria. • rubin shown the highest quantitative presence of fpc (832.49 ± 12.37 mg/l), twice as much as that found in the cabernet sauvignon control. • the highest content of anthocyanins in the grape must of the red varieties was found in the hybrid variety rubin (19.91 ± 0.48 mg/l). it was lowest in the cabernet sauvignon control. the results proved smooth growth dynamics in the content of anthocyanins in the order: international variety (cabernet sauvignon, control) < local variety (gamza) < hybrid variety (rubin). • in the red grapevine varieties, the grape must of the rubin hybrid shown significant and the highest antioxidant activity of the three studied varieties. it was four times higher than that found in the cabernet sauvignon control. a correlation was found between tpc, anthocyanin content and antioxidant activity in rubin. this proved the close connection between the phenolic composition and the antioxidant capacity of the variety. the grape must of the local variety gamza also shown higher antioxidant activity compared to the cabernet sauvignon control, but the difference between them was significantly smaller. 6. references [1]. dormandy t.l., an approach to free radicals, lancet, 2: 1010–1014, (2010). [2]. hertog m.g., kromhout d., aravanis c., blachburn h., buzina r., fidanza f., giampaoli s., jansen a., menotti a., nedeljkovic s., pekkarinen m., simic b.s., toshima h., feskens e.j.m., hollman p.c.h., katan m.b., flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study, archives of internal medicine, 155: 381–386, (1995). [3]. chobanova d., manual for enology exercises, academic press of university of food technologies, plovdiv, bulgaria, (2007). (bg) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 111 [4]. harbone j. b., "plant phenolics". in bell, e. a.; charlwood, b. v. (eds.). encyclopedia of plant physiology, volume 8 secondary plant products. berlin heidelberg new york: springerverlag. pp. 329–395, (1980). [5]. costa j.m., vaz m., escalona j., egipto r., lopes c., medrano h., chaves m.m., modern viticulture in southern europe: vulnerabilities and strategies for adaptation to water scarcity, agricultural water management, 164(1): 5-18, (2015). [6]. mazza g., fukumoto l., delaquis p., girard b., ewert b., anthocyanins, phenolics, and color of cabernet franc, merlot, and pinot noir wines from british, columbia journal of agriculture and food chemistry, 47: 4009-4017, (1999). [7]. teixeira a., eiras-dias j., castellarin s.d., gerós h., berry phenolics of grapevine under challenging environments, international journal of molecular sciences, 14(9): 18711-18739, (2013). [8]. ivanova v., stefova m., vojnoski b., dornyei a., mark l., dimovska v., identification of polyphenolic compounds in red and white grape varieties grown in r. macedonia and changes of their content during ripening, food research international, 44(9): 2851–2860, (2011). [9]. roubelakis-angelakis k.a., kliewer w.m., effects of exogenous factors on phenylalanine ammonia-lyase activity and accumulation of anthocyanins and total phenolics in grape berries, american journal of enology and viticulture, 37: 275–280, (1986). [10]. velkov e., encyclopedia of alcoholic beverages, ed. polygraphy, plovdiv, bulgaria. isbn 954-698-002-1, (1996). [11]. franco-banuelos a., galiciahernánde m.e., contreras-martínez c.s., carranza-télle j., carranza concha j., total phenolic content and antioxidant capacity of wine grapes grown in zacatecas, mexico, investigación y desarrollo en ciencia y tecnología de alimentos, 3: 477-482, (2017). [12]. aviram m., fuhrman b., wine flavonoids protect aganist ldl oxidation and atherosclerosis, annals of new york academy of science, 957: 146-161, (2002). [13]. parthasarathy s., rankin s.m., role of oxidized low density lipoprotein in atherogenesis, progress in lipid research, 31: 127143, (1992). [14]. romero i., paez a., ferruelo a., lujan m., berenguer a., polyphenols in red wine inhibit the proliferation and induce apoptosis of lncap cells, bju international, 89: 950-954, (2002). [15]. gris e.f., mattivi f., ferreira e.a., vrhovsek u., pedrosa, r.c., bordignon-luiz m.t., proanthocyanidin profile and antioxidant capacity of brazilian vitis vinifera red wines, food chemistry, 126: 213–220, (2011). [16]. sun b.s., ribes a., leandro, m.c., belchior a.p., spranger, m.i., stilbenes: quantitative extraction from grape skins, contribution of grape solids to wine and variation during wine maturation, analytica chimica acta, 563: 382–390, (2006). [17]. viala p., vermorel v., ampelographie, vol. 2, paris, macon protat freres imprimeurs, 51-54, 67-69, 99-104, 139-142, (1901). [18]. radulov l., babrikov d., georgiev s. ampelography with basics of winemaking. sofia, zemizdat, p. 187, (1992). (bg) [19]. roychev v., ampelography. plovdiv, academic publishing house of the agricultural university, pp. 576, (2012). (bg) [20]. turcovic z., ampelografski atlas, zagreb, poljoprivredni nakladni zavod, 72, (1963). [21]. avramov, briza к., posebno vinogradarstvo (ampelografia), novi sad, univerzitet u novom sadu, (1965). [22]. zirojevic d., poznavanje sorata vinove loze. vol. 1, beograd, nolit, 15-30, 119-126, 175182, 303-310, 343-350, (1974). [23]. csepregi p., zilai j., szolôfajtaink, budapest, mezоgazdasagi, kiadо, 114-115, 218219, 224-227, 278-279, (1960). [24]. nemeth m., ampelografi album, vol. 1, budapest, mezogazdasagi kiado, 157-164 (1967). [25]. kondarev m., kurtev p., tsankov b. and others. ampelography, plovdiv, “hr. danov ”, (1976). [26]. nedelchev n., georgiev i., raduchev s. and others., handbook of the winemaker, zemizdat, pp. 14, 32, 121-122.5, (1967). [27]. blaha j., ceskoslovenska ampelografi a, bratislava, orac, rolnicke vydavatelstvo, 41-45, 196-200, 219-225, 284-287, (1952). [28]. constantiueseu g., ampelografi a, bucuresti, еditura agrosilvica de stat, 193-195, 230-232, 235-236, 242-244, 250-252, 307-311, 314-317, (1958). [29]. georgiev h. viticulture guide, ruse, lithograph by ferenc jos alkalay, pp. 253, 256, 267.3, (1894). [30]. simeonov i ., concise economic description of the basic local grapevine varieties for food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dimitar dimitrov , tatyana yoncheva , vanyo haygarov , anatoli iliev, phenolic content and antioxidant activity of reg grapes from international, local and hybrid grapevine varieties grown in central northern bulgaria, food and environment safety, volume xx, issue 2 – 2021, pag. 101 – 112 112 the republic of bulgaria, p hytologia balcanica, 22 (2): 209 – 216, (2016). [31]. petkov g., biochemical and technological study of the varieties bouquet, rouen and rubin for the production of red wines, dissertation, institute of viticulture and enology, pleven, (1977). [32]. stoyanov n., study on the phenolic compounds of grapes and wines of the varieties cabernet sauvignon and mavrud. dissertation "phd", university of food technologies, plovdiv, 35-36, (2007). [33]. wang h., cao g., prior r.l., total antioxidant capacity of fruits, journal of agriculture and food chemistry, 44(3): 701-705, (1996). [34]. bitsch r., netzel m., frank t., strass g., bitsch i., bioavailability and biokinetics of anthocyanins from red grape juice and red wine, journal of biomedicine and biotechnology, 2004 (5): 293-298, (2004). 193 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 193 – 199 choosing fruit berry fillings for condensed canned milk with sugar natalija valeriivna ryabokon1, *dmitro viktorovich rindyuk1 1national university of food technologies, kiev, ukraine rel_dv@ukr.net *corresponding author received september 4th 2014, accepted september 29th 2014 abstract: the rheological and organoleptic properties of condensed canned milk with sugar and fruit fillings were determined. the syrups’ and jams’ usage in the technology of condensed canned milk with sugar reduces the viscosity of finished products and increase the length of the structures destruction in products with fruit jam. we studied the impact of fruit-berry jam on the indicators of conditional dynamic threshold fluidity and fluidity ratio of condensed canned milk with sugar and the possibility of losing the product’s fluidity during storage. in the ccm recipes with sugar and fruit berry fillings, the preference should be given to fruit-berry syrup. keywords: effective viscosity, fruit fillings, jam, rotational viscometer, syrup 1. introduction unbalanced diets, difficult psychoemotional stresses, lack of sleeping, bad habits and many other factors with the significant deficiency of biologically valuable food nutrients influence the health of individuals and the health of the nation in general. therefore, the focus on manufacturing the next-generation products with high nutritional, biological values is predictable in all sectors of food industry. dairy industry is no exception to this trend. significant contributions to developing of the dairy industry were made by such scientists as chekulaeva l.v. radayeva i., polansky k.k., l.v. golubev and others [1]. they invented major types of condensed canned milk with sugar that are represented in ukraine and are in demand among consumers. it should be noted that canned milk have a low nutritional value despite on the popularity of these products among people from the countries of former soviet union. the reasons for the low nutritional value are thermal processing of raw milk for a long time; high carbohydrate content; caloric; unbalanced chemical composition of condensed canned milk with sugar produced by classical technology [2, 3]. all mentioned factors encourage producers to insertion the untraditional materials in production and manufacturing products with combined composition. today among the untraditional raw material, used in the technology of condensed canned milk (ccm) with sugar, we can mention soy concentrates vegetable fats, fillers (coffee, cocoa, chicory, pectin, and tea), structure-, sugar substitutes, and sweeteners [2]. the raw materials of plant origin usage is a good way to cover the deficit of vitamins, minerals and other nutrients in the human diet. insertion of these materials food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 194 will enable manufacturers to expand the range of flavoring products on the ccm market, to satisfy needs of more customers. so developing the technology of condensed canned milk with adding plant origin fillers is relevant for implementation in industrial production. the nutritional value of processed fruit and berries and final product quality strongly depends on the quality of the feedstock. many factors influence the chemical composition of fruits and berries, their size, color and other technical indicators. it should be considered in usage the particular type of raw material for the production of various fillers. after the literary searching, we have established the basic criteria for selection of fruit berry fillings (fbf), which include organoleptic and rheological parameters of condensed canned milk with sugar and fruit and berry fillings (fig. 1). 2. matherial and methods selection by the organoleptic characteristics. choosing the organoleptic characteristics as criteria was caused by the fact that it is the main factor in food choosing for majority of consumers taste. rheological parameters were chosen since they provide the fullest characteristics of the structural and mechanical properties of developed products; moreover viscosity indicators are regulated by normative documents on canned condensed milk [4]. the indicators which are standardized by normative documentation for ccm with sugar also include indicators of microstructure, for example the size of the lactose crystals. below in the work (table 1) the influence of crystal size for lactose consistency in condensed canned milk and the quality of products will be considered. fig. 1. the main criteria for selection of fruit berry fillings at the first stage concentrated juice, syrup, sauce, jam, suburbs, jams from such fruits berries materials as cherries, apples, currants, raspberries, blueberries, rose, food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 195 pear, strawberry, hawthorn , apricot, orange were selected as fillers for condensed canned milk . mass fraction of filler was justified by the mild taste in the developed samples and visually unchanged viscosity that was equal 15%. at first, suitability of the mentioned fillers for the usage in the ccm technology with sugar was determined by the organoleptic characteristics. taking into account lack of scale for estimation of organoleptic characteristics for ccm with sugar and fruit berry syrup (fbs) [2, 3], the author developed a 10point scale. the evaluation criteria are listed in table 1. the organoleptic evaluation of ccm with sugar and fruit berry fillings is shown in fig. 2. it was identified that after 3 ... 6 days storage in the studied samples with concentrated juice, puree, suburbs started the processes of mold and fermentation. the reason for the processes was a significant amount of free moisture that the fillers contained. it becomes available to the microorganisms that increase their livelihoods after osmoanabiozu. considering and summarizing organoleptic properties, it was found that such fruit – berry fillings like jams and syrups are suitable for usage in the technology of condensed canned milk with sugar. selection by the rheological parameters. ccm is a long storage product during which it is necessary to ensure the stability of its structure and properties. by entering of fbf mass fraction of moisture, higher than the mass fraction of moisture in classic ccm, there exist a risk of unstable systems, tend for separation. table 1 assessment of organoleptic characteristics for ccm with sugar and fbf no. name of the evaluation criteria requirements for the product according to the criterion evaluation, scores 1 flavor and smell sweet and pure with an expressed flavor of filler without foreign tastes and odors 5 sweet and pure with a strong flavor of the filler, without foreign tastes and odors 4 sweet, pure, with a weakly expressed flavor of filler without foreign tastes and odors 3 sweet, pure, with no expressed flavor of filler without foreign tastes and odors 2 sweet, with no expressed flavor of filler with other defects of taste and smell 1-0 2 consistency homogeneous by all condensed mass, without impurities that are perceptible organoleptically 2 not homogeneous in all condensed mass of impurities that are perceptible organoleptically 0-1 3 color homogeneous by all condensed mass, which corresponds to the type of filler, natural shades 1 not homogeneous in all condensed mass, not natural shades 0 4 superficies glossy, smooth, net surface 2 with a matte shade, smooth, net surface 1 uneven surface with not shades of natural color 0 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 196 therefore, the new fillers insertion requires an examination of their influence on the structural and mechanical properties of the finished product. in accordance with the organoleptic characteristics the preference was given to jam and syrup. at this stage structural and mechanical properties and the ability to bundle in ccm with sugar and fbf were determined. taking into account that variability of tastes will not influence the rheology of finished product and considering its price accessibility and demand, the syrup of blackberry was chosen as an object of investigation. fig. 2.a. profile the organoleptic evaluation of condensed canned milk with sugar and fbf fig. 2.b. profile of the organoleptic evaluation of condensed canned milk with sugar and fbf 0 1 2 3 4 5 flavor and smell consistency color superficies ccm with sugar and concentrated juices ccm with sugar and fruit berry syrups ccm with sugar and pidvarka 0 1 2 3 4 5 flavor and smell consistency color superficies ccm with sugar and concentrated juices ccm with sugar and fruit berry syrups ccm with sugar and pidvarka food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 197 the effective viscosity is the main rheological indicator, which describes the structural and mechanical properties of the product. that’s why the effective viscosity indicator was determined on a rotary viscometer “reotest-2” for the samples, produced in the laboratory [5, 6, 7]. at the first stage of processing experimental data the shear stress system of p was calculated by the equation: p (1) where  constant of inner cylinder (shown in the passport of the device);  eaning of the scale on the indicator device. the next step in analyzing of received results was calculation of the dynamic viscosity. for the calculation the newton’s equation was used. it looks like this: 10   p (2) where  coefficient of dynamic viscosity, pа  s; p shear stress, pа;  shear rate, 1/s. 3. results and discussion from represented rheological viscosities curves the parameters η0 (most of viscosity with not destroyed structure) and ηm (minimal viscosity systems with almost destroyed structure) were determined for each sample. to display the status of the ccm system with sugar and fillers the dynamic viscosity that reflects the complexity of the process of the system flow under the influence of external forces was determined. the research results are presented in fig. 3. according to fig. 3 it was determined that the structure decreases in a very wide range of voltages (from 1000 to 5000 pa) with increasing shear rate of viscosity, and after the total destruction of structure that corresponds to the critical stress pcr, the viscosity remains constant [1]. fig. 3. rheological viscosity curves of condensed canned milk with sugar and fruit filling analysis of the rheological curves of test samples of ccm viscosity indicates that the largest ccm viscosity with sugar is equal to 6.6 pa·s, for ccm with sugar and jam 5.8, with syrup 5.4 pa·s; while the lowest viscosity that destroyed structure of the control sample was 3.7 pa·s, and for the condensed canned milk with sugar and fbs for both types the indicates were lower by 24% and were equal to 2.8 pa·s. the calculation results of basic rheological characteristics of the systems with different fbf are shown in table. 2. it is known that the value of the difference parameters (η0-ηm) characterizes the strength of formed systems of supramolecular structures. so we can conclude that supramolecular structure of ccm with jam and controls is the strongest (3.0 and 2.9 pa·s, respectively), and the addition of fruit-berry syrup makes it slightly weaker (η0-ηm =2,6 pа·s). the difference in the impact nature of different fruit-berry filling types on rheological indicators of the ccm can be explained by food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 198 different dynamic viscosity of the added fbf. in order to determine the stability of the samples of formed condensed canned milk with sugar and fruit-berry fillers comparing with the control, rheological fluidity curves were built on the next stage (fig. 4). depending on the character of the rheological fluidity curves it is possible to say that the structure of the systems can be classified as weakly structured (viscoplastic) type as pk1>0. analysis of rheological fluidity curves showed that the major parameters of conditional dynamic threshold fluidity (pk2) and the strength of the structural framework of supramolecular ties (рm) sample was characterized by the ccm with sugar and jam for which these indicators were higher by 8 and 6% comparing to classic ccm with sugar. it was identified that the syrup adding to ccm lowered the threshold fluidity of the product and weakened the strength of the structural frame, which is consistent with durability of formed supramolecular structure data and is explained by fbs chemical composition. table 2 basic rheological properties of condensed canned milk with sugar and fruit-berry fillings № name of the sample η0, pа·s ηm, pа·s η0-ηm, pа·s рk1 pk2 рm рk1/pk2 рm/pk2 1 ccm with sugar 6.6 3.7 2.9 138 4050 4900 0.034 1.21 2 ccm with sugar and fruit berry currant jam 5.8 2.8 3 180 4400 5200 0.041 1.18 3 ccm with sugar and fruit berry currant syrup 5.4 2.8 2.6 183 3350 4500 0.055 1.34 figure 4. rheological curves fluidity of condensed canned milk with sugar and fruit-berry fillings food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 natalija valeriivna ryabokon, dmitro viktorovich rindyuk, choosing of fruit berry fillings for condensed canned milk with sugar, food and environment safety, volume xiii, issue 3 – 2014, pag. 193 – 199 199 at the same time the types of fruit and berry fillings had no significant effect on the strength of structural connections (rk1/pk2) and the range of stress in which were the system’s destruction (rm/pk2) [8]. considering the mentioned threshold dependence of the dynamic fluidity from type of filling and importance of this indicator for long-term storage products, we determined the rate of fluidity next. fluidity is a characteristic of condensed canned milk with sugar, which is characterized by a coefficient (кf) that is measured by the mass of the product (m), which flows out of the hole with diameter defined by r in unit time (τ) [9]. к = , (3) it was defined that the rate of fluidity for condensed milk with sugar (control sample) was 7.0 ... 7.1 units; for ccm with sugar and fbs 6.9 ... 7.0; ccm with sugar and fruit-berry jam 6.2 ... 6.4. 4. conclusion the investigated canned condensed milk with sugar and fruit fillings can be attributed to weakly structured fluids in terms pk1> 0, which indicates the nature of the formed system, and is consistent with the data of ccm with sugar of classic range. the investigated fruit does not have a significant effect on the strength characteristics of condensed canned milk with sugar structure fillings in there amount of 15% of weight of the finished product. considering the impact of fruit-berry jam on the indicators of conditional dynamic threshold fluidity and fluidity ratio of ccm with sugar and possible losing the fluidity of the product during storage, the preference should be given to fruit-berry syrup in the ccm recipes with sugar and fbf development. 5. references [1]. heilbronn l., the effect of highand lowglycemic index energy restricted diets on plasma lipid and glucose profiles in type 2 diabetic subjects with varying glycemic control: translation from english by heilbronn l., noakes m, clifton p., p. 120, (2002). [2]. skorchenko t., pukhlyak a., ryabokon n., patent for a useful model #56598 mpk (2011) a23c9/00. method densified canned milk with fruit fillings. [3]. karel m., principles of food preservation: translation from english by m. karel, d. lund, p. 400, (2011). [4]. symonenko o., rheology viscous food systems based on milk. food industry #2, pp. 1214, (2012). [5]. skorchenko t., determination of rheological properties of condensed canned milk with sugar and fruit fillings. equipment and technology of food production. issue #27, donetsk, pp. 301-305, (2011). [6]. kuznetsov o., rheology of food: translation from english by kuznetsov a., voloshin e., sagitov r., p. 106, (2005). [7]. diagon v., rheology of dairy products (full course): translation from english by squint v., dunchenko n., merkulov m., p. 826, (2010). [8]. datta a., status of physics-based models in the design of food products, processes, and equiptment. comprehensive review in food science and food safety, p. 129, (2010). [9]. ceylan m., mathematical modelling of drying characteristics of tropical fruits. journal of food engineering. issue #75, pp. 193-196, (2011). 60 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 1 – 2014, pag. 60 67 dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum *inna mihaylivna zubareva1, nataliia borisivna mitina2, anastasia vadimivna vasilkova2 1national university of dnepropetrovsk, ukraine 2“dnepropetrovsk chemical-technological state university”, dnepropetrovsk, ukraine zubareva95@bk.ru, natalimitina_68@mail.ru *corresponding author received march 6th 2014, accepted 28th 2014 abstract. the need for formulations of biologically active substances of natural origin currently increasing.the industrial production of such products biotechnological method important, but limited by a number of unsolved problems and shortcomings.so, this paper studied the effect of the qualitative composition of culture media and substrates for biosynthetic activity and carotene producing filamentous proteins and red californian worm producing biologically active substances and feed protein.producers offered for mycelial growth medium containing a carbon source enzymatic hydrolysates flour of various grains, and as a source of nitrogen gluten or soy milk.found that filamentous organisms producers of carotenoids and proteins accumulate high amounts of both biomass and target metabolites on nutrient media containing hydrolysates of buckwheat, oat or corn flour in the presence of gluten. used for the cultivation of vermiculture modified sunflower husk, which eisenia foetida, as a producer of protein drugs and medical fodder has balanced essential amino acids, vitamins, enzymes and trace element composition of the biomass.these results show that the optimization of culture media and substrates provides increased biomass yield and biologically active substances in the few times producers of various taxonomic groups. key words: mucoraceous mold, enzymatic hydrolyzates, biomass, activity, carotene, higher edible fungus, vermiculture, nutrient media, substrata. 1. introduction requirement of different sectors of the national economy and medicine in bioactive substances, especially of the natural origin increasingly and stably rises nowadays, what is acknowledged by several interesting works [1-8]. however the problem of industrial productivity of specimens on the basis of biologically active substances remains unsolved, that is why actual. the difficulty of the given situation can be explained by imperfection or lack of elaborated technologies. those existent in many cases have a number of faults. the necessary conditions for successful realization of elaborated technologies of receiving biologically active substances are: presence of the select industrial producer of the appropriate target substance and raw material sources which is enough for growing of the given producer. though, main genetic properties food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 61 of the producers determine quality of the bioactive substance which is produced. whereas, quantity of the received target product depends not only on genetic of the definite producer, but also, in higher degree, on chemical composition of nutrient media and substrata on which this industrial producer is cultivated. such dependence refers to the producers of bioactive substances from different taxonomic groups. thus, lower mucoraceous molds from the class of zygomycetes blakeslea trispora and choanephora conjuncta are used as industrial producers of carotene substances [9, 10]. but the composition of synthesizable carotenoid and primary outgoing of one of them depend on the composition of zymotic nutrient media. for example, with presence of cellobioze in the nutrient medium, outgoing of carotenoids increases sevenfold [9]. presence of ion-substances in cultivated liquid provides primary synthesis betacarotenes, but pyridine-substances stimulate accumulation of lycopene [11, 12]. higher edible fungus from the class basidiomycetes pleurotus ostreatus potentially can be used for production of bioactive substances of the protein nature [6, 7, 13]. outgoing of protein is defined by qualitative and quantitative composition of the nutrient media and substrata on which the producer is grown. thus, in caprosome of oyster mushroom rising on natural substratum, for example, on wood, the quantity of protein is not more than 20%. on the liquid nutrient media optimized by the sources of nitrogen and carbon, content of protein can reach 30– 50% [13]. producers of biologically active substances of different chemical nature may be not only specimens of microorganisms and fungus, but also of animals. thus, private active components were isolated from the biomass of red californian worm eisenia foetida. they are capable to enforce the immunity of a human being, decrease a risk of cardiovascular and oncological conditions, increase resistance of organism to germ and viral diseases [8, 18, 19, 20]. the given organisms are producers of biologically active substances which have intracellular localization. however, it is known that the processes of accumulation of biomass of producers and synthesis of targeted bioactive substances in their cellular are stimulated by different nutrient components of media and substrata. whereas, when intercellular bioactive substances are received in the industrial conditions, it is economically expedient and important to accumulate both biomass and targeted products in them as much as possible. consequently, the given task is actual and may be solved by the way of optimization of qualitative compositions of nutrient media and substrata for appropriate producers. the purpose of the given work is identification of the dependence of intensity of synthesis of some biologically active substances from quality of nutrient media and substrata of proper producers of different taxonomical groups. 2. materials and methods as an object in experiments one used lower mucoraceous mold імв f-100019 variants (+), (–) blakeslea trispora (institute of microbiology and virology national academy of science of ukraine, kyiv) – producer of beta-carotene, higher edible fungus pleurotus ostreatus (pleurotus ostreatus) strain нк-35 (department of microbiology, institute of botany, kyiv) – as producer of protein substances, as well as vermiculture eisenia foetida (research institute “biotechnology” state university, dnepropetrovsk specification 3336406.00 2–95) – producer of biologically active food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 62 components, protein biomass. growing of mucoraceous mold blakeslea trispora was implemented in several stages. work culture was received by separate passage of museum swarms on the wart-agar nutrient medium and sufficient growing for 7 days. mother culture was received by the following also separate passage on the liquid nutrient media in flasks with capacity of 300 ml. the composition of mother nutrient media %): corn extract – 13, green treacle – 7. time of growing of the mother culture is three days on the termobiological termostatical shakers увмт-12-250, working at a speed of 220 – 240 rpm/min. the received (+), (–) mother inoculum was carried for combined growing in to zymotic flasks with capacity of 300 ml. correlation (+), (–) strains for combined cultivating is 4:1. the composition of the zymotic media is different. extract-treacle nutrient media contain (%): corn extract – 6, green treacle – 6, potassium dihydrogen phosphate – 0.05, thiamine chloride – 0.0002, vegetable oil – 4. alternative nutrient media include: enzyme hydrolyzate of different types of flour and shorts, as well as gluten or soya milk in different concentrations. all used nutrient media were under thermal sterilization at 120°с for 45 min [14]. duration of fermentation was 5 days on the microbiological shaker увмт-12-250, working in the mentioned above regime of hashing. the temperature on all stages of cultivation 24–26°с. at the end of fermentation the received cultural liquid (cl) was separated on centrifuge т–23 at 3000 rpm/min for 10 min. cl was analyzed according to quantity of biomass and beta-carotene using known methodology [14, 15]. the experiment was repeated 5 times. the culture of the fungus pleurotus ostreatus was kept on the wartagar nutrient media in flasks from which they were replanted into the shaking flasks with capacity of 300 ml, containing 50 ml enzyme nutrient medium. the composition of the experimental media was pointed above, рн of media – 6.8–6.9. to receive enzyme hydrolyzes domestic enzyme preparations were used «alphalad» and «glucolad» (plant of enzyme preparations, ladyzhsk c., ukraine) with concentration 1:1900 and 1:2000 respectively. temperature regime of hydrolyze 60°с for 10–15 min. duration of deep growing pleurotus ostreatus – 5 days on the microbiological thermostatic shaker, the speed of hashing – 220–240 rpm/min., temperature of cultivating 26–28°с. at the end of fermentation in cl the quantity of biomass and protein was determined [14, 16]. filamentous mass was separated from liquid fraction cl by the way of centrifuging at 3000 rpm/min for 15 min. the experiment was repeated 7 times. the received results were processed with help of methods of mathematical statistics [17]. cultivation of eisenia foetida was held on the substrate of modified sunflower husks (sh) grained till fractions 200–500 microns, wet with water in correspondence (hydromodule) 1:2. extract was laid on the fermentation in special capacities (plastic clamps) as high as 50–60 sm. the process of fermentation lasts 12–14 days under influence of enzymes of microorganisms introduced by microflora of sunflower husks. to increase aeration of fermenting mass, activation of microbiological work of microorganisms, leveling humidity about the whole volume, precaution of rotting in the depth of a clamp, hashing of substrate was held once a day. bioprocessing by the producer of the fermenting substrate is held 45 days with accommodation density eisenia foetida 5– 10 thousand specimens per 1 square meter and at an altitude of the lay of substrate of 30sm. humidity of substrate is kept at a level 70–80%, temperature – 20–25°с, рн 6.5–7.5. additional forage as thick as 5 sm added in 10–15 days after settlement of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 63 basic substrate with culture eisenia foetida. with accommodation density 100 hundred per square meter, selection of biomass in a quantity of 75–80% was done. quality and harmlessness of the biomass of population eisenia fоetida, adapted on the modified oil husks were determined with the help of the instruction of the ukrainian security council (usc–15) scientific council of un on protein problems and veterinary – sanitary regulations. biological worth of the biomass eisenia foetida was determined with the help of method of amino-acid score according to known methodology as well as by the degree of digestion and adaptation of protein [14, 16, 20, 21, and 22]. 3. results and discussion in biotechnology deep way of growing producers including filamentous is economically expedient and technologically reasonable. that is why to receive filamentous biomass blakeslea trispora and pleurotus ostreatus with higher content of biologically active substances, beta-carotene and protein respectively, liquid nutrient media were used. the media of different compositions are offered. for example, extract-treacle medium contains such wastes of starch-treacle producing as corn extract (the source of nitrogen and factors of growth) and green treacle (the source of carbon and energy). for comparison there were learnt the nutrient media including other sources of the development of producers of biological active substances. thus, так, as a source of carbon and energy were used wastes containing starch or secondary products of milling production, which were put to the fermenting, hydrolyze in advance, but as a source of nitrogen, gluten and soya milk were used. concentration of these components in the medium was determined by quantity of sugar in them (0.15%) and of nitrogen (0.22 – 0.24%) respectively. the received results on accumulation biomass and beta-carotene in mucoraceous mold blakeslea trispora, are introduced in the table 1. table 1 dependence of outgoing biomass and beta carotene in blakeslea trispora on the quality of nutrient media source of carbon gluten soya milk dry biomass, g/100ml content of beta carotene, mg/100ml dry biomass, g/100ml content of beta carotene, mg/100ml hydrolyzer of corn flour 2.98±0.13 67.67±3.37 2.77 ± 0.14 38.57 ± 1.92 hydrolyzer of buckwheat flour 3.10±0.15 51.09±2.54 2.95 ± 0.15 30.40 ± 1.51 hydrolyzer of wheat flour 2.26±0.13 21.26±1.95 2.03±0.13 16.53±1.65 hydrolyzer of wheat shorts 2.07±0.11 18.20±1.73 1.83±0.10 15.24±1.38 hydrolyzer of oatmeal flour 3.06±0.15 42.13±2.10 2.81 ± 0.14 33.06 ± 1.64 hydrolyzer of rie flour 2.76±0.13 39.26±1.95 2.48 ± 0.14 25.89 ± 1.48 the data of the table 1 indicate that the highest outgoing of the biomass containing carotene is observed on the media containing gluten and hydrolyzers of buckwheat, oatmeal, and corn flour, but the highest activity (quantity of beta-carotene) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 64 – on the medium with hydrolyzate of corn flour. analogical situation is found on the media with determined sources of carbon in combination with another source of nitrogen – soya milk. but on all investigated hydrolyzer far more quantity of mucoraceous mass is provided by the presence of gluten from 2.07 through 3.10 g/100 ml cl against presence of soya milk from 1.83 through 2.95 g/100 ml cl. content of beta-carotene is sure to be lower with presence of soya milk in hydrolyzer than with input of gluten. the difference between absolute amount of biomass on the media with gluten and hydrolyzer of buckwheat, oatmeal or corn flour is doubtful. concentration betacarotene on the medium with gluten and hydrolyzer of corn flour is 1.33 fold higher, than on the medium with gluten and hydrolyzer of buckwheat flour and 1.60 fold bigger, than on the media with gluten and hydrolyzer of oatmeal. consequently, analysis of the table 1 lets reveal optimal composition of the nutrient media providing high outgoing of biomass (in fact 70 mg/100 ml) and the biggest outgoing beta-carotene (about 70 mg/100 ml) in it: hydrolyzer of corn flour (0.15% sugar) in combination with gluten (0.22– 0.24% nitrogen). it is necessary to admit that content of biomass (2.68±0.130 g/100 ml) and beta-carotene (33.25±1.82 mg/100 ml) on the extract-treacle medium is lower than on the optimal one 1.1 fold and 2.06 fold respectively. the results on accumulation biomass and protein in higher edible fungus pleurotus ostreatus (oyster mushroom) strain нк-35 of fungus, received in the conditions of the given experiment are introduced in the table 2. table 2 dependence of outgoing of biomass and protein in pleurotus ostreatus on the quality of nutrient media source of carbon gluten soya milk dry biomass, g/100ml content of protein, % dry biomass, g/100ml content of protein, % hydrolyzer of corn flour 2.49±0.13 57.89±2.80 1.99±0.10 41.40±2.35 hydrolyzer of buckwheat flour 2.93±0.14 60.40±2.65 2.03±0.10 42.04±2.18 hydrolyzer of wheat flour 2.12±0.12 58.52±2.73 1.69±0.07 40.97±2.075 hydrolyzer of oatmeal flour 2.43±0.12 52.99±2.43 1.89±0.14 44.23±2.214 hydrolyzer of oatmeal flour 2.19±0.109 56.05±2.80 1.95±0.087 38.87±1.89 hydrolyzer of rie flour 2.28±0.114 55.16±2.75 2.00±0.100 43.55±2.17 out of demonstrate table it is clear shown that higher quantity of the deep biomass of oyster mushroom is accumulated on the media containing hydrolyzers of buckwheat, corn and wheat flour in combination with gluten. on the same media one can see also higher content of protein in biomass. combination of the pointed hydrolyzers with soya milk provides receiving of lower quantity of biomass than on gluten 1.4 fold; 1.2 fold and 1.2 fold respectively. this regularity is observed also in analysis on the content of protein in the deep biomass of fungus. thus, receiving of biomass pleurotus ostreatus with high content of protein (till 60%) is possible. for it fermenting hydrolyzers of different starch-containing raw may be applied. though, usage of of hydrolyzers of buckwheat, corn and wheat flour is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 65 optimal. with the given experiment such source as nitrogen acquired a great importance as a component of the nutrient medium for accumulation of biomass and protein of the oyster mushroom. thus, gluten provides big accumulation both biomass and protein in it against soya milk. but with presence of soya milk in the content of the nutrient medium, the very important property of floccus, namely, appearance of pleasant mushroom smell distinctive for oyster mushroom of natural growth. presence of proper smell and taste is necessary feature of the deep floccus as biotechnological product of food purpose. that was also found that growing of oyster mushroom on the extract-treacle medium doesn’t provide the highest outgoing protein-containing biomass of fungus. concentration of floccus in cl reaches 1.82±0.091 g/100ml, but content of protein is 45.99±2.01%, that is 1.5 fold and 1.3 fold lower than on the fermenting hydrolyzer of buckwheat flour in combination with gluten. efficiency of the frocess vermicultivation, except mentioned above is stipulated by biochemical high quality of the nutrient substrate, which is defined by many interconnected parameters, including physical condition of substrate. thus, to a considerable degree, nutrional value of substrate depends on the degree of grinding of the original phytogenous material. grinding sh leads to dimension of the degree of order of its submicroscopic structure. as a result of it valuable components of nutrition meal relieve out of the structure of husks and become more accessible for recycle by vermiculture. one time size, form and structure of the particles of modified sh are adoptable for productive assimilation by population eisenia foetida [23, 24, and 25]. the results of biochemical analyses showed that biomass eisenia foetida, cultivated on the modified sh, contains in a wellbalanced form necessary substances and vitamins– в1–0,03 mg/kg, в2–0,04 mg/kg, рр–0,9 mg/kg, d – 4–6 hundred ме, microelements of blood-making action, which respond to the requirements on the necessary substances for fattening of saplings of cattle and increase of immunity. there are also a set of enzymes producing by eisenia fоetida, lipids with components of higher fatty acids of both saturated and not saturated [25, 26]. veterinarian-sanitarian evaluation of biomass eisenia fоetida was held in three variants–fresh, boiled for 30 min, dried for three days (with breaks for 8 hours at night time). in all cases common bacterial insemination did not increase 500 germ bodies in 1g of biomass. at the same time pathogenic types of escherichia coli, forming toxins, as well as salmonella in tests weren’t found. biotest for toxins of botulism was also negative [21]. out of biomass eisenia fоetida, adapted on modified sh, dry powder could be received. preparation of dry biomass is friable, dark-grey powder with strong smell of the liver of cattle. when it is stored in the condition 18–22°с, and with 0°с, it has distinctive hygroscopicity. when it is stored without oxygen for 30 days, friability of the prepared product is kept what lets mix dry biomass with other fodders [22, 27]. there was investigated amino-acid composition of the dry biomass of the vermculture population of eisenia fоetida, adapted on modified sunflower husks. analisis showd that it consists of protein – 63.6%, lipids – 10.5–17.5%, nitrous extra active substances. amino-acid content of dry biomass of the culture eisenia fоetida is introduced in the table 3. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 66 table 3 amino-acid content of dry biomass of the culture eisenia fоetida adapted on modified sunflower husks amino-acid from content of protein, g from content in biomasseisenia fоetida, g lysine 6.27 3.99 histidine 2.09 1.33 arginine 6.62 4.21 asparagine acid 8.09 5.15 threonine 4.58 2.91 serine 4.28 2.72 glutamic acid 17.33 11.02 proline 3.73 2.37 glycine 3.43 2.18 cystine 11.67 7.42 valine 4.56 2.90 methionine 1.97 1.25 isoleucine 4.54 2.89 leucine 6.15 3.91 tyrosine 3.74 2.38 phenylalanine 4.03 2.56 alanine 5.98 3.80 biological adequacy of biomass eisenia fоetida was defined according to aminoacid score. there is (mg): isoleucine–71, leucine–97, lysine–63, methionine, cystine–214, phenylalanine and tyrosine– 122, threonine–72, valine–72 in 1g of protein which was selected out of biomass eisenia fоetida. against standard score scale content of the named amino-acids correspondingly consists (%): 177.5; 138.6; 114.5; 523; 203; 180; 144. the least quantity in biomass of lysine (score 114.5%), the most is sum of phenylalanine and tyrosine (score 523%). producer eisenia fоetida, cultivated on the modified subtract sh, is balanced according to amino-acid content and all not changeable amino acids, more saturated with microelements, vitamins, enzymes and other more active substances than the culture adapted on another types of substratum. 4. conclusions thus, at present experimental work was shown the dependence of accumulation of biomass and synthesis of some biologically active substances (carotene, protein, not changeable amino acids) on quality on nutrient media and substrata of the proper producers. this regularity is typical for the producers of the biologically active substances from different taxonomical groups: lower mucoraceous molds, higher edible fungus, specimens of cattle – vermiculture eisenia fоetida. consequently, the problem of receiving of biologically active preparations of different origin may be effectively solved even in the conditions of industrial production with the help of optimization of the composition of nutrient media and substrata. 5. references [1]. bykov v., monacov m. biotechnology. production of proteins. moscow: higher. school, 143, (1987) [2]. kinda v. perspectives of biologically active substances blakeslea trispora biomass in the agricultural sector of ukraine. biotechnology. education. science: proc. ext. 1 all-ukrainian scientific-practical conference. students and young food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 1 – 2014 inna mihaylivna zubareva, nataliia borisivna mitina, anastasia vadimivna vasilkova, dependence of the biosynthetic ability of the producers of biologically active substances on quality of nutrient medium and substratum, issue 1 2014, pag. 60 67 67 scientists kiev, 112-113, (2003) [3]. martynovsky v., zakharenko n. biomass fungus blakeslea trispora, as a source of β carotene and dietary substances. use carotenoids microbial origin in agriculture komplksi: proc. ext. intern. conf. young scientists and students. amounts, 100-105, (2002) [4]. zhyvoder o., kinda v. physiological aktyvnye compounds mytselyalnoho fungus blakeslea trispora. proc. ext. intern. conf. young scientists and students. amounts, 140-152, (2002) [5]. mhytaryants l., verteletskaya v., nezhenets e. using dietary supplements in the production of functional produktov appointment. proc. ext. intern. conf. young scientists and students. amounts, 159-162, (2002) [6]. kulik a.p., zubareva i.m. pleurotus ostreatus as a source of feed protein. problems of chemistry and chemical technology, 3. 51-53, (2006) [7]. lyapustina o. v., zubareva i.n., pozharsky v.y., sereda u.u. getting mycelial biomass of pleurotus ostreatus edible. chemistry and chemical technology: materials 1 intern. conf. young, lviv, 48-49, (2010) [8]. mitina n.b, zubareva i.m. research and selection of culture media, strains of producers to obtain biogas process followed vermikultyvuvannya. vopr. chemistry and chem. technology. 4, 24-26, (2012) [9]. yelinov n. fundamentals of biotechnology. st. petersburg. nauka, 600, (1995) [10]. o. kalinkevich, a.n. kalinkevich, v.d. chivanov, v.i. kindi. biochemical composition of fungal biomass blakeslea trispora. prirodnichy almanac. 13, 39-49, (2009) [11]. zubareva i.m., mitina n.b., babich y.v.. study ionones as stimulants development blakeslea trispora. issues. chemistry and chemical engineering. technology. 3, 47-49, (2012) [12]. feofilova e.p., tereshina v.m., memorsky a.s. regulation of synthesis of lycopene from the fungus blakeslea trispora mokorovogo pyridine derivatives. microbiology. 64, 734-740, (1995) [13]. velichko t.a., mitina n.b., weaver ai, shatalin db. optimization of culture media for the cultivation of pleurotus ostreatus proceedings onaft 2, 165-168, (2011) [14]. egorov n.s .guide to practical training in microbiology. moscow state university, 256, (1983) [15]. tereshina v.m., memorsky a.s., feofilova e.p. express a method of determining the content of lycopene and β-carotene. microbiology.63, 1111-1116, (1994) [16]. guidelines for zootechnical feed analysis. dnepropetrovsk: dgau, 47, (2007) [17]. frolov l.a., melnikov b.i., halivets j.d., mitina n.b. mathematical modeling and optimization technology objects inorganic substances. dnepropetrovsk: jour. foundation, 208, (2010) [18]. vasileva a. dietary supplements: a new word in the science of health. st. petersburg. nevsky prospekt, p. 192, 2001. [19]. kaminsky v.f. on the issue of solving the problem of protein. bulletin. agrarian. science. 12, 12-14. (2003) [20]. gorodnii n.m., kovalev v.b., miller i.a. vermiculture and its effectiveness. sel. host ˗ in; review. inform. ser. agriculture, agricultural chemistry with. x. reclamation. ukrniinti. 20, (1990) [21]. kucherenko m.e. modern methods of biochemical research. kiev. phyto ˗ center, 424, (2001) [22]. mitina n.b. research technologies of protein supplements. 1. prospects for feed protein questions of chemistry and hymycheskoy technology. 2, 44-48, (2009) [23]. kulik a.p., garmash s.n., polishchuk n.b. processing plant waste zoned ukraine to obtain drugs feed, veterinary, medical devices and biologically active substances (growth regulators). storage and processing of grain. 1, 810, (1999) [24]. mitina n.b., danyleyko t.s., kulik a.p. complex processing of natural polymers of plant origin. questions of chemistry and hymycheskoy technology. 1, 137-139, (2003) [25]. kulik a. p. produccing a feed additive on basis of sunflover shel. ecology of the indastrial regions. gorlovka˗kiev. p. 111–115. 1995. – [26]. mitina n.b., kalashnikov s.g., frolova l.a. research technologies of protein supplements. 2. resource base for vermitehnolohiy. questions of chemistry and hymycheskoy technology. 3, 32-34, (2009) [27]. mitina n.b., kulik a.p., kalashnikov s.g. research technologies of protein supplements. 3. biological purity raw materials for vermitehnolohiy. questions of chemistry and hymycheskoy technology. 5, 24-27, (2009) microsoft word 00 primele pagini 2_2023 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xxii, issue 2 30 june 2023 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania doi: https://doi.org/10.4316/fens.2021.023 205 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue3 2021, pag. 205 210 physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation * vlasios goulas , polina aresti department of agricultural sciences, biotechnology and food science, cyprus university of technology, lemesos, cyprus, vlasios.goulas@cut.ac.cy *corresponding author received 12th july 2021 2021, accepted 20th september 2021 abstract: the carob syrup has been attracted the interest of consumers as an alternative sweetener due to its superiority against refined ones. bioactive phytochemicals and polyols, vitamins and minerals are present in carob syrup; whereas sucrose, fructose and glucose are the main sugars. although the consumers prefer carob syrup for its special composition, the sucrose supplementation in carob syrup is a common adulteration abating its health benefits. the detection of adulteration in natural sweeteners and honey is usually required time-consuming and advanced analytical techniques. in the present study, the physicochemical properties of carob syrup were studied to pinpoint physicochemical parameters for detection of sucrose supplementation. more specific, moisture, fats, ph, color parameters and total soluble solids were determined. furthermore, sugar composition of carob syrups was studied using chromatographic separation. finally, the phenolic fraction and antioxidant activity of carob syrups were estimated. results showed significant differences in total soluble solids and moisture contents, but these parameters are linked with its preparation procedure. furthermore, an unexpected fluctuation was observed for d-pinitol and total phenolics contents. thus, both parameters were determined in genuine and sucrose-supplemented carob syrups. based on our findings the sucrose supplementation can be detected by d-pinitol content and sucrose to d-pinitol ratio. therefore, the present work demonstrated the potential of a simple chromatographic separation to detect the adulteration in a traditional mediterranean product. keywords: adulteration, sugars, d-pinitol, carob syrup, chromatography, phenolics 1. introduction the carob syrup is a traditional sweetener native to the mediterranean basin. it comprises a high proportion of sugars namely sucrose, fructose and glucose (> 65%) and small amounts of polyols [1]. it also contains additional nutritive compounds such as polyphenols, minerals, proteins and fibres. furthermore, the energy density and glycaemic index of carob syrup is lower than refined sweeteners as sucrose [2]. moreover, the traditional medicine describes the use of carob syrup to treat gastrointestinal and venereal diseases and to control rheumatic disorders [3-4]; whereas the health effects of many carob syrup constituents like phenolic compounds, fibres and polyols have been documented [5-6]. the aforementioned benefits of carob syrup and the increased demand for natural and healthy sweeteners have attracted the interest of consumers. the carob syrup is produced according to traditional method in cyprus. in particular, the chopped carob fruits were suspended with slightly warm water (1/3, w/v) to recover sugars as carob juice. subsequently, the carob juice is concentrated via boiling at least 66.5 °brix. this procedure is guided to the low yield of carob syrup due to the use of slightly warm water at the stage of maceration. the use of hot water increases the yield of sugar contents but it also recovers https://doi.org/10.4316/fens.2021.022 http://fens.usv.ro/index.php/fens food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 vlasios goulas, polina aresti, physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation, food and environment safety, volume xx, issue 3 – 2021, pag. 205 – 210 206 undesired astringent tannins. thus, some patents have been registered in order to transcend this drawback but the utilization of expensive chromatographic techniques is required. therefore, the adulteration of carob syrup using sucrose is widespread increasing the yield of syrup. carob fruits and its derivatives contain significant amounts of sucrose [1, 5], thus, it is difficult to detect the sucrose supplementation in carob syrup. the objective of the present study was to monitor physicochemical properties of cypriot carob syrup to discovery useful indexes for adulteration. then, the most promising properties were used to compare genuine carob syrups and sucrosesupplemented ones. 2. matherials and methods carob syrups at first, twenty-five commercial carob syrups were purchased from local markets for two successive years. for the preparation of genuine and sucrosesupplemented carob syrups, tilliria’ carob fruits were harvested at maturity stage from limassol and pafos districts. fruits were transferred to the laboratory and used for the preparation of carob syrup according to traditional method used by cypriot families (figure 1). fig. 1. preparation process of carob syrups according to the traditional method. proximate analysis the moisture contents of carob syrups were measured by a moisture analyzer (kern mls-50-3, kern & sohn gmbh, germany). the ph of samples was also measured by a benchtop ph meter (hi2211, hanna instruments, usa). fat contents were determined using bligh & dyer method. total soluble solids (brix) were determined using a portable digital refractometer (dr103l, sun instruments corp. usa). color carob syrups were placed in a uniform layer (0.5 cm) on a 5 cm diameter petri dish with the employment of a reflection colorimeter (cr-400, konica minolta sensing, inc., osaka, japan). the measurement head was over the dishes. color was calculated using the cie‐l* a* b* uniform colour space (cie‐lab), where l* indicates lightness, a* indicates hue on a green (‐) to red (+) axis, and b* indicates hue on a blue (‐) to yellow (+) axis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 vlasios goulas, polina aresti, physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation, food and environment safety, volume xx, issue 3 – 2021, pag. 205 – 210 207 total phenolic content and antioxidant activity total phenolics content of carob syrups was estimated by a colorimetric assay based on previous workd with some slight modifications [7]. more specific, 5 g of syrup were shaken with 5 ml of ethyl acetate for 5 min. then, the mixture was centrifuged for 10 min at 4700 rpm. the procedure was repeated three times and the filtrates were merged and used for total phenolic content and antioxidant activity assays. a 400 μl aliquot of sample was mixed with 500 μl of folin-ciocalteu reagent and 4.6 ml of deionised water. after mixing the contents for 3 min, 1 ml of saturated sodium carbonate and 3.5 ml of deionised water were added. samples were left to stand at room temperature for 60 min. absorbance measurements were taken at 725 nm. a standard curve of gallic acid was prepared and results expressed as mg gallic acid equivalents (gae) 100 g-1 carob syrup. ethyl acetate extracts of carob syrup was also used for the determination of antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (dpph) assay. in particular, 30 μl of extracts was reacted with 2 ml of dpph solution (0.3 mm) for 30 min at dark. subsequently, the absorbance of mixture was read at 517 nm and results are expresses as a μmol ascorbic acid equivalents (aae) 100 g-1 carob syrup by the preparation a standard curve of pure ascorbic acid [8]. sugar composition the samples were prepared according to previous work [1]. the sugar composition of carob syrups was analysed on a waters series hplc (waters corporation, milford, ireland) system equipped with vacuum degasser, binary pump, autosampler, thermostated column compartment, refractive index detector and empower software for data collection and analysis. the diluted samples were loaded on an aminex hpx-87p column (300 mm × 7.8 mm × 9 μm) at 80 °c. the flow rate was set at 0.5 ml min-1 and the injection volume was 20 μl as previous study describes for the separation of sucrose, glucose, fructose and d-pinitol in alfalfa biomass [9]. quantitation was performed by standard curves generated by chromatographic analysis of the standard solutions of the respective substances at various concentrations 3. results and discussion physicochemical properties of carob syrups physicochemical properties of carob syrups showed considerable differences between commercial samples. table 1 summarizes results of chemical analysis of twenty-five cypriot carob syrup that are available in local market. table 1. physico-chemical properties of commercial carob syrups produced in cyprus parameter mean median range moisture (%) 23.6 24.2 19.1-29.4 fat (%) 0.17 0.17 0.11-0.22 ph 4.57 4.58 4.39-4.80 l* 21.32 21.34 21.03-21.64 a* 0.34 0.18 0.11-1.20 b* 0.57 0.58 0.43-0.71 °brix 74.2 73.7 69.3-80.9 total phenolics* 912 960 300-1250 dpph activity** 129 129 103-161 *total phenolics are expressed as mg gallic acid equivalents 100 g-1 carob syrup ** dpph activity is measured as μmol ascorbic acid equivalents 100 g-1 carob syrup food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 vlasios goulas, polina aresti, physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation, food and environment safety, volume xx, issue 3 – 2021, pag. 205 – 210 208 total soluble solids of carob syrup were ranged between 69.3 brix and 80.9 brix; the median value of total soluble solids was found at 73.7 brix. previous studies also described similar total soluble solids for tunisian and turkish carob molasses [1, 10]. besides, the above concentration of sugars is required to avoid the growth of foodborne microorganisms. the moisture contents of carob syrups were inversely related with sugar contents; the median value of moisture was 24.2% w/w. the moisture content is linked with the evaporation process to concentrate carob juice. results also showed a low-fat content of carob syrups; the fat content was between 0.11 % w/w and 0.22 % w/w. it is expected as carob fruit contain small amounts of fats (~0.60% w/w) and the water is used as an extractor medium for recovery of hydrophilic sugars [11]. the ph of carob syrup is linked with soluble organic acids; there are no significant differences between carob syrups. similar ph values have also been found in tunisian carob molasses [10]. afterwards, the color values l∗, a∗, and b∗ were measured to designate the color space of carob syrups. more specific, the color is expressed as three values: l* for the lightness from black (0) to white (100), a* from green (−) to red (+), and b* from blue (−) to yellow (+). results demonstrated that only value a* had significant differences between studied carob syrups. in particular, carob syrups with high sugar contents had higher values of a*. on contrary, there are no considerable differences between values for l* and b* for carob syrup studied. carob syrups also contains significant amounts of phenolic compounds; whereas the gallic acid is the major constituent of phenolic fraction (>80%) [12]. results also showed a high phenolic content in carob syrup; its mean value was 960 mg gallic acid equivalents 100 g-1. however, a small number of samples had 3-fold lower phenolic contents. the latter cannot be explained by the diversity of phenolics in carob fruit. furthermore, the antioxidant activity of carob syrups was determined with the employment of dpph assay. the antioxidant activity of carob syrups was ranged from 103 to 161 μmol ascorbic acid equivalents 100 g-1. this assay revealed shorter variation between carob syrups than folin-ciocalteu assay. it can be attributed to the chemistry behind of each assay. the dpph assay measures the ability of antioxidants to scavenge free radicals; whereas folin-ciocalteu assay is based on the transfer of electrons in alkaline solution from the phenolic compounds to phosphomolybdic/ phosphotungstic acid complexes [8, 13]. the chromatographic separation of sugars and d-pinitol revealed interesting variances in carob syrups. sucrose was the main sugar in all samples, followed by fructose, glucose and d-pinitol. the sugar composition is in line with previous studies for mediterranean carob syrups [1, 10]. results demonstrated a fluctuation range of 18% for sucrose, 32% for fructose, 34% for glucose, and 75% for d-pinitol (table 2). the differences in d-pinitol in carob syrup are unexpected as a recent study reported that the d-pinitol content in cypriot carob varieties was from 53.20 mg g-1 and 54.58 mg g-1 [14]. taking into consideration that there are minor changes in the production of carob syrup, the d-pinitol content alone and/or in combination with sucrose content can be used as an index of sucrose supplementation in carob syrups. thus, we prepared genuine and sucrosesupplemented carob syrups to investigate the above hypothesis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 vlasios goulas, polina aresti, physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation, food and environment safety, volume xx, issue 3 – 2021, pag. 205 – 210 209 table 2. sugar composition of commercial carob syrups produced in cyprus. compound mean median range sucrose 368 374 332-404 fructose 158 156 125-184 glucose 151 148 115-189 d-pinitol 60 65 18-70 results are expressed as g 100 g-1 carob syrup comparison d-pinitol and sucrose content in carob syrups in a next step, genuine and sucrosesupplemented carob syrups were prepared and their sugar composition was studied. sucrose as an easy-available and low-cost sweetener, was added in carob juice to increase the yield of syrup. this addition of sucrose in natural sweetener as honey maple syrup as well as fruit juices is a common practice [15-17]. the supplemented sucrose was 27% and 42% of final sugar content of carob syrups. table 3 demonstrates a significant impact of sucrose on the chemical composition of carob syrup. table 3. effect of sucrose supplementation on sugar composition and total phenolic content in traditional made carob syrups pafos pafos 27% pafos 42% lemesos lemesos 27% lemesos 42% sucrose* 378.6±21.3 451.2±10.3 512±19.3 362.9±9.3 461.3±30.5 505.3±19.8 fructose* 138.5±6.2 109.3±3.8 84.3±6.3 162.4±5.8 116.1±14.9 102.1±7.2 glucose* 151.2±7.4 116.8±9.3 93.3±4.8 141.3±106.6 109.6±2.9 89.6±4.7 d-pinitol* 66.7±4.2 50.6±3.3 40.4±2.1 63.9±3.0 48.5±1.8 38.9±2.3 total sugars* 735.0 727.9 730.2 730.5 735.3 735.9 total phenolics** 1021±72 863±49 680±33 947±52 725±48 592±41 *results are expressed as g 100 g-1 carob syrup ** total phenolics are expressed as mg gallic acid equivalents 100 g-1 carob syrup in sucrose-supplements syrups, the dpinitol content decreased significantly; while the concentration of sucrose had a noteworthy increase. d-pinitol content is significant lower in sucrose supplemented syrups than genuine ones. the decrease in d-pinitol was 22-25% and 38-40% compared to genuine syrups when the added sugar was 27% and 42%, respectively. furthermore, the ratio sucrose to d-pinitol content also can be used to detect the sucrose supplementation. the above ratio is approximately 5.7 in genuine syrups; whereas the ratio sucrose to dpinitol grows up to 13 for sucrose supplemented syrups. as it is expected, the addition of sucrose also causing a drop in phenolic content in carob syrups. dhaouadi and co-workers also noticed a noteworthy decrease of phenolic content after sucrose supplementation [18]. specially, the total phenolic contents were decreased up to 23% and 38% for 27% and 42% sucrose supplementation. thus, this simple and rapid spectrophotometric assay can be also exploited as an additional index for sucrose supplementation. 4. conclusion carob syrup is a traditional sweetener in mediterranean basin with low glycemic index and health effects as it is consumed within their natural matrix. however, the carob syrup fraud and authenticity are an emerging issue as the addition of cheap food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 vlasios goulas, polina aresti, physicochemical properties of carob syrup reveal d-pinitol as an index of sucrose supplementation, food and environment safety, volume xx, issue 3 – 2021, pag. 205 – 210 210 sweeteners eg sucrose has been often used. in the present study, a knowledge base of physiochemical properties of cypriot carob syrup was build. our findings highlight that d-pinitol content, sucrose to d-pinitol ratio and total phenolic content can be utilized to detect sucrose supplementation in carob syrups. nevertheless, an integrated study with samples from whole island for three successive years is required to legislate national regulations and standards for this traditional product. 6. references [1] tetik, n., turhan, i., oziyci, h. r., karhan, m., determination of d-pinitol in carob syrup. international journal of food sciences and nutrition, 62: 572–576, (2011). [2] edwards, c. h., rossi, m., corpe, c. p., butterworth, p. j., ellis, p. r., the role of sugars and sweeteners in food, diet and health: alternatives for the future. trends in food science and technology, 56: 158–166. (2016). [3] lardos, a., the botanical materia medica of the iatrosophikon a collection of prescriptions from a monastery in cyprus. journal of ethnopharmacology, 104: 387–406, (2006) [4] lardos, a., heinrich, m., continuity and change in medicinal plant use: the example of monasteries on cyprus and historical iatrosophia texts. journal of ethnopharmacology, 150: 202– 214, (2013) [5] goulas, v., stylos, e., chatziathanasiadou, m. v., mavromoustakos, t. tzakos, a. g., (2016). functional components of carob fruit: linking the chemical and biological space. international journal of molecular sciences, 17: 1875, (2016) [6] nasar-abbas, s. m., e-huma, z., vu, t. h., khan, m. k., esbenshade, h., jayasena, v., carob kibble: a bioactive-rich food ingredient. comprehensive reviews in food science and food safety, 15: 63–72, (2016). [7] goulas, v., minas, i. s., kourdoulas, p. m., lazaridou, a., molassiotis, a. n., gerothanassis, i. p., manganaris, g. a., 1h nmr metabolic fingerprinting to probe temporal postharvest changes on qualitative attributes and phytochemical profile of sweet cherry fruit. frontiers in plant science, 6: 1–11, (2015) [8] orphanides, a., goulas, v. gekas, v., effect of drying method on the phenolic content and antioxidant capacity of spearmint. czech journal of food sciences, 31: 509-513, (2013) [9] duceppe, m. o., bertrand, a., pattathil, s., miller, j., castonguay, y., hahn, m. g., michaud, r., dubé, m. p., assessment of genetic variability of cell wall degradability for the selection of alfalfa with improved saccharification efficiency. bioenergy research, 5: 904–914, (2012) [10] tounsi, l., ghazala, i., kechaou, n., physicochemical and phytochemical properties of tunisian carob molasses. journal of food measurement and characterization, 14: 20–30, (2020) [11] avallone, r., plessi, m., baraldi, m., monzani, a., determination of chemical composition of carob (ceratonia siliqua): protein, fat, carbohydrates, and tannins. journal of food composition and analysis, 10: 166–172, (1997) [12] goulas, v. hadjisolomou, a., dynamic changes in targeted phenolic compounds and antioxidant potency of carob fruit (ceratonia siliqua l.) products during in vitro digestion. lwt food science & technology, 101: 269–275, (2019) [13] goulas, v., georgiou, e., utilization of carob fruit as sources of phenolic compounds with antioxidant potential: extraction optimization and application in food models. foods, 9, (2020) [14] christou, c., poulli, e., yiannopoulos, s. agapiou, a., gc–ms analysis of d-pinitol in carob: syrup and fruit (flesh and seed). journal of chromatography b: analytical technologies in the biomedical and life sciences, 1116: 60–64, (2019) [15] gad, h.a., ramadan, f.m., farag, m.a., authentication and quality control determination of maple syrup: a comprehensive review. journal of food composition and analysis, 100: 103901 (2021) [16] tosun, m., keles, f., investigation methods for detecting honey samples adulterated with sucrose syrup. journal of food composition and analysis, 101: 103941 (2021) [17] nair, s., venkatesh, s., athamaselvi, k.a., thakur, s., rapid estimation and quantification of sucrose content in fruit juices using fourier transform infrared– attenuated total reflectance (ftir–atr) spectroscopy. journal of food measurement and characterization, 10: 24–31 (2016) [18] dhaouadi, k., belkhir, m., akinocho, i., raboudi, f., pamies, d., barrajón. e., estevan, c., fattouch, s., sucrose supplementation during traditional carob syrup processing affected its chemical characteristics and biological activities. lwt food science and technology, 57: 1-8 (2014) 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2022.020 209 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 209 217 study of heat treatment effect on the biological value of pine nuts kernels *artur mykhalevych 1 , uliana kuzmyk 1 1educational and research institute of food technologies, national university of food technologies, 01601, 68 volodymyrska str., kyiv, ukraine, artur0707@ukr.net *corresponding author received 26rh july 2022, accepted 26th september 2022 abstract: the aim of the research was to determine the optimal modes of heat treatment of pine nut kernels in order to preserve their biological value to the optimal, which will allow in the future to use this type of raw material in the technology of various types of sauces with the addition of dry whey. the article analyzes the drying kinetics of pinus pinea linneus pine nut kernels for 120 minutes under 4 heat treatment regimes: 20, 40, 60 and 80 ˚c. the effect of heat treatment on the composition of essential amino acids, the content of vitamin c and β-carotene was studied. thus, the temperature of 20 and 40 ˚c ensures the maximum preservation of the biological value of the raw material, but it does not allow to achieve a significant decrease in the mass fraction of moisture, which will negatively affect the microbiological indicators of the raw material. during processing at 60 ˚с, the mass fraction of moisture in the kernels of pine nuts decreases from 7 to 5.28 %, which is likely to reduce water activity and positively affect the duration of storage. an increase in temperature to 80 ˚с significantly reduces the content of essential amino acids and β-carotene, as well as makes it impossible to achieve a technological effect (a significant decrease in moisture content) with the irrational use of energy resources. it was established that the optimal mode of heat treatment of pine nut kernels is drying at a temperature of 60 ˚с, which ensures the preservation of the biological value of the raw material and meets the recommendations for achieving satisfactory microbiological indicators. keywords: drying kinetics, moisture content, amino acid composition, vitamin c, β-carotene. 1. introduction today, the most important task of the food industry is the manufacturing of products with increased biological value in order to overcome the deficiency of protein, vitamins, minerals, dietary fiber in the human diet [1-2]. the most economically expedient is the development of technologies for food products of mass consumption [3-4], which, due to the use of plant raw materials, special methods of processing of individual ingredients or the product as a whole, will increase the amount of biologically active substances, protein and ensure food safety. nut raw materials are one of the largest branches of the agricultural sector, cultivated in most countries of the world [5]. the seeds of the siberian pine pinus pinea linneus, or as it is also called – pine nuts, are a source of biologically active substances and contain, on average, 13.69 % protein, 68.37 % fats, 13.08 % carbohydrates, 3.7 % dietary fiber, mineral and vitamin substances [6]. the fatty fraction of pine nut kernels is characterized by a high content of polyunsaturated fatty acids, such as linoleic and linolenic [7]. according to the amino acid profile, they are rich in arginine, lysine, methionine, and tryptophan [8]. pine nuts are widely consumed in eastern europe, asia, the usa and india. the most widely studied food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 210 representatives of nut raw materials are peanuts, almonds, walnuts, cashews and hazelnuts [9-10]. despite the high biological value of pine nuts, they have not been widely used in the production of food products [11], which is due to the issue of food safety and the need to find ways of their preliminary preparation [12]. at the same time, the seeds of the siberian pine still remain a promising raw material for use in the food industry [13], as it can perform the role of a flavor additive, which ensures the manufacturing of a product with original taste properties, as well as a fortifier capable of increasing the biological value. that is why it is promising to search and research different methods of processing pine nuts in order to avoid the risks of microbiological contamination in the case of their use in the recipe composition of food products [14]. microwave irradiation of nuts can cause the inactivation of pathogenic microorganisms under the influence of heat due to the destruction of the cell walls of bacteria. however, this mechanism and the factors affecting it have not been fully studied [15]. heat treatment is capable of reducing microbiological contamination, enzymatic and oxidative degradation, as well as facilitating its further processing, storage and quality, which will undoubtedly affect the shelf life of the finished product. in addition, such processing can create favorable conditions for the intensification of the fatty acid composition of nuts [16], which affects the texture of the food product in which they will be used. however, exposure to high temperatures over a long period of time leads to the loss of essential vitamins and other substances [17], while low temperatures may not have a significant effect on microbiological indicators [18]. thus, short-term preparation of nuts at a temperature of 200 ˚c allows obtaining a high-quality product due to the removal of 70...80 % of surface films, but at the same time, the mass fraction of protein decreases by 15...20 %, the content of fatty acids decreases to a level that does not contribute to their assimilation [19]. moisture-temperature processing allows to reduce the content of phytates to 45 % [20], however, this process is long-term, since only the soaking stage can take up to 10 hours, and the obtained raw materials have significantly worse indicators of microbiological purity. the aim of the study was to determine the optimal modes of heat treatment of pine nut kernels for the maximum preservation of biologically active substances, which will allow them to be used in the technology of various types of sauces with the addition of whey in the future. to achieve the goal, the following tasks were defined: 1. to study the kinetics of drying pine nut kernels at different heat treatment temperatures; 2. determine and compare the amino acid composition, vitamin c and β-carotene content of pine nut kernels before and after drying under different conditions; 3. choose the most appropriate mode of heat treatment, which contributes to the maximum preservation of biologically valuable substances in the kernels of pine nuts. 2. matherials and methods raw material for research siberian pine seeds pinus pinea linneus of the 2020 harvest were chosen as the raw material for research. the chemical composition according to the manufacturer's specification was as follows and met the requirementsща iso 6756: mass fraction of moisture 7 %, mass fraction of protein 13.8 %, mass fraction of fat 63.9 %, mass fraction of ash 2.44 %. pine kernels were shelled, weighed and placed on a metal sheet, which was then placed in a drying cabinet. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 211 the study of drying kinetics was carried out under 4 modes of heat treatment for 120 minutes: 1st sample 20 ˚с, 2nd sample 40 ˚с, 3rd sample 60 ˚с, 4th sample 80 ˚с. weighing of pine nut kernels was carried out at 10-minute intervals during the entire drying period. research methods study of drying kinetics when studying the drying process, an important aspect is the determination of physico-mechanical forms of moisture connection with the material. quantitatively, the content of moisture in the material is estimated by its humidity. relative humidity (ω, %) is distinguished, that is, the mass of moisture contained in the material (w, kg), attributed to the total mass of the sample (g, kg) [21]: and absolute humidity (ωc), which is determined in relation to 1 kg of absolutely dry matter in the material under study (gaс, kg): both relative and absolute humidity are expressed in fractions of a unit or in percentages. when analyzing the drying process, it is more convenient to use absolute humidity, because the amount of absolutely dry matter in the sample remains constant under any conditions. when calculating humidity according to the above formulas, its average value in this material is obtained. the values of relative ω and absolute ωc of humidity are connected by the following dependencies [22]: when studying the kinetics of drying, it is necessary to establish the influence of various external and internal factors on the speed of the process. the drying speed υ is determined by the decrease in humidity dωc by some an infinitesimal time interval dτ, i.e. [22-23]: determination of vitamin c content determination of vitamin c content in the kernels of pine nuts before and after drying was carried out by the titrometric method [24], which is based on the acid extraction of vitamin c (with solutions of hydrochloric, metaphosphoric, or a mixture of acetic and metaphosphoric acids) followed by titration with a solution of sodium 2.6-dichlorophenolindophenolate until a light pink color. the mass fraction of ascorbic acid (a) in percent was calculated according to the formula: where v1 is the volume of sodium 2.6dichlorophenolindophenolate solution used for titration of the sample extract, cm3; v2 is the volume of sodium 2.6dichlorophenolindophenolate solution used for the control test, cm3; t is titer of sodium 2.6dichlorophenolindophenolate solution, cm3; v3 is the volume of the extract obtained during the extraction of vitamin c from the sample product, cm3; v4 is the volume of extract used for titration, cm3; m weight of the product, g. determination of β-carotene content the content of β-carotene was determined by the accelerated uv-vis method [25], according to which an acetone extract was prepared from the kernels of pine nuts and scanned with a uv-vis spectrum at wavelengths from 350 to 600 nm. the content of β-carotene in the experimental food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 212 samples was calculated according to the calibration curve. determination of amino acid content the amino acid composition of pine nut kernels before and after heat treatment was determined by ion-exchange liquid column chromatography on the t339 amino acid analyzer in automatic mode [26-27]. statistical data processing statistical data processing was carried out using the statistica 10 program and microsoft excel 2016. construction of experimental drying curves was carried out in the microsoft excel 2016 editor. the results of the experimental part were obtained by conducting three times of research under the same conditions to ensure accuracy. 3. results and discussion kinetics of pine nut kernel drying on the basis of an analytical review of literature sources on the issue of drying various types of nuts, "soft" modes of heat treatment were determined, which will contribute to the maximum preservation of the protein fraction, in particular amino acids. the first stage was to study the kinetics of heat treatment of pine nut kernels during convective drying. based on the obtained data, drying curves were constructed at different processing temperatures (fig. 1). fig. 1 – drying curves of pine nut kernels at different processing temperatures as can be seen from fig. 1, the process of drying pine nut kernels depends on the temperature. drying at low temperatures 20 and 40 ˚с is long-term and there is no significant difference in the dynamics of the process. the drying kinetics of pine nut kernels at a temperature of 20 ˚с is the lowest, which is probably justified by strong bonds between water molecules [28], which require the expenditure of a greater amount of thermal energy to intensify the drying process. under such heat treatment modes, the value of moisture content during the entire duration of drying decreases by 11.4 % (at a temperature of 20 ˚с) and 18.7 % (at a temperature of 40 ˚с) from the initial value. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 213 the use of higher temperatures 60 and 80 ˚с accelerates the removal of free moisture from raw materials. this can be explained by the fact that the greater amount of heat that comes from the heated air to the food material in the drying chamber increases the rate of movement of moisture from the inside of the kernels to the surface [29], which significantly reduces the time to reach a constant humidity. at a temperature of 60 ˚c, the initial moisture content during the process decreases by 24.6 %, and at 80 ˚c by 33.2 %. a further decrease in drying efficiency is associated with the presence of hydrophobic lipid molecules [30], which act as a certain limiting factor when the maximum mass fraction of moisture in the food material is reached. thus, drying at a temperature of 80 ˚c is impractical from the point of view of using an excessive amount of energy resources for the possibility of significantly increasing the effect of the technological process. a characteristic feature of the drying curves of this type of raw material is the absence of a horizontal section at the beginning of the heat treatment process [31], which is called the period of constant drying speed (or the 1st period). this makes further use of traditional methods of calculating the duration of drying impossible. that is why it was decided to investigate the effect of temperature treatment under different regimes on the biological value. the effect of heat treatment on the biological value of pine nut kernels considering the fact that pine nuts are characterized by a high content of essential amino acids, the effect of heat treatment on their final content was investigated in order to determine the optimal mode of their processing, which will ensure a significant preservation of the amount of amino acids. the amino acid composition of pine nut kernels before and after drying under different modes of heat treatment is given in table 1. table 1 amino acid composition of pine nut kernels before and after heat treatment (р ≤ 0.05%, n = 3) the name of an essential amino acid content, mg/100 g before drying after drying 20 ˚с 40 ˚с 60 ˚с 80 ˚с tryptophan 107.542 101.412 94.548 88.657 75.28 isoleucine 522.708 500.541 480.854 449.083 405.956 valin 667.874 648.504 601.579 553.876 468.527 leucine 981.52 949.389 906.844 851.009 785.216 threonine 378.988 369.024 360.629 349.989 337.54 lysine 540.215 534.873 525.207 513.693 485.678 methionine + cysteine 259.058 237.01 219.924 196.551 140.851 phenylalanine + tyrosine 514.66 498.807 469.791 438.02 395.654 an increase in heat treatment temperature reduces the content of essential amino acids in pine nut kernels, which becomes more significant as it increases. the percentage of reduction in content for each essential amino acid at different processing temperatures is shown in fig. 2. thus, at a temperature of 20 ˚c, a decrease in the content of amino acids is observed from 0.99 to 8.51 %, depending on the essential amino acid, at 40 ˚c from 2.78 to 15.11 %, at 60 ˚c from 4.91 to 24.13 %, at 80 ˚c from 10.1 to 45.63 %. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 214 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 m a ss f ra c ti o n i n r e d u c ti o n o f a m in o a c id c o n te n t, % name of the essential amino acid 20 ˚с 40 ˚с 60 ˚с 80 ˚с fig. 2 dependence of the decrease in the content of essential amino acids of pine nut kernels on the processing temperature tryptophan, valine and methionine+cysteine undergo the greatest destruction, which is consistent with the data of the scientific literature [32]. the minimal effect on the content of amino acids occurs at heat treatment temperatures of 20 and 40 ˚с, while a significant decrease in biological components occurs at a temperature of 80˚с, which is explained by the partial denaturation of proteins [33] and the subsequent decrease in biological value. the content of vitamin c and β-carotene before and after heat treatment of pine nut kernels is illustrated in fig. 3. despite reports on the particular instability of vitamin c under thermal action on it in the temperature range of 70...90 ˚с [3435], it can be seen (fig. 3) that its decrease after drying of pine nut kernels at a temperature of 80 ˚с decreases by 30.96 % of the initial content, which is significant compared to the results of exposure to other regimes, but most of it still remains in the raw material after processing. the content of β-carotene significantly depends on the drying temperature: during processing at 20 ˚с, 10.71 % of the initial content is destroyed, at 40 ˚с – 25 %, at 60 ˚с – 42.86 %, and at 80 ˚с – 67.86 %, which corresponds to with well-known information about the effect of temperature on the stability of β-carotene [36]. taking into account the study of changes in the amino acid composition, the content of vitamin c and β-carotene in the kernels of pine nuts after heat treatment under different modes, the optimal treatment temperature is 60 ˚с, which allows reducing the mass fraction of moisture in the product from 7 to 5.28 %, which is more effective in compared with the processing temperature of 20 and 40 %. a decrease in the moisture content of pine nut kernels will contribute to a decrease in the water activity index [37], which will food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 215 have a positive effect on the microbiological state of the raw material, which requires additional scientific research. 0.852 0.824 0.769 0.717 0.602 0.028 0.025 0.021 0.016 0.009 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 before drying 20 ˚с 40 ˚с 60 ˚с 80 ˚с c o n te n t, m g /1 0 0 g mode of raw material processing vitamin c beta-carotene fig. 3 – content of vitamin c and β-carotene before and after heat treatment of pine nut kernels 4. conclusion the paper examines the kinetics of pine nut kernel drying under different modes of heat treatment and its effect on the biological value of the raw material. it was established that the best drying mode is a temperature of 60 ˚с, which ensures a significant decrease in the mass fraction of moisture in the raw material, helps to preserve a significant part of essential amino acids, vitamin c and β-carotene. the perspective of further scientific work consists in determination of unsaturated fatty acids content after processing, the development of original recipes of sauces using the kernels of pine nuts that have undergone heat treatment, and the study of the quality of the obtained products. 5. acknowledgments the research was carried out within the framework of research work (r&d) «implementation of resource-saving methods for modifying the functional and technological characteristics of milk whey in the technologies of food products for targeted purposes» (state registration number 0120u100868), ukraine. 6. references [1]. g. polishchuk, u. kuzmyk, t. osmak, m. kurmach, o. bass, analysis of the nature of the composition substances of sour-milk dessert with plant-based fillers. 6(11(114), easterneuropean journal of enterprise technologies, 2021, 68–73. https://doi.org/10.15587/17294061.2021.246309. [2]. g. polischuk, o. kochubeilytvynenko, t. osmak, u. kuzmyk, o. bass, a. mykhalevych, v. sapiga, scientific explanation of composition of acidophilic-whey ice cream, enriched with protein. 20 (1), food and environment safety, 2021, 13-20. https://doi.org/10.4316/fens.2021.002. [3]. a. mykhalevych, v. sapiga, g. polischuk, t. osmak, functional and technological properties of oat beta-glucan in acidophilic-whey ice cream. 21 (2), food and environment safety, 2022, 116-128. https://doi.org/10.4316/fens.2022.012. [4]. v. sapiga, g. polischuk, t. osmak (fedchenko), a. mykhalevych, m. maslikov, scientific explanation of the composition and technological modes of manufacture of dairy ice cream with vegetable puree. 7 (1), ukrainian journal of food science, (2019), 83-91. https://doi.org/10.24263/2310-1008-2019-7-1-10. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 216 [5]. fao. the future of food and agriculture – trends and challenges. rome. (2017). [6]. united states department of agriculture. national nutrient database for standard reference release 28; united states department of agriculture: washington, dc, usa. (2016). [7]. f. shahidi, p. ambigaipalan, phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects – a review. 18 (в), journal of functional foods, (2015), 820897. https://doi.org/10.1016/j.jff.2015.06.018. [8]. i. evaristo, d. batista, i. correia, p. correia & r. costa, chemical profiling of portuguese pinus pinea l. nuts. 90(6), journal of the science of food and agriculture, (2010), 1041– 1049. https://doi.org/10.1002/jsfa.3914. [9]. m. venkatachalam, s. k. sathe, chemical composition of selected edible nut seeds. 54(13), journal of agricultural and food chemistry, (2006), 4705–4714. https://doi.org/10.1021/jf0606959. [10]. a. luparelli, i. losito, e. de angelis, r. pilolli, f. lambertini & l. monaci, tree nuts and peanuts as a source of beneficial compounds and a threat for allergic consumers: overview on methods for their detection in complex food products. 11(5), foods, (2022), 728. https://doi.org/10.3390/foods11050728. [11]. s. l. bates, c. g. lait, j. h. borden & a. r. kermode, measuring the impact of leptoglossus occidentalis (heteroptera: coreidae) on seed production in lodgepole pine using an antibody-based assay. 95(4), journal of economic entomology, (2002), 770–777. https://doi.org/10.1603/0022-0493-95.4.770. [12]. m. bracalini, s. benedettelli, f. croci, p. terreni, r. tiberi & t. panzavolta, cone and seed pests of pinus pinea: assessment and characterization of damage. 106(1), journal of economic entomology, (2013), 229–234. https://doi.org/10.1603/ec12293. [13]. p.f. roversi, w.b. strong, v. caleca, m. maltese, g. sabbatini peverieri, l. marianelli, l. marziali & a. strangi, introduction into italy of gryon pennsylvanicum (ashmead), an egg parasitoid of the alien invasive bug leptoglossus occidentalis heidemann. 41, eppo bulletin, (2011), 72-75. https://doi.org/10.1111/j.1365-2338.2011.02439.x. [14]. a. karamysheva, l. trofimuk, n. priyatkin, m. arkhipov, l. gusakova, p. shchukina, n. staroverov & n. potrakhov, сomparative study of the fullness of dwarf siberian pine seeds pinus pumila (pall.) regel from places of natural growth and collected from plants introduced in northwestern russia by microfocus x-ray radiography to predict their sowing qualities. 65 (4), biological communications, (2020), 297-306. [15]. e. popeláˇrová, e. vlková, r. švejstil, l. kouˇrimská, the effect of microwave irradiation on the representation and growth of moulds in nuts and almonds. 11, foods, (2022), 221. https://doi.org/ 10.3390/foods11020221. [16]. t. belemets, i. radzievskaya, o. tochkova, n. yushchenko, u. kuzmyk & a. mykhalevych. evaluation of oxidity resistance of milk-containing products based on blending of vegetable oils. 1(3(57), technology audit and production reserves, (2021), 26–33. https://doi.org/10.15587/2706-5448.2021.225530. [17]. m. c. giannakourou, p. s. taoukis, effect of alternative preservation steps and storage on vitamin c stability in fruit and vegetable products: critical review and kinetic modelling approaches. 10(11), foods (basel, switzerland), (2021), 2630. https://doi.org/10.3390/foods10112630. [18]. m. cabello-olmo, m. oneca, p. torre, j. v. díaz, i. j. encio, m. barajas, m. araña, influence of storage temperature and packaging on bacteria and yeast viability in a plant-based fermented food. 9(3), foods, (2020), 302. https://doi: 10.3390/foods9030302. [19]. a. dyakonova, v. stepanova & e. shtepa, preparation of the core of walnut for use in the composition of soft drinks. 11(3), food science and technology, (2017). https://doi.org/10.15673/fst.v11i3.609. [20]. a. dyakonova, v. stepanova, usage of the nut raw materials and chia seeds to improve fatty acid composition of the smoothies. 5(4), ukrainian food journal, (2016), 713-723. https://doi.org/10.24263/2304-974x-2016-5-4-10. [21]. g.v. shljagun, kreponosova a.n., s.v. kozhokar'', vlijanie predvaritel'noj obrabotki na strukturu i obezvozhivanie jablok [influence of pre-treatment on the structure and dehydration of apples]. 7, pishh. prom-t', (1992), s.22-23. [22]. v. i. atanazevich, sushka pishhevyh produktov [food drying], spravochnoe posobie. m.: deli. (2000). [23]. i. guz''ova, v. atamanjuk, doslidzhennja kinetyky sushinnja cukativ z garbuza [study of drying kinetics of candied pumpkin]. 84(1), scientific works, (2020). 34-41. https://doi.org/10.15673/swonaft.v84i1.1866. [24]. g. f. m. ball, vitamin c. in: bioavailability and analysis of vitamins in foods. springer, boston, ma. (1998). https://doi.org/10.1007/9781-4899-3414-7_15. [25]. m. hagos, m. redi-abshiro, b. s. chandravanshi, e. e. yaya, development food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 artur mykhalevych, uliana kuzmyk, study of heat treatment effect on the biological value of pine nuts kernels, food and environment safety, volume xxi, issue 3 – 2022, pag. 209 – 217 217 of analytical methods for determination of βcarotene in pumpkin (cucurbita maxima) flesh, peel, and seed powder samples, international journal of analytical chemistry. vol. 2022, article, (2022). https://doi.org/10.1155/2022/9363692. [26]. o. grek, t. osmak, l. chubenko, a. mykhalevych. technological aspects of production of frozen dessert with protein-herbal component, journal of faculty of food engineering, (2019), 197-204. [27]. p. m. cummins, k. d. rochfort, b. f. o’connor, ion-exchange chromatography: basic principles and application. in: walls, d., loughran, s. (eds) protein chromatography. humana press, new york, ny. (2017). https://doi.org/10.1007/978-1-4939-6412-3_11. [28]. n. a. aviara, o. o. ajibola, thermodynamics of moisture sorption in melon seed and cassava. 55(2), journal of food engineering, (2002), 107-113. https://doi.org/10.1016/s0260-8774(02)00023-7. [29]. d. r. reis, f. b. brum, e. j. soares, j. r. magalhães, f. s. silva & a. g. porto, drying kinetics of baru flours as function of temperature. 22, revista brasileira de engenharia agrícola e ambiental, (2018), 713-719. https://doi.org/10.1590/1807-1929/agriambi. v22n10p713-719. [30]. c. ertekin, m. ziya firat, a comprehensive review of thin-layer drying models used in agricultural products. 57(4), critical reviews in food science and nutrition, (2017), 701-717. https://doi.org/10.1080/10408398.2014.910493. [31]. c. singh, n. saluja & r. k. sharma, a computation-driven, energy-efficient and hybrid of microwave and conventional drying process for fast gooseberry candy production. 53(4), journal of microwave power and electromagnetic energy, (2019), 259-275. https://doi.org/10.1080/08327823.2019.1677431. [32]. l. cai, a. cao, g. aisikaer & t. ying, influence of kernel roasting on bioactive components and oxidative stability of pine nut oil. 115, eur. j. lipid sci. technol., (2013), 556-563. https://doi.org/10.1002/ejlt.201200337. [33]. l. dyshluk, s. sukhikh, s. ivanova, i. smirnova, m. subbotina, a. pozdnyakova, e. neverov & s. garmashov, prospects for using pine nut products in the dairy industry. 6 (2), foods and raw materials, (2018), 264-280. [34]. h. s. farah, j. f. alhmoud, a. alothman, k. m. alqaisi, a. m. atoom, k. shadid, a. shakya, t. alqaisi, effect of ph, temperature and metal salts in different storage conditions on the stability of vitamin c content of yellow bell pepper extracted in aqueous media. 11(9), srp, (2020), 661-667. https://doi.org/10.31838/srp.2020.9.97. [35]. a. l. herbig, c. m. renard, factors that impact the stability of vitamin c at intermediate temperatures in a food matrix. 220, food chemistry, (2017), 444–451. https://doi.org/10.1016/j.foodchem.2016.10.012. [36]. n. thakur, heat stability and antioxidant potential of beta-carotene isolated from a fungal isolate. 24(5), bulgarian journal of agricultural science, (2018), 891–896. [37]. ş. karatas, i. pinarli, determination of moisture diffusivity of pine nut seeds, 19(3-4), drying technology, (2001), 701-708, https://doi.org/10.1081/drt-100103946. 1. introduction today, the most important task of the food industry is the manufacturing of products with increased biological value in order to overcome the deficiency of protein, vitamins, minerals, dietary fiber in the human diet [1-2]. the most economically exped... the seeds of the siberian pine pinus pinea linneus, or as it is also called – pine nuts, are a source of biologically active substances and contain, on average, 13.69 % protein, 68.37 % fats, 13.08 % carbohydrates, 3.7 % dietary fiber, mineral and vit... despite the high biological value of pine nuts, they have not been widely used in the production of food products [11], which is due to the issue of food safety and the need to find ways of their preliminary preparation [12]. at the same time, the see... that is why it is promising to search and research different methods of processing pine nuts in order to avoid the risks of microbiological contamination in the case of their use in the recipe composition of food products [14]. microwave irradiation of nuts can cause the inactivation of pathogenic microorganisms under the influence of heat due to the destruction of the cell walls of bacteria. however, this mechanism and the factors affecting it have not been fully studied [1... 4. conclusion doi: https://doi.org/10.4316/fens.2021.042 411 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 411 415 nutritional characterization of the peripheral layers of barley grains cultivated in algeria rawda benguella 1 , *samira meziani 1 , hayet mehida 1 , yamina belkessam 1 , kouider sennous 2 , abassia demmouche 1 1laboratory of biotoxicology, faculty of of life sciences and nature sciences, department of biology, university of sidi-bel-abbes, algeria, meziani_samira@ yahoo.fr 2laboratory of ecodevelopment of spaces, department of environmental science, faculty of natural and life sciences, djilali liabes university of sidi bel abbès, algeria *corresponding author: meziani_samira@yahoo.fr received 22th september 2021, accepted 30th december 2021 abstract: the purpose of this paper is to stady the different analyzes were carried out in this work on the peripheral layers (pl) of barley grains of the saida variety. barley is the most important cereal rich in primary metabolite than secondary, which are beneficial for human health. barley flour is based on peripheral layers which are generally intended for cattle feed, for their nutritional values linked to the supply of a food rich in vitamins, proteins, fibers etc. the analysis of polyphenols by liquid chromatography method at high pressure (rphplc-lc 2030c 3d), allowing the detection of a phenolic acid (syringic acid), the source of brut fibers with a gram of pl is 0.8%. mineralcontents assessed by the spectrophotometric technique of atomic absorption, reveals that calcium and magnesium are the most abundant with values of 2.8 (mg / kg) and 2.1 (mg / kg) respectively. keywords: wheat, peripheral layer, proteins, fibers, rp-hplclc-2030c 3d. 1. introduction cereals are plants cultivated mainly for its seeds, used in human and animal nutrition, often ground in the form of refined flour or more or less complete, but also in whole grains, we find such as wheat, barley, rye or dicotyledons such as sesame, quinoa, etc. particularly barley has very interesting biological properties, due to its richness in secondary metabolites which are beneficial to human health. studies have shown that barley contains phenolic compounds in free form and bound from the outer layer of the bran fraction [1] which have the potential to lower blood cholesterol and glucose levels and contribute to the intestinal microbial balance [2] [3] [4]. however, the peripheral layers of barley kernels (pl) have nutritional potential and contain most of the micronutrients such as fiber [5] and minerals which can greatly contribute to the increase of nutritional quality [6] and reduces of numerous diseases such as colon cancer, diabetes, obesity and cardiovascular disease [7] [8]. barley is rich in protein, complex carbohydrates and fat-soluble vitamin e (tocols) [2] [9] phenolic groups are the most abundant phenolic compounds in the walls of barley grains in cell-bound form, the main phenolic acids are ferulic acid, vanillic acid, syringic acid and p-coumaric acid [10] [11]. several studies have focused on milling barley in order to obtain products other than standard flour from cell walls rich in fiber and bioactive phytochemicals [10] [12]. the ain of this study is to conduct research on the peripheral layers of barley grains of the saida variety from the sidi bel abbes algeria region [13] through a more indepth study. an analysis of the polyphenols with the hplc method was mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 rawda benguella, samira meziani, hayet mehida, yamina belkessam, kouider sennous, abassia demmouche, nutritional characterization of the peripheral layers of barley grains cultivated in algeria, food and environment safety, volume xx, issue 4 – 2021, pag. 411 – 415 412 carried out, determination of the raw fibers, as well as the determination of some minerals by atomic absorption pectrophotometer. 2. matherials and methods plant material and extraction the objective of this research is to highlight the consumption of the peripheral layers of barley grains (hordeum vulgare) of the saïda variety cultivated in algeria. a manual dissection method under the binocular lens was adopted to obtain the peripheral layers (pl) at the mature stage; they were kept until analysis to be powdered. a quantity of 10 g of the pl was extracted twice with 200 ml of methanol by the infusion method. characterization of phenolic compounds by rp-hplc-lc-2030c 3d analysis the phenolic investigation for the crude extract of barley variety (saida) was carried out using a high performance liquid chromatography coupled with uv-vis detector rp-hplc-lc-2030c 3d (shimadzu prominence-i; lc-2030c 3d). the analytical were evaluated using a processing data system. the separation was achived on a supelco c18 column (25cm x4.6mm, 5 μm) at room temperature. the mobile phase consisted of water / acetic acid (ph = 3), phase b consisted of methanol / acetic acid (ph = 3). the gradient elution system was as follows at the minute 0.01 min the concentration of solvent b is 5% until the 2nd minute, from the 2nd minute up to the 40th minute the gradient will successively change from 5% to reach 100% of solvent b, then will remain 100% until the 45th and the length wave was 280 nm. phytochemicals were identified from a combination of retention time and spectral matching. mineral content by atomic absorption spectrophotometer regarding the mineral assay protocol [14] we used a brand atomic absorption spectrophotometer (shimadzu model aa7000). the samples were first mixed with 65% nitric acid, then 30% of hydrogen peroxide h2o2 was added, followed by heating at 90 to 120°c to remove all the solvent, after cooling, hydrolysis followed by filtration were carried out the samples were atomized by a flame (acetylene-air) and the reading was made at lamps and wavelengths specific to each chemical element, results were expressed as mg / kg of dw. raw fabers content (weende) the method is based on the solubilization of non-cellulose compounds in solutions of sulfuric acid and potassium hydroxide. 1g of each sample was added to 150 ml of 1.25 % sulfuric acid as well as 3-5 drops of the anti-foaming agent n-octanol, they were boiled for 30 min then the crucibles were washed 3 times with hot distilled water. then the same steps were repeated three times, but with 1.25% potassium hydroxide except that of the last wash which was with cold deionized water and 25 ml of acetone. the crucibles were removed and then weighed after drying at 105 ° for 1 hour, the content represents the crude fibers plus the ash content (f1), to remove the ash, the crucibles were placed in a muffle furnace at 550° for 3 hours and reweighed after cooling in a desiccator (f2) and the results were expressed in%. % crude fiber = (f1 f2 / f0) × 100 where: f1: gross fiber weight + ash f2: ash weight f0: weight after grinding food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 rawda benguella, samira meziani, hayet mehida, yamina belkessam, kouider sennous, abassia demmouche, nutritional characterization of the peripheral layers of barley grains cultivated in algeria, food and environment safety, volume xx, issue 4 – 2021, pag. 411 – 415 413 3. results and discussion results of phenolic compounds characterization by rp-hplc-lc2030c 3d analysis the analysis of total polyphenols of crude extract from the peripheral layers of barley grains (saida) was carried out by the rphplc-lc-2030c 3d method using 4 modes of identification. the chromatogram obtained (figure 1) showed a peak of syringic acid (4-hydroxy-3,5dimethoxybenzoic acid) within 280 nm. based on studies by lópez-perea et al., 2019 [15] on the quantification of phenolic acids in barley husks showed that ferulic acid and benzoic acid showed a higher concentration with 50% acetone; however, benzoic acid was observed at a high concentration with 80% methanol which is consistent with our results by detection of syringic acid. fig.1. hplc chromatogram of the crude extracts of peripheral layer of barley (saida) variety results of minerals the microelement content of the peripheral layers of the wheat grains obtained (figure2) shows that calcium is the most abundant mineral with the value of 2.79 mg / kg compared with 2.09 mg / kg of mg, 0.82 mg / kg of iron , 0.23 mg / kg of mn and 0.04 mg / kg of cu. afify et al., (2016) (16), asserted that sprouted barley have the highest levels of mg (0.83 ± 0.03 and 0.34 ± 0.01 mg 100g-1) which confirms our results, as well as the k and fe contents decreased by the germination of barley and these results are in agreement with that of hubner et al. (2010) (17), which are decreased from 3.7 to 2.8 mg 100g-1) after germination. svetlan and musa, (2016) [18] found that the cr and ni content of malted barley grains at 1.4 mg / kg, while the cu and fe content varieds between 0.7-6.8 mg / kg and 57.779.9 mg / kg respectively and they observed a strong increase in mineral and metallic elements during malting. platel et al., (2010) (19) showed that the iron (fe) and zinc (zn) content was higher in malted barley than in barley. youssef et al., 2013 [20], proved that the mineral content in barley was distributed as follows: ca = 120-160 mg / 100g, mg = 130-180 mg / 100g, iron = 3.27-39.9 mg / 100g cu = 0.550-0.985 mg / 100g and mn = 5.75-13.85 mg / 100g. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 rawda benguella, samira meziani, hayet mehida, yamina belkessam, kouider sennous, abassia demmouche, nutritional characterization of the peripheral layers of barley grains cultivated in algeria, food and environment safety, volume xx, issue 4 – 2021, pag. 411 – 415 414 fig.2. mineral composition of the peripheral layers of barley grains (saida) raw fiber content according to the obtained result the crude fiber content is 0.8%, they agree with montanuci et al., 2013 [21] who reported that the ash content of barley varieties varied from 1.73% to 2.47 % for the two varieties brs 195 and brs greta, as well as afify et al., 2016 [16] who showed that the ash content varied from 2.54 ± 0.07 to 1.90 ± 0.01% for bare barley and demonstrated that the crude fiber contents before and after germination were; 5.21 ± 0.11, 4.84 ± 0.13%. barley without shell is a cereal that can be considered as an energy source of food for livestock feed, according to yaghoubfar et al., 2013[22], the amount of crude fiber is the lowest (1.4%) compared to the other lines. sullivan et al, 2010 [18], confirmed that the amount of fiber, specifically insoluble fiber, in whole barley was significantly decreased by the peeling process. 4. conclusion in conclusion we can remark that the outer layers of barley kernels rich in fiber and minerals compared to wheat kernels, it may exhibit much more nutritional properties as whole kernels or as an addition to flour. the establishment of a diet rich in barley bran could replace synthetic antioxidants because of its richness in phenolic compounds which can play an important role in the prevention of certain diseases and valued the basic raw material for recipes of traditional bakery or various pasta products. more studies are needed to get more in-depth results and understand the recommended uses across multiple areas. 5. acknowledgments the authors acknowledge the university of sidi bel abbes, biology department at sidi bel abbes for the financial support of this research 6. references [1]. liu q.; yao h., antioxidant activities of barley seeds extracts. food chem., 102, 732–737. (2007) doi: 10.1016/j. foodchem. [2]. martínez m., motilva mj., lópez h., mc romero mp., vaculova k., ludwig ia., phytochemical composition and β-glucan food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 rawda benguella, samira meziani, hayet mehida, yamina belkessam, kouider sennous, abassia demmouche, nutritional characterization of the peripheral layers of barley grains cultivated in algeria, food and environment safety, volume xx, issue 4 – 2021, pag. 411 – 415 415 content of barley genotypes from two different geographic originsfor human health food production. food chem, 245, 61–70, (2018) [3]. komeili hr., sheikholeslami z., replacement effect of wheat flour with barley flour and hull-less barleyflour on the bread porosity and color. adv. agric. biol, 2, 39–43, (2014) [4]. cardona f., andrés-lacueva c., tulipani s., tinahones f.j., queipoortuño m.i., benefits of polyphenolson gut microbiota and implications in human health. journal nutr. biochem, 24, 1415–1422, (2013) [5] morrison i., m., & brice re., the digestion of untreated and ammonia-treated barley straw in an artificial rumen. animal feed science and technology journal, 10(2-3), 229-238, (1984) [6]. hemery y., rouau x., lullienpellerin v., barron c., abecassis j., dry processes to develop wheat fractions and products with enhanced nutritional quality. journal of cereal science, 46(3), 327-347, (2007) [7]. prinsen p., gutiérrez a., faulds c. b., del río jc.,comprehensive study of valuable lipophilic phytochemicals in wheat bran. journal of agricultural and food chemistry, 62(7), 1664-1673, (2014) [8]. sang s., zhu y., bioactive phytochemicals in wheat bran for colon cancer prevention. in wheat and rice in disease prevention and health, academic press, (pp. 121-129), (2014) [9]. abdel-aal e.sm., choo tm., differences in compositional properties of a hulless barley cultivar grown in23 environments in eastern canada. canadien plant science journal, 94, 807– 815, (2014) [10].idehe e., tang, y., sang s., bioactive phytochemicals in barley. food drug anal journal, 25, 148–161, (2017) [11] gamel th., abdel-aal, es phenolic acids and antioxidant properties of barley wholegrain and pearlingfractions. agric. food science journal, 21, 118–131, (2012) [12]. izydorczyk ms., mcmillan t., bazin s., kletke j., dushnicky l., dexter j., chepurna a, rossnagel b, milling of canadian oats and barley for functional food ingredients: oat bran and barley fibre-rich fractions.can. j. plant sci, 94, 573–586, (2014) [13]. benguella r., meziani s., ghueffari i., menadi n., chenni fz., labga l., &demmouche, a., bioactive compounds in the peripheral layers of barley and triticale species in the mature grain cultivated in algeria. journal of drug delivery and therapeutics, 10(4), 37-43, (2020) [14]. mahood he., estimation of essential elements and mineral in catharanthus roseus and its biological importance as a medicinal plant. plant cell biotechnology and molecular biology journal, 22(25), 1-7, (2021) [15]. lópez-perea p., guzmán-ortiz f a., román-gutiérrez ad., castro-rosas j., gómez-aldapac a., rodríguez-marín ml., torruco-uco jg, bioactive compounds and antioxidant activity of wheat bran and barley husk in the extracts with different polarity. international journal of food properties, 22(1), 646-658, (2019) [16] afify, a. e. m., abbas, m. s., abd ellattefi, b. m., ali, a. m. chemical, rheological and physical properties of germinated wheat and naked barley. international journal of chemtech research, 9(9), 521-531, (2016) [17] hübner, f., o’neil, t., cashman, k. d., & arendt, e. k.. the influence of germination conditions on beta-glucan, dietary fibre and phytate during the germination of oats and barley. european food research and technology, 231(1), 27-35, (2010) [18] sullivan p., o'flaherty j., brunton n., gee vl, arendt e. et gallagher e., composition chimique et microstructure des fractions d'orge broyées. recherche et technologie alimentaires européenne sjournal , 230 (4), 579595, (2010) [19]. platel, k., eipeson, s. w., & srinivasan, k. bioaccessible mineral content of malted finger millet (eleusine coracana), wheat (triticum aestivum), and barley (hordeum vulgare). journal of agricultural and food chemistry, 58(13), 8100-8103, (2010) [20]. youssef ma., elkhodary st., utilization of airborne gamma ray spectrometric data for geological mapping, radioactive mineral exploration and environmental monitoring of southeastern aswan city, south eastern desert, egypt. geophysical international journal, 195(3), 1689-1700, (2013) [21]. montanuci fd., jorge lmd., jorge rmm., kinetic, thermodynamic properties, and optimization of barley hydration. food science and technology journal, 33, 690-698, (2013) [22]. yaghoubfar, a., ghaffari, s., yousefi, a. determination nutritive value of hull-less barley cultivars used in poultry nutrition, 15-23, (2013) 1. introduction doi: https://doi.org/10.4316/fens.2023.005 50 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 50 58 fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species *nkonyeasua kingsley egun 1 , ovbokhan osavibie imadonmwiniyi 1 , vivian ezighodeomo iyoha 1 and ijeoma patience oboh 1 1department of animal and environmental biology, university of benin, benin city, nigeria, kenegun@yahoo.com. *corresponding author received 6th january 2023, accepted 26th march 2023 abstract: fish spoilage is one of the greatest challenges confronting the fishing industry globally, and efforts aimed at its reduction through various food processing methods have impacted on the nutritional composition of fishes. this study investigates the effects of oven-drying and smoke drying methods on the nutritional composition of fish species and their nutrient availability to meeting the required nutritional intake (rni) for consumers. results showed a significant variation in the nutritional composition of clarias gariepinus and tilapia zillii processed by oven drying and smoke drying. the increase in protein content of c. gariepinus (from 15.79% to 16.62%) and t. zillii (from 16.68% to 17.29%); and mineral contents in oven-dried samples as compared to the reduced protein content in c. gariepinus (from 15.79% to 14.62%) and t. zillii (from 16.68% to 13.76%) of smoked dried samples, makes oven-dried samples good sources of dietary protein and mineral elements for human consumption. potential contribution to rni showed that oven-dried and smoke dried c. gariepinus meet ≥ 25% of rnis for iron and zinc in children. oven-dried and smoke dried t. zillii meet ≥ 25% of the rni of iron in pregnant and lactating women (plw) and children; and meet ≥ 25% of the rni of zinc in children only. this indicate the need for increased per capital consumption of fish in nigeria. it is therefore recommended that the traditional method of smoke drying should be discouraged in lieu of its diminutive effect on nutritional composition of fishes, environmental pollution and health risk. keywords: clarias gariepinus; food processing; human nutrition; tilapia zillii; nutrient composition; recommended nutrient intakes (rnis) 1. introduction fish processing can be defined as procedures which are applied to the fish from time of harvest to the consumption period [1]. the processing and preservation of fresh fishes are important as they highly susceptible to deterioration immediately after harvest and also to prevent economic losses [2]. fish spoilage is one of the greatest problems affecting the fishing industry all over the world and efforts at reducing fish spoilage to the barest minimum through various preservation and processing techniques have impacted on the nutritional composition of fishes [3,4]. the nutritional value of harvested fish meat comprises the contents of moisture (66 81%), protein (16 21%), lipids (0.2 25%), ash (1.2 1.5%), vitamins, minerals and the caloric value of the fish [5 – 8]. the need for the knowledge of the effects of various fish processing methods of the nutritional mailto:kenegun@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 51 composition of fish is paramount. as nutritional studies have demonstrated that nutrient contents in fish that are required by the consumers are either increased [9,10] or reduced during processing, thereby impacting on the total nutritive value of the fish [11 – 15]. fish processing methods vary between different countries and within the same country depending on the species of fish used and the type of product desired. smoke drying is a method of fish processing which utilizes locally built fire place with firewood as the source of heat. it preserves and increases the shelf life of fish by drying, cooking, acting as an effective antioxidant, bacteriostatic and bactericidal agent [16,17]. the drawbacks to this method of fish processing include time consumption, deposits of vaporized chemical constituents of firewood on the fish, air pollution, health risk to fish processors and unregulated drying temperature resulting in the denaturing of important nutritional constituents of fish. while fish processing by oven drying is the use of drying ovens in the removal of moisture from fish. this method of fish processing is less time consuming, human and environmentally safe; and the heating temperature is regulated to preserve the integrity of important constituents such as protein and minerals, which can easily be denatured with heat. in nigeria, smoke drying is the commonly utilized method for fish processing; as smoked fish products are the commonest form of fish product available to consumers, which accounts for 61% of the 194,000 metric tons of dry fish produced in nigeria [18]. the smoke-drying method has been an age long traditional practice for processing fish and other aquatic organisms in order to improve their shelf life. increasing concerns on air pollution and wood consumption in the processing of food materials by smoking, has led to the introduction and usage of the oven-drying method as an alternative fish processing method. the poor technology assimilation and usage of the oven-drying method as an alternative to smoke drying among local fish processors, has necessitated this study to investigate the effects of oven-drying and smoke-drying methods on the nutritional composition of commercial fish species and their nutrient availability to meeting the required nutritional intake (rni) for adults, pregnant and lactating women (plw) and children in nigeria. 2. matherials and methods collection of fish samples: fresh fish samples of clarias gariepinus (burchell, 1822) and tilapia zillii (gervais, 1848) were procured from two (2) identified inland freshwater fish landing sites in edo state, nigeria. fish samples were collected monthly for a period of six (6) months, february to july, 2018. fish samples were preserved in ice blocks and transported to the central laboratory of the department of animal and environmental biology, faculty of life sciences, university of benin where they were properly identified using taxonomic guides [19]. in the laboratory, morphometric measurements of standard length, total length and body weight of all fish specimens were taken to the nearest 0.1cm and 0.1g using a metre rule and digital electronic weighing balance [mettler toledo (pl203 model)] respectively. experimental set-up: the fish samples were divided into three batches. this is important for valid conclusions to be drawn. each batch contained three (3) specimen each of clarias gariepinus and tilapia zillii batch i: fresh fish samples in batch i served as control for proximate and mineral analyses. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 52 batch ii: fish samples in batch ii were oven-dried at a temperature of 105oc for 6 hours using an electric oven [bran drying oven (dhg-9023a model)]. after drying, fish samples were placed in the desiccator to cool down and were immediately analysed for fat, protein and ash content. batch iii: fish samples were smoked dried by local fish farmers and sellers using locally built fireplace which utilizes firewood for heat production. analytical methods: nutritional composition analysis of moisture content, crude protein and crude fat were carried out as described in detail by aoac [20] and reksten et al.[21]. mineral contents were analysed using the inductively coupled plasma optical emission spectrometry (icp-oes) method [22]. calculation of potential contribution to recommended nutrient intakes (rni): the potential contribution of the nutritional content of fresh, oven dried and smoked samples of c. gariepinus and t. zilli to daily recommended nutrient intake (rni) for each nutrient as recommended for pregnant and lactating women (plw), adults/ adolescents (8 years and above) and children (1 – 7 years) was estimated as described by egun and oboh [23] and egun et al. [24]. the per capital consumption of fish as food in nigeria is 13.3 kg, which is equivalent to 36.4g portion per day [25]. the micronutrients of interest in this study are calcium, iron and zinc. these micronutrients were identified based on several reported literatures of their deficiencies especially among children, pregnant and lactating women (plw) in nigeria [26 – 31]. data analysis and presentation: all statistical analysis were computed using microsoft excel and statistical package for social sciences (spss) version 21. the composition values gotten from fish samples in batches i – iii were analysed for their range values, mean and standard errors. one way analysis of variance (anova) was used to test for significant difference (p< 0.01) between means and the source of significant differences identified using duncan’s multiple range (dmr) test. 3. results and discussion results the summary of the proximate and mineral composition of fresh, oven dried and smoked samples of c. gariepinus and t. zillii are presented in tables 1 and 2. in c. gariepinus, mean values for moisture content was 64.78% (fresh), 9.80% (ovendried) and 4.73% (smoked); protein content was 15.79% (fresh), 16.62% (oven-dried) and 14.62% (smoked); lipid content was 6.57% (fresh), 5.36% (ovendried) and 4.43% (smoked); ash content was 3.99% (fresh), 4.27% (oven-dried) and 6.05% (smoked) were recorded with observed significant difference (p < 0.01) between the means of the different treatments. for mineral contents, significant difference (p < 0.01) was observed in the mean contents of iron and magnesium, while there was no significant difference (p > 0.01) in the mean contents of zinc, calcium, sodium and potassium in the fresh, oven dried and smoked samples of c. gariepinus. in t. zillii, mean values for moisture content was 66.48% (fresh), 9.79% (ovendried) and 4.61% (smoked); protein content was 16.68% (fresh), 17.29% (oven-dried) and 13.76% (smoked); lipid content was 1.64% (fresh), 1.35% (ovendried) and 0.86% (smoked); ash content was 4.94% (fresh), 3.93% (oven-dried) and 4.58% (smoked) were recorded with observed significant difference (p < 0.01) between the means of the different food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 53 treatments. for mineral contents, significant difference (p < 0.01) was observed in the mean contents of iron, zinc, calcium, magnesium, sodium and potassium in the fresh, oven dried and smoked samples of t. zillii. the potential contribution of the fish samples from the various treatments to the recommended nutritional intake (rni) of calcium, iron and zinc in adults, pregnant and lactating women (plw), and children are presented in tables 3 to 5. fresh, ovendried and smoked samples of c. gariepinus and t. zillii did not meet ≥ 25% of the rni for calcium in adults, plw and children (table 3). table 1 summary of the proximate and mineral composition of fresh, oven-dried and smoked clarias gariepinus fresh oven dried smoked p value mean ± se mean ± se mean ± se moisture (%) 64.78 ± 0.84c 9.80 ± 0.09b 4.73 ± 0.08a p < 0.01 protein (%) 15.79 ± 0.21b 16.62 ± 0.14c 14.62 ± 0.15a p < 0.01 lipid (%) 6.57 ± 0.14c 5.36 ± 0.09b 4.43 ± 0.08a p < 0.01 ash (%) 3.99 ± 0.13a 4.27 ± 0.11a 6.05 ± 0.16b p < 0.01 iron (mg/100g) 6.17 ± 0.29b 4.81 ± 0.08a 4.43 ± 0.08a p < 0.01 zinc (mg/100g) 2.60 ± 0.12a 2.76 ± 0.06a 2.40 ± 0.06a p > 0.01 calcium (mg/100g) 4.21 ± 0.20a 5.24 ± 0.56a 4.84 ± 0.56a p > 0.01 magnesium (mg/100g) 3.31 ± 0.15a 3.47 ± 0.24ab 4.29 ± 0.30b p < 0.01 sodium (mg/100g) 4.61 ± 0.07a 6.08 ± 0.82a 6.66 ± 0.81a p > 0.01 potassium (mg/100g) 7.90 ± 0.14a 7.05 ± 0.33a 7.57 ± 0.41a p > 0.01 note: similar letters (superscripts) indicate values that are not significantly different from each other (p < 0.01) table 2 summary of the proximate and mineral composition of fresh, oven-dried and smoked tilapia zillii fresh oven smoked p value mean ± se mean ± se mean ± se moisture (%) 66.48 ± 0.89c 9.79 ± 0.25b 4.61 ± 0.20a p < 0.01 protein (%) 16.68 ± 0.26b 17.29 ± 0.37b 13.76 ± 0.70a p < 0.01 lipid (%) 1.64 ± 0.08b 1.35 ± 0.16b 0.86 ± 0.12a p < 0.01 ash (%) 4.94 ± 0.25a 3.93 ± 0.57a 4.58 ± 0.66a p > 0.01 iron (mg/100g) 8.19 ± 0.10b 8.46 ± 0.12b 7.56 ± 0.17a p < 0.01 zinc (mg/100g) 1.88 ± 0.10a 3.15 ± 0.10b 2.28 ± 0.07a p < 0.01 calcium (mg/100g) 2.56 ± 0.10a 6.73 ± 0.13c 5.86 ± 0.14b p < 0.01 magnesium (mg/100g) 2.50 ± 0.10a 3.47 ± 0.13b 4.27 ± 0.27c p < 0.01 sodium (mg/100g) 4.76 ± 0.11a 7.03 ± 0.18b 7.52 ± 0.22b p < 0.01 potassium (mg/100g) 9.86 ± 0.22b 7.37 ± 0.25a 8.09 ± 0.27a p < 0.01 note: similar letters (superscripts) indicate values that are not significantly different from each other (p < 0.01) table 3 potential contribution of fish in a standard portion, and to average daily rni (%) of calcium fresh oven dried smoked dried adults plw children adults plw children adults plw children average daily rni 1000 1040 700 1000 1040 700 1000 1040 700 clarias gariepinus 1.53 (0.15%) 2.11 (0.20%) 1.53 (0.22%) 1.91 (0.19%) 2.62 (0.25%) 1.91 (0.27%) 1.76 (0.18%) 2.42 (0.23%) 1.76 (0.25%) tilapia zillii 0.93 (0.09%) 1.28 (0.12%) 0.93 (0.13%) 2.45 (0.25%) 3.37 (0.32%) 2.45 (0.35%) 2.13 (0.21%) 2.93 (0.28%) 2.13 (0.30%) *assumed standard portion is equivalent to 36.4g/ day for nigerians *plw – pregnant and lactating women from a standard portion of 50g/day [32] *rni – recommended nutritional intake food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 54 fresh, oven-dried and smoked samples of c. gariepinus meet ≥ 25% of the rni of iron and zinc in children only. fresh, ovendried and smoked samples of t. zillii meet ≥ 25% of the rni of iron in plw and children; while oven-dried and smoked samples of t. zillii meet ≥ 25% of the rni of zinc in children only (tables 4 and 5). table 4 potential contribution of fish in a standard portion, and to average daily rni (%) of iron fresh oven – dried smoked dried adults plw children adults plw children adults plw children average daily rni 16 15 7 16 15 7 16 15 7 clarias gariepinus 2.24 (14%) 3.09 (20.6%) 2.24 (32%) 1.75 (10.94%) 2.41 (16.07%) 1.75 (25%) 1.61 (10.06%) 2.22 (14.80%) 1.61 (23%) tilapia zillii 2.98 (18.63%) 4.10 (27.33%) 2.98 (42.57%) 3.08 (19.25%) 4.23 (28.20%) 3.08 (44%) 2.75 (17.19%) 3.78 (25.20%) 2.75 (39.29%) *assumed standard portion is equivalent to 36.4g/ day for nigerians *plw – pregnant and lactating women from a standard portion of 50g/day [32] *rni – recommended nutritional intake table 5 potential contribution of fish in a standard portion, and to average daily rni (%) of zinc fresh oven dried smoked dried adults plw children adults plw children adults plw children average daily rni 10 7.9 3 10 7.9 3 10 7.9 3 clarias gariepinus 0.95 (9.50%) 1.30 (16.46%) 0.95 (31.67%) 1.01 (10.01%) 1.38 (16.07%) 1.01 (33.67%) 0.87 (8.70%) 1.20 (15.19%) 0.87 (29%) tilapia zillii 0.68 (6.80%) 0.94 (11.90%) 0.68 (22.67%) 1.15 (11.5%) 1.58 (20%) 1.15 (38.33%) 0.83 (8.30%) 1.14 (14.43%) 0.83 (27.67%) *assumed standard portion is equivalent to 36.4g/ day for nigerians *plw – pregnant and lactating women from a standard portion of 50g/day [32] *rni – recommended nutritional intake discussion fish processing and nutritional composition the main principle involved in fish processing by oven-drying or smoking is the creating of an unfavourable environment for microbial growth by reducing the fish moisture content through heating, thereby prolonging shelf life [33]. fish processing by oven-drying or smoking have been reported to enhance the flavour, taste, and promote digestibility of protein [9,10,34]. however, a substantive magnitude of nutrient loss occurs due to application of heat thereby decreasing the concentration of some nutrients especially protein [35]. the nutritional composition of c. gariepinus and t. zillii showed significant variation during processing by oven drying and smoking. the process of oven-drying and smoke drying significantly reduced the moisture and lipid contents in c. gariepinus and t. zillii. the low moisture and lipid content observed in oven-dried and smoked samples is of advantage as it safeguards fish samples from microbial attack and and rapid spoilage due to lipid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 55 dehydrogenation. protein content showed a remarked increase in oven-dried samples of the fishes when compared to the smoked samples which recorded the lowest. nutritionally, protein content is the most essential constituent which defines the wholesomeness and quality of fish meat. it is generally present in the range of 16 – 18% [36]. the significant increase in protein of c. gariepinus (from 15.79% to 16.62%) and t. zillii (from 16.68% to 17.29%); and their mineral contents iron, zinc and calcium in oven-dried samples, makes them good sources of dietary protein and mineral elements for human consumption. conversely, the reduced protein content in c. gariepinus (from 15.79% to 14.62%) and t. zillii (from 16.68% to 13.76%) makes the smoked samples of c. gariepinus and t. zillii inadequate sources of dietary protein. the increased ash content in oven-dried and smoked samples of c. gariepinus and t. zillii which is indicative of higher mineral contents is attributed to the decreased moisture content, concentration of chemical components in the fish samples and oxidation of polyunsaturated fatty acids (pufa) [37]. high ash content has been reported to lower bacterial and fungal activities leading to better shelf life in fishes [38]. potential contribution to required nutrient intake (rni) fishes provide a good supply of mineral elements which are essential to the proper growth and development of the human body in a readily usable form [39]. the insufficient intake of essential nutrients such as calcium, iron and zinc have been associated to major developmental health issues. the calcium content in the oven-dried and smoked samples of c. gariepinus and t. zillii did not provide the required proportional nutrient intake (≥ 25%) for adults, pregnant and lactating women (plw) and children (table 3). with reported clinical prevalence of hypocalcaemia among pregnant women in sub-sahara africa [40], and incidences of rickets among children particularly from poor households [41], the processed fish species should not be solely relied on to meet dietary calcium intake. reported high prevalence of iron deficiency anemia (ida) in plw especially in rural communities, and among children have been linked with adverse foetal, maternal and child development outcomes [30, 42 – 44]. iron content in oven-dried and smoked samples of c. gariepinus meet ≥ 25% of the rni for iron in children, while oven-dried and smoked samples of t. zillii meet ≥ 25% of the rni of iron in plw and children (table 4). this implies that the consumption of the processed fishes will contribute substantially to ameliorating ida in plw and children. zinc content in oven-dried and smoked samples of c. gariepinus and t. zillii meet ≥ 25% rni of zinc in children only (table 5). the reported clinical studies on the prevalence of maternal zinc deficiency (hypozincemia) among the socioeconomically disadvantaged plw, adults and children in developing countries such as nigeria [29,31,45,46], is indicative that the processed fish samples cannot be relied upon to meet dietary zinc requirements particularly for pregnant and lactating women. study insights the study has provided an insight on the advantage of oven-drying method in preserving and improving the nutritional quality of fish when compared to the smoked dried method of fish processing. therefore, there is need for increased knowledge awareness among local fish processors, and encouraging them to adopt food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 56 the oven-drying method through the provision of technology and equipment support. this will improve the nutritional quality of dried fish products available to consumers in the nigerian markets, and increase foreign income earnings. the nutrient availability in the processed fish samples to meeting the required nutrient intake (rni) for mineral elements in adults plw and children was found to be dependent on the per capita fish consumption. 4. conclusion this study has investigated the effect of oven-drying and smoke drying fish processing methods on the nutritional composition and nutrient availability of commercial fish species. the increased protein and mineral contents iron, zinc and calcium in oven-dried samples of c. gariepinus and t. zillii when compared to smoke dried samples makes them good sources of dietary protein and mineral elements for human consumption. it is therefore recommended that the traditional method of smoke drying should be discouraged in lieu of its diminutive effect on nutritional composition of fishes, environmental pollution and health risk. also, increased sensitization of local fish processors on the advantageous use of oven drying method and making the oven dryers available at affordable cost is expedient. increased per capita consumption of fish and fish products in nigeria is encouraged to increase nutrient availability in meeting the required nutrient intake (rni) for mineral elements in adults plw and children. 5. acknowledgments the authors are grateful to mr. peter uwaifo, mr, timothy agho and all the laboratory technicians at benin owena river basin/ university of benin joint analytical laboratory that have contributed to producing high-quality analytical data. disclosure of interest the authors report no conflict of interest. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 6. references [1]. aberoumand, a., studies on the effects of processing on food quantity of two selected consumed marine fishes in iran. international food research journal, 21(4): 1429 – 1432 (2014). [2]. holma, k. a., maalekuu, b. k., effect of traditional fish processing methods on the proximate composition of red fish stored under ambient room conditions. american journal of food and nutrition, 3(3): 73 – 82 (2013). [3]. okonta, a. a. and ekelem, j. k., a preliminary study of micro-organisms associated with fish spoilage in asaba, southern nigeria. proceedings of the 20th annual conference of the fisheries society of nigeria (fison), port harcourt, 14th-18th november, 557 – 560pp (2005). [4]. tao, w., linchun, m., influences of hot air drying and microwave drying on nutritional and odorous properties of grass carp (ctenopharyngodon idellus) fillets. food chemistry 110: 647-653 (2008). [5]. food and agriculture organization (fao)/who, vitamin and mineral requirements in human nutrition: report of a joint fao/whoexpert consultation. bangkok, thailand, turkish journal of fisheries and aquatic science, 12: 761 – 770 (1998). [6]. united state department of agriculture (usda), agricultural research service, national nutrition data base for standard reference. release 23. nutrition laboratory (2010). [7]. bashir, m. k., steven, s., pandit, r., the determinants of rural household food security in the punjab, pakistan: an econometric analysis. working paper 1203, school of agricultural and resource economics, university of western australia, crawley, australia (2012). online available at http://ageconsearch.umn.edu/bitstream/122526/2/w orking%20paper%201203.pdf. http://ageconsearch.umn.edu/bitstream/122526/2/working%20paper%201203.pdf http://ageconsearch.umn.edu/bitstream/122526/2/working%20paper%201203.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 57 [8]. womeni, h. m., tenyang, n., linder, m., tiencheu, b., villeneuve, p., tchouanguep mbiapo, f., the chemical composition, fatty acid, amino acid profiles and mineral content of six fish species commercialized on the wouri river coast in cameroon, rivista italiana delle sostanze grasse,91: 129–138 (2014). [9]. singh, r. p., heldman, d., introduction to food engineering. 5th edition. academic press. 892pp (2013). [10]. pigott, g. m., fish processing, a book by singh, r. p. encycopaedia britania, (2015). last updated 1-2-2015. retrieved 14 january, 2016. [11]. saliu, j. k., effect of smoking and frozen storage on the nutrient composition of some african fish. adv. nat. appl. sci.2 (1): 16 – 20 (2008). [12]. chukwu, o., shaba, i. m., effect of drying methods on proximate compositions of cat fish (clarias gariepinus). world j. agric. sci. 5(1): 114 – 116 (2009). [13]. osibona, a. o., comparative study of proximate composition, amino and fatty acids of some economical important fish species in lagos, nigeria. african journal of food science, 5(10): 581 – 588 (2011). [14]. mansur, m. a., rahman, s., khan, m. n. a., reza, m. d., kamrunnahar uga, s., study on the quality and safety aspect of three sun-dried fish. african journal of agricultural research 8: 5149 -5155 (2013). [15]. oboh, i. p., sanni, o. a., egun, n. k., wilfred-ekprikpo, c. p., the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae. food and environment safety, 18 (1): 60 – 69 (2019). [16]. ahmed, e.o., ali, m. e., kalid, r. a., taha, h. m., mahammed, a. a., investigating the quality changes of raw and hot smoked oreochromis niloticus and clarias lazara. pakistan journal of nutrition, 9(5): 481 – 484 (2010). [17]. daramola, j. a., kester, c. t., allo, o.o., biochemical changes of hot smoked african catfish (clarias gariepinus) samples from sango and ota markets in ogun state. the pacific journal of science and technology, 14(1): 380 – 386 (2013). [18]. ogbonnaya, c., ibrahim, m. s., effects of drying methods on proximate compositions of catfish (clarias gariepinus). world journal of agricultural science, 5(1): 114 – 116 (2009). [19]. idodo-umeh, g., freshwater fisheries of northern nigeria – taxonomy, ecological notes, diet and utilization (2003). isbn 978-8052-01-0. [20]. association of official analytical chemists, official methods of analysis of the association of official analytical chemists. 18th edition, washington d.c. usa (2010). [21]. reksten, a.m., bokevoll, a., frantzen, s., lundebye, a.-k., kogel, t., aakre, i., kjellevold, m., sampling protocol for the determination of nutrients and contaminants in seafood the eaf-nansen programme (2020). methodsx (submitted). [22]. american public health association (apa), american waterworks association (awa), water environment federation (wef), metals in water by icp/ms: section 3125. in: rice, e.w., baird, r.b., eaton, a.d., clesceri, l.s. (eds.), standard methods for the examination of water & wastewater. 22nd ed. (2012). american public health association, american waterworks association and water environment federation, washington, dc. [23]. egun, n. k., oboh, i. p., potential contribution of captured fishes to the recommended nutrient intakes (rnis): a case study of commercial fish species from ikpoba reservoir, edo state, nigeria. measurement: food, 5, (2022) https://doi.org/10.1016/j.meafoo.2021.100014 [24]. egun, n. k., olowo, u. c., oboh, i. p., potential contribution of selected wild fish species to the minerals intake of pregnant and lactating women, children and adults in rural riverine communities of edo state: insights and outcomes. measurement: food, 8, (2022). https://doi.org/10.1016/j.meafoo.2022.100063 [25]. world fish center, nigeria (2015). https://www.worldfishcenter.org/countrypages/nigeria. accessed 14 july 2019. [26]. awobusuyi, j.o., adedeji, o.o., [...], daniel, f.a., zinc deficiency in a semi urban nigerian community: prevalence and relationship with socioeconomic status and indices of metabolic syndrome. journal of public health, 22: 455 – 459 (2014). [27]. ekwochi, u., osuorah, d.i.c., odetunde, o.i., egbonu, i., ezechukwu, c.c., prevalence of iron deficiency anaemia in anaemic under-5 children in enugu, south east nigeria. nigerian journal of paediatrics 41(2):129 – 132 (2014). http://dx.doi.org/10.4314/njp.v41i2,10 [28]. olufunke, t.o., biobele, j.b., oluwagbemiga, o.a., iron deficiency among apparently healthy children aged 6 to 24 months in https://doi.org/10.1016/j.meafoo.2021.100014 https://doi.org/10.1016/j.meafoo.2022.100063 http://dx.doi.org/10.4314/njp.v41i2,10 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 2023 nkonyeasua kingsley egun, ovbokhan osavibie imadonmwiniyi, vivian ezighodeomo iyoha, ijeoma patience oboh, fish processing and nutrient availability: a study on the effect of drying methods on the nutritional content of selected fish species. food and environment safety, volume xxii, issue 1– 2023, pag. 50 – 58 58 ibadan, nigeria. pediatric hematology and oncology, 33: 338 – 346 (2016). [29]. ibeawuchi, a.n.e., onyiriuka, a.n., abiodun, p.o., high prevalence of zinc deficiency in rural nigerian preschool children: a community based cross-sectional study. rom. j. diabetes nutr metab dis., 24 (1): 031 – 039 (2017). [30]. olowole, o.b., agboola, a.a., dietary iron intake in nigeria. novel techniques in nutrition and food science, (2018). doi: 10.31031/ntnf.2018.01.000519. [31]. amadi, c., aleme, b. m., the prevalence of zinc deficiency among men with and without prostate cancer in port harcourt, nigeria. nutrition and cancer, 72 (6): 1018 – 1025 (2020). [32]. u.s. department of health and human services (hhs), u.s. department of agriculture (usda, 2015–2020 dietary guidelines for americans. 8th edition. december 2015. available at http://health.gov/dietaryguidelines/2015/guidelines/ [33]. ayolabi, i.c., fagade, e.o., mycological evaluation of smoked fish (ethmalosa fimbriata) from retail outlets in ago-iwoye, ogun state, nigeria. journal of life and physical sciences, 3 (2): 64 – 68 (2010). [34]. amoo, i. a., akinneye, j. o., arannilewa, s. t., effect of drying methods on the nutritional composition of three species of fish (bonga sp. sardinella sp. and heterotisniloticus). international journal of fisheries 2: 99 – 103 (2007). [35]. morris, a., barnett, a., burrows, o., effect of processing on nutrients content of foods, cajarticles, 37:160 – 164 (2004). [36]. ninan, g., chemical composition of fish and shell fish. in joseph, j., mathew, p.t., joseph, a. c. and muraleedharan, v. (eds). product development and seafood safety, p. 15-32 (2003). cochin, india: central institute of fisheries technology. [37]. daramola, j. a., fasakin, e. a., adeparusi, e. o., changes in physiochemical and sensory characteristics of smoked-dried fish species stored at ambient temperature. african journal of food. agriculture, nutrition and development 7(6): 12 – 22 (2007). [38]. oboh, i. p., sanni, o. a., egun, n. k., wilfred – ekprikpo, c. p., the effect of actellic dust treatment on the proximate and mineral composition of synodontis nigrita and tilapia mariae. food and environment safety 18 (1): 60 – 66 (2019). [39]. egun, n. k., olowo, u. c., oboh, i. p., potential contribution of selected wild fish species to the minerals intake of pregnant and lactating women, children and adults in rural riverine communities of edo state: insights and outcomes. measurement: food, 8 (2022). https://doi.org/10.1016/j.meafoo.2022.100063 [40]. ajong, a.b., kenfack, b., ali, i.m., yakum, m.n., telefo, p.b., prevalence and correlates of low serum calcium in late pregnancy: a cross sectional study in the nkongsamba regional hospital; littoral region of cameroon. plos one 14(11) (2009), e0224855. https://doi.org/10.1371/journal.pone.0224855 [41]. oginni, l.m., badru, o.s., sharp, c.a., davie, m.w.j., worsfold, m., knee angles and rickets in nigerian children. journal of pediatric orthopedics 24, 403 – 407 (2004) [42]. okafor, i.m., okpokam, d.c., antai, a.b., usanga, e.a., iron status of pregnant women in rural and urban communities of cross river state, south-south nigeria. niger. j. physiol. sci., 31: 121 – 125 (2016). [43]. ajepe, a.a., okunade, k.s., sekumade, a.i., daramola, e.s., beke, m.o., ijasan, o., et al., (2020). prevalence and foetomaternal effects of iron deficiency anaemia among pregnant women in lagos, nigeria. plos one, 15 (1), (2020). e0227965. https://doi.org/10.1371/journal.pone.0227965 [44]. ugwu, n.i., uneke, c.j., iron deficiency anemia in pregnancy in nigeria a systematic review. niger j clin pract [serial online] 23: 889 – 896 (2020). available from: http://www.njcponline.com/text.asp?2020/23/ 7/889/288872 [45]. darnton-hill, i., zinc supplementation during pregnancy: biological, behavioural and contextual rationale. july 2013. elibrary of evidence for nutrition actions (elena). https://www.who.int/elena/bbc/zinc_pregnancy/en/ assessed 10 th october, 2020. [46]. ejezie, f.e., nwagha, u.i., zinc concentration during pregnancy and lactation in enugu, south-east nigeria. ann med health sci res., 1(1): 69 – 76 (2011). pmid: 23209957 http://health.gov/dietaryguidelines/2015/guidelines/ https://doi.org/10.1016/j.meafoo.2022.100063 https://doi.org/10.1371/journal.pone.0224855 https://doi.org/10.1371/journal.pone.0227965 https://www.njcponline.com/text.asp?2020/23/7/889/288872 https://www.njcponline.com/text.asp?2020/23/7/889/288872 https://www.who.int/elena/bbc/zinc_pregnancy/en/ 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2021.026 235 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 235 246 evaluation of surface water quality and contamination status of the zeremnavallysub-basininthe skikda region (north-eastern of algeria) * nadjet zair 1, badra attoui 2 , abdelmonem miloudi 3 , abd erahman khachkhouche 4 1hydraulic and civil engineering department, university of el-oued, algeria. el-oued, algeria, nadjetzair@hotmail.fr 2laboratory of geology, badji mokhtar university of annaba.bp12, 23000 annaba, algeria,att.badra@yahoo.fr 3hydraulic and civil engineering department, university of el-oued, algeria, miloudi-abdelmonem@univ-eloued.dz 4hydraulic and civil engineering department, university of el-oued, algeria, abder03@hotmail.com *corresponding author received 5th may 2021, accepted 28th september 2021 abstract : the purpose of this paper isto assess the quality and status of organic and metal pollution of surface waterof zeremna vally sub bassin in the skikda. , used the water quality index (wqi), the organic pollution index (opi), the pollution index (pi) and the metal index (mi) according to the uses of drinking water, irrigation and aquatic life.the results of wqi revealed that 80% of the samples indicate a poor quality to non-drinking water, present a strong to very strong organic pollution for all use, except for the samples of the upstream of the study area (s1) and (s2) which present respectively good quality water and moderate pollution for all use, in s3 and s4 of the poor quality water with moderate organic pollution for drinking water and aquatic life and good for irrigation. the metal index (mi) and pollution index (pi) were calculated to assess the contamination of the water of zeremna vally by metals (cr +, pb + 2 and zn + 2).the values of mi and pi indicate aslight pollution of the water of the river, which is described as serious in the stations s9 and s10. this anthropic environmental degradation recorded downstream of the two urban communes due on the one hand to the intensive use of nitrogen and phosphate agricultural fertilizers and the absence of septic tanks in the neighboring anarchic settlements and on the other hand to the impact of leachates loaded with various pollutants from the zef-zef wilaya of skikda. keywords : surface water, wqi water quality index, organic pollution index opi, pollution index pi, metal index mi, zeremna vally. 1. introduction : water is a natural resource essential for life throughout the ecosystem [1,2].in algeria, the issue of water is one of the main environmental problems, due on the one hand to the health and economic consequences of water pollution and insufficient sanitation and, on the other hand, pressure exerted on resources due to the increase in water needs.the annual volume of wastewater discharges hasdoubled from 16.79 hm3 / year to 31.25 hm3 / year from 2015 to 2020 [3-5]. this is explained by the increase in the urban population, the increase in the supply and individual consumption of drinking water as well as the significant use of water by the industrial sector. the study region is experiencing rapid urban expansion with the production of large volumes of wastewater, posing a threat to the environment.in this region, themunicipalities located along the zeremna vally contribute for their part to the pollution of the receiving environment through the direct discharge of wastewater. the quantity of waste generated in the zeremna vally sub-basin is 340 ton / day[2,6]. the physico chemical characterization and quality control of http://fens.usv.ro/index.php/fens mailto:nadjetzair@hotmail.fr mailto:att.badra@yahoo.fr mailto:miloudi-abdelmonem@univ-eloued.dz food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 236 surface water in the zeremna vally provides a global view of the risks, in order to ensure the protection of resources and to determine the possible origin of alteration in the quality of the water. for the clear application of water quality, different methods used to assess the quality of surface water. the wqi global water quality index, organic pollution index (opi), metal quality index (mi) and pollution index (pi) were very useful tools to make the right decision and assess in a comparative way the water quality and the degree of pollution in time and space at the scale of the sub-watershed [7,8].the objective of the index is to transform complex data on water quality into information that is understandable and usable by the public [9]. this study is based on the use of indices of global water quality (wqi), organic pollution (poi), pollution(ip) and metal quality (mi) according to the physicochemical parameters of the water surface areas of the catchment area of the study region. the objective is to assess the quality and state of organic and metal pollution of surface water at ten stations located upstream downstream during a campaign in 2021.the final thematic distribution maps of the wqi, pi, mi, and poi were constructed using the gis geographic information system and golden surfer 13 logiciel. 2. materials and methods 2.1. study area the zeremna river subbasin is situated in algeria's northern east, and the coastal portion is the western part of the saf-saf bassin. about 36°-37° north latitude and 6°30' and 7° east longitude, the research region is situated in north-east algeria ;with an area of 168 km², the research area is part of the saf-saf river basin, and its natural boundaries are the mediterranean sea in the north, the eastern extension of the saf-saf river as well as the municipalities of hamadi krouma and hamrouch hamoudi, in the south the municipality of bouchtata mahmoud and the mounts ofksir-sasi in the west. the study area is divided into three main municipalities : bouchtata mahmoud, elhadaiek and skikda (fig.1). length of zeremna river is 30km, takes its source from the staiha mountains, an altitude of 550 m and flows into the mediterranean. the climate is a mediterranean type, with an average annual precipitation of 803 mm, and an average annual temperature of around 19.7 c °, during the period from 1994 to 2016[2,10],which allows the development of an abundant vegetation cover propagating on the ground by the density of the forests of cork oaks and variety of agricultural activies (citrus fields…) [3,5]. the study region presents terrains ranging from the paleozoic to the quaternary. this study region is dominated by schists, mica schists and gneisses, which gives the region soils rich in silt and clays this lithologically is reflected from the pedological point of view by soils characterized by a texture passing from silt to clay silt and a distribution of fine sands and clays which show the alluvial character of these soils [11, 12]. in the context of this study, the data used include data relating to the physicochemicalanalyzes of surface water from zeremna river. the sampling was carried out during the campaign spread over one month january 2021.the sampling stations were chosen according to the different activities identified in the study area (domestic, industrial and agricultural wastewater). thus, 10 water samples were taken from 10 sites in three municipalities spread along the zeremna river (fig. 2). two food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 237 stations were chosen upstream of the watershed near the sources of the zeremna river for stations s1 and s2. they represent reference stations far from anthropogenic domestic, industrial and agricultural inputs.stations s3, s4 and s5 are located respectively in the agricultural part ofmunicipalities the bouchtata mahmoudand elhadaiek.thestations s6, s7 and s8 are respectively located downstream of the urban municipality of skikda. the s8 station is located near the industrial area of skikda. finally, stations s9 and s10 are taken from a tributary of the main river downstream from the zef-zef public landfill which treats waste from the seven communes of the wilaya of skikda.water temperature, electrical conductivity and ph value weremeasured in situ, using hydrolab, model (multi set 430iwtw) and the biological oxygen demand after 5 days (bod5), ortho phosphates (po4 3-), ammonium (nh4) and nitrates (no3), cod, no2 -, zn, pb and cr analyzed in the laboratory using spectrophotometry. the evaluation and visualization of the results were carried out using excel 2010 software, aquakit software and software for the geographic information system gis (arc gis, qgis) and golden surfer 13 logiciel. fig. 1 the geographic situation of the study area fig.2. sample locations in study area 2.2. water quality index wqi : nine important parameters (ph, cod, ec, t°c, bod5, po4 3-, nh4 and no3 and no2) were selected to calculate the wqi water quality index. this index is a water quality classification technique based on the comparison of water quality parameters with algerian standards in our case of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 238 study.in other words, the wqi summarizes large amounts of water quality data in simple terms (excellent, good, poor, very poor, etc.).this method was originally proposed by horton (1965) and brown andal (1970) [13,14].in this study the wqi index was applied to estimate the influence of natural and anthropogenic factors on the basis of several parameters of the surface water chemistry of zeremna river.this index was calculated following the weighted arithmetic index method [14-17]. in this approach, a numerical value called relative weight (wi), specific to each physicochemical parameter, is calculated (table 1) according to the following formula : wi=k/si (1) where: k = constant of proportionality and can also be calculated using the following equation: k=1/σni=1(1/si) (2) n is the number of parameters ; si is the maximum value of the algerian surface water standard (2011)[18] for each parameter in mg/l except for ph, t°c and electrical conductivity.next, a quality rating scale (qi) is calculated for each parameter by dividing the concentration by the standard for that parameter and multiplying by 100 as in the following formula: qi= (ci/si) ×100(3) qi: quality assessment scale for each parameter. ci: the concentration of each parameter in mg/l. finlay, the global water quality index is calculated by the following equation : wqi= σni=1 qi×wi/ σni=1 wi (4) five quality classes can be identified according to the values of the wqi water quality index (table1). table 1. wqi water classification and possible uses [8] wqi class water quality possible uses 0-25 excellente drinking water, irrigation and industry >25 – 50 good drinking water, irrigation and industry >50 – 75 poor irrigation and industry >75 – 100 very poor irrigation ˃ 100 unsuitable for drinking purpose appropriate treatment required before use 2.3. calculation of the organic pollution index (opi) the organic pollution index (opi) of leclercq (2001) [19]was also used to assess the organic load in the river.the principle of the poi is to divide the values of the polluting elements in 05 classes (table 2).this index is obtained by means of the values of ammoniums, bod5andphosphates.the principle of the calculation is to divide the values of the three pollutants into five classes and to determine, from the values obtained in the study, the corresponding class number for each parameter using the average data in table 2.the final organic pollution index is the average of the pollution classes for all parameters (table 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 239 table 2 grid of organic pollution index classes (opi)[19] nh4 + (mg n /l) no2 (g n /l) po4 3 (g p /l) dbo5 (mg o2/l) opi organic pollution class 5 < 0.1 < 6 < 16 <2 5 – 4.6 null class 4 0.1 – 0.9 6 10 16 – 75 2 – 5 4.5 –4.0 slight class 3 1.0 – 2.4 11 – 50 76 – 250 5.1 – 10 3.9 – 3.0 moderate class 2 2.5 – 6.0 51 –150 251 900 10.1 –15 1.9 –1.0 strong class 1 >6 > 150 > 900 >15 very strong 2.4. calculation of the pollution index (pi) two different quality indices are used to determine the metal contamination of zeremna river. (1) pollution index (pi) is based on individual metal calculations and categorized into 5 classes (table 3) according thefollowing equation [20]. pi= [(ci/si)2max+ (ci/si) 2 min] 1/2 /2 (5) ci : the concentration of each element ; si : metal level accordingto national water quality criteria. (2) the metals index (mi) is based on a total trend assessment of current status. the higher the concentration of a metal compared to its respective mac value, the worse the water quality [5] (table 4). according to [21,22], the mi is calculated using the following formula : mi= σni=1ci/(mac) i(6) ci : the concentration of each element, mac : maximum allowable concentration. table 3 water pollution index categories (pi) [20] classes pi value categories 1 <1 null 2 1-2 slight 3 2-3 moderate 4 3-5 strong 5 >5 very strong table 4. classification of water quality based on the metal index (mi) [20] mi class water quality <0.3 i very pure 0.3–1.0 ii pure 1.0–2.0 iii slightly affected 2.0–4.0 iv moderately affected 4.0–6.0 v strongly affected >6.0 vi seriously affected food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 240 3. results and discussion 3.1. descriptive statistics the descriptive statistical characteristics of the physico-chemical variables used in this study concerned the minimum and maximum values, mean and standard deviation. the results show that the ph is slightly alkaline, and varies between 7.6 and 8.85. the temperature varies between 12.1 and 14.3°c. the mineralization of water is relatively high with an electrical conductivity varies between 556 and 7380 μs/cm (table 4).the concentration ofdissolved oxygen demand cod is very high and exceeded the permissible standards for the majority of stations, with a minimum value recorded upstream from the river. as regards nitrates and ammonium, the results increase respectively from 34 to 1280 and from 0.14 to 169.2 mg/l. the samples with high concentrations of ammonium and nitrates are accompanied by a high pollutant load (50 mg/l of bod5).the contents of heavy metals (pb, zn and cr) oscillate between 0.011 to 2.5 mg/l, 0.12 to 5.6 mg/ and 0.05 to 4 mg/l respectively (table 5). table 5. descriptive statistics of physico-chemical parameters related to the sampling campaign 2021 january 2021 ph ce(μs/cm) 25c° t°c no3 mg/l no2 mg/l nh4 mg/l po4 mg/l pb mg/l cr mg/l zn mg/l dbo5 mg/l dco mg/l means 8 2047.9 13.37 283.96 1.24 27.92 12.16 0.37 1.23 0.65 10.04 330.3 max 8.85 7380 14.3 1280 5.2 169.2 54 2.5 5.6 4 50 2020 min 7.6 556 12.1 34 0.09 0.14 2.5 0.011 0.12 0.05 0.5 20 standard deviation 0.42 2536.74 0.62 483.23 1.84 54.06 18.56 0.78 2.13 1.25 15.7 626.56 3.2. water quality index wqi : in this study, the overall quality of surface waters of zeremna river was assessed by the wqi method. the relative weight (wi) of each physico-chemical parameter and the proportionality constant k were first calculated using the maximum values of the algerian standard drinking water (2011)[18] of the physico-chemical parameters studied (table 6). indeed, 9 important parameters in the study of surface water quality : ph, t°c, ec, cod, nh4, no3, no2, po4 and bod5 were taken into account in the calculation of the wqi value.after the calculation of the overall quality index wqi using the resultsof physico-chemical analysis and the national standard values of algerian drinking water (2011)[18], the water quality class is determined for the 10 samples related to the 10 sampling stations. thus, the class of good quality was identified for the stations located in the mountains of bouchetata mahmoud (s1), (s2). the class of poor quality was recorded respectively in the stations of the commune of bouchtata mahmoud (s3) and the commune of elhadaiek (s4).thus the class of non-drinking water was found respectively at the commune of elhadaiek (s5), (s9) and (s10) and the commune of skikda (s6), (s7) and (s8). (fig.3). 3.3. organic pollution index opi : after the calculation of the organic pollution index opi using the results of analysis of the pollution indicator parameters (nh4 + . bod5. po4 3. no2 and no3), the water quality class is determined for the 10 samples related to the 10 sampling stations (fig.5). thus, three classes of organic pollution (moderate. strong and very strong organic pollution). the analysis followed in the different stations was the subject of a data processing by the establishment of thematic maps of organic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 241 pollution of surface waters of zeremna river during the sampling campaign. the class of moderate organic pollution was identified in the stations (s1). (s2). (s3) and (s4). the classes of strong and very strong organic pollution were observed respectively instations (s5). (s6). (s7). (s8). (s9) and (s10) (fig.4). table 6. weight of physico-chemical parameters and algerian standard of surface water quality fig 3 .thematic map of the water quality index wqi of surface waters of zeremna river during the campaign january 2021. 3.4. calculation of the pi pollution index the pi and mi index for drinking water was calculated using the guidelines of (algerian drinking water quality standards. 2011) [18]. the guidelines of (fao. 1994) [23] were used to calculate the pi and im value for irrigation water. aquatic environment protection lifetime was calculated using the guidelines of (ccme. 2007) [24].three metals (pb. zn and cr) are selected to assess the metal pollution of the study area.according to the pollution index (table 7) which is based on calculations of individual metals.weight of physicochemical parameters and algerian standards of surface water quality (2011) [18]. the measured metals show a differentdegree of pollution in the water of zeremna river for different uses. another index is used to estimate the metal pollution of the waters of zeremna riverfor different uses. the metal index refers to the evaluation of the trend of the current state by calculating all measured metals (table 8). fig .4. thematic map of the organic pollution index poi of surface waters of zeremna river during the campaign january 2021. parameters si (maximum standard algerian value) 1/si wi ph 9 0.11 0.017 t (°c) 25 0.04 0.006 cond (μs/cm) 2800 0.000 0.000 cod mg/l 30 0.14 0.022 nh4 mg/l 4 5 0.77 no3 mg/l 50 0.02 0.003 no2mg/l 0.1 0.33 0.051 po4mg/l 2 0.5 0.077 dbo5mg/l 7 0.33 0.051 σ=11.59 σ=0.29 k=1/σ (1/si) 0.086 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 242 3.5. metal index (mi) according to the metal indexvalues. 80% of the stations (8 stations) selected along zeremna river are moderately threatened by metal pollution for the protection of aquatic life (mi> 2), except for two stations (s9 and s10) highly affected or mi reaches 58.5 and 136 at station 9 and 55 and 94 at station 10 for irrigation and aquatic life, respectively. 3.6. discussion the deterioration of water quality from upstream to downstream rivers is thought to be mainly related to urban and industrial wastewater discharges and/or the impact of agricultural activities [25, 26, 27]. table 7 pollution index of metals measured in the water of zeremna river according to the indicative levels of drinking water, irrigation and aquatic life 2021. station ip drinking water drinking water effect ip irrigation irrigation effect ip life aquatic effect aquatic life pb+2 s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 zn+2 s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 cr+ s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 0.16 0.19 0.13 0.11 0.32 0.17 0.14 0.13 25.12 11.65 0.5 0.42 0.59 0.68 0.59 0.85 0.67 0.5 34.04 17.02 1.56 1.9 1.34 1.56 1.9 2.12 2.23 2.45 62.6 60.73 no effet no effect no effect no effect no effect no effect no effect no effect serious effect serious effect no effect no effect no effect no effect no effect no effect no effect no effect serious effect serious effect slight effect slight effect slight effect slight effect slight effect moderate effect moderate effect moderate effect serious effect serious effect 0.016 0.018 0.013 0.011 0.032 0.018 0.014 0.013 2.51 1.16 0.04 0.012 0.01 0.021 0.018 0.026 0.021 0.016 1.077 0.53 1.56 1.9 1.34 1.56 1.9 2.12 2.23 2.45 62.6 60.73 no effect no effect no effect no effect no effect no effect no effect no effect serious effect serious effect no effect no effect no effect no effect no effect no effect no effect no effect slight effect no effect slight effect slight effect slight effect slight effect slight effect moderate effect moderate effect moderate effect serious effect serious effect 1.39 1.56 1.13 1 2.78 1.56 1.22 1.33 217.97 101.14 0.5 0.5 0.7 0.8 0.7 1 0.8 0.6 40.19 20.09 1.56 1.9 1.34 1.56 1.9 2.12 2.23 2.45 62.6 60.73 slight effect slight effect slight effect slight effect moderate effect slight effect slight effect slight effect serious effect serious effect no effect no effect no effect no effect no effect slight effect no effect no effect serious effect serious effect slight effect slight effect slight effect slight effect slight effect moderate effect moderate effect moderate effect serious effect serious effect food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 243 table 8 metal index (mi) in the study area for the different uses drinking water, irrigation and aquatic life 2021 station drinking water irrigation aquatic life s1 0.53 pure 0.42 pure 2.4 moderate effect s2 0.83 pure 0.72 pure 2.7 moderate effect s3 1.36 slight effect 1.23 slight effect 2.6 moderate effect s4 1.58 slight effect 1.44 slight effect 3 moderate effect é s5 1.9 slight effect 1.74 slight effect 3.1 moderate effect s6 2.13 slight effect 1.95 slight effect 3.9 moderate effect s7 2.18 moderate effect 2.04 moderate effect 3.6 moderate effect s8 2.34 moderate effect 2.23 moderate effect 3.4 moderate effect s9 69 serious effect serious effect 58.5 serious effect 136.35 serious effect s10 60.32 serious effect 55.23 serious effect 94.16 serious effect the water quality index wqi during the january campaign, of the year 2021 indicates that the majority of the stations (s3, s4, s5, s6, s7, s8, s9 and s10), i.e. 80% of the stations remain of poor qualityto non-potable water (25<wqi≤75) and (wqi >100). this degradation of water quality is probably linked to domestic discharges in urban areas where the treatment plants are not all efficient and to individual sanitation in rural areas such as station s3. the two remaining stations representing 20% each of all stations, with a good quality (25 <wqi ≤50) for station s1 and s2 of the municipality of bouchtata mahmoud that are respected the various uses of water (drinking water,irrigationand protection of aquatic life), the class of excellent quality remains absent (fig.3). the increasing degree of water degradation between stations s4. s5. s6. s7 and s8 is related to the increasing demographic and urban impact of the municipality of elhadaiek and the municipality of skikda compared to that of bouchtata mahmoud, these waters used only for irrigation. the serious contamination of water between stations s9 and s10 is due to the impact of the public dumping of six communes of the wilaya of skikda.the state of organic pollution of surface waters of zeremna river, calculated from the organic pollution index opi, show that 40% of the stations (4 stations) have moderate organic pollution (3 ≤opi≤3.5) during the campaign of 2021 (january). the exception appears another time at the station (s9) and the station (s10) representing 20% of the stations, which indicate very strong organic pollution (opi =1) and the rest of the stations (4 stations) that displays waters with strong organic pollution (2≤opi≤2.25) (fig.4). this serious pollution is mainly related to the relatively high concentrations of bod5, cod, po4 , no3, nh4 and no2 which exceed the algerian standards, these serious contents are probably due to the effect of the public dump of skikda. the degradation of water quality at stations s5, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 244 s6, s7 and s8 respectively of the commune of elhadaiek and skikda is due to discharges of domestic and urban origin without any prior treatment. the moderate quality of water between the four stations upstream and the average along zeremna river is due to agricultural activities by leaching of fertilizers and individual domestic discharges from rural areas. the state of heavy metal pollution of surface waters of zeremna river, calculated from the pollution index pi and the index of metals mi, the first index pi show 80% of the stations (8 stations) have no pollution to moderate (0.1≤pi≤2.45) during the campaign of 2021 (january). the exception appears another time at the station (s9) and station (s10) representing 20% of stations, which indicate a very strong pollution (pi>5). this severe pollution is always related to the impact of the public landfill of skikda (zef-zef).the second index of metal pollution mi of surface waters of zeremna river presents a moderate effect for the protection of aquatic life in eight stations (2<mi<4) during the campaign of 2021 (january). at two stations (s9 and s10), which indicate a serious contamination (mi>6), this serious pollution is still related to the impact of discharges highly charged with various pollutants.often toxic and of generally unknown composition of the public landfill zef-zef of the wilaya of skikda.upstream of the study area (s1 and s2), there is no effect of heavy metals on the water of zeremna river either for drinking water use or for irrigation. in the middle part of zeremna river, there is a slight effect of heavy metals on water quality (s3. s4. s5 and s6) for drinking water and water for irrigation.downstream of the study area (s7 and s8) of the commune of skikda.a moderate effect of heavy metals on the waters of these stations is recorded; this average pollution is due to uncontrolled domestic discharges and industrial discharges of the industrial zone of the commune of skikda.the results of the different indices, namely the water quality index (wqi), the organic pollution index(opi), the pollution index (pi) and the metal index (mi) applied to the surface waters of zeremna river studied during the campaign of 2021 (january), were tested the degree of concordance and / or discordance between the different methods. the indices wqi., opi., pi and mi show a complete concordance among all stations. the deterioration of water quality is reflected by an important mineral and organic load of anthropic origin from the urban communes of elhadaiek and skikda in the study area. the comparaison with previous work also shows a good agreement with the different indices [3,10]. these waters are very polluted in these municipalities, because of the very high concentrations of cod. nh4. no2 and po4 which exceed the algerian standards and for any use. this degradation is mainly of anthropic origin in relation with the agricultural activities by leaching of the soils very charged in fertilizers and with the discharges of urban waste water. the increase in the degree of pollution in summer would be linked.without doubt, to effluents loaded with domestic and industrial wastewater from the various urban centers (elhadaiek and skikda) remain important, and to the impact of the public landfill (zef-zef) of skikda. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 245 4. conclusion the quality of surface and groundwater has been severely degraded, limiting the actual water potential and leading to significant health and ecological impacts. urban effluents and the use of agricultural and industrial chemicals.too often discharged without appropriate treatment into the receiving environment, are the main source of surface water quality degradation. agriculture participates in this degradation by the exaggerated use of nitrogen and phosphate fertilizers,pesticides, herbicides, etc., the phenomenon of water pollution. the monitoring of quality indices (wqi).organic pollution (opi), pollution (pi) and metal index (mi) of waters during this study shows that surface waters of zeremna vally sub-basin remain at 20% of good quality and without effect of heavy metals and organic pollution (opi) moderate for all uses. however, the urban stations show an increasing deterioration of the quality of their waters according to the demographic pressure of the communes. thus surface waters are of poor quality to non-drinking water with a strong organic pollution in the commune of elhadaiek and the commune of skikda and used only in irrigation. in exception the two stations of the arm of zeremna river s9 and s10 show a serious deterioration of the quality of their waters and a serious effect of heavy metals for any use according to the effect of the public discharge of skikda.after the evaluation and interpretation of the results we can propose some solutions that would reduce or limit the probleme of contribution of a number of different human behaviors and uses to environmental pollution.in particular surface water represented by zermna river.among these solution,we can mention:reduce the use of pesticides and supervise the use of fertilizers.stabilization of the landfill site and installation of a leachate drainage network.treatment of wastewater befor discharge to stop environmental degradation and pollution of surface and groundwater ;regularly control individual sanitation by municipalites. 5. references [1].tampo.l.. gnazou.m.. akpataku.v.. bawa.l.. djaneye-boundjou. g. application of statistical methods to the hydrochemical study of the waters of a tropical hydrosystem: case of the zio river watershed (togo). european scientific journal. 11(14): 204225. (2015). [2]. aher. d. n.. kele. v. d.. malwade. k. d.. shelke. m. d. lake water quality indexing to identify suitable sites for household utility: a case study jambhulwadi lake; pune (ms). int.journal of engineering research and applications. 6(5) : 16-21. (2016). [3]. directionof water ressource of skikda (2020).study report on the hydroagricultural development of the perimeter of the elhadaiek plain. skikda. [4]. anonymous 3 inspections of the environment wilaya of skikda; cegep. environmental impact study of the technical landfill center of skikda zef-zef.57 pages. (2002). [5]. cegep. report of study: study of impact on the environment of the center of technical intercommunal burying of skikda (zefzef); center of study and management of projects. alger. 52p. (2002). [6]. mate. information manual on urban solid waste management and disposal –alger. (2003). [7]. lalami. a. e. o.. merzouki.m.. el hillali.o.. maniar.s.. koraichi. s. i.. pollution of surface water in the city of fez.morocco: typology.origin and consequences. larhyss journal p-issn 1112-3680/e-issn 2521-9782. (9). (2011). [8]. el hmaidi a.. talhaoui a.. manssouri i.. jaddi h.. ousmana h.. contribution of the pollution index and gis in the assessment of the physico-chemical quality of the surface waters of moulouya river (ne. morocco). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 nadjet zair, badra attoui, abdelmonem miloudi, abd erahman khachkhouche, evaluation of surface water quality and contamination status of the zeremna vally sub-basin in the skikda region (north-eastern of algeria), food and environment safety, volume xx, issue 3 – 2021, pag. 235 – 246 246 la houille blanche. 3 : 45–54. https://doi.org/10.1051/lhb/2020028. (2020). [9]. bakan.g.. boke ozkoc.h.. tulek.s.. cuce.h.. integrated environmental quality assessment of kızılırmak river and its coastal environment. turk. j. fisheries aquat. sci. 10 : 453–462. (2010). [10]. bougloufm.. contribution to the management and valorization of solid and household waste in skikda (north-east algeria). (2014). [11]. villa. j m.. the alpine chain of eastern algeria and the algerian-tunisian confines. thesis doct; univ pierre et marie curie. paris vi. 655. (1980). [12]. boucenna. impact of public discharge on ground water quality case of zefzef (skikda) magister thesis university of annaba. (2007). [13]. horton. r. k..an index number system for rating water quality. journal of water pollution control federation. 37(3) : 300-306. (1965). [14]. brown. r. m.. mccclelland. n. i.. deininger. r. a.. tozer. r. g.. a water quality indexdo we dare water and sewage works. 117 : 339–343.(1970). [15]. brown r.m.. mcclelland n.i.. deininger r.a.. o’connor m.f.. a water quality index crashing the psychological barrier.in: thomas w.a. (eds) indicators of environmental quality. environmental science research. (1) : 173-182.. springer. boston. ma. https://doi.org/10.1007/978-1-4684-2856-8_15. (1972). [16] . chatterji.c.. raziuddin.m.. determination of water quality index of a degraded river in asanol industrial area. raniganj. burdwan. west bengal. nature. environment and pollution technology. 1 (2) : 181-189. (2002). [17] yidana. s. m.. yidana.a.. assessing water quality using water quality index and multivariate analysis. environmental earth sciences59(7) :1461-1473. (2010)https://doi.org/10.1007/s12665-009-0132-3. [18]official journal. standard of algerian surface water. (2011). [19] leclercq l.. running water: characteristics and means of study.in wetlands. conference proceedings organized in 1996 by the ministry of the walloon region as part of the world year of wetlands. legs. walloon region.dgrne: 67-82. (2001). [20]. caerio.s.. costa. m.h.. ramos. t.b.. fernandes.f.. silveira.n.. coimbra.a.. mederios.g.. painho.m.. assessing heavy metal contamination in sado estuary sediment: an index analysis approach. ecolog. indicators (5) : 151–169. (2005). [21]. tamasi.g.. cini.r.. heavy metals in drinking waters from mount amiata. possible risks from arsenic for public health in the province of siena. sci. total environ. 327 : 41–51. (2004). [22]. el-sayed. s.m.m.. physicochemical studies on the impact of pollution up on the river nile branches. egypt (m.sc. thesis). faculty of science. benha university. egypt. 248. (2011). [23]. fao (the food and agriculture organization of the united nations).. manual of irrigation techniques under pressure.(2008). [24]. ccme (canadian council of ministers of the environment).for the protection of aquatic life in : canadian environmental quality guidelines. 1999. canadian council of ministers of the environment. 1999. winnipeg. (2007). [25]. şener.ş.. şener.e.. davraz.a.. evaluation of water quality using water quality index (wqi) method and gis in aksu river (swturkey). science of the total environment. 584 : 131-144. (2017). [26]. izougarhane.m.. mansouri.d.. el ibaoui.h.. chakiri.s.. fadli.m.. physicochemistry and metal contents of the wadi sebou estuary waters during years of sand dredging/ 2007. 2014. 2015. 2016. european scientificjournal. 12(30) : 127-151. (2016).doi: 10.19044/esj.2016.v12n30p127. [27]. khamar m.. bouya d.. ronneau c.. metallic and organic pollution of water and sediments of a moroccan river by urban liquid discharges. water quality research journal. canada 35 (1) :147161. (2000). https://doi.org/10.1051/lhb/2020028 https://doi.org/10.1007/978-1-4684-2856-8_15 https://doi.org/10.1007/s12665-009-0132-3 doi: https://doi.org/10.4316/fens.2021.015 137 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag.137 148 development of enriched cupcakes and cookies with orangefleshed sweet potato (ipomeabatatas l.): sensory and nutritional evaluation *gisèle ahou yah koua 1 , konan evrad brice dibi 2 , edwige essoma akoa 1 , sébastien niamke 1 1faculty of biosciences, félix houphouët-boigny university, 22 bp 582 abidjan 22, abidjan, côte d’ivoire, kouayahgisele@yahoo.fr, 2national agronomic research center (cnra), bouaké, côte d’ivoire, *corresponding author received 26th march 2021, accepted 29th june 2021 abstract: in developing countries especially in côte d’ivoire child malnutrition is a major burden. this malnutrition is often associated with poor nutritional composition of the complementary foods introduced during the weaning period. the purpose of this study was to assess nutritional, microbiological, and sensory properties of cupcakes and cookies supplemented with orange-fleshed sweet potato (ofsp). the proximate and microbiological analysis of enriched products was performed using analytical standard methods. sensory evaluation of formulated cupcakes and cookies was based on nine-point hedonic scales. proximate composition showed significant improvements in the contents of ash (1.020.06 – 1.980.03 g/100g dw), fiber (5.390.13 – 7.640.47 g/100g dw), and provitamin a (1031 – 4641 g/100g dw) of enriched cupcakes and cookies compared to control. based on moderate daily consumption of two pieces of ofsp composite cupcakes (60g/piece) and cookies (44g/piece), children aged 1 to 3 years may cover at least 42 and 85 % of their rda for vitamin a, respectively. the microbial analysis indicated showed that enriched ofsp cupcakes and cookies were of acceptable microbiological quality. in addition, the enriched ofsp cupcakes and cookies were accepted and liked by the consumers excepted those containing 50% ofsp flour. thus, cupcakes and cookies formulated with 35% ofsp flour can serve as a valuable vehicle for improving the nutritional status of young children. keywords: cupcakes, cookies, ofsp, nutritional value, sensory acceptance. 1. introduction malnutrition continues to be a major public health issue in the world, especially in developing countries, due to insufficient quality and quantity food intake [1]. it affects many people in sub-saharan and causes death and morbidity in young children under 5 years [2]. despite progressive health system improvement high malnutrition rates in côte d'ivoire for children under 5 years old, constitutes a significant burden regarding economic and social development [3]. like other countries of sub-saharan africa, the major contributing factors to child malnutrition in côte d’ivoire are protein-energy and micronutrient deficiencies. micronutrient deficiency also called “hidden hunger” is often related to the nutritional composition of complementary foods introduced in the weaning period [5]. indeed, these complementary foods are mostly composed of starchy foods resulting in deficiencies of key nutrients such as http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 138 proteins, iron, zinc, calcium, vitamin a, and vitamin c [6]. numerous researches suggest that the use of local crops which are rich sources of micronutrients in food proven to be a viable and sustainable approach in tackling all forms of malnutrition in addition to promoting food security in long term [7]. currently, reports abound on the positive effects of orange-fleshed sweet potato (ofsp) on the well-being of people of some african countries like kenya, uganda, nigeria, and mozambique [8], [9]. indeed, orange-fleshed sweet potato (ofsp) is well known as a staple food that has several nutritional advantages with relatively low cost. it makes an important contribution to energy and micronutrient requirements in rural and low-income communities [10]. in addition, ofsp flour is considered as an excellent source of carotene (provitamin a), energy and minerals (ca, p, fe, zn, and k). it contribute also to add natural sweetness, color, flavor, and dietary fiber to processed foods products[8], [11]. thus, adding ofsp into wheat flour may be a valuable strategy for provitamin a fortification of baked products such as biscuits, cakes, cookies, considered as snack foods [12], [13]. primarily formulated by using refined wheat flour; snack foods represent an important part of human diet, generally consumed between meals. the snack foods are mainly consumed by young children and adolescents can help achieve an individual's rda within a day [14]. in recent years, substitution of wheat flour with non glutenflours from secondary crops such as cassava, cocoyam, sweet potato, maize, rice, millet, sorghum and soybean for bakery products processing has gained prominence in low-income countries[15], [16]. however, in côte d’ivoire, little researches have mentioned the use of flours from these secondary crops, especially the new varieties of ofsp for bakery foods (cupcakes and cookies) preparation. indeed, the utilization of ofsp cultivars is limited to water or steam cooking for traditional meals preparation. thus, the purpose of this study is to determine the nutritional value, microbial and sensory properties of two snack foods (cupcakes and cookies) developed by using composite flour from three new cultivars of ofsp. 2. matherials and methods 2.1. samples preparation the ofsp flours were obtained from roots of ofsp cultivars called “irene” (v1), “kabode” (v4) and “covington” (v5) harvested in experimental farm located in the north (two localities: korhogo and boundiali) and north-east (two localities: bondoukou and nassian) regions of côte d’ivoire. briefly, ofsp roots were peeled in thin slices and dried at 50°c in ventilated oven for 18h. dried ofsp chips were milled into flour. the other ingredients such as wheat flour and baked products ingredients (butter, sugar, salt, egg, milk, and baking powder) were purchased from a shopping mall (abidjan mall) in abidjan (côte d’ivoire). 2.2. flours and bakery products formulation for this study, different ratios of composite flour make from wheat flour and ofsp flour were used: 100:0 (control), 85:15, 75:25, 65:35, and 50:50 (w/w). cupcakes were prepared by mixing 100g of composite flour, butter (50 g), sugar (25g), milk powder (10g), baking powder (1g), water (25 ml), and pinch of salt. the formulation for cookies preparation was: composite flour (100 g), butter (75g), sugar (85g), eggs (10g), whole milk (15 ml) and baking powder (1g). the resulting dough was baked in an oven at 180°c for 5 min. the cupcakes and cookies were food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 139 immediately cooled and used for further analyses. all the baked products were vacuum-sealed in opaque zip-lock bags and stored at 4°c until required. 2.3. proximate composition evaluation the methods described in the official methods of analysis of aoac international were used to determine the contents of moisture, ash and proteins [17]. crude fat contents were evaluated by soxhlet extraction described by [18]. total fiber content was determined by the method described by [19]. carbohydrates were calculated by difference while energy value was determined using factors determined described by [20]. carotenoid content in the baked products was evaluated according to the method described by [21]. powdered samples (1 g) were mixed with 5 ml ethanol containing butylated hydroxytoluene (0.1%, w/v) and heated in water bath at 85˚c for 5 min. then, 400 µl koh in water (80%, w/v) was added for saponification and the suspension was mixed using a vortex for 20 s and heated again in a water bath at 85˚c for 5 min. the tubes containing the mixture reaction were placed in ice after introducing 3 ml distillated water and carotenoids were extracted two times with 4 ml of hexane. the suspension was centrifuged at 12000 rpm for 10 min and the supernatants were collected in a flask. the absorbance of combined extracts was measured at 450 nm using spectrophotometer (thermo scientifictm evolution 201 uv-visible spectrophotometer, france) and was used to quantify total carotenoids with a calibration curve of β-carotene as standard. the β-carotene values were used to calculate vitamin a contribution contributed to the diet, expressed as retinol equivalents (rae), where 12 μg βcarotene is converted to 1 μg rae [22]. 2.4. microbiology evaluation microbiology quality of ofsp cookies and cupcakes samples was analyzed using standard methods described by international organization standardization (iso). briefly, 10 g of each ofsp baked products were taken aseptically and homogenized in 90 ml tryptone salt in stomacher, and serial decimal dilutions (10-1 to 10-5) were performed. one milliliter (1 ml) of each dilution was poured plated in sterile petri dishes. the total viable bacterial counts (tvc) were carried out according to method [23], while the total mold and yeast count was determined by [24] method. the procedure [25] was used to count anaerobic sulfatereducing bacteria (asb) while total coliform bacteria count was carried with method[26]. detection of staphylococcusaureus and salmonella strains were done according to methods[27] and [28], respectively. the results of microorganisms count were expressed as log cfu/g. 2.5. sensory evaluation sensory evaluation of ofsp cupcakes and cookies was carried out using attributes including appearance, color, taste, aroma, texture, mouthfeel, and overall acceptability. a nine-point hedonic scale (1-dislike very much and 9-like very much) was used to evaluate for abovementioned sensory attributes [29]. the evaluation was undertaken by a panel of thirty mothers including breastfeeding mothers and non-breastfeeding mothers who have fed babies before. the samples were presented to each panelist in triplicates and average scores were reported. food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 140 2.6. statistical analysis all experiments were performed with three replications. the statistical analysis of collected data was performed by one-way analysis of variance (anova) using statistica software version 7.1 to determine differences among the treatments. significant differences among means of treatments were obtained by duncan’s multiple rank test at p<0.05. 3. results and discussion 3.1. proximate composition of the compsite ofsp flour baked products as shown in tables 1 and 2, there was a significant (p<0.05) difference in the nutritional properties of the enriched ofsp cupcakes and cookies. the results revealed that fat, proteins, carbohydrates content, and energy value decreased as level of ofsp flour increased. contrary to this moisture, ash, fiber, and β-carotene contents increased as proportion of ofsp flour increased in the formulation. the highest moisture content was found for cupcakes and cookies contained 50% ofsp flour. the reported results were similar to the findings of [30]. the increase of moisture content in the bakery products might be attributed to the higher water absorption capacity of the composite flour compared to wheat flour [31], [32]. however, lower moisture content of baked products is required for long time shelf life according to the report of [33]. based on the relatively high moisture content (14.18±0.04 to 16.79±0.04%), cupcakes could be considered as intermediatemoisture foods characterized by soft and tender eating properties[34]. moreover, the rapid consumption of these cupcakes is recommended to avoid microbial spoilage limiting their shelf life. fiber content in blended products is ranging from 5.38±0.16 to 7.64±0.0.26% and 5.39±0.13 to 7.70±0.20 % for cupcake and cookies, respectively. the crude fiber contents of baked products were significantly (p<0.05) higher than that of the control (1.520.22 and 2.510.12 g/100g dw). these trends could be attributed to the high content of fiber of ofsp flour than wheat as mentioned by [35]. similar result was reported by [36], [37] in other baked products. these results highlighted that consumption of one piece of enriched ofsp cupcake (54 g/piece) or two pieces of enriched ofsp cookies (44 g/piece) could contribute to 24.82-35.25% and 36.49-52.10% of fiber’s recommended dietary allowance (rda) for preschool children (1-3 years) that is estimated to 13 g/day. according to the reports of [38], foods containing more fiber have beneficial effects on the reduction of blood cholesterol and risk of colon cancer. moreover, dietary fiber enhances the rheological properties of doughs and overall acceptability of the resulting baked products [39]. the ash content of composite flour cupcake (1.64±0.10 to 1.98±0.03%) and cookies (1.02±0.06 to 1.71±0.05%) was significantly (p<0.05) higher as compared to those of the controls. results showed that as the ofsp flour proportion increases in the composite flours the ash contents of the blend increases. thus, the highest ash content was observed for composite flour cupcake (1.98±0.03%) and cookies (1.71±0.05%) made from 50% of ofsp flour and the lowest was for the controls. the same trend was recorded in the ash content of the composite cakes and cookies made with wheat-ofsp flour reported by [40], [41]. it can be concluded that substitution of wheat flour with ofsp flour in the blends could enhance the mineral intake of consumers of the final composite products developed for proper growth and development especially for the children. food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 141 table 1: proximate composition of cupcake made from wheat flour and ofsp flour and control (100g of dry weight) samples wheat:ofsp (w/w) moisture (%) ash (%) lipid (%) fiber (%) protein (%) control 100:00 14.18±0.04g 1.52±0.30d 18.97±0.45a 1.52±0.22h 12.11±0.66a irene 85:15 14.82±0.04efg 1.64±0.10cd 18.63±0.05bc 5.38±0.16g 11.37±1.03a 75:25 15.23±0.07cdef 1.71±0.02bcd 18.36±0.30c 5.51±0.69g 11.46±0.83ab 65:35 15.75±0.13bcd 1.83±0.02abc 17.90±0.02d 6.10±0.04ef 10.96±0.95abc 50:50 16.24±0.51ab 1.89±0.05ab 17.81±0.11d 6.19±0.06e 10.57±0.59abc kabode 85:15 14.54±0.04fg 1.64±0.10cd 18.78±0.13ab 5.73±0.21fg 11.33±1.21ab 75:25 15.04±0.48def 1.81±0.02abc 18.61±0.06bc 5.51±0.32g 11.44±0.36ab 65:35 16.00±0.56abc 1.96±0.22a 18.56±0.03bc 6.30±0.20de 10.84±0.61abc 50:50 16.17±0.15ab 1.98±0.03a 18.44±0.23c 6.29±0.12de 10.71±0.56abc covington 85:15 14.76±0.16efg 1.84±0.18abc 18.66±0.05bc 6.68±0.05cd 10.96±1.70abc 75:25 15.55±0.38bcde 1.86±0.08ab 18.59±0.10bc 7.11±0.08bc 10.56±0.77abc 65:35 16.02±1.12abc 1.89±0.07ab 18.52±0.02bc 7.64±0.26a 10.30±0.40bc 50:50 16.79±0.04a 1.94±0.04a 18.37±0.20c 7.32±0.18ab 9.60±0.28c (table continued) samples wheat:ofsp (w/w) carbohydrate (%) energy value (kcal) total provitamin a (μg) control 100:00 51.70±1.46a 425.99±0.87a irene 85:15 48.17±1.06b 405.77±0.92b 1138.68±14.051j 75:25 47.72±1.72bc 401.99±1.24cd 1421.21±8.45gh 65:35 47.45±1.08bc 394.80±0.43ef 1873.12±10.11e 50:50 47.29±0.97bc 391.76±2.01fg 2589.91±9.52b kabode 85:15 47.97±1.21b 406.28±1.26b 1031.04±6.36k 75:25 47.59±0.57bc 403.59±1.09bc 1269.38±10.12i 65:35 46.34±1.29bc 395.79±1.37e 1486.68±8.76g 50:50 46.42±0.53bc 394.45±10.34ef 1769.78±14.44f covington 85:15 47.10±1.61bc 400.16±0.88d 1332.39±8.58hi 75:25 46.33±1.19bc 394.91±1.74ef 2155.29±10.56d 65:35 45.64±0.68c 390.40±1.53gh 2443.88±6.83c 50:50 45.98±0.40bc 387.68±0.77h 3364.21±14.73a values are averages of triplicate replicates. data is represented as mean ± standard deviation. means in the same column with different superscript letters (a, b, c, d, e, etc…) are significantly different (p0.05). samples ratios are represented as wheat: ofspexpressed in proportion (w/w) expressed in weight (g) per weight (g). food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 142 table 2: proximate composition of cookies made from wheat flour and ofsp flour (100g of dry weight) samples wheat: ofsp (w/w) moisture (%) ash (%) lipid (%) fiber (%) protein (%) control 100:0 4.14±0.40k 1.01±0.10f 22.59±0.27a 2.51±0.12f 12.95±0.03a irene 85:15 4.58±0.06j 1.02±0.06f 21.83±0.27b 6.22±0.21cd 12.27±0.12abc 75:25 5.24±0.17h 1.18±0.06d 21.80±0.33b 6.73±0.22bc 12.21±0.12abc 65:35 6.33±0.04c 1.28±0.11c 21.67±0.31b 7.29±0.26a 11.49±0.80bc 50:50 6.32±0.04c 1.31±0.31c 20.67±0.44de 7.70±0.20a 11.13±1.06c kabode 85:15 5.47±0.15fg 1.16±0.12d 21.67±0.41b 5.39±0.13e 12.62±0.04ab 75:25 5.69±0.10e 1.17±0.04d 21.27±0.73bcd 6.15±0.11d 12.43±0.10abc 65:35 6.16±0.10d 1.44±0.15b 21.18±0.09bcde 6.53±0.30cd 11.82±0.59abc 50:50 7.91±0.08a 1.71±0.05a 20.48±0.39e 7.68±0.47a 11.50±0.06bc covington 85:15 4.79±0.12i 1.11±0.03e 21.48±0.36bc 6.03±0.14d 12.52±0.13ab 75:25 5.33±0.03gh 1.17±0.08d 21.05±0.33bcd 6.58±0.42cd 12.36±0.26abc 65:35 5.60±0.10ef 1.19±0.07d 21.05±0.36bcde 7.16±0.10ab 12.18±0.27abc 50:50 7.24±0.20b 1.42±0.11b 20.74±0.31cde 7.26±0.22ab 11.90±0.65abc (table continued) samples wheat: ofsp (w/w) carbohydrate (%) energy value (kcal) total provitamin a (µg) control 100:0 56.80±0.28a 482.35±0.28a irene 85:15 54.08±1.00b 461.86±1.00b 1055.67±11.14h 75:25 52.83±1.01bcd 456.37±0.90c 1512.91±10.05g 65:35 51.95±0.80cde 448.79±0.88d 1984.23±10.56f 50:50 52.87±0.19bcd 442.02±0.40e 2555.27±18.28d kabode 85:15 53.68±0.72b 460.28±1.49b 1009.57±8.38h 75:25 53.28±0.47bc 454.30±1.63c 1430.14±10.40g 65:35 52.87±0.50bcd 449.41±0.76d 1648.68±11.77g 50:50 50.71±0.58e 433.20±1.41f 2290.89±7.56e covington 85:15 54.08±0.25b 459.73±0.37b 2155.55±12.63ef 75:25 53.18±0.40bc 454.57±1.24c 2814.36±8.93c 65:35 52.81±0.22bcd 449.42±.1.54d 3588.98±10.55b 50:50 51.43±0.32de 440.00±1.20e 4640.72±12.43a values are averages of triplicate replicates. data is represented as mean ± standard deviation. means in the same column with different superscript letters (a, b, c, d, e, etc…) are significantly different (p0.05). samples ratios are represented as wheat:ofspexpressed in proportion weight (g) per weight (g) (w/w). as expected, the cupcake and cookies made of 100% wheat flour (control) had the highest protein content. this result could be attributed to the lower concentration of protein in ofsp flour compared to wheat flour. furthermore, substitution of wheat flour withofspflour showed a reduction of carbohydrate and food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 143 energy contents of cupcakes and cookies. the carbohydrate content of cupcakes and cookies were significantly (p<0.05) lower than that of control. as concern energy parameter, the highest value was observed for controls (425.99±0.87 kcal/100g dw and 482.35±0.28 kcal/100g dw, respectively for cupcakes and cookies). the lowest of energy was for 50% supplemented ofsp cupcakes (387.68±0.77 kcal/100g dw) and cookies (433.20±1.41 kcal/100g dw). this may be due to the lower contents of fat, proteins, and carbohydrates of different formulations when ofsp flour increases. the incorporation of ofsp flour in the processing of cupcakes and cookies contributed significantly to enhance vitamin a content of baked products. the provitamin a content increased from 1031.04±6.36 to 3364.21±14.73μg/100g dw of 50% ofsp cupcakes (table 1). as concern formulated cookies, provitamin a contents varied from 1009.57±8.38 to 4641.72±12.43μg/100g dw (table 2). these findings were consistent with results of [42], [43], which indicated that increase of provitamin a content in composite products (bread, cake, cookies, etc.) is essentially due to higher value of βcarotene of ofsp flour. based on the βcarotene conversion into vitamin a [22], consumption of one piece of enriched ofsp cupcakes (approximately 60g/piece) and two pieces of cookies (approximately 44g/piece) could meet 21.43% to 70.08% and 21.02% to 96.69% of vitamin a requirements of preschool children (1-3 years old) (figure 1 a and b). this suggests that consumption of enriched ofsp cupcakes and cookies could reduce vitamin adeficiency (vad) in children and other consumers (pregnant/lactating women), especially the populations of the north and nort-east, where the prevalence of vitamin a deficiency is estimated at 24%[4] (a) (b) fig. 1: estimated contribution (%) of ofsp composite flour snack foods to rda for vitamin a for children between 2 and 6 years old. rda: recommended dietary allowance; contribution was determined based on an assumed serving one piece of cupcake (approximatively 60g) or two pieces of cookie (approximatively 44g) for 2 and 3 years old, and two pieces of cupcake (approximatively 120g) or four pieces of cookie (approximatively 176g) for 3 and 6years old. cp: cupcake; co: cookie; v1, v4, v5: represent different ofsp cultivars v1: irene, v4: kabode, v5: covington 15; 25; 35; 50: represent different proportions of ofsp incorporated to wheat flour. food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 144 3.2. microbiologyqualityof the composite ofsp flour baked products the result of this study indicated that there was a significant difference (p<0.05) among the counts of aerobic colony count (acc) of all the baked products. these values ranged from 3.32±0.38 to 4.17±0.35 log cfu/g for cupcakes (table 3). for cookies, the value varied between 3.00±0.17 and 4.47±0.09 log cfu/g, with the highest value recorded for control and lowest value for cookie prepared with ofsp cultivar covington (table 4). spoilage bacteria (enterobacteria, asb,staphylococcus aureus, salmonella, …) were not detected in all the baked products formulated. the lower value of microorganisms count may be explained by the baking step which occurs at 180°c for 5 min. in addition, the absence of the hygiene indicators microorganisms (enterobacteriaceae) in the samples indicated a good cooking practice. table 3 microbial load on composite ofsp flour cupcakes (log10 cfu/g) cupcakes acc enterobacteria yeast/mould asb staph. aureus salmonella sp. irene 3.52±0.08 b 0 0 0 0 absence kabode 4.00±0.19a 0 0 0 0 absence covington 4.17±0.35a 0 0 0 0 absence control 3.32±0.38b 0 0 0 0 absence table 4 microbial load on composite ofsp flour cookies (log10 cfu/g) cookies acc enterobacteria yeast/mould asb staph. aureus salmonella sp irene 3.00±0.17 co 0 0 0 0 absence kabode 4.20±0.22a 0 0 0 0 absence covington 3.30±0.05co 0 0 0 0 absence control 4.47±0.09a 0 0 0 0 absence values are averages of triplicate replicates. data is represented as mean ± standard deviation. means in the same column with different superscript letters (a, b, c, …) are significantly different (p0.05). acc: aerobic colony count; asb: anaerobic sulphur-reducing bacteria; staph. aureus: staphylococcusaureus. 3.3. sensory evaluation of the of the composite ofsp flour baked products promote quality control and marketing of new products or recipes formulated, sensory evaluation constitute an important criteria to consider[44]. in this study, sensory attributes such as appearance, color, taste, aroma, texture, mouth-feel, and overall acceptability were considered. the result of sensory evaluation showed that cupcakes had less scores than that of the control for all the attributes except for colorand taste (table 5). these results were similar to those obtained by [35] who reported that cakes prepared with orangefleshed sweet potato, received a lower score than the control for sensory attributes. results also showed that the cupcakes were accepted by panelists food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 145 regarding color. this showed that panelists prefer orange color of cupcakes rather than the yellow color of the control. concerning appearance acceptability, a high score was obtained for the control cupcake followed by cupcakes supplemented with 15, 25, 35, and 50% ofsp. results also revealed that appearance acceptability increased with the addition of ofsp flour as previously reported by [36]. the taste acceptability scores of cupcakes ranged between 7.500.46 and 8.510.78. cupcakes supplemented with 50% ofsp flour recorded high score for all the three cultivars. this result may be attributed to natural sweet taste of ofsp flour that contributes to increase preference of panelists, as mentioned by [41]. however, the scores of texture and mouthfeel which reflect softness and chewiness of cupcakes decreased with addition of ofsp flour as shown in table 4. according to previous reports [45], substitution of wheat flour by ofsp flour contributes to add more compact texture. the overall acceptability score of cupcakes varied significantly. the maximum score value (8.250.37) was obtained by control cupcake; while minimum score value (5,740.07) was noted for cupcake containing 50% ofsp. as concern cookies, the sensory properties are indicated in table 6. the cookies supplemented with 15 to 35% of ofsp flour had the highest sensory rating. from all the sensory scores above, it was quite apparent that cupcakes and cookies containing up to 40% ofsp flours. table 5 sensory evaluation of cupcakes developed from composite flours. samples wheat: ofsp (w/w) appearance color taste aroma texture mouthfeel overall acceptability control 100:0 8,26±1.13a 6.06±0.17 f 7.50±0.46e 7.59±0.63a 8.36±1.31a 8.01±0.42a 8.25±0.37a irene 85:15 7,79±0.60bc 6.17±0.93 d 7.88±1.02bcde 7.39±0.77ab 6.98±0.77bc 7.53±1.22b 7.61±0.99b 75:25 7,04±1.14cd 6.39±0.78 cd 7.95±1.05bcde 7.07±0.80bcd 6.66±0.50cd 7.13±2.18bcd 7.22±1.30bcd 65:35 6,22±1.15f 7,67±0.65 bc 8.06±0.77abcd 6.93±1.10cd 6.30±0.40de 6.72±1.20de 6.98±0.86cd 50:50 5,41±2.36g 7.79±0.88 ab 8.25±0.89ab 5.89±0.41e 5.48±0.21f 5.06±2.04f 5.96±0.85e kabode 85:15 7,26±2.05cd 7.07±0.61 e 7.54±1.17de 7.25±1.12bc 7.19±0.82b 7.40±0.66bc 7.38±0.63bc 75:25 6,85±1.09cde 7.46±0.61 cd 7.70±1.20cde 7.09±0.37bcd 6.55±0.57cde 7.12±0.53bcd 7.15±1.12cd 65:35 6,41±1.27ef 7.73±0.75 ab 7.93±0.86bcde 6.88±1.08cd 6.15±0.88e 6.66±1.10ef 6.83±0.74d 50:50 5,20±2.28g 7.81±1.12 a 8.08±1.03abc 5.39±0.80f 5.04±0.28g 5.03±0.82f 5.70±1.04e covington 85:15 7,94±2.06cd 7.40±0.63 c 7.55±1.09de 7.45±1.05ab 7.21±1.05b 7.37±1.03bc 7.61±0.80b 75:25 7,33±1.19cd 7.48±0.55 ab 7.92±0.47bcde 6.88±0.69cd 6.97±0.90bc 7.19±0.93bc 7.40±1.07bc 65:35 6,88±2.14de 7.75±0.88 a 8.06±0.93abc 6.67±0.50d 6.94±1.10bc 6.97±2.09cde 7.30±0.57bc 50:50 5,16±1.22g 7.92±0.12 a 8.51±0.78a 5.37±0.83f 5.16±0.43g 5.43±0.71f 5.74±0.07e values are averages of triplicate replicates. data is represented as mean ± standard deviation. means in the same column with different superscript letters (a, b, c, d, e, etc…) are significantly different (p0.05). samples ratios are represented as wheat: ofsp expressed in proportion (w/w) expressed in weight (g) per weight (g). food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 146 table 6 sensory evaluation of cookies developed from composite flours. samples wheat: ofsp (w/w) appearance color taste aroma texture mouthfeel overall acceptability control 100:0 8.00±0.38a 6.37±0.70d 7.00±1.10e 8.02±0.33a 8.10±1.10a 8.48±0.33a 8.15±0.27a irene 85:15 7.88±0.33 ab 6.47±1.07cd 7.22±0.55de 7.59±0.47ab 7.91±0.25ab 7.74±0.27ab 7.77±0.30a 75:25 7.36±0.61 bcd 7.05±0.41abcd 7.50±1.14cd 6.81±0.44cd 7.49±0.14abc 7.13±0.14bc 7.53±0.37ab 65:35 7.08±0.77 cde 7.44±0.77ab 7.97±0.13ab 6.57±1.05de 7.07±0.14cd 6.60±0.25cd 7.02±0.42bc 50:50 6.72±1.07 e 7.80±0.13a 8.08±1.13a 5.47±0.09f 6.10±0.13ef 5.80±0.69d 5.72±0.37e kabode 85:15 7.60±1.06 abc 6.38±0.97d 7.64±0.63bc 7.34±0.47bc 7.08±0.53cd 7.89±0.57ab 7.98±0.49a 75:25 6.92±0.57 de 6.94±0.32bcd 7.90±0.57ab 7.12±0.80bcd 6.57±0.77de 7.52±1.10b 7.80±0.30a 65:35 6.57±0.53 e 7.04±0.54abcd 7.98±0.79ab 6.49±0.33de 6.00±0.39ef 6.45±0.13cd 6.45±0.29cd 50:50 6.01±0.74 f 7.27±0.75abc 8.19±0.47a 6.07±0.76ef 5.89±0.97ef 6.15±0.37d 5.68±0.35e covington 85:15 7.87±0.56 ab 6.77±0.54bcd 7.11±0.28e 7.13±0.21bcd 7.67±0.28abc 7.89±0.21ab 8.06±0.30a 75:25 7.69±0.31 ab 6.99±1.07abcd 7.67±0.23bc 6.83±0.24cd 7.17±0.23bcd 7.56±0.24b 7.83±0.25cd 65:35 6.90±0.24 de 7.33±0.15ab 8.11±0.08a 6.77±0.31cd 6.31±0.68e 6.33±0.31cd 7.06±0.40bc 50:50 6.56±0.23 e 7.78±0.24a 8.26±0.24a 6.00±0.37ef 5.35±0.24f 6.00±0.07d 5.84±0.50de values are averages of triplicate replicates. data is represented as mean ± standard deviation. means in the same column with different superscript letters (a, b, c, d, e, etc…) are significantly different (p0.05). samples ratios are represented as wheat: ofsp expressed in proportion weight (g) per weight (g) (w/w). 4. conclusion the findings of this study show that the nutritional composition of the cupcakes and cookies were improved due to ofsp incorporating, while the sensory attributes were overall acceptable. substitution of wheat flour with ofsp flours resulted in significant increase in pro-vitamin a carotenoid, ash, and fiber contents compared to the control. the sensory evaluation showed that ofsp cupcakes and cookies supplemented up to 35% ofsp were accepted by the panelists. moreover, the formulated cupcakes and cookies were of good microbiological quality. thus, ofsp flour could be recommended to develop complementary foods with improved nutritional properties. this would help fight against child malnutrition in côte d'ivoire. 5. acknowledgments this project was funded by academy of sciences, culture, arts and african diaspora (ascad, côte d’ivoire/abidjan). the authors are gratefully for this support. 6. references [1] fao/ifad/unicef/wfp/who, the state of food security and nutrition in the world 2019. safeguarding against economic slowdowns and downturns, rome, (2019), 214p. [2] unicef, malnutrition: a major cause of death in children, retreivedhttps://reliefweb.int/report/world/malnutrit ion-major-cause-death-children, (2019), pp. 1–5. [3] examen stratégique national : « faim zéro », côte d’ivoire, 120 p, (2020). [4] association of official analytical chemists (aoac), official methods of analysis, 16th edition, aoac inc ton, washington dc, (1997). https://reliefweb.int/report/world/malnutrition-major-cause-death-children https://reliefweb.int/report/world/malnutrition-major-cause-death-children food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 147 [5] allen l. h., causes of nutrition-related public health problems of preschool children: available diet, journal of pediatric gastroenterology and nutrition, 43: s8-s12, (2006). [6] bouis h., j. low, mcewan m., tanumihardjo s., biofortification: evidence and lessons learned linking agriculture and nutrition, fao/who, pp. 1–23, (2013). [7] neela s. fanta s.w., review on nutritional composition of orange‐fleshed sweet potato and its role in management of vitamin a deficiency, food science & nutrition, march, pp. 1–24, (2019). [8] low j.w., thiele g., understanding innovation: the development and scaling of orange-fleshed sweetpotato in major african food systems, agricultural systems, 179: 102770, (2020). [9] nigusse g., t. hadero, yoseph t. p., evaluation of nutritional, microbial and sensory properties of complementary food developed from kocho, orange-fleshed sweet potato (ipomoea batatas l.) and haricot bean (phaseolus vulgaris) for under five years children in boricha woreda, south ethiopia, journal of food processing, 85: 1–7, (2019). [10] koua g., zoue t., megnanou r.m., niamke s., nutritive profile and provitamin a value of sweet potatoes flours (ipomoea batatas l.) consumed in côte d’ivoire, journal of food research, 7: 36-48, (2018). [11] ridley s. c., lim m., heenan s., bremer p., evaluation of sweet potato cultivars and heating methods for control of maltose production, viscosity and sensory quality, journal of food quality, 28: 191–204, (2005). [12] laryea d., wireko-manu f. d., oduro i., formulation and characterization of sweetpotato-based complementary food, cogent food agriculture, 4: 1–15, (2018). [13] huffman s. l., piwoz e. g, vosti s. a., dewey k. g., babies, soft drinks and snacks: a concern in lowand middle-income countries? maternal & child nutrition, 10: 562– 574, (2014). [14] mu t.-h., sun h.-n., ma m.-m., sweetpotato snack foods, sweet potato, chemistry processing and nutrition, pp. 303-324, (2019). [15] chauhan a., saxena d. c., singh s., physical, textural, and sensory characteristics of wheat and amaranth flour blend cookies. cogent food agriculture, 2: 1125773, (2016). [16] aoac, official methods of analysis of aoac international, 19th edition, aoac internationnal, gaithersburg, maryland 208772417, usa, (2012), 17 p. [17] afnor, compendium of french standards, fats, oleaginous grains, derived products. afnor ed., paris, (1986), 527 p. [18] wolf, manual of fat analysis. azoulay edition, paris, (1968), 519 p. [19] fao, food energy-methods of analysis and conversion factors. fao edition, rome, (2002), 97 p. [20] howe j. a. tanumihardjo s. a., carotenoid-biofortified maize maintains adequate vitamin a status in mongolian gerbils, journal of nutrition, 136: 2562–2567, (2006). [21] institute of medicine, “nutrients summary” dietary reference intake (rdis): vitamins, (2011), pp. 2-5. [22] iso 4833-1, microbiology of the food chain horizontal method for the enumeration of microorganisms part 1: colony count at 30°c by the pour plate technique, 1st edition, genevia, (2013), 9p. [23] iso 21527-2, microbiology of food and animal feeding stuffs horizontal method for the enumeration of yeasts and moulds – part 2: colony count technique in products with water activity less than or equal to 0,95, afnor, saint-denis (2008), 15p. [24] nf v08-061, microbiology of food and animal fooding stuffs anaerobic enumeration of sulfite-reducing bacteria by colony count technique at 46 °c, 1st edition, volume 33, afnor, saintdenis, (2009), 11p. [25] nf iso 4832, microbiology of food and animal feeding stuffs horizontal method for the enumeration of coliforms colony-count technique, 1st edition, afnor, saint-denis, (2006), 10 p. [26] iso 6888-1, microbilogy of food and animal feeding stuffs routine method for enumeration of coagulase positive staphylococcus by colony-count technique at 37 °c part 1: technique with confirmation of the colonies, afnor, saint-denis, (1999), 15p. [27] iso 6579-1, microbiology of food and animal feeding stuffs — horizontal method for the detection of salmonellaspp, afnor, saint-denis, (2002), 39p. [28] xp v09-500. general guidelines for conducting hedonic tests in a sensory evaluation laboratory or in a controlled environment involving consumers, afnor, paris, (2012), 30p. [29] hadero t., substitution of sweet potato (ipomoea batatasl.) and soybean (glycine max.) flour with durum wheat (triticum durum) flour effect on physicochemical and sensory characteristics of cookies, food science and food and environment safety journal of faculty of food engineering, stefancel mare university suceava volume xx, issue 2 – 2021 gisèle ahou yah koua, konan evrad brice dibi, edwige essoma akoa, sébastien niamke, development of enriched cupcakes and cookies with orange-fleshed sweet potato (ipomea batatas l.): sensory and nutritional evaluation, food and environment safety, volume xx, issue 2 – 2021, pag. 137 – 148 148 quality management, 82: 22–29, (2019). [30] kidane g., abegaz k., mulugeta a., singh p., nutritional analysis of vitamin a enriched bread from orange flesh sweet potato and locally available wheat flours at samre woreda, northern ethiopia, current research in nutrition and food science, 1: 49–57, (2013). [31] wang j., rosell c. m., benedito de barber c., effect of the addition of different fibres on wheat dough performance and bread quality, food chemistry, 79: 221–226, (2002). [32] novotni d., ćurić d., galić k., škevin d., neđeral s., kraljić k., gabrić d., ježek d., influence of frozen storage and packaging on oxidative stability and texture of bread produced by different processes. lwt food science and technology, 44: 643–649, (2011). [33] cauvain s. p., cakes: nature of cakes, encyclopedia of food science and nutrition (second edition), (2003), pp. 751–756. [34] jemziya m. b. f., mahendran t., physical quality characters of cookies produced from composite blends of wheat and sweet potato flour, ruhuna journal of science, 8: 12-23, (2017). [35] andualem a., kebede a., abadi g. m., development of pro-vitamin a and energy rich biscuits: blending of orange-fleshed sweet potato (ipomea batatas l.) with wheat (triticum vulgare) flour and altering baking temperature and time, african journal of food science, 10: 79–86, (2016). [36] kolawole f. l., akinwande b. a., ade-omowaye b. i. o., physicochemical properties of novel cookies produced from orangefleshed sweet potato cookies enriched with sclerotium of edible mushroom (pleurotustuberregium). journal of the saudi society of agricultural science, 19: 174–178, (2018). [37] nirmala prasadi v. p., joye i. j., dietary fibre from whole grains and their benefits on metabolic health, nutrients, 12: 1–20, (2020). [38] reißner a.-m., beer a., struck s., rohm h., pre-hydrated berry pomace in wheat bread: an approach considering requisite water in fiber enrichment, foods, 9: 1600, (2020). [39] okorie s. u., onyeneke e.n., production and quality evaluation of baked cake from blend of sweet potatoes and wheat flour, academic research international, 3: 171–177, (2012). [40] teferra t. f., kurabachew h., tadesse t. f., nigusse g., nutritional, microbial and sensory properties of flat-bread (kitta) prepared from blends of maize (zea mays l.) and orange-fleshed sweet potato (ipomoea batatas l.) flours. int. j. food sci. nutr. eng., 5: 33–39, (2015). [41] korese j. k., chikpah s. k, hensel o., pawelzik e., sturm b., effect of orange-fleshed sweet potato flour particle size and degree of wheat flour substitution on physical, nutritional, textural and sensory properties of cookies. european food research and technology, 247: 889-905, (2021). [42] chikpah s. k., korese j. k., hensel o., sturm b., effect of sieve particle size and blend proportion on the quality properties of peeled and unpeeled orange fleshed sweet potato composite flours, foods, 9: 740, (2020). [43] sharif m. k., butt m. s., sharif h. r., nassir m., sensory evaluation and consumer acceptability, chapter 14, (2017), pp. 361-386. [44] sabanis d., tzia c., effect of rice, corn and soy flour addition on characteristics of bread produced from different wheat cultivars, food bioprocess technology, 2: 68–79, (2009) 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2021.005 43 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 1 2021, pag. 43 48 provision of children’s tourist groups with functional drinks * kristina belinska kamianetspodilskyi national ivan ohiienko university, suvorova str., 52, kamianets podilskyi, ukraine, 32300 kristina0612@ukr.net *corresponding author received 18th october 2020, accepted 30th march 2021 abstract: one of the main components of tourism is to provide tourist groups with a balanced diet. in addition to nutrition, drinking balance is important too. sweet carbonated drinks, which do not benefit the child's health, are especially common among children's tourist groups. therefore, it is advisable to develop drinks for children's tourist groups, which are characterized by useful properties, are tasty and have not a tendency to microbiological spoilage. the presented researches suggest developing milk dry drinks. however, it was proposed to use powdered sheep milk as a basis, because sheep milk powder contains much more essential substances needed by the child's body than cow milk. it is known that a significant number of children do not consume milk due to taste preferences. therefore, it was proposed to add cryopowders to the drink as an additional source of vitamins and minerals and as a flavor enhancer. in this work the research on establishment of a rational dosage of cryopowders to a compounding of dry drinks was carried out. sensory analysis and mathematical calculations showed that the rational dosage of cryopowders to the formulation was 10%. it was found that adding cryopowders to the milk drink improved taste of the finished product. in addition, the use of cryopowders increased children’s daily intake of vitamins and minerals. after conducted researches the terms at which the restored products are appropriate for consumption and do not carry the threat of food poisoning were established. keywords: milk drink, cryopowder, nutritional value, sheep milk. 1. introduction children's tourism is one of the most constant parts of the market of any country. however, the organization of children's recreation has its own specifics, requires special attention and careful preparation [1]. during the tourist trip it is important to provide children's groups with a balanced diet. today, many restaurants provide catering services for children's tourist groups. but the important fact remains that children need small snacks and drinks between main meals. during a tourist trip, the best product for regulating the water balance in the body is water. however, most children prefer carbonated sweet drinks and canned juice. it is known that such products are not useful for the body. and at a high temperature of the environment, which is typical for the summer period vacation time and the most favorable time for travel, there is microbiological spoilage of products. to solve this problem, it is necessary to explore possible ways to enrich children's drinks, justify the choice of the main component for drinks and explore the maximum possible expiration date of the restored product. of course, the most useful drink is milk. milk contains almost all known vitamins and minerals [2]. cow milk is commonly consumed, but other types of milk are much richer in substances which are essential for the baby's body. therefore, mailto:kristina0612@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 kristina belinska, provision of children’s tourist groups with functional drinks, food and environment safety, volume xx, issue 1 – 2021, pag. 43 – 48 44 for the preparation of beverages, it was proposed to use sheep's milk as a basis. but the use of milk during travel due to the threat of microbiological spoilage is possible only in dry form. therefore, the development of a milk drink for children's tourist groups based on milk powder was envisaged. compared to cow's milk powder, sheep's milk powder contains 9% more animal protein, more linoleic acid and twice more linolenic acid, 4.5 times more lactoferrin. in addition, sheep's milk powder contains twice more ascorbic acid than cow's milk powder, 1.6 times more thiamine and 4.6 times more nicotinic acid [3]. however, there was another problem a large number of children do not consume milk due to taste preferences. but at the same time they like milkshakes with flavors of tropical and domestic fruit. in addition, milk powder is characterized by poorer vitamin composition than liquid milk [4]. therefore, an important task was to develop milk drinks for children's tourist groups, which will be safe for children, will provide the child's body with essential substances and, most importantly, be delicious. in recent years, cryopowders have become very popular [5]. authors peresichny m., korzun v. [6] showed that the introduction of various types of cryopowders in the form of fillers in fermented milk drinks allowed creating new types of drinks with therapeutic and prophylactic action, which had delicious taste and high sensory characteristics. the use of cryopowders for sweet dishes and products allowed enriching them with vitamins, minerals, dietary fiber. due to their use, the chemical composition of food products was significantly improved and their biological value increased. these data allowed recommending cryopowders for use in production of dairy products. 2. matherials and methods sheep milk powder, cow milk powder, apricot, blackberry and raspberry cryopowders were used for the study. the chemical composition of cryopowders is presented in table 1. experimental studies included the search for and identification of rational ratios of components – milk base and cryopowder, study of organoleptic, technological indicators of finished beverages, assessment of their biological and nutritional value. table 1 chemical composition of cryopowders [7, 8] indicator cryopowder from apricot blackberry raspberry cellulose, g 15.3 35.0 27.14 vitamin content, mg/100g с 63.0 89.0 133.0 в1 0.1 0.1 0.11 в2 0.2 0.4 0.27 рр 4.0 9.0 3.2 the content of mineral substances, mg/100g k 1870 420 1193 ca 160 390 213 mg 170 320 117 p 3 89 197 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 kristina belinska, provision of children’s tourist groups with functional drinks, food and environment safety, volume xx, issue 1 – 2021, pag. 43 – 48 45 3. results and discussion organoleptic quality indicators of restored dried dairy products with cryopowders from apricot, blackberry and raspberry were determined in the study. the results of the study are presented in table 2. table 2 sensory characteristics of restored beverages dosage of g cryopowder / 100 g of the mixture consistence color taste flavor with cryopowder from apricot 5 liquid slightly pronounced orange hue slightly palpable apricot slightly noticeable aroma of apricot 10 pale orange moderately rich pleasant apricot 15 rich pale orange too rich apricot rich aroma with cryopowder from blackberry 5 liquid lilac moderately rich pleasant blackberry slightly noticeable aroma of blackberry 10 pale purple 15 deep purple with a red tinge oversaturated with a sour taste prominent with cryopowder from raspberry 5 liquid pale pink moderately rich pleasant raspberry slightly noticeable aroma of raspberries 10 pale pink 15 deep pink oversaturated with a sour taste prominent it was recommended to restore the dry mixture with drinking water at a temperature of 18-400c depending on the season. according to the recommendations for the restoration of milk powder, the dry drink was to be restored in the ratio – 1:10. studies indicated that increasing the dosage of cryopowders increased the intensity of taste, color and odor, and therefore the quality of finished drinks deteriorated. in the following experiment, changes in the chemical composition of dry milk drinks were determined depending on the amount of added cryopowder. the results are presented in table 3. table 3 chemical composition of dry milk drinks with cryopowders component, mg/100g cryopowder, per 100 g of dry mixture apricot blackberry raspberry 5 % 10 % 15 % 5 % 10 % 15 % 5 % 10 % 15 % cellulose, g 0.77 1.53 2.3 1.8 3.5 5.3 1.4 2.7 4.1 с, mg/100g 9.8 12.6 15.4 11.1 15.2 19.3 13.3 19.6 25.9 в1, mg/100g 0.31 0.3 0.29 0.31 0.3 0.29 0.31 0.3 0.29 в2, mg/100g 0.82 0.79 0.75 0.83 0.81 0.78 0.82 0.79 0.76 рр, mg/100g 3.24 3.28 3.32 3.49 3.78 4.07 3.2 3.2 3.2 k, mg/100g 93.8 187.29 280.77 21.3 42.29 63.27 59.95 119.59 179.22 ca, mg/100g 8.85 16.8 24.76 20.35 39.8 59.26 11.5 22.1 32.7 mg, mg/100g 8.57 17.07 25.56 16.07 32.07 48.06 5.92 11.77 17.61 p, mg/100g 0.56 0.69 0.82 4.86 9.29 13.72 10.26 20.09 29.92 according to the conducted researches it was established that with increase in a dosage of cryopowders the maintenance of essential substances in a finished product increased. consequently, the chemical composition of the finished products improved. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 kristina belinska, provision of children’s tourist groups with functional drinks, food and environment safety, volume xx, issue 1 – 2021, pag. 43 – 48 46 therefore, it was rational to add cryopowders to the mixture.in the amount of 10% to milk powder. however, it was necessary to calculate the ability of the developed products to meet the daily requirement of essential substances. for this purpose, it was proposed to make calculations per portion of the finished dish. since the primary goal of creating a product was to provide healthy drinks for children's tourist groups, and therefore the portion should be calculated for one consumption by one child, that is per one glass (table 4). therefore, when restoring the product in a ratio of 1:10, the weight of the dry mixture was taken per serving in the amount of 20 g, the weight of water – 200 g. therefore, further calculation was performed per one serving of dry drink. the daily requirement was taken into account for children aged 7-10 years. [9] thus, the expediency of using cryopowders from apricot, blackberry, and raspberry in the technology of dry milk drinks, which increased their biological value, was substantiated. the next stage of research was to determine the acidity of the drink after restoration and during storage (fig. 1). for this purpose, the dry beverage was restored with water (the temperature of 22 0c) at the temperature of the environment 22 0c. the acidity of the drink indicated its freshness and safety. sheep's milk was considered unfit for consumption in its native form with an acidity above 24 0t [10]. the results of the study indicated that cryopowders increased the acidity of the drink by 1 degree. it was found that after 3 hours after preparing, drinks with cryopowders from raspberry and blackberry were not appropriate for consumption. the acidity of these drinks exceeded the permissible norm. the suitability of the drink with cryopowder from apricot for consumption after 3 hours after preparation was due to the fact that the acidity of cryopowder from apricot was lower compared to other cryopowders. table 4 chemical composition of one portion of dry milk drink with cryopowders component dry drink based on sheep milk with cryopowder from apricot blackberry raspberry content per serving satisfaction of daily needs,% content per serving satisfaction of daily needs, % content per serving satisfaction of daily needs, % animal proteins, g 4.5 8.8 4.5 8.8 4.5 8.8 fats, g 5.76 8.2 5.76 8.2 5.76 8.2 carbohydrates, g 3.78 1.3 3.78 1.3 3.78 1.3 linoleic acid, g 0.7 5.8 0.07 5.8 0.07 5.8 linolenic acid, g 0.04 20.0 0.04 20.0 0.04 20.0 lactoferrin, g 0.13 0.13 0.13 cellulose, g 0.31 1.6 0.7 3.5 0.54 2.7 с, mg 2.52 4.2 3.04 5.1 3,92 6.5 в1, mg 0.06 6.0 0.06 6.0 0.06 6.0 в2, mg 0.16 13.3 0.16 13.3 0.16 13.2 рр, mg 0.66 4.4 0.76 5.1 0.64 4.3 k, mg 37.46 5.0 8.46 1.2 23.92 3.2 ca, mg 3.36 0.34 7.96 0.8 4.42 0.4 mg, mg 3.4 2.0 6.41 3.8 2.35 1.4 p, mg 0.14 0.01 1.86 0.2 4.02 0.4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 kristina belinska, provision of children’s tourist groups with functional drinks, food and environment safety, volume xx, issue 1 – 2021, pag. 43 – 48 47 in summer, when the temperature of the environment is about 300c or even higher, the accumulation of acidity in beverages is more intense. therefore, it was recommended to use the investigated drinks immediately after preparing and no later than 30 minutes after preparing. since these drinks were designed for children's tourist groups, it was proposed to produce these products packaged in plastic bags for every portion. this packaging will be convenient when preparing drink while traveling. 15 17 19 21 23 25 27 1 min 10 min 30 min 1 h 2 h 3 h a c id it y, d e g re e s o f t u rn e r time after restoration milk drink with cryopowder from apricot milk drink with cryopowder erom blackberry milk drink with cryopowder from raspberry restored sheep milk fig. 1. acidity of drinks after restoration 4. conclusion as a result of the conducted researches it was established that: the addition of cryopowders to milk powder led to an improvement of the organoleptic characteristics of the restored drink: the taste and aroma improved, a pleasant color appeared; the use of powdered sheep's milk as a basis for the drink increased the nutritional value: the amount of animal fats increased, the amount of polyunsaturated fatty acids increased, the amount of lactoferrin increased; production of milk drink with cryopowders improved the chemical composition: the daily requirement of vitamins and minerals was covered by 0.4… 13.2%; the use of cryopowders in the technology of dry milk drinks allowed covering the daily requirement of dietary fiber by 1.6… 3.5%; cryopowders affected the acidity of the restored beverage. therefore, it was established that the drink should be consumed immediately after preparing and no later than 30 minutes after preparing. 5. references [1] ivchenko l., kuzhyl s., petrenko m. features of children's tourism organization in ukraine, ekonomika ta derzhava, 1: 83-88, (2019). doi: 10.32702/2306-6806.2019.1.83 [2] serikova a., smolnikova f., rebezov m., okuskhanova e., temerbayeva m., gorelik o.. development of technology of fermented milk drink with immune stimulating properties. research journal of pharmaceutical, biological and chemical sciences, 9 (4): 495-500, (2018). https://doi.org/10.32702/2306-6806.2019.1.83 https://elibrary.ru/author_items.asp?authorid=806574 https://elibrary.ru/author_items.asp?authorid=715758 https://elibrary.ru/author_items.asp?authorid=419764 https://elibrary.ru/author_items.asp?authorid=774515 https://elibrary.ru/author_items.asp?authorid=1011721 https://elibrary.ru/author_items.asp?authorid=878171 https://elibrary.ru/contents.asp?id=35170042 https://elibrary.ru/contents.asp?id=35170042 https://elibrary.ru/contents.asp?id=35170042 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 1 – 2021 kristina belinska, provision of children’s tourist groups with functional drinks, food and environment safety, volume xx, issue 1 – 2021, pag. 43 – 48 48 [3] belinska k. falendysh n. perspectives of using sheep’s milk in baby food, research papers of the national university of food technologies, 21 (4): 231-236, (2015). [4] nadykta, v.d., shcherbakova, e.v., olkhovatov, e.a. (2017). technology of powdered food additives. polythematic network electronic scientific journal of the kuban state agrarian university, no. 131, pp. 659--671. [5] mansurov a., bocharov v., palchikov v., ratushny a. impact cryopowder ‘apple’ on the quality of whey beverage functional purpose, technologies of food and processing industry of aic – healthy food, 1: 48-55. (2019). [6] hachak, u., &vavrysevych, j. the use of cryopowder «pumpkin» in the technology of cheese masses with different fat content. scientific messenger of lnu of veterinary medicine and biotechnologies. series: food technologies 18 (2): 41-45, (2016). https://doi.org/10.15421/nvlvet6808 [7] yaralieva z., sovershenstvovanietechnologiikrioporoshkovizplod oviyagod, vyrashchyvaiemych v predgornychrayonachdagestana. kand, diss. [improving the technology of cryopowders from fruits and berries grown in the foothill regions of dagestan]. krasnodar, 2017. 141 [8] kasyanov, g.i., yaralieva, z.a., akhmedov, m.e. (2018). fruit and berry cryopowder technology. krasnodar: ecoinvest, 155 p. [9] about the statement of norms of physiological needs of the population of ukraine in the basic nutrients and energy. document z1206-17, september, 3, 2017. verchovna rada of ukraine. legislation of ukraine, (2017). available at: https://zakon.rada.gov.ua/laws/show/z120617?lang=en#text [10] balthazara cf, silva hla, vieira ah, neto rpc, cappato lp, coimbra pt.assessing the effects of different prebiotic dietary oligosaccharides in sheep milk ice cream. food res int91:38–46. (2017) https://doi.org/10.15421/nvlvet6808 https://zakon.rada.gov.ua/laws/show/z1206-17?lang=en#text https://zakon.rada.gov.ua/laws/show/z1206-17?lang=en#text doi: https://doi.org/10.4316/fens.2022.024 247 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 247 256 changes in texture profile of amaranth seeds subjected to sous vide treatment ovidiu procopeț 1 , *mircea oroian 1 1faculty of food engineering, stefan cel mare university, suceava, romania, m.oroian@fia.usv.ro, *corresponding author received 15th july 2022, accepted 15th september 2022 abstract: amaranth seeds represent an important category of pseudocereals that showed growth over time both in terms of cultivated areas and food products containing amaranth seeds. in different geographical areas, amaranth occupies an important place in terms of pseudocereal consumption, especially in latin america and china where amaranth cultivation has been documented for over 3000 years. due to the chemical composition in general but also due to the physical structure in particular, the cooking possibilities of amaranth seeds are limited to a certain extent. the purpose of this study was to examine and determine the impact of a new heat treatment method that can be applied to amaranth seeds in order to design and obtain new functional foods from a physical and sensory point of view. sous-vide technology is mostly used for cooking meat or vegetables. cereals and pseudocereals have so far not been considered suitable for sous-vide cooking due to their physical structure and low moisture content. in this study, the method adapted to these factors is described, which allows the heat treatment specific to sous-vide technology to be carried out for amaranth seeds as well. the sous vide treatment was applied at different temperature (75 °c, 80 °c and 85°c), times (60, 120, 180 and 240 min) and solid-liquid ratios (1/4, 1/5 and 1/6). the exposure time had a major influence on the textural characteristics up to the value of 180 minutes, after which the relevance of this parameter decreased. keywords: amaranth, texture, sous vide 1. introduction the amount of intake and form of the functional food should be as it is normally expected for dietary purposes [1]. therefore, it could not be in the form of pill or capsule just as normal food form [2] the physicochemical properties of food are strongly influenced by the cooking process, so different parameters such as aroma, color or texture represent important characteristics in consumer preferences. traditional heat treatments generally use high temperatures, which contributes to nutritional, flavor or color losses. sous-vide technology represents an alternative heat treatment model to other conventional heat treatments and an alternative to meet the growing demand for ready-to-eat foods [3]. by using sous-vide technology, we try to preserve the physical-chemical properties as close as possible to the natural state. the use of the expression "sous-vide" has its origin in the french language and can be translated as "sub-vide" or "under vacuum". using this expression, it is defined the process by which food is cooked at a stable temperature, in a vacuum, during a precisely set period of time [4]. sous-vide technology can be applied to almost all food categories. they are cooked at a low temperature ranged between 50 °c and 90 °c. a major advantage in using sous-vide technology is the high microbiological safety [5]. in addition to this fact, the use of sous-vide technology generates some improvements in terms of color and flavor food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 248 retention, but also the reduction of the deterioration of proteins, lipids, vitamins, and other compounds that are sensitive to high temperatures [6]. amaranth seeds have not been tested so far using sous-vide technology, however, due to their nutritional properties, there is a need to diversify food products based on amaranth seeds, especially by using new cooking techniques that can help preserving the amount of nutrients and vitamins. amaranth is a pseudocereal known to have a high nutritional value due to amino acids, fibers, trace elements, vitamins, flavonoids, phenolic compounds and polyunsaturated fatty acid [7,8]. antioxidants gained a considerable interest in food technology research. their availability in various diets and their high potential role in the fight against various diseases such as cancer and neurodegenerative or cardiovascular diseases were highlighted in a variety of studies [9,10]. amaranth seed meal is a known source of polyphenols and is recommended by doctors for use in balanced diets [11]. polyphenols are easily degraded in the intestine of humans and animals due to the abundance of the enzyme beta-glucosidase, which releases the aglyconic fragment of the molecules [12]. the increasingly acute lack of time has irreversibly changed eating habits but also economies and technologies that have transformed the food industry into an assembly. the sous-vide technique is one of the alternatives that can satisfy the growing consumer demand for ready-to-eat foods [13]. the sous-vide technique can be applied to almost all types of food. the most eloquent example to identify the advantages of using the sous-vide technique is cooking meat for a period of 4-12 hours at temperatures of 55-80 °c. thus, the juiciness of the meat is maintained and the flavor and texture are greatly improved [14]. traditionally, high temperatures are used to cook food, which contributes to loss of nutritional components, flavor or color. to solve this problem, different studies were carried out to identify cooking methods that use low temperatures [15]. using the sous-vide technique is a viable method that presents greater microbiological safety compared to conventional cooking [16]. in general, losses during traditional cooking are correlated with the degree of juiciness. by using the sou-vide technique, vitamins are preserved much better compared to cooking on the grill or in the oven, thus nutrient losses are reduced [17]. nutrient losses are low using the sous-vide technique due to the fact that the food is cooked evenly, despite the fact that the exposure time is longer. at the same time, the use of vacuum does not allow the food to dehydrate and thus does not result in losses, as happens with cooked food. the aim of this study was to evaluate the impact of sous-vide treatment of amaranth seed texture parameters. 2. materials and methods materials organic amaranth seeds (amaranthus cruentus) were procured from the driedfruits warehouse (timișoara, romania). the seeds were authenticated according to their botanical characteristics and stored in paper bags until processing. souse vide technique different samples were analyzed, and cooked at 75 °c, 80 °c and 85 °c for 60 minutes, 120 minutes, 180 minutes and 240 minutes. for each sample cooked at different temperature and time, different ratios of amaranth seeds/water of hydration (1/4, 1/5, 1/6) were also tested, where 1 represents the amount of 10 g of amaranth seeds and 4, 5 and 6 represents the measured value of water, respectively 40 ml of hydration water, 50 ml of hydration food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 249 water and 60 ml of hydration water in which the amaranth seeds were immersed and later emptied with the vessel in which they were hydrated. four samples were cooked by the sousvide method at a temperature of 75 °c but using different exposure times: 60 minutes, 120 minutes, 180 minutes and 240 minutes. the following samples were cooked at 80 °c and the same exposure times. the same was done with the last four samples from the 1/4 ratio series, at a temperature of 85 °c. for cooking with the sous-vide method, a specially designed cooking device model sirman (italy) was used (fig. 1). it is equipped with a recirculation pump, a thermostat and an electronic timer. the prepared and evacuated samples were placed in a gn1 wide opening vessel with a volume of 10 l, over which water was poured up to 2/3 of the vessel volume. the sous-vide cooking device was anchored to the edge of the vessel and was immersed with the lower part up to the mark in the water with samples. the recirculation pump with which the sous-vide cooking device is equipped helps to evenly distribute the temperature in the water in the vessel in which the samples are thermally processed. the time and temperature adjustment functions are shown on the device display and were set specifically for each sample to be analyzed. fig.1. sirman sous-vide cooking device after the programmed cooking time ended, the samples were removed from the pot of water. after cooling, the samples were removed from the glass containers, filtered and weighed. the water remaining after filtration was also measured. the same was done with the following 12 samples that were cooked at a temperature of 80 °c and the last 12 samples at a temperature of 85 °c using different time parameters and ratio of amaranth seeds/hydration water, depending on each sample. texture analysis the texture profile evaluation test was performed using a tvt 6700 texturometer (perten instruments, stockholm, sweden). a compression cylinder with a diameter of 35 mm was attached to this device. it was used to press with a determined force on the analyzed samples from all samples. for the analysis of the samples, they were dimensioned to a diameter of 30 mm and a height of 10 mm. the principle of the method is defined by the application of a force on samples of amaranth seeds cooked using sous-vide technology, in the preparation of which different and specific parameters of time, temperature and amaranth seed/hydration water ratio were used, inside the vessel in which the sample was made and which was subsequently emptied. the compression of the samples was carried out on the surface of a press table. using the software, the device was programmed to apply a compressive force to the sample twice. thus, the first compression was performed at a displacement equal to 75% of the sample height, applying a speed of 2 mm/s, after which the piston returned to the initial position. the second compression was performed at a cylinder displacement equal to 75% of the sample height, while also maintaining the compression speed. by using this method, the device records in real time the resistance force against the analyzed sample and at the same time food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 250 records the calculated parameters. the result consists in recording the values of the forces, but also the displacements and the calculation of the texture parameters derived from the integration of the two recorded values. experimental design and statistical analysis the three-level box–behnken design was adapted in this study to investigate and optimize the effect of the independent variables of temperature (x1), time (x2), and solid-liquid ratio (rsl) (x3) on hardness and adhesiveness. the coded levels of the variables are -1, 0 and 1 were 75 °c/120 min/ 1/6, 80 °c/180 min/ 1/5 and 85 °c/180 min/ ¼, respectively. all calculations and graphics were carried out using the statistical software design expert 11 (trial version, minneapolis, mn, usa). triplicate experiments were conducted in order to validate the optimal extraction conditions. the box–behnken design was based on a second-order (quadratic) polynomial response surface model using the following equation: 𝑦 = 𝑏0 + ∑ (𝑏𝑖𝑥𝑖) + ∑ (𝑏𝑖𝑖𝑥𝑖𝑖 2) +𝑛𝑖=1 𝑛 𝑖=1 ∑ (𝑏𝑖𝑗𝑥𝑖𝑥𝑗) 𝑛 𝑖𝑗=1 (1) where y is the predicted response (hardness and adhesiveness), xi stands for the coded levels of the design variable (temperature (x1), time (x2), and solid-liquid ratio (rsl) (x3)), b0 is a constant, bi is the linear effects, bii is the quadratic effects and bij is the interaction effects. 3. results and discussion 3.1. texture parameters evolution during the sours-vide treatment the evolution of the texture parameters is presented in fig. 2. the texture parameters studied (hardness and adhesiveness) were determined automatically from the displacement of the material (hardness represents the resisting force opposed by the sample after it was compressed; adhesiveness represents the resistance recorded when the piston detaches from the analyzed sample after the first compression. this parameter is calculated by integrating the negative area under the curve). based on the results obtained from the determinations made major differences were found between the influences of the parameters studied on amaranth seed texture. these differences are directly influenced by the parameters used in each individual sample. the exposure time and the temperature at which the amaranth seeds were cooked had a decisive role in identifying the best option for obtaining a food product with optimal textural parameters. temperature is an important parameter in modifying adhesiveness, while in hardness modifications the same temperature parameter (85 °c) makes the difference in achieving an optimal texture. according to the graphs, an increase in the adhesiveness can be observed at temperatures of 75 °c and 80 °c, while at the temperature of 85 °c the adhesiveness decreases steadily. the hardness was directly influenced by the exposure time where, according to the graphs, the time of 240 minutes represents the point of convergence of the values that form a common point, less in the case of the temperature of 85 °c and amaranth seed/water ratio for hydration 1/6 where the measured value was the lowest. the ratio of amaranth seeds/water for hydration is an important parameter due to the fact that during the sous-vide heat treatment process the starch in the composition of amaranth seeds gels. thus, the greater the amount of water, the more the starch granules have the opportunity to change their structure, a fact that directly leads to changes in texture. the exposure time had a major influence on the textural characteristics up to the value of 180 minutes, after which the relevance of this parameter for texture parameters decreased. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 251 fig. 2. changes in hardness over time, depending on temperature parameters and amaranth seed/water ratio used for hydration 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 0 50 100 150 200 250 300 h a rd n e ss ( g ) time (min) 75_1/4 75_1/5 75_1/6 0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0 1800.0 0 50 100 150 200 250 300 h a rd n e ss ( g ) time (min) 80_1/4 80_1/5 80_1/6 0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 0 50 100 150 200 250 300 h a rd n e ss ( g ) time (min) 85_1/4 85_1/5 85_1/6 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 252 fig. 3. adhesiveness changes depending on temperature parameters and amaranth seeds/water ratio used for hydration 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 a d h e si v e n e ss (j ) time (min) 75_1/4 75_1/5 75_1/6 0 100 200 300 400 500 600 0 50 100 150 200 250 300 a d h e si v e n e ss (j ) time (min) 80_1/4 80_1/5 80_1/6 0 100 200 300 400 500 600 0 50 100 150 200 250 300 a d h e si v e n e ss (j ) time (min) 85_1/4 85_1/5 85_1/6 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 253 3.2. box-behnken design and model fitting box-behnken design was applied to study the combined effects of the three parameters (temperature, time and rsl) on amaranth texture parameters (hardness and adhesiveness) during the sous-vide treatment. the regression coefficients of the two proposed model are presented in table 1; the two models were significant ones (p<0.0001), and the f-value was 10.34 for hardness and 9.4 for adhesiveness, respectively. the model developed for hardness and adhesiveness are significant in terms of anova and the regression of the two models are higher than 0.80. the parameters (temperature, time and rsl) presented significant linear effects of hardness and adhesiveness. the interaction effects between temperature and time, and time and rsl were significant for hardness and adhesiveness. the quadratic effects were not significant for the two parameters. 3.3. fitting of second order polynomial equations the relationship between the texture parameters and the sous-vide technique were assessed using a second order polynomial equations using the box behnken design. the equations which describe the correlation between the input and outputs are presented below: 𝐻𝑎𝑟𝑑𝑛𝑒𝑠𝑠 = 259.1 − 713.6 ∙ 𝑋1 − 744.4 ∙ 𝑋2 − 309.2 ∙ 𝑋3 + 676.1 ∙ 𝑋1 ∙ 𝑋2 + 807.6 ∙ 𝑋1 ∙ 𝑋3 + 184 ∙ 𝑋2 ∙ 𝑋3 + 861.1 ∙ 𝑋1 2 + 88.0 ∙ 𝑋2 2 + 230.4 ∙ 𝑋3 2 (2) 𝐴𝑑ℎ𝑒𝑠𝑖𝑣𝑒𝑛𝑒𝑠𝑠 = 398.6 + 67.2 ∙ 𝑋1 + 43.0 ∙ 𝑋2 − 2.8 ∙ 𝑋3 − 53.3 ∙ 𝑋1 ∙ 𝑋2 + 4.4 ∙ 𝑋1 ∙ 𝑋3 − 6.1 ∙ 𝑋2 ∙ 𝑋3 − 98.0 ∙ 𝑋1 2 − 9.8 ∙ 𝑋2 2 − 42.1 ∙ 𝑋3 2 (3) table 1. analysis of variance for hardness and adhesiveness modelling using box benhken design hardness adhesiveness source sum of squares df mean square fvalue pvalue sum of squares df mean square fvalue p-value model 4.3e+7 9 4.8e+6 10.4 < 0.0001 2.1e+5 9 23668.8 9.4 < 0.0001 atemperature 1.3e+7 1 1.3e+7 28.2 < 0.0001 85990.0 1 85990.0 34.1 < 0.0001 b-time 8.3e+6 1 8.3e+6 17.9 0.0004 15356.6 1 15356.6 6.1 0.0232 c-rsl 2.5e+6 1 2.5e+6 5.4 0.0310 2183.4 1 2183.4 0.87 0.3633 ab 4.3e+6 1 4.3e+6 9.3 0.0064 50170.5 1 50170.5 19.9 0.0003 ac 8585.7 1 8585.7 0.02 0.8932 12.3 1 12.3 4.9e3 0.9448 bc 9.4e+6 1 9.4e+6 20.3 0.0002 37881.7 1 37881.7 15.0 0.0010 a² 0.000 0 0.000 0 b² 9.4e+5 1 9.4e+5 2.1 0.1691 8698.6 1 8698.6 3.46 0.0786 c² 0.000 0 0.000 0 residual 8.8e+6 19 4.6e+5 47819.9 19 2516.8 lack of fit 8.8e+6 17 5.1e+5 47819.9 17 2812.9 pure error 0.000 2 0.000 0.000 2 0.000 cor total 5.2e+7 28 2.6e+5 28 model stdev 680.98 50.17 model r² 0.8318 0.8167 food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 254 3.4. surface response 3d plots in figure 4, 5 are presented the evolution of hardness and adhesiveness in function of temperature, time and rsl. in the case of combined influence of temperature and tine on hardness, it can be observed that at high temperature and times the hardness reaches the lowest values. in the case of the combined influence of temperature and rsl, the hardness reached the low values at high temperature and rsl. the time and rsl influences negatively the hardness. in the case of combined influence of temperature and time on adhesiveness, it can be observed that at the maximum time applied and medium temperature, the adhesiveness reached the high values, while at the lowest times the hardness reached the lowest values. in the case of the combined influence of temperature and rsl, the adhesiveness reached the low values at low temperature irrespective of the rsl applied. fig. 4. 3d evolution of hardness of amaranth seed submitted to sous-vide treatment food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 255 fig. 5. 3d evolution of adhesiveness of amaranth seed submitted to sous-vide treatment 4. conclusion considering the results obtained from the determinations differences were found in terms of the influence of the parameters studied on amaranth seed texture. these differences were directly influenced by the parameters used in each individual sample. the exposure time and the temperature at which the amaranth seeds were cooked had a decisive role in identifying the best option for obtaining a food product with optimal textural parameters. the hardness was directly influenced by the exposure time where, according to the graphs, the time of 240 minutes represents the point of convergence of the values that form a common point, less in the case of the temperature of 85 °c and amaranth seed/water ratio for hydration 1/6, where the measured value was the lowest. boxbehnken design was applied to study the combined effects of the three parameters (temperature, time and rsl) on amaranth texture parameters (hardness and adhesiveness) during the sous-vide treatment. the regression coefficients of the two proposed models were significant ones (p<0.0001), and the f-value was 10.34 for hardness and 9.4 for adhesiveness, respectively. the free parameters (temperature, time and rsl) presented significant linear effects of hardness and adhesiveness. food and environment safety journal of faculty of food engineering, ştefancel mare university suceava volume xxi, issue 3 – 2022 ovidiu procopeț, mircea oroian, changes in texture profile of amaranth seeds subjected to sous vide treatment, food and environment safety, issue 3 – 2022, pag.247 -256 256 5. acknowledgement this work was supported by romania national council for higher education funding, cnfis, project number cnfisfdi-2022-0259. 6. references [1] kato, h., suzuki k., bannai, m., and moore, d. r. protein requirements are elevated in endurance athletes after exercise as determined by the indicator amino acid oxidation method. plos one 11:e0157406, (2016). [2] rinaldi, m., dallasta, c., meli, f., morini, e., pellegrini, n., gatti, m., and chiavaro,e.,physicochemicaland microbiological quality of sous-vide-processed carrots and brussels sprouts. food bioprocess technol., 6, 3076-3087, (2013). [3] diaz, p., nieto, g., garrido, m. d., and banon,s., microbial, physical-chemical and sensory spoilage during the refrigerated storage of cooked pork loin processed by the sous vide method. meat sci., 80, 287–292,(2008). [4] scalbert, a., johnson i.t.; saltmarsh, m., polyphenols: antioxidants and beyond. am. j. clin. nutr. 81, 215s–217s,(2005). [5] barba dela rosa, a.p.; fomsgaard, i.s.; laursen, b.; mortensen, a.g.; olvera-martinez, l.; silva-sanchez, c.; mendoza-herrera, a.; gonzalezcastaneda, j.; de leon-rodriguez, a., amaranth (amaranthus hypochondriacus) as an alternative crop for sustainable food production: phenolic acids and flavonoids with potential impact on its nutraceutical quality. j. cereal sci. , 49, 117– 121,(2009). [6] pasko, p.; barton, h.; folta, m.; gwzdz, j. evaluation of antioxidant activity of amaranth (amaranthus cruentus) grain and byproducts (flour, popping, cereal). rocz. państw. zakł. hig. , 58, 35–40, (2007). [7] gorinstein, s.; vargas, o.j.m.; jaramillo, n.o.; salas i.a.; ayala, a.l.m.; arancibia-avila, p.; toledo, f.; katrich, e.; trakhtenberg, s. the total polyphenols and the antioxidant potentials of some selected cereals and pseudocereals. eur. food res. technol. 225, 321–328 ,(2007). [8] pasko, p.; sajewicz, m.; gorinstein, s.; zachwieja, z. analysis of selected phenolic acids and flavonoids in amaranthus cruentus and chenopodium quinoa seeds and sprouts by hplc. acta chromatogr., 20, 661–672, (2008). [9] lamothe l.m.; srichuwong, s.; reuhs, b.l.; hamaker, b.r., quinoa (chenopodium quinoa w.) and amaranth (amaranthus caudatus l.) provide dietary fibres high in pectic substances and xyloglucans. food chem., 167, 490–496,(2015). [10] kato, h., suzuki, k., bannai, m., and moore, d. r., protein requirements are elevated in endurance athletes after exercise as determined by the indicator amino acid oxidation method. plos one 11:e0157406,(2016). [11]renna,m.,gonnella, m., giannino, d.,santamaria, p.,quality evaluation of cookchilled chicory by conventional and sous vide cooking methods. journal of the science of food and agriculture 94 (4), 656-665, (2014). [12] diaz, p., nieto, g., garrido, m. d., and banon, s., microbial, physical-chemical and sensory spoilage during the refrigerated storage of cooked pork loin processed by the sous vide method. meat sci., 80, 287–292, (2008). [13] diplok, a. t., aggett, p. j., ashwell, m., bornet, f., fern, e. b., & roberfroid, m. b. scientific concepts of functional foods in europe: concensus document. british journal of nutrition, (suppl. 1), s1–s27, (1999). [14] siro, i., kapolna, e., kapolna b., lugasi, a., functional food. product development, marketing and consumer acceptance—a review appetite, 51, pp. 456-467, (2008). [15]manach, c., scalbert, a., morand c., remesy, c., and jimenez, l.,polyphenols: food sources and bioavailability. am j clin nutr 79:727-747.(2004) [16] aguilera, j., & whigham ld.,isot environ healt s, ;54(6), 573, (2018). [17] iborra-bernard, c.,tarrega, a., garcia-segovia, p., martinez-monzo, j., advantages of sous–vide cooked red cabbage: structural, nutritional and sensory aspects. lwt food science and technology, 56, 451-460, (2014). [18] botinestean, c., keenan, df., kerry jp., hamill, rm., the effect of thermal treatments including sous-vide, blast freezing and their combinations on beef tenderness of m. semitendinosus steaks targeted at elderly consumers. lwt food sci technol.;74:154– 159.doi: 10.1016/j.lwt.2016.07.026, (2016). 1. introduction table 1. analysis of variance for hardness and adhesiveness modelling using box benhken design doi: https://doi.org/10.4316/fens.2022.013 129 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 129 142 diversity, antibiogram and plasmid profile of microbial contaminants of some selected vegetables sold in bayelsa nigeria christiana n. opara 1 , uchenna c. okpe 1 , *christian k. anumudu 2 1department of microbiology, federal university otuoke, bayelsa state, nigeria 2school of chemical engineering, university of birmingham, united kingdom, c.k.anumudu@bham.ac.uk *corresponding author received 13th may 2022, accepted 29th july 2022 abstract: this study identified 12 microorganisms, including eight bacteria and four fungi associated with different vegetables; cabbage (brassica oleracea), ugu leaf (telfairia occidentalis), scent leaf (ocimum gratissimum), okra (abelmoschus esculentus), waterleaf (talinum fruticosum), uziza leaf (piper guineense), and bitter leaf (vernonia amygdalina) sold in otuoke market located in southern nigeria. cultural methods were used to quantitatively and qualitatively elucidate fungi and bacteria contaminants in the samples. the average bacteria concentration across all the vegetables was 3.88 x 109cfu/g. abelmoschus esculentus had the highest bacterial concentration of 9.03 (109) cfu/g, while brassica oleracea has the least bacterial load of 1.67 (109) cfu/g. the coliform count ranged from 0.5 to 2.63 (109) cfu/g for vernonia amygdalina and ocimum gratissimum respectively. mean coliform count was 8.43(109) cfu/g. the fungi count varied from 0.5 to 1.5 (103) cfu/g for ocimum gratissimum and vernonia amygdalina respectively. biochemical and morphological characterization identified enterobacter sp., escherichia coli, klebsiella sp., salmonella sp., serratia marcescens, micrococcus sp., proteus mirabilis and staphylococcus sp. as key bacterial contaminants of the vegetables. fungi species isolated from these vegetables include aspergillus niger, alternaria sp., rhizopus sp. and fusarium sp. the antibiotic susceptibility testing revealed that al the organism isolated were resistant to two or more antibiotics, including a 100% resistance to the penicillin family of antibiotics. furthermore, all the isolates contained plasmids with a range of 100-200kbp except salmonella sp., and the fungi; rhizopus, fusarium and alternaria. keywords: foodborne infection; food safety; public health; antibiotics resistance; food intoxication 1. introduction antimicrobial drug resistance is a global health challenge that sabotages the attainment of the sustainable development goal of good health [1]. this occurs when bacteria, viruses, fungi or parasites develop mechanisms that make antimicrobials ineffective over time. currently, the rate at which microorganisms acquire new resistance mechanisms outweighs the development of new drugs [2,3]. multidrug resistant microorganisms are widespread and have been isolated from all parts of the globe. in nigeria, several reports have highlighted a high prevalence of antimicrobial-resistant bacteria and this corresponds to the high burden of infectious diseases, including tuberculosis, diarrheal diseases, respiratory infections, urinary tract infections, zoonotic and nosocomial infections [4,5]. in nigeria, food and water pose a major contributing factor to disease burden, with the country http://www.fia.usv.ro/fiajournal mailto:c.k.anumudu@bham.ac.uk food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 130 ranked amongst those with the highest burden of foodborne illness with its concurrent negative effect on disabilityadjusted life years (dalys) [6]. various microbial agents are implicated in foodborne illnesses in the country, with the bacteria genera of salmonella, escherichia sp., shigella sp., and staphylococcus aureus being the most commonly encountered [7]. different food products can be contaminated with pathogenic microbial species and serve as a mode of entrance of such pathogens into the population, causing foodborne disease outbreaks. of importance are vegetables which receive minimal processing and are mainly consumed fresh. vegetables are an essential food group consumed daily by a large number of individuals. it may exclude foods derived from some plants such as nuts, and cereal grains but include savoury fruits such as tomatoes, flowers such as broccoli, and seeds such as pulses [8]. vegetables remain one of the essential parts of a balanced diet because of the many nutritional benefits associated to their consumption. they are rich sources of micronutrients, minerals, vitamins, and most importantly, antioxidants fibre. they are vital to human health, well-being and disease prevention [9]. recently, the level of public awareness with regards to the benefits of healthy eating habits has increased, prompting an increasing demand for and incorporation of fresh vegetables into diets [10]. the food and agriculture organization and the world health organization strongly recommend a daily intake of >400 g/day for fruits and vegetables in diets to promote good health [11]. despite the health benefits derived from consuming fresh vegetables, the risk of microbiological contamination in vegetables is of public concern due to the possibility of pathogenic microorganisms coming in contact with the fresh food products along the food chain, beginning from the vegetable farm to the dinner table [8]. microbiological contamination of vegetables can occur directly or indirectly, firstly through contact with soil, dust, and water, and secondly through punctures and open cuts of tissues of vegetables; thus, contaminations of vegetables may occur internally or externally during cultivation, harvest, packaging, and storage, transporting and marketing [9,12,13]. vegetables are rich in nutrients and serve as a suitable substrate for the growth of invading microorganisms, supporting their growth, leading to spoilage and unwholesomeness of the food. furthermore, because most vegetables are eaten fresh (not cooked) or slightly cooked, the risks of foodborne infections and intoxication increases as washing may not guarantee decontamination of pathogenic microbial contaminants which can easily enter the alimentary canal through the food. there have been many reported cases of foodborne disease outbreaks caused by consuming fresh vegetables. the most commonly implicated microorganisms are; listeria monocytogenes, escherichia coli o157:h7, and salmonella spp [14,15]. foodborne disease outbreaks lead to various illnesses, hospitalizations, deaths, and even food recalls, in all parts of the world [16,17]. of these, there are increasing cases of antimicrobial drugresistant foodborne pathogens with farreaching impacts on public health. a variety of factors influence the development of antibiotic resistance in bacteria. these include the use of antibiotics as growth promoters in farm animals, antibiotics abuse in humans and in food fraud cases where unapproved antibiotics are used in food preservation [18]. of paramount importance is the possession of plasmids [19]. plasmids are non-essential extrachromosomal dna food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 131 molecules located in the cell's cytoplasm and can replicate independently of the chromosomal dna [20]. because they can be passed to bacteria of the same or different genera through conjugation or transduction and are potential carriers of resistant genes, their rapid dissemination poses a threat to the rapid development of multidrug resistance in a population, thus necessitating the need for plasmid profiling in microbial isolates [21]. there is widespread belief and acknowledgement among most consumers that vegetables sold in the local nigerian markets are unwholesome. this belief is because vegetables are primarily produced and handled under unhygienic conditions and practice. these unhygienic practices include the application of manure obtained from animal faecal matter to soils during the cultivation of vegetables, irrigation of vegetable farms with heavily contaminated water, use of unclean water to wash vegetables, dressing vegetables on the bare floor and concrete slabs on the farm and at the grocery, and insufficient pre and postharvest inspections of vegetables [22]. in otuoke nigeria, river water is the most commonly used water source to irrigate vegetable farms. this is due to the high cost and unreliability of pipe-borne water supply. unfortunately, these water sources are heavily polluted, especially with pathogenic and toxigenic microorganisms which may contain plasmids that confers resistance to commonly utilised antibiotics [23-25]. in addition to conducting studies to ascertain the safety of foods sold in local markets such as otuoke based on microbial contamination, it is important to elucidate the antibiogram of isolated pathogens and analyse for the presence of plasmids which may be conferring such resistance. the present study, therefore, aimed at investigating the microbiological contaminations of vegetable samples including; cabbage (brassica oleracea), pumpkin leaf (telfairia occidentalis), scent leaf (ocimum gratissimim), okra (abelmoschus esculentus), waterleaf (talinum fruticosum), uziza leaf (piper guineense), and bitter leaf (vernonia amygdalina), sold in otuoke market, their antibiogram and plasmid profiles. 2. materials and methods 2.1 study area and sampling the study area, otuoke, is located in bayelsa state of nigeria. otuoke has one major market where vegetables and other groceries are sold. the collection of the vegetable samples followed a completely randomized experimental design. to minimize experimental error, the samples were collected randomly from each sampling unit in triplicates. a total of seven (7) vegetable species were collected randomly and in triplicates. these samples include brassica oleracea (cabbage), telfairia occidentalis (ugu leaf), ocimum gratissimum (scent leaf), abelmoschus esculentus (okra), talinum fruticosum (waterleaf), piper guineense (uziza leaf), and vernonia amygdalina (bitter leaf). the samples were put in clean containers, labelled and immediately transported to the laboratory for microbiological analyses. 2.2 samples preparation each vegetable was homogenized in phosphate buffered saline (pbs) into a working solution following the method described by yafetto et al. [26]. nutrient agar was used for total bacteria enumeration, macconkey agar to isolate coliforms, and potato dextrose agar (pda) media was used to isolate fungi from the vegetables. all the media used were commercially purchased (sigmaaldrich) and prepared according to the manufacturer's instructions. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 132 2.3 microbiological analysis the microbial quality of the vegetables was analysed following the methods described in the microbiological examination methods of food and water laboratory manual for aerobic mesophilic bacteria and coliforms [27]. standard pour plates of 10-fold serial dilution were prepared in three replicates for all vegetable samples. ninth dilutions were plated using the pour plate method for the triplicate samples of each vegetable for bacteria, while the third dilution factor was used for fungal isolation. after 24 hours of incubation at 370c, predominant bacteria colonies were subcultured using macconkey agar and nutrient agar plates. identification of bacteria cultures were done using standard biochemical tests, morphological characteristics and grams' reaction test [28]. 2.4 fungal isolation and identification the fungal isolates were identified using cultural and morphological features such as colony growth pattern, conidial morphology, and pigmentation [29]. 1 ml aliquot (103) of stock solution of the leaves was dispensed into a petri dish. 20ml of molten but cooled potato dextrose agar (pda) medium was added and evenly mixed with the stock solution using the pour plate method to identify fungi. the cultured plates were allowed to solidify and incubated at 25°c for seven days in an incubator (plus ii, gallenkamp, england). the technique of oyeleke and manga [30] was also adopted to identify the isolated fungi using cotton blue in lactophenol stain. a small portion of the aerial mycelia from the representative fungi cultures was removed and placed in a drop of lactophenol. the mycelium was well spread on the slide with a sterile needle. a coverslip was gently placed with little pressure to eliminate air bubbles. the slide was then mounted and viewed under the light microscope with ×10 and ×40 objective lenses. the morphological characteristics and appearance of the fungal organisms seen were identified according to published literature [31,32]. 2.5 antimicrobial susceptibility test antimicrobial susceptibility of the isolates was tested using the modified kirby-bauer multi discs diffusion method [33]. commercial antibiotic discs (rosco) containing the antibiotics were applied on muller hinton agar. the antibiotics evaluated for efficacy against the isolates include; pefloxacin(pef) 10ug, gentamycin(cn) 10ug, ampicillin(apx) 30ug, cefuroxime(z) 20ug, amoxicillin(am) 25ug, ceftriaxone(r) 25ug, ciprofloxacin(cpx) 10ug, streptomycin(s) 30ug, erythromycin(e) 10ug [34]. 2.6 plasmid profile analysis plasmid dna was isolated as previously described [35]. briefly, microbial cells were pelleted, and resuspended in e buffer with 40mm tris-hydroxide and 2mm edta. these were subsequently lysed, heated to 60°c for 30 minutes and digested with proteinase k. this was incubated at 37°c for 60 minutes with 1ml phenolchloroform-isoamy alcohol (sigmaaldrich), followed by centrifugation at 8000g for 7 minutes. plasmid profile analyses of the isolated strains were undertaken by electrophoresis of dna using 0.8 % agarose gel electrophoresis, stained with ethidium bromide and visualised by a uv transilluminator as described previously [36,37]. briefly, 1g of agarose powder was weighed out into 100ml of 1xtbe buffer and dissolved by boiling for 3-5 minutes using a magnetic stirrer and water bath. the media was allowed to cool at 50 degrees celsius and 10ul of ethidium bromide was added and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 133 gently mixed by swirling. the media was poured into the tank, ensuring that the comb was in place to obtain a gel thickness of about 4.5mm; bubbles were avoided. the media was allowed to stand for 20min to solidify, and then the comb was removed, and the tray placed in the electrophoresis tank. 1xtbe buffer was poured, ensuring that it covered the surface of the gel. 15-20ul of the sample with 2ul of the loading dye was mixed and carefully poured into the well created by the comb with marker and land, followed by the control. the electrode was connected, and electrophoresis was run for 60-100v until the loading dye migrated to the end of the gel field. the electrodes were turned off, and the gel was observed on the uvilluminator. 3. results and discussion 3.1 microbiological quality of vegetable samples a total of seven vegetables were screened for their microbial quality. the mean coliform, total aerobic bacteria and fungi count are presented in figure 1. fig. 1. mean microbial count of vagetable samples randomly sourced from otuoke market figure 1 shows that cabbage had the least viable bacteria count of 1.67x109 while okra (abelmoschus esculentus) had the highest total bacteria count of 2.7x109. the total bacteria count from okra (abelmoschus esculentus) was higher than previous reports [38] having a total viable count of 1.0x105. the total heterotrophic bacteria count recorded in this work is higher than the specified standard limit of bacteria in foods by the european commission ec no. 2073/2005 regulation on microbiological criteria for foods [39]. the aerobic bacteria load of cabbage 0 2 4 6 8 10 12 bitter leaf scent leaf cabbage water leaf uziza leaf okra ugu leaf m e a n m ic ro b ia l c o u n t (1 0 9 c f u /g ) heterotrophic bacteria count coliform count fungal count food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 134 (brassica oleracea) was the least with a count of 1.67x109, which is higher than what was reported by obiageli¸ et al. [40] at 1.8x107 in nigeria. it was observed from this study that there was microbial contamination of all the vegetable samples, and this can be attributed to poor hygiene, soil and environmental contamination. all vegetable samples recorded a high coliform count. the highest coliform count was recorded by scent leaf (ocimum gratissimum) 2.63x109 while the least was observed for bitter leaf (vernonia amygdalina) with a load count of 0.5x109. this result is higher than the load count of 1.8x109 reported in the research of ofor et al. [41]. this high level of microbial load suggests the possibility of faecal contamination through untreated animal manure, poor hygiene or polluted irrigation water. it could also result from human activities such as bathing, washing, and dumping refuse in the rivers where vegetable samples are washed. previous research [42] has recorded this level of microbial contamination in freshwater apple snails (pila ampullaceal) obtained from the same locale. based on the mean colony counts reported for this study, it is evident that the extent of microbiological contamination of the vegetables sold in the otuoke daily market is higher than permissible limits and their consumption can pose a threat to public health. this suggests that most of the vegetables sold in the otuoke community may be unwholesome and the food safety concerns among consumers in the metropolis are understandable. the high load of bacteria pathogen indicates poor sanitary conditions of vegetables. this is a growing issue as similar high microbial loads have previously been reported from other studies within markets in the southern region of nigeria [8,43,44]. thus, urgent action is needed to correct this and prevent the outbreak of foodborne illness within the region. 3.2 characterization of bacterial isolates the bacteria and fungi isolates were characterized based on morphological and biochemical characteristics. the complete identification was done by comparing viewed characteristics with known taxa [45]. the predominant bacteria isolates include enterobacter sp., escherichia coli, klebsiella sp., salmonella sp., serratia marcescens, micrococcus sp. proteus mirabilis and staphylococcus sp. the isolated bacteria contaminants are of public health significance. the presence of escherichia coli which is an indicator organism, suggests the possibility of faecal contamination of the samples [46]. escherichia coli has been implicated in other studies to be one of the leading causes of diarrheic infection and is one of the superbug organisms [47,48]. serratia marcescens, a pink-pigmented gramnegative bacterium of the family enterobacteriaceae, was isolated from waterleaf. this organism is notorious for its pathogenicity and toxigenicity, as reported in a previous study [49]. similarly, staphylococcus aureus contamination is a major public health issue because of its ability to cause a wide range of infections, especially foodborne intoxication [50]. staphylococcus aureus is a grampositive coccus, a normal flora of the skin and nasal passages; however, when consumed with vegetables becomes opportunistic, and this contamination is through infected vegetable handlers [14]. the two bacteria; escherichia coli and staphylococcus sp. have been significant contaminants of vegetables [5154]. similarly, [49] observed the presence of serratia mercescens while working with raw vegetables, with the potential to cause spoilage and gastrointestinal illness. the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 135 presence of salmonella sp. is an indication of polluted water used in washing vegetables. salmonella sp. is of high clinical importance in nigeria because many studies conducted within the country have reported the organism to be pathogenic, toxigenic, and multidrug resistant (mdr) [55-57]. 3.3 antibiotic susceptibility test of isolated bacteria groups the antibiotic susceptibility pattern of the isolates is presented in table 1. the isolates showed some level of resistance to all assayed antibiotics. the antibiotic which was most effective in inhibiting the isolates was pefloxacin (pef), with a sensitivity of 87.5%. whereas the least effective antibiotics were ampicillin (amp) and amoxicillin (am) to which all isolates were resistant. the most resistant isolate is klebsiella sp. which was resistant to 88.8% (8 out of 9) of assayed antibiotics. while the most sensitive organism is staphylococcus aureus which was sensitive to 55.5% (5 out of 9) of the assayed antibiotics. table 1 antibiotics sensitivity pattern of bacteria isolated from vegetables isolates cn (10ug) r (30ug) am (25ug) cpx (5ug) apx (30ug) pef (5ug) s (10ug) e (15ug) z (20ug) serratia marcescens r(10) r(0) r(0) s(42) r(0) s(34) r(0) r(0) i(16) enterobacter spp s(16) i(15) r(0) s(38) r(0) i(22) r(0) r(0) r(0) salmonella spp. i(14) r(8) r(0) s(40) r(0) s(32) r(0) r(0) s(20) proteus mirabilis s(16) r(0) r(0) s(40) r(0) s(36) i(10) r(10) s(22) escherichia coli r(12) r(12) r(0) s(36) r(0) s(42) s(20) r(10) r(0) klebseilla spp. r(12) s(22) r(0) r(0) r(0) r(0) r(0) r(0) r(0) staphylococc us spp. r(0) r(0) r(0) s(40) r(0) s(34) s(36) s(28) s(28) micrococcus spp r(12) r(14) r(0) s(38) r(0) s(36) r(0) r(0) i(16) key: pefloxacin(pef), gentamycin(cn), ampicillin(apx), cefuroxime(z), amoxicillin(am), ceftriaxone(r), ciprofloxacin(cpx), streptomycin(s), erythromycin(e). resistant (r), sensitive (s), intermediate (i). overall, it can be observed that all the gram-negative bacteria were sensitive to pefloxacin(pef) and ciprofloxacin (cpx), except klebsiella sp. a similar report of klebsiella sp. resistance to quinolone group antibiotics was recorded in a recent review [58]. the gram-positive bacteria, micrococcus sp. and staphylococcus sp., were most sensitive to ciprofloxacin (cpx). they were also food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 136 sensitive to pefloxacin (pef) and cefuroxime(z). similar reports of staphylococcus and micrococcus sensitivity to ciprofloxacin has been reported by [59,60]. all the bacteria isolates showed 100% resistance to penicillin (amoxicillin and ampicillin). penicillin resistance of gram-negative bacteria obtained in this study is consistent with reports from previous studies on this growing trend [61]. the resistance of serratia marcescens to multiple antibiotics utilised in this study is consistent with previous experimental results [62] which reported that serratia marcescens was only sensitive to ciprofloxacin (cpx) and pefloxacin (pef) with resistance to other commonly utilised antimicrobial agents. the result obtained from this assay shows a high prevalence of antimicrobial resistance with all isolated organisms being resistant to more than one antibiotic. this is consistent with the report on disease burden and antibiotic resistance by the nigerian ministry of health which found a high incidence of multi-drug resistance bacteria in nigeria, consequently increasing the potential mortality and morbidity associated with infectious and disease burden from these organisms [63]. 3.4 characterization of fungal isolates a total of 69 fungi isolates were obtained from assayed vegetables. these were characterised based on their microscopic and macroscopic attributes on potato dextrose agar (pda), including the colony growth pattern, conidial morphology, and pigmentation as presented in table 2. the technique of oyeleke and manga [30] was also adopted to identify the isolated fungi by comparing them with that of known taxa. the presence of fungi in vegetables sold in otuoke market is an indication of possible deterioration of the vegetable. isolated species include aspergillus niger (34.78%), alternaria spp. (15.94%), rhizopus sp., (26.09%) and fusarium spp. (23.19%). table 2 macroscopic and microscopic characteristics of fungal isolates. isolate macroscopic description (pda) microscopic morphological characteristics aspergillus niger colonies are initially white, becoming black with conidia production. hyphae are septate and hyaline. conidial heads are radiate. conidiophores are long, smooth, and transparent, becoming darker at the apex. the conidia are very rough. alternaria spp. the texture of the colony is downy to woolly, with the colour pale-grey to olive-brown on the surface. the reverse is brown to black, and the growth rate is rapid. hyphae septate appears brown. conidiophores are brown, septate, and branched. conidiophores are scarce. fusarium spp. fluffy white growth and dark violet pigmentation on the undersurface of the plate. conidiophores are short, and non-septate. the conidiophores have a slightly inflated appearance as their sides aren't parallel but slightly bulge out in the middle. rhizopus spp. the texture is deeply cottony, with a white colour which becomes greybrown on the surface. the growth rate is very rapid. hyphae is broad and septate; rhizoid and stolon present, sporangiophores brown, sporangia round; sporangiophores ovoid. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 137 the most isolated fungi specie is aspergillus niger (34.78%) which predominantly contaminated bitter leaf. the fungi isolated in this study are similar to previous reports [64,65] and highlight that green vegetables present suitable substrates that encourage fungi growth and food deterioration. the presence of a high fungi load in vegetables may be due to poor handling of the products and extended display in the marketplace. furthermore, fungi can gain entrance into the foods through improper post-harvest handling leading to bruising of the vegetables. some fungi pathogens isolated such as fusarium sp. and aspergillus sp. are of public health concern as they produce mycotoxins such as fumonisin, zearalenone and aflatoxin, which can cause a wide array of health challenges [66]. 3.5 plasmid dna present in isolates plasmid profiles of the bacteria and fungi isolates were carried out and visualized in a gel electrophoresis field as presented in figure 2. of the 12 isolates assayed, 8 showed the presence of plasmids, while four (4) isolates showed no visible band indicating the absence of plasmids. in contrast, the other isolates, enterobacter sp, micrococcus sp., aspergillus sp, serratia sp. and staphylococcus sp. showed the presence of multiple plasmids by the visible bands. m is a 1kb marker of standard molecular weight for calculating the molecular weight of the plasmid dna [67]. bacteria are known to develop drug resistance following external environmental pressure, through the direct introduction of antibiotics or resistant strains into the environment or transfer of resistance gene sequence via plasmids [68]. thus, the results obtained in this gel electrophoresis indicates that resistance to the assayed antibiotics may be mediated by plasmids. fig. 2: plasmid profile pattern of bacterial isolates in 0.5% agarose gel. plasmid profile photographic representation indicates the isolates and bands created by each isolate. each well is represented in codes, while m is a 1kb marker of standard molecular weight for calculation of the molecular weight of the plasmid dna. codes: 1enterobacter sp. 2salmonella sp. 3micrococcus spp. 4serratia sp. 5 proteus spp. 6staphylococcus spp. 7 escherichia coli. 8klebsiella sp. 9 aspergillus niger .10alternaria sp.11 rhizopus sp.12fusarium sp. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 138 all the isolated organisms possessed plasmids except salmonella sp., rhizopus sp, fusarium sp. and alternaria sp. several researches have shown that bacteria acquire resistance through plasmids [68,69], with others highlighting a positive correlation between the presence of plasmids and bacterial resistance [21]. there is evidence of horizontal gene transfer in diverse bacteria genera, including enterobacter isolates containing plasmids that confer resistance to antibiotics to other bacteria [70]. this is similar to studies conducted on serratia sp. [71]. additionally, the dna carried on plasmids can be integrated into bacterial dna, thus not only conferring the ability to resist antibiotics, but making the resistance genes fully inheritable [21]. in this study, plasmids were identified in the fungi isolates. although the function of plasmids has not been fully understood in fungi, studies have suggested that plasmids could confer a selective survival advantage to some fungi species of agricultural importance [72]. 4. conclusion this study highlighted the high microbial load of retail vegetables in otuoke, nigeria. the possible sources of these contaminations could be poor handling by sellers, extended stay in the market, use of animal manure for cultivation and washing vegetables with polluted water. the contaminating bacterial and fungal genera are of a public health concern as they are causative agents of numerous foodborne disease outbreaks and food spoilage. the presence of these organisms in retail vegetables can result in economic loss, reduced manpower, food shortages and pose serious food safety challenges, as vegetables are minimally cooked before eating. plasmid profile study results demonstrated presence of multiple plasmids especially in enterobacter sp. micrococcus sp. and alternaria sp. the presence of plasmids indicates the possibility of multidrug-resistant bacterial presence, as has been demonstrated by literature and in the antibiogram assay of this study in which all the isolates were shown to be resistant to two or more antibiotics. the result obtained in this study is of public health concern and highlights the need for systematic approach in the control of microbial contaminants in foods as this may be a potent source of antimicrobial drugresistant microbial strains into the population. similarly, the use of antimicrobials in nigeria needs to be further regulated to curb the spread of antimicrobial resistance. further research is required to determine all primary sources of antibiotic resistance in microbial contaminants of vegetables. the collation of data on the prevalence and type of antibiotic resistance in microorganisms isolated from vegetables may help direct public health efforts in the fight against multidrug resistant pathogens. it is recommended that vegetable farmers employ the use of pipe-borne water or chlorinated water instead of water from streams, wells, and storm drains to irrigate or wash their vegetables and observe good pre-and post-harvest handling practices for vegetables. furthermore, vendors should practice good personal and environmental hygiene at the markets. traders and consumers must thoroughly wash vegetables before sale and consumption, respectively. the vegetable producers (farmers) and retailers should improve storing, handling, transporting, and preserving their products to free them from pathogenic microorganisms. 5. acknowledgement the authors wish to acknowledge the help rendered by all technical staff of the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 139 microbiology laboratory of federal university otuoke towards the successful completion of this research article. funding: no funding was received for the preparation of this manuscript. conflict of interest: the authors declare no conflict of interests. author contributions: conceptualization and design; c.n.o methodology; u.c.o and c.n.o software and analysis; u.c.o and c.k.a writing—original draft preparation; u.c.o and c.n.o. writing— review and editing; c.k.a and u.c.o visualization; c.k.a. project administration; c.n.o. all authors have read and agreed to the published version of the manuscript. 6. references [1].gajdács, m.; urbán, e.; stájer, a.; baráth, z. antimicrobial resistance in the context of the sustainable development goals: a brief review. european journal of investigation in health, psychology and education 2021, 11, 71-82. [2].blazquez, j.; oliver, a.; gomezgomez, j.-m. mutation and evolution of antibiotic resistance: antibiotics as promoters of antibiotic resistance? current drug targets 2002, 3, 345-349. [3].durão, p.; balbontín, r.; gordo, i. evolutionary mechanisms shaping the maintenance of antibiotic resistance. trends in microbiology 2018, 26, 677-691. [4].sadauki, a.h.; olorukooba, a.a.; balogun, m.s.; dalhat, m.m.; waziri, h.; abdulaziz, m.m.; umeokonkwo, c.d.; hassan-hanga, f.; sabitu, k. nasal carriage of meticillin-resistant staphylococcus aureus among children living with hiv attending infectious diseases clinics in kano, nigeria. infection prevention in practice 2022, 100213. [5].elelu, n.; aiyedun, j.o.; mohammed, i.g.; oludairo, o.o.; odetokun, i.a.; mohammed, k.m.; bale, j.o.; nuru, s. neglected zoonotic diseases in nigeria: role of the public health veterinarian. pan african medical journal 2019, 32. [6].oduori, d.o.; kwoba, e.; thomas, l.; grace, d.; mutua, f. assessment of foodborne disease hazards in beverages consumed in nigeria: a systematic literature review. foodborne pathog. dis. 2022, 19, 118. [7].odo, s.e.; uchechukwu, c.f.; ezemadu, u.r. foodborne diseases and intoxication in nigeria: prevalence of escherichia coli 0157: h7, salmonella, shigella and staphylococcus aureus. journal of advances in microbiology 2021, 20, 84-94. [8].kuan, c.-h.; rukayadi, y.; ahmad, s.h.; wan mohamed radzi, c.w.; thung, t.-y.; premarathne, j.m.; chang, w.-s.; loo, y.-y.; tan, c.-w.; ramzi, o.b. comparison of the microbiological quality and safety between conventional and organic vegetables sold in malaysia. front. microbiol. 2017, 8, 1433. [9].eni, a.o.; oluwawemitan, i.a.; solomon, o.u. microbial quality of fruits and vegetables sold in sango ota, nigeria. african journal of food science 2010, 4, 291296. [10]. olaimat, a.n.; holley, r.a. factors influencing the microbial safety of fresh produce: a review. food microbiol. 2012, 32, 119. [11]. who. fruit and vegetables for health: report of the joint fao. 2005. [12]. bernstein, n.; sela, s.; nederlavon, s. assessment of contamination potential of lettuce by salmonella enterica serovar newport added to the plant growing medium. j food prot 2007, 70, 1717-1722, doi:10.4315/0362-028x-70.7.1717. [13]. de giusti, m.; aurigemma, c.; marinelli, l.; tufi, d.; de medici, d.; di pasquale, s.; de vito, c.; boccia, a. the evaluation of the microbial safety of fresh ready‐to‐eat vegetables produced by different technologies in italy. j. appl. microbiol. 2010, 109, 996-1006. [14]. beuchat, l.r. ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. microbes and infection 2002, 4, 413-423. [15]. maffei, d.f.; silveira, n.f.d.a.; catanozi, m.d.p.l.m. microbiological quality of organic and conventional vegetables sold in brazil. food control 2013, 29, 226-230, doi:https://doi.org/10.1016/j.foodcont.2012.06.0 13. [16]. scallan, e.; hoekstra, r.m.; angulo, f.j.; tauxe, r.v.; https://doi.org/10.1016/j.foodcont.2012.06.013 https://doi.org/10.1016/j.foodcont.2012.06.013 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 140 widdowson, m.-a.; roy, s.l.; jones, j.l.; griffin, p.m. foodborne illness acquired in the united states—major pathogens. emerging infectious diseases 2011, 17, 7. [17]. who. who estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007-2015; world health organization: 2015. [18]. koluman, a.; dikici, a. antimicrobial resistance of emerging foodborne pathogens: status quo and global trends. critical reviews in microbiology 2013, 39, 57-69. [19]. gama, j.a.; zilhão, r.; dionisio, f. impact of plasmid interactions with the chromosome and other plasmids on the spread of antibiotic resistance. plasmid 2018, 99, 8288. [20]. carroll, a.c.; wong, a. plasmid persistence: costs, benefits, and the plasmid paradox. can. j. microbiol. 2018, 64, 293-304. [21]. talukder, a.; rahman, m.; chowdhury, m.m.h.; mobashshera, t.a.; islam, n.n. plasmid profiling of multiple antibiotic-resistant pseudomonas aeruginosa isolated from soil of the industrial area in chittagong, bangladesh. beni-suef university journal of basic and applied science 2021, 10, 1-7. [22]. wachtel, m.r.; whitehand, l.c.; mandrell, r.e. association of escherichia coli o157: h7 with preharvest leaf lettuce upon exposure to contaminated irrigation water. j. food protection 2002, 65, 18-25. [23]. adetunde, l.; sackey, i.; dombiri, d.d.; mariama, z.w. potential links between irrigation water microbiological quality and fresh vegetables quality in upper east region of ghana subsistence farming. annual research & review in biology 2015, 347-354. [24]. donkor, e.s.; lanyo, r.; kayang, b.b.; quaye, j.; edoh, d.a. internalisation of microbes in vegetables: microbial load of ghanaian vegetables and the relationship with different water sources of irrigation. pakistan journal of biological sciences: pjbs 2010, 13, 857-861. [25]. keraita, b.; drechsel, p.; konradsen, f. using on-farm sedimentation ponds to improve microbial quality of irrigation water in urban vegetable farming in ghana. water science and technology 2008, 57, 519525. [26]. yafetto, l.; adator, e.h.; ebuako, a.a.; ekloh, e.; afeti, f.y. microbial quality of raw beef and chevon from selected markets in cape coast, ghana. journal of biology and life science 2019, 10, 78. [27]. da silva, n.; taniwaki, m.h.; junqueira, v.c.a.; de arruda silveira, n.f.; okazaki, m.m.; gomes, r.a.r. microbiological examination methods of food and water: a laboratory manual; crc press: 2018. [28]. cheesbrough, m. district laboratory practice in tropical countries, part 2; cambridge university press: 2005. [29]. tafinta, i.; shehu, k.; abdulganiyyu, h.; rabe, a.; usman, a. isolation and identification of fungi associated with the spoilage of sweet orange (citrus sinensis) fruits in sokoto state. nigerian journal of basic and applied sciences 2013, 21, 193-196. [30]. oyeleke, s.b.; manga, s. essentials of laboratory practicals in microbiology. minna, nigeria: tobest publishers 2008, 36-75. [31]. adebayo-tayo, b.; odu, n.; anyamele, l.; igwiloh, n.; okonko, i. microbial quality of frozen fish sold in uyo metropolis. nature and science 2012, 10, 71-77. [32]. anwer, s.s.; ali, g.a.; hamadamin, c.z.; jaafar, h.y. isolation and identification of fungi from fast food restaurants in langa bazar. international journal of environment, agriculture and biotechnology 2017, 2, 238822. [33]. bukhari, m.; iqbal, n.; naeem, s.; qureshi, g.; naveed, i.; iqbal, a.; khatoon, n. a laboratory study of susceptibility of methicillin resistant staphylococcus aureus (mrsa). pakistan journal of medical sciences 2004, 20, 229-233. [34]. akbar, a.; anal, a.k. prevalence and antibiogram study of salmonella and staphylococcus aureus in poultry meat. asian pacific journal of tropical biomedicine 2013, 3, 163-168. [35]. andrup, l.; barfod, k.k.; jensen, g.b.; smidt, l. detection of large plasmids from the bacillus cereus group. plasmid 2008, 59, 139-143. [36]. smith, s.; aboaba, o.; odeigha, p.; shodipo, k.; adeyeye, j.; ibrahim, a.; adebiyi, t.; onibokun, h.; odunukwe, n. plasmid profile of escherichia coli 0157: h7 from apparently healthy animals. african journal of biotechnology 2003, 2, 322-324. [37]. akingbade, o.; balogun, s.; ojo, d.; afolabi, r.; motayo, b.; okerentugba, p.; okonko, i. plasmid food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 141 profile analysis of multidrug resistant pseudomonas aeruginosa isolated from wound infections in south west, nigeria. world appl sci j 2012, 20, 766-775. [38]. arise, a.; arise, r.; akintola, a.; idowu, o.; aworh, o. microbial, nutritional and sensory evaluation of traditional sundried okra (orunla) in selected markets in south-western nigeria. 2012. [39]. european-commission. microbiological criteria. availabe online: https://ec.europa.eu/food/safety/biologicalsafety/food-hygiene/microbiological-criteria_en (accessed on 28th may). [40]. ozoude, t.; igbokoyi, r.; florence, o.; eleanya, e. evaluation of pathogenic microorganisms that cause onion bulb rots in selected markets in abuja, nigeria. microbiology research journal international 2019, 29, 1-6. [41]. ofor, m.; okorie, v.; ibeawuchi, i.; ihejirika, g.; obilo, o.; dialoke, s. microbial contaminants in fresh tomato wash water and food safety considerations in southeastern nigeria. life sci. j 2009, 1, 80-82. [42]. opara, c.; okpe, c. aerobic bacteria flora associated with freshwater apple snail (pila ampullaceal) in kolo creek, ogbia, bayelsa state. 2019. [43]. abakari, g.; cobbina, s.j.; yeleliere, e. microbial quality of ready-toeat vegetable salads vended in the central business district of tamale, ghana. international journal of food contamination 2018, 5, 1-9. [44]. goja, a.m.; mahmoud, m. microbial quality of some vegetables sold in ed dueimtwon, sudan. pakistan journal of biological sciences: pjbs 2013, 16, 585-588. [45]. collins, c.; lyne, p.; grange, j. microbiological methods. 5th. and 2004, 391, 282. [46]. ait-mouheb, n.; mange, a.; froment, g.; lequette, k.; bru-adan, v.; maihol, j.-c.; molle, b.; wery, n. effect of untreated or reclaimed wastewater drip-irrigation for lettuces and leeks on yield, soil and fecal indicators. resources, environment and sustainability 2022, 8, 100053. [47]. tsafack, h.n.; azeufack, j.; kenfack, s.; tamouf, s.j.; tchoffo, a.m.; tonssie, f.f.; wamba, f.; temgoua, e. physicochemical, bacteriological and parasitological quality of water used to wash vegetables in dschang, west cameroon: health risk assessment. journal of health and environmental research 2022, 8, 1-8. [48]. chigor, c.b.; ibangha, i.-a.i.; nweze, n.o.; onuora, v.c.; ozochi, c.a.; titilawo, y.; enebe, m.c.; chernikova, t.n.; golyshin, p.n.; chigor, v.n. prevalence of integrons in multidrug-resistant escherichia coli isolates from waters and vegetables in nsukka and enugu, southeast nigeria. environmental science and pollution research 2022, 1-8. [49]. akoachere, j.-f.t.k.; tatsinkou, b.f.; nkengfack, j.m. bacterial and parasitic contaminants of salad vegetables sold in markets in fako division, cameroon and evaluation of hygiene and handling practices of vendors. bmc research notes 2018, 11, 1-7. [50]. tambekar, d.; mundhada, r. bacteriological quality of salad vegetables sold in amravati city (india). journal of biological sciences 2006, 6, 28-30. [51]. bishop, h.g.; onyowoicho, g. escherichia coli and staphylococcus aureus contaminations of carrots sold within zaria, nigeria and their antibiotic susceptibility pro¦ les. open access journal of science 2017, 1, 110-113. [52]. itohan, a.m.; peters, o.; kolo, i. bacterial contaminants of salad vegetables in abuja municipal area council, nigeria. malaysian journal of microbiology 2011, 7, 111-114. [53]. beshiru, a.; okoh, a.i.; igbinosa, e.o. processed ready-to-eat (rte) foods sold in yenagoa nigeria were colonized by diarrheagenic escherichia coli which constitute a probable hazard to human health. plos one 2022, 17, e0266059. [54]. oluboyo, o.b.; olojede, o.g.; akinseye, f.j.; akele, y.r.; oluboyo, a.o.; adewumi, f.a. bacterial contamination of some vegetables sold in major markets in ado-ekiti, nigeria. int. j. adv. res 2019. [55]. akinyemi, k.; smith, s.; oyefolu, a.b.; coker, a. multidrug resistance in salmonella enterica serovar typhi isolated from patients with typhoid fever complications in lagos, nigeria. public health 2005, 119, 321327. [56]. akinyemi, k.; iwalokun, b.; foli, f.; oshodi, k.; coker, a. prevalence of multiple drug resistance and screening of enterotoxin (stn) gene in salmonella enterica https://ec.europa.eu/food/safety/biological-safety/food-hygiene/microbiological-criteria_en https://ec.europa.eu/food/safety/biological-safety/food-hygiene/microbiological-criteria_en food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 christiana n. opara, uchenna c. okpe, christian k. anumudu, diversity, antibiogram and plasmid profile of microbial contaminants of ready to eat vegetables sold in bayelsa nigeria, food and environment safety, volume xxi, issue 2 – 2022, pag.129 142 142 serovars from water sources in lagos, nigeria. public health 2011, 125, 65-71. [57]. ezekiel, c.; olarinmoye, a.; jnr, j.o.; olaoye, o.; edun, a. distribution, antibiogram and multidrug resistance in enterobacteriaceae from commercial poultry feeds in nigeria. african journal of microbiology research 2011, 5, 294-301. [58]. salmanov, a.g.; ushkalov, v.o.; shunko, y.y.; piven, n.; vygovska, l.m.; verner, o.m.; kushnirenko, s. one health: antibiotic-resistant bacteria contamination in fresh vegetables sold at a retail markets in kyiv. 2021. [59]. ajiboye, a.e. assessment of bacterial contamination in ready-to-eat fruits and vegetables sold at oja-oba market, ilorin, nigeria. african journal of biomedical research 2021, 24, 203-209. [60]. dajcs, j.j.; thibodeaux, b.a.; marquart, m.e.; girgis, d.o.; traidej, m.; o'callaghan, r.j. effectiveness of ciprofloxacin, levofloxacin, or moxifloxacin for treatment of experimental staphylococcus aureus keratitis. antimicrob. agents chemother. 2004, 48, 1948-1952. [61]. breijyeh, z.; jubeh, b.; karaman, r. resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. molecules 2020, 25, 1340. [62]. guerra, b.; junker, e.; schroeter, a.; malorny, b.; lehmann, s.; helmuth, r. phenotypic and genotypic characterization of antimicrobial resistance in german escherichia coli isolates from cattle, swine and poultry. journal of antimicrobial chemotherapy 2003, 52, 489492. [63]. egwuenu, a.; obasanya, j.; okeke, i.; aboderin, o.; olayinka, a.; kwange, d.; ogunniyi, a.; mbadiwe, e.; omoniyei, l.; omotayo, h. antimicrobial use and resistance in nigeria: situation analysis and recommendations, 2017. 2018. [64]. tsado, e.; adesina, o.; oyeleke, s. a survey on the bacterial load of selected fruits and leafy vegetables in minna metropolis of niger state, nigeria. 2013. [65]. udoh, i.p.; eleazar, c.i.; ogeneh, b.o.; ohanu, m.e. studies on fungi responsible for the spoilage/deterioration of some edible fruits and vegetables. advances in microbiology 2015, 5, 285. [66]. kłapeć, t.; wójcik-fatla, a.; farian, e.; kowalczyk, k.; cholewa, g.; cholewa, a.; dutkiewicz, j. levels of filamentous fungi and selected mycotoxins in leafy and fruit vegetables and analysis of their potential health risk for consumers. annals of agricultural and environmental medicine 2021, 28, 585-594. [67]. roberts, m.a.; crawford, d.l. use of randomly amplified polymorphic dna as a means of developing genus-and strainspecific streptomyces dna probes. appl. environ. microbiol. 2000, 66, 2555-2564. [68]. carattoli, a. plasmids and the spread of resistance. international journal of medical microbiology 2013, 303, 298-304. [69]. bennett, p. plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. british journal of pharmacology 2008, 153, s347-s357. [70]. vaidya, v.k. horizontal transfer of antimicrobial resistance by extended-spectrum β lactamase-producing enterobacteriaceae. journal of laboratory physicians 2011, 3, 037042. [71]. ugochukwu nmesirionye, b.; ugwu, c.t.; martins nworie, k. antibiotic susceptibility and plasmid profile of multidrug resistant uropathogenic serratia marcescens. journal of microbiology & infectious diseases 2022, 12. [72]. fitzpatrick, d.a. horizontal gene transfer in fungi. fems microbiol. lett. 2012, 329, 1-8. table 2 macroscopic and microscopic characteristics of fungal isolates. doi: https://doi.org/10.4316/fens.2022.016 171 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 171 184 evaluation of soil salinity of the fetzara lake region (north-east algeria) * faouzi zahi 1 , abdelmalek drouiche 1 , fethi medjani 2 , abdelmalek lekoui 1 , mohamed djidel 2 1 geological engineering laboratory, university of jijel, algeria, e-mail, zahi_faouzi@yahoo.fr, drouiche_malek@yahoo.fr, lekouim121@gmail.com 2 laboratory of geology of the sahara, university kasdi merbah ouargla, algeria, e-mail, medjanifethi@yahoo.fr, djidelm@yahoo.fr *corresponding author: received 24th june 2022, accepted 28th july 2022 abstract: fetzara lake, located at 18 km southwest of annaba city, is one of the largest lakes in the extreme northeastern algeria. in 2002, the ramsar convention classified it among the wetlands of international importance. in this study, we aim to evaluate soil properties of fetzara lake region, which are very sensitive to salinization phenomena, and their evolution according to depth. a sampling companion was carried out on the first two superficial levels 0 to 25 cm and 25 to 50 cm. a total of 24 samples, gathered from 12 sites distributed around the lake perimeter, were analyzed for their physical and physico-chemical properties. results show that the majority of the soils samples are classified as saline soils (esp ≤ 15%, ec > 4 ms cm-1), with a highly variable electrical conductivity (6.52 to 24.3 ms cm-1), reaching its maximum in both northeastern (wadi zied town) and southeastern part of the lake (cheurfa town). moreover, the soil solutions are characterized by the abundance of ions such as cl-, so4 2-, na+ and mg2+, giving a dominant sodium chloride facies in most of the analyzed samples. keywords: sodization, ionic exchanges, alkalinity, saline soils, soil quality, wetlands 1. introduction soil salinity becomes a major problem in global agriculture when soil and environmental factors contribute to the accumulation of salts in soil layers to a level that negatively affects crop production. worldwide, more than 800 million hectares of land are estimated to be affected by salt [1-2]. this is usually due to natural processes such as the accumulation of salt by rainfall or by the weathering of rocks, known as primary salinization. secondary salinization can result from human activities such as urbanization, agricultural practices and irrigation of cultivated land [3-9]. in the mediterranean region, salt accumulation is a process favoured by the ecological conditions of the region, controlled above all by soil and climate variability and the expected increase in irrigated areas and the scarcity of good quality water [10-13]. this accumulation and modification of the sorption complex (exchange phenomena) leads to a substantial degradation and transformation of the physical and chemical properties of soils, resulting in a reduction the productivity of many agricultural crops, including most vegetable crops, which present low tolerance to soil salinity [9, 14-16]. in algeria, several wetlands concentrated mainly in the northeastern part of the http://www.fia.usv.ro/fiajournal mailto:zahi_faouzi@yahoo.fr mailto:drouiche_malek@yahoo.fr mailto:medjanifethi@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 172 country, along the mediterranean coastal suffer from soil salinization [17]. the fetzara lake region is one of the most important wetlands in northeast algeria and has been classified as a ramsar wetland since 2002. its vast surface and its relatively temporal character make it a typical wetland of the mediterranean region [18] and an important ecosystem that requires surveillance of both its quality and quantity. several studies [7, 17, 19-24] have generally mentioned the salinization problem in fetzara lake region, especially in the northeastern and southeastern parts, but without providing details about the mechanisms controlling this phenomenon. therefore, a suitable study on the soil salinity of this region seems very useful and provides more information about this problem. the assessment of soil quality and salinization requires the determination of their physico-chemical properties, which are quantitatively measurable providing a complete description of the major processes of their functioning [16, 25-26]. the risk of soil degradation can be assessed by two important criteria, namely: the soluble salt content that reflects salinity in the strict sense while the percentage of exchangeable sodium in the soil reflects sodization [8-9, 14]. the sodization phenomenon is the process of sodium exchange on the sorption complex of clays and their progressive saturation in sodium ion [27]. the objective of this research is to study the quality and salinization of soils in the fetzara lake region, by interpreting the results obtained from the analysis of physicochemical parameters using analytical and statistical approaches (pca) as well as calculating indexes relating to the soil classification, i.e. the exchangeable sodium percentage (esp) and the sodium adsorption ratio (sar). 2. matherials and methods 2.1. study area fetzara lake is located in north-east algeria and distant of 18 km south-west of annaba city (fig. 1), covers an area of about 18600 ha stretching for 17 km long from east to west and 13 km wide from north to south [23]. the lake collects water from an important watershed of about 515 km2, it plays a major role in flood control, sediment and nutrient retention and recharge of the water table [28-29]. throughout the year, a main canal drains the water from the lake, which is evacuated into the oued meboudja, then into the oued seybouse, which in turn flows into the mediterranean sea. geologically, the fetzara region is characterized by two distinct lithologies, metamorphic and sedimentary. metamorphic formations outcrop in the edough massif and its surroundings area, they consist mainly of gneiss and metapelites (alumaceous shale unit). sedimentary formations occupy the rest of the study area and consist of marls, clays, cretaceous sandstone (cretaceous flysch), and oligo-miocene sandstone [30-31]. several studies on agricultural development have been carried out on the soils of the fetzara region [7, 20-21, 28]. these studies classified soils into four classes: poorly developed soils, vertisols, hydromorphic soils and halomorphic soils. 2.2. procedure the great variability of salt content in soil from one point to another has made more delicate both sampling and laboratory analyzes to study their spatial distribution. in the present case, soil sampling was carried out on the first two surficial levels (0 to 25 cm and 25 to 50 cm) where the most important ionic exchanges take place. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 173 a total of 24 samples were taken from twelve (12) points around fetzara lake (fig. 2). they were air dried, crushed and sieved to 2 mm to obtain the fine soil that will be object for all chemical, physicochemical and physical analyzes, namely: specific density, total porosity, total organic carbon (toc) and organic matter (om), ph, electric conductivity (ec) and soluble salts (ca2+, mg2+, na+, k+, cl-, hco3 -, so4 2-, co3 2-) ( table 1). fig. 1. location map of fetzara lake watershed fig. 2. soil sampling inventory map food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 174 table 1 physico-chemical soil analysis methods [32] parameters symbols unit méthods ph ph / ph meter electrical conductivity ec ms cm-1 conductimeter total organic carbon toc % modified anne method organic matter om % om = % toc * 1.72 specific density d g cm-3 paraffine total porosity p % p = (1da / dr) * 100 calcium, magnesium ca2+, mg2+ meq dm-3 complexometry chlorides cl meq dm-3 mohr with agno3. sulfates so4 2 meq dm-3 gravimetry with bacl2 bicarbonates, carbonates hco3 -, co3 2 meq dm-3 sulfuric acid (h2so4). sodium and potassium na+, k+ meq dm-3 flame emission spectrometry particle size measurement is one of the classical ways of studying soils, defining the texture, which in turn determines in part the physical and physicochemical properties of the environment [33]. the soil texture is the reflection of the proportion of sand particles, silt and clay found there [34-35]. these proportions determine the pores size and, largely, the ability to retain moisture and nutrients. soil particles less than 2 mm in size are divided into three categories: sand (the largest particle), silt and clay (the smallest). they are classified according to the ratios between different particles. for example, soils classified as "sandy" consist of more than 50 to 55% of sand, while "clayey" soils have a clay content of 20% or more [36]. the soil capacity to exchange cations is called cation exchange capacity (cec), arises because of the charge associated with clay particles and other soil components [34]. in this study, since the cec was not measured, an empirical equation proposed by us salinity laboratory [37] was used, the latter expressing a correlation between the percentage of exchangeable sodium (esp) and the values of the sodium adsorption ratio (sar) of soil solutions. it has been used by several authors [38-40] and can be written by the following formula (eq. 1): (eq. 1) this empirical equation satisfies the theoretical limit condition for high sar and esp values. however, the data used in the calculation of this ratio was sar < 65 and esp < 50. the sodium adsorption ratio (sar) was developed to describe empirically the imbalance between divalent cations (calcium and magnesium) and sodium for assessing the sodization risk of the sorption complex [8, 41]. it was calculated according to the following formula (eq. 2) where all concentrations are expressed in (meq dm-3): (eq. 2) there are different methods for classifying the saline soils; the american, french, russian and fao classification. among these classifications and the most used is the classification of ussl [37] (table 2), since it is based on easy-to-obtain parameters like electrical conductivity (ec) and exchangeable sodium percentage (esp). https://www.google.dz/search?hl=fr&tbo=p&tbm=bks&q=inauthor:%22cl%c3%a9ment+mathieu%22 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 175 table 2 classification of saline soils according to ussl [37] parameters ec ≤ 4 ms cm -1 ec > 4 ms cm -1 esp ≤ 15% non-saline and non-alkaline soils saline soil esp > 15% alkaline soils alkalinesaline soils 3. results and discussion 3.1. physical and chemical properties of soils soils in the fetzara region are weakly alkaline to acid ph (5.97 to 8.10) containing 0.4 to 3.47% of organic matter. they are characterized by an electrical conductivity ranging from 6.52 to 24.3 ms cm-1. total porosity shows an average value of 23% and 22% for the first and second level respectively, while the permeability coefficient is less than 2 cm hr-1. 3.1.1. particle size the obtained results of particle size analysis carried on soils from the fetzara lake region are plotted into the triangular diagram (fig. 3) in order to determine their textures. the majority of samples with average values of 69.34% for the first level (0-25 cm) and 58.67% for the second (2550 cm) show the dominance of silt fraction. this excess of silt fraction and clay deficiency conducts to a massive structure formation, and therefore the mediocre physical properties of soils [3435, 42]. the sandy fraction shows an average of 26.46% and 35.83% for the first and second level respectively. however, sandy fraction is greater than 50% in both the sample s4 (level 0-25 cm) located in the southern part of fetzara lake, near of wadi el hout, and s'4, s'8 (level 25-50 cm). soils with sandy fraction > 50% are generally, well ventilated, of low coherence, not or very weakly structured, poor in nutritive elements, and have a weak capacity of cationic exchange [34-35, 42]. the clay fraction shows low averages, 4.2% for the first level and 5.5% for the second level where the maximum value is registered in the south of berrahal town (18% sample s7). these clayey soils are chemically rich, but exhibit mediocre physical properties (impervious and weakly aerated soil) [34-35, 42]. 3.1.2. soil ph hydrogen potential (ph) is one of the most important properties of the soil solution [34], it reflects the content of free acid hydrogen ions in a soil solution generated either by pure water (phh2o) or potassium chloride-rich water (phkcl). the values of phh2o for the upper level (025 cm) vary between 6.37 and 7.67 with an average of 7.27. however, they oscillate between 7.02 and 8.10 with an average of 7.44 for the lower level (25-50 cm). accordingly, with reference to gaucher [43], these ph values could be qualified as weakly acidic to alkaline. the maximum values of phkcl reach 7.08 to 7.33, and 6.67 to 6.83 for the minimums for the first and second level respectively. obviously, phkcl < phh2o, the value of the difference indirectly reflects the biological state of the soil; (1) a difference of 0.2 to 0.4 indicates a heavy soil with a weakness of microbial activity. (2) a difference of 0.4 to 0.6 indicates normal microbial activity. (3) a difference of 0.6 to 0.8 indicates an excellent structure, conducive to a rapid organic matter transformation [32]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 176 fig. 3. soil texture of fetzara lake region: a first level, b second level for soils of the fetzara lake region, this difference fluctuates from one point to another, it varies from 0.3 to 0.8 (an average of 0.6) for the 24 samples, which shows that these soils are characterised by low to normal microbial activity. acidic or alkaline reaction of a soil is controlled by the preponderant presence of acidic salts, contained particularly in the humus, relative to basic salts outcome from limestone; it would be neutral when acids and bases equilibrate each other. 3.1.3. electrical conductivity (ec) measured electrical conductivities on the saturated paste extract (soil solution) vary between 6.52 and 24.3 ms cm-1 (average of 11.54 ms cm-1, n=12) for the first level (0-25 cm), and from 8.03 to 20.2 ms cm-1 (average of 13.96 ms cm-1, n=12) for the second (25-50 cm). the highest values exceeding 16 ms cm-1 are recorded at the stations 3, 6, 9 and 10 (fig. 4). the increase of the electrical conductivity according to depth has been observed in the majority of samples, except sample (s10), due to the trapping of salts in the silty parts of the upper level (fig. 4). soil classification proposed by ussl [37], enabled us to identify the distribution of samples by class (table 3). since the majority of soils in the fetzara lake region are affected by salinity with an evolution of the latter depending on the depth, three classes have been highlighted: 25% of samples are moderately saline soils (4 < ec ≤ 8 ms cm-1), 41.67% are saline soils (8 < ec ≤ 16 ms cm-1) and 33.33% are very salty soils (ec > 16 ms cm-1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 177 fig. 4. variation of soil electrical conductivity (ec) table 3 soil classification as a function of electrical conductivity ec (ms cm-1) level (0 – 25 cm) level (25 – 50 cm) appreciation ec ≤ 2 / / soils are not salty 2 < ec ≤ 4 / / soils are slightly salty 4 < ec ≤ 8 s1, s2, s4, s7, s8, s12 / soils are moderately salty 8 < ec ≤ 16 s5, s11 s1, s2, s4, s5, s7, s8, s11, s12 soils are salty ec > 16 s3, s6, s9, 10 s3, s6, s9, s10 soils are very salty 3.1.4. soluble salts soil salinity reflects an excessive concentration of soluble salts (saline soils), sodium adsorbed (sodium or alkaline soils) or alkaline-saline soils. the soluble salts concerned are mainly ca2+, mg2+, k+, na+, cl-, so4 2and hco3 -. the dynamic of these salts in soil is related to the hydrous and structural functioning of soils, to the water and geochemical conditions at the soil profile limits, to the supply of irrigation water and the fluctuations of the water table [44]. the main results of the chemical analyze performed on the extract of the saturated paste are summarized in the table below (table 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 178 table 4 chemical analyzes of soil solutions of the fetzara lake region levels soluble salts (meq dm -3 ) maximum minimum average standard deviation 0-25 cm ca2+ 14.40 2.80 6.18 3.51 mg2+ 31.00 0.60 13.27 10.61 na+ 29.90 1.24 13.86 7.10 k+ 3.66 0.64 1.88 0.90 cl240.00 40.00 105.00 67.27 so4 2 61.25 6.25 26.24 20.85 hco3 4.00 1.20 2.13 0.72 25-50 cm ca2+ 16.50 3.00 6.09 3.89 mg2+ 34.40 1.40 14.54 10.19 na+ 24.29 2.49 16.35 6.13 k+ 7.32 1.00 2.33 1.64 cl220.00 60.00 139.17 49.74 so4 2 61.98 14.50 31.09 15.96 hco3 3.60 0.60 2.29 0.82 3.2. chemical facies of soil solutions the most dominant chemical facies of the soil solutions in the fetzara lake region have been determined using the piper's triangle diagram (fig. 5), where the following remarks have been highlighted: on the anion’s triangle, the chlorides are dominant, plotted samples are grouped closely to chlorinated pole. they show 79.29% and 80.74% of the total sum of anions respectively for the two levels (0-25 cm and 25-50 cm). the sulphates are less important than chlorides, they exhibit 18.59% for the first level and 17.78% for the second. however, bicarbonates have the lowest percentage with a maximum of 4.5%. on the cation’s triangles, sodium is the most important ion, reaching 47.81% and 50.51% of the total sum of cations for the first and second level respectively. magnesium has percentages of 31.52% and 32.70%, while calcium exhibits the lowest percentages with 20.67% and 16.79% for the first and second level respectively. as a result, the soil solutions are characterized by a dominance of the sodium chloride facies for most soil samples (fig. 5), with the exception of samples s6, s'6, s9 and s'9 where the facies become magnesian chloride (fig. 5a). this character of the soil solutions allows to the successive precipitation of minerals, which modifies its composition and determines different soil evolution pathways as a function of the relative abundance of the different major ions in the starting solution. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 179 fig. 5. chemical facies of soil solutions: a first level, b second level 3.3. statistical study of chemical analyzes of soil solutions the principal component analysis (pca) was performed on 12 variables (ec, ph, ca2+, mg2+, na+, k+, cl-, hco3 -, so4 2-, moisture (h%), total organic carbon (toc) and organic matter (om)) from 24 samples (individuals). soil analysis has been interpreted by the software (spad version 5.5). for statistical data processing, the first three factorial axes (f1, f2 and f3) reach 74.53% of the total variance: f1= 45.89%, f2=18.01% and f3=15.14%. first factorial plane f1f2: the observation of the correlation circle formed by the two factorial axes f1 and f2 is presented in figure 6. the first axis groups the soluble salts (ca2+, mg2+, na+, cl-, so4 2and hco3 -) on its positive part; those are well correlated with the electrical conductivity (ec) while all the latter are opposed to the ph, which represents the soil acidity. according to mcgeorge [45-46], the ph decreases with increasing concentrations of nacl. he speculated that the ph of the soil increased as the salt concentration of the soil solution was reduced, due to increased hydrolysis of the sodium clay complex. salinity increases the ionic strength of the soil solution and consequently suppresses the activity coefficient of ions in solution. this would increase the ph values, but increasing ionic strength mainly decreases the ph values because salinity decreases the junction potential. as a result, this first axis reflects the salinization phenomena affecting some types of soil influenced by water intake of salt irrigation, climate type or specific hydrological conditions (low leaching near the aquifer water table). the second axis f2 (18.01% of the variance) contrasts carbon and organic matter with ph and potassium k+. this could be explained by two phenomena: (1) the dissolution of carbonates, which leads to co2 consumption causing the rise in ph. this type of mechanism allows the transient increase in ph leading to the dissolution of organic matter [47]. (2) the fixation of k+ by certain types of fine textured soils (clay texture, clay-silt ... etc.) [48]. the distribution of individuals on the plane (f1f2) allowed the discrimination of two groups of associations (fig. 6-b). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 180 the first group (g1) is characterized by highly mineralized solutions that oppose the second group (g2) which represents soil solutions of less mineralized basic character. fig. 6. principal component analysis of soil solutions according to plane f1f2 second factorial plane f1f3: (61% of the variance) (fig. 7), the first axis has been discussed above, reflects the overall concentration of soil solutions (axis of mineralization). the third axis represents in its positive part the soil solutions rich in potassium and an alkaline character. potassium could be fixed in soils by certain clay minerals (fig. 7-a). fig. 7. principal component analysis of soil solutions according to plane f1f3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 181 3.4. typology of saline soils the variation of the exchangeable sodium percentage (esp) as a function of the electrical conductivity measured on the saturated paste extract and according to the classification proposed by the ussl [37] is represented graphically in the following figure (fig. 8). fig. 8. classification of saline soils according to ussl [37] the majority of the soils analyzed from the fetzara lake region (about 87.5%), are classified as saline soils (esp ≤ 15%, ec > 4 ms cm-1) (fig. 8), these soils are characterized by a high content of salts [14, 47, 49-51], and are generally flocculated, which contributes to maintain a good soil structure and a good water and air circulation [14]. they are characterized by an electrical conductivity of the saturated paste extract > 4 ms cm-1, ph < 8.5 and an exchangeable sodium percentage < 15%. three samples of the lower level (25-50 cm) are part of the alkaline-saline soils (esp > 15%, ec > 4 ms cm-1) and are characterized by an electrical conductivity of the saturated paste extract > 4 ms cm-1 and ph > 8.5. according to marlet and job [47], they contain enough soluble salts and exchangeable sodium > 15%. this leads to low structural stability of the soil [48, 5052]. 4. conclusions pedological studies carried out on the soils of the fetzara lake region have led to identify four classes namely: poor soils, vertisols, hydromorphic soils and halomorphic soils. analytical results and their interpretation by the different methods led to the conclusion that the soils in the fetzara region are weakly alkaline to acid ph (5.97 to 8.10) containing 0.4 to 3.47% of organic matter. their average total porosity is about 23% for the first level (0-25 cm) and 22% for the second food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 182 (25-50 cm), while the permeability coefficient is less than 2 cm hr-1. the particle size analyzes show the dominance of the silty fraction in the majority of the samples, with an averages value of 69.34% and 58.67% for the first and second level respectively. the majority of soils are classified as saline soils (esp ≤ 15%, ec > 4 ms cm-1), with a very variable electrical conductivity (6.52 to 24.3 ms cm-1), reaches its maximum in the northeastern and southeastern parts of fetzara lake. the soil solutions are characterized by the abundance of ions (cl-, so4 2-, na+ and mg2+) conducting to a dominance of sodium chloride facies in the most samples analyzed. the principal component analysis (pca) indicates a strong correlation between soluble salts (ca2+, mg2+, na+, cl-, so4 2 and hco3 -) and electrical conductivity (ec), which corresponds to the salty soils of the south-east (cheurfa town) and the north-east (wadi zied town) of the lake. while the ensemble is opposed to the ph, which represents the soil's acidity, mainly observed in the north-west (berrahal town). this soil salinity would be mainly controlled by the seasonal variation of the lake water level, the strong evaporation of the water, particularly in summer, which causes the precipitation of mineral salts, and the phenomena of ion exchange between the sorption complex and the soil solutions. finally, in order to protect the soils of the fetzara lake region against salinization and to minimise their degradation, a continuous monitoring of the irrigation water quality and an improvement of the drainage system of the agricultural land around the lake are recommended. 5. acknowledgments thanks to the geomatics team of geology of the sahara laboratory, university kasdi merbah, ouargla, algeria, for collaboration. the authors appreciate the significant contributions made by the journal’s editors and reviewers to manage and review this article. 6. references [1]. fao (food and agriculture organization). “fao land and plant nutrition management service”, (2008). http://www.fao.org/ag/agl/agll/spush. [2]. bharti n, yadav d, barnawal d, maji d, kalra a., “exiguobacterium oxidotolerans, a halotolerant plant growth promoting rhizobacteria, improves yield and content of secondary metabolites in bacopa monnieri (l.) pennell under primary and secondary salt stress”. world journal of microbiology and biotechnology, 29: 379-387. (2013) [3]. barbiero l, valles v, regeard a, cheverry c., “residual alkalinity as tracer to estimate the changes induced by forage cultivation in a non-saline irrigated sodic soil”. agricultural water management, 50: 229-241, (2001) [4]. smedema lk, shiati k., “irrigation and salinity: a perspective review of the salinity hazards of irrigation development in the arid zone”. irrigation and drainage systems, 16 : 161174, (2002) [5]. ben hassine h., “effets de la nappe phréatique sur la salinisation des sols de cinq périmètres irrigués en tunisie”. étude et gestion des sols, 12 (4): 281-300, (2005) [6]. rengasamy p., world salinization with emphasis on australia. journal of experimental botany, 57 (5) : 1017-1023, (2006) [7]. djamaï r., “contribution à l’étude de la salinité des sols et des eaux du système endoréique du lac fetzara (nord-est algérien). approche géochimique et évolution spatio-temporelle des phénomènes”. thèse de doctorat d’état, institut national agronomique d’alger, algérie, (2007) [8]. kitamura y, yang sl, shimizu k., “secondary salinization and its countermeasures”. in: tsunekawa a., liu g., yamanaka n., du s. (eds) restoration and development of the degraded loess plateau, china". ecological research monographs. springer, tokyo, (2014) [9]. machado rma, serralheiro rp., “soil salinity: effect on vegetable crop growth. management practices to prevent and mitigate soil salinization”. horticulturae, 3 (2): 1-13, (2017) [10]. zalidis g, stamatiadis s, takavakoglou v, eskridge k, misopolinos n., “impacts of agricultural practices on soil and water quality in the http://www.fao.org/ag/agl/agll/spush food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 183 mediterranean region and proposed assessment methodology”. agriculture, ecosystems & environment, 88 (2): 137-146, (2002) [11]. aragües r., urdanoz v, çetin m, karda c, daghari h, ltifi w, lahlou m, douaik a., “soil salinity related to physical soil characteristics and irrigation management in four mediterranean irrigation districts”. agricultural water management, 98 (6): 959-966, (2011) [12]. abbas a, khan s, hussain n, hanjra ma, akbar s., “characterizing soil salinity in irrigated agriculture using a remote sensing approach”. physics and chemistry of the earth, parts a/b/c journals, 55-57: 43-52, (2013) [13]. li p, wu j, qian h., “regulation of secondary soil salinization in semi-arid regions: simulation research in the nanshantaizi area along the silk road, northwest china”. environmental earth sciences, 75: 698, (2016) [14]. ouardi j., “dégradation des sols et de la nappe des périmètres irrigués du tadla marocain par salinisation et sodisation”. thèse de doctorat, université cadi ayyad marrakech, maroc, (2005) [15]. bünemann ek, bongiorno g, bai z, creamer re, de deyn g, goede r…brussaard l., “soil quality a critical review”. soil biology and biochemistry, 120: 105125, (2018) [16]. nunes fc, alves lj, carvalho c, gross e, soares tm, prasad m., “soil as a complex ecological system for meeting food and nutritional security”. climate change and soil interactions, 9 : 229-269, (2020) [17]. fetnaci i, beddiar a, hamel t., le lac fetzara (nord-est algérien): biodiversité floristique et menaces potentielles. flora mediterranea, 29 : 227-245, (2019) [18]. boumezbeur a., “fiche descriptive sur les zones humides ramsar, lac de fetzara, wilaya de annaba (algérie) ”. direction générale des forêts, (2003) [19]. joleau l., “geological survey of bone (annaba)-calle (el kala)”. bulletin of the geological service of algeria, 2 (12) : 199, (1936) [20]. durand j., ”premiers résultats de l’étude des sols du lac fetzara”. document inédit ; service d’etude des sols, alger, (1950) [21]. djamaï r., “contribution à l’étude de la salinité des sols et des eaux du lac fetzara (annaba) ”. thèse de magister, institut national agronomique, alger, (1993) [22]. habes s, djabri l, hani a, bouhsina s, mudry j., “quantification des apports et des exores d’un lac : cas du lac fetzara, région de annaba (nord-est algérien)”. synthèse : revue des sciences et de la technologie, 24 : 21-28, (2012) [23]. zahi f, djamai r, chaab s, djabri l, drouiche a, medjani f., “dynamique de la nappe et qualités physico-chimiques des eaux souterraines du lac fetzara (nord-est algérien) ”. synthèse : revue des sciences et de la technologie, 26 : 86-95, (2013) [24]. halimi s, rechachi h, bahroun s, mizane e, daifallah t., “assessment of groundwater salinity and risk of soil degradation in quaternary aquifer system. example: annaba plain, algeria n-e”. journal of water and land development, 36 (3): 57-65, (2018) [25]. coll p., le velly r, le cadre e, villenave c., “la qualité des sols : associer perceptions et analyses des scientifiques et des viticulteurs”. étude et gestion des sols, 19 (2): 79-89, (2012) [26]. rahoui m, soudi b, badraoui m, marcoen jm, benzakour m., “situation actuelle de la qualité des sols et des eaux dans le périmètre irrigué des doukkala”. séminaire ‘intensification agricole et qualité des sols et des eaux’, rabat, 13-24, (2000) [27]. imadi sr, shah sw, kazi ag, azooz mm, ahmad p., “phytoremediation of saline soils for sustainable agricultural productivity”. plant metal interaction, 18: 455-468, (2016) [28]. zahi f., “contribution to hydrochemical study of the fetzara lake sub-watershed (northeast algeria). geochemical approach and functioning of soils and waters”. phd thesis, badji mokhtar university, annaba, (2014) [29]. mellouk k, aroua n., “the fetzara lake, a fragile wetland threatened by the urban growth of annaba city (western algeria)”. journal of mediterranean geography, 125: 133140, (2015) [30]. hammor d, lancelot j., “métamorphisme miocène de granites panafricains dans le massif de l’edough (nord-est de l’algérie)”. comptes rendus de l'académie des sciences, 327: 391-396, (1998) [31]. laouar r, boyce aj, ahmed-said y, ouabadi a, fallick ae, toubal a., “stable isotope study of the igneous, metamorphic and mineralized rocks of the edough complex, annaba, north-east algeria”. journal of african earth sciences, 35 (2) : 271-283, (2002) [32]. mathieu c, pieltain f., “analyse chimique des sols : méthodes choisies”. tec & doc, 387, (2003) [33]. gobat jm, grosvernier ph, matthey y, buttler a., “un triangle granulométrique pour les tourbes : analyse semiautomatique et représentation graphique”. science du sol, 29 (1) : 23-35, (1991) https://econpapers.repec.org/article/eeeagiwat/v_3a98_3ay_3a2011_3ai_3a6_3ap_3a959-966.htm https://econpapers.repec.org/article/eeeagiwat/v_3a98_3ay_3a2011_3ai_3a6_3ap_3a959-966.htm https://econpapers.repec.org/article/eeeagiwat/v_3a98_3ay_3a2011_3ai_3a6_3ap_3a959-966.htm https://econpapers.repec.org/article/eeeagiwat/v_3a98_3ay_3a2011_3ai_3a6_3ap_3a959-966.htm https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/author/7004011331/ron-g-m-de-goede https://www.sciencedirect.com/author/7004011331/ron-g-m-de-goede https://www.sciencedirect.com/science/article/pii/s0038071718300294#! https://www.sciencedirect.com/science/journal/00380717 https://www.sciencedirect.com/science/journal/00380717/120/supp/c https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/article/pii/b9780128180327000096#! https://www.sciencedirect.com/science/book/9780128180327 https://www.sciencedirect.com/science/book/9780128180327 https://www.sciencedirect.com/science/book/9780128031582 https://www.google.dz/search?hl=fr&tbo=p&tbm=bks&q=inauthor:%22cl%c3%a9ment+mathieu%22 https://www.google.dz/search?hl=fr&tbo=p&tbm=bks&q=inauthor:%22fran%c3%a7oise+pieltain%22 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 faouzi zahi, abdelmalek drouiche, fethi medjani, abdelmalek lekoui, mohamed djidel, evaluation of soil salinity of the fetzara lake region (north-east algeria), food and environment safety, volume xxi, issue 2 – 2022, pag. 171 – 184 184 [34]. pepper il, brusseau ml., “physicalchemical characteristics of soils and the subsurface”. environmental and pollution science (3rd edition), 2: 9-22, (2019) [35]. angst g, pokorný j, mueller cw, prater i, preusser s, kandeler e…angst s., “soil texture affects the coupling of litter decomposition and soil organic matter formation”. soil biology and biochemistry, 159:108302, (2021) [36]. ssds (soil survey division staff). “soil survey manual. united states department of agriculture”. agriculture handbook, 18: 603, (1993) [37]. ussl (u. s. salinity laboratory). “diagnosis and improvement of saline and alkali soils”. usda. handbook, 60, washington, 160, (1954) [38]. cheverry c., “exemple d’application des travaux de 1’us salinity laboratory (19631968) sur l’alcalinisation de sols soumis à l’action d’eaux bicarbonatées”. cahier orstom, série pédologique, 10 (2) : 193-203, (1972) [39]. saidi d, le bissonnais y, duval o, daoud y, tessier d., “estimation et signification de la capacité d’échange cationique des sols salés du cheliff (algérie) ”. etude et gestion des sols, 15 (4) :241-253, (2008) [40]. seilsepour m, rashidi m, khabbaz bg., “prediction of soil exchangeable sodium percentage based on soil sodium adsorption ratio”. american-eurasian journal of agricultural & environmental sciences, 5 (1): 1-4, (2009) [41]. marohn c, distell a, dercon g, wahyunto tr, noordwijk mv, cadisch g., “impacts of soil and groundwater salinization on tree crop performance in posttsunami aceh barat, indonesia”. natural hazards and earth system sciences, 12: 2879-2891, (2012) [42]. duchaufour p, faivre p, poulenard j, gury m., “introduction to soil science”. 7th edition, dunod, paris, france, (2018) [43]. gaucher g., “traité de pédologie agricole, le sol et ses caractéristiques agronomiques”, paris, dunod, (1968) [44]. douaoui a, gascuel-odoux c, walter c., “infiltrabilité et érodibilité de sols salinisés de la plaine du bas chéliff (algérie) ”. étude et gestion des sols, 4 (11): 379-392, (2004) [45]. mcgeorge, w.t. “the measurement and significance of hydroxyl-ion concentrations in alkaline-calcareous soils”. arizona agricultural experimental station technical bulletin, 57: 239271, (1935) [46]. mcgeorge wt., “factor contributing to the reaction of soils and their ph measurement”. arizona agricultural experimental station technical bulletin, 78: 95-126, (1938) [47]. marlet s, job jo., “processus et gestion de la salinité des sols. in : tiercelin j.r. traité d’irrigation”, 2ème édition. technique & documentation, lavoisier, (2006) [48]. assa ad., “phénomène de sélectivité d'échange cationique dans certains minéraux argileux : 2. les sites d'adsorption du potassium”. cahiers orstom. série pédologie, 14 (4) : 279286, (1976) [49]. aubert g., “observations sur les caractéristiques, la dénomination et la classification des sols salés ou salsodique”. cahier orstom, série pédologie, 20 (1) : 73-78, (1983) [50]. sparks dl., “the chemistry of saline and sodic soils”. environmental soil chemistry (2ed edition), 10: 285-300, (2003) [51]. yan n, marschner p, cao w, zuo c, qin w., “influence of salinity and water content on soil microorganisms”. international soil and water conservation research, 3 (4): 316323, (2015) [52]. vance wh, mckenzie bm, tisdall jm., “the stability of soils used for cropping in northern victoria and southern new south wales”. australian journal of soil research, 40 (4): 615624, (2002) https://www.sciencedirect.com/science/article/pii/b9780128147191000021#! https://www.sciencedirect.com/science/article/pii/b9780128147191000021#! file:///c:/users/kadir/appdata/roaming/microsoft/word/environmental%20and%20pollution%20science%20(3rd%20edition) file:///c:/users/kadir/appdata/roaming/microsoft/word/environmental%20and%20pollution%20science%20(3rd%20edition) https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/article/abs/pii/s0038071721001759?dgcid=rss_sd_all#! https://www.sciencedirect.com/science/journal/00380717 https://www.sciencedirect.com/science/journal/00380717/159/supp/c https://www.nrcs.usda.gov/wps/portal/nrcs/detail/national/nedc/training/soil/?cid=nrcs142p2_054262 https://www.nrcs.usda.gov/wps/portal/nrcs/detail/national/nedc/training/soil/?cid=nrcs142p2_054262 https://www.sciencedirect.com/science/article/pii/b9780126564464500104#! https://www.sciencedirect.com/science/book/9780126564464 https://www.sciencedirect.com/science/book/9780126564464 https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/article/pii/s209563391530112x#! https://www.sciencedirect.com/science/journal/20956339 https://www.sciencedirect.com/science/journal/20956339 https://www.sciencedirect.com/science/journal/20956339/3/4 1. introduction doi: https://doi.org/10.4316/fens.2023.001 5 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 5 14 influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value * anastasiia shevchenko 1 , svitlana litvynchuk 2 1 department of bakery and confectionery goods technologies, national university of food technologies, kyiv, ukraine, nastyusha8@ukr.net 2 department of physics, national university of food technologies, kyiv, ukraine, litvynchuk@nuft.edu.ua *corresponding author received 4 th january 2023, accepted 26th march 2023 abstract: the purpose of this paper is determination of the effect of rice flour on the conformational transformations in the structure of wheat bread, which contains lecithin, and its quality indicators. in the last decade, inflammatory bowel diseases, which became a global problem in the world, are a serious threat to human health. in the diet therapy of these diseases, it is recommended not to use a large quantity of dietary fiber. rice flour is a perspective raw material for production of bakery products for people with this disease, as it contains 8.5 times less dietary fiber than premium wheat flour. analysis of the chemical composition of bread without rice flour and with its addition showed a slight decrease in protein content by 4.4-10.5%, depending on the dosage (10-40% of wheat flour replacement), and fiber content – by 8.7-35.5%. it was confirmed that conformational changes occur in the structure of proteins and other nutrients of the product in bread making process. the changes of structural groups and their redistribution in the dough after kneading at once and after 3.5 hours of its fermentation and in bread when part of the wheat flour is replaced with rice flour were analyzed. the utilitarian coefficient, redundancy coefficient, dkas and the indicator of biological value indicated that it is rational to make a replacement of wheat flour with 10% or 20% of rice flour. keywords: ir-spectroscopy, wheat bread, rice flour, biological value, inflammatory bowel disease. 1. introduction food industry is structured into some branches and bread-making industry is one of the main ones. bread contains useful nutrients but it is mostly devoid of high nutritional value. nowadays in view of the unstable economic, ecological and political situation in the world the structure of nutrition changed. there is occurrence of alimentary and non-alimentary diseases and their amount increase. the rapid development of diseases of the gastrointestinal tract takes place and causes great concern [1]. in the last decade, inflammatory bowel diseases (ibd), which became a global problem in the world, are a serious threat to human health [2]. some studies indicate connection between the occurrence of ibd and the excess or deficiency of certain nutrients. if the special diet is not followed, the intestinal mucosa may become thinner and more permeable to pathogens and antigens, which leads to persistent inflammation. ibd is associated with intestinal dysbacteriosis, which is characterized by a generalized change in the diversity and abundance of bacterial species [3-4]. scientists who study the course and methods of treatment of ibd prevent from using a large number of dietary fibers in patients’ diet [5]. in view of this, it is mailto:litvynchuk@nuft.edu.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 6 necessary to develop approaches to diet therapy in context consuming the main groups of food products, such as bakery products. rice processing products, in particular rice flour, is a perspective raw material for production of bakery products for people with this disease as it contains a low amount of dietary fiber. rice flour is widely used in the technological process of making glutenfree bread. the effect of substitution wheat flour with rice flour on the change in structural characteristics and in vitro digestibility of low-gluten pretzels was studied [6]. after addition of rice flour, there was an increase in the swelling capacity (18.33±0.51) and bulk density (0.58±0.04 g/cm3), and a considerable decrease in the moisture absorption capacity (0.62±0.09 ml/g) and solubility index (6,72±0.17 %). infrared spectroscopy studies did not reveal differences in the main functional groups after the inclusion of rice flour, but its addition had a pronounced effect on the modulus of elasticity. characteristics of starch digestion in vitro showed an increase in starch digestibility by 7.23%. the content of amylose increased (27.3±1.45), as well as the content of resistant starch (6.12±0.97). the lowgluten pretzels have much higher mineral profile. the study showed that including rice flour for replacing wheat flour in an amount of up to 35% affected the quality characteristics of the developed pretzels with a low gluten content. water has a significant influence on the functional characteristics of raw materials and products [7]. scientists studied the influence of adding high-temperature water to different varieties of rice flour to evaluate the quality of bread. the rice flour had an amylose content of 12.1% to 24.5%, a damaged starch content – 2.4% to 5.5%, a protein content – 5.4% to 6.1%, and humidity in the range of 12.0%– 15.0%. the obtained results showed that regardless of the characteristics of the rice, bread made with the addition of warm water was fluffier and softer than bread made with cold water (5°c). the optimal water temperature and the degree of starch gelatinization increased for the variety of ror rice flour with a lower amylose content in case of the similar particle size [8]. in addition, in food production technologies, the use of activated water is becoming widespread to increase their efficiency [9]. it is important to include phospholipids in the nutrition of patients with ibd along with reducing the use of dietary fiber. phosphatidylcholine is a membrane phospholipid, which is one of the main ones, it takes part in the formation of tstructure of the protective layer of intestinal mucin [10]. phospholipids participate in biological processes in the body, maintaining cell membrane integrity, preventing neurological diseases, and regulating basic biological processes such as transmission of signals between cells. in human body phospholipids are concentrated in vital organs, such as the brain, liver, and kidneys [11]. phosphatidylcholine is found in large quantities in lecithin. soy lecithin became widely used in the food industry. sunflower lecithin is a promising alternative to soy lecithin, as it is a product without genetically modified organisms [12-13]. the effectiveness of using defatted sunflower lecithin for the production of gluten-free bakery products from rice flour with the use of flour starch modified with enzymes was studied [14]. there was determined that 1.0% of lecithin by mass of flour leads to the improvement of gas formation in rice dough and increases the porosity by 12.7% and the specific volume of ready bread by 16.2%. due to the presence of a hydrophobic group in the lecithin molecule, the addition of this raw matherial to the dough is compatible with adding sunflower oil in the amount of 3% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 7 to the mass of flour. in addition, the compatible use of vegetable oil and lecithin in rice bread tproduction allows to extend the freshness and shelf life of the products. however, there were not found studies of the effect of rice flour on the process of manufacturing bakery products from wheat flour in combination with lecithin, so this direction is relevant. previous studies established that the use of rice flour reduces gas retention by 3.720.4%, the viscosity of the dough system increases by 3.7-18.5%, and the specific volume and porosity of the samples decrease. the dimensional stability of bread is significantly reduced, which is associated with the features of the formation of the gluten framework in the dough samples with the addition of rice raw materials. the crumb acidity of the studied products is practically the same as in the control sample [15]. the aim of the work is to determine the effect of rice flour on the conformational transformations in the structure of wheat bread, which contains lecithin, and its quality indicators. 2. matherials and methods the main objectives of this study: 1. to determine the content of the main nutrients in raw materials; 2. to study the process of changing the structural components of bread with different dosage of additives; 3. to determine the chemical composition of bread with different dosages of additives; 4. to calculate indicators which characterize the biological value of bread with different dosages of additives. matherials premium wheat flour, rice flour and sunflower lecithin were used for research. samples of dough were prepared with the addition of salt (1.5% by weight of flour) and pressed baker's yeast (3% by weight of flour). lecithin was added in the amount of 3% to the mass of flour. this dosage was chosen due to the recommendations for the daily norm of lecithin for people with ibd [16]. wheat flour was replaced with rice in the amount of 10%, 20%, 30%, 40%. a sample without rice flour and lethitin was control sample. methods total protein content for determination total protein content there was used kjeldahl method. after mineralization of the sample titration technique was used. 1 g of the sample was hydrolyzed with 15 ml concentrated sulfuric acid in a heat block at 420°c for 2 h with two copper catalyst tablets. then the sample was cooled. after cooling distilled h2o was added using pipette to the hydrolysates before neutralization of the sample and its titration. the amount of protein was calculated taking into account the concentration of nitrogen in the product (g of proteins / 100 g of flour) [17]. fat the sample of the product is put in a thimble. during heating flask, the solvent (petroleum ether) is evaporated and moved to the condenser. it is reformed there into a liquid and collected in the extraction chamber where the sample is. in the process of passing solvent through the sample, the fats are extracted and carried into the flask. this continues from 6 to 24 hours. after finishing the extraction and evaporation of the solvent, the remained amount of lipid is measured [18]. fiber determination the content of total dietary fiber (tdf) in products was conducted using collaborative enzymatic-gravimetric method. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 8 amount of fiber was calculated as the difference between the mass of the residue and the mass of protein and ash [19]. near-infrared reflection spectroscopy to research the reflection spectra from shredded samples and a smooth surface in near infrared area in the range 1330 to 2370 nm infrapid spectrometer (labormim, hungary) was used. reference i0 is used to record the reflectance spectrum from it, then the spectrum is recorded from the researched sample. reflectivity of r characterizes the spectra, it is represented in relative units (i/i0 = r), depending on the wavelength in nm [20-21]. samples of dough immediately after kneading and after 3.5 hours of fermentation, raw matherials – rice and wheat flours, lecithin, and ready bread were researched and the intensity of reflection was measured in them. relative reflection coefficient was used to express the reflection intensity [22]. biological value of bread to characterizes the balance of all essential amino acids of proteins with respect to physiological norm utilitarian coefficient is used. it compares the protein composition of any food product based on its amino acid composition and inadequacy of the use in the body. it was calculated by the formula: where cmin is score of the first limited acid essential amino acid, units; aj is the mass fraction of the j-th of essential amino acid in the product, mg/g protein; aej is the mass fraction of the j-th of essential amino acid in the protein model, mg/g protein (fao/who scale, 2011). to determine the mass fraction of essential amino acids in 100 g product, which are not fully used by the body redundancy coefficient is determined. it was calculated by the formula: dcas is the average excess of essential amino acids compared to the least amino acid score of the limiting amino acid. dcas is calculated by the formula: where das is the difference in amino acid score for each essential amino acid compared to the amino acid score of a limiting amino acid, %; n – the number of essential amino acids. biological value was calculated by the formula: where, bv – biological value of the protein, % [23]. statistical аnalysis three parallel experiments were provided for each parameter to ensure the precision of the obtained results. standard deviation was taken into account for obtained and presented results. for performing graphical presentation of experimental data there was used standard statistical processing program – microsoft excel 2010. 3. results and discussion the properties of the dough system and the quality of bakery products are largely determined by the recipe components, especially peculiarities of their chemical composition. composition of main nutrients of rice flour and lecithin differs from wheat flour (table 1). protein content in rice flour is 1.8 times less than in wheat flour, but its amino acid composition is more complete. dietary fibers content in in rice flour is 8.5 times lower than wheat. it makes it a perspective raw material for the manufacturing bread for patients with ibd. almost the entire chemical composition of lecithin is represented by fat. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 9 the difference in the chemical composition of lecithin, rice flour, and wheat flour should affect the change in the basic structural units of the chemical composition of bread with these components in the recipe. to identify and estimate these components, it is recommended to use the reflection spectrum in the near infrared region [24]. table 1. chemical composition of rice flour and wheat flour of the highest grade indicator rice flour lecithin wheat flour of the highest grade protein, % 6.0±0.21 0.5±0.001 11.3±0.24 fat, % 1.4±0.01 95.3±1.1 1.1±0.01 carbohydrates, % 70.1±1.14 2.0±0.01 69.8±1.14 fiber, % 0.4±0.01 3.5±0.06 * results given as: m ± sd (mean ± standard deviation) of triplicate trials. in the process of baking high temperatures (220°c) are applied, under such conditions changes in the structure of protein composition and other substances of the product occur, which affects the intensity of reflection of product samples (figure 1). fig. 1. reflection spectra of bread with rice flour at different concentrations (10, 20, 30 and 40%). the reflectance spectrum of bread samples with different percentages of rice flour for replacement wheat flour showed peaks associated with protein functional groups at wavelengths of 1430, 1982, 2052 and 2292 nm. the peak at a wavelength of 1430 nm correlates with the absorptionstretching band of n-h groups. peaks at wavelengths of 1980 and 2052 nm can be correlated with the combined absorption band of n-h asymmetric stretching. peak at a wavelength of 2294 nm can be correlated with the combined absorption band of the stretching of the n-h and c=o groups corresponding to the wavelength of 2294 nm, which characterizes amino acids. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 10 in the entire analyzed range of wavelengths, the reflection spectra of the sample of bread without additives and samples with different dosages of rice flour turned out to be very similar. but the control sample had a bit higher reflectance intensity across the entire spectrum in comparison with the bread samples with the additive. this may be due to the greater presence of α-helical structures in the control sample than in the replacement samples. they are one of the structural elements which are responsible for dough viscosity. the differences and redistribution of structural groups in the dough immediately after kneading and after 3.5 hours of its fermentation and in bread with 40% wheat flour replaced with rice were analyzed. the reflection coefficient was converted to the spectral index, which is lg(1/r) and the spectra were obtained (figure 2). the functional groups o-h, n-h, s-h present in the studied samples were determined. fig. 2. changes and redistribution of structural groups in dough after kneading and after 3.5 hours of fermentation and in bread analyzing the spectra of the dough obtained after kneading, after 3.5 hours of fermentation and finished bread (control sample and with the maximum researched dosage of rice flour) the following reflectance maxima can be noted: 1460 nm, 1770 nm, 1930 nm, 2100 nm, 2270 nm and 2350 nm. the extremum at a wavelength of 1460 nm corresponds to the valence vibrations of the oh group, the first overtone (it is observed in all tested samples of dough and bread). the functional group of the first overtone of the s-h group appears at a wavelength of 1770 nm. at a wavelength of 1930 nm, it is possible to determine the moisture content food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 11 in the samples, i.e. the presence of water (the second overtone of o-h deformation oscillations). at a wavelength of 2100 nm, conformational changes of gluten proteins can be observed, the specified wavelength corresponds to the deformation vibrations of n-h, the second overtone. it was established that to a greater extent these transformations take place in the sample of bread with rice flour and the control sample of bread, which indicates that the protein structure changes significantly under the influence of high temperatures and to a greater extent in the sample with rice flour. dough samples during the fermentation process also undergo changes in the protein structure compared to samples immediately after kneading. the nutritional value of bread and provision of daily needs, when part of wheat flour is replaced with rice flour, was determined (table 2). the daily need for protein was taken at the level of 59.0 g, for fat – 60.0 g, for carbohydrates – 344.0 g, and for fiber – 25.0 g. table 2. nutritional value of bread samples with the replacement of part of wheat flour with rice flour indicators control sample sample with lecithin rice flour to replace wheat flour, % 10 20 30 40 protein, % 8.10±0.22 8.10±0.22 8.46±0.22 8.06±0.22 7.66±0.22 7.25±0.22 fat, % 1.09±0.01 3.06±0.02 3.08±0.02 3.11±0.02 3.13±0.02 3.15±0.02 carbohydrates, % 52.97±1.07 52.97±1.07 52.99±1.08 53.00±1.08 53.02±1.08 53.03±1.08 fiber, % 2.65±0.04 2.65±0.04 2.42±0.04 2.18±0.04 1.95±0.03 1.71±0.03 analysis of the chemical composition of bread without rice flour and with its addition showed a slight decrease in protein content by 4.4-10.5%, depending on the dosage, and fiber content by 8.735.5%. this is a positive factor due to the use of rice flour in dietary therapy for ibd. it was also calculated the degree of provision of the daily need for nutrients due to the consumption of 100 g of bread and the daily norm of bread in ukraine 277 g (according to "decree of the cabinet of ministers of ukraine" no. 656). the data are shown in table 3. table 3. ensuring the daily need for basic nutrients when replacing part of the wheat flour with rice flour indicators control sample sample with lecithin rice flour to replace wheat flour, % 10 20 30 40 integral score, % due to consumption of 100 g of bread protein 15.02 15.02 13.66 13.66 12.98 12.30 fat 1.82 5.10 5.18 5.18 5.21 5.25 carbohydrates 15.40 15.40 15.41 15.41 15.41 15.42 fiber 10.61 10.61 8.73 8.73 7.79 6.85 integral score, % due to consumption of 277 g of bread protein 24.54 24.54 23.43 22.32 21.21 20.10 fat 5.04 8.48 8.54 8.60 8.67 8.73 carbohydrates 42.65 42.65 42.67 42.68 42.69 42.70 fiber 29.38 29.38 26.78 24.17 21.57 18.97 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 12 the degree of providing the daily need for protein due to the consumption of 100 g and 277 g of bread in case of partial replacement of wheat flour with rice is lower than control sample by 9.0-18.1% and 4.5-18.1%, respectively. the body's supply of dietary fibers decreased by 17.735.4% and 8.8-35.4% when consuming 100 g and 277 g of bread per day, respectively. the utilitarian coefficient was calculated, which characterizes the balance of essential amino acids (eaa) in relation to the physiologically necessary norm. to characterize the total amount of eaa that the body uses for anabolic needs, the indicator of the comparative excess of essential amino acids (redundancy coefficient) and the amount of the excess amino acid composition of eaa (the coefficient of difference in the amino acid score of dcas) and the biological value were calculated (table 4). table 4. utilitarian coefficient, dcas, redundancy coefficient and biological value of bread in case of the partial replacement of wheat flour with different percentages of rice flour indicators control sample sample with lecithin rice flour to replace wheat flour, % 10 20 30 40 utilitarian coefficient 0.49±0.01 0.49±0.01 0.54±0.01 0.50±0.01 0.39±0.01 0.32±0.01 redundancy coefficient 37.25±1.11 37.25±1.11 30.28±1.07 35.90±1.17 55.18±1.22 74.91±1.26 dcas 0.47±0.01 0.47±0.01 0.64±0.01 0.98±0.01 1.61±0.01 2.31±0.01 biological value (%) 99.53±2.49 99.53±2.49 99.36±2.47 99.02±2.44 98.39±2.43 97.69±2.32 the analysis of the utilitarian coefficient showed that the degree of balance of essential amino acids in relation to the physiologically necessary norm is the highest in the sample with the replacement of 10% of wheat flour with rice. the coefficient of redundancy of samples shows that the protein of bread with 10% replacement is more fully used by the body for anabolic needs. as the replacement percentage increased, the dcas indicator increased too. this indicates that the amino acids contained in the control sample are more fully used by the body than when the supplement is applied. in general, the biological value of the control sample is slightly higher than the bread samples with rice flour, but this increase is insignificant. 4. conclusion in conclusion, rice flour is a perspective raw material for iuse in diet therapy of patients with ibd, as it contains 8.5 times less dietary fiber than wheat flour. analysis of the chemical composition of bakery products without rice flour and with its addition showed a slight decrease in protein content by 4.4-10.5%, depending on the dosage, and fiber content by 8.735.5%. it was confirmed that in the bread making process, conformational changes occur in the structure of proteins and other nutrients of the product. the utilitarian and redundancy coefficients, kras and the indicator of biological value indicate that it is rational to replace 10% or 20% of wheat flour with rice flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 13 5. references [1]. forbes a., escher j., hébuterne x., kłęk s., krznaric z., schneider s., bischoff c. clinical nutrition in inflammatory bowel disease, clinical nutrition, 36(2): 321-347, (2017). https://doi.org/10.1016/j.clnu.2016.12.02 [2]. cai z., wang s., li j. treatment of inflammatory bowel disease: a comprehensive review, frontiers in medicine: gastroenterology, 8, (2021). https://doi.org/10.3389/fmed.2021.765474 [3]. agus a., denizot j., thévenot j., martinez-medina m., massier s., sauvanet p., bernalier-donadille a., denis s., hofman p., bonnet r., billard e., barnich n. western diet induces a shift in microbiota composition enhancing susceptibility to adherent-invasive e. coli infection and intestinal inflammation, scientific reports, 6, 92016). https://doi.org/10.1038/srep19032 [4]. morgan x.c., tickle t.l., sokol h., gevers d., devaney k.l., ward d.v., reyes j.a., shah s.a., leleiko n., snapper s.b., bousvaros a., korzenik j., sands b.e., xavier r.j., huttenhower c. dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment, genome biology, 13(9), (2012). https://doi.org/10.1186/gb2012-13-9-r79 [5]. armstrong h., mander i., zhang z., armstrong d., wine e. not all fibers are born equal. variable response to dietary fiber subtypes in ibd, frontiers in pediatrics, 8, (2021). https://doi.org/10.3389/fped.2020.620189 [6]. jan n., naik h. r., gani g., bashir o., amin t., wani s. m., sofі s. a. infuence of replacement of wheat four by rice four on rheostructural changes, in vitro starch digestibility and consumer acceptability of low-gluten pretzels, food production, processing and nutrition 4:9, (2022). https://doi.org/10.1186/s43014-022-00088y [7]. bolshak yu. v., marynin a. i., sviatnenko r. s., shpak v. v. bioelektronika i zakonomirnosti nabuttia ozdorovchykh vlastyvostei pytnoiu vodoiu, zbahachenoiu molekulamy vodniu, naukovi pratsi natsionalnoho universytetu kharchovykh tekhnolohii, 27(5): 57-66, (2021). (in ukrainian). https://doi.org/10.24263/2225-2924-2021-27-5-9 [8]. saito k., okouchi m., yamaguchi m., takechi t., hatanaka y., kitsuda k., mannari t., takamura h. quality improvement of gluten-free rice flour bread through the addition of high-temperature water during processing, journal of food science, (2022). https://doi.org/10.1111/1750-3841.16333 [9]. ukrainets a. i., bolshak yu. v., sviatnenko r. s., prokhorenko zh. i. zastosuvannia fizychno zminenoi (aktyvovanoi) vody dlia pidvyshchennia efektyvnosti tekhnolohii kharchovoho vyrobnytstva ta polipshennia yakosti produktsii, naukovi pratsi natsionalnoho universytetu kharchovykh tekhnolohii, 24(5): 218224, (2018). (in ukrainian) [10]. stremmel w., vural h., evliyaoglu o., weiskirchen r. delayedrelease phosphatidylcholine is effective for treatment of ulcerative colitis: a meta-analysis, digestive diseases, 39: 508-515, (2021). https://doi.org/10.1159/000514355 [11]. nguyen m. t., van de walle d., petit c., beheydt b., depypere f., dewettinck k. mapping the chemical variability of vegetable lecithins, journal of the american oil chemists' society, 91: 1093–1101, (2014). https://doi.org/10.1007/s11746-014-2455-9 [12]. guiotto e. n., tomás m. c., diehl b. w. k. sunflower lecithin, polar lipids, 57–75, (2015). https://doi.org/10.1016/b978-1-63067-0443.50007-8 [13]. shevchenko a., drobot v. influence of food additive – sunflower lecithin on the properties of dough for manufacturing bakery products. proceedings of the іst international scientific and practical conference «problems and practical approaches to the production and regulation of the use of food additives in the european union countries and in ukraine», november 30, 2021. k.: nuft, 62-63б (2021). https://drive.google.com/file/d/1gf_qbecxwnvfrutwvdcb_5io5wvjpx5/view [14]. medvid i., shydlovska o., dotsenko v. the use of sunflower lecithin in the technology on glutenfree bread with enzymatic modification of flour starch, food and environment safety, 17(4): 352 – 362, (2018). http://dspace.nuft.edu.ua/jspui/bitstream/123456789 /28780/1/medvid.pdf [15]. drobot v. i., shevchenko a. o., litvynchuk s. i. vplyv rysovoho boroshna na strukturno-mekhanichni vlastyvosti tista ta yakist khliba, naukovi pratsi nukht, 27(5): 114-122, (2021). https://doi.org/10.24263/2225-2924-202127-5-15 (in ukrainian) [16]. partridge d., lloyd k. a., rhodes j. m., walker a. w., johnstone a. m., campbell b.j. food additives: assessing the impact of exposure to permitted emulsifiers on bowel and metabolic health – introducing the fadiets study, nutrition bulletin, 44(4): 329–349, (2019). https://doi.org/10.1111/nbu.12408 https://doi.org/10.1016/j.clnu.2016.12.02 https://doi.org/10.3389/fmed.2021.765474 https://doi.org/10.1186/gb-2012-13-9-r79 https://doi.org/10.1186/gb-2012-13-9-r79 https://doi.org/10.3389/fped.2020.620189 https://doi.org/10.1186/s43014-022-00088-y https://doi.org/10.1186/s43014-022-00088-y https://doi.org/10.1159/000514355 https://doi.org/10.1007/s11746-014-2455-9 http://dspace.nuft.edu.ua/jspui/bitstream/123456789/28780/1/medvid.pdf http://dspace.nuft.edu.ua/jspui/bitstream/123456789/28780/1/medvid.pdf https://doi.org/10.24263/2225-2924-2021-27-5-15 https://doi.org/10.24263/2225-2924-2021-27-5-15 https://doi.org/10.1111/nbu.12408 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 anastasiia shevchenko, svitlana litvynchuk, influence of rice flour on conformational changes in the structure of wheat bread and its nutritional value, food and environment safety, volume xxii, issue 1 – 2023, pag. 5 – 14 14 [17]. mæhre h. k., dalheim l., edvinsen g. k., elvevoll e. o., jensen i. protein determination—method matters, foods, 7(1): 5-14, (2018). https://doi.org/10.3390/foods7010005 [18]. shevchenko a., drobot v., galenko o. use of pumpkin seed flour in preparation of bakery products, ukrainian food journal, 11(1): 90-101, (2022). https://doi.org/10.24263/2304-974x-2022-11-1-10 [19]. mccleary b. v., devries j. w., rader j. i., cohen g., prosky l., mugford d. c., champ m., okuma k. determination of insoluble, soluble, and total dietary fiber (codex definition) by enzymaticgravimetric method and liquid chromatography: collaborative study, journal of aoac international, 95(3): 824–844, (2012). https://doi.org/10.5740/jaoacint.cs2011_25 [20]. niewitetzki o., tillmann p., becker h.c., mollers c. a new near-infrared reflectance spectroscopy method for highthroughput analysis of oleic acid and linolenic acid content of single seeds in oilseed rape (brassica napus l.), journal of agricultural and food chemistry, 58: 94–100, (2010). https://doi.org/10.1021/jf9028199. [21]. yip w.l., gausemel i., sande s.a., dyrstad k. strategies for multivariate modeling of moisture content in freeze-dried mannitolcontaining products by near-infrared spectroscopym, journal of pharmaceutical and biomedical analysis, 70: 202–211, (2012). https://doi.org/10.1016/j.jpba.2012.06.043. [22]. litvynchuk s., galenko o., cavicchi a., ceccanti c., mignani c., guidi l., shevchenko a. conformational changes in the structure of dough and bread enriched with pumpkin seed flour, plants, 11: 2762, (2022). https://doi.org/10.3390/plants11202762 [23]. grek o., onopriichuk o., tymchuk a. biological value in milk-protein concentrates with malt ingredients, ukrainian food journal, 8(3): 571-583, (2019). https://doi.org/10.24263/2304-974x-2019-8-3-13 [24]. baslar m., ertugay m.f. determination of protein and gluten quality-related parameters of wheat flour using near-infrared reflectance spectroscopy (nirs), turkish journal of agriculture and forestry, 35(2): 139–144, (2011). https://doi.org/10.3906/tar-0912-507. https://doi.org/10.3390/foods7010005 https://doi.org/10.24263/2304-974x-2022-11-1-10 https://doi.org/10.5740/jaoacint.cs2011_25 https://doi.org/10.1021/jf9028199 https://doi.org/10.1016/j.jpba.2012.06.043 https://doi.org/10.3390/plants11202762 https://doi.org/10.24263/2304-974x-2019-8-3-13 https://doi.org/10.3906/tar-0912-507 1. introduction 4. conclusion 323 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xix, issue 4 2020, pag. 323 331 a rapid method of determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc *alina lenuța crăciun 1 , gheoghe gutt 2 1faculty of food engineering, ”ștefan cel mare„ university, 720225 suceava, romania, alina.craciun@usm.ro , *corresponding author received 23th september 2020, accepted 29th december 2020 abstract: resveratrol (trans-3,5,4’-trihydroxystilbene) is a phytoalexin produced naturally by 72 different plant species especially grapevines, japanese knotweed and pines in response to some exogenous factors such as uv radioation, chemical stress and fungal parasites. with the discovery of the „french paradox” resveratrol had entered in the spotlight of researchers. the french paradox is a term generated in 1992 that refers to the cardioprotective effects of resveratrol that have been discovered based on data from french people that has shown an inverse correlation between wine consumption and the incidence of coronary heart disease (chd) despite the consumption of a diet in hight saturated fat. the aim of the present study is to determine the trans-resveratrol content in 11 romanian comercial wine samples by direct injection into the hplc instrument coupled with diode array detector after being filtered through 0.45 µm ptfe membrane filtres. the total resveratrol content in alayses samples varied from 0.03 mg/l to 6.64 g/l. pinot noir wines had the highest average resveratrol content, while the lowest was found for the sauvignion blanc variety. all wines analyzed contained resveratrol in variable quantities. this study could contribute to the analysis made on the variety of romanian wines for resveratrol content. keywords: trans-resveratrol; phytoalexin; wines; hplc; „french paradox” 1. introduction people are looking for an anti-aging cure for ages, but they didn’t know that this might be closer than expected. the concern for anti-aging substances did the object for many research over the years. it is well known that free radicals are responsible for aging and can contribute to various diseases. the antioxidants can neutralize the effect of those. among the substances with antioxidant effect are polyphenols, including resveratrol. resveratrol had entered in the spotlights of researchers once with the observation of „french paradox” [1]. coronary artery disease is the leading cause of death in western countries. high consumption of animal fats, especially meat, cheese and various dairy products, results in cardiovascular disease. in addition, the heavy use of tobacco products has an impact on coronary heart disease. the cardioprotective effects of resveratrol were discovered based on research that showed an inverse correlation between red wine consumption and the incidence of cardiovascular disease. scientists have discovered a phenomenon called the "french paradox", which is based on the hypothesis that a moderate consumption of red wine reduces the occurrence of cardiovascular disease [2,3]. all this is due to the inhibitory effect on cell membrane lipid peroxidation and protection against the oxidation of low-density lipoproteins, such as ldl, which influence the reduction of hdl cholesterol concentration [4]. resveratrol (3,4 ', 5 trihydroxystylbene) is a phenolic compound of plant origin, present in a variety of plant species, such as blackberries, grapes, peanuts and currants [33]. resveratrol is a phytoalexin http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 324 and some plants have been found to synthesize it in response to some exogenous stimuli, such as uv radiation, chemical stress and fungal parasites [6]. grapes and by-products are considered the most important sources of resveratrol [7]. resveratrol has two isomeric forms, transresveratrol and cis-resveratrol. the transisomer is present in all component parts of the vine, but in different amounts. the cisisomer is produced by uv irradiation of the trans isomer. it is generally absent or in small amount in grapes, but both isomers are present in varying amounts in commercial wines [8,9]. the concentration of resveratrol in wine increases during the fermentation process, but the amount present in the wine depends on the wine variety and the oenological conditions. the concentration of this compound is lower in white wines than in red wines due to the fact that the skin of the grapes is removed earlier during the production of white wines. this is confirmed by the fact that the concentration of resveratrol in rose wines is between white and red wines [10]. viticulture is one of the most important agricultural activities in the world, with approximately 7.4 million ha cultivated in the world in 2018 of which 4.3 million ha are cultivated in europe. the first country is represented by spain, with an area of 969,000 ha, followed by china with 875,000 ha and france with 793,000 ha [11]. romania ranks 10th in the world as an area cultivated with vines, approximately 180,000 hectares. a quarter of the counties where vines are cultivated account for over 65% of the total area, vrancea being the county with the largest area. among wineproducing countries, romania ranks 6th in europe and 12th in the world [12]. resveratrol has recently become the subject of intense research due to the fact that it is a powerful antioxidant, anticancer, anti-inflammatory, antiviral agent, provides protection against infections, prevents aging, reduces obesity, has neuroprotective and cardioprotective effect and protects against ischemia [12]. many analytical procedures have been developed for the determination of resveratrol in wine, which is based on the application of gas chromatography (gc), high purity liquid chromatography (hplc) and capillary electrophoresis [6]. for the determination of resveratrol, the most common technique used is high performance liquid chromatography (hplc) with various types of detectors, such as photodiode array detector (dad), electrochemical detectors, fluorimetric detectors and mass spectrometry. detection based on fluorimetry, electrochemistry and mass spectrometry can provide greater sensitivity and selectivity than the dad detector [11,13]. in the initial studies, in which the hplc technique was used as the resolution technique, step extractions were performed to separate the compounds while the detection sensitivity of the hplc technique was significantly improved using fluorimetric or electrochemical detectors. however, only the trans form of resveratrol was determined. more recently, improved hplc methods for determining cis, trans, and piceid forms have been reported. the generally used constituents of the mobile phase consist of various mixtures of methanol or acetonitrile with water and electrolytes, such as: acetic acid, formic acid or ammonium acetate. in general, the hplc method uses a normal phase or reverse phase column [14]. resveratrol can be easily determined by simply direct injection of the sample into the hplc system, when the separation is coupled to a dad detector. in any case, sometimes extraction and / or derivatization procedures are necessary due to the overlapping peaks [6]. in these food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 325 cases, the most used procedures are: liquid-liquid extraction, and dansyl chloride is used as a derivatizing agent [15-17]. in some cases the samples can be diluted with 12% alcoholic solutions in 1:3 ratio [10]. in this study, to determine the resveratrol content from comercial romanian wines was used a simple method by direct injection of the samples into hplc system coupled with dad detector. no dilution, derivatization and extraction procedures was needed. in this study, a simple method of hplc analysis coupled with dad detector was performed on samples of comercial romanian wines to determine the resveratrol content by direct injection of the samples into the hplc system. this study could contribute to the analysis made on the variety of romanian wines for resveratrol content. the results can be used in further for the correlation for coronary heart deseases, like the french paradox. 2. matherials and methods 2.1. wine samples eleven samples of comercial romanian wine, three white, one rosé and seven red, were purchased from a local store from suceava, romania. the selected wines have a controlled designation of origin in order to be able to identify the region where the raw material was grown. the viticultural regions that produced the raw material for the manufacture of these wines, except for one that is located in the cotnari area iasi, is located in the subcarpathian region. the climate of this unit is temperatecontinental transition. in the depression area the predominant is the low hilly floor with temperature values of 8-10 ° c and precipitation values of 600 700 mm / year. the predominant sector of climatic influence is continental (of aridity), with a minus of precipitations due to the circulation of the northeastern air masses that produces accentuated cooling in winter [18]. table 1. romanian commercial wine samples wine sample grape variety vintage vineyard region red1 pinot noir 2018 odobești, vrancea red 2 merlot 2018 cotești, vrancea red 3 cabernet sauvignon 2018 cotești, vrancea red 4 pinot noir 2019 odobești, vrancea red 5 fetească neagră 2018 odobești, vrancea red 6 merlot 2017 dealu mare, domeniile franco-române red 7 cabernet sauvignon 2018 dealu mare, domeniile dealu mare white 1 sauvignon blanc 2019 podgoria domnească white 2 grasa de cotnari 2019 cotnari, iași white 3 sauvignon blanc 2018 cotești, vrancea rosé merlot rosé 2018 dealu mare , domeniile franco-române food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 326 2.2. reagent and chemicals the trans-resveratrol standard (99% gc) was purchased from sigma-aldrich co. methanol and acetonitrile (lichrosolv for hplc) was obtained from merk. for preparation of the aquous solutions was used bidistilled and demineralised water. 2.3. standard solutions first a stock solution with a concentration of 1g /l was prepared. the resveratrol standard was initially dissolved in a minimal volume of methanol to guarantee a complete dissolution. before analysis, a set of standards with 0.025, 0.05, 0.075, 0.1, 0.5, 1, 2.5, 5, 10, 20, and 25 mg /l were prepared from the stock solution, diluted in methanol 99%. special care was taken in connection with the degradation of standard solutions, keeping them protected from exposure to air and light. standards were stored in brown glass containers at 200c. 2.4. analytical hplc procedure wine samples immediately after opening and standard solutions were filtered through 0.45 µm ptfe membrane filters and then injected (with a volume of 8 µl) into the hplc instrument (schimadzu, kyoto, japan) for analysis using an spdm-20a diode array detector. the separation was carried out on a phenomenex kinetex 2.6 µm biphenyl 100 å hplc column 150 x 4.6 mm thermostated at 200c. elution was carried out with a solvent system consisting of pure water (solvent a) and acetonitrile (solvent b) as previously described by marshall et al. [7] with modifications. binary gradient consisted of solvent a: pure water and solvent b: acetonitrile as follows: linear gradient from 0% to 10% b in 42 min, 10–40% b in 42.6 min, 40–90% b in 46.5 min. run time was 49.5 min. the analysis were made in triplicate and then the standard deviation was calculated. the solvent flow rate was of 0.5 ml/min. the determined of trans-resveratrol were carried out at at 306 nm detection wavelength. the obtained standard calibration curves showed high degrees of linearity (r2 > 0.99). data collection and subsequent processing were performed using the lc solution software version 1.21 (shimadzu, kyoto, japan). analyses were perform in duplicate and the quantification was made based on the peak area, using the external standard method. 3. results and discussion according to the specialized literature, more and more frequently, the methodology of sample analysis is much simplified compared to the data from previous years. thus, more and more authors recommend analysis for direct injection of samples into hplc, without pre-concentration, dilution or extraction steps [19,20]. as can be seen from fig.1, the standard chromatograms are free from interference, with the solvent and trans-resveratrol peaks clearly separated. in wine samples it is observed that the interferences are more significant, especially in the red wine samples. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 327 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min 0.0 0.5 1.0 1.5 2.0 ma u 306nm,4nm (1.00) (a) 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min 0 10 20 30 40 ma u 306nm,4nm (1.00) (b) 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ma u 306nm,4nm (1.00) (c) fig.1. chromatogram for : (a) trans-resveratrol standard solution; (b) red wine sample; (c) white wine sample food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 328 the trans-resveratrol standard was used as a standard, and the standard curve equation was used to determine the concentration in the wine samples: y = 117661x, where y is absorbance at 306nm (figure 2). y = 117661x r² = 0.9997 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 0 10 20 30 aria 306 nm trans-resveratrol mg/l fig. 2. standard carve of trans-resveratrol at 306 nm the concetration of trans-resveratrol ranged from 0.04 to 6,64 mg/l. all results are summarised in table 2. the concentration of resveratol is much higher in red wines than in white ones, and the content of resveratrol in rose wines is between the two. it can also be seen that the content of resveratrol in wines produced by the same grape variety can vary widely (pinot noir 3,14 – 6,64). how the concentration of resveratrol is known to be influenced by various factors (climate, temperature, growing conditions, uv irradiation, exposure to fungal and bacterial infections and production technology) these observation are expected [19]. as can be seen in table 1, the highest content in trans-resveratrol is in the wine of the pinot noir variety – red 4, and the lowest content is in the wine of the sauvignon blanc varietywhite 3. the average trans-resveratrol content in red wines is 4.31 mg/l and in white wines it is 0.07 mg/l. this study is in agreement with a previously reported study made on romanian wines [21]. table 2. trans-resveratrol content in romanian commercial wines wine sample resveratrol content (mg/l) red 1 pinot noir 3.14±020 red 2 merlot 6.17±0.41 red 3 cabernet sauvignon 2.39±016 red 4 pinot noir 6.64±0.26 red 5 fetească neagră 3.91±0.14 red 6 merlot 5.42±0.38 red 7 cabernet sauvignon 2.50±0.10 white 1 – sauvignon blanc 0.04±0.01 white 2 – grasă de cotnari 0.14±0.02 white 3 – sauvignon blanc 0.03±0.01 rose merlot rose 0.32±0.02 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 329 due to the fact that the concentration of resveratrol in wine is influenced by a multitude of factors (climate, temperature, growing conditions, uv irradiation, exposure to fungal and bacterial infections and production technology) [19], its concentration may differ even if the analysis is performed on the same wine of the same variety, produced under the same conditions but the raw material is harvested in different years [22]. in this sense, figure 3 shows the average amount of resveratrol in wines produced in various countries in certain years [23-33]. this classification is indicative, because the average results for a given year are taken into account. in order to be able to make a correct comparison it is necessary to make an average of the results from several years. as can be seen from figure 3., romanian wines have a high resveratrol content. according to the results obtained, they confirm this once again. as can be seen from figure 3., serbia has the highest concentration of resveratrol in wines. in the work published by souto et al. in 2001 the concentration of resveratrol in red wines produced in southern region of brazil varies between 0.82 to 5.75 mg/l.[21] the content of resveratrol in greek red wines ranged from 0.352 to 1.99 mg/l in the paper realised by geroginnaki-christopoulou et al. in 2006 [23]. according to the analyzes performed by ratola et al. in 2004 in the concentration of resveratrol in red wines from alentejo region from portugal ranged from 0.13 to 2.64 mg/l [10]. in montenegrin red wines the concentration of resveratrol ranged from 0.4 to 1.1 mg/l according to the work realised by šćepanović p.s. in 2018 [9]. 2.57 5.576 5.4 1.97 2 3 4 5 6 7 8 mg/l fig. 3. concentration of the trans-resveratrol in red wine differentiated according to countries [21-32]. following the analyzes, this paper shows that the concentration of resveratrol in romanian red wines varies between 2.506.64 mg/l. the results obtained in this study show that commercial romanian wines are a good source of resveratrol, as they have a higher concentration of resveratrol than wines from other countries. therefore, several analyzes can be performed for a clearer picture of the resveratrol concentration in romanian wines. 4. conclusion different types of romanian wine were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 330 analyzed to determine the trans-resveratrol content. the results of the analyzes are presented in table 1.the concetration of trans-resveratrol ranged from 0.04 to 6,64 mg/l. the concentration of resveratol is much higher in red wines than in white ones, and the content of resveratrol in rose wines is between the two. the content of resveratrol in wines can vary widely even if is produced by the same grape variety( pinot noir 3,14 – 6,64). how the concentration of resveratrol is known to be influenced by various factors (climate, temperature, growing conditions, uv irradiation, exposure to fungal and bacterial infections and production technology) these observation are expected.[31]the average trans-resveratrol content in red wines is 4.31 mg/l and in white wines it is 0.07 mg/l. this study is in agreement with a previously reported study made on romanian wines [18]. the use of the direct injection procedure in hplc with uv detector allowed to reduce the processing time of the samples. from the point of view of public health, the values obtained suggest that the physiological effects on romanian wine consumers may be significant, like the “french paradox”. however, transresveratrol represents only a small part of all the polyphenols present in wine 5. references [1]. lamuela-raventos r.m., romeroperez a.i., waterhouse a.l., lloret m., torre-boronat m.c, resveratrol and piceid levels in wine production and in finished wines. in wine nutritional and therapeutic benefits, r.t. watkins ed., chemical society symposium, , am. chem. soc. series: washington, u.s. 661, 56–68, (1997) [2]. vidavalur r., otani h., singal p., maulik n., significance of wine and resveratrol in cardiovascular disease: french paradox revisited, exp. clin. cardiol. 11(3), 217-225 ( 2006) [3]. liu b.l., zhang x., zhang w., new enlightenment of french paradox e resveratrol's potential for cancer chemoprevention and anticancer therapy, canc. biol. ther. 6(12), 1833-1836, (2007) [4]. frankel e., waterhouse a., kinsella j., inhibition of human ldl oxidation by resveratrol lancet, journal 341: 1103–1104 (1993) [5]. garcia, l., garcia, r., pacheco, g., sutili, f., souza, r. d., mansur, e., & leal, i. optimized extraction of resveratrol from arachis repens handro by ultrasound and microwave: a correlation study with the antioxidant properties and phenol contents. the scientific world journal, (2016) [6]. fabjanowicz m., platkawasylka j., namiesnik j., detection, identification and determination of resveratrol in wine. problems and chalenges trends in analytical chemistry, journal 103: 21-33 (2018) [7]. marshall d.a., stringer, s.j., spiers j.d., stilbene, ellagic acid, flavonol, and phenolic content of muscadine grape (vitis rotundifolia michx.) pharmaceutical crops, journal 3: 69-77 (2012) [8]. siemann e.h., creasy l.l., concentration of the phytoalexin resveratrol in wine, american journal of .enology and viticulture, journal 43: 49-52 (1992) [9]. šćepanović p.s., wendelin s., eder r., phenolic composition and varietal discrimination of montenegrin red wines (vitis vinifera var. vranac, kratošija,and cabernet sauvignon),european food research. and technology., journal 244: 2243-2254 (2018) [10]. ratola n., faria j. l., alves a., ., analysis and quantification of trans-resveratrol in wines from alemtejo region (portugal), food technology and biotechnology. journal 42 (2): 125-130 (2004) [11]. http://oiv.int/public/medias/6782/ oiv-2019-statistical-report-on-worldvitiviniculture.pdf international organisation of vine and wine intergovernmental organisation, 2019 statistical report on world vitiviniculture, accessed at 14.08.2020 [12]. changbin w., jian q., zhiling m., rapid determination of resveratrol and piceid in wine by high-performance liquid chromatography, earth and environmental science, journal 332 1-5 (2019) [13]. vinas p., lopez-erroz c., marinhernandez j., hernandezcordoba m., determination of phenols in wines by liquid chromatography with photodiode array and fluorescence detection, journal of chromatography a., journal 871: 85–93 (2000) [14]. adrian m., jeandet p., breuil a.c., levite d., debord s., bessis r, assay of http://oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf http://oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf http://oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xix, issue 4 – 2020 alina lenuța crăciun, gheorghe gutt, determination of trans-resveratrol in romanian commercial wines by reversed-phase hplc, food and environment safety, volume xix, issue 4 – 2020, pag. 323 – 331 331 resveratrol and derivated stilbenes in wines by direct-injection high performance liquid chromatography, american journal of enology and viticulture, journal 51: 37-4 (2000) [15]. fabjanowicz m., bistrazanowska m., namieśnik j., tobiszewski m., plotka-wasylka j., an analytical hierarchy process for selection of optimal procedure for resveratrol determination in wine samples, microchemical journal, journal 142: 126134, (2018) [16]. cvejic j.m., djekic v. s., petrovic v., atanackovic t.m., jovic m. s., brceski i. d., bukarica c. g., determination of transand cis-resveratrol in serbian comercial wines, journal of chromatographic science, journal 48: 229-234 (2010) [17]. laurian v., kiss b; sorin e.l., s. gocan, a rapid method for determination of resveratrol in wines by hplc-ms, journal of liquid chromatography & related technologies, journal 32(14): 2105 -2121, (2009) [18]. mcmurtrey k.d., minn j., pobanz k., shultz t.p., analysis of wines for resveratrol using direct injection high-pressure liquid chromatography with electrochemical detection, journal of agriculture and food chemestry, journal 42: 2077–2080 (1994) [19]. goldberg d.m., ng e., karumanchiri a., yan j., diamandis e.p., soleas g.j., assay of resveratrol glucosides and isomers in wine by direct injection highperformance liquid chromatography, journal of chromatography a, journal 708: 89–98 (1995) [20]. mattivi f., reniero f., s. korhammer s., , isolation, characterisation and evolution in red wine vinification of resveratrol monomers, journal of agriculture and food chemestry, journal 43: 1820–1823 (1995) [21]. jeandet p., bessis r., sbaghi m., meunier p., trollat p. , resveratrol content of wines of different ages: relationship with fungal disease pressure in the vineyard american journal of enology and viticulture, journal 46: 1–4 (1995) [22]. souto a., carneiro m., seferin m., senna m., conz a., gobbi k., determination of trans-resveratrol concentrations in brazilian red wines by hplc, journal of food composition and anaysis, journal 14: 441-445 (2001) [23]. kostadinovic s., wilkens a., stefova m., ivanova v., vojnovski b., mirhosseini, winterhalter p., stilbene levels and antioxidant activity of vranec and merlot wines from macedonia: effect of variety and enological practices, journal of food chemestry, journal 135: 3003-3009 (2012) [24]. gerogiannaki-christopoulou m., athanasopoulos p., kyriakidis n., gerogiannaki i., spanos m., journal of food control, journal 17: 700-706 (2006) [25]. bravo m., silva s., coelho a., vilas boas l., bronze m., trans-resveratrol in wines from the major greek red and white grape varieties, analytica chimica acta, journal 563: 84-92 (2006) [26]. ballus c.a., meinhart a.d., de oliveira r.g., teixeira g.h., optimization of capillary zone electrophoresis separation and online preconcentration of 16 phenolic compounds from wines produced in south america, food research international, journal 45: 136-144 (2012) [27]. lamuelaraventos r.m., waterhouse a.l., occurrence of resveratrol in selected california wines by new hplc method, journal of agriculture and food chemestry, journal 41: 521-523 (1993) [28]. mattivi f, nicolini g, analysis of polyphenols and resveratrol in italian wines, biofactors, journal 6: 445–448 (1997) [29]. radovic b., tesevic v., kodzulovic v., maras v., vitis, resveratrol concentration in vranac wines, journal 54: 169– 171 (2015) [30]. dourtoglou v.g., makris d.p., bois-dounas d., zonas c., trans-resveratrol concentration in wines produced in greece, journal of food composition and analysis, journal 12: 227–233 (1999) [31]. goldberg d.m., yan j., ng e. , . diamandis e.p., karumanchiri a, soleas g.j., waterhouse a.l., , a global survey of transresveratrol concentrations in commercial wines, american journal of enology and viticulture, journal 46: 159–165 (1995) [32]. lima m.t., waffo-teguo p., teissedre p.l., pujolas a., vercauteren j., cabanis j.c., merillon j.m., . determination of stilbenes (trans-astringin. cisand trans-piceid. and cis and trans-resveratrol) in portuguese wines, journal of agriculture and food chememestry, journal 47: 2666–2670 (1999) [33]. lamuela-raventos r.m., romeroperez a., waterhouse a.l., torreboronat m.c., direct hplc analysis of cis and trans-resveratrol and piceid isomers in spanish red vitis vinifera wines journal of agriculture and food chememestry, journal 43: 281–283 (1995) 1. introduction 134 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 134 – 146 importance of local cereal flours in the diet habitof consumers in the district of abidjan cote d’ivoire yevi d. nguessan1, *micael e. bedikou1, rose-monde megnanou1, sébastien l. niamke1 1laboratoire de biotechnologies, filière biochimie-microbiologie, unité de formation et de recherche en biosciences, université félix houphouët-boigny, cocody, abidjan-côte d’ivoire. 22 bp 582 abidjan 22 delphineyevi@yahoo.fr, bemicael@yahoo.fr *corresponding author received june 16th 2014, accepted june 29th 2014 abstract: the aim of this study is to gain understanding of the of the potential of local cereal flours in an urban context and the segment of the urban population concerned with traditional dishes made thereof in côte d’ivoire cereals are produced in the northern areas where they are traditionally consumed. these local cereals concerns: rice, maize, sorghum, millet and fonio. this survey showed that only maize along with millet flours were in greatest demand among the consumers in the districts of abidjan (p<0.05). ash-containing yellow maize and pure white maize flours are commonly consumed. the consumers surveyed, all ethnic groups combined, preferred white maize and millet flours more than yellow maize flour according to the pearson chi2 test (p<0.05). millet flour is predominantly cooked in the form of thin porridge, while the main form of preparing white and yellow maize flours is a dense paste (tô) eaten with sauce. the higher frequency of consuming millet flour derived product is linked to its availability as ready to eat food. the potential for convenient local cereal in the urban setting is real provided convenience and quality are met. keywords: cereals consumption, local dish, flours, survey, district of abidjan, côte d’ivoire 1. introduction in côte d’ivoire, cereals are traditionally grown in savannah zones (northern areas). their production concerns mainly oryzasativa (rice), zea mays (maize), sorghum bicolor (sorghum), pennisetumtyphoides (millet) and digitariaaexilis (fonio) [1]. these food crops produced by rural populations, essentially women, are usually intended for self-consumption however an important quantity is marketed [2]. traditionally, cereal flours were produced by hand pounding using wooden pestle and mortar, or by grinding with stones. these cereals, whether decorticated or not, are milled into flours which are cooked and consumed as a thin porridge or a dense paste called tô; flours can also be rolled into couscous. alcoholic and non-alcoholic beverages can be produced from flours of germinated cereals [3, 4]. traditional flours production however is time consuming, arduous and day-to-day chores [5]. these inconveniences have been the major pretext to adopt imported easy to cook foods such as refined rice and wheat flours, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 135 and to abandon local cereal flours production [6]. moreover urbanization has led to diversities in food consumption patterns due to increased food choice in markets and changes in lifestyles such as time constraints particularly caused by women’s employment [7]. people do not have much time to spare for long traditional meal preparation and would look for local convenient quality products [8]. urban consumers look for processed and pre prepared foods at a good economic cost [9]. this indicates that there are potential value-generating opportunities for local production if only adequate processing and eventually suitable marketing are provided [10]. african local cereal valorisation has made subject to plenty of research topics and over thousands of scientific articles have been published [11]. in many sub saharan areas, namely sahelian and coastal countries, tremendous progresses with regard to processing technologies and good hygiene practices are being achieved [1214]. at present, a wide range of easy to cook local cereal flour derived products with improving qualities [15] are available on the markets of countries like mali [16], senegal [17], niger [18], benin [19] and burkina faso [20]. in côte d’ivoire, no formal reports exist on local cereal flours production and consumption. the actual reports only referred to the existence of maize flours with ash, being sold on abidjan markets [21, 22]. this study aimed at getting on overview on the potential of local cereal flours in an urban context as well as the segment of the urban population concerned with traditional dishes consumption made thereof in this study, a survey was carried out to take inventory on the types of local cereal flours consumed and how they fit in the consumers eating habits in areas of the district of abidjan. 2. material and methods study area and selection of respondents the survey was carried out in nine (9) areas of the district of abidjan (côte d’ivoire, west africa), namely abobo, adjamé, attécoubé, cocody, marcory, koumassi, port-bouët, treichville and yopougon. respondents were customers in selling sites and persons at home that are head of household. consumers were randomly approached by leading questions to evaluate their appreciation of local cereals grains or flours. these particular selections have been made in order to gain reliable understanding of persons who consume local cereal flours, those who do not, and their motivations. in all, 2,700 people were interviewed during the survey with an overall mean of 300 persons per district. the main information collected concern: the sociodemographic status, the kind of local cereal flours consumed, the influence of ethnic origin on the preference of local cereals, the main dishes made out of these cereal flours, the frequency of consumption, the preferred form of cereals purchased (grains or flours), the purchase sites and the high regarded criteria at time of buying flours. statistical analysis data were collected under complete disjunctive matrix. multivariate statistical analysis (msa) by classification with tree diagram, euclidean distances metrics and single linkage distance methods were jointly used to analyse these data. pearson chi square (chi2) test was used to see correlation between frequencies at 5% level. anova (analysis of variance) was done and fisher’s least significant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 136 difference was used for means comparison at 5% level. spss 17.0 software was used for chi square (chi2) test while statistica 7.1 software was used for msa classification and anova. 3. results socio-demographic status of consumers the consumers surveyed were composed in majority of women (93%) with 73 % ivoirians and 23 % foreigners (table 1). the ivorian respondents were from different ethnic origins: 28% northern, 20% midland, 19% eastern, 16% western, 17% southern. regarding marital status, 61% were married, 34% single, 3% divorced and 3%widower. for education level, respondents were divided up in the following way: 30% were illiterate, 25% primary level, 27% secondary and 18% higher education level. table 1. characteristics of surveyed consumers gender nationality ethnic origin (ivorian only) marital status education level 93% women 73% ivorian 28% northern 61% married 30% illiterate 7% men 27% foreigner 20% midland 34% single 25% primary 19% eastern 3% divorced 27% secondary 17% western 3% widowed 18% high 16% southern local cereal flours consumption local cereals flours produced in côte d’ivoire and consumed by the surveyed population of abidjan are depicted in table 2. on the 7 types of flours listed, millet (67.48%) and white maize (65.07%) flours were by far the most consumed (p<0.05), and statistically in the same proportion. yellow maize flour consumption is also important (41.04%) but to a lesser extent than the latter. the consumptions of the flours of rice (4.44%), white and red sorghum (3 and 1.78%, respectively) and fonio (0%) were equally marginal to non-existent (table 2). table 2. local cereal flours consumption in areas of the district of abidjan types of flour means of consumers surveyed rate of consumption (%) millet 205.40a ± 61.55 68.47 white maize 195.22a ± 50.44 65.07 yellow maize 123.13b ± 54.39 41.04 rice 13.33c ± 11.38 4.44 white sorghum 9.00c ± 10.54 3.00 red sorghum 5.33c ± 06.46 1.78 fonio 0.00c ± 00.00 0.00 *means values having different superscript letters are significantly different at 5% level according to the fisher’s least significant difference (lsd). consumers were also questioned to evaluate their general appreciation of fonio and sorghum (table 3). the pearson chi2 test failed to show significant correlation between origins and consumption of red sorghum (p>0.05). for fonio and white sorghum appreciation, the chi2 test showed that ethnic origin had significant influence (p<0.05, table 3) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n iam ke , i m po r t a nc e o f l o c al c e r ea l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t r i ct of ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 137 table 3. influence of ethnic originson the general appreciation of sorghum and fonio. ethnic origin red sorghum(%) white sorghum(%) fonio(%) midland 0.52 0.58 0.77 eastern 0.56 0.37 0.81 northern 3.91 5.39 13.61 western 0.33 0.66 2.95 southern 0.00 0.00 0.39 foreigner 24.16 17.63 2.4 pearson chi 2 values 0.284 0.014 0.000 the respondents have identified 4 forms of maize flours (figure 2) which were the pure white and yellow maize in one hand and potash-containing white and yellow maize on the other hand. potash is an alkaline ash sold in the form of crystals. white maize was appreciated by 51% of respondents represents the dominant form at linkage distance varying from 30 to 32.8. potash-containing yellow maize (33%) is the second prevalent form pure yellow maize (7%) and potash-containing white maize (9%) are equally marginal. they are not commonly sold on market places. fig.1.tree diagram of the different forms of white and yellow maize consumption. origins of local cereals consumers these results bear out the fact that millet, white maize and yellow maize flours are the most known local cereal flours, and therefore the most consumed in abidjan districts. in view of the insignificant results of rice, sorghum and fonio flours t ree di agram for 4 variables si ngl e li nkage eucli dean di stances yel l ow maize-ash white mai ze-ash yell ow mai ze whi te mai ze 28,5 29,0 29,5 30,0 30,5 31,0 31,5 32,0 32,5 33,0 l in ka g e d is ta n ce food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n iam ke , i m po r t a nc e o f l o c al c e r ea l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t r i ct of ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 138 consumption, only maize and millet flours were considered for further analysis. the figure 2 illustrates proportion of foreigners and ethnic origin of ivorian consumers of local cereal flours. these results are reinforced by the pearson’s chi2 correlation test bearing out significant correlation (p< 0.05) between the ethnic group and the kind of cereal flours consumed (table 4). for white maize flours consumption, the foreigners (81.9%) were most numerous followed by northerners (72.5%) and easterners (68.5%); consumers originating from southern (58.1%) midland (52.1%) and western (47.2%) areas of côte d’ivoire were also quite important. the major consumers of millet flours are recruited among ivoirians, namely, northerners (81.5%) and southerners (71.9%); the foreigners (68.4%) are closely followed by persons from the midland (67.3%), eastern (64%) and western (62%) regions. the level of consumption of yellow maize flour is consistently below 50% for all the respondents regardless of their origins (table 4): the proportions range from 47.4% (northerners) to 20.5% (southerners). fig.2. local cereal flours consumption and ethnic origins of respondents. table 4. influence of consumers’ ethnic origins and consumption of maize and millet flours origin of respondents white maize (%) yellowmaize (%) millet (%) midland 52.10 31.20 67.30 eastern 68.50 31.20 64.00 northern 72.50 47.40 81.50 western 47.20 32.10 62.00 southern 58.10 20.50 71.90 foreigners 81.90 42.80 68.40 pearson’chi2 values 0.000 0.000 0.000 0 20 40 60 80 100 re sp on de nt s ( % ) ethnic origins of consumers white maize yellow maize millet food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 139 respondents common cereal flour dishes and frequency inventory of cereal flour dishes among the consumers surveyed has established that the most common traditional meals prepared from maize and millet flours were: thick porridge also known as tô in vernacular tongues, porridges, couscous, beverages, crumpets and infant meals(figure 3). tô and porridge represent the major forms of consumption of these local cereals because they clump together at the highest linkage distance (35) of the tree diagram. the tô made of white and yellow maize along with millet porridge consumers were more numerous with values of about 65% of the persons surveyed (table 5). at distance 15, the minor forms of consumption ranged from 10.04% to 21.52%and concern millet flours derived products such as couscous, crumpet, beverage and tô. white maize porridge and infant meals as well as yellow maize porridge are also classified in the same category. contrary to the previous group, most of the yellow maize meals consumption is met at the lowest distance (7), demonstrating a very marginal appreciation of this cereal in general (table5). fig. 3.tree diagram of consumption of traditional dishes from maize and millet flours. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 140 table 5. forms of millet and maize flours consumption forms of consumption tô porridge beverage couscous infant meal crumpet white maize 65.59% 19.89% 03.96% 05.41% 13.78% 06.96% yellowmaize 65.26% 11.11% 00.48% 01.81% 00.85% 01.56% millet 10.19% 65.22% 10.04% 21.52% 07.30% 12.59% the frequency of consumption of traditional dishes of millet and maize flours is presented in a tree diagram (figure 5). the weekly consumption of millet, white and yellow flours dishes is preponderant by considering the linkage distance around 34. on the daily frequency, millet consumption is quite important (linkage distance 33) while maize is less preponderant at linkage distance 20. fig. 4.tree diagram of the frequency of traditional maize and millet flour foods consumption. preferred buying forms of local cereals (raw grains or flours) the choice between raw grains and flours on the markets of abidjan is depicted in figure 5. at linkage distance 30, the tree diagram is divided into 2 groups with the dominant form being flours. consumers concerned with buying processed cereals were in the proportion of67% for millet, 72% for white maize and 77% for yellow food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 141 maize. the chi2 test showed significant correlation between marital status, educational background and buying processed grains (p<0.05, table 6). fig. 5. tree diagram of forms of buying cereals by the consumers surveyed in the districts of abidjan. table 6. chi2 valueson the preferred forms of local cereal crossed with education level and marital status of the surveyed consumers crossed parameters types of cereals cereal forms grain flour educational level white maize 0.000 0.028 yellow maize 0.002 0.031 millet 0.024 0.006 marital status white maize 0.000 0.000 yellow maize 0.000 0.000 millet 0.000 0.000 purchasing places of local cereal flours and choice criteria the main places identified were: market places, supermarkets and areas of growth referring to the tree diagram below (figure 6). market places are the first choice of the consumers surveyed (highest linkage distance, above 30). areas of production and supermarkets are marginal choices, at distance 5, in this study. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 142 fig. 6. buying places of local cereal flours. consumers’ motivation at the time of buying is recorded in figure 7. it takes into account trust in sellers and flours characteristics such as odour, colour, texture, price and availability. fig.7.purchasing criteria of local cereal flours in the market places. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 143 the criterion trust in sellers is distinctly separated from the others and is preeminent for the three types of cereals (aggregation distance about 23). colour is the next distinctive character, at 20, followed by odour. texture, availability price and taste clump together at distance 15; they are therefore minor criteria according to the survey. 4. discussion on the seven types of local cereal identified in cote d’ivoire, it appears from this survey that mainly maize and millet flours were more familiar to the consumers surveyed. if rice flour is not commonly used, sorghum and fonio, on the other side, are not generally named in the dietary habits of ivoirians [23]. sorghum and fonio, like millet, are staple foods in typical areas of côte d’ivoire, savannah regions, where they are traditionally grown [24, 25]. this study confirmed that fonio (in the form of hulled grain) and sorghum are more familiar to northerners and foreigners because of their background origins. however, sorghum and fonio, along with millet have potent nutritional and health benefits that are now being recognized [6, 26]. these local cereals could in fact be promoted on the basis of health claims. the availability of millet in the form of flours on market places is justified by consumer’s preference. actually millet is described as being tastier and more digestible than sorghum [27]. the popularity of maize flours may come from the agricultural performances of maize as well as its successful commercialization that both have contributed to the marginalization of sorghum and millet production [7]. indeed, maize is a nontraditional cereal which has been widely disseminated in savannah zones because of productivity and precocity [14, 28]. in côte d’ivoire, maize ranks 7th in agricultural production (over 500,000 tons) and 2nd behind rice in cereal production [29]. four types of maize flours were identified by respondents. the preferred maize flours were white maize followed by potash-containing yellow maize. potash is reported to improve texture and digestibility of yellow maize according to respondents. the preference of the surveyed consumers for white maize, is in accordance with a general trend observed in west africa, that is, white maize being more desirable for direct human food [30]. yellow maize is reportedly preferred for animal feed in many regions as it gives a yellow color to poultry, egg yolks and animal fat [31]. the persons surveyed in the districts of abidjan, all ethnic group combined, were concerned with local cereal flours consumption. there is no ethnical discrimination because the respondents demonstrated the same consumption pattern, meaning, important consumption of millet and white maize and lesser interest in yellow maize flour. this survey, even though led in an urban context, may indeed be revealing of an appreciable caloric contribution of millet which had been classified as not having any relevant importance in the ivoirians eating habits [23]. concerning the transformation of local cereal flours into dishes, the main forms were porridge for millet and tô for white and potashcontaining yellow maize. beside porridge consumption, millet is also much appreciated under the form of dèguè (data not shown). the range of recipes of the local cereal flours is very narrow and can be a major obstacle to their valorization [32]. maize utilization in côte d’ivoire, as shown in this survey, is comparative to eating habits of the neighboring sahelian food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 144 countries. in these regions, the meals from maize are mainly adaptation of dishes from traditional cereals like millet and sorghum [14]. in contrary, coastal countries, like benin, have developed about forty maize flours derived recipes [33]. such level of valorization has been described as the result of a satisfactory cultural integration of maize [34]. studies to develop new maize flours derived products that will better respond to consumers’ preferences for convenience, safety and improved nutritional values are possible. it was in this context that nixtamalization has been considered in senegal [35]. it is a process of cooking maize in a solution of limewater originating from latin america to produce staple breads called tortillas [36]. popularization of maize recipes from benin can also be a valuable option. the survey shows that millet and maize traditional meals are regularly consumed. however, when it comes to the daily to weekly consumption, millet is more important. this rate of consumption of millet food, in the form of porridge, may come from its availability as convenience foods (ready to eat) everywhere in the district of abidjan [37]. millet porridge is eaten as a breakfast, afternoon snack and is highly prized during ramadan. maize meals are eaten just as regularly, but at a more moderate level. as far as the main traditional dish of maize namely tô is concerned, it is commonly consumed in the family circle and this aspect maybe an inconvenient in the urban context where people increasingly consume meals outside of the home. the availability of local cereals under processed form is rather attractive to the consumers surveyed. respondents, in majority women, regardless of the educational background and marital statusbuy cereal flours because of gain of times provide by ease of cooking. buying local cereals flours at market places is self-evident for the majority of the consumers surveyed for it is a matter of habits, convenience and price for them. the informal way of selling local cereal flours limits the criteria of consumers’ choice to organoleptic considerations and mainly to trust in sellers. those who preferred raw cereals grains were preoccupied with the lack of hygiene and purity of the local cereal flours. they claimed that fresh flours are mixed with fermented and rancid flours. indeed, management quality with regards to local cereals products, mainly maize and millet, deserve particular attention giving their importance in the human daily consumption. some supermarkets of the districts of abidjan offer a variety of manufactured local dried cereal flours, well-conditioned. however it appears from the survey that most of the respondents are ignorant of local cereal flours being sold in supermarkets because of the lack of marketing policy from the processors. 5. conclusion flours of local cereals such as sorghum and fonio are not familiar to the population surveyed in the districts of abidjan. their consumption is rather localised to areas of growth. this survey have shown that local cereal flours of millet, pure white maize and potash-containing yellow maize are the most consumed in the districts of abidjan. the respondents, all ethnic origins combined, appreciate the three cereals in the same way, meaning, white maize and millet flours being the most prized. the main traditional dishes made out of millet and maize flours are respectively porridge and tô. the availability of the local cereals as convenient foods, easy to cook and ready to eat, is very attractive to the urban consumers surveyed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 145 6. references [1]. camara c. 1984. les cultures vivrières en république de côte-d'ivoire. annales de géographie, t. 93, n°518. pp. 432-453. [2]. anader 2009. la production vivrière: un enjeu national. le partenaire : bulletin de liaison de l’anader. volume 14. 12 p. [3]. aboua f., nemlin j., kossa a. andkamenan a. 1989. transformation traditionnelle de quelques céréales cultivées en côte d’ivoire. céréales en régions chaudes aupelf-ukef, eùs john libbeyeurotext, paris q 1989, pp. 223-228. [4]. aka s., n’dede t., djeni k., n’guessan f., yao k.c. , dje k.m. 2008. variabilité des propriétés physico-chimiques et dénombrement de la flore fermentaire du tchapalo, une bière traditionnelle de sorgho en côte d’ivoire. afrique science 04 (2) pp 274-286. [5]. cruz j.f., béavogui f. and dramé d. 2012. valoriser une céréale traditionnelle africaine, le fonio grain de sel. nº 58. pp 1618. [6]. ipgri 2003. actes du premier atelier sur la diversité génétique du fonio (digitariaexilisstapf.) en afrique de l’ouest.44p [7]. broutin c. 2011. une demande céréalière en forte croissance, sous l’influence des marchés urbains. grain de sel nº 54-56. [8]. delisle h. 1990. patterns of urban food consumption in developing countries: perspective from the 1980’s. in consultation with the food policy and nutrition division fao, rome. 85p. [9]. drenowski a. and popkin b.m. 1997. the nutrition transition: new trends in the global diet. nutrition reviews 55, 31-43. [10]. elbehri a. kaminski j., koroma s., iafrate m. , benalim., 2013. west africa foodsystems: an overview of trends and indicators of demand, supply, and competitiveness of staple food value chains, in: rebuilding west africa’s food potential, a. elbehri (ed.), fao/ifad. 503 p. [11]. sautier d. , o'deye m. 1989.mil, maïs, sorgho techniques et alimentation au sahel. paris, france, l'harmattan, 167p. [12]. seck, i. 1989. expériences de décorticage mécanique des céréales (mil, sorgho, maïs) an sénégal. céréales en régions chaudes aupelf-ukef, eùs john libbeyeurotext, paris q 19x9, pp. 273-282. [13]. thuillier c., hounhouigan d.j., devautour h., nago c.m. muchnik j. 1991. filières courtes et artisanat alimentaire au bénin. rapport de recherche. montpellier, france, mrt-cirad-fsa, 100 p. [14]. cirad 1995, production et valorisation du maïs à' l'échelon villageois en afrique de l'ouest. actes du séminaire «maïs prospère»25-28 janvier 1994, cotonou, bénin, cirad 1995 isbn 2-87614-206-6. [15]. souley b.g., gansari s. 2010. la dynamique des échanges régionaux des céréales en afrique de l’ouest projet de « renforcement de l’intégration agricole régionale en afrique de l’ouest» 111pp. [16]. kaminski j. elbehria. , samake m. 2013., an assessment of sorghum and millet in maliand implications for competitive and inclusive value chains. in: rebuilding west africa’s food potential, a. elbehri (ed.), fao/ifad pp 481-501. [17]. ndiakhere g. 2005. etude du marché des céréales transformées: couscous-sankalarrow-tiackry. 15p. [18]. 18 af 2008. ong afrique verte livret de l’apprenant des membres des op sur la transformation des céréales. thème 1: hygiène et techniques2ème édition, août 2008, 30p.www.afriqueverte.org. [19]. nago c.m. 1992. street foods in west africa.rome, italie, fao. 89p. [20]. 2006.ong afrique verte burkina. rapport d’étude sur l’élaboration d'une stratégie opérationnelle sur la commercialisation des céréales au burkina faso 77p. version finale. site internet: http://www.afriqueverte.org [21]. fusillier j.l. 1991. la filière maïs en côte-d'ivoire un exemple d'adaptation spontanée des appareils de production et de commercialisation à l'extension du marché intérieur. thèse, université montpellier 1, montpellier, france, 303 p. [22]. matty d. 1997. système de commercialisation des produits vivriers en côte d’ivoire étude pour le maïs (zeamays) et l’arachide (arachishypogaea). document de travail n°10. projet ‘renforcement des études agro-économiques à l’idessa.idessa k.u.leuven. [23]. reskass 2011. regional strategic analysis and knowledge support system.étude sur la consommation alimentaire en afrique de l’ouest, rapport de synthèse.77p. [24]. kouakou c.k., akanvou l., konan y.a., mahyao a. 2010., stratégies food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 y e vi d . ng u e s sa n, m i ca e l e. b e d ik ou , r o s e -m o nde me g n an ou , s é ba s t ie n l . n i am ke , i m po r t a nc e o f l o c al c e re a l f l ou r s i n t h e d i et h a bi t of c on s u m e rs i n t h e di s t ri ct o f ab id ja n c o t e d ’ i v o i r e, issue 2 2014, pag. 134 – 146 146 paysannes de maintien et de gestion de la biodiversité du maïs (zeamays l.) dans le département de katiola, côte d’ivoire.journal of applied biosciences 33: 2100 2109. [25]. n’zi g. , sylla b. , 1980., enquête de consommation alimentaire. acta trop. suppl. 11 ad. vol. 37(4), 34-40. [26]. dykes l., rooney l.w., 2006. sorghum and millet phenols and antioxidants.journal of cereal science 44, 236-251. [27]. fao, 1995.sorghum and millets in human nutrition.food and nutrition series, no.27. rome. 189p. [28]. iita 2012. maize. iita, research to nourish africa, 7 p. [29]. faostat, 2011.agricultural data.crops and products domain.http://faostat.fao.org/site/339/default.as px, rome. [30]. fao, 1997.white maize: a traditional food grain in developing countries. rome. 22 p. [31]. iita 2013.breakthroughs in maize breeding.review issue 10.9p. [32]. ndjame f., fall a. , griffon m. 1990. par quelle céréale remplacer le riz? quelques problèmes de la culture du maïs en basse-casamance. in economie des filières en régions chaudes. formation des prix et échanges agricoles. montpellier, france, cirad, p. 443-453. [33]. nago c.m. 1989. technologies traditionnelles et alimentation au bénin: aspects techniques, biochimiques et nutritionnels. abomey calavi, bénin, faculté des sciences agronomiques, université nationale du bénin, 222 p [34]. devautour h. , nago c.m. 1989. le maïs au sud-bénin. innovations technologiques et alimentation. ln céréales en régions chaudes: conservation et transformation. paris, france, aupelf/uref, edit.johnlibbeyeurotext, pp.167-177. [35]. mestres c. , ferré t. 1993. in:alimentation, techniques et innovations dans les régions tropicales. paris: muchnik josé (ed.).l'harmattan, p.429-451. [36]. fao, 2002. maïs en zones tropicales: amélioration et production. amazon france.rome. 382 p. [37]. soro-yao a.a., brou k., koussémon m. , djè k.m. 2014., international journal of agriculture innovations and researchvolume 2, issue 4, issn (online) 2319-1473 doi: https://doi.org/10.4316/fens.2023.013 135 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 135 145 study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria) ali nesrat¹, serhane brahmi¹, *chemseddine fehdi ¹, tarek djedid2, salim khachana2 1. department of earth sciences and universe, water and environment laboratory, echahid cheikh larbi tebessi university, tebessa 12002 algeria 2. faculty of technology, department of hydraulic and civil engineering, laboratory of exploitation and valorisation of saharan energy resources " levres " university of el-oued, 39000, algeria * corresponding author: fehdi@yahoo.fr received 3 rd december 2022, accepted 27th june 2023 abstract: the largest confined aquifers hydraulically continuous from the algeria atlas mountains as a recharge area to the tunisian chotts were discharge area. is being largely used in the valley of oued souf, southeastern of algeria and part of the northeastern sahara, composed with sedimentary series ranging from the lower cretaceous to the quaternary. these formations have favored several aquifer layers with variable hydrodynamic behavior depending on their facies. the high expansion of the agricultural field needs irrigation from groundwater aquifers in this area. the overexploitation of the shallow aquifer causes severe deterioration of groundwater quality and soil suffusion at arid areas. the high salinity (electrical conductivity exceeds 3600 ms.cm-¹) coupled with groundwater level decline pose serious problems for current irrigation and domestic water supplies. although, the multi-layers of complex terminal (ct) and continental intercalary (ci) aquifers is used to approve the water supplies. in aim to corroborate the hydrochemical process, and to investigate the water potentialities and mineralization of ct and ci groundwater aquifers, chemical tools namely major elements and trace elements are studied. the isotopic (δ 18o, δ 2h) hydrogeochemical study helps to identify and classify this deep underground water aquifers relationship. the samples isotopic signal shown a current recharge of the ct, according to the regional rainfall signal . keywords: aquifer, hydrogeochemical, isotope, terminal complex, oued souf, algeria. 1. introduction. in arid provinces, groundwater is the main water supply for populations, economics societies development in mena region [1], [2]. the oued souf valley in the north est algeria sahara is one of the major irrigated agriculture area in algeria. most of the agricultural activities use shallow aquifer groundwater through high pumping rates. the regions show a dominant problem of water scarcity, as result of the shallow groundwater overexploitation, climate variability and pollution risks [3], [4]. these acts have not provoked only depletion of the water resources but also the deterioration of the water quality [5], [6]. this poor-quality water can alter soil physicochemical properties causing soil degradation, reducing the productivity as consequence [7], [8], [9]. for permanent water exploitation, deep aquifers hosted in a large sedimentary series which embodies a huge stock of groundwater regard to climate variability and pollution risks [10], [11], [12]. the continental intercalaire (ci) aquifer of north africa is the ci aquifer in algeria, tunisia and libya, is considered as an area with a large artesian basin, from the west (grand erg septentrional) to the east (to the fezzan of libya) as well as to the southern sahara http://www.fia.usv.ro/fiajournal mailto:fehdi@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 136 basins [13]. although, the overlying aquifer of the complex terminal (ct) present a very important and large aquifer worldwide [14], [15]. these aquifers exhibit high vulnerability regards to their unknown proprieties, uncertain boundaries, and unpredictable hydrodynamic interactions with different reservoirs [16], [17]. for this aim, hydrochemical processes were integrated to determine the main factors and mechanisms controlling the chemistry of groundwater in the study area [18]. though, the stable oxygen and hydrogen isotopic (δ18o and δ2h) ratios properties in different groundwater have become an excellent way to characterize the endmember, of groundwater recharge and discharge, to identify the origins of water bodies, and their migration pathways, [19], [20]. as the first hydrogeological investigation, the mean objectives of this work is to show how isotopic tools and ion elements contribute to the study of water/rock interactions. the extent of any current recharge of these non-renewable water resources in this similar large sedimentary basin. fig.1. localization of the study area 1.1. geographical situation the study area is located in the northeast of algeria. located 700 km southeast of algiers. the oued souf vally extent approximately 10,000 km2 and lies between the longitudes 5°00´–10°00´e and the latitudes 29°00´–36°00´n. this basin is part of the north-west sahara aquifer system (nwsas), ranging from 700 to 1300 m elevation. the climate presents an alternation of a hot, dry and a cold, wet season. the average annual rainfall is lower than 71 mm, the average annual temperature is (+ 28.4°c) with a minimum of (16.3°c) in winter and a maximum of (+ 38°c) in summer. the area corresponds to a synclinal structure limited in the northeast by the algero-tunisian atlas, in the northwest with the algerian atlas mountains. the southern extension of the study area dominated with the sandy dunes of the oriental erg part of the great saharan platform (fig. 1). the drainage network is dendritic to sub-dendiritic reflecting less permeable bed rocks increasing high concentration of rainwater during flash flood times. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 137 the runoff component during flash flood times is driving its water to the endorhreic depression (sebkha or chott melrhir in algeria, and chott djerid in tunisia. 1.2. geological overview oued souf is located in the northeastern part of the saharan platform. it has been the subject of several geological studies data given in [21]. completed by geophysical logging [22], in some borehole’s sections, established from the drilling logs of the albian made by the [23]. these studies show the existence of a sedimentary terrain characterized by detrital facies. the mio-pliocene formations are covered by a considerable thickness of quaternary deposits, they appear in the arrangement of sandy dunes and anti-dunes expanding an immense erg which is part of the great eastern erg . it corresponds particularly to sub-horizontal and monotonic layers of phanerozoic [24]. the multi layers aquifers of ct and ci are hosted in the lower cretaceous shaly-sand and sandstone formations (from barremian to albian) covered and alternated with clay-rich layers. these layers have a thickness of hundreds of meters and are found in depths ranging from around 400 up to 2000 m below the surface [25]. 2. material and methods to study the hydrogeochemical characterization of this large groundwater aquifer, in the mio-pliocene (pontian), thirty samples from public piezometers and private water wells were collected. along the flow direction system, the water levels is measured and a multiparametric probe allows as the physicochemical information, ph, temperature (t), electrical conductivity (ec). other chemical parameters are made in the water and environment laboratory of tebessa university (table 1). the cation concentrations were stated and calibrated using atomic adsorption spectrophotometry, appropriate diluted standards. for accuracy the international used tools were checked. table 1: statistical parameters of chemical elements chemical parameter minimal values (mg/l) maximal values (mg/l) arithmetic average ecart type variation coefficient ca2+ 10.600 36.199 16.933 5.965 0.352 mg2+ 5.201 29.686 11.523 6.317 0.548 na+ 4.160 43.230 24.782 5.772 0.232 k+ 0.6122 1.444 1.080 0.255 0.236 cl8.364 36.869 25.342 5.067 0.199 so4-2 3.908 36.816 18.751 10.177 0.542 hco30.820 3.701 2.622 529 0.201 no30.067 4.591 1.475 0.821 0.556 conductivity 3070 7190 4061 679.820 0.167 the stable isotopes were analyzed by the integrated laboratory of water sciences of the ministry of higher education and scientific research (university of gabes; higher institute of sciences and techniques of water). the δ 2h ratio was analyzed using an isotope ratio mass spectrometer. the oxygen of 1 ml. of water sample was equilibrated with commercial co2 at a controlled temperature and then the δ18o water was analyzed by the equilibrated co2 using isotope ratio mass food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 138 spectrometer. the results of the isotopic analyses are reported with the standard notion (δ) in deviations per thousand (‰) with respect to the international standard v-smow. the analytical precision is ± 0.15‰ for δ18o and ± 0.15 ‰ for δ2h. 3. results and discussions 3.1. piezometry according to the availability and distribution of the sampling water wells in the prone area, a piezometric level campaign was conducted during the period of 2019. during this field work, the data allows us to draw the piezometric map of this aquifer in this period. this map shows a high piezometric level of the water table, located in mouih ouensa 59.25 m and ogla which marked a value of 58.3 m, a low piezometric level is located in the north-east of hassi khalifa area with a level of 15 to 20m. whereas, the piezometric level in the municipality of eloued varies between 35 and 40 m, it is noted that the piezometric surface is not regular (figure 2). the hydrodynamic study and the piezometric map of the terminal complex of the oued-souf valley, allowed us to that the groundwater flow direction is oriented in a general direction is south-west to north-east. the spacing of the piezometric curves is a function of the hydraulic gradient, which is stronger as the curves are tighter (i = 7.8x10-3), this is particularly the case in the central part of the region. it has been evaluated at (i = 2.2x10-3) in the s-e and n-e parts of the plain where the iso-piezo curves have a slightly wide spacing. this hydraulic gradient is directly related to the permeability of the environment, the flow velocity, and the flow rate. fig. 2. piezomeric map food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 139 3.2. hydrogeochemistry the study of water chemistry aims to identify the chemical facies of water, their quality, potability, as well as their suitability for irrigation. it also allows us to follow the spatial evolution of physicochemical parameters and to estimate their origin by correlating them with geology and piezometry. almost all the water wells of the ct aquifer are intended for drinking water supply and for the irrigation of agricultural fields. in order to be used, the water must meet certain standards that vary according to the type of use. for this purpose, a sampling of about thirty water points capturing the pontian aquifer and evenly distributed throughout the study area was carried out. the chemical analysis of the major elements shows that the ct water aquifer is characterized by the abundance of ions (na+, cl-, ca2+, hco-3). this diagram highlights the impact of geological facies in water quality; it also allows estimating the amount of the chemical elements and their classification. the diagram of the cation, we can comprehend that the water samples form essentially one large group, close to the potassium sodium pole (f17, f28, f34....) except for the water wells f100 and f98, which are close to the calcium pole. the wells represented in the middle of the triangle do not show dominance. in the anion diagram, only one group can be distinguished, close to the chloride pole (f13, f17, f18 and so on), except for the water points (f48, f45, f57 and f02) which are close to the sulfate pole. the wells represented in the middle of the triangle do not show dominance . 3.2.1. study of the ratios characteristic and the origin of the water chemistry the waters of the studied aquifer show a chloride sodium and sulfate sodium chemical facies, in connection with the lithological formations and a very high salinity, the electrical conductivity is generally high, it oscillates between 3070 µs/cm in the f70 wells and 7190 µs/cm in f28 with an average of 4061 µs/cm (table 1). 3.2.2. characteristic ratio the study of variation of these ratios allows to assess the chemical evolution of the water during its underground journey as well as the contact with the geological environment; to give indications on the zone of supply and the circulation of the groundwater and to explain some of the existing relationships between chemical elements, some graphs have been represented. a. rmg2+ vs rca2+ ratio the examination of figure 3, allows us to notice that all the values of the ratio are lower than 1 for all the samples. thus, the water is very rich in calcium, which explains the rapid dissolution of limestone compared to dolomites b. ratio r so42-/ r cl taking into account the dominance of chloride and sulfate facies, it is important to verify the relationship between these two elements. the graph shows a dispersion of points indicating a proportional evolution of the two elements having thus a common saliferous origin. in addition, the graph shows that some points have an excess of sulfates and others an excess of chlorides. this reflects the dominance of one facies over the other (figure 4). c. ratio r na+ / r so4-2 the graph shows a scattering of points indicating that the two chemical elements have different origins (figure 5). this relationship does not also confirm the existence of the sodium sulphate facies. this relationship shows a decrease in sodium while sulfates remain in evolution. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 140 d. ratio r na+ / r cl examination of this relationship shows a proportional evolution of chloride and sodium (figure 6). this relationship reflects the dissolution of a sodium chloride of saliferous origin. this relationship also confirms the existence of the sodium chloride facies. e. ratio r ca2+ / r cl examination of this relationship shows a proportional evolution of chloride and calcium (figure 7). this relationship confirms the existence of the chloridecalcium facies. note that this 'abnormal' facies is the result of a sign of pollution (cases f98 and f100) and/or a mixing of the waters of the superficial and deep aquifers. f. ration (rhco3/ r (so42+ cl-)/conductivity: the plot of the water points on this diagram shows a logarithmic decrease in the ratio as a function of electrical conductivity (figure 8). two poles are observed. one carbonate is characterized by a dominance of carbonate ions (μ<3000 μs/cm) and the other represents the evaporitic pole and asserts a dominance of sulfate and chloride ions. this pole appears for electrical conductivities above 3000 μs/cm. examination of the figure thus confirms that the electrical conductivity of the water is due to sulfates and chlorides with a slight dominance of sulfates. 3.3 origin and recharge mechanisms of groundwaters the use of stable isotopes is very important in the field evaluation of the water resources, [26], [27], [28]. stable isotopic oxygen and hydrogen in the groundwater is an active hydrological cycle derive from and reflect the initial isotopic composition of the recharging rainwater. within the coordinate system of δ18o and δ2h is possible to discern a meteoric line, the slope which is characteristic of some hydrological system [29]. in this paper the δ18o and δ2h values of precipitation samples, used to establish the local meteoric water lines, are obtained from the global network for isotopes in precipitation database managed by the aiea, for a period of 6 years (september 1992december 1998) for tunis–carthage and sfax meteorological stations (tunisia). the meteoric water lines defined for sfax and tunis (figure. 9) have been calculated using a least squares regression [30] : δ2h = (6,4± 0,5) δ18o+ (5,2 ±1,7); tunis (1992– 1998, n= 26) δ2h = (6,7± 0,3) δ18o+ (3,5 ±1,3); sfax (1992– 1998, n = 45) the water lines of tunis and sfax for the period 1992–1998 are close and seem to be representative of the precipitation isotopic content in tunisia. the comparison between the tunisia and the global meteoric water line [31], shows evidence of the evaporation that affects tunisian precipitation as the rain falls. 3.4 groundwater isotopic signatures stable isotope compositions of the sampling water wells (table 2). show that isotope values vary between -4.7291 and 6.2131 for water wells of the ct with a mean of -5.7526 ± 0.2‰ (n = 9) in 18o and from -44.9665 and – 37.2235 with a mean of -42.7555 (n = 9) in 2h. the oxygen-18 and deuterium contents for all the investigated ground waters are plotted both in the classical o18 – h2 diagram (fig. 9); in the global meteoric water line (d h2=8d o18+10), defined by [31], and the local meteoric water lines (tunis and sfax). the plot of data points in such diagram provides indication upon the origin and the possible recharge processes of groundwater. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 141 table.2 the composition of groundwater from the ct and ci aquifers n° sampling code altitudes (m) raw delta d raw delta 18o 1 7 68 -42.0726 -5.5325 2 17 100 -44.9665 -5.7843 3 25 82 -37.2235 -4.7291 4 39 94 -44.3208 -5.6658 5 48 56 -42.6954 -5.9996 6 58 61 -43.1057 -6.1261 7 66 30 -43.8134 -6.1152 8 81 67 -42.2472 -5.6084 9 98 29 -44.3544 -6.2131 10 ci 87 -67.6531 -8.2892 groundwater samples are plot close to the tunis and sfax meteoric line (fig. 9), and indicate no significant isotopic modifications by evaporation, which means that the recharge of the aquifer is quit rapid and the recharging meteoric water does not occupy the soil zone of the recharge area for a long time. the similarity of δ2h versus δ18o for both wells in the ct and ci groundwater samples is not unexpected. in the study area aquifers and suggests the recharge of the both aquifers (ct and ci) correspond to an old end-member in relation with palaeoclimatic recharge, which occurred during the late pleistocene and the early holocene humid periods. at high elevations (figure 10) and indicating, that groundwater is homogeneous at the aquifer of ct origin. the deuterium excess values vary very slightly, indicating that the catchment area of oued souf valley wells is not recharged by local originated fossilized water. the total dissolved solid values provide a good indication of the interaction time between the water and the aquifer formation. the ions concentrations increase from wells towards groundwater samples collected from wells of the ct at oued souf valley (fig. 11). whereas wells samples are represented with high cduc values, samples well the ci is represented with high δ18o values. it must be emphasized that, although water ci originates in higher recharge areas, their ion concentrations are lower than those of wells because of the interaction time of groundwater with shallow aquifer. fig. 3. ca2+ vs mg2+ relationship fig. 4. so42vs clrelationship food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 142 fig. 5. na+/so4-2 relationship fig. 6. na+ vs clrelationship fig.7. ca2+ vs clrelationship fig.8. hco3/ (so42+ cl-) vs conductivity relationship fig.9. δ 2h vs δ 18o relationship for groundwater fig. 10. δ18o and zs relationship food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 143 fig.11: δ18o and cduc relationship of water samples 4. conclusion the hydrogeochimical and isotopic study of the complex terminal (ct) groundwater shows that mineralization increases with the flow direction. this mineralization is part of the water and aquifer layers interactions. almost all the water wells of the ct aquifer are intended for drinking water supply and for the irrigation of agricultural fields. in order to be used, the water must meet certain standards that vary according to the type of use. the chemical analysis of the major elements shows that the ct water aquifer is characterized by the abundance of ions (na+, cl-, ca2+, hco-3). the water of the studied aquifer shows a chloride sodium and sulfate sodium chemical facies, in connection with the lithological formations and a very high salinity, the electrical conductivity is generally high, it oscillates between 3070 µs/cm in the f70 wells and 7190 µs/cm in f28 with an average of 4061 µs/cm. however, isotopic signal confirms the fossil nature of the water recharged throw discontinuity during the palaeoclimatic which occurred during the late pleistocene and the early holocene humid periods. 5. acknowledgments the authors would like to acknowledge the support of water and environment laboratory tebessa university, algeria. in addition, the cooperation of the international association of water resources in the southern mediterranean basin, tunisia. we would also like to acknowledge the technical support of douib lazhar and okba mohamed nadjib and djedid fatima from oued souf, algeria. 6. references [1]. hamed, y., hadji, r., redhaounia, b., zighmi, k., bâali, f., & el gayar, a. climate impact on surface and groundwater in north africa: a global synthesis of findings and recommendations. euromediterranean journal for environmental integration, 3(1), 1-15. (2018). [2]. brahmi, s., fehdi, c., hadji, r., brahmi, s., hamad, a., & hamed, y. karstinduced sinkhole detection using a tomography imaging survey, case of setifian high plain, ne algeria. geotechnical and geological engineering, 1-16. (2023). [3]. demdoum, a., hamed, y., feki, m., hadji, r., & djebbar, m. multi-tracer investigation of groundwater in el eulma basin (northwestern algeria), north africa. arabian journal of geosciences, 8(5), 3321-3333. (2015). [4]. nekkoub, a., baali, f., hadji, r., & hamed, y. the epik multi-attribute method for intrinsic vulnerability assessment of karstic aquifer under semi-arid climatic conditions, case of cheria plateau, ne algeria. arabian journal of geosciences, 13(15), 1-15. (2020). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 144 [5]. fehdi ch. rouabhia aek. baali f. the hydrogeochemical characterization of morsottel aouinet aquifer, northeastern algeria. environmental geology, springer-verlag, berlin; heidlerg. doi 10.1007/s00254-008-1667-4. (2008). [6]. besser h. redhaounia b. bedoui s. ayadi y. khelifi f geochemical. isotopic and statistical monitoring of groundwater quality: assessment of the potential environmental impacts of the highly polluted ci water in southwestern tunisia; journal of african sciences 153 (2019) 144-155; doi: 10.1016/j.jafrearsci.2019.03.001. (2019). [7]. ncibi, k., hadji, r., hamdi, m., mokadem, n., abbes, m., khelifi, f., ... & hamed, y. application of the analytic hierarchy process to weight the criteria used to determine the water quality index of groundwater in the northeastern basin of the sidi bouzid region, central tunisia. euro-mediterranean journal for environmental integration, 5(1), 1-15. (2020). [8]. besser, h., dhaouadi, l., hadji, r., hamed, y., & jemmali, h. ecologic and economic perspectives for sustainable irrigated agriculture under arid climate conditions: an analysis based on environmental indicators for southern tunisia. journal of african earth sciences, 177, 104134. (2021). [9]. hamad, a., abdeslam, i., fehdi, c., badreddine, s., mokadem, n., legrioui, r., ... & hamed, y. vulnerability characterization for multi-carbonate aquifer systems in semiarid climate, case of algerian–tunisian transboundary basin. international journal of energy and water resources, 6(1), 67-80. (2022). [10]. ncibi, k., hadji, r., hajji, s., besser, h., hajlaoui, h., hamad, a., ... & hamed, y. spatial variation of groundwater vulnerability to nitrate pollution under excessive fertilization using index overlay method in central tunisia (sidi bouzid basin). irrigation and drainage, 70(5), 1209-1226. (2021). [11]. hamad, a., hadji, r., boubaya, d., brahmi, s., baali, f., legrioui, r., ... & hamed, y. integrating gravity data for structural investigation of the youkous-tebessa and foussana-talah transboundary basins (north africa). euro-mediterranean journal for environmental integration, 6(2), 1-11. (2021). [12]. benmarce, k., hadji, r., zahri, f., khanchoul, k., chouabi, a., zighmi, k., & hamed, y. hydrochemical and geothermometry characterization for a geothermal system in semiarid dry climate: the case study of hamma spring (northeast algeria). journal of african earth sciences, 182, 104285. (2021). [13]. hamed, y., ahmadi, r., hadji, r., mokadem, n., dhia, h. b., & ali, w. groundwater evolution of the continental intercalaire aquifer of southern tunisia and a part of southern algeria: use of geochemical and isotopic indicators. desalination and water treatment, 52(10-12), 1990-1996. (2014). [14]. guendouz. a, moulla. a.s, edmunds. w.m, zouari. k, shand. p, mamou. a,: hydrogeology and isotopic evolution of water in the complexe terminal aquifer in the algerian sahara, hydrogeology journal, vol. 11, no. 4, pp.483-495. (2003) [15]. kamel, s., dassi, l., zouari, k., & abidi, b. geochemical and isotopic investigation of the aquifer system in the djerid-nefzaoua basin, southern tunisia. environmental geology, 49(1), 159-170. (2005). [16]. hamad, a., hadji, r., bâali, f., houda, b., redhaounia, b., zighmi, k., ... & hamed, y.. conceptual model for karstic aquifers by combined analysis of gis, chemical, thermal, and isotopic tools in tuniso-algerian transboundary basin. arabian journal of geosciences, 11(15), 1-16. (2018) [17]. brahmi, s., baali, f., hadji, r., brahmi, s., hamad, a., rahal, o., ... & hamed, y. assessment of groundwater and soil pollution by leachate using electrical resistivity and induced polarization imaging survey, case of tebessa municipal landfill, ne algeria. arabian journal of geosciences, 14(4), 1-13. (2021). [18]. kallel, a., ksibi, m., dhia, h. b., & khélifi, n. recent advances in environmental science from the euro-mediterranean and surrounding regions proceedings of euromediterranean conference for environmental integration (emcei-1), tunisia 2017. in conference proceedings emcei (p. 167). (2017). [19]. mokadem, n., demdoum, a., hamed, y., bouri, s., hadji, r., boyce, a., ... & sâad, a. hydrogeochemical and stable isotope data of groundwater of a multi-aquifer system: northern gafsa basin–central tunisia. journal of african earth sciences, 114, 174-191. (2016). [20]. mathuthu, j., mokhine, n. d., mkiva, n., nde, s. c., dennis, i., hendriks, j., ... & mathuthu, m. determining water isotope compositions for the iaea wico and north west villages, south africa. water, 13(20), 2801. (2021). [21]. busson, g., & cornée, a. the sahara from the middle jurassic to the middle cretaceous: data on environments and climates based on outcrops in the algerian sahara. journal of african earth sciences (and the middle east), 12(1-2), 85105. (1991). [22]. besbes, m., babasy, m., kadri, s., latrech, d., mamou, a., pallas, p., & food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana, the study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria). food and environment safety, volume xxii, issue 2 – 2023, pag. 135 – 145 145 zammouri, m. conceptual framework of the north western sahara aquifer system. shared aquifer resources. (2004). [23]. saibi, h., semmar, a., mesbah, m., & ehara, s. variographic analysis of water table data from the oued-souf phreatic aquifer, northeastern part of the algerian sahara. arabian journal of geosciences, 2, 83-93. (2009). [24]. askri, h., belmecheri, a., benrabah, b., boudjema, a., boumendjel, k., daoudi, m., ... & terkmani, m. geology of algeria. in well evaluation conference algeria (pp. 1-93). houston, tx: schlumberger. (1995). [25]. hamed, y., ahmadi, r., demdoum, a., bouri, s., gargouri, i., dhia, h. b., ... & choura, a. use of geochemical, isotopic, and age tracer data to develop models of groundwater flow: a case study of gafsa mining basin-southern tunisia. journal of african earth sciences, 100, 418-436. (2014). [26]. fritz j. fontes j. handbook of environmental isotope geochemistry. elsevier, amsterdam, (a'), p 21. (1980) [27]. saha, d., dwivedi, s. n., roy, g. k., & reddy, d. v. isotope-based investigation on the groundwater flow and recharge mechanism in a hard-rock aquifer system: the case of ranchi urban area, india. hydrogeology journal, 21(5), 1101.) (2013). [28]. hao, s., li, f., li, y., gu, c., zhang, q., qiao, y., ... & zhu, n. stable isotope evidence for identifying the recharge mechanisms of precipitation, surface water, and groundwater in the ebinur lake basin. science of the total environment, 657, 1041-1050.) (2019). [29]. fehdi, c., rouabhia, a., baali, f., & boudoukha, a. the hydrogeochemical characterization of morsott-el aouinet aquifer, northeastern algeria. environmental geology, 58, 1611-1620. (2009). [30]. celle-jeonatan.h., zouar k., travi y. and daoued, a. caractérisation isotopique des pluies en tunisie. essai de typologie dans la région de sfax. c.r.acad.sci.paris, sciences de la terre et des planètes. 333625-631. (2001). [31]. craig. h. standard for representing concentrations of deuterium and oxygcn-18 in natural waters. science. 133: 1833-1834. (1961) doi: https://doi.org/10.4316/fens.2022.028 292 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 292 302 general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish ahmed ayodeji ayeloja 1 and taofik oyedele dauda 2* 1department of aquaculture and fisheries, faculty of agriculture, university of ilorin, pmb 1515 ilorin, kwara state nigeria. ayeloja2@gmail.com 2institute of agriculture research and training, obafemi awolowo university, moor plantation ibadan, nigeria. todauda.iart@gmail.com *corresponding author received 10th november 2022, accepted 28th december 2022 abstract: this study aimed at assessing the effect of packaging materials and preservatives on nutrient composition of fish was carried out using 864 live clarias gariepinus with an average weight of 226g acquired from an earthen pond of a fish farm in omi-adio, ibadan, nigeria. the experiment was conducted using a 4 × 3 × 11 factorial experiment (4 preservatives, 3 packaging materials and 11 storage periods). the protein content of the fish decreased consistently from 77.538 (0days after smoking das) till it reaches 51.253 (140das). the variance of the protein however increases from 1.051 (for 0das) till 25.487 (140das). the moisture content of the fish increases consistently from 5.703 (for 0das) to 24.976 (for 140das). summary statistics of the packaging materials revealed that mean protein ranged between 59.817±0.94 obtained for the fish packed with basket and 65.897±0.659 (plastics). highest moisture content of 17.896 (±0.676) was obtained for fish packed with basket while the least moisture content of 13.160 (±0.427) was obtained for fish packed with plastic. all the nutrients returned negative covariates with the protein and the covariates generally ranges between 0.037(covxy of ash and crude fibre) and 91.206 (varx,x of protein). the general linear model analysis of the fish nutrients revealed that there exist significant difference in the protein, moisture content, ash, fat and crude fiber with 0.05):264 (3,f = 77.40, 460.24, 111.02, 395.77 and 2037.76 respectively. keywords: algorithmic, euthanized, eviscerated, therapeutic, deteriorate. 1. introduction fishery data analysis has two main goals – response prediction and information extraction and there exist two different approaches (data modeling and algorithmic modeling approach) for achieving these goals [1]. general linear model (glm) falls under the former and it predicts one variable, response variable from one or more other independent or explanatory variables [2]. general linear model (glm) can basically be found useful in testing almost any hypothesis about a numerically measurable dependent variable [3] and there are literally an infinite number of experimental and survey designs that can be analyzed using the glm. three statistical tools, analysis of variance, regression analysis and analysis of covariance are subsumed in the term general linear model. this is because all the statistical tools employed to solve data problem has an underlying model attached to it. fish, a nutritious food and source of high quality protein, is highly susceptible mailto:nigeria.%20ayeloja2@gmail.com mailto:todauda.iart@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 293 to deterioration [4]. similarly, it was established that the chemical composition of the fish enhances its ease of deterioration [5]. in view of this condition (of ease of deterioration) and importance of fish to food security, there have been concerted research efforts in the post harvest handling of fish (particularly storage). the knowledge of nutrient composition of smoked fish is essential for consumer acceptability and package presentation for fish optimal market. indeed, the nutrient composition of packaged products is a requirement for its endorsement by standard organization of nigeria (son) and other agencies for food control as well as in other countries of the world. extensive research has been conducted on fish nutrients’ composition but regular research in fish nutrient values is justified from the need to constantly monitor variability in fish nutrient composition. a big variation has been reported in the nutrient composition of fish by [6]. similarly, studies with the approach of the present research (glm) are scanty or unknown. the relationship between the fish nutrients compositions (response variables) and independent variables like packaging material – x1, preservatives – x2 and storage time – x3 are modeled using the glm. the present work thus seek to answer the research question – what are the statistical effect of independent variables (like preservatives, packaging materials and period) on the fish quality (which is the response variable)? the objectives of this study were therefore to assess the effect of packaging materials and preservatives on nutrient composition of fish. 2. matherials and methods eight hundred and sixty four (864) live catfish, clarias gariepinus, with an average weight of 226g were collected using drag net from an earthen pond, ire akari fish farm, omi-adio, ibadan oyo state, nigeria. the fish were conveyed in plastic bowls to the fish processing unit of the federal college of animal health and production technology (fcah&pt), moor plantation ibadan within 43 minutes. the fish collection was carried out between 07.00 and 09.00 am to reduce stress. six kilogram each of dried garlic (allium. sativum) bulb and ginger (zingiber officinale) rhizome bought at bodija market in ibadan oyo state nigeria were grinded into powder using sterile explosion proof warring blender (new hartford, ct). samples from powdered a. sativum and z. officinale were also collected and homogenized manually in a ratio 1:1 to give garlic–ginger homogenate. two hundred and eighty eight (288) transparent plastic (polyethylene terephthalate) and transparent zip-lock nylon bags (polythene) and twelve (12) plastic baskets were purchased from alesinloye market in ibadan oyo state nigeria. fish drying was done at the department of fisheries technology of the fcah&pt, moor plantation ibadan using the niomr smoking kiln. fish samples in plastic container were euthanized [7] by the addition of 20g of table salt/kg and left for 10 minutes. they were thereafter gutted, eviscerated, washed in potable water and were immersed in 15% brine for 5 min [8]. the research was conducted using 4 × 3 × 11 factorial experiment (4 preservatives, 3 packaging materials and 11 storage time). the preservatives are no preservatives (control); garlic (a. sativum); ginger (z. officinale) and mixture of garlic and ginger (a. sativum and z. officinale). the three packaging materials were basket, polythene (nylon), and polyethylene terephthalate (plastic) while 11 different storage periods were 0das, 14das, 28das, 42das, 56das, 70 das, 84 das, 98das, 112das, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 294 126das and 140das. the fish were spiced and packaged following the procedure of [9]. four preservative groups comprising of 216 pieces each were formed including zero preservative group and three other group of garlic, ginger, and garlic–ginger homogenate at ratio 1:1 for preservatives. the fishes were then smoked and seventytwo (72) samples of each treated smoked catfish were packaged in nylon (polythene) and plastic (polyethylene terephthalate), while others were left unpackaged in basket at ambient temperatures (25–36°c). the samples were thereafter stored for 140 days and sampled at 14 day intervals for organoleptic quality. the data obtained were analyzed using descriptive statistics (including mean, variance and standard error) using sas version 9. correlation analysis of the variables or nutrients as well as general linear model using 3 degree of freedom (for preservatives), 2df (for packaging materials) and 10 df for storage period. mean of significantly different sources of variation were partitioned using duncan multiple range test (dmrt). 3. results and discussion 3.1. results 3.1.1. summary statistics of the fish nutrients the protein content of the fish decreased consistently from 77.538 (0das) till it reaches 51.253 (at 140das– table 1 and figure 1). table 1. summary statistics of the fish nutrients (g/100g sample) over the storage time storage time (das) protein mc ash fat cf 0das mean± se 77.538±0.17 5.703±0.10 6.569±0.08 4.949±0.23 5.012±0.43 variance 1.051 0.329 0.257 1.894 6.714 14 das mean± se 75.965±0.27 7.094±0.16 6.863±0.08 4.525±0.26 5.553±0.47 variance 2.624 0.877 0.228 2.56 7.825 28 das mean± se 72.124±0.32 9.332±0.27 7.291±0.08 5.242±0.25 6.012±0.46 variance 3.676 2.551 0.258 2.182 7.777 42 das mean± se 68.4089±0.37 11.747±0.29 7.574±0.11 5.959±0.20 6.317±0.45 variance 4.995 3.11 0.427 1.493 7.214 56 das mean± se 64.789±0.38 14.364±0.28 7.972±0.12 6.330±0.22 6.545±0.45 variance 5.155 2.802 0.498 1.791 7.317 70 das mean± se 62.300±0.44 15.796±0.30 8.395±0.14 6.796±0.22 6.714±0.45 variance 7.107 3.199 0.743 1.772 7.34 84 das mean± se 58.585±0.55 18.101±0.49 8.649±0.17 7.667±0.23 7.003±0.43 variance 10.715 8.776 1.044 1.902 6.733 98 das mean± se 56.211±0.62 19.593±0.57 8.913±0.18 8.055±0.22 7.229±0.44 variance 13.889 11.724 1.18 1.736 6.928 112 das mean± se 53.559±0.68 21.439±0.65 9.239±0.16 8.336±0.22 7.426±0.44 variance 16.478 14.978 0.932 1.699 6.992 126 das mean± se 52.737±0.73 22.880±062 9.082±0.16 8.088±0.21 7.214±0.42 variance 19.017 13.862 0.923 1.533 6.268 140 das mean± se 51.253±0.84 24.976±0.75 8.947±0.16 7.720±0.23 7.105±0.41 variance 25.487 20.251 0.897 1.915 5.931 nb. das = days after smoking food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 295 fig. 1. protein content of fish as affected by both preservative and packaging materials (das = days after smoking), n=nylon, p = plastic and b = basket; garlc = garlic, gngr = ginger and 2gngr = garlic+ginger & cntrl = control. the decreasing protein index also continues until p4 (point 4) when it followed a cyclical pattern. the variance of the protein however increases steadily from 1.051 (for 0 das,) till 25.487 (140 das,). the moisture content of the fish unlike the protein content increases steadily from 5.703 (for 0 das) to 24.976 (for 140 das – table 1). the protein index returned a decreasing trend for the first 4 periods (42das) while a fluctuating trend was obtained for the remaining period (figure 2). figure 2. moisture content of fish as affected by both preservative and packaging materials (das = days after smoking). n=nylon, p = plastic and b = basket; garlc = garlic, gngr = ginger and 2gngr = garlic+ginger & cntrl = control. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 296 the implication is that the rate of decrease in protein content is not uniform while the moisture content index returned a randomly increasing trend (figure 3). the ash content of the fish was found to appreciate from 6.569 ± 0.08 (for 0das) until 9.239 ± 0.16 (for 112das) and drops till 8.9947 ± 0.16 on 140das. similarly, crude fiber of the fish rose from 4.949 ± 0.23 (for 0das) until 8.336 ± 0.22 in the 112das after which it decreases to 7.72 ± 0.23 in the 140das (table 1). mean crude fiber increases with increase in the period and it ranged between 5.012 ± 0.43 (0das) and 7.424 ± 0.44 (for 140das) after which it reduces to 7.105±0.41. the implication of these results is that the nutrient composition of the fish in terms of the ash content, crude fiber and fat can be guaranteed until 112das after the processing and that after this period the nutrient quality would begin to deteriorate. the summary statistics of the packaging materials revealed that mean protein ranged between 59.817±0.94 obtained for the fish packed with basket and 65.897±0.659 (plastics). highest moisture content of 17.896 (±0.676) was obtained for fish packed with basket while the least moisture content of 13.160 (±0.427) was obtained for fish packed with plastic (table 2). similar results were returned for the fat and crude fiber where the highest of both (6.736 and 6.934) were obtained for fish packed with basket. the least of both fat (6.667) and crude fiber (6.313) were obtained for fish packed with plastic (table 2). table 2. descriptive statistics of the fish nutrients according to packages. protein(%) mc(%) ash(%) fat(%) cf(%) packaging materials nylon mean± se 63.412±0.79 15.587±0.57 7.881±0.08 6.688±0.17 6.426±0.24 variance 82.275 42.636 0.837 3.584 7.408 plastic mean± se 65.897±0.659 13.160±0.427 7.970±0.089 6.667±0.181 6.312±0.236 variance 57.305 24.046 1.038 4.34 7.319 basket mean± se 59.817±0.940 17.896±0.676 8.556±0.131 6.736±0.144 6.934±0.235 variance 116.599 60.341 2.257 2.749 7.26 preservatives garlic + ginger mean± se 62.982±0.91 14.996±0.66 7.660±0.09 7.202±0.15 7.149±0.11 variance 82.628 42.507 0.781 2.173 1.221 garlic mean± se 62.724±0.89 15.037±0.60 7.924±0.10 5.272±0.19 8.989±0.15 variance 78.838 35.455 1.044 3.753 2.212 ginger mean± se 62.63±0.99 15.093±0.68 8.339±0.16 6.358±0.14 7.560±0.13 variance 97.468 42.287 2.658 2.025 1.587 control mean± se 68.832±1.04 17.064±0.77 8.620±0.09 7.955±0.15 2.530±0.10 variance 107.776 58.549 0.849 2.295 0.938 the implication of these results are that the fish packed with plastic materials could be adjudged the best since it had the highest protein content, least moisture content and fat. the summary statistics analysis of the preservatives (table 2) showed that the control (without any preservatives) maintained the highest of protein (68.832), mc (17.064), ash (8.620) and fat (7.955) while fish preserved with ginger returned closely higher values for mc (15.093) and ash (8.339). this was followed by garlic + ginger which was second best in both proteins (62.982) and fat (7.702) – table 2. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 297 the implication of this result is that addition of the preservatives enhances easy deterioration of the fish nutrients. the correlation analysis results showed that sixty percent (60%) of the bivariate correlations were positive while 40% were inversely correlated (table 3). table 3. correlation analysis of the nutrients protein mc ash fat cf protein 91.206 -62.820 -8.843 -11.345 -8.498 mc -0.971 45.874 6.042 8.247 2.867 ash -0.766 0.738 1.460 1.343 0.037 fat -0.631 0.647 0.591 3.540 -1.699 cf -0.328 0.156 0.011 -0.333 7.365 the bivariate correlation value, ri ranged between 0.011 (relationship between ash contents and crude fibre) and -0.971 (relationship between protein and moisture content). forty percent of the rij are greater than 0.5 while the remaining rij were generally low. the result of the variancecovariance analysis (table 3) indicated that generally 0.267 of the covariates (covxy) returned values greater than unity (covxy >10) while the remaining (0.733) were less than unity. all the nutrients returned negative covariates with the protein and the covariates generally ranges between 0.037(covxy of ash and crude fibre) and 91.206 which is the variance of the protein while none of the covariates was zero. the implications of these results are that the fish nutrients are not independent and that there exist little or minimal variability among the nutrients. 3.1.2. general linear model (glm) analysis the glm analysis of the fish nutrients revealed that there exist significant difference in the protein, moisture content, ash, fat and crude fiber returned by different preservatives. the 0.05):264 (3,f = 77.40, 460.24, 111.02, 395.77 and 2037.76 obtained respectively for protein, moisture content, ash, fat and crude fiber were significant (p<0.05table 4). in addition, mean protein obtained for the control (63.832) was significantly higher than that obtained for ginger +garlic (62.982table 4). mean protein obtained for ginger (62.630) was the least and was not significantly different from that obtained for garlic (62.724). the mean moisture content of the fish followed the trend control (17.064) > ginger (15.073) > garlic (15.037) > 2ginger (14.996). two classes (the control and other different treatments) were clearly defined from this partition (table 4). the ash content was grouped into four distinct group/classes. the control had significantly highest ash content of 8.620 and it was higher than that obtain for ginger which was 8.339. the least ash content was that of garlic+ginger (7.66) and it is significantly lower than the ash content of garlic (table 4). the glm analysis of the packaging materials used for the smoked fish indicated that significant difference exist between the mean of all the nutrients returned by different packaging materials except fat. this is because 0.05):264 (2,f = 3218.80, 3359.62, 109.29 and 37.95 obtained for protein, moisture content, ash and crude fiber were significant (p < 0.05 – table 4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 298 table 4. general linear model of the nutrients composition. parameters variables protein moisture content ash fat cf preservative materials f-statistics 77.40** 460.24** 111.02** 395.77** 2032.76** df 3 3 3 3 3 garlic 62.724c 15.037b 7.923c 5.272d 8.989a ginger 62.630c 15.093b 8.339b 6.358c 7.560b 2ginger 62.982b 14.996b 7.660d 7.202b 7.149c control 63.832a 17.064a 8.620a 7.955a 2.530d packaging materials f-statistics 3218.80** 3359.62** 109.29** 0.51 37.95** df 2 2 2 2 2 nylon 63.412b 15.587b 7.881b 6.688a 6.426b basket 59.817c 17.896a 8.556a 6.736a 6.934a plastic 65.897a 13.160c 7.970b 6.667a 6.312b ** significant @ 0.01 probability levels (p < 0.01), * significant @ 0.05 probability levels (p < 0.05) mean fat content (f2, 264:0.05 = 0.51) obtained for the packaging materials was however not significant (p < 0.05). dmrt partitioned the mean of both protein and moisture content into 3 significantly different packaging classes. the mean protein obtained for the fish packed with plastic (65.897) was significantly higher than that of the nylon (63.412). the least mean protein (59.817) was obtained for the fish packed with basket and was significantly different from others (table 4). the dmrt partitioning of the mean moisture content however reversed the partitioning trend of the protein for the moisture content. mean moisture content for the plastic packed fish was the least (13.160) and was significantly less than the moisture content obtained for the fish packed with nylon (15.587). the mean moisture content obtained for fish packed with basket was the highest (17.896 – table 4). dmrt however partitioned both the mean ash and mean crude fibre into two significantly different classes of similar trends. mean ash obtained for fish packed with basket was significantly higher (8.556) than the mean ash content obtained for fish with plastic (7.970) and nylon (7.881). similarly, mean crude fiber obtained for fish packed with basket (6.934) was significantly higher than those obtained for both nylon (6.426) and plastic (6.312) which falls within the same class (table 4). the glm analysis of the effects of the storage periods on fish nutrients quality (table 5) showed that there exist significant difference in the mean crude protein, moisture content, ash, fat content and crude fiber obtained for different storage periods. the f(10, 264:0.05) = 8394.09, 6194.90, 195.65, 206.38 and 55.82 obtained for crude protein, moisture content, ash, crude fat and crude fiber respectively were significant (p < 0.05). dmrt partitioned mean crude protein and mean moisture content into 11 significantly different classes. mean crude protein obtained for the 0das was significantly the highest (77.538) while mean crude protein obtained for the 140das was significantly the least 51.253 (table 5). dmrt however partitioned mean moisture content in a reverse order when compared to crude protein. mean moisture content obtained for the fish at the 0das was significantly the least (5.703) while food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 299 mean moisture content obtained for the 140das was significantly the highest (24.976). mean ash content were however partitioned into nine significantly different classes using dmrt. the partition was of the order )239.9(112....()28)863.6(14)569.6(0 dasdasdasdas  . the mean ash for the 126das (9.082) formed an intermediary class between 126das and 98das (table 5). similarly, mean fat was partitioned into eight significantly different classes. mean fat obtained for the 112das (8.336), 126das (8.088) and 98das (8.055) formed the highest significantly different class. this was followed by the mean fat returned for the 140das (7.720) and the mean fat obtained for the 42das remained the least 4.525 (table 5). mean crude fibers were partitioned into seven significantly different classes. mean crude fiber returned for the 112das was the highest but not significantly different from the crude fiber obtained for both 98das (7.229) and 126das (7.214). crude fiber obtained for the 0das (5.012) was significantly the least (table 5). table 5. general linear model (glm) analysis of the effects of storage period on fish nutrients parameters protein moisture ash fat crude fiber f(10, 264:0.05) 8394.09 6194.90 195.65 206.38 55.82 df 10 10 10 10 10 0das 77.538 a 5.703k 6.569i 4.949g 5.012g 14das 75.965 b 7.094j 6.863h 4.525h 5.553f 28das 72.124 c 9.332i 7.291g 5.242f 6.012e 42das 68.403 d 11.747h 7.574f 5.959e 6.317d 56das 64.789 e 14.364g 7.972e 6.330d 6.545cd 70das 62.299 f 15.796f 8.395d 6.796c 6.714c 84das 58.585 g 18.101e 8.649c 7.667b 7.003b 98das 56.211 h 19.593d 8.913b 8.055a 7.229ab 112das 53.559 i 21.439c 9.239a 8.336a 7.426a 126das 52.737 j 22.880b 9.082ab 8.088a 7.214ab 140das 51.253 k 24.976a 8.947b 7.720b 7.105b note: das = days after storage the least square mean analysis of the protein content of the fish decreases with storage period with the control (packaged with basket) maintaining the highest. fish seasoned with ginger and garlic as well as packaged with plastic maintained higher protein content after the control (figure 2). the least fish in term of protein content retention at the end of the experiment was the one packaged with basket and seasoned with ginger. the implication of this is that both the preservatives and the packaging materials have specific impact on the fish quality. also, analysis of the least square mean showed that moisture content on the other hand increases with increase in the period of the research. fish packaged with plastic and no preservatives had the highest moisture content while fish packaged with basket had the least. the least significant mean (lsm) analysis of the nitrogen returned a constant nitrogen values for each of the packaging materials by the preservatives over the research period (table 6). lsm for the nitrogen ranged between 12.24 for fish spiced with ginger and packaged with plastic and 13.04 (for fish spiced with ginger and packed with nylon. ash content showed a disparate trend from the nitrogen. the lsm analysis showed an increasing mean ash content over the period. the fish packaged with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 300 basket and spiced with ginger had the highest ash content of 11.24 at the end of the research (140das). it was however observed that the rate of increase differ from one treatment to another. table 6. least significant means of the fish nutrient content. nutrients 2gngr garlic ginger control period nylon plastic basket nylon plastic basket nylon plastic basket nylon plastic basket ash 0das 6.38 6.14 6.18 6.19 6.26 6.54 6.34 6.60 6.23 7.36 7.26 7.34 14das 6.72 6.54 6.54 6.53 6.69 7.15 6.39 6.67 6.89 7.46 7.26 7.53 28das 6.99 7.39 6.92 7.07 6.95 7.85 6.82 6.84 7.37 7.73 7.48 8.08 42das 7.08 7.53 6.95 7.14 7.12 8.09 7.22 7.31 8.13 7.84 8.13 8.35 56das 7.43 7.80 7.36 7.42 7.22 8.56 8.08 7.58 8.94 7.97 8.51 8.79 70das 7.93 8.37 7.73 7.87 7.40 8.99 8.14 7.82 10.44 8.48 8.76 8.81 84das 8.13 8.54 7.66 8.13 7.56 9.30 8.50 8.03 11.10 8.67 9.20 8.97 98das 8.13 8.87 7.74 8.41 7.66 9.31 8.41 8.40 11.57 9.24 9.36 9.52 112das 8.58 9.09 7.90 8.74 8.69 9.78 8.66 8.72 11.59 9.51 9.90 9.73 126das 8.43 8.94 7.87 8.57 8.60 9.48 8.43 8.41 11.47 9.31 9.78 9.68 140das 8.14 8.75 7.71 8.34 8.38 9.48 8.41 8.45 11.24 9.13 9.72 9.60 fat 0das 5.19 4.93 4.82 2.29 6.38 4.29 4.59 5.59 3.31 5.96 6.39 5.65 14das 5.63 4.78 4.77 2.29 1.29 4.70 4.51 5.59 3.32 5.86 6.39 5.16 28das 6.28 6.07 5.51 2.99 2.36 5.13 5.01 6.12 3.97 6.78 6.74 5.94 42das 6.63 6.16 6.71 4.62 3.16 5.99 5.71 6.75 5.00 6.88 7.18 6.71 56das 6.72 6.51 7.20 4.84 3.53 6.10 5.75 7.09 5.44 7.26 8.30 8.79 70das 7.24 7.02 7.73 5.44 3.93 6.52 5.97 7.27 6.20 7.66 8.70 7.87 84das 8.24 8.10 8.47 6.66 4.54 7.60 6.56 8.04 7.11 9.13 9.36 8.20 98das 8.24 8.68 8.60 7.04 5.16 7.77 7.08 8.14 7.42 9.88 9.62 8.70 112das 8.73 8.99 8.66 7.41 5.36 7.94 7.72 8.34 7.68 10.12 10.04 9.05 126das 8.40 8.88 8.66 7.13 5.29 7.50 7.70 7.75 7.49 9.66 9.45 9.14 140das 8.13 8.53 8.15 7.01 4.21 7.50 7.38 7.40 6.81 9.61 9.23 8.67 crude fiber 0das 7.20 5.53 6.98 8.61 6.02 4.57 6.62 5.32 7.42 1.08 1.03 1.42 14das 5.83 5.72 7.15 8.62 9.10 6.67 6.82 5.32 7.69 1.08 1.03 1.60 28das 6.27 6.20 7.52 9.06 9.54 7.32 7.13 5.85 8.19 1.58 1.61 1.88 42das 6.46 6.27 7.97 8.91 9.54 7.92 7.37 6.42 8.29 1.71 2.39 2.33 56das 7.20 6.40 8.40 9.33 10.02 7.92 7.59 6.42 8.64 2.07 2.54 2.63 70das 6.78 6.56 8.79 9.56 10.06 8.19 7.62 6.45 8.81 2.24 2.76 2.74 84das 7.04 6.88 8.77 9.66 10.30 8.41 7.62 6.87 9.17 2.90 3.27 3.01 98das 7.29 7.01 8.92 9.84 10.61 8.74 7.94 6.87 9.75 2.93 3.53 3.27 112das 7.50 7.12 9.07 10.02 10.80 9.25 8.20 7.05 9.85 2.99 3.63 3.64 126das 7.07 6.85 9.07 9.48 10.50 9.19 7.88 6.90 9.29 2.94 3.56 3.83 140das 6.92 6.70 8.77 9.20 10.26 9.19 7.87 6.86 9.21 2.88 3.56 3.62 note – das – days after storage 3.2. discussion. the essence of fish smoking is not only for presentation but preservation in case it is more than what can be consumed within acceptable quality guaranty period and for profit maximization. the goal of the present study has been the determination of shelf life of fish and nutrient quality of such fish using general linear model approach. it is noteworthy that crude protein and moisture content as well as other investigated nutrients of the fish are inversely related. also, the increasing trends of the moisture content of the fish can be hinged on the ability of smoked fish to absolve water or moisture from the surroundings and it is in agreement with [10]. similarly, the progressive increase in moisture content of the fish can food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 301 sufficiently cause decay and deterioration of the fish condition [11]. this increasing trend of the moisture content of the fish however, conflict with the [12]. fish smoked at different temperature (500c, 600c and 700c) were found to have declining moisture content when observed for 15 hours. the disparity in [12] and current study might be hinged on the length of storage or observation (140das against less than 1 day). the moisture content of the fish in our present study is far above 10 – 15% established by [13] and the fish still maintained its quality. declining crude protein as established in this present study is in line with [10], [14] and [15]. however, the declining rate of crude protein in [15] and the present study differ from each other. the difference could be hinged on differences in the species used in both cases. rana and chakraborty (2016) [15[ uses neotropius atherinoides while the present study uses clarias gariepinus. smoke-dried baim fish (6 month) was established to have better shelf-life than smoke-dried chapila (4 month) and kaika (4 month) fish [16]. the assumptions associated with the use of glm [17] include but not limited to; 1. response variable (which are the fish nutrients composition in our present study) is a continuous variable. 2. the error, ijk are normally distributed with mean zero. 3. equality of variance (though the variance are unequal but are minimal in our present study) for all values of i, j and k. 4. individual observations are independent. the data for the present study fulfilled some of these assumptions (continuous nature of the data, independent of observation and normal distribution of the error). 4. conclusion it can thus be concluded from the present study that baring any therapeutic, c. gariepinus can maintain its quality for 126 days after which its quality begin to deteriorate. although deterioration is an inevitable phenomenon in fish and fishery processing, the choice of appropriate packaging materials and preservatives would however enhance a longer shelve life. it is therefore recommended that the present study be extended to incorporate the effects of species on the shelf life of smoked fish. 5. references [1]. mackay, r. j. & oldford, r. w., scientific method, statistical method, and the speed of light. statist. sci. 15, 224–253, (2000). [2]. breiman, l., statistical modeling: the two cultures. statistical science, 16(3), 199–231, (2001). [3]. ware, m. e., chastain, j. d., computerassisted statistical analysis: a teaching innovation? teaching of psychology, 16, 222–227, (1989). [4]. amos, s.o., paulina, i., assessment of smoked fish quality using two smoking kilns and hybrid solar dryer on some commercial fish species in yola, nigeria. journal of animal research and nutrition, 2(1),1-7, (2017). [5]. bogard, j. r., thilsted, s. h., marks, g. c., wahab, m. a., hossain, m. a. r., jakobsen, j., stangoulis, j., nutrient composition of important fish species in bangladesh and potential contribution to recommended nutrient intakes. journal of food composition and analysis, 42, 120-133, (2015). [6]. dhaneesh, k.v., noushad, k.m., ajith-kumar, t.t., nutritional evaluation of commercially important fish species of lakshadweep archipelago, india. plos one 7(9), 1-7, (2012). [7] roth, b., oines, s., stunning and killing of edible crabs (cancer pagurus). journal of animal welfare. 19(3), 287 – 294, (2010). [8]. serkan, k., sevim, k., bekir, t. the effect of storage temperature on the chemical and sensorial quality of hot smoked atlantic bonito (sarda sarda, bloch, 1838) packed in aluminum foil. turkish journal of fisheries and aquaculture science. 10, 439-443, (2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ahmed ayodeji ayeloja, taofik oyedele dauda, general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish, food and environment safety, volume xxi, issue 4 – 2022, pag. 292 – 302 302 [9]. ayeloja, a.a., george, f.o.a., jimoh, w.a., shittu, m.o. , abdulsalami, s. a., microbial load on smoked fish commonly traded in ibadan, oyo state, nigeria. journal of applied sci. environ. manage. 22(4), 493 – 497, (2015). [10]. daramola, j.a., fasakin, e.a., adeparusi, e.o., changes in physicochemical and sensory characteristics of smokedried fish species stored at ambient temperature. african journal of food agriculture nutrition and development, 7(6),1 – 16, (2007). [11] olayemi, f. f., adedayo, m. r., bamishaiye, e. i. , awagu, e. f., proximate composition of catfish (clarias gariepinus) smoked in nigerian stored products research institute (nspri) developed kiln. int. j.fisheries aquaculture. 3(5), 96-98, (2011). [12]. idah, p. a., nwankwo, i., effects of smoke-drying temperatures and time on physical and nutritional quality parameters of tilapia. international journal of fisheries and aquaculture. 5(3), 29-34, (2013). [13]. jallow, s. s., identification of and response to drought by local communities in fulladu west district, the gambia. singapore journal of tropical geography. 16(1), 22-41, (1995). [14]. boran, g. , karaçam, h., seasonal changes in proximate composition of some fish species from the black sea. turkish journal of fisheries and aquatic sciences. 11, 01-05, (2011). [15]. rana, m.m. , chakraborty, effect of salt and smoke on quality and shelf life of saltsmoke-dried batashi (neotropius atherinoides) kept at different storage condition. research in agriculture, livestock and fisheries. 3(3),443 – 451, (2016). [16]. nahid, m.n, latifa, g.a., chakraborty, s.c., farid f.b., begum, m, shelf-life quality of smoke-dried freshwater sis fish; chapila (gudusia chapra, hamiltonbuchanan; 1822) kaika (xenentodon cancila, hamilton-buchanan; 1822) and baim mastacembelus pancalus, hamilton-buchanan; 1822) stored at laboratory condition (260-310c). journal of agriculture and veterinary science. 9(1), 23-32, (2016). [17]. gomez, k.a., a.a. gomez,. statistical procedures for agricultural research (2 ed.). john wiley and sons, newyork, 680p, (1984). 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2022.035 364 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 364 381 recovery of bioactive compounds from oilcakes a review *ancuța petraru 1 , sonia amariei 1 1faculty of food engineering, ștefan cel mare university, suceava, romania * ancuta.petraru@fia.usv.ro received 15th september 2022, accepted 26th december 2022 abstract: worldwide, food waste and by-product are concernig topics, due to the fact that annually large amount of these are generated from the food industry. their management should include first waste prevention, recycling, recovery and lastly disposal. the concerning awareness of consumers regarding healthy diets has led to an increase demand for foods containing bioactive compounds, namely proteins, dietary fibers and antioxidants with numerous health benefits. in other words, the food industry is heading towards innovations by using wastes as raw materials for the development of new products or applications. this paper provides a critical review of the possible ways to recovery the nutritive compounds from oilcakes obtained after the oilseed processing. keywords: circular economy, bioactive compounds, oilcake, protein, dietary fiber. 1. introduction the current global issues (overexploitation and mismanagement of resources, unsustainable consumption behaviour, climate change and degradation of the environment) caused by the impact of human activities, calls for a transition towards a circular economy. in this system the idea of waste is not contemplated because it is recycled and re-circulated within the production [1,2] . food wastes and by-products are the leftover edible materials (lost, discarded degraded or consumed by pests) generated from the food industry, in all phases, from agricultural production up to direct consumption into households [3]. the management of these poses a challenge due to the cost related to their handling. however, the increasing consciousness about environment issues and the legislative pressure require new methods for food waste recovery, rather than its disposal [4]. the most common practices of valorisation are animal feed, composting and biofuel conversion, but unfortunately they only provide a partial use of food industry waste [5]. lately the food waste was acknowledged as a rich source of various nutraceuticals and valuable compounds [6,7]. these compounds (pigments, fibers, minerals, proteins and antioxidants) can be reintegrated in the food, agricultural, cosmetic and pharmaceutical industries [8]. the extraction includes three stages: in the first the materials are subjected to a pretreatment to remove microbes without affecting biological activities, the second step is extraction trough classical (solvents, maceration, stream distillation) or novel/greed methods (pulsed electric field, supercritical fluid, microwave, ultrasound, high voltage electric discharge extractions, the final stage involve purification of the final product [9]. in order to ensure the protection of human health, the legal permissibility of the high valuable components as input substance is fundamental. they must undergo a safety assessment before their placement on the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 365 market. the bioactive compounds are separated from the food matrix trough operations without creating hazards and in the final must comply with the existing food regulations [4,10]. oilcakes are the principal by-products of the oil industry with many bioactive compounds, such as dietary fibers and proteins, that can be isolated and after consumption, provides positive health benefits and unique properties in the food industry [11,12]. this paper provides a critical review of the possible ways to valorisate the oilcake resulted from the oil industry and the methods used for the recovery of bioactive compounds. 2. extraction and identification of bioactive compounds from food waste and byproducts fig. 1. the main applications of bioactive compounds extracted from oilcakes 2.1. proteins during the last decade the rising world population and limited resources poses a challenge for protein supply, encouraging their searching in alternative sustainable and renewable sources [13]. the food agriculture organization has stated that proteins will be limited in the future so that a sufficient quantity and quality of proteins will become a fundamental right of every citizen. a balancing protein intake from both animal and vegetables sources is recommended [14]. the most widespread protein extraction methodology involves two steps process: an alkaline solubilization with a removal of insoluble material by centrifugation and an isoelectric precipitation (ph 4-5), followed by centrifugation and neutralization. alternative and improved version for this method have been developed. three methodology were developed: aqueous extraction, dry techniques and combined approaches [15]. aqueous extraction is a process in which water alone or in combination with acid, basic or saline agents is used as extraction medium. this extraction is followed by an isoelectric precipitation and then a membrane filtration technique for purification [16]. extraction with alkaline solution offers high protein yield, but at the same time has food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 366 some disadvantages such as low oil extraction yield, the presence of chemical contaminants in the end products and the loss of protein functionality [17,18]. at ph>12 the proteins suffer an irreversible denaturation and a decline in nutritive value [19]. alkaline extraction also can provoke maillard reaction, which causes the darkening of the final products color and negatively affects smell and taste [20]. acid extraction in comparison with basic extraction are less efficient regarding the protein content obtained in the final product [21]. taking into account all the previous observations an extraction with nacl at a moderate basic ph value could be a good alternative to obtain proteins with high yields without compromising their functionality and nutritional value. another drawback is the necessity to dry the final product, which increase the costs [15]. the dry methodologies includes always a milling or deagglomeration pre-treatment [22]. the procent of protein obtained (40%) by dry methods is less than those obtained by aqueous extraction (80%) [16]. the advantages of these methods are: low physical impact on the particles, preservation of the native structure, the involvement of less expenses. however, the expensive equipment is the major drawbacks [15]. enzyme treatments are an interesting approach due to the enzymes superior mode of action that allows the targeting towards specific components of the protein bonds. the cost of enzymes plus the difficulty to recycle the treatment stream works against this option [23]. the method used for protein extraction should achieve the following: obtaining of high yield, reduction of the number of purification steps, preservation of the nutritional and functional values, elimination of antinutrients and reduction of the environmental impact [15]. taking all these factors the aqueous extraction is the most preferable. 2.2. antioxidants, phenolic compounds and polyphenols antioxidants are compounds that at relatively low concentrations can inhibit or delay the oxidation process [24]. antioxidant functionality can be explained with the following mechanisms: scavenging free radicals that generate and distribute peroxidation, chelating metal ions that hasten the oxidation process, prevention of hydroxyl radical and interruption of the auto-oxidation reaction [25]. phenolic compounds are the largest group present in phytochemicals. the compounds include phenols with one aromatic ring, polyphenols with more than one aromatic ring and hydroxyl group and their derivates such as glycosides and esters. phenolic acids and their derivates (lignans, tannins, flavonoids) are the most important class of polyphenols [26]. there is no universal method for the extraction of antioxidants, but for a method to be suitable several requirements must be fulfilled such as selectivity, high extraction yield, possibility to recover solvent, the use of green solvent, mantaining the functionality of the recovered molecules [27]. the classical methods include maceration (solid-liquid extraction) and the use of solvents. the solvent used depends on the type of material and compounds that need to be recovered [10]. for flavonoids and proanthocyanidins, the extraction yield was improved when organic acids in combination with water were used [28,29]. these conventional methods present some limitations regarding the high amount of solvent and development time. to overcame these limitation green/innovative/alternative/sustainable processes (microwave, ultrasound, pressurized liquid, enzyme extractions) can food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 367 be used [10] . the identification and quantification of the recovered antioxidants is realized through high pressure liquid chromatography and spectrophotometric methods (uv-vis). the antioxidant capacity of the recovered compounds with 2,2-diphenyl-1picrylhydrazyl (dpph), ferric reducing antioxidant power (frap), 2,2’-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (abts), cupric reducing antioxidant capacity (cuprac), oxygen radical absorbance capacity (orac) and peroxide value [27]. the application and effectiveness of polyphenols in the functional foods, nutraceutical, and pharmaceutical industries depends on their stability, bioactivity and bioavailability [30]. moreover, many phenolic compounds have low water solubility and an unpleasant taste, that must be masked before their incorporation in foods or medicines [31]. the shelf life and stability of phenolics can be improved with microencapsulation (a process in which particles are coated with a homogeneous or heterogeneous matrix that act as a physical permeability barrier) [32]. 2.3. dietary fibers dietary fiber (df) includes a mixture of plant carbohydrate nonstarch polysaccharides, that enters in the cell wall composition [33]. fiber is an essential nutrient resistant to the human digestive enzymes in the small intestine [34]. depending on the water solubility, dietary fiber can be classified as soluble or insoluble [35]. soluble df include pectin (sugars from whole grain, legumes, fruits, etc.), inulin (agave, garlic, onion, artichoke, etc.), gums (sugar from beans, legumes, oatmeal, etc) and mucilage (from aquatic plants, cactus, aloe vera, food additives, etc.), whereas insoluble df include cellulose (from fruits, root vegetables, etc.), hemicellulose (sugars from cereal bran and grains), lignin (aromatic alcohols from vegetables) and resistant starch (uncooked potato, banana, legumes, oats) [36]. the soluble and insoluble forms vary in physicochemical and physiological properties [37]. soluble forms presents water solubility, viscosity and the ability to reduce blood lipids and glucose intestinal absorption [4]. the insoluble form is characterized by its porosity and density, and promotes the proper functioning of the intestinal tract (increase fecal bulk, decease intestinal transit time, enchance intestinal peristalsis) [38]. fibers are fractioned in individual constituents in order to eliminate unwanted compounds [39]. all these affects also the behavior of the fiber in the human body and in the food applications [33,40]. moreover the cost, time, yield, technological characteristics and lost functionalities occurring change considerably depending on the fractionation process applied [41]. df can be extracted through traditional (dry, wet, chemical, gravimetric, enzymatic, physical, microbial process or a combination of the latters) [42] or green/innovative methods (ethanol, water, steam, ultrasonic, hydrostatic pressure, and pulsed electric field extractions) [43]. the dry methods involve the disintegration by milling and air classification to separate the fiber fraction. wet milling methods (includes conventional, alkali and enzymatic) use water for fiber extraction and differs by the addition of reagents and conditions. physical methods were performed to preserve the structure of fibers. through microbial methods, a fermentation of the fibers is carried out with the help of microorganisms and enzymes [33]. an ideal extraction method should be environmentally friendly, safe, easy to perform and cost-effective. the chemicals, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 368 enzymes and equipments used to perform the majority of the previous presented methods tend to be very costly. the methods have different advantages and limitation presented in table 1. from all these methods wet-milling presents better extractibility, produce high quality fibers (purity 50-90%), it is cost effective and use small amounts of reagents and water [33,36,39]. table 1. advantages and limitations of fiber extractions methods extraction methods advantages limitations references dry processing -reduced water and energy consumption -it does not imply the use of chemicals -compared to wet milling the process is costeffective, environment friendly and high yield require repeated classification to purify fractions/reduces product recovery -suitable for plants that presents starch as their main storage material [44,45] wet milling -allows the obtaining of an appreciable amount of fibers -usage of water and minimal chemicals -time-consuming (36 hours) -very costly -usage of a large amounts of sulfur dioxide (so2), that is environmentally unfriendly and can produce severe respiratory disorders [42,46] alkali wet milling -produces a relatively environmentally acceptable stream of waste water -time-consuming -tedious -the method can damage the molecular structure of dietary fiber [36,46] modified wet milling -high purity products -it does not imply the use of reagents -it used less water -it is cost-effective -produce waste water [33] chemical -removal of starch and protein can be more complete -poor selectivity -difficulty in controlling extraction conditions -decrease in overall fibers physiological activity -creation of significant waste of strong acids and alkalis, unfriendly with the environment [39,47] enzymatic wet milling -alternative method to reduce so2 (at minimal levels that impart only antimicrobial properties) produced with the conventional wet milling -reduction of the processing time -possible so2 residues in final product [42] food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 369 enzymaticgravimetric -higher yield of fiber compared to enzymaticchemical methods -quick and easy to carry out -some insoluble polysaccharides, lignin and soluble polysaccharides are lost -resistance starch is not quantified in totality -the residues contain some nitrogenous material [48] enzymaticchemical -quick and easy to perform compared to enzymaticgravimetric methods -chemical residues in the obtained products -time-consuming -decrease in the dietary fiber yield (loss of polysaccharides during hydrolysis and chemical pre-treatment) [49,50] nonenzymaticgravimetric -provides fibers with high purity -poor selectivity -difficulty to control the extraction conditions [48] physical -the structure of the obtained fibers is mantained -unreliable [33,39] microbial -high selectivity -the structure of the obtained fibers is mantained -reaction conditions are easy to control -production of toxic substances [33,39] recently green extractions are widely used for extraction purposes. the innovative methods present advantages such as easy reproductibility, high quality extraction and low environmental impact [51,52]. however, there are some drawbacks for these modern extraction methods as follows: high temperature consumption (microwave extraction), separation issues (ultrasonic extraction) and lack of user friendliness (electric field extraction) [36]. 3. oilcake: potential source of bioactive compounds worldwide oilseed crops are the major grown agricultural commodities. oilseeds are crops grown all over the world mainly for the oil extraction. they are rich in proteins, fibers, fatty acids (mono and polyunsaturated), minerals, antioxidants and vitamins [53]. more than 61% of the total production in 2020/2021, was dominated by soybean, followed by rapeseed, peanut, sunflower, cottonseed and groundnut [54]. from oilseed processing (performed by either solvent extraction or mechanical pressing) results significant amounts of peels, oilcakes and oil sludge. oilcakes are the by-products resulted after the oil extraction from the oilseeds [55], either by mechanical (oil content around 6-7%) or solvent (<1% oil) extraction [56]. the chemical composition of seed oil cake depends on different factors, mainly extraction method, plant variety and growing conditions [40]. sesame, walnut, pumpkin and almond oil cake, despite the seed defatting process contain high amount of oil (5.10-48%) [4,57–65]. instead, olive, hemp and sunflower oil-cake are rich in fibers and carbohydrates [66–75]. the protein content is high in all oil-cakes, varying between a maximum of 60% in groundnut and a minimum of 24.79% in cottonseed [40,64,70,74]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 370 the most conventional practices of valorisation include animal feed, landfilling, and biofuel conversion [4]. they can be used as animal feed due to the rich content in protein and fibers. unfortunately, the presence of toxic/antinutritional compounds can affect both animals and humans [56]. landfilling through the decomposition leads to methane production and thus to water pollution. the conversion in energy is realized through incineration, this sparked concerns about the negative impact of the emissions [5]. new valorisation methods include the recapturing of valuable components for the development of new products and production of biopolymer films [76]. from oil press-cakes can be extracted proteins, antioxidants, phytochemicals, and dietary fiber. 3.1. proteins plant proteins from oilseeds and their byproducts gained increasing attention due to their relative low cost [77]. moreover, the proteins are gluten-free and represent an alternative for those of animal origin since they are easily digestible, non-toxic and high nutritive [78]. in the case of oilcakes when the material is defatted the aqueous extraction facilitates the recovery of proteins at high concentrations (80%). the use of full fat material led to a low protein content (40-50%). the use of solvents as ethanol, methanol or acetone also achieved low protein content in the concentrate (4560%) [15]. however, helling et al. [79] observed that with an aggressive alkaline solution with ph higher than 10 the fat can be dissolved. aqueous method facilitates the removal of antinutrients present in the samples, while in the dry methodologies the absence of an antinutritional removal step is a major drawbacks [80,81]. proteins from rapeseed contain a wellbalanced amino acid composition with large amounts of sulfur amino acids that exceed the requirement for adults and children. the dominating protein groups are napin and cruciferin, together represents 85-90%. in terms of nutritional value the protein ca be compared with those from soy [82]. rapeseed protein extracted with ultrasonic and ultrafiltration methods presented good functional properties (solubility, emulsifying capacity and stability) compared to soybean protein, indicating these proteins as a potential replacement of other proteins [83]. protein extracted from soybean and groundnut oilcakes can be absorb and digest easily and nutritionally are equivalent to animal protein. in the amino acidic profile can be observed the absence of methionine in soybean and the rich content in arginine found in groundnut. linseed oilcake proteins are rich in arginine, glutamic acid and aspartic acid. moreover, the proteins presents antifungal and emulsifying properties [84]. sunflower oilcakes proteins have high nutritional value and are easily digestible [85]. the essential amino acids present are cysteine, methionine, leucine, valine, isoleucine, tryptophan, alanine and phenylalanine [86]. from oilcake can be prepared protein hydrolysates, isolates (>90%) and concentrates (30-80%) [23]. the isolates are obtained by solubilization with alkali at high ph, isoelectric precipitation with acid and then washing and drying. isolates presented high water holding capacity, emulsion activity and stability. for this reasons they find application as emulsifier and functional ingredients [87]. protein isolates from canola cannot be used as food ingredients due to their poor technological and solubility abilities [88]. good water, fat, emulsifying and foaming properties were found in flaxseed and sesame isolates [65]. hydrolysates are obtained after the hydrolysis of protein isolates. through this food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 371 process the protein structure is modified (improvement of functionality, solubility, hydratation and gelling abilities) and a fragment of protein, known as bioactive peptone, is created. bioactive peptone presents biological, antioxidant, antithrombotic, hypercholesterolemic, immunomodulatory activities [56]. peptides from sesame and rapeseed presented antihypertensive, antioxidant and bile acid binding activities, while those obtained from peanut have antithrombotic activity [89–92]. 3.2. antioxidants oilcakes contains natural antioxidants (free, esterified, condensed phenolic acids, flavonoids and lignans) that play the role in reducing oxidative stress and thus preventing various types of cancers [56]. these compounds can be extracted with different methods (with organic or nontoxic solvents, high pressure, microwave and supercritical fluid) and used in the preparation of a variety of foods (bakery, beverages and extruded products) [10]. the principal antioxidants present in sunflower, rapeseed, coconut, mustard, cotton and sesame oilcakes are the phenolic compounds [56]. the major antioxidants in oilcakes are presented in table 2. table 2. major antioxidants in oilcakes oilcakes antioxidants references rapeseed/canola oilcake gallic acid, p-coumaric, catechin, caffeic acid, epicatechin, ferulic acid, quercetin, luteolin, sinapic acid [40,69,90,93,94] linseed/flaxseed oilcake tannic acid, p-coumaric, ferulic acid, lignans, phydroxybenzoic acid [40,56,93,94] peanut oilcake p-coumaric, caffeic acid [40,56,93] sunflower oilcake p-coumaric, catechin, caffeic acid, epicatechin, chlorogenic acid [40,69,93,95] sesame oilcake p-coumaric, ferulic acid, lignans [40,93,96,97] mustard oilcake p-coumaric, caffeic acid, ferulic acid, sinapic acid [40,56,93] palm oilcake p-coumaric, caffeic acid, ferulic acid, p-hydroxybenzoic acid [40,56,93,94] cottonseed oilcake ferulic acid, quercetin [40,93,97] hempseed oilcake caffeic acid, quercetin, luteolin [40,56,93] olive oilcake p-coumaric, quercetin, lignans, p-hydroxybenzoic acid [40,56,93,94] walnut oilcake gallic acid, ferulic acid, p-hydroxybenzoic acid [40,93,98] sesame oilcake contains phytochemicals (phenolic compounds, flavonoids, tocopherols, vitamins, carotenoids, lignans, pigments and steroids) with numerous benefits for health such as antioxidant, anticarcinogenic, antiproliferative, antimicrobial, anti-inflammatory, neuroprotective, and hypocholesterolemic activities [99]. 3.3. dietary fibers the increasing consumers awareness for the nutritional benefits of dietary fiber explained the demand for high fiber foods [100–102]. in order to meet this demand new alternative source of dietary fiber need to be studied, such low-cost source can be the oilcakes resulted from oilseed processing. df from oilcakes can be used as functional ingredient, supplements or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 372 additive in the food and pharmaceutical industry. sun et. al. [103] used a combined extraction method (ultrasonic and alkali) for the extraction of insoluble df. the results indicated that comparative to the classic methods the temperature can be reduced to 30˚c, the time to 10 min and the consumption of alkali can be reduced up to 95%. moreover, the physicochemical properties of dietary fibers are better than those obtained with the conventional methods because the ultrasound treatment changed the structure of df, increasing the amount of short-chain and surface area. zheng et al. [104] studied the physicochemical and functional properties of defatted coconut df that was subjected to acidic treatment, enzymatic hydrolysis and particle size distribution. the hydrolysis and particle size reduction caused structural modification, an increment in water holding capacity, water swelling capacity, soluble df content and a decrease in oil holding capacity, emulsion capacity and color. the opposite observations were obtained with acidic treatment. 4. bioactive compounds from oilcakes in foods the recovered compounds extracted from oilcakes can be re-utilized as food additives, functional foods, pharmaceuticals, cosmeceuticals and biopackaging. nowadays, consumers are concerned about a healthy diet, since nutrition is associated with many lifestyle diseases like obesity, cancers and diabetes [100]. plant proteins are used in foods due to their good fat/water absorbing, emulsifying, texture-modifying properties [105]. fibers are used in various food products to alter their consistency, rheological behavior, texture and sensory properties. moreover, fibers can be employed to improve shelf life due to their gel forming, water holding, fat mimetic and thickening abilities [33]. the addition of antioxidants to lipid-rich foods can reduce lipid-oxidative reactions. in foods are widely added synthetic antioxidants, such as butylated hydroxytoluene (bht), due to their efficacy and low cost. however, natural antioxidants are more accepted by consumers [106]. new food products can be developed with two categories of ingredients, the first includes new and alternative food ingredients and in the second enters the ingredients obtained by the valorisation of by-products. for a successful implementation, the marketing strategy applied need to include new regulations, customer education and clear transparency (better communication, adequate labelling) [107]. when ingredients with important benefits for consumers (functional ingredients) are introduced, the final products bears the name functional [56]. residues from the oil industry could be used as co-products for high value-added products, food additive or supplements [108]. 4.1. flaxseed oilcake (foc) foc is rich in omega 3 fatty acids, proteins, insoluble and soluble df, lignans, vitamins (a, c, d, e) and minerals with role in colon cancer prevention and reduction of cardiovascular diseases [109]. increasing interest for new vegetarian/vegan products for consumers with intolerance to dairies led to the production of kefir-like fermented beverages with different amounts of flaxseed cake. products with high percentages presented high viscosity (presence of mucilage and protein) and antioxidant activity (production of phenolic compounds and bioactive peptide [110]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 373 4.2. peanut oilcake (pnoc) pnoc is an excellent source of proteins with emulsion and foam properties. the high nutritional value makes it suitable for the improvement of the physicochemical properties of pasta and cookies extraction and evaluation of functional properties [111,112]. protein isolates from pnoc can form alone brittle films. for this reason it is necessary the introduction of glycerol or a crosslinking process to improve mechanical properties without influencing the barrier properties [113,114] 4.3. rapeseed oilcake (roc) sausages with canola protein concentrates in place of casein presented improved sensory properties (taste, texture and aroma) [90]. proteins from roc cannot be used alone in the formation of biopolymers films because they present bad mechanical and antimicrobial activities, therefore it is necessary to use emulsifiers and plasticizers. taking this into account proteins hydrolysate from roc with chitosan presented antimicrobial activity against staphylococcus aureus, bacillus subtilis, and escherichia coli [76,92]. 4.4. sunflower oilcake (sfoc) sfoc is a good source of antioxidants, proteins and fibers that can be used for emulsion applications. kreps et al. [115] compared the antioxidant potential of sfoc with a synthetic antioxidant and observed that the oilcake showed more stability during 56 day storage. the antioxidant potential is due to p-coumaric, catechin, epicatechin and chlorogenic acid. protein concentrates extracted from sfoc present the following properties: dissolvation appropriately in water, forming foam, emulsion at different ph and gel production. however, their enrichment with phenolic compounds limited their application in food because the phenolic acids react with the proteins decreasing solubility, digestibility, affect color and shelf-life of products [116]. when introduced this in biopolymer films, salgado et al. [117,118] obtained films with antioxidant properties, good properties (high water solubility) and the percentage of phenolic compound to aqueous phase was high. protein isolated from sfoc can be used to form films with good characteristics: good mechanical, barrier and adhesive characteristics, resistance to fats and organic solvents. further, the films are more elastic and resistance than those obtained with protein isolated from soybean oilcake [119–121]. 4.5. sesame oilcake (soc) soc as great potential in increasing nutritional values, sensory and physicochemical properties [12]. the lignans extracted from soc at lower amount (150 ppm) can be used as food additive for the improvement of oil stability [96]. the lipid oxidation time was reduced in butter when soc was used in comparison to when bht was used without changing sensory properties [122]. sesame meal introduced (5-20 ppm) in sunflower and soybean oil inhibited thermal deterioration and loss of polyunsaturated fatty acids due to the redox properties of the phenolic compounds [60] . flavonoids and phenolics acids extracted from soc and coc and bht were introduced in vanilla cake compositions to compare their potential to improve their chemical, microbial and oxidative stability. the natural oilcakes mantained the stability up to 13 days compared to the 11 days mantained by the synthetic food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 374 antioxidant. the natural antioxidants are stable thermally and mantained the sensory quality up to 12 days [123]. the introduction of soc flour in biscuits increase the resistance to microbial contamination, thus improving their shelflife [108]. the introduction of 10% sesame protein concentrate in extruded snacks improved organoleptic properties, color and protein content, while lowering the carbohydrate content [124]. sharma et al. [125] developed films with different sesame protein isolate (spi) and gum rosin (gr) ratios. the highest tensile strength and lowest wvp, moisture and solubility were found for the 80:20 ratio. the increase in gr percentage improved the optical properties (increase and decrease in films transparency and color). the addition of gr also improved the structure (porosity decreased and compactiness increased). considering all, the addition of gs to spi composite films improved their mechanical, optical, morphological and physical properties. 4.6. walnut oilcake (woc) food can act as a vector for bioactive ingredients. the incorporation woc as a replacement for wheat flour. increasing the level of oilcake enrichment led to a bread with a high antioxidant potential compared to the control [126]. the addition of walnut oilcake in macaroons improved their quality (decrease in carbohydrates, omega6/omega-3 ratio and energy). the same was observed for cakes. however, the addition of more than 10% for macaroons and 15% for cakes was not appreciated, due to the increase in phenolic and volatile compounds that affect the taste [127,128]. grosso et al. [106] evaluated the performance of polyphenols extracted from woc on the preservation of walnut oil obtaining good antioxidant properties against oxidation. 4.7. soybean oilcake (sboc) sboc is a source of high quality of proteins, essential amino acids, antioxidants (isoflavones, phenolic compounds), dietary fibers, minerals and fat that can be used in a food and other sectors [12]. biscuits and cookies with sboc have better antioxidant capacity, due to isoflavones content. the other nutritional compounds enchance the physicochemical, functional, nutritional and sensory properties [129,130]. for the production of films, protein extracted from sboc are superior in comparison to other plant proteins. the films are clean, smooth, flexible and presented adhesive ability. unfortunately, during long term storage, they suffer an aging that can be improved by crosslinking [15,76]. the direct incorporation of fiber from sboc produced films with poor filmforming properties, for this reason a dynamic pressure micro-fluidization treatment was performed. the final product presented excellent barrier and mechanical properties that can be used as packaging material for instant noodle, beverages, biscuits, sausages and candies [131]. 4.8. pumpkin oilcake (poc) poc is a source of natural macromolecules with film-forming ability. protein isolates obtained from poc were used as substrate for the development of biopolymer films. the resulted films had gas barrier properties 150-250 times better than the commercially polyethylene and polypropylene films [132]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 375 4.9. hempseed oilcake (hoc) worldwide hempseed, grown primarily for the production of fabrics and papers, are gaining increasing interest for oil production. industrial hemp although contains trace amount (<0.3%) of δ-9tetrahydrocannabinol (thc) is suitable for agricultural production [6]. the incorporation of oilcakes rich in antioxidants, essential fatty acids (desirable omega-6/omega-3 ratio), fiber, and minerals led to the development of healthy, non-caloric, and high nutritional snacks. the highest appreciation was achieved with the highest addition level [133]. the supplementation with 20% in bread formulation with hemp flour resulted in products with higher nutritional value (elevate intake of important nutrients such as proteins and macroand microelements, especially iron) [134]. 5. conclusion the food industry generates enormous amounts of wastes, which opens a research area with the role to manage and minimizate them efficiently to support the circular economy concept. the wastes remain currently underutilized. the main solutions for reducing wastes are conversion in energy, introduction in livestock and fish feedstuff, production of fertilizer and compost. another solution is the extraction of the maximum value (phenols, fibers, antioxidants, proteins, etc.) and they reintroduction in foods, pharmaceutics, cosmetics, and textiles. this represents a challenge, but at the same time could add more value to food, reducing disposal costs and risks caused by residues. bioactive compounds, extracted from oilcakes have beneficial health effects. the extraction processes depend on several factors: technique used, type of raw material and organic solvent used. conventional technique presents some drawbacks: use of large amounts of solvents, is time and energy consuming. for these reasons, there is an increasing interest for greener technologies, that are more friendly with the environment. moreover, the production of ingredients or additives from food wastes and byproducts may contribute to lower some nutritional problems. 6. acknowledgments this work was performed within the framework of the “decidedevelopment through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu/380/6/13/125031, supported by project co-financed from the european social fund through the 2014 – 2020 operational program human capital”. 7. references [1]. esposito b., sessa m.r., sica d., malandrino o., towards circular economy in the agri-food sector. a systematic literature review. sustainability (switzerland), 12, (2020). [2]. pesce m., tamai i., guo d., critto a., brombal d., wang x., cheng h., marcomini a., circular economy in china: translating principles into practice. sustainability (switzerland), 12, 1–31, (2020). [3]. ojha s., bußler s., schlüter o.k., food waste valorisation and circular economy concepts in insect production and processing. waste management, 118, 600–609, (2020). [4]. mateos-aparicio, i.; matias a.-t.r. of a., 2019 u., food industry processing by-products in foods. the role of alternative and innovative food ingredients and products in consumer wellness elsevier inc., (2019). doi:10.1016/b978-0-12816453-2.00009-7. [5]. otles s., despoudi s., bucatariu c., kartal c., food waste mangement, valorization, and sustainability in the food industry. in food waste recovery , ed. galanakis c.m., 3–23, elsevier inc., (2015). doi:10.1016/b978-0-12800351-0/00001-8. [6]. pojić m., mišan a., sakač m., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 376 hadnacrossed d signev t.d., šarić b., milovanović i., hadnacrossed d signev m., characterization of byproducts originating from hemp oil processing. journal of agricultural and food chemistry, 62, 12346–12442, (2014). [7]. galanakis c.m., recovery of high addedvalue components from food wastes: conventional, emerging technologies and commercialized applications. trends in food science and technology, 26, 68–87, (2012). [8]. c. fărcaş a., a. socaci s., m. diaconeasa z., introductory chapter: from waste to new resources. in food preservation and waste exploitation , ed. socaci, s.a.;fărcaş, a.c.; aussenac t., 1–11, intechopen, (2020). doi:10.5772/intechopen.89442. [9]. fierascu r.c., fierascu i., avramescu s.m., sieniawska e., recovery of natural antioxidants from agro-industrial side streams through advanced extraction techniques. molecules, 24, 1–29, (2019). [10]. baiano a., recovery of biomolecules from food wastes a review. molecules, 19, 14821– 14842, (2014). [11]. oreopoulou v., tzia c., utilization of plant by-products for the recovery of proteins, dietary fibers, antioxidants, and colorants. utilization of by-products and treatment of waste in the food industry, 209–232, (2007) doi:10.1007/978-0-38735766-9_11. [12]. abedini a., alizadeh a.m., mahdavi a., golzan s.a., salimi m., tajdar-oranj b., hosseini h., oilseed cakes in the food industry: a review on applications, challenges, and future perspectives. current nutrition & food science, 17, 1–20, (2021). [13]. sá a.g.a., moreno y.m.f., carciofi b.a.m., plant proteins as high-quality nutritional source for human diet. trends in food science and technology, 97, 170–184, (2020). [14]. campbell l., rempel c.b., wanasundara j.p.d., canola/rapeseed protein: future opportunities and directions—workshop proceedings of irc 2015. plants, 5, 281–284, (2016). [15]. arrutia f., binner e., williams p., waldron k.w., oilseeds beyond oil: press cakes and meals supplying global protein requirements. trends in food science and technology, 100, 88– 102, (2020). [16]. mullen a.m., álvarez c., pojić m., hadnadev t.d., papageorgiou m., classification and target compounds. food waste recovery: processing technologies and industrial techniques (2015). doi:10.1016/b978-0-12800351-0.00002-x. [17]. zaky a.a., shim j.h., abd el-aty a.m., a review on extraction, characterization, and applications of bioactive peptides from pressed black cumin seed cake. frontiers in nutrition, 8, 1–11, (2021). [18]. tabtabaei s., diosady l.l., aqueous and enzymatic extraction processes for the production of food-grade proteins and industrial oil from dehulled yellow mustard fl our. frin, 52, 547–556, (2013). [19]. manamperi w.a.r., wiesenborn d.p., chang s.k.c., pryor s.w., effects of protein separation conditions on the functional and thermal properties of canola protein isolates. 76, 266–273, (2011). [20]. rodrigues i.m., carvalho m.g.v.s., rocha j.m.s., increase of protein extraction yield from rapeseed meal through a pretreatment with phytase. journal of the science of food and agriculture, 97, 2641–2646, (2017). [21]. sari y.w., bruins m.e., sanders j.p.m., enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals. industrial crops and products, 43, 78–83, (2013). [22]. rivera j., siliveru k., li y., a comprehensive review on pulse protein fractionation and extraction : processes , functionality , and food applications. (2022) doi:10.1080/10408398.2022.2139223. [23]. singh r., langyan s., sangwan s., rohtagi b., khandelwal a., protein for human consumption from oilseed cakes : a review. 6, 1–12, (2022). [24]. francenia santos-sánchez n., salascoronado r., villanueva-cañongo c., hernández-carlos b., antioxidant compounds and their antioxidant mechanism. in antioxidants 1–28, (2019). doi:10.5772/intechopen.85270. [25]. abeyrathne e.d.n.s., nam k., huang x., ahn d.u., plant-and animal-based antioxidants’ structure, efficacy, mechanisms, and applications: a review. antioxidants, 11, (2022). [26]. mourtzinos i., goula a., polyphenols in agricultural byproducts and food waste. polyphenols in plants elsevier inc., (2019). doi:10.1016/b978-0-12-813768-0.00002-5. [27]. socaci s.a., rugină d.o., diaconeasa z.m., pop o.l., fărcaș a.c., păucean a., tofană m., pintea a., antioxidant compounds recovered from food wastes. 3–22,. [28]. dorta e., lobo m.g., gonzález m., optimization of factors affecting extraction of antioxidants from mango seed. 1067–1081, (2013) doi:10.1007/s11947-011-0750-0. [29]. dailey a., vuong q. v, effect of extraction solvents on recovery of bioactive compounds and antioxidant properties from macadamia ( macadamia tetraphylla ) skin waste. 1–10, (2015) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 377 doi:10.1080/23311932.2015.1115646. [30]. fang z., bhandari b., encapsulation of polyphenols a review. trends in food science and technology, 21, 510–523, (2010). [31]. munin a., edwards-lévy f., encapsulation of natural polyphenolic compounds; a review. pharmaceutics vol. 3 (2011). [32]. wang y., lu z., lv f., bie x., study on microencapsulation of curcumin pigments by spray drying. european food research and technology, 229, 391–396, (2009). [33]. maphosa y., jideani v.a., dietary fiber extraction for human nutrition—a review. food reviews international, 32, 98–115, (2016). [34]. nevara g.a., kharidah s., muhammad s., zawawi n., mustapha n.a., karim r., dietary fiber: fractionation, characterization and potential sources from defatted oilseeds gita. foods, 10, 1– 19, (2021). [35]. garcia-amezquita l.e., tejada-ortigoza v., serna-saldivar s.o., welti-chanes j., dietary fiber concentrates from fruit and vegetable by-products: processing, modification, and application as functional ingredients. food and bioprocess technology, 11, 1439–1463, (2018). [36]. hussain s., jõudu i., bhat r., dietary fiber from underutilized plant resources-a positive approach for valorization of fruit and vegetable wastes. sustainability (switzerland), 12, (2020). [37]. gupta p., premavalli k.s., in-vitro studies on functional properties of selected natural dietary fibers. international journal of food properties, 14, 397–410, (2011). [38]. capuano e., the behavior of dietary fiber in the gastrointestinal tract determines its physiological effect. critical reviews in food science and nutrition, 57, 3543–3564, (2017). [39]. yangilar f., the application of dietary fibre in food industry : structural features , effects on health and definition , obtaining and analysis of dietary fibre : a review. 1, 13–23, (2013). [40]. ancuţa p., sonia a., oil press-cakes and meals valorization through circular economy approaches: a review. applied sciences (switzerland), 10, 1–31, (2020). [41]. soukoulis c., aprea e., cereal bran fractionation: processing techniques for the recovery of functional components and their applications to the food industry. recent patents on food, nutrition & agriculturee, 4, 61–77, (2012). [42]. salehifar m., fadaei v., comparison of some functional properties and chemical constituents of dietary fibers of iranian rice bran extracted by chemical and enzymatic methods. african journal of biotechnology, 10, 18528– 18531, (2011). [43]. soquetta m.b., terra l. de m., bastos c.p., green technologies for the extraction of bioactive compounds in fruits and vegetables. cyta journal of food, 16, 400–412, (2018). [44]. wang j., suo g., de wit m., boom r.m., schutyser m.a.i., dietary fibre enrichment from defatted rice bran by dry fractionation. journal of food engineering, 186, 50–57, (2016). [45]. yáñez j.l., beltranena e., zijlstra r.t., dry fractionation creates fractions of wheat distillers dried grains and solubles with highly digestible nutrient content for grower pigs 1. 3416– 3425, (2014) doi:10.2527/jas2013-6820. [46]. eckhoff s.r., du l., yang p., rausch k.d., wang d.l., li b.h., tumbleson m.e., comparison between alkali and conventional corn wet-milling : 100-g procedures. 96–99, (1999). [47]. ma m., mu t., effects of extraction methods and particle size distribution on the structural , physicochemical , and functional properties of dietary fiber from deoiled cumin. food chemistry, 194, 237–246, (2016). [48]. e b.k.k., johansen h.n., glitse v., methods for analysis of dietary fibre advantage and limitations *. 185–206, (1997). [49]. dhingra d., michael m., rajput h., patil r.t., dietary fibre in foods: a review. journal of food science and technology, 49, 255–266, (2012). [50]. ma m., mu t., effects of extraction methods and particle size distribution on the structural , physicochemical , and functional properties of dietary fiber from deoiled cumin. food chemistry, 194, 237–246, (2016). [51]. chemat f., rombaut n., meullemiestre a., turk m., perino s., fabiano-tixier a.s., abert-vian m., review of green food processing techniques. preservation, transformation, and extraction. innovative food science and emerging technologies, 41, 357–377, (2017). [52]. jacotet-navarro m., rombaut n., deslis s., fabiano-tixier a.s., pierre f.x., bily a., chemat f., towards a ‘dry’ bio-refinery without solvents or added water using microwaves and ultrasound for total valorization of fruit and vegetable by-products. green chemistry, 18, 3106– 3115, (2016). [53]. mirpoor s.f., giosafatto c.v.l., porta r., biorefining of seed oil cakes as industrial costreams for production of innovative bioplastics. a review. trends in food science and technology, 109, 259–270, (2021). [54]. azenzem r., oilseeds development in morocco in the current international context. ocl oilseeds and fats, crops and lipids, 29, (2022). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 378 [55]. teh s.-s., morlock g., analysis of bioactive components of oilseed cakes by highperformance thin-layer chromatography(bio)assay combined with mass spectrometry. chromatography, 2, 125–140, (2015). [56]. gupta a., sharma r., oilseed as potential functional food ingredient. (2019). [57]. tarek-tilistyák j., juhász-román m., jeko j., máthé e., short-term storability of oil seed and walnut cake microbiological aspect. acta alimentaria, 43, 632–639, (2014). [58]. yasothai r., chemical composition of sesame oil cake – review. international journal of science, environment and technology, 3, 827–835, (2014). [59]. benítez benítez r., ortega bonilla r.a., franco j.m., comparison of two sesame oil extraction methods: percolation and pressed. biotecnoloía en el sector agropecuario y agroindustrial, 14, 10, (2016). [60]. mohdaly a.a.a., smetanska i., ramadan m.f., sarhan m.a., mahmoud a., antioxidant potential of sesame (sesamum indicum) cake extract in stabilization of sunflower and soybean oils. industrial crops and products, 34, 952–959, (2011). [61]. uppuluri k.b., dasari r.k.v.r., sajja v., jacob a.s., reddy d.s.r., optimization of lasparaginase production by isolated aspergillus niger c4 from sesame (black) oil cake under ssf using box-behnken design in column bioreactor. international journal of chemical reactor engineering, 11, 103–109, (2013). [62]. capellini m.c., chiavoloni l., giacomini v., rodrigues c.e.c., alcoholic extraction of sesame seed cake oil: influence of the process conditions on the physicochemical characteristics of the oil and defatted meal proteins. journal of food engineering, 240, 145–152, (2019). [63]. nagendra prasad mn, sanjay kr d.s.p., , neha vijay r.k. and n.s.s., a review on nutritional and nutraceutical properties of sesame. j. nutr. food sci., 02, (2012). [64]. das p., ghosh k., improvement of nutritive value of sesame oil cake in formulated diets for rohu, labeo rohita (hamilton) after bio-processing through solid state fermentation by a phytaseproducing fish gut bacterium. international journal of aquatic biology, 3, 89–101, (2015). [65]. elsorady m.e., characterization and functional properties of proteins isolated from flaxseed cake and sesame cake. croatian journal of food science and technology, 12, 77–83, (2020). [66]. contreras m. del m., romero i., moya m., castro e., olive-derived biomass as a renewable source of value-added products. process biochemistry, 97, 43–56, (2020). [67]. moftah o.a.s., grbavčić s., žuža m., luković n., bezbradica d., knežević-jugović z., adding value to the oil cake as a waste from oil processing industry: production of lipase and protease by candida utilis in solid state fermentation. applied biochemistry and biotechnology, 166, 348–364, (2012). [68]. cozea a., ionescu n., popescu m., neagu m., gruia r., comparative study concerning the composition of certain oil cakes with phytotherapeutical potential. revista de chimie, 67, 422–425, (2016). [69]. teh s.s., el-din bekhit a., birch j., antioxidative polyphenols from defatted oilseed cakes: effect of solvents. antioxidants, 3, 67–80, (2014). [70]. kristina montrimaitė e.m., possibilities of usage of oilcakes from non-traditional oil plants for development of health-friendly functional food products. food science and applied biotechnology, 1, 154–164, (2018). [71]. serrapica f., masucci f., raffrenato e., sannino m., vastolo a., barone c.m.a., di francia a., high fiber cakes from mediterranean multipurpose oilseeds as protein sources for ruminants. animals, 9, 1–11, (2019). [72]. jozinović a., ačkar d., jokić s., babić j., balentić j.p., banožić m., šubarić d., optimisation of extrusion variables for the production of corn snack products enriched with defatted hemp cake. czech journal of food sciences, 35, 507–516, (2017). [73]. tyapkova o., osen r., wagenstaller m., baier b., specht f., zacherl c., replacing fishmeal with oilseed cakes in fish feed – a study on the influence of processing parameters on the extrusion behavior and quality properties of the feed pellets. journal of food engineering, 191, 28– 36, (2016). [74]. jadhav m., kagalkar a., jadhav s., govindwar s., isolation, characterization, and antifungal application of a biosurfactant produced by enterobacter sp. ms16. european journal of lipid science and technology, 113, 1347–1356, (2011). [75]. lazaro e., benjamin y., robert m., the effects of dehulling on physicochemical properties of seed oil and cake quality of sunflower. tanzania journal of agricultural sciences, 13, 41– 47, (2014). [76]. popović s., hromiš n., šuput d., bulut s., romanić r., lazić v., valorization of by-products from the production of pressed edible oils to produce biopolymer films. cold pressed oils academic press, (2020). doi:10.1016/b978-0-12818188-1.00003-7. [77]. kaul tiku p., utilization of agri byfood and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 379 products for value addition as a protein source. agricultural research & technology: open access journal, 13, 32–34, (2018). [78]. bárta, jan;bártová, veronika bártová; jarošová, markéta; švajner, josef ; pavel smetana, pavel; jaromír kadlec , jaromír; filip , vladimír; kyselka, jan; bercíková, markéta; zdráhal, zbynek ; bjelková m.k.m., oilseed cake flour composition, functional properties species differences. foods, 10, 1–17, (2021). [79]. helling, j.j.; patterson, t.g.; campbell s.j., aqueous processing of oilseed press cake in google patents. [80]. samtiya m., aluko r.e., dhewa t., plant food anti-nutritional factors and their reduction strategies : an overview. 5, 1–14, (2020). [81]. das g., sharma a., sarkar p.k., conventional and emerging processing techniques for the post-harvest reduction of antinutrients in edible legumes. applied food research, 2, 100112, (2022). [82]. östbring k., malmqvist e., nilsson k., rosenlind i., rayner m., the effects of oil extraction methods on recovery yield and emulsifying properties of proteins from rapeseed meal and press cake. foods, 9, (2020). [83]. dong x., guo l., wei f., li j., jiang m., some characteristics and functional properties of rapeseed protein prepared by ultrasonication , ultrafiltration and isoelectric. 1488–1498, (2011) doi:10.1002/jsfa.4339. [84]. kajla p., sharma a., sood d.r., flaxseed—a potential functional food source. journal of food science and technology, 52, 1857–1871, (2015). [85]. kreps f., vrbiková l., schmidt š., industrial rapeseed and sunflower meal as source of antioxidants industrial rapeseed antioxidants and sunflower meal as source of. (2015). [86]. petraru a., ursachi f., amariei s., nutritional characteristics assessment of sunflower seeds, oil and cake. perspective of using sunflower oilcakes as a functional ingredient. plants, 10, (2021). [87]. teh s.s., bekhit a.e.d., carne a., birch j., effect of the defatting process, acid and alkali extraction on the physicochemical and functional properties of hemp, flax and canola seed cake protein isolates. journal of food measurement and characterization, 8, 92–104, (2014). [88]. tan s.h., mailer r.j., blanchard c.l., agboola s.o., canola proteins for human consumption: extraction, profile, and functional properties. journal of food science, 76, (2011). [89]. chatterjee r., dey t.k., ghosh m., dhar p., enzymatic modification of sesame seed protein, sourced from waste resource for nutraceutical application. food and bioproducts processing, 94, 70–81, (2015). [90]. aider m., barbana c., canola proteins: composition, extraction, functional properties, bioactivity, applications as a food ingredient and allergenicity a practical and critical review. trends in food science and technology, 22, 21– 39, (2011). [91]. yamada y., iwasaki m., usui h., ohinata k., marczak e.d., lipkowski a.w., yoshikawa m., rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin ip receptor followed by cck1 receptor. peptides, 31, 909–914, (2010). [92]. zhang s.b., in vitro antithrombotic activities of peanut protein hydrolysates. food chemistry, 202, 1–8, (2016). [93]. schmidt š., pokorný j., potential application of oilseeds as sources of antioxidants for food lipids a review. czech journal of food sciences, 23, 93–102, (2005). [94]. teh s.-s., bekhit a.e.-d.a., utilization of oilseed cakes for human nutrition and health benefits. in agricultural biomass based potential materials , ed. hakeem, k.r.; jawaid, m; alothman o.y., 191–229, springer international publishing:cham, (2015). doi:10.1007/978-3-31913847-3. [95]. pickardt c., eisner p., kammerer d.r., carle r., pilot plant preparation of light-coloured protein isolates from de-oiled sunflower (helianthus annuus l.) press cake by mild-acidic protein extraction and polyphenol adsorption. food hydrocolloids, 44, 208–219, (2015). [96]. şahin s., elhussein e.a.a., assessment of sesame (sesamum indicum l.) cake as a source of high-added value substances: from waste to health. phytochemistry reviews, 17, 691–700, (2018). [97]. senanayake c.m., algama c.h., wimalasekara r.l., weerakoon w.n.m.t.d.n., jayathilaka n., seneviratne k.n., improvement of oxidative stability and microbial shelf life of vanilla cake by coconut oil meal and sesame oil meal phenolic extracts. journal of food quality, 2019, (2019). [98]. slatnar a., mikulic-petkovsek m., stampar f., veberic r., solar a., identification and quantification of phenolic compounds in kernels, oil and bagasse pellets of common walnut (juglans regia l.). food research international, 67, 255–263, (2015). [99]. şahin s., elhussein e.a.a., valorization of a biomass: phytochemicals in oilseed by-products. phytochemistry reviews, 17, 657–668, (2018). [100]. de ridder d., kroese f., evers c., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 380 adriaanse m., gillebaart m., healthy diet: health impact, prevalence, correlates, and interventions. psychology and health, 32, 907–941, (2017). [101]. brandl b., rennekamp r., reitmeier s., pietrynik k., dirndorfer s., haller d., hofmann t., skurk t., hauner h., offering fiber-enriched foods increases fiber intake in adults with or without cardiometabolic risk: a randomized controlled trial. frontiers in nutrition, 9, 1–15, (2022). [102]. gustafson c.r., rose d.j., us consumer identification of the health benefits of dietary fiber and consideration of fiber when making food choices. nutrients, 14, 1–11, (2022). [103]. sun j., zhang z., xiao f., wei q., jing z., ultrasound-assisted alkali extraction of insoluble dietary fiber from soybean residues. iop conference series: materials science and engineering, 392, (2018). [104]. zheng y., li y., physicochemical and functional properties of coconut (cocos nucifera l) cake dietary fibres: effects of cellulase hydrolysis, acid treatment and particle size distribution. food chemistry, 257, 135–142, (2018). [105]. karaca a.c., low n., nickerson m., emulsifying properties of canola and fl axseed protein isolates produced by isoelectric precipitation and salt extraction. frin, 44, 2991– 2998, (2011). [106]. grosso a.l., asensio c.m., nepote v., grosso n.r., antioxidant activity displayed by phenolic compounds obtained from walnut oil cake used for walnut oil preservation. jaocs, journal of the american oil chemists’ society, 95, 1409–1419, (2018). [107]. stübler a.s., heinz v., aganovic k., development of food products. current opinion in green and sustainable chemistry, 25, 100356, (2020). [108]. prakash k., naik s.n., vadivel d., hariprasad p., gandhi d., saravanadevi s., utilization of defatted sesame cake in enhancing the nutritional and functional characteristics of biscuits. journal of food processing and preservation, 42, 1–10, (2018). [109]. łopusiewicz, ł.; drozłowska, e.; siedlecka, p.; bartkowiak, a.; sienkiewicz m.. z., non-dairy kefir-like fermented beverage based on flaxseed oil cake. 8–11, (2019). [110]. drozłowska e., valorization of flaxseed oil cake residual from cold-press oil production as a material for preparation of spray-dried functional powders for fooddrozłowska, emilia łopusiewiapplications as emulsion stabilizers. biomolecules, 10, (2020). [111]. jain a., prakash m., radha c., extraction and evaluation of functional properties of groundnut protein concentrate. (2015) doi:10.1007/s13197-015-1758-7. [112]. mridula d., gupta r.k., bhadwal s., khaira h., tyagi s.k., optimization of food materials for development of nutritious pasta utilizing groundnut meal and beetroot. journal of food science and technology, 53, 1834–1844, (2016). [113]. sun q., sun c., xiong l., mechanical, barrier and morphological properties of pea starch and peanut protein isolate blend films. carbohydrate polymers, 98, 630–637, (2013). [114]. reddy n., jiang q., yang y., preparation and properties of peanut protein films crosslinked with citric acid. industrial crops and products, 39, 26–30, (2012). [115]. kreps f., vrbiková l., schmidt š., industrial rapeseed and sunflower meal as source of antioxidants. journal of engineering research and applications, 4, 45–54, (2014). [116]. salgado p.r., ortiz s.e.m., petruccelli s., mauri a.n., functional food ingredients based on sunflower protein concentrates naturally enriched with antioxidant phenolic compounds. jaocs, journal of the american oil chemists’ society, 89, 825–836, (2012). [117]. salgado p.r., molina ortiz s.e., petruccelli s., mauri a.n., sunflower protein concentrates and isolates prepared from oil cakes have high water solubility and antioxidant capacity. jaocs, journal of the american oil chemists’ society, 88, 351–360, (2011). [118]. salgado p.r., drago s.r., molina ortiz s.e., petruccelli s., andrich o., gonzález r.j., mauri a.n., production and characterization of sunflower (helianthus annuus l.) protein-enriched products obtained at pilot plant scale. lwt food science and technology, 45, 65–72, (2012). [119]. ayhllon-meixueiro f., vaca-garcia c., silvestre f., biodegradable films from isolate of sunflower (helianthus annuus) proteins. journal of agricultural and food chemistry, 48, 3032–3036, (2000). [120]. šuput d., lazić v., popović s., hromiš n., bulut s., pezo l., banićević j., effect of process parameters on biopolymer films based on sunflower oil cake. journal on processing and energy in agriculture, 22, 125–128, (2018). [121]. nesterenko a., alric i., violleau f., silvestre f., durrieu v., a new way of valorizing biomaterials : the use of sun fl ower protein for α -tocopherol microencapsulation. frin, 53, 115–124, (2013). [122]. nadeem m., situ c., mahmud a., khalique a., imran m., rahman f., khan s., antioxidant activity of sesame (sesamum indicum food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 ancuța petraru, sonia amariei, recovery of bioactive compounds from oilcakesa rieview, food and environment safety, volume xxi, issue 4 – 2024, pag. 364 – 381 381 l.) cake extract for the stabilization of olein based butter. jaocs, journal of the american oil chemists’ society, 91, 967–977, (2014). [123]. mohdaly a.a.a., hassanien m.f.r., mahmoud a., sarhan m.a., smetanska i., phenolics extracted from potato , sugar beet , and sesame processing byproducts. international journal of food properties, 16, 1148–1168, (2013). [124]. sisay m.t., emire s.a., ramaswamy h.s., workneh t.s., effect of feed components on quality parameters of wheat–tef–sesame–tomato based extruded products. journal of food science and technology, 55, 2649–2660, (2018). [125]. sharma l., singh c., sesame protein based edible films: development and characterization. food hydrocolloids, 61, 139–147, (2016). [126]. pycia k., kapusta i., jaworska g., walnut oil and oilcake affect selected the physicochemical and antioxidant properties of wheat bread enriched with them. journal of food processing and preservation, 44, 1–11, (2020). [127]. bakkalbasi e., meral r., dogan i.s., bioactive compounds, physical and sensory properties of cake made with walnut press-cake. journal of food quality, 38, 422–430, (2015). [128]. pop a., paucean a., socaci s.a., alexa e., man s.m., muresan v., chis m.s., salanta l., popescu i., berbecea a., et al., quality characteristics and volatile profile of macarons modified withwalnut oilcake by-product. molecules, 25, 1–19, (2020). [129]. behera s., indumathi k., mahadevamma s., sudha m.l., oil cakes-a by-product of agriculture industry as a fortificant in bakery products. international journal of food sciences and nutrition, 64, 806–814, (2013). [130]. ghoshal g., kaushik p., development of soymeal fortified cookies to combat malnutrition. legume science, 2, 1–13, (2020). [131]. wan j., liu c., liu w., tu z., wu w., tan h., optimization of instant edible films based on dietary fiber processed with dynamic high pressure microfluidization for barrier properties and water solubility. lwt food science and technology, 60, 603–608, (2015). [132]. popović s., peričin d., vaštag ž., lazić v., popović l., pumpkin oil cake protein isolate films as potential gas barrier coating. journal of food engineering, 110, 374–379, (2012). [133]. radočaj o., dimić e., tsao r., effects of hemp (cannabis sativa l.) seed oil press-cake and decaffeinated green tea leaves (camellia sinensis) on functional characteristics of glutenfree crackers. journal of food science, 79, (2014). [134]. pojić m., dapčević hadnadev t., hadnadev m., rakita s., brlek t., bread supplementation with hemp seed cake: a byproduct of hemp oil processing. journal of food quality, 38, 431–440, (2015). 1. introduction zheng et al. [104] studied the physicochemical and functional properties of defatted coconut df that was subjected to acidic treatment, enzymatic hydrolysis and particle size distribution. the hydrolysis and particle size reduction caused structural ... 4. bioactive compounds from oilcakes in foods doi: https://doi.org/10.4316/fens.2021.030 274 journal homepage: http://fens.usv.ro/index.php/fens/index journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 274 281 determination of trace levels of nickel(ii) by cyclic voltammetry with spes from food products liliana anchidin-norocel1*, sonia amariei 2, gheorghe gutt 2 1faculty of medicine and biological sciences, stefan cel mare university of suceava, 720229 suceava, romania liliana.norocel@usm.ro 2faculty of food engineering, stefan cel mare university of suceava, 720229 suceava, romania *corresponding author received 23th june 2021, accepted 29th september 2021 abstract: detectable nickel ion levels are found in many foods, including cocoa beans, legumes, grains, nuts and seeds, crustaceans, saltwater fishes, and fruits. itis known that approximately 15% of the human population suffers from an allergy to dietary nickel. in addition to digestive problems, dietary nickel intolerance may lead to diffuse dermatitis and itching, fever, rhinitis, and headaches. increased nickel levels can affect liver function leading to hepatic failure. due to the harmful effects that nickel can have on human health, it is necessary to accurately measure nickel levels in foods and develop efficient detection protocols. the aim of this paper was to develop a chemosensor for the detection of nickel(ii) ions in food products and in this case bismuth oxide –spes (screen-printed electrodes) and graphene modified spes were used with very small amount of dimethylglyoxime and amino acid l-histidine which were deposited. a potentiostat was used to study the electrochemical properties of nickel standard solution with different concentration. the results obtained with aas (atomic absorption spectrometry) reference method for food samples were compared with the results obtained with the developed chemosensor. based on the results, the developed chemosensor that used bismuth oxide –spes with dimethylglyoxime was found efficient, having a linear response over a wide range (1–10 ppm) of nickel concentrations. keywords: electrochemical method, nickel allergy, screen printed electrodes. 1. introduction nickel (ni) is the fifth most abundant element on earth [1,2]. it is a silvery white hard, ductile, and malleable metal, with a density of 8.9 g/ml and a melting point of 1453 °c. nickel (ii) is the dominant inorganic species and has a wide ph and redox potential range (eh) [4-6]. elemental nickel is a group 10 transition metal, which is most commonly found in either the 0 or +2 oxidation states in biological materials, and less commonly in the -1 to +4 oxidation states. it is an indispensable metal for a number of industrial and clinical applications [3], and because nickel is abundant in the environment, it is commonly found in living tissues and many other kinds of organic matter. nickel performs many essential functions as a microtrace nutrient in many organisms, and specifically in the human body it is involved in the activation of urease, a metalloenzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia [7]. nickel is moderately toxic to the human metabolism when compared to other transition metals. however, higher levels of nickel ions in the human body can be toxic, affecting the lungs, brain and http://fens.usv.ro/index.php/fens/index food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 275 kidney, and may lead to other complications such as allergies, muscular damage, neurological disorders, and respiratory cancer. dietary intake of nickel is generally the main path of bioaccumulation [8-14]. the u.s. agency for toxic substance and disease registry estimates that the daily tolerable dietary intake of nickel for a 70 kg adult is 0.001–0.0024 mg/kg. about 540% of nickel present in foods and beverages is absorbed in the digestive tract, while ambient air when inhaled, absorption oscillates from around 20-35% [11]. the nickel content in most commercial vegetables is usually 10 mg/kg but in nickel-rich soils it can reach 100 mg/kg [15]. the greatest natural sources of dietary nickel are cocoa products, legumes, baking soda, nuts, and drinking water, with some of the highest mean concentrations of nickel measured in wild edible mushrooms (containing >10 mg/kg dry weight) [1619]. daily consumption of these foods in high amounts can increase nickel intake up to 900 mg. the level of nickel ions in these foods depends on where and how they are cultivated, specifically application of herbicides, pesticides and fertilizers, as well as nickel levels occurring in soil and air (natural or due to pollution), food processing methods, transport and storage conditions [20-24]. the link between dietary nickel bioaccumulation and human health is well-established [25-29], and therefore, it prompts the need for the development of efficient detection methods. biosensors hold great value because they can be selective to the analysis of interest, are both cost-effective and time-efficient, and can produce reliable data that benefits human health, namely that nickel detection is a preventative health care measure [30]. considering its toxic effects, precise quantification of nickel in our food supply is of great importance. the importance of determining the concentration of nickel ions in food products arises from its toxicity for human health. it is estimated that approximately 8–15% of women and 1–2% of men are sensitive to nickel [31-38]. total nickel content analysis is performed using flame atomic absorption spectrometry, graphite furnace atomic absorption spectrometry, inductively coupled plasma optical emission spectrometry, or mass spectrometry. these methods used to detect nickel in food and water samples, can be with or without preconcentration or separation steps [3941]. to streamline the sample preparation process, save time and materials, biosensors are increasingly used in the last years. biosensors are integrated devices capable of providing specific quantitative analyses by using a biological receptor in direct contact with a transducer [42-46]. a chemosensor is not a sensor, strictly speaking, as it is not a device, but it can be the active part of the device [47]. development of biosensors is largely to the success in glucose measurement for diabetics, where it dramatically improved patient care by providing a convenient, hygienic, compact method of selfmonitoring [48-51]. currently, biosensors applications have greatly expanded in detection of certain pathogens or even markers of cancer, cardiovascular disease and hormones. similar to its design in other fields, biosensors used in analyzing food compounds consist of a biologically active receiver, a transducer, and an electronic system for amplifying, processing and displaying data that provides specific analytical information that allows the recognition of a particular biological or chemical species. biosensors in the food industry are used to identify microorganisms, antibodies, heavy metals, alcohols, carbohydrates, vitamins and allergens [52, 53]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 276 biosensor’s technology has evolved greatly from in vitro studies based on the biosensitivity of organic beings to the highly sophisticated world of nanofabricated biosensors [54-56]. the application of this chemosensor solves the problem of the determination of nickel ions from foods which nowadays is difficult, expensive, requires specialized personnel and is limited to detection in a laboratory because there are no efficient in situ methods. 2. materials and methods reagents for the experimental part of developing and testing of chemosensor, nickel sulphate, dimethylglyoxime (dmg) (1%), amino acid l-histidine (1%), nitric acid, and ammonium buffer were purchased from sigma-aldrich (steinheim, germany). graphene and bismuth modified screen-printed electrodes were purchased from dropsens. all solutions were made using deionized water with a resistivity of 18.2 mω×cm (millipore, direct-q 3 uv). electrochemical measurements a metrohm autolab bipotentiostat µstat 300 controlled by dropview 8400 software was used for the electrochemical measurements. the testing conditions were: starting potential -0.1 v, switching potential 0.9 v, and scanning rate 0.1 v/s. a drop of sample was placed on the spe, then the potential was applied, and as a result, the ni-dmg complex was formed. response time was 60 seconds. immobilization of ligand on spe and development of chemosensor the ligand, dimethylglyoxime 1% and 1 µl amino acid l-histidine solution 1% were pipetted and immobilized on the surface of the working electrode of the spe. then the miniaturized electrode was used for nickel ions analyses by cyclic voltammetry. the development and testing of chemosensor were caried out according anchidin-norocel et al. (2021) [60]. testing the chemosensor on food products in this study, cocoa, cabbage, wine, and spinach samples were analyzed by the voltammetric method. the results obtained were compared with the atomic adsorption spectrometry (aas) values of the same samples. atomic adsorption spectrometry method nickel determination by aas was made using the method presented by salazar et al. (2011) [11] to prepare samples via acid digestion and calcination. 3. results and discussion the chemosensor development process had several stages, the first of which was to make a calibration curve with standard solution, to identify a linear regression higher and to use the most performant chemosensor for food samples. the concentration was reported at the maximum current density for each solution because the anodic and cathodic peaks are extremely small (figure 1). so, in order to obtain a calibration curve from the cyclic voltammograms, nickel solutions (1, 3, 5, 7, and 10 mg/l) were used (figure 2). following the analysis of the nickel solution, higher linear regression value (0.97) was obtained for spebi2o3/c dmgh2 and lower value (0.94) for cyclic voltammograms performed with spegph-dmgh2. because these results turned out to be better for spebi2o3/c dmgh2, analysis of food samples were made only for this chemosensor. by comparing the developed method with the reference method (aas) similar values were observed. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 277 a) b) fig. 1. the cyclic voltammogram obtained for nickel standard solutions with a) spe-gph-dmgh2 and b) spe bi2o3/c dmgh2 c) d) fig. 2. calibration curve obtained for nickel standard solutions with a) spe-gph-dmgh2 and b) spebi2o3/c dmgh2 figure 3 shows that the results obtained for two samples of cocoa, wine, spinach and cabbage analyzed with the developed chemosensor, had higher values than the aas method, suggesting that this biologic element may select from the matrix of food samples compounds another ion that interferes with nickel. so, we had to analyze the interferences that appear in the food sample matrices. this analitycal performance characteristic in this case can be called selectivity and is made in same condition (for an intermediary concentration of standard solution) for a lot of minerals standard solution (fe, na, ca, al, zn, cd, cu, k, pb). the results of this evaluation are presented in figure 4. the analytical performances of the chemosensor were evaluated also by sensitivity calculated according to norocel & gutt [58, 59]. sensitivity = m/a where m-slope of calibration curve (µa mm-1), a-area of active surface (cm2). the results obtained for sensitivity were 3.25 for spe-gph-dmgh2 and 1.32 for spebi2o3/c dmgh2. both the voltamogram and the calibration curve had shown that the values extracted can provide good results and can be used to calculate the nickel concentration of the standard solution. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 278 fig. 3. concentrations of nickel in food samples -s1, -s2 from final name of method used means samples 1 and samples 2 chemosensor reproducibility was analyzed by measuring the nickel-generated current of 5 mg/l in 10 ml deionized water. the chemosensor was tested on three different days with a triple analysis (n = 3). the total mean value was calculated and the relative standard deviation (rsd) provided analytical precision, and, the resulted reproducibility was 96.2% for spe-gphdmgh2 and 94.4% for spebi2o3/c dmgh2. fig. 4. selectivity evaluation of the developed chemosensor against the interference of mineral elements even if there are already many chemosensors for the determination of nickel ions, the difference between them is made by the analytical performance characteristics, respectively the detection limit, the sensitivity but also the dynamic range. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 279 4. conclusion clearly, from a public safety perspective, nickel needs to be measured in the food supply and used for environmental health assessments (e.g., air, soil, and water quality). chemosensors are ideal tools for the task of detecting analytes like nickel because they can be synthesized to have very high specificity and can be adapted for high-throughput processing. chemosensors are generally of lower cost and portable, which makes them valuable for small-scale and mobile/remote applications. most chemosensors used in the analysis of nickel ions found in the literature have no application data in certain industries and if both existing chemosensors and other new chemosensors were tried in identifying these ions in food, it would be very beneficial for people suffering from these allergies. the results obtained with the proposed chemosensor for determining nickel ion concentration in foods have proven to be effective both for the performance characteristics and for comparing the results with the reference method. the ligand used showed good sensitivity for the target analyte, thus recommending it as a possible mean for the quantification of nickel ions in the food samples analyzed. 5. acknowledgments this work was supported by a grant of the ministry of research, innovation and digitalization, cncs/uefiscdi, project number pn-iii-p1-1.1-pd-2019-1111, within pncdi iii. 6. references [1]. mattison, r. l., bowyer, a. a., & new, e. j. small molecule optical sensors for nickel: the quest for a universal nickel receptor. coord. chem. rev, (2020). 425, 213522. [2]. gopalapillai, y., fasfous, i. i., murimboh, j. d., yapici, t., chakraborty, p., & chakrabarti, c. l. determination of free nickel ion concentrations using the ion exchange technique: application to aqueous mining and municipal effluents. aquat geochem., 14(2), (2008), 99-116. [3]. li, j. j., hou, c. j., huo, d. q., shen, c. h., luo, x. g., fa, h. b., ... & zhou, j. detection of trace nickel ions with a colorimetric sensor based on indicator displacement mechanism. sens actuators b chem, 241, (2017), 1294-1302. [4]. mattison, r. l. fluorescent sensors to monitor ni (ii) in complex environments (doctoral dissertation, university of sydney), (2020). [5]. adegoke av, aliyu dh, akefe io, nyan se. a review on the metal complex of nickel (ii) salicylhydroxamic acid and its aniline adduct. j transl sci res, (2019) 2: 006. [6]. huang, d., & liu, j. functions and toxicity of nickel in plants: recent advances and future prospects. clean–soil, air, water, (2009), 37(4‐5), 304-313. [7]. muthuselvi, r. determination of nickel (ii) by spectrophotometry in micellar media. pharm anal chem, 3(3), (2017). [8]. d’alcamo, a., mansueto, p., soresi, m., iacobucci, r., blasca, f., geraci, g., ... & iacono, g. contact dermatitis due to nickel allergy in patients suffering from non‐celiac wheat sensitivity. nutrients, 9(2), 103, (2017). [9]. duda-chodak, a., & blaszczyk, u. the impact of nickel on human health. journal of elementology, 13(4), (2008), 685-693. [10]. kruszewski, b., obiedziński, m. w., & kowalska, j. nickel, cadmium and lead levels in raw cocoa and processed chocolate mass materials from three different manufacturers. j food compost anal., 66, (2018), 127-135. [11]. salazar, r. f. d. s., alcântara, m. a. k. d., & izário filho, h. j. evaluation of sample preparation methods and optimization of nickel determination in vegetable tissues. revista brasileira de ciência do solo, 35(1), (2011). 241248. [12]. verma, n., & singh, m. a bacillus sphaericus based biosensor for monitoring nickel ions in industrial effluents and foods. j autom methods manag chem., (2006). [13]. almaquer, f. e. p., ricacho, j. s. y., & ronquillo, r. l. g. simple and rapid colorimetric sensing of ni (ii) ions in tap water based on aggregation of citrate-stabilized silver nanoparticles. sustain. environ. res, 29(1), (2019), 1-10. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 280 [14]. antico, a., & soana, r. nickel sensitization and dietary nickel are a substantial cause of symptoms provocation in patients with chronic allergic-like dermatitis syndromes. allergy & rhinology, 6(1), 2015. [15]. kumari, s., chandrawal, a., kumar, m., & kumar, a. toxicity of cadmium and nickel in soil and vegetables. inter-national int j curr microbiol appl sci, 7(10), (2018), 23412352. [16]. smart, g. a., & sherlock, j. c. nickel in foods and the diet. food addit contam, 4(1), (1987), 61-71. [17]. onianwa, p. c., lawal, j. a., ogunkeye, a. a., & orejimi, b. m. cadmium and nickel composition of nigerian foods. food compost anal., 13(6), (2000), 961-969. [18]. reilly, c. metal contamination of food (pp. 40-60). london: applied science publishers. (1980). [19]. braga, m., quecchia, c., perotta, c., timpini, a., maccarinelli, k., di tommaso, l., & di gioacchino, m. systemic nickel allergy syndrome: nosologic framework and usefulness of diet regimen for diagnosis. int j immunopathol pharmacol., 26(3), (2013), 707-716. [20]. peeters, k., zuliani, t., žigon, d., milačič, r., & ščančar, j. nickel speciation in cocoa infusions using monolithic chromatography –post-column id-icp-ms and qtof-ms. food chem, 230, (2017), 327-335. [21]. dohnalova, l., bucek, p., vobornik, p., & dohnal, v. determination of nickel in hydrogenated fats and selected chocolate bars in czech republic. food chem., 217, (2017), 456-460. [22]. saito, m., arakaki, r., yamada, a., tsunematsu, t., kudo, y., & ishimaru, n. molecular mechanisms of nickel allergy. int. j. mol. sci., 17(2), 202, (2016). [23]. feyzullah tokay, development of uvvis spectrophotometric method for rapid nickel determination in original oil matrix, j. nat. appl. sci, volume 21, issue 2, 332-337, 2017, doi: 10.19113/sdufbed.67183 [24]. gideon ramtahal, ivan chang yen, et al., cost-effective method of analysis for the determination of cadmium, copper, nickel and zinc in cocoa beans and chocolates, j. food res., vol. 4, no. 1, 2015. [25]. ahlström, m. g., thyssen, j. p., menné, t., & johansen, j. d. consumer behaviour among nickel-allergic patients. acta derm venereol, 97(8-9), (2017), 1247-1248. [26]. alizadeh, k., & rad, n. a new optical sensor for selective monitoring of nickel ion based on a hydrazone derivative immobilized on the triacetyl cellulose membrane. j anal bioanal tech, (2016), 7(2). [27]. brandão, m. h. t., & gontijo, b. (2012). contact sensitivity to metals (chromium, cobalt and nickel) in childhood. an bras dermatol, 87(2), 269-276. [28]. babaee, s., pakdehi, s. g., & nabavi, a. s. an optical chemical sensor for determination of nickel in water and hydrogen peroxide samples. journal of new developments in chemistry, 1(2), 58, (2016). [29]. chen, b., xia, g., cao, x. m., wang, j., xu, b. y., huang, p., ... & jiang, q. w. urinary levels of nickel and chromium associated with dental restoration by nickel–chromium based alloys. international journal of oral science, 5(1), 44, (2013). [30]. efsa panel on contaminants in the food chain (contam). scientific opinion on the risks to public health related to the presence of nickel in food and drinking water. efsa journal, 13(2), 4002, (2015). [31]. lusi, e. a., di ciommo, v. m., patrissi, t., & guarascio, p. high prevalence of nickel allergy in an overweight female mansouri population: a pilot observational analysis. plos one, 10(3), e0123265. (2015). [32]. venter, c. food hypersensitivity: diagnosing and managing food allergies and intolerances. journal of allergy, (2012). [33]. valenta, r., hochwallner, h., linhart, b., & pahr, s. food allergies: the basics. gastroenterology, 148(6), (2015), 11201131. [34]. strinnholm, å. food hypersensitivity among schoolchildren: prevalence, health related quality of life and experiences of double-blind placebo-controlled food challenges (doctoral dissertation, umeå universitet). (2017). [35]. chakrabarty k., chakrabarty a.s. food hypersensitivity. in: textbook of nutrition in health and disease. springer, singapore, (2019). https://doi.org/10.1007/978-98115-0962-9_6 [36]. nasr, i. h. humaid al wahshi food intolerance versus food allergy, 2. (2017). https://scientonline.org/open-access/foodintolerance-versus-food-allergy.pdf [37]. kumar, r. v., rajvikram, n., rajakumar, p., saravanan, r., deepak, v. a., & vijaykumar, v. an accurate methodology to detect leaching of nickel and chromium ions in the initial phase of orthodontic treatment: an in vivo study. j. contemp. dent. pract., 17(3), (2016), 205-210. [38]. mansouri m. food allergy: a review, arch pediatr infect dis. (2015); 3(3):e22470. doi: 10.5812/pedinfect.22470. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 liliana anchidin-norocel, sonia amariei, gheorghe gutt, determination of trace levels of nickel(ii) by cyclic volt-ammetry with spes from food products, food and environment safety, volume xx, issue 3 – 2021, pag. 274–281 281 [39]. forzani, e. s., zhang, h., chen, w., & tao, n. detection of heavy metal ions in drinking water using a high-resolution differential surface plasmon resonance sensor. environ sci technol, 39(5), (2005), 1257-1262. [40]. gao, p. f., zhang, x. w., kuang, h. z., li, q. q., & li, y. study on simultaneous determination of ni, pb and cd by ion chromatography. in iop conference series: earth and environmental science (vol. 146, no. 1, p. 012068). (2018). iop pub-lishing. [41]. kumar, b. n., kanchi, s., sabela, m. i., bisetty, k., & jyothi, n. v. v. spectrophotometric determination of nickel (ii) in waters and soils: novel chelating agents and their biological applications supported by dft method. karbala international journal of modern science, 2(4), (2016), 239-250. [42]. soleymani, l., & li, f. mechanistic challenges and advantages of biosensor miniaturization into the nanoscale. acs sensors, 2(4), (2017), 458-467. [43]. patel, s., nanda, r., sahoo, s., & mohapatra, e. biosensors in health care: the milestones achieved in their development towards lab-on-chip-analysis. biochem. res. int, (2016). [44]. hasan, anwarul., review article recent advances in application of biosensors in tissue engineering, biomed res int volume (2014), article id 307519, 18 pages http://dx.doi.org/10.1155/2014/307519 [45]. odobasic, a., sestan, i., & begic, s. biosensors for determination of heavy metals in waters. in biosensors for envi-ronmental monitoring. intechopen. chapters. (2019). [46].https://www.intechopen.com/books/biosensorsfor-environmental-monitoring/biosensors-fordetermination-of-heavy-metals-in-waters [47]. rani, p. chemosensor and its applications. irjrr, 3, (2015), 1-10. [48]. bhatt, g., & bhattacharya, s. biosensors on chip: a critical review from an aspect of micro/nanoscales. journal of micromanufacturing, 2(2), (2019), 198-219. [49]. turdean, g. l., review article, design and development of biosensors for the detection of heavymetal toxicity, sage-hindawi access to research international journal of electrochemistry, volume 2011. [50]. mohanty, s. p., kougianos, e., biosensors: a tutorial review, 0278-6648/06, ieee, potentials, (2006). [51]. stamatin, i., nanomateriale aplicații în biosenzori, surse de energie, medicină, biologie, elemente de nanotehnologie, editura universității din bucurești, (2008). [52]. castillo-henríquez, l., brenesacuña, m., castro-rojas, a., corderosalmerón, r., lopretti-correa, m., & ve-ga-baudrit, j. r. biosensors for the detection of bacterial and viral clinical pathogens. sensors, 20(23), 6926, (2020). [53]. bahadir, e. b., mustafa kemal sezginturk, applications of commercial biosensors in clinical, food, environmental, and biothreat/biowarfare analyses, 0003-2697, elsevier, anal biochem, (2015). [54]. bhattarai, p., & hameed, s. basics of biosensors and nanobiosensors. nanobiosensors: from design to applications, 1-22, https://doi.org/10.1002/9783527345137.ch1 (2020). [55]. banerjee, a., maity, s., & mastrangelo, c. h. nanotechnology for biosensors: a review. arxiv preprint arxiv:2101.02430, (2021). [56]. chambers, j. p., et al, biosensor recognition elements, horizon scientific press, (2008). [57]. arora, r.k., and saini, r.p., biosensors: way of diagnosis, int. j. pharm. sci. res, ijpsr, (2013); vol. 4(7): 2517-2527. [58]. norocel, l., & gutt, g. screen-printed voltammetric biosensors for the determination of copper in wine. sensors, 19(21), 4618, (2019). [59]. norocel, l., & gutt, g. development and performance testing of an electrochemical sensor for determination of iron ions in wine. australian journal of grape and wine research, 25(2), (2019), 161-164. [60]. anchidin-norocel, l., amariei, s., & gutt, g. sensing of nickel (ii) ions by immobilizing ligands and using different spes. engineering proceedings, 6(1), 2, (2021). doi: https://doi.org/10.4316/fens.2021.034 314 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 314 321 extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin * mariana spinei 1 , mircea oroian 1 1faculty of food engineering, stefan cel mare university of suceava, suceava, romania *mariana.spinei@fia.usv.ro received 15th october 2021, accepted 16th december 2021 abstract: the purpose of this paper is the use of grape pomaces, obtained by processing two different vitis vinifera varieties (rară neagră and fetească neagră) from 2019 harvest, cultivated in the bugeac area, republic of moldova, as a source of unconventional pectin. pectin was extracted under the following conditions, citric acid used as extractant for 3 h of extraction, 125-200 µm of grape pomace particle size and solid to liquid ratio 1:10. the variables which were varied were temperature (70, 80 and 90 °c) and ph (1, 2 and 3). so, pectin yield and galacturonic acid content were determined under the influence of extraction temperature and ph. the obtained results demonstrated that the extraction conditions (temperature and ph) have a significant impact on the yield and purity of pectin from grape pomace. the highest influence on pectin yield was determined by ph 1 (15.54% and 9.96% for fetească and rară neagră grape pomace pectin, respectively) and temperature of 90 °c (7.34% and 7.56% for fetească and rară neagră grape pomace pectin, respectively). the same tendency is observed for galacturonic acid content. keywords: grape pomace, galacturonic acid, pectin recovery, citric acid extraction. 1. introduction pectin is the third group of complex polysaccharides which constitute a part of higher plant wall characterized by relatively extractability using different type of acids (mineral or organic) or chelators (ammonium oxalate, sodium citrate etc.) and a high content of galacturonic acid [1– 3]. pectin is found in the middle lamella of the cell wall (cw), with a gradual reduction from the primary cw to the plasma membrane [4]. the structure and firmness of the plant cw depend on the mechanical properties, orientation and link between cellulose and pectic substances [5]. although, some pectin molecules are linked to xyloglucan chains of the cw [6]. pectin accomplishes two different functions, as a thickening element on the cw and as a „cementing” component in the middle lamella of plant cw [7]. pectin is extracted from fruits, vegetables and their processing by-products (peel, pomace or seeds) [8,9]. presently, the main sources of commercially pectin are citrus peels and apple pomace which are suitable for specific applications in food industry [4,10,11]. due to source of pectin, extraction conditions (time, ph, temperature, solid to liquid ratio, acid type etc.) and technique (conventional and unconventional extraction), the pectin has different ability to form gels [8]. chen et al. [12] established that extraction parameters influence the pectin structure and properties. moreover, the temperature is a decisive factor [12]. the low temperature kept the pectin structure more intact, remaining more neutral sugars (arabinose, fucose, glucose, galactose, mannose etc.) and possibly more close to the initial molecule in the cw [12]. the ph is considered as one of the most sensitive parameter to note while extracting pectin [13,14]. commonly, low http://www.fia.usv.ro/fiajournal mailto:*mariana.spinei@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 315 acidic ph is essential for the hydrolysis of protopectin [14,15]. a compromise is often made between having a poor quality of pectin, but with higher yield at a lower ph and having a better quality of extracted pectin, but with low yield at a high ph; this trade-off is made between temperature and extraction time [14]. mainly, the reported values for ph range from 1 to 3, but optimal ph value used for pectin extraction is 2 [4]. due to the lack of information about how extraction factors influence grape pomace pectin yield and structure, we aimed to study the impact of ph and temperature on the yield and purity of pectin. 2. materials and methods 2.1. materials grape pomace was obtained by processing two different vitis vinifera varieties (rară neagră and fetească neagră) from 2019 harvest, cultivated in the bugeac area, republic of moldova. the grape pomace was dried at 50 °c until constant weight was achieved. the moisture content of dried grape pomace varied between 0.11% and 0.35%. then, dried pomace was powdered and separated it on the particle size interval of 125-200 µm using an analytical sieve shaker retsch as 200 basic (retsch gmbh, haan, germany). 2.2. extraction and purification of pectin initially, a sample of 10 g grape pomace powder was mixed with 100 ml of solvent (solid-liquid ratio of 1:10, w/v) acquired by adding citric acid to ultrapure (milli-q) water until a ph 1, 2 and 3 were achieved. then, the mixtures were kept in a water bath precisdig (jp selecta, barcelona, spain) at the different temperature (70, 80 and 90 °c) for 3 h. after extraction, the mixtures were cooled to room temperature, around 20-22 °c. firstly, the pectin was segregated by centrifugation at 3500 rpm for 35 min. then, the obtained supernatants were got through clean strainer, plased into the neck of a duran® laboratory glass bottle with pouring ring and screw cap. afterwards, ethyl alcohol (>96%, v/v) was added to supernatants in order to achieve 1:1 ratio (v/v). the mixtures were kept at 4-6 °c for 12 h to accomplish the precipitation. the precipitated pectin was separated by centrifugation at 4000 rpm for 30 min. the pectin was washed 3 times with ethyl alcohol (>96%, v/v) and dried in an oven with air circulation zhicheng zrd-a5055 (zhicheng, shanghai, china) at 50 °c until constant weight was achieved. 2.3. pectin yield pectin yield was calculated using eq. (1): where: – weight of dried pectin (g), – weight of dried grape pomace powder (g) [16,17]. 2.4. galacturonic acid content the galacturonic acid content (gala) of pectin was estimated using the sulfamate/m-hydroxydiphenyl method developed by filisetti-cozzi & carpita [18,19]. sample preparation was made according to miceli-garcia [20]. 3. results and discussion 3.1. effect of temperature on the pectin yield and galacturonic acid content in agreement with the results in fig. 1, yield of pectin was significantly influenced by extraction temperature. it is possible to food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 316 * fn – fetească neagră, rn – rară neagră. extraction conditions – ph 2, extraction time of 3 h and 125-200 µm of particle size. fig. 1 effect of the temperature on the pectin yield observe that yield varied between 4.13% and 7.34% for fetească neagră (fn) grape pomace pectin and between 4.58% and 7.56% for rară neagră (rn) grape pomace pectin depending on the extraction temperature. the obtained results showed that temperature has a major impact on the yield of pectin. a rising temperature from 50 to 95 °c utilizing citric acid at ph 2.5 or 4.0 significantly enhanced the yield of cocoa husks pectin, from 3.58% to 5.66% (extraction temperature of 50 °c and 95 °c, respectively at ph 2.5) and from 3.72% to (extraction temperature of 50 °c and 95 °c, respectively at ph 4.0) [21]. also, vriesmann et al. [22], méndez et al. [23] and gutöhrlein [24] demonstrated that increasing temperature significantly enhanced the pectin yield from cocoa husks by citric acid, watermelon rind waste by hydrochloric acid and pea hulls by citric acid, respectively. the heated acid solution facilitated to solubilize the pectin and other pectic compounds retained in the cw of plants (protopectin), thus increased of the pectin yield [21,25]. furthermore, a low value of temperature may be inadequate to allow the protopectin hydrolysis (waterinsoluble pectic substance) by different acids, thereby achieving a lower pectin yield [21]. the influence of the temperature on the galacturonic acid content (gala) is presented in fig. 2. therefore, temperature influenced the gala of grape pomace pectin from both grape pomace varities (fn and rn). the gala varied from 45.13 g/100 g to 63.72 g/100 g and from 33.92 g/100 g to 61.89 g/100 g for pectin of fn and rn grape pomace, respectively. this is in accordance with the research of chan & choo [21] who stated that the uronic acid content of cocoa husks pectin where higher at 95 °c than at 50 °c when was extracted with citric acid at different ph (2.5 or 4.0). also, garna et al. [26] demonstrated that 90 °c contributes to a higher gala obtained from apple pectin then 80 °c, with a sulphuric acid solution at ph 1.5 or 2.0. these results may be ascribed to the increased chemical hydrolysis of pectin sugars when temperature significantly enhances [27– 29]. chaharbaghi et al. [30], who analyzed the optimization of pectin extraction from pistachio hull, noted that the pectin yield food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 317 was enhanced with an increase in extraction temperature. thus, they obtained the highest yield of pectin from pistachio hull (23.42%), under the following conditions: ph 0.5, extraction time of 30 min, temperature of 90 °c and a solid to liquid ratio of 50 v/w [30]. * fn – fetească neagră, rn – rară neagră, gala – galacturonic acid content. extraction conditions – ph 2, extraction time of 3 h and 125-200 µm of particle size. fig. 2 effect of the temperature on the galacturonic acid content a higher temperature increased the yield of pectin due to diffusion of solvent into the structure of cw and so, this movement caused the increase of the mass of polysaccharides releasing from the cw’s particles into the acid solution [30]. further, these conclusions are related to the research data obtained from waste durian rinds and sour orange peel pectin by maran [31] and hosseini et al. [32], respectively. 3.2. effect of ph on the pectin yield and galacturonic acid content stronger acid conditions (ph of about 2) play an essential role in influencing the structure and properties of pectin [33]. fig. 3 presents the influence of ph on the pectin yield, and fig. 4 illustrates its impact on the galacturonic acid content of pectin. the yield varied between 6.28% and 15.54% for fn grape pomace pectin and between 6.38% and 9.96% for rn grape pomace pectin. thus, the highest influence on pectin yield was determined by ph 1 (15.54% and 9.96% for fn and rn grape pomace pectin, respectively). these results also are in line with the research of chaharbaghi et al. [30], who studied the extraction optimization of pectin from pistachio hull and obtained the highest pectin yield (23.42%) at ph 0.5. furthermore, ma et al. [34] examined the influence of extraction factors on the sugar beet pulp pectin, and noted that decrease in ph increased the pectin yield. this is possibly as a result of cw’s lysis in strong acidic solutions and thus, dissolve and release the pectin in acid extract [34]. also, raji et al. [35] obtained the maximum extraction yield of pectin from melon peel (29.48%) under ph 1, temperature of 90 °c after 200 min of citric acid extraction. the gala is a great indicator of pectin purity, thus that a direct relation between food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 318 this factor and purity of extracted pectin was occured [30,36]. moreover, according to fao/who the gala of pectin should not be less than 65% [37]. in current study, the data achieved from used citric acid extraction of pectin showed a gala range of 40.12-46.78 g/100 g and 39.57-78.16 g/100 g for fn and rn grape pomace pectin, respectively (fig. 4). the highest value of gala (78.16 g/100 g) was achieved for rn grape pomace pectin under the following conditions: ph 2, 90 °c, extraction time of 3 h and 125-200 µm of particle size. also, this result is nearly related to data acquired from sugar beet pulp (78.8 g/100 g) at ph 2 of citric acid solution, for 2 h of extraction by ma et al. [34]. * fn – fetească neagră, rn – rară neagră. extraction conditions – temperature of 90 °c, extraction time of 3 h and 125-200 µm of particle size. fig. 3 effect of the ph on the pectin yield * fn – fetească neagră, rn – rară neagră, gala – galacturonic acid content. extraction conditions – temperature of 90 °c, extraction time of 3 h and 125-200 µm of particle size. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 319 fig. 4 effect of the ph on the galacturonic acid content furthermore, yapo [38] presented similar results for gala (70.6%, w/w) extracted from pectin of yellow passion fruit rind under citric acid extraction at ph 1.8, for 60 min at temperature of 75 °c. the obtained data confirmed that ph as well as temperature can influence the yield and gala of extracted pectin from grape pomace. 4. conclusion the extraction conditions (temperature and ph) have a significant impact on the yield and purity of pectin from grape pomace. considering the high yield of pectin and its purity, it can be assured that grape pomace is an unconventional source of pectin. the highest influence on pectin yield was determined by ph 1 (15.54% and 9.96% for fn and rn grape pomace pectin, respectively) and temperature of 90 °c (7.34% and 7.56% for fn and rn grape pomace pectin, respectively). moreover, these results are in accordance with previous studies based on unconventional sources of pectin extraction. additional study about grape pomace pectin should be conducted to the influence of other extraction conditions with application of different extraction techniques. 5. acknowledgments this paper has been financially supported within the project entitled ”decide – development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu/380/6/13/125031, supported by project co-financed from the european social fund through the 2014-2020 operational program human capital”. 6. references [1]. harholt, j., suttangkakul, a., & vibe scheller, h., biosynthesis of pectin. plant physiology, 153, 384–395, (2010). [2]. wusigale, l., l., & luo, y., casein and pectin: structures, interactions, and applications. trends in food science and technology, 97, 391– 403, (2020). [3]. zhang, x., lin, j., pi, f., zhang, t., ai, c., & yu, s., rheological characterization of rg-i chicory root pectin extracted by hot alkali and chelators. international journal of biological macromolecules, 164, 759–770, (2020). [4]. picot-allain, m. c. n., ramasawmy, b., & emmambux, m. n., extraction, characterisation, and application of pectin from tropical and sub-tropical fruits: a review. food reviews international, 1–31, (2020). [5]. anees, m., gao, l., umer, m. j., yuan, p., zhu, h., lu, x., he, n., gong, c., kaseb, m. o., zhao, s., … & wenge, l., identification of key gene networks associated with cell wall components leading to flesh firmness in watermelon. frontiers in plant science, 12, (2021). [6]. fullerton, c. g., prakash, r., ninan, a. s., atkinson, r.g.; schaffer, r.j.; hallett, i. c., & schröder, r., fruit from two kiwifruit genotypes with contrasting softening rates show differences in the xyloglucan and pectin domains of the cell wall. frontiers in plant science, 11, (2020). [7]. induru, j., pectin-based nanomaterials in drug delivery applications. in biopolymer-based nanomaterials in drug delivery and biomedical applications, elsevier, pp. 87–117, (2021). [8]. cui, j., zhao, c., feng, l., han, y., du, h., xiao, h., & zheng, j., pectins from fruits: relationships between extraction methods, structural characteristics, and functional properties. trends in food science and technology, 110, 39– 54, (2021). [9]. fierascu, r. c., sieniawska, e., ortan, a., fierascu, i., & xiao, j., fruits by-products – a source of valuable active principles. a short review. frontiers in bioengineering and biotechnology, 8, (2020). [10]. moslemi, m. reviewing the recent advances in application of pectin for technical and health promotion purposes: from laboratory to market. carbohydrate polymers, 254, 117324, (2021). [11]. marques, c, sotiles, a. r., farias, f. o., oliveira, g., mitterer-daltoé, m. l., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 320 & masson, m. l., full physicochemical characterization of malic acid: emphasis in the potential as food ingredient and application in pectin gels. arabian journal of chemistry, 13, 9118–9129, (2020). [12]. chen, j., cheng, h.; zhi, z., zhang, h., linhardt, r. j., zhang, f., chen, s., & ye, x., extraction temperature is a decisive factor for the properties of pectin. food hydrocolloids, 112, 106160, (2021). [13]. liew, s. q., chin, n. l., & yusof, y. a., extraction and characterization of pectin from passion fruit peels. agriculture and agricultural science procedia, 2, 231–236, (2014). [14]. adetunji, l. r., adekunle, a., orsat, v., & raghavan, v. advances in the pectin production process using novel extraction techniques: a review. food hydrocolloids, 62, 239–250, (2017). [15]. kravtchenko, t. p., arnould, i., voragen, a. g. j., & pilnik, w., improvement of the selective depolymerization of pectic substances by chemical β-elimination in aqueous solution. carbohydrate polymers, 19, 237–242, (1992). [16]. dranca, f., & oroian, m., extraction, purification and characterization of pectin from alternative sources with potential technological applications. food research international, 113, 327–350, (2018). [17]. liew, s. q., ngoh, g. c., yusoff, r., & teoh, w. h., sequential ultrasound-microwave assisted acid extraction (umae) of pectin from pomelo peels. international journal of biological macromolecules, 93, 426–435, (2016). [18]. filisetti-cozzi, t. m. c. c., & carpita, n. c., measurement of uronic acids without interference from neutral sugars. analytical biochemistry, 197, 157–162, (1991). [19]. melton, l. d., & smith, b. g., determination of the uronic acid content of plant cell walls using a colorimetric assay. current protocols in food analytical chemistry, 00, (2001). [20]. miceli-garcia, l. g., pectinfrom apple pomace: extraction, characterization, and utilization in encapsulating alpha-tocopherol acetate, university of nebraska-lincoln, (2014). [21]. chan, s.-y., & choo, w.-s., effect of extraction conditions on the yield and chemical properties of pectin from cocoa husks. food chemistry, 141, 3752–3758, (2013). [22]. vriesmann, l. c., teófilo, r. f., & de oliveira petkowicz, c. l., extraction and characterization of pectin from cacao pod husks (theobroma cacao l.) with citric acid. lwt – food science and technology, 49, 108–116, (2012). [23]. méndez, d. a., fabra, m. j., gómezmascaraque, l., lópez-rubio, a., & martinez-abad, a., modelling the extraction of pectin towards the valorisation of watermelon rind waste. foods, 10, 738, (2021). [24]. gutöhrlein, f., drusch, s., & schalow, s., extraction of low methoxylated pectin from pea hulls via rsm. food hydrocolloids, 102, 105609, (2020). [25]. greve, l. c., mcardle, r. n., gohlke, j. r., & labavitch, j. m., impact of heating on carrot firmness: changes in cell wall components. journal of agricultural and food chemistry, 42, 2900–2906 (1994). [26]. garna, h., mabon, n., robert, c., cornet, c., nott, k., legros, h., wathelet, b., & paquot, m., effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol. journal of food science, 72, c001–c009, (2007). [27]. fraeye, i., deroeck, a., duvetter, t., verlent, i., hendrickx, m., & vanloey, a., influence of pectin properties and processing conditions on thermal pectin degradation. food chemistry, 105, 555–563, (2007). [28]. mao, g., wu, d., wei, c., tao, w., ye, x., linhardt, r. j., orfila, c., & chen, s., reconsidering conventional and innovative methods for pectin extraction from fruit and vegetable waste: targeting rhamnogalacturonan i. trends in food science and technology 94, 65–78, (2019). [29]. mao, y., lei, r., ryan, j., arrutia rodriguez, f., rastall, b., chatzifragkou, a., winkworth-smith, c., harding, s. e., ibbett, r., & binner, e., understanding the influence of processing conditions on the extraction of rhamnogalacturonan-i „hairy” pectin from sugar beet pulp. food chemistry x, 2, 100026, (2019). [30]. chaharbaghi, e., khodaiyan, f., & hosseini, s. s., optimization of pectin extraction from pistachio green hull as a new source. carbohydrate polymers, 173, 107–113, (2017). [31]. maran, j. p., statistical optimization of aqueous extraction of pectin from waste durian rinds. international journal of biological macromolecules, 73, 92–98, (2015). [32]. hosseini, s. s., khodaiyan, f., & yarmand, m. s., aqueous extraction of pectin from sour orange peel and its preliminary physicochemical properties. international journal of biological macromolecules, 82, 920–926, (2016). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 mariana spinei , mircea oroian, extraction temperature and ph as decisive factors for the yield and purity of grape pomace pectin, food and environment safety, volume xx, issue 4 – 2021, pag. 314 – 321 321 [33]. bai, l., zhu, p., wang, w., & wang, m., the influence of extraction ph on the chemical compositions, macromolecular characteristics, and rheological properties of polysaccharide: the case of okra polysaccharide. food hydrocolloids, 102, 105586, (2020). [34]. ma, s., yu, s., zheng, x., wang, x., bao, q., & guo, x., extraction, characterization and spontaneous emulsifying properties of pectin from sugar beet pulp. carbohydrate polymers, 98, 750– 753, (2013). [35]. raji, z., khodaiyan, f., rezaei, k., kiani, h., & hosseini, s. s., extraction optimization and physicochemical properties of pectin from melon peel. international journal of biological macromolecules, 98, 709–716, (2017). [36]. liang, r., chen, j., liu, w., liu, c., yu, w., yuan, m., & zhou, x., extraction, characterization and spontaneous gel-forming property of pectin from creeping fig (ficus pumila linn.) seeds. carbohydrate polymers, 87, 76–83, (2012). [37]. residue monograph prepared by the meeting of the joint fao/who expert committee on food additives (jecfa), 82nd meeting 2016. pectins, (2016). [38]. yapo, b. m., biochemical characteristics and gelling capacity of pectin from yellow passion fruit rind as affected by acid extractant nature. journal of agricultural and food chemistry, 57, 1572–1578, (2009). 1. introduction 2. materials and methods 2.1. materials 2.2. extraction and purification of pectin 2.3. pectin yield 2.4. galacturonic acid content 3. results and discussion 3.1. effect of temperature on the pectin yield and galacturonic acid content 3.2. effect of ph on the pectin yield and galacturonic acid content 4. conclusion 5. acknowledgments 6. references применение комплексоутворювачив для повышения эффективности мембранных процессов очистки стоковых вод food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 contents: 1. functional, pasting and sensory properties of complementary food from orange fleshed sweetpotatoroasted african yam bean-tigernut composite flour using response surface methodology omobolanle omowunmi olorode, emmanuel kehinde oke, pius alaba agbebi, adebola atinuke alabi, femi fidelis akinwande 273 291 2. general linear model (glm) analysis of the effects of packaging materials and preservatives on nutrient composition of smoked fish ahmed ayodeji ayeloja, taofik oyedele dauda 292 302 3. characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context chafia merah 303 312 4. comparison and risk assessment of trace metal content in factory-farmed broiler and free-range chicken meat sold in laguna, philippines monique t. estrella, aileen fritz t. samson, rafael a. espiritu 313 320 5. development of a new devıce for applyıng atmospherıc pressure cold plasma on foods tamer akan, sehban kartal, sühendan mol, serap coşansu, şehnaz yasemin tosun, didem üçok alakavuk, şafak ulusoy, hande doğruyol, kamil bostan 321 332 6. effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou 333 344 7. heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria udeme effiong jonah, cecilia friday mendie 345 353 8. recovery of hesperidin from lemon by-products using green techniques francisco lorca, david quintín, presentación garcía 354 363 9. recovery of bioactive compounds from oilcakesa review ancuța petraru, sonia amariei 364 381 10. author instructions i v 11. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 microsoft word 01 cuprins 2_2023 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2022 contents: 1. chemical and physical characterization of common bean (phaseolus vulgaris l.) landraces by north – north-western extremity of romania maria poroch seriţan, paula maria greculeac (galan), dumitriţa – sabina dobrincu, mihaela jarcău, alexandra-lăcrămioara matei, tudorița chicaroș 79 88 2. romanian traditional dishes and certified food products cristina ghinea, ancuta elena prisacaru 89 100 3. towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent) n’guettia kossonou, lessoy zoue, gisèle koua, sébastien niamke 101 108 4. functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio 109 121 5. chemical composition of animal feed and its influence on the milk quality julijana tomovska, ilmije vllasaku, elena josevska 122 134 6. study of hydrogeological functioning of complex terminal (ct) aquifer, using hydrogeochemical and isotopic tools: case study of oued souf region (southeast of algeria) ali nesrat, serhane brahmi, chemseddine fehdi, tarek djedid, salim khachana 135 145 7. assessment of groundwater vulnerability to pollution using god method in the soummam catchment area (northern algeria) selma kissar, kaddour khammoudj 146 158 8. author instructions i v 9. subscription information vi food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2022 doi: https://doi.org/10.4316/fens.2023.009 89 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 89 100 romanian traditional dishes and certified food products *cristina ghinea 1 , *ancuta elena prisacaru 1,2 1faculty of food engineering, stefan cel mare university of suceava, romania, e-mail, cristina.ghinea@fia.usv.ro, ancuta.prisacaru@fia.usv.ro 2suceava-botoșani regional innovative bioeconomy cluster association, romania *corresponding author received 15th march 2023, accepted 15th june 2023 abstract: the aim of this paper is to provide a description of romanian gastronomy and to analyze the current situation of certified food products. tastes, aromas, colors, combinations of romanian ingredients and traditional technologies have led to the appearance of traditional products that have been passed down from generation to generation. research focuses on linking existing natural resources to romanian cuisine from each region of the country. currently, at national level there are 725 certified traditional food products with unique characteristics, while at european level there are 11 products (meat, fish, cheese, fruit and vegetables) with protected geographical indication (pgi) designation and one product with protected designation of origin (pdo) (cheese), 41 wines with pdo, 13 wines with pgi and 9 spirits with geographical indication (gi). the largest number of traditional meat, dairy and bakery products were certified in the center region of the country, while the largest number of beverages and traditional fruit and vegetable products were certified in the north-west region. keywords: certification, gastronomy, romanian cuisine, traditional foods. 1. introduction food production is related to location, climate conditions, biodiversity, religion and beliefs, trade routes. since ancient times all communities developed the dishes based on diversity offered by nature [1]. thus, the natural resources, social, economic, and cultural development contribute to the traditional dishes [2]. romanian gastronomy was also influenced by these factors by combining old peasant recipes with recipes from the neighbors [3]. romanian cuisine includes meat (bacon, sausage, drums, wire, roast pork, and bone soup) and vegetables dishes, milk and cheese, fish, polenta, pies with cheese, meat, cabbage, wine, beer, plum brandy etc. over time, romanians began eating pork in the winter and poultry in the summer, while sheep and goat meat were consumed in smaller quantities since wool or milk were considered more important than meat [4]. romanian cuisine includes everyday food (breakfast, lunch, and dinner) and festive dishes connected with christian traditions especially holidays like christmas, easter and new year [3, 5]. all the historical regions of romania have common but also different dishes, having a very well-defined gastronomic identity [3]. the objectives of this paper are to provide a review of the location and characteristics of romania regions, a brief description of the gastronomy specific to romania and to identify the certified romanian food products. 2. romania: location and characteristics romania (with an area of 238,937 km2) is a largely rural country [7] and is located in south-eastern central europe [6]. romania's relief includes mountains (the carpathians with a maximum altitude of 2,544 m, the moldoveanu peak), hills and plateaus (the subcarpathians and the http://www.fia.usv.ro/fiajournal mailto:cristina.ghinea@fia.usv.ro mailto:ancuta.prisacaru@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 90 western hills, the moldavian, dobrogean and getic plateaus, but also the mehedinți plateau) and plains (the romanian plain and the western plain). the lowest region (below 10 m altitude) of the country is the danube delta [8]. romania benefits from all types of aquatic units: rivers, lakes, underground waters, sea waters [9]. according to prăvălie et al. [10] multiannual mean temperatures range between 11-12 °c in the south and fall below 1 °c in the mountains. carpathian mountains influence the temperate climate of the country, resulting precipitation > 1300 mm in mountain areas, while in extra-carpathian regions the precipitations are even as low as 400 mm [10]. in the mountainous area, annual average wind speeds of over 10 m/s were recorded, while in the black sea area, danube delta and northern dobrogea, were 9–10 m/s, and in the bârlad plateau were registered between 6 and 8 m/s. in the other areas of the country, the average annual wind speeds are lower [11]. in romania, there are 210 sunny days per year with three types of sunlight areas: greater than 1400 kwh/m2/year, between 1200 and 1400 kwh/m2/year, less than 1200 kwh/m2/year [12]. in romania, ecosystems (forest, grassland, freshwater and brackish ecosystems, marine and coastal ecosystems, and underground ecosystems) represent approximately 47% of the country's surface. at the national level, 89.1% of the forests are located in mountainous and hilly areas. coniferous forests represent 30.4% of the total forest area, and deciduous forests 69.6%. in romania there are beech forests (31.1%), followed by spruce (22.9%), various oak species (18.2%), fir (5%), pine (2.1%), other conifers (0.9%) and other deciduous (0.5%), the rest of the forests being made up of mixed forests. in the danube delta there are over 5,400 species of flora and fauna and 30 types of ecosystems. in 261 areas throughout the country, 783 types of habitats were identified. to date, 3795 species and subspecies of higher plants (3136 wild species, 623 cultivated species), 965 species of bryophytes (mosses), 8727 species of fungi (mushrooms), over 600 species of algae, including 35 marine species, have been recorded. also, a number of 33,802 animal species have been identified, of which 33,085 are invertebrates and 611 are vertebrate [13]. the 42 counties (including bucharest capital) of romania are grouped in the following regions: north-east (ne), south-east (se), south-muntenia (sm), south-west oltenia (sw), west (w) north-west (nw), centre (c) and bucharest-ilfov (bi) [6]. the historical provinces of romania were muntenia, oltenia, dobrogea, moldavia, bucovina, transylvania, maramureș, crișana, banat [14]. the ne region is part of the old historical region of moldova, and includes bucovina as well, south-east romania region includes part of the historical region of moldova (vrancea and galati counties), part of the historical region of muntenia (braila and buzau counties), as well as the historical region of dobrogea (constanta and tulcea counties), the south muntenia region is located in the southern part of romania and includes most of the historical region of muntenia. the sw oltenia region coincides with the old historical region of oltenia, west region includes mainly the banat region, while north-west development region includes crisana and maramures istorical regions. central region is located in the center of the country and includes the transilvania historical region. according to ins [15], in 2021, the total population was 22046917 inhabitants, of which 56.3% had residence in urban area and 43.7% in rural area. the highest number of inhabitants is found in the ne region, followed by the sm region (fig. 1). also, the highest percentage of women food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 91 living in rural areas are in these two regions. in 2014, the agricultural area of romania was 14630072 ha, and the arable area was 9395303 ha. southmuntenia and south-east regions have the largest agricultural areas and arable land (fig. 2). 40% of the total areas of 209417 ha of vineyards and viticultural nurseries are located in the se region of the country, while from the total of 196941 ha orchards and fruit nurseries approximately 41% are located in two regions: southmuntenia (20.90%) and south-west oltenia (20.16%). the regions rich in forests and other forest vegetation are centre and north-east, together accounting for about 37% of the total existing areas in 2014 (6734003 ha) [15]. the highest employment rate was observed in bucharest-ilfov, while the lowest was in north-east region in 2020 (fig. 3). fig. 1. inhabitants number by residence in different regions of the country (based on the data provided by ins [15]) a) b) fig. 2. distribution of a) agricultural area and b) arable land in romanian regions, in 2014 (calculated based on the data provided by ins [15]) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 92 fig. 3. employment rate by development regions (calculated based on the data provided by ins [15]) the oldest form of human settlement is village, which can be found in all geographical areas of romania. about 90% of the romania's surface is rural area and the majority of the rural population works predominantly in agriculture [16]. due to the large agricultural areas, good soil quality and temperate climate romania is an important producer of agrifood products [17]. in 2020, the live weight of animals intended for slaughter for cattle consumption was 172586 tons live weight. the regions that contributed more to the total number of cattle destined for slaughter were north-east with 29%, center with 16%, north-west with 15%, south muntenia with 14%. the west region contributes the most to the total live weight of animals destined for slaughter for consumption pigs (498,098 tons live weight in 2020) with 34%, followed by the nw and sm regions, each with 13%, by the se and ne regions with 12% and 11% respectively. the rest of the regions with contributions below 10% each. also in 2020, the live weight of animals destined for slaughter for sheep and goat consumption was 120,571 tons of live weight (with 22% contributed by the se region, followed by the center region with 20% and the nw region with 15%, while the other regions contribute with smaller percentages). the live weight of animals intended for slaughter, for poultry consumption was 663328 tons live weight. to this quantity, the south muntenia region had the largest contribution (with 25%), followed by the south-east and north-east regions, each with a 19% contribution. in romania, the total production of milk in 2020 was 42609 thousand hectoliters. the largest volumes of milk produced were registered in the ne, center and nw regions. the ne region is famous for cow's milk, while in the se region it ranks first in the production of sheep and goat milk. the most pieces of eggs were produced in the south muntenia region, 1,349 million pieces out of a total of 5,428 million pieces produced throughout the country in 2020. 30714 tons of honey were produced in romania in the same year. in the se and sw oltenia regions, the largest quantities of extracted honey were produced, 5652 tons and 5278 tons, respectively. in 2020, 6392369 tons of wheat (the regions that contributed the most to this amount were south muntenia with 28%, sw oltenia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 93 with 23% and the w region with 16%), 847241 tons of barley (sm, sw oltenia and se being the largest producers of barley), 196659 tons of oats (oats are grown on large areas especially in the nw region, but also in the ne and sw regions of oltenia), 10096689 tons of grain corn (the regions that contributed the most to this amount were sm with 18%, nw and ne, each with 16%) and 24,958 tons of rice (rice is grown only in the se and sm regions), were obtained. the sw oltenia, se muntenia and se regions are the largest producers of vegetables at the national level. sunflowers are cultivated more in the southern regions of the country, and in 2020, 2122865 tons of sunflowers were produced throughout the country. the high production of rapeseed was obtained in the south muntenia region, followed by the west and southeast regions. sugar beet and potatoes are mainly cultivated in the ne and central regions [15]. 3. gastronomy in romania gastronomy or food consumption plays an important role in country tourism and not only. the cuisine variety is influenced by different natural, historical and sociocultural factors like the relief, forest region, climate, seasonal changes, water source, soil type and agriculture. it is well knowing that romanian cuisine inherited numerous culinary habits from dacians, celts, greeks, turks, russians, bulgarians, hungarians, serbs, germans and austrians [4, 18]. romanian cuisine includes various dishes based on the local products (meat, milk, eggs, grain, vegetables, fruits), of these [19]: soups and borschts: vegetable cream soup (pumpkins, tomatoes, carrots, green beans, peas, peppers, potatoes, parsnips or leeks can be used), chicken soup with dumplings, vegetable soup (with potatoes, carrots, peppers, parsley, celery, onion, dill, etc.), fish soup with tomatoes, chicken peasant soup (chicken, carrot, parsnip, celery, red bell pepper, onion, boiled peas, oil, parsley, salt and pepper), beef soup, pork soup, lamb soup, tripe soup (beef bone, boiled beef belly, carrots, parsley root, celery, bay leaves, hot peppers, 20% sour cream, egg yolks, garlic, oil, salt and pepper to taste. to serve with: sour cream, hot peppers, pepper, vinegar or lemon juice) etc. vegetable dishes: stuffed peppers (for the filling minced meat, carrot, red bell pepper, green bell pepper, rice, onion, oil, salt, pepper to taste, fresh thyme. sauce tomato pulp, spoonful of flour, large onion, spoonful of paprika, oil, parsley, pepper), potato dish with mushrooms, spinach dish with garlic, pilaf with mushrooms, vegetable pot, bean dish, mashed vegetables („zacusca”), baked beans etc. salads: eggplant salad with tomatoes and peppers (ripe eggplant, bell pepper, red onion, tomato, oil, salt and pepper, parsley), cabbage salad with carrots, potato salad with meat, vegetable salad, baked zucchini salad, beetroot salad with horseradish, etc. steaks: grilled chicken wings, chicken legs with vegetables in the oven, roast beef in a pan, lamb roast with potatoes (lamb (leg and rib), oil, water, garlic, dried thyme, pepper, salt and paprika), pork neck with onions and potatoes etc. fish dish: fish borscht, fried fish with cornmeal, fish brine, fish plate (fish, onion, oil, garlic, red and green bell peppers, bay leaves, broth, small fresh tomatoes, fresh parsley, salt and pepper), carp with celery puree, marinated mackerel salad, carp roe salad etc. romanian cuisine includes dishes that can be prepared in entire country, and also specific foods belonging to romanian historical regions (with specific culture and customs) [5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 94 bucovina is famous for “sărmăluțe” in a nest of cream, pork sausages with beetroot and horseradish salad, trout “în fir de cetină”, chicken soup with homemade noodles, chicken or beef vegetable stews and pork chops. dishes made from forest mushrooms (“hribii, opintici”) are indispensable. for dessert, the most famous is the “cozonac”, followed by “poale-n brâu” pies, pancakes or “papanași” with cheese. also here you can find a wide range of alcoholic appetizer, made from fruit: cherries, blueberries, corned, raspberries. the traditional drink is double distilled brandy or “pălinca”. home beer is also a popular drink, which housewives brew from hops and corn kernels [20]. the inhabitants especially like to consume milk, cream and cheese. sour cream is consumed mostly with “tochitură de purcel”, with a “butcher's tochitura”, with “sarmale”, and even with a cheese pie. also, the borscht is prepared with sour cream. in this area are prepared “tocineii” (balls from bucovina “barabule” bucovina potatoes), trout or mushrooms with cream [21]. in moldavian cuisine are prepared „ciorbă de potroace”, „borşul moldovenesc”, „pârjoale moldoveneşti”, „alivenci”, „răcituri de pasăre”, „piftie de porc”, polenta, fish brine, „tochitură moldovenească”, pie with cighir, sausages (large intestine from pork stuffed with meat, onions, rice or groats), „poale’n brâu”, „cozonac” and „pască moldovenească”, pumpkin, cheese, apples and cabbage pies [22, 23]. the most important wines produced in moldavia are: “grasa de cotnari”, “feteasca alba”, “feteasca neagră”, “galbenă de odobeşti”, “zghihara de huşi”, “frâncuşa”, “băbeasca neagră”, tămâioasa românească” and “busuioaca de bohotin” [24]. dobrogea cuisine includes fish soups, belly soup, fish marinades, carp brine, fish “la proțap”, stuffed carp, mutton sausages, lamb in protap, saslac, kebab, moussaka, kefir, rice side dishes, but also baklava, “sarailii”, sweets with raisins. some examples of specific dobrogean dishes: fried fish, in the oven, on the grill, marinated, smoked catfish pastrami, fried turbot, danube carp brine, crumble on the grill or on the tray, pike marinated with vegetables in the oven, perch brine like in the danube delta, potato moussaka with fish, pike liver pate, fish sausage, filled pike, pike meatballs with rice, vegetables and seaweed, “sarmale” with fish, fishing borscht, fish meatballs in sauce, dobrogean “tochitură”, vareniki (dumplings with sweet cheese), cherdele (pies made from leavened dough, filled with a cheese composition), ghisman (it is prepared from corasla the first milk given by a cow after giving birth), nalangate (with yogurt, eggs, sugar, sodium bicarbonate, lemon or vanilla essence), dobrogean pie [25]. murfatlar wine is famous, and wines like chardonnay, pinot, riesling and muscat ottonel are produced in dobrogea [24]. muntenia traditional cuisine is characterized by tasty dishes like belly soup, beef and pork soup, “sarmale with polenta”, lamb stew, “ciulama”, „mititei”, grilled steaks and fish, pasta puddings, pies, pastry sweets with cream and chocolate. the traditional dishes are based on vegetables and meat and very appreciated in this country area are borscht of nettles, beans, mushrooms or chicken. steak with prunes and pie with cheese or pumpkin are prepared especially in winter. in muntenia, among the favorite dishes are roast chicken with quinces or apricots or duck with olives. rooster or goose stew is preferred by inhabitants, while pork stew is also prepared in winter [26]. “țuica” and wine are the alcoholic beverages produced muntenia, while non-alcoholic are ”socata” and syrups (of fir, rose, sour cherry) [27]. some famous wines such as “tămâioasa românească” and “feteasca food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 95 neagră” or “feteasca regală” from dealu mare or pitești brandy are obtained in this region [24]. in oltenia, the custom of cooking in clay pots is still preserved today. “sarmale” cooked in clay pots are very well know. fresh vegetables, poultry, beef and pork, fish or dairy products are used in oltenia cuisine. oltenia sausages, made from equal amounts of beef and pork, minced and mixed with garlic, pepper and salt, and sheep intestines which are stuffed with this paste, are smoked for two hours, are very famous. in oltenia villages are used more vegetables like nettles, stevia, sedge, green onion leaves for cooking. the soups are more sour, pickled with cabbage juice or tomato juice. chicken, vegetable, leek, loboda, stevia soups are preferred by oltenians. the leek has a place of honor in oltenia cuisine, and is prepared in most diverse forms. olives are also found in many dishes. desserts include doughnuts, puddings, pancakes [28]. “feteasca regală”, “băbeasca neagră”, sangiovese, “roșioara”, pinot noir, wines are produced in this region [24], while ”zaibăr” wine is the pride of each oltenian [27]. in transilvania cuisine, influenced by austro-hungarians, there are fatty, spicy and very tasty dishes based on pork. along with meat, vegetables, milk and cheeses are used. tarragon is a specific spice used in this region, especially in soups, soured with vinegar or cabbage juice, and dressings with egg yolk and cream. the sauces are white or seasoned with paprika, for color. the most famous dishes from transilvania are: “varza a la cluj cabbage á la cluj”, “branza de burduf”, “gulaș ardelenesc”, “ciorba de fasole în pâine”, “gomboți cu prune”, “vargabeles” [29, 30]. white wines “the white feteasca”, “the royal feteasca” or the delicious “muscat ottonel”, and red wines “babeasca” or “feteasca neagră” are obtained in transilvania [29]. similar to transilvanian cuisine, banatean cuisine receives austro-hungarian influences, but also influences from greek, italian and french cuisine. the dishes mainly consist of pork, with vegetables fried and stewed in lard and oil, with flour sauces and seasoned with pepper, thyme, paprika and cumin. the soups are soured with lemon, seasoned with tarragon and improved with cream. the sauces are made with breadcrumbs made from heated flour mixed with tomato or vegetable broth. sarmales from banat are prepared from meat minced with a cleaver, and are much larger compared to those specific to moldova. pork, cow, poultry and less fish are used a lot. the most popular banat dishes are: zacusca, veal soup, sarmale, stuffed onion, banat food with pork [31, 32]. pinot noir, cabernet sauvignon, merlot, big burgundy, “cadarcă de recaş”, italian riesling, sauvignon blanc, “creață de banat”, “majarcă” and steinschiller wines are obtained in this region [24]. crisana cuisine was influenced by hungarians and germans and is based on pork and vegetable dishes. products like pecica hearth pita, nădlac salami, chisindia brandy, măderat mustard are a real brand for this region [33]. aldești, groşii noi or chisindia brandy, miniș and pâncota wine are produced in crisana [33]. in maramures, dishes have an „ancestral taste” influenced by romanians, saxons and hungarians. this region is famous for zamuri, (soups) stews, papricaş, balmoş and gomboți (dumplings with plums). cauldron dishes are also prepared in this area, prepared over a fire with fir wood in the open air. cauldron stew, cauldron goulash and easter cauldron lamb are just a few examples [34, 35]. lard and cheese are used a lot, and there is no shortage of plum “horinca” and “țuica” boiled with honey [23]. in romania, there is a very taste and unique cuisine and the historical regions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 96 has a well-defined gastronomic identity [3, 22]. cuisine, culture and customs, house architecture and traditional costumes specific to each region contribute to romanian cultural heritage [5]. the cuisine is influenced by a number of material factors: geographical factor (relief, climate, vegetation, crops), historical and social factor (foreign occupations, historical experiences, social structures, exchanges trade), the economic factor (degree of productivity and prosperity). consumers will look for and privilege those foods, dishes and recipes that are part of their culinary tradition [36]. 4. certified food products due to the large agricultural areas, good soil quality and temperate climate, romania is an important producer of agrifood products [17]. at european level, romania has certified 11 products with the name protected geographical indication (pgi): “magiun de prune topoloveni” (class 1.6. fruit, vegetables and cereals fresh or processed), “novac afumat din ţara bârsei”, “scrumbie de dunăre afumată” and “salată cu icre de știucă de tulcea” (all three in class 1.7. fresh fish, molluscs, and crustaceans and products derived therefrom), “salam de sibiu”, “cârnaţi de pleşcoi” and “salinate de turda” (all three in class 1.2. meat products (cooked, salted, smoked, etc.)), “telemea de sibiu” and “caşcaval de săveni” (both in class 1.3. cheeses), “plăcintă dobrogeană” and “pită de pecica” (both in class 2.3. bread, pastry, cakes, confectionery, biscuits and other baker's wares), and one protected designation of origin (pdo) product namely “telemea de ibăneşti” (class 1.3. cheeses) [37]. as far as wines are concerned, 41 wines with pdo (such as cotnari, recaş, murfatlar, dealu mare or panciu) and 13 wines with pgi (like dealurile crişanei, dealurile sătmarului, dealurile transilvaniei, dealurile zarandului etc.) have been certified so far [37]. also, romania certified 9 spirit drinks with geographical indication (gi): “pălincă”, “țuică de argeș”, “horincă de cămârzana”, “țuică zetea de medieșu aurit” (product category: 9. fruit spirit) and “vinars segarcea”, “vinars târnave”, “vinars vrancea”, “vinars murfatlar” and “vinars vaslui” (product category: 4. wine spirit) [37]. the total number of traditional product certificates nationwide is 725, according to the national register of traditional products (rnpt) published on 6 july 2022. of these, 304 are meat and meat products, 131 are milk and milk products, 114 are bread, bakery and pastry products, 113 are fruit and vegetables, 28 are fish, 29 are beverages and 6 are other. the correlations between the traditional products and the main ingredients produced in the country regions are illustrated in figs. 4-7. according to fig. 4, the largest quantities of meat were produced in the following regions: beef in the north east region (ne), pork in the west region (w), sheep meat and goat meat in the south east region (se), and poultry meat in the south muntenia region (sm), while most traditional meat products were certified in the central region (c). it can be seen that although there are large meat producers, in these regions the certification of traditional products is lower. small productions of pork and mutton were obtained in the bucharest ilfov (bi) and sud oltenia (so) regions, while the north-west (nw) region ranks third in the production of beef, pork and mutton. a classification in descending order of the regions according to the number of certified traditional meat products would be the following c>nw>se>ne>so>sm>bi>w. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 97 40000 20000 0 160000 80000 0 20000 10000 0 120100806040200 160000 80000 0 b e e f (t o n s ) p o r k m e a t (t o n s ) m u tt o n ( to n s ) number of traditional meat products p o u lt r y m e a t (t o n s ) w so bi sm se ne cnw w so bi sm se ne c nw w so bi sm se ne c nw w so bi sm sene c nw fig. 4. correlation between traditional meat products and meat production by regions most dairy products were also certified in the center region according to fig. 5, instead the regions where the higher quantities of milk are obtained in the ne region (cow's milk) and the se region (sheep's milk). the center region is in second place in the production of cow and sheep milk. a descending ranking of regions according to the number of nationally certified traditional dairy products would be as follows: c>nw>sm>ne>w>se>so. 10000 8000 6000 4000 706050403020100 1200 1050 900 750 600 c o w m il k ( th o u s a n d h e c to li te r s ) number of traditional dairy products s h e e p m il k ( th o u s a n d h e c to li te r s ) w so sm se ne c nw w so sm se ne c nw fig. 5. correlation between traditional dairy products and milk production by regions the largest quantities of fruits were produced in the sm, nw and so regions, while the largest quantities of vegetables were obtained in the sm, so and ne regions. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 98 the region where the largest quantities of potatoes are produced is the central region, followed by the ne and nw regions. fig. 6 indicates that most traditional fruit and vegetable products were certified in the nw region. such products were also certified in the ne, c and w regions, and a smaller number of such products (under 10) were certified in the other regions. the regions where the largest amounts of grain are produced are sm, so and w, while the sm region produces the most eggs. according to fig. 7, the center region recorded the most traditional bakery products, followed by the nw and ne regions. 800000 400000 0 600000 450000 300000 403020100 300000 200000 100000 p o ta to e s ( to n s ) ve g e ta b le s ( to n s ) number of traditional fruit and vegetables products fr u it s ( to n s ) w so sm se nec nw w so sm se ne c nw w so sm se ne c nw fig. 6. correlation between traditional fruit and vegetable products and fruits and vegetable production by regions 4000000 2000000 0 10000 5000 0 706050403020100 1000 500 0 c e r e a ls ( to n s ) c o w m il k ( th o u s a n d h e c to li te r s ) number of traditional bakery products e g g s ( p ie c e s ) w so bi sm se ne c nw w so bi sm se ne cnw w so bi sm se ne cnw fig. 7. correlation between traditional bakery products and cereals, milk and eggs production by regions food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 99 classification of the regions according to the number of traditional bakery products in descending order is the following c>nw>ne>se>bi>w>sm> so. the results showed that the certification of traditional products is not specifically done in the large regions producing raw materials, so in the center region the most traditional meat, milk, bakery and fish products were registered, while in the northwest region there were most fruit and vegetable products, but also beverages, were registered. 5. conclusion traditional foods are prepared based on beliefs, behaviours, religions, values and social norms. each cuisine has unique characteristics and associated with cultural aspects can represents an attraction of tourists especially in rural areas. a wide variety of gastronomic riches were found in all country regions. certification of traditional products is a garantee of product quality, may increase by income of rural producers and depends on the pedoclimatic conditions of raw materials. even if they have a high price, their certification promotes them as natural, authentic and healthy products. rural producers must be encouraged and supported to obtain the certification of traditional products. 6. acknowledgments this paper was supported by competitiveness operational program – call: poc/975/1/1/large infrastructures – projects for innovation clusters, under the contract no. smis 2014+ 153837. 7. references [1]. özer ç., atay e., investigation of local cuisine culture in turkey by regions and the importance in rural tourism, international journal of social and economic sciences, 7:17-21, (2017) [2]. pipan a., gačnik a., role of cultural heritage in the development of gastronomy tourism in the rural area: case of the vipava valley, agricultura, 17:19-26, (2020) [3]. simoni s., teodoroiu f., țală m.l., țigu g., cultural and economic aspects of promoting romanian traditional gastronomy, basiq international conference, 582-589, (2017) [4]. voinea l., popescu d.v., negrea t.m., dina r., nutrient profiling of romanian traditional dishes—prerequisite for supporting the flexitarian eating style, information, 11:514, (2020) [5]. salanţă l.c., păucean a., tofană m., man s., pop c., romanian cuisine: culinary habits and local produce, journal of agroalimentary processes and technologies, 21:186-191, (2015) [6]. vrînceanu a., dumitrașcu m., kucsicsa g., site suitability for photovoltaic farms and current investment in romania, renewable energy, 187: 320-330, (2022) [7]. iorio m., corsale a., rural tourism and livelihood strategies in romania, journal of rural studies, 26:152-162, (2010) [8]. lungu m., antologia statelor lumii (a doua ediție) (anthology of the states of the world (second edition), ed. steaua nordului, constanța, (2004) [9]. pătru i., zaharia l., oprea r., geografia fizică a româniei–climă, ape, vegetație, soluri (physical geography of romania – climate, waters, vegetation, soils), ed. universitară, bucurești, (2006) [10]. prăvălie r., piticar a., roșca b., sfîcă l., bandoc g., tiscovschi a., patriche c. spatio-temporal changes of the climatic water balance in romania as a response to precipitation and reference evapotranspiration trends during 1961–2013, catena, 172:295-312, (2019) [11]. dragomir g., șerban a., năstase g., brezeanu a.i., wind energy in romania: a review from 2009 to 2016, renewable and sustainable energy reviews, 64:129-143, (2016) [12]. năstase g., șerban a., dragomir g., brezeanu a.i., bucur i., photovoltaic development in romania. reviewing what has been done, renewable and sustainable energy reviews, 94:523-535, (2018) [13]. cbd, strategia naţională şi planul de acţiune pentru conservarea biodiversităţii 2014 – 2020 (the national strategy and action plan for biodiversity conservation 2014-2020), available online at: https://www.cbd.int/doc/world/ro/ronbsap-v3-ro.pdf, (12 december 2022) [14]. cocoş r., schipor s., hervella m., cianga p., popescu r., bănescu c., food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 cristina ghinea, ancuta elena prisacaru, romanian traditional dishes and certified food products, food and environment safety, volume xxii, issue 2 – 2023, pag. 89 – 100 100 constantinescu m., martinescu a., raicu f., genetic affinities among the historical provinces of romania and central europe as revealed by an mtdna analysis, bmc genet., 18:20, (2017) [15]. ins, tempo ins database, national institute of statistics, romania. available online at: http://statistici.insse.ro/shop/?lang=en, (29 july 2022) [16]. academia română, cunoaşte românia (get to know romania), ed. economică, bucureşti, (2004) [17]. gavrilescu c., voicilaş d.m., competitiveness of the romanian agri-food trade and the new agricultural policies, zagadnienia ekonomiki rolnej, 4:106-116, (2014) [18]. sava c., cleşiu s.r., romanian gastronomy between traditionalism commerce, business and rural tourism, knowledge horizons – economics, 6:66–69, (2014) [19]. romanian cuisine, available online at: https://bucataria-romaneasca.ro/, (12 september 2022) [20]. local gastronomy. authentic bukovinian taste, available online at: https://sadova.ro/mancare-sadova-bucovina/, (12 september 2022) [21]. dănilă s., albu a., gastronomia din bucovina, available online at: http://tourisminbucovina.ro/gastronomie.pdf, (2014) [22]. muntean m.c., nistor c., nistor r., sarpe d., local cuisine – always an important decisional factor in international tourism, wseas transactions on business and economics, 4:381-391, (2010) [23]. nistor s., ghidul gastronomic al romaniei (romania's gastronomic guide), editura house of guides, (2004) [24]. nedelcu a., gastronomic and wine tourism – key elements for local economic development. case study romania, second international thematic monograph, thematic proceedings modern management tools and economy of tourism sector in present era, 142160, (2017) [25]. danube delta culinary recipes, available online at: https://www.infodelta.ro/category/retete-culinare/page/4/, (12 september 2022) [26]. muntenia cuisine, available online at: https://bucatarianationala.wordpress.com/2015/01/1 6/bucataria-din-muntenia/, (12 september 2022) [27]. smaranda s., traditional romanian food-as the result of history, nature, way of life and human ingenuity, lucrări științifice management agricol, seria i, 19:229-235, (2017) [28]. muntenia-oltenia, available online at: https://www.romanian-places.com/munteniaoltenia.html, (13 february 2023) [29]. păucean a., a geographical and historical overview of the transylvanian cuisine, bulletin uasvm food science and technology, 70:1-7, (2013) [30]. transilvania and banat cuisine, available online at: https://www.unileverfoodsolutions.ro/inspiratiebucatari/bucatariile-lumii/bucatariatraditionala/bucataria-din-transilvania-sibanat.html, (13 february 2023) [31]. pirvulescu i., bordean d.m., petroman i., petroman c., rujescu c., radu s., sirbulescu c., study on banat’s traditional gastronomy, lucrări ştiinţifice seria agronomie, 52: 732-736, (2009) [32]. traditional cuisine, available online at: https://www.unileverfoodsolutions.ro/inspiratiebucatari/bucatariile-lumii/bucatariatraditionala.html, (13 february 2023) [33]. aptr, crisana-bana, trasee naturale în zonele carstice din vestul muntilor apuseni (crisana-banat), mandrii de romania, ghid turistic (crisana-bana, natural routes in the karst areas of the western apuseni mountains (crisanabanat), proud of romania, tourist guide), (2012) [34]. maramures, available online at: https://www.romanian-places.com/maramures.html, (13 february 2023) [35]. the traditional maramures cuisine, https://marea-alpina.ro/bucataria-traditionalamaramureseana/, (13 february 2023) [36]. mitu s., traditional food and gastronomy in maramureș, memoria ethnologica, 78 – 79:82101, (2021) [37]. ec, eambrosia the eu geographical indications register, https://ec.europa.eu/info/foodfarming-fisheries/food-safety-andquality/certification/quality-labels/geographicalindications-register/, (2022) https://www.romanian-places.com/maramures.html https://marea-alpina.ro/bucataria-traditionala-maramureseana/ https://marea-alpina.ro/bucataria-traditionala-maramureseana/ 1. introduction 5. conclusion doi: https://doi.org/10.4316/fens.2021.038 352 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 352 359 analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria * omotade richard ogunremi 1 , enobong sunday ukpong 2 , aima airenobuwa nosa 2 1department of biological sciences, first technical university, ibadan, nigeria, tadeogunremi@yahoo.com 2department of biological sciences, samuel adegboyega university, ogwa, nigeria *corresponding author received 10th september 2021, accepted 20th deember 2021 abstract: education and regulations of hygienic practices are poorly carried out in the informal sector of food supply chain in developing countries, especially rural communities. therefore, this study was carried out to investigate microbiological status of fish and meat retail surfaces in ebelle market. four swab samples of fish and meat retail surfaces were collected and examined for microbiological load and profile. safety of selected isolates was assessed by haemolytic analysis and antibiotic susceptibility profile tests. the total bacterial counts from the surfaces were within the range of 5.06 6.40 log10 cfu/cm 2. coliforms were detected only in fish retail surface swab samples; ef1 (3.96 log10 cfu/cm 2) and ef2 (5.58 log10 cfu/cm 2). ef2 had a total e. coli count of 4.78 log10 cfu/cm 2. the identified species include bacillus sp. (23.08%), escherichia coli (7.69%), proteus sp. (15.38%), pseudomonas sp. (7.69%), salmonella sp. (97.69%), staphylococcus aureus (15.38%) and streptococcus sp. (23.08%). the total heterotrophic fungal counts were between 2.27 and 3.29 log10 cfu/cm 2. β-, αand γ-haemolysis was exhibited by 9, 2 and 2 isolates respectively. antibiotic susceptibility test showed that some isolates were multidrug resistant. the results obtained in this study indicate the retail surfaces are potential sources of food contamination. therefore it is pertinent for regulatory and public health authorities to establish cleaning and sanitation guidelines and train rural food vendor and retailers on the steps and importance of cleaning and sanitation. keywords: food safety, food contact surface, pathogens, antibiotics resistance, sanitation. 1. introduction microorganisms have evolved mechanisms of adhering and surviving on relatively dry surfaces, especially food contact surfaces [1, 2]. the nature of surfaces used in meat and fish retailing, storage and distribution include stainless steel, wood, plastic, concrete, and glass. these surfaces are subject to contamination by microorganisms and following failure to implement or comply with cleaning and sanitation guidelines, these surfaces are typically conditioned with nutrients and other organic materials that are sufficient for the attachment, viability and growth of microorganisms [2]. they develop microbial communities known as biofilms; microbial aggregates that are firmly attached to surfaces and embedded in exopolymers [3]. biofilms are difficult to detach and resistant to biocidal agents. therefore, conventional cleaning and sanitation regimes have not effectively decontaminated affected surfaces [3, 4]. biofilm-laden processing, storage and distribution surfaces represent an important source of contamination for a wide variety of food materials with pathogenic and spoilage microorganisms [4]. incidentaly, proper regulations and adherence to adequate hygienic and sanitation practices remains a challenge in the informal sector of the food supply chain in developing countries, especially the rural communities. this could be implicated in http://www.fia.usv.ro/fiajournal mailto:tadeogunremi@yahoo.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 353 the high incidence of foodborne infectious diseases in africa. the world health organization (who) estimates that 3 million infants die annually from the consumption of contaminated foods [2]. a recent estimate of global foodborne diseases with respect to incidence, mortality and disability adjusted life years (dalys) showed that africa has the highest burden of foodborne diseases per population [5]. dalys were attributed to foodborne diarrhoeal disease agents, particularly salmonella sp., shigella spp., escherichia coli and vibrio cholerae. there is a paucity of information on the microbiological status of food retailing surfaces in a typical nigerian rural community. it is imperative to generate the baseline data for increased awareness of public health inspectors and food supply regulators about the need to train rural food handlers on the guidelines for cleaning and sanitizing food contact surfaces and good hygienic practices (ghp) and for effective monitoring of activities in the food supply chain. therefore, this study was carried out to assess the burden and potential pathogenicity of microorganisms on fish and meat retail surfaces in ebelle market, nigeria. 2 materials and methods 2.1 samples swab samples were aseptically taken from a defined area (25 cm3) of fish and meat retail surfaces in ebelle market, igueben local government area, edo state, nigeria. the descriptions of the surfaces are presented in table 1. the samples were collected using sterile cotton swabs, immediately shaken and sealed in tube containing 10 ml of 0.85% physiological saline solution according to nsw food authority [6]. the swab samples were immediately transported to the microbiology laboratory, department of biological sciences, samuel adegboyega university, edo state for microbiological analysis. 2.2 enumeration and isolation of microorganisms a 1 ml of appropriate serial dilutions of each swab solution was pour-plated in appropriate media and incubated at appropriate condition to obtain the counts of different groups of microorganisms on the surfaces in cfu/cm2. total aerobic bacteria, coliform and escherichia coli counts were determined after incubation for 24 hours at 37℃, using nutirent agar, macconkey agar and eosine methylene blue agar respectively. fungal counts were determined after incubation for 72 hours at 30℃, using potatoes dextrose agar, supplemented with streptomycin. pure cultures of randomly selected bacterial colonies were obtained after repeated streaking on nutrient agar plates. the pure cultures were maintained on nutrient agar slants at 5 and renewed at two weekly intervals. 2.3 characterization and identification of bacterial isolates morphological examinations and biochemical tests were carried to determine the characteristics of pure bacterial cultures. they include; colony and cell morphology and gram reaction, catalase, oxidase, indole and sugar fermentation tests [7]. the isolates were identified by comparing their morphological and biochemical characteristics with the characteristics of reference organisms as described in bergey’s manual for determinative bacteriology. 2.4 haemolytic test fresh bacterial cultures were streaked in triplicate on blood agar plates (5 (w/v) defibrinated sheep blood) and incubated at 37 for 24 hours. the plates were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 354 examined for β-haemolysis (clear zones around colonies), α-haemolysis (green zone around colonies), and γ-haemolysis (no zones around colonies) [8]. 2.5 antibiotic susceptibility profile the antibiotic sensitivity profile of each bacterial isolate was determined using the agar disc diffusion method of kirby bauer. fresh culture of bacterial isolate was suspended in 0.85% sterilized physiological saline solution and adjusted to 0.5 mcfarland turbidity standard, equivalents to 1.5 x 108 cfu/ml. mueller-hinton agar plates were seeded with 0.2 ml of bacterial suspension by spread plate method with the aid of sterilized cotton swab and left to dry for 15 minutes at room temperature. commercially available antibiotics discs were aseptically placed on each seeded agar plates. the antibiotic discs were; ampiclox (apx-30 μg), amoxicillin (amx) (25μg), augmentin (au) (30μg), chloramphenicol (ch) (30μg), ciprofloxacin (cpx) (5μg), erythromycin (e-15 μg), gentamycin (gen-10 μg), ofloxacin (ofx) (5μg), perfloxacin (pef5 μg), rifampicin (ra-5 μg), septrin (sxt) (25μg), sparfloxacin (sfx) (5μg), streptomycin (str) (10μg) and zinnacef (z-25 μg) for gram-positive and/or gramnegative bacteria. the plates were incubated for 24 hours at 37℃. the resultant diameter of visible zones of inhibition were measured in millimeters (mm) and classified as resistant (r), intermediate (i) or sensitive (s) in accordance to the guidelines of the clinical and laboratory standard institute [9]. 2.6 statistical analysis data was collected in duplicate and presented in mean ± standard deviation of replicate values. counts were expressed in logarithmic units of microorganisms per centimeter square (log10 cfu cm -2) 3. results and discussion 3.1 nature of fish and meat retail surfaces food contact surfaces in informal sectors of food supply chain are major concerns as significant risk factors for foodborne diseases [10]. with reference to microbiological perspective, the nature of food contact surfaces can help prevent accumulation of microorganisms and potential contamination of foods. food contact surfaces should be smooth, nonabsorbent and easy to clean and sanitize. in ebelle market, the surfaces used for displaying and chopping fishes and meats were bare wooden or laid with papers or polyethylene (table 1). concerns with wooden and paper laid surfaces is the perviousness to moisture, roughness and difficulty cleaning them. thereby creating conditions that promote microbial growth and contamination of foods. table 1 characteristics of sampled retail surfaces in ebelle market retail surface name surface code description of surface ebelle meat retail surface 1 em1 bare wooden surface ebelle meat retail surface 2 em2 bare wooden surface ebelle fish retail surface 1 ef1 wooden surface covered with paper ebelle fish retail surface 2 ef2 wooded surface covered with polyethylene 3.2 analysis of microorganisms the total bacterial counts on the different sampled surfaces, including fish and meat retail surfaces ranged from 5.06 to 6.40 log10 cfu/cm 2. there were no significant differences in the total bacteria counts for different types of retail surfaces. the highest total bacteria count was recorded for em2, a meat retail surface. coliforms were detected only on fish retail surfaces, with counts ranging from 3.96 to 5.58 log10 cfu/cm 2. out of the retail surfaces food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 355 examined, only ef2, a fish retail surface was contaminated with escherichia coli. heterotrophic fungi were recorded for all the sampled surfaces, with counts ranging from 2.85 to 3.29 log10 cfu/cm 2 (table 2). the bacterial counts on fish and meat contact surfaces reported in this study are considered to be unacceptable, being significantly above the acceptable level of 3.0 cfu/cm2 as recommended by us public health service and the public health laboratory service (phls) in the uk [11, 12]. consistent with studies of food contact surfaces in meat and fish processing facilities by gounadaki et al. [13] and lani et al. [14] respectively, our results showed ˃5 log10 cfu/cm 2 and ~3 log10 cfu/cm 2 of bacteria and fungi respectively on fish and meat retailing surfaces in ebelle market. in a related study uzendu et al. [15] reported ˃5 log10 cfu/cm2 total heterotrophic bacterial count before and after beef processing on slaughter slabs and evisceration tables. besides, bacteria and molds greater than 4 log10 cfu/cm 2 were reported on all surfaces sampled in some butchers’ cold chain [16]. in agreement with this study, food contact surfaces in a child care center had generally low in e. coli and coliform counts [10]. table 2 microbial counts on meat and fish retail surfaces surface id counts (log10 cfu/cm 2) total bacteria total coliform e. coli total heterotrophic fungi em1 5.46 nil nil 2.85 em2 6.40 nil nil 2.92 ef1 5.06 3.96 nil 2.27 ef2 5.51 5.58 4.78 3.29 analysis of morphological and biochemical features of 13 bacterial isolates recovered from the enumeration agar plates and comparison with published reference databases revealed 7 species on fish and meat retail surfaces. the identified species include bacillus sp. (23.08%), escherichia coli (7.69%), proteus sp. (15.38%), pseudomonas sp. (7.69%), salmonella sp. (97.69%), staphylococcus aureus (15.38%) and streptococcus sp. (23.08%) (fig. 1). three of the 7 species, were found on fish retail surface, including bacillus sp., proteus sp. and staphylococcus aureus. this indicates poor hygienic practice and ineffective sanitation and that the surfaces used to retail fish and meat are sources of contamination with pathogenic and spoilage microorganisms. similar to this study, the reported bacterial pathogens and spoilers were previously reported from several food contact surfaces, including knives, chopping boards, holding tanks, pipes and milling, mixing, mincing and stuffing machines [2, 3, 16, 17, 18]. mohammed et al. [19] reported the occurrence of e. coli on food contact surfaces in a restaurant. pseudomonas spp., a prominent food spoilage microorganism was isolated from the surface of mixing machine used to produce keropok lekor’, a traditional processed fish product in malaysia [14] and meat cutting board in a brazilian hospital [20]. the most predominant bacterial species on fish and meat retail surfaces are bacillus sp. and streptococcus sp. respectively. the persistence of bacillus spp. on food contact surfaces is due to their resistance to desiccation, either in vegetative or spore form and biofilm production potentials food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 356 [21]. the predominance of bacillus spp. was previously reported for different meat contact surfaces of two abattoirs in ibadan, nigeria [15]. 0 5 10 15 20 25 bacillus sp. e. coli proteus sp. pseudomonas sp. salmonella sp. s. aureus streptococcus sp. p e rc e n ta g e o c c u rr e n c e ( % ) bacterial species meat retail surface fish retail surface fig. 1 percentage occurrence of bacterial species isolated from meat and fish retail surfaces 3.3 haemolytic properties of isolates nine isolates demonstrated β-haemolytic activities on blood agar, including bacillus sp. (2 strains), proteus sp. (1), pseudomonas sp. (1), salmonella sp. (1), staphylococcus aureus (2) and streptococcus sp. (2). escherichia coli em152 and proteus mirabilis em252 showed α-haemolysis while bacillus sp. ef141 and streptococcus sp. em261 exhibited γ-haemolysis. 3.4 antibiotic resistance pattern of isolates antibiotics susceptibility profiling of the 13 bacterial isolates revealed that 7, 5 and 2 out of 8 gram-positive bacterial strains and 0, 3 and 1 out of 5 gram-negative bacterial strains were resistant to amoxicillin, streptomycin and ciprofloxacin respectively (table 4 and 5). table 3 haemolytic activity of bacterial isolate isolated from meat and fish retail surfaces key: β-haemolysis: clear zones around colonies, αhaemolysis: green zone around colonies, γ –haemolysis: no zones around colonies. isolate code haemolysis staphylococcus aureus em151 β escherichia coli em152 α salmonella sp. em161 β pseudomonas sp. em241 β streptococcus sp. em251 β proteus mirabilis em252 α streptococcus sp. em261 γ streptococcus sp. em262 β bacillus sp. ef141 γ staphylococcus aureus ef143 β proteus vulgaris ef161 β bacillus sp. ef252 β bacillus sp. ef261 β food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 357 resistance was also recorded by 5 and 6 gram-positive bacterial strain against ampiclox and rifampicin. significant resistance to septrin and chloramphenicol were each found in 3 gram-negative bacterial strains. predominant proportions of the gram-positive and -negative strains were sensitive to ciprofloxacin, perfloxacin and gentamycin while exclusive sensitivity to amoxicillin was exhibited by gramnegative strains (4/5) (table 5). antibiogram of the bacterial strains to 10 antibiotics revealed multidrug antibiotic resistance (mar) index ranging from 0.1 to 0.5 (table 4 and 5). the highest level of mar index (0.5) was recorded for bacillus sp. ef141, having multidrug resistance pattern against amxr, apxr, cpxr, rar and strr and salmonella sp. em161 against chr, cpxr, sfxr, strr and sxtr. besides the implications of food spoilages and infections by microbial contaminants from the environment, microbial resistance to antibiotics is an important threat to human health, where treatment failures of foodborne infections are imminent. food contact surfaces are potential sources of resistant bacteria or genes, where contributory factors such as sub-optimal application of biocides and genetic exchanges within biofilm communities prevails [2]. the resistance to ampiclox, amoxicillin, chloramphenicol, rifampicin, septrin or streptomycin reported in this study was previously reported for bacterial isolates from food contact surfaces by lani et al. [14] and mohammed et al. [19]. table 4 antibiotics susceptibility and multidrug resistance profile of gram-positive bacterial isolate from meat and fish retail surfaces strain (8) antibiotics mar index pef gen apx z amx ra cpx str sxt e s. aureus em151 s s i i i i s r s i 0.1 streptococcus sp. em151 i i r i r r s r i i 0.4 streptococcus sp. em261 s s r s r i s i i i 0.2 streptococcus sp. em262 s s r r r r s i i i 0.4 bacillus sp. ef141 i i r i r r r r i s 0.5 s. aureus ef143 s s i i r r s r i s 0.3 bacillus sp. ef252 i s i i r r s r s s 0.3 bacillus sp. ef262 s s r i r r r s s s 0.4 resistant 5 1 7 6 2 5 intermediate 3 2 3 6 1 2 2 5 4 susceptible 5 6 1 6 1 3 4 key: perfloxacin (pef-5 μg), gentamycin (gen-10 μg), ampiclox (apx-30 μg), zinnacef (z-25 μg), amoxicillin (amx25 μg), rifampicin (ra-5 μg) ciprofloxacin (cpx-5 μg), streptomycin (str-10 μg), septrin (sxt-25 μg), erythromycin (e-15 μg), rresistant, iintermediate, ssusceptible, marmultiple antibiotics resistance food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 358 table 5 antibiotics susceptibility and multidrug resistance profile of gram-negative bacterial isolate from meat and fish retail surfaces strains (5) antibiotics mar index sxt ch sfx cpx amx aug gen pef ofx str e. coli em152 i i s s s r s i s s 0.1 salmonella sp. em161 r r r r i s s i i r 0.5 pseudomonas sp. em241 r r i s s i i s s r 0.3 proteus mirabilis em252 r r s s s i r s s r 0.4 proteus vulgaris ef161 i s s s s s s s s s 0 resistant 3 3 1 1 1 1 3 intermediate 2 1 1 1 2 1 2 1 susceptible 1 3 4 4 2 3 3 4 2 key: septrin (sxt-25 μg), chloramphenicol (ch-30 μg), sparfloxacin (sfx-5 μg), ciprofloxacin (cpx-5 μg), amoxicillin (amx-25 μg), augmentin (au-30 μg), gentamycin (gen-10 μg), perfloxacin (pef-5 μg), ofloxacin (ofx-5 μg), streptomycin (str-10 μg), rresistant, iintermediate, ssusceptible, marmultiple antibiotics resistance 4. conclusion this study revealed high microbial load and the presence of microbial species of public health and economic importance on the surfaces used to display and chop fish and meat in a nigerian rural market. this indicates the significance of these surfaces as sources of food contamination. therefore it is pertinent for regulatory and public health authorities to establish cleaning and sanitation guidlines for food contact surfaces in rural markets. rural food vendor and retailers should be adequately trained on the steps and importance of cleaning and sanitation. public health authorities should implement an effective monitoring programme, where routine verification for cleanliness and sanitation, including visual inspection and microbiological analysis of surface that come in contact with foods that are meant for pulic consumption are carried out. 5. references [1]. smoot, l.m. and pierson, m.d., effect of environmental stress on the ability of listeria monocytogenes scott a to attach to food contact surface, journal of food protection, 61 (10):1293-1298, (1998). [2]. barril, p.a., soto, s.a., jaureguiberry, m.v., gottardi, g., bascur, i., leotta, g.a. and oteiza, j.m., microbiological risk characterization in butcher shops from the province of neuquen, patagonia argentina, lwt food science and technology, 107: 35–40, (2019). [3]. schlegelová, j., babák, v., holasová, m., kons tantinová, l., necidová, l., šišák, f., vlková, h., roubal, p. and jaglic, z., microbial contamination after sanitation of food contact surfaces in dairy and meat processing plants, czech journal of food science, 28(5): 450–461, (2010). [4]. silva, s.n., teixeira, p., oliveira, r.r. and azeredo, j., adhesion to and viability of listeria monocytogenes on food contact surfaces, journal of food protection, 71(7):1379– 1385, (2008). [5]. world health organization, who estimates of the global burden of foodborne food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 omotade ogunremi, enobong ukpong, aima nosa, analysis and antibiogram study of microorganisms associated with fish and meat retail surfaces in ebelle, nigeria, food and environment safety, volume xx, issue 4 – 2021, pag. 352 359 359 diseases. foodborne disease burden epidemiology reference group 2007-2015, (2015). [6]. nsw food authority, environmental swabbing: a guide to method selection and consistent technique. nsw/fa/fi170/1303. www.foodauthority.nsw.gov.au. accessed august 2015. (2015). [7]. cheesebrough, m, discrete laboratory practice in tropical countries, part 1. cambridge second editions, published by press syndicate of the university of cambridge, chapter 5. 258. (2005). [8]. banwo, k., sanni, a. and tan, h., technological properties and probiotic potential of enterococcus faecium strains isolated from cow milk, journal of applied microbiology, 114: 229241, (2012). [9]. clinical and laboratory standard institute, performance standards for antimicrobial susceptibility testing. 28th ed. clsi supplement m100. wayne, pa: clinical and laboratory standards institute; 2018, (2018). [10]. cosby, c.m., costello, c.a., morris, w.c., haughton, b., devereaux, m.j., harte, f., and davidson, p.m., microbiological analysis of food contact surfaces in child care centers, applied and environmental microbiology, 74(22): 6918– 6922, (2008). [11]. herbert, m., donovan, t., manger, p., a study of the microbial contamination of working surfaces in a variety of food premises using the traditional swabbing technique and commercial contact slides. ashford, phls, (1990). [12]. food safety authority ireland (fsai), 3rd trimester national microbiological survey 2006 (06ns3): examination of the microbiological status of food preparation surfaces, final report 06ns3, (2006). [13]. gounadaki, a.s., skandamis, p.n., drosinos, e.h. and george-john e. nychas, g-j. e., microbial ecology of food contact surfaces and products of small-scale facilities producing traditional sausages, food microbiology, 25: 313–323, (2008). [14]. lani, m.n., peng, t.a., suhaili, z. and hassan, z., microbiological evaluation on raw materials and food contact surfaces of ‘keropok lekor’ premises in kuala nerus, terengganu and their prevalence of antibiotic resistant bacteria, malaysian applied biology, 46(3): 71–80, (2017). [15]. uzendu, c.j., banwo, k., ogunremi, o.r., ajao, o.s. and sanni, a.i., bacteriological quality of meat processing surfaces and antibiotic susceptibility patterns of isolates from selected abattoirs in ibadan, nigerian journal of science, 52(1): 1-7, (2018). [16]. diyoka, c.k., omba, i.k., lambo, g.n., ilunga, e.k., mposhy, p.e.m. and mjumbe, c.k., microbiological control of internal surfaces of appliances in the butcher’s cold chain in lubumbashi, open access library journal, 8: e7731, (2021). [17]. lani, m.n., azmi, m.f.m, ibrahim, r., alias, r. and hassan, z., microbiological quality of food contact surfaces at selected food premises of malaysian heritage food (‘satar’) in terengganu, malaysia, the international journal of engineering and science. 3(9): 66-70, (2014). [18]. ali, r., hayat, a., fatima, m. and muhammad noman, m., detection and enumeration of enteric bacteria associated with food handlers and surfaces of food manufacturing industry located in hub city, pakistan, world scientific news, 49(2): 192-203, (2016). [19]. mohammed, s.s.d. ayansina, a.d.v. mohammed, s.r. oyewole, o.a. and shaba, a.m., evaluation of food contact surfaces in selected restaurants of kaduna state university for the presence of escherichia coli and staphylococcus aureus, science world journal, 13(3): 45-49, (2018). [20]. pieniz, s., rodrigues, d.f., arndt, r.m., mello, j.f., rodrigues, k.l., andreazz, r., camargo, f.a.o. and brandelli, a., molecular identification and microbiological evaluation of isolates from equipments and food contact surfaces in a hospital food and nutrition unit, brazilian journal of biology, 79(2): 191-200, (2019). [21]. møretrø, t. and langsrud, s., residential bacteria on surfaces in the food industry and their implications for food safety and quality, comprehensive reviews in food science and food safety, 16: 1022-1041, (2017). http://www.foodauthority.nsw.gov.au/ 1. introduction doi: https://doi.org/10.4316/fens.2022.005 39 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 39 46 compositional energy values, macro and trace elements in the caridean species, macrobrachium macrobrachion (herklots, 1851) *rasheed olatunji moruf 1 , mosunmola florence akinwunmi 2 1department of fisheries and aquaculture, bayero university, kano, nigeria 2department of marine sciences, university of lagos, akoka, nigeria tunjimoruf@gmail.com *corresponding author received 24th february 2022, accepted 30th march 2022 abstract: edible crustaceans, such as prawns, are one of the most nutritious shellfish available to humans. meanwhile, the macro-micro nutrient content of seafood determines their nutritional value. the study assessed the energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion from the lagos lagoon in nigeria. the protein contents obtained from m. macrobrachion were 50.16±0.00 % and 56.20±0.01 % for exoskeleton and flesh samples respectively. crude fiber and total ash contents were significantly (p < 0.05) higher in exoskeleton compared to the flesh part. flesh sample had the highest proportion of energy contribution, which was from protein (pep = 63.8 %) while the least energy contribution was from fat (pef = 17.0 %) in exoskeleton sample. the value of total macro element was higher in exoskeleton sample (626.23 mg 100g-1) than in flesh sample (402.28 mg 100g-1). the highest percentage difference (% d) in mineral content between exoskeleton and flesh samples of m. macrobrachion was recorded in phosphorus with the value of 46.93 %. all the computed mineral ratios were comparably lower than the reference values. furthermore, all the minerals had calculated mineral safety index lower than the tabulated, showing positive percentage differences ranging from 74.67 to 90.61 % and 85.07 to 93.65 % for exoskeleton and flesh samples respectively. the trace metal result showed that iron, zinc and copper were the most abundant elements in exoskeleton and flesh of m. macrobrachion whereas chromium, lead and cadmium were the least abundant trace element detected. furthermore, most of the trace metal levels recorded in this study were below the maximum permitted concentrations. the nutrient composition recorded for m. macrobrachion implies that it could be a healthy diet option for health-conscious consumers. keywords: biochemical profile, trace metal, prawn, shellfish, nigeria 1. introduction prawns and shrimps are morphologically identical and closely related, belonging to the same order decapoda of the crustacea class and only differing in their gill structure [1], yet they are often used interchangeably. prawns are physically identical and closely related to shrimps, belonging to the same order decapoda of the class crustacea and only differing in their gill structure [1], though they are mostly used interchangeably. traditionally, shrimps/prawns have become one of the most highly traded seafood resources. they are recognized as an important commodity in the international fishery trade and there is evidence of a rise in the consumption of crustaceans worldwide [2-3]. marine shrimps dominate the production of typically farmed crustaceans in coastal aquaculture and are an important source of foreign exchange earnings for a number of developing countries, with catches reaching new peaks of over 336,000 tons in 2018 [4]. among caridean species, macrobrachium macrobrachion (brackish water prawn) has been identified as one of the most important economic resources in the world crustacean fishery and aquaculture farming industry. http://www.fia.usv.ro/fiajournal mailto:tunjimoruf@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 40 the prawn has an extensive distribution across the southern region of nigeria and has being described as one of the largest species in the genus macrobrachium [5]. according to akinwunmi and moruf [6], m. macrobrachion is commercially important and sustain viable artisanal fisheries in some water bodies within the region while providing direct and indirect employment. edible crustaceans such as prawn, crayfish, lobster and crab constitute one of the major sources of nutritious food for human nutrition, providing an important amount of dietary protein and lipid diet in many countries. they also contain several dietary minerals, which are essential, beneficial and play an important role in maintenance of physiological and biochemical activities in human beings [7-8]. these nutritive values of crustaceans depend upon their macromicro nutrient composition, such as carbohydrates, protein, amino acids, lipids, fatty acids, minerals and vitamins. this body composition is a good indicator of physiological condition of an organism [9]. the current wave of scientific literature which links diet with the incidence of certain diseases has brought seafood to the attention of the health-conscious public. crustaceans are known to dwell on sediment which is a sink for trace metals in aquatic ecosystems. meanwhile, crustaceans accumulate trace elements in their tissues whether or not these elements are essential to their metabolism [10-11]. hence, the need to assess the nutritive quality of prawn and associated species from the lagos lagoon in terms of its proximate composition and trace metals since these crustaceans are a major source of food and economically important to the coastal community. this study aimed to investigate the nutrient quality and energy values contributed by the nutrients in m. macrobrachion from the lagos lagoon as well as examine the presence of trace metals in the tissues of the species. this will serve to validate the current nutritional studies of west africa macrobrachium spp. in nigeria. 2. materials and methods 2.1 collection and preparation of specimens samples of m. macrobrachion were collected from fisher–folks who employed fish cages and woven cylindrical non-return valve traps for prawn capture, along makoko jetty of the lagos lagoon. the lagoon lies between latitudes 6º 26′ and 6º 39′n and longitudes 3º 29′ and 3º 50′e. lagos lagoon is a part of a continuous system of lagoons and creeks’ lying along the coast of nigeria and it is an open tidal estuary situated within the low-lying coastal zone of nigeria [12]. one hundred and sixty (160) fresh samples were collected over four months (march – june 2021). the samples were rinsed with ultrapure water to remove foreign particles and patted dry with paper towels. the samples were transported in a 10 l bucket to the laboratory and processed within 4 hours of the collection. the fresh samples were identified using identification guides [13]. routine body measurement (total prawn length) was determined using a 30 cm plastic ruler. the average length of the samples was 8.6 cm. each shrimp was separated into exoskeleton and flesh, dried under 60oc, grounded into powder and homogenized. 2.2 laboratory analysis methods of the association of official analytical chemists [14] were adopted for moisture, crude fibre, protein, fat, ash and carbohydrate analyses. by means of the already confirmed amount of proteins and fat, the energy content of the edible part of the organism was calculated. the energy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 41 values (kj 100g-1) of the specimen were estimated, multiplying the amount of proteins (%) by factor 17.16 and multiplying the amount of fat (%) by factor 38.96 and then calculating the number of the two already determined values [15]. using a flame photometer (model 405, corning, u.k), the macro minerals (sodium and potassium) were determined while the trace metals were determined using an atomic absorption spectrophotometer (perkin & elmer model 403, usa) [16]. mineral ratio and mineral safety index were calculated according to formulae described by santoso [17] and hatcock [18] respectively. 2.3 statistical analysis data were analyzed using the descriptive statistic spss (version 20). each value was a mean of six (6) replications. differences in means were tested using t-test with the significance level set at p<0.05. results were expressed as means, standard deviations (sd) and coefficient of variation per cent (cv %). 3. results 3.1 energy value contributed by nutrients in caridean species the proximate composition of exoskeleton and flesh of m. macrobrachion is shown in table 1. higher level of moisture content was noticed in the flesh than that of exoskeleton sample. the protein contents obtained from m. macrobrahion were 50.16±0.00 % and 56.20±0.01 % for exoskeleton and flesh samples respectively. fat content was higher in the flesh (9.04±0.00 %) than what was obtained in the exoskeleton (7.00±0.06 %). however, the contents of crude fiber, total ash and carbohydrate were higher in the exoskeleton sample with respective values of 2.02±0.12 %, 16.30±0.11 % and 6.37±0.05 %. significant (p < 0.05) differences were observed in crude fiber and total ash contents of exoskeleton and flesh of m. macrobrachion. the highest percentage proximate difference (%d) between exoskeleton and flesh samples of m. macrobrachion was recorded in crude fibre content with the value of 57.43 %. table 2 shows the result of the percentage energy contribution by nutrients. total metabolisable energy ranged between 1220.0 and 1381.0 kj100g-1 (289.0 – 328.0 kcal 100g-1). the highest proportion of energy contrition was from protein (pep = 63.8 %) in flesh sample, while the least energy contribution was from fat (pef = 17.0 %) in exoskeleton samples. similarly, the utilizable energy due to protein (uedp %: assuming 60% of protein energy utilization) was higher in flesh (38.3 kj/38.0 kcal) than in exoskeleton samples (33.6 kj/33.3 kcal) of m. macrobrachion. table 1 proximate composition of the caridean species, macrobrachium macrobrachion proximate (%) exoskeleton flesh p-value cv% d % d moisture 18.15±0.01 19.2±0.05 0.12 3.98 -1.05 -5.79 protein 50.16±0.00 56.2±0.01 0.15 8.03 -6.04 -12.04 crude fat 7.00±0.06 9.04±0.00 0.07 17.99 -2.04 -29.14 crude fiber 2.02±0.12 0.86±0.05 0.04* 56.96 1.16 57.43 total ash 16.30±0.11 9.34±0.12 0.03* 38.39 6.96 42.70 carbohydrate 6.37±0.05 5.36±0.01 0.09 12.18 1.01 15.86 keys: coefficient of variation per cent (cv %), difference between exoskeleton and flesh (d), percentage difference (% d). p < 0.05 with * indicate significant difference food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 42 table 2 compositional energy values in macrobrachium macrobrachion parameter unit exoskeleton flesh mean sd cv% total energy kj 100g-1 1220.0 1381.0 1300.5 113.8 8.8 kcal 100g-1 289.0 328.0 308.4 27.2 8.8 pef % (kj 100g-1) 17.0 (259) 22.3 (334) 19.7 3.8 19.2 % kcal 100g-1 17.4 (63) 22.9(81) 20.2 3.9 19.3 pec % (kj 100g-1) 7.1 (108) 6.1(91) 6.6 0.7 10.9 % kcal 100g-1 7.0 (25) 6.0(21) 6.5 0.7 10.8 pep % (kj 100g-1) 56.0 (853) 63.8 (955) 59.9 5.6 9.3 % kcal 100g-1 55.4 (201) 63.3 (225) 59.4 5.6 9.4 uedp% kj 33.6 38.3 35.9 3.3 9.3 kcal 33.3 38.0 35.6 3.4 9.4 keys: proportion of total energy due to fat (pef), proportion of total energy due to carbohydrate (pec), proportion of total energy due to protein(pep), utilization of energy value due to protein (uedp%), standard deviation (sd), coefficient of variation per cent (cv%). 3.2 macro elements in caridean species the result of the mineral content is contained in table 3. the total macro element was higher in exoskeleton sample (626.23 mg 100g-1) than in flesh sample (402.28 mg 100g-1). the highest percentage difference in mineral content between exoskeleton and flesh samples of m. macrobrachion was recorded in phosphorus with the value of 46.93 %. in exoskeleton samples, calcium (112.63±0.02 mg 100g-1) and phosphorus (303.91±0.05 mg 100g-1) were significantly higher (p<0.05) compared to that in flesh sample. table 3 macro elements in the caridean species, macrobrachium macrobrachion mg 100g-1 exoskeleton flesh p-value sd cv% % d calcium 112.63±0.02 76.18±0.05 0.03* 25.77 27.30 32.36 phosphorus 303.91±0.05 161.29±0.16 0.01* 100.85 43.36 46.93 magnesium 52.3±0.07 32.44±0.03 0.09 14.04 33.14 37.97 sodium 88.94±0.11 74.64±0.61 0.11 10.11 12.36 16.08 potassium 68.45±0.23 57.73±0.04 0.16 7.58 12.01 15.66 total 626.23 402.28 keys: standard deviation (sd), coefficient of variation per cent (cv %), percentage difference (d). p < 0.05 with * indicate significant difference all the computed mineral ratios as contained in table 4 were comparably lower than the reference values. ideally, ca/mg should be a 7.0:1 ratio of calcium relative to magnesium with a range of 3.0 to 11.0 being acceptable. the result of the study gave a ratio of 2.15:1 and 2.35:1. the milliequivalent ratio, [k/(ca+mg)] with the values of 0.68 and 0.80 for exoskeleton and flesh samples respectively, were also lower than the reference balance of 2.2. the values of the mineral safety index (msi) in the caridean species, m. macrobrachion are shown in table 5. the standard msi tabulated for the investigated minerals are ca (10), mg (15), p (10) and na (4.8). in this study, all the minerals had msi calculated values lower than the msi tabulated, thereby showing positive percentage differences, ranging from 74.6790.61 and 85.07-93.65 for exoskeleton and flesh samples respectively. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 43 table 4 elemental ratio in the caridean species, macrobrachium macrobrachion parameter reference ref. range exoskeleton flesh sd cv% ca/mg 7 3 to 11 2.15 2.35 0.14 6.12 ca/k 4.2 2.2 to 6.2 1.65 1.32 0.23 15.54 ca/p 2.6 1.5 to 3.6 0.37 0.47 0.07 17.07 na/k 2.4 1.4 to 3.4 1.3 1.29 0 0.35 na/mg 4 2 to 6 1.7 2.3 0.42 21.22 [k/(ca + mg)] 2.2 0.68 0.80 0.08 11.47 keys: standard deviation (sd), coefficient of variation per cent (cv %). source for reference value: watt [24] table 5 mineral safety index (msi) in the caridean species, macrobrachium macrobrachion mineral msitv exoskeleton flesh mean sd cv% msicv d %d msicv d %d ca 10 0.94 9.06 90.61 0.63 9.37 93.65 0.79 0.21 27.3 mg 15 1.96 13.04 86.93 1.22 13.78 91.89 1.59 0.53 33.14 p 10 2.53 7.47 74.67 1.34 8.66 86.56 1.94 0.84 43.36 na 4.8 0.85 3.95 82.21 0.72 4.08 85.07 0.79 0.1 12.36 keys: msi table value (msitv), msi calculated value (msicv), difference between msitv and msicv (d), percentage difference (% d), standard deviation (sd), coefficient of variation percent (cv%). 3.3 trace metal residues in caridean species the trace metal result showed that fe, zn and cu are the most abundant elements in exoskeleton and flesh of m. macrobrachion whereas cr, pb and cd are the least abundant trace elements detected (fig. 1). the rank profile of the trace metal levels in the exoskeleton part followed the pattern fe>zn>cu>mn> cr>pb>cd with respective mean concentration (mg 100g-1) of 11.95, 7.25, 5.58, 3.28, 1.10, 0.91 and 0.03. also, the ranking profile of trace metal concentration in flesh was fe>zn>cu>mn> pb> cr>cd with respective mean concentration (mg 100g-1) of 8.25, 6.11, 3.53, 1.53, 0.89, 0.85 and 0.02. 4. discussion the exoskeleton and flesh of m. macrobrachion had similar proximate compositions except for crude fiber and total ash, which were significantly higher in the exoskeleton. the higher carbohydrate content of exoskeleton indicates that it could be used to manage protein-energy malnutrition. a similar study on m. vollenhovenii body parts found that the exoskeleton had more crude fiber, ash, and carbohydrate content than the flesh [19]. the protein composition recorded in the present study was higher in value compared to that reported for m. vollenhovenii: 46.06 ± .38 % [20] and 41.94± 0.69 % [19], but comparable to that reported for m. macrobrachion (56.15 ± 3.82) from ovia river in edo state, nigeria [21]. the high content of protein recorded in this study indicated that m. macrobrachion is a good source of amino acids. high protein content in this species can be attributed to its omnivorous feeding habit. the total energy value contributed by nutrients was higher in the flesh part compared to the exoskeleton part of m. macrobrachion. furthermore, the highest proportion of energy contribution was from protein in flesh sample, while the least energy contribution was from fat in exoskeleton samples. the values of total metabolisable energy were higher than 457 kj 100g-1 reported for cardisoma amartum [22]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 44 fig. 1: trace element in the caridean species, macrobrachium macrobrachion all the investigated macro elements (minerals) were higher in exoskeleton than in the flesh. the reason may be due to the fact that most of these minerals such as calcium/magnesium are used to build up the shells. all the mineral elements were more positive towards the exoskeleton sample. this preponderance shows the mineral abundance in the exoskeleton part of the crustacean, similar to the report of moruf et al. [23] on the flesh and shell of callinectes amnicola from the coastal waters of lagos, nigeria. also in the present study, the result of macro elements concentration in both parts of m. macrobrachion indicates a decreasing order of calcium > magnesium > phosphorus > sodium > potassium. lawalare et al. [8] have reported a similar decreasing order of mineral content for the whole and fillet of guinean mantis shrimp, squilla aculeata calmani. mineral ratios revealed the significant balance between elements and provide details on many factors that can be represented by a disruption of their relationships [24]. in the present study, all the computed mineral ratios were comparably lower than the reference values [24]. the milliequivalent ratios [k/(ca+mg)] were also lower than the reference balance, contrary to adeyeye et al. [25], who reported a higher milliequivalent ratio of 12.8:1 to 14.7:1 on the furs of some selected domestic animals. in the present study, all the minerals had calculated mineral safety index values lower than the tabulated, indicating that the consumption of m. macrobrachion will not cause mineral overload thus, avoiding the possibility of secondary hypertension [2627]. this is in line with the report by moruf et al. [28] that ca, p and na in the royal spiny lobster (panulirus regius) would not constitute mineral overload to consumers. there are numerous factors affecting trace metal level in prawns such as parts of prawn, seasonal variation and sexes [29]. in this research, we focused on parts of prawn. the prawn exoskeleton contained the highest levels of trace metal residues is not surprising, as the exoskeleton at the head region contains hepatopancreas, an organ which is an essential metal storage site in decapod crustaceans [30]. according to silva et al. [31], accumulated cadmium is primarily concentrated in the hepatopancreas. thus, the exoskeleton of m. macrobrachion is a good indicator tissue for metal monitoring compared to the flesh part. this result compared well with those reported for decapod crustaceans from southwest nigeria [32-34]. furthermore, most of the trace metal levels recorded in this study were below the maximum permitted concentrations (mg/kg) for fe (425.5), zn (99.4), cu (73.3), mn (0.5), cr (5.0) cd (0.2) and pb (0.3) [35]. 0 2 4 6 8 10 12 14 fe zn cu mn cr cd pb c o n c e n tr a ti o n s (m g /1 0 0 g ) trace metals exoskeleton flesh food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 45 5. conclusion the study found that the concentrations of important nutrients in m. macrobrachion varied with respect to body parts. the exoskeleton had appreciable levels of macro minerals, whereas the flesh part had more compositional energy from nutrients. the presence of cd, cr, and pb in prawn tissues, on the other hand, indicates that the release of all of these trace metals into the aquatic medium as a result of anthropogenic activities must be controlled. 6. acknowledgments the author would like to thank prof. (mrs) aderonke omolara lawal-are, department of marine sciences, university of lagos, for the technical assistance provided in the study. the author would also like to acknowledge the editorial assistance rendered by hajia hawwau moruf (cln) of the department of library and information science, federal university dutsin-ma, katsina state, nigeria. 7. references [1]. ekpenyong e., williams io., osakpa u.u. variation in the proximate, energy and mineral compositions of different body parts of macrobrachium macrobranchion (prawn). journal of food research, 2(2): 150, (2013). [2]. manan h., ikhwanuddin m. triploid induction in penaeid shrimps aquaculture: a review. review in aquaculture,13: 619–631, (2020). [3]. moruf r.o., akinwunmi m.f., lawalare a.o. chemometric estimation of captured farfantepenaeus notialis, pérez-farfante 1967 (crustacea: penaeidae). acta sci pol technol aliment 20(3): 291–299, (2021). [4]. fao, the state of world fisheries and aquaculture. sustainability in action. food and agriculture organization of the united nations. rome, (2020). [5]. akinwunmi m., lawal-are a.o. occurrence, size composition and growth pattern of two caridean species from three interconnecting lagoons in south-west, nigeria. unilag journal of medicine, science and technology, 6 (1): 69-82, (2018). [6]. akinwunmi m.f., moruf r.o. aspects of the bionomics of brackish water prawn (macrobrachium macrobrachion: herklots 1851) in the interconnecting lagoons of south-western nigeria. egyptian journal of basic and applied science, 8 (1): 222–234, (2021). [7]. banu s.k., hareesh k., reddy m.s. evaluation of nutritional status of penaeid prawns through proximate composition studies. international journal of fisheries and aquatic studies, 4(1): 13-19, (2016). [8]. lawal-are a.o., moruf r.o., afolayan o.a. proximate composition, mineral profile and cholesterol level in whole and fillet of the guinean mantis shrimp, squilla aculeata calmani (holthuis, 1959) (crustacea: stomatopoda). albanian journal agricultural science, 17(3): 160165, (2018). [9]. siva-reddy k.v., babu k.r., raju m.r. proximate composition of the prawn, macrobrachium rosenbergii, from andrha pradesh coast, india. international journal current science, 8: 16-20, (2013). [10]. moruf r.o. target hazard quotient evaluation of selected trace elements in highly consumed crustacean species in lagos, nigeria. acta scienterium biological science, 43: 1-9, (2021). [11]. moruf r.o. seasonal heterogeneity and health risk assessment of metal contaminant in callinectes amnicola from epe lagoon, southwest, nigeria. journal material and environmental science, 13(1): 29-41, (2022). [12]. moruf r.o. lawal-are ao. size composition, growth pattern and condition factor of two portunid crabs, callinectes amnicola (de rochebrune) and portunus validus (herklots) off lagos coast, nigeria. nigerian journal of fisheries and aquaculture, 5(1): 15 – 21, (2017). [13]. powell c.b. fresh and brackish water shrimps of economic importance in niger delta. proceedings of 2nd annual conference of fisheries society of nigeria (fison), calabar: 254-284, (1983). [14]. aoac. official methods of analysis, 18th ed., association of official analytical chemists, washington, dc, usa, (2006). [15]. saveski a., kalevska t., stamatovska v., damjanovski d. chemical composition and energy value in the meat of the macedonian and ohrid trout. food and environ safety 16(1):40–46, (2017). [16]. gokoglu n., yerlikaya p. determination of proximate composition and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 rasheed olatunji moruf, mosunmola florence akinwunmi, compositional energy values, macro and trace elemental residues in the caridean species, macrobrachium macrobrachion (herklots, 1851), food and environment safety, volume xxi, issue 1 – 2022, pag. 39 – 46 46 mineral contents of blue crab (callinectes sapidus) and swim crab (portunus pelagicus) caught off the gulf of antalya. food chemistry, 80(4):495–498, (2003). [17]. santoso j., yoshie-stark y., suzuki t. comparative content of minerals and dietary fibers in several tropical seaweeds. bulletin of aquatic product technology, 9 (1): 1−11, (2006). [18]. hatcock j.n. quantitative evaluation of vitamin safety. pharmacy times, 104-135, (1985). [19]. ehigiator f.a.r., akise o.g. proximate composition, macro and trace mineral compositions of different body parts of macrobrachium vollenhovenii from ovia river in edo state, nigeria. tropical freshwater biology, 22: 1-11, (2013). [20]. asiru r.a., fafioye o.o. proximate composition of the giant river prawn, macrobrachium vollenhovenii from river osun, southwestern nigeria. journal of fisheries and livestock production, 6 (4): 285-288, (2018). [21]. ehigiator f.a.r., nwangwu i.m. comparative studies of the proximate composition of three body parts of two freshwater prawns’ species from ovia river, edo state, nigeria. australian journal of basic & applied science, 5(12): 2899-2903, (2011). [22]. moruf r.o., taiwo m.a., adebayo q. nutritional and functional attributes of raw and grilled crabmeat. agricultural science and technology, 13 (1): 83-90, (2021). [23]. moruf r.o., saba a.o., chukwuosazuwa j., elegbede i.o. seasonal variation in macro-micronutrient compositions of the flesh and shell of the portunid crab, callinectes amnicola (de rochebrune, 1883) from the coastal waters of southwest nigeria. agricultura, 102 (1-2): 200-209, (2019). [24]. watts d.l. htma mineral ratios. a brief discussion of their clinical importance. trace elem newsletter 21: 1-3, (2010). [25]. adeyeye e.i., adesina a.j., akinsola a.f., olagboye s.a. comparative assessment of the proximate, mineral, vitamin compositions and mineral safety index of some selected domestic animals’ furs. journal of bioinnovation, 6 (3): 462478, (2017). [26]. ademola o.a., abioye m.o.r. proximate composition, mineral content and mineral safety index of lablab purpureus seed flour. international journal of science and healthcare research, 2(4): 44-50, (2017). [27]. moruf r.o. biochemical profile, non‑enzymatic antioxidants and functional attributes of raw and thermally processed west african mud creeper, tympanotonos fuscatus var radula (linnaeus, 1758). bulletin of natural research center, 45(1): 1-8, (2021). [28]. moruf r.o., afolayan o.a., taiwo m.a., ogunbambo m.m. estimation of nutritional energy values, mineral ratio and mineral safety index in the royal spiny lobster, panulirus regius (de brito capello, 1864). croatian journal of food science and technology, 13 (1): 105-110, (2021). [29]. lee w.p., payus c., ali s.a.m., vun l.w. selected heavy metals in penaeus vannamei (white prawn) in aquaculture pond near likas lagoon, sabah, malaysia. international journal of environmental science and development, 8(7): 530533, (2017). [30]. heidarieh m., maragheh m.g., shamami m.a., behgar m., ziaei f., akbari z. evaluate of heavy metal concentration in shrimp (penaeus semisulcatus) and crab (portunus pelagicus) with inaa method. springerplus 2(1): 1-5, (2013). [31]. silva e., viana z.c.v., onofre c.r.e, korn m.g.a., santos v.l.c. distribution of trace elements in tissues of shrimp species litopenaeus vannamei (boone, 1931) from bahia, brazil. brazilian journal of biology, 76: 194-204, (2016). [32]. afolayan o.a., moruf r.o., lawalare a.o. bacterial contamination and heavy metal residues in frozen shellfish retailed within lagos metropolis, nigeria. science world journal, 15(1): 11-14, (2020). [33]. moruf r.o. metallic bioaccumulation in sesarma huzardii (decapoda: sesarmidae) from two estuarine creeks under different anthropogenic influences. polish journal of natural science, 36 (3): 271–282, (2021). [34]. moruf r.o., abubakar m.i., obiakara-amaechi a.i., sani i.m., akpan i.i. metal content and oxidative stress enzymes in aquatic crab, goniopsis cruentata (latreille, 1802) from tropical creeks adjacent western axis of the lagos lagoon. tropical journal of natural product research, 6(1):161-166, (2022). [35]. who (world health organization). report of 33rd meeting, joint fao/who joint expert committee on food additives, toxicological evaluation of certain food additives and contaminants no. 24, international programme on chemical safety, geneva (1996). doi: https://doi.org/10.4316/fens.2022.002 16 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 16 27 structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds). meiada khellou 1,2 , *zahra razkallah 3 , aziz laifa 2 , mohammed loudiki 4 , mountasser douma 4,5 and moussa houhamdi 3 1centre for scientific and technical research on arid regions; b.p.1682,07000 biskra, algeria 2 faculty of sciences, badji mokhtar university of annaba;b.p.12, 23000annaba,algeria, 3 laboratoire biologie, eau et environnement (lbee).faculté snv-stu, université 8 mai 1945 guelma,bp. 401 24000 guelma, algeria, zahra.razkallah@yahoo.fr 4 faculty of sciences semlalia, cadi ayyad university; b.p.511, 2390 marrakech, morocco 5 polydisciplinary faculty of khouribga (fpk),hassan premier university;b.p.145, 25000khouribga, morocco. *corresponding author: received 29th october 2021, accepted 25th march 2022 abstract: phytoplankton is a very important part of the ecosystem and is extremely susceptible to environmental variations. understanding and evaluating the effects of environmental changes on planktonic populations is central to freshwater ecology. in order to explore the phytoplankton diversity and abundance of two ponds in the arid region of megarine (lella fatma and zerzaim) district, oued righ, algeria, we collected phytoplanktonic samples during the period january-december 2016. the results showed that the ponds harbored a total of 58 species of phytoplankton. lella fatma had 55 species and zerzaim had 56 species. these species belonged to three phyla (bacillariophyta, euglenophyta, cyanobacteria) and 5 classes, and included 23 species of cyanophyceae and 21 species of bacillariophyceae. the latter was the most abundant class of phytoplankton community. the study reveals that the distribution equipoise in the megarine region is balanced with equity of 0.89 in lella fatma and 0.92 in zerzaim. the autumn season is the best period for phytoplankton in megarine 2016. keywords: richness, plankton, megarine, bacillariophyceae, cyanophyceae 1.introduction phytoplankton is a polyphyletic group with utmost variation in size, shape, color, type of metabolism, and life history traits [1].they represents the microscopic algal communities of water bodies and the pioneer of aquatic food chain. the productivity of an aquatic system is directly related to diversity of phytoplankton. they are source of food for zooplankton, fishes and other aquatic organisms. phytoplankton communities are sensitive to changes in their environment and therefore phytoplankton total biomass and many phytoplankton species are used as indicators of water quality [2, 3, 4]. according to harikrishnan et al., (1999) [5], the maintenance of a healthy aquatic ecosystem depends on the physicalchemical and the biological diversity of the ecosystem. physical-chemical parameters affect plankton distribution, occurrence and species diversity [6]. phytoplankton communities give more information on changes in water quality than mere nutrient concentrations or chlorophyll-a concentration. they are highly susceptible mailto:zahra.razkallah@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 17 to environmental disturbances such as changes in temperature, salinity, and turbidity [7]. here, we explore the diversity and composition of phytoplankton in an arid region where phytoplankton communities are rarely studied. thus, the current study represents a crucial baseline for future studies assessing biodiversity trends under climate change and anthropogenic stress [8, 9]. in addition, the fact that water is scarce in this arid region and used in agriculture, the local phytoplankton community needs to be documented because it is threatened by drought, agricultural pollution, and eutrophication [10, 11]. in algeria, diversity of phytoplankton in different freshwater systems along with their physico-chemical characteristics were studied by various scholars [12]. however, this study targets the phytoplankton populations of two ponds in the saharan region of algeria (lella fatma and zerzaim) of megarine, ouargla. 2.materials and methods 2.1.study area the study was carried out during 2016 in megarine region. megarine is a district in ouargla, algeria. it is one of the oasis in oued righ valley, north of the city of touggourt (fig.1). fig.1. picture of the ponds in megarine region. the region is characterized by a large area of palm plantations surrounding an oasis network that extends from sidi slimane to blidet ameur. it is limited to the north by sidi slimane, to the south by meggar and on the west by el alia. beyond the oasis is the arid and barren landscape of the sahara, featuring areas of sand dunes (ergs) and flat rocky plains (regs). the surface of megarine is about 285 km² [13]. the climatic conditions were characterized by a mean annual precipitation of 35.05 mm, a temperature of 23.3 °c, and a relative humidity of 42.8% [14]. the climate is hyper-arid with a long dry season. in megarine there are two small ponds namely lella fat ma and zerzaim. lella fatma is located at latitude 33°12′21″ north and longitude 06°05′54″ east and zerzaim lake is located at latitude 33°12′12″ north and longitude 06°05′50″ east. the two ponds are connected to each other by a natural trench but still distinct during the wet period (fig.2) (fig.3). https://en.wikipedia.org/wiki/touggourt food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 18 fig.2. geographic location of the two study ponds (2016). fig.3. geographic location of the localities where sampling was conducted in both ponds (2016). 2.2.sampling five sampling points were selected across the entire pond for an exhaustive representation of the phytoplanktonic communities (table 1),( fig.4). considering the shallow average depth of the water body, vertical variations of phytoplankton were very less likely. phytoplanktons were sampled from the surface water stratum lying between 0 and 40 cm; a suitable depth for the vital processes of phytoplankton (such as temperature, illumination, and oxygen). sampling was carried out monthly in the morning from 07:00 to 09:00 during 2016. this time of day is typically suitable for sampling because phytoplanktons are usually found on the surface [15]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 18 table 1. average morpho-dynamic characteristics of the studied ponds latitude longitude description s a m p li n g p o in ts l e ll a f a tm a p o n d f1 33°12′23,95″n 6°5′55,56″e near a run of the secondary drainage channel. presence of a few date palms. sandy soil. f2 33°12′24,20″n 6°5′53,40″e outlet point of a primary drain. the part of the soil near this point is bare and not very clayey. f3 33°12′20,31″n 6°5′52,66″e very close to a palm grove and the presence of phragmites. f4 33°12′18,4″n 6°5′55,07″e connection point with the second zerzaim pond, which is only visible during the rainy season. f5 33°12′18,64″n 6°5′56,92″e just near the point of discharge of domestic wastewater. z e r z a im p o n d z1 33°12′15,25″n 6°5′52,62″e connection point with the lella fatma pond. the connecting channel is covered with emerging plants. z2 33°12′13,44″n 6°5′51,81″e near a palm grove. z3 33°12′10,54″n 6°5′48,29″e near a palm grove. z4 33°12′11 ,58″n 6°5′47,86″e presence of aquatic plants (tamarix and phragmites). z5 33°11′13,49″n 6°5′49,78″e near a bare land with the presence of salt crusts. in order to obtain more accurate information, we repeated the collection of samples three times, both for qualitative and quantitative parameters. plankton samples were collected by filtering pond water through plankton net with 45μ mesh size. the quantitative collection of phytoplankton was carried out using a labelled dark sample 1l-bottle. the filtrate was immediately preserved in 4% formaldehyde for the identification of phytoplankton. in qualitative sampling, the contents of the collector are recovered from a dark glass sample bottle. qualitative sampling is not appropriate for accurate counts or biomass estimates [16]. diatom identification is based on microscopic examination of siliceous frustule [17]. we have made the specific identification of phytoplankton with the appropriate books and manuals. to get an overall idea of the spatio-temporal organization of the phytoplankton population in the study area we determined the parameters of mean abundance, which is the total number of cells (density). community structure analysis: three indices were used to obtain the estimate of the species diversity (h’), species richness (s), and species equitability (j). shannon and weaver (1963) [18] diversity index values were obtained using the following equation: 𝑯′ = − ∑ 𝒑𝒊 𝐥𝐨𝐠 𝟐 𝒑𝒊 𝑰=𝟏 (𝟏) where pi: = the proportion of species i relative to the total number of species in the site = richness h’= shannon and weaver diversity index species richness (d) was obtained using the equation 𝒅 = 𝑺 − 𝟏 ÷ 𝐥𝐨𝐠 𝟐 𝑵 (𝟐) where: d= margalef’s diversity index (1970)[19] s=no of species n= no of individuals. species equitability was determined by using the expression of pierlou (1966)[20]: 𝑱 = 𝑯′ 𝑰𝒏 𝑺 (𝟑) where : h’= shannon and weaver index j= equitability s= total no of species food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 20 lella fatma pond zerzaim pond f1 z1 f2 z2 f3 z3 f4 z4 f5 z5 fig.4. pictures of the sampling points in ponds (2016). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 21 3. results and discussions during the study period a total of 58 species of phytoplankton was recorded. they belong to three major phyla diatoms (bacillariophyta), cyanobacteria and euglenophyta. the phytoplankton community was dominated by diatoms. out of the total taxa identified, 34 species (58 %) were diatoms from 17 genera. the bluegreen algae included 23 species (39%) among 17 genera. phacus is the only representative genera of the euglenophyta (table 2 and 3) ( fig.5). table 2. phytoplankton class and species diversity in lella fatma and zerzaim ponds (2016). lella fatma pond zerzaim pond phylum class species diversity total number (cells/ml) percentage abundance (%) species diversity total number (cells/ml) percentage abundance (%) diatoms (bacillariophyta) mediophyceae 11 585 25.46 12 900 23.75 coscinodiscophyceae 0 0 0 1 90 0.76 bacillariophyceae 21 2720 37.43 21 4645 39.33 euglenophyta. euglenophyceae 1 15 0.20 1 45 0.38 cyanobacteria cyanophyceae 22 2680 36.88 21 4225 35.77 table 3. number and percentage of phytoplankton families (2016). megarine region lella fatma zerzaim no of spices % no of spices % no of spices % catenulaceae 3 5.17 3 5.45 3 5.36 3.57 1.79 amphipleuraceae 2 3.45 1 1.82 2 brachysiraceae 1 1.72 1 1.82 1 naviculaceae 2 3.45 2 3.64 2 3.57 pleurosigmataceae 1 1.72 1 1.82 1 1.79 cocconeidaceae 1 1.72 1 1.82 1 1.79 stephanodiscaceae 2 3.45 2 3.64 2 3.57 melosiraceae 1 1.72 0 0.00 1 1.79 cymbellaceae 1 1.72 1 1.82 1 1.79 gomphonemataceae 1 1.72 1 1.82 1 1.79 fragilariaceae 2 3.45 2 3.64 2 3.57 achnanthaceae 1 1.72 1 1.82 1 1.79 mastogloiaceae 2 3.45 2 3.64 2 3.57 bacillariaceae 11 18.97 11 20.00 11 19.64 surirellaceae 3 5.17 3 5.45 3 5.36 phacaceae 1 1.72 1 1.82 1 1.79 chroococcaceae 3 5.17 3 5.45 3 5.36 cyanobacteriaceae 1 1.72 1 1.82 0 0.00 microcystaceae 2 3.45 2 3.64 1 1.79 gomphosphaeriaceae 1 1.72 1 1.82 1 1.79 oscillatoriaceae 1 1.72 1 1.82 1 1.79 phormidiaceae 1 1.72 1 1.82 1 1.79 coelosphaeriaceae 4 6.90 3 5.45 4 7.14 merismopediaceae 1 1.72 1 1.82 1 1.79 leptolyngbyaceae 2 3.45 2 3.64 2 3.57 romeriaceae 1 1.72 1 1.82 1 1.79 pseudanabaenaceae 5 8.62 5 9.09 5 8.93 spirulinaceae 1 1.72 1 1.82 1 1.79 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 22 fig.5. phytoplankton species in megarine region (2016). species richness was variable in the study sites and between seasons (fig .6). fig.6. phytoplankton species in megarine region (2016). a : 1) amphora coffeaformis 2)amphora ovalis 3)amphora lineolata 4)amphiprora ( sp 5)frustuliarhomboides 6)brachysiraaponina 7)naviculahalophila 8)naviculamargalithie 9)pleurosigmaangulatum 10)cocconeisplacentula 11)cyclotellamenighiniana 12)cyclotellastriata 13)melosiradickiei 14)cymbellapusilla 15)gomphonemaangustatum 16)fragilariatenera 17)fragilariafasciculata 18)achnanthesminutissima 19)mastogloiabraunii20)mastogloiaelliptica 21)denticulakuetzingii 22)hantzschiaelogantula 23)nitzschiaconstricta 24)nitzschiafonticola roman 25)nitzschiavitrea 26)nitzschiageitleri 27)nitzschiatubicola 28)nitzschiapalea 29)nitzschia obtus e 30)nitzschia recta 31)nitzschiasigmoidea 32)campylodiscusclypeus 33)surirellastriatula 34)surirellaovata var. pinnata b) 1)phacusorbicularis. c: a)chroococcus minutes b)chroococcusturgidus c)chroococcuslimneticus d)cyanothece major e)gloeocapsasp f)microcystissp g)gomphosphaeriasalina h)oscillatoriachalybea i)phormidiumchalybeu m j)coelomoronpusillum k)coelosphareiumsp l)woronichiniakarelica m)woronichinianaegeliana n)merismopediawarmingiana o)leptolyngbyagranulifera p)planktolyngbyasp q)romeriasp r)jaaginemasubtilissimum s)pseudanabaena recta t)pseudanabaena galeata u)pseudanabaena mucicola v)pseudanabaena papillaterminata w)spirulina tenuior 61% 2% 37% zerzaim pond (bacillariophyta) euglenophyta. cyanobacteria 58% 2% 40% lella fatma pond (bacillariophyta) euglenophyta. cyanobacteria food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 23 fig.7. phytoplankton population in megarine region ponds’ (2016); density (a), species richness b), species diversity (c) and species equitability (d). species richness ranged between 4 and 33 in megarine ponds’. the highest richness of 33 species which represented 56% of all species were found in november at zerzaim pond, whereas the minimum species richness of four species was detected at lella fatma pond in december. the same results were found during the dry season when the species richness in zerzaim pond reached double lella fatma pond (fig.7b). according to their abundance in the pond, classes are listed as follows (table 2): bacillariophyceae were the most abundant class at the two ponds with 34 species, 39% and 37% respectively. mastogloia braunii, amphora coffeaformis, achnanthes minutissima and fragilaria tenera were the most abundant diatoms encountered during study period, whereas chroococcus minutus and chroococcus lemnticus were the highest abundant two species of bluegreen algae. although the number of euglenophyceae species was relatively low, this group’s total cell abundance represented the 0.2% and 0.38% of the total cells collected during the sampling period. they were represented by phacus orbicularis as the only representative of the class, which only appeared during the dry season. the last one was recorded as an indicator of moderately to strongly polluted water [21]. reynolds (1998) [22] suggested that the presence of euglenophytes is characteristic of eutrophic to hypereutrophic water bodies in tropical and temperate regions. the phytoplankton density (fig.7a) ranged between 65*103 and 1610*103cell/l at megarine region. the maximum density of 1610*103cell/l was observed in the november sample at zerzaim pond whereas the minimum density of 65*103 cell/l was reported in the december sample at lella fatma pond and in february sample with 380*103 cell/l at zerzaim. in general, higher phytoplankton biomass was recorded in the dry months than wet months (january and febreury). throughout the sampling period the shannon –waever (h’) diversity were higher (˃1.67) (fig.7c). the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 24 highest diversity values were recorded in october at lella fatma and in november at zerzaim (h’=4.59 and h’=4.54 respectively). the comparison of species evenness (j’) between months and sampling areas showed that the highest equitability was recorded during wet months (fig.7d). the greater values were observed in february and november at lella fatma (j’=0.93), whereas in february and september at zerzaim pond (j’=0.95). the period of high species richness corresponded with the period of lower evenness (fig .7). equitability (j) was relatively higher during all seasons (very close to 1) indicating reductions in the degree of dominance across the year. to understand the dynamics of the phytoplanktonic communities in the water of a saharan wetland’s, case of oued righ (lella fatma and zerzaim ponds), we surveyed two ponds of similar size, exposed to the the same climatic influence (temperature and rainfall) and human impact. we performed the sampling during 2016. the results of this early study showed that the phytoplankton community was dominated by diatoms in lella fatma and zerzaim ponds during all study period. the highest densities of phytoplanktons were usually found during dry period (1610*103cell/l in zerzaim). species density increased in the dry season and decreased in the rainy season, due to dilution. peak phytoplankton biomass, species richness and diversity were observed during the dry season in both ponds, which was attributed to favorable climatic and hydrologic conditions resulting from elevated temperature, solar irradiation and increased water retention time. such conditions tend to encourage algal development in lakes [23, 24]. the reasons for such elevation in biomass are more directly attributed to efficient utilization of light and nutrients [25]. qualitative and quantitative research reveals that the phytoplankton population's evolution and distribution are unpredictable and display major variations depending on the characteristics of the ecosystems in which they live. indeed the various groups of phytoplankton are sensitive to variations in ecological factors such as temperature, salinity, turbulence, and nutrients which are the precursors of the phenomena that govern the dynamics of phytoplankton groups. the environmental conditions at the origin of these variations are complex and specific to the considered geographic area, as well as to the species, responsible for these variations [26]. the temperatures also influence the entire ecosystem [27]. the dominant species was amphora coffeaformis, chroococcus lemnticus (diatoms), mastogloia braunii, chroococcus minutus(cyanophyceae); which composed on average 34% of the density in lella fatma and 27% in zerzaim pond. the genera pseudanabaena represented by four species (pseudanabaena recta; pseudanabaena papillaterminata ;pseudanabaena galeata ;pseudanabaena mucicola)recorded in the study site are considered potentially toxic [28]. the genera melosira represented by one species melosira dickiei and only in zerzaim pond during summer and autumn season. two phyla (diatoms and cyanobacteria) were detected during winter and spring seasons in two ponds (fig.8), whose diatoms represent more than 70% of species. according to patrick (1976) [29] diatom community affected by toxic pollution typically has a low diversity and low number of species, which is not our case. in summer, a third phylum was identified (euglunophycea) but with a very low frequency that does not exceed 2%. however, autumn season cyanobacteria were the most dominant with more than 56% of species. cyanobacteria tend to become dominant in turbid water because food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 25 they are superior competitors at low light intensity, and once their high biomass has created a turbid environment, other phytoplankton species compete less effectively. a previous study found high cyanobacteria density in water body due to its morphometric parameters and high nutrient availability, and its great capacity to reproduce and absorb nutrients [30, 31]. the shannon–wiener index values fluctuated between 1.67 and 4.59 in lella fatma and between 3.31 and 4.54 in zerzaim pond. a value of this index above 3 indicates clean water, whereas values lower than this would indicate pollution, noted that the latter was found to decrease with the increase in eutrophication [32, 33]. in the light of these results, we can conclude that lella fatma is more polluted than zerzaim pond. fig.8. evolution of the proportions of different taxonomic groups in lella fatma (a) , zerzaim ponds (b) (2016) 4. conclusion the ponds of megarine have interesting environmental characteristics enough to be the subject of much research. results from this study provide baseline information concerning the population density, species diversity and species richness of phytoplankton in ponds. the maximum diversity of phytoplankton observed during the season where temperature was high in the region. the phytoplankton species composition was found lower in lella fatma pond than zerzaim pond. the same for the population density was higher in zerzaim pond. these results are probably related to the location of the two ponds, of which zezaime is located in an area where palm cultivation is predominant compared to lella fatma, which makes it more vulnerable to organic pollution which generally affects the diversity of planktonic species. therefore, our results suggested that management efforts should be focused accordingly to check the deteriorating water food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 26 quality of these ponds. based on these observations, other researchers can develop concepts to monitor the water quality and biodiversity of different water bodies. 5. acknowledgements we would like to express our thanks to mr.halisyoucef, research director, at the center for scientific and technical research on arid regions (crstra, algeria) for the processing of species photo and his critical reading of the manuscript. we also tanks the mers (prfu number: d00l02un240120180001) and the dgrsdt for supporting and funding this work. 6. references [1].flynn, k. j., d. k. stoecker, a. mitra, j. a. raven, p. m. glibert, p. j. hansen, e. granéli & j. m. burkholder, misuse of the phytoplankton–zooplankton dichotomy: the need to assign organisms as mixotrophs within plankton functional types. journal of plankton research 35: 3–11, (2013). [2]. reynolds c.s., huszar v., kruk c., naselli-flores l., et melo, s., , towards a functional classification of the freshwater phytoplankton.journal of plankton research. 24 417-428,(2002). [3]. reynolds c.s., cyanobacterial waterblooms. adv. bot. res. 13, 67-143, (1987). [4].brettum p., and andersen t., the use of phytoplankton as indicators of water quality. niva-report 4818-2005norwegian institute for water research: 33pp, (2005) [5]. harikrishnan k., sabu thomas, sunil george, paul murugan. r., sathish mundayoor and das m.r.a study on the distribution and ecology of phytoplankton in the kuttanad wetland ecosystem, kerala. poll res. 18(3): 261-269,(1999). [6]. raymond, e.g. plankton and productivity in the oceans. 2nd (ed.) vol.1 pergamonpress 223-237, (1983) [7]. smayda, t. j.. patterns of variability characterizing marine phytoplankton, with examples from narragansett bay. – ices journal of marine science, 55: 562–573,(1998). [8]. khelifa r, deacon c, mahdjoub h, suhling f, simaika jp, et al. dragonfly conservation in the increasingly stressed african mediterranean-type ecosystems. frontiers in environmental science 9: 660163,(2021). [9]. khelifa r, mahdjoub h, baaloudj a, cannings ra, samways mj. effects of both climate change and human water demand on a highly threatened damselfly. scientific reports 11: 7725,(2021) [10]. khelifa r, mahdjoub h, baaloudj a, cannings ra, samways mj, remarkable population resilience in a north african endemic damselfly in the face of rapid agricultural transformation. insects 12: 353, (2021) [11]. khelifa r, mahdjoub h, samways mj .combined climatic and anthropogenic stress threaten resilience of important wetland sites in an arid region. science of the total environment 806: 150806, (2022) . [12].hamaidi ms, hamaidi f, zoubiri a, benouaklil f, dhan y. etude de la dynamique des populations phytoplanctoniques et résultats préliminaires sur les blooms toxiques à cyanobactéries dans le barrage de ghrib (ain deflaalgérie). – european journal of scientific research, 32, 369–380, (2009). [13].dubost, d. ecology, planning and agricultural development of algerian oases. – crstra, biskra, (2002). [14]. o. n. m. t. “office national of meteorology touggourt” ,climatic data of the year 2016. – onmt, touggourt , (2016). [15].ibelings, b.w., mur, l.r. and walsby, a.e., diurnal changes in buoyancy and vertical distribution in populations of microcystis in two shallow lakes. journal of plankton research 13: 419-436, (1991). [16].groga n.,.structure, functioning and dynamics of phytoplankton in lake taabo (ivory coast), thesis phd, inp toulouse, 224p, (2012). [17]. rumeau a. et coste m., introduction to the systematics of freshwater diatoms: for the practical use of a generic diatomic index, bull., fr., pechepiscic, 309: 1-69, (1988). [18]. shannon, c. e., & weaver, w. the mathematical theory of communication. urbanachampaign, il: university of illinois press, (1963). [19].margalef (r.), i n réseaux. seminario de ecologiamathematica.znv.pesq., 34 (l), 73-82, (1970). [20]. pielou, e. c.the measurement of diversity in different types of biological collections, journal of theoretical biology, elsevie. 13:131-144. 1967,(1966). [21].valadez. f, rosiles-gonzález .g & carmona .j, euglenophytes from lake chignahuapan, mexieo, cryptogamie, algologie, , 31 (3): 305-319,(2010). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 meiada khellou, zahra razkallah, aziz laifa, mohammed loudiki, mountasser douma and moussa houhamdi, structure and diversity of phytoplankton community in the water of a saharan wetland’s: case of oued righ, algeria (lella fatma and zerzaim ponds)., food and environment safety, volume xxi, issue 1 – 2022, pag. 16 – 27 27 [22].reynoldsc.s.,what factors influence the species composition of phytoplankton in lakes of different trophic status? hydrobiologia 369/370:1126,(1998). [23]. soares, m. c. s., huszar, v. & roland, f. phytoplankton dynamics in two tropical rivers with different degrees of human impact (southeast brazil). river research and applications, 23, 698-714,(2007). [24]. perbiche-neves, g., ferrareze, m. f., serafim-júnior, m., shirata, m. t., & lagos, p. e. d. influence of atypical pluviosity on phytoplankton assemblages in a stretch of a large sub-tropical river (brazil). biologia, 66, 33-41, (2011). [25].bukaveckas, p. a., macdonald, a., aufdenkampe, a., chick, j. h., havel, j. e., schultz, r., angradi t. r., bolgrien, d. w., jicha, t. m., & taylor. d. phytoplankton abundance and contributions to suspended particulate matter in the ohio, upper mississippi and missouri rivers.aquatic.sciences, 73, 419-436,(2011). [26].gailhard i. 2003.analysis of the spatiotemporal variability of coastal microalgal populations observed by the “phytoplankton and phycotoxins monitoring network”.thesis univ. mediterranean, aix-marseille ii, 293 p, (2003). [27].anderson, v. ; harrold, r. ; landblom, d. ; lardy, g. ; schatz, b. ; schroeder, j.w.,. a guide to feeding field peas to livestock: nutrient content and feeding recommendations for beef, dairy, sheep, swine and poultry. in: north dakota state university, extension service, as-1224: 11 p,(2002). [28]. zimba pv, huang is, je foley, ew linton. identification of a new-to-science cyanobacterium, toxifilum mysidocida gen. nov.& sp. nov. (cyanobacteria, cyanophyceae) ,journal of phycology ,53,188-197,(2017). [29].patrick, r.. the formation and maintenance of benthic diatom communities.proc. american philos. soc., 120(6):475-484,(1976). [30]. martins, n. r. dinâmica de algas e aspectoslimnológicosem um sistema de lagoas de estabilização de esgotossanitáriosemgoiâniagoiás..dissertação (mestrado) instituto de ciências biológicas, univerdidade federal de goiás, goiânia, (2003). [31].kajak, z. ecological characteristics of lakes in northeastern poland versus their trophic gradient. polish journal of ecology, 31, 495–530,(1983). [32]. rogozin, a. g. specific structural features of zooplankton in lakes differing in trophic status: species populations. ekologija (moscow), 6, 438– 443, (2000). [33]. paturej, e. assessment of the trophic state of the coastal lake gardno based on community structure and zooplankton-related indices. electronic journal of polish agricultural universities, biology, 9(2), 3–14,(2006) microsoft word 00 primele pagini 2_2021 stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xx, issue 2 30 june 2021 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) ispublished by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails:fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare university of suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de josuniversityof galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu ştefan cel mare universityof suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. klimentohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu ştefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of foodscience, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj-napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. kliment ohridski bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 contents: 1. evaluating an organisation’s management performance through the certification audit lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru 179 187 2. the effect of ultrasonic during nacl and kcl meat brining olga drăghici, alina toader, daniela oana andrăşescu 188 192 3. choosing fruit berry fillings for condensed canned milk with sugar natalija valeriivna ryabokon, dmitro viktorovich rindyuk 193 199 4. analysis methods of tasting as an instrument of marketing during quality forming of new grain products marina mardar 200 206 5. phy sic och emi cal m on it oring a nd per for ma n c e ev a luati on o f sbr mu ni cipal wa ste wat e r t rea t men t tsvet ko prok o po v, dash a miha ylo va, nik ola y mi halkov 207 213 6. study on the adhesion of lactobacillus plantarum strains with probiotic properties to mdck rositsa denkova, hristina strinska, zapriana denkova, georgi dobrev, daniel todorov, kirilka mladenova, stoyan shishkov 214 217 7. dete rmining a sen so ry p ro fi le o f p ast e ch e ese wi th spic es, using qua nt itati ve d es crip tiv e a naly sis micş uni ca ru su 218 223 8. influence ratio of water and oil phase of emulsions on viscosity, particle size and turbidity of products oksana lugovska, vasilij sidor 224 227 9. statistical studies on the consumer perception of fruit cristina-elena hrețcanu, alice – iuliana roșu 228 237 10. the op timu m densi ty for win t er rap e u nde r s uceav a p lat eau c on diti on s ioa n – căt ălin ene a, io a n gont a ri u 238 243 11. effect of soaking on the cooking quality and color parameters of common beans (phaseolus vulgaris l.) ana leahu, alice– iuliana rosu 244 251 12. study on roasting effect of leafy vegetables consumed in northern côte d’ivoire on nutritive and antioxidant properties patricia d. oulai, lessoy t. zoue, athanase k. otchoumou, sébastien l. niamke 252 – 262 13. virgin olive oil adulteration with other edible oils: influence of substitution on physicochemical properties mircea oroian, ana leahu 263 266 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 3 – 2014 14. fungal contamination and mycotoxins occurrence in peanut butters marketed in abidjan district (côte d’ivoire) zamblé a. boli, lessoy t. zoue, rose koffi-nevry, marina koussemon 267 275 15. microorganisms associated with traditional plantain-based food “dockounou” during spontaneous fermentation natia j. kouadio, bernadette g. goualie, honoré g. ouattara, sevérin k. a kra, sébastien l. niamke 276 – 282 16. author instructions i v 17. subscription information vi issn 1583-2295 stefan cel mare university of suceava journal foodandenvironmentsafety of the suceava university food engineering volume xxi, issue 3 30th september 2022 available online http://www.fia.usv.ro/fiajournal © publised by university publishing house of suceava aims and scope the scientific journal food and environment safetyis a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analeleuniversitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of thejournalwas made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishinghouse of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d,eng. sonia amariei(gutt),gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved http://www.fia.usv.ro/fiajournal mailto:fiajournal@fia.usv.ro http://www.usv.ro/ http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 http://www.fia.usv.ro/ mailto:gutts@fia.usv.ro mailto:fiajournal@fia.usv.ro editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare universityof suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare universityof suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania doi: https://doi.org/10.4316/fens.2022.009 82 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 82 93 bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling souad saidani 1, mohamed zairi 1, *samira meziani1, lahouaria labga1, ghania tiboura2, noreddine menadi 1, abassia demmouche1 1 faculty of natural and life sciences, djilali liabes university of sidi bel abbès, algeria. meziani_samira@yahoo.fr 2 faculty of life sciences, djilali liabes, university of sidi-bel-abbès, algeria. *corresponding author received 31th january 2022, accepted 30th march 2022 abstract: wheat bran is a by-product rich in bioactive compounds. however, little is known about the arrangement, composition and secondary metabolisms of the grain outer layers during grain development. to this end, the objective of this study is to evaluate the variations in cell wall structure and chemical composition of the outer layers during the cellularization phase, the actual filling of the grain and the maturation phase. morphological and structural aspects were evaluated by scanning electron microscopy; phytochemical study also was carried out: extracts from the peripheral layers (cp) of wheat grain (cultivar triticum aestivum hd1220) were analyzed to determine: total phenols content by folin-ciocâlteu method, flavonoids by aluminum trichloride and sodium hydroxide method; antioxidant power was evaluated by free radical scavenging (dpph) and iron reduction (frap). the results of histological analysis showed that the outer layers become thinner during grain development, the main tissues develop and differentiate synchronously; the phenolic content ranged from 47.58± 3.23 mg gae/g to 108.67± 10.7 mg gae/g wheat outer layers, the wheat outer layers contained 6.96±2.6135.297±8.68 mg eaq/g flavonoids. the contents of total phenols (tpc) and flavonoids (tfc) increased during development; the antioxidant activity, depended on the developmental phase of the peripheral layers; the antioxidant activity studied for 70% ethanolic extracts was highest in the peripheral layers of mature grains. these results underline that the peripheral layers of wheat bran derived from maternal tissues are constituted by a superposition of different tissues and are considered as an important source of bioactive compounds keywords: wheat,peripheral layers, histology, phenolic compounds, antioxidant activity. 1. introduction wheat is one of the main cereal crops second only to rice, which is consumed by nearly one third of the world's population. global wheat (triticum aestivum) production is estimated to be about 737 million tons [1, 2].it is rich in protein, minerals, vitamins and dietary fiber being considered a nutrient-rich grain [3] .caryopsis has three main parts: the bran part accounts for 13%~17%, the germ or embryo part accounts for 2%~3%, and the endosperm part accounts for 80%~85% rich in starch. the peripheral layer of the grain or wheat bran includes the pericarp, the testa, hyaline and the aleurone layer. a dense cell border aleurone layer envelops the endosperm, peripheral layer contains rich phytochemicals and minerals, including dietary fibre [4], however, wheat bran have been recognized by many researchers possess different antioxidant capacities. bioactives phytochemicals are not evenly distributed. the germ and bran parts usually contain higher concentrations of bioactive phytochemicals [5] they are becoming more and more popular around the world due to mailto:meziani_samira@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 83 their related health benefits. the bran part can act as a defense against different chronic diseases, particularly diabetes, which accounts for 3.2 million deaths worldwide every year [6]; studies have shown that adequate intake of cereals polyphenols, including phenolic acids, lignans, flavonoids and carotenoids, can prevent colon cancer, nervous system and cardiovascular diseases[1,7,8] grain filling and grain development are essential biological processes in the plant’s life cycle, eventually contributing to the final seed yield and quality in all cereal crops [9].wheat grain development is a complex process involving three successive stages: cellularization (i.e., cell division and differentiation), grain filling, and maturation/desiccation [10]. the first stage comprises cellularization and differentiation, leading to the development of both the embryo and endosperm. the grain filling stage is characterized by the onset of synthesis and accumulation of storage molecules such as starch and gluten proteins. the maturation/desiccation stage is characterized by dehydration of the grain, which gradually enters a quiescent state. the synthesis and accumulation of reserve substances occurs in the cells of the endosperm and the formation of protective structures of various types occurs in the integumentary tissues of the skin [11]. the duration of each stage varies, largely depending upon the genetic background and environmental conditions [12]. polyphenol compounds are synthesized by plants during their growth [13] mostly esterified to the arabinoxyl side groups of arabinoxylans [6] phenols are present in cereals in the free and conjugated forms [14]. most research focuses on specific developmental stages (especially grain filling), with little understanding of biochemical and molecular changes. herefore, this research aims to determine the structural aspects and concentration in bioactive compounds flavonoids, total phenolic compounds and the antioxidant activity in wheat outer layers during grain filling. 2. materials and methods developing grain stages samples of peripheral layers were fixed directly on stubs, coated with gold on the sample holder. digital images of topographical features of the samples were collected using a jsm 5800 scanning electron microscope (jeol, tokyo, japan) under the high vacuum/secondary electron imaging mode at an accelerating voltage of 10 kv and instrumental magnification 900×. plant materials for this study, peripheral layers (pl) were isolated from grains of soft wheat cv hd1220 .grains were harvested from the tassala station; seed production and demonstration farm technical institute of field crops (itgc) sidi bel abbes algeria ,at several developmental stages from cellularisation to maturation (fig 1). the endosperm and the outer layers were separated by hand and they were immediately frozen, and crushed in powder for further biochemical analyses (table1) table 1: some characteristics of the phases studied samples abbreviations harvest date characteristics 01 plp1 20.04.2020 phase 1:cellularisation phase 02 plp2 27.04.2020 phase 2:beginning of filling, milk stage 03 plp3 03.05.2020 phase 3: paste stage 04 plp4 12.05.2020 phase 4: vitreous caryopses 05 plp5 12.06.2020 phase5: maturation phase food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 84 fig 1. schematic trend of wheat grain caryopses harvested during grain filling. extraction of soluble phenolic compounds in our study, the extraction was performed by maceration in an organic solvent [15]. 5 g of sample were macerated twice in 50 ml ethanol (70%). after stirring for 24 hours to release bound and free phenolic compounds, the mixture was separated by filtration. then use a rotary evaporator to evaporate the extract to dryness at a temperature of about 45°c. weighing at this stage can calculate the yield of the extract [16]. determination of total phenolic compounds tpc after extraction, total phenolic compounds (tpc) were determined using folinciocâlteu’s phenol reagent [3]. briefly, 20 μl of extract, 1.58 ml of distilled water, and 100 μl of folin-ciocâlteu reagent were measured at 1 proportional dilution; and 300 μl 10% sodium carbonate, mixed and stored away from the light source. the absorbance was read at a wavelength of 765 nm. the calculation was based on the calibration curve using gallic acid. the concentration of phenols is expressed as μg gallic acid equivalent (gae)/mg sample. samples are analyzed in triplicate. determination of total flavonoid content (tfc) the colorimetric method was used to determine the content of flavonoids. in short, 0.5 ml of ethanol extract is diluted with 1 ml of distilled water. then, add 0.075 ml of 5% nano2 solution to the mixture. after 6 min add 0.15 ml of 10% alcl3 × 6h2o solution and let the mixture food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 85 stand for another 5 min. add half ml of 1 m naoh and distilled water to 2.5 ml. mix the solution thoroughly and immediately measure the absorbance of the blank (containing the extraction solvent instead of the sample) at 510 nm. the flavonoids are quantified using a calibration curve obtained by measuring the absorbance of a quercetin smear solution of known concentration, and the results are expressed in microgram equivalents of quercetin per mg of dry extract (mg eq/g). antioxidant activity ▪ dpph free radical scavenging activity the radical scavenging activity of the peripheral layers extracts was determined using dpph assay according to [17].a dpph radical solution with a volume of 0.025 mg ml-1 was prepared in methanol, and 1.950 μl of this solution was added to 50 μl of different concentrations of each extract. a negative control was prepared by adding 50 μl of methanol to 1,950 μl of dpph solution. after incubating for 30 min in the dark at room temperature, use a spectrophotometer to read the absorbance at 517 nm. the positive control is ascorbic acid. the lower absorbance of the reaction mixture indicates higher free radical scavenging activity. the scavenging percentage of dpph free radicals is calculated as follows: dpph (%) = (𝑨(𝒄𝒐𝒏𝒕𝒓𝒐𝒍)− 𝑨(𝒔𝒂𝒎𝒑𝒍𝒆) 𝑨𝒄𝒐𝒏𝒕𝒓𝒐𝒍∗ 𝟏𝟎𝟎 among them, dpph (%) is the reduction percentage of dpph, a control is the absorbance of the negative control, and a sample is the absorbance of the sample. the result is expressed as the average of the three measured values obtained for each sample. the percentage reduction of dpph depends on the concentration curve to graphically reveal the ic50 value that determines the concentration of 50% dpph free radicals ▪ ferricreducing antioxidant power (frap) the ability to reduce fe3+ was estimated by the method described by [18], which is based on the capacity of molecules to convert the oxidation form of iron fe3+ to its reduced state fe2+.10 μl of peripheral layers solution at varying concentrations was mixed with 40 μl of 100 mm potassium phosphate buffer (ph 6.6) and 50μl of 1% w/v potassium ferricyanide. the mixture was incubated at 50°c for 20 min, the reaction was terminated by adding 50 μl of trichloroacetic acid (10% w/v), 40 μl of water and 10 μl of 0.1% w/v fecl3 were then added. the absorbance was then measured at 700 nm. distilled water was used as negative control. the assay was done in triplicate. the intensity of the absorbance is proportional to the reducing power of the reaction mixture. the extract concentration providing 0.5 of absorbance (ec50) was calculated from the graph of absorbance at 700 nm against peripheral layers concentration. 3. results and discussion developing grain stages outer layers play key roles during grain development. images of morphological changes in the peripheral layers of soft wheat grain of cultivar hd1220 during grain filling observed under a scanning electron microscope (20 μm scale) are shown in figure 2. the substructures of peripheral layers are: the pericarp; the testa, the aleurone layer, the nucellus epidermis. the aleurone layer is the innermost layer attached to the pericarp tissue together with the hyaline layer and the testa layer [19]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 86 outer layers of developing endosperm have been studied in detail [20,21] ; fig 2. scanning electron microscopy graph of the peripheral layers and starchy endosperm of soft wheat kernel of cultivar hd1220 during grain filling (900 ×). al: aleurone layer. se: starchy endosperm. oep: outer epidermis of pericarp. mp: matrix proteins. ac: aleurone cell this study also allowed us to highlight the aggregation of starchy granules. all the main tissues (maternal and daughter) grow and differentiate synchronously (coordinately) until the kernel is fully matured (filling) [12]. observing the first phase plp1: developing grain mainly undergoes active cell division and differentiation, the cellularization stage is almost finished ,pericarp constitute most of the grain volume, aleurone cells are not yet differentiated, followed by beginning of grain filling at 17daa the milk stage’ the image shows the pericarp in continuous dark band, which wrap around the caryopses, starch granules begin to form on the inner side of the outer layers of the wheat caryopses, the maternal tissues gradually degenerate, resulting in a decrease in the thickness of the outer layers [22], the aleurone layer becomes recognizable [23].at 24 daa, corresponded to the beginning of starch accumulation, image reflect larger food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 87 polymorphic structures outer layers gradually thicken, such a modification usually happens due to the deposition of lignin [12].the aleurone cells form unicellular tissue layer surrounding the endosperm [23]. [24] reported that wheat aleurone is formed from surface endosperm cells and contains densely granular cytoplasms due to the accumulation of aleurone grains. regarding the starchy endosperm starchy granules are easily observed, regularly distributed and gradually increased in size; at 31 daa, the starchy granules showed holes in surface due to the dehydration and manipulation of the sample during the section preparation. at maturity, the peripheral layers derived from the maternal tissues are well differentiated and made up of a superposition of different tissues; from outside to the inner surface, it contains horizontal cells, tubular cells, and two continuous uniform film-like. structure corresponding respectively to the inner pericarp, the testa, and the nucellus epidermis and inner strip is the aleuron layer. results of photochemical analyzes yield extraction extraction is a very important step in the separation, identification and use of phenolic compounds. the extraction method depends on the extraction rate of phenolic compounds [25]. it allows us to calculate the yield of each water/ethanol extract. the yield determined relative to 5 g of dried and ground plant material is expressed as a percentage. the results obtained are shown in the (table 2) table 2. yield extraction, concentration of phenolic compounds in mg (eag)/g and total flavonoid compound mg eaq/g in peripheral layers during grain filling. samples yield in % tpc mg (eag)/g tfc mg eaq/g plp1 30.7 47.58± 3.23 6.96±2.61 plp2 43.91 77.86± 7.12 16.92±5.94 plp3 33.04 82.84± 2.38 24.643±1.707 plp4 33.4 88.99 ± 5.27 22.553±2.03 plp5 22.74 108.67± 10.7 35.297±8.682 tpc: total phenolic compound, tfc: total flavonoid compound, eag:acid gallic equivalent, eaq: quercetin acid equivalent. plp1: peripheral layer phase 1, plp2: peripheral layer phase 2, plp3: peripheral layer phase 3, plp4: peripheral layer phase 4, plp5: peripheral layer phase 5 results of total phenolic compounds (tpc) polyphenolic compounds are secondary metabolites synthesized by plants during its growth and are produced mostly in response to stress [14]. the total phenolics content of pl extracts was determined by folinciocâlteu assay and expressed in terms of microgram of gallic acid equivalent gae/mg [17]. using the standard curve of gallic acid (r2 = 0.997), results obtained from the extracts of the peripheral layers (pl) analyzed (fig 01) reveal that pl extracts of mature grains is the richest in phenolic compounds with 108,67± 10,7 mg (eag)/g followed by plp4 secondly with a content of 88,99 ± 5,27mg (eag)/g, plp3; plp2 extracts with a content of 82,84± 2,38 mg (eag) / g 77,86± 7,12 respectively and finally plp1 extract registers the lowest content with 47,58± 3,23 mg (eag) /g (table 2). at early developmental stages cellularisation stage polyphonols register the lowest content; outer layers constitute most of the grain volume and weight during this phase the pericarp contains chloroplasts food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 88 rich on chlorophyll indicating that polyphenols content was active in early grain filling stage. the total phenolics content of pl extracts demonstrated a close content between plp3 with 82,84± 2,38 mg(eag)/g content and plp4 of 88,99 ± 5,27 mg(eag)/g suggesting little differences in their metabolite level coincide with the accumulation of starch reserves in the endosperm. in contrast, pl extracts of mature grains register the highest content in phenolic compounds with 108, 67± 10, 7 mg (eag)/g. plp1: peripheral layer phase 1, plp2: peripheral layer phase 2, plp3: peripheral layer phase 3, plp4: peripheral layer phase 4, plp5: peripheral layer phase 5 fig 3. polyphenol and flavonoid content of wheat peripheral layers samples. previous work by [26] reported that the composition of grains is affected by the environmental and climatic conditions of crop growth, especially the temperature and water supply. the presence of the phenolics compounds in wheat bran is mainly covalently cross-linked with cell wall polymers [27]. furthermore [28] reported that the accumulation of polyphenolic compounds and the biosynthesis of flavonoids in wheat involve the expression of genes whose expression could be related to drought that in accordance with [6] who reported that the content of phenolic compounds depends on genetic components, as well as genetic interactions with the environment. the differentiation of the inner layer of the inner integument is associated with the synthesis and deposition of phenolic compounds, which gradually accumulate in the cytoplasm of these cells [12]. it is difficult to compare the results obtained in this study to those of previous studies because of the current study is the first investigation monitoring the antioxidant content and the antioxidant activity during grain development although previous work by [29] showed that the bran/germ fraction contributes 83% of the total phenolic content of the whole meal flour. actually, the total phenolic content of bran/germ fractions is 15to 18-fold higher than that of respective endosperm fractions and the 0 20 40 60 80 100 120 140 plp1 plp2 plp3 plp4 plp5 p o ly p h e n o ls m g e a g /m g f la v o n o id s m g e a q /g wheat samples polyphenols mg eag/mg flavonoids mg eaq/g food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 89 starchy endosperm contributes only 17% of the total phenolic content. results of total flavonoid content (tfc) flavonoids, a class of low-weight phenolic compounds, exert important role in plants, such as uv protection, as a defense pathogens and pests, as microbial signals, as auxins transport regulation and pigmentation [30].determined by the method used by tlili et al., 2015 flavonoid content for pl extracts was reported in mg equivalent quercetin per g of dry extract (r2 = 0.9921) .the flavonoid levels are expressed in table2. flavonoid concentration is 6,96±2,61 mg eaq/g (plp1) , 16,92±5,94 mg eaq/g ( plp2), 24,64±1,707 mg eaq/g ( plp3), 22;553±2,03 mg eaq/g (plp4), 35,297±8,682 mg eaq/g ( plp5). the results demonstrated that flavonoids concentrations ranging from 6, 96±2, 61 mg eaq/g to 35,297±8,682 mg eaq/g. it can be seen, from the results, that the lowest rate of flavonoids was found in pheripheral layers phase 1 with a rate of 6, 96±2, 61 mg eaq/g at increasing concentrations during wheat grain development. mature peripheral layer registered the highest rate oh flavonoids content with a rate of 35,297±8,682 mg eaq/g. regarding the flavonoids, the main reason for choosing this class of polyphenols is that flavonoids are the most important polyphenolic class, with more than 5,000 compounds already described (gomez 2006). flavonoids are a class of beneficial antioxidant substances of total phenolic compounds and some studies suggest that this class is more effective as antioxidants than vitamin c[31], [32] also reported that the flavonoids were detected bound to the cell wall of wheat. our results can be explained in the differences that exist in the accumulation of total flavonoid content during grain filling. in addition, [6] mentioned that the high concentration of flavonoids in wheat bran with ethanol, may be associated with flavonoid aglycone structure, which are easier to extract in ethanol,the response mechanism for flavonoid biosynthesis may be different between wheat cultivars [33]. determination of antioxidant activity dpph radicalscavenging activity the strong antioxidant activity of flavonoids and phenolic compounds and their ability to scavenge free radicals have received special attention [34]. the dpph free radical scavenging assay was widely used for evaluating the activity of natural antioxidant, because dpph radical can form stable molecules pairing with electrons or hydrogen radical. all peripheral layer extracts showed dpph scavenging activities. the ic50 (50% inhibitory concentration), also called ec50 (efficient concentration 50), is the concentration of the sample tested necessary to reduce 50% of the dpph radical. the ic50 are calculated graphically by percentages of inhibition as a function of the different concentrations of the extracts tested, a low value of the ic50 indicates a strong antioxidant activity. the ic50 values obtained are reported in (fig 4), ranging from 181,924 mg/ml to 345, 6 mg/ml with ascorbic acid used as (reference antioxidant) with ic50=35.326 mg/g. our results showed the lowest ic50 at early developmental stages with 345,6 mg/ml in plp1,than ic50:305,197 mg/ml for plp2; and the last extracts with values of ic50 224,6 mg/ml for plp3 extract and ic50: 193,83 mg/ml for plp4, respectively and finally ic50: 181,924 mg/ml for the mature pl extract. our results revels that our extracts between them have significant antioxidant potential, at increase in chelating activity during grain filling. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 90 the results of this study are in agreement with the work of luthria et al.,2015 [5] who reported that antioxidant capacity in wheat is associated to its content of polyphenolic compounds, due to the presence of phenolic acids located in the outer layers of the wheat grain .in addition [14] also reported that extracts with a higher content of phenolics possess a higher antioxidant activity. furthermore [35] reported that genotypes, extraction method, the choice of solvent used and also the dosage methods, the presence of hydroxyl groups as hydrogen donor constitute essential factors of these variations. the presence of antioxidant compounds and the level of antioxidant activity in wheat are significantly affected by the genotype, the growing area [36], and environmental conditions [37] fig. 4. histogram of the values of the inhibitory concentrations ic50 in mg / ml and iron reduction ec50 of the extracts by frap assay of the different samples plp1: peripheral layer phase 1, plp2: peripheral layer phase 2, plp3: peripheral layer phase 3, plp4: peripheral layer phase 4, plp5: peripheral layer phase 5 iron reduction: frap (ferric reducing antioxidant power) it is a measure of the ability of the substances in our extract to reduce ferric iron fe3+ to divalent iron fe2+. this is a fast, simple and reproducible technique [38]. the reducing ability of a compound can be used as an important indicator of its potential antioxidant activity [39]. previous work has shown that there is a direct relationship between antioxidant activity and the reducing ability of some plants [40]. in our work, we tested different pl extracts from hd 1220 wheat varieties through the frap method, with ascorbic acid used as (reference antioxidant) and the results obtained allowed us to draw a curve for each extract. we noticed that the iron reduction capacity is proportional to the increase in sample concentration [41, 42]. the results obtained show that the capacity of our extracts to reduce iron is lower than that of ascorbic acid for the five samples, but it is variable between the extract of the peripheral layers studied,the comparison of the antioxidant power of our extracts between them reveals that the peripheral layer of mature grains is the most active whereas the extract of plp1 sample had the lowest capacity iron reduction. if we classify our extracts according to the reduction power of iron compared to ascorbic acid, we will obtain the following order: ascorbic acid> plp5 > plp4> plp3> plp2> plp1.this indicates iron reduction capacity increased during grain food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 91 development due the fact that bran cumulate during its development phenolic compounds directly associated with antioxidant activity [43], that a high concentration in phenolic compounds of each extract increases its reducing power of iron. these results were confirmed by the dpph free radical scavenging assay; moreover [6] reported that the variation of antioxidant activity besides depending on the polarity of the phenolic compounds and flavonoids. it also depends on selectivity and action mechanism of radical used [32]. 4. conclusion the evaluation of bioactive compounds revealed the accumulation of a considerable amount of polyphenols and flavonoids, qualitatively and quantitatively during the filling of the wheat grain. it was also demonstrated that the accumulation of bioactive compounds, were affected by several factors, such as genetic components, environmental conditions and agronomic practices. it was established that antioxidant activity was found to increase throughout grain development especially in the final phase; soft wheat bran is an important source of antioxidants influenced by environmental factors characterized by grain cultivation and the varietal effect of wheat grain. 5. acknowledgments the authors acknowledge the university of sidi bel abbes, department of biology for the financial support of this study. 6. references [1]. ramadas s., kumar t.k, singleton g.p., wheat production in india: trends and prospects, in: recent advances in grain crops research, intechopen, 2019 [2]. singleton v.l., orthofer r., lamuela-raventos r.m., [14] analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent, in: methods in enzymology, academic press, 152– 178, (1999) [3]. onipe o. o., jideani a.i.o., beswa d., composition and functionality of wheat bran and its application in some cereal food products, int j food sci technol, 50: 2509–2518, (2015) [4]. deroover l., tie y., verspreet j., courtin c.m., verbeke k.., modifying wheat bran to improve its health benefits, critical reviews in food science and nutrition,60:1104–1122, (2020) [5]. luthria d.l., lu y., john k.m., bioactive phytochemicals in wheat: extraction, analysis, processing, and functional properties, journal of functional foods, 18:910–925, (2015) [6]. budhwar s., chakraorty m., sethi k., chatterjee a., antidiabetic properties of rice and wheat bran—a review, j. food biochem, 44, (2020) [7]. laddomada b., caretto s., mita g., wheat bran phenolic acids: bioavailability and stability in whole wheat-based foods, molecules, 20: 15666–15685, (2015) [8]. mateo anson n., van den berg r., havenaar r., bast a.,. haenen g. r. m. m, ferulic acid from aleurone determines the antioxidant potency of wheat grain ( triticum aestivum l.), j. agric. food chem, 56: 5589–5594 ,(2008) [9]. zhang s., ghatak a., bazargani m.m., bajaj p., varshney r.k., chaturvedi p., jiang d., weckwerth w., spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process, plant j. 107 669–687, (2021) [10]. nadaud i., girousse c., debiton c., chambon c., bouzidi m.f., martre p., branlard g., proteomic and morphological analysis of early stages of wheat grain development, proteomics,10:2901–2910,(2010) [11]. chaban i.a., gulevich a.a., smimova e.a., baranova e.n., morphological and ultrastructural features of formation of the skin of wheat (triticum aestivum l.) kernel, plants, 10: 25–38, (2021) [12]. jin x., fu z., ding d., li w., liu z., tang j., proteomic identification of genes associated with maize grain-filling rate, plos one. 8 e59353, (2013) [13]. vaher m., matso k., levandi t., helmja k., kaljurand m., phenolic compounds and the antioxidant activity of the bran, flour and whole grain of different wheat varieties, procedia chemistry, 2:76–82, (2010) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 92 [14]. kosik t., lacko-bartosova m., kobida l., free phenol content and antioxidant activity of winter wheat in sustainable farming systems, journal of microbiology, biotechnology and food sciences, 2021 :247–249, (2021) [15]. diouf p.n., stevanovic t., boutin y., the effect of extraction process on polyphenol content, triterpene composition and bioactivity of yellow birch (betula alleghaniensis britton) extracts, industrial crops and products,30: 297–303, (2009). [16]. tlili a., blondiaux e., frogneux x., cantat t., reductive functionalization of co 2 with amines: an entry to formamide, formamidine and methylamine derivatives, green chemistry,17: 157–168, (2015) [17]. brand-williams w., cuvelier m.-e., berset c., use of a free radical method to evaluate antioxidant activity, lwt-food science and technology, 28 :25–30, (1995) [18]. oyaizu m., studies on products of browning reaction antioxidative activities of products of browning reaction prepared from glucosamine, the japanese journal of nutrition and dietetics, 44 :307–315,(1986) [19]. mezia ni s., nadaud i., tasleemtahir a., nurit e., benguella r., branlard g., wheat aleurone layer: a site enriched with nutrients and bioactive molecules with potential nutritional opportunities for breeding, journal of cereal science,100: 103–225, (2021) [20]. philippe s., tranquet o., utille, l. saulnier j.-p., guillon f., investigation of ferulate deposition in endosperm cell walls of mature and developing wheat grains by using a polyclonal antibody, planta, 225: 1287–1299, (2007) [21]. palmer r., cornuault v., marcus s.e., knox j.p., shewry p.r., tosi p., comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain, planta,241 :669–685, (2015) [22]. mehdi c., virginie l., audrey g., axelle b., colette l., helene r., elisabeth j.,. fabienne g, mathilde f.a., cell wall proteome of wheat grain endosperm and outer layers at two key stages of early development, international journal of molecular sciences, 21: 239, (2020) [23]. gao x., ying r., zhao d., zhu j., variation in cell wall structure and composition of wheat grain based on geography and regulatory effect of cell wall on water mobility, functional plant biol, 47: 840, (2020). [24]. xiong f., yu x.r., zhou l., wang f., xiong a.s., structural and physiological characterization during wheat pericarp development, plant cell reports, 32:1309–1320 ,(2013) [25]. ivanišová e., ondrejovic m., šilhar s., antioxidant activity of milling fractions of selected cereals, nova biotechnologica et chimica, 11, (2012) [26]. shewry p.r., do ancient types of wheat have health benefits compared with modern bread wheat?, journal of cereal science, 79: 469–476, (2018) [27]. adom k.k., sorrells m.e., liu r.h., phytochemicals and antioxidant activity of milled fractions of different wheat varieties, j. agric. food chem, 53: 2297–2306, (2005) [28]. ma d., sun d., wang c., li y., guo t., expression of flavonoid biosynthesis genes and accumulation of flavonoid in wheat leaves in response to drought stress, plant physiology and biochemistry,80 :60–66, (2014) [29]. chateigner-boutin a.-l., lapierre c., alvarado c., yoshinagaa., barron c., bouchet b., bakan b., saulnier l., devaux m.-f, girousse c., guillon f., ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development, plant science, 276: 199–207, (2018) [30]. floegel a., kim d.-o., chung s.-j., koo s.i., o.k. chun, comparison of abts/dpph assays to measure antioxidant capacity in popular antioxidant-rich us foods, journal of food composition and analysis, 24: 1043–1048, (2011) [31]. winkel-shirley b., flavonoid biosynthesis. a colorful model for genetics, biochemistry, cell biology, and biotechnology, plant physiology, 126: 485–493, (2001) [32]. meziani s., saidani s., labga l., benguella r., bekhaled i., bioactive compounds and antioxidant potential of soft wheat and oat bran on the algerian market, the north african journal of food and nutrition research,4: 245–251, (2020) [33]. žilic s., serpen a., akillioglu g.,. jankovic m, v. gokmen, distributions of phenolic compounds, yellow pigments and oxidative enzymes in wheat grains and their relation to antioxidant capacity of bran and debranned flour, journal of cereal science, 56:652–658, (2012) [34]. fuentealba-sandoval c., pedreros a., fischer s., lopez m.d., influence of different water deficit levels during grain filling on yield and total polyphenols content in spring wheat cultivars, chil. j. agric. res, 80 :433–443,(2020) [35]. tapiero h., tew k.d., nguyen g.ba, mathe g., polyphenols: do they play a role in the prevention of human pathologies?, biomedicine & pharmacotherapy,56: 200–207, (2002) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 souad saidani, mohamed zairi, samira meziani, lahouaria labga, ghania tiboura, noreddine menadi, abassia demmouche, bioactive compounds and antioxidant activity of peripheral layers of soft wheat grown in algeria during seed filling, food and environment safety, volume xxi, issue 1 – 2022, pag. 82 93 93 [36]. martini d., taddei f., ciccoritti r., pasquini m., nicoletti i., corradini d., d’egidio m.g., variation of total antioxidant activity and of phenolic acid, total phenolics and yellow coloured pigments in durum wheat (triticum turgidum l. var. durum) as a function of genotype, crop year and growing area, journal of cereal science,65: 175–185,(2015) [37]. žilic s., hadzi-taskovicšukalovic v., dodig d., maksimovic v., kandic v., soluble free phenolic compound contents and antioxidant capacity of bread and durum wheat genotypes, genetika, 45: 87–100, (2013) [38]. karagozler a.a., erdag b., emek y.ç., uugun d.a., antioxidant activity and proline content of leaf extracts from dorystoechas hastata, food chemistry,111 :400–407 ,(2008) [39]. yang c.w., wang p., li c.y., shi d.c., wang d.l., comparison of effects of salt and alkali stresses on the growth and photosynthesis of wheat, photosynthetica,46: 107–114, (2008) [40]. yildirim a., mavi a., kara a.a., determination of antioxidant and antimicrobial activities of rumex crispus l. extracts, journal of agricultural and food chemistry, 49: 4083–4089, (2001) [41]. özturk m., aydigmus-özturk f., duru m.e., topcu g., antioxidant activity of stem and root extracts of rhubarb (rheum ribes): an edible medicinal plant, food chemistry, 103: 623– 630, (2007) [42]. su q.-s., tian y. zhang j.-g, zhang h., effects of allicin supplementation on plasma markers of exercise-induced muscle damage, il-6 and antioxidant capacity, european journal of applied physiology,103:275–283, (2008) [43]. iqbal s., bhanger m.i., anwar f., antioxidant properties and components of bran extracts from selected wheat varieties commercially available in pakistan, lwt-food science and technology, 40: 361–367, (2007) doi: https://doi.org/10.4316/fens.2022.017 185 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 185 189 influence of somatic cell count in raw milk on the quality of beaten cheese *ljubica trajkoska 1 , andrej stojkoski 1 , biljana trajkovska 1 , ljupche kochoski 1 1faculty of biotechnical scienes, university “st. kliment ohridski”, st. partizanska bb, bitola, north macedonia, info@fbn.uklo.edu.mk, ljubicaatrajkoska@gmail.com *corresponding author, received 10th february 2022, accepted 25th june 2022 abstract: the somatic cells count (scc) is an important indicator of the hygienic condition of milk. in general, any increase is due to infection of the mammary gland with mastitis, which causes a series of unwanted changes in milk composition and dairy products, respectively. the objective of our research was to determine the impact of somatic cell count on physic-chemical parameters (proteins, fats, moisture, dry matter, salt, and titration acidity), yield and sensory evaluation of beaten cheese. the research was conducted over a period of one year on milk processing capacity in the pelagonia region. a total of 24 samples of raw bulk milk were analyzed, which depending on the somatic cells count were divided into two categories (ѕсс≤400.000/ml and ѕсс≥400.001/ml). accordingly, raw bulk milks were used for production of beaten cheese. the results obtained from the conducted research indicate the fact that the changes in the somatic cells count have a significant impact on the physico-chemical parameters, yield and sensory scores on beaten cheese. high scc above 400.001/ml reduced the percentage of protein, fat, yield and the average sensory score of the beaten cheese. keywords: dairy, physic-chemical parameters, scc, yield 1. introduction beaten cheese is an autochthonous dairy product on the territory of the republic of macedonia which originates from the area of mariovo and was previously made from sheep's milk [1]. today, it is made in industrial conditions and from cow's milk [2]. in general, the composition and properties of beaten cheese are conditioned by the composition of the milk and the production process [3]. the nutritional characteristics of beaten cheese depend on the nutritional quality of the raw milk from which it is obtained [4] i.e., proteins and fats are largely transferred to the cheese. while the other parameters (percentage of salt, dry matter, moisture and titratable acidity) are individual and depend on the production process [2]. according to the [5], the milk intended for production of beaten cheese from a physico-chemical aspect should contain 12.5% dry matter, ph = 6.4-6.6 and °sh = 6.6-7.2, while in relation to hygienic condition scc≤400.000/ml and cfu≤100.000/ml. the objective of this research was to evaluate the influence of scc of raw milk in cheese yield, sensory evaluation, and physicochemical parameters. 2. materials and methods the research was conducted over a period of one year, in a dairy processing capacity in the pelagonija region. a total of 24 samples of raw bulk milks were analyzed. also, from every batch was obtained beaten cheese. mailto:info@fbn.uklo.edu.mk mailto:ljubicaatrajkoska@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 ljubica trajkoska, andrej stojkoski, biljana trajkovska, ljupche kochoski, influence of somatic cell count in raw milk on the quality of beaten cheese, food and environment safety, volume xxi, issue 2 – 2022, pag.186–189 186 milk samples were taken from bulk tank for determination of scc which was previously conserved with bronopol (broad spectrum microtabs ii preservative) as preservative in a 40 ml collecting bottle. samples were cold transported and stored under refrigeration (4 °c ± 1 °c). the analyses were performed on the same day. total scc was analyzed on the lactoscan scc apparatus (nova zagora, bulgaria). milk samples were divided into two categories according to scc, namely: i category where ѕcc≤400.000/ml and ii category where ѕcc≥400.001/ml. so, we analyzed two different groups of beaten cheese group 1 cheese obtained from raw milk with scc≤400.000/ml, and group 2 cheese obtained from raw milk with scc≥400.001/ml. the samples for analysis of beaten cheese (50g) were taken on the 18th day of production, placed in plastic cups, and transported to the laboratory where analyzes were performed. the determination of the percentage of proteins was done by the method of kjeldahl, while the percentage of fats was determined by the method of gerber. the titration acidity was determined by the method of soxhlet henkel. the percentage of nacl was determined by the mohr method. mettler toledo мј33 (greifensee, switzerland) was used to determine the percentage of moisture and dry matter. all chemical analyzes were performed according to the methods presented in [6]. the yield of the beaten cheese was calculated according to the following formula: where the result tells us how many kilograms of cheese are obtained from 100 liters milk [7]. for sensory analysis of beaten cheese the scoring method was used. panels of 30 untrained panelist from the staff and students of the faculty of biotechnical sciences who are familiar with beaten cheese were included in this evaluation. the qualitative parameters of the beaten cheese that were evaluated are color (1), taste (9), smell (5), texture (2) and appearance (3). additionally, the scores for the individual characteristics of the examined products were corrected with the “weight coefficient” (as a quantitative expression of the total product quality) [8] and their sum was expressed as a percentage "% of the maximum possible quality". when this value is divided by the sum of the weight coefficient (σ = 20), a weighted average value is obtained, i.e., a weighted grade. the obtained results are presented graphically (in the form of a spider web). мicrosoft office excel, (microsoft office corporation, usa) and spss 20 were used for statistical analysis of the data. the results were expressed in form of the mean ± sd (standard deviation). two-way anova was conducted to test if there was a significant difference between groups and p<0.05 was taken to be significant. 3. results and discussion in general, infection of the mammary gland with mastitis results in a decrease in milk synthesis and qualitative and quantitative changes in milk composition followed by changes in the ratio of proteins (decrease in casein and increase in whey protein), decrease in fats and lactose and increase in enzyme activity [9]. according to [10] when scc≤600.000/ml, the percentage of proteins is 3.27% and fats 4.21% and it continuously decreases to 3.19% and 4.11% when scc≤1.000.000/ml, i.e. 3.14% and 3.99% when scc≥1.000.000/ml. additionally, because proteins and fats are largely transferred to cheese [3] these negative changes further lead to qualitative and quantitative changes food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 ljubica trajkoska, andrej stojkoski, biljana trajkovska, ljupche kochoski, influence of somatic cell count in raw milk on the quality of beaten cheese, food and environment safety, volume xxi, issue 2 – 2022, pag.186–189 187 in the physico-chemical composition of cheese, yield and sensory scores [11;12] because of starter culture activity, coagulation time and thermostability of milk [10]. as a result of the increase of scc in ii category in raw milk there is a decrease in the percentage of proteins and fats content in cheese obtained from group 2 (table 1). when scc≤400.000/ml the percentage of proteins is 25.83%, and of fats 33.60% and it is respectively reduced to 25.10% and 33.06% when scc≥400.001/ml. similar results were presented by [4]. according to [13] when scc≤200.000/ml the percentage of proteins is 22.6%, and of fats 30%, while when scc≥600.000/ml they are reduced to 21.8% and 29%, respectively. additionally, because of the increase in the scc (scc≥400.001/ml), an increase in the percentage of moisture was observed. the percentage of moisture in group 1 is 41.79% and it increases to 42.93% in group 2, which according to [12] is a result of increasing the water holding capacity (whc) and a shorter coagulation period. table 1 physico-chemical changes in beaten cheese as a result of the increased number of scc ±sd cheese parameters proteins (%) fats (%) moisture (%) dry matter (%) salt (g) titration acidity (ºsh) group 1 (n=9) 25.83±2.46 33.60±0.17 41.79±4.49 58.21±0.78 6.33±0.55 58.83±2.07 group 2 (n=15) 25.10±3.25 33.06±1.77 42.93±1.69 57.07±0.98 6.54±0.34 58.13±3.58 according to the results obtained from this research (table 2) where beaten cheeses are produced under the same technological conditions, the yield is more favorable in group 1. namely the yield in group 1 is 9.69 kg, while in group 2 is 9.58kg. this only confirms the fact that the cheese yield is conditioned by the quality of the milk [3;9] and according to [12] it decreases from 1% to 4% depending on scc. according to [11] who performed the analysis on parmigiano reggiano cheese, when the scc ≤ 400.000/ml yield is 7.39 kg, while when scc ≥ 400.001/ml is 6.74 kg. additionally, according to the presented data of [12] when the scc increases from 240,000/ml to 640,000/ml the yield decreases by 3.26%. while, according to [9] when they produced white brine cheese from the raw milk with the scc≤600.000/ml, the yield is 17.64 kg and it continuously decreases to 15.89 kg when scc≤1.000.000/ml and 14.79 kg when scc≥1.000.000/ml. in accordance with this, which refers to different types of cheeses, it is important to note that the increase in the scc leads to a decrease in their yield. additionally, the yield of cheeses is influenced by the production process, added culture, salt and moisture loss during the ripening process [2]. table 2 changes in yield as a result of increasing total scc scc/ml average amount of daily processed milk (l) average daily production of cheese (kg) yield (kg) scc≤ 400.000/ml 1601.07 155.07 9.69 scc≥ 400.001/ml 1477.17 141.45 9.58 parallel to the increase in the scc in raw milk (scc≥400.001/ml) decrease in the average sensory scores of the analyzed properties of the beaten cheese (graph 1) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 ljubica trajkoska, andrej stojkoski, biljana trajkovska, ljupche kochoski, influence of somatic cell count in raw milk on the quality of beaten cheese, food and environment safety, volume xxi, issue 2 – 2022, pag.186–189 188 was observed. the increase in the scc (scc≥400.001/ml) has the most pronounced effect on the texture of the beaten cheese, i.e. when scc≤400.000/ml the average sensory score for this parameter is 4.73 and it is reduced when scc≥400.001/ml (4.23). similar findings were presented by [4]. the obtained results show that beaten cheese produced from milk in which scc≤400.000/ml would be better placed on the market because the appearance is an indicator of quality that the consumer uses to accept or reject the product [14]. increasing the scc (scc≥400.001/ml) leads to unwanted changes in appearance [12]. while, the color, taste and smell as sensory properties of beaten cheese, primarily depend on the evaluator [15] and on these parameters the increased number of somatic cells (scc≥400.001/ml) has no pronounced influence (graph 1). additionally, when scc≤400.000/ml the weighted average value is 4.35 while the percentage of maximum possible quality is 87.09% and 4.23 and 84.64% respectively, when scc≥400.001/ml. fig. 1. sensory analysis of the obtained beaten cheeses in general, an increase in the scc (scc≤400.000/ml) leads to unwanted changes in the physico-chemical composition, reduced yield and lower sensory scores which increase production costs and reduce the profit of milk processing capacities [13]. in addition, in our study no statistically significant differences were found between physicochemical and sensory characteristics in the groups beaten cheese obtained when scc≤400.000/ml (group 1) and when scc≥400.001/ml (group 2) which may be due to the smaller number of analyzed samples. 4. conclusion changes in the scc result with some differences between analyzed groups. on the other hand, there were no statistically significant differences in the parameters from groups 1 and 2 (p<0.05) on the physico-chemical composition of industrially obtained beaten cheese. that is, with the increase of the scc (scc≥400.001/ml) the percentage of proteins in the beaten cheese decreased from 25.83% to 25.10%, while the percentage of fats decreased from 33.60% of 33.06%. there was also an increase in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 ljubica trajkoska, andrej stojkoski, biljana trajkovska, ljupche kochoski, influence of somatic cell count in raw milk on the quality of beaten cheese, food and environment safety, volume xxi, issue 2 – 2022, pag.186–189 189 the percentage of moisture from 41.79% to 42.93%. no significant changes were observed in salt and titration acidity. as the scc increases, the yield decreases from 9.69kg to 9.58kg. additionally, as a result of the increased number of somatic cells (scc≥400.001/ml) there is a decrease in the average sensory score for texture from 4.73 to 4.23, and consequently to the percentage of maximum possible quality of 87.09% to 84.64%. no significant changes were found in the other sensory properties analyzed. 5. references [1]. goce t., traditional production of beaten cheese., third international scientific symposium „agrosym jahorina , 2012“, 10.7251/agsy1203524t, (2012) [2]. natasa d. m., zoran n., palasevski b., technology and chemical composition of beaten cheese in republic of macedonia. biotechnology in animal husbandry 24(1-2), p.139-147, (2008) [3]. mona, a. m. abd el-gawad, nawal, s. a., cheese yield as affected by some parameters review. acta sci. pol., technol. aliment. 10(2).2011,131-153 (2011) [4]. steven c. m., nicole h. m., david m. b., martin w., influence of raw milk quality on processed dairy products: how do raw milk quality test results relate to product quality and yield? journal of dairy science, 99(12), 10128– 10149, https://doi.org/10.3168/jds.2016-11172, (2016) [5]. official gazette of the republic of macedonia no. 96/2011, rulebook on requirements for raw milk quality, quality standards for consumed milk, dairy products and the use of their names, quality and activity of starter crops, silage and other specific substances and the manner of their use, the manner of additional labeling of milk and dairy products as well as the permissible deviation of the weight in relation to the declared one, (2011) [6]. biljana t., practicum in hygiene, control and supervision of milk and dairy products. university "st. kliment ohridski" bitola, faculty of biotechnical sciences bitola, bitola. isbn 978-608-4962-00-7, (2021) [7]. natalija d., uticaj kvaliteta mlijeka na randman proizvodnje travnickog sira. mljekarstvo 20 (1), (1970) [8]. jovanka v. p-r., jovanka g. l-p., "sensory properties and color measurements of dietary chocolates with different compositions during storage for up to 360 days." sensors 9, no. 3: 1996-2016 (2009) [9]. ljupche k., tatjana k., biljana t., influence of somatic cell count in raw milk on cheese production, agrar-es videkfejlesztesi szemle, 6 (1). pp. 18-23, issn 1788-5345, (2011) [10]. biljana t., vesna k-h., tatjana k., ljupche k., influence of somatic cells in raw milk on yield of dairy products., proceedings conference of agronomy students with international participation. 7, pp.142-148, issn: 1450-7323, (2011) [11]. piero f., michele f., massimo m., paolo f., andrea s., quantification of cheese yield reduction in manufacturing parmigiano reggiano from milk with non-compliant somatic cells count. foods (basel, switzerland). 9. 10.3390/foods9020212 (2020) [12]. mukta t., ahmed h., effect of somatic cell count on dairy products: a review. asian journal of medical and biological research. 1-9. 10.3329/ajmbr.v3i1.32030, (2017) [13]. mazgal g., vianna p., santos m., gigante m., (2017). effect of somatic cell count on prato cheese composition, journal of dairy science. 90. 630-6, 10.3168/jds.s00220302(07)71545-x, (2017) [14]. sonja b., almir t., senzorna analiza (interna skripta), univerziteta u sarajevo, poljoprivredno – prehrambeni fakultet, (2008) [15]. nikola t., senzorna analiza hrane, praktikum sa teorijskim osnovima, univerzitat u beagradu, poljoprivredni fakultet, (2016) https://doi.org/10.3168/jds.2016-11172 1. introduction 30 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiv, issue 12015, pag. 30 36 technological properties of sonochemically treated reconstituted milk olga krasulya 1 , *oksana kochubei-lytvynenko 2, vladymyr bogush 1, nataliya tihomirova 3 1moscow stateuniversity of technology and management named after k.g. razumovsky 31 talalihina str., moscow, russia okrasulya@mail.ru 2national university of food technologies, 68 volodymyrska str., kyiv, ukraine okolit@email.ua 3moscow state university of food production 33, talalihina str., moscow, russia techmol@inbox.ru *corresponding author e-mail: okolit@email.ua received january 22nd 2015, accepted march 31th 2015 abstract: the aim of this study was to examine the character of the sonochemical effects of treatment on technological and microbiological parameters of water and reconstituted milk.sonochemical processing performed in a reactor ultrasonic cavitation (ruc) with a piezoceramic ultrasonic transducer with power level at 45, 60, 80, 100 % of capacity (1000 w) and temperature (20 ± 2), (40 ± 2) and (50 ± 2) ° c.we reported on the effect of ultrasound for water pretreatment in the technology of reconstituted milk product. we showed that ultrasound (us) treatment of water changed its dissolving ability, due to destruction of water hydrogen-bonded network. it was experimentally confirmed that the sonochemical treatment poses an inactivating effect on the total microorganisms and fungi, and that the impact effect depends on the processing parameters power and temperature.overall, we showed that the sonochemical reactor reported here used for ultrasound water pretreatment can be successfully implemented in food industry and have positive effect on the quality of reconstituted milk as well as extended shelf life of the final product. keywords: reconstituted milk, sonochemical treatment, dissolving ability, microbiological properties. 1. introduction unconventional treatment methods enhancing the processing efficiency, shelflife, safety and functional properties of dairy products and other processed foods and food ingredients play an increasing role in modern industrial technologies. among various “emerging technologies” such as ultra-high pressure processing, pulsed electric fields, supercritical fluid extraction, microfluidization and ultraviolet light treatment, ultrasonication has been identified as a particularly promising technology for processing specific food materials, including dairy products [1]. the main idea of introduction of ultrasound technologies and sonochemical treatment in food industry lays in the fact that ultrasound treatment causes changes in functional and technological properties of liquid food systems (chemical, technological, physical, organoleptic etc.) thus contributing to a certain technological effect [2, 3]. http://www.fia.usv.ro/fiajournal mailto:okrasulya@mail.ru mailto:okolit@email.ua mailto:techmol@inbox.ru mailto:okolit@email.ua food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 31 many helpful reactions initiated by the ultrasound in food systems solutes, according to one of the leading experts in food sonochemistry m.ashokkumar (australia), are based on the cavit y treatment mechanisms during the denaturation of biopolymers in their colloidal solutes, restructuring of hydrated membranes of ions in true solutes, and even dispersion of sols phases, in other words in any process where the object of treatment is connections created by dipole-dipole and ion-dipole relations [1,4]. technology of the sonochemical water treatment can be implemented in the technology of dairy products from reconstituted and recombined dairy material, as well as in production of dairy, combined and milk constituting products [5-7]. majority of phenomena, caused by ultrasound treatment were studied in batch mode, on small amounts of model systems with the use of laboratory installations and modules. nonetheless, during the last decade continuous flow ultrasound systems, able to accomplish industry sized volumes if treatment, become more and more available.so, as an acoustic source of elastic fluctuations can be used germanmade hielscher systems gmbh ultrasound industrial processors. six such processors (500 w each) assembled together in one block can provide the needed water treatment for the material at room temperature for up to 2.5 m3/hour. russian scientists have suggested to use an industrial reactor ultrasonic cavitation of rcu type with a piezoceramic ultrasonic transducer (certificate c–ru– tm05.в.00020 тр 1002178, russia) as an alternative to mentioned ultrasound systems. productivity of the system is 0.36 m3/hour, which is able to satisfy industrial needs of enterprises with small amounts of produced goods. this work presents technological and microbiological indexes of water and dairy materials processed with the ruc. 2. matherials and methods the sonochemical reactor for water pretreatment is shown in figure 1. it’s technical characteristics – table 1. fig. 1.the reactor ultrasonic cavitation with a piezoceramic ultrasonic transducer table 1 technical specifications of the ruc with a crystal pick-up title characteristics measurement unit index ac power supply v 220 ± 10 frequency khz 20 ± 1.5 consumed power (max) w 1500 power control band % 30…100 running time (max) hour 8 following break time (min) m 10 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 32 ruc unit structurally consists of an electronic unit and the process with a fixed volume of the ultrasonic vibrating system, a centrifugal pump, assembled in a protective stainless steel casing. the electronic unit is an electronic generator a source of electrical oscillations with frequency 20 khz for excitation of mechanical vibrations of a piezoelectric transducer located in the ultrasonic vibrating system. it is important to note that reactor underwent “federal service for supervision of consumer rights and human welfare” and “state standards of russia” inspections and were permitted for food processing use. examinations of water after the sonochemical treatment was performed, as well as examinations of milk and milk formulas reconstituted on the treated water. only drinking (bottled) water was used for the experiments. ultrasound treatment of water was carried at operating frequency of 20 khz. sonochemical treatment was performed at capacities of 45, 60, 80 % from the ruc capacity (1000 w) and with water temperatures of (20 ±2), (40 ±2) и (50 ±2) °с. solubility index powder milk was determined according to iso 8156:2005(en). drinking and sonochemical reactor treated water was used to dissolve the dry milk products. this research presents average results of solvability index assessment (samples deviations ±0.03 сm3). the comparative dissolution speed is expressed as a percentage and appears to be a ratio of a dry product materials caught in a solution to a product sample weight. the concurrent specifications within-run precision was ± 1.5 %. microbial growth in water samples was studied plate-count methods. the storability forecast of drinking milk samples generated from the dry milk using sonochemical water treatment, has been conducted according to the specific conductivity (sc) index and determined correspondence connecting sc trend and microorganisms quantity changing rate. the аnion-7051 conductometer was programmed to measure sc (σj) and upload the results j = 1, 2, …100 of these measurements into computer memory every 12 minutes. the vectors σ1, σ2, σ3, σ4, presenting amounts of sc time trends (mсm/сm) point sets became available for the reconstructed milk samples, being processed using the traditional technologies (control) and sonochemical water treatment. the total microbial flora growth rates using every hour inoculation within 16hours period has been examined for the reconstructed milk test samples, stored at +8 and +22°с temperature. first the logarithms of the correspondences being obtained from the point sets have been taken and then they’ve been brought into proximity with the well-known least squares method. the approximation function n(t) of total microbial flora unit/сm3 containment is as follows: tt 203.0101807)n(  , (1) after functions form determination nn(t) (formula 1) out of formula:    80 0 2 005.0 )](nlg)(n[lg81 1 10 k knkt n   (2) where: a0.005 – student’s reciprocal distribution, the deviance confidence ranges have been calculated with the significance level at 99.5%. the table 2 presents their rates. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 33 table 2 the deviance confidence ranges deviance ε1 ε2 ε3 ε4 rate, unit/см3 10±0.187 10±0.192 10±0.115 10±0.141 the functions confidence intervals )(n 0.995 tn  of the microorganism’s growth within the tested reconstructed milk samples, prepared at different level of sonochemical water treatment have been determined according to the following formula: nnn tt   )(n)(n 0.995 (3) the water biological testing has been assessed according to the water toxicity express-determination method using “ecolum” luminescent bacterial test based upon the changes of the microorganisms luminous intensity, emerged during their lifetime. the luminous intensity has been registered by the “biotox-7” device. the microorganisms vitality index has been assessed in accordance to the luminous intensity during the fixed-time exposition of the tested solution with the test-object and control (distilled water) [8]. 3. results and discussion we suggest that pulses of pressure occurring due to oscillating cavitation bubble growth shift water self-organization equilibrium (h2o)n = n(h2o) to the right, thus, destroying three-dimensional hydrogen-bonded network (figure 2a). released in such a way so-called “free molecules” of water have higher possibility example, in a powdered milk (figure 2b). fig. 2. schematic representation of hydrogen-bonded network destruction due to cavitation effect of ultrasound (a) and released free water molecules incorporation into proteins (b). hydrogen-bonded network surface of the expanding cavitation bubble free water molecules a b binding water molecules amino acid parts of proteinsamino acid parts of proteins food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 34 increasing dissolving ability of water by the action of cavitation can effectively compensate natural water which was artificially removed for the purpose of prolonged storage of milk in the dried form. figure 3 shows how us treatment can improve dissolving ability of water. equal amounts of dry milk whey were added to regular water (figure 3a) and to the water which was preliminary sonicated (figure 3b). it can be clearly seen that us treatment of water significantly enhances whey dissolving. fig. 3. solubility of powdered whey in regular (a) and sonochemical treated water (b) during the approbation of ruc it was found that solubility index of dry milk and solutes, reconstituted on the treated water was lower by 0.1…0.2 cm3 than control, which signifies higher level of solubility of dry substances. intensification of the renewal processes with a sonochemical water treatment is also proved by the results of the relative dissolution speed determination. it’s increase is 18.0-45.0 in accordance with the processing power and dry milk products type. in the samples of reconstituted dairy material with the use of sonochemical water treatment a low level increase in mass portion of dry elements was found, including proteins, as well as increases in density and dynamic viscosity indexes. the study of structural-mechanical features showed positive changes of dynamic viscosity humectants ability of the milk clot in the samples created with the use of acoustic cavitation method, which can be considered an indirect sign of increase of the hydration level of the protein and its amount. it was experimentally proved that sonochemical treatment has a certain inactivation influence upon microorganisms. in which connection decrease in the overall population of microorganisms and moldy fungi was influenced by the intensity of the treatment and temperature of the treated water (figure 4 а,b). it was established that practically complete inactivation of microorganisms was observed in treatment when the capacity of the installation was 80 % from the ruc capacity and temperature of (50 ±2) °с. similar results were obtained in sonochemical treatment of the whole cow milk, maximal bacteriostatic effect was observed at 100 and 80% capacity levels. it should be mentioned that e. coli bacteria was absent in all samples of water treated in the reactor. we revealed that cavitation effects on water caused by acoustic wave could also influence the microbial growth. figure 5 shows the total microbial content of the milk samples which were prepared by dissolving of powdered milk in regular and sonicated in different regimes (variable acoustic energy) water. it shows that sonochemical water treatment increases products’ storing term compared to the traditional technology. in which connection, it was established that the speed of growing number of microorganisms was dependent on the durability of the sonochemical water treatment in other words on the number of acoustic energy received by the water. a b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 35 a b fig. 4 dependence of total microbial flora (a) and the colony count of fungi (b) of processing parameters sonochemical fig. 5. development of microflora in milk drinks obtained by sonochemical processing with an amplitude of sound pressure in the reactor of 2.5 bar and acoustic energy: 1 0 kj, 2 30 kj, 3 60 kj, 4 90 kj. we can assume that the sonochemical processing water causes damage to the molecular structure of cells and cell membranes, which in turn causes the morphological changes and destruction of cells and their partial or complete loss of viability. clearly, us treatment of water used for milk drink preparation significantly slows the bacterial growth, which gives the possibility to store the product for a longer time without reducing its quality. by means of mathematic processing of the received data was developed method of total microbial flora containment control in milk drinks by the index of specific electrical conductivity, which allows predicting shelf-life of these products depending on the level of use of sonochemical treatments. besides, according to the the ramc a.n.sysin human ecology and environment hygiene institute researches of cavitating water treatment on its state th e to ta l m ic ro bi al fl or a co nt en t, n , u ni ts /c m 3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiv, issue 1 – 2015 olga krasulya, oksana kochubei-lytvynenko, vladymyr bogush, nataliya tihomirova, technological properties of sonochemical treated reconstituted milk., food and environment safety, volume xiv, issue 1 – 2015, pag. 30 – 36 36 the sonochemical reactor treated water bacteriostatic specifications have been officially registered (table 3). table 3 in-water microorganisms lifetime range before and after sonochemical treatment besides 5 min sonochemical treatment “ecolum” microorganisms test lifetime rate untreated water (control) 1.760 2 hour 0.630 24 hours 0.849 therefore, optimization of the duration of water us treatment makes it possible to control the quality and the shelf life of the final product, as well as opens the opportunity to use sonochemistry on industrial level. 4. conclusions we reported on the effect of ultrasound for water pretreatment. we showed that us treatment of water is changing its dissolving ability, due to destruction of water hydrogen-bonded network. importantly, bacteriological properties of sonicated water has significantly improved, leading to much slower bacterial growth, which as it was found can be controlled by duration of us water treatment at fixed intensity. the obtained results allow us to recommend sonochemical treatment at a temperature of (50 ± 2) ° c and 80 % power as an alternative for the raw milk and dairy products thermization in order to inhibit microbial processes, improve the quality and extend the shelf life of both the raw materials and the end-product. overall, we showed that the sonochemical reactor reported here used for ultrasound water pretreatment can be successfully implemented in food industry and have positive effect on quality powdered milk as well as extend shelf life of the final product. 5. references [1] ashokkumar m., sunartio d., kentish s.e., mawson r., simons l., vilkhu k., versteeg c., modification of food ingredients by ultrasound to improve functionality, innovation food science emerging technolgies, 9: 155–160, (2008) [2] ashokkumar m., bhaskaracharya r., kentish s., lee j., palmer m., zisu b., the ultrasonic processing of dairy products, dairy science and technology, 90: 147-168, (2010) [3] mason t.j., paniwnyk l., lorimar j.p. the uses of ultrasound in food technology, ultrasonics sonochemistry, 3: 253–260, (1996) [4] wan j., mawson r., ashokkumar m., ronacher k., coventry m.j., roginski h., versteeg k. emerging processing technologies for functional foods, australian journal of dairy technology, 60: 167–169, (2005) [5] ashokkumar m, lee j, zisu b, bhaskarcharya r, palmer m, kenrish s., hot topic: sonication increases the heat stability of whey proteins, journal dairy science, 92:5353– 5356, (2009) [6] ashokkumar m., krasulya o., shestakov s., rink r., a new look at cavitation and the applications of its liquid-phase effects in the processing of food and fuel, applied physics research, volume 4, no. 1: 19-29, (2012) [7] shestakov s., krasulya o., artemova j., tikhomirova n. ultrasonic water treatment, dairy industry, 5: 39-42, (2011) [8] shestakov s., krasulya o. research and experience of sonochemical technology in the food industry, electronic journal “technical acoustics”, http://www.ejta.org (2011) [9] stekhin a., yakovleva g., structured water. nonlinear effects: 129-266, (2008) http://www.ejta.org 122 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 122 126 biogenic amine amount in ground pork and beef meat octavian baston1, *octavian barna1 1dunarea de jos university of galati, 111, domneasca street, 800201, galati, romania, octavian.baston@ ugal.ro , *octavian.barna@ yahoo.com * corresponding author received april 26th 2014, accepted may 14th 2014 abstract: the biogenic amine content in ground meat is always an actual issue to be studied. our objectives were to determine the initial amount of four biogenic amines (histamine, tyramine, serotonin and phenylethylamine) in raw ground pork and beef meat, and to determine the variation of biogenic amines along the aerobic refrigerated storage of ground meats. with the exception of serotonin, all the biogenic amines studied were under the detection limit at the first determination (day 0). all the biogenic amines accumulated in meats in time while refrigeration storage. the highest amount of biogenic amines was registered in ground beef on the last day of refrigerated storage (day 10). histamine did not exceed 12 mg/kg in ground beef meat and 10 mg/kg in ground pork meat on the last day of storage. tyramine was detected in both meats in lower amounts than histamine on the last day of refrigerated storage, whereas serotonin and phenylethylamine were found under 3.5 mg/kg and 2.5 mg/kg respectively. key words: amino acid decarboxilation, shelf life, consumer health, refrigerated storage. 1. introduction biogenic amines are called biologically active amines or bioactive amines because they have bio-activity in the human body. the biogenic amines occur naturally in organisms or they can be produced in fresh or processed foods by amino acids decarboxilation, aldehydes and ketones transamination or nitrogen compounds hydrolysis [1]. when fresh meat is spoiled by microorganisms, amino acid decarboxilation is the most important biogenic amines production pathway. the two most important factors for biogenic amines production in fresh foods are the food and the microorganism type. for the accumulation of biogenic amines in food, the most important factors are the existence of free amino acids and the presence of microorganisms with decarboxylasing activity. in the scientific literature bioactive amines are considered as biogenic (histamine, serotonin, tyramine, phenylethylamine, tryptamine, putrescine, cadaverine, agmatine) and naturally occurring amines (spermine and spermidine). histamine is an amine that is produced in food by decarboxilation of free histidine. it is an important amine to be studied because it has toxicological effects on humans such as headaches, sweating, burning nasal secretion, facial flushing, rashes, dizziness, oedema, urticaria, difficulty in swallowing, diarrhoea, respiratory distress, bronchospasm, increased cardiac output, tachycardia, extrasystoles [2]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 o c t a vi a n b as t on , o ct avia n b arn a, b i og e n i c am i ne am o u nt i n gr o u nd p o r k an d be e f m ea t , issue 2 2014, pag. 122 – 126 123 in fresh foods the amount of histamine should be low, under 5 mg/kg. histamine alone at low levels does not cause toxicological effects on humans, but the presence of putrescine and cadaverine can increase the poisoning effect [3]. tyramine is produced in food by decarboxilation of free tyrosine. it has a negative influence on human body, producing headaches, migraine, neurological disorders, nausea, vomiting, respiratory disorders, hypertension extrasystoles [2]. the toxic dose of tyramine is 100 mg/kg in normal individuals and 60 mg/kg in those under treatment with monoamine oxidase inhibitors [1]. serotonin is produced from tryptophan which is an amino acid that is transformed by tryptophan hydroxylase into 5hydroxytryptophan, which is enzymatically decarboxylated into 5hydroxytryptamine or serotonin. the serotonin syndrome in humans refers to tachycardia, hypertension, and hyperthermia [4]. the free amino acid phenylalanine is decarboxilated to phenylethylamine. the biogenic amine can produce headache, migraine, increased blood pressure, being toxic to humans at 30 mg/kg. [1]. ground meat is an important food, being used in romanian dishes in combination with vegetables, pasta, cheese, eggs, or as meatballs. therefore, it is used in traditional dishes such as „mici”, „sarmale”, „chiftele”, „perisoare”, or sausages, wherein the biogenic amines content of the main ingredient (ground meat) is very important for human health. the primary goal of this study is to determine the initial amount of four biogenic amines (histamine, tyramine, serotonin and phenylethylamine) in raw pork and beef ground meat and then to determine the variation of biogenic amines during the refrigerated storage of ground meats. 2. materials and methods sampling and refrigeration raw ground beef and pork meat were purchased directly from butcheries. all samples were put into an ice box for transportation to the laboratory. the samples were purchased from the same traders as ready to use ground fresh meat, on the production day. they were refrigerated at 4 ± 2 °c for ten days in dbk386 wd (beko, turkey) type refrigerator. they were analyzed every two days. the samples were stored aerobically, in plastic bags, packed after they had been purchased from butcheries. samples for analyses were taken aseptically from the original package. chemical analyses the chemical determinations were made on the same day of purchase. in total we analyzed 21 samples of ground pork meat and 19 samples of ground beef meat. the biogenic amine determinations were made in triplicates. chemical analyses of ground meats were made from 12 samples, in triplicates. moisture levels were determined by air drying in the oven at 100 °c according to aoac 950.46 method [5] using ule 400 oven (memmert, germany). the determination of biogenic amine amounts using high performance liquid chromatography was performed according to the method described in baston [6]. the calibration curves of the biogenic amines are linear, having r2 as follows: histamine r2 = 0.9981; tyramine r2 = 0.9986; serotonin r2 = 0.9977; phenylethylamine r2 = 0.9984.the concentration of each food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 o c t a vi a n b as t on , o ct avia n b arn a, b i og e n i c am i ne am o u nt i n gr o u nd p o r k an d be e f m ea t , issue 2 2014, pag. 122 – 126 124 biogenic amine was expressed in mg/kg dry weight. all the reagents were chromatographically grades. statistical data treatment the statistical analysis was made using microsoft office excel to determine the mean and standard deviations. 3. results and discussion four biogenic amines were determined: histamine, tyramine, serotonin and phenylethylamine. figure 1 shows the histamine variation for the meats studied. fig. 1 histamine variation in ground meats the initial amount of histamine was below the detection limit of the equipment in both studied meats. on the second day of refrigerated storage the average histamine amount is smaller in ground pork meat compared with ground beef meat which is two times higher. the amount of histamine has increased for the remaining storage days, on the tenth day the average amount of ground pork meat is 12.07 mg/kg d.w., and in ground beef meat is 10.02 mg/kg d.w. beginning with the sixth day of refrigeration, the histamine amount in ground pork meat was higher than the histamine amount found in ground beef meat. this was due to microbial transformation of the free histidine to histamine, being higher in ground pork meat. the initial low values of histamine in ground pork and beef meat are related to the values presented by min [7] and vidalcarou [8]. tyramine is a biogenic amine also produced by microbiota of the studied meats. the variation of tyramine is presented in figure 2. fig. 2 tyramine variation in ground meats the initial amount of tyramine in both studied meats was below the detection limit of the equipment. beginning with the second day of storage, tyramine was found only in ground pork meat, its presence being due to the microbiota that decarboxylated the corresponding amino acid. since the fourth day of refrigeration, 1.57± 0.37 mg/kg d.w. tyramine was found in ground beef meat. in both ground meats the amount of tyramine increased, but the maximum amount did not exceed 8 mg/kg on the tenth day of storage. our initial food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 o c t a vi a n b as t on , o ct avia n b arn a, b i og e n i c am i ne am o u nt i n gr o u nd p o r k an d be e f m ea t , issue 2 2014, pag. 122 – 126 125 findings comply with the values presented by vidal-carou [8]. the amount of serotonin in both types of ground meat is shown in figure 3. initially the amount of serotonin in ground pork meat was double than that found in beef. our findings came into contradiction with the ones found by min [9], because serotonin in ground beef was 1.6 mg/kg and in ground pork was 0 mg/kg. this difference is probably due to the meats microbiota and the slaughtering method of animals since serotonin was found in the body, where it is produced. serotonin is a biogenic amine that is accumulated during refrigerated storage of meats. fig. 3 serotonin variation in ground meats it was found in small amounts in meat because on the tenth day of storage the mean amount in ground pork meat was 3.33 mg/kg d.w., and in ground beef meat was 2.87 mg/kg d.w. according to hernández-jover [3], the amount of serotonin in meat and meat products in not high, being under 2 mg/kg. on the first four days of storage, respecting the validity term, the obtained results have not exceeded the limit. the last biogenic amine studied is phenylethylamine and its variation is presented in figure 4. fig. 4 phenylethylamine variation in ground meats being a biogenic amine that is produced by decarboxilation from the precursor amino acid phenylalanine, initially, phenylethylamine was not found in the ground meats. this is due to the fact that the meats had a small microbial load at the beginning and low amount of free precursor amino acid. our findings comply with those stated by min [9], because phenylethylamine was not found in ground beef and pork meat control samples. beginning with the second day of refrigerated storage, phenylethylamine, in a small quantity, was found only in ground pork meat. and it was found in ground beef meat beginning with the fourth day of storage, this is due to the low presence of free amino acid phenylalanine and low spoilage microbiota. on the last day of storage, the highest amount of phenylethylamine was found in ground pork meat, but it did not exceed 2.5 mg/kg d.w. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 o c t a vi a n b as t on , o ct avia n b arn a, b i og e n i c am i ne am o u nt i n gr o u nd p o r k an d be e f m ea t , issue 2 2014, pag. 122 – 126 126 4. conclusion the initial quantities of biogenic amines were low or under the detection limit. only the serotonin content in ground beef meat was identified for the initial determination. all the studied biogenic amine amounts have increased during the refrigerated storage. the highest amount of biogenic amines studied on the last day of storage was registered by histamine found in ground beef meat. after drawing a comparison between ground pork and beef meats, the highest amounts of biogenic amines was registered in beef meat on the last day of storage. this may be explained by the hygienic conditions of beef slaughter and meat storage, since the amount of biogenic amines is influenced by microbial contaminations and especially by bacterial types. as regards shelf life, the studied biogenic amines cannot be used individually as an indicator for the biogenic amine index. finally, the consumers’ health is not jeopardized by very small amounts of biogenic amines. 5. references [1] halász a., baráth á., simonsarkadi l., holzapfel w., biogenic amines and their production by microorganisms in food, trends in food science and technology, 5(2), 4249, (1994). [2] ladero v., calles-enriquez m., fernández m., alvarez m., toxicological effects of dietary biogenic amines, current nutrition and food science, 6(2), 145-156, (2010).[3] hernández-jover t., izquierdo-pulido m., veciana-nogués m.t., mariné-font a., vidal-carou m.c., biogenic amine and polyamine contents in meat and meat products, journal of agricultural and food chemistry, 45(6), 2098-2102, (1997). [4] boyer e.w., shannon m., the serotonin syndrome, new england journal of medicine, 352(11), 1112-1120, (2005). [5] association of official analytical chemists (aoac), official methods of analysis (18th ed.), washington dc, (2005). [6] baston o., stroia a.l., moise d., barna o., validation study of a hplc method able to measure biogenic amines in chicken meat, annals of the university dunarea de jos of galati fascicle vi--food technology, 1(31), (2008). [7] min j.s., lee s.o., jang a., jo c., park c.s., lee m., relationship between the concentration of biogenic amines and volatile basic nitrogen in fresh beef, pork and chicken meat, asian-australian journal animal science, 20(8), 1278-1284, (2007). [8] vidal-carou m. c., izquierdopulido m. l., martin-morro m.c., histamine and tyramine in meat products: relationship with meat spoilage, food chemistry, 37(4), 239-249, (1990). [9] min j.s., lee, s.o., jang, a., lee, m., kim, y., production of biogenic amines by microflora inoculated in meats, asian australasian journal of animal sciences, 17(10), 1472-1478, (2004). 154 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, stefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 154 159 types of synonymy in food engineering terminology *alice iuliana rosu1 faculty of food engineering, stefan cel mare university of suceava, romania alicer@fia.usv.ro *corresponding author received april 26th 2014, accepted may 25th 2014 abstract: being one of the most important procedures of language enrichment, accepted as such in lexicology, synonymy in specialized languages and on different language levels has been considered quite a controversial matter, taking into consideration the accuracy and univocity postulates. therefore, there are different opinions on this issue, claiming that synonymy is inadvisable because it hinders terminological unification and consequently it may lead to confusions [1]. in this respect, the phenomenon of synonymy goes against the need of brachylogy (accurate and precise locution), leading to erroneous interpretations of the message conveyed. a contrary opinion is expressed by s. marcus who claims that for each locution there is infinity of phrases with identical signification in the scientific language: the scientific locution is closed, namely it is independent of its receiver [2]. the aim of this paper is to emphasize that synonymy occurs even in the most unexpected activity fields, wherein the accuracy postulate is widely acknowledged, for example in food engineering discourse. thus, the main types of synonymy functioning in food engineering terminology are identified and their etymological sources are explained and illlustrated as well. key words: lexical synonymy, contextual analysis, food terminology, accuracy postulate. 1. introduction language is a system of signs organized on different levels, system that offers functional patterns, possibilities and oppositions to the speaker, out of which he can “choose” whatever he considers as being necessary and adequate to express on a certain moment and in a given language – his ideas [3].ch bally states that the plurality of locution means is the rational basis of synonymy [4]. but the selection of a term is not specific to a single level of language only, since this one may be conceived as a system involving interdependence relationships between its compartments. synonymy issue as a lexical phenomenon has been studied since antiquity, but at present it is being dealt with from different perspectives: morphologically, phonetically, phraseologically and syntactically. in terminology the existence of synonyms is closely connected with the problem of meaning of the term. therefore, one of the main objectives of normalization is exactly the exclusion of synonymous series. the univocity postulate, between denomination and notion, in the case of term, is theoretically valid whereas in reality, in practice, it is not. for one single and the same notion there may exist different denominations, thus making up synonymy series [5]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 alice iuliana rosu, types of synonymy in food engineering terminology, food and environment safety, volume xiii, issue 2 – 2014, pag. 154 – 159 155 lexical synonymy occurs even in the most unexpected activity fields, wherein the accuracy and univocity postulates are widely acknowledged, for example in food engineering discourse – i.e. the romanian food engineering terminology uses more than two heterogeneous terms to denominate the same chemical substance, compound or food additive carbohydrates – sugars – glucides (the last term is widely used in romanian especially), curcumin – natural yellow c1 3 curcuma yellow diferoilmethane, annato extract annatto – rucu – bixin – norbixin – e 160b. 2. materials and methods 2.1. materials two highly academic specialized manuals have been used in performing the semic and contextual analysis of synonyms; one is entitled meat biochemistry, microbiology and parasitology, whereas the other one is wine chemistry and analysis, both of them being considered as standards of authentic specialized terminology specific to food engineering discourse. the selection criteria used in making up the synonym taxonomy identified herein are the semantic and etymological one [6,7]. 2.2. method the first step to be taken in analyzing synonyms is by conducting a semic analysis which allows identifying synonymous series. the grouping of lexical units in synonymous series is done by establishing their semic structure and further on, by identifying their common semes. supplementary semes cancel semantic identity, the elements brought into relation being considered quasisynonyms. therefore, synonymous terms/ syntagms should have as many common semes as possible and as few differentiating ones as possible. a semic analysis has been conducted on short fragments of texts extracted from the previously mentioned manuals in order to identify the semantic features or elements of meaning shared by two or more similar terms and syntagms belonging to the same grammatical and lexical category. (1) polysaccharides are generically designated as ozydes (heavy sugars, with high molecule). they are complex sugars, resulted from condensation/polymerization of monosaccharides wherein a great number of glycoside rests participate in, being united between them by ethereal bonds (ch2-och2). (wca: 637) (2) staphylococci consist of over 30 species, producing enter-toxins (poisonous chemical) when they are growing. even though cooking may destroy the bacterial cells, it is unlikely to inactivate the toxin. for example, staphylococcus aureus produces a thermostable enter-toxin that remains in food products even after the bacteria have been destroyed. (mbmp: 299) (3) toxins from bacterial infections are delayed because the bacteria need time to multiply. as a result symptoms associated with intoxication are usually not seen until 12 – 72 hours or more after eating contaminated food. if the intoxication involves preformed toxins as is the case with staphylococci food poisoning, the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 alice iuliana rosu, types of synonymy in food engineering terminology, food and environment safety, volume xiii, issue 2 – 2014, pag. 154 – 159 156 symptoms occur within a few hours. (mbmp: 324) the terms polysaccharides, ozydes, heavy sugars, all contain the seme /complex sugars/. in other words, the field of a term denominations is seen as a single paradigm. the most important of the distinctive semes is “the molecular structure”, with two opposite values: “more sugar molecules” (+) ≠ “one single sugar molecule” (–), which can be divided into branched or linear forms. other differentiating seme is “taste”, which may be “sweet” (+) or “other taste” (–); (1) polysaccharides= [+macromolecular], [– monosaccharide], [–sweet] or ozydes =[+macromolecular ], [– monosaccharide], [+/– sweet] or sugars = [+macromolecular ], [+ monosaccharide ], [+/– sweet] . in other words, the synonymous terms polysaccharides, ozydes and heavy sugars can have at least three basic factors or semantic properties: they are macromolecular, but are or not monosaccharides and they are or not sweet. (2) staphylococci= [+microorganisms], [+ gram-positive], [– gramnegative], staphylococcus aureus [+microorganisms], [+ grampositive], [– gram-negative], or bacteria= [+microorganisms], [+/– gram-positive/negative], [+/– aerobic/anaerobic]. both terms staphylococci and bacteria contain the seme /microorganisms/. among their differentiating semes we can mention “classification” with two opposite values “grampositive” (+) and “gram-negative” (– ), “aerobic” (+) and “anaerobic” (–). (3) bacterial infections=[+bacterial], [– viral], [+/–short/long]or intoxication [+bacterial], [+ viral ] [+/–short/long] or food poisoning [+bacterial], [ +viral], [+/– short/long]. the series of partial synonymous terms bacterial infection, intoxication and food poisoning send to the same referent / foodborne diseases/. the most important of the distinctive semes is “the type of contamination or infection produced by the microorganisms in question”, with two opposite values: “bacterial”(+) ≠ “viral” (–), another one might be the “incubation period” with the opposites [short +] and [ – long]. the description of different relations existing between words and by which lexical units are defined is not possible without distributing them into classes. the grouping of synonyms into classes takes into consideration one single meaning; therefore, the terms selected are analyzed just in this sense. the first requirement to comply with, namely semantic identity, calls for the checking in this respect of the terms forming the class. this operation can be fulfilled by revealing the meaning components (features), the semes of the lexical units to be analyzed and pursuing to what extent they are shared by all the terms in the series. the examples provided above emphasize the common character of the main semes, those describing properly the semantic content of terms and allowing the classification into the same category: in (1) /complex sugars/, in (2) /microorganisms/ and in (3)/ food poisoning/. obviously the number of semes may vary depending on the complexity of the meaning analyzed, on the relationships established by this one with other lexical units in language and even on the formulation possibilities in metalanguage. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 alice iuliana rosu, types of synonymy in food engineering terminology, food and environment safety, volume xiii, issue 2 – 2014, pag. 154 – 159 157 according to the semantic criterion, the following classes of total synonymous terms have been identified: a) food additives : betain= beet-root red(e 163), ethylic ester of betaapocarotenoic acid =l-orange 9= e 160, cochineal=carminic acid= carmines e120, annatto extract=annatto=rucu=bixin=norb ixin=e 160 b, curcumin – natural yellow c1 3 curcuma yellow diferoilmethane; b) technological operations and processes: tightness= sealing, scalding = boiling, drying=desiccation=dehydration; c) chemical elements, substances and compounds: mercury (from latin mercurius)/ hydrargyrum (from greek)/quicksilver (common denomination), wistaria= glycin= glycol= α-amino-acetic acid, glucides(term used especially in romanian) =carbohydrates = sugars; cocarboxylase= tiamino – pirofosfat (tpp), polyalcohols= polyhidric alcohols, benzene polyhalide= phenyl polyhalide; d) vitamins: vitamin b1=thiamin, vitamin c= ascorbic acid, vitamin p=bioflavone, vitamin e= tocopherols, vitamin d=calcipherols, e) technological equipment and devices : converter=saccharizator, fluidmeter=viscosimeter, acidimeter=aerometer=densimeter =hydrometer; the second step in analyzing synonyms is contextual analysis. identical contexts of recurrence suppose identity of meaning, so the contextual analysis validates the semic one. in this stage, synonymy is determined by the substitution procedure: a lexical unit is replaced by another one, but the global meaning of the pattern must not change. therefore, context is essential when determining synonymy since semantic equivalence is emphasized by context, namely functionally. the context may be a minimal sequence of terms and as a sentence as well. to demonstrate the meaning identity of synonymous adjectives, most linguists make appeal to a nominal context; the substitution of adjectives should not lead to the change of the global meaning of nominal or adjectival syntagms. for example, in the following series of adjectival syntagms: genuine food=bio-food=functional food=organic food, there is one core-term food that is determined by adjectives different from the phonetics and spelling point of view, all of them (genuine =bio=functional =organic) conveying the same meaning and notion: high quality/healthy/ecological food product. but if we analyze the following set of nominal syntagms deterioration= poisoning = spoilage= alteration= rancidness when using the contextual analysis, there might be some inadequacy of use for specific types of foods, though all are generically partial synonyms. when specific mention is made about the type of food that may get altered or contaminated, then it is not possible to use any of the previously synonymous enumerated terms: fat rancidness or taste rancidness, but not bread or meat rancidness. therefore, the synonym substitutions limit the combinatory possibilities of a specialized term, being linguistically and comprehensively determined by context. simple terms are the most exposed to develop partial synonymy relationships which involve the existence of some supplementary ratios of overlapping (or hyperonimy), intersection and more rarely of incidence in the case of terminologies. examples of partially synonymous terms are provided as follows: coenzyme/enzyme cofactor; oscillation boom/oscillation food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 alice iuliana rosu, types of synonymy in food engineering terminology, food and environment safety, volume xiii, issue 2 – 2014, pag. 154 – 159 158 sieve; crystallizer/ evaporator; calcipherols /vitamin d; limestone /chalkstone/lime; gradient/factor/coefficient; fat oxidation /rancidness. specialists’ knowledge is required to establish exactly the type of ratio existing between partial terminological synonyms in the respective field. terminological syntagms presuppose a cumulus of specificity elements, which seemingly, facilitate the establishing of the ratio type existing between partial synonyms: resistivity/specific resistance; cellulose / macromolecular polyglucid; carbonated wine= effervescent wine. one may notice that the synonymy relationships, displayed by the elements of food engineering terminology are generated by more situations that mainly occur in diachronic plane: a) between a term borrowed from french or english and another one formed in the romanian language by means of internal creation: albirea făinii (formed in romanian, albi+re)/blanşarea făinii (in french blanchement de la farine)= flour bleaching; b) between borrowed terms, of different etymology: convertizor (from french convertisseur)=konverter (german) = converter (english), convertor (in romanian); c) between borrowed terms and their paraphrases, registered by dictionaries under the form of terminological syntagms: laser (from french, english laser)=quantic generator; 3. results and discussion context understood as a minimal sequence of terms, or more widely, as a text or stylisticfunctional variant is fundamental in determining synonymy as semantic equivalence is properly emphasized in context, so functionally. both types of analyses, the semic and contextual one, aim to emphasize the referent identity. synonymy occurs when the same complex of significances is rendered by different linguistic forms. if these ones develop synonymy relationships, then they bear and transmit, in communication between specialists, the same knowledge. it occurs between simple terms, between specialized syntagms or more frequently between terms of common language and the scientific ones, or between terms and syntagms. depending on the overlapping degree of semantic areas of the elements analyzed, more possible ratios may be distinguished. thus, total overlapping of significances is specific to the situations of total synonymy which, taking into consideration the univocity of lexical specialized elements, is natural to characterize the relationships of semantic equivalence of scientific and technical terms. total synonymy is displayed at the level of food engineering discourse especially when denominating chemical substances, elements of mendeleev table and food additives respectively. the phenomenon can be explained etymologically or justified by the need of borrowing terms from other languages to express technical and scientific objects, realities that do not exist in certain fields. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 alice iuliana rosu, types of synonymy in food engineering terminology, food and environment safety, volume xiii, issue 2 – 2014, pag. 154 – 159 159 the analyses conducted on samples of authentic food engineering texts reveal that first there are identities and differences when using synonymous terms, secondly that substituting one term by another one makes possible to test the meaning identity between them and thirdly that they aim at disambiguation by means of context of meanings of polysemous words (compulsory preliminary procedure), offering combinatory possibilities of a given lexical unit, established as against types of contexts. 4. conclusions the romanian food engineering terminology uses more than two heterogeneous terms or syntagms to denominate the same referent, displaying both total and partial lexical synonymy relationships, occurring mainly at the diachronic level. total overlapping of significances is specific to the situations of total synonymy which, taking into consideration the univocity postulate of lexical specialized elements, is natural to characterize the relationships of semantic equivalence of the scientific and technical terms herein analyzed. generally, in terminology the existence of synonyms is closely connected with the problem of meaning of the term. particularly, due to the accuracy of formulation and due to the univocal character of the elements it operates with, this specialized type of language provides identification of the referent through synonyms without difficulties. 5. references [1]. maneca c., compound words in presentday scientific and technical terminology, in studies and materials regarding word formation in romanian, vol. i, earpr publishing house, bucharest, (1959), p. 191202 [2]. marcus s., mathematical poetics, earsr publishing house, bucharest, (1970) [3]. coseriu e., lectures and conferences (1992 – 1993), stefan cel mare university publishing house, suceava (1994), p.57. [4]. bally c., traité de stylistique française, georg publishing house, geneva,1951, p.55. [5]. ploae hanganu m., the specific of terminology as science as against the other language sciences, lr, xliv, nr. 9-12, 1995, p. 529-532 [6]. banu c., meat biochemistry, microbiology and parasitology, agir publishing house, bucharest, (2006) [7]. tardea c., wine chemistry and analysis, ‘ion ionescu de la brad’ publishing house, iasi, (2007) doi: https://doi.org/10.4316/fens.2023.002 15 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 15 21 waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium * raghul m 1 1department of food technology, anna university, chennai, tamilnadu, india, raghulkrishvanth30@gmail.com *corresponding author received 16 december 2022, accepted 27th march 2023 abstract: the increased consumption of chicken eggs results in a tremendous volume of egg shell waste, which has an environmental impact. in the node of waste valorisation, egg shells can be utilized as a source of calcium supplementation in various food products. the purpose of this work was to make wheat biscuits supplemented with (1) egg shell powder and (2) calcium chloride extracted from egg shell. the supplemented biscuits were analysed for sensory and proximate analysis. the calcium content in both sources tends to increase, but much higher in calcium chloride formulated biscuits. from sensory scores, biscuits with egg shell powder in 15% and 20% concentrations have more sandy texture and taste, up to 10% concentration is more acceptable. whereas biscuits with calcium chloride in all concentrations (0.5, 1, 1.5, 2) does not marks any undesirable sensory properties. conclusively, fortifying biscuits with either egg shell powder or extracted calcium chloride might be an excellent source of calcium without any significant impact on the quality of biscuits. keywords: waste valorisation, chicken egg shells, calcium supplementation, fortified product. 1. introduction chicken eggshells are by products of chicken farms, hatcheries, household kitchens, and food processing plants that use eggs in their products. egg consumption has increased over the years due to cost effectiveness and increased dietary acceptance, which ultimately leads to increase in eggshell waste and the disposals poses serious environmental and health risks [1], the cost of disposal, accessibility of disposal locations, odour, insects, and abrasiveness are further constraints associated with the disposal of the egg shells [2]. as a novel method of waste valorisation, the egg shells waste can be converted into valuable products for the consumption of humans, which could alleviate its environmental impact [3]. egg shells are excellent source of calcium which has the capability to be utilised by humans [4]. eggshell calcium is regarded as the finest natural source of calcium, and it has been shown to boost bone mineral density in elderly adults struggling with osteoporosis [5]. calcium is one of the essential nutrients that is required for metabolic functions, and plays a pivotal role in sustaining bone and teeth health and other normal body functions. eggshells are the largest source of calcium with an approximate calcium content of 38% [6]. dairy products are an excellent source of calcium to meet the daily calcium requirement. the recommended intake of calcium ranges from 1200 to 1400 mg/day, which is difficult to meet for those with lactose intolerance. hence alternate source of calcium is much needed. chicken egg shells can be the best replacement, ideal mailto:raghulkrishvanth30@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 16 source of calcium supplementation, which is more easily accessible and cost effective. egg shells calcium can also be used as a cheap alternative for expensive calcium supplements [4]. [7] claimed that half of an eggshell might provide individuals with the daily recommended amount of calcium. with an approach to waste valorisation and to provide inexpensive calcium source the current study aims at evaluating the characterization of biscuits supplemented with egg shells powder and ca cl2 extracted from egg shells. 2. materials and methods materials: commercially available wheat flour (72% extraction), chicken eggs shells and all other necessary baking ingredients sugar, shortening agent, milk powder, baking powder, salt, eggs. preparation of eggshell powder: collected chicken eggshells were demembraned, rinsed with distilled water, boiled in hot water for 10mins to kill pathogens then dried, and made in to fine powder. powder was dried in oven and autoclaved. extraction of calcium chloride from eggshells: eggshells powder were mixed with a hydrochloric acid solution, left for 3 hr and stirred occasionally until no gas bubbles were observed. the mixture was centrifuged at 3500 rpm for 10 min at 4 °c. the supernatant was removed and heated to 110–115 °c until dried to yield crystals of calcium chloride [8]. formulation and preparation of formulated biscuits: (1) the powdered eggshell was added to wheat flour at various concentrations 0, 5, 10, 15, and 20% as shown in (table 1). (2) the extracted ca cl2 was added to wheat flour at 0, 0.5, 1, 1.5, and 2% as shown in (table 2). biscuit were prepared according to the creamery method, all the ingredients were blended and the dough is rested for a while. dough was sheeted using a wooden roller pin and the biscuits were cut into circular shape, and then baked at 220˚c for 15 min in the oven. the biscuits were allowed to cool for 30 minutes, packed, and stored in airtight container for further analysis. chemical analysis: chemical analysis of wheat flour, egg shell powder and formulated biscuits were determined according the procedures as described by association of official analytical chemist [9] for moisture, protein, fat, crude fibre, and ash contents moisture content were determined by using gravimetric method, protein by kjeldahl method, fat by soxhlet extraction method, ash by dry ashing method and fibre by enzymatic gravimetric method. all the analyses were carried out in triplicates. determination of mineral composition: minerals including iron, calcium, zinc, phosphorus, magnesium were determined using the method described by association of official analytical chemists [10]. calcium, magnesium, iron and zinc were determined using atomic absorption spectrophotometer (perkin elmer model 3300). sodium and potassium were determined using the standard flame emission photometer (jenway model pfp7). sensory analysis of formulated biscuits: sensory properties of the formulated biscuits were evaluated by twenty five trained panellists. the panellists evaluated biscuits for its colour, odour, taste, texture, flavour, and overall acceptability. each sensory attribute was rated on a 9 point hedonic scale: extremely liked ‒ 9, very much liked ‒ 8, moderately liked ‒ 7, slightly liked ‒ 6, neither liked nor disliked ‒ 5, slightly disliked ‒ 4, moderately disliked ‒ 3, very much disliked ‒ 2 and extremely disliked ‒ 1 [11]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 17 table1. treatment used in formulation of blend of wheat flour and egg shell powder (%) treatment wheat flour (g) egg shell powder (g) sugar (g) shortening (g) egg (g) milk powder (g) salt (g) baking powder (g) vanilla essence (drops) t0 100 0 60 20 40 5 1 .5 2 t1 95 5 60 20 40 5 1 .5 2 t2 90 10 60 20 40 5 1 .5 2 t3 85 15 60 20 40 5 1 .5 2 t4 80 20 60 20 40 5 1 .5 2 t0 – control biscuits with 100% wheat flour and 0% egg shell powder; t1 – biscuits with 95% wheat flour and 5% egg shell powder; t2 – biscuits with 90% wheat flour and 10% egg shell powder; t3 – biscuits with 85% wheat flour and 15% egg shell powder; t4 – biscuits with 80% wheat flour and 20% egg shell powder. table2. treatment used in formulation of blend of wheat flour and cacl2. treatment wheat flour (g) extracted cacl2 (g) sugar (g) shortening (g) egg (g) milk powder (g) salt (g) baking powder (g) vanilla essence (drops) t0 100 0 60 20 40 5 1 .5 2 t1 99.5 0.5 60 20 40 5 1 .5 2 t2 99 1 60 20 40 5 1 .5 2 t3 98.5 1.5 60 20 40 5 1 .5 2 t4 98 2 60 20 40 5 1 .5 2 t0 – control biscuits with 100% wheat flour and 0% cacl2; t1 – biscuits with 99.5% wheat flour and 0.5% cacl2; t2 – biscuits with 99% wheat flour and 1% cacl2; t3 – biscuits with 98.5% wheat flour and 1.5% cacl2; t4 – biscuits with 98% wheat flour and 2% cacl2. 3. results and discussion proximate analysis of wheat flour and eggshell powder: the proximate analysis of commercially available wheat flour and eggshell powder is reported in table 3. the wheat flour has the highest amount of protein (11.14%) and least amount of ash value (0.84%). these results are consistent with the findings of [12] and [13]. the ash content of egg shell powder records highest (93.26%) and has the lowest fat content (0.03%). the chemical compositions of eggshells were consistent with the analysis by [14]. table3. proximate analysis of wheat and egg shell powder. components wheat (%) egg shell powder (%) moisture 11.58a 0.78b protein 11.14a 2.43b fat 2.01a 0.03b fibre 0.41a ndb ash 0.84b 93.26a aindicates the highest value across the same row; nd defines not detected. mineral composition of the wheat flour and eggshell powder: the analysed composition of mineral content of wheat and egg shell powder is shown in table 4. the obtained data shows the egg shell powder is mainly composed of calcium (354.17 mg/100g), followed by magnesium. it to be noted the iron content is comparatively higher in egg shell (10.02 mg/100g) than in wheat flour (0.89 mg/100g). [15] reported values for egg shell powder were similar to the current findings. according to [16] the calcium content in egg shell powder is about (382 mg g-1) which is in closer range. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 18 table 4. mineral composition of wheat flour and egg shell powder. minerals (mg/100g) wheat flour egg shell powder ca 52.64b 354.17a mg 78.41b 275.14a p 120.14a 116.24b na 43.13b 78.24a k 198.74a 64.98b zn 0.66b 0.76a fe 0.89b 10.02a aindicates the highest value across the same row. impact of egg shell powder on the proximate analysis of biscuits: the proximate analysis of the formulated biscuit supplemented with egg shell powder is shown in table 5. significant increase in moisture content was observed. ash content increase with increase in concentration of shell powder, biscuits with 20% shell powder marks higher value (8.03%), which indicates higher mineral content in biscuits. no such significant effects were observed in fat, but a slight depletion in protein and fibre were observed. [17] reported biscuits supplemented with egg shell powder shows higher ash content. table 5. proximate analysis of formulated biscuits (wheat flour + egg shells powder). treatment moisture (%) protein (%) ash (%) fat (%) fibre (%) t0 3.98e 6.84a 0.45e 28.85a 0.18a t1 4.83d 6.67b 2.32d 28.77a 0.16b t2 5.67c 6.03c 4.37c 27.83b 0.15c t3 6.64b 5.86d 6.97b 27.04c 0.13d t4 7.09a 5.22e 8.03a 26.93d 0.13d aindicates the highest value across the same column; a-e different letter superscripts in the same column indicate statistical difference of p<0.05; t0 – control biscuits with 100% wheat flour and 0% egg shell powder; t1 – biscuits with 95% wheat flour and 5% egg shell powder; t2 – biscuits with 90% wheat flour and 10% egg shell powder; t3 – biscuits with 85% wheat flour and 15% egg shell powder; t4 – biscuits with 80% wheat flour and 20% egg shell powder. impact of egg shell powder on the mineral composition of formulated biscuits: mineral composition of formulated biscuits is shown in table 6. biscuits supplemented with egg shell powder increased the minerals content; it is observed that the concentration of calcium, magnesium, sodium, zinc and iron increased significantly, while with reduction of phosphorus and potassium contents. the level of calcium increases predominantly, the values of calcium obtained in this research are in consideration with the recommended daily allowance (rda) of 1000 to 1300 mg/day. studies by [14], [18], and [19] discovered a substantial rise in the calcium content of baked goods when eggshell was added. table 6. mineral composition of formulated biscuits (mg/100g) treatment ca mg p na k zn fe t0 48.14e 18.74e 78.21a 68.12e 180.53a 0.06a 0.27a t1 974d 22.08d 76.54b 113.76d 179.22a 0.18b 0.35b t2 1853c 29.34c 69.02c 149.12c 167.59b 0.34c 0.59c t3 2569b 38.69b 68.87c 158.23b 147.84c 0.58d 0.73d t4 3947a 44.12a 59.71d 169.77a 139.02d 0.67e 0.95e aindicates the highest value across the same column; a-e different letter superscripts in the same column indicate statistical difference of p<0.05 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 19 impact of cacl2 on the proximate analysis of biscuits: proximate analysis of ca supplemented biscuits was shown in table 7. when calcium chloride was supplemented the moisture content remains in the range as with control t0. protein, ash, fat and fibre content does not show any significant impact. the most significant effect was observed in the level of calcium, which increased from 48.14 in t0 control to 1243, 2547, 3698, 4729 mg/100g in t1, t2, t3 and t4, respectively. table7. proximate analysis of cacl2supplemented biscuits (wheat flour + cacl2). treatment moisture (%) protein (%) ash (%) fat (%) fibre (%) calcium (mg/100g) t0 3.98d 6.84b 0.45b 28.85a 0.18a 48.14e t1 4.03d 6.91a 0.45b 28.86a 0.18a 1243d t2 4.11c 6.79c 0.48ab 28.83a 0.17a 2547c t3 4.47b 6.80bc 0.51a 28.85a 0.15a 3698b t4 4.61a 6.77c 0.52a 28.85a 0.15a 4729a aindicates the highest value across the same column; a-e different letter superscripts in the same column indicate statistical difference of p<0.05; t0 – control biscuits with 100% wheat flour and 0% cacl2; t1 – biscuits with 99.5% wheat flour and 0.5% cacl2; t2 – biscuits with 99% wheat flour and 1% cacl2; t3 – biscuits with 98.5% wheat flour and 1.5% cacl2; t4 – biscuits with 98% wheat flour and 2% cacl2. sensory analysis of egg shell supplemented biscuits: sensory score of biscuits supplemented with egg shell powder is shown in figure 1. sensory analysis of biscuits supplemented with egg shell powder (t1 and t2) showed similar characteristics as control (t0), where as the biscuits containing 15% and 20% egg shells powder, has significant variation in taste, texture and flavour. fig.1. sensory score of biscuits supplemented with egg shell powder. sensory analysis of cacl2 supplemented biscuits: sensory score of biscuits supplemented with cacl2 is shown in figure 2. sensory analysis of biscuits supplemented with cacl2 at all concentrations (0.5, 1, 1.5, and 2) showed similarity with control t0. these are no such significant changes in the colour, texture, taste, odour, flavour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 20 fig.2. sensory score of biscuits supplemented with cacl2. it to be noted the biscuits containing higher amount of egg shell powder has sandy taste and flavour. comparing the texture sample t4 had more hardness. with increase in the concentration the colour of the biscuits darkened, studies by [20] reported that the darkening of biscuits may arise due the reaction between the reducing sugars and the amino acids. overall acceptability is higher up to 10% supplementation. 4. conclusion this study highlighted that both egg shell powder and calcium chloride extracted from egg shell increases the calcium content in the biscuits. on comparison the moisture content in the biscuits supplemented with egg shell powder is higher than biscuit with cacl2, which may reduce the shelf life of the product. even though egg shell powder increases the mineral profile other than calcium it may possess the risk of recontamination during storage. furthermore, the eggshell powder does not have any undesirable sensory attributes up to 10% supplementation and which meets the daily calcium intake up to 1300mg/day. biscuits containing cacl2tend to have more calcium as the concentration of cacl2increases. these findings suggest that both powder and extract can be utilized as a calcium supplement, with the extract being more desirable. valorization of egg shells reduces waste while also functioning as an inexpensive source of calcium to alleviate calcium deficiency. 5. references [1]. ajala e., eletta o., ajala m., oyeniyi s., characterization and evaluation of chicken eggshell for use as a bioresource, arid zone journal of engineering, technology and environment, 14: 26–40, (2018) [2]. phil g. &zhihoni m., high value products from hatchery waste. rirdc publication, (2009) [3]. waheed, m., yousaf, m., shehzad, a., inam-ur-raheem, m., khan, m.k.i., khan, m.r., ahmed, n., aadil, r.m., channeling eggshell waste to valuable and utilizable products: a comprehensive review. trends in food science and technology, 10: 78– 90, (2020) [4]. waheed m., butt m.s., shehzad a., adzahan n.m., shabbir m.a., suleria h.a.r., aadil, r.m., eggshell calcium: a cheap alternative to expensive supplements. trends in food science and technology, 91: 219–230, (2019) [5]. schaafsma z., van doormal j.j., muskiet f.a., hofstede g.j., pakan l. & vander v.e., positive effects of a chicken egg shell powder enriched vitamin-mineral supplement on femoral neck bone mineral food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 raghul m., waste valorisation of chicken egg shells and development of formulated biscuits with egg shell waste as a source of dietary calcium, food and environment safety, volume xxii, issue 1 – 2023, pag. 15 – 21 21 density in healthy late post-menopausal dutch women. br. j. nutr. 87:267-275, (2002) [6]. ray, s., barman, a. k., roy, p. k., & singh, b. k., chicken eggshell powder as dietary calcium source in chocolate cakes. the pharma innovation, 6(9, part a), 1, (2017) [7]. milbradt b.g., muller a.l.h., da silva j.s., lunardi j.r., milani l.i.g., de moraes flores e.m., callegaro m.d.g.k., emanuelli t., eggshell as calcium source for humans: mineral composition and microbiological analysis. ciencia rural, 45: 560– 567, (2015) [8]. thakur, r.j., shaikh, h., gat, y., roji b., effect of calcium chloride extracted from eggshell in maintaining quality of selected fresh-cut fruits. int j recycl org waste agricult 8 (suppl 1), 27–36, (2019) https://doi.org/10.1007/s40093-0190260-z [9]. aoac. association of official chemists. official methods of analysis, (15th edition). arlington, va. association of official analytical chemists, usa (2000) [10]. aoac. official methods of analysis, 15th ed. washington dc: association of official analytical chemists (2005) [11]. wichchukit, s. and o'mahony, m., the 9-point hedonic scale and hedonic ranking in food science: some reappraisals and alternatives. j. sci. food agric., 95: 2167-2178, (2015) https://doi.org/10.1002/jsfa.6993 [12]. ali m.a., halim m.a. bread made from wheat flour and white sesame flour in presences of natural improver. pakistan journal of nutrition, 12: 353–355, (2013) [13]. david, o., arthur, e., kwadwo, s. o., badu, e., & sakyi, p., proximate composition and some functional properties of soft wheat flour. international journal of innovative research in science, engineering and technology, 4(2), 753-758, (2015) [14]. ali m., badawy w.z., utilization of eggshells by product as a mineral source for fortification of bread strips. journal of food and dairy sciences, 8: 455–459, (2017) [15]. al-awwal, n.y. and ali, u.l., proximate analyses of different samples of egg shells obtained from sokoto market in nigeria. international journal of science and research, 4(3), pp.564–566, 2015 [16]. brun l.r., lupo m., delorenzi d.a., di loreto v.e., rigalli a., chicken eggshell as suitable calcium source at home. international journal of food sciences and nutrition, 64: 740– 743, (2013) [17]. hassan, n.m., chicken eggshell powder as dietary calcium source in biscuits, (2015) [18]. arnold, m., rajagukguk, y. v., sidor, a., kulczyński, b., brzozowska, a., suliburska, j., wawrzyniak, n., & gramza-michałowska, a., innovative application of chicken eggshell calcium to improve the functional value of gingerbread. international journal of environmental research and public health, 19(7), 4195, (2022) https://doi.org/10.3390/ijerph19074195 [19]. zerek, e., ersoy, n., & ersoy, g., determination of some nutritional and quality properties of eggshell powder added cookies. journal of food measurement and characterization, 16(5), 3315–3320, (2022) https://doi.org/10.1007/s11694-022-01419-w [20]. lee, kyong-ae & brennand, charlotte., physico‐chemical, textural and sensory properties of a fried cookie system containing soy protein isolate. international journal of food science & technology. 40. 501 – 508, (2005) https://doi.org/10.1007/s40093-019-0260-z https://doi.org/10.1007/s40093-019-0260-z https://doi.org/10.1002/jsfa.6993 https://doi.org/10.3390/ijerph19074195 https://doi.org/10.1007/s11694-022-01419-w 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2023.010 101 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 101 108 towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent) n’guettia kossonou 1 , *lessoy zoue 1,2 , gisèle koua 1 , sébastien niamke 1,2 1food biotechnology research unit, biosciences faculty, university félix houphouet-boigny, abidjan, côte d’ivoire, y.lessoy@yahoo.fr 2health, biology and environment doctoral school, university félix houphouet-boigny, abidjan, côte d’ivoire *corresponding author received 16th may 2023, accepted 28th june 2023 abstract: in côte d’ivoire, mango fruit plays an important economic role as the third major exported fruit after banana and pineapple. despite this economic importance, fungal diseases are one of the main issues in the mango value chain with colletotrichum gloeosporioides causing total postharvest losses estimated up to 20%. the aim of this study was to evaluate the antifungal activity of yeasts, lactic acid bacteria and bacillus strains isolated from fermented mango fruits as potential biocontrol agents of colletotrichum gloeosporioides. first, c. gloeosporioides was isolated from contaminated mangoes on potato agar medium, while fermented mangoes were used to isolate yeasts, bacillus and lactic acid bacteria on drbc, nutrient and mrs agar, respectively. afterwards, the in vitro antifungal activity of yeasts, bacillus and lactic acid bacteria against c. gloeosporioides was tested using dual culture and diffusion methods. the total isolates bateria and yeasts from fermented mango were: 238 yeasts, 214 bacillus and 252 lactic acid bacteria. the results of antifungal activity of isolated bacteria and yeasts against c. gloeosporioides were as follows: 78 yeasts isolates (33%) were able to inhibit c. gloeosporioides with 11% inhibition more than 40% mycelial growth. the bacillus isolates were less active (28% inhibition) of c. gloeosporioides with 2% inhibition greater than 60%. as for lactic acid bacteria, they were more active since 58% of isolates showed antifungal activity with 23% of which showed inhibition greater than 60%. based on the results lactic acid bacteria isolated from fermented mangoes can be used as valuable starters for anthracnose biocontrol. keywords: antagonistic microorganisms, biocontrol, colletotrichum gloeosporioides, mango 1. introduction mango (mangifera indica l.) is one of the most important and commonly eaten fruits in the tropical and subtropical areas. in côte d’ivoire, mango fruit plays an important economic role as the third major exported fruit after banana and pineapple. the main producing regions (korhogo, ferkessedougou, sinematiali, boundiali, odienne) of mango fruits are in northern part of côte d’ivoire and the most cultivated varieties are kent, keitt and amélie. despite the economic importance of mango in côte d’ivoire, fungal diseases are one of the main issues in the mango value chain, occurring during the production and handling stages [1]. fungal diseases are known as responsible for postharvest losses in mango production and they also constitute a health risk for consumers due to mycotoxins [2]. among the fungal diseases, anthracnose caused by colletotrichum gloeosporioides is considered as one of the most important that negatively impacts mango production http://www.fia.usv.ro/fiajournal mailto:y.lessoy@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 102 causing total losses estimated up to 20% [3,4]. to fight against fungal diseases, chemical fungicides are widely used as treatments to reduce fruit losses from anthracnose [5]. nevertheless, long-term, and excessive use of chemicals has negative consequences, such as long period of degradation, development of fungicide resistance, harmful effects on human health and the environment [6]. hence, researchers are working to develop alternative and less toxic strategies for the control of phytopathogens. among the different methods, biological control appears a promising option for the control of diseases of fungal origin [7,8]. furthermore, the biological control efficacy of microbial agents has been demonstrated against several postharvest diseases of fruit and vegetables [2]. biocontrol is essentially based on the use of living microorganisms and/or their metabolites to inhibit the growth or metabolism of phytopathogenic microorganisms [9]. compared to chemical agents, the use of antagonistic microorganisms has several advantages including specificity of action, absence of toxic and environmentally harmful residues, safer application and more economical production [10]. in addition, biocontrol is known as efficient solution in the short, medium and long term [11]. on the other hand, microbial agents with antagonistic activity used for the control of fungal plant pathogens are from several taxonomic groups such as bacteria, yeasts and filamentous fungi [12]. however, lactic acid bacteria, bacillus and yeasts are often used for food biopreservation [13]. the aim of this study was to evaluate the antifungal activity of yeasts, lactic acid bacteria and bacillus strains isolated from fermented mango fruits as potential biocontrol agents of colletotrichum gloeosporioides. 2. material and methods 2.1. sampling of mangoes plant material consists of mangoes (mangifera indica var kent), collected in three (03) producing regions of northern côte d'ivoire: korhogo (9° 25' 0.001" n 5° 37' 0.001" w), ferkéssédougou (9° 35' 60" n 5° 12' 0" w), sinématiali (9° 34' 59.999" n 5° 22' 59.999" w). mangoes were randomly collected in orchards during harvesting period (mid-may 2022). two groups of mangoes were collected: healthy mature mangoes for the first group and mangoes with symptoms of anthracnose for the second group (figure 1). the collected mangoes were put in separated boxes, labeled, and transported to biotechnology laboratory, félix houphouët-boigny university) for further analysis. fig. 1. photography of healthy mature kent mango (a) and contaminated kent mango with symptoms of anthracnose (b) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 103 2.2. isolation and morphological identification of colletotrichum gloeosporioides isolation of colletotrichum gloeosporioides was carried out according to pitt and hocking [14]. mangoes with symptoms of anthracnose (translucent, brown and blackish lesions spots) were washed with sterile distilled water and dried at ambient temperature. after drying, anthracnose lesions on mangoes surfaces were removed using a sterile scalpel and placed on potato dextrose agar (pda) medium supplemented with 0.1% chloramphenicol. the petri dishes were incubated at 30 °c for 3 to 5 days. the fungal isolates were subcultured on pda medium until pure cultures were obtained. then, pure cultures were incubated for 7 days at 30 °c and stored at 4 °c on agar slant for identification. macroscopic and microscopic characters of pure isolates were described by using identification keys of barnett and hunter [15] after observation under optical microscope at ×40 magnification (zeiss, germany). 2.3. fermentation of mangoes fifteen (15) ripe and healthy mangoes were cut into slices of small pieces of about 5 cm. the slices were crushed in a blender (moulinex, france) briefly for 5 to 10 seconds and the paste was fermented in sterile and hermetically sealed glass jars for 48 h at laboratory temperature (25 °c). 2.4. isolation and biochemical identification of lactic acid bacteria and bacillus from fermented mangoes twenty-five (25 g) grams of fermented mixture from mangoes slices were mixed in 225 ml sterile buffered peptone water. the whole mixture was stirred for 3 to 5 min for homogenization. afterwards, serial decimal dilutions (10-2 to 10-5) were carried out by using tryptone salt buffer as diluent. for lactic acid bacteria, an inoculum of 100 μl of each dilution was spread on mrs agar supplemented with 100 ppm of nystatin into petri dishes. afterwards, the plates were incubated at 30 °c for 24-72 h. isolation of bacillus sp. was done by inoculation of 100 μl of each dilution on nutient agar [16]. the plates were incubated at 30 °c for 24 to 72 h. biochemical identification of isolates of lactic acid bacteria and bacillus was based on gram staining, catalase and oxidase tests. 2.5. isolation and morphological identification of yeasts from fermented mangoes the isolation of yeasts from fermented mangoes was carried out by spreading 100 μl of each dilution (10-3 to 10-5) on dichloran rose bengal chloramphenicol (drbc) agar medium and the petri dishes were incubated at 30 °c for 24 h. the morphological identification of yeasts isolates was done by observation under optical microscope at ×40 magnification. 2.6. screening for antifungal activity of bacillus sp. and yeasts isolates the in vitro antifungal assay for bacillus and yeasts isolates was carried out based on the dual-culture method [16]. fungal specie (colletotrichum gloeosporioides) plugs of 6 × 6 mm diameter were placed at the center of pda medium and 5 µl of 24 hours bacillus and yeasts grown in nutrient broth were placed on the opposite four sides of the plates at 1.5 cm away from the fungal disc. plates containing the fungal plugs without bacillus or yeasts inoculation were used as control. petri dishes were incubated at 30 °c for 5 days and the growth diameter of colletotrichum gloeosporioides was determined. the percentage of inhibition of the fungal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 104 growth was calculated using the following formula: i = (r – r) x 100/r with i inhibition percentage; r mycelial ray of colletotrichum gloeosporioides in the control plate (mm); r mycelial ray of colletotrichum gloeosporioides in the plate with bacillus sp. or yeasts (mm) 2.7. screening for antifungal activity of lactic acid bacteria isolates lactic acid bacteria isolates were tested for their antifungal activity against colletotrichum gloeosporioides according to agar diffusion method [17]. for this, overnight grown of lactic acid bacteria strains were inoculated on mrs agar by using spot technique. the plates were incubated at 30 °c for 48 hours, and then overlaid with 10 ml of pda soft agar (pdb and 0.7% agar) containing 106 spores/ml of colletotrichum gloeosporioides. after the pda medium was added, the plates were incubated at 30 °c for 3-4 days. the zones of inhibition around the colonies of lactic acid bacteria were recorded and the percentage of inhibition of the fungal growth was calculated using the following formula: i = d x 100/d with d diameter of halo around the colonies and d diameter of growth of the control. 2.8. statistical analysis excel 2016 (microsoft corporation) was used for statistical analysis. 3. results and discussion 3.1. morphological characteristics of isolated colletotrichum gloeosporioides two macroscopic characteristics of colletotrichum gloeosporioides were obtained after isolation on potato dextrose agar (pda) medium (fig. 2). the first culture showed mycelia with a woolly texture with white color both for face and reverse (fig. 2.a and 2.b). the second culture highlighted mycelia with a cottony texture, with gray color both for face and reverse (fig. 2.c and 2.d). in addition, the cultures showed orange conidial mass in the center of plates. fig. 2. photography of macroscopic characteristics of colletotrichum gloeosporioides on potato dextrose agar (pda) medium. microbial spoilage is one of the most important factors in post-harvest fruits losses. mangoes, like most commercial fruits, are particularly susceptible to many microbial diseases [18]. among these diseases, anthracnose is one of the most widespread in tropical africa [19]. the results of our study were in agreement with those of several authors [20,21]. these authors have demonstrated that colletotrichum is the main fungi responsible for anthracnose contamination in mango fruits. anthracnose contamination is characterized by the development of deep, black rot spots or linear necrotic lesions on the fruit [1]. particularly, the specie c. gloeosporioides is the main causative agent of anthracnose in mango [22]. a b c d food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 105 3.2. morphological and biochemical characteristics of isolated bacteria and yeasts from fermented mango the total isolates bateria and yeasts from fermented mango were: 238 yeasts, 214 bacillus and 252 lactic acid bacteria. the lactic acid bacteria isolates were grampositive with a diversity of shape (cocci, coccobacillus, or bacillus) and arrangement mode (clump or chain), catalase negative and oxidase negative. as for bacillus strains, they were grampositive and exclusively rod-shaped. the yeasts showed a pink color on dichloranrose-bengal-chloramphenicol (drbc) agar medium. the isolation of fermentative microorganisms from mango showed a diversity of microbial groups with occurence of lactic acid bacteria and yeasts. these results were in agreement with those obtained by authors who showed that the surface of mango fruits is colonized by microorganisms mainly composed of gram-positive and gramnegative bacteria and yeast [23]. 3.3. in vitro antifungal activity of isolated bacteria and yeasts against colletotrichum gloeosporioides the results of antifungal activity of isolated bacteria and yeasts against c. gloeosporioides are shown in table 1 and figure 3. for yeasts, 78 isolates (33%) were able to inhibit c. gloeosporioides with 11% inhibition more than 40% mycelial growth. the bacillus isolates were less active (28% inhibition) against c. gloeosporioides with 2% inhibition greater than 60%. as for lactic acid bacteria, they were more active since 58% of isolates showed antifungal activity, and with 23% of this showed inhibition greater than 60%. table 1. inhibition percentage of isolated bacteria and yeasts against c. gloeosporioides isolates no inhbition against c. gloeosporioides inhibition against c. gloeosporioides i ˂ 40% 40% ˂ i ˂ 60% i ˃ 60% yeasts 160 (67%) 52 (22%) 26 (11%) 0 (0%) bacillus sp. 154 (72%) 37 (17%) 19 (9%) 4 (2%) lactic acid bacteria 106 (42%) 10 (4%) 78 (31%) 58 (23%) post-harvest diseases are usually controlled by the application of synthetic fungicides [12]. however, the use of synthetic products leads to various issues such as environmental pollution, toxicity, as well as the development of pathogens resistance. therefeore, it is essential to adopt alternative approaches as biocontrol for plant diseases management [24]. based on the antagonism tests, the microbial isolates presented inhibition percentage greater than 25%. moreover, 50% of lactic acid bacteria isolates inhibited c. gloeosporioides. this result is higher than that (18%) found by some authors [25]. this result could be explained by the fact that tested isolates were from the same ecological environment as c. gloeosporioides. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 106 fig. 3. photography of in vitro inhibition of c. gloeosporioides by yeast (a and b), bacillus (c and d), and lactic acid bacteria (e and f) isolates indeed, according to some authors [26], antagonistic microorganisms used to control plant diseases should be taken from ecological environment similar to those where the target disease is present. 4. conclusion the aim of this study was to evaluate the antifungal activity of yeast, lactic acid bacteria and bacillus strains isolated from fermented mango fruits as potential biocontrol agents of colletotrichum gloeosporioides. the results of the present study showed that yeasts, bacillus and lactic acid bacteria isolates presented inhibition percentage greater than 25% on colletotrichum gloeosporioides. thus, yeasts, bacillus and lactic acid bacteria isolated from fermented mangoes can be used as valuable starters for anthracnose biocontrol. before their use as starters, molecular identification of yeasts, bacillus and lactic acid bacteria isolates must be performed. 5. acknowledgments authors are grateful to the funds for science, technology and innovation (fonsti) of republic of côte d’ivoire for financial assistance through a research grant to the corresponding author lessoy zoue (project fonsti n°2 valomangues). 6. references [1]. archana t., gogoi r., kaur c., varghese e., sharma r., srivastav m., tomar m., kumar m., kumar a., bacterial volatile mediated suppression of postharvest anthracnose and quality enhancement in mango, postharvest biology and technology, 177, (2021) https://doi.org/10.1016/j.postharvbio.2021.111525 [2]. ranjith f., muhialdin b., arroo r., yusof n., mohammed n., hussin a., lacto-fermented polypeptides integrated with edible coatings for mango (mangifera indica l.) bio-preservation, food control, 134, (2022), https://doi.org/10.1016/j.foodcont.2021.108708 [3]. perumal a., nambiar r., periyar selvam sellamuthu p., rotimi s., use of modified atmosphere packaging combined with essential oils for prolonging post-harvest shelf life of mango (cv. banganapalli and cv. totapuri), lwt, 148, (2021), https://doi.org/10.1016/j.lwt.2021.111662. [4]. krishnapillai n., wilson wijeratnam w., sap volatile components in relation to susceptibility of anthracnose and aspergillus rot of mangoes (mangifera indica l.), the journal of horticultural science and biotechnology, 92, 206-213, (2016) https://doi.org/10.1080/14620316.2016.1249962 a c e b d f https://doi.org/10.1016/j.postharvbio.2021.111525 https://doi.org/10.1016/j.foodcont.2021.108708 https://doi.org/10.1016/j.lwt.2021.111662 https://doi.org/10.1080/14620316.2016.1249962 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 107 [5]. lima n., lima w., tovar-pedraza j. et al. comparative epidemiology of colletotrichum species from mango in northeastern brazil, eur j plant pathol 141, 679– 688 (2015). https://doi.org/10.1007/s10658-0140570-y [6]. ons l., bylemans d., thevissen k., cammue b., combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. microorganisms, 8, 19-30, (2020), https://doi.org/10.3390/microorganisms8121930 [7]. pu l., li l., chao-an l., characterization of competition for nutrients in the biocontrol of penicillium italicum by kloeckera apiculata, biological control, 67, 157-162, (2013), https://doi.org/10.1016/j.biocontrol.2013.07.011. [8]. lahlali r., aksissou w., lyousfi n., ezrari s., blenzar a., tahiri a., ennahli s., hrustić j., maclean d., amiri s., biocontrol activity and putative mechanism of bacillus amyloliquefaciens (sf14 and sp10), alcaligenes faecalis acbc1, and pantoea agglomerans acbp1 against brown rot disease of fruit, microbial pathogenesis, 139, (2020), https://doi.org/10.1016/j.micpath.2019.103914. [9]. carmona-hernandez s., reyespérez j.j., chiquito-contreras r.g., rincon-enriquez g., cerdan-cabrera c., hernandez-montiel l., biocontrol of postharvest fruit fungal diseases by bacterial antagonists: a review. agronomy, 9, (2019), https://doi.org/10.3390/agronomy9030121 [10]. bonaterra a., badosa e., daranas n., francés j., roselló g., montesinos e., bacteria as biological control agents of plant diseases. microorganisms, 2022, 10, 17-59 (2022), https://doi.org/10.3390/microorganisms10091759 [11]. cuthbert r., dick j., callaghan a., dickey j., biological control agent selection under environmental change using functional responses, abundances and fecundities; the relative control potential (rcp) metric, biological control, 121, 50-57, (2018). https://doi.org/10.1016/j.biocontrol.2018.02.008. [12]. dukare a., sangwan s., maheshwari h., guru p., khade y., vishwakarma r., chapter 15 utilization of antagonistic microbes for the eco-friendly management of fungal diseases of the harvested fruits during postharvest handling and storage, editor(s): ajay kumar, samir droby, food security and plant disease management, woodhead publishing, 307-322, (2021), https://doi.org/10.1016/b978-0-12-8218433.00015-5. [13]. salas m., mounier j., valence f., coton m., thierry a., coton e., antifungal microbial agents for food biopreservation-a review. microorganisms, 8, 2017, 10.3390/microorganisms5030037. pmid: 28698479; pmcid: pmc5620628 [14]. pitt j., hocking a., fungi and food spoilage. springer, 3rd edition, new york, usa, 524p, (2009). [15]. barnett h., hunter b., illustrated genera of imperfect fungi. mineapolis: burgress publishing company, minneapolis mn, 241 p., (1972). [16]. sun p., cui j., jia x., wang w., isolation and characterization of bacillus amyloliquefaciens l-1 for biocontrol of pear ring rot. horticultural plant journal, 3, 183–189, (2017). [17]. cheong e., sandhu a., jayabalan j., zwielehner j., bansal n., turner m., isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage mold penicillium commune and their potential as biopreservatives in cheese. food control, 46, 9197, (2014). [18]. zheng m., shi j., wang q., li y., antimicrobial effects of volatiles produced by two antagonistic bacillus strains on the anthracnose pathogen in postharvest mangoes. biological control, 65, 200-206, (2013). [19]. arauz l., mango anthracnose: economic impact and current options for integrated management. plant diseases, 84, 600-611, (2000). [20]. dembele d., kamara a., grechi i., silué n., n'goran n., yéo y., rey j., koné d., morphological characteristics and distribution of colletotrichum isolates morphotypes infecting mango (mangifera indica l.) in the north of côte d'ivoire. african journal of food, agriculture, nutrition and development, 20 :15837-15856, (2020). [21]. li q., bu j., shu j., yu z., tang l., huang s., guo t., mo j., luo s., solangi g., colletotrichum species associated with mango in southern china. scientific reports, 9, (2019). [22]. hernandez-montiel l., zuluetarodriguez r., angulo c., ruedapuente e., quinonez-aguilar e., galicia r., marine yeasts and bacteria as biological control agents against anthracnose on mango. journal of phytopathology, 165, 833-840, (2017). [23]. abadias m., usall j., anguera m., solsona c., viñas i., microbiological quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail https://doi.org/10.1007/s10658-014-0570-y https://doi.org/10.1007/s10658-014-0570-y https://doi.org/10.3390/microorganisms8121930 https://doi.org/10.1016/j.biocontrol.2013.07.011 https://doi.org/10.1016/j.micpath.2019.103914 https://doi.org/10.3390/agronomy9030121 https://doi.org/10.3390/microorganisms10091759 https://doi.org/10.1016/j.biocontrol.2018.02.008 https://doi.org/10.1016/b978-0-12-821843-3.00015-5 https://doi.org/10.1016/b978-0-12-821843-3.00015-5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 n’guettia kossonou, lessoy zoue, gisèle koua, sébastien, towards biocontrol of post-harvest anthracnose by antagonistic bacteria and yeast isolated from fermented mango (mangifera indica, var kent), food and environment safety, volume xxii, issue 2 – 2023, pag. 101 – 108 108 establishments. international journal of food microbiology, 123, 121-129, (2008). [24]. romanazzi g., lichter a., gabler f., smilanick j., recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. postharvest biology and technology, 63, 141-147, (2012). [25]. barrios-roblero c., rosasquijano r., salvador-figueroa m., gálvez-lópez d., vázquez-ovando a., antifungal lactic acid bacteria isolated from fermented beverages with activity against colletotrichum gloeosporioides. food bioscience, 29, 47-54, (2019). [26]. janisiewicz w., korsten l., biological control of postharvest diseases of fruits. annual review of phytopathology, 40, 411441, (2002). 1. introduction doi: https://doi.org/10.4316/fens.2022.025 257 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 257 264 influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes *ana batariuc 1 , ionica coţovanu 1 , silvia mironeasa 1 1faculty of food engineering, ştefan cel mare university, suceava, romania *corresponding author: ana.batariuc@usm.ro received 1st july 2022, accepted 28th september 2022 abstract: sorghum is the fifth most used cereal and contains many bioactive compounds which enhance the antioxidant capacity. the aim of this study was to investigate the antioxidant capacity and the total phenolic content by applying different organic solvents and extraction times on sorghum grain at different particle sizes which were subjected dry heat treatment. the antioxidant capacity values varied between 53.5% to 100%, and the total phenols content ranged from 6.58 mg gae/g to 54.90 mg gae/g for the sorghum flour particles sizes, indicating a significant increase when the dry heat treatment was applied. from the solvent used point of view, it can be seen a rise in antioxidant capacity and concentration of total phenols extracted in the following order: methanol > water > ethanol. also, when the particle size of sorghum flour decreased and extraction time increased, the values of antioxidant capacity and total polyphenols components increased. this study revealed the potential of different sorghum particles size from the total polyphenols and antioxidant capacity point of view and to use them as ingredients to raise the shelf life of food products. keywords: antioxidants; dry heat treatment; gluten-free; particle size; polyphenols; sorghum grains. 1. introduction sorghum is a gluten-free grain that contains bioactive components (phenolic acids, flavonoids, and anthocyanins), being a good cereal alternative for human consumption, especially for celiac patients [1,2]. many researchers have focused on the health benefits of sorghum phenolics and their antioxidant capacity, which found that the antioxidant capacity of sorghum is higher than that of any other grain [3]. sorghum grain contains from the outside to the inner: outer covering pericarp (5%), layer testa (1%), storage tissue endosperm (84%), and embryo germ (10%) [4]. testa is placed over the pericarp and under the endosperm, being unique through other cereals [5]. phenolic substances are mostly placed in the pericarp and testa [6], this fact is influenced by varieties and growing conditions [7]. the principal sorghum phenolic compounds are phenolic acids, flavonoids (anthocyanins, catechins, and leucoanthocyanidins), and condensed tannins [8]. phenolic compounds from sorghum grains manifest a great antioxidant capacity, which is mainly characterized by scavenging radicals [1,9]. the extraction yield, content, and profile of phenolics compounds in sorghum present significant differences when are used different extraction methods, such as refluxing extraction, water extraction, maceration extraction, soxhlet extraction, and organic solvent extraction [10,11]. various solvents including water, ethanol, methanol, ethyl ether, etc. were used for the phenol’s extraction [12-14]. furthermore, physical treatment methods applied to sorghum grains change the phenolic profile and antioxidant capacity. one of the methods that enhance the nutritional quality and phenolic http://www.fia.usv.ro/fiajournal mailto:ana.batariuc@usm.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 258 compounds of cereal grains is the thermal processes [15], such as baking, roasting, and extrusion which lead to physicochemical modification when starch, protein, and other, compounds interact, and maillard reactions are producing [16]. these modifications will improve the antioxidant characteristics, nutritional quality, organoleptic properties, inactivating of high temperature sensitive toxic components and enzyme inhibitors. these increases might be possible due to the damage to cellular components which release phenolic acids and hydrolyze the polyphenols in simple phenols [17,18]. some authors highlight an increase in vitamin e and a decrease in the carotenoids, flavanones, flavones, and proanthocyanidins when sorghum flours were treated with dry heat in a conventional oven [19]. contrarily, in the same study, was demonstrated that the antioxidant activity in treated flours with dry heat remained constant or increased [19]. this can be possible due to various bioactive substances which have different sensitivity to heat and the carotenoids, flavanones, flavones, and proanthocyanidins are more sensitive than vitamin e to heat. the processing technology of sorghum includes the partial removal of the germ, endosperm, and pericarp, through the decortication, malting, fermentation, roasting, flaking, and milling process [20]. for cereal grains, milling represents the operation of the bran and germ separation from the starchy endosperm which results in refined flour, because the pericarp, testa, and aleurone layers are all separated [21]. some studies reported higher flavonoid content in sorghum bran compared to other fractions [22] and an improvement of total polyphenols content when particle size decreased in sorghum fractions [9], in barley [23], because once the fiber matrix was broken, the phenolic components were released or exposed. these results demonstrated that grinding treatment could effectively improve the antioxidant capacity [24]. this leads to the conclusion that particle size has a great influence on compound present in grains such as minerals, vitamins, fibers, and phytochemicals. some authors stated that the bran particles represent a very valuable natural source of antioxidants due to phenolics, the bran being a a valueadded product in the preparation of functional food ingredients and/or for the enrichment of particular products [16]. the phenolic compounds from sorghum are bound to the cell wall and these makes it difficult to extract it [21], and are important to find the techniques that enhance polyphenol extraction at different temperature conditions and time. sorghum, one of the most diverse cereals in terms of the types and amounts of polyphenols [25], presents the following beneficial effects such as reducing the oxidative stress and chronic inflammation, prevention of some types of cancer, enhancement of glucose metabolism and reducing insulin resistance, improving lipid metabolism and changing the gut microbiota for good colon function [8,26]. it has been observed in the literature that recently, studies on sorghum have intensified, regarding bioactive compounds and their effects on health [27-29]. even though there are many studies about the phenolic compounds and antioxidant activity in the sorghum flour, from our knowledge are no reports about the total phenolic content and antioxidant activity of particle size of dry heat treatment sorghum grains at different temperatures, and the impact of the type of solvent and extraction time on them. the aim of this research was to assess the influence of dry heat treatment of sorghum grain on phenolic compounds food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 259 and antioxidant activity in different particle sizes of sorghum flour by using different organic solvents and extraction times. 2. materials and methods 2.1. materials the white sorghum seeds (es albanus hybrid) from harvest 2021, were purchased from the secuieni agricultural development research station (neamt, românia). the proximate composition includes: 11.50% moisture, 10.35% protein, 3.07% lipids, 1.16% ash, 8.35% dietary fiber and 65.56% carbohydrates. 2.2. sorghum treatment dry heat treatment of sorghum was performed at different temperatures (121 °c, 132 °c, and 140 °c) for 15 min in a binder ed53l convection oven (binder, tuttlingen, germany). the sorghum grains heat treated and untreated were milled into a laboratory machine (grain mill, kitchenaid, model 106 5kgm, benton harbor, mi, usa). then, the integral flour was sieved on three mesh (retsch vibratory sieve shaker as 200 basic, haan, germany), and was obtained three flour particle sizes large (l > 300 µm), medium (200 µm < m < 250 µm), small fractions (s < 200 µm). in this study, large particle size was selected from grains treated at 140 °c (lt), medium particle size from grains treated at 132 °c (mt) and small particle size from grains treated at 121 °c (st). the selection was made based on the results obtained in our previous research [9]. the untreated samples of sorghum grains sieved on the same three meshes, l, m, and s were coded as ln, mn, and sn and considered as control. 2.3. methods 2.3.1. antioxidant capacity to determine the antioxidant capacity of sorghum particle sizes was used the 2,2 diphenyl-1picrylhydrazyl (dpph) method at four different times (10, 15, 20, and 30 min). briefly, 2 ml of dpph reagent was mixed with 2 ml extract, prepared as reported in a previous study [9]. the absorbance was reading at 517 nm after incubation for 10, 15, 20, and 30 min at room temperature. the dpph scavenging activity was calculated using equation (1), where asample is the absorbance of the sample with sorghum flour extract and ablanck, is the absorbance reading without extract. (1) 2.3.2. total polyphenol content in order to determinate the total polyphenol content (tpc), 0.2 ml extract was mixed with 2 ml folin–ciocâlteu reagent and 1.8 ml sodium carbonate (7.5%) [30]. after resting at room temperature in the darkness for 10, 15, 20, and 30 min, the absorbance was read at 750 nm [30]. the calibration curve made with gallic acid had r2 = 0.99 and the equation y = 0.00949 x + 0.02950. 2.4. statistical analysis all assays were done in triplicate. statistically significant differences at a 95% confidence level were evaluated by means of one-way anova with tukey’s test, by using xlstat for excel 2021 version (addinsoft, new york, ny, usa). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 260 3. results and discussion 3.1. effect of dry heat treatment and extraction solvent type on the antioxidant capacity of sorghum particle size all three types of untreated and treated sorghum flour particle sizes were analyzed from an antioxidant activity point of view obtained after 10, 15, 20, and 30 min extraction times, and the results are presented in figure 1. the obtained results demonstrated that treatment temperatures remarkable influenced the antioxidant capacity of sorghum flour. fig. 1. effect of the water solvent on samples antioxidant capacity during phenolic compounds extraction with water, it is observed that particle sizes of sorghum flour obtained from dry heat-treated sorghum grains show higher values of antioxidant capacity. it can also be seen in figure 1 that the medium particle size has the highest antioxidant capacity, and when the extraction time increase, the antioxidant capacity increases proportionally. this fact can be possible due to higher temperatures during processing which leads to the enhance of total phenolic content and free radical scavenging capacity. similar results were obtained in other studies, which presented an increase in barley flour antioxidant capacity when was applied a roasted treatment to the grain [31,32]. in the case of ethanol extraction, there were significant differences observed between the particle sizes of thermally and non-thermally treated samples (figure 2). ethanol extraction showed an enhanced antioxidant capacity of the samples as the particle size decreased and the extraction time increased. sorghum phenolic types influenced the antioxidant activity of the samples. the increase of antioxidant activity with the diminishing of grain granularity was noticed by other authors. this fact can be due to the damage of the fiber matrix [33], and when was applied a heat treatment on sorghum grains it could lead to the release of phenolic compounds from glycosidic components and the hydrolyzation of higher phenolic components into smaller ones [34]. fig. 2. effect of the ethanol solvent on samples antioxidant capacity extraction with methanol of the phenolic compounds from the sorghum flour different particle sizes showed higher values, a similar trend with the water extraction. significant differences were observed between samples from the heat treatment applied, the size of the particle, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 261 and the extraction time used point of view (figure 3). fig. 3. effect of the methanol solvent on samples antioxidant capacity our results were similar to those obtained by the morais carddoso et al. [19]. the increase of antioxidant activity in the dry heat-treated samples can be explained by the formed compounds such as maillard reaction products [35], and the release of bound phenolic acids from cell walls during baking [36]. this variation in sorghum samples' dpph values can be based on the chemical extracts structure and on the radical–antioxidant reactions parameters. the value which presented higher antioxidant activity was with methanol extracts and this result can be based on the presence of higher content and synergistic action of antioxidant components with hydrophilic and hydrophobic nature [36]. the use of an alcoholic solvent could be more adequate for extraction of antioxidant compounds from sorghum [6]. 3.2. effect of dry heat treatment and extraction solvent type on the total polyphenol content of sorghum particle size the extraction of polyphenols from white sorghum flour was strongly influenced by the treatment applied to the grains, the sorghum flour particle size, and extraction time. the highest particle size values of polyphenols were found in the samples where the extraction time was applied for 20 and 30 min (figure 4). fig. 4. effect of the water solvent on samples total polyphenols contents statistical analysis of the data shows that the extraction solvent had a positive effect on the phenolic compounds in the following order: methanol > water > ethanol (figures 4, 5, and 6). the maillard reaction and chemical oxidation of phenols can be responsable for the increase of total polyphenols content. fig. 5. effect of the ethanol solvent on samples total polyphenols contents some authors demonstrated that heat enhanced the total phenols content [36]. some phenols can also be deposited in the cellular vacuoles [37], and processing food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 262 with high temperatures may release unavailable phenolics. fig. 6. effect of the methanol solvent on samples total polyphenols contents the processing conditions considerable influenced the phenolic compounds. the release of phenolic compounds depend of moisture content, time and temperature during extrusion processing [38]. based on the indicated results, we found significant differences (p < 0.05) on the total phenolic content of non-treated and heat-treated samples. this enhance can be explain by the hydrolization of conjugated phenolic moiety during the thermal process and a polymerization reaction which lead to the obtaining of phenols [16]. this suggests that even though the phenolic components are considered a major group of antioxidants, the nonphenolic components can also significantly contribute to the antioxidant activity of this cereal [6]. 4. conclusion the processing with dry heat treatment affected the antioxidant capacity and total polyphenols compounds of different particle-size sorghum flour depending on treatment temperature. the type of solvent and time of extraction that can facilitate the release of phenols highlighted an increased antioxidant activity in some sorghum particle sizes. the highest antioxidant capacity was found for medium fractions when methanol was used as extraction solvent at 10 min extraction time. 5. acknowledgment this work was performed within the framework of the “decide– development through entrepreneurial education and innovative doctoral and postdoctoral research, project code pocu/380/13/125031, supported by project cofinanced from the european fund through the 2014-2021 operational program human capital“. 6. references [1]. shen, s., huang, r., li, c., wu, w., chen, h., shi, j., chen, s., ye, x., phenolic compositions and antioxidant activities differ significantly among sorghum grains with different applications. molecules, 23, 1023, (2018). [2]. kumari, p.k., umakanth, a.v., narsaiah, t.b., uma, a., exploring anthocyanins, antioxidant capacity and a-glucosidase inhibition in bran and flour extracts of selected sorghum genotypes. food bioscience, 41, 100979, (2021). [3]. dlamini, n., taylor, j., rooney, l., the effect of sorghum type and processing on the antioxidant properties of african sorghum-based foods, food chemistry, 105, 1412–1419, (2007). [4]. slavin, j., whole grains and human health. nutrition research review, 17(1), 99–110, (2004). [5]. earp, c.f., mcdonough, c.m., rooney, l.w., microscopy of pericarp development in the caryopsis of sorghum bicolor (l.) moench. journal of cereal science, 39, 21–27, (2004). [6]. de cardoso, l.m., pinheiro, s.s., de carvalho, c.w.p., queiroz, v.a.v., de menezes, c.b., moreira, a.v.b., de barros, f.a.r., awika, j.m., martino, h.s.d., pinheiro-sant’ana, h.m., phenolic compounds profile in sorghum processed by extrusion cooking and dry heat in a conventional oven. journal of cereal science, 65, 220–226, (2015). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 263 [7]. dykes, l., peterson, g.c., rooney, w.l., rooney, l.w., flavonoid composition of lemon-yellow sorghum genotypes. food chemistry, 127, 173–179, (2011). [8]. taylor, j.r.n., belton, p.s., beta, t. duodu, k.g., increasing the utilization of sorghum, millets and pseudocereals: developments in the science of their phenolic phytochemicals, biofortification and protein functionality. journal of cereal science, 59, 257–275, (2014). [9]. batariuc, a., ungureanu-iuga, m., mironeasa, s., characterization of sorghum processed through dry heat treatment and milling. applied sciences, 12(15), 7630, (2022). [10]. billa, e., koullas, d.p., monties, b., koubios, e.g., structure and composition of sweet sorghum stalk components. industrial crops and products, 6, 297–302, (1997). [11]. awika, j. m., rooney, l. w., waniska, r. d., anthocyanins from black sorghum and their antioxidant properties. food chemistry, 90(1-2), 293-301, (2005). [12]. hong, s., pangloli, p., perumal, r., cox, s., noronha, l.e., dia, v.p., smolensky, d.a., comparative study on phenolic content, antioxidant activity and antiinflammatory capacity of aqueous and ethanolic extracts of sorghum in lipopolysaccharideinduced raw 264.7 macrophages. antioxidants, 9, 1297, (2020). [13]. gaytán-martínez, m., cabreraramírez, á.h., morales-sánchez, e., ramírez-jiménez, a.k.., cruz-ramírez, j., campos-vega, r., velazquez, g., loarca-piña, g., mendoza, s., effect of nixtamalization process on the content and composition of phenolic compounds and antioxidant activity of two sorghums varieties. journal of cereal science, 77, 1–8, (2017). [14]. iuga, m., ropciuc, s., mironeasa, s., antioxidant activity and total phenolic content of grape seeds and peels from romanian varieties. food and environment safety journal, 16(4), (2017). [15]. li, z., zhao, x., zhang, x., liu, h., the effects of processing on bioactive compounds and biological activities of sorghum grains. molecules, 27(10), 3246, (2022). [16]. ragaee, s., seetharaman, k., abdel-aal, e.s.m., the impact of milling and thermal processing on phenolic compounds in cereal grains. critical reviews in food science and nutrition, 54(7), 837-849, (2014). [17]. xiong, y., zhang, p., luo, j., johnson, s., fang, z., effect of processing on the phenolic contents, antioxidant activity and volatile compounds of sorghum grain tea. journal of cereal science, 85, 6–14, (2019). [18]. wu, l., huang, z., qin, p., ren, g., effects of processing on phytochemical profiles and biological activities for production of sorghum tea. food research international, 53, 678–685, (2013). [19]. de morais cardoso, l., montini, t. a., pinheiro, s. s., pinheiro-sant’ana, h. m., martino, h. s. d., moreira, a.v.b., effects of processing with dry heat and wet heat on the antioxidant profile of sorghum. food chemistry, 152, 210-217, (2014). [20]. mir, s.a., manickavasagan, a., shah, m.a., whole grains: processing, product development, and nutritional aspects; crc press: boca raton, fl, usa, isbn 1351104756, (2019). [21]. barros, f., dykes, l., awika, j. m., rooney, l. w., accelerated solvent extraction of phenolic compounds from sorghum brans. journal of cereal science, 58(2), 305-312, (2013). [22]. woo, k.s., seo, m.c., kang, j.r., ko, j.y., song, s.b., lee, j.s., oh, b.g., do park, g., lee, y.h., nam, m.h., et al., antioxidant compounds and antioxidant activities of the methanolic extracts from milling fractions of sorghum (sorghum bicolor l. moench). journal of the korean society of food science and nutrition, 39, 1695–1699, (2010). [23]. zhu, f., du, b., xu, b., super fi ne grinding improves functional properties and antioxidant capacities of bran dietary fi bre from qingke (hull-less barley) grown in qinghai-tibet plateau, china. journal of cereal science, 65, 43– 47, (2015). [24]. xu, q., huang, r., yang, p., wang, l., xing, y., liu, h., zhang, p., effect of different superfine grinding technologies on the physicochemical and antioxidant properties of tartary buckwheat bran powder. rsc advances, 11(49), 30898-30910, (2021). [25]. girard, a. l., awika, j. m., sorghum polyphenols and other bioactive components as functional and health promoting food ingredients. journal of cereal science, 84, 112-124, (2018). [26]. li, r., wang, q., peng, h., zhao, g., zhang, d., li, z. exploring the effect of microwave treatment on phenolic flavonoids, antioxidant capacity, and phenolic in vitro bioaccessibility of sorghum. international journal of food science & technology, 57(4), 2510-2522, (2022). [27]. li, z., zhao, x., zhang, x., liu, h., bioactive compounds and biological activities of sorghum grains. foods, 10(11), 2868, (2021). [28]. li, z., zhao, x., zhang, x., liu, h., the effects of processing on bioactive compounds food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 – 2022 ana batariuc, ionica coţovanu, silvia mironeasa, influence of dry heat treatment on antioxidant activity and total polyphenol content of different sorghum particle sizes, food and environment safety, volume xxi, issue 3 – 2022, pag. 257 – 264 264 and biological activities of sorghum grains. molecules, 27(10), 3246, (2022). [29]. nnamani, d.o., aleke, p.c., onyekere, p.f., sorghum byproducts as sources of nutraceuticals. in food and agricultural byproducts as important source of valuable nutraceuticals (pp. 203-213). springer, cham., (2022). [30]. singleton, v.l., rossi, j.a., colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. american journal of enology and viticulture, 16, 144–158, (1965). [31]. omwamba, m., and hu, q., antioxidant activity in barley (hordeumvulgare l.) grains roasted in a microwave oven underconditions optimized using response surface methodology. journal of food science, 75, 66-73, (2010). [32]. gallegos-infante, j. a., rochaguzman, n. e., gonzalez-laredo, r. f., & pulido-alonso, j., effect of processing on the antioxidant properties of extracts from mexican barley (hordeum vulgare) cultivar. food chemistry, 119(3), 903-906, (2010). [33]. zhu, f., du, b., xu, b., super fi ne grinding improves functional properties and antioxidant capacities of bran dietary fi bre from qingke (hull-less barley) grown in qinghai-tibet plateau, china. journal of cereal science, 65, 43– 47, (2015). [34]. almaiman, s.a., albadr, n.a., alsulaim, s., alhuthayli, h.f., osman, m.a., hassan, a.b., effects of microwave heat treatment on fungal growth, functional properties, total phenolic content, and antioxidant activity of sorghum (sorghum bicolor l.) grain. food chemistry, 348, 128979, (2021). [35]. michalska, a., amigo-benavent, m., zielinski, h., and del castillo, m. d., effect of bread making on formation of maillard reaction products contributing to the overall antioxidant activity of rye bread. journal of cereal science, 48, 123-132, (2008). [36]. dewanto, v., wu, x., adom, k.k., liu, r.h., thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. journal of agricultural and food chemistry, 50(10), 3010-3014, (2002). [37]. chism, g.w., haard, n.f., characteristics of edible plant tissues in food chemistry, (fennema, o. ed.), (1996). [38]. dimberg, l. h., molteberg, e.l., solheim, r., frølich, w., variation in oat groats due to variety, storage and heat treatment i: phenolic compounds. journal of cereal science, 24(3), 263-272, (1996). 1. introduction doi: https://doi.org/10.4316/fens.2022.032 333 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 333 344 effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste *m. vahid aïssi 1 , g. paul daniel time 1 , vénérande y. ballogou 2 , ifagbémi bienvenue chabi 3 , y. euloge kpoclou 1 , mohamed m. soumanou 2 1ecole des sciences et techniques de conservation et de transformation des produits agricoles, université nationale d'agriculture, sakété, benin republic, vahidaissi@yahoo.fr, 2unité de recherche en génie enzymatique et alimentaire, laboratoire d'etude et de recherche en chimie appliquée, univerité d'abomey calavi, abomey calavi, benin republic, 3laboratoire de valorisation et de gestion de la qualité des bio ingrédients alimentaires, faculté des sciences agronomiques, université d’abomey-calavi abomey calavi, benin republic *corresponding author received 1st september 2022, accepted 28th december 2022 abstract: musa paradisiaca, tectona grandis and thalia geniculata leaves used in the past as wrapping materials of olèlè (steamed dough of cowpea enriched to the shrimps and other condiments), are being abandoned in favor of metal cans. this situation contributes to the lost of identity of this traditional food product. two traditional processing methods which differ in the dehulling techniques are practiced to obtain olèlè also called magni magni or moin moin. the objective of this study was to determine the effect of processing methods and wrapping materials on sensory characteristics of olèlè and their consumer acceptability. for this purpose, sensory difference and overall acceptability tests and a sensory profile analysis were conducted. thus, olèlè differently wrapped was produced the day of the sensory evaluation and appreciated by sensory panelists. panelists clearly distinguished olèlè according to the processing method. sixteen representative descriptors were identified to profile olèlè. the leaves used for wrapping olèlè had a significant effect on its sensory characteristics noted by the consumer panel with clearly distinct sensory profiles (p ≤ 0.05). the overall acceptability test revealed that the olèlè wrapped in musa paradisiaca leaves is the most appreciated, followed respectively by those wrapped with tectona grandis leaves, thalia geniculata leaves and one packaged in recycled tin cans. keywords: vigna unguiculata l., moin moin, plant leaves, sensory profile, consumer acceptability, tradition. 1. introduction cowpea (vigna unguiculata) is a wellknown leguminous species. it is a very important legume crop in tropical africa and south and central america [1]. different parts of this crop such as the young leaves, green pods and dry seeds are eaten as different food recipes [2]. among these edible parts, cowpea seeds are more important. about 6.5 million tons of them are annually harvested worldwide especially in west africa [3]. in this region as well as in brazil, whole cowpea seeds are processed (dehulled/ground or not, boiled, fried, steamed, etc.) into various dishes and widely consumed by the populations. the steamed seasoned paste called moin-moin or olèlè in benin and nigeria is one of the most important cowpea seed dishes. this dish is also known as koki in cameroon [4], and abará, abala or olelé in brazil [5]. the traditional processing methods well described by several authors [5-7] are close, except little differences. after sorting, the cowpea seeds are dehulled by a wet process (washing-soaking and removal food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 334 of cowpea seed coat in water) [5] or by a dry process (reduction of the size of dry cowpea seeds using a mortar or a grain mill and removal of hulls through winnowing) [7]. wet or dry hulled cowpea seeds are ground in a grain mill. derived paste or flour is mixed with spices (chilli pepper, garlic, ginger, pepper etc.), salt, onion, water and red palm oil to obtain a fluid dough. in addition to those ingredients mentioned above, addition of cashew nut or peanut [5] and also other ingredients such as fish and boiled egg [6] is involved. the partial substitution of ground cowpea seeds by corn and yam flours is also experienced [8-9]. whatever the raw materials and the ingredients mixed, the slurry or liquid dough obtained is packaged in pouches made from plant leaves, in aluminium foil, in polyethylene bags, in metal cans and finally steamed to give an irreversible gel-pudding. the recycled tin cans are currently used by all the producers of olèlè interviewed in benin [10]. the use of plant leaves in food preparation and packaging is characteristic of the traditional practices and their perpetuation helps to keep alive the traditions and the diversity of leaves found in tropical regions of the world [11]. for instance, leaves from the species musa sapentium, musa paradisiaca, cola nitida and thaumatococcus daniellii have been used for centuries as natural food wrappers in nigeria [12]. during the cooking of olèlè, the slurry is steamed together with its wrapping materials. when the latter are leaves, their bioactive compounds can migrate into the paste. this phenomenon may not only improve the bioactive properties of olèlè, but also influence its sensory characteristics. with the growing interest of consumers in sustainable, safer, and healthier products [13], it is essential to understand how wrapping fashions can add advantageous properties to products. several studies have been conducted on the physicochemical, proximate and on sensory characteristics of olèlè from cowpea alone or blended with other flours. however, the research on the effect of leafpackaging, on their acceptance and on sensory characteristics of olèlè affected by the processing methods has not been investigated to date. this study therefore is conducted (1) to assess the consumer’s appreciation of olèlè’s sensory characteristics as are affected by processing methods on the one hand and by leaf-wrapping on the other hand and (2) to determine the sensory profile of olèlè wrapped with plant leaves. the obtained data will help in documenting the characteristics of olèlè preferred by consumers and give useful information for the cottage industry of olèlè production. 2. materials and methods plant material white cowpea seeds, other ingredients (palm oil, pepper, chili pepper, onion, garlic and salt) and wrapping materials (glass jar, recycled tin cans and the leaves from musa paradisiaca, tectona grandis and thalia geniculata) used in this study were purchased from a local market in porto-novo, benin republic. sample preparation ten different samples of olèlè were produced the day of the sensory evaluation. the samples included two types of olèlè made by using the process involving a wet dehulling [5] and a second one involving dry dehulling of the cowpea seeds and the addition of maize flour [7]. the two type of olèlè were packaged with five different wrapping materials: glass jar, recycled tin cans (the container currently and universally used by producers of olèlè) and the leaves from musa paradisiaca, tectona grandis and thalia food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 335 geniculata. the samples of olèlè packaged in the glass jar were used to evaluate the effect of processing method and the samples packaged in recycled tin cans were chosen as a reference to appreciate the effect of leaf-wrapping. the quantity of ingredients are : red palm oil (200 g), pepper (15 g), chili pepper (45 g), onion (50 g), ginger (15 g), garlic (20 g) and salt (35 g). they were used to process 1 kg of white cowpea seeds and did not vary for the two processing methods. but the quantity of water was 2 kg and 3 kg for the process involving wet dehulling and the process involving dry dehulling. the last processing method also implied an addition of 250 g of maize flour to the mixed paste before steaming. sample presentation fresh olèlè samples (approximately three hours after steaming) were divided into eight similar portions, served in disposable polystyrene containers with lid and presented to the taster. the presentation order of the samples was randomized and each sample was coded with 3-digit random numbers. mineral water in plastic bottles were provided to the panelists to rinse their palate between sample tests. sensory difference and overall acceptability tests a total of sixty (60) panelists who were interested in the sensory evaluation of olèlè were recruited among consumer knowing the product and used to eat it at least once a week in avrankou township in the south of benin republic, where the product is largely consumed. the number of panelists involved varied according to the type of analysis. thus, 36, 50, 32 and 30 panelists were solicited respectively for a triangle test, a paired preference test, a matching test and an analysis of the overall acceptability the first two days of the sensory evaluation. the triangle test presented previously [14] was carried out to identify if there is a difference between the olèlè resulting from the two different processing methods. three coded olèlè samples steamed in glass jar were presented to the panelists. among them, two olèlè samples were the same but are coded differently. for the paired preference test described earlier [14], samples of olèlè from the two processing methods were compared to determine which product is preferred based on the color, texture, aroma and taste. to perform the matching test, the panelists simultaneously and in a randomly chosen order received four controls coded respectively by the letters a (olèlè wrapped with leaves of thalia geniculata), b (olèlè wrapped with leaves of tectona grandis), c (olèlè wrapped with musa paradisiaca leaves) and d (olèlè packaged in recycled tin cans) and pairs of samples coded by different numbers: 502 and 258 for a, 111 and 322 for b, 333 and 129 for c then 610 and 412 for d. in the guideline provided, they were asked to assess each sample independently and then match the control samples (a, b, c, and d) to the coded samples. the evaluation of the overall acceptability of olèlè packed was carried out through a ranking test already presented [14]. panelists are asked to rank the coded samples according to acceptance from least acceptable to most acceptable for each given sensory parameter. thus, texture, color, aroma, and taste of the samples are classified in terms of acceptability, giving each sample a different rating even if it seemed comparable. the sample with the most acceptable appreciation was given a rank of 1, the next a rank of 2 and the next that seemed the least acceptable, a rank of 3. based on the responses of the panelists, totals per sensory parameter per sample (t) and differences between rank total pairs food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 336 (|x y|) were calculated according to the following formulas: (1) differences between rank total pairs (|x y|) were determined as follows: |x-y| = |t(x) – t(y)|, with x and y, the samples. (2) identification and selection of descriptors for establishing a sensory profile for the establishment of the sensory profile, the panel used consisted of 12 panelists (10 women and 2 men). the panelists are selected to participate based on ability to discriminate, communication skills, and task comprehension. the panelists recruited are directly used for the final panel due to their good level of knowledge of the product. moreover, they were mainly saleswomen of olèlè and daily consumers of the product. the sensory profile of olèlè was established based on the standardized procedure [15]. during the first session, the panelists generated descriptors that could describe the taste, aroma, texture, and color of olèlè. this list is reduced by grouping together the synonymous descriptors or antonyms and eliminating those that are poorly suited to describe olèlè. during session 2, panelists evaluated four samples of olèlè differing in the wrapping (thalia geniculata leaves, tectona grandis leaves, musa paradisiaca leaves and tin cans) but produced by the processing method involving the wet dehulling. the recycled tin cans were considered as a control sample or neutral wrapping. statistical analysis the results of triangle and preference tests were analyzed using a one-tailed binomial test and a two-tailed binomial test at the level of significance of 0.05. matching test results were analyzed with the pearson's chi² test. a principal component analysis (pca) was applied using r version 4.0.2 software, to the average scores per product and per attribute to determine the correlation between the various descriptors as described in the standardized procedure [15]. the overall acceptability test results were assessed by comparing the rank totals for all possible pairs of samples using the friedman test. 3. results and discussion discernment of olèlè sensory characteristics the number of responses relating to the discernment of the olèlè from the processing method involving wet dehulling of cowpea and the olèlè from the process involving dry dehulling of cowpea and the addition of maize flour are given in table 1. table 1: number of responses relating to the discernment of olèlè according to the processing method. number of panelists sample presentation order number of correct answers 6 a a (b) 5 6 a (b) a 6 6 (b) a a 6 6 b b (a) 5 6 b (a) b 6 6 (a) b b 5 total 36 33a a: olèlè from the process involving wet dehulling of cowpea seeds; b: olèlè from the process involving dry dehulling of cowpea seeds and the addition of maize flour; bold letter in brackets: intruder a: probability of 33 correct answers in 36 trials (p=l/3) was 0.001 according to one-tailed binomial test. thirty-three (33) panelists out of 36 were able to correctly identify the different olèlè samples from the two processes. the one-tailed binomial test applied to this result indicated that the level of significance was 0.001. it was concluded that olèlè from the production process involving wet dehulling of cowpea were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 337 therefore detectably different from olèlè produced according to the process involving dry dehulling of cowpea and the addition of maize flour. the difference between the olèlè from the two processes would be linked to variations of sensory attributes in general and particularly those related to the appearance such as color and texture. indeed, the appearance is the first characteristic of a product that can be perceived by the senses [16]. the number of responses relating to the discernment of olèlè packaged with different packaging were presented in table 2. table 2: number of responses relating to the discernment of olèlè packaged in different wrapping marerials. controls samples chisquared p-value 111 (b) 129 (c) 258 (a) 322 (b) 333 (c) 412 (d) 502 (a) 610 (d) a 1 4 5 0 3 0 17 2 54 2.352e-9 b 16 0 0 16 0 0 0 0 96 2.2e-16 c 0 14 3 0 12 0 3 0 57.5 4.754e-10 d 0 4 0 0 0 16 3 9 58.5 3.006e-10 a: olèlè wrapped with the leaves of thalia geniculata; b: olèlè wrapped with the leaves of tectona grandis, c: olèlè packaged with the leaves of musa paradisiaca, d: olèlè packaged in recycled tin cans. the letters in brackets designate the samples and those without the controls. twenty two (22) out of the 32 panelists correctly matched the sample of olèlè wrapped with the leaves of thalia geniculata to the control (table 2). the pearson test revealed that there is a significant dependence between the sample of olèlè wrapped with thalia geniculata leaves and its control (khi-2 = 54, p-value = 2.352e-09 < 0.05). this could be explained by the fact that the leaves of thalia geniculata conferred on olèlè distinctive sensory characteristics from those conferred by the leaves of tectona grandis, the leaves of musa paradisiaca and recycled tin cans. among these characteristics, the most remarkable was the soft texture of the olèlè packaged in the leaves of thalia geniculata. this texture of olèlè indicated that the pores of the leaves of thalia geniculata absorb water weakly. the distinction of olèlè wrapped in the leaves of thalia geniculata and its controls from the other samples would therefore be mainly favored by the less firm texture conferred by the leaves of thalia geniculata. all the panelists correctly matched olèlè wrapped with the leaves of tectona grandis to their control. the pearson test confirms this trend (khi-2 = 96, p-value = 2.2e-16 < 0.05). the success of the pairing test of olèlè wrapped with the leaves of tectona grandis, to its controls by all the panelists could mainly be explained by the firm and hard texture and the dark color conferred by the leaves of tectona grandis to the olèlè. moreover, 26 out of the 32 panelists correctly matched olèlè wrapped with the leaves of musa paradisiaca to the corresponding control. the pearson test showed that there is a significant dependence between the olèlè packaged with the leaves of musa paradisiaca sample and its controls (khi-2 = 57.5, pvalue = 4.754e-10 < 0.05). like olèlè wrapped in the other packaging, olèlè food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 338 wrapped with the leaves of musa paradisiaca has revealed sensory characteristics which are intrinsic and specific to these leaves. the olèlè packaged in these leaves had a very firm and smooth texture. it was also noted that 25 out of the 32 panelists correctly matched olèlè packaged in recycled tin canned samples to its controls with a significant dependence between the sample and its controls (khi-2 = 58.5; p-value = 3.006e-10 < 0.05). this could be explained by a lack of specific characteristics imparted by metal packaging to the product compared with the leaves. these results are consistent with those of other researchers who indicated that the ancient practice of using leaves as wrappers during the cooking of dishes like olèlè and okpra undoubtedly has a positive impact on their tastes, flavor and overall acceptance [17]. the contribution of plant leaves to the subtle flavor of food products wrapped in them and their potential to act on human health was mentioned previously by authors [18, 19]. sensory profile of olèlè to develop the sensory profile of olèlè wrapped with leaves from different plant species or packaged in recycled tin cans, sensory descriptors have previously been researched. table 3: sensory descriptors of olèlè. sensory parameter attribute description of the attribute taste/ flavor salty major mouthfeel that sodium chloride gives cooked musa paradisiaca leaf flavor associated with steamed musa paradisiaca leaf cooked tectona grandis leaf flavor associated with steamed tectona grandis leaf cooked thalia geniculata leaf flavor associated with steamed thalia geniculata leaf garlic flavor associated with garlic pepper flavor associated with pepper chilli pepper flavor associated with chilli pepper cowpea flavor associated with cooked cowpea color red intensity of the red appearence of olèlè orange-red intensity of the orange-red appearence of olèlè orange intensity of the orange appearence of olèlè yellow orange intensity of the yellow orange appearence of olèlè yellow intensity of the yellow appearence of olèlè yellowspotted with black intensity of the yellow spotted with black appearence of olèlè texture firm which has no cracks soft which can be easily cut hard which is difficult to penetrate smooth which does not offer roughness and mark rough which has cracks and stains aroma cowpea cooking smell associated with steamed cowpea seeds pepper spicy smell associated with pepper garlic ; sulfur smell associated with garlic cooked thalia geniculata leaves; cooking smell associated with steamed thalia geniculata leaf cooked musa paradisiaca leaves; cooking smell associated with steamed musa paradisiaca leaf cooked tectona grandis leaves; cooking smell associated with tectona grandis leaf onion sulfur smell associated with boiled onion bulb ginger spicy smell associated with boiled ginger rhizome thus, a list of 58 primary descriptors (not mentioned) were generated by the panelists to describe color, aroma, texture and taste of olèlè. a grouping of synonymous or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 339 antonymous primary descriptors has led to reducing these primary descriptors from 58 to 27 (table 3) and finally to 16 representative descriptors of olèlè. a principal component analysis (pca) was applied to the average intensity scores of olèlè differently packaged and affected by attribute to delineate the combinations between the olèlè samples and their representative descriptors. the results of this analysis (figure 1) indicated that the first two principal components (pc1 and pc2) respectively explained 46.47% and 31.34%, i.e. a total of 77.81% of the variations, which is sufficient to guarantee the interpretation precision. fig. 1: circle of correlations of the sensory descriptors and projection of olèlè in the axis systems (pc1 and pc2) (a) and (pc1 and pc3) (b). teakaro: aroma of cooked tectona grandis leaves ; thaliaaro: aroma of cooked thalia geniculata leaves ; teakflav : taste of cooked tectona grandis leaves ; thaliaflav: taste of cooked thalia geniculata leaves; htex: hard texture ; ftex: firm texture ; redcol: red color ; yelorcol: yellow-orange color; yelcol: yellow color; redyelcol : red-yellow color; flavcowpea : cooked cowpea flavor; aroon : aroma of onion; arogin: aroma of ginger; flavchi : taste of chilli pepper; banaaro: aroma of cooked musa paradisiaca leaves ; yelcolsb: yellow spotted with black ; couo : orange color ; couro : red-orange color; smoothapp: smooth appearance; stex : soft texture; banaflav: taste of cooked musa paradisiaca leaves ; othalia: olèlè packaged with the leaves of thalia geniculata; oteak: olèlè wrapped with the leaves of tectona grandis; obanana: olèlè wrapped with the leaves of musa paradisiaca; otin: olèlè packaged in recycled tin cans; pca: principal component analysis; pc1: first principal component; pc2: second principal component ; pc3: third principal component. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 340 however, the correlation analysis indicated that the descriptors: taste of cooked thalia geniculata leaves, yellow color, and aroma of cooked thalia geniculata leaf were not taken into account by either of these first two principal components (pc1 and pc2), but rather by the third (pc3) (figure 1). as a result, the first three main components comprising 100% of the initial variations will be retained to describe the links between the descriptors and olèlè differently packaged. on pc3, descriptors such as taste of cooked thalia geniculata leaf (thaliaflav), yellow color (yelcol), aroma of cooked thalia geniculata leaf (thaliaaro), and yellow spotted with black color (yelcolsb) described olèlè wrapped with the leaves of thalia geniculata. the sensory profile (figure 2) developed on the basis of pca results includes 16 sensory descriptors describing the color, aroma, texture and taste of olèlè wrapped with different packaging. it should be noted that strongly correlated and less discriminating descriptors were not considered. these included: salty taste, taste of pepper, aroma of pepper, flavor of garlic and aroma of garlic. olèlè wrapped by musa paradisiaca leaves had a taste of cooked musa paradisiaca leaf; color red orange; characteristic aroma of cooked musa paradisiaca leaf; hard, firm and soft texture and a smooth appearence (figure 2a). olèlè wrapped with tectona grandis leaves revealed a characteristic taste of cooked tectona grandis leaves; yellow spotted with black, aroma of cooked tectona grandis leaves, a hard and firm texture (figure 2b). fig. 2: profile of olèlè wrapped with the leaves of musa paradisiaca (a), tectona grandis (b), thalia geniculata (c) and recycled tin cans (d). teakaro: aroma of steamed tectona grandis leaves ; thaliaaro: aroma of steamed thalia geniculata leaves ; teakflav : taste of cooked tectona grandis leaves ; thaliaflav: taste of cooked thalia geniculata leaves; htex: hard texture ; ftex: firm texture ; flavcowpea : cooked cowpea flavor; arogin: ginger flavor; flavchi : taste of chilli pepper; banaaro: aroma of steamed musa paradisiaca leaves ; yelcolsb: yellow spotted with black ; couo : orange color ; couro : red-orange color; smoothapp: smooth appearance ; stex : soft texture; banaflav: flavor associated with steamed musa paradisiaca leaves. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 341 olèlè packaged using the leaves of thalia geniculata presented a characteristic taste of cooked thalia geniculata leaf; a color orange; aroma of cooked thalia geniculata leaf; a hard, firm and soft texture that is little noticed (figure 2c). olèlè packaged in recycled tin cans showed a characteristic, more intense chili taste; red orange color; ginger aroma; a soft texture slightly noticed (figure 2d). consumer acceptability of olèlè the total number of panelists preferring olèlè from one processing method over the other varied according to the sensory parameters (figure 3). fig. 3: judgment of olèlè from two processing methods according to sensory parameters. overall, 37 out of the 50 panelists preferred the color of olèlè sample from the processing method involving the wet dehulling of cowpea seeds. the statistical table of a two-tailed binomial test showed that the probability for x= 37 and n = 50 was 0.001 where x represents the number of panelists preferring a sample and n represents the total number of panelists participating in the test. therefore, the color of olèlè from the process involving the wet dehulling of cowpea seeds was significantly preferred to the color of olèlè from the process involving the dry dehulling of cowpea seeds and addition of the maize flour. indeed, the two operations, namely the dry dehulling of cowpea seeds and the addition of the maize flour led to a color of olèlè not appreciated by the consumers panel. moreover, at least 40 out of the 50 panelistss preferred the taste and the texture of olèlè from the process involving the wet dehulling of cowpea seeds. the statistical table of a two-tailed binomial test showed that the probability for x= 40 and n = 50 is 0.001. consequently, the taste and the texture of olèlè from the processing method involving the wet dehulling of cowpea seeds were significantly preferred to the taste and the texture of olèlè from the process involving a dry dehulling of cowpea seeds and an addition of the maize flour. likewise, the statistical table of a twotailed binomial test indicated that the probability for x= 39 panelists preferring the aroma of olèlè from the processing method involving wet dehulling of the cowpea seeds out of 50 panelists participating in the test was 0.001. therefore, the aroma of olèlè from the process involving the wet dehulling of cowpea seeds was significantly preferred for the aroma of olèlè as compared to the process involving the dry dehulling of cowpea seeds. the same trend was observed for the color, taste and texture of olèlè from the processing method involving the wet dehulling of cowpea seeds which were significantly preferred by the majority of panelists (p ≤ 0.05). as the similar ingredients used to produce olèlè were in the same proportions for the two processing methods, it can be concluded that the maize flour and the dry dehulling used to produce the second type of olèlè could be the cause of its rejection. a ranking test was conducted to compare four differently wrapped olèlè produced according to the processing method food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 342 involving wet dehulling based on the taste, the color, the aroma and the texture. the ranked values given to each olèlè sample were totalized and the calculated differences between rank total pairs were presented in table 4. table 4: difference between rank total pairs of packaged olèlè compared according to sensory parameters. sensory parameters differences between rank total pairs |a b| |ac| |a d| |b c| |b d| |c d| taste 3 6 29 3 32 35 color 16 6 42 22 26 48 texture 14 24 34 38 20 59 aroma 31 22 39 53 8 61 a: olèlè wrapped with the leaves of thalia geniculata; b: olèlè wrapped with the leaves of tectona grandis; c: olèlè packaged in recycled tin cans; d: olèlè wrapped with leaves of musa paradisiaca. the tabulated critical value at 5% level of significance for 30 panelists and four samples from the friedman test table was 26. the differences between rank totals of a: olèlè wrapped with thalia geniculata leaves and b: olèlè wrapped with tectona grandis leaves (a and b) and a and c: olèlè packaged in recycled tin cans (a and c) regarding the taste, the color and the texture were below the critical value of 26. this shows that the acceptability of aroma of a and that of b were significantly different (p ≤ 0.05). meanwhile, the sensory characteristics acceptability of a and b on the one hand and a and c on the other hand was not significantly different (p > 0.05). also, the acceptability obtained for the taste and the color of b and c and for the texture and the aroma of b and d did not differ significantly. the differences between rank totals of a and d on the one hand and c and d on the other hand corresponding to taste, color, aroma and texture were above the critical value of 26. so, the acceptability of the sensory characteristics of the aforementioned olèlè pairs (a and d) and (c and d) was significantly different (p ≤ 0.05). the same trend was observed for the aroma and the texture of olèlè wrapped with tectona grandis leaves and olèlè packaged in recycled tin cans and for the taste, the color and the aroma of olèlè wrapped with tectona grandis leaves and olèlè wrapped with musa paradisiaca leaves. the panelists found the sensory characteristics of olèlè wrapped with musa paradisiaca leaves more acceptable than those of olèlè wrapped with thalia geniculata leaves, tectona grandis leaves and in the tin cans. 4. conclusion the effect of the processing method on the one hand and the effect of the plant leaves used for the wrapping on the other hand on sensory characteristics of olèlè were highlighted in this study. olèlè obtained from the processing method involving wet dehulling of the cowpea seeds is more appreciated than that from the method involving dry dehulling of the cowpea seeds and the addition of the maize flour from the point of view of color, taste, aroma and texture. the sensory characteristics of olèlè varied depending on the material used for wrapping. the difference between the olèlè wrapped with different plant leaves was clearly noticeable by the panelists. the sensory profiles established have shown that leafwrapping has a remarkable influence on food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 343 the taste, color, texture and aroma of olèlè. although the overall acceptability test revealed that olèlè wrapped in musa paradisiaca leaves is the most appreciated, followed by olèlè wrapped with tectona grandis leaves, the one wrapped with thalia geniculata leaves and finally olèlè packed in tin cans, each olèlè wrapped with a different wrapping can have its niche or consumer segment. other studies on sensory characteristics and consumer acceptance of various olèlè produced by blending cowpea with other cereals or tubers powders will be interesting. 5. acknowledgments the authors gratefully thank the sensory panelists who collaborated on this study, the students mardochée deguenon and victor houngbo and the world academy of sciences (twas) and swedish international development cooperation agency (sida) for supporting the research through the grant 19-081 rg/che/af/ac_i – fr3240310154. 6. references [1]. amonsou e.o., houssou p.a., sakyidawson e., saalia f.k., dehulling characteristics, sensory and functional properties of flours from selected cowpea varieties. journal of the science of food and agriculture, 89: 1587– 1592, (2009). [2]. singh b. b., ehlers j. d., sharma b., freire-filho f. r., recent progress in cowpea breeding. in c. a. fatokun, s. a. trawali, b. b. singh, p. m. kormawa, & m. tamo (eds.), challenges and opportunities for enhancing sustainable cowpea production, nigeria: iita, ibadan, (2002) 22–40. [3]. ezin, v., tosse, a. g. c., chabi, i. b., ahanchede, a., adaptation of cowpea (vigna unguiculata (l.) walp.) to water deficit during vegetative and reproductive phases using physiological and agronomic characters. international journal of agronomy, (2021). https://doi.org/10.1155/2021/9665312 [4]. kaptso g., njintang n., nguemtchouin m., amungwa a., scher j., hounhouigan j., mbofung c., characterization of morphology and structural and thermal properties of legume flours: cowpea (vigna unguiculata l. walp) and bambara groundnut (vigna subterranea l. verdc.) varieties. international journal of food engineering, 12: (issue 2), 139-152 (2016). https://doi.org/10.1515/ijfe-2014-0146 [5]. cardoso l. a., greiner r., silva c. de s., maciel l. f., santos l. f. p., de almeida deusdelia t., small scale market survey on the preparation and physico-chemical characterstics of moin-moin: a traditional ready-toeat cowpea food from brazil. food sci. technol, campinas, 42, e59920, (2022) doi: https://doi.org/10.1590/fst.59920 [6]. madodé y.e., houssou p.a., linnemann a.r., hounhouigan d.j., nout m.j.r., van boekel m.a.j.s., preparation, consumption, and nutritional composition of west african cowpea dishes. ecology of food and nutrition, 50: 115–136, (2011). [7]. time g.p.d., aïssi m.v., houngbo y.v., chabi i.b., kpoclou y.e., portrait des procédés traditionnels de production du olèlè, une pâte de niébé cuite consommée au bénin. les cahiers du cbrsi, agriculture environnement et sciences de l’ingénieur, 19: 160-180, (2021). [8]. hussein j.b., ilesanmi j.o.y., aliyu h.m, akogwu v., chemical and sensory qualities of moimoi and akara produced from blends of cowpea (vigna unguiculata) and moringa oleifera seed flour. nigerian journal of technological research 15: (3), 15-23 (2020). [9]. otunola g. a., afolayan a. j., evaluation of the physicochemical, proximate, and sensory properties of moinmoin from blends of cowpea and water yam flour. food science & nutrition, 1–8, (2018). [10]. time g.p.d., aïssi m.v., deguenon m., chabi i.b., kpoclou y.e., ballogou v.y., diversité et raisons de choix des emballages utilisés pour le conditionnement du olèlè, une pâte cuite de niébé. science et technique, sciences naturelles et appliquées, spécial hors-série 6 : 205-220 (2022). [11]. ng c., plant leaves in food preparation and packaging. utar agriculture science journal, 1: (4), 34-39 (2015). [12]. adegunloye d.v., agarry o.o., adebolu t.t., adetuyi f.c., effect of leaf packaging on the microbiological assessment of https://doi.org/10.1155/2021/9665312 https://doi.org/10.1515/ijfe-2014-0146 https://doi.org/10.1590/fst.59920 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 m. vahid aïssi, g. paul daniel time, vénérande y. ballogou, ifagbémi bienvenue chabi, y. euloge kpoclou, mohamed m. soumanou, effect of traditional processing method and leaf-packaging on sensory characteristics of olèlè, a steamed cowpea paste, food and environment safety, volume xxi, issue 4 – 2022, pag. 333 – 344 344 some food items. african journal of biotechnology, 5: (5), 445-447, (2006). [13]. da silva rios d. a., nakamoto m. m., braga a. r. c., da silva e. m. c., food coating using vegetable sources: importance and industrial potential, gaps of knowledge, current application, and future trends. applied food research, 2: (1), 100073, (2022). https://doi.org/10.1016/j.afres.2022.100073. [14]. watts b.m., ylimaki g.l., jeffery l.e., elias l.g., basic sensory methods for food evaluation. ottawa, ont., idrc, 1989. x + 160 p.: ill. (1991). [15]. standard iso 11035. sensory analysis identification and selection of descriptors for establishing a sensory profile by a multidimensional approach, (1994). https://standards.iteh.ai/catalog/standards/sist/9332 ddf3-8396-449b-8dc7-208abd767576/iso-11035199 [16]. derndorfer e. colours and their influences on sensory perception of products. dlg expert report 3/(2017). https://www.dlg.org/en/food/topics/dlg-expertreports/sensory-technology/dlg-expert-report-32017 consulted on 12/07/2022. [17]. ibegbulem c.o., ezeibekwe i.o., ifekwe n.p., effects of pandenus candelabrum and musa paradisiaca leaf packagings on level of antinutrients in some nigerian local delicacies. journal of agriculture and food sciences, 2: (1), 25-31, (2004). [18]. lin f., luo b., long b. and long c. plant leaves for wrapping zongzi in china: an ethnobotanical study. journal of ethnobiology and ethnomedicine, 15 (63), 1-16, (2019). [19]. kora a. j. leaves as dining plates, food wraps and food packing material: importance of renewable resources in indian culture. bulletin of the national research centre, 43: (205), 1-15 (2019). https://doi.org/10.1016/j.afres.2022.100073 https://standards.iteh.ai/catalog/standards/sist/9332ddf3-8396-449b-8dc7-208abd767576/iso-11035-199 https://standards.iteh.ai/catalog/standards/sist/9332ddf3-8396-449b-8dc7-208abd767576/iso-11035-199 https://standards.iteh.ai/catalog/standards/sist/9332ddf3-8396-449b-8dc7-208abd767576/iso-11035-199 https://www.dlg.org/en/food/topics/dlg-expert-reports/sensory-technology/dlg-expert-report-3-2017 https://www.dlg.org/en/food/topics/dlg-expert-reports/sensory-technology/dlg-expert-report-3-2017 https://www.dlg.org/en/food/topics/dlg-expert-reports/sensory-technology/dlg-expert-report-3-2017 1. introduction the tabulated critical value at 5% level of significance for 30 panelists and four samples from the friedman test table was 26. the differences between rank totals of a: olèlè wrapped with thalia geniculata leaves and b: olèlè wrapped with tectona gra... 4. conclusion doi: https://doi.org/10.4316/fens.2021.016 149 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 2 2021, pag.149 159 the effects of sugar and fat substitution on the textural properties of the pie dough *dana huțu 1 , sonia amariei 1 1faculty of food engineering , stefan cel mare university of suceava, romania *corresponding author received 6th june 2021, accepted 29th june 2021 abstract: understanding the current technologies related to the food industry, which deal with the reduction of sugar in processed foods, research made on the subject and the consumers response to the reduction of sugar in food, has become a very important factor for food producers in the mission, to design and produce low sugar products. due to the multiple functions that sugar has in food, reducing it is a difficult task in itself, but progress is still being made in order to make the products acceptable to future consumers. in this quest, the process of identifying ways to incorporate apple puree as a healthy food ingredient in a diet may be a solution because it could offer many advantages for a healthy diet, mainly due to the reduction of sugar and respectively the fat, beneficial for both categories: adults and especially children, whose health may be affected by added sugar from pastries, for instance. in addition, apple puree brings an increased nutritional value to bakery and pastry products, through its composition which has a rich content of nutrients. apple puree could be considered an alternative source of dietary fiber needed for muscle strengthening and permanent burns in the body activity, which can be included in the daily diet a s a special food ingredient used in pastries. therefore, the potential exploitation of apple puree in the industry as a healthy food ingredient, due to its benefits, is higher ranked, with a high degree of interest, being more and more common in the bakery industry and selected functional foods, which allows it to have a promising future in this industry. keywords: apple puree,sugar, fat, substitution, texture, dough development 1. introduction there is an alarming increase in obesity and diseases related to obesity such as diabetes heart diseases and hypertension in developing children.although the risks of obesity can be increased due to genetics and environmental factors, the most frequent cause of obesity is energy imbalance [1]. sugar is high in calories and is found in a wide variety of foods and beverages. reducing or removing sugar from confectionery is an important research objective for the food industry,considering information as an important factor in consumer awareness of the diseases of civilization and the gouvernment's strategies for reducing sugar in high-sugar products [2]. in europe, some gouvernments have implemented taxes and recommendations for producers and consumers of sucroserich foods. for example, the irish gouvernment introduced a sugar tax in 2018, which increased the price of http://www.fia.usv.ro/fiajournal https://www.reverso.net/translationresults.aspx?lang=ro&sourcetext=exist%c4%83%20o%20cre%c8%99tere%20alarmant%c4%83%20a%20obezit%c4%83%c8%9bii%20%c8%99i%20a%20bolilor%20legate%20de%20obezitate%20cum%20ar%20fi%20diabetul%20zaharat,%20bolile%20de%20inim%c4%83%20%c8%99i%20hipertensiunea%20arterial%c4%83%20%c3%aen%20cazul%20copiilor%20afla%c8%9bi%20%c3%aen%20curs%20de%20dezvoltare.&action_form=translate&direction_translation=rum-eng-5 https://www.reverso.net/translationresults.aspx?lang=ro&sourcetext=exist%c4%83%20o%20cre%c8%99tere%20alarmant%c4%83%20a%20obezit%c4%83%c8%9bii%20%c8%99i%20a%20bolilor%20legate%20de%20obezitate%20cum%20ar%20fi%20diabetul%20zaharat,%20bolile%20de%20inim%c4%83%20%c8%99i%20hipertensiunea%20arterial%c4%83%20%c3%aen%20cazul%20copiilor%20afla%c8%9bi%20%c3%aen%20curs%20de%20dezvoltare.&action_form=translate&direction_translation=rum-eng-5 https://www.reverso.net/translationresults.aspx?lang=ro&sourcetext=exist%c4%83%20o%20cre%c8%99tere%20alarmant%c4%83%20a%20obezit%c4%83%c8%9bii%20%c8%99i%20a%20bolilor%20legate%20de%20obezitate%20cum%20ar%20fi%20diabetul%20zaharat,%20bolile%20de%20inim%c4%83%20%c8%99i%20hipertensiunea%20arterial%c4%83%20%c3%aen%20cazul%20copiilor%20afla%c8%9bi%20%c3%aen%20curs%20de%20dezvoltare.&action_form=translate&direction_translation=rum-eng-5 food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 150 beverages with a sugar content of > 5 g / 100 ml of beverage.in the uk, public health england has launched an action plan to combat childhood obesity. this strategy aims to encourage the food industry to reduce the sucrose content of a variety of products by 20% by 2020. these initiatives put pressure on the food industry to produce low-sucrose products, while consumers continue to require high quality products [2]. “added sugar” refers to sugar added to food before consumption, during preparation or during processing to enhance the flavour or texture of food, as opposed to the sugar inherent in a product [3]. added sugar and sugar naturally present in food do not differ chemically, but there is a great health concern about added sugar due to the addition of unnecessary calories [4]. in the last two decades, consumer focus on health has increased, leading to significant pressure for healthier food choices [5-7]. consumers generally have basic knowledge of nutrition when it comes to sugar, but what differentiates a healthy consumer from an unhealthy consumer is how they use their nutritional knowledge to make food choices [8]. many parents want low-sugar products for their children, and current studies emphasize the importance of developing healthy eating habits at an early age [9,10]. sigman-grant and hsieh (2005) found that consumers who selected low-sugar versions or a combination of low-sugar and whole-sugar products generally had more favorable diets [11]. apple puree contains a high concentration of dietary fiber, manufactured as a byproduct in the apple juice and cider industry. it has been used to increase the fiber content of pastries [12, 13]. it has been reported that the addition of apple puree can increase the perceived sweetness of some pastries [13]. apples are one of the most frequently consumed fruits, reaching an intake of about 20-30 kg a year for each citizen. apples come in over 7,500 varieties worldwide and are generally a good source of a variety of nutrients and non-nutrients [19]. in addition to simple carbohydrates mainly sugars apples are rich in vitamin c, a number of minerals, especially potassium, and also non-essential constituents such as polyphenols, fiber and additional phytochemicals such as triterpenes and phytosterols. many of these constituents have been associated through their mechanism of action and also in epidemiological studies with antiinflammatory and antioxidant properties, lowering the risk of developing diseases such as diabetes and cardiovascular complications, many of which are increasing. it is believed that apples contribute to a healthy diet, and the old saying "an apple a day keeps the doctor away" is believed to still contain a lot of wisdom [14]. 2. matherials and methods the determination of the texture profile of the pie dough using the perten tvt-6700 texturometer (perten instruments, sweden) was performed by applying a double compression of up to 50% of the initial sample height, using a cylindrical probe with a diameter of 45 mm., at a speed of 5.0 mm / s, a force of 20 g and a recovery period between compressions of 12 s [15]. in order to evaluate the texture of the pie dough, spheres of 50 g of dough were made which were then subjected to the double compression test, up to 50% of the initial height, using the perten tvt-6700 texturometer (perten instruments, sweden). and a cylindrical probe with a diameter of 35 mm, with the application of a speed of 5.0 mm / s, a force of 20 g and a recovery period between compressions of 12 s [16]. food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 151 the fermentation behavior of the pie was analyzed using the chopin reofermentometer (chopin rheo, type f4, villeneuve-la-garenne cedex, france) [17] according to chopin protocol by testing a pie dough sample under 2000 g cylindrical weight constraint at 30°c for 3 h. for this purpose, pie dough samples were mixed in a consistograph device. to 250 g sample 5 g salt and 7 g yeast of saccharomyces cerevisiae type were added. the dough development curve parameters such as maximum development reached by the dough, hm (mm) and the curve height of the end of the test, h (mm) were determined. also the gas production curve parameters were analyzed such as: total volume of gas production, (ml) total volume of carbon dioxide lost (ml), volume of carbon dioxide retention (ml), and retention coefficient (%). 3. results and discussion texture profile analysis texture profile analysis (apt) allows objective measurements of food texture parameters and is a major influencing factor on food acceptability. apt was designed as a two-cycle compression performed to simulate successive chewing (tuoc and glasgow, 2018). in addition, this test allows the evaluation of the texture parameters of the product such as cohesiveness, adhesiveness, firmness, fracturability both in various stages of the technological process and in the finished product stage [18]. sample preparation: m-sample obtained according to the manufacturing recipe without substitution of sugar and fat, p1 sample obtained by substituting 10% of the amount of sugar and fat, p2 sample obtained by substituting 20% of the amount of sugar and fat, p3 sample obtained by substituting 30% of the sugar and fat content, p4 sample obtained by substituting 40% of the amount of sugar and fat, p5 sample obtained by substituting 50% of the amount of sugar and fat [19]. the samples obtained by substituting 10%, 20%, 30% of the amount of sugar and fat with apple puree showed significantly lower hardness values than the control sample, instead, the hardness values of the samples obtained by replacing 40%, respectively 50% were approximately similar to those of the control sample. at the same time, in the case of stickiness, the samples with a lower percentage of sugar and fat substitution with apple puree showed values close to those of the control sample, on the other hand, the samples with a higher percentage of substitution showed lower values than witness. the elasticity may vary depending on the quality of the flour, the components of the dough and their proportion.as such, the values obtained for the elasticity of the samples varied from 0.9981 to 1.0011, the minimum extreme value being also the closest to the value of the elasticity of the control sample. the samples obtained by substituting 20%, respectively 50% of the amount of sugar and fat with apple puree showed a significantly higher elasticity than that of the control sample. in contrast, the samples with a substitution of 10% and 30% showed an elasticity closer to that of the control sample, while the sample obtained by substituting 40% of the amount of sugar and fat with apple puree had a higher elasticity, smallbut closest in value to the elasticitycontrol. the fibers present in apple puree contain – oh groups in the structure that can favour higher interactions with water through hydrogen bonds. due to the water solubility of sugars present in apple puree, the cohesiveness of the dough can be influenced. as a measure of the strength of the internal bonds that maintain the shape of the sample [20], the cohesiveness varied food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 152 between 1.04 and 1.58, extreme values corresponding to the control sample, respectively the sample obtained by substituting 20% of the amount of sugar and fat. substituting fat and sugar with apple puree has led to a higher cohesiveness of the dough, especially in the case of samples obtained by substituting 20% and 30% of the amount of sugar and fat in apple puree. fig.1. graphical representation of pie dough texture parameters: m-sample obtained according to the manufacturing recipe without substitution of sugar and fat, p1 sample obtained by substituting 10% of the amount of sugar and fat, p2 sample obtained by substituting 20% of the amount of sugar and fat, p3 sample obtained by substituting 30% of the sugar and fat content, p4 sample obtained by substituting 40% of the amount of sugar and fat, p5 sample obtained by substituting 50% of the amount of sugar and fat food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 153 dough rheological properties during fermentation the rheological properties of the dough during fermentation were determined using the rheofermentometer device. for the maximum height of gaseous production and the curve height of the end of the test parameters have been analyzed its behaviour during the 180 minutes fermentation period for every 30 minutes. table 2. rheofermentometer parameters of pie dough samples sample hm (mm) h (mm) h’m (mm) the total volume of gas production (ml) the volume of carbon dioxide lost (ml) volume of carbon of dioxide retention (ml) retention coefficient (%) control 43.2 37.6 52.8 1137 101 1036 91.1 sample 1 55.1 55.0 60.3 1084 3 1081 99.7 sample 2 57.2 57.1 57.7 1093 2 1091 99.8 sample 3 49.0 47.7 71.2 1431 24 1407 98.3 sample 4 41.1 39.9 47.1 905 3 902 99.7 sample 5 45.6 45.5 59.0 1087 4 1083 99.6 fig. 2. graphical representation of the total volume of gas removed the fermentation activity of yeast expressed by carbon dioxide emission is more intense in the dough in which 30% of the sugar and oil have been replaced. this being 20.55%, 24.25%, 23.62%, 36.76% and 24.04% higher than the control sample, sample 1, sample 2, sample 4 and sample 5 respectively. the maximum height of gaseous production during fermentation period are shown in the table 3. food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 154 table 3. maximum height of gaseous production values during fermentation period for the pie dough sample gas production is constantly increasing for all. this is due to the use of the old fermentable ingredients present in the dough. fig. 3. graphical representation of the maximum height of gaseous production during fermentation period for the pie dough sample the maximum height of gas production reaches maximum values in any of the moments of determination for the period of 3 hours in the case of the sample obtained by substituting 30% of the amount of sugar and fat, reaching the maximum value at the end of the fermentation period. the minimum values were recorded for the sample obtained by substituting 40% of the amount of sugar and fat. the samples obtained by substituting 10% and 20% of the amount of sugar and fat are those which show a fermentation activity similar to the control sample. sample maximum height of gaseous production, h’m (mm) time 30´ 60´ 90´ 120´ 150´ 180´ m 26.6 40.4 46.8 50.5 52.8 52.2 p1 12.6 32.7 44.7 50.7 56.4 60.3 p2 15.2 35.4 44.8 50 55 57.7 p3 29.6 49.9 58 63.3 66.1 71.2 p4 17.5 28 34.8 40.2 44.1 47.1 p5 13.6 32.9 44.8 51.5 56.4 59.0 food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 155 table 4. the curve height of the end of the test values during fermentation period for the pie dough samples at first the entire amount of gas produced is retained by the dough and the height increases rapidly. during fermentation. the hm value increases more slowly to a maximum value after which it stops. even if the production of gas continues during the fermentation only part of them are retained by the dough. the rest is lost. also. the value of the maximum growth height of the dough is correlated with the volume of gas dioxide lost. therefore. the dough made with 16.66g of sugar. 4.16g of puree and 33.32g of oil had a value of 57.2 mm. fig. 4. graphical representation of the curve height of the end of the test sample the curve height of the end of the test, h (mm) time 30´ 60´ 90´ 120´ 150´ 180´ m 6.3 17.9 30.6 40.4 43.2 37.6 p1 1.7 10 24 39.7 51 55 p2 2.5 11.9 26.2 41.7 54.4 57.1 p3 6.5 21.7 39.4 48.5 48.9 47.7 p4 4.2 12.7 24.1 35.8 40.6 39.9 p5 0.7 9 23.2 38.2 44.5 45.5 food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 156 food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the textural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 157 food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the text ural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 158 4. conclusion replacing some quantity of sugar and fat with apple puree has led to an increase in the nutritional. functional and technological value of pie. the nutritional value has increased due to the content of nutrients. especially fiber. a reduction of sugar between 10 and 50% was achieved in 5 samples of pie dough. the use of apple puree as an ingredient in substituting a percentage of the amount of sugar and fat produced a dough with physicochemical. textural. rheological properties and with fermentative behaviour approximately as the control sample depending on the percentage of sugar and fat substituted. therefore. for textural characteristics such as hardness. stickiness. elasticity. gumminess. chewability. adhesiveness and stiffness. the sample obtained by substituting 30% of the amount of sugar and fat is closest to the control sample. dough hardness. elasticity and cohesiveness were improved compared to the control sample. for the samples whose substitution was 10-30% of the amount of sugar and lipycositis fat. the gumminess and chewability kept their values around the control sample. therefore. the textural characteristics were improved for the sample obtained by substituting 30% of the amount of sugar and fat. both the total volume of co2 production and the maximum development reached by the dough, increased with the increase of the amount of sugar substituted. the maximum value for the total volume of co2 were recorded for the sample with a substitution of 30% of the amount of sugar and fat. 5. references [1]. o’sullivan. m.. & downey. l. (2017). sugar-refining. archaeology ireland. 31(4). 49-52. sigman‐grant. m. j.. & hsieh. g. (2005). reported use of reduced‐sugar foods and beverages reflect high‐quality diets. journal of food science. 70(1). s42-s46. [2]. mironeasa. s.. zaharia. d.. codină. g. g.. ropciuc. s.. & iuga. m. (2018). effects of grape peels addition on mixing. pasting and fermentation characteristics of dough from 480 wheat flour type. bull. uasvm food sci. technol. 75. 27. [3]. anton. s. d.. martin. c. k.. han. h.. coulon. s.. cefalu. w. t.. geiselman. p.. & williamson. d. a. (2010). effects of stevia. aspartame. and sucrose on food intake. satiety. and postprandial glucose and insulin levels. appetite. 55(1). 37-43. [4]. chen. m. f. (2011). the joint moderating effect of health consciousness and healthy lifestyle on consumers’ willingness to use functional foods in taiwan. appetite. 57(1). 253-262. [5]. chan. k.. prendergast. g.. grønhøj. a.. & bech‐larsen. t. (2011). danish and chinese adolescents' perceptions of healthy eating and attitudes toward regulatory measures. young consumers. [6]. küster. i.. & vila. n. (2017). healthy lifestyle and eating perceptions: correlations with weight and low-fat and low-sugar food consumption in adolescence. frontiers in life science. 10(1). 48-62. [7]. thompson. a. k.. & moughan. p. j. (2008). innovation in the foods industry: functional foods. innovation. 10(1). 61-73. [8]. yeon. j. y.. & lee. s. k. (2016). perception of sugar reduction. nutrition education. and frequency of snacking in children by the selfperceived sweet dietary habits of mothers in busan. nutrition research and practice. 10(5). 546. [9]. bohn. t.. & bouayed. j. (2020). apples: an apple a day. still keeping the doctor away?'. in nutritional composition and antioxidant properties of fruits and vegetables (pp. 595612). academic press. [10]. byrd-bredbenner. c.. mckinley. j.. martin-biggers. j.. & gager. e. food and environmentsafety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 2 – 2021 dana huțu, sonia amariei, the effects of sugar and fat substitution on the text ural properties of the pie doug, food and environment safety, volume xx, issue 2 – 2021, pag. 149 – 159 159 (2016). encouraging reductions in sugarsweetened beverage consumption: homestyles best drinks for families guide. journal of nutrition education and behavior. 48(7). s11-s12. [11]. struck. s.. gundel. l.. zahn. s.. & rohm. h. (2016). fiber enriched reduced sugar muffins made from iso-viscous batters. lwt-food science and technology. 65. 32-38. [12]. masoodi. f. a.. sharma. b.. & chauhan. g. s. (2002). use of apple pomace as a source of dietary fiber in cakes. plant foods for human nutrition. 57(2). 121-128. [13]. sudha. m. l.. baskaran. v.. & leelavathi. k. (2007). apple pomace as a source of dietary fiber and polyphenols and its effect on the rheological characteristics and cake making. food chemistry. 104(2). 686692. [14]. bohn. t.. & bouayed. j. (2020). apples: an apple a day. still keeping the doctor away?'. in nutritional composition and antioxidant properties of fruits and vegetables (pp. 595-612). academic press. [15]. iuga. m.. & mironeasa. s. (2020). potential of grape byproducts as functional ingredients in baked goods and pasta. comprehensive reviews in food science and food safety. 19(5). 2473-2505. [16]. ungureanu-iuga. m. (2020). byproducts ingredients in corn based pasta: effects on the technological and quality characteristics. scientific bulletin. series f. biotechnologies. 24(1). 97-106. [17]. rheofermentometer. c. (2005). tripette & renaud chopin. [18]. voinea. a.. stroe. s. g.. & codină. g. g. (2020). the effect of sodium reduction by sea salt and dry sourdough addition on the wheat flour dough rheological properties. foods. 9(5). 610. [19]. huțu d.. amariei s. (2021). high nutritional pastries for students in the context of current education dissertation paper. [20]. trinh. k. t.. & glasgow. s. (2012. september). on the texture profile analysis test. in proceedings of the chemeca (pp. 2326). [21]. sigman‐grant. m. j.. & hsieh. g. (2005). reported use of reduced‐sugar foods and beverages reflect high‐quality diets. journal of food science. 70(1). s42-s46 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2021.027 247 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 3 2021, pag. 247 255 extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power lahouaria labga 1 , ghania tiboura 2 , fatima zohra chenni 1 , dounia djebbouri 1 , nadjet ouafi 1 , souad saidani 1 , *samira meziani 1 1faculty of life science and nature, laboratory of biotoxicology, djillali liabes university, algeria * meziani_samira@yahoo.fr 2 faculty of life science and nature, laboratory of nutrition, pathology, agro biotechnology and health, djilali liabes. university of sidi-bel-abbes, algeria *corresponding author received 23th june 2021, accepted 29th september 2021 abstract: wheat is the second most consumed cereal for human consumption after rice due to the presence of high calories. wheat germ (2-3% of the grain) can be separated as a by-product when milling wheat. it is considered an important by-product and can be used in various applications such as food, and other biological purposes. it is considered a nutrient rich in vitamin and antioxidant included, vitamin b, minerals, polyphenols and flavonoids. the goal of our work is to estimate the total polyphenol composition and the evaluation of antioxidant potential present in pure and industrial common wheat germ. the manual dissection was carried out to produce pure wheat germ and based on the co-product of the flour mill for the treated wheat germ. the results obtained show that the total polyphenols represent values of 0.074 ± 0.001 mg (eag) / g and 0.062 ± 0.001 mg (eag) / g for the pure wheat germ and the treated wheat germ, respectively. a similarity in flavonoids was noted for the two varieties of common wheat (pure and treated) with a value of 0.022 ± 0.002 mg of ec / g dm. the evaluation of the antioxidant activity, show that the treated wheat germ has the best activity against the radical dpph with an ic50= 0.051 mg / ml compared to the pure wheat germ with ic50 = 0.361 mg / ml. wheat germ can be used as a protective agent, as a rich source of bioactive compounds with beneficial health effects. keywords: common wheat, wheat germ pure, polyphenols, antioxydant activity 1. introduction cereals play an important role in human nutrition, either for baking or as a raw material for baking flour. botanically, they belong to the gramineae family, which includes wheat, maize, rice, oats, etc., and are used in the production of flour, barley, millet, sorghum and rye. wheat is one of the main cereals and food ingredients in the world because of its ability to be milled into flour. wheat is an omnipresent cereal in the mediterranean diet due to its energetic and nutritional value. nowadays, western countries are more and more interested in adding a functional ingredient naturally enriched with bioactive molecules, interesting to incorporate in food formulas. cereals are considered as a main source of human and animal nutrition [1] according to [2] their production reaches up to 2 billion tons. wheat germ (wg) is widely recognized as a nutritious raw material to be incorporated into food product formulations or as a food in its own right. typical applications are in sprout-fortified bread, snacks and supplements, for breakfast cereals and for http://fens.usv.ro/index.php/fens mailto:meziani_samira@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 248 the production of wheat germ oil. wheat germ, containing about 8% to 14% oil (10% on average), is mainly used in the food, medical and cosmetic industries as a source of oil [3]. wheat germ, a nutritious byproduct of milling constituting 2.5 3.0 g / 100g of the cereal box is separated in a fairly pure form by using a germ separator or by appropriate adjustments in milling techniques [4]. wheat germ (embryonic axis and scutellum) accounts for about 2.5 to 3.8% of the total seed weight and is an important by-product of the milling industry [5]. is part of the wheat grain (triticum vulgaris) which contains the embryo of the future plant. it could therefore serve as a cheap source of raw material for food and oleochemical industries, and also give stable oil for various uses and applications, including shampoos, soaps and by-products,and salads and cooking oils. therefore, wheat germ, with its inherent nutritional value, could be a good alternative to processed vegetable oils in food products [6]. it is a major by-product of the wheat milling industry and is considered natural source of highly concentrated nutrients [7]. it is widely recognized as a nutritious raw material to be incorporated into food product formulations or as food in its own right. typical applications are in sproutenriched bread, snack foods and cereal supplements for breakfast and for the production of wheat germ oil. the latter, containing about 8% to 14% oil (on average 10%), is mainly used in the food, medical and cosmetic industries as a source of oil [8]. the stored raw sprout develops a rancid and bitter taste in a short time, due to the activity and unsaturated fat content of fresh sprouts. the low stability of the raw germ limits its uses; this problem could be overcome either by inactivating enzymes or by creating unfavorable conditions for enzymatic activity by appropriate means [9]. the objective of this study is to evaluate by a comparative study some phytochemical parameters in polyphenols and flavonoids for pure soft wheat germ and treated wheat germ. the antioxidant potential was also evaluated in this study. 2. matherials and methods 2.1. plant material in this study, a comparative study of the nutritional value of treated soft wheat germ and pure soft wheat germ (arz variety) by the analysis of phenolic compounds, flavonoids and the study of their antioxidant activity by the dpph test. for this experimental study, wheat germ samples are obtained in two different ways depending on the process of use; for pure soft wheat germ, it is degermination, a process obtained by a manual dissection method, while the treated germ comes directly from mills with advanced milling in our experimental part, all the analyses and the extraction method were carried out at the immunology and general biochemistry laboratories of the university of djillali liabes of sidi bel abbés. the plant material of our study includes two varieties of soft wheat, the arz variety from itgc of sidi bel abbés for the study of pure wheat germ and the soft wheat treated by l'o.a.i.c of sidi bel abbés for the study of treated wheat germ which was treated at the habour mill in oran (table 1). the wheat germ obtained must be kept at a temperature of 5° c in the fridge, in order to extend its shelf life and avoid rancidity (rotting) until the time of analysis. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 249 table 1. pure and processed common wheat 2.2. extraction of soluble phenolic compounds the sample preparation is done by maceration: a mass of 30g of each sample is macerated in 100ml of 70% ethanol, after the sample is stirred for 24 hours; the mixtures have been separated by simple filtration. the extracts are then evaporated to dryness using an evaporator of about 45°c. the extract will complete drying in the oven for 24 hours; so that the extract does not lose its phenolic compounds with water during evaporation. the conservation is done in the refrigerator. 2.3. the determination of total polyphenols in the extract 2.3.1. determination of total phenolic content using the folin-ciocalteu reagent, a volume of 200 μl of each extract was added, with a mixture decrypted [10] of 1 ml of folin-ciocalteu reagent diluted 10fold, and 800 μl of a 7.5% sodium carbonate (na2co3) solution. the tubes are shaken and stored for 30 min. the absorbance is read at 765 nm using a uv spectrophotometer (the level of polyphenols was expressed in microgram equivalent of gallic acid per milligram of extract (µg eag/mg e). a calibration curve was carried out in parallel under the same operating conditions using gallic acid at different concentrations (0 to 1000 µg/ml). 2.3.2. determination of flavonoid content the protocol used is based on the one described by [11], with some modifications. in a glass hemolysis tube, 0.5 ml of extract was mixed with 2 ml of distilled water and 0.15 ml of 5% nano2. after 6 minutes, 0.15 ml 10 % alcl3 was added. after 6 minutes a volume of 2 ml 4% naoh was added to the medium and adjusted with 0.2 ml distilled water. the absorbance is read off at 510 nm after stirring and incubation for 15 min. a methanolic quercitrin solution has been prepared. daughter solutions prepared from the stock solution at different concentrations between 100 and 900 µg/ml will be used to draw the calibration curve. 2.4. antioxidant activity 2.4.1. dpph radical scavenging activity the dpph radical scavenging activity was measured according to the protocol described by [12].a 50 µl solution of each methanolic extract at different concentrations was added to 1.95 ml of dpph (0.025g/l). at the same time, a negative control is prepared by mixing 50µl of methanol with 1.95 ml of the methanolic solution of dpph. the absorbance reading is taken against a blank prepared for each concentration at 515 nm after 30 min incubation in the dark and at room temperature. variety and origin pure common wheat arz itgc of sidi bel abbés soft wheat treaty treaty treaty by o.a.i.c; habour. oran food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 250 the positive control is represented by a solution of a standard antioxidant, ascorbic acid, whose absorbance was measured under the same conditions as the samples and for each concentration. dpph radical scavenging in percentage was calculated as follows: dpph (%)=100 (a (control) a (sample)) / a control where: dpph (%) is the percentage reduction in dpph, (acontrol) is the absorbance of the negative control, and (asample) is the absorbance of the sample. the results were expressed as the average of three measurements obtained for each sample. the ic50 value that determined the concentration that reduced the dpph radical by 50% was revealed graphically for each extract from the percentage dpph reduction versus concentration curve 2.5. statistical analysis all results were performed in three replicates. the results are expressed as average ± standard deviation. the data (the averages plus at least standard deviation as well as representations) are statistically analyzed using the statistical software excel 2010. 3. results and discussion 3.1. yield in dry extract extraction is a very important step in the isolation and identification of phenolic compounds. extraction methods depend on the extraction yield of the phenolic compounds. ethanol extracts (70% ethanol), recovered after evaporation, and was weighed to determine the resulting dry weight. these extracts contain the total polyphenols. extractions of the different cereals for the wheat varieties allowed us to calculate the yield of each extract. the table below shows the results for 30 g of dry plant matter expressed as a percentage. the results obtained are presented in table 2. table 2. physical parameters (yield, color, and physical appearance) in dry extract 3.2. result of total phenolic and flavonoid the dosage was carried out on alcoolic extracts f wheat germ after passing through an oven to eliminate the solvent in order to obtain dry extracts. the resulting dry weight contains the total polyphenols which are determined by the folinciocalteu test. the content of phenolic compounds in each extract was calculated from the calibration curve (y= 8.7218x0.002), of which y is the absorbance and x the concentration of the gallic acid solution), our results were expressed in milligrams gallic acid equivalent per gram of dry matter (eag/g dm) and the correlation coefficient (r²=0.997) the optical density measurement was performed at a wavelength of 760 nm. the results obtained are presented in table 3. sample variety treated common wheat germ pure tender wheat germ (arz) physical appearance sticky gel sticky gel colour pale yellow dark yellow return (%) 20.6 12.73 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 251 table 3 polyphenol content expressed in mg eag/g dry matter. variety pure common wheat germ (arz) treated common wheat germ total polyphenol content in mg eag/g 0.074±0.001 0.062±0.001 the results obtained from the wheat germ extracts (pure wheat germ and treated wheat germ analysed reveal that pure wheat germ (arz) is richer in total phenolic compounds 0.074±0.001mg (eag)/g than treated wheat germ (secondarily) with a content of 0.062±0.001mg (eag)/g. the main reason for looking for flavonoids in wheat germ is due to the fact that flavonoids are very well known for their antioxidant capacity.the flavonoid content of each extract was calculated from the calibration curve (y=4.6153x-0.0118), of which y is the absorbance and x the concentration of the catechin solution) our results were expressed in milligrams catechin equivalent per gram of dry matter (mg ec/g dm), the optical density measurement was performed at a wavelength of 510nm. the results obtained are presented in table 4. table 4 flavonoid content expressed in mg eag/g dry matter. fig.1. polyphenol and flavonoid content of wheat t germ from different varieties of soft wheat, gwp: pure wheat germ, gwt: processed wheat germ 3.3. free radical scavenging dpph. (2, 2-diphenyl-1-picrylhydrazyl) the natural antioxidant activity depends on various parameters, such as reaction mechanism, isolation procedures, purity of active compounds, as well as the test system and substrate to be protected by the antioxidant. sample variety pure common wheat germ (arz) treated common wheat germ flavonoid content expressed in mg ec/g dm 0.022±0.002 0.021±0.002 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 252 in our case, we evaluated the samples by dpph radical scavenging activity in order to estimate and measure their antioxidant activities. the anti-radical capacity cannot be measured directly but by monitoring the effect of reactivity in vitro. this activity has been evaluated by measuring the dpph radical scavenging power according to the method described by [13] 0.361 0.051 0.029 0 0.1 0.2 0.3 0.4 gwp gwt vitc ic 5 0 o f d if fe re n t v a ri é ty m g /m l variéty ic50 ic50 fig 2. antioxidant power of different extracts of soft wheat germ. gwp: pure wheat ger, gwt: processed wheat germ our results on the anti-radical activity presented in the curves were expressed by measuring the effective concentration ic50; the concentration that traps (inhibits) 50% of the dpph radical of each extract and ascorbic acid. this ic50 value is inversely proportional to the antioxidant power, each time the ic50 of the extract is close to the ic50 of the ascorbic acid. the stronger the antioxidant activity in the extra it from the comparison of ic50 of each variety studied and according to the results obtained with that of ascorbic acid, we notice that ic50 of the pure and treated wheat germ extracts are higher than ic50 of ascorbic acid with 0.029 mg/ml (used as a reference antioxidant), 0.051 mg/ml and 0.361 mg/ml, for the pure wheat germ extract (arz) and the treated wheat germ, respectively. the antioxidant capacity of a compound is higher when its ic50 is small, so the antioxidant capacity of ascorbic acid is higher than that of the extract of two wheat germs. in addition, the results also show that this same capacity is higher for pure wheat germ than for treated wheat germ.wheat germ is a by-product of wheat milling that has the potential as a food ingredient, a source of high quality protein, minerals, vitamins and antioxidants. several studies have indicated the superiority of wheat germ over other milling products as a rich nutritional supplement, [14]. the results of assaying total polyphenols and flavonoids in both wheat germ varieties show that wheat germ extracts contain moderate amounts of total polyphenols, particularly in the pure germ of the arz variety. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 253 no differences were found in flavonoids in the two germs used in our case. the marked values showed that the total polyphenol content. represents 0.84 mg/g wheat germ. in a study by [15] indicate that, ethanol extraction gave a polyphenol content ranging from 0.038 mg/g to 1.6 mg/g, from which he considers values ranging from average to good. our results are consistent with his work, hence the presence of considerable amounts of polyphenols in wheat germ, which are less present compared to other wheat grain tissues as indicated by several studies [16], studied the distribution of polyphenols in the milling function of wheat, and about 73% of the phenolic compounds in grains were found in wheat bran. this indicates that the wheat germ is less rich in polyphenols. this result is also confirmed by [17] who found that bran layers had the highest content of total phenolic compounds (total polyphenols and flavonoids). concerning flavonoids, the flavonoids in cereals were mainly concentrated in the outer layers of the grain compared to other parts of wheat grain [18]. a content of 0.45 mg/g to 1.07 mg/g and which vary according to the types of flavonoid compounds [19]. several parameters intervene and influence the quality and quantity of its compounds, the variety and/or species, the extraction method used as well as the extraction solvent can vary quantitatively the yield quality of the grain. the study by [20] show that flavonoid values in wheat germ are varied (changed) by the extraction solvent used. in addition, the quantity of polyphenols is considerable between the two varieties of the same species, it is less important compared to the tissues of the peripheral layers (aleurone layer, testa and hyaline layer). in the work for [21] confirm that the phenolic content that can be recovered in different samples of cereals is influenced by the polarity of the extraction solvents and the solubility of this compound in the solvent used for the extraction process. also [22] confirm that several factors can affect the content, composition and stability of phenolic compounds in wheat products depending on the ingredients, industrial or domestic processing. anti-oxidant properties are measured and highlighted by measuring the effective concentration (ic50). the results highlighted by the dpph test. the ic50 values obtained range from 0.029 mg/ml to 0.36 mg/ml with ascorbic acid used as (reference antioxidant) with ic50=0.029 mg/g. the antioxidant power of our extracts between them reveals that treated wheat germ is the most active with an ic50=0.051mg/ml followed by pure wheat germ with an ic50=0.3 mg/ml. comparison of the effective concentrations (ic50) of each of these two extracts shows that ic50 of our studied varieties and ascorbic acid (used as a reference antioxidant) are close, indicating that the anti-radical activity of the dpph trapping of our varieties is very important (very strong) with the highest value of ascorbic acid. several researches have been devoted to study the wheat germ and its derivatives and have proven that this part has a great antioxidant capacity through the presence of natural bioactive compounds. the highest free radical scavenging activity in wheat germ is a value of 0.42 mg/ml in 70% ethanol extract according to a study conducted by [23]. a similar trend has been observed in the study of antioxidant activity by [24]. our organism therefore reacts constantly to this permanent production of free radicals and we distinguish at the cell level two lines of defense unequally powerful to detoxify the cell [25]. these are exogenous molecules. unlike antioxidant enzymes, an food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 254 antioxidant molecule traps a single free radical. to be able to function again, this antioxidant molecule must therefore be regenerated by other systems [26]. 4. conclusion wheat germ (wg) is widely recognized as a nutritious raw material to be incorporated as a food ingredient and for the production of wheat germ oil. is a nutrient by-product of the milling process and is a natural source of highly concentrated nutrients. the composition total polyphenols and flavonoids in both wheat germ varieties gave that wheat germ extracts contain moderate amounts of total polyphenols, particularly in the pure germ of the arz variety. the antioxidant power reveals too that treated wheat germ is the most active that ic50 are close, indicating that the antiradical activity of the dpph trapping of our varieties is very important (very strong) different factors can influence the value of natural compounds present in the germ, preservation and storage methods, the method of growing the grain, the environment, exposure to heat and light, extraction methods and the solvent used may vary and affect the yield and value of the germ. to this end, it is interesting to extend our research toother varieties and other cereal species and to carry out further study based on advanced research techniques in order to improve nutritional and phytochemical quality. 5. acknowledgments the authors acknowledge the university of sidi bel abbes, biology department at sidi bel abbes for the financial support of this research 6. references [1]. slama, a, ben slam, m., bennaceur, m., zid ed. cereals in tunisia:production, drought effect and mechanisms of resistance. national research institute agronomic of tunisia (inrat). univ. elmanar. tunisia, p62 (2005). [2]. fao stat., statistical database of the food and agriculture organization of the united nations, (2007). [3]. zhu k.x, zhou h.m, qian h.f. proteins extracted from defatted wheat germ: nutritional and structural nutritional and structural properties. cereal chemistry, 83, 6975.http://dx.doi.org/10.1094/cc-83-0069, (2006). [4]. de vasconcelos m.c.b.m., bennett r., castro c., cardoso p., saavedra m.j., rosa e.a. study of the composition, stabilisation and transformation of industrial byproducts of wheat germ and maize. industrial crops and products, 42: 292-298, (2013). [5]. brandolini a et hidalgo a. wheat germ: not only a by-product, international journal of food sciences and nutrition: p 71-74. doi: 10.3109/09637486.2011.633898, (2012). [6]. awada.mahmoud, adel a. a. mohdaly, nady a. a. elneairy wheat germ: an overview of nutritional value, antioxidant potential, and antibacterial characteristics. food and nutrition sciences, 6, 265-277, (2015). [7]. ge y, sun a, ni y, cai t. some nutritional and functional properties of defatted wheat germ protein. j agric food chem, 48:6215–6218, (2001). [9]. rao, hp, kumar, gv, rao, rgcp and shurpaleker, sr studies on wheat germ stabilization. lebensmittelwissenschaft und technologie, 13, 302-307 the antioxidant activity and total phenolics in a nutritious snack food. int. j. food sci. technol, (1980). [10]. ali-rachedi f, meraghni s, touaibia n, sabrina m. quantitative analysis of phenolic compounds of an algerian endemic scabiosaatropurpurea sub. maritima l. bulletin de la société royale des sciences de liège, vol. 87, articles, p. 13 -21, (2018). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 3 – 2021 lahouaria labga, ghania tiboura, fatima zohra chenni, dounia djebbouri, nadjet ouafi, souad saidani, samira meziani, extraction and comparative study of phenolic compounds from tender and treated wheat germ and their antioxidant power, food and environment safety, volume xx, issue 3 – 2021, pag. 247 255 255 [11]. viuda-martos, m., mohamady, m. a., fernández-lópez, j., elrazik, k. a., omer, e. a., pérez-alvarez, j. a., &sendra, e. in vitro antioxidant and antibacterial activities of essentials oils obtained from egyptian aromatic plants. food control. 22(11):1715-1722, (2011). [12]. athamena s, chalghem i, kassahlaouar a, laroui s, khebri s .activity antioxidant and antimicrobial activity of extracts of cuminumcyminum l, lebanese science journal, vol. 11, no. 1, (2010). [13]. burits m, bucar f. antioxidant activity of nigella sativa essential oil.phytotheraphyresearch, 14 (2000): 323– 328.bakery products. j. cerealsci., 57, 312-318, (2013). [14]. truswell as. cereals and coronary heart disease.eur j clin nutr 56, 1-14, (2002). [15]. abor m. m. abd el-rahman. utilization of wheat germ as natural antioxidant and fat mimetic to increase shelflife in beef sausage and as lowering cholesterol in rats, middle east journal of agriculture research. p 555-563.115p. europeanscientific journal, (2015). [16]. abdel-aal, e.-s. m., hucl, p., sosulski, f. w., graf, r., gillot, c., pietrzak, l., screening of spring wheat for midge resistance in relation to ferulic acid content. j. agric. food chem. 49, 3559-3566, (2001) [17]. vaher m., matso k., levandi t., helmja k., kaljurand m. phenolic compounds and the antioxidant activity of the bran, flour and whole grain of different wheat varieties. procedia chemistry 2: 76-82, (2010). [18]. adom, k., liu, r: antioxidant activity of cereals. j. agric. food chem.50, 6182-6187, (2002). [19]. abor m. m. abd el-rahman. utilization of wheat germ as natural antioxidant and fat mimetic to increase shelflife in beef sausage and as lowering cholesterol in rats, middle east journal of agriculture research. p 555-563.115p.european scientific journal, (2015). [20]. king, a; young, g. characteristics and occurrence of phenolic phytochemicals. j. am. diet. assoc, (1962) [21]. alothman m, bhat r, karim a.a. antioxidant capacity and phenolic content of some tropical fruits from malaysia, extracted with different solvents. food chemistry.115, 785–788, (2009). [22]. abdel-aal, e.m.; rabalski, i. effect of cooking on free and bound phenolic acids in whole grains, (2013) [23]. ke-xue zhu, cai-xia lian, xiao-na guo, wei peng, hui-ming zhou. antioxidant activities and total phenolic content of various extracts from defatted wheat germ; food chemistry 126 1122-1126, (2011). [24]. nagai t, myoda t, nagashima t. antioxidative activities of water extract and ethanol extract from field horsetail (tsukushi) equisetum arvense l. food chemistry, 91, 389-394.2006, 41, 289–293, (2005). [25]. favier a., oxidative stress, conceptual and experimental interest in the understanding of disease mechanisms and therapeutic potential. chemical news, (2003), [26]. dacosta y., les phytonutriments bioactifs. yves dacosta, paris, 317p. king a,young, g , characteristicsandoccurrence of phenolic phytochemicals , j.am. diet.assoc.1962, (2003). 1. introduction 2.3. the determination of total polyphenols in the extract 2.3.1. determination of total phenolic content 2.3.2. determination of flavonoid content 3.1. yield in dry extract 4. conclusion 160 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 160 – 168 new starches from improved yellow colored cassava: enzymatic and acid sturdiness and potential of industrial utilization ginette g. doué1 , *micaël e. bédikou1 , gisèle a. koua1 , sévérin k. kra1 , sébastien l. niamké1 laboratoire de biotechnologies, filière biochimie-microbiologie, unité de formation et de recherche en biosciences, université félix houphouët-boigny, cocody, abidjan-côte d’ivoire. 22 bp 582 abidjan 22. bemicael@yahoo.fr *corresponding author received june 16th 2014, accepted june 26th 2014 abstract: native and gelatinized starches from eight improved cassava varieties were extracted and subjected to enzymatic and acid hydrolysis in order to highlight their potential of industrial application. the enzymatic hydrolysis showed that native starches were less hydrolyzed (8.40–15.80 10-2 ui/mg of proteins) than the gelatinized ones with values ranged from 1.14.102 ui (v23) to 6.93 102 ui/mg of proteins (v54). thin layer chromatography analysis showed glucose and a high molecular mass maltodextrin as the common products released from both starches. maltose and another low molecular mass maltodextrin were in addition observed when gelatinized starches were used. the hydrochloric acid solution had showed predominant conversion of v4, v54 and v55 with maximum values of reducing sugar products of about 70, 62, and 82 µmol, respectively. the starch variety v52 recorded the lowest value (36.23 µmol of reducing sugar). due to the relatively high degrees of hydrolysis and the wide range of industrial application of modified starches products (glucose, maltose and maltodextrins), v4, v54, and v55 could constitute new exploring sources of starch for the food and pharmaceutical industry while v52 could be suggested as a raw material for the paper industry and for biofuel production. keywords: acid conversion enzymatic hydrolysis industry modified cassava starch 1. introduction cassava (manihot esculenta crantz) is one of the most important food crops in the humid tropics, being particularly suited to low nutrient availability and able to survive drought [1]. initially grown as a substantial food crop, cassava root derivatives especially flour and starch are now used for various industrial purposes [2]. this utilization has been taking place in the so-called “newly opened economies” of china and vietnam, and to a lesser extent in latin america. starches can be classified as unmodified or modified. unmodified or native starches are produced through the separation of naturally occurring starch from grain or food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 161 root crops. irrespective of their source, native starches are undesirable for many industrial applications because of their inability to withstand processing conditions such as extreme temperatures. they yield pastes of poor stability which decreases products shelf life. native starches also showed high ability to retrograde, loss of viscosity, syneresis tendency and thickening power upon cooking [3]. in order to improve their desirable functional properties and overcome their limitation uses, native starches are often modified. modification of starches mainly consist in the alteration of physical and chemical characteristics. these processes enable to improve inherent poor structural and physicochemical properties of native starch thus tailor it to specific industrial applications [4]. nowadays, practically every industry in existence uses starch or its derivatives in one form or another [5]. in foods and pharmaceuticals, starch truly serves as a multifunctional ingredient [4]. indeed, it is used to influence or control such characteristics as texture, moisture, consistency and shelf stability. it can be used to bind, to densify, to opacify and to attract moisture in various products. as for the food industry, non-food industrial uses of cassava starch are also associated with larger scale firms. the textile industry, for example, uses cassava starch for sizing while the paper industry uses it for coating high quality paper. as all these industrial sectors develop, demand for cassava starch is likely to increase. despite their importance, a large proportion of these agro-resources are lost yearly due to nonavailability of appropriate technology and industry to harness these into various useful products [6]. nowadays, most of african countries have taken in earnest their abundantly produced cassava and have just started to process more expended food and non-food end-products [7]. in this context, plant breeders, agronomists and recently molecular biologists have made substantial improvements in cassava varieties and yields during the last two decades [8]. in côte d’ivoire, the national agricultural center has implemented several improved varieties for rural dissemination. recently, flours and starches of the new yellow colored cassava fleshes were extracted and extensively characterized through physicochemical and biochemical parameters [9,10]. in the present work, eight (8) of these starches were subjected to enzymatic and mild acid hydrolysis and their resulted products analyzed in order to explore the potential domains of industrial applications. 2. experimental 2.1. cassava varieties all cassava varieties used in this study were harvested at eleven months old and provided to us by the national agricultural research centre (cnra, adiopodoumé, côte dʼivoire 5°19'40'' latitude north and 4°23'00'' longitude west). as starches which are extracted, these varieties were encoded v64, v65, v66, v69, v71 and v73, and resulted from various crossbreeding’s of two already disseminated varieties: v4 (white coloured flesh) and v23 (yellow coloured flesh). 2.2. enzymatic source and amylolytic enzymes extraction ten active snails (achatina achatina) were denied water and food for three days to liquefy their initial bolus and then, their shells were cracked. the digestive fluid was collected with syringe, filtered through sterile cotton wool and centrifuged at 10,000 g for 15 min. the supernatant was food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 162 used as the crude extract source of amylolytic enzymes. 2.3. proteins estimation proteins concentration in the crude extract was measured by using the folinciocalteu’s phenolic reagent [11]. bovine serum albumin was used as the standard protein. 2.4 starch extraction fresh cassava roots were washed, peeled, chopped and then pulverized in a high speed blender. the cassava mash was suspended in ten times its volume of water, stirred for 5 min and filtered by using double fold cheesecloth. the filtrate was allowed to stand for 2 h and the top liquid was decanted and discarded. a 4% (w/v) nacl solution was added to the sediment and the mixture stirred for 5 min. filtration was carried out using sieves with reverse mesh sizes varying from 500 to 100 µm. after decanting the top liquid, starch paste was dried at 45 °c for 48 h, crushed to a fine powder and then stored for further treatments. 2.5. native and gelatinized starch substrate preparation both starch substrates were prepared at 1% (w/v). native starch substrates were obtained by dissolving 1 g of the above dried powder in 100 ml of distilled water. for gelatinized starches, the same protocol was initially followed in 50 ml of distilled water. then, the mixture was heated at 70 °c for 10 min and the residual solution adjusted to 100 ml with distilled water. 2.6. enzymatic and mild-acid hydrolysis the enzymatic hydrolysis were performed on both starch substrates. reactions were carried out at 37 °c with 2.5 µg of proteins in a 100 mm sodium acetate buffer (ph 5.6) for 24 hours. concerning the mild acid hydrolysis, only native starches were used. a 2.2 n hydrochloric acid solution was used at 25 °c to catalyse reactions for three (3) months. all the experiments were performed in triplicate under sporadic stirring with a total mixture volume of 200 µl by using a screw-cap glass cells. amylolytic enzymes activities were expressed as the amount of catalysts which release 1 µmol of reducing sugar from 1% (w/v) starch in a minute under the experimental conditions. for the acid conversion, activities were expressed in percent (mg of dextrose equivalent per 100 mg of starch). the initial velocities were graphically determined from the beginning rising side (linear part) of each hydrolysis profile by taking into account the earlier upward trend of reducing sugar production. 2.7. analysis of hydrolysed products at regular time intervals, 100 µl volume samples were withdrawn to quantify total reducing sugars [12], while 5 µl of the same solution were spotted onto thin layer chromatograph plates (model 60 f254, merck) to monitor the hydrolysis of native and gelatinized starches. tlc plates were run with butanol-acetic acid-water mixed at 9:3.75:2.25 (v/v/v), and sprayed with a naphto-resorcinol in ethanol and h2so4 20% (v/v) solution. then, sugar spots were visualized by heating plates at 110 °c for 5 min. 3. results and discussion 3.1. enzymatic hydrolysis the kinetics of the enzymatic hydrolysis of native and gelatinized starches are depicted in figure 1. these profiles obeyed the michaelis-menten kinetics which are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 163 characterized by logarithmic curves. so, they enabled us to determine the different initial velocities topped in table 1. these values reflect the strength of each starch overlooked amylolytic enzymes. the results indicated that, at the earlier stage of the reactions, and irrespective of the starch variety, native starches were less susceptible to enzymes attack than the gelatinized ones. concerning native starches, the best hydrolysis rate (15.80×10-2 ui/mg of proteins) was observed with v55. figure 1. kinetics of enzymatic hydrolysis at 37°c of native (a) and gelatinized (b) cassava starches by the digestive fluid of the giant snail achatina achatina. (v4, v23, v52, v53, v54, v55, v62 and v63, cassava starches) the other varieties are hydrolyzed to a lesser extent and in almost the same proportion with rates of between 8.40 and 10.37×10-2 ui/mg of proteins (table 1). unlike native starches, gelatinized starches had better tendency to hydrolysis of at least 1,000 times higher. v54 and v62 showed the highest values of respectively 6.93 and 3.26×102 ui/mg of proteins while v23 recorded the lower rate of conversion (1.14±1.03×102 ui/mg of proteins). the other gelatinized starches showed intermediate values of between 2.01 and 2.24×102 ui/mg of proteins (table 1). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 164 table 1. initial velocities of enzymatic hydrolysis of native and gelatinized cassava starches by the digestive fluid of the giant snail achatina achatina the observed variability at the hydrolysis rate of native starches may be related to the degree of damage in isolated starch granules. indeed, damage starch granules have larger surface area for enzymes hydrolysis than intact native starch granules with smooth surface structure lacking pinholes and channels, thus rendering them highly resistant to amylolytic enzymes attack [13]. the strong susceptibility of a native starch to enzymes attack could be also explained by both their relatively high amylopectin content and granules size [14]. indeed, the small granules would be more resistant to external influences and less inclined to the transformations therefore less subjected to enzymes attack. because most foods are consumed after cooking, the digestibility of ungelatinized starch granules is only applicable mostly for animal feed. so, the native starch v55 which is greatly hydrolysed by digestive enzymes could constitute an inexpensive and commodity feed material for livestock as substitute of maize starch usually used [15]. elsewhere, in the renewable energy industry, this starch variety could serve as an alternative substrate for selected yeasts in the production of bioethanol [16]. the difference at the hydrolysis behavior between native and gelatinized starch results in the modification of the native starch granules structure during gelatinization [17]. indeed, when native starches granules are gelatinized during a hydro thermal processing, the hydrogen bonds between starch chains are disrupted, allowing the granules to swell and then disintegrate. as a result, the availability of amylose and amylopectin chains to the digestive enzymes increases [18]. the great tendency to hydrolysis of the gelatinized starch v54 could also be linked to its higher solubility attribute partly to the high swelling it undergoes during gelatinization. also, this property could be due to its low proportion of amylose of about 14%, as already reported [10], which reduces the presence of inhibitors such as phosphate and promote gelatinization [19]. the potential gelling property of this starches lead to its use in the food industry as thickener and ambient products stabilizer. the other starches which have shown weak digestibility may be less gelatinized due to high proportion of small granules and amylose, and the large number of associative forces [20]. therefore, v54 could be more explore in infant diet preparations because of its high starch varieties initial velocities of amylolytic enzymes on native starches (10-2 ui/mg of proteins) initial velocities of amylolytic enzymes on gelatinized starches (102 ui/mg of proteins) v4 8.90±0.08 1.57±1.03 v23 10.37±0.08 1.14±1.03 v52 8.89±0.09 2.24±1.45 v53 8.65±0.07 2.03±2.06 v54 8.40±0.08 6.93±2.07 v55 15.80±1.03 2.21±2.44 v62 8.67±1.04 3.26±2.22 v63 11.37±1.06 2.01±1.03 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 165 degree of digestion. also, it could be incorporated into food ratio of sportsmen such as an ingredient of choice to substitute lipid foods in order to reduce prevalence of obesity and cardiovascular diseases [21]. as for the other gelatinized starches which have low digestibility rates, they could considered to be beneficial for dietary management of metabolic disorders, including diabetes and hyperlipidemia [22]. moreover, the low digestibility of these starches showed close resemblance with dietary fiber which is healthy and has previously been reported to reduce postprandial glycaemia [23]. the weaker associative bonds between the starch molecules in the granules also favours its hydrolysis. beyond influencing hydrolysis, cassava starch with weaker associative forces enables it to have better clarity when gelatinized, and such property has much relevant in food and textile applications. therefore, the great hydrolysed starches reported in this study for gelatinized starches v54 and v62 could promote their use as suitable materials in foods as pie fillings and in textile industry for sizing. the opposed hydrolysis behavior of v55 face to amylolytic enzymes when gelatinized, in comparison to its native form, remind us of two new qualities of starches namely resistant starch type 3 or 4 (rs3 or rs4). these kind of starches are formed through retrogradation such they are no longer well recognised by alpha-amylases, thus rendering them lower hydrolysed. in the same context, native starches such as v53, v54 and v62 that resist to enzymatic hydrolysis could be categorized as type 2 resistant starch (rs2) which escape digestion in the stomach and the small intestine so that they can be labelled as "dietary fibre". resistant starches are considered as ideal ingredients for the fibre fortification of low-calorie products. in nutrition, they have potential physiological benefits in relation to the biochemistry of the colon and glucose/insulin metabolism with healthy benefit to diabetics [24]. 3.2. thin layer chromatography products products spots on thin layer chromatograms of the enzymatic reaction mixtures are showed by figure 2. glucose and maltodextrin 1 were distinctly observed as the common products from both substrates (figures 2a and 2b). in addition to the latter products, maltose and another maltodextrin with relatively lower molecular mass (maltodextrin 2) were obtained for gelatinized starches (figure 2b). figure 2. thin layer chromatography of products from the reaction mixture of native (a) and gelatinized (b) cassava starches catalyzed by the digestive fluid of the giant snail achatina achatina. (v4, v23, v52, v53, v54, v55, v62 and v63, cassava starches; s, standard of starch; m, standard of maltose; g, standard of glucose). b a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 166 though starch truly serves as a multifunctional ingredient in the food industry, its released product such as glucose, maltose and maltodextrins also are essential and useful products for food confectionary industries [5,6] and widely applied in sugar, spirits, and textile as well as in brewing. for example, glucose syrup could serve as substrates for the production of a wide range of fermentation products such as monosodium glutamate [25], lysine for the animal feed industry [26] and ethanol for biofuel industry [16]. dextrins’ are used in backed goods and in confectionary where their main functions are viscosity control, softening, texturing and coating [6]. 3.3. thinned-acid starches the acid hydrolysis profiles of the studied cassava starches were shown in figure 3. the two-stage of hydrolysis pattern was quite obvious in all cases. a relatively fast hydrolysis rate during the first two weeks linked to the depolymerisation of the amorphous regions of starch granules. while the crystalline material is slowly degraded during the second stage [27]. figure 3. kinetic of the hydrolysis at 25°c of native cassava starches by a mild hydrochloric acid (2.2 n) solution. acid-thinned starches v4, v54 and v55 showed the highest amounts of reducing sugars with values of about 70.07, 61.52 and 82.58 µmol, respectively. these results corroborate those obtained for yam and wheat starches [28]. the strong hydrolysis rate of v4, v54 and v55 could suggest a predominant attack of their amorphous granule parts so that higher concentrations of these starches can be used without excessive thickening. such starch property are preferentially sought in the manufacture of glues and gums, and in the paper and textile industry for coating and sizing. also, their great conversion into lower molecular mass products could be used to improve printability and abrasion resistance on paper surfaces, as well as in calendar and size press applications [29]. 4. conclusion the present study has revealed that starches from eight improved yellow colored fleshes of cassava have variable sturdiness towards enzymatic and acid hydrolysis. gelatinized starches exhibited more enzymes attack than the native ones. starch v54 which is easily hydrolyzed under it gelatinized form, could be proposed as food dietary of sportsmen and workers. the other starches which were less hydrolyzed, could serve as raw materials in food products formulation for diabetics and persons who suffer from cardiovascular diseases. by considering the acid hydrolysis, the native starch v55 could be recommended in the production of biofuel. it could be conclude that, as native starches have limited industrial utilization, enzymatic and chemical modification opens new prospects for their use in various food, textile, pharmaceutics and papers industrial processes. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 167 5. acknowledgments this work was supported by a ph.d. grant to the first author. the authors are grateful to the ivorian national agricultural centre (cnra) for cassava varieties. 6. references [1]. burrell m.m, starch: the need for improved quality or quantity – an overview, j. exp. bot., 54. 451-456. (2003) [2]. demiate i.m., kotovicz v., cassava starch in the brazilian food industry, food sci. technol. (campinas), 31. 388-397, (2011) [3]. singh j., kaur l., mccarthy o.j., factors influencing the physico-chemical, morphological, thermal and rheological properties of some chemically modified starches for food applications-a review, food hydrocoll., 21. 1-22, (2007) [4]. miyazki m.r., hung p.v., maeda t., morita n., recent advances in application of modified starches for bread making, trends food sci. technol., 17. 591-599, (2006) [5]. akpa g.j., dagde k.k., modification of cassava starch for industrial uses, int. j. eng. technol., 2. 913-919, (2012) [6]. zainab a., modu s., falmata a.s., maisaratu t., laboratory scale production of glucose syrup by the enzymatic hydrolysis of starch made from maize, millet and sorghum, biokemistri, 23. 1-8, (2011) [7]. goletti f., rich k., wheatley c., the cassava starch industry in vietnam: can small firms survive and prosper?, int. food agribus. manage. rev., 2. 345-357, (2001) [8]. fregene m.a., suarez m., mkumbira j., kulembeka h., ndedya e., kulaya a., mitchel s., gullberg u., rosling h., dixon a.g., dean r., kresovich s., simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop, theor. appl. gen., 107. 1083-1093, (2003) [9]. koua g.a-y., megnanou r-m., kra k.s., n’zue b., tian-bi d.r., akpa e.e., niamke s.l., biochemical characterization of new varieties of yellow colored pulp cassava flours from côte d’ivoire, j. appl. biosci., 53. 3760-3772, (2012) [10]. doue g.g., megnanou r-m., beikou e.m., niamke l.s., physicochemical characterization of starches from seven improved cassava varieties: potentiality of industrial utilization, j. appl. biosci., 73. 6002-6011, (2014) [11]. lowry h.o., rosebrough n.j., farr l.a., randall r.j., protein measurement with the folin phenol reagent, j. biol. chem., 193. 265-275, (1951) [12]. bernfeld p., amylases α and β, method enzym., 1. 149-158, (1955) [13]. dhital s., shrestha a.k., gidle m.j., effect of cryomilling on starches: functionality and digestibility, food hydrocoll., 24. 152-163, (2010) [14]. sandstedt r.m., sitrahan d., veda s., abbott r.c., the digestibility of high amylose corn starches compared to that of other starches. the apparent effect of the ae gene and susceptibility to amylase action, cereal chem., 39. 123-131, (1962) [15]. cooper r.j., milton c.t., klopfenstein t.j., scott t.l., wilson c.b., mass r.a. effect of corn processing on starch digestion and bacterial crude protein flow in finishing cattle, j. anim. sci., 80. 797-804, (2002) [16]. duhan j.s., kumar a., tanwar s.k., bioethanol production from starchy part of tuberous plant (potato) using saccharomyces cerevisiae mtcc-170, afr. j. microbiol. res., 7. 5253-5260, (2013) [17]. amani n.g., tetchi f.a., coulibaly a., propriétés physico-chimique de l’amidon de gingembre (zingiber officinale roscoe) de côte d’ivoire, tropicultura, 22. 77-83, (2004) [18]. chung h.j., lim h.s., lim s.t., effect of partial gelatinization and retrogradation on the enzymatic digestion of waxy rice starch, j. cereal sci., 43. 353-359, (2006) [19]. nuwamanya e., baguma y., emmambux n., taylor j., rubaihayo p., physicochemical and functional characteristics of cassava starch in uganda varieties and their progenies, j. plant breed. crop sci., 2. 001-011, (2010) [20]. adejumo a.l., aderibigbe a.f., layokun s.k., cassava starch: production, physicochemical properties and hydrolysation a review, adv. food energy sect., 2. 8-17, (2011) [21]. miller j.c., miller, j.c., mcmillan p., steinbeck k., caterson i., carbohydrates: the good, the bad and the whole grain, asia pac. j. clin. nutr., 17. 16-19, (2008) [22]. wolever t.m., melhing c., highcarbohydrate-low-glycemic index dietary advice improves glucose disposition index in subjects with impaired glucose tolerance, br. j. nutr., 87. 477-487, (2002) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 g ine t t e g . d ou é 1 , m ic a ël e . b é di ko u , g is è le a. k o u a, s é vér i n k . k r a , sé b as t ie n l . n i am ké , n e w s t a r ch e s f r o m i m pr o v ed y e ll o w c o l or e d ca s s a va : e nz ym a t i c a nd a ci d s t u rd i ne s s and p o t e nt i al of i nd u s t r i a l u t i l i z at i on , fo od a nd e nv i r o nme nt s af e t y, issue 2 2014, pag. 160 – 168 168 [23]. nuttall f.q., dietary fiber in management of diabetes, diabetes, 42. 503-508, (1993) [24]. nugent a., health properties of resistant starch, br. nutr. found nutr. bull., 30. 27-54, (2005) [25]. nampoothiri k.m., pandey a., fermentation and recovery of l-glutamic acid from cassava starch hydrolysate by ion exchange resin column, rev. microbiol., 30. 258-264, (1999) [26]. moosavi-nasab m., ansari s., montazer z., fermentative production of lysine by corynebacterium glutamicum from different carbon sources, iran agric. res., 25. 99-106, (2007) [27]. robin j.p., mercier c., charbonniere r., guilbot a., lintnerized starches. gel filtration and enzymatic studies of insoluble residues from prolonged acid treatment of potato starch, cereal chem., 51. 389-406, (1974) [28]. dally l.i., sako a., koffi a., sidibe d., lia j.a., n’guessan a.g., influence des traitements acide et enzymatique sur la structure granulaire d’amidon d’igname «kponan» (dioscorea cayenensis rotundata. poir), j. sci. pharm. biol., 9. 17-26, (2008) [29]. lim w.j., liang y.t., seib p.a., cationic oat starch: preparation and effect on paper strength, cereal chem., 69. 237-239, (1992) doi: https://doi.org/10.4316/fens.2021.040 380 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xx, issue 4 2021, pag. 380 391 potential of romanian vine wastes as a natural resource of trans-resveratrol * alina lenuța crăciun 1 , gheorghe gutt 2 1faculty of food engineering, ”ștefan cel mare„ university, 720225 suceava, romania, alina.craciun@usm.ro , *corresponding author received 8th november 2021, accepted 22th december 2021 abstract: the methods for producing natural resveratrol are of big interest due to the numerous health benefits of this substance and its increasing use in functional foods, food supplements and parapharmaceutical preparations, especially with the recent permission of resveratrol as a novel food ingredient in the european union (commission implementing decision (eu) 2016/1190). since annually a large amount of waste is produced in vine pruning, the aim of this study was to evaluate the potential of vine shoots from ten grape varieties (fetească albă, fetească neagră, muscat ottonel, muscat de hamburg, tămâioasă, coarnă neagră, merlot, cabernet sauvignon, fetească regală and burgund mare) from two different locations to be used as a natural source of trans-resveratrol. to reach this goal, two different solvents were explored, two different storage periods, three different time of collecting of samples in two different years. moreover, a correlation between the type of vine (red or white) and trans-resveratrol content was analyzed. maceration solvent diethyl-ether:ethanol, 1:4 ratio, showed a higher yield of extraction of trans-resveratrol than methanol 80%. increasing the storage time by another 6 weeks positively affected the concentration of resveratrol for 5 samples, and for another 7 negatively. the time of collecting the samples had a high influence on the resveratrol content on shoots samples, highlighting the fact that autumn is the optimal time for harvesting. no correlation could be established among the grape varieties (red or white) and the trans-resveratrol content. the highest concentration of resveratrol was recorded for the fetească neagră variety – 2367.37 mg/kg. d.w. . keywords: trans-resveratrol; food supplements; phytoalexin; vine wastes; hplc. 1. introduction resveratrol (3,4’,5-trihydroxistilbene) is a phytoalexin synthesized by some plants in response to exogenous factors such as uv radiation, chemical stress and fungal parasites and its found in a wide variety of dietary sources including grapes, plums, berries and peanuts [1]. among foodstuffs the main source of resveratrol is red wine, and its moderate dietary consumption is considered to explain the so-called “french paradox” that in some parts of france the death rate caused by heart diseases is remarkably low in spite of high consumption of animal fats. [2]. resveratrol confers a variety of benefits to human health and well-being. this antioxidant has been widely explored as anti-inflammatory, anti-aging, cardio protection and anticancer agent [3]. despite the many health benefits of transresveratrol and the attraction to achieving those benefits by drinking red wine, wine is a poor source of the compound. a recent study has estimated that a daily average of 111 glasses of wine (11.1 liters) has to be consumed in order to reach the minimum dose that would exert therapeutic effects, if wine is the only dietary source of transresveratrol [4]. hence, the need to extract and purify the compound from other http://www.fia.usv.ro/fiajournal mailto:mariap@fia.usv.ro food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 381 sources to prepare trans-resveratrol formulations for research and medicinal purposes is apparent [3]. the impact of resveratrol intake to human body has been intensively study, with positive results, as a result resveratrol supplements are available on the market since 2012 [5], and it already used in cosmetic [6] and food industry as can be seen by the recent permission of resveratrol as a novel food ingredient in the european union (commission implementing decision (eu) 2016/1190) [7]. japanese knotweed (polygonum cuspidatum), an invasive herbaceous plant species is currently the prime source for the production of trans-resveratrol [3,8]. some studies suggest that additional compounds such as emodin present in unpurified or partially purified japanese knotweed root extracts may have laxative effects [9]. since the plant thrives even in heavily polluted environments and soils contaminated with heavy metals, cellular uptake of those contaminants by the root tissues poses further concerns regarding the safety of the commercial preparations of (e)-resveratrol made from root extracts [10]. recently, pruned wood from the vineyard has been shown to accumulate a broad spectrum of stilbenoids ranging from eresveratrol to complex stilbene oligomers [11]. therefore, grape canes represent a promising bioresource containing valuable bioactive compounds with potential applications in medicine because of multiple pharmacological properties of stilbenoids and in agriculture as an alternative source of anti-phytopathogenic substances [12,13]. moreover, the latest research in the field shows that resveratrol content in vine shoots can increase between 400% and 1400% by storage under determined conditions (at 20-25 °c for 6-8 weeks) [14]. viticulture is one of the most important agricultural activities in the world, with approximately 7.4 million hectares cultivated globally in 2018, of which 4.3 million hectares are cultivated in europe. the first country is represented by spain, with an area of 969.000 hectares, followed by china with 875.000 hectares and france with 793.000 hectares. romania ranks 10th in the world as an area cultivated with vines, approximately 180,000 hectares. a quarter of the counties where vines are grown account for over 65% of the total area, vrancea being the county with the largest area [15]. the annual average of by-products resulting from the cleaning of the vine is 1.3 kg of wood/live log, which results in 2x107 tons of wood harvested annually in the world. currently, the only use of this waste is as fertilizer for vines, by grinding and distribution on vineyards [16]. the aim of this study was to determine the potential of vine shoots as a raw material for extraction of resveratrol, a supplement that can be used in food, cosmetic and pharmacological industry. specifically, by improving the extraction and storage conditions, a significantly higher amount of resveratrol can be obtained. thus, from an economic point of view, shoots can be of great interest in obtaining resveratrolbased supplements. this research comes in addition to existent studies as an attempt to determine optimal conditions to extract resveratrol, conditions such as solvent for maceration, season of samples collecting, variety of vine and time of storage samples. 2. materials and methods 2.1. vine shoots samples fifteen samples of vine shoot were collected from two different regions. four samples (vitis vinifera variety merlot, fetească regală, cabernet sauvignon and burgund mare) were collected from region ștefănești, pitești food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 382 (47,6708864;26,2668288) on 30 may 2019, eleven samples were collected from region cotnari, iași (47,6708864;269361914), three of them on 07 october 2019 (vitis vinifera variety fetească albă, muscat de hamburg and muscat ottonel), four samples were colected on 20 may 2020 (tămâioasă, fetească albă, coarnă neagră and muscat ottonel) and four were colected on 07 november 2020 (muscat ottonel, fetească albă, fetească neagră and coarnă neagră). 2.2. reagent and chemicals the trans-resveratrol standard (99% gc) and diethyl ether was purchased from sigma-aldrich co. methanol, ethanol and acetonitrile (lichrosolv for hplc) was obtained from merk. for preparation of the aqueous solutions was used double distilled and demineralized water. the syringe filters with ptfe membrane of 0.22 µm dimension was purchased from pehnomenex. 2.3. standard solutions first a stock solution of resveratrol with a concentration of 1g /l was prepared. the resveratrol standard was initially dissolved in a minimal volume of methanol to guarantee a complete dissolution. before analysis, a set of standards were prepared from the stock solution, diluted in methanol 99%. special care was taken in connection with the degradation of standard solutions, keeping them protected from exposure to air and light. standards were stored in brown glass containers at 200c. 2.4. sample collection and preparation four sets of plant material, marked as i, ii, iii and iv were used in the study, as it follows: i. the samples collected on may 30, 2019 (vitis vinifera variety merlot, fetească regală, cabernet sauvignon and burgund mare) were kept for 6 weeks at 20 °c to increase the concentration of resveratrol, optimal parameters recommended by the latest research in the field [14]. after the 6 weeks, the samples were dried in a thermostat at 45 °c for 24h. then the samples were powdered in a laboratory grinder and macerated with a solution of 80% (v/v) methanol, 5 ml per g of the powdered material for 72 h at room temperature in darkness. after filtration through filter paper, the dry matter was macerated once more with the same amount of the solvent, and the two extracts were combined. until use in analyses the extracts were kept for several days in a refrigerator at 4 °c. ii. the samples collected on 7 october 2019 (vitis vinifera variety fetească albă, muscat de hamburg and muscat ottonel) were kept for 12 weeks at 20 °c to increase the concentration of resveratrol. after the 12 weeks, the samples were dried in a thermostat at 45 °c for 24h. then the samples were powdered in a laboratory grinder and the powdered material was split in two. the powdered material was macerated with the technique described above, with the difference that one sample was macerated with a solution of 80% (v/v) methanol, and one sample was macerated with a solution of diethylether:ethanol 96% in the ratio of 1:4. after that the samples were filtered and stored in same conditions described at i. iii. the samples collected on 20 may 2020 (vitis vinifera variety tămâioasă, fetească food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 383 albă, coarnă neagră and muscat ottonel) were split in two and one part was kept for 6 weeks at 20 °c and one part was kept for 12 weeks on the same conditions. after that, the samples were grinded, macerated and filtered with the technique described above, at point i, with the difference that one part was macerated with a solution of 80% (v/v) methanol, and one part was macerated with a solution of diethylether:ethanol 96% in the ratio of 1:4. iv. the samples collected on 07 november 2020 (vitis vinifera variety muscat ottonel, fetească albă, fetească neagră and coarnă neagră) were prepared with the technique described at iii. 2.5. analytical hplc procedure the extracts obtained were partially purified before hplc analysis. the extracts were first evaporated using a rotary evaporator near to dryness and then redissolved in diethyl ether, and the solution was washed 3 times with 5% sodium bicarbonate solution in a separatory funnel. the diethyl ether layer was recovered, evaporated on a rotary evaporator and redissolved in methanol. the obtained extract was filtered through 0.22 µm ptfe membrane filters prior to hplc analysis and then injected (with a volume of 8 µl) into the hplc instrument (schimadzu, kyoto, japan) for analysis using an spd-m-20a diode array detector. the separation was carried out on a phenomenex kinetex 2.6 µm biphenyl 100 å hplc column 150 x 4.6 mm thermostated at 20°c. elution was carried out with a solvent system consisting of pure water (solvent a) and acetonitrile (solvent b) as previously described by marshall et al. (2012) with modifications. binary gradient consisted of solvent a: pure water and solvent b: acetonitrile as follows: linear gradient from 0% to 10% b in 42 min, 10–40% b in 42.6 min, 40–90% b in 46.5 min. run time was 49.5 min [17]. the solvent flow rate was of 0.5 ml/min. the determination of transresveratrol was carried out at 306 nm detection wavelength. the obtained standard calibration curves showed high degrees of linearity (r2 > 0.9996). data collection and subsequent processing were performed using the lc solution software version 1.21 (shimadzu, kyoto, japan). analyses were performed in duplicate and the quantification was made based on the peak area, using the external standard method. 3. results and discussion 3.1. solvent extraction yield as it shown in table 1, solvent diethylether:ethanol 1:4 ratio have had a higher extraction yield in all cases. the increase in the amount of resveratrol extracted ranged from 5% (fetească albă from 0.20 mg/kg d.w. with methanol 80% to 0.21 mg/kg d.w.) to 1569% (tămâioasă from 0.36 mg/kg d.w. to 6.01 mg/kg d.w.). in most cases the amount of resveratrol extracted with this solvent was considerably higher. most studies recommend methanol 80% [18] or ethanol 80% [19, 20, 21] for extraction of resveratrol from vine shoots samples. gromova et al. (1975) used diethyl ether in the extraction of resveratrol from the bark of pinus sibirica [22] and piyaratne (2018) from the bark of spruce picea mariana [3]. although methanol is a polar organic solvent that provides a higher percentage of extractives, is not highly selective for polyphenols [23]. on the other hand, diethyl ether, who is a relatively nonpolar solvent, is more preferable because of the selectivity for polyphenols, including food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 384 trans-resveratrol. this solvent facilitates the extraction methodology for the next steps of purification. [3]. table 1. resveratrol content of the samples collected from the same area (cotnari, iași) extracted with methanol 80% and diethyl-ether:ethanol 1:4 ratio variety solvent extraction storage time of samples methanol (resveratrol mg/kg d.w.) diethyl-ether:ethanol 1:4 ratio (resveratrol mg/kg d.w.) fetească albă 107.09 178.65 12 weeks muscat ottonel 58.96 106.66 muscat de hamburg 51.27 106.99 tămăioasă 0.36 6.01 6 weeks coarnă neagră 1.42 1.57 muscat ottonel 1.51 12.52 fetească albă 0.37 1.89 tămâioasă 0.35 1.15 12 weeks coarnă neagră 0.21 0.28 muscat ottonel 0.24 0.66 fetească albă 0.20 0.21 fetească neagră 374.03 1676.39 6 weeks fetească albă 113.18 604.73 muscat ottonel 525.04 735.76 coarnă neagră 89.14 468.91 fetească neagră 820.26 2367.37 12 weeks fetească albă 92.98 524.37 muscat ottonel 315.19 525.04 coarnă neagră 157.19 261.21 the research made by aavikasaar et al. (2003) reported that dry extracts of vine stems obtained from its solution in 80% ethanol to be practically insoluble in diethyl ether. on the other hand, by adding diethyl ether to the solution extract 96.6% ethanol, at the diethyl-ether:ethanol ratio 1:4, the extract precipitate, resulting in a decrease of the content of dark brown matter, without the loss of stilbenoids [2]. in this study it was used this solvent as a solvent for maceration to extract resveratrol from vine shoots sample as an attempt to skip the precipitation step and to make the solvent more selective. unexpectedly, this solvent generated considerably higher resveratrol yields. 3.2. influence of storage time on resveratrol content recent publications gave rise to the hypothesis that trans-resveratrol accumulate in vine wastes during postharvest storage [14, 24]. houillé et al. (2015) reported a strong increase, approximately 40-fold, in the concentration of monomer transresveratrol during the first six weeks of storage at 20 °c of pruned grape canes. this phenomenon observed in eight different varieties was due to the induction of stilbenoid metabolism [14]. table 2 shows the vine shoot concentrations of resveratrol after 6 and 12 weeks of storage. the increase in resveratrol concentration is food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 385 recorded for 5 samples, varying between 64% (merlot 17.82 mg /kg d.w. to 29.28 mg /kg d.w.) and 119% (fetească neagră 374.03 mg /kg d.w. to 820.26 mg /kg d.w.) the decrease in resveratrol concentration is recorded for 7 samples, the decrease varying between 2.78% (tămâioasă 0.36 mg/kg d.w. to 0.35 mg/kg d.w.) and 98% (muscat ottonel 12.52 mg/kg d.w. to 0.24 mg/kg d.w.). ewald et al. (2017) reported an enormous increase (400 to 1400%) of transresveratrol during the 6 months of storage. after another 6 moth of storage the trans resveratrol decresed in all grape canes, except for regent variety [25]. the fact that post-pruning storage of vine shoots have a direct impact on the concentration of trans-resveratrol is confired by the study made by cebrián et al. (2017), who reported a significant increase of this monormer on the first 3 month of storage for the variety airén (77.10 to 151.60 mg/kg d.w.) and cencibel (50.41 to 224.83 mg/kg d.w) [26]. table 2. resveratrol content of vine shoots samples after 6 and 12 weeks of storage at 20 °c extracted with methanol 80% variety storage time sample set 6 weeks (resveratrol mg/kg d.w.) 12 weeks (resveratrol mg/kg d.w.) merlot 17.82 29.28 i cabernet sauvignon 20.58 23.39 fetească regală 17.74 3.13 burgund mare 6.75 443 tămâioasă 0.36 0.35 iii muscat ottonel 12.52 0.24 coarnă neagră 1.42 0.21 fetească albă 0.37 0.20 fetească neagră 374.03 820.26 iv fetească albă 113.03 92.98 muscat ottonel 149.98 315.48 coarnă neagră 89.14 157.19 accumulation of trans-resveratrol during storage period is due to activity of key genes involved in the stilbene biosynthesis. houille et al. (2015) reported that the presence of pal, 4cl and c4h, the three genes of the general phenylpropanoid pathway, were constitutively expressed over the 6 weeks of storage, and in addition, sts (forming e-resveratrol) was induced during the first 4 weeks of storage. therefore, vine shoots samples are still transcriptionally active during the storage period [14]. 3.3. influence of collecting period resveratrol concentration of the 3 sample sets of vine shoots varied considerably, as can be seen in table 3., with the highest concentrations being recorded for the sample set collected in november 2020. the lowest concentrations were recorded for the sample set collected in may 2020. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 386 the influence of the harvesting time can be seen clearly for the variety fetească albă and muscat ottonel. the most unfavorable harvest season would be spring, obtaining the lowest values of resveratrol. the most favorable season is autumn, especially november. resveratrol content may vary because of the difference in the microclimate and phytosanitary conditions in which vine grows [27]. ji et al. (2014) studied the distribution and season regularity of resveratrol in different tissues of vitis armurensis and shows that resveratrol contents are augmented gradually from january to september, and then decrease until january of the following year [28]. this study is in agreement with the result of this study regarding the fact that resveratrol reaches its maximum concentration in autumn. table 3. resveratrol content for vine shoots samples collected from the same area (cotnari, iași) extracted with diethyl-ether:ethanol 1:4 ratio after 12 weeks of storage 07 october 2019 (ii) 22 may 2020 (iii) 07 november 2020 (iv) variety resveratrol mg/kg d.w. variety resveratrol mg/kg d.w. variety resveratrol mg/kg d.w. fetească albă 178.65 fetească albă 0.51 fetească albă 524.37 muscat ottonel 109.66 muscat ottonel 0.66 muscat ottonel 525.04 muscat de hamburg 106.66 coarnă neagră 0.28 coarnă neagră 261.21 tămăioasă 1.15 fetească neagră 2367.37 the variation in trans-resveratrol concentration may also be influenced by the year of harvest, namely the climatic and growing conditions of that year. thus, in 2020, the highest concentrations of trans-resveratrol were recorded in the extracts of vine shoots. this is also confirmed by guerrero et. al (2016) who determined the trans resveratrol content for 4 vine shoots variety in 2014 and 2015 and showed that this was the most varying compound within the years. transresveratrol was more abundant in 2015 than in 2014, in all varieties [29]. 3.4. variety influence red wines, in generally, contain a high amount of trans-resveratrol, while in white wines trans-resveratrol occurs mostly in low levels [30 32]. the analysis was made to see if this ascertainment applies also to vine shoots samples from different varieties. the shoot samples were divided into 2 categories, depending on the variety of grapes, white or red to see if the resveratrol content can be correlated with the grape variety. more samples of both varieties, red and white, collected and processed under the same conditions needed to be studied to establish a correlation on the difference of transresveratrol content. the average concentration of transresveratrol is 82.3 mg/kg d.w. for white varieties and 221.73 mg/kg d.w. for red varieties. anastasiadi et al. (2012) found the average concentration of transresveratrol to be 149 mg/kg d.w. for red and 113 mg/kg d.w. for white varieties in vine shoots [33]. piñeiro et al. (2016) reported a trans-resveratrol content for vine shoots extracts who ranged between food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 387 37.4 1529 mg/kg d.w. for white varieties and 45.6 664.9 mg/kg d.w. for red varieties [34]. the results are much higher for red varieties in our study because the red variety who had the higher resveratrol content, fetească neagră (820.26 mg/kg d.w.), had 2.6 times more than the white variety muscat ottonel (315.48mg/kg d.w.), who had the higher resveratrol content among the white varieties. table 4. resveratrol content for vine shoots samples collected from the same area (cotnari, iași) extracted with methanol 80% after 12 weeks of storage white grape vines red grape vines sample set variety resveratrol mg/kg d.w. variety resveratrol mg/kg d.w. fetească albă 107.9 muscat de hamburg 51.27 ii muscat ottonel 58.96 tămăioasă 0.35 coarnă neagră 0.21 iii muscat ottonel 0.24 fetească albă 0.20 muscat ottonel 315.48 fetească neagră 820.26 iv fetească albă 92.98 coarnă neagră 157.19 3.5. potential of vine shoot wastes as a source for resveratrol extraction agricultural wastes represent a largely ignored source of high-value phytochemicals substances and valueadded industrial products that could contribute to sustainability objectives. summer and autumn trimming of vine is performed to maintain desired canopy shape best suited for optimal grape production. during this process considerable amount of green mass (portions of shoots, leaves and tendrils) is removed [35]. the global wine and table grape industry, with annual sales of >us$ 100 billion, generates large quantities of waste each year that is generally composted or burned for disposal. grapevines are pruned annually, and these wastes represent a potentially important global source for extraction of transresveratrol [36]. the resveratrol content of vine waste is between 0.20 – 2367.37 mg/kg d.w. for the varieties researched in this study. by optimizing extraction parameters and sample processing after collection the resveratrol extraction yield could be significantly improved. these findings are in agreement with other studies who reported a resveratrol content between 1300 and 4100 mg/kg d.w. in cane extracts [37]. in the work of cetin et al. (2011) resveratrol content varied between 9.5 and 39.4 μg/100g d.w [38]. aaviksaar et al. (2003) determined the presence of trans-resveratrol in estonian grape cultivars in concentrations between 100 and 4700 mg/kg d.w [2]. in the survey of trans-resveratrol contents of grape cluster rachis from nine vitis vinifera varieties, melzoch et al. (2001) found levels ranging from 7 to 480 mg/kg d.w [39]. higher levels of trans-resveratrol that is approaching to the results of this study were reported in china by zhang et al. (2011), who found concentrations between 570 and 1751 mg/kg fresh matter in oneyear-old canes [40]. püssa et al. (2006) reported a resveratrol content between food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 388 1100 and 3200 mg/kg d.w. for hybrid cultivars from estonia [41]. piñeiro et al. (2017) reported a resveratrol content for spanish vine shoots samples between 122 – 5361 mg/kg d.w [20]. this results confirmed that production of stilbene by grapevine largely depends on the grape cultivar, climate, extraction conditions and the expose to fungal parasites and physiological stresses such as uv light and heavy metals [42]. zhang et al. (2011) analyzed the transresveratrol content for 165 grape cane samples from three major grape production regions. of the cultivars analyzed, vitis vinifera (937.9 mg/kg fw –fresh weight) have had the higher amount of transresveratrol than hybrids of v. vinifera and v. lambrusca (571 mg/kg fw). among the grape vine varieties, the grape cane extracts coming from wine grapes vine (64.7 to 1,751.6 mg/kg fw) had a higher content of trans-resveratrol than table grapes vine (671.2 to 841.8 mg/kg fw) [40]. the extraction method is also critical to ensure a good recovery of stilbenoids and the range of extraction methods tends to bias the results [43]. furthermore, an accurate quantification of stilbenoids relies on calibration curves with pure stilbenoid standards because the quantification of stilbenoids as a resveratrol equivalent leads to an underestimation of the concentrations of resveratrol oligomers [11]. additionally, a disparity in stilbenoid composition of vine shoot samples has been observed during storage, the time elapsed between vine pruning and the phytochemical analyses. an increase in the transresveratrol concentration from 6 weeks to 12 weeks was observed. temperature of the storage of shoot samples have a high influence on the accumulation of transresveratrol. houille et al. (2015) reported a delay in accumulation for temperatures below 20 °c (15 °c and 5 °c). after a short heatshook treatment (2h at 65 °c) applied to vine shoot samples with the intent to denature proteins, inhibits the accumulation of trans-resveratrol during the storage period and suggests the involvement of active enzymes in this phenomenon. similarly, when canes were stored at −20 °c, trans-resveratrol was not induced [14]. 4. conclusion results of this study show that solvent diethyl-ether:ethanol 1:4 ratio used for extraction of resveratrol from vine shoots is better than methanol 80%, obtaining greater yields, up to 1569%. storing the shoots for another 6 weeks influenced both positively and negatively the amount of resveratrol. the increase or decrease in the amount of resveratrol may be due to the variety, cultivation conditions or chemical composition of the shoots at time of harvest. harvesting time of the shoots can significantly influence the amount of resveratrol available in the shoots. our findings show that november is the optimal harvesting time the vine waste as a prime material for resveratrol extraction, the variety fetească neagră having a resveratrol content of 2367.37 mg/kg d.w. the processing of waste in optimal conditions can increase the extraction efficiency much compared to their processing at the time of harvest. it could not be established a correlation between the color of grape variety and resveratrol content, as in the case of wines. these results suggest that romanian vine waste can be an excellent source for resveratrol extraction. further improvements can be done on establishing the optimal parameters for extracting resveratrol from vine shoots and purification methodologies. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 389 5. acknowledgment this paper has been financially supported within the project entitled “decidedevelopment through entrepreneurial education and innovative doctoral and postdoctoral research”, project code pocu/380/6/13/125031, project cofinanced from the european social fund through the 2014-2020 operational program human capital. 6. references [1]. al-jumaily, e. f., hamid, g. s., & ali, k. f. synthesis and total phenol content of new resveratrol derivative. open j adv drug deliv, 2(3), 320-329, (2014) [2]. aaviksaar, a., haga, m., pussa, t., roasto, m., & tsoupras, g. purification of resveratrol from vine stems. proceedingsestonian academy of sciences chemistry (vol. 52, no. 4, pp. 155-164). (2003) [3]. piyaratne, p. s. extraction and purification of (e)-resveratrol from the bark of maine's native spruces. (2018) [4]. lachenmeier, d.w., godelmann, r., witt, b., riedel, k., rehm, j. can resveratrol in wine protect against the carcinogenicity of ethanol? a probabilistic dose response assessment. int. j. cancer 134:144-153. (2014) [5]. heger, a., ferk, f., nersesyan, a., szekeres, t., kundi, m., wagner, k. h., & knasmüller, s. intake of a resveratrolcontaining dietary supplement has no impact on dna stability in healthy subjects. mutation research/genetic toxicology and environmental mutagenesis, 749(1-2), 82-86. (2012) [6]. jeandet, p., clément, c., tisserant, l. p., crouzet, j., & courot, é.. use of grapevine cell cultures for the production of phytostilbenes of cosmetic interest. comptes rendus chimie, 19(9), 1062-1070. (2016) [7]. commission implementing decision (eu) 2016/1190 of 19 july 2016 authorising the placing on the market of trans-resveratrol as a novel food ingredient under regulation (ec) no 258/97 of the european parliament and of the council (notified under document c(2016) 4567) c/2016/4567, available online on: https://eur-lex.europa.eu/legalcontent/ga/txt/?uri=celex:32016d1190 [8]. wang, d. g., liu, w. y., & chen, g. t. a simple method for the isolation and purification of resveratrol from polygonum cuspidatum. journal of pharmaceutical analysis, 3(4), 241-247. (2013) [9]. srinivas, g., babykutty, s., sathiadevan, p.p., srinivas p. molecular mechanism of emodin action: transition from laxative ingredient to an antitumor agent. med. res. rev. 27(5):591-608 (2007) [10]. berchová-bímová, k., soltysiak, j., vach, m. role of different taxa and cytotypes in heavy metals absorption in knotweeds (fallopia). sci. agric. bohem. 45(1):1118. (2014) [11]. lambert, c.; richard, t.; renouf, e.; bisson, j.; waffo-teguo, p.; bordenave, l.; ollat, n.; merillon, j.m.; cluzet, s. comparative analyses of stilbenoids in canes of major vitis vinifera l. cultivars. j. agric. food chem., 61, 11392−11399 (2013) [12]. schnee, s.; queiroz, e. f.; voinesco, f.; marcourt, l.; dubuis, p.h.; wolfender, j.-l.; gindro, k. vitis vinifera canes, a new source of antifungal compounds against plasmopara viticola, erysiphe necator, and botrytis cinerea. j. agric. food chem. 2013, 61, 5459−5467. (2013) [13]. moreira, m. m., barroso, m. f., porto, j. v., ramalhosa, m. j., švarcgajić, j., estevinho, l., & deleruematos, c. potential of portuguese vine shoot wastes as natural resources of bioactive compounds. science of the total environment, 634, 831-842. (2018) [14]. houillé b., besseau s., courdavault v., oudin a., glévarec g., delanoue g., guérin l., simkin a.j., papon n., clastre m., giglioliguivarc’h n., lanoue a., byosynthetic origin of resveratrol acummulation in grape canes during pastharvest storage, j. agric. food chem. 63, pg. 1631-1638, (2015) [15]. 2019 statistical report on world vitiviniculture, available online on: https://www.oiv.int/public/medias/6782/oiv-2019statistical-report-on-world-vitiviniculture.pdf https://eur-lex.europa.eu/legal-content/ga/txt/?uri=celex:32016d1190 https://eur-lex.europa.eu/legal-content/ga/txt/?uri=celex:32016d1190 https://www.oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf https://www.oiv.int/public/medias/6782/oiv-2019-statistical-report-on-world-vitiviniculture.pdf food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 390 [16]. angelov, g., boyadzhiev, l., & georgieva, s. useful bioactive substances from wastes: recovery of trans-resveratrol from grapevine stems. the open chemical engineering journal, 10(1) (2016) [17]. marshall, d.a., stringer, s.j., spiers, j.d. stilbene, ellagic acid, flavonol, and phenolic content of muscadine grape (vitis rotundifolia michx.) cultivars. pharm crops 3:6977. (2012) [18]. soural, i., vrchotová, n., tříska, j., balík, j., horník, š., cuřínová, p., & sýkora, j. various extraction methods for obtaining stilbenes from grape cane of vitis vinifera l. molecules, 20(4), 6093-6112. (2015) [19]. jesus, m. s., genisheva, z., romaní, a., pereira, r. n., teixeira, j. a., & domingues, l. bioactive compounds recovery optimization from vine pruning residues using conventional heating and microwave-assisted extraction methods. industrial crops and products, 132, 99-110. (2019) [20]. piñeiro, z., marrufo-curtido, a., vela, c., & palma, m. (2017). microwaveassisted extraction of stilbenes from woody vine material. food and bioproducts processing, 103, 18-26. (2017) [21]. romero-pérez, a. i., lamuelaraventós, r. m., andrés-lacueva, c., & de la torre-boronat, m. c. method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. effect of powdery mildew on the stilbene content. journal of agricultural and food chemistry, 49(1), 210-215. (2001) [22]. gromova, a. s., tyukavkina, n. a., luckiy, v. i., kalabin, g. a. & kushnarev, d. f. oxystilbenes in the bark of pinus sibirica. khim. prir. soedin., 1975, 677–682 (in russian). (1975) [23]. spigno, g., tramelli, l., & de faveri, d. m. effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics. journal of food engineering, 81(1), 200208. (2007) [24]. gorena, t., saez, v., mardones, c., vergara, c., winterhalter, p., & von baer, d. influence of post-pruning storage on stilbenoid levels in vitis vinifera l. canes. food chemistry, 155, 256–263. (2014) [25]. ewald, p., delker, u., & winterhalter, p. (2017). quantification of stilbenoids in grapevine canes and grape cluster stems with a focus on long-term storage effects on stilbenoid concentration in grapevine canes. food research international, 100, 326-331 (2017) [26]. cebrián, c., sánchez-gómez, r., salinas, m. r., alonso, g. l., & zalacain, a. effect of post-pruning vine-shoots storage on the evolution of high-value compounds. industrial crops and products, 109, 730-736 (2017) [27]. versari, a., parpinello, g. p., tornielli, g. b., ferrarini, r., & giulivo, c. stilbene compounds and stilbene synthase expression during ripening, wilting and uv treatment in grape cv. corvina. journal of agricultural and food chemistry, 49, 55315536. (2001) [28]. ji, m., li, q., ji, h., & lou, h. investigation of the distribution and season regularity of resveratrol in vitis amurensis via hplc–dad–ms/ms. food chemistry, 142, 61-65. (2014) [29]. guerrero, r. f., biais, b., richard, t., puertas, b., waffo-teguo, p., merillon, j.-m., & cantos-villar, e. grapevine cane's waste is a source of bioactive stilbenes. industrial crops and products, 94, 884– 892. (2016) [30]. ratola, n., luís faria, j., & alves, a. analysis and quantification of trans-resveratrol in wines from alentejo region (portugal). food technology and biotechnology, 42(2), 125-130. (2004) [31]. cvejic, j. m., djekic, s. v., petrovic, a. v., atanackovic, m. t., jovic, s. m., brceski, i. d., & gojkovicbukarica, l. c. determination of trans-and cisresveratrol in serbian commercial wines. journal of chromatographic science, 48(3), 229-234. (2010) [32]. pajović-šćepanović, r., wendelin, s., & eder, r. phenolic composition and varietal discrimination of montenegrin red wines (vitis vinifera var. vranac, kratošija, and cabernet sauvignon). european food research and technology, 244(12), 22432254. (2018) [33]. anastasiadi m., pratsinis h., kletsas d., skaltsounis a.-l., haroutounian s.a.: grape stem extracts: polyphenolic content and assessment of their in food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xx, issue 4 – 2021 alina lenuța crăciun, gheorghe gutt, potential of romanian vine wastes as a natural resource of trans-resveratrol, food and environment safety, volume xx, issue 4 – 2021, pag. 380 – 391 391 vitro antioxidant properties. lwt – food science and technology, 48: 316–322. (2012) [34]. piñeiro, z., marrufo-curtido, a., serrano, m. j., & palma, m. ultrasoundassisted extraction of stilbenes from grape canes. molecules, 21(6), 784 (2016) [35]. lachman, j., kotíková, z., hejtmánková, a., pivec, v., pšeničnaja, o., šulc, m., & dědina, m. resveratrol and piceid isomers concentrations in grapevine shoots, leaves, and tendrils. horticultural science, 43(1), 25-32. (2016) [36]. rayne s., karacabey e., mazza g. : grape cane waste as a source of transresveratrol and trans-viniferin: high-value phytochemicals with medicinal and anti-phytopathogenic applications. industrial crops and products, 27: 335–340. (2008) [37]. karacabey e., mazza g.: optimization of solid-liquid extraction of resveratrol and other phenolic compounds from milled grape canes (vitis vinifera). journal of agricultural and food chemistry, 56: 6318–6325 (2008) [38]. çetin, e. s., altinöz, d., tarçan, e., & baydar, n. g. chemical composition of grape canes. industrial crops and products, 34(1), 994-998 (2011) [39]. melzoch k., hanzlíková i., filip v., buckiová d., šmidrkal j.: resveratrol in parts of vine and wine originationg from bohemian and moravian vineyard regions. agriculturae conspectus scientificus, 66: 53–57 (2001) [40]. zhang, a., fang, y., li, x., meng, j., wang, h., li, h., ... & guo, z. (2011). occurrence and estimation of trans-resveratrol in one-year-old canes from seven major chinese grape producing regions. molecules, 16(4), 2846-2861. (2001) [41]. püssa t., floren j., kuldkepp p., raal a.: survey of grapevine vitis vinifera stem polyphenols by liquid chromatography-diode array detection-tandem mass spectrometry. journal of agricultural and food chemistry, 54: 7488–7494. (2006) [42]. gonzalez-barrio r., beltran d., cantos e., gil m.i., espin j.c., tomasbarberan f.a.: comparison of ozone and uvc treatments on the postharvest stilbenoid monomer, dimer, and trimer induction in var. “superior” white table grapes. journal of agricultural and food chemistry, 54: 4222–4228. (2006) [43]. karacabey, e.; bayindirli, l.; artik, n.; mazza, g. modeling solid−liquid extraction kinetics of trans-resveratrol and trans-εviniferin from grape cane. j. food process eng., 36, 103−112. (2013) 1. introduction comparison between the parameters of thhroufall and bulk deposition measured in twoa laboratories using the same methods doi: https://doi.org/10.4316/fens.2022.021 218 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 3 2022, pag. 218 229 comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods carmen iacoban 1 , marius curcă 1 , cosmin ilie cuciurean 1,2 , zitta csergo 3 , *amelia buculei 1,4 1"marin drăcea” national research-development institute in forestry, station câmpulung moldovenesc, romania 2 "ștefan cel mare" university of suceava, faculty of forestry, suceava, romania 3 ocolul silvic de regim gheorgheni sa, romania 4"ștefan cel mare" university of suceava, faculty of food engineering, suceava, romania *corresponding author received 5th august 2022, accepted 29th september 2022 abstract: among the main concerns of the researchers is the comparability of the analysis methods, in order to highlight possible errors. several statistical approaches and graphical tools are available to investigate sources of analytical error and for decision-making. in this article, we present an operative protocol for the comparison of the precision for two laboratories with the same quantitative analytical methods. this paper describes the importance of achieving sequential stages, including experimental design, familiarization with the method of optimal research analysis, quality evaluation, selection of sample, definition of acceptability criteria. sample measurement, data analysis and evaluation, final decision and reporting are also discussed and exemplified. the reason for performing a comparison experiment is to estimate the type and magnitude of systematic error between two laboratories with the same method and to judge if the two laboratories are identical within the inherent imprecision methods or within preset analytical quality specifications. finally, the purpose was to check if same ranges of concentrations and fluxes of pollutants are obtained using the same methods of analysis, but in two different labs. key words: validation, analytical error, pollutants 1. introduction atmospheric deposition of pollutants and nutrients are usually important factors in environmental monitoring, enabling massbudget studies, and evaluations of element cycling within terrestrial and aquatic ecosystems, reconstruction and prognosis of environmental biogeochemical trends [1]. statistical comparison of two analytical methods or laboratories is a persistent task and one of the most important steps in the method of validation process. an important question is why and in which way a correction can be made in case when a systematic error is indicated in the performed method comparison study. in order to receive harmonized results, it is than necessary to make a correction of the results obtained by one analyzer with respect to another [2]. when a new measurement method or device is developed it is important to test its agreement with other standardized or already established methods. the agreement is considered adequate when we might replace the old method with the new one. to test for agreement, the same samples are measured using both new and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 219 old method. in the case of destructive methods or where there are other reasons which prevent repeatability of measurement on the same samples (space and time variability), a larger number of samples is measured from the same population. within the measurement comparison approaches the agreement is evaluated by testing for significant systematic differences. the differences or bias can be fixed (same shift across the range of values) or proportional (shift related to the magnitude of a measured variable). what is sought for in method comparison studies is actually not the agreement but the bias [3] which results from the statistical foundations itself. a comparison between the inferential method and the throughfall method [4,5] is a cost-effective way to evaluate the inferential method; however, this procedure is limited to forest vegetation. in this procedure, the required measurements other than the inferential method related are wet-only deposition, throughfall, and stemflow. it is, therefore, desired that this procedure should be performed at a site where monitoring both for wet and dry deposition is conducted. while foliar extraction methods address processes on the scale of leaves, throughfall and watershed mass balance methods are needed to address deposition to individual trees, forest stands and small catchments. to the extent that deposited material is washed from the canopy by rain, the flux of an ion below the canopy in throughfall provides some information on dry deposition. throughfall methods are generally limited in space to forest plots or small catchments (<100 of m2). if locations exist where deposited so4 2is a conservative ion within an entire watershed, hydrologic mass balance methods may be used to derive mean deposition rates over larger spatial and possibly longer time scales. these calculations require that the hydrologic cycle of the system be well characterized and that significant sources and sinks of so4 2do not exist within the system, or are well quantified [6,1]. from a series of laboratory experiments, takenaka developed simple relationships illustrating that the fraction of the volatile ions emitted was dependent on the ratio of the difference between non-volatile cations and anions and the volatile ion concentrations [7]. the total mass reemitted and their rates were then dependent on the volume of the surface wetness and rate of drying. wentworth demonstrated the role of dew as a temporary reservoir for nh3 by measuring its volume and ion balance along with the dynamics of wetting and drying of the surface in relation to 62 atmospheric nh3 concentrations in a grass field. at their site, dew composition suggested that nearly all of nh4 + in the dew was emitted to the atmosphere as nh3 during evaporation [8]. studies of dew chemistry and reemission continues of volatile compounds should consider the modeling framework of burkhardt and, to the practical extent, include chemical analyses that will allow for extension of the results to this cuticular modeling framework [9]. other researchers have studied conventional throughfall collection methods that are labor intensive and analytically expensive to implement at broad scales. this study was conducted to test an alternative approach requiring infrequent sample collection and a greatly reduced number of chemical analyses. the major objective of the study was to determine nitrogen deposition measurements in bulk throughfall collected under pine (pinus sp.) canopies and in forest clearings were compared between co-located conventional throughfall solution collectors and ier throughfall food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 220 collectors using mixed bed ier columns [10]. previous studies have reported considerable and site-specific differences between these methods, complicating the interpretation of results [11, 12, 13, 14]. for the estimate the deposition, many researchers used canopy budget and the inferential method with correction factors [15,16]. the authors noticed that high precipitation amounts partly explained remaining differences in wet deposition and larger discrepancies were observed when dry deposition estimates are compared between the emissions-based approach and the other two approaches. the authors noted that the remaining differences for wet deposits are correlated with some large amounts of precipitation. larger discrepancies were also observed when dry deposition estimates are compared between the emission-based approach and the other two approaches. these differences occur due to weather conditions and their effects on tree species [17,18]. other researchers applying different methods to distinguish between dry deposition and canopy leaching and to add to the almost non-existing deposition measurements for dissolved cu, pb, mn, v, zn, ni and cd in two spanish forests differentially exposed to the urban and industrial environment. no significant differences in mean bulk deposition concentrations or fluxes were found between sites, indicating little differential effect of the urban/industrial environment on bulk precipitation chemistry. at both sites, throughfall and stemflow fluxes increased relative to bulk deposition for all elements, except for zn and cd. the relative contribution of leaching and dry deposition was evaluated through: the seasonal variability of throughfall, regressions of element fluxes on water flux in net throughfall [19, 20]. the authors also noted important aspects in the optimal estimation of the sample, related to the placement of the respective samples, the proper identification of the collection area. it was pointed out that the field intercomparison described by these researchers provided a good insight into the various aspects that contribute to the overall accuracy of measurements. [21]. a comparison study between two laboratories results of the same samples and methods is a new model research experiment. we purpose to study the constant systematic errors that are systematic deviations estimated as the average differences between laboratories. proportional systematic error means that the differences between the 2 experiments are proportionally related to the level of measurements. the higher the values, the lower the error. estimates of random error may help in assessing validity of the measurements by the individual laboratories and help identify unexpected test results arising from sample mix-ups, transposition errors, and other mistakes. the comparison between the values obtained in the two laboratories relates information concerning the comparability within the icp forests laboratories and not only. 2. matherials and methods before the measurements are conducted, is important to know analytical error that is allowable without compromising the test. it usually is very helpful to collect and write down information relating to both the new method and the comparative method. information on sample requirements, analytical process, reaction principles, calibration procedure, calculations, known interferences, and anticipated analytical performance is essential if unexpected or aberrant results occur. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 221 in this phase, a working procedure is established. in practical terms this means that one establishes sufficient working the competence with the method so one can correctly prepare reagents, set up the analyzer, calibrate the method and obtain test results. the quantification limits are based on analytical quality specifications. for quantitative and qualitative monitoring of atmospheric deposition, collectors were installed at 02.08.2018 and samples were collected during 16.08.2018– 03.08.2021, from the research area, located in the unit u.a. 35a, u.p. i, in the framework of composesoratul joseni location of the studied pilot is found in figure 1. the description of the studied plod and stand is given in table 1. fig. 1. location of the studied plot (u.a. 35 a, u.p. i, composesoratul joseni) table 1. description of the studied plod and of the stand (*amenajamentul ocolului silvic de regim gheorgheni sa, up, 2018-2021) studied plod u.a. 35 a area 12.9 ha gf 2-1b sup:a ts:3333; tp:1211; sol:3301 side medium wave ne exhibition inc.18g altitude 910-1010 m litter continue-normal flora type asparula-oxalis. natural fundamental superior productivity, relatively even aged trees current composition 6 mo 4br (mo=spruce, br=fir) sort mo tick and very tick timber, br tick and very tick exploitable age 110 years stand age 80 years according to barbu and iacoban [22], from incds marin drăcea, the working methodology consist of installation of collectors for precipitation in the open field and under the forest canopy. nine collectors for atmospheric deposition were food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 222 installed. out of the 9 collectors, 3 were installed in open land and 6 under the canopy. because the monitoring of atmospheric depositions was also carried out during the winter period, two types of collectors were used. the summer collectors are made of pvc gutters, equipped at one end with a collecting funnel, which directs liquid precipitation into a collection pe bag. each of them had an exposure surface of 0.1 m2. the gutters were fixed with a slope of approximately15 degrees, supported on wooden stakes, at a height of 0,8-1,0 m. a funnel was fixed at the lower end of the gutter, equipped with a hose, for collecting and directing the collected water in a bag installed in the ground, protected by a cover from the action of solar radiation. winter catchers are made of a frame with a diameter of 17 cm that supp orts the collection bag, so that solid precipitation falls directly into the bag, without tipping over. the methodology was implemented at ocolul silvic de regim gheorgheni sa, in the field and in the laboratory. precipitation during the growing season were collected twice a month, and during the cold season once a month. the samples were stored at maximum +4°c, in order to avoid changes in the chemical composition of samples, before analysis. for this study, aliquots of precipitation samples were sent from gheorgheni to the laboratory of incds “marin drăcea” câmpulung moldovenesc, in order to analyse the same parameters, with the same methods, in the two laboratories. the analysis of physicochemical parameters of the precipitation samples ph, conductivity, concentrations of cations: na+, n-nh4 +, k+, mg2+, ca2+ and anions: s-so4 2-, n-no3 și clwas the one applied within the icp forests program and is fully described by manny authors [22, 23]. in order to implement the icp forests methodology in the ocolul silvic de regim gheorgheni sa laboratory, reagents of analytical grade and prepared standards for instruments calibration were sent from the incds “marin drăcea” câmpulung moldovenesc laboratory. a separate sub-sample was taken, prior filtration, for the determination of ph and conductivity (as stated in iso 10523 and iso 7888). standardized analytical methods and procedures were used, preferably iso, specified by icp forests, in the two laboratories. for the determination of ion concentrations of cl-, s-so4 2-, n-no3 -, na+, n-nh4 +, k+, mg2+ and ca2+ from precipitation, an ion chromatography system dionex ics-3000, and an ion chromatography system dionex ics5000+, respectively, were used. the two instruments were equipped with systems for the simultaneous analysis of anions and cations. a total number of 110 samples was analysed: 56 bulk deposition and 54 throughfall samples. all reagents used were of high purity (merck or fluka). the determination of ph and conductivity were performed as soon as possible, after the sample’s reception in labs. samples were filtered and the concentrations of cations and anions were measured. comparative analytical methods and devices used are presented in table 2. internal clues of controlling and ensuring the quality of the analyses in the laboratory, according to the icp forests methodology, are: comparing the calculate conductivity with the measured conductivity, cheking the cation-anion balance, verifying that na/cl ratio is in the 0.5-1.5 range (but no more than 4.0); using of control charts for samples of known concentrations. the quality assurance and control (qa/qc) procedures further include the use of control charts for internal reference food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 223 material to check long-term comparability within national laboratories, as well as participation in periodic laboratory ring tests [24] and field inter-comparisons [25; 26] to check the international comparability. table 2. parameters, analytical methods and equipment used in the experiment 3. results and discussion precipitation is the most effective process in removing and transporting different ionic compounds, pollutants, and soluble gases from the atmosphere to the earth’s surface [27] and helps to understand the relative contribution of different sources of incds marin drăcea campulung ocolul silvic de regim gheorgheni sa analytical methods/standards no. parameter (um) equipment parameter (um) equipment 1. ph sianalyticstitrolineeasy titrator, tz3230 type, with 20 ml burette, n62 electrode, range 0-14 ph units ( -5...+100 °c) ph wtw ph meter, inolab type 7310, sentix 41 electrode, range: 0-14 ph units iso 10523 2. conductivity (µs/cm) wtw conductivity meter inolab 730p, with conductivity cell tetracon 325, range: 0-1000 µs/cm; 0-100 ms/cm conductivity (µs/cm) (0 50⁰c). sr en 27888:1997 3. k+ (mg/l) dionex ion chromatography dual system ics3000, for simultaneous determination of cations and anions with dp (dual pump), dc (compartement detector), and autosampler. the pumps ic work at a flow rate of 0,001 to 1,0 ml/min and operating pressures up to 41 mpa (6000 psi), recommended 35 mpa (5000 psi). k+ (mg/l) dionex ion chromatography dual system ics-5000+ thermoscientific), with dp (dual pump), eg (eluent generator), dc (compartement detector), and autosampler. the capillary pumps ic work at a flow rate of 0,001 to 3,0 ml/min and operating pressures up to 41 mpa (6000 psi), recommended 35 mpa (5000 psi). en 27888 4. ca2+ (mg/l) dionex ion cromatograf ics3000 ca2+ (mg/l) wtw conductivity meter, inolab 7110 with conductivity cell tetracon 325, range: 0-1000 µs/cm en iso 7980 5. mg2+ (mg/l) dionex ion cromatograf ics3000 mg2+ (mg/l) dionex ion chromatography dual system ics-5000+ en 15309 6. na+ (mg/l) dionex ion cromatograf ics3000 na+ (mg/l) dionex ion chromatography dual system ics-5000+ ( en 15309 7. n-nh4+ (mg/l) dionex ion cromatograf ics3000 n-nh4+ (mg/l) ion cromatograf dionex ics5000+ (thermoscientific), en iso 1491 8. cl(mg/l) dionex ion cromatograf ics3000 cl(mg/l) ioncromatograf dionex ics5000+ (thermoscientific), en iso 10304 9. n-no3 (mg/l) dionex ion cromatograf ics3000 n-no3 (mg/l) ioncromatograf dionex ics5000+ (thermoscientific), en iso 10304 10. s-so42 (mg/l) dionex ion cromatograf ics3000 ioncromatograf dionex ics5000+ (thermoscientific), en iso 10304 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 224 atmospheric pollutants. the composition of rainwater varies from site to site and region to region due to the difference of the local and remote emission sources [28] and reflects all the characteristics of air pollutants [29]. the amount of precipitation collected in the open field and under the forest canopy was calculated considering the reception surface of collectors. for this study, samples were split and half stored at the ocolul silvic de regim gheorgheni sa laboratory and half sent to the incds “marin drăcea” câmpulung moldovenesc laboratory. for 2018 and 2021, the periods of collection don’t cover all the years, so the calculated values of concentrations (fig.2) and fluxes (fig.3) are mean annual only for 2019 and 2020. calculating tf and bd (q·c·10-2, in kg ha-1) by multiplication of the precipitation quantity (q, in l m-2), the concentrations (c, in mg l-1) and the unity conversion factor 10-2. for the concentrations and fluxes of sso4 2 ion, the results obtained are lower in gheorgheni, but are comparable to those obtained at campulung. the values of the concentrations and fluxes of ca2+ ion measured and calculated in the two laboratories are comparable, except for 2018, under the canopy, when higher results were obtained in campulung than in gheorgheni. fig. 2. mean period concentrations of cations and anions determined in the same bulk deposition and throughfall samples in two laboratories from romania for the period 2018-2021 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 225 the values of the concentrations and fluxes of ions calculated under the forest canopy, for the ion of na2+ are comparable, except in 2019 under the canopy, when higher results were obtained in gheorgheni than in campulung. for mg2+ the values of the concentrations and fluxes are equal or lower in gheorgheni, except for 2019, under the canopy. higher values were highlighted for the nnh4 + ion at gheorgheni, due to the low stability of this ion. the tendency to transform to other compounds (n2, by reduction or no3 -, by oxidation) is high for ammonium ions. the highest differences are found in the years 2018, 2019 and 2021, for the results obtained in the open field, at gheorgheni, because the analysis was performed before those from campulung. fig. 3. depositions of cations and anions determined using the same bulk deposition and throughfall samples analyzed in two laboratories from romania for the period 2018-2021 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 226 the concentrations and fluxes of k+ ion calculated at the two laboratories are very close, both in open field and under the canopy. it is obvious that the results obtained at gheorgheni, for the n-no3 ion are systematically lower both in bulk deposition and in throughfall. the samples collected under the canopy may also contain other compounds that may interfere and change the obtained values. in the nh3–so2–liquid water system, nh3 neutralizes the h+ ions formed in the absorption of so2 and its subsequent oxidation to sulfate, and thus maintains the solubility of so2 and the rate of sulfate production. it also helps to maintain higher levels of ph, so the reaction of s (iv) with oxidizing agents, such as o3 proceeds at a faster rate, compared to that at lower ph values [30]. the declining concentrations of so2 cause less acidic so4 2−, therefore, a reduction in the reactions with nh3 will occur [31]. this may also be an explanation to the increased nh3 concentrations. the values for clconcentrations and fluxes are comparable excepting for 2021, when the results obtained at campulung are three times higher than at gheorgheni (0.97 comparing to 0.31 mg/l and 3.8 comparing to 1.2 kg/ha/year). this problem can be explained by the difficulties registered in the ion chromatograph operation from the anion’s subsystem, at gheorgheni laboratory. the measured precipitation composition data were quality checked using the ionic balance method and the calculated conductivity. the distribution of the measured ph and conductivity values showed that there are deviations of ± 10% reported to the values of the campulung laboratory that participated in the intercalibration exercise (fig.4). the rainwater ph of an unpolluted and clean atmosphere has a value around 5.6, as the dissolution of naturally existing co2 and other acidic species (nox; so2) occurs in the cloud droplets [32]. changes in the rainwater ph values, below or above this level, are mainly due to acidic or alkaline components. fig. 4. means weighted ph and conductivities, for the studied period 2018-2021 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 227 acid precipitation with ph values less than 5.0 is likely due to the influence of anthropogenic emissions of h2so4 and/or hno3 (seinfeld et al. 2006). ph values greater than 6.0 indicate inputs of alkaline substances into rainwater, like nh4 + and terrigenous components (ca2+, mg2+) derived from limestones and dolomites. the process of neutralization is also related with the anthropic emissions of nh3 and nh4 which react with the so4 2− and no3 − present in the atmosphere, and result in the forming of the ammonium sulfate (nh4) 2so4, and nitrate, nh4no3 [33]. 4. conclusion this experiment was very useful for transferring the icp forests methodology concerning atmospheric deposition from the incds “marin dracea” campulung moldovenesc laboratory to that from to the ocolul silvic de regim gheorgheni sa. procedure comparison as such may be very beneficial. for instance, they may be used to highlight the importance of correct handling techniques and be an incentive to evaluate the correctness of procedures currently used. performing studies comparing methods of analysis or checking the results obtained by two or more laboratories for applying the same methods of analysis at the same time is a major task, but also a very difficult one. as noted in this study, if no attention is paid to the difference between the comparison method and the procedure, this may lead to erroneous conclusions in the final results obtained. a proper approach to these procedures can lead to more effective monitoring of the analyzes performed, provoke a review of procedures, detect errors and increase motivation for developing new methods. the outcome of such an effort will be to ensure that harmozed results are reported. the results obtained revealed that the levels of concentrations and fluxes calculated with the values measured in the two laboratories are comparable and are within the same ranges considered at european level. the databases obtained in this study represent a novelty of the monitoring of atmospheric deposits and can be used in future research as a reference base. 5. acknowledgements the present paper was realized with the help of the research and innovation ministry (mci), through the nucleus program contracted by “marin drăcea” national research-development forestry institute within the pn 19070102 project and cresforlife project nr.4/15.01.2018, p_40_380/105506. 6. references [1]. kopaćek j., turek j., hejzla j., porcal p., bulk deposition and throughfall fluxes of elements in the bohemian forest (central europe) from 1998 to 2009, boreal environment research 16: 495–5081 issn 1239-6095 (print) issn 1797-2469, (2011). [2]. mocὰk j., balla b., bobrowschi a., blazicek p., present at the xvth slovak spectroscopic conference chem. pap.57(3)143146, (2003). [3]. ludbrook j., statistical techniques for comparing measurers and methods of measurement: a critical review. clinical and experimental pharmacology and physiology 29: 527-536. doi: 10.1046/j.1440-1681.2002. 03686.x, (2002). [4]. zimmermann f., plessow k., queck r., berhofer c., matschullat j., atmospheric nand s-fluxes to a spruce forest – comparison of inferential modeling and the throughfall method. atmospheric environment 40, 4782–4796., (2006). [5]. schimitt m., thoni l., waldner p., thimonier, a., total deposition of nitrogen on swiss long-term forest ecosystem research (lwf) plots: comparison of the throughfall and the inferential method. atmospheric environment 39, 1079–1091, (2005). [6]. avila a., rodrigo a., trace metal fluxes in bulk deposition, throughfall and stemflow at two evergreen oak stands in ne spain subject to https://www.sciencedirect.com/science/article/abs/pii/s1352231003008562#! food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 228 different exposure to the industrial environment, atmospheric environment,volume 38, issue 2, pages 171-180, https://doi.org/10.1016/j.atmosenv.2003.09.067, (2004) [7]. takenaka n., takayama k., ojiro n., shimazaki w., ohira k., soda h., suzue t., sadanaga y., bandow h., maeda y., the chemistry of drying an aqueous solution of salts. journal of physical chemistry a, 113, 12233-12242. (2009). [8]. wentworth g.r., murphy j.g., benedict k.b., bangs e.j., collett j.l., the role of dew as a night-time reservoir and morning source for atmospheric ammonia. atmospheric chemistry and physics, 16, 74357449 (2016) [9]. burkhardt j., flechard c.r., gresens f., mattsson, m., jongejan p.a.c., erisman j.w., weidinger t., meszaros r., nemitz e., sutton m.a., modelling the dynamic chemical interactions of atmospheric ammonia with leaf surface wetness in a managed grassland canopy. biogeosciences, 6, 67-84, (2009). [10]. fenn m., e., poth m. a., monitoring nitrogen deposition in throughfall using ion exchange resin columns: a field test in the san bernardino mountains, journal of environmetal quality, volume 33, issue 6, pg.2007-2014 https://doi.org/10.2134/jeq2004.2007, (2004)., [11]. astrua m., ichim d., pennecci f., pissani m., statistical techniques for assessing the agreement between two instruments. metrologia 44: 385 392. doi: 10.1088/0026-1394/44/5/015, (2007) [12]. bland jm., altman dg., agreement between methods of measurement with multiple observations per individual. journal of biopharmaceutical statistics 17: 571-582. doi: 10.1080/10543400701329422, (2007). [13]. clarke n., zlindra d, ulrich e, mosello r., derome j., derome k., kónig n., lövblad g, draaijers gpj., hansen k., thimonier a., waldner p., sampling and analysis of deposition. part xiv. in: “manual on methods and criteria for harmonized sampling, assessment, monitoring and analysis of the effects of air pollution on forests”. unece, icp forests, hamburg, germany, pp. 66. (isbn: 978-3-926301-03-1) [online] url: http://www.icpforests.org/manual.htm, (2010). [14]. purna j., sabyasachi d., nagendra p., throughfall and stemflow nutrient flux in deodar and oak forests, garhwal himalaya, india, water supply, 10.2166/ws.2021.009, 21, 4, (1649-1656), (2021) [15]. wei fang g., xueqin l., , estimating chemical exchange between atmospheric deposition and forest canopy in guizhou, china journal of environmetal quality, volume 33, issue 6, pg.332-340, https://doi.org/10.2134/jeq2012.0308. (2013). [16]. zhang g., zeng g.m., huang gh., jiang y-m, yao m-e., du c-y., jiang r., zhang c., deposition pattern of precipitation and throughfall in a subtropical evergreen forest in south-central china, journal of forest research, 10.1007/s10310-006-0232-9, 11, 6, (389-396), (2017). [17]. lindo z., jonathan a., whiteley a., 2011, old trees contribute bio-available nitrogen through canopy bryophytes, plant and soil, 10.1007/s11104-010-0678-6, 342, 1-2, (141148), [18]. ahrends b., schmitz a., prescher a-k., wehberg j., geupel m., henning a., henning m., comparison of methods for the estimation of total inorganic nitrogen deposition to forests in germany, frontiers in forest and global change. sec. forests and the atmosphere https://doi.org/10.3389/ffgc.2020.00103, (2020). [19]. arisci s., rogora m., marchetto a., dichiaro f., the role of forest type in the variability of doc in atmospheric deposition at forest plots in italy, environmental monitoring and assessment, 10.1007/s10661-011-2196-2, 184, 6, (3415-3425), (2011). [20]. zhang g., zeng g.m., jiang y.m., huang g.h., yao j.m., xiang r.j., zhang x.l., seasonal ionic exchange in two-layer canopies and total deposition in a subtropical evergreen mixed forest in central-south china, annals of forest science, 10.1051/forest:2006072, 63, 8, (887-896), (2006). [21]. bleeker a., draaijers g., van der veen d., erisman jw., mols h., fonteijn p., geusebroek m., field intercomparison of throughfall measurements performed within the framework of the pan european intensive monitoring program of eu/icp forest. environmental pollution 125 123-138. doi: 10.1016/s0269-7491(03)001428, (2003). [22]. barbu i., iacoban c. (2008). determinarea cantitativă și calitativă a fluxurilor de ioni poluanți (depuneri atmosferice) în ecosistemele forestiere. în: manual privind metodologia de supraveghere pe termen lung a stării ecosistemelor forestiere aflate sub acțiunea poluării atmosferice și a modificărilor climatice. ed. o., badea, pg. 57-64 https://www.academia.edu/44668462/ [23]. clarke n., žlindra d., ulrich e., m0sello, r., derome j., derome k., https://www.sciencedirect.com/journal/atmospheric-environment https://www.sciencedirect.com/journal/atmospheric-environment/vol/38/issue/2 https://doi.org/10.1016/j.atmosenv.2003.09.067 https://acsess.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=fenn%2c+mark+e https://acsess.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=poth%2c+mark+a https://doi.org/10.2134/jeq2004.2007 http://www.icp-forests.org/manual.htm http://www.icp-forests.org/manual.htm https://acsess.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=gao%2c+fang https://doi.org/10.2134/jeq2012.0308 https://www.frontiersin.org/people/u/862174 https://www.frontiersin.org/people/u/1013427 https://www.frontiersin.org/people/u/921599 https://www.frontiersin.org/people/u/1013577 https://doi.org/10.3389/ffgc.2020.00103 https://www.academia.edu/44668462/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 3 2022 carmen iacoban, marius curcă, cosmin ilie cuciurean, zitta csergo, amelia buculei, comparison between the parameters of throughfall and bulk deposition measured in two laboratories using the same methods, food and environment safety, volume xxi, issue 3 – 2022, pag. 218 – 229 229 koning n., gepert f., loyblad g., draaijers g.p.j., hansen k., thimonier, a., waldner p., verstraeten a., (2022): part xiv: sampling and analysis of deposition. version 2022-1. in: unece icp forests programme co-ordinating centre (ed.): manual on methods and criteria for harmonized sampling, assessment, monitoring and analysis of the effects of air pollution on forests. thünen institute of forest ecosystems, eberswalde, germany, 34 p. + annex [http://www.icpforests.org/manual.htm [24]. marchetto a., mosello r., tartari g., tornimbeni o., derome j., derome k., sorsa p., konig n., clarke n., ulrich e., kowalska a., in € fluence of qa/qc procedures on non-sampling error in deposition monitoring in forests. j. environ. monit. 11 (4), 745e750, (2009). [25]. draaijers g.p.j., bleeker a., van der veen erisman jw., mols h., fonteijn p., geusebroek m., field intercomparison of throughfall, stemflow and precipitation measurements performed within the framework of the pan european intensive monitoring program of eu/icp forests (tno-ecn report 2001/140), 221 p. þ ann p., (2001). [26]. zlindra d., eler k., clarke n., simoncic p., towards harmonization of forest deposition collectors case study of comparing collector designs, iforest 4: 218-225 url:http://www.sisef.it/iforest/show.php?id=586., (2011). [27]. keresztesi á., korodi a., boga r., petres s., ghita g, ilie m., chemical characteristics of wet precipitation in the eastern carpathians, romania. ecoterra 14(2):52–59, (2017) [28]. pu w., quan w., ma z., shi x., zhao x., zhang l., wang z., wang w. long-term trend of chemical composition of atmospheric precipitation at a regional background station in northern china. sci total environ 580:1340–1350. https://doi.org/10.1016/j.scitotenv.2016. 12.097, (2017) [29]. sakihama h., ishiki m., tokuyama a., chemical characteristics of precipitation in okinawa island, japan. atmos environ 42:2320– 2335, (2008). [30]. moller d., schieferdecker h., a relationship between agricultural nh3 emissions ant the atmospheric so2 content over industrial areas. atmos environ 19:695–700, (1985) [31]. butler t., vermeylen f., lechmann cm., likens ge., puchalscki m., increasing ammonia concentration trends in large regions of the usa derived from the nadp/amon network. atmos environ 146:132– 140, (2016) [32]. wang h., han g., chemical composition of rainwater and anthropogenic influences in chengdu, southwest china. atmos res 99: 190–196, (2011). [33]. singh s, sharma a., kulshrestha uc., relative contributions of nh3, no2, nh4+ and no3− to total nitrogen deposition at an agricultural site in the indogangetic plain of india. proceedings of the 2016 international nitrogen initiative conference, “solutions to improve nitrogen use efficiency for the world”, 4–8 december 2016, melbourne, australia. www.ini2016.com., (2016). http://www.sisef.it/iforest/show.php?id=586 http://www.ini2016.com/ 1. introduction 4. conclusion [5]. schimitt m., thoni l., waldner p., thimonier, a., total deposition of nitrogen on swiss long-term forest ecosystem research (lwf) plots: comparison of the throughfall and the inferential method. atmospheric environment 39, 1079–1091, (2005). [7]. takenaka n., takayama k., ojiro n., shimazaki w., ohira k., soda h., suzue t., sadanaga y., bandow h., maeda y., the chemistry of drying an aqueous solution of salts. journal of physical chemistry a, 113, 12233-12242. (2009). [9]. burkhardt j., flechard c.r., gresens f., mattsson, m., jongejan p.a.c., erisman j.w., weidinger t., meszaros r., nemitz e., sutton m.a., modelling the dynamic chemical interactions of atmospheric ammonia with leaf surface wetness in a managed gra... [11]. astrua m., ichim d., pennecci f., pissani m., statistical techniques for assessing the agreement between two instruments. metrologia 44: 385 392. doi: 10.1088/0026-1394/44/5/015, (2007) [12]. bland jm., altman dg., agreement between methods of measurement with multiple observations per individual. journal of biopharmaceutical statistics 17: 571-582. doi: 10.1080/10543400701329422, (2007). [18]. ahrends b., schmitz a., prescher a-k., wehberg j., geupel m., henning a., henning m., comparison of methods for the estimation of total inorganic nitrogen deposition to forests in germany, frontiers in forest and global change. sec. forests... [21]. bleeker a., draaijers g., van der veen d., erisman jw., mols h., fonteijn p., geusebroek m., field intercomparison of throughfall measurements performed within the framework of the pan european intensive monitoring program of eu/icp forest. envi... [27]. keresztesi á., korodi a., boga r., petres s., ghita g, ilie m., chemical characteristics of wet precipitation in the eastern carpathians, romania. ecoterra 14(2):52–59, (2017) [30]. moller d., schieferdecker h., a relationship between agricultural nh3 emissions ant the atmospheric so2 content over industrial areas. atmos environ 19:695–700, (1985) [31]. butler t., vermeylen f., lechmann cm., likens ge., puchalscki m., increasing ammonia concentration trends in large regions of the usa derived from the nadp/amon network. atmos environ 146:132–140, (2016) 99 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 2 – 2014, pag. 99 108 evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire armel f. zoro, *lessoy t. zoue, sébastien l. niamke laboratoire de biotechnologies, ufr biosciences, université félix houphouët-boigny, 22 bp 582, abidjan 22, côte d’ivoire, y.lessoy@yahoo.fr *corresponding author received 9th june, accepted 27th june 2014 abstract: african leafy vegetables are valuable sources of nutrients especially in rural areas where they contribute substantially to protein, minerals, vitamins, fibres and other beneficial nutrients. five leafy vegetable species (abelmoschus esculentus, celosia argentea, ipomea batatas, manihot esculenta and myrianthus arboreus) that are used as soup condiments in western côte d’ivoire were subjected to steam blanching in order to evaluate the effect of this processing method on their nutritive value and antioxidant properties. the result of the study revealed that longer time of blanching (higher than 15 min) caused negative impact by reducing nutritive value but positive impact by reducing anti-nutrients such as oxalates and phytates. the registered losses at 15 min were as follow: ash (3.78 – 55.81 %), proteins (0.1 10.79 %), vitamin c (1.04 – 79.70 %), carotenoids (47.91 66.28%) oxalates (5.51 – 33.33 %) and phytates (43.83– 78.16 %).the average reduction of polyphenols content at 15 min of blanching was 13.84 – 38.23 %. contrary to these reductions, a significant increase (0.4 – 29.94 %) of fibres content was observed in the studied blanched leafy vegetables. furthermore after 15 min of blanching time the residual contents of minerals were: calcium (228.62 – 402.39 mg/100g), magnesium (92.19 – 270.82 mg/100g), potassium (1255.24 – 2215.65 mg/100g), iron (17.24 – 43.48 mg/100g) and zinc (10.91 – 32.30 mg/100g). all these results suggest that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of ivorian population. keywords: antioxidant properties blanching processing leafy vegetables nutritive value 1. introduction african leafy vegetables (alvs) are indigenous or traditional vegetables whose leaves, young shoots and flowers are consumed [1,2]. agronomic characteristics of alvs include: short growth period with harvesting within 3-4 weeks; the ability to produce seed under tropical conditions; they respond well to organic fertilizers and can tolerate both biotic and abiotic stress [3]. socio-economic surveys conducted in various parts of africa indicate that alvs are important commodities in household food and nutrition security [4].there is empirical evidence that alvs have several advantages and values that include high micronutrient content, medicinal properties and contribute to food and nutrition security in sub-saharan africa [5]. indeed, alvs contain high levels of vitamin a, vitamin c, iron, calcium and protein and are a valuable source of nutrients in rural areas where they contribute substantially to protein, mineral and vitamin intake [6, 7, 8, 9]. they are compatible to use with starchy food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 100 staples and represent affordable nutrition to the poor sector of the population. fresh leaves of most alvs like vegetable amaranths (amaranthus), slenderleaf (crotalaria brevidens), spiderplant (chlorophytum comosum), vegetable cowpeas (vigna), pumpkin leaves (cucurbits) and jute mallow (corchorus) contain more than 100% of the recommended daily allowances for vitamins and minerals and 40% proteins for growing children and lactating mothers [10]. the high moisture content of fresh leafy vegetables renders them perishable and seasonal availability limits their utilization all round the year. hence, there is a need to preserve this nature's store house of nutrients through proper processing techniques for safe storage with efficient nutrient retention [11]. one common processing using before consumption of leafy vegetables is blanching which could be briefly described as the process of heating vegetables to a temperature high enough to destroy enzymes present in the tissue. it stops the enzyme action, sets the colour, and shortens the drying and dehydration time [12]. it is usually carried out in hot water or in steam; this technique is used by indigenous people to reduce or eliminate the bitterness of the vegetables and acid components that are common in leaves [13]. blanching affords also a series of secondary benefits, due to its washing action, such as elimination of off-flavors that may have been formed during the time between harvesting and processing, and removal of any residual pesticides [14]. blanching, however, has some adverse effects, such as pigment modifications, tissues softening and nutrient losses [15,16]. in côte d’ivoire, more than twenty (20) species of leafy vegetables belong to 6 botanical families, are consumed by populations through confectionary soups using boiling or blanched processing [17]. furthermore, the consumption of these leafy vegetables is linked to the region and ethno-botanical studies have stated that most people in western côte d’ivoire consume indigenous green leafy vegetables such as abelmoschus esculentus “gombo”, celosia argentea “soko”, ipomea batatas “patate”, manihot esculenta “manioc” and myrianthus arboreus “tikliti” [18,19,20]. earlier reports have highlighted the nutritive potential of these fresh leafy vegetables [7] but there is a lack of scientific data with regards to the effect of blanching processing on their physicochemical and nutritive characteristics. therefore, the purpose of this study is to conduct investigation on the effect of blanching on the nutritive value of these selected leafy vegetables in order to provide necessary information for their wider utilization and contribution to food security of ivorian populations. 2. material and methods 2.1. samples collection leafy vegetables (abelmoschus esculentus, celosia argentea, ipomea batatas, manihot esculenta and myrianthus arboreus) were collected fresh and at maturity from cultivated farmlands located at dabou (latitude: 5°19′14″ north; longitude: 4°22′59″west) (abidjan district). the samples were harvested at the early stage (between one and two weeks of the appearance of the leaves). these plants were previously authenticated by the national floristic center (university felix houphouët-boigny, abidjan-côte d’ivoire). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 101 2.2. samples processing the fresh leafy vegetables were rinsed with deionized water and the edible portions were separated from the inedible portion. the edible portions were chopped into small pieces (500 g) and allowed to drain at ambient temperature. each sample was divided into two lots. the first lot (raw, 250 g) was dried in an oven (memmert, germany) [21], ground with a laboratory crusher (culatti, france) equipped with a 10 μm mesh sieve. each sample was stored in a clean dry air-tight sample bottle in a refrigerator (4°c) until further analyses. the second lot (250 g) was steam blanched for 15, 25 and 45 min in a pressure cooker. the blanched samples were cooled, drained at ambient temperature and subjected to the same treatment using for raw samples. 2.3. chemicals all solvents (n-hexane, petroleum ether, acetone, ethanol and methanol) were purchased from merck. standards used (gallic acid, β-carotene) and reagents (metaphosphoric acid, folin-ciocalteu, dpph) were purchased from sigmaaldrich. all chemicals used in the study were of analytical grade. 2.4. nutritive properties 2.4.1. proximate analysis ash, proteins and lipids were determined using official methods [22]. for crude fibres, 2 g of dried powdered sample were digested with 0.25 m sulphuric acid and 0.3 m sodium hydroxide solution. the insoluble residue obtained was washed with hot water and dried in an oven (memmert, germany) at 100°c until constant weight. the dried residue was then incinerated, and weighed for the determination of crude fibres content. carbohydrates and calorific value were calculated using the following formulas [23]: carbohydrates: 100 – (% moisture + % proteins + % lipids + % ash + % fibres). calorific value: (% proteins x 2.44) + (% carbohydrates x 3.57) + (% lipids x 8.37). the results of ash, fibres, proteins, lipids and carbohydrates contents were expressed on dry matter basis. 2.4.2. mineral analysis the dried powdered samples (5 g) were burned to ashes in a muffle furnace (pyrolabo, france). the ashes obtained were dissolved in 10 ml of hcl/hno3, transferred into 100 ml flasks and the volume was made up using deionized water. the mineral composition of each sample was determined using an agilent 7500c inductively coupled argon plasma mass spectrometer (icp-ms). calibrations were performed using external standards prepared from a 1000 ppm single stock solution made up with 2% nitric acid. 2.4.3. anti-nutritional factors oxalates content was performed using a titration method [24]. one (1) g of dried powdered sample was weighed into 100 ml conical flask. a quantity of 75 ml of sulphuric acid (3 m) was added and stirred for 1 h with a magnetic stirrer. the mixture was filtered and 25 ml of the filtrate was titrated while hot against kmno4 solution (0.05 m) to the end point. phytates contents were determined using the wade’s reagent colorimetric method [25]. a quantity (1 g) of dried powdered sample was mixed with 20 ml of hydrochloric acid (0.65 n) and stirred for 12 h with a magnetic. the mixture was centrifuged at 12000 rpm for 40 min. an aliquot (0.5 ml) of supernatant was added with 3 ml of wade’s reagent. the reaction mixture was incubated for 15 min and absorbance was measured at 490 nm by using a spectrophotometer (pg instruments, england). phytates content was estimated using a calibration curve of sodium phytate (10 mg/ml) as standard food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 102 2.5. antioxidant properties 2.5.1 vitamin c and carotenoids determination vitamin c contained in analysed samples was determined by titration [26]. about 10 g of ground fresh leaves were soaked for 10 min in 40 ml metaphosphoric acidacetic acid (2%, w/v). the mixture was centrifuged at 3000 rpm for 20 min and the supernatant obtained was diluted and adjusted with 50 ml of bi-distilled water. ten (10) ml of this mixture was titrated to the end point with dichlorophenolindophenol (dcpip) 0.5 g/l. carotenoids were extracted and quantified by using a spectrophotometric method [27]. two (2) g of ground fresh leaves were mixed three times with 50 ml of acetone until loss of pigmentation. the mixture obtained was filtered and total carotenoids were extracted with 100 ml of petroleum ether. absorbance of extracted fraction was then read at 450 nm by using a spectrophotometer (pg instruments, england). total carotenoids content was subsequently estimated using a calibration curve of β-carotene (1 mg/ml) as standard. 2.5.2. polyphenols determination polyphenols were extracted and determined using folin–ciocalteu’s reagent [28]. a quantity (1 g) of dried powdered sample was soaked in 10 ml of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min. an aliquot (1 ml) of supernatant was oxidized with 1 ml of folin–ciocalteu’s reagent and neutralized by 1 ml of 20% (w/v) sodium carbonate. the reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm by using a spectrophotometer (pg instruments, england). the polyphenols content was obtained using a calibration curve of gallic acid (1 mg/ml) as standard. antioxidant activity antioxidant activity assay was carried out using the 2,2diphenyl-1-pycrilhydrazyl (dpph) spectrophotometric method [29]. about 1 ml of 0.3 mm dpph solution in ethanol was added to 2.5 ml of sample solution (1 g of dried powdered sample mixed in 10 ml of methanol and filtered through whatman no. 4 filter paper) and was allowed to react for 30 min at room temperature. absorbance values were measured with a spectrophotometer (pg instruments, england) set at 415 nm. the average absorbance values were converted to percentage antioxidant activity using the following formula: antioxidant activity (%) = 100 – [(abs of sample – abs of blank) x 100/abs positive control] 2.6 statistical analysis all the analyses were performed in triplicate and data were analyzed using excell and statistica 7.1 (statsoft). values were expressed as means ± standard deviation. 3. results and discussion the proximate composition of the blanched leafy vegetables examined in this study is presented in table 1. the ash content after 15 min of blanching ranged from 7.36 ± 0.01 % (m. esculenta) to 11.99 ± 0.02 % (c. argentea). besides the decrease rate at 15 min (3.78 – 55.81 %) the studied leafy vegetables may be considered as good sources of minerals when compared to values obtained for cereals and tubers [30]. blanching of all selected leafy vegetables resulted in a significant increase (0.4 – 29.94 %) in their crude fibres content at 15 min. indeed, the increased temperature during blanching leads to breakage of weak bonds between polysaccharides and the cleavage of glycosidic linkages, which may result in solubilization of the dietary fibre [31]. with regard to their fibres content at 15 min (15.84 – 29.78 %), adequate intake of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 103 blanched leafy vegetables could lower the risk of constipation, diabetes, colon and breast cancer [32]. as concern proteins content, blanching processing used in this study caused 0.1 to 10.79 % reduction after 15 min. this reduced protein contents of blanched leafy vegetables could be attributed to the severity of thermal process during blanching which leads to protein degradation [33]. with regards to their protein contents (21.52 ± 0.02; 15.08 ± 0.02 and 15.12 ± 0.02%) at 15 min, blanched leaves of m. esculenta, i. batatas and m. arboreus could be considered as non negligible sources in view to the minimal value (12%) recommended for protein foods [34]. the relatively low values of lipids contents at 15 min of blanching (2.11 – 7.58 %) in the studied cooked leafy vegetables corroborate the findings of many authors which showed that leafy vegetables are poor sources of fat [35]. the estimated calorific values agree with general observation that vegetables have low energy values due to their low crude fat and relatively high level of moisture [36]. table 1 proximate composition of raw and blanched leafy vegetables consumed in western côte d’ivoire ash (%) fibres (%) proteins (%) lipids (%) carbohydr ates (%) calorific value (kcal /100g) a. esculentus raw 11.90 ± 0.10 15.66 ± 0.05 9.19 ± 0.15 3.38 ± 1.59 59.87± 1.90 264.44± 2.51 15 min 11.45 ± 0.02 18.93 ± 0.25 9.18 ± 0.00 7.04 ± 0.08 48.19 ± 0.42 263.64 ± 2.63 25 min 11.16 ± 0.02 18.35 ± 0.02 9.15 ± 0.02 7.59 ± 0.16 48.66 ± 0.95 271.78 ± 5.31 45 min 11.06 ± 0.02 18.37 ± 0.06 9.15 ± 0.02 6.92 ± 0.03 47.99 ± 0.92 265.25 ± 5.77 m.esculenta raw 9.03 ± 2.12 26.23 ± 0.31 23.39 ± 0.71 4.09 ± 0.02 37.27 ± 3.16 224.35 ± 15.67 15 min 7.36 ± 0.01 26.34 ± 0.02 21.52 ± 0.02 7.58 ± 0.13 38.20 ± 0.31 252.31 ± 3.04 25 min 7.09 ± 0.09 26.87 ± 1.09 21.21 ± 0.00 8.15 ± 0.25 40.90 ± 0.73 256.17 ± 5.34 45 min 7.00 ± 0.05 26.88 ± 0.10 21.02 ± 0.00 7.84 ± 0.16 41.66 ± 0.23 254.91 ± 2.20 i. batatas raw 23.56 ± 2.13 21.5 ± 0.82 15.52 ± 0.40 2.63 ± 0.06 36.79 ± 3.41 232.91 ± 15.78 15 min 10.41 ± 0.11 24.62 ± 0.54 15.08 ± 0.02 3.02 ± 0.03 42.87 ± 0.73 224.85 ± 3.21 25 min 10.24 ± 0.08 23.25 ± 0.44 15.00 ± 0.02 3.87 ± 0.12 45.15 ± 0.54 236.01 ± 3.31 45 min 9.84 ± 0.05 23.55 ± 1.36 15.00 ± 0.02 3.63 ± 0.14 42.98 ± 1.30 232.59 ± 7.93 c. argentea raw 22.10 ± 0.75 30.83 ± 1.61 9.77 ± 0.10 1.79 ± 0.20 35.52 ± 0.6 165.62 ± 6.40 15 min 11.99 ± 0.02 29.78 ± 0.02 9.21 ± 0.00 4.71 ± 0.01 44.32 ± 1.49 244.47 ± 7.42 25 min 11.72 ± 0.12 22.80 ± 1.44 9.13 ± 0.00 4.59 ± 0.08 41.84 ± 2.70 234.93 ± 13.82 45 min 11.64 ± 0.10 22.09 ± 0.08 9.10 ± 0.02 4.75 ± 0.08 41.72 ± 1.51 237.00 ± 9.51 m. arboreus raw 11.73 ± 0,76 12.19 ± 0,73 16.95 ± 0.05 1.47 ± 0.02 56.70 ± 1.74 259.5 ± 5.26 15 min 9.14 ± 0.58 15.84 ± 1.10 15.12 ± 0.02 2.11 ± 0.18 49.80 ± 0.94 227.42 ± 4.90 25 min 8.92 ± 0.03 14.67 ± 0.83 15.08 ± 0.00 3.52 ± 0.07 48.59 ± 0.88 237.28 ± 4.50 45 min 8.56 ± 0.05 13.25 ± 0.65 15.00 ± 0.00 2.42 ± 0.13 52.31 ± 0.60 239.86 ± 3.48 values are mean of triplicate determination ± standard deviation mineral composition of blanched leafy vegetables used in this study is shown in table 2. the residual contents of minerals after 15 min of blanching were: calcium (228.62 – 402.39 mg/100g), magnesium (92.19 – 270.82 mg/100g), potassium (1255.24 – 2215.65 mg/100g), iron (17.24 – 43.48 mg/100g) and zinc (10.91 – 32.30 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 104 mg/100g). these observed reductions may be due to the losses of ashes as observed previously. considering the recommended dietary allowance (rda) for minerals [37]: calcium (1000 mg/day); magnesium (400 mg/day), iron (8 mg/day) and zinc (6 mg/day), consumption of 15 min blanched leafy vegetables could cover at least 50% rda. therefore, they could contribute substantially for improving human diet. calcium and phosphorus are associated for growth and maintenance of bones, teeth and muscles [38]. as concern magnesium, this mineral is known to prevent cardiomyopathy, muscle degeneration, growth retardation, congenital malformations and bleeding disorders [39]. iron plays important role in prevention of anemia while zinc is important for vitamin a and vitamin e metabolism [37, 40]. table 2. mineral composition of raw and blanched leafy vegetables consumed in western côte d’ivoire ca mg p k fe na zn a. esculentus raw 468.45±0.55 364.11±0.43 671.5 ± 0.79 1844.25 ± 8.22 130.95±0.15 35.76 ± 0.04 41.45 ±0.04 15 min 386.39±1.72 154.44±0.68 222.80±0.99 1695.10±14.98 41.32 ± 0.31 19.94 ± 0.08 32.30 ±0.28 25 min 374.41±2.81 151.23±1.33 176.53±1.32 1636.85 ± 1.94 34.11 ± 0.30 18.12 ± 0.16 25.34 ±0.11 45 min 376.46±3.32 149.49±1.12 166.21±1.46 1531.42±11.50 31.34 ± 0.13 16.75 ± 0.12 21.50 ±0.16 m.esculenta raw 296.66±0.46 229.45±0.35 759.81±1.18 2306.09 ± 3.61 48.69 ± 0.07 18.30 ± 0.02 45.48 ±0.31 15 min 248.61±0.47 92.19 ± 0.63 229.21±0.44 1405.93 ± 2.70 43.48 ± 0.30 9.53 ± 0.01 21.01 ±0.03 25 min 239.28±1.65 80.57 ± 0.15 201.81±1.40 1320.68 ± 9.16 42.52 ±0. 08 8.75 ± 0.06 27.46 ±0.24 45 min 194.75±1.77 56.61 ±0. 51 188.86±1.71 935.00 ± 8.50 29.26 ± 0.26 8.52 ± 0.07 36.76 ±0.07 i. batatas raw 898.83±0.53 501.75±0.30 494.76±0.29 1377.81 ± 0.22 53.54 ± 0.03 404.30±3.62 30.10 ±0.01 15 min 228.62±1.39 165.18±1.00 281.06±1.70 1362.73±12.20 19.74 ± 0.17 236.58±0.34 10.91 ±0.01 25 min 246.59 ± 2.2 161.55±1.44 266.89±2.39 1282.98± 2.13 18.67 ± 0.11 199.66±1.21 10.26 ±0.06 45 min 230.03±0.33 154.66±0.22 220.02±0.31 1280.07 ± 7.78 17.57 ± 0.02 139.69±0.08 10.20 ±0.09 c. argentea raw 788.02±0.50 981.31±0.62 650.37±0.41 4987.15 ± 3.19 285.31±0.18 42.26 ± 0.02 62.01 ±0.03 15 min 402.39±0.95 270.82±3.20 172.85±0.40 1255.24 ± 2.96 17.24 ± 0.20 34.64±0.041 26.58 ±0.06 25 min 407.75±4.90 249.91±0.59 151.07±1.81 1190.69±14.33 16.44 ± 0.03 32.58 ± 0.07 23.92 ±0.27 45 min 340.60±3.92 199.33±2.29 148.18±1.70 840.41 ± 9.68 13.76 ± 0.15 26.29 ±0.30 23.31 ±0.28 m. arboreus raw 436.64±0.52 354.23±0.42 283.19±0.34 2350.58 ± 2.83 79.54 ± 0.09 20.83 ± 0.02 75.20 ±0.09 15 min 293.81±3.53 213.89±2.46 269.64±7.78 2215.65±25.54 21.09 ± 0.24 18.34 ±0.04 22.43 ±0.25 25 min 277.74±3.20 205.23±2.47 232.31±0.91 1809.88±21.78 18.51 ± 0.04 15.85 ± 0.18 21.98 ±0.26 45 min 248.01±0.58 177.60±0.41 193.98±0.96 1688.05 ± 3.98 18.42 ± 0.22 15.19 ± 0.18 18.72 ±0.04 values are mean of triplicate determination ± standard deviation to predict the bioavailability of calcium and iron, anti-nutrients to nutrients ratios of blanched leafy vegetables were calculated (table 3). the calculated [phytates]/[ca] and [phytates]/[fe] ratios in all the blanched species were below the critical levels of 2.5 and 0.4 which are known to impair calcium and iron bioavailability [41,42]. the effect of blanching on anti-nutritional factors (oxalates and phytates) contents is depicted in figure 1. levels losses at 15 min were 5.51 – 33.33 % and 43.83– 78.16 % for oxalates and phytates, respectively. the reductions in oxalates and phytates contents during blanching could be advantageous for improving the health status of consumers. indeed, oxalates and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 105 phytates are anti-nutrients which chelate divalent cations such as calcium, magnesium, zinc and iron, thereby reducing their bioavailability [43]. therefore, blanching of leafy vegetables appears as a detoxification procedure by removing these anti-nutritional factors [44] table 3 anti-nutritional factors/mineral ratios of raw and blanched leafy vegetables consumed in western côte d’ivoire phytate/ ca phytate/ fe oxalate/ ca a. esculentus raw 0.07 0.28 1.66 15 min 0.02 0.27 1.50 25 min 0.02 0.23 1.46 45 min 0.01 0.22 1.34 m.esculenta raw 0.12 0.75 2.69 15 min 0.05 0.32 2.43 25 min 0.04 0.27 2.43 45 min 0.05 0.37 2.82 i. batatas raw 0.01 0.31 0.08 15 min 0.03 0.34 0.22 25 min 0.02 0.32 0.11 45 min 0.01 0.17 0.11 c. argentea raw 0.03 0.08 1.01 15 min 0.01 0.30 1.45 25 min 0.005 0.12 1.34 45 min 0.001 0.02 1.46 m. arboreus raw 0.05 0.31 1.19 15 min 0.04 0.66 1.67 25 min 0.04 0.68 1.16 45 min 0.04 0.63 1.24 blanching also resulted in a decrease of carotenoids and vitamin c contents in the studied leafy vegetables (figure 2). for carotenoids, losses at 15 min were estimated to 47.91 to 66.28%. the decrease in the concentration of the total carotenoids could be attributed to the oxidation and isomerization of β-carotene [45]. carotenoids are considered as sources of provitamin a in plants and their amount determine their bioavailability in human diet [27]. therefore, increased intake of blanched leafy with fat added, contributed significantly to improving the vitamin a status in children [46]. for vitamin c content, a significant reduction (1.04 – 79.70 %) was highlighted at 15 min during blanching processing (figure 2). this decrease in vitamin c content agrees with earlier findings on some tropical vegetables that reported 47.5 82.4% loss in vitamin c content during blanching [16]. it is important noting that ascorbic acid is heat labile and water-soluble antioxidant that promotes absorption of soluble iron by chelating or by maintaining the iron in the reduced form [47]. with regard to the decrease of vitamin c, consumption of blanched leafy vegetables may be supplemented with other sources of vitamin c such as tropical fruits to cover the daily need for humans (40 mg/day) as recommended by food agriculture organization [37]. figure 1: oxalates (a) and phytates (b) contents of raw and blanched leafy vegetables consumed in western côte d’ivoire 0 200 400 600 800 1000 o xa la te s (m g /1 00 g) a 0 10 20 30 40 p h yt at es (m g/ 10 0g ) b raw 15 min 25 min 45 min food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 106 the effect of blanching on polyphenols content and antioxidant activity of the selected leafy vegetables is depicted in figure 3. the losses of polyphenols contents at 15 min of cooking were 13.84 – 38.23 %. the decrease of the polyphenolic compounds content and overall antioxidant activity could be due to the heat lability of specific flavonoids [48]. indeed, flavonoids such as myricetin, quercetin, kaempferol, isorhamnetin and luteolin have been previously reported in leafy vegetables [49]. the negative impact of blanching on polyphenols content may affect the medicinal potentialities of leafy vegetables as previously mentioned [5]. therefore, other processing methods such as soaking, solar drying and refrigeration of leafy vegetables should be used to limit or avoid losses of polyphenols as observed during blanching. figure 2: vitamin c (a) and carotenoids (b) contents of raw and blanched leafy vegetables consumed in western côte d’ivoire figure 3: polyphenols contents (a) and antioxidant activity (b) of raw and blanched leafy vegetables consumed in western côte d’ivoire 4. conclusions african leafy vegetables (alvs) contain significant levels of nutrients that are essential for human health. the result of this study revealed that blanching at 15, 25 and 45 minutes decreased considerably the nutritional value of these leafy vegetables. nevertheless, the losses in anti-nutrients (oxalates, phytates) might have asserted a 0 10 20 30 40 50 60 70 v ita m in c (m g/ 10 0g ) a 0 1 2 3 4 5 c ar ot en o ïd s (m g /1 00 g) b raw 15 min 25 min 45 min 0 50 100 150 200 250 300 p o ly ph en ol s (m g /1 00 g) a 0 10 20 30 40 50 60 70 80 90 100 a nt io xy d an t a ct iv it y (% ) b raw 15 min 25 min 45 min food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 107 beneficial effect on bioavailability of minerals like calcium, iron and zinc. thus, the study suggests that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of ivorian population. 5. references [1] chweya j.a., eyzaguirre p.b. the biodiversity of traditional leafy vegetables. international plant genetic resources institute, rome, italy. (1999). [2] maundu p.m. the status of traditional vegetable utilization in kenya. in: l guarino (ed.). proceedings of the ipgri international workshop on genetic resources of traditional vegetables in africa: conservation and use. icraf-hq, nairobi, kenya. p. 66-75.(1997). [3] abukutsa-onyango m.o. market survey on african indigenous vegetables in western kenya. proceedings of the second horticulture seminar on sustainable horticultural production in the tropics. p. 39-46. (2002). [4] mnzava n.a. vegetable crop diversification and the place of traditional species in the tropics. proceedings of the ipgri international workshop on genetic resources of traditional vegetables in africa: conservation and use. icraf-hq, nairobi, kenya. p.1-15. (1997). [5]. schippers rr. african indigenous vegetables: an overview of the cultivated species. chatham, uk. natural resources institute /acpeu technical centre for agricultural and rural cooperation. (2002). [6] abukutsa-onyango m.o. unexploited potential of african indigenous vegetables in western kenya. maseno j. educ arts sci. 4. p. 103122. (2003). [7]. zoro a.f., zoue l.t., kra a.k., yepie a.e., niamke s.l. an overview of nutritive potential of leafy vegetables consumed in western côte d’ivoire. pak. j. nutr. 12. p. 949.956. (2013). [8] oulai p., zoue l., megnanou r.m., doue r., niamke s. (2014). proximate composition and nutritive value of leafy vegetables consumed in northern côte d’ivoire. eur. sci. j. 10. p. 212-227. (2014). [9] acho c.f., zoué l.t., akpa e.e., yapo v.g., niamké s.l. leafy vegetables consumed in southern côte d’ivoire: a source of high value nutrients. j. anim. plant sci. 20. p. 3159-3170. (2014). [10] chweya j.a. identification and nutritional importance of indigenous green leafy vegetables in kenya. acta hort. 153. p. 99-108. (1985). [11] gupta s., lakshmi j., prakash a.j. effect of different blanching treatments on ascorbic acid retention in green leafy vegetables. nat. prod. rad. 7. p.111-116. (2008). [12] canet w. quality and stability of frozen vegetables. in s. thorne (ed.). developments in food preservation. new york: elsevier science publishing inc. [13] akindahunsi a.a., oboh g. effect of some post-harvest treatments on the bioavailability of zinc from some selected tropical vegetables. la rivista italiana delle sostanze grasse. lxxvi. p. 285–287. (1999). [14] prestamo g., fuster c., risueno m.c. effects of blanching and freezing on the structure of carrots cells and their implications for food processing. j. sci. food agric. 77. p. 223–229. (1998). [15] murcia m.a., lopez-ayerra b., garcia-carmona f. effect of processing methods and different blanching times on broccoli: proximate composition and fatty acids. lebensmittel-wissenschaft technol. 32. p. 238–243. (1999). [16] oboh g. effect of blanching on the antioxidant properties of some tropical green leafy vegetables. lebensmittel-wissenschaft technol. 38. p. 513–517. (2005). [17]. cnra. socio-economical importance of leafy vegetables for the urban populations of côte d’ivoire, cnra ed. (2011). [18]. kouame n.m. contribution à l’étude des plantes spontanées alimentaires du department d’oumé (côte d’ivoire). mémoire de d.e.a d’ecologie tropicale. université de cocodyabidjan, côte d’ivoire, 122 p. (2000). [19]. fondio l., kouame c., n’zi j.c., mahyao a., agbo e., djidji a.h. survey of indigenous leafy vegetable in the urban and periurban areas of côte d’ivoire. acta hort. 752. p. 287-289. (2007). [20]. n’dri m.t., kouame g.m., konan e., traore d. plantes alimentaires spontanées de la région du fromager (centre-ouest de la côte d’ivoire): flore, habitats et organes consommés. sci. nat. 1. p. 61-70. (2008). [21]. chinma c.e., igyor m.a. micronutriments and anti-nutritional content of select tropical vegetables grown in south-east, nigeria. nig. food j., 25. p. 111-115. (2007). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xiii, issue 2 – 2014 armel f. zoro, lessoy t. zoue, sébastien l. niamke, evaluation of nutritive and antioxidant properties of blanched leafy vegetables consumed in western cote d’ivoire, volume xiii, issue 2 2014, pag. 99 – 108 108 [22]. aoac. official methods of analysis. association of official analytical chemists ed., washington dc. 684 p. (1990). [23]. fao. food energy-methods of analysis and conversion factors. fao ed, rome. 97 p. (2002). [24]. day r.a., underwood a.l. quantitative analysis. 5th ed. prentice hall. 701 p. (1986). [25]. latta m., eskin m. a simple method for phytate determination. j. agric. food chem. 28. p. 1313-1315. (1980). [26]. pongracz g., weiser h., matzinger d. tocopherolsantioxydant. fat sci. technol. 97. p. 90-104. (1971). [27]. rodriguez-amaya d.b. a guide to carotenoids analysis in foods. ilsi press, washington dc. 64 p. (2001). [28]. singleton v.l., orthofer r., lamuela-raventos r.m. analysis of total phenols and other oxydant substrates and antioxydants by means of folin-ciocalteu reagent. methods enzymol. 299. p. 152-178. (1999). [29]. choi c.w., kim s.c., hwang s.s., choi b.k., ahn h.j., lee m.z., park s.h., kim s.k. antioxydant activity and free radical scavenging capacity between korean medicinal plant and flavonoids by assay guided comparison. plant sci. 163. p. 1161-1168. (2002). [30]. antia b.s., akpan e.j., okon p. a., umoren i.u., nutritive and antinutritive evaluation of sweet potato (ipomea batatas) leaves. pak. j. nutr. 5. p. 166-168. (2006). [31]. svanberg s.m., nyman e.m. g., andersson l., nilsson r. effects of boiling and storage on dietary fiber and digestible carbohydrates in various cultivars of carrots. j. sci. food agric. 73. p. 245–254. (1997). [32]. ishida h., suzuno h., sugiyama n., innami s., todokoro t., maekawa a. nutritional evaluation of chemical component of leaves stalks and stems of sweet potatoes (ipomea batatas). food chem. 68. p. 359-367. (2000). [33]. lund d.b. effects of heat processing on nutrients. in: nutritional evaluation of food processing, (r. harries and e. karmas, eds). the avi publishing co. inc westport. p. 205 – 203. (1997). [34]. ali a. proximate and mineral composition of the marchubeh (asparagus officinalis). world dairy food sci. 4 p. 142-149. (2009). [35]. ejoh a.r., tchouanguep m.f., fokou e. nutrient composition of the leaves and flowers of colocasia esculenta and the fruits of solanum melongena. plant food hum. nutr. 49. p. 107-112. (1996). [36]. sobowale s.s., olatidoye o. p., olorode o.o., akinlotan j.v. nutritional potentials and chemical value of some tropical leafy vegetables consumed in south west nigeria j. sci. multidisciplinary res. 3. p. 55-65. (2011). [37]. fao. human vitamin and mineral requirements. fao ed. 361p. (2004). [38]. turan m., kordali s., zengin h., dursun a., sezen y. macro and micro-mineral content of some wild edible leaves consumed in eastern anatolia. plant soil sci. 53. p. 129-137. (2003). [39]. chaturvedi v.c., shrivastava r., upreti r.k. viral infections and trace elements: a complex trace element. curr. sci. 87. p. 15361554. (2004). [40]. trowbridge f. and martorell m. forging effective strategies to combat iron deficiency. j. nutri. 85. p. 875-880. (2002). [41]. hassan l.g., umar k.j. and umar z., antinutritive factors in tribulus terrestris (linn) leaves and predicted calcium and zinc bioavailability. j. trop. biosci. 7. p. 33-36. (2007). [42]. umar k.j., hassan l.g., dangoggo s.m., inuwa m., amustapha m.n. nutritional content of melochia corchorifolia (linn.) leaves. int. j. biol. chem. 1. p. 250-255. (2007). [43]. sandberg a.s. bioavailability of minerals in legumes. brit. j. nutr. 88. p. 281-285. (2002). [44]. ekop a.s., eddy n.o. comparative studies of the level of toxicants in the seed of indian almond (terminalia catappa) and african walnut (coula edulis). chem. class j. 2. p. 74-76. (2005). [45]. speek a.j., temalilwa g.r., schrijver j. determination of β-carotene content and vitamin a activity of vegetables by hplc. food chem. 19. p. 65-74. (1986). [46]. takyi e.e. children’s consumption of dark green leafy vegetables with added fat enhances serum retinol. j. nutr. 129. p. 1549–1554. (1999). [47]. yamaguchi t., mizobuchi t., kajinawa h., miyabe f., terao j., takamura h., matoba t. radicalscavenging activity of vegetables and the effect of cooking on their activity. food sci. technol. res. 7. p. 250–257. (2001). [48]. trichopoulou a., vasilopoulou e., hollman p., chamalides c., foufa e., kaloudis t., kromhout d., miskaki p., petrochilou i., poulima e., stafilakis k., theophilou d., nutritional composition and flavonoid content of edible wild greens and green pies: a potential rich source of antioxidant nutrients in the mediterranean diet. food chem. 70. p. 319323. (2000). [49]. wong s.p., leong l.p., koh j. h. antioxidant activities of aqueous extracts of selected plants. food chem. 99. p. 775-783. (2006). 179 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xiii, issue 3 – 2014, pag. 179 – 187 evaluating an organisation’s management performance through the certification audit *lucica brudiu1, mariana-liliana pǎcalǎ2 , constantin militaru3 1 s.c. vio-marprest s.r.l., department of quality management, 900581 constanta, romania; university politehnica of bucharest, faculty of engineering and management of technological systems, bucharest, romania; brudiu.lucica@yahoo.com 2 lucian blaga university of sibiu, department of agricultural science and food industry, 7-9 dr. i. ratiu st., 550012 sibiu, romania, tel.: +407-018-5438; fax: +4-026-921-2558; mariana.pacala@yahoo.com 3university politehnica of bucharest, faculty of engineering and management of technological systems, bucharest, romania; prof_militaru@yahoo.com *corresponding author received july 30th 2014, accepted august 29th 2014 abstract: the article proposes a strategy which reflects in the best way possible the evaluation of the integrated management system subjected to certification. for this purpose, 17 indicators of performance for the supply process of an organization have been defined and analysed so that the management performance of senior management can be improved during a certification cycle. the structure of the proposed strategy was tested in terms of applicability for the process of supply of an organization with an integrated management system. the case study "certification audit and evaluation of performance indicators for the supply process" has showed a decrease of non-compliant indicators and an even greater decrease of indicators that require analysis and what strategy to evaluate the performance indicators for the audited integrated management system is usable. the certification audit emphasizes the importance of integrated management system which is enlarged or minimized depending on how management addresses issues of responsiveness, adaptability and compliance, as well as its ability to pursue strategic objectives. keywords: integrated management, performance indicator, supply process 1. introduction a key objective of any organization is getting performance. implementing a system of performance indicators helps to assess the strategic objectives of the organization, so that at any moment one could be able to determine wheter it works the way it was established or not. a detailed knowledge of the state of the organization is not possible without a “system of performance indicators” [1] to inform management about the results obtained in all key activities and processes of the organization . implementation of the integrated management system in an organization begins by setting its specific performance indicators. by means of the integrated management system documents and records are generated by employees in order to give values of performance indicators. strategic objectives should be checked and measured objectively, otherwise the strategy remains a simple act. measuring the achievement of “strategic objectives” is done by evaluating the performance indicators [2]. the process of identification and definition of performance indicators should be established with the involvement of both “top management and the staff responsible” [3] for carrying out the processes in the organization. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 180 for each performance indicator the optimal value is defined and the period of time required to reach the indicator is established (in months, years, etc.). performance indicators are chosen so as to meet the strategic objective meaning that the optimal value reflects the strategic objective and the intermediate values to correspond to the achieved progress. identification, definition and implementation of the performance indicators should take into account their utility, namely they will not consider indicators whose calculation / evaluation requires offering too many resources (systems, work hours, spending surveys etc.) compared to the benefits for example, the classic performance indicators can be calculated based on the data stored in computer systems, such as financial data, production, quality statistics, etc. these are indicators that can be measured most frequently and are most easily determined. another type of performance indicators has the base of internal assessments or third party assesements. their advantage is that they can not be influenced, but have the disadvantage of being available only at longer intervals, and getting them can often cause high costs. indicators based on management's assessment of the organization on the functioning of the integrated management system can be used very well when they are comparable. it is clear that performance indicators need to be calculated in the same manner and on the same data in different processes of the organization. otherwise they lose consistency, the ability to reflect actual progress and ensure effective comparability. at the effective implementation of the integrated management the indicators will meet the following criteria: • indicator definition has a real basis ;is not purely theoretical; • indicator implementation costs benefits of measurement and monitoring outweigh the calculation of costs; • acceptance and responsabilisation are appropriated and taken out by the responsible managers to remain not only a bureaucratic exercise; • the capacity of the indicator to be defined, the indicator is a known name, a defined evaluation formula and used in the same way across the organization; • the evaluation/calculation frequency is defined. the certification of the integrated management system through certification auditing by a certification organism is also meant to evaluate performance indicators indentified by observing the measuring technique and monitoring the processes according to the referential requirements. the certification audit can find:  conformity – when a standard requirement is met;  non-conformity when a standard requirement is not met. the top management analysis of audit findings made after the certification audit allows the calculation of performance indicators’ values and the evaluation of the organisation status on meeting and improving organisational performance. without limiting the identification and use of performance indicators, we shall explain them for the supplying process from the integrated system management consisting of the quality management system according to standard sr en iso 9001: 2008[4], the food safety management system according to standard sr en iso 22000:2005[5], the enviroment management system according to standard sr en iso 14001:2005 [6] and occupational health and safety management system according to standard sr ohsas 18001: 2008[7]. the purpose of this article is to valorize the important role of the certification audit and food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 181 of the “audits for monitoring the integrated management system” 8] in the overall vision of the organisation and processes, by identifying improvement opportunities, which leads to optimizing the performance indicators of the supply process. 2. matherial and methods the case study consisted in testing and determining the degree of application of the evaluation strategy within an organisation in which the integrated management system for the supply process was implemented, functional and subjected to the certification process. this study has used for research the qualitative method: conceptual research and case study. as research tools we used the qualitative analysis of documents and observation as well as audit reports as support. the organisation involved in this study was chosen based on several factors:  it should have a documented and functional integrated management system;  it should have undergone a certification cycle for the integrated management system (quality, environment, food safety and occupational health and safety);  its field of activity should be the manufacturing of food products;  it should be a large organsation. in order to develop the model for evaluation and performance analysis, the following data of the organisation and input-data of the integrated management system and research have been identified, as shown in the table 1. the study was carried out for the supply process within the organisation in which the integrated management system is implemented and functional. table 1 values of data-input total no. of suppliers 90 value of supply costs (thousands lei) 800 no./value of products returned to supplier (thousands lei) 10 no. of orders not completed in due time 100 no. of suppliers with supply agreements 52 requirement 6.2. of referential standard “management system integration standard” [9] that is audited and connected to each integrated standard, appears in figure 1 figure 1. common requirements of standard for the supply process 3. results and discussion the calculation formula and optimal values have been determined for the performance indicators defined in this paper (table 2). the calculation formula is the percentage ratio between the determining measurements of each performance indicator. (1)  %100 b a i x where : ix – value of performance indicator, [%]; a – measured value at a certain moment, different from the initial moment; b – measured value at the initial moment. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 182 table 2. definition and optimal values of kpi for the supply process kpi designation kpi optimal level variables kpi 129 degree assessment suppliers 100% a no. evaluate suppliers b total no. of suppliers kpi 130 rate of steadily no of suppliers 90% a no.reevaluate suppliers maintained that accept b no.suppliers reassessed kpi 131 degree of acceptance suppliers 100% a no. suppliers accept b no. evaluate suppliers kpi 132 degree of uniqueness suppliers 5% a no. single supplier b total no. of suppliers kpi 133 degree of cost supply 20% a value of supply costs (thousands lei) b total cost value kpi 134 utilization degree 1 100% a the amount of raw materials used per month b the amount of monthly stock kpi 135 degree partial according to supply 0% a no. of noncompliance reports done for the supplied products noticed as non copliant at reception b total no. of non-conformities kpi 136 degree of compliance with food risk 0% a no. of noncompliance reports regarding food safety done for the supplied products noticed as non copliant at reception b total no. of non-conformities kpi 137 partial degree of compliance with food risk 0% a no. of noncompliance reports done for the supplied products noticed as non copliant at reception b no. noncompliance reports on food safety kpi 138 degree of return to supplier 0% a no./value of products returned to supplier (thousands lei) b no. / value products supplied kpi 139 auditing degree of suppliers 100% a no. second party audits conducted at suppliers b no. second party audits conducted at planned suppliers kpi 140 budget utilization rate of supply 100% a amount paid on goods supplied b value established by the budget kpi 141 level suppliers supply contracts 80% a no. of suppliers with supply agreements b total no. of suppliers kpi 142 degree of compliance of supplied products 100% a no. supply of products with documents attesting b no. total merchandise purchases kpi 143 assessment degree according to the reception 100% a requirements (qualified personnel + frequency analysis method of analysis + sample analysis) actually used b requirements (qualified personnel + frequency analysis method of analysis + sample analysis) set kpi 144 degree of honor purchase orders 5% a no. unfulfilled orders on time b no. orders issued kpi 145 share of noncompliant products obtained stocked with hidden flaws 0% a no. / assets obtained with non-compliant products supplied with hidden flaws b no. / value of non-conforming products obtained the following non-conformities were noticed for the supply process of the organisation, audited during the certification audit of year i:  not all suppliers have been evaluated. this way it was possible to calculate the value of kpi 129.  not all the previously accepted suppliers have been reevaluated. this way it was possible to calculate the value of kpi 130.  not all the second-party audits planned for the suppliers have been done. this way it was possible to calculate the value of kpi 139. food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 183  not all the supplied products have documents that assure quality. this way it was possible to calculate the value of kpi 142.  not all the responsible personnel are instructed in the “supplying” procedure. this way it was possible to calculate the value of kpi 143.  not all the orders to the suppliers have been delivered on time. this way it was possible to calculate the value of kpi 144.  not all methods of analysis of the products at the reception are likely to identify potential nonconformities. this way it was possible to calculate the value of kpi 145. following the analysis performed by the top management and relevant positions of the organisation involved in the certification process, based on the certification audit report and the nonconformity reports included in it, it was possible to calculate also the values of the indicators: kpi 131, kpi 132, kpi 133, kpi 134, kpi 135, kpi 136, kpi 137, kpi 138, kpi 141. this way, the values of the performance indicators for kpi 129-kpi 145 were calculated and shown in table 3. table 3 values of kpi of the certification cycle (year i, ii and iii) for the supply process kpi values for year i values for year ii values for year iii a b kpi, % a b kpi, % a b kpi, % kpi 129 78 90 87 92 100 92 110 110 100 kpi 131 69 78 8 84 92 91 96 110 87 kpi 132 6 90 7 6 100 6 6 110 5 kpi 133 800 1600 50 360 1600 23 320 1600 20 kpi 134 2 4 50 2.5 4 63 3.5 4 88 kpi 135 1 12 8 15 0 16 0 kpi 136 0 12 0 1 15 7 0 16 0 kpi 137 0 12 0 1 15 7 1 16 6 kpi 138 0.10 10 1 0.5 100 0.5 0 10 0 kpi 139 0 12 0 6 12 50 10 12 83 kpi 140 3 3.3 91 3.4 3.3 103 3.2 3.3 97 kpi 141 52 90 58 65 100 65 75 110 68 kpi 142 2.3 2.35 98 2.2 2.3 96 2.31 2.4 96 kpi 143 4 5 80 5 5 100 5 5 100 kpi 144 0.1 2.35 4 0.5 2.3 2 3.3 2.4 1 kpi 145 2 10 20 1 10 10 0 10 0 3.1. evaluation of performance indicators the next step in setting the strategy for the evaluation of management performance based on performance indicators is comparing their value determined during the certification audit to an optimal value. the optimal value of performance indicators was set as based on the organisational history, the analyses performed by the top management on the food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 184 evolution of the organisation in terms of organisational and financial strategy and meeting the requirements of the “management system integration standard” [9]. table 3 shows the optimal values of performance indicators. three degrees of evaluation were set for performance indicators based on the optimal value:  degree i conformity – if the value of the performance indicator is equal to the optimal value.  degree ii – non-conformity – if the value of the performance indicator is lower than the optimal value. in this case there shall be identified and analysed the cause of not reaching the optimal value and there shall be implemented the corrective action, sometimes the preventive action within a non-conformity report written by the responsible for the process considered to be non-conforming when the audit is performed. this nonconformity is not designed to affect the functioning of the integrated management system.  degree iii – requires analysis – if the value of the performance indicator is higher than the optimal value, in the case of indicators involved in evaluating food safety risk, product design, customer satisfaction or financial loss. this requires an analysis by all factors involved in the non-conforming process. throughout the development of the certification cycle of the integrated management system, consisting in the certification audit and the annual monitoring audits performed by auditors representing the certification organism, their impartiality has helped to determine the degrees of performance indicators. for the case study applied to the supply process within the integrated management system according to the classification of performance indicators into the three degrees, there resulted the situation presented in table 4 and in figure 2. table 4 classification of kpi in evaluation degrees from the certification cycle (year i, ii and iii) for the supply process within the organisation kpi optimal level, [%] year i interpretation year ii interpretation year iii interpretation kpi 129 100 87 nc 92 nc 100 c kpi 130 90 76 nc 88 nc 84 nc kpi 131 100 88 nc 91 nc 87 nc kpi 132 5 7 na 6 na 5 c kpi 133 20 50 na 23 na 20 c kpi 134 100 50 nc 63 nc 88 nc kpi 135 0 8 na 0 c 0 c kpi 136 0 0 c 7 na 0 c kpi 137 0 0 c 7 na 6 na kpi 138 0 1 na 0.5 na 0 c kpi 139 100 0 nc 50 nc 83 nc kpi 140 100 91 nc 103 na 97 nc kpi 141 80 58 nc 65 nc 68 nc kpi 142 100 98 nc 96 nc 96 nc kpi 143 100 80 nc 100 c 100 c kpi 144 5 4 nc 2 nc 1 nc kpi 145 0 20 na 10 na 0 c (c-conforming, nc-non-conforming, na-requires analysis) food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 185 the results of applying the strategy for evaluating management performance after the monitoring audit of year i, ii, respectively year iii, of the certification cycle were adapted and included in table 4. by keeping the same optimal values of the performance indicators, following the analysis performed by the top management, based on the report for the monitoring audit for year i, ii, respectively year iii, of the certification cycle, there has resulted the interpretation of performance indicators in table 4 and the graphic representation in figures 2 show the important role of the certification audit, the monitoring audits of year i, ii and year iii, by pushing the values of performance indicators towards the optimal value and improving the supplay process after each annual audit. by comparing the kpi values to the optimum value that had only 6 of the optimal values were achieved and it can be concluded that the supply takes place efficiently and effectively in a proportion of 35.3%. another purpose of the evaluation is to determine the performance management of all the causes that have generated employment performance indicators in levels of "non-compliant" and "needs analysis" in an analysis of the management. figure 2. kpi values for year i, ii and iii compared to optimal kpi values performance indicators were identified and adapted according to the strategy of evaluating management performance applied in the case study, as shown in the diagram in figure 3. 0 20 40 60 80 100 120 kpi 129 kpi 130 kpi 131 kpi 132 kpi 133 kpi 134 kpi 135 kpi 136 kpi 137 kpi 138 kpi 139 kpi 140 kpi 141 kpi 142 kpi 143 kpi 144 kpi 145 optimal values year 1 values year 2 values year 3 food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 186 figure 3. identification diagram for the system of indicators within organisations with certifiable or certified integrated management system 4. conclusion following the analysis and implemented corrections, corrective and preventive actions for non-conformities identified during the certification audit by auditors of the certification organism, the monitoring audits of year ii and iii of the certification cycle have shown that some of the performance indicators have reached optimal values and some indicators of the supply process have values that require analysis and correction within the nonconformity reports. by applying the stratgy suggested for improving management performance within an organisation, the audits of the certification cycle have shown a decrease of 20 % of non-conforming indicators and of 80 % of indicators that require analysis at the end of the certification cycle. a highly positive evolution was recorded for indicators kpi 139 degree auditing suppliers, kpi 142 degree of conformity assessment at reception. the certification of the integrated management system by performed audits supports the strategy for improving management performance which is a key element for developing an organisation. focusing on the certification of the integrated management system does not imply the need to use costly last generation equipment, but only team work at all organisational levels. the certification audit and the monitoring audits of the integrated management system have an important role in the overall vision of the organisation and processes, by identifying improvement opportunities and optimizing performance indicators. 5. acknowledgments we would like to thank the management team of s.c. safir s.a. for the permission to do the case study on the organisation and also to acknowledge the certification organism for letting us use the audit reports regarding the enterprise. define strategy define kpi identifiy kpi / monitor kpi implement kpi main actions • defining strategic objectives and functions • define how to achieve strategic objectives main actions • kpi identification • -measured • -can be measured • -need to be measured • define selected kpi • define optimum for selected kpi main actions • define data collectiong process • define monitoring process -steps • -roles and responsabilities measurements frequency • kpi reporting type main actions • kpi analisation and comparison to optimum • corelation with ims • optimum adjustment and definition of improvement actions of performance measurement sistem food and environment safety journal of faculty of food engineering, ştefan cel mare university – suceava volume xiii, issue 3 – 2014 lucica brudiu, mariana-liliana pǎcalǎ, constantin militaru, evaluating an organisation’s management performance through the certification audit, food and environment safety, volume xiii, issue 3 – 2014, pag. 179 – 187 187 6. references [1]. kaplan s.v. r., norton d.p., the balansed scorecard, harvard business school press, 1997; [2]. neely et al., designing, implementing and updating performance measurement systems, international journal of operations & production management vol. 20 iss: 7, 1995, 754 771; [3]. hăhăianu, l., how to motivate your employees to achieve outstanding performance, rentrop&straton, group publishing and consulting business, bucharest, 2002 (in romanian). [4]. sr en iso 9001:2008 quality management systems. requirements (in romanian). [5]. sr en iso 22000:2005 food safety management systems. requirements for any organization in the food chain(in romanian). [6]. sr en iso 14001:2005 environmental management systems. requirements with guidance for use (in romanian). [7]. sr ohsas 18001:2008 management systems occupational health and safety. requirements (in romanian). [8]. kanter r. m., borders management, meteor press publishing, bucharest, 2006 (in romanian). [9]. brudiu, l., pacala, m.-l., abdelrahim al ali (2013) systems management and integration, journal of management systems quality, 14(136), 9-13 (in romanian). doi: https://doi.org/10.4316/fens.2023.006 59 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 59 70 charcoal-based conservation methods’ impact on some functional properties of flours of three plantain varieties (musa sp.) loh tinndé charles sabli 1 , * wohi maniga 2 , souleymane coulibaly 3 , eugène jean parfait kouadio 1 1faculty of food science and technology, nangui abrogoua university, abidjan, côte d’ivoire, 2*faculty of biological sciences, peleforo gon coulibaly university, korhogo, côte d’ivoire, wmaniga@yahoo.fr, 3formerly laboratory of food technology, national center for agronomic research, côte d'ivoire, *corresponding author received 3rd january 2023, accepted 27th march 2023 abstract: the plantain contributes significantly to food security in sub-saharan africa. however, the sector is faced with several difficulties, in particular the lack of inexpensive conservation techniques accessible to all. in response, a conservation method combining charcoal and polyethylene bags was tested on the saci, big-ebanga, and orishélé varieties, harvested at the mature stage. the water and oil absorption capacity and the solubility index were determined according to standard methods. the results indicated a significant increase in these properties during storage. the water and oil absorption capacity and solubility index of fruit flour preserved in a control environment without polythene and charcoal are between 197.35% and 242.21%, 30.56% and 59.80%, and between 29.19% and 43.7 2%, respectively. plantain bananas stored in a control environment consisting of charcoal-free polyethylene packaging recorded water and oil absorption capacities and solubility index of between 214.12% and 241.19%, 35.86% and 59.21% then between 35.29% and 44.27%, respectively. fruit flours packed in polythene bags containing dry or moistened solid charcoal or dry or moistened charcoal powder have recorded water and oil absorption capacities and solubility between 215.11% and 241.14%, 35.90% and 59.51% and between 35.32% and 43.72%, respectively. charcoal preservation can be a solution approach to the problem of post-harvest loss. keywords: plantain flours, functional parameters, storage, polyethylene. 1. introduction plantain is one of the main food sources of significant income for producing countries [1]. the plantain is cultivated in more than 120 countries for an area of about 10 million hectares and a world production of nearly 106 million tons per year [2]. this makes it the second food crop in the world after cereals and the fourth cultivated food crop in the world after rice, wheat, and maize [3]. plantains are consumed in different forms of food depending on the state of maturity of the available bananas, including fries, boiled, roasted, mashed, etc. [4]. its average annual consumption per person in côte d'ivoire is around 75 kg [5]. plantain is a foodstuff characterized by a high carbohydrate content (more than 28 g per 100 g). although plantain is in high demand and sells very well on the ivorian market, its expansion faces several constraints. the most missing is the absence of conservation methods using inexpensive, practical, and accessible techniques, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 60 resulting in a lasting positive impact on food security. in fact, under normal ambient temperature conditions (30°c), the plantain ripens between 5 and 9 days after harvest, when physiological maturity is reached. temperature, oxygen, carbon dioxide (co2), and ethylene are factors that influence the ripening process of banana fruits. ethylene is the hormone that initiates all the processes involved in fruit ripening [6]. upon maturity, the fruit enters a period of senescence, which results in cellular disorganization and death. knowing that charcoal has been known for a long time for its ability to adsorb gases, it was used in this study to slow down fruit ripening by ethylene absorption. the objective of this work is to evaluate the impact of charcoal-based preservation methods on some functional parameters of plantain bananas. 2. matherials and methods material the plantain bananas that were the subject of this study come from a plantation in the locality of azaguié, about 50 km from abidjan in côte d'ivoire. these are three cultivars, namely big-ebanga, saci, and orishèlé. the fruits were harvested at optimum maturity according to the method described by gnakri and kamenan [7] and kouadio et al. [8]. it corresponds to 70 days for the orishélé variety and 80 days for those of saci and big-ebanga. as part of this study, 68 bunches were harvested including 25 bunches of the orishélé variety, 26 bunches of big-ebanga, and 17 bunches of saci. the preservation method consisted of packing four (4) plantain fingers in plastic bags containing either solid dry or moistened solid charcoal, or dry or moistened charcoal powder (figure 1). the wet charcoal powder used was extracted from a mixture of dry charcoal powder moistened to 1/5 (v/v) of its volume. solid coals immersed in water for a few seconds constituted the moistened solid coals used. the dimensions of the packaging bags were a function of the size of the plantain bananas and the mass of charcoal used according to the mass of the fingers of the plantain bananas. that is 7g of charcoal for 100g of plantain. hermetically sealed packages were stored at a temperature of 28°c. a: fruit of plantain bananas stored in packages containing coal powder; b: plantain banana fruit stored in packages containing solid coal fig 1: conservation methods for the fruits of the plantains studied . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 61 method flour production technology for flour production, 600 g of each plantain sample was peeled using a stainless-steel knife. the pulp obtained was washed and cut into slices (about 1 cm thick). these slices were dried in a ventilated oven (memmert, 854 scwachbach, west germany) at 45°c for 48 hours and then ground. the grinding was passed through a 200 um mesh sieve to obtain the flour. determination of water absorption capacity and water solubility index the water absorption capacity and the water solubility index of the plantain flours were respectively determined according to the methods of phillips et al. [9] and anderson et al. [10]. one (1) g of plantain flour (m0) was dissolved in 10 ml of distilled water in a centrifuge tube. this mixture was stirred for 30 min and then kept in a water bath (water bath hhw600, china) at 37° c for 30 min. it was then centrifuged (sigma 3-16p) at 4200 rpm for 20 min. the pellet obtained (m2) was weighed, and then dried (biobase bov-d70, china) at 105°c in an oven until a constant mass (m1) was obtained. the water absorption capacity and water solubility index were calculated according to the formula below. (1) (2) m0: mass of the sample taken m1: dry mass of the sample after passing through the oven m2: mass of the fresh pellet after centrifugation determination of oil absorption capacity the oil absorption capacity of flour has been determined according to the method described by sosulski [11]. a quantity of 1g of plantain flour (m0) was dissolved in 10 ml of oil. the mixture was agitated for 30 min at room temperature using a mechanical agitator (vortex genie k550ge, united states) and then, centrifuged (sigma 3-16p, germany) at 4200 rpm for 12 min. the pellet obtained was weighed (m1). the oil absorption capacity was calculated using the following formula: (3) m0: mass (g) of the sample taken m1: mass (g) of the fresh pellet of the sample after centrifugation statistical analysis statistical analysis of the data was carried out using the ibm spss statistics 21 software. the comparison of the means was made according to the tukey test at the 5% threshold. 3. results and discussion water absorption capacity the water absorption capacity of the saci variety (table 1) which was initially 197.35% increased (p ≤ 0.5) during storage to reach, after 30 days, 218.13% for sassi in polythene bags containing dry solid charcoal (sacss), 215.11% for sassi in polythene bags containing wet solid charcoal (sacsh), 216.71% for sassi in polythene bags containing dry powdered charcoal (sacps) and 217.05% for sassi in polythene bags containing wet charcoal powder (sacph). those of the control samples that are sassi without packaging (sa) and sassi in polythene bags without carbon (ssc), whose respective storage food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 62 times were 12 days and 24 days, recorded 213.90% and 214.12%, at the end of storage. for the big-ebanga variety (table 2), the values of the water absorption capacity of the fruits increased (p ≤ 0.5) as well and went from day 0 to day 30 from 230.48% to 240 .43% for big-ebanga in polythene bags containing dry solid charcoal (bcss), 240.74% for big-ebanga in polythene bags containing wet solid charcoal (bcsh), 240.79% for big-ebanga in polythene bags containing dry powdered charcoal (bcps) and 241.14% for big-ebanga in polythene bags containing wet charcoal powder (bcph). the bcph (241.14%) recorded the highest water absorption capacities while those of the bcss (240.74%) were the lowest. in addition, the control fruits of this variety that are big-ebanga without packaging (b) whose storage time was 12 days and big-ebanga in polythene bags without carbon (bsc) whose storage time was 24 days have obtained, respectively, at the end of storage, flours whose water absorption capacity was 242.21% and 241.19%. as for the fruits of the orishélé variety (table 3), the water absorption capacity increased (p ≤ 0.5), and the rates varied from day 0 to day 30 from 213.85% to 222, 59% for orishele in polythene bags containing dry solid charcoal (ocss), 222.31% for orishele in polythene bags containing wet solid charcoal (ocsh), 222.71% for orishélé in polythene bags containing dry powdered charcoal (ocps) and 222.51% for orishélé in polythene bags containing wet charcoal powder (ocph). at day 30, fruits of ocsh (222.31%) had the lowest rate of water absorption capacity and those of ocps (222.71%) had the highest rate. on the other hand, the control fruits of this variety have obtained, after respective conservation periods of 12 days for orishélé without packaging (o) and (24) days for orishélé in polythene bags without carbon (osc), flours whose water absorption capacities were 221.26% and 221 .61%. these rates are higher than those of sylvain et al. [12] who obtained rates whose highest value was 180.29% for plantain flour. the statistical analysis results indicated a significant difference (p ≤ 0.5) between the values of the water absorption capacity of the flours of the fruits from one storage environment to another. the high water absorption capacity could also be the result of the synthesis of hydrophilic constituents (amino acid, amylose, amylopectin) during ripening ([13], [14]), which contributed to increase the sites of interaction with water ([15], [16]). furthermore, diallo et al. [17] showed that the size of the starch grains and the high carbohydrate content in flour could promote greater water absorption. these high water absorption capacities of flours suggest that they can be incorporated with water, hence used in the formulation of certain foods such as sausages, pasta, and baked goods [18]. refined palm oil absorption capacity the refined palm oil absorption capacity of saci variety fruit flour (table 4) increases (p ≤ 0.5) during storage. the rate observed at the start of storage (day 0) is 30.56%. after 30 days of storage, this rate reached 36.13% for sacss, 36.26% for sacsh, 35.90% for sacps, and 36.82% for sacph. the highest refined palm oil absorption capacities of the saci variety at day 30 were those of the fruits of sacph (36.83%) and sacsh (36.26%), while the lowest rates were obtained from the fruits of the sacps (35.90%). the control samples sa and ssc whose respective storage times were 12 days and 24 days food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 63 recorded respective rates of 36.79% and 35.86%, at the end of storage. table 1: evolution of the water absorption capacity (wac) of the saci variety in six storage environments storage time (day) wac of sa (%) wac of sacss (%) wac of sacsh (%) wac of sacps (%) wac of sacph (%) wac of ssc (%) d0 197.35 ± 0.57 aa d4 201.63 ± 0.03 bf 198.02 ± 0.04ab 198.82 ± 0.03ae 197.98 ± 0.02aa 198.09 ± 0.05ac 198.70 ± 0.06ad d8 206.73 ± 0.02 cf 198.85 ± 0.04aa 199.12 ± 0.02bb 199.44 ± 0.02bc 203.21 ± 0.08be 199.72 ± 0.07bd d12 213.90 ± 0.14 de 200.34 ± 0.02aba 201.04 ± 0.02cc 201.03 ± 0.02cc 207.72 ± 0.04cd 200.47 ± 0.05cb d16 202.41 ± 0.03 bca 204.67 ± 0.02db 202.26 ± 1.12da 212.64 ± 0.06dc 201.78 ± 0.06da d20 205.15 ± 0.05 cb 207.47 ± 0.03ec 203.41 ± 0.03ea 213.72 ± 0.04ee 209.13 ± 0.09ed d24 209.85 ± 0.03 dc 209.81 ± 0.04fb 206.45 ± 0.03fa 215.01 ± 0.05fe 214.12 ± 0.08fd d28 212.11 ± 0.02 ec 211.62 ± 0.18gb 209.06 ± 0.03ga 216.24 ± 0.02gd d30 218.13 ± 0.02fb 215.11 ± 0.02ha 216.71 ± 0.03hab 217.05 ± 0.05hab these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. wac: water absorption capacity; sa: saci without packaging; sacss: saci in polythene bags containing dry solid carbon; sacsh: saci in polythene bags containing wet solid carbon; sacps: saci in polythene bags containing dry powdered charcoal; sacph: saci in polythene bags containing wet powdered charcoal; ssc: saci in polythene bags without carbon. table 2: evolution of the water absorption capacity (wac) of the big-ebanga variety in six storage environments storage time (day) wac of b (%) wac of bcss (%) wac of bcsh (%) wac of bcps (%) wac of bcph (%) wac of bsc (%) d0 230.48 ± 0.09 aa d4 234,53 ± 0.02 bf 230.77 ± 0.01ad 229.65 ± 0.02aa 231.02 ± 0.02ae 230.74 ± 0.02ac 230.64 ± 0.01ab d8 237,95 ± 0.01 cf 232.31 ± 0.03bd 230.26 ± 0.03ba 232.17 ± 0.02bc 231.12 ± 0.02bb 232.54 ± 0.01be d12 242.21 ± 0.03 df 232.89 ± 0.02cc 231.31 ± 0.02ca 232.36 ± 0.01cb 233.07 ± 0.01cd 233.73 ± 0.02ce d16 235.92 ± 0.03 dd 233.12 ± 0.15da 233.21 ± 0.03db 233.21 ± 0.02db 234.59 ± 0.01dc d20 237.06 ± 0.01 ee 235.16 ± 0.01eb 235.18 ± 0.02ec 236.54 ± 0.01ed 235.03 ± 0.02ea d24 237.83 ± 0.01 fb 237.51 ± 0.02fb 236.81 ± 0.02fa 239.23 ± 0.02fc 241.19 ± 0.03fd d28 238.09 ± 0.02 ga 239.91 ± 0.03gc 239.52 ± 0.02gb 241.03 ± 0.03gd d30 240.43 ± 0.04 ha 240.74 ± 0.02hb 240.79 ± 0.06hb 241.14 ± 0.02hc these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. wac: water absorption capacity; b: big-ebanga without packaging; bcss: big-ebanga in polythene bags containing dry solid charcoal; bcsh: big-ebanga in polythene bags containing wet solid charcoal; bcps: big-ebanga in polyethylene bags containing dry powdered charcoal; bcph: big-ebanga in polyethylene bags containing wet powdered charcoal; bsc: big-ebanga in polyethylene bags without carbon food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 64 table 3: evolution of the water absorption capacity (wac) of the orishele variety in six storage environments storage time (day) wac of o (%) wac of ocss (%) wac of ocsh (%) wac of ocps (%) wac of ocph (%) wac of osc (%) d0 213.85 ± 0.01 aa d4 217.24 ± 0.01 be 214.51 ± 0.01ad 214.14 ± 0.03ac 214.03 ± 0.05ab 213.31 ± 0.04aa 214.02 ± 0.02ab d8 219.45 ± 0.12 ce 214.64 ± 0.05bb 214.82 ± 0.05bc 215.17 ± 0.02bd 214.38 ± 0.19ba 214.43 ± 0.14ba d12 221.26 ± 0.02 df 216.57 ± 0.02ce 215.81 ± 0.02cc 215.92 ± 0.02cd 214.96 ± 0.03cb 214.89 ± 0.02ca d16 217.72 ± 0.02 de 217.01 ± 0.02dc 217.10 ± 0.01dd 216.02 ± 0.02db 215.83 ± 0.05da d20 219.01 ± 0.01 ee 217.24 ± 0.02ec 217.37 ± 0.01ed 216.44 ± 0.01ea 216.94 ± 0.03eb d24 219.91 ± 0.01 fd 219.13 ± 0.03fc 21761 ± 0.05fb 217.15 ± 0.02fa 221.61 ± 0.02ge d28 220.04 ± 0.01 gb 220.39 ± 0.01gd 219.123 ± 0.01ga 220.31 ± 0.04gc d30 222.59 ± 0.01 hc 222.31 ± 0.01ha 222.71 ± 0.02hd 222.51 ± 0.01hb these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. wac: water absorption capacity; o: orishele without packaging; ocss: orishele in polythene bags containing dry solid charcoal; ocsh: orishele in polythene bags containing wet solid charcoal; ocps: orishele in polyethylene bags containing dry powdered charcoal; ocph: orishele in polyethylene bags containing wet powdered charcoal; osc: orishele in polyethylene bags without carbon for the big-ebanga variety (table 5), the refined palm oil absorption capacity observed on day 0 was 45.45%. this rate increased (p ≤ 0.5) and reached, after 30 days of storage, respective rates of 49.49% for bcss, 49.32% for bcsh, 49.13% for bcps, and 48.91 % for bcph. bcss flours obtained the highest rate of refined palm oil absorption capacity (49.49%) while bcph flours (48.91%) and bcps flours (49.13%) recorded the lowest rates. in addition, the control fruits of this variety were obtained at the end of conservation flours with refined palm oil absorption capacity of 49.31% for the b and 49.17% for the bsc. as for the orishélé variety (table 6), the absorption capacity of refined palm oil which was 50.41% on day 0 increased (p ≤ 0.5) to respectively reach 59.51% for the ocss, 58.38% for ocsh, 58.07% for ocps and 58.13% for ocph, after 30 days of storage. the fruits of the ocss (59.51%) presented the highest refined palm oil absorption capacities while the lowest rates were obtained by the ocps (58.07%). on the other hand, the control fruits of this variety were obtained after respectively 12 days (o) and 24 days (osc) of conservation of the flours whose refined palm oil absorption capacities were 59.80% and 59.21%. statistical analysis indicates a significant difference (p ≤ 0.5) between the values of the refined palm oil absorption capacity of fruits from one storage environment to another. the increase in oil absorption capacity during storage could be attributed to the increase in protein content, which increases the hydrophobicity of flour [13]. indeed, the oil absorption capacity is the capacity of a protein to absorb and maintain oil in its structure. it can therefore be influenced by the lipophilic nature of proteins [19]. these results are lower than that (85.9%) obtained by medoua [20] on yam flours (dioscorea dumetorum) kept food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 65 for 2 days and then dehydrated in an oven at 45°c. flour with high oil absorption capacity assumes that it has good lipophilic constituents and can therefore be suitable for the production of sausages, cakes, and donuts [21]. table 4: evolution of the refined palm oil absorption capacity (rpoac) of the saci variety in six storage environments storage time (day) rpoac of sa (%) rpoac of sacss (%) rpoac of sacsh (%) rpoac of sacps (%) rpoac of sacph (%) rpoac of ssc (%) d0 30.56 ± 0.01 aa d4 31.05 ± 0.63 af 30.87 ± 0.06ad 30.76 ± 0.11ab 31.07 ± 0.03aa 31.17 ± 0.02ac 30.52 ± 0.02ae d8 33.42 ± 0.39 be 31.14 ± 0.22abc 30.85 ± 0.12abb 31.40 ± 0.27aba 31.64 ± 0.38aba 30.74 ± 0.23ad d12 36.79 ± 0.12 cf 31.41 ± 0.09bce 31.476 ± 0.43bca 31.73 ± 0.12bcc 32.06 ± 0.03bb 31.28 ± 0.23bd d16 31.65 ± 0.09 cd 32.07 ± 0.25cc 31.92 ± 0.09ca 32.28 ± 0.14bb 31.59 ± 0.16bd d20 33.95 ± 0.32 da 34.29 ± 0.06db 34.31 ± 0.27dc 34.36 ± 0.02cd 33.94 ± 0.13ca d24 34.49 ± 0.15 ea 34.71 ± 0.32deb 34.51 ± 0.03da 34.87 ± 0.35cdc 35.86 ± 0.05ed d28 34.72 ± 0.09 ec 35.09 ± 0.22ea 34.57 ± 0.04da 35.36 ± 0.12db d30 36.13 ± 0.13 fb 36.26 ± 0.06fc 35.90 ± 0.08eb 36.82 ± 0.52ea these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days accordin g to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. rpoac: refined palm oil absorption capacity; sa: saci without packaging; sacss: saci in polythene bags containing dry solid carbon; sacsh: saci in polythene bags containing wet solid carbon; sacps: saci in polythene bags containing dry powdered charcoal; sacph: saci in polythene bags containing wet powdered charcoal; ssc: saci in polythene bags without carbon. table 5: evolution of the refined palm oil absorption capacity (rpoac) of the big-ebanga variety in six storage environments storage time (day) rpoac of b (%) rpoac of bcss (%) rpoac of bcsh (%) rpoac of bcps (%) rpoac of bcph (%) rpoac of bsc (%) d0 45.45 ± 0.03 aa d4 46.64 ± 0.06 abe 45.42 ± 0.14ac 45.33 ± 0.09abc 45.21 ± 0.01aab 45.07 ± 0.08aa 45.58 ± 0.03ad d8 48.70 ± 0.03 bd 47.31 ± 0.01bc 45.69 ± 0.57aa 47.12 ± 0.03bc 46.11 ± 0.07bb 47.48 ± 0.05bc d12 49.31 ± 0.02 bd 47.45 ± 0.05cb 47.18 ± 0.06ca 47.16 ± 0.03ba 47.21 ± 0.02ca 47.75 ± 0.06cc d16 48.30 ± 0.01 dd 47.32 ± 0.02ca 48.21 ± 0.09cc 48.16 ± 0.03db 48.33 ± 0.01dd d20 48.61 ± 0.03 ee 48.08 ± 0.02da 48.39 ± 0.02dc 48.30 ± 0.02db 48.51 ± 0.01ed d24 49.21 ± 0.01 fc 49.22 ± 0.04ec 48.56 ± 0.06eb 48.44 ± 0.05da 49.17 ± 0.08fc d28 49.51 ± 0.01 ga 49.31 ± 0.01ea 49.07 ± 0.06fa 49.06 ± 0.09ea d30 49.49 ± 0.03 ga 49.32 ± 0.05ea 49.13 ± 0.03fa 48.91 ± 0.55fa these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. rpoac: refined palm oil absorption capacity; b: big-ebanga without packaging; bcss: big-ebanga in polythene bags containing dry solid charcoal; bcsh: big-ebanga in polythene bags containing wet solid charcoal; bcps: big-ebanga in polyethylene bags containing dry powdered charcoal; bcph: big-ebanga in polyethylene bags containing wet powdered charcoal; bsc: big-ebanga in polyethylene bags without carbon food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 66 table 6: evolution of the refined palm oil absorption capacity (rpoac) of the orishele variety in six storage environments storage time (day) rpoac of o (%) rpoac of ocss (%) rpoac of ocsh (%) rpoac of ocps (%) rpoac of ocph (%) rpoac of osc (%) d0 50.41 ± 0.02 aa d4 55.19 ± 0.02 bb 50.73 ± 0.04aa 50.87 ± 0.58aa 50.77 ± 0.10aa 50.81 ± 0.05aa 50.99 ± 0.51aa d8 57.11 ± 0.03 cc 51.56 ± 0.02bab 51.84 ± 0.02bab 51.45 ± 0.52ba 51.93 ± 0.08bab 52.05 ± 0.07bb d12 59.80 ± 0.02 df 56.30 ± 0.03ce 55.52 ± 0.03cb 56.02 ± 0.02cc 55.12 ± 0.02ca 56.21 ± 0.02cd d16 56.49 ± 0.02dd 56.36 ± 0.01dc 56.11 ± 0.02cb 55.31 ± 0.03da 56.61 ± 0.02ce d20 57.13 ± 0.01ed 56.70 ± 0.02dc 56.32 ± 0.02cb 56.11 ± 0.02ea 57.33 ± 0.01de d24 58.52 ± 0.02fd 58.22 ± 0.01ec 57.32 ± 0.02db 57.21 ± 0.01fa 59.21 ± 0.02ee d28 58.75 ± 0.02gd 58.28 ± 0.02ec 57.62 ± 0.01dea 58.12 ± 0.02gb d30 59.51 ± 0.02hd 58.38 ± 0.02ec 58.07 ± 0.01ea 58.13 ± 0.01hb these values are the means of three determinations for each parameter. the means ± standard deviati on, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant differen ce between storage media according to tukey. rpoac: refined palm oil absorption capacity; o: orishele without packaging; ocss: orishele in polythene bags containing dry solid charcoal; ocsh: orishele in polythene bags containing wet solid charcoal; ocps: orishele in polyethylene bags containing dry powdered charcoal; ocph: orishele in polyethylene bags containing wet powdered charcoal; cso: orishele in polyethylene bags without carbon solubility index the solubility index of flours from the fruits of the three plantain varieties saci (table 7), big-ebanga (table 8), and orishélé (table 9) increases significantly (p ≤ 0.05) during storage in all storage conditions for this study. the results also indicate a significant difference (p ≤ 0.5) between the values of the fruit solubility index from one storage condition to another. the solubility index of the saci variety recorded on day 0 is 29.19%. this rate increases and passes respectively, after 30 days of storage, to 35.91% for sacss, 36.35% for sacsh, 35.32% for sacps, and 36.35% for sacph. the highest solubility indices of the saci variety, after 30 days of storage, are obtained by the sacph (36.35%) and sacsh (36.35%) flours. sacps fruit flour (35.32%) recorded the lowest solubility index. the sa and ssc control samples, whose respective storage times were 12 days and 24 days, recorded rates of 35.22% and 35.29% at the end of storage. concerning the solubility index of the bigebanga variety, it evolved from 37.26% on day 0 to reach respectively, after 30 days of storage, rates of 43.11% for bcss, 43.81% for bcsh, 44.25% for bcps and 43.24% for bcph. the highest solubility index was obtained by bcps (44.25%) and the lowest rate was obtained by bcss (43.11%). furthermore, the control fruits of this variety obtained flours whose solubility indices were 43.72% and 44.27% for b and bsc respectively. the solubility index of the orishelé variety recorded on day 0 was 31.65%. this rate increased respectively, after 30 days of storage, to 39.31% for ocss, 37.84% for ocsh, 39.13% for ocps, and 39.12% for ocph. ocss flours (39.31%) obtain the highest solubility index of the orishelé variety, after 30 days of storage. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 67 table 7: evolution of the solubility index (si) of the saci variety in six storage environments storage time (day) si of sa (%) si of sacss (%) si of sacsh (%) si of sacps (%) si of sacph (%) si of ssc (%) d0 29.19 ± 0.01 aa d4 30.35 ± 0.08 bd 29.51 ± 0.01ab 29.42 ± 0.06ab 29.71 ± 0.03ac 29.81 ± 0.02ac 29.16 ± 0.02aa d8 33.18 ± 0.19 cc 31.77 ± 0.22bab 31.65 ± 0.03bab 31.99 ± 0.24bab 32.28 ± 0.38bb 31.38 ± 0.23ba d12 35.22 ± 0.12 dc 32.05 ± 0.09bca 32.14 ± 0.39bca 32.19 ± 0.04bcab 32.69 ± 0.03bcb 31.93 ± 0.23ca d16 32.29 ± 0.09bca 32.71 ± 0.25bcdc 32.55 ± 0.10cac 32.83 ± 0.08bcc 32.23 ± 0.16cda d20 32.59 ± 0.32cda 32.94 ± 0.06bcda 32.95 ± 0.28da 33.00 ± 0.02cda 32.58 ± 0.13da d24 33.14 ± 0.15da 33.16 ± 0.30cda 33.15 ± 0.03da 33.51 ± 0.35dea 35.29 ± 0.03fb d28 33.34 ± 0.06da 33.59 ± 0.03db 33.21 ± 0.04da 33.89 ± 0.11ec d30 35.91 ± 0.64ea 36.35 ± 1.19ea 35.32 ± 0.08ea 36.35 ± 0.39fa these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. si: solubility index; sa: saci without packaging; sacss: saci in polythene bags containing dry solid carbon; sacsh: saci in polythene bags containing wet solid carbon; sacps: saci in polythene bags containing dry powdered charcoal; sacph: saci in polythene bags containing wet powdered charcoal; ssc: saci in polythene bags without carbon. table 8: evolution of the solubility index (si) of the big-ebanga variety in six storage environments. storage time (day) si of b (%) si of bcss (%) si of bcsh (%) si of bcps (%) si of bcph (%) si of bsc (%) d0 37.26 ± 0.06 aa d4 40.79 ± 0.07 bf 38.91 ± 0.06ac 38.50 ± 0.01ab 39.08 ± 0.02ad 39.12 ± 0.03ae 38.42 ± 0.02aa d8 41.85 ± 0.03 cb 39.42 ± 0.57aa 39.15 ± 0.03ba 39.23 ± 0.01aa 39.65 ± 0.04ba 39.17 ± 0.01ba d12 43.72 ± 0.07 df 40.12 ± 0.01bd 39.61 ± 0.05cc 39.32 ± 0.06aa 40.59 ± 0.04ce 39.43 ± 0.02bb d16 40.30 ± 0.01 bd 39.92 ± 0.05db 39.54 ± 0.06aa 40.91 ± 0.09de 40.07 ± 0.02cc d20 41.09 ± 0.05 cc 40.27 ± 0.02ea 40.58 ± 0.09bb 41.04 ± 0.01ec 41.43 ± 0.02dd d24 41.41 ± 0.08 cc 40.73 ± 0.02fb 40.61 ± 0.15bca 41.71 ± 0.03fd 44.27 ± 0.56ee d28 42.08 ± 0.01 db 41.12 ± 0.04ga 41.17 ± 0.04ca 42.07 ± 0.05gb d30 43.11 ± 0.06ea 43.81 ± 0.06hab 44.25 ± 0.58db 43.24 ± 0.03ha these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. si: solubility index; b: big-ebanga without packaging; bcss: big-ebanga in polythene bags containing dry solid charcoal; bcsh: big-ebanga in polythene bags containing wet solid charcoal; bcps: big-ebanga in polyethylene bags containing dry powdered charcoal; bcph: big-ebanga in polyethylene bags containing wet powdered charcoal; bsc: big-ebanga in polyethylene bags without carbon food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 68 table 9: evolution of the solubility index (si) of the orishele variety in six storage environments storage time (day) si of o (%) si of ocss (%) si of ocsh (%) si of ocps (%) si of ocph (%) si of osc (%) d0 31.65 ± 0.08 aa d4 32.13 ± 0.09bcd 32.22 ± 0.05ade 31.89 ± 0.15abc 31.86 ± 0.05ab 32.41 ± 0.09ae 31.35 ± 0.15aa d8 36.42 ± 0.01 ce 32.64 ± 0.03abcd 32.46 ± 0.06abc 32.05 ± 0.10ab 32.70 ± 0.16ad 31.54 ± 0.02aba d12 39.74 ± 0.05 dc 32.94 ± 0.02bb 32.75 ± 0.16bb 32.17 ± 0.05aa 33.09 ± 0.25abb 31.98 ± 0.36bca d16 33.02 ± 0.06bb 32.99 ± 0.10bb 32.41 ± 0.08aa 33.66 ± 0.33bc 32.38 ± 0.16ca d20 36.21 ± 0.01ca 36.18 ± 0.21ca 36.10 ± 0.53ba 37.02 ± 0.01cb 35.91 ± 0.35da d24 36.42 ± 0.02cdab 36.62 ± 0.17cbc 36.73 ± 0.19cc 37.04 ± 0.02cd 38.21 ± 0.06de d28 36.81 ± 0.07dab 36.75 ± 0.01ca 37.02 ± 0.15cbc 37.14 ± 0.05cc d30 39.31 ± 0.51eb 37.84 ± 0.56da 39.13 ± 0.03db 39.12 ± 0.79db these values are the means of three determinations for each parameter. the means ± standard deviation, assigned different lowercase letters in the same column indicate a significant difference (p < 0.05) between the storage days according to tukey. means ± standard deviation with different capital letters in the same row indicates a significant difference between storage media according to tukey. ise: solubility index; o: orishele without packaging; ocss: orishele in polythene bags containing dry solid charcoal; ocsh: orishele in polythene bags containing wet solid charcoal; ocps: orishele in polyethylene bags containing dry powdered charcoal; ocph: orishele in polyethylene bags containing wet powdered charcoal; cso: orishele in polyethylene bags without carbon the fruits of ocsh (37.84%) and ocph (39.12%) recorded the lowest solubility indices. the control fruits of this variety recorded respective rates of 39.74% (o) and 38.21% (osc). indeed, the solubility index shows the affinity of flours to disperse in water and to give a homogeneous solution [17]. it also reflects the extent of starch degradation and measures the quantity of soluble substances released from starch granules [22]. the high solubility index percentage observed for flours at the end of storage could be due to the degradation of starch and fibers by amylolytic enzymes [23]. the solubility index is used to determine the ability of a product to dissolve following centrifugation. the high solubility indices of flours show that they can be ideal for the preparation of infant foods [24]. 4. conclusion functional properties such as water and oil absorption capacity and solubility index of plantain fruit flours increase during storage. flour from saci, big-ebanga and orishélé plantain varieties absorbs a large amount of water and oil. these flours have good availability to be used in pastry, bakery, and in the preparation of food porridges. 5. acknowledgments all our gratitude to the national center for agronomic research (cnra) for allowing us to collect samples well and to carry out certain analyses. we do not forget the biocatalysis and bioprocesses laboratory of nangui abrogoua university for allowing us to carry out our experiments. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 69 finally, our sincere thanks to all the authors of this work for their selflessness. 6. references [1]. inibap (international network for the improvement of banana and plantain), net working banana and plantain: inibap annual report 2001. montpellier, france: inibap, (2002) [2]. fao, faostat statistics database agriculture. roma: fao, (2004) [3]. fao, statistics production yearbook. rome, food and agriculture organization of the united nations, (2006) [4]. coulibaly s., caractérisation physicochimique, rhéologique et analyse des fruits de quelques cultivars de bananier (musa aab, aaaa, aaab), thèse de doctorat de l’université d’abobo-adjamé spécialité sciences et technologies des aliments, abobo-adjamé, 171 p, (2008) [5]. faostat, la situation des marchés des produits agricoles dans le monde. faostat, production yearbooks, 1(1): 346 p, (2004) [6]. johnson g.i., sharp j.l., oosthuyse s.a., postharvest technology and quarantine treatments. in: litz r.e., ed. the mango: botany, production and uses. wallingford, uk: cab international, 447-507, (1997) [7]. gnakry d., kamenan a., degré de maturité et conservation de la banane plantain (musa sp.), qualité physicochimique de l’amidon, cahiers des industries agroalimentaires, cahier scientifique et technique, 107: 251-256, (1990) [8]. kouadio kouakou k.a., coulibaly s., soro l.c., ocho-anin atchibri l., determination of the optimal point of cut fruit of plantain hybrids pita 3, pita 8 and varieties of plantain lorougnon and horn 1, international journal of agriculture and food science, 3: 16-21, (2013) [9]. phillips r.d., chinnan m.s., branch a.l., miller j., mcwatters k.h., effects of pre-treatment on functional and nutritional properties of cowpea meal, journal of food sciences, 53(3): 805-809, (1988) [10]. anderson r.a., conway h.f., pfeiffer v.f., griffin e.l., roll and extrusion cooking of grain sorghum grits, cereal science today, 26: 372-375, (1969) [11]. sosulski f.w., the centrifuge method for determining flour absorption in hard red spring wheat, cereal chemistry, 39: 344-350, (1962) [12]. sylvain d.b., doudjo s., giles l.n., ernest k. k., étude des comportements chimiques, fonctionnels et rhéologiques de mélanges de farines de blé (triticum aestivum), amande de cajou (anacardium occidentale l) et de banane plantain (musa paradisiaca), afrique science, 15(6): 143-155, (2019) [13]. igbabul b.d., amove j., okoh a., quality evaluation of composite bread produced from wheat, defatted soy and banana flours, international journal of nutrition and food sciences, 3(5): 471-476, (2014) [14]. khushnuma m., haroon r.n., syed z.h., abdul h.r., influence of soaking and germination on physico-chemical composition and functional properties of chickpea (var. skuast-233) flour, international journal of chemical studies, 5(5): 1048-1054, (2017) [15]. chinma c.e., adewuyi o., abu o.j., effect of germination in the chemical, functional and pasting properties of flours from brown and yellow varieties of tiger nut (cyperus esculentus). food research international, 42(8):1004-1009, (2009) [16]. elkhalifa a.e.o., bernhardt r., influence of grain germination on functional properties of sorghum flour, food chemistry, 121(2): 387-392, (2010) [17]. diallo k.s., koné k.y., soro d., assidjo n.e., yao k.b., gnakri d., caractérisation biochimique et fonctionnelle des graines de sept cultivars de voandzou [vigna subterranea (l.) verdc. fabaceae] cultivés en côte d'ivoire, european scientific journal, 11(27): 18577881, (2015) [18]. olaofe o., arogundad l.a., adeyeye e.i., falusi o.m., composition and food of the variegated grasshopper, tropical science, 38(4): 233-237, (1998) [19]. kinsella j.e., functional properties of soy proteins, journal of american chemistry society, 56(3): 242-258, (1979) [20]. medoua n.g.j.m., potentiels nutritionnels et technologique des tubercules durcis de l’igname dioscorea dumetorum (kunth) pax : etude du durcissement post-récolte et des conditions de transformation des tubercules durcis en farine, thèse de doctorat/ph. d. à l’université de yaoundé, spécialité sciences alimentaires et nutrition, 239 p., (2005) [21]. aremu m.o., olonisakin a., atolaye b.o., ogbu c.f., some nutritional and functional studies of prosopis africana, electron. j. environ. journal of agricultural and food chemistry, 5(6): 1640-1648, (2006) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 loh tinndé charles sabli, wohi maniga, souleymane coulibaly, eugène jean parfait kouadio , charcoal-based conservation methods’ impact on some functional properties of three varieties of plantain (musa ssp.), food and environment safety, volume xxii, issue 1 – 2023, pag. 59 – 70 70 [22]. kaushal p., kumara v., sharma h.k., comparative study of physicochemical, functional, antinutritional and pasting properties of taro (colocasia esculenta), rice (oryza sativa) flour, pigeonpea (cajanus cajan) flour and their blends, lwt-food science and technology, 48(1): 59-68, (2012) [23]. kumar a., ramakumar p., patel a.a., gupta v.k., singh a.k., influence of drying temperature on physico-chemical and techno-functional attributes of elephant foot yam (amorphophallus paeoniifolius) var. gajendra, food bioscience, 16(1): 11-16, (2016) [24]. mbofung c.m.f., aboubakar n.y., njintang a., bouba a., balaam f., physicochemical and functional properties of six varieties of taro (colocasia esculenta l.) flours, journal of food technology, 4(2): 135-142, (2006) 1. introduction refined palm oil absorption capacity the refined palm oil absorption capacity of saci variety fruit flour (table 4) increases (p ≤ 0.5) during storage. the rate observed at the start of storage (day 0) is 30.56%. after 30 days of storage, this rate reached 36.13% for sacss, 36.26% for sa... the highest refined palm oil absorption capacities of the saci variety at day 30 were those of the fruits of sacph (36.83%) and sacsh (36.26%), while the lowest rates were obtained from the fruits of the sacps (35.90%). the control samples sa and ssc ... for the big-ebanga variety (table 5), the refined palm oil absorption capacity observed on day 0 was 45.45%. this rate increased (p ≤ 0.5) and reached, after 30 days of storage, respective rates of 49.49% for bcss, 49.32% for bcsh, 49.13% for bcps, an... as for the orishélé variety (table 6), the absorption capacity of refined palm oil which was 50.41% on day 0 increased (p ≤ 0.5) to respectively reach 59.51% for the ocss, 58.38% for ocsh, 58.07% for ocps and 58.13% for ocph, after 30 days of storage.... statistical analysis indicates a significant difference (p ≤ 0.5) between the values of the refined palm oil absorption capacity of fruits from one storage environment to another. the increase in oil absorption capacity during storage could be attributed to the increase in protein content, which increases the hydrophobicity of flour [13]. indeed, the oil absorption capacity is the capacity of a protein to absorb and maintain oi... solubility index the solubility index of flours from the fruits of the three plantain varieties saci (table 7), big-ebanga (table 8), and orishélé (table 9) increases significantly (p ≤ 0.05) during storage in all storage conditions for this study. the results also in... concerning the solubility index of the big-ebanga variety, it evolved from 37.26% on day 0 to reach respectively, after 30 days of storage, rates of 43.11% for bcss, 43.81% for bcsh, 44.25% for bcps and 43.24% for bcph. the highest solubility index wa... the solubility index of the orishelé variety recorded on day 0 was 31.65%. this rate increased respectively, after 30 days of storage, to 39.31% for ocss, 37.84% for ocsh, 39.13% for ocps, and 39.12% for ocph. ocss flours (39.31%) obtain the highest s... the fruits of ocsh (37.84%) and ocph (39.12%) recorded the lowest solubility indices. the control fruits of this variety recorded respective rates of 39.74% (o) and 38.21% (osc). indeed, the solubility index shows the affinity of flours to disperse in water and to give a homogeneous solution [17]. it also reflects the extent of starch degradation and measures the quantity of soluble substances released from starch granules [22... doi: https://doi.org/10.4316/fens.2022.014 143 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 143 151 microbial synthesis of silver nanoparticles by enterococcus faecalis strain mi103 jane nsofor1, imade yolanda nsa1, busayo tosin akinyemi1, adewale kayode ogunyemi1, *matthew olusoji ilori1 1department of microbiology, university of lagos, nigeria milori@unilag.edu.ng, *corresponding author: received 1st january 2022, accepted 25th june 2022 abstract: silver nanoparticles (agnps) have recently attracted attention for their potential as a source of new antimicrobials, with the potential to better fight a wide range of pathogenic bacteria. nanomaterials are now commonly used for the production of industrial and domestic items. as a result of increase in the menace and threats to live by antibiotic-resistant bacteria, nanoparticles have been proposed as alternative for its control. enterococcus faecalis, strain mi103, was selected out of four isolates obtained from a soil sample and identified using 16s rrna gene sequencing method. the selected bacterial isolate synthesized agnps by reduction of silver nitrate (agno3) solution (incubated for 2 days at room temperature). colour development from pale yellow to brown was observed which indicated the extracellular production of agnps. agnps was further characterized by uv/vis spectroscopy, scanning electron microscopy (joel-jsm-7660f) (shape) and energy dispersive x-ray (edx) (composition). the synthesized agnps ranged in size of 90-100 nm and were of cylindrical shape. it was found that the highest quantity of agno3 was produced at ph 7. the synthesized agnps had strong bactericidal activity against mrsa. keywords: silver nanoparticles (agnps), antimicrobials, enterococcus faecalis, nanotechnology, nanoparticles, 16srrna 1. introduction the development of antibiotic resistance has posed a significant threat, first to health and wellness and then to food security globally. as a result of increasing abuse of antibiotics in health and industrial use, antibiotic resistance has significantly increased [1, 2]. the development of antimicrobial drug resistance is making it increasingly difficult to treat otherwise routine infections effectively and this has created a pressing need for new antimicrobials. the costs associated with antibiotic resistance are higher, hospital stays are longer, and mortality increases [3]. health care costs are rising due to antibiotic-resistant infections [4]. antibiotics used without a prescription results in the emergence and spread of resistance. actions must therefore be taken immediately to prevent infections and minor injuries from killing people around the world [5]. agnps have been proposed as an alternative to antibiotics in the race to curb the huge burden of infections that cannot be handled with the currently known antimicrobials. nanoparticles are molecules with a minimum dimension of 100 nanometers [6]. in addition to chemical and biological sensing [7], co2 capturing [8, 9] and drug delivery [10], nanoparticles have found numerous applications. a number of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 144 studies have shown that silver nanoparticles disrupt cell-walls and cellular respiration, as well as bind to them [11, 12, 13]. several studies have shown that nanosilver is bactericidal against gram-negative and gram-positive bacteria. however, the underlying bactericidal mechanism is still unknown [14, 15, 16]. in a study by morones et al. [15], four gram negative bacteria, e. coli, v. cholerae, p. aeruginosa and s. typhus, were found to exhibit antibacterial activities using silver nanoparticles and had been shown to attach to the surface of the cell membrane, adhere bacteria, and discharge silver ions. there have been other studies with gram positive bacteria, such as staphylococcus aureus [15]. by examining the antibacterial properties of synthesized silver nanoparticles against drug-resistant bacteria, this study aims to determine the effectiveness of the biotechnolgicaly generated nanoparticles. towards this end, methicillin-resistant staphylococcus aureus (mrsa) was used. 2. matherials and methods 2.1 collection of samples soil and water samples including loamy soil, oil contaminated sandy soil, seawater and tap water were collected in sterilized bottles from lagos mainland local government area (lga) of lagos state and transported immediately to microbiological laboratory for analysis. 2.2 isolation of bacteria and biosynthesis of silver nanoparticles. bacteria was isolated by 10-fold serial dilution. about 0.2 ml aliquot were plated on luria bertani (lb) plate agar. isolates were purified on lb agar medium in order to obtain pure cultures. the pure cultures were stored at 4°c on agar slant for further studies. the pure isolate was mass produced by cultivation on lb broth at 37°c for 24h on rotary shaker (150rpm). after incubation, cultures supernatants were obtained after centrifugation at 10,000 rpm for 10 min. one hundred millilitres of cell free supernatant were added to 50 ml of prepared 10 mm solution of agno3 (1.7g/l). three replicates were used for each strain. while cell free supernatant without agno3 served as control [17]. the flasks were left for 24 h at room temperature in the dark to prevent photo catalytic action. 2.3 characterization of silver nanoparticles extracellular synthesized silver nanoparticles (agnps) produced by cell free cells supernatant of strains of isolated bacterial strains, obtained after centrifuging at 10,000 rpm for 10 minutes, were rinsed several times and dried. they were then characterized by uv/vis spectroscopy in range of 200-700 nm [18]. the shape of the nanoparticles was determined by scanning electron microscopy (joel-jsm-7660f) and the composition of the nanoparticles was determined by energy dispersive xray (edx) [19]. 2.4 characterization of antibacterial activity of nanoparticles disc papers were soaked with 20 μl of 30 mg/l agnps suspensions and placed on the culture dish, incubated at 37°c for 24 h. for the control study, deionized water and generated ag suspension soaked disc was also placed on the dish. these were then placed on mueller hilton agar that contained mrsa with approximate density of initial inoculum of about 0.5 mc farland turbidity standard. sterilized cut filter papers that had been soaked in the cell-free water extract alone and agno3 were used as control. plates were incubated overnight at 35°c for 18 h and observed for the presence of zones of inhibition. 2.5 identification of synthesizing bacteria food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 145 the bacteria isolate that produced the largest quantity of silver nanoparticles was identified using 16srrna gene sequencing method. genomic dna (gdna) was extracted from the bacterial isolates using zymo research bacterial dna minipreptm kit (zymo research corp usa). the dna concentrations was determined using a nanodrop 2000c spectrophotometer (thermofisher). polymerase chain reaction (pcr) was used to amplify the 16s rrna gene of the isolate using the primer pair 27f (5′-agagtttgatcctggctcag-3) and 1492r 5’-ggttaccttgt tacgactt-3) [20] was done in an pielter thermal cycler (mj research series) under the following conditions: initial denaturation step was 95°c for 5 minutes followed by 30 amplification cycles of 30 seconds at 95°c; 1 minute at 50°c and 1 minute 30 seconds at 72°c; and then a final extension step of 10 minutes at 72°c. the amplification product was separated on a 1.5% agarose gel and electrophoresis was carried out at 80 v for 1 hour 30 minutes. after electrophoresis, dna bands were visualized by ethidium bromide staining. dna sequences were processed by basic local alignment search tool (blast) program (http://www.ncbi.nlm.nih.gov/blast). 2.6 effect of ph on nanoparticle production and efficacy the synthesizing agnps bacterial isolate was grown in lb broth at different ph 2, 7 and 9. the resulting suspensions were centrifuged and 100 ml of supernatant from each flask was inoculated with 50ml of 10mm of agno3 and incubated in darkness. production of nanoparticle was observed for in both flasks. 2.7 test of agnps against methicillinresistant staphylococcus aureus (mrsa) nanoparticles produced were tested against mrsa. sterile filter papers were cut, soaked in the different nanoparticles produced and placed on plates already swabbed with an inoculum of mrsa and left to incubate overnight. zones of inhibition were measured. 2.8 evaluation of antibacterial activity, minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of silver nanoparticles (agnps). kirby–bauer disk diffusion susceptibility test method described by bauer et al. [20] was employed for antibacterial activity of agnps against the mrsa pathogen. sterile cotton swab was used for spreading the bacterial strain on mueller-hinton agar (mha) (merck, germany). sterile blank antimicrobial susceptibility disk was used in the test. the agnps concentration of 10mg/ml, 5mg/ml, 2mg/ml and 1mg/ml was loaded into the disks and was used for the test. the bacteria strain was spread on the mueller-hinton agar (mha) (merck, germany) using sterile cotton swab. sterile blank antimicrobial susceptibility disk was used in the test. the antimicrobial efficacy determination by using method described by clsi m07-a8. here, observation for visible growth in the lb broth. the synthesized nanoparticles (4g) were suspended in 200 ml of lb broth using an ultrasonic homogenizer. the resulting (20 mg/ml) concentration was used as the stock concentrations. mic was carried by inoculating 1ml of peptone water containing mrsa of approximate density of a 0.5 mcfarland turbidity standard in all tubes. the tubes were incubated at 35°c for 24 hours and monitored for growth. the lowest concentration of silver nanoparticles where no visible growth is seen in the tubes is the mic endpoint. however, the turbidity before and after the incubation was noted. mbc was carried out by inoculating 0.1ml from tubes that showed no visible growth (no turbidity) onto lb agar and incubating http://www.ncbi.nlm.nih.gov/blast https://www.frontiersin.org/articles/10.3389/fmicb.2018.01555/full#b3 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 146 for 24 hours at 35°c. mbc endpoint is the lowest concentration that is able to kill 99.9% of bacterial population. this was obtained by noting the preand post incubated agar plates for the presence or absence of bacteria. 3. results 3.1 biosynthesis of silver nanoparticles among the four bacterial isolates, mi103 produced the highest quantity of brown precipitate (5g) indicating the presence of silver nanoparticles. one (one quarter) produced white precipitate and the other quarter of the (isolates) produced no precipitate. as a result of being the highest producer, mi103 was selected for further testing (table 1). a colour change from pale yellow to brown was observed which was an indication of the extracellular synthesis of agnps. the precipitate was obtained by centrifugation at 10,000 rpm for 10 min and oven dried. table 1: screening for production of silver nanoparticles 3.2 characterisation of silver nanoparticles synthesized silver nanoparticles were characterized using uv-vis spectrophotometry, scanning electron microscopy and energy dispersive x-ray (edx). figure 1 showed concentration of synthesized silver nanoparticles generated by isolate mi103 at different wavelengths. the absorbance obtained was within the range of 1.1 to 1.986 for synthesized silver nanoparticles. there was a steady rise in the absorbance readings between wave length of 200 nm to 300 nm, followed by a spike in absorbance readings at wavelengths between 380 nm and 426 nm until a decline occurred at 441 nm (figure 1). scanning electron micrograph (sem) of synthesized silver nanoparticles showed that the particles were cylindrical in shape, highly clustered, dense and colloidal in nature (figure 2). the nanoparticles are about 90100 nm (verify size by measuring a particle calculating with 15nm scale bar) in size. energy dispersive x-ray (edx) of silver nanoparticles showed that the particles contained several elements in the following concentrations: silver (51.85%), gold (2.70%), oxygen (20.40%), magnesium (2.44%), sodium (5.90%), silicon (3.75%), calcium (2.26%) and carbon (7.09%) (figure 3). fig. 1. concentration of synthesized silver nanoparticles generated from mi103 at different wavelengths 3.3 antibacterial efficacy, minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of silver nanoparticles (agnps) the antibacterial activity of agnps was determined against mrsa. the results for disk diffusion test, mic and mbc of the agnps are summarized in tables 2 and 3. 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 wavelength (nm) a b so rb an ce ( o .d ) organism code precipitate produced quantity mi101 brownish precipitate 0.8g mi102 whitish precipitate 0.5g mi103 brownish precipitate 5g mi104 no precipitate food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 147 fig. 2. electron micrograph of silver nanoparticles generated from isolate mi103 fig. 3. energy dispersive x-ray (edx) of mi103 generated silver nanoparticles for the disk diffusion test, the presence of clear zone around the agnps disk suggesting that the agnps possessed antibacterial activity which is able to inhibit the growth of the mrsa. the visible clear zone produced by agnps against mrsa bacteria is showed in supplementary plate 1. mrsa showed zones of inhibition ranging from 18 mm to 21 mm in diameter (table 2). thus, the antibacterial activity of ag nanoparticles was bactericidal. as showed in table 3, mrsa showed mic and mbc value of 5g/ml, respectively. in the study, the mic and mbc value showed that mrsa was susceptible to agnps. table 2. antimicrobial (antibacterial) sensitivity test (ast) of agnps against mrsa ag nanoparticle discs 1 2 3 4 5 6 7 8 9 10 zone of inhibition against mrsa (nm) 18 21 21 18 20 19 20 18 20 18 https://www.frontiersin.org/articles/10.3389/fmicb.2018.01555/full#f1 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 148 table 3. determination of mic and mbc of agnps mic (mg/ml) observation mbc (mg/ml) observation/any other description 20 clear 20 no growth 10 clear 10 no growth 5 clear 5 no growth 2 turbid 2 no growth 1 turbid 1 no growth table 4. effect of ph on production and antimicrobial activity of agnps generated by mi103 ph effect on production effect on antimicrobial activity growth in lb broth (after 24 hours) quantity of nanoparticles produced average zone of inhibition (mm) 2 clear 7 turbid 4.8g 25 9 slightly turbid 0.7g 10 3.4 silver nanoparticle synthesizing bacteria isolate mi103, the highest producer of ag nanoparticles, was identified as enterococcus faecalis strain mi103 based on 16s rrna gene sequencing method. the sequence (1506bp) was compared to sequences in genbank by blast. 3.5 ph and nanoparticle production/activity the optimal ph was obtained at 2 with no turbidity and precipitate observed in the lb after incubation in darkness. while at ph 7, the suspension was turbid and a brown precipitate formed. whereas at ph 9, less turbidity and small mass (0.7 g) of precipitate formed (table 3). mrsa were more sensitive to ag nanoparticles at ph 7 than at ph of 9 by a ~ 2.5-fold difference in their zones of inhibition, 25 mm and 10 mm respectively (table 3). the minimum inhibitory concentration of the synthesized nanoparticles was about 5 mg/ml (table 4) as turbidity was observed at concentrations below the mic indicating growth of mrsa. in addition, mbc of the synthesized nanoparticles is 5 mg/ml (table 4) as no growth was observed after plating on lb agar from concentrations of 5 mg/ml and above which initially showed no turbidity. 4. discussion research into alternative methods to cure infections has increased following reports of infections caused by antibiotic-resistant organisms. in this regard, nanoparticles have been used [21]. because of their large surface area to volume ratio, silver nanoparticles have the most remarkable antimicrobial properties of all nanoparticles. moreover, researchers are food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 149 interested in the growth of microbial resistance against metal ions, antibiotics, and the development of resistant strains [22, 23]. it is clear that these particles possess good antimicrobial properties; however, their exact mechanism of action remains unknown [24] plus the toxicological challenges that may result in vivo. there is evidence that agnps have action against a wide array of microbes due to the multiple mechanisms they utilize, including their ability to destroy drug-resistant bacteria, fungi, and viruses [25, 26, 27, 28, 29, 30]. it is true that there are various methods for synthesizing nanoparticles, but biological synthesis, which relies on plants or microbes, is environmentally and economically friendly [31]. a screen for microorganisms producing agnp revealed enterococcus faecalis to be the best candidate identified via the 16s rrna gene sequence. further characterization of biosynthesized agnps was achieved using uv-vis spectroscopy and the peak was located between 420-440 nm [32]. there has been well documented evidence that this peak is caused by an interaction between two metal nanoparticles of different sizes ranging from 2 to 100 nanometers [33]. this study supports the findings of jeevan et al. [17], where the peak was found between 420-430nm and [32], where the peak was at 420nm. it was reported [33] that extracts of parkia speciosa hassk pods were used in lieu of microbes, and the peak was found at 445 nm. sem studies showed that the nanoparticles were cylindrical, dense, and clustered. possibly, this is due to clumping and drying the samples prior to analysis. similarly, is [33] reports clumping. compared to agnps with sizes ranging from 10 to 49 nanometers, these nanoparticles are 90-100 nm in size and irregular shape of synthesized by pseudomonas aeruginosa (15-30nm) [34] and escherichia coli (20-30nm) [35]. this study used energy dispersive x-ray (edx) to examine agnps which contained several elements, with silver being the most abundant. several elements are present in agnps, but silver is the most abundant, and the study examined it using energy dispersive x-ray (edx). is [33] came up with the same findings. it was found that agnps were extremely effective against methicillin resistant staphylococcus aureus. in previous studies, silver nanoparticles were tested for antimicrobial activity against methicillin-resistant staphylococcus epidermidis, streptococcus pyogenes, salmonella typhi and klebsiella pneumonia [35], escherichia coli [17] and citrobacter sp. [36]. as a result, nanoparticles were found to be effective in destroying them. in this study, it was found that nanoparticles were produced in the highest quantity at ph 7. in contrast, silver nanoparticles were not produced at ph 2 or 9 when ph was tested. there is evidence that the mechanism of producing nanoparticles is dependent on an enzyme, and enzymes are sensitive to ph fluctuations. in the synthesis carried out by birla et al. [37], using fusarium oxysporum, maximum nanoparticle production was observed between ph 9 and 11, with lower production occurring at ph 7 and aggregate formation occurring between ph 3 and 5. it has been noted, however, that husseiny et al. [38] found a decrease in nanoparticle synthesis rate as ph increased, suggesting that reductases that are responsible for synthesis are less active at higher ph levels. the decrease in the efficacy of the nanoparticles produced against mrsa could also be attributed to the sensitivity to ph. the mode of antibacterial activity of the silver nanoparticles is thought to be bactericidal as there was no growth from zones of clearing indicating that the organisms had been killed as against the growth observed in the region where there was no clearing. the mic and mbc of the agnps were both found to be 5 mg/ml, which was similar to a food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 150 study by krishnan et al. [ 39]. 4. conclusion these results confirm that enterococcus faecalis synthesized silver nanoparticles are bactericidal and are effective against antibiotic-resistant pathogens, thus providing a more efficient alternative to antibiotics. 5. acknowledgments the authors are indebted to mr. james ugah, a technologist of department of microbiology, university of lagos, akoka, lagos, nigeria for his technical assistance during the course of the work. also, we thank, mr. olanewaju gabriel of the electron microscopy and bio-imaging laboratory, mechanical engineering of covenant for his assistance in preparing the scanning electron micrographs of enterococcus faecalis strain mi103. 6. references [1]. un (2001). who global strategy for containment of antimicrobial resistance. geneva, world health organization, 2001. http://www.who.int/drugresistance/who global strategy english.pdf. [2]. un (2005). who report, antibacterial drug resistance options for concerted action. norberg et al.archives.who.int/prioritymeds/.../antibacterial.do. [3]. holmberg sd.. solomon sl.. blake pa (1987) health and economic impacts of antimicrobial resistance. rev. infect. dis.9:10651078. [4]. metz m.. hlaes dm. (2014). eight more ways to deal with antibiotic resistance antimicrob agents chemother. 58(8): 4253–4256. [5]. who (2018). antibiotic resistance. available at: http://www.who.int/news-room/factsheets/detail/antibiotic-resistance. last accessed: 9th july, 2018. [6]. laurent s.. forge d.. port m.. roch a.. robic c.. vander elst l.. muller rn. (2010). magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. chem. rev. 110: 2574. [7]. barrak h.. saied t.. chevallier p, laroche g, m’nif a, hamzaoui ah (2016). synthesis, characterization, and functionalization of zno nanoparticles by n-(trimethoxysilylpropyl) ethylenediamine triacetic acid (tmsedta): investigation of the interactions between phloroglucinol and zno. arab. j. chem. 882: 1–21 [8]. ganesh m.. hemalatha p.. peng mm.. jang ht. (2013) one pot synthesized li, zr doped porous silica nanoparticle for low temperature co2 adsorption. arab. j. chem. 41: 2740–2779 [9]. ramacharyulu pvrk.. muhammad r.. praveen kumar j.. prasad gk.. mohanty p. (2015). iron phthalocyanine modified mesoporous titania nanoparticles for photocatalytic activity and co2 capture applications. phys. chem. chem. phys. 17: 26456– 26462. [10]. lee je.. lee n.. kim t.. kim j.. hyeon t. (2011) multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications. acc. chem. res. 44: 893–902. [11]. singh m.. singh s.. prasad s.. gambhir is. (2008) nanotechnology in medicine and antibacterial effect of silver nanoparticles. dig. j. nanomater. bios. 3: 115-122. [12]. durána n.. duránb m.. bispo de jesus m.. seabraf ab.. fávarob wj.. nakazatog g. (2016). silver nanoparticles. a new view on mechanistic aspects on antimicrobial activity nanomedicine 12(3):789-799. [13]. hamouda ra.. hussein mh.. aboelmagd ra.. bawazir ss. (2019). synthesis and biological characterization of silver nanoparticles derived from the cyanobacterium oscillatoria limnetica. sci rep 9:13071https://doi.org/10.1038/s41598-019-49444y [14]. wu d.. fan w... kishen a.. gutmann jl.. fan b. (2014). evaluation of the antibacterial efficacy of silver nanoparticles against enterococcus faecalis biofilm. j endod 40:285-90. [15]. morones jr.. elechiguerra j.. camacho a.. holt k.. kouri jb.. tapia j.. yacaman mj. (2005). the bactericidal effect of silver nanoparticles. nanotechnology 16:2346–2353 [16]. shrivastava s.. bera t.. roy a.. singh g.. ramachandrarao p.. dash d. (2007). characterization of enhanced antibacterial effects of novel silver nanoparticles. nanotechnology 18:1–9 [17]. jeevan p.. ramya k.. rena ae. (2012). extracellular biosynthesis of silver nanoparticles by culture supernatant of pseudomonas aeruginosa. indian j. biotechnol. 11: 72-76. [18]. abo-state m.. partila a. (2015). microbial production of silver nanoparticles by food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 jane nsofor, imade yolanda nsa, busayo tosin akinyemi, adewale kayode ogunyemi, matthew olusoji ilori, microbial generation of silver nanoparticles by synthesizing enterococcus feacalis strain mi103, food and environment safety, volume xxi, issue 2 – 2022, pag. 143 – 151 151 pseudomonas aeruginosa cell free extract. j. eco. heal. env. 3(3): 91-98 [19]. ayesha a. (2017). bacterial synthesis and applications of nanoparticles. nano. sci. nano. technol. 11(2):119 [20]. bauer aw.. kirby wm.. sherris jc.. turck m. (1966). antibiotic susceptibility testing by a standardized single disk method. am. j. clin. pathol. 45:493–496.. [21]. vallet-regí m.. gonzález isabel. zquierdo-barba b. (2019). nanomaterials as promising alternative in the infection treatment. int. j. mol sci. 20(15): 3806. doi: 10.3390/ijms20153806 [22]. durán n.. marcato priscyla d… de conti roseli alves oswaldo l. costab ftm.. brocchib m, (2010), potential use of silver nanoparticles on pathogenic bacteria, their toxicity and possible mechanisms of action. j braz chem soc 21(6):949–959 [23]. keat cl.. aziz a.. eid am.. elmarzugi na. (2015). biosynthesis of nanoparticles and silver nanoparticles. bioresour bioprocess 2:47 [24]. pelgrift ry.. friedman aj. (2013). nanotechnology as a therapeutic tool to combat microbial resistance. adv. drug deliv. rev. 65:1803–1815. [25]. sondi i.. salopek-sondi b. (2004). silver nanoparticles as antimicrobial agent: a case study on e. coli as a model for gram-negative bacteria. j. colloid interface sci. 275:177–182. doi: 10.1016/j.jcis.2004.02.012 [26]. pal s.. tak yk.. song jm. (2007). does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? a study of the gram-negative bacterium escherichia coli. appl. environ. microbiol. 73:1712–1720. doi: 10.1128/aem.02218-06 [27]. fayaz m.. balaji k.. kalaichelvan pt.. venkatesan r. (2009). fungal based synthesis of silver nanoparticles—an effect of temperature on the size of particles. colloids surf. b. biointerfaces 74:123–126 [28]. lara hh.. ayala-núnez nv.. del carmen ixtepan turrent l, padilla cr (2010). bactericidal effect of silver nanoparticles against multidrug-resistant bacteria. world j. microbiol. biotechnol. 26:615–621. doi: 10.1007/s11274-009-0211-3 [29]. samberg me.. orndorff pe.. monteiro-riviere na (2011). antibacterial efficacy of silver nanoparticles of different sizes, surface conditions and synthesis methods. nanotoxicology 5:244–253. doi: 10.3109/17435390.2010.525669 [30]. youj zhang y.. hu z. (2011). bacteria and bacteriophage inactivation by silver and zinc oxide nanoparticles. colloids surf. b biointerfaces 85:161–167. doi: 10.1016/j.colsurfb.2011.02.023 [31]. tillmann p. (2004) stability of silver nanoparticles in aqueous and organic media. j. matter. chem. 4:140-146. [32]. quinteros ma.. aiassa im.. martínez im.. dalmasso pr.. páez pl. (2016). silver nanoparticles: biosynthesis using an atcc reference strain of pseudomonas aeruginosa and activity as broad spectrum clinical antibacterial agents. int. j. biomater. 2016:1-7 [33]. is f (2016). green synthesis of silver nanoparticles using extract of parkia speciosa hassk pods assisted by microwave irradiation. j. adv. res. 7:961-969 [34]. du l.. jiang h.. liu x.. wang e. (2007). biosynthesis of gold nanoparticles assisted by escherichia coli dh5α and its application on direct electrochemistry of haemoglobin. electrochem. comm. 9(5):1165–1170. [35]. nanda a.. saravanan m. (2009). biosynthesis of silver nanoparticles from staphylococcus aureus and its antimicrobial activity against mrsa and mrse. nanomedicine 5: 452– 456. [36]. priya mm.. selvia bk.. paul ja. (2011). green synthesis of silver nanoparticles from the leaf extracts of euphorbia hirta and nerium indicum. dig. j. nanomater. bios. 6: 869 – 877. [37]. birla ss.. gaikwad sc.. gade ak.. rai mk.. (2013). rapid synthesis of silver nanoparticles from fusarium oxysporum by optimizing physicocultural conditions. sci. world j. 2013:1–12. [38]. husseiny sm.. salah ta.. anter ha. (2015). biosynthesis of size controlled silver nanoparticles by fusarium oxysporum, their antibacterial and antitumoral activities. beni suef univer. j. basic appl. sci. 4:225–31. doi: 10.1016/j.bjbas.2015.07.004. [39]. krishnan r.. arumugam v.. vasaviah sk. (2015). the mic and mbc of silver nanoparticles against enterococcus faecalis a facultative anaerobe. j. nanomed. nanotechnol. 6: 285. 1. introduction the development of antibiotic resistance has posed a significant threat, first to health and wellness and then to food security globally. as a result of increasing abuse of antibiotics in health and industrial use, antibiotic resistance has significa... doi: https://doi.org/10.4316/fens.2022.018 190 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 2 2022, pag. 190 197 nutritional and biological value of mushroom snacks *inna bobel 1 , greta adamczyk 2 , nataliia falendysh 3 1,2 institute of food technology and nutrition, university of rzeszow, zelwerowicza street 4, 35-601 rzeszów, poland, inna_3@ukr.net 3 educational and research institute of food technology, national university of food technologies, volodymyrska street 68, 01601 kyiv, ukraine *corresponding author, received 15th june 2022, accepted 28th july 2022 abstract: the paper presents the results of research and scientific substantiation of nutritional and biological value of developed new mushroom chips and snacks. taking into account the high popularity of snacks, the prospects for the use of artificially cultivated mushrooms due to the valuable chemical composition and useful properties, the global volume of their cultivation, such research is appropriate and relevant. to achieve this goal, the task was to investigate experimentally the chemical and amino acid composition, digestion of protein substances of developed chips and snacks based on edible mushrooms. the research was conducted using modern methods. on the basis of experimental researches, the increased consumer and biological value of the developed mushroom products, and also their low caloric content was proved. it was found that mushroom products increased the enzymatic hydrolysis of protein substances compared to fresh mushrooms at both pepsin and trypsin stage of their hydrolytic cleavage, which contributed to their increase digestibility. the conducted research and developed technologies of mushroom chips and snacks can be used both in the food concentrate branch and in other branches of the food industry. key words: oyster mushrooms, champignon mushrooms, mushroom products, chemical composition, amino acid composition, digestion. 1. introduction the rapid growth of modern living standards and at the same time the deterioration of the ecological situation in the world encouraged the development of food products of new generation using both traditional and non-traditional raw materials, which in addition to high quality and nutritional value will have protective properties. snacks are very popular all over the world. the biggest supporters of snack products are the generation of millennials. in order to meet this demand, opportune adaptation and an innovative approach by snack producers is needed. unfortunately, most of the raw materials used in the production of snacks are characterized by the high content of easily digestible carbohydrates, which leads to low biological value and high caloric content of products. an alternative to the most popular types of snacks, namely potato chips, are chips from other vegetable crops or fruit chips which are high-quality natural food products with high taste properties [1, 2]. a possible promising way to solve this problem is also the use of artificially cultivated edible mushrooms as the main raw material in the production of snack products. this choice of raw materials was due to the fact that mushrooms have a mailto:inna_3@ukr.net food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 191 valuable chemical composition and high protein content [3–10]. in recent years, there was an intensive development of industrial production of artificially cultivated mushrooms. today, world production of edible mushrooms is about 5 million tons per year. mushroom production increased annually by 13 18% in the last 20 years. сhampignon mushrooms and oyster mushrooms are the most popular among the population [11, 12]. however, only 20% of the total mushrooms grown is processed. the reasons that constrain their widespread use in production and promotion at the consumer market are the short shelf life and insufficient processing technologies. the analysis of scientific literature data showed that in case of the market economy to ensure consumer demand and constant sales, it was necessary to develop new technologies for processing mushrooms and expand the range of low-calorie snack products with high nutritional and biological value. taking into account the above-mentioned arguments, the development of snack products based on edible champignon mushrooms and oyster mushrooms and the study of their nutritional and biological value was appropriate and relevant. the purpose of this work was the research and scientific substantiation of nutritional and biological value of mushroom chips and snacks. to achieve this goal, the following tasks were formulated: to investigate experimentally the chemical and amino acid composition, digestion of protein substances of developed chips and snacks of high nutritional and biological value based on edible mushrooms; to determine the nutritional and biological value of mushroom chips and snacks. 2. materials and methods in laboratory studies, cultivated mushrooms of the oyster mushrooms (pleurotus ostreatus) and champignon mushrooms (agaricus bisporus), salt, citric acid monohydrate, sunflower oil, wheat flour of second grade and water were used. the objects of research were also finished products from these raw materials. developed technologies of mushroom chips and snacks involved obtaining products from whole mushrooms. in the production of chips, oyster mushrooms were the main raw material, and salt, citric acid, water, and refined sunflower oil were additional materials. the production of mushroom chips could be divided into the following main stages: preparation of raw materials for production, preparation of mushroom semi-finished products, cooling, mixing mushroom semi-finished products with oil, frying, cooling products, packaging. to prepare the mushroom semi-finished product, mushrooms were loaded into the cooking boiler and an acid-salt solution with a mass fraction of salt of 1% and citric acid of 0.02% was added. hydrothermal treatment of mushrooms was carried out at a temperature of 95 ± 5 °c, the duration of the process was 5 minutes. frying of the semi-finished product was carried out in a frying machine for 15 minutes at a temperature of 195 °c. in the production of snacks, the mushroom semi-finished product was additionally mixed with a breading mixture consisting of wheat flour of the second grade and salt. frying of the semi-finished product was carried out for 10 minutes at a temperature of 195 °c. oyster mushrooms or champignon mushrooms were used as the main raw materials for the production of mushroom appetizers. the technology of appetizers differed from the above technology of chips as instead of the frying process, the food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 192 drying process was carried out, and the use of oil was excluded. the obtained mushroom semi-finished product was dried at a temperature of 55 ± 3 °c. analytical, chemical, biochemical, experimental-statistical research methods were conducted with the use of modern devices and information technologies. the experimental part was performed in the laboratory of the university of rzeszow (poland), the national university of food technologies (ukraine) and in the o.a. palladin institute of biochemistry of the national academy of sciences of ukraine. the total nitrogen content was determined by the kjeldahl method [13]. a conversion factor of 6.25 was used to convert the total nitrogen content to protein. the amino acid composition of proteins was determined by ion-exchange liquid column chromatography using amino acid analyzer t339 (czech republic) [14] after preliminary acid hydrolysis of a portion of concentrated hcl for 24 hours at 106 °c. alkaline hydrolysis was performed to determine tryptophan in the samples, which was made with 5 mol/dm3 naoh solution at 60 °c for 2 hours in sealed ampoules. the fiber content was determined by the kurschner-hanakmethod with the modification by kogan; the content of dextrins was determined by the photoelectro coloremetric method; the content of reducing substances was determined by the iodometric method; the content of glycogen was determined by the preobrazhenskaya method and the content of starch was determined by the ewers polarimetric method [15]. the total carbohydrate content was determined as the sum of individually identified carbohydrates. nutritional and biological value was determined by calculation method. digestibility of protein substances of products was evaluated by the intensity of their hydrolysis by pepsin and trypsin in the in vitro system. 3. results and discussion the results of our previous comprehensive studies of chemical composition, quality indicators and technological properties of oyster mushrooms and champignon mushrooms, as well as the impact of technological processing on changes in their components showed the effectiveness and feasibility of using these raw materials to create new types of food concentrates [16, 17]. on the basis of the obtained results the technologies of appetizers «oyster mushroom» and «champignon mushroom», chips and snacks were developed. recipes, technological instructions, technical conditions for new types of food concentrates were developed and approved, the conclusion of the state sanitary-epidemiological examination was obtained. according to the results of this work, a patent of ukraine for a utility model was obtained. chemical composition and nutritional value of new mushroom products. food products should contain all the nutrients necessary for the normal functioning of the human body, and it was desirable that their ratio met the requirements of nutrition. in connection with the above, new mushroom products were created, namely: chips, snacks, appetizer "oyster mushroom", appetizer "champignon mushroom". they were evaluated for both nutritional and biological value. nutritional value reflected a set of properties of the food product that met the physiological needs of the human body in essential nutrients and energy. determination of the chemical composition and energy value of mushroom products was performed by calculation using the obtained experimental data. an integrated score of products was additionally found, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 193 and expressed the level of satisfaction of the human body's needs in essential nutrients. when calculating the nutritional value the norms of physiological needs of the population in basic nutrients and energy for men aged 18 29 years and group i of labor intensity were used. estimated data are given in table 1. it was established that the created mushroom food concentrates were characterized by the increased content of mass fraction of protein, which played an important role in human nutrition as it performed important functions in the human body and was a major component of cells of all organs and tissues. table 1 chemical composition and nutritional value of new mushroom products nutrient, energy value daily requirement chips snacks appetizer "oyster mushroom" appetizer "champignon mushroom" c o n te n t in 1 0 0 g in te g ra te d sc o re , % c o n te n t in 1 0 0 g in te g ra te d sc o re , % c o n te n t in 1 0 0 g in te g ra te d sc o re , % c o n te n t in 1 0 0 g in te g ra te d sc o re , % proteins, g 67 18.2 27.2 17.7 26.4 21.0 31.4 18.3 27.3 fats, g 68 7.7 11.3 9.8 14.4 2.8 4.1 1.8 2.7 carbohydrates, g 392 45.0 11.5 50.9 13.0 44.4 11.3 34.2 8.7 including fibers 8.6 8.3 9.5 7.1 energy value, kcal 2450 215 8.8 261 10.7 162 6.6 116 4.7 due to the use of 100 g of products, 26.4 31.4% of the body's daily protein needs were covered. mushroom products were also rich in fiber, which affected the regulation of physiological and biochemical processes in the digestive system, intensity and metabolism of proteins, fats and carbohydrates. the developed food concentrates were low in calories (115 261 kcal/100 g) compared to other products of the snack group [1]. studies identified 19 amino acids in the developed mushroom products. the amino acid composition of mushroom chips and snacks is presented in table 2. it was established that in the amino acid composition of the developed mushroom products included aspartic and glutamic acids, leucine, alanine, serine, proline prevailed. it contained less cystine, tryptophan and methionine. the biological value of the product reflected the degree of compliance of the amino acid composition of the protein to the needs of the human body in amino acids for its synthesis. table 2 amino acid content of food mushroom products amino acids amino acid content,% of dm chips snacks appetizer "oyster mushroom" appetizer "champigno n mushroom" alanine 0.79 0.56 1.70 2.57 arginine 0.69 0.49 1.05 0.83 aspartic acid 1.00 0.78 1.96 1.98 valine 0.64 0.49 0.82 0.95 gammaaminobutyric acid (gaba) 0.02 0.01 1.11 0.83 histidine 0.26 0.19 0.43 0.46 glycine 0.56 0.42 1.04 1.05 glutamic acid 1.69 2.18 6.12 2.88 isoleucine 0.39 0.31 0.73 0.82 leucine 0.99 0.71 1.39 1.67 lysine 0.64 0.53 1.05 1.31 methionine 0.21 0.16 0.40 0.46 proline 0.62 0.74 1.00 0.92 serine 0.76 0.57 1.26 1.45 tyrosine 0.38 0.24 0.66 0.58 threonine 0.64 0.38 1.09 1.35 tryptophan 0.14 0.13 0.24 0.23 https://en.wikipedia.org/wiki/alanine https://en.wikipedia.org/wiki/arginine https://en.wikipedia.org/wiki/aspartic_acid https://en.wikipedia.org/wiki/valine https://en.wikipedia.org/wiki/histidine https://en.wikipedia.org/wiki/glycine https://en.wikipedia.org/wiki/glutamic_acid https://en.wikipedia.org/wiki/isoleucine https://en.wikipedia.org/wiki/leucine https://en.wikipedia.org/wiki/lysine https://en.wikipedia.org/wiki/methionine https://en.wikipedia.org/wiki/proline https://en.wikipedia.org/wiki/serine https://en.wikipedia.org/wiki/tyrosine https://en.wikipedia.org/wiki/threonine https://en.wikipedia.org/wiki/tryptophan food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 194 phenylalanine 0.54 0.41 0.81 1.02 cystine 0.11 0.09 0.18 0.18 it was known that the most complete idea of the biological value of a protein was given by the method of comparing the composition of essential amino acids of this protein with the corresponding amino acid composition of the "ideal protein" by calculating the amino acid score. in table 3 it is shown the amino acid rate of the products. table 3 amino acid score of mushroom products name of amino acids the content in the ideal protein, mg/g chips snacks appetizer "oyster mushroom" appetizer "champignon mushroom" a m in o a c id c o n te n t, m g /1 g o f p ro te in a m in o a c id s c o re , % a m in o a c id c o n te n t, m g /1 g o f p ro te in a m in o a c id s c o re , % a m in o a c id c o n te n t, m g /1 g o f p ro te in a m in o a c id sc o re ,% a m in o a c id c o n te n t, m g /1 g o f p ro te in a m in o a c id s c o re , % valine 50 58.0 116.1 52.1 104.3 38.7 77.4 44.1 88.2 isoleucine 40 35.0 87.5 33.0 82.5 31.4 78.4 38.1 95.2 leucine 70 89.8 128.4 75.4 107.6 71.8 102.5 77.5 110.8 lysine 55 57.4 104.4 56.4 102.6 56.3 102.4 60.8 110.6 methionine + cystine 35 28.9 82.6 26.6 76.0 25.8 73.7 29.7 84.9 threonine 40 57.6 143.9 40.9 102.2 46.8 117.1 62.7 156.7 tryptophan 10 12.7 126.5 11.8 117.5 10.3 103.1 10.7 106.8 phenylalanine + tyrosine 60 82.9 138.1 69.2 115.3 60.2 100.3 74.3 123.8 the calculations showed that chips and snacks were low the following amino acids: isoleucine, methionine and cystine. amino acids valine, isoleucine, methionine and cystine were determined in lower quantities in appetizers. the first limiting amino acid of the products was the sum of sulfur-containing acids methionine and cystine. at the same time, the products were rich in tryptophan and lysine, the deficiency of which was acutely felt in many plant proteins. mushroom products had a high biological value of proteins, which indicated the prospects for the use of new food products. the biological value of chips was 82.6%, snacks 76.0%, appetizer "oyster mushroom" 73.7%, appetizer "champignon mushroom" 84.9%. thus, the obtained new mushroom food concentrates were characterized by high content of important nutrients and high biological value, which indicated the feasibility of using these food products. digestibility of mushrooms and food concentrates obtained on their basis. the biological value of proteins was determined not only by their amino acid composition, but also the degree of digestibility. the intensity of the process of protein breakdown in the human digestive tract depended on the activity of proteolytic enzymes and the susceptibility of food proteins to their action. during the research, the rate of digestion of protein substances of fresh oyster mushrooms and champignons was determined, as well as products based on them chips, snacks, appetizers "oyster mushroom" and "champignon mushroom". the degree of digestibility of protein substances of raw materials and products was evaluated by the intensity of their hydrolysis by the enzymes pepsin and trypsin in vitro. the intensity of https://en.wikipedia.org/wiki/phenylalanine https://en.wikipedia.org/wiki/cysteine food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 195 digestibility was evaluated by the increase of the number of final products of enzymatic hydrolysis of protein substances free amino acids in the model medium. analysis of the kinetics of the process of enzymatic hydrolysis of raw materials and products (figure 1) showed that in contrast to fresh mushrooms, for which the hydrolysis process was almost constant, mushroom products were characterized by a higher digestion rate at the pepsin stage. fig. 1. accumulation of free amino acids during hydrolysis of proteins of mushroom and their products in vitro: 1 – appetizer "oyster mushroom"; 2 – appetizer "champignon mushroom"; 3 – chips; 4 – snacks; 5 – fresh oyster mushroom; 6 – fresh champignon mushroom during the pepsin stage, 51.6% and 47.4% of free amino acids from their total amount were accumulated in fresh oyster mushrooms and champignons, respectively, and in chips and snacks this amount was 53.1% and 53.9%, respectively, in appetizers "oyster mushroom" and "champignon mushroom" 57.9% and 62.9%, respectively. it was found that the total amount of accumulated free amino acids in oyster products was much higher than in fresh mushrooms – for appetizer "oyster mushroom" this index was 3 times higher, for chips – 2.2 times higher, and for snacks 1.6 times higher. appetizer "champignon mushroom" had the best ability to the digestion, which accumulated 7 times more free amino acids compared to fresh mushrooms. digestion of proteins of fresh mushrooms was slightly slower compared to food products due to the higher resistance of native proteins to enzymes. in the process of production of mushroom products, protein substances underwent changes that affected their assimilation [18]. at insignificant denaturation of proteins, their attack by proteolytic enzymes amplified. heat treatment using harsh temperatures led to a certain decrease in protein absorption due to excessive compaction of protein molecules, which reduced the availability of peptide bonds of proteins to digestive enzymes. this explained the better ability to digest mushroom appetizers compared to chips and snacks. the accumulation of protein fractions of albumin and globulin in mushroom products caused an increase in their degree of assimilation. the process of assimilation was also influenced by the amino acid composition of products and the presence of other nutrients. fiber promoted faster passage of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 196 food by increasing intestinal peristalsis and some proteins did not have time to be hydrolyzed, in addition, it adsorbed proteinases and binded some amino acids, removing them from the body. when obtaining mushroom products there was a decrease in the degree of polymerization of fiber, which contributed to better digestibility of products. 4. conclusion in this study mushroom chips and snacks on the basis of pleurotus ostreatus and agaricus bisporus were developed and their composition and nutritional and biological values were determined. it was established that the obtained new mushroom products were characterized by high content of important nutrients and high biological value. the biological value of proteins of chips was 83.6%, of snacks 76.0%, of appetizer "oyster mushroom" 73.7%, of appetizer "champignon mushroom" 84.9%. it was found that mushroom products increased the enzymatic hydrolysis of protein substances compared to fresh mushrooms at both pepsin and trypsin stage of their hydrolytic cleavage, which contributed to their increased digestibility. the total amount of accumulated free amino acids in appetizer "oyster mushroom" was 3 times more than in fresh mushrooms, in appetizer "champignon mushroom" 7 times more, in chips 2.2 times more, in snacks 1.6 times more. the conducted research and developed technologies of mushroom chips and snacks can be used both in the food concentrate branch and in other branches of the food industry. in addition, such new types of mushroom products created opportunities for expanding the market for both artificially grown mushrooms and snack producers. 5. references [1]. hassen w. s., castetbon k., tichit c., péneau s., nechba a., ducrot p., méjean c. energy, nutrient and food content of snacks in french adults. nutrition journal, 17(1): 33, (2018). [2]. hess j. m, jonnalagadda s. s, slavin j. l. what is a snack, why do we snack, and how can we choose better snacks? a review of the definitions of snacking, motivations to snack, contributions to dietary intake, and recommendations for improvement. adv nutr.,7: 466-475, (2016). [3]. tagkouli d., kaliora a., bekiaris g., koutrotsios g., christea m., zervakis g., kalogeropoulos n. free amino acids in three pleurotus species cultivated on agricultural and agro-industrial by-products. molecules, 25 (17) : 4015, (2020). [4]. patel y., naraian r., singh v. medicinal properties of pleurotus species (oyster mushroom): a review. world journal of fungal and plant biology, 3: 1– 12, (2012). [5]. sadler m. nutritional properties of edible fungi. nutrition bulletin, 28: 305–308, (2003). [6]. ramos m., burgos n., barnard a., evans g., preece j., graz m., jiménez a. agaricus bisporus and its by-products as a source of valuable extracts and bioactive compounds. food chemistry, 292: 176-187, (2019). [7]. atila f., owaid m. n., shariati m. a. the nutritional and medical benefits of agaricus bisporus: a review. journal of microbiology, biotechnology and food sciences, 7: 281-286, (2017). [8]. reguła j., siwulski m. dried shiitake (lentinulla edodes) and oyster (pleurotus ostreatus) mushrooms as a good source of nutrient. acta scientiarum polonorum technologia alimentaria, 6: 135–142, (2007). [9]. hess j., wang q., gould t., slavin j. impact of agaricus bisporus mushroom consumption on gut health markers in healthy adults. nutrients, 10(10) : 1402, (2018). [10]. li s., liu h., wang w., wang x., zhang c., zhang j., jia l. antioxidant and anti-aging effects of acidic-extractable polysaccharides by agaricus bisporus. international journal of biological macromolecules, 106: 12971306, (2018). [11]. royse d. j., baars j., tan, q. current food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 2 – 2022 inna bobel, greta adamczyk, nataliia falendysh, nutritional and biological value of mushroom snacks, food and environment safety, volume xxi, issue 2 – 2022, pag.190 – 197 197 overview of mushroom production in the world. edible and medicinal mushrooms: technology and applications, wiley-blackwell, 592, (2017). [12]. manjit s., shwet k., sharma v. p. status and trends in world mushroom productioni. mushroom research, 26(1) : 1-20, (2017). [13] jiang b., tsao r., li y., miao m. food safety: food analysis technologies/ techniques. encyclopedia of agriculture and food systems. 464 p., (2014). [14] ozawa s., miyano h., ito m. advances in amino acid analysis and amino acid analyzer l-8900. hitachi sci. instrum. news, 6, 33-43, (2015). [15] iso 10520:1997. native starch— determination of starch content—ewers polarimetric method. (1997). [16]. zynchenko i., terletskaya v. derivative analysis of oyster mushrooms and champignons during their drying, scientific works “food science, engineering and technologies – 2013”, lx: 822-826, (2013). [17] zinchenko i., terletska v. changes in protein substances of mushrooms in the process of hydrothermal processing in the technology of mushroom snacks. scientific works of the national university of food technologies, 23 (2): 166-174, (2017). [18]. reis g. c., dala-paula b. m., tavano o. l., guidi l. r., godoy h. t., gloria m. b. a. in vitro digestion of spermidine and amino acids in fresh and processed agaricus bisporus mushroom. food research international, 137: 109616, (2020). stefan cel mare university of suceava journal food and environment safety of the suceava university food engineering volume xxii, issue 1 31 march 2023 available online http://www.fia.usv.ro/fiajournal © published by university publishing house of suceava aims and scope the scientific journal food and environment safety is a quarterly publication (4 issues per year) of the faculty of food engineering, stefan cel mare university of suceava, romania, that is indexed in the international databases: index copernicus journals master list, ulrich’s and chemical abstract (cas). also, it is acknowledged b+ by the main romanian funding organization for university and postgraduate research programmes national university research council (cncsis). the purpose of food and environment safety journal is to provide a means of rapid publication for significant contributions to the improvement and diversification of specific activities in the field of food engineering, food and environment safety, biotechnologies, chemical engineering, biosensors, agriculture and applied sciences as well. moreover, the journal assures promotion of research results in the field of food production and, from the nutritional and toxicological point of view, it underlines the importance of applying the best analyzing and control methods for consumer's protection, food technologies, industrial biotechnologies and environment protection as well to increase life quality. food engineering faculty journal has been published since 2000 like analele universitatii stefan cel mare suceava, sectiunea colegiul tehnic (issn 1583-2295). between 2002 and 2004 the journal was published biannually like analele universitatii stefan cel mare suceava, sectiunea inginerie alimentara (issn 1583-2295), then from 2005 till 2009, it was issued biannually like annals of suceava university food engineering (issn 1842-4597), indexed in index copernicus data base and acknowledged by the national university research council (cncsis), romania. in 2010, the food engineering faculty journal changed its name in food and environment safety (issn 2068 6609). the cover of the journal was made by niculai moroşan. available online: http://www.fia.usv.ro/fiajournal address for submission, e-mail fiajournal@fia.usv.ro cip description of national library the scientific journal food and environment safety (issn 2068 – 6609) is published by university publishing house of suceava, romania. mail address of the university publishing house of suceava: stefan cel mare university, suceava, universitatii str. no. 13, 720229, suceava, romania www.usv.ro, http://www.usv.ro/index.php/ro/1/editura%20usv/238/3/15 tel: +40 230 216 147, fax: +40 0230 520 080 editorial contact information: faculty of food engineering, stefan cel mare university, suceava, universitatii str., no. 13, 720229, suceava, romania, phone/fax: +40 230 520267, www.fia.usv.ro editor-in-chief: prof. ph.d, eng. sonia amariei (gutt), gutts@fia.usv.ro subscription information subscription information can be obtained from: the e-mails: fiajournal@fia.usv.ro the postal address: faculty of food engineering, stefan cel mare university -suceava, universitatii str., no. 13, 720229, suceava, romania copyright © university publishing house of suceava. all right reserved editor-in-chief: sonia amariei (gutt) ştefan cel mare university of suceava, romania editorial board petru alexe dunărea de jos university of galaţi, romania marcel avrămiuc ştefan cel mare universityof suceava, romania şerif baldiran amasya university, amasya, turkey elena-gabriela bahrim dunărea de jos university of galați, romania lidija basa university of ljubljana, slovenia zoiţa mărioara berinde technical university of cluj napoca north university center of baia mare, romania iulian bobe tyton bio sciences, darien, usa marija bodroža-solarov university novi sad, institute for food technology, serbia ioan chereji university of oradea, romania cristina-gabriela constantinescu stefan cel mare university of suceava, romania călina petruța cornea university of agronomic sciences and veterinary medicine in bucharest, romania toshiki enomoto ishikawa prefectural university, nonoichi, japan dirk flottman technical university of aalen, germany josé antonio gabaldón hernández catholic university of murcia (ucam), spain robert gravani cornell university, department of food science, usa oleksii gubenia national university of food technologies, kiev georg gutt ştefan cel mare university of suceava, romania nicoleta gabriela hădărugă banat university of agricultural sciences and veterinary medicine timisoara, romania cristina-elena hreţcanu ştefan cel mare university of suceava, romania elena joshevska university st. kliment ohridski, bitola, macedonia süleyman kaleli sakarya university, sakarya, turkey nijole kazlauskiene institute of ecology of nature research center, vilnius, lithuania ana leahu stefan cel mare university of suceava, romania anna maraz corvinus unversity of budapest, hungary angel martinez sanmartin centro tecnológico nacional de la conserva y alimentación, murcia, spain carmen moraru cornell university, department of food science, usa ileana denisa nistor vasile alecsandri university of bacău, romania mircea-adrian oroian ştefan cel mare university of suceava, romania mariana-atena poiană banat university of agricultural sciences and veterinary medicine timisoara, romania regina proskuviene vilnius pedagogical university, lithuania peter raspor university of ljubljana, slovenia gabriela râpeanu dunărea de jos university of galați, romania adrian riviş banat university of agricultural sciences and veterinary medicine timisoara, romania alice roşu ştefan cel mare universityof suceava, romania alexandru szép sapientia — hungarian university of transylvania, miercurea ciuc, romania carmen socaciu university of agricultural sciences and veterinary medicine cluj napoca, romania rodica sturza technical university of moldova, chisinau, moldova gagik torosyan state engineering university of yerevan, armenia never torosyan csro institute, yerevan, armenia yeva torosyan european regional educational academy, yerevan, armenia anka trajkovska petkoska university st. klimentohridski-bitola, r. north macedonia felicia tutulescu dragomir university of craiova, romania igor winkler bukovina medical state university, ukraine gheorghe zgherea dunărea de jos university of galaţi, romania doi: https://doi.org/10.4316/fens.2022.010 94 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 1 2022, pag. 94 106 2 preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) karima ould yerou 1 , *fatima zohra el kadi 2, khedoudja kanoun 3, hadj mostefa khelladi 4 , hanane benzahia 1 , sara bekhti 1 1faculty of sciences; mustapha stambouli university of mascara, algeria 2faculty of technology, djillali liabés university of sidi-bel-abbés, algeria elkadifatimazohra@yahoo.fr 3faculty of natural sciences and life ,djillali liabés university of sidi-bel-abbés, algeria. 4 faculty of natural sciences and life, university of oran 1, oran, algeria *corresponding author received 23th january 2022, accepted 30th march 2022 abstract: in the present work, the aqueous and methanolic extract of the underground part (comestible part) of brassica rapa (brassicaceae) was subjected to phytochemical and biological evaluation. the results show that the methanolic extract of b. rapa (mebr) presents a higher yield than the aqueous extract of b. rapa (aqebr) (24% vs 17%) respectively. the qualitative and quantitative phytochemical analysis revealed the presence of some chemical families. however, mebr showed higher contents of polyphenols, total flavonoids and condensed tannins compared to aqebr. the total antioxidant activity (tac) of mebr (5.32 ± 0.56 mg aae/gr de) was more powerful than that of aqebr (2.68 ± 0.33 mg aae/gr de). according to the dpph test, the ic50 values are (0.14±0.12 mg/ml vs 1.9±0.45 mg/ml) for mebr and aqebr respectively. on the other hand, the frap test confirmed that aqebr has antioxidant activity with a maximum of 1.41mg/ml, which is still higher than that of mebr 0.83 mg/ml. we conclude, that methanol was a more efficient extractor than water for b. rapa species, which allows a good yield, a high rate of secondary metabolites and a powerful antioxidant activity. b. rapa is a rich source of antioxidants that can eliminate free radicals and reduce the risk of chronic diseases. abbreviations: mebr: methanolic extract of brassica rapa, aqebr: aqueous extract of brassica rapa, tac: total antioxydant capacity, dpph: 2.2'-diphenyl-1pycrilhydrazyl, frap: ferric-reducing antioxidant power assay, ic50: inhibitory concentration 50. keywords: brassica rapa, mebr, aqebr, phenolic compounds, phytochemical screening, antioxidant activity. 1. introduction frequent consumption of cruciferous vegetables (340 genus and about 3700 species), such as broccoli, cauliflower, cabbages, radishes, turnips and kale reported to reduce the risk of developing cardiovascular disorders, cancer, heart attacks , strokes and diabetes [1]. . brassicas are the most important and widely consumed group of plants in the cruciferous family. in addition, brassica vegetables can be stored in their natural form for a prolonged time, and are available all year around .the turnip (brassica rapa) belongs to the brassicaceae family (cruciferous vegetables). among the different brassica species, turnip is one of the first to be domesticated [2]. the main constituents of this species are: glucosinolates, isothiocyanates [3], flavonoids [4 -5], indoles [6], sulphur and phenolic compounds [7-8], carbohydrates http://www.fia.usv.ro/fiajournal mailto:elkadifatimazohra@yahoo.fr food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 95 [9], volatile compounds (mainly terpenes, esters, aldehydes and ketones) [10-11] , and some well-known antioxidants ; such as vitamin c , vitamin e and carotenoids, as well as antioxidant enzymes ;such as catalase, superoxide dismutase and peroxidase [12]. the bitterness of turnip is related to glucosinolate (isothiocyanate) degradation compounds [13]. researchers have widely pointed out the therapeutic properties of turnip: hepatoprotective [9,14,15], antimicrobial, antitumor [16], antioxidant [11], anti-inflammatory [17], cardioprotective, lipid-lowering [18,19,20], anti-diabetic [21], nephroprotective [22] , and analgesic [23], as well as reducing obesity and metabolic syndrome. in view of the therapeutic and antioxidant virtues of the turnip, a phytochemical, biological and antioxidant evaluation of the underground part of brassica rapa was considered using two extraction methods (aqueous infusion and alcoholic maceration). 2. materials and methods 2.1. materials 2.1.1. plant material the choice of the plant drug was the brassica rapa variety which is commonly called turnip and widely consumed by the algerian population. fresh brassica rapa roots (rhizome) were collected in november 2018 from the local market of the ghriss town located at 19 km from the mascara province (western algeria). the ghriss plain is a part of the oued fékane watershed, which covers an area of 1,185 km2 , located in north-west algeria (fig 1), between 35° 07' and 35° 31' n latitude and between 0° 0' and 0° 26' e longitude [24]. fig 1: location of the ghriss plain [24] in the laboratory, the brassica rapa rhizomes were cleaned, peeled and ground and then dried at 38°c for 24 hours. after the drying process, the dried plant material was reduced to powder with an electric grinder (fig 2) and stored in sealed and hermetically containers for later use. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 96 fig 2: brassica rapa powder (original photo) 2.2. methods 2.2.1. moisture content the moisture content is defined as the weight loss during drying. for this purpose, 200 g of the vegetable, cut into small pieces, were weighed in clean capsules and then placed in the oven at 105°c +/5°c for a period of 48 h +/1 h until the constant weight was obtained. then allow the capsules to cool before weighing in a desiccator. repeat this process several times until a constant weight was attained [28]. 2.2.2. ash content the ash determination is based on the destruction of all organic matter under the effect of high temperature (500 ± 25°c). to perform this, the empty crucibles were weighed, 10 g of the sample was added to the crucibles, the empty crucibles were weighed and 10 g of the sample was added to them, then place them in a muffle furnace for 3-5h at 550°c. after removing from the oven, place these crucibles in a desiccator to cool. weigh the cooled crucibles, then reheat the crucibles again for a half hour or more. repeat this process until the weight remains constant [28]. 2.2.3. extracts preparation to prepare the extract, two extraction methods were adopted: aqueous infusion and alcoholic maceration .for the aqueous extract, 10 g of plant powder are introduced into 50 ml of boiling water and allowed to infuse for 15 minutes. after cooling, the liquid was then filtered and rinsed with a little hot water to obtain 50 ml of filtrate [25]. for the alcoholic extract, the protocol of [26] was adopted, which consists of macerating (10gr) of the plant powder in 100 ml of methanol (80%). the extraction was repeated 3 times with renewal solvent. the extracts obtained were filtered and then evaporated using a rotavapor at 45°c. two extracts were obtained: aqueous extract of brassica rapa = aqebr; and methanolic extract of brassica rapa = mebr. both extracts were stored in sealed glass vials at ±4°c before testing and analysis. 2.2.4. yield calculation the yield of the two extracts was determined using the following equation: r (%) = (m1 / m2) × 100 (i) r (%): yield in %; m1: mass of extract after evaporation of solvent; m2: mass of plant material used for extraction [27]. 2.2.5. phytochemical screening (qualitative) phytochemical tests consist in the identification of the different secondary metabolite families present in the edible part (roots) of brassica rapa by qualitative characterisation, using the standard procedures as described by [29].the results were expressed according to the reaction type: very positive: +++; moderately positive: ++; positive: +; negative: tannins to a test tube containing 1 ml of plant extract, 1 ml of a diluted aqueous solution of 1% fecl3 was added. the blue-black and blue-green color indicates the presence of hydrolyzable tannins and condensed tannins respectively [29] . food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 97 flavonoids to 5 ml of each extract, 1 ml of isoamyl alcohol, a few magnesium chips and a few drops of hydrochloric acid are added; the appearance of a pink or red colour indicates the presence of flavonoids [29] . coumarins (uv fluorescence) to 2 ml of extract we added 0.5 ml of 25% nh4oh. after mixture, the observance were made under uv at 366 nm. an intense fluorescence indicates the presence of coumarins[29]. free anthraquinones (bornträger test) we transfer 1 ml of each prepared extract into a test tube, then we added 1 ml of diluted nh4oh and stirred. the more or the less red coloration indicated the presence of free anthraquinones [29]. triterpenoids (salkowski’s test) a volume of 2 ml of chloroform was mixed with 1 ml of plant extract, and 3 ml of concentrated sulphuric acid (h2so4) was carefully added to the mixture, without stirring. the yellow color of the lower layer indicated the presence of triterpenoid [29]. free quinones the presence of free quinones was confirmed by adding a few drops of 10% naoh to 5 ml of extract, the coloring turns to yellow, red or purple indicates the presence of free quinones [29]. 2.2.4. secondary metabolite quantification polyphenols (folin-ciocalteu) the determination of total phenols was carried out according to the protocol established by [30], in a test tube 200 μl of the extract (1g dissolved in 10ml methanol), 1ml of 10-fold diluted folinciocalteu reagent, and 800 μl of the sodium carbonate solution na2co3 (7.5%) were added. after 30 min of incubation at room temperature, the absorbance was determined at 765 nm. a calibration range was established with gallic acid(y=0.592x+0.473 r²= 0. 9489) and the expression of the results were made in µg gallic acid equivalents per mg of dry matter (µg gae/ mg dm). flavonoids (aluminium trichloride) for the flavonoids determination, the aluminium trichloride colorimetric method was adopted [31]. a 500 µl of the extract was mixed with 1.5 ml of distilled water and then with 0.3 ml of a 5% sodium nitrite solution nano2. after 5 min, 3 ml of a 10% alcl3 solution was added. then, 6 min later, 1 ml of 4% naoh was added. after a 5 min rest time, the reaction volume was homogenised with a vortex and the absorbance was measured at 510 nm. to establish the calibration curve, quercetin was used as a standard (y=0.692x+1.471 r²= 0.967), the total flavonoid contents were expressed in µg quercetin equivalent / mg of dry matter (µg qe/ mg dm). condensed tannins (vanillin) the amount of tannins was estimated using the vanillin method described by [32]. in test tubes 50 µl of plant extract and 750 µl of 4% vanillin solution were introduced and mixed with the vortex, then 750 µl of concentrated hcl were added and incubated for 20 minutes. the absorbance reading was obtained at 550 nm, the calibration curve was prepared under the same conditions using catechin as standard (y=6.203x+0.394 r²=0.9843) and the results were expressed as µg catechin equivalent/mg of dry matter (µg ce/ mg dm). 2.2.5. antioxidant activity evaluation in order to estimate the antioxidant activity of the two studied extracts (aqebr and food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 98 mebr); three different tests were used: tac (total antioxidant capacity) , dpph (2.2'-diphenyl-1-pycrilhydrazyl) , frap (ferric-reducing antioxidant power assay) . total antioxidant capacity (tac) the total antioxidant capacity (tac) of the plant extracts was evaluated by the phosphomolybdenum method. this method was based on the reduction of molybdenum mo (vi) present as molybdate ions moo4 -2 to molybdenum mo (v) moo+2 in the presence of the extract to form a green phosphate/mo (v) complex at acidic ph. the stock solution (10 mg plant powder /10 ml methanol) was prepared. then, 1 ml of monosodium phosphate, 1 ml of sulphuric acid, 1 ml of molybdate and 500 µl of sample are introduced into a test tube, this solution was incubated at a temperature of 90°c for 90 min . the blank solution was prepared under the same conditions. the absorbance reading was obtained at 695 nm, the tac was expressed in milligram equivalents of ascorbic acid per gram of dry matter (mg eaa/ g dm). [33]. dpph test dpph radical scavenging activity was measured according to the protocol described by [34], in test tubes 30 μl of each extract (1mg/ml methanol) was introduced and 1.5 ml of the methanolic solution was added to dpph (4 mg /100ml). after vortexing, the tubes were stored in the darkness at room temperature for 30 minutes. the reading was performed by measuring the absorbance at 517 nm. the negative control consists of 1.5 ml of the methanolic solution of dpph and 30μl of methanol. the positive control was represented by an ascorbic acid solution; we calculate the inhibition percentages by the following formula: i % = ((ca ta)/ca) × 100 (ii) with: ca: the control absorbance; ta: the test absorbance. ic50 calculation ic50 (inhibitory concentration 50) is the concentration of the test sample required to reduce 50% of the dpph radical. ic50s were calculated graphically as inhibition percentages according to different extract tested concentrations [35]. frap test the protocol established by [36] was adopted, which consisted of removing 0.5 ml of each extract at different concentrations and adding 1.25 ml of a 0.2 m phosphate buffer solution (ph=6.6) and 1.25 ml of a 1% potassium ferricyanide k3fe(cn)6 solution and incubated at 50°c for 20 min, the tubes were then cooled to room temperature. 2.5 ml of 10% trichloroacetic acid (tca) were added to block the reaction, the tubes were centrifuged at 3000 rpm for 10 min. then the supernatant (1.25 ml) was added to 1.25 ml of distilled water, and 250 ml of a 0.1% iron chloride (fecl3) solution, the absorbance reading was made at 700 nm. the positive control was represented by an ascorbic acid solution. 2.2.6. statistical analysis each value is the mean of three replicates. values of different parameters were expressed as the mean ± standard deviation (sd). 3. results and discussion 3.1. moisture and ash content according to our results the turnip has a moisture content of 74%. our results were lower than those found by [37] which were 91.90%. according to [38], several factors (genetic, pedoclimatic, geographical, storage conditions, the plant's age, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 99 vegetative cycle and the maturation stage can influence the plant's water content. according to our results, turnip roots have an ash content of 10%, which indicated that brassica rapa is rich in mineral elements. in addition, the raw turnip contains a significant amount of potassium, 237 mg per 100 g [37]. fig 3: moisture and ash levels of brassica rapa roots 3.1. extraction yields after the extracts have been collected, the yields were calculated and represented in the (fig 4) 0 5 10 15 20 25 30 aqebr mebr y ie ld ( % ) extracts fig 4 : extraction yields of brassica rapa roots the highest yield was 24% for mebr compared to 17% for aqebr, this may be due to the differential solubility of the different phenolic compounds in the solvents and that this solubility is related to their degree of polymerisation, the interaction with other constituents and the used solvent type [27]. 3.3. phytochemical screening the preliminary evaluation of the brassica rapa phytochemical composition revealed the presence of some chemical groups (table 1). it can be seen that the two extracts mebr and aqebr are provided with different intensities of all the metabolites, except the anthraquinone class which is absent in both extracts. according to previous works, brassica species are rich in phenolic compounds, flavonoids, hydroxycinnamic acid, coumarins and terpenoids, which are very beneficial for human health. these components exert an antioxidant activity by inhibiting carcinogenesis and the production of reactive oxygen species (ros) [39] in addition, brassica rapa contains anthocyanins, responsible for red and purple pigmentation [40]. these data are comparable with our results, since the tests reveal the presence of flavonoids, coumarins and terpenoids. on the other hand, our results are synchronized with those of [41], who confirmed the presence of flavonoids, sulphur compounds and anthocyanins in the brassica rapa underground part. 3.4. secondary metabolites the total phenolic content (tpc), total flavonoid content (tfc) and condensed tannin content (ctc) were determined from the calibration curves of gallic acid, quercetin and catechin respectively. the tpc, tfc and ctc of the brassica rapa .l tubers aqueous and methanolic extracts of tubers are presented in (table 2). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 100 table 1: preliminary phytochemical analysis of brassica rapa n° phyto-chemical test extracts chemicals constituents tests mebr aqebr 1 tanins ferric chloride test + + 2 flavonoids shinoda test ++ ++ 3 coumarins fluorescence uv ++ ++ 4 free anthraquinones borntrager’s test 5 terpenoids salkowski test ++ + +++ 6 free quinones sulfuric acid test + +++ very positive: +++ ; moderately positive: ++; positive: +; negative: table 2: total phenolic, flavonoids and condensed tannins contents of brassica rapa tpc (𝜇g gae/mg dm) tfc (𝜇g qe/mg dm) ctc (𝜇g ce/mg dm) aqebr 0.12± 0.0009 0.015 ± 0.0005 0.007 ± 0.0003 mebr 1.48 ± 0.0009 0.15 ± 0.0005 0.11 ± 0.001 each value was expressed as means ± standard deviations for triplicate experiments. ce: catechin equivalent; dm: dry matter; gae: gallic acid equivalent; qe: quercetin equivalent; tfc: total flavonoid content; tpc: total phenolic content; ctc: condensed tannins content according to our results, the brassica rapa concentration of bioactive compounds (tpc, tfc, ctc) varied depending to the extractor type, of which extraction with methanolic maceration is more efficient than aqueous extraction .these results are similar to several works, including those of [42], who confirmed that the methanolic extract of b. rapa (root) had values of 0.3 µg gae / mg and of 0.0410.085 µg rutin equivalent / mg of fresh weight in polyphenols and flavonoids respectively. in addition, according to [37], the tpc of turnip was at 1.5 µg / mg. on the other hand, our results are considerably lower than those found by [43], who detected a tpc of 2.1 25.9 µg gae / mg of dw with 70 % ethanol. according to [44], the b. rapa subsp. rapifera .l aqueous extract contains 5.64 µg/mg of total phenols. according to the work of [45], the brassica rapa subsp. rapifer aqueous extract of showed an amount of phenolic and flavonoid compounds with values of 9.41 ± 0.18 𝜇g gae/mg and 1.01 ± 0.09 𝜇g qe/mg, respectively. the content of phenolic compounds varied significantly between the different parts of the turnip. for example, turnip green was revealed to contain 51.71 μmol/ g dw of phenolic compounds compared to 38.99 μmol/g dw in turnip top . the family, amount and concentration of different bioactive molecules in turnips are dependent on the development stage, as well as on biotic and abiotic factors. for example, norisoprenoids, terrpenes and aldehydes are highly concentrated at the germination stage (9-day-old turnip sprouts); whereas, at the maturity stage there is a decrease in sulphur and nitrogen compounds [11]. furthermore, herbivory pressure and high humidity always increased the abundance of secondary metabolites [46]. however, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 101 the choice of extraction solvent, ph, light and heat can influence the content of phenolic compounds [47]. several studies have confirmed the richness of turnip in glucosinolates, isothiocyanates, flavonoids, volatile substances [3, 4, 11, 48]. flavonoids are major components in turnips, present as glycosides. among the 35 flavonoids reported in this plant, kaempferol, quercetin and isorhamnetin are the most common aglycones [49]. flavonoids play an important role in uv protection, pigmentation and disease resistance. this explains their high concentration in the leaves and fruit epidermis. flavonoids detected in turnip include 27 flavonols, two flavanones and six chalcones [50, 51] . the presence of tannins suggests the ability of our plant to play a major function as an antioxidant agent [52]. this variation can be explained by the fact that the extraction of condensed tannins, depends on their chemical nature, the solvent used and the operating conditions 3.5. antioxidant activity 3.5.1. total antioxidant capacity (tac) the reducing capacity of a compound can serve as a significant indicator of its potential antioxidant activity [53]. the quantitative estimation of total antioxidant capacity was calculated according to the equation (y = 0.175x+0.125. r2= 0.9975). fig 5: total antioxidant capacity (tac) of different brassica rapa extracts. it can be seen that the tac of mebr is higher compared to aqebr with values of (5.32 ± 0.56 vs 2.68 ± 0.33 mg aae/gr de) respectively (fig 5). this suggests that the antioxidant activity increases with increasing polarity of the extracting solvent [43]. according to [54], the extraction time also participates in the increase of phytochemical content and antiradical activity . 3.5.2. dpph evaluation from our results, it can be seen that the inhibition percentage of dpph free radical increases with increasing the extract concentration. however, mebr demonstrated a strong antioxidant capacity compared to ascorbic acid. on the other hand, aqebr showed a moderate antiradical activity (fig 6). fig 6: antiradical activity (dpph test) of brassica rapa methanolic and aqueous extracts several researchers have widely pointed out the in vitro and in vivo antioxidant activity of turnip [9, 42, 55]. in addition, ethyl acetate extract from turnip roots exhibited higher free radical scavenging and lipoperoxidation inhibitory activity than other solvents (n-hexane, chloroform) [42]. however, [55] reported a inhibition percentage of dpph radical with 11.1186.3% using ethanol (70%) as an extractant solvent. on the other hand, [42] revealed a medium-low rate with 13-26% food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 102 of the methanolic extract of the brassica rapa underground part. in addition, further studies have revealed that turnip flower buds have the highest antioxidant capacity compared to other edible parts (leaves, stems and roots) with the dpph (1,1-diphenyl-2-picrylhydrazyl) radical scavenging test [8,56] . according to [57], the anti-free radical activity is dependent on the number, position and nature of substituents on the b and c rings (hydroxyl, metaxyl, glycosyl groups) and the degree of polymerization of phenolic compounds. ic50 (dpph test) according to our results, the methanolic extract showed the highest antiradical activity with an ic50 of 0.14±0.12 mg/ml compared to the aqueous extract with an ic50 of 1.9±0.45 mg/ml. fig 7 : ic50 of different brassica rapa extracts and ascorbic acid the results of [42] are synchronized with our, which they found that ic50 of ethanolic extract (70%) of the brassica. rapa subsp. rapifera .l edible part was 0.23-2.00 mg/ml. according to 8 , flower buds showed an ic50value of 0.94 mg/ml, followed by leaves and stems ic50=1.12 mg/ml, and then roots had the lowest antioxidant capacity with an ic50 value of 2.88 mg/ml. 3.5.3. frap evaluation the reduction of fe+3 is often used to study the ability of a substance to release electrons. this property constitutes an important mechanism of antioxidant action [58]. our results revealed that aqebr has the highest antioxidant power compared to mebr with maximum values of (1.41 mg/ml vs 0.83 mg/ml) respectively. however, ascorbic acid showed the highest antioxidant power with a maximum of 1.63mg/ml (fig 8). antioxidant activity depends not only on the concentration of phenolic molecules but also on the structure of these molecules. low or high antioxidant capacity may be due to the solvent's polarity, which modified the capacity to dissolve a selected group of antioxidant compounds, thus influencing the antioxidant activity evaluation [59]. the temperature strongly determined the level of enzyme activity involved in the polyphenol synthesis. therefore, different thermal conditions can induce a significant change in the final polyphenols concentration. other environmental factors could probably play a role in the process of polyphenol synthesis such as co2 content (c/n interaction) or the level of oxidative stress [60]. 4. conclusion from the results presented, it appears that methanol is a more efficient extractor than water, which mebr exhibited a higher antioxidant capacity and richness in secondary metabolites than aqebr .therefore, the selection of an appropriate solvent-based system remains one of the most important steps, in the optimization of the polyphenols, flavonoids and other antioxidant compounds extraction, for a better active ingredients valorization. furthermore, the results obtained in this food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 103 work are very promising; they indicate that the turnip brassica rapa can be a easily available food source of biologically active compounds. fig 8: antiradical activity of brassica rapa methanolic and aqueous extracts 5. acknowledgments the authors declare that they have no conflicts of interest in relation to this article. 6. references [1].ghanad m., nulit r., go r ., and seok cy., comparative study on antioxidant activity of brassica rapa var. parachinensis leaves and seeds. advances in nutrition and food science, 1-7. doi:10.4172/2472-1921-c1-006 , (2020) . [2].guo y., chen s., li z ., and cowling wa., center of origin and centers of diversity in an ancient crop, brassica rapa (turnip rape). journal of heredity, 105 (4):555-65.doi: 10.1093/jhered/esu021, (2014). [3].pierre ps., jansen jj., hordijk ca., van dam nm., cortesero am ., and dugravot s., differences in volatile profiles of turnip plants subjected to single and dual herbivory above and below ground. journal of chemical ecology, 37(4):368-377.doi: 10.1007/s10886-0119934-3, (2011). [4]. francisco m., moreno da., cartea me., ferreres f., garcía-viguera c ., and velasco p., simultaneous identification of glucosinolates and phenolic compounds in a representative collection of vegetable brassica rapa. journal of chromatographya, 1216(38):66116619.doi:10.1016/j.chroma.2009.07.055, (2009). [5]. jeong rh., wu q., cho jg., lee dy., shrestha s., lee m h., lee kt., choi ms., jeong ts., ahn em., chung hg., rho yd ., and baek n.i ., isolation and identification of flavonoids from the roots of brassica rapa spp. journal of applied biological chemistry , 56 (1): 23-27.doi: 10.3839/jabc.2013.005 , ( 2013). [6].wu q., bang mh., lee dy., cho jg., jeong rh., shrestra s., lee kt., chung hg., ahn em ., and baek ni ., new indoles from the roots of brassica rapa subsp. campestris. chemistry of natural compounds, 48 (2):251-253. doi: 10.1007/s10600-012-0221-5, (2012). [7].cartea me., francisco m., soengas p ., and velasco p., phenolic compounds in brassica vegetables. molecules, 16 (1): 251-280. doi :10.3390/molecules16010251 , (2011) [8]. fernandes f., valentãob p., sousa c., pereira j a ., seabra r m ., and andrade p b., chemical and antioxidative assessment of dietary turnip (brassica rapa var. rapa l). food chemistry, 105 (3):10031010.doi:10.1016/j.foodchem.2007.04.063 (2007). [9]. wu q., cho jg., lee ds., lee dy., song ny., kimy c., lee kt ., chung hg., choi ms., jeong ts., ahn em ., kim gs ., and baek ni., carbohydrate derivatives from the roots of brassica rapa ssp. campestris and their effects on ros production and glutamate-induced cell food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 104 death in ht-22 cells. carbohydrate research, 372: 9-14.doi: 10.1016/j.carres.2012.09.015, (2013). [10]. lee jg., bonnema g., zhang n., kwak jh., ric c., de vos rch ., and beekwilder j., evaluation of glucosinolate variation in a collection of turnip (brassica rapa) germplasm by the analysis of intact and desulfo glucosinolates. journal of agricultural and food chemistry, 61(16):39843993.doi:10.1021/jf400890p,(2013 . [11]. taveira m., fernandes f., de pinho pg., andrade pb., pereira ja., and valentao p., evolution of brassica rapa var. rapa l. volatile composition by hs spme and gc/it-ms. microchemical journal,93(2):140146.doi : 10.1016/j.microc.2009.05.011 , (2009). [12]. manchali s., murthy knc., and patil bs., crucial facts about health benefits of popular cruciferous vegetables. journal of functional foods, 4 (1):94-106.doi: 10.1016/j.jff.2011.08.004 , (2012). [13]. paul s., geng ca ., yang th ., yang yp ., and chen jj., phytochemical and health‐beneficial progress of turnip (brassica rapa). journal of food science, 84(1):19-30. doi: 10.1111/1750-3841.14417, ( 2018). [14]. gairola s., sharma j ., and bedi ys., a cross-cultural analysis of jammu, kashmir and ladakh (india) medicinal plant use. journal of ethnopharmacology, 155 (2): 925-986.doi: 10.1016/j.jep.2014.06.029, (2014). [15]. ullah s., khan mr., shah n a., shah sa., majid m ., and farooq ma., ethnomedicinal plant use value in the lakki marwat district of pakistan. journal of ethnopharmacology, 158 pta: 412-422 . doi: 10.1016/j.jep.2014.09.048 , (2014). [16]. hong jk., hwnag je., park th ., zang yx ., lee sc., kwon sj., mun jh., kim hu., kim ja ., jin m n., kim js., lee si., lim mh., hur y., lim co., and park bs., identification and characterization of the phytocystatin family from brassica rapa. journal of plant biotechnology, 35(4):317327.doi:10.5010/jpb.2008.35.4.317 ,(2008). [17]. shin js., noh ys., lee ys., cho yw., baek ni., choi ms., jeong ts., kang e., chung hg., and lee k t. arvelexin from brassica rapa suppresses nf-kb-regulated proinflammatory gene expression by inhibiting activation of ikb kinase. british journal of pharmacology, 164 (1):145-158.doi: 10.1111/j.1476-5381.2011.01351.x , (2011). [18]. bang mh., lee dy., oh yj ., han mw., yang h j., chung h g., jeong ts., lee kt., choi ms., and baek ni., development of biologically active compounds from edible plant sources xxii. isolation of indoles from the roots of brassica campestris ssp rapa and their hacat inhibitory activity. journal of the korean society for applied biological chemistry, 51 (1): 65-69, (2008). [19]. mirzaie h., johari h., najafian m., and kargar h., effect of ethanol extract of root turnip (brassica rapa) on changes in blood factors hdl, ldl, triglycerides and total cholesterol in hypercholesterolemic rabbits. advances in environmental biology, 6 (10):27962801 , (2012). [20]. al-snafi ae .,the pharmacological importance of bassica nigra and brasica rapa grown in iraq. journal of pharmaceutical biology, 5(4): 240-253 , (2015). [21]. daryoush m., bahram at., yousef d ., and mehrdad., n., protective effect of turnip root (brassica rapa. l) ethanolic extract on early hepatic injury in alloxanized diabetic rats. australian journal of basic and applied sciences, (7):748-756 , (2015). [22]. mohajeri d., gharamaleki mn., hejazi s., and nazeri m., preventive effects of turnip (brassica rapa l.) on renal ischemiareperfusion injury in rats. life science journal, 10 (1) :1165-1170, (2013). [23]. hosseini se., zahiri s ., and aqababa h., effect of alcoholic extract of brassica rapa root on formalin test pain in adult male rats. quarterly of the horizon of medical sciences, 19(3): 161-166 , (2013). [24]. baba-hamed k., bouanani a ., nasri a ., and bouanani r. la modélisation de la nappe alluviale de la plaine de ghriss (mascara nwalgérien).larhyss journal, 21 :143150 , (2015) [25]. djerroumi a., and nacef m., 100 plantes médicinales d’algérie. edition دار هومه .isbn10: 9961656235 .p 159, (2012). [26]. rebaya a., belghith si., baghdikian b., leddet vm., mabrouki f., olivier e ., cherif jk ., and trabelsi ayadi m., total phenolic, total flavonoid, tannin content, and antioxidant capacity of halimium halimifolium (cistaceae). journal of applied pharmaceutical science, 5 (01): 52-57.doi: 10.7324/japs.2015.50110 , ( 2015). [27]. falleh h., ksouri r., chaieb k., karray-bouraoui n., trabelsi n., boulaaba m ., and abdelly c., phenolic composition of cynara cardunculus l. organs, and their biological activities . comptes rendus biologies , 331(5): 372-379.doi: 10.1016/j.crvi.2008.02.008 , (2008). https://www.sciencedirect.com/science/journal/17564646 https://www.sciencedirect.com/science/journal/17564646 http://koreascience.or.kr/journal/smsmcm.page http://koreascience.or.kr/journal/smsmcm.page http://doi.or.kr/10.psn/adper0000315312 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 105 [28]. aoac.,the association of official analytical chemists. official methods of analysis. 17 th ed. maryland. u.s.a , (2000). [29]. trease ge., evans wc., pharmacognosy. 3th (ed). elbs/bailliere tindall, london. pp. (345-6, 535-6, 772-3), (1989). [30]. singleton v., and rossi j., colorimetry of total phenolic compounds with phosphomolybdic-phosphotungstic acid reagents. american journal of enology and viticulture, 16(3), 144-158.doi: www.ajevonline.org/content/16/3/144.full.pdf+html , (1965). [31]. kim do., chun ok., kim yj ., moon hy., and lee cy., quantification of polyphenolics and their antioxidant capacity in fresh plums. the journal of agricultural and food chemistry, 51(22):6509-6515. doi: 10.1021/jf0343074 , (2003). [32]. julkunen-tiitto, r., phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. journal of agricultural and food chemistry, 33 (2): 213-217.doi: 10.1021/jf00062a013, (1985). [33]. prieto p., pineda m ., and aguilar m., spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin e. analytical biochemistry, 269(2): 337-341.doi: 10.1006/abio.1999.4019 ,(1999). [34]. lopes-lutz d., alviano ds., alviano cs ., and kolodziejczyk pp., screening of chemical composition, antimicrobial and antioxidant activities of artemisia essential oils. phytochemistry, 69 (8):1732-1738.doi: 10.1016/j.phytochem.2008.02.014 , (2008). [35]. torres r., faini f., modak b., urbina f., labbé c ., and guerrero j ., antioxidant activity of coumarins and flavonols from the resinous exudate of haplopappus multifolius. phytochemistry, 67 (10): 984-987.doi: 10.1016/j.phytochem.2006.03.016 , (2006). [36]. oyaizu m., studies on products of browning reactions: antioxidative activities of product of browning reaction prepared from glucosamine. japan journal of nutrition, 44 (6):307-315.doi: org/10.5264/eiyogakuzashi.44.30 , (1986). [37]. ciqual. agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail. table de composition nutritionnelle des aliments. 2020. accessed on 26/08/2020 from the website https://ciqual.anses.fr/. [38]. ruiz-rodriquez bm ., morales p., fernandz-ruiz v., cortes sánchez mataa m., cámara m.,díez-marqués c., pardo-de-santayana m ., molina m ., and tardío j., valorization of wild strawberrytree fruits (arbutus unedo l.) through nutritional assessment and natural production data .food research international, 44 (5) :1244-1253.doi: 10.1016/j.foodres.2010.11.015, (2011). [39]. morales-lopez j., centenoálvarez m., nieto-camacho a., lopez mg., pérez-hernández perez e., nawaz h., shad ma ., and rauf a., optimization of extraction yield and antioxidant properties of brassica oleracea convar capitata var l. leaf extracts. food chemistry, 242: 182-187. doi: 10.1016/j.foodchem.2017.09.041 ,(2018). [40]. aires a., chapter 3 -brassica composition and food processing a2 preedy, victor, processing and impact on active components in food. academic press, san diego .17-25, (2015). [41]. rafatullah s., al-yahya m ., mossa j., galal a ., and el-tahir k., preliminary phytochemical and hepatoprotective studies on turnip brassica rapa l. international journal of pharmacology, 2 (6): 670-673.doi: 10.3923/ijp.2006.670.673 , (2006). [42]. iqbal s., younas u., chan kw., saeed z., shaheen ma., akhtar n ., and majeed a., growth and antioxidant response of brassica rapa var. rapa l.(turnip) irrigated with different compositions of paper and board mill (pbm) effluent. chemosphere, 91(8):11961202.doi: 10.1016/j.chemosphere.2013.01.035, (2013). [43]. ryu jp., kim dc., in mj .,chae hj ., and lee sd., antioxidant potential of ethanol extract of brassica rapa l. root. journal of medicinal plants research, 6 (9):1581-1584. doi : 10.5897/jmpr11.323 , (2012). [44]. anitha t., and divya dharsini r., studies on in-vitro antioxidant properties of brassica vegetables. international journal of pharmaceutical, chemical and biological sciences, 4 (4) :1061-1065 , (2014) . [45]. berdja s., smail l., saka b., neggazi s., haffaf em., benazzoug y., kacimi g., boudarene l., and aouichat bouguerra s. glucotoxicity induced oxidative stress and inflammation in-vivo and in-vitro in psammomys obesus: involvement of aqueous extract of brassica rapa rapifera. evidence-based complementary and alternative medicine , 6:114.doi: 10.1155/2016/3689208 , (2016). [46]. metz j., ribbers k., tielbörger k ., and müller c., long-and medium-term effects of aridity on the chemical defence of a widespread brassicaceae in the mediterranean. environmental and experimental botany, 105 : 39-45 .doi: 10.1016/j.envexpbot.2014.04.007 , (2018). https://ciqual.anses.fr/ https://www.sciencedirect.com/science/article/abs/pii/s0963996910004485#! https://www.hindawi.com/journals/ecam/ https://www.hindawi.com/journals/ecam/ food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 1 – 2022 karima ould yerou , fatima zohra elkadi , khedoudja kanoun , hadj mostefa khelladi , hanane benzahia, sara bekhti , preliminary phytochemical investigation and antioxidant potential of various extracts of dietary turnip (brassica rapa l.) , food and environment safety, volume xxi, issue 1 – 2022, pag. 94 – 106 106 [47]. akowuah ga., mariam a., and chin jh., the effect of extraction temperature on total phenols and antioxidant activity of gynura procumbens leaf. pharmacognosie magazine, 5(17): 81-85 . http://www.phcog.com/text.asp?2009/5/17/81/5799 1, (2009). [48]. padilla g., cartea me ., velasco p ., de haro a., and ordás a., variation of glucosinolates in vegetable crops of brassica rapa. phytochemistry, 68: (4) 536-545.doi: 10.1016/j.phytochem.2006.11.017,(2007). [49]. vallejo fa., tomás-barberán fa., and ferreres f., characterization of flavonols in broccoli (brassica oleracea l. var. italica) by liquid chromatography-uv diode array detection electrospray ionization mass spectrometry. journal of chromatography a, 1054, (1-2):181-193 .doi : 10.1016/j.chroma.2004.05.045,(2004). [50]. crozier a., jaganath ib., and clifford mn., phenols, polyphenols and tannins: an overview in crozier a, clifford m, ashihara h. (eds.), plant secondary metabolites: occurrence, structure and role in the human diet (pp. 1–24). uk, oxford: blackwell , (2006). [51]. pereira dm., valentão p., pereira ja., and andrade pb., phenolics: from chemistry to biology. molecules, 14 (6): 22022211.doi: 10.3390/molecules14062202 , (2009). [52]. ali-rachedi f., meraghni s., nourhene touaibia n., and sabrina m., analyse quantitative des composés phénoliques d'une endémique algérienne scabiosa atropurpurea sub. maritima l. bulletin de la société royale des sciences de liège, 87 :13-21 , (2018). [53]. apak r., güçlü k., özyürek m., and çelik se., mechanism of antioxidant capacity assays and the cuprac (cupric ion reducing antioxidant capacity) assay. microchim acta , 160: 413-419. doi : 10.25518/0037-9565.7398, (2008). [54]. nawaz h., shad m.a., and rauf a., optimization of extraction yield and antioxidant properties of brassica oleracea convar capitata var l. leaf extracts. food chemistry, 242, 182-187. doi: 10.1016/j.foodchem.2017.09.041, (2018) [55]. beltagy a., investigation of new antimicrobial and antioxidant activities of brassica rapa l. international journal of pharmacy and pharmaceutical sciences, 6 (6):84-88, (2014). [56]. chaudhary a., choudhary s., sharma u., vig ap., and arora s. in-vitro evaluation of brassica sprouts for antioxidant and antiproliferative potential. indian journal of pharmaceutical sciences, 78(5): 615-623 .doi: 10.4172/pharmaceutical-sciences.1000160, (2016). [57]. popovici j ., comte g ., bagnarol é ., alloisio n ., fournier p., bellvert f., and fernandez m. p., differential effects of rare specific flavonoids on compatible and incompatible strains in the myrica gale-frankia actinorhizal symbiosis. applied and environmental microbiology, 76(8): 2451-2460. doi:10.1128/aem.02667-09 , (2010). [58]. ebrahim zadeh ma., pourmorad f., and bekhradnia ar., iron chelating activity screening, phenol and flavonoid content of some medicinal plants from iran. african journal of biotechnology, 7(18): 3188 3192.doi: 10.4172/pharmaceutical-sciences.1000160, (2008). [59]. turkmen n., velioglu ys., sari f., and polat g., effect of extraction conditions on measured total polyphenol contents and antioxidant and antibacterial activities of black tea. molecules, 12(3): 484-496. doi : 10.3390/12030484 , (2007). [60]. sallas l., kainulainen p., utriainen j., holopainen t., and holopainen jk.,the influence of elevated o3 and co2 concentrations on secondary metabolites of scots pine (pinus sylvestris l.) seedlings. global change biology, 7(3): 303-311. doi: 10.1046/j.1365-2486.2001.00408.x , (2001). 1. introduction 4. conclusion doi: https://doi.org/10.4316/fens.2022.029 303 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 303 312 characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context *chafia merah mining laboratory,mining institute, echahid cheikh larbi tebessi university, tebéssa, algeria, chafia.merah@univ-tebessa.dz *corresponding author received 12th november 2022, accepted 29th december 2022 abstract: in the world the demand and consumption of raw materials and minerals is sharply increasing, which has encouraged countries around the world to intensify the mining of different materials and ores such as phosphate. in algeria, while phosphates are one of the country's most important natural resources; they are ranked third in the world in terms of proven reserves with 2,200,000 tones, mining of this ore is low. the bled el hadba deposit is one of the promising djebel onk’s deposits with 841 million tons of reserves and which starts mining in 2020. the ore is exploited by the open pit method, therefore a large quantity of waste rock will be evacuated to appropriate surfaces called waste rock dumps, and will consequently induce an increasingly serious destruction of the eco-environment. intelligent use of solid mine waste to compensate for resource scarcity and protect the environment appears to be a necessity. the main purpose of our research work involves the characterization and environmental management of sand waste rocks from bled el hadba phosphate deposit. in this context, we studied a series of runs: particle size analysis, cleanliness by the equivalent of sand, and chemical analysis. the results obtained are promising and encouraging for the use of sand waste from the mining of phosphates from bled el hadba in the glass and building materials industry. keywords: open pit mining, phosphate, sand, waste rock, environment. 1. introduction every day open pit mining produces large amounts of waste which occurs in several stages of the mining process and throughout the life of the mine, from the first exploration drilling project to the last processed material before mine closure. responsible mining can play a key role in developing a more sustainable mining industry. sustainable mining practices focus on resource sustainability, the environment and socio-economic benefits [1]. the waste generated is inevitable and has generally negative repercussions on the environment but some waste can be used safely in other applications, such as building materials. the reuse of mining waste as an alternative raw material in construction materials is a promising solution that not only equilibrates the deficit between production and consumption but also protects the environment. this solution makes it possible to reduce the volume of waste and to preserve the non-renewable natural resources which are intensively used in construction (clay, limestone, sand, etc.) [2,3]. in the same context, researches have studied total and partial substitution of phosphate waste rocks in ceramic tile manufacturing and they confirmed good adaptability for making traditional ceramic tiles [4]. mining waste includes materials resulting from the exploitation of minerals during the extraction, beneficiation and treatment processes. depending on the components and characteristics of the materials treated, the effects on the environment will be mailto:chafia.merah@univ-tebessa.dz food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 304 different. particular attention is needed to recover this waste and make it ecologically rational. the objective is to mobilize companies on clear environmental requirements and manage to organize construction sites differently [5]. for this, we must allow everyone to:  better know the different types of wastes and their disposal systems;  integrate waste management into the organization of the worksite and the contractual documents;  carry out concretely the wastes sort on the site. this approach can go further than the simple concept of waste management and deal with all the environmental impacts of construction sites. it will take the form of a clean or green project and thus becomes a part of the process of sustainable development, the aim of our work. in algeria, the economics of mining is in a state of great change. sales opportunities and prices are continuing to increase for most commodities. profits reached levels not seen in decades. in algeria, phosphates are one of the country's most important natural resources; that has been ranked third in the world in terms of proven reserves with 2,200,000 tons [6]. however, phosphate has been poorly exploited for over 45 years [7]. algeria has a significant deficit in building materials. facing the seriousness of the environmental problems due to the over exploitation of resources, the algerian government decided in 2001 to devote a significant financial envelope to achieve the objectives set out in the national action plan for the environment and sustainable development pnae -dd [8] relating to the management, control and elimination of waste which constitutes the starting point of this new environmental strategy. problems related to mining wastes the term "mining wastes" actually includes various products from the extractive industry and in particular mine tailings. these are generally geological materials too poor to be exploited in the technical and economic conditions of the moment [9, 10]. this economic distinction is variable over time. these waste rocks are products made up of soils and rocks excavated during the operation of a mine, after recovery of the commercially valuable part that constitutes the ore. depending on the types of mine tailings and their uses, they could be used directly or dressed in different sizes. many mining residues have been subject to financial and material "valorization" by being resold or reused as backfill material, or as fuel or raw material for the production of reconstituted fuel from dust. for example, we may cite [11]:  a very good material for the construction of roads, depending on the size of the waste rock, coarse can be used for the construction of the platform ,those of the fine size for the pavement of the road;  a very good material for construction of dams;  a very good material for beneficiating coarse and fine aggregate of concrete;  it could be used for making construction bricks when beneficiated to suitable size; and  to backfill the mined out area, subsidence area and other area needed to be filled. the open-pit mining of the bled el hadba phosphate deposit consists of removing the covering waste rock and accessing the deposit from the surface, the waste rock is composed mainly of sands. the aim of this work is the characterization of the sands resulting from the mining of the phosphates of bled el hadba, for their possible use in the glass industry or the field of construction. for this purpose, this paper reports results from laboratory tests analysis carried out to determine numerous characterizations of the material, such as particle size, food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 305 equivalent of sand as well as the chemical proprieties of the sand of mine. 2. materiel and methods 2.1 case study: description of the mining field phosphate deposits have two origins: sedimentary which produces 80% of the world production of natural phosphate and 20% of igneous ores often low in p2o5 which requires enrichment [12]. since the last century, the production of phosphate has grown significantly to provide mainly fertilizers to agriculture and to allow high yields in food production [13, 14]. phosphorus is produced by mining and beneficiation of phosphate ores [15]. the greatest production of phosphorus is destined for agricultural or food purposes with 82% for the manufacture of phosphate fertilizers, and 9% for animal feed and food additives. the rest 9% is used for processes in non-food industrial production: for example, phosphates are used for the manufacture of soaps, detergents, ceramics, etc. [16]. the djebel onk deposit is a sedimentary deposit [17], located about 100 km south of the city of tébessa, at the eastern end of the saharan atlas, close to the algeriantunisian border (fig.1). this mining basin contains five deposits: the djemi-djema deposit, djebel onk north, oued betita, kef essenoun and bled el hadba (fig.2). it is the natural geographic boundary between the high plateaus constantine and the saharan area [18, 19].it contains approximately half the phosphate reserves of algeria (estimated at 2 trillion tons of reserves) [20]. fig. 1. location map of the study area. bled el hadba deposit is located about 14 km to the east of djebel onk anticline. the phosphates of bled el hadba have long been recognized [21]. they have been food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 306 recognized by wells and galleries from the beginning of the century and the geology of the phosphatic zone is described by [22]. some data concerning the petrography of phosphates are provided by cayeux [23]. in 1962, ranchin [24], taking the data indicated in [22, 23] and reported the presence of four layers of phosphate. with the aim of developing its phosphate output, the algerian company society of mining phosphate (somiphos), aims to become a major supplier of phosphate. therefore, the company has planned to put in exploitation the layer of bled el hadba which represents one of the largest phosphates reserve in algeria. dmt estimated the value of proven and probable reserves at 841 mt with an average diluted p2o5 grade of 22.2% [25].lifetime is estimated at 34 years by taylor's formula developed in 1977[26]: 𝑡𝑙𝑖𝑓𝑒,𝑛 = 0.2 ∗ √𝑟𝑒𝑠𝑒𝑥𝑝 4 (1) where 𝑡𝑙𝑖𝑓𝑒,𝑛 denotes the lifetime in years and 𝑟𝑒𝑠𝑒𝑥𝑝 the total reserve tonnage expected in units of tone. fig. 2. map of the geographical and geological situation of the phosphate deposits in the djebel onk region [19]. horizontal stratification low dip stratification geogical outline western boundary of the main layer e 1b bbbbé we ster n bou nda ry of the mai n laye r montian: limetone, jumachelle, marl (tselja formation) e 1a w est er n bo un da ry of the ma in lay er danian: marl (el hania formation) e 6b-c we ster n bou nda ry of the mai n lay er maestrichtian: limestone compact (labiod formation) bled el hadba q quaternary: sands, silt, alluvium m miocene : sands,clays e 3 we ster n bou nda ry of the mai n laye r ypresian: marl, limestone with silex, some phosphate levels(metlaoui ss formation) e1 thaneti a: marl limestone,main llayer of phophates e 4 lutetian : variegated clays,formation with gypsum (formation souar) lutetian to ypresian: limestone,marl, limestone with silex nodule e 3-4 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 307 according to the study made by cerad (center for studies and applied research development) the direction of open pit mining is more favorable from the east towards the west because the layer of stripping varies from the east of 10 m to the west up to 100 m. 2.2 thickness of the sterile cover the sterile cover consists of the dolomitic limestones of the ypresian, surmounted by the sands of the miocene towards the west (fig.3).the thickness of the overlap increases progressively from east to west from the outcrop of the phosphate beam. on all the eastern band of the deposit, the cover, mostly limestone, is less than 1 m .towards the west, the appearance of miocene sands causes a rapid increase in the thickness of the cover from 30 m to 70 m. the maximum thickness of the cover is 123.80 geological ages log stratigraphique lithological description miocene 13.6 m ypersian 43.9 m soft yellow sands, powdery, finegrained to medium. thantian superior dolomitic limestone, yellow color, porous, neritic and weakly phosphates. phosphated beam 80 m the phosphate horizon. consisting of different sublayers, we distinguish: 43.9 62.2 m: dolomitic light brown phospharenites and phosphated coquinas. 62.2-70.2 m: slightly dolomitic brown phospharenites. 70.2-77 m: dolmotic and claylike phospharenites. 7781.70 m: clayey phospharenites + marls and clays fig. 3. the stratigraphic column of the bled el hadba deposit. according to the study carried out by the cerad (center for studies and applied research), the direction of the exploitation is more favorable from the east towards the west because the covering layer varies from the east 10 m to the west up to 100 m [27]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 308 2.3 description of the samples samples were taken from core drilling sph.01. the samples were collected from 3.40 to 184 m to cover the layer of sand. these samples are numbered, labeled and sorted by sampling interval. the total number of samples is 10. table 1. the geological description of the rocks of the sph.01 drills core and location of the samples. sample name intervals of geological description (m) geological description of rocks sam1 3.40 6.10 sandy clay of color brownish-yellow compact slightly and consolidated, residually foliated. sam2 6.10 17.10 quartz sand with medium and coarse grains of yellow soft brownish loose and friable sam3 17.10 21.00 compact, consolidated brownish-yellow sandy clay with thin, compact, hard green-yellow marl intercalations. sam4 21.00 68.00 quartz sand with fine grains of gray-brownish color interspersing a clay passage of centimeter to decimeter order. sam5 68.00 86.00 quartz sand with fine grains of gray-brownish color interspersing a clay passage of centimeter to decimeter order sam6 86.00 102.00 quartz sand with fine grains of gray-brownish color interspersing a clay passage of centimeter to decimeter order sam7 102.00 -114.00 quartz sand with fine grains of gray-brownish color interspersing a clay passage of centimeter to decimeter order. sam8 114.00 -140.00 quartz sand with fine grained greyish color. sam9 140.00 -165.00 quartz sand with fine grained greyish color sam10 165.00 -184.00 fine-grained quartz sand with a medium-sized fraction of grayyellowish 3. results and discussion sand samples collected in the area of bled el hadba are subjected to tests:  particle size analysis : the test is carried out according to the standard: nfp18-101 by screening on sieves of meshes 0.08 mm, 0.125 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm, 4 mm;  cleanliness by the equivalent of sand :the test is carried out according to standard nf en 933-8;  for the chemical analysis, samples are prepared according to the quartering method using sam1, sam2, sam3, sam4, sam5, sam6, sam7, sam8, sam9, and sam10 to obtain samp1, samp2, samp3, and samp4.the detail of the chemical analysis procedure is mentioned in the section 3.3. 3.1 particle size analysis to carry out the interpretation of the particle size analysis, the fineness module, the uniformity coefficient, and the curvature coefficient are introduced [28, 29]. sand of bled el hadba has a granular class of fine sand with maximum diameters ranging from 0.08 mm to 0.8 mm (fig.4). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 309 fig. 4. granulometric curve of the sands of bled el hadba. a. the fineness modulus (fm) fineness modulus is an empirical factor obtained by adding the cumulative percentages of aggregate retained on each of the standard sieves (0.125mm -0.25mm-0.50mm-1mm2mm-4mm) and dividing this sum by 100. fm: 1/100 σ cumulated refusal in% of sieves (0.125 mm 0.25 mm 0.50 mm 1 mm 2 mm 4 mm) the sands must have such a grain size that the fine elements are neither in excess nor in too small a proportion. coefficient of uniformity : 𝐶𝑢 = 𝑑60 𝑑10 (2) coefficient of curvature : cc= (𝑑30) 2 (𝑑10 ∗𝑑60 ) (3) where d60, d30 and d10 was the grain diameter at 60% , 30% and at 10% passing respectively. table 2. tests results of fineness module, coefficient of uniformity and curvature coefficient. sam10 sam9 sam8 sam7 sam6 sam5 sam4 sam3 sam2 sam1 0.98 0.96 1.03 0.95 1.03 1.03 1.02 1.04 1.05 1.49 fm 3.12 1.56 1.56 1.56 3.2 3.2 2.5 1.6 1.6 6.4 cu 3 1.5 1.5 1.5 0.8 0.8 0.93 0.6 0.6 1.6 cc the laboratory tests show that the sand of bled el hadba has a majority of fine grains. the fineness module is of very low average values for use in the manufacture of concretes. these values are between 0.95 and 1.49. given its coefficient of uniformity and the coefficient of curvature, it is poorly graded sand (sp) (table 2). 3.2 equivalent of sand according to the sand equivalent, 80% of the samples show that the sands of bled el hadba are clayey and 20% are very clean sands (table 3). -20 0 20 40 60 80 100 120 0.001 0.01 0.1 1 10 p a ss in g % sieve (mm) sam1 sam2 sam3 sam4 sam5 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 310 table 3. sand equivalent test result sam1 sam2 sam 3 sam4 sam5 sam6 sam7 sam8 sam9 sam10 es (%) 17.8 44 49 54 83 83 49 7 60 52 3.3 chemical analysis to carry out the chemical analysis, the samples must first be prepared in the laboratory. the homogenization operation of the 10 samples is carried out: sam1, sam2, sam3, sam4, sam5, sam6, sam7, sam8 and sam9 to obtain 4 samples. the 4 new samples now named: samp.1, samp.2, samp.3, samp .4 were obtained by quartering using a dividersampler which allows the division of the samples in a representative and reproducible way (fig.5.a). the mass of each of the samples sam1, sam2 , sam3 , sam4 , sam5 , sam6 , sam7 , sam8 , sam9, is 50 g. therefore, every three samples of the first 9 were combined to obtain 3 samples of 150 g each as mentioned in the (table4); in addition to the tenth sample sam10 which has a mass of 150 g. these 4 samples were crushed and reduced to a fine powder for chemical analysis (fig.5.b). table 4. samples after quartering and grinding sam10 sam9 sam8 sam7 sam6 sam5 sam4 sam3 sam2 sam1 samples samp.4 samp.3 samp.2 samp.1 samples after quartering and grinding fig. 5. chemical analysis, procedures and apparatus: (a) divider-sampler, (b) crusher the chemical analysis showed that the bled el hadba sand is 80.6% sio2which means that it can be used for the glass industry (table 5). table 5. results of chemical analysis tests samples samp.1 samp.2 samp.3 samp.4 elements content (%) total silica (si𝑂2) 81.6 81.6 80.20 79 iron oxide (fe2o3) 0.56 0.31 0.43 1.25 calcium oxide (cao) 7.85 2.24 1.12 1.12 magnesium oxide (mgo) 0.18 0.07 0.10 0.10 potassium oxide (k2o) 0.00602 0.00602 0.00602 0.00602 sodium oxide (na2o) 0.08088 0.06066 0.08088 0.08762 phosphoric anhydride (p2o5) 0.32 0.17 0.18 0.17 dosage of carbon dioxide (co2) 5.15 0.67 0.50 0.83 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 311 4. conclusion in this study, the laboratory tests showed that the sand of bled el hadba has a granular class of fine sand whose maximum diameters vary from 0.08mm to 0.8mm. the average values of the fineness module were between 0.95 and 1.49 very low for use in the manufacturing of concrete, what requires a valorization. in addition, the equivalent of sand showed that 80% of the samples are clay sands and 20% are very clean. in the other hand, chemical analysis have shown that bled el hadba sand is 80.6% sio2 which means it can be used for the glass industry. as this large mining complex is in preparation for the exploitation of phosphates, which automatically leads to a large quantity of sand waste. this generated waste has environmental impacts that raise deep concerns. as a result, and according to the encouraging results of this study, this waste can be valorized for a sustainable exploitation. while this preliminary classification of sands is promising to engage in a process of sustainable development, a detailed study should therefore be carried out to consider a beneficial use in order to maintain this land as a beautiful land (area) without wastes. 5. references [1]. chen j., jiskani im., jinliang c., yan h., evaluation and future framework of green mine construction in china based on the dpsir model, sustainable environment research, https://doi.org/10.1186/s42834-020-00054-8, (2020) [2]. zeghina si., bounouala m., chettibi m., benselhoub a., development of new composite cement based on waste rocks from djebel onk phosphate deposit (tebessaalgeria), naukovyi visnyk natsionalnoho hirnychoho universytetu. http://dx.doi.org/10.33271/nvngu/2020-2/107, (2020). [3]. mohammed belhadj ah., mahi a., kazi aouel mz., derbal r.,abdelhadi h., valorisation du déchet de marbre et de la pouzzolane naturelle dans les mortiers (valorization of waste marble and natural pozzolan in mortars), j. mater. environ. sci. 7 : 429-437, (2016) [4]. bordja w., el boudour eh., mouiya m., sbi s., daafi y., tamraoui y., alami j., phosphate waste rocks recycling in ceramic wall tiles : technical performance,ceramics international , 48 , https://doi.org/10.1016/j.ceramint.2022.06.272, (2022) [5]. aubertin m., bussiere b., bernier l., environnement et gestion des rejets miniers, cd rom, les presses internationales de polytechnique, (2002) [6]. usgs, mineral commodity summaries, accessed at http://mineralshttps://www.usgs.gov/centers/nmic/ mineral-commodity-summaries (3 september), (2020) [7]. tahchi b., les ressources de l’algérie outre terre, 47 : 9-11, (2016) [8]. republique algerienne democratique et populaire, ministere de l’amenagement du territoire et de l’environnement, plan national d’actions pour l’environnement et le développement durable (pnae-dd), (2002) [9]. bellenfant g ., guezennec ag., bodenan f., d’hugues p. , cassard d.,combining environmental management and metal recovery?, proceedings of the eighth international seminar on mine closure,australian centre for geomechanics, cornwall,571-82 , https://doi.org/10.36487/acg_rep/1352_48_belle nfant ,(2013) [10]. das r., choudhury i., waste management in mining industry ,indian j. sci. res. 4:139-142, (2013) [11]. zengxiang l., meifeng cai. , disposal methods on solid wastes from mines in transition from open-pit to underground mining, 7th international conference on waste management and technology, 16:715-721,(2012) [12]. cisse l., mrabet t., world phosphate production: overview and prospects, phosphorus research bulletin,15: 21-25, (2004) [13]. kauwenbergh sj ., stewart m., mikkelsen r., world reserves of phosphate rocka dynamic and unfolding story ,better crops, 97,(2013) [14]. carvalho fp, mining industry and sustainable development: time for change, food and energy security,6(2) :61–77,(2017) https://doi.org/10.1016/j.ceramint.2022.06.272 http://mineralshttps/www.usgs.gov/centers/nmic/mineral-commodity-summaries http://mineralshttps/www.usgs.gov/centers/nmic/mineral-commodity-summaries https://doi.org/10.36487/acg_rep/1352_48_bellenfant https://doi.org/10.36487/acg_rep/1352_48_bellenfant food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4 – 2022 chafia merah, characterization of waste rock from the phosphate mining field of bled el hadba , algeria in an environmental context, food and environment safety, volume xxi, issue 4 – 2022, pag. 303 – 312 312 [15]. szilas c., the tanzanian minjingu phosphate rock. possibilities and limitations for direct application,center for skov, landskab og planlægning/københavns universitet, 1‒187,(2002) [16]. heckenmüller m., narita d., klepper g., global availability of phosphorus and its implications for global food supply: an economic overview, kiel working paper ,1897,(2014) [17]. gebrehiwet r ., xiaohua d., zhonghua l., bob s., xiaonong h., huijuan b., dan y., hao w., ji l., yinghai l., gang x., kai w.,shijin x , environmental impact of phosphate mining and beneficiation: review, international journal of hydrology, 2: 424‒431, doi: 10.15406/ijh.2018.02.00106,(2018) [18]. cieslinski s., et al, travaux de prospection et d’évaluation des phosphates de la région de bir el ater, erem : 3–10, 14–26, 39–44, 62, 63, 73–84, unpublished, (1985), (1987) [19]. prian gp., cortiel p, etude de développement du gisement de phosphate de djebel onk (algérie), rapport d’expertise géologique, b.r.g.m. france :11–29, 133–149, 169–173, unpublished,(1993) [20]. dassamiour m., mezghache h., elouadi b., the use of three physicochemical methods in the study of the organic matter associated with the sedimentary phosphorites in djebel onk basin, algeria, arabian journal of geosciences ,6, (2011) [21]. thomas ph., sur les gisements de phosphates de chaux d’algérie, c. r. acad. sci .paris, 106 : 379 -382, (1988) [22]. dussert md., les gisements algériens de phosphate de chaux, annales des mines, dunod edition, paris, 288-290, (1924) [23]. cayeux l., phosphates de chaux sédimentaire de france (france métropolitaine et d’outre –mer), etude des gites minéraux de la france, imprimerie nationale, paris, trois tomes, tome2, algérie-tunisie, (1939), (1941), (1950) [24]. ranchin g., les phosphates de chaux sédimentaires de la région de djebel onk, etude géologique et minière de djemi-jema(algérie), serm paris, 2,(1963) [25]. dmt consulting gmbh allemagne., rapport du lot 3 bled el hadba et djebel onk sud, n°14/somiphos/dmt, algérie (2017) [26]. taylor hk, mine valuation and feasibility studies, in mineral industry costs, northwest mining assoc , spokane wa, usa, 1– 17,(1977) [27]. orgm., rapport final exploration des phosphates du gisement de bled el hadba, (2015) [28]. kossi ba., kouma n., seni t., ampah kcj., yaovi ek., ezoba n., caractérisation physique de sables silteux au togo, afrique science, 10 :53 – 69, (2014) [29]. hichour s., rachid e., ejjaouani lhoucine., caractérisation de sables fins marins de la région de casablanca (maroc) en tant que granulats pour béton, xiième journées nationales génie côtier – génie civil, cherbourg ,1009-1015, doi.org/10.5150/jngcgc.2012.111-h, (2012) https://doi.org/10.15406/ijh.2018.02.00106 1. introduction problems related to mining wastes 2. materiel and methods 2.1 case study: description of the mining field 2.3 description of the samples 3. results and discussion 4. conclusion 5. references doi: https://doi.org/10.4316/fens.2023.011 109 journal homepage: www.fia.usv.ro/fiajournal journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 2 2023, pag. 109 121 functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits kouadio alfred goude 1 , *kouassi martial-didier adingra 2 , koffi honoré kouame 3 , soguimondenin soro 1 , eugène jean parfait kouadio 1 1laboratoire de biocatalyse et des bio-procédés, université nangui abrogoua, abidjan, côte d’ivoire, 2laboratoire de biochimie alimentaire et de transformation des produits tropicaux, université nangui abrogoua, abidjan, côte d’ivoire, maxadingra@gmail.com, 3departement des sciences et techniques, université alassane ouattara, bouaké, côte d’ivoire *corresponding author received 16th january 2023, accepted 28th june 2023 abstract: this study aims to examine the effects of fermentation on the functional properties of placenta powder for their use in the food industry. cocoa placenta from freshly harvested pods was used for this study. a part was fermented, dried, crushed and sifted to obtain fermented cocoa placenta powder. the other part was not fermented but underwent the same steps to have an unfermented cocoa placenta powder. standard and referenced methods have made it possible to measure certain functional properties of these cocoa placenta powders. thus, the results showed that the fermentation increased the water absorption capacity (514.65%), the packed density (10.37g/ml), the clarity of the dough (2.13%) compared to to unfermented placenta powder which recorded rates of 496.50% for water absorption capacity, 9.34 g/ml for packed density and 1.86% for dough clarity. however, the solubility index of fermented cocoa placenta powder (15.45%) is lower than that of fermented powder (29.55%). these powders are able to easily disperse in water, giving a homogeneous solution. moreover, the wettability value of unfermented cocoa placenta powder (4.09s) is higher than that of fermented powder (2.09s). fermented cocoa placenta powder dissolves in water much faster than unfermented one. in addition, the oil absorption capacity (oac) was higher in unfermented cocoa placenta powder with unrefined palm oil (270%). the functional properties of these flours have shown their importance for each measured parameter and can be useful in food technology processes for the formulation of foods, particularly in biscuits and bread making. keywords: fermentation, functional caracteristics, cocoa, food industry, drying. 1. introduction cocoa (theobroma cacao) is highly prized to produce chocolate from its beans. but the other parts of this plant, which are the empty pods and the placenta, are rejected in nature, as are the by-products of the fruits after using the edible parts (pulps) and almonds [1, 2, 3]. thus, in most cocoa plantations in africa and particularly in côte d'ivoire, the placenta of the cocoa, which is considered waste, is rejected into nature, after the cocoa is shelled to extract the beans. however, fresh beans represent only 20% of the mass of the pod and the other 80% are made up of cocoa waste such as empty cocoa pods, the juice from the mucilage and the cocoa placenta [4, 5]. these by-products of the transformation of cocoa into market beans have been the subject of interesting valorization (manufacture of fertilizers, liqueurs, wines, marmalades, etc.) except for the cocoa placenta which has been little valorized [6, 7]. however, some studies have shown that empty pods (cocoa fruit skin) and cocoa http://www.fia.usv.ro/fiajournal mailto:maxadingra@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 110 placenta are rich in nutrients and antioxidants that could be useful in human and animal nutrition [8, 9, 10, 11]. cocoa fruit skin and cacoa placenta contains theobromine alkaloid (3,7 dimethylamine) which is a limiting factor in the use of cocoa waste as animal feed. to increase the nutritional value and reduce theobromine content the usual use of fermentation. fermentation is one of the technologies to increase the nutritional value of high fiber feed. fermentation can hydrolyze protein, fat, cellulose, lignin, and other polysaccharides, so the fermented material will have higher digestibility, fermentation will increase total digestible nutrient [12]. this work aims to contribute to the valorization of the cocoa placenta through its use in the food industry, particularly in biscuits. biscuits are food products generally made from a dough made from wheat flour, sweeteners, fat, water, and many other additives [13, 14, 15]. the wheat flour dough used in the manufacture of biscuits could be partly replaced by cocoa placenta powder dough easily available and accessible in the cocoa producing regions of côte d'ivoire. indeed, it is a question of proceeding to the formulation of foods enriched in nutrients by incorporating cocoa placenta powder for the manufacture of biscuits. however, several factors can influence the rheological characteristics of the dough and the quality of the biscuits. these include the ingredients used, the conditions of kneading, rest and molding after kneading, cooking and cooling [13, 14, 15, 16]. thus, after baking, the biscuits are able to retain their rheological, organoleptic and commercial qualities for a given time depending on their composition. normally, wheat flour is the main ingredient in cookies. other flours such as cassava, sweet potato, maize, rye, plantain, etc. can be added to this ingredient [14, 17]. the technological goal of this action is to obtain composite flours to reduce production costs and also to create new products with innovative organoleptic qualities [17]. for this study on the formulation of biscuits enriched with cocoa placenta, two types of cocoa placenta powder were used. indeed, according to gbogbri [18], fermentation is one of the important steps involved in the transformation of cocoa beans. this step is essential in determining the quality of the aroma of the cocoa. much of the world's cocoa supply undergoes fermentation, but significant quantities of cocoa are intentionally unfermented and immediately dried. these beans (fermented and unfermented) are commonly available commercially. thus, one batch of cocoa placenta powder that has undergone fermentation and another batch of unfermented cocoa placenta powder will be used for this study. the functional properties of fermented and unfermented cocoa placenta powders were measured to determine which type of cocoa placenta powder would best suit the biscuit formulation. 2. material and methods 2.1. material the plant material of this study is the cocoa placenta, which is extracted from cocoa pods at physiological maturity, freshly harvested from cocoa plantations in the center-west of côte d'ivoire. 2.2. methods 2.2.1 production method of cocoa placenta powders the cocoa placenta is subdivided into two batches. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 111 batch 1 consists of cocoa placenta fermented for 3 days with the beans using traditional fermentation methods, dried in an oven for 3 to 5 days and reduced to fermented cocoa placenta powder. the second batch consists of fresh unfermented cocoa placenta, dried in the oven for 3 to 5 days and reduced to powdered unfermented cocoa placenta. 2.2.2. methods for determining the functional properties of flours. water absorption capacity and water solubility index the water absorption capacity (wac) and the water solubility index (wsi) of fermented cocoa placenta and unfermented cocoa placenta flours were determined by the method of adebowale et al. [19] and phillips et al. [20] respectively. two grams of fermented or unfermented placenta flour were separately dissolved in 50 ml of distilled water contained in centrifuge tubes. after stirring for 30 min with a magnetic stirrer, the solutions were kept in a water bath at 37°c for 30 min. the resulting mixtures were centrifuged at 5000 rpm for 15 minutes and the pellets were weighed and then dried at 105°c to constant mass. the water absorption capacity was calculated by the following formula 1: (1) wac: water absorption capacity, m1: mass of dried pellet, m2: mass of the wet pellet. water solubility index was calculated by the following formula 2: (2) wsi: water solubility index, m0: mass of the flour, m1: mass of dried pellet. wettability the wettability of fermented cocoa placenta and unfermented cocoa placenta flours was determined according to the technique of onwuka [21]. one gram of flour was placed in a 25 ml graduated cylinder with a diameter of 1 cm. next, a finger was placed over the opening of the test tube to avoid spilling the sample. the finger used to close the test tube was placed at a height of 10 cm from the surface of a 600 ml beaker containing 500 ml of distilled water. the finger was removed and the sample was poured into the beaker. wettability was determined as the time required for the sample to become completely wet. bulk density, packed density and porosity the bulk density (bd) of fermented cocoa placenta and unfermented cocoa placenta flours was determined according to the method of narayana & narasinga [22]. as for the packed density (pd), it was determined using the modified technique of oladele and aina [23]. fifty grams of flour were placed in a 100 ml graduated cylinder. the initial volume of this sample was noted after leveling the surface with a spatula. then, the test tube was gently tapped on the bench until a constant volume was obtained. bulk density, tapped density and porosity were calculated according to the following mathematical relationships : (3) db: bulk density, ms: sample mass, v0: volume of sample noted after levelling. (4) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 112 dp: packed density, ms: sample mass, vt: volume of sample noted after tapping. (5) p: porosity, vo: volume of sample noted after good levelling, vt: volume of sample noted after tapping. foaming capacity the foaming capacity (fc) of fermented cocoa placenta and unfermented cocoa placenta flours was determined according to the method of coffman and garcia [24]. three grams of sample were transferred into a 50 ml graduated cylinder previously dried in an oven at 50°c. the flours were leveled and 30 ml of distilled water was added to the sample to facilitate the dispersion of the flour in the test tube. this volume was also noted (volume before homogenization); then the test tube was shaken vigorously by hand and the new volume was read on the test tube (volume after homogenization). the volume of the foam obtained was calculated by taking the difference between the volume after homogenization and the volume before homogenization. the sample was left to rest on the bench until the foam collapsed and at each time interval (every 10 min) the volume of the foam was determined. the foaming capacity (fc) of the mixed flours was calculated from the following formula 6 : (6) dough clarity the clarities of the doughs of fermented cocoa placenta and unfermented cocoa placenta flours were measured according to the method of craig et al. [25]. a mass of 0.2 g of flours of cocoa placenta was dissolved in 20 ml of distilled water contained in a centrifuge tube. the mixture was homogenized with a vortex, then heated in boiling water at 100°c for 30 min. during this heating, the mixture was homogenized every 5 min. after this heat treatment, the mixture was cooled on the bench for 10 min. the clarity of the sample paste was determined by measuring the transmittance at 650 nm on a spectrophotometer (pg instruments, england) against distilled water. oil absorption capacity oil absorption capacity (oac) of fermented cocoa placenta and unfermented cocoa placenta flours was tested using four different oils (refined palm oil, olive oil, sunflower oil and unrefined palm oil). the oil absorption capacity was determined according to the method of sosulski [26]. one gram of the placenta flour was dispersed in 10 ml of each oil. after stirring for 30 min using a magnetic stirrer, the mixture was centrifuged at 4500 rpm at 4°c for 10 min and the supernatant was collected and the pellet was recovered and weighed on a satorius precision balance. oac was calculated by the following formula: (7) oac: oil absorption capacity, mo: flour mass, m1: mass of pellet. hydrophilic lipophilic ratio the hydrophilic-lipophilic ratio (hlr), as defined by njintang et al. [27], was calculated as the ratio of water absorption capacity to oil absorption capacity. this ratio makes it possible to evaluate the comparative affinity of flours for water and oil. (8) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 113 hlr: hydrophilic-lipophilic ratio, wac: water absorption capacity, oac: oil absorption capacity dispersibility the dispersibility (d) of fermented cocoa placenta and unfermented cocoa placenta flours was determined according to the method described by mora-escobedo et al. [28]. 10 ml of distilled water were added to 1 g of flour contained in a graduated cylinder. the mixture was stirred thoroughly by hand for 2 min. flour dispersibility was defined as the difference between the total volume of particles just after manual stirring and the volume of deposited particles recorded at time t (min). (9) d: dispersibility, v0: volume of particles just after manual stirring, vt: volume of deposited particles recorded at time t (min). statistical analysis all experimental data obtained were subjected to analysis of variance (anova) procedure of spss version 15.0 (spss inc., 2006). the difference between mean values was determined by the least significant different test. significance was accepted at the 5% probability level. 3. results and discussion the results of some functional properties of fermented and unfermented cocoa placenta powder are shown in table 1. the wettability of unfermented cocoa placenta powder (4.09 s) was almost twice that of placenta from fermented cocoa (2.09 s). as for the apparent density, it is substantially equal for fermented and unfermented cocoa placenta powder with respective values of 0.69 and 0.64 g/ml. regarding the packed density, the clarity of the paste and the water absorption capacity, the values of the fermented cocoa placenta powder (10.37g/ml, 2.13%t, 514.65%) are higher than that of unfermented cocoa placenta (9.34g/ml, 1.86%t, 496.50%). the foaming capacity, porosity, and water solubility index of unfermented cocoa placenta powder with respective values of 11.62%, 26.56% and 29.55% showed higher values than fermented placenta powder (8.64% for foaming ability, 20.87% for porosity and 15.45% for wsi). statistical analysis showed that there is a significant difference between the values of wettability, tapped density, foaming ability, dispersibility, paste clarity, wac, porosity and wsi of powder from fermented cocoa placenta and that of placenta from unfermented cocoa at the 5% level. values are means ± standard deviation. means not sharing a similar letter in a row are significantly different (p ≤ 0.05) according to duncan's test. wac = water absorption capacity; wsi = water solubility index. the oil absorption capacity (oac) results reported in table 2 showed that the highest oac values were obtained with the unfermented cocoa placenta powders for each oil compared with those of the cocoa placenta fermented. oac was higher in unfermented cocoa placenta powder with unrefined palm oil (270%). the oac of unfermented cocoa placenta powder with olive oil (206.67%) is twice as high as that of fermented cocoa placenta (106.67%). on the other hand, the oac of cocoa placenta powder with refined palm oil (176.66%) is closer than that of fermented cocoa placenta (156.67%). it is the same for the oac of placenta powder of unfermented cocoa and fermented with food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 114 refined sunflower oil with respective values of 153.33% and 140.0%. table 1. some functional properties of fermented and unfermented cocoa placenta powders table 2. oil absorption capacity (oac) of fermented and unfermented cocoa placenta flours values are means ± standard deviation. means not sharing a similar letter in a row are significantly different (p ≤ 0.05), according to duncan's test. table 3 presents the results of the hydrophilic-to-lipophilic ratios (hlr) of the placenta powders of fermented or nonfermented cocoa as a function of the oils used. the results show that powders from fermented cocoa placenta have higher hlr than those from unfermented placenta for all oils used in this work. fermented cocoa placenta powder has twice the hlr value (5.11) than unfermented cocoa placenta powder (2.51) in the case of refined olive oil. table 3. hydrophilic to lipophilic ratio (hlr) of cocoa placenta flours values are means ± standard deviation. means not sharing a similar letter in a row are significantly different (p ≤ 0.05), according to duncan's test. parameter cocoa placenta powder unfermented fermented wac (%) 496.50±12.37a 514.65±43.58b wsi (%) 29.55±1.02b 15.45±4.73a wettability (s) 4.09±0.03b 2.09±0.03a bulk density (g/ml) 0.69±0.00a 0.64±0.00a packed density (g/ml) 9.34±0.89a 10.37±0.54b foaming capacity (%) 11.62±2.96b 8.64±2.08a dough clarity (%) 1.86±0.06a 2.13±0.04b porosity (%) 26.56±0.69b 20.87±0.42a oils used oil absorption capacity of cocoa placenta flours (%) unfermented fermented refined palm oil 176.66±6.55b 156.67±0.77a refined sunflower oil 153.33±0.77b 140.00±5.00a refined olive oil 206.67±15.27b 106.67±10.27a unrefined palm oil 270.00±5.00b 193.33±0.77a oils used hydrophilic to lipophilic ratio of flours from cocoa placenta unfermented fermented refined palm oil 2.82± 0.18a 3.28± 0.20b refined sunflower oil 3.24±0.20a 3.70± 0.54b refined olive oil 2.51± 0.38a 5.11± 1.07b unrefined palm oil 1.84± 0.02a 2.66± 0.24b food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 115 unfermented cocoa placenta flour disperses faster in water than fermented cocoa placenta flour. at a time, t = 10 minutes, the cocoa placenta flour has reached its maximum dispersibility (50%) while the fermented cocoa placenta flour continues to disperse (45% at t = 10 minutes) to stabilize at 46.66% at t=15 minutes. fig.1. dispersibility of fermented and unfermented cocoa placenta flours 4. discussion in order to evaluate the behavior of fermented or non-fermented cocoa placenta powders when making biscuits, the functional properties of these were determined. the water absorption capacity value of fermented cocoa placenta powder is higher than that of unfermented placenta powder. fermentation appears to increase the hygroscopic power of cocoa placenta powder. indeed, the water absorption capacity depends on the affinity of flour with water. also, water absorption capacity is an index of the maximum quantity of water that a food product would absorb and maintain. it is an essential parameter for certain product characteristics such as product wettability and starch retrogradation [29]. fermentation is a transformation process that reduces antinutritional compounds such as tannins, phytates that chelate minerals, proteins. fermentation thus favored their bioavailability by destruction of membrane cells and enzymatic activity [30]. fermentation therefore releases proteins, which are therefore more apt to absorb water. the high-water absorption observed in fermented cocoa placenta powder could be due to the presence of the amino group of amino acids present in the proteins contained in this fermented placenta powder. moreover, it could be due to the hydroxyl groups of the carbohydrates contained in the placenta powder of cocoa [31]. the work carried out by goude et al. [11] showed that the protein content of cocoa fermented placenta (8.4%) was higher than that of unfermented placenta (5.12%). the high wac value of the powders of cocoa placenta would be more related to their protein content than to their lipid content. this is because the availability of protein functional groups in flours is governed by the water absorption capacity which is an important property of flours used in pastry making [32]. afoakwa [33] indicates that proteins are mainly responsible for most of the water absorption. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 116 regarding the water solubility index, the fermentation caused a significant decrease in this index. indeed, from 29.55% in fresh cocoa placenta powder, this value decrease to 15.45% in fermented cocoa placenta powder. this would mean that powders or flours that have a low solubility index have an affinity with water. these powders or flours are able to disperse easily in water, giving a homogeneous solution. these results are lower than those obtained by karim et al. [34] during their work on cocoa bean powder. they obtained values between 34.39 and 40.83% for the water solubility index of the beans. also, the water solubility index obtained in this study are lower than those obtained by diallo et al. [35] for seed flours of voandzou cultivars grown in côte d'ivoire (43-46%). according to mbofung et al. [36], the water solubility index reflects the extent of starch degradation. proteins, sugars and lipids play an important role in this modification of the functional properties of cocoa placenta powder. the water solubility index plays an important role in the selection of flours for use in the food industry as thickeners. the wettability value of unfermented cocoa placenta powder is higher than that of fermented powder. fermented cocoa placenta powder dissolves in water much faster than unfermented one. indeed, the powder from fermented placenta would have smaller, lighter and more regular surfaces than those of unfermented cocoa powder. in fact, wettability describes the ability of flour or powder particles to absorb water on their surface, thus initiating reconstitution. wettability is the penetration of the liquid into the porous flour particles system by capillarity [37]. the effectiveness of this parameter is linked to the fact that food flours or powders from drying processes are normally reconstituted for consumption. thus, wettability can influence the overall reconstitution characteristics of cocoa powders. a higher wettability value obtained for powder from fresh (unfermented) cocoa is an indication of the difficulties of using this powder for food formulations, including rehydration, as it will float longer on the surface of cold water [38]. the low wettability recorded for fermented cocoa placenta powder could suggest its use in food formulations, particularly in the biscuit industry. as regards the bulk density, it is 0.69 g/ml for fresh cocoa placenta powder and 0.64 g/ml for fermented cocoa placenta powder. these values are substantially equal to the cocoa placenta powders studied. this would mean that fermentation did not significantly influence the bulk density of cocoa placenta powders. the values of bulk densities of cocoa flour grown in lôh-djiboua and indenie-djuablin regions (côte d'ivoire) (0.59 to 0.63 g/ml) are approximately equal to that of the placenta powder of fermented and non-fermented cocoa (0.64 ±0.0-0.69 g/ml) [34]. low bulk densities obtained for cocoa placenta powders are suitable for infant food formulation [39, 40]. they are important in the feed separation process such as sedimentation, centrifugation [41]. the bulk density is an important parameter for determining the easy ability of packaging and transportation of particulate or powdery foods [21, 42]. according to asiedu [43], the bulk density of cocoa placenta powders is affected by their moisture content. as for the packed density, it is higher for fermented cocoa placenta powder. unfermented cocoa placenta powder is said to be lighter than fermented cocoa placenta powder. the fermentation process increased significantly the packed loose density. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 117 the increase of packed density during fermentation is to be related to processing efficiency, particularly the particles size. certainly, small particle sizes may lead to high packed densities because of better particles disposition. in the other hand, large particles size flour could lead to low packed densities with high porosity [44]. the density of flours is important in determining the packaging requirement and material handling [45]. regarding porosity, the results showed that unfermented cocoa placenta powder (26.56%) is more porous than fermented cocoa placenta powder (20.87%). the porosity of a flour determines the ability of a flour to be easily packaged. this parameter allows you to have a clear idea about the transport of a large quantity of food. nutritionally, high porosity promotes the digestibility of food products, especially in children due to their immature digestive system [46]. porosities higher than 25% may be useful in infant food formulation. unfermented cocoa placenta powder has good porosity compared to fermented cocoa placenta powder. flour porosity is an important factor in the food industry. the foaming capacity of unfermented cocoa placenta powder is 11.62%. it is greater than the foaming capacity of fermented cocoa placenta which is 8.64%. according to sathe et al. [47], high foam capacity is related to high protein content in flours. this decrease in the foaming capacity of fermented cocoa placenta is related to fermentation. indeed, during fermentation, macromolecular proteins are degraded into small peptides and free amino acids under the action of microorganisms [48]. fermentation followed by drying reduced the foaming ability of cocoa placenta powder. indeed, during transformation processes unit operations such as fermentation, drying and cooking have an impact on the protein level and therefore on the foaming capacity of the flour or powder. this result has already been demonstrated by soro et al. [49] during the study on flours from fresh and boiled wild yam dioscorea praehensilis tubers. the latter obtained raw flour foam capacity of 36.92 ± 1.01% and after 40 min of cooking this value decreased to 11.79%. paste clarity is another important property of flour that governs which applications different flours may have for food processing. dough clarity of unfermented cocoa powder flour (1.86%) is lower than that of fermented cocoa placenta powder dough (2.13%). fermentation would have allowed a significant increase in the clarity of cocoa placenta paste. indeed, unfermented (fresh) cocoa placenta powder has a darker purple color compared to fermented placenta powder which is less colored. this would mean that the less colored fermented cocoa placenta powder has a better light transmittance than that of the (more colored) unfermented cocoa placenta powder. this study suggested that this situation could be the main reason for lower paste clarity of unfermented cocoa placenta powder compared to flours from fermented cocoa placenta powder. but the intrinsic structure of starch should not be neglected too. cross-linking of starch has been reported to reduce paste clarity in starch [50] and probably in entire flour. high paste clarity allows the application of flours and starches in confectionary and pie filling which require high clarity [51]. in this case, cooking may improve cocoa placenta flour technological potential in the food industry. when it comes to dispersibility, unfermented cocoa placenta powder disperses faster in water than fermented cocoa placenta flour. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 118 at a time t = 10 minutes, the unfermented cocoa placenta powder reached its maximum dispersibility (50%) while the fermented cocoa placenta powder stabilized at 46.66% at t = 15 minutes. dispersibility is a measure of the reconstitution of flour or starch in water, the higher the dispersibility the better the sample reconstitutes in water [52, 53] and gives a fine constituent during mixing [19]. the average value ranged from 46.66 to 50.0%. fermented cocoa placenta powder had the lowest average value while fresh cocoa placenta powder had the highest average value. the dispersibility of these two powder samples (fermented and unfermented) is low and this probably implies that the samples will tend to form lumps during preparation. according to compaore et al. [54], substances such as starch or potassium bi-carbonate (alkali) may be added to cocoa powders to prevent caking, neutralize the natural acids and astringents, with the purpose of improving its dispersibility. however, dispersibility observed for unfermented cocoa powder (fresh) hence that it will easily reconstitute to give a fine consistency to the dough during mixing compared to fermented cocoa placenta powder. the oil absorption capacity (oac) of cocoa placenta powder was tested using 4 different oils (refined palm oil, sunflower oil, olive oil and unrefined palm oil). the results showed that the highest oac values were obtained with unfermented cocoa placenta powders for each oil compared to fermented cocoa placenta powders. a significant difference at the 5% threshold of the oil absorption capacity in different powders was found. the fermentation of the cocoa placenta would lead to the reduction of the oac of the different types of powders produced. in general, the oac decreases with fermentation. thus, cocoa placenta flours could be considered products with a very high oac content. this characteristic is desired in food technology for the manufacture of preparations involving a mixture of oil such as in bakery products where oil is an important ingredient. the oil absorption capacity is high in cocoa placenta flours for the oils tested (refined palm oil, sunflower oil, olive oil and unrefined palm oil). the ability of fermented and unfermented cocoa placenta flours to bind oil makes them useful in food applications where optimal oil absorption is desired. these cocoa placenta powders would have potential functional applications in foods such as the production of pastries, sausages. according to suresh and samsher [55], the oil absorption capacity also makes these cocoa placenta flours suitable to facilitate flavor and mouthfeel enhancement when used in food preparation [56]. due to these properties, flours with good oac are used as a functional ingredient in foods such as sausages, whipped toppings, angel food cakes and sponges, chiffon desserts, etc. the values of the hydrophilic-lipophilic ratio obtained in this study are greater than 1. this information allows us to affirm that placenta powders of cocoa, fermented or not, have more affinity for water than for the various oils studied. this suggests that our powders should be used preferably for the formulation of products requiring high water absorption capacity. however, in refined oils (olive, sunflower, palm), fermented cocoa placenta powders have a higher affinity for these oils than unfermented (fresh) placenta powder. 5. conclusion fermentation has shown beneficial effects on the functional properties of cocoa placenta. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 119 the fermentation increased the water absorption capacity, the packed density as well as the clarity of the dough. however, the water solubility index, foaming capacity, porosity and oil absorption capacity were reduced with fermentation. these aspects involve the potential use of cocoa placenta powders (fermented and unfermented) as an ingredient in baby food formulations, cakes and bread products. finally, cocoa placenta flours (fermented and unfermented) could have multiple applications in food technology. 6. references [1]. adingra k. m.-d., konan k. h., kouadio e. j. p. & tano k. phytochemical properties and proximate composition of papaya (carica papaya l. var solo 8) peels. turkish journal of agriculture food science and technology, 5(6): 676-680, (2017). [2]. barral-martinez m., fragacorral m., garcia-perez p., simalgandara j., prieto m. a. almond byproducts: valorization for sustainability and competitiveness of the industry. foods, 10(8): 122, (2021). [3]. kossonou y. k., adingra k. m.-d., yao a. l. & tano k. biochemical characterization of mango kernel (mangifera indica) grown in côte d’ivoire and formulation of butter for cosmetic use. biotechnology journal international, 26(5): 33-44, (2022). [4]. erhart d. & poulain m. de l’agro écologie à la transformation locale : la quête de l’autonomie au service des producteurs, une filière équitable en bolivie, artisans du monde pour un commerce équitable. 1-60, (2014). [5]. kokou e. a. & ngo-samnick e. l. production et transformation du cacao, collection pro-agro, cta et isf 2014. pp. 44, (2014). [6]. cnra. centre national de recherche agronomique, côte d’ivoire 2013, 2012, pp. 52, (2012). www.cnra.ci/ (pages consultées le 21/11/2022) [7]. cirad (2015). cacao chez le porc en croissance finition, 2015, 15 p, http://pigtrop.cirad.fr (pages consultéesle 9/12/2022 [8]. laconi e. b. & jayanegara a. improving nutritional quality of cocoa pod (theobroma cacao) through chemical and biological treatments for ruminant feeding: in vitro and in vivo evaluation. asian australasian journal animal sciences, 1-8, (2014). [9]. quimbita f., rodriguez p. & edwin v. uso del exudado y placenta del cacao parala obtención de subproductos. departamento de ciencia de alimentos y biotecnología escuela politécnica nacional. revista tecnológica espol – rte, 26 (1): 8-15, (2013). [10]. anvoh k. y. b., bi a. z., gnakri d. production and characterization of juice from mucilage of cocoa beans and its transformation into marmalade. pakistan journal of nutrition, 8, 129– 133, (2009). [11]. goudé k. a., adingra k. m.-d., gbotognon o. j. & kouadio e. j. p. biochemical characterization, nutritional and antioxidant potentials of cocoa placenta (theobroma cacao l.). annals. food science and technology, 20(3): 603-613, (2019). [12]. anggorodi. ilmu makanan ternak umum. jakarta: pt gramedia, (1979). [13]. charun e. p. j & morel m-h. quelles caractéristiques pour une farine biscuitière ? influence de la dureté et de la composition biochimique des farines sur leur aptitude buscuitière. communication scientifique et technologiques. industries des céréales n° 125. novembre / décembre 2001, pp.2-16, (2001). [14]. badje s. d., soro d., niamketchi g. l. & koffi e. k. étude des comportements chimiques, fonctionnels et rhéologiques de mélanges de farines de blé (triticum aestivum), amande de cajou (anacardium occidentale l) et de banane plantain (musa paradisiaca). afrique science, 15(6): 143–155, (2019). [15]. soro s., koffi j. d. k., konan h. k. & kouadio e. j-p. appropriate ratio of wild yam (d. praehensilis) flour to add to wheat flour for breadmaking process. european journal of biology and biotechnology, 1(2): 1-11, (2020). [16]. makhlouf h. propriétés physicochimiques et rhéologiques de la farine et de l’amidon de taro (colocasia esculenta l. schott) variété sosso du tchad en fonction de la maturité et du mode de séchage. alimentation et nutrition. université de lorraine, (2018). [17]. vodouhe-egueh s., alidou c., aboudou k. & soumanou m. m. formulation de biscuits à base de farine de blé enrichie à la farine de patate douce à chair orange. afrique science, 13(6): 405-416, (2017). [18]. gbogbri g.-f. impact de la fermentation sur les propriétés antioxydantes, anti inflammatoires et immunomodulatrices du cacao. thèse en biochimie et physico-chimie alimentaires, université de montpellier, pp. 211, (2019). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 120 [19]. adebowale a. a., adegoke m. t, sanni s. a., adegunwa m. o. & fetuga g. o. functional properties and biscuit making potentials of sorghum-wheat flour composite. american journal of food technology, 7: 372-379, (2012). [20]. phillips r. d., chinnan m. s., branch a. l., miller j. & mcwatters k. h. effects of pre-treatment on functional and nutritional properties of cowpea meal. journal food science, 3: 805–809, (1988). [21]. onwuka g. i. food analysis and instrumentation: theory and practice. naphthali prints lagos, nigeria. (2005). [22]. narayana k. & narasimga r. n. m. s. functional properties of raw and heat processed winged bean flour. journal of food science, 47: 1534-1538, (1982). [23]. oladele a. k. & aina j. o. chemical composition and functional properties of flour from two varieties of tigernut (cyperus esculentus). african journal of biotechnology, 6(21): 24732476, (2007). [24]. coffman c. w. & garcia v. v. functional properties and amino acid content of protein isolate from mung bean flour. journal of food technology, 12: 473-484, (1977). [25]. craig s. a. s., maningat c. c., seib p. a., hoseney r. c. starch paste clarity. cereal chemistry, 66: 173-182, (1989). [26]. sosulski f. w. the centrifuge method for determining flour absorption in hard red spring wheat. cereal chemistry, 3 : 344-350, (1962). [27]. njintang n. y., mbofung c. m., waldron k. w. in vitro protein digestibility and physicochemical properties of dry red bean (phaseolus vulgaris) flour: effect of processing and incorporation of soybean and cowpea flour. journal of agricultural and food chemistry, 49(5): 246571, (2001) [28]. mora-escobedo r., roblesramirez m. c, ramon-gallegos e. & reza-aleman r. effect of protein hydrolysates from germinated soybeanon cancerous cells of the human cervix: an in vitro study. plant foods for human nutrition, 64: 271-278, (2009). [29]. siddiq m., rav r., harte j. b. & dolan k. d. physical and functional characteristics of selected dry bean (phaseolus vulgaricus (l.) flours. lwt-journal of food sciences technolology, 43: 232–237, (2010). [30]. fofona i., soro d., yeo m. a., koffi e. k. influence de la fermentation sur les caracteristiques physicochimiques et sensorielles de la farine composite à base de banane plantain et d’amande de cajou . european scientific journal, 13(30): 395-416, (2017). [31]. lawal o. s. & adewale k. o. effect of acelation and succinylation on solubility profile, water absorption capacity, oil absorption capacity and emulsifying properties of municina bean (mucuna prilens) protein concentrate. nahrung/food, 48(2)129-136, (2004). [32]. wolf w. j. soybean proteins: their functional, chemical physical. journal of agriculture and food chemistry, 18: 965-969, (1970). [33]. afoakwa j. c. water absorption capacity of legumes. b. sc thesis, departement of food science and technology, federal university of technology, owerri, nigeria. (1996). [34]. karim k. j. c., saki s. j., sea t. b., yoboue g. a. k. l. & soro y. r. functional characteristics of cocoa bean powder of the mercedes and theobroma cacao varieties from the lôh-djiboua and indenie-djuablin regions (côte d'ivoire). gsc biological and pharmaceutical sciences, 12(01): 62-71, (2020). [35]. diallo k. s., kone k. y., soro d., assidjo n. e & yao k. b. caractérisation biochimique et fonctionnelle des graines de sept cultivars de voandzou [vigna subterranea (l.) verdc. fabaceae] cultivés en côte d'ivoire. european scientific journal, 1(27): 288-304, (2015). [36]. mbofung c. m. f., aboubakar n. y. n., njintang y. n., abdou b. a. & balaam f. physicochemical and functional properties of six varieties of taro (colocasia esculenta l. schott) flour. journal food technology, 4: 135–146, (2006). [37]. barbosa-canovas g. v. & juliano p. physical and chemical properties of food powders, in: onwulata, c. (ed.), encapsulated and powdered foods. crc press, taylor & francis group, new york, (2005). [38]. nguyen d. q. etude comparative expérimentale des opérations d’atomisation et d’auto vaporisation: application à la gomme arabique et au soja. génie des procédés. thèse, université de la rochelle, (2014). [39]. abiodun o. a. & akinoso r. physical and functional properties of trifoliate yam flours as affected by harvesting periods and pre-treatment methods. journal of food processing & technology, 5(2): 1-5, (2014). [40]. jagannadham k., parimalavalli r., babu a. s. & rao j. s. a study on comparison between cereal (wheat) and non-cereal (chickpea) flour characteristics. international journal of current trends in research, 3(2): 70-76, (2014). [41]. lewis m. j. density and specific gravity of foods. in: physical properties of foods and food food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 2 – 2023 kouadio alfred goude, kouassi martial-didier adingra, koffi honoré kouame, soguimondenin soro, eugène jean parfait kouadio, functional properties of fermented and unfermented cocoa placenta powders (theobroma cacao l.) for the production of biscuits, food and environment safety, volume xxii, issue 2 – 2023, pag. 109 – 121 121 processing systems. ellis howard ltd, chichester, england, 53-57, (1987). [42]. oluwole o., akinwale t., adesioye t., odediran o., anuoluwatelemi j., ibidapo o., owolabi f., owolabi s. & kosoko s. some functional properties of flours from commonly consumed selected nigerian food crops. international research journal of agricultural and food sciences, 1(5): 92-98, (2016). [43]. asiedu j. j. processing tropical crops-a technological approach. macmillan education ltd, london and basingstoke, pp. 24-42, (1989). [44]. fagbemi t. n. effect of blanching and ripening on functional properties of plantain (musa aab) flour. plant food for human nutrition, 54: 261-269, (1999). [45]. ezeocha v. c. & ojimelukwe p. c. the impact of cooking on the proximate composition and anti-nutritional factors of water yam (dioscorea alata). journal of stored products and postharvest research, 3(13): 172–176, (2011). [46]. nelson-quartey f. c., amagloh f. k., oduro i. & ellis w. o. formulation of an infant food based on breadfruit (artocarpusaltilis) and breadnut artocarpuscamansi). actahorticulturae (ishs), 757: 212-224, (2008). [47]. sathe a. k., desphande s. s. & saluhke d. k. functional properties of lupin seed protein and protein concentrates. journal of food science, 42: 491-494, (1982). [48]. wang d., cheng f., wang y., han j., gao f., tian j., zhang k. & jin y. the changes occurring in proteins during processing and storage of fermented meat products and their regulation by lactic acid bacteria. foods, 11: 2427 (2022). https://doi.org/10.3390/ foods11162427. [49]. soro s., kouadio t. m., binate s, kouassi k. a., konan k. h. & kouadio e. j. p. functional properties of flours from fresh and boiled wild yam dioscorea praehensilis tubers. advances in research, 17(3): 1-13, (2018). [50]. kerr r. w. & cleveland f. c. orthophosphate esters of starch. us patent. 2. 884, 413, (1959). [51]. radley j. a. industrial uses of starch and its derivatives. in: radley ja (ed). applied science publishers, (1976). [52]. kulkarni k. d., kulkarni d. n., ingle u. m. sorghum maltbased weaning formulations: preparation, functional properties and nutritive value. food and nutrition bulletin, 13(4): 322–327, (1991). [53]. adebowale a. a., sanni l. o., onitilo m. o. chemical composition and pasting properties of tapioca grits from different cassava varieties and roasting methods. african journal of food science, 2: 77-82, (2008). [54]. compaore r. w., nikiema a. p., bassole n. h., savadogo a., hounhouigan d., mouecoucou j. & traore a. s. nutritional properties of enriched local complementary flours. advance journal of food science and technology, 3(1): 31-39, (2011). [55]. suresh c. & samsher s. assessment of functional properties of different flours. african journal of agricultural research, 8(38): 48494852, (2013). [56]. aremu m. o, olafe o. & akintayo e. t. compositional evaluation of cowpea and searlet runner bean varieties grows in nigeria. journal of food, agriculture and environment, 4: 3943, (2006). 1. introduction 4. discussion doi: https://doi.org/10.4316/fens.2022.033 345 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxi, issue 4 2022, pag. 345 353 heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria *udeme effiong jonah 1 , cecilia friday mendie 2 1department of zoology and environmental biology, michael okpara university of agriculture, umudike, abia state, nigeria.udemejonah@gmail.com 2 department of public health, national open university of nigeria, uyo study centre, akwa ibom state, nigeria. jmendie07@gmail.com *corresponding author received17thjuly 2022, accepted 28th december 2022 abstract: heavy metals in water pose detrimental consequence on health of the consumers and aquatic organisms. metallic pollutants in drinking water leads to dysfunction of organs and systems in the body with other associated health risk conditions. study on heavy metal content and health risk assessment of water from ikpe ikot nkon river was carried out between october 2020 and september 2021 (twelve months), to evaluate the suitability of the river water for human consumption. eight heavy metals were assessed in the water samples collected from three stations using atomic absorption spectrophotometer after digestion. the findings were compared with nigerian standard for drinking water quality. the mean concentration of some metals (mn, cr, ni, cd, fe and pb) exceeded the permissible limits due to anthropogenic activities and seasonal influences. the non-carcinogenic health risk assessment indicated that the chronic daily intake (cdi) and hazard quotient (hq) values for cr and fe exceeded oral toxicity reference dosage and threshold value of 1respectively. the hazard index (hi) was high in all the stations and above the threshold value (1). the results of some heavy metals (mn,cr,ni,cd,fe and pb), cdi and hq values for cr and fe, and the hi values calls for concern regarding their effects on human health, which could be detrimental to the people drinking from this river. based on the results, the water is not suitable for human consumption. keywords: heavy metals, health risk, human activities, pollution, river. 1. introduction the term heavy metal refers to any metallic element with high density and applies to the group of metals and metalloids with atomic density greater than 5g/cm3 [1, 2]. heavy metals in aquatic ecosystems have detrimental consequences on health of the consumers of water resource and aquatic organisms. human beings are exposed to heavy metals toxicity by consuming metallic contaminated animal and direct consumption of water containing metals in higher concentration [3]; which have been reported to be responsible forvarious biochemical disorders [4].some heavy metals are essential elements for healthy growth and development, but could result in detrimental health effect when the concentration consumed exceeded the recommended levels especially in drinking water [5]. study affirm that long exposure of metal like manganese have deleterious health effects on human being, ranging from kidney, liver and neurological disorder, hyperkeratosis and cardiovascular disease [6]. cadmium is associated with renal dysfunction and obstructive lung diseases, bone defects, osteoporosis and myocardic dysfunctions [4], while acute mailto:udemejonah@gmail.com mailto:jmendie07@gmail.com food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 346 exposure zinc lead to system dysfunctions which impaired growth in human. pollution of water with heavy metals should not be overlooked concern since it has sequences of health and environmental consequences [7]. many researchers reported the activities of human in industries, mining and agricultural sectors as the major contributors to heavy metal pollution in aquatic ecosystem [7, 8]. other sources of heavy metals in water bodies include leaching from metal contaminated soil, weathering of rocks and human activities (urbanization) [9, 10]. the intensity in the use of metal-based fertilizer in agricultural revolution has resulted in continued rise in the concentration of metal pollutants in fresh water bodies through surface runoff; leading to scarcity of good water for domestic uses [11]. the case is not different from ikpe ikot nkon river; it is subjected to agricultural activities on the banks and along with other anthropogenic activities that could add heavy metals into the water body. the water body served as the only source of water for irrigation and drinking for the nearby communities. hence, this study seeks to evaluate the concentration of some heavy metals and associated health risk of ikpe ikot nkon river vis-à-vis its suitability for human consumption. 2. materials and methods study area and sampling stations ikpe ikot nton river is located in ini local government area, northern part of akwa ibom state. geographically, the river lies between the latitude 5o 20’59.0’’n – 5o 22’42.4”n and longitude 7o 36’ 55.7’’e – 7o 49’27.5”e (figure 1).the region is characterized by tropical humid climate with distinct dry season (november march) and wet season(april october). the river is within low land area where runoffs from communities are high, and receives wastes from different anthropogenic sources. the sampling stations were chosen along the stretch of the river using the level and nature of human activities as a criterion for selection. station 1 (control station) was located upstream, close to ikot abia community; human activities in this station are minimal with only bathing and laundry and extraction of water for drinking purpose. station 2 was located (at the head bridge), about 2km downstream of station 1. the water in this station is also extracted for domestic uses and drinking purpose by the nearby communities. the observed human activities were farming on the left and right banks of the river, sand mining, landing and sales, lumbering, bathing, laundry. station 3 was located at (the head bridge of nkana), about 2km downstream of station 2. the observed activities were bridge construction, agricultural activities, intense fishing activities, and laundering. the inhabitants of the area and other nearby villages always extract water from this station for domestic uses, including drinking. samples collection and analyses water samples for the heavy metals evaluation were collected between october 2020 and september 2021 with 500ml polyethylene bottles and acidified with nitric acid (hno3) immediately after collection. the water samples were digested as described by [12, 13] using concentrated nitric acid and heavy metals concentration in each water sample were determined with atomic absorption spectrophotometer (unicam 939/959 model). all data were summarized in microsoft excel and subjected to statistically analysis using single factor anova while tukey pairwise posthoc was used to compare the means between the stations with significant difference set at p<0.05. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 347 human health risk assessment health risk assessment was carried out for non-carcinogenic using chronic daily intake (cdi), hazard quotient (hq) and hazard index (hi) as described by [14, 15].the chronic daily intake (cdi) of the heavy metals was calculated by the equation below: 𝐂𝐃𝐈 = 𝑪𝒘 × 𝑰𝑹 ×𝑬𝑭 × 𝑬𝑫 𝑩𝒘 × 𝑨𝑻 (1) where, cdi is the daily doses intake of heavy metal (mg/kg/day) to which consumers could be exposed to, cw is the concentration of heavy mental in the water sample (mg/l), ir is the ingestion rate (2l per day); ef is the exposure frequency (365 days / year), ed is the exposure duration (70 years); bw is the body weight of the exposed person (70.0kg) and at represent the averaging time in days (25,550 days). fig. 1: map of ini local government area showing the location of sampling stations hazard quotient (hq): the hazard quotient for non-carcinogenic risk was calculated using equation [16]: hq= 𝐶𝐷𝐼 𝑅𝐹𝐷 (2) where, cdi is the daily dose intake of heavy metals (mg/kg/day) and rfd represent oral reference dose of the contaminant (mg/kg/day). 1f hq value > 1, it indicates adverse non -carcinogenic effects, while value < 1 represents acceptable level. hazard index (hi): the hazard index of the heavy metals was determined by using equation below: hi = ∑ (𝐻𝑄)𝑖𝑛𝑖=1 (3) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 348 where, hi is the hazard index for the overall toxic risk and n is the total number of heavy metals considered. the noncarcinogenic adverse effect can be considered to be insignificant if hi <1. the hazard index is treated as the arithmetic sum of hq values [14]. 3. results and discussion heavy metal concentrations: heavy metals in aquatic ecosystems occurred naturally and through human induced activities in the environment [18]. some metals are essential for animals and human physiological activities, but could be detrimental when the concentration is higher or lower than the concentrations required by human and the recommended standard limits in drinking water [19].the range and mean values of the heavy metals are presented in table 1. table 1: the mean, range values of heavy metal and pollution index recorded from ikpe ikot nkon river (october 2020 and september 2021) heavy metals (mg/l) station 1 x ±s.e.m station 2 x ±s.e.m station 3 x ±s.e.m p value son (2015) manganese (mn) 0.13 ±0.08a (0.08 0.31) 0.24±0.02b (0.13 0.48) 0.16±0.10a (0.04 0.34) p<0.05 0.2 chromium (cr) 0.04±0.02a (0.01 – 0.14) 0.07±0.003b (0.01 0.16) 0.08±0.002b (0.02 – 0.12) p<0.05 0.05 nickel (ni) 0.04±0.01 (0.005 – 0.06) 0.04±0.01 (0.005 –0.08) 0.05±0.01 (0.002 –0.11) p>0.05 0.02 cadmium (cd) 0.01±0.00 (0.002 – 0.02) 0.01±0.00 (0.001 – 0.03) 0.01±0.00 (0.001– 0.04) p>0.05 0.003 copper (cu) 0.03±0.08a (0.001 – 0.08) 0.09±0.03b (0.02 – 0.16) 0.02±0.03a (0.001 – 0.18) p<0.05 1.0 iron (fe) 0.43±0.46a (0.36 – 1.00) 0.69±0.34b (0.33 – 1.67) 0.84±0.46c (0.28 – 1.14) p<0.05 0.3 lead (pb) 0.01±0.13 (0.00 – 0.10) 0.02±0.01 (0.00 – 0.46) 0.03±0.23 (0.006 – 0.16) p>0.05 0.01 zinc (zn) 0.45±0.05a (0.14-0.59) 1.17±0.03b 0.84 – 1.36 0.94±0.08c (0.28 – 1.01) p<0.05 3.0 x = mean, ±s.e.m = standard error of mean; means with different superscripts (a, b, c) in the same row indicate significantly difference at p <0.05. the mean values of copper (cu) and zinc (zn) were within the recommended limit set by son [17], while the others exceeded the acceptable limits. this is consistent with the findings of studies by [18] in river dilimi, jos north, plateau state, nigeria and [19] in ikwu river, umuahia, abia state, nigeria. the manganese (mn) ranged between 0.04 and 0.48 mg/l, with the highest mean value (0.24 mg/l) recorded in station 2 and the lowest in station 1 (0.13mg/l). station 2 was significantly (p < 0.05) higher than stations 1 and 3 and exceeded the standard limit of 0.2 mg/l set by son [17]. the mean values of mn in this study recorded exceeded the threshold (0.2 mg/l). the higher mean value recorded in station 2, suggests to intense anthropogenic activities ranging agricultural, sand mining, and laundering [19]. manganese (mn) is biological importance to living organisms including human during physiological process [23]. chromium (cr) values ranged from 0.01 0.16 mg/l, with the highest mean value (0.08 mg/l) recorded in station 3 and lowest (0.04 mg/l) in station 1. analysis of variance indicated that station 1 was significantly (p < 0.05) lower than stations 2 and 3.the higher value of cr observed in stations 2 and 3 suggests to influenced of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 349 anthropogenic activities and leaching from domestic wastes dumped near the stations. chromium (cr) is life-threatening if the ingested concentration is high. it is associated with anemia in human [20]. the mean values recorded are lower than the values reported by [10] and [21] and exceeded the values reported by [22] in benin river. statistical analysis showed significant differences in station 1 (p < 0.05). the mean values of nickel (ni) recorded were higher than 0.02 mg/l, the stipulated standard [17], with higher mean value in station 3(0.05 mg/l). similar values were reported [23] from obuburu river, kogi state. the observed values could be attributed to the sand mining activities in the stations. problems associated with ni toxicity include loss of hair in human, lung fibrosis, cardiovascular and kidney diseases if the concentration is high in drinking water [24, 25]. cadmium (cd) ranged between 0.001 and 0.04 mg/l. the same mean values (0.01 mg/l) were recorded across the stations. the mean values exceeded the stipulated limit of 0.003 mg/l [17]. however, [26] regarded cd as the most toxic metal in water even at low concentration. its concentrations in the drinking water should not be overlooked, as it associated with enormous health risk problems. the values could be attributed to the combined effects of human activities such agricultural, and intense sand mining activities in the stations. study [27] reported that the use of agrochemicals like pesticide, herbicide and fertilizer in agricultural activities near the water body is linked to the concentrations of cd in aquatic ecosystem. copper (cu) is an essential element for man, but pose adverse impact when the concentration is above the recommended limit of 1.0 [17]. acute exposure to drinking water contaminated with cu leads to alteration of brain function in human and kidney failure in young children. copper (cu) ranged from 0.001 to 0.18 mg/l, with the highest mean value (0.09 mg/l) recorded in station 2 and lowest (0.02 mg/l) in station 3. this is consistent with the findings of [23]in obuburu river, okene, kogi state, nigeria. the elevated value in station 2 could be linked to leaching from wastes dumped near the station and anthropogenic activities going on in the station. statistical analysis showed significant differences (p<0.05). iron (fe) ranged between 0.28 and 1.67 mg/l, with the highest mean value recorded in station 3 (0.84 mg/l) while lowest was recorded in station 1 (0.43 mg/l). the values recorded were the same with values reported by [28] in ossa river. most of the values exceeded 0.3 mg/l set by [17] in all the stations. high levels of fe in water samples may be due to the discharge of iron laden wastes and scraps into the water [13], and the fact that fe is more abundant in freshwater body than other metals, due to its high occurrence on earth [29, 30]. according to [31], fe in higher concentrations is associated with high risks of endocrine problems, cancer, heart diseases, arthritis, diabetes and liver disease. statistical analysis showed significant differences (p<0.05). lead (pb) is generally well-known to be a toxic element, having no biological importance to human being. according to [32] and [33], no concentration of pb in drinking water is considered safe for human consumption. the concentrations of pb exceeded 0.01 mg/l set by standard organization of nigeria [17]. the higher value in station 3 (0.03 mg/l) suggest to combine effects of anthropogenic activities going on in this station. high exposure to pb has been implicated in some learning disorders in children [34]. the levels of zinc recorded throughout the study were lower than acceptable limit (3.0 mg/l) for drinking water [17] and ranged food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 350 between 0.14 and 1.36 mg/l. the highest mean value (1.17 mg/l) was recorded in station 2. statistical analysis showed significant differences between the stations (p<0.05). study [35] reported that ingestion of zn contaminated water affect bone formation in children. with these, the concentration of zn in the water body should not be ignoring due to it antagonistic effects on the toxicity of other metals [35]. health risk assessment: chronic daily intake (cdi): thechronic daily intake (cdi) values recorded are presented in table 2. the values for cr and fe exceeded the oral toxicity reference dosage (rfd), while values for mn, ni, cd, pb and zn did not exceed oral toxicity reference dosage. manganese (mn) had cdi values ranging from 0.003 to 0.006 mg/kg/day, ni cdi ranged from 0.001 to 0.002 mg/kg/day, and cd was 0.0002 mg/kg /day across the stations while pb was 0.0002 to 0.008 mg/kg /day and zn ranged from 0.012 to 0.033 mg/kg /day. mn, ni, cd, pb and zn did not pose any health risk effects to the people using the river water for drinking, while cr and fe posed significant health risk to those exposed to drinking water from the river. chromium (cr) according to [36] and [37] is considered carcinogenic and genotoxic at higher concentrations. while [38] reported that kidney diseases, liver and tissue dysfunctions in human are as a result of long exposure to chromium in high concentration. however, the cdi values of fe corroborated with the values reported in related studies [15, 39]. iron in high concentrations is associated with venomous effect, leading to health risk and issues such as cancer, endocrine dysfunction, heart and liver diseases [31] table 2: chronic daily intake (cdi) and oral toxicity reference doses (rfd) for heavy metals from ikpe ikot nkon river (october 2020 september 2021) heavy metals (mg/l) *rfd (mg/kg/day) station 1 station 2 station 3 mn 0.14 0.003 0.006 0.004 cr 0.0003 0.001 0.002 0.002 ni 0.2 0.001 0.001 0.002 cd 0.0005 0.0002 0.0002 0.0002 fe 0.007 0.012 0.019 0.024 pb 0.0035 0.0002 0.0005 0.0008 zn 0.3 0.012 0.033 0.026 *source [14] hazard quotients (hq): the values recorded in station 1 were ranged from 0.005 to 3.34. in station 2, the hq values were between 0.005 and 6.67while station 3 was between 0.01 and 6.67(table 3).higher hq values were recorded for iron (fe) and chromium (cr) in all the stations that are greater than the threshold value (1.0).the hq values for mn, ni, cd, pb, and zn across the stations were lower than the threshold value (< 1.0). the results revealed that cr and fe are the major metals that contributed to the potential non-carcinogenic risk associated with drinking water from the river. this is attributed to geogenic source influenced by anthropogenic activities and surface runoffs. related studies reported high hq values for cr and fe [28, 29, 40]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 351 table 3: hazard quotients (hqs) and hazard index (hi) values for heavy metals from ikpe ikot nkon river (october 2020 -september 2021) heavy metals station 1 station 2 station3 mn 0.021 0.042 0.028 cr 3.34 6.67 6.67 ni 0.005 0.005 0.01 cd 0.4 0.4 0.4 fe 1.72 2.72 3.43 pb 0.06 0.14 0.23 zn 0.04 0.11 0.08 hi(ʃhq ) 5.58 10.08 10.85 hazard index (hi): the hi values recorded in all the stations exceeded the threshold value (1.0). higher values were recorded in stations 2(10.08)and 3(11.85) compared to 5.58recordedin station 1(table 3).the values could be attributed to the high metal, cdi and hq values recorded in the stations linked to high human activities; indicating that there is potential adverse health risk to humans exposed to drinking water from the river. similar findings were reported by [13] in qua iboe river estuary and [19] for adults in ikwu river, umuahia nigeria. 4. conclusion the study revealed that the river is polluted due to the concentration of heavy metals recorded. metals such as mn, cr, ni, cd, fe and pb exceeded the permissible standards to anthropogenic activities. water with high concentration of metals portends serious health challenge ranging from dysfunction of organs and systems in the body to other associated health risk conditions. the values of some heavy metals, cdi and hq values for cr and fe, and the hi values calls for concern regarding its effects on human health, which could have adverse effects on the people drinking from the river. based on the result of findings, the water is not suitable for human consumption. 5. acknowledgments the author acknowledges the contribution of mr ogbodo chinedu, department of geography, university of nigeria, nsukka nigeria for map production. 6. reference [1] oves, m., khan, m. s., zaidi, m. and ahmad, e., soil contaminations, nutritive value, and human health risk assessment of heavy metals: an overview. in: zaidi, a., wani, p. a, khan, m.s. (eds) toxicity of heavy metals to legumes and bioremediation. springer, vienna, 1-27 pp,(2012). [2]. nasrabadi, t., ruegner, h., sirdari, z. z., schwientek, m. and grathwohi, p.,using total suspended solids (tss) and turbidity as proxies for evaluation of metal transport in river water. applied geochemistry, 6; 1-9, (2016). [3]. popa, c. and petrus, m., heavy metals impact at plants using photoacoustic spectroscopy technology with tunable co2 laser in the quantification of gaseous molecules. microchemical journal, 134: 390-399., (2007). [4]. duruibe, j. o., ogwuegbu, m. o. and egwurugwu, j. n., heavy metal pollution and human biotoxic effects. international journal of physical sciences, 2 (5):112-118., (2007). [5]. prasanna, m. v., chitambaram, s., hameed, a. s. and srinivasamoorthy, k., hydrogeochemical analysis and evaluation of groundwater quality in the gadilam river basin, tamil nadu, india. journal of earth system science, 120 (1):85-98, (2011). [6]. farina, m., avila, d.s., rocha j.b.t., aschner, m., metals, oxidative stress and neurodegeneration: a focus on iron, manganese, and mercury. neurochemistry international, 62:575-594, (2013). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 352 [7]. ahmet, d., fevzi, y., tuna, a. l. and nedim, o., heavy metals in water, sediment and tissues of leuciscuscephalus from s stream in south eastern turkey. chemosphere, 1451-1458, (2006). [8]. anyanwu, e.d. and onyele, o. g., occurrence and concentration of heavy metals in a rural spring in southeastern nigeria.journal of applied science and environmental management, 22 (9): 1473-1478, (2018). [9]. raza, r. and singh, g., heavy metal contamination and its indexing approach for river water. international journal of environmental science and technology, 7 (4): 785-792,(2010). [10]. okon, s.u., atakpa, e. o. and ewaoboho, i., spatial and seasonal characteristics of heavy metals in ikpa river, niger delta, nigeria. international journal of fisheries and aquatic studies, 7(2): 193-199, (2019). [11]. umoren, i u. and onianwu, p. u., surface water quality status and chemical speciation of qua iboe river system, niger delta, nigeria. elixir pollution, 47:9064-9073, (2012) [12]. zhang, c., fundamental of environmental sampling and analysis. wiley, new york, 109pp., (2007). [13]. moses, e. a. and etuk, b. a., human health risk assessment of trace metals in water from qua iboe river estuary, ibeno, nigeria. journal of environmental and occupational science, 3 (4): 150-157, (2015).https://doi.org/10.5455/jeos.2015071412250 4 [14]. usepa iris, us environmental protection agency’s integrated risk information system; environmental protection agency region 1. united states environmental protection agency. washington dc, usa., (2011). [15]. onyele, o. g. and anyanwu, e. d., human health risk assessment of some heavy metals in a rural spring, southeastern nigeria. african journal of environment and natural science research, 1 (1): 15-23, (2018). [16]. usepa, guidance for performing aggregate exposure and risk assessments. office of pesticide programs.united state environmental protection agency,washington dc.usa., (1999). [17]. son, nigerian standard for drinking waterquality. nigerian industrial standard (nis 554-2015). standards organization of nigeria (son) abuja, nigeria, (2015). [18]. ezekiel, o. and dikam, k. i., assessment of concentration status of some heavy metals in water along river dilimi, jos north, plateau state, nigeria. indo. j. urban environ. tech., 4(10):29-44, (2020).https://doi.org/10.25105/urbanenvirotech.v4 i1.6768 [19]. anyanwu, e. d., adetunji, o. g. and nwoke, o. b., heavy metal content of water in ikwu river (umuahia, nigeria): pollution indices and health risk assessment approach. act aquatic turcica, (2022).https://doi.org/10.22392/actaquatr.1060806 [20]. fatemeh, f., amir, h. m., yadollah, j., carcinogenic and non-carcinogenic risk assessment of chromium in drinking water source: birjand, iran. research journal of environmental toxicology, 10:166-171, (2016). [21]. anyanwu, e. d. and umeham, s. n., an index approach to heavy metal pollution assessment of eme river, umuahia, nigeria. sustainability, agriculture, food and environmental research, 8 (x), (2020).https://doi.org/10.7770/safer-vonoart2067. [22]. ayobahan, s.u., ezenwa, i.m., orogun, e. e. uriri, j.e. and wemimo, i. j., assessment of anthropogenic activities on water quality of benin river. journal of applied science and environmental management, 18(4):629-636, (2014). [23]. emurotu, j. e. and habib, l. o., assessment of some water quality of obuburu river okene, kogi state, nigeria. journal of applied sciences and environmental management, 23 (11):1995-2002, (2019). [24]. salem, h. m., eweida, e. a., farag, a., heavy metals in drinking water and their environmental impact on human health. icehm, 542-556, (2000). [25]. salniknow, k. and denkhaus, e., nickel essentially, toxicity and carcinogenicity, critical review in oncology/hematology, 42(1):3556, (2002). [26]. chen, h., teng, y., lu, s., wang, y. and wang, j., contamination features and health risk of soil heavy metals in china. sci. total environ., 512-513:143-153,(2015). https://doi.org./10.1016/j.scitotenv.2015.01.025 [27]. ileperuma, o.a., environmental pollution in sri lanka: a review. j. of the national sci. found. of sri lanka, 28 (4):301-325, (2000).https://doi.org/10.4038/jnsfsr.v28i4.2644. . [28]. anyanwu, e.d. and nwachukwu, e. d., heavy metal content and health risk assessment of a south eastern nigeria river. applied water science, 10:210, (2020).https://doi.org./10.1007/s13201-020-01296y [29]. jonah, a. e., solomon, m. m. and ano, a.o., assessment of the physico-chemical properties and heavy metal status of water samples https://doi.org/10.5455/jeos.20150714122504 https://doi.org/10.5455/jeos.20150714122504 https://doi.org/10.25105/urbanenvirotech.v4i1.6768 https://doi.org/10.25105/urbanenvirotech.v4i1.6768 https://doi.org/10.22392/actaquatr.1060806 https://doi.org/10.7770/safer-vono-art2067 https://doi.org/10.7770/safer-vono-art2067 https://doi.org./10.1016/j.scitotenv.2015.01.025 https://doi.org/10.4038/jnsfsr.v28i4.2644 https://doi.org./10.1007/s13201-020-01296-y https://doi.org./10.1007/s13201-020-01296-y food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxi, issue 4– 2022 udeme effiong jonah, cecilia friday mendie, heavy metals content and health risk assessment of ikpe ikot nkon river, akwa ibom state, nigeria, food and environment safety, volume xxi, issue 4 – 2022, pag. 345 – 353 353 from ohii miri river in abia state, nigeria. merit research journal of environmental science and toxicology, 3(1); 1-11,(2015). [30]. ebong. g. a., udoessien, e. i. and ita, b.n., seasonal variations of some heavy metals concentration in qua iboe river estuary, nigeria. global journal of pure and applied science, 10 (4):611-618, (2004). [31]. elci, l., kartal, a. a. and soylak, m., solid phase extraction method for the determination of iron, lead and chromium by atomic absorption spectrometry using amberite xad-2000 column in various water samples. journal of hazardous materials, 153(1-2): 454461, (2008).https://doi.org/10.1016/j.jhazmat.2007.08.07 5 [32]. ferner, d. j., toxicity, heavy metals. emed. journal, 2 (5):1, (2001). [33]. young, r. a., toxicity profiles: toxicity summary for cadmium, risk assessment information system, rais, university of tennessee, (2005). [34]. muhammad, s., shah, m. t. and khan, s., health risk assessment of heavy metals and their source apportionment in drinking water of kohistan region, northern pakistan. microchemical journal, 98:334-343, (2011).https://doi.org/10.1016/j.microc.2011.03.00 3. [35]. bytyçi, p., fetoshi, o., durmishi, b. h., etemi, f.z., cadraku, h., ismaili, m. and abazi, a.s., status assessment of heavy metals in water of the lepenci river basin, kosova. j. ecol. engr., 19 (5):19-32, (2018).https://doi.org./10.12911/22998993/91273 [36]. paustenbach, d. j., finley, b.l., mowat, f. s. and kerger, b., human health risk and exposure assessment of chromium (vi) in tap water. j. toxic. environ. health. part a, 66 (14): 1295-1339, (2003).https://doi.org/10.1080/15287390306388. [37]. moffat, i., martinova, n., seidel, c. and thomson, c. m., hexavalent chromium in drinking water. journal awwa, 110 (5): e22e35, (2018). [38]. strachan, s., heavy metal. current anaesthesia and critical care, 21:44 –48, (2010). [39]. ekere, n. r., ihedioha, j. f., eze, i. s. and agbazue, v. e., health risk assessment in relation to heavy metals in water sources in rural regions of south east nigeria. international journal of physical sciences, 9 (6): 109-116, (2014).https://doi.org/10.5897/ijps2014.4125. [40]. anyanwu, e. d., adetunji, o. g. and nwachukwu, e. d., application of pollution indices and health risk assessment in the heavy metal content of a south-eastern nigeria river. pollution, 6 (4): 909-923, (2020).https://doi.org/10.22059/poll.2020.303140.8 20 . . https://doi.org/10.1016/j.jhazmat.2007.08.075 https://doi.org/10.1016/j.jhazmat.2007.08.075 https://doi.org/10.1016/j.microc.2011.03.003 https://doi.org/10.1016/j.microc.2011.03.003 https://doi.org./10.12911/22998993/91273 https://doi.org/10.1080/15287390306388 https://doi.org/10.5897/ijps2014.4125 https://doi.org/10.22059/poll.2020.303140.820 https://doi.org/10.22059/poll.2020.303140.820 doi: https://doi.org/10.4316/fens.2023.007 71 journal homepage: http://fens.usv.ro/index.php/fens journal of faculty of food engineering, ştefan cel mare university of suceava, romania volume xxii, issue 1 2023, pag. 71 78 effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus) *olaniyi alaba olopade1, henry eyina dienye1, glory chimburuoma denson1, vivian chiemela onyekwere1 1faculty of agriculture, university of port harcourt, choba, rivers state, nigeria olaniyi.olopade@uniport.edu.ng *corresponding author received 10th august 2022, accepted 28th march 2023 abstract: this study was designed to determine and compare the effects of smoking processes on the proximal composition, fatty acid profile, minerals, vitamins, total polycyclic aromatic hydrocarbons, and sensory characteristics of cultured clarias gariepinus. nine fatty acids were identified from the muscle of fish samples, with all nine fatty acids recorded for both smoked samples (hot and cold), while the raw sample had only eight. furthermore, the most abundant fatty acids in the smoked samples were palmitic acid, oleic acid, stearic acid, and palmitoleic acid. vitamins a, d, e, and k were higher in smoked samples than in raw samples, while vitamins b1, b2, and b3 were higher in raw samples than in smoke samples. raw, cold, and hot smoked samples had significantly different mineral profiles (p<0.05). iron, magnesium, and zinc were found in higher concentrations in the smoked samples examined. cold smoking had the highest value in terms of total polycyclic aromatic hydrocarbons, followed by hot smoking. the hot sample performed better in terms of color, flavor, tenderness, juiciness, texture, and overall acceptability, according to sensory evaluation results. based on the results of the study, hot-smoked c. gariepinus was nutritionally acceptable. keywords: clarias gariepinus, proximal composition, fatty acids, vitamins, minerals, sensory characteristics 1. introduction nigeria is african’s second-largest aquaculture producer, with an annual output of about 300, 000 tons dominated by catfish culture [1]. according to the catfish association of nigeria (cafan), in 2016, nigeria produced 370,000 metric tons of fish from aquaculture systems valued at over usd 1.3 billion [2]. the tremendous increase in production will impose additional challenges in the handling, storage, distribution and processing of fish. this is because fish is extremely perishable food especially in hot climates and tropical areas where cold preservation techniques are often missing. spoilage begins as soon as the fish dies and if fish is not sold fresh, preservation methods should be used to prevent or significanly slow spoilage (loss of quality, edibility or nutritive value) caused or accelerated by micro organisms. currently, smoking is the main method of fish preservation in the artisanal sector [3]. basically, smoking process preserve the fish, enhances flavor and impart good colour to the fish. there are several smoking methods such as hot, cold, liquid and electrostatic [4]. the two commonly used methods in nigeria are cold and hot smoking. cold smoking process requires that the fish never reach an internal cooking temperature. cold smoking mailto:olaniyi.olopade@uniport.edu.ng food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 72 smokehouse temperatures range from 20 and 30 °c (68 to 86 °f) [5]. in this temperature range, foods take on a smoked flavor but remain relatively moist. hot smoking temperatures range from 52 to 80 °c (126 to 176 °f) [5]. when food is smoked within this temperature range, it is fully cooked, moist, and flavorful. heat effects contribute to changes in the chemical composition and, hence, the nutritional value of processed foods [6]. the degrees of change are related to the species of fish as well as the presence or absence of activators and inhibitors [7]. thus, the present study attempted to determine the nutritional values of the cultured clarias gariepinus following hot and cold smoking treatments. 2. matherials and methods samples of clarias gariepinus with the average weight and length of sampled fish 881±20.00 g and 47.00±3.56 cm, respectively, were purchased from a fish farm. the fish samples were transported live to the laboratory and divided into three batches: two batches were prepared separately for cold and hot smoking, while the third was sacrificed immediately and analyzed for proximate, fatty acid, mineral, and vitamin compositions. the fish were then gutted, rewashed, and then separated into three groups: (i) the first group consisted of raw samples kept at -40 °c until analysis; (ii) the second group consisted of cold smoked samples partially dried for 2 hours in the industrial smoking chamber [8] with the temperature fluctuating between 30 and 35 °c and kept at -40 °c until analysis; and (iii) the third group consisted of a hot smoked sample using an industrial smoking chamber with a peripheral smoke generator [9]. proximate composition, moisture content, crude protein, fat and ash were determined for wet samples according to standard methods of the association of official agricultural chemists [10] as follows: (a). the moisture content was determined by oven drying samples at about 105ºc for about 8 to 10 hours until a constant weight was reached, then cooled in a desiccator and weighed again. the percentage of moisture content was calculated by the following equation: moisture percentage (%) = (weight lost/original weight of sample) x 100. (b) the ash content was determined by incineration of the samples for 6 h at 500– 600 °c in a muffle furnace. (c) the ash percentage (%) was calculated using the following equation: ash percentage (%) = (weight of ash/weight of sample) x 100. (d) crude protein was detected by the kjeldahl method [11] (e) crude lipid content was determined by the method presented in [12]. the samples (body tissue) were homogenized with a chloroform: methanol (2:1 v/v) mixture before being extracted using the method [12]. after the fat was extracted, it was esterified with 1% h2so4 and fatty acid methyl esters were prepared by following the procedure of [13]. identification and quantification of fatty acids were done using gas chromatography (hewlett packard 5890, usa). (f) the preparation of samples for mineral elements analysis followed a method described by [10]. approximately 5 g of each sample (wet weight) were placed in a teflon digestion vessel and double acid digested with nitric acid (hno3) and perchloric acid (hclo4). samples were then analysed for mineral contents of iron (fe), manganese (mn), zinc (zn), potassium (k), calcium (ca) magnesium (mg) and manganese (mn) using the atomic absorption spectrophotometer (shimadzu aas, aa-6300). (g) the sample preparation for vitamin analysis was done as per the method of food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 73 [14] were estimated the soluble fat vitamins a, d, e and k and the water soluble vitamins b1, b2, b6, b12 and c by using the high performance liquid chromatography (merk hitachi l-74000, usa) following the method [15]. polycyclic aromatic hydrocarbons (pahs) in the muscle ware quantified according to [16]. only solvents with a high degree of purity (98% hplc) were used; the glass material was washed with extranvr, then it was dried for 4 h at 100 c and rinsed with acetone and hexane. the pahs analysis was performed according to the method described by [17]. after the smoking operation, sensory analyses were performed by using the methods of [18]. sensory evaluations of the samples from the cold and hot processess were carried out with twenty trained individuals. the panelists evaluated each of the smoked fish for colour, flavour, tenderness, juiciness, texture and overall acceptability. a five-point hedonic scale [19] describing the score of attributes of the smoked fish samples as: very bad (1 point), bad (2 points), satisfactory (3 points), good (4 points), very good (5 points), excellent (6 points) was used. statistical analysis the descriptive statistics (mean and standard deviation) were conducted while statistical significance of differences (p < 0.05) was determined by analysis of variance (anova) with spss version 10.0 [20]. 3. results and discussion the proximate composition of fresh and smoked c. gariepinus is given in table 1. hot smoked samples had significantly higher percentages of crude protein content, crude fat content, ash content, and dry matter than cold smoked and raw samples (p < 0.05). results showed that hot smoked sample had higher protein content (67.84±0.06 %) than the cold smoked sample (53.13±0.06 %), while the raw sample had 17.03±0.06 %. the lipid contents of hot and cold smoked samples were 8.96±0.02 % and 4.41±0.02 %, respectively, while the raw sample was 0.77±0.02 %. the same observation was made on the ash content of the hot smoked samples, which had 7.88±0.02 followed by cold smoked samples (4.01±0.0) and raw samples (1.40±0.02). the lipid contents of hot and cold smoked samples were 8.96±0.02 % and 4.41±0.02 %, respectively, while the raw sample was 0.77±0.02%. the low nutritional quality in raw samples is a result of an inverse relationship between the water content and the content of fatty acids and protein; when the water content increases, the content of other nutrients decreases [21]. the proximate composition of raw and smoked fish analyzed in this study is similar to those obtained for the same species by [22]. in the raw stage, c. gariepinus had the highest moisture content (78.07±0.02) followed by cold (21.04±0.02) and hot (4.05±0.02) samples respectively. the main constituent of fish flesh is water, which usually accounts for about 80% of the weight of a fresh white fish fillet [23]. the decrease in moisture content occurring during smoking is the consequence of water loss during this process. furthermore, nitrogen free extract was significantly higher in cold smoked samples than in hot smoked samples and raw samples (p < 0.05). food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 74 table 1 promximate composition of raw, cold, and hot smoked samples of c. gariepinus promximate composition cold smoking hot smoking raw crude protein (%) 53.13±0.06 b 67.84±0.06 a 17.03±0.06 c crude fat (%) 4.41±0.02 b 8.96±0.02 a 0.77±0.02 c ash (%) 4.01±0.04 b 7.88±0.02 a 1.40±0.02 c moisture (%) 21.04±0.02 b 4.05±0.02 c 78.07±0.02 a dry matter (%) 78.97±0.02 b 95.95±0.02 a 21.93±0.02 c nitrogen free extract (%) 17.42±0.10 a 11.29±0.05 b 2.74±0.04 c means (±se) with different superscripts along same rows are significantly different (p<0.05). the fatty acid profile of raw, cold, and hot smoked c. gariepinus is shown in table 2. the number of fatty acids present in any fish species is quite high, but in this study, nine fatty acids were identified from the muscle of fish samples, with all nine fatty acids recorded for both smoked samples (hot and cold), while raw samples had eight fatty acids. the smoking method used showed a significant change in fatty acid composition, and this is in agreement with the results reported by [24]. five saturated fatty acids (sfa), two monounsaturated fatty acids (mufa) and two polyunsaturated fatty acids (pufa) were identified. this fatty acid pattern was significantly different compared to our earlier study of danube catfish composition (mufa>sfa>pufa) [25]. generally, palmitic acid, oleic acid, stearic acid, and palmitoleic acid were the most abundant fatty acids in all the three samples. the differences observed between samples of hot, cold, and raw samples were statistically significant (p < 0.05). in all the samples (raw, cold, and hot), palmitic acid had the highest percentage composition followed by oleic acid, while lauric acid was the lowest (0.02±0.01 g/100g) in the raw sample, and capric acid was the lowest (0.02±0.01 g/100g) in the smoked fish sample. the high level of palmitic acid in this study could be attributed to the fish diet and the general rearing conditions. however, smoked c. gariepinus had a significantly higher palmitic acid (27.26 ± 5.48 g/100g) than that of the raw sample (1.08±0.01 g/100g). a comparative study by [26] revealed that smoked clairas gariepinus has higher fatty acids than raw clairas gariepinus. table 2 fatty acids profile in clarias gariepinus fillets on raw, cold and hot smoking processes fatty acid hot smoking cold smoking raw capric acid c8:0 (sfa) (g/100g) 0.06±0.01 a 0.02±0.01 b 0.00±0.00 b lauric acid c12:0 sfa (g/100g) 0.09±0.02 a 0.12±0.01 a 0.02±0.01 b myristic acid c14:0 sfa (g/100g) 3.94±1.91 ab 5.82±0.03 a 0.30±0.02 b palmitic acid c16:0 sfa (g/100g) 27.26±5.48 a 32.70±0.03 a 1.08±0.01 b stearic acid c18:0 sfa (g/100g) 8.24±2.10 a 10.31±0.02 a 0.70±0.02 b palmitoleic acid c16:1 mufa (g/100g) 10.75±2.86 a 13.59±0.03 a 0.78±0.01 b oleic acid c18:1mufa (g/100g) 13.42±3.77 a 17.17±0.02 a 0.85±0.01 b linoleic acid c18:2 pufa (g/100g) 1.22±0.35 ab 1.55±0.02 a 0.35±0.01 b linolenic acid (ω3) c18:3 pufa(g/100g) 0.91±0.12 a 1.05±0.02 a 0.15±0.02 b means (±se) with different superscripts along same row are significantly different (p<0.05) food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 75 the vitamin compositions of smoked and raw samples of c. gariepinus are presented in table 3. the smoked samples (cold and hot) were statistically significantly higher (p < 0.05) than in the raw sample. in this study, vitamin a displayed high content in both raw and smoked study samples. hot smoked samples had higher levels of vitamins a (45.32±0.03), d (0.63±0.01), e (0.88±0.01), and k (0.35±0.01) than cold smoked samples. vitamins a (27.22±0.04), d(0.38±0.01), e (0.64±0.01) and k (0.23±0.02), while raw samples had the lowest levels of vitamins a (12.70±0.03), d (0.16±0.01) e (0.31±0.02) and k (0.14±0.01). table 3 vitamin content in raw and smoked c. gariepinus vitamins hot cold raw vitamins b1 (mg/100g) 0.04±0.01 b 0.08±0.01 b 0.12±0.01 a vitamins b2 (mg/100g) 0.01±0.00 c 0.02±0.00 b 0.03±0.00 a vitamins b3 (mg/100g) 0.91±0.02 c 1.35±0.01 b 1.70±0.01 a vitamine a(ug/100g) 45.32±0.03 a 27.22±0.04 b 12.70±0.03 c vitamins d(ug/100g) 0.63±0.01 a 0.38±0.01 b 0.16±0.01 c vitamins.e(ug/100g) 0.88±0.01 a 0.64±0.01 b 0.31±0.02 c vitaminsk(ug/100g) 0.35±0.01 a 0.23±0.02 b 0.14±0.01 c means (±se) with different superscripts along same rows are significantly different (p<0.05) there were significant differences (p < 0.05) in vitamin b2 and b3 contents between hot, cold, and raw samples, with the raw values having the highest values followed by the cold and hot samples, respectively. the vitamin b1 content of the raw was higher than that of the smoked samples (hot and cold) (p<0.05). [27] reported that the smoking method had a significant reduction in the vitamin content of some smoked fish samples. this could be due to the temperature of smoking, as some vitamins are heat unstable. smoke particles can react with nutrients in fish meat and may lead to loss of important nutrients and antioxidants [28]. the mineral compositions of raw, cold and hot smoked c. gariepinus are given in table 4. there was generally a significant difference (p<0.005) in the mineral profile between the raw, cold, and hot smoked fish samples, with the cold smoked fish samples having the highest mineral values than the hot and raw samples. similar changes in the content of essential nutrients in thermally treated fish were reported by [29]. iron, magnesium, and zinc were more abundant in the fish samples analysed than calcium, potassium, phosphorus, sodium, magnesium, and iron. the mineral content in this study followed the same pattern with the records for the cold smoked samples followed by the hot and raw samples, respectively. fresh fish contains a significant amount of minerals in general, but processed fish, such as dried fish, have higher values [30]. the total polycyclic aromatic hydrocarbon of the raw, cold, and hot smoked c.gariepinus is presented in table 5. the result shows that there are significantly different (p<0.05) across the various samples. it was observed that cold smoking had the highest value, followed by hot smoking. the results of this study revealed that cold smoking had a higher mean value than hot smoking, while no value was recorded for the raw sample. the direct contact of the product with combustion gases is definitely an important source of pah contamination [31]. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 76 fish samples with a shorter smoking time generated unexpectedly higher pah concentrations than those with a longer smoking time [32]. table 4 raw and smoked mineral compositions of c. gariepinus minerals hot cold raw manganese (mg/kg) 125.91±0.02 b 268.36±0.02 a 101.55±0.02 c calcium% 0.46±0.00 b 0.79±0.00 a 0.13±0.00 c phosphorus % 0.26±0.00 b 0.42±0.00 a 0.09±0.00 c magnesium% 0.19±0.00 b 0.24±0.00 a 0.09±0.00 c potassium % 0.22±0.00 b 0.41±0.00 a 0.10±0.00 c sodium % 0.18±0.00 b 0.31±0.00 a 0.09±0.00 c iron(mg/kg) 137.83±0.02 b 235.65±0.01 a 112.70±0.02 c zinc(mg/kg) 23.48±0.02 b 37.91±0.02 a 12.07±0.02 c means (±se) with different superscripts along same rows are significantly different (p<0.05) table 5 total polyaromatic hydrocarbon concentrations in the raw, cold, and hot smoked c. gariepinus sample total polyaromatic hydrocarbon (mg/kg) cold smoking 0.03±0.00 a hot smoking 0.01±0.00 b raw 0.00±0.00 c means (±se) with different superscripts along same column are significantly different (p<0.05) the organoleptic characteristics of hot and cold smoked samples of c. gariepinus are presented in tables 6 and 7. the result shows that there is no significant difference (p>0.05) across the various samples. the panelists' assessment rated the hot smoking higher than the cold smoking in terms of colour (1.75±0.50 vs.1.40±0.55), flavour (1.75±0.96 vs.1.40±0.55), tenderness (1.75±1.50 vs. 1.40±0.89), texture (1.75±1.50 vs. 1.40±0.55). also, in terms of overall acceptability and juiceness scores, hot smoking was also rated higher (1.75±0.96) than cold smoking (1.40±1.14). therefore, the hot smoking process presented better results for organoleptic traits. food colour helps to determine quality, degree of processing or spoilage level [33]. table 6 sensory evaluation of smoked c. gariepinus by sensory panelist variables cold hot t-value p-value colour 1.40±0.55 1.75±0.50 -0.99 0.36 flavour 1.40±0.55 1.75±0.96 -0.70 0.51 tenderness 1.40±0.89 1.75±1.50 -0.44 0.68 juiciness 1.40±1.14 1.75±0.96 -0.49 0.64 texture 1.40±0.55 1.75±1.50 -0.49 0.64 overall acceptability 1.40±0.55 1.75±1.71 -0.44 0.68 not significantly different (p>0.05) the result of the t test in table 7 showed that there was no significant difference in cold and hot smoking samples as determined by the sensory panelists. however, the hot smoked fish was more appreciated than cold smoked. food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 77 table 7 differences between cold and hot smoked c. gariepinus by sensory panelist variables mean std. deviation t-value p-value cold smoking 3.91 0.15 -1.63 0.14 hot smoking 4.07 0.20 not significantly different (p>0.05) 4. conclusion in conclusion, both hot and cold smoking processes had significant effects on proximal composition, fatty acids, vitamins, and mineral contents, in line with the findings of this study. the nutritional value of catfish was also highly improved by hot smoking as compared to cold smoking, and a comparison of the hot and cold smoking methods of c. gariepinus demonstrates that the hot method is preferable due to its superior organoleptic characteristics. polycyclic aromatic hydrocarbons were found to be less than 0.01 ppb for both cold and hot smoking, indicating that they are safe to consume. 5. references [1]. fao the state of world fisheries and aquaculture 2018‐meeting the sustainable development goals. licence: cc by-nc-sa 3.0 igo (2018). http://www.fao.org/3/i9540en/i9540en.pdf. [2]. businessday, n175bn worth of fish produced in 2016. http://www.businessdayonline.com/n175bn-worthfish-produced-2016/. [google scholar] (2017). [3]. olopade, o. a., taiwo, i. o. & agbato, d.a., effect of traditional smoking method on nutritive values and organoleptic properties of sarotherodon galilaeus and oreochromis niloticus international journal of applied agricultural and apicultural research ijaaar 9 (1&2): 91-97. (2013). [4]. nunes, m.l., defumação. in: ogawa, m.; maia, e.l. (eds.) manual de pesca ciência e tecnologia do pescado. são paulo: varela, 1999. p. 300-306. (1999). [5]. myrvold, n., modernist cuisine. the cooking lab. p. 143. isbn 978-0-9827610-0-7 (2011). [6]. garcía-arias m.t., alvarezpontes e., garcía-linares m.c., garcíafernández m.c. & sánchez-muniz f.j. cooking–freezing–reheating (cfr) of sardine (sardina pilchardus) fillets. effect of different cooking and reheating procedures on the proximate and fatty acid compositions. food chem, 83:349– 356. doi: 10.1016/s0308-8146(03)00095-5. (2003). [7]. goulas a.e. & kontominas m.c., effects of salting and smoking method on the keeping quality of chub mackerel (scomber japonicas): biochemical and sensory attributes. food chem., 93: 511520. (2005). [8]. machado, z.l. tecnologia de recursos pesqueiros. recife: minter e sudene. 277p. (1984). [9]. gonçalves a.a. & prenticehernández c., fumaça líquida: uma tecnologia para de fumar pescado. boletim sbcta, 32 (2), 189–199. (1998). [10]. aoac, association of official analytical chemists official methods of analysis. 17th edition. w. hortuntzed (ed.), washington, usa. 21447. (2000). [11]. matissek, r., schnepel, f.m., steiner, g., lebensmittelanaliytik springer verlag, berlin, heidelberg, p 440. (1989). [12]. bligh, e. g. & dyer, w. j., a rapid method of total lipid extraction and purification. can j biochem physiol, 37: 911-917. (1959). [13]. aoac, official methods of analysis. 13th edn. vol. 15, aoac, arlington, va., pp: 132-320(1995). [14]. sankar, t.v., susheela, m., anandan, r., asha, k.k. & mohanty, b.p., nutrient profiling of fish. icarcentral institute of fisheries technology, cochin, kerala, india. pp 1-61. (2010). [15]. sadasivam, s., & manickam, a, biochemical methods. 2nd ed. new delhi, india: new age international (p) ltd.; p. 179-186(1996). (1996). [16]. vallarino, a. & rendon-von osten, j., “comparison of organochlorine and pahs residues in terns eggs from two natural protected areas in the gulf of mexico,” marine pollution bulletin 116, no. 1-2: 48–55. (2017) http://www.fao.org/3/i9540en/i9540en.pdf http://www.businessdayonline.com/n175bn-worth-fish-produced-2016/ http://www.businessdayonline.com/n175bn-worth-fish-produced-2016/ http://scholar.google.com/scholar_lookup?hl=en&publication_year=2017&author=businessday&title=n175bn+worth+of+fish+produced+in+2016 https://en.wikipedia.org/wiki/isbn_(identifier) https://en.wikipedia.org/wiki/special:booksources/978-0-9827610-0-7 food and environment safety journal of faculty of food engineering, ştefan cel mare university suceava volume xxii, issue 1 – 2023 olaniyi alaba olopade, henry eyina dienye, glory chimburuoma denson, vivian chiemela onyekwere, effects of smoking processes on the nutritional value of cultured catfish (clarias gariepinus), food and environment safety, volume xxii, issue 1 2023, pag. 71 78 78 [17]. zhang y., wang f., wei h., wu z., zhao q. & jiang x., enhanced biodegradation of poorly available polycyclic aromatic hydrocarbons by easily available one, international biodeterioration & biodegradation, 84:72-78. (2013). [18]. amerina, m.a., pangborn, r.v. & roessler, e.b., principles of sensory evalution of food. new york: academic prees. 602 pp. (1965). [19]. clucas, i. j. & sutcliffe p. j., an introduction to fish handling and processing; tropical products institute isbn: o85954-124-x (1981). [20]. duncan, d. b., multiple range and multiple f test. biometrics.11:1-42(1955). [21]. efsa. opinion of the scientific panel on contaminants in the food chain [contam] related to the safety assessment of wild and farmed fish. efsa j. 3. (2005). [22]. fapohunda, o. o., & ogunkoya, m., effect of smoke-drying on the proximate composition of tilapia zillii, parachanna obscura and clarias gariepinus obtained from akure, ondo-state, nigeria. animal research international, 3(2), 478-480. (2006). [23]. murray, j. & burt, j.r., the composition of fish. torry advisory. note 38, torry research station, aberdeen. accessed on 31 august 2014. http://www.fao.org/wairdocs/ tan/x5916e/x5916e00.htm (1969). [24]. djopnang, d.j., tchoumbougnant, f., tomedi tem, womeni, h.m., tonfack d.f., prabhakar i. n.s., karuna, m. s. l., prasad, r. b. n., kemmo, s.s. & gouado, i., effects of boiling and smoking on proximate composition and oil quality of commercially important freshwater fish (chrysichthys nigrodigitatus) from nkam river in cameroon. j. food res., 7(6): 5969. doi: 10.5539/jfr.v7n6p59. (2018). [25]. stancheva, m., merdzhanova, a,, dobreva, d. & makedonski, l., common carp (cyprinus caprio) and european catfish (sillurus glanis) from the danube river as sources of fat soluble vitamins and fatty acids. czech j. of food sci., 32 (1): 16–24. (2014). [26]. saliu, l.m., akpabio c. j. & ogunsola m. o. (2013). comparative nutritional studies on fresh and smoked clarias gariepinus (cafish) and tilapia niloticus fish. european journal of experimental biology, 3(5):183-185. [27]. adeyeye, s.a.o, fayemi, o. e. & oyetoro, a. o. a. (2018). amino acid, vitain and mineral profiles of smoked fish as affected by smoking methods and fish types. journal of cullinary science and technology. 1-14. [28]. abraha b., admassu h., mahmud a., tsighe n., shui x.w. & fang y. (2018). effect of processing methods on nutritional and physicochemical composition of fish: a review. moj food process technol. 6:376–382. doi: 10.15406/mojfpt.2018.06.00191. [29]. bastías, j.m., balladares, p., acuña, s., quevedo, r. & muñoz, o. (2017). determining the effect of different cooking methods on the nutritional composition of salmon (salmo salar) and chilean jack mackerel (trachurus murphyi) fillets. plos one 12, e0180993. [30]. kinsella, j.e. (1986). food components with potential therapeutic benefits: the n-3 poly unsaturated fatty acids of fish oils. food technology 40(2): 89-97. [31]. purcaro, g., moret, s. & l. s. conte, (2013) “overview on polycyclic aromatic hydrocarbons: occurrence, legislation and innovative determination in foods,” talanta, 105: 292–305. [32]. hokkanen, m., luhtasela, u, kostamo, p., ritvanen, t., peltonen, k. & jestoi, m. (2018). critical effects of smoking parameters on the levels of polycyclic aromatic hydrocarbons in traditionally smoked fish and meat products in finland. hindawi journal of chemistry, article id 2160958, 14pp. [33]. clifford, m. n., tang, s. l. and eyo, a. a. (1980). smoking of food. proc. biochem., 8-11. 1. introduction 4. conclusion